UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Diabetes-induced alterations in isolated rat heart performance Vadlamudi, Rao Venkata Satya Veerabhadra 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1983_A1 V32.pdf [ 7.64MB ]
Metadata
JSON: 831-1.0095892.json
JSON-LD: 831-1.0095892-ld.json
RDF/XML (Pretty): 831-1.0095892-rdf.xml
RDF/JSON: 831-1.0095892-rdf.json
Turtle: 831-1.0095892-turtle.txt
N-Triples: 831-1.0095892-rdf-ntriples.txt
Original Record: 831-1.0095892-source.json
Full Text
831-1.0095892-fulltext.txt
Citation
831-1.0095892.ris

Full Text

DIABETES-INDUCED ALTERATIONS IN ISOLATED RAT HEART PERFORMANCE by Rao Venkata Satya Veerabhadra Vadlamudi B .Pharm. , Andhra U n i v e r s i t y , I n d i a , 1973 M.Pharm., Andhra U n i v e r s i t y , I n d i a , 1976 M . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF:THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES D i v i s i o n o f Pharmacology and Tox ico logy of the Facu l t y o f Pharmaceut ica l Sc iences We accept t h i s t h e s i s as conforming to the requ i red s tanda rd : THE UNIVERSITY OF BRITISH COLUMBIA March 1983 © Rao V.S. .V. Vadlamudi 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood th a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of 9\r^J\h^-^AJU^X^C^ SC\ZAAJ-ZS> The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT Chronic d i a b e t i c pa t i en t s have a h igher i nc idence of and m o r t a l i t y from c a r d i a c d i s e a s e . A wide spectrum o f c a r d i a c problems plague the c h r o n i c d i a b e t i c i n c l u d i n g coronary a r t e r y d i s e a s e , conges t i ve heart f a i l u r e and d i a b e t i c cardiomyopathy. Card iac d i sease i n the d i a b e t i c i s not s imp ly due to a c c e l e r a t e d a t h e r o s c l e r o s i s a l o n e , but i s a l so due to a combinat ion o f m ic roang iopa thy , autonomic neuropathy, and va r ious o ther f a c t o r s which produce b i o c h e m i c a l , f u n c t i o n a l and s t r u c t u r a l a l t e r a t i o n s in the hea r t . R e c e n t l y , c a r d i a c f u n c t i o n was s tud ied i n animals w i th expe r imen ta l l y - i nduced d iabe tes and c a r d i a c -dys func t i on was repor ted i n acute as we l l as ch ron i c phases o f exper imenta l d i a b e t e s . S ince c a r d i a c d isease i s a consequence o f l ong -s tand ing d iabetes i n d i a b e t i c p a t i e n t s , i n v e s t i g a t i o n o f myocard ia l f unc t i on a t var ious time po in ts a f t e r i n d u c t i o n o f exper imenta l d iabe tes would y i e l d i n fo rma t ion regard ing the development and p rog ress ion o f c a r d i a c dys func t i on i n d i a b e t e s . We. t he re fo re i n v e s t i g a t e d c a r d i a c f unc t i on and pharmacology i n i s o l a t e d per fused working hear ts ob ta ined from 7, 30, 100, 180, 240 and 360-day a l l o x a n and s t r e p t o z o t o c i n (STZ) d i a b e t i c and age-matched con t ro l h e a r t s . Diabetes was induced in the r a t by i n j e c t i n g e i t h e r a l l o x a n (65 or 40 mg/kg) or STZ (50 or 60 mg/kg) i n to the t a i l v e i n . D iabe t i c and age-matched con t ro l r a t s were s a c r i f i c e d a t va r ious time po in ts a f t e r the i nduc t i on o f d iabe tes and hear ts were i s o l a t e d and perfused on a working hear t appara tus . Card iac f u n c t i o n was s tud ied at va r ious l e f t a t r i a l f i l l i n g pressures and was expressed i n terms o f l e f t v e n t r i c u l a r developed pressure (LVDP), ra te o f r i s e o f l e f t i i i v e n t r i c u l a r pressure ( p o s i t i v e dP /d t ) and ra te o f d e c l i n e o f l e f t v e n t r i c u l a r pressure (negat ive d P / d t ) . Dose-response curves to carbachol and i sop ro te reno l were a l s o performed. Blood samples were c o l l e c t e d at the time o f s a c r i f i c e , serum was separated and analyzed fo r i n s u l i n and g lucose con ten t . Both a l l o x a n and STZ produced d iabe tes i n the r a t as shown by f a s t i n g hypo insu l inemia and hyperg lycemia . Card iac f u n c t i o n was not a l t e r e d i n 7-day a l l o x a n and STZ d i a b e t i c r a t s . Depressed f u n c t i o n a t var ious l e f t a t r i a l f i l l i n g pressures was seen i n hear ts i s o l a t e d from 30-day a l l o x a n d i a b e t i c ra t s but not i n 30-day STZ d i a b e t i c r a t s . Hearts i s o l a t e d from 100-day a l l o x a n and STZ d i a b e t i c r a t s , 180- and 360-day STZ d i a b e t i c r a t s and 240-day a l l o x a n d i a b e t i c r a t s , a l l e x h i b i t e d c a r d i a c f u n c t i o n a l a b n o r m a l i t i e s . Card iac f u n c t i o n a l abno rma l i t i e s observed i n d.iabeti.c r a t s were, depressed >LVOP and -posit ive«.and negat ive dP /d t a t high l e f t a t r i a l f i l l i n g p r e s s u r e s . D i a b e t i c r a t hear ts e x h i b i t e d no change in e i t h e r s e n s i t i v i t y or respons iveness to the negat ive i n o t r o p i c e f f e c t of carbachol a t 7 and 30 days a f t e r i nduc t i on o f the d i s e a s e . A sub-s e n s i t i v i t y to carbachol was observed in d i a b e t i c r a t hear ts a t 100 days a f t e r i n d u c t i o n o f d iabe tes as compared to age-matched con t ro l r a t h e a r t s . However, 180- and 240-day d i a b e t i c r a t hear ts e x h i b i t e d s u p e r s e n s i t i v i t y to the negat ive i n o t r o p i c e f f e c t o f c a r b a c h o l . I sopro-te reno l produced an i d e n t i c a l p o s i t i v e i n o t r o p i c e f f e c t i n con t ro l as we l l as d i a b e t i c r a t hear ts a t a l l o f the time po in ts s t u d i e d . However, the maximum changes produced by i s o p r o t e r e n o l i n negat ive dP /d t o f d i a b e t i c r a t hear ts were depressed a t va r ious t ime po in ts as compared to those i n age-matched con t ro l r a t h e a r t s . i v We a l s o s t u d i e d the e f f e c t o f i s o p r o t e r e n o l on the c y c l i c AMP content and phosphory lase a_ a c t i v i t y i n hear ts ob ta ined from 3 and 100 to 120 day con t ro l and d i a b e t i c r a t s . Basal c y c l i c AMP content and phosphory lase .a a c t i v i t y were not a l t e r e d in acute and ch ron i c d i a b e t i c and age-matched con t ro l r a t h e a r t s . I sopro te reno l produced s i m i l a r t ime- and dose-dependent changes in c y c l i c AMP content and p o s i t i v e and negat ive dP /d t i n i s o l a t e d perfused working hear ts obta ined from 3 and 100 to 120 day c o n t r o l and d i a b e t i c r a t s . However, i s o p r o t e r -enol caused a s i g n i f i c a n t l y g rea te r a c t i v a t i o n o f phosphory lase enzyme in hear ts i s o l a t e d from 3 and 100 to 120 day d i a b e t i c ra t s as compared to age-matched c o n t r o l s . D i a b e t i c r a t hear ts had a s i g n i f i c a n t l y h igher t o t a l phosphory lase a c t i v i t y a t 100 to 120 days as compared to age-matched c o n t r o l s . P ros tag land in E ] , a drug which inc reases c y c l i c AMP content w i thout a l t e r i n g phosphory lase a_ a c t i v i t y i n per fused ra t h e a r t s , i nc reased phosphory lase a^  a c t i v i t y i n acute as we l l as ch ron i c d i a b e t i c r a t hear ts but not i n con t ro l r a t h e a r t s . C h o l i n e r g i c muscar in i c recep to rs in the v e n t r i c l e s obta ined from 180-day con t ro l and STZ d i a b e t i c r a t s were s tud ied by per forming r a d i o l i g a n d b ind ing s t u d i e s . [ H]NMS was used as a r a d i o l i g a n d to s t e r e o s p e c i f i c a l l y l a b e l a l l o f the musca r in i c r ecep to r b ind ing s i t e s present i n the v e n t r i c u l a r membrane p r e p a r a t i o n . There was no change in e i t h e r the recep to r dens i t y or i n the b ind ing constants f o r an tagon is t s and agon is t s at the musca r in i c recep to r s i t e i n 180-day d i a b e t i c r a t hear ts as compared to c o n t r o l . V e n t r i c u l a r no radrena l ine content was est imated us ing an HPLC method, i n 180-day a l l o x a n and STZ d i a b e t i c and age-matched con t ro l r a t h e a r t s . There was no s i g n i f i c a n t change in the V noradrena l ine content o f d i a b e t i c r a t h e a r t s . Resu l t s ob ta ined i n the above s tud ies demonstrate tha t var ious f u n c t i o n a l , pharmacologica l and b iochemica l a l t e r a t i o n s occur i n the hear t i n exper imenta l d i a b e t e s . Depressed c a r d i a c performance was observed i n i s o l a t e d per fused d i a b e t i c r a t hear ts a t var ious time po in ts a f t e r the i nduc t i on o f d iabe tes and may represent the p r e c l i n i c a l v e n t r i c u l a r dys func t i on phase of a deve lop ing d i a b e t i c card iomyopathy. Changes no t i ced i n the s e n s i t i v i t y o f t h e . d i a b e t i c myocardium towards the negat ive i n o t r o p i c e f f e c t o f carbachol may represent va r ious stages of a parasympathet ic autonomic neuropathy o f the hear t i n d i a b e t e s . The una l te red p o s i t i v e i n o t r o p i c e f f e c t . o f H s o p r o t e r e n o l and unchanged noradrena l ine content i n d i a b e t i c r a t hear ts i n d i c a t e the absence o f a sympathet ic autonomic neuropathy. The depressed c a r d i a c r e l a x a n t e f f e c t (maximum changes produced in negat ive dP /d t ) o f i sop ro te reno l i n d i a b e t i c 2+ r a t hear ts suggest de fec ts i n c a r d i a c muscle r e l a x a t i o n , Ca hand l ing by -the sa rcop lasmic r e t i cu l um and perhaps ATP product ion and u t i l i z a t i o n . The enhanced s e n s i t i v i t y o f the ;phosphory lase enzyme to agon i s t s i n 2+ d i a b e t i c r a t hear ts may be an outcome o f a l t e r a t i o n s in Ca homeostasis and o ther acute metabo l i c derangements in the hear t caused by d i a b e t e s . A l l these changes cou ld c o n t r i b u t e to the pathogenesis o f a d i a b e t i c cardiomyopathy. John H. M c N e i l l , Ph.D. Thes is Supe rv i so r v i TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF FIGURES i x LIST OF TABLES x i v ACKNOWLEDGEMENTS xv INTRODUCTION 1 CLASSIFICATION AND ETIOLOGY OF DIABETES 1 PATHOPHYSIOLOGY OF DIABETES MELLITUS 5 THE HEART AND DIABETES 19 PRODUCTION OF EXPERIMENTAL DIABETES, ALLOXAN AND STREPTOZOTOCIN 29 METHODS 37 ANIMAL MODEL 37 WORKING HEART APPARATUS 38 HEART PERFUSION 42 CARDIAC FUNCTION CURVES 44 DOSE-RESPONSE CURVES TO CARBACHOL 47 DOSE-RESPONSE CURVES TO ISOPROTERENOL 48 CYCLIC AMP AND PHOSPHORYLASE ENZYME STUDIES 49 CYCLIC AMP DETERMINATION* 51 PHOSPHORYLASE a ASSAY 51 MUSCARINIC RECEPTOR BINDING EXPERIMENTS 52 ESTIMATION OF NORADRENALINE CONTENT IN VENTRICLES 54 SERUM ANALYSES 56 SERUM IMMUNOREACTIVE INSULIN DETERMINATION, 56 SERUM GLUCOSE DETERMINATION 57 v i i PAGE SERUM T 4 DETERMINATION 57 SERUM T 3 DETERMINATION 58 STATISTICAL ANALYSES 58 MATERIALS 59 RESULTS 60 FEATURES OF THE DIABETIC-RAT MODEL 60 CARDIAC PERFORMANCE OF CONTROL AND DIABETIC RATS 62 RESPONSIVENESS OF CONTROL AND DIABETIC HEARTS TO CARBACHOL 64 EFFECT OF ISOPROTERENOL ON POSITIVE dP/DT IN CONTROL AND DIABETIC RAT HEARTS 67 EFFECT OF ISOPROTERENOL ON NEGATIVE dP/DT IN CONTROL AND DIABETIC RAT HEARTS 68 TIME COURSE OF THE EFFECT OF ISOPROTERENOL ON CARDIAC CYCLIC AMP CONTENT, PHOSPHORYLASE ACTIVITY AND INOTROPY OF CONTROL AND DIABETIC RATS 69 MUSCARINIC RECEPTOR BINDING STUDIES IN CONTROL AND DIABETIC RAT HEARTS 74 LEFT VENTRICULAR NORADRENALINE CONTENT IN 180-DAY CONTROL AND DIABETIC RATS 75 DISCUSSION., 188 THE DIABETIC-RAT MODEL 188 CARDIAC FUNCTION IN DIABETIC AND CONTROL RATS 190 RESPONSIVENESS OF CONTROL AND DIABETIC HEARTS TO CARBACHOL 197 EFFECT OF ISOPROTERENOL ON CONTROL AND DIABETIC RAT HEARTS 201 v i i i PAGE CARDIAC CYCLIC AMP AND PHOSPHORYLASE SYSTEM IN EXPERIMENTAL DIABETES AND THE EFFECT OF ISOPROTERENOL AND PGE-, 207 MUSCARINIC RECEPTORS IN DIABETIC RAT HEARTS 214 CONCLUSIONS 215 BIBLIOGRAPHY 219 i x LIST OF FIGURES FIGURE-. PAGE 1. Schematic r ep resen ta t i on o f the working hear t apparatus 41 2. C a l c u l a t i o n o f p o s i t i v e and negat ive dP /d t from l e f t v e n t r i c u l a r pressure t r ace 46 3. E f f e c t o f changing a t r i a l f i l l i n g pressure on l e f t v e n t r i c u l a r pressure development i n 7 day con t ro l and d i a b e t i c ra t hear ts 80 4. E f f e c t o f changing a t r i a l f i l l i n g pressure on p o s i t i v e dP /d t i n 7 day con t ro l and d i a b e t i c r a t hear ts 82 5. E f f e c t o f changing a t r i a l f i l l i n g pressure on negat ive dP /d t i n 7 day con t ro l and d i a b e t i c r a t hear ts 84 6. E f f e c t o f changing a t r i a l f i l l i n g pressure on l e f t v e n t r i c u l a r pressure in 30 day con t ro l and d i a b e t i c r a t hear ts 86 7. E f f e c t o f changing a t r i a l f i l l i n g pressure on p o s i t i v e dP /d t i n 30 day con t ro l and d i a b e t i c r a t hearts 88 8. E f f e c t o f changing a t r i a l f i l l i n g pressure on negat ive dP /d t i n 30 day con t ro l and d i a b e t i c r a t hear ts 90 9. E f f e c t o f changing a t r i a l f i l l i n g pressure on l e f t v e n t r i c u l a r pressure in 100 day con t ro l and d i a b e t i c r a t hear ts 92 1 0 . , E f f e c t o f changing a t r i a l pressure on p o s i t i v e dP /d t i n 100 day con t ro l and d i a b e t i c r a t hear ts 94 11. E f f e c t o f changing a t r i a l f i l l i n g pressure on negat ive dP /d t i n 100 day con t ro l and d i a b e t i c r a t hear ts 96 12. E f f e c t o f changing a t r i a l f i l l i n g pressure on l e f t v e n t r i c u l a r pressure in 180 day con t ro l and d i a b e t i c r a t hearts 1 98 X FIGURE PAGE 13. E f f e c t o f changing a t r i a l f i l l i n g pressure on p o s i t i v e dP /d t i n 180 day con t ro l and d i a b e t i c r a t hear ts 100 14. E f f e c t o f changing a t r i a l f i l l i n g pressure on negat ive dP /d t i n 180 day con t ro l and d i a b e t i c r a t hear ts 102 15. E f f e c t o f changing a t r i a l f i l l i n g pressure on l e f t v e n t r i c u l a r pressure in 240 day con t ro l and d i a b e t i c r a t hear t 104 16. E f f e c t o f changing a t r i a l f i l l i n g pressure on p o s i t i v e dP /d t i n 240 day con t ro l and d i a b e t i c r a t hear ts 106 17. E f f e c t o f changing a t r i a l f i l l i n g pressure on negat ive dP /d t i n 240 day con t ro l and d i a b e t i c r a t hear ts 108 18. E f f e c t o f changing a t r i a l f i l l i n g pressure on l e f t v e n t r i c u l a r pressure i n 360 day con t ro l and d i a b e t i c r a t hear ts 110 19. E f f e c t o f changing a t r i a l f i l l i n g pressure on p o s i t i v e dP /d t i n 360 day con t ro l and d i a b e t i c r a t hear ts 112 20. E f f e c t o f changing a t r i a l f i l l i n g pressure on negat ive dP /d t i n 360 day con t ro l and d i a b e t i c r a t hear ts 114 21. E f f e c t o f the du ra t i on o f d iabetes on p o s i t i v e dP /d t at 15 cm 1^0 f i l l i n g pressure i n con t ro l and d i a b e t i c r a t hear ts 116 22. E f f e c t o f the du ra t i on o f d iabe tes on negat ive dP /d t at 15 cm l-^O f i l l i n g pressure in con t ro l and d i a b e t i c r a t hear ts H 8 23. Dose-response curve of carbachol on basal p o s i t i v e dP /d t i n 7 day con t ro l and d i a b e t i c r a t hear ts 120 x i FIGURE 24. Dose-response curve of carbachol on basal p o s i t i v e dP /d t i n 30 day con t ro l and d i a b e t i c r a t hear ts 25. Dose-response curve of carbachol on basal p o s i t i v e dP /d t i n 100 day con t ro l and d i a b e t i c r a t hear ts 26. Dose-response curve o f carbachol on basal p o s i t i v e dP /d t in 180 day con t ro l and d i a b e t i c r a t hear ts 27. Dose-response curve o f carbachol on basal p o s i t i v e dP /d t i n 240 day c o n t r o l . a n d d i a b e t i c r a t hear ts 28. Dose-response curve of carbachol on basal p o s i t i v e dP /d t i n 360 day con t ro l and d i a b e t i c r a t hear ts 29. Dose-response curve of i sop ro te reno l on p o s i t i v e dP /d t i n 7 day con t ro l and d i a b e t i c r a t hear ts 30. Dose-response curve of i sop ro te reno l on p o s i t i v e dP /d t i n 30 day con t ro l and d i a b e t i c r a t hear ts 31. Dose-response curve of i sop ro te reno l on p o s i t i v e dP /d t i n 100 day con t ro l and d i a b e t i c r a t hear ts 32. Dose-response curve o f i sop ro te reno l on p o s i t i v e dP /d t i n 180 day con t ro l and d i a b e t i c r a t hear ts 33. Dose-response curve o f i sop ro te reno l on p o s i t i v e dP /d t i n 240 day con t ro l and d i a b e t i c r a t hear ts 34. Dose-response curve o f i sop ro te reno l on p o s i t i v e dP /d t i n 360 day con t ro l and d i a b e t i c r a t hear ts 35. Dose-response curve of i sop ro te reno l on negat ive dP /d t i n 7 day con t ro l and d i a b e t i c r a t hear ts 36. Dose-response curve of i sop ro te reno l on negat ive dP /d t i n 30 day con t ro l and d i a b e t i c r a t hear ts XI 1 FIGURE , - PAGE 37. Dose-response curve o f i sop ro te reno l on negat ive dP /d t i n 100 day con t ro l and d i a b e t i c r a t hear ts 150 38. Dose-response curve of i sop ro te reno l on negat ive dP /d t i n 180 day con t ro l and d i a b e t i c r a t hear ts 152 39. Dose-response curve of i sop ro te reno l on negat ive dP /d t i n 240 day con t ro l and d i a b e t i c r a t hear ts 154 40. Dose-response curve of i sop ro te reno l on negat ive dP /d t i n 360 day con t ro l and d i a b e t i c r a t hear ts 156 41 . Time course o f the e f f e c t o f i sop ro te reno l on c a r d i a c c y c l i c AMP content i n 3 day con t ro l and d i a b e t i c r a t s 159 42. Time course o f the e f f e c t o f i sop ro te reno l on c a r d i a c c y c l i c AMP content i n 100-120 day con t ro l and d i a b e t i c ra t s 161 43. Time course of the e f f e c t o f i sop ro te reno l on c a r d i a c phosphory lase a_ a c t i v i t y i n 3 day con t ro l and d i a b e t i c r a t s 163 44. Time course of the e f f e c t o f i s o p r o t e r e n o l on c a r d i a c phosphory lase a_ a c t i v i t y i n 100-120 day con t ro l and d i a b e t i c ra ts 165 45. Tota l phosphory lase a c t i v i t y i n 3 and 100-120 day con t ro l and d i a b e t i c r a t hear ts 167 46. Time course of the e f f e c t o f i sop ro te reno l on the ra te o f l e f t v e n t r i c u l a r pressure r i s e in 3 day con t ro l and d i a b e t i c r a t hear ts 169 x i i i FIGURE PAGE 47. Time course o f the e f f e c t o f i sop ro te reno l on the ra te o f l e f t v e n t r i c u l a r pressure r i s e i n 100-120 day con t ro l and d i a b e t i c r a t hear ts 17.1 48. Time course o f the e f f e c t o f i sop ro te reno l on the ra te o f l e f t v e n t r i c u l a r pressure d e c l i n e in 3 day con t ro l and d i a b e t i c r a t hear ts 173 49. Time course o f the e f f e c t o f i sop ro te reno l on the ra te o f l e f t v e n t r i c u l a r pressure d e c l i n e in 100-120 day con t ro l and d i a b e t i c r a t hear ts I ? 5 50. Dose-response curve of i sop ro te reno l on c a r d i a c c y c l i c AMP content i n 100-120 day con t ro l and d i a b e t i c ra t s 1 7 7 51. Dose-response curve of i sop ro te reno l on c a r d i a c phosphory lase a^  a c t i v i t y i n 100-120 day con t ro l and d i a b e t i c r a t s 1 2 9 52. Dose-response curve o f i sop ro te reno l on the ra te o f l e f t v e n t r i c u l a r pressure development i n 100-120 day con t ro l and d i a b e t i c r a t hear ts 181 53. Dose-response curve o f i sop ro te reno l on the ra te o f l e f t v e n t r i c u l a r pressure d e c l i n e in 100-120 day con t ro l and d i a b e t i c rat. hear ts 183 x i v LIST OF TABLES TABLE PAGE I C e r t a i n fea tu res o f con t ro l and d i a b e t i c ra t s 7 6 I I C e r t a i n fea tu res o f con t ro l and d i a b e t i c r a t s used i n the time course study 7 7 I I I Features o f con t ro l and d i a b e t i c ra t s 7 8 I V Geometric mean E D ^ Q va lues of carbachol 1 3 1 V S e n s i t i v i t y o f the r a t myocardium to the p o s i t i v e i n o t r o p i c e f f e c t o f i sop ro te reno l (pD^ va lues ) 1 4 4 V I Card iac re laxant e f f e c t o f i sop ro te reno l 1 5 7 V I I E f f e c t o f PGE-| on c a r d i a c c y c l i c AMP and phosphory lase a_ a c t i v i t y i n con t ro l and d i a b e t i c ra t s 1 8 4 V I I I E f f e c t o f 1 0 " M indomethacin on i s o p r o t e r e n o l - i n d u c e d c a r d i a c phosphory lase a c t i v i t y i n 1 0 0 - 1 2 0 day con t ro l and d i a b e t i c r a t s 1 8 5 IX [ °H ] NMS b ind ing in 180 day con t ro l and d i a b e t i c r a t hear ts X L e f t v e n t r i c u l a r no rad rena l i ne content i n 180 day con t ro l and d i a b e t i c ra t s XV ACKNOWLEDGEMENTS I am deeply g r a t e f u l to Dr. John H. McNe i l l f o r h i s cons tant guidance and pa t ience throughout the present s tudy . I would l i k e to thank Dr. J.W. Wel ls f o r a l l o w i n g me to c a r r y out c h o l i n e r g i c recep to r b ind ing s tud ies i n h is l a b o r a t o r y a t the U n i v e r s i t y o f Toronto . I would a l s o l i k e to thank Dr. V l a d i m i r Pa la t y f o r c a r r y i n g out the HPLC de te rmina t ion o f n o r a d r e n a l i n e . I wish to thank my research committee members f o r t h e i r genuine i n t e r e s t and va luab le sugges t i ons . I would a l s o l i k e to express my s i nce re thanks to Dr. Gary Lopaschuk, Dr. Kath MacLeod, Mr. David H a r r i s , Ms. D a n i e l l e Wenkstern and to a l l my co l l eagues f o r t h e i r va luab le h e l p , encouragement and f r i e n d s h i p . My s p e c i a l thanks are due to Ms. Judy Wyne fo r her exper t t yp ing s k i l l s . The f i n a n c i a l support o f the Canadian Heart Foundation i s g r a t e f u l l y acknowledged. 1 INTRODUCTION CLASSIFICATION AND ETIOLOGY OF DIABETES: In recent y e a r s , the d isease d iabe tes has been recogn ized as a heterogeneous group of metabo l i c d i s o r d e r s c h a r a c t e r i z e d by i napp rop r i a te hyperglycemia and i n more severe cases w i th a c c e l e r a t e d l i p o l y s i s and excess i ve ketogenesis (Lefebvre and Luyckx, 1979). Most o f the va r ious forms of d iabe tes have a gene t i c background,; however, the gene t i c mechanisms invo l ved appear to vary ( S a l a n s , 1982) . R e c e n t l y , an i n t e r n a t i o n a l workgroup sponsored by the Nat iona l Diabetes Data group (1979) has developed a new c l a s s i f i c a t i o n f o r d iabe tes and o ther ca tego r i es o f g lucose i n t o l e r a n c e . Accord ing to t h i s c l a s s i f i c a t i o n , the heterogeneous syndrome d iabe tes has been d i v i d e d i n t o three major c l i n i c a l c l a s s e s and two s t a t i s t i c a l r i s k c l a s s e s ( C r a i g , 1980) . The new c l a s s i f i c a t i o n i s as f o l l o w s : C l i n i c a l c l a s s e s : 1. Diabetes m e l l i t u s (DM) 2 . Ges ta t i ona l d iabe tes (GDM) and 3. Impaired g lucose t o l e rance (IGT) S t a t i s t i c a l r i s k c l a s s e s : 1. Prev ious abnormal i t y o f g lucose t o l e rance (Pre AGT) and 2 . P o t e n t i a l abnormal i t y o f g lucose t o l e rance (Pot AGT) Diabetes m e l l i t u s (DM): DM has been f u r t h e r ca tego r i zed i n to three groups, which a r e : Type I: i nsu l i n -dependen t (IDDM) Type I I : non - insu l i n -dependen t (NIDDM) 2 and o ther types of d iabe tes a s s o c i a t e d w i th c e r t a i n c o n d i t i o n s and gene t i c syndromes. Type I: i nsu l i n -dependen t d iabe tes m e l l i t u s (IDDM): IDDM represen ts about 20% o f a l l the c l i n i c a l l y encountered cases o f d i a b e t e s . The usual age o f onset i s ch i ldhood or e a r l y adul thood and i t was the re fo re known p r e v i o u s l y as j u v e n i l e - o n s e t d i a b e t e s . IDDM i s u s u a l l y a s s o c i a t e d wi th no de tec tab le i n s u l i n a c t i v i t y i n the blood and i n s u l i n a d m i n i s t r a t i o n i s an abso lu te n e c e s s i t y f o r IDDM p a t i e n t s to prevent the development o f k e t o s i s . Total d e s t r u c t i o n o f panc rea t i c 3 - c e l l s i s a common f i n d i n g i n IDDM. I s l e t c e l l d e s t r u c t i o n may r e s u l t from autoimmunity or from a v i r a l i n f e c t i o n ( I r v i n e , 1977) . In most c a s e s , a combinat ion o f autoimmunity and v i r a l i n f e c t i o n appear to be i m p l i c a t e d in the development o f IDDM. A v i r a l invas ions o f panc rea t i c i s l e t c e l l s cou ld r e s u l t i n damage o f the i s l e t s wh ich , i n t u r n , would t r i g g e r an t ibody p roduc t ion and c y t o t o x i c i t y d i r e c t e d aga ins t i s l e t c e l l s ( C r a i g , 1980) . In most o f the cases o f newly diagnosed IDDM, i s l e t c e l l cy top lasmic a n t i b o d i e s were found and these a n t i b o d i e s d isappeared wi th i n c r e a s i n g du ra t i on of the d i sease (Lernmark and Baekkeskov, 1981) . Panc rea t i c i s l e t c e l l s u s c e p t i b i l i t y to v i r a l i n f e c t i o n i s thought to be g e n e t i c a l l y predetermined. IDDM has been shown to be f r e q u e n t l y a s s o c i a t e d w i th c e r t a i n h i s t o c o m p a t i b i l i t y an t igen (HLA) l i n k e d genes such as HLA-B8, BW15 and B18 (Cudworth and Woodrow, 1976) . However, s t u d i e s wi th i d e n t i c a l twins ( G o t t l i e b and Root , 1 968.; Pyke, 1979) show on ly about a 50% concordance fo r IDDM between t w i n s , sugges t ing a r o l e f o r nongenet ic f a c t o r s in the exp ress ion of t h i s d i s e a s e . For example, 3 environmental f a c t o r s such as v i r u s e s can p r e c i p i t a t e d iabe tes i n g e n e t i c a l l y s u s c e p t i b l e i n d i v i d u a l s ( S a l a n s , 1982) . Type I I : non - insu l i n -dependen t d iabe tes (NIDDM). The ma jo r i t y o f the c l i n i c a l l y encountered cases o f d iabe tes are o f the non - insu l i n -dependen t t ype . NIDDM was fo rmer ly r e f e r r e d to as ma tu r i t y -onse t d iabe tes owing to the age a t onse t , which u s u a l l y i s i n l a t e r adu l thood . NIDDM i s a s s o c i a t e d w i th e i t h e r normal or low l e v e l s o f c i r c u l a t i n g i n s u l i n and sometimes even w i th high l e v e l s . P a t i e n t s wi th NIDDM do not u s u a l l y r equ i re i n s u l i n therapy f o r the p reven t ion of k e t o a c i d o s i s as they are not k e t o s i s - p r o n e . Two types o f abno rma l i t i e s in i n s u l i n a c t i o n are demonstrable in NIDDM. F i r s t l y , l ack o f the f i r s t -phase (acute r e l e a s e o f i n s u l i n ) i n s u l i n response to an in t ravenous g lucose cha l l enge has been repor ted i n NIDDM p a t i e n t s (B runze l l e t a l . 1976). I t appears tha t g lucose i s a poor s t i m u l a t o r o f i n s u l i n r e l e a s e in these p a t i e n t s . Defects i n the h y p o t h e t i c a l 1 g l u c o r e c e p t o r ' on the 8 - c e l l have been proposed to be r e s p o n s i b l e f o r the i n a b i l i t y o f g lucose to r e l ease i n s u l i n (Robertson and Metz , 1979) . Second ly , i n s e n s i t i v i t y o f the t a rge t t i s s u e s ( l i v e r , ad ipose t i s s u e and muscle) to the a c t i o n o f i n s u l i n has been shown to be a.common fea tu re o f NIDDM (Reaven, 1980). The i n s e n s i t i v i t y to the a c t i o n o f i n s u l i n i s r e f e r r e d to as i n s u l i n r e s i s t a n c e and i s a common fea tu re o f o b e s i t y . Most o f the NIDDM pa t i en t s are obese ( C r a i g , 1980). I n s u l i n r e s i s t a n c e u s u a l l y r e s u l t s i n a compensatory r i s e i n c i r c u l a t i n g i n s u l i n l e v e l s i n p a t i e n t s w i th normal ly f u n c t i o n i n g B - c e l l s , thereby producing hype r insu l i nemia ( F l i e r e t a l . 1 9 7 9 ) . 4 Almost a l l cases o f NIDDM appear to have a gene t i c cause ( C r a i g , 1980) . S tud ies o f d iabe tes i n monozygotic twins show almost a 100% concordance f o r NIDDM between twins ( G o t t l i e b and Root , 1968; Pyke, 1979) . However, the gene t i c f a c t o r s tha t c o n t r i b u t e to the development o f NIDDM appear to be d i f f e r e n t from those which determine IDDM, as no a s s o c i a t i o n was found between HLA and NIDDM (Ganda and Soe ldne r , 1977) . Environmental f a c t o r s a l s o i n f l u e n c e the express ion of NIDDM. One o f the important environmental f a c t o r s f o r NIDDM i s o b e s i t y ( S a l a n s , 1982). Other types o f d i a b e t e s : Var ious types o f d iabe tes tha t develop secondary . to panc rea t i c d i s e a s e , c e r t a i n gene t i c syndromes, endocr ine d i so rde rs or due to t reatment w i th some hormones and drugs are grouped in t h i s c l a s s (Nat iona l Diabetes Data Group, 1979) . These types o f d iabe tes represent a very smal l p ropo r t i on o f c l i n i c a l l y encountered d iabe tes c a s e s . Ges ta t i ona l d iabe tes (GDM): Diabetes m e l l i t u s tha t t r a n s i e n t l y develops i n a p r e v i o u s l y non - d i abe t i c woman dur ing pregnancy owing to the complex metabo l i c changes occu r i ng i n pregnancy i s termed GDM. Usua l l y GDM d isappears a f t e r d e l i v e r y but these p a t i e n t s face an inc reased r i s k o f becoming over t d i a b e t i c s i n the fu tu re ( C r a i g , 1980). Impaired g lucose t o l e rance ( IGT) : Th is c l a s s i s comprised of cases which are in the b o r d e r l i n e 5 reg ion between d iabe tes and normal g lucose t o l e r a n c e . Formerly t h i s c l a s s was r e f e r r e d to as s u b c l i n i c a l d i a b e t e s , b o r d e r l i n e d iabe tes or l a t e n t d i a b e t e s . I n d i v i d u a l s i n t h i s c l a s s have a h igher r i s k o f becoming d i a b e t i c . On the other hand they may remain in the in te rmed ia te stage or become comple te ly normal . However, an inc reased prevalence of a t h e r o s c l e r o t i c d i sease has been repor ted to be a s s o c i a t e d wi th t h i s c l a s s , suggest ing the pathogenic nature o f IGT (Keen, 1970) . S t a t i s t i c a l r i s k c l a s s e s : Prev ious abnormal i t y o f g lucose t o l e rance (Pre -AGT) : I n d i v i d u a l s w i th normal g lucose t o l e r a n c e , but w i th a h i s t o r y o f impaired g lucose t o l e rance such as GDM belong in t h i s c l a s s . These i n d i v i d u a l s however, are a t a h igher r i s k f o r over t d i a b e t e s . P o t e n t i a l abnormal i t y o f g lucose t o l e rance (Po t -AGT) : I n d i v i d u a l s c a t e g o r i z e d in t h i s group have never shown impaired g lucose t o l e r a n c e , but have a s t a t i s t i c a l r i s k o f deve lop ing d i a b e t e s . Examples o f such persons i nc lude monozygotic twins of e i t h e r an i n s u l i n -dependent or a n o n - i n s u l i n dependent d i a b e t i c , f i r s t - d e g r e e r e l a t i v e o f a non- insu l i n -dependen t d i a b e t i c and persons wi th i s l e t c e l l an t i bod ies or HLA 1 inked genes. PATHOPHYSIOLOGY OF DIABETES MELLITUS: In d iabe tes m e l l i t u s , r ega rd less o f i t s e t i o l o g y , the major de fec t i s i n s u f f i c i e n t i n s u l i n a c t i o n on t a rge t t i s s u e s . S ince i n s u l i n regu la tes ca rbohydra te , f a t and amino a c i d metabol ism i n t a rge t t i s s u e s , i n the event o f inadequate i n s u l i n a c t i o n a number o f abno rma l i t i e s can 6 be p red i c ted to occu r . I n s u l i n exe r t s both shor t and long- term e f f e c t s on the metabo l ism, i n respons ive t a rge t t i s s u e s (Denton e t a l . 1981). The shor t - te rm e f f e c t s o f i n s u l i n are on carbohydrate and l i p i d metabol ism i n l i v e r , muscle and adipose t i s s u e , whereas the long- term e f f e c t s are main ly on p r o t e i n metabol ism and growth. The shor t - te rm e f f e c t s o f i n s u l i n i nc l ude enhancement o f g lucose uptake i n to muscle and ad ipose t i s s u e , s t i m u l a t i o n o f glycogen syn thes i s i n l i v e r and musc le , and syn thes i s o f f a t t y ac ids and t r i g l y c e r i d e s in l i v e r and adipose t i s s u e . I n s u l i n decreases g l y c o g e n o l y s i s and g luconeogenesis i n l i v e r and l i p o l y s i s i n ad ipose t i s s u e . I n s u l i n a l s o exer ts a few shor t - te rm e f f e c t s on p ro te in , metabol ism such as i n c r e a s i n g the amino a c i d uptake i n to muscle and adipose t i s s u e . The long- term e f f e c t s o f i n s u l i n i nc l ude i n c r e a s i n g the syn thes i s o f both general and s p e c i f i c p r o t e i n s , RNA and DNA, promotion of growth and i n h i b i t i o n o f p r o t e i n breakdown. During an ep isode o f acute i n s u l i n l a c k , a l l o f the above documented sho r t - t e rm i n s u l i n dependent processes are i n h i b i t e d and a number o f metabo l i c abno rma l i t i es take p l a c e . Glucose uptake i n to muscle and ad ipose t i s s u e i s reduced, r e s u l t i n g i n e leva ted blood g lucose l e v e l s . G lycogenes is i s impaired in l i v e r and musc le , a t the same time g l ycogeno l ys i s and g luconeogenesis i nc rease i n the l i v e r and l a rge q u a n t i t i e s o f g lucose are re leased i n to the blood s t ream, caus ing dramat ic i nc reases in blood g lucose l e v e l s . When the blood g lucose l e v e l s exceed the renal t h resho ld f o r g l u c o s e , g lucose s t a r t s to s p i l l i n to the u r ine caus ing g l u c o s u r i a . Increased amounts o f g lucose i n the u r ine cause osmot ic d i u r e s i s r e s u l t i n g i n a l o s s o f wa te r , e l e c t r o l y t e s and c a l o r i e s . This s i t u a t i o n g ives r i s e to the c h a r a c t e r i s t i c c l i n i c a l 7 symptoms such as p o l y u r i a , p o l y d i p s i a and po l yphag ia . In the absence o f i n s u l i n , syn thes i s o f f a t t y ac i ds and t r i g l y c e r i d e s i s reduced in the l i v e r and l i p o l y s i s becomes u n i n h i b i t e d in ad ipose t i s s u e . Large q u a n t i t i e s o f f a t t y ac ids are re leased i n to blood caus ing h y p e r l i p i d e m i a . P r o t e i n syn thes i s i s impaired and p r o t e i n breakdown i s inc reased dur ing prolonged i n s u l i n l a c k . Excess ive l i p o l y s i s and p r o t e i n breakdown r e s u l t i n weight l o s s , another c h a r a c t e r i s t i c symptom of IDDM. Compl icat ions: o f d iabe tes m e l l i t u s : Diabetes m e l l i t u s i s assoc i a t ed w i th severa l secondary c o m p l i c a t i o n s . C l i n i c a l and b iochemica l man i f es ta t i ons o f d iabe tes i nc l ude acute as we l l as ch ron i c comp l i ca t i ons (Fajans and F r e i n k e l , 1976) . Acute comp l i ca t i ons r e s u l t from e i t h e r a sudden and severe i n s u l i n d e f i c i e n c y or l o s s o f metabo l i c con t ro l due to severe s t r e s s or acute i l l n e s s or c e r t a i n a d d i t i o n a l drug t h e r a p i e s , wh i le ch ron i c comp l i ca t i ons o f d iabe tes appear s l ow l y and are consequences o f var ious b i o c h e m i c a l , 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 produced by ch ron i c hyperglycemia and o ther metabo l i c e r r o r s . Acute comp l i ca t i ons of d i a b e t e s : Acute metabo l i c comp l i ca t i ons o f ten occur i n d i a b e t i c s and i f not t rea ted in t ime , can lead to coma and dea th . There are two types o f acute c o m p l i c a t i o n s . 1. D i a b e t i c k e t o a c i d o s i s w i th or w i thout dehydra t ion and 2 . Hyperosmolar n o n - k e t o t i c d i a b e t i c coma. 8 D iabe t i c k e t o a c i d o s i s or d i a b e t i c coma r e s u l t s whenever there i s a profound i n s u l i n d e f i c i e n c y a s s o c i a t e d wi th changes i n o ther hormones such as inc reased l e v e l s o f growth hormone and glucagon ( F o s t e r , 1976) . The f o l l o w i n g metabo l i c events and t h e i r i n t e r -r e l a t i o n s h i p s are cons idered to be i nvo l ved i n the development o f k e t o a c i d o s i s . Unimpeded g luconeogenesis brought about by the above mentioned hormonal changes, r e s u l t s i n marked hyperglycemia fo l l owed by osmotic d i u r e s i s and dehyd ra t i on . Excess ive l i p o l y s i s i n adipose t i s s u e e l eva tes f ree f a t t y a c i d l e v e l s i n the b lood . Free f a t t y ac ids are taken up by l i v e r and o x i d i z e d to ketone bod ies , which are re leased in to the b l ood . Blood ketone l e v e l s g radua l l y i nc rease and cause metabo l i c a c i d o s i s l ead ing to coma. Death may r e s u l t from pe r i phe ra l v a s c u l a r c o l l a p s e and renal f a i l u r e . Hyperosmolar , non -ke to t i c d i a b e t i c coma i s another l i f e - t h r e a t e n i n g s i t u a t i o n which most commonly occurs in e l d e r l y d i a b e t i c p a t i e n t s ( F i e l d , 1976) . Very severe hyper-glycemia i s cons idered to be the main cause f o r the hype rosmo la r i t y . However, azotemia and hypernatremia a l so c o n t r i b u t e to the hyper-o s m o l a r i t y . Th is syndrome d i f f e r s from d i a b e t i c coma i n c e r t a i n a s p e c t s . I t i s more i n s i d i o u s in development than d i a b e t i c coma and ketogenesis i s comp le te ly absent due to e i t h e r an impaired r e l e a s e of f ree f a t t y ac i ds from adipose t i s s u e or a decreased hepa t i c syn thes i s of ketone bodies (Podo lsky , 1981). Plasma glucagon l e v e l s i n hyper-osmolar coma are h igher than those i n d i a b e t i c coma (L indsey e t a l . 1974). Hyperosmolar n o n - k e t o t i c coma i s f r equen t l y assoc i a t ed wi th a number o f neu ro l og i ca l s igns ranging from con fus ion to c o n v u l s i o n s . 9 Chronic comp l i ca t i ons o f d i a b e t e s : With the d i scove ry o f i n s u l i n i n 1921 (Bant ing e t a]_. 1 922) and the commercial a v a i l a b i l i t y o f va r ious forms o f pure i n s u l i n i n recent y e a r s , management o f the acute metabo l i c comp l i ca t i ons o f d iabe tes ceased to be a major problem f o r the c l i n i c i a n . However, the c l i n i c i a n has now been presented w i th an a r ray o f d e b i l i t a t i n g and l i f e - t h r e a t e n i n g comp l i ca t i ons which c h a r a c t e r i s t i c a l l y appear i n long- term d i a b e t i c s (Kanne l , 1978) . In general three major groups of a b n o r m a l i t i e s have been recogn ized i n ch ron i c d i a b e t i c s (Renold e t al_.'1 978) . These a r e : 1. A d i sease o f the smal l b lood v e s s e l s , which i s a l s o known as microangiopathy and 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 o p a t h y , nephropathy and perhaps cardiomyopathy. There appears to be 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 . 2 . A d isease o f medium and l a rge s i z e blood v e s s e l s , known as macroangio-pathy , which i s probably r e s p o n s i b l e f o r a c c e l e r a t e d a therogenes is and a number o f 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 myocard ia l i n f a r c t i o n , ce reb rovascu la r d i seases and hyper-t e n s i o n . Macroangiopathy i s d e f i n i t e l y more severe i n p a t i e n t s w i th marked hyperg lycemia , but has not been i d e n t i f i e d as a s i g n i f i c a n t l y p reva len t problem in d i a b e t i c s when compared to the n o n - d i a b e t i c popu la t ion (Renold et aj_. 1978) , and 3. A mix ture o f micro and macro-ang iopa th ies and c e r t a i n o ther metabo l i c abno rma l i t i e s g i v i n g r i s e to d i a b e t i c neu ropa th 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 s . 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 b iochemica l and patho-p h y s i o l o g i c a l processes under l y ing these secondary comp l i ca t i ons of long- term d iabe tes i s very impor tant i n order to prevent or t r e a t 10 them (Winegrad and Clements, 1976). Secondary comp l i ca t i ons have been found in both i nsu l i n -dependen t and non- insu l i n -dependen t d i a b e t i c s and a fea tu re common to these two types o f d iabe tes i s abnormal g lucose t o l e r a n c e . Therefore i t i s reasonable to assume tha t l ong -s tand ing hyperglycemia and o ther metabo l i c abno rma l i t i e s a s s o c i a t e d w i th i t , p lay a r o l e i n the pathogenesis o f the l a t e comp l i ca t i ons o f the d i a b e t i c . The f o l l o w i n g are some o f the pa thophys io l og i ca l processes that occur i n d iabe tes and probably are r e l a t e d to l ong -s tand ing hyperg lycemia . The po lyo l pathway: The po lyo l or s o r b i t o l pathway i s a minor pathway f o r g lucose by which non-phosphory la ted g lucose i s conver ted to f r uc tose through the format ion of an in te rmed ia te compound s o r b i t o l (Hers , 1956). The r e a c t i o n sequence i s as f o l l o w s : 1. D-glucose + NADPH + H + a 1 d o s e r educ tase^ s o r b i t o l + N A D P + 2. S o r b i t o l + NAD+ s o r b i t o l . dehydrogenase^ D _ f r u c t o s e + N A D P + H + The enzymes i nvo l ved i n t h i s pathway have been i d e n t i f i e d i n many t i s s u e s such as the a o r t a , b r a i n , k idney , l e n s , n e r v e s , p lacen ta and seminal v e s i c l e s (Gabbay, 1975) . Most o f these t i s s u e s do not r equ i r e i n s u l i n f o r g lucose uptake and dur ing hyperglycemia the i n t r a c e l l u l a r g lucose concen t ra t i on approaches tha t o f b l ood . The l a rge i n t r a -c e l l u l a r pool o f g lucose i n these t i s s u e s i s conver ted to s o r b i t o l . S ince s o r b i t o l i s unable to permeate through the c e l l membrane, l a rge q u a n t i t i e s o f s o r b i t o l accumulate i n t r a c e l 1 u l a r l y . S o r b i t o l can be conver ted to f r u c t o s e , but the net r e s u l t i s accumulat ion o f s o r b i t o l 11 and f r u c t o s e as f r uc tose i s metabo l ized ra the r poor l y i n some o f these t i s s u e s . Accumulat ion o f s o r b i t o l r e s u l t s i n h y p e r t o n i c i t y and osmot ic s w e l l i n g o f these c e l l s (Gabbay, 1973). I t has been demonstrated tha t among va r ious o ther t i s s u e s , l e n s , nerve and rena l p a p i l l a accumulate s u f f i c i e n t l y l a rge q u a n t i t i e s o f s o r b i t o l i n d iabetes (Gabbay, 1975) . Accumulat ion o f s o r b i t o l i n the lens has been shown to be accompanied wi th l o s s o f osmot ic i n t e g r i t y , e l e c t r o l y t e imbalance and va r ious o ther changes f i n a l l y r e s u l t i n g in l e n t i c u l a r o p a c i t y ( K i n o s h i t a e t al_. 1 962) . In d i a b e t e s , the a c t i v i t y o f the s o r b i t o l pathway has been demonstrated to be markedly e leva ted l ead ing to accumulat ion o f s o r b i t o l and f r uc tose i n the nerves(Ward e t a!..» 1972) , p a r t i c u l a r l y i n the Schwann c e l l s (Thomas and L a s c e l l e s , 1965) . Schwann c e l l s are r e s p o n s i b l e f o r the format ion and r e g u l a t i o n o f myel in i n the pe r i phe ra l nervous system, and accumulat ion o f p o l y o l s i n Schwann c e l l s somehow r e s u l t s i n suppress ion of mye l in p roduc t ion (Gabbay and O ' S u l l i v a n , 1968). These f i n d i n g s suggest a r o l e f o r the po lyo l pathway i n the pathogenesis o f d i a b e t i c neuropathy (Gabbay, 1975; G i a c h e t t i , 1981) . S o r b i t o l accumulat ion i n the kidney c e l l s i s high enough in d iabetes to cause osmot ic d i s tu rbances and t h i s pathway might c o n t r i b u t e to the development o f a d i a b e t i c nephropathy (Gabbay, 1975). Increased a c t i v i t y o f the s o r b i t o l pathway has a l s o been i m p l i c a t e d i n producing v a s c u l a r i n j u r y and microangiopathy (Wi l l iamson and K i l o , 1980). G l y c o s y l a t i o n o f p r o t e i n s : Prolonged hyperglycemia has been shown to be a s s o c i a t e d w i th inc reased l e v e l s o f g l y c o s y l a t e d hemoglobin (Bunn e t aj_. 1978) and 12 o ther serum p ro te ins (McFarland e t al_.- 1979). G l y c o s y l a t i o n o f hemoglobin i s a non-enzymat ic , nea r l y i r r e v e r s i b l e r e a c t i o n and i t s ra te i s dependent on blood g lucose concen t ra t i on (Koenig and Cerami , 1980; Jovanovic and Pe te r son , 1981). This r e a c t i o n i nvo l ves a d d i t i o n o f a molecule o f g lucose to the N-terminal v a l i n e o f the 8-cha ins o f hemoglobin. In general va r ious body p ro te ins such as serum a lbumin , plasma p r o t e i n s , i n s u l i n and lens c r y s t a l l i n e p r o t e i n undergo non-enzymatic g l y c o s y l a t i o n o f N-terminal l y s i n e groups i n a manner analogous to tha t o f hemoglobin (Rahbar, 1981) . G l y c o s y l a t i o n o f hemoglobin i nc reases w i th an i nc rease in the blood g lucose l e v e l and once syn thes i zed remains i n c i r c u l a t i o n u n t i l the end of 120-day l i f e span of the e r y t h r o c y t e s . Therefore de te rmina t ion o f g l y c o s y l a t e d hemoglobin should g ive an es t ima t i on of blood g lucose l e v e l s over a pe r iod o f prev ious e i gh t to twelve weeks (Koenig and Cerami , 1980) . Measurement o f g l y c o s y l a t e d hemoglobin l e v e l s i s p r e s e n t l y used as an accepted method f o r assess i ng the con t ro l o f d iabe tes in p a t i e n t s (Rahbar, 1981). Increased g l y c o s y l a t i o n o f hemoglobin and o ther body p ro te ins has been hypothes ized 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 ons o f d iabe tes (Renold et_ aj_. 1978). G l y c o s y l a t i o n o f hemoglobin has been shown to cause an i nc rease in the oxygen a f f i n i t y and a reduc t i on in the s e n s i t i v i t y to 2 ,3 -d iphosphog lyce ra te (Bunn and B r i e h l , 1970) , a r e g u l a t o r o f hemoglobin oxygenat ion (Benesch and Benesch, 1969) . Increased l e v e l s o f g l y c o s y l a t e d hemoglobin thus may cause t i s s u e hypoxia and other v a s c u l a r responses l ead ing to the development o f microangiopathy (Wi l l iamson and K i l o , 1980) . G lycosy -l a t i o n of. i n t r a c e l l u l a r p ro te ins might a l t e r t h e i r f unc t i on by b r i ng ing about changes i n t h e i r s o l u b i l i t y , a c t i v i t y and a n t i g e n i c i t y (Koenig 13 and Cerami , 1980). Widespread g l y c o s y l a t i o n o f va r ious p ro te i ns has been imp l i ca ted i n the pathogenesis o f d i a b e t i c c a t a r a c t s , t h i c k e n i n g o f g lomeru lar basement membrane and nephropathy, changes i n mye l in content and nerve metabol ism l ead ing to neuropathy, va r ious m ic ro -ang iopa th ies and marked i nc reases in ca rbohyd ra te -con ta in i ng connec t i ve t i s s u e i n the hear t r e s u l t i n g i n a cardiomyopathy (Renold e t a l . 1 9 7 8 ; Koenig and Cerami , 1980; Rahbar, 1981). C i r c u l a t i n g f a c t o r s and c i r c u l a t o r y changes: D i a b e t i c m ic roang iopa thy , a d i sease of smal l blood v e s s e l s , produces a l t e r a t i o n s in the m i c r o c i r c u l a t i o n o f va r ious organs and i s probably i nvo l ved i n the development o f nephropathy, r e t i nopa thy and cardiomyopathy. Diabetes has been shown to cause changes in the c i r c u l a t i n g f a c t o r s and hemorrheology and these changes might p lay a r o l e i n the pathogenesis o f microangiopathy ( M c M i l l a n , 1976) . V i s c o s i t y o f plasma and serum i s e leva ted i n d iabe tes and has been shown to be assoc i a t ed wi th an i nc rease i n red -b lood c e l l a g g r e g a b i l i t y ( M c M i l l a n , 1976; Schmid-Schonbein and V o l g e r , 1976). Red blood c e l l s a l s o e x h i b i t decreased d e f o r m a b i l i t y and these changes in red c e l l s cause a l t e r a t i o n s in b lood- rheo logy i n d i a b e t i c s (Schmid-Schonbein and V o l g e r , 1976). E leva ted blood v i s c o s i t y can be c o r r e l a t e d w i th plasma p r o t e i n changes. Acute-phase p ro te ins such as o q - a c i d g l y c o p r o t e i n , complement p ro te ins and f i b r i n o g e n are e leva ted i n d iabe tes w i th a concur rent decrease in serum a lbumin . These changes i n plasma p ro te i ns 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 iscous blood c rea tes r e s i s t a n c e to f low and as a r e s u l t pressure i n the m i c r o c i r c u l a t i o n inc reases ( M c M i l l a n , 1976) . Increased agg regab i1 i t y o f p l a t e l e t s 14 has been demonstrated i n d i a b e t i c s (Co lwe l l e t aj_. 1976) . Changes have a l s o been shown to occur i n va r ious p rope r t i es o f polymorpho-nuc lear leukocy tes and lymphocytes i n d iabe tes (Jones and P e t e r s o n , 1981) . C a p i l l a r y wa l l p e r m e a b i l i t y to smal l and l a rge molecules has been shown to i nc rease i n d iabe tes ( P a r v i n g , 1976; •:' B o l l i n g e r e t a l . 1982) . Due to the inc reased p e r m e a b i l i t y o f the m i c r o v a s c u l a t u r e , plasma p ro te ins leak out and dur ing t h i s process get depos i ted in the vesse l w a l l , thereby p rov id i ng the morpho log ica l bas is f o r the development o f a microangiopathy ( P a r v i n g , 1976) . The inc reased pe rmeab i l i t y o f m i c rovascu la tu re has been pos tu la ted to r e s u l t e i t h e r from an i nc rease o f h y d r o s t a t i c pressure i n the m i c r o c i r c u l a t i o n (Parv ing,1976) or from the t h i c k e n i n g o f c a p i l l a r y basement-membrane (Wi l l iamson and K i l o , 1976) . Basement membrane changes: One o f the c h a r a c t e r i s t i c % f e a t u r e s o f d i a b e t i c microangiopathy i s the t h i c k e n i n g o f basement membrane ( lamina densa) o f c a p i l l a r i e s tha t supply the g lomeru lus , r e t i n a , s k i n and s k e l e t a l muscle ( K e f a l i d e s , 1981). Hyperg lycemia , i n s u l i n d e f i c i e n c y and e leva ted l e v e l s o f plasma growth hormone are some o f the p o s s i b l e f a c t o r s tha t might c o n t r i b u t e to the pathogenesis o f basement membrane t h i c k e n i n g ( S p i r o , 1976 ; K e f a l i d e s , 1981) . There appears to be a good c o r r e l a t i o n between t h i c k e n i n g o f c a p i l l a r y basement membrane and the du ra t i on o f d iabe tes (Osterby , 1975) . Th icken ing o f c a p i l l a r y basement membrane cou ld r e s u l t from any one o f or combinat ions of the f o l l o w i n g mechanisms, 15 which i nc lude a) inc reased s y n t h e s i s , b) decreased deg rada t i on , c) d e p o s i t i o n o f serum p ro te ins i n basement membrane or d) i nc reased turnover o f basement membrane s y n t h e s i z i n g c e l l s ( K e f a l i d e s , 1977) . Basement membrane i s composed o f co l lagenous g l y c o p r o t e i n ma te r i a l w i th hyd roxy l ys i ne -1 inked carbohydrate un i t s ( S p i r o , 1967) . In d i a b e t e s , the basement membranes are found to have abnormal ly high hyd roxy l ys ine and hyd roxy l ys i ne -1 inked g l y c o s i d e content suggest ing an i nc rease i n c o l l a g e n - l i k e s u b u n i t s . Concommitant i nc reases in the a c t i v i t i e s o f the enzymes p ro toco l l agen l y s y l -hyd roxy lase , . l y s y l and p r o l y l hydroxy lases and g l y c o s y l t r a n s f e r a s e s have a l s o been found ( S p i r o , 1976 ; ; K e f a l i d e s , 1981) . The patho-p h y s i o l o g i c a l s i g n i f i c a n c e o f c a p i l l a r y basement membrane t h i c k e n i n g in d i a b e t i c microangiopathy i s not very c l e a r . W i l l i amson and K i l o (1977) have suggested tha t g r e a t l y th ickened c a p i l l a r y basement membrane might c o n t r i b u t e to de fec ts i n g lomeru lar f u n c t i o n and d i a b e t i c nephropathy, i nc reased v a s c u l a r p e r m e a b i l i t y l e a d i n g to de fec ts i n m i c r o c i r c u l a t i o n and probably d i a b e t i c r e t i n o p a t h y . C a p i l l a r y basement membrane t h i c k e n i n g , however, can be used as a use fu l index f o r de tec t i on o f the d e l e t e r i o u s changes produced by d iabe tes on the v a s c u l a r system. Other hormonal changes i n d i a b e t e s : Although d iabe tes i s cons idered as a d i s o r d e r p r i m a r i l y c h a r a c t e r i z e d by an abso lu te or r e l a t i v e d e f i c i e n c y of i n s u l i n i t i s a l s o a s s o c i a t e d w i th changes in va r ious o ther hormones such as g lucagon, growth hormone, s o m a t o s t a t i n , t h y r o i d hormones,, e t c . , 16 which may c o n t r i b u t e e i t h e r d i r e c t l y or i n d i r e c t l y to the patho-phys io logy o f the d i s e a s e . Glucagon, a po lypep t ide hormone sec re ted by the a - c e l l s o f panc rea t i c i s l e t s , i s a potent hyperglycemic f a c t o r and i t s plasma l e v e l s are g e n e r a l l y e leva ted in d iabe tes m e l l i t u s (Pek, 1977; . Lefebvre and Luyckx, 1979) . S ince d i s tu rbances i n glucagon s e c r e t i o n u s u a l l y accompany i n s u l i n d e f i c i e n c y , d iabe tes has r e c e n t l y been r e f e r r e d to as a bihormonal d i s o r d e r (Unger and O r c i , 1975). I t has been shown tha t i n d i a b e t e s , the i n h i b i t o r y e f f e c t of hyperglycemia on glucagon s e c r e t i o n i s l o s t and i n f a c t , i n c e r t a i n c a s e s , e leva ted blood g lucose l e v e l s may even i nc rease glucagon c o n c e n t r a t i o n s . Another c h a r a c t e r i s t i c f i n d i n g i n d iabe tes i s tha t a p r o t e i n meal or a r g i n i n e i n f u s i o n produces an exaggerated glucagon response (Fel i g e t aj_. 1 976; Unger,.1 978) . S ince glucagon has been shown to produce hyperglycemia by s t i m u l a t i o n o f g l y c o g e n o l y s i s and g luconeogenesis i n the l i v e r , hyperglucagonemia observed o c c a s i o n a l l y i n d iabe tes i s expected to c o n t r i b u t e to the hyperg lycemia(Pek, 1977) . Markedly e leva ted l e v e l s o f glucagon have been found i n d i a b e t i c k e t o a c i d o s i s ( M i l l e r e_t aj_. 1 973; ' F o s t e r , 1 976) and hyperosmolar non -ke to t i c coma (L indsey e t al_. 1 974) , and may be i nvo l ved in the pathogenesis o f these acute c o m p l i c a t i o n s . Growth hormone i s sec re ted by the a n t e r i o r p i t u i t a r y gland and i t g e n e r a l l y e x h i b i t s a n t a g o n i s t i c e f f e c t s to i n s u l i n on g lucose t o l e r a n c e . Growth hormone produces a reduc t i on in the uptake and u t i l i z a t i o n o f g lucose by va r ious t i s s u e s and thus cause a d iabe togen ic e f f e c t . Abnormal growth hormone s e c r e t i o n occurs i n d iabetes and i nsu l i n -dependen t d i a b e t i c s have been shown to have e leva ted l e v e l s 17 of growth hormone (Johansen and Hansen, 1969) . Increased serum growth hormone l e v e l s i n d iabe tes have been hypothes ized to be i nvo l ved i n the pathogenesis o f d i a b e t i c microangiopathy (Lundbaek, 1976). Growth hormone has a l s o been proposed to be one o f the causal fac to rs , , which cause c a p i l l a r y basement-membrane t h i c k e n i n g ( K e f a l i d e s , 1981) . Hypophysectomy or suppress ion of growth hormone has been shown to. produce an improvement o f r e t i nopa thy i n d i a b e t i c p a t i e n t s (Wright e t al_. 1 969) , thus sugges t ing a r o l e f o r growth hormone i n the deve lop-ment o f a r e t i n o p a t h y . Soma tos ta t i n , a hormone sec re ted by both the hypothalamus and the D - c e l l s o f the i s l e t s o f Langerhans, i s ab le to i n h i b i t r e l e a s e o f g lucagon, growth hormone and i n s u l i n . S tud ies w i th somatos ta t in have helped i n the understanding o f the r o l e o f glucagon in diabetes,-and somatos ta t in has even been t r i e d t h e r a p e u t i c a l l y i n order to suppress glucagon and growth hormone i n d i a b e t i c pa t i en t s but w i thout much success (Tzagourn i s , 1982) . However, the exact p h y s i o l o g i c r o l e o f somatos ta t in i n d iabe tes i s u n c l e a r . E leva ted somatos ta t in l e v e l s and an i nc rease i n the dens i t y o f somatos ta t in c o n t a i n i n g D - c e l l s have been demonstrated i n d i a b e t i c s as we l l as i n expe r imen ta l l y d i a b e t i c ra t s (Orc i e t a l . 1 976; t; Pate l e t al_. 1980) . Hypothyro id ism i s a f requent f i n d i n g in d i a b e t i c p a t i e n t s (Cooppan, 1982) . The t h y r o i d hormones thy rox ine (T4) and t r i i o d o t h y r o x i n e (T3) are produced by the t h y r o i d g land,and the t h y r o i d gland sec re tes about 8 to 10 t imes more T4, compared to T3. . In the l i v e r and k idney , T4' i s monodeiodinated to form T3 and reverse T3, a b i o l o g i c a l l y i n a c t i v e compound. Thyro id hormone metabol ism appears to be a l t e r e d i n 18 d iabe tes m e l l i t u s r e s u l t i n g i n a decreased product ion o f T 3 . D iabe t i c k e t o a c i d o s i s has always been found to be a s s o c i a t e d w i th low T3 l e v e l s (Cooppan, 1982). Recent ly a number o f repor ts have demonstrated tha t i n d i a b e t e s , most o f the monodeiodinat ion r e a c t i o n s are impaired r e s u l t i n g i n low serum l e v e l s o f T3 (Madsbad e t al_. 1981; P i t tman , et aj_. 1981) . However, the c o n t r i b u t i o n o f the prolonged low serum T3 syndrome to va r ious p a t h o l o g i c a l man i f es ta t i ons o f d iabe tes m e l l i t u s i s not known a t the present t ime (Pi t tman e t aj_. 1979) . C e l l u l a r abno rma l i t y : A l l o f the p a t h o l o g i c a l man i f es ta t i ons o f d iabe tes m e l l i t u s have been proposed to r e s u l t from an unde r l y i ng s o m a t i c - c e l l de fec t which may 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 enhanced c e l l death and rep len ishment , thereby caus ing an a c c e l e r a t e d somat ic c e l l t u rnove r . The inc rease i n somatic c e l l tu rnover ra te probably r e f l e c t s a high s u s c e p t i b i l i t y o f c e l l s to i n j u r i o u s events i n d iabe tes (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 basement-membrane t h i c k e n i n g , a t h e r o s c l e r o t i c l e s i o n s in l a rge v e s s e l s , behav ioura l a l t e r a t i o n s i n f i b r o b l a s t s e t c . tha t occur qu i te e a r l y i n d i a b e t i c s , have a l so been shown to occur i n n o n - d i a b e t i c s , but on ly w i th i n c r e a s i n g age. S tud ies i n v o l v i n g es t ima t i on of c o l l a g e n content and c r o s s - l i n k i n g have shown tha t d i a b e t i c c o l l a g e n has undergone a c c e l e r a t e d aging (Hamlin e_t a U 1 975) . I t has a l so been proposed tha t premature senescence o f 8 - c e l l s i s r espons ib l e fo r the development o f NIDDM ( C r a i g , 1980) . From the above documented ev idence , Renolds e_t al_. (1978) have suggested tha t d iabe tes i s a form o f premature c e l l u l a r senescence. 19 THE HEART AND DIABETES: Chronic d i a b e t i c s in general have a h igher i nc idence o f .and m o r t a l i t y from c a r d i a c d isease (Kanne l , 1978). A wide spectrum o f c a r d i a c problems plague the ch ron i c d i a b e t i c and i nc l ude coronary a r t e r y d i s e a s e , conges t i ve hear t f a i l u r e and the newly recogn ized c l i n i c a l e n t i t y , d i a b e t i c cardiomyopathy (Smi th , 1982). Coronary a r t e r y d i sease has been recogn ized as one o f the most common causes o f death in m idd le -aged , non- insu l i n -dependen t d i a b e t i c s (Knowles, 1978 and 1982) and i s est imated to be r e s p o n s i b l e f o r a t l e a s t 40-50% of d i a b e t i c deaths (Bennet t , 1982). Autopsy s tud ies comparing the f requency o f coronary a r t e r y d i sease i n d i a b e t i c s and n o n - d i a b e t i c s showed a preva lence ra te rang ing from 1 8 t o 75% i n d i a b e t i c p a t i e n t s (Knowles, 1978; . Sm i th , 1982) . A number o f ep idemio log i c s tud ies repor ted on the preva lence and i nc idence o f coronary a r t e r y d i sease in l i v i n g d i a b e t i c popu la t ions dur ing the fo l l ow-up p e r i o d s . The U n i v e r s i t y Group. D i a b e t i c Programme (UGDP) study i n d i c a t e d a preva lence ra te o f 9.5% and an inc idence ra te o f 12% fo r coronary a r t e r y d isease in d i a b e t i c s over a per iod o f 6.5 years (Knowles, 1978). In the Framingham s t u d y , a preva lence ra te o f 1.6% and an i nc idence ra te o f 17% have been repor ted f o r coronary a r t e r y d i sease i n the d i a b e t i c popu la t ion (Kannel et_ aj_. 1961 ; -Garc ia e_t al_. 1.974). S i m i l a r data were obta ined from var ious o ther popu la t ion s tud ies and l e d to the c o n c l u s i o n tha t the f requency o f coronary a r t e r y d i sease i n d i a b e t i c s was about two and o n e - h a l f t imes more than tha t i n n o n - d i a b e t i c s (Knowles, 1982) . These p o p u l a t i o n - s t u d i e s have a l s o shown tha t d iabe tes i nc reases the r i s k f o r coronary a r t e r y d i sease i n the female more than i n the male 20 (Kanne l , 1978; Benne t t , 1982) . Coronary a r t e r y d i sease i s c l i n i c a l l y mani fes ted i n the d i a b e t i c as angina p e c t o r i s , myocard ia l i n f a r c t i o n , sudden c a r d i a c death and o c c a s i o n a l l y as conges t i ve hear t f a i l u r e (Palumbo e_t al_. 1982) . Angina p e c t o r i s and acute myocard ia l i n f a r c t i o n are more common in the d i a b e t i c popu la t ion than in the general p o p u l a t i o n . Myocard ia l i n f a r c t i o n i s a l s o more severe i n d i a b e t i c s and r e s u l t s i n a h igher ra te o f i n - h o s p i t a l m o r t a l i t y , which again in the case o f female d i a b e t i c s i s c o n s i d e r a b l y h igher compared to male d i a b e t i c s (Partamian and B r a d l e y , 1965) . Recovery from acute myocard ia l i n f a r c t i o n i s a l s o poor i n d i a b e t i c p a t i e n t s (Smi th , 1982) . O c c a s i o n a l l y acute myocard ia l i n f a r c t i o n i n d i a b e t i c s i s not a s s o c i a t e d w i th any chest pain and o f f e r s a d i a g n o s t i c problem to the p h y s i c i a n (Brad ley .and S c h o n f e l d , 1962) . This type o f p a i n l e s s myocard ia l i n f a r c t i o n i s cons idered to be secondary to a d i a b e t i c neuropathy a f f e c t i n g the autonomic nerve con t ro l o f the hear t (Fearman e_t al_. 1 977) . Myocard ia l i n f a r c t i o n i s known to produce a s t a t e s i m i l a r to d iabe tes in a number o f p a t i e n t s owing to the increased production of C o r t i s o l , catecholamines, glucagon and growth hormone (Op ie , ejt aJL 1 979b).- The b e n e f i c i a l e f f e c t of promoting g lucose metabol ism i n the p lace o f f r ee f a t t y a c i d metabol ism on ischemic and i n f a r c t e d myocardium has been emphasized by Opie (1975) . In d i a b e t e s , myocard ia l metabol ism has been sub jec ted to va r ious a l t e r a t i o n s which r e s u l t i n a decrease i n g lucose metabol ism and an inc rease in f ree f a t t y a c i d metabol ism i n the hea r t . These changes would decrease the r e s i s t a n c e o f the d i a b e t i c myocardium to anox ia and ischemia and thus i nc rease the s e v e r i t y o f myocard ia l i n f a r c t i o n 21 (Opie et aj_. 1979 a and b ) . Coronary hear t d i sease o f the d i a b e t i c has been repor ted to be due to severe a t h e r o s c l e r o s i s which occurs a t an e a r l i e r age (Rub ie r , 1977). Disease o f l a rge blood v e s s e l s , a l s o known as macroangiopathy was cons idered to be r e s p o n s i b l e f o r the high preva lence of a t h e r o s c l e r o t i c hear t d i sease in d i a b e t i c s . However, many i n v e s t i g a t o r s have f a i l e d to d i f f e r e n t i a t e m o r p h o l o g i c a l l y macroangiopathy i n d i a b e t i c s from a t h e r o s c l e r o t i c changes tha t occur i n n o n - d i a b e t i c s , except tha t the a t h e r o s c l e r o t i c l e s i o n i n d i a b e t i c s i s much more f requent (Berkman and Zucker , 1978; S t o u t , 1979) . The inc reased frequency o f a the ro -s c l e r o s i s i n d i a b e t i c s can be a t t r i b u t e d to r i sk , f a c t o r s such as hype r tens ion , o b e s i t y , h y p e r c h o l e s t e r o l e m i a , h y p e r t r i g l y c e r i d e m i a and h y p e r l i p o p r o t e i n e m i a , which are more common to d i a b e t i c s :as compared to n o n - d i a b e t i c s (Rub ie r , 1977; Berkman and Zucker , 1978) . D iabe t i c s a l s o have a h igher i nc idence o f conges t i ve hear t f a i l u r e compared to n o n - d i a b e t i c s (Kanne l , 1978) . The i nc reased inc idence o f conges t i ve hear t f a i l u r e i s not always a consequence of c o - e x i s t i n g coronary hear t d i s e a s e . Ep idemio log i ca l data suggested tha t d i a b e t i c s devoid o f a t h e r o s c l e r o s i s or hyper tens ion have a l s o faced a f i v e - f o l d i nc reased r i s k f o r conges t i ve hear t f a i l u r e . The i nc reased r i s k f o r conges t i ve hear t f a i l u r e has a l s o been shown to be c o n f i n e d , more to i n s u l i n - t r e a t e d d i a b e t i c s (Kanne l , 1974; J a r r e t t , 1977) . In the absence o f a t h e r o s c l e r o s i s , i t i s be l i eved tha t conges t i ve heart f a i l u r e i s a consequence of myocard ia l smal l vesse l d i s e a s e , s i nce a number o f repor ts have po in ted to a p o s s i b l e r e l a t i o n s h i p between the ex ten t o f c l i n i c a l l y de tec tab le microangiopathy and the 22 degree of l e f t v e n t r i c u l a r dys func t i on (Shap i ro , 1982) . Evidence f o r the e x i s t e n c e of microangiopathy in the d i a b e t i c myocardium has been prov ided by recent repo r t s showing t h i c k e n i n g of myocard ia l c a p i l l a r y basement-membrane ( F i s c h e r e t al_. 1979) and c a p i l l a r y microaneurysms in the hear ts o f d i a b e t i c p a t i e n t s (Fac to r et al_. 1980). F i n a l l y , i t has a l s o been noted tha t ch ron i c d i a b e t i c s , p a r t i c u l a r l y the i n s u l i n -dependent' /ones, have been found to have obscure ca rd iomyopa th ies , which might a r i s e from e i t h e r a smal l vesse l d i s e a s e , impaired myocard ia l metabol ism or some other p a t h o l o g i c a l processes p e c u l i a r to d iabe tes (Kanne l , 1978). A number o f s t u d i e s have demonstrated the ex i s t ence o f myocard ia l dys func t i on i n d i a b e t i c p a t i e n t s i n the absence o f s i g n i f i c a n t coronary hear t d i sease (Regan et aj_... 1977; , D ' e l i a ejt aj_. 1 979) . Recent ly Ledet e_t al_. (1979) have c o r r e c t l y po inted out tha t the c a r d i a c d isease o f the d i a b e t i c i s not s imply due to a t h e r o s c l e r o s i s a l o n e , but i s a l s o due to o ther f a c t o r s such as microangiopathy and autonomic neuropathy which g ive r i s e to a s p e c i f i c type o f card iomyopathy. Evidence f o r the ex i s t ence o f a s p e c i f i c cardiomyopathy i n ch ron i c d i a b e t i c s has been prov ided by a number o f c l i n i c a l s t u d i e s . Rubier e t aj_. (1 972) have demonstrated a cardiomyopathy c h a r a c t e r i z e d by l e f t v e n t r i c u l a r hypertrophy and d i f f u s e myocard ia l f i b r o s i s i n ch ron i c d i a b e t i c p a t i e n t s . These p a t i e n t s d id not show any major coronary a r t e r y d i s e a s e , but had proven g l o m e r u l o s c l e r o s i s . A s i g n i f i c a n t l y high preva lence o f i d i o p a t h i c cardiomyopathy has been demonstrated i n ch ron i c d i a b e t i c pa t i en t s by Hamby e_t aj_. (1 974) . Func t iona l abno rma l i t i e s i n myocard ia l m i c r o c i r c u l a t i o n and c a p i l l a r y t r anspo r t owing to the development o f 23 microangiopathy have been proposed as the under l y ing cause f o r the ca rd iomyopa thy c o n d i t i o n . R e c e n t l y , a number o f c l i n i c a l s t u d i e s have demonstrated p r e c l i n i c a l l e f t v e n t r i c u l a r dys func t i on in d i a b e t i c s . Le f t v e n t r i c u l a r f u n c t i o n was s tud ied us ing the non - i nvas i ve s y s t o l i c t ime i n t e r v a l t echn ique . D i a b e t i c s have been shown to have a prolonged p r e - e j e c t i o n per iod ( P E P ) , a shortened l e f t v e n t r i c u l a r e j e c t i o n t ime (LVET) and an abnormal ly high PEP to LVET r a t i o (Ahmed et al_. 1 975; S e n e v i r a t n e , 1977). These abno rma l i t i es suggest a de fec t i n the ra te o f myocard ia l c o n t r a c t i o n . The abno rma l i t i es i n l e f t v e n t r i c u l a r f unc t i on were a t t r i b u t e d to p a t h o l o g i c a l a l t e r a t i o n s in the i n t e r s t i t i u m o f c a r d i a c muscle (Ahmed e_t al_. 1975) and to microangiopathy ( S e n e v i r a t n e , 1977). More r e c e n t l y Shapi ro e_t al_. (1981a) have s tud ied l e f t v e n t r i c u l a r f u n c t i o n in a l a rge group o f d i a b e t i c p a t i e n t s us ing non-i n v a s i v e techniques and found l e f t v e n t r i c u l a r dys func t i on in the ma jo r i t y o f c a s e s . These abno rma l i t i es were main ly i n d i a s t o l e and inc luded a s i g n i f i c a n t l y prolonged i sovo lumic r e l a x a t i o n t ime and a de lay i n m i t r a l va l ve opening r e l a t i v e to minimal dimension or a o r t i c va lve c l o s u r e . Abnormal s y s t o l i c t ime i n t e r v a l s have a l so been noted f r equen t l y i n these p a t i e n t s . P r e c l i n i c a l l e f t v e n t r i c u l a r dys func t i on has been observed in both i nsu l i n -dependen t and n o n - i n s u l i n dependent d i a b e t i c s ; however , i t was more common in i nsu l i n -dependen t d i a b e t i c s In another study Shapi ro et aj_. (1981b) demonstrated a c l o s e r e l a t i o n s h i p between abnormal l e f t v e n t r i c u l a r f u n c t i o n and the s e v e r i t y o f m ic ro -v a s c u l a r c o m p l i c a t i o n s . These authors have a l s o proposed tha t myocard ia l dys func t i on i n d iabetes, , .which, i n i t i a l l y i s seen as an impairment i n d i a s t o l i c r e l a x a t i o n * may occur f a i r l y e a r l y dur ing the course of the 24 d i s e a s e , and when more e x t e n s i v e , i s found both i n r e l a x a t i o n and c o n t r a c t i o n . Hemodynamic abno rma l i t i e s such as e leva ted e n d - d i a s t o l i c pressure and decreased e n d - d i a s t o l i c and s t roke volumes have been found in d i a b e t i c s wi thout hear t f a i l u r e (Ahmed and Regan, 1982) . H i s t o p a t h o l o g i c a l changes tha t are found to accompany a ca rd iomyopa thy c o n d i t i o n in d i a b e t i c s i n c l u d e , c a r d i a c d i l a t a t i o n and hypertrophy of muscle f i b r e s , e n d o t h e l i a l p r o l i f e r a t i o n , p e r i v a s c u l a r and i n t e r s t i t i a l f i b r o s i s , i nc reased accumulat ion o f p e r i o d i c a c i d - S c h i f f (PAS) p o s i t i v e or g l y c o p r o t e i n ma te r i a l and f oca l nec ros i s (Badeer and Zone ra i ch , 1978; Shapi ro et al_. 1981b; • Ahmed and Regan, 1982). A l l the above h i s t o p a t h o l o g i c a l changes occur independent ly o f coronary a t h e r o s c l e r o s i s . Autonomic neuropathy i s one of the major comp l i ca t i ons o f ch ron i c d iabetes and i s r e s p o n s i b l e f o r d i s tu rbances i n c a r d i o v a s c u l a r , g a s t r o i n t e s t i n a l , u r o g e n i t a l , endoc r i ne , r e s p i r a t o r y and thermo- regu la to ry systems (C la rke e t aj_. 1 979) . Autonomic neuropathy o f the h e a r t , a l though not f requent i n occu r rence , i s a s s o c i a t e d w i th a h igh m o r t a l i t y ra te (CIarke e_t al_. 1 979 ; Feldman, 1981) . Ca rd i ovascu la r abno rma l i t i e s owing to the l oss o f parasympathet ic a c t i v i t y are more f requent and have been repor ted to occur qu i t e e a r l y dur ing the development o f a d i a b e t i c autonomic neuropathy (Wheeler and Watk ins , 1 973; Bennett e t a l . 1975). The e a r l y de tec tab le fea tu res o f d e f e c t i v e parasympathet ic con t ro l o f the hear t are p e r s i s t e n t r e s t i n g t achyca rd ia (Rund les , 1945; .Wheeler and Watk ins , 1973) and l o s s o f bea t - to^bea t v a r i a t i o n or s inus arrhythmia dur ing deep b rea th ing (L loyd-Mostyn and Watk ins , 1975; Watkins and MacKay, 1980; • Feldman, 1981). The de fec t i n parasympathet ic i n n e r v a t i o n o f the d i a b e t i c heart has been shown o c c a s i o n a l l y to 25 progress to the ex ten t o f t o t a l vagal denerva t ion (L loyd-Mostyn and Watk ins , 1976) . In c o n t r a s t to parasympathet ic neuropathy, de fec ts in the sympathet ic i nne rva t i on o f the hear t occur very l a t e and are o f ten a s s o c i a t e d w i th o ther systemic comp l i ca t i ons such as d i a r rhea and impotence (Hosk ing , et_ al_. 1978; CIarke e_t al_. 1979) . O r t h o s t a t i c hypotension i s one o f the most s i g n i f i c a n t consequences o f a sympathet ic autonomic neuropathy o f the c a r d i o v a s c u l a r system (Hosking e t al_. 1978 ; CI arke e_t al_. 1979) . P a i n l e s s myocard ia l i n f a r c t i o n , which occurs f r e q u e n t l y i n ch ron i c d i a b e t i c s , i s ye t another consequence of an autonomic neuropathy o f the heart (C la rke et a l . 1979; Feldman, 1981) . Post-mortem h i s t o p a t h o l o g i c a l s t ud ies o f the autonomic nerves in d i a b e t i c s w i th neuropathy have revea led severe l o s s o f mye l ina ted axons, c o l l a g e n depos i t s and d is tended g ian t gang l i a wi th vacuoles in the vagus nerve and o ther sympathet ic nerves (Duchen et aj_. 1980). Recent ly a number o f s t u d i e s have been pub l i shed in which c a r d i a c f unc t i on was i n v e s t i g a t e d i n animals w i th expe r imen ta l l y - i nduced d i a b e t e s . Card iac f u n c t i o n a l a l t e r a t i o n s independent o f v a s c u l a r abno rma l i t i es have been demonstrated to occur i n dogs w i th ch ron i c a l l o x a n - i n d u c e d d iabe tes (Regan e t aj_. 1974). D i a b e t i c dog hear ts e x h i b i t e d a l t e r e d d i a s t o l i c pressure-volume r e l a t i o n s h i p s , which were a t t r i b u t e d to inc reased v e n t r i c u l a r wa l l s t i f f n e s s owing to accumu-l a t i o n o f PAS p o s i t i v e g l y c o p r o t e i n - 1 i k e m a t e r i a l . More r e c e n t l y , Regan e_t al_. (1981) have a l s o demonstrated tha t the above f u n c t i o n a l and s t r u c t u r a l a l t e r a t i o n s were not prevented by i n s u l i n - r e p l a c e m e n t . Card iac f u n c t i o n , metabol ism and b iochemis t ry ..have been s tud ied qu i te 26 e x t e n s i v e l y i n i s o l a t e d per fused working hear ts ob ta ined from r a t s wi th chemica l - i nduced d i a b e t e s . Acute a l l o x a n d i a b e t i c r a t hear ts have been repor ted to e x h i b i t a reduced t o l e rance to severe ischemia (Feuvray e_t al_. , 1978) or prolonged anoxia ( Ingebretsen .e_t al_. 1980) . I so l a t ed per fused working hear ts from acute a l l o x a n - d i a b e t i c r a t s have a l so been shown to have reduced a b i l i t i e s to develop normal s y s t o l i c ( a o r t i c ) pressure ( M i l l e r , 1979) , basal l e f t v e n t r i c u l a r pressure and ra te o f r i s e o f l e f t v e n t r i c u l a r pressure ( Ingebretsen e_t aj_. 1 980) i n response to inc reased c a r d i a c work. Reduct ions in c a r d i a c ou tpu t , s t roke work, peak l e f t v e n t r i c u l a r pressure and maximal ra te o f d e c l i n e in l e f t v e n t r i c u l a r pressure were observed i n i s o l a t e d per fused working hear ts ob ta ined from ch ron i c 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 , i n response to h igh a t r i a l f i l l i n g pressures (Penpargkul e t al_. 1980) . Fur thermore, l e f t v e n t r i c u l a r p a p i l l a r y muscles i s o l a t e d from ch ron i c 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 have been repor ted to e x h i b i t depress ions in the ra te o f r e l a x a t i o n and v e l o c i t y o f sho r ten ing a t va r ious loads (Fe in et a l . 1980). A number o f changes have been repor ted to occur i n the metabol ism and va r ious b iochemica l events in the hear ts o f expe r imen ta l l y d i a b e t i c an ima ls . M u l t i p l e abno rma l i t i e s occur i n carbohydrate and l i p i d metabol ism i n the hear t i n d i a b e t e s . Glucose uptake i n t o c a r d i a c c e l l s i s decreased and so i s g lucose phosphory la t ion and g l y c o l y s i s . As a r e s u l t f r ee g lucose accumulated w i t h i n the c e l l and c a r d i a c glycogen content i nc reased i n s p i t e o f decreased glycogen synthase a c t i v i t y (Opie. e_t al_. 1979a). L i p i d metabol ism i s inc reased i n d i a b e t i c hear ts owing main ly to the inc reased a v a i l a b i l i t y o f f ree f a t t y ac ids i n the 27 b lood . A marked inc rease in the t r i g l y c e r i d e content o f the d i a b e t i c myocardium has been repor ted (Paulson and C r a s s , 1980) . Feuvray et al_. (1979) have demonstrated inc reased t i s s u e l e v e l s o f t o t a l CoA, l ong -cha in acy l CoA and l o n g - c h a i n * a c y l c a r n i t i n e es te r s i n i s o l a t e d working hear ts from a l l o x a n - d i a b e t i c r a t s . Increased l e v e l s o f these in te rmed ia tes o f f a t t y a c i d metabol ism have been proposed to be invo lved in the development o f c a r d i a c f u n c t i o n a l d i s tu rbances (Farah and A l o u s i , 1981). Acute expe r imen ta l -d iabe tes has a l s o been found to r e s u l t i n reduced l e v e l s and produc t ion o f ATP ( A l l i s o n e_t aj_. 1 976) decreased p ro te i n syn thes i s (Wi l l i ams ej: aj_. 1980) and changes i n myosin components (D i l lmann , 1980) i n the hea r t . Var ious b iochemical processes have been found to be a l t e r e d in expe r imen ta l l y - i nduced d iabetes and these a l t e r a t i o n s may c o n t r i b u t e t o . t h e c a r d i a c dys func t i on observed i n d i a b e t i c an ima l s . Depressions 2+ in the a c t i v i t y o f m y o f i b r i l l a r basal and Ca s t imu la ted ATPases, a c t o -2+ myosin ATPase and Ca ATPase of pure myosin have been demonstrated in s t r e p t o z o t o c i n d i a b e t i c ra t s (D i l lmann , 1980; Malhot ra ejt al_. 1981; P ie r ce and _ D h a l l a , 1981) . Card iac sarcop lasmic r e t i cu l um from d i a b e t i c r a t s has been shown to e x h i b i t a decreased a b i l i t y to take up 2+ ca lc ium and a depressed a c t i v i t y o f the membrane-bound Ca ATPase (Penpargkul e_t aj_. 1981; Lopaschuk e_t al_. 1 983) . I t has been pos tu la ted tha t the e leva ted l e v e l s o f l o n g - c h a i n a c y l c a r n i t i n e s in d i a b e t i c 2+ 2+ hearts i n h i b i t Ca uptake and Ca ATPase a c t i v i t y o f sa rcop lasmic r e t i cu l um (Lopaschuk e t al_. 1983) . The a b i l i t y o f a c y l c a r n i t i n e es te r s 2+ to i n h i b i t Ca ATPase a c t i v i t y o f c a r d i a c sa rcop lasmic r e t i c u l u m ( P i t t s e t a l . 1978) and c a r d i a c N a + , K + , ATPase a c t i v i t y (Wood e t a l . 1977) 28 has p r e v i o u s l y been demonstrated. Very few repor ts have been pub l i shed thus f a r i n which the e f f e c t s o f exogenously admin is te red c h o l i n e r g i c and adrenerg ic drugs have been s tud ied on the hear t i n expe r imen ta l l y - i nduced d i a b e t e s . Foy and Lucas (1976 and 1978) have repor ted reduc t ions in the i n o t r o p i c e f f e c t s to exogenous adrenerg ic amines and the depressor response to a c e t y l c h o l i n e in both p i thed d i a b e t i c ra t s and in i s o l a t e d d i a b e t i c r a t a t r i a . These authors a l so repor ted tha t the ch rono t rop i c responses to i sop ro te reno l were reduced i n p i thed d i a b e t i c r a t s whereas they were inc reased i n i s o l a t e d r a t a t r i a l p r e p a r a t i o n s . Ingebretsen e t aj_. (1981) have repor ted no change i n the dose-dependent i n o t r o p i c e f f e c t o f i sop ro te reno l i n i s o l a t e d working hear ts from acute a l l o x a n - d i a b e t i c r a t s . The above documented s t u d i e s were performed wi th acute (7 to 14 day) d i a b e t i c r a t s . The on ly study in which ch ron i c d i a b e t i c ra t s have been used to study the e f f e c t s o f c a r d i o a c t i v e drugs i s by Tomlinson and Yusof (1981) . These i n v e s t i g a t o r s , us ing i s o l a t e d a t r i a ob ta ined from 7 to 8 month a l l o x a n - d i a b e t i c r a t s , have demonstrated no change in the responses o f d i a b e t i c a t r i a to no radrena l ine and a s u p e r s e n s i t i v i t y to the negat ive i n o t r o p i c e f f e c t o f a c e t y l c h o l i n e . Autonomic neuropathy has been demonstrated i n ch ron i c 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 by Schmidt et aj_. (1981) and Schmidt and Schapp (1982) , us ing u l t r a s t r u c t u r a l , h i s tochemica l and b iochemica l methods. Swol len degenerat ing unmyel in-ated te rmina l axons were f r e q u e n t l y found in the co lon and mesenter ic nerves of ch ron i c d i a b e t i c r a t s . Hi istochemical s t u d i e s showed an o v e r a l l decrease in the i n t e n s i t y o f catecholamine f l uo rescence and c h o l i n e s t e r a s e s t a i n i n g in ch ron i c d i a b e t i c r a t s . Decreased a c t i v i t i e s 29 of the enzymes dopamine g-hydroxy lase and c h o l i n e ace ty l t r a n s f e r a s e - . have a l so been found. Recent ly a number o f s t ud ies have been pub l i shed in which the e f f e c t s o f expe r imen ta l -d iabe tes on c a r d i a c c y c l i c AMP and phosphory lase were i n v e s t i g a t e d . Chaudhury and Shipp (1973) have demonstrated tha t basal c a r d i a c c y c l i c AMP l e v e l s were f o u r - f o l d h igher i n acute a l l o x a n -d i a b e t i c r a t s , whereas severa l o ther repor ts showed no changes in e i t h e r basal adeny la te c y c l a s e a c t i v i t y or basal c y c l i c AMP content i n hear ts i s o l a t e d from acute d i a b e t i c ra t s (Das, 1 973; Menahan e t aj_. 1977; Ingebretsen et al_. 1981; ; Mi T i e r ejb al_. 1981). However, Ingebretsen et al_. (1981) repor ted a 50 percent reduc t i on i n i s o p r o t e r e n o l - i n d u c e d inc reases in c a r d i a c c y c l i c AMP content i n acute a l l o x a n - d i a b e t i c r a t s , but M i l l e r e_t al_. (1981) f a i l e d to show such a reduc t i on i n the e f f e c t o f ep inephr ine on c a r d i a c c y c l i c AMP in a s i m i l a r d i a b e t i c r a t model . Basal c a r d i a c phosphory lase a^  a c t i v i t y has been shown to be una l te red in acute d i a b e t i c ra t s (Das, 1973; Ingebretsen ejt aj_. 1981 ; • M i l l e r e_t aj_. 1981) . However M i l l e r ejt aj_. (1981) have repor ted tha t ep inephr ine caused a s i g n i f i c a n t l y enhanced a c t i v a t i o n o f phosphory lase a^  i n acute d i a b e t i c r a t h e a r t s , and t h i s obse rva t i on has been p a r t i a l l y conf i rmed by Ingebretsen ejb aj_. (1981) , as these authors showed on ly a s l i g h t , but not s i g n i f i c a n t , enhancement i n the a c t i v a t i o n o f phosphory lase by i sop ro te reno l i n acute d i a b e t i c r a t h e a r t s . PRODUCTION OF EXPERIMENTAL DIABETES, ALLOXAN AND STREPTOZOTOCIN: Diabetes m e l l i t u s can be produced i n l a b o r a t o r y animals w i th chemical agents which cause immediate damage to the g - c e l l s o f the i s l e t s o f Langerhans. These chemical substances have been r e f e r r e d to as 30 3 - c y t o t o x i c agents or 3 - c y t o t o x i n s (Rerup, 1970). The most important o f these compounds are a l l o x a n and s t r e p t o z o t o c i n (STZ) . A l l oxan was f i r s t repor ted to have d iabe togen ic a c t i v i t y by Dunn and McLetchie i n 1943 and s i nce then has been w ide ly used to induce e x p e r i -mental d iabe tes in l a b o r a t o r y an ima l s . The d iabe togen ic e f f e c t o f STZ was f i r s t d i scovered by Rakieten e_t aj_. i n 1 963. A l l o x a n monohydrate: A l l oxan (2 ,4 ,5 ,6 - t e t raoxohexahyd ropy r im id ine ) i s u s u a l l y employed in i t s monohydrate form. I t i s f r e e l y s o l u b l e in water and i t s s t a b i l i t y i s pH and temperature dependent. A l l oxan i s qu i t e s t a b l e i n a c i d i c s o l u t i o n , but i s h i gh l y uns tab le i n neu t ra l s o l u t i o n a t room temperature, I t s h a l f - l i f e i n b i o l o g i c a l f l u i d s such as blood i s l e s s than 1 minute . A l l oxan has been repor ted to produce d iabe tes i n a lmost a l l o f the l abo ra to r y animal spec ies t es ted except the gu inea -p ig (Rerup, 1970) . A l l oxan has been shown to be e f f e c t i v e by a l l routes o f a d m i n i s t r a t i o n ; n e v e r t h e l e s s , t h e in t ravenous route i s most p re fe r red s i nce i t i s h i g h l y e f f e c t i v e and l e s s t o x i c (Lazarow and P a l a y , 1946) . The d iabe togen ic dose o f a l l o x a n i n r a t s v a r i e s between 30 to 70 mg/kg and i s sub jec t to v a r i a t i o n s i n mean s e n s i t i v i t y w i t h i n s t r a i n s and between l a b o r a t o r i e s (Rerup, 1 970).. A l l o x a n a d m i n i s t r a t i o n has been shown to produce a c h a r a c t e r i s t i c t r i p h a s i c response i n blood g lucose l e v e l s (Lundquis t and Rerup 1967) . These phases a r e : 1. An i n i t i a l hyperglycemia of shor t d u r a t i o n , probably due to inc reased g l y c o g e n o l y s i s in the l i v e r and i n s u l i n d e f i c i e n c y ; 2 . A t r a n s i e n t hypoglycemia which l a s t s up to 48 hours and o'ften r e s u l t s i n convu ls ions and dea th . Th is phase cou ld be due to e i t h e r a su rp lus 31 of i n s u l i n or a complete l ack o f g lucose product ion in the l i v e r and 3. A permanent hyperglycemia rep resen t i ng a l l o x a n d i a b e t e s . The mechanism o f a c t i o n o f the B - c y t o t o x i c e f f e c t o f a l l o x a n i s not very c l e a r . Recent ly severa l hypotheses have been put forward to e x p l a i n the h i g h l y s p e c i f i c B - c y t o t o x i c e f f e c t o f a l l o x a n . Accord ing to one of these hypotheses ( B o q u i s t , 1980) a l l o x a n i s supposed to pass the B - c e l l membrane, probably through a g lucose t r anspo r t system. Ins ide the 8 - c e l l , a l l o x a n appears to produce an inc reased concen t ra t i on o f i no rgan i c phosphate ( P i ) i n the cy toso l by i n h i b i t i n g a s u l f h y d r y l -dependent t r a n s p o r t system f o r phosphate in the m i t ochond r i a . The inc reased concen t ra t i on o f Pi i n the cy toso l i s be l i eved to produce 2+ a f a l l i n i n t r a c e l l u l a r pH and a change i n i n t r a c e l l u l a r Ca . A f a l l i n the c y t o s o l i c pH not on ly i nc reases the s t a b i l i t y o f a l l o x a n thereby p ro long ing i t s a c t i o n but a l s o i n h i b i t s the syn thes i s and g lucose -induced re l ease o f i n s u l i n . Blockade of m i tochondr ia l phosphate t r anspo r t may r e s u l t i n the i n h i b i t i o n o f NAD-dependent o x i d a t i v e r e a c t i o n s i n c l u d i n g o x i d a t i v e phospho ry l a t i ons . In the absence of energy p r o d u c t i o n , nec ros i s o f the B - c e l l occurs r e s u l t i n g i n the development o f d i a b e t e s . More r e c e n t l y a new and a l t e r n a t i v e hypothes is has been put forward by MaLaisse (1982) f o r the h i g h l y s e l e c t i v e B - c y t o t o x i c e f f e c t o f a l l o x a n . Th is hypothes is dea ls w i th two d i s t i n c t fea tu res o f a l l o x a n which may be r espons ib l e f o r the B - c y t o t o x i c e f f e c t . The f i r s t i s the r a p i d ra te at which a l l o x a n i s taken up by the panc rea t i c i s l e t c e l l s and the second i s the a b i l i t y o f a l l o x a n to undergo reduc t i on to d i a l u r i c a c i d i n the presence o f reduc ing agen ts . A l l oxan and d i a l u r i c a c i d then form a r e d u c t i o n - o x i d a t i o n c y c l e r e s u l t i n g i n 32 the genera t ion o f h i g h l y r e a c t i v e oxygen-con ta in ing f ree r a d i c a l s such as , ^ 2^2 a n c * ' I s l e t c e l l s are h ighly, s e n s i t i v e to the t o x i c e f f e c t s o f these f ree r a d i c a l s . This hypothes is p a r t l y e x p l a i n s why guinea p igs are the l e a s t s e n s i t i v e spec ies to the d iabe togen ic e f f e c t o f a l l o x a n s i nce guinea p ig i s l e t c e l l s have been shown to be a t l e a s t 5 t imes l e s s s e n s i t i v e than r a t i s l e t s to the t o x i c e f f e c t s o f t e r t - b u t y l hydroperox ide , a compound o f ten used as an exogenous peroxide (Ma ia isse-Lagae e t al_. 1981). S t r e p t o z o t o c i n (STZ) : STZ ( 2 - d e o x y - 2 - ( 3 - m e t h y l - 3 - n i t r o s o u r e a ) l - d - g l u c o p y r a n o s e ) i s a broad-spectrum a n t i b i o t i c i s o l a t e d from Streptomyces achromogenes (Herr e_t aj_. 1960). STZ has been repor ted to possess c a r c i n o g e n i c , ant i tumor and d iabe togen ic e f f e c t s i n a d d i t i o n to i t s a n t i b i o t i c p rope r t i es ( S r i v a s t a v a ejt aj_. 1 982) . STZ i s an uns tab le compound at e i t h e r room or r e f r i g e r a t o r temperatures and must be s to red below -20°C. I t i s f r e e l y s o l u b l e i n water , but i s very unstab le i n s o l u t i o n . STZ, however, i s qu i t e s t a b l e i n s o l u t i o n at pH 4.0 and low temperatures (Rerup, 1970) . STZ produces d iabe tes i n most o f the l a b o r a t o r y animal spec ies t e s t e d . In the r a t a s i n g l e in t ravenous i n j e c t i o n o f 50 mg/kg was repor ted to produce 100% d iabe tes (Rak ie ten e t a l . 1963). Due to the uns tab le nature o f STZ in s o l u t i o n , a r a p i d in t ravenous i n j e c t i o n i s the most p re fe r red route o f a d m i n i s t r a t i o n . STZ has been shown to produce a c h a r a c t e r i s t i c t r i p h a s i c response i n blood sugar l e v e l s which was s i m i l a r to tha t observed a f t e r a d m i n i s t r a t i o n o f a l l o x a n (Junod e_t al_. 1 969) . The d iabe togen ic e f f e c t o f STZ has been shown to be dose-dependent (Junod e_t al_. 1 969) and i n v e r s e l y 33 r e l a t e d to the age o f the animals ( M a s i e l l o et aj_. 1979) . The mechanism o f the d iabe togen ic a c t i o n o f STZ i s not known, and any of the f o l l o w i n g e f f e c t s o f STZ might be i nvo l ved i n i t s 3 - c y t o t o x i c i t y . Panc rea t i c i s l e t c e l l s e x h i b i t a marked s p e c i f i c i t y and c a p a c i t y to take up STZ. STZ i s be l i eved to be taken-up through a s p e c i f i ' c t r anspo r t system ( S r i v a s t a v a e t al_. 1982) and the g lucose moiety o f STZ i s necessary f o r the t r anspo r t process (Agarwa l , 1980). STZ has been shown to cause a marked d e p l e t i o n o f l i v e r and i s l e t NAD and NADH l e v e l s , w i t h i n 24 hours a f t e r a d m i n i s t r a t i o n and t h i s e f f e c t might c o n t r i b u t e to the d iabe togen ic a c t i o n (Rerup 1 970.; Agarwa l , 1980) as pret reatment w i th n ico t inamide has been shown to p ro tec t aga ins t STZ- induced d iabe tes (Schein e_t aj_. 1967) . H i s t o p a t h o l o g i c a l changes observed a f t e r the a d m i n i s t r a t i o n o f STZ i n c l u d e : hypert rophy o f B - c e l l Go lg i appa ra tus , nuc lea r pyknosis and cy top lasmic v a c u o l i z a t i o n r e s u l t i n g i n degenerat ion o f B - c e l l (Agarwal , 1980; S r i v a s t a v a et aj_. 1982). Comparison of a l l o x a n - and STZ- induced d i a b e t e s : Diabetes m e l l i t u s induced by e i t h e r a l l o x a n or STZ e x h i b i t e d the c l a s s i c a l symptoms o f t ype- I d iabetes (IDDM) such as p o l y d i p s i a , po l yphag ia , p o l y u r i a , g l y c o s u r i a and weight l o s s (Rerup , 1970). However, c e r t a i n d i f f e r e n c e s have been repor ted i n the metabo l i c a b n o r m a l i t i e s caused by a l l o x a n or s t r e p t o z o t o c i n . Mansford and Opie (1968) have demonstrated tha t a l l o x a n d iabe tes i s c h a r a c t e r i z e d by hyperg lycemia , e l eva ted f ree f a t t y a c i d l e v e l s i n the blood and k e t o s i s , whereas STZ d iabe tes on ly e x h i b i t e d hyperglycemia w i thout any changes i n blood f ree f a t t y a c i d and 34 ketone l e v e l s . On the c o n t r a r y , Junod e t aJL (1969) showed tha t the s e v e r i t y o f STZ-d iabetes i s dose-dependent and l a r g e r doses o f STZ produced d iabe tes wi th k e t o n u r i a . Recen t l y , Opie et al_. (1 979a) have r e p o r t e d . t h a t acute STZ- induced d iabe tes i s a s s o c i a t e d wi th e leva ted blood l e v e l s o f f ree f a t t y ac ids and ketone bodies and a l s o tha t Mansford and Opie (1968) have i n c o r r e c t l y con t ras ted acute a l l o x a n -d iabe tes w i th ch ron i c s t r e p t o z o t o c i n - d i a b e t e s in t h e i r s tudy . Furthermore H o f t e i z e r and Carpenter (1973) have repor ted tha t the c l i n i c a l courses o f a l l o x a n - and s t r e p t o z o t o c i n - i n d u c e d d iabe tes were almost s i m i l a r . From the above d i s c u s s i o n i t i s ev iden t tha t both a l l o x a n and STZ produce d iabe tes i n l abo ra to r y animals in a dose-dependent manner and the d i a b e t i c s t a t e produced by e i t h e r drug very c l o s e l y resembles the i nsu l i n -dependen t type o f d i a b e t e s . THE PURPOSE OF THE INVESTIGATION: From the in fo rmat ion documented thus f a r i n t h i s s e c t i o n , i t i s ev iden t tha t c a r d i a c f u n c t i o n , metabo l ism, b iochemis t ry and to some extent pharmacology have a l l been s tud ied in exper imenta l d i a b e t i c animal models. In most cases acute d i a b e t i c animal models were used in these s t u d i e s . N e v e r t h e l e s s , there have been a few s tud ies i n which ch ron i c d i a b e t i c animal models were used. In g e n e r a l , c a r d i a c comp l i ca t i ons of d i a b e t i c p a t i e n t s are a consequence of l ong -s tand ing d iabetes and r a r e l y appear i n the e a r l y phases of c l i n i c a l d i a b e t e s . In a recent rev iew , Farah and A lous i (1981) have po in ted out a need fo r the r e - e v a l u a t i o n o f the m o r p h o l o g i c a l , f u n c t i o n a l and b iochemica l c o n d i t i o n s i n hear ts ob ta ined from ch ron i c d i a b e t i c an ima l s , i ns tead 35 of those obta ined from animals w i th acute d i a b e t e s . We the re fo re i n v e s t i g a t e d c a r d i a c f u n c t i o n , pharmacology and c e r t a i n b iochemica l events in i s o l a t e d per fused hear ts ob ta ined from d i a b e t i c ra t s a t var ious t ime po in ts a f t e r i nduc t i on o f the d i s e a s e . We examined three important phases in the course of the exper imenta l d i s e a s e , namely a c u t e , moderately ch ron i c and ch ron i c phases, i n order to ob ta in i n fo rmat ion regard ing when c a r d i a c f u n c t i o n a l a l t e r a t i o n s appeared and whether these a l t e r a t i o n s progressed w i th the d i sease s t a t e . We chose the r a t as a model s i nce i t has been known f o r some time tha t a t h e r o s c l e r o t i c comp l i ca t i ons do not r e a d i l y develop i n t h i s model . In f a c t i n a recent repo r t Chobanian et_ al_. (1982) have pointed out tha t macrovascular d i sease was not common in ra t s made d i a b e t i c w i th e i t h e r a l l o x a n or s t r e p t o z o t o c i n . By us ing such a model , one would be ab le to i n v e s t i g a t e c a r d i a c f u n c t i o n a l a l t e r a t i o n s tha t might be o c c u r r i n g independent ly o f coronary a r t e r y d i sease and such f u n c t i o n a l a l t e r a t i o n s may represent the development o f a s p e c i f i c heart muscle d i s o r d e r a l s o known as d i a b e t i c cardiomyopathy. We used both a l l o x a n and s t r e p t o z o t o c i n to induce d iabe tes in the r a t . Most o f the acute s tud ies tha t have been pub l i shed thus f a r were done on a l l o x a n - d i a b e t i c r a t s and the ch ron ic s t u d i e s were done predominant ly on S T Z - d i a b e t i c r a t s . By employing both of these d iabe togen ic drugs to induce d i a b e t e s , we would be ab le to compare our r e s u l t s w i th almost a l l o f the p r e v i o u s l y pub l i shed in fo rmat ion on acute as we l l as ch ron i c d i a b e t i c animals rega rd less o f which d i abe to -genic drug was employed. At the same time we cou ld a l s o compare these 36 two models w i th regard to the onset and p rog ress ion o f c a r d i a c c o m p l i c a t i o n s . We used the i s o l a t e d per fused working hear t p repa ra t i on to study c a r d i a c performance and the e f f e c t s o f c a r d i o a c t i v e d rugs . The working hear t p repara t i on has advantages over the convent iona l Langendorf f p repa ra t i on s i n c e the c a r d i a c performance o f the working hear ts more n e a r l y approximates p h y s i o l o g i c a l c o n d i t i o n s and l e f t v e n t r i c u l a r f unc t i on can be est imated w i th more accu racy . F i n a l l y , we used age-matched ra t s as c o n t r o l s f o r the d i a b e t i c ra t s i n o rder to n u l l i f y the e f f e c t s o f aging on c a r d i a c f u n c t i o n . SPECIFIC AIMS: 1. To study c a r d i a c f u n c t i o n i n a l l o x a n and STZ d i a b e t i c and age-matched con t ro l r a t s a t va r i ous t ime -po in t s a f t e r the i n d u c t i o n o f d i a b e t e s . 2 . To study the negat ive i n o t r o p i c e f f e c t o f carbachol on con t ro l and d i a b e t i c r a t hear ts a t va r ious t i m e - p o i n t s . 3. To study the p o s i t i v e i n o t r o p i c and . .card iac, r e l a x a n t e f f e c t s of i sop ro te reno l -on con t ro l and - . d i a b e t i c ' r a t h e a r t s . 4,. To study the t ime-course of the e f f e c t o f i sop ro te reno l on c y c l i c AMP and the phosphory lase system in acute as we l l as ch ron i c d i a b e t i c and con t ro l r a t h e a r t s . 37 METHODS ANIMAL MODEL: Female Wis ta r r a t s weighing 175-200g, cor respond ing to 9-12 weeks of age, were used throughout the s tudy . The r a t s were a l lowed to recover from s t r e s s o f t r a n s p o r t a t i o n f o r a week before they were g iven any t reatment . The ra t s were randomly d i v i d e d i n to 3 groups, the f i r s t two groups were made d i a b e t i c and the t h i r d group was used as age-matched c o n t r o l s . Diabetes was induced by s i n g l e in t ravenous i n j e c t i o n o f e i t h e r a l l o x a n monohydrate or STZ d i s s o l v e d in 0.1 M c i t r a t e b u f f e r , pH 4 . 5 . Both a l l o x a n and s t r e p t o z o t o c i n are uns tab le in s o l u t i o n but are r e l a t i v e l y more s t a b l e in a c i d i c s o l u t i o n compared to e i t h e r bas i c or neu t ra l s o l u t i o n . S o l u t i o n s of a l l o x a n and STZ were f r e s h l y made before i n j e c t i n g and were kept on i c e . I n i t i a l l y d iabe tes was induced by us ing e i t h e r 45 mg/kg a l l o x a n or 60 mg/kg STZ. In the l a t t e r par t o f t h i s s tudy , these doses produced a high ra te o f m o r t a l i t y i n ra ts w i t h i n the f i r s t week of i n j e c t i o n owing to the produc t ion o f a very severe s t a t e o f d i a b e t e s . Seasonal v a r i a t i o n s in the mean s e n s i t i v i t y of Wis ta r ra t s towards these d iabe togen ic drugs cou ld have r e s u l t e d in such an e f f e c t . Dose response curves were performed to determine a s a f e r but e f f e c t i v e dose f o r these drugs and the new doses used were: 40 mg/kg a l l o x a n and 50 mg/kg STZ. S o l u t i o n s o f a l l o x a n c o n t a i n i n g e i t h e r 65 or 40 mg/ml and o f STZ c o n t a i n i n g e i t h e r 60 o r 50 mg/mj were p repared . Each r a t r ece i ved a f r a c t i o n o f a m l , i n 'the range of 0.17 to 0.2 m i s , depending on the body we igh t . Such smal l volumes helped in ach iev i ng rap id i n j e c t i o n s 38 wi thout caus ing major changes i n blood pH. Intravenous a d m i n i s t r a t i o n o f the drugs was made i n t o the r a t t a i l v e i n . Rats were anes the t i zed wi th e ther p r i o r to i n j e c t i o n . T a i l ve ins were d i l a t e d by app ly ing warm water to the t a i l , f o l l owed by the a p p l i c a t i o n o f 95% e thy l a l c o h o l . D iabetogenic drugs were r a p i d l y i n j e c t e d i n t o the d i l a t e d t a i l ve in us ing a 1 ml t u b e r c u l i n sy r i nge f i t t e d wi th a 25 1/2 gauge hypodermic need le . Rats i n the con t ro l group rece i ved app rop r i a te volumes o f c i t r a t e bu f fe r a l o n e . Rats recovered from e ther anes thes ia w i t h i n 5 minutes a f t e r i n j e c t i o n . The three groups o f r a t s were randomly d i v i d e d i n t o va r i ous se ts and were used a t va r ious t imes a f t e r i n j e c t i o n . Contro l and t r ea ted r a t s were housed s e p a r a t e l y and were given food (Pur ina Rat Chow) and water ad l i b i t u m . Produc t ion o f a d i a b e t i c s t a t e was diagnosed 48 hours a f t e r i n j e c t i o n by a p o s i t i v e t e s t f o r g l y c o s u r i a w i th L i l l y T e s t - t a p e . The d i a b e t i c s t a t e was moni tored by p e r i o d i c a l t e s t s f o r the presence o f g l u c o s u r i a t h e r e a f t e r . Blood samples were c o l l e c t e d at the time o f s a c r i f i c e , a l lowed to c l o t , c e n t r i f u g e d and the c l e a r serum was analyzed f o r serum g l u c o s e , immuno-r e a c t i v e i n s u l i n ( I R I ) , t hy rox ine (T4) and t r i i o d o t h y r o n i n e (T3) l e v e l s . WORKING HEART APPARATUS: The working hear t pe r fus ion apparatus used i n t h i s study i s a n o n - r e c i r c u l a t i n g type and i s a mod i f ied ve rs i on o f N e e l y ' s o r i g i n a l working hear t apparatus (Neely e_t a j \ 1967) as desc r ibed p r e v i o u s l y by Rodgers e t al_. (1981) . 39 The working hear t apparatus as shown in F igure 1, conta ined two jacke ted pr imary and two jacke ted f i l l i n g r e s e r v o i r s . One o f the pr imary r e s e r v o i r s i s connected through a 3-way stop cock to a 15 gauge s t e e l a o r t i c cannula or to one of the f i l l i n g r e s e r v o i r s . The o ther pr imary r e s e r v o i r i s connected to the second f i l l i n g r e s e r v o i r . The pr imary r e s e r v o i r connected to the a o r t i c cannula i s s i t u a t e d 45 cms above the a o r t i c cannu la . Both the f i l l i n g r e s e r v o i r s are connected to a 1,6 gauge s tee l pe r fus ion cannula and the r e s e r v o i r s are f i x e d such tha t they can be moved up and down in steps to prov ide va r ious f i l l i n g p r e s s u r e s . Bu f f e r l e v e l i n the jacke ted r e s e r v o i r s i s mainta ined a t a cons tant l e v e l w i th the help o f smal l g lass f l o a t v a l v e s . The a o r t i c cannula i s a l s o connected to a s e r i e s o f 1 ml p i p e t t e s , through a second 3-way stop cock . The 1 ml p i pe t t es are arranged to g ive va r ious combinat ions o f p i p e t t e s in p a r a l l e l and s e r i e s and served as an ex te rna l pe r i phe ra l r e s i s t a n c e . The per fusa te in the pr imary and f i l l i n g r e s e r v o i r s i s mainta ined at 37°C by means of D a Haake constant - tempera ture water r e c i r c u l a t i n g pump. The per fusa te in the r e s e r v o i r s i s a l s o c o n s t a n t l y aera ted wi th a mixture o f 95% oxygen and 5% carbon d i o x i d e . The per fusa te used i s the one desc r ibed p r e v i o u s l y by Chenoweth and Koe l l e (1946). The Chenoweth-Koel le (C-K) s o l u t i o n conta ined the f o l l o w i n g in m i l l i m o l a r q u a n t i t i t e s : N a C l , 120; KC1, 5 . 6 ; C a C l 2 ; 2 . 1 8 ; M g C l 2 , 2 . 1 ; NaHC0 3 , 19; g l u c o s e , 9 . 9 ; and EDTA, . 0 3 . • When aerated wi th 95% 02-5% C 0 2 mix ture at 37°C, t h i s s o l u t i o n gave a pH of 7 .4 . 40 FIGURE 1: Schematic r ep resen ta t i on o f the working hear t appara tus . The working hear t apparatus conta ined two jacke ted pr imary (1) and two jacke ted f i l l i n g r e s e r v o i r s ( 2 ) . One o f the pr imary r e s e r v o i r s i s connected through 3-way stop cocks (7) to a 15-gauge s t e e l a o r t i c cannula (3) or to one o f the f i l l i n g r e s e r v o i r s . The o ther pr imary r e s e r v o i r i s connected to the second f i l l i n g r e s e r v o i r . Both the f i l l i n g r e s e r v o i r s are connected to a 16 gauge s t e e l l e f t a t r i a l cannula ( 4 ) . The pr imary r e s e r v o i r connected to the a o r t i c cannula i s s i t u a t e d 45 cm above the cannu la . The f i l l i n g r e s e r v o i r s can be moved up or down in s teps to prov ide va r ious f i l l i n g pressures between 5 and 22.5 cm H^O. The a o r t i c cannula i s a l s o connected to a s e r i e s o f 1 ml p i p e t t e s , which serve as an ex te rna l pe r i phe ra l r e s i s t a n c e ( 8 ) . The per fusa te i n the r e s e r v o i r s i s c o n s t a n t l y aerated through the b u i l t - i n oxygenators (10) and i s main ta ined a t 37°C by means of a cons tant temperature water r e c i r c u l a t i n g pump (not shown i n the f i g u r e ) . The i s o l a t e d r a t hear t (12) i s a t tached ' to the a o r t i c cannula through the aor ta and per fused i n i t i a l l y i n the re t rograde manner a t 45 cm h^O f i l l i n g p ressu re . The l e f t v e n t r i c u l a r chamber i s connected to a pressure t ransducer (5) as desc r ibed i n the methods s e c t i o n . The l e f t , a t r ium i s cannu la ted wi th the a t r i a l cannula and the pe r fus ion i s swi tched to the working mode. In the working mode, pe r fusa te from f i l l i n g r e s e r v o i r s , entered the l e f t v e n t r i c u l a r chamber, pumped out through the aor ta i n to the pe r i phe ra l r e s i s t a n c e (as i n d i c a t e d by the a r rows ) . In t ra a o r t i c pressure i s recorded through the second pressure t ransducer (6) and the hear ts are paced a t 300 beats /min by p l a c i n g a b i p o l a r p la t inum e l e c t r o d e (11) on the l e f t a t r i u m . A o r t i c output i s determined by measuring the volume o f per fusa te f l ow ing out o f the a o r t i c o u t - f l o w system ( 9 ) . 41 42 HEART PERFUSION: Animals were s a c r i f i c e d by s tunn ing them wi th a blow to the head fo l l owed by d e c a p i t a t i o n us ing a g u i l l o t i n e . Hearts were q u i c k l y removed from the animals and were immediately p laced in aera ted C-K s o l u t i o n . The a o r t i c stump was l o c a t e d , separated from the sur rounding t i s s u e and a t tached to the 15 gauge a o r t i c pe r fus ion cannu la . Pe r fus ion was i n i t i a t e d i n the re t rograde manner through the aor ta a t 45 cm H^O (30 mm Hg) a o r t i c f i l l i n g p ressu re . Remnants o f the pulmonary ve in were l oca ted and a smal l p iece (3 cm in length) o f PE 90 tub ing was i n s e r t e d i n to the l e f t v e n t r i c l e through the pulmonary ve in and p u l l e d out through the apex o f the hear t such tha t one end o f the tube remained i n s i d e the l e f t v e n t r i c u l a r chamber. La te r on i n t h i s s tudy , a 20 gauge hypodermic needle was a t tached to the PE 90 tub ing and the needle was then i n s e r t e d i n to the l e f t v e n t r i c l e through the apex, i ns tead of i n s e r t i n g the PE 90 tube i n t o the l e f t v e n t r i c l e through the pulmonary v e i n . The second method was chosen to prevent any damage tha t may occur to the m i t r a l va l ve wh i le pass ing the PE 90 tube through i t . The l e f t a t r ium was then a t tached to the 16 gauge cannula through the pulmonary v e i n . Card iac work was i n i t i a t e d by sw i t ch i ng the pe r fus ion system from the re t rograde mode to the working hear t mode. In the working mode the per fusa te entered the l e f t v e n t r i c l e through the l e f t a t r ium and was pumped out i n to the a o r t i c stump. The a o r t i c ou t f low was r e d i r e c t e d i n to the ex te rna l pe r i phe ra l r e s i s t a n c e , which prov ided a r e s i s t a n c e of 340 PRU (pe r i phe ra l r e s i s t a n c e u n i t s ) or 45.32 x 104 dyne sec / cm^ . I n t r a - v e n t r i c u l a r pressure development 43 was measured by a t t a c h i n g a Statham P23AA , pressure t ransducer to the PE 90 tube. I n t r a a o r t i c pressure was measured w i th a second pressure t ransducer a t tached to the a o r t i c ou t f low system through a s i d e - a r m . A o r t i c and l e f t v e n t r i c u l a r pressure development and the f i r s t d e r i v a t i v e o f l e f t v e n t r i c u l a r pressure development were D recorded on a Grass 79D po lygraph . The f i r s t d e r i v a t i v e o f l e f t v e n t r i c u l a r pressure was obta ined through a Grass 7P20 d i f f e r e n t i a t o r . Hearts were e l e c t r i c a l l y s t imu la ted a t 300 beats /min by means o f a b i p o l a r p la t inum e l e c t r o d e p laced on the l e f t a t r ium wi th pu lses of 5 m sec du ra t i on and tw ice th resho ld vo l tage (1.4 V) from a Grass model SD9D s t i m u l a t o r . Each heart was e q u i l i b r a t e d f o r 15 min a t 15 cm H 2 0 l e f t a t r i a l f i l l i n g p ressu re . At 15 cm H 2 0 l e f t a t r i a l f i l l i n g p r e s s u r e , r a t hear ts developed peak l e f t v e n t r i c u l a r pressures in the range of 90-110 mm Hg which r e s u l t e d in mean a o r t i c pressures in the range o f 70-80 mm Hg. Under these c o n d i t i o n s adequate coronary pe r fus ion was a c h i e v e d . Coronary f low was measured dur ing the i n i t i a l 5 min per iod o f re t rograde pe r fus ion a t 45 cm H^O pe r fus ion p ressu re . Coronary f low was not measured dur ing the working hear t pe r fus ion mode s i nce a r t i f a c t u a l l y high f low ra tes were ob ta ined owing to l eakages . Card iac performance was expressed in terms o f l e f t v e n t r i c u l a r developed pressure (LVDP), ra te o f r i s e o f l e f t v e n t r i c u l a r pressure (+dP/dt) and ra te o f d e c l i n e o f l e f t v e n t r i c u l a r pressure ( - d P / d t ) . DP/dt was c a l c u l a t e d by hand by drawing a tangent l i n e to the pressure t race a t the po in t o f maximum s l o p e . The s lope of t h i s 44 l i n e i s normal ly c a l c u l a t e d by c o n s t r u c t i n g a r i g h t angle t r i a n g l e by dropping a v e r t i c a l and drawing a h o r i z o n t a l back to the tangent l i n e . However, as the reco rd ing system used i s c u r v i l i n e a r , t h i s method was mod i f ied s l i g h t l y . I t was assumed tha t the maximum p o s i t i v e s lope o f the pressure curve o c c u r r e d ' ^ approx imate ly 25 mm Hg and the negat ive s lope a t 75 mm Hg. At these d e f l e c t i o n s the reco rd ing pen i s about 6° and 2° r e s p e c t i v e l y from the h o r i z o n t a l . Therefore to c o r r e c t f o r t h i s the cons t ruc ted r i g h t angled t r i a n g l e s were a l s o t i l t e d a t 6° or 2° as shown in F igure 2. In the l a t t e r par t o f t h i s s tudy , dP /d t was obta ined us ing a microcomputer as p r e v i o u s l y desc r ibed by H a r r i s ejt aj_. (1982). B r i e f l y , a Mountain Hardware A/D and D/A conver te r was used to d i g i t i z e the l e f t v e n t r i c u l a r pressure pu lses from the Grass polygraph and the r e s u l t i n g d i g i t i z e d data was s to red to d i s k by an Apple II microcom-pu te r . The pressure t ransducer s i g n a l from the polygraph was sampled a t 667 Hz over 1.5 seconds. Th is r e s u l t e d i n data being c o l l e c t e d f o r s i x complete c a r d i a c p u l s e s . Three o f these were ana lyzed us ing va r ious curve f i t t i n g techniques to determine pu lse h e i g h t , p o s i t i v e and negat ive d P / d t , s t a r t and f i n i s h . CARDIAC FUNCTION CURVES: Card iac f unc t i on was est imated in i s o l a t e d per fused working hearts obta ined from 7, 30, 100, 180, 240 and 360 day a l l o x a n - and STZ-d i a b e t i c and age-matched con t ro l r a t s . Fo l low ing the 15 min e q u i l i -b r a t i o n per iod a t 15 cm HgO l e f t a t r i a l f i l l i n g p r e s s u r e , l e f t v e n t r i c u l a r f u n c t i o n was est imated by va ry ing the l e f t a t r i a l f i l l i n g 45 FIGURE 2. C a l c u l a t i o n o f p o s i t i v e and negat ive dP /d t from l e f t v e n t r i c u l a r pressure t race Tangent l i n e s (2 and 3) are drawn to the l e f t v e n t r i c u l a r pressure t race (1) a t the po in ts of maximum s l o p e , which are approx imate ly at 25 mm Hg fo r the p o s i t i v e s lope and 75 mm Hg fo r the negat ive s lope o f the pressure cu rve . S lopes o f these tangents are c a l c u l a t e d by c o n s t r u c t i n g r i g h t angled t r i a n g l e s by dropping v e r t i c a l s (4 and 5) and drawing h o r i z o n t a l s back to tangent l i n e s . S ince the reco rd ing pen i s about 6° a t 25 mm Hg and 2° a t 75 mm Hg from the h o r i z o n t a l , the r i g h t angled t r i a n g l e s A and B are t i l t e d by 6° and 2° r e s p e c t i v e l y . The s lope i s c a l c u l a t e d from the formula j. . . oppos i te s i de v e r t i c a l , S 1 ° P e o f t h e ^ t e n u s e = ad jacent s ide o r h o r i z o n t a l V e r t i c a l can be conver ted to pressure-' (mm' Hg) from cha r t c a l i b r a t i o n and h o r i z o n t a l to time (sec) from cha r t speed. 46 1 — £ 47 p r e s s u r e s , which in turn i n f l uenced the l e f t v e n t r i c u l a r p r e l o a d . During t h i s manoeuvre;, c o n d i t i o n s which i n f l u e n c e l e f t v e n t r i c u l a r a f t e r l o a d , such as the ex te rna l pe r i phe ra l r e s i s t a n c e were held cons tan t . L e f t a t r i a l f i l l i n g pressures were va r i ed by changing the he igh t o f the f i l l i n g r e s e r v o i r s from 5.0 to 22.5 cm H^O in 2.5 cm s t e p s . The f unc t i on curve was performed by i n i t i a l s tepwise reduc t i on o f f i l l i n g pressure from 15 to 5 cm H^O, fo l l owed by s tepwise increments up to 22.5 cm H^O and back to 15 cm H^O. At each f i l l i n g pressure hear ts were a l lowed to per fuse u n t i l s t a b l e pressure development was obta ined before changing to next f i l l i n g p ressu re . In general s t a b l e pressure development was achieved w i t h i n 2 min a f t e r changing the a t r i a l f i l l i n g pressure and a complete f u n c t i o n curve was u s u a l l y performed i n about 20 to 30 min . As hearts were per fused at 15 cm HgO f i l l i n g pressure dur ing the e q u i l i b r a t i o n p e r i o d , f i l l i n g pressures which were lower than 15 cm H^O were r e f e r r e d to as low work loads and f i l l i n g pressures h igher than 15 cm H^O were r e f e r r e d to as high work l o a d s . DOSE-RESPONSE CURVES TO CARBACHOL: Dose-response curves to ca rbamy lcho l ine (carbacho l ) were performed i n i s o l a t e d per fused working hear ts ob ta ined from a l l o x a n - and STZ-d i a b e t i c and con t ro l r a t s a t va r ious time po in ts a f t e r i nduc t i on o f the d i sease s t a t e . I s o l a t e d per fused working hearts were paced a t 300 beats /min by p l a c i n g two b i p o l a r p la t inum e l e c t r o d e s , one on the l e f t a t r ium and the o ther on the apex o f the hea r t . Each p a i r o f e l e c t r o d e s was connected to a Grass model SD9D s t i m u l a t o r . The s t i m u l a t o r s were connected such tha t each a t r i a l pu lse t r i g g e r e d 48 a separa te v e n t r i c u l a r pu lse 6 msec l a t e r . The pulses were o f 5 msec du ra t i on and were four t imes th resho ld vo l tage (2 .8 V ) . This method of pacing was used to overcome the A-V b lock tha t may occur a f t e r the a d m i n i s t r a t i o n o f c a r b a c h o l . Cumulat ive dose-response curves to carbachol were performed by adding i n c r e a s i n g concen t ra t i ons o f carbachol to the pe r fus ion medium. The concen t ra t i on o f carbachol which produced a 65 to 70 % reduc t ion in the basal ra te o f r i s e in l e f t v e n t r i c u l a r pressure or +dP/dt was cons idered as the maximum dose o f carbachol and doses g rea te r than t h i s were not t e s t e d . The _5 maximal concen t ra t i on o f carbachol was normal ly 1 x 10 M but i n a _5 few cases the maximal concen t ra t i on was 3 x 10 M . : S e n s i t i v i t i e s o f hear ts ob ta ined from con t ro l and d i a b e t i c ra ts towards the negat ive i n o t r o p i c e f f e c t o f carbachol . were compared by comparing the ED^g values f o r carbachol on these h e a r t s . The E D ^ Q va lue i s expressed as the geometr ic mean (wi th 95% conf idence i n t e r v a l s ) o f the dose of carbachol requ i red to produce a 30% reduc t ion in the basal +dP/dt and was c a l c u l a t e d as desc r ibed by Fleming e t al_ (1 972) . A f t e r the complet ion o f dose-response curve to c a r b a c h o l , each hear t was per fused w i th normal pe r fus ion bu f f e r to comple te ly wash out any r e s i d u a l c a r b a c h o l . Hearts which reached s t a b l e l e f t v e n t r i c u l a r pressure development a f t e r washing out carbachol were used f o r , i sop ro te reno l dose-response c u r v e s . DOSE-RESPONSE CURVE TO ISOPROTERENOL: Dose-response curves to i sop ro te reno l were performed in i s o l a t e d per fused working hear ts from d i a b e t i c and con t ro l ra ts a t va r ious time 49 po in ts a f t e r i nduc t i on o f d i a b e t e s . For t h i s s tudy , hear ts were paced at 300 beats /min a t twice t h resho ld vo l tage (1.4 V) w i th p la t inum e lec t r odes p laced on ly on the l e f t a t r i u m . Cumulat ive dose-response curves to i sop ro te reno l were performed by adding i n c r e a s i n g concen t ra t i ons of i sop ro te reno l (from 1 x 1 0 " ^ to 1 x 10"^ M) to the pe r fus ion medium. Changes produced by va r ious concen t ra t i ons o f i sop ro te reno l i n p o s i t i v e and negat ive l e f t v e n t r i c u l a r dP /d t were moni to red. The p o s i t i v e i n o t r o p i c e f f e c t o f i sop ro te reno l was expressed as changes produced by i sop ro te reno l i n the ra te o f r i s e o f l e f t v e n t r i c u l a r pressure and the c a r d i a c r e l axan t e f f e c t o f i sop ro te reno l was expressed as changes produced in the ra te o f d e c l i n e o f l e f t v e n t r i c u l a r p r e s s u r e . S e n s i t i v i t y o f the d i a b e t i c and con t ro l myocardium towards the p o s i t i v e i n o t r o p i c e f f e c t o f i sop ro te reno l were compared by determin ing pD2 v a l u e s . The pD^ va lue was de f ined as the negat ive log concen t ra t i on o f the drug requ i red to produce 50% of the maximum change produced i n +dP/dt . CYCLIC AMP AND PHOSPHORYLASE ENZYME STUDIES: I s o l a t e d per fused working hearts obta ined from 3 and 100-120 day d i a b e t i c and c o n t r o l r a t s were used to study the t ime-course o f the changes produced by an E D ^ Q concen t ra t i on (5 x 10 M) o f i s o p r o -te reno l on c a r d i a c c y c l i c AMP l e v e l s , phosphory lase a c t i v i t y and i n o t r o p y . Hearts were i s o l a t e d and per fused accord ing to the working hear t technique as desc r ibed under the s e c t i o n : Heart p e r f u s i o n . Fo l low ing the 15 min e q u i l i b r a t i o n per iod a t 15 cm H^0 l e f t a t r i a l f i l l i n g p ressu re , se ts o f con t ro l and d i a b e t i c hear ts were quick 50 f rozen before and a t var ious t ime po in ts ( 5 , 10, 20, and 40 sec) - 9 a f t e r the a d m i n i s t r a t i o n o f 5 x 10 M i s o p r o t e r e n o l , by the a p p l i c a t i o n o f tongs precoo led i n l i q u i d n i t r o g e n . I sopro te reno l was admin is te red by adding a s o l u t i o n o f i sop ro te reno l to the pe r fus ion medium to make -9 a f i n a l concen t ra t i on o f 5 x 10 M. Dose-response o f the e f f e c t o f i s o p r o t e r e n o l on c a r d i a c c y c l i c AMP l e v e l s , phosphory lase a c t i v i t y and ino t ropy was s tud ied in i s o l a t e d perfused working hear ts obta ined from 100-120 day con t ro l and STZ-d i a b e t i c r a t s . Hearts were qu ick f rozen e i t h e r before or 20 sec a f t e r -9 -8 a d m i n i s t r a t i o n o f an E D 2 Q dose (2 x 10 M) or an E D g 0 dose (1 x 10 M) o f i s o p r o t e r e n o l . The e f f e c t o f p ros tag land in E-j (PGE-j) on c a r d i a c phosphory lase a c t i v i t y i n con t ro l and d i a b e t i c r a t s was a l so s t u d i e d . I s o l a t e d perfused working hear ts from 3 and 100-120 day d i a b e t i c and con t ro l r a t s were qu ick f rozen 30 sec. a f t e r the a d m i n i s t r a t i o n o f 10"^ M PGE-j. A s tock s o l u t i o n o f PGE-j was prepared by d i s s o l v i n g an appropr ia te quan t i t y o f PGE-j i n abso lu te a l coho l to make a 10 M s o l u t i o n . S e r i a l d i l u t i o n o f the s tock s o l u t i o n to ob ta in 10~^ M s o l u t i o n was made in Chenoweth-Koel le b u f f e r . The e f f e c t of indomethacin pretreatment on i s o p r o t e r e n o l - i n d u c e d phosphory lase a c t i v a t i o n was s tud ied in i s o l a t e d working hear ts from 100-120 S T Z - d i a b e t i c and con t ro l r a t s . Hearts were per fused wi th bu f f e r c o n t a i n i n g 10"^ M indomethacin f o r 20 minutes before a d m i n i s t e r i n g -9 5 x 10 M i s o p r o t e r e n o l . Hear ts were qu ick f rozen 20 sec a f t e r the a d m i n i s t r a t i o n o f i s o p r o t e r e n o l . A s tock s o l u t i o n o f indomethacin was made i n abso lu te a l coho l and was s e r i a l l y d i l u t e d wi th Chenoweth-Koel le 51 bu f fe r to ob ta in the requ i red 10~° M c o n c e n t r a t i o n . A l l the f rozen hearts were s to red a t -80°C u n t i l they were ana lyzed fo r c y c l i c AMP content and phosphory lase a c t i v i t y . CYCLIC AMP DETERMINATION'-.: C y c l i c AMP content o f the myocard ia l t i s s u e was determined by a radioimmunoassay method acco rd ing to S t e i n e r e_t al_. (1972) , us ing . Becton-D. ick inson radioimmunoassay reagen ts . The assay i nvo l ved a 125 compe t i t i ve b ind ing r e a c t i o n between [ I ] - l a b e l l e d c y c l i c AMP and n o n - r a d i o a c t i v e c y c l i c AMP i n the s tandard or sample, f o r the b ind ing s i t e s present on the c y c l i c AMP-ant ibody. Rad ioac t i ve c y c l i c AMP tha t was bound to the ant ibody was separated by p r e c i p i t a t i o n wi th 60% sa tu ra ted ammonium s u l f a t e s o l u t i o n and was counted in a gamma coun te r . The c y c l i c AMP content o f the myocardium was expressed as femtomoles/mg t i s s u e wet we igh t . PHOSPHORYLASE a ASSAY: Phosphory lase a_ a c t i v i t y was measured in the d i r e c t i o n o f glycogen syn thes i s accord ing to a m o d i f i c a t i o n o f the method o f Cor i and Cor i (1940) which was p r e v i o u s l y desc r ibed by McNe i l l and Brody (1966) . The assay i nvo l ved an enzymatic r e a c t i o n c a t a l y z e d by phosphory lase , whereby g lucose - j -phospha te was conver ted to glycogen wi th the re l ease o f i no rgan i c phosphate. The i no rgan i c phosphate was est imated pho tome t r i ca l l y accord ing to the method o f F iske and Subbarow (1928) . Phosphory lase a^  a c t i v i t y was expressed as a percent o f t o t a l phosphorylase a c t i v i t y . Tota l phosphory lase a c t i v i t y was est imated 52 in the presence o f 5'-AMP and was expressed as micromoles o f i no rgan i c phosphate re leased per mg t i s s u e per, min..... Phosphory lase a_ a c t i v i t y was es t imated i n the absence o f 5'AMP. Most r e s u l t s are expressed as percent phosphorylase a_ which i s : phosphory lase a_ a c t i v i t y x 100 t o t a l phosphory lase a c t i v i t y MUSCARINIC RECEPTOR BINDING EXPERIMENTS: Musca r i n i c recep to r r a d i o l i g a n d b ind ing exper iments were c a r r i e d out on v e n t r i c u l a r t i s s u e obta ined from 180-day con t ro l and STZ d i a b e t i c r a t s . I s o l a t e d per fused working hear ts from con t ro l and d i a b e t i c ra t s were qu ick f rozen f o l l o w i n g the 15 min e q u i l i b r a t i o n per iod and were s to red a t -80°C u n t i l used f o r b ind ing exper iments . Frozen hear ts were suspended in co l d 0.32 M sucrose s o l u t i o n , a l lowed to thaw and v e n t r i c l e s were separated from a t r i a and other extraneous t i s s u e . Each v e n t r i c l e was suspended i n 10 ml o f co l d 0.32 M sucrose and was homogenized us ing a P o l y t r o n , s e t t i n g 7 f o r 20 s e c . Homogenate from 6 con t ro l or 6 d i a b e t i c r a t v e n t r i c l e s was combined and f u r t h e r homogenized in a g lass homogenizat ion tube us ing a t e f l o n pes t l e a t 200 rpm, w i th 8 down s t rokes and 8 ups t rokes . The homogenate was then d i s t r i b u t e d i n to p l a s t i c c e n t r i f u g e tubes-, loaded i n to a swinging bucket r o to r o f type SW28 #903 and c e n t r i f u g e d at 25,000 rpm fo r 30 min. The supernatant l i q u i d was d i sca rded and the crude membrane p e l l e t s used fo r the b ind ing exper iments . 53 The crude membrane p e l l e t was suspended in 10 ml o f Krebs-H e n s e l e i t s o l u t i o n c o n t a i n i n g i n mM: N a C l , 118; KC1, 4 . 7 ; NaHCO^, 25 ; C a C l 2 , 2 . 5 ; i .MgS0 4 , 1 .2 ; K H 2 P 0 4 > 1.2 and d e x t r o s e , 11.1 and was homogenized us ing a t e f l o n p e s t l e . The homogenate was f i l t e r e d through cheese c l o t h to remove connec t i ve t i s s u e . The p r o t e i n content o f the homogenate was determined us ing the Lowry 's p r o t e i n assay . The homogenate was d i l u t e d w i th K rebs -Hense le i t bu f fe r to ad jus t the p r o t e i n concen t ra t i on to approx imate ly 0.7 mg/ml. The f o l l o w i n g b ind ing exper iments were performed on the d i l u t e d membrane p r e p a r a t i o n : a . D i r e c t b ind ing or de te rmina t ion o f the s a t u r a t i o n b ind ing curve f o r the r a d i o l i g a n d . b. Compet i t ion by n o n - r a d i o a c t i v e an tagon is t f o r r a d i o l i g a n d l a b e l l e d b ind ing s i t e s . c . Compet i t ion by n o n - r a d i o a c t i v e agon is t f o r r a d i o l i g a n d l a b e l l e d b ind ing s i t e s and the e f f e c t o f guanine n u c l e o t i d e s . 3 T r i t i a t e d N-methylscopolamine ( [ H.]NMS.), a musca r in i c recep to r a n t a g o n i s t , was used as a r a d i o l i g a n d to s t e r e o s p e c i f i c a l l y and r e v e r s i b l y l a b e l muscar in i c recep to r s i t e s in the membrane p r e p a r a t i o n . For compet i t i on exper iments , n o n - r a d i o a c t i v e N-methylscopolamine was used as an an tagon is t and carbachol was used as an a g o n i s t . G u a n y l - 5 ' -y l - i m i d o - d i p h o s p h a t e (GMPPNP), a non-hydro lysab le d e r i v a t i v e o f GTP was used as a guanyl n u c l e o t i d e . A l l the po in ts i n each b ind ing experiment were c a r r i e d out i n q u a d r u p l i c a t e . In the d i r e c t b ind ing experiments, t o t a l b ind ing was 54 determined wi th va ry ing concen t ra t i ons o f [ H]NMS (10" - 10~ M) and n o n - s p e c i f i c b ind ing was determined in the presence o f a high -5 concen t ra t i on o f n o n - r a d i o a c t i v e NMS (10 \M) . In the compet i t i on 3 -7 experiments a s i n g l e concen t ra t i on o f [ H]NMS (1 x 10 M) was used in the presence o f va ry ing concen t ra t i ons o f e i t h e r a n o n - r a d i o a c t i v e an tagon is t ( 1 0 ~ ^ - 10"^ M NMS) or a n o n - r a d i o a c t i v e agon is t (10~^° -2 -4 - 1 0 M C a r b a c h o l ) . When a guanyl nuc leo t i de was inc luded 10 M GMPPNP was added. A l l the drugs were added to 0.5 ml Eppendorf tubes i n „5 y l q u a n t i t i e s . The b ind ing r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f 490-pi- o f the membrane p r e p a r a t i o n . The r e a c t i o n mix ture was incubated a t 30°C f o r 45 minu tes . The r e a c t i o n was stopped by rap id c e n t r i f u g a t i o n i n a Beckmann model bench top m i c r o c e n t r i f u g e f o r 5 minutes . The supernatant was decanted and the membrane p e l l e t was washed three t imes w i th 0.1 M NaCl s o l u t i o n . The p e l l e t s were s o l u b i l i z e d in 0.5 ml o f So luene , a t i s s u e s o l u b i l i z e r by keeping at 40°Ci f o r 12 hours . The d i s s o l v e d p e l l e t s were mixed w i th 5 ml o f Beckmann Ready So lve NA and counted us ing s tandard s c i n t i l l a t i o n count ing techn iques . ESTIMATION OF NORADRENALINE CONTENT IN VENTRICLES: Noradrena l ine content o f the hear ts ob ta ined from 180 day a l l o x a n -and S T Z - d i a b e t i c and con t ro l r a t s was determined us ing a s l i g h t m o d i f i c a t i o n o f the HPLC method desc r ibed by S tarke ejt aj_. (1981). I so la ted per fused working hear ts from d i a b e t i c and con t ro l r a t s were quick f rozen f o l l o w i n g a 30 min pe r fus ion and the f rozen hear ts were s to red a t -80°C u n t i l they were assayed f o r no radrena l ine con ten t . 55 Approx imate ly 100 mg of the f rozen v e n t r i c u l a r t i s s u e was weighed and homogenized i n 4 ml o f 0.4 M p e r c h l o r i c and (HCIO^) c o n t a i n i n g 0.2 ml o f 100 mM Na 2EDTA, 0.1 ml o f 10% N a 2 S 0 3 and 10 y l o f 25 yM dihydroxybenzylamine (DBA) as a marker , us ing a g lass homogenizat ion tube and g l ass pes t l e a t 1,000 RPM f o r 45 s e c . The homogenate was c e n t r i f u g e d a t 20,000 x g f o r 30 min and the p e l l e t was d i s c a r d e d . The c l e a r supernatant was mixed wi th 100 mg of alumina (A1 , ,0 3 , Woelm n e u t r a l , grade #1). The mix ture was s t i r r e d con t i nuous l y w i th a magnetic s t i r r e r bar and the pH of the mix ture was ad jus ted to 8.3 wi th 2 M t r i s b u f f e r . S t i r r i n g was cont inued f o r approx imate ly 5 min a f t e r a d j u s t i n g the pH to 8 . 3 . The mix ture was then t r a n s f e r r e d to a Sp in Sep m i c r o f i l t e r un i t and the alumina was a l lowed to s e t t l e . The supernatant was d i sca rded and the alumina column was washed 5 t imes w i th d i s t i l l e d water . A f t e r the 5th wash, water was comple te ly removed from the column by c e n t r i f u g a t i o n . Then the catecholamines tha t were adsorbed on the column were e lu ted tw ice wi th 0.5 ml o f 0.2 M HCIO^. The two 0.5 ml po r t i ons o f the e l u a t e were combined and analyzed fo r no radrena l ine content by a reverse phase HPLC method. A f u l l y capped 0DS ( o c t a d e c y l s i 1 i ca ) A l t e x column (5yM p a r t i c l e s , 25 cm long) was used f o r sepa ra t i on o f the ca techo lamines . The mobi le phase was a 5% methanol ic s o l u t i o n o f 0.1 M N a H 2 P 0 4 , 0.5 mM Na2EDTA and 0.5 mM Na-octane s u l f o n a t e , pH 3.0 (ad jus ted wi th H 3 P 0 4 ) . The f low ra te through the column was ad jus ted to 1 ml/min us ing an At tex model 100 A pump. A s tandard s o l u t i o n c o n t a i n i n g 25 yM noradrena l ine and 25 iiM DBA and samples f o r a n a l y s i s were i n j e c t e d i n 20 y l q u a n t i t i e s . Noradrena l ine and DBA were detected amperomet r i ca l l y us ing a BAS 56 model LC-4A de tec to r equ ipped, ; w i th a +0.7 V CP-0 e l e c t r o d e . Recovery from the alumina chromatogram was determined by comparing the DBA (marker) peak to tha t i n the S tandard . A l l va lues o f no radrena l ine were co r rec ted f o r incomplete recovery from columns. Noradrena l ine content was expressed as yg per g hear t t i s s u e . SERUM ANALYSES: The d i a b e t i c and con t ro l r a t s were withdrawn from food f o r 12-18 hrs p r i o r to s a c r i f i c e throughout t h i s s tudy . Blood samples were c o l l e c t e d a t the t ime of s a c r i f i c e . Blood samples were a l lowed to c l o t and c e n t r i f u g e d at 1000 rpm fo r 15 min to separate serum. The separated c l e a r serum was s to red below 0° C u n t i l ana lyzed fo r serum immunoreactive i n s u l i n , g l u c o s e , thy rox ine (T4) and t r i i o d o -thyron ine (T3) l e v e l s . SERUM IMMUNOREACTIVE INSULIN DETERMINATION'S I n s u l i n content o f the serum was measured by a radioimmunoassay, us ing the Bec ton -D ick inson i n s u l i n radioimmunoassay k i t . The assay 125 i nvo l ved a compe t i t i ve b ind ing r e a c t i o n between [ I ] l a b e l l e d i n s u l i n (porc ine i n s u l i n ) and the n o n - r a d i o a c t i v e i n s u l i n i n the standard (human i n s u l i n ) or serum sample ( r a t i n s u l i n ) . Cross r e a c t i v i t y between the ant ibody r a i s e d aga ins t porc ine i n s u l i n and r a t i n s u l i n i s 90% fo r the b ind ing s i t e s present on the ant ibody which were r a i s e d aga ins t porc ine i n s u l i n i n the guinea p i g . Bound and unbound r a d i o a c t i v e i n s u l i n were separated us ing dex t ran-coa ted c h a r c o a l . Dext ran-coated charcoa l has been repor ted to r e a d i l y adsorb f ree i n s u l i n but not ant ibody-bound i n s u l i n (Herber t et. a j_. , 1 965) . At the end of the i ncuba t i on p e r i o d , a suspension of dex t ran-coa ted 57 charcoa l was added to the i ncuba t i on m i x t u r e , vo r t exed , c e n t r i f u g e d and the ant ibody-bound r a d i o a c t i v i t y i n the supernatant was measured us ing a gamma coun te r . Serum immunoreactive i n s u l i n was expressed i n uU/ml . SERUM GLUCOSE DETERMINATION: Glucose content o f the serum was determined by an enzymat ic method us ing g lucose ox idase accord ing to Raabo and T e r k i l d s e n (1960) . During the i n i t i a l phase o f t h i s study serum g lucose was determined D wi th the Ames Blood Ana lyze r g lucose reagent k i t (Sunderland and Logan, 1970) . La te r on the Sigma g lucose assay k i t was used to determine serum g lucose s i nce the Sigma g lucose k i t i s l e s s expens i ve . The assay i nvo l ves an enzymat ic r e a c t i o n in which g lucose i s conver ted to g lucono lac tone in the presence o f g lucose o x i d a s e . Gluconolactone reac ts w i th water forming g l ucon i c a c i d and hydrogen pe rox ide . Hydrogen pe rox ide , i n the presence o f the enzyme pe rox i dase , o x i d i z e s the c o l o r l e s s reagent 0 - d i a n i s i d i n e to. a reddish-brown dye and the c o l o r developed was read a t 500 mM us ing a spect rophotometer . A 100 mg/100 ml g lucose standard s o l u t i o n was used as a re fe rence s tanda rd . Serum glucose l e v e l s were expressed in mg/dl (mg %). SERUM T4 DETERMINATION: T^ content o f the serum was determined by a radioimmunoassay D method us ing the Tetrabead -125 T^ d i a g n o s t i c k i t . The assay i nvo l ves compet i t i on between the r a d i o a c t i v e and n o n - r a d i o a c t i v e T^ i n the s tandard or serum sample f o r the b ind ing s i t e s present i n a l i m i t e d quan t i t y o f the T^ ant ibody immobi l i zed on beads. Separa t ion o f the bound and f ree r a d i o a c t i v e T. was ach ieved by removal o f the r e a c t i o n 58 l i q u i d from the beads. The r a d i o a c t i v i t y bound to the beads was es t imated us ing a gamma coun te r . Serum T^ content was expressed i n u g / d l . SERUM T 3 DETERMINATION: Serum T 3 content was determined by a charcoa l bead uptake method p us ing the Tr iobead -125 T 3 uptake k i t . The assay i nvo l ves p a r t i t i o n of the r a d i o l a b e l l e d T^ between the pr imary b ind ing s i t e s (serum p ro te ins such as thy rox ine b ind ing p r o t e i n , albumin e t c . ) i n the serum and secondary b ind ing s i t e s such as the charcoa l coated beads. The r a d i o a c t i v i t y taken up by the beads g ives an i n d i c a t i o n o f the t hy ro i d s t a t u s . In hypothyro id ism the pr imary b ind ing s i t e s are l e s s sa tu ra ted and they take up more r a d i o a c t i v i t y and the beads l e s s . Converse ly i n hype r thy ro id i sm, the pr imary b ind ing s i t e s are super-sa tu ra ted and so the beads take up more r a d i o a c t i v i t y . The r a d i o a c t i v e T 3 bound to the charcoa l coated beads was separated by removing the r e a c t i o n l i q u i d from the beads and was counted in a gamma coun te r . The charcoa l bead uptake was expressed as a percent o f the T 3 uptake va lue f o r the re fe rence con t ro l serum prov ided wi th the k i t . The percent T 3 bead uptake va lue prov ides an i n d i c a t i o n o f the t h y r o i d s ta tus and can not be d i r e c t l y t r a n s l a t e d i n to d e f i n i t e un i t s o f J^. STATISTICAL ANALYSES: A l l the r e s u l t s presented i n t h i s t h e s i s are expressed as mean ± standard e r r o r of the mean. Comparisons between con t ro l and d i a b e t i c groups were made by us ing the one-way a n a l y s i s o f v a r i a n c e . Whenever 59 a mul t ig roup comparison was made, a p o s t e r i o r i t e s t such as the Newman-Kuels t e s t f o r m u l t i p l e comparisons was then performed to determine s i g n i f i c a n t d i f f e r e n c e between groups. A m u l t i f a c t i o n a l two-way a n a l y s i s o f va r iance was used to determine the e f f e c t s o f t rea tment , t ime a f t e r t reatment and t h e i r i n t e r a c t i o n on the s e n s i t i v i t y o f the myocardium towards c a r b a c h o l . The l e v e l o f s i g n i f i c a n c e was se t a t p<0.05. MATERIALS A l l oxan monohydrate, l - a m i n o - 2 - n a p t h o l - 4 - s u l f o n i c a c i d reagen t , 5 ' -AMP, c a r b a c h o l , D-g lucose-1-phosphate , g l ycogen , D L - i s o p r o t e r e n o l , indomethac in , p ros tag land in E], s t r e p t o z o t o c i n and T r i s were a l l purchased from Sigma Chemical Company, S t . L o u i s , Mo. [ N]N-methyl scopo lamine, N-methyl scopolamine and guany l -5 ' - im idod iphospha te were generous g i f t s from Dr. J.W. W e l l s , Facu l t y o f Pharmacy, U n i v e r s i t y o f Toronto . Dihydroxybenzylamine and alumina were generous g i f t s from Dr. V. P a l a t y , Department o f Anatomy, U n i v e r s i t y o f B .C . A l l o f the chemica ls used to prepare va r ious bu f f e r s o l u t i o n s were of a n a l y t i c a l reagent grade. C y c l i c AMP radioimmunoassay reagents and i n s u l i n radioimmunoassay k i t s were obta ined from Beckton D ick inson and Company, Orangeburg, New York , U .S .A . Glucose assay k i t s were purchased from Ames Company, E l k h a r t , I nd iana , U .S .A . and Sigma Chemical Company, S t . L o u i s , Mo. , R R U .S .A . Tetrabead -125 T^ d i a g n o s t i c k i t s and Tr iobead -125 T^ uptake k i t s were bought from Abbott L a b o r a t o r i e s , North Ch icago , I I , U .S .A . The Spinsep m i c r o f i l t e r un i t s used i n catecholamine de termina t ions were obta ined from Mandel S c i e n t i f i c Company, C a l g a r y , A l b e r t a . 60 RESULTS FEATURES OF THE DIABETIC-RAT MODEL: A d m i n i s t r a t i o n o f e i t h e r a l l o x a n or STZ to ra t s r e s u l t e d in a d i a b e t i c s t a t e c h a r a c t e r i z e d by f a s t i n g hyperglycemia and hypo-i nsu l i nem ia throughout i n t h i s s tudy . Table I p resents f a s t i n g serum i n s u l i n and g lucose l e v e l s , body we igh t s , wet hear t we igh t s , hear t weight to body weight r a t i o s and coronary f low ra tes o f con t ro l and a l l o x a n - and STZ- t rea ted r a t s at the time po in ts s tud ied in the i n i t i a l par t o f t h i s s tudy . Serum i n s u l i n l e v e l s were s i g n i f i c a n t l y reduced in ra t s t rea ted w i th a l l o x a n and STZ, compared to c o n t r o l s a t a l l o f the time p o i n t s , except at 30 days where the serum i n s u l i n l e v e l s i n the STZ- t rea ted group 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 c o n t r o l s . Fas t ing serum g lucose l e v e l s were s i g n i f i c a n t l y e leva ted i n both the t rea ted groups at a l l o f the time po in ts s t u d i e d , i n c l u d i n g the 30 day STZ- t rea ted group. Body weight o f a l l o x a n and STZ 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 lower than c o n t r o l s a t a l l t ime -po in t s except a t 7 days a f t e r t rea tment . Wet hear t weights o f the d i a b e t i c animals were a l s o "'? s i g n i f i c a n t l y lower than c o n t r o l s a t a l l t ime po in ts except a t 7 days a f t e r t rea tment . The wet hear t weight to body weight r a t i o was una l te red i n a l l o x a n and STZ d i a b e t i c ra t s a t 7, 30 and 100 days a f t e r i nduc t i on o f d iabe tes whereas the r a t i o .was s i g n i f i c a n t l y l a r g e r i n d i a b e t i c animals a t 180, 240 and 360 days a f t e r t rea tment . Coronary f low per gram of hear t t i s s u e was una l te red in d i a b e t i c ra t s at any time po in t s t u d i e d . There was no m o r t a l i t y i n any group e i t h e r a t 7 or 30 days a f t e r t rea tment . At 100 days and t h e r e a f t e r some m o r t a l i t y was encountered. 61 S p e c i f i c a l l y , 5 out o f 15 r a t s i n j e c t e d w i th a l l o x a n d ied between 100 and 240 days . One con t ro l animal out o f 12 d ied before 100 days . There was no m o r t a l i t y i n e i t h e r group a t 180 days . Three out o f 12 animals d ied in both the con t ro l and STZ d i a b e t i c groups between 180 and 360 days . Table II presents some fea tu res o f 3 and 100-120 day a l l o x a n -and STZ- t rea ted and con t ro l r a t s used i n the time course study o f the e f f e c t o f i sop ro te reno l on c a r d i a c c y c l i c AMP con ten t , phosphory lase a c t i v i t y and i n o t r o p y . In t h i s study serum T4 and T3 l e v e l s were est imated in o rder to check the t h y r o i d s ta tus o f the d i a b e t i c r a t s . Body weights were s i g n i f i c a n t l y lower i n ra t s t rea ted w i th e i t h e r a l l o x a n or STZ a t 3 days and 100-120 days,- when compared to con t ro l ra ts a t the cor respond ing time p o i n t s . Fas t i ng serum g lucose l e v e l s were s i g n i f i c a n t l y e leva ted in a l l o x a n - and STZ- t rea ted r a t s a t 3 and 100-120 days compared to age-matched con t ro l r a t s . Serum T3 bead uptake va lues were s i g n i f i c a n t l y depressed i n d i a b e t i c r a t s a t both the time po in ts sugges t ing reduced serum T3 l e v e l s . Serum T4 l e v e l s were s i g n i f i c a n t l y lower i n a l l o x a n and STZ d i a b e t i c ra t s on ly a t 100-120 days a f t e r i nduc t i on o f d iabe tes but not a t 3 days . There was no m o r t a l i t y i n any group of r a t s e i t h e r a t 3 days or a t 100-120 days. Table I I I shows body weights and f a s t i n g serum glucose l e v e l s o f con t ro l and d i a b e t i c ra t s at va r ious time p o i n t s , tha t were used in the PGEi and indomethacin s tudy , musca r in i c recep to r b ind ing s tud ies or f o r hear t no radrena l ine content e s t i m a t i o n . Body weights o f d i a b e t i c ra t s were s i g n i f i c a n t l y lower compared to con t ro l a t a l l 62 of the t ime po in ts s tud ied in the above mentioned s t u d i e s . Fas t i ng serum g lucose l e v e l s were 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 r a t s compared to age-matched con t ro l r a t s . CARDIAC PERFORMANCE OF CONTROL AND DIABETIC RATS: Card iac performance was s tud ied a t va r ious l e f t a t r i a l f i l l i n g pressures in i s o l a t e d per fused working hear ts obta ined from con t ro l and d i a b e t i c r a t s at va r ious time po in ts a f t e r induc ing d i a b e t e s . Card iac performance i s expressed in terms of LVDP, p o s i t i v e dP /d t and negat ive d P / d t . F igures 3 , 4 and 5 i l l u s t r a t e LVDP, p o s i t i v e dP/d t and negat ive dP/d t a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts obta ined from 7 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . There were no d i f f e r e n c e s i n c a r d i a c performance between con t ro l and d i a b e t i c ra t s at t h i s time p o i n t . L e f t v e n t r i c u l a r pressure development, and p o s i t i v e and negat ive dP /d t o f i s o l a t e d per fused working hear ts from 30 day con t ro l and d i a b e t i c ra t s i s presented in F igures 6, 7 and 8 r e s p e c t i v e l y . Cont ro l and STZ d i a b e t i c r a t s e x h i b i t e d s i m i l a r c a r d i a c performance at va r ious l e f t a t r i a l f i l l i n g pressure at t h i s t ime p o i n t . However, a l l o x a n d i a b e t i c r a t hear ts e x h i b i t e d depressed l e f t v e n t r i c u l a r pressure development and p o s i t i v e d P / d t , when per fused at f i l l i n g pressures between 5 to 17.5 cm H2O and depressed negat ive dP /d t at f i l l i n g pressures between 10 and 22.5 cm H 2 O . I so l a t ed per fused working hear ts obta ined from 100-day a l l o x a n and STZ d i a b e t i c ra t s developed s i g n i f i c a n t l y lower l e f t v e n t r i c u l a r p r e s s u r e , and p o s i t i v e and negat ive dP /d t a t f i l l i n g pressures above 10 cm H£0 when compared to age-matched con t ro l r a t s as shown i n F igures 9, 10 and 11 r e s p e c t i v e l y . S i m i l a r depress ions i n c a r d i a c performance were seen i n i s o l a t e d 63 perfused working hear ts from 180-day STZ d i a b e t i c ra t s a t most o f the l e f t a t r i a l f i l l i n g pressures s tud ied (F igures 12, 13 and 14 ) . L e f t v e n t r i c u l a r pressure development was s l i g h t l y but not s i g n i f i c a n t l y depressed in 240-day a l l o x a n d i a b e t i c r a t hear ts (F igure 15 ) . However p o s i t i v e dP /d t was depressed a t a l l o f the f i l l i n g pressures s tud ied and negat ive dP /d t was depressed a t high f i l l i n g pressures in these h e a r t s , as i l l u s t r a t e d in F igures 16 and 17. In i s o l a t e d per fused working hear ts ob ta ined from 360-day STZ d i a b e t i c r a t s , there were no depress ions i n LVDP and negat ive dP /d t a t any f i l l i n g pressure s tud ied (F igures 18 and 20 ) . On the other hand p o s i t i v e dP /d t i n 360-day STZ d i a b e t i c r a t hear ts was s i g n i f i c a n t l y depressed compared to c o n t r o l s a t a l l o f the f i l l i n g pressures s tud ied except at 10 cm H20 (F igure 19 ) . F igure 21 demonstrates the changes produced in p o s i t i v e dP /d t w i th time in con t ro l and a l l o x a n and STZ d i a b e t i c r a t hear ts a t 15 cm H2O l e f t a t r i a l f i l l i n g p ressu re . Both con t ro l and d i a b e t i c r a t hear ts e x h i b i t e d s i m i l a r p o s i t i v e d P / d t , 7 days a f t e r i n d u c t i o n o f d i a b e t e s . A l l oxan d i a b e t i c r a t hear ts e x h i b i t e d a s i g n i f i c a n t d e c l i n e i n the ra te o f p ressure development, s t a r t i n g from 30 days a f t e r t reatment u n t i l the end of the s tudy , whereas STZ d i a b e t i c r a t hear ts e x h i b i t e d s i m i l a r p o s i t i v e dP /d t as c o n t r o l s a t 30 days a f t e r t reatment but t h e r e a f t e r showed a s i g n i f i c a n t d e c l i n e u n t i l the end o f the s tudy . The con t ro l r a t hear ts showed a s l i g h t d e c l i n e i n p o s i t i v e dP /d t w i th i n c r e a s i n g age. F igure 22 i l l u s t r a t e s the changes occu r i ng i n ra te o f l e f t v e n t r i c u l a r pressure d e c l i n e w i th t ime in con t ro l and d i a b e t i c r a t hear ts a t 15 cm H2O a t r i a l f i l l i n g p ressu re . Negat ive dP /d t was not 64 a l t e r e d by e i t h e r a l l o x a n or STZ d iabe tes a t 7 days a f t e r i n d u c t i o n . Hearts i s o l a t e d from a l l o x a n d i a b e t i c r a t s showed a p rog ress i ve d e c l i n e in negat ive dP /d t w i th t ime , s t a r t i n g from 30 days a f t e r i nduc t i on u n t i l 240 days which was the longes t t ime po in t s t u d i e d . A reduc t i on i n negat ive dP /d t was not observed at 30 days a f t e r i nduc t i on w i th STZ. STZ- induced d iabe tes produced a s i g n i f i c a n t reduc t i on i n negat ive dP /d t by 100 days , which p e r s i s t e d u n t i l 180 days and then s t a r t e d to r i s e towards the con t ro l va lue wi th t ime . At 360 days a f t e r t rea tment , the negat ive dP /d t o f hear ts obta ined from STZ d i a b e t i c r a t s was not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s . The negat ive dP /d t i n con t ro l r a t hear ts showed a marked d e c l i n e wi th i n c r e a s i n g age. RESPONSIVENESS OF CONTROL AND DIABETIC HEARTS TO CARBACHOL: Carbachol produced a dose-dependent reduc t i on i n the basal p o s i t i v e dP /d t o f i s o l a t e d per fused working hear ts obta ined from c o n t r o l , a l l o x a n and STZ d i a b e t i c ra t s a t a l l the t ime po in ts s t u d i e d . At 7 and 30 days a f t e r i n d u c t i o n o f d i a b e t e s , carbachol produced s i m i l a r reduc t ions in the basal p o s i t i v e dP /d t a t any dose employed in con t ro l as we l l as d i a b e t i c r a t hear ts (F igures 23 and 24 ) . Data are expressed as percent reduc t i on i n basal p o s i t i v e dP /d t produced by va r ious molar concen t ra t i ons o f c a r b a c h o l . [Basal p o s i t i v e dP /d t va lues a t 7 days were ( i n mm H g / s e c ) : con t ro l = 2425±97, a l l o x a n d i a b e t i c = 2283±132 and STZ d i a b e t i c = 2392±122. A f t e r the a d m i n i s t r a t i o n o f a maximum dose of carbachol these va lues were: con t ro l (10" 5 M) = 558±48, a l l o x a n d i a b e t i c (10~ 5M) = 566±39 and STZ d i a b e t i c (10" 5 M) = 539±60. Basal p o s i t i v e dP /d t va lues at 30 days were: con t ro l = 2737±59, a l l o x a n d i a b e t i c 2285±101 and 65 STZ d i a b e t i c = 2672±57. A f t e r the a d m i n i s t r a t i o n o f a maximum dose of carbachol these va lues were: con t ro l (10 M) = 679±62, a l l o x a n d i a b e t i c (10" 5 M) = 619±59 and STZ d i a b e t i c 596±52.Q At 100 days a f t e r t rea tment , carbachol produced s i g n i f i c a n t l y sma l l e r reduc t ions — 6 6 — 5 in basal +dP/dt development a t 10~ , 3 x 10" and 10~ M doses in a l l o x a n and STZ d i a b e t i c r a t hear ts as compared to con t ro l r a t h e a r t s . As a r e s u l t , the dose response curves to carbachol i n the d i a b e t i c r a t hear ts were s h i f t e d to the r i g h t sugges t ing tha t the d i a b e t i c r a t hearts were s u b s e n s i t i v e to carbachol (F igure 25 ) . [Basal +dP/dt va lues a t 100 days were ( i n mmHg/sec): con t ro l = 2584±126, a l l o x a n -d i a b e t i c = 2313±160 and STZ d i a b e t i c = 2302±138. A f t e r the admin is -t r a t i o n o f a maximum dose of carbachol these va lues were: con t ro l (10" 5 M) = 921±106; a l l o x a n - d i a b e t i c (3 x 10" 5 M) = 927±140 and STZ d i a b e t i c (3 x 10" 5 M) = 928+1330; STZ d i a b e t i c r a t hear ts a t 180 days and a l l o x a n d i a b e t i c r a t hear ts at 240 days e x h i b i t e d inc reased s e n s i t i v i t y to c a r b a c h o l . Reduct ions in basal +dP/dt development - 6 — 6 produced by 10" M and 3 x 10~ M doses o f carbachol were s i g n i f i c a n t l y g rea te r i n d i a b e t i c r a t hear ts compared to con t ro l h e a r t s . This inc reased s e n s i t i v i t y was r e f l e c t e d by the f a c t tha t dose-response . curves to carbachol i n d i a b e t i c r a t hear ts a t these two time po in ts were s h i f t e d to the l e f t as compared to con t ro l dose-response curve (F igures 26 and 27 ) . [Basal +dP/dt va lues a t 180 days were: con t ro l = 2295±73 and S T Z - d i a b e t i c = 1878±81. A f t e r the a d m i n i s t r a t i o n o f a maximum dose o f carbachol these values were: con t ro l (10 M) = 733±61 and STZ d i a b e t i c (3 x 10" 6 M) = 709±77. Basal +dP/dt va lues a t 240 days were: con t ro l = 2247±130, and a l l o x a n d i a b e t i c = 1844±142. A f t e r a 66 maximum dose of carbachol these va lues were: con t ro l (10 M) = 531±41 and a l l o x a n d i a b e t i c = 503±77]. At 360 days a f t e r t rea tment , both con t ro l and STZ d i a b e t i c r a t hear ts were l e s s respons ive to the negat ive i n o t r o p i c e f f e c t o f carbachol (F igure 28 ) . [Basal +dP/dt va lues at 360 days were: con t ro l = 2016±105 and S T Z - d i a b e t i c = 1931±54. A f t e r a _5 maximum dose of carbachol these va lues were: con t ro l (10 M) = 1212±282 and S T Z - d i a b e t i c (10 M) = 917±124]. The maximal concen t ra t i on o f carbachol (1 x 10 M) employed produced about a 70% reduc t i on in the basal +dP/dt development o f con t ro l r a t hear ts at e a r l i e r t ime p o i n t s , whereas t h i s concen t ra t i on produced on ly a 40% reduc t i on in basal +dP/dt development i n con t ro l hear ts a t 360 days. D i a b e t i c r a t h e a r t s , however, were s l i g h t l y but not s i g n i f i c a n t l y more s e n s i t i v e and respons ive to carbachol a t t h i s t ime p o i n t . Table IV presents E D 3 Q va lues f o r carbachol i n con t ro l and d i a b e t i c r a t hear ts a t a l l the time po in ts s t u d i e d . At 7 and 30 days of t rea tment , E D ^ Q va lues f o r carbachol i n d i a b e t i c r a t hear ts 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 c o n t r o l s . E D ^ g va lues f o r carbachol were s i g n i f i c a n t l y h igher i n 100 day d i a b e t i c r a t hear ts when compared to c o n t r o l s . At 180 and 240 days the E D ^ g va lues f o r carbachol i n d i a b e t i c r a t hear ts were s i g n i f i c a n t l y lower than con t ro l v a l u e s . At 360 days the E D ^ g va lue f o r carbachol i n d i a b e t i c r a t hear ts was s l i g h t l y but not s i g n i f i c a n t l y lower than c o n t r o l . E D ^ g va lues f o r carbachol on con t ro l r a t hear ts at va r ious time po in ts were a l l s i m i l a r except at 360 days , where the E D ^ Q value was s l i g h t l y but not s i g n i f i c a n t l y h igher than those at e a r l i e r t ime p o i n t s . 67 EFFECT OF ISOPROTERENOL ON POSITIVE DP/DT IN CONTROL AND DIABETIC RAT HEARTS: I sopro te reno l (1 x 1 0 " ^ - 1 x 10"^M) produced dose-dependent inc reases in p o s i t i v e dP /d t o f i s o l a t e d working hear ts obta ined from c o n t r o l , and a l l o x a n and STZ d i a b e t i c ra t s at a l l o f the time po in ts s t u d i e d . Maximum changes in p o s i t i v e dP /d t were observed a f t e r the -8 a d m i n i s t r a t i o n o f 3 x 10~ M i sop ro te reno l a t a l l t ime p o i n t s . I sopro te reno l produced s i m i l a r i nc reases i n p o s i t i v e dP /d t i n hear ts obta ined from 7 and 30 day con t ro l and d i a b e t i c ra t s (F igures 29 and 30) . At 100 days a f t e r the i nduc t i on o f d iabetes a l s o , i sop ro te reno l produced s i m i l a r changes in p o s i t i v e dP /d t i n con t ro l as we l l as d i a b e t i c ra t hear ts as dep ic ted in F igure 31 . D i a b e t i c r a t hear ts e x h i b i t e d s l i g h t but not s i g n i f i c a n t depress ion in basal p o s i t i v e d P / d t . F igures 32 and 33 show the dose response curves to i sop ro te reno l i n i s o l a t e d per fused working hear ts from 180 day con t ro l and STZ d i a b e t i c and 240 day con t ro l and a l l o x a n d i a b e t i c ra t s r e s p e c t i v e l y . Basal p o s i t i v e dP/d t was s i g n i f i c a n t l y depressed i n both 180 day STZ and 240 day a l l o x a n d i a b e t i c r a t hear ts compared to age-matched c o n t r o l s . However the maximum inc reases in p o s i t i v e dP /d t produced by i sop ro te reno l i n 180 and 240 day d i a b e t i c r a t hear ts were of t he ; same magnitude as i n age-matched c o n t r o l s . F igure 34 i l l u s t r a t e s the dose-response curve to i sop ro te reno l on p o s i t i v e dP /d t i n 360 day con t ro l and STZ-d i a b e t i c r a t h e a r t s . At t h i s t ime po in t a l s o there were no d i f f e r e n c e s in the e f f e c t o f i sop ro te reno l between con t ro l and d i a b e t i c r a t h e a r t s . The pD2 va lues f o r the p o s i t i v e i n o t r o p i c e f f e c t o f i sop ro te reno l i n d i a b e t i c and age-matched con t ro l r a t hear ts are presented i n Table V. 68 The s e n s i t i v i t y o f the r a t myocardium towards the i n o t r o p i c e f f e c t o f i sop ro te reno l was not a l t e r e d e i t h e r by d iabetes or w i th age. EFFECT OF ISOPROTERENOL ON NEGATIVE DP/DT IN CONTROL AND DIABETIC RAT HEARTS: I sopro te reno l (1 x 1 0 " ^ to 1 x 10 ^M) produced dose-dependent i nc reases i n negat ive dP /d t o f i s o l a t e d working hear ts from a l l o x a n and STZ d i a b e t i c and con t ro l r a t s a t a l l o f the time po in ts s t u d i e d . The maximum changes i n negat ive dP /d t were produced a f t e r a d m i n i s t r a t i o n -8 o f 3 x 10~ M i sop ro te reno l i n con t ro l as we l l as d i a b e t i c r a t hear ts at a l l t ime p o i n t s . The dose-response curve to i s o p r o t e r e n o l on negat ive dP /d t i n 7 day con t ro l and a l l o x a n and STZ d i a b e t i c r a t hear ts i s presented i n F igure 35. Maximum responses in negat ive dP /d t to i sop ro te reno l were s i g n i f i c a n t l y depressed i n a l l o x a n d i a b e t i c r a t h e a r t s . Basal negat ive dP /d t i n both the d i a b e t i c groups and maximum responses to i sop ro te reno l i n STZ d i a b e t i c r a t hear ts were not a l t e r e d at t h i s t ime p o i n t . At 30 days a f t e r i nduc ing d iabetes basal negat ive dP /d t i n con t ro l and d i a b e t i c r a t hear ts and maximum responses to i sop ro te reno l i n S T Z - d i a b e t i c r a t hear ts were una l te red (F igu re 36 ) . -8 Maximum change produced by 3 x 10 M i sop ro te reno l i n negat ive dP /d t i n a l l o x a n - d i a b e t i c hear ts was s i g n i f i c a n t l y depressed compared to tha t i n c o n t r o l . Maximum responses i n negat ive dP /d t to i s o p r o t e r e n o l were s i g n i f i c a n t l y depressed in both a l l o x a n and STZ d i a b e t i c r a t hear ts a t 100 days compared to age-matched c o n t r o l s (F igure 37 ) . Basal negat ive dP /d t was a l so s i g n i f i c a n t l y depressed in a l l o x a n and STZ d i a b e t i c ra t hearts a t t h i s t ime p o i n t . F igure 38 i l l u s t r a t e s the dose-response curves 69 to i sop ro te reno l i n con t ro l and STZ d i a b e t i c r a t hear ts a t 180 days a f t e r i nduc t i on o f d i a b e t e s . Both basal negat ive dP /d t and maximum responses i n negat ive dP /d t to i s o p r o t e r e n o l were s i g n i f i c a n t l y depressed i n STZ d i a b e t i c r a t h e a r t s . Dose-response curves to i sop ro te reno l on negat ive dP /d t i n 240 day con t ro l and a l l o x a n d i a b e t i c , r a t hear ts are p resen ted " in F igure 39. Basal negat ive dP /d t was s i g n i f i c a n t l y depressed in a l l o x a n d i a b e t i c r a t h e a r t s . Maximum responses i n negat ive dP /d t to i sop ro te reno l i n a l l o x a n r a t hear ts were s l i g h t l y but not s i g n i f i c a n t l y depressed compared to c o n t r o l s . At 360 days a f t e r i nduc t i on o f d iabe tes both basal negat ive dP /d t and maximum changes i n negat ive dP /d t produced by i sop ro te reno l were not a l t e r e d i n STZ>diabet ic r a t hear ts (F igure 4 0 ) . Table VI presents maximum changes in negat ive dP /d t produced a f t e r the a d m i n i s t r a t i o n -8 o f 3 x 10" M i sop ro te reno l i n con t ro l as we l l as d i a b e t i c r a t hearts a t a l l o f the t ime po in ts s t u d i e d . The maximum change produced by i s o p r o t e r e n o l i n hear ts obta ined from 7, 30, and 100 day a l l o x a n and 100 and 180 day STZ d i a b e t i c r a t s was s i g n i f i c a n t l y depressed when compared to tha t i n age-matched con t ro l h e a r t s . TIME COURSE OF THE EFFECT OF ISOPROTERENOL ON CARDIAC CYCLIC AMP CONTENT, PHOSPHORYLASE ACTIVITY AND IN0TR0PY OF CONTROL AND DIABETIC RATS: I so l a t ed per fused working hear ts obta ined from 3 day and 100-120 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s were quick f rozen before 70 and at va r ious t ime po in ts a f t e r the a d m i n i s t r a t i o n o f i s o p r o t e r e n o l and the f rozen hear t t i s s u e was ana lyzed fo r c y c l i c AMP content and phosphorylase a^  a c t i v i t y . EFFECT OF ALLOXAN - AND STZ-DIABETES ON ISOPROTERENOL-INDUCED CHANGES IN RAT MYOCARDIAL cAMP CONTENT AND PHOSPHORYLASE ACTIVITY: F igures 41 and 42 present the t ime course of the e f f e c t o f an E D ^ -9 concen t ra t i on o f i sop ro te reno l (5 x 10 M) on c a r d i a c c y c l i c AMP l e v e l s i n 3 day and 100-120 day con t ro l and d i a b e t i c ra t s r e s p e c t i v e l y . Both basal and i sop ro te reno l s t imu la ted l e v e l s of c y c l i c AMP were the same in con t ro l and d i a b e t i c r a t hear ts a t both time po in ts s t u d i e d . -9 F igure 43 presents the time course of the e f f e c t o f 5 x .10 M i sop ro te reno l o n . c a r d i a c phosphory lase a_ a c t i v i t y i n 3 day con t ro l and a l l o x a n - and STZ=diabet ic r a t h e a r t s . I sopro te reno l produced a time dependent a c t i v a t i o n o f phosphory lase enzyme in both con t ro l and d i a b e t i c r a t hear ts and maximal a c t i v a t i o n o f phosphory lase was observed a t 20 sees a f t e r the a d m i n i s t r a t i o n o f the d rug . Basal phosphory lase ^ a c t i v i t y was s l i g h t l y but not s i g n i f i c a n t l y h igher i n 3 day a l l o x a n and STZ d i a b e t i c r a t hear ts when compared to c o n t r o l s . I sopro te reno l caused s i g n i f i c a n t l y g rea te r a c t i v a t i o n o f phosphory lase at 10, 20 and 40 sec t ime po in ts i n 3 day a l l o x a n d i a b e t i c r a t hear ts when compared to c o n t r o l s . In 3 day STZ d i a b e t i c r a t hear ts i sop ro te reno l a l s o produced g rea te r a c t i v a t i o n o f phosphorylase enzyme. However, the changes were s i g n i f i c a n t on l y a t 20 sees a f t e r the a d m i n i s t r a t i o n o f d rug . The time course of the e f f e c t o f i s o p r o t e r e n o l on c a r d i a c phosphorylase .a a c t i v i t y i n 100-120 day con t ro l and d i a b e t i c ra t 71 hearts i s shown in F igure 44. Basal phosphorylase a_ a c t i v i t y i n a l l o x a n and STZ d i a b e t i c r a t hear ts was s l i g h t l y but not s i g n i f i c a n t l y h igher than tha t found in con t ro l r a t h e a r t s . I sopro te reno l produced a time dependent a c t i v a t i o n o f phosphory lase enzyme in con t ro l as we l l as d i a b e t i c r a t h e a r t s . The a c t i v a t i o n observed in a l l o x a n and STZ d i a b e t i c r a t hear ts at 20-40 sec a f t e r drug a d m i n i s t r a t i o n was s i g n i -f i c a n t l y g rea te r than in con t ro l ra t h e a r t s . F igure 45 presents t o t a l phosphorylase a c t i v i t y i n con t ro l and a l l o x a n and STZ d i a b e t i c r a t hear ts at 3 days and 100-120 days a f t e r t rea tment . Tota l phosphory lase a c t i v i t y was not s i g n i f i c a n t l y d i f f e r e n t i n 3 day d i a b e t i c r a t hear ts from c o n t r o l s . However, 100-120 day d i a b e t i c r a t hear ts e x h i b i t e d s i g n i f i c a n t l y h igher phosphory lase a c t i v i t y compared to age-matched con t ro l r a t s . EFFECT OF ALLOXAN AND STZ DIABETES ON ISOPROTERENOL-INDUCED CHANGES IN RAT HEART IN0TR0PY: The time course o f the e f f e c t o f i sop ro te reno l on p o s i t i v e dP /d t i n 3 day con t ro l and d i a b e t i c r a t hear ts i s shown in F igure 46 . Changes - 9 produced w i th t ime i n p o s i t i v e dP /d t by i sop ro te reno l (5 x 10 M) i n con t ro l and a l l o x a n and STZ d i a b e t i c r a t hear ts were s i m i l a r . F igure 47 shows the t ime course of the e f f e c t o f i sop ro te reno l on p o s i t i v e dP /d t i n 100-120 con t ro l and d i a b e t i c r a t h e a r t s . Basal p o s i t i v e dP /d t i n 100-120 day a l l o x a n and STZ d i a b e t i c r a t hear ts was s i g n i f i c a n t l y depressed compared to con t ro l r a t h e a r t s . The maximal changes produced in p o s i t i v e dP /d t by i sop ro te reno l (a t 20 and 40 sec) were not d i f f e r e n t i n 100-120 day d i a b e t i c r a t hear ts as compared to c o n t r o l s . However, the onset o f the p o s i t i v e i n o t r o p i c e f f e c t o f i sop ro te reno l appears to be 72 s lower i n d i a b e t i c r a t h e a r t s . The t ime course of the e f f e c t o f . i s o p r o t e r e n o l on negat ive dP/d t i n 3 day con t ro l and d i a b e t i c r a t hear ts i s shown in F igure 48. Isopro te reno l produced s i m i l a r t ime dependent changes i n negat ive dP /d t i n hear ts i s o l a t e d from 3 day con t ro l and a l l o x a n and STZ d i a b e t i c r a t s . F igure 49 i l l u s t r a t e s the time course o f the e f f e c t o f i sop ro te reno l on negat ive dP /d t i n 100-120 day con t ro l and d i a b e t i c r a t h e a r t s . Basal negat ive dP /d t was s i g n i f i c a n t l y depressed in 100-120 day a l l o x a n and STZ d i a b e t i c r a t h e a r t s . However, maximum changes i n negat ive dP /d t caused by i sop ro te reno l i n d i a b e t i c r a t hear ts were s l i g h t l y but 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 in con t ro l r a t h e a r t s . The onset o f the e f f e c t o f i sop ro te reno l on. negat ive dP /d t appears to be s lower in d i a b e t i c rat . h e a r t s . The dose-response curve o f i sop ro te reno l on c a r d i a c c y c l i c AMP content i n 100-120 day con t ro l and S T Z * d i a b e t i c ra t s i s shown in F igure 50. I sopro te reno l produced s i m i l a r dose-dependent i nc reases in c a r d i a c c y c l i c AMP content i n con t ro l as we l l as d i a b e t i c r a t s . F igure 51 presents the dose-response curve o f i sop ro te reno l on c a r d i a c phosphory lase enzyme in 100-120 day con t ro l and S T Z - d i a b e t i c r a t s . Basal phosphory lase a. a c t i v i t y was s l i g h t l y but not 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 r a t h e a r t s . I sopro te reno l produced s i g n i f i c a n t l y h igher a c t i v a t i o n o f phosphory lase enzyme i n d i a b e t i c r a t hear ts at any dose t e s t e d , when compared to tha t i n con t ro l r a t h e a r t s . The dose-response curve of i sop ro te reno l on p o s i t i v e dP /d t i n 100-120 day con t ro l and d i a b e t i c r a t hear ts i s i l l u s t r a t e d i n F igure 52. Basal p o s i t i v e dP /d t was s i g n i f i c a n t l y depressed i n S T Z - d i a b e t i c r a t hear ts compared to c o n t r o l s . However, i sop ro te reno l produced s i m i l a r dose-73 dependent changes in p o s i t i v e dP /d t o f both con t ro l and d i a b e t i c r a t h e a r t s . The dose-response curve o f i sop ro te reno l on negat ive dP /d t o f i s o l a t e d per fused working hear ts ob ta ined from 100-120 day c o n t r o l and STZ°d iabet ic r a t s i s shown i n F igure 53. I sopro te reno l produced dose-dependent i nc reases in negat ive dP /d t o f both con t ro l and d i a b e t i c r a t h e a r t s . Basal negat ive dP /d t was s i g n i f i c a n t l y depressed in 100-120 day STZ°d iabet ic r a t hear ts compared to c o n t r o l s . I sopro te reno l produced s i m i l a r changes i n negat ive dP /d t i n con t ro l and d i a b e t i c r a t h e a r t s . E f f e c t o f PGE-j on c a r d i a c phosphory lase a c t i v i t y i n con t ro l and d i a b e t i c r a t s : — 6 E f f e c t o f 10" M PGE-| on c a r d i a c phosphory lase a c t i v i t y was s tud ied in 3 day and 100-120 day con t ro l and S T Z j d i a b e t i c r a t h e a r t s . I so l a t ed per fused working hear ts were qu ick f r ozen before and 30 sec a f t e r the a d m i n i s t r a t i o n o f PGE-j and the f rozen t i s s u e was assayed fo r c y c l i c AMP content and phosphory lase a_ a c t i v i t y and the r e s u l t s are presented i n Table V I I . , PGE-j s i g n i f i c a n t l y i nc reased c y c l i c AMP content i n 3 day and 100-120 day con t ro l as we l l as d i a b e t i c r a t hear ts to a s i m i l a r e x t e n t . However, PGE^ s i g n i f i c a n t l y a c t i v a t e d phosphory lase a_ on ly i n d i a b e t i c r a t h e a r t s . To t e s t the p o s s i b i l i t y whether p r o s t a g l a n d i n - 1 i k e substances re leased by i s o p r o t e r e n o l a c t i v a t e phosphory lase i n the d i a b e t i c hear t and thereby p o t e n t i a t e the e f f e c t o f i sop ro te reno l on phosphory lase, i s o l a t e d working hear ts from 100-120 day con t ro l and STZ d i a b e t i c r a t s 74 were p re t rea ted w i th 10~DM indomethac in , a p ros tag land in syn thes i s i n h i b i t o r , fo r 20 minutes before i sop ro te reno l a d m i n i s t r a t i o n . Hearts -9 were f rozen 20 sees a f t e r the a d m i n i s t r a t i o n o f 5 x 10 M i s o p r o t e r e n o l and were assayed fo r phosphorylase a^  a c t i v i t y . Indomethacin pret reatment d i d . n o t a l t e r the e f f e c t o f i sop ro te reno l on con t ro l c a r d i a c phosphory lase ; however, i n the d i a b e t i c r a t hear ts indomethacin a c t u a l l y appeared to enhance the i s o p r o t e r e n o l e f f e c t on phosphory lase . I sopro te reno l caused a s i g n i f i c a n t l y g rea te r a c t i v a t i o n o f phosphorylase <a i n d i a b e t i c ra t hearts compared to con t ro l r a t hear ts as shown i n Table V I I I . MUSCARINIC RECEPTOR BINDING STUDIES IN CONTROL AND DIABETIC RAT HEARTS: To s determine whether the inc reased s e n s i t i v i t y to carbachol e x h i b i t e d by 180 day d i a b e t i c ra t hear ts was a s s o c i a t e d w i th changes a t the recep to r l e v e l , musca r in i c recep to r b ind ing s tud ies ! were c a r r i e d out on the v e n t r i c u l a r t i s s u e obta ined from 180 day con t ro l and d i a b e t i c r a t h e a r t s . The r e s u l t s are summarized in Table IX. 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 ro l and d i a b e t i c r a t hear ts e i t h e r i n the t o t a l number o f muscar in i c recep to r s i t e s or i n the a f f i n i t y o f these s i t e s 3 f o r [ H] NMS. N o n - r a d i o l a b e l l e d NMS competed fo r the b ind ing s i t e s 3 l a b e l l e d by [ H] NMS wi th s i m i l a r a f f i n i t i e s i n both the c o n t r o l and d i a b e t i c r a t h e a r t s . Carbachol e x h i b i t e d two a f f i n i t i e s , one a high a f f i n i t y and the o ther a low a f f i n i t y , f o r the musca r in i c recep to r c s i t e s . The low a f f i n i t y s i t e s represented about 60% o f the t o t a l b ind ing s i t e s . In the presence of GMPPNP, a non-hydro lyzab le analog of GTP, the high a f f i n i t y b ind ing s i t e s were conver ted to low a f f i n i t y b ind ing s i t e s . There were no d i f f e r e n c e s between con t ro l and d i a b e t i c 75 r a t hear ts w i th respec t to the a f f i n i t i e s o f carbachol f o r the muscar in i c recep to r s i t e s e i t h e r i n the presence or i n the absence of GMPPNP. LEFT VENTRICULAR NORADRENALINE CONTENT IN 180-DAY CONTROL AND DIABETIC RATS: Noradrena l ine content o f the l e f t v e n t r i c l e s obta ined from 180-day con t ro l and d i a b e t i c ra t s was measured us ing a HPLC mehtod, i n order to determine whether a sympathet ic neuropathy has developed i n d i a b e t i c r a t h e a r t s . The r e s u l t s are presented i n Table X. There were no s i g n i f i c a n t changes in no rad rena l ine content i n 180-day a l l o x a n and STZ d i a b e t i c r a t hear ts compared to con t ro l r a t h e a r t s . Table 1. Certain features of control and diabetic rats. SERUM SERUM BODY HEART HEART WEIGHT/ CORONARY TIME INSULIN GLUCOSE WEIGHT WEIGHT BODY WEIGHT FLOW IN DAYS TREATMENT pU/mL mg/dL 9 9 mg/g mL/ml n/G 7 Control (9) 17.5 ± 2.6 70.8 t 6.9 210.4 0.94 t 0.03 4.48 ± 0.11 4.1 ± 0.2 Al1oxan 7.5 ± 1.7* 200.8 t 35.5* 201.3 0.87 ± 0.03 4.35 ± 0.16 4.4 t 0.3 STZ (6) 8.8 ± 1-7* 200.7 ± 34.8* 201.4 0.87 ± 0.02 4.32 t 0.10 4.3 t 0.1 30 Control (10) 20.9 ± 2.5 100.3 ± 8.5 236.3 1.03 ± 0.03 4.38 t 0.16 3.7 i 0.2 Alloxan 13.1. ± 2.2* 383.2 i 92.9* 214.10* 0.85 1 0.04* 4.04 ± 0.19 3.1 t 0.1 STZ 9)m 16.7 t 0.9 252.8 ± 68.4* 222.5 0.88 t 0.02* 3.96 t 0.06 3.6 ± 0.2 100 Control [7) 11.2 t 0.7 88.9 ± 5.0 248.4 1.01 t 0.03 4.09 t 0.11 5.2 i 0.3 Alloxan 6) 5.1 ± 1.1* 528.8 ± 108.0* 215.11* 0.80 ± 0 07* 3.72 t 0.19 4.6 t 0.7 STZ 7) 5.2 ± 1.0* 581.5 t 26.8* 182.10* 0.79 t 0.06* 4.32 ± 0.18 5.6 ± 0.8 180 Control (8) 19.4 ± 1.3 137.0 t 21.9 312.8 1.41 t 0.05 4.55 ± 0.10 3.2 t 0.2 STZ 7) 9.4 ± 1.5* 349.3 ± 32.2* 211.9* 1.10 t 0.05* 5.23 ± 0.10* 3.1 ± 0.9 240 Control 5) 18.1 ± 2.4 132.6 i 15.1 324.8 1.41 t 0.06 4.36 t 0.16 3.2 t 0.1 Alloxan 8) 8.6 ± 1.9* 391.5 ± 89.7* 239.7* 1.20 t 0.04* 5.00 ± 0.21* 3.3 ± 0.3 360 Control 7) 18.1 ± 2.0 119.6 t 8.9 372.13 1.53 t 0.06 4.16 t 0.16 3.2 t 0.2 STZ 6) 10.9 t 1.6* 345.4 t 49.7* 249.10* 1.30 t 0.04* 5.29 ± 0.20* 3.1 ± 0.2 All the values are expressed as mean ± S.E. Numbers In parentheses are the number of rats used at that time point. Coronary flow was measured during the initial 5 min period of retrograde perfusion at 45 cm H20 aortic perfusion pressure. * Represents significant difference from control at p < 0.05. 77 TABLE II. Certain features of control and diabetic rats used in the time course study 3 DAYS 100-120 DAYS PARAMETER CONTROL ALLOXAN STZ CONTROL ALLOXAN STZ BODY WEIGHT 211*3.0 201*3.0* 199*3.0* 268*2.0 217*6.0* 223t5.0* G (30) (30) (30) (30) (30) (30) SERUM GLUCOSE 99*3.3 255*25.0* 188*.15.4* 89i3.0 415*30.7* 403i32.7* mg/dL (30) (30) (30) (30) (30) (30) SERUM T 4 4.3il.O 3.1*0.4 5.3*0.6 5.0*0.2 3.9*0.2* 3.9*0.2* V9/4L (6) (6) (4) (27) (24) (29) SERUM T3B 4 65.111.4 55.7*1.4* 57.3*3.4*59.7*0.8 47.9il.1* 49.3*0.9* * (6) (6) (4) (27) (24) (24) Seruir glucose. T^  (thyroxine) and T^ B (triiodothyronine bead uptake) were determined in blood collected at the time of sacrifice from 3 and 100-120 day control and diabetic rats. All the values are expressed as meantS.E.M. Numbers in parenthesis denote sample size. * - denotes significant difference from control at P<0.05. t - serum triiodothyronine content was expressed as % bead uptake. The values reported here were similar to those reported by Fein et al.(8). using a similar assay method. TABLE III. FEATURES OF CONTROL AND DIABETIC RATS. TITLE OF THE DURATION BODY WEIGHT (g) SERUM GLUCOSE mg/dL STUDY OF DIABETES CONTROL ALLOXAN STZ CONTROL ALLOXAN STZ PGE1 3 days 100-120 days 18U2.0 (6) 248±4.0 (6) 172+3.0* (6) 192±13.0* (6) 107.2±4.8 (6) 107.6±3.4 (6) 436.6±48.2* (6) 448.2±46.5* (6) INDOMETHACIN 100-120 days 243±6.0 (4) 200+7.0* (4) 123.8±5.1 (4) 398.3±46.9* (4) MUSCARINIC RECEPTOR BINDING 180 days 298+6.0 (6) 230+8.0* (7) 119.2±10.0 — (6) 560 ±36.4* (7) NORADRENALINE 180 days 310±10.0 268+9.0* 216±9.0* 114.0±11.1 336.9±42.0* 564.5±70.0* ^1 oo ESTIMATION All the values are expressed as mean S.E.M. Numbers 1n parentheses denote sample size. •Represent significant differences from control (p<0.05). 79 FIGURE 3 : Le f t v e n t r i c u l a r pressure development a t va r ious l e f t a t r i a l f i l l i n g pressures in i s o l a t e d per fused working hear ts obta ined from 7 day a l l o x a n - and STZ d i a b e t i c and con t ro l r a t s . A l l the va lues are expressed as the mean±S.E.M. Numbers i n parentheses a r e , t h e number o f animals used. LVDP or l e f t v e n t r i c u l a r developed pressure i s expressed as mm Hg on the y - a x i s . L e f t a t r i a l f i l l i n g pressure i s expressed as cm H 9 0 on the x - a x i s . 80 FILLING PRESSURE (cm H20) 81 FIGURE 4 : Rate o f l e f t v e n t r i c u l a r pressure development at var ious l e f t a t r i a l f i l l i n g pressures in i s o l a t e d perfused working hear ts obta ined from 7 day a l l o x a n and S T Z . d i a b e t i c and con t ro l r a t s . A l l the. va lues are expressed as the mean±S.E.M. Numbers in parentheses are the number o f r a t s used. +dP/dt or ra te o f l e f t v e n t r i c u l a r pressure development i s expressed in mm Hg/sec on the y - a x i s . L e f t a t r i a l f i l l i n g pressure i s expressed in cm H o 0 on the x - a x i s . 82 3000-1 I I 1 I I 1 » 5 10 15 20 FILLING PRESSURE (cm H20) 83 FIGURE 5 : Rate o f l e f t v e n t r i c u l a r pressure d e c l i n e a t va r ious l e f t a t r i a l f i l l i n g pressures in i s o l a t e d per fused working hear ts obta ined from 7 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . A l l the va lues are expressed as mean±S.E.M. Numbers i n parentheses are the number o f r a t s used. - d P / d t or ra te of l e f t v e n t r i c u l a r pressure d e c l i n e i s expressed in mm Hg/sec on the y - a x i s . L e f t a t r i a l f i l l i n g pressure i s expressed in cm H ? 0 on the x - a x i s . 84 FILLING PRESSURE (cm H 20) 85 FIGURE 6: L e f t v e n t r i c u l a r pressure development a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts from 30 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . A l l the po in ts are expressed as the mean±S.E.M. Numbers i n parentheses denote sample s i z e . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . LVDP i s expressed in mm Hg on the Y - a x i s . A t r i a l f i l l i n g pressure i s expressed in cm H 9 0 on the x - a x i s . 86 30 DAYS o-o CONTROL (9) A - A STZ (10) D - D ALLOXAN (7) 1 7n 1 ' ' r -1 ° 15 20 FILLING PRESSURE cm H 20 87 FIGURE 7: Rate o f r i s e o f l e f t v e n t r i c u l a r pressure a t va r ious l e f t a t r i a l f i l l i n g pressures in i s o l a t e d per fused working hearts from 30 day a l l o x a n and S T Z - d i a b e t i c and con t ro l r a t s . A l l the po in ts are expressed as the mean±S;.£vM. Numbers i n parentheses denote sample s i z e . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . P o s i t i v e dP /d t i s expressed in mm Hg/sec on the y - a x i s . A t r i a l f i l l i n g pressure i s expressed in cm FLO on the x - a x i s . 88 I • 1 i I ' r— 5.0 10.0 15.0 20.0 FILLING PRESSURE cm H 20 89 FIGURE 8: Rate o f d e c l i n e o f l e f t v e n t r i c u l a r pressure a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts from 30 day a l l o x a n s and S T Z - d i a b e t i c and con t ro l r a t s . A l l the po in ts are expressed as the mean±S.E.M. Numbers in parentheses denote sample s i z e . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . Negat ive dP /d t i s expressed in mm Hg/sec on the y - a x i s . A t r i a l f i l l i n g pressure i s expressed in cm H 9 0 on the x - a x i s . 90 300CH 2000H ioooH 0 I I 1 I I 1 I 5.0 10.0 15.0 20 0 FILLING PRESSURE cm H 20 91 FIGURE 9: Le f t v e n t r i c u l a r pressure development a t va r ious a t r i a l f i l l i n g pressures in i s o l a t e d per fused working hear ts obta ined from 100 day a l l o x a n - and S T Z d i a b e t i c and con t ro l r a t s . A l l the va lues are expressed as the meantS.E.M. Numbers i n parentheses are the number o f hear ts used. LVDP i s expressed in mm Hg on the y - a x i s . F i l l i n g pressure i s expressed i n cm H^O on the x - a x i s . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . 92 100 D a y s O—O CONTROL 01) I I I I I 10.0 15.0 200 FILLING PRESSURE c m H 2 0 9 3 FIGURE 10: Rate o f l e f t v e n t r i c u l a r pressure development a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts obta ined from 100 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . A l l the va lues are expressed as the mean+S.E.M. Numbers i n parentheses denote sample s i z e . P o s i t i v e dP /d t i s expressed as mm Hg/sec on the y - a x i s . F i l l i n g pressure i s expressed as cm H^O.on t h e . x - a x i s . A s t e r i s k s i n d i c a t e s i g n f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . 94 100 DAYS 1000-" « ' i I I I 100 15.0 20.0 FILLING P R E S S U R E c m of H 2 0 95 FIGURE 11 : Rate o f l e f t v e n t r i c u l a r pressure d e c l i n e a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts ob ta ined from 100 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . A l l the va lues are expressed as the meantS.E.M. Numbers in parentheses denote sample s i z e . Negat ive dP /d t i s expressed as mm Hg/sec on the y - a x i s . F i l l i n g pressure i s expressed i n cm H^O on the x - a x i s . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . 96 1 0 0 D A Y S —o C 0 N T R 0 L ( 1 1 ) F I L L I N G P R E S S U R E c m 97 FIGURE 12: L e f t v e n t r i c u l a r pressure development a t va r ious l e f t a t r i a l f i l l i n g pressures in i s o l a t e d per fused working hear ts from 180 day S T Z - d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers in parentheses denote the number o f hear ts used a t t h i s time p o i n t . LVDP i s expressed in mm Hg on the y - a x i s and f i l l i n g pressure i n cm H^O on the x - a x i s . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (one-way ANOVA). FILLING PRESSURE (cm H 20) 99 FIGURE 13 : Rate o f r i s e in l e f t v e n t r i c u l a r pressure a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts from 180 day STZ d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers in parentheses denote the number o f hear ts used a t t h i s time p o i n t . P o s i t i v e dP /d t i s expressed in mm Hg/sec on the y - a x i s and f i l l i n g pressure in cm H^O on the x - a x i s . A s t e r i s k s denote s i g n f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (one-way ANOVA). 100 FILLING PRESSURE (cm H 20) 101 FIGURE 14: Rate o f d e c l i n e in l e f t v e n t r i c u l a r pressure a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts from 180 day S T Z - d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean+S.E.M. Numbers i n parentheses denote the number o f hear ts used at t h i s t ime p o i n t . Negat ive dP /d t i s expressed i n mm Hg/sec on the y - a x i s and f i l l i n g pressure in cm H^O on the x - a x i s . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (one-way ANOVA). 102 FILLING PRESSURE (cm H 20) 103 FIGURE 15: L e f t - v e n t r i c u l a r pressure development a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hearts from 240 day a l l o x a n d i a b e t i c and c o n t r o l r a t s . A l l the po in ts represent the mean±S.E.M. Numbers in parentheses denote sample s i z e . LVDP i s expressed in mm Hg on the y - a x i s and f i l l i n g pressure i n cm H p 0 on the x - a x i s . 104 240 Days 120.0- • — • Control (7) o — o Alloxan (0) 90.0-X E E CL a > 60.0-30. QJ 5.0 10.0 15.0 Filling pressure cm 105 FIGURE 16: Rate o f l e f t v e n t r i c u l a r pressure development a t va r ious l e f t a t r i a l f i l l i n g pressures in i s o l a t e d per fused working hear ts from 240 day a l l o x a n d i a b e t i c and con t ro l r a t s . A l l the po in ts represen t the mean±S.E.M. Numbers in parentheses denote sample s i z e . P o s i t i v e dP /d t i s expressed in mm Hg/sec on the y - a x i s and f i l l i n g pressure in cm H^O on the x - a x i s . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (one-way ANOVA). 106 6.0 10.0 15.0 20.0 Filling pressure cm H 2 ° 107 FIGURE 17: Rate o f l e f t v e n t r i c u l a r pressure d e c l i n e a t va r ious l e f t a t r i a l f i l l i n g pressures in i s o l a t e d per fused working hear ts from 240 day a l l o x a n d i a b e t i c and con t ro l r a t s . A l l the po in ts represent the meantS.E.M. Numbers in parentheses denote sample s i z e . Negat ive dP /d t i s expressed i n mm Hg/sec on the y - a x i s and f i l l i n g pressure i n cm H^O on the x - a x i s . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (one-way ANOVA). 108 5.0 10.0 15.0 20.0 Filling pressure cm HgO 109 FIGURE 18: Le f t v e n t r i c u l a r pressure development a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts obta ined from 360 day STZ d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers i n pa ren thes is denote sample s i z e . LVDP i s expressed on the y - a x i s i n mm Hg and f i l l i n g pressure on the x - a x i s i n cm H ? 0 . no FILLING PRESSURE cm H 20 Ill FIGURE 19: Rate o f l e f t v e n t r i c u l a r pressure development a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts obta ined from 360 day STZ d i a b e t i c and con t ro l r a t s . Each po in t represents the mean±S.E.M. Numbers i n parentheses denote sample s i z e . P o s i t i v e dP /d t i s expressed on the y - a x i s i n mm Hg /sec . F i l l i n g pressure i s expressed on the x - a x i s i n cm H^O. A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (one-way ANOVA). 112 800J i i i i i i I I 5.0 10.0 15.0 20.0 FILLING PRESSURE cm H 20 113 FIGURE 20: Rate o f l e f t v e n t r i c u l a r pressure d e c l i n e a t va r ious l e f t a t r i a l f i l l i n g pressures i n i s o l a t e d per fused working hear ts obta ined from 360 day S T Z - d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers in parentheses denote sample s i z e . Negat ive dP /d t i s expressed i n mm Hg/sec on the y - a x i s and f i l l i n g pressure in cm H ? 0 on the x - a x i s . 114 115 FIGURE 21 : E f f e c t o f the du ra t i on o f d iabe tes on the ra te o f l e f t v e n t r i c u l a r pressure development i n i s o l a t e d per fused working hear ts obta ined from con t ro l and a l l o x a n and STZ. d i a b e t i c r a t s , a t 15 cm H^O l e f t a t r i a l f i l l i n g p ressu re . Each po in t represen ts the mean±S.E.M. Numbers in parentheses denote sample s i z e . A s t e r i s k s represent s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . Time a f t e r t reatment wi th the d iabe togen ic drug i s expressed in days i n log s c a l e on the x - a x i s . P o s i t i v e dP /d t i s expressed in mm Hg/sec on the y - a x i s . 116 +DP/DT AT 15 CM H20 FILLING PRESSURE (•) CONTROL (N=9) (•) ALLOXAN (N=7) («•) STZ (N=9) 3300-1 Tir€ AFTER TREATMENT (DAYS) 117 FIGURE 22: E f f e c t o f the du ra t i on o f d iabe tes on the ra te o f l e f t v e n t r i c u l a r pressure d e c l i n e i n i s o l a t e d per fused working hear ts obta ined from con t ro l and a l l o x a n and STZ d i a b e t i c r a t s , a t 15 cm H 2 0 l e f t a t r i a l f i l l i n g p ressu re . Each po in t rep resen ts the mean±S.E.M. Numbers in parentheses denote sample s i z e . A s t e r i s k s represent s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . Time a f t e r t reatment wi th the d iabe togen ic drug i s expressed i n days in log s c a l e on the x - a x i s . P o s i t i v e dP /d t i s expressed i n mm Hg/sec on the y - a x i s . 118 - D P / D T AT 1 5 CM H20 F I L L I N G PRESSURE (•) CONTROL (N=9) (•) ALLOXAN (N=7) («•) STZ (N=9) 3 0 0 0 - , 119 FIGURE 23 : Dose-response curve o f carbachol on basal p o s i t i v e dP /d t i n i s o l a t e d per fused work ing hear ts from 7 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers i n parentheses i n d i c a t e sample s i z e . Data are expressed as percent reduc t i on in basal +dP/dt ( y - a x i s ) produced by va r ious molar concen t ra t i ons of carbachol ( x - a x i s ) . 120 EFFECT OF CARBACHOL ON +DP/DT DEVELOPMENT 7 DATS (•) CONTROL tN-9) (•) ALLOXAN (N«91 MSTZ (N=8) -100 I I • 1 I I I I 1 1 1 | I I | • " ' M I T ] I 8 7 6 5 4 CARBACHOL -IM) 121 FIGURE 24: Dose-response curve o f carbachol on basal p o s i t i v e dP /d t i n i s o l a t e d per fused working hear ts from 30 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . Each po in t represents the mean±S.E.M. Numbers i n parentheses i n d i c a t e sample s i z e . Data are expressed as percent reduc t i on in basal +dP/dt ( y - a x i s ) produced by va r ious molar concen t ra t i ons o f carbachol ( x - a x i s ) . 122 E F F E C T OF CARBACHOL ON +DP/DT DEVELOPMENT 3 0 DATS (•) CONTROL (N=9) (•) ALLOXAN (N=7) («•) STZ (N=9) CARBACHOL - ( M ) 123 FIGURE 25: Dose-response curve o f carbachol on basal p o s i t i v e dP /d t i n i s o l a t e d perfused working hear ts from 100-day a l loxan- - and STZ d i a b e t i c and con t ro l r a t s . Each po in t represents the mean±S.E.M. Numbers in parentheses i n d i c a t e sample s i z e . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (Newman-Keuls t e s t ) . Data are expressed as percent reduc t i on in basal +dP/dt ( y - a x i s ) produced by var ious molar concen t ra t i ons o f carbachol ( x - a x i s ) . 124 EFFECT OF CARBACHOL ON +DP/DT DEVELOPMENT 100 DAYS (•) CONTROL CN=10) (•) ALLOXAN (N=8) H) STZ (N=10) 80 H 100 1 ' ' ""i •—i iiii»n 1—i 1111111 1—i 111mi « • -8 -7 -6 -5 -4 CARBACHOL CM) 125 FIGURE 26: Dose-response curve o f carbachol on basal p o s i t i v e dP /d t i n i s o l a t e d per fused working hear ts obta ined from 180 day STZ-d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers in parentheses i n d i c a t e sample s i z e . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from c o n t r o l s a t p<0.05 (one-way ANOVA). Data are represented as percent reduc t i on i n basal +dP/dt ( y - a x i s ) produced by va r ious molar concen t ra t i ons o f carbachol ( x - a x i s ) . 126 80 H •ICQ \ i i i ni| 1—i i i mi) 1—i i i mi] 1—i i 11 n«| '—• 1 • • i "| -8 -7 -6 -5 -4 CflRBflCHOL (M) r 127 FIGURE 27: Dose-response curve o f carbachol on basal p o s i t i v e dP /d t i n i s o l a t e d per fused working hear ts ob ta ined from 240 day a l l o x a n d i a b e t i c and con t ro l r a t s . Each po in t represents the mean±S.E.M. Numbers i n parentheses i n d i c a t e sample s i z e . A s t e r i s k s denote s i g n f i c a n t d i f f e r e n c e s from con t ro l a t p<0.05 (one-way ANOVA). Data are represented as percent reduc t i on i n basal +dP/dt ( y - a x i s ) produced by va r ious molar concen t ra t i ons of carbachol ( x - a x i s ) . 128 129 FIGURE 28: Dose-response curve o f carbachol on basal p o s i t i v e dP /d t i n i s o l a t e d per fused working hear ts ob ta ined from 360 day STZ d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers in parentheses i n d i c a t e sample s i z e . Data are presented as percent reduc t i on i n basal +dP/dt ( y - a x i s ) produced by va r ious molar concen t ra t i ons o f carbachol ( x - a x i s ) . 130 80 H 100 1 i I i i n ] 1 — i i I I i l l ) 1—I l i m i | 1 — i i i I n i | i — • i i 1111| -8 -7 -6 -5 -4 CflRBflCHOL (M) 131 TABLE IV. GEOMETRIC MEAN ED, n VALUES OF CARBACHOL TIME AFTER TREATMENT (DAYS) CONTROL ALLOXAN STZ 7 1.65xl0"6 1.45xl0"6 1.43xl0"6 (1.22-2.23)1 (1.16-1.85) (1.03-1.98) (9)* (9) (8) 30 1.67xl0"6 1.82xl0"6 1.57xl0"6 (1.36-2.03) (0.96-3.45) (1.11-2.22) (9) (7) (9) 100 1.57xlO' 6 5.91xl0" 6* 6.1x10 6 (1.00-2.46) (2.79-12.5) (3.08-12.1) (10) (8) (10) 180 1.85xl0"6 - 7.34xl0" 7* (1.48-2.3) - (4.77-11.2) (9) (7) 240 1.87xl0"6 8.16xl0' 7 * (1.64-2.13) (5.97-11.1) (6) (9) 380 3.02x10"6 - 1.43xl0"6 (0.65 - 14.1) - (0.46-4.4) (4) (5) All the values are expressed as the geometric mean with 95* confidence intervals of the dose of carbachol required to reduce basal dP/dt by 30X. E D30 v a 1 u e s w e r e obtained from Individual dose-response curves. Geometric mean and 95% confidence Intervals were calculated according to the method of Fleming et a ! (1972). l95X confidence limits 2number of hearts •signif icantly different from control at p<0.05 132 FIGURE 29: Dose-response curve o f i sop ro te reno l on p o s i t i v e dP /d t i n i s o l a t e d -perfused working hear ts from 7 day a l l o x a n ^ and STZ d i a b e t i c and con t ro l r a t s . A l l the po in ts are expressed as ithe mean±S.E.M. Numbers i n parentheses denote sample s i z e . P o s i t i v e dP /d t i n mm Hg/sec i s p l o t t e d aga ins t negat ive log< molar concen t ra t i ons o f i s o p r o t e r e n o l . C represents basal p o s i t i v e dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . 133 EFFECT OF ISOPROTERENOL ON +DP/DT 7 DATS (•) CONTROL (N=9) (•) ALLOXAN CN«=9) W STZ (N«6) SOOOn 4530 A c 10 9 8 ISOPROTERENOL -LOG M 134 FIGURE 30: Dose-response curve o f i sop ro te reno l on p o s i t i v e dP /d t i n i s o l a t e d per fused working hear ts from 30 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . A l l the po in ts are expressed as the mean±S.E.M. Numbers i n parentheses denote sample s i z e . P o s i t i v e dP /d t i n mm Hg/sec i s p l o t t e d aga ins t negat ive l o g ? rrfolar concen t ra t i ons of i s o p r o t e r e n o l . C represents basal p o s i t i v e dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . 135 EFFECT OF ISOPROTERENOL ON +DP/DT 30 DAYS (•) CONTROL (N=10) (•) ALLOXAN CN-6) H) STZ (N=9) 5000 n 1500 > • i i • »—i i 11111 1—i—i i 11111 i i i i 11 m c 10 9 8 7 ISOPROTERENOL -LOG M 136 FIGURE 31 : Dose-response curve o f i sop ro te reno l on p o s i t i v e dP /d t i n i s o l a t e d per fused working hear ts from 100 day a l l o x a n - and STZ d i a b e t i c and con t ro l r a t s . A l l the po in ts are expressed as the mean±S.E.M. Numbers in parentheses denote sample s i z e . P o s i t i v e dP /d t i n mm Hg/sec i s p l o t t ed aga ins t negat ive l og , , molar c o n c e n t r a t i o n s of i s o p r o t e r e n o l . C represen ts basal p o s i t i v e dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . 137 EFFECT OF ISOPROTERENOL ON +DP/DT (•1 CONTROL (N«=7) (•) ALLOXAN CN*6) H) STZ CN-=7) 100 DAYS 5000 n 4500 H 4000 H o u v> 3500 H Q 3000 H 2500 2000 H 1500 I i i i i 11111 i 10 9 ISOPROTERENOL i i 11111 8 -LOG M i—i i 11 m 138 FIGURE 32: Dose-response curve o f i s o p r o t e r e n o l on p o s i t i v e dP /d t i n i s o l a t e d perfused working hear ts from 180 day S T Z - d i a b e t i c and con t ro l r a t s . A l l the po in ts are expressed as the mean±S.E.M. Numbers i n parentheses denote sample s i z e . P o s i t i v e dP /d t i n mm Hg/sec i s p l o t t e d aga ins t negat ive l og molar concen t ra t i ons of i s o p r o t e r e n o l . C represents basal dP /d t p r i o r to i sop ro te reno l a d m i n i s r a t i o n . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s from con t ro l (p<0.05 one-way ANOVA). 139 EFFECT OF ISOPROTERENOL ON +DP/DT 180 DAYS (•) CONTROL (N=8) H) STZ (N=7) 4000 T 1000~i ' i i i — i — i i i n i j i — • — i i 111n 1 » i i 11111 c 10 9 8 7 ISOPROTERENOL -LOG M 140 FIGURE 33 : Dose-response curve o f i sop ro te reno l on p o s i t i v e dP /d t i n i s o l a t e d perfused working hear ts from 240 day a l l o x a n d i a b e t i c and con t ro l r a t s . A l l the po in ts are expressed as mean±S.E.M. Numbers in parentheses denote sample s i z e . P o s i t i v e dP /d t i n mm Hg/sec i s p l o t t ed aga ins t negat ive l og molar concen t ra t i ons of i s o p r o t e r e n o l . C represen ts basal p o s i t i v e dP /d t p r i o r to i sop ro te reno l admin is -t r a t i o n . A s t e r i s k s i n d i c a t e s i g n f i c a n t d i f f e r e n c e s from con t ro l (p<0.05 one-way ANOVA). 141 EFFECT OF ISOPROTERENOL ON +DP/DT 240 DAYS (•) CONTROL (N=5) (•) ALLOXAN (N=8) 142 FIGURE 34: Dose-response curve of i sop ro te reno l on p o s i t i v e dP /d t i n i s o l a t e d per fused working hear ts from 360 day STZ d i a b e t i c and c o n t r o l r a t s . A l l the po in ts are expressed as mean+S.E.M. Numbers i n parentheses denote sample s i z e . P o s i t i v e dP /d t i n mm Hg/sec i s p l o t t e d aga ins t negat ive l og molar concen t ra t i on o f i s o p r o t e r e n o l . C represents basal p o s i t i v e dP /d t p r i o r to i s o p r o t e r e n o l admin is -t r a t i o n . 143 EFFECT OF ISOPROTERENOL ON +DP/DT 360 DAYS (•) CONTROL (N«=7) (<«) STZ (N=5) 4000 n 1000 I i i i 1—i—i 111111 1—i—i 1111 n 1—i—i 111111 c 10 9 8 7 ISOPROTERENOL -LOG M Table V Sensitivity of the rat myocardium to the positive Inotropic effect of Isoproterenol (pD- values).* Treatment Time after treatment (days) 7 30 100 180 240 360 Control 8.291.06 8.32±.12 8.461.07 8.211.07 8.271.10 8.261.03 (9) (10) (7) (8) (5) (6) Alloxan 8.361.11 8.4U.08 8.431.10 - 8.261.06 (9) (6) (6) (8) STZ 8.341.07 8.391.15 8.531.13 8.151.04 - 8.34±.10 (6) (9) (7) (7) (6) All values are meaniS.E.M. Numbers In parenthesis denote sample size. *pD2 1s the negative log concentration of Isoproterenol required to produce 50% of the maximum change observed. 145 FIGURE 35: Dose-response curve o f i sop ro te reno l on negat ive dP /d t i n i s o l a t e d perfused working hear ts from 7 day a l l o x a n - and S T Z - d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers in parentheses denote sample s i z e . A s t e r i s k s i n d i c a t e 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 e f f e c t o f i s o p r o t e r e n o l on negat ive dP /d t from tha t i n con t ro l (p<0.05, Newman-Keuls t e s t ) . Negat ive dP /d t (mm Hg/sec) i s p l o t t e d aga ins t negat ive l og molar concen t ra t i on o f i s o p r o t e r e n o l . C represents basal negat ive dP /d t p r i o r to. i sop ro te reno l a d m i n i s t r a t i o n . 146 DRC OF ISO ON -DP/DT DEVELOPMENT 7 DAY5 (•) CONTROL CN=9) (•) ALLOXAN (N=9) H) STZ (N=6) 5303-1 1030 > | i i i 1 1 1 n | 1 — i — i 111111 1 —>—i 111111 C 10 9 8 7 ISOPROTERENOL - LOG DOSE 147 FIGURE 36: Dose-response curve o f i sop ro te reno l on negat ive dP/d t i n i s o l a t e d per fused working hearts from 30 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . Each po in t represents the mean±S.E.M. Numbers in parentheses denote sample s i z e . A s t e r i s k s i n d i c a t e 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 e f f e c t o f i sop ro te reno l on negat ive dP /d t from tha t i n con t ro l (p<0.05, Newman-Keuls t e s t ) . Negat ive dP /d t (mm Hg/sec) i s p l o t t e d aga ins t negat ive l og molar concen t ra t i on o f i s o p r o t e r e n o l . C represents basal negat ive dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . 148 DRC OF ISO ON -DP/DT DEVELOPMENT 30 DAYS (•) CONTROL (N«=10) (•) ALLOXAN (N*6) t [«) STZ (N«=9) 4500-1 3500 H 2500 H 1500 - T c 149 FIGURE 37: Dose-response curve o f i sop ro te reno l on negat ive dP /d t i n i s o l a t e d per fused working hear ts from 100-day .^alloxan and STZ d i a b e t i c and con t ro l r a t s . Each po in t rep resen ts the mean±S.E.M. Numbers i n parentheses denote sample s i z e . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s in the e f f e c t o f i sop ro te reno l on negat ive dP /d t from tha t i n con t ro l (p<0.05, Newman-Keuls t e s t ) . Negat ive dP /d t (mm Hg/sec) i s p l o t t e d aga ins t negat ive l og molar concen t ra t i on o f i s o p r o t e r e n o l . C represents basal negat ive dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . 1 5 0 151 FIGURE 38: Dose-response curve o f i sop ro te reno l on negat ive dP /d t i n i s o l a t e d per fused working hear ts from 180 day STZ d i a b e t i c and con t ro l r a t s . Each po in t represents the mean±S.E.M. Numbers in parentheses denote sample s i z e . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s in the e f f e c t o f i sop ro te reno l on negat ive dP /d t from tha t i n con t ro l (p<0.05, one-way ANOVA). Negat ive dP/d t (mm Hg/sec) i s p l o t t e d aga ins t negat ive log molar concen t ra t i ons of i s o p r o t e r e n o l . C represents basal negat ive dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . 152 DRC OF ISO ON -DP/DT DEVELOPMENT 180 DAYS (•) CONTROL (N=8) MSTZ (N*7) 4000-1 3000 H u UJ in V. o I 2000 H 1000- 1 1 I — I I I 1111 I I — I I I I 111 1 — I — I I I I I I I 10 9 8 7 ISOPROTERENOL - LOG DOSE 153 FIGURE 39: Dose-response curve o f i sop ro te reno l on negat ive dP /d t i n i s o l a t e d per fused working hear ts from 240 day a l l o x a n d i a b e t i c and con t ro l r a t s . Each po in t represen ts the meantS.E.M. Numbers i n parentheses denote sample s i z e . Negat ive dP /d t (mm Hg/sec) i s p l o t t e d aga ins t negat ive l og molar concen t ra t i on o f i s o p r o t e r e n o l . C represents basal negat ive dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . 1 5 4 DRC OF ISO ON -DP/DT DEVELOPMENT 240 DAYS (•) CONTROL (N=5) (•) ALLOXAN (N=8) C 10 9 8 7 ISOPROTERENOL - LOG DOSE 155 FIGURE 40: Dose-response curve o f i sop ro te reno l on negat ive dP /d t i n i s o l a t e d per fused working hear ts from 360 day STZ d i a b e t i c and con t ro l r a t s . Each po in t represents the mean±S.E.M. Numbers in parentheses denote sample s i z e . Negat ive dP /d t (mm Hg/sec) i s p l o t t e d aga ins t negat ive log molar concen t ra t i on o f i s o p r o t e r e n o l . C represents basal negat ive dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . 157 Table VI: Cardiac relaxant effect of isoproterenol TIME AFTER TREATMENT MAXIMUM 5 OF TREATMENT CHANGE CONTROL (IN DAYS) IN(-)dP/dt a (mm Hg/sec) control (9) 2725=300 100 7 alloxan (9) 1471:317* 54 STZ (7) 2089=274 77 control (9) 2692=330 100 30 alloxan (6) 1444=275* 55 STZ (9) 1993=256 74 control (7) 1750=185 100 100 alloxan ( £ ) 825=265* 47 STZ (7) 896=230* 51 control (8) 2207=270 100 1 8 0 STZ (7) 1308=256* ' 59 control (5) 2293=293 100 2 4 0 alloxan (8) 1685=274 74 control (7) 1437=217 100 3 6 0 STZ (5) 1066=232 74 All the values are mean=S.E.M. Numbers in parenthesis denote sample size a. maximum changes in -dP/dt produced by 3xlO*8M isoproterenol * Denotes significant difference from control at P*0.05. 158 FIGURE 41 : Time course of the e f f e c t o f i sop ro te reno l (5xlO"^M) on c y c l i c AMP content o f i s o l a t e d per fused working hear ts from 3 day a l l o x a n - and STZ d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers in parentheses denote sample s i z e . C y c l i c AMP content was est imated us ing a radioimmunoassay method. C y c l i c AMP content (femtomoles/mg t i s s u e ) i s p l o t t e d aga ins t t ime ( s e c ) . 159 3 DAYS (•) CONTROL (N°6) (•) ALLOXAN CN»6) (••) S T Z CN=6) 5x10~9M ISO 200 T 1 1 1 1 1 1 1 1 0 5 10 IS 20 25 30 35 40 T I M E ( S E C ) 160 FIGURE 42 : Time course o f the e f f e c t o f i sop ro te reno l (5x1 C ^ M ) on c y c l i c AMP content o f i s o l a t e d per fused working hear ts from 100-120 day a l l o x a n and STZ d i a b e t i c and con t ro l r a t s . Each, po in t represen ts the mean±S.E.M. Numbers in parentheses denote sample s i z e . C y c l i c AMP content was es t imated us ing a radioimmunoassay method. C y c l i c AMP content (femtomoles/mg t i s s u e ) i s p l o t t e d aga ins t t ime ( s e c ) . 161 100-120 DAYS (•) CONTROL (N-6) (•) ALLOXAN (N«6) («) STZ (N=6) 300 A 200 J 1 1 1 1 1 1 1 1 1 0 5 10 15 20 25 30 35 40 TIME (SEC) 162 FIGURE 43 : Time course of the e f f e c t o f i sop ro te reno l (5xlO~ 3 M) on phosphorylase a_ a c t i v i t y o f i s o l a t e d per fused working hear ts from 3 day a l l o x a n and S T Z - d i a b e t i c and con t ro l r a t s . Each po in t represents the mean+S.E.M. Numbers in parentheses denote sample s i z e . Phosphory lase a_ a c t i v i t y was measured i n the d i r e c t i o n o f glycogen s y n t h e s i s , and i s expressed as a % o f t o t a l phosphory lase a c t i v i t y on the y - a x i s . Time i s expressed in seconds on the x - a x i s . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l (p<0.05, Newman-Keuls t e s t ) . 163 3 DAYS (•) CONTROL (N=6) (•) ALLOXAN (N~6) MSTZ (N=6) 5x10'9M ISO ~r 5 - r -10 -r— 15 20 25 30 35 -1 40 TIME (SEC) 164 FIGURE 44: _ Q Time course o f the e f f e c t o f i sop ro te reno l (5x10. M) on phosphorylase a_ a c t i v i t y o f i s o l a t e d per fused working hear ts from 100-120 day a l l o x a n s and STZ»diabet ic and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers in parentheses denote sample s i z e . Phosphory lase a_ a c t i v i t y was measured i n the d i r e c t i o n o f g lycogen s y n t h e s i s , and i s expressed as a % of t o t a l phosphorylase a c t i v i t y on the y - a x i s . Time i s expressed i n seconds on the x - a x i s . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l (p<0.05, Newman-Keuls t e s t ) . 165 100-120 DAYS (•) CONTROL (N=6) (•) ALLOXAN (N=6) (<)STZ (N=6) 5X10"9M ISO 0 5 —r-10 — I 1 — 15 20 T I M E (SEC) 25 30 —r— 35 — i 40 166 FIGURE 45 : Tota l phosphory lase a c t i v i t y o f i s o l a t e d per fused working hear ts from 3 and 100-120 day a l l o x a n (AXN)o and S T Z - d i a b e t i c and con t ro l (CONT) r a t s . Each bar represen ts meantS.E.M. Numbers i n parentheses denote sample s i z e . Tota l phosphory lase a c t i v i t y was measured in the d i r e c t i o n o f glycogen syn thes i s i n the presence o f 5'-AMP and i s expressed as umoles o f phosphate l i b e r a t e d per mg t i s s u e per minute ( y - a x i s ) . A s t e r i s k s denote s i g n i f i c a n t d i f f e r e n c e s from con t ro l (p<0.05, Newman-Keuls t e s t ) . 167 TOTAL PHOSPHORYLASE ACTIVITY 3 DAYS (N=30) 100-123 DAYS (N«=30) .04-=c .03-XL CL in o x O-m o a: UJ m cr .02H or o m o £ ± 0-1 1 1—I 1—1 1 1 I I 1 I CONT flXN STZ CONT flXN STZ 168 FIGURE 46: Time course of the e f f e c t o f i sop ro te reno l (5x10 M) on the ra te o f l e f t v e n t r i c u l a r pressure r i s e o f i s o l a t e d per fused working hear ts from 3 day a l l o x a n - and STZ d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean S . E . M . Number i n parentheses denote sample s i z e . P o s i t i v e dP /d t (mm Hg/sec) i s p l o t t e d aga ins t t ime ( s e c ) . C represen ts basal p o s i t i v e dP /d t a f t e r 15 min e q u i l i b r a t i o n p e r i o d . 169 TIME COURSE OF ISO ON RATE OF LV PRESSURE DEVELOPMENT (•) CONTROL (N=6) (•) ALLOXAN (N=6) («•) STZ (N=6) 7000 n 3 DAYS 5x10"9M ISO TIME (SEC) 170 FIGURE 47: -9 Time course o f the e f f e c t o f i sop ro te reno l (5x10 M) on the ra te o f l e f t v e n t r i c u l a r pressure r i s e o f i s o l a t e d per fused working hear ts from 100-120 day a l l o x a n ^ and STZ-d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers i n parentheses denote sample s i z e . P o s i t i v e dP /d t (mm Hg/sec) i s p l o t t e d aga ins t t ime ( s e c ) . C represen ts basal p o s i t i v e dP /d t a f t e r 15 min e q u i l i b r a t i o n p e r i o d . A s t e r i s k s denote s i g n f i c a n t d i f f e r e n c e s from con t ro l (p<0.05 Newman-Keuls t e s t ) . 1 7 1 TIME COURSE OF THE EFFECT OF ISO ON +DP/DT 100-120 DAYS (•) CONTROL (N=6) 7000 -i 600JH UJ 5000 in x 45(53 3000 H 2000 (•) ALLOXAN (N=6) H) STZ (N«6) 5x10"9M ISO -i r C 0 T 5 10 " T-15 - T -20 " T -25 30 —r-35 40 TIME (SEC) 172 FIGURE 48: Time course o f the e f f e c t of i sop ro te reno l (5x10 M) on the ra te o f l e f t v e n t r i c u l a r pressure d e c l i n e o f i s o l a t e d perfused working hear ts from 3 day a l l o x a n and STZ^d iabe t i c and con t ro l r a t s . Each po in t r e p r e s e n t s the mean±S.E.M. Numbers in parentheses denote sample s i z e . Negat ive dP /d t (mm Hg/sec) i s p l o t t e d aga ins t t ime ( s e c ) . C represen ts basal negat ive dP /d t a f t e r 15 min e q u i l i b r a t i o n p e r i o d . 173 TIME COURSE OF ISO ON RATE OF LV PRESSURE DECLINE (•) CONTROL (N=6) (•) ALLOXAN (N=6) [<) STZ (N=6) SOflO-i LJ UJ in X H 4000 H o 0_ a i 3000 3 DAYS 5x1Cf 9M T c TIME (SEC) 174 FIGURE 49: Time course of the e f f e c t o f i sop ro te reno l (5x10- M) on the ra te o f l e f t v e n t r i c u l a r pressure d e c l i n e o f i s o l a t e d per fused working hearts from 100-120 day a l l o x a n ^ and S T Z - d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean S . E . M . Numbers in parentheses denote sample s i z e . Negat ive dP /d t (mm Hg/sec) i s p l o t t e d aga ins t t ime ( s e c ) . C represen ts basal negat ive dP /d t a f t e r 15.min e q u i l i b r a t i o n p e r i o d . 175 TIME COURSE OF THE EFFECT OF ISO ON -DP/DT 100-120 DAYS (•) CONTROL (N=6) (•) ALLOXAN (N=6) H)STZ (N=6) 2000- - i r C 0 "T~ 5 10 15 20 ~T 25 30 35 —I 40 TIME (SEC) 176 FIGURE 50: Dose-response curve of i sop ro te reno l on . the c y c l i c AMP content o f i s o l a t e d per fused working hear ts obta ined from 100-120 day S T Z - d i a b e t i c and con t ro l r a t s . Each po in t represen ts the mean±S.E.M. Numbers i n parentheses denote sample s i z e . C y c l i c AMP content (femtomoles/mg t i s s u e ) i s p l o t t e d aga ins t l og molar dose of i s o p r o t e r e n o l . C represen ts basal c y c l i c AMP con ten t . 177 DRC OF ISO ON CARDIAC CYCLIC RMP CONTENT (•) CONTROL (N=6) («)STZ CN<=6) 1833-1 100- 120 DAYS 933 H UJ in in ID s: v. m UJ _i o a: UJ o u 833 H 783 H c 683-u LJ 588 ^  488 J i i i | — i — i — i % | 2 X 1 0 " 8 5X10 -8 10~ 8 ISOPROTERENOL (M) 178 FIGURE 51 : Dose-response curve o f i sop ro te reno l on phosphory lase a_ a c t i v i t y i n i s o l a t e d per fused working hear ts from 100-120 day STZ=diabet ic and con t ro l r a t s . Each po in t represen ts the mean+S.E.M. Numbers in parentheses, denote sample s i z e . Phosphory lase ^ a c t i v i t y i s expressed as a % o f t o t a l phosphory lase a c t i v i t y and i s p l o t t e d aga ins t l og molar dose of i s o p r o t e r e n o l . A s t e r i s k s denote s i g n f i c a n t d i f f e r e n c e s from con t ro l (p<0.05, one-way ANOVA). C represen ts basal phosphorylase a_ a c t i v i t y . 179 DRC OF ISO ON CARDIAC PHOS A ACTIVITY (•) CONTROL (N«6) H) STZ (N=6) 40 n 100- 120 DAYS 35H 30-(X 5* O X ci-in o 25. £ 5? t-o t— h M 20 H in o 1C. £ 1 5 10-r _ r 2 x 10*M r ~ r H T — 1 — ' — 1 — I _ o 5x10*M 1 x 10°M ISOPROTERENOL CM) 180 FIGURE 52: Dose-response curve o f i sop ro te reno l on the ra te o f l e f t v e n t r i c u l a r pressure r i s e in i s o l a t e d per fused working hear ts from 100-120 day STZ d i a b e t i c and con t ro l r a t s . Each po in t represents the mean±S.E.M. Numbers i n parentheses denote sample s i z e . P o s i t i v e dP /d t (mm Hg/sec) i s p l o t t e d aga ins t l og molar concen t ra t i on o f i s o p r o t e r e n o l . C represents basal p o s i t i v e dP /d t p r i o r to i sop ro te reno l a d m i n i s t r a t i o n . A s t e r i s k represen ts s i g n i f i c a n t d i f f e r e n c e from con t ro l (p<0.05 one-way ANOVA). 181 DRC OF ISO ON RATE OF LEFT VEhlTRICULAR PRESSURE DEVELOPMENT (•) CONTROL (N=6) H) STZ (N«6) 7033-1 6333 A in 3: ¥ 5033' fl-ea 4030 A 3030 J 100- 120 DAYS * I c T o 1 ' r ~ Z o I 1 — ' — ' — I _ o 2 x 1 0 a M 5x10 a M 1 x 10BM ISOPROTERENOL (M) 182 FIGURE 53: Dose-response curve o f i sop ro te reno l on ra te o f l e f t v e n t r i c u l a r pressure d e c l i n e o f i s o l a t e d per fused working hear ts from 100-120 day STZ' d i a b e t i c and con t ro l r a t s . Each po in t represen ts mean±S.E.M. Numbers in parentheses denote sample s i z e . Negat ive dP /d t (mm Hg/sec) i s p l o t t e d aga ins t l og molar concen t ra t i on o f i s o p r o t e r e n o l . C represen ts basal negat ive dP /d t p r i o r to drug a d m i n i s t r a t i o n . A s t e r i s k s represent s i g n i f i c a n t d i f f e r e n c e from con t ro l (p<0.05 one-way ANOVA). 183 DRC OF ISO ON RATE OF LEFT VENTRICULAR PRESSURE DECLINE (•) CONTROL (N«6) H) STZ (N«6) 5533 n 4533H U4 o O I 3533-1 2533-100- 120 DAYS } """^—r-ZcT '—•— 1 I ISOPROTERENOL (M) 2 x 10°M 5x10 9 M 1 x ' l0 8 M 184 Table VII. Effect of PGE1 on cardiac cyclic AMP and phosphorylase a activity 1n control and diabetic rats. TREATMENT cAMP Phosphorylase a (femtomoles/mg tissue) (« of total phosphorylase) NO DRUG PGEi 10-fM NO DRUG PGEi 10*b M 100-120 days CONTROL 425*14.0 550=19.0* B.5±1.8 11.4*2.9 (6) (5) (6) (5) STZ DIABETIC 435=24.0 620127.0* 16.9=3.3 27.2*2.6* (6) (6) (6) (6) 3 days CONTROL 417±11.0 56U8.0* 6.7±1.8 n . i i . 7 (6) (6) (6) (6) STZ DIABETIC 414±26.0 57H27.0* 13.H2.8 29.6=3.2* (6) (6) (6) Isolated perfused working hearts from 100-120 and 3 day control and STZ diabetic rats were frozen before or 30 sees after the administration of 10"6 M PGE1 and the frozen hearts were analyzed for cAMP content and phosphorylase activity. All values are expressed as meaniS.E.M. Numbers 1n parenthesis denote sample size. * denotes significant difference from the basal (no drug) value at p<0.05. 185 Table VILI. Effect of 10 H Indomethacin on Isoproterenol-lnduced cardiac phosphorylase activity In 100-120 day control and diabetic rats. PHOSPHORYLASE a ACTIVITY (T. of total phosphorylase activity) NO DRUG ISOPROTERENOL 5 x 10"9 M ISOPROTERENOL 5 x 10"9 M 4 INDOMETHACIN 1 x 10"6 M CONTROL 8.5±1.8 (6) 19.7±1.6* (6) 22.611.7* (4) STZ DIABETIC 16.913.3 (6) 3«.8i3.2** (6) 50.614.0** (4) Isolated perfused working hearts from 100-120 day diabetic and control rats were frozen before or 20 sec after the administration of 5x10 M isoproterenol. Results In the last column were obtained from hearts which were perfused with buffer containing 10*6M indomethacin for 20 minutes before administering isoproterenol. All values are expressed as mean S.E.M. Numbers in parenthesis denote sample size. * denotes significant differences from basal (no drug) value at P<0.05 **denotes significant differences from basal (no drug) value and the corresponding value 1n control experiments at P<0.05. Table'X. [3H] NMS binding In 160 day control and diabetic rat hearts. TREATMENT DIRECT BINDING Cold NMS INHIBITION CARBACHOL INHIBITION CARBACHOL INHIBITION IN PRESENCE OF GMPPNP [R] t pmol/g pK P*, Fr 2 PK, P*2 fr2 CONTROL 57.3*9.8 9.571.1 9.31.03 6.121 .1 4.361.04 59% - 4.111.07 87% DIABETIC 61.8H4.8 9.551.1 9.41.06 6.231 .1 4.171.17 52% - 4.261.06 98% [R] t * Total amount of receptors. pK = negative log of dissociation constant pK, = high affinity dissociation constant pK? = low affinity dissociation constant Fr., •= Fraction of low affinity binding sites All values are meamS.E.M. of duplicate determinations 187 Table X. Left ventricular noradrenaline content in 180 day control and diabetic rats. Treatment Noradrenaline vg/G Control 0. .48 ± 0. 04 (6) STZ 0, .54 i 0. .04 (5) Al loxan 0 .44 i 0 .03 (4) All values are mean S.E.M. Numbers in parentheses denote sample size. 188 DISCUSSION Resu l t s ob ta ined in the present study demonstrate tha t i n j e c t i n g r a t s w i th e i t h e r a l l o x a n or STZ r e s u l t s i n a d i a b e t i c s t a t e c h a r a c t e r -i zed by h y p o i n s u l i n e m i a , hyperglycemia and weight l o s s and tha t these d i a b e t i c r a t s develop c a r d i a c f u n c t i o n a l and pharmacologica l abnormal-i t i e s w i th t ime. Card iac dys func t i on observed in d i a b e t i c ra t s i nc luded a decreased a b i l i t y o f i s o l a t e d per fused working hear ts to develop peak l e f t v e n t r i c u l a r pressure as we l l as a decrease i n maximum ra tes o f l e f t v e n t r i c u l a r pressure r i s e and d e c l i n e when hear ts from d i a b e t i c ra t s were exposed to high work l o a d s . The f o l l o w i n g pharmacolog ica l changes were observed : D i a b e t i c r a t hear ts e x h i b i t e d changes in t h e i r s e n s i t i v i t y towards the negat ive i n o t r o p i c e f f e c t o f c a r b a c h o l ; a decreased respons iveness towards the c a r d i a c r e l axan t e f f e c t (maximum changes produced i n negat ive dP /d t ) o f i s o p r o t e r e n o l ; and an inc reased s e n s i t i v i t y o f the c a r d i a c phosphory lase enzyme to i s o p r o t e r e n o l . THE DIABETIC-RAT MODEL: Rats t r ea ted w i th e i t h e r a l l o x a n or STZ developed almost i d e n t i c a l d i a b e t i c s t a t e s and e x h i b i t e d the c l a s s i c a l symptoms o f IDDM such as hyperg lycemia , h y p o i n s u l i n e m i a , g l u c o s u r i a , p o l y u r i a , p o l y d i p s i a , po lyphagia and weight l o s s . The a b i l i t y o f a l l o x a n and STZ to produce such a d i a b e t i c s t a t e has p r e v i o u s l y been repor ted by Rerup (1970). I n s u l i n l e v e l s , measured in the serum o f f as ted d i a b e t i c r a t s , were reduced to l e s s than approx imate ly 50% of con t ro l l e v e l s (Table I ) . The a v a i l a b i l i t y o f some f u n c t i o n a l i n s u l i n i n the d i a b e t i c ra t s e l i m i n a t e d the n e c e s s i t y f o r i n s u l i n therapy i n these r a t s f o r s u r v i v a l . Th is unt reated d i a b e t i c 189 ra t model cou ld p o s s i b l y represent a poor l y c o n t r o l l e d IDDM c o n d i t i o n . Fas t i ng serum g lucose l e v e l s were above 200 mg percent and i n d i c a t e d an over t d i a b e t i c s t a t e w i th f a s t i n g hyperg lycemia . In the l a t t e r par t o f t h i s s tudy , i n s u l i n l e v e l s were not measured and f a s t i n g serum g lucose l e v e l s were used as an i n d i c a t i o n o f the s e v e r i t y o f the d i s e a s e . Throughout t h i s study d i a b e t i c r a t s e x h i b i t e d lower body and wet hear t weights compared to c o n t r o l s . Loss o f body weight i n the d i a b e t i c r a t s cou ld r e s u l t from derangements i n p r o t e i n metabol ism such as decreased p r o t e i n syn thes i s and inc reased breakdown. A l l o f these fea tu res were c o n s i s t e n t w i th those p r e v i o u s l y repor ted i n a s i m i l a r d i a b e t i c r a t model by Penpargkul e_t aj_. (1980) and Fein ejt aj_. (1980) . D i a b e t i c r a t s e x h i b i t e d a hypothyro id s t a t e as i n d i c a t e d by reduced serum T3 l e v e l s at 3 days and reduced serum T3 and T4 l e v e l s a t 3 and 100 to 120 days a f t e r i nduc t i on o f the d isease (Table I I ) . I t has been p r e v i o u s l y shown tha t serum T3 and T4 l e v e l s are lower in d i a b e t i c p a t i e n t s as we l l as i n exper imenta l d i a b e t i c ra t s (Pi t tman e t al_. 1 979; 1981) . A decrease i n hepat ic T3 p roduc t ion owing to the i n h i b i t i o n o f monodeiodinat ion o f serum T4 i n the l i v e r has been demonstrated as e a r l y as 3 days a f t e r induc t ion , o f STZ d iabetes in the r a t (Pi t tman e_t a_l_. 1981) . Var ious other s tud ies have a l s o repor ted depressed t h y r o i d hormone l e v e l s i n the blood o f d i a b e t i c ra t s (Fe in et al_. 1980; D i l l m a n , 1982) . In the present study 75% o f the ra t s t rea ted w i th a l l o x a n and 95% of the ra t s t r ea ted wi th STZ became d i a b e t i c . The low inc idence of d iabe tes wi th a l l o x a n may be due main ly to the u l t r a sho r t h a l f - l i f e o f a l l o x a n i n body f l u i d s . M o r t a l i t y o f d i a b e t i c ra t s was seen on ly 190 at t ime po in ts longer than 100 days . I t i s d i f f i c u l t to a t t r i b u t e the m o r t a l i t y seen in d i a b e t i c ra t s to the s e v e r i t y o f the d i a b e t i c s t a t e s i nce an almost s i m i l a r i nc idence o f m o r t a l i t y was observed in the con t ro l group of r a t s . CARDIAC FUNCTION IN DIABETIC AND CONTROL RATS: Resu l t s obta ined in the present i n v e s t i g a t i o n demonstrate tha t c a r d i a c f u n c t i o n a l abno rma l i t i e s develop i n both a l l o x a n and STZ d i a b e t i c r a t s . The prominent a b n o r m a l i t i e s observed were depress ions i n peak l e f t v e n t r i c u l a r pressure development (100 and 180 days) and decreased ra tes o f l e f t v e n t r i c u l a r pressure r i s e (decreased myocard ia l c o n t r a c t i l i t y , a t 30, 100, 180, 240, and 360 days) and l e f t v e n t r i c u l a r pressure d e c l i n e (decreased c a r d i a c r e l a x a t i o n at 30, 100, 180, and 240 days) a t high a t r i a l f i l l i n g pressures (h igh work l o a d s ) . S i m i l a r c a r d i a c f u n c t i o n a l a l t e r a t i o n s have been repor ted i n hear ts obta ined from 8-week STZ d i a b e t i c r a t s (Penpargkul e t al_. 1980) . In our s tudy a l l o x a n - i n d u c e d d iabe tes produced c a r d i a c f u n c t i o n a l abno rma l i t i e s as e a r l y as 30 days a f t e r i n d u c t i o n , whereas the a l t e r a t i o n s produced by STZ d iabe tes were no t i ced on ly 100 days a f t e r t reatment (F igures 21 and 22 ) . A p o s s i b l e reason why a delayed onset o f c a r d i a c f u n c t i o n a l abno rma l i t i e s was observed i n STZ- induced d iabe tes may be t h a t , a t 30 days in t h i s s tudy , STZ t reatment r e s u l t e d in a r e l a t i v e l y m i ld form o f d i a b e t e s . STZ t reatment at 30 days d id not s i g n i f i c a n t l y reduce the i n s u l i n l e v e l s from con t ro l l e v e l s . However, a s i g n f i c a n t e l e v a t i o n in the serum g lucose l e v e l s was observed (Table I ) . The delayed onset o f f u n c t i o n a l abno rma l i t i e s i n STZ d i a b e t i c ra t s compared w i th a l l o x a n d i a b e t i c ra t s might not have r e s u l t e d owing 191 to the d i f f e r e n t forms o f d iabe tes induced by these two d rugs , but might have r e s u l t e d owing to a f a i l u r e o f the drug to produce as severe a form of the d i sease s t a t e . Recent ly Fein e t al_. (1980) repor ted tha t a b n o r m a l i t i e s in r e l a x a t i o n and v e l o c i t y o f sho r ten ing were observed i n p a p i l l a r y muscles i s o l a t e d from 5-week STZ d i a b e t i c r a t s . These authors used r a t s o f the same s t r a i n , sex and age and the same dose o f STZ as was used in our s tudy . However, a severe form o f d iabe tes was induced by STZ in F e i n ' s s tudy , as evidenced by the high blood g lucose va lues repor ted by these au tho rs . These r e s u l t s support our assumption tha t we d id not observe f unc t i ona l changes a t 30 days in STZ- t rea ted r a t s owing to the r e l a t i v e mi ldness o f the d i sease at t h i s time po in t i n t h i s s e r i e s o f an ima l s . Fur thermore, Opie e_t aj_. (1979a) have r e c e n t l y po in ted out tha t both a l l o x a n and STZ produce a lmost i d e n t i c a l d i a b e t i c s t a tes in the r a t . Peak l e f t v e n t r i c u l a r pressure development was depressed i n i s o l a t e d per fused working hear ts ob ta ined from 30- and 100-day a l l o x a n and 100- and 180-day STZ d i a b e t i c ra t s (F igures 6, 9 and 12 ) . Even though hear ts ob ta ined from 240- and 350-day d i a b e t i c r a t s developed lower l e f t v e n t r i c u l a r pressures as compared to c o n t r o l s , the decreases were not s i g n i f i c a n t l y d i f f e r e n t (F igures 15 and 18 ) . However, the ra tes a t which l e f t v e n t r i c u l a r pressure rose in 240- and 360-day d i a b e t i c r a t hear ts and dec l i ned i n 240-day d i a b e t i c r a t hearts were s i g n i f i c a n t l y s lower than in con t ro l hear ts (F igures 16, 17, 19 and 20) . I t appears tha t the f u n c t i o n a l a l t e r a t i o n s occur r ing in the hear t i n exper imenta l d iabe tes were more prominent i n the ra tes a t which l e f t v e n t r i c u l a r p ressure developed and d e c l i n e d , than 192 in the abso lu te amount o f p ressure deve loped. Depression i n the ra te o f r i s e o f l e f t v e n t r i c u l a r pressure by both a l l o x a n and STZ d iabe tes progressed w i th t ime . The decreased a b i l i t y o f the d i a b e t i c hear t to develop pressure may r e s u l t from a l t e r a t i o n s i n the c o n t r a c t i l e p ro te ins and i n the sa rcop lasmic r e t i c -ulum (SR) induced by d i a b e t e s . Some o f these a l t e r a t i o n s i nc l ude 2+ decreased actomyosin and myosin ATPase and m y o f i b r i l l a r basal and Ca -s t imu la ted ATPase a c t i v i t i e s . ^ D i l l m a n - 1 . 9 8 0 ; MaThotra et al_. 1981; P i e r c e and D h a l l a , 1981) , and a l t e r a t i o n s in v e n t r i c u l a r myosin components 2+ (Di l lman 1980). A d im in ished Ca t r anspo r t across c a r d i a c SR was a l s o repor ted in a l l o x a n and STZ d i a b e t i c ra t s (Penpargkul e t a l . 1981; Lopaschuk e_t al_. 1983) . I t has been shown tha t a bu i l d -up of in te rmed ia tes o f f a t t y a c i d metabol ism such as l o n g - c h a i n a c y l c a r n i t i n e s occur i n the d i a b e t i c myocardium (Feuvray e_t aj_. 1 979) . 2+ 2+ The d imin ished Ca t r a n s p o r t and Ca - ATPase a c t i v i t y o f the c a r d i a c SR obta ined from d i a b e t i c r a t s has been shown to be main ly due to an i nc rease i n the l e v e l s o f l o n g - c h a i n a c y l c a r n i t i n e s a s s o c i a t e d wi th the SR membrane (Lopaschuk ejb al_. 1983) . We d id not observe any a l t e r a t i o n s in c a r d i a c f u n c t i o n a t 7 days a f t e r i nduc t i on o f d iabe tes (F igures 3 , 4 and 5 ) . Our r e s u l t s do not agree wi th the r e s u l t s repor ted by M i l l e r (1979) , where a l l o x a n d i a b e t i c r a t hear ts e x h i b i t e d decreased a b i l i t i e s to develop normal s y s t o l i c pressure at high a t r i a l f i l l i n g p r e s s u r e s . In the above study 5 mM g lucose was used in the pe r fus ion medium, and when the g lucose concen t ra t i on was r a i s e d to 10 mM, d i a b e t i c hear ts performed s i m i l a r to c o n t r o l s . S i m i l a r e f f e c t s were observed by Ingebretsen et..aj_. (1980) , when they inc reased the g lucose 193 concen t ra t i on .of the per fusa te to 10 mM, in i s o l a t e d working hear ts from 3-week a l l o x a n d i a b e t i c r a t s . In the present study 10 mM glucose was present i n the per fusa te and might e x p l a i n why we d id not observe changes in the ra te o f l e f t v e n t r i c u l a r pressure development at 7 days. Depressions i n maximum negat ive dP /d t were observed i n a l l o x a n d i a b e t i c r a t s a t 30 days and i n STZ d i a b e t i c ra t s a t 100 days and these a l t e r a t i o n s progressed w i th t ime. Depression in maximum negat ive dP /d t was most prominent when hear ts were per fused a t high f i l l i n g p r e s s u r e s . Regan e t a]_. (1974) repor ted tha t i n dog, ch ron i c a l l o x a n d iabe tes r e s u l t e d i n a reduced v e n t r i c u l a r compl iance and inc reased v e n t r i c u l a r s t i f f n e s s due to accumulat ion o f g l y c o p r o t e i n i n the hea r t . S t i f f n e s s of. the v e n t r i c l e - cou ld r e s u l t i n depressed negat ive dP /d t development. In a p a r a l l e l s tudy conducted in our l a b o r a t o r y , c a r d i a c SR from 120-day a l l o x a n and STZ d i a b e t i c ra t s e x h i b i t e d reduced a b i l i t i e s 2+ to take up Ca , whereas c a r d i a c SR from 7-day a l l o x a n and STZ d i a b e t i c 2+ ra ts e x h i b i t e d normal Ca uptake pa t te rns (Lopaschuk e t aj_. 1983). In the present i n v e s t i g a t i o n , no changes i n negat ive dP /d t development were observed i n 7-day d i a b e t i c r a t h e a r t s , and a depressed maximum negat ive dP /d t development was observed a t 15 cm H 2 0 and h igher f i l l i n g pressures in hear ts i s o l a t e d from 100-day d i a b e t i c •rats.-.*- The decreased negat ive dP /d t development cou ld a l s o have r e s u l t e d from a decreased a b i l i t y 2+ of c a r d i a c SR to accumulate Ca The ra te o f d e c l i n e i n v e n t r i c u l a r pressure a t high f i l l i n g pressures was depressed i n 240- and 360-day con t ro l hear ts compared w i th younger con t ro l hear ts from o ther time p o i n t s . Though the maximum negat ive 194 dP/d t developments at high a t r i a l f i l l i n g pressures i n 360-day STZ d i a b e t i c hear ts were lower than the age-matched c o n t r o l s , the changes were not s i g n i f i c a n t . The ra tes o f d e c l i n e i n l e f t v e n t r i c u l a r pressure in 360-day STZ d i a b e t i c r a t hear ts were s i g n i f i c a n t l y depressed a t high f i l l i n g p r e s s u r e s , when compared wi th 180-day con t ro l r a t hear ts (hear t weight matched c o n t r o l s ) . A p ro longa t i on o f c a r d i a c muscle r e l a x a t i o n wi th age has been repor ted in the r a t ( W e i s f e l d t , l 9 8 0 ) . At t ime po in ts such as 240 and 360 days i n our s tudy , depress ions i n maximum negat ive dP /d t produced by d iabe tes were not r e a d i l y de tec tab le as the ca rd i ac r e l a x a t i o n i n c o n t r o l s was a l s o depressed owing to a l t e r a t i o n s induced by age. S ince the changes appear ing i n con t ro l hear ts due to age were not t o t a l l y d i f f e r e n t from those observed i n d i a b e t i c hear ts a t much e a r l i e r t ime p o i n t s , i t i s tempt ing to hypothes ize tha t d i a b e t i c r a t hear ts undergo an e a r l y ag ing p rocess . „ In f a c t , premature c e l l u l a r senescence owing to an under l y ing c e l l u l a r abnormal i t y has been proposed to be i nvo l ved in the pathogenesis o f va r ious pa tho log i c man i f es ta t i ons of d iabe tes (Renold. et aj[. 1978) . Hearts ob ta ined from 360-day STZ d i a b e t i c ra t s appeared to have performed be t t e r than those obta ined from 180-day d i a b e t i c r a t s , at 15 cm H^O a t r i a l f i l l i n g pressure (F igures 21 and 22) and at o ther h igher a t r i a l f i l l i n g p r e s s u r e s . The depress ion no t i ced i n the c a r d i a c performance i n 180-day d i a b e t i c r a t hear ts appeared to have g r a d u a l l y d isappeared by 360 days , i ns tead o f p r o g r e s s i v e l y i n c r e a s i n g . About 25% o f m o r t a l i t y has occur red both in the d i a b e t i c and con t ro l groups between 180 and 360 days . One p o s s i b i l i t y might be tha t the most seve re l y d i a b e t i c animals d ied before reach ing the 360-day mark and 1 9 5 t ha t the remain ing s u r v i v o r s , w i th m i l de r d i a b e t e s , e x h i b i t e d be t te r c a r d i a c performance. However, t h i s p o s s i b i l i t y appears to be h i gh l y u n l i k e l y s i nce va r i ous fea tu res o f 360-day d i a b e t i c r a t s were e s s e n t i a l l y the same as those«of the 180-day d i a b e t i c r a t s (Table I ) . Hearts obta ined from d i a b e t i c r a t s were sma l l e r than con t ro l hearts a t a l l t ime po in ts s tud ied except 7 days a f t e r i nduc t i on (Table I ) . A l l o xan d iabe tes has been shown tb i n h i b i t the ra te o f p r o t e i n syn thes i s i n r a t hear ts (Wi l l i ams e_t aj_. 1980). The depressed c a r d i a c f unc t i on i n the d i a b e t i c r a t s was not due to the sma l l e r s i z e o f the h e a r t , as sma l l e r hear ts from younger con t ro l r a t s from 7- and 30-day groups per fo rmed.be t te r than the l a r g e r hear ts o f 240- and 360-day c o n t r o l s . Fur thermore, sma l l e r s i z e d hear ts were shown to perform be t te r than l a r g e r hear ts i n the i s o l a t e d per fused working hear t system (Neely e_t al_. 1967) . I t should a l s o be noted tha t even though hearts obta ined from 180 - , 240- and 360-day d i a b e t i c r a t s were sma l l e r compared wi th c o n t r o l s , they were a c t u a l l y l a r g e r i n s i z e r e l a t i v e to body we igh t . Coronary f low ra tes were not a l t e r e d i n the d i a b e t i c r a t hear ts at any time po in t from con t ro l ra tes (Table I ) . Th is obse rva t i on suggests tha t the coronary vascu la tu re in the d i a b e t i c r a t hear ts has not been sub jec ted to any major c o m p l i c a t i o n s . S ince the f u n c t i o n a l a l t e r a t i o n s observed in the d i a b e t i c hear ts i n t h i s study cou ld have occurred wi thout major v a s c u l a r c o m p l i c a t i o n s , these a l t e r a t i o n s may i n d i c a t e the development o f a d i a b e t i c cardiomyopathy... In p a r t i c u l a r , the f i n d i n g s may i n d i c a t e the development o f the p r e c l i n i c a l l e f t v e n t r i c u l a r dys func t i on phase o f the cardiomyopathy. A number o f c l i n i c a l s t u d i e s have demonstrated p r e c l i n i c a l l e f t v e n t r i c u l a r 196 dys func t i on i n d i a b e t i c s . L e f t v e n t r i c u l a r dys func t i on was observed both in s y s t o l e and d i a s t o l e and cou ld represent de fec ts i n myocard ia l c o n t r a c t i o n as we l l as r e l a x a t i o n (Shapi ro e_t al_. 1981a and b ) . In the present s tudy , depress ions were observed i n the ra te o f l e f t v e n t r i c u l a r pressure development and d e c l i n e o f i s o l a t e d per fused working d i a b e t i c r a t h e a r t s , and these depress ions represent de fec ts i n myocard ia l c o n t r a c t i o n and r e l a x a t i o n . I n s u l i n l a c k and the va r ious metabo l i c abno rma l i t i es a s s o c i a t e d wi th i n s u l i n l ack might p r i m a r i l y be i nvo l ved i n the development o f va r ious b iochemical and s t r u c t u r a l a l t e r a t i o n s tha t cause myocard ia l dys func t i on in d i a b e t e s . Recen t l y , Fein e t a l . (1981) have demonstrated the a b i l i t y of 4 weeks of i n s u l i n t reatment to reverse the f u n c t i o n a l and b iochemica l a l t e r a t i o n s which occur i n 6-week STZ d i a b e t i c r a t h e a r t s . A recent study from our l abo ra to r y has shown tha t i n s u l i n t reatment cou ld not on ly reverse the f u n c t i o n a l a l t e r a t i o n s i n 6-week STZ d i a b e t i c r a t hear ts but cou ld a l s o prevent these a l t e r a t i o n s from o c c u r r i n g ( T a h i l i a n i e t a]_. 1983) . In the above two s t u d i e s , the e f f e c t o f i n s u l i n therapy was s tud ied i n d i a b e t i c ra ts a t the time of onset o f c a r d i a c d y s f u n c t i o n . S tud ies i n v o l v i n g i n s u l i n t reatment o f long- term d i a b e t i c animals w i th comple te ly developed c a r d i a c abno rma l i t i e s prov ided c o n f l i c t i n g r e s u l t s . Whi le Baandrup e t a l . (1981) have shown tha t i n s u l i n t reatment cou ld prevent the accumulat ion o f connec t ive t i s s u e and m u l t i p l i c a t i o n o f a r t e r i a l media c e l l s i n hear ts obta ined from 9-month STZ d i a b e t i c r a t s , Regan e_t aj_.' (1981) demonstrated the i n c a p a b i l i t y o f i n s u l i n supplementat ion to prevent c o l l a g e n accumulat ion and c a r d i a c dys func t i on from occu r i ng in 1-year a l l o x a n d i a b e t i c dogs. R e c e n t l y , i t has been demonstrated i n our l a b o r a t o r y tha t 4-week i n s u l i n t reatment d id not comple te ly reverse the c a r d i a c f unc t i ona l abno rma l i t i e s 197 tha t occur i n 5-month STZ d i a b e t i c r a t s ( T a h i l i a n i , 1983) . The e f f e c t i v e n e s s of prolonged i n s u l i n t reatment i n r e v e r s i n g the c a r d i a c dys func t i on o f 5-or 6-month d i a b e t i c r a t s i s ye t to be s t u d i e d . From these s t u d i e s i t i s apparent tha t i n s u l i n t reatment cou ld pro long the onset o f c a r d i a c f u n c t i o n a l abno rma l i t i es i n d i a b e t i c animals and cou ld a l s o reverse these abno rma l i t i e s back to normal , but on ly dur ing the e a r l y phases of development. However, i t sou ld be noted tha t c a r d i a c comp l i ca t i ons are observed in t rea ted as we l l as poor l y c o n t r o l l e d d i a b e t i c p a t i e n t s . RESPONSIVENESS OF CONTROL AND DIABETIC HEARTS TO CARBACHOL: Resu l t s ob ta ined i n the present i n v e s t i g a t i o n show tha t the responses of the d i a b e t i c myocardium to carbachol changes w i th the p rog ress ion of the d isease s t a t e . Hearts i s o l a t e d from shor t - te rm a l l o x a n and STZ d i a b e t i c ra t s (7 days and 30 days a f t e r i nduc t i on o f the d i s e a s e , F igures 23 and 24) e x h i b i t e d a s i m i l a r respons iveness and s e n s i t i v i t y to the negat ive i n o t r o p i c e f f e c t o f carbachol when compared to con t ro l r a t h e a r t s . As the d i sease s ta te p rog ressed , d i a b e t i c r a t hear ts (100 days a f t e r i n d u c t i o n , F igure 25) became s u b s e n s i t i v e to the negat ive i n o t r o p i c e f f e c t o f c a r b a c h o l . However, t h i s s u b s e n s i t i v e phase was t r a n s i e n t as hear ts obta ined from long- term d i a b e t i c r a t s (180^day STZ d i a b e t i c and 240-day a l l o x a n d i a b e t i c r a t s , F igures 26 and 27) were s u p e r s e n s i t i v e to the negat ive i n o t r o p i c e f f e c t o f carbachol . Both con t ro l and d i a b e t i c r a t hear ts appeared to be l e s s s e n s i t i v e to carbachol by 360 days (F igure 29 ) . At t h i s t ime -po in t d i a b e t i c hearts appeared to be s l i g h t l y more s e n s i t i v e than con t ro l h e a r t s . However, both con t ro l and d i a b e t i c hear ts were s l i g h t l y l e s s s e n s i t i v e to carbachol when compared to the r e s p e c t i v e groups a t e a r l i e r t i m e - p o i n t s . Basal p o s i t i v e dP /d t a t 15 cm H,,0 a t r i a l f i l l i n g pressure in 100-day d i a b e t i c r a t hear ts i n the c a r d i a c f u n c t i o n s tudy . Al though we d id not c o n s i s t e n t l y observe 198 s t a t i s t i c a l l y s i g n i f i c a n t depress ions i n p o s i t i v e dP /d t and 15 cm H^O f i l l i n g pressure i n 100-day d i a b e t i c r a t hear ts i n these s t u d i e s , we always observed t rends in tha t d i r e c t i o n . D i f f e rences in sample s i z e employed i n the va r ious exper iments probably account f o r t h i s i n c o n s i s t e n c y . To d a t e , there have been few data repor ted concern ing the e f f e c t s o f c h o l i n e r g i c agon i s t s on d i a b e t i c myocardium. Foy and Lucas (1976) repor ted tha t one-week a l l o x a n - and two-week STZ-induced d iabe tes produced a reduced s e n s i t i v i t y to the depressor e f f e c t o f a c e t y l c h o l i n e in p i thed r a t s . Very r e c e n t l y , Tomlinson and Yusof (1981) showed tha t i s o l a t e d l e f t a t r i a from 7 and 8 month a l l o x a n d i a b e t i c ra t s were supersen t i ve to the negat ive i n o t r o p i c e f f e c t o f a c e t y l c h o l i n e . Resu l t s obta ined i n the present i n v e s t i g a t i o n w i th 7-day and 30-day con t ro l and d i a b e t i c r a t hear ts demonstrated no change in the s e n s i t i v i t y o f d i a b e t i c myocardium towards the negat ive i n o t r o p i c e f f e c t o f c a r b a c h o l . Whi le our r e s u l t s appear to d isagree w i th those of Foy and Lucas (1976) i t should be noted tha t Foy and Lucas d id not d i r e c t l y study the negat ive i n o t r o p i c e f f e c t o f a c e t y l c h o l i n e . Resu l t s obta ined from long- term d i a b e t i c r a t hear ts (180-day STZ and 240-day a l l o x a n d i a b e t i c r a t s ) i n t h i s study agree w i th the f i n d i n g s o f Tomlinson and Yusof (1981). The s u p e r s e n s i t i v i t y e x h i b i t e d by the long- term d i a b e t i c myocardium "might i n d i c a t e the development o f an autonomic neuropathy. D i a b e t i c autonomic neuropathy w i th e a r l y vagal dys func t i on has been demonstrated c l i n i c a l l y i n long- term d i a b e t i c s (L loyd-Mostyn and Watk ins , 1975; Ewings e_t aj_. 1980) . De fec t i ve parasympathet ic i n n e r v a t i o n o f the hear t cou ld lead to a l a c k o f the neuro t ransmi t te r a c e t y l c h o l i n e and 199 to the development o f p o s t j u n c t i o n a l s u p e r s e n s i t i v i t y to exogenously admin is te red c h o l i n e r g i c d rugs . P a r t i a l and o c c a s i o n a l l y t o t a l vagal denerva t ion o f the hear t has been encountered c l i n i c a l l y i n ch ron i c d i a b e t i c p a t i e n t s (Wheeler and Watk ins , 1973; L loyd-Mostyn and Watk ins , 1976). P a t h o l o g i c a l changes such as severe l o s s o f mye l ina ted axons and excess d e p o s i t i o n o f c o l l a g e n have been demonstrated i n the vagus nerve dur ing postmortem examinat ion o f d i a b e t i c p a t i e n t s (Duchen et al_. 1980). S i m i l a r pa tho l og i ca l a l t e r a t i o n s a s s o c i a t e d w i th reduced c h o l i n e a c e t y l t r a n s f e r a s e a c t i v i t y have been repor ted i n c h o l i n e r g i c nerves i n n e r v a t i n g co lons i s o l a t e d from -5 to-.7* month STZ d i a b e t i c ra t s (Schmidt e t aj_. 1981) . Tomlinson and Yusof (1981) demonstrated a neuropathy o f the c a r d i a c vagus nerve i n 7- to 8 month a l l o x a n d i a b e t i c r a t s . These authors showed tha t s t i m u l a t i o n o f c h o l i n e r g i c nerves o f l e f t a t r i a f a i l e d to produce a negat ive i n o t r o p i c e f f e c t and u l t r a -s t r u c t u r a l l y demonstrated the absence o f c h o l i n e r g i c nerve endings i n the r i g h t a t r i a . The decreased s e n s i t i v i t y e x h i b i t e d by d i a b e t i c hear ts to carbachol a t 100 days a f t e r i nduc t i on o f d iabe tes i s d i f f i c u l t to e x p l a i n . During the i n i t i a l phase o f the d isease the d i a b e t i c myocardium e x h i b i t e d no changes in i t s s e n s i t i v i t y to carbachol suggest ing tha t there were no major a l t e r a t i o n s in the autonomic r e g u l a t i o n o f the hea r t . As the d i sease p rog ressed , hear ts f i r s t became t r a n s i e n t l y s u b s e n s i t i v e , before f i n a l l y becoming s u p e r s e n s i t i v e . The s u b s e n s i t i v e phase might represent an e a r l i e r s tage o f a deve lop ing autonomic neuropathy. Whi le changes were o c c u r r i n g i n the f u n c t i o n and s t r u c t u r e o f autonomic nerves owing to the severe metabo l i c derangement o f 200 d i a b e t e s , p h y s i o l o g i c a l compensatory mechanisms might come in to p l a y . An excess turnover o f the n e u r o t r a n s m i t t e r , a c e t y l c h o l i n e , f o r a sho r t du ra t i on r e s u l t i n g in p o s t j u n c t i o n a l recep to r d e s e n s i t i z a t i o n cou ld thus occu r . A r e c e n t l y pub l i shed repo r t by Stuesse e t a l . ( 1982 ) prov ides some evidence fo r the above assumpt ion. These authors showed tha t vagal nerve s t i m u l a t i o n r e s u l t e d in a more pronounced bradycard ia i n 7 to .9 week a l l o x a n r a t s when compared to con t ro l r a t s . A c t i v a t i o n o f a compensatory mechanism i n the vagal e f f e r e n t ne rves , r e s u l t i n g in a g rea te r response to nerve s t i m u l a t i o n i n the d i a b e t i c r a t s , was o f f e red as one o f the e x p l a n a t i o n s . Resu l t s ob ta ined in the present i n v e s t i g a t i o n demonstrate tha t the s e n s i t i v i t y o f the d i a b e t i c myocardium s h i f t s from one extreme to the o ther as the d i sease s t a t e p rog ressed . Such a phenomenon i s not a comple te ly uncommon f i n d i n g i n expe r imen ta l l y - i nduced d i a b e t i c r a t s . I t has been p r e v i o u s l y shown in STZ d i a b e t i c r a t s tha t shor t term d iabe tes produced hypotension but as the du ra t i on o f the d i sease inc reased r a t s became hyper tens ive (Jackson and C a r r i e r , 1981) . Hearts i s o l a t e d from con t ro l and d i a b e t i c ra t s a t 360 days were s l i g h t l y but not s i g n i f i c a n t l y l e s s s e n s i t i v e to carbachol when compared to con t ro l and d i a b e t i c r a t hear ts a t e a r l i e r t ime p o i n t s . This apparent decrease in s e n s i t i v i t y e x h i b i t e d by both con t ro l and d i a b e t i c r a t hear ts a t t h i s time po in t might be due to a g i n g . The e f f e c t o f ag ing on the respons iveness o f the hear t to c h o l i n e r g i c agon is t s has not been we l l documented a l though s tud ies on r a b b i t a t r i a show tha t the s e n s i t i v i t y to c h o l i n e r g i c agon is t s does decrease wi th age (Goldberg and Rober t s , 1980). 201 Di f fe rences in m o r t a l i t y d id not seem to a f f e c t the r e s u l t s . The g rea tes t m o r t a l i t y was seen in the 240-day a l l o x a n t r ea ted group (33%) but the same response to carbacho l was obta ined in the 180-day STZ t rea ted group i n which the m o r t a l i t y was z e r o . At 360 days the m o r t a l i t y i n both the STZ and con t ro l ra ts was e x a c t l y the same (25%). Therefore the r e s u l t s ob ta ined i n the present study are not due to the f a c t tha t on ly the s t ronger or l e s s d i a b e t i c animals su rv i ved f o r longer p e r i o d s . In c o n c l u s i o n , r e s u l t s ob ta ined wi th carbachol i n the present study demonstrate changes in the s e n s i t i v i t y o f the d i a b e t i c myocardium to c h o l i n e r g i c agents which may i n d i c a t e an autonomic neuropathy o f the hear t a s s o c i a t e d wi th vagal d y s f u n c t i o n . EFFECT OF ISOPROTERENOL ON CONTROL AND DIABETIC RAT HEARTS: Resu l ts ob ta ined demonstrate tha t n e i t h e r the s e n s i t i v i t y nor the respons iveness o f the d i a b e t i c myocardium to the p o s i t i v e i n o t r o p i c e f f e c t (changes produced in p o s i t i v e dP /d t ) o f i s o p r o t e r e n o l was a l t e r e d at any stage o f d iabe tes s tud ied (F igures 29 to 34 and Table V ) . However, the c a r d i a c r e l a x a n t e f f e c t (changes produced i n negat ive dP /d t ) o f i sop ro te reno l was a l t e r e d i n d i a b e t i c r a t h e a r t s . Maximum changes produced i n negat ive dP /d t by i sop ro te reno l were s i g n i f i c a n t l y depressed in 7->, 30- and 100-day a l l o x a n and 100- and 180rday S T Z - d i a b e t i c r a t hear ts (F igures 35 to 38). V e n t r i c u l a r noradrena l ine content d id not change in 180-day a l l o x a n - and S T Z - d i a b e t i c ra t s when compared to age-matched c o n t r o l s <.(Table X ) . . Prev ious repor ts concern ing the e f f e c t s o f B-adrenerg ic agon i s t s on the hear t i n expe r imen ta l l y - i nduced d iabe tes are c o n f l i c t i n g . Foy 202 and Lucas (1978) have demonstrated tha t the i n o t r o p i c responses to no radrena l ine and i sop ro te reno l were reduced wh i le the ch rono t rop i c responses to these agon i s t s inc reased i n i s o l a t e d spontaneously beat ing a t r i a from ra t s made d i a b e t i c w i th STZ two weeks p r i o r to use . However, Ingebretsen ejb (1980) repor ted no change in the i n o t r o p i c response of i s o l a t e d per fused working hear ts from acute a l l o x a n - d i a b e t i c ra t s to i s o p r o t e r e n o l . Furthermore, a very recent repor t by Tomlinson and Yusof (1981) showed tha t there were no d i f f e r e n c e s in the response to no radrena l ine o f i s o l a t e d a t r i a from 7 to 8 month a l l o x a n - d i a b e t i c and con t ro l r a t s . Resu l ts obta ined in the present i n v e s t i g a t i o n showed that the p o s i t i v e i n o t r o p i c e f f e c t o f i sop ro te reno l was not a l t e r e d i n i s o l a t e d working hear ts from a l l o x a n - and S T Z - d i a b e t i c r a t s at any time po in t a f t e r i n d u c t i o n o f the d i s e a s e . Our r e s u l t s w i th acute d i a b e t i c r a t hear ts appear to d isagree wi th those o f Foy and Lucas (1978) . The disagreement cou ld be due to c e r t a i n d i f f e r e n c e s between these two s t u d i e s . Foy and Lucas (1978) used i s o l a t e d spontaneously beat ing a t r i a from two week d i a b e t i c r a t s , whereas i s o l a t e d per fused working hear ts from 7- and 30-day d i a b e t i c r a t s were used in the present s tudy . Another d i f f e r e n c e between these two s tud ies i s i n the g lucose concen t ra t i on o f the per fusa te employed. Foy and Lucas (1978) used 5.6 mM glucose whereas 10 mM glucose has been used in t h i s s tudy . However, Ingebretsen e t al_. (1980) have demonstrated tha t i sop ro te reno l produced normal p o s i t i v e i n o t r o p i c responses in i s o l a t e d per fused working d i a b e t i c r a t hear ts per fused wi th 5.5 mM g l u c o s e . Resu l ts ob ta ined i n t h i s study w i th 6- and 8-month d i a b e t i c r a t hear ts agree w i th those repor ted by Tomlinson and Yusof (1981) us ing i s o l a t e d 203 a t r i a from 7 to 8 month a l l o x a n - d i a b e t i c r a t s . These authors d id not f i n d any changes i n the responses o f d i a b e t i c r a t a t r i a to n o r a d r e n a l i n e . Fur thermore, a recent study from our l a b o r a t o r y has demonstrated tha t l e f t a t r i a ob ta ined from 7-day and p a p i l l a r y muscles from 7- and 70-day d i a b e t i c r a t hear ts e x h i b i t e d no changes i n the maximum i n o t r o p i c responses to i sop ro te reno l when compared to c o n t r o l s (McCullough and M c N e i l l , 1983). Apart from producing a p o s i t i v e i n o t r o p i c e f f e c t , catecholamines are known to exer t a profound r e l a x i n g e f f e c t on the hear t (V i t t one e_t al_. 1981) . In the present s tudy , changes produced by i sop ro te reno l i n negat ive dP /d t were measured i n con t ro l and d i a b e t i c r a t hear ts and were used as "an index of the c a r d i a c r e l a x a n t e f f e c t o f i s o p r o t e r e n o l . Maximum changes produced i n negat ive dP /d t by i s o p r o t e r e n o l were depressed in d i a b e t i c r a t hear ts at va r ious t ime po in ts a f t e r the i nduc t i on o f the d isease (Table V I ) . R e c e n t l y , Fein e_t al_. (1980) have s tud ied the e f f e c t . o f no rad rena l ine on t ime to 1/2 r e l a x a t i o n (t ime requ i red to reach 50% o f t o t a l r e l a x a t i o n ) o f p a p i l l a r y muscles i s o l a t e d from 10-week d i a b e t i c and con t ro l r a t s . These authors repor ted tha t the t ime to 1/2 r e l a x a t i o n i n d i a b e t i c p a p i l l a r y muscles was longer than c o n t r o l s a t a l l the no rad rena l ine concen t ra t i ons t e s t e d . However, i n tha t study noradrena l ine seems to produce s i m i l a r changes i n time to 1/2 r e l a x a t i o n in con t ro l and d i a b e t i c p a p i l l a r y muscles as i n d i c a t e d by the p a r a l l e l upward s h i f t o f the no rad rena l ine dose-response curve in d i a b e t i c p r e p a r a t i o n s . In the present study d i a b e t i c r a t hear ts not on ly had a depressed basal negat ive dP /d t but a l s o e x h i b i t e d depressed responses to i s o p r o t e r e n o l . Calc ium b ind ing t o , and ca lc ium uptake by 204 the sa rcop lasmic r e t i c u l u m have been proposed to be one o f the main events tha t i n i t i a t e r e l a x a t i o n in the hear t ( K a t z , 1980) . A l t e r a t i o n s in ca lc ium b ind ing and ca lc ium uptake by the sa rcop lasmic r e t i cu l um cou ld a f f e c t c a r d i a c r e l a x a t i o n . I t has been repor ted p r e v i o u s l y tha t the c a r d i a c sa rcop lasmic r e t i cu l um from ch ron i c d i a b e t i c ra t s e x h i b i t e d a reduced a b i l i t y to accumulate and t r anspo r t ca l c ium (Penpargkul et a l . 1981; Lopaschuk e t aJL 1983). These abno rma l i t i e s in the sa rcop lasmic r e t i cu l um might be r e s p o n s i b l e f o r the depress ion observed on the e f f e c t o f i s o p r o t e r e n o l on negat ive d P / d t . However, decreases in the c a r d i a c r e l a x a n t e f f e c t o f -.isoproterenol were a l s o observed i n 7-day d i a b e t i c r a t h e a r t s ; a time po in t a t which the sa rcop lasmic r e t i c u l a r f u n c t i o n has been shown to be una l te red (Lopaschuk e t aj_. 1983) . Therefore abno rma l i t i e s in c a r d i a c sa rcop lasmic r e t i c u l a r f u n c t i o n may not s o l e l y be r e s p o n s i b l e f o r the observed depress ions in the e f f e c t o f i sop ro te reno l i n d i a b e t i c r a t h e a r t s . Regan ejb al_. (1 974) repor ted tha t inc reased v e n t r i c u l a r s t i f f n e s s and decreased v e n t r i c u l a r wa l l compl iance to be the major de fec ts i n ca rd i ac r e l a x a t i o n i n ch ron i c d i a b e t i c dogs. V e n t r i c u l a r wa l l s t i f f e n i n g cou ld a l s o r e s u l t i n the observed depress ions i n the e f f e c t o f i s o p r o t e r e n o l , but on ly i n ch ron i c d i a b e t i c r a t h e a r t s . A p o s s i b i l i t y remains t ha t the c y c l i c AMP cascade system might be depressed i n e x p e r i m e n t a l - d i a b e t e s , l ead ing to these a l t e r a t i o n s . Ingebretsen e_t aj_. (1981) have repor ted a f i f t y percent reduc t ion i n the i s o p r o t e r e n o l - i n d u c e d changes in the c a r d i a c c y c l i c AMP system i n acute d i a b e t i c r a t s . However, these authors a l s o repor ted tha t there were no changes i n the p o s i t i v e i n o t r o p i c e f f e c t o f i sop ro te reno l i n d i a b e t i c r a t h e a r t s . Fur thermore, i n the present s tudy , i s o p r o t e r e n o l 205 produced s i m i l a r t ime-dependent changes i n c a r d i a c c y c l i c AMP l e v e l s i n acute as we l l as ch ron i c d i a b e t i c r a t s , when compared to c o n t r o l s (F igures 41 and 4 2 ) . g -adrenerg ic recep to r dens i t y has been repor ted to be reduced by 30 percent i n d i a b e t i c r a t hear ts (Savarese and Be rkow i t z , 1979; Ingebretsen ejb al_. 1982) . However, a 30 percent reduc t i on i n B-adrenerg ic recep to r dens i t y might not r e s u l t i n the observed depress ion o f the e f f e c t o f i sop ro te reno l because: (1) Reduc-t i ons i n the e f f e c t o f i s o p r o t e r e n o l were on ly observed i n negat ive d P / d t , but not i n p o s i t i v e d P / d t . I f a lower number o f recep to rs were to account f o r a reduc t i on i n the maximum e f f e c t to i s o p r o t e r e n o l , then these reduc t ions should be seen both in p o s i t i v e and negat ive d P / d t . (2) I t has been shown p r e v i o u s l y tha t spare B-adrenerg ic recep to rs e x i s t in the hear t and tha t a s u b s t a n t i a l r educ t i on in the B-adrenerg ic recep to r d e n s i t y i s requ i red in order to cause a s h i f t i n the s e n s i t i v i t y to the a g o n i s t , w i thout a reduc t i on i n the maximum responses (Ven te r , 1979; S i e g l and M c N e i l l , 1982) . The re fo re , the observed reduc t ions i n the maximum negat ive dP /d t responses to i sop ro te reno l i n d i a b e t i c r a t hearts are probably not due to e i t h e r a reduc t i on in B- recep to r d e n s i t y or to changes in the c y c l i c AMP system. The depressed maximum e f f e c t o f i sop ro te reno l on negat ive dP /d t i n acute d i a b e t i c r a t hear ts might r e s u l t from the metabo l i c derangements o f acute d iabe tes l e a d i n g to a l t e r e d energy u t i l i z a t i o n (Feuvray e t a]_. 1 979) and decreased ATP produc t ion ( A l l i s o n e t al_. 1 976; M i l l e r , 1979) . Changes produced by i sop ro te reno l i n negat ive dP /d t i n 240- arid 360-day d i a b e t i c r a t hear ts were s l i g h t l y , but not s i g n i f i c a n t l y , depressed from c o n t r o l s . I t should be noted tha t the maximum changes 206 produced i n negat ive dP /d t i n con t ro l hear ts a t these t ime po in ts were lower compared to those a t e a r l i e r t ime p o i n t s . An age r e l a t e d decrease in the respons iveness o f the hear t (Lakat ta e t a K 1975) and blood vesse l s ( F l e i s h , 1981) to ad renerg ic agon is t s has been p r e v i o u s l y r e p o r t e d . S ince the maximum changes produced by i s o p r o t e r e n o l i n the negat ive dP /d t o f con t ro l r a t hear ts decreased w i th age, d i abe tes - i nduced reduc t ions in the e f f e c t o f i sop ro te reno l were not r e a d i l y apparent at the l a t e r t ime p o i n t s . Sympathet ic autonomic neuropathy o f the heart occurs in long term d i a b e t i c s , r e s u l t i n g in d i s tu rbances o f c a r d i o v a s c u l a r r e f l e x e s (Hosking et al_. 1978; C la rke et -a l_ . 1979). One o f the common fea tu res o f t h i s neuropathy i s degenerat ion o f the p o s t - g a n g l i o n i c nerve te rm ina ls l ead ing to d e p l e t i o n o f neu ro t ransmi t te r thus c r e a t i n g a s i t u a t i o n s i m i l a r to denerva t ion ( G i a c h e t t i , 1981) . Noradrena l ine content o f the hear t and a r t e r i a l system has been repor ted to be markedly decreased i n long- term d i a b e t i c s (Neubauer and C h r i s t e n s e n , 1976) . Evidence f o r degenerat ion o f sympathet ic nerves has been presented showing tha t d i a b e t i c p a t i e n t s were s u p e r s e n s i t i v e to exogenous norep inephr ine (Moorehouse e t aj_. 1966) . In the present study we determined v e n t r i c u l a r no radrena l ine content i n 180-day d i a b e t i c and con t ro l r a t s and found tha t i t was una l te red i n d i a b e t i c r a t hear ts (Table X ) . Our r e s u l t s , supported by p r e v i o u s l y pub l i shed r e p o r t s , show no changes i n the noradrena l ine content o f hear ts i s o l a t e d from d i a b e t i c r a t s (Head and Be rkow i t z , 1979; Kaul and Grewa l , 1980). Fur thermore, s e n s i t i v i t y o f the d i a b e t i c myocardium towards i s o p r o t e r e n o l was a l s o not a l t e r e d i n the present s tudy . These f i n d i n g s do not support the p o s s i b i l i t y o f the 207 development o f a sympathet ic autonomic neuropathy in the hear t under the c o n d i t i o n s used i n the present s tudy . In c o n c l u s i o n , expe r imen ta l l y - i nduced d iabetes d id not a l t e r e i t h e r the respons iveness or the s e n s i t i v i t y o f the myocardium towards the p o s i t i v e i n o t r o p i c e f f e c t o f i s o p r o t e r e n o l . However, maximum changes produced by i sop ro te reno l i n negat ive dP /d t o f i s o l a t e d working hear ts 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 depressed at va r ious t ime po in ts a f t e r i n d u c t i o n o f the d i s e a s e . V e n t r i c u l a r no rad rena l ine content i n 180-day d i a b e t i c r a t s was u n a l t e r e d , i n d i c a t i n g tha t there were no pa tho l og i ca l changes i n sympathet ic nerve te rm ina ls i n the hea r t . These r e s u l t s i n d i c a t e the absence of a sympathet ic d i a b e t i c neuropathy o f the hea r t . Acute metabo l i c derangements caus ing changes in energy product ion and u t i l i z a t i o n and abno rma l i t i es i n c a r d i a c sa rcop lasmic r e t i c u l a r f u n c t i o n may represen t the major under l y ing causes f o r the depressed c a r d i a c r e l a x a n t e f f e c t o f i sop ro te reno l i n d i a b e t i c r a t h e a r t s . CARDIAC CYCLIC AMP AND PHOSPHORYLASE SYSTEM IN EXPERIMENTAL DIABETES AND THE EFFECT OF ISOPROTERENOL AND PGE-j : Resu l t s ob ta ined in the present i n v e s t i g a t i o n demonstrate tha t i sop ro te reno l ( 5 X 1 0 _ 9 M ) causes a g rea te r a c t i v a t i o n o f the phosphory lase enzyme in i s o l a t e d per fused working hear ts from acute as we l l as ch ron i c d i a b e t i c ra t s when compared to age-matched c o n t r o l s (F igures 43 and 4 4 ) . Both acute and ch ron i c d i a b e t i c r a t hear ts e x h i b i t e d s l i g h t l y but not s i g n i f i c a n t l y h igher phosphory lase a_ a c t i v i t y when compared to con t ro l r a t h e a r t s , wh i l e t o t a l phosphory lase a c t i v i t y was s i g n i f i c a n t l y h igher 208 on ly i n ch ron i c d i a b e t i c r a t hear ts (F igure 4 5 ) . However, maximal changes produced by i sop ro te reno l i n c a r d i a c c y c l i c AMP con ten t , p o s i t i v e and negat i ve dP /d t development i n con t ro l and d i a b e t i c r a t s were a l l o f s i m i l a r magnitude (F igures 4 1 , 42 , 46 , to 4 9 ) . F u r t h e r , phosphory lase a^  was a c t i v a t e d by PGEi in both acute and ch ron i c d i a b e t i c r a t hear ts but not i n age-matched con t ro l r a t h e a r t s . I sopro te reno l produced i d e n t i c a l dose-dependent changes i n c a r d i a c c y c l i c AMP content and ino t ropy i n 100-120 day d i a b e t i c r a t hear ts compared to c o n t r o l s (F igures 50, 52 and 53 ) . However, i sop ro te reno l produced a s i g n i f i c a n t l y h igher a c t i v a t i o n o f phosphory lase i n 100 to 120-day d i a b e t i c r a t hear ts as compared to con t ro l (F igure 5 1 ) . Basal c y c l i c AMP l e v e l s were not a l t e r e d e i t h e r i n 3-day or i n 100 to 120-day d i a b e t i c r a t h e a r t s . Th is obse rva t i on does not agree wi th the f i n d i n g s o f Chaudhury and Shipp (1973) tha t basal c y c l i c AMP l e v e l s were f o u r - f o l d h igher i n 48-hour a l l o x a n d i a b e t i c r a t hear ts compared to c o n t r o l s . The d isc repancy between these two s tud ies can be to some extent a t t r i b u t e d to d i f f e r e n c e s i n the methodology employed. Chaudhury and Shipp (1973) s tud ied c y c l i c AMP content i n hear ts which were f r e e z e -clamped immediately a f t e r s a c r i f i c e , whereas in t h i s study c y c l i c AMP l e v e l s were s tud ied i n i s o l a t e d per fused working hear ts t ha t were f rozen f o l l o w i n g a 15 minute p e r f u s i o n . Recent ly seve ra l repor ts have appeared in the l i t e r a t u r e showing no changes in the basal c y c l i c AMP content o f i s o l a t e d per fused h e a r t s ' ( I n g e b r e t s e n e t aj_. 1981; M i l l e r et al_. 1981) , l i v e r (Yamashita e t a_l_. 1980) and kidney (Hoskins and Luong, 1981) , from acute a l l o x a n and STZ d i a b e t i c r a t s . Furthermore basal adeny la te c y c l a s e a c t i v i t y o f the hear ts from acute a l l o x a n (Menahan e t aj_. 1 977) 209 and STZ d i a b e t i c r a t s (Das, 1973b) has been shown to be u n a l t e r e d . These repor ts a l l suppor t our f i n d i n g tha t basal c y c l i c AMP l e v e l s are not a l t e r e d in acute d i a b e t i c r a t h e a r t s . The c y c l i c AMP system i n ch ron i c d i a b e t i c r a t hear ts had not been s tud ied p r e v i o u s l y . We observed tha t the basal c y c l i c AMP content was una l te red i n i s o l a t e d per fused hear ts from 100 to 120 day a l l o x a n and STZ d i a b e t i c r a t s . Isopro tereno l produced s i m i l a r changes i n the c y c l i c AMP content i n i s o l a t e d per fused hear ts from acute as we l l as ch ron i c d i a b e t i c and con t ro l r a t s (F igures 41 and 42 ) . . E x i s t i n g l i t e r a t u r e on the e f f e c t o f i sop ro te reno l on c y c l i c AMP content i n acute d i a b e t i c hear ts i s not i n agreement. Ingebretsen e t al_. (1981) have shown tha t there was a f i f t y percent reduc t i on in the a c t i v a t i o n o f c y c l i c AMP and c y c l i c AMP-dependent p r o t e i n k inase system by i sop ro te reno l i n acute d i a b e t i c r a t hear ts compared to c o n t r o l s ; whereas M i l l e r e t al_. (1981) repor ted no such reduc t i on in the ep inephr ine - induced a c t i v a t i o n o f c y c l i c AMP i n acute a l l o x a n d i a b e t i c r a t h e a r t s . Card iac 3 -adrenerg ic recep to r dens i t y has been shown to be decreased in d i a b e t i c r a t s (Savarese and Be rkow i t z , 1977; Ingebretsen e_t a K 1982). However, Ingebretsen .et . a l . (1981) have repor ted no changes i n maximum p o s i t i v e i n o t r o p i c e f f e c t o f i sop ro te reno l i n i s o l a t e d per fused hear ts from acute d i a b e t i c r a t s . In the present study we d id not observe any s i g n i f i c a n t changes in the i s o p r o t e r e n o l - i n d u c e d maximum inc reases i n p o s i t i v e and negat ive dP /d t development (F igures 46 , 47 , .48 and 49) i n hearts from acute and ch ron i c d i a b e t i c ra t s and these changes were assoc i a t ed w i th normal c a r d i a c c y c l i c AMP responses to i s o p r o t e r e n o l . S ince the maximum i n o t r o p i c responses to i sop ro te reno l were not depressed i n acute as we l l as ch ron i c d i a b e t i c r a t h e a r t s , i t i s reasonable to assume tha t va r ious 210 events occu r r i ng between the recep to r and the i n o t r o p i c e f f e c t are normal . I t seems h i g h l y u n l i k e l y to have, i n d i a b e t i c r a t h e a r t s , a decrease i n the t o t a l number o f B-adrenerg ic recep to rs and a reduc t i on in the B-adrenerg ic a g o n i s t - s t i m u l a t e d a c t i v a t i o n o f the - c y c l i c AMP system and ye t have a normal maximum p o s i t i v e i n o t r o p i c e f f e c t . Fur ther suppor t ing our data i s the repor t ( Ingebretsen ejt aj_. 1982) showing tha t the basal myocard ia l adeny la te c y c l a s e a c t i v i t y and i t s responses to i sop ro te reno l were una l te red i n acute a l l o x a n d i a b e t i c r a t s . Very few s tud ies have been pub l i shed in which the glycogen phos-phory lase enzyme i n hear ts i s o l a t e d from d i a b e t i c ra t s has been s t u d i e d . Das (1973a) repor ted tha t there were no changes in the c a r d i a c basal phosphory lase a^  and t o t a l phosphory lase a c t i v i t i e s i n STZ d i a b e t i c r a t s . M i l l e r ejt aj_. (1981) showed tha t acute a l l o x a n d iabe tes d id not a l t e r basal c a r d i a c phosphory lase a c t i v i t y but r e s u l t e d i n a s i g n i f i c a n t l y g rea te r a c t i v a t i o n o f phosphory lase by ep ineph r i ne . In the present study we observed tha t i sop ro te reno l caused a 2 to 3 - f o l d h igher a c t i v a t i o n o f phosphory lase a_ enzyme i n acute and ch ron i c d i a b e t i c r a t hear ts , , as compared to age-matched con t ro l r a t hear ts (F igures 43 and 44 ) . Our obse rva t i ons on c a r d i a c phosphory lase enzyme l e v e l s from acute d i a b e t i c r a t s thus agree w i th those repor ted by M i l l e r et_ al_. (1981) and we have now extended these obse rva t i ons to ch ron i c d i a b e t i c r a t hear ts as w e l l . Tota l phosphory lase a c t i v i t y was s i g n i -f i c a n t l y g rea te r i n 100 to 120 day d i a b e t i c ra t hear ts as compared to age-matched con t ro l hear ts (Figure, . 4 5 ) , whereas basal phosphory lase a^  a c t i v i t y was s l i g h t l y but not 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 r a t h e a r t s . The g rea te r a c t i v a t i o n o f phosphorylase a_ enzyme by i sop ro te reno l 211 cou ld p a r t l y r e s u l t from the i nc rease i n t o t a l phosphory lase a c t i v i t y . However, t h i s i s not the case w i th acute d i a b e t i c hear ts as t o t a l phosphory lase a c t i v i t y i n these hear ts was not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . Phosphory lase can be a c t i v i a t e d by ca l c ium (F r i esen 2+ et al_. 1969) as we l l as by c y c l i c AMP. Ca uptake by sa rcop lasmic r e t i cu l um has been shown to be depressed i n ch ron i c d i a b e t i c r a t hearts (Penpargkul e t al_. 1981; Lopaschuk e_t a]_. 1983) . Th is may 2+ lead to a s i t u a t i o n where a b u i l d up o f cy top lasmic f ree Ca concen-t r a t i o n over normal concen t ra t i ons cou ld occur i n the myocardium, p a r t i c u l a r l y - w h e n the hear t was s t imu la ted by a 3 -adrenerg ic a g o n i s t . 2+ The inc rease i n cy top lasmic f ree Ca cou ld then cause an a c t i v a t i o n o f phosphory lase which would be most apparent f o l l o w i n g i sop ro te reno l induced i nc reases i n i n t r a c e l l u l a r c a l c i u m . A l t e r a t i o n s in c y c l i c AMP-mediated ca lc ium f l u x i n the d i a b e t i c myocardium as suggested by M i l l e r e t al_. (1981) are ye t another p o s s i b i l i t y c o n t r i b u t i n g to the phosphory lase response in ch ron i c d i a b e t i c r a t h e a r t s . The nature o f the inc reased s e n s i t i v i t y o f phosphory lase to i sop ro te reno l i n acute d i a b e t i c r a t hear ts i s more d i f f i c u l t to e x p l a i n . 2+ Ca t r anspo r t ac ross t h e . s a r c o p l a s m i c r e t i cu l um has been shown to be una l te red i n acute d i a b e t i c r a t hear ts (Lopaschuk ejt al_. 1983) . Hence 2+ the c o n t r i b u t o r y e f f e c t o f an e leva ted cy top lasmic Ca concen t ra t i on may not o c c u r . M i l l e r e t al_. (1981) suggested tha t a d i a b e t e s - r e l a t e d a - e f f e c t , which was not present i n the normal h e a r t , may p lay a c o n t r i b u t o r y e f f e c t but s i nce i sop ro te reno l i s e s s e n t i a l l y a pure B-agon is t t h i s p o s s i b i l i t y i s a l s o u n l i k e l y . Another p o s s i b i l i t y cou ld be tha t changes occur i n the phosphory lase enzyme caused by acute metabo l i c derangement produced by the i n d u c t i o n o f a d i a b e t i c 212 s t a t e . The inc reased s e n s i t i v i t y o f phosphorylase enzyme to i sop ro te reno l may thus be r e l a t e d to the s e v e r i t y o f the acute d i a b e t i c phase. F igure 43 shows tha t i s o p r o t e r e n o l - a c t i v a t e d phosphory lase was s i g n i f i c a n t l y g rea te r i n hear ts ob ta ined from acute a l l o x a n d i a b e t i c ra t s a t 10, 20 and 40 seconds a f t e r a d m i n i s t r a t i o n as compared to con t ro l r a t h e a r t s . In hear ts obta ined from acute STZ d i a b e t i c ra t s s i g n i f i c a n t l y g rea te r a c t i v a t i o n was seen on l y a t 20 seconds. This d i f f e r e n c e between a l l o x a n and STZ t rea ted r a t s may be due to the s e v e r i t y o f the induced d i a b e t i c s t a t e . In t h i s study a l l o x a n r e s u l t e d in a more severe acute d i a b e t i c s t a t e as i n d i c a t e d by the f a s t i n g serum glucose l e v e l s (Table I I ) . S ince s i m i l a r i nc reases i n c y c l i c AMP, i n acute as we l l as ch ron i c d i a b e t i c r a t h e a r t s , r e s u l t e d in a s i g n i f i c a n t l y g rea te r a c t i v a t i o n o f phosphory lase compared to tha t i n con t ro l r a t h e a r t s ; the p o s s i b i l i t y remains tha t the s e n s i t i v i t y o f the phosphory lase enzyme to. c y c l i c AMP-mediated a c t i v a t i o n cou ld have been inc reased due to d i a b e t e s , as has been suggested p r e v i o u s l y ( Ingebretsen e_t al_. 1981) . Fur thermore, the s e n s i t i v i t y o f phosphory lase k inase enzyme from acute d i a b e t i c r a t hear ts to ep inephr ine - induced a c t i v a t i o n has been shown to be inc reased when compared to tha t i n c o n t r o l s ( M i l l e r e t al_. 1981) . PGE-j e leva ted c y c l i c AMP content i n con t ro l and d i a b e t i c r a t hear ts to a s i m i l a r e x t e n t , but a c t i v a t e d phosphory lase a_ i n a d i a b e t i c r a t hear ts on ly (Table V I I ) . I t has been shown p r e v i o u s l y tha t PGE-j can e leva te c y c l i c AMP content w i thout a l t e r i n g phosphory lase a^  a c t i v i t y i n per fused ra t hear ts (Kee l y , 1 977; Vadlamudi and McNeil 1, 1981) . Hypo-t h e t i c a l compar tmenta l i za t ion o f the c a r d i a c c e l l w i th respec t to c y c l i c n u c l e o t i d e a c t i o n has been proposed to be r espons ib l e f o r such a s p e c i f i c e f f e c t o f PGE-j (Hayes and Brun ton , 1982) . The e x p e r i m e n t a l l y -induced d i a b e t i c s t a t e seems to have d i s rup ted these hypo the t i ca l compartments in the myocardium r e s u l t i n g in the a c t i v a t i o n o f phosphory lase 213 by PGE-j. Such an e f f e c t might a l s o be r espons ib l e f o r the enhanced a c t i v a t i o n o f phosphory lase by i s o p r o t e r e n o l i n the d i a b e t i c myocardium. The obse rva t i on tha t indomethacin d i d not b lock the enhanced a c t i v a t i o n o f phosphory lase by i sop ro te reno l i n d i a b e t i c r a t hear ts r u l es out the p o s s i b l e involvement o f p r o s t a g l a n d i n - 1 i k e subs tances , r e l eased by 3 -adrenerg ic recep to r s t i m u l a t i o n , i n a c t i v a t i n g phosphory lase . A very recent repo r t by Sha f f e r and Ma l i k (1982) has shown tha t $1-adrenerg ic recep to r s t i m u l a t i o n indeed caused a r e l e a s e o f p r o s t a -g land ins in i s o l a t e d per fused r a b b i t hear ts and a l s o tha t these p ros tag land ins d id not i n f l u e n c e the mechanical e f f e c t s produced by the adrenerg ic s t i m u l i . Indomethacin, i n f a c t , appeared to enhance the e f f e c t o f i s o p r o t e r e n o l a c t i v a t i n g phosphory lase i n d i a b e t i c but not i n con t ro l h e a r t s . There i s no known i n t e r a c t i o n between the two drugs and the enhancement i s thus d i f f i c u l t to e x p l a i n . Thyro id f u n c t i o n has been shown to be depressed i n e x p e r i m e n t a l l y -induced d iabe tes (Pi t tman e t aj_. 1981) . As shown in Table 2 , Tg l e v e l s were depressed i n acute as we l l as ch ron i c d i a b e t i c ra t s but T^ l e v e l s were depressed on ly in ch ron i c d i a b e t i c r a t s . These r e s u l t s agree wi th p r e v i o u s l y pub l i shed r e s u l t s (Fe in et al_. 1 981; Di 11 man 1982). A l t e r e d t h y r o i d f u n c t i o n has been shown to cause va r ious changes in the respon-s iveness o f the myocardium towards the e f f e c t s o f catecholamines (Kunos, 1981) . Myocard ia l adeny la te c y c l a s e a c t i v i t y , and i t s respon-s iveness to a g o n i s t s , was shown to be depressed in hypothyro id animals (Levey e_t al_. 1 969; Brodde et_ al_. 1979). Me tabo l i c e f f e c t s o f ca techo-lamines have been repor ted to be depressed in hypothyro id r a t hear ts ( F a i n , 1981) . The changes due to hypothyro id ism seem to be i n the 214 oppos i te d i r e c t i o n to those observed i n t h i s s tudy . Hence, the changes observed in the c a r d i a c glycogen phosphory lase system in expe r imen ta l l y - i nduced d iabetes are almost c e r t a i n l y not the r e s u l t o f the hypothyro id s t a t e produced by d i a b e t e s . In c o n c l u s i o n , expe r imen ta l l y - i nduced d iabetes d id not a l t e r e i t h e r the basal or the i s o p r o t e r e n o l - s t i m u l a t e d c y c l i c AMP l e v e l s i n the hea r t . However, the respons iveness of the c a r d i a c phosphory lase enzyme towards i sop ro te reno l was inc reased in d i a b e t e s . This e f f e c t was observed as e a r l y as 3 days a f t e r i nduc t i on o f d iabe tes and p e r s i s t e d i n 100- to .120-day d i a b e t i c r a t s . Fur thermore, the c a r d i a c phosphory lase enzyme in d i a b e t i c ra t s was a c t i v a t e d by PGE] , a compound which does not a c t i v a t e phosphory lase i n normal r a t h e a r t s . The nature and p h y s i o l o g i c a l s i g n i f i c a n c e of t h i s i nc reased respons iveness o f phosphory lase enzyme i n d i a b e t i c r a t hear ts i s ye t to be determined. 2+ Severe metabo l i c derangements and a l t e r a t i o n s i n Ca homeostasis produced by d iabetes may be the under l y ing causes f o r t h i s e f f e c t . MUSCARINIC RECEPTORS IN DIABETIC RAT HEARTS: Resu l t s from muscar in i c recep to r b ind ing s t u d i e s demonstrated no changes e i t h e r i n the number or the a f f i n i t y o f musca r i n i c recep to rs i n the hear ts obta ined from 180-day STZ d i a b e t i c ra t s compared to c o n t r o l s . These r e s u l t s suggest tha t the s u p e r s e n s i t i v i t y e x h i b i t e d by the 180-day d i a b e t i c myocardium towards the negat ive i n o t r o p i c e f f e c t o f carbachol i s not a r e s u l t o f the increased.number o f r e c e p t o r s . The s u p e r s e n s i t i v i t y o f the d i a b e t i c myocardium cou ld r e s u l t from changes in p o s t - r e c e p t o r even ts . U n f o r t u n a t e l y , the consequences o f 2 1 5 muscar in i c recep to r s t i m u l a t i o n i n the hear t are not we l l unders tood. Therefore i t i s very d i f f i c u l t to make any assumptions on the nature o f the changes occu r i ng in pos t -musca r i n i c r e c e p t o r / e v e n t s . R e c e n t l y , L a t i f p o u r et al_. (1983) have demonstrated a 30% decrease in the number o f musca r in i c receptors i n v e n t r i c l e s ob ta ined from 180-day d i a b e t i c r a t s . These r e s u l t s d isagree wi th the f i n d i n g s of t h i s s tudy iand a l s o make i t d i f f i c u l t to e x p l a i n the s u p e r s e n s i t i v i t y e x h i b i t e d by d i a b e t i c r a t hear ts towards c a r b a c h o l . However, a number o f methodolog ica l d i f f e r e n c e s e x i s t between the two b ind ing s t u d i e s . We used pooled v e n t r i c u l a r p repara t ions obta ined from d i a b e t i c and con t ro l hear ts tha t were per fused on working hear t appara tus , f rozen and s to red a t -70°C f o r over 8 months, f o r the b ind ing s tud ies and L a t i f p o u r e_t aj_.. (1983) • used f r e s h , non-per fused , i n d i v i d u a l v e n t r i c l e s i n t h e i r b ind ing s t u d i e s . The r a d i o l i g a n d s , i ncuba t i on c o n d i t i o n s , and methods used to separa te bound r a d i o a c t i v i t y from f ree were a l l d i f f e r e n t i n these two s t u d i e s . These methodolog ica l d i f f e r e n c e s cou ld p a r t l y e x p l a i n the d i f f e r e n c e s in r e s u l t s between these two s t u d i e s . In any c a s e , both s t u d i e s agree tha t musca r i n i c recep to r number and /or a f f i n i t y are not i nc reased i n v e n t r i c l e s from 6-month d i a b e t i c r a t s . CONCLUSIONS: Expe r imen ta l l y - i nduced d iabe tes in the r a t produced var ious t ime dependent changes in c a r d i a c f u n c t i o n , pharmacolog ica l responses and b iochemical even ts . Card iac f u n c t i o n was not a l t e r e d in 7-day a l l o x a n and STZ d i a b e t i c 216 ra t s compared to con t ro l r a t s . Card iac f u n c t i o n was depressed in 3 0 - , 100- and 240-day a l l o x a n d i a b e t i c and 100 - , 180- and 360-day STZ d i a b e t i c r a t s when compared to age-matched c o n t r o l s . Card iac f u n c t i o n a l abno rma l i t i es observed in d i a b e t i c r a t s inc luded a decreased a b i l i t y to develop peak l e f t v e n t r i c u l a r pressure and a decrease in maximum ra tes o f l e f t v e n t r i c u l a r pressure r i s e and d e c l i n e , when i s o l a t e d per fused working hear ts from d i a b e t i c ra t s were exposed to high a t r i a l f i l l i n g p r e s s u r e s . These f u n c t i o n a l a b n o r m a l i t i e s may r e f l e c t de fec ts i n myocard ia l c o n t r a c t i l i t y and r e l a x a t i o n induced by the d i a b e t i c s t a t e . These f i n d i n g s may a l s o represen t the development o f the p r e c l i n i c a l l e f t v e n t r i c u l a r dys func t i on phase o f d i a b e t i c card iomyopathy, which i s o f ten observed i n ch ron i c d i a b e t i c p a t i e n t s . Responsiveness o f the d i a b e t i c myocardium to the negat ive i n o t r o p i c e f f e c t o f carbacho l was not a l t e r e d a t 7 and 30 days a f t e r i nduc t i on o f d i a b e t e s . A s u b s e n s i t i v i t y to carbachol was e x h i b i t e d by d i a b e t i c r a t hear ts at 100 days , which was u l t i m a t e l y conver ted to a s u p e r s e n s i t i v i t y by 180 days and t h i s s u p e r s e n s i t i v i t y p e r s i s t e d in the d i a b e t i c myocardium t h e r e a f t e r . The changes i n the s e n s i t i v i t y o f d i a b e t i c r a t hear ts to the negat ive i n o t r o p i c e f f e c t o f carbachol may represent va r ious stages in the development o f a parasympathet ic d i a b e t i c neuropathy. D i a b e t i c r a t hear ts d id not e x h i b i t any changes in e i t h e r the s e n s i t i v i t y or i n the respons iveness to the p o s i t i v e i n o t r o p i c e f f e c t o f i s o p r o t e r e n o l . However, maximum changes produced by i sop ro te reno l i n negat ive dP /d t ( ca rd i ac r e l a x a n t e f f e c t ) were depressed i n d i a b e t i c r a t hear ts a t va r ious t ime po in ts a f t e r the i nduc t i on o f d i a b e t e s . The change in the respons iveness o f the d i a b e t i c myocardium to the c a r d i a c 217 r e l a x a n t e f f e c t o f i sop ro te reno l might r e s u l t from metabo l i c derangements in the hear t caus ing changes i n energy produc t ion and u t i l i z a t i o n and/or abno rma l i t i es i n c a r d i a c sa rcop lasmic r e t i c u l a r f u n c t i o n . L e f t v e n t r i c u l a r no radrena l ine content was unchanged i n ch ron i c d i a b e t i c r a t hear ts i n d i c a t i n g the absence o f any sympathet ic nerve damage owing to the development o f a sympathet ic neuropathy. Basal c y c l i c AMP content and phosphory lase a_ a c t i v i t y were not a l t e r e d in acute as we l l as ch ron i c d i a b e t i c r a t h e a r t s . I sopro te reno l (5 x 10"9M) produced s i m i l a r t ime- and dose-dependent changes in c y c l i c AMP content and p o s i t i v e and negat ive dP /d t i n i s o l a t e d per fused working hear ts obta ined from 3 and 100 t o ' 1 2 0 ' d a y - a l l o x a n .and-STZ. d i a b e t i c ra t s as compared to c o n t r o l s . However, i sop ro te reno l caused a s i g n i f i -c a n t l y g rea te r a c t i v a t i o n o f phosphory lase in hear ts ob ta ined from 3- and ,100. to-120 day d i a b e t i c r a t s , when compared to age-matched c o n t r o l s . D iabe t i c r a t hear ts from 100 to 120-day animals had s i g n i f i c a n t l y h igher t o t a l phosphory lase a c t i v i t y as compared to age-matched c o n t r o l s . PGE-j, a drug which i nc reases c y c l i c AMP content w i thout a l t e r i n g phosphory lase _a a c t i v i t y i n per fused r a t h e a r t s , i nc reased phosphory lase a^  a c t i v i t y i n acute as we l l as ch ron i c d i a b e t i c r a t hear ts but not i n con t ro l r a t h e a r t s . The nature and p h y s i o l o g i c a l s i g n i f i c a n c e o f the enhanced respons iveness of the phosphory lase enzyme i n d i a b e t i c r a t hear ts i s 2+ u n c l e a r . Acute metabo l i c derangements and a l t e r a t i o n s i n Ca homeostasis caused by d iabe tes cou ld p o s s i b l y r e s u l t i n the enhanced respons iveness o f phosphory lase to a g o n i s t s . Musca r i n i c recep to r b ind ing s tud ies performed on v e n t r i c l e s i s o l a t e d from 180-day con t ro l and d i a b e t i c ra t s revea led no changes 218 e i t h e r i n the t o t a l dens i t y or i n the a f f i n i t y o f musca r in i c recep to rs in 180-day d i a b e t i c r a t h e a r t s . These r e s u l t s suggest tha t the s u p e r s e n s i t i v i t y e x h i b i t e d by the 180-day d i a b e t i c myocardium to carbachol i s not a r e s u l t o f i nc reased dens i t y o f musca r in i c recep to rs in the h e a r t , but cou ld r e s u l t from a l t e r a t i o n s in events beyond the recep to r l e v e l . 219 REFERENCES Agarwa l , M .K . , 1980. S t r e p t o z o t o c i n : Mechanisms of a c t i o n . FEBS L e t t . 120: 1-3. Ahmed, S . S . , A . J . Ghazanfar , R.M. Narang• • and T . J ; Regan,. . 1975, P r e c l i n i c a l abnormal i t y o f l e f t v e n t r i c u l a r f u n c t i o n i n d iabe tes m e l l i t u s . Amer. Heart J . 89 : 153-158. Ahmed, S . S . and T . J . Regan, 1982, D i a b e t i c cardiomyopathy: D iagnos is and c l i n i c a l s i g n i f i c a n c e . P r a c t i c a l C a r d i o l . 8 : 76-86 . A l l i s o n , T . B . , S . P . B r u t t i g , M.F. Crass I I I , R .S . E l i o t and J . C . Sh ipp , 1976, Reduced h igh-energy phosphate l e v e l s i n r a t hear t I. E f f e c t s o f a l l o x a n d i a b e t e s . Am. J . P h y s i o l . 230 ( 6 ) : 1744-1750. Baandrup, U . , T . Ledet and R. Rasch , 1981. Exper imental d i a b e t i c ca rd iopa thy preventab le by i n s u l i n t rea tment . Laboratory i n v e s t i -g a t i o n . 45 : 169-173. Badeer , H .S. and S . Zone ra i ch , 1978. Pathogenesis o f cardiomyopathy in d iabe tes m e l l i t u s . I n : Diabetes and the Hear t , Ed . Zone ra i ch , S . , Char les C. Thomas, P u b l . S p r i n g f i e l d , pp. 26-45 . B a n t i n g , F . G . , C . H . B e s t , J . B . C o l l i p , W.R. Campbell and A . A . F l e t c h e r , 1922. Panc rea t i c e x t r a c t s i n the t reatment o f d iabe tes m e l l i t u s . Can. Med. Assoc . \ . Jv 12 : 141-146. Benesch, R. and R . E . Benesch, 1969. I n t r a c e l l u l a r o rgan ic phosphates as r e g u l a t o r s o f oxygen r e l e a s e by hemoglobin. Nature 221: 618-622. Bennet t , P . H . , 1982. Diabetes and hear t d i sease - the magnitude o f the problem. I n : C l i n i c a l Card io logy and D iabe tes , V o l . 1, Par t 1: Fundamental c o n s i d e r a t i o n s i n c a r d i o l o g y and d i a b e t e s . Ed. S c o t t , R .C. Futura P u b l i s h i n g C o . , New York , pp. 3-12. Bennet t , T . , D . J . Hosk ing , and J . R . Hampton, 1975. C a r d i o v a s c u l a r con t ro l i n d iabe tes m e l l i t u s . B r . Med. J . I I : 585-587. Berkman, J . I . and D. Zucker , 1978. Pathology o f a t h e r o s c l e r o t i c coronary a r t e r y d i sease and m isce l laneous d i a b e t e s - a s s o c i a t e d d i s e a s e s . I n : Diabetes and the hea r t . Ed. Zone ra i ch , S . , Char les C. Thomas, P u b l . S p r i n g f i e l d , pp. 19-25 . B o l l i n g e r , A . , J . F rey , K. J a g e r , J . F u r r e r , J . S e g l i a s and W. S i e g e n t h a l e r , 1982. Pa t te rns o f d i f f u s i o n through s k i n c a p i l l a r i e s in p a t i e n t s w i th long- term d i a b e t e s . N. E n g l . J . Med. 307: 1305-1310. B o q u i s t , L. 1980. A new hypothes is f o r a l l o x a n d i a b e t e s . Acta Pa th . M i c r o b i o l . Scand. S e c t . A , 88: 201-209. 220 B r a d l e y , R .F . and A. S c h o n f e l d , 1962. Diminished pain i n d i a b e t i c p a t i e n t s w i th acute myocard ia l i n f a r c t i o n . G e r i a t r i c s 17: 322-326. Brodde, O . E . , H . J . Schumann, and J . Wagner, 1979. Decreased respons iveness o f the adeny la te c y c l a s e system on l e f t a t r i a from hypothyro id r a t s . MoT. Pharmacol . 17: 180-186. B r u n z e l l , J . D . , R .P . Rober tson , R .L . L e r n e r , W.R. Hazza rd , J.W. E n s i n c k , E . L . Bierman and D. P o r t e , 1976. R e l a t i o n s h i p s between f a s t i n g plasma g lucose l e v e l s and i n s u l i n s e c r e t i o n dur ing in t ravenous g l u c o s e - t o l e r a n c e t e s t s . J . C l i n . E n d o c r i n o l . Metab. 42 : 222-229. Bunn, H.F . and H.F. B r i e h l , 1970. The i n t e r a c t i o n o f 2 ,3 -d iphosphog lyce ra te w i th va r i ous human hemoglobins. J . C l i n . I nves t . 49 : 1088-1095. Bunn, H . F . , K.H. Gabbay and P.M. G a l l o p , 1978. The g l y c o s y l a t i o n o f hemoglobin: Relevance to d iabe tes m e l l i t u s . Sc ience 200: 21-27. Chaudhur i , S . N . and J . C . Sh ipp , 1973. C y c l i c AMP in hear ts o f a l l o x a n -d i a b e t i c r a t s . I n : Recent Advances i n S tud ies on Card iac S t r u c t u r e and Metabo l ism. Myocard ia l Metabo l ism, B a l t i m o r e , U n i v e r s i t y Pa rk , V o l . 3 , pp. 319-329. Chenoweth, M.B. and E . S . K o e l l e , 1946. An i s o l a t e d hear t pe r f us i on system adapted to the de te rmina t ion o f non-gaseous m e t a b o l i t e s . J . Lab. C l i n . Med. 31 : 600-608. Chobanian, A . V . , E.R. A r q u i l l a , T . B . C l a r k s o n , H.A. Eder , C . F . Howard, J r . , T . J . Regan and J . R . W i l l i a m s o n , 1982. C a r d i o v a s c u l a r c o m p l i c a t i o n s . Diabetes 31 (Supp l . 1 ) : 54-64. C l a r k e , B . F . , D . J . Ewing, and I.W. Campbe l l , 1979. D i a b e t i c autonomic neuropathy. D iabe to l og ia 17: 195-212. C o l w e l l , J . A . , P .V . Ha lushka , K. S r j i , J . L e v i n e , J . Sagel and R.M.G. N a i r , 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 iabe tes m e l l i t u s . Diabetes 25 (Supp l . 2 ) : 826-831. Cooppan, R . , 1982. Recogn i t ion o f hypothyro id ism 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 o r i , G.T. and G .F . C o r i , 1940. The k i n e t i c s o f enzymat ic syn thes i s o f glycogen from g lucose-1 -phospha te . J . B i o l . Chem. 135: 733-756. 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. Cudworth, A . G . and J . C . Woodrow, 1976. Genet ic s u s c e p t i b i l i t y in d iabe tes m e l l i t u s : A n a l y s i s o f HLA a s s o c i a t i o n . B r . Med. J . 2 : 846-848. 221 Das, I. 1973a. S tud ies on glycogen metabol ism i n normal and d i a b e t i c r a t hea r t . Can. J . Biochem. 51 : 637-641. Das, I. 1973b. E f f e c t o f d iabe tes and i n s u l i n on r a t hear t a d e n y l c y c l a s e , c y c l i c AMP phosphodiesterase and c y c l i c AMP. Horm. Metab. Res. 5 : 330-333. D ' e l i a , J . A . , L .A . Weinrauch, R.H. Hea l y , J . A . L i b e r t i n o , R . F . B rad ley and O.S. L e l a n d , J r . , 1979. Myocard ia l dys func t i on w i thout coronary a r t e r y d i sease i n d i a b e t i c rena l f a i l u r e . Am. J . C a r d i o l . 43 : 193-199. Denton, R . M . , R.W. Brownsey and G . J . Belsham, 1981. A p a r t i a l view o f the mechanism o f i n s u l i n a c t i o n . D i a b e t o l o g i a , 21 : 347-362. D i l lmann , W.H. 1980. Diabetes m e l l i t u s induced changes in c a r d i a c myosin o f the r a t . D iabe tes , 29: 579-582. <Di 1 lmann,, W.,H:. 1982. In f luence o f t h y r o i d hormone a d m i n i s t r a t i o n on myosin ATPase a c t i v i t y and myosin isoenzyme d i s t r i b u t i o n i n the hear t o f d i a b e t i c r a t s . Metabo l i sm, 31 : 199-204. Duchen, L .W. , A. A n j o r i n , P . J . Watkins and J . D . MacKay, 1980. Pathology o f autonomic neuropathy in d iabe tes m e l l i t u s . Annals o f In t . Med. 92: 301-303. Dunn, J . S . and N.G.B . McLe tch ie , 1943. Exper imental a l l o x a n d iabe tes i n the r a t . Lancet I I : 384. Ewing, D . J . , I.W. Campbell and B . F . C l a r k e , 1980. Assessment o f c a r d i o -v a s c u l a r e f f e c t s i n d i a b e t i c autonomic neuropathy and p rognos t i c i m p l i c a t i o n s . Annals o f In t . Med. 92: 308^311. F a c t o r , S . M . , E.M. Okun and T. Minase, 1980. C a p i l l a r y microaneurysms i n the human d i a b e t i c hea r t . New Eng. J . Med. 302: 384-388. F a i n , J . N . 1981. Catechol amine- thy ro id hormone i n t e r a c t i o n s in l i v e r and adipose t i s s u e . L i f e Sc iences 28: 1745-1754. Fa jans , S . S . and N. F r e i n k e l , 1976. The problem of d iabe tes m e l l i t u s . I n : Diabetes m e l l i t u s . Ed. Fa jans , S . S . DHEW p u b l i c a t i o n s (NIH) , Bethesda, pp. 1-7. Farah , A . E . and A ..A. A l o u s i , . 1981. The ac t i ons o f i n s u l i n on c a r d i a c c o n t r a c t i l i t y . L i f e S c i . 29 : 975-1000. Fearman, I., E. F a c c i o , J . M i l e c , R. Nunez, M. J a d z i n s k y , D. Fox and M. Rapapaort , 1977. Autonomic neuropathy and p a i n l e s s myocard ia l i n f a r c t i o n i n d i a b e t i c pa t i en t s - h i s t o l o g i c a l ev idence o f t h e i r r e l a t i o n s h i p . D i abe t es , 26: 1147-1158. F e i n , F . S . , L . B . K o r n s t e i n , J . E . S t robeck , J . M . Capasso, and E .H . Sonnenb l i ck , 1980. A l t e r e d myocard ia l mechanics i n d i a b e t i c r a t s , C i r c . Res. 47 : 922-933. 222 F e i n , F . S . , J . E . S t robeck , A. M a l h o t r a , J . Scheuer and E .H . Sonnenb l i ck , 1981. R e v e r s i b i l i t y o f cardiomyopathy w i th i n s u l i n i n r a t s . C i r c . Res. 49 : 1251-1261 . Feldman, J . M . 1981. Diagnos is o f c a r d i o v a s c u l a r autonomic neuropathy i n the d i a b e t i c p a t i e n t . P r a c t i c a l C a r d i o l . 7: 101-116. F e l i g , P . , J . Wahren, R. Sherwin and R. Hend le r , 1976. I n s u l i n , glucagon and somatos ta t in i n normal phys io logy and d iabetes m e l l i t u s . Diabetes 25 : 1091-1099. Feuvray, D. , J . A . Ide l l -Wenger and J . R . Nee ly , 1979. E f f e c t s o f ischemia on r a t myocard ia l f unc t i on and metabol ism i n d i a b e t e s . C i r c . Res. 44: 322-329. F i e l d , J . B . 1976. Hyperosmolar coma. I n : Diabetes M e l l i t u s . Ed. Fa jans , S.S. , .DHEW p u b l i c a t i o n s , Bethesda, pp. 133-141. F i s c h e r , V .W. , H.B. Barner and M.L. Lesk iw , 1979. C a p i l l a r y basal laminar t h i ckness in d i a b e t i c human myocardium. D iabe tes , 28: 713-719. F i s k e , C .H . and Y. Subbarow, 1925. The c o l o r i m e t r i c de te rmina t ion o f phosphorus. J . B i o l . Chem. 66: 375. F l e i s c h , J . H . 1981. A g e - r e l a t e d decrease i n beta adrenoceptor a c t i v i t y o f the c a r d i o v a s c u l a r sys tem. T IPS, 2 : 337-338. F leming, W.W., D.P. W e s t f a l l , I .S . De l a Lande and L . B . J e l l e t t , 1972. Log-normal d i s t r i b u t i o n o f e q u i e f f e c t i v e doses o f norep inephr ine and a c e t y l c h o l i n e i n severa l t i s s u e s . J . Pharmacol . Exp. Ther. 181: 339-345. F l i e r , J . S . , C R . Kahn and J . Roth , 1979. Recep to rs , a n t i r e c e p t o r a n t i b o d i e s and mechanisms o f i n s u l i n r e s i s t a n c e . New Eng. J . Med. 300: 413-419. F o s t e r , D.W. 1976. D i a b e t i c coma. I n : Diabetes M e l l i t u s . Ed. Fa jans , S . S . DHEW p u b l i c a t i o n s (NIH) Bethesda, pp. 123-132. Foy, J . M . and P.D. Lucas , 1976. E f f e c t o f exper imenta l d i a b e t e s , food d e p r i v a t i o n and gene t i c o b e s i t y on the s e n s i t i v i t y o f p i thed r a t s to autonomic agen ts . B r . J . Pharmacol . 57: 229-234. Foy, J . M . and P.D. Lucas , 1978. Comparison between spontaneously beat ing a t r i a from con t ro l and 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 . J . Pharm. Pharmacol . 30: 558-562. F r i e s e n , A . J . D . , N. O l i v e r and G. A l l e n , 1969. A c t i v a t i o n o f c a r d i a c glycogen phosphory lase w i th c a l c i u m . Am. J . P h y s i o l . 217: 445-450. Gabbay, K.H. 1973. The s o r b i t o l pathway and the comp l i ca t i ons of d i a b e t e s . New E n g l . J . Med. 288: 831-836. 223 Gabbay, K . H . , 1 975. Hyperg lycemia , polyol metabo l ism, and comp l i ca t i ons o f d iabe tes m e l l i t u s . Ann. Rev. Med. 26: 521-536. Gabbay, K .H. and J . B . O ' S u l l i v a n , 1968. The s o r b i t o l pathway. Enzyme l o c a l i z a t i o n and content in normal and d i a b e t i c nerve and c o r d . Diabetes 17: 239-243. Ganda, O.P . and S . S . Soe ldne r , 1977. G e n e t i c , acqu i red and r e l a t e d f a c t o r s i n the e t i o l o g y of d iabe tes m e l l i t u s . A r c h . I n t e r n . Med. 137: 461-469. G a r c i a , J . J . , P.M. McNamara, T. Gordon and W.B. Kanne l , 1974. M o r b 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 teen year f o l l ow-up s tudy . D iabe tes , 23: 105-111. G i a c h e t t i , A . , 1981. D i a b e t i c neu ropa th ies : Pathogenet ic mechanisms and t he rapeu t i c p e r s p e c t i v e s . Pharmacol . Res. Com. 13: 101-118. Go ldberg , P . B . and Rober t s , J . , 1980. Pharmacology. In : Ag ing , v o l . 12, The Aging Hear t . Ed. W e i s f e l d t , M . L . , Raven P r e s s , New York , pp. 215-246. G o t t l i e b , M.S. and H.F. Root , 1968. Diabetes m e l l i t u s in t w i n s . Diabetes 17: 693-704. Hamby, R . I . , Zone ra i ch , S . and Sherman, M.D. , 1974. D i a b e t i c c a r d i o -myopathy. JAMA 229: 1749-1754. Haml in , C . R . , R.R. Kohn and J . H . L u s c h i n , 1975. Apparent a c c e l e r a t e d ag ing o f human c o l l a g e n i n d iabetes m e l l i t u s . D iabe tes , 24: 902-904. H a r r i s , D . P . , L .M. M a r r i o t t and J . H . M c N e i l l . Microcomputer a c q u i s i t i o n and a n a l y s i s system f o r the i s o l a t e d working hear t p r e p a r a t i o n . J . Pharmacol . Methods ( i n p r e s s ) . Hayes, J . S . and L . L . Brun ton , 1982. Funct iona l compartments in c y c l i c n u c l e o t i d e a c t i o n . J . C y c l . N u c l . Res. 8 : 1-16. Head, R . J . and B.A. B e r k o w i t z , 1979. Concen t ra t ion and f u n c t i o n of dopamine i n normal and d iseased blood v e s s e l s . I n : Advances i n the B i o - S c i e n c e s , v o l . 20. Pe r i phe ra l dopaminergic r e c e p t o r s , Ed. Jean-Louis Imbs and Jean Schwar tz , Pergamon P r e s s , Ox fo rd , pp. 173. He rbe r t , V . , K .S . Lau and F.W. P a i r e n t , 1965. Coated charcoa l immuno-assay of i n s u l i n . J . C l i n . E n d o c r i n o l . 25 : 1375-1384. He r r , R . R . , T . E . E b l e , M.E. Bergy and H.K. Jahnke, 1960. I s o l a t i o n and c h a r a c t e r i z a t i o n o f s t r e p t o z o t o c i n . A n t i b i o t . Ann. 236-240. Hers , H . G . , 1956. Le mecanisme de l a t rans fo rma t ion de g lucose en f r uc tose par l e s v e s i c u l e s semina les . B ioch im. B iophys . A c t a . 22: 202-203. 224 H o f t i e z e r , V. and A .M. Carpen te r , 1973. Comparison o f s t r e p t o z o t o c i n and a l l o x a n - i n d u c e d d iabe tes in 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 o f 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. Hosk ing , D . J . , T. Bennet t , and J . R . Hampton, 1978. D i a b e t i c autonomic neuropathy. D iabe tes , 27: 1043-1054. Hosk ins , B. and H.B. Luong, 1981. C y c l i c nuc leo t i de and c y c l i c n u c l e o t i d e phosphodiesterases i n k idneys from ra t s w i th exper imenta l d i a b e t e s . Res. Com. Chem. P a t h . Pharmacol . 33 : 381-384. Ingebre tsen , C . G . , P. Moreau, C .Hawe lu -Johnson and W.R. Ingebre tsen , J r . 1980. Performance o f d i a b e t i c r a t h e a r t s ; e f f e c t o f anox ia and inc reased work. Am. J . P h y s i o l . 239: H614-H620. Ingebre tsen , W.R. J r . , C. P e r a l t a , M. Mousher, L .K . Wagner and C .G . Ingebre tsen , 1981. Diabetes a l t e r s the myocard ia l cAMP-pro te in k inase cascade system. Am. J . P h y s i o l . 240: H375-H382. Ingebre tsen , C . G . , C. Hawelu-Johnson and W.R. Ingebre tsen , 1982. Myocard ia l adeny la te c y c l a s e i n d i a b e t i c r a t s : E f f e c t s o f GppNHP, i s o p r o t e r e n o l , NaF and f o r s k o l i n . The Pharmaco log is t , 24: 157 ( A b s t r . H351). I r v i n e , W . J . , 1977. C l a s s i f i c a t i o n o f i d i o p a t h i c d i a b e t e s . Lance t , I: 638-642. Jackson , C .V . and G.O. C a r r i e r , 1981. 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 i e s to no radrena l ine i n the long- term exper imenta l d i a b e t i c r a t . J . Auton. Pharmacol . 1: 399-405. J a r r e t t , J . , 1977. Diabetes and the hea r t : Coronary hear t d i s e a s e . C l i n . E n d o c r i n o l . Metab. 6: 389-402. Johansen, K. and Aa. P. Hansen, 1969. High 24-hour l e v e l o f serum growth hormone i n j u v e n i l e d i a b e t i c s . B r . Med. J . 2 : 356-357. Jones , R .L . and C M . P e t e r s o n , 1981. Hematologic a l t e r a t i o n s in d iabe tes m e l l i t u s . Am. J . Med. 70: 339-352. Jovanov i c , L. and C M . Pe te r son , 1981. The c l i n i c a l u t i l i t y o f g l y c o s y l a t e d hemoglobin. Am. J . Med. 70: 331-338. Junod, A . , A . E . Lambert, W. S tau f fache r and A . E . Reno ld , 1969. Diabetogenic a c t i o n o f s t r e p t o z o t o c i n : R e l a t i o n s h i p o f dose to metabo l i c response. J . C l i n . I nves t . 48: 2129-2139. Kanne l , W . B . , 1978. Role o f d iabe tes in c a r d i a c d i s e a s e : Conc lus ions from popu la t ion s t u d i e s . I n : Diabetes and the hea r t . Ed. Zone ra i ch , S . , Char les C. Thomas, S p r i n g f i e l d , pp. 97-112. Kanne l , W . B . , T .R. Dawber, A. Kagan, N. Revotske, and J . S t o k e s , 1961. Factors o f r i s k i n the development o f coronary hear t d i sease - s i x - y e a r f o l l ow-up of expe r i ence . Ann. I n t e r n . Med. 55 : 33-50. 225 Kanne l , W . B . , M. Huor t land and W.P.. C a s t e l l i , 1974. Role o f d iabe tes in conges t i ve hear t f a i l u r e : The Framingham s tudy . Am. J . C a r d i o l . 34: 29-34. K a t z , A . M . , 1980. Re lax ing e f f e c t s o f catecholamines in the hea r t . T IPS, 1: 434-436. K a u l , C . L . and R .S . Grewa l , 1980. Increased u r i n a r y e x c r e t i o n o f catecholamines and t h e i r me tabo l i t es i n 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 . Pharmacol . 21 : 223-228. K e e l y , S . L . , 1977. A c t i v a t i o n o f cAMP-dependent p r o t e i n k inase wi thout a cor respond ing i nc rease in phosphory lase a c t i v i t y . Res. Commun. Chem. P a t h o l . Pharmacol . 18: 283-290. Keen, H . , 1970. Minimal d iabe tes and a r t e r i a l d i s e a s e , preva lence and the e f f e c t o f t rea tment . I n : E a r l y D iabe tes : A Symposium. Eds. C a m e r i n i - D a v i s , R.A. and C o l e , H .S. Academic P r e s s , New York , pp. 437-442. K e f a l i d e s , N . A . , 1977. B iochemis t ry o f v a s c u l a r d i sease i n d iabe tes m e l l i t u s . I n : D iabe tes , Ed. B a j a j , J . S . Excerpta Med ica , Amsterdam, pp. 575-587. K e f a l i d e s , N . A . , 1981. Basement membrane research in d iabe tes m e l l i t u s . Co l lagen R e l . Res. 1: 295-299. K i n o s h i t a , J . H . , L .O. Me ro l a , K. Satoh and E. Dikmak, 1962. Osmotic changes caused by the accumulat ion o f d u l c i t o l i n the lenses o f r a t s fed w i th g a l a c t o s e . Nature , 194: 1085-1087. Knowles, H . C . , J r . , 1 9 7 8 . Coronary a r t e r y d i sease in d i a b e t e s : I t s development, course and response to t rea tment . I n : Diabetes and the Hear t . Ed. Zone ra i ch , S . and Char les C. Thomas, S p r i n g f i e l d , pp. 113-122. Knowles, H . C . , 1982. The na tu ra l h i s t o r y o f coronary hear t d i sease and d iabe tes m e l l i t u s . I n : C l i n i c a l Card io logy and D iabe tes , v o l . 1, par t 2 : Fundamental c o n s i d e r a t i o n s i n c a r d i o l o g y and d i a b e t e s . Koen ig , R . J . and A. Cerami , 1980. Hemoglobin A i c and d iabe tes m e l l i t u s . Ann. Rev. Med. 31 : 29-34. Kunos, G . , 1981. Modulat ion o f ad renerg ic r e a c t i v i t y and adrenoceptors by t h y r o i d hormones. I n : Adrenoceptors and Catecholamine A c t i o n , Ed. Kunos, G . , John Wi ley and Sons, 1: 297-333. L a k a t t a , E . G . , G. G e r s t e n b l i t h , C . S . A n g e l l , N.W. Shock and M.L. W e i s f e l d t , 1975. Dimin ished i n o t r o p i c response o f aged myocardium to ca techo lamines . C i r c . Res. 36: 262-269. L a t i f p o u r , J . G . , C .V . Jackson and J . H . M c N e i l l , 1983. A l t e r a t i o n s in c a r d i a c autonomic recep to rs in s t r e p t o z o t o c i n (STZ)- induced d iabe tes in the r a t . Fed. Proceed. 42 : 1358, Abs t . # 6245. 226 Lazarow, A. and S . L . P a l a y , 1946. The produc t ion and course o f a l l o x a n d iabe tes i n the r a t . J . Lab. C l i n . Med. 31 : 1004-1015. Ledet , T . , Neubauer, B . , C h r i s t e n s e n , N . J . , Lundbaek, K . , 1979. D i a b e t i c c a r d i o p a t h y , D iabe to log ia 16: 207-209. Le febv re , P . J . and A . S . Luyckx, 1979. Glucagon and d i a b e t e s : A r e a p p r a i s a l . D iabe to log ia -16 : 347-354. Lernmark, A. and S . Baekkeskov, 1981. I s l e t c e l l a n t i b o d i e s - t h e o r e t i c a l - and p r a c t i c a l i m p l i c a t i o n s . D iabe to l og ia 21 : 431-435. Levey, G . S . , C L . Ske l ton and S . E . E p s t e i n , 1969. Decreased myocard ia l adeny la te c y c l a s e a c t i v i t y i n hypo thy ro id ism. J . C l i n . I nves t . 48: 2244-2250. L i ndsey , C . A . , C R . Faloona and R. Unger, 1974. Plasma glucagon i n non-k e t o t i c hyperosmolar coma. J . Amer. Med. Assoc . 229: 1771-1773. L loyd -Mos tyn , R . H . , and P . J . Watk ins , 1975. De fec t i ve i n n e r v a t i o n o f the hear t i n d i a b e t i c autonomic neuropathy. B r i t . Med. J . 3 : 15-17. L loyd -Mos tyn , R.H. and P . J . Watk ins , 1976. Tota l c a r d i a c denerva t ion in d i a b e t i c autonomic neuropathy. Diabetes 25: 748-751. Lopaschuk, G . D . , S . Katz and J . H . M c N e i l l , 1982. The e f f e c t o f a l l o x a n and s t r e p t o z o t o c i n - i n d u c e d d iabe tes on ca l c ium t r anspo r t i n r a t c a r d i a c s a r c o -p lasmic r e t i c u l u m . The p o s s i b l e involvement o f l o n g - c h a i n a c y l c a r n i t i n e s . Can. J . P h y s i o l . Pharmacol , ( i n p r e s s ) . Lundbaek, K . , 1 976. Growth hormone's r o l e i n d i a b e t i c m ic roang iopa thy . D iabe tes , 25 (Supp l . 2 ) : 839-844. Lundqu is t , I. and C. Rerup, 1967. On the development o f a l l o x a n d iabe tes in m ice . Eur . J . Pharmacol . 2 : 35 -41 . McCul lough, A . L . and J . H . M c N e i l l , 1983. Chronic d iabe tes decreases the ouabain i n o t r o p i c . r e s p o n s e i n r a t l e f t a t r i a and p a p i l l a r y musc les . Gen. Pharmacol , ( i n p r e s s ) . McFar land , K . F . , E.W. Ca ta lano , J . F . Day, S . R . Thorpe and J.W. Baynes, 1979. Nonenzymatic g l u c o s y l a t i o n o f serum p ro te ins i n d iabe tes m e l l i t u s . Diabetes 28: 1011-1014. M c M i l l a n , 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 m ic roang iopa thy . Diabetes 25 (Supp l . 2 ) : 858-864. M c N e i l l , J . H . and T .M. Brody, 1966. The e f f e c t o f a n t i h i s t a m i n e s , coca ine and rese rp i ne on amine induced r a t c a r d i a c phosphory lase a c t i v a t i o n . J . Pharmacol . Exp. Ther . 152: 478-487. Madsbad, S . , P. Laurberg , J . Weeke, H. Orskov, O.K. Faber , C. B i n d e r , T. Krarup and L. Regeur, 1981. Very e a r l y changes i n c i r c u l a t i n g T3 and rT3 dur ing development o f metabo l i c derangement i n d i a b e t i c p a t i e n t s . Acta Med. Scand. 209: 385-387. 227 MaLa isse , W . J . , 1982. A l l o x a n t o x i c i t y to the panc rea t i c 3 - c e l l . A new hypo thes i s . Biochem. Pharmacol . 31 : 3527-3534. M a l a i s s e - L a g a e , F . , A. Sener and W . J . MaLa isse , 1981. FEBS L e t t . 133: 181-182. Ma lho t ra , A . , S . Penpargku l , F . S . F e i n , E .H . Sonnenbl ick and J . Scheuer , 1981. The e f f e c t o f s t r e p t o z o t o c i n - i n d u c e d d iabetes in r a t s on c a r d i a c c o n t r a c t i l e p r o t e i n s . C i r c . Res. 49: 1243-1250. Mansford , K . R . L . and L. Op ie , 1968. Comparison o f metabo l i c abno rma l i t i e s in d iabe tes m e l l i t u s induced by s t r e p t o z o t o c i n or by a l l o x a n . Lancet I: 670-671. M a s i e l l o , P . , A . A . De P a o l i and E. Be rgamin i , 1979. In f luence o f age on the s e n s i t i v i t y o f the r a t to s t r e p t o z o t o c i n . Hormone Res. 11 : 262-274. Menahan, L . A . , S . N . Chaudhur i , H.E. Weber and J . C . Sh ipp , 1977. Lack o f ep inephr ine s t i m u l a t i o n o f r a t hear t adeny la te c y c l a s e in exper imenta l d i a b e t e s . Horm. Metab. Res. 9: 527-528. M i l l e r , T . B . J r . , 1979. Card iac performance o f i s o l a t e d per fused hear ts from a l l o x a n - d i a b e t i c r a t s . Am. J . P h y s i o l . 236: H808-H812. M i l l e r , T . B . J r . , M. P r a d e r i o , C. Wolleben and J . Bu l lman, 1981. A h y p e r s e n s i t i v i t y o f glycogen phosphory lase a c t i v a t i o n i n hear ts o f d i a b e t i c r a t s . J . B i o l . Chem. 256: 1748-1753. Moorehouse, J . A . , S . A . Car te r and J . Doupe, 1966. Vascu la r responses i n d i a b e t i c pe r i phe ra l neuropathy. B r i t . Med. J . 1: 883-888. M u l l e r , W . , G . Faloona and R.H. Unger, 1973. Hyperglucagonemia i n d i a b e t i c k e t o a c i d o s i s : I t s preva lence and s i g n i f i c a n c e . Am. J . Med. 54: 52-57. Nat iona l Diabetes Data Group, 1979. C l a s s i f i c a t i o n and d iagnos i s o f d iabetes m e l l i t u s and o ther ca tego r i es o f g lucose i n t o l e r a n c e . Diabetes 28: 1039-1057. Nee ly , J . R . , H. L i e b e r m e i s t e r , E . J . B a t t e r s b y , and H.E. Morgan, 1967. E f f e c t o f p ressure development on oxygen consumption by the i s o l a t e d r a t hea r t . Am. J . P h y s i o l . 212: 815-822. Neubauer, B. and N . J . C h r i s t e n s e n , 1976. Norep inephr ine , ep inephr ine and dopamine contents o f the c a r d i o v a s c u l a r system i n long- term d i a b e t i c s . D iabe tes , 25: 6-10. Op ie , L . H . , 1975. Metabol ism of f ree f a t t y a c i d s , g lucose and c a t e c h o l a -mines in acute myocard ia l i n f a r c t i o n . R e l a t i o n to myocard ia l ischemia and i n f a r c t s i z e . Am. J . C a r d i o l . 36: 938-953. 228 Op ie , L . H . , M . J . Tansey and B.M. K e n n e l l y , 1979a. The hear t i n d iabe tes m e l l i t u s . Par t I. B iochemica l bas i s f o r myocard ia l d y s f u n c t i o n . S . A f r . Med. J . 56: 207-211. Op ie , L . H . , M . J . Tansey and B.M. K e n n e l l y , 1979b. The heart i n d iabe tes m e l l i t u s . Pa r t I I . Acute myocard ia l i n f a r c t i o n and d i a b e t e s . S . A f r . Med. J . 56: 256-261. O r c i , L . , D. Bae tens , C. Rufener , M. Amherdt, M. Ravazzo la , P. S t u d e r , F. Ma la isse -Lagae and R.H. Unger, 1976. Hypertrophy and h y p e r p l a s i a o f s o m a t o s t a t i n - c o n t a i n i n g D - c e l l s i n d i a b e t e s . P roc . Nat . Acad. S c i . 73 : 1338-1342. 0 s t e r b y , R . , 1975. E a r l y phase i n the development o f d i a b e t i c g lomeru lo-pathy. A q u a n t i t a t i v e e l e c t r o n m ic roscop i c s tudy . Acta Med. Scand. 574 ( S u p p l . ) : 3 -82. Palumbo, P . J . , L .R . Elveback and D.C. C o n n o l l y , 1982. Coronary hear t d i sease and conges t i ve hear t f a i l u r e in the d i a b e t i c : Ep idemio log i ca l a s p e c t s . The Rochester Diabetes P r o j e c t . I n : C l i n i c a l Card io logy and D iabe tes , v o l . 1, par t 1: Fundamental Cons ide ra t i ons i n Card io logy and D iabe tes . Ed. S c o t t , R .C. Futura P u b l i s h i n g C o . , New York , pp. 13-28. Par tamian, J . O . and R .F . B r a d l e y , 1965. Acute myocard ia l i n f a r c t i o n i n 258 cases o f d i a b e t e s . Immediate m o r t a l i t y and f i v e - y e a r s u r v i v a l . N. Eng. J . Med. 273: 455-461. P a r v i n g , H . H . , 1976. Increased m ic rovascu la r p e r m e a b i l i t y to plasma p ro te ins in s h o r t - and long- term j u v e n i l e d i a b e t i c s . Diabetes 25 (Supp l . 2 ) : 884-889. P a t e l , Y . C . , T. Wheatley and H.H. Z ingg , 1980. Increased blood somato-s t a t i n concen t ra t i on in 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 . L i f e S c i . 27 : 1563-1570. Pau l son , D . J . and M.F. Crass I I I , 1980. Myocard ia l t r i a c y l g l y c e r o l f a t t y a c i d compos i t ion i n d iabe tes m e l l i t u s . L i f e S c i . 27: 2237-2243. Pek, S . , 1977. Glucagon and d i a b e t e s . C l i n i c s i n E n d o c r i n o l , and Metab. 6: 33-344. Penpargku l , S . , T. S c h a i b l e , T. Y i p i n t s o i and J , Scheuer , 1980. The e f f e c t o f d iabe tes on performance and metabol ism o f r a t h e a r t s . C i r c . Res. 47 : 911-921. Penpargku l , S . , Fj.S. F e i n , E .H . Sonnenbl ick and J . Scheuer , 1981. Depressed c a r d i a c sa rcop lasmic r e t i c u l a r f u n c t i o n from d i a b e t i c r a t s . J . M o l . C e l l . C a r d i o l . 13 : 303-309. 229 P i e r c e , G.N. and N.S. D h a l l a , 1981. Card iac m y o f i b r i l l a r ATPase a c t i v i t y i n d i a b e t i c . r a t s . J . M o l . C e l l . C a r d i o l . 13 : 1-7. P i t tman , C . S . , A. K. Suda, J . B . Chambers, J r . and G.Y. Ray, 1979. Impaired 3 , 5 , 3 ' - t r i i o d o t h y r o n i n e (T3) p roduc t ion i n d i a b e t i c p a t i e n t s . Metabol ism 28: 333-338. P i t tman , C . S . , R.H. L i ndsay , 0. Senega, J . B . Chambers, J r . and J . B . H i l l , J r . , 1981. The e f f e c t o f d iabetes m e l l i t u s on the 3 , 5 , 3 ' - t r i i o d o t h y r o n i n e p r o d u c t i o n . L i f e S c i . 28: 1677-1682. P i t t s , B. J r . , C A . Ta te , B. Van W ink le , J . M . Wood and M.L. Entman, 1978. P a l m i t y l c a r n i t i n e i n h i b i t i o n o f the ca lc ium pump in c a r d i a c sa rcop lasmic r e t i c u l u m : A p o s s i b l e r o l e i n myocard ia l i s chem ia . L i f e S c i . 23 : 391-402. Podo lsky , S . , 1981. The e l d e r l y c a r d i a c p a t i e n t : Typ i ca l cand idate f o r hyperosmolar n o n - k e t o t i c d i a b e t i c coma. P r a c t i c a l C a r d i o l . 7: 91-100. Pyke, D .A . , 1979. D iabe tes : The gene t i c connec t i on . D iabe to l og ia 17: 333-343. Raabo, E. and T . C . T e r k i l d s e n , 1960. On the enzymat ic de te rmina t ion o f blood g l u c o s e . Scand. J . C l i n . Lab. I nves t . 12 : 402-407. Rahbar, S . , 1981. G l y c o s y l a t e d hemoglobins. Texas Rep. B i o l . Med. 40: 373-385. R a k i e t e n , N . , M.L. Rak ie ten and M.V. Nadkarn i , 1963. S tud ies on the d iabe togen ic a c t i o n o f s t r e p t o z o t o c i n ( N S C T 3 7 9 1 7 ) . Cancer Chemother. Rep. 29: 91-98. Reaven, G . M . , 1980. I nsu l i n - i ndependen t d iabetes m e l l i t u s ; me tabo l i c c h a r a c t e r i s t i c s . Metabo l i sm, 29: 445-454. Regan, T . J . , P .O. E t t i n g e r , M. I . Khan, M.U. J e s r a n i , M.M. Lyons, H.A. Oldewurte l and M. Weber, 1974. A l t e r e d myocard ia l f u n c t i o n and metabol ism in ch ron i c d iabetes m e l l i t u s wi thout ischemia in dog. C i r c . Res. 35 : 222-237. Regan, T . J . , M.M. Lyons, S . S . Ahmed, G .E . Lev inson , H.A. O l d e w u r t e l , M.R. Ahmad and B. Ha ide r , 1977. Evidence fo r cardiomyopathy in f a m i l i a l d iabe tes m e l l i t u s . J . C l i n . I nves t . 60: 885-889. Regan, T . J . , C . F . Wu, C.K. Yeh, H.A. Oldewurtel and B. Ha ide r , 1981. Myocard ia l compos i t ion* and f u n c t i o n in d i a b e t e s . The e f f e c t s o f ch ron i c i n s u l i n use. C i r c . Res. 49 : 1268-1277. Reno ld , A . E . , D.H. M i n t z , W.A. Mu l l e r and G .F . C a h i l l , J r . , 1978. Diabetes m e l l i t u s . I n : The metabo l ic bas is o f i n h e r i t e d d i s e a s e . Eds. Stombury, J . B . . , J . B . Wyagaarden, and D.S. F r e d r i c k s o n . M c G r a w - H i l l , New York , pp. 79-109. 230 Rerup, C , 1970. Drugs producing d iabe tes through damage o f 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. Rober tson, R .P . and S . A . Metz , 1979. P r o s t a g l a n d i n s , the g luco recep to r and d i a b e t e s . New Eng. J . Med. 301: 1446-1447. Rodgers, R . L . , K.M. MacLeod, and J . H . M c N e i l l , 1981. Responses o f r a t and guinea p ig hear ts to g lucagon. Lack o f ev idence f o r a d i s s o c i a t i o n between changes i n myocard ia l c y c l i c 3 ' , 5 ' - a d e n o s i n e monophosphate and c o n t r a c t i l i t y . C i r c . Res. 49 : 216-225. Rub ie r , S . , 1977. Card iac man i f es ta t i ons o f d iabe tes m e l l i t u s . Ca rd i ovasc . Med. 2 : 823-835. Rub ie r , S . , D lugash, J . , Yuceogh i , Y . Z . , Kumral , T . , Branwood, A.W. and Grishman, A . , 1972. A new type o f cardiomyopathy a s s o c i a t e d wi th d i a b e t i c g l o m e r u l o s c l e r o s i s . Amer. J . C a r d i o l . 30 : 595-602. Rund les , R.W., 1945. D i a b e t i c neuropathy. 0 Medic ine 24: 110-160. S a l a n s , S . B . , 1982. Diabetes M e l l i t u s . A d i sease tha t i s coming i n t o f o c u s . J . Amer. Med. Assoc . 247: 590-594. Sava rese , J . J . and B.A. B e r k o w i t z , 1979. B-adrenerg ic recep to r decrease i n d i a b e t i c r a t h e a r t s . L i f e Sc iences 25: 2075-2078. S c h e i n , P . S . , D.A. Cooney and M.L. Vernon, 1967. The use o f n i co t inamide to modify the t o x i c i t y o f s t r e p t o z o t o c i n d iabe tes w i thout l o s s o f ant i tumor a c t i v i t y . Can. Res. 27 : 2324-2332. Schmidt , R . E . , J . S . Nelson and E.M. Johnson, 1981. Exper imental d i a b e t i c autonomic neuropathy. Am. J . P a t h o l . 103: 210-225. Schmidt , R .E . and D.W. Scharp , 1982. Axonal dystrophy in exper imenta l d i a b e t i c autonomic neuropathy. Diabetes 31 : 761-770. S e n e v i r a t n e , B . I . B . , 1977. D i a b e t i c cardiomyopathy: The p r e c l i n i c a l phase. B r . Med. J . 1: 1444-1446. S h a f f e r , J . E . and K.U. M a l i k , 1982. Enhancement o f p ros tag land in output dur ing a c t i v a t i o n o f beta-1 adrenoceptors i n the i s o l a t e d r a b b i t hea r t . J . Pharmacol . Exp. Therap. 223: 729-735. S h a p i r o , L . M . , A . P . Howat, and M.M. C a l f e r , , 1981a. L e f t v e n t r i c u l a r f u n c t i o n "in d iabe tes m e l l i t u s I: Methodology, and preva lence and spectrum o f a b n o r m a l i t i e s . B r . Heart J . 45 : 122-128. S h a p i r o , L . M . , B .A. Leather-dale, . J . Mackinnon, R .F . F l e t c h e r , 1981b. L e f t v e n t r i c u l a r f unc t i on i n d iabe tes m e l l i t u s I I : R e l a t i o n between c l i n i c a l f ea tu res and l e f t v e n t r i c u l a r f u n c t i o n . B r . Heart J . 45 : 129-132. 231 S h a p i r o , L . M . , 1982. S p e c i f i c hear t d i sease i n d iabetes m e l l i t u s . B r i t . Med. J . 284: 140-141. S i e g l , P . K . S . and J . H . M c N e i l l , 1982. Antagonism w i th d ibenamine, D-600, and RO 3-7894 to es t imate d i s s o c i a t i o n constants and recep to r reserves f o r c a r d i a c adrenoceptors i n i s o l a t e d r a b b i t p a p i l l a r y musc les . Can. J . P h y s i o l . Pharmacol . 60: 1131-1137. Smi th , J . W . , 1982. Heart d i sease in d iabetes m e l l i t u s . I n : Management o f Diabetes M e l l i t u s . Ed. B r e s s l e r , R. and D.G-. Johnson; John Wright PS'G I n c . , Bos ton , pp. 285-298. S p i r o , R . G . , 1967. S tud ies on the renal g lomeru lar basement membrane: Nature o f the carbohydrate un i t s and t h e i r attachement to the pept ide p o r t i o n . J . B i o l . Chem. 243: 1923-1932. S p i r o , R . G . , 1976. I n v e s t i g a t i o n s i n to the b iochemica l bas is o f d i a b e t i c basement-membrane a l t e r a t i o n s . Diabetes 25 (Supp l . 2 ) : 909-913. S r i v a s t a v a , L . M . , P . S . Bora and S .D . B h a t t , 1982. D iabetogenic a c t i o n o f s t r e p t o z o t o c i n . T IPS: 376-378. S t a r k e , K . , L. Hedler and A. S t e p p e l e r , 1981. Metabol ism o f endogenous exogenous no radrena l ine i n gu inea -p ig a t r i a . Naunyn Schmiedeberg 's A r c h . Pharmacol . 317: 193-198. S t e i n e r , A . L . , A . S . P a g l i a r i , L .R . Chase and D.M. K r i p h i s , 1972. Rad io-immunoassay f o r c y c l i c nuc leo t i des I I : Adenosine 3 ' :5 ' -monophosphate i n mammalian t i s s u e and body f l u i d s . J . B i o l . Chem. 247: 1114-1120. S t o u t , R.W., 1979. Diabetes and a t h e r o s c l e r o s i s , the r o l e o f i n s u l i n . D iabe to log ia 16: 141-150. S t u e s s e , S . L . , D.W. W a l l i c k and S . Mace, 1982. Vagal con t ro l o f hear t pe r iod i n a l l o x a n d i a b e t i c r a t s . L i f e S c i . 31 : 393-398. Sunder land , M . L . E . and J . E . Logan, 1971. An assessment o f the Ames/BMI blood a n a l y z e r . C l i n . Biochem. 4 : 22-28. T a h i l i a n i , A . G . , 1983. E f f e c t s o f i n s u l i n o r t h y r o i d t reatment on d iabe tes - i nduced myocard ia l a b n o r m a l i t i e s . M.Sc. t h e s i s , U n i v e r s i t y o f B r i t i s h Columbia, Vancouver, B . C . T a h i l i a n i , A . G . , R . V . S . V . Vadlamudi and J . H . M c N e i l l , 1983. Preven t ion and r e v e r s a l o f a l t e r e d myocard ia l f unc t i on in d i a b e t i c ra ts by i n s u l i n t rea tment . Can. J . P h y s i o l . Pharmacol , ( i n p r e s s ) . Thomas, P .K . and R.G. L a s c e l l e s , 1965. Schwann-ce l l a b n o r m a l i t i e s i n d i a b e t i c neuropathy. Lancet 1: 1355-1357. 232 Toml inson, D.R. and A . P . M . Yusof , 1981. On the s t r u c t u r a l and f u n c t i o n a l i n t e g r i t y o f autonomic nerves in ra t s w i th long- term d iabe tes m e l l i t u s induced by a l l o x a n . B r . J . Pharmacol . 74: 184P. Tzagou rn i s , M . , 1982. I n s u l i n , g lucagon, growth hormone and s o m a t o s t a t i n . The i r r e l a t i o n to d iabe tes and hear t d i s e a s e . I n : C l i n i c a l c a r d i o l o g y and d i a b e t e s , v o l . 1, par t 1: Fundamental Cons ide ra t i ons i n Card io logy and D iabe tes , Ed. S c o t t , R .C. Futura P u b l i s h i n g C o . , New York , pp. 171-190. Linger, R . H . , 1978. Role o f glucagon i n the pathogenesis o f d i a b e t e s : The Sta tus o f the Con t roversy . Metabo l i sm, 27: 1691-1709. Vadlamudi , R . V . S . V . and J . H . M c N e i l l , 1981. Card iac e f f e c t s o f p r o s t a -g land ins E] and F"|a Can. J . P h y s i o l . Pharmacol . 59 : 473-478. Ven te r , J . C , 1979. High e f f i c i e n c y c o u p l i n g between be ta -ad rene rg i c recep to rs and c a r d i a c c o n t r a c t i l i t y : D i r e c t ev idence f o r " spa re " be ta -ad rene rg i c r e c e p t o r s . MoT. Pharmacol . 16: 429-440. V i t t o n e , L . , A. G r a s s i , L. Ch iappe, M. Argel and H.E. C i n g o l a n i , 1981. Re lax ing e f f e c t o f pharmacolog ica l i n t e r v e n t i o n s i n c r e a s i n g cAMP i n r a t hea r t . Am. J . P h y s i o l . 240: H441-H447. Vracko, R. and E . P . B e n d i t t , 1974. Man i f es ta t i ons o f d iabe tes 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 ing c e l l d e f e c t . Am. J . P a t h o l . 75 : 204-224. Ward, J . D . , R.W.R. Baker and B .H . Dav i s , 1972. E f f e c t o f blood sugar con t ro l on the accumulat ion o f s o r b i t o l and f r uc tose i n nervous t i s s u e s . Diabetes 21 : 1173-1178. Watk ins , P . J . and J . D . MacKay, 1980. Card iac denerva t ion in d i a b e t i c neuropathy. Med. A n n a l . I n t . Med. 92: 304-307. W e i s f e l d t , M . L . , 1980. L e f t v e n t r i c u l a r f u n c t i o n . I n : A g i n g , v o l . 12. The Aging Hear t . Ed. M.L. W e i s f e l d t , Raven P r e s s , New York , pp. 297-316. Wheeler , T. and P . J . Watk ins , 1973. Card iac denerva t ion in d i a b e t e s . B r . Med. J . 4 : 584-586. W i l l i a m s , I . H . , B . H . L . Chua, R.H. Sahms, D. S i e h l and H.E. Morgan, 1980. E f f e c t o f d iabe tes on p r o t e i n tu rnover i n c a r d i a c musc le . Am. J . P h y s i o l . 239: E178-E185. W i l l i a m s o n , J . R . and C. K i l o , 1976. Basement-membrane t h i c k e n i n g and d i a b e t i c m ic roang iopa thy . Diabetes 25 (Supp l . 2 ) : 925-927. 233 W i l l i a m s o n , J . R . and C. K i l o , 1977. Current s ta tus o f c a p i l l a r y basement-membrane d isease in d iabe tes m e l l i t u s . Diabetes 26: 65-73 . W i l l i a m s o n , J . R . and C. K i l o , 1980. Vascu la r comp l i ca t i ons i n d iabe tes m e l l i t u s . New E n g l . J . Med. 302: 399-400. Winegrad, A . I . and R .S . Clements, J r . , 1 9 7 6 . Demonstrable metabo l i c abno rma l i t i e s in d iabe tes m e l l i t u s tha t may c o n t r i b u t e to the pathogenesis o f s p e c i f i c l a t e c o m p l i c a t i o n s . I n : Diabetes m e l l i t u s , Ed. Fa jans , S . S . DHEW P u b l i c a t i o n s (NIH), Bethesda, pp. 173-193. Wood, J . M . , B. Bush, B . J . R . P i t t s and A. Schwar tz , 1977. I n h i b i t i o n o f bovine hear t N a + , K + -ATPase by p a l m i t y l c a r n i t i n e and p a l m i t y l - C o A . Biochem. B iophys . Res. Commun. 74: 677-684. Wr igh t , A . D . , E.M. Kohner, N.W. Oak ley , M. Har tog , G .F . J o p l i n and T .R. F r a s e r , 1969. Serum growth hormone l e v e l s and the response of d i a b e t i c r e t i nopa thy to p i t u i t a r y a b l a t i o n . B r . Med. J . 2 : 346-348. Yamashi ta , K . , S . Yamashi ta , H. Yasuda and E. Ogata, 1980. A decreased response o f c y c l i c adenosine monophosphate concen t ra t i ons to glucagon i n l i v e r s l i c e s from s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c r a t s . Diabetes 29: 188-192. PUBLICATIONS PAPERS: 1. Vadlamudi , R . V . S . V . and M c N e i l l , J . H . (1981) . Card iac e f f e c t s o f p ros tag land ins E-j and F^a. Can. J . P h y s i o l . Pharmacol . 59 ( 5 ) : 473-478. 2. Vadlamudi , R . V . S . V . , Rodgers, R .L . and M c N e i l l , J . H . (1982) . The e f f e c t o f ch ron i c a l l o x a n and s t r e p t o z o t o c i n induced d iabe tes on i s o l a t e d r a t hear t performance. Can. J . P h y s i o l . Pharmacol . 60 ( 7 ) : 902-911 . 3 . Vadlamudi , R . V . S . V . and M c N e i l l , J . H . (1983) . E f f e c t o f a l l o x a n - and s t r e p t o z o t o c i n - i n d u c e d d iabe tes on i s o l a t e d r a t hear t respons iveness to c a r b a c h o l . J . Pharmacol . Exp. Therap. 225 ( 2 ) : ( i n p r e s s ) . 4 . Vadlamudi , R . V . S . V . and M c N e i l l , J . H . (1983) . E f f e c t o f exper imenta l d iabe tes on r a t c a r d i a c cAMP, phosphory lase and i n o t r o p y . Ara. J . P h y s i o l . 244: ( i n p r e s s ) . 5 . 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) . P reven t ion and r e v e r s a l o f a l t e r e d myocard ia l f u n c t i o n i n d i a b e t i c ra ts by i n s u l i n t rea tment . Can. J . P h y s i o l . Pharmacol , ( i n p r e s s ) . 6. Vadlamudi , R . V . S . V . and M c N e i l l , J . H . (1983) . E f f e c t o f exper imenta l d iabe tes on i s o l a t e d r a t hear t respons iveness to i s o p r o t e r e n o l Can. J . P h y s i o l . Pharmacol , (submit ted f o r p u b l i c a t i o n ) . 7. Lopaschuk, G . D . , T a h i l i a n i , A . G . , Vadlamudi , R . V . S . V . , K a t z , S . and M c N e i l l , J . H . (1983) . Card iac sa rcop lasmic r e t i c u l u m f u n c t i o n i n i n s u l i n - or c a r n i t i n e - t r e a t e d d i a b e t i c r a t s . Am. J . P h y s i o l , (submit ted f o r p u b l i c a t i o n ) . ABSTRACTS: 8. Vadlamudi , R . V . S . V . and M c N e i l l , J . H . (1979) . Card iac e f f e c t s o f p ros tag land ins E-j and F^a. The Pharmaco log i s t , 21 ( 3 ) : 257. 9. Vadlamudi , R. and M c N e i l l , J . H . (1980) . Card iac f u n c t i o n i n normal and d i a b e t i c r a t s . P roc . West. Pharmacol . Soc . 23 : 29 -31 . 10. Vadlamudi , R . V . S . V . , R i e d e l , B . E . and M c N e i l l , J . H . (1980). Myocard ia l f u n c t i o n i n expe r imen ta l l y induced d i a b e t e s . P r o c . Can. Fed. B i o l . Soc . 23 : 91 . 11 . Vadlamudi , R . , and M c N e i l l , J . H . (1981) . E f f e c t o f ch ron i c s t r e p t o z o t o c i n induced d iabe tes on c a r d i a c performance i n r a t s . P r o c . West. Pharmacol . Soc . 24 : 73-77. 12. Vadlamudi , R . V . S . V . and M c N e i l l , J . H . (1981) . E f f e c t o f sho r t term exper imenta l d iabe tes on c a r d i a c performance i n the r a t . P r o c . Can. Fed. B i o l . Soc . 24: 193. PUBLICATIONS, c o n t i n u e d : 13. M c N e i l l , J . H . and Vadlamudi , R . V . S . V . (1981) . Func t iona l and pharmacolog ica l changes in r a t hear ts induced by ch ron i c d i a b e t e s . Presented a t the E igh th I n t e r n a t i o n a l Congress o f Pharmacology, Tokyo, Japan. J u l y , 1981. 14. Vadlamudi , R . V . S . V . and M c N e i l l , J . H . (1981) . Long-term exper imenta l d iabe tes induced changes i n c a r d i a c f u n c t i o n i n the r a t . The Pharmaco log is t 23 ( 3 ) : 220. 15. Vadlamudi , R . V . S . V . , Wenkstern, B.M. and M c N e i l l , J . H . (1982). Rat c a r d i a c c y c l i c AMP and phosphory lase a^  l e v e l s i n long- te rm exper imenta l d i a b e t e s . Federa t ion P r o c . 41 ( 5 ) : 1735. 16. Vadlamudi , R . V . S . V . , Wenkstern, B.M. and M c N e i l l , J . H . (1982) . I sopro te reno l induced changes i n c y c l i c AMP, phosphory lase and ino t ropy in normal and d i a b e t i c r a t h e a r t s . P roc . Can. Fed. B i o l . Soc . 2 5 : 89. 17. M c N e i l l , J . H . and Vadlamudi , R . V . S . V . (1983) . E f f e c t s o f acute and ch ron i c exper imenta l d iabe tes on r a t c a r d i a c c y c l i c AMP and phosphory lase a^  l e v e l s . Federa t ion P r o c . 42 ( 5 ) : 1358. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095892/manifest

Comment

Related Items