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Evidence for the functional expression of inducible nitric oxide synthase in vascular smooth muscle from… Bardell, Andrea Lee 2000

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E V I D E N C E  INDUCIBLE  FOR  T H E  FUNCTIONAL EXPRESSION OF  NITRIC OXSDE  S M O O T H  M U S C L E  SYNTHASE IN VASCULAR  FROM DIABETIC RATS by  ANDREA L. BARDELL B S c . M c G i l l University, 1998  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES FACULTY OF PHARMACEUTICAL SCIENCES Division of P h a r m a c o l o g y and T o x i c o l o g y W e accept this thesis a s conforming tcxthe required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A J U L Y 2000 © A n d r e a L e e Bardell , 2QOO  In presenting degree  this thesis  in partial fulfilment  of the  requirements  at the University of British Columbia, I agree that the  for an  advanced  Library shall make it  freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes department  or  by  his  or  her  may be granted  representatives.  It  is  by the head of my  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of ? l w r t \ ( A ( * ~ \ ^ C t x \ The University of British Columbia Vancouver, Canada  Date t  DE-6 (2/88)  im.  IPSO  ^Ctg^CltS  Abstract Nitric oxide (NO) h a s important physiological a n d pathophysiological functions in V S M . T h e p u r p o s e of the present r e s e a r c h w a s to investigate the hypothesis that N O S activity is altered in arteries from diabetic rats. C o n c e n t r a t i o n - r e s p o n s e c u r v e s to N A , in the p r e s e n c e of d e x a m e t h a s o n e (0.1 uM) to prevent in vitro induction of i N O S , w e r e obtained in superior m e s e n t e r i c arteries from rats 12-14 w e e k s following S T Z - i n j e c t i o n a n d their a g e a n d g e n d e r m a t c h e d controls. Endothelial denudation or preincubation with n o n - s e l e c t i v e N O S inhibitors, L N M M A or L-NIO (300uJvl), p r o d u c e d a n i n c r e a s e in N A sensitivity (as reflected by the N A p D value) in arteries from both control a n d S T Z - d i a b e t i c rats. 2  Following endothelial denudation, L N M M A or L-NIO still i n c r e a s e d the N A p D v a l u e 2  in diabetic but not control arteries. T h e selective n N O S inhibitor 7 - N I N A h a d no effect o n N A r e s p o n s e s of e n d o t h e l i u m - d e n u d e d control or S T Z - d i a b e t i c arteries. H o w e v e r , the selective i N O S inhibitor, EIT (10LIM), p r o d u c e d a n i n c r e a s e in the N A p D v a l u e in 2  e n d o t h e l i u m - d e n u d e d diabetic but not control arteries. Immunohistochemistry r e v e a l e d that e N O S is present in the endothelial cell m o n o l a y e r of both control a n d diabetic arteries. N o positive s i g n a l for n N O S w a s obtained in either control or diabetic arteries. H o w e v e r , immunostaining for i N O S indicated the p r e s e n c e of i N O S throughout all layers of diabetic but not control arteries. Quantitative m e a s u r e m e n t of cytosolic N O S activity indicated no significant c a l c i u m - d e p e n d e n t ( n N O S ) activity in control or diabetic arteries at any time following S T Z - i n j e c t i o n . Similarly, no c a l c i u m - i n d e p e n d e n t ( i N O S ) activity w a s detected in control arteries. H o w e v e r , significant i N O S activity w a s detected in 12-14 w e e k S T Z -  diabetic arteries. T h e s e data s u g g e s t the novel finding that i N O S is functionally e x p r e s s e d in V S M of arteries from 12-14 w e e k S T Z - d i a b e t i c rats. T i m e c o u r s e studies indicate that the induction of i N O S o c c u r s s o m e t i m e after 8 w e e k s of d i a b e t e s . Further studies will be required to establish the significance of i N O S induction in diabetic V S M .  TABLE OF CONTENTS Page  Abstract  ii  Table of Contents  iv  List of Tables  vii  List of Figures  viii  List of Abbreviations  x  Acknowledgements  xii  Dedication  xiii  Introduction A. O v e r v i e w  1  B. Nitric O x i d e  2  C. N O S isoforms  3  D.  M e c h a n i s m of i N O S induction in V S M  6  E.  Potential contributors to i N O S induction in d i a b e t e s  8  F.  Potential c o n s e q u e n c e s of i N O S induction in V S M  11  G. STZ-induced diabetes  12  H.  15  Rationale and Hypothesis  Specific objectives of the present investigation  16  iv  Materials and Methods A. Materials  18  B. Methods 1.  Experimental a n i m a l s  19  2. 3. 4. 5. 6. 7. 8.  Preparation of isolated m e s e n t e r i c arteries Preparation of solutions for p h a r m a c o l o g i c a l experiments C u m u l a t i v e c o n c e n t r a t i o n - r e s p o n s e c u r v e s to N A Immunohistochemistry Quantitative m e a s u r e m e n t of N O S activity B l o o d g l u c o s e a n d insulin determination Statistical a n a l y s i s  19 20 20 21 22 23 24  Results A. G e n e r a l characteristics of control a n d diabetic rats  25  B. P h a r m a c o l o g i c a l investigation of the effects of the non-selective N O S inhibitors L N M M A a n d L-NIO  27  C.  Effects of the selective n N O S inhibitor 7 - N I N A in endotheliumd e n u d e d arteries  34  D. Effects of i N O S inhibitors in e n d o t h e l i u m - d e n u d e d arteries 1. A m i n o g u a n i d i n e 2. EIT  38 42  E. Investigation of reversibility of L-NIO a n d EIT  46  F.  P h a r m a c o l o g i c a l investigation of the time-course of i N O S induction in diabetic V S M  52  G.  Immunohistochemical a n a l y s i s of 12-14 w e e k S T Z - d i a b e t i c a n d control m e s e n t e r i c arteries  58  H. Quantitative m e a s u r e m e n t of cytosolic N O S activity at different time-points following STZ-injection  75  V  Discussion  81  Summary and Conclusions A. Summary B. Conclusions and future research  References  94 9  7  9 8  vi  List of Tables Table  Page  1  Summary of general characteristics of control and STZ-diabetic rats  26  2  Sensitivity and maximum responses to N A of endotheliumintact and endothelium-denuded control and 12-14 week STZ-diabetic mesenteric arteries in the absence and presence of L N M M A or L-NIO (300uM)  29  3  Sensitivity and maximum responses to NA of endotheliumdenuded control and 12-14 week STZ-diabetic arteries in the absence and presence of 7-NINA (100uJv1)  35  4  Sensitivity to N A of endothelium-intact and endotheliumdenuded control and 12-14 week STZ-diabetic mesenteric arteries in the absence and presence of aminoguanidine (AG) (1mM)  39  5  Sensitivity to N A of endothelium-denuded control and 12-14 week STZ-diabetic mesenteric arteries in the absence and presence of EIT  43  6  Effect of co-incubation of N O S inhibitors with L-arginine or D-arginine on NA p D values in mesenteric arteries from control and 12-14 week STZ-diabetic rats  47  7  Sensitivity and maximum responses to NA of endotheliumdenuded control and STZ-diabetic mesenteric arteries 2-3 weeks or 6-8 weeks following STZ-injection in the absence and presence of EIT (10uM)  53  8  Cytosolic N O S specific activity of control and STZ-diabetic superior mesenteric arteries 2-3 weeks, 6-8 weeks, and 12-14 weeks following STZ-injection  76  2  vii  List of Figures Figure  Page  1  Cumulative concentration-response curves to N A of endothelium-intact control and 12-14 week STZ-diabetic superior mesenteric arteries in the absence and presence of L N M M A or L - N I O (300LLM each)  30  2  Cumulative concentration-response curves to N A of endothelium-denuded control and 12-14 week STZ-diabetic superior mesenteric arteries in the absence and presence of L N M M A or L - N I O (300LIM each)  32  3  Cumulative concentration-response curves to N A of endothelium-denuded control and 12-14 week STZ-diabetic superior mesenteric arteries in the absence and presence  36  of 7 - N I N A ( 1 0 0 u . M )  4  Cumulative concentration-response curves to N A of endothelium-intact and endothelium-denuded control and 12-14 week STZ-diabetic superior mesenteric arteries in the absence and presence of A G (1mM)  40  5  Cumulative concentration-response curves to N A of endothelium-denuded control and 12-14 week STZ-diabetic superior mesenteric arteries in the absence and presence of EIT(IOuM)  44  6  Cumulative concentration-response curves to N A of endotheliumintact superior mesenteric arteries from control rats in the absence and presence of L - N I O (300u.M), L - N I O (300u.M) + D-arginine (1mM), or L - N I O (300p:M) + L-arginine (1mM)  48  7  Cumulative concentration-response curves to N A of endotheliumdenuded superior mesenteric arteries from 12-14 week S T Z diabetic rats in the absence and presence of L - N I O (300u.M), L - N I O (300uM) +D-arginine (1mM), or L - N I O (300p.M) + L-arginine (1mM)  50  8  Cumulative concentration-response curves to N A of endotheliumdenuded control and 2-3 week STZ-diabetic superior mesenteric arteries in the absence and presence of E I T (10u,M)  54  9  Cumulative concentration-response curves to NA of endotheliumdenuded control and 6-8 week STZ-diabetic superior mesenteric arteries in the absence and presence of EIT (10u,M)  56  10  Immunostaining of endothelium-intact control and 12-14 week STZ-diabetic superior mesenteric arteries with the monoclonal anti-eNOS antibody  60  11  Immunostaining of endothelium-intact control and 12-14 week STZ-diabetic superior mesenteric arteries and mouse brain with the polyclonal anti-nNOS antibody  63  12  Immunostaining of endothelium-intact control and 12-14 week STZ-diabetic superior mesenteric arteries with the polyclonal anti-iNOS antibody  67  13  Immunostaining of endothelium-intact control and 12-14 week STZ-diabetic superior mesenteric arteries and rat spleen with the monoclonal anti-macrophage (clone ED2) antibody  72  14  Calcium dependent N O S specific activity of the cytosolic fraction of control and STZ-diabetic arteries 2-3 weeks, 6-8 weeks, and 12-14 weeks following STZ-injection  77  15  Calcium independent N O S specific activity of the cytosolic 79 fraction of control and STZ-diabetic arteries 2-3 weeks, 6-8 weeks, and 12-14 weeks following STZ-injection  16  Chemical structures of L-arginine, LNMMA, and L-NIO  84  ix  List of Abbreviations  ACh  acetylcholine  AG  aminoguanidine  AGEs  advanced glycosylation end-products  BH  (6R)-5,6,7,8-tetrahydrobiopterin  4  cGMP  guanosine 3',5' cyclic monophosphate  DNA  deoxyribonucleic acid  EDRF  endothelium derived relaxing factor  EIT  S-ethylisothiourea  eNOS  endothelial nitric oxide synthase  ET-1  endothelin-1  FAD  flavin adenine dinucleotide  FMN  flavin mononucleotide  HDL  high-density lipoproteins  IFN-y  interferon gamma  IL-1  interleukin-1  iNOS  inducible nitric oxide synthase  KCI  potassium chloride  LDL  low-density lipoproteins  L-NIO  N -(1 -iminoethyl)L-ornithine  LNMMA  N -monomethyl-L-arginine  LPS  lipopolysaccharide  uM  micromolar  5  G  mg  milligram  mM  millimolar  mmol  millimoles  NA  noradrenaline  NADPH  nicotinamide adenine dinucleotide phosphate  NF-KB  nuclear factor - kappa B  7-NINA  7-nitroindazole  nNOS  neuronal nitric oxide synthase  NO  nitric oxide  NOS  nitric oxide synthase  PE  phenylephrine  PKC  protein kinase C  pmol  picomoles  RAGE  receptor for advanced glycosylation end-products  ROS  reactive oxygen species  s.e.m.  standard error of the mean  SOD  superoxide dismutase  STZ  streptozotocin  TNF-a  tumour necrosis factor alpha  VSM  vascular smooth muscle  VSMC  vascular smooth muscle cells  xi  Acknowledgements I would sincerely like to thank and extend my gratitude to my supervisor Dr. Kathleen MacLeod for her expert guidance, patience, example, encouragement and overall support. I would also like to thank the members of my supervisory committee, Dr. Brian Rodrigues, Dr. John McNeill, Dr. Ismail Laher, and Dr. Keith McErlane for their useful suggestions and valuable input in this research project. I would also like to thank Lili Zhang and Violet Yuen for their constant support and advice. I wish to acknowledge Dr. Fadi Khadour for his expert instruction in setting up the N O S assay and Julie Chow for her assistance with the immunohistochemistry. Furthermore, my thanks go out to Dr. Yi He and Denise Galipeau for their advice, instruction, and friendly company.  D e d i c a t e d to m y  mother,  w h o has been the strongest influence i n m y for a l l o f h e r love a n d e n d l e s s  life,  support.  xiii  Introduction  A . Overview  Diabetes mellitus can be considered as a cluster of syndromes, characterized by insulin deficiency (type 1 diabetes mellitus) or insulin resistance (type 2 diabetes mellitus). The diabetic state is associated with hyperglycemia and altered metabolism, including increased utilization of free fatty acids (Rodrigues et al, 1997). Diabetic patients are at greater risk of developing long-term pathophysiological complications including peripheral neuropathy, nephropathy, and cardiovascular complications, resulting in an increased risk of premature morbidity and mortality. Hyperglycemia has been recently identified as an independent risk factor for the development of cardiovascular disease (Diabetes Control and Complications Trial Research Group, 1993). Diabetes has been reported to be associated with altered vascular reactivity to neurotransmitters and circulating hormones (Christlieb et al, 1976). Endothelial dysfunction (as reflected as an imbalance in the release of, or sensitivity to, endothelial-derived vasoconstrictors and vasodilators) has been proposed as an important contributor to diabetes-induced vascular smooth muscle (VSM) dysfunction (Taylor et al., 1992). Endothelium-derived relaxing factor (EDRF), which is nitric oxide (NO) produced by the endothelial subtype of nitric oxide synthase (eNOS), is an important vasodilator (Furchgott, 1999). Abnormal release of or response to NO has been proposed as a contributor to vascular and endothelial dysfunction in the diabetic state (Huszka et al., 1997; Cohen, 1995). Previous studies of N O S activity in diabetic  l  V S M have focused on e N O S . Although E D R F may be of primary importance under normal conditions, another constitutive subtype of N O S , nNOS, and the inducible subtype of N O S , iNOS, may be expressed in vascular smooth muscle cells (VSMC) under pathological conditions (Boulanger et al., 1998; Gonzalez-Fernandez et al., 1998). The present study was undertaken to investigate N O S activity in diabetic VSM.  B. Nitric oxide  Since its discovery as E D R F , a great deal of research has been conducted on the physiologic and pathophysiologic roles of NO in the cardiovascular system (Palmer et al, 1987; Ignarro, 1999). NO diffuses readily across cell membranes (Kerwin et al, 1995) and mediates vasodilation by binding to soluble guanylate cyclase, resulting in increased production of c G M P (Nakatsu and Diamond, 1989). A n increase in intracellular c G M P levels leads to decreased intracellular calcium levels, resulting in vasorelaxation (Chabaud et al, 1994). NO also reacts with sulfhydryl groups, forming S-nitrosothiols, which may serve as cellular NO carriers or active intermediates in NO cell signaling (Ignarro et al, 1981). NO plays important roles in the control of vascular tone, platelet aggregation, and V S M proliferation (Marin and Rodriguez-Martinez, 1997). However, if there is a high concentration of reactive oxygen species (ROS) present in the local environment of NO production, overproduction of NO can result in tissue dysfunction and damage (Bhagat, 1996).  2  C . Nitric oxide synthase enzymes  NO is synthesized from the terminal guanidino nitrogen atom of L-arginine and molecular oxygen by the nitric oxide synthase (NOS) family of enzymes, with N A D P H , tetrahydrobiopterin (BH ), F M N , FAD, and calmodulin as cofactors. The N O S 4  enzymes are cytochrome P450 enzymes, which differ from all other mammalian cytochrome P450 mono-oxygenases in that they contain both the reductase and oxygenase domain (Nathan and Xie, 1994). All of the N O S isoforms have two binding domains. The N-terminal domain contains the oxygenase activity and binds heme, B H , and the substrate, L-arginine. The C-terminal domain contains the reductase 4  activity and binds N A D P H , F A D , and FMN. The calcium regulatory protein calmodulin binds between the two domains and may be involved in electron transfer between the domains (Abu-Soud et al, 1994). The two major classes of N O S are the constitutive subtypes and the inducible subtype. The constitutive N O S subtypes, e N O S and nNOS (also known as N O S 3 and NOS1 respectively) are calcium-dependent and are subject to regulation by phosphorylation by protein kinase C (PKC) (Nakane et al, 1991). The inducible subtype, iNOS (or NOS2), is calcium-independent and is regulated primarily at the transcriptional level (Morris and Billiar, 1994).  eNOS e N O S is a calcium-dependent constitutive subtype of N O S which was originally purified and cloned from vascular endothelial cells but may also be expressed in platelets, neutrophils, and cardiac myocytes under certain conditions (Michel and  3  Feron, 1997). The e N O S subypte of N O S has a N-myristoylation site that causes its localization to the cell membrane, thus e N O S is present predominantly in the particulate fraction (Forstermann et al, 1991; Busconi and Michel, 1993). Previous studies of the role of nitric oxide in the cardiovascular system in diabetes have focused on NO derived from e N O S (EDRF). Numerous studies have suggested that the diabetic state is associated with a decreased release of or decreased responsiveness to endothelial-derived NO (Huszka et al, 1997; Cohen, 1995). Proposed explanations for the decreased responsiveness to E D R F in diabetes include accelerated inactivation of NO due to increased oxidative stress, enhanced activation of P K C (resulting in phosphorylation and inhibition of eNOS), and increased production of vasoconstrictor prostanoids or ET-1 that counteract the effect of N O (Tesfamariam and Cohen, 1992; Takeda et al, 1991).  nNOS Another calcium-dependent subtype of N O S is nNOS. nNOS was originally purified from peripheral neurons but is now known to be expressed in the brain, sympathetic ganglia, peripheral nitrergic nerves (nonadrenergic, noncholinergic nerves), adrenal glands, skeletal muscle, and, under certain conditions in V S M (Gath et al, 1996; Marin and Rodriguez-Martinez, 1997; Brophy et al, 2000). nNOS activity in the peripheral nervous system is associated with relaxation of smooth muscle and, in the brain, is associated with NO-mediated synaptic plasticity (Kerwin et al, 1995). The expression of nNOS in the uterine artery has been reported during pregnancy and is thought to be involved in the re-distribution of cardiac output with pregnancy (Garvey et al, 1994). Further evidence for the expression of nNOS has been reported  4  in bovine carotid arteries (Brophy et al, 2000) and in carotid arteries from spontaneously hypertensive rats (Boulanger et al, 1998). n N O S is predominantly a cytosolic enzyme (Forstermann et al, 1991).  iNOS i N O S was originally isolated from an immunoactivated macrophage cell line but is now known to be induced in a wide variety of cell types including cardiac myocytes, glial cells, endothelial cells, and V S M cells (Marin and Rodriguez-Martinez, 1997). i N O S is similar to the constitutive N O S isoforms in that it contains a calmodulin regulatory sequence. However, calmodulin is tightly bound to iNOS, thus its activity is independent of stimulation by calcium (reviewed in Schulz and Triggle, 1994). Unlike the constitutive isoforms, iNOS is not subject to regulation by phosphorylation (Marin and Rodriguez-Martinez, 1997). Once induced, iNOS synthesizes a prolonged and increased release of NO as compared to e N O S and nNOS (Moncada and Higgs, 1995). Although it is widely accepted that cytokine-stimulated induction of iNOS in macrophages is an important element of the inflammatory response (Wei et al, 1995), iNOS may be functional in other tissue types under pathological conditions, including disease states such as diabetes. Induction of iNOS has been recently demonstrated in cardiac myocytes from rats with streptozotocin-induced diabetes (Smith et al, 1997). In addition, evidence for iNOS activity has recently been reported in platelets from type 1 and type 2 diabetic patients (Tannous et al., 1999). It is possible that induction of iNOS occurs in diabetic tissues as a result of pathophysiological changes associated with chronic diabetes.  5  D. M e c h a n i s m of i N O S induction in V S M  Apart from potential limitations of substrate and cofactor availability, iNOS activity does not appear to be regulated once the enzyme is expressed. Therefore, iNOS activity is predominantly regulated at the level of D N A transcription. Gene transcription and induction of iNOS is inhibited by glucocorticoids, including dexamethasone (Knowles et al, 1990).  Regulation of i N O S expression in V S M C  appears to be very complex, involving several factors acting alone or synergistically. Distinct species differences have been reported for the transcriptional control of iNOS expression in V S M C (Paul et al, 1997). Furthermore, significant differences in regulation of i N O S induction have been reported for V S M C from the same species but different vascular origin (Kolyada et al, 1996). The iNOS gene has been cloned and sequenced from the mouse, rat, and human. A sequence homology of 47-66% of the promoter region has been reported between species (DeVera et al, 1996). The promoter region of all three genes (mouse, rat, and human) includes several putative elements including  NF-KB,  IFN^y,  IL-13, and TNF-responsive elements (DeVera et al, 1996). Induction of iNOS in V S M may occur synergistically in response to various stimuli including IL-13, TNF-ot, INF^y, IL-6, and/or L P S or oxidative stress induced activation of  NF-KB  (Hecker et al, 1999).  Transcriptional control of iNOS in vascular smooth muscle may also be regulated by protein kinase C (PKC). Individual P K C isoforms may regulate iNOS expression in both a positive and negative manner with distinct cell-type specific differences (Hecker et al, 1999). In a recent study, activation of P K C - a and PKC-e 6  was associated with enhanced LPS-induced iNOS expression in R A W 264.7 macrophages and attenuated iNOS induction in rat aortic smooth muscle cells (Paul et al, 1997). In contrast, in the same study, it was suggested that activation of P K C < may contribute to iNOS induction in rat aorta, but may inhibit iNOS induction in the macrophage cell line (Paul et al, 1997). The mechanism by which iNOS gene expression in V S M is regulated by individual P K C isoforms in different cell types remains to be investigated.  7  E. Potential contributors to iNOS induction in diabetes  Formation of Advanced Glvcosylation End-products (AGEs)  Glucose can form glucosylation products with amino groups from proteins or amino acids by a non-enzymatic process. Early glucosylation products may undergo a complex series of irreversible chemical arrangements, including autoxidation of the Amadori product (a 1-amino-1-deoxyketose formed by the reaction of reducing sugars with protein amino groups), to form advanced glycosylation end-products (Brownlee, 1994). Chronic diabetes is associated with an increased accumulation of A G E s (Schleicher and Nerlich, 1996). A highly significant correlation exists between A G E accumulation and severity of diabetic microvascular damage in the retina, kidney, and peripheral nerve (Beisswenger et al, 1995). Vascular endothelial cells express an AGE-specific receptor, designated receptor for A G E s (RAGE), which appears to be a member of the Ig receptor superfamily (Neeper et al, 1992). The R A G E receptor appears to mediate signal transduction through the generation of reactive oxygen species (ROS) (Brownlee, 2000). R O S are generated by A G E binding and are reported to activate  NF-KB  (Bierhaus et al, 1997). Interaction of A G E s with R A G E induces changes in gene expression, including upregulation of various cytokines including T G F - p and IL-1 (Vlassara et al, 1988). Thus, AGE-mediated upregulation of cytokines may contribute to iNOS induction in V S M .  Oxidative stress  The diabetic state has been reported to be associated with increased levels of oxidative stress. Oxidative stress occurs when antioxidant defenses, including superoxide dismutase (SOD) in a cell are overwhelmed due to increased exposure to oxidants (Darley-Usmar and White, 1997). Increased generation of oxygen-derived free radicals has been reported in diabetes (Giugliano et al, 1996). Increased lipid peroxidation and elevated levels of superoxide anion have been reported in aorta from STZ-induced diabetic rats (Chang et al, 1993). Elevation of levels of reactive oxygen species (ROS) may also occur due to the formation of A G E s with long-term hyperglycemia and poor metabolic control. The oxidant, peroxynitrite, which is the product of the interaction of N O with R O S , may also contribute to oxidative stress. The consequences of oxidative stress may include modulation of antioxidant and free radical scavenger levels and activation of stress responsive transcription factors, including  NF-KB  (Li and Karen, 1999). Therefore, activation of  NF-KB  with  increased oxidative stress may contribute to iNOS induction in V S M . It has also been suggested that increased production of free radicals in aorta from STZ-induced diabetic rats leads to accelerated inactivation of NO (Hattori et al, 1991).  Altered activation of P K C isoforms There is some evidence for enhanced P K C activation with chronic diabetes although this remains an area of controversy. Previous studies from our laboratory have demonstrated enhanced PKC-mediated contractile responses to noradrenaline  9  (NA) in mesenteric arteries from male rats with STZ-induced diabetes of 12 weeks duration (Harris and MacLeod, 1988), providing indirect evidence for enhanced P K C activation with chronic diabetes. Direct evidence for diabetes-induced P K C activation has also been reported. Activation of the (32 isoform of P K C is enhanced in the aorta and heart from diabetic rats (Inoguchi et al, 1992). In addition, prolonged high ambient glucose concentrations activate P K C in cultured V S M C in vitro (Williams and Schrier, 1992). Thus, enhanced activation of P K C with long-term diabetes may also potentiate iNOS induction in V S M .  10  F. Potential consequences of iNOS induction in VSM  Induction of iNOS in V S M would result in an overproduction of NO, as iNOS synthesizes 10-50 fold more NO than the constitutive N O S subtypes (Moncada and Higgs, 1995). Any increase in production of NO has potential for adverse effects, particularly under conditions of oxidative stress. NO itself is not cytotoxic although highly chemically reactive, possessing a very short half-life (5-10 seconds in vitro) (Ignarro, 1990). NO may react rapidly with oxygen derived free radicals to produce peroxynitrite ( O N O O ) . Peroxynitrite can result in oxidation of lipids and the modification of proteins in the cell by nitration of tyrosine residues (Koppenol et al, 1992). The oxidation of lipoproteins, including low-density lipoproteins (LDLs) by peroxynitrite has been implicated in the formation of atherosclerotic lesions (Leeuwenburgh et al, 1997). Peroxynitrite may disrupt signal transduction events in the cell by modification of tyrosine residues and carbohydrates (Darley-Usmar and White, 1997). Furthermore, peroxynitrite has been reported to generate lipid products with vasoconstrictive properties (Elliot et al, 1998). Other consequences of iNOS induction in V S M are possible. For instance, alteration of NO levels may result in an imbalance in the release of other endotheliumderived factors including endothelin-1 (ET-1) as there is a likely in vivo interplay between ET-1 and NO (Warner, 1999). Induction of iNOS in V S M may also act to compensate for a decrease in the release of, or responsiveness to, endothelial derived NO, which has been suggested to occur in diabetes in experimental animals (Durante et al, 1988).  n  G . Streptozotocin (STZ)-induced diabetes  STZ-model of diabetes  The present study was aimed at investigating the effects of chronic diabetes on N O S activity in V S M . The S T Z model of type I diabetes was utilized, since a single bolus injection of S T Z consistently results in chronic hyperglycemia and hypoinsulinemia (Rakieten et al, 1963). S T Z [2-deoxy-2-(3-methyl-3-nitrosourea) 1-D-glucopyranose] binds with high affinity to membrane receptors on pancreatic-p cells. The mechanism of p-cell destruction by S T Z involves free radical generation, methylation, and nitric oxide production (reviewed in Rodrigues, 1999). Destruction of pancreatic p-cells results in permanent hypoinsulinemia in these animals and produces characteristics associated with the diabetic state including hyperglycemia, polydypsia, polyuria, cataracts, and decreased body weight gain. The severity of the diabetic state induced by S T Z is dose-dependent and may vary between species, route of administration (intravenous or intraperitoneal), and animal age and weight. However, numerous studies have indicated that a single bolus injection of at least 40mg/kg administered intravenously to male rats consistently results in a permanent hypoinsulinemic and hyperglycemic state (reviewed in Rodriguez, 1999).  12  Altered vascular reactivity associated with long-term STZ-induced  diabetes  Changes in vascular reactivity that occur in the STZ-model of diabetes have been investigated in various arteries and vascular beds. Numerous investigations, including studies from our laboratory, have demonstrated that maximum responsiveness to a-adrenoceptor stimulation is enhanced in aorta and mesenteric arteries from STZ-diabetic rats of 12-14 week duration (MacLeod 1985, Abebe et al, 1990). Enhanced contractile responses of 12-14 week STZ-diabetic arteries are thought to arise from direct stimulation of ai-adrenoceptors (Abebe et al, 1990). Furthermore, the enhanced responsiveness to on -adrenergic receptor stimulation is not thought to be due to a nonspecific increase in contractile responsiveness of S T Z diabetic arteries, since no significant difference has been reported between control and diabetic arteries in response to potassium-induced depolarization (MacLeod 1985; Abebe et al, 1994). Additional studies from this laboratory have indicated that the enhanced contractile responses to NA of STZ-diabetic aorta and mesenteric arteries are associated with an increase in phosphoinosotide turnover (Abebe and MacLeod, 1992). Altered phosphoinositide metabolism may result in overproduction of the second messengers, inositol 1,4,5-trisphosphate (IP3) and diaglycerol (DAG), which may in turn result in increased calcium release from intracellular stores and/or enhanced activation of P K C (Abebe and MacLeod, 1990). It has been suggested that in addition to increased contractile responsiveness, N O production and release may be altered in arteries from STZ-diabetic rats. A number of studies have shown that endothelial production of NO or vascular relaxation in response to A C h is impaired in arteries from long-term STZ-diabetic rats  13  (Fortes et al, 1983; Durante et al, 1988). However, studies from this laboratory have reported no differences in rat aorta between control and 12 week STZ-diabetic in c G M P levels in the absence and presence of A C h (Harris and MacLeod, 1988). There is further controversy regarding NO production in diabetes since increased release of NO has also been proposed to occur (Bhardwaj and Moore, 1988; Langenstroer and Pieper, 1992). An increased release of NO may be masked by inactivation of NO in a local environment of elevated free radicals (Langenstroer and Pieper, 1992).  14  H. Rationale and hypothesis  Rationale Long-term hyperglycemia that occurs in chronic diabetes is associated with changes in vascular reactivity, endothelial and vascular dysfunction, and vascular deterioration. Other changes that are associated with long-term hyperglycemia, including cytokine production resulting from A G E formation and increased oxidative stress, and activation of P K C , may result in induction of iNOS gene transcription. Since alterations in N O S expression and NO production may result in various effects, investigation of N O S activity in vascular smooth muscle in the diabetic state may be of particular importance in understanding the etiology of endothelial and vascular dysfunction associated with chronic diabetes mellitus.  Hypothesis It is hypothesized that iNOS is functionally expressed in V S M of rats with chronic diabetes.  15  Specific objectives of the present investigation  1. To investigate pharmacologically N O S activity of isolated endothelium-intact and endothelium-denuded superior mesenteric arterial rings from control and 12-14 week STZ-diabetic rats by obtaining cumulative concentration-response curves to NA in the absence and presence of the non-selective N O S inhibitors L N M M A and L-NIO.  2. To determine which subtype of N O S is active in diabetic V S M by obtaining cumulative concentration response curves to NA in endothelium-denuded arterial rings in the absence and presence of the selective n N O S inhibitor 7-nitroindazole or the selective iNOS inhibitors aminoguanidine or S-ethylisothiourea.  3. To investigate the time course of iNOS induction by obtaining cumulativeconcentration response curves to NA of endothelium-denuded arteries in the absence and presence of EIT, 2-3 weeks and 6-8 weeks following STZ-injection.  16  4. To perform immunohistochemical analysis for the detection of the individual N O S subtypes, e N O S , nNOS, and iNOS, in control and 12-14 week STZ-diabetic rat superior mesenteric arteries. Furthermore, to investigate whether macrophage infiltration is the source of iNOS in diabetic V S M by immunohistochemical staining for macrophage specific protein.  5. To quantitatively measure nNOS and iNOS activity of control and STZ-diabetic arteries 2-3 weeks, 6-8 weeks, and 12-14 weeks following injection.  17  i  Materials a n d M e t h o d s  A. Materials  Male Wistar rats weighing 190-220g were obtained from the Animal Care Center, U B C . The Periodochrom® glucose assay kit was obtained from Boehringer Mannheim (Mannheim, Germany). The rat insulin radioimmunoassay kit was obtained from Linco Research Inc. (St. Charles, MO). L-NIO, EIT, and 7-NINA were obtained from Tocris Ltd. (Ballwin, MO). The polyclonal anti-iNOS antibody and L N M M A were obtained from Calbiochem (La Jolla, CA). Polyclonal anti-nNOS and monoclonal antie N O S antibodies were obtained from Transduction Laboratories (Franklin Lakes, NJ). Monoclonal anti-macrophage (clone ED2) antibody was obtained from Serotec Inc. (Raleigh, NC). The A B C Vector kit and the DAB reagent were obtained from Vector Inc. (Burlingame, CA). [ C]-L-Arginine was obtained from Amersham Pharmacia 14  Biotech Inc. (Piscataway, NJ). The BioRad protein reagent was obtained from BioRad (Richmond, CA). All other chemicals were obtained from Sigma Chemical (Oakville, Ontario, Canada).  18  B.  Methods  1. Experimental Animals Male Wistar rats weighing 190-220g were obtained from the Animal Care Centre, U B C , and were treated according to the Guidelines of the Canadian Council for Animal Care. Bolus injection of streptozotocin (STZ) (55mg/kg IV) was administered via the tail vein under light halothane anaesthesia 2-3 weeks, 6-8 weeks or 12-14 weeks prior to use. Control rats received citrate buffer vehicle (0.1 u.M, pH 4.5). Both diabetic and control rats were allowed access to food and water ad libitum. STZ-treated animals were considered diabetic and retained for experiments if their blood glucose was greater than 16mmol/L, 7 days following STZ-injection.  2. Preparation of isolated mesenteric arterial rings Rats were deeply anaesthetized with an intraperitoneal injection of pentobarbitol (65mg/kg). The chest cavity was opened and blood was taken by cardiac puncture. The superior mesenteric artery was then carefully removed and placed in a Petri dish containing cold Kreb's solution of composition (mM): NaC1113, KCI 4.7, NaHCO-3, 25.0, CaCI , K H P 0 1.2, M g S 0 1.2, and dextrose 11.5, pH 7.4, 2  2  4  4  continuously aerated with 95% 0 - 5 % C 0 . Water-soluble dexamethasone (0.1 uM) 2  2  was added to the Kreb's solution to prevent iNOS induction in vitro during the course of the experiment. Tissues were cleaned of excess fat and connective tissue and cut into two 4mm rings. The endothelium was either kept intact or removed by careful rubbing of the vessel lumen. Ring preparations of mesenteric arteries were placed  19  individually in isolated tissue baths containing 20ml Kreb's solution continuously aerated with 95% 02-5% C02and maintained at 37°C. Isometric contractions were measured with force-displacement transducers connected to a Grass model 7E polygraph. Tissue preparations were equilibrated for 90 minutes under a resting tension of 1g, which was previously found to be optimal for both control and diabetic arteries (MacLeod 1985). During the equilibration period, the Kreb's solution was replaced every 20 minutes.  3. Preparation of solutions for pharmacological experiments The Kreb's solution and solutions of NA, acetylcholine (ACh), phenylephrine (PE), water-soluble dexamethasone, aminoguanidine (AG), S-ethylisothiorea (EIT), 7nitroindazole (7-NINA), N -monomethyl-L-arginine (LNMMA), N -(1-iminoethyl)LG  5  ornithine (L-NIO), L-arginine, L-Citrulline, and potassium chloride (KCI) were prepared in distilled water. Ascorbic acid (4mg/ml) was added to NA stock solutions to reduce oxidation.  4. Cumulative concentation-response curves to NA Endothelial status was assessed by determining the ability of acetylcholine (10" M) to relax a precontraction to phenylephrine (3x1 O^M). Arteries were then 5  washed 3 times with Kreb's solution and allowed to re-equilibrate for 60 minutes before a concentration-response curve to NA was obtained. The tissues were again washed 3 times and allowed to re-equilibrate for 45 minutes, following which one  arterial ring of each diabetic and control pair was incubated with one of the following antagonists (L-NIO (300uM), aminoguanidine (1mM), 7-NINA (lOOuM), or EIT (5uM or 10u.M)). In some experiments, L-arginine (1mM) or D-arginine (1mM) was added with the antagonist. Subsequently, a second concentration-response curve to N A was obtained. The other arterial ring of the pair remained untreated and served as a control to determine whether any changes in reactivity occurred during the course of the experiment. No significant time-dependent changes in the N A response were detected (data not shown). After the second NA concentration-response curve, the tissues were washed and allowed to re-equilibrate for 30 minutes. Finally, the maximum response to KCI was determined in the presence of phentolamine (10" M). 5  Contractile responses to N A of each arterial ring were expressed as a percent of the maximum response of the same ring to KCI.  5.  Immunohistochemistry Superior mesenteric arteries were excised and cleaned as described above.  Arteries were then fixed in 10% neutral buffered formalin followed by paraffin processing through increasing grades of ethyl alcohol, xylene, and Paraplast. Tissue blocks were sectioned at 3um and the luminal artery cross sections were mounted on positively charged slides. Endogenous peroxidase activity was quenched with 3% (w/v) aqueous hydrogen peroxide for ten minutes and slides were rinsed with water. Background staining was minimized with 2 % normal goat serum in Tris buffered saline (TBS). Sections were incubated with the primary antibody (polyclonal anti-iNOS, n N O S , or  21  monoclonal anti-eNOS 1:2500 dilution in T B S with 1 % (w/v) B S A or monoclonal antimacrophage ED2 1:1000 dilution) overnight in a humid chamber. The primary antibody was rinsed off with T B S and sections were incubated with a biotinylated species-specific secondary antibody (1:150 dilution in TBS) for 1 hour at room temperature. The secondary antibody was rinsed off with T B S and the streptavidinbiotin peroxidase complex (ABC Kit, Vector Inc) was applied for 1 hour at room temperature. The A B C reagent was rinsed off with T B S and sections were stained with DAB reagent (60mg/100ml T B S , 500uJ DAB intensifier, 100u.l 30% hydrogen peroxide) for 10 minutes. Sections were rinsed with tap water and counterstained with 0.1% (w/v) Nuclear Fast Red in 5% (w/v) aluminum sulfate. Slides were rinsed with tap water, dehydrated in alcohol, cleared in xylene and mounted in resinous mounting medium. Paraffin embedded sections of mouse brain and rat spleen were also processed and served as positive controls for detection of n N O S and macrophages respectively. Photographs were taken with a photomicroscope at 50X magnification.  6. Quantitative measurement of N O S activity Control and diabetic mesenteric arteries were excised and cleaned as described above and flash frozen in liquid nitrogen. Isolated arteries were stored at 70°C until assayed. The assay procedure requires 80mg tissue/sample, so 4-6 cleaned mesenteric arteries were pooled for each sample. Tissues were crushed with a mortar and pestle under liquid nitrogen. The frozen dry weight was obtained and homogenization buffer containing H E P E S (10mM), N a E D T A (0.1 mM), DL2  22  dithiothreitol (DTT) (1mM), type-S trypsin inhibitor (10u.g/ml), leupeptin hemisulfate (10u.g/ml), and aprotinin (2u.g/ml) was added at a ratio of 1mg tissue: 4uJ homogenization buffer. Phenylmethylsulfonyl fluoride (PMSF) was added at a ratio of 1uL P M S F : 100u\l homogenization buffer and samples were homogenized by sonication. The sample was then centrifuged at 16,000xg for 20min at 4°C and the supernatant (consisting of the cytosolic fraction containing iNOS and nNOS) was retained on ice. N O S activity of the supernatant was quantitated by measuring the formation of radiolabeled [ C]-L-citrullinefrom [ C]- L-arginine as previously 14  14  described (Knowles et al., 1990a). For each sample, the supernatant was incubated in cocktail buffer of composition (mM): L-Valine 50, B H 0 . 0 1 . N A D P H 0.1, L-arginine 4  0.018, L-citrulline 1, DTT 1.05, [ C]- L-arginine 0.002, for 30 minutes at 37°C. 14  Samples were incubated in duplicate in cocktail buffer alone or in the presence of either E G T A (1mM) or E G T A plus L N M M A (1mM each) to determine the level of calcium-dependent and calcium-independent N O S activity. [ C]-L-citrulline was 14  separated from [ C]- L-arginine by cation-exchange chromatography using activated 14  A G 50W-X8 resin and quantified by liquid-scintillation counting. Protein content of the cytosolic fraction was measured with the BioRad protein reagent with bovine serum albumin used as a standard.  7. Blood Glucose and Insulin Determination Plasma glucose levels were measured by colorimetric enzyme assay, while plasma insulin was measured by radioimmunoassay, both using commercially available kits (Periodochrom® glucose assay kit and Linco rat insulin radioimmunoassay kit respectively).  23  8. Statistical Analysis NA concentration-response curves were analyzed by non-linear regression analysis using Graphpad Prism software for the determination of p D (-log 2  EC50)  values and maximum contractile responses (Rmax). All values are expressed as mean ± standard error of the mean (s.e.m.). Except where indicated, statistical significance was evaluated by two-way A N O V A followed by Newman-Keuls post-hoc tests for multiple comparisons and considered significantly different if p<0.05.  24  Results  A. General characteristics of control and diabetic rats  Two-3 weeks, 6-8 weeks, and 12-14 weeks following injection, STZ-treated rats had significantly increased plasma glucose levels and decreased plasma insulin levels compared to their age and gender-matched vehicle-injected controls (Tablel). 6-8 weeks and 12-14 weeks following injection, STZ-diabetic rats had significantly reduced body weight as compared to control (Table 1). The STZ-diabetic rats also exhibited other symptoms associated with the disease including polyuria and osmotic diarrhea. Detection of cataracts in STZ-diabetic rats was time-dependent, and most notable after 6-8 weeks following injection.  25  Table 1  Summary of general characteristics of control and STZ-diabetic rats  Rats  Weeks  Body weight  Plasma  Plasma insulin  following  (g)  glucose  (ng/ml)  injection  (mmol/l)  Control, N=20  2-3  285+8  8.71±0.06  3.6±0.4  Control, N=18  6-8  439+6  8.74+0.10  4.3±0.4  Control, N=39  12-14  556+5  8.65±0.10  8.9+0.7  STZ-diabetic,  2-3  266±8  22.95+0.24*  0.5+0.1*  6-8  344±9*  23.33±0.28*  0.6±0.1*  12-14  343±7*  23.65±0.39*  0.5+0.1*  N=20 STZ-diabetic, N=18 STZ-diabetic, N=39  All values are mean ± s.e.m. *p<0.05 as compared to corresponding control values (One-way ANOVA)  26  B. Pharmacological investigation of the effects of the non-selective N O S inhibitors L N M M A and L-NIO  In order to investigate whether functional N O S activity could be detected in superior mesenteric arteries of control and 12-14 week STZ-diabetic rats, cumulative concentration-response curves to NA were obtained in endothelium-intact and endothelium-denuded vessels in the absence and presence of the non-selective N O S inhibitor L N M M A (300uM). In untreated arteries, the maximum contractile response to NA of diabetic rat mesenteric arteries was found to be significantly greater than that of control arteries, although no significant difference in the NA p D values could be detected (Table 2). 2  Neither endothelial-denudation nor pharmacological inhibition of N O S had any significant effect on maximal contractile responses to N A of either control or diabetic arteries (Table 2, Fig. 1-4). Preincubation of endothelium-intact vessels with L N M M A resulted in a leftward shift in the NA concentration-response curve in both control and diabetic arteries (Fig. 1) associated with a significant increase in the NA p D values (Table 2). Removal 2  of the endothelium also resulted in a significant increase in NA p D values in both 2  control and diabetic arteries (Table 2). Incubation of endothelium-denuded control arteries with L N M M A had no further effect on the NA response (Fig.2A, Table 2). However, L N M M A produced a further leftward shift in the NA response and a significant increase in NA p D values of endothelium-denuded diabetic arteries 2  (Fig.2B; Table 2).  27  A significant difference in NA p D values was observed between endothelium2  intact and endothelium-denuded STZ-diabetic arteries following preincubation with L N M M A (Table 2). It was hypothesized that the disparity in NA p D values between 2  treated endothelium-intact and endothelium-denuded diabetic arteries was due to insufficient diffusion of L N M M A to the V S M when the endothelium remained intact. In order to investigate this hypothesis, cumulative concentration-response curves to NA were obtained in endothelium-intact and endothelium-denuded control and 12-14 week STZ-diabetic mesenteric arteries in the absence and presence of L-NIO (300u,M ), another nonselective N O S inhibitor that has been reported to be transported intracellularly more efficiently (Kerwin et al, 1995). A s seen with LNMMA, preincubation of endothelium-intact vessels with L-NIO resulted in an increase in NA p D values in both control and diabetic arteries (Fig 1; 2  Table 2). However, the NA p D value in diabetic arteries in the presence of LNIO was 2  significantly greater than that in control arteries (Table 2). Incubation of endotheliumdenuded control arteries with L-NIO produced no further effect on the NA responses (Fig 2A, Table 2). A s observed with LNMMA, L-NIO produced a significant increase in NA p D values of endothelium-denuded diabetic arteries (Fig 2B; Table 2). 2  However, unlike LNMMA, no significant difference in N A p D values was observed 2  between endothelium-intact and endothelium-denuded STZ-diabetic arteries following preincubation with L-NIO (Table 2).  28  Table 2  Sensitivity (pD2) and maximum responses (Rmax) to NA of endothelium-intact and endothelium-denuded control and 12-14 week STZ-diabetic mesenteric arteries in the absence and presence of L N M M A or L-NIO (300wVI)  Animals  Control  Control  Diabetic  Diabetic  Endo.  Intact  Denud.  Intact  Denud.  Rmax  Rmax  Rmax  PD2  PD2  PD2  Untreated  LNMMA  L-NIO  Untreated  LNMMA  L-NIO  N=16  N=10  N=6  N=16  N=10  N=6  151.6  148.5  150.5  6.51  7.01  7.08  ±2.1  ±2.8  ±1.8  ±0.04  ±0.11  157.8  157.4  163.5  7.31  7.21  7.26  ±1.1  ±2.4  ±2.0  ±0.07  ±0.03  ±0.04  194.6  195.2  207.7  6.58  7.18  8.39  ±4.5  ±3.0  ±2.1  ±0.05  ±0.10 8.15  a  a  a  198.6  196.3  199.9  7.32  ±1.7  ±2.3  ±2.5  ±0.04  a  a  a  b  b  ±0.03  c  c  ±0.12°  C  8.38  ±0.10 ' c  d  ±0.15  C  All values are mean ± s.e.m. a  p<0.05 compared to correspoding control values  b  p<0.05 compared to endothelium-intact values  c  p<0.05 compared to untreated values  d  p<0.05 compared to treated endothelium-intact values  29  Figure 1  Cumulative concentration-response curves to NA of endothelium-intact control (A) and 12-14 week STZ-diabetic (B) superior mesenteric arteries in the absence ( V , N=16) and presence of 300uM L N M M A ( • , n=10) or 300uJv1 L-NIO (#, n=6). Each point represents the mean ± s.e.m.  30  A  300 -,  untreated LNMMA LNIO  8  7  6  4  -log[NA] M  B 300 v  •  O CD  E w 200 E o  untreated LNMMA LNIO  Q.  § or  100H  8  7  6  -log[NA] M 31  Figure 2  Cumulative concentration-response curves to NA of endothelium-denuded control (A) and 1 2 - 1 4 week STZ-diabetic (B) superior mesenteric arteries in the absence ( V , N=16) and presence of 300uM L N M M A ( • , n=10) or 300u.M L-NIO ( • , n=6). Each point represents the mean ± s.e.m.  32  A 300-n  untreated LNMMA L-NIO  8  7  6  -log[NA] M  B 300-i  untreated LNMMA L-NIO  O  E to 200 E O Q.  ss a:100-^  8  7  6  -log[NA] M 33  C. Effects of the selective nNOS inhibitor 7-NINA in endothelium-denuded arteries  In order to investigate whether nNOS is the subtype of N O S contributing to the observed increase in NA-sensitivity in endothelium-denuded 12-14 week STZ-diabetic arteries with L N M M A or L-NIO, cumulative concentration-response curves to NA were obtained in endothelium-denuded vessels in the absence and presence of the n N O S inhibitor 7-NINA (100u,M). Pre-incubation with 7-NINA had no effect on NA responses of either control or diabetic endothelium-denuded mesenteric arteries (Fig.3, Table 3).  34  Table 3  Sensitivity (pD2) and maximum responses (Rmax) to NA of endothelium-denuded control and 12-14 week STZ-diabetic mesenteric arteries in the absence and presence of 7-NINA (100uJv1)  Animals  Rmax  Rmax  pD2 value  pD2 value  untreated  7-NINA  untreated  7-NINA  Control  166.1+4.1  171.7+4.1  7.34+0.09  7.28+0.07  Diabetic  225.8± 9.9*  223.5+5.3*  7.69+ 0.20  7.56+ 0.10  N=4 All values are mean i s . e . m . * P<0.05 as compared to corresponding control values  35  Figure 3 Cumulative concentration-response curves to N A of endothelium-denuded control (A) and 12-14 week STZ-diabetic (B) superior mesenteric arteries in the absence (V, N=4) and presence of 100uM 7-NINA (•, N=4). Each point represents the mean ± s.e.m.  36  A  B  37  D. Effects of i N O S inhibitors in e n d o t h e l i u m - d e n u d e d arteries  In order to investigate whether i N O S is the subtype of N O S contributing to the i n c r e a s e in N A sensitivity in e n d o t h e l i u m - d e n u d e d 12-14 w e e k S T Z - d i a b e t i c arteries with L N M M A or L-NIO, cumulative concentration-response c u r v e s to N A w e r e obtained in e n d o t h e l i u m - d e n u d e d arteries in the a b s e n c e a n d p r e s e n c e of the i N O S inhibitors, a m i n o g u a n i d i n e (1mM) or EIT (5|iM or 10u,M).  1.  Aminoguanidine  A m i n o g u a n i d i n e (1mM) h a d no effect o n N A sensitivity in e n d o t h e l i u m - d e n u d e d control arteries (Figure 4 , T a b l e 4). A significant i n c r e a s e in N A p D 2 v a l u e s w a s o b s e r v e d in e n d o t h e l i u m - d e n u d e d a n d endothelium-intact S T Z - d i a b e t i c arteries following preincubation with a m i n o g u a n i d i n e (Figure 4 , T a b l e 4). H o w e v e r , a m i n o g u a n i d i n e a l s o p r o d u c e d a significant i n c r e a s e in N A p D 2 v a l u e s in endothelium-intact control arteries s u g g e s t i n g that a m i n o g u a n i d i n e is also inhibiting e N O S at this concentration (Figure 4, T a b l e 4).  38  Table 4  Sensitivity (pD2 values) to NA of endothelium-intact and endothelium-denuded control and 12-14 week STZ-diabetic mesenteric arteries in the absence and presence of aminoguanidine (AG) (1mM)  Animals  Endothelial-status  PD2  pD2  Untreated  AG  Control  Intact  5.92+0.19  6.89+0.26*  Control  Denuded  7.49+0.18**  7.59+0.20**  Diabetic  Intact  6.04+0.14  7.11+0.18*  Diabetic  Denuded  7.37+0.21**  8.32+0.38*'**  N=6 All values are mean i s . e . m . * P<0.05 as compared to untreated values ** P<0.05 as compared to corresponding endothelium-intact values  39  Figure 4  C u m u l a t i v e concentration-response c u r v e s to N A of endothelium-intact (open s y m b o l s , N=4) a n d e n d o t h e l i u m - d e n u d e d ( c l o s e d - s y m b o l s , N=8) control (A) a n d 1 2 14 w e e k S T Z - d i a b e t i c (B) superior m e s e n t e r i c arteries in the a b s e n c e (circles) a n d p r e s e n c e (squares) of 1 m M A G . E a c h point represents the m e a n ± s . e . m .  40  A  300-1  ° • ° •  8  7  untreated,endo untreated, no endo A G , endo A G , no endo  6  -log[NA] M  B  300-1  untreated, endo untreated, no endo A G , endo A G , no endo  8  7  6  -log[NA] M  41  2.  S-ethylisothiourea  Since aminoguanidine did not appear to be acting as a selective inhibitor of iNOS, cumulative concentration-response curves to NA were obtained in endothelium-denuded arteries in the absence and presence of the highly selective i N O S inhibitor S-ethylisothiourea (EIT) (5u.M or 10u.M). EIT (5u.M) had no effect on NA sensitivity in either control or diabetic arteries. Similarly, no significant increase in sensitivity to NA was obtained in endotheliumdenuded control vessels following preincubation with 10u,M EIT (Figure 5,Table 5). However, 10u.m EIT produced a leftward shift in the N A concentration-response curve, and a significant increase in NA p D values in endothelium-denuded diabetic 2  mesenteric arteries (Fig.5, Table 5). To investigate whether EIT (IOLUVI) also inhibits e N O S , cumulative concentration-response curves to NA were obtained in endothelium-intact control arteries. N A pD2 values in endothelium-intact arteries were 6.04±0.41 in the absence, and 6.14+0.33 in the presence of 10LIM EIT (N=3, P>0.05).  42  Table 5  Sensitivity (pD2 values) to N A of endothelium-denuded control and 12-14 week S T Z diabetic mesenteric arteries in the absence and presence of EIT  Animals  EIT  pD2 untreated  PD2  EIT  Concentration Control, N=4  5uM  7.14+0.21  7.38+0.19  Control, N=5  10u,M  7.40+0.09  7.39+0.19  Diabetic, N=4  5uM  7.42+0.21  7.38±0.13  Diabetic, N=11  10uM  7.43+0.23  8.40+0.13*  All values are mean ± s.e.m. *P<0.05 as compared to untreated values  43  Figure 5  C u m u l a t i v e concentration-response c u r v e s to N A of e n d o t h e l i u m - d e n u d e d control (A, N=5) a n d 12-14 w e e k S T Z - d i a b e t i c (B, N=11) superior m e s e n t e r i c arteries in the a b s e n c e ( • ) a n d p r e s e n c e of 1 0 u M E I T ( B ) . E a c h point represents the m e a n ± s.e.m.  44  A  B  45  E. Investigation of reversibility of L-NIO and EIT  To determine if the increase in NA sensitivity seen with L-NIO was due to competitive inhibition of NOS, cumulative concentration-response curves to NA were obtained in endothelium-intact control mesenteric arteries treated with L-NIO alone or in the presence of L-arginine or D-arginine (1mM each). L-arginine abolished the leftward shift in the NA concentration-response curve and the increase in N A p D  2  values due to L-NIO, while D-arginine had no effect on the NA response in the presence of L-NIO (Table 6, Fig.6). NA concentration response curves were also obtained in endothelium-denuded diabetic arteries treated with EIT (10u.M) alone or in the presence of L-arginine or Darginine to verify that the increase in N A p D values with EIT in diabetic vessels was 2  due to competitive inhibition of NOS. A s was found with L-NIO, the effect of EIT on the NA response was abolished by co-incubation with L-arginine but was not affected by D-arginine (Table 6, Fig.7).  46  Table 6 Effect of co-incubation of N O S inhibitors with L-arginine or D-arginine on NA p D values in mesenteric arteries from control and 12-14 week STZ-diabetic rats.  Mesenteric Arteries  Inhibitor  pD (untreated)  pD (inhibitor)  Control (endotheliumintact) Diabetic (endotheliumdenuded)  L-NIO  6.29±0.08  7.11+ 0.07*  EIT  7.22+0.08  8.07± 0.12*  2  2  pD (inhibitor + Larginine) 6.28±0.11  pD (inhibitor + Darginine) 7.13±0.09*  7.46±0.32  8.13±0.16*  2  2  2  N=5 in each group. All values are mean + s.e.m. * P<0.05 compared to untreated values  47  Figure 6  Cumulative concentration-response curves to NA of endothelium-intact superior mesenteric arteries from control rats in the absence (•) and presence of 300u,M LNIO (A) (A), 300uM L-NIO + 1mM D-arginine (•) (B), or 300uM L-NIO + 1 m M L arginine (•)  (C). Each point represents the mean ± s.e.m. (N=5 animals)  48  -log[NA] M  Figure 7  Cumulative concentration-response curves to NA of endothelium-denuded S T Z diabetic superior mesenteric arteries in the absence (•) and presence of 10jiM EIT (•) (A), 10uM EIT + 1mM D-arginine (•) (B), or 10|iM EIT + 1mM L-arginine ( • ) (C). Each point represents the mean ± s.e.m. (N=5 animals)  50  F. Pharmacological investigation of the time-course of iNOS induction in diabetic V S M To investigate the time course of iNOS induction in V S M of STZ-diabetic rats, cumulative concentration-response curves to NA in the absence and presence of EIT (10LLM) were obtained in endothelium-denuded control and diabetic arteries 2-3 weeks and 6-8 weeks following STZ-injection. No significant differences in sensitivity (pD2) or responsiveness (Rmax) were observed between untreated control and diabetic arteries 2-3 weeks following S T Z injection (Table 7; Fig.8). NA pD2 values of 6-8 week STZ-diabetic arteries were also not significantly different from control (Table 7; Fig.9). A s a group, 6-8 week S T Z diabetic arteries had significantly greater maximum responses to NA than control (2way A N O V A ) . However, individual comparisons using Newman-Kuehl's post-hoc tests failed to show significant differences. Preincubation with EIT had no significant effect on NA responses in endotheliumdenuded 2-3 week or 6-8 week STZ-diabetic or control arteries (Table 7; Fig.8,9).  52  Table 7 Sensitivity (pD2) and maximum responses (Rmax) to NA of endothelium-denuded control and STZ-diabetic mesenteric arteries 2-3 weeks or 6-8 weeks following S T Z injection in the absence and presence of EIT (10u.M)  Animals  Time-point  Rmax  Rmax  PD2  PD2  untreated  EIT  untreated  EIT  Control  2-3 weeks  164±9.4  167.6+7.4  7.24±0.15  7.33+0.12  Control  6-8 weeks  155.2+5.8  155.6+4.4  7.06+0.1  7.14+0.08  Diabetic  2-3 weeks  162.2+4.3  160.4+.4.8  7.36±0.08  7.49±0.10  Diabetic  6-8 weeks  172.9+7.3  169.7+8.0  7.21±0.13  7.46±0.18  N=6  All values are mean + s.e.m.  53  Figure 8 Cumulative concentration-response curves to N A of endothelium-denuded control (A) and 2-3 week STZ-diabetic (B) superior mesenteric arteries in the absence (•) and presence of 10LIM EIT(B). Each point represents the mean ± s.e.m., N=7  54  A  0) (0 c  300n  untreated  o Q.  0) O  QL  EIT 200-  o E 3  E 100X (0  s  55 8  7  6  -log[NA] M  B  300  untreated EIT  8  7  6  -log[NA] M  55  Figure  9  Cumulative concentration-response curves to N A of endothelium-denuded control (A) and 6-8 week STZ-diabetic (B) superior mesenteric arteries in the absence (•) and presence of  IOWVI  EIT(B).  Each point represents the mean ± s.e.m.,  N=7  56  A  B  57  G . Immunohistochemical analysis of 12-14 week STZ-diabetic and control mesenteric arteries  Immunohistochemical analysis was performed in order to investigate whether the individual N O S subtypes could be detected in V S M from control or 12-14 week STZ-diabetic rats. In all immunostains, nuclei are indicated by dark pink dots and positive signals for antigen detection are indicated by dark black/purple dots.  eNOS Immunostaining of mesenteric arteries for e N O S indicated that e N O S was expressed only in the endothelial cell monolayer of both control and STZ-diabetic arteries (Fig.10).  nNOS Immunostaining for nNOS produced no positive signal in either control or diabetic arteries (Fig.11 A-D). The anti-nNOS antibody used did produce a positive signal in the positive control (mouse brain sections) at the same dilution (Fig.11E).  iNOS There was a striking difference between control and STZ-diabetic arteries in immunostaining for iNOS. A strong positive signal for iNOS was observed in the medial, adventitial, and intimal layers of the superior mesenteric arteries from S T Z diabetic but not control rats (Fig. 12).  58  Macrophage To determine whether macrophage infiltration is the source of iNOS expression in the diabetic mesenteric arteries, control and diabetic arteries were incubated with a specific antibody to rat macrophage (clone ED2). No positive staining for macrophages were obtained in control or diabetic arteries (Fig.13A,B). The antimacrophage (clone ED2) antibody used did produce a positive signal in the positive control (rat spleen sections) at the same dilution (Fig.13C).  59  Figure 10  Immunostaining of endothelium-intact control (A,B) and 12-14 week STZ-diabetic (C,D) superior mesenteric arteries with the monoclonal anti-eNOS antibody. Scale bar: 100u.m  60  62  Figure 11  Immunostaining of endothelium-intact control (A,B) and 12-14 week STZ-diabetic (C,D) superior mesenteric arteries and mouse brain (E), with the polyclonal anti-nNOS antibody.  Scale bar: 100um  63  64  E  66  Figure 12  Immunostaining of endothelium-intact control (A-C) and 12-14 week STZ-diabetic (DG) superior mesenteric arteries with the polyclonal anti-iNOS antibody.  Scale bar: 10Ourn  67  (  69  Figure 13  Immunostaining of endothelium-intact control (A) and 12-14 week STZ-diabetic (B) superior mesenteric arteries and rat spleen (C) with the monoclonal anti-macrophage (clone ED2) antibody.  Scale bar: 100um  72  A  B  c  71  H. Quantitative measurement of cytosolic N O S activity at different time points following STZ-injection  To investigate N O S activity in control and STZ-diabetic superior mesenteric arteries at 2-3 weeks, 6-8 weeks, and 12-14 weeks following STZ-injection, the citrulline assay for quantitative analysis of N O S activity was performed. Cytosolic calcium-dependent (nNOS) and independent (iNOS) activity was determined. No significant calcium-dependent (nNOS) activity was observed in either control or diabetic arteries at any time point following STZ-injection (Fig. 14, Table 8). Similarly, no significant calcium-independent activity was detected in control mesenteric arteries at any time point (Fig. 15, Table 8). Furthermore, no calciumindependent activity was observed in STZ-diabetic arteries at 2-3 weeks or 6-8 weeks following STZ-injection (Fig. 15, Table 8). However, a significant and marked elevation in calcium-independent (iNOS) activity was detected in superior mesenteric arteries from 12-14 week STZ-diabetic rats (Fig. 15, Table 8).  75  Table 8  Cytosolic N O S specific activity of control and STZ-diabetic superior  mesenteric  arteries 2-3 weeks, 6-8 weeks, and 12-14 weeks following STZ-injection  Animals  Time-point  Calcium-  Calcium-  dependent activity  independent  (pmol/min/mg)  (pmol/min/mg)  Control, N=6  2-3 weeks  0.39±0.37  0.92+0.39  Control, N=5  6-8 weeks  2.17±0.27  1.47±0.87  Control, N=7  12-14 weeks  0.24±0.39  1.43+0.39  Diabetic, N=6  2-3 weeks  1.70±0.94  0.66±0.40  Diabetic, N=6  6-8 weeks  0.24+0.23  2.31±0.63  Diabetic, N=7  12-14 weeks  0.25+0.46  18.06±4.11*  * P<0.05 as compared to background levels and corresponding control levels All values are mean ± s.e.m.  76  Figure 14  Calcium-dependent N O S specific activity of the cytosolic fraction of control (solid bars) and STZ-diabetic (open bars) 2-3 weeks, 6-8 weeks, and 12-14 weeks following STZ-injection. Each bar represents the mean±s.e.m.  77  20-,  I Control I Diabetic  £\E  :> 3  ts s  15H  £ E  10H  < Q. o i  CO ^ 0) O  O E Z a2-3 weeks  6-8 weeks  12-14 weeks  time after STZ-injection  Figure 15 Calcium-independent N O S specific activity of the cytosolic fraction of control (solid bars) and STZ-diabetic (open bars) 2-3 weeks, 6-8 weeks, and 12-14 weeks following STZ-injection. Each bar represents the mean+s.e.m. * P<0.05 compared to control  79  30  n  • I  £ E Q-'E  := 10CO o  O E Z Q.  2-3 weeks  6-8 weeks  12-14 weeks  Control I Diabetic  Discussion  The results of the present investigation provide evidence for the novel finding that iNOS is functionally expressed in V S M of superior mesenteric arteries from 12-14 week STZ-diabetic rats, but not their age and gender matched controls. Furthermore, induction of iNOS does not occur in diabetic arteries until a time point after 6-8 weeks following STZ-injection. Induction of iNOS in STZ-diabetic arteries and the elevated NO levels that result may be implicated in the cardiovascular dysfunction associated with chronic diabetes mellitus. In the present study, endothelium-intact mesenteric arteries from 12-14 week STZ-diabetic rats exhibited an increased maximum responsiveness (when normalized for the maximum response of the same preparation to KCI) with no change in sensitivity to NA compared to responses of age and gender-matched control rats. No difference in maximum responses was observed between control and 2-3 week S T Z diabetic arteries. These data are consistent with previous reports from our laboratory, which have found increased maximum responses of 3 month diabetic arteries to N A but not KCI, with little or no change in NA p D value (reviewed in Subramanian and 2  MacLeod, 1999). Neither endothelial-denudation nor preincubation with N O S inhibitors produced any significant effect on maximum responses in control or diabetic arteries suggesting that the enhanced maximum responsiveness to NA in long-term diabetic arteries is independent of the endothelial-cell layer and of NO. Endothelial denudation or non-selective inhibition of N O S with L N M M A or LNIO resulted in a leftward shift of the concentration-response curves to NA in both control and diabetic arteries, suggesting that NO release in these arteries normally  81  limits NA sensitivity. However the magnitude of the shift produced by L-NIO in endothelium-intact arteries was significantly greater in 12-14 week STZ-diabetic arteries. The L N M M A or L-NIO-induced increase in sensitivity to N A in control arteries is likely due primarily to inhibition of e N O S from the endothelial cell layer, since N O S inhibition produced no further effect on NA sensitivity in these vessels following endothelial denudation. In contrast, the presence of a leftward shift in the NA concentration-response curve with LNMMA or L-NIO in endothelium-denuded 12-14 week STZ-diabetic arteries suggests the presence of N O S activity in diabetic V S M . Following preincubation with LNMMA, the NA sensitivity of endotheliumdenuded 12-14 week STZ-diabetic arteries was significantly greater than that of endothelium-intact (treated) diabetic arteries. However, there was no difference in N A p D values between treated endothelium-intact and treated endothelium-denuded 122  14 week STZ-diabetic arteries with L-NIO. This maybe due to differences between the two inhibitors (Figure 16). Both L N M M A and L-NIO act to inhibit N O S activity by competing with L-arginine for the active site on the N O S enzyme (Kerwin et al, 1995). However, L-NIO may diffuse through the endothelial cell layer to the V S M more efficiently than L N M M A and may also compete with L-arginine for uptake into the cell at its transporter, the cationic y+ transporter (Kerwin et al, 1995). It is unlikely that the observed increase in N A sensitivity produced by L-NIO is caused by a non-specific effect of the compound, since the stereospecificity of the interaction of L-NIO with N O S and/or competition with L-arginine for intracellular transport was confirmed by reversal of the leftward shift of the concentration-response curve with L-arginine but not D-arginine.  82  Figure 16  Chemical structures of L-Arginine (A), LNMMA (B) and L-NIO (C)  L-arginine  H rV >=NH z  LNMMA  NH  H  H  H ^NH^  L-NIO  NH COOH  Results from the present study do not provide evidence for a significant difference in e N O S activity or expression between control and diabetic arteries at any time point following STZ-injection. Endothelial-denudation produced increases in pD2 values of similar magnitude in control and diabetic arteries. Furthermore, immunohistochemical analysis revealed a positive signal for e N O S in the endothelial cell layer in both control and diabetic arteries. These observations are consistent with previous findings from our laboratory, that there is no significant difference in the maximum ACh-induced relaxation or in the ACh-induced increase in c G M P levels between control and STZ-diabetic arteries (Harris and MacLeod, 1988). However, numerous studies from other laboratories have reported impaired endotheliumdependent relaxation in isolated arterial rings from STZ-diabetic rats. Explanations for reported impairments of endothelium-dependent vasodilation include enhanced c G M P metabolism, reduced activity of cGMP-dependent protein kinase, decreased availability of L-arginine, and increased inactivation of NO by free radicals (Kamata et al, 1989; Hattori etal, 1991; Taylor et al, 1992; Rodriguez-Manas etal, 1998). Further controversy in this area comes from reports from other laboratories that endothelialderived NO is enhanced in diabetic arteries (Bhardwaj and Moore, 1988; Langenstroer and Pieper, 1992). The reasons for these discrepancies remain unclear at present. Although endothelial-derived NO (from eNOS) may be of primary importance under normal conditions, both nNOS and iNOS may be expressed in V S M under certain conditions. The expression of nNOS in V S M has been reported in uterine arteries during pregnancy (Garvey et al, 1994) and in carotid arteries from  85  spontaneously hypertensive rats (Boulanger et al, 1998). However, in the present study, incubation of endothelium-denuded control and 12-14 week STZ-diabetic arteries with 100wVI 7-NINA had no effect on NA responses. At this concentration, 7NINA has been reported to be a selective inhibitor of nNOS in arterial ring preparations (Moore et al., 1993). In support of the pharmacological experiments, no positive immunostaining for nNOS was observed in either control or diabetic arteries, although a positive signal was present in the positive control (mouse brain). Furthermore, quantitative measurement of N O S activity of the cytosolic fraction (containing nNOS and iNOS) revealed no significant calcium-dependent activity in 2-3 week, 6-8 week, or 12-14 week control or STZ-diabetic superior mesenteric arteries. Therefore, it seems unlikely that nNOS is the subtype of N O S present in diabetic mesenteric arteries. iNOS has also been reported to be expressed in V S M under various pathological conditions including exposure to inflammatory cytokines and endothelial injury (Schulz and Triggle, 1994; Hanson et al, 1994; Gonzalez-Fernandez et al, 1998). The results of the present study provide substantial evidence that iNOS is the subtype that is expressed in V S M from STZ-diabetic rats. Aminoguanidine (1mM) has been used as a selective inhibitor of i N O S in arterial preparations (Tilton et al, 1993; Joly et al, 1994). In the present study, preincubation of endothelium-denuded 12-14 week STZ-diabetic arteries with 1mM aminoguanidine produced an increase in sensitivity to N A similar to that seen with LNIO or LNMMA, suggesting the presence of N O S activity in V S M of 12-14 week S T Z diabetic arteries. However, the results of the present study also indicate that at the concentration used (1mM), aminoguanidine is not selective for iNOS. Preincubation  86  of endothelium-intact control arteries with aminoguanidine (1mM) produced a significant increase in NA sensitivity, suggesting that aminoguanidine also inhibits e N O S at this concentration. EIT has been reported to be 40-50 fold more selective for iNOS than for nNOS or e N O S (Nakane et al., 1995). In arterial ring preparations, EIT has been reported to inhibit iNOS at concentrations ranging from 2u.M-30u.M (Southan et al, 1995). EIT (5LIM) produced no effect on N A responses in our experimental conditions. However, EIT (10u,M) mimicked the increase in NA sensitivity seen with L-NIO in endotheliumdenuded 12-14 week STZ-diabetic mesenteric arteries. In the present investigation, EIT (10u.M) had no effect on NA responses in endothelium-intact control arteries, providing evidence that EIT is selective for iNOS over e N O S at this concentration in mesenteric arteries. L-arginine but not D-arginine reversed the leftward shift in the concentration-response curve to NA, confirming that EIT acts as a stereoselective competitive inhibitor of NOS. EIT had no effect on NA responses in endotheliumdenuded control arteries from rats of any age, suggesting that iNOS activity is not present in V S M from control rats. Similarly, EIT had no effect on responses to NA of endothelium-denuded 2-3 week or 6-8 week STZ-diabetic mesenteric arteries, suggesting that iNOS is not induced in STZ-diabetic arteries until after 6-8 weeks following STZ-injection. Immunohistochemical analysis demonstrated a strong positive signal for iNOS expression in mesenteric arteries from 12-14 week STZ-diabetic but not control rats. Immunostaining for iNOS protein was observed in the intimal, medial, and adventitial layers of diabetic arteries.  87  Further evidence for the functional expression of iNOS in 12-14 week S T Z diabetic arteries was obtained from quantitative measurement of cytosolic N O S activity. Levels of calcium-independent (iNOS) activity in 12-14 week STZ-diabetic arteries were significantly increased above both background and levels in control arteries. No significant calcium-independent N O S activity was observed in control arteries at any time point. Similarly, no significant calcium-independent activity was detected in the cytosolic fraction of 2-3 week or 6-8 week STZ-diabetic arteries, further suggesting that iNOS is not functionally expressed in diabetic V S M until after 6-8 weeks following STZ-injection. The possibility that the presence of iNOS in the 12-14 week STZ-diabetic arteries was due to its induction in vitro seems unlikely. All experiments were conducted in the presence of dexamethasone, at a concentration (0.1 pjvl) that has been reported to inhibit iNOS induction in vitro (Knowles et al., 1990b). Furthermore, iNOS induction in vitro has been reported to require a time period of hours (Zheng et al., 1997) whereas isolated arteries obtained for immunohistochemical analysis or for quantitative N O S assay were fixed in formalin or flash frozen in liquid nitrogen respectively within minutes of excision. Finally, it is unlikely that iNOS was induced in 12-14 week STZ-diabetic arteries as a result of experimental procedures, since no evidence for the presence of iNOS in control arteries or 2-3 week or 6-8 week S T Z diabetic arteries, treated in the exact same manner, was obtained in the present study. Immunohistochemical results from the present study suggest that iNOS is present throughout all layers of superior mesenteric arteries from 12-14 week S T Z diabetic rats. iNOS appears to be induced in the tunica intima in endothelial cells. It is  assumed that iNOS is expressed in V S M cells in the tunica media. However, the specific cell types that express iNOS in the tunica media and tunica adventitia cannot be stated with certainty from results of the present study since no double antibody immunohistochemical analysis for the co-detection of iNOS with specific cell types was performed. Macrophage infiltration has been recently identified as a source of iNOS expression in smooth muscle preparations under certain conditions (Zheng et al., 1997). However, it is not likely that the iNOS detected in V S M of 12-14 week STZ-diabetic arteries in the present study is due to macrophage infiltration as no positive staining above background was detected for the macrophage-specific ED2 antibody in either control or diabetic arteries. Therefore, iNOS induction in 12-14 week STZ-diabetic arteries is likely due to pathophysiological changes that occur with the chronic diabetic state. The factors contributing to iNOS induction in diabetic arteries remain unclear. However, AGE-mediated alterations in cytokine production, increased oxidative stress and/or enhanced P K C activation could all be implicated in the process of iNOS induction in long-term diabetes (DeVera et al., 1996, Paul et al., 1997; Hecker et al, 1999). As described in the Introduction, the formation of A G E s with chronic hyperglycemia may result in A G E s binding to their receptor, R A G E , resulting in the generation of R O S and alterations in gene expression (Brownlee, 2000). A G E s have been shown to upregulate various cytokines that are involved in iNOS gene transcription, including TGF-f3, IL-1, and  NF-KB  (Vlassara et al, 1988). Preliminary  data from our laboratory indicate that levels of glycosylated hemoglobin, a measure of A G E formation, increase in STZ-diabetic rats with time. Increased A G E formation and  89  the enhanced cytokine production that may result over time may help to explain why iNOS is not expressed in STZ-diabetic rats until after 8 weeks following induction of diabetes. Increased oxidative stress has also been associated with chronic diabetes and may result in the activation of factors involved in iNOS induction, including  NF-KB  (Guigliano et al, 1996; Li and Karen, 1999). In addition, long-term diabetes may be associated with enhanced activation of specific P K C isoforms, which may also potentiate cytokine-induced iNOS expression in V S M . The individual P K C isoforms that are elevated in different diabetic tissues remains to be elucidated. The mechanism by which iNOS is induced in diabetic V S M also remains unclear. A s described in the Introduction, the promoter region of the iNOS gene in mouse, rat, and human contains several putative binding elements, including response elements to transcription factors that may be elevated in long-term diabetes, including  NF-KB  and IL-1 p (DeVera et al, 1996). Induction of iNOS by individual  cytokines varies depending on the tissue, which may be due to variable expression of cytokine receptors or cell-specific differences in iNOS gene transcription factors (Kolyada et al, 1996). Although the underlying mechanism remains unclear, activation of P K C has been reported to potentiate iNOS induction in V S M , depending on the isoform of P K C and the individual blood vessel (Hecker et al, 1999; Paul et al, 1997). The individual isoforms of P K C capable of potentiation (or attenuation) of iNOS induction in different tissues and cell types also remains to be determined. Once induced, iNOS synthesizes a prolonged and increased release of N O as compared to e N O S and nNOS (Moncada and Higgs, 1995). Any increase in NO production has potential for free radical mediated damage, particularly under  90  conditions of oxidative stress where peroxynitrite is formed more readily (Snyder and Bredt, 1992). Since there has been considerable recent evidence that there is an increase in the generation of oxygen-derived free radicals in diabetes, increased NO production in diabetic arteries has potential for considerable damage. In other conditions in which the expression of iNOS in all three arterial layers has been demonstrated, the overproduction of NO and subsequent peroxynitrite formation have been reported to contribute to vascular damage and to atherosclerosis (Luoma and Yla-Herttuala, 1999; Gutterman, 1999). Peroxynitrite itself has been reported to have vasoconstrictor properties (Elliot et al, 1998). Furthermore, oxidation of LDL and other plasma lipids by peroxynitrite results in the production of various potentially detrimental substances, including lipid peroxides, modified aldehydes, and lipids with vasoconstrictor properties (DarleyUsmar and White, 1997). Peroxynitrite has been shown to react with plasma lipids and lipoproteins (including LDL) to produce vasoconstrictor isoprostanes (Moore et al, 1995). The mechanism of isoprostane action remains unclear although it has been suggested that isoprostanes may be involved in the alteration of cyclo-oxygenasedependent signal transduction pathways (Moore et al, 1995). Increased plasma levels and urinary excretion of isoprostanes have been reported in type I and type II diabetic patients (Gopaul et al, 1995; Davi et al, 1999). The vasoconstrictor properties of peroxynitrite and isoprostane in vivo remain to be determined. However, one potential implication of iNOS induction in human diabetic arteries may be an increase in blood pressure and/or peripheral resistance in response to the vasoconstrictor actions of peroxynitrite and/or isoprostanes. In humans, iNOS has been detected in V S M cells and megakaryocytes from  91  atherosclerotic lesions (Butterly et al, 1996). Oxidation of LDL by peroxynitrite is thought to be involved in the initiation of macrophage infiltration and inflammatory response in the process of atherosclerotic lesion formation in humans (Graham et al, 1993). It is not fully understood why experimental STZ-diabetic rats do not appear to develop atherosclerosis. However, rats have higher levels of high density lipoproteins (HDL) than LDL (Bennani-Kabchi et al, 2000). Therefore, unlike human arteries, levels of oxidized LDL in rat arteries may not be sufficient to stimulate atherogenesis. In humans, peroxynitrite-mediated nitration of tyrosine residues results in a stable, highly immunogenic adduct, which also has been implicated in an inflammatory response and the initiation of atherogenesis (Darley-Usmar and White, 1997). Therefore, if iNOS is expressed in V S M in humans with diabetes, there is significant potential for an increase in the formation of atherosclerotic lesions. Abnormal production of NO may also contribute to endothelial dysfunction due to an imbalance in the release of other endothelium-derived factors, including endothelin-1 (ET-1) (Kiff et al, 1991). ET-1 is a potent vasoconstrictor whose production has been reported to be both increased (Takeda et al, 1991) and decreased (Wu and Tang, 1998) in experimental diabetes. NO may be involved in the regulation of ET-1 release, since inhibition of N O S increases ET-1 release (Kiff et al., 1991), while ET-1 may stimulate the release of NO (Warner et al., 1999). Results from the present study would predict a decrease in ET-1 release from diabetic mesenteric arteries, since induction of iNOS would result in overproduction of NO. The consequences of iNOS induction in diabetic V S M need not be entirely detrimental. Increased NO production could act in a protective manner, by limiting the enhancement of vasoconstrictor responses of diabetic arteries. This is supported by  92  the observation of this study, that the sensitivity of diabetic arteries to NA is not significantly different from control in the absence of L-NIO, but is enhanced in it's presence. The induction of iNOS in V S M of STZ-diabetic arteries may also help to explain why these animals have generally not been reported to be hypertensive. Induction of iNOS could help to compensate for the decreased release of or responsiveness to endothelial-derived NO that has been commonly reported in diabetic arteries. Although the overproduction of NO by iNOS may contribute to atherogenesis as a result of peroxynitrite production, NO itself acts to prevent atherosclerosis by inhibition of platelet aggregation and V S M proliferation (Moncada and Higgs, 1995; Yates et al, 1992). Whether induction of iNOS in diabetic V S M results in detrimental or protective effects remains unclear but may be dependent on levels of oxygen derived free radicals in the local environment of NO production.  93  Summary and Conclusions  A. Summary 1. STZ-diabetic rats exhibited characteristics associated with the diabetic state including hyperglycemia, hypoinsulinemia, decreased body weight gain, polyuria, and osmotic diarrhea.  2. Maximum responsiveness to NA but not sensitivity was significantly greater in 1214 week STZ-diabetic superior mesenteric arteries as compared to control regardless of the status of the endothelium or the absence or presence of N O S inhibitors. No difference in N A responses were observed between 2-3 week or 68 week STZ-diabetic arteries and their age and gender-matched controls.  3. Endothelial-denudation or preincubation of endothelium-intact control arteries with L-NIO or L N M M A produced an increase in NA sensitivity, suggesting that NO derived from the endothelial cell layer normally limits NA sensitivity. Following endothelial-denudation, no further change in N A sensitivity was observed in control superior mesenteric arteries following preincubation with LNIO, LNMMA, aminoguanidine, EIT, or 7-NINA, suggesting that N O S activity is not present in V S M from control rats.  4. Endothelial-denudation or incubation of 12-14 week STZ-diabetic arteries with LNIO or L N M M A produced an increase in NA sensitivity. However, in superior mesenteric arteries from 12-14 week STZ-diabetic rats, L N M M A or L-NIO  94  produced a further increase in NA sensitivity following endothelial denudation, suggesting the presence of N O S activity in V S M in diabetic arteries.  5. L-NIO produced a greater increase in NA sensitivity than L N M M A in endotheliumintact 12-14 week STZ-diabetic arteries. There was no observed difference between the two inhibitors in NA responses in endothelium-denuded arteries, suggesting that L-NIO may be more efficiently transported through the endothelial cell layer.  6. 7-NINA had no significant effect on NA responses in endothelium-denuded 12-14 week STZ-diabetic arteries, suggesting that nNOS is not the subtype of N O S present in diabetic V S M . Furthermore, no positive signal for nNOS protein was obtained in either control or 12-14 week STZ-diabetic arteries, suggesting the lack of nNOS expression in these arteries.  7. EIT (10u.M) produced a significant increase in NA sensitivity in endotheliumdenuded 12-14 week but not 2-3 week or 6-8 week STZ-diabetic mesenteric arteries, suggesting that iNOS is functionally expressed in diabetic V S M , but not until after 8 weeks following S T Z injection.  8. Immunohistochemical analysis indicated e N O S expression in the endothelial cell monolayer of both control and 12-14 week STZ-diabetic superior mesenteric arteries.  95  9. Immunohistochemical staining for iNOS protein indicated extensive iNOS expression in 12-14 week STZ-diabetic arteries but not control. The presence of iNOS protein in diabetic V S M was not due to macrophage infiltration, since immunostaining for macrophages produced no positive signal.  10. Quantitative measurement of cytosolic N O S activity revealed no significant calcium-dependent N O S (nNOS) activity in control, 2-3 week, 6-8 week, or 12-14 week STZ-diabetic rats.  11. No calcium-independent N O S (iNOS) activity was obtained in control, 2-3 week, or 6-8 week STZ-diabetic arteries. However, there was a marked elevation in calcium-independent N O S activity in 12-14 week STZ-diabetic rats, providing further evidence for the functional expression of iNOS in 12-14 week STZ-diabetic arteries.  96  B. Conclusion and Future experiments  Conclusion The results of the present study demonstrate the novel finding that iNOS is functionally expressed in V S M from rats with chronic STZ-induced diabetes, at a time when vasoconstrictor responsiveness is also enhanced. Induction of iNOS in diabetic V S M may play a role in the cardiovascular complications associated with diabetes mellitus. Future research Further investigation will be necessary to determine whether iNOS is expressed in chronic STZ-diabetic animals in other tissue types and/or V S M from different arteries. Immunohistochemical detection for the presence of iNOS in other arteries and arterial beds of chronic STZ-diabetic rats should be performed. In addition, investigation of iNOS activity in arteries from other species that are susceptible to atherosclerosis, such as rabbits or guinea pigs with STZ-induced diabetes, may also be of interest to investigate the effects of iNOS induction on atherogenesis. 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