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Role of altered signalling pathways in abnormal vasoconstrictor responses in mesenteric arteries from… Irem, Mueed 2005

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ROLE OF ALTERED SIGNALING PATHWAYS IN ABNORMAL VASOCONSTRICTOR RESPONSES IN MESENTERIC ARTERIES FROM STZ-DIABETIC RATS by IREM MUEED B.Sc, Punjab University, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES PHARMACEUTICAL SCIENCES THE UNIVERSITY OF BRITISH COLUMBIA November 2005 © Irem Mueed, 2005 ABSTRACT Cardiovascular complications are recognized to be the major cause of morbidity and mortality associated with diabetes mellitus. One of the most common features of vascular dysfunction in well-established diabetes is the enhanced reactivity of blood vessels to vasoconstrictors. We and others have consistently found that contractile responses of arteries from rats with well-established diabetes to stimulation of G-protein coupled receptors (GPCRs), such as ai-adrenoceptor (ai-AR) and endothelin-1 (ET-1) receptors, are enhanced. Previous studies from this lab have demonstrated that the increased contractile responses of arteries from chronic (12-14 weeks) streptozotocin- (STZ-) diabetic rats to cci-AR stimulation result from a change in the signal transduction process downstream from the receptor. Protein kinase C (PKC) has been suggested to contribute to enhanced contractile responses of arteries from STZ-diabetic rats to stimulation of GPCRs. This was investigated in the present study by comparing the effects of the PKC inhibitors, Ro-318220 and calphostin C on contractile responses of mesenteric arteries from diabetic and age-matched control rats to the cci-AR agonist, norepinephrine (NE) and to ET-1. Since translocation of P K C to the membrane is considered a hallmark of its activation, the effects of N E and ET-1 on particulate (membrane) levels of three isoforms of P K C (PKCoc, 8 and s) that have been implicated in contraction were determined. The effect of N E on phosphorylation of CPI-17, a substrate for P K C , was also investigated. Contractile responses of endothelium-denuded arteries from diabetic rats to N E were enhanced, but were normalized by the P K C inhibitors. In contrast, no change in contractile responses of diabetic arteries to ET-1 could be detected, and PKC inhibition attenuated ET-1 responses to a similar extent in both control and diabetic tissues. NE produced a small translocation of PKCs in control arteries, but a significant translocation of P K C a and a much larger translocation of PKCe in diabetic arteries. ET-1 increased translocation of P K C a , 8 and 8 to the same extent in both control and diabetic arteries. NE significantly enhanced CPI-17 phosphorylation in diabetic, but not control, arteries and this was blocked by calphostin C. In addition to PKC, the RhoA/Rho kinase (RhoK) signaling pathway is also believed to play an important role in vasoconstriction. In the next part of this study, the role of RhoK in enhanced contractile responses of diabetic mesenteric arteries to stimulation of a i -ARs was ii investigated. The selective RhoK inhibitors, Y-27632 and H-1152, produced greater inhibition of contractile responses to phenylephrine (PE) in diabetic than control mesenteric arteries and normalized the difference between them. Contractile responses to ET-1 were not different between control and diabetic arteries, and were not affected by RhoK inhibition. Since translocation of RhoK from the cytosolic to the membrane fraction is considered a marker of enzyme activity, the effects of PE on particulate levels of the two RhoK isoforms (ROCK I and II) were determined. A maximum concentration of PE produced significant translocation of ROCK I and II that was inhibited by Y-27632 in both control and diabetic arteries. The PE-induced translocation of ROCK II was significantly greater in diabetic tissues. PE also produced significant translocation of PKC a, 5 and s in diabetic but not control arteries. Y-27632 blocked the translocation of these isoforms in diabetic arteries but had no effect on ET-1 induced translocation of PKC isoforms in control arteries. These data suggest that increased activation of the PKC/CPI-17 as well as the RhoK pathway contribute to the enhanced contractile responses of diabetic mesenteric arteries to a t -AR stimulation. They further suggest that there is an interaction between the RhoK and PKC pathways on stimulation of oti-ARs in diabetic mesenteric arteries, and that RhoK may be upstream of PKC. On the other hand, RhoK does not appear to contribute to contractile responses to ET-1 in either control or diabetic mesenteric arteries; this may be the reason why vasoconstrictor responses to this agonist are not altered in diabetic arteries. 111 Abstract Table of Contents List of Tables List of Figures List of Abbreviations 1. INTRODUCTION 1 1.1 D I A B E T E S 1 1.2 D I A B E T E S A N D C A R D I O V A S C U L A R D I S E A S E 2 1.3 E X P E R I M E N T A L M O D E L S OF D I A B T E T E S 3 1.4 A B N O R M A L I T I E S I N T H E D I A B E T I C V A S C U L A T U R E 5 1.5 P R E V I O U S F I N D I N G S F R O M O U R L A B 7 1.6 G - P R O T E I N C O U P L E D R E C E P T O R S 7 1.7 G P C R s A N D A S S O C I A T E D S I G N A L T R A N S D U C T I O N P A T H W A Y S 10 1.8 V A S C U L A R S M O O T H M U S C L E C O N T R A C T I O N 12 1.9 T H E P R O T E I N K I N A S E C F A M I L Y 14 1.10 H Y P E R G L Y C E M I A I N D U C E D A C T I V A T I O N O F P K C 16 1.11 V A S O R E A C T I V I T Y A N D P K C A C T I V A T I O N I N D I A B E T E S 19 1.12 P K C A N D C A L C I U M S E N S T I Z A T I O N 20 1.13 RhoA A N D Rho K I N A S E 22 1.14 R h o A / R h o K A N D C A R D I O V A S C U L A R D I S E A S E 24 1.15 R h o A / R h o K A N D D I A B E T E S 25 iv T A B L E O F C O N T E N T S Page i i iv v i i i ix x i i i 1.16 RhoA/RhoK AND CALCIUM SENSITIZATION 27 1.17 CALCIUM SENSITIZATION BY ARACHIDONIC ACID 28 1.18 CALCIUM SENSITIZATION BY TYROSINE KINASES 29 1.19 HYPOTHESIS AND OBJECTIVES 31 2. M A T E R I A L S A N D M E T H O D S 34 2.1 MATERIALS 34 2.1.1 Materials used to make the Krebs Buffer 34 2.1.2 Materials used in the tissue bath contractility studies 34 2.1.3 Materials used in Western Blotting 34 2.2 METHODS 35 2.2.1 Control and Diabetic Rats 35 2.2.2 Tissue Bath Studies 35 2.2.3 Bio-Rad Protein Assay - 36 2.2.4 Western Blotting 37 2.2.5 Western Blotting: Total levels of PKC and RhoK isoforms 38 2.2.6 Western Blotting: Particulate levels of PKC and RhoK isoforms 39 2.2.7 Western Blotting: Total and phosphorylated levels of CPI-17 40 2.2.8 Statistical Analyses 41 3. R E S U L T S 44 3.1 CHARACTERISTICS OF CONTROL AND DIABETIC ANIMALS 44 3.2 ROLE OF PKC/CPI-17 PATHWAY IN ENHANCED CONTACTILE 45 RESPONSES OF MESENTERIC ARTERIES FROM DIABETIC v RATS TO a, -AR STIMULATION' 3.2.1 Effect of PKC Inhibitors on Contractile Responses to NE and ET-1 45 3.2.2 Effect of Diabetes on Levels of PKC Isoforms in Mesenteric 53 Arteries 3.2.3 Effect of NE and ET-1 on Particulate Levels of PKC Isoforms 54 in Mesenteric Arteries 3.2.4 Effect of PdBu and NE on Phosphorylation of CPI-17 57 3.3 CONTRIBUTION OF RhoK TO THE ENHANCED CONTRACTILE 60 RESPONSES OF DIABETIC MESENTERIC ARTERIES TO ai-AR STIMULATION: INTERACTION WITH PKC 3.3.1 Effect of RhoK & PKC Inhibitors on Contractile Responses to PE 60 and ET-1 3.3.2 Effect of Diabetes on Levels of RhoK Isoforms in Mesenteric Arteries 68 3.3.3 Effect of PE and ET-1 on Particulate Levels of RhoK Isoforms in 69 Mesenteric Arteries 3.4 ROLE OF TKs AND cPLA 2 IN ENHANCED CONTRACTILE 75 RESPONSES OF MESENTRIC ARTERIES FROM DIABETIC RATS TO ai-AR STIMULATION 3.4.1 Effect of TKs and cPLA 2 Inhibitors on Contractile Responses to 75 PE and ET-1 4. D I S C U S S I O N 82 4.1 OVERVIEW 82 4.2 CONTRACTILE RESPONSES TO ai-AR STIMULATION 83 4.3 CONTRACTILE RESPONSES TO ET-1 85 4.4 BASAL LEVELS OF PKC ISOFORMS 86 4.5 BASAL LEVELS OF RhoK ISOFORMS 88 4.6 PARTICIPATION OF PKC IN ot,-AR AND ET-1 RECEPTOR 89 STIMULATION vi 4.7 PHOSPHORYLATION OF CPI-17 93 4.8 PARTICIPATION OF RhoK IN a , -AR AND ET-1 RECEPTOR 95 STIMULATION 4.9 K + DEPOLARIZATION-INDUCED RhoA/RhoK A C T I V A T I O N 98 4.10 AGONIST-INDUCED CHANGES IN CYTOSOLIC (SOLUBLE) 98 L E V E L S OF P K C A N D RhoK ISOFORMS 4.11 INTERACTION OF P K C A N D RhoK SIGNALING P A T H W A Y S 99 4.12 POTENTIAL MECHANISMS LEADING TO E N H A N C E D RhoK 103 PARTICIPATION IN a i -AR-MEDIATED VASOCONSTRICTION IN MESENTERIC ARTERIES F R O M DIABETIC RATS 4.13 P H A R M A C O L O G I C A L INHIBITION OF TKs A N D c P L A 2 104 5. SUMMARY AND CONCLUSIONS 108 6. FUTURE DIRECTIONS 110 7. REFERENCES 112 vii LIST OF TABLES Page 3.1 Characteristics of Control and Diabetic Rats 44 3.2 Effect of PKC Inhibitors on NE and ET-1 -Induced Contractions 52 3.3 Effect of RhoK and PKC Inhibitors on PE-Induced Contractions 66 3.4 Effect of TKs and C P L A 2 Inhibitors on PE-Induced Contractions 81 viii LIST OF FIGURES Page 1.1 Diagrammatic Representation of the Structure of PKC Isoforms 15 1.2 Principal Sources of DAG Production 17 1.3 Diagrammatic Representation of the Structure of RhoK 23 1.4 Regulation of Activation of RhoK 24 2.1 Preliminary experiments for the selection of optimal loading concentration. 42 A Optical density vs. protein concentration curve for particulate 42 levels of PKCa B Optical density vs. protein concentration curve for particulate 42 levels ofPKCs 2.2 A Total levels of actin in control and diabetic arteries 43 B Particulate levels of actin in the absence and presence of agonist 43 in particulate fractions of control and diabetic arteries 3.1 A Effect of Ro-318220 on Maximum KC1 Contractions in Control and 46 Diabetic Mesenteric Arteries B Effect of Ro-318220 on NE Concentration Response Curves in 46 Control and Diabetic Mesenteric Arteries 3.2 A Effect of Calphostin C on Maximum KC1 contractions in Control and 48 Diabetic Mesenteric Arteries B Effect of Calphostin C on NE Concentration Response Curves in 48 Control and Diabetic Mesenteric Arteries 3.3 A Effect of Rottlerin on Maximum KC1 Contractions in Control and 49 Diabetic Mesenteric Arteries B Effect of Rottlerin on NE Concentration Response Curves in 49 Control and Diabetic Mesenteric Arteries ix 3.4 Effect of Ro-318220 on ET-1 Concentration Response Curves in 50 Control and Diabetic Mesenteric Arteries 3.5 Effect of Calphostin C on ET-1 Concentration Response Curves in 50 Control and Diabetic Mesenteric Arteries 3.6 Effect of Rottlerin on ET-1 Concentration Response Curves in 51 Control Mesenteric Arteries 3.7 Total Levels of PKC a, (32, 5 and s in Control and Diabetic Mesenteric Arteries 53 3.8 A Particulate levels of PKCa in the Absence and Presence of NE 55 in Control and Diabetic Arteries B Particulate levels of PKC8 in the Absence and Presence of NE 55 in Control and Diabetic Arteries C Particulate levels of PKCe in the Absence and Presence of NE 55 in Control and Diabetic Arteries 3.9 A Particulate levels of PKCa in the Absence and Presence of ET-1 56 in Control and Diabetic Arteries B Particulate levels of PKC8 in the Absence and Presence of ET-1 56 in Control and Diabetic Arteries C Particulate levels of PKCs in the Absence and Presence of ET-1 56 in Control and Diabetic Arteries 3.10 Levels of phosphorylated CPI-17 in Mesenteric Arteries from Control Rats 58 in the Untreated State and in the Presence of PdBu 3.11 A Total Levels of CPI-17 in Control and Diabetic Arteries 5 8 B Levels of Phosphorylated CPI-17 in Mesenteric Arteries from Control 59 and Diabetic Rats in the Untreated State, in the Presence of NE and NE + Calphostin C 3.12 A Effect of Y-27632 on KC1 Contractions in Control and 62 Diabetic Mesenteric Arteries B Effect of Y-27632 on PdBu contractions in Control and 62 Diabetic Mesenteric Arteries C Effect of Y-27632 on PE Concentration Response Curves in 62 Control and Diabetic Mesenteric Arteries x 3.13 A Effect of H-1152 on KC1 Contractions in Control Mesenteric Arteries 63 B Effect of H- l 152 on PE Concentration Response Curves in 63 Control and Diabetic Mesenteric Arteries 3.14 Effect of Ro-318220 on PE Concentration Response Curves in 64 Control and Diabetic Mesenteric Arteries 3.15 Effect of Calphostin C on PE Concentration Response Curves in 64 Control and Diabetic Mesenteric Arteries 3.16 Effect of Rottlerin on PE Concentration Response Curves in 65 Control and Diabetic Mesenteric Arteries 3.17 Effect of Y-27632 on ET-1 Concentration Response Curves in 67 Control and Diabetic Mesenteric Arteries 3.18 Effect of H- l 152 on ET-1 Concentration Response Curves in 67 Control Mesenteric Arteries 3.19 Total Levels of ROCK I and II in Control and Diabetic Mesenteric Arteries 68 3.20 A Particulate levels of ROCK in the Untreated State, Presence of PE 71 and PE + Y-27632 in Control and Diabetic Arteries • B Particulate levels of ROCK II in the Untreated State, Presence of PE 71 and PE + Y-27632 in Control and Diabetic Arteries 3.21 A Particulate levels of ROCK I in the Absence and Presence of ET-1 72 in Control and Diabetic Arteries B Particulate levels of ROCK II in the Absence and Presence of ET-1 72 in Control and Diabetic Arteries 3.22 A Particulate levels of PKCa in the Untreated State, Presence of PE 73 and PE + Y-27632 in Control and Diabetic Arteries B Particulate levels of PKC5 in the Untreated State, Presence of PE 73 and PE + Y-27632 in Control and Diabetic Arteries C Particulate levels of PKCs in the Untreated State, Presence of PE 73 and PE + Y-27632 in Control and Diabetic Arteries 3.23 A Particulate levels of PKCa in the Untreated State, Presence of ET-1 74 and ET-1 + Y-27632 in Control Arteries xi B Particulate levels of PKCS in the Untreated State, Presence of ET-1 74 and ET-1 + Y-27632 in Control Arteries C Particulate levels of PKCs in the Untreated State, Presence of ET-1 74 and ET-1 + Y-27632 in Control Arteries 3.24 A Effect of Tyrphostin A23 on KC1 Contractions in Control and 76 Diabetic Mesenteric Arteries B Effect of Tyrphostin A23 on PE Concentration Response Curves in 76 Control and Diabetic Mesenteric Arteries 3.25 A Effect of Genistein on KC1 Contractions in Control Mesenteric Arteries 77 B Effect of Genistein on PE Concentration Response Curves in 77 Control and Diabetic Mesenteric Arteries v 3.26 A Effect of A A C O C F 3 on KC1 Contractions in Control and 78 Diabetic Mesenteric Arteries B Effect of A A C O C F 3 on PE Concentration Response Curves in 78 Control and Diabetic Mesenteric Arteries 3.27 Effect of Tyrphostin A23 on PE Concentration Response Curves in 79 Control and Diabetic Mesenteric Arteries 3.28 Effect of Genistein on PE Concentration Response Curves in 79 Control and Diabetic Mesenteric Arteries 3.29 Effect of AACOCF3 on PE Concentration Response Curves in 80 Control and Diabetic Mesenteric Arteries The proposed signal transduction pathways activated in response to 102 stimulation of cci-AR and ET-1 receptors in diabetic mesenteric arteries xn LIST OF ABBREVIATIONS [Caz+]j intracellular calcium concentration 5-HT 5-hydroxytryptamine 7TM seven transmembrane AA Arachidonic acid ACh acetylcholine ADA American Diabetes Association AG II angiotensin II AGE advanced glycation end products aPKC atypical protein kinase C Ca 2 + calcium ion CaD caldesmon CaP calponin CICR Ca2+-induced Ca 2 + release CPI-17 protein kinase C potentiated inhibitor protein of 17 kDa cPKC conventional protein kinase C C P L A 2 Cytosolic Ca dependent phospholipase A 2 CV cardiovascular CVD cardiovascular disease DAG diacylglycerol DM diabetes mellitus • EDCF endothelium derived constricting factors EDRF endothelium derived relaxing factors ET-1 endothelin-1 GAP GTPase activating protein GDI guanine nucleotide dissociation inhibitor GDP guanosine 5'-diphosphate GDPpS guanosine 5-0- (2-thiodiphosphate), a G-protein inhibitor GEF guanine nucleotide exchange factor GPCR G-protein coupled receptor G-protein heterotrimeric guanine nucleotide binding protein GTP guanosine 5"-triphosphate GTPyS guanosine 5"-0- (3-thiotriphosphate), a non-hydrolysable GTP analogue IDDM insulin dependent diabetes mellitus IP3 inositol 1, 4, 5 trisphosphate iPLA 2 Cytosolic Ca 2 + independent phospholipase A 2 KC1 potassium chloride LPA lysophosphatidic acid MAB mesenteric arterial bed MAP mitogen activated protein MLC2o regulatory light chains of myosin MLCK myosin light chain kinase MLCP myosin light chain phosphatase M YPT 110-13 OkDa regulatory subunit of MLCP Xlll NE norepinephrine NEFA non-esterified fatty acid NIDDM non-insulin dependent diabetes mellitus NO nitric oxide NOS nitric oxide synthase nPKC novel protein kinase C PA phosphatidic acid PC phosphatidylcholine pD2 - log E C 5 0 PdBu phorbol 12, 13 dibutyrate PDK-I phosphoinositide dependent kinase I PE phenylephrine PIP2 phosphatidylinositol 4, 5 bisphosphate PIP5K phosphatidylinositol 4-phosphate 5-kinase PKC protein kinase C PLA 2 phospholipase A 2 PLC phospholipase C PLD phospholipase D PMA phorbol 12-myristate 13-acetate PPlc 37-38kDa catalytic subunit of MLCP PS phosphatidylserine PTX pertussus toxin RACKS receptors for activated C-kinase RGS regulators of G-protein signaling RhoK Rho kinase ROS reactive oxygen species Ser serine SNP sodium nitroprusside sPLA2 secretory phospholipase A 2 SR sarcoplasmic reticulum STZ streptozotocin Thr threonine TK tyrosine kinase TXA 2 thromboxane A 2 UT untreated VGCC voltage gated calcium channel cti-AR alphai -adrenoceptor P-AR beta-adrenoceptor xiv 1. INTRODUCTION 1.1 DIABETES Diabetes mellitus (DM) is a heterogeneous group of disorders that are characterized by abnormally high levels of glucose in the blood. Hyperglycemia is usually due to defects in insulin secretion or resistance to the actions of insulin or a combination of these. D M affects approximately 100 million people worldwide, and by the year 2010 this number is expected to increase to 221 million (Amos et al, 1997). Intensive research showed that there were different types of D M with varying etiologies and there was a need for appropriate classification and systematic categorization for proper management of the disease. In 1980, the World Health Organization (WHO) endorsed the classification developed by an international group referred to as the National Diabetes Data Group (NDDG). According to this, two major forms of D M were identified: insulin dependent D M (IDDM, Type 1 DM) and non-insulin dependent D M (NIDDM, Type 2 DM) (World Health Organization, 1980). However in 1996 - 1997, the American Diabetes Association (ADA) proposed some changes to this classification scheme (American Diabetes Association Expert Committee, 1997). According to the new system, the terms insulin dependent D M and non-insulin dependent D M as well as their acronyms, IDDM and NEDDM, were eliminated and instead the two major kinds of D M were simply referred to as Type 1 and Type 2 D M . Type 1 DM involves destruction of pancreatic (5-cells, either immune mediated or due to unknown etiologies, and comprises about 5 - 10% of all cases of diabetes (Harris, 2004). It usually begins early in life, affecting children and adolescents, and is often called juvenile diabetes. However it has also been shown to affect adults (> 30 years of age) (Melton et ah, 1983; Laakso & Pyorala, 1985). The primary cause of immunologic (3-cell destruction is the development of antibodies to the pancreatic islet cells and to insulin. The initiation of p- cell autoimmunity may be induced in any person at any time (Knip, 1997). Amongst the etiologic agents that initiate and subsequently accelerate P-cell destruction are environmental factors (e.g. viral infections). Type 2 DM involves impaired insulin-mediated glucose disposal (insulin resistance) and defects in insulin secretion. It comprises about 90% of all cases of diabetes (Harris, 2004). It mostly affects adults, starting off with symptoms of insulin resistance and eventually leading 1 to a decline in insulin secretion. Although there is a strong genetic basis for this disease, lifestyle choices play a very important role in the development of Type 2 DM. There is a strong correlation between a sedentary lifestyle, physical inactivity, high calorie intake and obesity (especially central obesity) and increased incidence of Type 2 D M (Chisholm et al, 1997; Gerich, 1998). 1.2 DIABETES AND CARDIOVASCULAR DISEASE Both Type 1 and Type 2 D M are independent risk factors for the development of cardiovascular (CV) complications such as coronary heart disease, stroke, peripheral arterial disease and cardiomyopathy (Grundy et al, 1999; Creager et al, 2003). Cardiovascular disease (CVD) occurs at a significantly higher rate in diabetic patients as compared to the general population (Kowalska et al, 2001; Hu et al, 2002; Norhammar et al, 2002). Not only is there an increased incidence of CV pathologies but also lower survival outcomes, significantly increasing the mortality and morbidity associated with this disease (Nesto, 2004). CVD is the major cause of death in more 65% of the diabetic population. Most of the CV complications of diabetes are associated with tissue damage in the macro-and microvasculature. Macrovascular complications occur in the large blood vessels such as peripheral arteries of the lower limbs (e.g. femoral, popliteal), cerebral and coronary arteries and lead to the development of atherosclerosis, peripheral vascular occlusive disease and myocardial infarction. Microvascular disease affects the small arteries such as those supplying the kidneys, retina and nerves. Many of the long-term complications of diabetes stem from microvascular complications such as retinopathy, nephropathy and neurovascular defects associated with autonomic neuropathy. Clinical studies have shown that tight glycemic control with the administration of insulin results in significant retardation of the development and progression of microvascular complications (Diabetes Control and Complications Trial Research Group, 1993; UK Prospective Diabetes Study (UKPDS) Group, 1998). Although multiple biochemical mechanisms have been proposed to explain how hyperglycemia leads to development of diabetic vascular complications (as reviewed by King & Brownlee, 1996; Cooper et al, 2001; Setter et al, 2003), the four major proposed mechanisms are: 2 1) Increased glucose metabolism through the polyol pathway. Through this pathway glucose is reduced to sorbitol by aldose reductase, coupled with oxidation of NADPH to NADP. The sorbitol is then oxidized to fructose by sorbitol dehydrogenase coupled with reduction of NAD to NADH. Persistent hyperglycemia can lead to accumulation of sorbitol (which can increase osmotic stress and cell permeability), fructose, vasodilatory prostaglandins and unbound NADH. However in vivo studies using polyol pathway inhibitors have been disappointing overall, showing little improvement in diabetes-induced retinopathy or nephropathy (Sorbinil Retinopathy Trial Research Group, 1990; Engerman et al, 1994) 2) Increased production of advanced glycation end products (AGEs) by non-enzymatic and spontaneous reactions between reducing sugars and amino groups on proteins, lipids and nucleic acids. AGEs can contribute to basement membrane thickening of various tissues as well as alteration in the functional properties of several matrix molecules (e.g. collagen). This can result in altered structure and function of intact vessels. A number of clinical studies have shown that AGE inhibitors like aminoguanidine can prevent chronic manifestations of diabetes in the microvasculature (Eldenstein & Brownlee, 1992; Brownlee, 1995; Brownlee, 2001). 3) Generation of reactive oxygen species (ROS) by a number of mechanisms such as auto-oxidation of glucose and AGEs, increased activity of oxidative enzymes by enhanced NADH from the polyol pathway as well as enhanced activation of PKC. In fact overproduction of ROS such as superoxide is the common consequence of polyol pathway activation, formation of glycoxidation products and accumulation of AGEs. ROS can increase microvascular permeability and damage protein and mitochondrial DNA (Baynes, 1991; Taylor, 2001). It has also been shown that antioxidant levels are decreased in diabetes (Jennings et al., 1987). Antioxidant supplementation in animal models and humans has been shown to ameliorate some of diabetes-induced microvascular abnormalities (Nagamatsu et al, 1995; Bursell et al, 1999). 4) Increased activation of the DAG/PKC pathway (discussed in section 1.10) 1.3 EXPERIMENTAL MODELS OF DIABETES Intensive research conducted over the last few decades has enhanced our knowledge about the pathophysiology of DM. However despite significant progress, this disease at best can be 3 treated but is usually refractory to prevention, particularly with respect to Type 1 DM. Animal models of DM have allowed investigators to acquire more information about all aspects of this disease, as well as test new and innovative therapies for treating it. Type 1 DM can be induced in animals with the help of toxins and infectious agents (e.g. streptozotocin (STZ), synthesized from streptomycetes acromogenes) that act as selective pancreatic P cell cytotoxins. This model is of research interest because environmental factors can often trigger the onset of Type I DM in humans (Akerblom et al, 2002; Hawa et al, 2002). STZ is taken up by pancreatic p cells via the glucose transporter GLUT 2 (Schnedl et al, 1994) and induces p-cell death primarily due to alkylation of DNA (as reviewed by Szkudelski, 2001). In addition to this, STZ generates reactive oxygen species such as the superoxide anion and is also a nitric oxide (NO) donor, and these effects can contribute to DNA fragmentation and damage (as reviewed by Szkudelski, 2001). A number of studies have shown that the diabetogenic effects of STZ are related to its dose (Junod et al, 1969). An intermediate dose of STZ, 50 - 70 mg/kg injected intravenously can induce Type 1 DM that is stable over a long time (Ar'Rajab & Ahren, 1993). Lower doses of STZ (30 - 40 mg/Kg) induce a transient diabetic state with complete recovery after a few days (Ar'Rajab & Ahren, 1993). Junod et al (1969) showed that when STZ was administered at a dose of 55 - 65 mg/kg (a dose commonly used in a lot of cardiovascular studies including the present investigation), rats developed hyperglycemia but were able to survive without insulin supplementation. At a higher dose of 100 mg/kg, rats were able to survive only with insulin treatment (Junod et al, 1969). Within 7 - 1 0 hours after STZ administration, there is massive P-cell necrosis associated with increased serum insulin levels and hypoglycemia. Within 1 - 28 days pancreatic insulin levels are reduced to less than 5% of normal values and a state of prolonged hyperglycemia develops (Junod et al, 1967). Rats treated with STZ display many features seen in human patients with uncontrolled diabetes mellitus, such as hyperglycemia, hypoinsulinemia, development of cataracts, increased urinary glucose levels and consequently polyuria, polydipsia and weight loss. Studies focused on vascular dysfunction in the STZ diabetic rat are usually carried out within a few months of STZ treatment and therefore reflect the effects of relatively acute insulin deficiency in newly diagnosed Type I patients (as reviewed by Tomlinson et al, 1992). The continued use of this model is evidence of the fact that it can provide valuable information 4 about the underlying pathophysiological conditions contributing to this debilitating disease and can be used for the development of treatments besides insulin therapy, which can arrest or inhibit the development of these conditions. 1.4 ABNORMALITIES IN THE DIABETIC VASCULATURE The cardiovascular complications of diabetes mellitus have been shown to arise in part from altered reactivity of vascular smooth muscle to neurotransmitters and circulating hormones like norepinephrine (NE) and angiotensin II (AG II). While it has been well established that diabetes leads to abnormalities in vascular function, studies conducted on vascular reactivity in Type I diabetic animal models have not all been in agreement with each other and there have been conflicting reports on whether responsiveness is increased or decreased. We and others have consistently found that contractile responses of arteries from rats with well-established diabetes to stimulation of G-protein coupled receptors (GPCRs), such as alpha adrenoceptors (a-ARs) and endothelin (ET) receptors, are enhanced (MacLeod, 1985; Abebe & MacLeod, 1991a; Abebe & MacLeod, 1992; Inoguchi et al, 1992; Ishikawa et al, 2004; Yousif et al, 2004). Significantly elevated responses have been observed in the aorta (Hattori et al, 1995), mesenteric resistance (Taylor et al, 1994), renal (Inazu et al, 1991), carotid (Llorens et al, 2004) and coronary (Tickerhoof et al, 2003) arteries from diabetic animals on stimulation by agonists when compared to corresponding age-matched controls. However, in contrast, some studies have shown that diabetic arteries exhibit attenuated responses to agonist-induced stimulation (James & Hodgson, 1995; Makino & Kamata, 2000a; Misurski et al, 2001). The precise reason for this discrepancy is not clear. One possibility could be differences in the severity and duration of diabetes, which has been shown to significantly affect the development of vascular dysfunction. Enhanced reactivity to agonists has usually been observed after a relatively long-term diabetic state (> 12 weeks) (Jackson & Carrier, 1981; MacLeod & McNeill, 1985; Van Buren et al, 1998). Similarly most studies conducted on Type I diabetic patients have shown vasoconstrictor responses to be increased (Christlieb et al, 1976; Drury et al, 1984; Eichler et al, 1992; Mclntyre et al, 2001), although decreased reactivity has also been reported (McNally et al, 1994). Another commonly reported vascular abnormality in Type I diabetic patients as well as experimental animal models is the development of endothelial dysfunction (as reviewed by De Vriese et al, 2000). The integrity of the endothelium is usually assessed by measuring 5 vasodilatation in response to agents like acetylcholine (ACh) or bradykinin. Arteries like the aorta (Tesfamariam et al, 1993; Pieper & Siebeneich, 1998) and the superior mesenteric artery (Heygate et al, 1995; Palmer et al, 1998) isolated from STZ-diabetic animals exhibit impaired relaxation to ACh with no change in vasodilator responses to sodium nitroprusside (SNP, endothelium-independent vasodilator). However, impaired endothelium-dependent vasodilatation is not consistently observed in diabetic vessels. Our lab reported that ACh-induced vasodilatation in aorta and mesenteric arteries from STZ-diabetic rats was not significantly different from the corresponding controls (Harris & MacLeod, 1988). Similarly it has been shown that endothelium-dependent relaxation is preserved in aorta (Head et al, 1987; Wakabayashi et al, 1987) and renal arteries (Yousif et al, 2002) from diabetic animals. The reason for this discrepancy is not known but may be because of differences in the species used, the strain of rats, or the duration of the diabetes. Endothelial dysfunction in diabetes is believed to arise predominantly from an imbalance in availability and production of endothelium derived relaxing factors (EDRFs) and endothelium derived constricting factors (EDCFs) (as reviewed by De Vriese et al, 2000). A hyperglycemia-induced increase in steady-state levels of reactive oxygen species results in inactivation of NO (an EDRF) (Pieper et al, 1992; Giugliano et al, 1996). At the same time there can also be enhanced production of EDCFs such as prostanoids, which can oppose the action of EDRFs (Mayhan et al, 1991; Shimizu et al, 1993). Another factor that can contribute to endothelial dysfunction in diabetes is impaired release of endothelium-derived hyperpolarizing factor (EDHF), which has been suggested to be a major determinant of vascular tone in small arteries (Garland et al., 1995). For example, attenuation of endothelium-dependent relaxation in mesenteric arteries from STZ-diabetic rats was suggested to be due to a diminished contribution of EDHF (Fukao et al, 1997; Makino et al, 2000b; Wigg et al, 2001). In most cases the abnormal vasoreactivity can be restored to normal by administration of insulin (MacLeod, 1985; Takiguchi et al, 1989; Tanz et al, 1989; Hodgson & King, 1992), suggesting that hyperglycemia and associated metabolic abnormalities predominantly mediate vascular dysfunction in diabetes . 6 1.5 PREVIOUS FINDINGS FROM OUR LAB Our lab has consistently reported that contractile responses to ai-AR stimulation in diabetic arteries are significantly elevated as compared to age and gender matched controls. The enhanced responses are due to the diabetic state as insulin treatment can prevent or reverse these effects (MacLeod, 1985). Intensive investigation of the factors that could be contributing to this revealed that the enhanced responses of diabetic arteries were not due to: 1) A generalized increase in the reactivity of diabetic tissues, because contractile responses to potassium depolarization were not significantly elevated (MacLeod, 1985). 2) Release of endothelium-derived vasoactive factors, since enhanced responses were also observed in the absence of a functional endothelium (Harris & MacLeod, 1988). 3) The development of autonomic neuropathy or changes in (3 receptor function or extraneuronal uptake mechanisms (Weber & MacLeod, 1994). 4) Differences in total cti-AR number or affinity or change in G-protein (G2/3 and Gq/n) levels (Weber & MacLeod, 1997). These results suggest that significantly enhanced contractile responses in diabetic arteries are due to an intrinsic alteration at the level of the smooth muscle, such as increased efficiency of coupling of the ai-AR to its signal transduction pathway. 1.6 G-PROTEIN COUPLED RECEPTORS G proteins are heterotrimeric guanine nucleotide binding proteins that serve as transducers for GPCRs. These receptors constitute the largest, most ubiquitous and most versatile family of membrane receptors. They are characterized by a signature seven transmembrane (7TM) configuration with three extracellular loops and three intracellular cytoplasmic loops (hence GPCRs are also referred to as heptahelical or serpentine receptors). G-proteins consist of three subunits, a, P and y, of which Ga is responsible for GDP and GTP binding. Stimulation of GPCRs with their cognate agonists results in the formation of a transient high affinity complex of agonist-activated receptor and G-protein. GDP is released from the G-protein and is replaced by GTP, promoting its dissociation into the GTP bound Ga-subunit and a GPy dimer, both of which have the capacity to activate a number of different effector systems and second messenger cascades (as reviewed by Pierce et al, 2002). Regulators of G-protein 7 signaling (RGS) mediate hydrolysis of GTP to GDP, leading to the reassociation of the subunits and termination of the activation cycle. To date 16 a, 5 p and 12 y proteins have been identified. G proteins are generally referred to by their a subunits. So for example the Gq complex contains Gaq. Four different a subunit families have been recognized (as reviewed by Landry & Gies, 2002) -1) Gs: including a s and a0if, which couple to stimulation of adenylyl cyclase. 2) G,: including a n , 0CJ2, (XJ3, a 0 i , C t 0 2 , Ot t i , 0 C T 2 , & z and C t g u s t , which couple to inhibition of adenylyl cyclase and activation of G-protein coupled inwardly rectifying potassium channels (GIRK). 3) Gq: including a q , a n , a.14, OC15 and 0ti6, which couple to stimulation of phospholipase Cp (PLCP). 4) G12: including (X\2 and a n , which couple to stimulation of Rho guanine nucleotide exchange factors (GEFs). The two GPCRs that are most pertinent to this project are the cti-AR and the ET receptor. The oti-ARs play an important role in regulating the vascular and cardiac effects of the endogenous sympathomimetic catecholamines, epinephrine and N E and mediate the vasoconstrictor actions of exogenously administered a -AR agonists (as reviewed by Guimaraes & Moura, 2001). They constitute three pharmacologically defined subtypes: a ) A -AR, aiB-AR and OCID-AR. A l l three oti ARs have been shown to be expressed in smooth muscles of different rat arteries (Faure et al, 1994; Scofield et al, 1995). While these subtypes share structural homology, they exhibit considerable functional heterogeneity as well as differences in tissue distribution and affinities for agonists and antagonists. The CCIB-A R has been shown to be predominantly expressed on the cell surface (Hrometz et al, 1999) whereas the CCID-AR was detected mainly intracellularly (McCune et al, 2000). cciA-AR was detected both on the cell surface as well as intracellularly (Hirasawa et al, 1997; McCune et al, 2000). In vascular smooth muscle, oti-ARs mediate two important effects: vasoconstriction and vascular growth (as reviewed by Varma & Deng, 2000). Arterial vasoconstriction is mediated 8 primarily by postjuctional oci-ARs, although depending on the artery, different subtypes participate in this response (Hrometz et al, 1999). For'example it has been shown that the CCID-AR is predominantly activated in the aorta, femoral and iliac artery (Piascik et al, 1995; Buckner et al, 1996; Gisbert et al, 2003), ociA-AR in the renal and caudal artery (Hrometz et al, 1999; Gisbert et al, 2003) and CCIB-AR and aip-AR in the mesenteric artery (Hussain & Marshall, 2000). a,-AR stimulation by catecholamines has been shown to trigger not only immediate responses but also long term actions such as enhanced growth and proliferation in vascular smooth muscle cells (Hu et al, 1996; Xin et al, 1997). Abnormally increased sympathomimetic activity due to elevated plasma catecholamine levels has been shown to produce vascular hypertrophic effects via increased a-actin mRNA and total cell protein (Chen et al, 1995) and increased D N A synthesis (van Kleef et al, 1992). Mitogenesis in vascular smooth muscle due to cci-AR stimulation is primarily due to tyrosine phosphorylation of several proteins (Hu et al, 1999). The hypertrophic effects of cti-ARs in arterial smooth muscle cells have been shown to be predominantly mediated by the ctie and a ] D subtype (Chen et al, 1995; Xin et al, 1997). There are two kinds of E T receptors, E T A and ETB in mammalian cells and they are stimulated by three endogenous isoforms of E T , E T - 1 , E T - 2 and E T - 3 (as reviewed by Sokolovsky, 1995; Sugden, 2003). The E T A receptor has high affinity for ET-1 and E T - 2 but a low affinity for E T - 3 whereas the E T B receptor has equal affinity for all three E T isoforms. ETA receptors are located primarily on vascular smooth muscle cells and regulate vasoconstriction and cellular proliferation and mitogenesis (Schiffrin, 1995). These effects are opposed by stimulation of ETB receptors that are predominantly expressed by vascular endothelial cells. Their activation leads to generation of prostacyclin and NO by stimulation of nitric oxide synthase (NOS) and helps to prevent apoptosis (Niwa et al., 2000). Therefore the vasoregulatory action of ETs can be controlled by a number of different factors such as number and type of receptors activated, receptor affinity and the kind of tissue being stimulated. To a lesser extent, ETB receptors are also expressed in vascular smooth cells and can mediate vasoconstriction like the E T A receptors. In pathological conditions there could be upregulation of ETB receptors on vascular smooth muscle and together with E T A 9 receptors, they could amplify the vasoconstrictive and mitogenic effects of ET (Haynes et al, 1995). 1.7 GPCRs AND ASSOCIATED SIGNAL TRANSDUCTION PATHWAYS Both cti-ARs (otiA, OCIB and OCID) (as reviewed by Zhong & Minneman, 1999; Varma & Deng, 2000) and ET receptors (ETA and ETB) (as reviewed by Sokolovsky, 1995; Sugden, 2003) have been shown to couple to the pertussus toxin (PTX)-insensitive G q/n G protein. Activation of this G protein leads to the stimulation of the phosphoinositide-specific phospholipase CP (PLCP) that hydrolyzes the membrane phospholipid, phosphatidylinositol 4, 5 bisphosphate (PIP2). This leads to the production of two second messengers: diacylglycerol (DAG) and inositol 1, 4, 5 trisphosphate (IP3). DAG remains in the cell membrane and mediates the activation of protein kinase C (PKC). IP3 diffuses into the cytoplasm and binds to receptors present on a number of intracellular organelles, primarily the sarcoplasmic reticulum (SR), and mediates Ca 2 + release. As far as cci-ARs are concerned, studies have suggested that there are differences in the coupling of the different receptor subtypes to their second messenger systems. ctiA-ARs have been shown to be the most effective activators of phosphoinositide hydrolysis and have been reported to be much more efficiently coupled to the activation of PKC than OCIB or OCID-ARS (Schwinn et al, 1995; Theroux et al, 1996; Taguchi et al, 1998). The cti-ARs (as reviewed by Seasholtz et al, 1999; Fukata et al, 2001) and ET receptors (Gohla et al, 2000) have also been shown to couple to the PTX-insensitive G12/13 G-protein. These G-proteins regulate the activity of the small monomeric GTPase Rho via direct activation and regulation of RhoGEFs (guanine nucleotide exchange factors) (as reviewed by Sah et al, 2000). In the inactive state, Rho is bound to GDP and GDI (guanine nucleotide dissociation inhibitor). Activation of RhoGEFs by G12/13 catalyzes nucleotide exchange on Rho, replacing GDP with GTP, thereby activating Rho. Since RhoGEFs facilitate RhoA activation, they can regulate the signaling duration and intensity of GPCRs. The two GEFs identified in vascular smooth muscle that have been shown to be activated by G12/13 are p l l 5 R h o G E F and PDZ-RhoGEF (Hart et al, 1998; Fukuhara et al, 1999). Inactivation of 10 small G-proteins is regulated by GAPs (GTPase activating proteins) that accelerate their intrinsic GTPase activity. Stimulation of ai-ARs and ET receptors has also been shown to increase the activity of phospholipase D (PLD) and phospholipase A 2 (PLA2) (as reviewed by Sokolovsky, 1995; Varma & Deng, 2000). PLD hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA), which is then converted into DAG. This signaling pathway provides an additional source of DAG in addition to the rapid but transient increase in DAG following agonist induced PI hydrolysis. The activated PKC resulting from PLD-induced DAG formation further enhances PLD activation. This produces a sustained increase in DAG formation and hence prolonged maintenance of PKC activation, a pathway that could be useful for long-term cellular responses such as cell growth (Nishizuka, 1992; Eskildsen-Helmond et al, 1996). PLA 2 represents a superfamily of enzymes broadly classified into (as reviewed by Balsinde et al, 2002): 1) Secretory (sPLA2) 2) Cytosolic Ca 2 + dependent (cPLA2) 3) Cytosolic Ca 2 + independent (iPLA2) On activation, PLA 2 catalyzes the hydrolysis of phospholipids into a free fatty acid and a lysophospholipid. Arachidonic acid (AA) production from phospholipids is primarily mediated via the catalytic activity of PLA 2 . sPLA 2 isoforms are low molecular weight extracellular enzymes (~ 14kDa), which require millimolar concentrations of Ca 2 + for activation (Balsinde et al., 1999). They do not exhibit fatty acid selectivity in vitro and so are not specific for phospholipids containing AA. Both iPLA 2 and cPLA 2 are intracellular enzymes and have been shown to have similar molecular weights (~ 85 kDa) and catalytic mechanisms (Balsinde & Dennis, 1997). iPLA 2 isoforms are Ca 2 + independent whereas cPLA 2 isoforms require micromolar concentrations of Ca 2 + for activation and are selective for AA-containing phospholipids. Free AA is the precursor of eicosanoids, which include the prostaglandins (produced via the cyclooxygenase reaction) and the leukotrienes (produced via the lipoxygenase reaction) (Smith, 1992). Stimulation of cti-ARs (Toma et al, 1995; Ward et al, 1995; Jin et al, 1996) and ET receptors (Ohanian et al, 1997) has also been associated with increased phosphorylation of proteins on tyrosine residues. Traditionally, activation of tyrosine kinases (TKs) has been 11 associated with long-term effects such as oncogenesis, cell growth and proliferation. However recent evidence has shown that TKs can be phosphorylated within seconds in response to vasoconstrictor hormones, and can influence vascular tone (Hughes & Wijetunge, 1998). These are usually non-receptor TKs, e.g. Src kinases that are present in the cytosol and, unlike receptor TKs whose activity is regulated by extracellular ligands; they usually do not have extracellular recognition domains for ligands. However, their activity can be regulated at an intracellular level by GPCRs that themselves lack intrinsic TK activity. One of the mechanisms used by GPCRs to signal through non-receptor TKs is via adaptor/scaffolding proteins that facilitate interaction between receptor and their downstream signaling effectors (Luttrell et al, 1999; Imamura et al, 2001). 1.8 VASCULAR SMOOTH MUSCLE CONTRACTION The principle trigger initiating contraction in smooth as well as striated (cardiac and skeletal) muscle is an increase in the intracellular calcium concentration ([Ca2+]i). In the resting state, vascular smooth muscle has a negative membrane potential, with [Ca2+]i ranging from 120-270 nmol. Membrane depolarization (positive potentials) can result in the opening of voltage-gated calcium channels (VGCC) and the influx of Ca 2 + from the extracellular space. This in turn also leads to Ca2+-induced Ca 2 + release (CICR) from the sarcoplasmic reticulum (SR). An increase in [Ca2+]j to 500-700 nmol triggers the binding of Ca 2 + to calmodulin, a multi-functional Ca 2 + binding protein. This in turn induces a conformational change in the calmodulin molecule as a result of which it binds to the catalytic sub-unit of myosin light chain kinase (MLCK) and activates it. M L C K then phosphorylates serine 19 on the regulatory light chains of myosin (MLC20, 20kDa) and allows the actomyosin ATPase to be activated by actin. The myosin cross bridges slide over the actin filaments, as a result of which there is development of contractile force. A fall in the [Ca2+]j inactivates MLCK and allows myosin light chain phosphatase (MLCP) to dephosphorylate MLC20, producing relaxation (as reviewed by Somlyo & Somlyo, 1994; Horowitz et al, 1996c). The development of contraction that is dependent on changes in membrane potential and [Ca2+]i is referred to as electromechanical coupling. Another physiologically important component of the contractile process is pharmacomechanical coupling (Somlyo & Somlyo, 1968) that operates via the ability of intracellular signaling pathways to affect force without changes in membrane potential. An important mechanism of pharmacomechanical coupling 12 is the regulation of Ca 2 + sensitivity. The development of force is not always proportional to the [Ca2+]j and neurohumoral stimulation of smooth muscle can increase the MLCK/ MLCP activity ratio, hence enhancing the sensitivity of the contractile proteins to Ca 2 + . This is referred to as Ca 2 + sensitization, that is the development of tension without change in [Ca2+]j. It has been shown that agonist-induced stimulation of GPCRs, including ai-AR, ET, thromboxane A 2 (TXA 2) and AT receptors, can induce Ca 2 + sensitization, and this can be mimicked by GTPyS (a non-hydrolysable GTP analogue) and is inhibited by GDPpS (a G-protein inhibitor) (Kitazawa et al, 1989; Himpens et al., 1990; Iizuka et al., 1997), suggesting that G-proteins are a vital factor in this process. The principal mechanisms for GPCR-mediated Ca 2 + sensitization have been suggested to be via inhibition of MLCP and inhibition of actomyosin ATPase by thin filament associated proteins. Inhibition of MLCP MLCP is a heterotrimeric complex composed of a 37 - 38kDa catalytic subunit (PPlc), a 110-130 kDa regulatory subunit (MYPT) and a 20kDa subunit of unknown function. While the catalytic subunit is common to all type I phosphatases, it is MYPT that confers the high selectivity of MLCP for phosphorylated myosin. It enhances the catalytic activity of PPlc and targets the entire MLCP complex towards phosphorylated myosin. Initially characterized as an unregulated 'house-keeping enzyme', it was soon established that MLCP is a G-protein regulated enzyme (Somlyo et al., 1989) and inhibition of MLCP is the major mechanism for GPCR mediated Ca 2 + sensitization (Somlyo & Somlyo, 2003; Ito et al, 2004). The inhibition of MLCP results in increased M L C 2 u phosphorylation and enhanced contractile force at a given [Ca2+]j. MLCP is predominantly inhibited via phosphorylation of MYPT and the site most commonly phosphorylated by a number of different kinases (such as ZIP-like kinase (Borman et al, 2002), integrin-linked kinase (Deng et al, 2001) and Rho kinase (RhoK) (Feng et al, 1999a) is Thr 696. However only RhoK-induced inhibition of MLCP has been shown to play a significant role in GPCR-mediated Ca 2 + sensitization and contraction in vascular smooth muscle (discussed in section 1.16). MLCP can also be inhibited by other mechanisms of action. It was shown that A A dissociated the MLCP holoenzyme and since the isolated PPlc is much less active compared to the MYPT-PPlc complex towards phosphorylated myosin, its activity was significantly 13 attenuated (Gong et al, 1992). MLCP can also be inhibited via phosphorylated CPI-17, a predominant PKC substrate (discussed in detail in section 1.23) (Eto et al, 1995; Eto et al, 1997). Inhibition of actomyosin ATPase by thin filament associated proteins Smooth muscle thin filaments are regulated by a number of proteins and the two most well characterized are caldesmon (CaD) and calponin (CaP). Both CaD and CaP have been shown to bind to actin and inhibit actomyosin ATPase under physiological conditions in vitro. They can compete with myosin for binding sites on actin and inhibit actomyosin ATPase by a number of mechanisms. CaD has been shown to decrease the affinity between actin and myosin as a result of which the ATPase cannot be activated (Hemric & Chalovich, 1988). It has also been shown to decrease the rate of reaction of the ATPase cycle (Hemric et al, 1994). CaP on the other hand does not affect the affinity between actin and myosin. Instead it can produce a conformational change in the actin molecule as a result of which there is a significant decrease in the activation rate of the ATPase (Horiuchi & Chacko, 1991; Noda et al, 1992). Phosphorylation of CaD and CaP by kinases such as PKC and mitogen activated protein (MAP) kinases results in 'disinhibition' of actomyosin ATPase activity, leading to enhanced contractile force without change in [Ca2+]j (as reviewed by Horowitz et al, 1996c). However while the role of CaD and CaP in GPCR-mediated Ca 2 + sensitization has been demonstrated in vitro, their in vivo significance and functions have yet to be established. The agonist-G-protein complex is physically separated from the myosin-bound MLCP and the thin filament associated CaD and CaP (as reviewed by Somlyo & Somlyo, 1994). PKC, RhoK, AA and TKs can serve as messengers that can communicate the Ca 2 + sensitization signals to MLCP and/or CaD and CaP. 1.9 THE PROTEIN KINASE C FAMILY PKC was identified in bovine cerebellum by Nishizuka and colleagues in 1977 (Inoue et al, 1977; Takai et al, 1977). Two years later it was found that PKC was activated by phosphatidylserine (PS) and DAG in a Ca 2 + dependent manner (Takai et al, 1979). More than 25 years later, intensive research has revealed PKC to be a large extended superfamily and not a single protein. At present the PKC family comprises of 12 known isoforms, which differ in structure, function and co-factor requirements. In fact the PKC family is the largest serine/threonine kinase family known, which could explain the many signal transduction 14 functions that have been attributed to this family (as reviewed by Webb et al, 2000). The 12 isoforms are divided into three families on the basis of co-factor requirements: 1) The conventional PKCs (cPKC: a, pi, Pn, y) that require PS, DAG and Ca 2 + for activation 2) The novel PKCs (nPKC: 8, 8, n, 9, p) that are Ca2+-independent and require PS and DAG for activation 3) The atypical PKCs (aPKC: C, x, X) that only require PS for activation The PKC isoforms share a highly conserved catalytic domain (containing the ATP (C3) and substrate (C4) binding site) and so have similar substrate specificities (Fig. 1.1). However they tend to differ in their membrane targeting regulatory domains, which contain the binding sites for PS (Cl), DAG (Cl) and Ca 2 + (C2). Figure 1.1 R E G U L A T O R Y DOMAIN C A T A L Y T I C DOMAIN PS DAG binding binding Ca 2 + ATP Substrate-site site binding site binding site binding site cPKCs VI C l V2 C2 V3 C3 V4 C4 V5 PS DAG binding binding ATP Substrate-site site binding site binding site nPKCs VI C l V2/ V3 C3 V4 C4 . V5 binding ATP Substrate-site binding site binding site aPKCs VI C l V2/ V3 C3 V4 C4 V5 Diagrammatic representation of the structure of PKC isoforms (Modified from Webb et al, 2000) In the unstimulated state, PKC isoforms are present mainly in the cytosol, whereas their activators are hydrophobic and reside in the particulate fraction. When the PKC molecule is inactive, the regulatory domain covers the catalytic site preventing access to the protein 15 substrate. The critical events in the regulation of PKC activity are phosphorylation by phosphoinositide dependent kinase I (PDK-I) (Dutil et al, 1998) and at least one subsequent autophosphorylation in the catalytic domain (Le Good et al, 1998). These phosphorylations are essential for PKC to be activated by second messengers (Dutil et al, 1998; Dempsey et al, 2000). Upon receptor stimulation and subsequent Ca 2 + mobilization (critical for activation of cPKCs), PKC translocates from the cytosolic to the membrane fraction (Zidovetzki & Lester, 1992). The availability of DAG and PS is essential for activation of cPKCs and nPKCs and facilitate their penetration through the cell membrane (Lester et al, 1990). This results in a conformational change in the PKC molecule, such that the regulatory domain is unmasked from the catalytic site, allowing substrate entry and catalysis to occur, hence activating PKC for downstream signaling. Translocation of PKC isoforms from the cytosolic to membrane fraction is often used as an index of their activation (Taggart et al, 1999). With the help of immunofluorescence techniques, it was shown that agonist-induced stimulation of PKC is associated with isozyme-selective localization in the cell (Mochly-Rosen et al, 1990; Disatnik et al, 1994) and this is achieved by binding of PKC to specific anchoring proteins (Mochly-Rosen & Gordon, 1998). These are referred to as RACKS (receptors for activated C-kinase). RACKS bind to PKC in the presence of activators and help to regulate its intracellular compartmentalization and localization to its sites of action. To date two RACKS have been characterized: 1) RACK1 - found to be selective for PKC p2 (Ron et al., 1994) 2) p'-COP - found to be selective for PKC s (Csukai et al, 1997) 1.10 HYPERGLYCEMIA-INDUCED ACTIVATION OF PKC A number of mechanisms have been put forward to explain vascular dysfunction in diabetes (discussed in section 1.2). One of the major pathways implicated in the pathogenesis of hyperglycemia-induced vascular injury is DAG-mediated PKC activation (as reviewed by King & Brownlee, 1996; Idris et al, 2001). DAG is generated from 4 major sources (Fig. 1.2): 1) Receptor-mediated, PLC-catalyzed hydrolysis of PIP2, resulting in the production of IP3 and DAG 16 2) PLD-mediated hydrolysis of PC to PA, which is then converted to DAG by phosphatidic acid phosphohydrolase 3) PLA 2 mediated release of non-esterified fatty acids (NEFAs) from precursor lipids (e.g. PC) which are then converted into DAG 4) de novo synthesis or direct metabolism of glucose to DAG. Figure 1.2 Phosphatidic Acid (PA) ^ P L D \ Phosphatidycholine (PC) PLA 2 • / Non Esterified Fatty Diacylglycerol (DAG) + Acids (NEFAs) Principal Sources of DAG Production (Modified from Idris et al, 2001) A number of studies have shown that hyperglycemia stimulates the DAG-PKC pathway in a variety of microvascular and macrovascular tissues (Craven & DeRubertis, 1989; Inoguchi et al, 1992; Inoguchi et al, 1994). This was primarily shown to be due to enhanced de novo synthesis of DAG (Inoguchi et al, 1994; Xia et al, 1994). Under conditions of hyperglycemia, the amount of glucose metabolized through glycolysis is increased and glycolytic intermediates such as glyceraldehyde-3-phosphate and dihydroxyacetone phosphate are converted into PA. This is then converted to DAG through the action of phosphatidic acid phosphohydrolase. In addition to this, there has been evidence to indicate that PC turnover is also significantly elevated in diabetes (Okurama et al, 1991; Li et al, 1994). The increased activation of PKC resulting from elevation of DAG levels has been shown to be one of the major factors contributing to functional and structural abnormalities leading to diabetic micro- and macroangiopathy (as reviewed by Meier & King, 2000; Idris et al, 2001; Waye; al, 2001). de novo Phosphoinositides 17 One of the ways found to ameliorate hyperglycemia induced DAG-PKC activation in the vasculature was administration of d-ot-tocopherol (vitamin E). Vitamin E has been suggested to indirectly modulate the PKC signal transduction pathway in diabetes in a number of ways. It does not affect already-activated PKC (Kunisaki et al, 1995). However it can enhance DAG kinase activity, which reduces DAG levels by increasing its metabolism to PA (Lee et al, 1999a). Vitamin E has also been shown to inhibit PKC by preventing its translocation to the cell membrane (Boscoboinik et al., 1991) and by activating phosphatase type 2A activity, which can dephosphorylate and inactivate PKC (Azzi et al., 2000). Some studies have shown that vitamin E supplementation can produce a significant reduction in the development and progression of vascular complications by a combination of both its anti-oxidant properties and its inhibitory effect on the DAG-PKC pathway (Bursell et al, 1999; Way et al, 2001; Wigg et al, 2004). However at the same time a number of investigations have reported that vitamin E supplementation had no effect on cardiovascular complications in diabetic patients (Gazis et al, 1999; Yusuf et al, 2000) and in some cases also had a worsening effect (Economides et al, 2005). Hyperglycemia-induced PKC activation is isoform and tissue specific, and out of the 12 known isoforms of PKC, it has been shown that there is preferential activation of PKCp 2 as well as increased expression of this isoform in a number of different cardiovascular tissues (Inoguchi et al, 1992; Guo et al, 2003; Tickerhoof et al, 2003; Wigg et al, 2004). PKC isoforms play a very important role in cell signaling, and nonspecific inhibition of PKC in vivo is liable to have major detrimental effects. Therefore attempts have.been made to develop isoform selective inhibitors and one of the most promising compounds is LY333531, a bisindolymaleimide that can be administered orally and has been shown to selectively inhibit PKCp. Treatment of diabetic animals with LY333531 produced significant amelioration of diabetes-induced cardiomyopathy and nephropathy (as reviewed by Idris et al, 2001; Way et al, 2001). This suggests that PKCp plays a critical role in the development of cardiovascular complications in diabetes. In addition to PKCp, enhanced activity and/or expression of other PKC isoforms have also been reported in other diabetic tissues. For example, in the diabetic rat retina PKC a, P and s were increased in the membrane and cytosolic fractions (Shiba et al, 1993). In the diabetic rat glomeruli, the expression of PKCa and P i in the membrane fraction was enhanced and 18 this was associated with an increased phosphorylation of these isoforms, suggesting a state of activation (Koya et al, 1997). A number of reasons could explain the differential activation of PKC isoforms in diabetes (Ishii et al, 1998; Way et al, 2001). For example, there could be differences in the sensitivity of the PKC isoforms to DAG, there could be increased DAG production at specific subcellular sites accessible to only certain PKC isoforms pools or the rates of protein synthesis and degradation of the isoforms could be different. 1.11 VASOREACTIVITY AND PKC ACTIVATION IN DIABETES Enhanced contractility of vascular smooth muscle in Type I diabetic animal models has been reported frequently, and a number of studies have implicated increased activation of PKC as the underlying reason for this observation. Significantly increased contractions to ET-1, 5-hydroxytryptamine (5-HT) and the thromboxane A 2 mimetic, U-46619, in aortas from 8-12 week diabetic rats were suggested to be due to a greater influx of Ca 2 + through transmembrane Ca 2 + channels resulting from increased PKC-activated processes (Hattori et al, 1995; Hattori et al, 1999). However these conclusions were based on pharmacological inhibition of PKC and Ca 2 + channels and did not involve direct measurement of [Ca2+]j or PKC activation. Resistance arteries from 4 week diabetic hamsters exhibited significantly enhanced contractions to prostaglandin F 2 a (PGF 2 a) and an enhancement of the PKC pathway was implicated in the augmented contractility (Georgescu & Popov, 2003). Increased aortic reactivity to NE in 6-7 week diabetic Wistar rats was attributed to a PKC-mediated increase in DAG kinase activity (Nobe et al, 2002). Coronary arteries from 6-week diabetic rats exhibited significantly greater contractile responses to ET-1, which were normalized following treatment with a selective PKC inhibitor (Tickerhoof et al, 2003). Significantly enhanced sensitivity to PE-induced contractions in a 4-week diabetic mouse model was reversed by treatment with LY333531 (10 mg/kg/day), suggesting that this was due to enhanced PKC|3 activation (Nangle et al, 2003). Consistent with these reports, our lab also has preliminary data to implicate PKC in significantly enhanced contractile responses to NE in arteries from 12-14 week diabetic rats. It was shown that NE mediated stimulation of phosphoinositide metabolism was significantly elevated in diabetic arteries, resulting in an increased production of JP3, total inositol phosphates and phosphatidic acid (Abebe & MacLeod, 1991a; Abebe & MacLeod, 1991b). Moreover the enhanced responses were 19 normalized following treatment with the relatively non-specific PKC inhibitor staurosporine (Abebe & MacLeod, 1990). 1.12 PKC AND CALCIUM SENSITIZATION A role for PKC in Ca 2 + sensitization was suggested when phorbol esters such as phorbol 12, 13 dibutyrate (PdBu), which are structural analogs of DAG and cause direct activation of PKC, increased contractile force without a significant increase in [Ca2+]j (Jiang & Morgan, 1987; Jiang & Morgan, 1989; Gailly et al, 1997; Miura et al, 1997). Agonists that stimulate the phosphoinositide pathway leading to activation of PKC also have the ability to sensitize the contractile proteins to Ca 2 + (Jiang & Morgan, 1989; Horowitz et al, 1996c; Kitazawa et al, 1999; Kanashiro & Khalil, 2001; Wickman et al, 2003). Of the twelve known PKC isoforms, the three that have been most frequently implicated in Ca 2 + sensitization and contraction in response to stimulation of GPCRs in vascular smooth muscle are PKC a, 8 and 8 (Lee et al, 1999b; Eto et al, 2001). Proposed mechanisms by which PKC may induce Ca 2 + sensitization are: 1) Direct phosphorylation of MYPT (within residues 40-296) which diminishes its affinity for PPlc (Toth et al, 2000), hence decreasing the activation of MLCP. However this has only been demonstrated in vitro and its physiological relevance has yet to be established. 2) Activation of PLA 2 and production of A A (Gong et al, 1995; Parsons et al, 1996; Miura et al, 1997), which has the ability to enhance MLC phosphorylation via a number of mechanisms (discussed in section 1.18). 3) Inhibitory phosphorylation of CaP and CaD (Horowitz et al, 1996a; Horowitz et al, 1996b; Pohl et al, 1997; Sohn et al, 2001). PKC-mediated reversal of the inhibitory action of CaP and CaD on actomyosin ATPase has been demonstrated in vitro and there is conflicting evidence regarding the importance of this mechanism under physiological conditions. In one study, phosphopeptide maps of caldesmon phosphorylated in intact porcine carotid arteries suggested that PKC was not the phosphorylating kinase (Adam et al, 1989). Moreover increasing evidence suggests that MAP kinase is the most likely candidate to induce CaD phosphorylation under physiological conditions (Adam et al, 1993). In some studies calponin phosphorylation was undetectable in response to agonist-induced contraction in a 20 number of different smooth muscles (Barany & Barany, 1993; Adam et al, 1995). The importance of PKC-catalyzed phosphorylation of CaP and CaD in agonist-2"|_ induced Ca sensitization requires further investigation. 4) Phosphorylation of CPI-17, the most widely accepted mechanism of Ca 2 + sensitization by PKC (see below). In 1995, Eto and colleagues reported that a heat stable protein, referred to as CPI-17 (short for: PKC potentiated inhibitor protein of 17kDa) was able to inhibit both the catalytic and regulatory subunit of MLCP when it was phosphorylated by PKC at Thr 38 (Eto et al, 1995). Phosphorylation at Thr 38 increased the inhibitory potency of CPI-17 >1000 fold. This investigation unveiled the signaling pathway between PKC and inhibition of MLCP. Cloning and sequencing analysis of CPI-17 revealed that it was a novel protein with a unique amino acid sequence distinct from all other known proteins. It was also unique in its mechanism of action in that it did not phosphorylate MLCP but rather inhibited both the catalytic and regulatory subunits of the phosphatase (Eto et al, 1995). In fact recent studies have shown that phosphorylated CPI-17 forms a stable inactive complex with MYPT and PPlc, which allows MLC phosphorylation and contraction to increase (Eto et al, 2004). CPI-17 was shown to be exclusively expressed in smooth muscle but Northern and Western blotting experiments showed that expression of CPI-17 tended to vary depending on whether smooth muscle was vascular or visceral (Woodsome et al, 2001). It was shown that CPI-17 content was greater in vascular (e.g. aorta) than in visceral (e.g. vas deferens) smooth muscle and within these tissues, that the tonic muscle phenotype contained more CPI-17 than the phasic muscles. Contractile force generated by PdBu was found to be directly proportional to the total amount of CPI-17 in a muscle and was associated with increases in CPI-17 and MLC phosphorylation, suggesting that PKC physiologically mediated Ca 2 + sensitization by phosphorylating CPI-17 (Woodsome et al, 2001). It was initially suggested that PKCa and 8 were the principle isoforms involved in mediating CPI-17 phosphorylation in response to agonist-induced stimulation of smooth muscle, with the latter being a more efficient kinase (Eto et al, 2001). However it has been shown that all PKC isoforms can bind to CPI-17 in vitro, and that PKCa and s are equally efficient in phosphorylating it (Zemlickova et al, 2004). 21 Initially believed to be a PKC substrate, a number of kinases, including RhoK, ZIP-like kinase, integrin-linked kinase and PKN have now been shown to phosphorylate CPI-17 in vitro (as reviewed by Somlyo & Somlyo, 2003). However whether the CPI-17 phosphorylation mediated by these kinases plays a physiological role in Ca 2 + sensitization and contraction in vascular smooth muscle has yet to be shown. 1.13 RhoA AND Rho KINASE The Rho (Ras homologous member) GTPases form a subgroup of the Ras superfamily of low molecular weight (20-30 kDa) G-proteins. In mammalian cells at least 10 distinct proteins of this sub-group have been identified: RhoA, RhoB, RhoC, RhoD, RhoE, RhoG, Rac (Ras related C3 botulinum toxin substrate) 1 and 2, Cdc42 (cell division cycle 42) and TC10 (Van Aelst & D'Souza-Schorey, 1997; as reviewed by Seasholtz et al, 1999). These proteins have a conserved primary structure and share 50-55% structural homology. Most of the functions of the Rho family are based on studies conducted on RhoA. These proteins have been implicated in membrane trafficking, regulation of cytoskeletal reorganization, cell growth control and development. In the inactive GDP bound state, RhoA is predominantly cytosolic. On activation, when GDP is replaced with GTP, there is an increase in membrane associated RhoA with a corresponding decrease in cytosolic RhoA levels (as reviewed by Seasholtz et al, 1999). A key step in the activation of RhoA is the attachment of geranylgeraniol, which is an isoprenoid intermediate of cholesterol biosynthesis. This posttranslational lipid modification, referred to as geranylgeranylation, is essential for membrane binding. Translocation to the particulate (membrane) fraction is often used as an index of RhoA activation (Taggart et al, 1999). The downstream effectors of RhoA include protein kinase N (PKN), RhoK, citron, citron kinase, rhophilin, rhotekin and pl40mDia (as reviewed by Fukata et al, 2001). Of these the first and most well characterized RhoA effector is RhoK, particularly with respect to the effects of RhoA on vascular smooth muscle contractility. RhoK is a serine/threonine kinase that exists in two isoforms: ROCK I (also known as ROKP, 160 kDa) and ROCK II (ROKoc, 150 kDa). RhoK contains an amino-terminal catalytic domain, and a C-terminal regulatory domain which contains the Rho binding site (RBD) and a pleckstrin homology (PH) domain (Fig. 1.3) (as reviewed by Riento & Ridley, 2003). Both ROCK I and II share a 92% similarity in their kinase domains, and there is very 22 little evidence to show that they phosphorylate different substrates. Some substrates that have been shown to be phosphorylated by PdioK include MYPT, MLC, adducin, the ERM family (ezrin, radixin, moesin), LIM kinase and vimentin. In the inactive state, the regulatory site binds to the catalytic domain, forming an autoinhibitory loop (Fig. 1.4). Binding of RhoA induces a conformational change in the kinase which results in the disruption of the negative regulatory interaction between the catalytic and regulatory domain (Fig. 1.4) (as reviewed by Riento & Ridley, 2003). RhoA binds to RhoK only in the activated GTP bound form and on binding enhances its kinase activity (Ishizaki et al, 1996; Matsui et al, 1996). Like RhoA, RhoK translocates to the membrane on activation and this is often considered as a marker of enzyme activity (Obara et al, 1999; Miao et al, 2002b; Kandabashi et al, 2003). Figure 1.3 CATALYTIC DOMAIN Kinase REGULATORY DOMAIN RBD P H CRD Diagrammatic Representation of the Structure of RhoK. The amino-terminal kinase region constitutes the catalytic domain whereas the Rho binding domain (RHD) and the pleckstrin homology domain (PH) containing an internal cysteine rich domain (CRD) constitute the regulatory subunit of RhoK. (Modified from Riento & Ridley, 2003) 23 Figure 1.4 Inactive RhoK Active RhoK Regulation of Activation of RhoK. In the inactive state, the regulatory domain negatively regulates the kinase activity. However binding of GTP bound RhoA disrupts this interaction resulting in activation of the enzyme. (Modified from Riento & Ridley, 2003) 1.14 RhoA/RhoK AND CARDIOVASCULAR DISEASE Under normal physiological conditions, RhoA/RhoK serve a number of homeostatic functions that are essential for normal cell growth and development. However their sustained activation in pathological conditions can have detrimental consequences. So much so that they have come to be regarded as a formidable force in the field of cardiovascular disease (as reviewed by Shimokawa, 2002; Wettschureck & Offermanns, 2002). Increased activation of the RhoA/RhoK pathway has been found in various conditions of abnormal vascular smooth muscle contractility. A link between RhoA/RhoK and hypertension was established by the ground-breaking work of Uehata et al. (Uehata et al, 1997). They found that administration of Y-27632, a selective RhoK inhibitor, was able to correct hypertension in several experimental hypertensive rat models without a significant effect on blood pressure of normotensive rats. RhoK inhibition by fasudil also improved forearm circulation in humans, suggesting that enhanced activation of this pathway was a significant contributor to increased peripheral vascular resistance in human hypertension (Masumoto et al, 2001). 24 RhoA/RhoK activation has also been implicated in vasospasm. RhoK inhibitors significantly improved cerebral vasospasm after subarachnoid hemorrhage in humans as well as experimental animal models (Shibuya et al, 1992; Sato et al, 2000b). Coronary artery spasm is a common feature of ischemic heart diseases such as angina pectoris, myocardial infarction and ventricular arrhythmias. Inhibition of RhoK significantly ameliorated symptoms of angina in humans and experimental animal models (Utsunomiya et al, 2001; Masumoto et al, 2002). For example, in a dog model of effort angina, hydroxyfasudil protected the myocardium subjected to pacing-induced ischemia by increasing regional blood flow in the absence of any inotropic or chronotropic effects (Utsunomiya et al, 2001). Similarly in patients with vasospastic angina, fasudil effectively prevented myocardial ischemia resulting from ACh-induced coronary artery spasm (Masumoto et al, 2002). Lung diseases such as bronchial asthma are associated with airway inflammation and hyper responsiveness. Elevated bronchial levels of RhoA/RhoK and enhanced activation of this pathway were reported in animals suffering from airway hyper-reactivity (Chiba et al, 1999; Chiba et al, 2001; Hunter et al, 2003; Sylvester, 2004). The main treatment for asthmatic patients is administration of p2-AR antagonists, whose beneficial effects can be limited after chronic use due to desensitization (Johnson, 1998). Inhibition of RhoA/RhoK could serve as a therapeutic alternative for these patients. Increased activation/expression of RhoA/RhoK has also been implicated in pre-term labor (increased uterine contractions) (Tahara et al, 2002), glaucoma (increased intraocular pressure) (Honjo et al, 2001; Rao et al, 2001) and erectile dysfunction (decreased intracavernosal pressure) (Chitaley et al, 2001; Mills et al, 2001). Most vascular pathologies are characterized by vascular smooth muscle proliferation and migration as well as vascular remodeling which involves the rearrangement of cellular and extracellular components. In addition to mediating enhanced vascular smooth muscle contractility, RhoA/RhoK have also been shown to participate in vascular remodeling processes. RhoA/RhoK influence cell cycle progression and cell proliferation by a number of mechanisms, for example, by reducing the expression of cell cycle inhibitors, which leads to increased DNA synthesis (Olson et al, 1998; Laufs et al, 1999) or by increased phosphorylation of extracellular-regulated kinase (ERK) (Welsh et al, 2001; Roovers et al, 2003). This increased vascular smooth muscle cell migration and proliferation contributes to 25 the pathophysiological progression of vascular diseases including hypertension, and could participate in the pathogenesis of arteriosclerosis/atherosclerosis. 1.15 RhoA/RhoK AND DIABETES The role of the RhoA/RhoK pathway has been investigated in various diabetic complications. It has been shown that diabetes mellitus adversely affects endothelial and nitrergic relaxation in the corpus cavernosum of humans and animals leading to erectile dysfunction. Increased expression of RhoA and ROCK II was detected in the STZ diabetic rat penis (Bivalacqua et al, 2004) and RhoK antagonism was shown to have a beneficial therapeutic effect in diabetic mice suffering from erectile dysfunction (Buyukafsar & Un, 2003). Over expression of ROCK I mediating increased contractility to ET-1 was proposed to be causing the increased tone of the corpus cavernosum smooth muscle from chronic diabetic rabbits, which leads to erectile dysfunction in this model (Chang et al, 2003). Increased activation of RhoA has been implicated in diabetic renal injury. It was shown that the renal cortex of STZ diabetic rats had significantly increased levels of membrane bound RhoA without any change in total RhoA expression (Massey et al, 2003). This was accompanied by renal pathologies such as hypertrophy, proteinuria and glomerulopathy. This was supported by another report where inhibition of RhoA by statins prevented high glucose induced proliferation of mesangial cells, an early development in diabetic nephropathy (Danesh et al, 2002). Upregulation of basal RhoA expression and activity was found in basilar arteries from STZ-diabetic rats, and it was suggested to be involved in cerebral vascular pathogenesis during diabetes mellitus, such as stroke and cerebral edema (Miao et al, 2002a). However unlike the DAG/PKC pathway that has been intensively investigated with respect to peripheral arterial dysfunction in diabetes, there is very little information regarding the role of the RhoA/RhoK pathway in abnormal vasoreactivity in diabetes. Increased activation of this pathway has been implicated in the enhanced contractile responsiveness to thrombin in aortic smooth muscle cells from a type II diabetic model, the Goto-Kakizaki rat, due to loss of insulin-mediated inhibition of the pathway (Begum et al, 2000). However whether the RhoA/RhoK pathway also contributes to abnormal vasoconstrictor responses in hypoinsulinemic STZ-diabetic rats has not yet been investigated. 26 1.16 RhoA/RhoK AND CALCIUM SENSITIZATION GPCRs have been shown to be highly effective activators of the RhoA/RhoK pathway in vascular smooth muscle. Agonists like ET, phenylephrine (PE), thrombin, TXA 2 , and lysophosphatidic acid (LPA) have been shown to stimulate the activity of this pathway leading to enhanced contractile force via increased Ca 2 + sensitization of the contractile apparatus (as reviewed by Somlyo et al, 1999; Somlyo & Somlyo, 2000). Inhibition of RhoA by bacterial exoenzymes such as EDIN (epidermal differentiation inhibitor from staphylococcus aureus) or exoenzyme C3 (Clostridium botulinum) by ADP-ribosylation (which blocks the biological activity of RhoA and inhibits its translocation to the plasma membrane) results in significant attenuation of agonist induced Ca 2 + sensitization and force development in smooth muscle (Gong et al, 1996; Otto et al, 1996; Fujihara et al, 1997; Lucius et al, 1998). Similarly inhibition of RhoK by selective inhibitors like Y-27632 significantly reduces contraction produced by excitatory agonists (Uehata et al, 1997; Fu et al, 1998; Yoshii et al, 1999). These observations have established RhoA/RhoK as being major effectors in the regulation of pharmacomechanical coupling in smooth muscle. The predominant molecular mechanism by which RhoA regulates Ca 2 + sensitization induced by agonist stimulation is the activation of its downstream effector, RhoK. Although GTP bound RhoA can interact with MYPT, this association was found to have no significant effect on MLCP activity (Kimura et al, 1996). However RhoK has been suggested to produce Ca 2 + sensitizing effects via a number of different mechanisms: 1) Phosphorylation of MYPT resulting in inhibition of MLCP, an increase in the MLCK/MLCP activity ratio and enhanced MLC phosphorylation. RhoK has been shown to phosphorylate MYPT at Thr 696 in vivo in response to a number of different agonists, suggesting that MLCP is predominantly a RhoK substrate (Kimura et al, 1996). In one study, point mutation at Thr 696 on the MYPT subunit resulted in complete loss of RhoK mediated inhibition of MLCP, suggesting that it was the major inhibitory site (Feng et al, 1999a). RhoK has also been shown to phosphorylate two other sites on MYPT in vitro, Thr 853 and Ser 852. Thr 853 lies within a region that has been identified as a myosin-binding domain (Johnson et al., 1997) and can affect targeting function of MLCP (Velasco et al, 2002). Phosphorylation of this site by RhoK decreases binding of MLCP to MLC and could be one of the mechanisms 27 responsible for RhoK mediated Ca 2 + sensitizing effects. Data regarding the relevance of Ser 852 phosphorylation by RhoK has been conflicting. In one study RhoK did not produce any detectable phosphorylation of this site (Velasco et al, 2002) while in another, Ser 852 phosphorylation was only partially blocked by RhoK inhibitors (Kawano etal, 1999). 2) Direct phosphorylation of MLC20 at serine 19 (the site that is also phosphorylated by MLCK) leading to enhanced activation of actomyosin ATPase by actin (Amano et al, 1996; Kureishi et al, 1997; Feng et al, 1999b). Whether RhoK induces M L C 2 0 phosphorylation under physiological conditions has yet to be established. 3) Phosphorylation of CPI-17 at Thr 38 (demonstrated in vitro) which converts it into a potent MLCP inhibitor (discussed in section 1.12) (Koyama et al, 2000). 4) Inhibitory phosphorylation of CaP (demonstrated in vitro and physiological importance not established). RhoK induced phosphorylation of calponin at a number of sites and inhibited its binding to actin (Kaneko et al, 2000). 1.17 CALCIUM SENSITIZATION BY AA It has been shown in a wide variety of tissues that agonist-induced A A release is primarily via stimulation of CPLA2, which translocates to the particulate fraction on activation (Anderson et al, 1997; Muthalif et al, 1998; Trevisi et al, 2002). A A release induced by NE (LaBelle & Polyak, 1998) and ET-1 (Trevisi et al, 2002) in rat tail artery was inhibited by treatment with A A C O C F 3 , an inhibiter selective for CPLA2 (Street et al, 1993). However in these studies the effect of A A C O C F 3 on NE and ET-1-induced contraction was not investigated. In a recent study PE-induced A A release in vascular smooth muscle was found to be via stimulation of iPLA 2 (Guo et al, 2003). A number of studies have implicated A A in agonist-induced Ca 2 + sensitization. It was shown that ONO-RS-082 (a non-isoform specific PLA 2 inhibitor) and BEL (a selective iPLA 2 inhibitor) significantly attenuated PE-induced Ca 2 + sensitization and A A production in permeabilized rabbit femoral artery (Gailly et al, 1997) and rabbit portal veins (Guo et al, 2003), respectively. Similarly quinacrine (a non-isoform specific P L A 2 inhibitor) inhibited 5-hydroxytryptamine- (5-HT-), but not PE-, induced Ca sensitization in permeabilized rabbit mesenteric artery (Parsons et al, 1996). The different mechanisms by which A A has been suggested to mediate GPCR-induced Ca sensitization are discussed below. 28 1) AA was shown to directly inhibit MLCP (discussed in section 1.8) in permeabilized smooth muscle resulting in increased MLC phosphorylation (Gong et al, 1992; Gong et al, 1995; Gailly et al, 1996). However this has only been demonstrated in permeabilized rabbit femoral arteries. 2) AA can stimulate RhoK activation independent of RhoA (Fu et al, 1998; Feng et al, 1999b; Araki et al, 2001). This was demonstrated in permeabilized smooth muscle when treatment with Y-27632 resulted in the loss of A A induced Ca 2 + sensitizing effects. A A activated RhoK by binding to its PH domain and releasing the autoinhibition mediated by the interaction between the C-terminal regulatory domain and the catalytic site. 3) A A can stimulate the activation of aPKCs (Nakanishi & Exton, 1992; Gailly et al, 1997). In bovine kidney, PKC^ was found be activated by A A and other unsaturated fatty acids (Nakanishi & Exton, 1992). In vascular smooth muscle there is one study showing that inhibition of aPKCs by a pseudopeptide inhibitor resulted in significant attenuation of AA-induced Ca 2 + sensitization of force and MLC20 phosphorylation in rabbit femoral artery (Gailly et al, 1997). 1.18 CALCIUM SENSITIZATION BY TKs TKs have been shown to participate in GPCR-mediated contraction via modulation of ion channels leading to an increase in [Ca2+]j. TK inhibitors have prevented agonist induced Ca 2 + influx via VGCC (Gould et al, 1995; Toma et al, 1995) as well as Ca 2 + release from intracellular stores (Liu & Sturek, 1996). However at the same time, activation of TKs following agonist-induced stimulation of GPCRs have also increased myofilament force sensitivity to Ca 2 + . It was shown that Ca 2 + sensitization mediated by PE and histamine (Sasaki et al, 1998) and ET-1 (Ohanian et al, 1997) was significantly blocked by TK inhibitors in permeabilized mesenteric arteries. In other studies TKs were shown to play a role in both Ca 2 + dependent and independent contractile processes in response to classical vasoconstrictors in mesenteric resistance (Toma et al, 1995) and human omental (Martinez et al, 2000) arteries. The degree of physiological importance of TKs in modulating GPCR-mediated Ca 2 + sensitivity is uncertain but some potential mechanisms linking tyrosine phosphorylation to this process have been suggested: 29 1) Increased activation of PLD, leading to enhanced production of DAG and a resultant increase in PKC activation (Ward et al, 1995; Jinsi et al, 1996). However these studies are based on the effects of TK inhibition on PLD activation in response to agonist-induced stimulation in vascular smooth muscle, and do not show how this affects agonist-induced Ca 2 + sensitization. 2) Increased activation of MAP kinases, which require phosphorylation of tyrosine and threonine residues for complete activation (Ward et al, 2002). MAP kinases have the ability to phosphorylate and inhibit CaD (Adam et al, 1995; Dessy et al, 1998). 3) There is no direct evidence suggesting activation of RhoK by TKs but one study has suggested that there is an interaction between them. Sphingosylphosphorylcholine (SPC)-induced Ca 2 + sensitization and contraction was suggested to be mediated by RhoK as well as Src family protein tyrosine kinases (SrcPTKs) (Nakao et al, 2002). PP1 (a SrcPTKs kinase-inhibitor) and eicosapentaenoic acid (EPA, a SrcPTKs translocation-inhibitor) but not Y-27632 abolished SPC-induced activation of SrcPTKs. PP1 and EPA also inhibited activation and translocation of RhoK in response to SPC, suggesting that SrcPTKs maybe acting upstream of RhoK. 30 1.19 HYPOTHESIS AND OBJECTIVES CVD is the major burden of DM and vascular complications significantly enhance the morbidity and mortality associated with this disease. In spite of many advances and breakthroughs in our understanding of this debilitating disease, there is a paucity of data related to the pathogenesis of vascular disease in diabetes and this has hindered the development of new preventative and therapeutic strategies for the treatment of diabetic vascular disorders. Abnormal vasoreactivity in the diabetic vasculature has been the subject of much investigation. Our lab has consistently reported that vasoconstrictor responses to stimulation of cti-ARs are significantly elevated in mesenteric arteries isolated from STZ-induced diabetic rats as compared to corresponding controls. In order to investigate if this was due to increases in mean cytosolic [Ca2+]j, changes in [Ca2+]j and contractile tension in response to stimulation with NE were measured in mesenteric arteries from control and diabetic rats (Chow et al, 2001). The results demonstrated that the enhanced sustained contractile responses of mesenteric arteries from diabetic rats to a maximum concentration of NE were not associated with a greater increase in Ca 2 + influx or mean cytosolic Ca 2 + . This suggests that enhanced vasoreactivity of diabetic arteries is due an increase in the Ca 2 + sensitivity of the contractile proteins. Diabetes-induced increases in the expression and/or activity of some PKC isoforms have been demonstrated in various cardiovascular tissues and based on functional studies, PKC has been suggested to be involved in abnormal vasoconstrictor responses in arteries from STZ-diabetic rats. However, the effect of diabetes on agonist-induced changes in PKC isoforms involved in Ca 2 + sensitization has not been reported. Moreover, the contribution of RhoK, TKs and CPLA2 to abnormal vasoconstrictor responses in tissues from Type I diabetic rats has not been investigated. Since PKC, RhoK, TKs and CPLA2 have been shown to participate in GPCR-mediated Ca sensitization and contraction, we hypothesized that one or more of these pathways play a role in the altered contractile responses to a r A R and ET-1 receptor stimulation in mesenteric arteries from diabetic rats. It is also possible that these pathways are not activated independent of each other but in fact interact at various points to mediate abnormal vasoconstrictor responses in diabetic arteries. 31 In order to investigate the role of PKC in ai-AR and ET-1 receptor-mediated contractile responses in mesenteric arteries from diabetic rats, functional and biochemical studies were conducted. In the functional studies, pharmacological experiments were carried out to investigate the effects of calphostin C and Ro-318220 (non-isoform selective PKC inhibitors) and rottlerin (an inhibitor selective for PKC8) on contractile responses to NE and ET-1 in mesenteric arteries from control and diabetic rats. Since activation of PKC has been shown to involve its translocation from the cytosol to isoform-specific binding sites at cell membranes, changes in particulate (membrane) levels of the isoforms are often used as an index of their activation. Therefore in the biochemical studies, western blotting experiments were conducted to measure the change in the particulate levels of the PKC isoforms, a, 8 and s, in response to a maximum concentration of NE and ET-1 in mesenteric arteries from control and diabetic rats. PKC-mediated CPI-17 phosphorylation has been proposed to be the primary mechanism by which PKC increases Ca 2 + sensitization in vascular smooth muscle. To investigate if activation of PKC was associated with increased phosphorylation of this downstream target, the effects of NE on the phosphorylation of CPI-17 in the absence and presence of the PKC inhibitor, calphostin C, in control and diabetic mesenteric arteries were measured by western blotting. In order to investigate the role of RhoK in oti-AR and ET-1 receptor-mediated contractile responses in mesenteric arteries from diabetic rats, functional and biochemical studies were conducted. Functional studies consisted of pharmacological experiments investigating the effects of specific RhoK inhibitors, Y-27632 and H-l 152, on contractile responses to PE and ET-1 in mesenteric arteries from control and diabetic rats. Like PKC, translocation of RhoK isoforms from the cytosol to the membrane fraction is considered a marker of the enzyme activity. Therefore in the biochemical studies, western blotting experiments were conducted to measure the degree of translocation of the RhoK isoforms, ROCK I and ROCK II in response to a maximum concentration of PE and ET-1 in mesenteric arteries from control and diabetic rats. To investigate the possibility of an interaction between the PKC/RhoK pathways, the effect of RhoK inhibition by Y-27632 on agonist-induced changes in levels of PKC isoforms in the particulate fraction was also determined. In order to investigate the role of TKs and CPLA2 in PE and ET-1-induced contractile responses in mesenteric arteries from control and diabetic rats, pharmacological experiments 32 were conducted. In these studies the effects of structurally different TK inhibitors, tyrphostin A23 and genistein, and cPLA 2 inhibitor, AACOCF 3 , on contractile responses to PE and ET-1 in mesenteric arteries from control and diabetic rats were investigated. 33 2. MATERIALS AND METHODS 2.1 MATERIALS 2.1.1 Materials used to make the Krebs buffer: Sodium chloride (NaCl), potassium chloride (KG), sodium bicarbonate (NaHC03), calcium chloride (CaCb), potassium dihydrogen phosphate (KH2PO4), magnesium sulphate (MgSCU) and dextrose were purchased from Sigma (St. Louis, MO). 2.1.2 Materials used in the tissue bath contractility studies: Heparin (Hepalean from porcine intestinal mucosa) was obtained from Organon Teknika, Toronto, Canada. NE, PE, ET-1, acetylcholine (Ach), phorbol 12,13 dibutyrate (PdBu), phentolamine, Ro-318220 (3-[l-[3-(amidinothio)propyl-lH-indol-3yl]-3-(l-methyl-lH-indol-3-Bisindolylmaleimide IX methanesulphonate), rottlerin and calphostin C, A A C O C F 3 , genistein and tyrphostin A23 were purchased from Sigma (St. Louis, MO). Y-27632 ((+)-(R)-trans-4-(l -aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate) and H-1152 ((S)-(+)-2-methyl-l-[(4-methyl-5-isoquinoline)sulphonyl]-homopiperazine) were purchased from Calbiochem (La Jolla, CA): 2.1.3 Materials used in Western Blotting: Tris (hydroxymethyl) aminomethane (Tris-HCI), 2-mercaptoethanol, sodium dodecyl sulphate (SDS), glycine, protein assay dye reagent, acrylamide, bis N,N ' methylene-bis-acrylamide and temed were purchased from Bio-Rad (Hercules, CA). Polyoxyethylenesorbitan monolaurate (Tween 20), ethylene glycol-bis(|3-aminoethyl ether)-N^jN'jN'-tetraacetic acid (EGTA), EDTA, sodium fluoride (NaF), leupeptin, aprotinin, deoxycholic acid, NP-40 were purchased from Sigma (St. Louis, MO). 4-(2-aminoethyl)benzenesulfonylflouride (AEBSF) was purchased from Calbiochem (La Jolla, CA). Hyperfilms and enhanced chemiluminescence (ECL) detection kits were purchased from Amersham Pharmacia, Piscataway, N.J. The primary antibodies for PKC and RhoK isoforms (rabbit polyclonal) and actin (goat polyclonal) and secondary antibodies (goat anti-rabbit IgG-HRP and donkey anti-goat IgG-HRP) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The primary 34 antibodies for CPI-17 and phospho-CPI-17 (Thr38) (rabbit polyclonal) were purchased from Upstate (Charlottesville, VA). 2.2 METHODS 2.2.1 Control and Diabetic Rats Male Wistar rats weighing between 150-175g were either obtained from the University of British Columbia Animal Care Unit or from Charles River Laboratories Inc., Montreal, Quebec. They were housed and treated in accordance with the guidelines of the Canadian Council on Animal Care. Rats were randomly divided into two groups. In one group, diabetes was induced by injection of 60mg/kg STZ (Sigma, St. Louis, MO) into the lateral tail vein of rats lightly anesthetized with halothane. The other group received the citrate buffer vehicle and served as the age and weight matched control group. STZ-treated rats with blood glucose levels of 13mmol/L or greater, measured with an Ames glucometer 1 week after injection, were considered diabetic and were kept for experiments. The blood for this preliminary glucose measurement was taken by nicking the end of the rat's tail with a scapel and bleeding just enough to cover a glucometer strip. Control and diabetic rats were housed separately and given free access to food (rat chow, LabDiet 5001, PMI Nutrition International, Inc., Brentwood MO) and water. Twelve to fourteen weeks later, animals were weighed and given an overdose of sodium pentobarbital. Blood was collected by cardiac puncture with a syringe containing heparin (0.1ml of 1000USP units/ml) and then centrifuged in an eppendorf microcentrifuge for 15 minutes at 4°C. The plasma was removed with a Pasteur pipette, divided into aliquots and stored at -20°C for later assay of plasma insulin and glucose levels. 2.2.2 Tissue Bath Studies The superior mesenteric artery was excised, placed in Krebs solution (composition in mM: NaCl 124, KC1 4.7, NaHC0 3 25, CaCl 2 2.5, K H 2 P 0 4 1.2, MgS0 4 1.2 and dextrose 11.5) at room temperature and cleaned of fat and connective tissue. The artery was then cut into 4mm segments. The endothelium was removed from each segment by rubbing the internal lumen of the vessel gently against a thin wire. The rings were then suspended in isolated tissue baths using triangular hooks, one of which was attached to a fixed tissue support, while the other was connected to a Grass FT.03 force displacement transducer which in turn was attached to a Grass polygraph (Model 7E, Grass Instruments Co., Quincy, Mass.). The rings 35 were equilibrated for 90 minutes under lg resting tension in Krebs solution continuously aerated with 95% 02/5% CO2 and maintained at a temperature of 37°C. Tissues were allowed to equilibrate for 60 minutes with the bathing solution being changed every 10-15 minutes. The loss of the endothelium in the tissues was first confirmed by demonstrating their inability, when pre-contracted with 3uM PE, to relax in response to lOuM Ach. The tissues were then washed for 1 hour and cumulative concentration response curves to NE, PE and ET-1 were obtained in the presence or absence of: 1) Ro-318220, 3uM for 30 minutes 2) Calphostin C, 3uM for 30 minutes 3) Rottlerin, 10 uM for 30 minutes 4) Y-27632, l uM for 15 minutes 5) H-1152, 0.1 uM for 15 minutes 6) AACOCF3, 10 uM for 30 minutes 7) Genistein, 10 uM for 30 minutes 8) Tyrphostin A23, 10 uM for 30 minutes Only one agonist and one antagonist were tested in each tissue. The maximum response to KC1 (0.1M) in the presence of l u M phentolamine was also measured. In some cases the effect of the inhibitors on KC1 and PdBu induced contractions was also determined. 2.2.3 Bio-Rad Protein Assay Protein content of freshly prepared samples was determined using the Bio-Rad® assay system, which is based upon the protein dye binding method of Bradford. Bovine serum albumin (BSA) was used as a protein standard. Standard curves were performed in triplicate, using at least 5 different protein concentrations ranging from 3 - 15u.g. Triplicates of lOul of each sample solution/tube were used to measure total protein levels. The EGTA buffer was used for the blank and 2.5ml of the diluted and filtered reagent was added to each tube and vortexed. The mixture was allowed to react for 5 minutes at room temperature after which 200ul of each standard and sample tube was pipetted into separate microtiter plate wells. The absorbance was measured at 595nm and the protein content of the unknown sample solutions was calculated from the linear standard curve. 36 2.2.4 Western Blotting While the specific tissue treatments and extraction procedures for identification of the various proteins of interest are discussed in Section 2.2.4, 2.2.5 and 2.2.6, this section will deal with the general aspects of the procedure. Preparation of the polvacrylamide eels Separating gel (11%): (mixture for two gels) 30 / 0.8% acrylamide / bisacrylamide = 29.4 ml Separating gel buffer (1.5M Tris-HCI, pH 8.8, 0.4% SDS) = 26.4 ml Distilled water = 24.2 ml 20% ammonium persulphate = 160pl Temed = 28 pi Stacking gel: (mixture for two gels) 30 / 0.8% acrylamide / bisacrylamide = 4 ml Stacking gel buffer (0.5M Tris-HCI, pH 6.8, 0.4% SDS) = 7.5 ml Distilled water = 18.5 ml 20% ammonium persulphate = 90ul Temed = 30 pi The separating gel was poured in the glass plates and sealed with 70% ethanol. It was allowed to set for at least an hour, after which the ethanol was poured off and the gel was repeatedly rinsed with distilled water. The combs (15 wells) were inserted into the glass plates and the stacking gel was poured in. It was allowed to set for at least an hour after which the combs were removed. The wells were repeatedly flushed with running buffer (1.4% glycine, 0.3% Tris and 0.1% SDS). After the protein samples were poured into the wells, the gels were run at constant current (16 - 18 m Amp overnight). Transfer of Separated Proteins from Gel to Membrane The sponges, filter papers, membrane (nitrocellulose or PVDF) and gel were wetted in transfer buffer (0.3% Tris, 0.01% SDS and 20% methanol). The gel was assembled in a 'sandwich' - sponge - filter paper - membrane - gel - filter paper - sponge. Membrane transfer was conducted at 4°C in transfer buffer for three hours at 4 Amp. The transferred proteins were visualized by reversibly staining the membranes with Ponceau (0.2g Ponceau 37 in 5% glacial acetic acid), a protein stain for serum proteins separated by agarose gel and cellulose acetate electrophoresis. Excess stain was rinsed off with water. Detection by Enhanced Chemiluminescence (ECL) Each membrane was treated with the respective primary and secondary antibodies and then allowed to soak in ECL western blotting detection reagent (equal volumes of solution 1 and 2 mixed together) for one minute at room temperature. The membrane was wrapped in saran wrap. It was placed in a film cassette with a sheet of film on top of it and the cassette was closed. Exposure time varied from 5 - 1 5 seconds. The film was then processed using the GBX developer/fixer pack (Sigma). It was thoroughly washed with distilled water and allowed to dry. Densitometric Analysis ofImmunoblots The immunoblots were quantified with a pdi 420 oe scanning densitometer (pdi Inc., Huntington Station, NY) connected to a personal computer using pdi Quantity One® Ver. 3.0 software. The background was subtracted from each film. A contour was drawn around each band and the contour quantity (contour area x optical density) was obtained for each band. 2.2.5 Western Blotting: Total levels of PKC and RhoK isoforms Mesenteric arteries from control and diabetic animals were cleaned, denuded and frozen in liquid nitrogen. The arteries were powdered (with the help of pestle and mortar) and placed in 240pl ice-cold EGTA buffer (composition: Tris HCI 20mM, mercaptoethanol 50mM, EGTA 5mM, EDTA 2mM, NaF lOmM, AEBSF ImM, leupeptin 25pg/ml, aprotinin 2pg/ml, NP40 1%, SDS 0.1% and deoxycholic acid 0.5%). The mixture was sonicated on ice and then centrifuged at 100,000g for 1 hour at 4°C. The supernatant was collected and total protein content was determined using the Bradford assay. Sample dilution buffer was added (5 X) to equal amounts of sample protein (20ug) and the mixture was vortexed and boiled for 4 minutes. The boiled protein was stored at -70°C to be later used for separation by gel electrophoresis. Samples of total protein (20pg) were subjected to SDS-PAGE on 11% polyacrylamide gels and the resolved proteins were electrophoretically transferred to nitrocellulose membranes, as described in section 2.2.4. The membranes were then blocked with 5% non-fat milk/0.05% Tween-TBS for 2 hours at room temperature. They were then incubated with the appropriate PKC isoform-specific (rabbit polyclonal, 1:500) or RhoK isoform specific (rabbit polyclonal, 38 1:180) primary antibody overnight at 4°C. Actin was used as an internal control and membranes were incubated with actin primary antibody (goat polyclonal, 1:200) in the manner described above. In preliminary experiments, each of the antibodies for PKCa, p2, 8, and s were tested against the purified standard for each isoform, and no cross-reactivity was detected. The same dilution of each PKC and RhoK isoform antibody and the same amount of protein were used for all the control and diabetic samples. These were found in preliminary experiments to give signals in the linear range of the optical density vs. protein concentration curve (Fig. 2.1). Immune complexes were detected following incubation of membranes with horseradish peroxidase conjugated anti-rabbit (for PKC and RhoK) or anti-goat (for actin) secondary antibody for 2 hours at room temperature (1:20,000 in 3% non-fat milk) using an enhanced chemiluminescence detection kit. Band intensity was analyzed by densitometry and normalized for actin on the same membrane to correct for any inaccuracies that might have occurred during gel loading and transfer. Total actin levels were not significantly different in control and diabetic mesenteric arteries (Fig. 2.2A). As an alternative to actin, some of the membranes were analyzed by the method described by Ping (Ping et al. 1997). Following Ponceau staining, the most predominant band on the membrane was quantified with the help of an image scanning densitometer. Each PKC or RhoK signal was normalized to the corresponding Ponceau stain signal. The results corrected by this method and by actin were found to be similar. Total levels of PKC isoforms in diabetic arteries were expressed as a percent of levels of the same isoform in control arteries. 2.2.6 Western Blotting: Particulate levels of PKC and RhoK isoforms Following the equilibration period, artery rings from control and diabetic animals were divided into 7 groups -1) Untreated 2) NE, 30 uM for 5 minutes 3) ET-1, 0.03 uM for 7 minutes 4) Y-27632, 1 uM for 15 minutes 5) PE, 30 uM for 5 minutes in the absence of Y-27632 6) PE, 30 uM in the presence of Y-27632 (1 uM for 15 minutes) 39 7) ET-1, 0.03 J J M in the presence of Y-27632 (1 uM for 15 minutes) Once the peak sustained contractile response was reached, the rings were quickly rinsed in ice-cold Krebs solution, blotted, frozen in liquid nitrogen and stored at -70°C. The frozen rings were powdered then sonicated in EGTA buffer (without detergents), and then centrifuged at 100,000g for 1 hour. The supernatant was retained as the soluble fraction, and the pellets were re-suspended in the EGTA buffer containing NP-40 1%, SDS 0.1% and deoxycholic acid 0.5%. Following centrifugation at 100,000g for 1 hour, the supernatant was collected and used as the particulate fraction. The protein content of the particulate fraction was determined using the Bradford assay. Equal amounts of protein (20u.g) were subjected to SDS-PAGE on 11% polyacrylamide gels and the resolved proteins were electrophoretically transferred to a nitrocellulose membrane. Membranes were treated with PKC or RhoK isoform-specific antibodies and results were normalized for actin as described above. Neither diabetes nor treatment with agonist had any effect on particulate levels of actin (Fig. 2.2B). Particulate levels of PKC and RhoK isoforms following NE, PE and ET-1 treatment were calculated relative to particulate levels in untreated control tissues. 2.2.7 Western Blotting: Total and phosphorylated levels of CPI-17 In preliminary experiments, artery rings from control animals were either left untreated or treated with PdBu (3 uM for 10 minutes). To investigate the effect of NE on CPI-17 phosphorylation, artery rings from control and diabetic animals were divided into 3 groups: 1) Untreated 2) NE, 30 uM for 5 min 3) Calphostin C, 3 uM for 35 min, with 30 uM NE being added for the last 5 min. The rings were then frozen by immersion in acetone containing 10% trichloroacetic acid (TCA) and lOmM dithiothreitol (DTT) cooled with dry ice, and stored at -70°C. Frozen tissues were gradually warmed to -20°C, then 4°C and finally washed several times with ice-cold acetone to remove the TCA. The tissues were ground in a motor-driven glass homogenizer (Kontes) in EGTA buffer containing 20mM DTT and 0.5% SDS. They were sonicated on ice and then centrifuged at 10,000g for 15 minutes at 4°C. The supernatant was collected and equal amounts of protein were subjected to SDS-PAGE on 16% polyacrylamide gels. 40 The resolved proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% non-fat milk/0.05% Tween-TBS overnight at 4°C and then incubated with the appropriate anti-CPI-17 (0.2ug/ml) or anti-phospho-CPI-17 (Thr38, 2u,g/ml) (rabbit polyclonal) primary antibody for 3 h at room temperature. Actin was used as an internal control and membranes were incubated with actin primary antibody as described above. Membranes were incubated with the horseradish peroxidase conjugated anti-rabbit (1:12500 for total CPI-17 and 1:7575 for anti-phospho-CPI-17 in 3% non-fat milk) or anti-goat (for actin, 1:20,000 in 3% non-fat milk) secondary antibody for two hours at room temperature. Levels of phosphorylated CPI-17 were expressed as a percent of total CPI-17 levels in each group. 2.2.8 Statistical Analyses All data are presented as the mean ± S.E.M. V indicates the number of animals in each group. The concentration response curves were analyzed by non-linear regression using GraphPad Prism version 4.00 (GraphPad Software, San Diego, CA) for calculation of pD 2 (-log EC50) values and maximum responses (Rmax). Statistical significance was evaluated by one-way or two-way ANOVA followed by Newman-Keuls post hoc test for multiple comparisons, using Number Cruncher Statistical System (NCSS, Kaysville, Utah). A p<0.05 was considered statistically significant. 41 Figure 2.1.A R 2 = 0.9613 0 10 20 30 40 Amount of protein (ug) Figure 2.1.B 1 R 2 = 0.9388 0 10 20 30 40 Amount of protein (ug) . Figure 2.1 Preliminary experiments for the selection of optimal loading concentration. Optical density vs. protein concentration for particulate levels of PKCa (A) and PKCs (B). 42 Figure 2.2.A Control Diabetic 3(H CO 'E D 20-ro 10-< Control Diabetic Figure 2.2.B 30 c 20 to S 10-| < Control Diabetic UT + Agonist UT + Agonist C-UT C+Agonist D-UT D+Agonist Figure 2.2 Total levels of actin in control and diabetic arteries (A). Particulate levels of actin in the absence (UT) and presence of agonist in particulate fractions of control (C) and diabetic (D) arteries (B) 43 3. RESULTS 3.1 CHARACTERISTICS OF CONTROL AND DIABETIC ANIMALS Some characteristics of rats used in the present investigation are shown in Table 3.1. Diabetic rats weighed significantly less than their corresponding controls, and had much lower serum insulin levels, associated with significant hyperglycemia. STZ-treated rats also exhibited other symptoms of diabetes including polydipsia, polyuria, development of cataracts and increased food intake. However, there was no significant difference in the cross-sectional areas of arteries from the two groups of animals. TABLE 3.1 Characteristics of Control and Diabetic Rats Body Weight (g) Blood Glucose (mmol/L) Serum Insulin (ng/ml) Mesenteric Artery Cross-Sectional Area (mm2) Control (n=30) 533.9 ± 6.6 8.2 ± 0.1 5.0 ± 0.4 0.35 ± 0.02 Diabetic (n=30) 344.9+ 9.4* 22.9 ± 0.6* 0.3 ± 0.03* 0.35 + 0.02 Values represent mean ± S.E.M of number of animals in brackets. *p<0.05 compared to control value. (One-way ANOVA) 44 3.2 ROLE OF PKC/CPI-17 PATHWAY IN ENHANCED CONTACTILE RESPONSES OF MESENTERIC ARTERIES FROM DIABETIC RATS TO a r A R STIMULATION 3.2.1 Effect of PKC Inhibitors on Contractile Responses to NE and ET-1 Consistent with our previous report, mesenteric arteries from diabetic rats exhibited enhanced contractile responses to NE (Fig. 3.1.B, 3.2.B, 3.3B, Table 3.2) without a corresponding increase in contractile responses to depolarization with 0.1M KC1 in the presence of l u M phentolamine. The response to KC1 was 0.6 ± O.lg in the control and 0.7 ± O.lg in the diabetic mesenteric arteries. Incubation of mesenteric arteries with 3uM Ro-318220 for 30 min had no significant effect on the contractile response to 0.1M KC1 plus l uM phentolamine (Fig. 3.1.A), but completely abolished the contractile response to direct activation of PKC with phorbol 12,13 dibutyrate (PdBu) (data not shown). In mesenteric arteries from control rats, Ro-318220 had no significant effect on the NE Rmax, but produced a significant shift in the NE concentration-response curve, resulting in a decrease in the NE pD 2 value (Fig. 3.LB, Table 3.2). Ro-318220 had a greater inhibitory effect on contractile responses of mesenteric arteries from diabetic rats to NE, producing a significant decrease in both the pD 2 value and Rmax to NE. In the presence of this antagonist, there was no significant difference between control and diabetic arteries in NE pD 2 values or Rmax (Table 3.2). 45 Figure 3.1.B 8 7 6 5 NE [-logM] Figure 3.1 Effect of the non-isoform selective PKC inhibitor Ro-318220 (Ro, 3uM for 30min) on maximum KC1 contractions (A, n=3) and NE concentration response curves (Fig. B, n=13) in control and diabetic mesenteric arteries. The response to KC1 was 1.7 ± 0.1 g in the control mesenteric arteries. 46 Similar results were obtained with calphostin C, a second non-isoform selective PKC inhibitor. In preliminary experiments, incubation of mesenteric arteries with 3uM calphostin C for 30 min had little effect on the contractile response to 0.1M KC1 in the presence of phentolamine (Fig. 3.2.A). As was found with Ro-318220, calphostin C was without effect on the NE Rmax, but produced a small but significant decrease in the NE pD 2 value in mesenteric arteries from control rats (Fig. 3.2.B, Table 3.2). Calphostin C produced only a small shift in the NE response in mesenteric arteries from diabetic rats, and in the presence of this antagonist, the NE pD 2 value remained greater than that in control arteries (Table 3.2). However, this antagonist depressed the NE Rmax in mesenteric arteries from diabetic rats, and in the presence of calphostin C there was no significant in the NE Rmax between arteries from the two groups of animals. The effect of rottlerin, an inhibitor reported to be specific for PKC5 (Gschwendt et al., 1994), on contractile responses to NE in control and diabetic mesenteric arteries was very different from that of Ro-318220 and calphostin C. While lOuM rottlerin for 30 min did not affect the contractile response to 0.1M KC1 in the presence of phentolamine in diabetic arteries, it significantly depressed KCl-induced contractions in control tissues (Fig. 3.3.A). For this reason the concentration was not increased any further. Rottlerin had no significant effect on the NE Rmax or pD 2 in control mesenteric arteries (Fig. 3.3.B). It produced a significant shift in the NE response in mesenteric arteries from diabetic rats. However this antagonist did not significantly affect the NE Rmax in diabetic arteries and in the presence of rottlerin, the Rmax continued to be significantly higher when compared to the control arteries (Table 3.2). In contrast to NE, neither the maximum contractile response nor the sensitivity of mesenteric arteries from diabetic rats to ET-1 were significantly different from control (Fig. 3.4 and 3.5, Table 3.2). Incubation with Ro-318220 resulted in marked attenuation of maximum responses of both control and diabetic arteries to ET-1, without affecting the ET-1 pD2 value (Fig. 3.4, Table 3.2). Similar results were obtained with calphostin C (Fig. 3.5), which significantly decreased the ET-1 Rmax in both control and diabetic arteries (Table 3.2) without altering the sensitivity to this agonist. There was no significant difference between control and diabetic arteries in response to ET-1 in the presence of either antagonist. 47 The effect of rottlerin on the ET-1 response in control arteries was also investigated. While it had no affect on the contractile responses to NE, rottlerin significantly attenuate the ET-1 Rmax (Fig. 3.6, Table 3.2). Figure 3.2.A C-UT C+CC D-UT D+CC Figure 3.2.B 8 7 NE [-logM] Figure 3.2 Effect of the non-isoform selective P K C inhibitor calphostin C (CC, 3uM for 30min) on maximum KC1 contractions (A, n=2) and N E concentration response curves (B, n=8) in control (C) and diabetic (D) mesenteric arteries. 48 Figure 3.3.A C-UT C+Rott D-UT D+Rott Figure 3.3.B Figure 3.3 Effect of rottlerin, the PKC inhibitor specific for PKCS, (Rott, lOuM for 30min) on maximum KC1 contractions (A, n=4) and NE concentration response curves (B, n=8) in control (C) and diabetic (D) mesenteric arteries. * p<0.05 compared to the corresponding untreated. The response to KC1 was 1.1 ± 0.1-g in the control mesenteric arteries. 49 Figure 3.4 Figure 3.4 Effect of the non-isoform selective P K C inhibitor Ro-318220 (Ro, 3uM for 30min, n=4) on ET-1 concentration response curves in control (C) and diabetic (D) mesenteric arteries. Figure 3.5 3- - • -C-UT -*-D-UT S 2--D-C+CC -a-D+CC £ o Tensi Tensi 11 10 9 8 ET-1[-logM] 6 Figure 3.5 Effect of the non-isoform selective P K C inhibitor calphostin C (CC, 3uM for 30min, n=5) on ET-1 concentration response curves in control (C) and diabetic (D) mesenteric arteries. 50 Figure 3.6 F i g u r e 3.6 Effect of rottlerin, the PKC inhibitor specific for PKC8, (Rott, lOuM for 30min, n=5) on ET-1 concentration response curves in control (C) mesenteric arteries. 51 TABLE 3.2 Effect of PKC Inhibitors on NE and ET-l-Induced Contractions CONTROL DIABETIC Rmax pD2 Rmax pD 2 (g) (- log EC50) (g) (- logECso) NORADRENALINE Untreated 1.4 ± 0.1 6.8 ±0.1 1.9 ± 0.1* 7.1 ±0.1* Ro-318220 1.2 ±0.1 6.4 ±0.1® 1.4 ±0.1® 6.6 ± 0.04® Untreated 1.5 + 0.1 6.8 ±0.1 1.9 ±0.1* 7.1 ±0.1* Calphostin C 1.4 ±0.2 6.5 ±0.1® 1.2 + 0.1® 6.9 ±0.1*® Untreated 1.5 ±0.1 6.9 ± 0.04 2.0 ±0.1* 7.1 ±0.1 Rottlerin 1.6 ±0.2 6.9 ±0.1 2.0 ±0.2* 6.6 ±0.2® ENDOTHELIN-1 Untreated 1.6 ±0.2 8.2 ±0.1 1.9 ±0.3 8.2 ± 0.2 Ro-318220 0.8 ±0.1® 8.0 ±0.1 0.7 ± 0.2® 8.0 ±0.1 Untreated 1.7 ±0.1 8.4 ±0.2 1.9 ±0.2 8.3 ±0.1 Calphostin C 0.8 ± 0.2® 8.1 ±0.2 0.7 ±0.1® 8.1 ±0.2 Untreated 1.5 ± 0.1 8.9 ±0.3 Rottlerin 1.0 + 0.1® 8.7 ±0.3 * p<0.05 compared to corresponding control value ®p<0.05 compared to corresponding untreated value (Two-way ANOVA followed by Newman Keuls post-hoc test) 52 3.2.2 Effect of Diabetes on Levels of PKC Isoforms in Mesenteric Arteries PKCa, p2, 5, and s were all detected in mesenteric arteries from both control and diabetic rats (Fig. 3.7). No significant differences in total levels of PKCa or PKC8 were detected between control and diabetic arteries. However, levels of PKCp 2 and PKCs in diabetic arteries were increased to 195 ± 26.9% and 140 ± 13.2%, respectively, of levels in control arteries. Figure 3.7 C D C D C D C D O 200 o 55 100-3 0 0 n PKCbt PKCft PKCS PKGB PKC a P K C P ; P K C 5 PKC, Figure 3.7 Total levels of PKC a, p2, 5 and s in control (open bars) and diabetic (closed bars) mesenteric arteries (n=5). Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.5), centrifuged and 20 u.g of total protein was loaded onto 11% polyacrylamide gels for separation of proteins. Results were expressed as a percent of levels of the isoform in control mesenteric arteries. * p<0.05 compared to the corresponding control fraction (One-way ANOVA). 53 3.2.3 Effect of NE and ET-1 on Particulate Levels of PKC Isoforms in Mesenteric Arteries Treatment with 30uM NE for 5 minutes, the time required for the sustained contractile response to this agonist to reach its maximum in both control and diabetic arteries, had no significant effect on particulate levels of PKCa in control arteries, but resulted in a marked increase in levels of PKCa in the particulate fraction of diabetic mesenteric arteries, to 190 ± 22% of levels in untreated control arteries (Fig. 3.8.A). NE treatment had no detectable effect on particulate levels of PKCp 2 (data not shown) or PKC5 (Fig. 3.8.B) in mesenteric arteries from either control or diabetic rats. However, NE did result in a small but significant increase of PKCe in the particulate fraction of control mesenteric arteries, to 125 ± 8% of those in untreated arteries (Fig. 3.8.C). Particulate levels of PKCs in untreated diabetic arteries were significantly greater than those in untreated control arteries, and treatment with NE resulted in a further significant increase in PKCs particulate levels, to 230 ± 19% of control. The NE-induced increase in PKCs in the particulate fraction was significantly greater in diabetic than in control mesenteric arteries. Treatment with 0.03u,M ET-1 for 7 minutes, the time required for the sustained contractile response to this agonist to reach its maximum, resulted in similar increases in particulate levels of PKCa in mesenteric arteries from both control and diabetic rats. Particulate levels of PKCa were increased by ET-1 to 192 ± 15% in control and to 180 ± 12% in diabetic arteries of levels in untreated control arteries (Fig. 3.9.A). Like NE, ET-1 had no significant effect on particulate levels of PKCp 2 (data not shown). However, ET-1 produced significant increases in particulate levels of PKCS in both control and diabetic arteries (Fig. 3.9.B). The ET-1-induced increase in particulate PKCS was significantly less in diabetic (117 ± 7%) than in control (153 ± 5%) arteries. In mesenteric arteries from control rats, ET-1 treatment resulted in a significant increase in levels of PKCs in the particulate fraction, to 166 ± 7% of levels in the untreated control (Fig. 3.9.C). A similar increase in particulate levels of PKCs in response to ET-1, to 181 ± 8% of control, was also detected in diabetic mesenteric arteries. 54 Figure 3.8 Control Diabetic C -UT C -NE D-UT D-NE B Control Diabetic rj \s e ^||ggg|||gigg| ^ ^ ^ ^ ^ ^^^^mg ^ ^ g ^ ^ ^ ^ r l v L u «™^^™ nmmmmm ^^^^mmm w V o k D a UT N E U T N E 300-i 200-100-0-C -UT C - N E D-UT D-NE C Control Diabetic PKCs 84kDa UT N E U T N E I O 300 200H 100 C-UT C-NE D-UT D-NE Figure 3.8 Particulate levels of P K C a (A), 5 (B) and s (C) in the absence (UT) and presence of N E (n=5) in control (C) and diabetic (D) arteries. Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.6), centrifuged and 20 pg of the particulate fraction was loaded onto 11% polyacrylamide gels for separation of proteins. * p<0.05 compared to all the other corresponding groups; + p<0.05 compared to the corresponding control basal group. (Two-way A N O V A followed by Newman Keuls post-hoc test) 55 Figure 3.9 5 A P K C a 300-2 0 0 -100-Control Diabetic UT ET UT ET 77kDa 78kDa 6 5? 2 0 0 -100 -C PKCs 300-2 0 0 -Control ET 100-C - U T C - E T D - U T D - E T Diabetic 84kDa UT ET C - U T C - E T D - U T D - F T Figure 3.9 Particulate levels of PKCa (A), 8 (B) and s (C) in the absence (UT) and presence of ET-1 (n=5) in control (C) and diabetic (D) arteries. Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.6), centrifuged and 20 pg of total protein was loaded onto 11% polyacrylamide gels for separation of proteins. * p<0.05 compared to the corresponding basal group; + p<0.05 compared to the corresponding control basal group; @ p<0.05 compared to the corresponding control ET-1 treated group (Two-way ANOVA followed by Newman Keuls post-hoc test). 56 3.2.4 Effect of PdBu and NE on Phosphorylation of CPI-17 We investigated whether stimulation of arteries with PdBu and NE was associated with increased phosphorylation of the downstream target of PKC, CPI-17. In preliminary experiments, treatment of control arteries with PdBu (3 uM for 10 minutes) produced a significant increase in CPI-17 phosphorylation levels (Fig. 3.10). No significant differences in total protein levels of CPI-17 were detected between control and diabetic mesenteric arteries (Fig. 3.11.A). NE had no significant effect on the phosphorylation of CPI-17 in control arteries (Fig. 3.1 l.B). Levels of phosphorylated CPI-17 were lower in untreated diabetic than in untreated control arteries, although this difference was not statistically significant. However, NE produced a significant increase in CPI-17 phosphorylation in diabetic mesenteric arteries, and in the presence of NE, levels of phosphorylated CPI-17 were significantly greater in diabetic than in control arteries. The NE-induced increase in CPI-17 phosphorylation in diabetic arteries was blocked by the same concentration of calphostin C (3 |oM) that normalized the contractile response of diabetic mesenteric arteries to NE (Fig. 7B). 57 Figure 3 .10 Figure 3 .10 Levels of phosphorylated CPI-17 in mesenteric arteries from control (C) rats in the untreated state (UT) and in the presence of PdBu (3uM for 10 min). Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.7), centrifuged and 20 pg of total protein was loaded onto 16% polyacrylamide gels for separation of proteins. * p<0.05 compared to C-UT (One-way ANOVA). Figure 3.1 L A Control Diabetic 10-, c/> 'E >» ro < Control Diabetic Figure 3.11.A Total levels of CPI-17 in control and diabetic arteries (n=3) Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.7), centrifuged and 20 pg of total protein was loaded onto 16% polyacrylamide gels for separation of proteins 58 Figure 3.11.B Control Diabetic wmm —mm, ^pMttktb * * ini.iwi.jiim 100- U T N E N E + C C U T N E N E + C C N T -rj a so 0s 0 C - U T C - N E C - N E + C C D-UT D-NE D-NE + C C Figure 3.1 l .B Levels of phosphorylated CPI-17 in mesenteric arteries from control (C) and diabetic (D) rats in the untreated state (UT), in the presence of NE (30uM for 5 min) and in the presence of NE plus the non-isoform specific PKC inhibitor, calphostin C (NE + CC, 3uM calphostin C for 35 min with 30 uM NE being added for the last 5 min) (B, n=3). Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.7), centrifuged and 20 u.g of total protein was loaded onto 16% polyacrylamide gels for separation of proteins. * p<0.05 compared to C-NE, C-NE + CC, D-UT and D-NE + CC (Two-way ANOVA followed by Newman Keuls post-hoc test). 59 3.3 CONTRIBUTION OF RhoK TO THE ENHANCED CONTRACTILE RESPONSES OF DIABETIC MESENTERIC ARTERIES TO a r A R STIMULATION: INTERACTION WITH PKC 3.3.1 Effect of RhoK & PKC Inhibitors on Contractile Responses to PE and ET-1 As previously reported for NE, contractile responses of diabetic arteries to PE were significantly greater than control (Fig 3.12.C, 3.13.B, 3.14, 3.15, 3.16 and Table 3.3). This enhanced response was observed in the absence of any significant difference in the maximum response to KC1 (0.1M in the presence of lOuM phentolamine) between the control and diabetic tissues (1.18±0.07 vs. 1.18±0.08g, control vs. diabetic). Treatment with the RhoK inhibitor Y-27632, l uM for 15 minutes, had no significant effect on maximum KC1 (Fig. 3.12.A) or PdBu (3.12.B) induced contractions. Used at the same concentration for the same time period, it significantly decreased the sensitivity of control mesenteric arteries to PE, but had no significant effect on the Rmax (Fig. 3.12.C, Table 3.3). In arteries from diabetic rats, Y-27632 significantly attenuated the PE Rmax and also shifted the concentration response curve resulting in a significant decrease in the PE pD 2. In the presence of Y-27632, the difference in the PE response between the control and diabetic arteries was abolished. Similar results were obtained with a second RhoK inhibitor, H- l 152 (Fig. 3.13.B, Table 3.3). Used at 0.1 uM for 15 minutes, H- l 152 had no significant effect on the maximum KC1 response (Fig. 3.13.A). However like Y-27632, in control mesenteric arteries it significantly reduced the PE pD 2 without affecting the PE Rmax. It had a greater inhibitory effect on diabetic arteries, as it significantly attenuated both the sensitivity and maximum contractile responses to PE. After treatment with the inhibitor, the significant difference in PE response between control and diabetic arteries was abolished. In order to confirm that inhibition of PKC normalized enhanced contractile responses of diabetic arteries to PE (as previously reported for NE), we investigated the effect of the PKC inhibitors, calphostin C and Ro-318220 on contractile responses of arteries from control and 60 diabetic rats to PE. Ro-318220, used at 3uM for 30minutes, produced a small but significant shift in the control PE concentration response curve without affecting the Rmax (Fig. 3.14, Table 3.3). However it had a greater inhibitory effect in diabetic tissues, significantly attenuating the PE Rmax and pD2, and normalizing responses of diabetic arteries to PE. Similar results were obtained with the other PKC inhibitor calphostin C. At a concentration of 3uM, calphostin C produced a small but significant decrease in the PE pD2 in control mesenteric arteries without affecting the PE Rmax (Fig. 3.15, Table 3.3). In diabetic tissues, calphostin C produced significant attenuation of both the PE Rmax and pD 2 and abolished the difference in PE response between control and diabetic arteries. When we investigated the effect of rottlerin on PE induced contractions in control and diabetic arteries, the results were different from those observed with NE. Rottlerin produced a significant shift in the PE pD 2 in control mesenteric arteries without affecting the PE Rmax. However in diabetic arteries, it significantly attenuated both the PE Rmax and pD 2 and normalized the enhanced response to PE. Unlike the a i-AR responses, RhoK inhibition did not significantly affect contractile responses to ET-1 in both control and diabetic mesenteric arteries. The concentration of Y-27632 that significantly attenuated NE-induced contractile responses in control mesenteric arteries, had no significant effect on ET-1 Rmax (222.5±18.2 vs. 190.3±14.7, absence vs. presence of Y-27632) and pD 2 (8.4±0.1 vs. 8.2±0.1, absence vs. presence of Y-27632) (Fig. 3.17). Similarly in diabetic mesenteric arteries, Y-27632 little inhibitory effect on ET-1 Rmax (227.2±12.9 vs. 197.6±19.2, absence vs. presence of Y-27632) and pD 2 (8.2±0.1 vs. 8.3±0.2, absence vs. presence of Y-27632) (Fig. 3.17). The effect of H-1152 (Fig. 3.18) was investigated only in control mesenteric arteries. Like Y-27632, it had almost no inhibitory effect on ET-1-induced vasoconstriction. 61 Figure 3.12.A Figure 3.12.B C-UT C+Y D-UT D+Y 400-— 300-O x ^ 200-100-J L 7 6 5 PdBu [-logM] Figure 3.12 Effect of the RhoK inhibitor Y-27632 (Y, l u M for 15min) on KC1 (A, n=6) and PdBu (B, n=4) contractions in control and diabetic mesenteric arteries. The response to KC1 was 1.3 + O.lg in the control mesenteric arteries. Figure 3.12.C 300n — • - C - U T — D-UT —D— C+Y £ 200q D+Y Max ^ 100-•A A A 10 8 7 Pe[-logM] Figure 3.12.C Effect of the RhoK inhibitor Y-27632 (Y, l u M for 15min, n=6) on PE concentration response curves in control (C) and diabetic (D) mesenteric arteries. 62 Figure 3.13.A C-UT C-H Figure 3.13.B Pe[-logM] Figure 3.13 Effect of the RhoK inhibitor H - l 152 (H, 0.1 uM for 15min) on maximum KC1 contractions (A, n=3) and PE concentration response curves (B, n=8) in control (C) and diabetic (D) mesenteric arteries. The response to KC1 was 0.9 ± O.lg in the control mesenteric arteries. 63 Figure 3.14 3 Pe[-logM] Figure 3.14 Effect of the non-isoform selective PKC inhibitor Ro-318220 (Ro, 3uM for 30min, n=7) on PE concentration response curves in control (C) and diabetic (D) mesenteric arteries. Figure 3.15 3 Pe[-logM] Figure 3.15 Effect of the non-isoform selective PKC inhibitor calphostin C (CC, 3uM for 30min, n=6) on PE concentration response curves in control (C) and diabetic (D) mesenteric arteries. 64 Figure 3.16 Pe [-logM] Figure 3.16 Effect of rottlerin, the PKC inhibitor specific for PKCS, (Rott, lOpM for 30min, n=6) on PE concentration response curves in control (C) and diabetic (D) mesenteric arteries 65 TABLE 3.3 Effect of RhoK and PKC Inhibitors on PE-Induced Contractions CONTROL DIABETIC Rmax pD2 Rmax pD 2 ' (%MaxKCl) (- logEC5o) (% Max KC1) (- log EC50) Untreated 155.9 + 13.4 7.0 ±0.1 182.5 ±3.7* 7.4 ±0.1* Y-27632 119.8 + 8.5 5 .6±0.1 @ 126.6 ± 11.3® 5.8 ±0.1® Untreated 197.3 ±3.7 6.8 ±0.1 232.3 ± 15.9* 7.1 ±0.1 H-1152 172.6 ± 12.7 5.9 ± 0.1® 184.5 ±7.8® 6.1 ±0.1® Untreated 174.9 ±7.4 6.6 ±0.2 204.6 ±2.5* 7.0±0.1* Ro-318220 152.1 ±9.1 6.2 ±0.1® 169.0 ±6.0® 6.5 ±0.2® Untreated 135.4 ±6.6 6.6 ±0.1 208.0 ± 11.3* 6.9 ±0.1 Calphostin C 138.9 ± 10.2 6.0 ±0.1® 160.3 ± 14.2® 6.1 ±0.1® Untreated 159.7 ± 11.7 6.8 ±0.1 221.8 ±9.1* 7.0 ±0.1 Rottlerin * A C 142.3 ± 11.5 6.1 ±0.1® 148.7 ± 16.4® 6.5 ±0.1® * p<0.05 compared to corresponding control value ' @p<0.05 compared to corresponding untreated value (Two-way ANOVA followed by Newman Keuls post-hoc test) 66 Figure 3.17 Figure 3.17 Effect of the RhoK inhibitor Y-27632 (Y, l u M for 15min, n=6) on ET-1 concentration response curves in control (C) and diabetic (D) mesenteric arteries. Figure 3.18 11 10 9 8 7 6 ET[-logM] Figure 3.18 Effect of the RhoK inhibitor H-l 152 (H, 0.1 uM for 15min, n=5) on ET-1 concentration response curves in control (C) mesenteric arteries. 67 3.3.2 Effect of Diabetes on Levels of RhoK Isoforms in Mesenteric Arteries Total levels of the RhoK isoforms in diabetic arteries were not significantly different from those in control arteries (Fig. 3.19). The total levels of ROCK I in diabetic arteries were 105.2 ± 9.0% and those of ROCK II were 106.3 ± 7.8% of levels in control tissues. Figure 3.19 C I) C D 200- R O C K I R O C K II o 100-O R O C K I R O C K II Figure 3.19 Total levels of ROCK I and II in control (open bars) and diabetic (closed bars) mesenteric arteries (n=5). Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.5), centrifuged and 20 ug of total protein was loaded onto 11% polyacrylamide gels for separation of proteins. Results were expressed as a percent of levels of the isoform in control mesenteric arteries. 68 3.3.3 Effect of PE and ET-1 on Particulate Levels of RhoK Isoforms in Mesenteric Arteries Particulate levels of ROCK I and II in untreated arteries from control and diabetic rats were not significantly different (Fig. 3.20.A and B). Treatment with a maximum concentration of PE (30uM) for the time required to produce a sustained contraction produced a significant increase in levels of ROCK I in the particulate fraction in both control and diabetic mesenteric arteries (Fig. 3.20. A). The particulate levels of ROCK I increased to 132 ± 14.0% in control tissues, and to 152 ± 6.7% in diabetic tissues, of levels in untreated control arteries. The increment in ROCK I particulate levels was not significantly different between arteries from control and diabetic rats. PE also induced significant translocation of ROCK II in both control (137 ± 13.2%) and diabetic (187 ± 18.5%) arteries (Fig. 3.20.B). However the increase in ROCK II levels in diabetic tissues was significantly greater than control. Treatment of both control and diabetic tissues with Y-27632, l uM for 15 minutes, did not affect basal particulate levels of ROCK I or II (data not shown). However, it did abolish the PE-induced increases in the particulate levels of these isoforms in arteries from both control and diabetic rats (Fig. 3.20.A and B). Consistent with the contractility data, treatment of tissues with 0.03 uM ET-1, had no effect on the particulate levels of ROCK I or II (Fig. 3.21 .A and B) in either control or diabetic arteries. Because both inhibition of RhoK and inhibition of PKC normalized the PE response in diabetic arteries, we investigated the effect of Y-27632 on PE-induced increases in particulate levels of PKC isoforms in control and diabetic arteries. Stimulation of control tissues with 30uM PE had no effect on the particulate levels of PKCa. However in the diabetic tissues, PE significantly increased particulate levels of PKCa by 148 ± 7.3% compared to the untreated control (Fig. 3.22.A). PE had no effect on particulate levels of PKC5 in control arteries but it also significantly increased particulate levels of this isoform to 145 ± 5.9% in diabetic arteries (Fig. 3.20.B). In diabetic arteries basal particulate levels of PKCs were significantly increased to 141 ± 8.6% of control (Fig. 3.22.C). Stimulation with PE had no effect on levels of PKCs in control tissues. However in diabetic arteries, PE induced a further significant increment in particulate levels' of PKCe to 219 ± 11.9%. Treatment of both control and diabetic arteries with Y-27632, l u M for 15 minutes, did not 69 affect basal particulate levels of PKCa, 8 and e (data not shown). However PE-induced increases in these PKC isoforms in diabetic arteries were abolished by Y-27632 (Fig. 3.22.A, B and C). In order to determine whether this effect of Y-27632 was due to direct inhibition of PKC, the effect of the inhibitor on ET-1 induced translocation of PKC isoforms was investigated. In control arteries, ET-1 induced significant increases in particulate levels of PKCa, Sand s (Fig. 3.23.A, B and C). Incubation of Y-27632 had no significant effect on these changes. 70 Figure 3.20 Control Diabetic A U T PE PE+Y U T PE PE+Y 1"^  ^ .) C. I^v 1 '^B^MI^BB' (^(mUj IWMMMI^ ^vtfli^ WMMM^ fc l c t ^ ^ ^ u i / \ i» r*\ ,^  ^^^^^^^ ^^S^^^^^^^ 1 \JV/iVJL-'£l 200-1 C-UT C-PE C-PE+Y D-UT D-PE D-PE+Y Control Diabetic B U T PE PE+Y U T PE PE+Y C -UT C -PE C-PE+Y D-UT D-PE D-PE+Y Figure 3.20 Particulate levels of ROCK I (A) and ROCK II (B) in the absence (UT) and presence of PE (n=6) in control (C) and diabetic (D) arteries. Some tissues were treated with the RhoK inhibitor, Y-27632 prior to stimulation with PE. Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.6), centrifuged and 20 u.g of total protein was loaded onto 11% polyacrylamide gels for separation of proteins. * p<0.05 compared to the corresponding basal group. + p<0.05 compared to the corresponding control group (Two-way ANOVA followed by Newman Keuls post-hoc test). 71 Figure 3.21 Control Diabetic UT ET UT ET C-UT C-ET D-UT D-ET B ROCK II 200-Control UT ET Diabetic UT ET 150kDa c3 100 C-UT C-ET D-UT D-ET Figure 3.21 Particulate levels of ROCK I (A) and R O C K II (B) in the absence (UT) and presence of ET-1 (n=5) in control (C) and diabetic (D) arteries. Arteries were ground in EGTA buffer (for composition see Results - Section 2.2.6), centrifuged and 20 pg of total protein was loaded onto 11% polyacrylamide gels for separation of proteins. 72 Figure 3.22 Cont ro l Diabet ic A UT P E PE+Y U T P E P E +Y pi' ( . ^ ^ ^ ^ ^ M M B t e **mm^^ - "'71-rv 1 IN. V . U . W W B W ^ ^ ^ ' < VNNMMWMfc / / K U u 200-fc-D (j, 100 X B C-UT C-PE C-PE+Y D-UT D-PE D-PE+Y Cont ro l Diabe t ic U T PE PE+Y U T P E P E + Y 7 8 k D a C-UT C-PE C-PE+Y D-UT D-PE D-PE+Y Cont ro l Diabe t ic C U T P E PE+Y U T P E P E + Y P K C s 300-200H => I o 100H 8 4 k D a C U T r.pc r.pP4.v n. i iT n.pp n . P C i V Figure 3.22 Particulate levels of P K C a (A), 5 (B) and e (C) in the absence (UT) and presence o f P E (n=5-6) in control (C) and diabetic (D) arteries. Some tissues were treated with the R h o K inhibitor, Y-27632 prior to stimulation with P E . Arteries were ground in E G T A buffer (for composition see Results - Section 2.2.6), centrifuged and 20 ug o f total protein was loaded onto 11% polyacrylamide gels for separation of proteins. * p<0.05 compared to the corresponding basal group. + p<0.05 compared to the corresponding control group. # p<0.05 compared to the corresponding PE-treated group (Two-way A N O V A followed by Newman Keul ' s post hoc test). 73 Figure 3.23 A P K C a 300 200H 0 100 B P K C S 300-1 2004 5 6 300-1 2004 ioo4 U T E T E T + Y 7 7 k D a C-UT U T 7 8 k D a 8 4 k D a C-UT C-ET C-ET+Y Figure 3.23 Particulate levels o f P K C a (A), 8 ( B ) and 8 (C) in the absence (UT) and presence of ET-1 (n=5) in control (C) arteries. Some tissues were treated with the R h o K inhibitor, Y-27632 prior to stimulation with ET-1. Arteries were ground in E G T A buffer (for composition see Results - Section 2.2.6), centrifuged and 20 pg o f total protein was loaded onto 11% polyacrylamide gels for separation of proteins. * p<0.05 compared to the corresponding basal group (One-way A N O V A ) . 74 3.4 ROLE OF TKs AND cPLA 2 IN ENHANCED CONTRACTILE RESPONSES OF MESENTRIC ARTERIES FROM DIABETIC RATS TO a,-AR STIMULATION 3.4.1 Effect of TKs and cPLA? Inhibitors on Contractile Responses to PE and ET-1 Contractile responses to PE in mesenteric arteries from diabetic rats were significantly enhanced as compared to the corresponding controls (Fig. 3.24.B, 3.25.B, 3.26.B and Table 3.4). TK inhibitors, tyrphostin A23 and genistein were used at concentrations at which they had no significant effect on KC1 induced contractions (Fig. 3.24.A and 3.25.A). In control arteries, tyrphostin A23 (lOuM for 30min.) did not affect the PE response (Fig. 3.24.B). Genistein (lOuM for 30min.) significantly shifted the PE concentration response curve without affecting the Rmax (Fig. 3.25.B, Table 3.4). Neither inhibitor had any significant effect on diabetic arteries. The inhibitors were unable to abrogate the significant difference in Rmax between control and diabetic arteries. The cPLA 2 inhibitor, A A C O C F 3 was used at a concentration at which it did not have a significant effect on the contractile responses to KC1 (Fig. 3.26.A). A A C O C F 3 (lOuM for 30min.) did not significantly affect the pD2 or the Rmax to PE in either control or diabetic arteries (Fig. 3.26.B, Table 3.4). The diabetic Rmax remained significantly greater than the control in the presence of the inhibitor. The contractile responses to ET-1 were not significantly different between control and diabetic arteries (Fig. 3.27, 3.28, 3.29 and Table 3.4). Tyrphostin A23, genistein and A A C O C F 3 , did not have any significant effect on the ET-1 concentration-response curves in either control or diabetic arteries. 75 Figure 3.24.B Pe[-logM] Figure 3.24 Effect of the TK inhibitor, Tyrphostin A23 (T, 1 OuM for 30min) on maximum KC1 contractions (A, n=6) and PE concentration response curves (B, n=6) in control (C) and diabetic (D) mesenteric arteries. The response to KC1 was 0.7 ± 0.02g in the control mesenteric arteries. 76 Figure 3.25.A C-UT C+G Figure 3.25.B Pe[-logM] Figure 3.25 Effect of the TK inhibitor genistein (G, lOuM for 30min) on maximum KC1 contractions (A, n=4) and PE concentration response curves (B, n=6) in control (C) and diabetic (D) mesenteric arteries. The response to KC1 was 1.0 ± 0.02g in the control mesenteric arteries. 77 Figure 3.26.A Pe[-logM] Figure 3.26 Effect of the cPLA 2 inhibitor AACOCF 3 (AA, lOuM for 30min) on maximum KC1 contractions (A, n=3) and PE concentration response curves (B, n=4) in control (C) and diabetic (D) mesenteric arteries. The response to KC1 was 0.6 ± 0.02g in the control mesenteric arteries. 78 Figure 3.27 ET[-logM] Figure 3.27 Effect of the TK inhibitor Tyrphostin A23 (T, lOuM for 30min) on ET-1 concentration response curves (n=3) in control (C) and diabetic (D) mesenteric arteries. Figure 3.28 ET[-logM] Figure 3.28 Effect of the TK inhibitor genistein (G, lOuM for 30min) on ET-1 concentration response curves (n=3) in control (C) and diabetic (D) mesenteric arteries. 79 Figure 3.29 11 10 9 8 7 6 ET-1[-logM] Figure 3.29 Effect of the cPLA 2 inhibitor AACOCF 3 (AA, lOuM for 30min) on ET-1 concentration response curves (n=4) in control (C) and diabetic (D) mesenteric arteries 80 TABLE 3.4 Effect of TKs and cPLA 2 Inhibitors on PE-Induced Contractions CONTROL DIABETIC Rmax pD2 Rmax pD 2 (% Max K C 1 ) (- logEC5o) (% Max K C 1 ) (- log EC 5 0) PHENYLEPHRINE Untreated 168.2 + 5.9 7.0 ±0.1 218.8 ± 12.2* 7.3 ±0.1 Genistein 182.0 + 7.4 6.4 ± 0.2 229.4 ± 10.7* 6.8 ± 0.2 Untreated 168.2 ± 5.9 7.0 ±0.1 218.8 ± 12.2* 7.3 ±0.1 Tyrphostin A23 185.5 ±7.4 7.0 ±0.1 211.5 ±6.2* 7.2 ± 0.2 Untreated 170.6 ±9.7 6.8 ±0.2 249.7 ± 18.9* 7.4 ±0.1 A A C O C F 3 195.0 ± 14.1 6.3 ±0.3 236.4 ± 14.5* 6.9 ±0.2 ENDOTHELIN-1 Untreated 201.2 ±9.8 8.4 ±0.2 220.6 ±9.8 8.3 ±0.2 Genistein 192.9 ± 8.8 8.2 ±0.2 230.6 ±25.5 8.2 ±0.2 Untreated 235.4 ± 16.3 8.3 ±0.1 244.9 ± 16.3 7.1 ±0.1 Tyrphostin A23 215.3 ±21.9 8.1 ±0.1 231.4 ± 12.4 8.2 ±0.1 Untreated 217.5 ± 12.2 8.1 ±0.2 242.1 ±26.3 8.1 ±0.1 A A C O C F 3 •1* ./-v i~\ j-182.2 ± 11.7 8.6 ±0.1 220.2 ± 16.0 8.3 ±0.1 p<0.05 compared to corresponding control value Two-way ANOVA followed by Newman-Keuls post-hoc test. 81 4. DISCUSSION 4.1 OVERVIEW Epidemiological and pathological data have shown that diabetes is an independent risk factor for the development of CVD in both men and women (McGill et al, 1998; Wilson et al, 1998). Diabetic women tend lose their inherent protection against development of CVD (Brezinka et al, 1994; Wilson et al, 1998). Vascular diseases, such as coronary, cerebrovascular and peripheral arterial disease, are major causes of disability and death in diabetic patients (Nesto, 2004) and the processes that lead to the changes in the peripheral vascular system in diabetes have been the subject of intense investigation. A better understanding of the underlying mechanisms leading to vascular dysfunction could unmask new strategies to decrease the incidence of cardiovascular morbidity and mortality in diabetic patients. One of the most common findings in both experimental models of diabetes and human diabetic subjects is functional changes in the sensitivity of the vascular smooth muscle to vasoactive substances (Kamata et al, 1992). Structural and functional deterioration of large and small blood vessels leading to abnormalities in vascular reactivity can contribute significantly to the development of CVD in diabetes (McVeigh et al, 1996). Abnormalities in the diabetic vasculature can compromise blood flow in a number of organs such as the kidney, retina, peripheral arteries and nerves (as reviewed by Koya & King, 1998). Enhanced vascular reactivity in diabetes is observed in the absence of autonomic neuropathy and hypertension (Christlieb et al, 1976; Drury et al, 1984; Tuck et al, 1990), which suggests that it is due to hyperglycemia-associated biochemical changes and can contribute to the eventual development of cardiovascular dysfunction in diabetes (Weidmann et al, 1985a; Weidmann et al, 1985b; Tuck et al, 1990). The purpose of this study was to characterize the biochemical abnormalities underlying enhanced vasoconstrictor responses of mesenteric arteries from STZ-diabetic rats, a widely used rodent model of Type 1 diabetes. Rats injected with STZ demonstrate many of the features observed in patients suffering from uncontrolled Type 1 diabetes (as reviewed by Tomlinson et al, 1992). Histological studies have shown that STZ-induced toxicity is primarily observed in the pancreatic P-cells and not in any other organs (Junod et al, 1967). 82 Moreover prolonged exposure of tissues to STZ is unlikely since the serum half-life of the drug is reported to be 15 minutes (Rossini et al, 1977). Most of the abnormalities observed in the cardiovascular system in STZ-diabetic rats are prevented by correction of hyperglycemia with insulin treatment (Fein et al, 1981; Tahilani et al., 1983; MacLeod, 1985; Hopfner et al., 1998). Therefore it can be concluded that the cardiovascular changes observed in the STZ-diabetic rat are due to metabolic derangements such as hyperglycemia, hyperlipidemia and hypoinsulinemia rather than due to a direct effect of STZ. 4.2 CONTRACTILE RESPONSES TO cti-AR STIMULATION It has been frequently reported that arteries from Type 1 diabetic animal models exhibit enhanced reactivity to ai-AR stimulation. Contractile responses to NE have been shown to be significantly elevated in aorta (Nobe ct al., 2002) and renal artery (Inazu et al., 1991) from STZ-induced diabetic rats as well as in the tail artery from alloxan-induced rats (Fiol de Cuneo et al., 1988) and carotid artery from alloxan-induced rabbits (Agrawal et al., 1987). Similarly PE- and methoxamine-induced contractions were significantly elevated in STZ-induced diabetic rat aorta (Otter et al, 1994; Zhu et al., 2001) and alloxan-induced rabbit carotid artery (Agrawal et al., 1987). Contractile responses to PE and NE were also significantly elevated in aorta obtained from STZ-induced (Nangle et al., 2003) and alloxan-induced (Abe et al, 2003) diabetic mice respectively. Animal models of Type 2 diabetes have also been shown to exhibit enhanced contractile responses to aj-AR stimulation. For example PE-induced contractions were significantly elevated in aorta and mesenteric artery of ob/ob mice (a spontaneous mouse model of NIDDM characterized by obesity, hyperglycemia and hyperinsulinemia) (Piercy & Taylor, 1998; Okon et al, 2003) and in cremaster muscle arterioles of Otsuka-Long-Evans-Tokushima fatty (OLETF) rats (a rat model of NIDDM that naturally develops insulin resistance) (Yoshida et al, 2003). Small mesenteric arteries from diabetic (db/db) mice (a model of Type 2 diabetes also) exhibited significantly elevated contractions to PE (Pannirselvam et al., 2003, Pannirselvam et al., 2005). However not all studies have reported enhanced aj-AR vasoconstrictor responses in arteries from Type 1 diabetic animal models. There have been reports of depressed contractile responses to NE in aorta (Pfaffman et al, 1982; Head et al., 1987) and methoxamine in perfused mesenteric arterial bed (MAB) (Misurski et al., 2001) obtained from STZ-induced 83 diabetic rats. Some investigators have reported unchanged vasoconstrictor responses to oti-AR agonists in diabetic arteries (Beenan et al., 1996; Kam et al., 1996; Torffvit et al., 1997). The discrepancy between the different studies could be due a number of factors such as differences in the species and strains of animals used, the diabetogenic agent used, the duration of diabetes or the presence of endothelium in some studies. Our lab has reported that aorta, mesenteric and caudal arteries from STZ-diabetic rats exhibit significantly enhanced contractile responses to ai-AR stimulation as compared to age and gender matched controls. In the present study, maximum contractile responses to NE and PE in mesenteric arteries from STZ-diabetic rats were also significantly enhanced. However increased sensitivity to oti-AR stimulation (as evidenced by significantly greater pD 2 values) was not always observed in the diabetic mesenteric arteries. This is also consistent with previously published work from our lab as significantly greater NE pD 2 values in diabetic arteries were observed in some studies (Harris & MacLeod, 1988; Weber & MacLeod, 1994) but not in others (Abebe & MacLeod, 1990). Enhanced contractile responses to NE and PE have been reported frequently in the mesenteric vasculature from diabetic rats. Vasoconstrictor responses and sensitivity to NE were significantly enhanced in the MAB (Yousif et al, 2003; Yousif et al, 2004) and mesenteric resistance arteries (3 rd branch) (Van Buren et al, 1998) from STZ-diabetic Wistar rats, respectively. Vascular sensitivity to PE was significantly elevated in mesenteric arterioles (3rd and 4 t h branch) from STZ-diabetic rats (Ishikawa et al, 2004) whereas maximum responses to PE were significantly greater in terminal branches of ileal mesenteric arteries from STZ-induced Sprague Dawley rats (Dresner etal, 1997). Our lab has shown that contractile responses to NE in mesenteric arteries are predominantly mediated by the a r A R (Abebe et al, 1990). This is also true for contractile responses to PE, since prazosin (an ai-AR antagonist) significantly attenuated these contractions, whereas yohimbine (an a 2 - A R antagonist) and timolol (a P-AR antagonist) had no effect on these responses (data not shown). While ai-AR-mediated vasoconstrictions have been reported to be increased, decreased or unchanged in isolated arteries from STZ-diabetic rats, many studies have shown that pressor responses to vasoactive agents like NE and methoxamine are depressed in vivo (Hebden et al., 1987; Makino & Kamata, 1998). A number of reasons have been suggested to contribute 84 to this observation. Hyperglycemia-induced impairment of sympathetic function (Maeda et al, 1995a; Maeda et al, 1995b) can reduce the pressor responses to ai-AR agonists. Makino and Kamata (1998) suggested that depressed pressor levels to methoxamine in STZ-diabetic rats were due to alterations in prostanoid turnover, that is increased production of PGI2 and decreased formation of TXA 2 . A number of studies have shown that the inducible isoform of nitric oxide synthase (iNOS) is activated in diabetic tissues (Smith et al, 1997; Bardell & MacLeod, 2001; Cheng et al, 2004a; Ishikawa et al, 2004) and it could play a significant role in cardiovascular dysfunction in diabetes. Cheng and Pang (2004b) showed that the maximum pressor response and mean circulatory filling pressure (index of body venous tone) in response to NE was significantly attenuated in 3-week STZ-diabetic rats. Selective inhibition of iNOS by 1400W restored arterial and venous constriction in response to NE administration. It was also shown that NE-induced left ventricular contractility in vivo was attenuated in 3-week STZ-diabetic rats and treatment with 1400W resulted in partial restoration of the depressed cardiovascular contractile function in response to NE (Cheng et al, 2004a). 4.3 CONTRACTILE RESPONSES TO ET-1 A number of studies have reported that ET-1-induced contractions are enhanced in arteries from Type 1 diabetic animal models. For example, aorta from STZ-induced diabetic rats (Hopfher et al, 1998; Hattori et al, 1999; Murat et al, 1999) and carotid and basilar arteries from alloxan-induced diabetic rabbits (Alabadi et al, 2004; Llorens et al, 2004) exhibited significantly elevated contractile responses to ET-1. Coronary arteries isolated from Sprague-Dawley rats made diabetic by partial pancreatectomy (Tickerhoof et al, 2003) also exhibited enhanced vasoreactivity to ET-1. However in other studies, contractile responses of aorta from STZ-diabetic rats to ET-1 have been reported to be depressed (Fulton et al, 1991; Hodgson & King, 1992; Tada et al, 1994; Utkan et al, 1998). In one study vasoconstriction to ET-1 was unchanged in perfused hindquarters of STZ-diabetic rats (James & Hodgson, 1995). Reasons for these variable results are not completely understood but could be due to the differences in the methodologies used to measure vasocontractile responses. It has also been shown that there are regional differences in vascular responses to ET-1 (Gardiner et al, 1989), which could lead to variable data in vessels isolated from different vascular beds. 85 In the mesenteric vasculature from diabetic rats there have been reports of enhanced (Yousif et al, 2003; Yousif et al, 2004) as well as depressed (Makino & Kamata, 2000a; Misurski et al, 2001) contractile responses to ET-1. In our studies, contractile responses to ET-1 (both Rmax and pD2) were not significantly different in mesenteric arteries from control and diabetic animals, which is consistent with another study reporting similar results (Arikawa et al, 2001). One explanation for the variability between our study and those quoted above could be the preparation used, as we conducted our experiments on the superior mesenteric artery while the perfused MAB was used by the other investigators. 4.4 BASAL LEVELS OF PKC ISOFORMS Increased levels and activity of specific isoforms of PKC occur in the cardiovascular system in diabetes, and evidence suggests that this may contribute to the cardiovascular dysfunction associated with this condition (as reviewed by Meier & King, 2000; Way et al, 2001). The isoform that has most frequently been found to be altered in the vasculature is PKCp 2. For instance, an increase in total PKC activity was found to be associated with increased particulate levels of PKCp 2 but not PKCa in aorta from STZ-diabetic rats (Inoguchi et al, 1992). Increased total levels of PKCp 2 were reported in coronary arteries (Tickerhoof et al, 2003) and in femoral and small mesenteric arteries (Wigg et al, 2004) from diabetic rats. There was significant upregulation of PKCp 2 at the transcriptional and translational levels in aorta from STZ-diabetic pigs (Guo et al, 2003). Consistent with these studies, we detected a two-fold increase in total PKCP 2 levels in superior mesenteric arteries from diabetic rats. There was also a smaller but significant increase in total PKCs levels and particulate levels of this isoform were also enhanced in diabetic tissues. However, there was no change in expression of PKCa or 5 in the diabetic arteries. This contrasts with a recent report of increased expression of PKCa and p2, but not PKCs, in diabetic coronary arteries (Tickerhoof et al, 2003). Whether this reflects a difference due to the longer duration of diabetes in the present study, or to differences between arteries is not clear. The significant involvement of PKCp 2 in hyperglycemia-mediated cardiovascular dysfunction was strongly suggested by experiments using LY333531, an inhibitor specific for PKCp (Jirousek et al, 1996). Enhanced PKCp 2 activation was shown to contribute to diabetes-induced cardiomyopathy, as treatment with LY333531 significantly improved 8 6 functional and morphological abnormalities associated with cardiomyopathy in diabetic mice (Wakasaki et al., 1997; Takeishi et al., 1998). Renal function and abnormalities in the retinal circulation in diabetic rats were also ameliorated by treatment with LY333531 (Ishii et al., 1996). Treatment with LY333531 prevented impairment of endothelium-dependent vasodilatation caused by experimental hyperglycemia in intact, healthy humans (Beckman et al, 2002). Similarly in STZ-induced diabetic rats, LY333531 produced significant improvement in abnormalities associated with neuronal function, neural tissue perfusion and endothelium-dependent vasodilatation (Cotter et al, 2002). In the latter study PE-induced contractile responses were not enhanced in the MAB and LY333531 treatment had no effect on contractility. But in another study, significantly greater sensitivity to PE in aorta from STZ-diabetic rats was normalized following treatment of rats with LY333531 (Nangle et al, 2003). Similar to the results of our study, PKCs levels have previously been shown to be increased in particulate fractions of diabetic tissues such as the rat kidney (Kang et al, 1999), glomeruli (Babazono et al, 1998) mesangial cells (Shiba et al, 1993) and cardiomyocytes (Malhotra et al, 1997), suggesting enhanced activation of this isoform. However the precise role of this isoform in hyperglycemia-induced cardiovascular dysfunction is not clearly understood. PKCs has been shown to participate in hyperglycemia-induced enhancement of endothelial cell permeability (Hempel et al, 1997). In addition, Malhotra et al (1997) suggested that activation of PKCs might participate in the cardiac dysfunction seen in chronic diabetes. A number of mechanisms have been proposed to lead to hyperglycemia-induced PKC activation. The de novo synthesis of DAG is significantly enhanced in diabetes as a result of which glucose is directly metabolized to DAG (Inoguchi et al, 1994) (discussed in Introduction, section 1.10). In addition to this, it has been suggested that accumulation of AGEs leading to activation of AGE receptors can lead to activation of PKC (Li et al, 1996). STZ-diabetic rats were shown to exhibit enhanced PKC activation in the retina, glomeruli and mesenteric artery after 8 and 24 weeks of induction of diabetes (Osicka et al, 2001). Aminoguanidine, an agent that leads to inhibition of AGEs, partially prevented activation of PKC in the retina and completely inhibited PKC activation in the mesenteric artery from 8-week diabetic rats. In 24-week STZ-diabetic rats, it completely prevented PKC activation in 87 the retina, glomeruli and mesenteric artery. These effects were observed in the absence of any significant effects on blood glucose levels (Osicka et al, 2001). Similarly AGEs were shown to lead to enhanced activation of PKCa in cultured human mesangial cells leading to increased collagen mRNA expression (Kim et al, 2001). These changes were inhibited by treatment of the cells with aminoguanidine. Increased activation of PKC has also been suggested to arise from AG II-induced activation of A T i receptors (Malhotra et al, 1997). Mesenteric arteries from STZ-diabetic rats were shown to exhibit significantly elevated levels of angiotensin-converting enzyme (ACE), suggesting that the activity of renin-angiotensin system and tissue levels of AG II were increased (Cooper et al, 1994). Ramipril, an ACE inhibitor, prevented increases in PKC activation in the retina, glomeruli and mesenteric artery in 8 and 24-week STZ-diabetic rats (Osicka et al, 2001). Treatment of STZ-diabetic rats with L-158,809, an AG II receptor antagonist, prevented translocation of PKCs in cardiomyocytes (Malhotra et al, 1997). 4.5 BASAL LEVELS OF RhoK ISOFORMS Dysregulation of RhoA/RhoK expression and/or activity has been shown to participate in vascular pathophysiologies. For example, RhoA activity was significantly increased in a number of different models of hypertension, such as stroke-prone spontaneously hypertensive rats (SHR), renal hypertensive rats and deoxycorticosterone acetate (DOCA)-salt hypertensive rats (Seasholtz et al, 2001; Seko et al, 2003; Moriki et al, 2004). We found no evidence for increased expression of RhoK isoforms in diabetic mesenteric arteries. However, increased expression and/or activity of components of the RhoA/RhoK pathway have been reported in other vascular tissues from Type I and II diabetic animals. For example, upregulation of basal RhoA expression and activity was found in basilar arteries from STZ-diabetic rats (Miao et al, 2002a). Increased expression of RhoA (membrane) and ROCK II (cytosol) in the corporal tissue of STZ-diabetic Sprague-Dawley rats was suggested to be the reason for erectile dysfunction in this model (Bivalacqua et al, 2004). Over expression of ROCK I was proposed to mediate increased contractility to ET-1 in corpus cavernosum smooth muscle from chronic diabetic rabbits (Chang et al, 2003). In aortic smooth muscle cells isolated from Goto-Kakizaki rats (a highly inbred strain of Wistar rats that spontaneously develop Type II diabetes), the basal RhoA activity and membrane levels were significantly greater when compared to the corresponding controls (Sandu et al, 2000). 88 4.6 PARTICIPATION OF PKC IN ct,-AR AND ET-1 RECEPTOR STIMULATION Selectivity of PKC Inhibitors Although there are a large number of inhibitory compounds for PKC, there are very few that exhibit selectivity for PKC alone or for individual PKC isoforms. Inhibitors of PKC are broadly classified into groups according to their site of interaction with the PKC molecule (Way et al, 2000): 1) Inhibitors of the regulatory domain, that target the phospholipid or DAG-binding site 2) Inhibitors of the catalytic domain, that target the substrate binding or ATP-binding site. , Calphostin C is a non-isoform selective PKC inhibitor that acts by inhibiting the regulatory domain of the PKC molecule, competing at the binding site for diacylglycerol and phorbol esters (Kobayashi et al, 1989). Ro-318220 and rottlerin on the other hand inhibit PKC by acting on the catalytic domain, competing at the ATP binding site. Ro-318220, a non-isoform selective PKC inhibitor, belongs to the family of bisindolylmaleimides derived from the microbial alkaloid staurosporine (Wilkinson et al, 1993). Although less potent than the parent compound, Ro-318220 shows more selectivity for PKC compared to other kinases. Rottlerin (mallotoxin) is a naturally occurring product derived from Mallotus Philippines is. It inhibited the activity of PKCS with an IC50 of 3pM while the other isoforms were at least one order of magnitude less sensitive to this inhibitor (Gschwendt et al, 1994). The IC50 of the different PKC isoforms increased in the following order § > a, p, y > r\, C^, s (Gschwendt et al, 1994). The selectivity of rottlerin for PKCS has been controversial. Davies et al (2000) found that even at a concentration of 20uM rottlerin had no inhibitory effect on activity of PKCa and 8. Nonetheless, rottlerin has continually been used as a PKC inhibitor with relative selectivity for the § isoform (De Witt et al, 2001; Nishizawa et al, 2003; Jin et al, 2004). De Witt et al (2001) showed by immunohistochemical analysis that PMA- and AG II-induced localization of PKCS in feline pulmonary arterial smooth muscle cells was decreased by treatment with rottlerin. Nishizawa et al (2001) showed by Western blotting that PKCS and a were translocated to the particulate fraction in canine basilar arteries on development 89 of angiographic vasospasm, and that the translocation of only PKC8 was inhibited by rottlerin. Calphostin C, Ro-318220 and rottlerin have been frequently used to investigate the role of PKC in contractile processes in vascular smooth muscle (Dallas & Khalil, 2003; Nishikawa et al, 2003; Tan et al, 2004). In order to establish selectivity, the effects of various concentrations of the PKC inhibitors on KC1 induced contractions were investigated at in control and diabetic mesenteric arteries. Ro-318220 and calphostin C, used at a concentration of 3uM, had no significant effect on the contractile responses to K+-depolarization, suggesting that their inhibitory effects were not due to nonspecific actions. However, at a concentration of lOuM, rottlerin produced a small but significant attenuation of KCl-induced contractions in mesenteric arteries from control, not diabetic, rats suggesting that some of its actions might arise from non-specific inhibition of the contractile process. At the same time, Ro-318220 abolished the contractile response of mesenteric arteries to PdBu, suggesting that it is effectively blocking the activation of PKC. Pharmacological Inhibition and Translocation of PKC in Response to arAR Stimulation Treatment of mesenteric arteries from control rats with Ro-318220 and calphostin C produced a small decrease in the NE pD2 values but had no effect on the Rmax to this agonist. However, rottlerin had no effect on NE responses. NE produced only a small translocation of PKCs, with no change in PKCa or 8. The non-isoform selective PKC inhibitors had similar effects on PE-induced contractions in control arteries. However, like Ro-318220 and calphostin C, rottlerin also produced a small decrease in the sensitivity to PE. Unlike NE, PE produced no detectable translocation of any PKC isoform. The fact that the PKC inhibitors did not have a prominent effect on contractile responses to ai-AR agonists, and that agonist-induced translocation of PKC isoforms was not very pronounced suggests that PKC does not play an important role in ai-AR mediated vasoconstriction in mesenteric arteries from control rats. Our results are consistent with other studies in which the PKC inhibitors did not significantly affect PE or NE-induced contractions in mesenteric arteries (Sasaki et al, 1998; Fetscher et al, 2001). However at the same time other studies conducted on the mesenteric vasculature have implicated PKC in Ca 2 + sensitization and contraction in response to ai-AR stimulation 90 (Buus et al, 1998; Shirasawa et al, 2003). The reasons for the discrepancy between these studies is not clear but could be due differences in the selectivity of the PKC inhibitors used, differences in experimental animals or in the methodologies used to measure vasocontractile responses. While PKC may not contribute to a large extent to the contractile responses of control mesenteric arteries to NE or PE, it appears to mediate the enhanced contractile responses of diabetic mesenteric arteries to ai-AR stimulation. Both Ro-318220 and calphostin C produced greater inhibition of NE-induced contractions in diabetic than control arteries, and normalized the significant difference in Rmax values found in the absence of antagonist. However, treatment of diabetic arteries with rottlerin produced only a small decrease in NE pD 2 values and the Rmax continued to be significantly greater than the control arteries in the presence of the inhibitor. The enhanced contractile responses to NE were associated with translocation of PKCa and s (greater than in mesenteric arteries from control rats), suggesting that the enhanced NE-induced contractile responses may be mediated by increased activation of these isoforms. Consistent with the functional data obtained with rottlerin, there was no detectable translocation of PKCS in response to NE in diabetic arteries. As was the case with NE, Ro-318220 and calphostin C were able to normalize the significantly greater PE-induced contractions in diabetic arteries. However there was a difference in the effects of rottlerin with respect to the ai-AR agonists in diabetic mesenteric arteries. Rottlerin normalized contractile responses to PE in diabetic tissues and abolished the significant difference in Rmax existing prior to inhibitor treatment. The enhanced contractile responses to PE were associated with translocation of PKCa, 8 and s. The significant translocation of PKC8 in response to PE is consistent with the effect of rottlerin on contractile responses to this agonist. The greater inhibitory effect of rottlerin on PE-induced contractions and the significant translocation of PKCS in response to PE suggests that PKCS may play a more prominent role in PE-, than in NE-induced contractions in diabetic arteries. The reasons for this difference are not clear. It is possible that there are differences in the ai-AR subtype activated by PE or NE as a result of which the signal transduction pathways activated vary between the agonists. It has also been suggested that the magnitude and efficiency of pharmacomechanical 91 coupling varies between different agonists (Somlyo & Somlyo, 1968). The relative importance of PKCS in PE and NE-induced contractile responses in diabetic mesenteric arteries requires further investigation. There have been some studies investigating the role of PKC in ai-AR-mediated responses in diabetic tissues. For example, in one study significantly elevated NE responses in aorta from STZ-induced diabetic rats were normalized following treatment with staurosporine (Kawasaki, 1997). Normalization of significantly elevated cti-AR responses in mesenteric arteries from diabetic rats by pharmacological inhibition of PKC has been previously reported by our lab (Abebe & MacLeod, 1990) and has been confirmed in this study. To the best of our knowledge, this is the first study that demonstrates enhanced ai-AR-mediated translocation of specific PKC isoforms in diabetic tissues. The elevated contractile response to ai-AR agonists in diabetic arteries may be mediated by increased activation of one or more of these isoforms. This may result from increased production of DAG as PIP2 breakdown was shown to be enhanced in response to a maximum concentration of NE in mesenteric arteries from diabetic rats (Abebe & MacLeod, 1991a; Abebe & MacLeod, 1991b). Pharmacological Inhibition and Translocation of PKC in Response to ET-1 Receptor Stimulation The PKC inhibitors produced a much greater attenuation of ET-1 than PE or NE-induced contractions in mesenteric arteries from control rats. Ro-318220 and calphostin C reduced the ET-1 Rmax by more than 50% while rottlerin decreased it by 33%. The significant inhibition produced by the PKC inhibitors together with the much greater translocation of all three PKC isoforms by ET-1 suggests that under normal circumstances, the ET-1 signaling pathway may be more effectively coupled to PKC than the ai-AR pathway. In contrast to the ai-AR agonists, there was no evidence for an enhancement of the contractile response to ET-1 in diabetic mesenteric arteries, nor was there any indication that the contractile response of diabetic mesenteric arteries to ET-1 was more dependent on PKC than that of control arteries, since both Ro-318220 and calphostin C produced a similar magnitude of block of contractile responses to ET-1 in control and diabetic mesenteric arteries, and ET-1 produced similar translocation of PKCa and s. PKCS translocation was 92 actually lower in response to ET-1 in diabetic than in control arteries, although the reasons for this are not clear at this point. The results of our study are in contrast to previous reports in aorta (Hattori et al, 1995; Kawasaki, 1997) and coronary (Tickerhoof et al, 2003) arteries of diabetic rats, which indicated that enhanced vasoconstrictor responsiveness to ET-1 could be blocked by inhibition of PKC. This could be due to the development of tissue-specific changes in reactivity to ET-1 during diabetes, as a result of which different diabetic vascular tissues respond variably to ET-1-induced vasoconstrictor actions (Gardiner et al, 1989). Differences in the effects of diabetes on contractile responses to stimulation of GPCRs in different arteries have previously been reported. For example, contractile responses to U-46619 and ET-1 were unchanged in the,perfused hindquarters of 6-week STZ-diabetic rats whereas 5-HT-induced contractions were attenuated (James & Hodgson, 1995). Similarly in mesenteric resistance vessels from 40-week STZ-diabetic rats sensitivity to NE was significantly enhanced but contractile responses to serotonin and vasopressin were unchanged (Van Buren et al, 1998). 4.7 PHOSPHORYLATION OF CPI-17 PKC mediated phosphorylation of CPI-17 has been shown to participate in contractile responses to GPCR agonists such as PE (Hayashi et al, 2001), histamine (Eto et al, 2001) and ET-1 (Kitazawa et al, 2003). Evidence suggests that CPI-17 is the most important downstream mediator of the Ca 2 + sensitizing effects of PKC (as reviewed by Somlyo & Somlyo, 2000). For instance, it was shown that in vascular smooth muscle, demembranation with Triton-X-100 resulted in significant depletion of PKC and CPI-17, with a corresponding loss in Ca sensitization of contraction and MLC phosphorylation in response to PKC activators (Kitazawa et al, 1999). This was restored by addition of exogenous PKC and CPI-17 and not by the former alone. CaP, CaD, myosin and MLC were shown to be much less efficient substrates for PKC when compared to CPI-17. The importance of CPI-17 for G-protein/PKC-mediated Ca sensitization in vascular smooth muscle was also established by another elegant study conducted by Kitazawa and colleagues (Kitazawa et al, 2004). In this study they showed that smooth muscle (such as artery, gizzard and small intestine) from chickens was deficient in CPI-17, and consequently contraction and Ca sensitization in response to GTPyS, PdBu, histamine, PE and ET-1 were significantly attenuated when 93 compared to "CPI-H-ridf tissues from rabbits and pigeons. Addition of recombinant CPI-17 to permeabilized chicken smooth muscle resulted in restoration of PdBu-and GTPyS-induced 2+ Ca sensitization. There is not a lot of information about how the expression and/or activation of CPI-17 vary with disease states associated with vascular dysfunction, such as diabetes. However in one study the role of CPI-17 in gastrointestinal (GI) motility disorders such as inflammatory bowel disease, Crohn's disease and ulcerative colitis was investigated (Ohama et al, 2003). In this study, long-term treatment of ileal muscle from Wistar rats with interleukin-ip (a pro-inflammatory cytokine that plays an integral role in GIT disorders) resulted in attenuated agonist-induced contraction and this was associated with a decrease in total levels and phosphorylation of CPI-17. In the present study, there was no detectable increase in phosphorylation of CPI-17 in response to stimulation with NE in mesenteric arteries from control rats, which is consistent with a minor role for PKC in ai-AR-mediated responses. However in contrast, treatment of diabetic arteries with NE produced an almost three-fold increase in CPI-17 phosphorylation and this increase was blocked by the same concentration of calphostin C (3 uM) that normalized the contractile response of diabetic mesenteric arteries to NE. These data suggest that the significantly greater contractile responses to NE in diabetic arteries arise from PKC-dependent phosphorylation and activation of CPI-17. While all PKC isoforms have been shown to bind to CPI-17 in vitro, PKCa, 8 and s have the greatest ability to phosphorylate and activate it (Eto et al, 2001; Zemlickova et al, 2004). Although RhoK has also been shown to phosphorylate and activate CPI-17 (Koyama et al, 2000), it seems unlikely that it is doing so in mesenteric arteries from control or diabetic rats. This is because in spite of evidence of participation of RhoK in ai-AR-mediated responses in both control and diabetic mesenteric arteries, there was no detectable phosphorylation of CPI-17 in control arteries and calphostin C treatment completely blocked CPI-17 phosphorylation in diabetic tissues. It has not been experimentally demonstrated how activated PKC in the membrane fraction can phosphorylate CPI-17, which is located in the cytosol. Eto et al (2001) suggested that CPI-17 lies close to the plasma membrane where activated PKC is present and can readily phosphorylate it. Once phosphorylated, CPI-17 can diffuse to the myofilaments to inhibit MLCP. A similar idea was put forward by Ruegg (1999) who suggested that CPI-17 shuttles 94 between membrane-bound active PKC and MLCP. It is also possible that activated PKC in the membrane may be in rapid equilibrium with cytosolic PKC. In the cytosolic compartment the quantity of activated PKC may be low but could still be sufficient to cause CPI-17 phosphorylation (Ruegg, 1999). 4.8 PARTICIPATION OF RhoK IN cti-AR AND ET-1 RECEPTOR STIMULATION Selectivity of RhoK Inhibitors Most of the RhoK inhibitors that are used in cardiovascular studies, such as Y-27632, H-1152 and HA-1077 act by inhibiting the ATP-binding site in the catalytic domain of the RhoK molecule (Takami et al, 2004). There are some agents that act by inhibiting the substrate-binding site in the catalytic domain but they are usually characterized by a high degree of hydrophilicity (Takami et al, 2004). The ATP-competitive agents on the other hand are less hydrophilic and accordingly exhibit good membrane permeability and absorption. Introduced by Narumiya and colleagues, Y-27632 (Kj = 0.2 - 0.3 pM) selectively inhibits both isoforms of RhoK approximately 200 times more potently than cPKCs and PKA and 2000 times more potently than MLCK (Uehata et al, 1997; Ishizaki et al, 2000). H-l 152 is a novel RhoK inhibitor (K, = 0.0016 pM) (Sasaki et al, 2002) that has been shown to have a better inhibitory profile than its parent compound HA-1077 and to be approximately 10 times more potent than Y-27632 at inhibiting RhoK (Shum et al, 2003). H- l 152 inhibits RhoK approximately 400 times more potently than PKA, and 6000 times more potently than cPKCs and M L C K (Sasaki et al, 2002). Y-27632 and H-l 152, used at concentrations of l uM and 0.1 uM respectively, had no effect on contractile responses of mesenteric arteries to K + - depolarization, suggesting that their effects were not due to non-specific actions on smooth muscle. Two independent studies have reported the nPKCs, 5 (Eto et al, 2001) and 8 (Uehata et al, 1997) to be sensitive to inhibition by Y-27632. We therefore investigated the effect of this inhibitor on direct activation of PKC with PdBu and found that the concentration of Y-27632 used in this investigation had no significant effect on contractile responses of control or diabetic mesenteric arteries to PdBu. Pharmacological Inhibition and Translocation of RhoK 95 The significant shift in the PE concentration-response curve produced by both the RhoK inhibitors in mesenteric arteries from control rats is consistent with a role for this pathway in contractile responses to ai-AR stimulation in this tissue. This is supported by the observation that PE increased levels of both ROCK I and II in the particulate fraction, and this was abolished by Y-27632. Other studies conducted on the rat mesenteric vasculature have also implicated RhoK in Ca 2 + sensitization and contraction in response to ai-AR stimulation (VanBavel et al., 2001; Altmann et al., 2003). However in the rabbit mesenteric artery, contractile responses to PE were unaffected by inhibition of RhoA by C3 exoenzyme (Sasaki et al, 1998). This suggests that coupling of the RhoA/RhoK pathway to apAR-mediated Ca 2 + sensitization mechanisms could differ depending on the species being investigated. The role of the RhoA/RhoK pathway in abnormal vascular reactivity in Type 2 D M has been investigated in a few studies. In vascular smooth muscle cells isolated from diabetic Goto-Kakizaki rats, thrombin increased RhoK activity and this was associated with an increase in MYPT phosphorylation and inhibition of MLCP (Begum et al., 2000). This was proposed to be partly due to the loss of insulin-mediated inhibition of RhoK. Very recently it was reported that RhoK mediated endothelial dysfunction in cerebral arterioles isolated from db/db mice (a genetic model of Type 2 DM) by enhancing basal superoxide levels, probably via inhibition of nitric oxide synthase (Didion et al., 2005). However whether the RhoA/RhoK pathway also contributes to abnormal vasoconstrictor responses in animal models of Type 1 D M has not yet been investigated. Our results show that the RhoK inhibitors, like the PKC inhibitors, produced a greater attenuation of PE responses in mesenteric arteries from diabetic rats and eliminated the significant difference in contractile responses of control and diabetic arteries to PE. In addition to significant translocation of ROCK I, there is greater translocation of ROCK II in response to PE in diabetic than in control arteries, suggesting that increased activation of RhoK contributes to the enhanced contractile response of diabetic arteries to cci-AR stimulation. The RhoA/RhoK pathway has been implicated in ET-1-induced Ca 2 + sensitization and contraction in vascular smooth muscle such as the rabbit aorta (Sakurada et al., 2001) and basilar artery (Miao et al., 2002b), human mammary artery (Batchelor et al., 2001) and gerbil spiral modiolar artery (Scherer et al., 2002). While there is not a lot of information regarding 96 the role of this pathway in abnormal vasoconstrictor responses to ET-1 in diabetes, in one study significantly elevated ET-1-induced vasoconstriction in corpus cavernosum smooth muscle from alloxan-induced diabetic rabbits was suggested to be mediated by RhoK, specifically ROCK I (Chang et al, 2003). However the results of this study were based on pharmacological inhibition of RhoK by Y-27632 and measurement of total ROCK I expression, while ET-1-induced activation or translocation of ROCK I were not determined. In the present investigation no participation of RhoK in contractile responses of control and diabetic mesenteric arteries to ET-1 was found, since concentrations of the antagonists that significantly blocked the PE response had no effect on responses to ET-1. The lack of effect of RhoK inhibitors bn ET-1-induced contraction is consistent with the lack of detectable translocation of RhoK isoforms following stimulation with this agonist in both control and diabetic arteries. Our results are similar to a recent report showing that ET-1-induced vasoconstriction in the rat mesenteric artery bed was resistant to RhoK inhibition by Y-27632 and HA-1077 (Buyukafsar et al, 2004). In another study, sub-threshold concentrations of ET-1 induced potentiation of NE responses in perfused rat mesenteric arteries and RhoK was suggested to participate in this process (Matsumura et al, 2001). However, the role of RhoK in ET-1-induced contractions was not determined in that investigation. The results of the present investigation suggest that oti-ARs couple more efficiently to the RhoK than the PKC pathway, while ET-1 receptors couple more effectively to the PKC pathway in mesenteric arteries from control rats. Receptor agonists have been shown to couple to different Ca2+ sensitization pathways in a particular vascular tissue. Variations in the contribution of the PKC and RhoK signaling pathways to contractile responses to stimulation of different receptors have been reported. For instance, in porcine coronary artery, the Ca sensitizing effect of carbachol was suggested to be primarily due to RhoA-independent tyrosine phosphorylation, with no participation of PKC whereas that of ET-1 was proposed to be mediated by PKC and RhoA-dependent and -independent tyrosine phosphorylation, based on pharmacological inhibition of these pathways (Sato et al, 2000a). In another study also conducted on the porcine coronary artery, RhoK was shown to play a more prominent role in U-46619-induced Ca sensitization than PKC (Nobe & Paul, 2001). RhoK was also shown to participate to a greater extent in NE- and A G II-induced contraction when compared to PKC in the rat renal artery (Bauer & Parekh, 2003). This may be due to 97 variations in the ability of agonists and receptors to activate G-proteins of the Gq/n and G12/13 families (Gohla et al, 2000). 4.9 K + DEPOLARIZATION-INDUCED RhoA/RhoK ACTIVATION There is evidence to show that K + depolarization of vascular smooth muscle can sensitize the contractile proteins to Ca 2 + , and this process has been shown to involve the activation of RhoA/RhoK (Mita et al, 2002; Sakamoto et al, 2003; Urban et al, 2003). For example, stimulation of rabbit femoral artery with KC1 resulted in significant translocation of ROCK II to the caveolae, which are invaginations of the cell membrane (Urban et al, 2003) and KC1 significantly increased activities of both RhoA and RhoK in airway smooth muscle (Janssen et al, 2004). This activation is Ca 2 + dependent since removal of extracellular Ca 2 + or use of Ca 2 + channel blockers abolishes KCl-induced RhoA/RhoK activation (Sakurada et al, 2003; Janssen et al, 2004). It is unlikely that in our studies K+-depolarization is associated with RhoK activation because the concentration of Y-27632 that blocked PE-induced translocation of ROCK I and II did not have a significant effect on KCl-induced contractions in both control and diabetic mesenteric arteries. It is possible that activation of the RhoA/RhoK pathway by depolarization takes place only in some smooth muscles. Somlyo and Somlyo (2003) suggested that participation of the RhoA/RhoK pathway in KCl-induced contractions might represent constitutively active RhoA and/or RhoK or activation of this pathway due to very high [Ca2+]j, since translocation of RhoK was evident only at 30 uM free Ca 2 + or more (Gong et al, 1997). 4.10 AGONIST-INDUCED CHANGES IN CYTOSOLIC (SOLUBLE) LEVELS OF PKC AND RhoK ISOFORMS In our study, we have seen varying effects of ai-AR agonists and ET-1 on particulate levels of PKC and RhoK isoforms (discussed in detail in section 4.6 and 4.9). However the agonist-induced increases in particulate levels of these isoforms were not associated with corresponding decreases in the cytosolic fractions (data not shown). This been previously reported for both PKC and RhoA/RhoK. For instance, PE induced significant translocation of PKCs in cardiomyocytes but there was no detectable decrease in soluble fractions of this isoform (Puceat et al, 1994; Wang et al, 2003). Similarly, oxyhemoglobin-mediated cerebral vasoconstriction was associated with significant increases in particulate levels of 98 PKCa and s and RhoA but the corresponding soluble fractions were not significantly decreased (Wickman et al, 2003). Reactive oxygen species induced by treating rat aortic rings with xanthine-xanthine oxidase also resulted in a significant increase in RhoA particulate levels without any detectable decrease in the soluble fraction (Jin et al, 2004), while stimulation of smooth muscle cells from rabbit colon with ACh resulted in a significant increase in RhoA and ROCK II in the particulate fraction without any significant changes in the soluble fraction (Patil et al., 2004). A possible explanation for these observations is the greater amount of protein present in the soluble as compared to the particulate fraction, as a result of which decreases in soluble protein levels of PKC or RhoK are relatively difficult to detect as compared to increases in the particulate levels (Henry et al., 1996). 4.11 INTERACTION OF PKC AND RhoK SIGNALING PATHWAYS It has been suggested that interaction between the different kinase pathways activated on stimulation of GPCRs may be a key signaling event of Ca 2 + sensitization of the contractile apparatus in vascular smooth muscle (Sasaki et al., 1998; Martinez et al., 2000). Crosstalk between the RhoK and PKC pathways has been demonstrated to occur in a number of cell systems. For example, translocation and activation of PKC in mammalian endothelial and bronchial epithelial cells was blocked by inhibition of the Rho GTPases, RhoA and/or Cdc42 (Hippenstiel et al, 1998). Conversely in another study, PKC inhibition or depletion blocked RhoK activation in cultured human umbilical vein endothelial cells (Barandier et al, 2003). RhoA association with PKCa in the membrane fraction was essential for optimal enhancement of AP-1 transcriptional activity in Jurkat T cells (Chang et al, 1998). A direct interaction between PKCa and RhoA, demonstrated in an in vitro assay, was shown to lead to the activation of the former (Slater et al, 2001). Stimulation of rabbit rectosigmoid smooth muscle cells with ACh resulted in translocation and significant complexing of RhoA and PKCa in the membrane fraction (Bitar et al, 2002). Two studies have investigated interactions between the PKC and RhoK pathway in intact artery preparations. PKC and RhoK appeared to be acting along the same intracellular signaling pathway leading to interleukin (3-induced coronary artery vasospasm in a porcine model (Kandabashi et al, 2003). Since PdBu-induced contraction was significantly attenuated by hydroxyfasudil (a RhoK inhibitor) and RhoA was significantly translocated to the membrane fraction on stimulation with the phorbol, PKC was suggested to be located upstream of RhoK. Similarly, 99 in another study conducted on the rabbit aorta, fasudil (a RhoK inhibitor) inhibited 12-deoxyphorbol 13-isobutyrate-induced contraction and phosphorylation of MLC 2o, also suggesting that PKC is located upstream of RhoK in that artery (Shimomura et al, 2004). In our studies, PKC but not RhoK appeared to participate in the unchanged contractile responses of diabetic mesenteric arteries to ET-1, so we reasoned that RhoK might be upstream of PKC on stimulation of cti-ARs. This possibility is supported by the finding that a concentration of Y-27632 that completely inhibited PE-induced RhoK translocation in both control and diabetic mesenteric arteries, also completely prevented PE-induced translocation of PKC isoforms in diabetic mesenteric arteries. We do not believe that this is due to direct inhibition of PKC because: 1) The concentration of Y-27632 used in this investigation had no significant effect on contractile responses of control or diabetic mesenteric arteries to direct activation of PKC with PdBu. 2) Y-27632 had no inhibitory effect on contractile responses to ET-1, which are markedly reduced by inhibition of PKC. 3) Y-27632 was completely without effect on the ET-1 induced translocation of PKC a, 8 and s. This suggests that the effects of Y-27632 on the PE-induced translocation of PKC isoforms do not arise from direct inhibition of PKC by this antagonist, but result from the inhibition by Y-27632 of PE-induced increases in PKC activity. At the present time we have no direct evidence to suggest how increased activation of RhoK might lead to increased activation of PKC in mesenteric arteries from diabetic rats. However, our lab has found that oti-AR stimulated phosphoinositide hydrolysis is also enhanced in diabetic arteries, since breakdown of PIP2, and production of total inositol phosphates and IP3 are all increased (Abebe & MacLeod, 1991a; Abebe & MacLeod, 1991b; Abebe & MacLeod, 1992). There is evidence that small GTPases of the Rho family especially RhoA associate with phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to regulate the production of PIP2 (Ren & Schwartz, 1998). PIP5Ks exist in multiple isoforms, which can be divided into type-I and type-II on the basis of their biochemical and chromatographic profiles (as reviewed by Anderson et al, 1999). Type-I PIP5Ks phosphorylate phosphatidylinositol 4-phosphate (PI4P), which is the final step in PIP2 synthesis in vivo and therefore regulate supply of the 100 substrate for PLC. It was found that in HEK-293 cells, type-I PEP5K isoforms were positively regulated by RhoA resulting in enhanced PIP2 synthesis (Weernink et al., 2004). In Cos-7 cells stimulation of GPCRs by thrombin led to the activation of type-I P IP5K and its translocation to the cell membrane and this was dependent on GTP-bound RhoA (Chatah & Abram, 2001). The RhoA-induced increase in P IP5K activity in HEK-293 cells was mediated by RhoK as treatment with HA-1077 prevented the increment in PJP2 cellular levels (Oude Weernink et al., 2000). Since PIP2 levels in the cell are limited and are rapidly depleted on sustained agonist stimulation, RhoA/RhoK mediated regulation of this phospholipid has been suggested to have significant implications on PKC signaling by receptors coupled to PLC (Sah et al, 2000). However the regulation of inositol lipid kinases by RhoA and RhoK have been investigated in relation to their effects on the actin cytoskeleton, as the Rho family have been shown to be key regulators of the cytoskeletal organization (Etienne-Manneville & Hall, 2002), and it is not clear whether a similar pathway exists in vascular smooth muscle. The signal transduction pathways in mesenteric arteries from STZ-diabetic rats as suggested by the results of our study are summarized in Figure 4.1. The dashed line shows the proposed interaction between the PKC and the RhoK pathway. We hypothesized that the enhanced activation of RhoK, especially ROCK II, mediates the increased participation of PKC in cti-AR-induced vasocontractile responses by increasing cellular PIP2 levels in diabetic mesenteric arteries. This explanation is speculative at present since we do not have experimental evidence to support it. Whether RhoK contributes to the regulation of PIP2 production requires further investigation. 101 Figure 4.1 G a,-AR 2/13 > PLC * G. q / l l G. ETA / B-R q / l l RhoA PIP, > DAG + IP, i RhoK MLCP MLCK MLC20-P CONTRACTION The proposed signal transduction pathways activated in response to stimulation of aj-AR and ET-1 receptors in diabetic mesenteric arteries. Abbreviations: a r A R - alphai adrenoceptor, CPI-17 - PKC potentiated inhibitor protein of 17kDa, DAG - diacylglycerol, ET-1 - endothelin-1, IP 3 - inositol trisphosphate, MLC 2 o -regulatory light chains of myosin, M L C 2 0 - P - phosphorylated regulatory light chains of myosin, M L C K - myosin light chain kinase, M L C P - myosin light chain phosphatase, PIP 2 - phosphatidylinositol bisphosphate, PKC - protein kinase C, PLC - phospholipase C, RhoK - Rho kinase 102 4.12 POTENTIAL MECHANISMS LEADING TO ENHANCED RhoK PARTICIPATION IN cti-AR-MEDIATED VASOCONSTRICTION IN MESENTERIC ARTERIES FROM DIABETIC RATS There is no experimental evidence concerning the mechanisms responsible for enhanced participation of RhoK in PE-induced vasoconstriction in mesenteric arteries from diabetic rats at present. However a number of possible mechanisms can be proposed. For instance, cti-ARs in vascular smooth muscle have been shown to couple to the G12/13 G-protein, which regulate the activity of RhoA/RhoK (as reviewed by Sah et al., 2000). It has been suggested that an increase in the number of G-proteins or in the efficiency of their coupling to the receptor can result in enhanced vasocontractile responses (Raymond, 1995). A number of studies have shown that diabetes can affect G-protein function and expression. For instance, Gj G-protein levels were decreased in diabetic hepatocytes (Bushfield et al., 1990; Caro et al., 1994) and its functioning was compromised in diabetic adipocytes (Green & Johnson, 1991). Although previous studies from our lab have shown that levels of Gq/n and GJ2/3 G-proteins and their coupling efficiency to oti-ARs in diabetic arteries are not altered, it is possible that there are differences in G12/13 G-protein functioning and/or expression in mesenteric arteries from diabetic rats. Increased levels and/or coupling efficiency of this G-protein to oti-ARs could explain the enhanced participation of RhoK to PE-induced vasoconstriction in diabetic arteries. It is also possible that chronic diabetes is associated with abnormalities in regulators of Rho GTPases such as RhoGEFs and RhoGDI, which are positive and negative modulators of RhoA, respectively. Ying et al (2004) showed that RhoGEF (PDZ-RhoGEF, pi 15 RhoGEF and LARG) expression levels were significantly elevated in aorta from stroke-prone spontaneously hypertensive rats (SHRSP), and this was suggested to contribute to enhanced vasoconstrictor responsiveness in hypertension. Since RhoGEFs facilitate activation of RhoA and hence regulate signaling via GPCRs, it is possible that overexpression of these regulatory proteins could increase PE-induced RhoK activation in mesenteric arteries from diabetic rats. Another possible explanation for enhanced participation of RhoK in PE-induced vasoconstriction in diabetic arteries is hyperglycemia-induced generation of ROS. ROS have been suggested to be important modulators of vascular tone and some studies have shown 103 that even after removal of the endothelium, ROS can affect vascular smooth muscle contractility (Auch-Schwelk et al, 1989; Rodriguez-Martinez et al, 1998; Yang et al, 1999). This has been suggested to be via Ca2+-independent stimulation of signal transduction pathways such as the MAP kinase or TK pathway (Jin et al, 1991; Yang et al, 1999; Pelaez et al, 2000). In a recent study, Jin et al (2004) showed that ROS induced activation of the RhoA/RhoK pathway in rat aorta. ROS have been suggested to participate in vascular abnormalities in DM (Ceriello et al, 1993) and some studies have demonstrated a role for ROS in the development of enhanced ai-AR responses in diabetic animal models (Chang et al., 1993, Kanie & Kamata, 2000). It is possible that enhanced oxidative stress in mesenteric arteries from chronic diabetic rats in our study mediates increased aj-AR vasoconstriction via enhanced activation of RhoK. 4.13 PHARMACOLOGICAL INHIBITION OF TKs AND cPLA 2 Functional studies implicating TKs in GPCR-mediated contractile processes have used low molecular weight TK inhibitors that are categorized depending on their mechanism of action. One class of TK inhibitors acts by interacting with the ATP-binding site; the most commonly used inhibitor from this class is the naturally occurring quercetin derivative, genistein. The other class of inhibitors, which includes the synthetic analogues of erbstatin, the tyrphostins, acts by interacting with the substrate-binding site (Casnellie, 1991). Inactive analogues for each class of inhibitors have also been developed, namely daidzein and tyrphostin A l . TKs have been implicated in GPCR-mediated contractile responses in the mesenteric vasculature. Arginine vasopressin-induced contractions were significantly attenuated in resistance mesenteric arteries from 6-week, but not 21-week, spontaneously hypertensive rats by treatment with tyrphostin A23 (Endemann et al, 2002). NE induced tyrosine phosphorylation of a number of different proteins in small mesenteric arteries from adult female Sprague Dawley rats and this was inhibited by treatment with tyrphostin A23 (Ward et al, 2002). In the same vessels, tyrphostin A23 significantly attenuated ET-l-induced contractions (Ohanian et al, 1997). In rat mesenteric resistance arteries, genistein and tyrphostin A23 produced a concentration-dependent ( luM - lOOpM) relaxation of NE-induced contractions (Toma et al, 1995). In rabbit permeabilized mesenteric artery, histamine- and PE-induced Ca 2 + sensitization was significantly attenuated by genistein and tyrphostin A25 (Sasaki et al, 1998). However there are also reports of TKs not participating 104 in GPCR-mediated contractions in mesenteric arteries. For example in rat mesenteric resistance arteries, U-46619-induced contractions were unaffected by tyrphostin A25 (Bolla et al., 2002) and in permeabilized rabbit mesenteric arteries genistein did not affect ET-1-induced Ca 2 + sensitization (Sato et al, 2000a). The lack of effect of TK inhibitors in our studies could be due differences in the preparation used, as most of the studies described above have utilized the resistance arteries in the mesenteric vasculature whereas we have used the superior mesenteric vessels. There could also be differences in coupling of TKs to GPCRs depending on species and gender of the experimental animal (Sato et al., 2000a). The concentration of genistein and tyrphostin A23 used in the present investigation (lOuM) has been shown to attenuate agonist-induced responses in vascular smooth muscle (Filipeanu et ai, 1995; Toma et al., 1995; Fetscher et al, 2001; Endemann et al, 2002). At this concentration, genistein and tyrphostin A23 had no significant effect on KCl-induced contractile responses, suggesting that their effects were not due to non-specific actions. At higher concentrations they significantly attenuated KC1-mediated responses (data not shown). It has been suggested that tyrphostins may require up to 16 hours to attain their optimal inhibitory effect in intact cells (Lyall et al., 1989). However in most studies the effects of TK inhibitors in intact vessels have been investigated following an incubation period of 10 - 30 minutes (Toma et al., 1995; Fetscher et ai, 2001). There is not a lot of information regarding the role of TKs in abnormal vasoconstrictor responses in Type I diabetic animal models. However in one investigation, insulin-mediated potentiation of NE-induced contraction in rabbit facial artery was suppressed by pretreatment with genistein (10 uM) (Henrion & Laher, 1994). In our studies TK inhibition did not normalize ai-AR mediated responses in mesenteric arteries from diabetic rats. This preliminary evidence suggests that TKs do not participate in abnormal vasoconstrictor responses in diabetic tissues. However more experimental evidence, such as measurement of PE-induced tyrosine phosphorylation, is required before the role of TKs in GPCR-mediated responses in diabetic tissues can be determined. CPLA2 has been frequently implicated in GPCR-mediated responses using A A C O C F 3 , which has been suggested to be a selective CPLA2 inhibitor (Street et al., 1993; L i & Cathcart, 1997). In the perfused rat MAB, A A C O C F 3 significantly attenuated histamine- and ACh-induced decreases in perfusion pressure in response to cirazoline (Adeagbo & Henzel, 1998). 105 This inhibition was due to attenuation of cPLA2-mediated production of AA, which in turn led to release of endothelium derived hyperpolarizing factor (EDHF). AACOCF 3 significantly attenuated NE- (Labelle & Polyak, 1998) and ET-1-induced (Trevisi et al, 2002) AA release in rat tail artery. In rat aorta, A A C O C F 3 significantly decreased ET-1-induced prostacyclin production (Oriji et al, 1996). NaF, a nonselective activator of heterotrimeric G-proteins, increased A A and prostacyclin production in bovine aortic endothelial cells and this was inhibited by treatment with A A C O C F 3 (Rosenstock et al, 1996). The concentration of A A C O C F 3 used in the present investigation (10 uM) has been used to selectively inhibit cPLA 2 in vascular smooth muscle (Xia et al., 1995; Labelle & Polyak, 1998; Silfani & Freeman, 2002). At this concentration, A A C O C F 3 had no significant effect on KCl-induced contractile responses, suggesting that its effects were not due to non-specific actions. The fact that A A C O C F 3 had no effect on PE and ET-1-induced contractile responses in mesenteric arteries from control rats suggests that cPLA 2 is not activated in response to these agonists in our preparation. However it is not possible to rule out a role of other classes of PLA 2 in ai-AR and ET-1 receptor-mediated responses in superior mesenteric arteries. This can be investigated using BEL, an inhibitor suggested to be selective for iPLA 2 (Balsinde & Dennis, 1998), LY311727, an inhibitor suggested to be selective for sPLA 2 (Schevitz et al, 1995) or non-isoform selective PLA 2 inhibitors like quinacrine (Mukherjee et al, 1994) or ONO-RS-082 (Billah & Anthes, 1990). The role of cPLA 2 in abnormal vasoconstrictor responses in Type I diabetes has not been investigated in detail. However there is evidence to show that AA-induced production of prostaglandins can affect vascular responses. For example vasodilator responses to bradykinin were attenuated in the isolated perfused kidney of STZ-induced diabetic rats and this was due to decreased renal PLA 2 activity and attenuated release of prostaglandin E 2 (Quilley et al, 1992). Similarly in diabetic rat aorta, NE- and U-46619-induced prostacyclin production was significantly attenuated due to decreased PLA 2 activity (Jeremy et al, 1987). In our studies, A A C O C F 3 did not normalize significantly elevated PE responses in mesenteric arteries from diabetic rats, suggesting that cPLA 2 does not participate in the enhanced oci-AR-induced contractions in diabetic tissues. The role of cPLA 2 and A A in 106 GPCR-mediated Ca + sensitization and contraction in diabetic arteries is controversial and requires further investigation. 107 5. SUMMARY AND CONCLUSIONS Contractile responses of endothelium-denuded mesenteric arteries from diabetic rats to NE were enhanced, but were normalized by the PKC inhibitors, Ro-318220 and calphostin C. In contrast, no change in contractile responses of diabetic arteries to ET-1 could be detected, and PKC inhibition attenuated ET-1 responses to a similar extent in both control and diabetic tissues. A maximum concentration of NE produced a small translocation of PKCs in control arteries, but a significant translocation of PKCa and a much larger translocation of PKCs in diabetic arteries. ET-1 increased translocation of PKCa, 5 and s in both control and diabetic arteries and the degree of translocation was no greater in diabetic tissues. NE significantly enhanced CPI-17 phosphorylation in diabetic, but not control, arteries and this was blocked by PKC inhibition. These data suggest that increased activation of the PKC/CPI-17 pathway mediates the enhanced contractile response to NE in mesenteric arteries from diabetic rats. On the other hand, although PKC appears to contribute to a greater extent to ET-1 than to NE-induced contractions in control mesenteric arteries, no evidence was found for increased activation of this pathway in response to ET-1 in mesenteric arteries from diabetic rats. This suggests that significantly elevated NE-induced contractions result from a selective change in ai-AR-mediated signaling in the diabetic arteries. The selective RhoK inhibitors, Y-27632 and H-l 152, produced greater inhibition of contractile responses to PE in diabetic than control mesenteric arteries and normalized the difference between them. Contractile responses to ET-1 were not affected by RhoK inhibition. A maximum concentration of PE produced significant translocation of ROCK I and II that was inhibited by Y-27632 in both control and diabetic arteries. The PE-induced translocation of ROCK II was significantly greater in diabetic tissues. ET-1 produced no detectable translocation of RhoK isoforms. These data suggest that activation of RhoK, especially ROCK II, in addition to PKC, is required for the enhanced vasoconstrictor responses of diabetic mesenteric arteries to ai-AR stimulation. On the other hand, RhoK does not appear to contribute to contractile responses to ET-1 in either control or diabetic mesenteric arteries. 108 PE produced significant translocation of PKC a, 5 and s in diabetic but not control arteries. Y-27632 blocked the translocation of these isoforms in diabetic arteries but had no effect on ET-1 induced translocation of PKC isoforms in control arteries. These data suggest that there is an interaction between the RhoK and PKC pathways on stimulation of a t-ARs in diabetic mesenteric arteries and that increased activation of RhoK may mediate'enhanced activation of PKC. Although increased participation of PKC in enhanced contractile responses of diabetic arteries to stimulation of GPCRs has been proposed, this is the first demonstration to our knowledge that RhoK may also be required and may be functioning upstream of PKC. Preliminary pharmacological data suggests that TKs and C P L A 2 do not participate in PE or ET-1 induced responses in either control or diabetic mesenteric arteries. 109 6. FUTURE DIRECTIONS It would be helpful to identify the ai-AR subtype (ai A , a i s or ctio) that is involved in mediating the enhanced contractile responses to NE and PE in diabetic arteries. While selective agonists are only available for the a i A - A R ( A - 6 1 6 0 3 and oxymetazoline), selective antagonists for every subtype are available (e.g. WB 4101 and KMD 3213 for CCIA-AR, CEC for a ) B -AR and BMY 7378 for a ] D -AR). These could be used in tissue bath contraction studies to identify the subtype mediating enhanced ai-AR responses in diabetic arteries. The number of the different subtypes and their binding affinity to PE/NE can be determined by saturation binding assays using the selective antagonists for the different subtypes. To investigate which subtype is mediating the enhanced activation of RhoK and PKC following oti-AR stimulation in diabetic arteries, the effect of subtype-selective antagonists on PE-induced translocation of RhoK and PKC isoforms should be determined. We have proposed that RhoK mediates enhanced PKC activation in response to a i -AR stimulation in diabetic arteries via increased activation of P I P 5 K . In order to confirm this we need to determine if increased activation of RhoK results in enhanced stimulation of P I P 5 K activity and if this promotes ai-AR-mediated phosphoinositide hydrolysis and DAG production in diabetic mesenteric arteries. Agonist-induced translocation of PKC and RhoK isoforms is regarded as an index of their activation. To further confirm a role for PKC and RhoK in enhanced ai-AR vasoconstriction in diabetic mesenteric arteries, direct activity measurements can be carried out for PKC (Kanashiro & Khalil, 2001) and RhoK (Murthy et al, 2003) following stimulation with NE/PE. Activity should be measured in the absence and presence of selective inhibitors to confirm specificity of the assay The mechanism of RhoK-mediated Ca sensitization in control and diabetic mesenteric arteries should be determined. This can be done by measuring MYPT phosphorylation (if any) at Thr 696 and Thr 850 following a i - A R stimulation. Since CPI-17 also mediates MYPT phosphorylation and inhibition, the effect of RhoK inhibition on the degree of phosphorylation of MYPT should also be investigated. 110 The reasons for the enhanced PE-induced RhoK activation in diabetic arteries are speculative at present (discussed in section 4.13). In order to investigate the underlying mechanisms, levels of G12/13 G-protein and its interaction with cti-ARs in diabetic mesenteric arteries can be determined by Western blotting and competition binding experiments, respectively. To determine if increased expression of RhoGEFs is mediating enhanced PE-induced RhoK activation in diabetic mesenteric arteries, its expression levels can be measured by RT-PCR and Western blotting. 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