Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Physiology of small contractile arteries : Ca²⁺-sensitization in myogenic tone and glucose transport… Gaudreault, Nathalie 2004

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2005-994767.pdf [ 23.07MB ]
Metadata
JSON: 831-1.0099840.json
JSON-LD: 831-1.0099840-ld.json
RDF/XML (Pretty): 831-1.0099840-rdf.xml
RDF/JSON: 831-1.0099840-rdf.json
Turtle: 831-1.0099840-turtle.txt
N-Triples: 831-1.0099840-rdf-ntriples.txt
Original Record: 831-1.0099840-source.json
Full Text
831-1.0099840-fulltext.txt
Citation
831-1.0099840.ris

Full Text

Physiology of small contractile arteries Ca -sensitization in myogenic tone and glucose transport i n endothelial cells. 2+  by  Nathalie Gaudreault B.Sc, Laval University, 1996 M.Sc, Laval University, 1999  A thesis submitted in partial fulfilment of The requirements for the degree of Doctor of Philosophy In  The Faculty of Graduate Studies Department of Physiology  The University of British Columbia December 2004 © Nathalie Gaudreault, 2004  Abstract Recent evidence suggests the involvement of C a  2 +  sensitization in the  development of myogenic tone, possibly mediated by P K C and Rho-kinase. To investigate this, rat cerebral arteries were mounted on a pressure myograph, and pressure-induced constrictions and changes in intracellular [Ca The ratio of the change in diameter to change in  [Ca ]i 2+  2+  were recorded.  ]i  was greater for pressure-  induced constriction compared with constriction produced by depolarization with 60 m M KPSS. Pressure-induced constriction in depolarised arteries was not associated with further increases in  [Ca ]i 2+  but was abolished by selective  inhibitors of P K C , and Rho kinase. These data suggest that in addition to increases in  [Ca ]i, 2+  enhanced myofilament C a  2 +  sensitivity, mediated by P K C and  Rho kinase activation, occurs during myogenic tone development.  Although  it is  well  established  that  elevated  intracellular glucose  concentration leads to endothelial dysfunction, how and why glucose tends to accumulate i n E C s remains poorly understood. The effects of hyperglycaemia on the expression and subcellular distribution of G L U T - 1 to 5 and SGLT-1 in ECs of rat microvasculature were examined. We found, through immu^ohistochemistry and fluorescence microscopy that all transporters except SGLT-1 were expressed preferentially at the cell-to-cell junction and on the abluminal side of these cells. Hyperglycaemia,  significantly  downregulated  GLUT-1,  3,  4  and  5  and  dramatically upregulated G L U T - 2 ; leaving SGLT-1 unchanged.  To determine the functionality of these glucose transporters in ECs, glucose uptake was monitored with a fluorescent glucose analog in live coronary  ii  arteries. The arteries were mounted in an arteriograph chamber on the stage of a confocal microscope. Results show a dense accumulation of glucose at the ECs periphery, as predicted by the subcellular distribution of the transporters. In addition, an increase in glucose uptake was observed in the presence of insulin.  We conclude that the high susceptibility to glucose toxicity of ECs may be the result of the subcellular organization of their GLUTs, and the increased expression of GLUT-2. The asymmetric subcellular organisation of GLUTs may facilitate transcellular glucose exchange between the blood and the cells of the vascular wall. Finally, it was demonstrated that the endothelium of coronary arteries is insulin sensitive.  iii  Contents Abstract  ii  Contents  iv  List of Tables  viii  List of Figures  ix  List of Abbreviations  xii  Acknowledgements  xvi  Preamble  .  xviii  Chapter 1 Ca -sensitization mechanisms in myogenic tone 2+  1.1 Introduction  1 1  1.1.1 The contractile apparatus  1 3  1.1.2 C a  2 +  regulation  1.1.3 C a  2 +  sensitization  ,  1.1.4 P K C  7 9  1.1.5 Rho-associated kinase  13  1.1.6 Myogenic tone  18  1.1.7 Signalling mechanisms in myogenic tone  19  1.1.8 Ca -sensitization in myogenic tone  27  1.1.9 Hypothesis  28  1.1.10 Pressure myography  28  1.1.11 C a  29  2+  2 +  measurements  1.2 Methods  30  1.2.1 Arterial diameter measurements  30  1.2.2 Measurements of [Ca ]s  32  1.2.3 Experimental procedures  33  1.2.4 Expression of the results and statistical analysis  34  1.2.5 Drugs and solutions  35  2+  1.3 Results  35  1.3.1 Myogenic tone in rat cerebral resistance arteries  iv  35  1.3.2 Effect of 60 m M KPSS on myogenic tone  37  1.3.3 Calphosfin C inhibits vascular tone  39  1.3.4 Effect of calphostin C and Y-27632 in depolarized arteries  42  1.3.5 Y-27632 inhibits vascular tone  46  1.3.6 Y-27632 inhibits C a  47  2 +  entry  1.4 Discussion  50  1.4.1 Ca -sensitization contribution to myogenic response  50  1.4.2 Ca -sensitization is mediated by P K C and Rho-kinase  51  1.4.3 Activation of Rho-kinase by membrane depolarization  52  1.4.4 Summary  55  2+  2+  Chapter 2 Characterization of glucose transporters in endothelial cells of small contractile arteries 56 2.1 Introduction  56  2.1.1 The glucose paradox  56  2.1.2 The endothelium  59  2.1.3 Hyperglycaemia  61  2.1.4 Glucose transporters  65  2.1.5 Hypothesis  73  2.1.6 Native endothelium preparation  73  2.1.7 Fluorescence microscopy and deconvolution  74  2.1.8 S T Z animal model  75  2.2 Methods  77  2.2.1 Animals  77  2.2.2 Tissue preparation  77  2.2.3 ImmuTioWstochemistry....  78  2.2.4 Acetylated L D L uptake  81  2.2.5 Western blots  81  2.2.6 Immuriostaining controls  83  2.2.7 3D Image acquisition, deconvolution and analysis  84  2.2.8 Data quantification and analysis  85  2.3 Results  86  2.3.1 Weight and blood glucose  86  2.3.2 Identification of endothelial cells  87  2.3.3 Antibody specificity  90  2.3.4 Immunolocalization  96  v  2.3.5 Effect of long-term hyperglycaemia  ;  2.4 Discussion  106 109  2.4.1 Immimostaining  110  2.4.2 G L U T - 1  Ill  2.4.3 G L U T - 2  112  2.4.4 G L U T - 3 , 4 , and 5  112  2.4.5 SGLT-1  113  2.4.6 Other transporters  113  2.4.7 Cell junction labelling  114  2.4.8 Similarity amongst vascular beds  114  2.4.9 Effect of hyperglycaemia  115  2.4.10 Intracellular glucose accumulation  117  Chapter 3 Measurements of glucose uptake in endothelial cells 3.1 Introduction  119 119  3.1.1 Glucose as a principal source of energy  119  3.1.2 Glucose metabolism in E C  120  3.1.3 Regulation of glucose uptake in E C  120  3.1.4 Glucose metabolism in S M C  125  3.1.5 Regulation of glucose uptake in S M C s  126  3.1.6 Aims of the present study and hypothesis  128  3.1.7 In vivo fluorescence microscopy  129  3.1.8 Fluorescent glucose analog  130  3.2 Methods  132  3.2.1 Cell culture  133  3.2.2 IiruTiunocytochernistry  133  3.2.3 3D Image acquisition, deconvolution and analysis  134  3.2.4 Measurements of 2-NBDG uptake in H C A E C s  135  3.2.5 Measurements of 2-NBDG uptake in ECs of intact vessels  136  3.2.6 Experimental protocols  137  3.2.7 Fluorescence quantification and analysis  140  3.3 Results  142  3.3.1 E C identification in cultured H C A E C s  142  3.3.2 2-NBDG uptake in H C A E C s  152  3.3.3 2-NBDG uptake in native endothehum of intact coronary artery.... 157 3.4 Discussion  163  vi  3.4.1 G L U T s and SGLT-1 identification in H C A E C s  163  3.4.2 2 - N B D G uptake in H C A E C s  165  3.4.3 Inhibitory effect of D-glucose and cytochalasin B in H C A E C s  166  3.4.4 Effect of insulin in H C A E C s  167  3.4.5 2-NBDG uptake in native E C  169  3.4.6 Inhibitory effect of D-glucose and cytochalasin B in native E C  170  3.4.7 Effect of insulin in native E C  171  3.4.8 Difference in responses between H C A E C s and native ECs  171  Epilogue  173  References 179 Appendix 1 Calphostin C and Y-27632 non-interaction with Fura-2 222 Appendix 2 Western blots negative controls  224  Appendix 3 FWHM and image depth measurements  225  Appendix 4 D-glucose blunts insulin effects in HCAECs  227  Appendix 5 Modulation of PKC and SGLT  228  vii  List of Tables Table 2.1 Summary of the principal characteristics of the sugar transporters  69  Table 2.2 List of the primary antibodies  79  viii  List of Figures Chapter 1 Figure 1.1 C a  regulation and Ca -dependent contraction in S M C s  7  Figure 1.2 Ca -dependent and Ca -independent activation of P K C  10  Figure 1.3 Ca -sensitization mechanisms in smooth muscle contraction  16  2 +  2+  2+  2+  2+  Figure 1.4 Effects of mtralurninal pressure on rat cerebral arteries diameter and 340/380 fluorescence ratio  37  Figure 1.5 Effects of intraluminal pressure on depolarised cerebral arteries  39  Figure 1.6 Effect of calphostin C on myogenic tone and 340/380 ratio  41  Figure 1.7 Effect of calphostin C on pre-contracted arteries  42  Figure 1.8 Effects of calphostin C on depolarized and pressurized arteries  44  Figure 1.9 Effects of Y-27632 on depolarised and pressurized arteries  45  Figure 1.10 Extent of Y-27632 inhibition of vascular tone  47  Figure 1.11 Effect of Y-27632 on depolarised arteries  48  Figure 1.12 Effect of different pharmacologic inhibitors of C a  2 +  sensitization  49  Chapter 2 Figure 2.1 Daily morning weight and blood glucose measurements  87  Figure 2.2 Identification of ECs  89  Figure 2.3 E C and S M C identification in coronary artery tissue lysate  90  Figure 2.4 Determination of antibody specificity with Western blots  91  Figure 2.5 Immunohistochemistry demonstrating antibody specificity  93  Figure 2.6 Secondary antibody cross-reactivity controls  95  Figure 2.7 Competitive blocking peptide controls  96  Figure 2.8 Digital manipulations schematic of the quantitative analysis  98  Figure 2.9 G L U T - 1 subcellular distribution in ECs  99  ix  Figure 2.10 G L U T - 2 subcellular distribution in ECs  100  Figure 2.11 G L U T - 3 subcellular distribution in E C s  101  Figure 2.12 G L U T - 4 subcellular distribution in ECs  102  Figure 2.13 G L U T - 5 subcellular distribution in ECs  103  Figure 2.14 SGLT-1 subcellular distribution in E C s  104  Figure 2.15 Effect of hyperglycaemia on the Abluminal: Luminal ratio  107  Figure 2.16 Effect of hyperglycaemia on transporters subcellular distribution. 108 Figure 2.17 Effect of hyperglycaemia in the expression of the transporters  109  Chapter 3 Figure 3.1 Time course of 2 - N B D G uptake; experimental protocols  140  Figure 3.2 Dual Imaging of phase contrast and fluorescence in H C A E C s  141  Figure 3.3 Identification of E C s in culture of H C A E C s  143  Figure 3.4 G L U T - 1 in H C A E C  144  Figure 3.5 G L U T - 2 in H C A E C  145  Figure 3.6 G L U T - 3 in H C A E C  146  Figure 3.7 G L U T - 4 in H C A E C  147  Figure 3.8 G L U T - 5 in H C A E C  148  Figure 3.9 SGLT-1 in H C A E C  149  Figure 3.10 Secondary antibody controls in H C A E C s  150  Figure 3.11 Secondary antibody cross-reactivity controls in H C A E C s  151  Figure 3.12 Rate of fluorescent glucose uptake in H C A E C s  154  Figure 3.13 D-glucose inhibition of 2 - N B D G uptake in H C A E C s  155  Figure 3.14 Effect of cytochalasin B on 2 - N B D G uptake in H C A E C s  156  Figure 3.15 Effect of insulin on 2 - N B D G uptake in H C A E C s  157  Figure 3.16 Phase contrast and fluorescence in whole vessel preparation  159  Figure 3.17 Rate of 2 - N B D G uptake in E C s of intact coronary artery  160  Figure 3.18 Effect of D-glucose on 2-NBDG uptake in native ECs  161  Figure 3.19 Effect of cytochalasin B on 2 - N B D G uptake in native ECs  162  Figure 3.20 Effect of insulin on 2 - N B D G uptake in native ECs  162  Appendices Figure A l Spectrum of the 340/380 ratio  223  Figure A 2 Histogram of the 340/380 ratio  223  Figure A 3 Secondary antibody negative controls  224  Figure A 4 Lateral fluorescence intensity distribution  226  Figure A 5 Axial fluorescence intensity distribution  226  Figure A 6 Effect of D-glucose on insulin stimulation in H C A E C s  227  Figure A 7 Regulation by P K C of the 2-NBDG uptake in H C A E C s  229  Figure A 8 Effect of Phloridzin on 2 - N B D G uptake in H C A E C s  230  xi  List of Abbreviations AA  Arachidonic acid  ANOVA  analysis of variance  ADP  adenosine diphsphate  AGE  advanced glycation end-product  ATP  adenosine triphosphate  BBB  blood brain barrier  BAEC  bovine aortic endothelial cells  CaM  calmodulin  CCD  charge-coupled device  CFP+  carbonium ions  CPI-17  P K C targeted protein phosphatase-! inhibitor of 17KDa  DABCO  l,4-diazabicyclo[2.2.2] octane (triemylenediamine)  DAG  Diacylglycerol  DAPI  4^6-diamidmo-2-phenylindole dihydrochloride  DCCT  Diabetes Control and Complications Trial  2DG  2-deoxy-D-glucose  DMSO  dimethyl sulfoxide  EC(s)  endothelial cell(s)  EDTA  ethylenediaminetetraacetic acid  EGM-2  endothelial growth medium-2  EGF  endothehal growth factor  EGTA  ethylene glycol-bis(2-aminoethylether)-N N N' N'-tetraacetic acid  eNOS  endothehal nitric oxide synthase  EPR  exhaustive proton reassignment  FFA  free fatty acid  FITC  fluorescein isothiocyanate  FRET  fluorescence resonance energy transfer  GAPDH  glyceraldehydes-3-phosphate dehydrogenase  /  xii  /  /  GDP  guanosine diphosphate  GLUT  facilitative glucose transporter  GTPyS  non-hydrolysable GTP analogue  HCAEC(s)  .  human coronary artery endothelial cell(s)  HDL  high density lipoprotein  hEGF  epidermal growth factor, recombinant  HETE  hydroxyeicosatetraenoic acid  hFGF  fibroblastic growth factor, recombinant  HMIT  H -myo-inositol cotransporter  Hoechst  trihydrochloride, trihydrate  HUVEC  human umbilical vein-derived ECs  IGEPAL  Ethoxylate octylphenol  IGF  Insulin-like growth factor  ILK  Integrin-linked kinase  IPs  Ionositol-1,4-5-triphosphate  iPA2  Phospholipase A 2 isoform  Kca  Ca -activated potassium channel  kDa  kilo Dalton  Km  Michaelis constant=[substrate] at which the velocity(V) is V2V  LDL  low density lipoprotein  LGCC  ligand-gated Ca channel  Lyso PC  lysophospholipids choline  MAP-kinase  mitogen-activated protein-kinase  MBS  myosin binding site  MDG  a-methyl-D-glucose  MLC  myosin light chain  MLCK  myosin light chain kinase  MLCP  myosin light chain phosphatase  Mono  monoclonal  MYPT1  myosin phosphatase target 1  NADPH  nicotinamide adenine dinucleotide phosphate  2-NBDG  2-[N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl) amino]-2-deoxyglucc  +  2+  2+  xiii  6-NBDG  6-[N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl) amino]-2-deoxyglucose  n.d.  not determined  NF-kB  nuclear f actor-kB  NIH  National Institute for Health-  NO  nitric oxide  30MG  3-O-methyl-D-glucose  PA  phosphatidic acid  PARP  poly ADP-ribose polymerase  PAI  plasminogen activator inhibitor  PBS  phosphate buffered saline  PC-PLC  phosphatidylcholine-specific phospholipase C  PC-PLD  phosphatidylcholine-specific phospholipase D  PDGF  platelet-derived growth factor  PI-PLC  phosphatidylinositol 4,5,-diphosphate  PI3-kinase  phosohatidylinositol-3 kinase  PKM  protein kinase M  PLA  phospholipase A  PMCA  Ca -ATPase pump  poly  polyclonal  PSF  point-spread-function  PSS  physiological salt solution  RAGE  receptor for A G E  RhoA-GEF  RhoA-guanine nucleotide exchanger factor  RhoA-GTP  RhoA-guanosine triphosphate  Rho-GDI  Rho-guanine nucleotide dissociation inhibitor  R3-IGF  insulin-like growth factor-1  RyR  ryanodine receptor  SAC  stretch-activated cation channel  SERCA  sarcoplasmic reticulum Ca -ATPase pump  SMC(s)  smooth muscle cell(s)  SM22  smooth muscle specific protein 22KDa  SGLT  sodium-dependent glucose transporter  2+  2+  XIV  SE  standard error  soc  store operated Ca -entry mechanisms  SPC  sphingosyl phosphorylcholine  SR  sarcoplasmic reticulum  STOC  spontaneous transient outward current  STZ  streptozotocin  TBS-T  tris-buffered saline-tween  TGF  United Kingdom Prospective Diabetes Study  TLC  Thin layer chromatography  TNF-cx  Tumour necrosis factor-a  Trp  Transient receptor potential channel  Tween  Polyoxyethylene sorbitan monolaurate  UKPDS  transforming growth factor  VE  vascular Endothelial  VEGF  vascular endothelial growth factor  VGCC  voltage-gated Ca channel  VLDL  very low density lipoprotein  Vmax  maximal transport velocity  ZIP  Zipper-interacting protein  2+  2+  XV  Acknowledgements Many people have contributed, throughout the past 6 years, to this Ph.D. Some have provided intellectual guidance and technical help but others, even from a considerable distance, have provided me with unfailing moral support. It is therefore with great pleasure that I take this opportunity to express my gratitude to each of them.  I would like to express my sincere gratitude to my supervisor Dr. E d Moore, for giving me the opportunity to do my Ph.D. in his laboratory, for his patience and understanding, and most of all for giving me the freedom and latitude to pursue my own goals and ideas. I must also thank E d for his many revisions of my manuscripts including this thesis, for correcting my English style and grammar, and offering suggestions for improvement. I am also indebted to Dr.  David  Scriven for his  detailed  and  constructive  comments  on  my  manuscripts, and for his broad computer knowledge, which he so generously shared.  I am deeply grateful to Dr. Ismail Laher for the many opportunities he provided to learn new techniques, meet people from the scientific community, and participate in various activities with members of his laboratory. Special thanks to Dr. G u y Lagaud for sharing his expertise in pressure myography with me, and for the opportunity to participate to his project.  I would like to thank Dr. Raymond Pederson and Jennifer Martin for providing the streptozotocin and help with the animal injections, and Dr.  xvi  Kenneth Baimbridge for the primary neuron cultures. I am also grateful to Dr. Christopher Mcintosh for his critical reading of the manuscript presented in chapter 2 and valuable discussions. I would also like to thank all members of my Ph.D. committee, Dr. Pederson, Dr. Laher and Dr. McNeill, for guiding my work and providing me with valuable comments on my thesis. For their financial support, I thank the Heart and Stroke Foundation of British Columbia and the Yukon.  I  would  like  to  thank  Tim Blanche from  the  Department of  Ophthalmology, who provided technical and intellectual support in the development of techniques used to measure fluorescent glucose uptake in cells of the vascular wall. I would also like to thank Pauline Dan and Jodene Eldstrom for sharing with me their expertise and knowledge, for their help and friendship.  This thesis represents a significant scientific accomplishment in my life and has been made a lot easier by all the people who supported me and believed in me. I would like to thank my parents, Normand and Mariette for their unconditional love, encouragement and constant involvement in my life in spite of our geographical separation.  Finally, I thank Tim for sharing this adventure with me, for his inspiration, love and support, and for helping me be a better scientist and a better person.  xvii  Preamble The prevalence of cardiovascular diseases, associated with coronary atherosclerosis, is eminent in diabetics, such that this population is more at risk for cardiovascular diseases than non-diabetics. This increased susceptibility to cardiovascular diseases in diabetic patients has been associated, amongst other risk factors, with early and poorly controlled elevated blood sugar levels. The endotheUum, the first line of protection of the vascular wall, has been shown to be dysfunctional in persons with diabetes, an impairment attributed to glucose toxicity.  Despite a strong association between glucose toxicity and endothehal dysfunction,  very  little  is  known  about  glucose  transport regulation in  endothehal cells (ECs). A critical question remains; how and why does glucose accumulate in ECs?  The ultimate goal was to study the regulation of glucose transport in E C s of a whole vessel preparation because E C s and smooth muscle cells (SMCs) function as a unit and modulate each other's actions. The development of an experimental design and protocol that preserves this close structural relationship was therefore the objective of this study.  Over the course of this Ph.D. I have been given the opportunity to work in the laboratory of Dr. Ismael Laher in the Department of Pharmaceuticals and Therapeutics. In close collaboration with Dr. G u y Lagaud, a post-doctoral fellow, I learned different techniques for studying, in vitro, the physiology of small  xviii  contractile arteries. M y work with Dr. Lagaud concerned the involvement of Ca -sensitization mechanisms in the myogenic response of the rat cerebral 2+  arteries. This work was published in the American Journal of Physiology in (Lagaud et al,  2002  2002). Chapter 1 presents the parts of this published material,  which I was involved with; the data collection, analyses and part of the redaction. Complementary data further to this work are also included in Chapter 1, and will be submitted for publication in the near future.  In  the  second  part of this  Ph.D.,  having acquired techniques  for  microdissection and isolation of blood vessels in Dr. Laher's laboratory, I undertook a study of glucose transport regulation in ECs of intact arteries of the rat. I succeeded in exposing the cells of interest by opening freshly dissected vessel longitudinally without damage approach, I investigated  to the endothelium. With this  new  the expression and subcellular distribution of the  different glucose transporter isoforms of the ECs using immunohistochemistry and fluorescence microscopy in Dr. Edwin Moore's laboratory in the Department of Physiology. This work is presented in Chapter 2 and was recently accepted for publication in Diabetologia.  In the third part of my Ph.D. I combined the in vitro techniques of pressure myography from Dr. Laher's laboratory with confocal fluorescence microscopy to measure, in real time, glucose uptake in individual ECs of the intact vascular wall. The use of a fluorescent glucose analog provided the ideal tool for studying glucose kinetics of individual cells in their native environment. Chapter 3 is a logical extension of chapter 2 in that it presents a preliminary assessment of the functionality of the transporter isoforms identified in intact coronary arteries. It also includes the initial experiments performed in cultured ECs as groundwork  xix  for the ultimate experiments in whole vessels. This work will also shortly be submitted for publication.  Bonne lecture!  xx  Chapter 1  Ca -sensitization mechanisms in myogenic tone 2+  1.1 Introduction  1.1.1 The contractile apparatus  A link between smooth muscle contraction and increased cytoplasmic calcium concentration was first established in 1965 (Filo et al, 1965). The "sliding filament theory" originally exclusively attributed to skeletal muscle (Hill, 1970), was then found applicable to smooth muscle cells (SMCs). Skeletal muscle cells have cytoskeletal anchoring proteins composed of thick myosin-containing and thin actin / tropomyosin - containing filaments. These proteins are organized into repeating contractile units called sarcomeres and their alignment gives cardiac and skeletal muscle cells their characteristic striated appearances. SMCs lack this organized striation even though they also contain myofibrils comprising thick and thin filaments. These filaments are mostly, but not exclusively, aligned along the long axis of the cell, and allow shortening of the S M C s in more than one direction. The functional unit of the thick filament is a hexamer composed of two 205kDa myosin heavy chains, each of which has two myosin light chains (MLC), one of 20kDa (MLC20kDa) and one of 17kDa (MLCi7kDa). Each heavy chain contains an ammo-terminal 'head group' and a carboxyl-terminal 'tail'. Both 17 and 20kDa light chains bind to the heavy chains at the junction between the head and tail domains. The sites of adenosine triphosphate (ATP) hydrolysis and actin binding sites are located at the N-terminus of the heavy chain on the myosin head, and A T P hydrolysis is actin dependent. The N-terminus is also referred to  1  as the actin-activated Mg -ATPase activity site (Allen and Walsh, 1994; Somlyo 2+  and Somlyo, 1994; Walsh et al., 1995). For both skeletal and smooth muscle, contraction occurs by the formation and breakdown of cross-bridges between the myosin head and actin filaments. The affinity of myosin for actin increases upon hydrolysis of A T P to adenosine diphosphate (ADP). When A D P dissociates from the myosin head, it induces a conformational change leading to the sliding of the filaments. Subsequent binding of A T P to the myosin head reduces its affinity for actin, allowing it to detach in preparation for the formation of a new crossbridge.  Dissimilarities  in  contractile  regulatory  mechanisms  distinguish  contraction in striated versus smooth muscles. Whereas in skeletal muscle C a  2 +  binds to troponin to activate cross-bridge cycling, troponin is absent from smooth muscle. Indeed, in SMCs, C a  2 +  binds to calmodulin (CaM), which  activates myosin light chain kinase (MLCK), an enzyme responsible for myosin phosphorylation, and consequently activates cross-bridge cycling. The level of MLC20kDa  phosphorylation regulates the number of cross-bridges cycling and  therefore the shortening velocity of smooth muscle (Murphy, 1976; Somlyo and Somlyo, 1976; Dillon et al, 1981; Wingard et al, 2001). Calcium is the main regulator of force development in skeletal muscle. A rise in intracellular calcium concentration  ([Ca ]i) 2+  induces a contraction, which is maintained until  [Ca ]i 2+  is  decreased. The excitation-contraction coupling process is comparatively simple in skeletal muscle. It involves a neuronal action potential inducing membrane depolarization, triggering C a SMCs, increased  [Ca ]i 2+  2 +  release from the sarcoplasmic reticulum (SR). In  is achieved through C a  2 +  release from the SR and C a  entry from the extracellular fluid, although sustained  2  [Ca ]i 2+  2 +  is dependent on a  supply of extracellular Ca (Dillon et al, 1981). While skeletal muscle contraction 2+  is induced uniquely through electromechanical coupling, smooth muscle contraction also occurs independently of changes in membrane potential, also referred to as pharmacomechanical coupling.  1.1.2 Ca regulation 2+  Measurements of  [Ca ]i 2+  following pharmacomechanical stimulus-induced  contraction in smooth muscle revealed an intriguing discrepancy between  [Ca ]i 2+  and tone maintenance (Morgan and Morgan, 1982). It soon became apparent that smooth muscle contraction occurred at surprisingly low levels of [Ca  2+  ]i.  During  smooth muscle contraction, three types of Ca profiles can be recorded. The most 2+  common, for example due to a-adrenergic receptor activation, involves peak Ca increases during force development followed by a decline in  [Ca ]i 2+  2+  to near basal  levels during force maintenance. A second profile is observed with KC1 depolarization that produces parallel changes in [Ca Finally, a third category of prostaglandins  [Ca ]i 2+  which produce  undetectable changes in [Ca  2+  ]i  2+  ]i  and force development.  changes is observed with agents such as  sustained  levels of tone with small or  (Morgan et al, 1992).  From these observations, it has been concluded that smooth muscles have two phases of contraction. The first is associated with force development and involves a high rate of energy consumption while the second component occurs during tone maintenance and involves a slow rate of energy consumption. Murphy and coworkers in 1981 proposed the "Latch bridge hypothesis" in one of the first attempts to explain the mechanism of prolonged smooth muscle tone  3  (Murphy, 1976; Dillon et al, 1981; Guth and Junge, 1982). The "latch bridge" refers to an attached, non-cycling or lower rate cycling cross-bridge maintaining force developed during contraction. This static cross-bridge is associated with low phosphorylation levels of  MLC20kDa  concomitant with high force generation  and a reduced contraction rate and ATP consumption. This capacity of smooth muscle to sustain contraction with low energy utilization is well served in the functioning of the airways of the lungs, coordinated contraction of the different layers of smooth muscle in the uritogenital system, the peristaltic movements of the gastrointestinal tract and the regulation of blood pressure by the vasculature where constant tone is required for prolonged periods.  At first glance, smooth muscle contraction is preceded by an increase in [Ca ]i. 2+  Smooth muscle depolarization is the main stimulus for Ca  occurs through voltage-gated Ca  2+  2+  entry which  channels (VGCC, L - and T-type), mechanical  stretch that activates Ca -permeable ion channels and stretch-activated cation 2+  channels (SAC). Other important pathways of Ca Ca  2+  channels (LGCC) and store operated Ca  2+  2+  entry involve ligand-gated  entry mechanisms (SOC). L G C C  encompass both P2X receptors (an ATP operated cation channel) and G-proteincoupled receptors. SOC involve indirect interaction between the SR and the plasma membrane. They are believed to be responsible, upon depletion of the internal store, for the activation of a Ca -permeable cation conductance at the 2+  plasma membrane. Ca induced Ca release from the SR, mediated by inositol2+  2+  1,4,5-triphosphate ( I P 3 ) and ryanodine receptors (RyR), also contributes to the rise in [Ca  2+  ]i,  although to a lesser extent. RyR mediated Ca release may also act 2+  as a negative feedback process leading to relaxation. Localized Ca RyR, termed "Ca  2+  2+  release by  sparks" activates Ca -activated potassium ( B K or K c ) 2+  a  4  channels in the vicinity of RyR. The efflux of K mediated by K c +  a  channel opening  hyperpolarizes the cell membrane and thus closes V G C C (Patterson et al, 2002) and reduces C a  entry. A decrease in  2 +  [Ca ]i 2+  leads to relaxation. C a  2+  extrusion  mechanisms include the plasma membrane Ca -ATPase pumps (PMCA) and the 2+  Na /Ca +  2+  exchanger using respectively A T P and N a entry to move C a +  the electrochemical gradient. C a  2 +  store repletion by the sarcoplasmic reticulum  Ca -ATPase pump (SERCA) and mitochondrial C a 2+  decrease in  [Ca ]i 2+  against  2 +  2+  uptake contribute to the  (Sanders, 2001).  Thus, mechanical stretch, autonomic nervous system stimuli, hormones, and various transmitters can trigger contraction. A t high molecules of C a  2 +  [Ca ]i 2+  (500-700nM), 4  bind to C a M and are believed to increase its affinity for the  enzyme M L C K (Allen and Walsh, 1994; Walsh et al, 1995; Horowitz et al, 1996b). The activated C a ( 4 ) - C a M - M L C K complex catalyses the phosphorylation of the 2+  MLC20kDa  high  on residue Ser-19. Phosphorylated myosin binds to actin filaments with  affinity,  permitting  actin-dependent  A T P hydrolysis.  The  resulting  hydrolysis of A T P drives the sliding of myosin along the actin filaments and, hence, the contraction of the muscle. Following a decrease in dissociates  from  phosphorylation of  CaM, MLC20kDa.  inactivating  MLCK  and  [Ca ]i,  preventing  2+  Ca  2+  further  Myosin light chain phosphatase (MLCP) activity  then dominates, dephosphorylating  MLC2okDa,  reducing cross-bridge cycling and  resulting in muscle relaxation (Allen and Walsh, 1994; Walsh et al,  1995;  Horowitz et al, 1996b). Figure 1.1 summarizes the basic molecular aspects of contraction and relaxation in smooth muscle. A different model, wherein C a M remains associated with M L C K , has been proposed by Wilson et al. in 2002 (Wilson et al, 2002) and was based on the earlier proposals by Kretsinger et al  5  (Persechini and Kretsinger, 1988; Kretsinger, 1992; Kawasaki et al, 1998). In this model, C a M is bound to the contractile apparatus in the absence of C a . In 2+  response to a rise in [Ca ]i, C a 2+  2+  binds to C a M inducing a conformational change  that activates M L C K (Van Lierop et al, 2002; Wilson et al, 2002). The basis of this proposal stemmed from experiments wherein purified C a M was add to a C a M depleted permeabilized rat-tail arterial preparation in the absence of C a . 2+  Following a rapid washout of the unbound C a M , a Ca -dependent contraction 2+  was observed, confirming that a pool of tightly bound C a M to the Triton-X-100 insoluble fraction supported the Ca -dependent mediated contraction (Wilson et 2+  al,  2002). Wilson et al. also suggested that M L C K  was  the  myofilament  component to which C a M bound. This proposed mechanism gives C a M a similar function as that assumed  by troponin in skeletal muscle. More  Geguchadze et al., using a fluorescent biosensor and fluorescence  recently, resonance  energy transfer (FRET) detection, found no evidence of C a M binding without activating M L C K at minimal [Ca ]> (Geguchadze et al, 2004). A s the F R E T 2+  experiments were performed in transfected HEK-293 cell lines, these results may indicate that the Ca -independent anchorage of C a M to M L C K is specific to 2+  contractile SMCs. O n the other hand, it is possible that the C a M is permanently bound to a component of the myofilament in close proximity but not directly associated with M L C K . Further investigations will be required to determine the exact site of attachment of C a M (Wilson et al, 2002).  6  F i g u r e 1.1  Ca  2 +  regulation and Ca -dependent contraction i n S M C s . 2+  Increased cytoplasmic [Ca ] occurs mainly through C a 2+  2+  release from the sarcoplasmic  reticulum (SR) mediated by Inositol-l,4,5-triphosphate (IP3) and ryanodine receptor (RyR) stimulation. Extracellular C a [Ca ]i. C a 2+  2+  2+  entering the cell also contributes to the increased  entry occurs through the activation of L-type and T-type voltage-gated C a  channels(VGCC), ligand-gated C a  2+  channels (LGCC), stretch-activated cation channels  (SAC) and also through store operated C a [Ca ]i, 4 molecules of C a 2+  2 +  2+  2+  entry mechanisms (SOC). Following a rise i n  b i n d to C a M . The (Ca )4-CaM complex translocates to the 2+  contractile elements, binds and activates M L C K . In blue, new proposed models show C a M is already associated w i t h the contractile elements. C a  binds to C a M inducing a  2+  conformational change that activates M L C K . The activated ( C a ) 4 - C a M - M L C K complex 2+  phosphorylates myosin light chain  and initiates cross-bridge cycling. C a  (MLC20kDa)  extrusion from the cell occurs through the N a / C a +  2+  2+  exchanger and plasma membrane  Ca -ATPase p u m p ( P M C A ) . The sarcoplasmic reticulum Ca -ATPase p u m p (SERCA) 2+  2+  and mitochondrial C a  2+  uptake contributes to the extrusion of C a from the cytoplasm. A 2+  lower [Ca ]i deactivates the (Ca )4-CaM-Kinase complex and reduces the rate of 2+  2+  MLC20kDa  phosphorylation. Dephosphorylation of  MLC20kDa  by myosin light chain  phosphatase ( M L C P ) induces relaxation.  1.1.3 Ca sensitization 2+  Typically, force generation by SMC involves a rapid rise in [Ca  2+  ]i.  During  sustained contraction, this fast elevation in [Ca ]i gradually fades so that levels 2+  remain only slightly above resting values in order to maintain the force  7  developed. Ligands binding to al-adrenergic and muscarinic G-protein coupled receptors can, under some conditions, induce contraction without any apparent changes in [Ca ]i (Morgan and Morgan, 1982; Somlyo and Somlyo, 1994), also 2+  suggesting the existence of modulatory mechanisms increasing C a  2+  sensitivity of  the contractile apparatus.  The increased C a  2+  sensitivity occurring during sustained contraction is  associated with M L C P inhibition (Kitazawa et al, 1991; Somlyo and Somlyo, 2000). M L C P is composed of three subunits, a catalytic subunit (37kDa, PP-1C), a regulatory subunit or myosin binding site (MBS) or myosin phosphatase target 1 (MYPT1) subunit (110-130kDa) and a subunit of unknown function (20kDa) (Allen and Walsh, 1994; Fukata et al,  2001). When the M B S subunit  is  phosphorylated on residue Thr-695, M L C P is prevented from binding to and dephosphorylating modulators  of  MLC20kDa,  MLCP  activity  so  that  have  contraction  is  been identified.  prolonged.  Potential  Signalling  molecules  including RhoA/Rho-associated kinase (Uehata et al, 1997; Fukata et al, 2001), protein kinase C (PKC) and arachidonic acid (AA) inactivate M L C P and cause sustained contraction at lower [Ca ]i (Fu et al, 1998; Solaro, 2000; Somlyo and 2+  Somlyo, 2000). The phosphorylation of M Y P T subunit is associated with its translocation to the plasma membrane. A s shown by Shin et al., M L C P translocates to the plasma membrane upon agonist stimulation, where catalytic and  targeting M Y P T subunits dissociate when phosphorylated by Rho-kinase  (Shin et al, 2002). Thus, Ca -sensitization is a result of M L C P inhibition and the 2+  consequent increase in M L C phosphorylation.  8  1.1.4 P K C  P K C was first discovered by Nishizuka in 1977 (Inoue et al, 1977; Takai et al,  1977).  P K C with  its  10  isoforms  constitute  the  largest  family  of  serme/mreonine specific kinases (Webb et al, 2000). Ligands stimulating Gocq activate  three  main  phospholipid  groups.  Phosphoinositide-specific  phospholipase C (PI-PLC) catalyses the formation of the second messengers IP3 and 1,2, -diacylglycerol ( D A G ) through the hydrolysis of phosphatidylinositol 4,5,- diphosphate (PIP2). IP3 promotes C a while  D A G is  a  potent  activator  of  2 +  release from the sarcoplasmic SR  P K C (Andrea and  Walsh,  1992).  Phosphatidylcholine-specific phospholipases C (PC-PLC) and D (PC-PLD) are also activated upon ligand binding to G-protein coupled receptors. P C - P L C and P C - P L D generate only D A G upon hydrolysis and are therefore more likely to be involved in Ca -independent mechanisms. D A G promotes the translocation of 2+  inactive P K C from the cytosol to the plasma membrane where it becomes activated, as shown in Figure 1.2. P K C can also be activated by arachidonic, linoleic, and oleic acids (Webb et al, 2000). Since activated P K C is thought to be a membrane-associated protein, its access to contractile proteins would be limited. One suggested mechanism by which activated P K C could physically interact with  the  contractile  apparatus  is  through  calpain  hydrolysis.  P K C is  proteolytically cleaved by calpain to release its catalytic subunit, protein kinase M (PKM) that can phosphorylate proteins in the cytosol (Tapley and Murray, 1984; Pontremoli et al, 1990).  9  Relaxation  F i g u r e 1.2  Contraction  Ca -dependent and Ca -independent activation of P K C . 2+  2+  Activation of phosphoinositide-specific-phospholipase C (PI-PLC) and not phosphatidylcholine-specific-phospholipase C (PC-PLC) and phosphatidylcholinespecific-phospholipase D (PC-PLD) lead to C a release. Therefore Ca -independent isoforms of P K C are more likely to be activated through the P C - P L C and P C - P L D pathways. Both phosphatidic acid (PA) and phosphatidylinositol-4,5-biphosphate ( P I P 2 ) produce 1,2-diacylglycerol ( D A G ) , which i n turn activates P K C . P I P 2 also generates I P 3 leading to C a release from the SR. Other k n o w n activators of P K C are A A and i P L A 2 through free fatty acid (FFA) and the lysophospholipid choline (Lyso PC). P K C activation induces the release of its catalytic subunit, protein kinase M (PKM). P K M phosphorylates CPI-17 leading to inhibition of M L C P . P K M is also believed to phosphorylate a smooth muscle specific protein of 22kDa (SM22) but its functional role is still undetermined. Integrin-linked-kinase (ILK) induces Ca -independent contraction through CPI-17, inhibition of M L C P and direct phosphorylation of M L C . M L C is also phosphorylated by Zip-kinase i n a Ca -independent manner. 2+  2+  2+  2+  2+  Comparable force generation is correlated with the levels of  MLC20KDa  phosphorylation in both intact and skinned smooth muscle preparations. Changes in Ca -sensitivity induced by PKC activators occur in both intact and a2+  toxin permeabilised smooth muscle preparations (Weber et al, 1999). However, force produced by a-adrenergic agonists is lower in skinned preparations, possibly due to the disruption of a PKC-dependent signal pathway that usually  10  leads to M L C P inhibition. Evidence for this comes from biochemical analysis of Triton-XlOO-treated rat-tail arterial smooth muscles. In these preparations, the content of P K C a, (3, C, and CPI-17 (see below) is significantly decreased, while other proteins such as calponin, caldesmon, M L C K and P P l m seem to be retained (Kitazawa et al, 1999; Weber et al, 2000).  Recent studies implicate an important regulatory role for caveolin in P K C a and R h o A translocation from the cytosol to the plasma membrane. Using a peptide capable of interacting with the caveolin-1 scaffolding domain (the site of interaction with signalling molecules),  Taggart et al. were able to inhibit  carbachol-induced translocation of P K C a and R h o A in rat uterine smooth muscle (Taggart et al,  2000). Moreover, caveolin-1  knockout mice have  decreased  responses to angiotensin II, endothelin-1, and phorbol ester (Drab et al, 2001).  Force generation in smooth muscle can occur at very low levels of [Ca ]i 2+  (Katsuyama and Morgan, 1993; Weber et al, 1999). M L C K seems to be the kinase responsible for M L C phosphorylation; however, because of its considerably reduced activity at low [Ca ]i, its singular activity cannot entirely explain the 2+  force generated and the amount of M L C K phosphorylated during sustained contraction maintained at low [Ca ]i (Somlyo and Somlyo, 2003). Ligand binding 2+  to G-protein coupled receptors activates P C - P L C or P C - P L D , generating D A G , which in turn activates Ca -independent isoforms of P K C . Both PKCe and P K C ^ 2+  have been identified  in vascular smooth muscles (Horowitz et al,  1996a;  Throckmorton et al, 1998). In saponin-permeabilized single ferret aortic SMCs, the application of a constitutively active form of P K C s (and not P K C Q stimulates contraction independently of MLC2OKD phosphorylation and extracellular [Ca ]i 2+  3  11  (Horowitz et al, 1996a). Moreover, phorbol esters activate PKCe. but not P K C £ while phenylephrine induces PKCs translocation from the cytosol to the plasma membrane and P K C ^ translocation from perinuclear localization to the interior of the nucleus. Thus, PKCs has been suggested as the isoform responsible for Ca -independent regulation of vascular tone. 2+  Both MLC20KDa and M L C K are potential targets for P K C . P K C catalyses the phosphorylation  of  serine  1,  2  and  threonine  9  of  MLC20KDa,  however  phosphorylation at these residues results in either no effect or a 50% reduction of the actomyosin ATPase activity (Nishikawa et al,  1983; Ikebe et al,  1985).  Furthermore, phosphorylation of M L C K by P K C decreases its affinity for C a 2+  C a M , thus indirectly reducing MLC2OKD phosphorylation at serine 19 (Ikebe et al, 3  1985; Nishikawa et al, 1985). Therefore, the role of P K C in Ca -sensitization 2+  cannot be attributed to a direct interaction with MLC2OKD  3  or M L C K  since  phosphorylation of the targeted sites by P K C is more likely to result in relaxation rather than a sustained contraction (Umemoto et al, 1989; Andrea and Walsh, 1992; Webb et al, 2000).  P K C can enhance contraction by indirectly inhibiting M L C P activity. CPI17 is a novel, 17kDa, smooth muscle specific inhibitor of protein phosphatase-1 that has been identified as a P K C substrate. In its phosphorylated form, CPI-17 inhibits the catalytic subunit of M L C P (Somlyo and Somlyo, 2000; Fukata et al, 2001). In the presence of both P K C a and CPI-17, MLC20KDa phosphorylation is increased and contraction maintained at constant [Ca ]i (Kitazawa et al, 1999). 2+  CPI-17  phosphorylation  is  increased  in  response  to  receptor-mediated  stimulation (histamine and phenylephrine) but not following K depolarization. +  12  Levels of CPI-17 phosphorylation are reduced when arteries are incubated with inhibitors of Rho-kinase and P K C (Kitazawa et al, 2000; Fukata et al, 2001). Muscarinic receptor (m3) activating signalling pathways through Rho-kinase and P K C may act cooperatively in order to inhibit M L C P activity and induce a sustained contraction (Murthy et al, 2003). Recent reports have identified SM22 as a potential substrate of P K C . SM22 is an abundant smooth muscle specific protein of 22KDa,  which may bind  to  actin, however,  at this  time  its  physiological function is not known (Morgan and Gangopadhyay, 2001).  Other possible substrates of PKCe are the smooth muscle specific thin filament associated proteins calponin and caldesmon (Horowitz et al, 1996a). Caldesmon phosphorylation as well as myristoylated, alanine-rich C-kinase substrate (MARCKS) phosphorylation have been observed following phorbol 12,13-dibutyrate (PDBu) stimulation of bovine carotid arterial smooth muscles (Throckmorton et al, 1998). Unphosphorylated caldesmon and calponin are both associated with actin and act as inhibitors of cross-bridges cycling activity. U p o n phosphorylation, their affinity for actin is decreased and cross-bridge cycling activity restored. Their phosphorylation may contribute to the development of force induce by Ca -independent P K C isoform. 2+  1.1.5 Rho-associated kinase  Agonists binding to both Gal2/13 (Gohla et al, 2000) and G a q (Chikumi et al, 2002) protein-coupled receptors induce the dissociation of heterotrimeric G protein complexes, allowing the activation of the small monomeric G protein, RhoA. Endogenous activators of RhoA include growth factors,  13  cytokines,  integrins, and hormones such as bradykinin and lysophosphatidic acid (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998). The activation of R h o A is believed to induce its translocation to the plasma membrane. The translocation of a GFPtagged  RhoA  from  the  cytosol  to  the plasma membrane, upon  agonist  stimulation, has been demonstrated in porcine tracheal smooth muscles by Miyazaki et al. (Miyazaki et al, 2002). This translocation of the GFP-tagged RhoA coincides with increased myosin phosphorylation and sustained contraction (Miyazaki et al, 2002). In the cytosol, RhoA is present in a complex with a guanosine  diphosphate  (GDP) and a Rho-guanine nucleotide  dissociation  inhibitor (Rho-GDI). Activation of a RhoA-guanine nucleotide exchanger factor (RhoA-GEF) by the Ga-subunit leads to the dissociation of G D P and subsequent formation of a RhoA-guanosine triphosphate (RhoA-GTP) complex. R h o A - G T P translocates to the plasma membrane and activates a serine/threonine kinase termed  Rho-kinase  (Somlyo  and  Somlyo,  2000).  Activated  Rho-kinase  phosphorylates and inactivates the regulatory subunit of M L C P independently of changes in the C a - C a M - M L C K pathway (Kureishi et al, 1997; Uehata et al, 2 +  1997; Iizuka et al, 1999; Solaro, 2000; Somlyo and Somlyo, 2000; Sward et al, 2000; Fukata et al, 2001).  Two indirect mechanisms linking Rho-kinase to the inhibition of M L C P have been proposed. In the first instance, CPT17, when phosphorylated by Rhokinase on residue Thr-38, blocks the activity of M L C P (Koyama et al, 2000). A second and more recently proposed player, a MYPTl-associated kinase ( M H O kinase),  referred  to  as  zipper-interacting  protein  (ZlP)-like  kinase,  phosphorylates M L C P at its inhibitory site (MacDonald et al, 2001). MacDonald et al. suggest that ZIP-Iike kinase may play a role of intermediate in the  14  inhibition of M C L P by Rho-kinase. Despite the lack of a direct phosphorylation of ZIP-like kinase by Rho-kinase, the authors suggest that a link between the two kinases is present since the activation of ZIP-like kinase is inhibited by Y-27632 (Rho-kinase inhibitor) in vivo. Thus, ZIP-like kinase is a putative candidate linking the plasma membrane Rho-kinase and M L C P  associated  with the  contractile apparatus (MacDonald et al, 2001) see Figure 1.3.  Rho-kinase is also modulated by several intracellular molecules and pathways. A A , produced by P L C activation subsequent to receptor binding to Gaq, also induces C a A2 (iPLA2)  2 +  sensitization in vitro. Formation of A A by phospholipase  is Ca -independent in smooth muscles (Guo et al, 2003). A A inhibits 2+  M L C P either by directly activating Rho-kinase or by causing the dissociation of MBS from the catalytic subunit of M L C P , so reducing its activity (Somlyo and Somlyo, 2000; Fukata et al,  2001). The activation of Rho-kinase by A A is  produced by the binding of A A to its c-terminus that acts as an inhibitory site. The release of the autoinhibitory site from the catalytic unit containing the R h o A binding'site, leads to activation of Rho-kinase (Araki et al, 2001).  15  Figure  1.3 Ca -sensitization mechanisms in smooth muscle contraction. 2+  R h o A is located i n the cytosol and is associated w i t h guanosine diphosphate (GDP) and guanine nucleotide dissociation inhibitor (GDI) molecules. The RhoA/Rho-kinase pathway is activated by ligand binding to G-protein coupled receptors (Gal2/13 and Gaq)). R h o A dissociation from G D P is activated by Rho guanine nucleotide exchanger factors (GEF). Sphingosylphosphorylcholine (SPC) acting through the Src family of tyrosine kinase also stimulates G E F . The reverse reaction is catalyzed by a GTPaseactivating protein (GAP). Rho-guanosine triphosphate (Rho-GTP) is translocated to the plasma membrane and activates Rho-kinase. Rho-kinase inhibits M L C P through direct phosphorylation and by activating a novel zipper-interacting protein (zip)-like kinase. P K C targeted protein phosphatase-1 inhibitor of 17kDa (CPI-17) is phosphorylated by both P K C and Rho-kinase and i n turn inhibits M L C P activity. Arachidonic acid (AA),a product of the Ca -independent phospholipase A2 isoform (iPLA2), also inhibits M L C P directly and indirectly by activating Rho-kinase. R n d l and M g prevent the translocation of R h o A to the plasma membrane and therefore play an important role i n vascular relaxation and associated pathologies. 2+  2 +  A newly identified member of the Rho family, Rndl, which is constitutively bound to GTP, regulates the RhoA/Rho kinase pathway (Loirand et al, 1999). Rndl is able to prevent Ca  2+  sensitization induced by carbachol or  GTPyS (a non-hydrolysable GTP analogue). It is thought that Rndl is able to  16  interact directly with RhoA without targeting M L C K or M L C P (Loirand et al, 1999) . R n d l expression in smooth muscles is increased by sex hormones such as estrogen and progesterone (Loirand et al, 1999). These steroid hormones also decrease vascular contractile activity by stimulating the production of N O (Mendelsohn  and  Karas,  1999;  Mendelsohn, 2000; Varbiro  et  al,  2000).  Accordingly, R n d l as part of the RhoA-Rho-kinase pathway may also play an important role in the protective cardiovascular effects induced by steroid hormones. M g Mg  2 +  2 +  can also regulate R h o A activation. A t normal concentrations,  reinforces the G D P - R h o A binding interaction, decreasing the dissociation  rate, and therefore reducing the activation of RhoA. Moreover, a low [Mg ]i 2+  increases the activity of G E F allowing an increased formation of Rho-GTP and activation of Rho-kinase (Maesaki et al, 1999; Shimizu et al, 2000; Zhang et al, 2000) . Some studies have linked hypomagnesaemia with hypertension (Kawano et al, 1998), leading to a possible role for M g  2 +  related RhoA/Rho-kinase activity  in the pathogenesis of hypertension.  A n additional activator of Rho-kinase is sphingosylphosphorylcholine (SPC), a product of deacylation of spingomyelin, which is the most abundant lipid in the cell membrane. Sphingosylphosphorylcholine is able to increase the Ca  2 +  sensitivity of the contractile apparatus, an effect that is blunted by Y-27632  (Todoroki-Ikeda et al,  2000; Shirao et al,  2002). Shirao et al. have  also  demonstrated in primary cultures of S M C s that S P C induces translocation of cytosolic Rho-kinase to the plasma membrane (Shirao et al, 2002). Thus, it would appear that SPC is an endogenous activator of Rho-kinase during  Ca 2+  independent smooth muscle contraction, although its production and temporal relationship to C a  2 +  sensitization has yet to be confirmed.  17  More recently, Sakurada et al. have shown a novel pathway of activation for R h o A and Rho-kinase in vascular smooth muscle. They demonstrate that both high KC1 and receptor agonist stimulation induce Ca -dependent RhoA2+  Rho-kinase mediated contraction (Sakurada et al, 2003). Noradrenaline and KC1 induced a contraction that was accompanied with a 4 to 5 fold increase in the GTP-bound form of RhoA. The removal of extracellular C a contraction and R h o A  2 +  abolished both the  stimulation. The use of Rho-kinase inhibitors also  implicated Rho-kinase in this process. Both Y-27632 and HA1077 suppressed (by 60 to 70%) noradrenaline and KC1 induced contraction and inhibited M L C phosphorylation. Similar observations with the thromboxane A 2 mimetic U46619, a receptor agonist which like noradrenaline activates P L C to mobilise C a the SR, suggest that Gaq  2 +  from  mediates the  Ca -dependent RhoA/Rho-kinase  activation (Sakurada et al, 2003). Thus, C a  is likely playing an active role in  2 +  2+  Ca -sensitization in concert with other Ca -independent mechanisms. Further 2+  2+  investigation will be needed to determine which Ca -dependent signalling 2+  molecules are involved in the activation of the RhoA/Rho-kinase pathway.  1.1.6 Myogenic tone  In the absence of neuronal, chemical, and hormonal stimulation, the small arteries of the circulation exist in a state of partial constriction. The intrinsic tone is produced in response to transmural pressure, a phenomenon first described in 1902 by Bayliss and referred to as myogenic tone (Bayliss, 1902). A myogenic response has been observed in a wide variety of small arteries and arterioles from different vascular beds and its strength has been shown to be dependent on the diameter of the vessel. The strongest myogenic response has been recorded  18  from vessels of intermediate diameter (Davis, 1993). Differences in the strength of the myogenic response are also found between vascular beds. For example, cerebral (Lagaud et al, 1999) and skeletal muscle arteries (Watanabe et al, 1993) have a stronger myogenic response  than mesenteric arteries of a similar  diameter. The main purpose of such inherent contractile ability of the vascular smooth muscle is attributed to the maintenance of a basal level of vascular tone but it also contributes to the autoregulation of blood flow. In order to meet metabolic requirements, regional vasoactive factors (nitric oxide,  metabolite  accumulation, p H changes, constrictor and dilator mechanisms) modulate the underlying basal constriction of resistance arteries and thus reduce or enhance the perfusion of organs, muscles and tissues, independently of each other (Davis and Hill, 1999). The basal tone of resistance arteries contributes substantially to the total peripheral resistance encountered by the heart and is therefore one of the factors that determines the systemic blood pressure. It is only since the development  of the pressure myograph in 1984  that the properties and  characteristics of the myogenic tone have been systematically studied (Halpern et al, 1984). It is therefore not surprising that a great deal of effort is still underway aimed at understanding the mechanisms of myogenic tone.  1.1.7 Signalling mechanisms in myogenic tone  The initiation of the myogenic response is triggered by an increase in the intraluminal pressure. It has been suggested that the increase in pressure stretches the vascular smooth muscles, altering the vessel wall tension. The wall tension (T) can be calculated from the Laplace relation (T = P x r), where P is the intraluminal pressure and r the vessel radius. Experiments with bat wing and cat  19  mesenteric arteries have demonstrated that as P increases, the radius of the vessel is adjusted in order to keep T constant (Johnson, 1989). Subsequent observations established a correlation between the vascular wall tension and the level of both [Ca ]i and M L C phosphorylation (Zou et al, 1995), providing strong 2+  evidence that the wall tension acts as the mechanostimulus in the myogenic response.  The theory of mechanotransduction implies that a sensor element is responsible for transducing changes in wall tension to constriction of the vessel via intracellular signalling pathways. The site and nature of this sensor element remains to be identified. Growing evidence suggests that a group of adhesion molecules,  the integrins, play a central role in this  mechanotransduction  mechanism. The emergent interest in the integrins comes from the mechanical link they form between the extracellular matrix and the cytoskeleton. External stress on the integrins directly affects the cytoskeleton of the cell, activating intracellular signalling and more importantly induces the phosphorylation of proteins associated with the cytoskeleton (Davis et al, 2001; Martinez-Lemus et al, 2003). Despite that, integrins, through their molecular function, represent the ideal sensor element; there is still little direct evidence of their participation in the myogenic response.  Previous work has shown that synthetic peptides containing amino acid sequences specific to the avpVintegrins induce a vasodilation in myogenically active rat cremaster arterioles (Mogford et al,  1996). This vasodilation was  inhibited by antibodies targeting the (33 subunit of the integrins (Pierschbacher and Ruoslahti, 1987; Mogford et al., 1996). Conversely, the activation of as|3i  20  (Mogford et al, 1997) and  ou|3i  (Waitkus-Edwards et al, 2002) integrins enhance  the myogenic response. Although these findings support a role for the integrins in the myogenic response,  they also indicate that multiple integrins and  downstream intracellular pathways may be involved. The integrins have also been associated with the regulation of  [Ca ]i 2+  (D'Angelo et al, 1997b). The binding  of appropriate ligands to the integrins can initiate C a  2+  entry into SMCs (Xie et al,  1998), an essential event in the generation of a myogenic response. Recently proposed mechanisms implicate the modulation of L-type V G C C by the integrin ligands (Martinez-Lemus et al, 2003).  The integrins may also modulate the myogenic tone through collaboration with the cytoskeleton (Davis et al, 2001). The integrins are located at a site of assemblage for several components of the cytoskeleton. The compressible and elastic structural components of the cytoskeleton are a potential mechanical stress sensor itself. Thus, mechanical strain has been shown to induce a reorganisation of the cytoskeleton elements (Smith et al,  1997), providing  evidence for the active participation of the cytoskeleton in response to external mechanical stress. Moreover, depolymerization of the microtubules has been shown to enhance contractility in response to agonist stimulation (Leite and Webb, 1998; Paul et al, 2000).  Downstream targets of the interaction between the extracellular matrix and the integrins are also potential modulators of myogenic tone. Several nonreceptor protein kinases; focal adhesion kinase  (FAK),  MAPK  and other  signalling proteins such as members of the Rho family of small GTPases are recruited to the plasma membrane and produce Ca -dependent and C a 2+  21  2+  sensitization integrins  signalling mechanisms  link  these cytoskeleton  in response  to mechanical stress. The  anchored cascades of  kinases  with  the  extracellular matrix (Hall, 1998; Karandikar and Cobb, 1999), again adding to the evidence that integrins are potentially the site of initiation of mechanical stressed induced myogenic  tone.  Further investigations  are needed  to clarify the  mechanism of action of the integrins but to this day, they remain the most plausible candidates  to fulfill  the role of a sensor element in the signal  transduction pathway in the myogenic response.  As in conventional S M C contraction, the myogenic response is largely dependent on Ca -entry to induce M L C phosphorylation and the eventual 2+  vasoconstriction. The removal of extracellular C a  2 +  abolishes the development of  myogenic tone (Uchida and Bohr, 1969, Osol, 1985 #622; McCarron et al, 1989; Kuo et al, 1990). Furthermore, the loss of myogenic tone observed with the use of inhibitors of V G C C (Knot and Nelson, 1995; Setoguchi et al, contributors of Ca -entry in S M C , re-enforces 2+  extracellular C a  2+  1997), major  the general agreement  that  is critical to the maintenance of myogenic tone.  Numerous studies have also established that an increase in transmural pressure induces a vascular S M C depolarization (Harder, 1984; Harder et al, 1985; Harder et al, 1987; Knot and Nelson, 1995; Wesselman et al, 1997; Knot and Nelson, 1998). The proposed signalling events leading to S M C membrane depolarization include the activation of the S A C , C F channel, K c channel a  intracellular C a  2+  store and A A pathway.  22  A  hypothetical pathway,  involving S A C channel, suggest that  activation of inward current of N a , K , and C a +  +  2+  the  is induced by a stretch  deformation of the S M C s (Kirber et al, 1988; Davis et al, 1992; Setoguchi et al, 1997; W u and Davis, 2001). While the C a  2 +  entry could itself generate the  contraction, the influx of cations is more likely to produce a membrane depolarization and the subsequent activation of the L-type V G C C , increasing the influx of C a . In agreement with this sequence of events, the inhibition of L-type 2+  V G C C prevents the myogenic response without affecting the stress-induced depolarization (Knot and Nelson,  1995). The importance of S A C in  the  development of myogenic tone is however questionable. First, V G C C and K c  a  channels are also activated by increased intraluminal pressure. Secondly, the demonstration of S A C activation was performed only in single isolated S M C using a variant of the patch clamping technique to induce a stretch or pressure stimulus, neither of which is likely to mimic the wall tension changes produced in the myogenic response. A specific blocker for the S A C would provide direct evidence of such mechanisms in a whole vessel preparation (Schubert and Mulvany, 1999), but unfortunately, one does not exist. Nevertheless,  recent  evidence implicates the activation of transient receptor potential 6 (Trp6) channel in response to mechanotransduction mechanism in resistance arteries. It was demonstrated that pressure induced depolarization and myogenic constriction were both reduced by the use of antisense R N A for the Trp channel 6 (Welsh et al, 2002). These Trp channels 6, which exhibit biophysical properties similar to that of the cation current found in vascular SMCs, may therefore represent an important modulator of the myogenic tone.  23  In addition, CI" channel activation has also been suggested to participate in the membrane depolarization following pressure-induced myogenic tone (Nelson et al, 1997). The opening of CI" channels and consequent efflux of CI could potentially cause the membrane depolarization induced by an increase in transmural pressure. Despite the presence of volume-regulated CI" channel in the vascular SMCs, the lack of a specific inhibitor for this channel and controversy existing, for the moment, regarding the occurrence of a concomitant cation current, prevents a clear understanding of the role of the CI" channel in the regulation of the myogenic tone (Welsh et al, 2000).  The increase in [Ca ]i, due to the activation of V G C C is theorized to , 2+  enhance the frequency of C a  2 +  released events mediated by RyR (Ca  2+  sparks),  and activates in turn the K c a channels. The efflux of K mediated by the opening +  of K c a channels, hyperpolarizes the cell membrane and thus closes the V G C C (Patterson et al, 2002) and reduces C a  2 +  influx. This activation of K c channels is a  thought to be a negative feedback mechanism regulating S M C membrane depolarization and myogenic tone (Brayden and Nelson, 1992; Knot and Nelson, 1995). C a  2+  sparks have also been closely associated  with the synchronised  opening of groups of K c channel producing spontaneous transient outward a  currents (STOCs). In coronary artery, these S T O C s have been shown to occur following stretch-activated membrane depolarization (Wu and Davis, 2001). This feedback mechanism is thought to protect the cells from excess Ca -entry and 2+  limit the degree of contraction. More recently, a decrease in the sensitivity of the K c a , induced by the deletion of its pVsubunit, has been shown to impair the coupling between C a  2 +  sparks and the activation of K c a channel, consequently  increasing arterial tone and blood pressure (Brenner et al, 2000). Therefore, the  24  regulation of K c channel sensitivity to STOCs is likely to play a key role in the a  development of hypertension.  In SMCs, one of the functions of the SR is to the regulate Ca -entry. This is 2+  based on the observation that when the SR is empty, C a  2+  influx is mobilised  primarily to replenish the SR store and only subsequently becomes available to the contractile machinery. After restoration, the SR emits sequential vectorial release of C a  2 +  regulating the membrane depolarization and thus the opening of  the V G C C and level of Ca -entry. This regulating pathway has been described 2+  by V a n Breemen and referred to as the superficial buffer-barrier theory (van Breemen et al, 1995). Its implication in the myogenic response has yet to be directly demonstrated. Further evidence implicating Ca -induced Ca -release in 2+  2+  myogenic tone comes from studies showing that the depletion of intracellular Ca -store with ryanodine and the inhibition of SR Ca -ATPase pump, both 2+  2+  enhanced the myogenic response (Watanabe et al, 1993). Ca -entry is closely 2+  associated with the degree of emptying of the SR, and is thought to rely on a physical coupling between the SR and plasma membrane C a release of messengers enhancing C a  2 +  influx. The influx in C a  2+  2 +  channel or the induced by the  depleted store of the SR are referred to as capacitative C a - entry or store2+  mediated C a  2+  entry mechanisms. Evidence, of such opening of non-selective  cation channels following Ca -store depletion have been found in resistance 2+  arteries (Trepakova et al, 2000; Trepakova et al, 2001). The activation of such non-selective cation channel produces a membrane depolarization responsible for the stimulation of V G C C and subsequent Ca -entry. The channel responsible 2+  for the capacitative Ca -entry current has yet to be identified but is thought to be 2+  derived from the Trp channel gene family.  25  Growing evidence  indicates the participation of several intracellular  second messengers in the myogenic response. Inhibitors of tyrosine kinase, tyrosine phosphatase (Masumoto et al, 1997), G protein (Osol et al, 1993),  i P L A 2  (Kauser et al, 1991), P L C (Osol et al, 1993) and cytochrome P-450 (Kauser et al, 1991), have all been shown to modulate, to some extent, the myogenic response.  The  activation of P L A and P L C liberates  A A . The latter can be  metabolized by the enzyme P-450 4 A to form 20-hydroxyeicosatetraenoic acid (20-HETE) (Imig et al, 1996). 20-HETE induces vasoconstriction accompanied with depolarization. Moreover, 20-HETE is a potent inhibitor of Kca, and may therefore induce membrane depolarization by inhibiting the K outward current +  in S M C s (Ma et al, 1993). The inhibition of K c by 20-HETE has been suggested to a  be mediated by P K C activation (Lange et al, 1997). More recently, 20-HETE induced contraction has also been associated with the increased activation of Rho-kinase (Randriamboavonjy et al, 2003).  Inhibitors of P L C such as U-73122 abolish myogenic tone. In accordance with the involvement of P L C activation in myogenic response, increased levels of I P 3 and D A G have been measured in response to step increases in transmural pressure (Narayanan et al, 1994). A role for I P 3 in myogenic tone has yet to be demonstrated, however, D A G is a potent activator of P K C , which may be associated with Ca -sensitization mechanisms. 2+  26  1.1.8 Ca -sensitization in myogenic tone 2+  In addition to depolarization induced Ca -entry via L-type V G C C , 2+  previous studies have indicated the participation of other regulatory mechanisms in the myogenic response. A s pressure-induced constriction produces greater change  in  the  arterial diameter  to  [Ca ]i ratio 2+  than  does  KCl-induced  depolarization, the involvement of Ca -sensitization pathways in the mechanism 2+  of myogenic tone has been suggested (Laporte et al, 1994; D'Angelo et al, 1997a; VanBavel et al, 1998; Dessy et al, 2000; Lagaud et al, 2002; Yeon et al, 2002). P K C as well as Rho-kinase are potential regulators of sustained myogenic tone (Lagaud et al, indolactam)  of  2002; Yeon et al, P K C enhance  the  2002). Specific activators myogenic  response  (phorbol-ester,  while  inhibitors  (staurosporine, calphostin C) reduce it (Hill et al, 1990; Osol et al, 1991; Laporte et al, 1994; Miller et al, 1997). In addition, pressure-induced activation of P K C induces a vasoconstriction without any further increase in Ca -entry or M L C 2+  phosphorylation (Hill et al, 1990; Laporte et al, 1994). The Rho-kinase inhibitor Y-27632 also reduces myogenic tone in a concentration-dependent  manner  (VanBavel et al, 2001; Schubert et al, 2002). Evidence of P K C and Rho-kinase participation has been reinforced by observations of their translocation to the plasma membrane during development of vascular myogenic tone (Dessy et al, 2000; Yeon et al, 2002). More recently, Bolz et al. demonstrated that sphingosine kinase also modulates myogenic tone via the RhoA/Rho-kinase pathway (Bolz et al, 2003). While it is well accepted that both P K C and Rho-kinase participate in the myogenic response, their contribution to the Ca -sensitization pathway 2+  requires further understanding.  27  1.1.9 Hypothesis  In the present study, we tested the hypothesis that P K C and Rho-kinase regulation of pressure-activated myogenic tone occur independently of changes in membrane potential. A steep relationship between the level of extracellular K  +  and the membrane potential regulates the pressure-induced vasoconstriction in cerebral arteries. This is explained by the feedback mechanism involving the Kca channels, whereby the initial pressure-induced depolarization activates the L type V G C C leading to an increase in [Ca ]i. The loaded SR spontaneously 2+  releases C a  2 +  (sparks) which in turn activates the K  c a  channels producing a  membrane hyperpolarisation which in turn prevents further C a and  Nelson,  1998). Ca -sensitization, 2+  as  a regulator of  2 +  influx (Knot  pressure-induced  constriction, was investigated under conditions in which changes in the activity of Kca channels were prevented by pre-treating cerebral arteries with a solution of 60 m M K . A t this concentration of external K , the membrane potential of +  +  smooth muscles in rat cerebral arteries is clamped at approximately -21 m V , and further activation of the V G C C is unlikely to occur (Knot and Nelson, 1998).  1.1.10 Pressure myography  The pressure myograph system has been used for the in vitro study of vessels of 75 to 250 u m diameter under pressure (Halpern et al, 1984). This approach reflects conditions that approximate the in vivo milieu of the vessel more closely than other methods such as ring segments or strips of arteries suspended in a tissue bath between two wires connected to a force transducer (Furchgott and Zawadzki, 1980). Furthermore, this technique exposes the arterial wall to a real transmural pressure. The system allows both static and dynamic  28  experiments. In addition, the incorporation of a video analyser permits the simultaneous recording of wall thicknesses and the inside diameter, both of which are necessary for the calculation of wall tension. The pressure myograph consists  of  two  microcannulae,  axially  oriented  in  a  tissue  bath. The  microcannulae face each other, and their tips of approximately 60 u m in diameter, approach the bottom glass coverslip at a 45 angle. The isolated arterial Q  segments are mounted between the cannulae and secured with fine surgical suture. The distal cannula is clamped while the other is connected to a pressure transducer and a peristaltic pump from which a pressure servo unit controls the intraluminal pressure. The tissue bath is placed on the stage of an inverted microscope, allowing the pressurized vessel to be transilluminated through the glass bottom coverslip. A n in-line video camera captures the image of the arterial diameter, which is projected and measured on a video monitor.  1.1.11 C a  2+  measurements  The use of a C a  2 +  sensitive fluorescent indicator (Fura-2) and a ratio  fluorescence spectrometer coupled to an inverted microscope, a vessel chamber and a dimension analysis system allow the simultaneous recording of changes in vascular tone and in cytosolic free C a . When C a 2+  2+  binds to Fura-2 the excitation  spectrum is shifted from 340-350nm to 380-390 nm wavelengths. concentration of C a  2+  Thus, the  is measured from the ratio of amplitudes of a pair of  excitation wavelengths (Tsien, 1988). This prevents  variations induced by  inconsistency in dye loading, cell thickness, local optical path length, and changes in illumination from influencing the calculation. The use of fluorescent indicators offers the possibility of visualisation by imaging, and produces a much  29  faster response than C a  2 +  sensitive microelectrodes. It is possible to record signals  from smaller cells than it was  with previous  absorbance indicators and  chemiluminescent proteins. These fluorescent indicators can be calibrated and are less sensitive to bleaching. Moreover, the biggest advantage is that cells can be loaded with these fluorescent dyes without any disruption of the plasma membrane (Cobbold and Rink, 1987; Tsien, 1988). Fura-2 A M is membrane permeant, and therefore diffuses easily into the cytosol of the cells. Intracellular Fura-2 A M is quickly de-esterified by cytosolic esterase, and converted to an anionic membrane impermeant Fura-2, trapped in the cell. A photomultiplier is positioned behind an adjustable aperture and used as a photodetector.  1.2 Methods  A l l chemicals were purchased from Sigma-Aldrich Ltd. (Oakville, O N , Canada) unless otherwise stated. Animal handling was done in accordance with the guidelines of the Canadian Council on Animal Care and the Principles of laboratory animal care ( N I H publication no. 85-23, revised 1985).  1.2.1 Arterial diameter measurements  Male  Sprague-Dawley  rats  (200  -  300g)  were  anaesthetized  with  intraperitoneal injections of a mixture of sodium pentobarbital (Somnotol, 30 mg/kg) and heparin (Hepalean, 500 U/kg), and then killed by decapitation. The brain was excised and transferred to a dissection dish filled with ice-cold physiological salt solution (PSS). A small branch (0.6-1.0 m m long) of distal second-order middle cerebral arteries (inner diameter of 100-200um)  was  dissected from surrounding connective tissues, and transferred to the myograph  30  chamber (Living Systems Instrumentation, Burlington, V T ) . The chamber was filled with oxygenated PSS heated to 37°C.  The proximal end of the artery was fed onto the tip (diameter of -60 um) of a glass microcannula and tied with single strands (20 um) of 4-0 braided nylon suture; the perfusion pressure was then gently raised to clear the vessel of blood. The  distal end of the artery was  then similarly mounted to the  outflow  microcannula. After several minutes of perfusion, the distal outflow cannula was closed, and the transmural pressure was slowly increased to 60 rrtmHg by using an electronic pressure servo system (Living Systems Instrumentation). Thus, pressure-induced constrictions were recorded under conditions of no flow.  The  PSS in the vessel chamber was  continuously  re-circulated by  superfusion (MasterFlex, Cole-Parmer Instrumentation Co., Vernon Hills, IL) around the pressurized artery at a flow of 20-25 ml/min passing through an external reservoir that was bubbled with a gas mixture of 95% O 2 , 5% C O 2 . A p H micro-probe was positioned in the bath and used to adjust the reservoir gassing rate such to maintain the p H at 7.4 + 0.04.  A heating pump  (NESLAB  Instruments, inc., Portsmouth, N H ) connected to a glass heat exchanger warmed the PSS to 37°C.  The arteriograph containing a cannulated pressurized artery was placed on the stage of an inverted microscope (Olympus 1X70, UAPO/340 20X objective, Melville, NY) with a C C D (monochrome black and white) video camera (XC73/73CE, Sony) attached to a viewing tube. The arterial preparation was allowed to equilibrate for 60 min. Arterial dimensions were viewed on a U L T R A K K M -  31  12A monitor (Carrolton, TX) and measured using a video dimension analyser system  V94  (Living  Systems  Instrumentation)  that  provides  automatic  continuous readout measurements of luminal diameter and wall thickness. The information is up-dated every 17 ms, and the precision of the measurement is within 1%. Cerebral myogenic tone developed  diameter  spontaneously  and consistently during equilibration, resulting in significantly reduced luminal diameter. Once attained, it remains stable for hours unless perturbed by changes in transmural pressure or the addition of vasoactive compounds (Skarsgard et al, 1997).  1.2.2 Measurements of [Ca ]i 2+  Arteries were loaded with Fura-2, a Ca -sensitive fluorescent dye. Fura2+  2 A M (lOul of 1 m M stock solution, Molecular Probes, Eugene, OR) was premixed with an equal volume of a 0.01% solution of pluronic acid (Pluronic F-127, Molecular Probes) and 0.05% anhydrous dimethylsulfoxide (DMSO) diluted in PSS to yield a final concentration of 5 u M . The cannulated middle cerebral artery mounted in the myograph chamber, was incubated in the Fura-2AM/PSS loading solution for 1 hour at ~30 C, followed by a washout period of 30 min at 37 C and Q  Q  10 m m H g of intraluminal pressure.  Excitation was achieved by fluorescence microscopy using a 75-W xenon light source and a filter wheel rotating at -50 H z and containing 340- and 380-nm filters  (High-speed  Multi-Wavelength  Illuminator,  Photon  Technology  International, Monmouth Junction, NJ). Fluorescence emission was detected with a photomultiplier detection system 810/814 (Photon Technology International).  32  The 340/380 ratios were obtained at a rate of 20 points/sec of the .510 n m emission using  Felix quantitative  ratio fluorescence  software  (Photon Technology  International). Motion artefacts were typically limited to <10% in the individual fluorescence signals and were not detectable in the 340/380 ratio.  1.2.3 Experimental procedures  After the development of myogenic tone during the equilibration period (60 min), the relation between pressure and vessel diameter was studied. Intravascular pressure was decreased to 10 m m H g and then raised in 20 m m H g steps from 20 to 100 m m H g , while corresponding changes in vessel diameter and 340/380 ratio were measured. A t each step, diameter was monitored for 5-10 min until steady state was achieved. The protocol was repeated and the results averaged. After the study of the relation between pressure and vessel diameter in the absence of any compounds, transmural pressure was lowered to 20 m m H g , a manoeuvre that places the vessel below the lower limit of the pressure range for myogenic tone. Luminal diameter was allowed to stabilize for 15-20 min before pharmacologic agents were added into the bath to study their effects on pressure-induced myogenic tone.  The effects of calphostin C (1 uM) and a Rho kinase inhibitor, Y27632 (1 uM) on the pressure-diameter responses were examined under conditions where further changes in membrane potential were prevented with 60 m M KPSS (Knot and Nelson, 1998). The activators and inhibitors were used at concentrations previously described by others, and us, as selective for their intracellular targets in isolated arterial preparations (Henrion and Laher, 1993; Osol et al,  33  1993;  Gokina and Osol, 1998; Bakker et al, 1999; Kandabashi et al, 2000; Matrougui et al, 2001). Inhibitors were added to supervising buffer and allowed to circulate for 20 min until a new steady state diameter was reached. This was followed by reassessment of the constriction and 340/380 ratio due to the change  in  transmural pressure. A t the conclusion of each experiment, the superfusion solution was changed to a calcium-free PSS that contained 2 m M Ethylene glycolbis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and no CaCh. Vessels were incubated for 20 min and then the pressure steps were repeated to obtain the "passive" diameter of each vessel at each pressure value in order to calculate the percentage of myogenic constriction.  1.2.4 Expression of the results and statistical analysis  Myogenic tone at each pressure was expressed as a percent decrease in diameter from the "passive" diameter (% constriction) or as a percent decrease in myogenic response (% inhibition) using the equations below.  % constriction = 100 x [(DCa-free - DPSS) / DCa-free]  % i n h i b i t i o n 100 x [1- (DCa-free - DPSS) / (DCa-free - DPSS + inhibitor ]  Where D is the arterial diameter in the indicated solution. The K -induced +  increase in 340/380 ratio was measured in each tissue. Therefore, in the present study the changes in 340/380 ratio were normalized to the change in the ratio produced by 60 m M KPSS in the same vessel. A l l results are expressed as mean ± SE of n experiments. One vessel was taken from each animal. Statistical  34  evaluation was done by A N O V A followed by Newman-Keuls tests. Means were considered significantly different when P < 0.05.  1.2.5 Drugs and solutions  The ionic composition of the PSS was (in mM): N a C l 119, KC1 4.7, KHzPCu 1.18, N a H C O s 24, M g S 0 4 - 7 H 0 1.17, CaCh. 1.6, glucose 5.5 and E D T A 0.026. The 2  solution of depolarizing high K solution was made by equimolar substitution for +  N a C l . Calphostin C was purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA) and vasopressin from Sigma (Ontario, Canada). Y-27632 was a gift from Welfide Corporation (Osaka, Japan). Y-27632 was dissolved in deionised water (NANOpure). Calphostin C was dissolved in D M S O (total of 0.0001% of D M S O in Krebs buffer solution). The effects of D M S O had been previously tested in our laboratory and did not alter the pressure-diameter relation  or  the  vascular responses  to  norepinephrine and  acetylcholine.  Comparative constrictor responses were obtained from control arteries and arteries loaded with Fura-2-AM. Interactions between Fura-2 and calphostin C or Y-27632 were absent (Appendix 1).  1.3 Results  1.3.1 Myogenic tone in rat cerebral resistance arteries  Cerebral arteries developed  graded myogenic constrictions over the  physiologic intraluminal pressure range of 40-100 m m H g . Figure 1.4A shows a representative trace of the steady state response of middle cerebral arteries (mean of diameter at rest 169.5 ± 7.7 um; n = 15) to increases in intraluminal  35  pressure. Figure 1.4B shows that myogenic tone of middle cerebral arteries is associated with a small change in the 340-to-380nm (340/380) fluorescence ratio of Fura 2 from 1.60 ± 0.31 at 20 m m H g to 2.57 ± 1.79 at 60 m m H g . However, no significant change in the 340/380 ratio was obtained when the pressure was further increased (from 60 m m H g to 100 m m Hg). Data summarized in Figure 1.4C are normalized to 60 m M KPSS and show a significant changes at each transmural pressure in 340/380 ratio with greatest changes recorded at 60 (34.6 ± 4.1%; n = 24) and 100 m m H g (43.5 ± 5.8 %; n = 24). Removal of the endothelium or pre-treatment of vessels with L - N A M E (200 uM) and indomethacin (10 uM) have been tested previously in this laboratory and did not change pressureinduced constriction (Lagaud et al, 1999).  36  100 (mmHg)  Q CM B  Pressure (mm Hg)  Figure 1.4 Effects of intraluminal pressure on rat cerebral arteries diameter and 340/380 fluorescence ratio. Original traces showing myogenic tone development (A) and accompanying changes in 340/380 fluorescence ratio (B) in response to elevation of mtralurninal pressure from 10 to 100 mrnHg. (C): Histograms showing the normalised change in 340/380 fluorescence ratio in response to elevation of mtralurninal pressure from 10 to 100 rruriHg. Values are means ± S.E, (n=). Each bar is significantly different from the others, P < 0.001.  1.3.2 Effect of 60 m M KPSS on myogenic tone  A  series of experiments were designed to examine primary events  independent of depolarization in pressure-induced myogenic tone of cerebral arteries. Thus,  the role of raised K  +  37  concentrations on pressure-induced  contraction (Figure 1.5A, B and C) was investigated. Step-wise increases of transmural pressure (20 to 100 m m Hg) in the presence of 60 m M KPSS, which clamps the arteries at -21mV (depolarized cerebral arteries) (Knot and Nelson, 1998),  produced myogenic  responses  (Figure  1.5A).  Thus,  simultaneous  recordings of diameter and [Ca ]iin Figure 1.5A and B show that pre-treatment 2+  of cerebral arteries with 60 m M KPSS induced a contraction. The diameter was reduced to 48.80 ± 4.2% (n = 7 at 20 mmHg) of its original value which was associated with a transient increase in 340/380 fluorescence ratio followed by a plateau phase. Step-wise increases of transmural pressure above 20 m m H g in the presence of 60 m M KPSS produced a myogenic response without any significant changes in 340/380 fluorescence ratio (Figure 1.5B). These results suggest that additional mechanisms, independent of the increase in [Ca ]i, may be important 2+  components of myogenic contraction.  Data summarized in Figure 1.5C are normalized to 60 m M KPSS and show that 60 m M KPSS caused a significant increase in [Ca ]i at 20 m m H g (88.3 ± 3.2%, 2+  n = 6 ) compared with responses to pressure of 60 m m H g (54.4 ± 7.3%, n = 6) and 100 m m H g (48.6 ± 8.1%, n = 6). The pressure-induced constriction caused by 60 and 100 m m H g was accompanied by a transient increase in 340/380 fluorescence ratio (Figure 1.5C). A t higher K concentrations such as 100 m M , the arteries were +  maximally contracted so that increases in transmural pressure did not produce additional myogenic tone and the arterial diameter was maintained.  38  100 (mm Hg)  60 mM KPSS  B  o  '•5  2  o  00  100  200  £2  300  400  Time (s)  o O  1Q0  (6) +  ooo  §5  (6)  o •o  • LBL.BLBLJ J  U  L  I  U  '  • L B L B U B L U 1 J U L J • LM_«LJIL U L I L t l • L a l .  a  ! m\...  20 mm Hg 60 mm Hg 100 mm Hg 60 mM KPSS  Figure 1.5 Effects of intraluminal pressure on depolarised cerebral arteries. Original traces show that changes in mtralurninal pressure from 20 to 100 mm H g caused transient myogenic tone (A), and accompanying changes in 340/380 fluorescence ratio (B). Normalized 340/380 fluorescence ratio in response to elevation of mtralurninal pressure from 20 to 100 mmHg on depolarized arteries (60 m M KPSS) in the same vessel (C). Values are means + S.E. **P < 0.01, significantly different from response obtained at 60 rrtrnHg. +P < 0.001, significantly different from response obtained at 100 mmHg; (n=).  1.3.3 Calphostin C inhibits vascular tone  The effect of the P K C inhibitor, calphostin C , on the activity of cerebral arteries was tested. Calphostin C inhibits myogenic tone induced by 60 m m H g intraluminal pressure (Figure 1.6A). The arteries subjected to 60 m m H g were  39  allowed to equilibrate for an hour after which they gradually contracted. The sustained contraction was reversed by the addition of calphostin C , resulting in dilation of greater amplitude than that induced by the initial increase of pressure to 60 m m H g . The vasodilation induced by calphostin C occurred independently of any changes in [Ca ]i, while the dilation induced by the removal of C a 2+  2+  from  the solution (0 Ca ) was accompanied with a decrease in 340/380 ratio (Figure 2+  1.6B). Calphostin C also inhibited the tone developed in arteries pre-treated with indolactam ( l u M ; a P K C activator), while it did not alter the constriction induced by a 60 m M KPSS depolarising solution (Figure 1.7).  40  60 mmHg  2+  1 uM  OCa'  Calphostin  500-  (60 mmHg)  3 400E ro Q 300-  200  B  1.751.50  1.004 0.75 0.50 0.25H 0.00  1000  2000  3000  4000  6000  5000  Time (sec)  Figure 1.6  C on myogenic tone and 340/380 ratio. Representative traces showing the inhibition of myogenic tone by calphostin C in rat cerebral arteries. Increases in intraluminal pressure to 60 mmHg induced a vasodilation followed by a transient vasoconstriction. The addition of calphostin C induced a vasodilation followed by a plateau phase. Further removal of Ca in the bath dilated the artery to its maximum (A). The increase in intraluminal pressure was accompanied with an increase in the 340/380 fluorescence ratio which remain unchanged with the addition of calphostin C until it decreased following the removal of extracellular Ca (B). Effect of calphostin  2+  2+  41  I  I Calphostin C (1 uM)  F i g u r e 1 . 7 Effect of calphostin C on pre-contracted arteries. Histograms showing the effects of 60 mmHg increase in pressure ( | |), indolactam (1 uM; ) and 60 mM KPSS ( ) in the absence and in the presence of calphostin C (1 uM). *P < 0.001, significantly different from pressure-induced contraction at 60 mrnHg. #P < 0.001, significantly different from the relative control response induced by indolactam (1 uM); (n=).  1.3.4 Effect of calphostin C and Y-27632 in depolarized arteries.  Pharmacological inhibitors were used on depolarized cerebral arteries to determine whether the ability of arteries to maintain their diameters in the absence of pressure-induced depolarization is mediated by P K C or Rho-kinase Ca -sensitization pathways. Figure 1.8A and B show representative traces of the 2+  effects of a P K C inhibitor, calphostin C on cerebral arteries placed in a depolarizing solution. In the presence of calphostin C (1 uM), application of 60 m M KPSS resulted in a significant contraction of the cerebral artery. Step-wise increases of transmural pressure in the presence of 60 m M KPSS and calphostin C resulted in a loss of myogenic tone development (Figure 1.8A). This was  42  accompanied by a transient elevation of 340/380 fluorescence ratio followed by a plateau phase (Figure 1.8B). Data in Figure 1.8C are normalized to 60 m M KPSS and summarize the effects of 60 m M KPSS on the 340/380 fluorescence ratio, in the presence of calphostin C (1 uM).  A commonly used inhibitor of Rho-kinase, Y-27632, was also examined in rat cerebral resistance arteries as shown in Figure 1.9. In the presence of Y-27632 (1 uM), application of 60 m M KPSS resulted in cerebral artery contraction (Figure 1.9A). Step-wise increases of transmural pressure failed to induce myogenic tone development in the presence of 60 m M KPSS and Y-27632 (Figure 1.9A). This was accompanied by an elevation of 340/380 fluorescence ratio in response to 60 m M KPSS, followed by a plateau phase, in which there was no change in response to l u M of Y-27632 (Figure 1.9B). Data in Figure 1.9C are normalized to 60 m M KPSS and summarize the effects of 60 m M KPSS, in the presence of Y 27632 (1 uM).  43  100 (mmHg)  B  C  20 mm Hg 60 mm Hg 100 mm Hg  60 mM KPSS/1 uM Calphostin  Figure 1.8 Effects of calphostin C on depolarized and pressurized arteries. Original traces show that in the presence of calphostin C (1 uM), 60 mM KPSS caused a vasoconstriction (A) accompanied with an increase in 340/380 fluorescence ratio (B). Changes in mtraluminal pressure from 20 to 100 mm Hg increased the vessel diameter (A) without changes in 340/380 fluorescence ratio (B). Data in C show the 340/380 fluorescence ratio normalized to the change in the ratio produced by 60 mM KPSS in the same vessel; values are means ± S.E. *P < 0.01, significantly different from response obtained at 60 mmHg. #P < 0.001, significantly different from response obtained at 100 mmHg; (n=).  44  A 100 (mm Hg)  60 mM KPSS  B o £2 ©  100  200  300  Time (s) 100  20 m m H g  60 mm Hg  100 m m H g  60 mM KPSS/1 uM Y-27632  Figure 1.9 Effects of Y-27632 on depolarised and pressurized arteries. Original traces show that in the presence of Y-27632 (1 uM), 60 m M KPSS caused a vasoconstriction (A) accompanied with an increase in 340/380 fluorescence ratio (B). Subsequent changes in mtraluminal pressure from 20 to 100 mm H g increased the vessel diameter (A) without changes in 340/380 fluorescence ratio. Data in C show the 340/380 fluorescence ratio normalized to the change in the ratio produced by 60 m M KPSS in the same vessel; values are means ± S.E; (n=).  Unlike, calphostin C , Y-27632 significantly inhibited the transient rise in 340/380 fluorescence ratio caused by the application of 60 m M KPSS (Figure 1.8B and Figure 1.9B). This was also reflected in the normalised 340/380 ratio at 20  45  m m H g which in the case of calphostin C (Figure 1.8) is comparable to the effect of 60 m M KPSS by itself (Figure 1.5) while in contrast, the addition of Y-27632 produced a reduced normalised ratio (Figure 1.9). The specificity of the Rhokinase inhibitor Y-27632 was therefore further examined.  1.3.5 Y-27632 inhibits vascular tone  In order to determine to what extent Rho-kinase plays a role in contraction induced by 60 m M KPSS, we compared the effects of Y-27632 on cerebral arteries pre-contracted with three different stimuli; pressure induced myogenic tone, 60 mM  KPSS  induced  contraction  and  G-protein mediated  constriction  by  vasopressin. In pre-contracted arteries, the addition of Y-27632 (0.1-3.0 uM) induced a concentration dependent vasodilation in cerebral arteries (Figure 1.10). Myogenic tone was inhibited at all concentrations of Y-27632 used, with 22±3% inhibition occurring with 0.1 u M Y-27632 and 83±6 % with 3.0uM Y-27632 (n=4; Figure 1.10). The vasoconstriction due to vasopressin (0.1 u M ; n=4) and 60 m M KPSS (n=8) were both studied at a pressure of 20 m m H g , a pressure at which myogenic  tone  is  absent  (Meininger et  al,  1991;  Laporte  et  al., 1994).  Vasoconstrictor responses to both vasopressin and 60 m M KPSS were blunted by Y-27631 with the highest concentration of Y-27632 (3.0uM) reducing the response to vasopressin by 60±5% and that to 60 m M KPSS by 33±4% (Figure 1.10). Although the vasoconstriction to 60 m M KPSS was also reduced by Y-27632 in a concentration dependent manner, the extent of inhibition was significantly lower (p<0.05) than that occurring with either myogenic or vasopressin induced tone at each concentration (0.1-3.0uM) of the Rho-kinase inhibitor (Figure 1.10).  46  100r  CM CO CD CM  >-  ZL d  CM CO CO  CM CO CD  CM CO CD  CM CO CD  CM CO CD  CM CO CD  CM  CM  CM  CM  CM  CM  ZL CO d  ZL  ZL  o  3. O CO  3.  r-  r--  r^- r-~  > >- >-  >- >-  o o  CO  CM CO CD 1^ CM  CM CO CD  CM CO CD  CM  CM  ZL CO d  ZL O  ZI  >- >- >- >d  T — '  o  CO  Figure1.10  Extent of Y-27632 inhibition of vascular tone. Myogenic tone was induced at an intraluminal pressure of 60 mmHg ( | | ). Vasoconstriction in response to both vasopressin (0.1 uM; 77777}) and KPSS (60 mM; EOQWO were determined in the absence of myogenic tone (intraluminal pressure=20 mmHg). Significant differences in the inhibitory responses induced by the same concentration of Y-27632 were observed between pre-contracted arteries with 60 m M KPSS and those pre-contracted with both mtralurninal pressure of 60 mmHg and vasopressin (* p<0.05, A N O V A ; n=4-8).  1.3.6 Y-27632 inhibits C a  2 +  entry  Since constriction to high K occurs via the activation of V G C C , changes in +  intracellular C a luM)  2+  in response to 60 m M KPSS before and after Y-27632 (60 min,  were measured in the absence  of myogenic response (intraluminal  pressure of 20 mmHg). A typical recording is shown in Figure 1.11. Exposure to 60 m M KPSS induced a rapid constriction followed by a moderate but constant vasoconstriction  accompanied by  a rapid  and  sustained  rise  in 340/380  fluorescence ratio, reflecting an increase in free intracellular C a . In the presence 2+  47  of Y-27632, 60 m M KPSS induced a rapid transient constriction followed by a reduced vasoconstriction. The rapid rise in intracellular C a  2 +  was also reduced by  Y-27632.  1nM Y-27632  300 PSS Wash 3 mM KPSS  200  400  (4-7  600  K*)  PSS Wash (4.7 mM K )  60mMKPSS  i  800  1000  1  1200  1  1400  1  1600  1  1800  Time (sec)  Figure 1.11 Effect of  Y-27632  on depolarised arteries.  Representative trace of the changes i n diameter (A) and i n 340/380 fluorescence ratio (B) i n cerebral arteries submitted to 60 m M KPSS and 1 u M Y-27632 i n the absence of myogenic tone (mtralurriinal pressure=20 m m H g ) .  48  Receptor-independent  Ca -entry induced with 60 m M KPSS under 2+  control conditions (n=7) raised the 340/380 fluorescence ratio to 1.8±0.06 (Figure 1.12). In 'the presence of Y-27631  ( l u M ) and 60 m M KPSS, the 340/380  fluorescence ratio was significantly decreased to 1.4+0.09 (p<0.05; Figure 1.10). However, the 340/380 fluorescence ratio induced by 60 m M KPSS was unaltered by pre-treatment with an inhibitor of protein kinase C (calphostin C , l u M ; 340/380 ratio=1.8±0.1; Figure 1.12).  2.0 n o 1.5  2  o ©  0.5 0.0  Control  Y-27632  Calphostin C  60 mM KPSS  F i g u r e 1 . 1 2 Effect of different pharmacologic inhibitors of Ca  2+  sensitization. Pre-contracted cerebral arteries with 60 mM KPSS induced increase in the 340/380 fluorescence ratio before (control) and after incubation with inhibitors of Rho-kinase (1 uM, Y-27632), and protein kinase C (1 uM, calphostin C). * p<0.05, ANOVA; (n=).  49  1.4 Discussion  Growing interest in the mechanism of myogenic constriction has increased our understanding of its cellular basis and added to our knowledge of the various signalling pathways involved. To this day, it is generally accepted that the principal mechanism of pressure-induced myogenic constriction involves a depolarization followed by the entry of extracellular C a . Then again, pressure2+  induced constriction has been associated with significantly lower increases in [Ca ]i when compared to high K -induced constriction (VanBavel et al, 1998). 2+  +  Thus, a second underlying mechanism of the myogenic tone implies that in addition to an elevation of  [Ca ]i, 2+  an increase in myofilament Ca -sensitivity 2+  may also occur. This study explored the interaction of these two pathways in the response of arterial diameter to increases in transmural pressure. Observations that pressure-induced constriction is maintained in cerebral arteries incubated in a depolarising solution (60 m M KPSS) are reported. In addition, the activation of P K C and Rho-kinase leading to Ca -sensitization is shown to participate in the 2+  pressure-induced constriction in depolarised cerebral arteries.  1.4.1 Ca -sensitization contribution to myogenic response 2+  Several studies report that myogenic tone is only dependent on Ca -entry 2+  via the L-type V G C C , and does not require any contribution by mechanisms that change the Ca -sensitivity (Brayden and Nelson, 1992; Nelson et al, 2+  1995;  McCarron et al, 1997; Knot and Nelson, 1998). To determine the primary events in pressure-induced myogenic tone in cerebral arteries, a series of experiments were performed in which the arteries were incubated in a depolarizing solution (60 m M KPSS) and changes in diameter and 340/380 fluorescence ratio produced  50  by  increased transmural pressure were measured. Under such conditions,  cerebral arteries were still able to produce a myogenic response i.e. arteries maintained a relatively constant diameter with increases in transmural pressure (Figure. 1.5). The fact that myogenic contraction still occurred in a 60 m M KPSS solution when presumably, the membrane potential is close to the theoretical K  +  equilibrium potential (Casteels et al, 1977; Droogmans et al, 1977; Knot and Nelson, 1998) suggests that K channel activity alone cannot account for change +  in membrane potential that occurs during myogenic tone. Others have proposed that  activation  of  CI" channels  may  be  involved  in  pressure-induced  depolarization of vascular S M C s of rat cerebral artery (Nelson et al, 1997). However, it has been reported that the effects induced by Cl~ channel inhibitors may be due to inhibition of L-type V G C C (Doughty et al, 1998). Step-wise increases of transmural pressure in depolarized arteries induced a transient contraction without a significant change in [Ca ]i. A t this external [K ], the 2+  +  membrane potential of S M C s of the rat cerebral arterial wall is approximately -21 mV, and further changes in membrane potential are unlikely to occur (Knot and Nelson, 1998). Therefore, the observed myogenic contraction is most likely due to an increase in Ca -sensitivity of the myofilaments. The possible involvement of 2+  the P K C and/or Rho-kinase pathways  in pressure-induced constriction of  depolarised arteries was then investigated.  1.4.2 Ca -sensitization is mediated by P K C and Rho-kinase 2+  Despite an elevated [Ca ]i generated by membrane depolarization with 60 2+  m M KPSS, cerebral arteries subjected to increased transmural pressure dilated in the presence of inhibitors of both P K C and Rho-kinase. Under conditions where  51  no  additional  Ca -entry 2+  through  VGCC  is  possible,  Ca -sensitization 2+  mechanisms are likely responsible for the constriction. If Ca -sensitization 2+  mechanisms are prevented by the use of P K C and Rho-kinase inhibitors, the ability of the artery to actively respond to an increase in intraluminal pressure is consequently compromised. This could explain the passive response of the cerebral arteries dilating to increased intraluminal pressure in the presence of both 60 m M KPSS and inhibitors of P K C or Rho-kinase.  These results provide further evidence that P K C plays an active role in myogenic tone of small contractile arteries by modulating intracellular C a 2+  sensitivity (Laher and Bevan, 1989; H i l l et al, 1990; Osol et al, 1991; Osol et al, 1993; Narayanan et al, 1994; Lange et al, 1997; Gokina and Osol, 1998). In addition, they implicate Rho-kinase as a mediator of C a  2+  sensitization in  myogenic contraction. These findings are consistent with previous studies, which associate stretch-induced redistribution of P K C and RhoA membrane with C a  2 +  to the plasma  sensitization (Gong et al, 1997; Dessy et al, 2000; Yeon et al,  2002). Furthermore, stretch-activated P K C and Rho-kinase are correlated with an increased phosphorylation of MLC2OKD  3  (Yeon et al,  2002), confirming their  involvement in force generation.  1.4.3 Activation of Rho-kinase by membrane depolarization  The  inhibition of the myogenic  response  by P K C and Rho-kinase  inhibitors in depolarised arteries provides indirect evidence that intracellular signalling leading to vasoconstriction of pressurised arteries involves a series of phosphorylation reactions, which ultimately decreases the activity of M L C P . The  52  activation of such pathways is often associated with ligands binding to G-protein coupled receptors  (Somlyo  and Somlyo, 2000). In accordance with these  observations, Y-27632 significantly reduced the contraction induced by both intraluminal  pressure  and  vasopressin  (Figure  1.10).  However,  higher  concentration of Y-27632 (3 uM) reduced, to a lesser extent but significantly, the vasoconstriction induced by 60 m M KPSS. What seemed to be a non-specific interaction of Y-27632 at an unrelated site, may in fact demonstrate depolarization in S M C leads to both an increase in  [Ca ]i 2+  that  and Ca -sensitization. 2+  Recently, similar observations have been reported in isolated S M C s where sustained contraction induced by 60 m M KC1 depolarization are practically abolished by both Rho-kinase inhibitor, Y-27632 and HA-1077 (Mita et al, 2002; Sakurada et al, 2003). The consequent reduction in force has been associated with the decrease in  MLC20KDa  phosphorylation and the loss in Ca -sensitization (Mita 2+  et al, 2002). Both agonist (noradrenaline) and KC1 depolarization increase the GTP-bound active form of RhoA (Sakurada et al, 2003). KC1 activation of R h o A is however  Ca -dependent 2+  and  has  been  further  associated  phosphorylation at Thr695, a well-known inhibitory site of the  with  MLCP  phosphatase  (Sakurada et al, 2003). Taken together, these latter observations suggest that a similar Ca -sensitization pathway as the one described for agonist-induced 2+  contraction (Kitazawa et al, 1991) is involved in KC1 depolarization-induced contraction. While agonists activate RhoA through binding of G-protein coupled receptor, C a M and CaMKII have been implicated in the KC1 depolarization induced R h o A activation (Sakurada et al, 2003). Bolz et al. have demonstrated that sphingosine kinase also modulates myogenic tone via the RhoA/Rho-kinase pathway (Bolz et al, 2003). Since the activity of sphingosine kinase increases with depolarization and the activation of V G C C (Alemany et al, 2001), sphingosine  53  kinase could also be potentially responsible for Rho-kinase activation in KPSS depolarised arteries. Taken together, these results indicate that Ca -sensitization 2+  mediated by Rho-kinase can be induced by membrane depolarization in addition to the classical activation through ligand binding to G-protein coupled receptor and the newly proposed stretch-induced mechanisms.  In the present study, Y-27632 simultaneously  decreased  the 340/380  fluorescence ratio recorded from the 60 m M KPSS depolarised arteries (Figure 1.11). While these observations, although obtained in whole arterial preparation, go against previous reports in S M C strips of rat tail artery (Mita et al, 2002), they imply that Y-27632 interacts with unrelated sites and may indirectly be capable of blocking Ca -entry. This latter observation seems to detract from the 2+  uncontrolled use of Y-27632 as a specific inhibitor of Rho-kinase. Y-27632 is a selective inhibitor of the Rho kinase and acts as a competitive inhibitor of A T P binding. It is ~200 times more selective for inhibiting Rho-kinase than P K C , cyclic AMP-dependent protein kinase and myosin light chain kinase (Uehata et al, 1997). While Y-27632 reduced the 340/380 fluorescence ratio in depolarised arteries, inhibitors of P K C (calphostin C) did not, which indicates that the suggested inhibition of Ca -entry is unique to Rho-kinase or its inhibitor. The 2+  possibility of an undesirable interaction between Y-27632 and Fura-2 has also been ruled out (Appendix 1). This suggests two possibilities: a potential lack of selectivity of Y-27632 or a novel role for Rho-kinase in Ca -entry mechanisms. 2+  Further investigations will be required in order to elucidate the downstream mechanisms following the activation of Rho-kinase in depolarised arteries.  54  1.4.4 Summary  In summary, it was demonstrated that pressure induced myogenic tone in cerebral  arteries  involves  Ca -sensitization 2+  mechanisms.  This  increase  in  intracellular Ca -sensitivity is hypothesised to be mediated by P K C and/or Rho2+  kinase. A s the arteries were depolarised with 60 m M KPSS, additional pressureinduced changes in C a  2 +  and K channel activity were minimised. Therefore, the +  inhibition of myogenic tone in depolarised arteries by both P K C and Rho-kinase inhibitors is attributed to the inhibition of Ca -sensitization pathways of the 2+  myogenic responses. Whereas C a  2 +  entry via V G C C is an essential component of  pressure-induced constriction of small arteries, these results provide evidence that other intracellular events are able to maintain active constriction in response to pressure changes under conditions where additional Ca -entry into the cell is 2+  minimal. These results also support the participation of both P K C and Rhokinase in Ca -sensitization of myogenic responses. In addition, the activation of 2+  Rho-kinase by membrane depolarization has been demonstrated. Downstream effectors of Rho-kinase depolarization-induced activation remain to be identified, but they may be involved in indirect control of Ca -entry mechanisms. 2+  55  Chapter 2  Characterization of glucose transporters in endothelial cells of small contractile arteries 2.1 Introduction  2.1.1 The glucose paradox  In 1921-22, the discovery of insulin by Banting and Best changed the course of one of today's most prominent metabolic disorders (Banting and Best, 1990). Diabetes Mellitus, first described by the sweetness of the urine, is characterised by elevated plasma glucose levels resulting from defects in insulin secretion, insulin action or both (Kernohan et al, 2003). Increased life expectancy acquired with insulin supplements, and the development of new methods,  have  allowed  diabetes  to  reach  today  epidemic  diagnostic  proportions.  Worldwide, the diabetic population is estimated to increase each year by 5.5 million and is expected to attain 300 million by the year 2025 (King et al, 1998). Since fatal ketosis is no longer the predetermined fate of diabetic patients, new complications have emerged from long-term diabetes, regardless of its aetiology. Chronic hyperglycaemia can lead to renal failure, blindness, defective nerve conduction and impaired wound healing but, by far, the major cause of morbidity and mortality is attributed to cardiovascular diseases associated with atherosclerosis (Haffher et al, 1990; Anonymous, 1995; Wolffenbuttel and van Haeften, 1995; Anonymous, 1999; Haffner et al,  2000). In fact, myocardial  infarction and stroke are more common among people with diabetes than in those without (Barrett-Connor et al, 1991; Manson et al, 1991; Koskinen et al,  56  1992). Thus, diabetes mellitus has become an epidemic and a prime risk factor for cardiovascular diseases.  In the diabetic population, well known cardiovascular risk factors such as hypertension, dyslipidemia (increased level of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol and decreased level of high  density  lipoprotein  (HDL)),  obesity,  hyperinsulinaemia  and  insulin  resistance seem to be exacerbated (Howard, 1996a). Hyperglycaemia, a metabolic abnormality of diabetes, could potentially precipitate the onset of macrovascular disease by itself. Hyperglycaemia has been shown to inhibit the production of N O in arterial ECs (Williams et al, 1998) and to stimulate the production of plasminogen activator inhibitor-1 (PAI-1) (Du et al,  2000). In addition, by  reducing the expression of hepatic perlecan heparan sulphate proteoglycan, hyperglycaemia contributes to the elevation of atherogenic cholesterol-enriched apolipoprotein  B-containing  residual  particles  (Ebara  et  al,  2000).  More  importantly, hyperglycaemia is known to be directly toxic for the ECs (Sank et al, 1994; Haller, 1997; Cosentino and Luscher, 1998; Williams et al, 1998; Aronson and Rayfield, 2002; Lee et al, 2002), the first line of protection of the vascular wall. Dysfunction of the ECs has been identified as the first step in the pathogenesis of atherosclerosis in non-diabetics (Lusis, 2000). Both the Diabetes Control and Complications Trial (DCCT) (Anonymous, 1995) and the United Kingdom Prospective previously  Diabetes  attributed the  Study  (UKPDS)  high prevalence  of  (Anonymous,  microvascular  1998),  have  complications  (degeneration of the retina, renal glomerulus and vasa nervorum of peripheral nerves) to hyperglycaemia. However, the same studies failed to reach statistical significance in the case of macrovascular disease (atherosclerosis associated with  57  larger blood vessels), despite a trend showing its reduction with tight control of the glycaemia (Anonymous, 1998). A d d i n g to this fact is that more effects on the reduction of the risks associated with macrovascular diseases were observed with treatments targeting dyslipidaemia or hypertension. H o w can such a discrepancy be explained? If hyperglycaemia is the triggering event in the development of macrovascular diseases, then why is the effect of glucose lowering drugs so minimal? A few plausible explanations have been put forward. Firstly, it is possible that the approaches used to lower the level of lipid cholesterol and reduce elevated blood pressure are simply more efficient than the one used to lower blood glucose. Secondly, the available glucose lowering therapies may not produce enough impact on the macrovascular state or thirdly, their administration may come too late into the process and/or be of too short duration to reverse the undesirable effect of long-term hyperglycaemia (Libby and Plutzky, 2002). Despite all the progress made towards a cure for diabetes, important aspects in the early stage of the disease remain unidentified. Earlier interventions may represent the only key solution to prevent the end-point of a long degenerative process induced by the toxic effects of hyperglycaemia.  O n one hand, patients with insulin-dependent diabetes mellitus (Type 1), experience elevated blood glucose levels for decades before any signs of macrovascular diseases are manifested (Jacobs et al, 1991). O n the other hand, diagnoses  of non-insulin dependent  diabetes  mellitus (Type 2) are  often  pronounced at a time where vascular diseases are already present (Medalie et al, 1975;  McPhillips  et  al,  1990).  Abundant  evidence  also  indicates  that  macrovascular diseases are initiated in the pre-diabetic state. Random episodes of hyperglycaemia, in fasting or post-glucose-challenge, are also associated with  58  increased  cardiovascular risk factors  in non-diabetic  subjects (Temelkova-  Kurktschiev et al, 2000). In addition, elevated blood glucose, after a glucose challenge, is highly correlated with atherosclerosis in people at high risk for diabetes Type 2 (Yamasaki et al, 1995; Hanefeld et al, 2000). Post-prandial changes are likely to occur before any changes in fasting plasma glucose can be detected and are responsible for the pre-diabetic onset of macrovascular diseases (Haller, 1997; Hanefeld and Temelkova-Kurktschiev, 2002; Lee et al, 2002). Thus, the pathogenesis of macrovascular disease in diabetes is insidious, takes time to develop and is triggered by early events in the progression of the diseases. In the face of hyperglycaemia-induced macrovascular diseases, we proposed to take a step back and investigate the events leading to the high predisposition of the endothelium to glucose toxicity.  2.1.2 The endothelium  The endothelium is the first line of protection for the vascular wall since it occupies a strategic position between the blood and underlying tissue. The role and function of ECs varies between vascular beds; in capillaries ECs regulate gas and nutrient exchange or prevent the passage of harmful substances, while in larger vessels they also control vascular tone by releasing vasoconstrictors and vasodilators (Davis and Hill, 1999). In small contractile vessels deprived of the vasa vasorum (small blood vessels irrigating the walls of large blood vessels), such as coronary arteries, the ECs must allow transport between the blood and cells of the vascular wall, but they must also protect the vessel's integrity, through selective permeability. Elucidating the processes whereby ECs mediate the exchange of plasma molecules with the vascular wall, while at the same time  59  safeguarding its functional integrity, is important for understanding how E C dysfunction relates to systemic diseases such as diabetes, in which the main causes of morbidity and mortality are vascular diseases (Grundy et al, 1999).  A healthy endothelium contributes to the homeostasis of the vascular wall. This can be achieved through the release of antithrombotic factors, preventing  thrombocyte,  leukocyte  and erythrocyte  adhesion,  vasodilating  substances such as N O and prostaglandins and contracting factors such as endothelin and thromboxane (Badimon et al, 1992; Haller, 1997). In isolated vessels, any injury to the endothelium is reflected by an altered endothelial vasoreactivity (Badimon et al, 1992). It is therefore not surprising that endothelial injury  or  dysfunction  leads  to  increased  vessel  permeability,  disturbed  coagulation and fibrinolysis (Schror, 1997), impaired vascular reactivity (French, 1966; Ross and Glomset, 1976; Goldstein and Brown, 1977), and precede any atherogenic process (Badimon et al, 1992; Lusis, 2000).  Impaired endothelial function is also observed in coronary and peripheral circulation of diabetics (Williams et al, 1998). In animal models, diabetes has been associated with an increased frequency of ECs death and transendothelial transport of macromolecules (Lin et al, 1993). Endothelial dysfunction identified in both diabetic patients and animals has also been linked with a reduced bioavailability of N O (Cosentino and Luscher, 1998; Mompeo et al, 1998). A decreased production of N O , results in an unbalanced vascular tone with increased contractility, and consequent amplification of the shear stress on the endothelium. In both diabetic patient and animals, endothelial dysfunction has  60  been attributed to the toxic effect of hyperglycaemia (Sank et al, 1994; Cosentino and Luscher, 1998; Lee et al, 2002).  2.1.3 Hyperglycaemia  The growing evidence that hyperglycaemia represents on its own a major risk factor for the development of both micro- and macrovascular disease has stimulated a great interest in the molecular mechanisms by which an elevated concentration of glucose can lead to endothelial dysfunction. From a large body of evidence, four main hypotheses have been put forward. A s high plasma glucose saturates the ability ofECsto metabolize glucose, the excess is catabolised or reduced by other, potentially toxic, pathways. First, the polyol pathway converts glucose to sorbitol via the enzyme aldose reductase (Greene et al, 1987; Brolin and Naeser, 1988; Burg and Kador, 1988; Simmons and Winegrad, 1989). A t first, the accumulation of sorbitol was thought to produce osmotic damage to the cell, since sorbitol does not diffuse easily across cell membrane. However, the relative accumulation of sorbitol in the cell is far less than other glucose derivatives, and therefore, cannot account for the increased osmotic pressure (Van den Enden et al, 1995). Sorbitol production is also accompanied by a decrease in nicotinamide adenine dinucleotide phosphate ( N A D P H ) , which interferes  with antioxidation reactions  (Kashiwagi et al,  1994), ultimately  reducing the availability of N O , a potent vasodilator (Harlan et al, 1984; Rosen and Freeman, 1984; Hunt et al, 1990; Tesfamariam and Cohen, 1992; Kashiwagi et al, 1994). Sorbitol is itself converted to fructose via sorbitol dehydrogenase and cofactor N A D . A n increased ratio of N A D H / N A D +  phosphate  dehydrogenase  +  inhibit glyceraldehyde-3-  ( G A P D H ) and raises the concentration of triose  61  phosphate, both contributing to the exacerbation of the flux and reflux of glucose through the polyol pathway and the formation of advanced glycation endproducts (AGEs).  Second, intracellular hyperglycaemia induces the formation of A G E s (Degenhardt et al, 1998). A t first, this was believed to occur through nonenzymatic  reactions  between  glucose  and  extracellular  macromolecules  (Brownlee et al, 1988; Hogan et al, 1992), a process found later on to be much slower than the actual formation of intracellular A G E from glucose-derived dicarbonyl precursors. In E C , the intracellular formation of A G E s occurs within a week of hyperglycaemia (Giardino et al, 1994). Three main A G E precursors: glyoxal, 3-deoxyglucosone and methylglyoxal are obtained respectively form glucose  auto-oxidation,  decomposition  of  the  Amadori  products,  and  fragmentation of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (Thornalley, 1990; Wells-Knecht et al, 1995). The A G E precursors react with amino groups of intracellular and extracellular proteins to form A G E s . These A G E s impair cell replication, increase  formation of lipoprotein and immune  reaction adhesion complexes (Brownlee et al, 1985; Cerami et al, 1988), disturb the coagulation process, increase endothelin (a potent vasoconstrictor) release, (Brownlee,  1992), and alter  the  elasticity  of  large  vessels by  enhancing  intermolecular cross-linking of type 1 collagen (Huijberts et al, 1993). In addition, the A G E s can bind to A G E receptors such as the receptor for A G E (RAGE), which induces, through a cascade of cellular signalling events, the production of reactive oxygen species (Yamagishi et al, 1998). In ECs, binding of ligands to A G E receptors has been shown to induce the expression of pro-coagulatory and  62  pro-inflammatory molecules and increase the permeability of the capillary walls (Doi et al, 1992; Schmidt et al, 1995; Abordo et al, 1996; L u et al, 1998).  Third, excess glucose increases the production of diacylglycerol (DAG), which in turn activates P K C (Lee et al, 1989; Inoguchi et al, 1994; King et al, 1996). P K C activation can also be enhanced through A G E receptors and through the polyol pathway (Keogh et al, 1997; Portilla et al, 2000). Increased levels of D A G and P K C have been found in diabetic animal models. Exposure of cultured vascular cells to hyperglycaemia and the isolation of vascular tissue from diabetic animals has revealed the predominance of the activation of P K C p isoform in these cells (Ishii et al, 1998). P K C is believed to exert an inhibitory function on endothelial nitric oxide synthase (eNOS) production or on its enzymatic activity (Hirata et al, 1995; Ohara et al, 1995), with the end result being impaired vascular relaxation. P K C also increases endothelin-1 -stimulated MAP-kinase activity (Tomlinson, 1999). In E C s and SMCs, P K C enhanced the activity of vascular endothelial growth factor (VEGF) which increases vascular wall permeability, and induces  the overexpression  the  of fibrinolytic  inhibitor PAI-1 and the activation of nuclear factor (NF)-KB.  Fourth and last, is the increased flux through the hexosamine pathway, which affects both gene expression and protein function. In this pathway, an intermediate of glycolysis, fructose-6-phosphate, is converted to glucosamine-6phosphate by the enzyme glutamine: fructose-6-phosphate  amidotransferase.  Glucosamine-6-phosphate participates in the synthesis of proteoglycan and the formation of O-linked glycoproteins. A n increased flux through the hexosamine  63  pathway produces changes in gene expression such as transforming growth factor-a (TGF-a), TGF-01 and PAI-1 (Kolm-Litty et al, 1998; D u et al, 2000).  Specific inhibitors for each of these pathways have been shown to improve the deleterious effect  caused by intracellular hyperglycaemia in  cultured cells and diabetic animal models. Many attempts have been made to unify all four hypotheses into one single interlinked process. Despite the growing evidence that each of these pathways affects the other, a consensus has yet  to  be  reached.  Among  the  proposed  unified  theories  stands  the  overproduction of superoxide by the mitochondrial electron-transport chain, the MAPKs  serving as  glucose  transducers, the  redox  imbalance (increased  tissue/ratio of free cytosolic N A D H / N A D ) , and the increased production of +  V E G F . The ultimate goal of each unified theory is to develop an effective inhibition of a single pathway that reduces all glucotoxic effects induced by hyperglycaemia.  Hyperglycaemia has long been associated with impaired endothelial functions (Aronson and Rayfield, 2002). A s most studies have focused on the mechanism  by  which  elevated  intracellular glucose  induces  endothelial  dysfunction (Brownlee, 2001; Sheetz and King, 2002), very few have raised the questions of how and why ECs accumulate toxic levels of glucose. It is only recently that the regulation of glucose transport in E C s has been examined (Mann et al, 2003), and most of this work has been in the blood-brain and bloodretina barriers (Olson and Pessin, 1996; Barrett et al, 1999; Joost and Thorens, 2001). The integrity of E C s of small contractile vessels is also affected by hyperglycaemia, and the coronary arteries are among the most susceptible to  64  atheroma, but few studies have directly examined glucose transporters in these vessels (Mann et al, 2003). Despite having the same embryonic origin, there is a strong heterogeneity between ECs of different vascular beds, and even those within the same vascular bed are known to have morphological, physiological and biochemical differences (Garlanda and Dejana, 1997; Ghitescu and Robert, 2002). Data derived from one vascular bed may not be applicable to the rest of the vasculature.  2.1.4 Glucose transporters  Since glucose is a hydrophilic polar molecule, which cannot freely penetrate the double lipid layer of the plasma membrane, a system of carriers, composed of transmembrane proteins, is used to carry glucose in and out of the cells. Over the last 15 years or so, several laboratories have been investigating the molecular structure and mechanisms of the regulation of these proteins involved in the transport of glucose. Two distinct systems of glucose transport have been identified. Glucose can cross the plasma membrane through facilitated diffusion, or be carried through by a secondary active transport system involving the cotransport of N a . +  The passive transport of glucose is mediated by the facilitative glucose transporters (GLUTs) family. The mechanism of facilitated diffusion involves a 12-transmembrane spanning regions protein forming a pore through the plasma membrane. This pore allows the free movement of glucose in both inward and outward directions, with a net movement of glucose down a concentration gradient. The first G L U T was isolated in 1977 from ghost erythrocytes by  65  Kasahara and Hinkle (Kasahara and Hinkle, 1977). Nearly ten years later, Mueckler et al. sequenced  the first isoform of the large family of  GLUTs  (Mueckler et al, 1985). The protein structure analysis has revealed five regions that possess both hydrophobic surface, linked to the plasma membrane, and hydrophilic surface, allowing the pore formation. The proposed mechanism of diffusion involves the binding of glucose to the pore complex by means of weak hydrogen bonds (Mueckler et al, 1985). The transport of glucose inside the cell is believed to occur in four steps. The first step corresponds to the binding of glucose to the extracellular site of the transporter. The attachment of glucose would then induce a change in the conformation of the transporter, allowing glucose to occupy the intracellular binding site on the transporter. The release of glucose into the cytosol  causes the  transporter to take back its original  conformation (Appleman and Lienhard, 1989).  Following the cloning of G L U T - 1 , four more isoforms were discovered (GLUT-2 to GLUT-5) and extensively studied (Mueckler, 1994). A sixth isoform was identified, but later revealed to be a pseudogene not expressed as a protein (Kayano et al, 1990), and a putative seventh isoform was a cloning artefact (Waddell et al, 1992; Burchell, 1998). With the completion of the H u m a n Genome Project, new members of the G L U T family, sharing between 28 and 65 % identity in terms of amino acid sequence and all carrying the sugar transporter signature (consisting of highly conserved glycine and tryptophan residues) were found. In total, 13 members of the gene family of the solute carrier 2A (gene symbol SLC2A) have been identified (Joost and Thorens, 2001). Each G L U T has now been renamed according to its gene symbol approved by the H u m a n Genome Organization Gene Nomenclature Committee (Joost et al, 2002). Under this new  66  nomenclature system, the G L U T s family has been divided in three distinct classes based on sequence similarity. Class I is characterised by a glutamine in helix 5 and an STSIF-motif in the extracellular loop 7, and comprises G L U T - 1 to 4. These isoforms have been well characterised and are often, with G L U T - 5 , referred to as the classical G L U T s . They differ from each other by their tissue distribution, affinity for glucose and hormonal regulation. Class II lacks a tryptophan following  a conserved  region  (GPXXXP)  in helix  10,  which  corresponds to the binding site for cytochalasin B and forskolin in G L U T - 1 . Class II includes G L U T - 5 and three other related isoforms, G L U T - 7 , G L U T - 9 and GLUT-11. Both G L U T - 5 and GLUT-11 have been shown to transport fructose with greater affinity than glucose. Class III unlike the two previous classes, has a shorter extracellular loop 1, and the glycosylation site is in the larger loop 9. Class III includes the last five isoforms, G L U T - 6 , G L U T - 8 , GLUT-10, and  GLUT-12,  the H -coupled myo-inositol cotransporter (HMIT1). In addition, four +  pseudogenes have been identified, of which three of them, including the previously named G L U T - 6 , share between 80 and 95% similarity with G L U T - 3 (Joost and Thorens, 2001; Joost et al, 2002). Each transporter has unique kinetics, and different rates for the transport of sugar into and out of cells.  Glucose can also be carried by a secondary active transporter, the sodiumdependent glucose transporter (SGLT). The active cotransport of glucose was first described by Crane in 1960 (Crane, 1960). The S G L T protein forms a pore across the plasma membrane from which both glucose and water can be transported. The pore, which contains the glucose-binding site, is formed of four transmembrane helices in close proximity to the C-terminus of the protein. The N-terminal domain has been proposed to act as a N a binding or translocation +  67  site. The interaction between the N - and C-terminal domains is thought to result in the cotransport of N a  +  and glucose (Wright et al, 1998). Glucose can be  transported against its concentration gradient using the energy generated by the Na -electrochemical gradient provided by the activity of the N a / K ATPase. The +  +  +  high-affinity SGLT-1 was cloned in 1987 from rabbit intestine (Hediger et al, 1987). SGLT-1 active transport of glucose has been well characterised in the epithelial cells of the brush border of the small intestine and proximal tubules of the kidneys (Wright et al, 1998; Wright, 2001). A low affinity SGLT-2 has been cloned from the pig kidney epithelial cells. It has 76% identity to its homologue SGLT-1. While most of the kinetic properties are similar between SGLT-1 and SGLT-2, a reduced NaVghicose coupling (from 2:1 to 1:1) has been found in the case of SGLT-2 (Mackenzie et al, 1996). SGLT-2 is predominantly located on the apical membrane of convoluted proximal tubule epithelial cells of the kidney (Wallner et al, 2001). A low affinity SGLT-3 has also been identified in both small intestine and renal epithelial cells (Kong et al, 1993; Mackenzie et al, 1994). The stoichiometry of this third S G L T protein is similar to that of SGLT-1 with a coupling of 2 N a for 1 glucose (Diez-Sampedro et al, 2001). SGLT-3 shares 76 to +  89% similarity with SGLT-1. Three more SGLT-like transporters are part of the same gene family (gene symbol SLC5A), identified from the H u m a n Genome Project, and remain to be completely characterised (Kong et al, 1993; W o o d and Trayhurn, 2003). Table 2.1 summarise the principal characteristics of each isoform of the G L U T and S G L T families.  68  Table 2.1  Summary of the principal characteristics of the sugar transporters.  Isofoim  M.W. (~kDa)  Glucose affinity (-Km)  Other affinity (~Km)  GLUT-I  45 55-65  20mM 30MG 5-7mM 2DG  17mM galactose >5M fructose mannose  Most tissue, erythrocytes, EC of blood barrier, brain  GLUT-2  56  42mM 30MG 7-16mM 2DG  66mM fructose 36-86mM galactose  liver, intestine, pancreatic p-cell  GLUT-3  48  6-8mM galactose  Brain, adipocyte  GLUT-4  50  GLUT-5  50-55  GLUT-6  46  GLUT-7  10mM 30MG 1-2mM 2DG 2-4mM 30MG 5mM 2DG  Tissue expression  muscle, adipocyte, heart intestine, testis, kidney, adipocyte, muscle spleen, leukocyte, brain, adipocyte n.d. testis, brain, blastocyst, adipocyte, muscle liver, kidney liver, pancreas, muscle, heart  6-14mM fructose  Functional characteristics  Asymmetric distribution  basal transport  basolateral membrane  trans-epithelial transport glucose sensor neuronal transporter insulin-sensitive transporter  basolateral membrane  fructose transporter  apical membrane  not responsive to insulin n.d. not responsive to insulin  n.d.  n.d.  Low affinity, high km n.d.  n.d.  GLUT-8  37-50  2mM 2DG  maybe fructose  GLUT-9  59  n.d.  n.d.  GLUT-I 0  57  0.3mM 2DG  GLUT-11  45  n.d.  maybe fructose  heart, muscle  GLUT-I 2  50  n.d.  n.d.  heart, prostate, muscle, adipocyte, small intestine  HMIT  67-83  No hexose transport  0.1 mM myo-, scillomuco-, chiro-inosito  Brain, adipocyte  Transport of H / myo-inositol  SGLT-1  71-77  0.4mM MOG  Galactose  kidney proximal tubule (straight part), intestine  Na*-dependent transport  SGLT-2  73  2mM MDG  Transport only glucose  Kidney proximal convoluted tubule  n.d.  may be a fructose transporter Insulin-sensitive transporter +  Kidney Independent transporter Na -dependent transport n.d. n.d. n.d.  apical membrane apical membrane  kidney, intestine, Transport only liver, spleen glucose n.d. n.d. n.d. SGLT4 n.d. n.d. n.d. n.d. SGLT-5 n.d. n.d. n.d. n.d. SGLT-6 n.d. Class I, Class II, Class III, 3-O-methyl-D-glucose (30MG), 2-deoxy-D-glucose (2DG), a-methyl-D-glucose (MDG), not determined (n.d.). (Thorens etal., 1988; James etal., 1989; Gould etal., 1992; Bell etal., 1993; Mueckler, 1994; Thorens, 1996; Walmsley ef at, 1998; Hirsch and Rosen, 1999; Carayannopoulos et al., 2000; Phay ef al., 2000; Dawson etal., 2001; Doege etal., 2001; Joost and Thorens, 2001; Wright, 2001; Rogers etal., 2002; Mann etal., 2003; Wood and Trayhurn, 2003). +  SGLT-3  73  6mM MDG  69  The tissue and subcellular distribution, as well as the kinetic properties of the isoforms, are critical for determining the net glucose transport rate, and play a central role in the regulation of intracellular glucose concentration. What then is known of the tissue and subcellular distribution of the G L U T s and SGLTs in the vasculature system? Despite the evidence implicating glucose toxicity in the prevalence of both micro- and macro-vascular disease in diabetes, it is only recently that an interest in the regulation of glucose transport in E C and S M C has been shown (Mann et al, 2003).  GLUT-1  was  thought  for the longest time to be the only glucose  transporter present in the ECs (Pardridge et al, 1990). In both ECs and SMCs, it regulates the basal transport of glucose (Charron et al, 1989; Pardridge et al, 1990; Pekala et al, 1990; Farrell and Pardridge, 1991; Hemmila and Drewes, 1993; Lutz and Pardridge, 1993; Maher et al, 1993; Sank et al, 1994; Cornford et al, 1995; Bolz et al, 1996; Gaposchkin and Garcia-Diaz, 1996; Urabe et al, 1996; Leino et al, 1997; Shi et al, 1997; Vannucci et al, 1997; Cornford et al, 1998b; Takagi et al, 1998; Dobrogowska and Vorbrodt, 1999; Nualart et al, 1999; Simpson et al, 1999). Of all vascular tissue, the BBB has been by far the E C source in which glucose transport regulation has been the most studied and characterised. This is not surprising given the importance of glucose as a primary source of energy for the brain and the tight junctions of the BBB, regulating the passage of substances to the brain (Garlanda and Dejana, 1997). Asymmetric distribution of G L U T - 1 has been reported in the BBB (Farrell and Pardridge, 1991; Cornford et al, 1998b) and in cerebral microvasculature (Bolz et al, 1996; Dobrogowska and Vorbrodt, 1999; Hirsch and Rosen, 1999). In each case, G L U T - 1 was found in greater abundance on the abluminal membrane (Farrell and Pardridge, 1991; Bolz et al,  70  1996; Cornford et al, 1998a; Dobrogowska and Vorbrodt, 1999; Hirsch and Rosen, 1999). The presence of G L U T - 3 has been reported on human intraplacental microvessel ECs (Hauguel-de M o u z o n et al, 1997). G L U T - 3 m R N A has also been found through in situ hybridization in E C s of brain microvessels following a long period of reperfusion after ischemia (Urabe et al, 1996) and in ECs of human brain tumour vessels (Nishioka et al, 1992). G L U T - 3 protein expression  was  detected only in the tumour vessels while it was not in the post-ischemic cortical ECs (Nishioka et al,  1992; Urabe et al,  1996). G L U T - 3 , if expressed in the  endothelium, is likely to play a role in the cells in critical need for glucose. Insulin-sensitivity as well as the presence of G L U T - 4 has also been reported in ECs of calf retinal (King et al,  1983)  and brain microvessels  (Frank and  Pardridge, 1981), in forebrain of bovine BBB (McCall et al, 1997), and in fat and muscle capillary ECs of the rat (Vilaro et al, 1989). However, in cultured cardiac ECs (Thomas et al, 1995), cultured aortic E C s (McCall et al, 1997), and human muscle capillary ECs (Friedman et al, 1991) studies have reported the absence of G L U T - 4 . The role of G L U T - 4 in the vascular system is still controversial and may be specific to E C of only certain vascular beds. G L U T - 5 has been detected in cerebral microvasculature ECs (Mantych et al, 1993a; Mantych et al, 1993b), but its functionality and role in these cells remain to be determined. A n SGLT-1-like glucose transporter has been identified on the luminal membrane of brain capillaries  and,  was  shown  to  be  mostly  functional  during period  of  hypoglycaemia (Matsuoka et al, 1998; Nishizaki and Matsuoka, 1998). To our knowledge, the presence of G L U T - 2 and the novel G L U T isoforms in ECs has not yet been assessed (Mann et al, 2003).  71  G L U T expression can be altered in diabetes. In diabetic animal models, down-regulation (Asada et al., 1998; Hajduch et al., 1998; Hirsch and Rosen, 1999), as well as up-regulation (Vannucci et al., 1997; Vannucci et al., 1998; Reagan et al., 1999) of G L U T expression has been reported in a variety of tissues. Yet again, in ECsof the vasculature very little is known regarding the regulation of glucose transporters (Mann et al, 2003). The expression of G L U T - 1 is reduced in brain capillary ECs of STZ-induced diabetic rats but not in post-prandial hyperglycaemia (Pouliot and Beliveau, 1995). Similar observations in bovine retinal and aortic ECs support the lack of effect of acute hyperglycaemia on G L U T - 1 expression (Kaiser et al, 1993; Mandarino et al, 1994; Vinals et al, 1999). Accordingly, it has been shown that ECs are unable to decrease their rate of glucose transport in ambient elevated glucose concentration (Kaiser et al, 1993; Giardino et al, 1994; Howard, 1996b). Since ECs are highly specialized cells and play a major role in the transport of glucose from the blood through the vascular walls, especially in small vessels deprived of vasa vasorum this inability to down-regulate glucose uptake could be responsible for their high susceptibility to glucose toxicity.  While a short-term elevation of glucose concentration seems to have no effect on the expression of G L U T - 1 , hypoglycaemia has been show to up-regulate G L U T - 1 expression in bovine brain microvascular ECs (Takakura et al, 1991), rat heart (Hirsch and Rosen, 1999) and BBB ECs (Simpson et al, 1999). The upregulation of G L U T - 1 following a period of hypoglycaemia is thought to compensate for the low ambient glucose concentration. The observations of an increased rate of glucose uptake in ECs exposed to glucose starvation support this idea (Takakura et al, 1991; Gaposchkin and Garcia-Diaz, 1996). A s only  72  limited studies have found other G L U T isoforms in ECs, an even smaller number have looked at the effect of diabetes and hyperglycaemia on their expression. In bovine BBB, diabetes was shown to have no effect on the expression of G L U T - 4 (Mantych et al, 1993b), while in rat placental vasculature, G L U T - 3 expression has been up-regulated by both diabetes and elevated glucose concentration (Mantych et al, 1993b).  2.1.5 Hypothesis  Given the EC's susceptibility to hyperglycaemia, we hypothesized that the expression  and subcellular distribution of the  transporters would  favour  intracellular glucose accumulation, and that there would be an inappropriate adaptation to the diabetic state.  2.1.6 Native endothelium preparation  In this study, we examined the expression and subcellular distribution of the classical G L U T s (1-5) and SGLT-1 in en face preparations of rat coronary, cerebral,  renal  and  mesenteric  artery  endothelia.  Because  it  has  been  demonstrated that the endothelial cells and S M C s are functioning as a unit, the structural relation between them should be preserved in any experimental design. The ECs, when cultured, tend to lose their specialized  properties  acquired during differentiation. The ECs of the BBB develop tight junctions and express selective transporters to protect the brain against toxic and harmful substances. These properties are found only in brain capillary ECs and are therefore specific to the brain. This organ-specificity is believed to be determined and  maintained by  their surrounding environment  73  and interaction  with  neighbouring cells. For example, the loss of expression of G L U T - 4 and G L U T - 1 after the third and tenth passage respectively of cultured brain ECs has been previously reported (McCall et al, 1997). Specialization of the endothelium can be organ-specific or function-specific, expressing different phenotypes even in the same organ. Markers of these phenotypes are mainly enzymes and proteins produced and expressed by different types of ECs (Garlanda and Dejana, 1997). For these reasons, freshly dissected vessels from four different vascular beds were used in this investigation. The coronary, cerebral, renal and mesenteric arteries have been selected for their variable levels of susceptibility to damage induced by hyperglycaemia and prevalence of vascular disease (Taylor et al, 1994; Mavrikakis et al, 1998; Perler and Becker, 1998). Moreover examining the specific structure of ECs from different vascular beds will allow the identification of possible differences in their specialized properties.  2.1.7 Fluorescence microscopy and deconvolution  The utilisation of a 3D microscopy imaging system provides a precise evaluation of the location and disposition of the transporters, and with a greater resolution than is possible with a conventional fluorescent microscope. Other biochemical methods based on cell fractionation disrupt the architecture of the cell.  On  the  other  hand,  microscopy can localise  immunohistochemistry  proteins  with a greater  coupled  with  electron  resolution, however  the  uncertainty of the labelling once the tissue has been embedded for sectioning renders this technology problematic. Although confocal microscopy provides a more straightforward approach, it has been demonstrated that image restoration generates greater resolution for discretely organized objects from which the  74  emitted fluorescence intensity is low (Carrington et al, 1989; Carrington et al., 1995). Deconvolution is superior because out of focus light is not rejected, as is the case with a confocal microscope whose pinhole is closed so as to maximize resolution, but is instead collected and used to reconstruct the original image (Moore et al, 1990). Moreover, our imaging system is equipped with a cooled charge-coupled device (CCD) camera (the image detector) with a quantum efficiency 5X higher than the image detector in a confocal. When this quantum efficiency is added to the loss of signal through the confocal pinhole, our imaging system becomes approximately 50X more sensitive than a confocal microscope. In addition, the high quantum efficiency of the cooled C C D camera means that total light exposure of the specimen can be reduced, which in turn minimizes photo bleaching and photo damage which are inherent problems in confocal microscopy. The use of this system in conjunction with immunohistochemistry, and on an intact fixed vessel preserved the molecular and structural organisation of the G L U T s and SGLTs examined.  2.1.8 STZ animal model  The effect of long-term hyperglycaemia on the expression and subcellular distribution of the G L U T in ECs was examined in the streptozotocin (STZ) diabetic rat. This is the predominant animal model chosen for the study of diabetes. Streptozotocin (2-deoxy-2-(3-methyl-3-nitrosurea)  1-d-glucopyranose)  (a+P), was first isolated in 1959 from Streptomyces achromogenes (Herr et al., 1959). This broad-spectrum antibiotic agent is an N-nitroso derivative of D glucosamine, composed of a cytotoxic moiety, 1-methyl 1-nitrosurea, positioned on  carbon-2  of  2-deoxy-D-glucose  (Rerup,  75  1970). The  administration of  streptozotocin has been shown to induce hyperglycaemia, polydipsia, polyuria, weight loss, albuminuria and glomerular ultra structural changes comparable to those seen in diabetes type I (Tomlinson et al, 1992). Streptozotocin produces its diabetogenic actions through cytotoxic destruction of the P-cell of the islets of Langerhans of the pancreas (Junod et al, 1969; Tomlinson et al, 1992). Previous studies have shown that S T Z toxicity is restricted to cells expressing G L U T - 2 (Schnedl et al, 1994). The transport of the toxin by G L U T - 2 but not G L U T - 1 has been recognized as a requirement for the destruction of the neuroendocrine cells. STZ achieves an efficient killing of the cells by initiating the fragmentation of the D N A . The cytotoxic portion of STZ generates free-alkylating radicals, carbonium ions (CH ) and nitric oxide (NO), all agents reducing the level of cellular 3+  nucleotides and related compounds (Yamamoto et al, 1981; Uchigata et al, 1982). Burkart et al. identified N A D  +  depletion caused by poly ADP-ribose polymerase  (PARP) activation, as the metabolic event leading to cell death. This study demonstrated that knockout PARP-/- mice were completely resistant to the STZ toxin. The STZ-treated knockout mice were normoglycaemic, maintained a normal level of secreted insulin, and preserved their islet ultrastructure (Burkart et al, 1999). It has been previously demonstrated that a stable hyperglycaemia for up to 3 months, can be induced with intravenous or intraperitoneal injection of STZ at doses exceeding 40mg/kg (50mg/kg to 70mg/kg) and can be restored within weeks with insulin treatment at the 50mg/kg S T Z dose but not with higher doses of STZ (Ar'Rajab and Ahren, 1993). Moreover, with lower doses of STZ, a small but sufficient number of pancreatic p-cells still secrete insulin and prevent ketoacidosis  thus allowing the animal to survive without insulin  supplementation (Junod et al, 1969).  76  2.2 Methods  A l l chemicals were purchased from Sigma-Aldrich Ltd. (Oakville, O N ) unless otherwise stated. Animal handling was done in accordance with the guidelines of the Canadian Council on Animal Care and the Principles of laboratory animal care (National Institute of Health (NIH) publication no. 85-23, revised 1985).  2.2.1 Animals  Male Wistar rats (200-235 g; University of British Columbia Animal Care Centre, Vancouver, B.C.) were given STZ (50 mg/kg, dissolved in saline) or saline (1 ml/100 g) via injection through the tail-vein. Animals were sacrificed 8 weeks later with a peritoneal injection of pentobarbital (30 mg/kg). During this period, bi-weekly morning measurements glucometer,  of tail-vein blood glucose (One Touch II  Lifescan Canada L T D , Burnaby, B.C.) and body weight were  recorded.  2.2.2 Tissue preparation  Segments of approximately 0.6 to 1.0 m m in length of septal coronary, middle cerebral, second order interlobar renal and mesenteric arteries, which are 100 um to 250 um in diameter, were carefully dissected.  The tissue was  immersed in ice-cold oxygenated (95% Oil 5% C O 2 ) Krebs buffer solution (in mmol/1: 119 N a C l , 4.7 KC1, 1.18 KH2PO4, 1.17 M g S 0 , 24.9 NaCOs, 11.1 dextrose, 4  2.5 CaCh, p H 7.4) during the dissection.  77  The vessel was cut longitudinally to expose the luminal side of the vessel, avoiding damage to the endothelium, and then fixed in 2% paraformaldehyde dissolved in phosphate buffered saline, (PBS; in mmol/1: 137 N a C l , 8 NaFfcPCk, 2.7 KC1, 1.5 KH2PO4; p H 7.4) for 10 min. Fixation was quenched with a 10-min rinse in 100 mmol/1 glycine (pH 7.4) and the cells were permeabilized with 0.06% saponin in PBS for 10 min at room temperature followed by 3 x 10-min rinses with PBS.  2.2.3 Immunohistochemistry  Non-specific  binding sites were  blocked  by  incubating  the  fixed,  permeabilized tissue in 10% donkey serum dissolved in PBS for two hours at room temperature. Vessels were then incubated overnight at 4 ° C with two antibodies raised in different species; one against a glucose transporter isoform (GLUT-1 to 5 or SGLT-1), and one against an E C or membrane marker (Vascular Endothelial (VE)-cadherin, von Willebrand factor, or caveolin-1). The optimal antibody concentrations  were determined empirically. A l l antibodies  were  diluted in antibody buffer (in mmol/1: 75 N a C l , 18 Na3 citrate with 2% donkey serum, 1% bovine serum albumin, 0.06% saponin, and 0.02% NaN3). The antibody sources, concentrations, epitope, and suppliers are listed in Table 2.2.  78  T a b l e 2.2 List of the primary antibodies.  1° ANTIBODY  Mouse human VonWillebrand Factor (mono IgG) Goat human VE-cadherin (C-19) (poly IgG) Mouse caveolin-1 (mono IgM) Mouse a-actin (mono IgG) Rabbit GLUT-1 (H-43) (poly IgG)  IMMUNOHISTO-  WESTERN  CHEMISTRY  BLOTTING  ug/ml  ug/ml  40  -  6.6  0.2  80  -  Intracellular domain, Carboxy terminus  Santa Cruz Biotechnology, Santa Cruz, CA Transduction Laboratories, Lexington, KY  5.8  —  1  10  Dilution 1:500  10  0.8  30  5  Rabbit GLUT-5 (poly IgG)  10  2  Rabbit SGLT-1 (poly IgG)  10  -  Goat GLUT-3 (M-20) (poly IgG) Mouse GLUT-4 (mono IgG)  Supplier  Serotec Inc., Raleigh, NC  dilution 1:500  Rabbit GLUT-2 (Neat serum)  EPITOPE  a  a  Intracellular domain, carboxy terminus, aa 218-260 Intracellular domain, carboxy terminus, 25 aa Intracellular domain, Carboxy terminus Intracellular domain Intracellular domain, carboxy terminus, aa 490-502 Extracellular domain, aa 402-420  'The exact antibody concentration has not been determined.  79  Santa Cruz Biotechnology, Santa Cruz, CA  East Acres Biologicals, Southbridge, MA Santa Cruz Biotechnology, Santa Cruz, CA Biogenesis, inc. Kingston, NH Alpha Diagnostic, Int. San Antonio, TX Alpha Diagnostic, Int. San Antonio, TX  Excess antibody was removed by 3 x 10 min rinses in antibody wash solution (in mmol/1: 75 N a C l , 18 Na3 citrate with 0.06% saponin). The tissue was then incubated for 2 hours with two antibodies that had been solid-phase reactivity  donkey  affinity-purified  secondary  adsorbed to minimize species cross-  (Jackson ImmunoResearch Laboratories, West  Grove,  PA). The  secondary antibodies were conjugated to either fluorescein isothiocyanate (FITC) or Texas Red and diluted in antibody buffer. Following incubation with the secondary antibodies, the tissue was rinsed 3 x 10 min in antibody wash solution. The cells' nuclei were then labelled with D A P I (4', 6-diamidino-2-phenylindole dihydrochloride; 0.3 umol/1, Molecular Probes, Eugene, OR), for 5 min, followed by 3 x 10 min rinses in PBS. The labelled vessels were mounted on a coverslip in D A B C O (l,4-diazabicyclo[2.2.2] octane (triethylenediamine) mounting medium (90% glycerol, 10% 10X PBS, 2.5% triethylenediamine, and 0.02% NaN3). To avoid compression of the vessel the edge of the coverslips were suspended on a thin layer of clear nail polish that had been applied to the slide. Small subresolution  beads  were  added  to  the  DABCO  (TetraSpeck  Fluorescent  microsphere standards, 0.22um diameter, Molecular Probes). These fluoresce at all the wavelengths used (DAPI, FITC and Texas Red) and enabled alignment of the data sets.  Two  control experiments were conducted. In the first, vessels were  labelled with a primary antibody and an inappropriately targeted secondary antibody. In the second, the primary antibody was omitted.  The adherens junctions, of which VE-cadherin is an integral part, separate the luminal from the abluminal sides of the cell (Leach et al, 1993; Firth, 2002).  80  We used the position of this molecule to optically dissect the cells into luminal and abluminal sides. This was not possible for the G L U T - 3 transporter, whose antibody was also raised in goats. In this case, we used mouse  anti-von  Willebrand factor to mark the endothelial cells, and used the contact points of neighbouring cells to demarcate the luminal-abluminal boundary (Farrell and Pardridge, 1991).  2.2.4 Acetylated L D L uptake  Arteries were dissected in Krebs solution as described above and cut longitudinally to expose the endothelial cells. The arteries were then incubated for 4 hours at 3 7 ° C in an incubator equilibrated with 5% C02, immersed in culture medium (GIBCO RPMI medium 1640, Invitrogen, Burlington, O N ) to which 1% serum replacement and 10 ug/ml of acetylated low density lipoprotein labelled with l,l'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (Dil-Ac-LDL; Biomedical Technologies Inc., M A ) were added. After a 10-min wash in fresh media, without D i l - A c - L D L , the artery was incubated for 5 min with a nucleic acid stain (Hoechst 33342, trihydrochloride, trihydrate, 200 ug/ml; Molecular Probes). The vessel was washed for 10 min in fresh media, and then mounted open on a coverslip in fresh media, sealed to the slide with clear nail polish, and imaged immediately.  2.2.5 Western blots  Tissue lysate and homogenate were obtained from rat septal coronary artery, human coronary artery endothelial cells ( H C A E C s ,  see  chapter 3),  epididymal adipose tissue, heart ventricle, jejunum, and brain cortex. After  81  dissection, the arteries were longitudinally opened as described above. This procedure exposes, primarily, the endothelium to the lysis buffer (0.025 mol/1 Phosphate  buffer  pH  7.4,  150  mmol/1  NaCl,  2.5  mmol/1  ethylenediaminetetraacetic acid (EDTA), 0.5% ethoxylate octyl phenol (IGEPAL), 10% Glycerol) in which the tissue was incubated for 30 min on ice. With this procedure most of the endothelial proteins were present in the lysate with some contamination of S M C proteins which could not be avoided (Figure 2.3). H C A E C s grown in a T-25 flask (see chapter 3), were rinsed with ice cold PBS. While kept on ice, 0.8 ml of lysis buffer was added to the flask and the cells scraped off with a scraper. The lysate was then vortexed and incubated on ice for 30 min A l l other tissues were dissected and homogenized, in the same ice-cold lysis buffer, using two 20-second pulses of a Polytron (type PT 10 O D ) at medium to high speed and further incubated on ice for 30 min The homogenized tissues and lysed H C A E C s were centrifuged at 4 C for 10 min at a lOOOg to Q  remove any cellular debris. Protein concentrations were determined using a modified Lowry reagent protein assay kit (Pierce, Rockford, IL). Two parts of tissue lysate or homogenate were combined with one part of sample buffer (3X concentrate, 0.175 mol/1 Tris, 44% Glycerol, 15% p-2-mercapto-ethanol, 7% SDS, 0.01% Bromophenol Blue, p H 6.8) and boiled for 5 min (GLUT-2 gave better results with the omission of the boiling step). Tissue samples and the prestained protein ladder (unboiled; Fermentas, Burlington, O N ) were loaded and resolved on a 5% stacking and 12.5% transferred  to  nitrocellulose  ruruTing  S D S - P A G E and the proteins were  membrane.  Antibody  incubations  with  the  membranes were performed at 37 C with gentle agitation. Non-specific binding Q  sites were blocked with 10% non-fat dry milk in Tris-buffered saline Tween (TBST; 50 mmol/1 Tris, 0.09% N a C l , and 0.01% Tween (Polyoxyethylene  82  sorbitan  monolaurate); p H 7.6). The membranes were then incubated with primary antibodies diluted in 5% non-fat dry milk in TBS-T, rinsed with TBS-T and further incubated with the appropriate horseradish peroxidase  conjugated  secondary antibodies (1:20000, either anti-mouse, anti-rabbit or anti-goat; Jackson ImmunoResearch Laboratories) also diluted in 5% non-fat dry milk in TBS-T. After a final rinse in TBS-T, the membranes were treated with the Western Lightning chemiluminescence Reagent Plus detection system (Perkin Elmer Life Sciences,  Boston, M A ) and developed  on Kodak BioMax film (Amersham  Biosciences Inc. Baie D'Urfe, PQ). Control experiments were performed without the primary antibodies and demonstrate specificity of the labelled single band for each protein probed (Appendix 2).  A  BLAST  search  (National  Center for  Biotechnology  Information,  Washington, D C ) was conducted to ensure that the antibodies used in this study only targeted the proteins of interest.  2.2.6 Immunostaining controls  Antibody specificity was also tested by labelling tissues in which the glucose transporters had been previously characterized (Sato et al,  1996;  Thorens, 1996; Concha et al, 1997). A s positive and negative immunostaining controls, red blood cells, pancreatic islets, neuronal primary culture, epididymal adipose tissue and segments of the jejunum, were labelled with antibodies against G L U T - 1 , 2, 3, 4 and 5 and SGLT-1. Blood samples were collected from rat-tail vein, heparinized (10%) and centrifuged for 5 min at 17000g. The plasma was removed and the erythrocytes fixed and labelled as described above. Whole  83  pancreas, epididymal adipose tissue and segments of the jejunum were dissected from rats in Krebs solution, embedded in Tissue Tek (Sakura, Torrance, C A ) and flash frozen in liquid nitrogen-cooled isopentane at -60°C. Cross-sections of these tissues were cut on a cryostat, thaw-mounted  on Superfrost slides (VWR,  Edmonton, AB), fixed, and labelled as described above. Primary cultures of rat neurons  were  obtained  from  Dr.  Kenneth  Baimbridge,  Department  of  Physiology, U B C , Vancouver, B.C. (Abdel-Hamid and Baimbridge, 1997; Krebs et al, 2003). The neurons were fixed, and labelled as described above.  2.2.7 3D Image acquisition, deconvolution and analysis  A series of two-dimensional fluorescent images was acquired through the depth of the cell with a standard N i k o n Diaphot 200 inverted microscope equipped  for epifluorescence  (100W  H g illumination, 60X oil  immersion  objective, N A 1.4, 4X adaptor). The pixel size was 100 x 100 n m and the Z spacing was 250 nm. Images were recorded with a C C D camera with a SITe SI502AB chip,  peak  quantum  efficiency  of  80%,  with  a  16-bit  dynamic  range  (Photometries, Tucson, A Z ) . For each wavelength, a three-dimensional data set was acquired using narrow bandpass filters specific for Fluorescein, Texas Red, and D A P I (XF22, XF43, XF06; Omega Optical, Brattleboro, V T ) . Images were background and dark current subtracted and corrected for photobleaching as previously described (Moore et al, 1993). A flat field was used to correct for nonuniform illumination and camera sensitivity across the field of view. Images were  deconvolved  using  the  algorithm  developed  by  Carrington et  al.  (Carrington et al, 1995) with an empirically determined point-spread-function (PSF) on an exhaustive proton reassignment (EPR) client-server  84  (Scanalytics,  Billerica, M A ) . Small fluorescent microspheres (100 n m diameter, Molecular Probes), of the appropriate wavelength, were used to measure the microscope's PSF. After deconvolution, each image was thresholded by partitioning the image grey-scale histogram based on visual inspection of both the image and its histogram. The background was segmented from the image by assigning a zero value to all pixels with a grey level lower than the threshold. Deconvolved and thresholded images were aligned using the fiduciary markers. A l l images are 3D reconstruction of the indicated depth.  2.2.8 Data quantification and analysis  Each imaged cell was isolated from the en face endothelium of an intact vessel through digital dissection (using cell junction marker VE-Cadherin, Figure 2.2B). To quantify the distribution of the G L U T s and SGLT-1, the 3D images of a single cell were divided into a series of non-overlapping 5 u m thick segments along their long axis. Each segment was then divided into a luminal and abluminal section using VE-cadherin and neighbouring cells as guides. Then, the number of labelled voxels on both sides was counted for the whole volume of the segment. The total number of labelled voxels on each side was summed along the length of the entire cell and expressed as a ratio of the number of abluminal to luminal voxels. The data are presented as mean ± Standard Error (SE) and compared using analysis of variance ( A N O V A ) with multiple comparisons as required.  85  2.3 Results  2.3.1 Weight and blood glucose  The mean weights and morning blood glucose concentrations of the control Wistar rats at sacrifice were 493 + 10 g and 3.90 ± 0.03 mmol/1 respectively (n=5). STZ-diabetic rats (n=8) had a significantly lower mean weight at sacrifice than the control animals (367 ± 11 g, p<0.05) and significantly elevated morning blood glucose concentrations (18.90 + 0.72 mmol/1, p<0.05). Daily morning weight and blood glucose measurements confirmed that the diabetic state of the S T Z injected rats was sustained throughout the experiment (Figure 2.1).  86  A  750 -i  oH 0  1  5  1  1  1  1  1  1  1  1  1  10  16  20  25  30  35  40  45  50  Days  Figure 2.1 Daily morning weight and blood glucose measurements. Morning weight (A) and blood glucose level (B) of Wistar rats following Streptozotocin (50mg/kg;  • STZ) or Saline (lml/lOOg;  ' Control) injection. The error bars are hidden  by the symbols.  2.3.2 Identification of endothelial cells  ECs were distinguished from other cells of the vascular wall using morphological criteria and specific markers. The nuclei of E C s are oriented parallel to the lumen due to the shear stress produced by blood flow (Galbraith et al, 1998), whereas S M C nuclei are oriented perpendicularly. This is reflected in the fusiform shape of the DAPI-labelled nuclei and in their orientation, parallel to the long axis of the vessel, as shown in Figure 2.2A.  87  In addition, ECs were identified by immunolabelling with two different E C markers: VE-cadherin (Figure 2.2B) and von Willebrand factor (used with G L U T - 3 labelling; Figure 2.2D). Both antibodies react positively with the top monolayer of the vascular wall where labelled nuclei run parallel to the lumen.  To determine if the endothelium was well preserved during the dissection procedures, cell viability was tested by visualizing A c - L D L uptake (Netland et al., 1985). Fluorescence was observed in all cells of the top layer of freshly dissected vessels incubated with A c - L D L for four hours. This demonstrates that the ECs were intact and viable immediately prior to being fixed (Figure 2.2C).  88  _ .  . . .  •3  *;  D  f *  :  v-  •  i t  ' * '.1 "  i  •  i  F i g u r e 2.2 Identification of ECs Deconvolved images of en face septal coronary artery ECs. A ) Smooth muscle cell (SMC) and endothelial cell (EC) nuclei are indicated. The white arrow indicates the blood flow orientation. The image is 10 um deep, scale bar 8 um (A-C). B) VE-cadherin (red) and nuclei (blue). The image is 1.25 um deep. C) A c - L D L (red) and nuclei (blue). The image is 1.25 um deep. D) von Willebrand factor (red) and nuclei (blue). The image is 3.25 um deep. Scale bar 2 um.  89  2.3.3 A n t i b o d y specificity  A n t i b o d y specificity w a s first tested u s i n g W e s t e r n Blots. T h e presence of E C a n d S M C proteins i n the lysate w a s d e t e r m i n e d w i t h V E - c a d h e r i n a n d  a-  actin antibodies i n b o t h vessel p r e p a r a t i o n a n d the c u l t u r e d H C A E C s (Figure 2.3). P o s i t i v e controls ( H C A E C s , fat, heart, b r a i n a n d j e j u n u m lysates) w e r e u s e d to establish the m o l e c u l a r w e i g h t b a n d i d e n t i f i e d b y the different antibodies. Single bands, for G L U T - 1 (-62 k D a ) , G L U T - 2 (-72 k D a ) , G L U T - 3 (-47 k D a ) , G L U T - 4 (~47kDa) a n d G L U T - 5 (-55 k D a ) , w e r e f o u n d f r o m e n face  septal  c o r o n a r y artery lysate (Figure 2.4). T h e S G L T - 1 a n t i b o d y d i d not recognize denatured  p r o t e i n a n d w e w e r e u n a b l e to p r o d u c e a W e s t e r n Blot for this  antibody.  170 130~ 100_  Vessel HCAEC  !~ Vessel HCAEC }QQ~ 70 — 55 — 40  70 _ 55_  33 _  40_  24 _ 17_  Figure2.3  E C and S M C identification in coronary artery tissue lysate.  Rat tissue lysates from septal coronary artery blood vessel and cultured H C A E C s 30 ug of protein, were separated by 12% S D S - P A G E . After transfer of the proteins to nitrocellulose, detection was performed using antibodies against A ) VE-cadherin and B) S M C specific a-actin. The position of molecular weight markers is indicated on the left side i n k D a .  90  A  Vessel H C A E C  Fat  100_ 70 _  B  Vessel HCAEC  Gut  100 — 70_  55 _ 40  Heart  Brain  40  40_  33 _  Vessel HCAEC  55 _ i  55_  _  c  100_ 70_  33_  33_ Vessel HCAEC  Fat  100_ 70_  100. 70. 55. 40.  55 _ 40 _ 33 _  Vessel HCAEC  Gut  33.  Figure2.4  Determination of antibody specificity with Western blots.  Rat tissue lysates from septal coronary artery blood vessel, H C A E C s , epididymal adipose tissue, heart, jejunum and brain cortex. 30 pg (A-C) or 45 pg (D and E) of protein, were separated by 12% S D S - P A G E . After transfer of the proteins to nitrocellulose, detection was performed using antibodies against A ) G L U T - 1 , B) G L U T 2, C) G L U T - 3 , D) G L U T - 4 , and E) G L U T - 5 . The position of molecular weight markers is indicated on the left side i n k D a .  D u e to their s i m i l a r i t y i n m o l e c u l a r w e i g h t , the W e s t e r n blot c o u l d not determine the i s o f o r m specificity of the antibodies u t i l i z e d . W e therefore  used  p r o t o c o l s established b y others, a n d l a b e l l e d cells a n d s u b c e l l u l a r structures for which  the  glucose transporters h a d  been  well  characterized;  erythrocytes,  pancreatic islets, neurons, a d i p o s e tissue a n d e p i t h e l i a l cells of the j e j u n u m (Sato et al, 1996; Thorens, 1996; C o n c h a et al, 1997). G L U T - 1 (Figure 2.5A), G L U T - 2 (Figure 2.5B) a n d G L U T - 5 (Figure 2.5E) w e r e f o u n d i n erythrocytes. T h e side v i e w of the images (see insets), demonstrates the characteristic b i c o n c a v e shape of the cell, i n d i c a t i n g that the l a b e l w a s p r e d o m i n a n t l y o n the m e m b r a n e . G L U T 3 (Figure 2.5C), G L U T - 4 (Figure 2.5D) a n d S G L T - 1 (Figure 2.5F) w e r e absent, i n  91  agreement with  previous  studies (Concha et al,  1997). Cross-sections  of  pancreatic islets showed the presence of G L U T - 2 (Figure 2.5H) but not G L U T - 1 (Figure 2.5G) in accordance with earlier data (Sato et al, 1996). We also show positive controls for G L U T - 3 (Figure 2.51) labelling in neurons and G L U T - 4 (Figure 2.5J) labelling in adipocytes (Figure 2.51 and J), as shown by other authors (Leino et al, 1997). Lastly, we looked at the distribution of G L U T - 2 , G L U T - 5 and SGLT-1 in epithelial  cells of the jejunum, a tissue for which the glucose  transporter organization is well established (Thorens, 1996). Our results show a mostly basolateral distribution for G L U T - 2 (Figure 2.5K), G L U T - 5 present on both  the  apical  and basolateral  membranes  (Figure  2.51)  and  an  apical  distribution for SGLT-1 (Figure 2.5M), confirming published results (Blakemore et al, 1995).  92  Figure2.5  Immunohistochemistry demonstrating antibody specificity.  A l l images were deconvolved and represent a 3D reconstruction of whole cells or section of cells or tissues from rats. A-F) Erythrocytes (3.75 [am deep), scale bar 5 um, stained for A ) G L U T - 1 , B) G L U T - 2 , C) G L U T - 3 , D) G L U T - 4 , E) G L U T - 5 , and F) SGLT-1. The insets i n A , B and E are cross-sections (average of 2.5 u m deep) of a side view of the erythrocytes. G and H ) Cross-sections of pancreatic islet (0.75 u m deep), scale bar 20 um, stained for G) G L U T - 1 and H ) G L U T - 2 . I) Neuronal primary culture labelled w i t h G L U T - 3 . The image is 8 u m deep and the scale bar is 20 um. The inset is a cross-section of a side view of the neuron body. J) Cross-section of epididymal adipose tissue (10 u m deep) labelled with GLUT-4, scale bar is 20 um. K - M ) Jejunum epithelial cell crosssections (0.5 u m deep, apical surface facing up) stained for K ) G L U T - 2 , L) G L U T - 5 and M ) SGLT-1, scale bar is 5 um.  N o immunostaining was observed when primary antibodies were applied in combination with a secondary antibody targeting an irrelevant species. Figure 2.6 shows ECs labelled with a single primary antibody and two  secondary  antibodies. The relevant secondary antibody produced labelling in ECs (Figure 2.6A&B for FITC, I for Texas Red). The irrelevant secondary antibodies, in each  93  case, did not produce any labelling in E C s (Figure 2.6G for FITC and C & F for Texas Red). The middle column in Figure 2.6B, E , and H , shows, as a landmark, the nucleus of each cell labelled with DAPI.  Figure 2.7 shows G L U T - 3 (Figure 2.7A), G L U T - 5 (Figure 2.7D) and S G L T 1 (Figure 2.7G) labelling. G L U T - 3 antibody's immunoreactivity was partly abolished by equal volume of competitive blocking peptides (Figure 2.7B) and completely by 5X excess (Figure 2.7C). Similarly, G L U T - 5 and SGLT-1 antibody's immunoreactivity  was  partly  abolished  by  5X  excess  (Figure  2.7E&H,  respectively) and mostly by 10X excess of the blocking peptide (Figure 2.7F&I respectively).  94  F i g u r e 2 . 6 Secondary antibody cross-reactivity controls. Images are 3D reconstruction of coronary artery ECs, view of the cells from the lumen. ECs are labelled with a single primary antibody, rabbit anti-GLUT-1 (A-C), mouse antivon Willebrand Factor (D-F) and VE-cadherin (G-I) and dual secondary antibody, donkey anti-rabbit FITC and donkey anti-goat Texas Red (A-C).  95  Figure2.7  Competitive blocking peptide controls.  Images are 3D reconstruction of coronary artery E C , view of the cells from the lumen. ECs labelled w i t h G L U T - 3 (A), G L U T - 5 (D) and SGLT-1 (G) antibodies. Each respective antibodies were also pre-incubated w i t h equal volume (B) 5 X excess volume (C, E & H ) , and 10 X excess volume (F & I) of competitive blocking peptide prior to labelling. Scale bar is 5 um.  2.3.4 I m m u n o l o c a l i z a t i o n  T h e relative d i s t r i b u t i o n of G L U T - 1 (green), V E - c a d h e r i n (red), a n d the n u c l e u s (blue) i n a c o r o n a r y artery E C is d i s p l a y e d i n F i g u r e 2.8. T h e adherens junctions, as l a b e l l e d b y V E - c a d h e r i n , are d i s c o n t i n u o u s , as expected for a n e n d o t h e l i u m e x p e r i e n c i n g shear stress ( N o r i a et al, 1999). T h e transporters w e r e  96  distributed in discrete clusters, mostly around the periphery of the nucleus and close to the edge of the cell. From the side view (Figure 2.8B), it can be seen that the bulk of the nucleus was on the abluminal side of the cell, and that clusters of G L U T - 1 were located on both the luminal and abluminal sides. Additional details were observed by rotating the image presented in Figure 2.8A 90 degrees about the X-axis and extracting three 0.5 u m thick cross-sections from the indicated locations (Figure 2.8C, D and E). These cross-sections demonstrated that G L U T - 1 was located in the same region of the cell as was VE-cadherin, and that when split into luminal and abluminal segments using VE-cadherin as a guide (Figure 2.8F and G), the majority of the transporters appeared to be on the abluminal side.  En face 3D data set and cross-sections (comparable to Figure 2.8C, D and E) of E C from all four vascular beds labelled for G L U T - 1 (Figure 2.9), G L U T - 2 (Figure 2.10), G L U T - 3 (Figure 2.11); G L U T - 4 (Figure 2.12), G L U T - 5 (Figure 2.13) and SGLT-1 (Figure 2.14) are shown. The distributions of G L U T - 2 , 3, 4, 5 and S G L T appeared similar to that of G L U T - 1 ; the transporters were distributed in discrete clusters present on both sides of the cell, and had a high density in proximity to the cell-to-cell junctions. The arrangement was the same in each of the vascular beds.  97  Abluminal  °U  90  Figure2.8  ^ Digital manipulations schematic of the quantitative analysis.is.  Coronary E C labelled w i t h antibodies specific for G L U T - 1 (green), VE-cadherin (red) and nuclei w i t h D A P I (blue), scale bar is 2 u m i n each direction. A ) 3D reconstruction, view of the cells from the lumen. The white arrow indicates a small section of an adjacent cell. B) Side view obtained from a 90° rotation about the Y-axis, luminal surface on the right. C-E) Half micron thick cross-sections were obtained from (A) at the points indicated by the yellow arrows, and then rotated 90° about the X-axis, luminal surface on top. The yellow dashed line i n D indicates where this cell segment was divided into luminal (F) and abluminal (G) sections. White pixels indicate colocalization between V E cadherin and G L U T - 1 (A, B), or between G L U T - 1 and D A P I (C, D , G  98  Coronary Cerebral  Renal  Mesenteric  Coronary Cerebral  Figure2.9  Renal  Mesenteric  G L U T - 1 subcellular distribution in ECs.Cs. E C of coronary (A, E), cerebral (B, F), renal (C, G), and mesenteric (D, H ) arteries labelled with antibodies specific for G L U T - 1 (green), VE-cadherin (red) and nuclei w i t h D A P I (blue). A - D ) 3D reconstruction, view of the cells from the lumen. The white arrow indicates a small section of an adjacent cell. E-H) Half-micron thick cross-sections were obtained from (A-D), and then rotated 90° about the X-axis, luminal surface on top. White pixels indicate colocalization between G L U T - 1 and D A P I (A-D) or between V E cadherin and G L U T - 1 (E-H). Scale bar is 2 u m i n each direction. I) Plot of the total number of illuminated voxel per cell (mean ± S.E.), each bar is the average of 4 cells from 3 rats for each vascular bed. J) Plot of the ratio of the number of abluminal to luminal voxels per cell (mean ± S.E.).  99  Coronary  Renal  Cerebral  Mesenteric  5 CO wooo  o I—  Coronary  Cerebral  Renal  Mesenteric  Coronary  Cerebral  Renal  Mesenteric  F i g u r e 2 . 1 0 GLUT-2 subcellular distribution in ECs. E C of coronary (A, E), cerebral (B, F), renal (C, G), and mesenteric (D, H ) arteries labelled w i t h antibodies specific for G L U T - 2 (green), VE-cadherin (red; E) or caveolin (red; F-H) and nuclei D A P I (blue). A - D ) 3D reconstruction, view of the cells from the lumen. The white arrow indicates a small section of an adjacent cell. E-H) Half-micron thick crosssections were obtained from (A-D), and then rotated 90° about the X-axis, luminal surface on top. White pixels indicate colocalization between G L U T - 2 and D A P I (A-D) or between VE-cadherin or caveolin and G L U T - 2 (E-H). Scale bar is 2 p m i n each direction. I) Plot of the total number of illuminated voxel per cell (mean i S.E.), each bar is the average of 4 cells from 3 rats for each vascular bed. J) Plot of the ratio of the number of abluminal to luminal voxels per cell (mean ± S.E.).  100  Coronary  Renal  Cerebral  Mesenteric  J  I.I ~T  Coronary  Cerebral  Renal  Mesentenc  Coronary  Cerebral  Renal  Mesenteric  F i g u r e 2 . 1 1 GLUT-3 subcellular distribution in ECs. E C of coronary (A, E), cerebral (B, F), renal (C, G), and mesenteric (D, H ) arteries labelled w i t h antibodies specific for G L U T - 3 (green), caveolin (red) and nuclei with D A P I (blue). A - D ) 3D reconstruction, view of the cells from the lumen. The white arrow indicates a small section of an adjacent cell. E-H) Half-micron thick cross-sections were obtained from (A-D), and then rotated 90° about the X-axis, luminal surface on top. White pixels indicate colocalization between G L U T - 3 and D A P I (A-D) or between caveolin and G L U T - 3 (E-H). Scale bar is 2 u m i n each direction. I) Plot of the total number of illuminated voxel per cell (mean ± S.E.), each bar is the average of 6 cells from 3 rats for each vascular bed. J) Plot of the ratio of the number of abluminal to luminal voxels per cell (mean ± S.E.). The ratio of cerebral artery was significantly higher than the ratio of coronary and renal arteries * (p<0.01).  101  Coronary  Coronary  Cerebral  Cerebral  Renal  Renal  Mesenteric  Mesenteric  Coronary  Cerebral  Renal  Mesenteric  F i g u r e 2 . 1 2 GLUT-4 subcellular distribution in ECs. E C of coronary (A, E), cerebral (B, F), renal (C, G), and mesenteric (D, H ) arteries labelled with antibodies specific for G L U T - 4 (green), VE-cadherin (red; E-G) or caveolin (red; H ) and nuclei w i t h D A P I (blue). A - D ) 3D reconstruction, view of the cells from the lumen. The white arrow indicates a small section of an adjacent cell. E-H) Half-micron thick cross-sections were obtained from (A-D), and then rotated 90° about the X-axis, luminal surface on top. White pixels indicate colocalization between G L U T - 4 and D A P I (A-D) or between VE-cadherin or caveolin and G L U T - 4 (E-H). Scale bar is 2 u m i n each direction. I) Plot of the total number of illuminated voxel per cell (mean + S.E.), each bar is the average of 5 cells from 2 rats for each vascular bed. J) Plot of the ratio of the number of abluminal to luminal voxels per cell (mean ± S.E.).  102  Coronary  Coronary  Cerebral  Renal  Cerebral  Renal  Mesenteric  Mesenteric  Coronary  Cerebral  Renal  Mesenteric  F i g u r e 2 . 1 3 GLUT-5 subcellular distribution in ECs. E C of coronary (A, E), cerebral (B, F), renal (C, G), and mesenteric (D, H ) arteries labelled with antibodies specific for G L U T - 5 (green), VE-cadherin (red) and nuclei with D A P I (blue). A - D ) 3D reconstruction, view of the cells from the lumen. The white arrow indicates a small section of an adjacent cell. E-H) Half-micron thick cross-sections were obtained from (A-D), and then rotated 90° about the X-axis, luminal surface on top. White pixels indicate colocalization between G L U T - 5 and D A P I (A-D) or between V E cadherin and G L U T - 5 (E-H). Scale bar is 2 u m i n each direction. I) Plot of the total number of illuminated voxel per cell (mean ± S.E.), each bar is the average of 6 cells from 4 rats for each vascular bed. J) Plot of the ratio of the number of abluminal to luminal voxels per cell (mean ± S.E.).  103  P. Coronary  - o *  >  Cerebral  Renal  Mesenteric  5000  Coronary  Cerebral  Renal  Mesenteric  Figure2.14  Coronary  Cerebral  Renal  Mesenteric  S G L T - 1 subcellular distribution in ECs. E C of coronary (A, E), cerebral (B, F), renal (C, G), and mesenteric (D, H ) arteries labelled with antibodies specific for SGLT-1 (green), VE-cadherin (red) and nuclei w i t h D A P I (blue). A - D ) 3D reconstruction, view of the cells from the lumen. The white arrow indicates a small section of an adjacent cell. E-H) Half-micron thick cross-sections were obtained from (A-D), and then rotated 90° about the X-axis, luminal surface on top. White pixels indicate colocalization between SGLT-1 and D A P I (A-D) or between V E cadherin and SGLT-1 (E-H). Scale bar is 2 u m i n each direction. I) Plot of the total number of illuminated voxel per cell (mean ± S.E.), each bar is the average of 5 cells from 2 rats for each vascular bed. J) Plot of the ratio of the number of abluminal to luminal voxels per cell (mean ± S.E.).  104  The quantification of this distribution and a comparative analysis between the different vascular beds is presented. Figure 2.9 to Figure 2.141 show the total number of illuminated voxels for each artery expressed as the mean per cell, and its standard error. When the numbers of illuminated voxels for GLUT-1 were compared across the different vascular beds, no significant differences  were  found (Figure 2.91). The numbers of illuminated voxels were also comparable between each artery for G L U T - 2 (Figure 2.101), G L U T - 3 (Figure 2.111), G L U T - 4 (Figure 2.121), G L U T - 5 (Figure 2.131), and SGLT-1 (Figure 2.141).  We also determined the ratio of abluminal to luminal illuminated voxels for each transporter in all four arteries. The ratio for G L U T - 1 in the coronary E C shown in Figure 2.9A and E was 10.6 and in a total of 7 coronary artery ECs examined the abluminal: luminal ratio was 8.9 ± 1.4 (mean ± S.E.), implying that the majority of G L U T - 1 was on the abluminal side (Figure 2.9J). We found a similar subcellular distribution in cerebral (6.0 ± 0.8), renal (4.9 ± 1.0) and mesenteric (11.1 ± 2.4) arteries (Figure 2.9J). G L U T - 2 was also mostly located on the abluminal side of the cells (Figure 2.10). We found comparable abluminal: luminal ratio amongst coronary (5.3 ± 0.7), cerebral (5.7 ± 1.1), renal (3.7 ± 1.0) and mesenteric (4.9 ± 1.6) arteries (Figure 2.101). A s for the other transporters, no significant differences were observed between vascular beds with the exception of G L U T - 3 , for which an A N O V A followed by Bonferroni's multiple comparison test indicated a significant higher ratio for the cerebral artery (4.8 ± 0.7) in comparison to both coronary (2.1 ± 0.3) and renal arteries (2.3 ± 0.3; p<0.01), but not to mesenteric artery (4.1 ± 0.5; Figure 2.11J). GLUT-4, G L U T - 5 and SGLT-1 had a low ratio in all vascular beds examined, ranging from 3.4 ± 0.6 to 1.1 ± 0.2 (Figure 2.12 to Figure 2.14J).  105  The abluminal: luminal ratio was also compared between transporters for each vascular bed. In coronary artery, an analysis of variance of the mean abluminal: luminal ratios were significant (p < 0.001) and Bonferroni's multiple comparisons test indicated that the distributions fell into three different groups (Figure 2.15). SGLT-1 had an abluminal: luminal ratio that was not significantly different from 1, and was therefore symmetrically distributed. G L U T - 3 , 4 and 5 were more concentrated on the abluminal side with ratios that ranged from 2.1 to 3.5 and were not significantly different from each other. G L U T - 1 had the most asymmetric distribution with a ratio of 8.9, which was significantly different from G L U T - 3 , 4, 5 and SGLT-1 (p<0.001), followed by G L U T - 2 with a ratio of 5.4 significantly different from SGLT-1 (p<0.05). Although a similar trend was found in the other three vascular beds, only the mesenteric showed a G L U T - 1 ratio significantly different from G L U T - 2 (p<0.05), 3 (p<0.05), 4 (p<0.01), 5 (p<0.01), and SGLT-1 (p<0.001).  2.3.5 Effect of long-term hyperglycaemia  We  looked at the effect of hyperglycaemia on the expression  and  subcellular distribution of the G L U T s and SGLT-1 in coronary arteries (Figure 2.16). Sustained hyperglycaemia (8 weeks) produced a reduction in the labelling for G L U T - 1 (Figure 2.16A vs B), G L U T - 3 (Figure 2.161 vs J), G L U T - 4 (Figure 2.16M  vs N) and slightly less for G L U T - 5 (Figure 2.16Q vs R), but caused a  dramatic increase in labelling for G L U T - 2 (Figure 2.16E vs F). SGLT-1 labelling was  not  distinctively  changed  by  hyperglycaemia  (Figure 2.16U  vs V).  Quantification of the total number of voxels labelled for G L U T - 1 , 3, 4 and 5 revealed a significant reduction ranging from 77 to 95%, while the number of  106  voxels labelled for G L U T - 2 was increased by 222% (Figure 2.17). SGLT-1 was also reduced by hyperglycaemia but to a lesser extent (39%). These changes did not significantly  affect the abluminal: luminal distributions of any of  the  transporters (see Figure 2.16, G L U T - 1 (C vs D), G L U T - 2 (G vs H), G L U T - 3 (K vs L), G L U T - 5 (S vs T) and SGLT-1 (W vs X)) except G L U T - 4 (Figure 2.160 vs P) where there was a significant shift towards the luminal side of the cell (Figure 2.15), in spite of a 77% reduction in the total number of illuminated voxels (Figure 2.17).  +3 1098-  765H  43210-  ft* *  *  ** *  *  nil  ft GLUT-1  GLUT-2  GLUT-3  GLUT-4  GLUT-5  SGLT  F i g u r e 2 . 1 5 Effect of hyperglycaemia on the Abluminal: Luminal ratio. Plot of the ratio of the number of abluminal to luminal voxels per cell (mean ± S.E.) for both control I I and diabetic animals I 1 (each bar is the average of 7 cells from 4 rats). *, **, + marks ratios that are significantly different from GLUT-1, G L U T - 2 or SGLT1, respectively. # indicates those transporters where the ratio is significantly different between the diabetic and the control group.  107  Non-treated  f  •  STZ-treated  D  Non -treated  G  STZ-treated  H  Non-treated  -  STZ-treated  1  t  GLUT-1  GLUT-2  GLUT-3  GLUT-4  GLUT-5  SGLT  Figure2.16  Effect of hyperglycaemia on transporters subcellular  distribution. Representative E C labelled w i t h antibodies specific for a glucose transporter (green), V E cadherin (red) (except G L U T - 3 caveolin (red)) and nuclei w i t h D A P I (blue). G L U T - 1 (A to D), G L U T - 2 (E to H ) , G L U T - 3 (I to L), G L U T - 4 ( M to P), G L U T - 5 (Q to T) and SGLT-1 (U to X). First and third rows: 3D reconstruction of an en face preparation, view of the cells from the lumen, each are 4 to 5 u m thick. Second and fourth rows: cross-sections, luminal side up, 0.5 u m thick. E C from Non-treated and STZ-treated rats alternate respectively from left to right. Scale bar is 2 um.  108  20000-,  "8 c  1 «  - 3  100004,  «_ o o > »  ft  15 o  GLUT-1  B  !  fL  GLUT-2  * GLUT-3  GLUT-4  GLUT-5  SGLT  300  >  "8  re c  200  £  3  =  c  100-1  0 D) C  a  € o  0  -100 GLUT-1  GLUT-2  GLUT-3  GLUT-4  GLUT-5  SGLT-1  F i g u r e 2 . 1 7 Effect of hyperglycaemia in the expression of the transporters. A ) Plot of the total number of uluminated voxels per cell (mean ± S.E.) for both control I 1 and diabetic animals I 1 . B) Plot of the percentage change i n the number of illuminated voxels per cell i n the diabetic compared to the control (from A ) . * (p<0.05) and ** (p<0.0005) indicates a significant change.  2.4 Discussion  Our principal findings are that the ECs of the coronary artery express G L U T - 1 to 5 and SGLT-1, and that long-term hyperglycaemia induced by STZ  109  had a profound effect on their expression. We observed a downregulation of G L U T - 1 , 3, 4, and 5, and a three-fold increase of G L U T - 2 in the ECs of STZdiabetic rats. We examined an en face endothelial preparation because ECs structure is maintained by the surrounding environment and by interactions with neighbouring cells (Redmond et al, 1995; Garlanda and Dejana, 1997). In particular, ECs, when cultured, have a tendency to lose the ability to express most of the G L U T and S G L T isoforms (McCall et al, 1997; Mamchaoui et al, 2002). By studying the endothelium of freshly dissected vessels, we preserved the cellular architecture of the ECs as close to the native state as possible. The endothelial markers allowed us to readily visualize the cells in the intact vessel, and  the A c - L D L uptake indicated that they remained viable throughout the  dissection (Figure 2.2).  2.4.1 Immunostaining  We have interpreted an increase or decrease in the number of voxels labelled for a given transporter as increases or decreases in that transporter protein expression.  We cannot  draw any  conclusions  about  the  relative  expression levels between the transporters since we do not know the affinity of either the primary or the secondary antibodies for their epitopes. The specificity of the antibodies used in these studies has been thoroughly tested. First, B L A S T searches confirmed that the sequences of the epitopes recognized by the antibodies are unique to the proteins. Second, we obtained positive and negative controls for each antibody using immunohistochemistry in well-characterized cells and tissues. Third, Western blots produced single bands of the appropriate molecular weight for each of the G L U T s (Thorens et al, 1988; James et al, 1989;  110  Gould et al, 1992; Mantych et al, 1993b; Hirsch and Rosen, 1999). Fourth, control peptides for G L U T - 3 , 5 and SGLT-1 blocked the labelling. Together, these results indicate that the antibodies are specific for each glucose transporter isoform.  A l l of the G L U T s and SGLT-1 were distributed in discrete clusters. This organised distribution may reflect the capacity of the transporters to target specific subcellular sites and tether or anchor to cytoskeletal proteins, as has been observed for G L U T - 1 (Bunn et al, 1999). While most labelling surrounded the nucleus and the edge of the cells, a small amount seems to be located within the nucleus. A plane by plane analysis revealed that this labelling is located in deep folds of the nuclear envelope, typical for an EC's nucleus (Woolf, 1982), and constitutes perinuclear and not intranuclear labelling.  2.4.2 G L U T - 1  In agreement with previous studies (Mann et al, 2003), we found G L U T - 1 in ECs. We also found that it is asymmetrically distributed, with a greater abundance on the abluminal side. A similar distribution of G L U T - 1 has been previously reported in the BBB (Bolz et al,  1996; Cornford et al,  1998a;  Dobrogowska and Vorbrodt, 1999), where G L U T - 1 has long been considered the main glucose transporter (Pardridge et al, 1990). G L U T - l ' s high affinity, intrinsic asymmetry (glucose extrusion from the cell can be 20 times faster than its uptake (Gould and Seatter, 1997; Kinne, 1997), and high abluminal density suggests its participation in the maintenance of a constant transport of glucose from the cytosol of ECs to the interstitial space in the vascular wall.  Ill  2.4.3 G L U T - 2  To our knowledge, we are the first to report the presence of G L U T - 2 in the ECs of a small artery. A basolateral distribution of G L U T - 2 has been found in epithelial cells of kidney proximal tubules and small mtestine (Takata, 1996; Thorens, 1996; Wallner et al., 2001) where it enables the rapid extrusion of accumulated intracellular glucose, facilitating its transfer to the interstitium. If transcellular glucose transport occurs across the endothelium  of coronary  arteries, then the low-affinity/high-capacity of G L U T - 2 and its high abluminal density would contribute to the transfer of glucose from the blood to the cells of the vascular wall. Thus, G L U T - 1 may be responsible for glucose extrusion at basal concentrations, while G L U T - 2 could potentially protect ECs from elevated intracellular glucose accumulation during brief periods of hyperglycaemia.  2.4.4 G L U T - 3 , 4, and 5  We also found that G L U T - 3 , 4 and 5 were present in these ECs. Other groups have previously reported the presence of both G L U T - 3 (Mantych et al, 1993b; Urabe et al, 1996) and G L U T - 5 (Mantych et al, 1993b) in brain ECs. The presence of G L U T - 4 has been previously found in ECs of the blood barrier (retinal and brain) (Frank and Pardridge, 1981; King et al, 1983; McCall et al, 1997) and in fat and muscle capillary ECs (Vilaro et al, 1989), while it was absent from cultured cardiac (Thomas et al, 1995), cultured aortic (McCall et al, 1997), and human muscle capillary ECs (Friedman et al, 1991). These contradictory findings may be explained in part by the heterogeneity found between ECs of different vascular beds and vessels of different calibres and by the fact that ECs, when cultured, have a tendency to lose the ability to express most of the G L U T  112  and S G L T isoforms (McCall et al, 1997; Mamchaoui et al, 2002) and the resulting reduced expression is likely to be interpreted as non significant.  2.4.5 SGLT-1  We found SGLT-1 in ECs of small contractile arteries. The presence of SGLT-1, also previously reported in bovine cortical artery ECs (Nishizaki et al, 1995; Matsuoka et al, 1998; Nishizaki and Matsuoka, 1998), raises the question of the role it plays in the cells of the vascular wall. A s a permanent N a gradient is +  maintained between the blood and the cytosol of the cell, one would expect a constant activation of S G L T in the ECs even during periods of low blood glucose level. Nishizaki et al. have shown that low glucose levels enhance the activity of S G L T in bovine cortical artery ECs (Nishizaki and Matsuoka, 1998). Thus, the activity of SGLT-1  may be of greater importance during stresses such as  hypoglycaemia in ECs, in which case it's unique capacity to transport glucose against its concentration gradient may serve the cells of the vascular wall during prolonged periods of starvation.  2.4.6 Other transporters  In addition to the G L U T isoforms that we studied, there are another seven known isoforms (GLUT6 - 12) (Joost and Thorens, 2001), at least five other isoforms of S G L T (Wright, 2001) as well as the H M I T (Joost and Thorens, 2001). Antibodies to some of these transporters became available after this study was completed, and for this reason, we have not examined their role in the vascular EC.  113  2.4.7 Cell junction labelling  A  surprising result was  the preferential localization of all glucose  transporter isoforms towards the edges of the cell, near the cell-to-cell junctions. This cell-to-cell junction labelling is mostly noticeable in the case of G L U T - 1 (Figure 2.9A-D), but was also observed with the other isoforms (best examples in Figure 2.11C, Figure 2.12A&C, Figure 2.13B, Figure 2.14A&D). A t these regions of the cells, the luminal and abluminal membranes are at their closest apposition. This, combined with the preferential distribution of the G L U T s on the abluminal side, suggests that there could be transcellular transport of glucose through ECs at their narrowest points. We cannot rule out the possibility that there is paracellular glucose transport, particularly given the discontinuous  adherens  junctions which are a characteristic of endothelia exposed to shear stress (Noria et al., 1999), and given that glucose transport across the capillaries in the microvasculature is thought to be almost exclusively paracellular (Michel and Curry, 1999). Nevertheless, our results indicate that ECs possess the molecular architecture to promote rapid transcellular transport of glucose to the cells of the vascular wall, and with SGLT-1, G L U T - 1 and G L U T - 2 on the luminal side, to maintain transcellular transport across a wide range of glucose concentrations.  2.4.8 Similarity amongst vascular beds  We found a comparable expression for each G L U T isoform and SGLT-1 in all four vascular beds examined. The subcellular distribution of each isoform was also comparable between the different vascular beds with the exception of G L U T - 3 in cerebral ECs found with greater abundance on the abluminal side than in other blood vessels. A s each the arteries used in this study have a  114  comparable diameter, wall thickness and function, it is not surprising to find a similarity between the expression  and subcellular distribution of  glucose  transporters in each of these arteries.  2.4.9 Effect of hyperglycaemia  Hyperglycaemia had profound effects on all of the G L U T s , significantly reducing the number of labelled voxels for all except G L U T - 2 , which was dramatically increased. The decrease in G L U T - 1 staining is consistent with reports indicating that the expression of this transporter is downregulated in rat heart endothelium following STZ-induced diabetes (Hirsch and Rosen, 1999). There are no reports of the effects of prolonged hyperglycaemia on the expression of G L U T - 3 , 4 and 5 in the endothelium, but our results indicate that their expression is also reduced. In cultured bovine aortic endothelial cells (BAEC) exposed to hyperglycaemia for 48h, the expression of G L U T - 1 and its Vmax are both reduced (Alpert et al, 2002) a decrease which has been attributed to the enhanced production of 12-hydroxyeicosatetraenoic acid (12-HETE). There is no evidence to show whether the changes observed in the other G L U T s are due to the same mechanism. Nevertheless,  it may represent an effective  regulatory mechanism for the endothelium to protect itself from hyperglycaemia, were it not for G L U T - 2 . We observed a 165% increase in the number of voxels stained for G L U T - 2 in STZ-treated rats (Figure 2.17). This is the first report concerning the endothelium, but our results are consistent with the dramatic increases in G L U T - 2 expression seen in rat intestinal enterocytes (Corpe et al, 1996) and kidney proximal tubule cells (Marks et al, 2003) in response to STZinduced diabetes. The presence of G L U T - 2 , and its increase in diabetes, would be  115  expected to have profound effects on the endothelial cell's ability to regulate their glucose uptake.  STZ-treatment is a well-established animal model of Type 1 diabetes. It may be argued that the observed changes in the G L U T isoforms are due to the effect of the S T Z toxin, rather than to hyperglycaemia. Previous studies have shown that STZ toxicity is restricted to cells expressing G L U T - 2 (Schnedl et al, 1994), and that its effect, either no change or a decrease in the level of G L U T - 2 protein expression (Wang and Gleichmann, 1998; Burkart et al, 1999), is the opposite of that observed here. Although G L U T - 2 may facilitate the transport of STZ in cells, it is not necessarily targeted by the toxin. Burkart et al. identified NAD  +  depletion caused by P A R P activation, as the metabolic event leading to  cell death. The study went on to demonstrate that knockout PARP-/- mice were completely resistant to the S T Z toxin. The STZ-treated knockout mice were normoglycaemic, maintained a normal level of secreted insulin, and preserved their islet ultrastructure. In addition, the level of G L U T - 2 protein expression found in the islets was unaffected by S T Z (Burkart et al, 1999). It is only following multiple doses of STZ (after 3 to 5 doses of 40 mg/kg/day), that Wang et al. observed a decrease in G L U T - 2 protein levels in mice pancreatic beta-cells (Wang  and  Gleichmann,  1998),  which  preceded  the  development  of  hyperglycaemia and beta-cell destruction. In other tissues expressing G L U T - 2 (liver, intestine and kidney proximal tubules), an increase in the protein level is observed following STZ-induced hyperglycaemia. This effect is reversed by insulin (Thulesen et al, 1997), insulin-like growth factor (Asada et al, 1998), or is abolished by overnight fasting (Marks et al, 2003), mdicating that the transporter upregulation is a direct effect of hyperglycaemia and/or hypoinsulinaemia.  116  Taken together, it is most likely that the increased level of G L U T - 2 observed in our study is attributable to hyperglycaemia and/or hypoinsulinaemia rather than any direct effect of the STZ toxin.  2.4.10 Intracellular glucose accumulation  Our results seem to indicate that the ECs have a range of transporters that should protect them against accumulating intracellular glucose, as well as an adaptive response to hyperglycaemia, (a reduction in the expression of G L U T - 1 , 3, 4 and 5), that should reduce glucose uptake under these conditions. W h y then do ECs have a high susceptibility to glucose toxicity? Despite appearances, we think that the subcellular organization of the G L U T s , and the inability to downregulate hyperglycaemia  GLUT-2,  may  regardless  be  of  unfavourable  whether  for  glucose  the  cell  transport  exposed through  to this  endothelium is largely transcellular or paracellular. The high abluminal density of G L U T - 2 may compromise the net extrusion rate of glucose on the abluminal side if the glucose concentration in the interstitial space remains elevated. This is likely to occur if the rate of glucose uptake by the SMCs is lower than the rate of extrusion by the E C , which is probable, given that SMCs are reported to downregulate glucose uptake far better than ECs (Howard, 1996b; Vinals et al, 1999). The high abluminal density of G L U T - 2 increases the possibility of glucose reuptake down its concentration gradient from the abluminal surface of ECs. The counter productive increase  in G L U T - 2  expression,  if this represents  the  production of functional protein, would exacerbate this problem, and may explain why ECs are so susceptible to hyperglycaemic damage. If this hypothesis is correct, it follows that reducing the expression of G L U T - 2 in the endothelial  117  cell may have beneficial effects. In support of this, recent clinical trials using an inhibitor of PKC-beta, which controls the transport of GLUT-2 to the membrane (Helliwell et al, 2000), have  demonstrated  a significant  improvement in  endothelial function that might be explained, in part, by a reduction of GLUT-2 trafficking (Beckman et al, 2002).  Our results suggest that vascular ECs are particularly susceptible to glucose toxicity due to their inability to decrease the expression of G L U T - 2 during prolonged hyperglycaemia. Further studies will be required to determine the specific functional role of each transporter isoform.  118  Chapter 3 Measurements of glucose uptake in endothelial cells. 3.1 Introduction  3.1.1 Glucose as a principal source of energy  Carbohydrates are the body's principal source of energy. Some cells, like neurons and erythrocytes, depend almost exclusively on glucose and do not have the capacity to store it in the form of glycogen. It is therefore of great importance to maintain a basal level of plasma glucose to prevent hypoglycaemia, which could  lead  to  nervous  system  dysfunction.  Secondary  functions  of  the  carbohydrates include the formation of extracellular complexes with proteins and lipids of the plasma membrane (glycocalyx),  and the participation of  pentoses in the formation of nucleic acids. In adipose tissue, glucose is also essential to the formation of the glycerol molecule, an integral component of triglycerides,  which are necessary for lipid  storage. While  carbohydrates  constitute almost half of the energy from a North American diet, it is still possible to maintain health with a diet poor in carbohydrates since the body can provide most of its required energy through the oxidation of lipids and amino acids. 200g of carbohydrate per day is sufficient to maintain a basal plasma glucose level, below this, the body starts to catabolise stored lipids and proteins (Brosnan, 1999; Macdonald, 1999). Glucose is also the main source of energy for the cells of the vascular wall.  119  3.1.2 Glucose metabolism in E C  ECs actively metabolise glucose in order to fulfil their energy demands. With normoglycaemia, the catabolism of other substrates such as palmitate, lactate and certain amino acids (L-glutamine, L-alanine and L-arginine) is minimal and glucose constitutes the main source of energy (Krutzfeldt et al, 1990). ECs sustain their energy requirements through glycolysis (Gerritsen and Burke, 1985; Krutzfeldt et al, 1990). It is only during periods of hypoglycaemia that the Krebs cycle becomes the prominent pathway for glucose metabolism. Thus, A T P is primarily generated through the glycolytic pathway and ECs consume a low level of O 2 . It is therefore not surprising that ECs adapt quite easily to hypoxic conditions and remain functional despite prolonged periods of substrate depletion (Mertens et al, 1990; Culic et al, 1999).  3.1.3 Regulation of glucose uptake in E C  Despite  all the  evidence  linking  glucose  toxicity  to  an  increased  susceptibility to cardiovascular diseases in the diabetic population, very little is known about the regulation of glucose uptake in vascular ECs. Kaiser et al. were amongst the first to provide insights regarding the potential impact of a differential regulation of glucose transport between ECs and S M C s (Kaiser et al, 1993). They reported that a 24 hour exposure to elevated glucose (22 m M ) had negligible effects on the rate of 2-deoxyglucose (2DG) uptake (room temperature) in bovine aortic ECs, whereas the same treatment significantly decreased the Vmax of glucose transport in bovine S M C s (Kaiser et al, 1993).  120  Other studies on the effect of an elevated ambient glucose concentration in cultures of bovine GM7373 (Giardino et al, 1994), and mouse microvessel, E C s (Vinters et al, 1985) have even reported an increased rate of glucose uptake in response to exposure to high glucose. Conversely, a study in human umbilical vein-derived E C s ( H U V E C )  has shown that a high glucose concentration  decreased the rate of 2 D G uptake at 37°C. However, this study also shows that the concentration-response curve for the inhibition of 2 D G uptake by D-glucose was significantly shifted in H U V E C s in comparison to other cell types; the concentration of D-glucose for half-maximal inhibition of the rate of 2DG uptake in H U V E C s was 4 to 8 fold greater than that of glial cells (Walker et al, 1988), myoblasts L6 (Walker et al, 1989) and adipocytes 3T3-L1 (Tordjman et al, 1990). Taken together, this suggests that glucose uptake in vascular E C s is regulated differently than in other cell types.  The inhibitory effect on the rate of glucose uptake in H U V E C exposed to elevated ambient glucose was also accompanied by a dramatic decrease in the ratio of phosphorylated 2 D G to free 2 D G . The decrease in the rate of glucose uptake was therefore attributed to the inhibition of glucose phosphorylation rather than to the decrease in transport (Vinals et al, 1999). This indicates that glucose uptake in ECs is limited by its phosphorylation rate not its transport rate. This is in accordance with previous studies which showed that elevated glucose concentrations had neither an effect on the expression of G L U T - 1 (Kaiser et al, 1993; Giardino et al, 1994; Vinals et al, 1999) nor on the rate of uptake of 3-0methylglucose ( 3 0 M G , a non-metabolizable glucose analog) (Takakura et al, 1991;  Kaiser  et  al,  1993;  Vinals  et  al,  1999). Thus,  when  exposed  to  hyperglycaemia, E C s reduce glucose utilisation rather than glucose transport.  121  This may explain in part, the lack of inhibition of glucose uptake in ECs in the presence of elevated glucose concentrations at room temperature, where the rate of metabolism is slowed down (Kaiser et al, 1993).  In contrast to elevated glucose, a low glucose concentration up-regulates glucose transport in ECs. Glucose starvation has been shown to increase the Vmax for 3 0 M G transport and G L U T - 1 protein expression in bovine microvessel ECs (Takakura et al, 1991). Similarly, the rate of 2 D G uptake was also increased in primary cultures of brain and adrenal capillaries, and aortic ECs, deprived of glucose  for periods of 48 hours (Gaposchkin and Garcia-Diaz,  1996). In  accordance with these results, chronic hypoglycaemia but not hyperglycaemia, increases  the transendothelial transport of glucose  (Simpson et al,  across the rodent BBB  1999). Thus, in ECs, hypoglycaemia up-regulates  glucose  transport while hyperglycaemia inhibits glucose metabolism, with little effect on transport.  Like the presence of G L U T - 4 in ECs (see Chapter 2) the effect of insulin on ECs remains controversial. O n one hand, several studies have reported that the exposure to insulin produces no effect on the rate of glucose uptake in brain microvessel ECs (Drewes et al, 1988; Takakura et al, 1991), bovine retinal ECs (Betz et al, 1983), cultured cardiac ECs (Thomas et al, 1995), and H U V E C s (Corkey et al, 1981). O n the other hand, others have observed an increased 2 D G and/or 3 0 M G transport in response to insulin in ECs of mouse microvessels (Vinters et al, 1985), rabbit coronary microvessels (Gerritsen and Burke, 1985), bovine retinal microvessels (Allen and Gerritsen, 1986), rat capillary (Kwok et al, 1989), and human omental microvessels (Abe et al, 1990).  122  The inconsistency in the observed effect of insulin on E C s may be due to the presence of different regulatory mechanisms amongst vascular beds of different calibres and functions. For instance, the effect of insulin on glucose uptake seems to be limited to the ECs of microvessels such as those from adipose tissue (Bar et al, 1988) and the retina (King et al, 1983) with no effect on ECs of larger vessels like the pulmonary artery and aorta (King et al, 1983; Bar et al, 1988). It is also possible that specific conditions need to be met in order to detect the effect of insulin on ECs of certain vascular beds. Thus, Gerritson at al. have demonstrated that glucose deprivation is required to enhance the effect of insulin on 2 D G uptake in rabbit coronary microvessel ECs (Gerritsen et al, 1988). Taken together, this may explain in part, the lack of insulin effect observed by studies performed in ECs of larger vessels and certain microvessels.  Estrogen (Shi et al, 1997), V E G F (Sone et al, 2000) and T N F - a (Pan et al, 1995) are also known to increase glucose uptake in ECs through stimulation of G L U T - 1 protein expression. IGF-1 has also been shown to stimulate glucose uptake in bovine retinal ECs (Bar et al, 1989; DeBosch et al, 2001; DeBosch et al, 2002). The increase in glucose uptake in response to IGF-1 was shown to be dependent on the activation of phosphatidylinositol-3 kinase (PI3-kinase), P K C and  mitogen-activated  protein kinase (MAP-kinase) (DeBosch et al,  2001;  DeBosch et al, 2002). P K C has also been implicated in the regulation of glucose transport in dog and human BBB (Drewes et al, 1988). Although the authors have suggested that IGF-1 produces the increase in glucose transport through the translocation of G L U T - 1 to the plasma membrane, other transporter isoforms are likely  to be  involved. PI3-kinase  participates  in the  signalling  pathway  responsible for the enhanced glucose transport induced by insulin through  123  G L U T - 4 translocation to the plasma membrane (Sakaue et al, 1997). In addition, the activation of PI3-kinase and P K C have been shown to induce translocation of G L U T - 2 to the plasma membrane of intestinal epithelial cells (Helliwell et al, 2000; Helliwell et al, 2003).  Despite the presence of G L U T - 5 in human BBB (Mantych et al, 1993b) and as shown by the present study in H C A E C s and rat coronary, cerebral, mesenteric and renal ECs (see Chapter 2), the transport of fructose in ECs has yet to be demonstrated. The presence of G L U T - 5 in the BBB remains particularly puzzling as fructose is not considered an energy substrate for the adult brain (Nualart et al, 1999). Fructose could potentially be metabolised by the ECs themselves, but a clear demonstration of its uptake by the ECs, using a labelled fructose, needs to be performed.  The coupling of glucose and N a transport by an SGLT-like transporter +  has been previously demonstrated in brain microvessels (Nishizaki et al, 1995; Nishizaki and Matsuoka, 1998). More recently, SGLT-1 was identified at the luminal surface of the BBB (Elfeber et al, 2004a), heart and skeletal muscle capillaries, and in cultured coronary ECs (Elfeber et al, 2004b). Nishizaki et al. simultaneously recorded glucose-evoked N a currents and monitored glucose +  uptake with radio labelled 2 D G in cultured brain microvessel E C s . Glucoseevoked N a current and 2 D G uptake were enhanced by a 30-min pre-incubation +  in glucose free media and the addition of cytochalasin B, but inhibited by an increased concentration of glucose, or by phloridzin (an S G L T specific inhibitor), dinitrophenol (an inhibitor of energy metabolism), the removal of extracellular Na  +  (Nishizaki and Matsuoka, 1998). Elfeber et al. used a non-re-circulating  124  hindlimb perfusion method and measured glucose consumption by the skeletal muscles to demonstrate that phlorizin blunted the insulin-induced increase of glucose extraction from the perfusate (Elfeber et al, 2004b). Because S G L T is not expressed in skeletal muscle, the inhibitory effect was attributed to a block of transcellular and/or paracellular transport at the capillary level. In addition, the same authors showed in ECs of BBB that S G L T expression is up-regulated after 1 day of ischemic occlusion followed by reperfusion (Elfeber et al, 2004a). Taken together, these results show that S G L T actively co-transports glucose in ECs, and that low cytosolic glucose concentrations enhance its activity. Thus, S G L T may play an important role in the maintenance of an adequate supply of glucose to the ECs in periods of stress such as hypoglycaemia and ischemia.  The information available on the regulation of glucose uptake in E C s indicates that ECs are affected by the ambient glucose concentration. The rate of uptake in E C s is likely modulated by the plasma, cytosolic and interstitial glucose concentrations. The interstitial space contained in the vascular wall is also probably influenced by the S M C rate of glucose uptake. So what do we know about glucose metabolism and the regulation of glucose uptake in SMC?  3.1.4 Glucose metabolism in S M C  The glycolytic pathway is also the principal route of glucose metabolism and A T P generation in SMCs (Hardin and Pauley, 1995). The glycolytic and gluconeogenic pathways have been shown to be compartmentalised in SMCs. This means that intermediates from the glycolytic pathway do not diffuse uniformly in the cytosol and consequently do not become substrates for other  125  pathways (Lloyd and Hardin, 1999). This indicates the possibility of coupling between  glucose  transporters and the enzymes of each of the  respective  metabolic pathways. This coupling could facilitate the compartmentalisation process by localising pools of intracellular glucose. Such localised pools of glucose may in turn be responsible for the greater capacity of the S M C s to regulate their rate of glucose uptake.  While S M C s also store excess glucose, the glycogen content is rapidly depleted in the absence of extracellular glucose. Contracted S M C s derive about 10 % of their energy needs from glycogen, while glucose transported from the blood accounts for up to 50% of the total energy consumption (Allen and Hardin, 2000). A t rest, glucose metabolism increases while the oxidation of fatty acids such as acetate and octanoate decreases (Barron et al, 1998).  3.1.5 Regulation of glucose uptake in S M C s  The rate of glucose transport in S M C s is inversely proportional to the extracellular glucose concentration (Kaiser et al, 1993; Fujiwara and Nakai, 1996; Howard, 1996b). Accordingly, Vmax for glucose uptake is decreased by high ambient glucose concentrations, with no changes in the transporter affinity (Kaiser et al, 1993; Howard, 1996b). These changes in glucose transport activity have been correlated with a decrease in the amount of G L U T - 1 in S M C s (Kaiser et al, 1993; Quinn and McCumbee, 1998). Although the decrease in glucose transport in response to an elevated extracellular glucose concentration may have represented a mechanism of protection for the SMCs, it was demonstrated  126  that the intracellular glucose  concentration remained abnormally elevated  (Howard, 1996b).  Vascular S M C s from the rat aorta and posterior vena cava, as well as several cell lines, have been shown to be insulin-sensitive (Falholt et al, 1985; Standley and Rose, 1994; Banz et al, 1996), and to express G L U T - 4 (Charron et al, 1989; Banz et al, 1996). Interestingly, a high glucose concentration has been shown to attenuate the effect of insulin in rat vascular SMCs, an effect attributed to a reduced activity of the glucose transporter system (Fujiwara and Nakai, 1996). Insulin-sensitivity and G L U T - 4 expression have also been shown to be decreased in aortic S M C s of hypertensive rats (Atkins et al, 2001). This indicates that the transport and metabolism of glucose in S M C s is likely to be affected by metabolic disorders such as diabetes and hypertension, which could have repercussions for the ECs.  Insulin enhancing drugs such as metformin and troglitazone have been shown to stimulate glucose uptake and G L U T - 1 expression in human and bovine aortic S M C s (Sasson et al, 1996; Kihara et al, 1998). Amongst other modulators of glucose transport, angiotensin II (Low et al, 1992; Quinn and McCumbee, 1998), IGF-1 (Standley and Rose, 1994), endothelial growth factor (EGF) (Low et al, 1992), and platelet-derived growth factor (PDGF) (MacKenzie et al, 2001) have been shown to stimulate glucose uptake in SMCs.  Thus, differences in glucose needs and transport regulation exist between ECs and SMCs. These differences may account for the increased susceptibility of the ECs to glucose toxicity.  127  3.1.6 Aims of the present study and hypothesis  The main goal of this study was to examine, in an intact vessel, the regulation of glucose transport in individual ECs. The initial step towards this goal was to determine the appropriate experimental conditions for recording fluorescent glucose uptake in live cells. This was done using cultured cells, a much simpler preparation than the intact vessel.  First, the presence and subcellular distribution of glucose transporters, not previously characterised in primary culture of human coronary artery ECs (HCAEC),  was  assessed  through  immunocytochemistry  and  fluorescence  microscopy. It was hypothesised that the same glucose transporter isoforms previously identified in rat coronary artery ECs, G L U T - 1 , 2, 3, 4, 5 and SGLT-1, (see Chapter 2) would be detected in H C A E C s .  Second, to elucidate the functional role of the glucose transporter isoforms identified in these ECs, the uptake of a fluorescent glucose analog was examined, in the presence of different compounds known to regulate glucose transport. This also enabled us to establish experimental conditions that would be applied to the intact vessel. It was hypothesised that well-known competitive inhibitors of glucose uptake such as D-glucose and cytochalasin B, would inhibit the accumulation of the fluorescent glucose analog in ECs. It was also hypothesised that insulin, because it stimulates the translocation of G L U T - 4 to the plasma membrane, would increase the accumulation of the fluorescent glucose analog in ECs.  128  3.1.7 In vivo fluorescence microscopy  The pressure myograph technique presented in Chapter 1 was coupled to confocal microscopy. The arteriograph chamber allows the study of small blood vessels under both perfusion and supervision. The bottom cover glass of the arteriograph chamber permits the imaging of the juxtaposed  vessel wall.  Changes of solutions and the addition of compound to the perfusate were performed with a small peristaltic pump used in an open system without disturbing the equilibrium of the vessel's basal tone. Such perfusion systems have been previously described and used into study the effect of intraluminal flow on myogenic tone (Falcone, 1995; Henrion et al, 1997), and allows complete control over flow, pressure, temperature and oxygenation.  A confocal microscope has a narrow depth of field, which is needed to distinguish the fluorescence of the endothelial layer from that of the S M C layer. Although a wide field microscope coupled to deconvolution could potentially provide a better axial resolution (see chapter 2), the deconvolution algorithm was developed for punctate sources of light, and is not suitable for a uniform field of fluorescence,  like that obtained with the fluorescent glucose analog in the  vascular wall. A 20X objective suitable for both phase contrast and fluorescence was chosen, and its long working distance (160 um) ensured that images could be obtained through the vessel wall (~ 30 um). Phase contrast was used to determine the boundary of each cell in the field of view.  129  3.1.8 Fluorescent glucose analog  Deoxyglucose (2DG) uptake assays were developed by Sokoloff in 1977 (Sokoloff et al., 1977). The difference between 2 D G and D-glucose is at the C-2 position where the hydroxyl group of D-glucose has been replaced by a hydrogen atom. 2 D G is transported into cells by glucose transporters and competes with D-glucose for the enzyme hexokinase, (phosphorylates glucose to produce  glucose-6-phosphate).  isomerization  of  2DG  into  The missing hydroxyl fructose-6-phosphate,  group prevents  which  aborts  the  further  metabolism through the glycolytic pathway. The 2 D G remains trapped in the cell, as it does not appear to be a substrate for the enzyme  glucose-6-  phosphatase. Previous studies have also demonstrated that the exit of radioactive 2DG before its phosphorylation is negligible, and it remains in the cytosol for a minimum of 30 min (Goodner et al., 1980; Horn and Goodner, 1984; Vallerand et al., 1987). Thus, when 2 D G is labelled with a radioactive atom or with a fluorescent molecule, it becomes a useful tool for the imaging and quantification of glucose uptake in cells or tissues.  Two fluorescent glucose analogs, the 2- and 6-[N-(7-nitrobenz-2-oxa-l,3diazol-4-yl)amino]-2-deoxyglucose (2-NBDG and 6-NBDG), are now available for measurements of glucose uptake in live individual cells. 6-NBDG was the first fluorescent hexose derivative to be synthesised to study the nature of the glucose transport system in erythrocytes (Speizer et al, synthesised  from  6-amino-6-deoxyglucose  1985). The compound was  hydrochloride  and  4-chloro-7-  nitrobenz-2-oxa-l,3-diazol (NBD-C1). When cells were exposed to 6-NBDG, thin layer chromatography (TLC) analyses of intracellular lysates identified a single  130  spot  of  fluorescence,  indicating that  the  compound is  not  metabolised  intracellularly (Speizer et al, 1985; Shimada et al, 1994). Modifications at neither the C-2 nor at the C-6 positions prevents transport across the plasma membrane, however, a modification at the C-6 position prevents the phosphorylation of the 2DG (Yoshioka et al, 1996c). Consequently, both glucose analogs are taken up by the cell but only the 2-NBDG is trapped in the cell while the 6-NBDG can move freely in and out of the cell.  Yoshioka et al. have introduced the N B D molecule at the C-2 position in order to monitor further metabolic events. 2-NBDG was synthesised by reacting a molecule of D-glucosamine and NBD-C1 (Yoshioka et al, 1996c). T L C analysis revealed 2 spots of fluorescence in the cytoplasmic fraction of Escherichia coli cells (E. coli) treated with the 2-NBDG compound. The first spot of fluorescence was comparable to the one found in the extracellular fraction. The second fluorescence spot found in the cytoplasmic faction was thought to be a phosphorylated derivative of the 2 - N B D G (Yoshioka et al, 1996c). The fate of the 2-NBDG was further examined in E . coli and it was confirmed that the 2-NBDG is rapidly converted into 2-NBDG-6-phosphate after is incorporation into cells. In E. coli, the 2-NBDG-6-phosphate was later decomposed into a non-fluorescent derivative (Yoshioka et al, 1996b). Thus, it was concluded that the fluorescence intensity recorded from these cells may reflect a dynamic equilibrium between the incorporation and decomposition of 2-NBDG  (Yoshioka et al,  1996b).  Additional observations showed that 2 - N B D G is incorporated only in living cells, that it is not completely metabolised (remains fluorescent in the cytosol for a minimum of 10 min) and its accumulation does not cause a lethal effect in E . coli (Yoshioka et al, 1996a; Yoshioka et al, 1996c). 2-NBDG has also been used to  131  measure glucose uptake in a variety of mammalian cell types including isolated SMCs (Lloyd et al, 1999), pancreatic p-cell (Yamada et al, 2000), enterocytes (Roman et al, 2001), cardiomyocytes (Ball et al, 2002), neurons and astroglia cells (Itohetal, 2004).  The uptake of 2 - N B D G and 6-NBDG has been shown to be inhibited by D glucose and cytochalasin-B, suggesting that the uptake is mediated by glucose transporters (Speizer et al, 1985; Lloyd et al, 1999; Yamada et al, 2000). D-glucose competes with both fluorescent glucose analogs for the binding site on the glucose transporter. L-glucose, its stereoisomer, is not transported in the cells and thus does not inhibit 2 - N B D G uptake (Lloyd et al, 1999). Cytochalasin B is a metabolite  of  the  fungus  Helminthosporum  dermatoideum  and  a  well-  characterised inhibitor of the stereospecific transport of D-glucose in a variety of cell types (Gould and Seatter, 1997). Cytochalasin B has been shown to inhibit the efflux rather than the influx of D-glucose, which suggests an endofacial binding site for this molecule. Cytochalasin B inhibits all isoforms of the G L U T s with the exception of G L U T - 5 which is insensitive to its effect (Burant and Bell, 1992; Burant et al, 1992).  3.2 Methods  A l l chemicals were purchased from Sigma-Aldrich Ltd., unless otherwise stated. Animal handling was done in accordance with the guidelines of the Canadian Council on Animal Care and the Principles of laboratory animal care (National Institute of Health (NIH) publication no. 85-23, revised 1985).  132  3.2.1 Cell culture  Cryopreserved H C A E C s (third passage) were obtained from a male donor; 29 years old, with no known diabetic or cardiovascular conditions (Cambrex Bio Science Walkersville, inc., Walkersville, M D ) . The cells were resuspended in Endothelial Growth Medium-2 (EGM-2; Cambrex Bio Science Walkersville,  inc.)  containing  supplements  and  growth  factors  (hydrocorticosterone, hEGF (epidermal growth factor, recombinant), FBS (foetal bovine serum), V E G F , hFGF-B (fibroblastic growth factor, recombinant), R3-IGF( insulin-like  growth  factor)  -1,  ascorbic  acid,  heparin  and  gentamicin/amphotericin-B) and seeded at a density of 5000 cells/cm in a T-25 2  flask. H C A E C s (4th and 5th passages) were plated on sterile Lab-Tek chambered coverglass (#1 borosilicate, 25 X 56 mm, Nalge Nunc international, Naperville, IL),  coated  with  measurements,  APES  (3-aminopropyltriethoxysilane)  for glucose  uptake  or on a coverslip (#1 borosilicate, 25 X 25 mm) in six-well  multidishes for both immunocytochemistry and the preparation of whole cell lysate. The cells were cultured for 96 to 120 hrs, or until they reached 90% confluence at 3 7 ° C in a humidified incubator gassed with 5% CCh-95% air.  3.2.2 Immunocytochemistry  H C A E C s were fixed in 2% paraformaldehyde dissolved in PBS for 10 min. Fixation was quenched with a 10-min rinse in 100 mmol/1 glycine ( p H 7.4) and the cells were permeabilized with 0.06% saponin in PBS for 10 min at room temperature followed by 3 x 10-min rinses with PBS. Non-specific binding sites were blocked by mcxibating the fixed, permeabilized tissue in 10% donkey serum dissolved in PBS for one hour at room temperature. The cells were then  133  incubated for 4 hours with two antibodies raised in different species; one against a glucose transporter isoform (GLUT-1 to 5 or SGLT-1), and one against V E cadherin. The same antibody concentrations used with the vessel preparation were applied to the monolayer of H C A E C s . A l l antibodies were diluted in antibody buffer. The antibody sources, concentrations, epitope, and suppliers are listed in Table 2.2.  Excess antibody was removed by 3 x 10-min rinses in antibody wash solution. The tissue was then incubated for 2 hours with two donkey affinitypurified secondary antibodies that had been solid-phase adsorbed to minimize species cross-reactivity (Jackson ImmunoResearch Laboratories). The secondary antibodies were conjugated to either fluorescein isothiocyanate (FITC) or Texas Red and diluted in antibody buffer. Following incubation with the secondary antibodies, the tissue was rinsed 3 x 10 min in antibody wash solution. The cells' nuclei were then labelled with D A P I (0.3 umol/1, Molecular Probes), for 5 min, followed by 3 x 10-min rinses in PBS. The labelled cells were mounted on a slide in D A B C O mounting medium. Small sub-resolution beads were added to the D A B C O ; these fluoresce in all wavelengths and are used to align the image stacks. Two control experiments were conducted. In the first, the cells were labelled with a primary antibody and an inappropriately targeted secondary antibody. In the second, the primary antibody was omitted.  3.2.3 3D Image acquisition, deconvolution and analysis  As described in chapter 2, a series of two-dimensional fluorescent images was acquired through the depth of the cell with a standard Nikon Diaphot 200  134  inverted microscope equipped for epifluorescence (60X oil immersion objective, N A 1.4, 2X adaptor). The pixel size was 200 x 200 n m and the Z spacing was 250 nm. Images were recorded with a C C D camera. For each wavelength, a threedimensional data set was acquired using narrow bandpass filters specific for Fluorescein, Texas Red, and DAPI. Images were background and dark current subtracted and corrected for photobleaching. A flat field was used to correct for non-uniform illumination and camera sensitivity across the field of view. Images were  deconvolved  using  the  algorithm developed  by  Carrington  et  al.  (Carrington et al., 1995) with an empirically determined PSF on an EPR clientserver. After deconvolution, each image was thresholded. The threshold values were determined from control experiments where the cells were labelled with a primary antibody and an inappropriately targeted secondary antibody or where the primary  antibody was  omitted. The remaining fluorescence  intensity  acquired from these control experiments was used, as a threshold value, to apply to each image collected. The background was segmented from the image by assigning a zero value to all pixels with a grey level lower than the threshold. Deconvolved and thresholded images were aligned using the fiduciary markers. A l l images are 3D reconstruction of the indicated depth.  3.2.4 Measurements of 2 - N B D G uptake in H C A E C s  The experimental conditions for measuring 2 - N B D G uptake were first determined in cultured cells. H C A E C s were mounted on the stage of a Nikon inverted microscope Diaphot-TMD and superfused at a flow rate of 7.5 ml/hr with oxygenated Krebs buffer (pH maintained at 7.35 to 7.45 by continual oxygenation; room temperature). The microscope was attached to an MRC-600  135  laser confocal imaging system (Bio-Rad, Hercules, C A ) . The MRC-600, equipped with a mixed-gas (krypton-argon) laser, was used with a FITC filter block (BHS, excitation filter 488 n m DF10, 515 n m emission barrier) and neutral density (ND) filter #1 (transmits 10% of the incident light). Dual recordings of phase contrast and fluorescence were performed using a 20X objective (Fluor 20X, Ph3DL, N . A . 0.75). Phase contrast was used to determine the position and the boundary of each cell and glucose uptake was monitored using the fluorescently  tagged  glucose analog 2 - N B D G (Yoshioka et al, 1996a; Yoshioka et al, 1996b; Yoshioka et al, 1996c). The emitted fluorescence was collected in confocal mode (confocal aperture of 1.55 m m diameter, 4X zoom), with an estimated lateral resolution of 3.7 um and an axial resolution of 9.3 u m with an image depth of 16.9 um. The resolution and depth of field were determined with a fluorescent  bead  (Appendix 3). A n image was acquired for a group of cells observed in the field of view. The fluorescence collected corresponded to the amount of 2 - N B D G sequestered  intracellularly. The average fluorescence  intensity per cell was  quantified over time.  3.2.5 Measurements of 2 - N B D G uptake in ECs of intact vessels  Male Wistar rats (200-235 g; University of British Columbia Animal Care Centre,  Vancouver, B.C.) were  sacrificed  with  a peritoneal  injection  of  pentobarbital (30 mg/kg). Segments of approximately 0.6 to 1.0 m m in length of septal coronary arteries were carefully dissected. The tissue was immersed in icecold oxygenated (95% Oil 5% C O 2 ) Krebs buffer solution during the dissection. The vessels were mounted on glass cannulae in an arteriograph chamber (Living System Instrumentation) providing both intraluminal flow and superfusion of  136  the adventitial tissue with oxygenated Krebs solution (pH of 7.35 to 7.45) at room temperature. The adventitial surface was continuously superfused at 20-25 ml/min with oxygenated Krebs buffer containing 5 m M D-glucose. Intraluminal flow  of  Krebs buffer  solution  was  maintained at  5.5  ml/hr  (a  normal  physiological rate) and constant pressure using a small peristaltic pump (model P720, tubing of 0.093" internal diameter; Instech Laboratories, Inc., Plymouth Meeting, PA). A windkessel (a "T" connector with a closed end containing trapped air) was placed in the output line of the pump in order to reduce to a minimum the frequency and amplitude of the pulses generated by the pump. The intraluminal pressure was monitored with a pressure transducer (PT/F, Flow-thru monolithic chip, Luer fittings; Living System Instrumentation) and kept constant at 20 m m H g . The chamber was mounted on the stage of the microscope. Phase contrast was used to locate the axial position of ECs in the vascular wall and to determine the position and the boundary of each cell. Glucose uptake was monitored using the fluorescently tagged glucose analog 2N B D G . The lumen was perfused with I m M 2 - N B D G in Krebs solution for various times followed by a 10-min wash period (Krebs solution). Images of E C fluorescence were acquired after each 10-min wash. The fluorescence collected corresponded to the intracellular sequestered 2-NBDG. The average fluorescence intensity per cell was quantified over time.  3.2.6 Experimental protocols  The time course of 2-NBDG accumulation in H C A E C s was examined. These experiments were performed to determine the conditions (time and concentration) from which to measure 2 - N B D G in its initial rate of uptake, before  137  equilibrium is reached. H C A E C s were superfused with a Krebs solution containing a total of 5 m M glucose; between 0.1 to 3 m M of 2-NBDG and the rest was of L-glucose. The rate of uptake was obtained from consecutive exposures to 2-NBDG, adapted from the protocols used by others (Lloyd et al, 1999; Yamada et al, 2000; Roman et al, 2001). The cells were exposed to a supervision of 2N B D G for 5 min, then washed with a Krebs solution containing 5 m M D-glucose for a subsequent 5 min. Following this wash, an image of the cells was acquired. Thereafter, the cells were further superfused with 2 - N B D G for another 5 min, washed for 5 min and an image was acquired. This represented an accumulation of intracellular 2 - N B D G for a total of 10 min. The superfusion with 2-NBDG was then repeated twice more for consecutive 10 and 15 min periods, each followed by 5-min wash and image acquisition. Thus, the same cells were imaged at a total time of exposure to 2-NBDG of 5, 10, 20, and 35 min (Figure 3.1A). The same protocol was repeated with concentration of 0.1, 0.5 and 1 m M . A second protocol was used where the cells were superfused with 2-NBDG for 5 min, washed for 5 min and imaged. This was repeated 6 times so that the cells were imaged at a total time of exposure to 2 - N B D G of 5, 10, 15, 20, 25, and 30 min (Figure 3.1B). This protocol was used with concentrations of 2 and 3 m M 2N B D G . The comparison between the rate of uptake of 0.1 m M of 2 - N B D G and 6N B D G was obtained from consecutive superfusion of 2 - N B D G of 5, 5,10, 10, 15, and 15 min, each followed by a 5-min wash and image acquisition. Thus, the same cells were imaged for a total time of exposure to 2-NBDG of 5,10, 20, 30, 45 and 60 min (Figure 3.1C). In each case, the image acquisition consisted of both phase contrast and fluorescence recordings.  138  The effect of D-glucose (4 and 9 mM), cytochalasin B (10 uM) and insulin (2.5 uU/ml; Humulin R, human biosynthetic Hl-210; Eli Lilly Canada Inc., Toronto, O N ) were tested on a 20-min exposure to a Krebs solution containing a total of 5 m M glucose; 1 m M 2 - N B D G and 4 m M L-glucose (9 m M L-glucose were compared  with  9 m M D-glucose).  A  10  and 20-min  pre-incubation with  cytochalasin B and insulin respectively, in a Krebs solution containing 4 m M L glucose, preceded the addition of 2 - N B D G to the solution. The effect of insulin (2.5 uU/ml) was also tested in H C A E C s on a 30-min exposure to a Krebs solution containing 1 m M 2 - N B D G and 4 m M D-glucose preceded by a 20-min preincubation with insulin and 5 m M D-glucose.  In intact vessels, the lumen was perfused with a Krebs solution containing a total of 5 m M glucose; 1 m M 2-NBDG and 4 m M L-glucose. Each exposure to 2N B D G was followed by a 10-min wash with a Krebs solution containing 5 m M D-glucose. Phase contrast and fluorescence images were acquired after each wash. To determine the rate of uptake of 2-NBDG, the vessels were perfused with 2 - N B D G for consecutive 5, 5, 10, 10, 15, and 15 min, each followed by a wash and image acquisition. Thus, the same cells were imaged for a total time of exposure to 2 - N B D G of 5, 10, 20, 30, 45 and 60 min (Figure 3.1D). For all other experiments, the vessels were exposed to 2 - N B D G for a period of 20 min in which the effect of D-glucose (replaced by the same amount of L-glucose), cytochalasin B (10 uM) and insulin (2.5 pU/ml) were tested. After each 20 min of perfusion with 1 m M 2-NBDG, the vessels were perfused with the washing solution for 10 min and imaged. Pre-incubations of 10 min and 20 min with cytochalasin B and insulin, respectively, in a Krebs solution containing 4 m M L glucose, were also performed prior to exposure to 2-NBDG.  139  A 2-NBDG W  2-NBDG W -> -4I—  0  U  t  2-NBDG W •4 •  h 5  2-NBDG W  — I  a  a  W  -h 15  2-NBDG W  2-NBDG W  i  30  a W  2-NBDG  20  30  a  2-NBDG W 2-NBDG  h  40  35  T15  2-NBDG W  45  a T5  20  30  a T10  40  a T30  45  2-NBDG W  I  65  50  a  70  1  85  Time (min)  image acquisition W  h->  90 Time (min)  a  a  a  T45  T60  2-NBDG W  70  a T30  T20  2-NBDG  -I  image acquisition  T30  2-NBDG W  60  60  a  a  50  55  T25  -t15  r  50  a  2-NBDG W  35  H  Time (min)  W  T20  T20  T10  T5  TO  TO  2-/VeD6  10  0  a  a T10  1J  T0  0  -t-  a  a  2-NBDG W  25  20  T35  T20 W  15  55  50  a  T10  2-NBDG W  10  35  H  5  B  0  w  2-NBDG •  ~i  a  I  W < 30  20  15  10  0  — I  2-NBDG •  85  2-NBDG  -i 95  image acquisition  W  1  1—•  110  120 Time (min)  a  a  T45  T60  Figure 3.1 Time course of 2-NBDG uptake; experimental protocols.  image acquisition  A) Protocol used for concentration of 0.1, 0.5, and 1 m M 2-NBDG in HCAECs. B) Protocol used for concentration of 2 and 3 m M 2-NBDG in HCAECs. C) Protocol used for comparisons between 0.1 m M 2-NBDG and 6-NBDG. D) Protocol used for 1 m M 2NBDG in vessel preparation. 2-NBDG: period of incubation with 2-NBDG, W: period of wash, T0-T60: represents a time of total exposure to the fluorescent glucose analog at which an image was collected.  3.2.7 Fluorescence quantification and analysis  The data collected were analysed with Scion Image Beta 4.02 for Windows XP (2000 Scion Corporation, N I H , U S A ) . Each cell boundary was determined  140  u s i n g phase contrast i m a g e . T h e freehand selection t o o l w a s u s e d to define a r e g i o n o n the phase contrast i m a g e f r o m w h i c h the average  fluorescence  per  p i x e l (for each i n d i v i d u a l cell) w a s m e a s u r e d (Figure 3.2). T h e b a c k g r o u n d fluorescence  r e c o r d e d p r i o r to 2 - N B D G i n c u b a t i o n w a s subtracted f r o m this  v a l u e . T h e m e a s u r e d intensity per p i x e l f r o m each cell a n a l y s e d w a s a v e r a g e d a n d c o m p a r e d b e t w e e n different c o n d i t i o n s . T h e same analysis w a s p e r f o r m e d w i t h the data collected f r o m the intact vessel. T h e data are presented as m e a n ± SE a n d are c o m p a r e d u s i n g A N O V A a n d t-test as r e q u i r e d .  Figure 3.2  Dual Imaging of phase contrast and fluorescence in HCAECs.  Both phase contrast (A) and 2 - N B D G fluorescence (B) images were acquired from H C A E C s following 2 - N B D G incubation (20 min) and a 5-min wash. A n individual cell boundary was determined on the phase contrast image from which the average fluorescence intensity per pixel was measured (Scion Image; scale bar is 25 um).  141  3.3 Results  3.3.1 E C identification in cultured H C A E C s  As presented in Chapter 2, Western blots were performed on H C A E C s . In the H C A E C lysate, VE-cadherin was present at the expected molecular weight of 130 K D a while no band was found for the S M C specific protein a-actin (Figure 2.3). H C A E C s were also fixed and labelled with VE-cadherin and DAPI. V E cadherin was found at the cell-to-cell junction of adjacent cells (Figure 3.3A & Figure 3.7A) but was absent where a cell was without a neighbour (Figure 3.4A) and  discontinued in area that had not reached complete confluence (Figure  3.5A,Figure 3.8A, and Figure 3.9A). From a side view it could also be observed that VE-cadherin is in close proximity to the coverslip and that the bulk of the nuclei mass are above the VE-cadherin (Figure 3.3B). The morphology of the H C A E C s was found to be different from that of ECs of the intact coronary artery. H C A E C s were not elongated and their overall size was bigger than that of an E C from a native vessel (a typical rat native E C is ~3um wide and ~12um long in comparison to ~18um wide by ~50um long for a H C A E C ) .  142  A  [J i  W' i k  V  - \ *  txV* '  ^sr#{  <  1 & \  K \ - '  \ \  K  ;  J  V;  -  . • *  *  B  illjb|M«i4H rip Figure 3.3  Identification of ECs i n culture of H C A E C s .  Deconvolved images of H C A E C s . VE-cadherin (red) and nuclei (blue). A ) En face view, the image is 6.5 um deep. B) Side view obtained from the 3D image in A , rotated 90° about the X-axis. The image is 68 um deep. Scale bar is 8 um.  W e h a v e s h o w n p r e v i o u s l y that each of the classical G L U T s a n d S G L T - 1 w e r e f o u n d i n the H C A E C lysate, at the s a m e M . W . as that f o u n d i n the n a t i v e e n d o t h e l i u m lysate, w i t h the e x c e p t i o n of G L U T - 2 for w h i c h a l o w e r M . W . i n HCAECs  was  found  immunocytochemistry,  (Figure  2.4).  We  then  assessed,  through  the s u b c e l l u l a r d i s t r i b u t i o n o f each o f these glucose  transporter isoforms. A representative cell is s h o w n l a b e l l e d w i t h  GLUT-1  (Figure 3.4), G L U T - 2 (Figure 3.5), G L U T - 3 (Figure 3.6), G L U T - 4 (Figure 3.7),  143  /  C  B  E  D  Figure 3.4  G L U T - I in H C A E C Deconvolved images of H C A E C labelled with antibodies specific for G L U T - 1 (green), VE-cadherin (red) and nuclei w i t h D A P I (blue). A ) En face view of the full 3D reconstruction of the cell, 7.5 u m deep. The arrow points to an area without an adjacent cell and without VE-cadherin. B & C ) Central planes (0.750 u m deep) obtained from A . B) G L U T - 1 and D A P I labelling, C) only G L U T - 1 labelling. D & E) Side view obtained from a 90° rotation about the X-axis of the image i n A , 2 u m deep cross-sections; coverslip at the bottom of the images. D) G L U T - 1 , VE-cadherin and D A P I labelling, E) only G L U T - 1 labelling. Scale bar is 8 um.  G L U T - 5 (Figure 3.8), a n d S G L T - 1 (Figure 3.9). E a c h of the transporters w a s seen o n the p e r i p h e r y of the cell, m o r e l i k e l y o n the p l a s m a m e m b r a n e . A l l of the transporters h a d a u n i f o r m d i s t r i b u t i o n above a n d b e l o w the nucleus, w i t h n o indication  of the  asymmetric  subcellular  distribution  found  i n the  native  e n d o t h e l i u m . A l s o m i s s i n g i n the H C A E C s is the distinctive tendency of the G L U T s a n d S G L T - 1 to locate at the cell-to-cell junctions f o u n d p r e v i o u s l y i n the native e n d o t h e l i u m (Figure 3.4 to F i g u r e 3.9A). A dense perinuclear l a b e l l i n g w a s f o u n d for m o s t transporters w i t h the exception of G L U T - 4 a n d G L U T - 5 (Figure 3.7B&C and Figure 3.8B&C). Figure 3.4B&C, Figure 3.5B&C, Figure 3.6B&C, and F i g u r e 3 . 9 B & C s h o w , f r o m a t h i n section taken f r o m the m i d d l e of the cell, the l a b e l l i n g s u r r o u n d i n g the nucleus, a n d the lack of l a b e l l i n g i n s i d e the n u c l e u s of the same section. G L U T - 4 w a s the o n l y i s o f o r m w i t h a h i g h prevalence of l a b e l l i n g located i n the nuclear area (Figure 3.7B & C ) . In the case of G L U T - 1 , 2, 3 and S G L T - 1 , labelling was observed  i n the cytosol (Figure 3 . 4 D & E ,  144  Figure  3 . 5 D & E , F i g u r e 3 . 6 D & E a n d F i g u r e 3.9D & E). G L U T - 4 a n d 5 w e r e not f o u n d i n the cytosol of H C A E C s (Figure 3 . 7 D & E a n d F i g u r e 3.8D & E).  lit  Figure 3.5  GLUT-2 in H C A E C Deconvolved images of H C A E C labelled w i t h antibodies specific for G L U T - 2 (green), VE-cadherin (red) and nuclei with D A P I (blue). A ) En face view of the full 3D reconstruction of the cell, 8 u m deep. The arrows point to discontinuous contacts between adjacent cells. B & C) Central planes (1 u m deep) obtained from A . B) G L U T - 2 and D A P I labelling, C) only G L U T - 2 labelling. D & E) Side view obtained from a 90° rotation about the X-axis of the image i n A , 4 um deep cross-sections, coverslip at the bottom of the images. D) G L U T - 2 , VE-cadherin and D A P I labelling, E) only G L U T - 2 labelling. Scale bar is 8 um.  145  B  A  c ;.,>.;-:•  \  -  •* •  * »' i  E  D  Figure 3.6  i  ' Ii  I M F * ! 7 "'if f * i* MI i  G L U T - 3 in H C A E C  Deconvolved images of H C A E C labelled with antibodies specific for G L U T - 3 (green), VE-cadherin (red) and nuclei w i t h D A P I (blue). A ) En face view of the full 3D reconstruction of the cell, 8.5 u m deep. B & C) Central planes (1 u m deep) obtained from A . B) G L U T - 3 and D A P I labelling, C) only G L U T - 3 labelling. D & E) Side view obtained from a 90° rotation about the X-axis of the image in A , 4 u m deep cross-sections, coverslip at the bottom of the images. D) G L U T - 3 , VE-cadherin and D A P I labelling, E) only G L U T - 3 labelling. Scale bar is 8 um.  F i g u r e 3.10 s h o w s secondary a n t i b o d y l a b e l l i n g i n H C A E C s . The d o n k e y anti-rabbit F I T C (Figure 3.10A), anti-mouse F I T C (Figure 3.10B), anti-goat F I T C (Figure 3.10C) a n d anti-goat Texas r e d (Figure 3.10D) failed to label the H C A E C s o n their o w n . N o i m m u n o s t a i n i n g w a s o b s e r v e d w h e n p r i m a r y antibodies w e r e a p p l i e d i n c o m b i n a t i o n w i t h a secondary  a n t i b o d y targeting a n  irrelevant  species. F i g u r e 3.11 s h o w s H C A E C s l a b e l l e d w i t h a single p r i m a r y a n t i b o d y a n d t w o secondary antibodies. T h e relevant secondary a n t i b o d y p r o d u c e d l a b e l l i n g i n H C A E C s (Figure 3 . 1 1 C & F for Texas R e d a n d G , J, M , P, a n d S for F I T C ) . The irrelevant secondary antibodies, i n each case, d i d not p r o d u c e a n y l a b e l l i n g i n H C A E C s (Figure 3 . 1 1 A & D for F I T C a n d I, L , O , R, a n d U for Texas R e d ) . T h e  146  m i d d l e c o l u m n i n F i g u r e 3.11B, E , H , K , N , Q , a n d T, s h o w s , as a l a n d m a r k , the n u c l e u s of each cell l a b e l l e d w i t h D A P I .  A  E  D  Figure 3.7  C  B  GLUT-4 in H C A E C  Deconvolved images of H C A E C labelled w i t h antibodies specific for G L U T - 4 (green), VE-cadherin (red) and nuclei w i t h D A P I (blue). A ) En face view of the full 3D reconstruction of the cell, 9 u m deep. B & C) Central planes (1.25 um deep) obtained from A . B) G L U T - 4 and D A P I labelling, C) only G L U T - 4 labelling. D & E) Side view obtained from a 90° rotation about the X-axis of the image in A , 4 u m deep crosssections, coverslip at the bottom of the images. D) G L U T - 4 , VE-cadherin and D A P I labelling, E) only G L U T - 4 labelling. Scale bar is 8 um.  147  Figure 3.8  G L U T - 5 in H C A E C  Deconvolved images of H C A E C labelled w i t h antibodies specific for G L U T - 5 (green), VE-cadherin (red) and nuclei w i t h D A P I (blue). A ) En face view of the full 3D reconstruction of the cell, 8.5 u m deep. The upper arrow points to discontinuous contacts between adjacent cells, the lower arrow points to an area without adjacent cell and without VE-cadherin. B & C) Central planes (1 u m thick) obtained from A . B) G L U T - 5 and D A P I labelling, C) only G L U T - 5 labelling. D & E) Side view obtained from a 90° rotation about the X-axis of the image i n A , 4 u m deep cross-sections, coverslip at the bottom of the images. D) G L U T - 5 , VE-cadherin and D A P I labelling, E) only G L U T - 5 labelling. Scale bar is 8 um.  148  Figure 3.9  S G L T - I in H C A E C Deconvolved images of H C A E C labelled w i t h antibodies specific for SGLT-1 (green), VE-cadherin (red) and nuclei w i t h D A P I (blue). A ) En face view of the full 3D reconstruction of the cell, 6.5 u m deep. The arrows point to discontinuous contacts between adjacent cells. B & C) Central planes (4.8 um deep) obtained from A . B) SGLT-1 and D A P I labelling, C) only SGLT-1 labelling. D & E) Side view obtained from a 90° rotation about the X-axis of the image i n A , 4 um deep cross-sections, coverslip at the bottom of the images. D) SGLT-1, VE-cadherin and D A P I labelling, E) only SGLT-1 labelling. Scale bar is 8 um.  149  Figure 3.10 Secondary antibody controls in  HCAECs.  Images are 3D reconstruction of H C A E C s , en face view of the cells. H C A E C s are labelled w i t h D A P I (blue) and a single secondary antibody (FITC: green or Texas Red: red); A ) donkey anti-rabbit FITC, B) donkey anti-mouse FITC, C) donkey anti-goat FITC and D) donkey anti-goat Texas Red. Images are 5.5 u m deep; scale bar is 8 um.  150  A  B  C  D  E  F  G  H  1  J  K  L  M  N  0  P  Q  R  S  T  U  •  Figure 3.11  Secondary antibody cross-reactivity controls in H C A E C s .  Images are 3D reconstruction of H C A E C s , en face view of the cells. H C A E C s are labelled w i t h a single primary antibody: A - F ) anti-VE-cadherin, G-I) rabbit anti-GLUT-1, J-L) rabbit anti-GLUT-2, M - O ) mouse anti-GLUT-4, P-R) rabbit anti-GLUT-5, S-U) rabbit antiSGLT, and dual secondary anti-body : A - C , G - L &P-U) donkey anti-rabbit FITC (green, 1 column) and donkey anti-goat Texas Red (red, 3 column), D-F & M - O ) donkey antimouse FITC (green, 1 column) and donkey anti-goat Texas Red (red, 3 column). D A P I (blue) is shown i n 2 column. Images are 5.5 u m deep; scale bar is 8 um. st  rd  st  rd  nd  151  3.3.2 2 - N B D G uptake in H C A E C s  The  immunocytochemistry  identified  six  different  transporters  in  H C A E C s . The next step was to test whether these transporters were functional. To proceed, a concentration and time point from the linear portion of the initial rate of uptake of 2-NBDG was determined. Then, we examined the regulation of 2-NBDG uptake in the presence of compounds known to enhance or inhibit specific G L U T and S G L T isoforms.  First, it was determined that the fluorescent images should be acquired following a 5-min wash, during which non-incorporated 2-NBDG was removed. The possibility of imaging the cytosol of H C A E C s in the presence of extracellular 2-NBDG was rejected due to insufficient axial resolution. The image depth was -17 um while the thinnest cytosolic section of H C A E C s was ~2 um, therefore the image was dominated by extracellular fluorescence.  It is impossible to predict, a priori, how much 2-NBDG to use or for how long the cells should be incubated with the compound before recording. The initial  series of experiments  were therefore  designed  to determine  these  parameters. We tested concentrations of 2-NBDG ranging from 0.1 m M to 3 m M , adding enough L-glucose to bring the total glucose concentration to the normal physiological value of 5 m M . The uptake of 1 m M 2-NBDG was linear (r =0.99) 2  over 30 min (Figure 3.12A), while at 0.1 m M the uptake was linear (r =0.996) over 2  60 min (Figure 3.12D). A t higher concentrations of 2-NBDG (3 mM), the uptake remained linear over time (r =0.86) but increased in variability (Figure 3.12B). 2  The plot of the rate of uptake of 2-NBDG at concentrations of 0.1, 0.5, 1, 2, and 3  152  m M 2-NBDG  (Figure 3.12C) was better described by a single exponential  (r =0.95) then a linear regression (r =0.93). Nevertheless, for concentrations below 2  2  1 m M , the rate of 2-NBDG uptake was more accurately linear (r = 0.98) to the 2  concentration (see inset in Figure 3.12C). The rate of uptake of 2-NBDG and 6NBDG  were  also  compared  in  HCAECs  to  determine  the  effect  of  phosphorylation on the rate of uptake (Figure 3.12D). The rate of uptake of the 6N B D G was 35% higher than the rate of uptake of the 2-NBDG. Significant differences in the average fluorescence intensity accumulated over time in H C A E C s were found at 20 min of exposure to the 2-NBDG and 6-NBDG, and beyond. From these observations, it was decided that subsequent experiments would be performed at concentration of 1 m M 2-NBDG and for an incubation time of 20 min. This concentration (1 mM) was selected because of the greater precision of the linearity over time of the fluorescence recorded. The time point (20 min) was chosen because it was clearly on the linear portion of the curve and was not approaching saturation of the transporters and/or that of hexokinase.  153  B  A  Figure 3.12 Rate of fluorescent glucose uptake in HCAECs A) Plot of 1 m M 2-NBDG uptake over time (r =0.99), average of 12 cells from 3 different experiments B) Plot of 3 m M 2-NBDG uptake over time (r =0.86), average of 18 cells from 5 different experiments. C) Plot of the rate of uptake versus the concentration of 2NBDG, single exponential (r =0.95), each rate is the average of 10 cells from 1 to 5 different experiments. The inset shows a linear relation between the rate of uptake of 2NBDG and concentration below 1 m M (r = 0.98). D) Plot of 0.1 m M 2-NBDG (open circle) and 6-NBDG (closed circle) uptake over time (r =0.99 and 0.97 respectively), each plot is the average of 15 and 6 cells from 3 and 2 different experiments respectively. 2  2  2  2  2  To demonstrate that the uptake of 2 - N B D G by H C A E C s occurs through G L U T s and/or S G L T , we assessed the competitive inhibitory effect of D-glucose and cytochalasin B on the rate of uptake of 2-NBDG. Concentrations of 4 m M and 9 m M of D-glucose reduced significantly the accumulation of fluorescence  154  intensity in H C A E C s by 55% (p<0.05) and 76% (pO.OOl) respectively (Figure 3.13). These were compared to values obtained in solution where the same concentration of D-glucose was replaced by L-glucose.  15-,  Figure 3.13 D-glucose  inhibition of 2 - N B D G uptake i n H C A E C s . Effect of 4 mM l l and 9 m M L-glucose and 4 mM l l and 9 mM • • Dglucose on the rate of uptake of 1 m M 2-NBDG over a period of 20 min. Each bar is the average of 9 cells from 2 different experiments. Significant differences are indicated; * p<0.05 (4 mM) and ** pO.OOl (9 mM).  Cytochalasin B, a commonly used inhibitor of glucose transport by G L U T s , also inhibited the rate of 2-NBDG uptake in H C A E C s . The fluorescence intensity accumulated in H C A E C s over 15 min of exposure to 1 m M 2-NBDG was significantly reduced by 35% (p<0.05) in the presence of 10 u M cytochalasin B (Figure 3.14). These experiments were done at 15 min incubation with 2 - N B D G rather than 20 min because of the effects of cytochalasin B on the cell cytoskeleton, changing their shape and the consequent fluorescence intensity, independently of the accumulation of 2-NBDG in the cell.  155  8 c  15-,  © -r  S 3  a m 10-  Figure 3.14  Effect of cytochalasin B on 2-NBDG uptake i n HCAECs.  Uptake of 2-NBDG (1 mM) over 15 min, in the absence I  I or presence H B of 10 u M  cytochalasin B. Each bar is the average of 8 and 7 cells respectively from 2 different experiments; (* PO.05).  The effect of insulin on the rate of uptake of 2-NBDG was tested in H C A E C s to determine the functionality of G L U T - 4 . A concentration of 2.5 u U / m l of  insulin significantly increased, by 64% (p<0.0001), the accumulation of  fluorescence intensity in H C A E C s incubated for 20 min in a Krebs solution containing 4 m M L-glucose (Figure 3.15A). Although the same concentration of insulin only produced a 31% (p<0.05) increase in accumulated  fluorescence  intensity in H C A E C s incubated for 30 min in a Krebs solution containing 4 m M D-glucose (Figure 3.15B). In addition, we observed no effect of insulin on the average fluorescence intensity accumulated in H C A E C s exposed to a lower ratio of 2 - N B D G and D-glucose (0.1 m M 2 - N B D G in presence of 5 m M D-glucose, see Appendix 4).  156  A  B  Figure 3.15  Effect of insulin on 2-NBDG uptake i n HCAECs  Uptake of 1 m M 2-NBDG in the absence I  I or presence  I  I of 2.5 uU/ml of insulin.  A) over 20 min in the presence of 4 m M L-glucose, each bar is the average of 18 and 33 cells from 2 different experiments respectively, (** P< 0.0001). B) over 30 min in the presence of 4 m M D-glucose, each bar is the average of 16 and 15 cells from 2 and 3 different experiments respectively; (* PO.05).  3.3.3 2 - N B D G uptake in native endothelium of intact coronary artery.  2 - N B D G uptake was then measured in the endothelium of native coronary arteries. Figure 3.16 shows a schematic representation of a vessel from which phase contrast and confocal fluorescence images were acquired at different focal planes through the vessel wall. Phase contrast was used to image a small section of the vessel wall touching the coverslip of the arteriograph chamber (Figure 3.16B).  Individual ECs were  identified  through phase  contrast  by  their  characteristic longitudinal fusiform shape oriented parallel to the blood flow (Figure 3.16C). Phase contrast was also used to locate the axial position of ECs and S M C s in the vascular wall (Figure 3.16 D to G left). The lumen of this vessel was perfused with 1 m M 2-NBDG for 40 min, followed by a 10-min wash with Krebs solution containing 5 m M D-glucose. The fluorescence emitted by trapped  157  intracellular  2-NBDG  and  the  phase  contrast  images  were  acquired  simultaneously. While no fluorescence could be detected from both the lumen (Figure 3.16D right) and the coverslip (Figure 3.16G right), a strong fluorescence at the periphery of each E C was observed (Figure 3.16E right). From the S M C layer, strong fluorescence in long strips, oriented perpendicularly to the blood flow were also observed (Figure 3.16F, right). A s shown by the fluorescence acquired from the E C and S M C layers, 16 um apart, it is possible to distinguish the fluorescence accumulated from one layer of cells to the other with the 20X objective, which has an axial resolution of 9.3 u m and an image depth of 16.9 um.  From the above experiment, it was shown that emitted fluorescence could be measured in ECs, without interference from light emitted by the S M C s in the vascular wall of a coronary artery. Before proceeding with further experiments on the regulation of glucose uptake in the native endothelium we examined the rate of uptake of 2 - N B D G in ECs of the vascular wall. The plot of the uptake of 1 m M of 2 - N B D G over 60 min (Figure 3.17) was better described by a single exponential (r =0.98) then a linear regression (r =0.94). Nevertheless, for the first 2  2  20 min, the rate of uptake was more accurately linear over time (see inset from Figure 3.17, r =0.99). Time points beyond 20 min also revealed an increased in 2  variability of the fluorescence intensity as shown by the error bars on Figure 3.17. The effects of D-glucose, cytochalasin B and insulin on 2 - N B D G uptake were therefore tested on periods of 20 min of exposure to concentration of 1 m M of 2N B D G . This time point is in the linear portion of the curve from which one can effectively  approximate the tangent  to the curve and sensitively  variations in the initial rate of uptake.  158  measure  Figure 3.16 Phase contrast and fluorescence i n whole vessel  preparation. Representative coronary artery mounted on glass cannulae i n an arteriograph chamber. A) Schematic representation of the vessel i n relation to the coverslip and the 20X objective. B) Phase contrast image of the vessel attached to the cannulae; scale bar is 100 um. C) Higher magnification of the inset from B; scale bar is 25 um. D to G) simultaneous recording of phase contrast (left) and fluorescence (right) after 40 rnin incubation with 1 m M 2 - N B D G and 10-min wash; scale bar is 25 um. D) lumen imaged at 52 u m above the cover slip. E) ECs imaged at 32 u m above the coverslip. F) S M C s imaged at 16 u m above the coverslip. G) Image at the coverslip. In red, E C (E) and S M C (F) boundaries.  159  ? 80  (0  w 60 O 8  4  0  c  <D  O  (0 <D  i_  20 ]  O 10  > (0 <  15  20  25  Time (min)  20  60  40  Time (min)  Figure 3.17 Rate of 2-NBDG uptake in ECs of intact coronary artery. Representative rate of 2-NBDG uptake from a rat coronary artery. The vessel was perfused with a solution containing 1 m M 2-NBDG and 4 m M of L-glucose for cumulative periods of 5, 10 and 15 min, each followed by 10-min wash and image acqiusition. The plot is the average of 5 cells, single exponential (r = 0.98). The inset shows a linear uptake over time points below 20 min (r =0.99). 2  2  To demonstrate that the uptake of 2 - N B D G occurred also through G L U T s and/or SGLTs transporter proteins in E C s of an intact arteries, we repeated the competitive  inhibitory assay performed in H C A E C s  with D-glucose  and  cytochalasin B, this time in the cannulated vessel, expecting to find similar results. The average fluorescence intensity accumulated in the endothelium of intact coronary artery over 20 min was reduced significantly by 41% (p<0.0001) in vessels perfused with a solution containing 4 m M D-glucose in comparison to a vessel perfused with a solution containing L-glucose.  160  100-,  Figure 3.18 Effect of D-glucose  on 2 - N B D G uptake i n native ECs.  Uptake of 1 m M 2-NBDG in ECs of intact coronary artery in presence of 4 m M L-glucose i  i or D-glucose W W . Each bar is the average of 37 and 24 cells respectively,  from 3 different experiments; * pO.0001.  Similarly, the addition of 10 u M of cytochalasin B reduced significantly by 27% (pO.0001) the fluorescence intensity recorded in ECs of intact arteries, accumulated over 20 min of perfusion with a solution containing 1 m M of 2N B D G and 4 m M of L-glucose (Figure 3.19).  Lastly, we tested the effect of insulin in the endothelium of intact coronary artery to determine the functionality of G L U T - 4 previously identified in these cells. The addition of 2.5 u U / m l of insulin increased significantly by 23% (p<0.05) the fluorescence intensity of the ECs of intact arteries perfused with a solution of 1 m M 2 - N B D G and 4 m M L-glucose for 20 min (Figure 3.20).  161  Figure 3.19 Effect of cytochalasin B on 2 - N B D G uptake i n native  ECs.  Uptake of 1 m M 2-NBDG in a Krebs solution containing 4 mM L-glucose in ECs of intact coronary artery in the absence I  l or in the presence of 10 u M cytochalasin B w  I.  Each bar is the average of 31 and 20 cells respectively, from 3 different experiments; * pO.0001.  Figure 3.20 Effect of insulin on 2 - N B D G uptake i n native  ECs. Uptake of 1 m M 2-NBDG in a Krebs solution containing 4 mM L-glucose in ECs of intact coronary artery in the absence I 1 or in the presence of 2.5 uU/ml of insulin H i - Each bar is the average of 18 and 19 cells respectively, from 2 different experiments; * p<0.05.  162  3.4 Discussion  This study examined the regulation of glucose transport in both cultured and native ECs. We describe for the first time the measurement of glucose uptake in individual cells of an intact microvessel. We first showed the presence and symmetrical distribution of G L U T - 1 , 2, 3, 4, 5, and SGLT-1 in H C A E C s . Secondly, we used the fluorescent glucose analog 2 - N B D G to quantify glucose uptake in ECs. While the different variables and technical difficulties were more easily identified in experimentation with cultured cells, measurements in individual cells of an intact vascular wall were also demonstrated. Results from experiments with H C A E C s and an intact vessel indicate that coronary artery ECs are insulinsensitive.  3.4.1 G L U T s and SGLT-1 identification in H C A E C s  We used H C A E C s at the 4  th  passage for immunocytochemistry. Previous  studies have shown that ECs tend to reduce the expression of G L U T isoforms, other than G L U T - 1 , when cultured beyond the 4  th  passage (McCall et al, 1997;  Mamchaoui et al, 2002). The H C A E C s were fixed when they reached 80% confluence as this type of cell can become irreversibly contact-inhibited and detached from the coverslip if allowed to reach confluence. In this regard, V E cadherin labelled only neighbouring cell-junctions (Figure 3.3). Most cells showed  discontinuous attachments  to their neighbours and therefore V E -  cadherin labelling also appeared discontinuous (Figure 3.4A, Figure 3.5A, Figure 3.8A  and Figure 3.9A). This is in agreement with previous reports that V E -  cadherin  is  expressed  only  at  the  intercellular boundaries  monolayers of ECs (Dejana and Plantier, 1996).  163  of  confluent  We have previously identified G L U T - 1 , 2, 3, 4, 5, and SGLT-1 in rat coronary artery ECs (see chapter 2). In the present study, we extend these results to the H C A E C s . We used the same antibodies (Table 2.2) for which the specificity had been previously well characterised (see chapter 2). A s expected, we found a symmetric distribution of each transporter isoform examined. A s the ECs in culture tend to lose their characteristics, it is not surprising that the asymmetrical distributions, found previously in rat native coronary endothelium, was missing in H C A E C s . It is possible that in a native environment, the blood flow on one side and the interaction of the S M C s on the other side produces an asymmetric distribution. Accordingly, laminar flow has been previously shown to induce polarity of secretion in cultured bovine aortic ECs (Grimm et al,  1988). In  addition, the lack of neighbouring cells with which to form tight junctions may represent a key missing element for the development of polarity. Thus, others have shown that a disturbance of the BBB tight junctions was associated with a reduced polarity of G L U T - 1 distribution in a model of stroke-prone spontaneous hypertensive rat (Lippoldt et al, 2000).  In rat coronary artery ECs we have found a distinct tendency of the transporters to localise at the cell-to-cell junctions. This subcellular organisation was not observed in H C A E C s . Transporters did not concentrate at the boundary of either confluent or non-confluent cells, and this may also be linked to the loss of polarity of the H C A E C s in culture.  Each transporter isoform was located all around the periphery of the ECs, presumably on the cell membrane and in the cytosol of the cell, potentially in vesicles, being recycled, or in the process of translocation to or from the plasma  164  membrane. In certain cases, such as shown with G L U T - 2 (Figure 3.5D) and G L U T - 4 (Figure 3.7D), the transporters were located in the nuclear area. A s this labelling tends to coincide with deep holes in the nucleus, we consider these transporters to be located between the deep folds of the nuclear envelope and the plasmalemma (Fricker et al,  1997). This morphology is typical for an EC's  nucleus (Woolf, 1982).  3.4.2 2 - N B D G uptake in H C A E C s  In this study, concentrations of 2-NBDG ranging from 0.1 to 3 m M were tested in H C A E C s . The uptake was linear for a minimum of 30 min, regardless of the 2 - N B D G concentration. The rate of uptake also increased in a concentration dependent manner (Figure 3.12). This indicated that no saturation of 2-NBDG transport was reached during this time and within this range of concentrations.  The rate of uptake of the 6-NBDG was 35% higher than the rate of uptake of 2-NBDG. Significant differences in the accumulation of 2-NBDG and 6-NBDG occurred after 20 min of exposure to each of the fluorescent glucose analogs. A s the 6 - N B D G is not phosphorylated, it can be transported out of the cell. Conversely, 2 - N B D G is phosphorylated by hexokinase, and is trapped in the cell. Therefore, a faster accumulation of 2-NBDG was expected.  One explanation for this discrepancy could be that phosphorylation, in H C A E C s inhibits glucose uptake. This has been previously observed in ECs exposed to elevated glucose concentrations (Vinals et al, 1999). It is also possible that the transporters have different affinities for 2-NBDG and 6 - N B D G . Loaiza et  165  al. have measured the rate of uptake of both 2-NBDG and 6 - N B D G in cultures of astrocytes in a near zero-trans entry condition (Loaiza et al, 2003); meaning that the initial intracellular glucose concentration was minimal and therefore what was measured from this rate of uptake is the sugar binding affinity with negligible effects of the phosphorylation step (Gould and Seatter, 1997). The rate of uptake of 2 - N B D G was shown to be about 60% slower than that of 6-NBDG in these astrocytes. In the present study, lower concentrations than that used in astrocytes, for both 2-NBDG and 6-NBDG were used (O.lmM vs 0.3 m M ) . Thus, the difference in the rate of uptake between 2-NBDG and 6 - N B D G in H C A E C s is more likely attributable to a difference in affinity for the binding site of the glucose transporters between the two glucose analogs than an inhibitory effect on the part of the hexokinase. One last possibility, as reported previously in E . Coli (Yoshioka et al, 1996b), is that following its phosphorylation, 2-NBDG is further metabolised into a non-fluorescent  compound. This transformation  however was not reported in studies in mammalian cells (Lloyd et al, 1999; Yamada et al, 2000; Roman et al, 2001). From these observations, a concentration of 1 m M 2-NBDG and a maximal time of incubation of 20 min, were selected for further experiments.  3.4.3 Inhibitory effect of D-glucose and cytochalasin B in H C A E C s  The uptake of 2 - N B D G was significantly inhibited by both D-glucose and cytochalasin B in H C A E C s . This confirmed that the 2-NBDG uptake in ECs occurs through a system of glucose transporters in agreement with previous reports from other cell types (Yoshioka et al, 1996a; Lloyd et al, 1999; Yamada et al, 2000). L-glucose, the stereoisomer of D-glucose is not transported in the cell  166  and was therefore used in control assays to maintain an equivalent osmotic pressure. Although no statistical differences  were found between the 55%  inhibition with 4 m M and 75% inhibition with 9 m M , the trend implied a concentration dependent inhibition by D-glucose on the uptake rate of 2-NBDG, as previously described by others (Yoshioka et al, 1996a).  The inhibitory effect of cytochalasin B, although well documented in other glucose transport assays, was very difficult to demonstrate with a fluorescent glucose analog and confocal microscopy. Cytochalasin B depolymerises F actin and consequently changes the cell shape (Iglic et al, 2001; Pedersen et al, 2001). This cell shape change can increase or decrease the fluorescence  intensity  independently of the accumulated 2 - N B D G . The period of incubation with Cytochalasin B was therefore reduced to 15 min. Cells whose shapes were significantly affected by cytochalasin B treatment even with this shorter period of incubation were not imaged and/or considered for analysis.  3.4.4 Effect of insulin in H C A E C s  We observed a significant increase of 64% in 2 - N B D G uptake in the presence  of 2.5  uU/ml  of insulin in H C A E C s .  These results  reflect  the  functionality of G L U T - 4 in H C A E C s . Other studies have also reported insulin sensitivity of the vascular endothelium (Gerritsen and Burke, 1985; Vinters et al, 1985; Allen and Gerritsen, 1986; Kwok et al, 1989; A b e et al, 1990) and the presence of G L U T - 4 in ECs of various microvascular beds (Frank and Pardridge, 1981; King et al, 1983; Vilaro et al, 1989; McCall et al, 1997), still, this is not generally accepted, and remains highly controversial.  167  Other studies have reported in other vascular beds, mostly of greater calibre but also in BBB, that ECs are not sensitive to insulin (Corkey et al, 1981; Betz et al, 1983; Drewes et al, 1988; Takakura et al, 1991; Thomas et al, 1995). The present results may therefore not be extended to all vascular endothelia. O n the other hand as we report insulin sensitivity in both human and rat coronary artery and since others have previously described similar responses in rabbit coronary artery ECs (Gerritsen and Burke, 1985; Gerritsen et al, 1988), it is likely that ECs from coronary arteries of most other species are responsive to insulin and express G L U T - 4 .  In the present study, 2.5 u U / m l insulin produced a greater increase in 2NBDG  uptake in H C A E C s pre-incubated and exposed to a Krebs solution  containing L-glucose than in H C A E C s pre-incubated and exposed to a Krebs solution containing D-glucose (Figure 3.15). This is in agreement with previous observations, by Gerritson at al., that glucose deprivation enhances the effect of insulin on 2 D G uptake in coronary microvessel ECs (Gerritsen et al, 1988). Although, this previous study found that glucose deprivation was necessary to obtain a significant effect of insulin, we found a lower but still significant effect of insulin in H C A E C s pre-incubated in D-glucose. This discrepancy may be explained by the longer exposure to both insulin and 2 - N B D G (30 min) in the present study compared to the previous one (6 min uptake of radiolabeled 2DG). Since coronary ECs accumulate unphosphorylated (between 30-60%) 2DG, it has been suggested that the limiting step for glucose uptake in ECs is not glucose transport but glucose phosphorylation (Betz et al, 1983; Gerritsen et al, 1988). This may potentially  explain the increased  sensitivity  of insulin in ECs  previously deprived of glucose. ECs pre-incubated with D-glucose, especially in  168  the presence of insulin, are likely to experience a lower phosphorylation rate for any subsequent transported labelled 2 D G , due to saturation of hexokinase. The consequent  increased in intracellular unphosphorylated labelled 2 D G may  increase its transport out of the cell or inhibit its entry. O n the other hand, in cells deprived of glucose, the labelled 2 D G is not facing competition for hexokinase, and a faster rate of phosphorylation increases its accumulation. Taken together, this may explain in part, the lack of insulin effect observed by other studies performed in ECs of larger vessels and certain microvessels.  3.4.5 2-NBDG uptake in native E C  The recording of 2 - N B D G uptake in ECs of an intact, perfused, vessel was demonstrated in this study. A s a distance of approximately 16 u m separates the ECs from the S M C s in the wall of a rat coronary artery, the  respective  intracellular fluorescence was easily distinguished and separated from one another with the 20X objective.  The subcellular localisation of 2-NBDG in an intact coronary artery was found most prominently at the periphery of the ECs. The cytosol of native ECs is mainly occupied by the nucleus, this may explain in part the peripheral accumulation of 2-NBDG. In addition, the subcellular accumulation of 2-NBDG co-localizes with the prevalent distribution of G L U T isoforms at the cell-to-cell junction of ECs (see Chapter 2). Such compartmentalised organisation may serve a purpose. ECs accumulate free (non-phosphorylated) 2 D G even in the presence of physiological concentrations of glucose (Betz et al, 1983; Gerritsen et al, 1988), non-phosphorylated glucose can be transported out of the cells. Taken together,  169  the accumulation of 2-NBDG at the periphery of the cells where the cytosol is at its thinnest, with the high prevalence of glucose transporters in the same subcellular area and the possibility that this 2-NBDG is mostly free, suggests the involvement  of transcellular transport of glucose from the blood to  the  interstitium of the vascular wall. Further experiments are needed to test this possibility, but the implications of such a process may explain to some extent the high susceptibility of ECs to glucose toxicity.  Then again, in the ECs of the intact vessel, the uptake of 1 m M 2 - N B D G remained linear for a minimum of 20 min and began to level off after 30 min of exposure to the 2-NBDG, contrary to the linear uptake of 1 m M 2-NBDG observed in H C A E C s . This may, on the one hand, represent a saturation of the transporters. O n the other hand, looking back at the differences  in  the  distribution of the G L U T s and S G L T observed between H C A E C s and E C s of an intact coronary artery, this may be a reflection of transcellular transport of glucose. A s suggested by the asymmetric distribution of the transporters, which are higher in numbers on the abluminal side in native ECs (see chapter 2), an accumulation of non-phosphorylated 2-NBDG in a narrow cytosolic space is likely to produce a driving force for its extrusion on the abluminal side of the ECs.  3.4.6 Inhibitory effect of D-glucose and cytochalasin B in native E C  The uptake of 2-NBDG was also significantly inhibited by both D-glucose and cytochalasin B in native ECs. This confirmed that the 2-NBDG uptake in ECs of an intact vascular wall also occurs through a system of glucose transporters in  170  agreement with previous observations in cultured H C A E C s . In comparison to the effect produced in the H C A E C s , cytochalasin B did not change noticeably the shape of the native ECs. The environment of the intact vessel could have made such change in EC's shape difficult to observe. It is also possible that the surrounding  environment has  compensated  for this  effect  and provided  scaffolding for the ECs deprived of an intact cytoskeleton.  3.4.7 Effect of insulin in native E C  A n increase of 23% in 2 - N B D G uptake was observed in the presence of 2.5 uTJ/ml of insulin in E C s of an intact vessel. These results confirm previous observations in H C A E C s and reflect the functionality of G L U T - 4 also identified in ECs of intact rat coronary artery (see Chapter 2).  3.4.8 Difference in responses between H C A E C s and native ECs  The  same response  to D-glucose, cytochalasin B and insulin were  observed in both H C A E C s and native ECs, although in average, a 10% lower inhibitory response and a 40 % lower stimulating response were observed in native ECs. These lower responses in native ECs, may be related to a reduced sensitivity and/or increased signal to noise ratio of the system. This may be accounted for by the increased light diffraction from the other cell layers and components of the vascular wall. It may also be due to the further distance from the coverslip at which the ECs of the vascular wall were imaged in comparison to that of the H C A E C s directly apposed to the coverslip. This lower effect may also be attributed to distinctions between differences between rat and human tissue.  171  culture and native cells and/or to  O n the other hand, the 40% lower uptake rate stimulated by insulin in native ECs compared to cultured H C A E C s may be explained by an increased transcellular transport of glucose in presence of insulin. In accordance, we have found previously a higher distribution of G L U T - 4 on the abluminal side of native ECs. A s glucose is taken up from the blood on the luminal side, a translocation of GLUT-4  to the abluminal plasma membrane mediated by insulin would  stimulate mainly transport of glucose out of the cell in the interstitial space between ECs and SMCs. This glucose could then be used to feed the S M C s of the vascular wall.  Interestingly, G L U T - 4 was downregulated in native ECs submitted to chronic hyperglycaemia and hypoinsulinaemia (see Chapter 2). This downregulation of G L U T - 4 was also accompanied with a significant reduction in the ratio of abluminal to luminal G L U T - 4 . It is therefore possible that transcellular transport of glucose mediated by insulin is reduced in diabetes.  Since coronary vascular diseases are strongly associated with diabetes and ECs are highly susceptible to glucose toxicity, it is of great importance to consider the implications of insulin therapies on the accumulation of intracellular glucose in vascular ECs. Further studies in this regard will need to be pursued in order to determine the impact of insulin on the regulation of glucose uptake by the endothelium of diabetic patients, and determine if insulin treatment restores this potential transcellular transport of glucose or if it increases intracellular glucose uptake in native ECs.  172  Epilogue The clear association between glucose toxicity in E C s and an increased prevalence of cardiovascular diseases in the diabetic population has motivated this research, the question of interest being: how and why glucose accumulates in E C . The difficulty of harvesting and culturing ECs without losing most of their morphological and behavioural characteristics has been a major obstacle in endothehal research. Therefore, the ultimate goal of the present study was to use an experimental design and protocol that preserved the native environment of the ECs and their close structural relationship with the SMCs.  Initial research, on the myogenic tone of small contractile arteries conducted in Dr. Laher's laboratory, has greatly influenced the approach and means employed to measure, in real time, glucose uptake in individual ECs of an intact vascular wall. In addition, the development of a fluorescent glucose analog provided a good tool for studying glucose uptake in individual cells in their native environment. The specific aims and hypothesis of this study were also formulated with regards to previous findings from immunohistochemistry work, which identified six different sugar transporter isoforms in ECs of intact coronary arteries.  While measurements of 2-NBDG uptake were successfully achieved in both cultured and native ECs, due to time constraints and the difficult nature of these experiments, several mteresting questions and hypotheses have not yet been  addressed  or  tested.  In  addition,  173  unexpected  limitations  of  this  experimental approach were encountered and often made it difficult to quantify measurements of glucose uptake in live cells. The following is a discussion of unexplored avenues and hypotheses that could potentially be tested with the experimental setting presented in chapter 3.  One of the aims for developing this methodology was to measure 2-NBDG uptake in ECs and SMCs in the same vessel, at the same time. In the first attempt, a continuous perfusion of 2-NBDG and an image acquisition every minute from four different focal planes representing the lumen, ECs, SMCs, and the coverslip, was done. No washing steps were included prior to the image acquisition for the entire period (30 min) of perfusion with 2-NBDG. The fluorescence recorded from the EC and SMC layers increased over time, reached a plateau phase and decreased at a similar rate during the subsequent washing period. To our astonishment, the addition of D-glucose or cytochalasin B increased the fluorescence intensity of each layer. These observations made us suspect that a non-negligible amount of 2-NBDG was possibly in the interstitial space, surrounding the cells or trapped between the EC and SMC layers. Following this observation, a more cautious and systematic approach was used. A washing step was included after each period of incubation with the 2-NBDG prior to the image acquisition and we focused on the EC layer as a priority. Nevertheless, these observations possibly constitute evidence for the transcellular transport of glucose in ECs of small contractile arteries. A first piece of evidence is the report, in a previous study, that non-phosphorylated cytosolic 2DG is present in ECs at a physiological glucose concentration (Betz et al, 1983). This means that intracellular accumulated glucose can be transported out of the ECs. A n additional piece of evidence comes from our studies showing asymmetric  174  distribution of the G L U T s  and SGLT-1  in ECs of intact arteries  and a  concentration of the transporter at the edge of the cell-to-cell junction where the cell thickness is minimal. One way to examine this possibility would be to use simultaneously a fluorescent L-glucose analog (not-transported by the G L U T s and SGLTs) with the 2-NBDG to measure and compare how much glucose penetrates the  vascular wall  through paracellular transport and through  transcellular transport. Talks with The Molecular Probes Co. about the possibility of synthesising such a compound were undertaken but had to be terminated, as the chemical required for the synthesis of a fluorescent analog of L-glucose, L glucosamine, was unavailable at that time.  One of the most important findings of this work is that coronary ECs are insulin-sensitive. For a long time, this possibility was ignored due to conflicting reports in various cultured cell lines and preparations. We provided evidence for the presence of G L U T - 4 in both ECs of intact rat coronary artery and in culture from human coronary artery. In addition, we showed that in both preparations, insulin stimulates the uptake of the fluorescent glucose analog 2-NBDG. Because of the importance of these results and the current controversial status of insulin sensitivity of the endothelium, additional experiments were initiated to further demonstrate that the increase in 2 - N B D G observed following insulin stimulation was indeed a reflection of G L U T - 4 translocation to the plasma membrane. Insulin increases glucose uptake through the translocation of G L U T - 4 via a signalling pathway kinase-1  (PDK-1)  mediated  by  PI3-kinase,  and protein kinase  3-phosphoinositide-dependent  B (PKB) (Khan and Pessin,  2002).  Wortmannin, a PI3-kinase inhibitor, has been previously shown to inhibit both G L U T - 4 translocation and glucose uptake (Yu et al, 1999). Wortmannin was  175  therefore used to test the participation of G L U T - 4 in the insulin response observed in ECs. Our results from these studies were inconclusive due to other unexpected effects of wortmannin on the cell shape. H C A E C s changed shape; they shrank and retracted from each other, when incubated with wortmannin. This change increased the axial thickness of the cell and therefore produced an increase in fluorescence intensity unrelated to glucose uptake. PI3-kinase has been shown to play a significant role in the regulation of cell volume, an effect shown to be inhibited by both wortmannin and an independent IP3-kinase inhibitor, LY294002 (Feranchak et al, 1998). Thus, in this experimental setting, where  a stable cell shape  is required for accurate quantification of  the  fluorescence, a compound like wortmannin cannot be used. A good alternative to wortmannin may potentially be indinavir, an H I V protease inhibitor. Indinavir has been shown to directly and specifically inhibit G L U T - 4 translocation without any effects on intracellular signalling (Murata et al., 2000).  G L U T - 2 was found to be expressed in both rat intact coronary artery endothelium and human coronary cultured ECs. Its functionality was tested in preliminary experiments with both an activator and an inhibitor of P K C , presented in Appendix 5. The inhibition of P K C , but not its activation, produced an effect on 2 - N B D G uptake in the presence of 20 m M D-glucose. A s Calpostin C inhibits all P K C isoforms, which could contribute to the inhibition of G L U T - 4 translocation, it would therefore be preferable to use a specific inhibitor of the PKCp  isoform, shown  previously to be  specific  to G L U T - 2  translocation  signalling (Helliwell et al., 2003). In addition, other activators of P K C such as phorbol ester could be used at basal glucose concentrations (Drewes et al, 1988).  176  Although G L U T - 5 was identified in both rat and human coronary ECs, the functionality of this transporter was not assessed. A t the time of the study, a fluorescent fructose analog was not available. The synthesis of such a compound would enable the observation and quantification of fructose uptake in individual ECs and S M C s of an intact vascular wall.  SGLT-1 was also identified in both rat and human ECs. Its functionality has been investigated at a basal glucose concentration with the specific inhibitor phloridzin in H C A E C s (Appendix 5). N o effects on the uptake of 2-NBDG were observed. A s previously reported, S G L T activity is enhanced by hypoglycaemic and hypoxic conditions. It would therefore be worthwhile to test the effect of phloridzin under these conditions in ECs. In addition, changes in ambient N a  +  concentration may potentially activate SGLT-1 and should therefore be tested in coronary ECs.  Finally, important changes in the expression of the glucose transporters of the rat coronary artery ECs were observed with long-term diabetes. It would be of great interest to determine the impact of these changes induced by diabetes, on ECs glucose uptake. The synthesis of a fluorescent L-glucose analog could also permit, if used simultaneously with the 2-NBDG, testing the possibility of an interstitial accumulation of 2-NBDG glucose due to a slower rate of uptake from the S M C s and/or to the increased E C s transcellular and/or paracellular transport of 2-NBDG in hyperglycaemic conditions. The role of G L U T - 2 upregulation in diabetes is also of particular interest and may potentially be addressed with the use of a PKCp-specific inhibitor in diabetic animals. A l l of these avenues  177  represent potential future directions to be pursued with the measurements of glucose uptake in a live vessel preparation.  178  References Abdel-Harriid, K. M. and Baimbridge, K. G.: The effects of artificial calcium buffers on calcium responses and glutamate-mediated excitotoxicity in cultured hippocampal neurons. Neuroscience, 81, (3): 673-87,1997. Abe, M., Ono, J., Sato, Y., Okeda, T. and Takaki, R.: Effects of glucose and insulin on cultured human microvascular endothelial cells. Diabetes Res Clin Pract, 9, (3): 287-95,1990.  Abordo, E. A., Westwood, M. E. and Thornalley, P. J.: Synthesis and secretion of macrophage colony stimulating factor by mature human monocytes and human monocytic THP-1 cells induced by human serum albumin derivatives modified with methylglyoxal and glucose-derived advanced glycation endproducts. Immunol Lett, 53, (1): 7-13,1996. Alemany, R., Kleuser, B., Ruwisch, L., Danneberg, K., Lass, H., Hashemi, R., Spiegel, S., Jakobs, K. H. and Meyer zu Heringdorf, D.: Depolarisation induces rapid and transient formation of intracellular sphingosine-l-phosphate. FEBS Lett, 509, (2): 239-44, 2001. Allen, B. G. and Walsh, M. P.: The biochemical basis of the regulation of smoothmuscle contraction. Trends Biochem Sci, 19, (9): 362-8,1994. Allen, L. A. and Gerritsen, M. E.: Regulation of hexose transport in cultured bovine retinal microvessel endothelium by insulin. Exp Eye Res, 43, (4): 679-86, 1986. Allen, T. J. and Hardin, C. D.: Influence of glycogen storage on vascular smooth muscle metabolism. Am J Physiol Heart Circ Physiol, 278, (6): H1993-2002, 2000. Alpert, E., Gruzman, A., Totary, H., Kaiser, N., Reich, R. and Sasson, S.: A natural protective mechanism against hyperglycaemia in vascular endothelial and smooth-muscle cells: role of glucose and 12-hydroxyeicosatetraenoic acid. Biochem J, 362, (Pt 2): 413-22,2002.  Andrea, J. E. and Walsh, M. P.: Protein kinase C of smooth muscle. Hypertension, 20, (5): 585-95,1992.  179  Anonymous: The relationship of glycemic exposure ( H b A l c ) to the risk of development  and progression  of retinopathy in the  complications trial. Diabetes, 44, (8):  diabetes control and  968-83,1995.  Anonymous: Intensive blood-glucose  control with sulphonylureas or insulin  compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). U K Prospective Diabetes Study (UKPDS) Group [published  erratum  appears  in  comments]. Lancet, 352, (9131):  Lancet  1999  Aug  14;354(9178):602]  [see  837-53,1998.  Anonymous: Diabetes mellitus: a major risk factor for cardiovascular disease. A joint editorial statement by the American Diabetes Association; The National Heart,  Lung,  and  Blood  Institute;  The  Juvenile  Diabetes  Foundation  International; The National Institute of Diabetes and Digestive  and Kidney  Diseases; and The American Heart Association. Circulation, 100, (10):  1132-3,  1999. Appleman, J. R. and Lienhard, G . E . : Kinetics of the purified glucose transporter. Direct measurement of the rates of interconversion of transporter conformers. Biochemistry, 28, (20): 8221-7,1989. Araki, S., Ito, M . , Kureishi, Y., Feng, J., Machida, H . , Isaka, N . , Amano, M . , Kaibuchi, K., Hartshorne, D . J. and Nakano, T.: Arachidonic acid-induced C a  2 +  sensitization of smooth muscle contraction through activation of Rho-kinase. PfTugers Arch, 441, (5): 596-603,  2001.  Aronson, D . and Rayfield, E . J.: H o w hyperglycemia promotes atherosclerosis: molecular mechanisms. Cardiovasc Diabetol, 1, (1): 1., 2002. Ar'Rajab, A . and Ahren, B.: Long-term diabetogenic effect of streptozotocin in rats. Pancreas, 8, (1): 50-7,1993. Asada, T., Takakura, S., Ogawa, T., Iwai, M . and Kobayashi, M . : Overexpression of glucose transporter protein 5 in sciatic nerve of  streptozotocin-induced  diabetic rats. Neurosci Lett, 252, (2): 221-4, 1998. Atkins, K . B., Johns, D., Watts, S., Clinton Webb, R. and Brosius, F. C , 3rd: Decreased vascular glucose transporter expression and glucose uptake in D O C A salt hypertension. J Hypertens, 19, (9): 2582-7, 2001.  180  Badimon, L . , Badimon, J. J., Penny, W., Webster, M . W., Chesebro, J. H . and Fuster, V.: Endothelium and atherosclerosis. J Hypertens, 10, (2): S43-50,1992. Bakker, E . N . , Kerkhof, C . J. and Sipkema, P.: Signal transduction in spontaneous myogenic tone in isolated arterioles from rat skeletal muscle. Cardiovasc Res, 41, (1): 229-36., 1999. Ball, S. W., Bailey, J. R., Stewart, J. M . , Vogels, C . M . and Westcott, S. A . : A fluorescent  compound  for  glucose  uptake  measurements  in  isolated  rat  cardiomyocytes. Can J Physiol Pharmacol, 80, (3): 205-9,2002. Banting, F. G . and Best, C . H . : Pancreatic extracts. 1922. J Lab Clin M e d , 115, (2): 254-72,1990. Banz, W . J., Abel, M . A . and Zemel, M . B.: Insulin regulation of vascular smooth muscle glucose transport in insulin-sensitive and resistant rats. H o r m Metab Res, 28, (6): 272-5, 1996. Bar, R. S., Booth, B. A . , Boes, M . and Dake, B. L . : Insulin-like growth factorbinding proteins from vascular endothelial cells: purification, characterization, and intrinsic biological activities. Endocrinology, 125, (4): 1910-20,1989. Bar, R. S., Siddle, K., Dolash, S., Boes, M . and Dake, B.: Actions of insulin and insulinlike growth factors I and II in cultured microvessel endothelial cells from bovine adipose tissue. Metabolism, 37, (8): 714-20,1988. Barrett, M . P., Walmsley, A . R. and Gould, G . W.: Structure and function of facilitative sugar transporters. Curr O p i n Cell Biol, 11, (4): 496-502., 1999. Barrett-Connor, E . L . , Cohn, B. A . , Wingard, D . L . and Edelstein, S. L.: W h y is diabetes mellitus a stronger risk factor for fatal ischemic heart disease in women than in men? The Rancho Bernardo Study. Jama, 265, (5): 627-31,1991. Barron, J. T., Barany, M . , G u , L . and Parrillo, J. E.: Metabolic fate of glucose in vascular smooth muscle during contraction induced by norepinephrine. J M o l Cell Cardiol, 30, (3): 709-19,1998. Bayliss, W . M . : O n the local reactions of the arterial wall to changes in internal pressure. J Physiol, 28:220-231,1902.  181  Beckman, J. A . , Goldfine, A . B., Gordon, M . B., Garrett, L . A . and Creager, M . A . : Inhibition of protein kinase C-beta prevents impaired endothelium-dependent vasodilation caused by hyperglycemia in humans. Circ Res, 90, (1): 107-11, 2002. Bell, G . I., Burant, C . F., Takeda, J. and Gould, G . W.: Structure and function of mammalian facilitative sugar transporters. J Biol Chem, 268, (26): 19161-4,1993. Betz, A . L . , Bowman, P. D . and Goldstein, G . W.: Hexose  transport in  microvascular endothelial cells cultured from bovine retina. Exp Eye Res, 36, (2): 269-77, 1983. Blakemore, S. J., Aledo, J. C , James, J., Campbell, F. C , Lucocq, J. M . and Hundal, H.  S.: The G L U T 5 hexose transporter is also localized to the basolateral  membrane of the human jejunum. Biochem J, 309, (Pt 1): 7-12., 1995. Bolz, S., Farrell, C . L . , Dietz, K . and Wolburg, H . : Subcellular distribution of glucose transporter (GLUT-1) during development of the blood-brain barrier in rats. Cell Tissue Res, 284, (3): 355-65,1996. Bolz, S. S., Vogel, L., Sollinger, D., Derwand, R., Boer, C , Pitson, S. M . , Spiegel, S. and Pohl, U . : Sphingosine kinase modulates microvascular tone and myogenic responses through activation of RhoA/Rho kinase. Circulation, 108, (3): 342-7, 2003. Brayden, J. E . and Nelson, M . T.: Regulation of arterial tone by activation of calcium-dependent potassium channels. Science, 256, (5056): 532-5,1992. Brenner, R., Perez, G . J., Bonev, A . D., Eckman, D . M . , Kosek, J. C , Wiler, S. W., Patterson, A . J., Nelson, M . T. and Aldrich, R. W.: Vasoregulation by the betal subunit of the calcium-activated potassium channel. Nature, 407, (6806): 870-6, 2000. Brolin, S. E . and Naeser, P.: Sorbitol in aortic endothelium of diabetic rats. Diabetes Res, 8, (2): 59-61,1988. Brosnan, J. T.: Comments on metabolic needs for glucose and the role of gluconeogenesis. Eur J Clin Nutr, 53 Suppl 1: S107-11,1999. Brownlee,  M . : Glycation  products  and  the  complications. Diabetes Care, 15, (12): 1835-43,1992.  182  pathogenesis  of  diabetic  Brownlee, M . : Biochemistry and molecular cell biology of diabetic complications. Nature, 414, (6865): 823-20., 2001. Brownlee, M . , Cerami, A . and Vlassara, H . : Advanced glycosylation  end  products in tissue and the biochemical basis of diabetic complications. N Engl J M e d , 318, (20): 2325-22, 1988. Brownlee, M . , Vlassara, H . and Cerami,  A . : Nonenzymatic  glycosylation  products on collagen covalently trap low-density lipoprotein. Diabetes, 34, (9): 938-41, 1985. Bunn, R. C , Jensen, M . A . and Reed, B. C : Protein interactions with the glucose transporter binding protein G L U T 1 C B P that provide a link between G L U T 1 and the cytoskeleton. M o l Biol Cell, 10, (4): 819 32,1999. Burant, C . F. and Bell, G . I.: Mammalian facilitative glucose transporters: evidence  for similar substrate  recognition sites in functionally monomeric  proteins. Biochemistry, 31, (42): 10414-20,1992. Burant, C . F., Takeda, J., Brot-Laroche, E . , Bell, G . I. and Davidson, N . O.: Fructose transporter in human spermatozoa and small intestine is G L U T 5 . J Biol Chem, 267, (21): 14523-6,1992. Burchell, A.: A re-evaluation of G L U T 7 [letter]. Biochem J, 331, (Pt 3): 973,1998. Burg, M . B. and Kador, P. F.: Sorbitol, osmoregulation, and the complications of diabetes. J Clin Invest, 81, (3): 635-40,1988. Burkart, V . , Wang, Z . Q., Radons, J., Heller, B., Herceg, Z . , Stingl, L., Wagner, E . F. and Kolb, H . : Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta-cell  destruction and diabetes development  induced by  streptozocin. Nat Med, 5, (3): 324-9, 1999. Carayannopoulos, M . O., Chi, M . M . , C u i , Y., Pingsterhaus, J. M . , McKnight, R. A . , Mueckler, M . , Devaskar, S. U . and Moley, K . H . : G L U T 8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst. Proc Natl Acad Sci U S A , 97, (13): 7313-8, 2000. Carrington, W . A . , Forgarty, K. E . and Fay, F. S.: The handbook of biological confocal microscopy. N e w York, 1989.  183  Carrington, W . A . , Lynch, R. M . , Moore, E . D., Isenberg, G . , Fogarty, K . E . and Fay, F. S.: Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. Science, 268, (5216): 1483-7,1995. Casteels, R., Kitamura, K., Kuriyama, H . and Suzuki, H . : Excitation-contraction coupling in the smooth muscle cells of the rabbit main pulmonary artery. J Physiol, 271, (1): 63-79,1977. Cerami, A . , Vlassara, H . and Brownlee, M . : Role of advanced  glycosylation  products in complications of diabetes. Diabetes Care, 11, (Suppl 1): 73-9, 1988. Charron, M . J., Brosius, F. C . d., Alper, S. L . and Lodish, H . F.: A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci U S A , 86, (8): 2535-9,1989. Chikumi, H . , Vazquez-Prado, J., Servitja, J. M . , Miyazaki, H . and Gutkind, J. S.: Potent activation of RhoA by Galpha q and Gq-coupled receptors. J Biol Chem, 277, (30): 27230-4, 2002. Cobbold, P. H . and Rink, T. J.: Fluorescence and bioluminescence  measurement  of cytoplasmic free calcium. Biochem J, 248, (2): 313-28,1987. Concha, II, Velasquez, F. V . , Martinez, J. M . , Angulo, C , Droppelmann, A . , Reyes, A . M . , Slebe, J. C , Vera, J. C . and Golde, D . W.: H u m a n erythrocytes express G L U T 5 and transport fructose. Blood, 89, (11): 4190-5., 1997. Corkey, R. F., Corkey, B. E . and Gimbrone, M . A . , Jr.: Hexose transport in normal and SV40-transformed human endothelial cells in culture. J Cell Physiol, 106, (3): 425-34,1981. Cornford, E . M . , Hyman, S., Black, K . L . , Cornford, M . E . , Vinters, H . V . and Pardridge, W . M . : H i g h expression of the G l u t l glucose transporter in human brain hemangioblastoma endothelium. J Neuropathol Exp Neurol, 54, (6): 842-51, 1995. Cornford, E . M . , Hyman, S., Cornford, M . E., Damian, R. T. and Raleigh, M . J.: A single glucose transporter configuration in normal primate brain endothelium: comparison with resected human brain. J Neuropathol Exp Neurol, 57, (7): 699713,1998a.  184  Cornford, E . M . , Hyman, S., Cornford, M . E . , Landaw, E . M . and DelgadoEscueta,  A . V.: Interictal  seizure  resections show  two  configurations  of  endothelial G l u t l glucose transporter in the human blood-brain barrier. J Cereb Blood Flow Metab, 18, (1): 26-42,1998b. Corpe, C . P., Basaleh, M . M . , Affleck, J., Gould, G . , Jess, T. J. and Kellett, G . L . : The regulation of G L U T 5 and G L U T 2 activity in the adaptation of intestinal brush-border fructose transport in diabetes. Pflugers Arch, 432, (2): 192-201,1996. Cosentino, F. and Luscher, T. F.: Endothelial dysfunction in diabetes mellitus. J Cardiovasc Pharmacol, 32, (Suppl 3): S54-61., 1998. Crane, R. K.: Intestinal absorption of sugars. Physiol Rev, 40: 789-825,1960. Culic, O., Decking, U . K . and Schrader, J.: Metabolic adaptation of endothelial cells to substrate deprivation. A m J Physiol Cell Physiol, 276, (5 Pt 1): C1061-8, 1999. D'Angelo,  G.,  Davis,  mechanotransduction  M.  J.  and  Meininger,  G.  A.:  Calcium  and  of the myogenic response. A m J Physiol Heart Circ  Physiol, 273, (1 Pt 2): H175-82., 1997a. D'Angelo, G . , Mogford, J. E., Davis, G . E . , Davis, M . J. and Meininger, G . A . : Integrin-mediated reduction in vascular smooth muscle  [Ca ]i 2+  induced by R G D -  containing peptide. A m J Physiol Heart Circ Physiol, 272, (4 Pt 2): H2065-70, 1997b. Davis, M . J.: Myogenic response gradient in an arteriolar network. A m J Physiol Heart Circ Physiol, 264, (6 Pt 2): H2168-79, 1993. Davis, M . J., Donovitz, J. A . and H o o d , J. D.: Stretch-activated single-channel and whole cell currents in vascular smooth muscle cells. A m J Physiol Heart Circ Physiol, 262, (4 Pt 1): C1083-8,1992. Davis, M . J. and Hill, M . A . : Signaling mechanisms underlying the vascular myogenic response. Physiol Rev, 79, (2): 387-423,1999. Davis, M . J., W u , X., Nurkiewicz, T. R., Kawasaki, J., Davis, G . E., Hill, M . A . and Meininger, G . A . : Integrins and mechanotransduction of the vascular myogenic response. A m J Physiol Heart Circ Physiol, 280, (4): H1427-33, 2001.  185  Dawson, P. A . , Mychaleckyj, J. C , Fossey, S. C Mihic, S. J., Craddock, A . L . and v  Bowden, D . W.: Sequence  and functional analysis  of GLUT10: a glucose  transporter in the Type 2 diabetes-linked region of chromosome 20ql2-13.1. M o l Genet Metab, 74, (1-2): 186-99, 2001. DeBosch, B. J., Baur, E., Deo, B. K . , Hiraoka, M . and Kumagai, A . K.: Effects of insulin-like growth factor-1 on retinal endothelial cell glucose transport and proliferation. J Neurochem, 77, (4): 1157-67, 2001. DeBosch, B. J., Deo, B. K . and Kumagai, A . K.: Insulin-like growth factor-1 effects on bovine retinal endothelial cell glucose transport: role of M A P kinase. J Neurochem, 81, (4): 728-34, 2002. Degenhardt, T. P., Thorpe, S. R. and Baynes, J. W.: Chemical modification of proteins by methylglyoxal. Cell M o l Biol (Noisy-le-grand), 44, (7): 1139-45,1998. Dejana, E . and Plantier, J.-L.: Molecular organization of endothelial cell to cell junctions.; in Vascular Endothelium: responses to injury. 167-171. New York, 1996. Dessy, C , Matsuda, N . , Hulvershorn, J., Sougnez, C . L . , Sellke, F. W . and Morgan, K . G.: Evidence for involvement of the PKC-alpha isoform in myogenic contractions of the coronary microcirculation. A m J Physiol Heart Circ Physiol, 279, (3): H916-23, 2000. Diez-Sampedro, A . , Eskandari, S., Wright, E . M . and Hirayama, B. A . : Na -to+  sugar stoichiometry of SGLT3. A m J Physiol Renal Physiol, 280, (2): F278-82, 2001. Dillon, P. F., Aksoy, M . O., Driska, S. P. and Murphy,  R. A . : Myosin  phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science, 211, (4481): 495-7,1981. Dobrogowska, D . H . and Vorbrodt, A . W.: Quantitative immunocytochemical study of blood-brain barrier glucose transporter (GLUT-1) in four regions of mouse brain. J Histochem Cytochem, 47, (8): 1021-30,1999. Doege, H . , Bocianski, A . , Scheepers, A . , Axer, H . , Eckel, J., Joost, H . G . and Schurmann, A . : Characterization of human glucose transporter (GLUT)  11  (encoded by SLC2A11), a novel sugar-transport facilitator specifically expressed in heart and skeletal muscle. Biochem J, 359, (Pt 2): 443-9, 2001.  186  Doi, T., Vlassara, H . , Kirstein, M . , Yamada, Y., Striker, G . E . and Striker, L . J.: Receptor-specific increase in extracellular matrix production in mouse mesangial cells by advanced glycosylation end products is mediated via platelet-derived growth factor. Proc Natl Acad Sci U S A , 89, (7): 2873-7,1992. Doughty, J. M . , Miller, A . L . and Langton, P. D.: Non-specificity of chloride channel blockers in rat cerebral arteries: block of the L-type calcium channel. J Physiol, 507 ( Pt 2): 433-9, 1998. Drab, M . , Verkade, P., Elger, M . , Kasper, M . , Lohn, M . , Lauterbach, B., Menne, J., Lindschau, C., Mende, F., Luft, F. C., Schedl, A . , Haller, H . and Kurzchalia, T. V . : Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science, 293, (5539): 2449-52., 2001. Drewes, L . R., Broderius, M . A . and Gerhart, D . Z.: Phorbol ester stimulates hexose uptake by brain microvessel endothelial cells. Brain Res Bull, 21, (5): 7726,1988. Droogmans,  G.,  Raeymaekers,  L.  and  Casteels,  R.:  Electro-  and  pharmacomechanical coupling in the smooth muscle cells of the rabbit ear artery. J Gen Physiol, 70, (2): 129-48,1977. D u , X. L., Edelstein, D., Rossetti, L., Fantus, I. G., Goldberg, PL, Ziyadeh, F., W u , J.  and  Brownlee,  M . : Hyperglycemia-induced  mitochondrial  superoxide  overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing S p l glycosylation. Proc Natl Acad Sci U S A , 97, (22): 12222-6, 2000. Ebara, T., Conde, K., Kako, Y., Liu, Y., X u , Y., Ramakrishnan, R., Goldberg, I. J. and Shachter, N . S.: Delayed catabolism of apoB-48 lipoproteins due to decreased heparan sulfate proteoglycan production in diabetic mice. J Clin Invest, 105, (12): 1807-18, 2000. Elfeber, K., Kohler, A . , Lutzenburg, M . , Osswald, C , Galla, H . J., Witte, O . W . and Koepsell, H . : Localization of the Na -D-glucose cotransporter SGLT1 in the +  blood-brain barrier. Histochem Cell Biol, 121, (3): 201-7, 2004a. Elfeber, K., Stumpel, F., Gorboulev, V . , Mattig, S., Deussen, A . , Kaissling, B. and Koepsell, H . : Na(+)-D-glucose  cotransporter in muscle capillaries  increases  glucose permeability. Biochem Biophys Res Commun, 314, (2): 302-5, 2004b.  187  Falcone, J. C : Endothelial cell calcium and vascular control. M e d Sci Sports Exerc, 27, (8): 2265-9, 1995. Falholt, K., Cutfield, R., Alejandro, R., Heding, L . and Mintz, D.: The effects of hyperinsulinemia on arterial wall and peripheral muscle metabolism in dogs. Metabolism, 34, (12): 2246-9, 1985. Farrell, C . L . and Pardridge, W . M . : Blood-brain barrier glucose transporter is asymmetrically  distributed  on  brain  capillary  endothelial  lumenal  and  ablumenal membranes: an electron microscopic immunogold study. Proc Natl Acad Sci U S A , 88, (13): 5779-83, 1991. Feranchak,  A.  P.,  Roman,  R.  M . , Schwiebert,  E . M . and  Fitz,  J. G . :  Phosphatidylinositol 3-kinase contributes to cell volume regulation through effects on A T P release. J Biol Chem, 273, (24): 14906-11,1998. Filo, R. S., Bohr, D . F. and Ruegg, J. C : Gycerinated skeletal and smooth muscle: calcium and magnesium dependence. Science, 147:1581-1583,1965. Firth, J. A.: Endothelial barriers: from hypothetical pores to membrane proteins. J Anat, 200, (6): 541-8, 2002. Frank, H . J. and Pardridge, W . M . : A direct in vitro demonstration of insulin binding to isolated brain microvessels. Diabetes, 30, (9): 757-61,1981. French, J. E . : Atherosclerosis in relation to the structure and function of the arterial intima, with special reference to the endothelium. Int Rev Exp Pathol, 5: 253-353,1966. Fricker, M . , Hollinshead, M . , White, N . and Vaux, D.: Interphase nuclei of many mammalian  cell  types  contain  deep,  dynamic,  tubular  membrane-bound  invaginations of the nuclear envelope. J Cell Biol, 136, (3): 531-44,1997. Friedman, J. E . , Dudek, R. W., Whitehead, D . S., Downes, D . L . , Frisell, W . R., Caro, J. F. and Dohm, G . L.: Immunolocalization of glucose transporter G L U T 4 within human skeletal muscle. Diabetes, 40, (1): 250-4,1991. Fu, X., Gong, M . C , Jia, T., Somlyo, A . V . and Somlyo, A . P.: The effects of the I^o-kinase inhibitor Y-27632 on arachidonic acid-, GTPgammaS-, and phorbol ester-induced Ca -sensitization of smooth muscle. FEBS Lett, 440, (1-2): 183-7., 2+  1998.  188  Fujiwara, R. and Nakai, T.: Effect of glucose, insulin, and insulin-like growth factor-I on glucose transport activity in cultured rat vascular smooth muscle cells. Atherosclerosis, 127, (1): 49-57,1996. Fukata, Y., Amano, M . and Kaibuchi, K.: Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci, 22, (1): 32-9, 2001. Furchgott, R. F. and Zawadzki, J. V.: The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288, (5789): 373-6, 1980. Galbraith,  C . G . , Skalak, R. and Chien,  reorganization  of  the  endothelial  cell  S.: Shear stress induces  cytoskeleton.  Cell  spatial  Motility &  the  Cytoskeleton, 40, (4): 317-30,1998. Gaposchkin, C . G . and Garcia-Diaz, J. F.: Modulation of cultured brain, adrenal, and aortic endothelial cell glucose transport. Biochim Biophys Acta, 1285, (2): 255-66,1996. Garlanda, C . and Dejana, E.: Heterogeneity of endothelial cells. Specific markers. Arterioscler Thromb Vase Biol, 17, (7): 1193-202,1997. Geguchadze, R., Zhi, G . , Lau, K . S., Isotani, E . , Persechini, A . , Kamm, K . E . and Stull, J. T.: Quantitative  measurements  of  Ca( )/calmodulin binding and 2+  activation of myosin light chain kinase in cells. FEBS Lett, 557, (1-3): 121-4,2004. Gerritsen, M . E . and Burke, T. M . : Insulin binding and effects of insulin on glucose uptake  and metabolism  in cultured rabbit coronary  microvessel  endothelium. Proc Soc Exp Biol Med, 180, (1): 17-23,1985. Gerritsen, M . E., Burke, T. M . and Allen, L . A.: Glucose starvation is required for insulin stimulation of glucose uptake and metabolism in cultured microvascular endothelial cells. Microvasc Res, 35, (2): 153-66, 1988. Ghitescu, L . and Robert, M . : Diversity in unity: the biochemical composition of the endothelial cell surface varies between the vascular beds. Microsc Res Tech, 57, (5): 381-9., 2002. Giardino, I., Edelstein, D . and Brownlee, M . : Nonenzymatic glycosylation in vitro and in bovine endothelial cells alters basic fibroblast growth factor activity. A  189  model for intracellular glycosylation in diabetes [see comments]. J Clin Invest, 94, (1): 110-7,1994. Gohla, A . , Schultz, G . and Offermanns, S.: Role for G(12)/G(13) in agonistinduced vascular smooth muscle cell contraction. Circ Res, 87, (3): 221-7, 2000. Gokina, N . I. and Osol, G.: Temperature and protein kinase C  modulate  myofilament Ca2+ sensitivity in pressurized rat cerebral arteries. A m J Physiol Heart Circ Physiol, 274, (6 Pt 2): H1920-7, 1998. Goldstein, J. L . and Brown, M . S.: Atherosclerosis: the low-density lipoprotein receptor hypothesis. Metabolism, 26, (11): 1257-75,1977. Gong, M . C , Fujihara, H . , Somlyo, A . V . and Somlyo, A . P.: Translocation of rhoA associated with Ca2+ sensitization of smooth muscle. J Biol Chem, 272, (16): 10704-9,1997. Goodner, C: J., Horn, F. G . and Berrie, M . A.: Investigation of the effect of insulin upon regional brain glucose metabolism in the rat in vivo. Endocrinology, 107, (6): 1827-32, 1980. Gould, G . W., Brant, A . M . , Kahn, B. B., Shepherd, P. R., McCoid, S. C . and Gibbs, E. M . : Expression of the brain-type glucose transporter is restricted to brain and neuronal cells in mice. Diabetologia, 35, (4): 304-9,1992. Gould,  G . W . and Seatter, M . J.: Introduction to the facilitative  glucose  transporter familly.; in Facilitative Glucose Transporter. 1-37. New York, 1997. Greene, D . A . , Lattimer, S. A . and Sima, A . A . : Sorbitol, phosphoinositides, and sodium-potassium-ATPase in the pathogenesis of diabetic complications. N Engl J M e d , 316, (10): 599-606,1987. Grimm, J., Keller, R. and de Groot, P. G.: Laminar flow induces cell polarity and leads to rearrangement of proteoglycan metabolism in endothelial cells. Thromb Haemost, 60, (3): 437-41,1988. Grundy, S. M . , Benjamin, I. J., Burke, G . L., Chait, A . , Eckel, R. H . , Howard, B. V., Mitch, W., Smith, S. C , Jr. and Sowers, J. R.: Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association. Circulation, 100, (10): 1134-46, 1999.  190  Guo, Z . , Su, W., M a , Z . , Smith, G . M . and Gong, M . C.: Ca2+-independent phospholipase  A 2 is  required for  agonist-induced  Ca2+ sensitization  of  contraction in vascular smooth muscle. J Biol Chem, 278, (3): 1856-63, 2003. Guth, K. and Junge, J.: Low C a  2 +  impedes cross-bridge detachment in chemically  skinned Taenia coli. Nature, 300, (5894): 775-6., 1982. Haffner, S. M . , Mykkanen, L . , Festa, A . , Burke, J. P. and Stern, M . P.: Insulinresistant prediabetic subjects have more atherogenic risk factors than insulinsensitive prediabetic subjects: implications for preventing coronary heart disease during the prediabetic state. Circulation, 101, (9): 975-80, 2000. Haffner, S. M . , Stern, M . P., Hazuda, H . P., Mitchell, B. D . and Patterson, J. K.: Cardiovascular risk factors in confirmed prediabetic individuals. Does the clock for coronary heart disease start ticking before the onset of clinical diabetes? [see comments]. Jama, 263, (21): 2893-8,1990. Hajduch,  E . , Darakhshan, F. and Hundal,  adipocytes:  GLUT5  expression  and  the  H . S.: Fructose uptake  effects  of  in rat  streptozotocin-induced  diabetes. Diabetologia, 41, (7): 821-8,1998. Hall, A . : Rho GTPases and the actin cytoskeleton. Science, 279, (5350): 509-14., 1998. Haller, H . : Postprandial glucose and vascular disease. Diabet M e d , 14 Suppl 3: S50-6,1997. Halpern, W., Osol, G . and Coy, G . S.: Mechanical behavior of pressurized in vitro prearteriolar vessels determined with a video system. Annals of Biomedical Engineering, 12, (5): 463-79,1984. Hanefeld, M . , Koehler, C , Henkel, E . , Fuecker, K., Schaper, F. and TemelkovaKurktschiev, T.: Post-challenge  hyperglycaemia relates more strongly  than  fasting hyperglycaemia with carotid intima-media thickness: the R I A D Study. Risk Factors in Impaired Glucose Tolerance for Atherosclerosis and Diabetes. Diabet Med, 17, (12): 835-40, 2000. Hanefeld,  M . and  Temelkova-Kurktschiev, T.: Control  of  post-prandial  hyperglycemia-an essential part of good diabetes treatment and prevention of cardiovascular complications. Nutr Metab Cardiovasc Dis, 12, (2): 98-107, 2002.  191  Harder, D . R.: Pressure-dependent  membrane depolarization in cat middle  cerebral artery. Circ Res, 55, (2): 197-202,1984. Harder, D . R., Gilbert, R. and Lombard, J. H . : Vascular muscle cell depolarization and activation in renal arteries on elevation of transmural pressure. A m J Physiol Renal Physiol, 253, (4 Pt 2): F778-81,1987. Harder, D . R., Smeda, J. and Lombard, J.: Enhanced myogenic depolarization in hypertensive cerebral arterial muscle. Circ Res, 57, (2): 319-22, 1985. Hardin, C . D . and Pauley, R. G.: Metabolism and energetics of vascular smooth muscle.; in Physiology and Pathophysiology of the Heart. 1069-1086.1995. Harlan, J. M . , Levine, J. D., Callahan, K . S., Schwartz, B. R. and Harker, L . A . : Glutathione redox cycle protects cultured endothelial cells against lysis by extracellularly generated hydrogen peroxide. J Clin Invest, 73, (3): 706-13,1984. Hauguel-de Mouzon, S., Challier, J. C , Kacemi, A . , Cauzac, M . , Malek, A . and Girard, J.: The G L U T 3 glucose transporter isoform is differentially expressed within human placental cell types. J Clin Endocrinol Metab, 82, (8): 2689-94,1997. Hediger, M . A . , Coady, M . J., Ikeda, T. S. and Wright, E . M . : Expression cloning and c D N A sequencing of the NaVghicose co-transporter. Nature, 330, (6146): 37981,1987. Helliwell, P. A . , Richardson, M . , Affleck, J. and Kellett, G . L . : Stimulation of fructose transport across the intestinal brush-border membrane by P M A is mediated by G L U T 2 and dynamically regulated by protein kinase C . Biochem J, 350 Pt 1:149-54, 2000. Helliwell, P. A . , Rumsby, M . G . and Kellett, G . L.: Intestinal sugar absorption is regulated by phosphorylation and turnover of protein kinase C betall mediated by  phosphatidylinositol  3-kinase-  and mammalian  target  of rapamycin-  dependent pathways. J Biol Chem, 278, (31): 28644-50, 2003. Hemmila, J. M . and Drewes, L . R.: Glucose transporter (GLUT1) expression by canine brain micro vessel endothelial cells in culture: an immunocytochemical study. A d v Exp M e d Biol, 331:13-8, 1993. Henrion, D., Dechaux, E., Dowell, F. J., Maclour, J., Samuel, J. L., Levy, B. I. and Michel, J. B.: Alteration of flow-induced dilatation in mesenteric  192  resistance  arteries of L - N A M E treated rats and its partial association with induction of cyclo-oxygenase-2. Br J Pharmacol, 121, (1): 83-90, 1997. Henrion, D . and Laher, I.: Effects of staurosporine and calphostin C , two structurally unrelated inhibitors of protein kinase C , on vascular tone. C a n J Physiol Pharmacol, 71, (7): 521-4., 1993. Herr, R. R., Eble, T. E . , Bergy, M . E . and Jahnke, H . K.: Isolation and characterization of streptozotocin. Antibiot A n n u , 7:236-40,1959. Hill, M . A . , Falcone, J. C . and Meininger, G . A . : Evidence for protein kinase C involvement in arteriolar myogenic reactivity. A m J Physiol Heart Circ Physiol, 259, (5 Pt 2): H1586-94., 1990. Hill, T. L.: Sliding filament model of muscular contraction. V . Isometric force and interfilament spacing. J Theor Biol, 29, (3): 395-410., 1970. Hirata, K., Kuroda, R., Sakoda, T., Katayama, M . , Inoue, N . , Suematsu, M . , Kawashima, S. and Yokoyama, M . : Inhibition of endothelial nitric oxide synthase activity by protein kinase C . Hypertension, 25, (2): 180-5,1995. Hirsch, B. and Rosen, P.: Diabetes mellitus induces long lasting changes in the glucose transporter of rat heart endothelial cells. H o r m Metab Res, 31, (12): 64552,1999. Hogan, M . , Cerami, A . and Bucala, R.: Advanced glycosylation endproducts block the antiproliferative effect of nitric oxide. Role in the vascular and renal complications of diabetes mellitus. J Clin Invest, 90, (3): 1110-5,1992. Horn, F. G . and Goodner, C . J.: Insulin dose-response characteristics among individual muscle and adipose tissues measured in the rat in vivo with 3[H]2deoxyglucose. Diabetes, 33, (2): 153-9,1984. Horowitz, A . , Clement-Chomienne, O., Walsh, M . P. and Morgan, K . G.: Epsilonisoenzyme of protein kinase C induces a Ca(2+)-independent  contraction in  vascular smooth muscle. A m J Physiol Cell Physiol, 271, (2 Pt 1): C589-94,1996a. Horowitz, A . , Menice, C . B., Laporte, R. and Morgan, K. G.: Mechanisms of smooth muscle contraction. Physiol Rev, 76, (4): 967-1003,1996b. Howard, B. V . : Macrovascular Complications of Diabetes Mellitus; in Diabetes Mellitus. 792-797. Philadelphia, 1996a.  193  Howard, R. L.: Down-regulation of glucose transport by elevated extracellular glucose concentrations in cultured rat aortic smooth muscle cells does not normalize intracellular glucose concentrations. J Lab Clin M e d , 127, (5): 504-15, 1996b. Huijberts, M . S., Wolffenbuttel, B. H . , Boudier, H . A . , Crijns, F. R., Kruseman, A . C , Poitevin, P. and Levy, B. I.: Aminoguanidine treatment increases elasticity and decreases fluid filtration of large arteries from diabetic rats. J Clin Invest, 92, (3): 1407-11,1993. Hunt, J. V., Smith, C . C . and Wolff, S. P.: Autoxidative glycosylation and possible involvement of peroxides and free radicals in L D L modification by glucose. Diabetes, 39, (11): 1420-4,1990. Iglic, A . , Veranic, P., Batista, U . and Kralj-Iglic, V.: Theoretical analysis of shape transformation of V-79 cells after treatment with cytochalasin B. J Biomech, 34, (6): 765-72, 2001. Iizuka, K., Yoshii, A . , Samizo, K., Tsukagoshi, H . , Ishizuka, T., Dobashi, K., Nakazawa, T. and Mori, M . : A major role for the rho-associated coiled coil forming  protein  kinase  in  G-protein-mediated  Ca  2+  sensitization  through  inhibition of myosin phosphatase in rabbit trachea. Br J Pharmacol, 128, (4): 92533, 1999. Ikebe, M . , Inagaki, M . , Kanamaru, K . and Hidaka, H . : Phosphorylation of smooth muscle myosin light chain kinase by Ca -activated, phospholipid-dependent 2+  protein kinase. J Biol Chem, 260, (8): 4547-50,1985. Imig, J. D., Zou, A . P., Stec, D . E . , Harder, D . R., Falck, J. R. and Roman, R. J.: Formation and actions of 20-hydroxyeicosatetraenoic acid in rat renal arterioles. A m J Physiol Regul Integr Comp Physiol, 270, (1 Pt 2): R217-27,1996. Inoguchi, T., Xia, P., Kunisaki, M . , Higashi, S., Feener, E . P. and King, G . L . : Insulin's effect on protein kinase C and diacylglycerol induced by diabetes and glucose in vascular tissues. A m J Physiol Endocrinol Metab, 267, (3 Pt 1): E369-79, 1994. Inoue, M . , Kishimoto, A . , Takai, Y. and Nishizuka, Y.: Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. II. Proenzyme and its activation by calcium-dependent protease from rat brain. J Biol Chem, 252, (21): 7610-6., 1977.  194  Ishii, H . , Koya, D . and King, G . L.: Protein kinase C activation and its role in the development of vascular complications in diabetes mellitus. J M o l M e d , 76, (1): 21-31, 1998. Itoh, Y., Abe, T., Takaoka, R. and Tanahashi, N . : Fluorometric determination of glucose utilization in neurons in vitro and in vivo. J Cereb Blood Flow Metab, 24, (9): 993-1003, 2004. Jacobs, J., Sena, M . and Fox, N . : The cost of hospitalization for the late complications of diabetes in the United States. Diabet M e d , 8 Spec No: S23-9, 1991. James,  D.  E . , Strube,  M.  and  Mueckler,  characterization of an insulin-regulatable  M . : Molecular  cloning  and  glucose transporter. Nature,  338,  (6210): 83-7, 1989. Johnson, P. C.: The myogenic response in the microcirculation and its interaction with other control systems. J Hypertens Suppl, 7, (4): S33-9; discussion S40,1989. Joost, H . G . , Bell, G . I., Best, J. D., Birnbaum, M . J., Charron, M . J., Chen, Y. T., Doege, H . , James, D . E., Lodish, H . F., Moley, K . H . , Moley, J. F., Mueckler, M . , Rogers, S., Schurmann, A . , Seino, S. and Thorens, B.: Nomenclature of the GLUT/SLC2A  family of sugar/polyol  transport facilitators.  A m J Physiol  Endocrinol Metab, 282, (4): E974-6, 2002. Joost, H . G . and Thorens, B.: The extended GLUT-family of sugar/polyol transport facilitators: nomenclature,  sequence characteristics,  and  potential  function of its novel members (review). M o l Membr Biol, 18, (4): 247-56, 2001. Junod, A . , Lambert, A . E., Stauffacher, W . and Renold, A . E.: Diabetogenic action of streptozotocin: relationship of dose to metabolic response. J Clin Invest, 48, (11): 2129-39, 1969. Kaiser, N . , Sasson, S., Feener, E . P., Boukobza-Vardi, N . , Higashi, S., Moller, D . E., Davidheiser, S., Przybylski, R. J. and King, G . L . : Differential regulation of glucose transport and transporters by glucose in vascular endothelial and smooth muscle cells. Diabetes, 42, (1): 80-9,1993. Kandabashi, T., Shimokawa, PL, Miyata, K., Kunihiro, I., Kawano, Y., Fukata, Y., Higo, T., Egashira, K., Takahashi, S., Kaibuchi, K . and Takeshita, A.: Inhibition of myosin phosphatase by upregulated rho-kinase plays a key role for coronary  195  artery spasm in a porcine model with interleukin-lbeta. Circulation, 101, (11): 1319-23., 2000. Karandikar, M . and Cobb, M . H . : Scaffolding and protein interactions in M A P kinase modules. Cell Calcium, 26, (5): 219-26,1999. Kasahara, M . and Hinkle, P. C : Reconstitution and purification of the D-glucose transporter from human erythrocytes. J Biol Chem, 252, (20): 7384-90,1977. Kashiwagi, A . , Asahina, T., Ikebuchi, M . , Tanaka, Y., Takagi, Y., Nishio, Y., Kikkawa, R. and Shigeta, Y.: Abnormal glutathione metabolism and increased cytotoxicity caused by  H2O2  in human umbilical vein endothelial cells cultured in  high glucose medium. Diabetologia, 37, (3): 264-9, 1994. Katsuyama,  H . and  Morgan,  K . G . : Mechanisms  of  Ca(2+)-independent  contraction in single permeabilized ferret aorta cells. Circ Res, 72, (3): 651-7., 1993. Kauser, K., Clark, J. E . , Masters, B. S., Ortiz de Montellano, P. R., M a , Y. H . , Harder, D . R. and Roman, R. J.: Inhibitors of cytochrome P-450 attenuate the myogenic response of dog renal arcuate arteries. Circ Res, 68, (4): 1154-63,1991. Kawano, Y., Yoshimi, H . , Matsuoka, H . , Takishita, S. and Omae, T.: Calcium supplementation in patients with essential hypertension: assessment by office, home and ambulatory blood pressure. J Hypertens, 16, (11): 1693-9., 1998. Kawasaki, H . , Nakayama, S. and Kretsinger, R. H . : Classification and evolution of EF-hand proteins. Biometals, 11, (4): 277-95., 1998. Kayano, T., Burant, C . F., Fukumoto, H . , Gould, G . W., Fan, Y. S., Eddy, R. L . , Byers, M . G . , Shows, T. B., Seino, S. and Bell, G . I.: H u m a n facilitative glucose transporters. Isolation, functional characterization, and gene localization of c D N A s encoding an isoform (GLUT5) expressed in small intestine, kidney, muscle, and adipose tissue and an unusual glucose transporter pseudogene-like sequence (GLUT6). J Biol Chem, 265, (22): 13276-82,1990. Keogh, R. J., Dunlop, M . E . and Larkins, R. G . : Effect of inhibition of aldose reductase on glucose flux, diacylglycerol formation, protein kinase C , and phospholipase A 2 activation. Metabolism, 46, (1): 41-7,1997.  196  Kernohan, A . F  v  Perry, C . G . and Small, M . : Clinical impact of the new criteria  for the diagnosis of diabetes mellitus. Clin Chem Lab M e d , 41, (9): 1239-45, 2003. Khan, A . H . and Pessin, J. E.: Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways. Diabetologia, 45, (11):  1475-83,  2002. Kihara, S., Ouchi, N . , Funahashi, T., Shinohara, E., Tamura, R., Yamashita, S. and Matsuzawa, Y.: Troglitazone enhances glucose uptake and inhibits mitogenactivated protein kinase in human aortic smooth muscle cells. Atherosclerosis, 136, (1): 263-8,1998. King, G . L., Buzney, S. M . , Kahn, C . R., Hetu, N . , Buchwald, S., Macdonald, S. G . and Rand, L . I.: Differential responsiveness to insulin of endothelial and support cells from micro- and macrovessels. J Clin Invest, 71, (4): 974-9,1983. King, G . L . , Kunisaki, M . , Nishio, Y., Inoguchi, T., Shiba, T. and Xia, P.: Biochemical and molecular mechanisms in the development of diabetic vascular complications. Diabetes, 45, (Suppl 3):  S105-8,1996.  King, PL, Aubert, R. E . and Herman, W . H . : Global burden of diabetes, 1995-2025: prevalence, numerical estimates, and projections. Diabetes Care, 21, (9):  1414-31,  1998. Kinne, R. K.: Endothelial and epithelial cells: general principles of selective vectorial transport. Int J Microcirc Clin Exp, 17, (5): 223-30., 1997. Kirber, M . T., Walsh, J. V . , Jr. and Singer, J. J.: Stretch-activated ion channels in smooth muscle: a mechanism for the initiation of stretch-induced contraction. Pflugers Arch, 412, (4):  339-45,1988.  Kitazawa, T., Eto, M . , Woodsome, T. P. and Brautigan, D . L.: Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem, 275, (14): 9897-900, 2000. Kitazawa, T., Masuo, M . and Somlyo, A . P.: G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci U S A , 88, (20): 9307-10,  1991.  197  Kitazawa, T., Takizawa, N . , Ikebe, M . and Eto, M . : Reconstitution of protein kinase C-induced contractile C a  2 +  sensitization in triton X-100-demembranated  rabbit arterial smooth muscle. J Physiol, 520, (Pt 1): 139-52., 1999. Knot, H . J. and Nelson, M . T.: Regulation of membrane potential and diameter by voltage-dependent K channels in rabbit myogenic cerebral arteries. A m J Physiol +  Heart Circ Physiol, 269, (1 Pt 2): H348-55,1995. Knot, H . J. and Nelson, M . T.: Regulation of arterial diameter and wall [Ca ] in 2+  cerebral arteries of rat by membrane potential and intravascular pressure. J Physiol, 508, (Pt 1): 199-209., 1998. Kolm-Litty, V., Sauer, U . , Nerlich, A . , Lehmann, R. and Schleicher, E . D.: H i g h glucose-induced transforming growth factor betal production is mediated by the hexosamine pathway in porcine glomerular mesangial cells. J Clin Invest, 101, (1): 160-9,1998. Kong, C . T., Yet, S. F. and Lever, J. E.: Cloning and expression of a mammalian NaVamino  acid  cotransporter  with  sequence  similarity  to  NaVghicose  cotransporters. J Biol Chem, 268, (3): 1509-12,1993. Koskinen, P., Manttari, M . , Manninen, V., Huttunen, J. K., Heinonen, O . P. and Frick, M . H . : Coronary heart disease incidence in N I D D M patients in the Helsinki Heart Study. Diabetes Care, 15, (7): 820-5,1992. Koyama, M . , Ito, M . , Feng, J., Seko, T., Shiraki, K., Takase, K., Hartshorne, D . J. and Nakano, T.: Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett, 475, (3): 197-200., 2000. Krebs, C , Fernandes, H . B., Sheldon, C , Raymond, L . A . and Baimbridge, K . G.: Functional N M D A receptor subtype 2B is expressed in astrocytes after ischemia in vivo and anoxia in vitro. J Neurosci, 23, (8): 3364-72, 2003. Kretsinger, R. H . : Calmodulin and myosin light chain kinase: how helices are bent. Science, 258, (5079): 50-1., 1992. Krutzfeldt, A . , Spahr, R., Mertens, S., Siegmund, B. and Piper, H . M . : Metabolism of exogenous substrates by coronary endothelial cells in culture. J M o l Cell Cardiol, 22, (12): 1393-404, 1990.  198  Kuo, L., Chilian, W . M . and Davis, M . J.: Coronary arteriolar myogenic response is independent of endothelium. Circ Res, 66, (3): 860-6,1990. Kureishi, Y., Kobayashi, S., Amano, M . , Kimura, K., Kanaide, H . , Nakano, T., Kaibuchi, K . and Ito, M . : Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem, 272, (19): 12257-60,1997. Kwok, C . F., Goldstein, B. J., Muller-Wieland, D., Lee, T. S., Kahn, C . R. and King, G . L . : Identification of persistent defects in insulin receptor structure and function capillary endothelial cells from diabetic rats. J Clin Invest, 83, (1): 127-36, 1989. Lagaud, G . , Gaudreault, N . , Moore, E . D., V a n Breemen, C . and Laher, I.: Pressure-dependent  myogenic  constriction  of  cerebral  arteries  occurs  independently of voltage-dependent activation. A m J Physiol Heart Circ Physiol, 283, (6): H2187-95., 2002. Lagaud, G . J., Skarsgard, P. L., Laher, I. and van Breemen, C : Heterogeneity of endothelium-dependent  vasodilation  in  pressurized  cerebral  and  small  mesenteric resistance arteries of the rat. J Pharmacol Exp Ther, 290, (2): 832-9, 1999. Laher, I. and Bevan, J. A . : Staurosporine, a protein kinase C inhibitor, attenuates Ca2+-dependent stretch-induced vascular tone. Biochem Biophys Res Commun, 158, (1): 58-62., 1989. Lange,  A.,  Gebremedhin,  Hydroxyeicosatetraenoic  D.,  Narayanan,  acid-induced  J.  and  vasoconstriction  Harder, and  D.:  inhibition  20of  potassium current in cerebral vascular smooth muscle is dependent on activation of protein kinase C . J Biol Chem, 272, (43): 27345-52,1997. Laporte, R., Haeberle, J. R. and Laher, I.: Phorbol ester-induced potentiation of myogenic tone is not associated with increases in C a Ca  2+  2 +  influx, myoplasmic free  concentration, or 20-kDa myosin light chain phosphorylation. J M o l Cell  Cardiol, 26, (3): 297-302., 1994. Leach, L., Clark, P., Lampugnani, M . G., Arroyo, A . G., Dejana, E. and Firth, J. A . : Immunoelectron characterisation of the inter-endofhelial junctions of human term placenta. J Cell Sci, 104 (Pt 4): 1073-81,1993.  199  Lee, I. K., Kim, H . S. and Bae, J. H . : Endothelial dysfunction: its relationship with acute hyperglycaemia and hyperlipidemia. Int J Clin Pract Suppl, (129): 59-64., 2002. Lee, T. S., Saltsman, K . A . , Ohashi, H . and King, G . L . : Activation of protein kinase C by elevation of glucose concentration: proposal for a mechanism in the development of diabetic vascular complications [published erratum appears in Proc Natl Acad Sci U S A 1991 N o v 1;88(21):9907] [see comments]. Proc Natl Acad Sci U S A , 86, (13): 5141-5,1989. Leino, R. L., Gerhart, D . Z . , van Bueren, A . M . , McCall, A . L . and Drewes, L . R.: Ultrastructural localization of G L U T 1 and G L U T 3 glucose transporters in rat brain. J Neurosci Res, 49, (5): 617-26, 1997. Leite, R. and Webb, R. C : Microtubule disruption potentiates phenylephrineinduced vasoconstriction in rat mesenteric arterial bed. Eur J Pharmacol, 351, (1): Rl-3,1998. Libby, P. and Plutzky, J.: Diabetic macrovascular disease: the glucose paradox? Circulation, 106, (22): 2760-3, 2002. Lin, S. J., Hong, C . Y., Chang, M . S., Chiang, B. N . and Chien, S.: Increased aortic endothelial death and enhanced transendothelial macromolecular transport in streptozotocin-diabetic rats. Diabetologia, 36, (10): 926-30,1993. Lippoldt, A . , Kniesel, U . , Liebner, S., Kalbacher, H . , Kirsch, T., Wolburg, H . and Haller, H . : Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously  hypertensive  rat blood-brain barrier  endothelial cells. Brain Res, 885, (2): 251-61, 2000. Lloyd, P. G . and Hardin, C . D.: Role of microtubules in the regulation of metabolism in isolated cerebral microvessels. A m J Physiol Cell Physiol, 277, (6 Pt 1): C1250-62, 1999. Lloyd, P. G., Hardin, C . D . and Sturek, M . : Examining glucose transport in single vascular smooth muscle cells with a fluorescent glucose analog. Physiol Res, 48, (6): 401-10,1999. Loaiza, A . , Porras, O . H . and Barros, L . F.: Glutamate triggers rapid glucose transport  stimulation  in astrocytes  as  evidenced  microscopy. J Neurosci, 23, (19): 7337-42, 2003.  200  by  real-time  confocal  Loirand, G., Cario-Toumaniantz, C., Chardin, P. and Pacaud, P.: The Rho-related protein R n d l inhibits C a  2 +  sensitization of rat smooth muscle. J Physiol, 516, (Pt  3): 825-34,1999. Low, B. C . , Ross, I. K . and Grigor, M . R.: Angiotensin II stimulates glucose transport activity in cultured vascular smooth muscle cells. J Biol Chem, 267, (29): 20740-5,1992. L u , M . , Kuroki, M . , Amano, S., Tolentino, M . , Keough, K., Kim, I., Bucala, R. and Adamis, A . P.: Advanced glycation end products increase retinal vascular endothelial growth factor expression. J Clin Invest, 101, (6): 1219-24,1998. Lusis, A . J.: Atherosclerosis. Nature, 407, (6801): 233-41, 2000. Lutz, A . J. and Pardridge, W . M . : Insulin therapy normalizes G L U T 1 glucose transporter m R N A but not immunoreactive transporter protein in streptozocindiabetic rats. Metabolism, 42, (8): 939-44,1993. M a , Y. H . , Gebremedhin, D., Schwartzman, M . L . , Falck, J. R., Clark, J. E . , Masters, B. S., Harder, D . R. and Roman, R. J.: 20-Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal arcuate arteries. Circ Res, 72, (1): 126-36,1993. Macdonald, I. A . : Carbohydrate as a nutrient in adults: range of acceptable intakes. Eur J Clin Nutr, 53 Suppl 1: S101-6, 1999. MacDonald, J. A . , Borman, M . A . , Muranyi, A . , Somlyo, A . V . , Hartshorne, D . J. and Hay stead, T. A . : Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci U S A , 98, (5): 2419-24., 2001. Mackenzie, B., Loo, D . D., Panayotova-Heiermann, M . and Wright, E . M . : Biophysical characteristics of the pig kidney NaVglucose cotransporter SGLT2 reveal a common mechanism for SGLT1 and SGLT2. J Biol Chem, 271, (51): 32678-83, 1996. Mackenzie, B., Panayotova-Heiermann, M . , Loo, D . D., Lever, J. E. and Wright, E . M . : S A A T 1 is a low affinity NaVglucose cotransporter and not an amino acid transporter. A reinterpretation. J Biol Chem, 269, (36): 22488-91,1994.  201  MacKenzie, C . J., Wakefield, J. M . , Cairns, F., Dominiczak, A . F. and Gould, G . W.: Regulation of glucose transport in aortic smooth muscle cells by c A M P and c G M P . Biochem J, 353, (Pt 3): 513-9, 2001. Maesaki, R., Ihara, K., Shimizu, T., Kuroda, S., Kaibuchi, K . and Hakoshima, T.: The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of P K N / P R K 1 . M o l Cell, 4, (5): 793-803., 1999. Maher, F., Simpson, I. A . and Vannucci, S. J.: Alterations in brain glucose transporter proteins, G L U T 1 and G L U T 3 , in streptozotocin diabetic rats. A d v Exp M e d Biol, 331: 9-12, 1993. Mamchaoui, K., Makhloufi, Y. and Saumon, G : Glucose transporter gene expression in freshly isolated and cultured rat pneumocytes. Acta Physiol Scand, 175, (1): 19-24., 2002. Mandarino, L . J., Finlayson, J. and Hassell, J. R.: H i g h glucose downregulates glucose transport activity in retinal capillary pericytes but not endothelial cells. Invest Ophthalmol Vis Sci, 35, (3): 964-72,1994. Mann, G . E . , Yudilevich, D . L . and Sobrevia, L.: Regulation of amino acid and glucose transporters in endothelial and smooth muscle cells. Physiol Rev, 83, (1): 183-252., 2003. Manson, J. E . , Colditz, G . A . , Stampfer, M . J., Willett, W . C , Krolewski, A . S., Rosner, B., Arky, R. A . , Speizer, F. E . and Hennekens, C . H . : A prospective study of maturity-onset diabetes mellitus and risk of coronary heart disease and stroke in women. Arch Intern M e d , 151, (6): 2142-7, 1991. Mantych, G . J., Hageman, G . S. and Devaskar, S. U . : Characterization of glucose transporter isoforms in the adult and developing human eye. Endocrinology, 133, (2): 600-7,1993a. Mantych, G . J., James, D . E . and Devaskar, S. U . : Jejunal/kidney glucose transporter isoform (Glut-5) is expressed in the human blood-brain barrier. Endocrinology, 132, (1): 35-40,1993b. Marks, J., Carvou, N . J., Debnam, E . S., Srai, S. K . and Unwin, R. J.: Diabetes increases facilitative glucose uptake and G L U T 2 expression at the rat proximal tubule brush border membrane. J Physiol, 553, (Pt 1): 137-45, 2003.  202  Martinez-Lemus, L . A . , W u , X., Wilson, E., Hill, M . A . , Davis, G . E., Davis, M . J. and Meininger, G . A . : Integrins as unique receptors for vascular control. J Vase Res, 40, (3): 211-33, 2003. Masumoto, N . , Nakayama, K., Oyabe, A . , Uchino, M . , Ishii, K., Obara, K . and Tanabe, Y.: Specific attenuation of the pressure-induced contraction of rat cerebral artery by herbimycin A . Eur J Pharmacol, 330, (1): 55-63,1997. Matrougui, K., Tanko, L . B., Loufrani, L . , Gorny, D., Levy, B. I., Tedgui, A . and Henrion, D.: Involvement of Rho-kinase and the actin filament network in angiotensin II-induced contraction and extracellular signal-regulated  kinase  activity in intact rat mesenteric resistance arteries. Arterioscler Thromb Vase Biol, 21, (8): 1288-93, 2001. Matsuoka, T., Nishizaki, T. and Kisby, G . E . : Na -dependent and phlorizin+  inhibitable transport of glucose and cycasin in brain endothelial  cells. J  Neurochem, 70, (2): 772-7,1998. Mavrikakis, M . E., Sfikakis, P. P., Kontoyannis, D., Horti, M . , Kittas, C., Koutras, D . A . and Raptis, S. A . : Macrovascular disease of coronaries and cerebral arteries in streptozotocin-induced diabetic rats. A controlled, comparative study. Exp Clin Endocrinol Diabetes, 106, (1): 35-40,1998. McCall, A . L., van Bueren, A . M . , Huang, L., Stenbit, A . , Celnik, E . and Charron, M . J.: Forebrain endothelium expresses G L U T 4 , the insulin-responsive glucose transporter. Brain Research, 744, (2): 318-26,1997. McCarron, J. G., Crichton, C . A . , Langton, P. D., MacKenzie, A . and Smith, G . L . : Myogenic contraction by modulation of voltage-dependent calcium currents in isolated rat cerebral arteries. J Physiol, 498 (Pt 2): 371-9,1997. McCarron, J. G., Osol, G . and Halpern, W.: Myogenic responses are independent of the endothelium in rat pressurized posterior cerebral arteries. Blood Vessels, 26, (5): 315-9,1989. McPhillips, J. B., Barrett-Connor, E . and Wingard, D . L . : Cardiovascular disease risk factors prior to the diagnosis of impaired glucose tolerance and non-insulindependent diabetes mellitus in a community of older adults. A m J Epidemiol, 131, (3): 443-53,1990.  203  Medalie, J. H . , Papier, C . M . , Goldbourt, U . and Herman, J. B.: Major factors in the development of diabetes mellitus in 10,000 men. Arch Intern M e d , 135, (6): 811-7,1975. Meininger, G . A . , Zawieja, D . C . , Falcone, J. C., Hill, M . A . and Davey, J. P.: Calcium measurement  in isolated  arterioles during myogenic  and  agonist  stimulation. A m J Physiol Heart Circ Physiol, 261, (3 Pt 2): H950-9,1991. Mendelsohn, M . E.: Mechanisms of estrogen action in the cardiovascular system. J Steroid Biochem M o l Biol, 74, (5): 337-43, 2000. Mendelsohn, M . E . and Karas, R. H . : The protective effects of estrogen on the cardiovascular system. N Engl J Med, 340, (23): 1801-11,1999. Mertens, S., Noll, T., Spahr, R., Krutzfeldt, A . and Piper, H . M . : Energetic response of coronary endothelial cells to hypoxia. A m J Physiol Heart Circ Physiol, 258, (3 Pt 2): H689-94, 1990. Michel, C . C . and Curry, F. E.: Microvascular permeability. Physiol Rev, 79, (3): 703-61,1999. Miller, F. J., Jr., Dellsperger, K. C . and Gutterman, D . D.: Myogenic constriction of human coronary arterioles. A m J Physiol Heart Circ Physiol, 273, (1 Pt 2): H25764,1997. Mita, M . , Yanagihara, H . , Hishinuma, S., Saito, M . and Walsh, M . P.: Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase. Biochem J, 364, (Pt 2): 431-40, 2002. Miyazaki, K., Yano, T., Schmidt, D . J., Tokui, T., Shibata, M . , Lifshitz, L . M . , Kimura, S., Tuft, R. A . and Ikebe, M . : Rho-dependent agonist-induced spatiotemporal change in myosin phosphorylation in smooth muscle cells. J Biol Chem, 277, (1): 725-34, 2002. Mogford, J. E., Davis, G . E. and Meininger, G . A.: R G D N peptide interaction with endothelial  alpha5betal  integrin  causes  sustained  endothelin-dependent  vasoconstriction of rat skeletal muscle arterioles. J Clin Invest, 100, (6): 1647-53, 1997.  204  Mogford, J. E., Davis, G . E., Platts, S. H . and Meininger, G . A . : Vascular smooth muscle alpha v beta 3 integrin mediates arteriolar vasodilation in response to R G D peptides. Circ Res, 79, (4): 821-6,1996. Mompeo, B., Popov, D., Sima, A . , Constantinescu, E . and Simionescu, M . : Diabetes-induced structural changes of venous and arterial endothelium and smooth muscle cells. J Submicrosc Cytol Pathol, 30, (4): 475-84,1998. Moore, E . D., Becker, P. L . , Fogarty, K . E . , Williams, D . A . and Fay, F. S.: C a  2 +  imaging in single living cells: theoretical and practical issues. Cell Calcium, 11, (2-3): 157-79, 1990. Moore, E . D., Etter, E . F., Philipson, K . D., Carrington, W . A . , Fogarty, K . E . , Lifshitz, L . M . and Fay, F. S.: Coupling of the N a / C a +  2+  exchanger, N a / K pump +  +  and sarcoplasmic reticulum in smooth muscle. Nature, 365, (6447): 657-60., 1993. Morgan, J. P. and Morgan, K . G.: Vascular smooth muscle: the first recorded C a  2 +  transients. Pflugers Arch, 395, (1): 75-7,1982. Morgan, K . G . and Gangopadhyay, S. S.: Invited review: cross-bridge regulation by thin filament-associated proteins. J A p p l Physiol, 91, (2): 953-62., 2001. Morgan, K. G . , Khalil, R. A . , Suematsu, E . and Katsuyama, H . : Calciumdependent and calcium-independent pathways of signal transduction in smooth muscle. Jpn J Pharmacol, 58, (Suppl 2): 47P-53P., 1992. Mueckler, M . : Facilitative glucose transporters. Eur J Biochem, 219, (3): 713-25, 1994. Mueckler, M . , Caruso, C , Baldwin, S. A . , Panico, M . , Blench, I., Morris, H . R., Allard, W . J., Lienhard, G . E . and Lodish, H . F.: Sequence and structure of a human glucose transporter. Science, 229, (4717): 941-5,1985. Murata, H . , Hruz, P. W . and Mueckler, M . : The mechanism of insulin resistance caused by H I V protease inhibitor therapy. J Biol Chem, 275, (27): 20251-4, 2000. Murphy, R. A.: Contractile system function in mammalian smooth muscle. Blood Vessels, 13, (1-2): 1-23., 1976. Murthy, K. S., Zhou, H . , Grider, J. R., Brautigan, D . L., Eto, M . and Makhlouf, G . M . : Differential signalling by muscarinic receptors in smooth muscle: r e mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Racl and p21-  205  activated kinase 1 pathway, and m3-mediated M L C 2 0 (20 kDa regulatory light chain  of  myosin  II)  phosphorylation  via  Rho-associated  kinase/myosin  phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway. Biochem J, 374, (Pt 1): 145-55, 2003. Narayanan, J., Imig, M . , Roman, R. J. and Harder, D . R.: Pressurization of isolated renal arteries increases inositol trisphosphate and diacylglycerol. A m J Physiol Heart Circ Physiol, 266, (5 Pt 2): H1840-5,1994. Nelson, M . T., Cheng, H . , Rubart, M . , Santana, L . F., Bonev, A . D., Knot, H . J. and Lederer, W . J.: Relaxation of arterial smooth muscle by calcium sparks. Science, 270, (5236): 633-7,1995. Nelson, M . T., Conway, M . A . , Knot, H . J. and Brayden, J. E.: Chloride channel blockers inhibit myogenic tone in rat cerebral arteries. J Physiol, 502, (Pt 2): 25964,1997. Netland, P. A . , Zetter, B. R., Via, D . P. and Voyta, J. C : In situ labelling of vascular endothelium  with fluorescent  acetylated  low  density  lipoprotein.  Histochem J, 17, (12): 1309-20,1985. Nishikawa, M . , Hidaka, H . and Adelstein, R. S.: Phosphorylation of smooth muscle  heavy  meromyosin  by  calcium-activated,  phospholipid-dependent  protein kinase. The effect on actin-activated MgATPase activity. J Biol Chem, 258, (23): 14069-72,1983. Nishikawa, M . , Shirakawa, S. and Adelstein, R. S.: Phosphorylation of smooth muscle myosin light chain kinase by protein kinase C . Comparative study of the phosphorylated sites. J Biol Chem, 260, (15): 8978-83., 1985. Nishioka, T., Oda, Y., Seino, Y., Yamamoto, T., Inagaki, N . , Yano, H . , Imura, H . , Shigemoto, R. and Kikuchi, H . : Distribution of the glucose transporters in human brain rumors. Cancer Res, 52, (14): 3972-9,1992. Nishizaki, T., Kammesheidt, A . , Sumikawa, K., Asada, T. and Okada, Y.: A sodium- and energy-dependent glucose transporter with similarities to SGLT1-2 is expressed in bovine cortical vessels. Neurosci Res, 22, (1): 13-22., 1995. Nishizaki, T. and Matsuoka, T.: Low glucose enhances NaVglucose transport in bovine brain artery endothelial cells. Stroke, 29, (4): 844-9,1998.  206  Noria, S., Cowan, D . B., Gotlieb, A . I. and Langille, B. L.: Transient and steadystate effects of shear stress on endothelial cell adherens junctions. Circ Res, 85, (6): 504-14,1999. Nualart, F., Godoy, A . and Reinicke, K.: Expression of the hexose transporters G L U T 1 and G L U T 2 during the early development of the human brain. Brain Research, 824, (1): 97-104,1999. Ohara, Y., Sayegh, H . S., Yamin, J. J. and Harrison, D . G.: Regulation of endothelial constitutive nitric oxide synthase by protein kinase C . Hypertension, 25, (3): 415-20,1995. Olson, A . L . and Pessin, J. E.: Structure, function, and regulation of  the  mammalian facilitative glucose transporter gene family. A n n u Rev Nutr, 16: 23556,1996. Osol, G . , Laher, I. and Cipolla, M . : Protein kinase C modulates basal myogenic tone in resistance arteries from the cerebral circulation. Circ Res, 68, (2): 359-67., 1991. Osol, G . , Laher, I. and Kelley, M . : Myogenic tone is coupled to phospholipase C and G protein activation in small cerebral arteries. A m J Physiol Heart Circ Physiol, 265, (1 Pt 2): H415-20., 1993. Pan, M . , Wasa, M . and Souba, W . W.: Tumor necrosis factor stimulates system X A G - transport activity in human endothelium. J Surg Res, 58, (6): 659-64, 1995. Pardridge, W . M . , Boado, R. J. and Farrell, C . R.: Brain-type glucose transporter (GLUT-1) is selectively localized  to the blood-brain barrier. Studies with  quantitative western blotting and in situ hybridization. J Biol Chem, 265, (29): 18035-40,1990. Patterson, A . J., Henrie-Olson, J. and Brenner, R.: Vasoregulation at the molecular level: a role for the betal subunit of the calcium-activated potassium (BK) channel. Trends Cardiovasc M e d , 12, (2): 78-82., 2002. Paul, R. J., Bowman, P. S. and Kolodney, M . S.: Effects of microtubule disruption on force, velocity, stiffness and [Ca(2+)](i) in porcine coronary arteries. A m J Physiol Heart Circ Physiol, 279, (5): H2493-501, 2000.  207  Pedersen, S. F., Hoffmann, E . K . and Mills, J. W.: The cytoskeleton and cell volume regulation. Comp Biochem Physiol A M o l Integr Physiol, 130, (3): 385-99, 2001. Pekala, P., Marlow, M . , Heuvelman, D . and Connolly, D.: Regulation of hexose transport in aortic endothelial cells by vascular permeability factor and tumor necrosis factor-alpha, but not by insulin. J Biol Chem, 265, (30): 18051-4,1990. Perler, B. A . and Becker, G . J.: Vascular intervention, A clinical Approch. New York, 1998. Persechini, A . and Kretsinger, R. H . : Toward a model of the calmodulin-myosin light-chain kinase complex: implications for calmodulin function. J Cardiovasc Pharmacol, 12, (Suppl 5): Sl-12., 1988. Phay, J. E., Hussain, H . B. and Moley, J. F.: Cloning and expression analysis of a novel member of the facilitative glucose transporter family, SLC2A9 (GLUT9). Genomics, 66, (2): 217-20, 2000. Pierschbacher, M . D . and Ruoslahti, E.: Influence of stereochemistry  of the  sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J Biol Chem, 262, (36): 17294-8,1987. Pontremoli, S., Melloni, E . , Sparatore, B., Michetti, M . , Salamino, F. and Horecker, B. L.: Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases. J Biol Chem, 265, (2): 706-12., 1990. Portilla, D., Dai, G . , Peters, J. M . , Gonzalez, F. J., Crew, M . D . and Proia, A . D.: Etomoxir-induced PPARalpha-modulated enzymes protect during acute renal failure. A m J Physiol Renal Physiol, 278, (4): F667-75, 2000. Pouliot, J. F. and Beliveau, R.: Palmitoylation of the glucose transporter in bloodbrain barrier capillaries. Biochim Biophys Acta, 1234, (2): 292-6,1995. Quinn, L . A . and McCumbee, W . D.: Regulation of glucose transport by angiotensin II and glucose in cultured vascular smooth muscle cells. J Cell Physiol, 177, (1): 94-102,1998. Randriamboavonjy, V . , Busse, R. and Fleming, I.: 20-HETE-induced contraction of  small  coronary  arteries  depends  Hypertension, 41, (3 Pt 2): 802-6, 2003.  208  on  the  activation  of  Rho-kinase.  Reagan, L . P., Magarinos, A . M . , Lucas, L . R., van Bueren, A . , McCall, A . L . and McEwen, B. S.: Regulation of G L U T - 3 glucose transporter in the hippocampus of diabetic rats subjected to stress. A m J Physiol Endocrinol Metab, 276, (5 Pt 1): £ 8 7 9 - 8 6 , 1999. Redmond, E. M . , Cahill, P. A . and Sitzmann, J. V.: Perfused transcapillary smooth muscle and endothelial cell co-culture— a novel in vitro model. In Vitro Cell Dev Biol A n i m , 31, (8): 601-9., 1995. Rerup, C . C : Drugs producing diabetes through damage of the insulin secreting cells. Pharmacol Rev, 22, (4): 485-518,1970. Rogers, S., Macheda, M . L . , Docherty, S. E . , Carty, M . D., Henderson, M . A . , Soeller, W . C , Gibbs, E . M . , James, D . E . and Best, J. D.: Identification of a novel glucose transporter-like protein-GLUT-12. A m J Physiol Endocrinol Metab, 282, (3): E733-8, 2002. Roman, Y., Alfonso, A . , Louzao, M . C , Vieytes, M . R. and Botana, L. M . : Confocal microscopy  study  of the  different  patterns  of 2 - N B D G  uptake  in rabbit  enterocytes in the apical and basal zone. Pflugers Arch, 443, (2): 234-9, 2001. Rosen, G . M . and Freeman, B. A . : Detection of superoxide  generated  by  endothelial cells. Proc Natl Acad Sci U S A , 81, (23): 7269-73,1984. Ross, R. and Glomset, J. A . : The pathogenesis of atherosclerosis (second of two parts). N Engl J Med, 295, (8): 420-5,1976. Sajan, M . P., Bandyopadhyay, G . , Kanoh, Y., Standaert, M . L., Quon, M . J., Reed, B. C , Dikic, I. and Farese, R. V.: Sorbitol activates atypical protein kinase C and G L U T 4 glucose transporter translocation/glucose transport through proline-rich tyrosine  kinase-2,  the  extracellular  signal-regulated  kinase  pathway  and  phospholipase D . Biochem J, 362, (Pt 3): 665-74, 2002. Sakaue, H . , Ogawa, W., Takata, M . , Kuroda, S., Kotani, K., Matsumoto, M . , Sakaue, M . , Nishio, S., Ueno, H . and Kasuga, M . : Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarityinduced glucose uptake in 3T3-L1 adipocytes. M o l Endocrinol, 11, (10): 1552-62, 1997. Sakurada, S., Takuwa, N . , Sugimoto, N . , Wang, Y., Seto, M . , Sasaki, Y . and Takuwa, Y.: Ca2+-dependent activation of Rho and Rho kinase in membrane  209  depolarization-induced  and  receptor  stimulation-induced  vascular  smooth  muscle contraction. Circ Res, 93, (6): 548-56, 2003. Sanders, K . M . : Invited review: mechanisms of calcium handling in smooth muscles. J A p p l Physiol, 91, (3): 1438-49., 2001. Sank, A . , Wei, D., Reid, J., Ertl, D., Nimni, M . , Weaver, F., Yellin, A . and Tuan, T. L.: H u m a n endothelial cells are defective in diabetic vascular disease. J Surg Res, 57, (6): 647-53,1994. Sasson, S., Gorowits, N . , Joost, H . G . , King, G . L . , Cerasi, E . and Kaiser, N . : Regulation by metformin of the hexose transport system in vascular endothelial and smooth muscle cells. Br J Pharmacol, 117, (6): 1318-24, 1996. Sato, Y., Ito, T., Udaka, N . , Kanisawa, M . , Noguchi, Y., Cushman, S. W . and Satoh, S.: Immunohistochemical localization of facilitated-diffusion  glucose  transporters in rat pancreatic islets. Tissue Cell, 28, (6): 637-43., 1996. Schmidt, A . M . , Hori, O., Chen, J. X., Li, J. F., Crandall, J., Zhang, J., Cao, R., Yan, S. D., Brett, J. and Stern, D.:. Advanced glycation endproducts interacting with their endothelial receptor induce expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human endothelial cells and in mice. A  potential  mechanism for the accelerated vasculopathy of diabetes. J Clin Invest, 96, (3): 1395-403,1995. Schnedl, W . J., Ferber, S., Johnson, J. H . and Newgard, C . B.: STZ transport and cytotoxicity. Specific enhancement in GLUT2-expressing cells. Diabetes, 43, (11): 1326-33,1994. Schror, K.: Blood vessel wall interactions in diabetes. Diabetes, 46, (Suppl 2): S115-8,1997. Schubert, R., Kalentchuk, V . U . and Krien, U . : Rho kinase inhibition partly weakens  myogenic  reactivity  in rat small  arteries  by  changing  calcium  sensitivity. A m J Physiol Heart Circ Physiol, 283, (6): H2288-95., 2002. Schubert, R. and Mulvany, M . J.: The myogenic response: established facts and attractive hypotheses. Clinical Science, 96, (4): 313-26, 1999.  210  Setoguchi, M  v  Ohya, Y., Abe, I. and Fujishima, M . : Stretch-activated whole-cell  currents in smooth muscle cells from mesenteric resistance artery of guinea-pig. J Physiol, 501 ( Pt 2): 343-53,1997. Sheetz, M . J. and King, G . L . : Molecular understanding of hyperglycemia's adverse effects for diabetic complications. Jama, 288, (20): 2579-88., 2002. Shi, J., Zhang, Y. Q. and Simpkins, J. W.: Effects of 17beta-estradiol on glucose transporter 1 expression and endothelial cell survival following focal ischemia in the rats. Exp Brain Res, 117, (2): 200-6,1997. Shimada, M . , Kawamoto, S., Hirose, Y., Nakanishi, M . , Watanabe, H . and Watanabe,  M . : Regional  differences  in  glucose  transport  in  the  mouse  hippocampus. Histochem J, 26, (3): 207-12,1994. Shimizu, T., Ihara, K., Maesaki, R., Kuroda, S., Kaibuchi, K. and Hakoshima, T.: A n open conformation of switch I revealed by the crystal structure of a Mg -free 2+  form of R H O A complexed with G D P . Implications for the G D P / G T P exchange mechanism. J Biol Chem, 275, (24): 18311-7., 2000. Shin, H . M . , Je, H . D., Gallant, C , Tao, T. C , Hartshorne, D . J., Ito, M . and Morgan, K . G.: Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle. Circ Res, 90, (5): 546-53, 2002. Shirao, S., Kashiwagi, S., Sato, M . , Miwa, S., Nakao, F., Kurokawa, T., TodorokiIkeda, N . , Mogami, K., Mizukami, Y., Kuriyama, S., Haze, K., Suzuki, M . and Kobayashi, S.: Sphingosylphosphorylcholine is a novel messenger for Rhokinase-mediated C a  2+  sensitization in the bovine cerebral artery: unimportant  role for protein kinase C . Circ Res, 91, (2): 112-9, 2002. Simmons, D . A . and Winegrad, A . I.: Mechanism of glucose-induced (Na , K ) +  +  ATPase inhibition in aortic wall of rabbits. Diabetologia, 32, (7): 402-8,1989. Simpson, I. A . , Appel, N . M . , Hokari, M . , Oki, J., Holman, G . D., Maher, F., Koehler-Stec, E . M . , Vannucci, S. J. and Smith, Q. R.: Blood-brain barrier glucose transporter: effects of hypo- and hyperglycemia revisited. J Neurochem, 72, (1): 238-47, 1999.  211  Skarsgard, P., van Breemen, C . and Laher, I.: Estrogen regulates myogenic tone in pressurized cerebral arteries by enhanced basal release of nitric oxide. A m J Physiol, 273, (5 Pt 2): H2248-56,1997. Smith, P. G., Garcia, R. and Kogerman, L . : Strain reorganizes focal adhesions and cytoskeleton in cultured airway smooth muscle cells. Exp Cell Res, 232, (1): 22736, 1997. Sokoloff, L . , Reivich, M . , Kennedy, C , Des Rosiers, M . H . , Patlak, C . S., Petti grew, K . D., Sakurada, O. and Shinohara, M . : The [ C]deoxy glucose method 14  for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem, 28, (5): 897-916,1977. Solaro, R. J.: Myosin light chain phosphatase: a Cinderella of cellular signaling. Circ Res, 87, (3): 273-5, 2000. Somlyo, A . P. and Somlyo, A . V.: Ultrastructural aspects of activation and contraction of vascular smooth muscle. Fed Proc, 35, (6): 1288-93., 1976. Somlyo, A . P. and Somlyo, A . V.: Signal transduction and regulation in smooth muscle. Nature, 372, (6503): 232-6, 1994. Somlyo, A . P. and Somlyo, A . V.: Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol, 522, (Pt 2): 277-85, 2000. Somlyo, A . P. and Somlyo, A . V.: C a nonmuscle  myosin  II: modulated  2 +  by  sensitivity G  proteins,  of smooth muscle and kinases,  and  myosin  phosphatase. Physiol Rev, 83, (4): 1325-58, 2003. Sone, H . , Deo, B. K . and Kumagai, A . K.: Enhancement of glucose transport by vascular endothelial growth factor in retinal endothelial cells. Invest Ophthalmol Vis Sci, 41, (7): 2876-84, 2000. Speizer, L . , Haugland, R. and Kutchai, H . : Asymmetric transport of a fluorescent glucose analogue by human erythrocytes. Biochim Biophys Acta, 815, (1): 75-84, 1985.  212  Standley, P. R. and Rose, K . A . : Insulin and insulin-like growth factor-1 modulation of glucose transport in arterial smooth muscle cells: implication of G L U T - 4 in the vasculature. A m J Hypertens, 7, (4 Pt 1): 357-62,1994. Sward, K., Dreja, K., Susnjar, M . , Hellstrand, P., Hartshorne, D . J. and Walsh, M . P.: Inhibition of Rho-associated kinase blocks agonist-induced C a  2+  sensitization  of myosin phosphorylation and force in guinea-pig ileum. J Physiol, 522, (Pt 1): 33-49, 2000. Taggart, M . J., Leavis, P., Feron, O . and Morgan, K . G.: Inhibition of PKCalpha and rhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res, 258, (1): 72-81, 2000. Takagi, PL, King, G . L . and Aiello, L . P.: Hypoxia upregulates glucose transport activity through an adenosine-mediated increase of G L U T 1 expression in retinal capillary endothelial cells. Diabetes, 47, (9): 1480-8,1998. Takai, Y., Kishimoto, A . , Inoue, M . and Nishizuka, Y.: Studies on a cyclic nucleotide-independent protein kinase and its proenzyme in mammalian tissues. I. Purification and characterization of an active enzyme from bovine cerebellum. J Biol Chem, 252, (21): 7603-9., 1977. Takakura, Y., Kuentzel, S. L . , Raub, T. J., Davies, A . , Baldwin, S. A . and Borchardt, R. T.: Hexose uptake in primary cultures of bovine brain microvessel endothelial cells. I. Basic characteristics and effects of D-glucose and insulin. Biochim Biophys Acta, 1070, (1): 2-20,1991. Takata, K.: Glucose transporters in the transepithelial transport of glucose. J Electron Microsc (Tokyo), 45, (4): 275-84., 1996. Tapley, P. M . and Murray, A . W.: Modulation of Ca -activated, phospholipid2+  dependent protein kinase in platelets treated with a tumor-promoting phorbol ester. Biochem Biophys Res Commun, 122, (1): 258-64,1984. Taylor, P. D., Oon, B. B., Thomas, C . R. and Poston, L . : Prevention by insulin treatment of endothelial dysfunction but not enhanced noradrenaline-induced contractility  in mesenteric  resistance  arteries  from  streptozotocin-induced  diabetic rats. Br J Pharmacol, 111, (1): 35-42, 1994. Temelkova-Kurktschiev, T., Henkel, E . , Schaper, F., Koehler, C , Siegert, G . and Hanefeld, M . : Prevalence and atherosclerosis risk in different types of non-  213  diabetic hyperglycemia. Is mild hyperglycemia an underestimated evil? Exp Clin Endocrinol Diabetes, 108, (2): 93-9, 2000. Tesfamariam, B. and Cohen, R. A . : Free radicals mediate  endothelial  cell  dysfunction caused by elevated glucose. A m J Physiol Heart Circ Physiol, 263, (2 Pt 2): H321-6, 1992. Thomas, J., Linssen, M . , van der Vusse, G . J., Hirsch, B., Rosen, P., Kammermeier, H . and Fischer, Y.: Acute stimulation of glucose transport by histamine in cardiac microvascular endothelial cells. Biochim Biophys Acta, 1268, (1): 88-96, 1995. Thorens, B.: Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes. A m J Physiol Gastrointest Liver Physiol, 270, (4 Pt 1): G541-53., 1996. Thorens, B., Sarkar, H . K., Kaback, H . R. and Lodish, H . F.: Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and beta-pancreatic islet cells. Cell, 55, (2): 281-90,1988. Thornalley, P. J.: The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life. Biochem J, 269, (1): 1-11, 1990. Throckmorton, D . C , Packer, C . S. and Brophy, C . M . : Protein kinase C activation during Ca -independent vascular smooth muscle contraction. J Surg Res, 78, (1): 2+  48-53,1998. Thulesen, J., Orskov, C , Hoist, J. J. and Poulsen, S. S.: Short-term insulin treatment  prevents  the  diabetogenic  action  of  streptozotocin  in  rats.  Endocrinology, 138, (1): 62-8, 1997. Todoroki-Ikeda, N . , Mizukami, Y., Mogami, K., Kusuda, T., Yamamoto, K., Miyake, T., Sato, M . , Suzuki, S., Yamagata, H . , Hokazono, Y. and Kobayashi, S.: Sphingosylphosphorylcholine induces Ca(2+)-sensitization  of vascular smooth  muscle contraction: possible involvement of rho-kinase. FEBS Lett, 482, (1-2): 8590, 2000. Tomlinson, D . R.: Mitogen-activated protein kinases as glucose transducers for diabetic complications. Diabetologia, 42, (11): 1271-81,1999.  214  Tomlinson, K . C , Gardiner, S. M . , Hebden, R. A . and Bennett, T.: Functional consequences  of  streptozotocin-induced  diabetes mellitus,  with particular  reference to the cardiovascular system. Pharmacol Rev, 44, (1): 103-50,1992. Tordjman, K . M . , Leingang, K. A . and Mueckler, M . : Differential regulation of the HepG2 and adipocyte/muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation. Biochem J, 271, (1): 201-7,1990. Trepakova, E . S., Csutora, P., Hunton, D . L . , Marchase, R. B., Cohen, R. A . and Bolotina, V . M . : Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells. J Biol Chem, 275, (34): 26158-63, 2000. Trepakova, E . S., Gericke, M . , Hirakawa, Y., Weisbrod, R. M . , Cohen, R. A . and Bolotina, V . M . : Properties of a native cation channel activated by C a  2 +  store  depletion in vascular smooth muscle cells. J Biol Chem, 276, (11): 7782-90, 2001. Tsien, R. Y.: Fluorescence measurement and photochemical manipulation of cytosolic free calcium. Trends Neurosci, 11, (10): 419-24,1988. Uchida, E . and Bohr, D . F.: Myogenic tone in isolated perfused resistance vessels from rats. A m J Physiol—Legacy Content, 216, (6): 1343-50,1969. Uchigata, Y., Yamamoto, PL, Kawamura, A . and Okamoto, H . : Protection by superoxide  dismutase,  catalase,  and poly(ADP-ribose) synthetase inhibitors  against alloxan- and streptozotocin-induced islet D N A strand breaks and against the inhibition of proinsulin synthesis. J Biol Chem, 257, (11): 6084-8,1982. Uehata, M . , Ishizaki, T., Satoh, PL, Ono, T., Kawahara, T., Morishita, T., Tamakawa, H . , Yamagami, K., Inui, J., Maekawa, M . and Narumiya, S.: Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature, 389, (6654): 990-4,1997. Umemoto, S., Bengur, A . R. and Sellers, J. R.: Effect of multiple phosphorylations of smooth muscle and cytoplasmic myosins on movement in an in vitro motility assay. J Biol Chem, 264, (3): 1431-6., 1989. Urabe, T., Hattori, N . , Nagamatsu, S., Sawa, H . and Mizuno, Y.: Expression of glucose transporters in rat brain following transient focal ischemic injury. J Neurochem, 67, (1): 265-71, 1996.  215  Vallerand, A . L., Perusse, F. and Bukowiecki, L . J.: Cold exposure potentiates the effect of insulin on in vivo glucose uptake. A m J Physiol Endocrinol Metab, 253, (2 Pt 1): E179-86,1987. V a n Aelst, L . and D'Souza-Schorey, C : Rho GTPases and signaling networks. Genes Dev, 11, (18): 2295-322., 1997. van Breemen, C , Chen, Q . and Laher, I.: Superficial buffer barrier function of smooth muscle sarcoplasmic reticulum. Trends Pharmacol Sci, 16, (3): 98-105, 1995. Van den Enden, M . K., Nyengaard, J. R., Ostrow, E., Burgan, J. H . and Williamson, J. R.: Elevated glucose levels increase retinal glycolysis and sorbitol pathway metabolism. Implications for diabetic retinopathy. Invest Ophthalmol Vis Sci, 36, (8): 1675-85,1995. V a n Lierop, J. E., Wilson, D . P., Davis, J. P., Tikunova, S., Sutherland, C , Walsh, M . P. and Johnson, J. D.: Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40). J Biol Chem, 277, (8): 6550-8., 2002. VanBavel, E . , van der Meulen, E . T. and Spaan, J. A . : Role of Rho-associated protein kinase in tone and calcium sensitivity of cannulated rat mesenteric small arteries. Exp Physiol, 86, (5): 585-92., 2001. VanBavel, E., Wesselman, J. P. and Spaan, J. A.: Myogenic activation and calcium sensitivity of cannulated rat mesenteric small arteries. Circ Res, 82, (2): 210-20, 1998. Vannucci, S. J., Koehler-Stec, E . M . , L i , K., Reynolds, T. H . , Clark, R. and Simpson, I. A . : G L U T 4 glucose transporter expression in rodent brain: effect of diabetes. Brain Research, 797, (1): 1-11,1998. Vannucci, S. J., Maher, F. and Simpson, I. A . : Glucose utilization and glucose transporter proteins G L U T - 1 and G L U T - 3 in brains of diabetic (db/db) mice. A m J Physiol Endocrinol Metab, 272, (2 Pt 1): E267-74, 1997. Varbiro, S., Nadasy, G . L., Monos, E., Vajo, Z . , Acs, N . , Miklos, Z . , Tokes, A . M . and Szekacs, B.: Effect of ovariectomy and hormone replacement therapy on small artery biomechanics  in angiotensin-induced  Hypertens, 18, (11): 1587-95, 2000.  216  hypertension  in rats. J  Vilaro, S., Palacin, M . , Pilch, P. F., Testar, X. and Zorzano, A . : Expression of an insulin-regulatable glucose carrier in muscle and fat endothelial cells. Nature, 342, (6251): 798-800, 1989. Vinals, F., Gross, A . , Testar, X., Palacin, M . , Rosen, P. and Zorzano, A . : H i g h glucose  concentrations  inhibit  glucose  phosphorylation,  but not glucose  transport, in human endothelial cells. Biochim Biophys Acta, 1450, (2): 119-29., 1999. Vinters, H . V . , Beck, D . W., Bready, J. V . , Maxwell, K., Berliner, J. A . , Hart, M . N . and Cancilla, P. A . : Uptake of glucose analogues into cultured cerebral microvessel endothelium. J Neuropathol Exp Neurol, 44, (5): 445-58,1985. Waddell, I. D., Zomerschoe, A . G . , Voice, M . W . and Burchell, A . : Cloning and expression of a hepatic microsomal glucose transport protein. Comparison with liver plasma-membrane glucose-transport protein G L U T 2. Biochem J, 286, (Pt 1): 173-7,1992. Waitkus-Edwards, K . R., Martinez-Lemus, L . A . , W u , X., Trzeciakowski, J. P., Davis, M . J., Davis, G . E . and Meininger, G . A.: alpha(4)beta(l) Integrin activation of  L-type calcium  channels  in vascular  smooth  muscle  causes  arteriole  vasoconstriction. Circ Res, 90, (4): 473-80, 2002. Walker, P. S., Donovan, J. A . , V a n Ness, B. G . , Fellows, R. E . and Pessin, J. E.: Glucose-dependent regulation of glucose transport activity, protein, and m R N A in primary cultures of rat brain glial cells. J Biol Chem, 263, (30): 15594-601,1988. Walker, P. S., Ramlal, T., Donovan, J. A . , Doering, T. P., Sandra, A . , Klip, A . and Pessin, J. E.: Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line. J Biol Chem, 264, (11): 6587-95,1989. Wallner, E . I., Wada, J., Tramonti, G . , Lin, S. and Kanwar, Y. S.: Status of glucose transporters in the mammalian kidney and renal development. Ren Fail, 23, (3-4): 301-10., 2001. Walmsley, A . R., Barrett, M . P., Bringaud, F. and Gould, transporters  from  bacteria,  parasites  and  mammals:  relationships. Trends Biochem Sci, 23, (12): 476-81,1998.  217  G . W.: Sugar  structure-activity  Walsh, M . P., Kargacin, G . J., Kendrick-Jones, J. and Lincoln, T. M . : Intracellular mechanisms involved in the regulation of vascular smooth muscle tone. Can I Physiol Pharmacol, 73, (5): 565-73., 1995. Wang, Z . and Gleichmann, H . : G L U T 2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice. Diabetes, 47, (1): 50-6,1998. Watanabe, J., Karibe, A . , Horiguchi, S., Keitoku, M . , Satoh, S., Takishima, T. and Shirato, K.: Modification of myogenic intrinsic tone and  [Ca ]i 2+  of rat isolated  arterioles by ryanodine and cyclopiazonic acid. Circ Res, 73, (3): 465-72,1993. Webb, B. L . , Hirst, S. J. and Giembycz, M . A . : Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis. Br J Pharmacol, 130, (7): 1433-52, 2000. Weber, L. P., Seto, M . , Sasaki, Y., Sward, K. and Walsh, M . P.: The involvement of protein kinase C in myosin phosphorylation and force development in rat tail arterial smooth muscle. Biochem J, 352, (Pt 2): 573-82, 2000. Weber,  L . P.,  phosphorylation  V a n Lierop, of  myosin  J. E . and in  rat  Walsh,  caudal  M . P.: Ca -independent  artery  2+  and  chicken  gizzard  myofilaments. J Physiol, 516, (Pt 3): 805-24., 1999. Wells-Knecht, K . J., Zyzak, D . V . , Litchfield, J. E., Thorpe, S. R. and Baynes, J. W.: Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates  in the autoxidative  modification of proteins by  glucose.  Biochemistry, 34, (11): 3702-9,1995. Welsh, D . G., Morielli, A . D., Nelson, M . T. and Brayden, J. E.: Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ Res, 90, (3): 248-50, 2002. Welsh, D . G., Nelson, M . T., Eckman, D . M . and Brayden, J. E.: Swelling-activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure. J Physiol, 527 Pt 1:139-48, 2000. Wesselman, J. P., Schubert, R., VanBavel, E . D., Nilsson, H . and Mulvany, M . J.: KCa-channel blockade prevents sustained pressure-induced depolarization in rat mesenteric small arteries. A m J Physiol Heart Circ Physiol, 272, (5 Pt 2): H2241-9, 1997.  218  Williams, S. B., Goldfine, A . B., Timimi, F. K., Ting, H . PL, Roddy, M . A . , Simonson,  D.  C . and  endothelium-dependent  Creager,  M . A . : Acute  hyperglycemia  attenuates  vasodilation in humans in vivo. Circulation, 97, (17):  1695-701,1998. Wilson, D . P., Sutherland, C . and Walsh, M . P.: C a  2+  activation of smooth muscle  contraction: evidence for the involvement of calmodulin that is bound to the triton insoluble fraction even in the absence of C a . J Biol Chem, 277, (3): 22862+  92., 2002. Wingard, C . J., Nowocin, J. M . and Murphy, R. A . : Cross-bridge regulation by Ca(2+)-dependent phosphorylation in amphibian smooth muscle. A m J Physiol Regul Integr Comp Physiol, 281, (6): R1769-77., 2001. Wolffenbuttel, B. H . and van Haeften, T. W.: Prevention of complications in noninsulin-dependent diabetes mellitus (NIDDM). Drugs, 50, (2): 263-88,1995. Wood, I. S. and Trayhurn, P.: Glucose transporters ( G L U T and SGLT): expanded families of sugar transport proteins. Br J Nutr, 89, (1): 3-9, 2003. Woolf, N . : Pathology of atherosclerosis. London, 1982. Wright, E . M . : Renal Na(+)-glucose cotransporters. A m J Physiol Renal Physiol, 280, (1): ¥10-8,  2001.  Wright, E . M . , Loo, D . D., Panayotova-Heiermann, M . , Hirayama, B. A . , Turk, E., Eskandari, S. and Lam, J. T.: Structure and function of the  NaVglucose  cotransporter. Acta Physiol Scand Suppl, 643: 257-64,1998. W u , X. and Davis, M . J.: Characterization of stretch-activated cation current in coronary smooth muscle cells. A m J Physiol Heart Circ Physiol, 280, (4): H175161, 2001. Xie, L., Clunn, G . F., Lymn, J. S. and Hughes, A . D.: Role of intracellular calcium ([Ca2+]i) and tyrosine phosphorylation in adhesion of cultured vascular smooth muscle cells to fibrinogen. Cardiovasc Res, 39, (2): 475-84,1998. Yamada, K., Nakata, M . , Horimoto, N . , Saito, M . , Matsuoka, H . and Inagaki, N . : Measurement of glucose uptake and intracellular calcium concentration in single, living pancreatic beta-cells. J Biol Chem, 275, (29): 22278-83, 2000.  219  Yamagishi, S., Fujimori, PL, Yonekura, H . , Yamamoto, Y . and Yamamoto, PL: Advanced glycation endproducts inhibit prostacyclin production and induce plasminogen activator inhibitor-1 in human microvascular endothelial  cells.  Diabetologia, 41, (12): 1435-41,1998. Yamamoto, H . , Uchigata, Y. and Okamoto, H . : Streptozotocin and alloxan induce D N A strand breaks and poly(ADP-ribose) synthetase in pancreatic islets. Nature, 294, (5838): 284-6,1981. Yamasaki, Y., Kawamori, R., Matsushima, H . , Nishizawa, PL, Kodama, M . , Kubota, M . , Kajimoto, Y . and Kamada, T.: Asymptomatic hyperglycaemia is associated with increased intimal plus medial thickness of the carotid artery. Diabetologia, 38, (5): 585-91,1995. Yeon, D . S., K i m , J. S., A n n , D . S., Kwon, S. C , Kang, B. S., Morgan, K. G . and Lee, Y. PL: Role of protein kinase C - or RhoA-induced Ca(2+) sensitization in stretch-induced myogenic tone. Cardiovasc Res, 53, (2): 431-8., 2002. Yoshioka, K., O h , K. B., Saito, M . , Nemoto, Y. and Matsuoka, PL: Evaluation of 2[N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino]-2-deoxy-D-glucose, fluorescent  a  new  derivative of glucose, for viability assessment of yeast Candida  albicans. A p p l Microbiol Biotechnol, 46, (4): 400-4,1996a. Yoshioka, K., Saito, M . , O h , K. B., Nemoto, Y., Matsuoka, PL, Natsume, M . and Abe, PL: Intracellular fate of 2-NBDG, a fluorescent probe for glucose uptake activity, in Escherichia coli cells. Biosci Biotechnol Biochem, 60, (11): 1899-901, 1996b. Yoshioka, K., Takahashi, H . , Homma, T., Saito, M . , O h , K . B., Nemoto, Y. and Matsuoka, PL: A novel fluorescent  derivative of glucose applicable to the  assessment of glucose uptake activity of Escherichia coli. Biochim Biophys Acta, 1289, (1): 5-9,1996c. Yu, B., Poirier, L . A . and Nagy, L . E.: Mobilization of G L U T - 4 from intracellular vesicles by insulin and K(+) depolarization in cultured H9c2 myotubes. A m J Physiol Heart Circ Physiol, 277, (2 Pt 1): E259-67,1999. Zhang, B., Zhang, Y., Wang, Z . and Zheng, Y.: The role of M g  2 +  cofactor in the  guanine nucleotide exchange and G T P hydrolysis reactions of Rho family G T P binding proteins. J Biol Chem, 275, (33): 25299-307., 2000.  220  Zou, H . , Ratz, P. H . and Hill, M . A . : Role of myosin phosphorylation and  [Ca ]i 2+  in myogenic reactivity and arteriolar tone. A m J Physiol Heart Circ Physiol, 269, (5 Pt 2): H1590-6,1995.  221  Appendix 1 Calphostin C and  Y-27632  non-interaction with Fura-2  The possibility of an interaction between Fura-2 and both Calphostin C and Y-27632 were tested on a Cary Eclipse fluorescence spectrometer (Varian Canada Inc., Mississauga, O N ) . Fura-2 pentapotassium salt (0.1%) wad added to samples of PSS with and without 1.6 m M C a C h . Calphostin C and Y-27632 were added to sample of PSS with both C a  2 +  and Fura-2. Each sample was placed in a  fluorescence cuvette in the multicell holder of the Cary Eclipse fluorescence spectrophotometer. The temperature of the solution inside the cuvette was set to 37 ° C . Using the scan application, each solution was repeatedly excited at 340 and 380 nm. The scan speed was set to 600 nm/min for best signal to noise ratio. The  emission scans were recorded for Fura-2 between 450 and 600nm of  wavelength  with a P M T (800 volts). For each sample, four replicas were  averaged. The ratio of the corrected spectra from the 340 and 380 excitations were calculated for each sample and compared. The 340/380 ratio spectra of Fura-2 obtained in presence of C a  2 +  is not affected by neither calphostin C nor Y -  27632 (Figure A l ) . These data demonstrated that Calphostin C and Y-27632 do not interact with Fura-2. Moreover, when compared at the wavelength of 510 n m (the  emission  collected  during  physiological  experiment  with  the  photomultiplier detection system from Photon Technology International and using Felix quantitative ratio fluorescence software), the 340/380 ratio of the 0 Ca  2 +  solution was significantly lower than all other solution containing 1.6mM  Ca  2+  (p<0.001). We found no significant difference in the 340/380 ratio between  solution without and with Y-27632 (1 uM) or calphostin C (1 uM) (Figure A2).  222  i  50 o  40" H  00 C O  o  30 H  C O  •5 20 CO  i  DC  10 H 500  450  600  550  Emission wavelength (nm)  Figure A l Spectrum of the 340/380 ratio Solution with 0 Ca  2+  - , 1.6mM Ca  1.6mM Ca and 1 u M calphostin C 2+  S  30  , 1.6mM Ca  2+  2+  and 1 u M Y-27632  , and  :  H  C O  «  20 H  Figure A2 Histogram of the 340/380 ratio. Recorded at a wavelength emission of 510 nm. 0 Ca [ ~1 ,1.6mM Ca H, 1.6 m M C a and 1 p M Y-27632 ^B, 1.6 m M Ca and 1 p M calphostin C • • . * marks ratios significantly different from others (pO.001; A N O V A followed by Bonferroni test). 2+  2+  2+  223  2+  Appendix 2 Western blots negative controls  Control experiments were performed concomitantly with the data shown in Figure 2.4. In these control experiments, the same amount of protein (30 pg to 45 pg) from rat septal coronary artery (vessel), human coronary artery ECs (HCAECs), epididymal adipose tissue (Fat), heart, jejunum (GUT) and brain cortex was loaded and resolved on SDS-PAGE and transferred to nitrocellulose membrane. A l l incubations with the membranes were performed at 37 C with Q  gentle agitation. Non-specific binding sites were blocked with 10% non-fat dry milk in Tris-buffered saline Tween (TBS-T; 50 mmol/1 Tris, 0.09% NaCl, and 0.01% Tween; p H 7.6). The membranes were then incubated in 5% non-fat dry milk in TBS-T without the primary antibody, rinsed with TBS-T and further incubated with the appropriate horseradish peroxidase conjugated secondary antibodies (1:20000), also diluted in 5% non-fat dry milk in TBS-T (Figure A3).  Anti-rabbit Vessel HCAEC  A  g Gut  Fat  Heart  Anti-mouse Vessel HCAEC  100_ 55~ 40  _  33  _  24  1 7 _  Figure A 3 Secondary antibody negative controls A) anti-rabbit, B) anti-mouse, and C) anti-goat.  224  Q Fat  Anti-goat Vessel HCAEC  Brain  Appendix 3 FWHM and image depth measurements  The lateral and axial resolution of the Fluor 20X, N . A . 0.75 were measured using a Tetraspeck sphere; 200 n m diameter. A Z-stack of 2D images of the bead were acquired using the same system settings as those used for both the H C A E C s and the intact vessel (confocal aperture of 1.55 m m in diameter, pixel size of 156 n m and Z step of 500 n m (satisfied Nyquist criteria), N D filter #1, BHS filter (488 n m excitation wavelength), and mode of acquisition (gain 10, black level 4.8)). The fluorescence intensity was plotted as a function of distance from the brightest pixel in both the X and Y (Figure A4) and Z (Figure A5) directions. A n estimate of the full width half max (FWHM) of 3.7 p m and 9.3 p m for the lateral and axial dimensions respectively was calculated from the Gaussian curve (r of 0.90 and 0.88 respectively). The image depth, corresponding to the axial 2  depth from which light will be collected, calculated from the same Gaussian curve was estimated to be 16.9 pm.  225  2 0 0  1  -I  1 8 0 -  -  8  -  6  -  4  -2  0  2  4  6  Distance (i*m)  F i g u r e A4 Lateral fluorescence intensity distribution. Plot of the fluorescence intensity as a function of the lateral distance from the brightest pixel. The solid line represents the Gaussian fit. The dashed line indicates the position of X-Y coordinates at half of the maximal intensity.  1 6 0  -  =  1 4 0 -  &  1 2 0  \  -20  - 1 0  0  1 0  2 0  Distance (nm)  F i g u r e A5 Axial fluorescence intensity distribution. Plot of the fluorescence intensity as a function of the axial distance from the brightest pixel. The solid line represents the Gaussian fit. The dashed line indicates the position of Z coordinates at half of the maximal intensity.  226  Appendix 4 D-glucose blunts insulin effects in HCAECs  The effect of 2.5 p U / m l of insulin were investigated, on a 20-min exposure to a Krebs solution containing 0.1 m M 2 - N B D G and 5 m M D-glucose. A 20-min pre-incubation with 5 m M D-glucose and 2.5 p U / m l of insulin preceded the addition of 2 - N B D G to the solution. Insulin had no effect on the rate of accumulation of 2 - N B D G in H C A E C s in these conditions (Figure A6).  Figure A6 Effect of D-glucose on insulin stimulation in HCAECs. Uptake of 0.1 m M 2 - N B D G over 20 min, i n the absence I I or presence H M of 2.5 p U / m l of insulin, i n a Krebs solution containing 5 m M D-glucose. Each bar is the average of 14 and 17 cells from 3 different experiments respectively.  227  Appendix 5 Modulation of PKC and SGLT  We also performed a preliminary investigation on the effect of indolactan (1 pM), calphostin C (1 p M ; Biomol Research Laboratories, Inc.), and phloridzin (phloretin 2'-8-D-glucoside; 50 pM), known to activate, and inhibit G L U T - 2 and S G L T respectively, on a 20-min exposure to a Krebs solution containing a total of 5 m M (for phloridzin) or 20 m M (for indolactam and calphostin C) D-glucose and 0.1 m M of 2-NBDG. For each experiment, a 10-min pre-incubation with the respective compound preceded the addition of 2 - N B D G to the solution.  First,  the  regulation of  glucose uptake by  P K C (involved  in  the  translocation of G L U T - 2 to the plasma membrane) was assessed in the presence of 20 m M of D-glucose in H C A E C s . P K C activation with 1 p M indolactan had no significant effect on the average fluorescence intensity recorded after a 20 min exposure to 0.1 m M 2-NBDG. O n the other hand, the inhibition of P K C with 1 p M calphostin C reduced significantly by 37% (p<0.05) the accumulation of fluorescence intensity in H C A E C s (Figure A7).  228  15-, <D O  c  F i g u r e A 7 Regulation by P K C of the 2 - N B D G uptake i n H C A E C s . Uptake of 0.1 m M 2-NBDG over 20 min, in a solution of 20 m M D-glucose, in the absence CE2 or in the presence of 1 p M Indolactam^^ or 1 p M Calphostin C . Each bar is the average of 10, 16 and 9 cells respectively, from 3 different experiments. Significant differences between control and indolactant are indicated as follow; * p<0.05.  Secondly, phloridzin, a specific inhibitor of SGLT, was tested in order to determine the functionality of the Na -dependent co-transporter in H C A E C s . +  Phloridzin produced no significant inhibition on the average accumulation of fluorescence intensity in H C A E C s exposed to 0.1 m M 2-NBDG, at a basal concentration of 5 m M of D-glucose, for 20 min (Figure A8).  229  Figure A 8 Effect of Phloridzin on 2-NBDG uptake in HCAECs. Uptake of 0.1 mM 2-NBDG in presence of 5 mM D-glucose for a period of 20 min, in the absence |  1 or in the presence of 50 p M Phloridzin  . Each bar is the average of 20  and 21 cells respectively, from 4 different experiments.  The regulation of glucose transport by P K C has been previously reported in ECs. Phorbol ester, IGF-1 and V E G F have been shown to mediate through P K C , glucose uptake increases in brain microvessel and retinal ECs (Drewes et al, 1988; Sone et al, 2000; DeBosch et al, 2001). The effect of phorbol ester was reported to be greatest in primary or first passage cells and to be diminished or lost completely in older culture (beyond the 4th passage) (Drewes et al, 1988). IGF-1 was shown to induce glucose uptake in the absence of an increase in G L U T - 1 transcript or protein level (DeBosch et al, 2001). However, the increased in glucose uptake following V E G F stimulation also associated with no change in total G L U T - 1 protein content was shown to be accompanied with a 75% translocation of G L U T - 1 from the cytosol to the plasma membrane (Sone et al, 2000). Both studies performed in retinal ECs have identified PKCp as the isoform responsible for the regulation of glucose uptake in response to IGF-1 and V E G F stimulations. Interestingly, the traffic of G L U T - 2 from the cytosol to the brush-  230  border of epithelial cells has also been shown to be mediated by PKCp (Helliwell et al, 2000). In contrast, in signalling pathways modulated by insulin and sorbitol, the activation of atypical P K C isoforms were associated with G L U T - 4 translocation from the cytosol to the plasma membrane of adipocytes and myocytes  (Sajan et al,  2002). A s each of these transporter isoforms were  identified in H C A E C s and previously in rat coronary artery ECs, we therefore cannot rule out the possibility that the inhibitory effect on 2 - N B D G uptake, produced by calphostin C , is the result of the blockade of the translocation of G L U T - 1 , G L U T - 2 , G L U T - 4 or a combination of them to the plasma membrane.  In this study, experiments performed with calphostin C and indolactam were done in presence of 20 m M of D-glucose. G L U T - 2 unlike other transporter isoforms has a low affinity for glucose and a high K m value, making it the most efficient transporter in presence of high glucose concentration. A s indolactam, the P K C activator, did not increase any further 2 - N B D G uptake in H C A E C s , it is possible that G L U T - 2 , had already been translocated to the plasma membrane, through P K C activation, in response to the elevated glucose concentration. In agreement  with  this  concept,  calphostin  C , the  P K C inhibitor, reduced  significantly the uptake of 2 - N B D G in presence of 20 m M D-glucose.  Phloridzin, the S G L T specific inhibitor, was used in presence of much lower concentration of D-glucose (5 m M ) in H C A E C s . Although, we and others have previously identified SGLT-1 in ECs (Nishizaki and Matsuoka, 1998; Elfeber et al, 2004a; Elfeber et al, 2004b), 50 p M of phloridzin produced no significant effect on the uptake of 2 - N B D G in H C A E C s . A previous confocal microscopy study has shown an inhibition of 2 - N B D G by phloridzin as low as 35% in rabbit  231  enterocytes (Roman et ah, 2001). Since SGLT-1 is likely to play a more important role in enterocytes than in vascular ECs, the inhibitory effect of phloridzin on 2N B D G in ECs was expected to be very low. A s shown by Nishizaki et al., the activity of S G L T is greatly enhanced by low cytosolic glucose concentrations in brain micro vessel ECs (Nishizaki and Matsuoka, 1998). Since S G L T is more likely to  participate  in glucose  uptake  in  ECs in  periods  of  stress such  as  hypoglycaemia and ischemia, such conditions may need to be reproduced in ECs in order to determine the functionality of SGLT-1.  232  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0099840/manifest

Comment

Related Items