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Studies on the role of cAMP and its activation of cAMP-dependent protein kinase in the mediation of vascular… Zammit, Danielle Christine 1997

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STUDIES ON THE ROLE OF cAMP AND ITS ACTIVATION OF cAMP-DEPENDENT PROTEIN KINASE IN THE MEDIATION OF VASCULAR SMOOTH MUSCLE RELAXATION  by DANIELLE CHRISTINE ZAMMIT  B.Pharm. (Hons.), University of Malta, 1992  A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in The Faculty of Graduate Studies Division of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard  University of British Columbia April, 1997 © Danielle C. Zammit, 1997  In  presenting  degree  at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be her  for  It  publication of this thesis for financial gain shall not  is  granted  by the  understood  head of  that  copying  my or  be allowed without my written  permission.  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  J * ? ^  / f f f  .  ^  ^  ^  ^  "  \  *  ABSTRACT  The adenosine 3',5'-cyclic monophosphate (cAMP) activation of cAMPdependent protein kinase (PKA), and its resultant effect on vascular smooth muscle tone was investigated. Isoproterenol (ISO) and prostaglandin Ei ( P G E ^ have both been shown to elevate cAMP in vascular smooth muscle, the increase being accompanied by relaxation with ISO and contraction with  PGET.  The  activation of different pools of PKA by these drugs has been suggested to explain a similar observation in cardiomyocytes, where both ISO and  PGEi  elevated cAMP, yet only ISO increased contractility. To determine whether the circumstances in vascular smooth muscle were analogous to those in cardiac tissue, the study investigated the elevation of cAMP and the activation of PKA in subcellular fractions of vascular preparations treated with 1uM ISO and 10uM  PGEL  A 1uM forskolin (FSK)/ 10uM PGE^  combination was used as a positive control for cAMP measurement. An integral component was the use of the rabbit aorta medial strip, consisting of only smooth muscle cells. It was not possible to observe PKA activation in any of these experiments. Studies in aortic strips denuded of the endothelium demonstrated a large increase in cAMP levels in response to the FSK/PGEi combination. ISO had no effect on cAMP levels in helical strips, although it relaxed phenylephrine-induced contractions. Contrary to observations in helical strips, no difference in cAMP levels was recorded in any cell fraction from the medial strips, with any of the  ii  drugs used. ISO relaxed medial strips, but cAMP assays present no evidence of an increase in cAMP being related to the relaxation of vascular smooth muscle. The results obtained are not sufficient to either support, or negate, the hypothesis that cAMP elevation and the subsequent activation of PKA plays an important role in vascular smooth muscle relaxation. The data suggest that a) it is possible to measure cAMP levels in different subcellular fractions of vascular smooth muscle and b) differences exist between the tunica media and the adventitia with respect to their functional and cAMP responses to these drugs. Further investigation of the functional compartmentation of cAMP and PKA in the cell, and the signalling systems in the different layers of the blood vessel wall may elucidate the role of the cAMP/PKA system in the control of vascular tone.  iii  TABLE OF CONTENTS Page  ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  vii  LIST OF FIGURES  viii x  LIST OF ABBREVIATIONS ACKNOWLEDGEMENT  xiii  DEDICATION  xiv  1.0  INTRODUCTION  1  1.1  Calcium-dependent mechanism of vascular smooth muscle contraction 2  1.2  Signal transduction by beta-adrenergic agonists  3  1.2.1 Adenylyl cyclase  3  1.2.2 Cyclic AMP  7  1.2.2.1  Structure  7  1.2.2.2  cAMP phosphodiesterases  8  1.2.3 Cyclic AMP-dependent protein kinase  1.3  9  1.2.3.1  Cyclic AMP activation of holoenzyme  11  1.2.3.2  Regulatory subunit  12  1.2.3.3  Catalytic subunit  13  1.2.4 PKA substrates and putative consequences of phosphorylation  15  Cyclic AMP and PKA: Role in vascular smooth muscle relaxation  18  iv  1.4  1.3.1 Evidence for the involvement of cAMP and PKA in vascular smooth muscle relaxation  18  1.3.2 Evidence inconsistent with a role for cAMP in smooth muscle relaxation  19  Aspects of cellular signalling to be considered  21  1.4.1 Cellular homogeneity of the preparation  22  1.4.2 Compartmentation of cAMP  22  1.4.3 Compartmentation of PKA  24  1.4.3.1  Cellular localization of PKA  24  1.4.3.2  Translocation of the catalytic subunit  25  1.4.4 Activation of cGMP-dependent protein kinase by cAMP  25  Summary and rationale for proposed research  27  1.5.1 Hypothesis  28  1.5.2 Specific objectives of the present investigation  28  2.0  MATERIALS AND METHODS  30  2.1  Chemicals and materials  30  2.2  Drug Solutions  32  2.3  Animals  32  2.4  Preparation of rabbit aorta  32  2.4.1  Helical strips  33  2.4.2 Medial strips  33  Tissue-bath studies  34  1.5  2.5  v  2.6  Measurement of cAMP  36  2.6.1 Tissue extraction  36  2.6.2 Cyclic AMP assay  38  PKA measurements  39  2.7.1 Tissue extraction  39  2.7.2 PKA assay  40  2.8  Protein estimation  41  2.9  Statistical analyses  41  3.0  RESULTS  42  3.1  Nature of tension change  42  3.2  PKA studies  50  3.3  Cyclic AMP studies  51  4.0  DISCUSSION  65  4.1  Nature of drug-induced tension change in vascular smooth muscle  66  4.2  Activation of cAMP-dependent protein kinase  67  4.3  Cyclic AMP elevation in vascular smooth muscle  69  4.4  Future experiments  72  5.0  CONCLUSION  75  6.0  BIBLIOGRAPHY  77  2.7  vi  LIST OF TABLES  1.  Treatment of aortic strips following contraction with 1 p.M phenylephrine  2.  Effects of prostaglandin E<\ on rabbit aorta helical strips and medial strips, pre-treated with forskolin  vii  LIST OF FIGURES  Page  1.  Intracellular events leading to a-adrenergic-stimulated vascular smooth muscle contraction  4  2.  Schematic diagram of membrane-bound adenylyl cyclase  5  3.  Activation-deactivation cycle of G-proteins  7  4.  Chemical structure of adenosine 3',5'- cyclic monophosphate  8  5.  Regulation of cAMP-PDE  10  6.  Activation of PKA by cAMP  12  7.  Schematic representation of the putative phosphorylation sites of PKA in vascular smooth muscle as they relate to the whole of this signal transduction pathway  17  8.  Photograph of medial strip tissue  34  9.  Schematic representation of cAMP extraction process  38  10.  Calculation of the PKA activity ratio  41  11.  Protocol for drug treatment, and response observed, of rabbit aorta helical strips and medial strips  43  12.  Effect of 1 uM isoproterenol on the phenylephrine-induced contraction in helical strips and medial strips  45  13.  Effect of 10 u,M prostaglandin Ei on the phenylephrineinduced contraction in medial strips as compared with that of 65% ethanol  47  14.  Effects of 1 u,M isoproterenol and a combination of 1 u.M forskolin and 10 u,M prostaglandin E-\ on cAMP levels in the soluble fraction of rabbit aorta helical strips  52  15.  Effects of 1 (iM isoproterenol and a combination of 1 uTvl forskolin and 10 uM prostaglandin E-\ on cAMP levels in the particulate fraction of rabbit aorta helical strips  54  viii  Page  16.  Effects of 1 uJvl isoproterenol and a combination of 1 uJvl forskolin and 10 uJvl prostaglandin on total tissue cAMP levels in helical strip preparations  56  17.  Effects of 1 u.M isoproterenol (ISO), 10 uM prostaglandin Ei, and a combination of 1 u.M forskolin and 10 u.M prostaglandin Ei on cAMP levels in the soluble fraction of rabbit aorta medial strips  59  18.  Effects of 1 u.M isoproterenol, 10 u.M prostaglandin Ei, and a combination of 1 u.M forskolin and 10 uJvl prostaglandin E^ on cAMP levels in the particulate fraction of rabbit aorta medial strips  61  19.  Effects of 1 u.M isoproterenol, 10 uM prostaglandin Ei, and a combination of 1 \iM forskolin and 10 uJvl prostaglandin E^ on total tissue cAMP levels in medial strip preparations  63  ix  LIST OF ABBREVIATIONS AC  adenylyl cyclase  AN OVA  analysis of variance  ATP  adenosine 5'- triphosphate  [y- P] ATP  ATP in which the phosphorus at the y position is radioactive  Arg  arginine  BSA  bovine serum albumin  [Ca ],  intracellular calcium ion concentration  cAMP  adenosine 3',5'-cyclic monophosphate  CaM  calmodulin  cGMP  guanosine 3',5'-cyclic monophosphate  DAG  diacylglycerol  DTT  dithiothreitol  EDTA  ethylenediaminetetraacetic acid  EIA  enzymeimmunoassay  Eth  ethanol  FSK  forskolin  G-protein  heterotrimeric guanine nucleotide binding protein  HEPES  N -[2-hydroxyethyl] piperazine - N'- [2 - ethanesulfonic acid]  HS  helical strips  IBMX  3-isobutyl-1-methylxanthine  i.e.  id est (that is)  IP  inositol-1,4,5-trisphosphate  32  2+  3  ISO  isoproterenol  i.v.  intravenous(ly)  MLCK  myosin light chain kinase  MLCP  myosin light chain phosphatase  M  Molar  MS  medial strips  NO  nitric oxide  Pi  inorganic phosphate  PDE  phosphodiesterase  PE  phenylephrine  PGEi  prostaglandin  PGE  prostaglandin E  2  2  prostaglandin l  2  phosphatidylinositol-4,5-bisphosphate  PGI PIP  2  2  PKA  cAMP-dependent protein kinase  PKC  protein kinase C  PKG  cGMP-dependent protein kinase  PL  phospholamban  PLC  phospholipase C  PM  plasma membrane  PSS  physiological salt solution  SEM  standard error of the mean  xi  SR  sarcoplasmic reticulum  TK  tyrosine kinase  xii  ACKNOWLEDGEMENT I want to express my sincere gratitude and appreciation to Dr. Jack Diamond, my supervisor, for his unending patience, guidance and understanding in making my first experience of graduate studies a memorable one. His kindness and support were always present. I would also like to thank the members of my supervisory committee, Drs. Kathleen MacLeod, Roger Brownsey, Gail Bellward and Keith McErlane. I truly appreciate their constructive advice and suggestions regarding my research. I extend my thanks to Dr. Stelvio Bandiera, who generously provided access to his spectrophotometer plate reader and his microtitre plate shaker, and to Dr. Keith McErlane who allowed me the use of his nitrogen-drying equipment. I would also like to acknowledge the technical help of Dr. Rob Thies. I appreciate Dr. Mary Todd, in the Department of Anatomy, Faculty of Medicine, taking the time to help me get acquainted with the blood vessel wall. Additional thanks go to Drs. Heiko Prade and Steve Howard, post-doctoral fellows in the Faculty of Chemistry, for their help in getting the extraction process set up. Thanks and appreciation to Mr. Ali Tabatabaei, for his help and guidance in animal handling. Special thanks for their friendship, and acknowledgement of all they taught me in making Dr. Jack Diamond's laboratory such an interesting place: Dr. Karen MacDonell, Dr. Lakshman Sandirasegarane, Dr. Ashwin Patel and Mr. James Hennan. I express my appreciation to all the other members of the Faculty of Pharmaceutical Sciences, all the graduate students and the staff, for making me feel welcome and coming to my aid when it was needed. I would like to thank the International Pharmaceutical Federation Foundation for Education and Research for financial support in the form of a fellowship, and the Heart and Stroke Foundation of B.C. and Yukon for generously funding the research. I also appreciate the continual moral support and generous financial assistance which my family extended to me.  xiii  To  my sister, Claudine my mother, Tonia and my father, Ronnie,  for making me feel as if Malta was just around the  xiv  1.0  INTRODUCTION It was first recognized that catecholamines could cause smooth muscle  relaxation at the beginning of the twentieth century [Dixon, 1903; Hooker, 1911]. Adrenergic receptors for these compounds may be classified into a and p receptors, each having two subtypes. In vascular smooth muscle, ct-adrenergic agonists  generally  produce  vasoconstriction,  and  p-adrenergic  agonists,  vasodilation [reviewed by Hoffman and Lefkowitz, 1990]. Adenosine 3',5'-cyclic monophosphate (cAMP) was first isolated in 1957 by two groups working separately [Rail et. al., 1957; Cook et. al., 1957]. It is an ubiquitous molecule involved in a variety of physiological processes.  Further studies [Rail and  Sutherland, 1958; Sutherland and Rail, 1958] on the effects of glucagon and adrenaline resulted in the suggestion that cAMP is the intracellular mediator of some of the events mediating p-adrenergic responses [reviewed by Sutherland and Rail, 1960].  cAMP is produced from adenosine triphosphate (ATP) by  adenylyl cyclase [Murad et. al., 1962] and is believed to exert its effects via activation of cAMP-dependent protein kinase (PKA), which, in turn, catalyzes the serine and/or threonine phosphorylation of specific proteins [reviewed by Flockhart and Corbin, 1982].  It is believed to play a major role in vascular  smooth muscle relaxation, brought about by the PKA-mediated decrease in cytoplasmic calcium ion concentration, and a considerable amount of evidence for this role has accumulated over the years. Notwithstanding all the evidence, both the effect of cAMP in vascular smooth muscle, and the mechanism by which it is exerted, remain issues of much controversy. Reports contradicting the  1  relaxant role of cAMP have appeared in the literature, suggesting that the steps involved in this sequence of events need to be further elucidated [reviewed by Diamond, 1978; Hardman, 1984; Murray, 1990].  1.1  CALCIUM-DEPENDENT  MUSCLE  MECHANISM  OF VASCULAR SMOOTH  CONTRACTION  Some possible mechanisms involved in mediating contraction of vascular smooth muscle by adrenergic agonists are illustrated in Figure 1.  An a-  adrenergic agonist, such as phenylephrine (PE), causes G-protein-mediated activation of phospholipase C (PLC), which then catalyses the production of inositol-1,4,5-trisphosphate  (IP )  and  3  diacylglycerol  (DAG)  from  phosphatidylinositol-4,5-bisphosphate ( P I P 2 ) [reviewed by Abdel-Latif, 1986].  IP3  then binds to the IP3 receptors on the sarcoplasmic reticulum (SR) to release C a , mediating the initial phase of the contraction [Somlyo et al.. al., 1985]. 2+  Together with C a , DAG activates protein kinase C (PKC) and is thought to 2+  cause a delayed tonic contraction [Rasmussen et. al., 1987]. The increase in [Ca ]i 2+  will activate ryanodine receptors and give rise to the phenomenon of  calcium-induced-calcium-release.  An increase in free  [Ca ]i 2+  is necessary for  contraction to take place. Following this increase, 4 C a  2+  ions bind to calmodulin (CaM) to induce a  conformational change enabling its association with myosin light chain kinase (MLCK), the enzyme responsible for phosphorylation of myosin (Figure 1). When phosphorylated, myosin is able to bind to actin, developing force, hence  2  contraction. This sequence of events cannot occur when the [Ca ]\ decreases 2+  and C a  2+  dissociates from CaM. A lowering of [Ca ]\ thus produces relaxation. 2+  Regulation of [Ca ]j is very tightly controlled both electromechanically and 2+  pharmacomechanically [reviewed by Walsh, 1993].  1.2  SIGNAL TRANSDUCTION BY BETA-ADRENERGIC AGONISTS A beta-adrenergic agonist, such as isoproterenol, is generally thought to  bring about its effect by stimulating adenylyl cyclase to produce cAMP.  This  mechanism may also be employed by other agonists, such as PGEi.  1.2.1 Adenylyl Cyclase Adenylyl cyclase (AC) is the membrane bound enzyme responsible for the conversion of Mg-ATP to cAMP. This reaction may be controlled by hormones, neurotransmitters and other regulatory molecules involved in metabolism, gene transcription and memory. The adenylyl cyclases [Drummond, 1984] are a family of Type I - VIII glycoproteins, seven of which have so far been identified in mammalian tissues [Krupinski et. al., 1992]. They are incorporated into a variety of protein structures, most notably associated with the plasma membrane.  3  PE  CONTRACTION  Figure 1. Intracellular events leading to a-adrenergic-stimulated vascular smooth muscle contraction. See text for details. PE, phenylephrine; a, aadrenergic receptor; Gq, GTP-binding protein, subtype q; PLC, phospholipase C; P I P 2 , phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; PKC, protein kinase C; PM, plasma membrane; IP3, inositol 1,4,5-trisphosphate; SR, sarcoplasmic reticulum; R, ryanodine receptor; CaM, calmodulin; MLCK, myosin light chain kinase; Pi, inorganic phosphate; MLCP, myosin light chain phosphatase.  4  The cytosolic part of the mammalian isoform consists of a short aminoterminal motif, and two cytosolic domains, punctuated by two intenselyhydrophobic structures (Figure 2), each hypothesized to contain 6 transmembrane helices [reviewed by Tang and Gilman, 1992]. The catalytic domains, C1a and C2a, bind the substrate.  Both sites are thought to be required for  catalytic activity, none being detectable when half the molecule is expressed by itself [Krupinski et. al., 1989; Tang et. al., 1991]. The lipid-soluble diterpene, forskolin (FSK), is a potent activator of AC. It is thought to bind to hydrophobic pockets in the catalytic region of the molecule [Zhang et. al., 1997].  Figure 2. Schematic diagram of membrane-bound adenylyl cyclase. PM, plasma membrane; Mi, M 2 , hydrophobic trans-membrane helices; N, short amino-terminal motif; C, catalytic subunit, (adapted from Tang et. al., 1991).  5  AC is coupled to cell-surface receptors of the 7-trans-membrane-spanning domain type, via heterotrimeric, guanine-nucleotide-binding regulatory proteins (G-proteins) possessing GTPase activity.  Binding of a p-adrenergic agonist to  the cell-surface receptor activates adenylyl cyclase via stimulatory G-protein (G ), s  amplifying the response through up to eight G molecules [Brandt and Ross, s  1986]. Inhibitory G-proteins may mediate inhibition of AC activity [reviewed by Tang and Gilman, 1992;  Birnbaumer and Birnbaumer, 1995].  G-proteins,  heterotrimers consisting of a,p and y subunits, are continually in an activationdeactivation cycle. The a-subunit (Ga) activates AC, while the Py-subunit (GPy) may have some separate, inherent activity [reviewed by Birnbaumer and Birnbaumer, 1995]. In the inactive state, G a is associated with GDP and has a high affinity for GPy (Figure 3).  A Ga.GDP.GPy complex is formed, which  interacts with the receptor when this is stimulated by a p-adrenergic ligand, causing the release of GDP, with the subsequent binding of GTP. This guanine nucleotide exchange is the rate-limiting step of the cycle and the activated receptor will stabilize the complex in the absence of guanine nucleotides. GTP binding is dependent on the presence of Mg , which will promote exchange by 2+  de-stabilizing the complex. In its absence, Py-subunits will inhibit dissociation of GDP from G a [Codina et. al., 1984]. The complex will release Ga-GTP, which has a high affinity for the effector, which it will activate. Hydrolysis by an intrinsic GTPase in G a will liberate P,. The receptor and GPy re-enter the cycle [reviewed by Conklin and Bourne, 1993; Eschenhagen, 1993]. The effector in this case is AC, with the message transmitted being to increase production of cAMP.  6  Figure 3. Activation-deactivation cycle of G-proteins. E, effector; R, padrenergic receptor; Pi, inorganic phosphate; Ga, a-subunit; GPy, Py-subunit; GDP, guanosine diphosphate; GTP, guanosine triphosphate. See text for details, (adapted from Conklin and Bourne, 1993). 1.2.2  Cyclic AMP Adenosine 3',5'- cyclic monophosphate (cAMP) was first isolated by  Sutherland and Rail in 1958. Since then, its presence has been detected in a diversity of tissues and implicated in a variety of physiological processes.  1.2.2.1  Structure This nucleotide is a purine derivative consisting of an adenosine moiety  cyclized with a phosphate group, by means of two ester linkages (Figure 4).  7  NH2  Figure 4. Chemical structure of adenosine 3',5'- cyclic monophosphate.  1.2.2.2  cAMP Phosphodiesterases  The effect of cAMP is terminated when the cyclic nucleotide is hydrolyzed to adenosine 5'-monophosphate (5'-AMP) by specific phosphodiesterases. The family of PDE enzymes is currently an area of active research. There are four recognized families that are capable of hydrolyzing cAMP.  The regulation of  phosphodiesterase (PDE) activity is, in itself, a complex issue  [reviewed by  Beltman et. al., 1993]. Figure 5 represents the major pathways thought to be involved in PDE regulation.  Cyclic AMP degradation generally occurs rapidly  through the activation of constitutive enzymes, while a persistently high intracellular cAMP concentration will induce PDE activity by supposedly increasing expression of cAMP-PDE mRNA. An increase in intracellular calcium is related to activation of PDE by Ca-CaM. PDE can also be activated by a  8  signalling pathway that is inhibited by ISO  [reviewed by Hakim, 1995].  Phosphorylation by PKA may be a general mechanism for dampening and terminating the hormonal signal through accelerated degradation of cAMP [Gettys et. al., 1987]. PDE isozymes types III and IV are low-affinity cAMP phosphodiesterases present in smooth muscle. Type III is inhibited by cGMP. There are a number of PDE inhibitors for different PDE isozymes, mainly important for their clinical uses. They include theophylline, caffeine and the alkylxanthines [reviewed by Hall, 1993].  1.2.3  Cyclic AMP- dependent protein kinase Cyclic AMP is believed to mediate p-induced relaxation of vascular  smooth muscle via the activation of PKA [Walsh et. al., 1968; Silver, et. al., 1982]. The protein kinase family of enzymes catalyzes the phosphorylation of proteins, thus altering the equilibrium between active and inactive conformational states. Protein kinases may be of two general classes, one class transferring phosphate to tyrosine, and the other class transferring phosphate to serine or threonine [reviewed by Krebs, 1986].  g  PM AC TK 5'AMP  Ca-CaM  ^  ^  ^  cAMP  ^  >  5'AMP  ^ ^ " ^ M ^ ^  3 }  CaMK— RAPID R E G U L A T I O N via phosphorylation  cAMP-PDE mRNA DELAYED REGULATION via modulation of c A M P - P D E gene  cAMP-PDEl gene 'nucleus  Figure 5. Regulation of cAMP-PDE, (adapted from Hakim, 1995). R, receptor; G , Gj, stimulatory and inhibitory G-proteins; AC, adenylyl cyclase; PM, plasma membrane; TK, tyrosine kinase; PK, non-specific protein kinase; cAMP-PDE, cAMP phosphdiesterase; Ca-CaM, calcium-calmodulin; CaM, calmodulin; CaMK, calmodulin kinase; 5'AMP, 5'adenosine monophosphate; +, stimulated; , inhibited; P, phosphorylated. s  PKA  provided the first clues about phosphorylation and its role in  regulating the responses of the eucaryotic cell.  It is the simplest and best  biochemically understood kinase, making it a suitable candidate to be a model for this structurally diverse family of enzymes.  Cyclic GMP-dependent protein  kinase (PKG), PKC, MLCK and protein tyrosine kinases all share the same conserved structural features, yet differ to a great extent, depending on the 10  nature of their individual substrates and the ligands that activate them [reviewed by Taylor, 1989].  1.2.3.1  Cyclic AMP activation of holoenzvme PKA is an inactive tetramer composed of two regulatory subunits (R) and  two catalytic subunits (C), the amino acid sequences of which were obtained using the purified subunits [reviewed by Francis and Corbin, 1994]. Holoenzyme activation involves the ternary complex of cyclic AMP, R and C [Builder et. al., 1980], according to the equation: RC 2  2  + 4cAMP  <=>  cAMP»R C 2  2  <=>  R «cAMP + 2 C 2  Two molecules of cAMP bind to each R subunit of PKA, causing the affinity of R for C to decrease, resulting in the dissociation of the tetramer into dimeric R and two catalytically active monomers of C (Figure 6). The inhibition of C by R being released, the now activated catalytic site catalyses the phosphorylation of a protein or enzyme responsible for vascular smooth muscle relaxation [reviewed by Swillens and Dumont, 1976].  11  p -Adrenoceptor) 2  Cyclic A M P  Mg-ATP  AMPc +  2.(C). +  4.CAMP a  P'  ACTIVE CATALYTIC SUBUNITS  y  Adenosine- © - ©  Substrate  QAMP  (RJ^FT  AMPc  3AMP  (Pj)-Substrate a  p  Adenosine- © - ©  Figure 6. Activation of PKA by cAMP. See text for details.  1.2.3.2  Regulatory subunit  Type I and Type II holoenzymes are distinguished on the basis of their R subunit.  There are two major mammalian classes of R, each of which is  subdivided into isoforms a and p. Type I [Titani et. al., 1984], consisting of 379 amino acids, is similar in domain substructure and amino acid sequence to Type II [Takio et. al., 1984], which is the predominant isoform in cardiac muscle and  12  consists of 400 amino acids. Studies suggest that activation of either type can elicit a given physiological response [Van Sande et. al., 1989]. The 100 residues closest to the amino terminus are believed to include the region for oligomeric association, while two, tandem, gene-duplicated sequences at the carboxyterminus, correspond to two cAMP-binding sites [Takio et. al., 1984], 'slow' site A and 'fast' site B. Site B is carboxy-terminal to site A [reviewed by Taylor et. al., 1990]. Both cAMP binding sites are involved in activation of PKA; kinetic studies show that cAMP binds first to site B and induces a conformational change, which enables site A to have a cAMP molecule bound to it. The occupation of site A is required for dissociation of the catalytic subunit [0greid and D0skeland, 1981a, 1981b] from the 'hinge region', at amino acids 90-100. Responsiveness of the Type II isoenzyme to cAMP is altered by the state of phosphorylation of its regulatory  subunit  [Hofmann  et.  al.,  1975].  Data  suggest  that  the  phosphorylation site of the R subunit contains an arginine residue involved in the binding of this subunit to C [Corbin et. al., 1978]. The R subunit also contains substrate sites for other kinases, the physiological significance of which is not yet known, but may be important for interaction with other proteins.  1.2.3.3  Catalytic subunit  Three different forms of mammalian C subunit have been described. The cDNA for Ca [Uhler et. al., 1986a] and for Cp code for proteins that are 91% identical. The Cp isoform is found mostly in the brain, although it is also detected in other tissues [Uhler et. al., 1986b].  Both these forms of C are capable of  13  participating in the regulation of transcription [Maurer, 1989]. Data indicate that Cy is also an active PKA catalytic subunit, possibly with different peptide recognition determinants [Beebe et. al., 1992]. The catalytic subunit contains 2 lobes, from amino acid 25-120 and 128310 [reviewed by Walsh and Van Patten, 1994]. There is a cleft between the two, wherein lies the catalytic site. Binding of the substrate causes closure of the cleft, resulting in a different topographical relationship between the lobes. The catalytic site typically phosphorylates peptides or proteins with the -Arg-Arg-XSer-Y- consensus sequence, where X represents a small residue, and Y a large hydrophobic group [Kemp et. al., 1977]. The other functional sites of this subunit are the Mg-ATP binding site and the peptide binding site. The Mg-ATP binding site is near the amino-terminus, and includes a glycine-rich loop to aid the binding of the phosphate in ATP, by allowing the polypeptide to turn in conformation with the approach of the pyrophosphate moiety [Wierenga and Hoi, 1983]. Stereochemical techniques have shown that Mg  2+  is a ligand that is preferred by this enzyme [Granot et. al., 1979]. Arg  chelates the M g  2+  184  ion and assists in juxtaposing the y-phosphate with the  substrate serine for effective transfer. Lysine 72 was shown to be essential for enzyme action, causing irreversible inhibition of C when covalently modified [Zoller et. al., 1981]. Carboxyl groups act as a general base to pull the proton off the serine or threonine in the substrate. In the substrate-binding site, they may also play a role in the recognition of the substrate [Yoon and Cook, 1987].  14  1.2.4  PKA substrates and putative consequences of phosphorylation The cAMP stimulation of PKA is thought to bring about vascular smooth  muscle relaxation by decreasing intracellular C a  2+  concentration.  The  mechanisms by which this could be brought about (see Figure 7) may include the following multiple sites of regulation: 1. C a  2+  efflux, by extrusion along the ATP-dependent C a  2+  pump in the plasma  membrane, was suggested to be enhanced by cAMP and PKA at high [Ca ], 2+  [Kattenburg and Daniel, 1984], a proposal inconsistent with earlier studies by Kreye and Schlicher [1980], whose studies with nitrovasodilators were not in favour of a close correlation between C a  2+  pumps and phosphorylation. It must  be noted, however, that the drugs they used are more likely to act via PKG than via PKA. 2.  Enhanced sequestration of C a  2+  into intracellular stores [Brockbank and  England, 1980; Raeymaekers et. al., 1990] was discerned by using ouabain to block the Na /K pump [Mueller and van Breemen, 1979]. +  +  [1984] inhibited the uptake of C a  2+  Suematsu et. al.,  by the plasma membrane, but not by the  sarcoplasmic reticulum, to show the latter to be enhanced by PKA. associated phosphorylation  of phospholamban (PL),  PKA  responsible for the  regulation of the sarcoplasmic reticulum Ca /Mg ATPase, has been observed 2+  2+  in the heart [Kranias et. al., 1988]. The endoplasmic reticulum of many smooth muscles contain PL.  In vascular smooth muscle, PL was shown to be  phosphorylated by PKA [Raeymaekers and Jones, 1986].  15  3. Increased activity of the Na /K pump: activation of the Na /K ATPase will +  alter the Na /Ca +  2+  +  +  +  exchange process, due to an increase in the Na gradient  [Scheid et. al., 1979; Webb and Bohr, 1980]. 4. From experiments that excluded the mechanism in (3), above, inhibition of Ca  2+  influx was thought to be the manner by which p-receptor stimulation  reduced intracellular free C a , to promote relaxation of smooth muscle [Meisheri 2+  and van Breemen, 1982]. 5. A direct effect at the level of the contractile proteins [Kerrick and Hoar, 1981; Adelstein et. al., 1982], by phosphorylation of MLCK [Silver and DiSalvo, 1979; Bhalla et. al., 1982] has not been excluded, but is not thought to be the primary mechanism of cAMP-induced vasodilation [Gerthoffer et. al., 1984; Jones et. al., 1984]. Besides being phosphorylated by PKA, the Ca -activated K channel has 2+  +  also been reported to be stimulated directly by cAMP [Minami et. al., 1993]. PKA has also been demonstrated to regulate the function of isolated p-adrenergic receptors by phosphorylation, associated with desensitization of the adenylyl cyclase system [Benovic et. al., 1985].  16  Figure 7. Schematic representation of the putative phosphorylation sites of PKA in vascular smooth muscle as they relate to the whole of this signal transduction pathway. PKA, cAMP-dependent protein kinase; MLCK, myosin light chain kinase; SR, sarcoplasmic reticulum; PL, phospholamban; P, phosphorylation; +, stimulation; -, inhibition; AC, adenylyl cyclase; G , stimulatory G-protein; ATP, adenosine triphosphate; R, receptor; PM, plasma membrane; [Ca ]j, intracellular calcium ion concentration. The numbered arrows refer to the mechanisms described in the text. s  2+  17  1.3  CYCLIC AMP AND PKA: ROLE IN VASCULAR SMOOTH MUSCLE  RELAXATION 1.3.1  Evidence for the involvement of cAMP and PKA in vascular smooth  muscle relaxation The assumption that an elevation in cAMP levels, with PKA activation and subsequent phosphorylation of protein, leads to intracellular events that result in a decrease in [Ca ],, in turn responsible for the relaxant effect, has been 2+  accepted for many years. In vascular tissue, relaxation of the vascular smooth muscle cells translates into vasodilation. The criteria proposed by Sutherland et. al. [1968] for a cyclic nucleotide to be considered capable of mediating the effect of an agonist, have all been fulfilled to a certain extent by cAMP in the mediation of p-adrenergic vascular smooth muscle relaxation. Isoproterenol was shown to increase cAMP levels by stimulation of adenylyl cyclase in a cell-free system from bovine coronary artery [Kukovetz et. al., 1978]. The hormone-induced increase in cAMP level has been correlated with relaxation [Collins and Sutter, 1975], and decreased contractility [Honeyman et. al., 1978] in smooth muscle cells in a temporal and dose-dependent manner. Exogenously applied cAMP derivatives are thought to penetrate the cell more easily than cAMP and be hydrolyzed less quickly by PDE. Dibutyryl cAMP and divalenyl cAMP were able to inhibit a-adrenergic contractions [Kreye and Schultz, 1972].  Kramer and Wells [1979] synthesized a series of xanthine  derivatives designed to selectively inhibit the hydrolysis of cAMP. The order of  18  potency of PDE inhibitors  to  cause relaxation was similar to the order of  inhibition of the enzyme. Evidence in support of the hypothesis that p-induced relaxation of vascular smooth muscle involves the cAMP activation of PKA has been obtained in bovine coronary artery [Silver et. al., 1982] and in the canine saphenous vein [Kikkawa et. al., 1986].  Forskolin, a p-adrenergic-receptor-independent activator of  adenylyl cyclase, produced time- and dose-dependent increases in PKA activation [Lincoln and Fisher-Simpson, 1983; Silver et. al., 1985] and cAMP levels [Lincoln and Fisher-Simpson, 1983], which correlated with relaxation in rat aorta.  1.3.2  Evidence inconsistent with a role for cAMP in smooth muscle  relaxation Although the hormonally-induced elevation of cAMP levels in vascular smooth muscle is generally thought to lead to relaxation, various investigators have reported observations that have cast doubt on the sufficiency of cAMP in satisfying these criteria [reviewed by Murray, 1990]. This becomes increasingly apparent when considering reports in the literature, which do not show a good correlation between vascular smooth muscle relaxation and increases in cAMP levels and PKA activity. ISO, PGEi and FSK were all able to increase cAMP levels in PEcontracted rabbit aortic rings. However, ISO and FSK produced relaxation, while treatment with PGEi contracted the arterial muscles further.  19  Although pre-  treatment with FSK potentiated the PGEHnduced cAMP elevation up to a 30-fold increase of basal levels, P G E i still produced contraction of the tissues partially relaxed by FSK. ISO was able to relax this contraction with no further increase in cAMP [Vegesna and Diamond, 1986a].  A dose of FSK lower than 1uM was  unable to relax helical strips from bovine coronary artery, although it caused an increase in cAMP and activation of PKA. ISO and higher doses of FSK produced good correlations of these parameters with relaxation of the tissues [Vegesna and Diamond, 1984]. Consistent with a role for cAMP as a mediator of vascular smooth muscle relaxation, PGb elevated cAMP levels and relaxed bovine coronary artery strips. On the contrary, in rabbit aortic rings, PGb-induced cAMP elevation was accompanied by contraction. Although ISO is believed to exert its actions via the cAMP-mediated pathway, it was able to relax the PGb-induced contraction with no further increase in cAMP [Vegesna and Diamond, 1986b]. Moreover, when cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP were used in rat vas deferens, both were shown to increase PKA activity, but only dibutyryl cAMP caused inhibition of contraction, indicating that PKA activation is not responsible for the relaxant effects of cAMP analogs in some smooth muscle [Hei et. al., 1991]. Similarly, in the rat thoracic aorta, PKA activation by cAMP analogs was not well correlated with relaxation [MacDonell and Diamond, 1994]. In uterine smooth muscle,  PGE2  was able to increase cAMP to the same  extent as ISO, yet it caused contraction, which was antagonized by ISO and FSK [Do Khac et. al., 1986]. The possibility that p-adrenergic-linked relaxation may be  occurring  through  both  cAMP-dependent  20  and  cAMP-independent  mechanisms was supported by the studies of Xiong et. al. [1994], who suggested that ISO may be acting partly via a direct action at the membrane level, by modulating the L-type C a  1.4  2+  channel.  ASPECTS OF CELLULAR SIGNALLING TO BE CONSIDERED The multisubstrate nature of PKA [reviewed by Walsh and Van Patten,  1994] requires it to be very specifically regulated.  It has a wide variety of  substrates, all playing diverse roles, all integral to the smooth functioning of the cell, and consequently, the physiological processes the particular cell is involved in. It is clear from the above section that the response obtained seems to have an element of tissue specificity, suggesting that different vascular smooth muscle preparations may have alternative signalling pathways being triggered. It is also possible that the activated kinase phosphorylates different proteins, depending on which ones are present in that particular preparation, thus resulting in a different outcome.  Besides its inherent amino acid sequence and  three-dimensional structure, the parameters of a protein that influence its susceptibility to phosphorylation by PKA include the immediate environment of the target serine residue and any covalent modification that the protein has undergone. The location of the kinase relative to its potential substrate, and vice versa, is also a determining factor.  21  1.4.1 Cellular homogeneity of preparation In vascular preparations which are contracted by  PGEi  in spite of  increased cAMP levels, it may be that PGEi is increasing cAMP levels and activating PKA in non-muscle cells, thereby having no effect on tension. In this way, contractile effects exerted on the smooth muscle cell could not be overcome by the increase in cAMP.  However, it has been shown that PGEi directly  stimulates adenylyl cyclase in isolated vascular smooth muscle cells [Oliva et. al., 1984]. The use of the aortic tunica media should facilitate the study of the function of the vascular smooth muscle cell in the modulation of vascular tone. By definition, it is the middle layer of the vascular wall, the outer layer being the adventitia, and the tunica intima consisting of the one-cell thick endothelium. The only cell type present in the mammalian tunica media is the vascular smooth muscle cell [Rhodin, 1980]. These cells are arranged in helical fashion around the vessel, occupying the main part of the vascular wall [Rhodin, 1962].  1.4.2 Compartmentation of cAMP The criteria described earlier in this introduction, which traditionally have been used to establish cAMP as a mediator of a biochemical action, assume the cytoplasm to be a uniform soluble environment and do not take cellular compartmentation into account. action has been  reviewed  The compartmentation of second messenger  by Harper  et. al. [1985].  The functional  compartmentation of cAMP may be the reason that the increase in cAMP level  22  and subsequent activation of PKA leads to a different response to the different pharmacological agents used. For example, PGEi could be increasing cAMP in a compartment of the vascular smooth muscle cell that does not influence the contractile activity of the cell. Evidence for such compartmentation has been described in cardiac tissue. Both ISO and PGEi were shown to increase cAMP levels in cardiomyocytes, but only ISO had a positive inotropic effect.  The elevation of cAMP in different  cellular compartments, reviewed by Hayes and Brunton in 1982, has been postulated to explain this differential response of cardiomyocytes to ISO and PGEL  [Hayes et. al., 1980a; Buxton and Brunton, 1983]. In these studies, ISO,  but not PGEi, was shown to elevate cAMP in a particulate fraction in the cardiomyocytes. Elevation of cAMP in the particulate fraction in response to ISO, has also been reported by other investigators [Aass et. al., 1988; Zhang and MacLeod, 1996], thus providing further support for the importance of particulate cAMP in the control of cardiac contractility. Cyclic AMP elevation does not always lead to phosphorylation of a specific protein, and depends on i) the accessibility of the kinase to cAMP in the specific compartment and ii) the location of the activated kinase with respect to its substrate. This is described further in the section below. Moreover, not all the phosphorylation that occurs is necessarily contributing to the observed effect and the agonist may be generating another message via a different pathway.  23  1.4.3 1.4.3.1  Compartmentation of PKA Cellular localization of PKA  It may be that selective activation of the different isoenzymes (Type I and Type II) of PKA may lead to distinct functional responses. Type II isoenzyme contains an autophosphorylation site which Type I does not [Rosen and Erlichman, 1975], having instead a high-affinity MgATP binding site [Hofmann et. al., 1975], There is no evidence for cAMP binding to proteins other than Type I and Type II isozymes of the regulatory subunit [Ekanger et. al., 1985].  Type I PKA is  distributed uniformly throughout the cytoplasm , while Type II isoenzymes are found in membrane structures, their location being determined by interaction with anchoring or adaptor proteins. The localization of the kinase relative to the substrate is one of the cellular parameters that dictates the efficacy of the protein in question as a substrate. Akinase anchoring proteins (AKAP) are a group of proteins which, as their name implies, target Type II PKA to specific subcellular locations [reviewed by Coghlan et. al., 1993; Scott and McCartney, 1994], such as microtubules [Nigg et. al., 1985], the cytoskeleton and specific membrane components. From computeraided studies on the sequences of Rll-anchoring protein sequences, the Scott group suggested that an amphipathic helix motif is necessary for interaction with RM [Carr et. al., 1991]. The existence of a high-affinity, 85 kDa, Rlip-binding protein has been demonstrated to localize the regulatory subunit to the Golgi apparatus of human lymphoblasts [Rios et. al., 1992]. Although Type I PKA is generally considered a cytosolic enzyme [Meinkoth et. al., 1990], recent studies  24  have reported the association of Type I regulatory subunit with cardiomyocyte sarcolemma in porcine ventricles [Robinson et. al., 1996].  1.4.3.2  Translocation of the catalytic subunit  Following studies which excluded protein kinase isozyme profiles as an explanation for the different responses observed, Hayes et. al., [1980a] proposed a mechanism to explain the differential responses of cardiac myocytes to ISO and PGEi, by which distinct pools of PKA were being activated by these agents. This proposal was in agreement with an earlier suggestion of a mechanism possibly involving translocation of the catalytic subunit from the membrane to the cytoplasm [Corbin et. al., 1977]. Once the catalytic subunit detaches from the regulatory subunit, it could make its way to the cytosol and exert its action there, thereby increasing soluble PKA activity. This was demonstrated in cardiac tissue by Buxton and Brunton [1983], who showed that ISO increased cAMP levels in both soluble and particulate fractions, and increased PKA activity in the soluble fraction, with a concomitant decrease in activity remaining in the particulate fraction. On the other hand, PGEi increased both cAMP and PKA activity in the soluble fraction, but produced no significant change in either cAMP or PKA, in the particulate fraction.  1.4.4  Activation of cGMP-dependent protein kinase by cAMP Guanosine  3':5'-cyclic  monophosphate  (cGMP)  is  another  cyclic  nucleotide which has been suspected to act as a second messenger in a variety  25  of physiological responses [reviewed by Lincoln, 1989].  It is synthesized from  GTP via the action of guanylyl cyclase and exerts its action by activation of cGMP-dependent protein kinase (PKG).  Phosphorylation by PKG is thought to  be responsible for the vascular relaxant effects of the nitrovasodilator group of compounds, thought to act via nitric oxide (NO) as an intermediary [Rapoport et. al., 1983].  NO, which stimulates guanylyl cyclase to produce cGMP, is also  thought to be part of, or the same chemical entity as endothelium-derived relaxing factor [Palmer et. al., 1987; Ignarro et. al., 1987]. Studies with cAMP and cGMP analogs by Francis et. al. [1988] indicate that the ability of these compounds to relax smooth muscle preparations may be due to their activation of PKG. The authors do not completely exclude a role for PKA, as this would have been activated by the high intracellular concentration of the cAMP derivatives. This group proceeded to provide the first direct evidence of PKG activation by elevated cAMP levels in coronary artery strips exposed to high doses of ISO and FSK [Jiang et. al., 1992], without elevation of cGMP levels. However, with both agents, increase in PKA activity ratio was higher than that in PKG activity ratio. Cyclic AMP-dependent responses were surprisingly lost when PKG was lost from passaged aortic cells: FSK decreased [Ca ]/, but not in the cells that 2+  had been depleted of PKG [Lincoln et. al., 1990].  In contrast, PKG and PKA  have been proposed to act separately and in concert to decrease [Ca ], to 2+  induce vascular smooth muscle relaxation [Murthy and Makhlouf, 1995].  26  1.5  SUMMARY AND RATIONALE FOR PROPOSED RESEARCH It is generally assumed that the activation of PKA is responsible for cAMP-  induced vascular relaxation, yet the evidence presented in the above introduction clearly indicates that the precise functions of cAMP and PKA in mediating this outcome still remain to be elucidated. An increase in PKA activity ratio has been correlated with relaxation in a variety of vascular preparations. ISO and PGEi have both been shown to increase cAMP levels in vascular smooth muscle. However, ISO causes relaxation, while tissue further.  PGEi  contracts the  When PGEi is combined with the adenylyl cyclase activator,  forskolin, the cAMP levels have been shown to be considerably increased above basal, yet once again, PGEi will cause a contraction on top of the relaxation induced by forskolin. ISO relaxed this contraction with no further increase in cAMP [Vegesna and Diamond, 1986a]. These events lead to an investigation into what really goes on to produce vascular smooth muscle relaxation. Why do both ISO and PGEi increase cAMP levels, yet lead to opposing changes in vascular smooth muscle tension? The present study was intended to be an exploration into the basic nature of the cAMP activation of PKA and its resultant effect on vascular smooth muscle tone. One plausible explanation for these conflicting data might be similar to that suggested by Buxton and Brunton [1983] to clarify a similar observation in cardiomyocytes.  These authors concluded that different pools of PKA were  being activated by ISO and PGEi, involving the translocation of the catalytic subunit from the particulate to the soluble fraction. PGEi was shown to be unable  27  to increase cAMP in the particulate fraction, and it was suggested that activation of the kinase in that fraction was necessary for the production of a change in contractility. Another possible explanation is that ISO-induced relaxation is due to cAMP activation of PKG, which is somehow unable to be activated by PGEi.  1.5.1  Hypothesis The current investigation was designed to test the hypothesis that the  elevation of cAMP and the subsequent activation of PKA play an important role in the relaxation of vascular smooth muscle.  1.5.2  Specific objectives of the present investigation To determine whether the circumstances in vascular smooth muscle were  analogous to events occurring in cardiac tissue, experiments were carried out in an attempt to realize the following objectives: 1. To investigate the nature of the tension change produced by ISO and PGEi, when applied to a steady-state contraction induced by phenylephrine 2. To investigate the activation of PKA by these drugs and explore the possibility of translocation of the catalytic subunit as a mechanism for the different pharmacological responses to ISO and PGEi 3. To measure the levels of cAMP produced by these drugs, and investigate the compartmentation of this cyclic nucleotide in relation to the response obtained. An integral part of the study was the use of a vascular smooth muscle preparation consisting of essentially only smooth muscle cells, which responded  28  functionally to contractile and relaxant agents. The direct comparison of tension change with cyclic nucleotide elevation and kinase activation was an important component of the research design.  29  2.0  MATERIALS AND METHODS  2.1  Chemicals and Materials Chemicals and materials were obtained form the following sources:  Amersham International pic (Little Chalfont, Buckinghamshire, England) BIOTRAK™cAMP enzymeimmunoassay (EIA) system (dual range) BDH Inc. (Toronto, Ontario, Canada) ethylenediaminetetraacetic acid ethanol (95%) D-glucose, anhydrous di-potassium hydrogen orthophosphate sodium fluoride sodium hydrogen carbonate sulphuric acid Bio-Rad Laboratories (Hercules, California, U.S.A.) protein assay kit DuPont NEN Research Products (Boston. MA, U.S.A.) [y- P] adenosine 5'-triphosphate, 2 mCi/ ml 32  Fisher Scientific (Fairlawn, NJ, U.S.A.) aluminium foil calcium chloride magnesium acetate magnesium sulphate o-phosphoric acid  30  potassium chloride potassium dihydrogen phosphate ScintiVerse® scintillation fluid Medigas (Vancouver, British Columbia, Canada) 5% carbon dioxide/ 95% oxygen MTC Pharmaceuticals (Cambridge, Ontario, Canada) sodium pentobarbital injection, 65mg/ml Sioma Chemical Co. (St. Louis, MO, U.S.A.) adenosine 3':5'-cyclic monophosphate (sodium salt) L-ascorbic Acid DL-dithiothreitol (Cleland's Reagent) forskolin f3-glycerophophate (disodium salt) guanosine 3':5'-cyclic monophosphate (sodium salt) N -[2-hydroxyethyl] piperazine - N'- [2 - ethanesulfonic acid] (HEPES) 3-isobutyl-1 -methylxanthine (-)-isoproterenol hydrochloride kemptide lithium sulphate microcystin - LR L-phenylephrine hydrochloride prostaglandin Ei (synthetic) Triton® X-100  31  Whatman Ltd. (Maidstone. Kent, England) P81 phosphocellulose paper  2.2  DRUG SOLUTIONS Phenylephrine (PE) and isoproterenol (ISO) solutions were made up in  distilled water. ISO was dissolved in the presence of ascorbic acid (1 mg/ml) to reduce oxidation. Forskolin (FSK) and prostaglandin Ei (PGEi) were dissolved in 65% ethanol. The light-sensitive PGEi solution was prepared and kept in darkness. Stock solutions (10 mM) of FSK and PGEi were stored at -70°C and removed 45 minutes before use.  All drug solutions were made up in their  respective diluents and added to the baths to give the desired final concentration. Control strips were treated with the appropriate volume of water or 65% ethanol. Neither diluent had any effect on tension.  2.3  ANIMALS The New Zealand white rabbits (2-3 months old) used in this study were  obtained from the Animal Care Facility, University of British Columbia, and housed with free access to water and food.  2.4  PREPARATION OF RABBIT AORTA New Zealand white rabbits of either sex, weighing 2-3Kg, were sacrificed  using 65mg/Kg sodium pentobarbital injected through the ear vein.  After  exsanguination, the descending thoracic aorta was dissected out and placed into  32  a freshly-prepared, oxygenated (pH 7.4), physiological salt solution (PSS) with the following composition (mM): KCI (4.7), K H P 0 (1.19), M g S 0 (1.19), CaCI 2  4  4  2  (1.26), NaCI (118), D-glucose (11), N a H C 0 (25). The aorta was then trimmed 3  free of loosely adhering fat and connective tissue and used to prepare helical strips or medial strips.  2.4.1  Helical Strips The endothelium was removed by inserting a glass rod of approximately  the same diameter throughout the length of the aorta. Following removal of the rod, the aorta was held at one end using a pair of forceps, and cut in helical fashion along its length, to form one long piece of tissue when unravelled. It was then cut into strips, each approximately 5 mm wide and 14 mm long.  2.4.2  Medial Strips The tunica media was prepared according to the method described by  Fallier-Becker et. al. (1990). The aorta was pinned down and cut longitudinally between the exits of the intercostal arteries to expose the endothelial surface of the vessel. The endothelium was removed by gentle rubbing with the blunt edge of a pair of forceps. Using two pairs of fine forceps the medial layer was gently teased away from the adventitia, and then gently peeled off into strips approximately 4 mm wide and 10 mm long. Light microscopy of hematoxylin and eosin-stained tissues determined the cellular homogeneity of the medial strips (Figure 8).  33  Figure 8. Photograph of medial strip tissue. Magnification is X 400.  2.5  TISSUE-BATH STUDIES The aortic strips were suspended for 60 minutes under 1 g resting  tension in 20 ml tissue-baths containing PSS of the composition described  34  above.  The PSS was replaced with fresh solution every 15 minutes during  equilibration and bubbled with 5% C 0 in 0 to maintain the pH at approximately 2  2  7.4. Water jackets around the baths ensured that the temperature was kept at 37°C. Each strip was contracted with 1 u.M PE to ensure its viability, as well as its ability to sustain a contraction. The PE was washed out after 15 minutes. Following relaxation of the tissues to baseline tension, the strips were contracted once more with 1 u.M PE. After 15 minutes, at steady-state contracture, control muscles were exposed to an appropriate volume of water or 65% ethanol, and test muscles were exposed to different drugs for a pre-determined duration of time (see Table 1).  Drugs used included 1 uM ISO, 10 uM PGE^ or a  combination of 1 u.M FSK with 10 u.M PGEi, previously shown to synergistically increase cAMP well above control levels [Vegesna and Diamond, 1986]. Experiments using PGEi were all carried out with the lights switched off and the respective tissue-baths wrapped in aluminium foil, to minimize the amount of light that reached the light-sensitive drug. The tissues were frozen using liquidnitrogen-cooled clamps and kept at -70°C until assayed for cAMP or PKA activity.  35  Helical Strips Treatment  Medial Strips Time  Treatment  (minutes)  water  2  6 5 % ethanol  5/10  ISO  water 6 5 % ethanol  2  FSK/PGET  5 / 1 0  Time  (minutes)  2 10  ISO  2  PGET  10  FSK/PGEi  5 / 1 0  Table 1. Treatment of aortic strips following contraction with 1 u,M phenylephrine 2.6  MEASUREMENT OF CYCLIC AMP The measurement of cAMP in total homogenate, soluble and particulate  fractions of homogenized tissue was done according to a procedure modified from that used in cardiac tissue [Aass et. al., 1988; Zhang and MacLeod, 1996].  2.6.1  Tissue Extraction Similarly-treated tissues, with similar responses, were pooled to a total  weight ranging from 30 - 50 mg and powdered in a liquid-nitrogen-cooled Teflon™ capsule using a ProMix™ dental amalgam mixer for 20 s at high speed. They were then homogenized in 20 volumes of ice-cold 50 mM potassium phosphate buffer (pH 7) containing 4 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM iso-butyl-methyl-xanthine (IBMX), 50 uM cGMP [Ekanger et. al., 1985] and 20% L i S 0 (saturated) [Aass et. al., 1988]. 2  4  36  An aliquot of homogenate, from each set of pooled tissues, was removed and placed into a polypropylene micro test tube with twice its volume of 95% ethanol [Bansinath et. al., 1994], vortexed gently, and used for homogenate cAMP estimation. The rest of the homogenate was centrifuged at 40,000 x g for 15 minutes at 4°C in a Heraeus Sepatech Contifuge 28 RC. An aliquot of the supernatant was removed and added to twice its volume of 95% ethanol and vortexed gently.  The rest of the supernatant was removed.  The pellet was  washed with homogenization buffer, disrupted, and re-constituted in ice-cold buffer/ 95% ethanol (1:2). The supernatant and pellet mixtures were used for estimation of soluble and particulate cAMP respectively. The ethanol mixtures were centrifuged at 2000 x g for 15 minutes at 4°C in a Heraeus Sepatech Contifuge 28 RC. The supernatants were removed into a 12 x 75 mm glass test tube and evaporated to dryness under a nitrogen stream. They were kept at -70°C until assayed for cAMP, within 24 hours. The pellets were washed with homogenization buffer, re-suspended in 200u.l homogenization buffer supplemented with 0.1% Triton® X-100, left to stand on ice, and vortexed gently every 10 minutes for 30 minutes and again at the end of 1 hour, at which point they were used for protein estimation. This process enabled the expression of results on a pmol cAMP-per-mg protein basis. A schematic representation of this process is presented in Figure 9.  37  HOMOGENATE  Centrifugation 40,000 x g, x 15min. @ 4°C supernatant  aliquot + 95% ethanol (2 vols.)  pellet  aliquot + 95% ethanol (2 vols.)  washed broken up and reconstituted in buffer/95% ethanol (1:2)  (soluble)  (total)  (particulate)  Centrifugation 2,000 x g x 15 min. @ 4°C pellet  supernatant  I  reconstituted (1hr./0.1% Triton X-100)  dried under N2 stream  protein assay cAMP Assay  Figure 9. Schematic representation of cAMP extraction process.  2.6.2  Cyclic AMP assay Measurement of cAMP was done using a cAMP enzymeimmunoassay  (EIA) kit, commercially available from Amersham LIFE SCIENCE®. De-ionized water was used to dilute the assay buffer concentrate present in this kit. The  38  assay buffer was in turn used to make up the other components, as well as to reconstitute the dried tissue extracts, further diluted with de-ionized water. Homogenate and soluble fraction extracts from helical strips were diluted fivefold, while particulate fractions were diluted two-fold. All extracts from medial strip tissue were diluted five-fold.  The dilution factor had been previously  determined using range-finder experiments.  2.7  PKA MEASUREMENTS Frozen tissues were assayed for PKA activity within 4 weeks. Soluble  and particulate fractions were assayed for PKA activity using a procedure based on that described by Giembycz and Diamond (1990a), modified from the method suggested by Corbin and Reimann (1974).  2.7.1  Tissue extraction Similarly-treated (and similarly-responding) tissues were pooled to a total  weight ranging from 50 - 70 mg and powdered in a liquid-nitrogen-cooled Teflon™ capsule using a ProMix™ dental amalgam mixer for 20 s at high speed. They were then homogenized in 10 volumes of ice-cold 10 mM HEPES buffer (pH 7.4) containing EDTA (10 mM), IBMX (0.5 mM), dithiothreitol (10 mM) and NaCI (150 mM).  The soluble fraction consisted of the supernatant from  cehtrifugation of the homogenate at 31,000 x g at 4°C for 15 minutes. After being washed with homogenization buffer, the pellet was re-suspended in a similar volume of buffer containing 0.1% Triton® X-100, and agitated every 10  39  minutes for 30 minutes and then again at the end of 1 hour. Centrifugation at the same conditions produced a supernatant containing the particulate fraction. The fractions were kept on ice and assayed for PKA activity as soon as possible after their preparation.  2.7.2  PKA assay The PKA assay was initiated by adding 20  JLLI  of the fraction to be  assayed to 50 uJ of a reaction cocktail consisting of: HEPES buffer (10 mM) at pH 7.4, Kemptide (71 uM) as a substrate for PKA, 3-glycerophosphate (35 mM), magnesium acetate (10 mM), ATP (150 u.M), microcystin-LR (100 nM), and 32  [y-  P] ATP (0.5 uCi/ tube). The mixture was vortexed and incubated at 30°C for 8  minutes. A 50 u.l aliquot was then spotted onto Whatman P81 phosphocellulose paper, pre-coated with 1 mM ATP and 10 mM  K H 2 P O 4 .  The papers received 4  successive washes of 10 minutes each in 0.5% o-phosphoric acid before being allowed to dry and placed in scintillation vials.  Quantitation of the bound  radioactivity was determined by adding 3 ml scintillation fluid (ScintiVerse®) and counting in a liquid scintillation counter. A "no-substrate" blank was present for each sample.  An aliquot of each fraction was used for protein estimation,  enabling the activity of PKA to be expressed as picomoles of phosphate incorporated into the kemptide substrate per  minute per mg  Activation of the kinase was described in terms of the Activity Ratio:  40  of protein.  Activity Ratio =  activity in absence of added cAMP activity in the presence of enough cAMP to maximally activate the enzyme  Figure 10. Calculation of the PKA activity ratio.  2.8  PROTEIN ESTIMATION Protein levels were estimated using a commercially available (Bio-Rad)  dye-binding microassay based on the principle that various concentrations of protein produce a differential colour change of an acidic solution of Coomassie Brilliant Blue G-250 dye [Bradford, 1976]. Bovine serum albumin was used as a protein standard.  2.9  STATISTICAL A N A L Y S E S The statistical tests that were used are specified in each case.  Non-  parametric tests were employed when the data did not fit a normal distribution, or the groups being compared did not have equal variances.  41  3.0  RESULTS  3.1  NATURE OF TENSION CHANGE Endothelium-denuded muscle strips from rabbit thoracic aorta, with or  without the presence of the adventitia, were contracted with PE before being exposed to ISO, PGEi and FSK according to the protocol described in Table 1, in the previous section. Figure 11 depicts a representation of this protocol and the responses obtained with these agents.  The  nature of the  responses  corresponded with earlier studies [Vegesna and Diamond, 1986a], which had raised the question of functional compartmentalization of cAMP and PKA in vascular smooth muscle. As expected, while control treatments, water and 65% ethanol, caused no or minimal response respectively,  ISO  relaxed both  preparations (Figure 12), and PGEi caused a further contraction in medial strips (Figure 13). In the helical strips, PGEi also contracted strips that were partially relaxed by FSK.  Surprisingly, in the medial strips, the FSK-induced partial  relaxation was followed by a further relaxation when PGEi was added to the tissue-baths (Table 2). These results demonstrate that, following contraction with PE, both medial and helical strips were relaxed by 1 uJvl ISO, and medial strips were contracted by 10 uM PGEi.  On the other hand, a number of FSK-treated medial strips  responded differently to 10 u.M PGEi (i.e. they relaxed).  42  Figure 11.  Protocol for drug treatment, and response observed, of rabbit  aorta helical strips and medial strips. The traces show each type of treatment protocol. In each case, the tissue was contracted with phenylephrine (PE) and allowed to reach steady-state for 15 minutes, a) Water (H 0) was added, and 2  the tissue was frozen after 2 minutes, b) 65% ethanol (Eth) was added and the tissue was frozen after 10 minutes, c) 1 u,M isoproterenol (ISO) was added and the tissue was frozen after 2 minutes, d) 10 u.M prostaglandin Ei (PGEi) was added and the tissue was frozen after 10 minutes, e) 1 u.M forskolin (FSK) was added and partially relaxed the tissue. After 5 minutes, 10 u,M PGEi was added, and reversed the response.  The tissue was frozen 10 minutes later.  particular trace emphasizes the contractile response of  PGEi.  This  It under-  represents the prominent relaxation that is generally produced by FSK. f) The tissue was treated exactly the same as above, but the trace shows an example of the PGEi treatment causing no contraction following partial relaxation by FSK. Traces a), b), and c) represent protocols done in both types of tissue. Trace d) is from an experiment using medial strips, while traces e) and f) contrast the response obtained by using the FSK/PGEi combination in helical and medial strips respectively.  The time-points for the protocol were chosen to duplicate  conditions present in previous studies, which had correlated cAMP elevation to response in a time-dependent fashion.  43  44  Figure 12. Effect of 1 u,M isoproterenol (ISO) on the phenylephrine-induced contraction in helical strips (HS) and medial strips (MS). presented as the percent  relaxation, by  contractions in each preparation.  ISO,  of  The response is  phenylephrine-induced  Numbers in bars indicate sample numbers.  The responses of the two preparations are significantly different (*) from each other and from control tissues, Mann-Whitney test, p < 0.05.  45  % Relaxation o  o  ro o  w o  1  1  1  -k  HS  -  *»• o  *  *  Figure 13. Effect of 10 uM prostaglandin Ei (PGEi) on the phenylephrineinduced contraction in medial strips as compared with that of 65% ethanol (Eth). Numbers in bars indicate sample numbers. The response is presented as the percent increase in the PE-induced contractions on addition of the drug or the vehicle (Eth) respectively.  10 uM PGEi caused a significant (*) contraction,  Mann-Whitney test, p < 0.0001  47  % Contraction  00  Table 2. Effects of PGE on rabbit aorta helical strips and medial strips, pre-treated with FSK. The responses are represented in terms of the percentage of the PE contraction which was modulated on addition of the drugs. 1  HELICAL STRIPS  MEDIAL STRIPS  Drug  Response  n  Response  n  FSK (1 uM) +  relaxation  6  relaxation  15  P G E T (10UM)  contraction  6  contraction relaxation none  3  Total  10  2  15  6  The responses are presented in terms of the change in the P E contraction which was produced on addition of the drugs. F S K , forskolin. P G E ^ prostaglandin E  49  v  3.2  PKA STUDIES Similarly-treated medial strips were pooled and assayed for PKA activity  within 4 weeks of being frozen, according to the method described in Section 2.7. Comparison of the activity ratio of soluble PKA between control tissues and tissues treated with ISO (1 uM) or PGEi (10 uJvl) would indicate whether both these drugs were activating PKA. Past reports have indicated that both of these drugs increase cAMP levels in rabbit aorta [Vegesna and Diamond, 1986a]. Due to the time that passes between preparation of the particulate fraction and measurement of activity, the particulate activity ratio is not a reliable indication of PKA activation in this fraction. However, translocation of the catalytic subunit of PKA from the particulate to the soluble fraction would be identified by observing a lower particulate percentage of total activity remaining in the treated tissues than in controls, together with a corresponding increase in soluble activity ratio. Buxton and Brunton [1983] correlated these measurements to the cAMP elevation in each fraction, to explain the differential responses to ISO and PGEi in an analogous situation in cardiomyocytes. In the present set of experiments, it was not possible to record a significant increase in PKA activity ratio over basal levels, with either ISO or PGEi. The major source of concern was the persistently high activity ratio obtained in control tissues. The basal activity ratio in phenylephrine-contracted strips from rabbit aorta in these studies was 0.34, as opposed to the earlierreported 0.2 [Vegesna and Diamond, 1986c]. Exposing both helical (n=2) and medial (n=4)  strips pre-treated with 1  uJvl  50  FSK to 10 u,M PGEi, a combination  reported to synergistically increase cAMP levels [Vegesna and Diamond, 1986a], was still not successful in demonstrating PKA activation. It was thus inferred that the assay method was not working. Following unsuccessful attempts to improve the assay by decreasing the centrifugation time, and by employing a modification of the procedure previously used successfully in the same laboratory [MacDonell and Diamond, 1994], the differential responses of ISO and PGEi were investigated by exploring the compartmentation of cAMP.  3.3  CYCLIC AMP STUDIES  In a pilot study carried out in helical strips obtained from 6 rabbits, the FSK/PGET  combination significantly increased cAMP above basal in the soluble  fraction (Figure 14).  Interestingly though, the increase in the particulate fraction  (Figure 15) was not significant. I S O had no significant effect on cAMP levels in either fraction.  51  Figure 14.  Effects o f 1 u.M isoproterenol (ISO) and a combination o f 1 u.M  f o r s k o l i n a n d 10 p.M prostaglandin Ei ( F S K / P G E 1 ) o n c A M P levels in the s o l u b l e fraction of rabbit aorta helical strips. Effect of treatment is expressed  in pmol cAMP/mg protein. Numbers in bars indicate sample numbers.  The  FSK/PGE combination significantly (*) increased cAMP levels above basal, 1way ANOVA, Dunnett's test, P < 0.05.  52  1500 1250 O Q.  1000  O)  E  IS o £ Q_  750 500 250  Basal  ISO  53  FSK/PGE1  Figure 15. Effects of 1 uM isoproterenol (ISO) and a combination of 1 u,M forskolin and 10 p.M prostaglandin  (FSK/PGE1) on cAMP levels in the  particulate fraction of rabbit aorta helical strips.  Effect of treatment is  expressed in pmol cAMP/mg protein. Numbers in bars indicate sample numbers. The drugs did not significantly change cAMP levels above basal, 1-way ANOVA.  54  1500 1250 1000  750 _  500 250 0  Basal  ISO  55  FSK/PGE1  Figure 16. Effects of 1 uM isoproterenol (ISO) and a combination of 1 (iM forskolin and 10 p.M prostaglandin Ei (FSK/PGE1) on total tissue cAMP levels in helical strip preparations. Numbers in bars indicate sample numbers. A significant (*) increase in cAMP above basal was obtained with the FSK/PGE combination, Kruskal-Wallis ANOVA on ranks, p < 0.05.  56  4000  Basal  ISO  57  FSK/PGE1  Using the above results as an indication that cAMP could be measured in different fractions of homogenized aortic tissue, the experiment was repeated in the tunica media obtained from 9 rabbit aortas. Unexpectedly, the observations recorded in the helical strips could not be repeated in the medial strips.  No  significant increase in cAMP (Figure 17, 18) over basal levels was found with any of the drugs or combinations used. Total tissue cAMP content in each treatment sample is depicted in Figures 16 and 19. Once again, a significant elevation of cAMP was registered in the helical strips when treated with the FSK/PGE combination, yet not reproduced in the medial strips. The basal cAMP level is not significantly different (p = 0.416) between the two tissue preparations, Student's t - test. These results indicate that, as in past studies [Vegesna and Diamond, 1986a], cAMP levels were significantly increased by the combinations of FSK and PGEi in intact rabbit aorta muscle strips. On the other hand, there is also a strong suggestion that none of the drugs used are successful in elevating the cAMP level above basal when the strips are denuded of the adventitia.  58  Figure 17.  Effects of 1 u.M isoproterenol (ISO), 10 u,M prostaglandin Ei  (PGE1), and a combination of 1 u,M forskolin and 10 u.M prostaglandin Ei (FSK/PGE1) on cAMP levels in the soluble fraction of rabbit aorta medial strips. Effect of treatment is expressed in pmol cAMP/mg protein. Numbers in bars indicate sample numbers. There is no significant difference between any treatment and basal levels of cAMP, 1-way ANOVA on ranks, Dunn's test.  59  60  Figure 18. Effects of 1 uM isoproterenol (ISO), 10 p.M prostaglandin Ei (PGE1), and a combination of 1 iiM forskolin and 10 u,M prostaglandin E^ (FSK/PGE1) on cAMP levels in the particulate fraction of rabbit aorta medial strips. Effect of treatment is expressed in pmol cAMP/mg protein. Numbers in bars indicate sample numbers. There is no significant difference between any of the groups, 1-way ANOVA on ranks, Dunn's test.  61  800  62  Figure 19.  Effects of 1 u.M isoproterenol (ISO), 10 p.M prostaglandin Ei  (PGE1), and a combination of 1 p.M forskolin and 10 LIM prostaglandin Ei (FSK/PGE1) on total tissue cAMP levels in medial strip preparations. Numbers in bars indicate sample numbers. There was no significant difference between any of the groups, Kruskal-Wallis ANOVA on ranks.  63  64  4.0  DISCUSSION This study was carried out to investigate the hypothesis that the elevation  of cAMP and the subsequent activation of PKA play an important role in the relaxation of vascular smooth muscle. The results obtained are not sufficient to either support or negate this hypothesis.  The data do suggest a) that it is  possible to measure cAMP levels in different subcellular fractions of vascular smooth muscle, and b) that there are differences between the tunica media and the adventitia with respect to their functional response to tension-modulating drugs and the corresponding increases in cAMP levels. The medial strip is a vascular smooth muscle preparation which is functionally responsive to PE, ISO,  PGEi and FSK, yet eliminates the  confounding influences of cellular events occurring in non-smooth muscle cells. This preparation was used in these studies in an attempt to decrease sources of variation, but it may have raised more questions than it answered. The other preparation used in these studies, the helical strip, still retains the adventitia. It was used in an attempt to reproduce the tension change and cAMP measurements that the hypothesis of this study was based on, and in preliminary efforts to measure cAMP in different subcellular fractions of vascular smooth muscle. There do not appear to be any other studies in the literature which a) use a "pure" intact muscle preparation that is functionally responsive to contractile and relaxant drugs and b) measure cAMP in subcellular fractions of vascular smooth muscle tissue.  65  4.1  NATURE OF DRUG-INDUCED TENSION CHANGE IN VASCULAR  SMOOTH MUSCLE Both of the rabbit aorta preparations used in this study were contracted by PE and relaxed by ISO.  PGEi produced a definite contraction in medial strips,  on top of that induced by PE.  These responses correspond well to those  reported in rabbit aortic rings in a study by Vegesna and Diamond [1986a], who also reported that pre-treatment with FSK potentiated the effect of PGEi on cAMP levels, elevating these up to 30 times basal amounts. In that study, FSK produced a relaxation which was partially reversed on addition of PGEi.  In the  present study, similar results were obtained in rabbit aortic helical strips, but not in medial strips. In the medial strips, PGEHnduced contraction was occasionally observed (in 3 of 15 preparations), but in the majority of preparations, the FSKinduced relaxation was either unaffected or potentiated by PGEi.  The only  apparent explanation for this occurrence would be based on the assumption that events happening in non-muscle cells are responsible for their contraction in response to PGEi. Nevertheless, the opposing functional responses of PE-contracted medial strips to ISO and PGEi established the basis for an investigation into the cyclicnucleotide-related events involved in their mediation. It is difficult to explain the observation that the degree of relaxation induced in the medial strips was significantly greater than that in the helical strips, exposed to the same dose of ISO (1 uM). Once again, one possibility is that the presence of the adventitial cells is somehow diminishing the muscle's ability to relax.  66  Of course, the  element of co-incidence cannot be excluded, but it may be interesting to compare the [Ca ] in adventitial and muscle cells at rest, as well as during contraction and 2+  relaxation. The orientation of muscle cells may be different between the two preparation, and may influence the extent of responses. The tunica media and the adventitia of the thoracic aorta are quite different from each other with respect to their cellular component. The adventitia contains no smooth muscle cells, in addition to the fibroblasts and macrophages that are present.  Its other components are myelinated and non-myelinated  nerves, collagenous fibrils and vasa vasorum. It serves as a layer that carries nutrients to the smooth muscle cells of the media [Rhodin, 1980].  4.2  ACTIVATION OF cAMP-DEPENDENT PROTEIN KINASE Difficulties were encountered in trying to detect an increase in activity ratio  above basal, even in muscle strips treated with a combination of FSK and PGEi, known to elevate cAMP levels 30-fold [Vegesna and Diamond, 1986a] and to increase PKA activity ratios up to 230% above the basal level [Vegesna and Diamond, 1986c].  It is extremely unlikely that such a great increase in cAMP  would not activate PKA. Incidentally, this increase in cAMP was not mirrored in the medial strips. Nevertheless, it was still not possible to measure activation of the kinase when the assay was repeated in helical strips. The only other explanation for the failure to measure activation of the kinase is that some factor (or factors) in the assay procedure or reagents must be interfering with the measurement of kinase activation.  67  To this end, the  centrifugation time was decreased from 15 to 5 minutes, in the hope that less kinase activity would be lost, or less kinase would be newly-activated in the control tissues, by cAMP possibly released during homogenization. The change in the centrifugation time did not reveal different results in the present study. It has been found that too vigorous a homogenization can shift the cAMP/PKA equilibrium such that more free catalytic subunit is released and that this subunit is more susceptible to denaturation than the holoenzyme [Corbin, 1983]. The composition of the homogenization buffer was designed to prevent the degradation of cAMP and inactivation of the kinase. EDTA chelates C a  2+  ions (which enhance the action of PDE), thus working in conjunction with the PDE inhibitor, IBMX, to prevent cAMP degradation.  On the other hand, PDE  inhibitors themselves have been reported to increase cAMP levels [Kramer and wells, 1979].  Reverting to a homogenization buffer that had previously been  used successfully [MacDonell and Diamond, 1994] still did not provide a mechanism by which PKA activation could be estimated. Corbin [1983] also described potential aspects of the assay procedure which may introduce an element of uncertainty, including the non-specific activation of other kinases in the cell, the presence of  phosphoprotein  phosphatases and the release of sequestered cAMP. The substrate, kemptide, has been shown to be preferable to histone I la as a substrate for estimating PKA activity  in  guinea-pig  lung  [Giembycz  and  Diamond,  1990a].  Beta-  glycerophosphate and microcystin-LR were included in the reaction mixture to inhibit the de-phosphorylation of kemptide by phosphatases. Modification of the  68  cocktail in accordance with previous successful measurements [MacDonell and Diamond, 1994] did not make a difference to the outcome of the assay, although it did suggest that the HEPES buffer may be ruled out as a factor inhibiting the kinase.  Lowering the concentration of cAMP added to maximally activate the  kinase and lowering the concentration of Mg acetate still made no difference. Thus, at the present time, we have no explanation for our inability to measure activation of PKA in vascular tissues known to have elevated levels of cAMP.  4.3  CYCLIC AMP ELEVATION IN VASCULAR SMOOTH MUSCLE In helical strips, total tissue cAMP levels were significantly increased by  the FSK/PGEi combination, up to 10-fold the basal level. This is in agreement with the previous results [Vegesna and Diamond, 1986a].  Changes in cAMP  levels were also detected in subcellular fractions from helical strips of rabbit aorta.  To the best of our knowledge, the measurement of cAMP in different  subcellular fractions of vascular smooth muscle tissue has not been reported before, so a comparison with other previous reports cannot be made. It can only be assumed that the measurements that have been made previously, represent, to a large extent, the cAMP that is extracted from the soluble fraction of the cell. The FSK/PGEi combination produced a ten-fold cAMP increase in the soluble fraction. A significant ISO-induced cAMP increase was not detected in either fraction. ISO has been reported to inhibit contractions in other types of smooth muscle with little effect on cAMP levels [Nesheim et. al., 1975; Marshall and Fain, 1985].  69  The helical strips were used in pilot studies on the measurement of cAMP in subcellular fractions because they are easier and faster to work with. The use of the combination of FSK and PGEi was a positive control to ensure that the procedure could detect a known increase in cAMP.  It would have been very  interesting to have PGEi as a test drug in these studies, as it was in subsequent studies with the medial strips. Knowing the subcellular distribution and increase of cAMP in vascular smooth muscle tissue on exposure to PGEi could have enabled a comparison to be made with that obtained for ISO.  In this way, the  compartmentalization of this cyclic nucleotide may or may not have been implicated in the different responses of vascular smooth muscle to the drugs. Buxton and Brunton [1983] carried out a similar study in cardiomyocytes, where they demonstrated that hormonally-specific compartmentation of PKA within the cardiomyocyte was responsible for the different influences caused by these drugs.  Both 1 u,M ISO and 10 uM PGEi increased soluble cAMP levels  and the activity ratio of soluble PKA, but only ISO resulted in a change in cAMP levels in the particulate fraction and a translocation of protein kinase activity from the particulate to the soluble fraction. To explore the possibility of subcellular compartmentation of cAMP in vascular smooth muscle, similar studies were repeated in rabbit aorta medial strips exposed to ISO and to  PGEi.  The FSK/PGEi combination was also used  to indicate whether cAMP was being measured or not. Figures 17, 18 and 19 in Section 3.3 demonstrate that it was not possible to detect an elevation of cAMP levels with any of the drugs, in either fraction. On a pmol cAMP/g tissue basis,  70  the basal cAMP level was higher than that in tissues treated with 1 u,M ISO, 10 uM PGEi and the FSK/PGEi combination. Although these results represent tissue sampled from 9 rabbit aortas, they are most anomalous.  FSK is an adenylyl cyclase activator, which should be  increasing cAMP levels well above basal, even if it weren't combined with PGEi. The medial strip is essentially a "purer" tissue than the helical strips, and one might expect less variation in response to drugs in a tissue where the only cell present was the vascular smooth muscle cell.  Relying solely on these results  would imply that either 1) cAMP elevation does not occur in the vascular smooth muscle part of the blood vessel wall and/or 2) cAMP is not responsible for the observed tension changes with these drugs. No significant difference was noted between the basal level of total cAMP in medial strips versus helical strips. Since the same assay procedure was used in both preparations, there may be a factor influencing the measurement of cAMP in medial strips that was either not present, or not as consequential, in the helical strips, or vice versa. The extraction procedure used was modified from that used by Aass et. al., [1988], who described the potential sources of error when making estimates of this nature. This procedure was also adapted for use by Zhang and MacLeod [1996]. L i S 0 was used to precipitate bound cAMP, as it has been reported to 2  4  be just as effective as (NH ) S0 [D0skeland et. al., 1977], without interfering 4  2  4  with cAMP analysis the way (NH ) S0 did [Aass et. al., 1988]. The presence of 4  2  4  the sulphate, together with the low temperatures that were maintained, served to stabilize the protein kinase and prevent the loss of cAMP due to heat evolved  71  during the homogenization process [Terasaki and Brooker, 1977].  In order to  minimize, during homogenization and subsequent stages, artefactual binding of cAMP to sites that were vacant in the intact cell, cGMP was included in the homogenization medium [Ekanger, et. al., 1985], at a low concentration which would not interfere with the cAMP EIA. In an effort to lessen the overestimation of cAMP bound due to contamination of pellets with cAMP from the supernatants, the surface of the pellets was washed once with homogenization buffer prior to extraction of cAMP. The effect of potential proteolysis was not checked. The EIA is based on a cAMP-antibody-binding principle. This, and the high ionic strength recommended [Daskeland et. al., 1977] for assaying cAMP, may introduce a potential limitation of these measurements when such a high concentration of  I J 2 S O 4  (2.4M) is used. Although this salt was present in similar  concentrations during the assays of both types of muscle strip preparations, the possibility of its interference cannot be completely excluded. There is still no explanation for the reported observation that ISO and PGEi both increase cAMP levels in vascular smooth muscle, with the former causing relaxation and the latter, contraction. Further studies need to be carried out in this regard.  4.4  FUTURE EXPERIMENTS The current measurements of cAMP did not reflect the involvement of this  cyclic nucleotide in vascular smooth muscle relaxation that has been suggested to be present.  They neither support nor negate the suggestion that beta-  72  adrenergic relaxation is mediated via cAMP.  Further studies should include a  similar research design to the current study as well as an investigation into differences in cellular signalling with or without the presence of the adventitia. The endothelium, although only one-cell thick, is a complex layer, very rich in intracellular signal transduction  pathways.  Its mechanical removal and  subsequent microscopy to establish the extent of its removal was assumed to be enough guarantee of its absence in the present studies. In future experiments, it may be worthwhile to test each tissue pharmacologically for the presence of the endothelium before exposing it to cAMP-elevating agents. There are various ways to improve the above studies and produce more meaningful results.  It would still be desirable to measure PKA activation and  cAMP elevation in the same tissues that produce a tension change. Recording the tension change on a g/mm basis may enable direct correlations between 2  tension change, cAMP elevation and PKA activation. The reliability of the cAMP extraction procedure needs more investigation, especially with respect to the effect of the lithium sulphate in each type of preparation. The PKA assay itself might be made functional by carrying out a detailed optimization of conditions for measurement of PKA activity in medial strip tissue.  Specific considerations  would include the necessity and concentration of each ingredient in the homogenization buffer and the reaction cocktail, as well as temperature and time courses. An alternative method may be to bypass this PKA assay altogether and focus on monitoring the cAMP activation of PKA and the subcellular localization of the latter by means of fluorescence imaging [Adams et. al., 1991].  73  Another  consideration that may have meaningful implications is the measurement of cGMP and PKG activation in the same tissues that the cAMP-dependent measurements are being carried out in.  Cyclic AMP and cGMP have been  proposed to act separately and in concert to result in relaxation brought about by a decrease in intracellular calcium ion concentration [Murthy and Makhlouf, 1995].  In this way, a more accurate representation of intracellular cyclic-  nucleotide-dependent events in achieving vascular smooth muscle relaxation, might be obtained.  74  5.0  CONCLUSION The novel aspects of this study were a) the use of intact tissue,  functionally responsive to relaxant and contractile drugs, where the cellular component consisted only of vascular smooth muscle and b) the measurement of cAMP levels in subcellular fractions of vascular smooth muscle tissue. The elevation of cAMP demonstrated to occur in helical strips which were exposed to a combination of 1 u.M FSK and 10 u.M P G E i , is the only observation that may be considered to be in support of the original hypothesis, in view of the relaxant response to forskolin. It is not backed up by the subsequent contractile response to  PGEi  or the failure to assay PKA activation.  The other observations made in this study do not present sufficient evidence to be able to support the hypothesis that cAMP and its subsequent activation of PKA play an important role in mediating vascular smooth muscle relaxation: 1.  No significant increase in ISO-induced cAMP levels was recorded in any  fraction in either of the two preparations, which both relaxed on exposure to this drug.  This presents no evidence of an increase in cAMP being related to  relaxation of vascular smooth muscle. 2. Studies with the medial strip preparation produced very anomalous results when cAMP was assayed, data which emphasize the diversity of intracellular events that occur in different cell types, which should be characterized before being applied to this hypothesis.  75  3. The investigation of PKA activation has not added to the current knowledge, mainly due to the fact that it was not possible to measure activation of the kinase under conditions of elevated cAMP. 4. 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