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Alpha₁-adrenoceptor-mediated phosphoinositide breakdown and inotropic responses in right ventricles of… Xiang, Hong 1990

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ALPHAx-ADRENOCEPTOR-MEDIATED PHOSPHOINOSITIDE BREAKDOWN AND INOTROPIC RESPONSES IN RIGHT VENTRICLES OF STREPTOZOTOCIN-DIABETIC RATS By HONG XIANG M.D., Beijing Medical University, 1984 M.Sc, University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Division of Pharmacology and Toxicology Of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1990 © Hong Xiang, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmaceutical Sciences The University of British Columbia Vancouver, Canada Date March 9, 1990 DE-6 (2/88) A B S T R A C T The morbidity of and the morta l i ty from cardiac disease are higher i n d i a b e t i c pa t i en t s . C l i n i c a l and experimental evidence suggests that diabetes- induced changes at the l e v e l of myocardium can, at l eas t p a r t i a l l y , contr ibute to these cardiac problems. The mechanism(s) involved i n t h i s d i a b e t i c cardiomyopathy i s s t i l l unclear , but one defect appears to occur i n the alphai-adrenoceptor system. A l t e r e d myocardial s e n s i t i v i t y and responsiveness to alpha^-adrenoceptor agonists have been reported i n experimental diabetes m e l l i t u s . St imulat ion of alpha]_-adrenoceptors i s known to produce a p o s i t i v e ino trop ic e f f ec t and has been recent ly shown to st imulate the h y d r o l y s i s of phosphoinosi t ides . To evaluate the p o s s i b i l i t y that the changes i n the i n o t r o p i c responsiveness to alpha^-adrenoceptor s t imulat ion i n the d i a b e t i c heart could be l inked to a l t ered a lphai-adrenoceptor-st imulated phosphoinosit ide turnover and further to the development of d i a b e t i c cardiomyopathy, we studied c o n t r a c t i l i t y and receptor-s t imulated phosphoinosit ide turnover fo l lowing norepinephrine ( in the presence of propranolol) s t imulat ion i n r i g h t v e n t r i c l e s from male Wistar ra t s (200-225 g) which were made d i a b e t i c with s treptozotoc in (55 mg/kg, i . v . ) . Rats were s a c r i f i c e d s i x weeks a f t er the induct ion of diabetes . Diabet ic ra t s were character ized by decreased body weight ga in , hypoinsulinemia, hyperglycemia and hyper l ip idemia . i i i S t imulat ion of alpha^-adrenoceptors by norepinephrine ( in the presence of propranolol) i n r i g h t v e n t r i c l e s re su l t ed i n the formation of i n o s i t o l monophosphate (measured with a radio isotope method) and i n o s i t o l 1 ,4 ,5-trisphosphate (measured with an i n o s i t o l 1 ,4 ,5- tr isphosphate p r o t e i n binding assay k i t ) i n a t ime- and concentrat ion-dependent manner i n both contro l and d i a b e t i c r a t s . The increase i n i n o s i t o l 1 ,4 ,5-tr isphosphate l e v e l s preceded the increase i n the alpha^-adrenoceptor-mediated p o s i t i v e i n o t r o p i c e f f e c t . Diabet ic hearts showed a greater maximum i n o t r o p i c response to norepinephrine s t imulat ion and a lso had a higher i n o s i t o l 1 ,4 ,5-tr isphosphate l e v e l s . However, with the radio isotope method, a decreased i n o s i t o l monophosphate formation was shown i n d i a b e t i c hearts compared with c o n t r o l s . Omega-3 f a t t y acids supplementation (PromegaR, 0.5 ml/kg/day) had no s i g n i f i c a n t e f fec t on the changes i n norepinephrine-st imulated i n o s i t o l monophosphate formation i n d i a b e t i c hearts . In the presence of the cyclooxygenase i n h i b i t o r indomethacin or the thromboxane synthetase i n h i b i t o r imidazole , the norepinephrine-st imulated p o s i t i v e i n o t r o p i c e f fec t and i n o s i t o l 1 ,4 ,5-tr isphosphate formation were s i g n i f i c a n t l y increased i n contro l hearts , but were unal tered i n the hearts from d i a b e t i c s . The add i t i on of the p r o s t a c y c l i n synthetase i n h i b i t o r tranylcypromine reduced the norepinephrine-st imulated p o s i t i v e i n o t r o p i c e f f ec t and i v i n o s i t o l 1 ,4 ,5-tr isphosphate formation only i n d i a b e t i c hearts and had no e f fec t i n the c o n t r o l s . While i n o s i t o l 1 ,4 ,5-tr isphosphate may be able to mediate only t rans i ent ino trop ic e f fec ts produced by alpha^-adrenoceptor s t imula t ion , d i a c y l g l y c e r o l may provoke a sustained p o s i t i v e ino trop ic e f fec t by a c t i v a t i n g slow C a 2 + channels through s t imulat ion of pro te in kinase C. Our r e s u l t s showed that the d i a b e t i c hearts had a higher pro te in kinase C a c t i v i t y i n the membrane f r a c t i o n compared with contro l s and t h i s was accompanied by a decrease i n c y t o s o l i c p r o t e i n kinase C a c t i v i t y . The present study suggests that the increases i n i n o s i t o l 1 ,4 ,5-tr isphosphate l e v e l s and the membrane f r a c t i o n pro te in kinase C a c t i v i t y may be impl icated i n the increased i n o t r o p i c responsiveness to alphai-adrenoceptor s t imulat ion i n the hearts of the s t rep tozo toc in -d iabe t i c r a t s . The increases i n i n o s i t o l 1 ,4 ,5- tr isphosphate l e v e l and pro te in kinase C a c t i v i t y could induce C a 2 + overload i n the d i a b e t i c heart which might be involved i n the development of d i a b e t i c cardiomyopathy. The r e s u l t s from the omega-3 f a t t y ac id study ind icate that the changes i n cardiac alphai-adrenoceptor-mediated i n o s i t o l phosphates formation cannot contr ibute to the prev ious ly described improved cardiac funct ion of omega-3 f a t t y a c i d - t r e a t e d s t rep tozo toc in -d iabe t i c r a t s . The nature and p h y s i o l o g i c a l s i g n i f i c a n c e of the enhanced p o s i t i v e i n o t r o p i c e f f ec t and i n o s i t o l 1 ,4 ,5-tr isphosphate formation i n the c o n t r o l heart V with the add i t ion of indomethacin and imidazole i s s t i l l unc lear . The e f fec t of tranylcypromine may ind ica te the p a r t i c i p a t i o n of prostaglandins i n mediating the enhanced a l p h a i - i n o t r o p i c e f fec t of norepinephrine i n the d i a b e t i c heart . John H. M c N e i l l , Ph.D. Dean, Facul ty of Pharmaceutical Sciences Thesis Supervisor v i T A B L E O F C O N T E N T S Page ABSTRACT i i LIST OF TABLES X LIST OF FIGURES xi LIST OF ABBREVIATIONS XVi ACKNOWLEDGEMENT xix 1 INTRODUCTION 1 1.1 OVERVIEW 1 1.1.1 Insulin-Dependent Diabetes Mellitus (IDDM) 4 1.1.2 Non-Insulin-Dependent Diabetes Mellitus (NIDDM) 6 1.2 PATHOPHYSIOLOGY OF DIABETES AND ASSOCIATED HEART DISEASE 7 1.2.1 Pathophysiology of Diabetes 7 1.2.2 Heart Disease and Diabetes 10 1.2.2.1 Sarcolemma 12 1.2.2.2 Sarcoplasmic reticulum 14 1.2.2.3 Mitochondria 15 1.2.2.4 Contractile proteins 17 1.2.2.5 Receptors 18 1.3 CARDIAC ALPHA!-ADRENOCEPTORS AND PHOSPHOINOSITIDE METABOLISM 21 1.3.1 Receptor-stimulated Phosphoinositides Metabolism 21 1.3.2 Myocardial Alphai-adrenoceptors and their Positive Inotropic Effect on the Heart 29 1.3.3 Myocardial Alphai-Adrenoceptors and Phosphoinositide Hydrolysis 32 1.3.4 Alpha-Adrenoceptor Agonists 34 1.3.5 Prazosin as a Selective Alpha--Adrenoceptor Blocker 35 1.4 CARDIAC ALPHAx-ADRENOCEPTORS AND PHOSPHOINOSITIDE METABOLISM IN DIABETES 36 1.4.1 Diabetes and Cardiac Alpha-Adrenoceptor Functions 36 1.4.2 Diabetes and Cardiac Phosphoinositide Metabolism 37 1.5 OMEGA-3 FATTY ACID 41 v i i v i i i Page 2 2 2 .7.1 .7.2 .7.3 Preparation of Membrane and Cytosolic Fractions DEAE Cellulose Column Assay of Protein Kinase C Activity 61 63 64 2 2 2 2 2 .8 .8.1 .8.2 .8.3 .8.4 BLOOD ANALYSIS Plasma Glucose Determination Plasma Insulin Determination Plasma Triglyceride Determination Plasma Cholesterol Determination 65 65 65 65 65 2 .9 STATISTICAL ANALYSIS 65 3 RESULTS 67 3 . 1 THE BASAL LEVEL OF INOSITOL MONOPHOSPHATE (IPi) FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 67 3 .2 TIME COURSE OF ALPHAi~ADRENOCEPTOR-MEDIATED INOSITOL MONOPHOSPHATE (IPi) FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 67 3 .3 CONCENTRATION-RESPONSE CURVES FOR ALPHA^-ADRENOCEPTOR-MEDIATED INOSITOL MONOPHOSPHATE (IP].) FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 69 3 .4 EFFECT OF PRAZOSIN AND PROPRANOLOL ON NOREPINEPHRINE-STIMULATED INOSITOL MONOPHOSPHATE (IP].) FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 70 3 .5 EFFECT OF PRAZOSIN, PROPRANOLOL AND DMSO ALONE ON INOSITOL MONOPHOSPHATE (IP].) FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 71 3 . 6 EFFECT OF OMEGA-3 FATTY ACID ON CARDIAC ALPHAx-ADRENOCEPTOR-MEDIATED INOSITOL PHOSPHATES FORMATION IN CONTROL AND DIABETIC RATS 71 3 .7 EFFECT OF ALPHA^-ADRENOCEPTOR STIMULATION ON POSITIVE INOTROPIC EFFECT AND INOSITOL 1,4,5-TRISPHOSPHATE [INS(1,4,5)P3] FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 73 IX Page 3.8 TIME COURSE OF ALPHA^-ADRENOCEPTOR-MEDIATED POSITIVE INOTROPISM AND INOSITOL 1,4,5-TRISPHOSPHATE [INS(1,4,5)P3] FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 76 3.9 CONCENTRATION DEPENDENCE OF ALPHA^-ADRENOCEPTOR-MEDIATED POSITIVE INOTROPIC EFFECT AND INOSITOL 1,4,5-TRISPHOSPHATE [INS(1,4,5)P3] FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 78 3.10 INFLUENCE OF INDOMETHACIN ON ALPHAi~ ADRENOCEPTOR-MEDIATED POSITIVE INOTROPIC EFFECT AND INOSITOL 1,4,5-TRISPHOSPHATE [INS(1,4,5)P3] FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 80 3.11 INFLUENCE OF IMIDAZOLE ON ALPHA^-ADRENOCEPTOR-MEDIATED POSITIVE INOTROPIC EFFECT AND INOSITOL 1,4,5-TRISPHOSPHATE [INS(1,4,5)P3] FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 82 3.12 INFLUENCE OF TRANYLCYPROMINE ON ALPHA^-ADRENOCEPTOR-MEDIATED POSITIVE INOTROPIC EFFECT AND INOSITOL 1,4,5-TRISPHOSPHATE [INS(1,4,5)P3] FORMATION IN CONTROL AND DIABETIC RAT VENTRICLES 84 3.13 PROTEIN KINASE C ACTIVITY IN CONTROL AND DIABETIC RAT VENTRICLES 86 DISCUSSION 181 SUMMARY AND CONCLUSIONS 214 6 REFERENCES 218 X L I S T O F T A B L E S Page 1. Classification of diabetes mellitus and a l l i e d categories of glucose intolerance. 5 2. General features of experimental rats (Time Course Study). 91 3. Plasma l i p i d profile of the experimental rats (Time Course Study). 94 4. General features of experimental rats (Concentration-Response Study). 99 5. Plasma l i p i d profile of the experimental rats (Concentration-Response Study). 102 6. General features of control and diabetic rats 6 weeks after administration of streptozotocin. 131 7. Effect of prazosin on alphai-adrenoceptor-mediated inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] formation and inotropic responses in control and diabetic right ventricles. 142 8. % Change in inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] levels at different time points of norepinephrine stimulation in control and diabetic right ventricles. 147 9. % Change in inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] formation stimulated by different concentrations of norepinephrine in control and diabetic right ventricles. 152 10. General features of control and diabetic rats 6 weeks after administration of streptozotocin (Tranylcypromine Study). 173 11. Plasma l i p i d profile of the experimental rats (Tranylcypromine Study). 174 12. Protein kinase C activity of control and diabetic right ventricles. 179 13. Protein kinase C activity of control and diabetic right ventricles with or without norepinephrine (NE) stimulation. 180 L I S T O F F I G U R E S xi Page 1. Pathophysiological alterations in carbohydrate, l i p i d and protein metabolism in diabetes mellitus. 8 2. Inositol phospholipid metabolism. 24 3. Biosynthesis of the products of arachidonic acid 30 4. Basal inositol monophosphate (IPi) formation in right ventricles of control and diabetic rats. 89 5. Time course of body weight changes in control and six-week diabetic rats (Time Course Study). 92 6. Right ventricular wet weights of control and STZ-diabetic rats (Time Course Study). 95 7. Time course of the effect of norepinephrine (10 fj,Vl) on inositol monophosphate (IPi) formation in right ventricles from control and diabetic animals. 97 8. Time course of body weight changes in control and six-week diabetic rats (Concentration-Response Study). 100 9. Right ventricular wet weights of control and STZ-diabetic rats (Concentration-Response Study). 103 10. Effect of different concentrations of norepinephrine (in the presence of propranolol) on inositol monophosphate (IPi) production in control and diabetic right ventricles. 105 11 Effect of prazosin (10 fM) and propranolol (10 /uM) on norepinephrine-stimulated inositol monophosphate (IP].) formation in right ventricles of control and diabetic rats. 107 12 Effect of prazosin, propranolol or DMSO alone on basal inositol monophosphate (IPi) formation in right ventricles of control and diabetic rats. 109 13. Body weights of control and STZ-diabetic rats treated with omega-3 fatty acid. I l l x i i Page 14. Plasma g l u c o s e l e v e l s o f c o n t r o l and STZ-d i a b e t i c r a t s t r e a t e d w i t h omega-3 f a t t y a c i d . 113 15. Plasma i n s u l i n l e v e l s o f c o n t r o l and STZ-d i a b e t i c r a t s t r e a t e d w i t h omega-3 f a t t y a c i d . 115 16. Plasma t r i g l y c e r i d e l e v e l s o f c o n t r o l and STZ-d i a b e t i c r a t s t r e a t e d w i t h omega-3 f a t t y a c i d . 117 17. Plasma c h o l e s t e r o l l e v e l s o f c o n t r o l and STZ-d i a b e t i c r a t s t r e a t e d w i t h omega-3 f a t t y a c i d . 119 18. R i g h t v e n t r i c u l a r wet weights of c o n t r o l and S T Z - d i a b e t i c r a t s t r e a t e d w i t h omega-3 f a t t y a c i d . 121 19. E f f e c t o f omega-3 f a t t y a c i d on c a r d i a c n o r e p i n e p h r i n e - s t i m u l a t e d i n o s i t o l monophosphate (IP i ) formation i n c o n t r o l and d i a b e t i c r a t s . 123 20. E f f e c t o f omega-3 f a t t y a c i d on c a r d i a c n o r e p i n e p h r i n e - s t i m u l a t e d i n o s i t o l b isphosphate ( IP2) formation i n c o n t r o l and d i a b e t i c r a t s . 125 21. E f f e c t o f omega-3 f a t t y a c i d on c a r d i a c n o r e p i n e p h r i n e - s t i m u l a t e d i n o s i t o l t r i s p h o s p h a t e ( IP3) formation i n c o n t r o l and d i a b e t i c r a t s . 127 22. E f f e c t o f omega-3 f a t t y a c i d on c a r d i a c n o r e p i n e p h r i n e - s t i m u l a t e d i n o s i t o l t e t r a k i s p h o s p h a t e ( IP4) formation i n c o n t r o l and d i a b e t i c r a t s . 129 23. Con c e n t r a t i o n - r e s p o n s e curves f o r the e f f e c t o f n o r e p i n e p h r i n e ( i n the presence o f p r o p r a n o l o l ) on change i n t e n s i o n (% of maximum) i n r i g h t v e n t r i c l e s o f c o n t r o l and d i a b e t i c r a t s . 132 24. Estimated PD2 v a l u e s f o r no r e p i n e p h r i n e ( i n the presence o f p r o p r a n o l o l ) i n c o n t r o l and d i a b e t i c r i g h t v e n t r i c l e s . 134 25. Concentration-response curves f o r the e f f e c t o f no r e p i n e p h r i n e ( i n the presence o f p r o p r a n o l o l ) on A change i n t e n s i o n i n c o n t r o l and d i a b e t i c r i g h t v e n t r i c l e s . 13 6 x i i i Page 26. A Change in tension in response to 10~2 M norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. 138 27. Norepinephrine (10 mM)-stimulated changes in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] levels in right ventricles from control and STZ-diabetic rats. 140 28. Time course of positive inotropic effects mediated by alphai-adrenoceptor stimulation in right ventricles from control and diabetic rats. 143 29. Time course of the effect of norepinephrine (10 /iM) on inositol 1,4,5-trisphosphate [Ins(l,4,5 ) P 3 ] formation in right ventricles of control and diabetic rats. 145 30. Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on change in tension (% of maximum) in right ventricles of control and diabetic rats. 148 31. Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation in right ventricles of control and STZ-diabetic rats. 150 32. Correlation curves for the concentration-responses of alphai-adrenoceptor-mediated positive inotropic effect and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation in control and diabetic right ventricles. 153 33. Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on change in tension (% of maximum) in right ventricles of control and diabetic rats. Influence of the cyclooxygenase inhibitor indomethacin (10 /nM) . 155 34. Influence of indomethacin on the estimated PD2 values for norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. 157 35. Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on A change in tension in right ventricles of xiv Page control and diabetic rats. Influence of indomethacin. 159 36. A Change in tension in response to 10~2 M of norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Influence of indomethacin. 161 37. Norepinephrine (10 mM)-stimulated changes in inositol 1,4,5-trisphosphate [ I n s ( l , 4/5 ) P 3 ] levels in right ventricles of control and diabetic rats. Influence of indomethacin. 163 38. Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on change in tension (% of maximum) in right ventricles of control and diabetic rats. Influence of the thromboxane synthetase inhibitor imidazole (10 /LtM) . 165 39. Influence of imidazole on the estimated P D 2 values for norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. 167 40. A Change in tension in response to 10~2 M of norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Influence of imidazole. 169 41. Norepinephrine (10 mM)-stimulated changes in inositol 1,4,5-trisphosphate [Ins(l,4,5 ) P 3 ] levels in right ventricles of control and diabetic rats. Influence of imidazole. 171 42. A Change in tension in response to 10~2 M of norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Influence of the prostacyclin synthetase inhibitor tranylcypromine (1.25 x 10~4 M). 175 43. Norepinephrine (10 mM)-stimulated changes in inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] levels in right ventricles of control and diabetic rats. Influence of tranylcypromine. 177 44. The inositol phospholipid cycle replenishes the supply of second messengers, Ins(1,4,5)P3 and DG. 186 The possible involvement of arachidonic acid metabolites in the changes in contractility and Ins(1,4,5 ) P 3 formation in control and diabetic hearts. L I S T O F A B B R E V I A T I O N S AA arachidonic acid AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate CDP-DG cytidine-5'-diphospho-l,2-diacyl-sn-glycerol CK solution Chenoweth-Koelle solution cpm counts per minute DG diacylglycerol DM diabetes mellitus -dP/dT rate of l e f t ventricular pressure decline dpm disintegrations per minute GDM gestational diabetes mellitus GppNHp guanosine 5'-[BY-imido]triphosphate GTP guanosine 5'-triphosphate 12-HETE 12-hydroxyeicosatetraenoic acid HHT 17-carbon hydroxy acid HLA human leukocyte antigen 12-HPETE 12-hydroperoxyeicosatetraenoic acid HPLC high performance liquid chromatography IDDM insulin-dependent diabetes mellitus IGT impaired glucose tolerance Inositol myo-inositol Ins(l)P D-inositol 1-phosphate Ins(3)P D-inositol 3-phosphate Ins(4)P D-inositol 4-phosphate x v i i Ins(l, 3)P2 D-inositol 1,3-bisphosphate Ins(l, 4)P2 D-inositol 1,4-bisphosphate Ins(3, 4)P2 D-inositol 3,4-bisphosphate Ins(l, 3,4)P3 D-inositol 1,3,4-trisphosphate Ins(l, 4,5)P3 D-inositol 1,4,5-trisphosphate Ins(l: 2cyc)P D-inositol 1,2-cyclic phosphate Ins(l: 2cyc,4)P2 D-inositol 1,2-cyclic-4-bisphosphate Ins(l: 2cyc,4,5)P3 D-inositol 1,2-cyclic-4,5-trisphosphate Ins(l, 3,4,5)P4 D-inositol 1,3,4,5-tetrakisphosphate Ins(l, 3,4,6)P4 D-inositol 1,3,4,6-tetrakisphosphate Ins(l, 3,4,5,6)P5 inositol 1, 3,4,5,6-pentakisphosphate Ins(l, 2,3,4,5,6)P6 inositol 1, 2,3,4,5,6-hexakisphosphate IP! inositol monophosphate IP2 inositol bisphosphate IP3 inositol trisphosphate IP4 inositol tetrakisphosphate kg kilogram min minute(s) ml m i l l i l i t r e mmol/1 millimoles per l i t r e MRDM malnutrition-related diabetes mellitus MW molecular weight NE norepinephrine NIDDM non-insulin-dependent diabetes mellitus PA phosphatidic acid PGD2 prostaglandin D2 PGE2 prostaglandin E2 x v i i i 6-Keto PGFla 6-keto prostacyclin F]_ a PGF2a prostaglandin F2 a PGH2 prostaglandin H2 PGI2 prostacyclin PI phosphatidylinositol Ptdlns(4)P phosphatidylinositol 4-phosphate PtdIns(4,5)P2 phosphatidylinositol 4,5-bisphosphate S.E.M. standard error of the mean SR sarcoplasmic reticulum STZ streptozotocin TXA2 thromboxane A2 TXB2 thromboxane B2 /iU/ml microunits per m i l l i l i t r e WHO World Health Organization xix ACKNOWLEDGEMENT I wish to express my deepest gratitude to my supervisor, Dr. John H. McNeill for moral support and encouragement, knowledgeable direction and patience which have enabled me to complete the work. I would like to express my sincere thanks to my supervisory committee members, Dr. Keith McErlane, Dr. Jack Diamond, Dr. Kath Macleod, Dr. David Godin and Dr. Clayton Heyliger for their valuable suggestions and constructive comments. I would also like to thank Mr. Bruce Allan and Dr. John Langlands for their invaluable support and technical instructions. I am indebted to the British Columbia Heart Foundation and Medical Research Council for the financial support they have provided me throughout the study. Also my thanks to a l l my laboratory colleagues for their valuable help, encouragement and friendship. Hong Xiang DEDICATION TO MY PARENTS, TO MY AUNT, TO MY PROFESSOR WEI-CHIN LIU, 1 INTRODUCTION 1.1. OVERVIEW. The pancreas, a flattened elongate organ lying against the posterior wall of the upper abdomen, is both an exocrine and an endocrine gland. The exocrine role of the pancreas i s carried out by cel l s within the tubular and acinar units of the gland. These cells secrete enzymes that catalyze the digestion of proteins, carbohydrates and fats (Luckmann and Sorensen, 1987). The endocrine functions of the pancreas are carried out by several hundred thousand microscopic nests of ce l l s called the is l e t s of Langerhans, which are scattered randomly through the pancreas and account for only 1-2% of i t s weight. The is l e t s contain several c e l l types including A cel l s secreting glucagon, B ce l l s secreting insulin, D cel l s secreting somatostatin and F cel l s secreting pancreatic polypeptide. Most of the pancreatic i s l e t c e l l s (60-80%) are of the B type (Steinberg, 1985). Insulin i s a 6000 MW polypeptide composed of an alpha chain and a beta chain linked together by disulfide bridges. The insulin molecule can exist as a monomer, dimer or hexamer (3 dimers). Two molecules of zinc are coordinated in the hexamer which is presumably the form stored in the B c e l l . The biologically active form of the hormone i s thought to be the monomer (Cahill, 1971). Main stimulants to insulin secretion are blood glucose, amino acids, gastrointestinal peptide hormones (especially gastric 2 inhibitory peptide, GIP), ketone bodies, glucagon, parasympathetic and beta-adrenergic stimulation. Factors which inhibit insulin release include somatostatin, alpha-adrenergic stimulation, as well as pharmacological factors such as B c e l l poisons alloxan and streptozotocin (Steinberg, 1985). Normal insulin secretion by a pancreas is about 30-60 units/day (Cahill, 1971). Insulin has important effects on carbohydrate, fat and protein metabolism as well as on ion transport and growth development. With regard to carbohydrate metabolism, insulin reduces the rate of release of glucose from l i v e r and increases the rate of uptake of glucose into a l l insulin-sensitive tissues, notably muscle and adipose tissue. As for l i p i d metabolism, insulin reduces the rate of release of free fatty acids from adipose tissue and stimulates de novo fatty acid synthesis and also the conversion of fatty acids to triglycerides in l i v e r . In protein metabolism, insulin stimulates transport of free amino acids across the plasma membrane in l i v e r and muscle; as well, i t stimulates protein biosynthesis and reduces release of amino acid from muscle (Steinberg, 1985). A l l of the metabolic effects of insulin are initiated by the interaction of the insulin molecule with a highly specific receptor on the plasma membrane (Stadie et a l . , 1953; Kohn et a l . , 1981). The insulin receptor has been partially purified and characterized (reviewed by Czech, 1981). It i s a membrane protein of high molecular weight (about 350,000) 3 made up of four disulfide-linked subunits: two identical alpha chains of about 125,000 daltons and two beta chains of 45,000-90,000 daltons. It i s clear that insulin receptor activation provokes increases in tyrosine kinase activity and autophosphorylation of the insulin receptor (Kahn and White, 1988), but subsequent molecular events that couple the insulin-receptor interaction to the regulation of cellular metabolism remain uncertain. Recent work by Sa l t i e l et al.(1986) has indicated that inositol phosphate-glycan, produced by the insulin-sensitive hydrolysis of a glycosyl-phosphatidylinositol in the plasma membrane, may be involved in the signal transduction system of insulin. This hydrolysis reaction, which is catalyzed by a specific phospholipase C, also results in the production of 1,2-diacylglycerol that may regulate protein kinase C. Both inositol phosphate-glycan and protein kinase C may mediate the effects of insulin. Diabetes mellitus comprises an etiologically and c l i n i c a l l y heterogeneous group of disorders characterized by an absolute or relative deficiency of insulin and subsequent disorders in the metabolism of carbohydrate, fat and protein. These abnormalities account for both the acute and chronic complications of the disease. According to the World Health Organization (WHO) classification adopted in 1985 (WHO Study Group, 1985), diabetes mellitus and a l l i e d categories of glucose intolerance are classified and characterized as shown in 4 Table 1 (Brunner and Suddarth, 1988). As indicated, two common types of diabetes mellitus are insulin-dependent diabetes mellitus (IDDM) (type I) and non-insulin-dependent diabetes mellitus (NIDDM) (type I I ) . 1.1.1. Insulin-Dependent Diabetes M e l l i t u s (IDDM). Insulin-dependent diabetes mellitus i s clearly associated with an absolute deficiency of insulin secretion. This marked decrease in insulin secretory capacity i s due to a specific loss of pancreatic B c e l l mass (Kahn, 1985). The etiology of IDDM i s not completely understood. Genetic predisposition, viruses and/or autoimmune factors have been suggested to be involved in the pathogenesis of disease (Craig, 1980). Genetically, a significantly increased frequency of certain human leukocyte antigens (HLA) has been recognized among type I diabetes (Cudworth and Woodrow, 1975; Nerup, 1978). It has been suggested that the antigens are determinants of the molecular nature of some beta c e l l surface proteins, some of which may have unique defects that render the c e l l more susceptible to certain insults (Nerup, 1978). The characteristically abrupt appearance of type I diabetes could also be the result of an infection with a diabetogenic virus. A variant of Coxsackie B4 virus has been identified as being able to damage the pancreatic beta ce l l s (Yoon et a l . , 1979). A large number of experiments carried out in animal models also seem to implicate viruses Table 1. C l a s s i f i c a t i o n of diabetes mel l i tus and a l l i e d categories of glucose into lerance. 5 Category C l i n i c a l Charac te r is t ics CLINICAL CLASSES DIABETES-MELLITUS (DM) Insulin-dependent diabetes mel l i tus (IDDM) (5-102 of a l l diabetes) Any age but usual ly young Mostly thin at diagnosis Causes nay be genet ic , v i r a l or abnormal immune responses Often have I s le t c e l l antibodies L i t t l e or no endogenous i n s u l i n Need i n s u l i n to preserve l i f e Ketosis-prone Non-insulin-dependent diabetes mel l i tus (NIDDM) (90-952 of a l l diabetes) Non-Obese - 202 of NIDDM Obese - 802 of NIDDM Any age, usual ly over -40 but occas iona l ly under 21 Mostly obese at diagnosis Causes may be genet ic , obesi ty or environmental fac tors No i s l e t c e l l ant ibodies Varying amounts of endogenous i n s u l i n present, often higher than normal l eve ls May need i n s u l i n to avoid hyperglycemia Rare k e t o s i s , except i n s t ress or i n f e c t i o n Non-ketotic hyperosmolar coma Malnut r i t ion- re la ted diabetes mel l i tus (MRDM) (a) F ibrocalculous pancreatic diabetes Any age but usual ly below 30 Mostly th in Causes may be high cassava intake combined with inadequate prote in intake Require i n s u l i n Absence of ketos is (b) P ro te in -de f i c ien t pancreat ic diabetes Any age but usua l ly below 35 Extreme degrees o f wasting and emaciation Causes may be malnutr i t ion P a r t i a l res istance to i n s u l i n Absence of ketos is Other types of diabetes associated with ce r ta in condit ions and syndromes; pancreat ic d isease, disease of hormone e t io logy , drug or chemical-induced condi t ions , abnormalit ies of i n s u l i n or i t s receptors , . cer ta in genetic syndromes, e tc . IMPAIRED GLUCOSE TOLERANCE (IGT) (non-obese, obese, or associated with cer ta in condi t ions or syndromes) Blood glucose l e v e l s between normal and that of diabetes Above normal s u s c e p t i b i l i t y to a therosc lero t ic disease Renal and r e t i n a l complications usual ly not s i g n i f i c a n t GESTATIONAL DIABETES MELLITUS (GDM) Begins or i s recognized during or a f te r pregnancy Above normal r i s k of per ina ta l complications Glucose intolerance t rans i tory but frequently recurs 502 go on to develop overt diabetes within 15 years 802 go on to develop overt diabetes a f te r 20 years B. STATISTICAL RISK CLASSES (Subjects with normal glucose tolerance but substant ia l ly increased r i sk of developing diabetes. ) PREVIOUS ABNORMALITY OF GLUCOSE TOLERANCE POTENTIAL ABNORMALITY OF GLUCOSE TOLERANCE 6 in the pathogenesis of diabetes (Craighead, 1975; Yoon and Notkins, 1983). The viruses may cause direct cytolytic infection of pancreatic beta ce l l s or trigger autoimmune responses (Yoon et a l . , 1987). Autoimmune responses may also be important in the type I diabetes. Circulating i s l e t c e l l antibodies capable of destroying beta c e l l s in the pancreatic i s l e t s are found in type I diabetics (Lernmark et a l . , 1978). In a l l , type I diabetes i s a heterogeneous group of disorders with varying etiologies. Viruses and autoimmunity may be the possible causes of a beta c e l l destruction in genetically predisposed individuals. 1.1.2. Non-Insulin-Dependent Diabetes M e l l i t u s (NIDDM). Non-insulin-dependent diabetes mellitus accounts for 90-95% of the diabetic population. A large number (between 60 and 90%) of those having NIDDM are obese and exhibit hyperinsulinism and associated insulin resistance. They usually have considerable preservation of the beta c e l l mass (Maclean and Ogilvie, 1955; Westermark and Wilander, 1978). It has been suggested that, in type II diabetes, either a "post-receptor defect", a decreased a f f i n i t y of the receptors for insulin (Bar et a l . , 1978), or a "down regulation" of the insulin receptors as a consequence of permanantly increased levels of insulin (Kahn et a l . , 1977) might be possible factors in the pathogenesis of the disease. 7 1.2. P A T H O P H Y S I O L O G Y O F D I A B E T E S A N D A S S O C I A T E D H E A R T D I S E A S E . 1.2.1. P a t h o p h y s i o l o g y o f D i a b e t e s . Regardless of the etiology of diabetes mellitus, absolute or relative deficiency of insulin can result in abnormal metabolism of carbohydrate, l i p i d and protein, leading to a series of c l i n i c a l symptoms (Figure 1). Glucose uptake into muscle and adipose tissue, and glycogenesis in l i v e r and muscle are reduced. At the same time, glycogenolysis and gluconeogenesis increase. A l l of these changes result in markedly elevated blood glucose levels and glucosuria occurs when the blood glucose levels exceed the renal threshold for glucose. Increased amounts of glucose in the urine cause osmotic diuresis giving rise to the symptom of polyuria, followed by polydipsia and polyphagia due to metabolic imbalance. Lipids are mobilized resulting in hyperlipidemia. Insulin deficiency also causes the net breakdown of muscle protein and weight loss follows. These disturbances in carbohydrate, l i p i d and protein metabolism in diabetes are known to result in a large number of acute and chronic complications which affect various systems and organs. Acute complications include ketoacidosis (McGarry and Foster, 1977) and non-ketotic diabetic coma (Arieff and Caroll, 1972) (Figure 1), most of which are reversible with insulin therapy. Much more common now are chronic complications including retinopathy and 8 F i g u r e 1 Pathophysiological alterations in carbohydrate, l i p i d and protein metabolism in diabetes (modified from Steinberg, 1985) . INSULIN DEFICIENCY J.Glucose Uptake G^lycogenesH ^ Glycoqenolysls fGluconeogenesis T Activity of Hormone-sensitive Lipase .[.Rate of Free Fatty Acids (FFA) Re-Estertf1caUon Hyperglycemia Glya :osur1a Y > -Polyuria Y Dehydration ana Electrolyte Depletion f Hypovolemia Y Hypotension Y Por Tissue Perfusion Coma Polyphagia and Polydipsia Lactlcactdenla fPlasma FA t ?Hepatic FA Uptake Triglyceride Synthesis \ Fatty Liver 4* Lipoprotein Lipase 4. Very LOK Oenslty Lipoproteins (VLDl) and Chylomicron Clearance f VLDL Secretion I Llpemla Loss of Stimulus for Protein Synthesis Decreased Rate of Amino Acid Transport Net Protein. Breakdown Weight Uss Muscle Hastlno Amlnoacldentia Gluconeoqenesls Urinary K+ and Urea 10 cataract formation which may lead to blindness in patients with long-standing diabetes (Davis, 1974), nephropathy (Balodimos, 1971), neuropathy (Sibley, 1982), an increased incidence of infection (Brunner and Suddarth, 1988), and cardiovascular diseases. 1.2.2. Heart Disease and Diabetes. Cardiovascular disease represents one of the major complications of the diabetic state. C l i n i c a l studies have shown that the incidence of cardiovascular disease i s two to three fold greater among diabetics than among non-diabetics (Kannel and McGee, 1979) , and i t now accounts for approximately 80% of a l l diabetic deaths (Kannel, 1978). The pathogenesis of cardiac disease in diabetes i s uncertain. It i s often associated with an increase in atherosclerosis but i t may also be due to a combination of factors including microangiopathy, macroangiopathy and autonomic neuropathy (Ledet et a l . , 1979). Recently, i t has become apparent that cardiac disease associated with diabetes i s not always associated with the above factors since they do not occur in a significant number of diabetic patients, and various studies have now suggested that a cardiomyopathy may also be a causal factor in producing the increase in morbidity and mortality of diabetics. For example, c l i n i c a l l y and pathologically, Hamby et a l . (1974) reported l e f t ventricular dilation and hypertrophy in the 11 absence of large coronary artery disease or systemic hypertension in 16 diabetic patients. A shorter l e f t ventricular ejection time and a longer pre-ejection period were shown in diabetic subjects without myocardial ischemia or other cardiovascular disease (Ahmed et a l . , 1975). Post-mortem examination by Regan et a l . (1977) showed nine out of eleven diabetics had no significant coronary artery disease, although six had had heart failure at death. In experimental animal studies, a primary myocardial abnormality in diabetes was f i r s t shown in dog, a species known to have a low incidence of spontaneous arterial disease (Regan et a l . , 1974). The end-diastolic volume and the stroke volume response were significantly less in eleven-month alloxan-diabetic dogs, associated with a two-fold greater end-diastolic pressure increment, suggesting a decreased ventricular compliance in the absence of coronary artery disease. Similar abnormalities in ventricular function were reported in 18-month diabetic rhesus monkeys (Haider et a l . , 1978). Fein et al.(1985) examined papillary muscle function in rabbits made diabetic with alloxan and demonstrated a markedly prolonged duration of isometric and isotonic contraction and relaxation. Studies with rats also showed a slowing of relaxation as well as a depression of shortening velocity in l e f t ventricular papillary muscles from severely diabetic rats (Fein et a l . , 1980). Using the isolated working heart preparation, a decreased rate of contraction and relaxation at higher f i l l i n g pressures 12 (Penpargkul et a l . , 1980; Vadlamudi et a l . , 1982) and afterloads (Ingebretsen et a l . , 1980) have been reported in streptozotocin- or alloxan-diabetic rats. Although these c l i n i c a l and experimental data have strongly suggested that diabetes mellitus i s associated with the development of a cardiomyopathy—diabetic cardiomyopathy, the mechanism(s) involved are s t i l l unclear. However, increasing evidence suggests that the cardiomyopathy associated with diabetes may be due to an alteration of myocardial enzyme systems and subcellular organelles such as sarcolemma, sarcoplasmic reticulum, mitochondria, contractile proteins as well as receptors. 1.2.2.1. Sarcolemma. The contraction and relaxation cycle of the heart i s generally viewed as the consequence of raising and lowering the intracellular concentration of free calcium. Ca2 + movements in the cardiac c e l l can be modified at a number of sites: sarcolemma (Langer, 1984), sarcoplasmic reticulum (Fabiato, 1983), mitochondria (Carafoli, 1985), and at myofilaments (Fabiato and Fabiato, 1975; Lab et a l . , 1984). The sarcolemma, a surface membrane of the c e l l , i s composed of the glycocalyx (the basement membrane) and the plasmalemma. The glycocalyx i s the exterior layer of the sarcolemma and contains polysaccharides which may be associated with l i p i d s (glycolipids) or with proteins (glycoproteins). S i a l i c acid residues, located at the ends 13 of short branched carbohydrate chains attached to proteins, are the site for calcium binding at the basement membrane. The plasmalemma, on the other hand, i s a bilayered structure and contains phospholipids (Nayler et a l . , 1984). In addition to their role as major structural constituents of c e l l membranes, phospholipids bind large quantities of calcium (Philipson et a l . , 1980). Several enzymatic and nonenzymatic systems are also associated with this l i p i d bilayer which, together with phospholipids, are involved, either directly or indirectly, in the regulation of myocardial calcium transport and contractility. These include Na+-K+-ATPase, Ca2+-Mg2+-ATPase (Ca2+-pump), adenylate cyclase, Na+-Ca2+-exchanger (Schwartz, 1974; Dhalla et a l . , 1978; Caroni and Carafoli, 1981; Philipson et a l . , 1982). The membrane-bound enzymes are also involved in transmembrane methylation of phospholipids (Strittmatter et a l . , 1981), which alters membrane f l u i d i t y and, in turn, influences several enzyme systems (Hirata and Axelrod, 1980). Thus, both glycocalyx and plasmalemma bind large quantities of calcium which are believed to play a strategic role in the beat-to-beat control of the heart. When the cardiac c e l l i s depolarized, there i s an influx of calcium through the sarcolemma as well as a release of calcium from sarcolemmal stores, which results in contraction. Relaxation, on the other hand, i s partly achieved by calcium efflux through the sarcolemma (Dhalla et a l . , 1978). 14 In diabetic myocardium, defects in sarcolemmal enzymatic ac t i v i t i e s and composition have been observed. A depression of Na+-K+-ATPase activity was reported in STZ-diabetic hearts (Ku and Sellers, 1982; Pierce and Dhalla, 1983). Decreased Na+-Ca2+-exchanger (Makino et a l . , 1987) and Ca2+-pump act i v i t i e s (Heyliger et a l . , 1987) have also been shown. It i s possible that the changes in enzyme act i v i t i e s as well as ion transport systems associated with cardiac sarcolemma during diabetes might be par t i a l l y induced by phospholipid alteration. In fact, phospholipid methylation has been reported to be depressed in the diabetic rat heart (Ganguly et a l . , 1984). Also a decrease in the s i a l i c acid residues of the sarcolemma has been shown (Pierce et a l . , 1983). Parallel with these changes, a significant loss in the a b i l i t y of the cardiac sarcolemma to bind calcium was found in chronic STZ-diabetic rats (Pierce et a l . , 1983). These alterations in the composition and enzyme ac t i v i t i e s of the sarcolemma could change i t s a b i l i t y to transport calcium effectively and the cardiac contraction and relaxation process may thus be altered. 1 . 2 . 2 . 2 . S a r c o p l a s m i c r e t i c u l u m . Sarcoplasmic reticulum (SR) i s an intracellular organelle and occupies about 2% of each ventricular myocyte (David et a l . , 1979). Ca2 + transport by SR i s another major mechanism by which intracellular Ca2 + levels and thereby contractility of the heart are regulated. This i s 15 accompanied by a K -sensitive Ca^ -dependent ATPase on the SR membrane (Maclennan, 1970), which represents up to 90% of the total protein content of the membrane and which transports Ca2 + from the cytoplasmic space into the vesicles of SR with a high velocity and a f f i n i t y (Chamberlain et a l . , 1983) and thus i s involved in the relaxation of the heart. The Ca2+-dependent ATPase appears to be regulated either directly via ionic control (such as K+) at allo s t e r i c sites (Jones et a l . , 1977) or indirectly through phosphorylation of phospholamban, the protein closely associated with the ATPase protein in the membrane, by calmodulin (Katz, 1980) and/or cAMP-dependent protein kinase (LaRaia and Morkin, 1974). In the diabetic rat heart, the a b i l i t y of the SR to transport calcium i s impaired (Penpargkul et a l . , 1981; Lopaschuk et a l . , 1983a; Ganguly et a l . , 1983). This may explain the impairment of relaxation in the diabetic heart. As a consequence of this lowered uptake of Ca , the amount of calcium available for release during the following beats may be lower than normal which may cause defects in contraction. This calcium-transport defect may be due to biochemical changes in the diabetic heart (Lopaschuk et a l . , 1983b). 1 . 2 . 2 . 3 . M i t o c h o n d r i a . Mitochondria occupy a large portion (33%) of each myocyte (David et a l . , 1979) and are the chief source of the 16 myocardial energy (as ATP) that the cel l s need to survive and function. This energy can be distributed to a number of energy-utilizing systems of the c e l l , such as Na+-K+-ATPase and the Ca2+-pump of sarcolemma and Ca2+-dependent ATPase of sarcoplasmic reticulum. Besides generation of ATP as i t s main function, cardiac mitochondria have also been reported to actively accumulate calcium (Carafoli and Crompton, 1978). It i s generally believed that mitochondrial calcium transport does not play a major role in the regulation of cytoplasmic calcium during the contraction-relaxation cycle. It may, however, help to relax the myocardium when intracellular Ca* concentrations become extremely high or in certain pathological states such as myocardial ischemia (Katz, 1977). Studies have also revealed a Na+-induced 2 + Ca*" -release as well as spontaneous calcium release from mitochondria (Goring et a l . , 1977), though the physiological significance of this calcium release has not been established. A depression in calcium uptake activity was observed in the diabetic mitochondria (Pierce and Dhalla, 1985). A defect in phosphorylation of creatine in the presence of succinate or malate was also shown in alloxan-diabetic heart (Goranson and Erulker, 1949) which may lead to a reduction in ATP levels (Haugaard and Haugaard, 1964; Pieper and Murray, 1987). Both low tissue ATP levels and intracellular calcium accumulation may eventually impair normal cellular 17 function and may thus be involved in the pathogenesis of cardiomyopathy during diabetes. 1 .2 .2 .4 . C o n t r a c t i l e p r o t e i n s . The contractile apparatus of the cardiac c e l l consists of myofilaments. The thick filament i s composed of myosin molecules. Myosin has the a b i l i t y to s p l i t ATP, i.e., i t acts as an ATPase. When myosin combines with actin, i t forms an actomyosin complex that i s enzymatically even more active in i t s a b i l i t y to s p l i t ATP. Myosin i t s e l f can be separated into three isozyme components—, V 2 , V 3 , among which the V]_ form i s thought to be the fastest of the myosin isoenzymes with regard to ATP hydrolysis as well as cross-bridge formation. The thin filament i s composed of actin molecules (Braunwald et a l . , 1984). A number of studies have shown a diabetes-induced depression of myosin and actomyosin ATPase activity in rat hearts (Dillman, 1980; Malhotra et a l . , 1981; Pierce and Dhalla, 1981; Garber et a l . , 1983). This depression was suggested to be due to altered myosin isoenzyme distribution. In non-diabetic rats, myosin ATPase i s present predominantly in the most active form (about 72% of the total) and about 13% in the V 3 form (slowest form) . In diabetic rat hearts, however, the V3 form predominates while the content i s low (Dillman. 1980). Depression of myosin ATPase activity has also been demonstrated in the alloxan-diabetic rabbit (Bhimji et a l . , 1985). However, 18 normal rabbits predominantly have the V 3 form, so the mechanism for depression of myosin ATPase would be different from that seen in rats. Various studies have demonstrated the presence of hypothyroidism in diabetic patients and animals (Dillman, 1980; Pittman et a l . , 1979). The shift of myosin ATPase isoenzymes and depression of myosin ATPase are thought to occur as result of the diabetes-induced hypothyroidism (Yazaki and Raben, 1975; Flink et a l . , 1979; Dillmann, 1982). Since activity of myosin ATPase i s known to correlate closely with contractility (Scheuer and Bhan, 1979), i t can be hypothesized that diabetes induces depression of myosin ATPase in the heart which then leads to altered contractility. 1 . 2 . 2 . 5 . R e c e p t o r s . Evidence currently available indicates that induction of diabetes has a profound effect on myocardial muscarinic and adrenergic receptor mediated events. With regard to muscarinic receptor responses, c l i n i c a l l y , persistent resting tachycardia (Rundles, 1945; Wheeler and Watkins, 1973) and loss of beat-to-beat variation (sinus arrhythmia) during deep breathing (Lloyd-Mostyn and Watkins, 1975; Feldman, 1981) are some of the characteristic symptoms of defective vagal control of the heart in diabetic patients. Partial and occasionally total vagal denervation of the heart has been encountered in chronic diabetic patients (Wheeler and Watkins, 1973). Pathological changes such as 19 severe loss of myelinated axons and excess deposition of collagen have been demonstrated in the vagus nerve during postmortem examination of diabetic patients (Duchen et a l . , 1980). The defective parasympathetic (vagal) control i s an early detectable feature of cardiac autonomic neuropathy in diabetics, which i s , at least p a r t i a l l y , responsible for disturbances in the cardiovascular system in diabetics. Experimentally, an i n i t i a l (up to 100 days of STZ or alloxan-diabetes) reduction in sensitivity to carbachol was noted followed by a supersensitivity to carbachol which lasted up to 360 days of diabetes (Vadlamudi and McNeill, 1983a). This supersensitivity in long-term diabetics has also been reported by Tomlinson and Yusof (1981) who showed that isolated l e f t atria from 7- to 8-month alloxan-diabetic rats were supersensitive to the negative inotropic effect of acetylcholine. Defective parasympathetic innervation of the heart could lead to a lack of the neurotransmitter acetylcholine and to the development of postjunctional supersensitivity to exogeneously administered cholinergic drugs. The myocardial beta-adrenergic receptor system is also affected by the induction of diabetes. However, reports concerning beta-adrenergic effects in the experimentally-induced diabetic heart are conflicting. Foy and Lucas (1978) have demonstrated that the inotropic responses to isoproterenol were reduced while the chronotropic responses to isoproterenol increased in isolated spontaneously beating 20 atria from 2-week STZ diabetic rats. Ramanadham and Tenner (1986) also showed a decreased sensitivity of ventricular tissue to the inotropic effects of isoproterenol in chronic diabetic rats. However, Ingebretsen et a l . (1980) reported no change in the inotropic response of isolated perfused working hearts from acute alloxan-diabetic rats to isoproterenol. Furthermore, results from Vadlamudi and McNeill (1984) showed that the positive inotropic effect of isoproterenol was not altered in isolated working hearts from alloxan- or STZ-diabetic rats at any time points after induction of the disease. Apart from producing a positive inotropic effect, beta-agonists are known to exert a profound relaxing effect on the heart. In this aspect, maximum changes produced in -dP/dT by isoproterenol were depressed in diabetic rat hearts at various time points after the induction of the disease (Vadlamudi and McNeill, 1984). The changes in the beta-adrenergic system in diabetes also include decreases in beta-adrenoceptor density (Savarese and Berkowitz, 1979) and decreases in isoproterenol stimulated adenylate cyclase activity (Atkins et a l . , 1985). The responses of the cardiac cyclic AMP system to isoproterenol have been reported to be decreased in acute diabetic rats (Ingebretsen et a l . , 1981) or to be unaltered in both acute and chronic diabetic rats (Vadlamudi and McNeill, 1983b). 21 1.3. C A R D I A C A L P H A 2 - A D R E N O C E P T O R S A N D P H O S P H O I N O S I T I D E M E T A B O L I S M . 1.3.1. R e c e p t o r - s t i m u l a t e d P h o s p h o i n o s i t i d e s M e t a b o l i s m . Over thirty years have now passed since the original report by Hokin and Hokin (1953) of receptor-stimulated turnover of inositol l i p i d s . Since then, the phosphoinositides have enjoyed periods of interest, neglect, controversy and f i n a l l y acceptance as important intermediaries in biological signalling processes in a wide variety of systems. The pivotal ' contributions which resulted in this tumultuous history came from a number of laboratories. The hypothesis (Michell, 1975) that the phosphoinositides somehow served to couple receptors to cellular calcium mobilization provoked considerable research and criticism. However, further progress in understanding the exact role of inositol l i p i d turnover in receptor mechanisms was hindered by an imprecise knowledge of the biochemical pathway involved. Only in the early 1980's did the experimental evidence begin to indicate that the i n i t i a l reaction in stimulated phosphoinositide turnover was the breakdown of not the major known inositide, phosphatidylinositol, but rather a minor phosphorylated derivative, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] (Abdel-Latif et a l . , 1977; Kirk et a l . , 1981). Berridge (1983) realized that the water soluble product of this reaction, D-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], was a li k e l y candidate for a second 22 messenger to activate the release of Ca^ from intracellular stores. Shortly, Berridge and others (Streb et a l . , 1983) demonstrated that this molecule in fact had the predicted biological activity; Ins(l,4,5)P3 in micromolar concentration rapidly released Ca2 + from a non-mitochondrial store in permeabilized pancreatic acinar c e l l s . This result was quickly confirmed in a number of different laboratories (Berridge, 1986). A parallel story evolved from the work in Nishizuka's laboratory (Nishizuka, 1983; Nishizuka, 1984a; Nishizuka, 1984b) which demonstrated that the other product of inositol l i p i d breakdown, diacylglycerol (DG) was also a cellular messenger; this intermediate of phospholipid metabolism was shown to be a potent and specific activator of a ubiquitous protein kinase which Nishizuka designated as C-kinase. Ptdlns (4, 5) P2 i s hydrolysed by phospholipase C under the stimulation of receptors to form Ins(1,4,5)P3 and DG. There are indications that receptor coupling to phospholipase C might involve a G-protein mechanism similar to the adenylate cyclase system. In e l e c t r i c a l l y permeabilized platelets, the effects of thrombin on secretion were potentiated by guanine nucleotides (Haslam and Davidson, 1984). More recently, several studies have demonstrated an activation by guanine nucleotides of phospholipase C in membranes or in permeable cel l s (Cockcroft and Gomperts, 1985; Smith et a l . , 1986). Further, activation of phospholipase C shows the same 23 relative sensitivity to guanine nucleotide analogs as for regulation of adenylate cyclase: GTP-gamma-S>GppNHp>GTP (Merritt et a l . , 1986). In many systems, receptor activation of phospholipase C i s potently inhibited by pertussis toxin (Ui, 1986). However, phospholipase C-linked receptors have been shown to be insensitive to pertussis toxin in the exocrine pancreas (Merritt et a l . , 1986). These data suggest that the coupling of receptors to phospholipase C involves a guanine nucleotide dependent regulatory protein which i s similar, but not identical to the proteins which regulate adenylate cyclase. The differential sensitivity to pertussis toxin may indicate that different G-proteins are involved in the regulation of inositol l i p i d metabolism in different systems. Only six years ago, the formation of inositol t r i s - and bisphosphates in response to the activation of receptors was demonstrated (Berridge et a l . , 1983). At that time a rather simple biochemical pathway was described involving the sequential dephosphorylation of Ins(1,4,5)P3 to D-inositol 1,4-bisphosphate [Ins(l,4)P2], to D-inositol 1-phosphate [Ins(l)P] and fi n a l l y to free inositol by a lithium-sensitive inositol 1-phosphatase. Today, the picture of inositol phosphate metabolism i s quite complex (Figure 2) , which i s largely due to the power of high performance liquid chromatography (HPLC) analyticl procedures that can separate inositol phosphates with only subtle structural differences. Ins(l,4,5)P3 i s dephosphorylated by an extremely active 5-24 F i g u r e 2 Inositol phospholipid metabolism. DG, diacylglycerol (1-stearoyl 2-arachidonyl-sn-glycerol); PA, phosphatidic acid; CDP-DG, cytidine-5'-diphospho-l,2-diacyl-sn-glycerol; Ptdlns(4,5) P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; Ptdlns, phosphatidylinositol; Ins(l:2cyc,4,5)P3, D-inositol 1,2-cyclic-4,5-trisphosphate; Ins(l,2cyc,4)P2, D-inositol 1,2-cyclic-4-bisphosphate; Ins(1,2cyc)P, D-inositol 1,2-cyclic phosphate; Ins(l)P, D-inositol 1-phosphate; Ins(l,4)P2, D-inositol 1,4-bisphosphate; Ins(4)P, D-inositol 4-phosphate; Inositol, myo-inositol; Ins(1,4,5)P3, D-inositol 1,4,5-trisphosphate; Ins(l,3,4,5)P4, D-inositol 1,3,4,5-tetrakisphosphate; Ins(1,3,4)P3, D-inositol 1,3,4-trisphosphate; Ins(3,4)P2, D-inositol 3,4-bisphosphate; Ins(3)P, D-inositol 3-phosphate; Ins(1,2,3,4,5,6)Pg, inositol 1,2,3,4,5,6-hexakisphosphate; Ins(1,3,4,5,6)P5, inositol 1,3,4,5,6-pentakisphosphate; Ins(1,3,4,6)P4, D-inositol 1,3,4,6-tetrakisphosphate; Ins(l,3)P2, D-inositol 1,3-bisphosphate. 26 phosphatase to Ins(l,4)P2 (Downes et a l . , 1982). Ins(l,4)P2 thus formed i s dephosphorylated almost exclusively to D-inositol 4-phosphate [Ins(4)P] by a relatively non-specific inositol polyphosphate 1-phosphatase (Inhorn et a l . , 1987). In addition to the dephosphorylation of Ins(l,4,5)P3 by the 5-phosphatase, there exists in most tissues thus far examined a 3-kinase which transfers a phosphate from ATP to the 3 position of Ins(l,4,5)P3 to form D-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] (Irvine et a l . , 1986). This molecule i s then dephosphorylated by, probably, the same 5-phosphatase which degrades Ins(1,4,5)P3 to form an isomeric inositol trisphosphate, D-inositol 1,3,4-trisphosphate [Ins(l,3,4)P3]. Ins(l,3,4)P3 i s then dephosphorylated by the inositol polyphosphate 1-phosphatase to D-inositol 3,4-bisphosphate [Ins(3,4)P2] (Inhorn et a l . , 1987), and to a lesser extent to D-inositol 1,3-bisphosphate [Ins(l,3)P2] (Bansal et a l . , 1987) by an enzyme that has not been well characterized. These bisphosphates are then dephosphorylated primarily to a mixture of Ins(l)P and D-inositol 3-phosphate [Ins(3)P]. The complexity of this metabolic pathway suggests that some biological function may be regulated by one of these metabolites. For example, Ins(1,3,4,5)P4 has been shown to either potentiate the action of Ins(1,4,5)P3 or modulate Ca2 + entry (Irvine and Moor, 1986; Morris et a l . , 1987). The metabolism of the inositol phosphates i s further complicated by the demonstration that in vitro, the soluble 27 product of phospholipase C action on Ptdlns (4,5) P2 i s a mixture of Ins(1,4,5)P3 and i t s cyclic derivative D-inositol 1,2-cyclic-4,5-trisphosphate [Ins(1:2cyc,4,5)P3] (Connolly et a l . , 1986). In platelets, Ins(l:2cyc,4,5)P3 can also mobilize intracellular Ca2 + with about the same potency as the non-cyclic variety (Wilson et a l . , 1985). Ins(l:2cyc,4,5)P3 i s not a substrate for the 3-kinase, and is only slowly dephosphorylated by the 5-phosphatase (Connolly et a l . , 1987). Thus, i f formed in vivo, this compound might cause persistent activation of Ca2 +-mobilization. Reports on the production of Ins(1:2cyc,4,5)P3 in cel l s are somewhat conflicting; i t appears that on brief stimulation l i t t l e of the cyclic derivative i s formed (Hawkins et a l . , 1987), but i t accumulates on prolonged stimulation (Sekar et a l . , 1987) probably due to i t s slow metabolism. Estimation from kinetics of Ins(l,4,5)P3 and Ins(1:2cyc,4,5)P3 turnover in parotid gland suggest that as l i t t l e as 1% of the Ptdlns (4, 5) P2 phospholipase C product may be cyclic (Hughes et a l . , 1988). Although Ins(1:2cyc,4,5)P3 can mobilize Ca2 + (Wilson et a l . , 1985), i t s potency i s probably at least an order of magnitude less than Ins(l,4,5)P3 (Willcocks et a l . , 1989). In addition, the cellular turnover of Ins(1:2cyc,4,5) P3 i s so slow in the parotid gland that i t s role in regulating cellular calcium metabolism i s doubtful. Inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P5] and inositol 1,2,3,4,5,6-hexakisphosphate 28 [Ins(1,2,3,4,5,6)Pg] are also present in most mammalian c e l l s , but their levels do not change noticeably on stimulation (Heslop et a l . , 1985). The inositol phospholipid metabolism pathway i s stimulated by a variety of receptors. Most of these receptors are Ca2 + mobilizing receptors, such as muscarinic, alphai-adrenergic, histamine (Hi), serotonin (5-H2), peptidergic [vasopressin(Vi), angiotensin I I , bradykinin, substance P and f-methionyl-leucyl-phenylalanine], thrombin, thyrotropin-releasing hormone, glucose, platelet-activating factor, l i g h t , etc. Recent findings suggest that certain receptors which do not function through Ca2 + mobilization can lead to Ptdlns(4,5)P2 hydrolysis. These receptors are linked to ion channels. Examples of this class of receptors are the nicotinic acetylcholine receptor, a cation channel, and receptors of glutamate which control chloride channels. The receptor-coupled phosphoinositide breakdown occurs in a wide variety of tissues, including: (a) those which are excitable, e.g., smooth muscle, brain, and sympathetic ganglion; (b) those which are nonexcitable, e.g., l i v e r hepatocytes, platelets, and neutrophils; and (c) those which are either endocrine, e.g., adrenal medulla and pineal, or exocrine, e.g., pancreas and parotids. Most agents that enhance phosphoinositide turnover also increase release of arachidonic acid (AA) and subsequent synthesis of cyclooxygenase and lipoxygenase products— eicosanoids (prostaglandins, thromboxane, leukotrienes, 29 etc.). The biosynthesis pathway of the products of AA i s summarized in Figure 3. 1 . 3 . 2 . M y o c a r d i a l A l p h a ^ a d r e n o c e p t o r s a n d T h e i r P o s i t i v e I n o t r o p i c E f f e c t o n t h e H e a r t . Both in vivo and in vitro pharmacologic experiments have demonstrated the presence of alpha-adrenoceptors in the myocardium (Benfey, 1973; Williams and Lefkowitz, 1978; Levy and Martin, 1979). The physiological role of alpha-adrenoceptors in control of cardiac function i s , however, unclear. Mammalian cardiac tissues have pre- and post-junctional alpha-adrenoceptors (Benfey, 1980). There i s evidence that pre-junctional alpha2-adrenoceptors modulate cardiac noradrenergic transmission (Docherty and McGrath, 1979; Drew, 1980), and that post-junctional alpha--adrenoceptors may alter the inotropic responses of the heart. In isolated cardiac tissue (atria or ventricle) from various species (guinea pig, rabbit, rat, cat, dog, human, etc.), activation of post-junctional alphai-adrenoceptors produces positive inotropic responses (Wagner and Brodde, 1978; Endoh, 1982). In general, alpha-adrenoceptor stimulation has no effect on chronotropic response under normal conditions (Wagner and Brodde, 1978). The alphai-adrenoceptor-mediated positive inotropic effect i s characterized by a slower rate of increase in contraction and relaxation compared with that mediated by beta-adrenoceptors (Osnes et a l . , 1985). Unlike the beta-3 0 F i g u r e 3 Biosynthesis of the products of arachidonic acid. 12-HPETE, 12-hydroperoxyeicosatetraenoic acid; 12-HETE, 12-hydroxy eicosatetraenoic acid; PGI2, prostacyclin; 6-Keto PGFia , 6-keto prostacyclin Fa^ ; PGH2, prostaglandin H2; PGE2, prostaglandin E2; PGD2, prostaglandin D2; PGF2cx , prostaglandin F2a /' HHT, 17-carbon hydroxy acid; TXA2, thromboxane A2; TXB2, thromboxane B2. Arachidonic Acid 32 adrenoceptor-mediated effect, the alphai-adrenoceptor-mediated effect i s not associated with shortening of the time-to-peak tension and relaxation time, and the duration of contraction may be prolonged, as seen in heart preparations of rats and guinea pigs (Bruckner et a l . , 1978; Osnes et a l . , 1978). In contrast to beta-adrenoceptor stimulation, the alphai-adrenoceptor-mediated effect occurs only when the rate of beating i s relatively slow (Hamakawa et a l . , 1973). Besides, alphai-adrenoceptor stimulation does not alter adenylate cyclase activity or cyclic AMP concentration (Osnes and Oye, 1975; Osnes, 1978). Thus, the alphai-adrenoceptor-mediated positive inotropic effect observed in isolated heart preparations differs greatly from the beta-adrenoceptor mediated effect. In patients an alphai-adrenoceptor-mediated inotropic effect has not been described, but may be obtainable particularly in the presence of beta-adrenoceptor blockade. 1 . 3 . 3 . M y o c a r d i a l A l p h a i - a d r e n o c e p t o r A n d P h o s p h o i n o s i t i d e H y d r o l y s i s . The primary molecular mechanism involved in alpha^-adrenoceptor-induced positive inotropic effect i s largely unknown. It has, at least in part, been attributed to an increase in slow calcium inward current (Bruckner and Scholz, 1984), to an increased calcium sensitivity of the myofibrils (Endoh and Blinks, 1984; Blinks and Endoh, 1986) and to an increased PI turnover (Brown et a l . , 1985; Otani 33 et a l . , 1986; Poggioli et a l . , 1986; Schmitz et a l . , 1987a; Scholz et a l . , 1987; Scholz et a l . , 1988). Limited evidence further suggests that PIP2 i s a substrate, and that IP3 i s formed by activation of phospholipase C following alpha^-adrenoceptor stimulation. Hydrolysis of PIP2 produces two presumed intracellular second messengers, IP3, which releases calcium from intracellular stores and DG, which activates protein kinase C. However, the role of IP3 as an intracellular calcium mobilizing agent in skeletal muscle, as well as in cardiac muscle is a matter of controversy (Hirata et a l . , 1984; Movsesian et a l . , 1985; Scherer and Ferguson, 1985; Vergara et a l . , 1985; Volpe et a l . , 1985; Fabiato, 1986; Nosek et a l . , 1986). Protein kinase C is present in the heart (Kuo et a l . , 1980; Wise et a l , 1982b), and a variety of myofibrillar, SR, and plasma membrane proteins are putative substrates for this enzyme (Wise et a l . , 1982a; Katoh et a l . , 1983; Movsesian, 1984; Lim et a l . , 1985; Lindemann, 1986). It is not yet known whether alpha^-adrenergic stimulation in the heart activates protein kinase C to catalyze relevant protein phosphorylations. Also, there i s no direct evidence showing that diacylglycerol levels are increased in cardiac slices or myocytes exposed to alpha^-adrenergic agonists. An active metabolite of diacylglycerol i s arachidonic acid, which is a substrate of the enzyme cyclooxygenase to form prostaglandins (Nishizuka, 1984b). The involvement of these metabolites in alpha^-34 adrenergic stimulation in the heart i s also poorly understood. 1 . 3 . 4 . A l p h a - A d r e n o c e p t o r A g o n i s t s . In studies concerning alpha-adrenergic inotropic effects, phenylephrine, methoxamine, epinephrine and norepinephrine have often been used as agonists. Among them, phenylephrine has been mostly used since i t has a relatively weak beta-stimulating property compared to i t s alpha-adrenoceptor stimulating one (Furchgott, 1972; Osnes et a l . , 1973; Scholz, 1980). The small beta effect of phenylephrine i s easily overcome by a beta-receptor blocker like propranolol without interfering either with mechanical or electrophysiological effects of the alpha-stimulation (Osnes et a l . , 1973; Bruckner et a l . , 1978). Methoxamine, which i s devoid of detectable beta-receptor agonist ac t i v i t y , also evokes alpha-adrenoceptor-mediated inotropic effects (Endoh, 1982). But the alpha-effect of methoxamine seems to be somewhat atypical because i t sometimes develops very slowly compared to the alpha-effect of phenylephrine (Skomedal et a l . , 1982; Siegl and McNeill, 1980). Besides, methoxamine has a marked negative inotropic action at large concentrations (Siegl and McNeill, 1980). Both phenylephrine and methoxamine preferentially stimulate alphai-adrenoceptors compared to alpha2-adrenoceptors (Starke, 1981). 35 A prerequisite for implicating an alpha-adrenergic mechanism in heart i s that i t can be mimicked by the endogenous agonists epinephrine and norepinephrine. Both agonists e l i c i t alpha as well as beta inotropic effects in rat papillary muscles. For epinephrine, the maximal increase in contractility after alpha-stimulation (epinephrine in the presence of propranolol) was about 60% of the corresponding increase after beta-stimulation (epinephrine in the presence of prazosin), while for norepinephrine, they are of the same magnitude (Skomedal and Osnes, 1983). Epinephrine was slightly less potent at the alpha-receptors compared to the beta-receptors while norepinephrine was about 10 times less potent at the alpha-receptors compared to the beta-receptors. At alphai receptors, epinephrine i s equal to or more potent than norepinephrine (Skomedal et a l . , 1984). Moreover, norepinephrine i s the chemical mediator liberated by mammalian postganglionic adrenergic nerves and therefore mediates physiological and metabolic responses that follow stimulation of sympathetic nerves. For the reasons l i s t e d above, norepinephrine was employed in the present study. 1.3.5. Prazosin as a Selective Alphai-Adrenoceptor Blocker Alphai-adrenoceptor blockers are crucial agents in demonstrating an alpha-adrenoceptor-mediated effect. However, obvious d i f f i c u l t i e s exist when alpha-receptor blockers like phentolamine and phenoxybenzamine are used 36 since they possess properties in addition to alpha-adrenoceptor blockade (Iversen, 1973; Starke et a l . , 1971). Both substances were shown to inhibit neuronal and extraneuronal uptake mechanisms, and w i l l probably interfere with the presynaptic alpha2-adrenoceptors regulating neurotransmitter release (Langer, 1974; Starke and Endoh, 1976; Starke, 1981). These effects are most apparent for agonists that are most influenced by these mechanisms (eg. the endogenous catecholamines). As the endogenous catecholamines are very potent stimulators of the beta-adrenoceptors, a blockade of postsynaptic alpha-effects may be masked or even reversed by a 'potentiation' of the beta effects by these alpha-blockers. Compared with the alpha-blockers mentioned above, prazosin has been shown to have very high a f f i n i t y and selectivity for the alpha^-subtype of alpha-adrenoceptors (Davey and Massingham, 1976). By using prazosin, i t i s therefore possible to avoid interference by mechanisms other than the alphas-receptors. Thus, prazosin was employed in our study. 1 .4. C A R D I A C A L P H A i - A D R E N O C E P T O R S A N D P H O S P H O I N O S I T I D E M E T A B O L I S M I N D I A B E T E S . 1 .4 .1 . D i a b e t e s a n d C a r d i a c A l p h a - a d r e n o c e p t o r F u n c t i o n s . Cardiac adrenoceptors are affected in both number and function by diabetes. In acute diabetes, an enhanced inotropic response of diabetic atria (Canga and Sterin-37 Borda, 1986), ventricle (Wald et a l . , 1988) and isolated heart (Downing et a l . , 1983) to methoxamine has been reported accompanied by a reduced number of alpha-adrenoceptor binding sites and an increase in their a f f i n i t y constants (Wald et a l , 1988). Similarly in chronic diabetes, i t has been demonstrated that both inotropic and chronotropic responses to phenylephrine (Jackson et a l . , 1986) in the presence of propranolol, and methoxamine (Goyal et a l . , 1987) are enhanced in a t r i a . Recently, supersensitivity to phenylephrine in the ventricles of chronic diabetic animals was also shown in our lab (unpublished results). On the contrary, a decrease in the contractile force generation by methoxamine (Heyliger et a l . , 1982) and norepinephrine (Sunagawa et a l . , 1987) was observed in isolated papillary muscles and isolated hearts of chronic diabetic animals. The number of alpha-adrenoceptors was shown to be decreased in the ventricles of chronic diabetic rats (Heyliger et a l . , 1982; Latifpour and McNeill, 1984) without changes in the a f f i n i t y constants. 1.4.2. D i a b e t e s a n d C a r d i a c P h o s p h o i n o s i t i d e M e t a b o l i s m There are very limited data demonstrating that changes in alpha-adrenoceptor-stimulated phospholipid metabolism occur in diabetic heart. Wald et a l . (1988) have made some very interesting observations with regard to alpha-adrenoceptor-stimulated phosphoinositide hydrolysis in diabetic ventricles. Alpha-adrenoceptor supersensitivity 38 was shown in hearts from acute diabetic rats accompanied by an increase in receptor a f f i n i t y and a decrease in receptor number. Inhibition of phospholipase C by 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) blocked the ventricular response to methoxamine in non-diabetic as well as in diabetic hearts. Synthetic DG potentiated the inotropic action of the alpha-agonist in normal ventricles and increased the a f f i n i t y with a decreased number of alpha-adrenoceptor sites in normal ventricles, producing values of K<j and Bmax similar to those of the acute diabetic heart. Inhibitors of protein kinase C partially reduced the supersensitivity of alpha-agonists in diabetic ventricles and prevented the stimulatory action of DG upon the positive inotropic effect of methoxamine in normal ventricles. These results provide the f i r s t evidence that alpha-adrenoceptor-mediated inotropic stimulation i s secondary to receptor-mediated hydrolysis of phosphoinositides in diabetic heart as well as in normal heart. In the acute diabetic state, the supersensitivity to alpha-agonists could be due to high activity of phospholipase C (with an increase in DAG production and protein kinase C activation) which induces alterations in the membrane alpha-adrenergic receptors. This i s supported by data from Okumura et a l . (1988) who showed that 1,2-diacylglycerol content was significantly elevated in diabetic ventricles. Diacylglycerol i s metabolized to arachidonic acid which is the substrate required for cyclooxygenase to produce 39 prostacyclin. It i s therefore important to note that an increased prostacyclin release from perfused hearts of acutely diabetic rats has been reported (Rosen and Schror, 1980). In addition, an increased PI response coupled with alphai-adrenoceptors has been shown in cardiomyopathic hamsters (Iwakura et a l . , 1988). On the contrary, a decrease in the metabolites of the PI pathway has also been demonstrated. Rosen et a l . (1983) reported reduced prostacyclin synthesis by the diabetic rat heart. Bydlowski et a l . (1985) showed a decrease in the myocardial synthesis of prostacyclin in diabetic offspring. Also, reduced arachidonic acid levels have been observed in heart muscle of the diabetic rat (Holman et a l . , 1983; Gudbjarnason et a l . , 1987). Besides alphai-adrenoceptor, stimulation of muscarinic receptor in the heart i s also associated with an increase in phosphoinositide turnover (Brown and Brown, 1983; Brown et a l . , 1985). Recently, a few studies have reported the changes in muscarinic receptor-mediated phosphatidylinositol hydrolysis in diabetic animals. It was shown by Bergh et a l . (1988) that the incorporation of myo-inositol amounts to about 65% in acute diabetes and 80% in chronic diabetes compared to age-matched controls. They further studied muscarinic receptor-stimulated phosphatidylinositol breakdown in diabetes. It was observed that in the acute form of diabetes with ketosis, the release of IP^ was significantly reduced whereas in chronic diabetes no defects 40 in receptor-mediated coupling to the phosphoinositides were apparent. An important finding by Canga et al (1985) indicated that during diabetes arachidonic acid metabolism in the heart i s shifted towards thromboxanes and lipoxygenase metabolites, while in normal atria i t i s directed towards prostaglandin production. Since these substances affect cardiac function the authors examined the response to methoxamine in the presence and absence of inhibitors of thromboxane and prostacyclin synthesis. F i r s t , Canga and Sterin-Borda (1986) showed that inhibitors of the cyclo-oxygenase pathway for arachidonic acid metabolism blocked the a t r i a l response to methoxamine in non-diabetic as well as acute diabetic a t r i a , indicating the involvement of cyclo-oxygenase metabolites in the alpha-adrenoceptor-mediated positive inotropic response. Results further suggest that in acute diabetes, the response to alpha-adrenoceptor stimulation may be mediated by oxidative products generated via thromboxane synthetase and lipoxygenase a c t i v i t i e s , whereas in normal a t r i a , the action of methoxamine may involve the release of prostacyclin. We therefore conducted a series of experiments to study the effects of inhibitors of cyclooxygenase, thromboxane synthetase and prostacyclin synthetase (indomethacin, imidazole and tranylcypromine) on alphaj-adrenoceptor-mediated inotropic response in chronic diabetic hearts. As well, Ins(1,4,5)P3 formation in these hearts was studied. 41 1.5. OMEGA-3 FATTY ACID. Epidemiologic studies suggest that the consumption of fish by humans may offer protection against coronary heart disease (Bang and Dyerberg, 1972; Dyerberg and Bang, 1978; Kromann and Green, 1980; Kagawa et a l . , 1982; Kromhout et a l . , 1985). The mechanisms of this protection have not been ful l y elucidated; however, the omega-3 fatty acid content of the fish may be at least partly responsible. Dietary supplementation with polyunsaturated omega-3 fatty acids has been shown to have potential beneficial effects on platelet function, plasma l i p i d levels, blood pressure and glycemic control. It has been demonstrated that dietary fis h o i l contributes to changes in eicosanoid metabolism leading to decreased platelet aggregation (Croft et a l . , 1987). Also, omega-3 fatty acid supplementation has been associated with lowering of plasma l i p i d and blood pressure in normal subjects (Lorenz et a l . , 1983). In patients with hypertriglyceridemia, omega-3 fatty acid supplementation was associated with decreased serum triglyceride and cholesterol levels (Phillipson et a l . , 1985), lowering of blood pressure (Singer et a l . , 1985), and improvement of insulin sensitivity during glucose tolerance testing (Singer et a l . , 1985). Besides the above beneficial effects of fish o i l , i t has been reported that fish o i l supplementation may also 42 have some effects on l i p i d composition of myocardial membrane, as well as cardiac function and response to adrenoceptor stimulation. It was shown that dietary fats can greatly influence the l i p i d composition of cellular membranes in the myocardium (Charnock et a l . , 1984). For example, omega-3 fatty acid enriched diets have been shown to change the acyl composition of myocardial membrane phospholipids, such that there i s a replacement of omega-6 by omega-3 fatty acids (Hock et a l . , 1987). Additionally, dietary supplementation with an omega-3 fatty acid enriched diet may have a protective effect on the heart during an ischemic episode, as myocardial creatine kinase loss following coronary artery ligation i s reduced following omega-3 fatty acid supplementation (Hock et a l . , 1987). DeDeckere and Ten Hoor (1980) reported increased coronary flow and l e f t ventricular work after 1 and 4 weeks, respectively, in isolated hearts of rats fed sunflower seed o i l vs. lard (saturated). Further, i t was shown that omega-3 fatty acid feeding results in reduced cardiac responsiveness to alpha-adrenoceptor stimulation (Reibel et a l . , 1988). In other studies the contractile response to isoprenaline was found to be diminished in isolated hearts of rats fed sunflower seed o i l vs. saturated diets (Dryden et a l . , 1982; Hoffman et a l . , 1982). Diabetes mellitus i s associated with a high incidence of coronary artery disease and heart failure. Diabetic cardiomyopathy frequently coexists with other known risk 43 factors, such as abnormalities of lipoprotein metabolism and hypertension. Experimentally, the cardiomyopathy i s present after six weeks of streptozotocin-induced diabetes in the rat and i s manifest as impaired l e f t ventricular function. In type I diabetes, omega-3 fatty acid consumption has been associated with an improvement of insulin sensitivity (Popp-Snijders et a l . , 1987). Omega-3 fatty acid treatment in type I diabetes also has variable effects on plasma triglyceride concentrations as these l i p i d s have been reported to increase (Illman et a l . , 1986), decrease (Miller et a l . , 1987; Mori et a l . , 1988) or remained unchanged (Haines et a l . , 1986; Black et a l . , 1989) after omega-3 fatty acid treatment/supplementation, and to have similarly variable effects on plasma cholesterol. Recently, Black et al (1989) have shown omega-3 fatty acid treatment attenuates the development of diabetes-induced cardiomyopathy in streptozotocin-induced model of type I diabetes in the rat. The improved isolated working heart function (systolic and diastolic function) of the omega-3 fatty acid-treated diabetic rats was associated with improved sarcoplasmic reticulum transport activi t y . Cardiovascular effects of omega-3 fatty acids in human type II diabetes include reductions in both systolic and diastolic blood pressures (Kasim et a l . , 1988). Experimental diabetes mellitus i s also associated with altered responsiveness to alpha^-adrenergic receptor stimulation in the heart (Heyliger et a l . , 1982; Jackson et 44 a l . , 1986). Alpha^-adrenergic receptor stimulation in the heart has been shown to increase phosphoinositide metabolism (Brown et a l . , 1985; Scholz et a l . , 1988). Further, we have recently shown that alpha^-adrenergic receptor-mediated phosphoinositide metabolism i s altered in the diabetic heart (unpublished results). However, i t i s not known whether omega-3 fatty acid treatment has any beneficial effect on the cardiac response to alphai-adrenoceptor stimulation with respect to phosphoinositide metabolism in diabetic rats. Therefore, to further define possible mechanisms for the improved cardiac performance of omega-3 fatty acid-treated STZ-induced diabetic rats, we investigated the effects of omega-3 fatty acid supplementation on alphai-adrenoceptor-stimulated inositol phosphates formation in STZ-diabetic rats. 1 . 6 . C H E M I C A L L Y - I N D U C E D A N I M A L M O D E L S O F D I A B E T E S M E L L I T U S . It has long been known that chemicals can produce diabetes in laboratory animals. Among these agents are alloxan, streptozotocin, cyproheptadine, hexamethylmelamine, etc. Cyproheptadine and hexamethylmelamine can produce diabetes only in rat and mouse (Fischer, 1985), which has limited their widespread use. Alloxan was f i r s t reported to have diabetogenic activity by Dunn and McLetchie in 1943. Alloxan, a cyclic urea analog, i s taken up by pancreatic cel l s and rapidly reduced to form dialuric acid. This product undergoes auto-oxidation to yield detectable amounts 45 of H2O2, superoxide anion (C>2~) and hydroxyl free-radicals ("OH). These reduced species of oxygen, particularly the extremely reactive 'OH radical, are believed to i n i t i a t e the alloxan-based attack on beta ce l l s (Malaisse, 1982). The basis for the specificity of the attack on beta ce l l s by alloxan i s not clear. At present, a speculative explanation includes relatively high concentrations of the agent with relatively low levels of the protective substances that deactivate reduced forms of oxygen (Fischer, 1985). Streptozotocin has now, however, replaced alloxan as the primary compound used to produce experimental, insulin-dependent diabetes in laboratory animals. This i s due to the lower mortality rate seen in streptozotocin-induced diabetic animals (Hoftiezer and Carpenter, 1973), the greater selectivity of streptozotocin for beta c e l l s (Junod et a l . , 1969) and the longer half l i f e of streptozotocin in the body (Agarwal, 1980). Streptozotocin contains a N-methylnitrosourea moiety and i s an alkylating agent. This property i s capable of i n i t i a t i n g i t s cytotoxic action. The glucose moiety (2-deoxy D-glucose) present in the streptozotocin molecule may direct the alkylating agent to the pancreatic beta c e l l which may have a special a f f i n i t y for glucose. Further, streptozotocin has been shown to concentrate in pancreatic i s l e t s (Johnson and Tjalve, 1978), which may also account for the cell-selective action of streptozotocin. Both alloxan and streptozotocin share the 46 ab i l i t y to produce beta-cell necrosis after single doses to laboratory animals. 1 . 7 . P U R P O S E O F T H E P R E S E N T I N V E S T I G A T I O N . The purpose of this thesis was to investigate the changes in alphai-adrenoceptor-stimulated phosphoinositide metabolism in the diabetic heart and i t s involvement in the diabetes-induced alterations in the alphai-adrenoceptor-mediated positive inotropic effect. The following are the major objectives of the investigation: (1) To determine the time course and concentration-responsiveness of alphai-adrenoceptor-stimulated inositol monophosphate (IPi) formation (using a radioisotope method) in right ventricles of control and diabetic rats. (2) To determine the effect of omega-3 fatty acid supplementation on alphai-adrenoceptor-mediated inositol phosphates formation in right ventricles of control and diabetic rats. (3) To study the time course and concentration responsiveness of alphai-adrenoceptor-mediated positive inotropic effect and Ins(1,4,5)P3 formation [using an Ins(l,4,5)P3 protein binding assay kit] in right ventricles of control and diabetic rats. (4) To study the influence of indomethacin on alpha^-adrenoceptor-mediated positive inotropic effect and Ins(1,4,5)P3 formation in right ventricles of control and diabetic rats. 47 (5) To study the influence of imidazole on alpha^-adrenoceptor-mediated positive inotropic effect and Ins(l,4,5)P3 formation in right ventricles of control and diabetic rats. (6) To study the influence of tranylcypromine on alphai-adrenoceptor-mediated positive inotropic effect and Ins(l,4,5)P3 formation in right ventricles of control and diabetic rats. (7) To study protein kinase C activity in right ventricles of control and diabetic rats. 48 M A T E R I A L S A N D M E T H O D S 2 . 1 . M A T E R I A L S . 2 . 1 . 1 . A n i m a l s . Male Wistar rats weighing between 200-225 g were used throughout the study. The rats were obtained from Charles River Canada, Montreal, Canada. 2 . 1 . 2 . C h e m i c a l s . The following chemicals were purchased from Sigma Chemical Co.: adenosine 5'-triphosphate(disodium salt) (Na2ATP), adenosine 5'-triphosphate(tris salt) (t r i s ATP), ammonium formate, ascorbic acid, DEAE (diethylamino ethyl) cellulose anion exchanger, dithiothreitol, DMSO (dimethyl sulfoxide), EDTA (ethylenediaminetetraacetic acid), EGTA [ethyleneglycol-bis-(6-aminoethyl ether) N,N,N',N'-tetraacetic acid], HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), histone (type III-S, lysine-rich), imidazole, indomethacin, lithium chloride (LiCl), norepinephrine (arterenol), phenylmethylsulfonyl fluoride (PMSF), potassium phosphate (KH2P04), prazosin, propranolol, sodium azide, sodium formate, sodium orthovanadate (Na3V04), sodium tetraborate, streptozotocin, sucrose, tranylcypromine, Triton X-100 and Trizma base. The following chemicals were purchased from BDH: calcium chloride (CaCl2), chloroform (CHCI3), diethyl ether, glucose, hydrochloric acid (HCl), magnesium chloride 49 (MgCl2), methanol, potassium chloride (KC1), sodium bicarbonate (NaHCC^), sodium chloride (NaCl), sodium hydroxide (NaOH) and trichloroacetic acid. Leupeptin was obtained from Calbiochem Co. Phosphatidylserine (PS) (beef brain) and D-l-stearoyl-2-arachidonyl glycerol (DG) were obtained from Serdary Co. Analytical grade anion exchange resin (AG 1-X8, 100-200 mesh, formate form) was purchased from Bio-Rad Laboratories. Aquasol and myo-[2-3H]inositol (15.2 Ci/mmol) were purchased from New England Nuclear Research Products. [Y-32P]ATP (30 Ci/mmol) was purchased from Amersham. 2 . 1 . 3 . A s s a y K i t s . Insulin radioimmunoassay kits and Ins(1,4,5)P3 [ H] assay kits were purchased from Amersham. Glucose assay k i t s , cholesterol assay kits and triglycerol assay kits were purchased from Boehringer-Mannheim. 2 . 2 . I N D U C T I O N O F S T R E P T O Z O T O C I N D I A B E T E S I N R A T S . Male Wistar rats (175-200 g) were used. A l l animals were anesthetized transiently with diethyl ether to allow injection of either STZ or i t s vehicle into the t a i l vein. Diabetes was induced by a single intravenous injection of STZ (55 mg/kg) dissolved in citrate buffer (pH 4.5). The citrate buffer was made by mixing 0.1 M c i t r i c acid and 0.1 M sodium citrate until a pH of 4.5 was obtained. Control 50 rats were injected with citrate buffer alone. A l l rats injected with STZ survived and were housed two to three per cage with Lobound (R) grade corncob bedding (Paxton Processing, Paxton, IL.). A l l rats had access to Purina rat chow and water ad libitum. The lights were l e f t on from 6 a.m. to 6 p.m., keeping in mind that the rats are nocturnal animals. The room temperature was maintained at 25"C. Diabetes was detected three days later by estimating the extent of glucosuria with the aid of enzymatic test strips (Tes-Tape, E l i L i l l y , Toronto, Canada). Those rats displaying glucosuria greater than 2% were used as diabetic rats. The animals were sacrificed six weeks after diabetes induction. The duration of 6-week diabetes was chosen based on a report from our laboratory which showed that alterations in cardiac performance occur six weeks after the onset of diabetes (Tahiliani et a l . , 1983). At the time of death, whole blood (arterial and venous) samples were collected in heparinized tubes from non-fasting animals and centrifuged at 1300 x g for 20 min to separate c e l l s from plasma. The plasma obtained was stored at -20°C unt i l assayed for glucose, insulin, triglyceride and cholesterol. Hearts were excised and used for inositol phosphates radioisotope assay or for function studies in the isolated tissue bath. 2 . 3 . M E A S U R E M E N T O F I N O S I T O L P H O S P H A T E S W I T H T H E R A D I O I S O T O P E M E T H O D . 51 2 . 3 . 1 . R i g h t V e n t r i c l e P r e p a r a t i o n a n d P h o s p h o i n o s i t i d e L a b e l l i n g . Inositol phosphates formation was measured using a modified method of Brown and Brown (1983) and Jones et a l . (1988). Control and diabetic animals were sacrificed by decapitation. The hearts were excised quickly and rinsed in a beaker containing oxygenated Chenoweth-Koelle (CK) solution consisting of (mM): NaCl (120); KC1 (5.6); CaCl2 (2.18); MgCl2 (2.1); NaHC03 (19), and glucose (10). After expressing the blood from the intact heart, the atria and connective tissues were removed and the right ventricle was obtained. The right ventricle was dissected into two pieces (approximately 0.1 g wet weight per piece) and transferred to continuously oxygenated tubes containing CK solution in a shaking waterbath (37 C). Following an equilibration period of 30 min, the solution was removed and replaced with fresh solution containing 4 /iCi/mmol myo- [2-3H] inositol in 4.7 ml CK buffer. After 60 min of incubation with myo-[2-3H] i n o s i t o l , LiCl (10 mM) (an inhibitor of inositol 1-phosphatase) and the beta-adrenoceptor antagonist propranolol (10 /iM) were added and tissues were incubated for another 15 min. In the time course study, the alpha^-adrenoceptor agonist norepinephrine (10 /iM) was then added and tissues were incubated for time periods ranging from 5 to 30 min. In the concentration-response study, norepinephrine at concentrations ranging from 10- 7 to 10~4 M was added and tissues were incubated for another 15 52 minutes. In the omega-3 fatty acid treatment study, norepinephrine (10 /iM) was added and tissues were incubated for 15 min. In studies involving prazosin (10 fxH) (a concentration determined in a preliminary experiment to not affect the basal tension) and DMSO, these were added at the same time as L i C l . 2 . 3 . 2 . D e t e r m i n a t i o n o f [ 3 H ] i n o s i t o l M o n o p h o s p h a t e ( I P ^ ) . At the end of the incubation period, tissues were rinsed in 3 ml of ice-cold 12% trichloroacetic acid (TCA), blotted, weighed, and then transferred to 1.3 ml of chloroform ( C H C 1 3 ) : methanol (MeOH): water (H20) (5:10:4). Tissues were homogenized for 1 min in a glass homogenizer. The homogenate was transferred to a glass v i a l , and the homogenizer was rinsed with a CHCI3 (0.342 ml)/water (0.069 ml) mixture. A two-phase system was obtained with the addition of the mixture to the glass v i a l , making a solution of C H C I 3 : MeOH: H20 (10:10:5). Samples were centrifuged at 1600 x g at 4°C for 20 min. A 0.65 ml aliquot of the aqueous upper phase was transferred to a column containing approximately 175 mg of anion exchange resin (AG 1-X8, 100-200 mesh, formate form). Columns were washed with a total of 9.6 ml water to remove myo-[2-3H]inositol. Labelled myo-inositol monophosphate [3H-IPi] was eluted with 8 ml of 5 mM sodium tetraborate/15OmM sodium formate. 53 2 . 3 . 3 . D e t e r m i n a t i o n o f [ ^ H ] I n o s i t o l B i s p h o s p h a t e ( I P 2 ) / [ 3 H ] I n o s i t o l T r i s p h o s p h a t e ( I P 3 ) a n d [ 3 H ] I n o s i t o l T e t r a k i s p h o s p h a t e ( I P 4 ) . In experiments where inositol bisphosphate (IP2) and inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4) were measured, the column was further washed with an additional 32 ml of the same sodium tetraborate/sodium formate solution. IP2 was eluted with 8 ml of 300 mM ammonium formate (AF)/100 mM sodium formate (SF) followed by an 8 ml wash with this same buffer. IP3 was eluted with 8 ml 750 mM AF/100 mM SF followed by an 8 ml wash with this same buffer. IP4 was eluted with 8 ml of 1 M AF/100 mM SF. Radioactivity in a l l of the eluted fractions was quantified by liquid s c i n t i l l a t i o n counting. 2 . 4 . O M E G A - 3 F A T T Y A C I D T R E A T M E N T . The treatment protocol chosen to study effects of omega-3 fatty acid supplementation was conducted with Promega (Warner-Lambert, Morris Plains, NJ), a refined f i s h -o i l product concentrated in long-chain polyunsaturated omega-3 fatty acids. The dose-response study of the effect of Promega on isolated heart function in control and diabetic rats demonstrated an ameliorative effect of Promega at 0.5 ml/kg/day and 1.0 ml/kg/day (Black et a l . , 1988). Therefore, Promega was administered to control and diabetic rats at a dose of 0.5 ml/kg/day. Promega was obtained in bulk form; therefore, to prevent oxidation of the highly 54 unsaturated l i p i d s , aliquots of the stock was drawn and stored in glass vials under nitrogen at -80°C u n t i l they were used. Control and diabetic rats were given Promega between 0830 and 0930 daily for the duration of the treatment. Promega was administered to the rats via oral gavage with a 75-mm stainless-steel tube. Promega treatment was initiated 2 week after STZ or citrate buffer injection and was continued for 4 week, after which the animals were sacrificed. Hearts were removed and blood samples collected for further assays. 2 . 5 . M E A S U R E M E N T O F C O N T R A C T I L E F O R C E I N I S O L A T E D T I S S U E B A T H . 2 . 5 . 1 . T i m e C o u r s e a n d C o n c e n t r a t i o n - R e s p o n s e C u r v e s o f I n o t r o p i c R e s p o n s e s t o N o r e p i n e p h r i n e . (1) Tissue Preparations Isolation of cardiac tissues and measurement of the time course and concentration-response curves were done according to the method of Ramanadham and Tenner (1983). At the time of k i l l i n g , the heart was excised and immediately placed in a beaker containing ice-cold oxygenated CK solution (pH 7.4). After expressing blood from the heart, a t r i a l tissue, fat and connective tissue were trimmed o f f . The right ventricle was then carefully dissected and transferred to another beaker containing cold fresh CK solution. Each right ventricle tissue was further dissected into three to four triangular shaped pieces. One end of 55 each right ventricular piece was attached to a tissue-electrode assembly with two platinum electrodes. The other end was mounted to a Grass FT.03 force-displacement transducer that was connected to a Grass Model 79D polygraph four-channel recorder (Quincy, Mass., USA). The tissue preparations were equilibrated at 37°C for 60 min in 20-ml tissue baths of CK solution under constant oxygenation (95% O2 and 5% CO2) . During this period, CK solution in the tissue bath was replaced every 15 min. Resting tensions in right ventricular pieces of 1 g were maintained throughout the experiment. After 30 min of equilibration, the right ventricular pieces were stimulated e l e c t r i c a l l y via the punctate platinum electrodes (at a frequency of 1.0 Hz, 2 to 8 V, and a duration of 5 ms) . At the end of the equilibration period, basal developed force in right ventricular tissues was recorded. (2) Time Course and Concentration-Response Curves of Inotropic Responses to Norepinephrine After the recording of basal developed force in right ventricular tissues, propranolol (10 jiM) was added to the solution and the tissues were allowed to equilibrate an additional 15 min. Propranolol was added to block the beta inotropic effect of norepinephrine. Norepinephrine was prepared as a stock solution in ascorbic acid (0.1 g/dl). The appropriate dilutions (10~6 to 10~2 mol/1) were made in CK solution. Positive inotropic responses to increasing concentrations of norepinephrine 56 were obtained by a cumulative increase in the total concentration in the tissue bath. The recordings were made when the responses had reached a steady-state level at each concentration. In general, stable responses were achieved within 3 min after addition of the drug. Tissues were immediately freeze-clamped when the whole concentration-response curves were finished. The tissues were stored at -80°C unt i l assayed for Ins(l,4,5)P3 levels. In the study where the time course of Ins(l,4,5)P3 formation was measured, inotropic responses to 10~5 mol/1 of norepinephrine on isolated right ventricles were recorded at each chosen time point (0, 15, 30, 60 and 180 sec) and tissues were freeze-clamped at these time points for the measurement of Ins(1,4,5)P3. In the study where the Ins(l,4,5)P3 level at each concentration of norepinephrine was measured, inotropic responses to cumulative additions of norepinephrine in isolated right ventricular tissues were recorded when the responses had reached a steady-state le v e l . Tissues were freeze-clamped when the steady-state level was obtained at each concentration of norepinephrine. Sensitivity of the right ventricular tissues towards the positive inotropic effect of norepinephrine was determined by estimated PD2 values. The PD2 value i s defined as the negative log concentration of the drug required to produce 50% of the maximum change in tension. 57 2 . 5 . 2 . C o n c e n t r a t i o n - R e s p o n s e C u r v e s o f I n o t r o p i c R e s p o n s e s t o N o r e p i n e p h r i n e i n t h e P r e s e n c e o f I n d o m e t h a c i n . Indomethacin was dissolved in 70% ethanol. The drug was diluted in the tissue bath to achieve the fi n a l concentration of 10 jxM, a concentration determined in a preliminary experiment to not affect the basal tension. After a 60 min equilibration period, isolated right ventricular pieces were incubated for 15 min with propranolol and the cyclooxygenase inhibitor indomethacin before increasing concentrations of norepinephrine were added. Following the determination of the concentration-responses curves, tissues were freeze-clamped in liquid nitrogen and stored at -80"C prior to the measurement of Ins(l,4,5)P3. 2 . 5 . 3 . C o n c e n t r a t i o n - R e s p o n s e C u r v e s o f I n o t r o p i c R e s p o n s e s t o N o r e p i n e p h r i n e i n t h e P r e s e n c e o f I m i d a z o l e . Imidazole was dissolved in d i s t i l l e d water. The drug was diluted in the tissue bath to achieve the fin a l concentration of 1 mM (Canga and Sterin-Borda, 1986). At this dilution, the drug i t s e l f had no effect on inotropic responses. After a 60 min equilibration period, isolated right ventricular pieces were incubated for 15 min with propranolol and the thromboxane synthetase inhibitor imidazole before increasing concentrations of norepinephrine were added. Following the determination of the concentration-responses curves, tissues were freeze-clamped 58 in liquid nitrogen and stored at -80"C prior to the measurement of Ins(1,4,5)P3. 2 . 5 . 4 . C o n c e n t r a t i o n - R e s p o n s e C u r v e s o f I n o t r o p i c R e s p o n s e s t o N o r e p i n e p h r i n e i n t h e P r e s e n c e o f T r a n y l c y p r o m i n e . Tranylcypromine was dissolved in d i s t i l l e d water. The drug was diluted in the tissue bath to achieve the fi n a l concentration of 1.25 x 10~4 M, a concentration determined in a preliminary experiment to not affect the basal tension. After a 60 min equilibration period, isolated right ventricular pieces were incubated for 15 min with propranolol and the prostacyclin synthetase inhibitor tranylcypromine before increasing concentrations of norepinephrine were added. Following the determination of the concentration-responses curves, tissues were freeze-clamped in liquid nitrogen and stored at -80°C prior to the measurement of In s ( l , 4 , 5 ) P 3 . 2 . 6 . D E T E R M I N A T I O N O F I N O S I T O L 1 , 4 , 5 - T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] L E V E L S W I T H T H E I N S ( 1 , 4 , 5 ) P 3 P R O T E I N B I N D I N G A S S A Y K I T . 2 . 6 . 1 . E x t r a c t i o n o f I n s ( l , 4 , 5 ) P 3 . The frozen right ventricular strips were weighed and put in 0.8 ml 6% of trichloroacetic acid (TCA) on ice immediately. The tissues were then homogenized with a polytron PT-10 homogenizer for 10 sec at setting 5. The homogenates were centrifuged at 6000 x g for 15 min at 4°C. 59 The supernatants were transferred into glass tubes, to which 2 ml of H2O-saturated diethyl ether was added. The tubes were shaken well and the top layers were removed. The diethyl ether extraction procedure was repeated 5 times. The tubes were then put in a rack which was placed in warm tap water for a few minutes to evaporate diethyl ether. A 100 f/,1 portion of the ether-extracted supernatant was used for subsequent analysis. 2 . 6 . 2 . M e a s u r e m e n t o f Ins(1,4,5)P3. Ins(l,4,5)P3 levels were measured using a protein binding assay k i t (Amersham). The assay i s based on the competition between unlabelled Ins(1,4,5)P3 and a fixed quantity of tritium labelled Ins(1,4,5)P3 for a limited number of binding sites on a bovine adrenal binding protein preparation. With fixed amounts of binding protein and radioactive ligand, the amount of radioactive ligand bound to the binding protein w i l l be inversely proportional to the concentration of added non-radioactive ligand. The bound Ins(1,4,5)P3 is then separated from free ligand by centrifugation, which brings the binding protein preparation to the bottom of the tube. The free fraction in the supernatant can then be discarded by decantation leaving the bound fraction adhering to the tube. Measurement of the radioactivity in the tube enables the amount of unlabelled Ins(1,4,5)P3 in the sample to be determined by interpolation from the standard curve. 60 A l l reagents were allowed to equilibrate to 4°C. Once prepared, a l l reagents were kept on ice prior to performing the assay. Reagent preparation: Assay buffer was allowed to thaw and was mixed thoroughly. Standard solution was also allowed to thaw and was mixed thoroughly. A 100 /xl aliquot of tracer was removed from the v i a l and added to 11 ml of d i s t i l l e d water in a glass vessel. The contents were thoroughly mixed. The binding protein solution was allowed to thaw and was mixed well by inversion and swirling. Preparation of working standards: Eight glass tubes (containing 0.19, 0.38, 0.76, 1.5, 3.1, 6.2, 12.5, 25 pmol) were labelled. D i s t i l l e d H2O, 1.5 ml, was added into the tube marked 25 pmol. Then 500 /xl of d i s t i l l e d H2O was added into the remaining marked standard tubes. Exactly 100 /xl of standard solution was transferred into the tube marked 25 pmol and vortexed thoroughly. A 500 /xl aliquot from the 25 pmol tube was transferred to the 12.5 pmol tube and vortexed thoroughly. This dilution was repeated successively with the remaining tubes. One hundred aliquots from each serial dilution gave 8 standard levels of Ins(l,4,5)P3 ranging from 0.19-25 pmol per tube. Assay procedure: Polypropylene tubes in duplicate for total counts (TC), non-specific binding tubes (NSB), zero standard tubes (Bo), standards and samples were labelled and placed in an ice-bath. A 100 /xl aliquot of assay buffer was pipetted into a l l tubes. D i s t i l l e d H2O, 100 /xl, was put 61 into the Bo tubes and 200 /xl of H2O into TC tubes. A 100 Ml aliquot of each standard solution was put into the appropriately labelled tubes and 100 /xl of the stock standard was put into the NSB tubes. A 100 /zl aliquot of sample was put into the appropriate tubes. A 100 /xl aliquot of tracer was put into a l l tubes and 100 /xl of binding protein was put into a l l tubes except TC. A l l tubes were vortexed thoroughly and incubated for 15 min on ice. A l l tubes except TC were centrifuged at 1000 x g for 1.5 min at 4°C. After centrifugation, the tubes were placed carefully into racks and the supernatants were discarded. A 200 /xl aliquot of H2O was added to each tube except for TC tubes. The tubes were vortex mixed thoroughly to resuspend the pelle t . A 180 /xl aliquot of the mixture was transferred into the s c i n t i l l a t i o n tubes and 2 ml of s c i n t i l l a n t was added to each tube. Radioactivity was measured in a liquid s c i n t i l l a t i o n counter. 2 . 7 . P R O T E I N K I N A S E C A N A L Y S I S The preparation of membrane and cytosolic fractions, preparation of DEAE cellulose column and assay of protein kinase C activity were performed by a modification of the method of Kikkawa et a l . (1982) . 2 . 7 . 1 . P r e p a r a t i o n o f M e m b r a n e a n d C y t o s o l i c F r a c t i o n s Experiments were carried out on male Wistar rats (200-225 g) . Diabetes was induced as described in section 2.2. The animals were sacrificed by decapitation six weeks after 62 induction of diabetes. The hearts were excised immediately and rinsed in a beaker containing oxygenated CK solution. After expressing the blood from the intact heart, the atria and connective tissues were removed and the right ventricle was obtained. The right ventricle was dissected into four pieces (approximately 0.05-0.08 g wet weight per piece). Where norepinephrine was used, each right ventricular piece was suspended in a tissue bath and stimulated with 10 mM norepinephrine (in the presence of 10 jiM propranolol) . Tissues were frozen in liquid nitrogen when the inotropic responses reached a steady level. Otherwise, each right ventricular piece was quickly frozen right after dissecting with Wollenberger clamps precooled in liquid nitrogen, weighed, and pulverized to a fine powder with a precooled mortar and pestle. The procedures for the isolation of membrane and cytosolic fractions were done at 4°C. The powdered tissue from each right ventricular piece was thawed and homogenized in 5 ml of buffer A [20 mM HEPES, 0.25 M sucrose, 2 mM EGTA, 0.005% leupeptin, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4] with a hand homogenizer. Homogenates were centrifuged at 100,000g for 60 min. The resulting supernatants were saved as cytosolic fraction and put on ice until use. The pellets were rehomogenized with a hand homogenizer in 3 ml of 0.075% (v/v) Triton X-100 in buffer A. Homogenates were centrifuged at 100,000g for 60 min. The resulting 63 supernatants were saved as membrane fraction and put on ice until use. 2 . 7 . 2 . D E A E C e l l u l o s e C o l u m n Two grams of DEAE cellulose anion exchange resin was added to 100 ml of 0.1 N HC1 and the mixture swirled. The slurry was allowed to settle for 10 min and the top layer was decanted. The procedure was repeated another four times unt i l the top layer was clear. The mixture was then applied to Whatman No. 1 f i l t e r paper (Whatman International Ltd., England). The f i l t e r paper was then washed in order with 140 ml each of H20, 0.1 N NaOH, H20 and 0.1 N HCl. The powder was scratched into a beaker containing 100 ml of 0.1 N HCl, which was neutralized with Trizma base to pH 7.4. The mixture was allowed to settle and 50 ml of the top layer was decanted. The rest was swirled and 4.5 ml of the slurry was applied to the column preequilibrated with 0.6 ml buffer B (20 mM Trizma base, 0.5 mM EGTA and 0.5 mM EDTA). A total of 20 ml of buffer B was further applied to the column. The column was stored at 4°C with 3 ml of buffer B in i t . On the day of use, another 6 ml of buffer B containing 2 mM dithiothreitol was run through the column. Cytosolic and membrane preparations were then applied to the columns. The column was washed with another 4 ml of buffer B containing 2 mM dithiothreitol, and the enzyme was eluted by 2 ml buffer B containing 2 mM dithiothreitol and 0.15 M 64 NaCl. A 25-/il aliquot was analyzed for protein kinase act i v i t y . The column was regenerated by running through the column 20 ml of buffer B containing 1 M NaCl, and stored in 1 ml of buffer B containing 0.02% sodium azide. 2 . 7 . 3 . A s s a y o f P r o t e i n K i n a s e C A c t i v i t y Protein kinase C activity was assayed by measuring the incorporation of 3 2P from [y -32P]ATP into lysine-rich histone. A 25 of the sample was preincubated with buffer C (20 mM HEPES, 2.4 mg histone, 10 mM dithiothreitol, 20 mM MgCl2, 2 mM EGTA, 1 mM Na3V04, and 0.004% leupeptin) with or without 1.75 mM CaCl2, 0.14 mg phosphatidyl serine (PS) or 14.4 /xg D-l-stearoyl-2-arachidonyl glycerol (DG) for 2.5 min at 30°C. PS and DG were f i r s t dried under nitrogen and resuspended in 20 mM HEPES by sonication for 20 min at room temperature. The reaction was initiated by the addition of [ Y - 3 2 P ] A T P (5 x 105 dpm) and allowed to proceed at 30°C for 1 min. The reaction was terminated by the addition of 1 ml of 5% trichloroacetic acid containing 2.5 mM Na2ATP and 5 mM KH2PC>4. The incubation solution was f i l t e r e d through Whatman GF/C f i l t e r paper (Whatman International Ltd., England) . The test tubes were washed twice each with 1 ml of 5% trichloroacetic acid solution. The f i l t e r papers were then washed three times each with 1 ml of 5% trichloroacetic acid. The f i l t e r papers were put into s c i n t i l l a t i o n v ials containing 5 ml aquasol, and were counted in a liquid 65 s c i n t i l l a t i o n counter. Protein kinase activity was expressed as pmol Pi/mg tissue/min. Specific protein kinase C activity was calculated by subtracting the protein kinase activity in the presence of 1.75 mM CaCl2 alone from the protein kinase activity in the presence of 1.75 mM CaCl2, 0.14 mg phosphatidylserine (PS) and 14.4 jug D-l-stearoyl-2-arachidonyl glycerol (DG). 2 . 8 . B L O O D A N A L Y S I S . 2 . 8 . 1 . P l a s m a G l u c o s e D e t e r m i n a t i o n . Plasma glucose levels were determined by the glucose oxidase method using a Sigma glucose k i t . 2 . 8 . 2 . P l a s m a I n s u l i n D e t e r m i n a t i o n . Plasma immunoreactive insulin was assayed by the radioimmunoassay method using the Amersham insulin radioimmunoassay k i t . 2 . 8 . 3 . P l a s m a T r i g l y c e r i d e D e t e r m i n a t i o n . Plasma triglyceride was determined using a Boehringer-Mannheim diagnostic reagent k i t . 2 . 8 . 4 . P l a s m a C h o l e s t e r o l D e t e r m i n a t i o n . Plasma cholesterol was determined using a Boehringer-Mannheim diagnostic reagent k i t . 2 . 9 . S T A T I S T I C A L A N A L Y S I S . 66 Results are expressed as mean ± S.E.M. (standard error of the mean). The s t a t i s t i c a l differences between mean values of the various groups were evaluated by two way analysis of variance followed by Newman-Keul's test. For isolated tissue function curves, repeated measures of analysis of variance was used. A l l other analyses were carried out using one way analysis of variance where appropriate. The level of s t a t i s t i c a l significance was set at a probability of less than 0.05 (p<0.05). 67 R E S U L T S 3 . 1 . T H E B A S A L L E V E L O F I N O S I T O L M O N O P H O S P H A T E ( IP ] . ) F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . Rat right ventricular muscles were labelled with [3H] inositol and incubated in CK solution containing 10 /iM LiCl and 10 /iM propranolol in the absence of norepinephrine for 3 0 minutes. The basal levels of IP^ formation in the right ventricles of control and diabetic animals were not significantly different from each other (Figure 4). 3 . 2 . T I M E C O U R S E O F A L P H A i - A D R E N O C E P T O R - M E D I A T E D I N O S I T O L M O N O P H O S P H A T E ( I P i ) F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . (1) General features of the experimental rats. The general features of the experimental rats are presented in Table 2. As shown, the gain in body weight by diabetic rats was significantly less than that of controls. The time course of weight changes over the 6-week period shows that the significant difference in body weight gain started as early as 3 days after the STZ injection and became greater with the increased duration of diabetes (Figure 5). An elevated urinary glucose (>2%) was found in diabetic rats throughout the study period. No detectable glucose was present in the urine of control animals. Other symptoms usually associated with the diabetic state such as polyuria and polyphagia were seen in the diabetic rats. 68 Plasma glucose levels were elevated markedly in the diabetic rats accompanied by a depression in plasma insulin levels (Table 2). A plasma l i p i d profile of the rats used in this study is shown in Table 3. Plasma triglyceride and cholesterol were markedly elevated in the STZ-diabetic rats compared with controls. In addition, right ventricular weights were reduced in diabetic animals compared with controls (Figure 6). (2) Time course of alphai-adrenoceptor-mediated IP^ formation. The time course for production of IPi in response to norepinephrine stimulation in the presence of LiCl and 10 uM propranolol i s shown in Figure 7. In both control and diabetic rats, stimulation of alpha^-adrenoceptors by norepinephrine in right ventricles resulted in the production of IP^ in a time dependent manner. Right ventricles from diabetic rats, however, showed decreased IP^ production in response to norepinephrine compared to controls at time periods longer than 10 minutes. At the 5 minute period, there was no significant difference in norepinephrine stimulated IP^ production between the hearts of control and diabetic animals. 69 3.3. C O N C E N T R A T I O N - R E S P O N S E C U R V E S F O R A L P H A ^ - A D R E N O C E P T O R -M E D I A T E D I N O S I T O L M O N O P H O S P H A T E ( I P i ) F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . (1) General features of the experimental rats The general features of the diabetic rats were quite characteristic of the disease and are summarized in Table 4. Diabetic rats exhibited severe hyperglycemia and hypoinsulinemia. Body weight gain was significantly less in the diabetic group compared with control rats. The body weight difference progressed with the duration of the disease (Figure 8) . Plasma glucose levels were elevated significantly in the diabetic rats accompanied by a depression in plasma insulin levels compared with controls (Table 4). Plasma triglyceride and cholesterol were significantly increased in the diabetic group compared with controls (Table 5). Right ventricular weights were significantly reduced in the diabetic rats compared with controls (Figure 9). (2) Concentration-response curves for alphai-adrenoceptor-mediated IPi formation. The concentration-response curves for the effect of alphai-adrenoceptor stimulation on the formation of IPi in control and diabetic animals are shown in Figure 10. Addition of norepinephrine (in the presence of propranolol) to right ventricles of the control rats markedly increased 70 the accumulation of [JH]IPi in a concentration-dependent manner. In contrast, hearts from diabetic rats showed decreased IPi formation compared with controls at norepinephrine concentrations higher than 10~5 M . Therefore, the maximum IPi formation in response to norepinephrine stimulation in the diabetic heart was reduced. 3 . 4 . E F F E C T O F P R A Z O S I N A N D P R O P R A N O L O L O N N O R E P I N E P H R I N E -S T I M U L A T E D I N O S I T O L M O N O P H P O S P H A T E ( I P i ) F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . Norepinephrine stimulates beta- as well as alpha-adrenoceptors on the heart. Therefore additional pharmacological studies were performed to define the nature of the receptor through which catecholamines stimulate phosphoinositide hydrolysis. Propranolol did not antagonize the stimulatory effect of norepinephrine on IP^ formation in either control or diabetic animals (Figure 11) , indicating that the adrenoceptor coupled to phosphoinositide metabolism is not the beta-adrenoceptor. If phosphinositide hydrolysis i s mediated by the cardiac alphai-adrenoceptor, the hydrolysis should be blocked by antagonizing the receptors. To test this hypothesis, we used prazosin as an alphai-adrenoceptor antagonist. When the right ventricles labelled with [3H] inositol were exposed to 10 /uM prazosin, the formation 71 of IPi induced by 10 /iM norepinephrine was significantly inhibited (Figure 11) . 3 . 5 . E F F E C T O F P R A Z O S I N , P R O P R A N O L O L A N D DMSO A L O N E O N I N O S I T O L M O N O P H O S P H A T E ( IP ] . ) F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . As mentioned above, propranolol and prazosin were used in the experiments to explore the nature of the receptor through which norepinephrine stimulates phosphinositide hydrolysis. DMSO was used to dissolve prazosin. In order to examine the effect of these agents alone on basal IP^ formation (unstimulated IP^ formation), prazosin, propranolol and DMSO were employed separately. Results show that these agents did not affect basal IP^ production in either control and diabetic hearts (Figure 12). 3 . 6 . E F F E C T O F O M E 6 A - 3 F A T T Y A C I D O N C A R D I A C A L P H A } . -A D R E N O C E P T O R - M E D I A T E D I N O S I T O L P H O S P H A T E S F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T S . (1) General features of the experimental rats. Figures 13, 14 and 15 show the general characteristics of animals used in this study. The diabetic rats exhibited reduced body weights compared with a l l controls (Figure 13). Plasma insulin levels of the diabetic rats were about 50% of the controls (Figure 15) and their glucose values increased about fourfold relative to controls (Figure 14) . Omega-3 fatty acid treatment did not seem to have any significant 72 effects on either control or diabetic rats with respect to body weights, plasma glucose or insulin levels. Plasma triglyceride (Figure 16) and cholesterol (Figure 17) were significantly higher in the untreated diabetic rats compared with a l l controls. In contrast, the hypertriglyceridemia of diabetes was significantly reduced in the diabetic treated group. However, the elevated plasma cholesterol of diabetic rats was not affected by the treatment. Wet right ventricular weights were decreased in the diabetic animals (Figure 18). Omega-3 fatty acid treatment had no effect on the right ventricular weights of either control or diabetic rats. (2) Effect of omega-3 fatty acid treatment on cardiac alphai-adrenoceptor-mediated inositol phosphates metabolism. The effect of omega-3 fatty acid on alpha^-adrenoceptor-stimulated IPi formation i s presented in Figure 19. IPi formation was measured under different concentrations of norepinephrine stimulation (10~6, 10~5 and 10~4 M) . As shown, stimulation of IP^ production due to norepinephrine was concentration-dependent in a l l groups. However, the increase in IP^ formation was significantly lower in both untreated and treated diabetic groups compared with controls when the hearts were stimulated with higher concentrations of norepinephrine (10~5 and 10~4 M). 73 Norepinephrine stimulated IP^ formation did not appear to be affected by omega-3 fatty acid in the ventricles from control or diabetic animals. When [3H]IP2 was measured, i t was noted that IP2 formation in response to 10~4 M of norepinephrine was significantly lower in the diabetic heart compared with controls (Fig. 20). Omega-3 fatty acid had no effect on IP2 production in response to 10~4 M of norepinephrine in diabetic rats. At norepinephrine concentrations lower than 10~4 M, there were no significant differences in norepinephrine-stimulated IP2 formation in any of the groups. In this experiment, we also showed that [ H]IP3 and [3H]IP4 were formed in response to alphai-adrenoceptor stimulation (Figure 21 and 22). Concentration-dependent increases in IP3 and IP4 in response to norepinephrine were evident in most cases. However, differences in IP3 or IP4 formation between untreated and treated, or control and diabetic rat hearts were not evident. This i s probably due to the large standard error and the low specific activity of the myo[2-3H] inositol (4 liCi/mmol) employed. 3 . 7 . E F F E C T O F A L P H A 2 . - A D R E N O C E P T O R S T I M U L A T I O N O N P O S I T I V E I N O T R O P I C E F F E C T A N D I N O S I T O L 1 , 4 , 5 - T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . (1) General features of the experimental rats. 74 Rats injected with STZ exhibited symptoms characteristic of the diabetic state. Diabetic rats did not gain as much weight as age-matched controls and had significantly lower body weights at the time of sacrifice (Table 6) . This occurred despite the fact that diabetic animals had dramatically elevated f l u i d (polydipsia) and food (hyperphagia) intake. Diabetic rats also had increased fecal and urine output which were not measured quantitatively. Urine glucose was qualitatively assessed three days after STZ injection using Tes-Tape. On a scale of 1+ (0.1%) to 4+ (2.0%), i t was found to be greater than 2% for diabetic rats. No detectable glucose was present in the urine of control animals. Plasma glucose levels of diabetic animals were found to be elevated by three-fold (Table 6) . On the other hand, plasma insulin levels measured at the time of sacrifice were significantly depressed in diabetic animals (Table 6). (2) Effect of alphai-adrenoceptor stimulation on positive inotropic effect in control and diabetic rat ventricles. Figure 23 shows the effect of increasing concentrations of norepinephrine (in the presence of propranolol) on the change in tension (% of maximum) in right ventricles obtained from control and diabetic rats. It can be seen that norepinephrine induced a concentration-dependent 75 increase in percentage of maximum change in tension in both groups of right ventricles. However, concentration-response curves to norepinephrine obtained from diabetic rats were shifted to the l e f t of the corresponding age-matched control groups, indicative of supersensitivity to the inotropic effect of norepinephrine. The increase in sensitivity to norepinephrine was reflected by higher estimated PD2 values (3.26 ± 0.19) obtained in the diabetic group relative to the age-matched control group (2.43 ± 0.21) (Figure 24). When data were expressed as A change in tension in response to increasing concentrations of norepinephrine, similar results were obtained (Figure 25) . Norepinephrine produced a positive inotropic effect in both groups of right ventricles. The increase in tension was concentration-dependent and significantly greater in the diabetic group compared with controls. At the highest concentration employed (10~2 M) , A change in tension in right ventricles from diabetic animals was shown to be most prominently increased compared with controls (Figure 26). (3) Effect of alphai-adrenoceptor stimulation on Ins(1,4,5)P3 formation in control and diabetic rat ventricles. When the concentration-response curves were completed, tissues were immediately freeze-clamped. Ins(l,4,5)P3 levels were determined using a protein binding assay. It i s shown that norepinephrine induced a significantly greater increase in Ins(l,4,5)P3 levels in diabetic right ventricles (2.98 ± 0.19) compared with controls (0.56 ± 0.17) (Figure 27) , which paralleled the A change in tension at 10~2 M of norepinephrine. (4) Effect of prazosin on alphai-adrenoceptor-mediated Ins(1,4,5)P3 formation and inotropic responses in control and diabetic right ventricles. To assess the role of alpha-adrenoceptors in the contractile effect of norepinephrine, ventricles were exposed to prazosin (in the presence of propranolol) before addition of norepinephrine. The results shown in Table 7 indicate that in the presence of prazosin, the positive inotropic action of norepinephrine was antagonized in both control and diabetic right ventricles. As well, norepinephrine-stimulated Ins(1,4,5)P3 formation was blocked by the addition of prazosin. 3 . 8 . T I M E C O U R S E O F A L P H A ^ - A D R E N O C E P T O R - M E D I A T E D P O S I T I V E I N O T R O P I S M A N D I N O S I T O L 1 , 4 , 5 - T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . Figure 28 depicts the time course of norepinephrine-stimulated inotropism in control and diabetic rat hearts. Stimulation of alphai-adrenoceptors in the right ventricles of rats resulted in a biphasic inotropic response. The i n i t i a l decrease in contractile force appeared about 10 sec 77 after the stimulation and reached a maximum level at 30 sec. The contractile force then started to increase at about 45 to 60 sec, reaching a maximum level at 3 min. The contractile force developed in the diabetic heart [Figure 28 (b)] was significantly higher compared with controls [Figure 28 (a)]. The level of Ins(l,4,5)P3 in both control and diabetic rat hearts increased significantly within 15 sec after the stimulation, reached maximum at 60 sec and gradually declined thereafter (Figure 29) . Starting from 30 sec, Ins(l,4,5)P3 levels in the diabetic hearts were significantly higher compared with controls. When Ins(l,4,5)P3 formation was expressed as percentage change of Ins(l,4,5)P3 over basal levels (basal level=100%) at different time points of norepinephrine stimulation, i t was shown that % change in Ins(1,4,5)P3 levels in response to norepinephrine stimulation for the time periods longer than 30 sec was also significantly greater in diabetic ventricles compared with controls (Table 8). Correlation coefficients for the time course of alpha^-adrenoceptor-mediated positive inotropic effect and Ins (1,4,5) P3 formation were 0.33 for the controls and 0.39 for the diabetics, which were not significantly different from each other. The low correlation coefficients might be due to transient negative inotropic effect induced by norepinephrine. The correlation between tissue Ins(l,4,5)P3 levels and the extent of the transient negative inotropic 78 effect as documented by the present study may suggest that the transient negative inotropic effect of norepinephrine may also be triggered by Ins(l,4 , 5 )P3 formation. One possible mechanism by which Ins(1,4,5)P3 formation causes a negative inotropic effect i s Ca2 + overload induced by intracellular Ca2 + mobilization. In fact, caffeine and Ca2 + lonophore, which could cause release of intracellular Ca , produced negative inotropic effects following a transient positive inotropic effect (Otani et a l . , 1988a). It is also possible that alphai-adrenoceptor-mediated reduction of cardiac cAMP levels may be associated with the transient negative inotropic effect. 3 . 9 . C O N C E N T R A T I O N D E P E N D E N C E O F A L P H A J . - A D R E N O C E P T O R -M E D I A T E D P O S I T I V E I N O T R O P I C E F F E C T A N D I N O S I T O L 1 , 4 , 5 -T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . (1) Concentration responsiveness of alphai-adrenoceptor-mediated positive inotropic effect in control and diabetic rats. The effect of increasing concentrations of norepinephrine (in the presence of propranolol) on the change in tension (% of maximum) in right ventricles obtained from control and diabetic rats i s shown in Figure 30. Norepinephrine induced a concentration-dependent increase in percentage of maximum change in tension in both groups of right ventricles. However, concentration-response 79 curves to norepinephrine obtained from diabetic right ventricles were shifted to the l e f t of the corresponding age-matched controls, indicative of supersensitivity to the inotropic effect of norepinephrine in diabetic animals. (2) Concentration responsiveness of alphai-adrenoceptor-mediated inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] formation in control and diabetic rats. Tissues were freeze-clamped following stimulation by different concentrations of norepinephrine. Ins(1,4,5)P3 was determined using a protein binding assay. Norepinephrine induced a concentration-dependent increase in Ins(1,4,5)P3 levels in right ventricles from control rats (basal 0.22 ± 0.09; peak 0.66 ± 0.09) (Figure 31). In right ventricles of diabetic animals, Ins(l,4,5)P3 levels also increased in norepinephrine concentration-dependent manner and were significantly greater compared with control hearts. The peak level of Ins(l,4,5)P3 formation in diabetic hearts was 1.13 ± 0.13. The basal levels of Ins(l,4,5)P3 formation were similar in right ventricles of both control and diabetic groups (control, 0.22 ± 0.09; diabetic, 0.30 ± 0.08). When Ins(l,4,5)P3 formation was expressed as percentage change of Ins(l,4,5)P3 over basal levels (basal level=100%) at different concentrations of norepinephrine stimulation, i t was shown that % change in Ins(l,4,5)P3 levels in response to 10~6 to 10~3 M of norepinephrine stimulation was 80 significantly greater in diabetic ventricles compared with controls (Table 9). Correlation curves for the concentration-responses of alphai-adrenoceptor-mediated positive inotropic effect and Ins(l,4,5)P3 formation (Figure 32) indicated that the positive inotropic effect and Ins(1,4,5)P3 formation were indeed correlated with each other in control and diabetic right ventricles. 3 . 1 0 . I N F L U E N C E O F I N D O M E T H A C I N O N A L P H A ^ - A D R E N O C E P T O R -M E D I A T E D P O S I T I V E I N O T R O P I C E F F E C T A N D I N O S I T O L 1 , 4 , 5 -T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . (1) Influence of indomethacin on concentration responsiveness of alphai-adrenoceptor-mediated positive inotropic effect. Figure 33 depicts concentration-response curves for the change in tension (% of maximum) evoked by norepinephrine in right ventricles of control and diabetic rats. Norepinephrine produced a positive inotropic effect in both control and diabetic groups. The increase in tension was concentration-dependent and was significantly greater in the diabetic group than in controls. In the presence of the cyclooxygenase inhibitor indomethacin, the positive inotropic effect induced by norepinephrine was significantly increased in control right ventricles. In fact, the change in tension (% of maximum) 81 in control right ventricles pretreated with indomethacin was similar to that in diabetic right ventricles. On the other hand, indomethacin failed to modify the contractile stimulatory action of norepinephrine in the diabetic right ventricles. The increase in sensitivity to norepinephrine in diabetic right ventricles was reflected by higher estimated PD2 values obtained in the diabetic group (3.25 ± 0.23) relative to the age-matched control group (2.43 ± 0.25) (Figure 34) . Estimated PD2 values in the control group treated with indomethacin were significantly increased (3.51 ± 0.25) compared with controls, whereas estimated PD2 values remained elevated in the diabetic group pretreated with indomethacin (3.36 ± 0.23). When data were expressed as A change in tension in response to increasing concentrations of norepinephrine stimulation, norepinephrine also produced a positive inotropic effect in a l l groups of right ventricles (Figure 35). The increase in tension was concentration-dependent, and was significantly greater in the diabetic group compared with controls. In the presence of indomethacin, A change in tension induced by norepinephrine was significantly increased in control right ventricles. However, indomethacin was unable to modify A change in tension to norepinephrine in the right ventricles from diabetic animals. 82 At the highest norepinephrine concentration employed (10 * M) , A change in tension in diabetic right ventricles was increased compared with controls (Figure 36) . The tension change remained the same in the diabetic right ventricles pretreated with indomethacin. On the other hand, the change in tension increased in the control group treated with indomethacin. (2) Influence of indomethacin on alphai-adrenoceptor-stimulated Ins(l,4,5)P3 formation in control and diabetic rats. At the end of the concentration-response curves, right ventricles were freeze-clamped and Ins(l,4,5)P3 levels were measured in these tissues. The changes in Ins(l,4,5)P3 corresponded with the above changes in the tension development. Ins(1,4,5)P3 levels were elevated in the diabetic group (2.98 ± 0.59) as well as in the diabetic group pretreated with indomethacin (3.36 ± 0.42) compared with the control group (0.56 ± 0.17) (Figure 37). Ins(l,4,5)P3 levels in the control group treated with indomethacin (2.79 ± 0.68) were, however, similar to the Ins(l,4,5)P3 values in diabetic hearts. 3 . 1 1 . I N F L U E N C E O F I M I D A Z O L E O N A L P H A x - A D R E N O C E P T O R - M E D I A T E D P O S I T I V E I N O T R O P I C E F F E C T A N D I N O S I T O L 1 , 4 , 5 -T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . 83 (1) Influence of imidazole on concentration responsiveness of alphai-adrenoceptor-mediated positive inotropic effect. The exposure of control right ventricles to the thromboxane synthetase inhibitor imidazole increased the positive inotropic effect of norepinephrine to values similar to those in the diabetic group (Figure 38). However, i t should be noted that imidazole did not modify significantly the tension development in the diabetic group. The increase in sensitivity to norepinephrine in diabetic right ventricles was reflected by higher estimated PD2 values obtained in the diabetic group (3.25 ± 0.18) relative to the age-matched control group (2.43 ± 0.20) (Figure 39) . Estimated PD2 values in the control group treated with imidazole were significantly increased (3.70 ± 0.25) compared with the control group, whereas estimated PD2 values remained elevated in the imidazole-treated diabetic group (4.11 ± 0.22). Similar results were shown when data were expressed as A change in tension (Figure 40) . At 10 * M of norepinephrine stimulation, the diabetic group as well as the diabetic group pretreated with imidazole had enhanced positive inotropic effect compared with the control group. Interestingly, imidazole enhanced the positive inotropic effect significantly in the control animals. 84 (2) Influence of imidazole on alphai-adrenoceptor-stimulated Ins(l,4,5)P3 formation in control and diabetic rats. At the end of the concentration-response curves, right ventricles were freeze-clamped immediately and Ins(l,4,5)P3 levels were measured in these tissues using a protein binding assay. Ins(1,4,5)P3 levels were significantly elevated in the control group treated with imidazole (3.67 ± 0.27) compared with the control group (0.56 ± 0.17) (Figure 41). Also, Ins(l,4,5)P3 levels were increased in the diabetic group (2.98 ± 0.27). Ins(l,4,5)P3 levels in the diabetic group treated with imidazole (1.51 ± 0.21) were higher than the ones in the control group but were lower than the ones in the imidazole-treated control group as well as the diabetic group. 3 . 1 2 . I N F L U E N C E O F T R A N Y L C Y P R O M I N E O N A L P H A ^ - A D R E N O C E P T O R -M E D I A T E D P O S I T I V E I N O T R O P I C E F F E C T A N D I N O S I T O L 1 , 4 , 5 -T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] F O R M A T I O N I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . (1) General features of control and diabetic rats 6 weeks after administration of streptozotocin. Injecting rats with STZ resulted in a diabetic state, with an elevated urine glucose (>2%) throughout the study period. No detectable glucose was present in the urine of control animals. Parameters used to assess the diabetic state of animals are summarized in Table 10. At the time of 85 sac r i f i c e , the diabetic rats exhibited reduced body weights compared with controls. Plasma glucose levels were markedly elevated in STZ-injected rats accompanied by a depression in plasma insulin levels. Table 11 shows the plasma l i p i d profile of the experimental rats. Plasma triglyceride and cholesterol were significantly higher in the Wistar diabetic group compared with controls. (2) Influence of tranylcypromine on concentration responsiveness of alphai-adrenoceptor-mediated positive inotropic effect in control and diabetic rats. . . . — p A Change in tension in response to 10 M norepinephrine obtained from the diabetic group was significantly elevated compared with the control group (Figure 42). Incubation with tranylcypromine, a prostacyclin synthetase inhibitor, reduced the inotropic effect of norepinephrine in diabetic right ventricles, but failed to modify i t in the control right ventricles. (3) Influence of tranylcypromine on alphai-adrenoceptor-stimulated Ins(l,4,5)P3 formation in control and diabetic rats. Changes in Ins(l,4,5)P3 levels (Figure 43) corresponded with the changes in tension development discussed above. The diabetic group showed a significant increase in Ins(l,4,5)P3 levels (2.98 ± 0.15) compared with controls 86 (0.41 ± 0.13). The prostacyclin synthetase inhibitor tranylcypromine diminished norepinephrine-induced Ins(1,4,5)P3 formation in diabetic preparations (0.50 ± 0.13) without altering norepinephrine-induced Ins(1,4,5)P3 formation in the controls (0.48 ± 0.15). 3 . 1 3 . P R O T E I N K I N A S E C A C T I V I T Y I N C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S . Table 12 shows the distribution of protein kinase C in cytosolic and membrane fractions in the right ventricles of control and diabetic rats. In the control hearts, the basal kinase activity was similar in the cytosolic and membrane fractions. The activated kinase activity (kinase activity in the presence of Ca2 +, PS and DG) was higher in the cytosol fraction compared with the membrane fraction. The specific protein kinase C activity was also higher in the cytosol fraction compared with the membrane fraction. In the diabetic hearts, the basal kinase activity was also similar in the cytosolic and membrane fractions. However, the activated kinase activity was higher in the membrane fraction compared with the cytosolic fraction. As well, the specific protein kinase C activity was higher in the membrane fraction compared with the cytosolic one. Therefore, the activated kinase activity as well as the specific protein kinase C activity in the cytosolic fraction in the diabetic hearts was significantly lower than that in the control hearts, whereas the activated kinase activity 87 and the specific protein kinase C activity in the membrane fraction in the diabetic hearts was significantly higher than that in the control hearts. It was also noted that both basal kinase activity and the kinase activity in the presence of Ca2 + alone in the membrane fraction were higher in the diabetic hearts compared with controls. Total protein kinase C activity, calculated as the sum of the cytosolic and membrane fractions, was not significantly different between diabetic and control animals. It i s also demonstrated in Table 12 that Ca2 + and phosphatidylserine are required for protein kinase C activity in both the cardiac cytosolic and membrane fractions. It can be seen that when Ca2 + alone was added, there was no significant change in the kinase ac t i v i t y , either in the cytosolic or membrane fractions. Both the cytosolic and membrane fractions showed a significant increase in kinase activity when both Ca^ and phosphatidylserine were added to the incubation mixture. A significant amount of protein kinase C activity in the membrane fraction was detected by the addition of EGTA and Triton X-100, since both EGTA and Triton X-100 were 2 + found to be essential in the exposure of the Ca. - and phosphatidylserine-binding sites of protein kinase C in cardiac membrane preparations (Yuan and Sen, 1986). Triton X-100, having l i t t l e or no effect when used alone, has been found to enhance the a b i l i t y of EGTA to solubilize a large amount of the enzyme from the membrane fraction (Katoh and 88 Kuo, 1982). Triton X-100 at the concentration of 0.075% was chosen since i t has been reported to be the optimum one required to expose the kinase C activity (Yuan and Sen, 1986) . EGTA was used at a concentration of 2 mM since higher concentrations (up to 10 mM) did not release additional enzyme (Katoh and Kuo, 1982). Table 13 demonstrats the protein kinase C activity of control and diabetic rat ventricles with or without norepinephrine stimulation. Results showed that protein kinase C activity was not altered significantly by norepinephrine stimulation. F i g u r e 4 Basal inositol monophosphate (IPi) formation in right ventricles of control and diabetic rats. Tissues were incubated with propranolol (10 /iM) for 30 min. IP^ levels were then measured using the radioisotope method. Results are mean ± S.E.M. n, number of ventricles. 90 91 T a b l e 2 G E N E R A L F E A T U R E S O F E X P E R I M E N T A L R A T S ( T i m e C o u r s e S t u d y ) Control Diabetic (n=15) (n=15) I n i t i a l Body Weight (g) 228±3 221±3 Final Body Weight (g) 458±8 362±8* A Body Weight (g) 230±8 141±8* Plasma Glucose (mmol/1) 5.49±0.18 20.45±0.23* Plasma Insulin (/xU/ml) 49±6 25±4* * Significantly different from controls; P<0.05. 92 F i g u r e 5 Time course of body weight changes in control and six-week diabetic rats. Points represent mean ± S.E.M. n, number of hearts. (Time course study) * Significantly different from controls, p<0.05. 94 T a b l e 3 P L A S M A L I P I D P R O F I L E O F T H E E X P E R I M E N T A L R A T S ( T i m e C o u r s e S t u d y ) Control Diabetic (n=15) (n=15) Plasma triglyceride 1.38±0.13 3.92±0.15* (mmol/1) Plasma cholesterol 1.77±0.22 3.31±0.23* (mmol/1) * Significantly different from controls; P<0.05. 95 F i g u r e 6 Right ventricular wet weights of control and STZ-diabetic rats. Results are mean ± S.E.M. for the number of ventricles shown in parentheses. (Time course study) * Significantly different from controls, p<0.05. 96 0 .400 0 .300 D O 0 .200 c > CP 0 .100 0 .000 Contro l (n= 1 5) Diabet ic (n= 1 5) F i g u r e 7 Time course of the effect of norepinephrine (10 JUM) on inositol monophosphate (IP3.) formation in right ventricles from control and diabetic animals. Norepinephrine was applied 15 min after addition of propranolol (10 f/M) . Tissues were incubated for the fixed time period and IP^ levels were measured using the radioisotope method. Points represent mean ± S.E .M. n, number of ventricles. * Significantly different from controls, p<0.05. 99 T a b l e 4 G E N E R A L F E A T U R E S O F E X P E R I M E N T A L R A T S ( C o n c e n t r a t i o n - R e s p o n s e S t u d y ) Control Diabetic (n=15) (n=15) I n i t i a l Body Weight (g) 202±2 200±2 Final Body Weight (g) 423±10 327±10* A Body Weight (g) 221±10 127±10* Plasma Glucose (mmol/1) 5.99±0.19 20.21±0.24* Plasma Insulin (/xU/ml) 44±5 25±3* * Significantly different from controls; P<0.05. 100 F i g u r e 8 Time course of body weight changes in control and six-week diabetic rats. Points represent mean ± S.E.M. n, number of rats. (Concentration-response study) * Significantly different from controls, p<0.05. 101 STZ injection T a b l e 5 P L A S M A L I P I D P R O F I L E O F T H E E X P E R I M E N T A L R A T S ( C o n c e n t r a t i o n - R e s p o n s e S t u d y ) Control (n=15) Diabetic (n=15) Plasma triglyceride (mmol/1) 1.28±0.13 3.74±0.14* Plasma cholesterol (mmol/1) 1.69±0.13 2.58±0.16* * Significantly different from controls; P<0.05. F i g u r e 9 Right ventricular wet weights of control and STZ-diabetic rats. Results are mean ± S.E.M. for the number of ventricles shown in parentheses. (Concentration-response study) * Significantly different from controls, p<0.05. 104 0 .400 sz 0 .300 3 O 0 . 2 0 0 -c <D > SZ CP 'cc 0 . 1 0 0 0 .000 C Z l Cont ro l ( n = 1 5 ) B f l Diabet ic ( n = 1 5 ) 105 F i g u r e 10 Effect of different concentrations of norepinephrine (in the presence of propranolol) on inositol monophosphate (IPi) production in control and diabetic right ventricles. Norepinephrine was applied 15 min after addition of propranolol (10 juM) . The time of exposure to norepinephrine was 15 min. IP^ levels were measured using the radioisotope method. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p<0.05. 106 JZ CP CD CP E E Q-O Q_ i f 70 60 50 4 0 3 0 20 10 0 fl Con t ro l (n = 6) • D iabet ic (n = 6) 1 0 ~ 7 1 0 - 6 5 * 1 0 - 6 1 0 ~ 5 Norepinephr ine (M) 10" Figure 11 Effect of prazosin (10 /xM) and propranolol (10 /xM) on norepinephrine-stimulated inositol monophosphate (IPi) formation in right ventricles of control and diabetic rats. Norepinephrine (10 /xM) was applied 15 min after addition of prazosin or propranolol. The time of exposure to norepinephrine was 15 min. IP^ levels were measured using the radioisotope method. Results are mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from basal values from control rats, p<0.05. + Significantly different from basal values from diabetic rats, p<0.05. 108 cn cu <: CP E \ E ex u QL BASAL NE NE+PRAZ NE+PROP F i g u r e 12 Effect of prazosin, propranolol or DMSO alone on basal inositol monophosphate (IPi) formation in right ventricles of control and diabetic rats. Tissues were incubated with prazosin (10 /xM) for 15 min and IP^ levels were measured using the radioisotope method. Results are mean ± S.E.M. for the number of ventricles shown in parentheses. B A S A L P R A Z O S I N P R O P R A N O L O L D M S O [^H] IP-| ( c p m / m g wet we igh t ) O O F i g u r e 13 Body weights of control and STZ-diabetic rats treated with omega-3 fatty acid (Promega, 0.5 ml/kg/day). Results are mean ± S.E.M. for the number of animals shown in parentheses. * Significantly different from controls, p<0.05. + Significantly different from controls treated with omega-3 fatty acid, p<0.05. 112 cn cn CD O CD 8 0 0 600 4 0 0 -2 0 0 -Contro l S S 3 Cont ro l with f i s h - o i l I X I Diabet ic with f ish—oil 113 F i g u r e 14 Plasma glucose levels of control and STZ-diabetic rats treated with omega-3 fatty acid (Promega, 0.5 ml/kg/day). Results are mean ± S.E.M. for the number of animals shown in parentheses. * Significantly different from controls, p<0.05. + Significantly different from controls treated with omega-3 fatty acid, p<0.05. 114 (n = 9) CZD Cont ro l BBS Diabet ic KSS Cont ro l with f i s h - o i l CKJ Diabet ic with f i s h - o i l F i g u r e 1 5 Plasma insulin levels of control and STZ-diabetic rats treated with omega-3 fatty acid (Promega, 0.5 ml/kg/day). Results are mean ± S.E.M. for the number of animals shown in parentheses. * Significantly different from controls, p<0.05. + Significantly different from controls treated with omega-3 fatty acid, p<0.05. 117 F i g u r e 16 Plasma triglyceride levels of control and STZ-diabetic rats treated with omega-3 fatty acid (Promega, 0.5 ml/kg/day). Results are mean ± S.E.M. for the number of animals shown in parentheses. * Significantly different from controls, p<0.05. + Significantly different from controls treated with omega-3 fatty acid, p<0.05. + Significantly different from diabetics, p<0.05. 118 o (D o o E CO Q_ Contro l KSS Contro l with f i s h - o i l Diabet ic I X I Diabet ic with f ish—oil 119 F i g u r e 17 Plasma cholesterol levels of control and STZ-diabetic rats treated with omega-3 fatty acid (Promega, 0.5 ml/kg/day). Results are mean ± S.E.M. for the number of animals shown in parentheses. * Significantly different from controls, p<0.05. + Significantly different from controls treated with omega-3 fatty acid, p<0.05. 120 o E CD CO <D O XL o o E C/) 3 Con t ro l SSS Cont ro l with f i s h - o i l Diabet ic I X I Diabet ic with f ish—oil F i g u r e 18 Right ventricular wet weights of control and STZ-diabetic rats treated with omega-3 fatty acid (Promega, 0.5 ml/kg/day) . Results are mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p<0.05. + Significantly different from controls treated with omega-3 fatty acid, p<0.05. 122 0 .400 cn 0 .300 CD o .y 0 . 2 0 0 c CD > CT> or CD 0 .100 0 .000 I I Con t ro l K53 Cont ro l with f i s h - o i l Diabet ic XH Diabetic with f ish —oil F i g u r e 1 9 Effect of omega-3 fatty acid (Promega, 0.5 ml/kg/day) on cardiac norepinephrine-stimulated inositol monophosphate (IPi) formation in control and diabetic rats. Norepinephrine was applied 15 min after addition of propranolol (10 fiM) . The time of exposure to norepinephrine was 15 min. IP^ levels were measured using the radioisotope method. CON, controls; DIA, diabetics; F, omega-3 fatty acid-treated. Points represent mean ± S.E.M. n, number of ventricles. * Significantly different from untreated controls, p<0.05. + Significantly different from treated controls, p<0.05. 124 CO II c x x x x x x x x * x x x x x x x + J V ' W v w N Z \y\y v \ z ^ZZZZZZZZZZZZZZ H X X X X X X X X X X X X ^ ^zzzzzzzzzzzzz. o CD o in ~ T ~ o —r_ o —r~ o CM ~ T " o I < < Li_ I O O O O (}/w }8M 6LU/LUC IO) Ldi [H ]^ 125 F i g u r e 2 0 Effect of omega-3 fatty acid (Promega, 0.5 ml/kg/day) on cardiac norepinephrine-stimulated inositol bisphosphate ( I P 2 ) formation in control and diabetic rats. Norepinephrine was applied 15 min after addition of propranolol (10 /xM) . The time of exposure to norepinephrine was 15 min. IP2 levels were measured using the radioisotope method. CON, controls; DIA, diabetics; F, omega-3 fatty acid-treated. Points represent mean ± S.E.M. n, number of ventricles. * Significantly different from untreated controls, p<0.05. + Significantly different from treated controls, p<0.05. 126 CO Ii c xxxxx^ t n x x x x x : *-¥///////// H X X X X X X X S ^-Y//////////ZZZZL X X X X X X X X ] o CN —r~ LO — r ~ o l O Lt_ ! < < Q I z : o o :z o o I o LU 10 I o L U 2 CO I o • (yw }9M 6LU/LUC1O) [ L ^ ] F i g u r e 21 Effect of omega-3 fatty acid (Promega, 0.5 ml/kg/day) on cardiac norepinephrine-stimulated inositol trisphosphate ( I P 3 ) formation in control and diabetic rats. Norepinephrine was applied 15 min after addition of propranolol (10 i M ) . The time of exposure to norepinephrine was 15 min. I P 3 levels were measured using the radioisotope method. CON, controls; DIA, diabetics; F, omega-3 fatty acid-treated. Points represent mean ± S.E.M. n, number of ventricles. 128 CO II c H X X X 5 x x x x X X X X X x s H X X X X X ^/////////7Z\ H : o CM LO O I LL. I < < I O O O O O I O LU m I o LU CO I o • ( } M }8M 6 LU/LUC J O ) [f-j^] 129 F i g u r e 22 Effect of omega-3 fatty acid (Promega, 0.5 ml/kg/day) on cardiac norepinephrine-stimulated inositol tetrakisphosphate (IP4) formation in control and diabetic rats. Norepinephrine was applied 15 min after addition of propranolol (10 /xM) . The time of exposure to norepinephrine was 15 min. IP4 levels were measured using the radioisotope method. CON, controls; DIA, diabetics; F, omega-3 fatty acid-treated. Points represent mean ± S.E.M. n, number of ventricles. pH] I P 4 (cpm/mg wet wt) • m o I CD m o I cn m o I K X X X X o o z I K X X X X X > > I 1 . o ro o S X X X X H : X X X X H II CD OCT T a b l e 6 G E N E R A L F E A T U R E S O F C O N T R O L A N D D I A B E T I C R A T S 6 W E E K S A F T E R A D M I N I S T R A T I O N O F S T R E P T O Z O T O C I N Control Diabetic (n=12) (n=12) Body Weight (g) Plasma glucose (mmol/1) Plasma insulin (/iU/ml) 456±8 365±8* 6.0410.13 18.13±0.15* 45±5 27±3* * Significantly different from controls; P<0.05. F i g u r e 2 3 Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on change in tension (% of maximum) in right ventricles of control and diabetic rats. Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /xM) . At each concentration, the inotropic response to norepinephrine was allowed to stabilize before i t was recorded. The stabilization time was approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p < 0.05. 133 - 7 - 6 - 5 - 4 - 3 - 2 -1 N o r e p i n e p h r i n e ( l og M) 134 F i g u r e 2 4 Estimated PD2 values for norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Results are mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from PD2 values for norepinephrine in control rat hearts, p<0.05. 136 F i g u r e 2 5 Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on A change in tension in control and diabetic right ventricles. Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /xM) . At each concentration, the inotropic response to norepinephrine was allowed to stabilize before i t was recorded. The stabilization time was approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p<0.05. 1.000 0 .800 0 .600 J •o Contro l (n = 6) a Diabetic (n = 6) * T / A 0 .400 0 .200 H 0 .000 •7 A ' T -o-J . T _0' -O X o - 6 - 5 - 4 - 3 Norepinephr ine (log M) T -2 - 2 F i g u r e 2 6 A Change in tension in response to 10 M norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /iM) . The inotropic response to norepinephrine was allowed to stabilize before i t was recorded. The stabilization time was approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p<0.05. 140 F i g u r e 2 7 Norepinephrine (10 mM)-stimulated changes in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] levels in right ventricles from control and STZ-diabetic rats. Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 MM) . At the end of the determination of concentration-response curves, tissues were freeze-clamped and Ins(l,4,5)P3 levels were determined using a protein binding assay k i t . The basal Ins(l,4,5)P3 levels [Ins(1,4,5)P3 levels in the absence of norepinephrine] were 0.19 ± 0.02 in control right ventricles and 0.18 ± 0.02 in diabetics. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p<0.05. 142 T a b l e 7 E F F E C T O F P R A Z O S I N O N A L P H A ^ - A D R E N O C E P T O R - M E D I A T E D I N O S I T O L 1 , 4 , 5 - T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 / I P 3 ] ( P M O L / M G T I S S U E ) F O R M A T I O N A N D I N O T R O P I C R E S P O N S E S (G) I N C O N T R O L A N D D I A B E T I C R I G H T V E N T R I C L E S . Control (n=8) Diabetic (n=8) IP3 A Change IP3 A Change in tension in tension Basal 0.20±0.08 - 0.30±0.08 NE 0.76±0.10 0.38±0.10 2.98±0.19* 0.72±0.08* Prazosin + NE 0.22±0.09 0.02±0.00 0.31±0.07 O.OliO.OO Rat right ventricles were treated with prazosin (10 jxM) for 15 min in the presence of propranolol (10 tiM) and exposed to 10 mM norepinephrine (NE) for 3 min. * Significantly different from controls; p<0.05. F i g u r e 2 8 Time course of positive inotropic effects mediated by alphai-adrenoceptor stimulation in right ventricles from control (a) and diabetic (b) rats. Norepinephrine (NE) (10~3 M) was applied to the tissue bath 15 min after addition of propranolol (10 /iM) . 144 F i g u r e 2 9 Time course of the effect of norepinephrine (10 /xM) on inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation in right ventricles of control and diabetic rats. Norepinephrine (10 /xM) was applied to the tissue bath 15 min after addition of propranolol (10 /xM) . Tissues were freeze-clamped at indicated times after addition of norepinephrine. Ins(l,4,5)P3 levels were determined using a protein binding assay k i t . The basal values of Ins(l,4,5)P3 [Ins(1,4,5)P3 levels in the absence of norepinephrine] at zero time were 0.17 ± 0.02 in the control group and 0.18 ± 0.02 in the diabetic group. The basal levels over the time periods studied did not change significantly compared with the basal values at zero time. Points represent mean ± S.E.M. n, the number of ventricles. * Significantly different from controls at the same time point, p<0.05. 146 CD D U) CO CP ^E 75 E Gi-r o Q_ "in" 2 .500 2 .000 1.50CH 1.000 £ 0 .500 0 .000 4 O O Contro l A - A Diabet ic n = 7 -i 1 1 1 i i 0 15 30 45 60 75 90 105 1 2 0 1 3 5 1 5 0 1 6 5 1 8 0 1 9 5 Time (sec ) 147 T a b l e 8 % C H A N G E I N I N O S I T O L 1 , 4 , 5 - T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] L E V E L S A T D I F F E R E N T T I M E P O I N T S O F N O R E P I N E P H R I N E S T I M U L A T I O N I N C O N T R O L A N D D I A B E T I C R I G H T V E N T R I C L E S . % Change in Ins(1,4,5)P3 Control Diabetic (n=7) (n=7) 0 sec 100 100 15 sec 492±25 607±70 3 0 sec 537±95 774±31* 60 sec 858±11 911±35* 180 sec 609±26 872188* Norepinephrine (10 »uM) was applied to the tissue bath 15 min after addition of propranolol (10 /xM) . Tissues were freeze-clamped at indicated time periods of norepinephrine stimulation. Ins(l,4,5)P3 was determined using a protein binding assay k i t . Values are expressed as mean ± S.E.M. n, number of right ventricles. Basal level=100 %. * Significantly different from controls at the same time point; P<0.05. 148 F i g u r e 3 0 Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on change in tension (% of maximum) in right ventricles of control and diabetic rats. Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 [iM) . At each concentration, the inotropic response to norepinephrine was allowed to stabilize before i t was recorded. The stabilization time was approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p<0.05. 149 150 F i g u r e 3 1 Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] formation in right ventricles from control and diabetic rats. Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 izM) . Tissues were freeze-clamped when the steady inotropic responses to each concentration of norepinephrine were obtained. Ins(l,4,5)P3 levels were determined using a protein binding assay k i t . The basal values of Ins(l,4,5)P3 [Ins(1,4,5)P3 levels in the absence of norepinephrine] over the concentration range studied did not change significantly compared with basal levels shown on the graph. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p < 0.05. 151 CD CO CO E o E Q_ 0_ i f ? CO c 2 .000 1.500 1.000 0 .500 0 .000 Contro l (n = 5) Diabet ic (n=5) I I Basa l 1 0 - 6 1 0 ~ 5 1 0 ~ 4 1 0 - 3 Norepinephr ine (M) 152 T a b l e 9 % C H A N G E I N I N O S I T O L 1 , 4 , 5 - T R I S P H O S P H A T E [ I N S ( 1 , 4 , 5 ) P 3 ] F O R M A T I O N S T I M U L A T E D B Y D I F F E R E N T C O N C E N T R A T I O N S O F N O R E P I N E P H R I N E I N C O N T R O L A N D D I A B E T I C R I G H T V E N T R I C L E S . % Change in Ins(1,4,5)P3 Control Diabetic (n=5) (n=5) 10"6 M 110±11 165±10* 10"5 M 176±46 310±49* 10~4 M 195±33 336±15* 10"3 M 331±18 376±17* Norepinephrine (10 juM) was applied to the tissue bath 15 min after addition of propranolol (10 /iM) . Tissues were freeze-clamped when the steady inotropic responses to each concentration of norepinephrine stimulation were obtained. Ins(l,4,5)P3 was determined using a protein binding assay k i t . Values are expressed as mean ± S.E.M. n, number of right ventricles. Basal level=100 %. * Significantly different from controls; P<0.05. 153 F i g u r e 32 Correlation curves for the concentration-responses of alphai-adrenoceptor-mediated positive inotropic effect and inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] formation in control and diabetic right ventricles. r , correlation coefficient. 154 cr> E 2 .000 1.500 o Con t ro l (n=5) r = 0 . 9 6 • D iabet ic (n=5) r =0.79 o E Q_ Q-m CO c 1.000 0 .500 H 0 .000 0 . 0 0 0 0 .200 0 .400 0 .600 0 . 8 0 0 1.000 A Change in tens ion (g) 155 F i g u r e 33 Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on change in tension (% of maximum) in right ventricles of control and diabetic rats. Influence of the cyclooxygenase inhibitor indomethacin (10 nK). Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /zM) and indomethacin. At each concentration, the inotropic response to norepinephrine was recorded after i t was stabilized which took approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from the other three groups, p<0.05. 156 o o Control (n = 6) • • Control with Indomethacin Norepinephrine (log M) F i g u r e 34 Influence of indomethacin on the estimated PD2 values for norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Results are mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from PD2 values for norepinephrine in control rat ventricles, p<0.05. 158 Contro l l\\N Contro l with i ndomethac in I X 1 Diabet ic with i ndome thac in F i g u r e 3 5 Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on A change in tension in right ventricles of control and diabetic rats. Influence of indomethacin (10 /xM) . Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /zM) and indomethacin. At each concentration, the inotropic response to norepinephrine was recorded after i t was stabilized which took approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from the other three groups, p<0.05. 160 cn cu cn c o sz o <. 1 .200 1 .000 o o Contro l • • Contro l with Indomethocin A A Diabet ic * Diabet ic with Indomethacin -7 6 -5 - 4 - 3 Norepinephr ine (log M) -2 (n = 6) - 1 161 F i g u r e 3 6 A Change in tension in response to 10~2 M of norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Influence of indomethacin (10 /xM) . Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /xM) and indomethacin. The inotropic response to norepinephrine was recorded after i t was stabilized which took approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls without indomethacin, p<0.05. 162 CP CD CT> c o sz o < 2 . 0 0 0 1.500 1 .000-0 .500 0 .000 (n = 6) C o n t r o l D i a b e t i c ESS C o n t r o l wi th i n d o m e t h a c i n C X J D i a b e t i c wi th i n d o m e t h a c i n 163 F i g u r e 37 Norepinephrine (10 mM)-stimulated changes in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] levels in right ventricles of control and diabetic rats. Influence of indomethacin (10 /xM) . Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 juM) and indomethacin. At the end of the determination of concentration-response curves, tissues were freeze-clamped and Ins(1,4,5)P3 levels were determined using a protein binding assay k i t . The basal Ins(1,4,5)P3 levels [Ins(1,4,5)P3 levels in the absence of norepinephrine] were 0.14 ± 0.02 in the control group, 0.12 ± 0.01 in the control with indomethacin group, 0.13 ± 0.02 in the diabetic group, and 0.12 ± 0.02 in the diabetic with indomethacin group. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls without the inhibitor, p<0.05. S 164 cu CO CO 5 4 -o E C L 3 Q_ to" CO c 0 Cont ro l K \ N Cont ro l with indomethac in (n = 6) BOB Diabet ic X I Diabet ic with i ndomethac in 165 Figure 38 Concentration-response curves for the effect of norepinephrine (in the presence of propranolol) on change in tension (% of maximum) in right ventricles of control and diabetic rats. Influence of the thromboxane synthetase inhibitor imidazole (10 uM). Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /iM) and imidazole. At each concentration, the inotropic response to norepinephrine was recorded after i t was stabilized which took approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from the other three groups, p<0.05. 166 x o E M — O c o CO c CD CD cr> c o o 100- , 8 0 60 4 0 20 A 0 - 7 o o Cont ro l • • Cont ro l with Imidazole A A Diabet ic A Diabet ic with Imidazole (n = 6) - 5 - 4 - 3 Norepinephr ine (log M) - 2 F i g u r e 3 9 Influence of imidazole on the estimated PD2 values for norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Results are mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from PD2 values for norepinephrine in control rat ventricles, p<0.05. 168 o 1 Contro l K\N Contro l with imidazo le Diabetic X I Diabetic with im idazo le F i g u r e 40 A Change in tension in response to 10~* M of norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Influence of imidazole. Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /xM) and imidazole. The inotropic response to norepinephrine was recorded after i t was stabilized which took approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls without imidazole, p<0.05. 170 2 .000 1 .500-1.000 0 . 5 0 0 -0 .000 Contro l l \ \ N Cont ro l with im idazo le B O Diabet ic "XI Diabet ic with im idazo le F i g u r e 4 1 Norepinephrine (10 mM)-stimulated changes in inositol 1,4,5-trisphosphate [Ins(l,4,5)P3] levels in right ventricles from control and diabetic rats. Influence of imidazole (10 /xM) . Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 juM) and imidazole. At the end of the determination of concentration-response curves, tissues were freeze-clamped and Ins(l,4,5)P3 levels were determined using a protein binding assay k i t . The basal values of Ins(l,4,5)P3 [Ins(1,4,5)P3 levels in the absence of norepinephrine] were 0.14 ± 0.01 in the control group, 0.13 ± 0.02 in the control with imidazole group; 0.13 ± 0.01 in the diabetic group and 0.12 ± 0.02 in the diabetic with imidazole group. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from controls, p<0.05. + Significantly different from controls with imidazole and diabetic group, p<0.05. 172 CD CP E \ o E C L t O Q_ in 3 (n = 6) * + 0 l l Cont ro l N \ N Cont ro l with imidazole B i Diabet ic I X I Diabet ic with im idazo le T a b l e 10 G E N E R A L F E A T U R E S O F C O N T R O L A N D D I A B E T I C R A T S 6 W E E K S A F T E R A D M I N I S T R A T I O N O F S T R E P T O Z O T O C I N ( T r a n y l c y p r o m i n e S t u d y ) Control Diabetic (n=6) (n=6) Body Weight (g) Plasma glucose (mmol/1) Plasma insulin (iiU/ml) 457±8 7.25±0.61 48±6 347±8* 25.27±0.61* 26±5* * Significantly different from controls; P<0.05. 174 T a b l e 11 P L A S M A L I P I D P R O F I L E O F T H E E X P E R I M E N T A L R A T S ( T r a n y l c y p r o m i n e S t u d y ) Control (n=6) Diabetic (n=6) Plasma triglyceride (mmol/1) 1.54±0.39 3.73±0.39* Plasma cholesterol (mmol/1) 1.70±0.15 2.51±0.16* * Significantly different from controls; P<0.05. F i g u r e 42 A Change in tension in response to 10 M of norepinephrine (in the presence of propranolol) in control and diabetic right ventricles. Influence of the prostacyclin synthetase inhibitor tranylcypromine (1.25 x 10~4 M) . Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 /iM) and tranylcypromine. The inotropic response to norepinephrine was recorded after i t was stabilized which took approximately 3 min. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from the other three groups, p<0.05. 176 2 .000 1.500H 1.000 0 .500 ^ 0 .000 I I Cont ro l K\N Cont ro l with t rany lcypromine I X I Diabetic with t rany lcypromine F i g u r e 43 Norepinephrine (10 mM)-stimulated changes in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] levels in right ventricles of control and diabetic rats. Influence of tranylcypromine (1,25 x 10~4 M) . Norepinephrine was applied to the tissue bath 15 min after addition of propranolol (10 juM) and tranylcypromine. At the end of the determination of concentration-response curves, tissues were freeze-clamped and Ins(1,4,5)P3 levels were determined using a protein binding assay k i t . The basal values of Ins(l,4,5)P3 [Ins(1,4,5)P3 levels in the absence of norepinephrine] were 0.13 ± 0.01 in the control group, 0.12 ± 0.01 in the control with tranylcypromine group, 0.12 ± 0.02 in the diabetic group, and 0.13 ± 0.01 in the diabetic with tranylcypromine group. Points represent mean ± S.E.M. for the number of ventricles shown in parentheses. * Significantly different from the other three groups, p<0.05. 178 <u if) cn E o E ex. ro Q_ to c CZJ Cont ro l ESS Cont ro l with t ranylcypromine I X I Diabet ic with t rany l cyp romine 179 T a b l e 12 P R O T E I N K I N A S E C A C T I V I T Y O F C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S ( p m o l P i / m g t i s s u e / m i n ) Control (n=8) Diabetic (n=8) Protein Kinase Activity Cytosol -Ca,-PS,-DG 12.2±2.3 16.1±1.4 +Ca,-PS,-DG 16.2±2.3 16.2±1.4 +Ca,+PS,+DG 38.2±2.3* 20.5±1.4+ Membrane -Ca,-PS,-DG 13.7±1.6 20.7±2.6+ +Ca,-PS,-DG 15.5±1.6 20.3±2.6+ +Ca,+PS,+DG 21.5±1.6* 37.8±2.6*+ Protein Kinase C Activity Cytosol 22.0±1.9 4.4±1.3+ Membrane 5.9±2.4 17.611.8+ Total Activity 27.4±2.9 22.3±2.8 PS, phosphatidylserine. DG, D-l-stearoyl-2-arachidonyl glycerol. * Significantly different from "-Ca2+,-PS,-DG" values in the same preparations in control or diabetic animals, p<0.05. + Significantly different from the relative control values, p<0.05. 180 T a b l e 13 P R O T E I N K I N A S E C ( P K C ) A C T I V I T Y O F C O N T R O L A N D D I A B E T I C R A T V E N T R I C L E S W I T H O R W I T H O U T N O R E P I N E P H R I N E ( N E ) S T I M U L A T I O N . (pmol Pi/mg tissue/min) Control Diabetic With NE Without With NE Without (n=4) NE (n=8) (n=4) NE (n=8) PK Activity Cytosol -Ca~ ,-PS,-DG +Ca2+,-PS,-DG +Ca2+,+PS,+DG 11. 14. 36. 2±2. 3±1. 0±2. 1 9 0* 12. 16. 38. 2±2. 2±2. 2±2. 3 3 3* 15. 16. 21. 2±2. 3±2. 1±2. 1 1 1+ 16. 16. 20. 1±1. 2±1. 5±1. 4 4 4+ Membrane -Ca2+,-PS,-DG +Ca2+,-PS,-DG +Ca2+,+PS,+DG 13. 14. 20. 0±1. 5±1. O i l . 7 7 7* 13. 15. 21. 7±1. 5±1. 5±1. 6 6 6* 19. 20. 38. 4±2. 5±2. 6±2. 3+ 3+ 3*+ 20. 20. 37. 7±2. 3±2. 8±2. 6+ 6+ 6*-PKC Activity Cytosol 21. 7±1. 8 22. 0±1. 9 4. 8±1. 5+ 4. 4±1. 3+ Membrane 5. 6±1. 2 5. 9±2. 4 17. 9±2. 0+ 17. 6±1. 8+ Total Activity 27. 5±2. 1 27. 4±2. 9 22. 8±2. 6 22. 3±2. 8 Norepinephrine (10 mM) , where used, was applied to the tissue bath 15 min after addition of propranolol (10 /xM) . Tissues were frozen in liquid nitrogen for the subsequent measurement of PKC activity on the same day. PS, phosphatidylserine. DG, D-l-stearoyl-2-arachidonyl glycerol. * Significantly different from l,-Ca2+,-PS,-DG" values in the same preparations in control or diabetic animals, p<0.05. + Significantly different from the relative control values, p<0.05. 181 D I S C U S S I O N 1. Alphai-adrenoceptor-mediated Inositol Phosphates Formation and Positive Inotropic Effect in the Control Rat Heart Extensive studies have been made of hormone-stimulated phosphatidylinositol turnover in many different tissues, but detailed studies on alphai-adrenoceptor-mediated phosphatidylinositol hydrolysis in heart tissue have been limited. The observations presented here demonstrate that stimulation of alpha^-adrenoceptors with norepinephrine increased the formation of IP^ and IP2 (measured by a radioisotope method) and Ins(1,4,5)P3 [measured by a Ins(l,4,5)P3 protein binding assay kit] in rat right ventricles, which i s in general agreement with the data from embryonic chick heart cells (Brown and Jones, 1986); neonatal rat cardiomyocytes (Karliner and Simpson, 1986); rat cardiomyocytes (Brown et a l . , 1985; Kaku et a l . , 1986); mouse atria (Scherer and Sastre, 1985); rat papillary muscles (Otani et a l . , 1986); rat ventricles (Poggioli et a l . , 1986); and intact perfused rat hearts (Woodcock et a l . , 1987; Woodcock et a l . , 1988). The molecular mechanism by which the activated receptor couples to phospholipase C-mediated hydrolysis of phosphoinositide i s unknown. Recent evidence (Cockcroft and Gomperts, 1985; Gonzales and Crews, 1985; Straub and Gershengorn, 1986) suggests that a guanine nucleotide-182 binding protein (G-protein) regulates phospholipase C acti v i t y , a molecular mechanism possibly analogous to that by which the activity of adenylate cyclase i s regulated (Gilman, 1984). The hypothesis that a G-protein regulates phospholipase C activity in the heart i s supported by the observation that guanosine-5'-0-(3-thiotriphosphate) (G T P Y S ) and other guanine nucleotides stimulate the accumulation of [3H]IP3 in permeabilized chick heart ce l l s (Jones, 1988). Masters et a l . (1985) have previously shown in the intact chick heart c e l l that pertussis toxin blocks muscarinic receptor-mediated inhibition of adenylate cyclase, but does not block muscarinic stimulation of PI hydrolysis. Pertussis toxin pretreatment also f a i l s to block GTPrS-stimulated IP^ formation in the permeabilized heart. Recently, i t was shown that pertussis toxin could not inhibit the alphai-adrenoceptor-mediated effect on IPi production in the heart (Schmitz et a l . , 1987b), nor could i t influence the alphai-adrenoceptor-mediated positive inotropic response (Bohm et a l . , 1987). The putative G-protein to regulate phosphlipase C in the heart would therefore appear to be a protein other than G± or GQ, both of which can be ribosylated by pertussis toxin (Martin et a l . , 1985). Our results showed that norepinephrine (in the presence of propranolol) resulted in the formation of IPi and Ins(1,4,5)P3 in a time and concentration dependent manner. The increase in Ins(l,4,5)P3 preceded the increase in the 183 alphai-adrenoceptor-mediated positive inotropic effect. Moreover, stimulation of alphai-adrenoceptors increases force of contraction and Ins(l,4,5)P3 approximately to the same extent and a l l effects were blocked by the alpha^-adrenoceptor antagonist prazosin. This provided evidence that the alphai-adrenoceptor-mediated increase in Ins(l,4,5)P3 and force of contraction may be related causally. The molecular mechanism of the alphai-adrenoceptor-mediated positive inotropic effect in the heart is poorly understood. It has been suggested to be partly due to a cAMP-independent increase in transmembrane slow calcium inward current and an increase in calcium sensitivity of the myofibrils. Recently, the involvement of the phosphoinositide pathway with the resulting increase in IP3 in the positive inotropic effect of alphai-adrenoceptor agonists has aroused great interest. Micromolar concentrations of IP3 have been shown to release calcium from a nonmitochondrial intracellular calcium store in pancreatic ce l l s (Streb et a l . , 1983). This i n i t i a l observation has been confirmed for many other permeabilized cel l s (e.g. hepatocytes, GH3 c e l l s , Swiss 3T3, neutrophils, smooth muscles and macrophages) and microsomes (Berridge and Irvine, 1984; Burgess et a l . , 1984; Joseph et a l . , 1984; Dawson and Irvine, 1984). In isolated cardiac (Movsesian et a l . , 1985) and skeletal muscle (Scherer and Ferguson, 1985) some authors failed to observe an effect of exogenously added IP3 on calcium release from the sarcoplasmic 184 reticulum. But there i s also evidence that IP3 i s indeed able to release calcium from skeletal (Vergara et a l . , 1985; Volpe et a l . , 1985) as well as from cardiac sarcoplasmic reticulum (Hirata et a l . , 1984; Fabiato, 1986; Nosek et a l . , 1986). Further, there i s evidence that IP3 thereby increases force of contraction in skeletal (Vergara et a l . , 1985; Volpe et a l . , 1985) and cardiac muscle (Nosek et a l . , 1986). As mentioned above, alphai-adrenoceptor-stimulated phosphoinositide breakdown has been observed in several different preparations of the heart. However, i t was only recently that the relationship between alpha^-receptor-linked phosphoinositide breakdown and the regulation of contractile activity in cardiac muscle has been studied. The f i r s t evidence was provided by Scholz et a l . (1987) who showed that stimulation of alphai-adrenoceptors in rat l e f t auricles increased force of contraction and IP3 formation to approximately the same extent. Subsequently, i t was observed that the increase in IP3 preceded the alphai-adrenoceptor-induced increase in force of contraction in cardiac muscle (Schmitz et a l . , 1987a). Furthermore, Otani et a l . (1988a) showed that alpha^-adrenoceptor stimulation of rat l e f t ventricular papillary muscles by phenylephrine in the presence of propranolol resulted in rapid inositol phosphates formation and a triphasic inotropic response in a concentration-dependent manner. The alphai-adrenoceptor antagonist prazosin or the 185 phospholipase C inhibitor neomyocin inhibited PIP2 hydrolysis and blocked a l l components of the inotropic responses. Furthermore, they suggest that IP3 may produce transient inotropic effects by mobilizing intracellular calcium which is supported by the fact that combined addition of 2,3-diphosphoglyceric acid (a competitive inhibitor of IP3 phosphatase) with phenylephrine doubled the IP3 formation and potentiated the i n i t i a l phases of inotropic responses, as well as the fact that the transient inotropic responses were insensitive to calcium entry blockers. On the other hand, diacylglycerol, along with some cofactors, may provoke a sustained positive inotropic effect by potentiating slow calcium channels through activation of protein kinase C (Figure 46). This i s evidenced by the fact that 4-phorbol-12,13-dibutyrate (PDBU) at concentrations similar to those in stimulating protein kinase C activity in vitro (Nishizuka, 1984a), in combination with caffeine or A23187 resulted in dose-dependent potentiation of an alphai-adrenoceptor-mediated sustained positive inotropic effect, which was blocked by the calcium channel blockers, nifedipine or Mn^ . The relationship between PI hydrolysis and alphai-adrenoceptor-mediated positive inotropic effect was also studied by Scholz et a l . (1988) using e l e c t r i c a l l y driven rat l e f t auricles. It was shown that the decrease in phosphatidylinositol bisphosphate and increase in IP3 preceded the increase in force of contraction. 186 F i g u r e 4 4 The inositol phospholipid cycle replenishes the supply of second messengers, Ins(l,4 ,5)P3 and DG. PLC, phospholipase C; Ptdlns, phosphatidylinositol; CDP-DG, cytidine - 5 ' -diphospho-l ,2-diacyl-sn-glycerol; DG, diacylglycerol; Ins(l)P, D-inositol 1-phosphate; Ins(4)P, D-inositol 4-phosphate; Ptdlns(4)P, phosphatidylinositol 4-phosphate; Ins(l,4)P2, D-inositol 1,4-bisphosphate; Ptdlns(4,5) P2 , phosphatidylinositol 4,5-bisphosphate; Ins(1,4,5)P3, D-inositol 1,4,5-trisphosphate; Ins(l ,3,4 )P3, D-inositol 1,3,4-trisphosphate; Ins(l ,3,4 ,5 )P3, D-inositol 1,3,4,5-tetrakisphosphate. A l l N A A D H L~LTJ r u A T P K C D P - D G N 1.2-DG I n o s i t o l l n s ( U P 4. + c y c l i c IP 0 l n s ( 4 ) P « -recep lo r G-protein P L C P L C P L C 1 A T P >. P t d - l n j < \ P t d - l n s ( 4 j P t * P f d - Ins C4.5)P 2 1,2-DG l n * < M > P 2 In* 0.4.5) P 3 • c y c l i c I P 2 + c y c l i c IP3 l n » 0 . 3 . 4 ) P 3 + cyc l ic IP3 <-C Q + + a r a c h i d o n a t e K i n a s e - C ER P l a s m a M e m b r a n e © A T P \ C a " ft Ins (1,3.4.5) P 4 + cyc l i c I P 4 188 Very recently, Kohl et al.(1989) provided evidence for alphai-adrenoceptor-mediated increase of IP3 in the nonfailing human heart, which is associated with a similar extent of increase in the force of contraction, further suggesting a physiological role of IP3 hydrolysis in the regulation of myocardial contractility. A few problems were encountered in the measurement of inositol phosphates with the radioisotope method. F i r s t , with the specific activity of the myo[2-3H]inositol (4 jLiCi/mmol) employed in our study, large standard errors and very low a c t i v i t i e s of IP3 and IP4 were found. Secondly, unless high performance liquid chromatography (HPLC) separation of inositol phosphates is used, any quantitation of the IP3 fraction may consist of two isomers, Ins(l,4,5)P3 and Ins(l,3,4)P3 (Irvine et a l . , 1984), and also may contain their cyclic forms (Wilson et a l . , 1985) as well as the Ins(l,3,4,5)P3 (Batty et a l . , 1985). In fact, Ins(l,3,4)P3 accumulation predominates over Ins(l,4,5)P3. Therefore, [3H]IP3 accumulation i s a poor index of Ins(l,4,5)P3 formation. Thirdly, there are problems with agonist-stimulated incorporation of [3H]inositol into the phosphoinositides (Chilvers et a l . , 1988). Fourthly, the radioisotope method has proven to be arduous and time-consuming . Since only Ins(1,4,5) P3 has been demonstrated to release Ca2 + in several tissues whereas other inositol phosphates were ineffective in this respect, i t i s necessary 189 to specifically measure Ins(1,4,5)P3. Therefore, we employed an Ins(1,4,5)P3 protein binding assay k i t in our study. The method provides a simple, rapid, sensitive and specific assay for the measurement of intracellular Ins(1,4,5)P3. The Ins(l,4,5)P3 protein binding assay k i t was developed using a microsomal preparation of bovine adrenal cortex which demonstrates Ins(l,4,5)P3~specific binding (Palmer et a l . , 1989). The microsomal preparation has been used to quantitate Ins(l,4,5)P3 in acid extracts of rat hepatocytes. Direct evidence for the presence of high a f f i n i t y , intracellular Ins(l,4,5)P3 binding sites has been obtained in a number of systems - bovine adrenal cortex homogenate (Baukal et a l . , 1985), permeabilised rabbit neutrophils (Spat et a l . , 1986), permeabilised guinea pig hepatocytes (Spat et a l . , 1986), l i v e r microsomal fractions and plasma membrane fractions (Guillemette et a l . , 1987), bovine adrenal cortex microsomes (Guillemette et a l . , 1987), bovine anterior pituitary microsomes (Guillemette et a l . , 1987), rat cerebellar membranes (Willcocks et a l . , 1987), and rat cerebral cortex membranes (Lo and Hughes, 1987). In addition, Willcocks et a l . (1987) have shown that the sites in rat cerebellum are stereospecific for Ins(1,4,5)P3. More recently, the purification of an Ins(l,4,5)P3 binding protein, which may be the receptor from rat cerebella, has been reported (Supattapone et a l . , 1988). A prerequisite for a second messenger role of Ins(1,4,5)P3 in the alphai-adrenoceptor-mediated increase in 190 force of contraction i s that the norepinephrine-induced increase in Ins(l,4,5)P3 should precede the increase in force of contraction. As shown in Figure 30 and 31, whereas the increase in Ins(l,4,5)P3 could already be detected at 15 sec the increase in force of contraction did not start before 45 sec. Thus, i t i s evident that the increase in Ins(1,4,5)P3 preceded the increase in force of contraction. Norepinephrine seemed to be more potent in increasing force of contraction (effect significant at 10~5 M; Figure 33) than in enhancing Ins(l,4,5)P3 in which a pronounced and significant effect was observed only at 10 M norepinephrine (Figure 34). This, however, does not necessarily suggest a dissociation between both parameters but may indicate that, i f both events are related causally, a relatively small increase in Ins(l,4,5)P3 i s capable of producing a relatively great increase in contractile force. We carried out experiments to study PI hydrolysis by using isolated rat hearts, in which alphai-adrenoceptors are relatively rich (Mukherjee et a l . , 1983; Bristow et a l . , 1988). A potent alpha-adrenoceptor antagonist, [3H]-dihydroergocryptine ([3H]DHE), has been shown to bind to the particulate membrane fraction derived from the rat myocardium (Williams and Lefkowitz, 1978; Wei and Sulakhe, 1979) at sites which have the characteristics expected of alpha-adrenoceptors. In addition, [3H]WB4101 has been reported to selectively label postsynaptic alphai-adrenoceptors in the rat heart (Yamada et a l . , 1980). In 191 fact, although there i s a substantial amount of physiological evidence to support the presence of alpha-receptors in the hearts of various species - including rat, guinea pig, rabbit, and dog - to the present, most of the binding studies have been done in rat (Mugge et a l . , 1985) and guinea pig hearts (Karliner et a l . , 1979). Furthermore, i t was shown that rat cardiac ventricular tissue contains a relative larger number of alphai-adrenoceptors than guinea-pig ventricular muscle (Rugevics and Schumann, 1987). It i s important to note that there may be a wide range of species variation in the extent of PI elevation after myocardial alpha-adrenoceptor activation, since in the rabbit papillary muscle IP turnover i s increased much less than that in the rat heart by activation of alpha adrenoceptors (Hiramoto and Endoh, 1988). Ventricular tissue of the rat was employed in the present study and may offer several advantages. F i r s t , rat ventricular alpha-adrenergic receptor density i s greater than in the a t r i a l region (Wei and Sulakhe, 1979) . Therefore, alpha-adrenergic receptor-mediated effects of catecholamines may be more readily seen with the rat ventricle compared to a t r i a l preparations. Secondly, compared with a t r i a l muscle, ventricular preparations are less affected by the variations in experimental conditions (Endoh, 1982) . In the measurement of inositol phosphates formation using the radioisotope method, lithium was employed, which 192 is based on the report by Berridge et a l . (1982) that IP3 turnover i s very rapid and that lithium, by inhibiting inositol 1-phosphatase, could be used as an amplification technique to f a c i l i t a t e the measurement of phosphoinositide products. In fact, without lithium, no or only small stimulatory effects of agonist on IP3 content are detectable (Batty and Nahorski, 1985). The rapid generation of IP3 in tissues and i t s degradation by sequential dephosphorylation to inositol i s , in particular, characteristic of a cellular second messenger (Nohorski and Batty, 1986). On the other hand, a dissociation between an increase in IP3 and an increase in force of contraction with the addition of lithium has been suggested because i t appears that the presence of lithium i s required to produce any measurable increase in inositol phosphate formation induced by phenylephrine. But lithium did not enhance the positive inotropic effect of phenylephrine (Scholz et a l . , 1988). Based on the published data on a variety of tissues (e.g. brain and salivary glands)(Berridge et a l . , 1982) including the heart (Brown and Brown, 1983; Scholz et a l . , 1988) , a concentration of 10 mM lithium and an incubation time of 15 minutes were employed in the radioisotope method because at this concentration lithium i t s e l f had only a small effect on force of contraction, but made the alpha^-adrenoceptor-mediated stimulation of PI turnover easily detected. Higher concentrations of lithium and longer times of incubation with lithium suppressed the phenylephrine-193 induced increase in inositol phosphates (Batty and Nahorski, 1985), suggesting a complex effect of lithium on phosphoinositide metabolism. 2. Changes in Alphai-adrenoceptor-induced Phosphoinositide Breakdown and Positive Inotropic Effect in the Diabetic Rat Heart It i s now well known that there are abnormalities in the myocardium in experimental as well as in c l i n i c a l human diabetes. The mechanism(s) involved in this diabetic cardiomyopathy i s s t i l l not clear, but one defect appears to occur in the alphai-adrenoceptor system. In diabetes, i t has been reported that both the sensitivity and responsiveness of the diabetic myocardium to alpha adrenoceptor agonists are altered. Generally, increased sensitivity and responsiveness of a t r i a l (Jackson et a l . , 1986; Goyal et a l . , 1987) and ventricular preparations (unpublished results from our laboratory) in response to alphai-adrenoceptor agonists have been reported in diabetes, although a decrease in the contractile force generation by methoxamine in isolated papillary muscles has also been shown (Heyliger et a l . , 1982). We found that the diabetic right ventricles were supersensitive (increase in PD2 values) and showed a higher maximum response to norepinephrine stimulation when compared with controls, which i s in accord with previous observations from most authors. 194 It i s now well known that stimulation of alpha^-adrenoceptors causes the hydrolysis of phosphoinositides in a variety of tissues including the heart. It i s therefore possible that changes in alphai-adrenoceptor stimulated phosphoinositides turnover in the diabetic heart may be involved in the changes in the responsiveness to alpha^-adrenoceptor stimulation. Results from our study show that norepinephrine induced a time- and concentration-dependent increase in Ins(l,4,5)P3 content in both control and diabetic myocardium. Ins(1,4,5)P3 levels in the diabetic heart were significantly higher than in the control heart. Since Ins(l,4,5)P3 has been suggested to be one of the important intracellular second messengers, the data may indicate the involvement of Ins(l,4,5)P3 and, possibly, the resulting increase in Ca2 + release in the supersensitivity of the diabetic myocardium to alpha-adrenoceptor agonists. The increase in Ins(1,4,5)P3 may exacerbate Ca2 + overload in the diabetic state which could contribute to the development of diabetic cardiomyopathy. These postulated effects of Ins(l,4,5)P3 and Ca2 + on the diabetic heart require further investigation. As suggested by Otani et a l . (1988), Ins(l,4,5)P3 may only be able to produce transient inotropic effects by mobilizing intracellular Ca2 +, while DG may provoke a • 2 + sustained positive inotropic effect by activating slow Ca' channels through stimulation of protein kinase C. Protein kinase C i s present in the heart (Kuo et a l . , 1980; Wise et 195 a l . , 1982a), and a variety of myofibrillar, sarcoplasmic reticular and plasma membrane proteins are putative substrates for this enzyme (Lim et a l . , 1985; Lindemann, 1986). We therefore measured diacylglycerol-stimulated protein kinase C activity in the diabetic heart. Results showed that the diabetic heart had a higher activated protein kinase C acitvity in the membrane fraction compared with controls, accompanied by a decrease in cytosolic protein kinase C act i v i t y . Moreover, total enzyme activity of the soluble plus membrane fraction did not d i f f e r between the hearts from diabetics and controls when expressed on the basis of tissue weight. Thus, the increase in membranous protein kinase C appeared to be due to translocation of the enzyme from the cytosolic to the membrane fraction as suggested by May et a l . (1985). Nevertheless, alternative interpretations of these findings must be considered. Several isoenzymic forms of protein kinase C have been described that d i f f e r in their tissue distribution and subcellular localization (Yoshida et a l . , 1988). Each isozyme i s known to be encoded by a different gene and thus may be diff e r e n t i a l l y expressed. We cannot exclude the possibility that the shift in subcellular distribution of protein kinase C that we observed in the hearts from diabetic rats reflects an altered pattern of protein kinase C isozyme expression. The observed increase in the membranous protein kinase C activity has also been shown in diabetic nerve. Also, Lee et a l . (1989) have reported that 196 the membranous pool of protein kinase C activity was increased 100% by elevation of the glucose level in cultured bovine retinal capillary endothelial c e l l s . Recent models envision the activated protein kinase C as a component of a quaternary complex consisting of the enzyme, Ca2 +, acid phospholipid (such as phosphatidylserine) and DG, presumably associated with a cellular membrane structure (Garong et a l . , 1986). Thus, the observed increase in membranous protein kinase C in the diabetic ox heart may be due to increased membranous DG, Ca and/or acidic phospholipid levels. It might be postulated that the increased responsiveness of the diabetic heart to alpha^-adrenoceptor stimulation may enhance the activity of phospholipase C, resulting in the increase in phosphoinositides turnover and the increase in the level of DG which i s known to be an important second messenger to activate protein kinase C. In fact, an increase in DG level in the hearts of diabetic Wistar rats has recently been reported (Okumura et a l . 1988). Nevertheless, the relationship between the increase in responsiveness to alphai-adrenoceptor stimulation and the increase in DG formation in the diabetic heart requires further investigation. DG, in addition to being derived from the breakdown of phosphoinositides, can also be synthesized de novo from the glycolytic intermediates dihydroxyacetone phosphate and glycerol 3-phosphate (Peter-Riesch et a l . , 1988) by stepwise acylation. Hyperglycemia in diabetes may 197 therefore enhance the glycolytic pathway with a resulting increase in DG levels which further stimulates protein kinase C. There is also the possibility of an effect of glucose to enhance phospholipase C-mediated hydrolysis of phosphatidylcholine (Besterman et a l . , 1986), or phospholipase C-induced hydrolysis of inositol phospholipids (Dunlop et a l . , 1984). This i s supported by the fact that elevation of glucose concentration can cause an elevation of membranous protein kinase C activity in cultured bovine retinal capillary endothelial ce l l s (Lee et a l . , 1989). However, insulin treatment failed to normalize DG content in the diabetic myocardium (Okumura et a l . , 1988). Therefore, i t i s not l i k e l y that DG i s activated by an increase in glucose levels in the diabetic heart. . . . . . o+ Diabetes mellitus i s associated with an altered Ca* disposition. It has been shown that the a b i l i t y of the sarcoplasmic reticulum to take up Ca2 + i s impaired in the diabetic heart (Penpargkul et a l . , 1981; Lopaschuk et a l . , 1983). Glucose has been shown to increase cytosolic Ca2 + in pancreatic i s l e t s (Nilsson et a l . , 1988) and recent evidence suggests that an increase in cytosolic Ca2 + alone may be sufficient to trigger protein kinase C activation (Ho et a l . , 1988). There remains the possibility that increases in cytosolic Ca2 + in diabetes may contribute to the observed activation of protein kinase C. Whatever the mechanisms by which protein kinase C i s activated in the heart of diabetic rats, this finding i s of 198 considerable interest and potential importance. The increase in protein kinase C activity associated with the membrane fraction, an index of enzyme activation (Kim et a l . , 1988), may be involved in the sustained increase in the positive inotropic effect of the diabetic heart. However, since alphai-adrenoceptor stimulation did not further increase protein kinase C activity in these hearts, the involvement of protein kinase C in the supersensitivity of the diabetic hearts to alpha^-adrenoceptor stimulation requires further investigation. The mechanisms involved in the supersensitivity of the diabetic heart to alphai-adrenoceptor stimulation and in the increased inositol phosphates formation are s t i l l unclear. A number of po s s i b i l i t i e s could be forwarded. F i r s t l y , since treatment of diabetic rats with T3 were able to abolish the increased sensitivity to alpha-agonists (Goyal et a l . , 1987), changes in alpha-adrenoceptors may be the result of hypothyroidism. Secondly, the supersensitivity to alpha-agonist could be due to high activity of phospholipase C with an increase in DG production and protein kinase C act i v i t y . This i s supported by the fact that, in the acute diabetic state, inhibition of phospholipase C blocked the ventricular response to methoxamine in control as well as in diabetic hearts. Synthetic DG potentiated the inotropic action of the alpha-agonist in control ventricles. On the other hand, inhibition of protein kinase C parti a l l y reduced the supersensitivity to alpha-agonists in diabetic 199 ventricles and prevented the stimulatory action of DG on the positive inotropic effect of methoxamine in control ventricles (Wald et a l . , 1988). Thirdly, recent evidence indicates that a rise in cytosolic Ca2 + can also activate phospholipase C (Eberhard and Holz, 1988). It i s known that there i s a decreased uptake of Ca into diabetic SR (Penpargkul et a l . , 1981). The resulting increase in intracellular Ca2 + could then activate phospholipase C. Fourthly, in diabetes the response to alpha-adrenoceptor stimulation could be mediated by oxidative products generated from arachidonic acid metabolism. With the radioisotope method, however, we showed a decrease in IP^ and IP2 formation in the diabetic heart compared with controls. This appears to contradict the results obtained with the use of Ins(l,4,5)P3 protein binding assay k i t s . There are several possible explanations for this discrepancy. F i r s t l y , the radioisotope method is not as sensitive a method as the Ins(1,4,5)P3 protein binding assay k i t and the measurement may thus not be as accurate. Secondly, there could be a reduced metabolism from Ins(1,4,5)P3 to IPi in the diabetic state. In insulin-secreting 6-cells, i t has been shown that endogeneous rate of Ins(l,4,5)P3 production i s not fast enough to account for the rapid increased rate of lns(l,4,5)P3 formation that has been measured in response to glucose (Turk et a l . , 1986). Therefore i t has been suggested that some of the glucose metabolites, particularly 2,3-diphosphoglyceric acid and 200 fructose 1,6-bisphosphate, which increase markedly in the presence of glucose, may act to inhibit Ins(1,4,5)P3 5-phosphatase that degrades Ins(l,4,5)P3 (Rana et a l . , 1986). It i s possible that the increased glucose level as well as i t s metabolites in the diabetic heart may inhibit Ins(1,4,5)P3 5-phosphatase, which may then result in an increase in Ins(l,4,5)P3 and a decrease in IP2 and IPi levels. Thirdly, IP^ can also be produced from the breakdown of Ptdlns and PtdIns(4)P (via IP2)• Similarly, IP2 can also be produced directly from the breakdown of Ptdlns(4)P. It i s possible that, in diabetic hearts, the breakdown of Ptdlns(4,5)P2 i s much greater than the breakdown of Ptdlns(4)P and Ptdlns, which could result in an increase in Ins(l,4,5)P3 formation with a decrease in IP2 and IPi formation. 3. Effects of Omega-3 Fatty Acid Treatment on Cardiac Alphai-adrenoceptor-mediated Inositol Phosphates Formation We found that omega-3 fatty acid supplementation had no effect on the plasma glucose level of STZ-diabetic rats compared with the untreated diabetics. Plasma glucose levels in the treated nondiabetics were also not affected which i s in accord with previous observations by Black et a l . (1988). There are however also reports describing a decrease in plasma glucose level (Popp-Snijders et a l . , 1987) or worsening of hyperglycemia (Friday et a l . , 1987; 201 Kasim et a l . , 1988) by omega-3 fatty acid treatment in type II diabetic patients. We also found that omega-3 fatty acid treatment decreased plasma triglyceride levels in diabetic rats compared with untreated diabetics. This response i s similar to that observed in nondiabetic normolipidemic (Sanders and Roshanai, 1983) and nondiabetic hypertriglyceridemic individuals (Sanders et a l . , 1985). In the present study, omega-3 fatty acid supplementation did not affect plasma cholesterol level of the diabetic rats. Kasim et a l . (1988) have shown similar results. However, the data are in contrast with previous studies (Harris et a l . , 1983; Phillipson et a l . , 1985) which reported decreases in serum cholesterol and triglyceride levels during omega-3 fatty acid treatment. However, these previous studies were carried out in patients with either familial hypertriglyceridemia or familial combined hyperlipidemia. There are also reports that LDL cholesterol i s increased by omega-3 fatty acid supplementation in subjects with hyperlipidemia (Simons et a l . , 1985) or insulin-dependent diabetes (Haines et a l . , 1986). Although a suitable explanation for the variable effects of omega-3 fatty acid supplementation on plasma cholesterol concentrations cannot at this time be provided, the variable response to omega-3 fatty acids may relate to the population studied, the dose and/or duration of supplementation. 202 The decrease in serum triglyceride levels observed in the present study could be attributed to decreased hepatic triglyceride synthesis (Nestel et a l . , 1984), and/or accelerated triglyceride clearance due to increased LPL activity (Kasim et a l . , 1988). An important finding was that some of the changes during omega-3 fatty acid supplementation were persistent, e.g. those on blood pressure and serum apo B levels, whereas others were transient, e.g. plasma LPL activity (Kasim et a l . , 1988). A previous study (Simons et a l . , 1985) reported that the most prominent changes in the l i p i d p r ofile during omega-3 fatty acid treatment were in the early phase of the study. Currently, most of the studies in the literature are limited to 4-6 weeks duration. An obvious question i s the degree of compliance. Therefore, i t i s possible that the long term effects of omega-3 fatty acid may d i f f e r from their short term effects. Our results show that dietary omega-3 fatty acid supplementation had no effect on cardiac alpha^-adrenoceptor-mediated inositol phosphates production in diabetic or non-diabetic Wistar rats. This could be related to several factors. It i s known that, in relative terms, the arachidonic acid content i s decreased and omega-3 fatty acid content i s often increased in the tissue phospholipids of diabetic rats (Holman et a l . , 1983). If this i s the case, additional supplementation with omega-3 fatty acids may be of l i t t l e benefit. Secondly, potentially deleterious 203 effects of omega-3 fatty acids have been reported, including increased plasma glucose (Friday et a l . , 1987; Kasim et a l . , 1988), glycosylated hemoglobin (Glauber et a l . , 1988; Schectman et a l . , 1988), plasma total cholesterol and low density lipoprotein cholesterol (Haines et a l , 1986; Stacpoole et a l . , 1988). These adverse effects of omega-3 fatty acids have almost invariably been achieved with large doses (4-10 g/day). In the present study, a relatively small dose of omega-3 fatty acid was employed. However, the dose of omega-3 fatty acids that w i l l yield the optimal ratio of beneficial to deleterious effects i s unknown. Whether small quantities taken over a prolonged period w i l l produce the same effects as large quantities consumed over a brief period i s also unknown. It i s possible that the potential beneficial effects of the omega-3 fatty acid treatment employed in the present study was negated by putative adverse effects of the omega-3 fatty acid treatment, such that a favourable effect of omega-3 fatty acid on alphai-adrenoceptor-stimulated PI turnover in the diabetic heart was prevented. Thirdly, i t may be d i f f i c u l t to predict the effect of omega-3 fatty acids on the diabetic heart and i t s metabolism, because the pathogenesis of these complications involves multiple variables. In summary, we have shown that dietary omega-3 fatty acid supplementation did not alter the reduced alphai-adrenoceptor-stimulated inositol phosphates formation in STZ-diabetic rats. These observations indicate that the 204 changes in cardiac alphai-adrenoceptor-mediated inositol phosphates formation cannot contribute to the previously described improved cardiac function of omega-3 fatty acid treated STZ-diabetic rats. 4. Effect of Indomethacin, Imidazole and Tranylcypromine on Alphai-adrenoceptor-mediated Ins(l,4,5)P3 Formation and Positive Inotropic Effect in Control and Diabetic Rat Heart. Besides activating protein kinase C, DG can be converted to AA. AA, a fatty acid bound to membrane phospholipids, i s metabolized by cyclooxygenase, thromboxane synthetase and prostacyclin synthetase to form thromboxanes and prostaglandins, and i s metabolized by lipoxygenase to form leukotrienes. There i s evidence that one or more metabolites of AA can produce positive inotropic effects (Otani et a l . , 1988b) and these could be implicated in the pathophysiological alterations observed in diabetes mellitus (Chen and Robertson, 1979; Metz, 1981). Indeed, studies with rats rendered diabetic with STZ showed an altered metabolism of platelet AA (Johnson and Harrison, 1979) . We therefore proposed a study to investigate the possibility that some products derived from AA metabolism could be involved in the altered cardiac alphabet feet of norepinephrine. Our results have shown that the cyclooxygenase inhibitor indomethacin had no effect on either alphai-adrenoceptor-mediated positive inotropic 205 effect or Ins(1,4,5)P3 formation in the diabetic heart, but did have a stimulatory effect on both the function and Ins(l,4,5)P3 production in control hearts. The thromboxane inhibitor imidazole produced effects similar to indomethacin. In contrast, the prostacyclin synthetase inhibitor tranylcypromine reduced the alphai-adrenoceptor-mediated positive inotropic effect and Ins(l,4,5)P3 formation only in the diabetic heart and had no effect in the controls. The enhancement by indomethacin and imidazole of the alphai-adrenoceptor-mediated positive inotropic effect in the control heart could be attributed to the increase in the level of cytosolic arachidonic acid and in this way an enlarged quantity of calcium could be mobilized from the sarcoplasmic reticulum. In favour of this assumption is the observation that arachidonic acid, which might result from the diacylglycerol metabolism, is able to mobilize Ca from isolated organelles such as mitochondria and sarcoplasmic reticulum (Hokin, 1985; Zeitler et a l . , 1986). It could be that, in control hearts, the arachidonic acid level i s relatively low (Figure 45a). With the addition of indomethacin and imidazole, arachidonic acid levels increase and this could be involved in the increased inotropic responses to alphai-adrenoceptor stimulation. It i s also possible that thromboxane under normal condition may have an inhibitory effect on alphai-inotropic-effeet of the heart. F i g u r e 4 5 The possible involvement of arachidonic acid metabolites in the changes in contractility and Ins(l,4,5)P3 formation in control and diabetic hearts. 207 CONTROL HEARTS Alpha y-Ad renoceptor Stimulation Ptdlns(4,5)P 2 Turnover DG Arachidonic Acid j | C a 2 * Release from SR j | PGH 2 31 o T Y ; 208 DIABETIC HEARTS Alpha^-Adrenoceptor Stimulation Ptdins(4,5)P 2 Turnover DG f f \ t PKC Activation ff Conversion of PI to PIP 2 f f —>-lns(1,4,5)Pgff Arachidonic Acid ff Ci* Release from SB\\ Indomethacin PGH Tranylcypromine 1~~ " Imidazole H 1 PGi 2ff T X A , t cAMP \ \ Protein Kinase G Activation | | I Gp Inhibition JJ Ptdlns(4,5)P 2 Hydrolysis ff lns(1,4,5)P3ff 209 With the addition of indomethacin or imidazole, the inhibition was withdrawn and alphai-adrenoceptor-mediated positive inotropic effect was therefore enhanced. Further, decreased breakdown of arachidonic acid with the addition of indomethacin or imidazole might increase DG levels which might increase Ptdlns(4,5)P2 formation and i t s turnover. Ins(1,4,5)P3 might thus be increased. However, the exact mechanism involved in the effect of indomethacin or imidazole on the control heart with regard to both function and Ins(l,4,5)P3 formation i s s t i l l not clear. Previously, indomethacin has been shown to enhance the effect of isoproterenol, activating phosphorylase in diabetic but not in control hearts (Vadlamudi and McNeill, 1983b). There is also no interpretation for this observation. In the diabetic heart, however, indomethacin and imidazole did not have any significant effect on either function or Ins(1,4,5)P3 formation. This could suggest that thromboxane formation did not play an important role in alphai-inotropic-effeet in the diabetic heart. In fact, recent finding showed that in vivo TXA2 biosynthesis i s not altered in diabetes (Alessandrini et a l . , 1988). The effect of tranylcypromine may indicate the participation of prostaglandins in mediating the enhanced alphai~effeet of norepinephrine in the diabetic state. In previous studies by Sterin-Borda et a l . (1979, 1980), a positive inotropic action of prostacyclin (PGI2) in rat isolated atria has been reported. Further, there are 210 reports of an enhanced release of prostacyclin from the perfused heart of diabetic rats (Rosen and Schror, 1980) and from coronary arteries of acutely diabetic dogs (Sterin-Borda et a l . , 1981). Stimulation of prostaglandin synthesis by ischemia and reperfusion has been demonstrated in isolated rat hearts (Karmazyn, 1986). An increase in the concentration of prostaglandins ( P G E 2 and P G F 2 a) in coronary sinus blood has been shown to accompany a decrease in the ventricular multiple response threshold following coronary artery occlusion and release in dogs (Miyazake et a l . , 1982). In addition, according to Koltai et a l . (1988), the diabetic coronaries are more sensitive to the contractile effect of P G F 2 a than controls. However, i t must be mentioned that prostaglandins produce a wide variety of actions on the heart. The effects are quite complex and are dependent on such factors as type of prostaglandin, animal species and experimental format. Prostaglandins are able to modify transmembrane ionic permeabilities. Prostaglandins have been shown to act as Ca2 + ionophores in cardiac as well as other tissues (Karmazyn, 1986) which may, in part, explain the effect of prostaglandins on the diabetic heart. P G E 2 has been shown to inhibit sympathetic neurotransmission in the perfused rabbit heart (Hedqvist et a l . , 1970). This may suggest that the positive inotropic effect of norepinephrine in the diabetic heart i s mainly due to prostaglandins rather than to norepinephrine i t s e l f . 211 Until now the mechanism(s) of the enhanced positive inotropic effect mediated by alpha^-adrenoceptors in the diabetic heart has been unclear. The metabolic pathway of AA or distribution of metabolites from AA may be altered in the diabetic heart, favouring the production of prostacyclins. The supersensitivity and enhanced maximum response to norepinephrine of the diabetic heart could be partly explained by this increase in production of prostaglandins. It i s also possible that arachidonic acid level i s relatively high in the diabetic state, which could result in an increase in Ca release from SR and therefore an increase in contractility. Tranylcypromine i s well known as a monoamine oxidase inhibitor (Baldessarini, 1985). The resulting increased monoamine level in the central nervous system has been suggested to be the mechanism involved in the use of tranylcypromine as an antidepressant. The importance of monoamine oxidase inhibition in the heart i s not clear. The possibility can not be excluded that changes in the monoamine levels may be involved in the effect of tranylcypromine on alphai-adrenoceptor-mediated inotropic effect. However, we feel this possibility i s unlikely. Reduced cAMP levels are observed in ischemic cardiac tissue after treatment with PGI2 (Roesen et a l . , 1981). Decreased cAMP levels could result in a decrease in protein kinase G activation, which may result in a decrease in 212 inhibition of G protein (Gp protein) involved in Ptdlns(4,5)P2 turnover. Ins(l,4,5)P3 levels may therefore increase. In this regard, the addition of tranylcypromine could decrease Ins(l,4,5)P3 levels. It also could be that, with the addition of tranylcypromine, arachidonic acid levels increased and DG levels also increased. The resulting activation of protein kinase C by DG may inhibit Ptdlns(4,5)P2 hydrolysis and Ins(l,4,5)P3 levels may therefore decrease. Our results regarding inotropic responses of control and diabetic hearts to alphai-adrenoceptor stimulation in the presence of indomethacin, imidazole and tranylcypromine are different from the data from Canga and Sterin-Borda (1986), which could be due to a number of reasons. F i r s t l y , we used right ventricles of the rats while they employed right atria of the rats. Secondly, in our study, diabetes was induced by a single i.v. injection of STZ at 55 mg/kg and the duration of diabetes was 6 weeks. In contrast, they used 3-day diabetic rats which were made diabetic with a single i.p. injection of STZ at 100 mg/kg. Further, in our study, STZ was dissolved in citrate buffer which had a pH value of 4.5, whereas they dissolved STZ in citrate buffer with pH 4.8. A l l these variations in species, STZ dose and injection route and duration of diabetes may have some impact on the responses of the heart to alphai-adrenoceptor stimulation. 213 An important point with respect to the generalization of this study to intact animals and humans is that the experiments were conducted with isolated right ventricles. Prostaglandin production may be changed due to this intervention. Therefore, the importance of endogeneous prostaglandins in the involvement of the cardiac response to alphai-adrenoceptor stimulation in the diabetics cannot necessarily be inferred. S U M M A R Y A N D C O N C L U S I O N S Wistar rats made diabetic with streptozotocin are characterized by hypoinsulinemia, hyperglycemia and hyperlipidemia. Using a radioisotope method, i t was shown that stimulation of alphai-adrenoceptors by norepinephrine (in the presence of propranolol) in right ventricles resulted in the production of inositol monophosphate (IPi) in a time- and concentration-dependent manner in both control and diabetic rats. Right ventricles from diabetic rats, however, showed decreased IPi formation in response to norepinephrine as compared with controls. Omega-3 fatty acid supplementation had no significant effect on the decrease in norepinephrine-stimulated IPi and inositol bisphosphate (IP2) formation in diabetic rat ventricles. The data indicate that the changes in cardiac alphai-adrenoceptor-mediated inositol phosphates formation cannot contribute to the previously described improved cardiac function of omega-3 fatty acid-treated STZ-diabetic rats. Right ventricles from diabetic rats were supersensitive (increase in PD2 values) and showed a greater maximum inotropic response to alphai-adrenoceptor stimulation by norepinephrine (in the presence of propranolol) when compared with stimulation of control right ventricles. Using an inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] protein binding assay k i t , i t was shown that norepinephrine induced a time- and concentration-dependent increase in Ins(l,4,5)P3 contents in right ventricles of both control and diabetic rats. The increase in Ins(1,4,5)P3 levels preceded the increase in the contractility of the heart. Ins(l,4,5)P3 levels in the diabetic right ventricles were significantly higher than those in the controls. These observations suggest that the changes in Ins(l,4,5)P3 levels may be involved in the increased inotropic responsiveness to alphai-adrenoceptor stimulation in the diabetic heart. The Ca2 + overload induced by Ins(l,4,5)P3 could further be involved in the development of diabetic cardiomyopathy. In the presence of the cyclooxygenase inhibitor indomethacin, the positive inotropic effect of norepinephrine was significantly increased in control but not diabetic right ventricles. Similarly, Ins(1,4,5)P3 levels were elevated in control right ventricles treated with indomethacin as compared with untreated controls, and remained elevated in the 216 diabetic right ventricles treated with indomethacin compared with untreated diabetics. 7. The thromboxane synthetase inhibitor imidazole had similar effects as indomethacin on alphai-adrenoceptor-mediated positive inotropic effect and Ins(1,4,5)P3 formation in control and diabetic right ventricles. The nature and physiological significance of the enhanced positive inotropic effect and Ins(l,4,5)P3 formation in the control heart with the addition of indomethacin and imidazole are s t i l l unclear. 8. Inhibition of prostacyclin synthetase by tranylcypromine reduced the positive inotropic effect of norepinephrine only in diabetic right ventricles and had no effect in the control preparations. Corresponding with the above functional changes, tranylcypromine diminished norepinephrine-induced Ins(l,4,5)P3 formation in diabetic preparations without altering i t in the controls. The effect of tranylcypromine may indicate the participation of prostaglandins in mediating the enhanced alpha^-inotropic-effeet of norepinephrine in the diabetic heart. 9. Diabetic right ventricles had a higher activated protein kinase C activity in the membrane fraction as 217 compared with controls and this was accompanied by a decrease in cytosolic protein kinase C a c t i v i t y . The increase in protein kinase C activity associated with the membrane fraction may be involved in the sustained increase in alphai-adrenoceptor-mediated positive inotropic effect of the diabetic heart. 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