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Noradrenaline-induced vascular contractility and phosphoinositide metabolism in streptozotocin-diabetic… Abebe, Worku 1990

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NORADRENALINE-INDUCED VASCULAR CONTRACTILITY AND PHOSPHOINOSITIDE METABOLISM IN STREPTOZOTOCIN-DIABETIC RATS by WORKU ABEBE B.Pharm., H a i l e S e l a s s i e I U n i v e r s i t y , 1974 M . S c , U n i v e r s i t y o f B rad fo rd , 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DIVISION OF PHARMACOLOGY AND TOXICOLOGY (FACULTY OF PHARMACEUTICAL SCIENCES) We accept t h i s t h e s i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1990 ( c ) Worku Abebe, 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 Sc iences The University of British Columbia Vancouver, Canada Date 12 October, 1990 DE-6 (2/88) ABSTRACT A l t e r a t i o n s i n the r e a c t i v i t y of blood ve s se l s to neu ro t ran sm i t te r s and c i r c u l a t i n g hormones have been suggested to cause, or c o n t r i b u t e t o , some of the c a r d i o v a s c u l a r comp l i ca t i on s a s s oc i a ted w i th d i abetes m e l l i t u s . Previous s tud ie s have demonstrated t ha t aortae and mesenter ic a r t e r i e s from male r a t s w i th s t r e p t o z o t o c i n (STZ)- induced d iabetes o f 12 weeks du ra t i on are more respons ive to the c o n t r a c t i l e e f f e c t s o f no rad rena l i ne (NA). The increased respons iveness o f the d i a b e t i c a r t e r i e s to NA has been repor ted to r e s u l t from s t i m u l a t i o n of a\-adrenoceptors. Con t r ac t i on of v a s cu l a r smooth muscle induced by NA v i a a\-adrenoceptors has been shown to be a s soc i a ted w i th enhanced metabolism o f pho spho ino s i t i de s . The purpose of the present study was to i n v e s t i g a t e whether the enhanced c o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s to NA are a s soc i a ted w i th a l t e r a t i o n s i n phospho inos i t i de metabol ism. There fo re , we compared changes i n c o n t r a c t i l i t y and phospho inos i t i de metabolism i n response to NA i n aortae and mesenter ic a r t e r i e s from male r a t s w i th STZ-induced d iabetes o f 12-14 weeks du ra t i on and t h e i r age-matched c o n t r o l s . To f u r t h e r i n v e s t i g a t e the s p e c i f i c i t y and mechanisms o f a l t e r a t i o n s i n these responses, experiments were a l so conducted us ing p o r t a l v e i n s , and s e l e c t i v e a j - ad renocepto r agon i s t s , potassium c h l o r i d e and a d i r e c t a c t i v a t o r o f p r o t e i n k inase C (PKC). Maximum c o n t r a c t i l e responses but not s e n s i t i v i t i e s o f aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s to NA were s i g n i f i c a n t l y enhanced compared w i th responses of a r t e r i e s from con t r o l r a t s . In c o n t r a s t , i i responses o f p o r t a l ve ins from con t r o l and d i a b e t i c r a t s to NA were not d i f f e r e n t from each o the r . In a d d i t i o n , c o n t r a c t i l e response of d i a b e t i c a r t e r i e s to KC1 were not a l t e r e d . C o n t r a c t i l e responses o f both aortae and mesenter ic a r t e r i e s i n C a 2 + - f r e e medium t o maximum concen t ra t i on s of NA, PE and METH were s i g n i f i c a n t l y g r ea te r i n p repa ra t i on s from d i a b e t i c than from c o n t r o l r a t s . Fo l l ow ing r e a d d i t i o n of C a 2 + , responses of d i a b e t i c a r t e r i e s to the a-adrenoceptor agon i s t s were a l s o s i g n i f i c a n t l y g r ea te r than c o n t r o l s . In the absence of e x t r a c e l l u l a r Ca , c o n t r a c t i o n s of d i a b e t i c aortae to a submaximum concen t ra t i on o f NA (10" ^M) were a l s o s i g n i f i c a n t l y increased compared w i th c o n t r o l , wh i l e the responses o f d i a b e t i c mesenter ic a r t e r i e s to t h i s c oncen t r a t i on of NA were not s i g n i f i c a n t l y d i f f e r e n t from those of c o n t r o l p r epa r a t i on s . Upon r e a d d i t i o n of C a 2 + , c o n t r a c t i l e responses of both a r t e r i e s from d i a b e t i c r a t s were s i g n i f i c a n t l y g r ea te r than those from c o n t r o l s . These data i n d i c a t e tha t the maximum c o n t r a c t i l e responses of d i a b e t i c aortae and mesenter ic a r t e r i e s to a j - ad renocepto r s t i m u l a t i o n are a s soc i a ted w i th inc reased r e l ea se of i n t r a c e l l u l a r C a 2 + . However, cq -adrenoceptor -mediated C a 2 + i n f l u x may a l so be enhanced i n a r t e r i e s from d i a b e t i c r a t s p a r t i c u l a r l y i n response to submaximum concent ra t i on s o f NA. A maximum concen t ra t i on of NA (10"^M) induced a r a p i d , t r a n s i e n t decrease i n the l e v e l of [ 3 2 P ] - p h o s p h a t i d y l i n o s i t o l 4,5-b i sphosphate ( [ " ^P ] - P td I n s ( 4 , 5 )P2 ) , and a time-dependent inc rease i n the format ion of [ 3 2 P ] - p h o s p h a t i d i c a c i d ( [ 3 2 P ] - P A ) and [ 3 H ] - i n o s i t o l phosphates i n aortae from both c on t r o l and d i a b e t i c r a t s . The i nc rease i n [ H] -i n o s i t o l phosphate product ion was s e l e c t i v e l y b locked by p r a z o s i n . In i i i both types o f a o r t i c p repa ra t i on s , NA a l so induced a r a p i d , t r a n s i e n t and c o n c e n t r a t i o n - r e l a t e d e l e v a t i o n o f i n o s i t o l 1 ,4 ,5 - t r i sphosphate ( I n s ( l , 4 , 5 )P3 ) content dur ing c o n t r a c t i o n . The breakdown o f [ 3 2 P ] -Ptd Ins (4 ,5 )P2, and format ion of [ 3 2 P ] - P A , [ 3 H ] - i n o s i t o l phosphates and I n s ( l , 4 , 5 )P3 i n response to the maximum concen t ra t i on of NA were s i g n i f i c a n t l y g r ea te r i n d i a b e t i c than i n c on t r o l ao r tae . S t i m u l a t i o n of a|-adrenoceptors by a maximum concen t r a t i on o f NA (3xlO"^M) i n mesenter ic a r t e r i e s r e s u l t e d i n a t ime-dependent i nc rease i n the format ion of [ 3 H ] - i n o s i t o l phosphates i n both c o n t r o l and d i a b e t i c r a t s . NA a l so caused a r ap i d and concentrat ion-dependent e l e v a t i o n of I n s ( l , 4 ,5 )P3 content i n mesenter ic a r t e r i e s from both groups of an imals . The inc reases i n [ 3 H ] - i n o s i t o l phosphates and I n s ( l , 4 ,5 )P3 l e v e l s i n d i a b e t i c mesenter ic a r t e r i e s i n response to the maximum concen t r a t i on o f NA were s i g n i f i c a n t l y h igher than i n c on t r o l p r epa ra t i on s . In c o n t r a s t , I n s ( l , 4 ,5 )P3 product ion i n mesenter ic a r t e r i e s i n response to a submaximum concen t ra t i on of NA (10"^M) was not s i g n i f i c a n t l y d i f f e r e n t between con t r o l and d i a b e t i c p r epa r a t i on s . NA a l s o induced a time-dependent inc rease i n the p roduct ion of [ 3 H ] - i n o s i t o l phosphates i n both c on t r o l and d i a b e t i c p o r t a l v e i n s . The NA-induced format ion of [ 3 H ] - i n o s i t o l phosphates was not s i g n i f i c a n t l y d i f f e r e n t c o n t r o l and d i a b e t i c p o r t a l ve ins w i th a shor t pe r i od o f exposure (0.5 min) to the agon i s t suggest ing t ha t the extent of occurrence of e a r l y b iochemical events i n c l u d i n g the h y d r o l y s i s o f Ptd Ins(4,5)P2 i n response to NA i s s i m i l a r i n both types o f p o r t a l v e i n s . i v The r o l e o f PKC i n mediat ing enhanced c o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s to NA was a l s o i n v e s t i g a t e d . The PKC i n h i b i t o r , s taurospor ine abo l i shed the d i f f e r e n c e i n the magnitude of c o n t r a c t i o n observed between c o n t r o l and d i a b e t i c a r t e r i e s i n response to NA. Maximum c o n t r a c t i l e responses o f mesenter ic a r t e r i e s but not aortae or po r t a l ve ins from d i a b e t i c r a t s to the PKC a c t i v a t o r , PDB, were s i g n i f i c a n t l y enhanced compared w i th c o n t r o l s . The enhanced c o n t r a c t i l e responses of d i a b e t i c mesenter ic a r t e r i e s to PDB were abo l i shed i n the presence of s t au ro spo r i ne , i n the presence of C a 2 + - c h a n n e l b l o c k e r s , or i n the absence of e x t r a c e l l u l a r C a 2 + . These r e s u l t s i n d i c a t e tha t phospho inos i t i de metabolism i s enhanced i n aortae and mesenter ic a r t e r i e s from STZ-induced d i a b e t i c r a t s i n response to NA, v i a a\-adrenoceptor s t i m u l a t i o n . The enhancement may c o n t r i b u t e to the increased c o n t r a c t i l e respons iveness of these ve s se l s to NA. In a d d i t i o n , increased a c t i v a t i o n o f PKC-mediated processes , which are dependent on the presence o f e x t r a c e l l u l a r C a 2 + , may f u r t h e r c o n t r i b u t e to the enhanced c o n t r a c t i l e responses of d i a b e t i c mesenter ic a r t e r i e s to NA. However, increased i n f l u x o f e x t r a c e l l u l a r Ca^"1" by o ther mechanisms may a l so c o n t r i b u t e to the enhanced c o n t r a c t i o n of d i a b e t i c a r t e r i e s to submaximum concent ra t i on s o f NA. Diabetes does not seem to induce such changes i n p o r t a l v e i n s . Kath leen M. MacLeod, Ph.D. Thes i s Superv i so r v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES i x LIST OF FIGURES x LIST OF ABBREVIATIONS x i v ACKNOWLEDGEMENTS xv i 1. INTRODUCTION 1 1.1. DIABETES MELLITUS: AN OVERVIEW 1 1.1.1. The r o l e o f i n s u l i n 1 1.1.2. C l a s s i f i c a t i o n and e t i o l o g y o f d iabetes 3 1.1.3. Pathophys io logy of d iabetes 7 1.2. VASCULAR COMPLICATIONS OF DIABETES 10 1.3. INFLUENCE OF DIABETES ON THE REACTIVITY OF BLOOD VESSELS 19 1.3.1. A l t e r e d r e a c t i v i t y to neuro-humoral and r e l a t e d substances 19 1.3.2. Mechanisms o f increased r e a c t i v i t y t o neuro-humoral and r e l a t e d substances 22 1.4. VASCULAR SMOOTH MUSCLE CONTRACTION 24 1.4.1. Sources and r e g u l a t i o n of c e l l u l a r ca l c i um 24 1.4.2. C o n t r a c t i l e mechanisms 30 1.5. PHOSPHOINOSITIDE METABOLISM: GENERAL VIEW 33 1.5.1. Phospho inos i t i des and the phospho inos i t i de c y c l e 34 1.5.2. I n o s i t o l phosphates 41 1.5.2.1. Metabol ism 41 1.5.2.2. Calc ium m o b i l i z a t i o n 43 1.5.3. 1 , 2 -D i a c y l g l y ce r o l (DG) and PKC a c t i v a t i o n 46 1.6. ROLE OF PHOSPHOINOSITIDE METABOLISM IN VASCULAR SMOOTH MUSCLE CONTRACTION 49 1.7. OBJECTIVES OF THE PRESENT STUDY 55 v i 2. MATERIALS AND METHODS 58 2.1. ANIMALS 58 2.2. ASSAY KITS 58 2.3. DRUGS AND CHEMICALS 58 2.4. KREBS SOLUTION 60 2.5. INDUCTION OF DIABETES 60 2.6. TISSUE PREPARATION 61 2.7. CONTRACTION STUDIES 62 2.8. [ 3 2 P ] LABELLING STUDIES 65 2.9. [3H]-MYOINOSITOL LABELLING STUDIES 67 2.10. DETERMINATION OF INS(1,4,5)P3 69 2.11. ANALYSIS OF DATA AND STATISTICAL METHODS 71 3. RESULTS 73 3.1. GENERAL FEATURES OF EXPERIMENTAL ANIMALS 73 3.2. CONTRACTILE RESPONSES TO NA, PE, CALCIUM CHLORIDE AND POTASSIUM CHLORIDE 73 3.3. CALCIUM DEPENDENCE OF CONTRACTILE RESPONSES TO a-ADRENOCEPTOR AGONISTS 79 3.4. EFFECTS OF NA ON [ 3 2 P ] INCORPORATION INTO PTDINS(4,5)P2 AND PA 80 3.5. NORADRENALINE-INDUCED [ 3H]-INOSITOL PHOSPHATE ACCUMULATION 82 3.6. NORADRENALINE-INDUCED INS(1,4,5)P 3 PRODUCTION 86 3.7. CONTRACTILE RESPONSES TO PDB IN THE PRESENCE OF EXTRACELLULAR CALCIUM 90 3.8. EFFECTS OF STAUROSPORINE ON CONTRACTILE RESPONSES OF ARTERIES TO NA AND PDB 91 v i i 3.9. EFFECTS OF EXTRACELLULAR CALCIUM REMOVAL ON PDB RESPONSES 94 3.10. EFFECTS OF CALCIUM CHANNEL BLOCKERS ON PDB RESPONSES 94 3.11. CONTRACTILE RESPONSES TO SODIUM FLUORIDE PLUS ALUMINIUM CHLORIDE 95 4. DISCUSSION 160 5. SUMMARY AND CONCLUSIONS 194 6. REFERENCES 197 v i i i Table LIST OF TABLES Page 1 Summary of major c a tego r i e s o f d i abe te s m e l l i t u s and r e l a t e d g lucose i n t o l e r a n c e s . 5 2 General f ea tu re s of exper imental an imal s . 74 3 Agon i s t pD2 va lues and maximum c o n t r a c t i l e responses i n ao r tae , mesenter ic a r t e r i e s and p o r t a l ve in s from c o n t r o l and d i a b e t i c r a t s . 76 4 CaCl2 pD£ values and maximum c o n t r a c t i l e responses i n the presence of 4 x 10~ 2 M KC1 or 1 0 " 6 M NA i n aortae from con t r o l and d i a b e t i c r a t s . 78 5 Basal [ 3 2 P ] l a b e l l i n g of phospho l ip id s i n aortae from con t r o l and d i a b e t i c r a t s . 81 6 Basal [ 3 H] -myo i no s i t o l l a b e l l i n g o f t o t a l phospho l ip id s i n ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins from con t r o l and d i a b e t i c r a t s . 83 7 Basal accumulat ion of t o t a l [ 3 H ] - i n o s i t o l phosphates i n ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins from con t r o l and d i a b e t i c r a t s . 84 8 PDB pD2 values and maximum c o n t r a c t i l e responses i n the presence and absence of e x t r a c e l l u l a r C a 2 + i n ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins from con t r o l and d i a b e t i c r a t s . 92 9 NaF pD£ values and maximum c o n t r a c t i l e responses i n the presence of 10"^ AICI3 i n aortae and mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s . 96 ix LIST OF FIGURES Figure Page 1 E f f e c t s o f i n s u l i n d e f i c i e n c y on carbohydrate, f a t and p r o t e i n metabol ism. 9 2 Pathways of phospho inos i t i de metabol ism. 37 3 Concentrat ion- response curves f o r NA-induced c o n t r a c t i o n s o f ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins from con t r o l and d i a b e t i c r a t s . 99 4 Time-course of the c o n t r a c t i l e responses to 10" 5 M NA i n aortae from con t r o l and d i a b e t i c r a t s . 101 Time-course of the c o n t r a c t i l e responses to 3xlO"^M NA i n mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s . 103 6 Concentrat ion- response curves f o r PE-induced c o n t r a c t i o n s o f aortae and mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s . 105 7 Concentrat ion- response curves f o r KC1-induced c o n t r a c t i o n s o f aortae and mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s . 107 8 Concentrat ion- response curves f o r CaCl2- induced c o n t r a c t i o n s of aortae from con t r o l and d i a b e t i c r a t s i n the presence o f 4 x l 0 " 2 M KC1 and 10" 6 M NA. 109 9 Concentrat ion- response curves f o r CaCl2- induced c o n t r a c t i o n s o f po r t a l ve ins from c o n t r o l and d i a b e t i c r a t s i n the presence of 4x lO " 2 M KC1. I l l 10 C o n t r a c t i l e responses of aortae from c o n t r o l and d i a b e t i c r a t s to NA i n C a 2 + - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA and f o l l o w i n g a d d i t i o n of x Figure Page 3.5 mM C a C l 2 . 113 11 C o n t r a c t i l e responses of aortae from c o n t r o l and d i a b e t i c r a t s to maximum concent ra t i on s o f PE and METH i n C a 6 - f r e e Krebs s o l u t i o n c on t a i n i n g 1 mM EGTA and f o l l o w i n g a d d i t i o n 3.5 mM CaCl2. 115 12 C o n t r a c t i l e responses o f mesenter ic a r t e r i e s from p . con t r o l and d i a b e t i c r a t s to NA i n Ca - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA and f o l l o w i n g a d d i t i o n of 3.5 mM CaCl2. 117 13 C o n t r a c t i l e responses o f mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s to maximum concent ra t i on s o f PE and METH in C a 2 + - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA and f o l l o w i n g a d d i t i o n of 3.5 mM CaCl2- 119 14 Time-course of NA-induced [ 3 2 P ] l a b e l l i n g of Ptd Ins(4,5)P2 and PA i n aortae from con t r o l and d i a b e t i c r a t s . 121 15 Time-course of NA-induced product ion o f t o t a l [ 3 H ] - i n o s i t o l phosphates i n aortae from c o n t r o l and d i a b e t i c r a t s . 123 16 E f f e c t s of yohimbine and p razo s i n on NA-induced t o t a l [ 3 H ] - i n o s i t o l phosphate p roduct ion i n aortae from con t r o l and d i a b e t i c r a t s . 125 17 Time-course of NA-induced product ion of t o t a l [ 3 H ] - i n o s i t o l phosphates i n mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s . 127 18 E f f e c t s of p razo s i n on NA-induced t o t a l [ 3 H ] - i n o s i t o l phosphate product ion i n mesenter ic x i Figure Page a r t e r i e s from con t r o l and d i a b e t i c r a t s . 129 19 Time-course o f NA-induced product ion of t o t a l [ 3 H ] - i n o s i t o l phosphates i n p o r t a l ve ins from con t r o l and d i a b e t i c r a t s . 131 20 Time-course of I n s ( l , 4 ,5 )P3 product ion and c o n t r a c t i o n s i n response to 10'^M NA i n aortae from con t r o l and d i a b e t i c r a t s . 133 21 Time-course of I n s ( l , 4 ,5 )P3 product ion and c o n t r a c t i o n s i n response to 10"^M NA i n aortae from con t r o l and d i a b e t i c r a t s . 135 22 Concentrat ion- response curves f o r NA-induced I n s ( l , 4 ,5 )P3 product ion i n aortae from c o n t r o l and d i a b e t i c r a t s . 137 23 Time-course o f NA-induced product ion of I n s ( l , 4 , 5 )P3 i n mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s . 139 24 Concentrat ion-dependent I n s ( l , 4 ,5 )P3 p roduct ion i n response to NA i n mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s . 141 25 Basal I n s ( l , 4 ,5 )P3 l e v e l s i n p o r t a l ve ins from con t r o l and d i a b e t i c r a t s . 143 26 Concentrat ion- response curves f o r PDB-induced c o n t r a c t i o n s of ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins from con t r o l and d i a b e t i c r a t s . 145 27 E f f e c t s of s tau rospor ine on NA-induced c o n t r a c t i o n s o f aortae and mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s . 147 x i i Figure Page 28 E f f e c t s of s taurospor ine on PDB-induced c o n t r a c t i o n s of aortae and mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s . 149 29 E f f e c t s of s tau rospor ine on KC1-induced c o n t r a c t i o n s o f aortae and mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s . 151 30 Concentrat ion- response curves f o r PDB-induced c o n t r a c t i o n s of aortae and mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s i n C a 2 + - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA. 153 31 E f f e c t s of verapamil and n i f e d i p i n e on PDB-induced c o n t r a c t i o n s of aortae and mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s . 155 32 Concentrat ion- response curves f o r NaF-induced c o n t r a c t i o n s of aortae and mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s i n the presence o f 10" 5 M A1C1 3 . 157 33 C o n t r a c t i l e responses to NaF plus 10" 5 M AICI3 i n aortae and mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s i n C a 2 + - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA. 159 x i i i LIST OF ABBREVIATIONS CaM Calmodul i n cAMP C y c l i c adenosine 3 ' ,5 ' -monophosphate CDP-DG C y t i d i n e diphosphate 1 , 2 - d i a c y l g l y c e r o cpm Counts per minute DG 1,2- D i a c y l g l y c e r o l DMSO Dimethyl s u l f o x i d e EDRF Endothel ium-der ived r e l a x i n g f a c t o r ER Endoplasmic r e t i c u l u m FCPD F i b r o c a l c u l u s panc rea t i c d i abetes g Gram(s) Gpp(NH)P Guanosine 5 ' - [/*Tr-imido]-triphosphate GTP Guanosine t r i pho spha te hr Hour(s) IDDM Insu l in-dependent d i abetes m e l l i t u s I n o s i t o l Myo ino s i t o l I n s ( l ) P I n o s i t o l 1-phosphate Ins(3)P I n o s i t o l 3-phosphate Ins(4)P I n o s i t o l 4-phosphate I n s ( l , 3 ) P 2 I n o s i t i o l 1,3-bisphosphate I n s ( l , 4 ) P 2 I n o s i t o l 1,4-bisphosphate I n s ( 3 , 4 )P 2 I n o s i t o l 3,4-b isphosphate I n s ( l , 3 , 4 ) P 3 I n o s i t o l 1 ,3 ,4 - t r i sphosphate I n s ( l , 4 , 5 ) P 3 I n o s i t o l 1 ,4 ,5 - t r i sphosphate I n s ( l : 2 c y c ) P I n o s i t o l 1 , 2 - c y c l i c phosphate I n s ( l : 2 c y c , 4 ) P 2 I n o s i t o l 1 , 2 - c y c l i c - 4-b isphosphate I n s ( l : 2 cyc ,4 ,5 )P3 I n o s i t o l 1 , 2 - c y c l i c - 4 , 5 - t r i s p h o s p h a t e I n s ( l , 3 , 4 , 5 ) P 4 I n o s i t o l 1 , 3 ,4 ,5 - te t rak i s -phospha te I n s ( l , 3 , 4 , 6 ) P 4 I n o s i t o l 1 , 3 ,4 ,6 - te t rak i s -phospha te I n s ( l , 3 , 4 , 5 , 6 ) P 5 I n o s i t o l 1 ,3,4,5,6-pentak i s -phosphate I n s ( l , 2 , 3 , 4 , 5 , 6 ) P 6 I n o s i t o l 1,2,3,4,5,6-hexak is -phosphate kg K i logram M Molar mCi M i l l i c u r i e /iCi M i c r o c u r i e METH Methoxamine mg M i l l i g r a m (s) xiv min Minute (s) ml M i l l i l i t r e mm M i l l i m e t e r mM, mmol/1 M i l l i m o l e s per l i t r e MLC Myosin l i g h t cha in MLC.P Phosphorylated myosin l i g h t cha in MLCK Myosin l i g h t cha in k inase NA Noradrena l ine NIDDM Noninsul in-dependent d i abetes m e l l i t u s PA Phosphat id i c a c i d PDB Phorbol 12, 13 -d ibu ty ra te PDPD P r o t e i n d e f i c i e n t panc rea t i c d i abetes PE Phenylephr ine PKC P r o t e i n k inase C PLC Phosphol ipase C pmol Picomole P td ln s Phosphat idy l i n o s i t o l Ptd Ins (4)P Phosphat idy l i n o s i t o l 4- phosphate P td ln s (4,5)P2 Phosphat idy l i n o s i t o l 4 ,5 - b isphosphate ROC Receptor-operated ca l c i um channels sec Second(s) SEM Standard e r r o r o f the mean SR Sarcoplasmic r e t i c u l u m STZ S t r ep t o zo t o c i n TPA 12 -0 - te t racdecanoy l -phorbo l 13, ace ta te VOC Vo l tage-operated ca l c i um channels WHO World Health O rgan i za t i on xv ACKNOWLEDGEMENTS I wish to express my deepest g r a t i t u d e to my supe r v i s o r Dr. Kath leen M. MacLeod f o r her constant guidance, i n t e r e s t , encouragement and pa t ience throughout the present study. I would a l s o l i k e to thank members o f my research committee f o r t h e i r i n t e r e s t and va l uab l e suggest ions . I am a l s o g r a t e f u l to Dr. C h r i s t i n e N icho l and Dr. John Langlands f o r t h e i r i n v a l uab l e t e c h n i c a l a s s i s t ance and suggest ions . The f i n a n c i a l support of the B.C. and Yukon Heart Foundat ion, and WUSC Local Committee i s g r a t e f u l l y acknowledged. Worku Abebe xvi INTRODUCTION 1.1. DIABETES MELLITUS: AN OVERVIEW Diabetes m e l l i t i i s i s a heterogeneous group o f metabo l i c d i s o r de r s c h a r a c t e r i z e d by the presence o f hyperglycemia and i n more severe cases w i th a c ce l e r a t ed l i p o l y s i s and exces s i ve ketogenes i s (Lefebvre and Luyckx, 1979). These metabo l i c abno rma l i t i e s can be a t t r i b u t e d to a r e l a t i v e i n s u l i n d e f i c i e n c y , as a r e s u l t of e i t h e r an abnormal i ty i n i n s u l i n s yn the s i s and/or r e l e a s e , or a decreased response to i n s u l i n at i t s s i t e o f a c t i o n . 1.1.1. The role of insulin I n s u l i n i s a powerful hormone which p lays a major r o l e i n the metabolism o f carbohydrates , f a t s and p r o t e i n s . I t i s s yn the s i zed , s to red and re lea sed by the /3-cel ls of the i s l e t s of Langerhans of the pancreas. The i n s u l i n molecule i s a 5700 da l ton po l ypep t i de c o n s i s t i n g of an a - cha in and a /3-chain un i ted by d i s u l f i d e bonds. The molecule can e x i s t as a monomer, dimer or hexamer (3 d imer s ) . Two z i n c atoms are coord inated i n the hexamer which i s probably the form s to red i n the /}-c e l l s o f the pancreas. The b i o l o g i c a l l y a c t i v e form o f the hormone i s be l i e ved to be the monomer and seems to r e s i d e i n the e n t i r e molecu la r s t r u c t u r e r a t h e r than s p e c i f i c amino a c i d groupings w i t h i n the molecule (Addison - Wesley, 1983; C a h i l l , 1971). Factors which s t i m u l a t e i n s u l i n r e l e a s e i n c l ude g lucose , amino a c i d s , g lucagon, g a s t r o i n t e s t i n a l pept ide l hormones (eg. g a s t r i c i n h i b i t o r y p e p t i d e ) , ketone bod ies , and /?-adrenerg i c and parasympathet ic s t i m u l a t i o n . Main i n h i b i t o r s of i n s u l i n r e l ea se i n c l ude somatostat in and a -adrenerg i c s t i m u l a t i o n . The e f f e c t s o f i n s u l i n on carbohydrate metabolism i n c l ude r educ t i on i n the r a t e of r e l ea se o f g lucose from the l i v e r and inc rease i n the r a t e o f uptake o f g lucose i n t o i n s u l i n - s e n s i t i v e t i s s u e s such as muscle and adipose t i s s u e s . On f a t metabol ism, i n s u l i n decreases the r e l ea se o f f a t t y ac id s from adipose t i s s u e s , and s t imu l a te s de novo s yn thes i s of f a t t y ac ids and convers ion o f f a t t y a c i d to t r i g l y c e r i d e s i n the l i v e r . With regard to p r o t e i n metabol ism, i n s u l i n promotes the t r an spo r t of amino ac ids across the plasma membrane i n the l i v e r and muscle, and a l s o s t imu la te s p r o t e i n s ynthes i s and reduces the r e l ea se of amino ac id s from muscle ( S te inbe rg , 1985). The hormone a l s o has e f f e c t s on o ther c e l l u l a r processes such as s t i m u l a t i o n of RNA and DNA syn thes i s and enhancement o f ion t r an spo r t (eg. K + uptake i n t o c e l l s ) . I t i s now be l i e ved tha t almost a l l of the metabo l i c e f f e c t s of i n s u l i n are i n i t i a t e d by the i n t e r a c t i o n o f the hormone w i th a s p e c i f i c r ecep to r on the plasma membrane (Kahn et a l . , 1989). The i n s u l i n r ecep to r has been p a r t i a l l y p u r i f i e d and c h a r a c t e r i z e d (Czech, 1981). I t i s a t e t r a m e r i c g l y c o p r o t e i n of high molecu lar weight c o n s i s t i n g o f two a - subun i t s o f Mr 135,000 and two 0 - subun i t s o f Mr 95,000 l i n k e d by d i s u l f i d e bonds (Kahn et a l . , 1989). The a - subun i t conta in s the i n s u l i n b ind ing s i t e , and the /J-subunit i s a transmembrane p r o t e i n and i s t h e r e f o r e l i k e l y to be the subunit i nvo l ved i n i n t r a c e l l u l a r s i g n a l l i n g 2 (Kahn et a l . , 1989). I t i s now e s t a b l i s h e d tha t a c t i v a t i o n o f the i n s u l i n r ecep to r leads to inc reases i n t y r o s i n e k inase a c t i v i t y and autophosphory la t ion o f the i n s u l i n r e c e p t o r ; however, subsequent mo lecu la r events coup l i ng the i n t e r a c t i o n o f i n s u l i n w i t h i t s r e cep to r to the r e g u l a t i o n o f c e l l u l a r metabolism are not c l e a r l y understood (Kahn et a l . , 1989). I t has r e c e n t l y been suggested t ha t i n o s i t o l phosphate g lycan (which conta in s glycosamine) and 1 , 2 - d i a c y l g l y c e r o l (DG) generated by i n s u l i n - s e n s i t i v e phosphol ipase C-mediated h y d r o l y s i s of g l y c e r y l - phosphat idy l i n o s i t o l i n the plasma membrane are i nvo l ved i n the s i gna l t r an sduc t i on system of i n s u l i n ( S a l t i e l et a l . , 1990). The DG produced i s be l i e ved to s t imu l a te s p e c i f i c forms o f p r o t e i n k inase C (PKC) which may mediate the e f f e c t s o f i n s u l i n (Be r r i dge , 1987; S a l t i e l , 1990). The i n o s i t o l phosphate g lycan re lea sed by i n s u l i n has been shown to mimic c e r t a i n a c t i on s of i n s u l i n i n s u b c e l l u l a r p repa ra t i on s (eg. r e g u l a t i o n of phosphorylase a, pyruvate k inase , cAMP l e v e l s e t c . ) but the extent to which i t produces the a c t i on o f i n s u l i n i n i n t a c t c e l l s or i n v i vo remains unc lear ( S a l t i e l , 1990). 1.1.2. Class i f icat ion and etiology of diabetes I t i s now recogn ized tha t there i s more than one cause o f d i abetes m e l l i t u s , and a g reat deal o f v a r i a t i o n e x i s t s i n pa t t e rn and course of the d i s ea se . Although most of the var ious forms o f d i abetes have a gene t i c background, the genet i c mechanisms i nvo l ved appear to vary (Sa lans , 1982). Recogniz ing the d i v e r s i t i e s i n the nature o f the 3 d i s ea se , i n 1971 an i n t e r n a t i o n a l workgroup sponsored by the Nat iona l Diabetes Data Group developed a new c l a s s i f i c a t i o n f o r d i abetes and other c a tego r i e s o f g lucose i n t o l e r a n c e (Brunner and Suddarth, 1988). Table 1 summarizes the major ca tego r i e s along w i th cu r r en t te rmino logy , o l d l a b e l s , and major c l i n i c a l c h a r a c t e r i s t i c s . The two most common types o f d iabetes are in su l in -dependent d iabetes m e l l i t u s (IDDM, Type I) and non insu l in -dependent d iabetes m e l l i t u s (NIDDM, Type I I ) . IDDM i s a s soc i a ted w i th an abso lute d e f i c i e n c y o f i n s u l i n s e c r e t i o n . Th i s has been shown to be r e l a t e d to a s p e c i f i c l o s s o f panc rea t i c /3-cel ls (Kahn, 1985). The e t i o l o g y o f IDDM i s not we l l understood, but i t has been suggested to i nvo l ve gene t i c p r e d i s p o s i t i o n , v i r u s e s and/or autoimmune f a c t o r s ( C r a i g , 1980). G e n e t i c a l l y , a p o s i t i v e c o r r e l a t i o n between c e r t a i n human leucocy te ant igens (HLA) and d iabetes has been demonstrated. I t has been proposed tha t the ant igens are determinants o f some /J -ce l l su r face p r o t e i n s , some o f which may have de fec t s render ing the c e l l s more s u s c e p t i b l e to c e r t a i n i n s u l t s (Nerup, 1978). The c h a r a c t e r i s t i c a l l y abrupt occurrence of IDDM cou ld a l s o be a consequence o f an i n f e c t i o n w i th a d iabetogen ic v i r u s i n g e n e t i c a l l y predisposed i n d i v i d u a l s . The i n f e c t i o n might cause an autoimmune (ant i gen -ant ibody ) r e a c t i o n . In t h i s regard , a v a r i a n t of Coxsack ie B4 v i r u s has been i d e n t i f i e d as being able to damage the panc r ea t i c /J - ce l l s (Yoon et a l . , 1979). S tud ies c a r r i e d out i n animal models a l s o appear to i n d i c a t e the involvement of v i r u s i n the pathogenesis of d iabetes m e l l i t u s , e i t h e r by d i r e c t c y t o l y t i c i n f e c t i o n and d e s t r u c t i o n of 4 Table 1. Summary of major categories of diabetes me l l i t u s and re la ted glucose into lerances. Current category Previous category C l i n i c a l features IDDM (Type I) (5-10% of a l l diabetes) Juveni le diabetes Juveni le onset diabetes Ketosis-prone diabetes B r i t t l e diabetes A l l ages, but often young Mostly underweight Often with i s l e t c e l l antibodies L i t t l e or no endogenous i n s u l i n Ketosis-prone Dependence on i n s u l i n to preserve l i f e NIDDM (Type I I ) (90-95% o f a l l d i a b e t e s ) Maturity onset diabetes Adult onset diabetes K e t o s i s re s i s tant diabetes Stable diabetes Maturity onset diabetes of youth A l l ages, but often > 40 Mostly obese Often high endogenous i n su l i n l eve l s May need i n s u l i n to avoid hyperglycemia Nonketotic hyperosmolar coma Impaired g l u c o s e t o l e r a n c e Borderl ine diabetes L a t e n t diabetes Subc l in ica l diabetes Chemical diabetes Asymptomatic diabetes S l i g h t l y above normal glucose leve l s Susceptible to a therosc le rot i c diseases G e s t a t i o n a l d i a b e t e s G e s t a t i o n a l diabetes Associated with pregnancy High r i s k of per inata l complications Glucose intolerance t r an s i t o r y but frequently occurs. Diabetes mel l i tus associated with other condit ions Secondary diabetes Pancreatic diseases Disease of hormone et io logy Drug or chemical t o x i c i t y Certain genetic syndromes Previous abnormality of glucose tolerance Latent diabetes Prediabetes Previous h i s tory of hyperglycemia Potent ia l abnor-mal i ty of glucose to lerance. Potent ia l diabetes Prediabetes No h i s tory of glucose intolerance Modified from Brunner and Suddarth, 1988. 5 panc rea t i c /3 -ce l l s , or by t r i g g e r i n g an autoimmune response (Cra ighead, 1978). Autoimmune f a c t o r s generated independent ly o f v i r a l i n f e c t i o n may a l so be important i n IDDM. C i r c u l a t i n g i s l e t c e l l a n t i bod i e s capable of complexing w i th j S - ce l l s i n the pancreas i s l e t s are found i n p a t i e n t s w i th IDDM (Lernmark et a l . , 1978). NIDDM has been suggested to o r i g i n a t e as a r e s u l t o f some i n s u l i n r ecep to r d e f e c t , e i t h e r due to a decreased a f f i n i t y o f the recep to r f o r i n s u l i n , a po s t recep to r d e f e c t , or a down r e g u l a t i o n of the i n s u l i n r ecep to r (Kahn et a l . , 1977; Bar et a l . , 1978; Berger and B e r c h t o l d , 1985). In NIDDM, there i s con s ide rab le p re se r va t i on o f the panc r ea t i c /?-c e l l s . Th i s c l a s s of d iabetes represents the m a j o r i t y of the d i a b e t i c p o p u l a t i o n . Most (60-90%) of these d i a b e t i c s are obese and e x h i b i t h y p e r i n s u l i n i s m and a s soc ia ted i n s u l i n r e s i s t a n c e . As shown i n Table I, bes ides the above de sc r i bed pr imary syndromes, there are other types of d iabetes which r e s u l t from other c o n d i t i o n s or syndrome (Nat iona l Data Group, 1979). These represent l e s s than 5% o f the d i a b e t i c popu l a t i on . Recent l y , another c l a s s of d iabetes m e l l i t u s , m a l n u t r i t i o n - r e l a t e d d iabetes m e l l i t u s , has been recogn ized by the World Hea l th O rgan i za t i on (WHO) Study Group (1985) as a major c l i n i c a l s ubc la s s , rank ing w i th IDDM and NIDDM in some t r o p i c a l c o u n t r i e s . M a l n u t r i t i o n - r e l a t e d d iabetes m e l l i t u s i s f u r t h e r d i v i d e d i n t o two subc lasses depending upon the cause and c l i n i c a l f ea tu re s of the d i s ea se : f i b r o c a l c u l o u s p anc r ea t i c d iabetes (FCPD) and p r o t e i n - d e f i c i e n t panc rea t i c d iabetes (PDPD). 6 FCPD i s a s soc i a ted w i th reduced s e c r e t i o n o f i n s u l i n due to damage to the panc rea t i c i s l e t s . I t u s u a l l y occurs below the age o f 30 years and p a t i e n t s are g r o s s l y underweight. Although the d i sease i s not a s soc i a ted w i th k e t o s i s , treatment r equ i r e s the a d m i n i s t r a t i o n of i n s u l i n . High cassava (cyanogenic food) i n take combined w i th inadequate p r o t e i n consumption has been suggested to be a cau sa t i ve f a c t o r f o r FCPD. The key f ea tu re s of PDPD i nc l ude r e s i s t a n c e to the development o f k e t o s i s , p a r t i a l r e s i s t a n c e to the a c t i on of i n s u l i n , extreme degree o f wast ing and emac ia t i on , and an onset of symptoms before the age of 35 yea r s . P a t i e n t s w i th t h i s type o f d iabetes are a l s o c h a r a c t e r i s t i c a l l y underweight. M a l n u t r i t i o n i s be l i e ved to be the cau sa t i ve f a c t o r f o r PDPD. 1.1.3. Pathophysiology of diabetes The d e f i c i e n c y i n the product ion or a c t i o n of i n s u l i n o c c u r r i n g i n d iabetes can r e s u l t i n abnormal metabolism of carbohydrates , f a t s and p r o t e i n s . The e f f e c t s produced are summarized i n F igure 1. The uptake of g lucose i n t o muscle and adipose t i s s u e s , and g l ycogenes i s i n l i v e r and muscle are decreased, wh i l e g l y cogeno l y s i s and g luconeogenes is are i n c rea sed . These changes r e s u l t i n s i g n i f i c a n t e l e v a t i o n o f blood g lucose l e v e l s , l ead ing to g l y c o s u r i a when blood g lucose l e v e l s exceed the rena l t h re sho ld f o r g lucose . The increased g lucose i n the u r i ne can induce osmotic d i u r e s i s r e s u l t i n g i n p o l y u r i a , f o l l owed by p o l y d i p s i a . 7 F igure 1 E f f e c t s of i n s u l i n d e f i c i e n c y on carbohydrate, f a t and p r o t e i n metabolism (modi f ied from S te i nbe rg , 1985). 8 I N S U L I N D E F I C I E N C Y C a r b o h y d r a t e m e t a b o l i s m F a t m e t a b o l i s m t G l u c o s e u p t a k e t G l y c o g e n o l y s i s ) G l u c o n e o g e n e s i s ( L i p o p r o t e i n H y p e r g l y c e m i a . I n s u l i n - s e n s i t i v e . F r e e f a t t y a c i d ] - j p a s e ' l i p a s e a c t i v i t y r e - e s t e r i f i c a t i o n n, + -G l y c o s u r i a P o l y u r i a J. P l a s m a f r e e f a t t y a c i d 1 V e r y l o w d e n s i t y l i p o p r o t e i n a n d c h y l o m i c r o n c l e a r a n c e P r o t e i n m e t a b o l i s m t A m i n o a c i d t r a n s p o r t J P r o t e i n s y n t h e s i s N e t p r o t e i n b r e a k d o w n . G l u c o s e , u p t a k e i n p e r i p h e r y D e h y d r a t i o n & e l e c t r o l y t e l l o s s A c i d o s i s • T H y p o v o l e m i a J . H y p o t e n s i o n \ R e d u c e d t i s s u e p e r f u s i o n Anur ia L a c t i c a c e d e m i a \ / Coma & d e a t h t K e t o n e m i a t K e t o n u r i a t H e p a t i c f r e e f a t t y a c i d u p t a k e | ) V e r y l o w d e n s i t y 1 i p o p r o t e i n , T r i g l y c e r i d e s y n t h e s i s t E l e c t r o l y t e l o s s H y p o v o l e m i a F a t t y l i v e r H y p o k a l e m i a M u s c l e w a s t i n g A m i n o a c i d e m i a K + a n d w a t e r l o s s G l u c o n e o g e n e s i s U r e a a n d u r i n a r y K + T L i p c r a i a The a s soc i a ted metabo l i c imbalance a l so causes po l yphag ia . The increased metabolism o f f a t i n d iabetes produces h y p e r l i p i d e m i a . The d e f i c i e n c y of i n s u l i n a l s o r e s u l t s i n a net breakdown of muscle p r o t e i n and subsequent l o s s o f weight. The abno rma l i t i e s i n carbohydrate, f a t and p r o t e i n metabolism o c c u r r i n g i n d iabetes can r e s u l t i n both s ho r t - term and long-term comp l i ca t i on s a f f e c t i n g va r ious organs and systems. The sho r t - te rm e f f e c t s i n c l ude ke toac i do s i s and non - ke t o t i c d i a b e t i c coma (Young and Brad ley , 1967; S te i nbe rg , 1985). These e f f e c t s are o f ten r e v e r s i b l e w i th i n s u l i n a d m i n i s t r a t i o n . The long-term comp l i ca t i on s o f d iabetes i n c l ude r e t i nopa thy and c a t a r a c t f o rmat ion , which lead to b l i ndnes s i n p a t i e n t s w i th l ong - s tand ing d iabetes (Davis, 1974), neuropathy ( S i b l e y , 1982), nephropathy (Balodimos, 1971), an increased i nc idence o f i n f e c t i o n (Brunner and Suddarth, 1988) and c a r d i o v a s c u l a r d i seases such as hyper tens ion , coronary heart d i s ea se , a t h e r o s c l e r o s i s and cardiomyopathy (Garc i a et a l . , 1974; K r ow le s k i , 1977; Co lwe l l et a l . , 1979; A l t u r a et a l . , 1980; Brunner and Suddarth, 1988). Chronic comp l i ca t i on s of d i abetes are now r e l a t i v e l y more common due to the commercial a v a i l a b i l i t y o f i n s u l i n to t r e a t acute comp l i ca t i on s more e f f e c t i v e l y . 1.2. VASCULAR COMPLICATIONS OF DIABETES With the d i s cove ry o f i n s u l i n i n 1921 (Bant ing et a l . , 1922) and the commercial a v a i l a b i l i t y of va r i ous forms o f pure i n s u l i n i n recent yea r s , management of acute metabo l i c comp l i ca t i on s of d i abetes ceased to 10 be a major problem. However, i n the i n t e r ven i n g t ime , i t has become apparent t ha t d i a b e t i c s have increased m o r t a l i t y and a reduced l i f e span compared to the general p opu l a t i on . I t i s now recogn ized t ha t the development o f c a r d i o v a s c u l a r comp l i ca t i on s i s the pr imary reason f o r the inc reased m o r t a l i t y i n both IDDM and NIDDM (Garc i a et a l . , 1974; Krowlewski et a l . , 1977; A l t u r a et a l . , 1980). In f a c t , i t has been repor ted tha t 40-80% of the human d i a b e t i c popu l a t i on e x h i b i t s s i g n i f i c a n t e l e v a t i o n i n a r t e r i a l blood pres sure , and c a r d i o v a s c u l a r d i seases such as hyper tens ion , a t h e r o s c l e r o s i s , coronary heart d i sease and s t roke account f o r approximately 80% of a l l d i a b e t i c deaths (Kannel and McGee, 1979; Ga rc i a et a l . , 1974; Krowlewski et a l . , 1977; A l t u r a et a l . , 1980). There fo re , i n the past two decades a g reat deal o f e f f o r t has been d i r e c t e d towards i n v e s t i g a t i n g the m a n i f e s t a t i o n s , o r i g i n , p r ophy l ax i s and treatment o f the va s cu l a r comp l i ca t i on s of d iabetes m e l l i t u s . In a d d i t i o n to a general i nc rease i n the i nc idence of c a r d i o v a s c u l a r d i s o rde r s i n d i abe te s , changes i n the va s cu l a r system occur which appear to be r e l a t i v e l y s p e c i f i c to the d i a b e t i c s t a t e . Depending upon the progress of the d i s ea se , these d i s o r de r s can a f f e c t the f unc t i on s o f a number of organs i n the body, e s p e c i a l l y those o f the eyes, k idneys , heart and nervous system ( A l t u r a et a l . , 1980; Brunner and Suddarth, 1988). Two major groups of va s cu l a r d i s o rde r s are recogn ized to be s p e c i f i c a l l y a s soc i a ted w i th d i abe te s : microangiopathy and 1 1 macroangiopathy (Renold et a l . , 1987; Brunner and Suddarth, 1988). Microangiopathy i s a d i sease o f the small blood v e s s e l s , which i s thought to be the under l y ing de fec t i n d i a b e t i c r e t i n opa th y , nephropathy and perhaps cardiomyopathy. There i s evidence demonstrat ing a d i r e c t c o r r e l a t i o n between microangiopathy and the degree and du ra t i on o f hyperg lycemia. Macroangiopathy i s p r i m a r i l y a d i sease o f medium and l a r ge blood v e s s e l s . I t i s be l i e ved to c o n t r i b u t e to a number of c a r d i o v a s c u l a r d i seases such as coronary a r t e r y d i sease and myocardia l i n f a r c t i o n , ce reb rova scu la r d i seases and hyper tens ion . Macroangiopathy has been found to be more severe i n p a t i e n t s w i th marked hyperglycemia (Renold et a l . , 1978). A mixture of m i c ro - and macroangiopathy and c e r t a i n o ther metabo l i c abno rma l i t i e s g i ve r i s e to d i a b e t i c neuropath ies , g e s t a t i o n a l problems and d i a b e t i c s e n i l e c a t a r a c t . Most of these va s cu l a r comp l i ca t i on s can occur i n both IDDM and NIDDM and appear to i n vo l ve a number o f under l y ing me tabo l i c , s t r u c t u r a l and f u n c t i o n a l a l t e r a t i o n s ( A l t u r a et a l . , 1980). Although l ong - s tand ing hyperglycemia and other metabo l i c abno rma l i t i e s are known to be a s soc i a ted w i th the va s cu l a r comp l i ca t i on s of d i abe te s , i t i s u n l i k e l y t ha t a s i n g l e f a c t o r cou ld account f o r a l l of them. The i d e n t i f i c a t i o n o f the s p e c i f i c processes under l y ing these comp l i ca t i on s of d i abetes i s important i n order to prevent or t r e a t them ( A l t u r a et a l . , 1980). The f o l l o w i n g are some o f the pa thophy s i o l o g i ca l processes t ha t may be a s soc i a ted w i th changes i n the va s cu l a r system i n d i abe te s . 12 (a) L i p i d s and phospho l ip id s : I t has been demonstrated tha t a r t e r i e s from d i a b e t i c sub jec t s con ta in e leva ted amounts o f c h o l e s t e r o l and c e r t a i n phospho l ip id s ( A l t u r a et a l . , 1980). Pulmonary a r t e r i e s from s t r e p t o z o t o c i n ( STZ ) -d i abe t i c r a t s have a l so been shown to have a high i nc idence of t r i g l y c e r i d e depos i t s i n the media and i n t ima ( R e i n i l a et a l . , 1977). Two major f a c t o r s have been proposed to account f o r the inc reased a r t e r i a l wa l l l i p i d content i n d i abe te s : decreased membrane-bound l i p o p r o t e i n l i p a s e ( R e i n i l a et a l . , 1977); and high a c t i v i t y o f acy l CoA hydro lase (Hashimoto and Dayton, 1977), an enzyme which i s important i n the synthes i s of c h o l e s t e r o l . These changes i n l i p i d and pho spho l i p i d contents of a r t e r i a l wa l l may r e s u l t i n a l t e r a t i o n s i n the f u n c t i o n a l p r o p e r t i e s of blood v e s s e l s . (b) Glucose metabolism and the po l yo l pathway : I t has been shown tha t v a s cu l a r smooth muscle o f d i a b e t i c animals e x h i b i t s impaired g lucose metabolism when con t r o l and d i a b e t i c ve s se l s i n v i t r o are exposed to the same concent ra t i on s of g lucose (Wertheimer and Bentor, 1960). I r r e s p e c t i v e of the type of d i abe te s , g lucose uptake and i t s convers ion to CO2, glycogen and l i p i d s i n a o r t i c smooth muscle have been found to be depressed (Chobanian et a l . , 1974; Mulcaky and Winegrad, 1962; U r r u t i a et a l . , 1962). In d i a b e t i c r a b b i t a o r t a , hexokinase, but not phosphohexose isomerase or phosphohexokinase, a c t i v i t y i s a l so reduced (Wertheimer and Bentor, 1962). These data suggest t ha t the decreased g lucose u t i l i z a t i o n , at l e a s t i n a o r t i c t i s s u e , may be due to impaired g lucose phosphory l a t i on . S ince va s cu l a r smooth muscles have a 13 very small re serve of c r e a t i n e and glycogen ( A l t u r a and A l t u r a , 1978), they are h i g h l y dependent upon the u t i l i z a t i o n o f f r e e g lucose f o r normal f u n c t i o n . A l t e r a t i o n s i n g lucose a v a i l a b i l i t y and u t i l i z a t i o n i n v a s cu l a r muscle has been suggested to r e s u l t i n concomitant a l t e r a t i o n s i n v a s cu l a r r e a c t i v i t y ( i . e . c o n t r a c t i l e responses to va soac t i ve agent s ) , and e x c i t a t i o n - c o n t r a c t i o n coup l i ng phenomena i n these c e l l s ( A l t u r a and A l t u r a , 1978; A l t u r a et a l . , 1980). Exposure o f r a b b i t ao r ta to an e leva ted g lucose concen t r a t i on has been shown to be a s soc ia ted w i th an increase i n a c t i v i t y o f the po l yo l pathway, accompanied by increased water content and decreased oxygen consumption (Morr i son et a l . , 1972). With regard to the po l yo l pathway, a great deal o f g lucose i s converted to s o r b i t o l or f r u c t o s e i n d iabetes v i a t h i s pathway (McMi l l an , 1975; Pe t r i de s et a l . , 1977). S o r b i t o l i s formed v i a the enzyme aldose reductase. Aldose reductase i s present i n a r t e r i a l smooth muscle and many other body t i s s u e s such as nerve, lens and rena l p a p i l l a (McMi l l an , 1975). I t i s be l i e ved tha t the products o f the po l yo l pathway have adverse e f f e c t s i f they accumulate i n c e l l s at high concen t r a t i on s , and can a l so exer t osmotic e f f e c t s . Changes i n e x t r a c e l l u l a r o smo la r i t y are known to exer t severa l d i f f e r e n t e f f e c t s on v a s cu l a r smooth muscle c e l l s , i n c l u d i n g a l t e r a t i o n s i n vasomotor tone, e l e c t r i c a l p r o p e r t i e s , v a s cu l a r r e a c t i v i t y and ion contents ( A l t u r a et a l . , 1980). In a d d i t i o n , increased a c t i v i t y of the po l yo l pathway i n nerves of d i a b e t i c s has been shown to be r e l a t e d to impairment of nerve 14 conduct ion (Gabby, 1975) which may have an i n f l u e n c e on the c a r d i o v a s c u l a r system (eg. depress ion of vasomotor r e f l e x e s ) . (c) P r o t e i n g l y c o s y l a t i o n : Prolonged hyperglycemia has been demonstrated to be a s soc ia ted w i th increased content o f g l u c o s y l a t e d hemoglobin and other serum p ro te i n s (Bunn et a l . , 1978). Th i s has been suggested to p lay a r o l e i n the pathogenesis o f the l a t e comp l i ca t i on s of d i abe te s . Th is c o n d i t i o n causes t i s s u e hypoxia and o ther v a s cu l a r e f f e c t s c o n t r i b u t i n g to the development of microangiopathy (Wi l l iamson and K i l o , 1980). Furthermore, widespread g l y c o s y l a t i o n o f p r o t e i n s has been found to be a s soc i a ted w i th the pathogenesis o f d i a b e t i c c a t a r a c t , nephropathy, neuropathy and cardiomyopathy (Renold et a l . , 1978; Rahbar, 1981). (d) C i r c u l a t i n g substances and c i r c u l a t o r y e f f e c t s : D iabetes has been observed to be a s soc i a ted w i th changes i n c e r t a i n c i r c u l a t i n g substances and hemorrheology. These changes are a l s o be l i e ved to be i nvo l ved i n the pathogenesis of microangiopathy (McM i l l an , 1976). The v i s c o s i t y o f plasma and serum i s increased i n d iabetes and t h i s has been shown to be l i n k e d to an inc rease i n red blood c e l l a g g r e g a b i l i t y (McM i l l an , 1976). Red blood c e l l s a l so e x h i b i t reduced d e f o r m a b i l i t y which may cause changes i n blood rheology i n d i a b e t i c s (McM i l l an , 1976). E levated blood v i s c o s i t y can be r e l a t e d to plasma p r o t e i n changes which a l s o cause a l t e r a t i o n s i n hemorrheology and plasma v i s c o s i t y . V i scous blood causes r e s i s t a n c e to f l ow and thus increased pressure i n the c i r c u l a t i o n (McM i l l an , 1976). In a d d i t i o n , increased a g g r e g a b i l i t y of 15 p l a t e l e t s as we l l as changes i n the p r o p e r t i e s o f polymorphonuclear leukocytes and lymphocytes have been shown in d i a b e t i c s (Jones and Peter son, 1981). > Diabetes i s a l so a s soc i a ted w i th increased p e r m e a b i l i t y of c a p i l l a r y w a l l s . Because o f t h i s , plasma p r o te i n s l eak out and are depos i ted i n the vesse l w a l l , thereby p r o v i d i n g the morpholog ica l bas i s f o r the development of microangiopathy (Pa rv ing , 1976). Increased h y d r o s t a t i c pressure or t h i c ken i n g of c a p i l l a r y basement membrane has been proposed to c o n t r i b u t e to the increased p e r m e a b i l i t y of the m ic rova scu l a tu re (Pa rv ing , 1976). A number of repor t s show tha t d iabetes i s a l s o a s soc i a ted w i th changes i n va r i ous types of hormones such as t h y r o i d hormones, growth hormone, glucagon and somatos ta t in , which may c o n t r i b u t e d i r e c t l y or i n d i r e c t l y to the pathophys io logy of the d i s ea se . While the l e v e l s of t h y r o i d hormones decrease, those of the other hormones have been found to i nc rease i n d iabetes (Cooppan, 1982). (e) Basement membrane and r e l a t e d changes i n v a s cu l a r wa l l : Th icken ing o f basement membrane of c a p i l l a r i e s t ha t supply p a r t i c u l a r l y the g lomeru lus , r e t i n a , s k i n and s k e l e t a l muscle i s one o f the c h a r a c t e r i s t i c f ea tu re s of d i a b e t i c microangiopathy ( K e f a l i d e s , 1981). P o s s i b l e f a c t o r s that might c o n t r i b u t e to the pathogenesis of basement membrane t h i c k e n i n g i n c l ude hyperg lycemia, i n s u l i n d e f i c i e n c y and e l eva ted l e v e l s of growth f a c t o r s ( K e f a l i d e s , 1981). S tud ies demonstrated tha t there i s a good c o r r e l a t i o n between t h i c k e n i n g of 16 c a p i l l a r y basement membrane and the du ra t i on of d i a b e t e s . C a p i l l a r y basement membrane t h i c k e n i n g has been used as a u se fu l index f o r the d e t e c t i o n of the d e l e t e r i o u s changes produced by d iabetes on the v a s c u l a t u r e . Although t h i c ken i n g o f c a p i l l a r y basement membrane has been shown to r e s u l t from var ious f a c t o r s , i t s p a t h o l o g i c a l s i g n i f i c a n c e i n d i a b e t i c microangiopathy i s not c l e a r . I t i s , however, conce i vab le t ha t i t cou ld impa i r l o c a l oxygenation and t i s s u e n u t r i t i o n and thereby r e s u l t i n a l t e r a t i o n s not on ly the f u n c t i o n of the m i c r o va s cu l a t u re , but i n v a s cu l a r r e a c t i v i t y as we l l ( A l t u r a , et a l . , 1980). Other s t r u c t u r a l changes i n v a s cu l a r wa l l i n d i abetes i n c l ude increased content o f co l l a gen (a ub iqu i tous p r o t e i n p r o v i d i n g mechanical s t r e n g t h ) ; i nc reased ca l c ium content and c a l c i f i c a t i o n o f e l a s t i c t i s s u e ; and increased p e r m e a b i l i t y to plasma p r o t e i n s and other l a r ge molecules ( A l t u r a et a l . ; 1980). I t has been suggested t ha t these s t r u c t u r a l changes cou ld i n f l uence the respons iveness ( c o n t r a c t i l e f o r ce generated i n response to a g iven concen t ra t i on of agon i s t ) o f v a s cu l a r smooth muscle to va soac t i ve agents ( A l t u r a et a l . . , 1980). ( f ) Endo the l i a l damage : Endo the l i a l damage has been shown to occur i n both c l i n i c a l and exper imental d iabetes (Co lwe l l e t a l . , 1983). Th i s p lays a r o l e i n c i r c u l a t o r y changes of d i abe te s . E ndo the l i a l death and regenera t i on have been shown to cause basement membrane t h i c k e n i n g i n animal models. Endo the l i a l r e p l i c a t i o n and m ig ra t i on i n t o the areas o f decreased c a p i l l a r y pe r fu s i on and microaneurysm format ion have been seen i n d i a b e t i c r e t i nopa thy . I n tac t e n d o t h e l i a l c e l l s have the a b i l i t y 17 to prevent p l a t e l e t adhesion and aggregat ion (Co lwe l l et a l . , 1976). There fo re , damage of the e n d o t h e l i a l c e l l s i n d i abetes may lead to a decrease i n ant iagg regatory p r o p e r t i e s . The importance of e n d o t h e l i a l i n t e g r i t y to the maintenance of normal v a s cu l a r r e a c t i v i t y has been recogn ized p r i m a r i l y from the po in t of view of the a b i l i t y o f the e n d o t h e l i a l c e l l s to produce a r e l a x i n g substance known as endothel ium -de r i ved r e l a x i n g f a c t o r (EDRF) which a c t i v a t e s the r e l a x a t i o n of v a s cu l a r smooth muscle c e l l s (Furchgot t , 1983). Damage to the e n d o t h e l i a l c e l l s i n d iabetes would t he re f o re be expected to r e s u l t i n l o s s o f r e l a x a t i o n , and i t has been suggested tha t t h i s cou ld i n f l u e n c e the r e a c t i v i t y of blood ves se l s to va soac t i ve substances. (g) Other c e l l u l a r changes : The pa t ho l o g i c a l man i f e s t a t i on s of d i abetes have a l so been proposed to r e s u l t from an under l y i ng somatic de fec t which cou ld be acqu i red g e n e t i c a l l y . The de fec t i n somatic c e l l s i s c h a r a c t e r i z e d by a c ce l e r a ted somatic c e l l tu rnover which may r e f l e c t the high s u s c e p t i b i l i t y of c e l l s to an i n j u r i o u s event i n d i abetes (Vracko and B e n d i t t , 1974). Most o f the c e l l u l a r l e s i o n s such as a t h e r o s c l e r o t i c l e s i o n s , basement membrane t h i c k e n i n g e t c . t ha t occur r e l a t i v e l y e a r l y i n d i abe te s , have been demonstrated to occur i n non-d i a b e t i c s , but on ly w i th i n c r ea s i n g age. On the ba s i s o f va r i ous exper imenta l r e s u l t s , i t has been proposed tha t d i abetes i s a form of premature c e l l u l a r senescence (Renolds et a l . , 1978). 18 1.3. INFLUENCE OF DIABETES ON THE REACTIVITY OF BLOOD VESSELS 1.3.1. Altered react iv i ty to neuro-humoral and related substances A l t e r e d r e a c t i v i t y o f va s cu l a r smooth muscle to neu ro t ran sm i t te r s and c i r c u l a t i n g hormones has been proposed by many i n v e s t i g a t o r s to under ly some o f the va s cu l a r comp l i ca t i on s (eg. hyper tens ion , coronary heart d i seases ) a s soc ia ted w i th d iabetes m e l l i t u s . Th i s i s p a r t i c u l a r l y apparent i n view o f the f a c t t ha t the maintenance o f c i r c u l a t o r y homeostasis depends to a l a r ge extent on the respons iveness of the pe r i phe r a l blood ve s se l s to the sympathetic nervous system and c i r c u l a t i n g hormones ( A l t u r a et a l . , 1980). In support of t h i s hypothes i s , a number of s tud ie s conducted on human sub jec t s i n d i c a t e t ha t both i n the presence and absence of va s cu l a r d e t e r i o r a t i o n d i a b e t i c p a t i e n t s w i thout s igns of autonomic neuropathy are more respons ive to va soac t i ve substances such as noradrena l ine (NA) and ang io ten s i n II ( C h r i s t l i e b et a l . , 1976; Weidmann et a l . , 1979). In an attempt to f u r t h e r i n v e s t i g a t e the i n f l u e n c e o f d iabetes on va s cu l a r r e a c t i v i t y , many s tud ie s have been conducted i n d i a b e t i c animal models. However, the r e s u l t of these s t ud i e s have been i n c o n s i s t e n t . A number of i n v e s t i g a t o r s have repor ted an inc rease i n r e a c t i v i t y of i s o l a t e d v a s cu l a r p repara t ions from d i a b e t i c animals t o va r i ou s agon i s t s (Brody and Dixon, 1964; Cseuz et a l . , 1973 Owen and C a r r i e r , 1980; Jackson and C a r r i e r , 1981; MacLeod and M c N e i l l , 1981; 1982; 1985; Scarborough and C a r r i e r , 1983; 1984; MacLeod, 1985; Agrawal and M c N e i l l , 19 1987; de Cuneo, 1988; Agrawal et a l . , 1987; H a r r i s and MacLeod, 1988; Friedman, 1989; White and C a r r i e r , 1988; 1990), w h i l e others have observed decreased r e a c t i v i t y ( S u l l i v a n and Sparks, 1979; Pfaffman et a l . , 1980; Tu r l ap t y et a l . , 1980; Mue l l e r et a l . , 1982; Pfaffman et a l . , 1982; Ramanadham et a l . , 1984; Longhurst and Head, 1985; Head et a l . , 1987). In a d d i t i o n , other workers have repor ted no change i n r e a c t i v i t y of blood ve s se l s from d i a b e t i c animals (Sjogren and Edvinsson, 1988; Jackson and C a r r i e r , 1981; Tak iguchi et a l . , 1988). In most of these s tud ie s there i s a l a c k o f con s i s tency i n the methodologies employed, and thus i t appears t ha t the d i f f e r i n g r e s u l t s obta ined cou ld be due to many f a c t o r s . There may be spec ies d i f f e r e n c e s i n the e f f e c t s of d iabetes on va s cu l a r r e a c t i v i t y . There were a l so d i f f e r e n c e s i n the d iabetogen ic agents used and t h e i r dose and route of a d m i n i s t r a t i o n . A l l o x an and STZ have been commonly used as d iabetogen ic agents. Both agents induce immediate and r e l a t i v e l y s e l e c t i v e damage to the panc rea t i c i s l e t fi - c e l l s f o l l o w i n g i n j e c t i o n o f a s i n g l e dose, r e s u l t i n g i n a ch ron i c d i a b e t i c s t a t e (Rerup, 1970). Although the re i s some cont rover sy as to which k ind of human d iabetes i s mimicked by the syndromes produced by e i t h e r a l l o x a n or STZ, the d iabetes caused by both agents appears to be s i m i l a r i n c l i n i c a l course, i s not i n s u l i n -dependent and i s c h a r a c t e r i z e d by hyperglycemia ( o ccu r r i n g w i t h i n 24 -48 hours a f t e r i n j e c t i o n ) , reduced plasma i n s u l i n l e v e l s , reduced body weight g a i n , p o l y d i p s i a , p o l y u r i a , g l y c o s u r i a , po l yphag ia , d i a r r h e a , c a t a r a c t s , h y p e r l i p i d i m i a , t r i g l y c e r i d e m i a e t c . (Rerup, 1970; H o f t i e z e r 20 and Carpenter , 1973). In a d d i t i o n to producing p anc r ea t i c d e s t r u c t i o n , a l l o x a n , p a r t i c u l a r l y at h igher than d iabetogen ic doses, has a l s o been repor ted to produce t o x i c e f f e c t s on other organs i n c l u d i n g the kidney and l i v e r (Rerup, 1970). Because of these a d d i t i o n a l t o x i c e f f e c t s o f a l l o x a n and the added advantage of STZ having a longer du r a t i on o f a c t i o n , STZ i s now becoming i n c r e a s i n g l y favored i n exper imental s t u d i e s . However, due to i t s low s t a b i l i t y , the r a p i d IV i n j e c t i o n of STZ i s thought to be the s imp le s t , most dependable route o f a d m i n i s t r a t i o n of the drug (Rerup, 1970). The i n f l u e n c e of d iabetes on va s cu l a r r e a c t i v i t y a l s o appears to vary w i th the du ra t i on of the d i a b e t i c s t a t e and w i th the va s cu l a r p repa ra t i on s t u d i e d , as we l l as the sex o f the animal used. For example, MacLeod and McNe i l l (1985) demonstrated tha t responses o f ao r tae , but not p o r t a l v e i n s , from female r a t s w i th STZ-induced d iabetes to NA were a l t e r e d 3 months, but not 7 days a f t e r the i nduc t i on o f d i a b e t e s . Using male r a t s t r e a t e d w i th STZ, Owen and C a r r i e r (1980) a l s o observed tha t the respons iveness o f aortae to NA was increased a f t e r 1 month of d i a b e t e s , but not a f t e r 14 - 20 days. On the other hand, the respons iveness o f mesenter ic a r t e r i e s from STZ t r e a t e d male r a t s to NA was found to be enhanced a f t e r 42-43 weeks of d i abe te s , but not a f t e r 4-5 weeks. In these s tud ie s the animals w i th ch ron i c d i abe te s were a l s o found to be hyper tens ive (Owen and C a r r i e r , 1980). Although v a r i a t i o n s i n r e a c t i v i t y o f blood ve s se l s cou ld be demonstrated depending upon the c ond i t i o n s employed, g e n e r a l l y , most 2 1 s t ud i e s i n d i c a t e tha t wh i l e acute or e a r l y stage o f d i abetes induces a tendency towards h y p o r e a c t i v i t y , ch ron i c or l ong - s tand ing d iabetes u s u a l l y induces h y p e r r e a c t i v i t y of a r t e r i a l smooth muscle ( A l t u r a et a l . , 1980). :1.3.2. Mechanisms of increased react iv i ty to neuro-humoral and related substances The mechanisms r e spon s i b l e f o r the increased r e a c t i v i t y of blood ve s se l s from d i a b e t i c animals to neuro-humoral substances have been examined by a number of i n v e s t i g a t o r s . In recent yea r s , MacLeod and co-workers s tud ied the mechanisms o f increased a r t e r i a l r e a c t i v i t y o c c u r r i n g i n male r a t s w i th STZ - induced d iabetes o f 3 months d u r a t i o n . I t was found tha t both aortae and mesenter ic a r t e r i e s i s o l a t e d from d i a b e t i c r a t s responded to NA w i th g rea te r inc reases i n t en s i on than d i d the corresponding t i s s u e s from age - matched con t r o l animals (MacLeod, 1985). E i t h e r l i t t l e (Ha r r i s and MacLeod, 1988) or no change (MacLeod, 1985) i n the s e n s i t i v i t y (expressed as the pD 2 or - l o g ED50 va lue) o f e i t h e r aortae or mesenter ic a r t e r i e s from d i a b e t i c r a t s to NA was de tec ted . The increased respons iveness o f d i a b e t i c blood ve s se l s to NA cou ld be prevented from o c c u r r i n g , or reversed once e s t a b l i s h e d , by t r e a t i n g the d i a b e t i c animals w i th i n s u l i n (MacLeod, 1985) suggest ing t ha t the inc reased respons iveness i s a r e s u l t of the d i a b e t i c s t a t e , and not due to some other e f f e c t , such as a d i r e c t a c t i o n o f STZ. At the same t ime, responses of K + - d e p o l a r i z e d p repa ra t i on s to i n c r e a s i n g 22 concen t ra t i on s of e x t r a c e l l u l a r Ca^ were not a l t e r e d (MacLeod, 1985), i n d i c a t i n g t ha t the increased respons iveness to NA i s not the r e s u l t of a g e n e r a l i z e d inc rease i n the c o n t r a c t i l i t y o f the d i a b e t i c blood v e s s e l s . Cons i s ten t w i th these ob se rva t i on s , s i m i l a r f i n d i n g s were a l so repor ted by other i n v e s t i g a t o r s who demonstrated enhanced r e a c t i v i t y of va r i ou s types o f blood ves se l s from d i a b e t i c r a t s to NA, o ther a-adrenoceptor agon i s t s (eg. pheny lephr ine, methoxamine, c l o n i d i n e , guanbenz and 0-HT 920) and se ro ton in (Brody and Dixon, 1964; Owen and C a r r i e r , 1980; 1985; Jackson and C a r r i e r , 1981; Scarborough and C a r r i e r , 1983; 1984; Agrawal and M c N e i l l , 1987; Agrawal et a l . , 1987; de Cuneo et a l . , 1988; White and C a r r i e r , 1988; Friedman, 1989). Subsequent i n v e s t i g a t i o n s by Ha r r i s and MacLeod (1988), showed t ha t the enhanced c o n t r a c t i l e respons iveness of the d i a b e t i c a r t e r i e s to NA was not due to a decrease i n the r e l ea se of EDRF but was a consequence o f s t i m u l a t i o n of a j - ad renocepto r s (Abebe et a l . , 1990). In agreement w i th these r e s u l t s , White and C a r r i e r (1988) repor ted tha t enhanced c o n t r a c t i l e responses o f mesenter ic a r t e r i e s from r a t s w i th ch ron i c STZ - induced d iabetes to NA and other a-adrenoceptor s t imu lan t s are mediated by a\-adrenoceptors . On the bas i s o f these f i n d i n g s , i t was proposed tha t the enhanced c o n t r a c t i l e responses of the d i a b e t i c a r t e r i e s to NA r e s u l t from a change i n e i t h e r the a\-adrenoceptors themselves, or i n the coup l i ng o f these recepto r s w i th the c o n t r a c t i l e response (MacLeod, 1985; H a r r i s and MacLeod, 1988; Abebe et a l . , 1990). 23 1.4. VASCULAR SMOOTH MUSCLE CONTRACTION 1.4.1. Sources and regulation of ce l lu lar calcium I t i s g e n e r a l l y accepted tha t c o n t r a c t i o n o f v a s cu l a r smooth muscle depends p r i m a r i l y on the concen t ra t i on o f i n t r a c e l l u l a r f r e e C a 2 + , o f t en r e f e r r e d to as a c t i v a t o r C a 2 + ( Bo l t on , 1979). In the r e s t i n g s t a t e , the i n t r a c e l l u l a r C a 2 + c oncen t ra t i on ranges from 10"^ to 10"^ M and t h i s l e v e l i s not s u f f i c i e n t to achieve a c t i v a t i o n of the c o n t r a c t i l e mechanism (Rasmussen and Ba r r e t , 1984; Morgan and Morgan, 1985; Sommervi l le and Hartshorne, 1986). On the other hand, the e x t r a c e l l u l a r C a 2 + c oncen t ra t i on i s g r ea te r than 1 0 " 3 M (Daniel et a l . , 1983). Desp i te the l a r ge concen t ra t i on g r ad i en t s , due to the poor p e r m e a b i l i t y o f the plasma membrane to t h i s i o n , on ly very small amounts o f C a 2 + en te r the c e l l s i n the r e s t i n g s t a t e . C a 2 + s l ow l y en t e r i n g the c e l l under t h i s c o n d i t i o n ( C a 2 + l eak ) i s e f f i c i e n t l y removed by homeostat ic mechanisms, and thus the i n t r a c e l l u l a r Ca^ concen t r a t i on does not i nc rease high enough to i n i t i a t e c o n t r a c t i o n . For c o n t r a c t i o n to be induced, the i n t r a c e l l u l a r C a 2 + c oncen t ra t i on must r i s e , ranging from fou r to s i x f o l d (Rasmussen and Ba rne t t , 1984). There fo re , i t i s of v i t a l importance tha t the smooth muscle c e l l has the necessary mechanisms f o r r e g u l a t i n g the en t ry and removal of Ca , and thus modulat ing c o n t r a c t i l i t y . Evidence from p h y s i o l o g i c a l and pharmacolog ical s t ud i e s i n d i c a t e s t ha t a c t i v a t o r C a 2 + r equ i red to support c o n t r a c t i o n can be de r i ved from 24 e i t h e r the e x t r a c e l l u l a r compartment or the i n t r a c e l l u l a r Ca^ s t o r e s . The r e l a t i v e c o n t r i b u t i o n o f C a 2 + from these sources i s dependent upon the v a s cu l a r smooth muscle type, the nature and concen t r a t i on o f the agon i s t t e s t e d and the components (phas ic or t o n i c ) o f the response measured (Bo l t on , 1979; T r i g g l e , 1983; McGrath et a l . , 1989). A l a r ge p ropo r t i on o f the e x t r a c e l l u l a r C a 2 + i n smooth muscle i s bound to d i f f e r e n t b ind ing s i t e s on the ex te rna l membrane s u r f a ce . These b ind ing s i t e s c o n s i s t of severa l n e ga t i v e l y charged p r o t e i n s , phospho l i p id s and s i a l i c a c i d res idues ( Ishiyama et a l . , 1979). E x t r a c e l l u l a r C a 2 + needed f o r c o n t r a c t i o n can ga in access to the cytoplasm v i a two sets of d i f f e r e n t and independent Ca^ channels l o c a t e d on the plasma membrane : vo l tage -opera ted ca l c i um channels (VOC) and recep to r -ope ra ted ca l c i um channels (ROC). VOC respond p r i n c i p a l l y to changes i n vo l tage across the membrane, such as r educ t i on i n membrane p o t e n t i a l r e s u l t i n g from e l e v a t i o n of the K + c oncen t r a t i on i n the e x t r a c e l l u l a r medium (Bo l ton , 1979). These channels are s u s c e p t i b l e to blockade by a group of drugs known as ca l c ium channel b l o c k e r s . In a d d i t i o n , the c o n t r a c t i o n induced by C a 2 + en t r y through VOC can be e l i m i n a t e d i n C a 2 + - f r e e s o l u t i o n i n d i c a t i n g tha t the c o n t r a c t i o n i s s o l e l y due to the i n f l u x of C a 2 + i n t o the c e l l . The c o n t r a c t i o n process i n v o l v i n g VOC has been r e f e r r e d to as e l ec t r o -mechan i ca l coup l i ng (Somlyo and Somlyo, 1968). ROC respond p r i n c i p a l l y to hormones and neu ro t ran sm i t te r s which act through recepto r s on the plasma membrane. These channels remain 2 5 ope ra t i ve when the membrane i s depo l a r i z ed and thus are d i s t i n g u i s h e d from VOC based on t h e i r a d d i t i v i t y . In a d d i t i o n , ROC are r e l a t i v e l y l e s s s e n s i t i v e to blockade by C a 2 + channel b locke r s ( Bo l t on , 1979). Furthermore, most agon i s t s i n c l u d i n g NA, which a c t i v a t e ROC, can apparent l y a l s o cause r e l ea se of C a 2 + from i n t r a c e l l u l a r s to re s so tha t t r a n s i e n t phas ic c o n t r a c t i l e responses to these agon i s t s can be generated even i n the absence o f e x t r a c e l l u l a r C a 2 + . Con t rac t i on s induced by such agon i s t s i n the presence o f e x t r a c e l l u l a r C a 2 + thus c o n s i s t o f two components : an i n i t i a l phas ic component and a delayed sus ta ined component. Release o f C a 2 + from i n t r a c e l l u l a r s to re s c o n t r i b u t e s to the i n i t i a l phas ic component, w h i l e i n f l u x of e x t r a c e l l u l a r C a 2 + appears important f o r the sus ta ined t o n i c phase of the c o n t r a c t i l e responses. The process of c o n t r a c t i o n induced wi thout a l t e r a t i o n s i n membrane p o t e n t i a l and i n v o l v i n g en t ry of C a 2 + through ROC and/or r e l ea se o f C a 2 + from sequestered s to re s i s r e f e r r e d to as pharmaco-mechanical coup l i ng (Somlyo and Somlyo, 1968). There i s a great deal o f unce r t a i n t y as to the l o c a t i o n o f the i n t r a c e l l u l a r C a 2 + pool i nvo l ved i n v a s cu l a r smooth muscle c o n t r a c t i o n . The inner su r face of the plasma membrane has been suggested to be a major s torage s i t e f o r r e l ea se of C a 2 + i n t r a c e l l u l a r l y (Daniel e t a l . , 1983). However, an i n c r ea s i n g body o f data i n d i c a t e t ha t sa rcop lasmic r e t i c u l u m (SR) i n smooth muscle, although present i n small amounts, i s the major i n t r a c e l l u l a r s to re r e g u l a t i n g C a 2 + f l u x dur ing c o n t r a c t i o n (Somlyo, 1985). Recogniz ing the p h y s i o l o g i c a l importance o f the SR as a 26 source of a c t i v a t o r Ca , r e c e n t l y g r ea te r i n t e r e s t has been d i r e c t e d towards determin ing the s i gna l t r an sduc t i on mechanisms r e spon s i b l e f o r t r i g g e r i n g the r e l ea se of C a 2 + from such sources f o l l o w i n g agon i s t -p i p i r ecep to r i n t e r a c t i o n . While there i s evidence f o r C a £ - induced Ca r e l ea se mechanisms i n some va s cu l a r smooth muscles (Sa ida and Van Breemen, 1984), a t t e n t i o n has been pa id mainly to s tudy ing the r o l e o f p i phospho ino s i t i de metabolism i n r e g u l a t i n g i n t r a c e l l u l a r Ca r e l ea se ( M i c h e l ! , 1975; St reb et a l . , 1983; Somlyo et a l . , 1988). There i s some exper imental evidence i n d i c a t i n g tha t the i n t r a c e l l u l a r C a 2 + s torage s i t e s are i n c l o se p rox im i t y to the plasma membrane suggest ing a p o s s i b l e f u n c t i o n a l l i n k between the storage s i t e s and ROC ( I t o et a l . , 1982; Putney, 1986; Van Breemen and Sa ida , 1989). In o rder to prevent the b u i l d up o f C a 2 + i n the cytoplasm and thus ma inta in c e l l i n t e g r i t y , i t i s of major importance tha t c e l l s have con t r o l mechanisms which r egu l a te and minimize changes i n i n t r a c e l l u l a r C a 2 + c o n c e n t r a t i o n . Two mechanisms a s soc ia ted w i th the plasma membrane f o r e x t r u s i o n o f C a 2 + are the sodium-calc ium ( N a + - C a 2 + ) exchange process and the C a 2 + - ATPase e f f l u x pump. Moreover, a t h i r d process of C a 2 + removal operates w i t h i n the c e l l t o sequester C a 2 + i n t o i n t r a c e l l u l a r s torage s i t e s . The N a + - C a 2 + exchange i s a mechanism whereby cy top la smic C a 2 + i s extruded aga in s t i t s e l ec t rochemica l g rad ien t by energy d r i v en from inward movement of Na + down i t s e l e c t r ochemica l g r ad i en t ( B l a u s t e i n , 1977). A s imple exchange of 3 Na + f o r 1 C a 2 + extruded has been suggested 27 by Baker (1972). However, under p h y s i o l o g i c a l c o n d i t i o n s , the extent of the c o n t r i b u t i o n of the N a + - C a 2 + exchange f o r removal o f cy top la smic Ca i s not c l e a r . I t has been suggested tha t u n l i k e c a r d i a c muscle where the system removes the bulk o f the cy top la smic C a 2 + (Caroni et a l . , 1980), the N a + - C a 2 + exchange process i n v a s cu l a r smooth muscle f unc t i on s p r i m a r i l y as a back up system when the C a 2 + c oncen t r a t i on reaches t o x i c l e v e l s (Van Breemen, 1975). The plasma membrane C a 2 + pump i s a magnesium-dependent C a 2 + - H + -p • ATPase, d e r i v i n g i t s energy from ATP, and t r a n s p o r t i n g Ca*- out o f the c e l l aga ins t a l a r ge concen t ra t i on g rad ien t (Daniel et a l . , 1983; Grover et a l . , 1980). N i g g l i et a l . (1982) repor ted t ha t i n human e r y th rocy te membranes, the C a 2 + pump operates n o n - e l e c t r o g e n i c a l l y , whereby each C a 2 + extruded i s balanced by the ent ry o f two protons ( H + ) . The r e s u l t i n g a l t e r a t i o n s i n the pH of the cytoplasm are counter -ba lanced by p . an inward movement o f OH" exchanged f o r HCO3", C I " or HPO4 . The Ca 2 + -ATPase e f f l u x pump has been shown to be a sub s t ra te f o r PKC, and i t s a c t i v a t i o n by PKC has been suggested to be i nvo l ved i n a feedback mechanism p r o t e c t i n g the c e l l from prolonged exposure to e l eva ted i n t r a c e l l u l a r C a 2 + dur ing c o n t r a c t i o n r e s u l t i n g from phospho ino s i t i de metabolism (Lagast et a l . , 1984). Un l i ke the Na -Ca f c exchange, the presence and f unc t i on of the Ca 2 + -ATPase e f f l u x pump has been we l l documented (Daniel et a l . , 1983). p i With regard to i n t r a c e l l u l a r s eques t ra t i on o f Ca , the re i s a p 1 s t rong evidence demonstrat ing the involvement of the SR as a Ca*-28 s torage s i t e . Permeab i l i zed p repara t i ons which a l l ow man ipu la t ion of i n t r a c e l l u l a r ca l c i um and microsomal f r a c t i o n s obta ined by c e l l f r a c t i o n a t i o n have been used f o r the i d e n t i f i c a t i o n of the SR as a C a 2 + storage s i t e . Accumulat ion of C a 2 + i n the SR i s accomplished by a C a 2 + -Mg 2 + -ATPase, and has been shown to occur at low concen t ra t i on s of C a 2 + and when m i tochondr i a l uptake i s i n h i b i t e d (Burgess, 1983). In con t r a s t to the C a 2 + e x t r u s i o n mechanism a c t i n g at the plasma membrane, the C a 2 + -ATPase uptake system at the SR f unc t i on s independent ly o f ca lmodu l in (CaM) and i s a high a f f i n i t y removal mechanism, ope ra t i ng at low concen t ra t i on s o f cy top la smic C a 2 + (Raeymaekers et a l . , 1985). I t p rov ides the major mechanism of i n t r a c e l l u l a r ca l c i um s e q u e s t r a t i o n . The mi tochondr ia have a l s o been i m p l i c a t e d i n the i n t r a c e l l u l a r s eque s t r a t i on of c a l c i um; however, they appear to f u n c t i o n main ly when the concen t r a t i on of C a 2 + i s e x c e s s i v e l y h i gh . I t has been suggested tha t the mi tochondr ia p lay a r o l e under pa thophy s i o l o g i c a l c ond i t i o n s when the i n t r a c e l l u l a r C a 2 + c oncen t ra t i on reaches t o x i c l e v e l s (Daniel e t a l . , 1983). Calc ium has a l so been shown bound to the i nner plasma membrane, but the i d e n t i t y and f u n c t i o n o f t h i s pool are l e s s c l e a r (Danie l et a l . , 1983). 1.4.2. Contractile mechanisms The cu r ren t understanding of smooth muscle c o n t r a c t i l e mechanisms i s based on the s l i d i n g f i l amen t theory o r i g i n a l l y proposed f o r s k e l e t a l muscle (Huxley and Hanson, 1954). In t h i s proposal the ba s i c concept i s 29 t ha t the f i l amen t s of myosin and a c t i n d o n ' t change i n l eng th themselves and t ha t the development of ten s i on i s r e l a t e d to the degree of i n t e r d i g i t a t i o n between the two types of f i l a m e n t s . However, i t has subsequently become ev ident tha t the c e l l u l a r and mo lecu la r mechanisms under l y i ng f o r ce development and maintenance i n s k e l e t a l and smooth muscle are d i f f e r e n t , although they both share the p r o te i n s myosin, a c t i n and tropomyos in. In a d d i t i o n , smooth muscle conta in s the major p r o t e i n s caldesmon, g e l s o l i n , f i l a m i n and desmin which are e i t h e r absent or scarce i n s k e l e t a l muscle (Rasmussen et a l . , 1987). These p r o te i n s have been proposed to p lay important r o l e s i n smooth muscle c o n t r a c t i o n . Var ious mechanisms have been suggested f o r the r e g u l a t i o n o f ac t in -myos in i n t e r a c t i o n , and thus c o n t r a c t i o n , by C a 2 + i n smooth muscle. One of the most w ide l y accepted mechanisms i n v a s cu l a r smooth muscle i s t ha t of Ca 2 + -CaM- induced myosin phosphory la t ion ( A d e l s t e i n and Hathaway, 1979). Th i s model proposes tha t when the i n t r a c e l l u l a r Ca*-concen t r a t i on r i s e s above the th re sho ld va lue (> 10~^M), ex ten s i ve b ind ing of C a 2 + to CaM occurs forming an a c t i v a t e d Ca 2 + -CaM complex. The Ca*- -CaM complex then a c t i v a t e s a 130,000 da l t on myosin l i g h t cha in k inase (MLCK) l ead ing to the phosphory la t ion o f p a r t i c u l a r s e r i ne re s idues on the 20,000 da l t on myosin l i g h t cha in (MLC) ( B a i l i n , 1984). The phosphory lated myosin then combines w i th a c t i n and undergoes a complex i n t r a c e l l u l a r arrangement r e s u l t i n g i n c r o s s - b r i d ge format ion and muscle c o n t r a c t i o n ( Ade l s t e i n and E i senberg, 1980). Accord ing to t h i s model, on ly when phosphory la t ion o f MLC occurs can a c t i n a c t i v a t e 30 myosin ATPase and c r o s s - b r i d ge format ion take p l a c e . Dephosphory lat ion of MLC and thus t e rm ina t i on of c r o s s - b r i d ge c y c l i n g are achieved by a Ca 2 + - i ndependen t MLC phosphatase present i n the smooth muscle (Pato and A d e l s t e i n , 1980). In c on t r a s t to the above model, Ebashi (1980) proposed an a l t e r n a t i v e mechanism whereby smooth muscle c o n t r a c t i o n can occur wi thout MLC phosphory l a t i on . Accord ing to t h i s theo ry , C a 2 + and a c t i n b ind ing p r o te i n s known as l e i t o n i n can cause a c t i n a c t i v a t i o n o f myosin ATPase wi thout phosphory l a t i on . Other i n v e s t i g a t o r s (Walters and Marston, 1981) have a l so proposed other mechanisms such as a dual r e g u l a t o r y system i n v o l v i n g phosphory la t ion of p r o t e i n s a s soc i a ted w i th both a c t i n and myosin f i l a m e n t s . Although a l t e r n a t i v e mechanisms have been proposed f o r the i n t e r a c t i o n between a c t i n and myosin, most o f the evidence repor ted to date i n d i c a t e s tha t phosphory la t ion o f MLC i s an important event mediat ing smooth muscle c o n t r a c t i o n . I m p l i c i t i n t h i s model are the assumptions t ha t dur ing sus ta ined phase of c o n t r a c t i o n the re i s a su s ta ined i nc rease i n i n t r a c e l l u l a r C a 2 + c oncen t ra t i on and a sus ta ined i nc rea se i n the extent o f MLC phosphorylaton (MLC.P). However, recent s t ud i e s have demonstrated tha t n e i t h e r of these assumptions i s c o r r e c t . Morgan and Morgan (1983, 1984), us ing aequorin as an i n t r a c e l l u l a r C a t T i n d i c a t o r , have shown tha t the agon i s t - i nduced r i s e i n i n t r a c e l l u l a r f r e e C a 2 + c oncen t ra t i on i s t r a n s i e n t , l a s t i n g on ly a few minutes even when the c o n t r a c t i l e response i s s u s t a i ned . The C a c c oncen t ra t i on 3 1 remained on ly s l i g h t l y g r ea te r than the basal va lue t h e r e a f t e r . S i m i l a r l y , S i l v e r and S t u l l (1984), Aksoy et a l . (1982) and G e r t h o f f e r and Murphy (1983) have a l l demonstrated tha t the amount o f MLC.P r i s e s r a p i d l y dur ing an agon i s t - i nduced response but then f a l l s g r a d u a l l y to basal va lues dur ing sus ta ined phase o f the c o n t r a c t i l e response. The obse rva t ion o f su s ta ined c o n t r a c t i o n i n the face o f t r a n s i e n t i nc reases i n the i n t r a c e l l u l a r C a 2 + c oncen t ra t i on and i n the content of MLC.P gave r i s e to the l a t c h br idge hypothes i s f o r tens ion maintenance ( D i l l o n et a l . , 1981). In t h i s mechanism, a new type of l o n g - l a s t i n g ac t in -myos in br idge ( i e . the l a t c h br idge or s l owly c y c l i n g c ro s sb r i dge ) i s formed a f t e r dephosphory lat ion o f MLC. I t has been po s tu l a ted t ha t l a t c h br idge format ion i s more s e n s i t i v e to C a 2 + than the i n i t i a l a c t i v a t i o n of MLCK and t h i s has been suggested to e x p l a i n why l a t c h br idges form a f t e r the concen t r a t i on o f Ca*- f a l l s back c l o s e to i t s basal va lue (Rasmussen et a l . , 1989). Recent developments i n smooth muscle research i n v o l v i n g u l t r a s t r u c t u r a l a n a l y s i s of the arrangement o f a c t i n , myosin and i n te rmed ia te p r o t e i n s , and the r o l e of phospho ino s i t i de metabolism and PKC po i n t to a new exp lana t i on f o r the sus ta ined phase of smooth muscle c o n t r a c t i o n . 1.5. PHOSPHOINOSITIDE METABOLISM: GENERAL VIEW The concept tha t changes i n phospho inos i t i de metabol ism are i n t e g r a l l y i nvo l ved i n the ac t i on s of ca lc ium-mediated neu ro t ran sm i t te r s and hormones arose out of the p ioneer ing s t ud i e s of Hokin and Hokin i n 32 1953. However, the f u n c t i o n o f t h i s system remained a mystery u n t i l 1975, when M i c h e l l recogn ized tha t phospho inos i t i de h y d r o l y s i s , y i e l d i n g l i p o p h i l i c DG and a water s o l ub l e i n o s i t o l phosphate, i s a r e c e p t o r -p i s t imu l a ted r e a c t i o n tha t somehow causes the r i s e i n c y t o s o l i c Ca^ concen t r a t i on t ha t mediates many o f the e f f e c t s o f c e l l s t i m u l a t i o n . In 1979, N i sh i zuka and h i s co l leagues showed tha t DG formed i n t h i s r e a c t i o n serves as a c e l l u l a r second messenger i n t ha t i t a c t i v a t e s a novel p r o t e i n k inase , PKC, which i s a l s o the t a r g e t of te rpeno id tumor promoters such as 12 -0 - tet radecanoy l phorbol 13-acetate (TPA) and phorbol 12 ,13 -d ibuty ra te (PDB) (N i sh i zuka , 1984). The r e c o g n i t i o n i n 1981 t ha t phosphat idy l i n o s i t o l 4,5 -b isphosphate (Ptd Ins (4,5)P2) i s the l i p i d hydro lyzed i n i t i a l l y i n response to s t i m u l a t i o n (M i che l l et a l . , 1981) revea led tha t i n o s i t o l 1 ,4 ,5 - t r i sphosphate ( I n s ( l , 4 , 5 )P3 ) i s the w a t e r - s o l u b l e product of the r e c e p t o r - a c t i v a t e d phosphol ipase C (PLC). I t was then q u i c k l y shown that t h i s molecule i s an i n t r a c e l l u l a r second messenger r e spon s i b l e f o r r a i s i n g the c y t o s o l i c Ca*- c oncen t r a t i on w i t h i n s t imu l a ted c e l l s by caus ing r e l ea se i n t o the c y to so l o f the C a c sequestered w i t h i n a nonmitochondr ia l i n t r a c e l l u l a r membrane compartment (S t reb e t a l . , 1983; Ber r idge and I r v i n e , 1984). S ince these i n i t i a l ob se r va t i on s , the phospho inos i t i de s i g n a l l i n g system has been recogn ized as an important i n te rmed ia ry i n i n t r a c e l l u l a r s i g n a l l i n g i n a wide v a r i e t y o f b i o l o g i c a l systems. There has been i n t e n s i v e work on i t i n many l a b o r a t o r i e s i n the l a s t 10 years and t h i s has achieved s t a r t l i n g l y r a p i d progress . 33 1.5.1. Phosphoinositides and the phosphoinositide cycle The phospho inos i t i de s are minor components (<10%) o f membrane phospho l i p id s e x i s t i n g i n th ree forms as phosphat idy l i n o s i t o l ( P t d l n s ) , phosphat idy l i n o s i t o l 4-phosphate (Ptd Ins(4)P) and Ptd Ins(4,5)P2 i n a r a t i o o f approx imately 90:5:5 (Rasmussen, 1986). They are h i g h l y a c t i v e m e t a b o l i c a l l y and are r a p i d l y i n t e r c o n v e r t i b l e by app rop r i a te k inases and phosphatases which are a l s o present i n the membrane. Ptd Ins (4)P and Ptd Ins(4,5)P2 are formed v i a sequent ia l phosphory la t ion o f P td ln s by the enzymes P td ln s k inase and Ptd Ins(4)P k inase , r e s p e c t i v e l y . Phosphatase enzymes dephosphorylate the po l yphospho inos i t i de s so t ha t even when unst imulated these i n o s i t o l l i p i d s are c on s t an t l y t u r n i n g over i n a so c a l l e d f u t i l e c y c l e (Abdel - L a t i f , 1986). Although P td ln s i s l o ca ted i n severa l s u b c e l l u l a r membranes (mainly i n endoplasmic r e t i c u l u m ; ER), the other two forms are l o ca ted l a r g e l y i n plasma membrane. In t h i s membrane, they are thought to be l o ca ted i n i t s i nner l e a f l e t , where they are s u s c e p t i b l e to h y d r o l y t i c c leavage by a s p e c i f i c PLC (a phosphodiesterase tha t c leaves the i n o s i t o l phosphate from the g l y c e r o l backbone) to form DG and i n o s i t o l phosphates ( A b d e l - L a t i f , 1986). Although a l l th ree forms of the phospho inos i t i des are sub s t ra te s f o r PLC, accumulat ing evidence i n d i c a t e s tha t the i n i t i a l r e a c t i o n i n r e c e p t o r - s t i m u l a t e d phospho inos i t i de metabolism i n vo l ve s the s e l e c t i v e breakdown o f P td l n s ( 4 , 5)P2 ( A b d e l - L a t i f et a l . , 1977; K i r k et a l . , 1981; Chuang, 1989). The h y d r o l y s i s of P td lns and Ptd Ins (4)P i s be l i e ved to be an event t r i g g e r e d by the m o b i l i z a t i o n of ca l c i um i n response to 34 Ins(1,4,5)P3 produced by s t imulus -mediated breakdown o f Ptd Ins (4,5)P2-Changes i n the membrane l i p i d microenvironment as a r e s u l t of Ptd Ins(4,5)P2 h y d r o l y s i s may a l so c o n t r i b u t e to the change i n sub s t ra te s p e c i f i c i t y from PtdIns(4,5)P2 to Ptd Ins(4)P and P td ln s (Chuang, 1989). S ince the m a j o r i t y of P td ln s i s l o ca ted i n the ER, i t i s a l s o p o s s i b l e t ha t some P td ln s h y d r o l y s i s may take p lace i n t h i s o r g a n e l l e , secondary to the s t i m u l a t i o n o ccu r r i n g i n the plasma membrane. With few except i on s , the breakdown o f the phospho inos i t i de s i s accompanied by compensatory r e s y n t h e s i s . The breakdown and r e s yn the s i s process can be cons idered as two l i n k e d metabo l i c c y c l e s as i l l u s t r a t e d i n F igure 2. In one o f the c y c l e s , phospho inos i t i de h y d r o l y s i s g i ve s r i s e to the format ion o f i n o s i t o l phosphates which are u l t i m a t e l y converted to f r ee i n o s i t o l . Free i n o s i t o l , by r e a c t i n g w i th c y t i d i n e d iphosphate -1 ,2 -d i a c y l g l y c e r o l (CDP-DG) i n the presence o f the enzyme CDP-DG i n o s i t o l p h o s p h a t i d y l t r a n s f e r a s e , i s then re syn thes i zed i n t o P td ln s and hence back to Ptd Ins (4)P and P td ln s (4 ,5 ) P2. CDP-DG i s formed as a r e s u l t of combinat ion of c y t i d i n e t r i phosphate w i th phosphat id i c a c i d (PA). The s y n t h e t i c enzyme, Ptd lns synthetase, i s present i n ER and the s yn thes i s of new Ptd ln s i s be l i e ved to take p lace i n t h i s o r g a n e l l e . The newly s yn thes i zed P td ln s i s t r an spor ted to the plasma membrane by p r o t e i n exchange (Chuang, 1989). In the second c y c l e , the other product o f pho spho ino s i t i de breakdown, DG, i s r a p i d l y converted to PA by DG k inase . PA i s then converted to CDP-DG to serve i n the s yn thes i s o f P td ln s as noted above. Thus the phospho inos i t i de l i p i d s are r e s yn the s i zed wi thout 3 5 F igure 2 Pathways o f phospho inos i t i de metabolism (modi f ied from Heagerty and Ol lerenshaw, 1987). 36 11 DG t DG t PtdIns^z=!rPtdIns(4)P ==^PtdIns(4,5)P 2 1^(1,2,3,4,5,6^ • Ins(l)P^*- Ins(l:2cyc)P •Ins(4)P -« Ins(l,4)P2-*--Ins(l:2cyc4)P2- •Ins(l:2cyc4,5)P3 Ins(l,3,4,5,6)P5 • rns(l,4^)P3-^ns(l,3,4,5)P4. •Ins(3)P Ins(3,4)P2 Ins(l,3,4)P3 •Ins(l)P Ins(l,3)P2 Ins(l,3,4,6)P4 37 the need f o r an exhaust ive supply of p recu r so r s . DG may a l s o be hydro lyzed by DG l i p a s e to form a rach idon i c a c i d and monoacy l g l y ce ro l . There are a l so a d d i t i o n a l pathways f o r PA metabol ism, the major ones i n v o l v i n g i t s h y d r o l y s i s to l y s opho spha t i d i c a c i d and a r ach i don i c a c i d by a s p e c i f i c phosphol ipase A 2 . The product ion o f a r a ch i don i c a c i d may lead to the format ion o f va r ious e i cosano ids which themselves may s t imu l a t e the generat ion of i n o s i t i d e - d e r i v e d second messengers (Abde l -L a t i f , 1986). Under normal r e s t i n g c ond i t i o n s p h o s p h o d i e s t e r a s e c leavage of Ptd Ins(4,5)P2 does not take p lace to any g reat e x ten t . However, combinat ion o f a C a 6 - m o b i l i z i n g agon i s t w i th a r e cep to r on the plasma membrane induces a marked inc rease i n PLC a c t i v i t y w i th the d i v e r s i o n of Ptd Ins(4,5)P2 out of the f u t i l e c y c l e . In doing so, as p r e v i o u s l y po inted out , two i n t r a c e l l u l a r second messengers, I n s ( l , 4 , 5 )P3 and DG are produced. A number of i n v e s t i g a t o r s have proposed t ha t s p e c i f i c GTP-b ind ing p r o te i n s known as G-prote ins may r egu l a te the recep to r - coup led PLC a c t i v i t y (Gomperts, 1983; Cockc ro f t and Gomperts, 1985; L i tosh et a l . , 1985; Smith et a l . , 1985). Many s tud ie s i n pe rmeab i l i zed c e l l s (Cockc ro f t and Gomperts, 1985; M e r r i t t et a l . , 1986) and i s o l a t e d membranes ( L i t o s ch et a l . , 1985) have demonstrated tha t non-hydro lyzab le GTP analogues such as GTP7S or GPP(NH)P s t imu l a t e PLC-mediated Ptd Ins(4,5)P2 breakdown and I n s ( l , 4 ,5 )P3 fo rmat i on . In a d d i t i o n , i n some c e l l t ypes , PLC a c t i v i t y has been shown to be i n h i b i t e d by p e r t u s s i s t o x i n , which i s known to i n a c t i v a t e Gi ( U i , 1986). However, s e n s i t i v i t y 38 to the t o x i n i s by no means un i ve r s a l as many systems i n which GTP7S s t imu l a te s Ptd Ins(4,5)P2 h y d r o l y s i s are not p e r t u s s i s t o x i n - s e n s i t i v e ( M e r r i t t et a l . , 1986). Vascu la r smooth muscle appears to belong to the l a t t e r group, s i nce ang io tens in II s t i m u l a t i o n of PLC i s mediated v i a p e r t u s s i s t o x i n - i n s e n s i t i v e GTP-binding p r o t e i n ( G r i e n d l i n g e t a l . , 1988). In pe rmeab i l i zed va s cu l a r smooth muscle c e l l s , GTP7S has been found to inc rease I n s ( l , 4 ,5 )P3 format ion i n a dose-dependent manner (Somylo et a l . , 1988). These experiments suggest t ha t a guanine n u c l e o t i d e b ind ing p r o t e i n d i s t i n c t from G-j modulates PLC a c t i v i t y i n v a s cu l a r smooth muscle c e l l s . Fur ther evidence f o r the ope ra t i on of a G-p r o t e i n i n the phospho inos i t i de s i g n a l l i n g system has come from s tud ie s us ing f l u o r i d e which can a c t i v a t e the system i n va r i ou s c e l l types i n c l u d i n g v a s cu l a r smooth muscle (Sternweiss and Gi lman, 1982; Cockc ro f t and T a y l o r , 1987). In the presence o f A l 3 + , f l u o r i d e forms a complex, A IF4 " , which i s able to i n t e r a c t w i th the GDP s i t u a t e d on the a - subun i t of G -p ro te i n s , r e s u l t i n g i n a c t i v a t i o n by mimicking GTP at i t s b ind ing s i t e (Cockc ro f t and Tay l o r , 1987; Blackmore et a l . , 1985; Harden, 1989). As w i th other PLC a c t i v a t i n g agents, f l u o r i d e has been repor ted to induce i n o s i t o l phosphate product ion and c o n t r a c t i o n o f v a s cu l a r smooth muscle p repa ra t i on s (Zeng et a l . , 1989). Desp i te the va r i ou s f i n d i n g s , i t should be noted tha t the i d e n t i t y and p r e c i s e c h a r a c t e r i s t i c s of the G-p ro te in s i nvo l ved i n the coup l i ng of a g o n i s t - r e c e p t o r b ind ing to PLC s t i l l remain to be d e f i n e d . 39 The phospho inos i t i de s i g n a l l i n g pathway- can be t r i g g e r e d by a c t i v a t i o n o f a v a r i e t y of PLC-coupled r e cep to r s . Almost a l l o f these recepto r s are C a 2 + - m o b i l i z i n g recepto r s and i n c l ude a j - a d r e n e r g i c , mu sca r i n i c , H j - h i s t am ine , V j - v a s o p r e s s i n , ang io tens in I I , 5-HT2-s e r o t o n i n , b r a d y k i n i n , t h y r o t r o p i n - r e l e a s i n g hormone, g lucose , substance P, thrombin, p l a t e l e t - a c t i v a t i n g f a c t o r and growth f a c t o r s ( A b d e l - L a t i f , 1986). Recent f i n d i n g s a l so i n d i c a t e tha t c e r t a i n recepto r s which d o n ' t f u n c t i o n through C a 2 + m o b i l i z a t i o n can lead to phospho inos i t i de h y d r o l y s i s (eg. n i c o t i n i c a c e t y l c h o l i n e recepto r s and glutamate r e c e p t o r s ) . The recepto r - coup led phospho inos i t i de tu rnover has been shown to occur i n a wide v a r i e t y of t i s s u e s i n c l u d i n g those which are e x c i t a b l e (eg. smooth muscle, b ra in and sympathet ic g ang l i o n ) , nonexc i t ab l e (eg. l i v e r hepatocytes, p l a t e l e t s , and n e u t r o p h i l s ) , and those which are e i t h e r endocr ine (eg. adrenal medul la and p i n e a l ) , or exoc r i ne (eg. pancreas, p a r o t i d s and bowfly s a l i v a r y g l and s ) . 1.5.2. Inositol phosphates 1.5.2.1. Metabolism I t was demonstrated by Berr idge and co-workers i n 1983 tha t i n o s i t o l t r i s - and bisphosphates are formed i n response to a c t i v a t i o n of su r face membrane r e cep to r s . A r e l a t i v e l y s imple b iochemica l pathway was env i s i oned at t ha t t ime, i n v o l v i n g the stepwise dephosphory la t ion of I n s ( l , 4 , 5 )P3 to i n o s i t o l 1,4-bisphosphate ( I n s ( l , 4 )P2 ) to i n o s i t o l 1-40 phosphate ( I n s [ l ] P ) , and f i n a l l y to f r e e i n o s i t o l . P r e s e n t l y , the p i c t u r e of i n o s i t o l phosphate metabolism has been found to be exceed ing ly complex (F igure 2) and t h i s i s a t t r i b u t a b l e to the a v a i l a b i l i t y o f s o p h i s t i c a t e d techniques tha t can de tec t metabo l i t e s w i th r e l a t i v e l y s ub t l e s t r u c t u r a l d i f f e r e n c e s . In the metabo l i c process , I n s ( l , 4 , 5 )P3 i s dephosphorylated to I n s ( l , 4 )P2 by 5-phosphatase as o r i g i n a l l y suggested (Downes et a l . , 1982), but i t now appears t ha t I n s ( l , 4 )P2 i s dephosphorylated p r i m a r i l y to Ins(4)P by a 1-phosphatase ( Inhorn et a l . , 1987). Besides the 5-phosphatase, most t i s s u e s con ta in 3 -k inase , which t r a n s f e r s a phosphate from ATP to the 3 p o s i t i o n of I n s ( l , 4 , 5 )P3 to form i n o s i t o l 1 ,3 ,4 ,5 - te t rak i sphosphate ( I n s ( l , 3 , 4 ,5 )P4 ) ( I r v i n e et a l . , 1986). I n s ( l , 3 ,4 ,5 )P4 i s then dephosphorylated (presumably by the same 5-phosphatase tha t metabo l i zed I n s ( l , 4 ,5 )P3 ) to form an i somer i c i n o s i t o l t r i s pho spha te , I n s ( l , 3 , 4 ) P3 . Th i s metabo l i t e i s then dephosphorylated by the same 1-phosphatase t ha t degrades I n s ( l , 4 )P2 to Ins(3,4)P2 (Inhorn et a l . , 1987) and to a l e s s e r extent I n s ( l , 3 )P2 i s a l s o formed (Bansal et a l . , 1987) by an enzyme t ha t has not ye t been as we l l c h a r a c t e r i z e d . The bisphosphate molecules thus formed are dephosphorylated mainly to a mixture of I n s ( l ) P and Ins(3)P s te reo i somers . I t has a l s o been shown tha t I n s ( l , 3 , 4 )P3 can be phosphory la ted, to a l i m i t e d extent , by 6-k inase to form I n s ( l , 3 , 4 ,6 )P4 (Shears et a l . , 1987). The complex i ty of the i n o s i t o l phosphate pathway suggests t ha t some b i o l o g i c a l f unc t i on s may be regu l a ted by one or more of these m e t a b o l i t e s . 4 1 Fur ther comp l i c a t i n g the i n o s i t o l phosphate metabo l i c pathway i s the demonstrat ion tha t PLC i n v i t r o acts on Ptd Ins(4,5)P2 to produce a mixture of I n s ( l , 4 ,5 )P3 and i n o s i t o l c y c l i c 1 :2 ,4 ,5 - t r i sphosphate ( I n s ( l : 2 c y c , 4 , 5 ) P 3 ) , a d e r i v a t i v e w i th the 1-phosphate c y c l i z e d between the 1- and 2- hydroxy l s (Connol ly et a l . , 1986). I n s ( l : 2cyc ,4 ,5 )P3 has been shown to apparent l y m o b i l i z e i n t r a c e l l u l a r C a 2 + w i th s l i g h t l y l e s s potency than i t s n o n c y c l i c analog in p l a t e l e t s (Wilson et a l . , 1985). I n s ( l : 2cyc ,4 ,5 )P3 i s not a sub s t ra te f o r the 3 -k inase , and i s dephosphorylated on ly s l ow ly by the 5-phosphatase (Connoly et a l . , 1987; Hawkins et a l . , 1987). In f a c t , r epo r t s o f the product ion o f I n s ( l : 2cyc ,4 ,5 )P3 are somewhat c o n f l i c t i n g ; i t appears t ha t w i th b r i e f s t i m u l a t i o n , l i t t l e o f the c y c l i c d e r i v a t i v e i s formed, but i t accumulates w i th prolonged s t i m u l a t i o n presumably because of i t s r a t h e r slow r a t e o f metabolism (Hawkins et a l . , 1987; Sekar et a l . , 1987). K i n e t i c s tud ie s i n p a r o t i d g land a l s o suggest tha t as l i t t l e as 1% of the PLC product may be i n c y c l i c form (Hughes et a l . , 1988). I n o s i t o l pentakisphosphate ( I n s ( l , 3 , 4 , 5 , 6 )Ps ) and hexakisphosphate ( I n s ( l , 2 , 3 , 4 , 5 , 6 )Ps ) are a l s o present i n most mammalian t i s s u e s . However, t h e i r l e v e l s do not change n o t i c e a b l y on s t i m u l a t i o n (Heslop e t a l . , 1985). Whether any f u n c t i o n a l r e l a t i o n s h i p e x i s t s between the C a 2 + s i g n a l l i n g mechanism and h igher i n o s i t o l phosphates i s not ye t known. 42 1.5.2.2. Calcium mobilization In a l l systems so f a r s t ud i ed , r e c e p t o r - a c t i v a t e d ca l c i um m o b i l i z a t i o n i nvo l ve s an i n i t i a l phase o f C a 2 + r e l ea se from i n t r a c e l l u l a r s to res f o l l owed by more prolonged phase o f Ca f c ent ry (Putney et a l . , 1981; Putney, 1987). The a v a i l a b l e evidence now s t r ong l y suggests tha t the i n t e r n a l r e l ea se i s s i g n a l l e d by I n s ( l , 4 ,5 )P3 (Be r r i dge , 1983; 1986). C a 2 + i s r e lea sed from a d i s c r e t e i n t r a c e l l u l a r p i pool t ha t does not i n c l ude a l l the nonmitochondr ia l sequestered Ca (Tay lo r et a l . , 1985). The s i z e of t h i s pool has been demonstrated to be regu la ted i n c e l l s by a GTP-dependent mechanism (Mulhaney et a l . , 1987, 1988). The o r g ane l l e from which I n s ( l , 4 ,5 )P3 r e l ea se s C a 2 + i s be l i e ved to be a component of the SR/ER. The mechanism by which I n s ( l , 4 ,5 )P3 re l ea se s C a 2 + appears to i n vo l ve i t s i n t e r a c t i o n w i t h a s p e c i f i c membrane r ecep to r (Spat et a l . , 1986 ; Supattapone et a l . ; 1988). B ind ing o f Ins(1,4,5)P3 to i t s r ecep to r inc reases C a 2 + e f f l u x from the i n t r a c e l l u l a r pool by opening a C a 2 + channe l , which may be c l o s e l y a s soc i a ted w i th the r e cep to r . Recent l y , a high a f f i n i t y b ind ing s i t e f o r I n s ( l , 4 , 5 )P3 has been p u r i f i e d from bovine b ra i n and o ther organs (Worley et a l . , 1987). Th is p r o t e i n has been found to have many o f the same p r o p e r t i e s as the p u t a t i v e I n s ( l , 4 , 5 ) P 3 r e c e p t o r , a lthough no f u n c t i o n a l data are yet a v a i l a b l e to conf i rm the i d e n t i t y o f the b ind ing s i t e w i t h the I n s ( l , 4 ,5 )P3 recepto r i nvo l ved i n mediat ing i n t r a c e l l u l a r C a 2 + r e l e a s e . The high a f f i n i t y b ind ing o f I n s ( l , 4 , 5 )P3 to t h i s molecule 43 has prov ided the bas i s f o r a commerc ia l ly a v a i l a b l e assay k i t f o r I n s ( l , 4 , 5 )P3 (see M a t e r i a l s and Methods). I t has been mentioned tha t upon recep to r a c t i v a t i o n , I n s ( l : 2cyc ,4 ,5 )P3 i s a l so re lea sed i n some c e l l s . Th i s compound has a l s o been suggested to serve an i n t r a c e l l u l a r second messenger f u n c t i o n (Wilson et a l . , 1985). In v i t r o I n s ( l : 2cyc ,4 ,5 )P3 appears to be on ly somewhat l e s s potent than I n s ( l , 4 ,5 )P3 i n r e l e a s i n g i n t r a c e l l u l a r Cac (Wilson et a l . , 1985; I r v i ne et a l . , 1986). The s lower i n a c t i v a t i o n o f I n s ( l : 2 cyc ,4 ,5 )P3 observed i n most c e l l s compared w i th t ha t of Ins(1,4,5)P3 suggests tha t i f i t i s formed i n c e l l s , i t s e f f e c t s on C a 2 + m o b i l i z a t i o n might be more sus ta ined than those o f I n s ( l , 4 , 5 ) P3 . On the other hand, i n panc rea t i c m i n i l o b u l e s , Dixon and Hokin (1987) determined t h a t , upon musca r in i c recepto r s t i m u l a t i o n , the l e v e l s o f I n s ( l , 4 , 5 )P3 inc reased r a p i d l y and then f e l l to a new e leva ted steady s t a t e , whereas I n s ( l : 2cyc ,4 ,5 )P3 increased s l ow l y . A c co rd i n g l y , i t has been suggested tha t I n s ( l , 4 , 5 )P3 cou ld be r e spon s i b l e f o r i n t r a c e l l u l a r C a 2 + r e l ea se dur ing short per iods o f s t i m u l a t i o n , whereas both I n s ( l , 4 , 5 )P3 and I n s ( l : 2 cyc , 4 , 5 )P3 cou ld c o n t r i b u t e almost e q u a l l y to C a 2 + r e l ea se dur ing prolonged s t i m u l a t i o n (Dixon and Hokin, 1987; Muallem et a l . , 1988). However, s i nce I n s ( l : 2cyc ,4 ,5 )P3 i s formed i n r e l a t i v e l y small q u a n t i t i e s and has very slow c e l l u l a r tu rnover , i t s r o l e i n r e g u l a t i n g c e l l u l a r ca l c i um metabolism i s s t i l l doubt fu l (Putney et a l . , 1989). Compared w i th C a 2 + r e l e a s e , the r e g u l a t i o n o f C a 2 + e n t r y , the second phase of C a 2 + m o b i l i z a t i o n , i s poo r l y understood. There i s 44 i n d i r e c t evidence tha t i n o s i t o l phosphates may a l s o p lay a r o l e i n the en t ry of C a 2 + (Putney et a l . , 1989). Recent l y , ev idence has been presented suggest ing tha t I n s ( l , 3 ,4 ,5 )P4 would act as a s p e c i f i c s i gna l f o r C a 2 + en t r y i n sea u rch in eggs and i n l a c r i m a l g land c e l l s ( I r v i n e and Moor, 1986; 1987; Mooris et a l . , 1987). However, s i n ce I n s ( l , 3 , 4 ,5 )P4 when i n j e c t e d alone d i d not produce any e f f e c t , and s i nce C a 2 + r e l e a s e seemed to be a p r e r e q u i s i t e f o r a c t i v a t i o n o f C a 2 + e n t r y , I r v i ne and Moor (1986, 1987) concluded tha t the emptying of the I n s ( l , 4 , 5 ) P3 - r e gu l a t ed C a ' pool was necessary f o r a c t i v a t i o n of Ca en t ry by I n s ( l , 3 , 4 , 5 ) P 4 . Furthermore, i n view o f the r a p i d tu rnover of I n s ( l , 3 , 4 ,5 )P4 i n va r i ous t i s s u e s , many i n v e s t i g a t o r s have suggested tha t t h i s compound may a l so have a s i m i l a r r o l e to p lay i n r e g u l a t i n g the i n t r a c e l l u l a r C a 2 + c oncen t ra t i on i n other systems ( I r v i n e and Moor, 1986; Mo r r i s et a l . , 1987; Heathers et a l . , 1988; P i j uan and L i t o s c h , 1988). In a d d i t i o n to the above proposal f o r the involvement o f i n o s i t o l phosphates i n C a 2 + e n t r y , the a c t i v a t i o n o f PKC by DG or phorbol e s t e r s has a l so been shown to s t imu l a te the i n f l u x o f C a 2 + i n t o c e l l s (Gleason and F l a im , 1986; Chiu et a l . , 1987) (see below). There i s a l s o evidence demonstrat ing t ha t C a 2 + ent ry i n t o c e l l s i n response to a c t i v a t i o n of receptor -med ia ted events can occur independent ly of phospho ino s i t i de metabol ism (Chiu et a l . , 1987) and i t has been suggested t ha t t h i s may i n v o l v e , at l e a s t i n p a r t , a c t i v a t i o n of G-prote ins which are d i r e c t l y 4 5 coupled to Ca channels on the plasma membrane (Minneman, 1988). 1.5.3. 1,2-Diacylglycerol (DG) and PKC activation The other major product o f phospho inos i t i de h y d r o l y s i s which has a second messenger f unc t i on i s DG. DG i s normal ly present i n very low concen t ra t i on s i n c e l l membranes. Un l i ke I n s ( l , 4 ,5 )P3 which i s produced as a r e s u l t of h y d r o l y s i s of Phtd Ins(4,5)P2, DG i s formed from a l l th ree forms o f the phospho inos i t i des i n response to C a 2 + - m o b i l i z i n g s t i m u l i ( A b d e l - L a t i f , 1986; G r i e n d l i n g et a l . , 1987) (F igure 2 ) . I t has been demonstrated t ha t the product ion o f DG i n v a s cu l a r smooth muscle i s b i pha s i c and sus ta ined i n response to c e r t a i n agon i s t s such as ang io tens in II (Alexander et a l . , 1985; G r i e n d l i n g et a l . , 1986; 1987). The b i pha s i c format ion of DG appears to r e s u l t from sequent ia l h y d r o l y s i s i n i t i a l l y of PtdIns(4,5)P2 and Ptd Ins (4)P and subsequently of P t d l n s . The h y d r o l y s i s of o ther phospho l ip id s to form DG has a l s o been repor ted to occur i n other systems ( L o f f e l h o l z , 1989). DG a c t i v a t e s PKC, a ub iqu i tous enzyme present i n a l l organs, r e s u l t i n g i n phosphory la t ion of s p e c i f i c p r o t e i n s and generat ion o f p h y s i o l o g i c a l responses. The a c t i v a t i o n of PKC i s a complex process t ha t r equ i r e s ca l c i um and a pho spho l i p i d as a c o f a c t o r , phosphat idy l s e r i ne being the most e f f e c t i v e pho spho l i p i d . I t appears that DAG increases the a f f i n i t y o f PKC f o r c a l c i um. K i n e t i c a n a l y s i s of the enzyme a c t i v i t y suggests t ha t on ly a small amount o f DG can cause a r e l a t i v e l y l a r ge i nc rease i n the a f f i n i t y 46 of PKC f o r c a l c i um, s t i m u l a t i n g the enzyme wi thout any major a l t e r a t i o n s i n i n t r a c e l l u l a r ca l c ium concen t ra t i on from basal (Ka ibuchi et a l . , 1981). DG appears to be s p e c i f i c f o r PKC s i nce both monoacyl- and t r i a c y l g l y c e r o l have not been found to a c t i v a t e the enzyme (Kaibuchi et a l . , 1981). Most o f the mechan i s t i c a c t i on s of PKC have been determined based on s tud i e s us ing phorbol e s t e r s such as TPA and PDB. Phorbol e s t e r s , l i k e DG, have been repor ted to b ind to the enzyme r e s u l t i n g i n increased a f f i n i t y f o r ca l c i um and pho spho l i p i d , and subsequent a c t i v a t i o n (Castagna et a l . , 1982; Cooper et a l . , 1982; D i v i g i l l i o et a l . ; 1984). These compounds o f f e r the advantage of being l e s s r e a d i l y hyd ro l y zab le than DG. PKC occupies a p i v o t a l p o s i t i o n i n the b iochemica l pathways tha t r e l a y i n fo rmat i on i n t o c e l l , because i t i s ab le to i n f l u e n c e the c e l l u l a r responses to numerous s t i m u l i . These i n c l ude those coupled to Ptd Ins(4,5)P2 h y d r o l y s i s , as we l l as growth f a c t o r s and those coupled to the a c t i v a t i o n o f c y c l i c nuc l eo t i de dependent p r o t e i n k ina se s . Th is c on t r o l may be exerted at d i f f e r e n t l e v e l s o f the s i g n a l l i n g pathways, w i t h e i t h e r a p o s i t i v e or negat ive r e s u l t a n t e f f e c t (N i s h i zuka , 1988). The fo rmat ion o f an a c t i v e enzyme complex at the plasma membrane, by the coo rd ina te a c t i o n o f phosphat idy l s e r i n e , DG, and C a 2 + has imp l i ed tha t the pr imary t a r ge t s of PKC are membrane p r o t e i n s . In t h i s r e spec t , i t i s becoming c l e a r tha t a key f u n c t i o n of the enzyme i s i t s a b i l i t y to modulate the movement of c e r t a i n ions (eg. Ca2+) i n t o and out o f the 47 c y t o s o l i c compartment of the c e l l , and thus to r egu l a te the c e l l u l a r processes t ha t depend on t h i s . A l t e r a t i o n s o f the a c t i v i t y o f "exchange"( eg. Na+-H+ exchange) and "pump" (eg. Ca2+-ATPase e f f l u x pump, Na+-K+-ATPase pump) p ro te i n s by PKC are we l l documented (N i s h i zuka , 1986). Recent l y , a wealth o f i n fo rmat i on has become a v a i l a b l e t ha t demonstrates tha t PKC i s able to modulate the a c t i v i t y of the numerous, pha rmaco log i ca l l y d i s t i n c t types o f ion channels t ha t are expressed i n most, i f not a l l , c e l l s (eg. ca l c i um and potassium channe l s ) . Severa l i n v e s t i g a t o r s have po inted out tha t the I n s ( l , 4 ,5 )P3 and DG pathways, the two arms of the phospho inos i t i de c y c l e , cou ld i n t e r a c t e i t h e r c o o p e r a t i v e l y or s y n e r g i s t i c a l l y to a f f e c t the metabolism and p h y s i o l o g i c a l a c t i on s of these messenger molecules (Be r r i dge , 1984; N i s h i z u ka , 1984; Rasmussen et a l . , 1985; A b d e l - L a t i f , 1986). A s y n e r g i s t i c i n t e r a c t i o n between C a 2 + and PKC has been repor ted to u n d e r l i e the agon i s t - i nduced changes i n the a c t i v a t i o n of va r i ou s c e l l s . I t has been suggested tha t an important consequence o f having t h i s b i f u r c a t i n g s i gna l pathway i s t ha t i t p rov ides the v e r s a t i l i t y necessary to i n t roduce s ub t l e v a r i a t i o n s i n the c on t r o l system (Be r r i dge , 1984). Thus, f o r example, one pathway phosphory lates p r o t e i n s by a c t i v a t i o n o f CaM by the e leva ted i n t r a c e l l u l a r C a 2 + c oncen t r a t i on brought about by the I n s ( l , 4 ,5 )P3 - i nduced C a 2 + r e l ea se mechanism, and other pathway phosphory lates s p e c i f i c p r o te i n s by a c t i v a t i o n of PKC. The r e l a t i v e importance o f each pathway may a l so vary w i th t ime, i n t ha t I n s ( l , 4 ,5 )P3 48 may be r e spon s i b l e f o r i n i t i a t i o n whereas DG may be more important i n ma in ta i n i ng the response (Kajima et a l . , 1983; Zawal ich et a l . , 1983; Be r r i dge , 1984). 1.6. ROLE OF PHOSPHOINOSITIDE METABOLISM IN VASCULAR SMOOTH MUSCLE CONTRACTION A s u b s t a n t i a l amount of data i n d i c a t e s tha t the phospho ino s i t i de s i g n a l l i n g system p lays an important r o l e i n v a s cu l a r smooth muscle c o n t r a c t i o n induced by a c t i v a t i o n of C a 2 + - m o b i l i z i n g recepto r s such as a\-adrenergic, V j - v a s o p r e s s i n , 5-HT2-serotonin and ang i o ten s i n I I . As w i th most other systems, the second messengers generated as a r e s u l t o f s t i m u l a t i o n of phospho inos i t i de breakdown appear to p a r t i c i p a t e i n a cascade of events t ha t c o n t r i b u t e to the f i n a l c o n t r a c t i l e response. The c o n t r a c t i o n o f va s cu l a r smooth muscle i n response to va r ious C a 2 + - m o b i l i z i n g agon i s t s i s u s u a l l y composed of two phases : a r a p i d phas ic component fo l l owed by a s lower more sus ta ined t o n i c component. Th i s b i pha s i c response has been hypothes ized to be l i n k e d to the two known i n t r a c e l l u l a r second messengers of the pho spho ino s i t i de system-I n s ( l , 4 , 5 )P3 and DG. I t has been suggested tha t I n s ( l , 4 , 5 )P3 - i nduced C a 2 + r e l e a s e from the SR i s r e spon s i b l e f o r the phas ic response (Streb et a l . , 1983; Joseph et a l . , 1984; Semastu et a l . , 1984; A lexander et a l . , 1985; Somlyo et a l . , 1985; Campbell et a l . , 1985; Hashimoto et a l . , 1986; G r i e n d l i n g et a l . , 1987), wh i l e DG a c t i v a t i o n o f PKC i s i nvo l ved i n the t o n i c c o n t r a c t i o n (Forder et a l . , 1985; Campbell e t a l . , 1985; 49 Gleeson and F l a im, 1986; G r i e n d l i n g et a l . , 1987). The i n t r a c e l l u l a r r e l ea se o f C a 2 + by I n s ( l , 4 ,5 )P3 i s be l i e ved to induce c o n t r a c t i o n by a c t i v a t i n g the CaM pathway o f the c o n t r a c t i l e process ( A b d e l - L a t i f , 1986; Rasmussen et a l . , 1987). I t , t h e r e f o r e , appears t ha t t h i s e f f e c t o f I n s ( l , 4 , 5 )P3 i s r e spon s i b l e f o r the t r a n s i e n t i nc rease i n c y t o s o l i c C a 2 + and myosin phosphory la t ion observed dur ing the e a r l y pe r i od of c o n t r a c t i o n a f t e r agon i s t s t i m u l a t i o n (Rasmussen et a l . , 1987). A c t i v a t i o n o f PKC by DG, on the other hand, has been repor ted to r e s u l t i n the phosphory la t ion of va r ious s t r u c t u r a l and r e g u l a t o r y p ro te i n s i n c l u d i n g caldesmon, desmin, synemin and a group of low molecu la r weight c y t o s o l i c p r o t e i n s , which have been proposed to be i nvo l ved i n mediat ing the t o n i c c o n t r a c t i l e response (Rasmussen et a l . , 1987). S ince t h i s process has been observed to take p lace without s u b s t a n t i a l i nc reases i n the average i n t r a c e l l u l a r f r ee C a 2 + c oncen t ra t i on and MLC.P, Rasmussen et a l . (1987) suggested tha t t h i s can o f f e r an a l t e r n a t i v e exp l ana t i on f o r the sus ta ined c o n t r a c t i o n to the l a t c h br idge hypothes i s o r i g i n a l l y forwarded ( D i l l o n et a l . , 1981). The conc lu s i on tha t phospho inos i t i de metabolism i s i nvo l ved i n v a s cu l a r smooth muscle c o n t r a c t i o n i s based on the f i n d i n g s of severa l i n v e s t i g a t o r s . C a 2 + - m o b i l i z i n g agon i s t s have been repor ted to induce the pho sphod ie s te ra t i c breakdown of Ptd Ins(4,5)P2 i n a wide v a r i e t y of p repa ra t i on s (Alexander et a l . , 1985; Campbell et a l . , 1985; 1986; Hashimoto et a l . , 1986; P i j uan and L i t o s c h , 1988). Th i s has been found to be a s soc i a ted w i th a r ap i d product ion o f I n s ( l , 4 , 5 )P3 and DG or i t s 50 phosphory lated product, PA (Alexander et a l . , 1985; Campbell et a l . , 1985; 1986; Hashimoto et a l . , 1986; G r i e n d l i n g et a l . , 1987; P i j uan and L i t o s c h , 1988). In t h i s regard , wh i l e the format ion o f I n s ( l , 4 , 5 )P3 has been found to be t r a n s i e n t , the product ion o f DG has been shown to be s u s t a i ned . I n s ( l , 4 ,5 )P3 has been demonstrated to r e l ea se [ 4 5 C a 2 + ] accumulated i n t o the SR o f s apon in - t rea ted s i n g l e c e l l suspensions (Suematsu et a l . , 1984), to produce a c o n t r a c t i o n i n saponin t r e a t ed skinned muscle t i s s u e s (Somlyo et a l . , 1985; 1988; Hashimoto et a l . , 1986) and to r e l ea se C a 2 + from the SR, as est imated us ing f u r a - 2 ( l i n o , 1987) . In a d d i t i o n , Campbell et a l . (1985) c o r r e l a t e d NA-induced phospho ino s i t i de metabolism w i th the agon i s t - i nduced C a 2 + f l u x and c o n t r a c t i o n i n r a b b i t a o r t a . These authors demonstrated a c l o se c o r r e l a t i o n between agon i s t - induced s t i m u l a t i o n o f Ptd Ins(4,5)P2 breakdown and Ca^ r e l e a s e , and showed tha t the t ime-cour se o f these events p a r a l l e l s the t ime-course o f the r a p i d phase o f t en s i on development i n the c o n t r a c t i l e response of t h i s t i s s u e . A c o r r e l a t i o n between Ptd Ins(4,5)P2 h yd r o l y s i s and format ion o f I n s ( l , 4 , 5 )P3 and C a 2 + r e l ea se (measured us ing quin 2) has a l s o been shown i n c u l t u r e d r a t a o r t i c smooth muscle c e l l s i n response to ang io ten s i n II (Alexander et a l . , 1985). These e f f e c t s of ang io tens in II were repor ted to be tempora l l y c o r r e l a t e d w i th i n i t i a t i o n o f c o n t r a c t i o n i n r a t ao r ta ( G r i e n d l i n g et a l . , 1987). In another study, Doctrow and Lowenstein (1985), us ing a s u b c e l l u l a r f r a c t i o n prepared from c a l f ao r t a smooth muscle which conta in s c o n t r a c t i l e p r o t e i n s and membranes, demonstrated 5 1 tha t adenosine and c e r t a i n o f i t s analogs, which decrease c o n t r a c t i o n i n t h i s t i s s u e , a l so decrease the phosphory la t ion of P td ln s and MLC, suggest ing a p o s s i b l e r e l a t i o n s h i p between phospho ino s i t i de metabolism and MLC pho spho ry l a t i on . There are a l so r epo r t s demonstrat ing t ha t v a s cu l a r smooth muscle i s enr i ched w i th PKC and tha t a c t i v a t i o n o f PKC i n t h i s muscle r e s u l t s i n phosphory la t ion o f va r ious p r o t e i n s which may be i nvo l ved i n the sus ta ined c o n t r a c t i o n of v a s cu l a r smooth muscle (Rasmussen et a l . , 1987). Moreover, phorbol e s t e r s , agents which have been repor ted to a c t i v a t e PKC i n a f a sh ion s i m i l a r to tha t o f the endogenously produced a c t i v a t o r , DG (Castagna et a l . , 1982; Cooper et a l . , 1982; Kikkawa et a l . , 1983; N iedel et a l . , 1983; D i v i g i l l i o et a l . , 1984), have been demonstrated to induce c o n t r a c t i o n o f v a s cu l a r smooth muscle (Danthu lur i and Deth, 1984; Rasmussen et a l . , 1984; Forder et a l . , 1985; Sybertz et a l . , 1986; Chiu et a l . , 1987; S inger and Baker, 1987; Swamura et a l . , 1987; Kha l i 1 and Van Breemen, 1988). The c o n t r a c t i l e responses to phorbol e s t e r s are r e l a t i v e l y slow and su s t a i ned . I t i s from such observat ions tha t a connect ion between a c t i v a t i o n o f PKC and generat ion o f t en s i on has developed. Phorbol e s te r - i nduced c o n t r a c t i o n have been found to occur i n many p repara t ions wi thout any s i g n i f i c a n t change i n c y t o s o l i c ca l c ium concen t r a t i o n , and on ly a s l i g h t i nc rease i n MLC.P, t ha t i s s i m i l a r to tha t induced by agon i s t s dur ing the t o n i c phase of c o n t r a c t i o n (DeFeo and Morgan, 1985; Cha t t e r j ee and Tejada, 1986). Th i s f u r t h e r suggests tha t the agon i s t - i nduced sus ta ined phase o f v a s cu l a r 52 smooth muscle c o n t r a c t i o n i s dependent on PKC a c t i v a t i o n . An i n t e r a c t i o n between C a 2 + and PKC a c t i v a t o r s i n producing smooth muscle c o n t r a c t i o n has a l s o been found. The c o n t r a c t i l e responses to phorbol e s t e r s alone are slow to develop, but i n the presence o f ca l c i um ionophores (eg. A23187) and ca l c i um channel agon i s t s (eg. BAY K 8644), the PKC a c t i v a t o r s produce a more r a p i d c o n t r a c t i o n o f g r ea te r magnitude. Fur ther support f o r an s y n e r g i s t i c i n t e r a c t i o n between phorbol e s t e r s 7+ and C a ' has emerged from the demonstrat ion t ha t removal of e x t r a c e l l u l a r C a 2 + or a d d i t i o n of ca l c ium channel b l ocke r s (eg. verapami l ) e i t h e r abo l i shes or reduces the phorbol e s t e r - i nduced c o n t r a c t i l e responses ( J iang and Morgan, 1987; S inger and Baker, 1987). These observat ions l ed to the suggest ion tha t a s y n e r g i s t i c i n t e r a c t i o n of the endogenous PKC a c t i v a t o r , DG, and C a 2 + on PKC cou ld be i nvo l ved i n the t o n i c phase of v a s cu l a r smooth muscle c o n t r a c t i o n i n response to C a 2 + - m o b i l i z i n g agon i s t s ( A b d e l - L a t i f , 1986). S t imu la ted h y d r o l y s i s o f Ptd Ins(4,5)P2 may e l eva te the c y t o s o l i c C a 2 + c oncen t r a t i on by r a p i d l y caus ing i t s r e l e a s e from the SR, which may i n tu rn p rov ide an e f f e c t i v e c o n d i t i o n f o r DG a c t i v a t i o n of PKC. Recent evidence i n d i c a t e s that PKC a c t i v a t i o n by DG or phorbol e s t e r s may inc rease the s e n s i t i v i t y o f the c o n t r a c t i l e p r o t e i n s f o r C a ' ( J i ang and Morgan, 1987; Itoh et a l . , 1988; Nishimura et a l . , 1989). Th i s conc l u s i on i s p a r t l y based on the observat ions t ha t phorbol e s t e r s s i g n i f i c a n t l y reduce the C a 2 + t r a n s i e n t f o r agon i s t s w i thout modi fy ing the ampl itude of c o n t r a c t i o n , t ha t phorbol e s t e r s (with 53 phosphat idy l s e r i ne ) i nc rease the ampl itude o f c o n t r a c t i o n s evoked by va r ious concent ra t i on s o f C a 2 + and I n s ( l , 4 ,5 )P3 i n sk inned muscle s t r i p s ( I toh et a l . , 1988), t ha t the r a t i o of f o r ce to i n t r a c e l l u l a r C a 2 + concen t r a t i on i s h igher dur ing c o n t r a c t i o n s induced by r ecep to r agon i s t s than dur ing those induced by high K + (Morgan and Morgan, 1984; DeFeo and Morgan, 1985; Sato et a l . , 1988), and tha t a -ad rene rg i c agon i s t s and GTP7S enhance the c o n t r a c t i l e respons iveness o f ot-toxin pe rmeab i l i zed v a s cu l a r smooth muscle p repara t ions to C a 2 + (Nishimura et a l . , 1988). Although PKC a c t i v a t i o n i s be l i e ved to be r e spon s i b l e f o r t h i s e f f e c t , the exact mechanism f o r the increased s e n s i t i v i t y of the c o n t r a c t i l e p r o t e i n s to C a 6 i s not known. The recent suggest ion t ha t PKC may act through phosphory la t ion o f a p r o t e i n phosphatase i n h i b i t o r and i n h i b i t i o n o f MLC phosphatase i s a t t r a c t i v e , but remains to be expe r imen ta l l y determined (Somlyo and Himpens, 1989). Another p o s s i b i l i t y i s p o t e n t i a t i o n r e s u l t i n g from some e f f e c t o f PKC on a t h i n f i l a m e n t - a s s o c i a t e d , C a 2 + - s e n s i t i v e r e gu l a t o r y mechanism (Marston and Smith, 1985). Whatever the mechanism might be, the C a 2 + - s e n s i t i z i n g e f f e c t o f PKC i n v a s cu l a r smooth muscle may be r e spon s i b l e f o r the s y n e r g i s t i c i n t e r a c t i o n between C a 2 + and DG or phorbol e s t e r s dur ing su s ta ined c o n t r a c t i o n . C o n t r a c t i l e responses of v a s cu l a r smooth muscle to phorbol e s t e r s have been shown to be a s soc ia ted w i th increased i n f l u x of e x t r a c e l l u l a r C a 2 + ( A b d e l - L a t i f , 1986; Heagerty and Ol lerenshaw, 1987; F i s h et a l . , 54 1988; McGrath e t a l . , 1989). Th is e f f e c t has been suggested to be due to phosphory la t i on of ca l c ium channels on plasma membrane by PKC ( F i s h , 1988). However, the r o l e of C a 2 + i n f l u x i n c o n t r a c t i l e responses to phorbol e s t e r s appears to vary con s i de rab l y . Thus, c o n t r a c t i l e responses have been found to be t o t a l l y or p a r t i a l l y dependent on the i n f l u x of e x t r a c e l l u l a r C a 2 + v i a d i h y d r o p y r i d i n e - s e n s i t i v e C a 2 + channels i n both the r a t ao r ta and r a b b i t ear a r t e r y (Danthulur i and Deth, 1984; Forder et a l . , 1985; Chiu et a l . , 1987). In a d d i t i o n , i n the r a b b i t ao r ta phorbol e s t e r - i nduced C a 2 + i n f l u x occurs thorough d i h y d r o p y r i d i n e -r e s i s t a n t channels (Gleason and F l a im, 1986). In both the r a b b i t and r a t ao r tae , a major p o r t i o n of the c o n t r a c t i o n appears to be r e s i s t a n t to the l ower ing o f e x t r a c e l l u l a r C a 2 + by severa l orders of magnitude ye t t h i s cou ld not be a t t r i b u t a b l e to the r e l ea se o f i n t r a c e l l u l a r C a 2 + (Chiu et a l . , 1987). Th is l a t t e r e f f e c t has been suggested to be the r e s u l t o f the a b i l i t y o f phorbol e s t e r s , l i k e DG, to a c t i v a t e PKC at r e s t i n g l e v e l s o f i n t r a c e l l u l a r C a 2 + . 1.7. OBJECTIVES OF THE PRESENT STUDY A l t e r a t i o n s i n r e a c t i v i t y o f blood ve s se l s to neu ro t ran sm i t te r s and c i r c u l a t i n g hormones have been proposed to cause, or c o n t r i b u t e t o , some of the va s cu l a r c omp l i c a t i on s , such as hyper tens ion , t ha t are a s soc i a ted w i th d iabetes m e l l i t u s . Enhanced a r t e r i a l c o n t r a c t i l i t y to the neu ro t r an sm i t te r substance, NA, has been p r e v i o u s l y de sc r i bed i n exper imenta l d iabetes m e l l i t u s . Th i s e f f e c t of NA on d i a b e t i c t i s s u e s 5 5 has been found to be mediated by a\-adrenoceptors. A l pha j - ad renocep to r -mediated phospho inos i t i de metabolism i s be l i e ved to be f u n c t i o n a l l y l i n k e d to v a s cu l a r smooth muscle c o n t r a c t i o n . There fo re , the o v e r a l l o b j e c t i v e o f the present study was to t e s t the hypothes i s t ha t the enhanced c o n t r a c t i l e responses observed i n a r t e r i e s from d i a b e t i c r a t s to NA are a s soc i a ted w i th increased phospho ino s i t i de metabol i sm. Experiments were conducted us ing i s o l a t e d aortae and mesenter ic a r t e r i e s from r a t s w i th ch ron i c STZ-induced d iabetes and age-matched c o n t r o l s . P o r t a l ve ins were a l s o used i n the study f o r comparison purposes. While NA was used as a t e s t substance, other va soac t i ve agents were a l s o employed f o r re fe rence purposes or to i n v e s t i g a t e mechanisms. The research was designed to achieve the f o l l o w i n g s p e c i f i c o b j e c t i v e s : (a) to determine whether enhanced c o n t r a c t i l e responses o f a r t e r i e s from d i a b e t i c r a t s to NA and the a\-adrenoceptor s e l e c t i v e agon i s t s phenylephr ine (PE) and methoxamine (METH) are a s soc i a ted w i th inc reased r e l ea se of i n t r a c e l l u l a r C a 2 + or are dependent on the presence of e x t r a c e l l u l a r C a 2 + . (b) to determine whether NA-induced inc reases i n pho spho ino s i t i de metabol i sm, assessed by measuring the breakdown of [ 3 2 P ] - P t d I n s ( 4 , 5 ) P 2 , and format ion o f [ 3 2 P ] - P A , [ 3 H ] - i n o s i t o l phosphates and I n s ( l , 4 , 5 )P3 are enhanced i n a r t e r i e s from d i a b e t i c r a t s . (c) to determine i f c o n t r a c t i l e responses mediated by PKC a c t i v a t i o n are increased i n d i a b e t i c a r t e r i e s . 56 (d) to determine whether G-prote in-mediated c o n t r a c t i l e responses are enhanced i n a r t e r i e s from d i a b e t i c r a t s . 57 MATERIALS AND METHODS 2.1. ANIMALS Male Wi s ta r r a t s (Char les R i ve r Canada, Mont rea l , Canada) weighing 190-220g were used i n the present study. These animals were chosen i n order to extend prev ious work i n our l abo r a t o r y which used the same type of an imal s . 2.2. ASSAY KITS Glucose assay k i t s were obta ined from Boehr inger Mannheim. I n s ( l , 4 ,5 )P3 assay k i t s were purchased from Amersham. 2.3. DRUGS AND CHEMICALS The f o l l o w i n g chemicals were purchased from BDH Chemicals : sodium c h l o r i d e (NaCl ) , l i t h i u m c h l o r i d e ( L i C l ) , potassium phosphate (KH2PO4), ca l c i um c h l o r i d e (CaCl2) , magnesium s u l f a t e (MgS04), potass ium c h l o r i d e (KC1), sodium b icarbonate (NaHC03), sodium f l u o r i d e (NaF), aluminium c h l o r i d e (A IC I3), ammonium hydroxide (NH4OH), potass ium o x a l a t e , ch lo ro fo rm, d i e t h y l e the r , h y d r o c h l o r i c a c i d (HC1), methanol, t r i c h l o r o a c e t i c a c i d (TCA), dex t ro se , methyl a ce t a t e , n -propano l , i od i ne and e t h a n o l . The f o l l o w i n g were obta ined from Sigma Chemical Co. (S t . Lou i s , MO, U.S.A.) : a c e t y l c h o l i n e hyd roch l o r i de , ammonium formate, m y o i n o s i t o l , a s co rb i c a c i d , d imethyl s u l f o x i d e (DMSO), phorbol 12, 13-58 d i b u t y r a t e (PDB), 4o -phorbo l , e t h y l eneg l y co l b is - (/J-aminoethyl e ther ) N, N, N ' , N ' - t e t r a a c e t i c a c i d (EGTA), ( - )no radrena l i ne hyd roch lo r i de (NA), phenylephr ine hyd roch lo r i de (PE) , p razo s i n h yd roch l o r i de , s t r e p t o z o t o c i n (STZ), t i m o l o l h yd roch l o r i de , des ipramine, hyd roco r t i s one , sodium formate, sodium t e t r a b o r a t e , yohimbine h yd r o ch l o r i de , n i f e d i p i n e , ve rapami l , phosphat idy l i n o s i t o l ( P t d l n s ) , phosphat idy l i n o s i t o l 4-phosphate (P td In s (4 )P ) , phosphat idy l i n o s i t o l 4 ,5 -bisphosphate (P td Ins (4 ,5 )P2) , phosphat idy l s e r i n e , phospha-t i d y l e t h a n o l a m i n e , phosphat id i c a c i d (PA), p h o s p h a t i d y l c h o l i n e , and sph ingomyel in. Methoxamine hyd roch lo r i de (METH) was purchased from Burroughs Wellcome Co. ( L a s a l l e , Quebec, Canada), s tau ro spor ine from Calbiochem Corpora t ion (La J o l l a , CA, U.S.A.) and phentolamine mesylate from CIBA Canada (Do rva l , Quebec, Canada). Anion exchange r e s i n (AG 1-X8, formate form, 100-200 mesh) was from Bio-Rad Labo ra to r i e s (M i s s i s sauga, On ta r i o , Canada). Precoated s i l i c a gel 60 t h i n l a y e r chromatography p l a t e s and high-performance t h i n l a y e r p l a t e s were obta ined from Merck (Darmstadt, Germany). [ 3 2 P ] sodium orthophosphate (~200mCi/mmol) and m y o - [ 3 H ] - i n o s i t o l (10-20 Ci/mmol) were purchased from Amersham. Aquasol was from New England Nuc lear . Stock s o l u t i o n s of NA, PE and METH were prepared f r e sh d a i l y i n Krebs s o l u t i o n or de i on i zed water con ta i n i n g 1 mg/ml a s co rb i c a c i d . PDB, 4a-phorbol and s taurospor ine were d i s s o l v e d i n DMS0, and verapamil and 59 n i f e d i p i n e i n e t hano l . Stock s o l u t i o n s o f a l l drugs were d i l u t e d w i th Krebs s o l u t i o n or de i on i zed water to app rop r i a te c oncen t r a t i on s . The f i n a l DMSO or ethanol concen t ra t i on i n the bath ing medium was wi thout e f f e c t on c o n t r a c t i l e responses. Experiments i n v o l v i n g PDB, 4a -phorbo l , ve rapami l , n i f e d i p i n e and s taurospor ine were performed i n t i s s u e baths p ro tec ted from l i g h t . 2.4. KREBS SOLUTION Unless otherwise s t a t e d , a l l i ncubat ions o f t i s s u e were c a r r i e d out i n Krebs s o l u t i o n of the f o l l o w i n g compos i t ion (mM) : NaCl (113), KC1 (4 .7 ) , NaHC0 3 (25), C a C l 2 ( 2 .5 ) , KH 2 P0 4 ( 1 .2 ) , MgS0 4 (1.2) and dext rose (11.5) . T imolo l (10 '^M), des ipramine ( lO '^M) and hydrocor t i sone (10"^M) were a l s o added to the Krebs s o l u t i o n to b lock the e f f e c t s o f ^ -adrenoceptor s , and neuronal and extraneurona l uptake, r e s p e c t i v e l y . 2.5. INDUCTION OF DIABETES Rats were l i g h t l y anes the t i zed w i th ether to a l l ow i n j e c t i o n of e i t h e r STZ or i t s v e h i c l e i n t o the l a t e r a l t a i l v e i n . Diabetes was induced w i th a s i n g l e i n j e c t i o n o f STZ (60 mg/kg body weight) d i s s o l v e d i n c i t r a t e b u f f e r (pH 4.5) made by mix ing 0.1 M sodium c i t r a t e and 0.1 M c i t r i c a c i d . Age-matched con t r o l animals were i n j e c t e d w i th the c i t r a t e b u f f e r v e h i c l e a lone. The STZ-t reated and c o n t r o l r a t s were caged s epa ra te l y and housed i n a s i m i l a r environment mainta ined at 25°C. A l l 60 animals were g iven f r ee access to normal l a bo r a t o r y d i e t (Pur ina r a t chow) and water. Three days a f t e r i n j e c t i o n , r a t s were monitored f o r the development o f g l y c o s u r i a us ing enzymatic t e s t s t r i p s (Tes - tape, E l i L i l l y , Toronto, On ta r i o , Canada). Animals e x h i b i t i n g g l y c o s u r i a >3+ were cons idered to be d i a b e t i c . 2.6. TISSUE PREPARATION Twelve to 14 weeks a f t e r i n j e c t i o n , r a t s were weighed and k i l l e d by s tunning f o l l owed by d e c a p i t a t i o n . Th is du r a t i on was chosen based on r epo r t s from our l a bo r a t o r y tha t enhanced a r t e r i a l c o n t r a c t i l i t y to NA can be detected 12 weeks a f t e r STZ i n j e c t i o n (MacLeod, 1985; H a r r i s and MacLeod, 1988). At the time of s a c r i f i c e , blood samples were c o l l e c t e d f o r serum g lucose assay. Plasma obta ined by sp inn ing the blood samples (2000 x g f o r 20 min) was s to red at -20°C u n t i l assayed f o r g lucose . The t h o r a c i c a o r t a , the supe r i o r mesenter ic a r t e r y and the hepat i c p o r t a l ve in were r a p i d l y removed from each r a t and p laced i n Krebs s o l u t i o n . The ve s se l s were c a r e f u l l y trimmed f r e e of f a t and connect i ve t i s s u e s and cut i n t o r i n g s . Care was taken to ensure tha t the e n d o t h e l i a l l a y e r was not damaged dur ing t i s s u e p r e p a r a t i o n . The f u n c t i o n o f the endothel ium was o c c a s i o n a l l y conf irmed by mon i to r ing the a b i l i t y o f a r t e r i a l p repa ra t i on s p recont rac ted w i th 10"^M or 10"^M NA to r e l a x i n response to 10" 6 M a c e t y l c h o l i n e (Furchgot t , 1983). 6 1 2.7. CONTRACTION STUDIES Rings o f ao r t a (5-6mm) and mesenter ic a r t e r y (2-3mm), and s p i r a l s t r i p s o f p o r t a l ve in (about 2 mm i n width and 20 mm i n length ) from c o n t r o l and d i a b e t i c r a t s were prepared f o r i s o m e t r i c ten s i on r e co rd i ng s . Each a r t e r i a l r i n g was suspended between the bases of two L-i shaped s t a i n l e s s s t ee l w i r e s . One w i re was attached to a f i x e d t i s s u e support i n a 20 ml t i s s u e bath con ta i n i n g Krebs s o l u t i o n mainta ined at 37°C and oxygenated w i th 95% 0 2 and 5% C 0 2 . The other w i re was connected by means of a s i l k thread to a Grass FT.03 f o r c e d isp lacement t r an sduce r . The s t r i p s of p o r t a l ve in were suspended i n the t i s s u e bath w i th the a i d of s i l k thread t i e d to each end o f the s t r i p s , one end being attached to the t i s s u e support and the other to a t ran sducer . C o n t r a c t i l e responses were recorded on a Beckman model 611 dynograph or a Grass model R7 po lygraph. Rings o f ao r ta were p laced under a r e s t i n g t en s i on o f 2 g, whereas r i n g s o f mesenter ic a r t e r y and s t r i p s o f p o r t a l ve in were p laced under r e s t i n g tens ions of l g and 0.75g, r e s p e c t i v e l y . These r e s t i n g tens ions were found i n p r e l i m i n a r y experiments to be optimum f o r both c on t r o l and d i a b e t i c blood v e s s e l s . The muscle p repa ra t i on s were a l lowed to e q u i l i b r a t e f o r 90-120 min before experiments were i n i t i a t e d . During t h i s p e r i o d , the bath ing s o l u t i o n was changed every 20-30 min. During the exper iments, drugs were removed from the t i s s u e baths by severa l washes and ten s i on was a l lowed to r e t u rn to b a s e l i n e . T i s sues were a l lowed to e q u i l i b r a t e i n Krebs s o l u t i o n f o r at 62 l e a s t 60 min between drug t reatments . In the absence o f drugs, the r e s t i n g tens ion s were mainta ined throughout the exper iment. (a) Concentration-response curves : Concent rat ion- response curves were obta ined by a cumulat ive inc rease i n the t o t a l c oncen t r a t i on of agon i s t . Each a d d i t i o n was made a f t e r the c o n t r a c t i l e response to the prev ious concen t ra t i on reached a maximum l e v e l . Concent rat ion- response curves were cons t ruc ted f o r NA and PDB i n the th ree types o f blood ve s se l s from both con t r o l and d i a b e t i c r a t s . Concent rat ion- response curves to PE and KC1 were a l so obta ined i n aortae and mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s . C o n t r a c t i l e responses to CaCl2 were measured i n aortae and po r t a l ve ins incubated w i th 4x l0~ 2 M KC1 ( s u b s t i t u t e d f o r an equimolar amount of NaCl) and 10"^M phentolamine (to b lock the e f f e c t s o f any n e u r a l l y r e l ea sed NA). CaCl2 c o n t r a c t i l e responses were a l so obta ined i n a o r t i c p repa ra t i on s pre incubated f o r 15 min i n C a 2 + - f r e e medium con ta i n i n g 10~ 6M NA. Aortae and mesenter ic a r t e r i e s were t r e a t e d w i th the b i o l o g i c a l l y i n a c t i v e phorbol e s t e r , 4a-phorbol (10" 8 M-10" 4 M) f o r 30 min (Castagna et a l . , 1982). Concen t r a t i on -response curves to NaF were a l so determined i n aortae and mesenter ic a r t e r i e s i n the presence o f 10" 5 M AICI3. As repor ted p r e v i o u s l y (Marc et a l . , 1988; Zeng et a l . , 1989), and determined i n p r e l i m i n a r y exper iments, c o n t r a c t i l e responses to NaF were g r ea te r i n the presence than i n the absence of 10" 5 M A lC I 3 . (b) Contractile responses to single concentrations of agonists: C o n t r a c t i l e responses to s i n g l e maximum concent ra t i on s o f NA, PDB and 63 KC1 were obta ined i n aortae and mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s . Responses to these concent ra t i on s o f the agon i s t s were a l s o obta ined i n a r t e r i e s p re t rea ted w i th the p u t a t i v e PKC i n h i b i t o r , s tau ro spor ine (5x lO" 5 M) f o r 25 min (Tamaoki et a l . , 1986). Experiments i n v o l v i n g KC1 were c a r r i e d out i n the presence o f phentolamine (10~ 7 M). C o n t r a c t i l e responses to PDB (3xlO"^M) were a l s o assessed i n a r t e r i a l r i n g s pre incubated w i th verapamil and n i f e d i p i n e (3x lO" 6 M) f o r 15 min. (c) Determination of Ca^+ dependence of responses: C o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s to s i n g l e concen t ra t i on s o f NA, PE and METH were measured i n the absence o f e x t r a c e l l u l a r C a 2 + i n order to es t imate the magnitude of i n t r a c e l l u l a r C a 2 + r e l e a s e i n c on t r o l and d i a b e t i c a r t e r i e s . Responses to NaF p lus 10'^M A lC I3 were a l so measured under s i m i l a r c o n d i t i o n s . In these exper iments, f o l l o w i n g the e q u i l i b r a t i o n pe r iod i n Krebs s o l u t i o n , a r t e r i e s were f u r t h e r incubated i n C a ' - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA f o r 15 min before adding a s i n g l e concen t ra t i on of agon i s t . When the c o n t r a c t i l e response had returned to the basal l e v e l , 3.5 mM CaCl2 was added i n the presence o f agon i s t (except NaF) to determine the dependency o f the c o n t r a c t i l e response on i n f l u x of e x t r a c e l l u l a r C a 2 + . Concent rat ion- response curves to PDB were a l s o measured i n ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins i n C a 2 + - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA f o l l o w i n g a 30 min e q u i l i b r a t i o n pe r iod i n t h i s s o l u t i o n . In a l l exper iments , CaCl2 omit ted i n the C a 2 + - f r e e s o l u t i o n was rep laced by NaCl to ma inta in o s m o l a r i t y . 64 2.8. [J'P] LABELLING STUDIES •so [ P] l a b e l l i n g s tud ie s were c a r r i e d out i n a o r t i c p repa ra t i on s as de sc r i bed by A b d e l - L a t i f (1986). Aortae i s o l a t e d from c o n t r o l and d i a b e t i c r a t s were c leaned o f f a t and connect i ve t i s s u e . Ten mm r i n g s were cut l o n g i t u d i n a l l y to expose the inner s u r f a ce , t a k i n g care not to damage the endothel ium l i n i n g the ve s s e l s . The t i s s u e segments were then e q u i l i b r a t e d f o r 2 hours i n Krebs s o l u t i o n at 37°C under an atmosphere of 95% O2 and 5% CO2 i n a shaking water bath. The muscles were then incubated i n the presence or absence of NA ( l o " 5 M ) , i n Krebs s o l u t i o n con ta i n i n g 30 /iCi/ml [ 3 2 P ] sodium orthophosphate. The r e a c t i o n was terminated at va r ious t imes w i th the a d d i t i o n o f an equal volume o f 10% i c e - c o l d TCA. The t i s s u e s were then immediately t r a n s f e r r e d i n t o a mixture o f 2 ml i c e - c o l d ch lo ro fo rm, methanol and HC1 (200:200:1, by volume). Subsequently, each segment was removed, b l o t t e d dry and weighed as q u i c k l y as p o s s i b l e and then homogenized i n the ch l o ro fo rm, methanol and HC1 mixture f o r 1-2 min w i th a t e f l o n - c o a t e d Po t te r -E l veh jem homogenizer at s e t t i n g 60 - 70. The homogenate was t r a n s f e r r e d to a g l a s s tube and c e n t r i f u g e d at 3000xg f o r 15 min at 4°C. The supernatant was r e t a i n e d wh i l e the re s idue was r e - e x t r a c t e d w i th another s o l u t i o n of ch l o ro fo rm, methanol and HC1 (400:200:0.5, by volume). The e x t r a c t s were combined and evaporated to dryness under a stream of n i t r ogen gas. The re s i due was resuspended i n 3 ml ch lo ro form and washed 3 t imes w i th 1 ml o f 0.1 M HC1. Fo l l ow ing each a c i d wash, the samples were mixed by v o r t e x i n g , c e n t r i f u g e d and the upper a c i d l a y e r removed and saved. The 65 combined upper phases were washed w i th 3 ml ch l o ro fo rm, mixed by v o r t e x i n g , and c e n t r i f u g e d . The lower ch lo ro fo rm phase was then added back to the o r i g i n a l ch lo ro form l a y e r and evaporated to dryness under n i t r ogen gas. The phospho l i p id re s idue was resuspended i n a small volume o f ch lo ro fo rm (100 /il) and the phospho l ip id s separated by one-d imens ional t h i n l a y e r chromatography. Ptd Ins(4,5)P2 and Ptd Ins (4)P were separated on a s i l i c a gel 60 high-performance t h i n l a y e r p l a t e ( l a y e r t h i c kne s s 25 /xM) accord ing to Akhtar et a l . (1983). Before use, the p l a t e s were impregnated w i th potassium o x a l a t e , by development i n methanol and water (2:3, by volume) c on t a i n i n g 1% potass ium o x a l a t e , d r y i ng at room temperature and a c t i v a t i o n at 110°C f o r 15 min. Chromatograms were developed i n a chromatographic chamber w i th ch lo ro fo rm :methanol :4M NH4OH (9:7:2, by volume). P td ln s and PA were separated on a s i l i c a gel 60 t h i n l a y e r p l a t e ( l a y e r t h i c kne s s 25 fiM) us ing the so l ven t system methyl a ce t a te : n -propano l : ch lo ro fo rm: methanol : 0.25% KC1 (25: 25: 25: 10: 1, by volume) as de sc r i bed by B ianca et a l . (1986). The phospho l ip id s were v i s u a l i z e d by exposing the p l a t e s to i o d i n e . A f t e r the spots were marked w i th p e n c i l , the s t a i n was a l lowed to fade away by storage overn ight i n a fume hood. Spots corresponding to known standards were scraped i n t o i n d i v i d u a l s c i n t i l l a t i o n v i a l s and t h e i r r a d i o a c t i v e contents determined by l i q u i d s c i n t i l l a t i o n count ing a f t e r a d d i t i o n o f 10 ml Aquaso l . 66 2.9. [3H]-MYOINOSITOL LABELLING STUDIES The accumulat ion of t o t a l [ 3 H ] - i n o s i t o l phosphates i n blood ve s se l s was determined accord ing to Ber r idge et a l . (1982) w i th a s l i g h t m o d i f i c a t i o n . Ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins obta ined from c o n t r o l and d i a b e t i c r a t s were c a r e f u l l y c leaned and cut l o n g i t u d i n a l l y to expose the inner s u r f ace . The segments were e q u i l i b r a t e d f o r 2 hours i n Krebs s o l u t i o n at 37°C under an atmosphere of 95% O2 and 5% CO2 i n a shaking water bath. The muscles were then pre incubated f o r va r ious per iods of t ime, up to 4 hours, w i th 6 /iCi/ml [ 3 H] -myo i no s i t o l i n oxygenated (95% 02/5%C02) Krebs s o l u t i o n at 37°C. A f t e r washing th ree t imes w i th warm oxygenated Krebs s o l u t i o n , the a r t e r i e s were t r a n s f e r r e d i n t o f r e s h Krebs s o l u t i o n wi thout the l a b e l l e d i n o s i t o l and f u r t h e r incubated f o r a pe r iod of 30 min, 10 mM L i C l being present f o r the l a s t 15 min. Fo l l ow ing t h i s , t i s s u e s were t r e a t ed w i th NA (10"^M i n aortae and p o r t a l v e i n s , and 3x 10"^M i n mesenter ic a r t e r i e s ) d i s s o l v e d i n Krebs s o l u t i o n con ta i n i n g un labe led i n o s i t o l (5 mM f i n a l concent ra t i on ) and i n cuba t i on cont inued f o r 0.5 to 20 min. Some p repa ra t i on s were exposed to lO '^M p razo s i n or yohimbine f o r 15 min before i n cuba t i on w i th NA f o r 20 min. Basal accumulat ion of [ 3 H ] - i n o s i t o l phosphates was determined i n the absence of NA, p razos in or yohimbine. At the end of the i n c u b a t i o n , t i s s u e s were r a p i d l y t r a n s f e r r e d i n t o a s o l u t i o n o f 2 ml i c e - c o l d ch lo ro fo rm: methanol: HC1 (100:200:1, by volume). Subsequently, the muscles were r a p i d l y b l o t t e d dry and weighed and then homogenized i n the ch l o ro fo rm: methanol: HC1 s o l u t i o n f o r 1-2 min w i t h a t e f l o n - c o a t e d 67 Pot te r -E l veh jem homogenizer at s e t t i n g 60-70, The homogenate was t r a n s f e r r e d to a g l a s s tube, and ch lo ro form (1 ml) and water (1.2 ml) were added. The e x t r a c t was c e n t r i f u g e d at 3000xg f o r 15 min at 4°C to separate the phases. Two ml of the aqueous phase formed was removed f o r de te rminat i on of t o t a l [ 3 H ] - i n o s i t o l phosphates, w h i l e the remainder o f the e x t r a c t was r e t a i ned f o r measurement of i n c o r p o r a t i o n o f [ H ] -myo ino s i t o l i n t o phospho l ip id s (see below). The 2 ml aqueous e x t r a c t r e t a i ned was n e u t r a l i z e d (pH 7-8) w i th 0.1M NaOH. I t was then brought up to 5 ml w i th d i s t i l l e d water and app l i ed to a column con t a i n i n g l g o f Bio-Rad AG l - x8 r e s i n (formate form, 100-200 mesh) f o r de te rminat i on o f t o t a l [ 3 H ] - i n o s i t o l phosphates by anion exchange chromatography. The column was s e q u e n t i a l l y washed w i th 50 ml o f water and 30 ml of 60 mM sodium formate / 5 mM disodium t e t r a b o r a t e to remove f r e e [ H ] - i n o s i t o l and [ 3 H ] - g l y c e r o p h o s p h o i n o s i t o l , r e s p e c t i v e l y . Tota l [ 3 H ] - i n o s i t o l phosphates were e l u t ed w i th 14 ml of 1M ammonium formate / 0.1M fo rmic a c i d . In p r e l i m i n a r y experiments i n which e l u t i o n p r o f i l e o f the i n o s i t o l phosphates was determined, t h i s volume o f the e luant was found to remove almost a l l of the [ 3 H ] - i n o s i t o l phosphates app l i ed to the column. A l i q u o t s (1ml) c on t a i n i n g t o t a l [ 3 H ] - i n o s i t o l phosphates were p laced i n l i q u i d s c i n t i l l a t i o n v i a l s , 10 ml o f Aquasol was added and v i a l s were counted f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n counter . For dete rminat ion of the basal i n c o r p o r a t i o n of [ J H ] - m y o i n o s i t o l i n t o t o t a l pho spho l i p i d s , l i p i d s were e x t r a c t ed based on the method desc r ibed by A b d e l - L a t i f (1986). One ml of the lower o rgan ic phase o f 68 the reserved e x t r a c t was c o l l e c t e d i n a c lean g l a s s tube and the in te rphase mixed w i th 1.5 ml of ch lo ro fo rm: methanol: HC1 (200:100:1 by volume). Two ml o f the lower phase formed was combined w i th the o r i g i n a l o rgan ic e x t r a c t and 2 ml o f ch loro form added. The combined e x t r a c t was then washed tw ice w i th 5 ml of 0.1M HC1. Four ml o f the e x t r a c t were evaporated to dryness under a stream o f n i t rogen gas. The re s i due was resuspended w i th 1 ml of ch lo ro form and a l i q u o t s counted f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n counter t o determine t o t a l [ 3 H ] -i n o s i t o l l i p i d s . 2.10. DETERMINATION OF INS(1,4,5)P3 I n s ( l , 4 , 5 )P3 l e v e l s were measured i n ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins from con t r o l and d i a b e t i c r a t s us ing a p r o t e i n b ind ing assay as desc r ibed below. Rings of ao r ta were set up and e q u i l i b r a t e d i n i s o l a t e d t i s s u e baths as exp la ined above. At va r ious t imes (0-120 sec) a f t e r the a d d i t i o n of NA (10~ 7 - 10 " 4 M), t i s s u e s were f rozen w i th clamps precoo led i n l i q u i d n i t r o g e n . The tens ion was monitored at the same time to determine the c o n t r a c t i l e e f f e c t of NA. The f rozen t i s s u e s were s to red at -80°C u n t i l assayed f o r I n s ( l , 4 ,5 )P3 l e v e l s . Untreated t i s s u e s were a l s o f rozen to determine basal I n s ( l , 4 ,5 )P3 l e v e l s . In t h i s way, t ime-cour se o f a c t i o n and concent ra t ion - response curves t o NA f o r I n s ( l , 4 , 5 )P3 product ion and c o n t r a c t i o n were determined. For dete rminat ion of I n s ( l , 4 ,5 )P3 l e v e l s i n mesenter ic a r t e r i e s and p o r t a l v e i n s , muscles c leaned of f a t and connect i ve t i s s u e , were 69 opened l o n g i t u d i n a l l y and e q u i l i b r a t e d f o r 2 hours i n Krebs s o l u t i o n , mainta ined at 37°C and oxygenated w i th 95% O2 and 5% CO2, i n a shaking water bath . The t ime-course of a c t i o n and concent ra t i on - re sponse r e l a t i o n s h i p s to NA were determined i n mesenter ic a r t e r i e s by f r e e z i n g the t i s s u e s i n l i q u i d n i t rogen at va r i ous t imes (0-60 sec) a f t e r the a d d i t i o n of NA (10~ 6 or 3 x lO " 5 M) . Untreated mesenter ic a r t e r i e s and p o r t a l ve ins were a l so f rozen f o r dete rminat ion o f basal I (1,4,5)P3 l e v e l s . The f rozen samples of ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins were weighed and homogenized i n 6% i c e - c o l d TCA i n a p o l y t r o n PT-10 homogenizer f o r 15 sec at s e t t i n g 5. The homogenates were c e n t r i f u g e d at 8000xg f o r 15 min at 4°C. The supernatants were c o l l e c t e d i n c lean g l a s s tubes and washed fou r t imes w i th f ou r volumes o f water sa tu ra ted d i e t h y l e ther to remove the TCA. A l i q u o t s (100 /il) o f the e ther -e x t r a c t ed samples were then used f o r Ins (1,4,5)P3 assay. I n s ( l , 4 ,5 )P3 assay was performed by a p r o t e i n - b i n d i n g method us ing an assay k i t obta ined from Amersham. Th i s assay system i s based on the p r i n c i p l e o f compet i t i on between un labe led I n s ( l , 4 , 5 )P3 and a f i x e d q u a n t i t y o f [ 3 H] l a b e l l e d I n s ( l , 4 ,5 )P3 f o r a l i m i t e d amount o f b ind ing s i t e s on a bovine adrenal b ind ing p r o t e i n p r e p a r a t i o n . With f i x e d amounts o f b ind ing p r o t e i n and r a d i o a c t i v e I n s ( l , 4 , 5 ) P3 , the amount o f l a b e l l e d I n s ( l , 4 ,5 )P3 bound to the b ind ing p r o t e i n i s i n v e r s e l y p r opo r t i o na l to the concen t ra t i on of un labe led I n s ( l , 4 , 5 )P3 i n the assay sample. In b r i e f , the assay procedure i nvo l ve s the a d d i t i o n o f 100 /il of 70 b ind ing p r o t e i n p repa ra t i on to 100 /zl o f t r i s b u f f e r (pH 9.0) to which 100 /il o f a s e r i e s o f Ins (1,4,5)P3 standards ( rang ing from 0.19 to 25 pmol) or sample, and l a b e l l e d I n s ( l , 4 ,5 )P3 were added s e q u e n t i a l l y . The mixture was vortex-mixed and then a l lowed to reach to e q u i l i b r i u m wi th i ncuba t i on f o r 15 min on i c e . The bound I n s ( l , 4 ,5 )P3 was separated from f r e e I n s ( l , 4 ,5 )P3 by c e n t r i f u g a t i o n at lOOOxg f o r 3 min at 4°C, s e t t l i n g the b ind ing p r o t e i n p repa ra t i on to the bottom o f the c o n t a i n e r . The supernatant c on ta i n i n g the f r e e f r a c t i o n was d i s ca rded by decanta t i on l e a v i n g the bound f r a c t i o n adher ing to the c o n t a i n e r . The p e l l e t was resuspended i n 200 /j1 of water and a 190 /il a l i q u o t o f the suspension was t r a n s f e r r e d i n t o a s c i n t i l l a t i o n v i a l and counted f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n counter a f t e r a d d i t i o n of 2 ml o f Aquaso l . A standard curve was cons t ruc ted f o r each I n s ( l , 4 ,5 )P3 assay and the amount o f I n s ( l , 4 ,5 )P3 i n each sample was determined by i n t e r p o l a t i o n from t h i s curve. 2.11. ANALYSIS OF DATA AND STATISTICAL METHODS C o n t r a c t i l e responses to agon i s t s were determined as the inc rease i n t en s i on (g) per cross - s e c t i o n a l area o f t i s s u e . The est imated c r o s s - s e c t i o n a l area of each p repa ra t i on was c a l c u l a t e d us ing the formula : C r o s s - s e c t i o n a l area (mm2) = weight (mg) / length (mm) x den s i t y (mg/mm3). The d e n s i t y of the p repa ra t i on was assumed to be 1.05 mg/mm3 (Wyse, 1980). 7 1 The amount of r a d i o a c t i v i t y i n phospho l i p id s and i n o s i t o l phosphates was expressed as counts per min (cpm) per mg wet t i s s u e weight . I n s ( l , 4 , 5 )P3 l e v e l s were c a l c u l a t e d as pmol per mg o f wet t i s s u e weight. The s e n s i t i v i t i e s o f blood ves se l s to agon i s t s f o r c o n t r a c t i o n and I n s ( l , 4 , 5 )P3 product ion were determined by c a l c u l a t i n g pD£ ( - log ED50) va lues by n o n - l i n e a r reg re s s i on ana l y s i s o f the concen t r a t i on - response curves us ing a l l the data p o i n t s . Resu l t s are repor ted as mean + SEM. Mean va lues are c a l c u l a t e d based on the number of animals used i n each exper iment. S t a t i s t i c a l d i f f e r e n c e s between con t r o l and d i a b e t i c p repa ra t i on s wered compared us ing one-way or two-way ana l y s i s of va r i ance (ANOVA) f o l l owed by Neuman-Keul 's t e s t . Resu l t s were cons idered to be s i g n i f i c a n t l y d i f f e r e n t i f P < 0.05. 72 RESULTS 3.1. GENERAL FEATURES OF EXPERIMENTAL ANIMALS Twelve to 14 weeks a f t e r i n j e c t i o n , the STZ- t reated d i a b e t i c r a t s e x h i b i t e d decreased body weight and e leva ted serum g lucose l e v e l s compared w i th age-matched c o n t r o l s (Table 2 ) . The c r o s s - s e c t i o n a l areas o f aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s were a l s o sma l l e r than those from con t r o l an imals . However, no d i f f e r e n c e s i n c r o s s -s e c t i o n a l areas o f p o r t a l ve ins were found between c o n t r o l and d i a b e t i c r a t s (Table 2 ) . In a d d i t i o n , the STZ-t reated r a t s mani fested other symptoms of d iabetes i n c l u d i n g g l y c o s u r i a , d i a r r h e a , p o l y u r i a and c a t a r a c t s . 3.2. CONTRACTILE RESPONSES TO NA, PE, CALCIUM CHLORIDE AND POTASSIUM CHLORIDE In the absence o f drugs, aortae and mesenter ic a r t e r i e s from both c o n t r o l and d i a b e t i c r a t s were qu ie scent , wh i l e p o r t a l ve ins from both groups of animals were spontaneously a c t i v e . No s i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and d i a b e t i c p o r t a l ve ins i n e i t h e r the magnitude or the frequency o f the spontaneous c o n t r a c t i l e a c t i v i t y cou ld be de tec ted . NA induced a concentrat ion-dependent inc rease i n c o n t r a c t i l e f o r ce o f ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins from both c o n t r o l and d i a b e t i c r a t s (F igure 3 ) . The maximum c o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s but not po r t a l ve ins from d i a b e t i c r a t s to the 73 Table 2. General features of experimental animals Body weight Serum Cross s e c t i o n a l area (mm2) (g) g lucose Aortae Mesenter ic Po r t a l (mmol/1) a r t e r i e s ve ins Cont ro l 521±6 6.1±0.1 1.34±0.04 0.38+0.02 0.3H0.01 (186) (186) (186) (186) (45) D i a b e t i c 339±8* 27 .3±0.5 * 0 .92±0.03* 0.30±0.02 * 0.35±0.01 (186) (186) (186) (186) (45) Values are mean ± SEM . Numbers i n pa renthes i s i n d i c a t e the number o f animals. * S i g n i f i c a n t l y d i f f e r e n t from corresponding c on t r o l (P < 0.05) . 74 agon i s t were s i g n i f i c a n t l y g r ea te r than the corresponding responses obta ined i n c on t r o l p repara t i ons (Table 3 ) . Diabetes d i d not a l t e r the s e n s i t i v i t i e s (as r e f l e c t e d by pD 2 va lues ) o f any of the blood ve s se l s to NA (Table 3 ) . The t ime-course of c o n t r a c t i o n of aortae to a c oncen t r a t i on of NA (10" 5M) producing maximum c o n t r a c t i l e response i s shown i n F igure 4. In both c on t r o l and d i a b e t i c ao r tae , the t ime-course o f the NA response was s i m i l a r , c o n s i s t i n g of an i n i t i a l r a p i d r i s e i n t en s i on dur ing the f i r s t 20-30 sec, f o l l owed by a s lower inc rease i n ten s i on which was sus ta ined f o r a pe r i od o f at l e a s t 10 min. The magnitude o f the c o n t r a c t i l e response of aortae from d i a b e t i c r a t s to NA was s i g n i f i c a n t l y g r ea te r than c on t r o l 20 sec a f t e r NA a d m i n i s t r a t i o n , and remained s i g n i f i c a n t l y e leva ted over the e n t i r e pe r iod measured (F igure 4 ) . As w i th ao r tae , the t ime-course of c o n t r a c t i o n o f mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s to a maxiumum concen t r a t i on of NA ( 3x l 0 " 5 M) was a l so s i m i l a r (F igure 5 ) . I t c on s i s t ed o f an i n i t i a l r a p i d r i s e i n ten s i on dur ing the f i r s t 20-30 sec, f o l l owed by a s lower i nc rease i n ten s i on which was sus ta ined f o r a pe r i od o f at l e a s t 5 min. The c o n t r a c t i l e response was s i g n i f i c a n t l y g r ea te r i n d i a b e t i c than i n c o n t r o l mesenter ic a r t e r i e s 20 sec a f t e r NA a d m i n i s t r a t i o n and remained s i g n i f i c a n t l y e leva ted over the t ime per iod measured (F igure 5 ) . C o n t r a c t i l e responses of aortae and mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s to the a j - s e l e c t i v e agon i s t PE were a l so measured. PE, l i k e NA, caused c o n t r a c t i o n o f a r t e r i e s from both c on t r o l 7 5 Table 3. Agon i s t pD2 va lues and maximum c o n t r a c t i l e responses i n ao r t ae , mesenter i c a r t e r i e s and p o r t a l ve ins from c o n t r o l and d i a b e t i c r a t s . pD2 values C o n t r a c t i l e response (g/mm2) Contro l D i a b e t i c Cont ro l D i a b e t i c Ao r ta NA PE KC1 6.88±0.06 6.30±0.08 1.82+0.07 Mesenter ic a r t e r y NA 6.40±0.11 PE 6.03±0.14 KC1 1.78±0.04 Po r t a l ve in NA 6.40±0.10 6.55±0.14 6.52±0.06 1.70+0.00 6.19±0.11 5.98±0.11 1.88+0.04 6.48+0.10 2.12±0.18 1.57±0.25 1.10+0.27 3.60±0.45 4.3510.58 1.9510.27 3.0910.30 3.21+0.38 2.7010.18* 1.5510.11 5.2510.53* 6.4610.58* 2.0510.42 3.3210.34 Values are the mean 1 SEM o f 6-12 observat ions * S i g n i f i c a n t l y d i f f e r e n t from corresponding con t r o l (P < 0.05) 76 and d i a b e t i c r a t s i n a concentrat ion-dependent manner. (F i gure 6 ) . The maximum c o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s to PE were s i g n i f i c a n t l y enhanced i n d i a b e t i c ve s se l s compared w i th c on t r o l t i s s u e s (Table 3 ) . However, as w i th the e f f e c t o f NA, s e n s i t i v i t i e s of the a r t e r i e s from the two groups o f animals to PE d i d not d i f f e r from each other s i g n i f i c a n t l y (Table 3 ) . To assess the s p e c i f i c i t y o f the a c t i o n of NA and PE i n producing enhanced c o n t r a c t i l e responses i n d i a b e t i c aortae and mesenter ic a r t e r i e s , c o n t r a c t i l e responses o f a r t e r i e s to KC1 were measured. The magnitude o f the maximum c o n t r a c t i o n and s e n s i t i v i t i e s of the two types of blood ve s se l s from con t r o l and d i a b e t i c r a t s to KC1 were s i m i l a r (F i gure 7; Table 3 ) . To f u r t h e r c h a r a c t e r i z e the NA and KC1-induced responses, c o n t r a c t i o n s o f aortae incubated i n C a 2 + - f r e e Krebs s o l u t i o n from c o n t r o l and d i a b e t i c r a t s to i n c r ea s i n g concen t ra t i on s o f CaCl2 were measured i n K + - d e p o l a r i z e d p repara t i ons or i n the presence o f NA (10" ^M). As w i th the KC1 responses, maximum c o n t r a c t i o n and s e n s i t i v i t i e s of the K + - d e p o l a r i z e d aortae from con t r o l and d i a b e t i c r a t s to CaCl2 were s i m i l a r (F i gure 8, Table 4 ) . In c o n t r a s t , the maximum c o n t r a c t i l e response (but not s e n s i t i v i t y ) to CaCl2 i n the presence o f NA was g r ea te r i n d i a b e t i c than i n c on t r o l aortae (F igure 8; Table 4 ) . For comparison purposes, c o n t r a c t i l e responses to C a C l 2 were a l s o determined i n K + - d e p o l a r i z e d po r t a l ve ins from con t r o l and d i a b e t i c r a t s (F igure 9 ) . No s i g n i f i c a n t d i f f e r e n c e s i n the magnitude o f maximum c o n t r a c t i l e 77 Table 4. CaCl2 pD2fivalues and maximum contracti le responses in the presence of 4xlO" zM KC1 or 10"° M NA in aortae from control and diabetic rats. pD2 va lues Maximum c o n t r a c t i l e response Contro l D i a b e t i c Cont ro l D i a b e t i c KC1 3.00+0.28 3.1110.30 2.1010.20 2.5010.23 NA 3.2010.11 3.3010.22 1.1810.12 1.8010.21* Values are the mean l SEM of 4-6 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c on t r o l (P < 0.05). 78 responses or s e n s i t i v i t i e s were detected between c o n t r o l and d i a b e t i c p o r t a l v e i n s . 3.3. CALCIUM DEPENDENCE OF CONTRACTILE RESPONSES TO a-ADRENOCEPTOR AGONISTS C o n t r a c t i l e responses to s i n g l e concent ra t i on s o f NA and PE, and to another s e l e c t i v e a i - ad renocepto r agon i s t , METH, were measured f o l l o w i n g i ncuba t i on of aortae and mesenter ic a r t e r i e s i n C a 2 + - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA f o r 15 min. Th i s r e s u l t e d i n a r a p i d , phas ic c o n t r a c t i o n . When the ten s i on had re tu rned to basal 7+ l e v e l s , 3.5 mM C a ' was added i n the presence o f the agon i s t s , r e s u l t i n g i n a l a r g e , sus ta ined c o n t r a c t i o n . In both the absence and presence o f 7+ e x t r a c e l l u l a r Ca , the magnitudes of the c o n t r a c t i l e responses o f aortae from d i a b e t i c r a t s to both 1 0 " 6 and 10" 5 M NA were s i g n i f i c a n t l y g r ea te r than c o n t r o l (F igure 10). S i m i l a r l y , under both c o n d i t i o n s , c o n t r a c t i l e responses o f d i a b e t i c aortae to a maximum concen t r a t i on o f PE (10"^ M) and METH (3xl0"^M) were found to be s i g n i f i c a n t l y g r ea te r than c o n t r o l (F igure 11). In the absence of C a 2 , responses to 3xl0"^M but not 10"°M NA were s i g n i f i c a n t l y g r ea te r i n d i a b e t i c than i n c on t r o l mesenter ic a r t e r i e s (F igure 12). Fo l l ow ing r e - a d d i t i o n o f Ca , c o n t r a c t i l e responses o f d i a b e t i c mesenter ic a r t e r i e s to both concen t ra t i on s o f NA were s i g n i f i c a n t l y g r ea te r than c o n t r o l (F i gure 12). C o n t r a c t i l e responses of mesenter ic a r t e r i e s i n the absence as we l l as i n the presence of e x t r a c e l l u l a r C a 2 + to maximum concen t ra t i on s of PE 79 (3xlO"°M) and METH (10"^M) were s i g n i f i c a n t l y g r ea te r i n p repa ra t i on s from d i a b e t i c than from con t r o l r a t s (F igure 13). 3.4. EFFECTS OF NA ON [32P] INCORPORATION INTO PTDINS(4,5)P2 AND PA. The e f f e c t s of NA on [ 3 2 P ] i n c o r p o r a t i o n i n t o Ptd Ins(4,5)P2 and PA were i n v e s t i g a t e d i n a o r t i c p repara t i ons from c o n t r o l and d i a b e t i c r a t s . I n i t i a l l y , the basal i n co rpo r a t i o n o f [ 3 2 P ] i n t o phospho ino s i t i de s and PA was s tud ied i n order t o determine i f there were d i f f e r e n c e s between con t r o l and d i a b e t i c aortae i n the basal l a b e l l i n g o f the pho spho l i p i d s . The r e s u l t s demonstrate that the i n c o r p o r a t i o n o f [ 3 2 P ] i n t o phospho inos i t i de s and PA increased as a f u n c t i o n of t ime over 4 hours i n cuba t i on i n both con t r o l and d i a b e t i c aortae (Table 5 ) . No evidence of s a t u r a t i o n was seen through the 4 hours o f l a b e l l i n g , and there were no s i g n i f i c a n t d i f f e r e n c e s between con t r o l and d i a b e t i c aortae i n the r a t e o p of [ P] l a b e l l i n g o f the phospho l ip id s over t h i s t ime. An i ncuba t i on pe r i od of 30 min was u t i l i z e d f o r subsequent exper iments. The t ime-course o f the e f f e c t o f 10" 5 M NA on [ 3 2 P ] l a b e l l i n g o f Ptd Ins(4,5)P2 and PA i s shown i n F igure 14. In both c o n t r o l and d i a b e t i c ao r tae , NA caused a r a p i d decrease i n the l e v e l of [ 3 2 P ] i n c o r p o r a t i o n found i n Ptd Ins (4 ,5 )P2, although the magnitude o f the decrease was on ly s l i g h t i n c o n t r o l p r epa ra t i on s . The NA-induced decrease i n [ 3 2 P ] -Ptd Ins(4,5)P2 seemed to be maximal w i t h i n 0.5 min a f t e r the a d d i t i o n of the agon i s t , the e a r l i e s t t ime po in t examined. The l e v e l o f [ 3 2 P ] i nco rpo ra ted i n t o PtdIns(4,5)P2 returned nea r l y t o basal w i th 2 min of 80 Table 5. Basal [ 3 2 P ] l a b e l l i n g of phospholipids (cpm / mg wet weight) i n aortae from control and d i abe t i c r a t s . Time PtdIns(4,5)P 2 PtdIns(4)P Ptdlns PA (n = 7) (n - 6) (n = 6) (n = 4) 10 min Control 250 ± 26 65 ± 28 28 ± 9 10 ± 3 Di abet ic 191 ± 28 49 ± 5 35 ± 13 11 ± 2 30 min Control 386 ± 26 70 ± 7 47 ± 9 31 ± 8 D iabet ic 343 ± 45 64 ± 8 49 ± 6 27 ± 2 1 hr Control 875 ± 80 159 ± 6 152 ± 29 59 ± 7 D iabet ic 683 ± 114 166 ± 30 158 ± 29 45 ± 13 2 hr Control 1465 ± 192 324 ± 36 502 ± 70 97 ± 21 D iabet ic 1384 ± 156 272 ± 32 419 ± 54 72 ± 7 4 hr Control 2334 ± 110 530 ± 30 1234 ± 85 198 ± 30 D iabet ic 2001 ± 360 431 ± 75 1057 ± 240 139 ± 31 Tissues were incubated with 30/xCi/ml [ 3 2 P]-sod ium orthophosphate and the react ion terminated at the various times ind icated above. Phospholipids were extracted, separated by th in layer chromatography and t h e i r r a d i o a c t i v i t y determined as described in methods. Values are mean ± SEM. 81 exposure to NA. The d e p l e t i o n of [i6P] l a b e l l e d P td ln s (4,5)P2 was s i g n i f i c a n t l y g r ea te r i n aortae from d i a b e t i c r a t s than c o n t r o l s , 0.5 and 1 min a f t e r the a d d i t i o n of NA (F igure 14). In c o n t r a s t , NA caused a time-dependent s t i m u l a t i o n o f [ 3 2 P ] i n c o r p o r a t i o n i n t o PA over a pe r i od o f 2 min i n both c o n t r o l and d i a b e t i c aortae (F igure 14). The i n co rpo r a t i o n o f [ 3 2 P ] i n t o PA was s i g n i f i c a n t l y h igher i n d i a b e t i c aortae than i n c o n t r o l s 2 min a f t e r a d d i t i o n o f NA. 3.5. NORADRENALINE-INDUCED [3H]-INOSITOL PHOSPHATE ACCUMULATION The accumulat ion of [ 3 H ] - i n o s i t o l phosphates i n response to NA was determined i n ao r tae , mesenter ic a r t e r i e s and p o r t a l ve ins from both c o n t r o l and d i a b e t i c r a t s . However, p r i o r to t e s t i n g the e f f e c t of NA, basal l a b e l l i n g of t o t a l phospho l ip id s w i th [ 3 H] -myo i no s i t o l and the accumulat ion o f t o t a l [ H ] - i n o s i t o l phosphates were assessed over a pe r i od o f 4 hours. The r e s u l t s demonstrated tha t the basal l a b e l l i n g of t o t a l phospho l ip id s and accumulat ion o f t o t a l [ 3 H ] - i n o s i t o l phosphates were l i n e a r w i th time over a pe r i od of 4 hours i n both c on t r o l and d i a b e t i c aortae (Tables 6 and 7 ) . No s i g n i f i c a n t d i f f e r e n c e s between c o n t r o l and d i a b e t i c aortae were found i n the l a b e l l i n g o f phospho l ip id s w i t h [ 3 H] -myo i no s i t o l or i n the accumulat ion o f [ 3 H ] - i n o s i t o l phosphates over t h i s p e r i o d . In both mesenter ic a r t e r i e s and p o r t a l ve ins from c o n t r o l and d i a b e t i c r a t s , the accumulat ion of basal t o t a l [ 3 H ] - i n o s i t o l phosphates a l s o increased w i th t ime. While basal [ 3 H ] - i n o s i t o l 82 Table 6. Basal [ JH]-myoinositol label l ing of total phospholipids (cpm / mg wet weight) in aortae, mesenteric arteries and portal veins from control and diabetic rats. Time Aortae Mesenter ic Po r t a l (hr) a r t e r i e s ve ins Cont ro l D i a b e t i c Contro l D i a b e t i c Cont ro l D i a b e t i c (n = 16) (n = 16) (n = 10) (n = 10) (n = 5 ) (n = 5) 0.5 624± 680± 20 38 2.0 2375± 2370± 313 289 4.0 3425± 3688± 3088± 337 298 138 5812+. 14335± 18626±. 635 1141 1270 T i s sues were incubated w i th 6/xCi/ml [ 3 H] -myo i no s i t o l and r e a c t i o n terminated at the var ious t imes i n d i c a t e d above. Tota l phospho l ip id s were e x t r a c t ed and r e a d i o a c t i v i t y determined as desc r ibed i n methods. Values are mean ± SEM. * S i g n i f i c a n t l y d i f f e r e n t from corresponding c on t r o l (P < 0.01) . 83 Table 7. Basal accumulat ion o f t o t a l [ J H ] - i n o s i t o l phosphates (cpm / mg wet weight) i n ao r t ae , mesenter ic a r t e r i e s and p o r t a l ve in s from c o n t r o l and d i a b e t i c r a t s . Time Aor ta Mesenter ic P o r t a l ve ins (hr) a r t e r i e s (n = 10) (n = 6) (n = 4) 0.5 Cont ro l 24 ± 2 D i a b e t i c 30 ± 2 1 Cont ro l 73 ± 6 108 + 4 D i a b e t i c 99 ± 4* 172 ± 21 2 Contro l 38 ± 3 108 ± 9 218 + 37 D i a b e t i c 41 ± 5 182 ± 6* 270 + 45 4 Contro l 102 ± 21 231 ± 23 353 ± 32 D i a b e t i c 183 ± 36 483 ± 79* 506 + 60 T i s sues were incubated w i th 6/iCi/ml [^H]-myoinos i to l and the r e a c t i o n terminated at the var ious t imes i n d i c a t e d above. To ta l [ j H ] - i n o s i t o l phosphates were determined by anion exchange chromatography as desc r ibed i n methods. Values are mean ± SEM. * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l (P < 0.05). 84 phosphate accumulat ion was s i g n i f i c a n t l y g r ea te r i n d i a b e t i c than i n c on t r o l mesenter ic a r t e r i e s at a l l t imes measured, i t was s i g n i f i c a n t l y g r ea te r i n p o r t a l ve ins from d i a b e t i c r a t s than i n c o n t r o l p repa ra t i on s on ly a f t e r 4 hours of l a b e l l i n g . The basal l a b e l l i n g o f t o t a l phospho l ip id s w i th [ 3 H] -myo i no s i t o l a f t e r a 4 hour i n cuba t i on pe r i od was s i g n i f i c a n t l y g r ea te r i n d i a b e t i c mesenter ic a r t e r i e s and p o r t a l ve ins than the va lues obta ined i n corresponding c o n t r o l blood v e s s e l s . In subsequent exper iments, a 4 hour pe r iod o f l a b e l l i n g w i th [ 3 H ] -myo ino s i t o l was used to examine the e f f e c t of NA on t o t a l [ 3 H ] - i n o s i t o l phosphate accumulat ion. The t ime-course of t o t a l [ 3 H ] - i n o s i t o l phosphate p roduct ion i n aortae i n response to 10" 5 M NA s t i m u l a t i o n i s shown in F igure 15. In aortae from both groups of an imals , NA s t imu la ted the r a p i d product ion of [ J H ] - i n o s i t o l phosphates w i t h i n 0.5 min. More prolonged exposure to the agon i s t caused a time-dependent inc rease i n the p roduct ion of [ 3 H ] -i n o s i t o l phosphates over 20 min i n p repara t i ons from both groups o f an imal s . No s i g n i f i c a n t d i f f e r e n c e i n basal [ 3 H ] - i n o s i t o l phosphate accumulat ion was found between aortae from con t r o l and d i a b e t i c r a t s . However, the p roduct ion of [ 3 H ] - i n o s i t o l phosphates i n response to NA was s i g n i f i c a n t l y g r ea te r i n aortae from d i a b e t i c r a t s than i n c o n t r o l p repa ra t i on s at a l l t imes of i n cuba t i on . Pretreatment w i t h p razo s i n (10" 7 M ) markedly decreased the product ion o f [ 3 H ] - i n o s i t o l phosphates i n response to NA i n both c on t r o l and d i a b e t i c aortae (F igure 16). In 8 5 c o n t r a s t , yohimbine (10~'M) had almost no i n h i b i t o r y e f f e c t on NA-induced [ 3 H ] - i n o s i t o l phosphate product ion (F igure 16). NA-induced t o t a l [ 3 H ] - i n o s i t o l phosphate accumulat ion i n mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s i s i l l u s t r a t e d i n F igure 17. As w i th aor tae , i n both types o f mesenter ic a r t e r i e s , 3xlO"^M NA s t imu l a ted the r a p i d product ion o f [ 3 H ] - i n o s i t o l phosphates w i t h i n 0.5 min. More prolonged exposure (20 min) r e s u l t e d i n increased product ion of [ 3 H ] - i n o s i t o l phosphates i n both p r epa r a t i on s . The accumulat ion o f [ 3 H ] - i n o s i t o l phosphates i n response to NA was s i g n i f i c a n t l y g r ea te r i n mesenter ic a r t e r i e s from d i a b e t i c r a t s than i n con t r o l p r epa ra t i on s . Pretreatment o f mesenter ic a r t e r i e s w i th p razo s i n (10" 7 M) markedly i n h i b i t e d the NA-induced accumulat ion of [ 3 H ] - i n o s i t o l phosphates i n both c on t r o l and d i a b e t i c ve s se l s (F igure 18). NA (10" 5 M ) a l so induced a r a p i d and t i m e - r e l a t e d enhancement of the accumulat ion o f t o t a l [ 3 H ] - i n o s i t o l phosphates i n p o r t a l ve ins from c o n t r o l and d i a b e t i c r a t s (F igure 19). The inc rease i n the amount of [ 3 H ] - i n o s i t o l phosphates was s i g n i f i c a n t l y g r ea te r i n p o r t a l ve in s from d i a b e t i c r a t s than i n con t r o l 20 min but not 0.5 min a f t e r the a d d i t i o n o f the agon i s t . 3.6. NORADRENALINE-INDUCED INS(1,4,5)P3 PRODUCTION In o rder t o determine whether the enhanced accumulat ion of t o t a l [ 3 H ] - i n o s i t o l phosphates i n blood ve s se l s from d i a b e t i c r a t s was a s soc i a ted w i th increased product ion of the b i o l o g i c a l l y a c t i v e i n o s i t o l 86 t r i s pho spha te , the abso lute t i s s u e mass of I n s ( l , 4 , 5 )P3 was measured us ing a p r o t e i n b ind ing assay system. In ao r tae , both I n s ( l , 4 ,5 )P3 l e v e l and c o n t r a c t i o n i n response to NA were measured i n the same p repa ra t i on s i n o rder to assess the c o r r e l a t i o n between b iochemical and f u n c t i o n a l responses more d i r e c t l y . I n s ( l , 4 , 5 )P3 l e v e l and c o n t r a c t i l e responses o f aortae were determined at va r i ous t imes a f t e r the a d m i n i s t r a t i o n o f 10" 5 M NA (F igure 20). In c on t r o l ao r tae , t h i s concen t ra t i on o f NA induced a t ime-dependent inc rease i n I n s ( l , 4 ,5 )P3 l e v e l which reached a maximum at 20 sec a f t e r NA a d m i n i s t r a t i o n . Th i s was a s soc i a ted w i th a r a p i d inc rease i n t e n s i o n . The I n s ( l , 4 ,5 )P3 content returned to c l o s e to basal va lues 60 sec a f t e r NA a d m i n i s t r a t i o n and remained at t h i s l e v e l over a pe r i od of 120 sec. However, the c o n t r a c t i l e response increased s l i g h t l y over t h i s t ime. Basal l e v e l s of I n s ( l , 4 ,5 )P3 i n aortae from c o n t r o l and d i a b e t i c r a t s were not s i g n i f i c a n t l y d i f f e r e n t from each o the r . In aortae from d i a b e t i c r a t s , 10" 5 M NA a l s o induced a r a p i d i nc rease i n I n s ( l , 4 , 5 )P3 l e v e l and t e n s i o n . However, the maximum inc rease i n I n s ( l , 4 , 5 )P3 l e v e l i n d i a b e t i c aortae was detected 30 sec a f t e r a d m i n i s t r a t i o n of NA. I n s ( l , 4 ,5 )P3 l e v e l de c l i n ed to c l o s e to basal va lue a f t e r t h i s t ime, although the c o n t r a c t i l e response increased s l i g h t l y . Two way a n a l y s i s of va r iance demonstrated t ha t d i abetes had a s i g n i f i c a n t e f f e c t compared to c on t r o l on the format ion o f I n s ( l , 4 , 5 )P3 i n response to 10"^M NA over the time per iod assayed. The peak I n s ( l , 4 , 5 )P3 l e v e l measured i n d i a b e t i c aortae i n response to NA was 87 a l s o s i g n i f i c a n t l y g r ea te r than the peak l e v e l a t t a i n e d i n response to the agon i s t i n c on t r o l p repa ra t i on s . The ten s i on developed i n response to NA i n aortae from d i a b e t i c r a t s at the t ime o f the peak I n s ( l , 4 ,5 )P3 was a l s o s i g n i f i c a n t l y g rea te r than that o f c o n t r o l ao r tae . The t ime-course o f a c t i on o f 10"^M NA on I n s ( l , 4 ,5 )P3 accumulat ion and c o n t r a c t i o n was a l s o assessed i n aortae i n o rder to determine whether i t was s i m i l a r to that of 10" 5 M NA (F igure 21). In both c o n t r o l and d i a b e t i c ao r tae , 10"^ M NA induced inc reased I n s ( l , 4 ,5 )P3 accumulat ion and c o n t r a c t i o n , w i th pa t te rn s s i m i l a r to those induced by 10" 5 M NA, although the magnitudes of the responses to 10" 6 M NA were r e l a t i v e l y sma l l e r . Ana l y s i s of va r i ance (two-way) conf i rmed tha t a s i g n i f i c a n t l y enhanced product ion o f I n s ( l , 4 , 5 )P3 was induced i n d i a b e t i c aortae compared to c on t r o l i n response to 10"^M NA over the 60 sec pe r i od assayed. However, the peak Ins (1,4,5)P3 contents measured i n the two groups of animals i n response to t h i s c oncen t r a t i on o f the agon i s t were not s i g n i f i c a n t l y d i f f e r e n t from each o the r . On the other hand, the I n s ( l , 4 ,5 )P3 l e v e l detected i n d i a b e t i c aortae 30 sec a f t e r NA a d d i t i o n was s i g n i f i c a n t l y g r ea te r than tha t detected i n c o n t r o l aortae at t h i s t ime p o i n t . C o n t r a c t i l e responses o f the d i a b e t i c aortae to 10" NA at the time o f the maximum inc rease i n I n s ( l , 4 , 5 )P3 were s i g n i f i c a n t l y g r ea te r than those of the c on t r o l ao r tae . F igure 22 shows the concent ra t ion - response curves to NA f o r I n s ( l , 4 , 5 )P3 accumulat ion i n a o r t i c r i n g s from con t r o l and d i a b e t i c r a t s at the t ime of t h e i r r e s p e c t i v e maximum inc rease i n I n s ( l , 4 , 5 ) P 3 ( i e . 20 88 sec a f t e r NA a d m i n i s t r a t i o n i n c on t r o l ao r tae ; 30 sec a f t e r NA a d m i n i s t r a t i o n i n d i a b e t i c a o r t ae ) . In aortae from both c o n t r o l and d i a b e t i c r a t s , NA caused a concentrat ion-dependent i nc rease i n I n s ( l , 4 , 5 )P3 accumulat ion. The maximum inc rease i n I n s ( l , 4 , 5 )P3 measured i n d i a b e t i c aortae was s i g n i f i c a n t l y g r ea te r than tha t obta ined i n c o n t r o l p repa ra t i on s (2.07 ± 0.35 pmol/mg i n c o n t r o l and 3.80 ± 0.40 pmol/mg i n d i a b e t i c a o r t a e ) . However, the s e n s i t i v i t i e s (as r e f l e c t e d by pD 2 va lues ) of the a r t e r i e s from the two groups o f animals to NA d i d not d i f f e r from each other (NA pD 2 va lues f o r I n s ( l , 4 , 5 )P3 p roduct ion were 5.92 ± 0.02 i n c on t r o l and 6.00 ± 0.10 i n d i a b e t i c a o r t a e ) . The t ime-course of I n s ( l , 4 ,5 )P3 accumulat ion i n mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s i n response to 3X10"^M NA i s i l l u s t r a t e d i n F igure 23. In mesenter ic a r t e r i e s from both c o n t r o l and d i a b e t i c r a t s , 3x 10" 5 M NA induced a r a p i d r i s e i n I n s ( l , 4 , 5 )P3 l e v e l which reached a peak 20 sec a f t e r NA a d m i n i s t r a t i o n . The l e v e l of I n s ( l , 4 , 5 )P3 then dec l i n ed s l i g h t l y over a pe r i od o f 60 sec. Basal I n s ( l , 4 , 5 )P3 accumulat ion i n mesenter ic a r t e r i e s from both groups o f animals d i d not d i f f e r s i g n i f i c a n t l y , although i t tended to be increased (by 138.5 ± 23.3%) i n d i a b e t i c mesenter ic a r t e r i e s . However, the i nc reases i n I (1,4,5)P3 i n response to NA were s i g n i f i c a n t l y g r ea te r i n mesenter ic a r t e r i e s from d i a b e t i c r a t s than i n c o n t r o l p r epa r a t i on s . I n s ( l , 4 , 5 )P3 l e v e l was a l so measured i n response to 10"^M NA i n mesenter ic a r t e r i e s from con t r o l and d i a b e t i c r a t s 20 sec a f t e r i t s a d m i n i s t r a t i o n (F igure 24). For comparison purposes responses to 3x l0"^M 89 NA were a l s o obta ined under s i m i l a r c o n d i t i o n s . Basal I n s ( l , 4 , 5 )P3 l e v e l i n d i a b e t i c mesenter ic a r t e r i e s again tended to be inc reased (by 89.8 ± 3.6%) compared w i th those o f c on t r o l a r t e r i e s , a lthough the d i f f e r e n c e was not s t a t i s t i c a l l y s i g n i f i c a n t . In both c o n t r o l and d i a b e t i c a r t e r i e s , both concent ra t i on s of NA r e s u l t e d i n enhanced accumulat ion o f I n s ( l , 4 , 5 ) P3 , although the enhancement was g r ea te r w i th 3x lO " 5 M NA. While 3x lO " 5 M NA induced s i g n i f i c a n t l y g r ea te r I n s ( l , 4 , 5 ) P 3 p roduct ion i n d i a b e t i c than i n c on t r o l mesenter ic a r t e r i e s , no s i g n i f i c a n t d i f f e r e n c e s i n I n s ( l , 4 ,5 )P3 l e v e l were detected between c o n t r o l and d i a b e t i c a r t e r i e s i n response to 10'^M NA (F igure 24). F igure 25 i l l u s t r a t e s the basal l e v e l of I n s ( l , 4 , 5 )P3 i n p o r t a l v e i n s . The basal l e v e l of I n s ( l , 4 ,5 )P3 i n p o r t a l ve ins from c o n t r o l and d i a b e t i c r a t s were not s i g n i f i c a n t l y d i f f e r e n t from each o the r . 3.7. CONTRACTILE RESPONSES TO PDB IN THE PRESENCE OF EXTRACELLULAR CALCIUM C o n t r a c t i l e responses of aortae and mesenter ic a r t e r i e s to the PKC a c t i v a t o r , PDB, were measured i n order to i n v e s t i g a t e whether PKC-mediated responses are a l t e r e d i n these blood ve s se l s from d i a b e t i c r a t s . C o n t r a c t i l e responses of p o r t a l ve ins to PDB were a l s o assessed f o r comparison purposes. PDB caused r e l a t i v e l y s l ow ly deve lop ing , concentrat ion-dependent c o n t r a c t i o n s of blood ves se l s from both c on t r o l and d i a b e t i c r a t s . The magnitudes of the maximum c o n t r a c t i o n s and s e n s i t i v i t i e s o f aortae from 90 c o n t r o l and d i a b e t i c r a t s to PDB were s i m i l a r (F igure 26, Table 8 ) . In c on t r a s t to the ao r tae , maximum c o n t r a c t i l e responses o f mesenter ic a r t e r i e s to PDB were enhanced i n d i a b e t i c ve s se l s compared to c o n t r o l t i s s u e s i n the presence of 2.5 mM C a 2 + ( F i gure 26; Table 8 ) . However, as w i th the ao r tae , s e n s i t i v i t i e s of the mesenter ic p repa ra t i on s from the two groups of animals to PDB d i d not d i f f e r from each o ther (Table 8 ) . A r t e r i e s from both c on t r o l and d i a b e t i c r a t s d i d not c on t r a c t i n response to the i n a c t i v e phorbol e s t e r , 4a-phorbol ( 1 0 " 8 - 1 0 " 5 M, n=3) (data not shown). Ne i the r the magnitude o f the maximum c o n t r a c t i o n nor the s e n s i t i v i t i e s of p o r t a l ve ins to PDB were d i f f e r e n t between con t r o l and d i a b e t i c p repara t i ons (F igure 26; Table 8 ) . 3.8. EFFECTS OF STAUROSPORINE ON CONTRACTILE RESPONSES OF ARTERIES TO NA AND PDB The e f f e c t s of PKC i n h i b i t o r , s t au ro spo r i ne , on maximum c o n t r a c t i l e responses of aortae and mesenter ic a r t e r i e s to NA (10"^M i n aortae or 3xlO"^M M i n mesenter ic a r t e r i e s ) was assessed (F igure 27) i n order to i n v e s t i g a t e whether the enhanced c o n t r a c t i l e respons iveness of the d i a b e t i c a r t e r i e s to the agon i s t was the r e s u l t o f i nc reased a c t i v a t i o n of PKC-mediated mechanisms. In the absence o f s t au ro spo r i ne , NA induced g rea te r maximum c o n t r a c t i l e responses i n both aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s than i n c o n t r o l p r epa r a t i on s . Pretreatment o f a r t e r i e s from both groups of animals w i th Table 8. PDB pD£ values and maximum contracti le responses in the presence and absence of extracellular C a z + in aortae, mesenteric arteries and portal veins from control and diabetic rats pDg va lues C o n t r a c t i l e response (q/mmM Contro l D i a b e t i c Cont ro l D i a b e t i c Ao r ta 2.5 mM C a 2 + 6.90±0.06 0 C a 2 + 6.03±0.07 Mesenter ic a r t e r y 2.5 C a 2 + 6.91±0.09 0 C a 2 + 6.09+0.20 Po r t a l ve in 2.5 C a 2 + 7.30±0.27 7.39±0.24 2.39±0.30 2.85±0.50 Values are the mean ± SEM o f 6-10 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding con t r o l (P < 0.05) . 6.73+0.04 6.05±0.08 3.96±0.23 1.24+0.81 4.50±0.59 1.15+0.22 6.77±0.12 5.83±0.09 4.61±0.46 1.45+0.11 6.26±0.58 1.30+0.14 92 s tau ro spo r i ne (5x 10"°M) caused marked i n h i b i t i o n o f the maximum c o n t r a c t i l e responses to NA. S taurospor ine appeared to exe r t a g r ea te r i n h i b i t o r y e f f e c t on responses of mesenter ic a r t e r i e s . In the presence of s t au ro spo r i ne , the d i f f e r e n c e i n c o n t r a c t i l e responses observed between c o n t r o l and d i a b e t i c a r t e r i e s to NA was abo l i s hed . Responses of a r t e r i e s to PDB were a l so measured a f t e r pretreatment w i th s tau ro spor ine (F igure 28). In the absence o f s t au ro spo r i ne , c o n t r a c t i l e responses o f mesenter ic a r t e r i e s , but not ao r tae , from d i a b e t i c r a t s to 3xlO"^M PDB, a concen t ra t i on producing the maximum response, were s i g n i f i c a n t l y g rea te r than c o n t r o l . S tau ro spo r ine , o (5xlO~°M) a l s o produced marked i n h i b i t i o n o f c o n t r a c t i o n s o f both a r t e r i e s from con t r o l and d i a b e t i c r a t s to PDB, w i th g rea te r antagonism o f responses o f mesenter ic a r t e r i e s . The i n h i b i t o r y e f f e c t of s t au ro spo r i ne abo l i shed the d i f f e r e n c e i n c o n t r a c t i l e responses to PDB found between c o n t r o l and d i a b e t i c mesenter ic a r t e r i e s . Con t rac t i on s o f a r t e r i e s induced by KC1 were a l s o measured i n the presence o f s tau ro spor ine i n order to assess the s p e c i f i c i t y of the a c t i o n o f the i n h i b i t o r i n antagon iz ing PKC-mediated responses to NA and PDB (F igure 29). The magnitudes of c o n t r a c t i o n s o f unt reated a r t e r i e s from c o n t r o l and d i a b e t i c r a t s to KC1 were s i m i l a r . In c on t r a s t to i t s a c t i o n aga ins t NA and PDB, s tau rospor ine (5x lO" 8 M) caused r e l a t i v e l y l e s s i n h i b i t i o n of c o n t r a c t i o n s induced by KC1 (8x lO" 2 M) i n both aortae and mesenter ic a r t e r i e s from con t r o l and d i a b e t i c an imal s . In the 93 presence o f s t au ro spo r i ne , no d i f f e r e n c e s were observed i n c o n t r a c t i l e responses o f a r t e r i e s from con t r o l and d i a b e t i c r a t s to KC1. 3.9. EFFECTS OF EXTRACELLULAR CALCIUM REMOVAL ON PDB RESPONSES To assess the r e l a t i v e importance o f e x t r a c e l l u l a r C a 2 + i n PKC-mediated c o n t r a c t i l e responses i n c on t r o l and d i a b e t i c blood v e s s e l s , responses to PDB were a l so obta ined i n C a 2 + - f r e e s o l u t i o n c on t a i n i n g 1 mM EGTA f o l l o w i n g a 30 min e q u i l i b r a t i o n pe r i od i n t h i s s o l u t i o n . PDB produced concentrat ion-dependent c o n t r a c t i o n s of aortae and mesenter ic a r t e r i e s incubated i n C a 2 + - f r e e medium. In c o n t r a s t , no responses to PDB were obta ined i n p o r t a l ve ins i n the absence o f e x t r a c e l l u l a r C a 2 + (data not shown). The magnitude o f the responses of the a r t e r i e s to PDB under these c o n d i t i o n s was markedly reduced compared to the responses observed i n the presence of 2.5 mM C a 2 + (F igure 30; Table 8 ) . In C a 2 + - f r e e s o l u t i o n , no d i f f e r e n c e s were observed i n the magnitude o f c o n t r a c t i l e responses or s e n s i t i v i t i e s of c on t r o l and d i a b e t i c aortae to PDB. S i m i l a r l y , c o n t r a c t i l e responses and s e n s i t i v i t i e s o f d i a b e t i c mesenter ic a r t e r i e s to PDB i n C a 2 + - f r e e s o l u t i o n d i d not d i f f e r from those i n c o n t r o l ve s se l s (F igure 30; Table 8 ) . 3.10. EFFECTS OF CALCIUM CHANNEL BLOCKERS ON PDB RESPONSES In order to assess the i n f l u e n c e of C a 2 + en t r y blockade on c o n t r a c t i l e responses o f a r t e r i e s from con t r o l and d i a b e t i c r a t s to PDB, the e f f e c t s o f the C a 2 + channel b l o c ke r s , verapamil and n i f e d i p i n e , on 94 PDB responses were eva luated i n Krebs s o l u t i o n c on t a i n i n g 2.5 mM Ca^ (F igure 31) . C o n t r a c t i l e responses of mesenter ic a r t e r i e s , but not ao r t ae , from d i a b e t i c r a t s to 3 x l 0 " 6 M PDB were s i g n i f i c a n t l y enhanced compared w i th those from c o n t r o l s i n the absence o f the C a z + channel b l o c k e r s . Pretreatment of a r t e r i e s w i th 3 x l 0 " 6 M verapamil or n i f e d i p i n e r e s u l t e d i n i n h i b i t i o n of responses of both a r t e r i e s from d i a b e t i c and con t r o l r a t s to 3 x l 0 " 6 M PDB, although these e f f e c t s were g r ea te r i n d i a b e t i c mesenter ic p repa ra t i on s . The i n h i b i t o r y e f f e c t s o f the C a z + channel b l ocke r s abo l i shed the d i f f e r e n c e between c o n t r o l and d i a b e t i c mesenter ic a r t e r i e s i n c o n t r a c t i l e responses to PDB. 3.11. CONTRACTILE RESPONSES TO SODIUM FLUORIDE PLUS ALUMINIUM CHLORIDE C o n t r a c t i l e responses of aortae and mesenter ic a r t e r i e s to NaF i n the presence of 10" 5 M AlC I3 were measured to determine i f G - p r o t e i n -mediated c o n t r a c t i o n s are a l t e r e d i n a r t e r i e s from d i a b e t i c r a t s . A combinat ion o f NaF and A lC I3 e l i c i t e d a s l ow ly deve lop ing c o n c e n t r a t i o n -dependent c o n t r a c t i o n o f a r t e r i e s from both c on t r o l and d i a b e t i c r a t s i n the presence o f 2.5 mM C a z + (F igure 32) . In both p r epa r a t i on s , the magnitude o f the maximum response but not s e n s i t i v i t y was s i g n i f i c a n t l y g r ea te r i n d i a b e t i c than i n con t r o l a r t e r i e s (Table 9 ) . NaF (3x 10~ZM) p lus 10"^M AlC I3 a l s o induced c o n t r a c t i o n o f both c o n t r o l and d i a b e t i c a r t e r i e s i n C a z + - f r e e medium (F igure 33) . However, the magnitude of the c o n t r a c t i o n generated under these c o n d i t i o n s was markedly reduced compared w i th the responses obta ined i n the presence of 9 5 Table 9. NaF pD2 values and maximum contracti le responses in the presence of 10" 5M AT CI 3 in aortae and mesenteric arteries from control and diabetic rats. pD£ values Maximum c o n t r a c t i l e ~ response (q/mrn^)" Contro l D i a b e t i c Cont ro l D i a b e t i c Ao r ta 2.24±0.20 2.25±0.13 2.46±0.21 3.13±0.17* Mesenter ic a r t e r y 2.10±0.11 2.24±0.15 2.91±0.40 4 .78±0.52* Values are the mean ± SEM of 9-10 ob se rva t i on s . S i g n i f i c a n t l y d i f f e r e n t from corresponding con t r o l (P < 0.05) . 96 e x t r a c e l l u l a r Ca . In the Ca - f r e e medium, the c o n t r a c t i l e responses were a l so s i g n i f i c a n t l y g rea te r i n a r t e r i e s from d i a b e t i c r a t s than i n c on t r o l p repara t ions (F igure 33) . 97 F igure 3 Concentrat ion- response curves f o r NA-induced c o n t r a c t i o n s of (a) ao r tae , (b) mesenter ic a r t e r i e s and (c) p o r t a l ve ins from con t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po in t represent the mean ± SEM o f 10-12 ob se r va t i on s . 98 a 6.0 5.0 4.0 3.0 2.0 1.0 0.0 6.0 5.0 4.0 3.0 T T • 1 1 J/1 T/* - X J 5-?"? 2.0 A ° 'S o 1.0 -j 1 i 0.0 » St i ]AJ J/i O -©«n^B 1 1 t_ 6.0 5.0 A 4.0 A 3.0 2.0 1.0 0.0 • 4 1 I o 1 o o 9 8 7 6 5 4 9 8 7 6 5 4 NORADRENALINE (- log M) • • f 1 1 1 1 9 8 7 6 5 4 Figure 4 The t ime-course of the c o n t r a c t i l e response to 10'^M NA i n aortae from con t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po in t represents the mean ± SEM o f 8 ob se r va t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 1 0 0 1 0 1 F igure 5 The t ime-course of the c o n t r a c t i l e response to 3xlO"^M NA i n mesenter ic a r t e r i e s from con t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po in t represents the mean ± SEM o f 9 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 102 103 F igure 6 Concentrat ion- reponse curves f o r PE-induced c o n t r a c t i o n s o f (a) aortae and (b) mesenter ic a r t e r i e s from c o n t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po i n t represent s mean ± SEM of 6-11 ob se rva t i on s . 104 E E c o c 7.0 n 6.0' 5.0 4,0 3.0 2.0 1.0 0.0 7.0 n r/ 9 8 7 6. 5 4 PHENYLEPHRINE (-log M) F igure 7 Concentrat ion- response curves f o r KC1-induced c o n t r a c t i o n s o f (a) aortae and (b) mesenter ic a r t e r i e s from c o n t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po in t represent s the mean ± SEM o f 4-6 ob se r va t i on s . 1 0 6 Tension (g/mm2) Figure 8 Concentrat ion- response curves f o r CaC l^ - induced c o n t r a c t i o n s of aortae from con t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s i n the presence of (a) 4xl0~2 M KC1 and (b) 10"^M NA. Each po in t represents mean ± ' SEM of 4-6 ob se r va t i on s . 108 F igure 9 Concentrat ion- response curves f o r C a C l i n d u c e d c o n t r a c t i o n s of p o r t a l ve ins from con t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s i n the presence of 4x lO " 2 M KC1. Each po i n t represents the mean ± SEM o f 6 ob se r va t i on s . 1 1 0 Tension (g/mm2) F igure 10 C o n t r a c t i l e responses of aortae from con t r o l (open columns) and d i a b e t i c (hatched columns) r a t s to NA i n (a) Ca^ - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA and (b) f o l l o w i n g a d d i t i o n of 3.5 mM CaCl2- Each column represent s the mean ± SEM o f 6-11 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 112 F igure 11 C o n t r a c t i l e responses of aortae from con t r o l (open columns) and d i a b e t i c (hatched columns) r a t s to maximum concen t ra t i on s o f PE (10 ' 5 M) and METH (3x lO ' 5 M) i n (a) C a 2 + -f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA and (b) f o l l o w i n g a d d i t i o n of 3.5 mM CaCl2- Each column represents the mean ± SEM o f 11-12 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 114 1 1 5 Figure 12 C o n t r a c t i l e responses of mesenter ic a r t e r i e s from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s to NA i n (a) C a 2 + - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA and (b) f o l l o w i n g a d d i t i o n o f 3.5 mM C a C ^ . Each column represent s the mean ± SEM of 4-11 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 1 1 6 117 Figure 13 C o n t r a c t i l e responses of mesenter ic a r t e r i e s from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s to maximum concent ra t i on s of PE (3xlO~ 5M) and METH (10'^M) i n (a) C a d - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA and (b) f o l l o w i n g a d d i t i o n of 3.5 mM CaCl2• Each column represent s the mean ± SEM of 11 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 118 Tension (g/mm2) F igure 14 Time-course of NA (10 " 5 M)- induced [ 3 2 P ] l a b e l l i n g of Ptd Ins(4,5)P2 and PA i n aortae from con t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . Each column represent s the mean ± SEM of 5-9 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 1 2 0 1 2 1 Figure 15 Time-course of NA (10 '^M)- induced product ion o f t o t a l [ 3 H ] -i n o s i t o l phosphates i n aortae from con t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . Each column represent s the mean ± SEM o f 5-10 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 122 1200 en E \ E CL O CO c u o JZ CL V) o sz Q . O c 900 600 -300 -s 1 I 0.5 1 T i m e ( m i n ) I I 20 123 Figure 16 E f f e c t s of yohimbine (YOH, 10" 7M) and p razo s i n (PZ, 10" 7M) on NA (10 " 5 M)- induced t o t a l [ 3 H ] - i n o s i t o l phosphate p roduct ion i n aortae from con t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . T i s sues were p r e t r ea ted w i th antagon i s t s f o r 15 min before i ncuba t i ng them w i t h NA f o r 20 min. Each column represents mean ± SEM o f 5-15 ob se rva t i on s . S i g n i f i c a n t l y d i f f e r e n t from correspond ing c o n t r o l . 124 CD E \ E Q . o CO c u Q . CO o . CL CO o c 1200 900 -600 300 Basal NA Y0H + NA PZ + NA 1 2 5 Figure 17 Time-course of NA (3x lO " 5 M)- induced product ion o f t o t a l [ H ] - i n o s i t o l phosphates i n mesenter ic a r t e r i e s from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . Each column represents mean ± SEM of 6 ob se r va t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 126 2500 E D l CO <1> a sz CL CO o Q . CO o c 2000 -1500 1000 500 0 1 s 0 0.5 TIME (min) 20 127 F igure 18 E f f e c t o f p razo s i n (PZ, 10" 7M) on NA (3x lO " 5 M)- induced t o t a l [ J H ] - i n o s i t o l phosphate product ion i n mesenter ic a r t e r i e s from con t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . T i s sues were p re t r ea ted w i th p razo s i n f o r 15 min before i ncuba t i ng them wi th NA f o r 20 min. Each column represent the mean ± SEM of 6 ob se r va t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 128 129 Figure 19 Time-course of NA (10"^M)-induced product ion o f t o t a l [ 3 H ] -i n o s i t o l phosphates i n p o r t a l ve ins from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . Each column represents the mean ± SEM of 5 ob se r va t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 1 3 0 Inositol phosphates (cpm/mg) o o o In o < -x-O O O o o o o o o T en o o o Figure 20 Time-course of I n s ( l , 4 ,5 )P3 product ion and c o n t r a c t i o n i n response to 10"^M NA i n aortae from c o n t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po in t represent s the * mean ± SEM of 5-15 ob se rva t i on s . S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 132 133 F igure 21 Time-course of I n s ( l , 4 , 5 )P3 product ion and c o n t r a c t i o n i n response to 10"°M NA i n aortae from con t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po in t represent s the mean ± SEM of 4 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 134 F igure 22 Concentrat ion- response curves f o r NA-induced I n s ( l , 4 , 5 )P3 product ion i n aortae from con t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Contro l t i s s u e s were incubated w i th NA f o r 20 sec and d i a b e t i c t i s s u e s f o r 30 sec. Each po i n t represent s the mean ± SEM of 7-9 ob se r va t i on s . S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 1 3 6 137 Figure 23 Time-course of NA (3xlO"^M)- induced p roduc t i on o f I n s ( l , 4 , 5 ) P 3 i n mesenter ic a r t e r i e s from c o n t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po i n t represent s the mean ± SEM of 4 ob se r va t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 138 7 139 Figure 24 Concentrat ion-dependent I n s ( l , 4 ,5 )P3 p roduct ion i n response to NA i n mesenter ic a r t e r i e s from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . T i s sues were incubated w i th NA f o r 20 sec. Each column represent s the mean ± SEM of 4 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c on t r o l 1 4 0 141 Figure 25 Basal I n s ( l , 4 ,5 )P3 l e v e l s i n po r t a l ve ins from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . Each column represents the mean ± SEM o f 4 ob se r va t i on s . 142 21 CP E o E D_ 1 -F igure 26 Concentrat ion- response curves f o r PDB-induced c o n t r a c t i o n s of (a) ao r tae , (b) mesenter ic a r t e r i e s and (c) p o r t a l ve ins from con t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s . Each po in t represents the mean ± SEM of 9-10 ob se rva t i on s . 144 Tension ( g / m m 2 ) 1 4 5 F igure 27 E f f e c t s of s tau rospor ine (STAU) on NA-induced c o n t r a c t i o n s o f (a) aortae and (b) mesenter ic a r t e r i e s from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . T i s sues were incubated w i th 5x lO " 8 M s taurospor ine f o r 25 min before the a d d i t i o n of NA (10~^M i n aortae or 3xlO"^M i n mesenter ic a r t e r i e s ) . Each column represents the mean ± SEM of 8-9 ob se r va t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 1 4 6 147 F igure 28 E f f e c t s of s tau rospor ine (STAU) ond PDB-induced c o n t r a c t i o n s of (a) aortae and (b) mesenter ic a r t e r i e s from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . T i s sues were incubated w i th 5xlO"^M s taurospor ine f o r 25 min before the a d d i t i o n to 3xlO"^M PDB. Each column represents the mean ± SEM of 5-10 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 148 149 Figure 29 E f f e c t s of s tau rospor ine (STAU) on KC1-induced c o n t r a c t i o n s of (a) aortae and (b) mesenter ic a r t e r i e s from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . T i s sues were incubated w i th 5xlO"^M s taurospor ine f o r 25 min before the a d d i t i o n of 8x l 0~ 2 M KC1. Each column represents the mean ± SEM of 5-10 obse rva t i on s . 1 5 0 1 5 1 F igure 30 Concentrat ion- response curves f o r PDB-induced c o n t r a c t i o n s of (a) aortae and (b) mesenter ic a r t e r i e s from c o n t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s i n C a 2 + - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA. Each po in t represent s the mean + SEM o f 6 ob se rva t i on s . 152 Tension ( g/mm2) o b o In 00 o 9 \ IE O CJJ o I o CO P > —1 m o o In - e - n i o o II o Ln 153 F igure 31 E f f e c t s of verapamil (VER) and n i f e d i p i n e (NIF) on PDB-induced c o n t r a c t i o n s of (a) aortae and (b) mesenter ic a r t e r i e s from con t r o l (open columns) and d i a b e t i c (hatched columns) r a t s . T i s sues were incubated w i th 3x lO " 6 M of the b l ocke r s f o r 15 min before the a d d i t i o n of 3x lO " 6 M PDB. Each column represents the mean ± SEM of 8-16 ob se r va t i on s . S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 154 1 5 5 F igure 32 Concentrat ion- response curves f o r NaF-induced c o n t r a c t i o n s of (a) aortae and (b) mesenter ic a r t e r i e s from c o n t r o l (open c i r c l e ) and d i a b e t i c ( f i l l e d c i r c l e ) r a t s i n the presence of 10'^M Al CI3. Each po in t represents the mean ± SEM o f 9 ob se r va t i on s . 156 Tension (g/mm ^ 157 Figure 33 C o n t r a c t i l e responses to 3x 10" 2 M NaF p lus 10~ 5M AICI3 i n (a) aortae and (b) mesenter ic a r t e r i e s from c o n t r o l (open columns) and d i a b e t i c (hatched columns) r a t s i n C a d - f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA. Each column represent s the mean ± SEM of 6 ob se rva t i on s . * S i g n i f i c a n t l y d i f f e r e n t from corresponding c o n t r o l . 158 Tension (g/mm2) DISCUSSION Contractile responses The r e s u l t s of the present i n v e s t i g a t i o n demonstrate t ha t aortae and mesenter ic a r t e r i e s from male r a t s w i th STZ-induced d iabetes are more respons ive to the c o n t r a c t i l e e f f e c t s o f NA than are the corresponding a r t e r i e s from age-matched con t r o l r a t s . Th i s obse rva t ion i s c o n s i s t e n t w i th prev ious r epo r t s from t h i s l a b o r a t o r y (MacLeod, 1985; H a r r i s and MacLeod, 1988) and w i th the r e s u l t s o f o ther i n v e s t i g a t o r s who used i n t a c t animals as we l l as i s o l a t e d t i s s u e s from va r ious exper imental d i a b e t i c models (Brody and Dixon, 1964; Jackson and C a r r i e r , 1981; Scarborough and C a r r i e r , 1984; Agrawal et a l . , 1987; Agrawal and M c N e i l l , 1987; White and C a r r i e r , 1988). The inc reases i n t en s i on i n response to NA i n d i a b e t i c a r t e r i e s observed i n the present study were not accompanied by changes i n r e a c t i v i t y to KC1 or o f K + -depo l a r i z ed p repara t i ons to increased e x t r a c e l l u l a r C a 2 + . There fo re , the enhanced respons iveness of the d i a b e t i c a r t e r i e s to NA does not appear to r e s u l t from a gene ra l i z ed increase i n the c o n t r a c t i l i t y of the a r t e r i a l smooth muscle but r a t he r r e f l e c t s more s e l e c t i v e changes i n the events l ead i ng to the NA response. S ince the i nc rease i n t en s i on of a r t e r i e s from d i a b e t i c r a t s i n response to NA occurred i n the absence of any a l t e r a t i o n s i n s e n s i t i v i t y (expressed as the pD2 va lue) to t h i s agon i s t , i t appears u n l i k e l y tha t a change i n the d e n s i t y or the a f f i n i t y of the ce-adrenoceptor themselves occurred i n blood ve s se l s from 160 d i a b e t i c r a t s . A more l i k e l y p o s s i b i l i t y i s t ha t there i s an a l t e r a t i o n i n the coup l i ng o f the a - recep to r s to the m o b i l i z a t i o n o f C a 2 + . However, t h i s should be conf i rmed by measuring the den s i t y and a f f i n i t y of these recepto r s by r a d i o l i g a n d b ind ing assay i n the c on t r o l and d i a b e t i c blood v e s s e l s . The r e s u l t s of the present i n v e s t i g a t i o n a l s o show tha t i n c on t r a s t to the response of aortae and mesenter ic a r t e r i e s , c o n t r a c t i l e responses of p o r t a l ve ins from con t r o l and d i a b e t i c r a t s to NA were not d i f f e r e n t from each o the r . Th is obse rva t ion i s a l s o i n agreement w i th prev ious r epo r t s from t h i s and other l a b o r a t o r i e s (Tur lapaty e t a l . , 1980; MacLeod and M c N e i l l , 1985). These r e s u l t s suggest t ha t the d i abe te s - i nduced a l t e r a t i o n s i n c o n t r a c t i l i t y of blood ve s se l s i n response to NA are s p e c i f i c to a r t e r i a l p r epa ra t i on s . I t i s g e n e r a l l y agreed tha t a-adrenoceptor a gon i s t s , i n c l u d i n g NA, produce t h e i r e f f e c t s by s t i m u l a t i n g a\-adrenoceptors i n r a t ao r ta and mesenter ic a r t e r y , and there i s l i t t l e evidence f o r f u n c t i o n a l a 2 -recepto r s i n these t i s s u e s (Digges and Summers, 1983; Agrawal e t a l . , 1984). Based on the dete rminat ion of pA 2 va lues f o r a\- and a 2 -s e l e c t i v e antagon i s t s i n the presence of va r i ous a -adrenoceptor agon i s t s i n c l u d i n g NA, r e c e n t l y i t was demonstrated tha t the enhanced responses of a r t e r i e s from d i a b e t i c r a t s to a-adrenoceptor agon i s t s are mediated v i a the same popu la t i on of recepto r s as those present i n a r t e r i e s from nond iabe t i c r a t s , which have the c h a r a c t e r i s t i c s o f a\-adrenoceptors (Abebe et a l . , 1990). The f i n d i n g s i n the present i n v e s t i g a t i o n that c o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s to the s e l e c t i v e 1 6 1 a\-adrenoceptor agon i s t s , PE and METH, were g r ea te r i n d i a b e t i c than i n c o n t r o l p repa ra t i on s are c on s i s t en t w i th these ob se r va t i on s . A c t i v a t i o n of v a s cu l a r smooth muscle i n response to c q -adrenoceptor s t i m u l a t i o n i s a s soc i a ted w i th i nc reases i n the concen t r a t i on o f i n t r a c e l l u l a r f r ee C a 2 + , and the r i s e i n f r e e C a z + concen t r a t i on i s a major determinant o f the t en s i on developed (Bo l t on , 1979). I t i s g e n e r a l l y be l i e ved tha t s t i m u l a t i o n o f v a s cu l a r c q -adrenoceptors r e s u l t s in m o b i l i z a t i o n of C a 2 + v i a two pathways : the r e l ea se of C a 2 + from i n t r a c e l l u l a r s to res and the i n f l u x of e x t r a c e l l u l a r C a 2 + through ROC (Cauvin et a l . , 1982; Cauvin and Ma l i k , 1984). The r e l ea se of C a 2 + from i n t r a c e l l u l a r s to re s i s thought to be r e spon s i b l e f o r the i n i t i a l f a s t phas ic component, w h i l e the i n f l u x of e x t r a c e l l u l a r C a 2 + c o n t r i b u t e s to the s lower, su s ta ined component o f the c o n t r a c t i l e response of a r t e r i a l smooth muscle to a-adrenoceptor agon i s t s i n c l u d i n g NA. In the present i n v e s t i g a t i o n , the t ime-cour se of the c o n t r a c t i l e response to NA demonstrated tha t wh i l e both components o f the c o n t r a c t i o n cou ld be detected i n both aortae and mesenter ic a r t e r i e s , the magnitudes of both components were g r ea te r i n a r t e r i e s from d i a b e t i c r a t s than i n c on t r o l p r epa ra t i on s . However, s i n ce both responses i n these experiments were measured i n the presence o f e x t r a c e l l u l a r C a 2 + , the r e l a t i v e importance o f C a 2 + from i n t r a c e l l u l a r and e x t r a c e l l u l a r sources to the c o n t r a c t i l e responses cou ld not be de f i ned p r e c i s e l y based on these da ta . There fo re , i n subsequent exper iments, the c o n t r i b u t i o n of i n t r a c e l l u l a r C a 2 + r e l e a s e and i n f l u x 162 of e x t r a c e l l u l a r Ca^ to the enhanced c o n t r a c t i o n s observed i n a r t e r i e s from d i a b e t i c r a t s were assessed. C o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s to NA, PE and METH cou ld be detec ted i n C a 2 + -f r e e Krebs s o l u t i o n con ta i n i n g 1 mM EGTA. Th i s c o n t r a c t i o n i s be l i e ved to r e s u l t from the r e l ea se o f C a 2 + from i n t r a c e l l u l a r s t o r e s , probably from the SR (Deth and Van Breemen, 1974; Karak i e t a l . 1979; T u r l a and Webb, 1990). The magnitude o f c o n t r a c t i l e responses o f both aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s i n C a 2 + - f r e e medium to maximal concen t r a t i on o f each agon i s t t e s t ed was s i g n i f i c a n t l y g r e a t e r than the corresponding response i n c on t r o l p r epa ra t i on s . S i m i l a r r e s u l t s were obta ined i n aortae from d i a b e t i c r a t s i n response to a submaximum concen t r a t i on o f NA (10"^ M), although no s i g n i f i c a n t i nc rease i n c o n t r a c t i l e response to t h i s concen t ra t i on o f NA cou ld be detected i n d i a b e t i c mesenter ic a r t e r i e s i n the absence o f e x t r a c e l l u l a r C a 2 + . These f i n d i n g s suggest tha t the r e l ea se of C a 2 + from i n t r a c e l l u l a r s to re s i s enhanced i n a r t e r i e s from d i a b e t i c r a t s at l e a s t i n response to maximal concen t ra t i on s o f a-adrenoceptor agon i s t s . I t i s l i k e l y t ha t t h i s enhanced r e l e a s e of C a 2 + c on t r i bu te s to the inc reased c o n t r a c t i l e responses o f a r t e r i e s from d i a b e t i c r a t s to a -adrenoceptor s t i m u l a t i o n observed i n t he .p re sence o f e x t r a c e l l u l a r C a 2 + . However, the r e s u l t s a l s o i n d i c a t e t ha t t h i s cannot be the on ly f a c t o r i n vo l ved because (a) c o n t r a c t i l e responses o f d i a b e t i c mesenter ic a r t e r i e s to a submaximal concen t r a t i on o f NA (10"^ M) were s i g n i f i c a n t l y g r ea te r than c on t r o l i n 163 the presence o f e x t r a c e l l u l a r C a z + , but not i n i t s absence, and (b) i f c o n t r a c t i l e responses i n C a 2 + - f r e e medium are expressed as a percentage of corresponding responses obtained i n the presence o f e x t r a c e l l u l a r 2+ Ca , f o r each o f the agon i s t s used, the va lues are e i t h e r s i m i l a r or s l i g h t l y l e s s i n d i a b e t i c than i n c on t r o l a r t e r i e s . For i n s t ance , the response of c o n t r o l and d i a b e t i c aortae to 10" 5 M NA i n the absence of e x t r a c e l l u l a r C a 2 + was 54 ± 4% and 47 ± 3%, r e s p e c t i v e l y , o f the c o n t r a c t i l e responses measured i n the presence o f C a 2 + . Th i s ob se rva t i on suggests t ha t even when the re l ea se of i n t r a c e l l u l a r C a 2 + i s i nc reased i n a r t e r i e s from d i a b e t i c r a t s compared w i th c o n t r o l s , i t c o n t r i b u t e s p r o p o r t i o n a l l y the same or l e s s to the o v e r a l l c o n t r a c t i l e responses i n d i a b e t i c than i n c o n t r o l t i s s u e s i n the presence o f e x t r a c e l l u l a r C a 2 + . There fo re , i t appears that e x t r a c e l l u l a r C a 2 + i s necessary f o r the f u l l expres s ion of enhanced c o n t r a c t i l e responses of a r t e r i e s from d i a b e t i c r a t s to cq-adrenoceptor s t i m u l a t i o n by agon i s t s l i k e NA. Th i s suggests tha t the i n f l u x o f e x t r a c e l l u l a r C a 2 + i n response to t h i s s t i m u l a t i o n may a l s o be inc reased i n d i a b e t i c a r t e r i e s compared w i t h c o n t r o l p r epa r a t i on s . I t appears that the increased i n f l u x o f e x t r a c e l l u l a r C a 2 + i n d i a b e t i c a r t e r i e s i s s p e c i f i c a l l y a s soc i a ted w i t h ROC but not w i th VOC s i nce c o n t r a c t i l e responses of d i a b e t i c a r t e r i e s t o KC1 or K + -depo l a r i z ed p repa ra t i on s from d i a b e t i c r a t s to i n c r e a s i n g concen t r a t i on s o f e x t r a c e l l u l a r C a 2 + were not a l t e r e d . Although i n f l u x o f e x t r a c e l l u l a r C a 2 + has been shown to c o n t r i b u t e to the su s ta ined c o n t r a c t i l e responses of v a s cu l a r smooth muscle to a-adrenoceptor agon i s t s ( Bo l t on , 1979), at 164 present there i s no general agreement as to the mechanism(s) by which t h i s process i s brought about. D i r e c t a c t i v a t i o n o f PKC has been shown to promote the i n f l u x of C a 2 + i n v a s cu l a r smooth muscle (Gleason and F l a im, 1986; Chiu et a l . , 1987). Furthermore, ev idence has been presented demonstrat ing tha t i n a r t e r i a l smooth muscle C a 2 + i n f l u x i n response to a j - ad renocepto r s t i m u l a t i o n i s at l e a s t p a r t i a l l y independent of PtdIns(4,5)P2 h y d r o l y s i s (Chiu et a l . , 1987) and PKC a c t i v a t i o n ( K h a l i l and Van Breemen, 1988). A l t e r n a t i v e l y , i t has been suggested tha t C a 2 + i n f l u x may be c o n t r o l l e d by d i r e c t a c t i v a t i o n of C a 2 + channels by G-prote ins (Minneman, 1988). Based on r e s u l t s obta ined us ing nonvascular t i s s u e s , i t has a l so been proposed t ha t products of phospho ino s i t i de breakdown such as I n s ( l , 3 ,4 ,5 )P4 may be i nvo l ved i n C a 2 + i n f l u x i n v a s cu l a r smooth muscle i n response to a\-adrenoceptor s t i m u l a t i o n ( I r v i n e and Moor, 1986; Minneman, 1988; P i j uan and L i t o s c h , 1988; McGrath et a l . , 1990). Phosphoinositide metabolism As noted i n the I n t r oduc t i on , i t i s now w ide l y accepted t ha t the h y d r o l y s i s o f phospho inos i t ides i s i nvo l ved i n v a s cu l a r smooth muscle c o n t r a c t i o n i n response to a\-adrenoceptor s t i m u l a t i o n . The products of t h i s h y d r o l y s i s , p a r t i c u l a r l y I n s ( l , 4 , 5 )P3 and DG have been suggested to be i nvo l ved i n mediat ing both the i n i t i a l phase and the delayed su s ta ined components o f the c o n t r a c t i l e responses. In view o f the observed inc rease i n c o n t r a c t i l e respons iveness o f a r t e r i e s from 1 6 5 d i a b e t i c r a t s to cq-adrenoceptor s t i m u l a t i o n , the p o s s i b i l i t y t ha t the phosopho inos i t i de messenger system i s augmented i n these t i s s u e s was i n v e s t i g a t e d . I n i t i a l l y we used the method o f l a b e l l i n g i n o s i t o l phospho l ip id s w i t h [ 3 2 P ] and/or [ 3 H] -myo i no s i t o l i n order to examine phospho ino s i t i de metabolism by measuring the breakdown o f [ 3 2 P ] - P t d I n s ( 4 , 5 ) P 2 , and format ion of [ 3 2 P ] - P A and/or t o t a l [ 3 H ] - i n o s i t o l phosphates. The r e s u l t s show tha t i n unst imulated aortae the l a b e l l i n g of phospho inos i t i de s w i th [ 3 2 P ] and [ 3 H] -myo i no s i t o l was s i m i l a r i n p repa ra t i on s from con t r o l and d i a b e t i c r a t s . The basal va lues of [ P I -PA and [ 3 H ] - i n o s i t o l phosphates a l s o showed t ha t aortae from both groups o f r a t s were i n a s i m i l a r r e s t i n g s t a t e . These observat ions suggest tha t the pool s i z e of the phospho inos i t i des l a b e l l e d w i th [ 3 2 P ] and [ 3 H ] -m y o i n o s i t o l , and the turnover of these phospho l ip id s i n the unst imulated aortae d i d not d i f f e r between the two groups of an imal s . On a d d i t i o n of 10"^M NA to aortae from c o n t r o l r a t s , there was a r a p i d ( w i t h i n 0.5 min) decrease i n the l e v e l of Ptd Ins(4,5)P2 l a b e l l i n g , w h i l e at the same time the product ion of [ 3 2 P ] - P A i n c rea sed . Th i s was accompanied by increased product ion o f [ 3 H ] - i n o s i t o l phosphates. These data are g e n e r a l l y i n agreement w i th prev ious r epo r t s (Legan et a l . , 1985; Campbell et a l . , 1986; Heagerty et a l . , 1986; Chiu et a l . , 1 987 ; P i j uan and L i t o s c h , 1988) and suggest tha t NA a c t i v a t e d PLC i n aortae from c o n t r o l r a t s , l ead ing to the breakdown of Ptd Ins(4,5)P2 and format ion o f p h y s i o l o g i c a l l y a c t i v e metabo l i c products i n c l u d i n g 166 I n s ( l , 4 , 5 )P3 and DG. However, the magnitude o f the decrease i n Ptd Ins(4,5)P2 l a b e l l i n g i n response to NA found i n aortae i n the present i n v e s t i g a t i o n was l e s s than has p r e v i o u s l y been repor ted (Campbell et a l . , 1986; P i j uan and L i t o s c h , 1988). The reason f o r t h i s d i f f e r e n c e between our r e s u l t s and those o f others i s not c l e a r ; however, i t may be r e l a t e d to d i f f e r e n c e s i n exper imental c ond i t i o n s used. In a d d i t i o n , our data may r e f l e c t the inherent d i f f i c u l t i e s a s soc i a ted w i th d e t e c t i o n of Ptd Ins(4,5)P2 breakdown, s i nce t h i s i s u s u a l l y accompanied by compensatory r e s yn the s i s ( I r v i n e , 1986). Although DG i s known to be produced by h y d r o l y s i s of other phospho l ip id s (eg. p h o s p h a t i d y l c h o l i n e ) , i t i s u n l i k e l y tha t t h i s c on t r i bu ted to the inc reased [ 3 2 P ] - P A , because the time course of t h i s e f f e c t has been shown to be s lower than t ha t of DG product ion by PtdIns(4,5)P2 h y d r o l y s i s ( L o f f e l h o l z , 1989). Our f i n d i n g s are c on s i s t en t w i th prev ious r epo r t s demonstrat ing tha t on ly a t r a n s i e n t i nc rease i n the h y d r o l y s i s o f [ 3 2 P ] - P t d I n s ( 4 , 5 ) P 2 i n response to NA i n a r t e r i e s can be detected (Campbell et a l . , 1985; P i j uan and L i t o s c h , 1988). However, our observat ion tha t the p roduct ion o f t o t a l [ 3 H ] - i n o s i t o l phosphates i n the presence o f the i n o s i t o l monophosphate phosphatase i n h i b i t o r , L i C l , cont inued to i nc rease f o r much longer per iods (at l e a s t 20 min) suggests tha t the pathway l e ad i n g to the fo rmat ion of these metabo l i t e s i s s t i l l a c t i v e dur ing t h i s t ime. L i C l i s w ide l y used to f a c i l i t a t e the measurement o f i n o s i t o l phosphates (Ber r idge et a l . , 1982; Fox et a l . , 1985; Legan et a l . , 1985; Rapoport, 1987). As the NA-induced increase i n [ 3 H ] - i n o s i t o l phosphate p roduct ion 167 was markedly reduced by the cq-adrenoceptor an tagon i s t , p r a z o s i n , but not by the a^-adrenoceptor an tagon i s t , yohimbine, i t appears to be the consequence o f s t i m u l a t i o n o f cq -adrenoceptor s , as repor ted i n severa l o ther s t ud i e s (Campbell et a l . , 1985; Legan et a l . , 1 985 ; Chiu et a l . ,1987). NA (10" 5 M) a l so induced a r a p i d decrease i n [ 3 2 P ] - P t d I n s ( 4 , 5 ) P 2 l e v e l s and an inc rease i n [ 3 2 P ] - P A and [ 3 H ] - i n o s i t o l phosphate p roduct ion i n aortae from d i a b e t i c r a t s . While the t ime-cour se o f these e f f e c t s was s i m i l a r to tha t seen i n c on t r o l ao r tae , our r e s u l t s demonstrate tha t both the breakdown of [ 3 2 P ] - P t d I n s ( 4 , 5 ) P 2 , and format ion o f [ 3 2 P ] - P A and [ 3 H ] - i n o s i t o l phosphates were s i g n i f i c a n t l y g r ea te r i n aortae from d i a b e t i c than from c o n t r o l r a t s . Th i s suggests tha t NA induced g rea te r h y d r o l y s i s of Ptd Ins(4,5)P2 i n aortae from d i a b e t i c r a t s , r e s u l t i n g i n increased format ion o f the metabo l i c products o f t h i s pho spho l i p i d . Because the NA-induced [ 3 H ] - i n o s i t o l phosphate p roduct ion i n the d i a b e t i c ve s se l s was reduced nea r l y to basal l e v e l s by p r a z o s i n , i t appears t ha t the g rea te r accumulat ion of [ H] -i n o s i t o l phosphates i n these t i s s u e s i n the presence of NA was mediated v i a cq-adrenoceptor a c t i v a t i o n . As w i th ao r tae , the a d d i t i o n of NA ( 3 x l 0 " 5 M) to mesenter ic a r t e r i e s from con t r o l r a t s induced a r a p i d (w i t h i n 0.5 min) and t ime -dependent i nc rease i n [ 3 H ] - i n o s i t o l phosphate accumulat ion. Th i s i s a l so c o n s i s t e n t w i th the repo r t s of o ther i n v e s t i g a t o r s who used mesenter ic a r t e r i e s (Hashimoto et a l . , 1986; E id and Champlain, 1988). S ince t h i s 168 e f f e c t o f NA was markedly i n h i b i t e d by p r a z o s i n , i t appears to be mediated s p e c i f i c a l l y by a\-adrenoceptors as repor ted p r e v i o u s l y (Hashimoto et a l . , 1986; E id and Champlain, 1988). S i m i l a r l y , NA ( 3 X 1 0 - 5 M) a l s o induced a r a p i d and t i m e - r e l a t e d i nc rease i n [ 3 H ] - i n o s i t o l phosphate accumulat ion i n mesenter ic a r t e r i e s from d i a b e t i c r a t s , w i th e f f e c t s s i g n i f i c a n t l y g r ea te r than those observed i n c o n t r o l p r epa r a t i on s . The NA-induced [ 3 H ] - i n o s i t o l phosphate accumulat ion i n d i a b e t i c mesenter ic a r t e r i e s was blocked by p r a z o s i n , suggest ing t ha t the enhanced [ 3 H ] - i n o s i t o l phosphate p roduct ion i n these t i s s u e s i s a l so mediated v i a a ] -adrenoceptor s t i m u l a t i o n . As the basal [ 3 H ] - i n o s i t o l phosphate accumulat ion i n d i a b e t i c mesenter ic a r t e r i e s was g r ea te r than t ha t o f c o n t r o l p repa ra t i on s , i t i s not c l e a r from these data i f the enhanced product ion o f [ 3 H ] - i n o s i t o l phosphates observed i n d i a b e t i c t i s s u e s i n the presence of NA was due to increased uptake o f [ H ] -m y o i n o s i t o l . The f i n d i n g tha t [ 3 H ] - i n o s i t o l phosphate p roduct ion cou ld be detected as e a r l y as 0.5 min a f t e r NA a d m i n i s t r a t i o n i n mesenter ic a r t e r i e s suggests the occurrence o f e a r l y b iochemica l events i n these t i s s u e s , p o s s i b l y r e s u l t i n g i n the p roduct ion and accumulat ion o f p h y s i o l o g i c a l l y a c t i v e products such as I n s ( l , 4 , 5 ) P3 . The r e l a t i v e l y l a r g e inc rease i n t o t a l [ 3 H ] - i n o s i t o l phosphates measured 20 min a f t e r NA a d m i n i s t r a t i o n was l i k e l y to be mainly due to i n o s i t o l monophosphate accumulat ion as a r e s u l t of i n h i b i t i o n o f i n o s i t o l monophosphate 169 phosphatase by L i C l present i n the i ncubat i on medium (Ber r idge et a l . , 1982; Fox e t a l . , 1985; Legan et a l . , 1985; Rapoport, 1987). The dete rminat ion of t o t a l [ 3 H ] - i n o s i t o l phosphates ( i n the presence of L i C l ) i n p o r t a l ve ins i n response to NA (10"^ M) a l so demonstrated a time-dependent inc rease i n both c o n t r o l and d i a b e t i c p r epa r a t i on s . Although there was a r a p i d (w i t h i n 0.5 min) p roduct ion of [ 3 H ] - i n o s i t o l phosphates i n response to NA i n both c o n t r o l and d i a b e t i c p o r t a l v e i n s , the values measured were not s i g n i f i c a n t l y d i f f e r e n t from each o the r . Th i s suggests tha t the extent of occurrence o f e a r l y b iochemica l events , i n c l u d i n g the h y d r o l y s i s o f Ptd Ins(4,5)P2 and fo rmat ion of I n s ( l , 4 , 5 ) P3 , i s s i m i l a r i n c o n t r o l and d i a b e t i c p o r t a l v e i n s . On the other hand, the obse rva t ion t ha t a s i g n i f i c a n t l y g r ea te r accumulat ion of t o t a l [ 3 H ] - i n o s i t o l phosphates cou ld be measured i n d i a b e t i c than i n c on t r o l p o r t a l ve ins w i th 20 min i n cuba t i on w i th NA may r e f l e c t a g r ea te r format ion of i n o s i t o l phosphates (eg. I n s ( l ) P and P t d l n s ( l , 4 ) P 2 ) o ther than I n s ( l , 4 , 5 ) P3 . These i n o s i t o l phosphates may be produced from Ptd ln s and Ptd Ins(4)P whose h y d r o l y s i s has been repor ted to be delayed compared w i th tha t o f Ptd Ins(4,5)P2 (Chuang, 1989). The r ad i o i s o t ope l a b e l l i n g method employed to measure i n o s i t o l phosphates i s a s soc ia ted w i th c e r t a i n problems. F i r s t , us ing t h i s method, i t i s not p o s s i b l e to s p e c i f i c a l l y measure the t i s s u e contents o f the b i o l o g i c a l l y a c t i v e forms of the i n o s i t o l phosphates. Second, e q u i l i b r i u m l a b e l l i n g of phospho inos i t i de pools cou ld not be a s ce r t a i ned us ing t h i s method. I t i s p o s s i b l e tha t m u l t i p l e pools of 1 7 0 phospho ino s i t i de s w i th d i f f e r e n t r a te s o f l a b e l l i n g w i th r ad i o i s o t ope s may e x i s t i n the p repara t i ons we used. T h i r d , pharmacolog ica l i n t e r v e n t i o n , such as i n h i b i t i o n o f i n o s i t o l phosphate degradat ion by L i C l was r equ i r ed to produce measurable accumulat ion o f i n o s i t o l phosphates. In subsequent exper iments, these problems were c ircumvented by d i r e c t l y measuring the mass of I n s ( l , 4 , 5 )P3 i n un labe led blood ve s se l s us ing a p r o t e i n b ind ing assay system. Th i s method has been found to be s imp le , r a p i d and s e n s i t i v e , and measures the l e v e l s o f I n s ( l , 4 , 5 )P3 w i th minimum i n t e r f e r e n c e from other i n o s i t o l phosphates (Palmer et a l . , 1989). Using t h i s method, a t ime - and c o n c e n t r a t i o n -dependent e f f e c t o f NA on t i s s u e contents o f I n s ( l , 4 , 5 )P3 was determined i n a r t e r i e s from con t r o l and d i a b e t i c r a t s . In a o r t i c p r epa r a t i o n s , t h i s was accompanied by measurement of c o n t r a c t i l i t y i n response to the agon i s t , thus p e r m i t t i n g the assessment o f both b iochemica l and f u n c t i o n a l responses i n the same p repa ra t i on s . Our data show tha t the a d d i t i o n of NA to aortae from c o n t r o l r a t s induced a r a p i d t r a n s i e n t e l e v a t i o n of I n s ( l , 4 , 5 )P3 content dur ing c o n t r a c t i o n . Prev ious s tud ie s us ing t r a chea l smooth muscle ( C h i l v e r s et a l . , 1989; Langlands et a l . , 1989) and c a r d i a c p repa ra t i on s (Heathers et a l . , 1988) have a l s o demonstrated s i m i l a r changes i n the mass o f I n s ( l , 4 , 5 )P3 f o l l o w i n g agon i s t s t i m u l a t i o n . These data suggest that I n s ( l , 4 , 5 )P3 may p lay an important r o l e i n the i n i t i a t i o n o f c o n t r a c t i o n induced by agon i s t s l i k e NA but may not c o n t r i b u t e to the maintenance o f the c o n t r a c t i l e response (Somlyo et a l . , 1988; C h i l v e r s e t a l . , 1989; 1 7 1 Langlands et a l . , 1989). As po inted out e a r l i e r , i nc reased i n f l u x of C a 2 + , p o s s i b l y v i a r e c e p t o r - l i n k e d C a 2 + channels and a c t i v a t i o n o f PKC by DG, which i s produced by h y d r o l y s i s o f Ptd Ins(4,5)P2 and other pho spho l i p i d s , may be r e spon s i b l e f o r the maintenance o f c o n t r a c t i o n . Whi le i nc reased format ion of I n s ( l , 4 ,5 )P3 i n response to NA and other agon i s t s i s known to r e s u l t from a c t i v a t i o n of PLC, the decrease has been suggested to a r i s e from i t s enhanced metabolism to I n s ( l , 4 )P2 and/or I n s ( l , 3 , 4 ,5 )P4 by C a 2 + - a c t i v a t e d I n s ( l , 4 , 5 )P3 5-phosphatase and/or I n s ( l , 4 , 5 )P3 -3 - k i na se present i n the c y to so l (Biden and Wol lheim, 1986; I r v i ne and Moor, 1987; Putney et a l . , 1989). Although a feedback r e gu l a t o r y e f f e c t of PKC i s known to be ope ra t i v e i n v a s cu l a r smooth muscle (Alexander et a l . , 1985), the d e c l i n e i n I n s ( l , 4 ,5 )P3 observed does not appear to be r e l a t e d to a d e s e n s i t i z a t i o n phenomenon. Th i s i s because i n t i s s u e s l a b e l l e d w i th [ 3 H] -myo i no s i t o l and s t imu l a ted w i th NA ( i n the presence o f L i C l ) , [ 3 H ] - i n o s i t o l phosphates accumulated l i n e a r l y w i th t ime over a pe r i od of at l e a s t 20 min. I t should be noted tha t the NA pD2 f o r IP3 p roduct ion i n c on t r o l ao r ta was s i g n i f i c a n t l y l e s s than the agon i s t pD2 va lue f o r c o n t r a c t i o n . Other i n v e s t i g a t o r s (Campbell et a l . , 1985; Fox et a l . , 1985; Rapoport, 1987; Jones et a l . , 1988) have a l so repor ted s i m i l a r d i s c repancy between EC50 va lues f o r NA-induced c o n t r a c t i l e and b iochemica l responses i n v a s cu l a r p r epa ra t i on s . One po s s i b l e exp l ana t i on f o r t h i s d i sc repancy i s t ha t the assays which measure the b iochemical responses are not as s e n s i t i v e as t ha t which de tec t s c o n t r a c t i l i t y changes. On the other 172 hand, the r e s u l t s observed may r e f l e c t the phenomenon of a m p l i f i c a t i o n between second messenger product ion and f u n c t i o n a l responses, t ha t i s , a r e l a t i v e l y small change i n t i s s u e I n s ( l , 4 ,5 )P3 and/or DG may have a l a r ge e f f e c t on c o n t r a c t i l e responses (Jones et a l . , 1988). In t h i s r ega rd , the s y n e r g i s t i c i n t e r a c t i o n between I n s ( l , 4 , 5 ) P 3 / C a 2 + and DG/PKC in producing c o n t r a c t i l e responses i s be l i e ved to p lay an important r o l e . NA a l s o induced a r a p i d t r a n s i e n t e l e v a t i o n o f I n s ( l , 4 , 5 )P3 content dur ing c o n t r a c t i o n of aortae from d i a b e t i c r a t s . However, the t ime-course f o r I n s ( l , 4 ,5 )P3 increase i n response to NA i n these ve s se l s was d i f f e r e n t from tha t recorded i n c on t r o l p r epa r a t i o n s . As 10"^ and 1 0 " 5 M NA induced s i m i l a r pa t te rns of I n s ( l , 4 , 5 )P3 accumulat ion w i th t ime , the a l t e r e d t ime-course of I n s ( l , 4 ,5 )P3 accumulat ion observed i n d i a b e t i c aortae w i th NA a d m i n i s t r a t i o n was not r e l a t e d to the concen t r a t i on o f the agon i s t used. As w i th c on t r o l p r epa r a t i on s , the NA pD 2 va lue f o r I n s ( l , 4 ,5 )P3 product ion i n d i a b e t i c aortae was markedly reduced compared w i th i t s pD 2 va lue f o r c o n t r a c t i o n determined i n these t i s s u e s , r e f l e c t i n g tha t the s e n s i t i v i t i e s to NA f o r c o n t r a c t i o n and I n s ( l , 4 , 5 )P3 product ion are a l so d i f f e r e n t i n the d i a b e t i c ao r tae . Comparison of the NA pD 2 va lue f o r Ins(1,4,5)P3 product ion i n c o n t r o l and d i a b e t i c ao r tae , however, demonstrates tha t there was no s i g n i f i c a n t d i f f e r e n c e i n s e n s i t i v i t y to the agon i s t between the two types of a r t e r i e s . Th is obse rva t ion i s i n agreement w i th the f u n c t i o n a l data repor ted here and p r e v i o u s l y (MacLeod, 1985; White and C a r r i e r , 1988). 173 As i n the c o n t r a c t i l i t y exper iments, maximum I n s ( l , 4 , 5 )P3 accumulat ion i n the present i n v e s t i g a t i o n was s i g n i f i c a n t l y g r ea te r i n d i a b e t i c than i n c on t r o l ao r tae . These f i n d i n g s are a l so i n agreement w i th our observat ions t ha t phospho inos i t i de metabol ism, as assessed by increased h y d r o l y s i s o f [ 3 2 P ] - P t d I n s ( 4 , 5 ) P 2 , and format ion o f [ 3 2 P ] - P A and [ 3 H ] -i n o s i t o l phosphates, was enhanced i n aortae from d i a b e t i c r a t s i n response to 1 0 " 5 M NA. The t ime-course o f a c t i on o f NA determined w i t h r a d i o i s o t o p e l a b e l l i n g and p r o t e i n b ind ing assay methods demonstrates tha t although I n s ( l , 4 , 5 )P3 l e v e l s de c l i n ed a f t e r the i n i t i a l r i s e , the p roduct ion of [ J H ] - i n o s i t o l phosphates i nc rea sed . As mentioned e a r l i e r , wh i l e the d e c l i n e i n the l e v e l s of Ins(1,4,5)P3 may be due to i t s metabol ism p o s s i b l y i n t o I n s ( l , 4 )P2 and/or I n s ( l , 3 , 4 , 5 )P4 , the i nc rease i n the p roduct ion o f [ J H ] - i n o s i t o l phosphates i s be l i e ved to be due to the i n h i b i t i o n o f t h e i r metabolism by L i C l . I t i s l i k e l y t ha t t o t a l [ 3 H ] -i n o s i t o l phosphates detected a f t e r i ncubat i on of t i s s u e s w i th NA f o r 0.5 min ( i n the presence of L i C l ) con s i s t ed of I n s ( l , 4 , 5 )P3 s i nce the inc rease i n [ J H ] - i n o s i t o l phosphates measured at t h i s t ime was accompanied by [ 3 2 P ] - P t d I n s ( 4 , 5 ) P 2 breakdown. In a d d i t i o n , the p roduct ion o f o ther i n o s i t o l phosphates (eg. Ins (1)P and I n s ( l , 4 )P2 ) has been shown to be delayed (Chuang, 1989). Although L i C l at the concen t r a t i on (10 mM) and i ncubat i on pe r iod (15 min) used i n the present study has been repor ted to act on i n o s i t o l monophosphate phosphatase, the re are severa l r epo r t s i n d i c a t i n g tha t i t may a l s o act on other 174 enzyme systems and decrease the metabolism o f I n s ( l , 4 , 5 )P3 (Ber r idge et a l . , 1982; Bat ty and Nahorsk i , 1985). The r e s u l t s o f the present i n v e s t i g a t i o n a l s o demonstrate tha t NA induced r a p i d and concentrat ion-dependent i nc reases i n I n s ( l , 4 ,5 )P3 content i n c o n t r o l mesenter ic a r t e r i e s . These data suggest tha t I n s ( l , 4 , 5 )P3 may a l so p lay an important r o l e i n the i n i t i a t i o n o f t en s i on development by agon i s t s l i k e NA. However, i n c on t r a s t to our data i n aortae and the r e s u l t s of o ther s tud ie s (Heather e t a l . , 1988; C h i l v e r s et a l . , 1989; Langlands et a l . , 1989), a f t e r reach ing a peak 20 sec a f t e r NA a d m i n i s t r a t i o n , the l e v e l s of I n s ( l , 4 , 5 )P3 i n the r a t mesenter ic a r t e r y dec l i n ed on ly s l i g h t l y over the pe r i od o f t ime assayed. Th i s suggests that the generat ion o f I n s ( l , 4 , 5 )P3 i s g r ea te r and / or i t s i n a c t i v a t i o n i s l e s s i n mesenter ic a r t e r y than i n other v a s cu l a r and non-vascu la r p repa ra t i on s . The l a t t e r p o s s i b i l i t y i s supported by the observat ion o f Somlyo et a l . (1985) t ha t the i n a c t i v a t i o n o f exogenously added I n s ( l , 4 ,5 )P3 i n r a b b i t main pulmonary a r t e r i e s i s s low. S ince NA induced c o n t r a c t i l e responses i n mesenter ic a r t e r i e s l a s t i n g more than 10 min, the sus ta ined NA-induced I n s ( l , 4 ,5 )P3 product ion i n these t i s s u e s may a l s o at l e a s t p a r t l y be r e spon s i b l e f o r the su s ta ined c o n t r a c t i o n . Somlyo et a l . (1985) suggested t ha t t o n i c c o n t r a c t i o n i n r a b b i t pulmonary a r t e r i e s can be induced by i n t r a c e l l u l a r C a 2 + r e l e a s e mediated by I n s ( l , 4 ,5 )P3 wi thout the need f o r i n f l u x of e x t r a c e l l u l a r C a 2 + . However, i f I n s ( l , 4 ,5 )P3 i s i n vo l ved i n the NA-induced sus ta ined c o n t r a c t i o n of the r a t mesenter ic a r t e r y , i t appears 1 7 5 to r e q u i r e the presence of e x t r a c e l l u l a r C a ' t o produce t h i s e f f e c t s i nce i n C a 2 + - f r e e medium c o n t r a c t i l e responses o f mesenter ic a r t e r i e s to NA were found to be r a p i d and phas i c . NA a l s o induced a r a p i d and c o n c e n t r a t i o n - r e l a t e d e l e v a t i o n o f I n s ( l , 4 , 5 ) P 3 content i n d i a b e t i c mesenter ic a r t e r i e s , w i t h the peak o c c u r r i n g at a t ime s i m i l a r to tha t o f c o n t r o l a r t e r i e s . I t can be seen t ha t the peak I n s ( l , 4 , 5 ) P 3 time observed i n d i a b e t i c mesenter ic a r t e r y i n response to NA was d i f f e r e n t from tha t observed i n d i a b e t i c ao r tae . The reason f o r t h i s d i f f e r e n c e between the two types o f d i a b e t i c p repa ra t i on s i s not known. The r e s u l t s , however, f u r t h e r demonstrate tha t d i abetes may produce d i f f e r e n t e f f e c t s on d i f f e r e n t v a s cu l a r t i s s u e s . Cons i s tent w i th t h i s , we a l so observed d i f f e r e n t i a l e f f e c t of d iabetes on PKC-mediated c o n t r a c t i l e responses i n aortae and mesenter ic a r t e r i e s (see below). The present r e s u l t s show tha t I n s ( l , 4 , 5 ) P 3 l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t between mesenter ic a r t e r i e s from c o n t r o l and d i a b e t i c r a t s i n response to 1 /JM NA. However, as w i th the ao r tae , the peak I n s ( l , 4 , 5 ) P 3 accumulat ion was s i g n i f i c a n t l y g r ea te r i n mesenter ic a r t e r i e s from d i a b e t i c r a t s than c on t r o l r a t s i n response to a concen t r a t i on o f NA ( 3 X 1 0 - 5 M) producing the maximum c o n t r a c t i l e responses. The r e s u l t s obta ined i n mesenter ic a r t e r y w i th the r a d i o i s o t o p e l a b e l l i n g and p r o t e i n b ind ing assay methods are g e n e r a l l y i n good agreement. Using both methods, basal i n o s i t o l phosphate l e v e l s were found to be h igher i n d i a b e t i c than i n c o n t r o l mesenter ic a r t e r i e s , 1 7 6 although the d i f f e r e n c e i n I n s ( l , 4 ,5 )P3 determined us ing the p r o t e i n b ind ing assay system was not s t a t i s t i c a l l y s i g n i f i c a n t . There fo re , i t appears t ha t the d i f f e r e n c e i n basal and NA-s t imulated [ 3 H ] - i n o s i t o l phosphate accumulat ion found between con t r o l and d i a b e t i c p repa ra t i on s was main ly due to the a v a i l a b i l i t y of d i f f e r e n t t i s s u e concen t ra t i on s o f the metabo l i t e s r a t h e r than to d i f f e r e n t i a l i n c o r p o r a t i o n o f [ 3 H ] -myo ino s i t o l i n the plasma membrane. The obse rva t ion t ha t NA induced a r a p i d i nc rease i n I n s ( l , 4 ,5 )P3 content i n mesenter ic a r t e r i e s suggest t ha t the p roduct ion o f I n s ( l , 4 ,5 )P3 may c o n t r i b u t e to the accumulat ion o f [ ^ - i n o s i t o l phosphates detected 0.5 min a f t e r NA . adm in i s t r a t i on . In a d d i t i o n , the f a c t t ha t the maximum In s ( l , 4 ,5 )P3 produced was g rea te r i n d i a b e t i c mesenter ic a r t e r i e s than i n c on t r o l suggests tha t inc reased p roduct ion of I n s ( l , 4 ,5 )P3 a l so c o n t r i b u t e s to the enhanced accumulat ion o f [ 3 H ] - i n o s i t o l phosphates detected 30 sec a f t e r 3x10"^ M NA a d d i t i o n i n mesenter ic a r t e r i e s from d i a b e t i c r a t s . The r e s u l t s of the present i n v e s t i g a t i o n a l s o show tha t basal I n s ( l , 4 , 5 )P3 measured i n d i a b e t i c po r t a l ve ins was not d i f f e r e n t from tha t measured i n c on t r o l p r epa ra t i on s . Th is i s i n c on t r a s t to the r e s u l t s obta ined w i th the r ad i o i s o t ope l a b e l l i n g method and suggests t ha t the enhanced basal t o t a l [ 3 H ] - i n o s i t o l l i p i d s and [ 3 H ] - i n o s i t o l phosphates observed i n d i a b e t i c po r t a l ve ins was at l e a s t p a r t l y due to inc reased uptake o f [ 3 H] -myo i no s i t o l i n t o these t i s s u e s . The inc reased uptake o f [ 3 H] -myo i no s i t o l i n t o the d i a b e t i c p o r t a l ve ins may c o n t r i b u t e 177 to the inc rease i n [ J H ] - i n o s i t o l phosphates measured i n these p repa ra t i on s i n the presence of NA. Correlation between functional and biochemical responses The r e s u l t s o f our study show tha t pho spho ino s i t i de metabolism was enhanced i n aortae from d i a b e t i c r a t s i n response to 10"^ M NA a c t i n g at -adrenoceptors . Th i s may be r e s p o n s i b l e , at l e a s t i n p a r t , f o r the inc reased c o n t r a c t i l e responses of these t i s s u e s to the agon i s t . I t has been shown tha t the enhanced c o n t r a c t i l e responses o f d i a b e t i c aortae to NA are a l s o mediated by a j -ad renocepto r s t i m u l a t i o n (Abebe et a l . , 1990). The c o n t r a c t i l e responses o f v a s cu l a r smooth muscle incubated i n the absence of e x t r a c e l l u l a r C a 2 + t o NA a c t i n g at a\-adrenoceptors are be l i e ved to r e s u l t from In s ( l , 4 ,5 )P3 - i nduced i n t r a c e l l u l a r C a ' r e l ea se (S t reb et a l . , 1983; Joseph et a l . , 1984; Hashimoto et a l . , 1986; Somlyo et a l . , 1988). In the present study, c on s i s t en t w i th the increased p roduct ion of I n s ( l , 4 ,5 )P3 and [ 3 H ] - i n o s i t o l phosphates, c o n t r a c t i l e responses to 1 0 " 6 and 1 0 " 5 M NA i n the absence of e x t r a c e l l u l a r C a 2 + were s i g n i f i c a n t l y increased i n d i a b e t i c aortae compared to c o n t r o l s . p I Thi s suggests tha t enhanced I n s ( l , 4 ,5 )P3 - i nduced i n t r a c e l l u l a r C a ' r e l ea se may c o n t r i b u t e to the enhanced c o n t r a c t i l e responses of d i a b e t i c aortae to these concent ra t i on s of NA i n the presence o f e x t r a c e l l u l a r C a 2 + . I t should be noted tha t wh i l e peak I n s ( l , 4 ,5 )P3 l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t between con t r o l and d i a b e t i c aortae i n response to 10"^ M NA, there was a more sus ta ined accumulat ion o f I n s ( l , 4 ,5 )P3 i n 178 d i a b e t i c than i n c on t r o l aortae suggest ing g r ea te r p roduct ion of the metabo l i t e i n these t i s s u e s . I t i s t he r e f o r e p o s s i b l e t ha t t h i s may c o n t r i b u t e to the enhanced c o n t r a c t i l e responses found i n aortae from d i a b e t i c r a t s i n response to t h i s concen t ra t i on o f the agon i s t . A c t i v a t i o n o f PKC by DG has been suggested to c o n t r i b u t e to the sus ta ined component of the c o n t r a c t i l e responses to NA mediated v i a c q -adrenoceptor s t i m u l a t i o n (Campbell et a l . , 1985; Hashimoto e t a l . , 1986; Rapoport, 1987). There fo re , the enhanced sus ta ined c o n t r a c t i l e response of the d i a b e t i c aortae to NA i n the presence of e x t r a c e l l u l a r Ca , cou ld r e s u l t at l e a s t i n pa r t from increased a c t i v a t i o n o f PKC by enhanced product ion of DG ( r e f l e c t e d by increased fo rmat ion o f [ 3 2 P ] - P A ) i n these v e s s e l s . Th is conc lu s i on i s supported by our f i n d i n g s tha t the PKC i n h i b i t o r s tau rospor ine abo l i shed the d i f f e r e n c e i n magnitude of su s ta ined c o n t r a c t i l e response o f c on t r o l and d i a b e t i c aortae to NA (see below). The augmented i n o s i t o l phosphate accumulat ion observed i n mesenter ic a r t e r i e s from d i a b e t i c r a t s i n response to 3X10"^ M NA a l so suggests tha t p o t e n t i a t i o n of the phospho ino s i t i de pathway may be r e s p o n s i b l e , at l e a s t i n p a r t , f o r the enhanced maximum c o n t r a c t i l e responses o f these blood ves se l s to NA. As w i th the ao r tae , i n mesenter ic a r t e r y , phas ic c o n t r a c t i o n s induced by NA (mediated v i a a\-adrenoceptor s t i m u l a t i o n ) i n C a 2 + - f r e e medium are a l s o be l i e ved to r e s u l t from i n t r a c e l l u l a r C a 2 + r e l ea se by I n s ( l , 4 ,5 )P3 (Hashimoto et a l . , 1986). In the present i n v e s t i g a t i o n , the phas ic response to 3x10"^ 179 M NA i n the absence o f e x t r a c e l l u l a r C a ' + was s i g n i f i c a n t l y g r ea te r i n d i a b e t i c than i n c on t r o l mesenter ic a r t e r i e s . Th i s i n d i c a t e s t ha t inc reased I n s ( l , 4 ,5 )P3 - i nduced i n t r a c e l l u l a r C a t T r e l e a s e may c o n t r i b u t e to the enhanced c o n t r a c t i o n s of d i a b e t i c mesenter ic a r t e r i e s to 3xlO"^M NA i n the presence o f e x t r a c e l l u l a r C a 2 + . In c o n t r a s t , the phas ic c o n t r a c t i o n s induced by 10" 6 M NA i n C a 2 + - f r e e medium were not s i g n i f i c a n t l y d i f f e r e n t between con t r o l and d i a b e t i c mesenter ic a r t e r i e s . Th i s i s c on s i s t en t w i th the I n s ( l , 4 ,5 )P3 l e v e l s , which were a l s o not s i g n i f i c a n t l y d i f f e r e n t between con t r o l and d i a b e t i c mesenter ic a r t e r i e s i n response to t h i s concen t ra t i on o f NA. The enhanced maximum t o n i c c o n t r a c t i l e response detected i n d i a b e t i c mesenter ic a r t e r i e s i n response to 3x10"^ M NA cou ld a l s o r e s u l t , at l e a s t i n p a r t , from increased a c t i v a t i o n of PKC by enhanced product ion o f DG i n these t i s s u e s , although DG or i t s phosphorylated product , PA, was not measured i n mesenter ic a r t e r i e s . Th i s p o s s i b i l i t y i s a l s o supported by our data tha t s t au ro spo r i ne abo l i shed the d i f f e r e n c e i n magnitude o f su s ta ined c o n t r a c t i o n observed i n con t r o l and d i a b e t i c mesenter ic a r t e r i e s i n response to 3x 10"^ M NA (see below). The obse rva t ion tha t the c o n t r a c t i l e responses o f d i a b e t i c mesenter ic a r t e r i e s to 1 0 " 6 M NA were s i g n i f i c a n t l y g r ea te r than c o n t r o l i n the presence of e x t r a c e l l u l a r C a 2 + but not i n i t s absence, wh i l e I n s ( l , 4 , 5 )P3 product ion was not s i g n i f i c a n t l y i n c rea sed , may r e f l e c t the c o n t r i b u t i o n of C a 2 + i n f l u x to the enhanced c o n t r a c t i o n of the d i a b e t i c mesenter ic a r t e r i e s i n response to t h i s c oncen t r a t i on of 1 8 0 NA. Th i s e f f e c t cou ld be produced as a r e s u l t o f enhanced PKC-mediated processes o c cu r r i n g i n the presence of e x t r a c e l l u l a r C a 2 + (see below) and/or due to other mechanisms independent o f phospho ino s i t i de metabolism or PKC a c t i v a t i o n (Chiu et a l . , 1987; Kha le l and Van Breman, 1988). I t should be noted tha t although q u a l i t a t i v e agreement was found between the r e s u l t s of the c o n t r a c t i l i t y s tud ie s and the measurements of phospho ino s i t i de metabolism i n c on t r o l and d i a b e t i c aortae and mesenter ic a r t e r i e s , q u a n t i t a t i v e l y the inc reases i n pho spho ino s i t i de metabolism i n response to maximum concent ra t i on s of NA were r e l a t i v e l y g r ea te r than the enhancement of the c o n t r a c t i l e responses to t h i s agon i s t i n d i a b e t i c a r t e r i e s compared to c o n t r o l . The reason f o r these apparent d i s c r epanc i e s i s not known. S i m i l a r d i s c r epanc i e s have been repor ted r e c e n t l y i n o ther s tud ie s (McGrath et a l . , 1989; T u r l a and Webb, 1990). Although the c o n t r i b u t i o n of the e f f e c t o f d i f f e r e n c e s i n exper imental c o n d i t i o n s used i n the f u n c t i o n a l and the b iochemica l s t ud i e s to the d i s c r epanc i e s observed between some of the r e s u l t s (eg. i n mesenter ic a r t e r y ) cannot be exc luded, these data suggest tha t NA i n the concen t ra t i on s used can induce g rea te r pho spho ino s i t i de metabolism than i s needed f o r c o n t r a c t i o n i n a r t e r i e s from d i a b e t i c r a t s . However, the i m p l i c a t i o n s o f t h i s are not known. The l a c k o f d i f f e r e n c e i n i n o s i t o l phosphate p roduct ion between c o n t r o l and d i a b e t i c p o r t a l ve ins i n response to NA 0.5 min a f t e r i t s a d m i n i s t r a t i o n cou ld o f f e r an exp lana t i on f o r the l a c k o f d i f f e r e n c e i n 1 8 1 the NA-induced c o n t r a c t i l i t y between the two types o f blood v e s s e l s . In f a c t , s i n ce no c o n t r a c t i l e response to NA was detected i n C a 2 + - f r e e s o l u t i o n i n these t i s s u e s , the r o l e of products o f phospho ino s i t i de metabolism i n the c o n t r a c t i l i t y o f the r a t p o r t a l ve ins may be l e s s important . Th i s appears to be supported by our f i n d i n g o f reduced c o n t r a c t i l e responses of p o r t a l ve ins from both c o n t r o l and d i a b e t i c r a t s to the PKC a c t i v a t o r PDB compared w i th the responses to NA. As found i n our ao r ta and mesenter ic a r t e r y exper iments, and as repor ted i n severa l o ther s tud ie s (Sybertz et a l . , 1986; Chiu et a l . 1987), c o n t r a c t i l e responses o f va s cu l a r smooth muscle to PDB are g e n e r a l l y g r ea te r than the responses to NA (see a l s o below). However, f u r t h e r s t ud i e s are needed to c l a r i f y t h i s s ub j e c t . The data presented here are not c on s i s t en t w i th a recent r epo r t from another l a b o r a t o r y tha t c o n t r a c t i l e responses o f aortae from r a t s w i th STZ-induced d iabetes o f 28 days du ra t i on to NA and se ro ton in were a s soc i a ted w i th decreased metabolism of phospho inos i t i de s (as assessed by measuring the product ion o f [ 3 H ] - i n o s i t o l monophosphate) (Legan, 1989). Moreover, reduced metabolism o f phospho inos i t i de s i n d i a b e t i c t i s s u e s i n response to PE was a l so repor ted by the same author, although no changes i n c o n t r a c t i l e respons iveness to t h i s agon i s t was de tec ted . The reasons f o r the d i f f e r e n c e s between our r e s u l t s and those of Legan (1989) are not c l e a r . However, i t i s p o s s i b l e tha t they may be due to d i f f e r e n c e s i n the du ra t i on and s e v e r i t y o f the d iabetes induced, and/or to v a r i a t i o n s i n methodologies used to measure responses. 182 Possible explanations for altered responsiveness The reasons f o r the increased phospho inos i t i de metabolism observed i n aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s i n response to NA i n the present i n v e s t i g a t i o n are not ye t c l e a r . S ince both b iochemica l and f u n c t i o n a l responses were measured i n the presence o f a ^ - ad rene rg i c b l o c k e r , and b locker s of neuronal and extraneurona l uptake, the enhancement detected i n d i a b e t i c t i s s u e s were not due to reduced uptake of NA or reduced a c t i v a t i o n of /J-adrenoceptors i n these p r epa r a t i on s . Although the endothel ium has been repor ted to modulate c o n t r a c t i o n and phospho ino s i t i de turnover i n va s cu l a r smooth muscle (Legan and S i s son , 1990), a prev ious r epo r t from t h i s l a bo r a t o r y (Ha r r i s and MacLeod, 1988) demonstrated tha t the f unc t i on of the endothel ium i s unchanged i n aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s compared to c o n t r o l . In a d d i t i o n , the enhanced respons iveness o f the d i a b e t i c a r t e r i e s i s not due to a gene ra l i z ed inc rease i n the c o n t r a c t i l i t y o f the blood ves se l s or to t o x i c e f f e c t s of STZ (MacLeod, 1985). Furthermore, the acute treatment o f i s o l a t e d a r t e r i a l p repara t ions w i th i n s u l i n or g lucose at concent ra t i on s equ i va l en t to those present i n serum from c o n t r o l or d i a b e t i c r a t s , r e s p e c t i v e l y , d i d not i n f l u e n c e the c o n t r a c t i l e respons iveness of the a r t e r i e s to NA (data not shown). Th i s suggests tha t the enhanced responses to NA measured i n d i a b e t i c a r t e r i e s r e l a t i v e to c o n t r o l under i n v i t r o cond i t i on s were not r e l a t e d to acute e f f e c t s of i n s u l i n r educ t i on or g lucose excess. O v e r a l l , the r e s u l t s suggest 183 t ha t d i abetes s p e c i f i c a l l y a f f e c t s the a-adrenoceptor-PLC complex and r e l a t e d mechanisms on the plasma membrane of a r t e r i a l smooth muscle c e l l s t o induce enhanced c o n t r a c t i l i t y i n response to NA. As po in ted out i n the I n t r o d u c t i o n , the e f f e c t s of d iabetes on v a s cu l a r smooth muscle are complex and no s i n g l e f a c t o r appears to be r e spon s i b l e f o r the observed r e s u l t s i n the present study. In a d d i t i o n to being hyperglycemic and underweight (malnour i shed), STZ-induced d i a b e t i c r a t s are a l s o hypo insu l inemic and have g r e a t l y e l eva ted serum l i p i d l e v e l s (Rodrigues and M c N e i l l , 1987). They a l so have changes i n the l e v e l s o f hormones such as reduced t h y r o i d hormone content (Cooper, 1982; T a h i l i a n i et a l . , 1983). Any of these changes cou ld p o t e n t i a l l y c o n t r i b u t e to the a l t e r e d NA-induced phospho inos i t i de tu rnover and c o n t r a c t i o n . Moreover, there are repo r t s demonstrat ing tha t d i abetes can a l s o induce changes i n membrane phospho l i p id (eg. P t d l n s , phosphat idy lethano lamine, phosphat idy l s e r i n e , sphingomyel in and PA) and c h o l e s t e r o l contents i n va r i ous systems i n c l u d i n g a r t e r i e s , k idney, l i v e r , e r y t h r ocy te s and p l a t e l e t s (Heveke, 1955; R e i n i t e et a l . , 1977; A l t u r a et a l . , 1980; C a r a f o l i , 1984; Levy et a l . , 1988; Wali et a l . , 1988). Th i s e f f e c t of d iabetes has been proposed to have an i n f l u e n c e on the a c t i v i t i e s of va r i ous enzymes or p r o te i n s which are i n t e g r a l pa r t s of the membrane (Levy et a l . , 1988). I t i s p o s s i b l e t ha t the NA-induced enhanced phospho inos i t i de metabolism found i n d i a b e t i c a r t e r i e s i s r e l a t e d to inc reased a c t i v i t y of PLC and/or G-prote in s i n vo l ved i n the coup l i ng o f (^-adrenoceptors to PLC. Our f i n d i n g o f inc reased 184 c o n t r a c t i l e responses o f a r t e r i e s from d i a b e t i c r a t s to NaF p lus A l CI 3 suggests t ha t G-prote in -mediated responses are inc reased i n these p r epa r a t i on s . I t has been shown tha t i n i n t a c t t i s s u e s and membrane p repa ra t i on s from a number of t i s s u e s , PLC a c t i v i t y can be s t imu l a ted by the f l u r o a l u m i n a t e complex A IF4 ' formed when A l 3 + and F" ions are present i n s o l u t i o n together (Sternweiss and Gi lman, 1982). AIF4" i s be l i e ved to s t imu l a t e PLC a c t i v i t y by a c t i v a t i n g the G-p ro te in s d i r e c t l y v i a the GTP b ind ing s i t e (Blackmore et a l . , 1985; Harden, 1989). In a d d i t i o n , AIF4" has been demonstrated to induce both c o n t r a c t i o n and i n o s i t o l phosphate format ion i n v a s cu l a r and nonvascular smooth muscle p repa ra t i on s (Marc et a l . , 1988; Zeng et a l . , 1989; H a l l et a l . , 1990). Th i s e f f e c t i s be l i e ved to be accompanied by inc reased fo rmat ion o f DG. These observat ions t he r e f o r e prov ide support f o r the involvement of phospho ino s i t i de metabolism i n A lF4 " -med iated c o n t r a c t i o n s o f v a s cu l a r smooth muscle v i a a c t i v a t i o n o f G-prote ins coupled to PLC and suggest tha t the enhanced c o n t r a c t i o n o f a r t e r i e s from d i a b e t i c r a t s to NaF p lus A lC I3 i s mediated at l e a s t i n par t through t h i s mechanism. In a d d i t i o n to a c t i v a t i n g PLC-coupled G -p ro te i n s , AIF4" has been repor ted to a c t i v a t e other G-prote ins which are coupled to C a 2 + channels (Zeng et a l . , 1 989 ; Minneman, 1988). There fo re , i t i s p o s s i b l e t ha t pa r t o f the inc reased c o n t r a c t i l e responses observed i n d i a b e t i c a r t e r i e s i n the presence o f e x t r a c e l l u l a r C a 2 + may be mediated by a c t i v a t i o n o f these G-p r o t e i n s . Our r e s u l t s show tha t c o n t r a c t i o n s o f d i a b e t i c a r t e r i e s to NaF p lus A lC I3 were a l so enhanced i n C a 2 + - f r e e medium compared to c o n t r o l s . 1 8 5 Thi s suggests tha t increased In s ( l ,4 ,5 )P3 -med ia ted i n t r a c e l l u l a r C&6+ r e l ea se and / or increased a c t i v a t i o n o f PKC by enhanced product ion of DG may c o n t r i b u t e to the enhanced c o n t r a c t i l e responses o f d i a b e t i c a r t e r i e s to NaF p lus A lC I 3 . Desp i te i t s delayed and slow e f f e c t , NaF i n the presence o f A lC I3 has been repor ted to r e s u l t i n the prolonged accumulat ion o f I n s ( l , 4 ,5 )P3 due to the i n h i b i t o r y a c t i o n of NaF on i n o s i t o l phosphate phosphatases (Ha l l c he r and Sherman, 1980; Storey et a l . , 1984). Our proposal o f a l t e r e d G-p ro te in a c t i v i t y i n a r t e r i e s from d i a b e t i c r a t s i s p a r t l y supported by the recent observat ions t ha t the a c t i v i t y of G s a s soc i a ted w i th adenylate c y c l a se i s i nc reased wh i l e tha t of Gi i s decreased i n va r i ous p repara t i ons (eg. l i v e r , r e t i n a and adipose t i s s u e ) from STZ-induced d i a b e t i c r a t s (Had j i con s tan t i nou et a l . , 1988; Lynch et a l . , 1989). However, i t i s not ye t c l e a r whether s i m i l a r changes occur i n the G-prote ins coupled w i th PLC. Furthermore, a l t e r a t i o n s i n a c t i v i t y of G s and G-j p r o t e i n s i n v a s cu l a r smooth muscle cou ld c o n t r i b u t e to changes i n c o n t r a c t i l e responses of d i a b e t i c a r t e r i e s to A IF4 " . Th is p o s s i b i l i t y remains to be i n v e s t i g a t e d . In a d d i t i o n to the NA-induced enhanced phospho ino s i t i de tu rnover , our data show tha t the l e v e l s o f basal [ 3 H ] - i n o s i t o l l i p i d s or [ 3 H ] -i n o s i t o l phosphates i n d i a b e t i c mesenter ic a r t e r i e s were g r ea te r than those o f c o n t r o l . Th is f i n d i n g was oppos i te to the r e s u l t s repor ted f o r nerves from d i a b e t i c r a t s (Gabby, 1975) and suggests t ha t the re i s no myo ino s i t o l d e f i c i e n c y at l e a s t i n these v a s cu l a r p r epa r a t i on s . The reasons f o r these d i s c r epanc i e s between va s cu l a r and nerve t i s s u e s are 1 8 6 not known, but the r e s u l t s f u r t h e r i n d i c a t e t ha t d i abetes can induce d i f f e r e n t e f f e c t s on d i f f e r e n t types o f t i s s u e s . PKC-mediated responses One o f the major products of phospho inos i t i de metabol ism w i th a second messenger f u n c t i o n i s DG. This compound, by a c t i v a t i n g PKC has been proposed to be i nvo l ved i n the delayed su s ta ined c o n t r a c t i o n of a r t e r i e s i n response to a c t i v a t i o n o f C a 2 + - m o b i l i z i n g recepto r s (Campbell et a l . , 1985; DeFeo and Morgan, 1985; Cha t t e r j ee and Tejada, 1986; Drenth et a l . , 1989). D e r i v a t i v e s of phorbol e s t e r which are be l i e ved to mimic the a c t i o n of DG can a l so a c t i v a t e PKC i n v a s cu l a r beds and cause c o n t r a c t i o n . In view of the observed inc reases i n c o n t r a c t i l i t y and phospho inos i t i de metabolism i n aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s i n response to NA, i n the next s e r i e s o f experiments we i n v e s t i g a t e d the p o s s i b i l i t y t ha t d i abetes induces changes i n c o n t r a c t i l e mechanisms r e s u l t i n g from a c t i v a t i o n o f PKC. In these exper iments, we used the phorbol e s t e r , PDB, as a PKC a c t i v a t o r (Castagna et a l . , 1982), and the m i c r o b i a l a l k a l o i d , s t au ro spo r i ne , as a PKC i n h i b i t o r (Tamaoki et a l . , 1986) to probe the s t a tu s o f PKC-mediated c o n t r a c t i l e mechanisms. Our f i n d i n g tha t c o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s to NA were i n h i b i t e d by s tau rospor ine suggests t ha t at l e a s t pa r t o f the NA response o f these a r t e r i e s i s mediated v i a a c t i v a t i o n of PKC. Th i s obse rva t ion i s c on s i s t en t w i th the view t ha t PKC a c t i v a t i o n i s 187 i n vo l ved i n the t o n i c c o n t r a c t i l e responses of v a s cu l a r smooth muscle to agents l i k e NA which s t imu l a te the phospho inos i t i de messenger system (Campbell e t a l . , 1985; DeFeo and Morgan, 1985; Cha t t e r j ee and Tejada, 1986; Heagerty and Ol lerenshaw, 1987; Drenth et a l . , 1988). Recent l y , i t was suggested tha t s tau rospor ine may b lock c o n t r a c t i l e responses of a r t e r i e s to agon i s t s , i n c l u d i n g those caus ing PKC a c t i v a t i o n , by a PKC-independent mechanism, p o s s i b l y i n v o l v i n g i n h i b i t i o n of myosin l i g h t cha in k inase (Ruegg and Burgess, 1989). I f t h i s were the case, s tau ro spor ine would be expected to b lock KC1-induced c o n t r a c t i o n s , which are be l i e ved to be a s soc ia ted w i th a c t i v a t i o n o f myosin l i g h t cha in k ina se , and occur independent ly of PKC a c t i v a t i o n (Nakaki et a l . , 1985; T u r l a and Webb, 1990). However, the concen t ra t i on of s t au ro spo r i ne used i n the present i n v e s t i g a t i o n caused much g r ea te r i n h i b i t i o n of c o n t r a c t i l e responses of a r t e r i e s to NA and the PKC a c t i v a t o r , PDB, than to KC1. Th i s suggests tha t the i n h i b i t o r was r e l a t i v e l y more s e l e c t i v e i n i n h i b i t i n g PKC-mediated responses. There fo re , i t seems l i k e l y t ha t the c o n t r a c t i l e responses o f a r t e r i e s to NA measured i n the present study are mediated, at l e a s t p a r t l y , by PKC a c t i v a t i o n . The f a c t t ha t the enhanced c o n t r a c t i l e responses of a r t e r i e s from d i a b e t i c r a t s to NA were abo l i shed i n the presence of s tau rospor ine suggests t ha t at l e a s t pa r t o f the increased respons iveness i s the r e s u l t o f enhanced a c t i v a t i o n of PKC i n these t i s s u e s . However, these data do not demonstrate whether t h i s r e s u l t s from increased a c t i v a t i o n o f PKC by 188 i nc reased product ion o f DG, and/or from increased respons iveness of c o n t r a c t i l e processes to a c t i v a t i o n o f PKC. PDB was used i n the present i n v e s t i g a t i o n to f u r t h e r probe the s t a tu s o f PKC-mediated c o n t r a c t i l e mechanisms because o f i t s g r ea te r water s o l u b i l i t y than the commonly used ana log, TPA. Con t r a c t i on of v a s cu l a r smooth muscle by a c t i v e phorbol e s t e r s such as PDB or TPA, i n the absence and presence of e x t r a c e l l u l a r C a 2 + i s a we l l documented phenomenon (Danthu lur i and Deth, 1984; Kojima et a l . , 1984; Rasmussen et a l . , 1984; Gleason and F l a im, 1986; M i l l e r et a l . , 1986; Chiu et a l . , 1987; S inger and Baker, 1987; Swamura et a l . , 1987). The assumption tha t the c o n t r a c t i o n s induced by these agents are mediated by PKC i s supported by a v a r i e t y of evidence i n c l u d i n g the l a c k of c o n t r a c t i l e e f f e c t i n response to i n a c t i v e phorbol analogs, blockade o f the a c t i o n of phorbol e s t e r s by PKC i n h i b i t o r s and a l a c k of repor ted n o n s p e c i f i c a c t i on s of phorbol e s te r s ( M i l l e r et a l . , 1986 ; S inger and Baker, 1987). Moreover, there are s tud ie s showing tha t the phorbol e s t e r r ecep to r and PKC are the same p r o t e i n (Kikkawa et a l . , 1983; N iedel et a l . , 1983). In the present study, we have shown tha t the b i o l o g i c a l l y i n a c t i v e phorbol e s t e r , 4o;-phorbol, (Castagna et a l . , 1982) d i d not cause c o n t r a c t i o n s o f a r t e r i e s . In a d d i t i o n , c o n t r a c t i l e responses o f a r t e r i e s from both c o n t r o l and d i a b e t i c r a t s to PDB were i n h i b i t e d by s t au ro spo r i ne . S taurospor ine a l s o abo l i shed the d i f f e r e n c e i n c o n t r a c t i l e responses observed between d i a b e t i c and con t r o l mesenter ic a r t e r i e s to PDB. These data suggest tha t the c o n t r a c t i l e responses of a r t e r i e s to PDB observed 189 i n the present i n v e s t i g a t i o n are a l s o mediated by PKC a c t i v a t i o n , and the inc reased respons iveness of mesenter ic a r t e r i e s from d i a b e t i c r a t s to t h i s agent i s the r e s u l t of enhanced a c t i v a t i o n o f PKC-mediated mechanisms i n these t i s s u e s . A pa r t o f the c o n t r a c t i l e response of both a r t e r i e s to PDB was dependent on the presence of e x t r a c e l l u l a r C a 2 + , s i n ce C a 2 + - f r e e s o l u t i o n c on t a i n i n g EGTA or the C a 2 + channel b l ocke r s verapamil or n i f e d i p i n e caused a reduc t i on i n the magnitude of the PDB c o n t r a c t i o n s . The obse rva t i on tha t the extent o f r educ t i on o f the PDB response by verapamil or n i f e d i p i n e was not as g reat as t ha t observed i n C a 2 + - f r e e , EGTA s o l u t i o n i s i n agreement w i th prev ious r epo r t s t ha t a s u b s t a n t i a l component of the PDB response i s r e s i s t a n t to C a 2 + channel b l ocke r s (Sybertz e t a l . , 1986; Chiu et a l . , 1987). S i m i l a r C a 2 + dependencies of c o n t r a c t i l e responses to PDB and TPA have been observed by a number o f o ther i n v e s t i g a t o r s i n r a t aortae and mesenter ic a r t e r i e s as we l l as i n other types o f blood ves se l s (Rasmussen et a l . , 1984; Baraban, 1985; Gleason and F l a im, 1986; Chiu et a l . , 1987; S inger and Baker, 1987; T u r l a and Webb, 1987). Phorbol 12, 13 -d ibu ty ra te has a l s o been shown to s t i m u l a t e the i n f l u x of [ ^ C a 2 + ] i n r a b b i t and r a t aortae (Gleason and F l a im , 1986; Chiu e t a l . , 1987). These data suggest t ha t a c t i v a t i o n o f PKC w i th phorbol e s te r s leads to opening of C a 2 + channels a l l o w i n g the i n f l u x of e x t r a c e l l u l a r C a 2 + i n t o smooth muscle c e l l s . The r e s u l t s of the present i n v e s t i g a t i o n demonstrate tha t the enhanced respons iveness of mesenter ic a r t e r i e s from d i a b e t i c r a t s to PDB was dependent on the 1 9 0 a v a i l a b i l i t y or ent ry of e x t r a c e l l u l a r Ca , s i n ce i n the absence o f e x t r a c e l l u l a r C a 2 + or i n the presence o f verapamil or n i f e d i p i n e , the magnitude of the PDB-induced c o n t r a c t i o n s were s i m i l a r i n c o n t r o l and d i a b e t i c mesenter ic a r t e r i e s . Fur ther evidence suppor t ing t h i s obse rva t ion was obta ined from experiments showing t ha t upon the r e -a d d i t i o n of C a 2 + t o a C a 2 + - f r e e medium, d i a b e t i c mesenter ic a r t e r i e s p r e t r ea ted w i th PDB cont rac ted more than con t r o l a r t e r i e s (data not shown). These observat ions suggest tha t PDB-mediated a c t i v a t i o n o f PKC i n mesenter ic a r t e r i e s from STZ -d i abe t i c r a t s may r e s u l t i n increased i n f l u x o f C a 2 + through c e l l membrane C a 2 + channel s , thereby l ead i ng to inc reased ten s i on development. In t h i s regard , i t i s p o s s i b l e t ha t the inc reased Ca*- i n f l u x i n response to PDB i s due to i nc reased a c t i v a t i o n p, o f Ca channels by PKC-dependent phosphory la t ion ( F i s h e t a l . , 1986; Be r r i dge , 1987; Heagerty and Ol lerenshaw, 1987) and/or an increased p + number o f Ca^ channels a v a i l a b l e f o r a c t i v a t i o n by PKC, i n the d i a b e t i c v e s s e l s . However, o ther exp lanat ions i n c l u d i n g (a) t ha t the C a 2 + s e n s i t i v i t y o f PKC may be increased i n the presence of PDB to a g rea te r extent i n the d i a b e t i c v e s s e l s , and/or (b) augmentation o f o ther C a 2 + -dependent c e l l u l a r events o ccu r r i n g subsequent to PKC a c t i v a t i o n i n d i abetes cannot be exc luded. I t i s not c l e a r why d iabetes induced enhanced c o n t r a c t i l e responses of mesenter ic a r t e r i e s to d i r e c t a c t i v a t i o n o f PKC by PDB i n the presence of e x t r a c e l l u l a r C a 2 + w i thout a f f e c t i n g the responses i n ao r tae . One p o s s i b l e exp lana t i on f o r t h i s v a r i a t i o n i n t i s s u e 1 9 1 respons iveness i s t ha t d i f f e r e n t types o f PKC may be present i n the two types o f blood v e s s e l s , w i th d i f f e r e n t i a l respons iveness to d i abe te s . A l t e r n a t i v e l y , the number and/or the nature of C a 2 + channels a c t i v a t e d by PKC may be a f f e c t e d by d iabetes i n a d i f f e r e n t manner i n the two k inds o f v a s cu l a r t i s s u e s , and/or other events o c c u r r i n g subsequent to PKC a c t i v a t i o n may be i n f l uenced d i f f e r e n t l y by d i abetes i n the two types o f v e s s e l s . These p o s s i b i l i t i e s appear to be p a r t l y supported by the r epo r t s of o ther i n v e s t i g a t o r s who have shown tha t d i f f e r e n c e s i n PKC a c t i v i t y e x i s t i n d i f f e r e n t v a s cu l a r beds (Wagner et a l . , 1987; S i l v e r et a l . , 1988). Our data i n d i c a t e tha t c o n t r a c t i l e responses to NA mediated v i a PKC a c t i v a t i o n are enhanced, although those r e s u l t i n g from d i r e c t a c t i v a t i o n of PKC are una l t e r ed , i n aortae from d i a b e t i c r a t s . Th is suggests tha t the increased c o n t r a c t i o n s observed i n aortae from d i a b e t i c r a t s i n response to NA may r e s u l t , at l e a s t i n p a r t , from increased a c t i v a t i o n o f PKC by enhanced product ion of DG i n these t i s s u e s . Th i s i s supported by our data tha t there was g rea te r pho spho ino s i t i de turnover i n d i a b e t i c aortae than i n c on t r o l p repa ra t i on s i n response to NA. On the other hand, c o n t r a c t i l e responses to both d i r e c t a c t i v a t i o n of PKC, and to a c t i v a t i o n o f PKC i n response to NA, were enhanced i n mesenter ic a r t e r i e s from d i a b e t i c r a t s . S ince enhancement o f NA-induced phospho inos i t i de turnover was a l s o observed i n mesenter ic a r t e r i e s from d i a b e t i c r a t s , both increased a c t i v a t i o n of PKC by enhanced product ion of DG, and increased respons iveness of 192 c o n t r a c t i l e mechanisms to PKC a c t i v a t i o n , may c o n t r i b u t e to the increased c o n t r a c t i l e responses o f d i a b e t i c mesenter ic a r t e r i e s to NA. For comparison purposes, responses o f p o r t a l ve in s from con t r o l and d i a b e t i c r a t s to PDB were a l so i n v e s t i g a t e d . In c on t r a s t to the observat ions made i n aortae and mesenter ic a r t e r i e s , the magnitude of the PDB response was l e s s than tha t o f the NA response i n p o r t a l ve ins from con t r o l and d i a b e t i c r a t s . The PDB responses o f p o r t a l ve ins from the two groups of r a t s were not a l so s i g n i f i c a n t l y d i f f e r e n t from each o the r . Furthermore, i n p o r t a l ve ins from both c on t r o l and d i a b e t i c r a t s no responses to PDB were detected i n C a 2 + - f r e e s o l u t i o n . These data suggest t ha t PKC a c t i v a t i o n by PDB p lays a l e s s important r o l e i n c o n t r a c t i o n of p o r t a l v e i n s . I t i s p o s s i b l e tha t t h i s cou ld be a f a c t o r c o n t r i b u t i n g to the l a c k of enhancement of c o n t r a c t i l e respons iveness of p o r t a l ve ins from d i a b e t i c r a t s to NA, although the p roduct ion of [ 3 H ] -i n o s i t o l phosphates was found to be s i g n i f i c a n t l y inc reased i n these t i s s u e s w i th prolonged (20 min) exposure to NA. 193 SUMMARY AND CONCLUSIONS 1. C o n t r a c t i l e responses o f aortae and mesenter ic a r t e r i e s but not p o r t a l ve ins from r a t s w i th s t r e p t o z o t o c i n (STZ)- induced d iabetes of 12 - 14 weeks du ra t i on to noradrena l ine (NA), s e l e c t i v e c q -adrenoceptor agon i s t s and AIF4" were enhanced. The enhanced maximum c o n t r a c t i l e responses to cq-adrenoceptor s t i m u l a t i o n appear to be a s soc i a ted w i th increased r e l ea se o f i n t r a c e l l u l a r C a 2 + . However, e x t r a c e l l u l a r C a 2 + was requ i red f o r f u l l expres s ion o f enhanced c o n t r a c t i l e responses to these agon i s t s , suggest ing t ha t a-adrenoceptor-mediated C a 2 + i n f l u x i s enhanced i n a r t e r i e s from d i a b e t i c r a t s . In c o n t r a s t , responses o f a r t e r i e s from d i a b e t i c r a t s to KC1 or K + - d e p o l a r i z e d d i a b e t i c p repa ra t i on s to e x t r a c e l l u l a r C a 2 + were unchanged. 2. Aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s demonstrated enhanced phospho inos i t i de metabolism as assessed by increased h y d r o l y s i s of [ 3 2 P ] - p h o s p h a t i d y l i n o s i t o l (4 ,5 ) -b i sphosphate , and format ion o f [ 3 2 P ] - p h o s p h a t i d i c a c i d , [ 3 H ] - i n o s i t o l phosphates and i n o s i t o l ( 1 ,4 ,5 ) t r i sphosphate ( I n s ( l , 4 , 5 )P3 ) i n response to maximum concen t r a t i on o f NA. The product ion of I n s ( l , 4 ,5 )P3 i n response to a submaximum concen t ra t i on of NA was a l so enhanced i n aortae but not i n mesenter ic a r t e r i e s from d i a b e t i c r a t s r e l a t i v e to c o n t r o l . The NA-induced enhanced phospho inos i t i de metabolism i n d i a b e t i c a r t e r i e s was 194 mediated v i a a\-adrenoceptor s t i m u l a t i o n . The a l t e r a t i o n s i n phospho ino s i t i de metabolism observed i n d i a b e t i c a r t e r i e s i n response to NA were c on s i s t en t w i th the enhanced c o n t r a c t i l e responses of these t i s s u e s to the agon i s t i n C a 2 + - f r e e medium. 3. NA induced enhanced sus ta ined c o n t r a c t i o n s of aortae and mesenter ic a r t e r i e s from d i a b e t i c r a t s were i n h i b i t e d by the p r o t e i n k inase C (PKC) i n h i b i t o r , s t au ro spo r i ne , suggest ing the involvement o f PKC a c t i v a t i o n i n these c o n t r a c t i o n s . 4. C o n t r a c t i l e responses o f mesenter ic a r t e r i e s but not aortae or p o r t a l ve ins from d i a b e t i c r a t s to the PKC a c t i v a t o r , phorbol 12, 13-d i b u t y r a t e (PDB), were i nc rea sed . Th i s e f f e c t was a s soc i a ted w i th inc reased i n f l u x of C a 2 + through c e l l membrane C a 2 + channel s . 5. The r e s u l t s suggest that enhanced phospho ino s i t i de metabolism i n aortae and mesenter ic a r t e r i e s from STZ-induced d i a b e t i c r a t s i n response to NA, v i a a j - ad renocepto r a c t i v a t i o n , may c o n t r i b u t e to the increased c o n t r a c t i l e respons iveness of these ve s se l s to NA. This appears to be l i n k e d to increased a c t i v i t y o f G -prote in s and/or subsequent steps a s soc ia ted w i th the phospho ino s i t i de system. In a d d i t i o n , increased a c t i v a t i o n of PKC-mediated processes , which are dependent on the presence o f e x t r a c e l l u l a r C a 2 + , may f u r t h e r c o n t r i b u t e to the enhanced c o n t r a c t i l e responses o f d i a b e t i c 1 9 5 mesenter ic a r t e r i e s to NA. However, increased i n f l u x of C a " by other mechanisms such as those o c cu r r i n g independent o f phospho ino s i t i de tu rnover or PKC a c t i v a t i o n may c o n t r i b u t e to the enhanced c o n t r a c t i o n s o f d i a b e t i c a r t e r i e s to submaximum concen t ra t i on s of NA. Diabetes does not seem to induce such changes i n p o r t a l v e i n s . I t i s proposed t ha t the a l t e r a t i o n s i n phospho ino s i t i de metabolism desc r i bed may under ly at l e a s t some of the f u n c t i o n a l d e t e r i o r a t i o n of v a s cu l a r smooth muscle i n d i abe te s . 1 9 6 REFERENCES A b d e l - L a t i f , A.A., Akhtar , R., and Hawthorne, J . N . 1977. A c e t y l c h o l i n e i nc reases the breakdown of t r i p h o s p h o i n o s i t i d e o f r a b b i t i r i s muscle p r e l a b e l l e d w i th [ 3 Z P ]pho spha te . Biochem. J . 162: 61-73. A b d e l - L a t i f , A.T. 1986. Ca l c i um-mob i l i z i n g r e c e p t o r s , polyphospho-i n o s i t i d e s , and the generat ion o f second messengers. Pharmacol Rev. 38: 227-272. Abebe, W., H a r r i s , K.H., and MacLeod, K.M. 1990. Enhanced c o n t r a c t i l e responses of a r t e r i e s from d i a b e t i c r a t s to a\-adrenoceptor s t i m u l a t i o n i n the absence and presence o f e x t r a c e l l u l a r Ca . J . Ca rd iovasc . Pharmacol. 16: 239-248. Addison-Wesley. 1983. Manual o f Nurs ing P r a c t i c e . Addison-Wesley Pub l i s h i n g Company, C a l i f o r n i a , pp 454-464. A d e l s t e i n , R.S., and E i senberg, E. 1980. Regu la t ion and k i n e t i c s o f the ac t in -myos in i n t e r a c t i o n . Ann. Rev. Biochem. 49: 921-956. A d e l s t e i n , R.S., and Hathaway, D.R. 1979. Role o f ca l c i um and c y c l i c adenosine 3 ' : 5 ' monophosphate i n r e g u l a t i n g smooth muscle c o n t r a c t i o n . Am. J . P h y s i o l . 44: 783-787. Agrawal, D.K., and M c N e i l l , J . H . 1987. Vascu la r responses to agon i s t s i n r a t mesenter ic a r t e r y from d i a b e t i c r a t s . Can. J . P h y s i o l . Pharmacol. 65: 1484-1490. Agrawal, D.K., B h i m j i , S., and M c N e i l l , J . H . 1987. E f f e c t o f ch ron i c exper imental d iabetes on va s cu l a r smooth muscle f u n c t i o n i n r a b b i t c a r o t i d a r t e r y . J . Card iovasc . Pharmacol. 9: 584-593. Agrawal, D.K., T r i g g l e , C.R., and D a n i e l , E.E. 1984. Pharmacological c h a r a c t e r i z a t i o n of the po s t s ynapt i c a lpha adrenoceptors i n v a s cu l a r smooth muscle from canine and r a t mesenter ic v a s cu l a r beds. J . Pharmacol. Exp. Ther. 229: 831-838. Akhtar , R.A., T a f t , W.C., and A b d e l - L a t i f , A.A. 1983. E f f e c t s o f ACTH on po l ypho spho ino s i t i de metabolism and p r o t e i n phosphory la t ion i n r a b b i t i r i s s u b c e l l u l a r f r a c t i o n . J . Neurochem. 41: 1460-1468. Aksoy, M.P., Mras, S., Kamm, K.E., and Murphy, R.A. 1983. C a 2 + , cAMP and changes i n myosin l i g h t cha in phosphory la t i on dur ing c o n t r a c t i o n of smooth muscle. Am. J . P h y s i o l . 245: C255-C270. 197 Alexander , R.W., Brock, T.A., Gimbrone, M.A., and R i t tenhouse, S.E. 1985. Ang io tens in inc reases i n o s i t o l t r i s pho spha te and ca l c i um i n v a s cu l a r smooth muscle. Hypertens ion, 7: 447-451. A l t u r a , B.M., and A l t u r a , B.T. 1978. Magnesium and v a s cu l a r tone and r e a c t i v i t y . Blood Ves se l s , 15: 5-16. A l t u r a , B.M., Halevy, S., and Tu r l apa t y , P.D.M.V. 1980. Vascu la r smooth muscle i n d iabetes and i t s i n f l u e n c e on r e a c t i v i t y o f blood v e s s e l s . In: The m i c r o c i r c u l a t i o n of d i abe te s . Ed i ted by C. Dav i s , Karger, Ba se l , pp. 118-150. B a l i n g , G. 1984. S t r u c tu re and f u n c t i o n o f a calmodul in-dependent smooth muscle myosin l i g h t cha in k inase . E x p e r i e n t i a , 40: 1185-1188. Baker, P.F. 1972. Transport and metabolism o f ca l c i um ions i n nerve. Prog. B iophys. Mol . B i o l . 24: 177-223. Balodimos, M.C. 1971. D i a b e t i c nephropathy. In: J o s l i n ' s Diabetes M e l l i t u s . Ed i ted by A. Marble, P. White, R.F. B rad ley , and R.F. K r a l l , Lea and Feb iger , P h i l a d e l p h i a , pp. 526-561. Bang, H.O., and Dyerberg, J . 1972. Plasma l i p i d s and l i p o p r o t e i n s i n Green land ic west coast Eskimos. Acta Med. Scand. 192: 85-94. Bansa l , V.S., Inhorn, R.C., and Majerus, P.W. 1987. The metabolism of i n o s i t o l 1 ,3 ,4 - t r i sphosphate to i n o s i t o l 1,3-bisphosphate. J . B i o l . Chem. 262: 9444-9447. Bar, R.S., Mugges, M., Roth, J . , Kahn, C.R., Havrankova, J . , and Imperto-McGinley, J . 1978. I n s u l i n r e s i s t a n c e , acanthuses n i g r i c a n s and normal i n s u l i n recepto r s i n a young woman: evidence f o r a po s t recep to r d e f e c t . J . C l i n . E n d o c r i n o l . Metab. 47: 620-625. Baraban. J .M . , Gould, R . J . , Peroutka, S . J . , and Snyder, S.H. 1985. Phorbol e s t e r e f f e c t s on neurot ransmi s s ion : i n t e r a c t i o n w i th neu ro t ran sm i t te r s and ca l c i um ion smooth muscle. Proc. N a t l . Acad. S c i . U.S.A. 82: 604-607. Ba t t y , I.R., and Nahork i , S.R. 1985. D i f f e r e n t i a l e f f e c t s o f l i t h i u m on musca r in i c recepto r s t i m u l a t i o n o f i n o s i t o l phosphates i n r a t ce reb ra l co r tex s l i c e s . J . Neurochem. 45: 1514-1521. Bant ing , F.G., Best , C.H., C o l l i p , J . B . , Campbel l , W.R., and F l e t c h e r , A.A. 1922. Panc rea t i c e x t r a c t s i n the treatments of d iabetes m e l l i t u s . Can. Med. Assoc. J . 12: 141-146. 198 Berger, M., and B e r c h t o l d , P. 1983. I n s u l i n t r an spo r t and a c t i o n at t a r g e t c e l l s . In: Diabetes M e l l i t u s . Ed i ted by A. Marb le, L. K r a l l , R.F. B rad ley , A.R. C h r i s t l i e b , and J . S . Soe ldner . Lea and Feb iger , P h i l a d e l p h i a , pp. 82-109. Be r r i dge , M.J. 1983. Rapid accumulat ion o f i n o s i t o l t r i s pho spha te r e vea l s t ha t agon i s t s hydro lyse po l yphospho ino s i t i de s i n s tead o f phosphat idy l i n o s i t o l . Biochem. J . 212: 849-858. Be r r i d ge , M.J. 1984. I n o s i t o l t r i sphospha te and d i a c y l g l y c e r o l as second messengers. Biochem. J . 220: 345-360. Be r r i dge , M.J. 1986. I n o s i t o l phosphates as second messengers. In: Phospho inos i t i des and Receptor Mechanisms. Ed i ted by J.W. Putney, J r . , A lan R. L i s s , Inc. , pp. 25-46. Be r r i dge , M.J. 1987. I n o s i t o l t r i sphospha te and d i a c y l g l y c e r o l : two i n t e r a c t i n g second messengers. Ann. Rev. Biochem. 56: 159-193. Be r r i dge , M . J . , and I r v i n e , R.F. 1984. I n o s i t o l t r i s pho spha te , a novel second messenger i n c e l l u l a r s i gna l t r a n s d u c t i o n . Nature, 312: 315-321. Be r r i dge , M . J . , Dawson, R.M., Downes, C P . , Hes lop, J . P . , and I r v i n e , R.F. 1983. Changes i n the l e v e l s o f i n o s i t o l phosphates a f t e r agonist-dependent h y d r o l y s i s of membrane pho spho i no s i t i de s . Biochem. J . 212: 473-482. Be r r i d ge , M . J . , Downes, C P . , and Hanley, M.R. 1982. L i t h i um a m p l i f i e s agonist-dependent phosphat idy l i n o s i t o l responses i n b r a i n and s a l i v a r y g lands . Biochem. J . 206: 587-595. B ianca , V.D., Togn i , P.D., Gozskowiak, M., V i c e n t i n e , L.M., and V i r g i l i o , F.D. 1986. C y c l i c AMP i n h i b i t i o n o f p h o s p h o i n i s i t i d e tu rnover i n human n e u t r o p h i l s . Biochem. Biophys. A c t a , 886: 441-447. B iden, T . J . , and Wal lheim, C B . 1986. Calc ium regu l a te s the i n o s i t o l t r i s / t e t r a k i s p h o s p h a t e pathway i n i n t a c t and broken p repa ra t i on s o f i n s u l i n - s e c r e t i n g RINmSF c e l l s . J . B i o l . Chem. 261: 11931-11939. B l a u s t e i n , M.P. 1977. Sodium ions , ca l c i um i on s , b lood pressure r e g u l a t i o n and hyper tens ion : A reassessment of a hypothes i s . Am. J . P h y s i o l . 232: C165-C173. 199 Blackamoor, P.F., Bocck ino, S.B., Waynick, L.E., and Exton, J . H . Role o f a guanine nuc l eo t i de b ind ing p r o t e i n i n the h y d r o l y s i s o f hepatocyte phosphat idy l i n o s i t o l 4,5-b i sphosphate by c a l c i u m -m o b i l i z i n g hormones and the c on t r o l o f c e l l c a l c i um. J . B i o l . Chem. 260: 1477-1483. Brody. M . J . , and Dixon, R.L. 1964. Vascu la r r e a c t i v i t y i n exper imental d i abetes m e l l i t u s . C i r c . Res. 14: 494-501. Brunner, L.S., and Suddarth, D.S. 1988. Assessment and management of p a t i e n t s w i th d iabetes m e l l i t u s . In: Textbook o f M e d i c a l - S u r g i c a l Nurs ing (Chapter 36) , Ed i ted by L.S. Brunner, and D.S. Suddarth, 6th Ed. , J . B . L i p p i n c o t t Company, P h i l a d e l p h i a , pp. 898-941. Bunn, H.F., Gabby, K.H., and Ga l l op , P.M. 1978. The g l y c o s y l a t i o n of hemoglobin: Relevance to d iabetes m e l l i t u s . S c i ence , 200: 21-27. Burgess, G.M., McKinney, J . S . , I r v i n e , R.F., and Putney, J.W. 1985. I n o s i t o l 1 ,4 ,5 - t r i sphosphate and i n o s i t o l 1 ,3 ,4 - t r i sphosphate and format ion i n Ca - m o b i l i z i n g hormone s e n s i t i v e c e l l s . Biochem. J . 232: 237-243. C a h i l l , G.F. 1971. Phys io logy of i n s u l i n i n man. D iabetes , 20: 785-799. Campbel l , M.D., Deth, R.C., Payne, R.A., and Honeyman, T.W. 1985. Phospho inos i t i de h y d r o l y s i s i s c o r r e l a t e d w i th agon i s t - i nduced ca l c i um f l u x and c o n t r a c t i o n i n the r a b b i t a o r t a . Eur. J . Pharmacol. 116: 129-136. Campbel l , M.D., Raju, N.D., and R i cha rd , C D . 1986. D i f f e r ence s i n pho spho l i p i d i n co rpo r a t i o n o f 3 Z P r e l e van t to c q - r e c e p t o r coup l i ng events i n r a t and r a b b i t a o r t a . Biochem. Biophys. Res. Commun. 141: 1213-1221. C a r a f o l i , E. 1984. Ca l c i um- t r an spo r t i n g system of plasma membranes, w i t h s p e c i a l a t t e n t i o n to t h e i r r e g u l a t i o n . In: Advances i n c y c l i c nuc l eo t i de and p r o t e i n phosphory la t ion r e sea r ch . Ed i ted by P.R. Greengerd, R.P. P a o l e t t i , and S.N. N i c o s i a . Raven Pres s , New York, pp. 543-549. Ca ron i , P. R e i n l i b , L., and C a r a f o l i , E. 1980. Charge movements dur ing the N a + - C a z + exchange i n heart sarcolemma v e s i c l e s . Proc. N a t l . Acad. S c i . U.S.A. 77: 6354-6358. Castagna, M., Takaz, Y., K a i buch i , K., Sano, K., Kikkawa, U., and N i s h i z u ka , Y. 1982. D i r e c t a c t i v a t i o n o f ca l c i um a c t i v a t e d 200 phospho l i p i d dependent p r o t e i n k inase by tumor promoting phorbol e s t e r s . J . B i o l Chem. 257: 7847-7851. Cauv in, C , and Ma l i k , S. 1984. Induct ion o f C a z + i n f l u x and i n t r a c e l l u l a r C a z r e l ea se i n i s o l a t e d r a t ao r t a and mesenter ic r e s i s t a n c e ves se l s by noradrena l ine a c t i v a t i o n o f alpha-1 r e cep to r s . J . Pharmacol. Exp. Ther. 220: 413-418. Cauv in, C , Loutzenh i se r , R., Hwang, 0 . , and Van Breemen, C. 1982. a\-adrenoceptors induce C a z + i n f l u x and i n t r a c e l l u l a r C a z + r e l ea se i n i s o l a t e d r a b b i t a o r t a . Eur. J . Pharmacol. 84: 223-235. C h a t t e r j e e , M., and Tejada, M. 1986. Phorbol e s t e r induced c o n t r a c t i o n i n chem ica l l y - s k i nned va s cu l a r smooth muscle. Am. J . P h y s i o l . 251: C356-C361. C h i l v e r s , E.R., C h a l l i s , R .A .J . , Barnes, P . J . , and Nahor sk i , S.R. 1989. Mass changes of i n o s i t o l ( 1 , 4 , 5 , ) t r i s pho spha te i n t r a c h e a l i s muscle f o l l o w i n g agon i s t s t i m u l a t i o n . Eur. J . Pharmacol. 164: 587-590. Ch iu , A.T. , Bozar th , J .M . , and Timmermans, P.B.M.W.M. 1987. R e l a t i o n s h i p between phosphat idy l i n o s i t o l tu rnover and C a z + m o b i l i z a t i o n induced by alpha-1 adrenoceptor s t i m u l a t i o n i n the r a t a o r t a . J . Pharmacol. Exp. Ther. 240: 123-127. Ch iu , A.T. , Boza r th , J .M . , Forsythe, M.S., and Timmermans, P.B.M.W.M. 1987. C a z + u t i l i z a t i o n i n the c o n t r a c t i o n of r a t ao r t a to s t i m u l a t i o n of p r o t e i n k inase C by phorbol d i b u t y r a t e . J . Pharmacol. Exp. Ther. 242: 934-939. Chobanian, A.V., G e r r i t s e n , G.C., Brecher, P. I., and McCombs, L. 1974. A o r t i c g lucose metabolism i n the d i a b e t i c hamster. D i a b e t o l o g i a , 10; 589-593. C h r i s t l i e b , A.R., Janka, H.V., and Solano, A. 1976. Vascu la r r e a c t i v i t y to ang io tens in II and to norep inephr ine i n d i a b e t i c s u b j e c t s . D iabetes , 25: 268-274. Chuang, D. 1989. Neurot ransmi t te r recepto r s and phospho ino s i t i de tu rnove r . Ann. Rev. Pharmacol. T o x i c o l . 29: 71-110. C o c k c r o f t , S., and Gomperts, B.D. 1985. Role o f guanine n u c l e o t i d e b ind ing p r o t e i n i n the a c t i v a t i o n of po l ypho spho ino s i t i de phosphodiesterase. Nature, 314: 534-536. C o c k c r o f t , S., and Tay l o r , J .A . 1987. F luoroa luminate mimics guanosine 5 ' - [ r - t h i o ] - t r i p ho spha te i n a c t i v a t i n g the p o l y p h o s p h o i n i s i t i d e 2 0 1 phosphodiesterase of hepat i c membranes. Biochem. J . 241: 409-414. C o l w e l l , J . A . , Halushka, P.V., S a r j i , K., Lev ine , J . , Sega l , J . , and N a i r , R.M.G. 1976. A l t e r e d p l a t e l e t f u n c t i o n i n d iabetes m e l l i t u s . D iabetes , 25: 826-831. C o l w e l l , J . A . , Halushka, P.V., S a r j i , K.E., L o p e s - V i r e l l a , M.F., and Sega l , J . 1979. Vascu la r d i seases i n d i abe te s . A r ch . I n t . Med. 139: 225-230. C o l w e l l , J . A . , Winocow, P.D., and Ho lushta, P.V. 1983. E f f e c t s o f ageing on the regenera t i ve c apac i t y of the panc r ea t i c /J - ce l l o f the r a t ingemar swenine. D iabetes , 32(Suppl . 2 ) : 14-19. Connoly, T.M., Bansa l , V.S., Bross, T .E . , I r v i n e , R.F., and Majerus, P.W. 1987. The metabolism o f t r i s - and tet raphosphates o f i n o s i t o l by 5-phosphomonoesterase and 3-k inase enzymes. J . B i o l . Chem. 262: 2146-2149. Cooper, R.A., Brunwald, A.D., and Kuo, A.L. 1982. Phorbol e s t e r s i nduc t i on o f leukemic c e l l d i f f e r e n t i a t i o n i s a membrane-mediated process . Proc. N a t l . Acad. S c i . U.S.A. 79: 2865-2869. Cooppan, R. 1982. Recogn i t ion o f hypothyro id i sm i n d i a b e t e s . P r a c t . C a r d i o l . 8: 113-115. C r a i g , J.W. 1980. C l i n i c a l i m p l i c a t i o n s o f the new d iabetes c l a s s i f i c a t i o n . Post Grad. Med. 68: 122-133. Cra ighead, J . E . 1978. The r o l e o f v i r u se s i n the pathogenesis o f p anc rea t i c d i sease and d iabetes m e l l i t u s . In: Progress i n Medical V i r o l o g y . Ed i ted by J . L . Me ln i ck , S. Krager, and M. Bassek. P a r i s , London, New York, Sydney, pp. 162-216. Creuz, R., Wenger, T .H. , Kunoz, G., and S z e n t i v a n y i , M. 1973. Changes o f adrenerg ic r e a c t i o n pat te rns i n exper imental d i abetes m e l l i t u s . Endocr ino logy, 93: 752-755. Czech, M.P. 1981. I n s u l i n a c t i o n . In: Diabetes M e l l i t u s . Ed i ted by J . S . S k y l e r , and G.F. C a h i l l , Yorke Medical Books, New York, pp. 64-72. D a n i e l , E.e., Grover, A.K., and Kwan, C.Y. 1983. Ca lc ium. In: B iochemis t ry o f smooth muscle. Ed i ted by N.L. Stephens, v o l . 3, CRC Pres s , Boca Raton, F l o r i d a , pp. 1-88. 202 D a n t h u l u r i , N.R., and Deth, R.C. 1984. Phorbol e s t e r - i nduced c o n t r a c t i o n of a r t e r i a l smooth muscle and i n h i b i t i o n of a -adrenerg i c response. Biochem. Biophys. Res. Comm. 125: 1103-1109. Dav i s , M.D. 1974. D e f i n i t i o n , c l a s s i f i c a t i o n and course o f d i a b e t i c r e t i n opa th y . In: D i a b e t i c Ret inopathy. Ed i ted by J.R. Lyn, W.E. Snyder, and A. Va s s i e r , Grune and S t r a t t o n , New York, pp. 145-170. De Cuneo, M.F., Ru i z , R.D., Lacuara, J . L . , and de To r re s , R.T. 1988. C o n t r a c t i l i t y and pharmacological r e a c t i v i t y of i s o l a t e d va s cu l a r smooth muscle from d i a b e t i c r a t s . Pharmacol. 36: 228-237. Defeo, T.T. , and Morgan, K.G. 1985. Ca l c i um- fo r ce r e l a t i o n s h i p s as detected w i th acquor in i n two d i f f e r e n t v a s cu l a r smooth muscles o f the f e r r e t . J . P h y s i o l . (Lond.) 369: 269-282. Deth, R., and Van Breemen, C. 1974. R e l a t i v e c o n t r i b u t i o n o f C a ' i n f l u x and c e l l u l a r r e l ea se dur ing drug- induced a c t i v a t i o n of the r a b b i t a o r t a . P f l uge r s A rch . 348: 13-22. D iges, K.G., and Summers, R.J . 1983. C h a r a c t e r i z a t i o n o f o f po s t s ynap t i c a-adrenoceptors i n r a t a o r t i c s t r i p s and p o r t a l v e i n s . Br. J . Pharmacol. 79: 655-665. D i l l o n , P.F., Aksoy, M.O., D r i s k a , S.P., and Murphy, R.A. 1981. Myosin phosphory la t ion and the c r o s s - b r i d ge c y c l e i n a r t e r i a l smooth muscle. Sc ience, 211: 495-497. D i v i r g i l l i o , F., Lew, D.P., and Pozzan, T. 1984. P r o t e i n k inase C a c t i v a t i o n of p h y s i o l o g i c a l processes i n human n e u t r o p h i l s at v a n i s h i n g l y small c y t o s o l i c C a 2 + l e v e l s . Nature, 310: 691-693. D ixon, J . F . , and Hokin, L.E. 1987. I n o s i t o l 1 , 2 - c y c l i c 4 ,5-t r i s pho spha te i s formed i n the r a t p a r o t i d g land on muscar in i c s t i m u l a t i o n . Biochem. Biophys. Res. Commun. 149: 1208-1213. Dixon, J . F . , and Hokin. L.E. 1985. The format ion o f i n o s i t o l 1,2-c y c l i c phosphate on agon i s t s s t i m u l a t i o n of phospho ino s i t i de breakdown i n mouse panc rea t i c m i n i l o b u l e s . J . B i o l . Chem. 260: 16068-16071. Doctrow, S.R., and Lowenstein, J .M. 1985. Adenosine and 5 - c h l o r o - 5 ' -deoxyadenosine i n h i b i t s the phosphory la t ion of phosphat idy l i n o s i t o l and myosin l i g h t cha in i n c a l f ao r t a smooth muscle. J . B i o l . Chem. 260: 3469-3476. 2 0 3 Downes, C P . , Mussat, M . C , and M i c h e l l , R.H. 1982. The i n o s i t o l t r i s pho spha te phosphomonoesterase of the human e r y t h r o c y te membrane. Biochem. J . 203: 169-177. Drenth, J . P . H . , N ish imura, J . , Noua i l h ta s , V.L .A. , and Van Breemen, C 1989. Receptor-mediated C-k inase a c t i v a t i o n c o n t r i b u t e s to a l pha -adrenerg ic tone i n r a t mesenter ic r e s i s t a n c e a r t e r y . J . Hypertens ion, 7 (Suppl 4 ) : S41-S45. Ebash i , S. 1980. Regu lat ion o f muscle c o n t r a c t i o n . Proc. R. Soc. London. 207: 259-286. E i d , H., and Champlain, J .D. 1988. Increased i n o s i t o l monophosphate p roduct ion i n c a r d i o v a s c u l a r t i s s u e s of DOCA-salt hyper tens ive r a t s . Hypertens ion, 12: 122-129. F i s h , R.D., S p e r t i , G., C o l u c c i , W.S., and Clapham, D.E. 1988. Phorbol e s t e r inc reases the d i h y d r o p y r i d i n e - s e n s i t i v e ca l c i um conductance i n a v a s cu l a r smooth muscle c e l l l i n e . C i r c . Res. 62: 1049-1054,. Forder, J . , S c r i a b i n e , A., and Rasmussen, H. 1985. Plasma membrane ca l i um f l u x , p r o t e i n k inase C a c t i v a t i o n and smooth muscle a c t i v a t i o n . J . Pharmacol. Exp. Ther. 235: 267-273. Fox, A.W., A b e l , P.W., and Minneinan, K.P. 1985. A c t i v a t i o n o f c q -adrenoceptors inc reases [ 3 H ] i n o s i t o l metabol ism i n r a t vas deferens and caudal a r t e r y . Eur. J . Pharmacol. 116: 145-152. Friedmann, J . J . 1989. Vascu la r s e n s i t i v i t y and r e a c t i v i t y to norep inephr ine i n d iabetes m e l l i t u s . Am. J . P h y s i o l . 256: HI134-H1138. Furchgot t , R.F. 1983. Role of endothel ium in responses o f v a s cu l a r smooth muscle. C i r c . Res. 53: 557-573. Gabby, K.H. 1975. Hyperglycemia, po l yo l metabol ism, and comp l i c a t i on of d iabetes m e l l i t u s . Ann. Rev. Med. 26: 521-536. G a r c i a , M . J . , McNamara, P.M., Gordon, T., and K i n n e l l , W.B. 1974. Mo rb i d i t y and m o r t a l i t y i n d i a b e t i c s i n the Framingham p o p u l a t i o n : s i x t e e n - y e a r f o l l o w up study. D iabetes , 23: 104-111. G e r t h o f f e r , W.T., and Murphy, R.A. 1983. C a 2 + , myosin phosphory la t ion and r e l a x a t i o n of a r t e r i a l smooth muscle. Am. J . P h y s i o l . 245: C271-C277. 204 Gleason, M.M., and F l a im, S.F. 1986. Phorbol e s t e r c on t r a c t s r a b b i t t h o r a c i c ao r ta by i n c r ea s i n g i n t r a c e l l u l a r Ca and by a c t i v a t i n g ca l c i um i n f l u x . Biochem. Biophys. Res. Comm. 138: 1362-1369. Gompertz, B.D. 1983. Involvement o f guanine n u c l e o t i d e - b i n d i n g p r o t e i n i n the ga t i ng o f C a z + by r e cep to r s . Nature, 306: 64-66. G r i e n d l i n g , K.K., Berk, B.C., Gimbrone, M.A., and A lexander , R.W. 1987. Regu lat ion o f ang io tens in 1 1 - d i a c y l g l y c e r o l fo rmat ion by i n t r a c e l l u l a r pH i n c u l t u r e d va s cu l a r smooth muscle c e l l s . C i r c u l a t i o n (Abs t r . ) 76: 244. G r i e n d l i n g , K.K., Berk, B.C., Socorro, L., Tsuda, T., De l a f on t a i ne , P., and A lexander, R.W. 1988. Secondary s i g n a l l i n g mechanisms i n ang io ten s i n I I - s t i m u l a t e d va s cu l a r smooth muscle c e l l s . C l i n . Exp. Pharmacol. P h y s i o l . 15: 105-112. G r i e n d l i n g , K.K., R i t tenhouse, S.E., Brock, T.A., E k s t e i n , L.S., Gimbrone, M.A., and A lexander, R.W. 1986. Susta ined d i a c y l g l y c e r o l format ion from i n o s i t o l pho spho l i p i d i n ang io tens in I I - s t i m u l a t e d va s cu l a r smooth muscle c e l l s . J . B i o l . Chem. 261: 5901-5906. Grover, A.K., Kannan, M.S., and D a n i e l , L. 1980. Canine t r a chea l i s membrane f r a c t i o n a t i o n and c h a r a c t e r i z a t i o n . C e l l Ca lc ium, 1: 135-140. H a d i j i c o n s t a n t i n o u , M., Qu, J . X . , F e t t e r s , S.E.M., and Ne f f , N.H. 1988. Apparent l o s s o f Gi p r o t e i n a c t i v i t y i n the d i a b e t i c r a t i n a . Eur J . Pharmacol. 149: 193-194. H a l l , I.P., Donaldson, J . , and H i l l , S . J . 1990. modulat ion o f f l uo roa l um ina te - i nduced i n o s i t o l phosphate format ion by inc reases i n t i s s u e c y c l i c AMP content i n bovine t r a chea l smooth muscle. Br. J . Pharmacol. 100: 646-650. H a l l c h e r , L.M., and Sherman, W.R. 1980. The e f f e c t s o f l i t h i u m ion and o ther agents on the a c t i v i t y of myo- inos i to l - 1 -phosphata se from bovine b r a i n . J . B i o l . Chem. 255: 10896-10901. Harden, T.K. 1989. The r o l e of guanine nuc l eo t i de r e g u l a t o r y p r o t e i n s i n r e c e p t o r - s e l e c t i v e d i r e c t i o n of i n o s i t o l s i g n a l l i n g . In: I n o s i t o l l i p i d s i n c e l l s i g n a l l i n g . Ed i ted by R.H. M i c h e l l , A.H. Drummond, and C P . Downes. Academic Press , London, pp. 113-133. H a r r i s , K.H., and MacLeod, K.M. 1988. In f luence o f the endothel ium on c o n t r a c t i l e responses of a r t e r i e s from d i a b e t i c r a t s . Eur. J . Pharmacol. 153: 55-64. 205 Hashimoto, S., and Dayton, S. 1977. In f luence o f d i e t a r y s t a tu s and d iabetes on a o r t i c acyl-CoA hydro lase a c t i v i t y . A t h e r o s c l e r o s i s , 26: 289-296. Hashimoto, T., H i r a t a , M., I t oh , T., Kanmura, Y., and Kuriyama, H. 1986. I n o s i t o l 1 ,4 ,5 , - t r i sphosphate a c t i v a t e s pharmacomechanical coup l i n g i n smooth muscle of the r a b b i t mesenter ic a r t e r y . J . P h y s i o l . (Lond.) 370: 605-618. Hawkins, P.T., B e r r i e , C P . , M o r r i s , A . J . , and Downes, C P . 1987. I n o s i t o l 1 , 2 - c y c l i c 4 , 5 - t r i s pho spha te i s not a product of musca r in i c r e c e p t o r - s t i m u l a t e d phosphat idy l i n o s i t o l 4 ,5 -biphosphate h y d r o l y s i s i n r a t p a r o t i d g lands . Biochem. J . 243: 211-218. Head, R . J . , Longhurst, P.A., Panek, R.L., and S t i t z e l . 1987. A c o n t r a s t i n g e f f e c t o f the d i a b e t i c s t a t e upon the c o n t r a c t i l e responses o f a o r t i c p repara t i ons from the r a t and r a b b i t s . Br. J . Pharmacol. 91: 275-286. Heagerty, A.M., and Ol lerenshaw, J.D. 1987. The phospho ino s i t i de s i g n a l l i n g system and hyper tens ion . J . Hypertens ion, 5: 515-524. Heagerty, A.M., Ol lerenshaw, J .D . , and Swales, J .D. 1986. Abnormal v a s cu l a r phospho inos i t i de h y d r o l y s i s i n the spontaneously hyper tens i ve r a t . Br. J . Pharmacol. 89: 803-807. Heathers, G.P., Co r r , P.B., and Rubin, L . J . 1988. T ran s i en t accumulat ion o f i n o s i t o l ( 1 , 3 , 4 , 5 , ) t e t r ak i s pho spha te i n response to cq -ad rene rg i c s t i m u l a t i o n i n adu l t c a r d i a c myocytes. Biochem. Biophys. Res. Commun. 156: 485-493. Hes lop, J . P . , I r v i n e , R.F., T a s h j i a n , A .H. , and Be r r i dge , M.J . 1985. I n o s i t o l t e t r a k i s - and pentakisphosphates i n GH4 c e l l s . J . Exp. B i o l . 119: 395-401. H o f t i e z e r , V., and Carpenter, A.M. 1973. Comparison o f s t r e p t o z o t o c i n and a l l o xan - i nduced d iabetes i n the r a t , i n c l u d i n g vo lumet r i c q u a n t i t a t i o n of the panc rea t i c i s l e t s . D i a b e t o l o g i a , 9: 178-184. Hok in, M.R.* and Hokin, L.E. 1953. Enzyme s e c r e t i o n and i n c o r p o r a t i o n o f P i Z i n t o phospho l ip id s of pancreas s l i c e s , J . B i o l . Chem. 203: 967-977. Hughes, A.R., Takemura, H., and Putney, J.W., J r . 1988. K i n e t i c s of i n o s i t o l 1 ,4 ,5 - t r i sphosphate and i n o s i t o l c y c l i c 1:2,4,5-t r i pho spha te metabolism i n i n t a c t r a t p a r o t i d a c i n a r c e l l s : 206 R a l a t i o n s h i p to ca l c i um s i g n a l l i n g . J . B i o l . Chem. 263: 1314-1319. Huxley, H.E., and Hanson, J . 1954. Changes i n the c ross s t r i a t i o n of muscle dur ing c o n t r a c t i o n and s t r e t c h and t h e i r s t r u c t u r a l i n t e r p r e t a t i o n . Nature, 173: 973-976. l i n o , M. 1987. Calc ium dependent i n o s i t o l t r i s pho spha te - i nduced ca l c i um r e l ea se i n the gu inea -p ig t a e n i a c o l i . Biochem. Biophys. Res. Commun. 142: 47, 52. Inhorn, R.C., Bansal V.S., and Majerus, P.W. 1987. Pathway f o r 1,3,4-t r i pho spha te and 1,4-biphosphate metabol ism. Proc. N a t l . Acad. S c i . U.S.A. 84: 2170-2174. I r v i n e , R.F., and Moor, R.M. 1986. M i c r o - i n j e c t i o n o f i n o s i t o l 1 ,3 ,4 ,5 - te t rak i sphosphate a c t i v a t e s sea u r ch in eggs by a mechanism dependent on ex te rna l Ca . Biochem. J . 240: 917-920. I r v i n e , R.F., and Moor, R.M. 1987. I n o s i t o l ( l , 3 , 4 , 5 ) t e t r a k i s pho spha te - i nduced a c t i v a t i o n of sea u r ch in eggs r equ i r e s the presence of i n o s i t o l t r i pho spha te . Biochem. Biophys. Res. Commun. 146: 284-290. I r v i n e , R.F., Le tcher , A . J . , Heslop, J . P . , and Be r r i dge , M.J. 1986. The i n o s i t o l t r i s / t e t r a k i s p h o s p h a t e pathway- demonstrat ion o f I n s ( l , 4 , 5 )P3 3-k inase a c t i v i t y i n animal t i s s u e s . Nature, 320: 631-634. I r v i n e . R.F. 1986. The s t r u c t u r e , metabolism and a n a l y s i s o f i n o s i t o l l i p i d s and i n o s i t o l and phosphates. In: Phospho inos i t i de s and recep to r mechanisms. Ed i ted by J.W. Putney, J r . A lan R. L i s s , Inc . , New York, PP. 89-107. Ishiyama, Y., Yabu, H., and M i y a zak i , E. 1975. Changes i n c o n t r a c t i l i t y and ca l c ium b ind ing of gu inea -p i g t a e n i a - c o l i by treatment w i th enzymes which hydro lyze s i a l i c a c i d . Jap. J . P h y s i o l . 25: 719-727. I t oh , T., Izumi, H., and Kuriyama, H. 1982. Mechanisms o f r e l a x a t i o n induced by mesenter ic a r t e r y . J . P h y s i o l . (Lond.) 326: 475-493. I t oh , T., Kubota, Y., and Kuriyama, H. 1988. E f f e c t s o f a phorbol e s t e r on a c e t y l c h o l i n e - i n d u c e d C a z + m o b i l i z a t i o n and c o n t r a c t i o n i n the porc ine coronary a r t e r y . J . P h y s i o l . (Lond.) 240: 917-920. 2 0 7 Jackson, C.V., and C a r r i e r , G.O. 1982. S u p e r s e n s i t i v i t y o f i s o l a t e d mesenter ic a r t e r y to noradrena l ine i n the long- term exper imental d i a b e t i c r a t . J . Auton. Pharmacol. 1: 399-405. J i a n g , M . J . , and Morgan, K.G. 1987. I n t r a c e l l u l a r ca l c i um l e v e l s i n phorbol e s te r - i nduced c o n t r a t i o n o f v a s cu l a r muscle. Am. J . P h y s i o l . 253: H1365-H1371. Jones, A.W., Ge i sbuh le r , B.B., Shukla, S.D., and Smith, J .M. 1988. A l t e r e d b iochemical and f u n c t i o n a l responses i n ao r ta from hyper tens ive r a t s . Hypertens ion, 11: 627-634. Jones, R.L., and Peterson, C M . 1981. Hematologic a l t e r a t i o n s i n d iabetes m e l l i t u s . Am. J . Med. 70: 339-352. Joseph, S.K., Thomas, A .P . , W i l l i a m s , R . J . , I r v i n e , R.F., and W i l l i amson , J.R. 1984. M y o - i n o s i t o l 1 ,4 ,5 - t r i sphosphate : a second messenger f o r the hormonal m o b i l i z a t i o n o f i n t r a c e l l u l a r C a z + i n l i v e r . J . B i o l . Chem. 259: 3077-3081. Kahn, C R . 1985. Pathophys io logy o f d iabetes m e l l i t u s : An overv iew. In: J o s l i n ' s Diabetes M e l l i t u s (12th Ed.) Ed i ted by A. Marb le, P. White, R.F. B rad ley , and L.P. K r a l l , Lea and Feb iger , P h i l a d e l p h i a , pp.43-50. Kahn, C.R., Ka ra s i k , A., Rothenberg, P., Becker, J . , and White, M.F. 1989. How i n s u l i n works- shedding some more l i g h t i n t o the b lack box. In: Diabetes 1988, Ed i ted by R.G. La rk ing s , P.Z. Zur imet, and D.J. Chisholm. Excerpta Medica, Amsterdam, New York, Oxford, pp. 3-6. Kahn, C.R., Meghesi, K., and Ba r r , R.S. 1977. Receptors f o r pept ide hormones. Ann. I n te rn . Med. 86: 205-219. K a i b u c h i , k., T aka i , Y., and N i s h i z uka , Y. 1981. Cooperat ive r o l e s of va r i ous membrane phospho l ip id s i n the a c t i v a t i o n of c a l c i u m -a c t i v a t e d pho spho l i p i d - dependent p r o t e i n k i na se . J . B i o l . Chem. 256: 7146-7149. Kannel , W.B., and McGee, D.L. 1979. Diabetes and c a r d i o v a s c u l a r r i s k f a c t o r s . The Framingham Study. C i r c u l a t i o n , 59: 8-13. K a r a k i , H., Kubota, H., and Urakawa, N. 1979. M o b i l i z a t i o n o f s to red ca l c i um f o r phas ic c o n t r a c t i o n induced by norep inephr ine i n r a b b i t a o r t a . Eur. J . Pharmacol. 56: 237-245. K e f a l i d e s , N.A. 1981. Basement membrane research i n d i abetes m e l l i t u s . Co l lagen R e l . Res. 1: 295-299. 208 K h a l i l , R.A., and Van Breemen, C. 1988. Susta ined c o n t r a c t i o n o f v a s cu l a r smooth muscle: ca l c ium i n f l u x or C-k inase a c t i v a t i o n ? J . Pharmacol. Exp. Ther. 244: 537-541. Kikkawa, U., Taka i , Y., Tanaka, Y., Myake, R., and N i s h i z u k a , Y. 1983. P r o t e i n k inase C as a p o s s i b l e r ecep to r p r o t e i n o f tumor-promoting phorbol e s t e r s . J . B i o l . Chem. 258: 11442-11445. K i r k , C . J . , Creba, J . A . , Downes, C P . , and M i c h e l l , R.H. 1981. Hormone-stimulated metabolism of i n o s i t o l l i p i d s and i t s r e l a t i o n s h i p s to hepat i c recepto r f u n c t i o n . Biochem. Sco. Trans. 9: 377-411. Koj ima, I., Koj ima, K., K r e u t t e r , D., and Rasmussen, H. 1984. The temporal i n t e g r a t i o n o f the a ldosterone s ec re to r y response to ang io tens in occurs v i a two i n t r a c e l l u l a r pathways. J . B i o l . Chem. 259: 14448-14457. K r a f t , A.S. , and Anderson, W.B. 1983. Phorbol e s t e r s i nc rease the amount of Ca , phosphol ip id-dependent p r o t e i n k inase a s soc i a ted w i th plasma membrane. Nature, 301: 621-623. K r o l e w s k i , A.S . , Czyzyk, A. , Janecko, D., and Kopczynsk i , J . 1977. M o r t a l i t y from c a r d i o v a s c u l a r d i seases among d i a b e t i c s . D i a b e t o l o g i a , 13: 345-350. Langlands, J .M . , Rodger, I.M., and Diamond, J . 1989. The e f f e c t of M & B 22948 on methacol ine and h i s tamine- induced c o n t r a c t i o n and i n o s i t o l 1 ,4 ,5 - t r i sphosphate l e v e l s i n gu inea -p i g t r a chea l t i s s u e . Br. J . Pharmacol. 98: 336-338. Lefebvre , P . J . , and Luyckx, A.S. 1979. Glucagon and d i a b e t e s ; A r e a p p r a i s a l . D i a b e t o l o g i a , 16: 347-354. L e f f e l h o l z , K. 1989. Receptor r e g u l a t i o n i n c h o l i n e pho spho l i p i d h y d r o l y s i s . Biochem. Pharmacol. 38: 1543-1549. Legan, E. 1989. E f f e c t s of s t r ep t o zo t o c i n - i nduced hyperglycemia on a g o n i s t - s t i m u l a t e d phosphat idy l i n o s i t o l tu rnover i n r a t a o r t a . L i f e S c i . 45: 371-378. Legan, E., and S i s son . J .A . 1990. Method to denude r a t a o r t i c endothel ium wi th saponin f o r phospho inos i t i de a n a l y s i s i n v a s cu l a r smooth muscle. J . Pharmacol. Methods, 23: 31-39. 209 Legan, E., Chernow, B., P a r i l l o , J . , and Roth, B . J . 1985. A c t i v a t i o n o f phosphat idy l i n o s i t o l tu rnover i n r a t ao r t a by cq -ad rene rg i c r ecep to r s t i m u l a t i o n . Eur. J . Pharmacol. 110: 385-390. Lernmark, A., Freedman, Z.R., Hofmann, C , Rubenste in, A .H. , S t e i n e r , D.F., Jackson, R.L., Winter, R . J . , and Fraisman, H.S. 1978. I s l e t - c e l l su r face an t i bod ie s i n j u v e n i l e d i a b e t e s . N. Eng l . J . Med. 299: 375-380. Levy, J . , Suzuk i , Y., A v i o l i , L.V., Grunberger, G., and Gav in , J .R. 1988. Plasma membrane phospho l i p id content i n n o n - i n s u l i n -dependent s t r e p t o z o t o c i n - d i a b e t i c r a t s - e f f e c t o f i n s u l i n . D i a b e t o l o g i a , 31: 315-321. L i t o s c h , I., W a l l i s , C , and F a i n , J . N . 1985. 5-hydroxytreptamine s t imu l a te s i n o s i t o l phosphate product ion i n a c e l l - f r e e system from b l o w f l y s a l i v a r y g lands. J . B i o l . Chem. 260: 5464-5471. Logar s t , H., Pozzan, T., Waldvogel, F.A., and Lews, P.D. 1984. Phorbol m y r i s t a t e acetate s t imu la te s TPA-dependent ca l c i um t r an spo r t by the plasma membrane v e s i c l e s . J . C l i n . Invest . 73: 878-885. Longhurst, P.A., and Head, R.J. 1985. Responses o f the i s o l a t e d perfused mesenter ic va s cu l a tu re from d i a b e t i c r a t s : The s i g n i f i c a n c e of appropr i a te c on t r o l t i s s u e s . J . Pharmacol. Exp. Ther. 235: 45-49. Lynch, C . J . , Blackmore, P.F., Johnson, E.H., Wange, R.L., Krone, P.K., and Exton, J . H . 1989. Guanine nuc l eo t i de b ind ing p r o te i n s and adenylate c y c l a se i n l i v e r s of s t r e p t o z o t o c i n - and BB/Wor-diabet ic r a t s . J . C l i n . Invest . 83: 2050-2062. MacLeod, K.M. 1985. The e f f e c t o f i n s u l i n treatment on changes i n v a s cu l a r r e a c t i v i t y i n c h r o n i c , exper imental d i abe te s . D iabetes , 34: 1160-1167. Macleod, K.M., and M c N e i l l , J . H . 1981. The e f f e c t s of a l l o x a n or s t r e p t o z o t o c i n on responses o f r a t v a s cu l a r smooth muscle to va soac t i ve agents. Proc. West. Pharmacol. Soc. 24: 69-71. Macleod, K.M., and M c N e i l l , J . H . 1982. Alpha adrenoceptor-mediated responses i n ao r ta from three month s t r e p t o z o t o c i n d i a b e t i c r a t s . Proc. West. Pharmacol. Soc. 25: 245-247. MacLeod, K.M., and M c N e i l l , J . H . 1985. The i n f l u e n c e o f ch ron i c exper imental d iabetes on c o n t r a c t i l e responses o f r a t i s o l a t e d blood v e s s e l s . Can. J . P h y s i o l . Pharmacol. 63: 52-57. 2 1 0 Marc, S. L e i be r , D., and Harbon, S. 1988. F luoroa luminate mimic mu sca r i n i c - and oxy toc in - recepto r -med ia ted generat ion o f i n o s i t o l phosphates and c o n t r a c t i o n i n the i n t a c t gu inea -p i g myometrium. Biochem. J . 255: 705-713. Marston, S.B., and Smith, C.W. 1985. The t h i n f i l a m e n t s o f smooth muscle. J . Muscle Res. C e l l M o t i l . 6: 669-670. McGrath, J . C . , Brown, C M . , and Wi l son , V.G. 1989. A lpha -adrenoceptors : a c r i t i c a l rev iew. Med. Res. Rev. 9: 407-533. McM i l l a n , D.E. 1975. D e t e r i o r a t i o n o f the m i c r o c i r c u l a t i o n i n d i abe te s . D iabetes , 24: 944-957. McM i l l an , D.E. 1976. Plasma p r o t e i n changes, blood v i s c o s i t y and d i a b e t i c microangiopathy. D iabetes , 25(Suppl. 2 ) : 852-864. M e r r i t t , J . E . , Tay l o r , C.W., Rubin, R.P., and Putney, J.W. 1986. Evidence suggest ing tha t a novel guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n couples recepto r s to phposphol ipase C i n exoc r i ne pancreas. Biochem. J . 236: 337-343. M i c h e l l , R.H. 1975. I n o s i t o l phospho l ip id s and c e l l su r face recep to r f u n c t i o n . Biochem. Biophys. A c t a , 415: 81-147. M i c h e l l , R.H., K i r k , C . J . , Jones, L.M., Downes, C P . , and Creba, J .A . 1981. The s t i m u l a t i o n of i n o s i t o l l i p i d metabol ism tha t accompanied ca l c ium m o b i l i z a t i o n i n s t imu l a ted c e l l s : de f i ned c h a r a c t e r i s t i c s and unanswered ques t i on s . P h i l Trans. R. Soc. Lond. 296: 123-137. M i l l e r , J .R . , Hawkins, D.J . , and We l l s , J . N . 1986. Phorbol d i e s t e r s a l t e r the c o n t r a c t i l e responses o f porc ine coronary a r t e r y . J . Pharmacol. Exp. Ther. 239: 38-42. Minneman, K.P. 1988. a ] - ad rene r g i c r ecep to r subtypes, i n o s i t o l phosphates, and sources o f c e l l Ca . Pharmacol. Rev. 40: 87-119. Morgan, J . P . , and Morgan, K.G. 1983. Vascu la r smooth muscle: The f i r s t recorded C a z + t r a n s i e n t . P f l uge r s A rch . 395: 75-75. Morgan, J . P . , and Morgan, K.G. 1984. A l t e r a t i o n s o f cy top la smic i o n i z e d ca l c i um l e v e l s i n smooth muscle by v a s o d i a l a t o r s i n the f e r r e t . J . P h y s i o l . (Lond.) 357: 539-551. 2 1 1 Mor i son, A.D., Clements, R.S., and Winegard, A . I . 1972. E f f e c t s of e l eva ted g lucose concent ra t i on s on the metabolism o f the a o r t i c w a l l . J . C l i n . Invest . 51: 3114-3123. M o r r i s , A .P . , G a l l a c h e r , D.V., I r v i n e , R.F., and Peter son, O.H. 1987. Synergism o f i n o s i t o l t r i s pho spha te and te t r ak i s pho spha te i n a c t i v a t i n g Ca -dependent K + channels . Nature, 330: 653-655. Muallem, S., Pandol, S . J . , and Baker, T.G. 1988. Two components of hormone-evoked ca l c i um re l ea se from i n t r a c e l l u l a r s to re s of panc rea t i c a c i na r c e l l s . Biochem. J . 255: 301-307. M u e l l e r , S.M., Mue l l e r , R.M., and E r t e l , P . J . 1982. Sympathetic and va s cu l a r dy s f unc t i on i n e a r l y exper imental j u v e n i l e d iabetes m e l l i t u s . Am. J . P h y s i o l . 243: H139-H144. Mulcahy, P.D., and Winegrad, A. 1962. E f f e c t s of i n s u l i n and a l l o x a n d iabetes on g lucose metabolism i n r a b b i t a o r t i c t i s s u e . Am. J . P h y s i o l . 203: 1038-1042. Mul laney, J . M . , Church, S.H., Ghosh, T.K., and G i l l , D.L. 1987. I n t r a c e l l u l a r ca l c i um uptake a c t i v a t e d by GTP: evidence f o r a p o s s i b l e guan ine -nuc leo t ide induced transmembrane conveyance o f i n t r a c e l l u l a r c a l c i um. J . B i o l . Chem. 262: 13865-13872. Mul laney, J .M . , Yu, M., Ghosh, T.K., and G i l l , D.L. 1988. Calc ium ent ry i n t o the i n o s i t o l 1 , 4 , 5 - t r i pho spha te - r e l ea s ab l e ca l c i um pool i s mediated by a GTP-regulatory mechanism. Proc. N a t l . Acad. S c i . U.S.A. 85: 2499-2503. Nakak i , J . , Roth, B.L., Chuang, D.M., and Costa, E. 1985. Phas ic and t o n i c components of 5-HT2 receptor -mediated r a t ao r ta c o n t r a c t i o n : p a r t i c i p a t i o n of C a ^ + channels and phosphol ipase C. J . Pharmacol. Exp. Ther. 234: 442-446. Nat iona l Diabetes Data Group. 1979. C l a s s i f i c a t i o n and d i agno s i s o f d iabetes m e l l i t u s and other ca tego r i e s o f g lucose i n t o l e r a n c e . D iabetes , 28: 1039-1057. Nerup, J . 1978. HLA s tud ie s i n d iabetes m e l l i t u s : a rev iew. Adv. Med. D i s o rd . 9: 263. N i e d e l , J . E . , Kunn, L . J . , and Vandenbark, G.R. 1983. Phorbol d i e s t e r r e cep te r c o p u r i f i e s w i th p r o t e i n k inase C. Proc. N a t l . Acad. S c i . U.S.A. 80: 36-40. p i N i g g l i , V., S i g e l , E., and C a r a f o l i , E. 1982. The p u r i f i e d C a t _ r pump o f human e r y t h r o c y te membrane c a t a l y z e s an e l e c t r o n e u t r a l Ca - H + 2 1 2 exchange i n r e c o n s t i t u t e d lyposomal systems. J . B i o l . Chem. 257: 2350-2356. N i sh imura, J . , K h a l i l , R.A., and Van Breemen, C. 1987. Agon i s t - i nduced v a s cu l a r tone. Hypertens ion, 13: 835-844. N i s h i z u ka , Y. 1984a. The r o l e o f p r o t e i n k inase C i n c e l l su r face s i gna l t r an sduc t i on and tumor promotion. Nature, 308: 693-698. N i s h i z u ka , Y. 1984b. Turnover of i n o s i t o l phospho l i p id s and s i gna l t r a n s d u c t i o n . Sc ience, 225: 1365-1370. N i s h i z u ka , Y. 1986. S tud ies and pe r spec t i ve of p r o t e i n k inase C. Sc ience , 233: 305-312. N i s h i z u ka , Y. 1988. The molecu lar hererogen i ty o f p r o t e i n k inase C and i t s i m p l i c a t i o n s f o r c e l l u l a r r e g u l a t i o n . Nature, 334: 661-665. Owen, M.P., and C a r r i e r , G.O. 1980. Calc ium dependence of nonepinephr ine- induced va scu l a r c o n t r a c t i o n i n exper imental d i abe te s . J . Pharmacol. Exp. Ther. 212: 253-258. Palmer, S., Hughes, K.T., Lee, D.Y., and Wakelam, M.J.O. 1989. Development o f a nove l , I n s ( l , 4 , 5 ) P 3 - s p e c i f i c b ind ing assay. I t s use to determine the i n t r a c e l l u l a r concen t r a t i on o f I n s ( l , 4 , 5 )P3 i n unst imulated and va sop re s s i n - s t imu l a ted r a t hepatocytes . C e l l u . S i g n a l l i n g , 1: 147-156. Pato, M.D., and A d e l s t e i n , R.S. 1980. Dephosphory lat ion o f the 20,000-da l t on l i g h t cha in of myosin by two d i f f e r e n t phosphatases from smooth muscle. J . B i o l . Chem. 255: 6535-6538. P e t r i d e s , P., Weiss, L., L o f f l e r , G., and Wei land, O.H. 1978. Diabetes m e l l i t u s : Theory and P r a c t i c e . Urban and Schwarzenberg, Ba l t imo re . Pfaf fman, M.A., B a l l , C.R., Darby, A., and Hi lman, R. 1982. I n s u l i n r e v e r s a l of d i abetes - i nduced i n h i b i t i o n o f v a s cu l a r c o n t r a c t i l i t y i n the r a t . Am. J . P h y s i o l . 242: H490-H495. Pfaf fman, M.A., Hi lman, R., and Darby, A. 1980. C o n t r a c t i l e and r e l a x i n g a c t i v i t y o f a r t e r i a l smooth muscle from s t r e p t o z o t o c i n -d i a b e t i c r a t s . Res. Commun. Chem. P a t h o l . Pharmacol. 30 (2 ) : 283-299. P i j u a n , V., and L i t o s c h , I. 1988. Norepinephr ine s t imu l a t e s the p roduct ion of i n o s i t o l t r i pho sphate and i n o s i t o l t e t rek i spho spha te i n r a t a o r t a . Biochem Biophys. Res. Commun. 156: 240-245. 2 1 3 Porv ing , H.H. 1976. Increased m ic rova scu l a r p e r m e a b i l i t y to plasma p r o t e i n i n s ho r t - and long-term j u v e n i l e d i a b e t i c s . D iabetes , 25(Suppl . 2 ) : 884-889. Putney, J.W. 1986. A model f o r r e cep to r - r e gu l a t ed ca l c i um e n t r y . C e l l Ca lc ium. 7: 1-12 Putney, J.W. 1987. Ca l c i um-mob i l i z i n g r e cep to r s . Trends i n Pharmacol. S c i . 8: 481-486. Putney, J.W., P o g g i o l i , J . , and Weiss, S . J . 1981. Receptor r e g u l a t i o n o f ca l c i um r e l ea se and ca l c ium p e r m e a b i l i t y i n p a r o t i d g land c e l l s . P h i l . Trans. R. Soc. Lond. B 296: 37-45. Putney, J.W., Takemura, H., Hughes, A.R., Horstman, D.A., and Thastrup, A.O. 1989. How do i n o s i t o l phosphates r e gu l a t e ca l c i um s i g n a l i n g ? FASEB J . 3: 1899-1905. Raeymaekers, L., Wuytack, F., and Ca s t ee l s , R. 1985. S u b c e l l u l a r f r a c t i o n a t i o n of Dig stomach smooth muscle. A study o f the d i s t r i b u t i o n of Ca - M g z + ATPase a c t i v i t y i n plasma membrane and endoplasmic r e t i c u l u m . Biochem. Biophys. A c t a , 815: 441-454. Rahbar, S. 1981. G l yco sy l a ted hemoglobins. Texas Rep. B i o l . Med. 40: 373-385. Ramanadhan, S., and Tenner, T.E. 1984. A l t e r a t i o n s i n a o r t i c and t a i l a r t e r y r e a c t i v i t y to agon i s t s a f t e r STZ t reatment . Can. J . P h y s i o l . Pharmacol. 62: 418-423. Rapoport, R.M. 1987. E f f e c t s of norep inephr ine on c o n t r a c t i o n and h y d r o l y s i s o f phosphat idy l i n o s i t o l s i n r a t a o r t a . J . Pharmacol. Exp. Ther. 242: 188-194. Rasmussen, H. 1986. The ca l c i um messenger system. N. Eng. J . Med. 314: 1164-1170. Rasmussen, H., and B a r r e t t , P.Q. 1984. Calc ium messenger system. An i n t e g r a t ed view. P h y s i o l . Rev. 64: 938-984. Rasmussen, H., B a r r e t t , P., Takuwa, Y., and A p f e l d o r f , W. 1987. Calc ium in the r e g u l a t i o n of a ldosterone s e c r e t i o n and v a s cu l a r smooth muscle c o n t r a c t i o n . Hypertens ion (Suppl I ) , 1-23-1-26. Rasmussen, H., Forder, J . , Koj ima, I., and S c r i a b i n e , A. 1984. TPA-induced c o n t r a c t i o n o f i s o l a t e d r a b b i t v a s cu l a r smooth muscle. Biochem. Biophys. Res. Comm. 122: 776-784. 2 1 4 Rasmussen, H., Takuwa, Y., and Park, S. 1987. P r o t e i n k inase C i n the r e g u l a t i o n of smooth muscle c o n t r a c t i o n . FASEB J . 1: 177-185. Rasmussen, H., Zawa l i ch , W., and Koj ima, I. 1985. Ca and cAMP i n the r e g u l a t i o n o f c e l l f u n c t i o n . In: Calc ium and C e l l Phys io logy . Ed i ted by D. Marme. Sp r i n ge r -Ve r l a g . New York, pp. 1-17. R e i n i l a , A. , K o i v i s t o , V.A., and Akerbom, H.K. 1977. L i p i d s i n the pulmonary a r t e r y and the lungs o f severa l d i a b e t i c r a t s . A h i s tochemica l and chemical study. D i a b e t o l o g i a , 13: 305-310. Renold, A .E . , M i n t z , D.H., M u l l e r , W.A., and C a h i l l , G.F. 1978. Diabetes m e l l i t u s . In: The metabo l i c bas i s o f i n h e r i t e d d i s ea se . Ed i ted by J . B . Stombury, J . B . Wyagaarden and D.S. F r ed r i c k s on . McGraw-H i l l , New York, pp. 79-109. Rerup, C.C. 1970. Drugs producing d iabetes through damage o f the i n s u l i n s e c r e t i n g c e l l s . Pharmacol. Rev. 22: 485-518. Reugg, U.T., and Burgess, G.M. 1989. S tau ro spor ine , K-252 and UCN-01: potent but n o n s p e c i f i c i n h i b i t o r s o f p r o t e i n k inase C. Trends i n Pharmacol. S c i . , 10: 218-220. Rodr igues, B., and MCNe i l l , J . H . 1987. Comparison o f c a r d i a c f u n c t i o n i n male and female d i a b e t i c r a t s . Gen. Pharmacol. 18: 421-423. Sa ida , K., and Van Breemen, C. 1984. C h a r a c t e r i s t i c s o f the n o r a d r e n a l i n e - s e n s i t i v e C a 2 + s t o re i n v a s cu l a r smooth muscle. Blood Ve s se l s , 21: 43-52. Sa lans , S.B. 1982. Diabetes M e l l i t u s . A d i sease t ha t i s coming i n t o f ocu s . J . Am. Med. Assoc. 247: 590-594. S a l t i e l , A.R. 1990. Second messengers of i n s u l i n a c t i o n . D i a b e t i c Care, 13: 244-256. Sato, K., Ozak i , H., and K a r a k i , H. 1988. Changes i n c y t o s o l i c ca l c ium l e v e l i n v a s cu l a r smooth muscle s t r i p measured s imu l taneous l y w i th c o n t r a c t i o n us ing f l u o r e s c e n t ca l c ium i n d i c a t o r f u r a - 2 . J . Pharmacol. Exp. Ther. 246: 294-300. Scarborough, N.L., and C a r r i e r , G.0. 1984. N i f e d i p i n e and a l pha -adrenoceptors i n r a t ao r ta I I . Role of e x t r a c e l l u l a r ca l c i um i n enhanced a lpha-2 adrenoceptor-mediated c o n t r a c t i o n i n d i abe te s . J . Pharmacol. Exp. Ther. 231: 603-608. 2 1 5 Scarborough, N.L., and C a r r i e r , G.O. 1983. Increased a lpha-2 adrenoceptor-mediated va s cu l a r c o n t r a c t i o n i n d i a b e t i c r a t s . J . Auton. Pharmacol. 3: 177-183. Sekar, M.C., Dixon, J . F . , and Hokin, L.E. 1987. The format ion of i n o s i t o l 1 , 2 - c y c l i c 4 ,5 - t r i sphospha te and i n o s i t o l 1 , 2 - c y c l i c 4-bisphosphate on s t i m u l a t i o n o f mouse panc rea t i c m i n i l o b u l e s w i th ca rbamy lcho l i ne . J . B i o l . Chem. 262: 340-344. Shears, S.B., Pa r ry , J . B . , Tang, E.K.Y., I r v i n e , r . F . , M i c h e l l , R.H., and K i r k , C . J . 1987. Metabol ism of D -myo- inos i to l 1,3,4,5-te t rak i spho spha te by r a t l i v e r , i n c l u d i n g the s yn thes i s o f a novel isomer of myo - i no s i t o l t e t r ak i s pho spha te . Biochem. J . 246: 139-147. S i b l e y , W.A. 1982. The neuropathies of d i abetes m e l l i t u s . In: Management of Diabetes M e l l i t u s . Ed i ted by R. B r e s s f e r , and D.J. Johnson. John Wright, Boston, B r i s t o l , London, pp. 249-269. S i l v e r , P . J . , and S t u l l , J . P . 1984. Phosphory la t ion o f myosin l i g h t cha in and phosphorylase i n t r a chea l smooth muscle i n response to KC1 and ca rbacho l . Mol . Pharmacol. 25: 267-274. S i l v e r . P . J . , Lepore, R.E., Cumiskey, W.R., K i e f e r , D., and H a r r i s , A.L. 1988. P r o t e i n k inase C a c t i v i t y and r e a c t i v i t y to Phorbol e s t e r i n v a s cu l a r smooth muscle from spontaneously hyper tens i ve r a t s (SHR) and normotansive Wis tar Kyoto r a t s (WKY). Biochem. Biophys. Res. Comm. 84: 272-277. S inger , H.A., and Baker, K.M. 1987. Calc ium dependence o f phorbol 12, 13 -d ibu ty ra te - i nduced f o r ce and myosin l i g h t cha in phosphory la t ion i n a r t e r i a l smooth muscle. J . Pharmacol. Exp. Ther. 243: 814-820. S jogren, J.W., and Edvinsson, L. 1988. Vasomotor changes i n i s o l a t e d coronary a r t e r i e s from d i a b e t i c r a t s . Acta P h y s i o l . Scand. 134: 429-436. Smith, C D . , Lane, B.C., Kusaka, I., Verghess, M.W., and Snyderman, R. 1985. Chemoattachment recepto r - i nduced h y d r o l y s i s of phosphat idy l i n o s i t o l 4,5-b isphosphate i n human polymorphonuclear l eukocyte membranes: requirements f o r a guanine n u c l e o t i d e r e g u l a t o r y p r o t e i n . J . B i o l . Chem. 260: 5875-5878. Somylo, A . P . , and Himpens, B. 1989. C e l l ca l c ium and i t s r e g u l a t i o n i n smooth muscle. FASEB J . 3: 2266-2267. 2 1 6 Somylo, A .P . , Walker, J.W., Goldman, Y .E . , Trentham, D.R., Kobayosk i , S., K i tazawa, T., and Somylo, A.V. 1988. I n o s i t o l t r i s pho spha te , ca l c i um and muscle c o n t r a c t i o n . P h i l o s . Trans. R. Soc. Lond. B i o l . S c i . 320: 399-414. Somylo, A.V. , and Somylo, A.P. 1968. E lec t ro -mechan i ca l and pharmaco-mechanical coup l i ng i n v a s cu l a r smooth muscle. J . Pharmacol. Exp. Ther. 159: 129-145. Somylo, A.V., Bond, M., Somylo, A .P . , and Scarpa, A.P. 1985. I n o s i t o l t r i s pho spha te ( InsP3)- induced ca l c i um r e l ea se and c o n t r a c t i o n i n v a s cu l a r smooth muscle. Proc. N a t l . Acad. S c i . U.S.A. 82: 5231-5235. Spat, A. , B rad fo rd , P.G., McKinney, J . S . , Rubin, R.P., and Putney, J.W., J r . 1986. A s a tu rab le recepto r f o r 3 2 P - i n o s i t o l - t r i s p h o s p h a t e i n hepatocytes and n e u t r o p h i l s . Nature, 319: 514-516. S t e i nbe r g , D. 1985. The endocr ine pancreas. In: Best and T a y l o r ' s P h y s i o l o g i c a l Bas i s o f Medical P r a c t i c e (Chapter 49) , 11th Ed. Ed i ted by J . B . West. W i l l i ams and W i l k i n s , Ba l t imo re , pp. 818-833. S te rnwe i s , P.C., and Gi lman, A.G. 1982. Aluminium: A requirement f o r a c t i v a t i o n o f the r e gu l a t o r y components o f adeny late c y c l a s e by f l u o r i d e . Proc. N a t l . Acad. SC i . U.S.A. 79: 4888-4891. S torey , D . J . , Shears, S.B., K i r k , C . J . , and M i c h e l l , R.H. 1984. Stepwise enzymatic dephosphory lat ion o f i n o s i t o l 1,4,5-t r i s pho spha te to i n o s i t o l i n l i v e r . Nature, 312: 374-376. S t reb , M., I r v i n e , R.F., Be r r i dge , M . J . , and Schu l z , I. 1983. Release of C a 2 + from a nonmitochondr ia l i n t r a c e l l u l a r s t o re i n panc rea t i c a c i na r c e l l s by i n o s i t o l - 1 , 4 , 5 - t r i s p h o s p h a t e . Nature, 306: 67-69. Suematsu, E., H i r a t a , M., Hashimoto, T., and Kuriyama, H. 1984. I n o s i t o l 1 ,4 ,5 - t r i sphosphate re lea se s C a 2 + from i n t r a c e l l u l a r s torage s i t e s i n skinned s i n g l e c e l l s o f po rc ine coronary a r t e r y . Biochem. Biophys. Res. Commun. 120: 481-485. S u l l i v a n , S., and Sparks, H.V. 1979. Diminished c o n t r a c t i l e response o f aortas from d i a b e t i c r a b b i t s . Am. J . P h y s i o l . 236: H301-H306. Summerv i l le , L.E., and Hartshorne, D.J. 1986. I n t r a c e l l u l a r ca l c i um and smooth muscle c o n t r a c t i o n . C e l l Ca lc ium, 7: 353-364. 2 1 7 Suppattapone, S., Worley, P.F., Baraban, J .M . , and Snyder, S.H. 1988. S o l u b i l i z a t i o n , p u r i f i c a t i o n , and c h c a r a c t e r i z a t i o n o f an i n o s i t o l t r i s pho spha te r e cep to r . J . B i o l . Chem. 263: 1530-1534. Swamura, M., Kobayashi , Y., Nara, Y., H a t t o r i , K., and Yomori, Y. 1987. E f f e c t o f e x t r a c e l l u l a r ca l c i um on va s cu l a r c o n t r a c t i o n induced by phorbol e s t e r . Biochem. Biophys. Res. Comm. 145: 492-501. S ybe r t z , E . J . , De s i de r i o , D.M., T e t z l o f f , G., and Ch iu , P.J.S. 1986. Phorbol d i b u t y r a t e c o n t r a c t i o n s i n r a b b i t a o r t a : ca l c ium dependence and s e n s i t i v i t y to n i t r o v a s o d i a l a t o r s and 8 - B r - c y c l i c GMP. J . Pharmacol. Exp. Ther. 239: 78-83. T a h i l i a n i , A .G. , Vadlamudi, R.V.S.V., and M c N e i l l , J . H . 1983. Prevent ion and r eve r s a l of a l t e r e d myocardia l f u n c t i o n i n d i a b e t i c r a t s by i n s u l i n t reatment. Can. J . P h y s i o l . Pharmacol. 61: 516-523. Tak i guch i , Y., Satoh, N., Hashimoto, H., and Nakashima, M. 1988. Changes i n v a s cu l a r r e a c t i v i t y i n exper imental d i a b e t i c r a t s : comparison w i th hypothyro id r a t s . Blood Ve s se l s , 25: 250-260. Tamaoki, T. , Nomoto, H., Takamashi, I., Kato, Y., Morimoto, M., and Tomito, F. J 9 8 6 . S taurospor ine , a potent i n h i b i t o r of p h o s p h o l i p i d / C a z + dependent p r o t e i n k inase . Biochem. Biophys. Res. Comm. 135: 397-402. T a y l o r , C.W., and Putney, J.W. 1985. S i ze of the i n o s i t o l 1,4,5-t r i s p h o s p h a t e - s e n s i t i v e ca l c ium pool i n gu inea -p i g hepatocytes . Biochem. J . 232: 435-438. T r i g g l e , D.J. 1983. Ca lc ium, the c on t r o l of smooth muscle f u n c t i o n and broncheal h y p e r r e a c t i v i t y . A l l e r g y , 38: 1-9. T u r l a , M.B., and Webb, C. 1990. Augmented phospho ino s i t i de metabolism i n aortas from g e n e t i c a l l y hyper tens ive r a t s . Am. J . P h y s i o l . 258: H173-H178. Tu r l apa t y , P.D.M.V., Lum, G., and A l t u r a , B.M. 1980. Vascu la r respons iveness and serum biochemical parameters i n a l l o x a n d iabetes m e l l i t u s . Am. J . P h y s i o l . 239: E412-E421. U i , M. 1986. P e r t u s s i s t o x i n as probe of r ecep to r coup l i ng to i n o s i t o l l i p i d metabol ism. In: Phospho inos i t i de and Receptor Mechanisms. Ed i ted by J.W. Putney, J r . A lan R. L i s s , Inc. , New York, pp. 163-196. 2 1 8 U r r u t i a , G., Beai/en, D.W., and C a h i l l , G.F. 1962. Metabol ism of g l u c o s e - C l - C i n r a t ao r ta i n v i t r o . Metabol i sm, 11: 530-534. Van Breemen, C , and Sa ida , K. 1989. C e l l u l a r mechanisms r e g u l a t i n g [ C a ' + ] i n smooth muscle. Ann. Rev. P h y s i o l . 51: 315-329. Van Breemen, C , Wuytack, F., and Ca s t ee l s , R. 1975. S t i m u l a t i o n of 4 t > C a e f f l u x from smooth muscle c e l l s by metabo l i c i n h i b i t i o n and high K + - d e p o l a r i z a t i o n . P f l uge r s A rch . 359: 183-196. Vracko, R., and B e n d i t t , E.P. 1974. Man i f e s t a t i on s o f d iabetes m e l l i t u s - t h e i r p o s s i b l e r e l a t i o n s h i p s to an under l y i ng c e l l d e f e c t . Am. J . P a t h o l . 75: 204-224. Wagner, B., Schachte le , C , and Marme, D. 1987. Phorbol 12,13-d i bu t y r a t e - i nduced c o n t r a c t i o n of i s o l a t e d r a b b i t v a s cu l a r smooth muscle. Eur. J . Pharmacol. 140: 227-232. W a l i , R.K., J a f f e , S., Kumar, D., and K a l r a , V.K. 1988. A l t e r a t i o n s i n o r g a n i z a t i o n o f phospho l ip id s i n e r y t h r ocy te s as f a c t o r i n adherence to e n d o t h e l i a l c e l l s i n d iabetes m e l l i t u s . D iabetes , 37: 104-111. Wa l te r s , M., and Marston, S.B. 1981. Phosphory la t ion o f ca l c i um i o n -regu la ted t h i n f i l amen t s from va scu l a r smooth muscle. Biochem. J . 197: 127-139. Weidmann, P., B e r e t t a - P i c o l i , C , Keusch, G., G luck, Z., Mujag ic , M., Grimm, M., Meier , A., and Z e i g l e r , W.H. 1979. Sodium-volume f a c t o r , c a r d i o v a s c u l a r r e a c t i v i t y and hyper tens ive mechanisms of d i u r e t i c therapy i n m i l d hypertens ion a s soc i a ted w i th d iabetes m e l l i t u s . Am. J . Med. 657: 779-784. Wertheimer, H.E., and Bontor, V. 1960. Age and hormone i n f l u ence s on a o r t i c t i s s u e metabol ism. A rch . K r e i s l a u f f o r s c h , 33: 25-33. White, R.D., and C a r r i e r , G.0. 1988. Enhanced v a s cu l a r a -ad rene rg i c neu roe f f e c t o r system in d i abe te s : importance of c a l c i u m . Am. J . P h y s i o l . 255: H1036-H1042. WHO Study Group. 1985. Diabetes m e l l i t u s . World Hea l th O r gan i z a t i on , Geneva, pp. 17-24 (Tech. Rep. Ser. No. 727). W i l l i amson , J .R . , and K i l o , C. 1980. Vascu la r comp l i ca t i on s i n d i abetes m e l l i t u s . New Eng l . J . Med. 302: 399-400. Wi l son , D.B., Conno l l y , T.M., Bross, T .E . , Majerus, P.W., Sherman, W.R., T y l e r , A .N. , Rubin, L . J . , and Brown, J . E . 1985. I s o l a t i o n and 2 1 9 c h a r a c t e r i z a t i o n o f the i n o s i t o l c y c l i c phosphate products of po l ypho spho ino s i t i de c leavage by phosphol ipase C. P h y s i o l o g i c a l e f f e c t s i n pe rmeab i l i zed p l a t e l e t s and Limulus photoreceptor c e l l s . J . B i o l . Chem. 260: 13496-13501. Wolf, M., Lev ine , H., Mays, W.S., Cuatrecases, P., and Sahyoun, N. 1985. A model f o r i n t r a s e l l u l a r t r a n s l o c a t i o n o f p r o t e i n k inase C i n v o l v i n g synergism between C a ' + and phorbol e s t e r s . Nature, 317: 546-549. Worley, P.F., Barban, J .M . , Supattopone, S., W i l son , V.S. , and Snyder, S.H. 1987. C h a r a c t e r i z a t i o n o f i n o s i t o l t r i s pho spha te recep to r b ind ing i n b r a i n : Regu lat ion by pH and c a l c i um. J . B i o l . Chem. 262: 12132-12136. Wyse, D.G. 1980. On v t h e n o r m a l i z a t i o n ' of a c t i v e developed f o r ce of i s o l a t e d h e l i c a l s t r i p s of muscular and e l a s t i c a r t e r i e s f o r v a r i a t i o n i n wa l l t h i c k n e s s . J . Pharmacol. Methods, 4: 313-326. Yoon, J.W., A u s t i n , M., Onoseda, T., and Notk in s , A.L. 1979. V i r u s -induced d iabetes m e l l i t u s : i s o l a t i o n of a v i r u s from the pancreas o f a c h i l d w i th d i a b e t i c k e t o a c i d o s i s . N. Eng l . J . Med. 300: 1173-1179. Young, E., and B rad l y , R.F. 1967. Cerebra l edema w i th i r r e v e r s i b l e coma i n severe d i a b e t i c k e t o a c i d o s i s . N. Eng l . J . Med. 276: 665-669. Zawa l i ch , W., Brown, C , and Rasmussen, H. 1983. I n s u l i n s e c r e t i o n : Combined e f f e c t s of phorbol e s t e r and A23187. Biochem, Biophys. Res. Commun. 117: 448-455. Zeng. Y.Y., Ben i s h i n , C.G., and Pang, P.K.T. 1989. Guanine n u c l e o t i d e b ind ing p r o t e i n may modulate ga t i ng o f ca l c i um channels i n v a s cu l a r smooth muscle. I. S tud ies w i th f l u o r i d e . J . Pharm. Exp. Ther. 250: 343-357. 220 

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