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UBC Theses and Dissertations

A study on three models of hypertension : genetic, mineralocorticoid, and ethanol-induced Chan, Thomas C. K. 1982

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A STUDY ON THREE MODELS OF HYPERTENSION: " GENETIC, MINERALOCORTICOID, AND ETHANOL-INDUCED by THOMAS C. K. CHAN B . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1977 A T H E S I S SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f P h a r m a c o l o g y , F a c u l t y o f M e d i c i n e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r 1982 0 Thomas C. K. C h a n , 1982 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 o f B r i t i s h Columbia, I agree t h a t 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 r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e 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 o f 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 t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f Pharmacology, Faculty of Medicine The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date March 16, 1983 )E-6 (3/81) ( i i ) ABSTRACT The Okamoto-Aoki s t ra in of spontaneously hypertensive rats (SHR) i s probably the most studied animal model of hypertension. Previous experience from our laboratory indicated that the vascular smooth muscle of the SHR i s hyper-reactive to pressor agents in v i t r o , and that the s e n s i t i v i t y to calcium i s also altered in these t i ssues. Since there is evidence for sodium ion and sodium pump abnormalities in erythrocytes of hypertensive patients and animals, the decision was made to use the erythrocyte membrane as a model for studying calcium-membrane interact ions in the SHR. The Ca + + /Mg + + ATPase a c t i v i t i e s (both high and low a f f i n i t y ) in the erythro-cyte membrane from SHR were measured and found to be s i gn i f i c an t l y elevated when compared to those of normotensive controls (WKY). Other erythrocyte membrane enzymes are not d i f fe rent between the two groups. Passive calcium in f lux into intact erythrocytes at 4°C was examined using ^ C a as tracer and a s i gn i f i cant increase in the passive permeability to calcium across the SHRs erythrocyte membrane was found when compared to the WKYs. Other abnorm-a l i t i e s in the SHR erythrocyte include a reduction in osmotic f r a g i l i t y and increased levels of membrane cho lestero l , phosphatidyl choline and sphingo-myelin with marginal decreases in phosphatidyl ethanolamine and phosphatidyl serine. These f indings indicate that structural and compositional a l t e r a -t ions are present in the SHR erythrocyte membrane and these may be related to the increased passive permeabil ity to calcium and the possibly compensa-tory increase in Ca + + /Mg + + ATPase a c t i v i t y described e a r l i e r . Studies ( i i i ) on t h e e r y t h r o c y t e membranes o f D O C A / s a l t h y p e r t e n s i v e (DOCA) r a t s r e v e a l e d no m a j o r d i f f e r e n c e s i n t h e a c t i v i t i e s o f t h e membrane enzymes b e t w e e n t h e DOCA and n e p h r e c t o m i z e d c o n t r o l s (NC) a n i m a l s . F u r t h e r m o r e , no d i f f e r e n c e was o b s e r v e d i n p a s s i v e c a l c i u m p e r m e a b i l i t y b e t w e e n t h e e r y t h r o c y t e s f r o m b o t h g r o u p s o f a n i m a l s . C o n t r a r y t o what was f o u n d i n t h e SHR, o s m o t i c f r a g i l i t y o f t h e DOCA e r y t h r o c y t e s i s s l i g h t l y h i g h e r t h a n t h a t o f NC's a t a l l NaCl c o n c e n t r a t i o n s s t u d i e d . No g r o s s d i f f e r e n c e s i n membrane c h o l e s -t e r o l and p h o s p h o l i p i d p r o f i l e were d e t e c t e d b e t w e e n t h e e r y t h r o c y t e membranes f r o m t h e two g r o u p s o f animals'. T h e s e f i n d i n g s c a n be v i e w e d as s u p p o r t i v e e v i d e n c e f o r t h e h y p o t h e s i s t h a t t h e membrane a b n o r m a l i t i e s o b s e r v e d i n t h e SHR e r y t h r o c y t e a r e p r o b a b l y g e n e t i c a l l y d e t e r m i n e d and n o t a c o n s e q u e n c e o f e l e v a t e d a r t e r i a l p r e s s u r e . I n t h e t h i r d model o f h y p e r t e n s i o n s t u d i e d , r a t s were g i v e n i n c r e a s i n g * amounts o f e t h a n o l i n t h e i r d r i n k i n g w a t e r o v e r p e r i o d o f 12 weeks, and a m i l d b u t s i g n i f i c a n t ( a p p r o x . 20%) e l e v a t i o n o f s y s t o l i c p r e s s u r e was . o b s e r v -a b l e b e g i n n i n g a t week 4 and p e r s i s t i n g t h r o u g h week 12. U r i n e i o n a n a l y s i s r e v e a l e d t h a t t h e e t h a n o l - t r e a t e d a n i m a l s h a v e r e d u c e d 24 h o u r u r i n a r y o u t p u t and s i g n i f i c a n t s o d i u m r e t e n t i o n . T h e s e f i n d i n g s p r o m p t e d us t o 14 s t u d y t h e p l a s m a v o l u m e i n t h e a n i m a l s u s i n g C - m e t h y l - Y - g l o b u l i n s as an i n d i c a t o r . T h e p l a s m a v o l u m e o f t h e e t h a n o l - t r e a t e d a n i m a l s a v e r a g e d 2 0 % h i g h e r t h a n t h a t o f t h e c o n t r o l a n i m a l s . P l a s m a c a t e c h o l a m i n e a n a l y s i s r e v e a l e d a s i g n i f i c a n t i n c r e a s e i n t h e l e v e l s o f c i r c u l a t i n g n o r a d r e n a l i n e i n t h e e t h a n o l - t r e a t e d a n i m a l s , b u t no d i f f e r e n c e s i n a d r e n a l i n e and d o p a -mine l e v e l s c o u l d be f o u n d when c o m p a r e d t o c o n t r o l s . I n v i t r o v a s c u l a r smooth m u s c l e r e s p o n s i v e n e s s and e r y t h r o c y t e membrane p r o p e r t i e s a r e n o t i * The t e r m e t h a n o l i s u s e d i n t e r c h a n g e a b l y w i t h a l c o h o l i n t h e t e x t . ( i v ) d i f f e r e n t b e t w e e n t h e two g r o u p s o f a n i m a l s . T h e s e r e s u l t s s u g g e s t t h a t t h e m i l d b l o o d p r e s s u r e e l e v a t i o n in t h e e t h a n o l - t r e a t e d a n i m a l s may be a s s o c i -a t e d w i t h b o t h p l a s m a v o l u m e e x p a n s i o n and e l e v a t e d l e v e l s o f c i r c u l a t i n g n o r a d r e n a l i n e , and i s p r o b a b l y n o t t h e r e s u l t of. i n c r e a s e d v a s c u l a r r e s p o n -s i v e n e s s . Membrane a l t e r a t i o n s o b s e r v e d i n SHR were n o t p r e s e n t i n t h e e r y t h r o c y t e s o f t h e e t h a n o l - t r e a t e d a n i m a l s . To f u r t h e r i n v e s t i g a t e t h e c o n f o u n d i n g f a c t o r s r e s p o n s i b l e f o r t h e b l o o d p r e s s u r e e l e v a t i o n i n e t h a n o l - t r e a t e d a n i m a l s , b o t h c o n t r o l s and a l c o h o l i c a n i m a l s were s u b j e c t e d t o h e a t s t r e s s b e g i n n i n g a t t h e 4th week o f e t h a n o l t r e a t m e n t , and a s t r a t i f i e d d i s t r i b u t i o n i n t h e mean b l o o d p r e s s u r e i s o b s e r v e d a t week 12. T h e c o n t r o l u n s t r e s s e d (CU) a n i m a l s h a v e t h e l o w e s t p r e s s u r e s , t h e c o n t r o l s t r e s s e d ' (CS) a n i m a l s h a v e s i g n i f i c a n t l y h i g h e r p r e s s u r e s t h a n CU g r o u p s , t h e a l c o h o l i c u n s t r e s s e d (AU) a n i m a l s have t h e second h i g h e s t p r e s s u r e v a l u e s , w i t h t h e a l c o h o l i c 'Stressed (AS) h a v i n g t h e h i g h e s t mean b l o o d p r e s s u r e o f a l l . P l a s m a v o l u m e i s n o t d i f f e r e n t among t h e two c o n t r o l (CU, CS) and t h e two a l c o h o l i c (AU, AS) g r o u p s . T h e p l a s m a n o r a d r e n a l i n e l e v e l s c o r r e l a t e d v e r y w e l l w i t h t h e e x t e n t o f b l o o d p r e s s u r e e l e v a t i o n and f o l l o w e d t h e same s t r a t i f i e d d i s t r i b u t i o n w i t h CU a n i m a l s h a v i n g t h e l o w e s t and AS a n i m a l s h a v i n g t h e h i g h e s t l e v e l s . T h e s e r e s u l t s s u g g e s t t h a t e t h a n o l and s t r e s s may i n t e r a c t t o p r o d u c e h y p e r t e n s i o n , and t h a t e l e v a t e d p l a s m a n o r a d r e n a l i n e l e v e l s may p l a y a r o l e i n t h e b l o o d p r e s s u r e e l e v a t i o n i n d u c e d by s t r e s s . (v) L I S T OF CONTENTS A b s t r a c t i i L i s t o f C o n t e n t s v L i s t o f T a b l e s i x L i s t o f F i g u r e s ' x i A c k n o w l e d g e m e n t s x i i i C h a p t e r I - INTRODUCTION I . H i s t o r i c a l B a c k g r o u n d N 1 I I . T h e C e n t r a l N e r v o u s S y s t e m and H y p e r t e n s i o n 3 I I I . T h e R e n i n - A n g i o t e n s i n S y s t e m and H y p t e r t e n s i o n 7 I V . T h e A d r e n a l C o r t e x , I t s Hormones, and H y p e r t e n s i o n 11 V. P r o s t a g l a n d i n s and H y p e r t e n s i o n - . 15 V I . A n i m a l M o d e l s o f H y p e r t e n s i o n 17 V I I . I n t r o d u c t i o n and S t a t e m e n t o f P r o b l e m s 24 C h a p t e r I I - METHODS AND MATERIALS I . P r e p a r a t i o n o f A n i m a l s A. G e n e t i c h y p e r t e n s i o n 28 B. D O C A / s a l t h y p e r t e n s i o n N 28 C. P h a r m a c o l o g i c a l model o f a l c o h o l i s m i n t h e ^ r a t 29 I I . Model o f S t r e s s 30 I I I . E r y t h r o c y t e P r e p a r a t i o n s 31 IV. Membrane Enzyme A s s a y s 31 ( v i ) V . Membrane C o m p o s i t i o n a l S t u d i e s A. T o t a l p h o s p h o l i p i d e s t i m a t i o n 34 B. P h o s p h o l i p i d p r o f j l e 34 C. C h o l e s t e r o l d e t e r m i n a t i o n 35 V I . O s m o t i c F r a g i l i t y o f E r y t h r o c y t e s 36 V I I . P a s s i v e C a l c i u m I n f l u x M e a s u r e m e n t s i n I n t a c t E r y t h r o c y t e s A. C a l c i u m l o a d i n g 36 B. P r e p a r a t i o n o f r e d c e l l s f o r LSC 37 C. N e u r a m i n i d a s e d i g e s t i o n 38 D. S i a l i c a c i d m e a s u r e m e n t s 1 38 V I I I . L a n g e n d o r f f ' s P e r f u s i o n i n C h r o n i c A l c o h o l - T r e a t e d A n i m a l s A. P r e p a r a t i o n and i s o l a t i o n o f h e a r t s , 39 B. S t a r l i n g c u r v e s o f t h e i s o l a t e d p e r f u s e d h e a r t s 39 C. Dose r e s p o n s e r e l a t i o n s h i p t o IPNA 40 I X . P l a s m a V olume M e a s u r e m e n t s 41 X. P l a s m a C a t e c h o l a m i n e M e a s u r e m e n t s A. Sample p r e p a r a t i o n 42 B. H i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y w i t h 42 e l e c t r o c h e m i c a l d e t e c t i o n X I . M u s c l e B a t h S t u d i e s 43 X I I . S t a t i s t i c a l A n a l y s i s 44 C h a p t e r I I I - RESULTS I . G e n e t i c Model o f H y p e r t e n s i o n A. T i m e c o u r s e o f h y p e r t e n s i o n , d e v e l o p m e n t 45 ' B. E r y t h r o c y t e membrane enzyme a s s a y s 45 ( v i i ) C. ^ C a l c i u m i n f l u x m e a s u r e m e n t s 52 D. O s m o t i c f r a g i l i t y m e a s u r e m e n t s 54 E. E r y t h r o c y t e membrane c o m p o s i t i o n a l a n a l y s e s 54 I I . E x p e r i m e n t a l Model o f H y p e r t e n s i o n ( D O C A / s a l t ) A. T i m e c o u r s e o f h y p e r t e n s i o n d e v e l o p m e n t 58 B. E r y t h r o c y t e membranes enzyme a s s a y s 60 C. C a l c i u m i n f l u x m e a s u r e m e n t s 60 D. O s m o t i c f r a g i l i t y m e a s u r e m e n t s 60 E . E r y t h r o c y t e membrane c o m p o s i t i o n a l a n a l y s e s 63 I I I . P h a r m a c o l o g i c a l Model o f A l c o h o l i s m A. E t h a n o l c o n s u m p t i o n and f o o d i n t a k e 63 B. T o t a l c a l o r i c i n t a k e e s t i m a t i o n 65 C. Body and h e a r t w e i g h t m e a s u r e m e n t s 65 D. B l o o d p r e s s u r e m e a s u r e m e n t s 70 E . C a r d i a c p e r f o r m a n c e 70 F. P l a s m a and u r i n e i o n a n a l y s e s ' 77 6. P l a s m a v o l u m e m e a s u r e m e n t s 80 H. I n v i t r o v a s c u l a r smooth m u s c l e r e s p o n s i v e n e s s 80 I . E r y t h r o c y t e membrane c h a r a c t e r i z a t i o n 82 IV. C h r o n i c A l c o h o l and H e a t S t r e s s A. O b s e r v a t i o n s on t h e a n i m a l s ' 86 B. T i m e c o u r s e o f b l o o d p r e s s u r e d e v e l o p m e n t 87 C. P l a s m a v o l u m e m e a s u r e m e n t s 87 D. P l a s m a c a t e c h o l a m i n e d e t e r m i n a t i o n s 91 ( v i i i ) \ C h a p t e r IV - DISCUSSION j I . G e n e t i c s and H y p e r t e n s i o n . 93 I I . Membrane A l t e r a t i o n s i n t h e SHR 95 I I I . S t u d i e s o f E r y t h r o c y t e s Membranes f r o m D O C A / s a l t 98 H y p e r t e n s i v e R a t s I V . C o m p a r i s o n o f t h e Two M o d e l s o f H y p e r t e n s i o n 99 V. A l c o h o l and C a r d i o v a s c u l a r D i s e a s e s 103 V I . A n i m a l M o d e l s o f A l c o h o l i s m 105 V I I . C h r o n i c E t h a n o l C o n s u m p t i o n and C a r d i o v a s c u l a r 106 F u n c t i o n i n t h e R a t V I I I . S t r e s s and C h r o n i c E t h a n o l C o n s u m p t i o n 111 I X . G e n e r a l C o n c l u s i o n s 114 C h a p t e r V - B i b l i o g r a p h y - 118 A p p e n d i x { ( i x ) L I S T OF TABLES T a b l e 1. R e a g e n t T a b l e 33 2. B a s e l i n e m e a s u r e m e n t s o f d i f f e r e n t g r o u p s o f a n i m a l s 46 3. C a + + / M g + + A T P a s e a c t i v i t i e s i n t h e e r y t h r o c y t e g h o s t s 47 f r o m t h e g r o u p s o f r a t s 4. A c t i v a t i o n e n e r g i e s f o r C a + + / M g + + A T P a s e s 51 5. N a + / K + A T P a s e a c t i v i t i e s i n e r y t h r o c y t e g h o s t s f r o m 51 t h r e e g r o u p s o f r a t s 6. P a s s i v e c a l c i u m i n f l u x i n t o e r y t h r o c y t e s f r o m SHRs and WKYs 55 7. WKY and SHR e r y t h r o c y t e membrane c o m p o s i t i o n a l a n a l y s e s 57 8. B l o o d p r e s s u r e s and A T P a s e a c t i v i t i e s o f NC and DOCA 59 a n i m a l s a t Week 4 9. P a s s i v e c a l c i u m i n f l u x i n t o e r y t h r o c y t e s f r o m DOCA and 61 NC a n i m a l s 10. Membrane c o m p o s i t i o n a l a n a l y s e s on e r y t h r o c y t e s f r o m DOCA 64 and NC a n i m a l s , 1 1 . E s t i m a t e d a v e r a g e d a i l y c a l o r i c i n t a k e o f t h e c o n t r o l and 67 a l c o h o l - t r e a t e d a n i m a l s 12. R e l a t i o n s h i p o f h e a r t t o body w e i g h t s i n c o n t r o l and' 69 a l c o h o l - t r e a t e d a n i m a l s 1 3 . C a r d i a c p e r f o r m a n c e d a t a 72 14. I o n a n a l y s e s o f p l a s m a f r o m a l c o h o l - t r e a t e d and c o n t r o l r a t s 78 15. I o n a n a l y s e s o f u r i n e f r o m a l c o h o l - t r e a t e d and c o n t r o l r a t s 79 (x) 16. A c t i v i t i e s of erythrocyte membrane enzymes from chronic 83 a l coho l - t reated and control animals 17. Cholesterol and phospholipid analyses of erythrocyte 84 membrane and plasma from chronic a l coho l - t rea ted and contro l rats 18. Various measurements taken at Week 12 on groups of 89 a l coho l - t rea ted and contro l rats 19. PIasma volume . 90 20. Plasma catecholamines 92 ( x i ) L I S T OF FIGURES F i g u r e 1. The R e n i n - a n g i o t e n s i n s y s t e m 9 2. O u t l i n e o f hormone s y s t h e s i s i n t h e z o n a g l o m e r u l o s a o f t h e a d r e n a l c o r t e x 12 3. C a l c i u m d e p e n d e n c e c u r v e s o f t h e e r y t h r o c y t e membrane C a + + / M g + + A T P a s e f r o m WKYs and SHRs 49 4. A r r h e n i u s p l o t s o f t h e C a + + / M g + + A T P a s e i n a t y p i c a l e x p e r i m e n t 50 5. 45 ++ T i m e c o u r s e o f Ca u p t a k e i n t o e r y t h r o c y t e s f r o m WKYs and SHRs 53 6. O s m o t i c f r a g i l i t y o f e r y t h r o c y t e s f r o m WKYs and SHRs 56 7. O s m o t i c f r a g i l i t y o f e r y t h r o c y t e s f r o m NCs and DOCAs 62 8. D a i l y f o o d i n t a k e o f c o n t r o l and e t h a n o l - t r e a t e d a n i m a l s 66 9. B o d y w e i g h t s o f c o n t r o l and e t h a n o l - t r e a t e d a n i m a l s 68 10. S y s t o l i c b l o o d p r e s s u r e d e v e l o p m e n t o f b o t h e t h a n o l - t r e a t e d and c o n t r o l a n i m a l s 71 1 1 . T y p i c a l S t a r l i n g c u r v e s f r o m one i s o l a t e d p e r f u s e d h e a r t e x p e r i m e n t 74 12. D o s e - r e s p o n s e o f t h e i s o l a t e d p e r f u s e d h e a r t s t o IPNA i n t h e p r e s e n c e o f 2.5 mM C a + + 75 13. D o s e - r e s p o n s e o f t h e i s o l a t e d p e r f u s e d h e a r t s ^ t o IPNA i n t h e p r e s e n c e o f 0.2 mM C a + + 76 (xi i ) D o s e - r e s p o n s e c u r v e s o f i s o l a t e d l o n g i t u d i n a l p o r t a l v e i n s t r i p s t o n o r a d r e n a l i n e i n t h e p r e s e n c e o f 2.5, 0.4, and 0.2 mM e x t e r n a l c a l c i u m C o n t i n u o u s b l o o d p r e s s u r e t r a c i n g f r o m a c h r o n i c t a i l a r t e r y c a n n u l a i m p l a n t i n a m a l e W i s t a r r a t s u b j e c t e d t o h e a t s t r e s s B l o o d p r e s s u r e d e v e l o p m e n t o f f o u r g r o u p s o f r a t s d u r i n g t h e 12-week p r o t o c o l ( x i i i ) ACKNOWLEDGEMENTS The a u t h o r w o u l d l i k e t o t h a n k D r . M o r l e y C. S u t t e r f o r h i s c o n t i n u a l s u p p o r t and c o n f i d e n c e ; D r s . D. V. G o d i n and C. C. Y. P a n g f o r t h e i r many h e l p f u l s u g g e s t i o n s and e n c o u r a g e m e n t s ; D r . R. A. W a l l f o r h i s a d v i c e on HPLC t e c h n q u e s ; and t h e members o f t h e S u p e r v i s o r y C o m m i t t e e ( D r s . J . G. F o u l k s , V. P a l a t y , B. D. R o u f o g a l i s and M. J . A. W a l k e r ) f o r t h e i r a d v i c e and s u p p o r t t h r o u g h o u t t h e p r o j e c t . The e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f Mr. G e o r g e C h u a , M r s . T h e r e s e Ng, M i s s M a u r e e n G a r n e t t , M r s . R o b i n a Kay, M i s s V i c k i M ontgomery, M r s . C a r l a C u m b e r b i r c h , and Mr. R a l p h L u i , i s g r a t e f u l l y a c k n o w l e g e d . T h e a u t h o r i s e s p e c i a l l y g r a t e f u l t o M i s s T r a c y S l o c o m b e f o r h e r e x c e l -l e n t s e c r e t a r i a l h e l p i n p r e p a r i n g t h i s document.. - 1 -INTRODUCTION I . H i s t o r i c a l B a c k g r o u n d E l e v a t i o n s o f a r t e r i a l p r e s s u r e h a v e b e e n r e c o g n i z e d f o r many d e c a d e s by t h e l i f e i n s u r a n c e i n d u s t r y as an u n f a v o u r a b l e r i s k on t h e m o r t a l i t y r a t e . The e x p e r i e n c e o f a g r o u p o f 26 l a r g e i n s u r a n c e c o m p a n i e s , w i t h o v e r 4 m i l l i o n p o l i c i e s i s s u e d f r o m 1935 t o 1953 were s u m m a r i z e d i n t h e s t u d y o f t h e S o c i e t y o f A c t u a r i e s r e l e a s e d i n 1959 ( 1 ) . T h i s s t u d y r e v e a l e d a p o s i -t i v e c o r r e l a t i o n b e t w e e n e l e v a t e d b l o o d p r e s s u r e and i n c r e a s e d m o r t a l i t y and t h a t t h e i n c r e a s e d r i s k seemed t o c l o s e l y p a r a l l e l t h e d e g r e e o f b l o o d p r e s -s u r e e l e v a t i o n . K a n n e l and h i s c o l l e a g u e s p r o v i d e d f u r t h e r p r o o f f o r t h i s k i n d o f a s s o c i a t i o n , c o n c l u d i n g t h a t t h e r i s k o f . d e a t h i n c r e a s e s i n p r o p o r -t i o n t o t h e l e v e l o f s y s t o l i c b l o o d p r e s s u r e e l e v a t i o n and t h a t t h e r e i s "no c r i t i c a l v a l u e o f b l o o d p r e s s u r e t o i n d i c a t e w here l e t h a l h y p e r t e n s i o n b e g i n s " ( 2 ) . P a u l r e p o r t e d s i m i l a r f i n d i n g s s h o w i n g a s t r o n g c o r r e l a t i o n b e t w e e n t h e d e g r e e o f d i a s t o l i c p r e s s u r e e l e v a t i o n and i n c r e a s e d m o r t a l i t y r a t e s ( t o t a l as w e l l as c a r d i o v a s c u l a r - r e l a t e d ) ( 3 ) . T h e s e and o t h e r more r e c e n t s t u d i e s h a v e b o r n e o u t t h e f a c t t h a t t h e r e i s no c l e a r c u t d i v i s i o n b e t w e e n o p t i m a l and p a t h o l o g i c a l b l o o d p r e s s u r e s . M o r t a l i t y r i s e s as b o t h s y s t o l i c and d i a s t o l i c b l o o d p r e s s u r e r i s e . S i n c e , i n m o s t ' p o p u l a t i o n s , t h e mean s y s t o l i c p r e s s u r e i n c r e a s e s n o n - 1 i n e a r l y w i t h age ( 4 , 5 ) , t h e d i a s t o l i c p r e s s u r e i s u s e d i n most o f t h e c u r r e n t c l a s s i f i c a t i o n s o f h y p e r t e n s i o n b e c a u s e t h e i n c r e a s e s o b s e r v e d were s m a l l e r and more c o n s i s t e n t . G e n e r a l l y , p a t i e n t s w i t h d i a s t o l i c p r e s s u r e above 90 mm Hg a r e c o n s i d e r e d m i l d l y h y p e r -t e n s i v e ; t h o s e w i t h p r e s s u r e s a b o v e 105 mm Hg a r e c o n s i d e r e d m o d e r a t e l y h y p e r t e n s i v e ; t h o s e w i t h p r e s s u r e s a b o v e 115 mm Hg a r e c o n s i d e r e d s e v e r e l y - 2 -h y p e r t e n s i v e . P a t i e n t s w i t h a d i a s t o l i c p r e s s u r e a bove 130 mm Hg a r e c o n s i -d e r e d m a l i g n a n t l y h y p e r t e n s i v e and a r e u s u a l l y t r e a t e d as m e d i c a l e m e r g e n -c i e s ( 6 ) . H y p e r t e n s i o n i s now r e c o g n i s e d as one o f t h e most i m p o r t a n t r i s k f a c t o r s i n t h e p a t h o g e n e s i s o f c a r d i o v a s c u l a r and c e r e b r o v a s c u l a r d i s e a s e s i n man ( 7 ) . E s s e n t i a l h y p e r t e n s i o n i s a c o n d i t i o n i n w h i c h no s p e c i f i c A e t i o l o g y h a s b e e n i d e n t i f i e d and i s r e s p o n s i b l e f o r o v e r 8 0 % o f h y p e r t e n s i o n d i a g n o s e d ( 8 ) . A l t h o u g h i t has been shown q u i t e c o n c l u s i v e l y t h a t p a t i e n t s w i t h d i a s t o l i c p r e s s u r e s a b o v e 104 mm Hg b e n e f i t f r o m a n t i h y p e r t e n s i v e t r e a t m e n t s i n t e r m s o f r e d u c e d m o r b i d i t y and m o r t a l i t y ( 9 - 1 1 ) , h y p e r t e n s i o n i s s t i l l one o f t h e most u n d e r - t r e a t e d d i s e a s e s o f o u r t i m e . Conway e s t i m a t e d t h a t o n l y 1 5 % o f t h e h y p e r t e n s i v e p o p u l a t i o n i s a d e q u a t e l y t r e a t e d by t h e i r p h y s i c i a n s ( 1 2 ) . T h e r e a r e s e v e r a l r e a s o n s f o r t h i s p a r a d o x e x i s t i n g i n t h e ' m i d s t o f a m e d i c a l l y and s c i e n t i f i c a l l y a d v a n c e d s o c i e t y s u c h a s o u r s . F i r s t and f o r e m o s t , i s t h e i n s i d i o u s n a t u r e o f h y p e r t e n s i o n . M o st p a t i e n t s w i t h e l e v a t e d b l o o d p r e s s u r e a r e c o m p l e t e l y a s y m p t o m a t i c and go on n o r m a l l y w i t h t h e i r d a i l y r o u t i n e . T h e i r c o n d i t i o n s a r o u s e a t t e n t i o n o n l y when t h e y go f o r p e r i o d i c m e d i c a l e x a m i n a t i o n s , s u c h as t h o s e t o c o m p l y w i t h i n s u r a n c e o r m i l i t a r y r e q u i r e m e n t s ( 1 3 ) . A s e c o n d r e a s o n f o r u n d e r t r e a t m e n t o f e l e v a -t e d b l o o d p r e s s u r e i s o u r l a c k o f k n o w l e d g e c o n c e r n i n g t h e e t i o l o g y o f h y p e r -t e n s i o n . P h y s i c i a n s a r e i n a d i l e m m a i n t r y i n g t o f i n d a r a t i o n a l t r e a t m e n t f o r a c o n d i t i o n whereby, t h e s u s t a i n i n g m e c h a n i s m s a r e l a r g e l y unknown, r e a l i z i n g t h e f a c t t h a t o n c e i n i t i a t e d , a n t i h y p e r t e n s i v e m e d i c a t i o n s and , t h e i r t o x i c i t i e s w i l l s t a y w i t h t h e p a t i e n t f o r l i f e . The t h i r d r e a s o n f o r u n d e r t r e a t m e n t o f h y p e r t e n s i o n a l s o r e f l e c t s o u r l a c k o f k n o w l e d g e i n t h e a r e a . A l t h o u g h t h e r e i s ample e v i d e n c e f o r i m p r o v e d p r o g n o s i s i n m o d e r a t e l y h y p e r t e n s i v e p a t i e n t s (104 mm Hg) r e c e i v i n g t r e a t m e n t , t h e r e a r e s t i l l - 3 -a r g u m e n t s as t o w h e t h e r t h e m i l d l y h y p e r t e n s i v e p a t i e n t (90-104 mm Hg) s h o u l d be a g g r e s s i v e l y t r e a t e d , as t h e r e i s c o n f l i c t i n g e v i d e n c e r e g a r d i n g i m p r o v e d p r o g n o s i s w i t h t r e a t m e n t i n t h i s g r o u p ( 1 4 , 1 5 ) . M o r e o v e r , t h e r e i s s t i l l c o n s i d e r a b l e a r g u m e n t r e g a r d i n g t h e e n d - p o i n t o f a n t i h y p e r t e n s i v e t h e r a p y . I t i s a d i f f i c u l t b a l a n c e i n d e e d t o j u g g l e m i n i m a l t o x i c i t i e s , l o w e s t p o s s i b l e d o s e o f d r u g ( s ) , and maximal r e d u c t i o n o f b l o o d p r e s s u r e . T h e " s t e p p e d c a r e a p p r o a c h " recommended by t h e J o i n t N a t i o n a l C o m m i t t e e on D e t e c t i o n , E v a l u a t i o n and T r e a t m e n t o f H i g h B l o o d P r e s s u r e ( 6 ) a d d r e s s e d t h e p r o b l e m d i r e c t l y and made t h e r a p e u t i c r e g i m e n s more c o n s i s t e n t among p h y s i -c i a n s . T h i s a p p r o a c h has l a r g e l y e v o l v e d e m p i r i c a l l y f r o m e x p e r i e n c e a c c u m u l a t e d by c e n t r e s a l l o v e r t h e w o r l d . We a r e s t i l l f a r away f r o m t h e i d e a l s i t u a t i o n w h e r e b y we c a n t r e a t o r p r e v e n t h y p e r t e n s i o n a t i t s c a u s e — an a s p i r a t i o n t h a t i s i m p o s s i b l e t o a t t a i n u n t i l we c a n u n d e r s t a n d t h e m e c hanisms s u s t a i n i n g t h e h y p e r t e n s i v e s t a t e i n p a t i e n t s . I n o r d e r t o .gain some i n s i g h t i n t o t h e a e t i o l o g y o f h y p e r t e n s i o n , a b r i e f r e v i e w o f what a r e c u r r e n t l y a c c e p t e d as p o s s i b l e f a c t o r s p l a y i n g a r o l e i n h y p e r t e n s i o n i s i n o r d e r . 11. T h e C e n t r a l N e r v o u s S y s t e m and H y p e r t e n s i o n In t h e e a r l y t w e n t i e s , K a r p l u s and K r e i d l e showed t h a t t h e y c o u l d d r a m a -t i c a l l y i n c r e a s e t h e b l o o d p r e s s u r e i n c a t s by s t i m u l a t i n g t h e h y p o t h a l a m u s e l e c t r i c a l l y ( 1 6 ) . T h e s e f i n d i n g s s p a r k e d a h o s t o f r e s e a r c h a c t i v i t i e s w h e r e b y d i f f e r e n t g r o u p s o f w o r k e r s mapped t h e h y p o t h a l a m u s and t h e m i d b r a i n i n b o t h a n a e s t h e t i z e d and c o n s c i o u s a n i m a l s , t a b u l a t i n g t h e a r e a s w here sym-p a t h e t i c r e s p o n s e s c o u l d be e l i c i t e d ( 1 7 , 1 8 ) . F o l k o w and Uvnas b o t h f o u n d - 4 -t h a t s t i m u l a t i o n o f t h e p e r i f o r n i c a l r e g i o n i n t h e l a t e r a l h y p o t h a l a m u s and. p a r t s o f t h e tegmentum and c e n t r a l g r e y m a t t e r i n t h e m i d b r a i n e l i c i t e d a c o m p l e x s y m p a t h e t i c d i s c h a r g e p a t t e r n l e a d i n g t o a t r a n s i e n t e l e v a t i o n i n b l o o d p r e s s u r e and i n c r e a s e d t h e h e a r t r a t e , s t r o k e v o l u m e , and c a r d i a c o u t p u t . V a s o c o n s t r i c t i o n was a l s o o b s e r v e d i n s e v e r a l v a s c u l a r b e d s s u c h as t h o s e o f t h e k i d n e y , t h e i n t e s t i n e and t h e s k i n ' ( 1 9 , 2 0 ) . T h e s e c a r d i o v a s c u -l a r e f f e c t s were l a t e r a s s o c i a t e d w i t h a c o l l e c t i o n o f b e h a v i o u r a l t r a i t s known as t h e " d e f e n s e b e h a v i o u r " ( 2 1 ) . A t t e m p t s t o p r o d u c e a l o n g e r l a s t i n g o r p e r m a n e n t p r e s s o r r e a c t i o n i n a n i m a l s by c h r o n i c r e p e t i t i v e h y p o t h a l a m i c s t i m u l a t i o n h a v e been l a r g e l y u n s u c c e s s f u l ( 2 2 , 2 3 ) . More r e c e n t l y , s e v e r a l g r o u p s o f w o r k e r s f o u n d t h e t e c h n i q u e o f o p e r a n t c o n d i t i o n i n g ' u s e f u l i n p r o d u c i n g more s u s t a i n e d and more r e p r o d u c i b l e b l o o d p r e s s u r e e l e v a t i o n s i n v a r i o u s p r i m a t e s ( 2 4 , 2 5 ) . T h e s e t y p e s o f c o n d i t i o n i n g p r o d u c e b o t h e m o t i o n a l and p h y s i c a l s t r e s s e s i n t h e a n i m a l s s t u d i e d and may p r o v e u s e f u l i n t h e s t u d y o f e x p e r i m e n t a l h y p e r t e n s i o n i n t h e l a b o r a t o r y . E f f o r t s i n m a r s h a l l i n g t h e p o s s i b l e a s s o c i a t i o n b e t w e e n human e s s e n t i a l h y p e r t e n s i o n and h y p o t h a l a m i c " d e f e n s e r e a c t i o n " h a v e b e e n p r o m i s i n g b u t f a r f r o m c o n v i n c i n g . B r o d and h i s c o w o r k e r s showed t h a t a c u t e e m o t i o n a l s t r e s s i n d u c e d i n humans by m e n t a l a r i t h m e t i c r e s u l t e d i n haemodynamic p a t t e r n s s i m i l a r t o t h o s e s e e n i n t h e c a t a f t e r h y p o t h a l a m i c s t i m u l a t i o n ( 2 6 ) . C o m p a r a b l e p a t t e r n s were a l s o o b s e r v e d i n s e v e r a l p a t i e n t s w i t h b o r d e r l i n e h y p e r t e n s i o n ( 2 7 ) . P a r a l l e l o b s e r v a t i o n s , h o w e v e r , we're n o t s e e n i n p a t i e n t s w i t h s u s t a i n e d e s s e n t i a l h y p e r t e n s i o n , w here h e a r t r a t e s and c a r d i a c o u t p u t s r e m a i n e d n o r m a l ( 2 8 ) . T h e s e e s s e n t i a l h y p e r t e n s i v e p a t i e n t s , n o n e t h e l e s s , d i d h a v e a h i g h r e n a l v a s c u l a r r e s i s t a n c e w h i c h p a r a l l e l e d t h e o b s e r v a t i o n s made i n e x p e r i m e n t a l a n i m a l s . T h e a bove f i n d i n g s and o t h e r s i m i l a r r e s u l t s a r e s u g g e s t i v e t h a t t h e s y m p a t h e t i c n e r v o u s s y s t e m i s i n v o l v e d i n t h e i n i t i -a t i o n o f h y p e r t e n s i o n . - I - 5 -Evidence for the sympathetic nervous system playing a ro le in the maintainance of hypertension also has come i nd i r e c t l y from studies^on the haemodynamics of humans and animals during sleep. Zanchetti studied sleep patterns of cats and found that when the animals entered a stage of "desyn-chronized sleep" ( s imi lar to REM sleep in humans), there were large reduc-tions in both sy s to l i c and d i a s t o l i c pressures (29). He used th i s sleep stage as a model of natural suppression of sympathetic a c t i v i t y . In cats made hypertensive by b i l a t e r a l renal artery stenosis, he found that the removal of baroreceptor regulation by s inoaort ic deafferentation from one group resulted in larger drops in blood pressure during desynchronized sleep in these animals, while no differences between the groups could be observed during wakefulness (30,31). The fact that s inoaort ic deafferentation r e s u l -ted in non-hypertensive blood pressure values in "desynchronized sleep" was thought to provide evidence .for the sympathetic nervous system playing a ro le in the maintainance of hypertension. This type of argument is st imula-t ing and the experimental evidence i s in teres t ing , but to conclude from the above mentioned observations that the sympathetic nervous system plays a ro le in hypertension i s a l i t t l e premature. F i r s t of a l l , renovascular hypertension is not primary hypertension of unknown aetiology. Therefore, conclusions drawn from a model of secondary experimental hypertension and re la t ing these to mechanisms of essential hypertension are at best sugges-t i v e . Secondly, the v a l i d i t y of "desynchronized sleep" as a model of sympa-thet ic suppression has not been proven; and f i n a l l y , other homeostatic mechanisms also responsible for the regulation of blood pressure (e.g. renin-angiotensin) were not monitored simultaneously and therefore the i r r e l a t i ve ro le ( i f any) in th i s model of hypertension is unknown. - 6 -P e r h a p s t h e r e s u l t s f r o m c a t e c h o l a m i n e m e a s u r e m e n t s i n h y p e r t e n s i v e p a t i e n t s p r o v i d e more s u p p o r t f o r s y m p a t h e t i c i n v o l v e m e n t i n h y p e r -t e n s i o n . T h e r e h a v e b e e n r e p o r t s f r o m s e v e r a l i n v e s t i g a t o r s s i n c e 1965 c l a i m i n g i n c r e a s e d u r i n a r y l e v e l s o f c a t e c h o l a m i n e s and t h e i r m e t a b o l i t e s i n h y p e r t e n s i v e i n d i v i d u a l s ( s e e r e v i e w by de C h a m p l a i n ; 3 2 ) . T h e s e f i n d i n g s , t h o u g h i n t e r e s t i n g , do n o t p r o v i d e t h e e v i d e n c e n e e d e d b e c a u s e t h e v a l i d i t y o f u r i n e c a t e c h o l a m i n e l e v e l s as an a c c u r a t e i n d e x o f s y m p a t h e t i c a c t i v i t y has n o t b e e n e s t a b l i s h e d . U r i n a r y l e v e l s m e r e l y r e p r e s e n t t h e g r o s s summa-t i o n o f a l l e v e n t s i n t h e b o d y and t h e r e i s no a c c u r a t e way t h a t one c a n d e t e r m i n e t h e c o n t r i b u t i o n f r o m c a r d i o v a s c u l a r s y m p a t h e t i c f i b e r s v e r s u s t h a t o f o t h e r o r g a n s . T h e same p r o b l e m h o l d s t r u e f o r some o f t h e more r e c e n t r e p o r t s w h e r e b y t h e c i r c u l a t i n g l e v e l s o f c a t e c h o l a m i n e s i n t h e p l a s m a were m e a s u r e d a c c u r a t e l y u s i n g r a d i o e n z y m a t i c o r c h r o m a t o g r a p h i c t e c h n i q u e s . N o n e t h e l e s s , i t i s i n t e r e s t i n g t o n o t e t h a t t h e r e i s good a g r e e -ment among s e v e r a l g r o u p s o f w o r k e r s s t u d y i n g u n t r e a t e d e s s e n t i a l h y p e r t e n -s i v e p a t i e n t s ( 3 3 - 3 8 ) . A l l o f t h e s t u d i e s r e p o r t e d e l e v a t e d a v e r a g e d l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s o r n o r a d r e n a l i n e i n p a t i e n t s w i t h e s s e n t i a l , h y p e r t e n s i o n . F u r t h e r a n a l y s e s r e v e a l e d t h a t t h e h y p e r t e n s i v e p a t i e n t s c o u l d be c l a s s i f i e d i n t o two s u b g r o u p s , w i t h 4 0 % o f them i n t h e " h y p e r - a d r e -n e r g i c " g r o u p and t h e r e s t i n t h e " n o r m o - a d r e n e r g i c " g r o u p w h i l e no s u c h d i f f e r e n c e s were s e e n i n n o r m o t e n s i v e c o n t r o l s ( 3 9 ) . A l t h o u g h t h e number o f p a t i e n t s s t u d i e d was r e l a t i v e l y s m a l l , t h e o b s e r v a t i o n t h a t t h e r e was a g r o u p o f h y p e r t e n s i v e p a t i e n t s w i t h i n c r e a s e d b a s a l s y m p a t h e t i c t o n e seems t o be v a l i d . T h e p a t h o 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 t h i s o b s e r v a t i o n , h o w e v e r , r e m a i n s t o be e l u c i d a t e d . I n a d d i t i o n t o h i g h e r l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s , - t h e " h y p e r - a d r e n e r g i c " p a t i e n t s a l s o showed e n h a n c e d . s y m p a t h e t i c r e s p o n s e s as e v i d e n t f r o m t h e much l a r g e r i n c r e a s e s i n c a t e c h o l a m i n e ' l e v e l s when t h e p a t i e n t s c h a n g e d f r o m t h e s u p i n e t o t h e e r e c t - 7 -p o s t u r e ( 4 0 ) . T h e s e i n c r e a s e s were b r o u g h t back t o n o r m a l l e v e l s w i t h e - a n t a g o n i s t t r e a t m e n t . I n t e r e s t i n g l y e n o u g h , b o t h E s l e r and de C h a m p l a i n n o t i c e d t h a t B - b l o c k a d e was much more e f f e c t i v e i n r e d u c i n g t h e b l o o d p r e s s u r e o f p a t i e n t s with- h i g h c i r c u l a t i n g l e v e l s o f n o r a d r e n a l i n e and c a t e c h o l a m i n e s ( 4 1 - 4 3 ) . I n summary, t h e a b o v e m e n t i o n e d f i n d i n g s f r o m e x p e r i m e n t a l h y p e r t e n s i o n i n a n i m a l s and e s s e n t i a l h y p e r t e n s i o n i n humans seem t o s u g g e s t t h a t t h e s y m p a t h e t i c n e r v o u s s y s t e m i s a s s o c i a t e d w i t h h y p e r -t e n s i o n . I n t h e 2 5 - 4 0 % o f h y p e r t e n s i v e p a t i e n t s who h a v e h i g h l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s , t h e a s s o c i a t i o n i s s t r o n g and f a i r l y c o n c l u s i v e . I n t h e r e m a i n i n g m a j o r i t y , t h e f a c t o r s r e s p o n s i b l e f o r t h e i n i t i a t i o n and m a i n t e n a n c e o f h y p e r t e n s i o n r e m a i n unknown, and more r e s e a r c h i s r e q u i r e d b e f o r e one c a n u n d e r s t a n d t h e o t h e r m e c h a n i s m s and t h e i r i n t e r p l a y i n h y p e r -t e n s i o n . I I I . The R e n i n - A n g i o t e n s i n S y s t e m and H y p e r t e n s i o n A s f a r back as 1836, B r i g h t , made t h e a s s o c i a t i o n b e t w e e n r e n a l d i s e a s e and c a r d i a c h y p e r t r o p h y ( 4 4 ) . T h u s , t h e i d e a t h a t p a t h o l o g i c a l c h a n g e s i n t h e k i d n e y c o u l d c a u s e h y p e r t e n s i o n i s n o t new. V o l h a r d and F a h r were amongst t h e f i r s t t o v o i c e t h e i r s u p p o r t and d i d much t o f u r t h e r t h e k n o w l e d g e i n t h i s a r e a ( 4 5 ) . A t t e m p t s were made s u b s e q u e n t l y t o t r y t o p r o d u c e h y p e r t e n s i o n by a l t e r i n g r e n a l f u n c t i o n , w i t h m i n i m a l s u c c e s s and maximal c o n t r a d i c t i o n ( s e e r e v i e w by G o l d b l a t t ; 4 6 ) . G o l d b l a t t ' s g r o u p i n 1934 u s e d a model w h e r e b y dogs were made p e r s i s t e n t l y h y p e r t e n s i v e by r e n a l a r t e r i a l c o n s t r i c t i o n u s i n g s i l v e r c l a m p s ( 4 7 ) . T h e s e r e s u l t s were amply c o n f i r m e d by o t h e r l a b o r a t o r i e s , and t h e f a c t t h a t r e m o v a l o f a l a r g e - 8 -p o r t i o n o f t h e n e r v o u s s y s t e m r e s p o n s i b l e f o r v a s o m o t o r r e g u l a t i o n d i d n o t p r e v e n t o r a b o l i s h t h e h y p e r t e n s i o n p r o d u c e d s u g g e s t e d t h a t h u m o r a l mechan-i s m s were i n v o l v e d ( 4 8 - 5 1 ) . T h e r e f o r e , t h e s e a r c h f o r t h e p r e s s o r s u b s t a n c e , p o s s i b l y a s s o c i a t e d w i t h t h e k i d n e y , was o n . A f t e r some e x p e r i m e n t a t i o n w i t h g r a f t i n g k i d n e y s f r o m h y p e r t e n s i v e a n i m a l s t o n e p h r e c t o m i z e d n o r m o t e n -s i v e a n i m a l s , i t was c l e a r t h a t t h e i s c h a e m i c k i d n e y s s e c r e t e d a p r e s s o r s u b s t a n c e t h a t r e p r o d u c i b l y e l e v a t e d t h e a r t e r i a l b l o o d p r e s s u r e i n t h e r e c i p i e n t s ( 5 2 ) . In 1939, a s u b s t a n c e c a l l e d h y p e r t e n s i n was e x t r a c t e d f r o m r e n a l b l o o d o f i s c h a e m i c k i d n e y s ( 5 3 ) . T h i s was a p o t e n t v a s o c o n s t r i c t o r w i t h p r o p e r t i e s d i f f e r e n t f r o m t h o s e o f o t h e r p r e s s o r a g e n t s i n c l u d i n g r e n i n . F u r t h e r work by t h e same g r o u p o f r e s e a r c h e r s showed t h a t r e n i n was r e l e a s e d by t h e i s c h a e m i c k i d n e y s w h i l e h y p e r t e n s i n was f o r m e d i n t h e b l o o d f r o m a p r e c u r s o r c a l l e d h y p e r t e n s i n o g e n ( 5 4 ) . A t a b o u t t h e same t i m e , P age and H e l m e r a l s o r e p o r t e d a new p r e s s o r s u b s t a n c e f o r m e d by t h e a c t i o n o f r e n i n on p l a s m a p r o t e i n s and c a l l e d i t a n g i o t o n i n ( 5 5 ) . I t d i d n o t t a k e l o n g b e f o r e t h e d i f f e r e n t g r o u p s r e a l i z e d t h a t h y p e r t e n s i n and a n g i o t o n i n w ere t h e same p r e s s o r s u b s t a n c e , and i n 1958 P age and B r a u n - M e n e n d e z a g r e e d on a new name f o r t h e p r e s s o r s u b s t a n c e r e l e a s a b l e by r e n i n — t h e y c a l l e d i t a n g i o t e n s i n . T o g e t h e r , t h e two s u b s t a n c e s r e p r e s e n t a h u m o r a l b l o o d p r e s s u r e - r e g u l a t i n g s y s t e m o f r e n a l o r i g i n . R e n i n , a p r o t e o l y t i c enzyme f i r s t d i s c o v e r e d by T i g e r s t e d t and Bergmanm i n 1898 ( 5 6 ) , i s s y n t h e s i z e d , s t o r e d and r e l e a s e d m a i n l y by t h e e p i t h e l i a l c e l l s o f t h e j u x t a g l o m e r u l a r a p p a r a t u s i n t h e k i d n e y . T h i s enzyme a c t s on t h e c i r c u l a t i n g o ^ - g l o b u l i n s y n t h e s i z e d by t h e l i v e r t o p r o d u c e a d e c a p e p -t i d e a n g i o t e n s i n I , w h i c h i s f u r t h e r d e g r a d e d i n t o an o c t a p e p t i d e by t h e enzyme p e p t i d y l d i p e p t i d a s e ( c o n v e r t i n g e n z y m e ) . T h e o c t a p e p t i d e i s h i g h l y a c t i v e and was c a l l e d a n g i o t e n s i n I I . F u r t h e r e n z y m a t i c d e g r a d a t i o n o f t h i s - 9 -FIGURE 1. The Ren in-Ang iotens in System o^ -G lobu l i n (Renin subs t ra te ) ren in • Ang io tens in I (Decapeptide) conver t ing enzyme ANGIOTENSIN II (OCTAPEPTIDE) ang iotens inase A (aminopeptidase) y ANGIOTENSIN I I I (HEPTAPEPTIDE) ang iotens i nases y FRAGMENTS (INACTIVE) ) - 10 -p e p t i d e b y a m i n o p e p t i d a s e y i e l d e d t h e h e p t a p e p t i d e a n g i o t e n s i n I I I , an a c t i v e m e t a b o l i t e t h a t has r o u g h l y h a l f t h e p o t e n c y o f a n g i o t e n s i n I I i n p r e s s o r a c t i v i t y and e q u i p o t e n t i n s t i m u l a t i o n o f a l d o s t e r o n e s e c r e t i o n (57) . , T h e d e g r a d a t i o n p r o d u c t s o f a n g i o t e n s i n I I I were f o u n d t o be i n a c t i v e ( s e e F i g u r e 1 f o r p a t h w a y ) . T h e r o l e o f t h e r e n i n - a n g i o t e n s i n s y s t e m i n t h e i n i t i a t i o n and m a i n t e n -a n c e o f h y p e r t e n s i o n has been a c o n t r o v e r s i a l a r e a f o r t h e p a s t t h i r t y y e a r s . I n a n i m a l s w i t h r e n o v a s c u l a r h y p e r t e n s i o n , p l a s m a r e n i n a c t i v i t y was shown t o i n c r e a s e o n l y t r a n s i e n t l y i m m e d i a t e l y a f t e r r e n a l a r t e r i a l c l a m p i n g ( 5 8 ) . I n j e c t i o n s o f SQ 20881 ( a n a n g i o t e n s i n - c o n v e r t i n g enzyme i n h i b i t o r ) i n t o u n i n e p h r e c t o m i z e d ' d ogs p r e v e n t e d t h e d e v e l o p m e n t o f h y p e r t e n s i o n by s u b s e q u e n t r e n a l a r t e r y s t e n o s i s , and r e v e r s e d t h e a c u t e h y p e r t e n s i o n i n d u c e d b y t h i s same t e c h n i q u e ( 5 9 ) . T h e s e k i n d s o f d a t a p r o v i d e d some e v i d e n c e f o r t h e i n v o l v e m e n t o f t h e r e n i n - a n g i o t e n s i n s y s t e m i n t h e i n i t i a -t i o n o f r e n o v a s c u l a r h y p e r t e n s i o n i n e x p e r i m e n t a l a n i m a l s . T h e e v i d e n c e f r o m s t u d i e s on p a t i e n t s i s more c o n t r o v e r s i a l . L a r a g h ' s g r o u p , f o r e x a m p l e , f o u n d t h a t w i t h i n a p o p u l a t i o n o f p a t i e n t s w i t h p r i m a r y h y p e r t e n s i o n , t h e r e e x i s t e d a s u b - g r o u p o f i n d i v i d u a l s ( r e p r e s e n t i n g 30 % o f t h e p a t i e n t s ) w i t h low p l a s m a r e n i n a c t i v i t y , w h i l e t h e r e s t o f t h e p a t i e n t s a l l e x h i b i t e d n o r m a l t o h i g h r e n i n a c t i v i t y . T r e a t i n g t h e p a t i e n t s w i t h t h e a n g i o t e n s i n c o n v e r t i n g enzyme i n h i b i t o r y (SQ 14225) r e v e a l e d t h a t t h e low r e n i n g r o u p r e s p o n d e d v e r y p o o r l y , w h i l e t h e h i g h - n o r m a l r e n i n g r o u p showed l a r g e r e d u c -t i o n s i n b l o o d p r e s s u r e ( 6 0 ) . M a c G r e g o r and c o w o r k e r s f o u n d , h o w e v e r , t h a t t h e p e r c e n t a g e b l o o d p r e s s u r e r e d u c t i o n i n d u c e d b y t h e c o n v e r t i n g enzyme i n h i b i t o r was t h e same i n h y p e r t e n s i v e p a t i e n t s and n o r m o t e n s i v e c o n t r o l s ( 6 1 ) , t h e r e b y a r g u i n g t h a t t h e i n v o l v m e n t o f a n g i o t e n s i n i n t h e m a i n t e n a n c e o f b l o o d p r e s s u r e was s i m i l a r i n b o t h n o r m o t e n s i v e c o n t r o l s and h y p e r t e n s i v e p a t i e n t s . T h i s - 11 -a r g u m e n t has i t s own p r o b l e m s , t h e f i r s t o f w h i c h i s w h e t h e r t h e p e r c e n t a g e r e d u c t i o n i n b l o o d p r e s s u r e i s more i m p o r t a n t t h a n t h e a b s o l u t e b l o o d p r e s s u r e v a l u e s r e c o r d e d by o t h e r w o r k e r s . The s e c o n d q u e s t i o n i s w h e t h e r t h e a s s u m p t i o n t h a t s i m i l a r p e r c e n t a g e d r o p a f t e r c o n v e r t i n g enzyme b l o c k a d e r e p r e s e n t s a s i m i l a r d e g r e e o f r e n i n - a n g i o t e n s i n i n v o l v e m e n t , k e e p i n g i n m i n d t h a t o t h e r h o m e o s t a t i c m e c h a n i s m s were a l s o shown t o be a l t e r e d i n h y p e r t e n s i v e p a t i e n t s . I n summary, o u r c u r r e n t k n o w l e d g e s e e m s - t o s u p p o r t t h e i d e a t h a t t h e r e n i n - a n g i o t e n s i n s y s t e m d o e s p l a y a n a e t i o l o g i c r o l e i n a s u b - g r o u p o f h y p e r -t e n s i v e p a t i e n t s . T h e e x a c t n a t u r e o f i n v o l v e m e n t i s s t i l l unknown, and t h e i n t e r a c t i o n b e t w e e n t h i s and o t h e r 1 h o m e o s t a t i c m e c h a n i s m s i s c u r r e n t l y u n d e r i n v e s t i g a t i o n by many g r o u p s . I V . The A d r e n a l C o r t e x , I t s Hormones, and H y p e r t e n s i o n T h e r e l a t i o n s h i p o f t h e a d r e n a l c o r t e x t o l e v e l s o f b l o o d p r e s s u r e has b e e n amply d o c u m e n t e d o v e r t h e y e a r s , w i t h p a t i e n t s d i s p l a y i n g f r a n k h y p e r -t e n s i o n i n C u s h i n g ' s d i s e a s e , and h y p o t e n s i o n i n a d r e n a l i n s u f f i c i e n c y . A c l e a r e t i o l o g i c a l r o l e f o r s t e r o i d s i n h y p e r t e n s i o n , h o w e v e r , was n o t e s t a b l i s h e d u n t i l s e v e r a l g r o u p s o f w o r k e r s d e m o n s t r a t e d t h a t 1 1 - d e o x y - c o r -t i c o s t e r o n e a c e t a t e (DOCAJ c a n c a u s e h y p e r t e n s i o n i n r a t s and i n p a t i e n t s w i t h a d r e n a l i n s u f f i c i e n c y ( s e e r e v i e w by G e n e s t ; 6 2 ) . A f t e r S i m p s o n ' s d i s c o v e r y o f a l d o s t e r o n e i n 1953 ( 6 3 ) , G e n e s t and c o w o r k e r s r e p o r t e d e l e v a t e d a l d o s t e r o n e l e v e l s i n u r i n e f r o m p a t i e n t s w i t h m a l i g n a n t h y p e r t e n -s i o n ( 6 2 ) . L a r a g h ' s g r o u p c o n f i r m e d t h e s e f i n d i n g s and a l s o p o i n t e d o u t t h a t i n c r e a s e d a l d o s t e r o n e s e c r e t i o n was a l s o n o t e d i n some p a t i e n t s w i t h - 12 -FIGURE 2. Outline of hormone synthesis in the zona glomerulosa of the adrenal cortex. j . Acetate Cholesterol Pregnenolone •1 Progesterone I Deoxycorticosterone i Corticosterone I CH20H C = 0 - 13 -e s s e n t i a l ^ h y p e r t e n s i o n ( 6 4 ) . T h e s e r e s u l t s c a u s e d waves o f c o n t r o v e r s i e s , w i t h s e v e r a l ' g r o u p s u n a b l e t o d e t e c t a n y d i f f e r e n c e s i n a l d o s t e r o n e s e c r e t i o n b e t w e e n c o n t r o l and h y p e r t e n s i v e p a t i e n t s . On s u b s e q u e n t r e - e x a m i n a t i o n , G e n e s t ' s g r o u p c o n c l u d e d t h a t t h e r e was i n d e e d a s m a l l b u t c o n s i s t e n t e l e v a -t i o n o f a v e r a g e a l d o s t e r o n e l e v e l s i n p a t i e n t s w i t h e s t a b l i s h e d e s s e n t i a l h y p e r t e n s i o n ( 6 5 , 6 6 ) . T h e a v e r a g e s e c r e t i o n r a t e s were l a r g e l y u n c h a n g e d w h i l e t h e r a t e o f e l i m i n a t i o n seemed t o be r e t a r d e d i n h y p e r t e n s i v e s . In~ c o n t r a s t , t h e s e d i f f e r e n c e s were n o t o b s e r v e d i n p a t i e n t s w i t h e a r l y l a b i l e h y p e r t e n s i o n , b u t t h e c o n s i d e r a b l e s p r e a d o f v a l u e s r e c o r d e d c o u l d mean t h a t a l d o s t e r o n e was i n d e e d i n v o l v e d i n some o f t h e h y p e r t e n s i v e p a t i e n t s s t u d i e d . A l d o s t e r o n e i s s y n t h e s i z e d f r o m c o r t i c o s t e r o n e by t h e c e l l s o f t h e z o n a g l o m e r u l o s a i n t h e a d r e n a l c o r t e x ( 6 7 ; s e e F i g u r e 2 f o r s y n t h e t i c p a t h w a y ) . N o r m a l l y , i t s b i o s y n t h e s i s and s e c r e t i o n i s r e g u l a t e d by t h e r e n i n - a n g i o t e n -s i n s y s t e m , ACTH and p l a s m a c o n c e n t r a t i o n s o f p o t a s s i u m and s o d i u m . B o t h a n g i o t e n s i n I I and ACTH a c t d i r e c t l y on t h e z o n a g l o m e r u l o s a t o s t i m u l a t e t h e c o n v e r s i o n o f c h o l e s t e r o l t o p r e g n e n o l o n e , t h e r e b y f a c i l i t a t i n g f u r t h e r c o n v e r s i o n t o a l d o s t e r o n e ( 6 7 ) . P o t a s s i u m has a l s o b e e n shown t o a c t d i r e c t l y on t h e a d r e n a l c o r t e x t o s t i m u l a t e a l d o s t e r o n e s e c r e t i o n ( s e e M u l l e r ; 6 8 ) , i n a d d i t i o n t o i t s s t i m u l a t o r y e f f e c t on t h e c o n v e r s i o n o f d e o x y c o r t i s c o s t e r o n e t o c o r t i c o s t e r o n e ( 6 9 , 7 0 ) , and c o r t i c o s t e r o n e t o a l d o -s t e r o n e ( 7 1 , 7 2 ) . The m e c h a n i s m o f s t i m u l a t i o n as w e l l as t h e i m p o r t a n c e o f e a c h o f t h e s e s t e p s i n t h e o v e r a l l a l d o s t e r o n e r e s p o n s e i s s t i l l u n d e r i n v e s t i g a t i o n . S o d i u m d e f i c i e n c y was f o u n d t o s t i m u l a t e a l d o s t e r o n e s e c r e -t i o n i n a l l mammals and t h e r e i s g e n e r a l a g r e e m e n t t h a t t h i s e f f e c t i s m e d i a t e d p r i n c i p a l l y by t h e r e n i n - a n g i o t e n s i n s y s t e m ( 6 8 ) . T h e r e i s a l s o e v i d e n c e , h o w e v e r , s u g g e s t i n g t h a t d e c r e a s e d p l a s m a s o d i u m c o n c e n t r a t i o n i t s e l f may h a v e a d i r e c t e f f e c t on t h e a d r e n a l s t o s t i m u l a t e a l d o s t e r o n e - 14 -s e c r e t i o n ( 7 3 , 7 4 ) . The d i r e c t e f f e c t s o b s e r v e d s o f a r a r e f a i r l y s m a l l and t r a n s i e n t , w h i l e t h e l a r g e r e d u c t i o n s i n s o d i u m r e q u i r e d i n d i c a t e d t h a t u n d e r n o r m a l c i r c u m s t a n c e s p l a s m a s o d i u m c o n c e n t r a t i o n by i t s e l f may n o t p l a y a m a j o r r o l e i n t h e c o n t r o l o f a l d o s t e r o n e s e c r e t i o n . When a d m i n i s t e r e d a c u t e l y , a l d o s t e r o n e c a u s e s a r e d u c t i o n o f r e n a l s o d i u m i o n e x c r e t i o n and an i n c r e a s e i n p o t a s s i u m and h y d r o g e n i o n e x c r e t i o n . When a l d o s t e r o n e i s g i v e n c h r o n i c a l l y i n l a r g e d o s e , t h e r a t e o f s o d i u m e x c r e t i o n e v e n t u a l l y r e t u r n s t o n o r m a l w h i l e p o t a s s i u m and h y d r o g e n i o n e x c r e t i o n r e m a i n e d a c c e l e r a t e d ( 7 5 ) . I n t h e p r e s e n t s t a t e o f a f f a i r s , c o n c l u s i v e e v i d e n c e f o r a l d o s t e r o n e p l a y i n g an a e t i o l o g i c a l r o l e i n e s s e n t i a l h y p e r t e n s i o n i s l a c k i n g . However, t h e r e s u l t s t o d a t e seem t o s u g g e s t a s t r o n g a s s o c i a t i o n b e t w e e n t r a n s i e n t i n a p p r o p r i a t e e l e v a t i o n s o f p l a s m a a l d o s t e r o n e and s t a b l e u n c o m p l i c a t e d e s s e n t i a l h y p e r t e n s i o n ( 6 2 , 6 4 - 6 6 ) . The a d r e n a l c o r t i c a l hormone 1 1 - d e o x y -c o r t i c o s t e r o n e (DOC) was a l s o f o u n d t o be e l e v a t e d b o t h i n t h e p l a s m a ( 7 6 ) and t h e u r i n e ( 7 7 ) o f h y p o k a l e m i c e s s e n t i a l h y p e r t e n s i v e s b u t t h e s e f i n d i n g s w ere n o t d u p l i c a t e d i n u n c o m p l i c a t e d e s s e n t i a l h y p e r t e n s i o n s ( 7 7 , 7 8 ) . C o r t i c o s t e r o n e s e c r e t i o n r a t e s were a l s o i n v e s t i g a t e d i n e s s e n t i a l h y p e r t e n -s i o n and no a b n o r m a l i t i e s c o u l d be f o u n d . T h e r e f o r e , t o s u m m a r i z e , t h e a d r e n a l , t o g e t h e r w i t h i t s h o r m o n e s , a r e i m p o r t a n t f a c t o r s i n b l o o d p r e s s u r e , v o l u m e , and i o n r e g u l a t i o n i n t h e b o d y . A l d o s t e r o n e i s t h e o n l y hormone f o u n d t o have some a s s o c i a t i o n w i t h human e s s e n t i a l h y p e r t e n s i o n and o n g o i n g r e s e a r c h may o n e d a y p r o v i d e us w i t h i n f o r m a t i o n r e g a r d i n g i t s i n v o l v e m e n t , i t s m e c h a n i s m , and i t s i m p o r t a n c e i n h y p e r t e n s i o n . - 15 -V. P r o s t a g l a n d i n s and H y p e r t e n s i o n I n t h e i r a t t e m p t s t o i d e n t i f y f a c t o r s t h o u g h t t o be i n v o l v e d i n t h e p a t h o g e n e s i s o f e s s e n t i a l h y p e r t e n s i o n , r e s e a r c h e r s h a v e g e n e r a l l y t r i e d t o f i n d t h e p r e s s o r a g e n t s o r s y s t e m s r e s p o n s i b l e . S e v e r a l g r o u p s o f w o r k e r s -n o t i c e d t h e d i f f i c u l t i e s i n v o l v e d i n t r y i n g t o g a i n d i r e c t e v i d e n c e i n c r i m i -n a t i n g c e r t a i n p r e s s o r s y s t e m s i n h y p e r t e n s i o n . T h e y d e c i d e d t o work b a c k -w a r d s , a r g u i n g t h a t a d e f i c i e n c y i n t h e m e c h a n i s m s o r a g e n t s w h i c h a r e a n t i -h y p e r t e n s i v e o r v a s o d i l a t o r y i n n a t u r e c o u l d a l s o l e a d t o p a t h o l o g i c a l e l e v a t i o n s o f b l o o d p r e s s u r e . I n t h e e a r l y t h i r t i e s , G o l d b l a t t , von E u l e r , and K u r z r o k i n d e p e n d e n t l y o b s e r v e d a d e p r e s s o r s u b s t a n c e i n e x t r a c t s o f s h e e p s e m i n a l v e s i c l e s and human semen. Von E u l e r ( 7 9 ) l a t e r c h a r a c t e r i z e d t h e l i p i d s o l u b l e compounds and c a l l e d them p r o s t a g l a n d i n s ( P G s ) . I t was o v e r t w e n t y y e a r s l a t e r when B e r g s t r o m and S j o v a l l s e p a r a t e d t h e s e i n t o two c l a s s e s , n a m e l y PGEs and P G Fs, and f o u n d t h a t PGEs have b o t h " b l o o d p r e s s u r e r e d u c i n g " and " n o n - v a s c u l a r smooth m u s c l e - s t i m u l a t i n g " a c t i v i t i e s ( 8 0 ) , w h i l e PGFs o n l y h a v e " n o n - v a s c u l a r smooth m u s c l e - s t i m u l a t i n g a c t i v i t y " ( 8 1 ) . P r o s t a g l a n d i n s a r e u b i q u i t o u s compounds f o u n d i n most t i s s u e s o f t h e human body. T h e i r e f f e c t s a r e h i g h l y v a r i a b l e and g e n e r a l i z a t i o n s o f t h e i r a c t i o n s a r e i n a p p r o p r i a t e and o f t e n m i s l e a d i n g . We a r e o n l y c o n c e r n e d a b o u t p r o s t a g l a n d i n s ' i n v o l v e m e n t i n h y p e r t e n s i o n , so o n l y t h e e f f e c t s o f p r o s t a -g l a n d i n s on t h e k i d n e y i s b e i n g a d d r e s s e d i n t h i s s e c t i o n . P r o s t a g l a n d i n s ( m o s t l y PGE2) h a v e been f o u n d t o be s y n t h e s i z e d i n t h e k i d n e y by t h e i n t e r s t i t i a l c e l l s o f t h e r e n a l m e d u l l a ( s e e r e v i e w by M c G i f f ; 8 2 ) . T h e r e i s a l s o new e v i d e n c e s u g g e s t i n g t h a t t h e r e n a l c o r t e x i s c a p a b l e o f s y n t h e -s i z i n g . p r o s t a g l a n d i n s , and PGI2 i s t h e p r e d o m i n a n t compound ( 8 3 ) . B o t h P G E ? and P G I ? a r e p o w e r f u l d i l a t o r s o f t h e r e n a l e f f e r e n t a r t e r i o l e s - 16 -( a n d t h u s t h e y a r e s p e c i f i c p h y s i o l o g i c a l a n t a g o n i s t s o f a n g i o t e n s i n I I ) , and P G ^ m i g h t p l a y an i m p o r t a n t r o l e i n m e d i a t i n g r e n i n r e l e a s e d i r e c t l y f r o m t h e m a c u l a d e n s a and i n d i r e c t l y v i a r e n a l b a r o r e c e p t o r s ( 8 2 , 8 3 ) . L e e p r o p o s e d t h a t r e n o m e d u l l a r y P G A £ o r PGE^ c a n f u n c t i o n as a n t i h y p e r t e n -s i v e hormones i n i n t a c t a n i m a l s , and t h a t e i t h e r a g e n e t i c o r a c q u i r e d d e f i -c i e n c y i n p r o s t a g l a n d i n p r o d u c t i o n c a n r e s u l t i n h y p e r t e n s i o n ( 8 4 ) . T h e r e i s no d i r e c t e v i d e n c e s o f a r t o s u p p o r t t h i s h y p o t h e s i s f r o m s t u d i e s on h y p e r t e n s i v e p a t i e n t s . I n t h e . r a t , A r m s t r o n g ' s g r o u p r e p o r t e d a r e l a t i v e d e f i c i e n c y i n PG c a t a b o l i s m i n t h e k i d n e y s o f New Z e a l a n d s t r a i n o f h y p e r -t e n s i v e r a t s ( N Z ) , w h i c h i s o f some i m p o r t a n c e b e c a u s e PGs a r e t h o u g h t t o m e d i a t e v a s o c o n s t r i c t i o n i n t h e NZ h y p e r t e n s i v e a n i m a l s ( 8 5 ) . T h e s i g n i f i -c a n c e o f t h e s e f i n d i n g s i n human h y p e r t e n s i o n i s n o t e s t a b l i s h e d , and more r e c e n t r e s u l t s d o x n o t s u p p o r t t h e i n v o l v e m e n t o f r e n a l PGs i n t h e i n i t i a t i o n o f h y p e r t e n s i o n i n t h e s e a n i m a l s ( 8 6 ) . T h e r e f o r e , t o c o n c l u d e , i n f u s e d PGs do h a v e a n t i h y p e r t e n s i v e a c t i v i t i e s i n human ( 8 7 , 8 8 ) , b u t as y e t t h e r e i s no e v i d e n c e t o s u p p o r t t h e i r p r e v e n t i v e r o l e i n t h e i n i t i a t i o n o r t h e m a i n t e n -a n c e o f h y p e r t e n s i o n . T h e r e a r e many more s y s t e m s and m e c h a n i s m s t h a t a r e t h o u g h t t o be i n v o l -v e d i n e s s e n t i a l h y p e r t e n s i o n . V i r t u a l l y any a g e n t o r m e c h a n i s m t h a t c a n a l t e r a r t e r i a l b l o o d p r e s s u r e d i r e c t l y o r i n d i r e c t l y has t h e p o t e n t i a l t o be i n v o l v e d a e t i o l o g i c a l l y i n e s s e n t i a l h y p e r t e n s i o n . I t i s p a i n f u l l y o b v i o u s t o w o r k e r s i n t h e f i e l d t h a t h y p e r t e n s i o n i s m e r e l y a m a n i f e s t a t i o n o f a c o l l e c t i o n o f p a t h o l o g i c a l a b n o r m a l i t i e s i n t h e haemodynamic and h o m e o s t a t i c s t a t u s o f t h e b o d y . In t h e n o r m a l p h y s i o l o g i c a l s t a t e , t h e s e m e c h a n i s m s i n t e r a c t w i t h one a n o t h e r t o r e g u l a t e p l a s m a v o l u m e , i o n c o n t e n t , b l o o d p r e s s u r e , h e a r t r a t e and c a r d i a c o u t p u t . The s y m p a t h e t i c n e r v o u s s y s t e m , - 17 -f o r e x a m p l e , r e g u l a t e s r e n i n s e c r e t i o n , w h i c h i n t u r n d i c t a t e s t h e l e v e l o f a n g i o t e n s i n I I i n t h e b o d y . T h e s e c r e t i o n o f a l d o s t e r o n e and p r o s t a g l a n d i n s i s m e d i a t e d by t h e r e n i n a n g i o t e n s i n s y s t e m as w e l l as t h e s y m p a t h e t i c i n p u t . T h e r e f o r e i t i s o b v i o u s t h a t u n l e s s a l l t h e s e p a r a m e t e r s a r e m o n i t o r e d o r c o n t r o l l e d s i m u l t a n e o u s l y i n s t u d i e s o f e s s e n t i a l h y p e r t e n s i o n , t h e r e s u l t s o b t a i n e d may n o t be m e a n i n g f u l . One o f t h e m a j o r p r o b l e m s r e m a i n s " t h a t e t h i c a l l y , many o f t h e more c o n c l u s i v e e x p e r i m e n t s c a n n o t be c a r r i e d o u t on human s u b j e c t s . T h e u s e o f a n i m a l m o d e l s o f h y p e r t e n s i o n u n d o u b t a b l y has been v e r y h e l p f u l b o t h i n e x p e r i m e n t a l u s e o f a n t i h y p e r t e n -s i v e d r u g s and i n i n v e s t i g a t i o n a l a t t e m p t s i n u n d e r s t a n d i n g t h e m e c h a n i s m s o f h y p e r t e n s i o n . The r e s u l t s and c o n c l u s i o n s drawn f r o m a n i m a l s t u d i e s , h o w e v e r , s h o u l d be t a k e n w i t h c a u t i o n u n t i l p a r a l l e l o b s e r v a t i o n s a r e f o u n d and c o n f i r m e d i n humans. V I . A n i m a l M o d e l s o f H y p e r t e n s i o n The f o l l o w i n g s e c t i o n w i l l a t t e m p t t o b r i e f l y s u r v e y t h e a n i m a l m o d e l s o f h y p e r t e n s i o n a v a i l a b l e . One o f t h e e a r l i e s t a t t e m p t s t o p r o d u c e e x p e r i -m e n t a l h y p e r t e n s i o n was t h a t o f K o c h and M i e s , who s e v e r e d t h e c a r o t i d s i n u s and a o r t i c a r c h n e r v e s t o and f r o m t h e b a r o r e c e p t o r s a t t h o s e l o c a t i o n s ( 8 9 ) . T h e r e were a r g u m e n t s i n t h e s u b s e q u e n t y e a r s as t o w h e t h e r s i n o a o r t i c d e n e r -v a t i o n ' c o u l d i n d e e d p r e c i p i t a t e c h r o n i c h y p e r t e n s i o n , w i t h s e v e r a l g r o u p s r e p o r t i n g n e g a t i v e r e s u l t s . More r e c e n t s t u d i e s u s i n g c o n s c i o u s d o g s seemed t o show t h a t t h e r e i s a m o d e r a t e r i s e i n a r t e r i a l p r e s s u r e a f t e r s i n o a o r t i c d e n e r v a t i o n b u t t h e h y p e r t e n s i o n d e v e l o p e d i s v e r y l a b i l e , w i t h t h e r e p r o d u -c i b i l i t y o f m e a s u r e m e n t s b e i n g h i g h l y d e p e n d e n t on e x t r a n e o u s v a r i a b l e s s u c h as t i m e o f t h e d a y and t h e v a r i o u s e n v i r o n m e n t a l s t i m u l i p r e s e n t ( 9 0 , 9 1 ) . F u r t h e r i n f o r m a t i o n was g a i n e d f r o m r a b b i t s s u b j e c t e d t o s i m i l a r ^ s u r g i c a l p r o c e d u r e s and h e r e , t h e a r t e r i a l p r e s s u r e s were e l e v a t e d by an a v e r a g e o f 3 5 % . ( 9 2 ) . C o n t i n u o u s m o n i t o r i n g o f t h e a n i m a l s t h r o u g h o u t t h e c o u r s e o f t h e e x p e r i m e n t r e v e a l e d t h a t t h e r e was an i n i t i a l r i s e i n c a r d i a c o u t p u t w h i c h r e t u r n e d t o n o r m a l w i t h i n a month. T o t a l p e r i p h e r a l r e s i s t a n c e and h e a r t r a t e were i n c r e a s e d s i g n i f i c a n t l y i n a m a j o r i t y o f a n i m a l s o v e r t h i s t i m e p e r i o d . S y s t e m i c b l o o d f l o w was a l s o m o n i t o r e d and i n i t i a l l y an i n c r e a s e d r e n a l v a s c u l a r r e s i s t a n c e w i t h n o r m a l b l o o d f l o w was o b s e r v e d . T h e i n c r e a s e d b l o o d p r e s s u r e i n t h e s e a n i m a l s was a c c o m p a n i e d by an e l e v a t i o n i n r e n a l v a s c u l a r r e s i s t a n c e ( 9 2 ) . T h i s and o t h e r t y p e s o f h y p e r t e n s i o n i n v o l v i n g t h e a l t e r a t i o n o f CNS o r p e r i p h e r a l n e r v o u s f u n c t i o n a r e g e n e r a l l y c l a s s i -f i e d as " n e u r o g e n i c h y p e r t e n s i o n " and f i n d i n g s i n t h e s e m o d e l s l e n d s u p p o r t t o t h e p r o p o s e d i n v o l v e m e n t o f n e u r a l and r e n a l f a c t o r s i n h y p e r t e n s i o n . The- r e p r o d u c i b i l i t y o f r e s u l t s , h o w e v e r , i s n o t v e r y good and c o n f l i c t i n g e v i d e n c e i s p l e n t i f u l . F u r t h e r m o r e , t h e m e c h a n i s m s d e s c r i b e d h e r e i n t h e s e a n i m a l s do n o t n e c e s s a r i l y r e f l e c t t h o s e t h a t a r e i m p o r t a n t i n e s s e n t i a l h y p e r t e n s i o n . A n o t h e r model o f n e u r o g e n i c h y p e r t e n s i o n r e l a t i n g t o s i n o a o r t i c d e n e r v a -t i o n i s t h e newer model o f p r o l o n g e d s t e l l a t e g a n g l i o n s t i m u l a t i o n i n u n a n a e s t h e t i z e d d o g s . E a r l i e r work on a n a e s t h e t i z e d d ogs had shown t h a t s t i m u l a t i o n o f t h e s t e l l a t e g a n g l i a r e s u l t e d i n an i n c r e a s e d c a r d i a c o u t p u t and a p r o l o n g e d e l e v a t i o n i n b l o o d p r e s s u r e ( 9 3 ) . More r e c e n t r e s u l t s f r o m c o n s c i o u s d ogs seem t o i n d i c a t e t h a t t h e i n i t i a l t r a n s i e n t r i s e i n b l o o d p r e s s u r e was e x c l u s i v e l y a s s o c i a t e d w i t h i n c r e a s e d c a r d i a c o u t p u t . By t h e - 19 -end o f t h e f i r s t week, h o w e v e r , t h e p e r s i s t e n t e l e v a t i o n o f b l o o d p r e s s u r e was a t t r i b u t a b l e t o i n c r e a s e d p e r i p h e r a l r e s i s t a n c e , w h i l e o t h e r c a r d i o v a s -c u l a r p a r a m e t e r s r e t u r n e d t o n o r m a l . The e l e v a t e d b l o o d p r e s s u r e r e t u r n e d t o c o n t r o l l e v e l s w i t h a - a d r e n e r g i c b l o c k a d e , t h u s l e n d i n g s u p p o r t t o t h e i d e a o f s y m p a t h e t i c i n v o l v e m e n t i n t h i s model o f h y p e r t e n s i o n ( 9 4 ) . H y p e r t e n s i o n r e s u l t i n g f r o m | o p e r a n t c o n d i t i o n i n g has a l r e a d y b e e n d i s c u s s e d e a r l i e r ( 2 4 , 2 5 ) . I t i s i n t e r e s t i n g , h o w e v e r , t o n o t e t h a t t h i s t y p e o f h y p e r t e n s i o n i s a s s o c i a t e d w i t h a n o r m a l ' c a r d i a c o u t p u t and an i n c r e a s e d t o t a l p e r i p h e r a l r e s i s t a n c e ( 9 5 ) . A l s o , t h e t i m e c o u r s e o f t h e e l e v a t i o n p a r a l l e l s t h e r e d u c t i o n o f b l o o d f l o w t o many o r g a n s s u c h as t h e k i d n e y , s k e l e t a l m u s c l e s and t h e v i s c e r a ( e x c l u d i n g t h e l i v e r ) . T h i s i s a u n i q u e f e a t u r e s i n c e most o t h e r m o d e l s o f h y p e r t e n s i o n a r e n o t a s s o c i a t e d w i t h a r e d u c t i o n o f b l o o d f l o w . B r a i n s t e m l e s i o n s were a l s o u s e d t o p r o d u c e h y p e r t e n s i o n . Doba and R e i s p r o d u c e d b i l a t e r a l s t e r e o t a x i c e l e c t r o l y t i c l e s i o n s a t s p e c i f i c n u c l e i i n t h e , b r a i n s t e m o f r a t s ( n u c l e u s t r a c t u s s o l i t a r i i a t t h e l e v e l o f t h e o b e x ) and r e p o r t e d d r a s t i c c h a n g e s i n t h e i r s y s t e m i c haemodynamic f u n c t i o n s i n c l u d i n g an e l e v a t i o n o f b l o o d p r e s s u r e w i t h o u t a p p r e c i a b l e c h a n g e i n h e a r t r a t e ( 9 6 ) . T h i s a c u t e h y p e r t e n s i v e r e s p o n s e was a s s o c i a t e d w i t h an i n c r e a s e i n p e r i p h e r a l r e s i s t a n c e , a d e c r e a s e i n a b d o m i n a l a o r t i c b l o o d f l o w , and an i n c r e a s e o f c e n t r a l v e n o u s p r e s s u r e . C a r d i a c o u t p u t was d r a s t i c a l l y r e d u c e d and was f o u n d t o be t h e r e s u l t o f r e d u c e d s t r o k e v o l u m e and i n c r e a s e d v e n t r i c u l a r e n d - d i a s t o l i c p r e s s u r e . I f l e f t u n t r e a t e d , t h e a n i m a l s r a p i d l y d e v e l o p e d c o n g e s t i v e h e a r t f a i l u r e . A l p h a - a d r e n e r g i c b l o c k a d e r e v e r s e d a l l t h e symptoms. W h i l e t h i s i s s u g g e s t i v e o f s y m p a t h e t i c i n v o l v e m e n t i n t h i s f o r m o f h y p e r t e n s i o n , one a l s o s h o u l d n o t f o r g e t t h e e f f e c t s o f a b l o c k a d e - 20 -on o t h e r p r e s s o r and d e p r e s s o r s y s t e m s . A b e t t e r u n d e r s t a n d i n g o f t h e n e u r o g e n i c m e c h a n i s m s i n v o l v e d i n m a i n t a i n i n g b l o o d p r e s s u r e i s n e e d e d b e f o r e one c a n hope t o d i s s e c t o u t t h e m e c h a n i s m s u n d e r l y i n g t h e v a r i o u s f o r m s o f n e u r o g e n i c h y p e r t e n s i o n d i s c u s s e d h e r e . We h a v e p r e v i o u s l y m e n t i o n e d t h e model o f r e n a l a r t e r i a l h y p e r t e n s i o n d e s c r i b e d by G o l d b l a t t ' s g r o u p i n 1934 ( 4 7 ) . S i n c e t h e n , many a t t e m p t s were made t o t r y t o i d e n t i f y t h e s e q u e n c e o f e v e n t s u n d e r l y i n g t h e d e v e l o p m e n t o f h y p e r t e n s i o n i n t h i s m o d e l . P a g e ' s g r o u p f o u n d t h a t t h e e l e v a t e d p r e s s u r e was a s s o c i a t e d w i t h a n o r m a l c a r d i a c o u t p u t and t h e r e f o r e a s c r i b e d t h e h y p e r t e n s i o n t o i n c r e a s e d p e r i p h e r a l r e s i s t a n c e ( 9 7 ) . L a t e r s t u d i e s u s i n g more s o p h i s t i c a t e d m e thods c o n f i r m e d t h e s e f i n d i n g s b u t i n t h e e a r l y p h a s e o f h y p e r t e n s i o n a f e w o f t h e a n i m a l s d i d h a v e an i n c r e a s e d c a r d i a c o u t p u t w h i c h l a t e r r e t u r n e d t o n o r m a l ( 9 8 ) . T h e s e o b s e r v a t i o n s were g e n e r a l l y a c c e p t e d , and t h e m e c h a n i s m p o s t u l a t e d was t h a t e a r l y i n h y p e r t e n s i o n t h e r e was an e x p a n s i o n i n p l a s m a v o l u m e due t o s o d i u m r e t e n t i o n r e s u l t i n g f r o m r e s t r i c t e d b l o o d f l o w t o t h e k i d n e y , and c o n s e q u e n t l y a c t i v a t i o n o f t h e r e h i n - a n g i o t e n s i n - a l d o s t e r o n e s y s t e m t h e r e b y l e a d i n g t o i n c r e a s e d b l o o d v o l u m e c a r d i a c o u t p u t . W i t h t i m e , an a d a p t i v e i n c r e a s e i n t h e k i d n e y ' s v a s c u l a r r e s i s t a n c e w o u l d a l l o w t h e c a r d i a c o u t p u t t o r e t u r n t o n o r m a l ( 9 9 ) . T h i s i s v e r y much s t i l l a h y p o t h e s i s , , and d i s a g r e e m e n t s a r e p l e n t i f u l . The e x a c t m e c h a n i s m s u s t a i n i n g t h i s f o r m o f h y p e r t e n s i o n r e m a i n s u n p r o v e n b u t i t seems t h a t s y m p a t h e t i c p r o c e s s e s and t h e r e n i n - a n g i o t e n s i n s y s t e m a r e i n v o l v e d ( s e e s e c t i o n I I o f I n t r o d u c t i o n ) . A n o t h e r f o r m o f k i d n e y - r e l a t e d h y p e r t e n s i o n was t h a t p r o d u c e d f r o m a p e r i n e p h r i c h u l l i n d u c e d b y w r a p p i n g c e l l o p h a n e o r s i l k a r o u n d t h e k i d n e y ( 1 0 0 ) . T h i s i s one o f t h e m o r e - e x t e n s i v e l y s t u d i e d m o d e l s and t h e mechan-- 21 -i s m s i m p l i c a t e d i n b l o o d p r e s s u r e e l e v a t i o n i n t h i s model were e s s e n t i a l l y s i m i l a r t o t h o s e o f G o l d b l a t t ' s r e n a l a r t e r i a l h y p e r t e n s i o n ( 1 0 1 ) . • G e n e t i c h y p e r t e n s i o n i n a n i m a l s has b e e n u s e d as a model o f human e s s e n - ' t i a l h y p e r t e n s i o n f o r o v e r t w e n t y y e a r s . S m i r k and H a l l r e p o r t e d i n 1958 i n i t i a t i n g a s t r a i n o f h y p e r t e n s i v e r a t s t h r o u g h b r o t h e r - s i s t e r i n b r e e d i n g ( 1 0 2 ) . T h i s s t r a i n has come t o be known as t h e New Z e a l a n d s t r a i n , and t h e r a t s seemed t o have a r e d u c e d p l a s m a v o l u m e r e s u l t i n g i n a h i g h e r h a e m a t o -c r i t v a l u e . T h e e x t r a c e l l u l a r f l u i d v o l u m e and e x c h a n g e a b l e s o d i u m c o n c e n -t r a t i o n s a l s o were l o w e r i n t h e s e a n i m a l s , g i v i n g e v i d e n c e o f a l a c k o f i n v o l v e m e n t o f s o d i u m - r e t a i n i n g m e c h a n i s m s . F u r t h e r e x p e r i m e n t a t i o n seemed t o s u g g e s t t h a t i n c r e a s e d c a r d i a c o u t p u t was n o t n e c e s s a r y f o r t h e d e v e l o p -ment o f t h i s f o r m o f h y p e r t e n s i o n ( 1 0 2 ) . I f t h a t i s i n d e e d t h e c a s e , t h i s may r e p r e s e n t a h y p e r t e n s i v e s t a t e w h e r e b y o n l y an e n d o g e n o u s n e u r o g e n i c i n v o l v e m e n t i s r e q u i r e d f o r t h e i n i t i a t i o n and m a i n t a n e n c e . o f h y p e r t e n s i o n , and t h i s may p r o v i d e a good o p p o r t u n i t y f o r f u t u r e e f f o r t s i n t r y i n g t o d i s s e c t o u t t h e v a r i o u s n e u r o g e n i c m e c h a n i s m s i n v o l v e d i n b l o o d p r e s s u r e r e g u l a t i o n . A n o t h e r s t r a i n o f g e n e t i c a l l y h y p e r t e n s i v e r a t s was d e s c r i b e d b y B i a n c h i ' s g r o u p , now known as t h e M i l a n h y p e r t e n s i v e s t r a i n . T h e s e a n i m a l s d i f f e r e d f r o m t h e New Z e a l a n d h y p e r t e n s i v e s t r a i n by h a v i n g a s t r o n g k i d n e y i n v o l v e m e n t i n t h e p r o g r e s s i o n o f h y p e r t e n s i o n ( 1 0 3 ) . I n a s e r i e s o f k i d n e y c r o s s - t r a n s p l a n t a t i o n s , t h e s e w o r k e r s h a v e d e m o n s t r a t e d t h e i m p o r t a n c e o f t h e k i d n e y i n c o n t r o l i n g t h e l e v e l o f a r t e r i a l p r e s s u r e i n t h e s e a n i m a l s ( 1 0 4 ) . More r e c e n t d a t a f r o m t h e c a r e f u l m o n i t o r i n g o f t h e s e a n i m a l s r e v e a l e d t h a t t h e e a r l y p h a s e o f h y p e r t e n s i o n d e v e l o p m e n t * i s a s s o c i a t e d w i t h s - 22 -a r e d u c t i o n o f r e n i n a c t i v i t y i n t h e p l a s m a , b u t o n c e t h e h y p e r t e n s i o n was e s t a b l i s h e d , t h e p l a s m a r e n i n a c t i v i t y r e t u r n s t o c o n t r o l l e v e l s . I n c r e a s e d o u t p u t o f h y p o - o s m o l a r u r i n e and s o d i u m r e t e n t i o n was a l s o a s s o c i a t e d w i t h t h e i n i t i a l r i s e i n b l o o d p r e s s u r e , p r o v i d i n g f u r t h e r p r o o f f o r r e n a l i n v o l v e m e n t i n t h i s f o r m o f h y p e r t e n s i o n ( 1 0 4 ) . More work on e l u c i d a t i n g t h e t i m e c o u r s e o f haemodynamic f u n c t i o n s w i l l u n d o u b t a b l y p r o v i d e b o t h v a l u a b l e i n f o r m a t i o n on t h i s f o r m o f h y p e r t e n s i o n as w e l l as a b a s i s f o r c o m p a r i n g t h i s w i t h o t h e r m o d e l s o f h y p e r t e n s i o n . P e r h a p s t h e most w e l l known and w e l l s t u d i e d model o f g e n e t i c h y p e r t e n -s i o n i s t h e s p o n t a n e o u s l y h y p e r t e n s i v e r a t s (SHR) f r o m K y o t o d e v e l o p e d by Okamoto and A o k i i n 1963 ( 1 0 5 ) . T h i s s t r a i n o f r a t s has been a c c e p t e d w o r l d w i d e as t h e b e s t a n i m a l model o f human e s s e n t i a l h y p e r t e n s i o n , w i t h v e r y s i m i l a r t i m e c o u r s e and haemodynamic f u n c t i o n s ( 1 0 6 ) . F r o h l i c h ' s g r o u p s t u d i e d t h e s e a n i m a l s e x t e n s i v e l y and c o n c l u d e d t h a t c o n t r a r y t o o t h e r m o d e l s o f h y p e r t e n s i o n , i n c r e a s e d c a r d i a c o u t p u t was n o t r e q u i r e d f o r t h e d e v e l o p m e n t o f h y p e r t e n s i o n , and t h a t t h e d e v e l o p m e n t o f h y p e r t e n s i o n was u n a l t e r e d by a r e d u c t i o n i n c a r d i a c o u t p u t . F u r t h e r m o r e , t h e r e n a l b l o o d f l o w r e m a i n e d n o r m a l ( o r i n c r e a s e d ) t h r o u g h o u t t h e d e v e l o p m e n t o f h y p e r t e n -s i o n ( 1 0 7 ) . L e f t v e n t r i c u l a r h y p e r t r o p h y was e v i d e n t a t a v e r y e a r l y age i n t h e SHRs and p e r s i s t e d t h r o u g h o u t l i f e , and h e a r t r a t e i n c r e a s e d i n p a r a l l e l t o b l o o d p r e s s u r e e l e v a t i o n ( 1 0 8 ) . B a r o r e c e p t o r m e c h a n i s m s were s t u d i e d and a " r e s e t t i n g " phenomenon was o b s e r v e d , h o w e v e r , t h i s a l t e r a t i o n ^ i s n o t a d i r e c t c a u s e o f t h e h y p e r t e n s i o n ( 1 0 9 , 1 1 0 ) . P l a s m a r e n i n a c t i v i t y and r e n a l r e n i n e x c r e t i o n were f o u n d t o be s l i g h t l y e l e v a t e d i n t h e SHRs ( 1 1 1 ) , b u t t h e r e was no e v i d e n c e f o r an a l t e r e d s o d i u m b a l a n c e i n t h e s e a n i m a l s . More - 23 -d e t a i l e d work on i n t r a v a s c u l a r and e x t r a v a s c u l a r f l u i d v o l u m e d i s t r i b u t i o n on SHRs o f v a r i o u s a g e s w i l l be h e l p f u l i n o u r u n d e r s t a n d i n g o f t h e haemody-n a m i c a l t e r a t i o n s i n t h i s a n i m a l model o f e s s e n t i a l h y p e r t e n s i o n . V a s c u l a r r e s p o n s i v e n e s s i s an a r e a t h a t i s w e l l e x p l o r e d i n t h e SHRs, b u t c o n t r o v e r s i e s a r e p l e n t i f u l . T h i s i s p e r h a p s a r e f l e c t i o n o f t h e d i f f e r e n t m e t h o d o l o g i e s and d i f f e r e n t s y s t e m s ( e . g . i n v i v o v s . i n v i t r o ) u s e d i n t h e v a r i o u s s t u d i e s . H i s t o r i c a l l y , M e n d l o w i t z i n 1957 r e p o r t e d e n h a n c e d v a s c u l a r r e s p o n s e t o n o r a d r e n a l i n e (NA) i n t h e r e s i s t a n c e v e s s e l s i n t h e f i n g e r s o f h y p e r t e n s i v e p a t i e n t s and p o s t u l a t e d a g e n e t i c d e f e c t i n NA t u r n o v e r ( 1 1 2 ) . S i n c e d o s e - r e s p o n s e c u r v e s c o u l d n o t be o b t a i n e d , t h e a l t e r n a t i v e e x p l a n a t i o n o f i n c r e a s e d v a s c u l a r r e s p o n s i v e n e s s c a n n o t be r u l e d o u t h e r e . I n " t h e l a t e 1 9 5 0 s , D o y l e ' s g r o u p r e p o r t e d v a s c u l a r smooth m u s c l e h y p e r s e n s i t i v i t y , i n h y p e r t e n s i v e s b u t t h i s was o f a n o n - s p e c i f i c n a t u r e b e c a u s e e n h a n c e d f l o w r e s t r i c t i o n r e s p o n s e s were n o t e d t o i n t r a - a r t e r i a l i n j e c t i o n s o f NA, a n g i o t e n s i n (Ang I I ) , and s e r o t o n i n (5HT) ( 1 1 3 , 1 1 4 ) . S i n c e t h e n , a h o s t o f r e p o r t s have a p p e a r e d i n t h e l i t e r a t u r e c o m m e n t i n g on v a s c u l a r s u p e r s e n s i t i v i t y i n human h y p e r t e n s i v e s , b u t some w o r k e r s c o u l d n o t d e m o n s t r a t e s u p e r s e n s i t i v i t y i n a r t e r i a l s t r i p s t e s t e d - i n v i t r o ( 1 1 5 ) . R e s u l t s f r o m s t u d i e s made on SHRs and WKYs were l e s s c o n t r o v e r s i a l . V a s c u l a r smooth m u s c l e p r e p a r a t i o n s t h a t were u s e d g e n e r a l l y f e l l i n t o one o f s u b c e l -l u l a r f r a c t i o n s ; i s o l a t e d v e s s e l s t r i p s and m e s e n t e r i c a r t e r i a l t r e e s ; c o m p l e t e i s o l a t e d v a s c u l a r b e d s o r t o t a l d e n e r v a t e d c a r d i o v a s c u l a r s y s t e m s . E a c h s y s t e m has i t s own a d v a n t a g e s and s h o r t - c o m i n g s , b u t t h e y a l l s e r v e d t o e n l i g h t e n us w i t h some g e n e r a l p r i n c i p l e s . A l t e r e d r e s p o n s i v e n e s s was n o t e d i n most o f t h e SHR v a s c u l a r smooth m u s c l e p r e p a r a t i o n s s t u d i e d when c o m p a r e d t o a p p r o p r i a t e c o n t r o l s ( 1 1 7 - 1 2 0 ) . T h e r e a l s o seemed t o be d i f f e r e n c e s i n - 24 -t h e s y m p a t h e t i c c o n t r o l o f v a s c u l a r smooth m u s c l e s i n SHR. C o l l i s ' g r o u p , f o r e x a m p l e , f o u n d an e x a g g e r a t e d n e u r o g e n i c r e s i s t a n c e r e s p o n s e i n r e n a l v a s c u l a r bed o f y o u n g SHRs and a l s o d e m o n s t r a t e d i n c r e a s e d NA r e l e a s e p e r p u l s e . T h e u n d e r l y i n g m e c h a n s i s m s o f t h e s e o b s e r v a t i o n s a r e s t i l l l a r g e l y unknown ( 1 2 2 ) . . , F u r t h e r work on t h e e f f e c t s o f r e g i o n a l s y m p a t h e t i c a c t i v a t i o n on v a s c u -l a r b e d s may g i v e us some i n s i g h t i n t o p o s s i b l e a r e a s o f a b n o r m a l i t i e s i n t h e v a s o c o n s t r i c t o r s y s t e m — one o f t h e most i m p o r t a n t f a c t o r s i n t h e r e g u -. l a t i o n o f s y s t e m i c r e s i s t a n c e and h e n c e t h e f i n a l a r t e r i a l b l o o d p r e s s u r e . V I I . I n t r o d u c t i o n and S t a t e m e n t o f P r o b l e m s . E a r l i e r work f r o m o u r l a b o r a t o r y ( 1 1 9 , 1 6 1 ) as w e l l as o t h e r s ( 1 6 2 , 1 6 3 ) seems t o i n d i c a t e t h a t t h e a b n o r m a l v a s c u l a r r e a c t i v i t y i n t h e SHR i s a s s o -c i a t e d w i t h c a l c i u m i o n b e i n g h a n d l e d d i f f e r e n t l y i n t h e v a s c u l a r smooth m u s c l e s o f t h e s e a n i m a l s c o m p a r e d t o n o r m o t e n s i v e c o n t r o l s . T h e v a s c u l a r smooth m u s c l e o f t h e p o r t a l v e i n s f r o m t h e SHR b e h a v e s as i f t h e r e i s an i n c r e a s e i n t h e amount o f c a l c i u m a v a i l a b l e t o t h e c o n t r a c t i l e a p p a r a t u s , and we s u s p e c t t h a t t h i s may be t h e r e s u l t o f an i n c r e a s e i n t h e p e r m e a b i l i t y o f t h e m u s c l e ' s membrane t o c a l c i u m . T h e r e i s ample d o c u m e n t a t i o n i n t h e l i t e r a t u r e r e g a r d i n g a l t e r e d i o n i c p e r m e a b i l i t y t i s s u e s f r o m t h e SHR. B o t h J o n e s ( 1 2 3 ) and F r i e d m a n ( 1 2 4 ) n o t i c e d i n c r e a s e d s o d i u m and p o t a s s i u m perme-a b i l i t i e s i n v a s c u l a r smooth m u s c l e o f SHRs. Hermsmeyer r e p o r t e d , e l e c t r o -p h y s i o l o g i c a l ^ , s i m i l a r f i n d i n g s i n t h e SHR t a i l a r t e r i e s . ( 1 2 5 ) , and A l t m a n ' s g r o u p n o t i c e d membrane a b n o r m a l i t i e s i n v i s c e r a l smooth m u s c l e s - 25 -) f r o m SHRs ( 1 2 6 ) . A l l o f t h e r e s u l t s s u g g e s t an i n c r e a s e i n t h e p a s s i v e i o n i c p e r m e a b i l i t y i n t h e SHR smooth m u s c l e s s t u d i e d . T h i s i s a l s o c o n s i s -t e n t w i t h t h e f i n d i n g s o f a German g r o u p some t w e n t y y e a r s ago who o b s e r v e d i n c r e a s e d s o d i u m p e r m e a b i l i t y i n e r y t h r o c y t e s f r o m p a t i e n t s w i t h p r i m a r y ( b u t n o t s e c o n d a r y ) h y p e r t e n s i o n . P e r h a p s more i n t e r e s t i n g was t h e i r o b s e r -v a t i o n o f a l t e r e d p e r m e a b i l i t y i n t h e n o r m o t e n s i v e r e l a t i v e s o f p a t i e n t s w i t h e s s e n t i a l h y p e r t e n s i o n as w e l l ( 1 2 8 ) , s u g g e s t i n g a s t r o n g g e n e t i c c omponent i n t h e phenomena o b s e r v e d . More r e c e n t l y , G a r a y ' s g r o u p r e p o r t e d t h e e x i s t a n c e o f a b n o r m a l i t i e s i n , s o d i u m and p o t a s s i u m i o n f l u x e s a c r o s s r e d c e l l membranes o b t a i n e d f r o m p a t i e n t s w i t h e s s e n t i a l h y p e r t e n s i o n ( 1 2 9 ) . P o s t n o v ' s g r o u p i n R u s s i a a l s o f o u n d i n c r e a s e d p a s s i v e s o d i u m p e r m e a b i 1 i t i y i n b o t h human and SHR e r y t h r o c y t e s , as w e l l as a d e f e c t i v e s o d i u m pump 'system ( 1 3 0 , 1 3 1 ) . More r e c e n t l y , t h i s same g r o u p r e p o r t e d a l t e r e d c a l c i u m i o n b i n d i n g c a p a c i t y i n e r y t h r o c y t e s f r o m b o t h h y p e r t e n s i v e s u b j e c t s and a n i m a l s ( 1 3 2 ) . I n t h e f i r s t p a r t - o f t h i s t h e s i s , a t t e m p t s were made t o d e a l w i t h some o f t h e p o s s i b l e m e c h a n i s m s u n d e r l y i n g t h e a p p a r e n t e r y t h r o c y t e membrane a b n o r m a l i t i e s m e n t i o n e d a b o v e . Our w o r k i n g h y p o t h e s i s i s t h a t t h e r e may be a g e n e t i c a l l y d e t e r m i n e d a l t e r a t i o n i n t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r -t i e s o f c e l l membranes i n t h e SHR. I f t h i s i s i n d e e d t h e c a s e , t h e n membrane a b n o r m a l i t i e s n eed n o t be r e s t r i c t e d t o v a s c u l a r smooth m u s c l e , b u t a l s o s h o u l d be f o u n d i n o t h e r membrane s y s t e m s i n t h e a n i m a l . T h i s i s i n d e e d s u p p o r t e d b y t h e f a c t t h a t membrane a l t e r a t i o n s h a v e been n o t e d i n v i s c e r a l smooth m u s c l e , a d i p o c y t e s as w e l l as e r y t h r o c y t e s ( 1 3 2 ) o f SHRs. S i n c e c a l c i u m i o n s a r e v i t a l f o r c o n t r a c t i o n o f v a s c u l a r smooth m u s c l e , we have a t t e m p t e d t o d e t e c t d i f f e r e n c e s i n c a l c i u m h a n d l i n g i n t h e SHRs a t t h e - 26 -membrane l e v e l , u s i n g t h e e r y t h r o c y t e as a model membrane s y s t e m . Red c e l l s f r o m SHR and DOCA h y p e r t e n s i v e a n i m a l s were s c r e e n e d f o r s t r u c t u r a l and f u n c t i o n a l a b n o r m a l i t i e s w i t h p a r t i c u l a r e m p h a s i s on c a l c i u m f l u x e s . T h e o b s e r v a t i o n s r e p o r t e d i n t h e R e s u l t s s e c t i o n may g i v e some i n s i g h t i n t o t h e p o s s i b l e i n t e r - r e l a t i o n s h i p b e t w e e n w i d e s p r e a d a b n o r m a l i t i e s i n c a l c i u m h a n d l i n g and t h e v a s c u l a r r e s p o n s i v e n e s s i n o u r h y p e r t e n s i v e a n i m a l . P a r a l -l e l e x p e r i m e n t s were p e r f o r m e d on e r y t h r o c y t e s f r o m D O C A / s a l t h y p e r t e n s i v e a n i m a l s t o c o n f i r m e d t h a t t h e membrane a l t e r a t i o n s o b s e r v e d a r e n o t c h a n g e s s e c o n d a r y t o e l e v a t e d b l o o d p r e s s u r e . C h r o n i c e t h a n o l c o n s u m p t i o n has been i m p l i c a t e d as a c a u s a t i v e f a c t o r i n a v a r i e t y o f c a r d i o v a s c u l a r a b n o r m a l i t i e s i n man and t h e r e a r e s e v e r a l e x c e l l e n t r e v i e w s w r i t t e n on t h i s a r e a ( 1 3 3 - 1 3 5 ) . T h e r e i s a l o n g s t a n d i n g s u s p i c i o n t h a t c h r o n i c e t h a n o l c o n s u m p t i o n c a n p r e c i p i t a t e h y p e r t e n s i o n i n man ( 1 4 0 , 1 4 1 ) . More r e c e n t l y , e p i d e m i o l o g i c a l s t u d i e s on a l c o h o l i c s and t h e g e n e r a l p o p u l a t i o n l e n d s s u p p o r t t o t h i s a s s o c i a t i o n by s h o w i n g a s t r o n g c o r r e l a t i o n b e t w e e n e t h a n o l c o n s u m p t i o n and b l o o d p r e s s u r e e l e v a t i o n ( 1 3 4 , 2 2 3 , 2 2 4 ) . C o n t r o v e r s i e s i n t h i s a r e a a r e p l e n t i f u l b e c a u s e r e s u l t s f r o m human s t u d i e s a r e v e r y d i f f i c u l t t o i n t e r p r e t . V a r i a b l e s s u c h as s t r e s s , s m o k i n g h a b i t s , n u t r i t i o n a l s t a t u s , g e n e t i c p r e d i s p o s i t i o n and p o o r q u a n t i -t a t i o n o f c o n s u m p t i o n may i n f l u e n c e b l o o d p r e s s u r e and may l i m i t t h e u s e f u l -n e s s o f t h e r e s u l t s so o b t a i n e d . We have a t t e m p t e d t o b y p a s s t h e s e p r o b l e m s by p r o d u c i n g a p h a r m a c o l o g i c a l model o f a l c o h o l i s m i n t h e r a t where most o f t h e v a r i a b l e s m e n t i o n e d c a n be c o n t r o l l e d o r m o n i t o r e d . U s i n g o u r r a t model o f a l c o h o l i s m , we i n v e s t i g a t e d t h e e f f e c t o f a l c o h o l c o n s u m p t i o n on c a r d i o -v a s c u l a r f u n c t i o n and b l o o d p r e s s u r e . S e v e r a l c a r d i o v a s c u l a r a b n o r m a l i t i e s were i d e n t i f i e d and a t t e m p t s w e r e made t o e l u c i d a t e some o f t h e u n d e r l y i n g ) - 27 -m e c h a n i s m s r e s p o n s i b l e f o r t h e a b n o r m a l i t i e s o b s e r v e d . R e c e n t l y , good e v i d e n c e h as a l s o been o b t a i n e d s u g g e s t i n g t h a t a c u t e and c h r o n i c a l c o h o l c o n s u m p t i o n a l t e r s p l a s m a membrane s t r u c t u r e and f u n c t i o n i n r o d e n t s ( 1 3 6 - 1 3 9 ) . We h a v e a l s o p a r t i a l l y c h a r a c t e r i z e d t h e membrane p r o p e r t i e s o f t h e e r y t h r o c y t e f r o m a l c o h o l t r e a t e d a n i m a l s i n s e a r c h f o r a b n o r m a l i t i e s t o compare w i t h t h o s e o b s e r v e d i n t h e SHR r e d c e l l membrane. I t i s g e n e r a l l y a c c e p t e d t h a t h y p e r t e n s i o n c a n be a m a n i f e s t a t i o n o f many u n d e r l y i n g a b n o r -m a l i t i e s . I n t h i s t h e s i s , we w i l l e x a m i n e and c r o s s - c o m p a r e t h e p o s s i b l e u n d e r l y i n g m e c h a n i s m s r e s p o n s i b l e f o r e l e v a t i n g t h e b l o o d p r e s s u r e s i n t h e s e v e r a l a n i m a l m o d e l s o f h y p e r t e n s i o n . T h e r e s u l t s may r e v e a l some o f t h e me c h a n i s m s t h a t a r e i m p o r t a n t i n a l l o f t h e m o d e l s s t u d i e d . More i m p o r t a n t -l y , i n f o r m a t i o n may be g a i n e d f r o m t h e s e e x p e r i m e n t s on t h e i m p o r t a n c e o f i n t e r a c t i o n s b e t w e e n t h e v a r i o u s i d e n t i f i e d m e c h a n i s m s i n p r e c i p i t a t i n g t h e f i n a l h y p e r t e n s i v e s t a t e i n t h e a n i m a l s . METHODOLOGY I . P r e p a r a t i o n o f A n i m a l s A. G e n e t i c h y p e r t e n s i o n . M a l e Okamoto and A o k i s t r a i n SHRs and t h e i r n o r m o t e n s i v e c o n t r o l s (WKYs) w e r e o b t a i n e d f r o m C h a r l e s R i v e r when t h e y were 8 weeks o l d . T h e a n i m a l s were g i v e n P u r i n a r a t chow and t a p w a t e r ad l i b i t u m . B l o o d p r e s s u r e and h e a r t r a t e were m o n i t o r e d w e e k l y b y t h e t a i l c u f f m e thod, a l l t h e p r e s s u r e v a l u e s r e p o r t e d were t h e mean o f t h r e e c o n s i s t e n t ( l e s s t h a n 5% v a r i a t i o n ) r e a d i n g s . R a t s were s a c r i f i c e d a t 9 weeks and 14 weeks o f age i n w e i g h t and age m a t c h e d ( a s c l o s e l y as p o s s i b l e ) p a i r s , w i t h a f i n a l f e m o r a l a r t e r i a l p r e s s u r e r e a d i n g t a k e n u n d e r l i g h t e t h e r a n a e s t h e s i a j u s t p r i o r t o s a c r i f i c e . I n some o f t h e l a t e r e x p e r i m e n t s , m a l e W i s t a r r a t s o f t h e same age were a d d e d as a d d i t i o n a l c o n t r o l s , t h e r e f o r e age m a t c h e d t r i p l e t s were u s e d when i n d i c a t e d . ( W i s t a r s a r e g e n e r a l l y h e a v i e r t h a t WKYs and SHRs o f t h e same age m a k i n g w e i g h t - m a t c h i n g i m p o s s i b l e i n t h e t r i p l e t s t u d i e s ) . B. D O C A / s a l t h y p e r t e n s i o n . M a l e W i s t a r r a t s w e i g h i n g 250 g ( C h a r l e s R i v e r ) were s u b j e c t e d t o u n i l a -t e r a l n e p h r e c t o m y . One week a f t e r t h e s u r g e r y , t h e r a t s were l i g h t l y a n a e s -t h e t i z e d w i t h e t h e r f o r c a n n u l a t i o n o f t h e f e m o r a l a r t e r y . F e m o r a l a r t e r i a l p r e s s u r e was r e c o r d e d w i t h a. s t a t h a m p r e s s u r e t r a n s d u c e r l i n k e d t o a G r a s s p o l y g r a p h . T h e n t h e r a t s were r a n d o m l y d i v i d e d i n t o two g r o u p s . One g r o u p o f r a t s ( t r e a t e d r a t s ) was i n j e c t e d s u b c u t a n e o u s l y t w i c e w e e k l y w i t h DOCA (15 mg/kg) ( S i g m a ) d i s s o l v e d i n sesame o i l and t h e i r d r i n k i n g w a t e r was r e p l a c e d w i t h 1% s o d i u m c h l o r i d e . T h e o t h e r g r o u p o f r a t s ( n e p h r e c t o m i z e d - 29 -c o n t r o l r a t s ) was i n j e c t e d t w i c e w e e k l y w i t h t h e v e h i c l e o n l y and was g i v e n t a p w a t e r . A t 4 weeks a f t e r g i v i n g D O C A / s a l t o r t h e v e h i c l e , c o n t r o l and t r e a t e d p a i r s o f r a t s were r a n d o m l y t a k e n f o r s t u d i e s . (A t h i r d g r o u p o f i n t a c t W i s t a r s f r o m t h e same s h i p m e n t were k e p t as e x t r a b l o o d p r e s s u r e c o n t r o l s , and t h e y were f e d s t a n d a r d f o o d and w a t e r . ) C. P h a r m a c o l o g i c a l model o f a l c o h o l i s m i n t h e r a t . In o u r p h a r m a c o l o g i c a l model o f a l c o h o l i s m , e i g h t - w e e k - o l d m a le W i s t a r r a t s ( C h a r l e s R i v e r ) w e i g h i n g a p p r o x i m a t e l y 250 gm were o b s e r v e d f o r two weeks p r i o r t o e t h a n o l t r e a t m e n t , and m e a s u r e m e n t s s u c h as body w e i g h t , b a s a l f o o d and f l u i d c o n s u m p t i o n were r e c o r d e d . B l o o d p r e s s u r e and .h e a r t r a t e were m e a s u r e d w e e k l y u s i n g t h e e x t e r n a l t a i l c u f f m e t hod. A t t h e o n s e t o f t h e e x p e r i m e n t , r a t s were a l l o c a t e d r a n d o m l y t o " t r e a t m e n t " and " c o n t r o l " g r o u p s . T h e a n i m a l s i n t h e " t r e a t m e n t " g r o u p r e c e i v e d 5% e t h a n o l ( d o u b l e d i s t i l l e d , v / v ) i n t h e i r d r i n k i n g w a t e r a d . l i b i t u m f o r t h e f i r s t week, t h e n 10% ( v / v ) e t h a n o l f o r t h e n e x t two weeks. A f t e r t h e t h i r d week, t h e y were g i v e n 20* ( v / v ) e t h a n o l f o r t h e r e s t o f t h e t r e a t m e n t p e r i o d . T o t a l t r e a t -ment t i m e was 12 weeks and t h e " c o n t r o l " g r o u p r e c e i v e d d o u b l e d i s t i l l e d w a t e r t h r o u g h o u t t h i s p e r i o d . B o t h g r o u p s were f e d w i t h P u r i n a r a t chow ( p u l v e r i z e d t o i m p r o v e t h e a c c u r a c y o f w e i g h i n g ) and d a i l y f l u i d and f o o d c o n s u m p t i o n were m o n i t o r e d . V i t a l p a r a m e t e r s m e n t i o n e d e a r l i e r were m o n i -t o r e d w e e k l y and b l o o d e t h a n o l l e v e l s w ere a s s a y e d w i t h C a l b i o c h e m ' s " E t h a n o l A s s a y S t a t " k i t b i - w e e k l y on b l o o d o b t a i n e d f r o m t h e c o n s c i o u s r a t by v e n o u s p u n c t u r e t h r o u g h t h e t a i l v e i n . P l a s m a and u r i n e s a m p l e s were c o l l e c t e d a t v a r i o u s t i m e s and s e n t o u t t o c l i n i c a , l l a b s f o r i o n a n a l y s e s . A t t h e end o f t h e 12 weeks, t r e a t m e n t and c o n t r o l p a i r s ( w e i g h t m a t c h e d t o w i t h i n 10 g) were u s e d f o r m e a s u r e m e n t s and l a t e r s a c r i f i c e d . A f i n a l f e m o r a l a r t e r i a l - 3 0 -) p r e s s u r e r e a d i n g was r e c o r d e d f r o m e a c h a n i m a l u n d e r l i g h t e t h e r a n a e s t h e s i a and an ECG r e c o r d i n g was a l s o t a k e n f o r a p p r o x i m a t e l y 10 m i n u t e s d u r i n g t h a t t i m e ( s u b c u t a n e o u s l e a d - I I ) . I I . M o del o f S t r e s s I n o r d e r t o i n v e s t i g a t e t h e e f f e c t s o f s t r e s s on t h e c a r d i o v a s c u l a r s y s t e m i n c h r o n i c a l c o h o l t r e a t e d a n i m a l s , we have d e v i s e d a s t r e s s model f o r o u r a n i m a l s . To e x a m i n e t h e e f f e c t s o f s t r e s s on t h e t h e b l o o d p r e s s u r e o f t h e a n i m a l s i n t h e c o n s c i o u s s t a t e w i t h o u t t h e i n f l u e n c e o f a n a e s t h e s i a and s u r g i c a l i n t e r v e n t i o n , a p o l y e t h y l e n e c a t h e t e r ( P E 5 0 ) was i n s e r t e d i n t o t h e t a i l a r t e r y o f t h e a n i m a l u n d e r e t h e r a n a e s t h e s i a and e x t e r i o r i z e d a t t h e neck by p a s s i n g t h r o u g h a s u b c u t a n e o u s c h a n n e l a l o n g t h e back o f t h e a n i m a l . T h e c a t h e t e r was k e p t p a t e n t b y f l u s h i n g w i t h h e p a r i n i z e d s a l i n e (20 I U / m l ) . A t 24-48 h o u r s a f t e r s u r g e r y , t h e c a t h e t e r was l i n k e d t o a p r e s s u r e t r a n s d u c e r o u t s i d e t h e c a g e and a c o n t i n u o u s b l o o d p r e s s u r e t r a c i n g r e c o r d e d on a G r a s s p o l y g r a p h . A f t e r e x p e r i m e n t i n g w i t h many methods o f s t r e s s i n c l u d i n g r e p e a t e d e x p o s u r e s t o i n v o l u n t a r y r e s t r a i n t , swimming, blow d r y i n g and l o u d n o i s e , we f o u n d t h a t i r r a d i a t i o n w i t h a 150 w a t t h e a t lamp f o r a s h o r t p e r i o d most r e p r o d u c i b l y e l i c i t e d a t r a n s i e n t r i s e i n a r t e r i a l p r e s s u r e i n t h e a n i m a l s t e s t e d . (We h a v e made t h e a s s u m p t i o n h e r e t h a t t r a n s i e n t b l o o d p r e s s u r e e l e v a t i o n s d e n o t e d p h y s i c a l s t r e s s i n t h e a n i m a l s . ) F o r t h e c h r o n i c e t h a n o l and s t r e s s m o d e l s , a n i m a l s were p r e p a r e d as m e n t i o n e d a b o v e i n M e t h o d o l o g y S e c . I C . A t week 4, t h e 2 g r o u p s o f a n i m a l s were f u r t h e r d i v i d e d r a n d o m l y i n t o u n s t r e s s e d and s t r e s s e d g r o u p s . The s t r e s s e d g r o u p s ( b o t h e t h a n o l - t r e a t e d and c o n t r o l ) t h e n r e c e i v e d h e a t i r r a d i a t i o n - 31 -d a i l y (5 min o f h e a t a l t e r n a t i n g w i t h 5 min o f r e s t ) f o r 1 h r f o r t h e r e m a i n d e r o f t h e e x p e r i m e n t a l p e r i o d . A t week 12, one a n i m a l f r o m e a c h g r o u p ( w e i g h t m a t c h e d ) was s a c r i f i c e d f o r p h y s i o l o g i c a l and b i o c h e m i c a l s t u d i e s . I I I . E r y t h r o c y t e P r e p a r a t i o n s U s u a l l y 10 ml o f b l o o d was w i t h d r a w n f r o m e t h e r a n a e s t h e t i z e d a n i m a l s t h r o u g h c a r d i a c p u n c t u r e u s i n g ACD ( s t a n d a r d a c i d - c i t r a t e - d e x t r o s e ) as t h e a n t i c o a g u l a n t . A f t e r i n i t i a l c e n t r i f u g a t i o n ( 2 , 0 0 0 x g f o r 10 min) and r e m o v a l o f p l a s m a and b u f f y c o a t , t h e r e d c e l l s were washed t w i c e w i t h c o l d ( 4 ° C ) i s o t o n i c s a l i n e . I n p r o c e d u r e s where i n t a c t e r y t h r o c y t e s were r e q u i r e d , t h e s a m p l e s were t a k e n and u s e d a t t h i s p o i n t . Red c e l l membranes were p r e p a r e d by s u b j e c t i n g t h e washed e r y t h r o c y t e s t o t h e m o d i f i e d s t e p w i s e h a e m o l y s i s p r o c e d u r e s o f S c h r i e r ( 1 42) u s i n g s o d i u m c h l o r i d e s o l u t i o n s o f 0.08, 0.06, 0.04 and 0.02 m o l a r i t y , w i t h a p a c k e d c e l l : h a e m o l y s i n g s o l u t i o n r a t i o o f 1:50. A f i n a l wash w i t h 10 mM T r i s (pH 7.4) was i n c l u d e d t o e l i m i n a t e r e s i d u a l h a e m o g l o b i n . T h e membranes were t h e n q u i c k f r o z e n a t , - 8 0 ° C and s t o r e d a t - 2 0 ° C . E n z y m a t i c and p r o t e i n a n a l y s i s were p e r f o r m e d w i t h i n 24 h o u r s . « I V . Membrane Enzyme A s s a y s The m a g n e s i u m - d e p e n d e n t , c a l c i u m - s t i m u l a t e d a d e n o s i n e t r i p h o s p h a t a s e ( C a + + / M g + + A T P a s e ) a c t i v i t i e s w e r e a s s a y e d u s i n g t h e p r o c e d u r e o f Q u i s t and R o u f o g a l i s ( 1 4 3 ) . The E G T A - C a + + b u f f e r s y s t e m u s e d g ave f r e e C a + + - 32 -i o n c o n c e n t r a t i o n s i n t h e r a n g e o f 0.16-279 uM. A f t e r s e v e r a l s e r i e s o f e x p e r i m e n t s , t h e D i p h a s i c c a l c i u m d e p e n d e n c e c u r y e i n d i c a t e d t o us t h a t t h e s p e c i f i c a c t i v i t y l e v e l e d o f f a t 2.7 uM and 179 uM e x t e r n a l c a l c i u m c o n c e n -t r a t i o n , t h e r e f o r e t h o s e two p o i n t s were t a k e n as h i g h and low a f f i n i t y a c t i v i t i e s r e s p e c t i v e l y ( s e e T a b l e 1 ) . R e a c t i o n s were t e r m i n a t e d by t h e a d d i t i o n o f 1.0 ml o f c o l d 1 0 % TCA and c e n t r i f u g a t i o n ( 1 , 0 0 0 x g f o r 5 min) a t low t e m p e r a t u r e . T h e i n o r g a n i c p h o s p h a t e l i b e r a t e d t o t h e s u p e r n a t a n t was m e a s u r e d by t h e F i s k e - S u b b a R o w method ( 1 4 4 ) . ++ ++ E s t i m a t i o n o f a c t i v a t i o n e n e r g y f o r t h e Ca /Mg A T P a s e was p e r f o r -med by i n c u b a t i n g t h e r e a c t i o n m i x t u r e s m e n t i o n e d above a t v a r i o u s t e m p e r a -t u r e s ( 1 0 ° C , 2 0 ° C , 3 0 ° C , 3 7 ° C and 4 2 ° C ) and c o n s t r u c t i n g A r r h e n i u s p l o t s ( L o g g s p e c i f i c a c t i v i t y vs_. 1 / T ) . T h e a c t i v a t i o n e n e r g y ( E a ) was c a l c u l a -t e d f r o m t h e s l o p e o f t h i s p l o t and t h e e q u a t i o n u s e d was: l n ( S . A . ) = _1 E a + c o n s t a n t T R ^ Na/K A T P a s e a c t i v i t y was a s s a y e d u s i n g a s i m i l a r p r o t o c o l e x c e p t t h a t 250 u m o l e s o f NaCl' and 90 u m o l e s o f KC1 were u s e d i n t h e p l a c e o f c a l c i u m ( 1 4 2 ) . A c e t y l c h o l i n e s t e r a s e was a s s a y e d s p e c t r o p h o t o m e t r i c a l l y a t room t e m p e r a t u r e b y q u a n t i f y i n g t h e amount o f t h i o c h o l i n e r e l e a s e d f r o m a c e t y l -t h i o c h o l i n e u s i n g 5 , 5 - d i t h i o - 2 - n i t r o b e n z o i c a c i d (DTNB) as t h e s u b s t r a t e . T h e r e a c t i o n m i x t u r e c o n t a i n e d 2.6 ml T r i s b u f f e r (0.2 M, pH 8 . 0 ) , 0.1 ml a c e t y l t h i o c h o l i n e (30 mM), 0.1 ml DTNB (10 mM, d i s s o l v e d i n 0.1 M T r i s b u f f e r , pH 8.0) and 0.2 ml membrane s u s p e n s i o n ( 0 . 2 - 0 . 4 mg/ml). T h e r e a c t i o n was i n i t i a t e d w i t h a c e t y l t h i o c h o l i n e , and r a t e s were c a l c u l a t e d f r o m t h e i n c r e a s e o f a b s o r b a n c e a t 412 nm d u r i n g t h e f i r s t 5 m i n u t e s o f t h e r e a c t i o n , w i t h r e a d i n g s b e i n g t a k e n e v e r y 30 s e c o n d s . N o n - e n z y m a t i c h y d r o l y s i s o f s u b s t r a t e was c o r r e c t e d f o r i n a l l a s s a y s ( 1 4 5 ) . - 33 -TABLE 1. R e a g e n t T a b l e F i n a l C o n e . (mM) B a s a l H i g h Low B l a n k I B l a n k I I A f f i n i t y A f f i n i t y T r i s (pH 7.4) 55.0 55.0 55.0 - 55.0 55.0 ATP 2.0 2.0 2.0 2.0 2.0 Mg 6.4 6.4 6.4 6.4 6.4 Ca (yM) 0 2.7 179.0 2.7 179.0 EGTA" 0.1 0.1 . 0.1 0.1 0.1 Membrane ( i n a p p r o x . mg o f p r o t e i n ) 0.8 0.4 0.4 0 0 H2O as r e q u i r e d t o make up 3.0 ml f i n a l v o l u m e 1. Enzyme R e a c t i o n . I n c u b a t i o n t u b e s w e r e s e t up as above and e q u i l i b r a t e d i n w a t e r b a t h s a t 3 7 ° C . Membranes were added t o s t a r t t h e r e a c t i o n . 1.0 mL o f 10% c o l d TCA was a d d e d t o e a c h t u b e t o s t o p t h e r e a c t i o n a f t e r 1.0 h o u r and p r o t e i n p r e c i p i t a t e s were s e d i m e n t e d a t 2,000 x g f o r 10 m i n . 2. C o l o u r R e a c t i o n . 3.0 ml s u p e r n a t a n t f r o m e a c h t u b e i n ( 1 ) was m i x e d w i t h 5% m o l y b d a t e and h^O. R e a c t i o n was s t a r t e d by a d d i n g 0.2 m l o f ANS s o l u t i o n t o e a c h t u b e . P h o s p h a t e c o n c e n t r a t i o n s w ere e s t i m a t e d f r o m a b s o r b a n c e a t 660 nm a f t e r 15.0 min i n c u b a t i o n f r o m a c a l i b r a t i o n c u r v e made w i t h s t a n d a r d p h o s p h a t e s o l u t i o n s . - 34 -V. Membrane C o m p o s i t i o n S t u d i e s A. T o t a l p h o s p h o l i p i d e s t i m a t i o n . E r y t h r o c y t e membranes o b t a i n e d as i n M e t h o d o l o g y S e c . I I I . a b o v e were u s e d i n t h i s a s s a y . B r i e f l y , 25 y l o f membrane was added t o 1.5 ml o f 70 p e r c h l o r i c a c i d i n a c e n t r i f u g e t u b e and h e a t e d f o r 30 min i n a s a n d b a t h a t 2 3 0 ° C . A f t e r c o o l i n g t o room t e m p e r a t u r e , t h e s a m p l e was d i l u t e d w i t h 7.5 ml o f d o u b l e d i s t i l l e d w a t e r and m i x e d t h o r o u g h l y . I t was t h e n c e n t r i f u g e d f o r 5 m i n a t 2,000 x g. An a l i q u o t o f 4.5 ml was t a k e n and added t o a r e a c t i o n m i x t u r e c o n t a i n i n g 0.5 ml d o u b l e d i s t i l l e d w a t e r , 0.2 ml 5% ammonium m o l y b -d a t e , and 0.2 ml ANS ( s e e A p p e n d i x f o r i n g r e d i e n t s ) . T h e m i x t u r e was h e a t e d i n a b o i l i n g w a t e r b a t h f o r 8 m i n , c o o l e d and t h e a b s o r b a n c e was r e c o r d e d a t 830 nm u s i n g a Beckmann A c t a I I s p e c t r o m e t e r ( 1 4 6 ) . T h e r e a d i n g was c o m p a r e d t o a p h o s p h o r u s s t a n d a r d ( S i g m a ) o f 20 y g / m l , and r e s u l t s were r e p o r t e d i n y g phophorus/mg membrane p r o t e i n . T o t a l p l a s m a p h o p h o l i p i d s were a s s a y e d s i m i l a r l y e x c e p t t h a t 1.0 ml o f s a m p l e was u s e d f o r t h e i n i t i a l s t e p and t h e r e s u l t s w ere r e p o r t e d as y g p h o s p h o r u s / m l p l a s m a . B. P h o s p h o l i p i d p r o f i l e . F i v e h u n d r e d y l o f r e d . c e l l membrane was e x t r a c t e d w i t h 2.0 ml o f c h l o r o f o r m / m e t h a n o l ( 2:1 v / v ) s o l v e n t and l e f t s t a n d i n g on i c e f o r 30 m i n . T h e n i t was c e n t r i f u g e d a t 6,000 x g f o r 5 m i n . The s u p e r n a t a n t was s a v e d and t h e p e l l e t was e x t r a c t e d a s e c o n d t i m e as b e f o r e . T h e s u p e r n a t a n t was t h e n p o o l e d and washed t h r e e t i m e s w i t h 0.5 ml o f 0.75 s o d i u m c h l o r i d e s o l u t i o n . T h e o r g a n i c p h a s e was t h e n e v a p o r a t e d t o d r y n e s s u n d e r n i t r o g e n and r e d i s s o l v e d i n 200 y l o f t h e same c h l o r o f o r m / m e t h a n o l s o l v e n t . T h e s a m p l e was t h e n s p o t t e d o n t o a S i l i c a g e l t h i n l a y e r c h r o m a t o g r a p h i c p l a t e \ \ - 35 -( B r i n k m a n n F254, 25 mm t h i c k n e s s , a c t i v a t e d e a r l i e r b y h e a t i n g a t 1 5 0 ° C f o r 30 min) and d e v e l o p e d i n a s o l v e n t s y s t e m c o n t a i n i n g 25 p a r t s c h l o r o f o r m , 15 p a r t s m e t h a n o l , 4 p a r t s a c e t i c a c i d , and 2 p a r t s w a t e r by v o l u m e . Amino p h o s p h o l i p i d s p o t s were l o c a l i z e d w i t h n i n h y d r i n s p r a y ( B a k e r ) and t h e r e m a i n i n g p h o p h o l i p i d s were v i s u a l i z e d w i t h i o d i n e v a p o r . I n d i v i d u a l p h o s -p h o l i p i d s were q u a n t i f i e d by e x t r a c t i o n f r o m t h e s i l i c a g e l u s i n g t h e p r o c e -d u r e d e s c r i b e d i n M e t h o l o d o l o g y S e c . VA a b o v e . R e s u l t s were r e p o r t e d as p e r c e n t a g e o f t o t a l p h o s p h o l i p i d p h o s p h o r u s . F o r p l a s m a p h o s p h o l i p i d p r o f i l e , 1.0 ml o f s a m p l e was u s e d i n s t e a d i n t h e i n i t i a l e x t r a c t i o n and r e s u l t s were a l s o e x p r e s s e d as p e r c e n t a g e o f t o t a l p h o s p h o l i p i d p h o s p h o r u s i n p l a s m a as m e n t i o n e d i n Sec.'VA. C. C h o l e s t e r o l d e t e r m i n a t i o n . G l a c i a l a c e t i c a c i d ( 2 . 0 ml) was u s e d t o d i s s o l v e 50 y l o f membranes i n a g l a s s c e n t r i f u g e t u b e . T h e t i m e r e q u i r e d f o r t h i s p r o c e s s was a b o u t 30 min a t room t e m p e r a t u r e w i t h p e r i o d i c s t i r r i n g . T h e n 1.3 ml o f Zak w o r k i n g r e a g e n t ( s e e A p p e n d i x ) was added c a r e f u l l y t o t h e s o l u t i o n s o as t o u n d e r l a y t h e g l a c i a l a c e t i c a c i d . R e a c t i o n was s t a r t e d b y ^ m i x i n g t h e two l i q u i d p h a s e s ( c a u t i o u s l y ) on a v o r t e x m i x e r f o r 10 s e c . T h e c o l o u r was a l l o w e d t o d e v e l o p f o r 30 min i n room t e m p e r a t u r e and t h e a b s o r b a n c e a t 565 nm was r e c o r d e d ( 1 4 7 ) . R e s u l t s were r e p o r t e d as y m o l . c h o l e s t e r o l / m g membrane p r o t e i n . F o r p l a s m a c h o l e s t e r o l a s s a y , 10 y l s a m p l e s were u s e d f o r t h e i n i t i a l s t e p and r e s u l t s were r e p o r t e d as ymol./ml p l a s m a . - 36 -/ V I . O s m o t i c F r a g i l i t y A p p r o x i m a t e l y 1.0 ml o f washed e r y t h r o c y t e s was u s e d f o r t h i s p r o c e d u r e . T h e c e l l s w ere washed o n c e i n a c o l d s o l u t i o n o f T r i s (15 mM, pH 7.0) i n i s o t o n i c s a l i n e and r e s u s p e n d e d i n 10 ml o f c o l d T r i s s a l i n e s o l u t i o n ( t o a h a e m a t o c r i t o f a b o u t 6 % ) . A l i q u o t s (0.2 m l ) o f t h i s c e l l s u s p e n s i o n were added t o 3.8 ml o f s o d i u m c h l o r i d e s o l u t i o n s o f v a r y i n g c o n c e n t r a t i o n s ( 0.07 t o 0.04 M) made up i n 15 mM T r i s b u f f e r (pH 7 . 0 ) . S a m p l e s were c e n t r i f u g e d f o r 1 min a t 40,000 x g a f t e r a 15 min i n c u b a t i o n a t room t e m p e r a t u r e and t h e e x t e n t o f h a e m o l y s i s a t e a c h s o d i u m c h l o r i d e c o n c e n t r a t i o n was a s s e s s e d f r o m t h e a b s o r b a n c e o f t h e s u p e r n a t a n t a t 540 nm. R e s u l t s were e x p r e s s e d as p e r c e n t o f t h e a b s o r b a n c e o f an e q u a l a l i q u o t o f c e l l s l y s e d i n d i s t i l l e d w a t e r . R e p r o d u c i b i l i t y o f t h i s p r o c e d u r e i s v e r y h i g h i f t h i s r e g i m e n i s f o l l o w e d r i g i d l y ( s e e r e f . 1 4 8 ) . ( T h i s p o r t i o n o f membrane f u n c t i o n a l and s t r u c t u r a l a n a l y s e s was a c c o m p l i s h e d i n c o l l a b o r a t i o n w i t h D r . D a v i d V. G o d i n , D e p a r t m e n t o f P h a r m a c o l o g y , U.B.C.) V I I . P a s s i v e C a l c i u m I n f l u x M e a s u r e m e n t s i n I n t a c t E r y t h r o c y t e s . A. C a l c i u m l o a d i n g . S a m p l e s o f i n t a c t r e d c e l l s were o b t a i n e d as o u t l i n e d i n s e c t i o n I I I , and were s p l i t i n t o two ' p o r t i o n s . One p a r t was s u b j e c t e d t o n e u r a m i n i d a s e d i g e s t i o n a t 3 7 ° C w h i l e t h e o t h e r was i n c u b a t e d w i t h s a l i n e a t t h e same t e m p e r a t u r e ( s e e b e l o w f o r m e t h o d o l o g y ) . A f t e r t h e p r e t r e a t m e n t , 1.0 ml o f p a c k e d r e d c e l l s was added t o 4.0 ml o f c o l d ( 4 ° C ) " l o a d i n g " s o l u t i o n - 37 -45 c o n t a i n i n g N a C l 75 mM, T r i s 83 mM, KC1 4.5 mM, M g C l 2 1.2 mM, C a C l 2 3.0 mM (10 v i C i / m l ; New E n g l a n d N u c l e a r ) , and EDTA 23 pM 'at pH 7.4. T h e c e l l s were h a r v e s t e d a f t e r 20 h o u r s o f i n c u b a t i o n a t 4 ° C w i t h c o n t i n u o u s s t i r r i n g . An a l i q u o t o f t h e c e l l s u s p e n s i o n was d i l u t e d w i t h 10 v o l u m e s o f c h i l l e d , n o n - r a d i o a c t i v e l o a d i n g s o l u t i o n c o n t a i n i n g 3.0 mM c a l c i u m . A f t e r t h o r o u g h m i x i n g , t h e s a m p l e s were c e n t r i f u g e d at. 2,000.g f o r 15 min and t h e s u p e r n a t a n t d i s c a r d e d . T h i s w a s h i n g p r o c e d u r e was r e p e a t e d , and t h e a f i n a l wash u s i n g c o l d ( 4 ° C ) c a l c i u m - f r e e " l o a d i n g " s o l u t i o n ( w i t h 1.0 mM EDTA) was i n c l u d e d t o e n s u r e t o t a l e x t e r n a l c a l c i u m e x t r a c t i o n . T h e n one a l i q u o t ( 0 . 1 ml) o f p a c k e d r e d c e l l s was u s e d i n t h e l i q u i d s c i n i t i l l a t i o n c o u n t i n g w i t h a s e c o n d 0.1 ml a l i q u o t b e i n g t a k e n f o r manual r e d c e l l c o u n t . To t e s t 45 t h e c o n t r i b u t i o n o f f r e e i n t r a c e l l u l a r C a t o o u r t o t a l c o u n t , 1.5 yM o f t h e c a l c i u m i o n o p h o r e A23187 ( S i g m a ) was i n c l u d e d i n t h e f i n a l wash i n t h e a b o v e p r o c e d u r e i n i t i a l l y and o v e r 9 5 % o f t h e t o t a l c o u n t s was c o n s i s t e n t l y f o u n d t o be r e l e a s e d i n t o t h e s u p e r n a t a n t by t h e i o n o p h o r e . B. P r e p a r a t i o n o f r e d c e l l s f o r L i q u i d S c i n t i l l a t i o n C o u n t i n g ( L S C ) . G l a s s LSC v i a l s (22 ml) w i t h p o l y e t h y l e n e l i n e d c a p s were u s e d i n t h i s 45 p r o c e d u r e . An a l i q u o t o f 0.1 ml o f Ca l o a d e d r e d c e l l s was p l a c e d i n t h e v i a l and 0.5 ml o f a 1:2 P r o t o s o l : e t h a n o l m i x t u r e was added t o t h e v i a l and m i x e d g e n t l y . T h e v i a l was c a p p e d t i g h t l y and i n c u b a t e d a t 6 0 ° C i n a s h a k i n g w a t e r b a t h f o r 1 h r . A f t e r c o o l i n g t o room t e m p e r a t u r e , 0.5 ml o f 30 h y d r o g e n p e r o x i d e ( F i s h e r ) was added d r o p w i s e t o t h e v i a l . When t h e f o a m i n g had s u b s i d e d , t h e v i a l was c a p p e d l o o s e l y and i n c u b a t e d a t 6 0 ° C f o r an a d d i t i o n a l 30 m i n . A f t e r c o o l i n g , 15 ml o f B i o f l u o r (New E n g l a n d N u c l e a r ) s c i n t i l l a t i o n c o c k t a i l was added' and m i x e d . v i g o r o u s l y . T h e n 0.5 ml o f 0.5 N h y d r o c h l o r i c a c i d ( F i s h e r ) was added and m i x e d t h o r o u g h l y a g a i n . T h e v i a l - 38 -was t h e n c o u n t e d i n a P a c k a r d 3385 c o u n t e r a t 7°C f o r 10 min ( i n t r i p l i c a t e ) u s i n g a g a i n o f 6% and a window o f 5 0 - 1 0 0 0 . T y p i c a l c o u n t i n g e f f i c i e n c y was 6 5 % u s i n g t h i s p r o c e d u r e (N.E.N. T e c h n i c a l B u l l e t i n ) . C. N e u r a m i n i d a s e d i g e s t i o n . B e f o r e t h e i n t a c t r e d c e l l s were u s e d f o r c a l c i u m l o a d i n g s t u d i e s , one p o r t i o n o f t h e c e l l s was s u b j e c t e d t o n e u r a m i n i d a s e t r e a t m e n t t o remove t h e s i a l i c a c i d r e s i d u e s on t h e c e l l s u r f a c e . B r i e f l y , .washed r e d c e l l s p r e p a r e d as d e s c r i b e d i n S e c t i o n I I I were added t o an i n c u b a t i o n s o l u t i o n c o n t a i n i n g 145 mM N a C l , 5 mM C a C l 2 , and 0.3 mM NaHC0 3 a t pH 7.2. F o r e v e r y 1.0 ml o f p a c k e d e r y t h r o c y t e s , 3.0 ml o f t h e i n c u b a t i o n s o l u t i o n was u s e d . Neuram-i n i d a s e (1 I U / m l , f r o m B e h r i n g w e r k e A . G . ) . w a s added a t a c o n c e n t r a t i o n o f 0.08 IU p e r ml o f p a c k e d c e l l s w h i l e c o n t r o l s a m p l e s r e c e i v e d e q u i v a l e n t v o l u m e s o f n o r m a l s a l i n e . S a m p les were t h e n i n c u b a t e d f o r 30 min a t 3 7 ° C , c e n t r i f u g e d a t 2,000 x g f o r 5 min and t h e s u p e r n a t a n t d i s c a r d e d . The c e l l s w e re washed t w i c e w i t h i s o t o n i c s a l i n e and o n c e w i t h n o r m a l s a l i n e - T r i s (15 mM, pH 7.4) s o l u t i o n . T h e n , 1.0 ml o f t h e s e p a c k e d c e l l s were u s e d i n t h e c a l c i u m l o a d i n g p r o c e d u r e m e n t i o n e d a b o v e i n S e c . V I I - A . D. S i a l i c a c i d m e a s u r e m e n t s . S a m p l e s t a k e n f r o m b o t h n e u r a m i n i d a s e t r e a t e d and u n t r e a t e d r e d c e l l s w ere h a e m o l y s e d i n 10 mM T r i s a t pH 7.4. T h e h a r v e s t e d membranes were h y d r o -l y s e d i n 0.1 N h^SO^ a t 8 0 ° C f o r 30 min and s u b j e c t e d t o t h e s i a l i c a c i d a s s a y a c c o r d i n g t o W a r r e n ( 1 4 9 ) . N e u r a m i n i d a s e t r e a t m e n t t y p i c a l l y r e m o v e d more t h a n 80% o f t h e m e m b r a n e - a s s o c i a t e d s i a l i c a c i d i n o u r s a m p l e s . - 39 -V I I I . L a n g e n d o r f f s P e r f u s i o n i n H e a r t s f r o m C h r o n i c A l c o h o l T r e a t e d A n i m a l s A. P r e p a r a t i o n and i s o l a t i o n o f h e a r t s . A t t h e end o f t h e 12-week a l c o h o l t r e a t m e n t p e r i o d , w e i g h t - m a t c h e d ( t o w i t h i n 10 g) t r e a t m e n t and c o n t r o l p a i r s o f r a t s were u s e d and s a c r i f i c e d . The a n i m a l s were a n a e s t h e t i z e d w i t h e t h e r , and s u b c u t a n e o u s ECG e l e c t r o d e s w ere i m p l a n t e d t o m o n i t o r h e a r t r a t e and t h e o c c u r r e n c e o f a r r h y t h m i a s . F e m o r a l a r t e r i a l p r e s s u r e r e a d i n g s were m e a s u r e d t h r o u g h a c a n n u l a ( P E 5 0 ) l i n k e d t o a G o u l d / S t a t h a m P23ID p r e s s u r e t r a n s d u c e r . A l l r e c o r d i n g s were d i s p l a y e d on a G r a s s model 7D p o l y g r a p h . A f t e r a l l t h e n e c e s s a r y m e a s u r e -ments were made, a b l o o d s a m p l e ( 2 . 0 ml) was t a k e n t h r o u g h t h e a r t e r i a l c a n n u l a f o r t h e d e t e r m i n a t i o n o f b l o o d a l c o h o l c o n t e n t and p l a s m a c a t e c h o l a -m i n e c o n c e n t r a t i o n . A f t e r 50 I.U. o f h e p a r i n was i n j e c t e d i n t r a - a r t e r i a l l y i n t o t h e a n i m a l , t h e h e a r t was q u i c k l y r e m o v e d by s t e r n a l t h o r a c o t o m y and i m m e d i a t e l y p l u n g e d i n t e r c o l d ( 4 ° C ) K r e b ' s - R i n g e r s o l u t i o n ( s e e A p p e n d i x ) t o s t o p t h e c o n t r a c t i o n s . The h e a r t s were t r i m m e d o f e x c e s s f a t and c o n n e c t i v e t i s s u e , b l o t t e d and w e i g h e d p r i o r t o t h e o n s e t o f p e r f u s i o n . B. / S t a r l i n g c u r v e s i n i s o l a t e d p e r f u s e d h e a r t s . The e x c i s e d h e a r t s were p e r f u s e d a t a c o n s t a n t f i l l i n g p r e s s u r e o f 90 cm H2O w i t h K r e b ' s - R i n g e r ' s o l u t i o n s a t u r a t e d w i t h 95 % 0^ - 5% C0^ a t 3 7 ° C (pH 7.4) w i t h a m o d i f i e d L a n g e n d o r f f p r o c e d u r e ( 1 5 0 ) . T h e p e r f u s i o n f l o w f o r e a c h h e a r t u n d e r r e s t i n g c o n d i t i o n s was 15.5 ± 0.7 m l / m i n . I n t r a v e n t r i -c u l a r p r e s s u r e ( I V P ) was m e a s u r e d w i t h a s a l i n e - f i l l e d l a t e x b a l l o o n (maximum i n f l a t a b l e v o l u m e o f 2.0 ml) t h r e a d e d t h r o u g h ah i n c i s i o n on t h e l e f t a t r i u m i n t o t h e l e f t v e n t r i c l e , and m o n i t o r e d u s i n g a S t a t h a m P23 ID p r e s s u r e t r a n s -- 40 -d u c e r l i n k e d v i a a c a n n u l a ( P E 5 0 ) t o t h e b a l l o o n . A f t e r a l l o w i n g 15 min o f e q u i l i b r a t i o n f r o m t h e i n i t i a t i o n o f p e r f u s i o n , a S t a r l i n g c u r v e was o b t a i n e d f o r e a c h h e a r t . T h i s was a c c o m p l i s h e d b y i n c r e a s i n g t h e e n d d i a s t o l i c p r e s s u r e (EDP) t h r o u g h i n f l a t i o n o f t h e i n t r a v e n t r i c u l a r b a l l o o n ( 0 - 7 0 mmHg, i n 10 mm i n c r e m e n t s ) and m o n i t o r i n g t h e c o r r e s p o n d i n g I V P . S i n c e t h e autonomous h e a r t r a t e s v a r i e d i n e a c h h e a r t , a s e c o n d S t a r l i n g r e l a t i o n s h i p was o b t a i n e d f r o m e a c h h e a r t u n d e r e l e c t r i c a l p a c i n g (240 p u l s e s / m i n , 1.5 V, 2.0 msec d u r a t i o n ) t h r o u g h a p a i r o f p l a t i n u m e l e c t r o d e s a t t a c h e d t o t h e l e f t a t r i u m . IVP was c a l c u l a t e d as end s y s t o l i c p r e s s u r e ( E S P ) m i n u s e n d d i a s t o l i c p r e s s u r e ( E D P ) . C. Dose r e s p o n s e r e l a t i o n s h i p t o i s o p r o t e r e n o l ( I P N A ) . F o l l o w i n g t h e d e t e r m i n a t i o n o f t h e s e c o n d S t a r l i n g c u r v e , h e a r t s were a l l o w e d t o e q u i l i b r a t e f o r 15 min a t an EDP o f 10 mmHg. IPNA (± I s o p r o t e r e -n o l f r o m Sigma) was i n f u s e d f r o m two s t o c k s o l u t i o n s (3 x 10^ and 3 x 10 M) i n t o t h e p e r f u s a t e t o p r o v i d e f i n a l c o n c e n t r a t i o n s o f -9 -6 1 x 10 t o 1 x 10 M and t h e IVP a t e a c h d o s e was r e c o r d e d . T h e e f f e c t s o f c a l c i u m i o n c o n c e n t r a t i o n i n t h e p e r f u s a t e on t h e d o s e r e s p o n s e , c u r v e s o f t h e h e a r t s -were e x p l o r e d i n a r a n d o m i z e d manner b y u s i n g two c o n c e n t r a t i o n s o f c a l c i u m ( 2 . 5 mM and 0.2 mM). T h e h e a r t s were e l e c t r i c a l l y p a c e d t h r o u g h o u t t h e d o s e r e s p o n s e p r o t o c o l . A t t h e end o f t h e e x p e r i m e n t , a l a s t S t a r l i n g r e l a t i o n s h i p was o b t a i n e d f r o m e a c h h e a r t ( u n p a c e d ) t o q u a n t i f y t h e e x t e n t o f m y o c a r d i a l d e t e r i o r a t i o n . T y p i c a l l y , l e s s t h a n 10% r e d u c t i o n i n b o t h t h e maximum IVP ( a t t h e same EDP) and t h e autonomous h e a r t r a t e was o b s e r v e d c o m p a r e d w i t h m e a s u r e m e n t s made a t t h e o n s e t o f p e r f u s i o n , and t h e w h o l e p r o t o c o l was c o m p l e t e d w i t h i n 2 ' h r . The h e a r t s were b l o t t e d and w e i g h e d a f t e r e a c h e x p e r i m e n t . The v e n t r i c l e s - 41 -w ere t h e n e x c i s e d , w e i g h e d , and d r i e d o v e r n i g h t i n a c o n v e c t i o n o v e n . T h e d r y w e i g h t s o f t h e v e n t r i c l e s w ere a l s o r e c o r d e d . ( F o r more d e t a i l s , s e e r e f . 151.) T h e IVP v a l u e s r e p o r t e d i n t h e d o s e r e s p o n s e r e l a t i o n s h i p were c a l c u l a t e d f r o m I V P T p N A - I V P n o d r u g . IX. P l a s m a Volume M e a s u r e m e n t s A n i m a l s t o be u s e d f o r p l a s m a v o l u m e m e a s u r e m e n t s were p r e p a r e d and a n a e s t h e t i z e d as d e s c r i b e d i n M e t h o d o l o g y S e c . I C . T h e t e c h n i q u e o f " i n d i c a t o r d i l u t i o n " was i m p l e m e n t e d . A c a n n u l a was i n t r o d u c e d i n t o t h e j u g u l a r v e i n t h r o u g h a s m a l l s u p e r f i c i a l i n c i s i o n on t h e r i g h t v e n t r a l c e r v i c a l r e g i o n . R a d i o a c t i v e l y - l a b e l l e d gamma g l o b u l i n ( m e t h y l - ^ C , New E n g l a n d N u c l e a r ) o f 150,000 M.W. was i n j e c t e d as a b o l u s (0.1 y C i t o t a l ) v i a t h e j u g u l a r c a n n u l a and b l o o d s a m p l e s ( 0 . 5 ml) were t a k e n f r o m t h e f e m o r a l a r t e r i a l c a n n u l a a t 2.0, 5.0 and 10.0 min a f t e r t h e i n j e c t i o n . T h e s e were c e n t r i f u g e d a t 2,000 x g f o r 5 min and 0.2 ml o f p l a s m a was w i t h -drawn and m i x e d w i t h 15.0 ml o f B i o f l u o r and c o u n t e d . P l a s m a v o l u m e was c a l c u l a t e d f r o m t h e f o l l o w i n g e q u a t i o n : P l a s m a v o l u m e = Dose g i v e n ( c a l c u l a t e d cpm) C o n c e n t r a t i o n i n P l a s m a (cpm/ml) V a l u e s were n o r m a l i z e d t o t h e body w e i g h t o f t h e a n i m a l s i n q u e s t i o n and e x p r e s s e d as mL/kg. \ - 42 -X. P l a s m a C a t e c h o l a m i n e M e a s u r e m e n t s A. Sample p r e p a r a t i o n . A n i m a l s w e r e p r e p a r e d , and a n a e s t h e t i z e d as d e s c r i b e d i n S e c t i o n I C . A f t e r a l l t h e n e c e s s a r y m e a s u r e m e n t s were t a k e n , 2.0 ml o f b l o o d was w i t h -drawn u n d e r l i g h t e t h e r a n a e s t h e s i a v i a t h e f e m o r a l a r t e r i a l c a n n u l a . The s a m p l e was c e n t r i f u g e d , t h e p l a s m a q u i c k l y f r o z e n and s t o r e d a t - 8 0 ° C , and t h e r e d c e l l s w i t h t h e b u f f y c o a t d i s c a r d e d . When a n a l y s e s were c a r r i e d o u t , 1.0 ml o f p l a s m a and 25 y l o f 100 ng/ml d i h y d r o x y b e n z y l a m i n e (DHBA, i n 0.1 M HCIO^) were c o m b i n e d i n a s c r e w - c a p c o n i c a l g l a s s c e n t r i f u g e t u b e c o n t a i n i n g 100 y l o f 10 EDTA. To each' t u b e , 50 mg o f a c i d - w a s h e d a l u m i n a (AA0, B i o a n a l y t i c a l s y s t e m s ) was a d d e d . T h e n 1.0 ml o f 1.5 M T r i s b u f f e r - (pH 8.7) was a d d e d t o e a c h m i x t u r e and t h e t u b e s were c a p p e d t i g h t l y , v o r t e x e d and a g i t a t e d on a r e c i p r o c a l s h a k e r f o r 5 m i n . A f t e r t h e a l u m i n a was a l l o w e d t o s e t t l e , t h e s u p e r n a t a n t was r e m o v e d by a s p i r a t i o n and t h e a l u m i n a was washed t w i c e w i t h d o u b l e d i s t i l l e d w a t e r . C a t e c h o l s were t h e n d e s o r b e d f r o m t h e a l u m i n a by a d d i n g 200 y l o f 0.1 M H C 1 0 4 t o t h e s a m p l e and v o r t e x i n g t h o r o u g h l y . T h i s m i x t u r e was t r a n s f e r e d t o a c e n t r i f u g a l f i l t e r (MF-1, BAS) and a f t e r c e n t r i f u g i n g f o r 5 min a t 15,000 x g, t h e c l e a r a c i d f i l t r a t e were u s e d i n t h e f o l l o w i n g h i g h p e r f o r m a n c e l i q u i d c h r o m a t o -g r a p h i c p r o c e d u r e ( H P L C ) . B. , H i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y w i t h e l e c t r o c h e m i c a l d e t e c t i o n . T h e HPLC s y s t e m u s e d i n t h e s e p a r a t i o n and d e t e c t i o n o f c a t e c h o l s c o n s i s t e d o f a Beckman model 110A pump, a 50 o r 100 y l f i x e d l o o p i n j e c t o r and a v 4 . 6 mm x 125 mm r e v e r s e p h a s e c o l u m n p a c k e d w i t h ODS H y p e r s i l ( S h a n d o n - 43 -S c i e n t i f i c ) . An i s o c r a t i c s o l v e n t s y s t e m c o n t a i n i n g 0.05 M p o t a s s i u m f o r m a t e (pH a d j u s t e d t o 4.30 w i t h f o r m i c a c i d ) , 200 mg/l -sodium o c t a n e s u l f o n a t e ( A l d r i c h ) , and 60 mg/l EDTA ( F i s h e r ) was u s e d i n t h e s e p a r a t i o n . T h e f l o w r a t e was m a i n t a i n e d a t 1.0 m l / m i n . A m p e r o m e t r i c d e t e c t i o n was e f f e c t e d u s i n g a, c a r b o n p a s t e d e t e c t o r e l e c t r o d e ( T L - 3 , B i o a n a l y t i c a l S y s t e m s I n c . ) p a c k e d w i t h a g r a p h i t e : n u j o l p a s t e ( C P - 0 , B A S ) . The e l e c t r o d e p o t e n t i a l was m a i n t a i n e d a t +0.60 V v_s. a A g / A g C l • r e f e r e n c e e l e c t r o d e (BAS) u s i n g an LC-3 a m p e r o m e t r i c d e t e c t o r ( B A S ) . C h r o m a t o g r a m s were r e c o r d e d on a s t r i p c h a r t r e c o r d e r ( 1 5 2 , 1 5 3 ) . T y p i c a l r e c o v e r y was i n t h e r a n g e o f 7 5 % . V a l u e s r e p o r t e d were i n ng/ml p l a s m a , c a l c u l a t e d f r o m peak h e i g h t r a t i o s u s i n g t h e f o l l o w i n g e q u a t i o n : r 4. 4. • £ r 4. u i ( C a t e c h o l / D H B A ) , r . , C o n c e n t r a t i o n o f C a t e c h o l = v ' s a m p l e x [ c a t e c h o l ] . , , ( C a t e c h o l / D H B A ) s t a n d a r d s t a n d a r d ( T h e HPLC d e t e r m i n a t i o n s o f c a t e c h o l a m i n e s were p e r f o r m e d i n c o l l a b o r a -t i o n w i t h D r . R i c h a r d A. W a l l , D e p a r t m e n t o f P h a r m a c o l o g y , U.B.C.) X I . M u s c l e B a t h S t u d i e s A n i m a l s were p r e p a r e d and a n a e s t h e t i z e d as m e n t i o n e d i n M e t h o d o l o g y S e c t i o n I . A l o n g i t u d i n a l p o r t a l v e i n s t r i p and a h e l i c a l a o r t i c s t r i p ( p r e p a r e d by d i s s e c t i o n , a t an a n g l e o f 15° r e l a t i v e t o t h e c i r c u l a r a x i s o f t h e a r t e r y ) were p r e p a r e d f r o m e a c h r a t . One s t r i p f r o m a c o n t r o l and one c o r r e s p o n d i n g s t r i p f r o m a t r e a t e d r a t were mounted i n random o r d e r t o s t a i n l e s s s t e e l hooks a f f i x e d t o o n e end o f a g l a s s r o d p l a c e d i n t h e same o r g a n b a t h f o r i s o m e t r i c r e c o r d i n g w i t h G r a s s FT-03-C f o r c e - d i s p l a c e m e n t t r a n s d u c e r s a t a p a s s i v e f o r c e o f 5 FnN f o r t h e p o r t a l v e i n s t r i p s and 10 mN f o r t h e a o r t i c s t r i p s . T h e s t r i p s were a l l o w e d t o e q u i l i b r a t e and 37 C f o r a t l e a s t 1 h r i n n o r m a l K r e b ' s s o l u t i o n . T h e s o l u t i o n was c o n t i n u o u s l y b u b b l e d w i t h a g a s m i x t u r e o f 5 % CC^ and 9 5 % C^. A f t e r e q u i l i b r a t i n g t h e s t r i p s , c u m u l a t i v e d o s e s o f n o r a d r e n a l i n e (NA) were added t o t h e b a t h s -9 -4 t o p r o v i d e f i n a l d r u g c o n c e n t r a t i o n s f r o m 10 t o 10 M. E a c h d o s e o f NA was a l l o w e d t o r e m a i n i n t h e b a t h f o r 3-6 min i n t h e c a s e o f t h e a o r t i c s t r i p s , and f o r 2 min i n t h e c a s e o f t h e p o r t a l v e i n s s t r i p s t o o b t a i n a s t e a d y s t a t e r e s p o n s e b e f o r e t h e a d d i t i o n o f t h e n e x t d o s e o f d r u g . A f t e r o b t a i n i n g ^ a c u m u l a t i v e d o s e - r e s p o n s e c u r v e t o NA, t h e C a + + c o n c e n t r a t i o n o f t h e b a t h f l u i d was a l t e r e d . T h e s t r i p s were i n c u b a t e d f o r 30 min i n t h e a l t e r e d Ca s o l u t i o n b e f o r e o b t a i n i n g a n o t h e r d o s e - r e s p o n s e c u r v e t o NA. T h e s e q u e n c e o f t h e Ca c o n c e n t r a t i o n s s t u d i e d i n m i l l i m o l e s o f Ca was: 2.5, 0.2 and 0.4. A t t h e end o f t h e e x p e r i m e n t , t h e l e n g t h s o f t h e s t r i p s were m e a s u r e d by a v e r n i e r c a l i p e r ( r e s o l u t i o n : * 0.05 mm). T h e s t r i p s were t h e n r e m o v e d by c u t t i n g away t h e t i e d e n d s , b l o t t e d and d r i e d o v e r n i g h t a t 9 0 ° C . The d r y w e i g h t o f t h e s t r i p s w ere o b t a i n e d t h e f o l l o w i n g d a y . C o n t r a c t i l e f o r c e o f , t h e m u s c l e was r e p o r t e d as s t r e s s ( f o r c e / c r o s s - s e c t i o n a l a r e a , p mN/mm ) . T h e a p p a r e n t c r o s s - s e c t i o n a l a r e a was e s t i m a t e d f r o m t h e d r y w e i g h t and t h e m e a s u r e d l e n g t h o f t h e s t r i p ( d r y w e i g h t / l e n g t h ) . X X I . S t a t i s t i c a l A n a l y s i s . V a l u e s g i v e n a r e mean ± S.D. u n l e s s o t h e r w i s e s t a t e d . S t u d e n t ' s t - t e s t was e m p l o y e d i n t h e a n a l y s i s o f two s a m p l e means, w i t h t h e l e v e l o f s i g n i f i -c a n c e c h o s e n as 9 5 % (P < 0 . 0 5 ) . F o r v a l u e s where n o r m a l d i s t r i b u t i o n c a n n o t be a ssumed, t h e , W i l c o x o n t w o - s a m p l e r a n k t e s t ( M a n n - W h i t n e y U t e s t ) was u s e d w i t h t h e same l e v e l o f s i g n i f i c a n c e b e i n g c h o s e n . RESULTS I . G e n e t i c Model o f H y p e r t e n s i o n A. T i m e c o u r s e o f h y p e r t e n s i o n d e v e l o p m e n t . A n i m a l s were o b t a i n e d f r o m t h e b r e e d i n g f a r m a t e i g h t weeks o f age" and w ere w e i g h t m a t c h e d . W i s t a r r a t s ( a n a d d i t i o n a l c o n t r o l g r o u p ) o f t h e same age t e n d e d t o be h e a v i e r s o t h a t w e i g h t - m a t c h i n g was n o t p o s s i b l e . A t 9 weeks o f a g e , SHRs a l r e a d y showed a s l i g h t l y e l e v a t e d s y s t o l i c b l o o d p r e s -s u r e c o m p a r e d t o t h e WKYs and Wi s t a r s , and t h e d i f f e r e n c e s were f u r t h e r m a g n i f i e d w i t h t i m e . A t 14 weeks, t h e mean b l o o d p r e s s u r e o f t h e SHRs was f o u n d t o be e l e v a t e d by a b o u t 3 0 % c o m p a r e d t o t h e c o n t r o l s (WKY and W i s t a r ) and t h a t t h e i r body w e i g h t s were s i g n i f i c a n t l y l o w e r t h a n t h e a n i m a l s f r o m t h e two c o n t r o l g r o u p s . T h e mean h e a r t r a t e s o f t h e SHRs a l s o were s i g n i f i -c a n t l y h i g h e r t h a n t h e o t h e r g r o u p s s t u d i e d ( S e e T a b l e 2 ) . B. E r y t h r o c y t e membrane enzyme a s s a y s . A c t i v i t i e s o f s e v e r a l enzymes were s t u d i e d and c h a r a c t e r i z e d i n t h e e r y t h r o c y t e g h o s t s p r e p a r e d f r o m t h e v a r i o u s g r o u p s o f r a t s . A s i g n i f i c a n t i n c r e a s e i n t h e C a + + / M g + + A T P a s e a c t i v i t i e s was f o u n d i n t h e membranes f r o m SHRs, and t h i s i n c r e a s e was p r e s e n t i n a n i m a l s f r o m b o t h t h e 9- and 14-week age g r o u p s ( s e e T a b l e 3 ) . I n t e r e s t i n g l y e n o u g h , t h e b a s a l ( M g + + d e p e n d e n t ) A T P a s e a c t i v i t y was a l s o f o u n d t o be s l i g h t l y e l e v a t e d i n t h e SHRs f r o m b o t h age g r o u p s . T h e c a l c i u m c o n c e n t r a t i o n d e p e n d e n c e c u r v e s f o r ++ ++ t h e C a /Mg A T P a s e seemed t o i n d i c a t e t h a t i n a d d i t i o n t o t h e d i f f e r -++ ++ e n c e i n s p e c i f i c a c t i v i t y , t h e SHR Ca /Mg A T P a s e may a l s o h a v e TABLE 2. B a s e y l i n e m e a s u r e m e n t s ! o f d i f f e r e n t g r o u p s o f a n i m a l s 9 weeks 14 weeks M e a s u r e m e n t s WKY (n= =12) SHR (n= =12) W i s t a r (n= =6) WKY (n=24) ' SHR (n=24) W i s t a r (n=6) Body w e i g h t ( g ) 210 ± 7 206 ± 8 221 ± 6b 295 ± 16 280 ± 14b 312 ± 14 S y s t o l i c p r e s s u r e (mmHg) 126 ± 6 140 ± gb 128 ± 8 129 ± 10 168 ± l i b 125 ± 10 D i a s t o l i c p r e s s u r e (mmHg) 96 ± 5 i 102 ± 6 95 ± 8 98 ± 12 115 ± 10b - 96 ± 8 Mean p r e s s u r e (mmHg) 110 ± 6 121 ± 9 112 ± 8 114 ± 12 142 ± 12b 116 ± 10 H e a r t r a t e ( b p m ) £ 356 ± 18 382 ± 25^. 342 ± 15 362 ± 22 414 ± 24b 355 ± 15 — V a l u e s a r e mean ± S.D. — S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (WKY) v a l u e s , (p < 0 . 0 5 ) . — bpm = b e a t s p e r m i n u t e TABLE 3. C a + + / M g + + A T P a s e a c t i v i t i e s i n t h e e r y t h r o c y t e g h o s t s f r o m t h r e e g r o u p s o f r a t s . 9 weeks 14 weeks A c t i v i t y 9 . WKY (n=12) SHR (n=12) W i s t a r (n=6) WKY (n=24) SHR (n=24) W i s t a r (n=6) B a s a l 1.01 ± 0.12 1.14 ± 0 . 1 1 £ 0.97 ± 0.10 1.10 ± 0.14 1.26 ± 0 . 1 8 £ 1.02 ± 0.11 H i g h A f f i n i t y ^ 1.12 ± 0.28 1.48 ± 0 . 4 0 £ 1.08 ± 0.19 1.14 ± 0.32 1.59 ± 0 . 3 8 £ 1.10 ± 0.28 Low A f f i n i t y 1.97 ± 0.33 2.42 ± 0 . 4 2 £ 1.90 ± 0.34 2.02 ± 0.48 2.51 ± 0 . 4 2 £ 1.92 ± 0.40 - Enzyme a c t i v i t y i s r e p o r t e d asymp/T.Pi/mg p r o t e i n - hr -"*", v a l u e s a r e mean ± S.D. - V a l u e s g i v e n a r e s p e c i f i c a c t i v i t i e s w i t h b a s a l v a l u e s ( C a + + i n d e p e n d e n t ) s u b t r a c t e d . - S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (WKY) v a l u e s , . P < 0.05 - 48 -i n c r e a s e d a f f i n i t y f o r Ca when c o m p a r e d w i t h t h e enzyme o f t h e c o n t r o l a n i m a l s as e v i d e n t f r o m t h e s h i f t o f t h e C a + + d e p e n d e n c e c u r v e t o l o w e r C a + + c o n c e n t r a t i o n s ( s e e F i g . 3 ) . A r r h e n i u s p l o t s f o r t h e C a + + / M g + + A T P a s e f r o m SHRs and WKYs we r e c o n s t r u c t e d and a t y p i c a l s e t o f c u r v e s i s shown i n F i g . 4. A c t i v a t i o n e n e r g i e s were c a l c u l a t e d f r o m t h e s l o p e o f t h e c u r v e s and i t was f o u n d t h a t t h e h i g h a f f i n i t y enzymes f r o m t h e SHRs have s m a l l b u t s i g n f i c a n t l y e l e v a t e d a c t i v a t i o n e n e r g y v a l u e s when c o m p a r e d t o t h e WKYs' ( s e e T a b l e 4 ) , w h i l e t h e low a f f i n i t y enzyme showed a s i m i l a r t r e n d , t h e d i f f e r e n c e s were n o t s t a t i s -t i c a l l y s i g n i f i c a n t . T h i s i s g e n e r a l l y t a k e n as an i n d i c a t i o n o f some s u b t l e c h a n g e s i n membrane p h o s p h o l i p i d e n v i r o n m e n t s u r r o u n d i n g t h e enzymes i n q u e s t i o n . O t h e r membrane enzyme a c i t i v i t e s were a l s o s t u d i e d . No d i f f e r e n c e s i n t h e N a + / K + A T P a s e a c t i v i t i e s were n o t e d o t h e r t h a n a s l i g h t l y e l e v a t e d b a s a l a c t i v i t y i n SHR membranes when c o m p a r e d t o c o n t r o l s ' ( s e e T a b l e 5 ) . D i f f e r e n c e s i n some o f t h e enzyme a c t i v i t i e s ~ w e r e o b s e r v e d b e t w e e n WKY and W i s t a r r a t s . T h e s e c a n be v i e w e d as an i n d i c a t i o n t h a t WKY and W i s t a r s a r e g e n e t i c a l l y q u i t e d i f f e r e n t . Membrane a c e t y l c h o l i n e s t e r a s e a c t i v i t i e s i n e r y t h r o c y t e s f r o m t h e d i f f e r e n t SHRs were s i m i l a r . T h i s s u g g e s t s t h a t t h e a l t e r a t i o n s s e e n i n C a + + / M g + + A T P a s e a c t i v i t y w e r e n o t t h e r e s u l t o f some g e n e r a l i z e d membrane p r o t e i n (enzyme) a l t e r a t i o n as s e e n i n c e r t a i n d i s e a s e s t a t e s s u c h as m u s c u l a r d y s t r o p h y where most o f t h e membrane enzymes were a f f e c t e d ( 1 4 8 ) . T h e n e x t s t e p was t o i n v e s t i g a t e t h e p o s s i b l e m o l e c u l a r b a s i s f o r t h e s p e c i f i c a l t e r a t i o n i n t h e C a + + / M g + + A T P a s e a c t i v i t i e s . One p o s s i b i l i t y t o be c o n s i d e r e d was a g e n e t i c d e f e c t i n t h e SHRs t h a t s p e c i f i c a l l y c o d e d f o r an a l t e r a t i o n i n t h e membrane enzyme i n q u e s t i o n . V - 49 -i F i g u r e 3. C a l c i u m d e p e n d e n c e c u r v e s o f t h e e r y t h r o c y t e membrane Ca /Mg'' A T P a s e f r o m WKY and SHR . E a c h p o i n t r e p r e s e n t s t h e mean s p e c i f i c a c t i v i t y o f enzymes f r o m 8 a n i m a l s , and t h e b a r s d e n o t e one s t a n d a r d d e v i a t i o n f r o m t h e mean. E x c e p t f o r t h e p o i n t s a t 1 0 ~ 7 M C a + + , a l l SHR v a l u e s a r e s i g n i f i c a n t l y h i g h e r t h a n WKY v a l u e s (P < 0 V 0 5 ) . - 50 -;F igure 4. A r rhen ius p l o t s of the Ca /Mg ATPases i n a t y p i c a l e x p e r i -ment. The l i n e s are generated by " l e a s t - s qua re r e g r e s s i o n " and the SHR enzymes c o n s i s t e n t l y have a steeper s lope than the WKY enzymes. TABLE 4. A c t i v a t i o n e n e r g i e s f o r C a + + / M g + + A T P a s e s ( K c a l / m o l e ) Mean * S.D. (n = 6) WKY SHR H i g h a f f i n i t y Low a f f i n i t y 11.2 ± 2.4 11.4 ± 3.7 13.8 ± 2 . 6 1 12.8 ± 3.7 1 S t u d e n t s ' t - t e s t , p < 0.05 +. + -TABLE 5. Na /K A T P a s e a c t i v i t i e s i n e r y t h r o c y t e g h o s t s f r o m t h r e e g r o u p s o f r a t s . 9 weeks 14 weeks A c t i v i t y , WKY (n=12) SHR (n=12) W i s t a r (n=6) WKY (n=24) SHR (n=24) W i s t a r (n=6) B a s a l S t i m u l atedJ2-1.12 ± 0.06 1.18 ± 0.05& 1.04 ± 0.03a 1.17 ± 0.07 1.27 ± 0.093- 1.10 ± 0.042-0.79 ± 0.08 0.76 ± 0.06 0.84 ± 0.05 0.84 ± 0.07 0.88 ± 0.06 0.92 ± 0 . 0 6 1 1 S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (WKY) v a l u e s , P < 0.05 — V a l u e s g i v e n were s p e c i f i c a c t i v i t i e s w i t h b a s a l a c t i v i t y s u b t r a c t e d , u n i t s a r e i n , i p n i o i e s P i mg p r o t e i n - h r - 52 -S u c h a s p e c i f i c g e n e t i c a b e r r a t i o n was t h o u g h t t o be u n l i k e l y s i n c e h y p e r -t e n s i o n i s g e n e r a l l y r e g a r d e d as a m u l t i f a c t o r i a l p o l y g e n i c i n h e r i t e d a b n o r -m a l i t y ( 1 5 6 , 1 5 7 ) . We t h e r e f o r e c o n s i d e r e d t h e p o s s i b i l i t y t h a t c a l c i u m i o n was h a n d l e d a b n o r m a l l y i n t h e e r y t h r o c y t e s o f t h e SHRs, and t h e r e i s ample d o c u m e n t a t i o n i n t h e l i t e r a t u r e f o r a b n o r m a l i t i e s o f i o n p e r m e a b i l i t i e s i n h y p e r t e n s i o n ( s e e I n t r o d u c t i o n S e c t i o n V I I ) . I n o r d e r t o i n v e s t i g a t e t h i s f u r . t h e r , o u r f i r s t s t e p was t o s t u d y t h e p a s s i v e c a l c i u m f l u x e s a c r o s s t h e e r y t h r o c y t e membrane. i C. C a l c i u m i n f l u x m e a s u r e m e n t s . I n i t i a l l y , a t t e m p t s were made t o l o a d i n t a c t r e d c e l l s i n t h e c o l d ( 4 ° C ) w i t h r a d i o a c t i v e c a l c i u m ( ^ C a ) and q u a n t i t a t e t h e r a t e o f p a s s i v e i n f l u x as t h e amount o f r a d i o a c t i v i t y a c c u m u l a t e d i n s i d e t h e r e d c e l l s w i t h t i m e . T h e r e d c e l l membrane, h o w e v e r , i s h i g h l y i m p e r m e a b l e t o C a + + and o u r c o u n t s i n s i d e t h e c e l l s were q u i t e low and t h e r e f o r e c o u n t i n g e r r o r was h i g h . I n o r d e r t o i m p r o v e t h e r e l i a b i l i t y o f c o u n t i n g ( i . e . t o i n c r e a s e t h e c o u n t s i n s i d e t h e c e l l s ) , L a n g e r ' s method o f p r e d i g e s t i n g t h e c e l l s w i t h n e u r a m i n i d a s e (154), was u s e d . ( L a n g e r ' s g r o u p p r o p o s e d t h a t s i a l i c a c i d r e s i d u e s on t h e c e l l s u r f a c e o f . c u l t u r e d h e a r t c e l l s a t t r a c t d i v a l e n t c a t i o n s , f o r m i n g an e l e c t r o p o s i t i v e s h e l l and s l o w i n g down f u r t h e r c a t i o n i n f l u x . ) F o l l o w i n g n e u r a m i n i d a s e p r e t r e a t m e n t , t h e e r y t h r o c y t e s were t a k i n g up t w i c e t h e amount o f r a d i o a c t i v e c a l c i u m as b e f o r e b u t we f o u n d t h a t t h e 45 t i m e c o u r s e o f Ca i n f l u x was u n c h a n g e d i n c e l l s f r o m b o t h g r o u p s ( s e e F i g u r e 5 ) . I n p r e s e n c e and a b s e n c e o f n e u r a m i n i d a s e , t h e r e was an i n i t i a l r a p i d i n f l u x p h a s e w h i c h was f o l l o w e d by a p r o l o n g e d s l o w u p t a k e p h a s e where 45 t h e c e l l s were s t i l l t a k i n g up Ca e v e n a f t e r 35 h o u r s * The l o a d i n g t i m e was a r b i t r a r i l y s e t a t 20 h o u r s b e c a u s e t h e c o u n t s were t o o low t o be r e l i a -b l e when s h o r t e r l o a d i n g t i m e s were u s e d . T h e r e d c e l l s f r o m t h e SHRs - 53 -7 r o o o s a. o 5 H 4 h 3 I-0 « -..• pretreated untreated 10 1 2 14 16 18 20 22 24 T I M E ( h r s ) 45 ++ F i g u r e 5. Ti m e c o u r s e o f Ca u p t a k e i n t o e r y t h r o c y t e s f r o m W i s t a r r a t s . T h e r e i s an i n i t i a l r a p i d v u p t a k e p h a s e l a s t i n g up t o 10 h r s and t h e n a s l o w u p t a k e p h a s e t h a t p e r s i s t s b e y o n d 72 h o u r s . N e u r a m i n i d a s e p r e t r e a t -ment d o e s n o t a f f e c t t h e t i m e c o u r s e o f t h e u p t a k e b u t d o e s i n c r e a s e t h e a b s o l u t e amount o f c a l c i u m i n f l u x t h r o u g h o u t t h e two p h a s e s o f u p t a k e . - 54 -showed s i g n i f i c a n t l y h i g h e r p a s s i v e c a l c i u m i n f l u x t h a n t h o s e o f t h e WKYs', and n e u r a m i n i d a s e t r e a t m e n t seemed t o f u r t h e r e n h a n c e t h e d i f f e r e n c e ( s e e T a b l e 6 ) . S i n c e t h e f u n c t i o n a l a b n o r m a l i t i e s i n SHR membranes were d e m o n s t r a t e d b y i n c r e a s e d C a + + / M g + + A T P a s e a c t i v i t i e s and i n c r e a s e d C a + + p e r m e a b i l i t y , t h e n e x t s t e p was t o s t u d y t h e m e c h a n i s m r e s p o n s i b l e f o r t h e s e i n c r e a s e s . O s m o t i c f r a g i l i t y m e a s u r e m e n t s were u n d e r t a k e n t o e x p l o r e t h e p o s s i b i l i t y t h a t t h e o b s e r v e d p e r m e a b i l i t y i n c r e a s e m i g h t be t h e r e s u l t o f some g e n e r a l -i z e d s t r u c t u r a l a l t e r a t i o n o f t h e r e d c e l l membrane o f t h e SHR. D. O s m o t i c f r a g i l i t y m e a s u r e m e n t s . The a b i l i t y o f i n t a c t ' r e d c e l l s t o w i t h s t a n d h y p o t o n i c c o n d i t i o n s was s t u d i e d b y s u b j e c t i n g t h e c e l l s t o s o l u t i o n s o f d e c r e a s i n g i o n i c s t r e n g t h ( 0 . 0 6 5 , 0.060, 0.055 and 0.045 M N a C l ) and q u a n t i f y i n g t h e e x t e n t o f h a e m o l -y s i s b y m e a s u r i n g t h e c o n c e n t r a t i o n o f h a e m o g l o b i n r e l e a s e d i n t o t h e medium. We f o u n d t h e r e d c e l l s f r o m t h e SHRs t o be s i g n i f i c a n t l y more r e s i s t a n t t o h y p o t o n i c l y s i s a t a l l N a C l c o n c e n t r a t i o n s s t u d i e d ( s e e F i g u r e 6 ) . T h i s o b s e r v a t i o n s u g g e s t e d t h e p o s s i b i l i t y o f a l t e r e d membrane s t r u c t u r a l c h a r a c -t e r i s t i c s , t h e r e f o r e e r y t h r o c y t e membrane c h e m i c a l c h a r a c t e r i s t i c s w e r e n e x t e x a m i n e d . E. E r y t h r o c y t e membrane c o m p o s i t i o n a l a n a l y s e s . P l a s m a and e r y t h r o c y t e membranes were s u b j e c t e d t o d e t a i l e d c o m p o s i t i o n a l a n a l y s e s as m e n t i o n e d i n M e t h o d o l o g y S e c t i o n V. Membrane c h o l e s t e r o l c o n t e n t was f o u n d t o be s i g n i f i c a n t l y e l e v a t e d i n SHR membranes when c o m p a r e d t o WKYs 1 ( T a b l e 7 ) . I n t e r e s t i n g l y , p l a s m a c h o l e s t e r o l c o n t e n t was s i g n i f i c a n t l y l o w e r i n t h e SHRs. T o t a l p h o s p h o l i p i d c o n t e n t was f o u n d t o be t h e same i n b o t h - 55 -TABLE 6. P a s s i v e C a l c i u m I n f l u x i n t o e r y t h r o c y t e s f r o m SHRs and WKYs No P r e t r e a t m e n t N e u r a m i n i d a s e p r e t r e a t m e n t mean * S.D. (n = 8) mean * S.D. ( n = 8 ) WKY 0.40 ± 0.05 1.02 ± 0.09 SHR 0.58 ± 0 . 0 6 - 1.46 ± 0 . 1 2 -JL U n i t s a r e i n n m o l e s - h r ~ l 1~1 b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (WKY) v a l u e s (p < 0.01, S t u d e n t ' s t - t e s t ) - 56 -Hemolysis % 1 0 0 - i 90 H 80H 70 H 6 0 H 5 0 H 4 0 H 3 0 H 2 0 H 1 0 H OSMOTIC FRAGILITY ( n = 6 WKY SHR 0 0 4 5 0 0 5 5 0 0 6 0 0 0 6 5 M Na CI Figure 6. Osmotic f r a g i l i t y of erythrocytes from WKY and SHR .. The amount of haemolysis is expressed as percent of a reference value obtained by incubating the red c e l l s in d i s t i l l e d water. Values show are mean of s ix pa irs of animals with bars i nd i ca t ing one standard dev i a t i on . A l l SHR values are s i g n i f i c a n t l y lower than WKY values (P < 0.05). / - 57 -TABLE 7. WKY and SHR^. e r y t h r o c y t e membrane c o m p o s i t i o n a l a n a l y s e s Mean ± S.D. WKY (n = 24) SHR (n = 24) Membrane (ym./mg)- 0.52 ± 0.02 0.61 ± 0 . 0 4 ^ 1. C h o l e s t e r o l P l a s m a (ym/ml ) 4 57.9 ± 5.0 . 48.5 ± 7.6 -Membrane (yO/mg) 23.2 ± 1 . 4 22.8 ± 1.2 / 2. T o t a l P h o s p h o l i p i d s P l a s m a ( y g / m l ) 9.6 ± 1.6 8.4 ± 1.8 3. P - e t h a n o l a m i n e 28.0 ± 2.6 25.5 ± 2.0 4. P - s e r i n e p e r c e n t o f 11.7 ± 2.0 10.0 ± 1.9 t o t a l p h o s p h o l i p i d s * 5. P - c h o l i n e 6. S p h i n g o m y e l i n 42.6 ± 2.7 46.8 ± 1.5^ 14.0 ± 1.8 16.0 ± 1.2-- S t u d e n t ' s t - t e s t , p < 0.05 - A n i m a l s a r e 14 week o l d , age matched, c ym = y m o l e s - 58 -g r o u p s o f a n i m a l s and t h i s i s t r u e b o t h f o r t h e e r y t h r o c y t e membranes, and f o r t h e p l a s m a f r o m t h e ' a n i m a l s . P h o s p h o l i p i d p r o f i l e s were o b t a i n e d and t h e r e seemed t o be a s m a l l b u t s i g n i f i c a n t i n c r e a s e s i n t h e p e r c e n t a g e o f p h o s p h a t i d y l c h o l i n e and s p h i n g o m y e l i n i n t h e membranes f r o m SHRs, w i t h c o r r e s p o n d i n g d e c r e a s e s ( t h o u g h s t a t i s t i c a l l y n o t s i g n f i c a n t ) i n p h o s p h a t i d y l e t h a n o l a m i n e and p h o s p h a t i d y l s e r i n e . T h i s s h i f t o f p h o s p h o l i p i d c o m p o s i -t i o n , t o g e t h e r w i t h t h e a l t e r e d c h o l e s t e r o l c o n t e n t , p r o v i d e d good e v i d e n c e f o r s t r u c t u r a l a l t e r a t i o n s i n SHR e r y t h r o c y t e membranes. I I . E x p e r i m e n t a l M odel o f H y p e r t e n s i o n ( D O C A / s a l t H y p e r t e n s i o n ) A. T i m e c o u r s e o f h y p e r t e n s i o n d e v e l o p m e n t . A n i m a l s s u b j e c t e d t o u n i l a t e r a l n e p h r e c t o m y t e n d e d t o g a i n w e i g h t a t a s l o w e r r a t e t h a n i n t a c t c o n t r o l s . T h e b l o o d p r e s s u r e , h o w e v e r , was n o t a f f e c t e d b y t h e s u r g i c a l m a n o e u v r e , and p r e - t r e a t m e n t ( D O C A / s a l t ) s y s t o l i c b l o o d p r e s s u r e s were 118 ± 4 and 117 ± 5 mm Hg f o r n e p h r e c t o m i z e d and i n t a c t r a t s (C) r e s p e c t i v e l y . The n e p h r e c t o m i z e d a n i m a l s were d i v i d e d i n t o c o n t r o l (NC) and t r e a t m e n t (DOCA) g r o u p s . A l t h o u g h .the b l o o d p r e s s u r e s r e m a i n e d e s s e n t i a l l y s i m i l a r d u r i n g t h e f i r s t 2 weeks o f DOCA t r e a t m e n t , t h e DOCA g r o u p showed s i g n i f i c a n t l y e l e v a t e d p r e s s u r e s b y t h e t h i r d week and t h i s t r e n d was f u r t h e r m a g n i f i e d by t h e f o u r t h week ( s e e T a b l e 8 ) . No d i f f e r e n -c e s i n t h e b l o o d p r e s s u r e v a l u e s were o b s e r v e d b e t w e e n t h e i n t a c t W i s t a r s (C) and t h e n e p h r e c t o m i z e d c o n t r o l s ( N C ) . In o r d e r t o f i n d o u t w h e t h e r t h e a b n o r m a l i t i e s s e e n i n SHR e r y t h r o c y t e s a r e i n d u c e d b y t h e h i g h b l o o d p r e s -s u r e , t h e e r y t h r o c y t e s f r o m DOCA and NC r a t s were s u b j e c t e d t o s i m i l a r f u n c t i o n a l and s t r u c t u r a l a n a l y s e s as t h o s e o u t l i n e d f o r SHR. - 59 -TABLE 8. B l o o d p r e s s u r e s and A T P a s e a c t i v i t i e s o f NC and DOCA a n i m a l s a t Week 4 N e p h r e c t o m i z e d C o n t r o l - DOCA-. ( n = 8) ( n = 8 ) Mean a r t e r i a l B.P. (mm Hg) , 112 ± 5 141 ± 6-1.14 ± 0.09 2.12 ± 0.14 N a + / K + A T P a s e - ( s t i m u l a t e d ) 0.84 ± 0.07 0.9.3 ± 0.07 - V a l u e s g i v e n a r e mean ± S.D. - E n z y m e a c t i v i t i e s a r e e x p r e s s e d as s p e c i f i c a c t i v i t i e s . V a l u e s a r e n e t s t i m u l a t e d a c t i v i t i e s w i t h b a s a l a c t i v i t i e s s u b t r a c t e d . - S i g n i f i c a n t l y d i f f e r e n t t h a n c o n t r o l v a l u e (P < 0 . 0 5 ) . C a + + / M g + + A T P a s e s ^ H i g h A f f . 1.18 ± 0.22 Low A f f . 2.09 ± 0.31 - 60 -B. E r y t h r o c y t e membrane enzyme a s s a y s . E r y t h r o c y t e membranes were p r e p a r e d and t h e enzymes were a s s a y e d as d e s c r i b e d i n t h e S H R / W K Y , e x p e r i m e n t s . T h i s t i m e , h o w e v e r , no d i f f e r e n c e s i n t h e C a + + / M g + + A T P a s e s i n t h e membranes f r o m t h e DOCA and t h e NC f a n i m a l s were d e t e c t e d ( s e e T a b l e 8 ) . T h e N a + / K + A T P a s e a c t i v i t i e s were a l s o e x a m i n e d and a g a i n no s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d . A t r e n d t o w a r d s an e l e v a t i o n i n enzyme a c t i v i t i e s i n t h e membranes f r o m DOCA a n i m a l s was n o t e d when compa r e d t o NC v a l u e s ( 0.05 < P < 0 . 1 ) . T h e s e p o i n t s w i l l be d e a l t w i t h i n t h e D i s c u s s i o n s e c t i o n . Membrane a c e t y l c h o l i n e s t e r a s e a c t i v i -t i e s w ere s i m i l a r i n b o t h g r o u p s o f a n i m a l s . C. C a l c i u m i n f l u x m e a s u r e m e n t s . T h e t i m e c o u r s e o f C a + + u p t a k e i n t h e DOCA e r y t h r o c y t e s was s i m i l a r t o t h a t o f t h e SHRs and WKY. I t was e s s e n t i a l l y a b i p h a s i c u p t a k e w i t h an i n i t i a l r a p i d u p t a k e l a s t i n g s e v e r a l h o u r s and a p r o l o n g e d s l o w i n f l u x c o m p o n e n t . T h e e r y t h r o c y t e s f r o m t h e DOCA g r o u p d i d n o t show any d i f f e r e n c e s i n t h e i r C a + + u p t a k e when compa r e d t o t h e c o n t r o l (NC) e r y t h r o c y t e s e i t h e r w i t h o r w i t h o u t n e u r a m i n i d a s e p r e - t r e a t m e n t ( s e e T a b l e 9 ) . T h e a b s o l u t e v a l u e s o f c a l c i u m u p t a k e r a t e s were c o m p a r a b l e t o . t h e v a l u e s o f t h e WKYs ( T a b l e 6) and l o w e r t h a n t h o s e o b s e r v e d e a r l i e r i n t h e SHRs ( s e e R e s u l t s S e c t i o n I C ) . D. O s m o t i c f r a g i l i t y m e a s u r e m e n t s . I n t a c t e r y t h r o c y t e s f r o m t h e DOCA h y p e r t e n s i v e a n i m a l s h a v e v e r y s i m i l a r o s m o t i c f r a g i l i t y p r o f i l e when c o m p a r e d t o t h e i r c o n t r o l s ( N C ) . I f a n y t h i n g , t h e h y p e r t e n s i v e a n i m a l s a c t u a l l y showed a m a r g i n a l l y g r e a t e r f r a g i l i t y t h a n t h e i r n o r m o t e n s i v e c o n t r o l s ( s e e F i g . 7 ) , w h i c h was c o n t r a r y t o what was - 61 '-TABLE 9. P a s s i v e c a l c i u m i n f l u x i n t o e r y t h r o c y t e s f r o m DOCA and NC a n i m a l s No p r e t r e a t m e n t N e u r a m i n i d a s e t r e a t e d , mean^ * S.E. (n=4) mean- * S.E. (n=4) C o n t r o l (NC) 0.43 ± 0.04 1.12 ± 0.13 DOCA 0.45 ± 0.04 1.09 ± 0.09 U n i t s a r e i n n m o l e s - h r * - 1 * - 6 2 -O S M O T I C FRAG IL ITY ( n = 6 ) H e m o l y s i s 0.045 0.055 0.060 0.065 M N a C l Figure 7. Osmotic f r a g i l i t y of erythrocyte from NC and DOCA .. The amount of haemolysis i s expressed as percent of a reference value obtained by incubating the red c e l l s -in d i s t i l l e d water. Values shown are mean of s ix pair of animals with bars ind icat ing one standard deviat ion. Values at 0.055, 0.060, and 0.065 M NaCl are s i g n i f i c an t l y d i f fe rent between the two ( groups of animals (P < 0.05).-- 63 -f o u n d i n t h e SHR/WKY e x p e r i m e n t s ( s e e R e s u l t s S e c t i o n I D ) . T h e r e i s no i n d i c a t i o n so f a r o f any f u n c t i o n a l a l t e r a t i o n s i n t h e e r y t h r o c y t e membranes f r o m t h e s e DOCA a n i m a l s . I n o r d e r t o f i n d o u t i f s t r u c t u r a l a l t e r a t i o n s ' a r e p r e s e n t i n DOCA membranes, c o m p o s i t i o n a l a n a l y s e s were a l s o p e r f o r m e d . E . E r y t h r o c y t e membrane c o m p o s i t i o n a l a n a l y s e s . P l a s m a s a m p l e s and e r y t h r o c y t e membranes f r o m DOCA h y p e r t e n s i v e a n d n e p h r e c t o m i z e d c o n t r o l a n i m a l s were s u b j e c t e d t o t h e same c h e m i c a l a n a l y s e s as f o r t h e SHRs and WKYs. M a r g i n a l l y e l e v a t e d p l a s m a c h o l e s t e r o l l e v e l s were o b s e r v e d as w e l l . a s a s m a l l d e c r e a s e i n t h e p e r c e n t a g e o f p h o s p h a t i d y l e t h a n o l a m i n e c o n t e n t i n t h e membranes f r o m t h e DOCA a n i m a l s ( s e e T a b l e 1 0 ) . A l l o t h e r a n a l y s e s i n d i c a t e d t h a t t h e r e a r e no g r o s s d i f f e r e n c e s o b s e r v a b l e b e t w e e n t h e two g r o u p s o f a n i m a l s . T h i s i s i n good a g r e e m e n t w i t h t h e o t h e r r e s u l t s f r o m s t u d i e s on t h e membranes t h a t t h e r e i s no m a j o r a l t e r a t i o n d e t e c t a b l e i n t h e e r y t h r o c y t e membrane o f DOCA a n i m a l s . I I I . P h a r m a c o l o g i c a l Model o f A l c o h o l i s m A. E t h a n o l c o n s u m p t i o n and f o o d i n t a k e . The a l c o h o l - t r e a t e d r a t s consumed on t h e a v e r a g e 0.8 ± 0.3 g o f e t h a n o l d a i l y d u r i n g t h e f i r s t week and i n c r e a s e d t h e i r i n t a k e t o 5.0 ± 1.2 g d a i l y by t h e end o f t h e 12-week e x p e r i m e n t a l p e r i o d . T h e s e a n i m a l s consumed 22 ± 5 g o f R a t Chow d a i l y b e f o r e t r e a t m e n t , b u t r e d u c e d t h e i r i n t a k e t o 15 ± 4 g p e r d a y by t h e end o f Week 12. The c o n t r o l g r o u p , on t h e o t h e r h a n d , i n c r e a s e d t h e i r f o o d c o n s u m p t i o n f r o m an a v e r a g e o f 21 ± 5 g a t Week 1 t o 30 ± 9 g p e r d a y p e r a n i m a l a t Week 12. P l a s m a e t h a n o l l e v e l s i n t h e t r e a t e d - 64 -TABLE 10. Membrane c o m p o s i t i o n a l a n a l y s e s on e r y t h r o c y t e s f r o m DOCA and NC a n i m a l s . MEAN ± S.D. NC (n = 6) DOCA (n = 6) Membrane (ytn/mg)- 0.39 ± 0.04 ' 0.41 ± 0.05 1. C h o l e s t e r o l 2. T o t a l P h o s p h o l i p i d s 3. P - e t h a n o l a m i n e 4. P - s e r i n e 5. P - c h o l i n e 6. S p h i n g o m y e l i n P l a s m a (p.m/ml)-Membrane (y.g/mg) P l a s m a (y;g/ml) p e r c e n t o f t o t a l p h o s p h o l i p i d s 37.4 ± 4.5 8.21 ± 0.60 26.2 ± 2.2 11.2 ± 2.4 44.4 ± 3.0 14.5 ± 1.9 42.6 ± 4 . 3 -19.0 i 1.8 19.9 ± 2.0 7.98 ± 1.7 23.5 ± 1 . 8 ^ 12.6 ± 2.1 44.2 ± 2.3 15.6 ± 2.2 - p < 0.05, S t u d e n t ' s t - t e s t — ym = y m o l e s - 65 -a n i m a l s a v e r a g e d 8.7 ± 2.2 mg/dl a t week 1, 86.0 ± 10.0 mg/dl a t week 4, and 29.6 ± 5.0 mg/dl a t week 12. No e t h a n o l was d e t e c t a b l e i n t h e p l a s m a o f c o n t r o l a n i m a l s a t a n y t i m e t h r o u g h o u t t h e s t u d y p e r i o d . B. T o t a l C a l o r i c i n t a k e e s t i m a t i o n . The e t h a n o l consumed by t h e t r e a t e d a n i m a l s r e p r e s e n t e d e x t r a c a l o r i e s i n a d d i t i o n t o t h o s e p r o v i d e d by t h e i r r e g u l a r f o o d i n t a k e . C a r e f u l m o n i -t o r i n g o f f l u i d and f o o d c o n s u m p t i o n , h o w e v e r , i n d i c a t e d t h a t d u r i n g t h e c o u r s e o f i n c r e a s i n g e t h a n o l c o n s u m p t i o n , t h e t r e a t e d a n i m a l s a l s o r e d u c e d t h e i r f o o d i n t a k e s i m u l t a n e o u s l y ( s e e F i g . 8 ) . The t o t a l c a l o r i e s consumed d a i l y w ere c a l c u l a t e d t o be r o u g h l y e q u i v a l e n t , t h e r e b y n e g a t i n g t h e need t o i m p l e m e n t any b a l a n c i n g m e a s u r e s ( s e e T a b l e 1 1 ) . E t h a n o l was assumed t o c o n t a i n 7.0 K c a l / g and r a t ' c h o w 5.0 K c a l / g i n o u r c a l c u l a t i o n s . C. Body and h e a r t w e i g h t m e a s u r e m e n t s . E v e n t h o u g h t h e d a i l y c a l o r i c c o n s u m p t i o n o f t h e two g r o u p s o f a n i m a l s was s i m i l a r , t h e a l c o h o l - t r e a t e d a n i m a l s seemed t o g a i n w e i g h t a t a s l o w e r r a t e t h a n t h e c o n t r o l a n i m a l s t h r o u g h o u t t h e t r e a t m e n t p e r i o d ( s e e F i g u r e 9) r e s u l t i n g i n s i g n i f i c a n t l y l o w e r b o d y w e i g h t a t t h e end o f week 12 ( T a b l e 1 2 ) . T h e r e was no d i f f e r e n c e i n mean h e a r t w e i g h t s b e t w e e n t h e two g r o u p s and t h e r a t i o s o f w h o l e h e a r t w e i g h t t o body w e i g h t were a l s o s i m i l a r , H owever, we f o u n d a s m a l l b u t s i g n i f i c a n t l y h i g h e r v a l u e i n t h e r a t i o o f v e n t r i c l e w e i g h t t o w h o l e h e a r t w e i g h t i n t h e t r e a t e d a n i m a l s as c o m p a r e d t o c o n t r o l s ( s e e T a b l e 1 2 ) . The w a t e r c o n t e n t s i n t h e v e n t r i c u l a r m u s c l e s were s i m i l a r i n b o t h g r o u p s . - 66 -Week s Figure 8. Daily food intake of control and ethanol-treated animals through-out the 12-week treatment period. Values are mean intake of 12 animals in each group with bars indicat ing one standard deviat ion. The control animals consumed s i g n i f i c an t l y more food da i ly than ethanol-treated animals from Week 2 through Week 12 (P < 0.05). x -- 67 -TABLE 1 1. E s t i m a t e d ^ a v e r a g e d a i l y c a l o r i c i n t a k e o f t h e c o n t r o l and a l c o h o l - t r e a t e d a n i m a l s Week 1 Week 7 Week 9 Week 12 C o n t r o l 105.0 K c a l 126.5 K c a l 135.2 K c a l 150.0 K c a l T r e a t e d 115.6 K c a l 120.6 K c a l 128.1 K c a l 1 1 0 . 0 . K c a l A V a l u e s a r e c a l c u l a t e d f r o m t h e a v e r a g e amount o f e t h a n o l consumed and t h e a v e r a g e amount o f R a t Chow i n g e s t e d . - 68 -550 r 500 cn OJ >> T3 O CQ 450 400 350 h 300 h 250 L Figure 9. Bodyweights of c o n t r o l and e t hano l - t r e a t ed animals dur ing the 12-week p r o t o c o l . Values are mean of 24 animals from each group w i th bars i n d i c a t i n g one standard d e v i a t i o n . Contro l animals are s i g n i f i c a n t l y heav ier than e t hano l - t r e a t ed animals from week 5 through week 12 (P < 0.05) . - 69 -TABLE 12. R e l a t i o n s h i p o f h e a r t t o body w e i g h t s i n c o n t r o l and a l c o h o l - t r e a t e d a n i m a l s C o n t r o l r a t s — A l c o h o l - t r e a t e d r a t s — Body W e i g h t 468 ± 20 g 450 ± 11 g^-H e a r t W e i g h t 1.94 ± 0.21 g 1.90 ± 0.30 g H e a r t W e i g h t Body W e i g h t 0.0041 ± 0.002 0.0042 ± 0.0002 V e n t r i c u l a r W e i g h t H e a r t W e i g h t 0.654 ± 0.017 0.673 ± 0.014^ 1 Mean ± SD; n = 16. b p < 0.05 S t u d e n t ' s t t e s t . - 70 -D. B l o o d p r e s s u r e m e a s u r e m e n t s . T h e t i m e c o u r s e o f t h e b l o o d p r e s s u r e d e v e l o p m e n t i n b o t h c o n t r o l and t r e a t e d g r o u p s was m o n i t o r e d c l o s e l y and a s i g n i f i c a n t e l e v a t i o n i n s y s t o l i c b l o o d p r e s s u r e s ' was a p p a r e n t i n t h e a l c o h o l - t r e a t e d g r o u p b y Week 4 ( s e e F i g . 1 0 ) . T h e a l c o h o l - t r e a t e d a n i m a l s had s y s t o l i c p r e s s u r e r e a d i n g s t h a t were c o n s i s t e n t l y h i g h e r ( 2 0 % ) ' t h a n t h e c o n t r o l s t h r o u g h o u t t h e r e m a i n d e r o f t h e t r e a t m e n t p e r i o d . T h e a b s o l u t e v a l u e s o f t h e s y s t o l i c b l o o d p r e s s u r e were n o t e l e v a t e d t o what m i g h t be c l a s s i f i e d as h y p e r t e n s i v e l e v e l s , b u t t h e 2 0 % e l e v a t i o n o b s e r v e d i s s t a t i s t i c a l l y s i g n i f i c a n t and o t h e r c a r d i o v a s -c u l a r a b n o r m a l i t i e s were n o t e d c o n c u r r e n t l y ( s e e f o l l o w i n g s e c t i o n s ) . I t was t h e r e f o r e d e c i d e d t o s t u d y t h e model f u r t h e r i n hope o f i d e n t i f y i n g some u n d e r l y i n g m e c h a n i s m s r e s p o n s i b l e f o r t h e e l e v a t e d p r e s s u r e s o b s e r v e d . B e f o r e c o n s i d e r i n g t h e s e ' e x p e r i m e n t s , we w o u l d l i k e t o q u a n t i t a t e some c a r d i a c f u n c t i o n p r o p e r t i e s o f t h e s e a n i m a l s and s e e i f c h r o n i c e t h a n o l c o n s u m p t i o n i s a s s o c i a t e d w i t h a b n o r m a l i t i e s i n t h e h e a r t s o f o u r t r e a t e d a n i m a l s . However, we h a v e a l r e a d y m e n t i o n e d t h e h i g h e r v e n t r i c l e / w h o l e h e a r t w e i g h t r a t i o s i n .our t r e a t e d a n i m a l s . T h e c a r d i a c p e r f o r m a n c e i n  v i t r o was a l s o s t u d i e d . E. C a r d i a c p e r f o r m a n c e i n a l c o h o l t r e a t e d r a t s . Mean r e s t i n g h e a r t r a t e s i n b o t h g r o u p s o f a n i m a l s was 350 ± 15 b e a t s / m i n (bpm) d u r i n g t h e p r e t r e a t m e n t p e r i o d . A t t h e end o f t h e t r e a t m e n t p e r i o d , t h e a l c o h o l - t r e a t e d g r o u p showed a s i g n f i c a n t r e d u c t i o n i n t h e mean r e s t i n g h e a r t r a t e ( s e e T a b l e 1 3 ) . T h i s t r e n d was a l s o o b s e r v e d i n t h e i s o l a t e d h e a r t s p e r f u s e d a t 10 mm Hg end d i a s t o l i c p r e s s u r e ( E D P ) . ECG r e s u l t s were s i m i l a r i n b o t h g r o u p s w i t h r e s p e c t t o a r r h y t h m i a s ( b o t h h a v e l e s s t h a n one a r r h y t h m i c b e a t p e r m i n u t e o v e r a 1 0 - m i n u t e p e r i o d ) , b u t t h e P-R i n t e r v a l s 1 4 0 r •—• 1 3 0 CD I E E d ) 1 2 0 3 in m 0) k_ Q. 1 1 0 "O O o m 1 0 0 9 0 * — L - 2 _L _L 4 6 Weeks 10 C o n t r o l A l c o h o l i c 12 Figure 10. S y s t o l i c blood pressure development of both the e t h a n o l - t r e a t e d and con t r o l an imals . Values given are the mean of two t y p i c a l groups, each with 12 animals, and the bars i n d i c a t e one standard d e v i a t i o n . The e t h a n o l -t r ea ted animals having s i g n i f i c a n t l y higher pressure than the con t r o l animals beginning at week 4 and the pressure remained e levated f o r the remainder of the p ro toco l (P < 0.05). TABLE 13. C a r d i a c p e r f o r m a n c e d a t a - C o n t r o l r a t s - A l c o h o l - t r e a t e d r a t s — R e s t i n g h e a r t r a t e i n v i v o 360 ± 10 b p m £ - 320 ± 15 bpmb I s o l a t e d r e s t i n g h e a r t r a t e 152 ± 19 bpm 135 ± 20 bpm 5. Peak IVP/wt on S t a r l i n g c u r v e 168.6 ± 14.0 mmHg 166.5 *• 17.0 mmHg w i t h no p a c i n g 9 (EDP = 28.8 ± lOmmHg)! 9 (EDP = 33.8 ± 8 mmHg) Peak IVP/wt on S t a r l i n g c u r v e 131.0 ± . 1 2 . 0 mmHg 127.0 ± 11.0 mmHg w i t h p a c i n g ( 2 4 0 / m i n ) 9 (EDP = 15.0 ± 10 mmHg) 9 (EDP = 13.0 ± 9 mmHg) P e r c e n t r e d u c t i o n o f p e a k IVP/wt due t o p a c i n g 22 24 P e r c e n t r e d u c t i o n o f o p t i m a l EDP due t o p a c i n g ' 48 ' 62 1 Mean ± SD; n = 1 6 . b. p < 0.05, S t u d e n t ' s t - t e s t £ bpm = b e a t s p e r m i n u t e ^ EDP a t w h i c h t h e peak IVP/wt was o b s e r v e d ( o p t i m a l E D P ) . i n t h e a l c o h o l - t r e a t e d a n i m a l s were i n c r e a s e d b y a b o u t 2 2 % . S t a r l i n g c u r v e s o b t a i n e d f r o m p e r f u s e d h e a r t s a l l o w e d t o b e a t s p o n t a n e o u s l y were n o t d i f f e r -e n t b e t w e e n t h e two g r o u p s o f a n i m a l s . T o a c c o u n t f o r t h e d i f f e r e n c e s i n v e n t r i c u l a r mass, a l l i n t r a v e n t r i c u l a r p r e s s u r e s ( I V P ) v a l u e s r e p o r t e d were n o r m a l i z e d as a r a t i o o f IVP t o v e n t r i c l e w e i g h t ( I V P / w t ) . A s a m p l e i l l u s -t r a t i o n o f a t y p i c a l s e t o f S t a r l i n g c u r v e s i s g i v e n i n F i g . 1 1 . U n d e r e l e c t r i c a l p a c i n g , t h e h e a r t s f r o m b o t h g r o u p s showed a r e d u c t i o n i n peak IVP and o p t i m a l EDP. T h e h e a r t s f r o m t h e a l c o h o l - t r e a t e d a n i m a l s , h o w e v e r , show a g r e a t e r d r o p i n EDP a t w h i c h peak IVP was o b s e r v e d ( s e e T a b l e 1 3 ) . Dose r e s p o n s e r e l a t i o n s h i p s f o r i s o p r o t e r e n o l (IPNA) were e x a m i n e d a t 2+ two d i f f e r e n t c o n c e n t r a t i o n s o f Ca . S t a r l i n g c u r v e s were n o t c o n s t r u c t e d 2+ i n 0.2 mM Ca b e c a u s e i s o l a t e d p e r f u s e d h e a r t s do n o t n o r m a l l y c o n t r a c t 2+ s p o n t a n e o u s l y i n t h e p r e s e n c e o f low [ C a ] . T h e s p o n t a n e o u s I V P / w e i g h t 2+ v a l u e s ( i . e . b e f o r e t h e a d d i t i o n o f IPNA) i n t h e p r e s e n c e o f 2.5 mM Ca w i t h p a c i n g were t h e same i n b o t h g r o u p s ( c o n t r o l = 106 ± 10 mmHg/g, a l c o h o l t r e a t e d = 106 ± 7 mmHg/g). In t h e p r e s e n c e o f 0.2 mM Ca , ho w e v e r , a h i g h l y s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e two g r o u p s was o b s e r v e d ( c o n t r o l = 0.6 ± 0.5 mmHg/g, a l c o h o l t r e a t e d = 3.9 ± 0.9 mmHg/g, P < 0.005, S t u d e n t ' s t - t e s t ) . T h e d o s e - r e s p o n s e r e l a t i o n s h i p s t o IPNA o f h e a r t s f r o m b o t h g r o u p s 2+ a r e i l l u s t r a t e d i n F i g s . .12 and 1 3 . U n d e r n o r m a l c o n d i t i o n s ( 2 . 5 mM Ca ) t h e h e a r t s f r o m a l c o h o l - t r e a t e d a n i m a l s seemed t o r e s p o n d l e s s w e l l t o h i g h e r d o s e s o f IPNA when c o m p a r e d t o c o n t r o l s ( F i g . 1 2 ) . T h i s t r e n d , h o w e v e r , was c o m p l e t e l y r e v e r s e d u n d e r t h e r e d u c e d c a l c i u m c o n d i t i o n (0.2 mM 2+ Ca ) . The h e a r t s f r o m a l c o h o l - t r e a t e d a n i m a l s r e s p o n d e d s i g n i f i c a n t l y b e t t e r t o a l l IPNA c o n c e n t r a t i o n s when compa r e d w i t h c o n t r o l s a t t h i s l o w e r c a l c i u m - i o n c o n c e n t r a t i o n ( F i g . 1 3 ) . T h i s k i n d o f r e s u l t s u g g e s t e d , t h a t - 74 -Sta r l ing C u r v e s 200 r-180 160 A l c o h o l i c C o n t r o l IVP w7 140 V." 120 ( C o n t r o l ! p a c e d ) 100 * . A I c o h o l i c ( p a c e d ) o t _1_ 10 20 30 40 50 E D P (mmHg) 60 70 Figure 11. Typical Starling curves from one isolated perfused heart experi-ment. When le f t to beat spontaneously, the heart from the ethanol-treated rat has similar contractile activity as the control 's but have a higher optimal EDP (at peak IVP). When e lectr ica l ly paced at 240/min, the "alcoho-l i c heart" develops less pressure than the "control heart", particularly at high EDP values. To account for differences in heart s ize, a l l IVP values are normalized to the weight of the heart. - 75 -200 r > 1 9 0 1 8 0 7 0 1 6 0 1 5 0 140 130 120 1 _L C o n t r o l A l c o h o l i c - 9 - 8 - 7 - 6 L o g [ I P N A ] ( M ) F igure 12. Dose-response of the i s o l a t e d perfused hearts to IPNA in the ++ presence of 2.5 mM Ca . Each po in t represent s the mean IVP/weight values of 16 hearts w i th the e r r o r bar i n d i c a t i n g one standard d e v i a t i o n . The d i f f e r e n c e i n responses to the two h ighest doses of IPNA i s s i g n i f i c a n t (P < 0.05). - 76 -L o g C l P N A j ( M ) i F i g u r e 13. D o s e - r e s p o n s e o f t h e i s o l a t e d p e r f u s e d h e a r t t o IPNA i n t h e . p r e s e n c e o f 0.2 mM C a + + . E a c h p o i n t r e p r e s e n t s t h e mean I V P / w e i g h t v a l u e s o f 1 6 ' h e a r t s w i t h t h e e r r o r b a r i n d i c a t i n g one s t a n d a r d d e v i a t i o n . The d i f f e r e n c e i n r e s p o n s e s a t a l l d o s e s o f IPNA i s s i g n i f i c a n t (P < 0 . 0 1 ) . - 77 -t h e r e may be a b n o r m a l i t i e s i n c a l c i u m i o n h a n d l i n g i n t h e h e a r t s o f t h e a l c o h o l - t r e a t e d a n i m a l s . T h i s p o s s i b i l i t y w i l l be c o n s i d e r e d i n t h e D i s c u s -s i o n s e c t i o n . F. P l a s m a and u r i n e i o n a n a l y s e s . P l a s m a and u r i n e s a m p l e s ^ c o l l e c t e d a t t h e b e g i n n i n g o f t h e 1 2 t h week and p r e p a r e d as d e s c r i b e d i n M e t h o d o l o g y S e c t i o n IC were s e n t o u t t o two d i f f e r -e n t c l i n i c a l l a b o r a t o r i e s f o r a n a l y s e s . R e s u l t s f r o m t h e p l a s m a a n a l y s e s a r e shown i n T a b l e 14. None o f t h e i o n s s t u d i e d a p p e a r e d t o be d i f f e r e n t b e t w e e n t h e 2 g r o u p s o f a n i m a l s . ( T h e r e was a s l i g h t d e c r e a s e i n t h e mean + K c o n c e n t r a t i o n i n t h e p l a s m a o f a l c o h o l i c r a t s b u t t h e d i f f e r e n c e was o n l y m a r g i n a l l y s i g n i f i c a n t , ,p < 0 . 1 ) . T h e e t h a n o l l e v e l s d e t e c t e d i n t h e p l a s m a were v e r y d i f f e r e n t as e x p e c t e d and t h e a b s o l u t e v a l u e s a g r e e d v e r y w e l l w i t h v a l u e s o b t a i n e d f r o m o u r e n z y m a t i c a s s a y ( C a l b i o c h e m K i t ) . U r i n e i o n a n a l y s e s r e v e a l e d some v e r y i n t e r e s t i n g r e s u l t s ( s e e T a b l e 1 5 ) . T h e 24 h o u r mean u r i n e o u t p u t was s i g n i f i c a n t l y l o w e r i n t h e t r e a t e d a n i m a l s , as was t h e s o d i u m i o n c o n c e n t r a t i o n i n t h e i r u r i n e ( r e d u c e d by 5 0 % ) ; B o t h / c a l c i u m and magnesium c o n c e n t r a t i o n s were s i g n i f i c a n t l y e l e v a t e d i n t h e u r i n e o f t h e t r e a t e d a n i m a l s w i t h t h e magnesium i o n c o n c e n t r a t i o n s h o w i n g a 4 - f o l d i n c r e a s e c o m p a r e d t o c o n t r o l v a l u e s . P o t a s s i u m and p h o s p h a t e l e v e l s were n o t d i f f e r e n t b e t w e e n t h e two g r o u p s . The r e d u c t i o n o f s o d i u m e x c r e t i o n and u r i n e v o l u m e o b s e r v e d i n t h e a l c o h o l - t r e a t e d a n i m a l s s u g g e s t e d t h a t i n t h e s e a n i m a l s , i n c r e a s e d b l o o d v o l u m e may be one o f t h e m e c h a n i s m s i n v o l v e d i n l e a d i n g an e l e v a t i o n i n t h e b l o o d p r e s s u r e . T h e p l a s m a v o l u m e s o f t h e two g r o u p s o f a n i m a l s were t h e r e f o r e e s t i m a t e d u s i n g t h e method o f i n d i c a t o r d i l u t i o n t o v e r i f y o u r s u s p i c i o n o f v o l u m e e x p a n s i o n i n t h e a l c o h o l - t r e a t e d a n i m a l . - 78 -TABLE 14. I o n a n a l y s e s o f p l a s m a f r o m . a l c o h o l - t r e a t e d and c o n t r o l r a t s MEAN ± S.D. 1 Compounds A n a l y s e d C o n t r o l (n = 12) A l c o h o l i c s (n 1. N a + 140.8 ± 1.4 i mM 139.6 ± 1.2 mM 2. K + 7.2 ± 1.4 mM 5.9 ± 2.6 mM 3. C a + + 5.1 ± 0.3 mM 5.1 ± 0.2 mM 4. M g + + ' 1.9 ± 0.2 mM 2.1 ± 0.2 mM 5. ' Pi- 5.8 ± 0.9 mM 5 . 6 ' ± 0.4 mM 6. ETOH N.D.- 28.6 ± 11 mg £ N.D. = none d e t e c t a b l e £" S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l v a l u e (p < 0.05) j TABLE 1 5 . Ion a n a l y s e s o f u r i n e f r o m a l c o h o l - t r e a t e d and c o n t r o l r a t s Mean ± S.D. C o n t r o l (n = 12) A l c o h o l i c s ( n = 12) Volume (24 h r ) Compounds A n a l y s e d N a + K + Ca Mg P i ++ ++ 12.9 ± 2.0 ml 119 ± 12 meq/1 280 ± 14 meq/1 1.8 ± 0.4 meq/1 11.8 ± 2.4 meq/1 99 ± ' 1 5 meq/1 10.8 ± 3.1 ml a 56 ± 12 meq/13-249 ± 32 meq/1 3.5 ± 1.2 m e q / i a 44.2 ± 6.8 meq/1^-102 ± 30 meq/1 § S t u d e n t s ' t - t e s t , p < 0.05 - 80 -G. P l a s m a v o l u m e m e a s u r e m e n t s . E s t i m a t i o n o f t o t a l p l a s m a v o l u m e was p e r f o r m e d u s i n g m e t h y l - Y g l o b u l i n s . The a d v a n t a g e o f t h i s i n d i c a t o r o v e r o t h e r s ( s u c h as E v a n s B l u e o r a l b u m i n ) i s t h a t a - g l o b u l i n s a r e n o t c l e a r e d o r p a r t i t i o n e d f r o m t h e p l a s m a w i t h i n t h e t i m e i n t e r v a l r e q u i r e d f o r e q u i l i b r i u m ( 2 3 6 ) . T h u s , t h e u s u a l p r o c e d u r e o f e x t r a p o l a t i o n back t o "0 t i m e " i s n o t r e q u i r e d w h i c h e l i m i n a t e s t h e e r r o r s i n h e r e n t i n t h a t p r o c e d u r e . S a m p l e s t a k e n a t 2, 5, and 10 min c o n t a i n e d i d e n t i c a l amounts o f r a d i o a c t i v i t y and t h e r e s u l t s were t r a n s l a t e d i n t o 49.1 ± 3.2 m l / k g f o r t h e c o n t r o l s and 58.9 ± 3.3 m l / k g f o r t h e t r e a t e d a n i m a l s . T h e d i f f e r e n c e s o b s e r v e d a r e h i g h l y s i g n i f i c a n t (p < 0.001; n = 6) and t h i s i n c r e a s e i n p l a s m a v o l u m e i n t h e a l c o h o l - t r e a t e d g r o u p may be c a u s a l l y r e l a t e d t o t h e e l e v a t i o n o f b l o o d p r e s s u r e o b s e r v e d . H. I n v i t r o v a s c u l a r smooth m u s c l e r e s p o n s i v e n e s s . A n o t h e r m e c h a n i s m t h a t c o u l d be p a r t i a l l y r e s p o n s i b l e f o r b l o o d p r e s s u r e e l e v a t i o n i s i n c r e a s e d v a s c u l a r r e s p o n s i v e n e s s t o v a s o p r e s s o r s . We e x a m i n e d t h e c o n t r a c t i l e r e s p o n s e s ,of h e l i c a l a o r t i c s t r i p s and p o r t a l v e i n s f r o m b o t h g r o u p s o f a n i m a l s t o n o r a d r e n a l i n e i n a e r a t e d and h e a t e d m u s c l e b a t h s . T h e r e was no d i f f e r e n c e i n t h e c o n t r a c t i l e r e s p o n s e s o f t h e a o r t i c s t r i p s t o n o r a d r e n a l i n e , u n d e r v a r i o u s c a l c i u m c o n c e n t r a t i o n s , between c o n t r o l and e t h a n o l - t r e a t e d a n i m a l s . The p o r t a l v e i n s t r i p s f r o m b o t h g r o u p s r e s p o n d e d s i m i l a r l y i n b a t h i n g medium c o n t a i n i n g n o r m a l amount o f C a + + ( 2 . 5 mM) b u t t h e s t r i p s f r o m a l c o h o l - t r e a t e d , a n i m a l s showed a' t r e n d t o w a r d s r e d u c e d r e s p o n s i v e n e s s a t 0.4 mM C a + + . T h e r e s p o n s e t o n o r a d r e n a l i n e was s i g n i f i -c a n t l y r e d u c e d i n t h e s t r i p s f r o m a l c o h o l - t r e a t e d a n i m a l s a t low (0.2 mM) e x t e r n a l C a + + ( s e e F i g . 1 4 ) . T h e s e f i n d i n g s do n o t s u p p o r t t h e i d e a o f i n c r e a s e d v a s c u l a r r e s p o n s i v e n e s s as an u n d e r l y i n g m e c h a n i s m f o r t h e b l o o d i - 81 -| Portal Veins C a * * 0 . 2 -7 -6 -8 -7 -6 -5 L o g 1 0 CNAl F i g u r e 14. D o s e - r e s p o n s e c u r v e s o f i s o l a t e d l o n g i t u d i n a l p o r t a l v e i n s t r i p s t o n o r a d r e n a l i n e i n t h e p r e s e n c e o f 2.5 mM, 0.4 mM, and 0.2 mM e x t e r n a l c a l c i u m . V a l u e s a r e mean o f 8 s t r i p s (n = 8 ) w i t h b a r s i n d i c a t i n g one s t a n d a r d d e v i a t i o n . A t 0.2 mM C a + + , t h e r e s p o n s e s o f t h e " c o n t r o l s t r i p s " a r e s i g n i f i c a n t l y h i g h e r t h a n t h e " a l c o h o l i c s t r i p " f r o m 10"^ M t h r o u g h 1 0 ~ 4 M n o r a d r e n a l i n e (P < 0 . 0 5 ) . p r e s s u r e e l e v a t i o n o b s e r v e d i n t h e a l c o h o l - t r e a t e d a n i m a l s . T h e y do s u g g e s t , h o w e v e r , t h a t c a l c i u m i o n s a r e h a n d l e d d i f f e r e n t l y i n t h e t i s s u e s o f t h e c h r o n i c - a l c o h o l a n i m a l s ( s e e a l s o R e s u l t s s e c t i o n H I E f o r comments on i s o l a t e d p r e f u s e d h e a r t s ) . I . E r y t h r o c y t e membrane c h a r a c t e r i z a t i o n . ^ In o r d e r t o f i n d o u t w h e t h e r t h e a b n o r m a l c a l c i u m h a n d l i n g o b s e r v e d i n t h e v a s c u l a r smooth m u s c l e s and i s o l a t e d p e r f u s e d h e a r t s o f t h e a l c o h o l - t r e a -t e d a n i m a l s r e f l e c t s a g e n e r a l i z e d membrane d e f e c t , we d e c i d e d t o i n v e s t i g a t e some o f t h e p r o p e r t i e s o f e r y t h r o c y t e membrane m e n t i o n e d e a r l i e r . Membrane A T P a s e s were s t u d i e d , and t h e o n l y d i f f e r e n c e o b s e r v e d was t h e s l i g h t l y e l e v a t e d a c t i v i t y o f t h e h i g h a f f i n i t y C a + + / M g + + A T P a s e i n t h e a l c o h o l - t r e a t e d r a t s . No d i f f e r e n c e was d e t e c t a b l e i n t h e N a + / K + A T P a s e f r o m b o t h g r o u p s o f a n i m a l s ( s e e T a b l e 1 6 ) . P a s s i v e c a l c i u m i n f l u x and o s m o t i c f r a g i l i t y were e x a m i n e d i n i n t a c t e r y t h r o c y t e s f r o m b o t h g r o u p s and no d i f f e r e n c e s c o u l d be o b s e r v e d . C o m p o s i t i o n a l a n a l y s e s r e v e a l e d t h a t b o t h membrane and p l a s m a c h o l e s t e r o l c o n t e n t s were l o w e r i n t h e a l c o h o l - t r e a t e d r a t s as c o m p a r e d t o c o n t r o l s ( s e e T a b l e 1 7 ) . T o t a l p h o p h o l i p i d c o n t e n t s and p h o s p h o l i p i d p r o f i l e s however, were v i r t u a l l y i d e n t i c a l i n b o t h g r o u p s o f a n i m a l s . T h e r e s u l t s s u g g e s t t h a t t h e r e i s no g r o s s s t r u c t u r a l a b n o r m a l i -t i e s i n t h e e r y t h r o c y t e membranes o f t h e a l c o h o l - t r e a t e d r a t s . T h e r e i s t h e p o s s i b i l i t y o f a l t e r e d membrane f l u i d i t y , as s u g g e s t e d by t h e l o w e r membrane c h o l e s t e r o l c o n t e n t o b s e r v e d and t h i s c o u l d r e s u l t i n c e r t a i n a l t e r a t i o n s o f membrane enzyme a c t i v i t y ( C a + + / M g + + A T P a s e i n t h i s c a s e ) . T h e r e w i l l be f u r t h e r e l a b o r a t i o n on t h i s t o p i c l a t e r i n t h e D i s c u s s i o n s e c t i o n . - 83 -TABLE 16. A c t i v i t i e s o f e r y t h r o c y t e membrane enzymes f r o m c h r o n i c a l c o h o l - t r e a t e d and c o n t r o l a n i m a l s Mean^- ± S.D. Enzymes A n a l y s e d 1. C a + + / M g + + A T P a s e : B a s a l H i g h A f f i n i t y £ Low A f f i n i t y ? . 2. N a + / K + A T P a s e s : B a s a l S t i m u l a t e d ^ . C o n t r o l (n = 12) 0,92 ± 0.11 1.42 ± 0.40 2.16 ± 0.38 1.13 ± 0.24 0.78 ± 0.21 A l c o h o l i c s ( n = 12) 0.98 ± 0.21 1.97 ± 0.32^ 2.41 ± 0 . 3 3 1.06 ± 0.22 0.84 ± 0.19 U n i t s a r e i n y m o l e s P i hr-mg p r o t e i n S i g n i f i c a n t l y d i f f e r e n t f r o m C o n t r o l v a l u e s , p < 0.05 V a l u e s g i v e n a r e s p e c i f i c a c t i v i t i e s w i t h b a s a l a c t i v i t i e s s u b t r a c t e d b c - 84 -TABLE 17. C h o l e s t e r o l and p h o s p h o l i p i d a n a l y s e s o f e r y t h r o c y t e membrane and p l a s m a f r o m c h r o n i c a l c o h o l - t r e a t e d and c o n t r o l r a t s Mean ± S.D. Compounds A n a l y s e d C o n t r o l (n = 8) A l c o h o l i c s (n = 8 ) 1. C h o l e s t e r o l Membrane (ym/mg)- 0.59 ± 0.04 0.52 ± 0 . 0 3 ^ P l a s m a (ym/mg)- 76.3 ± 5.2 66.2 ± 4.8^-Membrane (yfl/mg) 24.4 ± 3.0 22.1 ± 1 . 0 2. T o t a l P h o s p h o l i p i d s P l a s m a (yg/mg) 9.2 ± 1.1 8.3 ± 0.8 3. P - e t h a n o l a m i n e | 25.7 ± 1.4 25.6 ± 2.0 4. P - s e r i n e p e r c e n t o f t o t a l 10.2 ± 1.3 11.4 ± 2.3 p h o s p h o l i p i d s 5. P - c h o l i n e 6. S p h i n g o m y e l i n 44.1 ± 2.6 43.4 ± 3.4 18.7 ± 2.3 17.9 ± 1.4 — S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l v a l u e s P < 0.05 — ym = y m o l e s - 8 5 -mmHg ie:r>—. Blood Pressure min. F i g u r e 1 5 . C o n t i n u o u s b l o o d p r e s s u r e t r a c i n g f r o m a c h r o n i c t a i l a r t e r y c a n n u l a i m p l a n t i n a m a l e W i s t a r r a t . T h e t h i c k b l a c k b a r s a r e o f 5 min d u r a t i o n , and d e n o t e i r r a d i a t i o n w i t h a 150 Watt h e a t lamp. The " s q u a r e - w a v e l i k e " r e s p o n s e , o f t h e b l o o d p r e s s u r e t o t h e i r r a d i a t i o n p e r s i s t s b e y o n d 180 min w i t h o u t any s i g n s o f r e d u c e d r e s p o n s e . I V . C h r o n i c A l c o h o l and H e a t S t r e s s A. O b s e r v a t i o n s on t h e a n i m a l s R a t s were r a n d o m l y a l l o c a t e d t o f o u r g r o u p s a t Week 4 o f t h e a l c o h o l -t r e a t m e n t p r o t o c o l c o m p r i s i n g o f : c o n t r o l u n s t r e s s e d ( C U ) ; c o n t r o l s t r e s s e d ( C S ) ; a l c o h o l i c u n s t r e s s e d ( A U ) ; and a l c o h o l i c s t r e s s e d (AS) as d e s c r i b e d i n M e t h o d o l o g y S e c t i o n V. A l l m e a s u r e m e n t s made p r i o r t o Week 4 i n d i c a t e d t h a t t h e a n i m a l s d i d c o n f o r m t o p r e v i o u s l y r e p o r t e d r e s u l t s r e g a r d i n g body w e i g h t , f l u i d and f o o d i n t a k e , b l o o d e t h a n o l c o n t e n t , p l a s m a and u r i n e i o n a n a l y s i s , and b l o o d p r e s s u r e v a l u e s . I r r a d i a t i o n w i t h a 150 w a t t h e a t lamp most r e p r o d u c i b l y e l i c i t e d a t r a n s i e n t b l o o d p r e s s u r e e l e v a t i o n r e s p o n s e w h i c h d i d n o t d i m i n i s h o v e r t i m e ( i . e . no a d a p t i o n t o t h e s t r e s s was a p p a r e n t ) . F i g u r e 15 i l l u s t r a t e d t h e t y p i c a l b l o o d p r e s s u r e t r a c i n g o f a r a t w i t h a c h r o n i c c a n n u l a i m p l a n t e d i n t h e t a i l a r t e r y . The b l o o d p r e s s u r e r o s e s h a r p l y w h e n e v e r t h e a n i m a l was s u b j e c t e d t o t h e h e a t , and r e t u r n e d t o n o r m a l when t h e lamp was t u r n e d o f f . T h e c o r e t e m p e r a t u r e ( m e a s u r e d v i a a r e c t a l p r o b e ) o f t h e a n i m a l s was 39.8 ± 0.9°C ( n = 4 ) a f t e r one h o u r o f h e a t s t r e s s i n g . By Week 12, o n l y h a l f o f t h e a n i m a l s i n t h e AS g r o u p s u r v i v e d t h e p r o t o c o l . T h e c a u s e o f d e a t h i n t h i s g r o u p a p p e a r e d t o be c a r d i a c f a i l u r e , as e v i d e n t f r o m t h e p u l m o n a r y e f f u s i o n and v e n o u s c o n g e s t i o n ; o b s e r v e d a t a u t o p s y . T h e s u r v i v i n g a n i m a l s i n t h i s g r o u p were g e n e r a l l y v e r y i r r i t a b l e and s o m e t i m e s showed s e i z u r e - l i k e m o t o r a c t i v i t y . T h e CS g r o u p on t h e o t h e r h a n d , s u r v i v e d t h e p r o t o c o l w i t h no o b v i o u s p r o b l e m s . T a b l e 18 l i s t s some o f t h e w e i g h t s and m e a s u r e m e n t s on t h e f o u r g r o u p s o f a n i m a l s t a k e n a t Week 12. A l l f o u r g r o u p s consumed r o u g h l y s i m i l a r amounts o f f o o d e n e r g y d a i l y . T he AU and AS a n i m a l s were s i g n i f i c a n t l y l i g h t e r a t Week 12. T h e CS a n i m a l s a l s o t e n d e d t o be l i g h t e r t h a n t h e CUs, b u t no s t a t i s t i c a l s i g n i f i c a n c e c o u l d be f o u n d . E t h a n o l c o n s u m p t i o n was s i m i l a r b e t w e e n t h e AU and t h e , A S a n i m a l s ( s e e T a b l e 1 8 ) . B. T i m e c o u r s e o f b l o o d p r e s s u r e d e v e l o p m e n t . S y s t o l i c b l o o d p r e s s u r e o f t h e a n i m a l s was m o n i t o r e d w e e k l y v i a t h e t a i l c u f f and a f i n a l f e m o r a l a r t e r i a l p r e s s u r e was t a k e n a t Week 12. F i g u r e 16 shows t h e w e e k l y mean s y s t o l i c b l o o d p r e s s u r e v a l u e s o f t h e a n i m a l s t h r o u g h -o u t t h e e x p e r i m e n t . A t week- 4, a d i f f e r e n c e i n t h e b l o o d p r e s s u r e was a l r e a d y n o t i c e d between t h e c o n t r o l s and t h e a l c o h o l - t r e a t e d a n i m a l s . D u r i n g t h e s t r e s s i n g p r o t o c o l (Week 4 t o Week 12) b o t h t h e AS and CS g r o u p s showed a g r a d u a l r i s e i n t h e i r s y s t o l i c p r e s s u r e s , r e a c h i n g a p l a t e a u a t Week 8. By Week 12, t h e mean b l o o d p r e s s u r e s o f t h e f o u r g r o u p s o f a n i m a l s were a l l d i f f e r e n t f r o m e a c h o t h e r , w i t h t h e a l c o h o l i c s t r e s s e d g r o u p h a v i n g t h e h i g h e s t b l o o d p r e s s u r e v a l u e s ( s e e T a b l e 1 8 ) . The mean h e a r t r a t e s o f t h e CS g r o u p were h i g h e r w h i l e t h e AU g r o u p s were l o w e r t h a n t h e c o n t r o l ' s ( C U ) . C. P l a s m a V o l u m e M e a s u r e m e n t s . S i n c e t h e r e was good e v i d e n c e t h a t c h r o n i c a l c o h o l i n g e s t i o n and s t r e s s seemed t o h a v e an a d d i t i v e e f f e c t on b l o o d p r e s s u r e e l e v a t i o n i n o u r a n i m a l s , a t t e m p t s t o e l u c i d a t e t h e u n d e r l y i n g m e c h a n i s m ( s ) were made. T h e f i r s t p o s s i b i l i t y c o n s i d e r e d was f u r t h e r p l a s m a v o l u m e e x p a n s i o n . We u s e d t h e same p r o c e d u r e o f i n d i c a t o r d i l u t i o n m e n t i o n e d e a r l i e r t o d e t e r m i n e t h e p l a s m a v o l u m e i n a l l f o u r g r o u p s o f a n i m a l s . T a b l e 19 l i s t s t h e mean v a l u e s o f t h e p l a s m a v o l u m e s o f t h e 4 g r o u p s o f a n i m a l s , n o r m a l i z e d t o t h e i r body w e i g h t s . T h e a l c o h o l i c s t r e s s e d and a l c o h o l i c u n s t r e s s e d g r o u p s b o t h showed - 88 -1 0 0 1 1 1 : 1 1 1 1 1 -2 0 2 4 6 8 10 12 Weeks F i g u r e 16. B l o o d p r e s s u r e d e v e l o p m e n t o f f o u r g r o u p s o f r a t s d u r i n g t h e 12-week p r o t o c o l . H e a t s t r e s s was i m p l e m e n t e d a t t h e 4 t h week and c o n t i n u e d u n t i l t h e e n d o f t h e p r o t o c o l . V a l u e s a r e mean o f 12 a n i m a l s e x c e p t f o r AS g r o u p , where a t week 12, t h e r e were o n l y 7 a n i m a l s l e f t i n t h e g r o u p . The s t a n d a r d d e v i a t i o n s f o r t h e b l o o d p r e s s u r e v a l u e s a r e ± 6 mm Hg f o r t h e " c o n t r o l " (CU, CS) and ± 1 1 mm Hg f o r t h e " a l c o h o l i c s " (AU, A S ) . - 89 -TABLE 18. V a r i o u s m e a s u r e m e n t s t a k e n a t Week 12 on t h e g r o u p s o f a l c o h o l - t r e a t e d and c o n t r o l r a t s . M e a s u r e m e n t s CU CS AU AS' 1. Body w e i g h t ( g ) 459 ± 18 442 ± 15 440 ± lc£ 428 ± 1 2 -2. E t h a n o l c o n s u m p t i o n ( g / d a y ) 5.4 ± 1.0 5.9 ± 1.3 3. E s t i m a t e d c a l o r i c i n t a k e ^ ( K c a l / d a y ) • 138 130 112 108 4. Mean b l o o d p r e s s u r e (mm Hg) ' 98 ± 6 110 ± 8 ^ 112 ± 1 1 - 134 ± l O 6 -5. H e a r t r a t e (bpm) 382 ± 14 424 ± 2 0 ^ 340 ± 2 2 - 386 ± 24 - V a l u e s g i v e n a r e mean ± S.D., n = 12 e x c e p t f o r AS where n = 7. - S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (CU) v a l u e s , P < 0.05 - S i n c e t h e v a l u e s a r e o n l y e s t i m a t e s and n o t m e a s u r e m e n t s , no s t a n d a r d d e v i a t i o n s a r e g i v e n and no s t a t i s t i c a l a n a l y s e s were p e r f o r m e d . - 90 -TABLE 19. PLASMA VOLUME (ml/kg) Group No. Mean ± S.D. 1. Control unstressed (CU) 49.8 ± 2.2 , 2. Control stressed (CS) 50.2 ± 1.9 3. Alcoholic unstressed (AU) 59.7 ± 2.1a 4. Alcoholic stressed (AS)- 59.9 ± 2.0^ -_a Significantly different from (CU) ; p 0.05 (n=l*) control values b AS group,, n=6 - 91 -i n c r e a s e d p l a s m a volume c o m p a r e d t o t h e two c o n t r o l g r o u p s . T h e r e was, h o w e v e r , no d i f f e r e n c e b e t w e e n t h e two a l c o h o l i c g r o u p s t h e m s e l v e s , p o i n t i n g t o t h e f a c t t h a t t h e f u r t h e r e l e v a t i o n o f b l o o d p r e s s u r e i n t h e AS g r o u p i s u n l i k e l y t h e r e s u l t o f p l a s m a volume e x p a n s i o n . D. P l a s m a c a t e c h o l a m i n e d e t e r m i n a t i o n s . To f u r t h e r i n v e s t i g a t e t h e u n d e r l y i n g m e c h a n i s m f o r t h e a d d i t i o n a l b l o o d p r e s s u r e e l e v a t i o n o b s e r v e d i n AS a n i m a l s , we d e c i d e d t o e s t i m a t e t h e b a s a l s y m p a t h e t i c t o n e i n t h e 4 g r o u p s o f a n i m a l s by m e a s u r i n g p l a s m a c a t e c h o l a -m i n e s p r i o r t o s a c r i f i c e u s i n g a HPLC s y s t e m and t h e r e s u l t s a r e l i s t e d i n T a b l e R19. T h e n o r a d r e n a l i n e (NA) l e v e l s were s i g n i f i c a n t l y h i g h e r i n t h e p l a s m a o f b o t h g r o u p s o f a l c o h o l - t r e a t e d a n i m a l s (AS and A U ) . T h e r e i s a l s o a m a r g i n a l e l e v a t i o n i n t h e c o n t r o l s t r e s s e d ( CS) a n i m a l s . T h e a l c o h o l i c -s t r e s s e d g r o u p seemed t o have t h e h i g h e s t l e v e l o f NA among a l l f o u r g r o u p s o f a n i m a l s . P l a s m a a d r e n a l i n e (A) l e v e l s were t h e same i n a l l f o u r g r o u p s . T h e r e i s an i n t e r e s t i n g o b s e r v a t i o n t h a t t h e p l a s m a d o p a m i n e l e v e l was t h e l o w e s t i n AS a n i m a l s c o m p a r e d t o t h e t h r e e o t h e r g r o u p s o f r a t s . Our r e s u l t s t h e r e f o r e s u g g e s t t h a t ' i n c r e a s e d s y m p a t h e t i c n e r v o u s a c t i v i t y may p l a y a r o l e i n t h e f u r t h e r e l e v a t i o n o f b l o o d p r e s s u r e i n t h e AS a n i m a l s . TABLE 20. P l a s m a c a t e c h o l a m i n e s ( n g / m l ) Mean ± S.E.M. G r o u p No. n NA- A - D / £ 1. C o n t r o l u n s t r e s s e d (CU) 6 0.48 ± 0.04 0.18 ± 0.03 0.11 ± 0.03 2. C o n t r o l s t r e s s e d ( CS) 6 0.52 ± 0.03 0.17 ± 0.03 0.08 ± 0.01 3. A l c o h o l u n s t r e s s e d (AU) 6 0.59 ± 0.06^- 0.15 ± 0.04 0.10 ± 0.02 4. A l c o h o l s t r e s s e d (AS) 4 0.66 ± 0 . 0 7 - 0.16 ± 0.02 0.05 ± 0.02-— N.A. = n o r a d r e n a l i n e — A. = a d r e n a l i n e — D.A. = d o p a m i n e — S i g n i f i c a n t l y d i f f e r e n t f r o m GP 1. p < 0.05 - 93 -DISCUSSION I . G e n e t i c s and H y p e r t e n s i o n I t was o v e r 30 y e a r s ago t h a t P age f i r s t p u t f o r t h h i s " m o s a i c t h e o r y " f o r t h e a e t i o l o g y o f h y p e r t e n s i o n ( 1 5 5 ) . He p o s t u l a t e d t h a t c h e m i c a l and n e u r a l f a c t o r s as w e l l as v a s c u l a r e l a s t i c i t y , c a r d i a c o u t p u t , b l o o d v i s c o -s i t y , v a s c u l a r c a l i b e r , b l o o d v o l u m e and v a s c u l a r r e a c t i v i t y a l l i n t e r a c t w i t h e a c h o t h e r t o r e g u l a t e t h e u l t i m a t e t i s s u e p e r f u s i o n p r e s s u r e ana p e r i -p h e r a l r e s i s t a n c e . T h i s i d e a o f a m u l t i f a c t o r i a l a p p r o a c h i n h y p e r t e n s i o n r e s e a r c h d i d n o t r e c e i v e much s u p p o r t and r e c o g n i t i o n u n t i l t h e l a t e s i x t i e s when i n v e s t i g a t o r s s l o w l y r e a l i z e d t h a t t h e many u n i t a r y c o n c e p t s o f p r i m a r y h y p e r t e n s i o n a l l l e d t o dead e n d s . In a r e c e n t r e v i e w , F o l k o w s u p p o r t e d t h e " m o s a i c t h e o r y " and l i s t e d 10 f a c t o r s t h a t a r e g e n e r a l l y a c c e p t e d b y a u t h o r -i t i e s as b e i n g i n v o l v e d i n p r i m a r y h y p e r t e n s i o n . G e n e t i c p r e d i s p o s i t i o n , a f a c t o r t h a t Page l e f t o u t i n h i s o r i g i n a l " m o s a i c t h e o r y " , t o p p e d t h e l i s t ( 1 5 6 ) . T h e r e a s o n why P a g e o v e r l o o k e d t h e g e n e t i c f a c t o r i n h i s t h e o r y i s n o t i m m e d i a t e l y a p p a r e n t , b u t o n e c a n p o s t u l a t e t h a t 30 y e a r s ago, t h e o n l y i n f o r m a t i o n a v a i l a b l e on h y p e r t e n s i o n was t h a t o b t a i n e d f r o m human s t u d i e s b e c a u s e t h e s e v e r a l s t r a i n s o f h y p e r t e n s i v e r a t s t h a t a r e i n u s e t o d a y were d e v e l o p e d some 10 y e a r s a f t e r h i s o r i g i n a l p r o p o s a l . Humans, u n l i k e h y p e r -t e n s i v e r a t s t r a i n s , a r e n o t h i g h l y i n b r e d — t h e r e f o r e i n d i v i d u a l v a r i a t i o n s w i l l t e n d t o mask t h e e f f e c t s o f t h e g r o u p o f p r e d i s p o s i n g g e n e s f o r h y p e r -t e n s i o n , m a k i n g g e n e t i c s t u d i e s a l m o s t i m p o s s i b l e . A g e n e t i c " d e v i a t i o n " g e n e r a l l y means t h a t c e r t a i n m a c r o m o l e c u l a r c o n s t i -t u e n t s , w h e t h e r e nzymes, membrane c o m p o n e n t s , o r c a r r i e r m o l e c u l e s , a r e d i f f e r e n t i n d e s i g n and f u n c t i o n f r o m t h e norm. The d e v i a t i o n s h o u l d be - 94 -r e g a r d e d as a " d e f e c t " o n l y when f u n c t i o n a l c h a r a c t e r i s t i c s a r e a l t e r e d so as t o j e o p a r d i z e t h e w e l l b e i n g o f t h e o r g a n i s m i n q u e s t i o n . T h e g e n e t i c d e v i a t i o n t h a t p r e d i s p o s e s c e r t a i n i n d i v i d u a l s t o h y p e r t e n s i o n ( a n d t h e a c c o m p a n y i n g i n c r e a s e d m o r t a l i t y and m o r b i d i t y ) c a n c e r t a i n l y be r e g a r d e d as a " f a u l t " o r " d e f e c t " . The s p o n t a n e o u s l y h y p e r t e n s i v e r a t s (SHR) d e v e l o p e d by Okamoto and A o k i and o t h e r h y p e r t e n s i v e r a t s t r a i n s a r e u s e f u l i n a t t e m p t s t o i d e n t i f y t h e g e n e t i c d e f e c t s a s s o c i a t e d w i t h b l o o d p r e s s u r e e l e v a t i o n i n t h e s e a n i m a l s b e c a u s e t h e i r d e v e l o p m e n t was by s t r i c t i n b r e e d i n g , w h i c h r e n d e r e d them g e n e t i c a l l y more u n i f o r m . I n a r e c e n t a r t i c l e , T r i p p o d o and F r o h l i c h ( 1 5 7 ) r e v i e w e d and d i s c u s s e d a l l t h e known c a r d i o v a s c u l a r a b n o r m a l -i t i e s i n SHR ( a n d comp a r e d them t o e s s e n t i a l h y p e r t e n s i o n i n h u m a n s ) . I n c r e a s e d p e r i p h e r a l r e s i s t a n c e , i n c r e a s e d v a s c u l a r r e a c t i v i t y and e l e v a t e d s y m p a t h e t i c t o n e a r e a l t e r a t i o n s t h a t were c o n s i s t e n t l y m e n t i o n e d . I f we c o n s i d e r t h e s e " a b n o r m a l i t i e s " c a r e f u l l y , o n e o f t h e "common d e n o m i n a t o r " i s t h e c a l c i u m i o n . I t i s g e n e r a l l y a c c e p t e d t h a t c a l c i u m i s t h e a g e n t t h a t a c t i v a t e s t h e c o n t r a c t i l e a p p a r a t u s i n smooth m u s c l e ( 1 5 8 - 1 6 0 ) . E x p e r i e n c e ^ f r o m o u r l a b o r a t o r y ( 1 1 9 , 1 6 1 ) as w e l l as o t h e r s ' ( 1 6 2 , 1 6 3 ) c o n f i r m e d t h a t c a l c i u m i s h a n d l e d d i f f e r e n t l y i n v a s c u l a r smooth m u s c l e o f t h e SHR when c o m p a r e d t o c o n t r o l s . The use o f t h e e r y t h r o c y t e membrane as a model membrane s y s t e m f o r t h e s t u d y o f d r u g a c t i o n s h as p r o v e n t o be v e r y u s e f u l ( 1 4 2 , 1 6 4 , 1 6 5 ) . T h e r e ^ s good e v i d e n c e t h a t i n c e r t a i n d i s e a s e s t a t e s where g e n e r a l i z e d membrane d e f e c t s w e r e o b s e r v e d , p a r a l l e l d e f e c t s were d e t e c t e d i n t h e e r y t h r o c y t e membrane as w e l l ( 1 6 6 - 1 6 8 ) . Our s t u d i e s o f t h e e r y t h r o c y t e membrane o f t h e SHRs h a v e a l l o w e d a d e t a i l e d i n v e s t i g a t i o n o f membrane s t r u c t u r a l and f u n c t i o n a l a b n o r m a l i t i e s i n c l u d i n g a l t e r a t i o n s i n membrane C a + + h a n d l i n g w h i c h may p r o v i d e some i n s i g h t i n t o t h e m o l e c u l a r m e c h a n i s m s u n d e r l y i n g t h e h y p e r t e n s i o n i n t h e s e a n i m a l s . - 95 -I I . Membrane A l t e r a t i o n s i n t h e SHR S i n c e c a l c i u m m o d u l a t e s t h e c o n t r a c t i l i t y o f v a s c u l a r smooth m u s c l e , and t h i s i o n was f o u n d t o be a b n o r m a l l y h a n d l e d i n b l o o d v e s s e l s f r o m SHRs, we d e c i d e d t o s t u d y t h e i n t e r a c t i o n o f c a l c i u m w i t h e r y t h r o c y t e s f r o m t h e s e a n i m a l s . T h e a c t i v i t y o f t h e m a g n e s i u m - d e p e n d e n t c a l c i u m - s t i m u l a t e d A T P a s e ( C a + + / M g + + A T P a s e ) i s b e l i e v e d t o r e f l e c t a c t i v e c a l c i u m i o n t r a n s p o r t a c t i v i t y i n t h e e r y t h r o c y t e and t h e r e f o r e i s an i m p o r t a n t r e g u l a t o r o f i n t r a c e l l u l a r c a l c i u m i o n c o n c e n t r a t i o n ( 1 6 9 - 1 7 1 ) . Our e x p e r i m e n t a l d a t a i n d i c a t e t h a t t h e s p e c i f i c a c t i v i t y o f t h e Ca /Mg A T P a s e i s s i g n i f i -c a n t l y h i g h e r i n e r y t h r o c y t e membranes f r o m SHRs when comp a r e d t o t h o s e f r o m WKYs. T h i s s u g g e s t s t h a t C a + + may be e x t r u d e d a t a f a s t e r r a t e i n t h e r e d c e l l s o f t h e SHR. T h e i n c r e a s e d enzyme a c t i v i t y c o u l d e i t h e r a r i s e f r o m a s p e c i f i c g e n e t i c - a l t e r a t i o n s i n t h e enzyme, o r a r i s e as a s e c o n d a r y ( o r c o m p e n s a t o r y ) e f f e c t a s s o c i a t e d w i t h some u n d e r l y i n g a b n o r m a l i t y . T h e s e c o n d e x p l a n a t i o n i s deemed more p l a u s i b l e b e c a u s e t h e r e s u l t s o f e x p e r i -ments on v a s c u l a r smooth m u s c l e f r o m t h e SHR s u g g e s t t h a t t h e r e may be an i n c r e a s e d a v a i l a b i l i t y o f c a l c i u m i n s i d e t h e smooth m u s c l e c e l l r e s u l t i n g i n an i n c r e a s e d r e a c t i v i t y o f c o n t r a c t i l e a p p a r a t u s ( 1 6 1 - 1 6 3 ) . T h e f u r t h e r o b s e r v a t i o n t h a t t h e n o n - c a l c i u m s t i m u l a t e d b a s a l A T P a s e s a c t i v i t y was a l s o e l e v a t e d i n t h e SHR membrane s u g g e s t s t h a t maybe o t h e r enzyme a c t i v i t i e s s u c h as a c i d a n d / o r a l k a l i n e p h o s p h a t a s e s a r e a l s o e l e v a t e d . T h e s e f i n d i n g s a r e p e r h a p s o f r e l e v a n c e t o t h e f i n d i n g s o f b o t h t h e J a p a n e s e g r o u p who u s e d a r t e r i a l l y s o s o m a l a c i d p h o s p h a t a s e as h y p e r t e n s i v e m a r k e r s and f o u n d t h e a c t i v i t y o f t h i s enzyme t o be e l e v a t e d i n SHR a o r t a e ( 1 7 2 ) , and o f D a n i e l ' s g r o u p , who r e p o r t e d i n c r e a s e d a l k a l i n e p h o s p h a t a s e a c t i v i t y i n t h e m e s e n t e r i c a r t e r i e s o f t h e SHR ( 1 9 4 ) . T h e c a l c i u m d e p e n d e n c y c u r v e s shown v i n F i g u r e 3 - 96 -i n d i c a t e t h a t t h e SHR enzymes have s i g n i f i c a n t i n c r e a s e s i n V . A s w e l l , J 3 max ++ a h i g e r a f f i n i t y o f t h e enzyme f o r C a i s e v i d e n t f r o m t h e s h i f t o f t h e EDgQ v a l u e s t o w a r d s l o w e r C a + + c o n c e n t r a t i o n s . A c t i v a t i o n e n e r g y s t u d i e s - i n d i c a t e t h a t t h e i n c r e a s e d C a + + / M g + + A T P a s e a c t i v i t y s e e n i n t h e SHR membrane i s n o t a r e s u l t o f l o w e r e d a c t i v a t i o n e n e r g y b e c a u s e t h e v a l u e s o b t a i n e d f r o m t h e SHR enzyme a r e s i g n i f i c a n t l y h i g h e r t h a n t h a t o f t h e WKYs'. I n o r d e r t o r u l e o u t a s p e c i f i c g e n e t i c e r r o r i n p r o t e i n b i o s y n -t h e s i s (enzyme i n t h i s c a s e ) i n t h e SHR, we a s s a y e d o t h e r membrane enzymes and f o u n d t h e i r a c t i v i t i e s t o be t o t a l l y s i m i l a r t o t h a t o f t h e WKYs'. The s l i g h t l y e l e v a t e d b a s a l A T P a s e a c t i v i t y o b s e r v e d i n t h e N a + / K + A T P a s e a s s a y i s p r o b a b l y a r e f l e c t i o n o f i n c r e a s e d p h o s p h a t a s e a c t i v i t y m e n t i o n e d e a r l i e r ( s e e T a b l e 5 ) . T h e h y p o t h e s i s ' t h a t t h e i n c r e a s e d C a + + / M g + + A T P a s e a c t i v i t y i s a s e c o n d a r y a l t e r a t i o n i n r e s p o n s e t o s o m e . p r i m a r y u n d e r l y i n g membrane d e f e c t p o s s i b l y " i n v o l v i n g c a l c i u m , was t e s t e d b y s t u d y i n g t h e p a s s i v e c a l c i u m f l u x a c r o s s t h e e r y t h r o c y t e membrane. The r e s u l t s p r o v i d e d s u p p o r t f o r t h i s h y p o t h e s i s b e c a u s e SHR e r y t h r o c y t e membrane s h o w e d , a 4 0 % i n c r e a s e i n p a s s i v e c a l c i u m i n f l u x when comp a r e d t o t h a t o f t h e WKY. To make t h e l i q u i d s c i n t i l l a t i o n c o u n t i n g r e s u l t s ( c a l c i u m l o a d i n g ) more r e l i a b l e , we t r i e d t o i n c r e a s e c a l c i u m i n f l u x by e n z y m a t i c a l l y r e m o v i n g s i a l i c a c i d r e s i d u e s f r o m t h e o u t s i d e r e d c e l l s u r f a c e . T h i s t e c h n i q u e was f i r s t u s e d by L a n g e r t o s t u d y c a l c i u m e x c h a n g e i n c u l t u r e d h e a r t c e l l s ( 1 5 4 ) . We h a v e a d o p t e d , t h e i r t e c h n i q u e and managed t o i n c r e a s e d o u r c a l c i u m l o a d i n g b y 2 - f o l d ( s e e R e s u l t s S e c . I C ) . N e u r a m i n i d a s e - t r e a t e d SHR e r y t h r o c y t e s d e m o n s t r a t e d a s i m i l a r i n c r e a s e ( 4 0 % ) i n p a s s i v e c a l c i u m i n f l u x when c o m p a r e d t o e n z y m e - t r e a t e d c o n t r o l e r y t h r o c y t e s . I t seems r e a s o n a b l e t o c o n c l u d e t h a t t h e SHR e r y t h r o -c y t e membrane i s more p e r m e a n t ( o r l e a k y ) t o c a l c i u m i o n when c o m p a r e d t o - 97 -WKY e r y t h r o c y t e membrane, and t h a t s i a l i c a c i d r e s i d u e s a r e p r o b a b l y n o t r e s p o n s i b l e f o r t h e d i f f e r e n c e b e t w e e n r e d c e l l s o f SHR and WKY. The i n c r e a s e i n t h e C a + + / M g + + A T P a s e a c t i v i t y o b s e r v e d may w e l l be a s e c o n -d a r y c o m p e n s a t o r y c h a n g e r e s u l t i n g f r o m t h e e l e v a t e d l e v e l s o f c a l c i u m i n s i d e t h e SHR e r y t h r o c y t e . K l i n g e r ' s g r o u p s r e c e n t l y r e p o r t e d t h a t i n c r e a s e d c a l c i u m c o n c e n t r a t i o n s c a n t r a n s f o r m t h e Ca /Mg A T P a s e i n t h e e r y t h r o c y t e f r o m l ow a f f i n i t y t o h i g h a f f i n i t y a c t i v i t y ( 1 7 3 ) . T h i s c o u l d be one m e c h a n i s m r e s p o n s i b l e 1 f o r t h e i n c r e a s e d enzyme a c t i v i t y demon-s t r a t e d h e r e and i s c o n s i s t e n t w i t h t h e o b s e r v a t i o n t h a t t h e SHR enzyme showed a h i g h e r a p p a r e n t a f f i n i t y f o r c a l c i u m ( s e e R e s u l t s s e c . I B ) . A n o t h e r p o s s i b i l i t y i s t h a t t h e SHR e r y t h r o c y t e s r e s p o n d e d t o t h e e l e v a t e d i n t r a c e l -l u l a r c a l c i u m c o n c e n t r a t i o n s b y i n c r e a s i n g t h e amount o f c a l m o d u l i n p r e s e n t , t h e r e f o r e e f f e c t i n g a h i g h e r r a t e o f e x t r u s i o n o f c a l c i u m f r o m i n s i d e t h e ++ ++ c e l l s by s t i m u l a t i o n o f t h e Ca /Mg ATPase.. E x p e r i m e n t s i n v o l v i n g c a l m o d u l i n a s s a y s a r e c u r r e n t l y i n p r o g r e s s i n o u r l a b o r a t o r y i n o r d e r t o d e t e r m i n e i f t h e r e i s any d i f f e r e n c e i n b o t h t h e amount and t h e e f f i c a c y o f c a l m o d u l i n p r e s e n t i n t h e e r y t h r o c y t e s o f t h e SHR when c o m p a r e d t o t h e WKY's., The p o s s i b l e b a s i s o f t h e a l t e r e d c a l c i u m p e r m e a b i l i t y i n SHR membranes was i n v e s t i g a t e d f u r t h e r . Our f i n d i n g s o f d e c r e a s e d o s m o t i c f r a g i l i t y and a l t e r e d p h o s p h o l i p i d c o m p o s i t i o n ( s e e R e s u l t s s e c . I'D, E) t o g e t h e r w i t h t h e e a r l i e r r e s u l t s i n d i c a t e d c l e a r l y t h a t t h e SHR membrane d i f f e r s i n f u n c t i o n and s t r u c t u r e f r o m t h e WKY membrane. I t seems p o s s i b l e t h a t t h e s e a l t e r a -t i o n s i n t h e SHR membrane a r e r e s p o n s i b l e f o r t h e i n c r e a s e d c a l c i u m p e r m e a -b i l i t y o b s e r v e d , s i n c e t h e r e i s ample d o c u m e n t a t i o n i n t h e l i t e r a t u r e t h a t a l t e r a t i o n s o f p h o s p h o l i p i d c o m p o s i t i o n c a n r e s u l t s i n p e r m e a b i l i t y c h a n g e s i n l i p i d membranes ( 1 7 4 , 1 7 5 ) . - 98 -I I I . S t u d i e s on E r y t h r o c y t e Membranes f r o m D O C A / s a l t H y p e r t e n s i v e R a t s A s e c o n d model o f h y p e r t e n s i o n , t h e D O C A / s a l t h y p e r t e n s i v e r a t (DOCA), was s t u d i e d u s i n g a s i m i l a r p r o t o c o l t o t h a t u s e d i n t h e SHR/WKY e x p e r i m e n t s . Our . p r e v i o u s e x p e r i e n c e w i t h t h i s model o f h y p e r t e n s i o n i n d i c a t e d t h a t t h e v a s c u l a r smooth m u s c l e s i n t h e s e a n i m a l s , u n l i k e t h o s e i n t h e SHR, a r e n o t h y p e r - r e a c t i v e ( 1 7 6 ) . I t i s t h e r e f o r e u n l i k e l y t h a t t h e h y p e r t e n s i o n o b s e r v e d i n t h e DOCA a n i m a l s i s t h e r e s u l t o f a g e n e r a l i z e d a l t e r a t i o n i n v a s c u l a r smooth m u s c l e r e s p o n s i v e n e s s . S e v e r a l g r o u p s h a v e p o s t u l a t e d t h a t t h e s o d i u m r e t e n t i o n c a u s e d by e x c e s s s a l t i n t a k e a n d N m i n e r a l o c o r t i c o i d t r e a t m e n t i s t h e p r i m a r y m e c h a n i s m r e s p o n s i b l e f o r b l o o d p r e s s u r e e l e v a t i o n i n t h i s model ( 1 7 7 ) . T h i s h y p o t h e s i s i s s u p p o r t e d b y t h e f a c t t h a t i n c r e a s e s i n e x t r a c e l l u l a r f l u i d v o l u m e , b l o o d v o l u m e and c a r d i a c o u t p u t a r e o b s e r v e d a t t h e o n s e t o f h y p e r t e n s i o n i n t h i s model ( 1 7 8 , 1 7 9 ) . T h i s d i f f e r e n c e i n t h e m e c h a n i s m o f b l o o d p r e s s u r e e l e v a t i o n p r o v i d e s a n o n - g e n e t i c h y p e r t e n s i v e model f o r m e m b r a n e o l o g i c a l s t u d i e s and a l l o w s c o m p a r i s o n o f t h e r e s u l t s t o t h o s e o f t h e SHR, as w e l l as a means o f a s s e s s i n g t h e p o s s i b l e e f f e c t s o f e l e v a t e d b l o o d p r e s s u r e on t h e b i o c h e m i c a l a l t e r a t i o n s o b s e r v e d i n t h e SHR. L The membrane bound enzyme a c t i v i t i e s o f t h e DOCA r a t s and t h e , i r n e p h r e c -t o m i z e d c o n t r o l s (NC) were s t u d i e d and no d i f f e r e n c e i n t h e s p e c i f i c a c t i v i -t i e s o f Ca /Mg A T P a s e and a c e t y l c h o l i n e s t e r a s e b e t w e e n t h e two g r o u p s o f a n i m a l s was d e t e c t e d ( s e e R e s u l t s s e c . I I B ) . • T h e N a + / K + A T P a s e a c t i v i t y was m a r g i n a l l y h i g h e r i n t h e DOCA a n i m a l s ( s e e T a b l e 8 ) . T h i s i s c o n s i s t e n t w i t h t h e p r o p o s e d a l t e r a t i o n s i n s o d i u m m e t a b o l i s m i m p l i c a t e d i n D O C A / s a l t h y p e r t e n s i o n ( 1 8 0 , 1 8 1 ) , and c o u l d be t h e r e s u l t o f i n c r e a s e d i n t r a c e l l u l a r s o d i u m s e c o n d a r y t o s o d i u m r e t e n t i o n ( 1 8 1 ) . T h e i n c r e a s e d N a + / K + A T P a s e a c t i v i t y c o u l d be an a d a p t i v e c h a n g e o f t h e c e l l s i n an - 99 -a t t e m p t t o e x t r u d e more s o d i u m . O t h e r f u n c t i o n a l s t u d i e s , s u c h as p a s s i v e c a l c i u m i n f l u x and o s m o t i c f r a g i l i t y , d i d n o t r e v e a l any m a j o r d i f f e r e n c e b e t w e e n t h e DOCA and NC a n i m a l s . C o n t r a r y t o what was o b s e r v e d i n t h e SHR, t h e r e d c e l l s f r o m t h e DOCA h y p e r t e n s i v e a n i m a l s a c t u a l l y had a m a r g i n a l l y i n c r e a s e d f r a g i l i t y when compared- t o c o n t r o l s . C o m p o s i t i o n a l a n a l y s e s o f t h e membrane a g a i n r e v e a l e d no g r o s s d i f f e r e n c e s when c o m p a r e d t o t h e c o n t r o l s ( s e e R e s u l t s s e c . V E ) , l e n d i n g s u p p o r t t o t h e h y p o t h e s i s t h a t h y p e r t e n s i o n i n t h e DOCA a n i m a l s i s n o t a s s o c i a t e d w i t h any g r o s s membrano-l o g i c a l c h a n g e s i n t h e r e d c e l l . I V . C o m p a r i s o n o f t h e Two M o d e l s o f H y p e r t e n s i o n . I t i s o b v i o u s f r o m t h e f o r e g o i n g d i s c u s s i o n t h a t t h e e r y t h r o c y t e membrane f r o m t h e SHR i s s t r u c t u r a l l y and f u n c t i o n a l l y d i f f e r e n t f r o m t h a t o f t h e WKY. . T h e s e d i f f e r e n c e s may r e s u l t i n t h e i n c r e a s e d p a s s i v e c a l c i u m p e r m e a -b i l i t y o b s e r v e d i n t h e membrane o f t h e SHR. To c o u n t e r t h i s e l e v a t i o n o f i n t r a c e l l u l a r c a l c i u m , t h e c a l c i u m pump ( C a + + / M g + + A T P a s e ) may be s t i m u -l a t e d t o e f f e c t h i g h e r r a t e o f c a l c i u m e x t r u s i o n f r o m i n s i d e t h e c e l l ( t h e m e c h a n i s m by w h i c h t h e c a l c i u m pump i s s t i m u l a t e d i s s t i l l u n d e r i n v e s t i g a -t i o n ) . S i n c e h i g h l e v e l s o f i n t r a c e l l u l a r c a l c i u m a r e d a m a g i n g t o t h e r e d c e l l ( 1 8 2 ) , an i n c r e a s e i n i t s e x t r u s i o n r a t e i s o f e v o l u t i o n a r y i m p o r t a n c e t o t h e SHR. Our f i n d i n g t h a t none o f t h e a b n o r m a l i t i e s f o u n d i n t h e SHR membrane were o b s e r v e d i n t h e DOCA membranes l e n d s s u p p o r t t o t h e h y p o t h e s i s t h a t h i g h a r t e r i a l p r e s s u r e a l o n e d i d n o t c a u s e t h e a l t e r a t i o n s s e e n , and t h a t a g e n e t i c d e f e c t i s t h e most l i k e l y e x p l a n a t i o n f o r t h e a b n o r m a l i t i e s s e e n i n SHR membranes. - 100 -T h e r e h a v e b e e n numerous r e p o r t s o f o t h e r a b n o r m a l i t i e s i n t h e h a n d l i n g o f i o n s b y e r y t h r o c y t e s i n h y p e r t e n s i o n . M e y e r ' s g r o u p r e p o r t e d a r e d u c t i o n i n n e t N a + e x t r u s i o n i n e r y t h r o c y t e s o f t h e SHR and s e v e r a l o t h e r s t r a i n s o f h y p e r t e n s i v e r a t s , a r g u i n g t h a t i n t h e m a t u r e a n i m a l , t h i s i s a r e f l e c t i o n o f a r e d u c t i o n i n s o d i u m pump a c t i v i t y ( 1 8 3 ) . T h i s same g r o u p a l s o r e p o r t e d a r e d u c t i o n i n t h e n e t s o d i u m e f f l u x f r o m e r y t h r o c y t e s o f h y p e r t e n s i v e p a t i e n t s and t h e i r i m m e d i a t e r e l a t i v e s ( 1 8 4 ) . T h e y s u g g e s t e d t h a t a d e f e c -t i v e s o d i u m / p o t a s s i u m c o - t r a n s p o r t s y s t e m i s r e s p o n s i b l e f o r t h e s o d i u m l o a d i n g o b s e r v e d and p o s t u l a t e d t h a t t h i s t r a i t i s i n h e r i t e d t h r o u g h a s i n g l e g e n e by an a u t o s o m a l d o r m i n a n t mode ( 1 8 4 ) . T h e y a l s o s u g g e s t e d t h a t t h e r e d u c e d s o d i u m p o t a s s i u m c o - t r a n s p o r t phenomenon c o u l d be u s e d as a g e n e t i c m a r k e r f o r i d e n t i f y i n g p a t i e n t s who a r e a t r i s k f o r d e v e l o p i n g h y p e r t e n s i o n . . T h e s e v i e w s , h o w e v e r , a r e n o t s h a r e d by a n o t h e r g r o u p o f w o r k e r s who d i d f i n d l o w e r c o - t r a n s p o r t a c t i v i t y i n h y p e r t e n s i o n b u t w i t h t r e m e n d o u s o v e r l a p w i t h t h e n o r m o t e n s i v e v a l u e s ( 1 8 5 ) , t h e r e f o r e , t h e y c o n c l u d e d t h a t c o - t r a n s p o r t a c t i v i t y d o e s n o t seem t o be a u s e f u l g e n e t i c m a r k e r f o r h y p e r t e n s i o n . D i s a g r e e m e n t s s u c h as t h i s a r e p l e n t i f u l . M e y e r ' s g r o u p f o r e x a m p l e , as m e n t i o n e d e a r l i e r , f o u n d l o w e r Na /K A T P a s e a c t i v i t y i n t h e e r y t h r o c y t e membrane o f t h e SHR ( 1 8 6 ) , b u t B e r g l u n d ' s g r o u p r e p o r t e d t h a t no s i g n i f i c a n t d i f f e r e n c e i n t h i s enzyme a c t i v i t y c o u l d be d e t e c t e d ( 1 8 7 ) . Wood's g r o u p r e p o r t e d , u s i n g A T P - d e p e n d e n t R u b i d i u m - 8 6 u p t a k e , t h a t t h e s o d i u m pump i s more a c t i v e i n t h e e r y t h r o c y t e s o f h y p e r t e n -s i v e p a t i e n t s ( 1 8 8 ) . Our own r e s u l t s t e n d t o s u p p o r t B e r g l u n d ' s c o n c l u s i o n t h a t N a + / K + A T P a s e a c t i v i t y i s n o t a l t e r e d i n t h e e r y t h r o c y t e o f t h e SHR. T h e d i f f e r e n c e b e t w e e n t h e r e s u l t s o b t a i n e d b y t h e v a r i o u s l a b o r a t o r i e s i s p r o b a b l y a r e f l e c t i o n o f t h e d i f f e r e n t m e t h o d o l o g y u s e d i n t h e enzyme - 101 -a s s a y s . T h e r e i s t h e o t h e r p o s s i b i l i t y t h a t t h e a n i m a l s u s e d b y t h e v a r i o u s g r o u p s , a r e g e n e t i c a l l y d i f f e r e n t s i n c e t h e r e i s e v i d e n c e o f p h e n o t y p i c d i s s i m i l a r i t i e s b e t w e e n t h e SHRs p u r c h a s e d f r o m d i f f e r e n t s u p p l i e r s ( 1 8 9 ) . Our d e m o n s t r a t i o n t h a t t h e r e a r e s t r u c t u r a l and f u n c t i o n a l a b n o r m a l i t i e s i n t h e e r y t h r o c y t e membranes o f t h e SHR i s s u p p o r t e d by M e y e r ' s g r o u p i n t h e i r f l u o r e s c e n c e p o l a r i z a t i o n s t u d y u s i n g d i p h e n y l h e x t r i e n e (DPH) as a p r o b e ( 1 9 0 ) . T h e i n c r e a s e d f l u o r e s c e n c e p o l a r i z a t i o n o b s e r v e d i n v e r y y o u n g SHRs ( b e f o r e b l o o d p r e s s u r e e l e v a t i o n ) as w e l l as i n a d u l t r a t s s u p p o r t e d t h e h y p o t h e s i s t h a t t h e membrane a l t e r a t i o n s a r e p r o b a b l y g e n e t i c a l l y d e t e r -m i n e d and n o t a c o n s e q u e n c e o f e l e v a t e d b l o o d p r e s s u r e . Our c o n c l u s i o n t h a t t h e r e a r e a b n o r m a l i t i e s i n c a l c i u m h a n d l i n g i n t h e SHR e r y t h r o c y t e s i s p a r t i a l l y s u p p o r t e d b y M e y e r ' s g r o u p who r e p o r t e d i n c r e a s e d p a s s i v e c a l c i u m i n f l u x i n t o A T P - d e p l e t e d r e d c e l l s f r o m SHRs. T h e y a l s o o b s e r v e d a r e d u c t i o n i n c a l c i u m b i n d i n g s i t e s i n t h e SHR membranes. C o n t r a r y t o o u r r e s u l t s , t h e y f o u n d t h e Ca /Mg A T P a s e a c t i v i t y t o be t h e same i n t h e membranes f r o m b o t h g r o u p s o f a n i m a l s ( 1 9 1 ) . O d d l y e n o u g h , t h e A T P - d e p e n d e n t C a + + u p t a k e i n t o i n s i d e - o u t v e s i c l e s ( a n o t h e r e s t i m a t i o n o f t h e c a l c i u m pump a c t i v i t y ) was f o u n d t o be l o w e r i n t h e i r SHR s a m p l e s , and no a t t e m p t s were made by t h e a u t h o r s t o e x p l a i n t h i s a p p a r e n t d i s p a r i t y i n t h e i r r e s u l t s . On c l o s e r e x a m i n a t i o n o f t h e i r d a t a , i t i s e v i d e n t t h a t t h e r a t e o f t h e c a l c i u m d e p e n d e n t ATP h y d r o l y s i s i s q u i t e s l o w , a b o u t o n e - h a l f o f o u r v a l u e s . T h i s may be an i n d i c a t i o n o f membrane a n d / o r enzyme damage s u s t a i n e d d u r i n g t h e p r e p a r a t i v e p h a s e , w h i c h i s a p o s s i b i l i t y i n v i e w o f t h e i r p r e p a r a t i v e p r o c e d u r e s ( S t e c k and K a n t ; 1 9 2 ) . T h e m o d i f i e d s t e p - w i s e h a e m o l y s i s p r o c e d u r e we e m p l o y e d i n p r e p a r i n g t h e e r y t h r o c y t e membrane i s m i l d e r t h a n o t h e r , s t a n d a r d p r o c e d u r e s ( 1 4 2 ) . T h e r e s u l t s f r o m M e y e r ' s g r o u p on t h e A T P - d e p e n d e n t C a + + u p t a k e i n t o i n s i d e - o u t v e s i c l e s s h o u l d a l s o be t a k e n - 102 -w i t h c a u t i o n s i n c e t h e c a l c i u m pump i n t h e v e s i c l e s was n o t s t u d i e d , i n i t s n a t i v e s t a t e , and t h e h i g h e r p a s s i v e p e r m e a b i l i t y o f t h e SHR membrane t o c a l c i u m c a n r e s u l t i n a p a s s i v e l e a k o f t h e a c c u m u l a t e d c a l c i u m b a c k • o u t i n t o t h e medium, t h e r e b y p r o d u c i n g an a p p a r e n t d e c r e a s e i n m e a s u r e d t r a n s p o r t a c t i v i t y . Our f i n d i n g s c o n c e r n i n g t h e SHR e r y t h r o c y t e c a l c i u m pump and i t s r e l a t i o n t o h y p e r t e n s i o n a r e s t r e n g t h e n e d by d a t a f r o m G o d f r a i n d ' s g r o u p who r e c e n t l y d e m o n s t r a t e d - t h e e x i s t e n c e o f a c a l m o d u l i n - s t i m u l a t e d c a l c i u m pump i n t h e p l a s m a membrane o f t h e r a t a o r t a s i m i l a r t o t h a t o f t h e e r y t h r o c y t e ' s ( 1 9 3 ) . D a n i e l ' s g r o u p have s u c c e s s f u l l y i s o l a t e d and c h a r a c t e r i z e d a v i a b l e p l a s m a membrane p r e p a r a t i o n f r o m t h e m e s e n t e r i c a r t e r i e s o f SHRs and WKYs. T h e y r e p o r t e d e n h a n c e d a l k a l i n e p h o s p h a t a s e a c t i v i t y as w e l l as a d e f e c t i v e " c a l c i u m r e g u l a t o r y m e c h a n i s m " i n t h e p l a s m a membrane o f t h e SHR ( 1 9 4 - 1 9 6 ) . T h e i r r e s u l t s l e n d s u p p o r t t o o u r h y p o t h e s i s t h a t s i m i l a r membrane m e c h a n i s m s a r e o p e r a t i v e i n t h e v a s c u l a r smooth m u s c l e and t h e e r y t h r o c y t e o f t h e SHR. F u r t h e r m o r e , G i m b l e and h i s c o - w o r k e r s c o m p a r e d t h e Ca /Mg A T P a s e s and c a l c i u m t r a n s p o r t i n b o t h r a t and human e r y t h r o c y t e s and c o n c l u d e d t h a t t h e p r o p e r t i e s o f t h e two s y s t e m s a r e d i r e c t l y c o m p a r a b l e b e t w e e n t h e two s p e c i e s ( 1 9 7 ) . T h e r e f o r e o u r o b s e r v a t i o n s i n t h e r a t e r y t h r o c y t e s may w e l l be r e l e v a n t t o human c e l l s as w e l l . F u t u r e s t u d i e s on e r y t h r o c y t e s o b t a i n e d f r o m u n t r e a t e d e s s e n t i a l h y p e r t e n s i v e p a t i e n t s may p r o v i d e f u r t h e r s u p p o r t f o r t h e h y p o t h e s i s t h a t g e n e t i c a l l y d e t e r m i n e d membrane a l t e r a t i o n s a r e e t i o l o g i c a l l y r e l a t e d t o h y p e r t e n s i o n . I £ p a r a l l e l a l t e r a t i o n s a r e s e e n i n p a t i e n t s , t h e e r y t h r o c y t e membrane may p r o v i d e a c o n v e n i e n t and r e l i a b l e m a r k e r f o r i d e n t i f y i n g t h o s e a t r i s k o f d e v e l o p i n g h y p e r t e n s i o n and a l l o w m e d i c a l m o n i t o r i n g o r t r e a t m e n t a t an e a r l i e r s t a g e . I f i n c r e a s e d c a l c i u m p e r m e a b i l i t y i s i n d e e d i n v o l v e d i n h y p e r t e n s i o n , t h e n a g e n t s t h a t c a n r e d u c e - 103 -t h e i n f l u x o r i n c r e a s e t h e e x t r u s i o n o f c a l c i u m may p r o v e t o be t h e a n t i h y -p e r t e n s i v e d r u g o f t h e f u t u r e . T h e g r o u p o f a n t i - a n g i n a l d r u g s known as t h e " c a l c i u m a n t a g o n i s t s " i s now f o u n d t o a l s o h a v e a n t i h y p e r t e n s i v e p r o p e r t i e s . V e r a p a m i l , f o r e x a m p l e , d e m o n s t r a t e d s u b s t a n t i a l l y g r e a t e r l o c a l v a s o d i l a t o r e f f e c t i n p a t i e n t s w i t h e s s e n t i a l h y p e r t e n s i o n t h a n i n n o r m o t e n s i v e s u b j e c t s c o m p a r e d t o n i t r o p r u s s i d e w h i c h d i d n o t h a v e d i f f e r e n t i a l e f f e c t s b e t w e e n t h e two g r o u p s o f p a t i e n t s ( 2 4 9 ) . T h i s f i n d i n g i s c o n s i s t e n t w i t h t h e h y p o -t h e s i s t h a t i n c r e a s e d c a l c i u m i n f l u x i s e t i o l o g i c a l l y r e l a t e d t o e s s e n t i a l h y p e r t e n s i o n . V. A l c o h o l and C a r d i o v a s c u l a r D i s e a s e P h y s i c i a n s i n t h e 1 9 t h c e n t u r y were t a u g h t i n m e d i c a l s c h o o l s t h a t " t o o much a l c o h o l i s bad f o r t h e h e a r t as w e l l as f o r t h e l i v e r " . S t e e l l s t a t e d i n 1892 t h a t a l c o h o l was a c a u s e f o r " m u s c l e f a i l u r e o f t h e h e a r t " ( 2 0 1 ) . O s i e r (202) and P r i c e ( 2 0 3) b o t h w r o t e i n t h e i r b o o k s t h a t a l c o h o l c a u s e d d e g e n e r a t i v e c h a n g e s i n t h e h e a r t and a r t e r i e s . T h e s e f i n d i n g s , h o w e v e r , f a d e d i n t o t h e b a c k g r o u n d when Wenckebach d e s c r i b e d c a r d i a c b e r i - b e r i i n a l c o h o l i c s who were m a l n o u r i s h e d ( 2 0 4 ) , and t h i a m i n e d e f i c i e n c y was t h e n t h o u g h t t o be t h e s o l e c a u s e o f c a r d i o v a s c u l a r d i s e a s e s i n a l c o h o l i c s . I t was n o t u n t i l t h e e a r l y s i x t i e s t h a t t h e d i r e c t e f f e c t o f a l c o h o l on t h e c a r d i o v a s c u l a r s y s t e m was r e d i s c o v e r e d ( 2 0 5 , 2 0 6 ) . E a r l i e r r e s e a r c h e f f o r t s i n t o t h e e f f e c t o f a l c o h o l c o n s u m p t i o n h a b i t s on c a r d i o v a s c u l a r f u n c t i o n s f a i l e d t o p r o v i d e much i n s i g h t b e c a u s e i n v e s t i g a t o r s had t r i e d t o l a b e l a l c o h o l as b e i n g "good" o r " b a d " f o r t h e h e a r t i n g e n e r a l . More r e c e n t l y , a t t e m p t s were b e t t e r f o c u s e d and c h r o n i c a l c o h o l c o n s u m p t i o n i n man has been - 104 -f o u n d t o be a s s o c i a t e d w i t h numerous a b n o r m a l i t i e s s u c h as c a r d i a c f i b r o s i s ( 2 0 7 ) , c o r o n a r y c a p i l l a r y d i s o r d e r s ( 2 0 8 ) , s u b c e l l u l a r a b e r r a t i o n s ( 2 0 9 ) , r e d u c e d c a r d i a c c o n t r a c t i l i t y ( 2 1 0 ) , a b n o r m a l v e n t r i c u l a r f u n c t i o n ( 2 1 1 ) , and many ' o t h e r s . One o f t h e e a r l i e s t p i e c e s o f e v i d e n c e a s s o c i a t i n g a l c o h o l c o n s u m p t i o n ' w i t h h y p e r t e n s i o n was s u p p l i e d by L i a n i n 1 9 1 5 . " He n o t i c e d an i n c r e a s e i n t h e p r e v a l e n c e o f h y p e r t e n s i o n among F r e n c h s e r v i c e m e n who d r a n k more t h a n 2.5 l i t e r s o f w i n e p e r d a y ( 2 1 2 ) . T h e s u b s e q u e n t y e a r s were f i l l e d w i t h s t u d i e s c l a i m i n g a l c o h o l t o be a s s o c i a t e d w i t h e i t h e r h y p e r t e n -s i o n ( 1 4 0 , 2 1 3 ) o r h y p o t e n s i o n ( 2 1 4 , 2 1 5 ) . T h e r e a s o n f o r s u c h d i s p a r i t y o f r e s u l t s i s p r o b a b l y a l a c k o f u n d e r s t a n d i n g f o r t h e o t h e r c o n f o u n d i n g f a c t o r s t h a t c a n i n f l u e n c e b l o o d p r e s s u r e i n t h e s u b j e c t s , as w e l l as b i a s e d s u b j e c t p o p u l a t i o n s b e i n g c h o s e n f o r t h e s t u d i e s . I n t h e l a t e s i x t i e s , r e s u l t s f r o m b e t t e r d e s i g n e d s t u d i e s became a v a i l a b l e , b u t t h e c o n t r o v e r s i e s r e m a i n e d . T h e r e were many g r o u p s c l a i m i n g p o s i t i v e c o r r e l a t i o n s b e t w e e n a l c o h o l i s m and h y p e r t e n s i o n ( 2 1 6 - 2 1 9 ) , b u t j u s t as many c l a i m e d no c o r r e l a -t i o n was d e t e c t a b l e ( 2 2 0 - 2 2 2 ) . The m a j o r p r o b l e m c o n f r o n t i n g t h e r e s e a r c h e r s i s t h a t v a r i a b l e s s u c h as s t r e s s , n u t r i t i o n a l s t a t u s , s m o k i n g h a b i t s , g e n e t i c d e t e r m i n a n t s , and q u a n t i t a t i o n o f e t h a n o l c o n s u m p t i o n a r e e x t r e m e l y d i f f i c u l t t o c o n t r o l ( o r m o n i t o r ) s i m u l t a n e o u s l y i n t h e p a t i e n t s . T h e r e i s a l s o t h e p r o b l e m o f e t h i c a l c o n s i d e r a t i o n w h e r e b y many o f t h e more d e f i n i t i v e e x p e r i -m e nts c a n n o t be r e a d i l y p e r f o r m e d on humans. T h e s e l i m i t a t i o n s t h e r e f o r e make a n i m a l m o d e l s o f a l c o h o l i s m an a t t r a c t i v e a l t e r n a t i v e t o human e x p e r i -m e nts i n a l c o h o l r e s e a r c h . - 105 -V I . A n i m a l M o d e l s o f A l c o h o l i s m A n i m a l s m o d e l s o f a l c o h o l i s m h a v e s t e a d i l y g a i n e d i m p o r t a n c e i n many a r e a s o f a l c o h o l r e s e a r c h . T h e u s e o f r o d e n t s i n a l c o h o l f e e d i n g e x p e r i m e n t s h a s a l o n g - s t a n d i n g h i s t o r y , b u t t h e e a r l i e r m o d e l s f a i l e d e i t h e r t o p r o d u c e m e a s u r e a b l e b l o o d e t h a n o l c o n c e n t r a t i o n s o r t o i n d u c e s t a t e s o f a b s o l u t e p h y s i c a l d e p e n d e n c e ( 1 9 7 , 1 9 8 ) . A r e c e n t s u r v e y r e v e a l e d t h a t l e s s t h a n o n e - t h i r d o f a l l t h e p u b l i c a t i o n s on r o d e n t m o d e l s r e p o r t e d b l o o d e t h a n o l c o n c e n t r a t i o n s i n t h e i r a n i m a l s , and t h o s e who d i d i n c l u d e t h i s i n f o r m a t i o n g a v e q u e s t i o n a b l y h i g h v a l u e s ( 1 9 9 ) . C u r r e n t a n i m a l m o d e l s o f a l c o h o l i s m g e n e r a l l y f a l l i n t o one o f t h e t h r e e g e n e r a l t y p e s : i n v o l u n t a r y , s e m i - v o l u n t a r y , and v o l u n t a r y . In t h e v o l u n t a r y m o d e l s , a n i m a l s a r e a l l o w e d t o s e l e c t b e t w e e n e t h a n o l and an a l t e r n a t i v e ( u s u a l l y w a t e r ) , and t h e e t h a n o l i s consumed e i t h e r o r a l l y o r i n f u s e d , i n t r a v e n o u s l y o r i n t r a g a s t r i -c a l l y . T h i s model i s g e n e r a l l y u s e d i n p y c h o l o g i c a l and b e h a v i o u r a l e x p e r i -m e n t s . I n t h e i n v o l u n t a r y m o d e l s , t h e r e s e a r c h e r has t o t a l c o n t r o l o f b o t h t h e d o s e and t h e s c h e d u l e o f e t h a n o l a d m i n s t r a t i o n , and t h e m e t h o d s u s e d i n c l u d e i n h a l a t i o n , o r a l i n t u b a t i o n , i n t r a g a s t r i c o r i n t r a v e n o u s i n f u s i o n s , and s u s t a i n e d - r e l e a s e i m p l a n t s . T h e s e m i - v o l u n t a r y model i s s u c h t h a t t h e a n i m a l s consume e t h a n o l t h r o u g h e i t h e r t h e d a i l y f l u i d o r f o o d s u p p l y a t t h e i r own p a c e . The i n v o l u n t a r y and s e m i - v o l u n t a r y m o d e l s a r e i d e a l f o r p h a r m a c o l o g i c a l s t u d i e s w h e r e d e m o n s t r a t i o n o f p s y c h o l o g i c a l d e p e n d e n c e i s n o t r e q u i r e d ( 1 9 0 ) . T h e p h a r m a c o l o g i c a l model we e m p l o y e d i n o u r s t u d i e s f a l l s i n t o t h e s e m i - v o l u n t a r y c a t e g o r y . T h e a n i m a l s we s t u d i e d had a peak b l o o d e t h a n o l l e v e l o f 86.0 ± 10.0 mg/dL a t t h e 4 t h week w h i c h t a p e r e d o f f t o 26.9 ± 5.5 mg/dL a t t h e end o f ,the 1 2 t h week, s u g g e s t i n g enzyme i n d u c t i o n . C o n t r a r y t o what i s known i n - 106 -humans, r a t enzymes c a n be i n d u c e d by e t h a n o l so t h a t i t i s m e t a b o l i z e d f o u r t i m e s f a s t e r t h a t t h e i n i t i a l r a t e ( I s r e a l , Y., p e r s o n a l c o m m u n i c a t i o n ) . The a d v a n t a g e t o t h e f e e d i n g method we u s e d i s i t s s i m p l e and i n e x p e n s i v e a d m i n i s t r a t i o n , m a k i n g t h e t r e a t m e n t o f l a r g e g r o u p s o f a n i m a l s p o s s i b l e . The m i n i m a l m a n i p u l a t i o n o f t h e a n i m a l s a l s o r e d u c e s v a r i a b l e s s u c h as s t r e s s and t h e r e s u l t i n g n u t r i t i o n a l i m b a l a n c e s a s s o c i a t e d w i t h o t h e r m o d e l s ( e . g . , f o r c e d f e e d i n g ) . V I I . C h r o n i c E t h a n o l C o n s u m p t i o n and C a r d i o v a s c u l a r F u n c t i o n i n t h e R a t The r e p e a t e d b l o o d p r e s s u r e m e a s u r e m e n t s t a k e n o v e r t h e 12-week p e r i o d i n o u r a l c o h o l model g i v e e v i d e n c e t h a t a s m a l l b u t c o n s i s t e n t e l e v a t i o n i n t h e s y s t o l i c p r e s s u r e i s a l w a y s d e t e c t e d i n t h e a l c o h o l - t r e a t e d g r o u p o f a n i m a l s . T h e mean s y s t o l i c p r e s s u r e r e p o r t e d d o e s n o t c o n v e y t h e f u l l p i c t u r e b e c a u s e i n a g r o u p o f 12 r a t s , t h e r e were a l w a y s 2 t o 3 a n i m a l s t h a t d i d n o t show any e l e v a t i o n i n b l o o d p r e s s u r e t h r o u g h o u t t h e t r e a t m e n t p e r i o d , t h e r e b y r e d u c i n g t h e g r o u p ' s mean v a l u e . T h e s e f i n d i n g s a r e q u i t e c o n s i s t e n t w i t h r e s u l t s f r o m human s t u d i e s w h e r e b y 1 0 % t o 1 5 % o f t h e s u b j e c t s i n a p o p u l a t i o n d i d n o t show b l o o d p r e s s u r e e l e v a t i o n w i t h e v e n " h e a v y " c h r o n i c a l c o h o l c o n s u m p t i o n ( 2 1 7 , 2 2 3 ) . A n o t h e r i n t e r e s t i n g o b s e r v a -t i o n i s t h a t d u r i n g one o f t h e p r e l i m i n a r y e x p e r i m e n t s where f e m a l e r a t s w ere u s e d i n s t e a d o f m a l e s , o n l y one a n i m a l i n t h e g r o u p o f 10 c o n s i s t e n t l y showed a 2 0 % e l e v a t i o n i n b l o o d p r e s s u r e f r o m week 5 t o week 12. T h i s s e x - r e l a t e d d i f f e r e n c e i n t h e b l o o d p r e s s u r e r e s p o n s e t o c h r o n i c a l c o h o l c o n s u m p t i o n i s a l s o e v i d e n t i n some c l i n i c a l s t u d i e s w here b l o o d p r e s s u r e i s f o u n d t o be i n d e p e n d e n t o f e t h a n o l c o n s u m p t i o n i n f e m a l e s ( 2 1 7 , 2 2 4 ) . - 107 -T h e c a r d i a c p e r f o r m a n c e o f t h e e t h a n o l - t r e a t e d and c o n t r o l a n i m a l s was a s s e s s e d i n v i t r o u s i n g a m o d i f i e d L a n g e n d o r f f i s o l a t e d p e r f u s e d h e a r t p r e p a r a t i o n . As m e n t i o n e d e a r l i e r i n t h e R e s u l t s s e c t i o n ( I I I C ) , t h e r e i s e v i d e n c e f o r v e n t r i c u l a r h y p e r t r o p h y i n t h e a l c o h o l - t r e a t e d a n i m a l s . A p a r a l l e l c o n d i t i o n i s f o u n d i n humans. ' One o f t h e e a r l i e s t d o c u m e n t a t i o n s o f s u c h a l c o h o l i c c a r d i o m e g a l y was by B o l l i n g e r i n 1884, and he c a l l e d i t t h e " M u n i c h B e e r H e a r t Syndrome" ( 2 2 5 ) . I t i s p o s s i b l e t h a t t h e v e n t r i c u l a r h y p e r t r o p h y i n o u r e t h a n o l - t r e a t e d a n i m a l s i s t h e r e s u l t o f i n c r e a s e d c a r d i a c work due t o t h e e l e v a t e d a r t e r i a l p r e s s u r e and i n c r e a s e d p l a s m a v o l u m e a s s o c i a t e d w i t h t h e e t h a n o l c o n s u m p t i o n . The r e d u c t i o n i n mean r e s t i n g h e a r t r a t e i n t h e a l c o h o l i c r a t s b o t h i n v i v o and i n v i t r o i s v e r y l i k e l y a r e s u l t o f c o n d u c t i o n a b n o r m a l i t i e s i n t h e h e a r t s o f t h e s e a n i m a l s as e v i d e n t f r o m t h e p r o l o n g e d P-R i n t e r v a l s ( s e e R e s u l t s s e c . H I E ) . T h e s e f i n d i n g s a r e i n a g r e e m e n t w i t h r e s u l t s f r o m a l c o h o l i c p a t i e n t s ( 1 3 3 ) and a l c o h o l i c d o gs (226) w h e r e t h e e l e c t r i c a l c o n d u c t i o n i n t h e h e a r t was f o u n d t o be i m p a i r e d . S t a r l i n g c u r v e s f r o m t h e two g r o u p s o f h e a r t s were s i m i l a r when t h e h e a r t s were a l l o w e d t o b e a t a t t h e i r s p o n t a n e o u s r a t e s . T h e s l i g h t l y h i g h e r optimum E D P V ( F i g . 11) may r e f l e c t t h e e f f e c t o f v e n t r i c u l a r h y p e r t r o p h y on t h e c o n t r a c t i l i t y (more p r e s s u r e i s r e q u i r e d now t o s t r e t c h t h e m u s c l e t o d e g r e e t h e same f o r c e ) o f t h e v e n t r i c l e s 'from e t h a n o l - t r e a t e d a n i m a l s . E l e c t r i c a l p a c i n g o f t h e h e a r t s f r o m b o t h g r o u p s d r o p p e d t h e maximum IVP and optimum EDP. T h e r e i s , h o w e v e r , a g r e a t e r r e d u c t i o n o f peak IVP and o p t i m a l EDP i n t h e a l c o h o l - t r e a t e d g r o u p when c o m p a r e d t o c o n t r o l s ( s e e F i g . 11 and T a b l e 1 3 ) . The m e c h a n i s m r e s p o n s i b l e f o r t h e s e a l t e r a t i o n s i s unknown, b u t one c a n p o s t u l a t e t h a t t h e c o n d u c t i o n a b n o r m a l i t y i n t h e h e a r t s o f t h e a l c o -h o l - t r e a t e d a n i m a l s m e n t i o n e d e a r l i e r may p l a y a r o l e i n r e d u c i n g c a r d i a c - 108 -c o n t r a c t i l i t y u n d e r s t r e s s f u l c o n d i t i o n s by a l t e r i n g t h e " f r e q u e n c y - c o n t r a c -t i 1 t y " r e l a t i o n s h i p i n t h e h e a r t s . T h e d i r e c t e f f e c t o f c h r o n i c e t h a n o l c o n s u m p t i o n on r e d u c i n g t h e v e n t r i c u l a r c o n t r a c t i l i t y i s a n o t h e r p o s s i b l e c a u s e f o r t h e d i f f e r e n c e s o b s e r v e d b e c a u s e v e n t r i c u l a r f u n c t i o n i s f o u n d t o be d e p r e s s e d by a c u t e ( 2 1 0 ) and c h r o n i c .(211) e t h a n o l i n g e s t i o n i n humans w i t h o u t c o - e x i s t i n g c o n d u c t i o n a b n o r m a l i t i e s . Dose r e s p o n s e c u r v e s o f t h e i s o l a t e d p e r f u s e d h e a r t s t o i s o p r o t e r e n o l (IPNA) r e v e a l e d f u r t h e r d i f f e r e n c e s b e t w e e n t h e h e a r t s f r o m t h e two g r o u p s . The h e a r t s f r o m a l c o h o l - t r e a t e d a n i m a l s p e r f o r m e d p o o r l y a t t h e h i g h e r d o s e s o f IPNA i n t h e p e r f u s a t e c o n t a i n i n g p h y s i o l o g i c a l amounts (2.5 mM) o f C a + + . T h e r e was a 2 0 % d r o p i n t h e maximum IVP d e v e l o p e d by t h e h e a r t s o f c h r o n i c a l l y - t r e a t e d a n i m a l s when c o m p a r e d t o c o n t r o l s . T h i s f u r t h e r s u b s t a n t i a t e s t h e h y p o t h e s i s t h a t h e a r t s f r o m " a l c o h o l i c " a n i m a l s p e r f o r m l e s s w e l l u n d e r s t r e s s f u l c o n d i t i o n s , w h i c h a r e r e p r e s e n t e d h e r e by e l e c t r i c a l s t i m u l a t i o n a t a h i g h e r r a t e and p h a r m a c o l o g i c a l , s t i m u l a t i o n a t h i g h d o s e s o f IPNA. T h e s e o b s e r v a t i o n s a r e i n a g r e e m e n t w i t h t h e r e s u l t s o b t a i n e d by a n o t h e r g r o u p o f w o r k e r s who f o u n d t h a t h e a r t s f r o m c h r o n i c a l c o h o l i c r a t s a r e l e s s r e s p o n s i v e t o d o b u t a m i n e , a p - j - a d r e n e r g i c a g o n i s t ( 2 2 7 ) . More r e c e n t l y , t h i s same g r o u p o f w o r k e r s c h a r a c t e r i z e d t h e B - r e c e p t o r s i n t h e h e a r t s o f c h r o n i c a l c o h o l and c o n t r o l r a t s and f o u n d no d i f f e r e n c e i n t h e d e n s i t y and a g o n i s t a f f i n i t y o f t h e 3 - r e c e p t o r s f r o m c a r d i a c membranes o f e i t h e r g r o u p o f a n i m a l s . T h e y t h e r e f o r e p r o p o s e d a b i o c h e m i c a l a b n o r m a l i t y w i t h i n t h e e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g s y s t e m i n t h e h e a r t s o f c h r o n i c a l c o h o l i c r a t s ( 2 2 8 ) . T h i s i s a v i a b l e e x p l a n a t i o n f o r t h e r e d u c e d c o n t r a c t i l i t y i n a d d i t i o n t o t h e p o s s i b l e a l t e r a t i o n s i n e l e c t r i c a l and c o n t r a c t i l e f u n c t i o n s m e n t i o n e d e a r l i e r . F u r t h e r b i o c h e m i c a l c h a r a c t e r i z a t i o n may p r o v i d e some a n s w e r s t h a t a r e l a c k i n g a t t h e moment. When t h e low c a l c i u m p e r f u s a t e was u s e d (0.2 mM), s e v e r a l i n t e r e s t i n g p h e n o -- 109 -mena were o b s e r v e d . The h e a r t s f r o m t h e a l c o h o l - t r e a t e d a n i m a l s r e t a i n e d a c e r t a i n amount o f s p o n t a n e o u s c o n t r a c t i l e a c t i v i t y w h i l e t h e h e a r t s f r o m c o n t r o l a n i m a l s were q u i e s c e n t . The r e s p o n s e s o f t h e " a l c o h o l i c " h e a r t s w e r e s i g n i f i c a n t l y h i g h e r a t a l l c o n c e n t r a t i o n s o f IPNA when c o m p a r e d t o c o n t r o l h e a r t s ( s e e F i g . 1 3 ) . One p o s s i b l e e x p l a n a t i o n f o r t h i s phenomenon i s t h a t t h e h e a r t s f r o m a l c o h o l - t r e a t e d a n i m a l s e i t h e r u t i l i z e C a + + more e f f i c i e n t l y o r h a v e an i n c r e a s e d a v a i l a b i l i t y o f c a l c i u m t o t h e i r c o n t r a c t i l e a p p a r a t u s t h r o u g h i n c r e a s e d p e r m e a b i l i t y o r i n c r e a s e d i n t r a c e l l u l a r s t o r a g e . The r e s u l t s a r e c o n s i s t e n t w i t h t h e f i n d i n g s o f s e v e r a l g r o u p s o f r e s e a r c h e r s who h a v e r e p o r t e d t h a t c h r o n i c a l c o h o l c o n s u m p t i o n a l t e r s " c a l c i u m t r a n s p o r t " i n t h e h e a r t ( 2 2 7 - 2 3 0 ) . A t t h e p r e s e n t t i m e , t h e a l t e r a t i o n i n c a l c i u m t r a n s p o r t i s deemed more l i k e l y a r e s u l t o f p e r m e a b i l i t y c h a n g e s b e c a u s e t h e r e i s a b u n d a n t e v i d e n c e l i n k i n g a l t e r e d p h o s p h o l i p i d c o m p o s i t i o n and membrane s t r u c t u r e t o a c u t e and c h r o n i c e t h a n o l e x p o s u r e ( 1 3 6 - 1 3 9 ) . F u r t h e r e x p e r i m e n t a t i o n i s i n p r o g r e s s i n o u r l a b o r a t o r y t o t r y t o c h a r a c t e r i z e t h e m y o c a r d i a l p l a s m a membrane and membranes o f t h e m i t o c h o n d r i a and s a r c o l e m m a f r o m t h e h e a r t s o f c o n t r o l and a l c o h o l - t r e a t e d a n i m a l s . T h e s e r e s u l t s may p r o v i d e more i n s i g h t i n t o t h e n a t u r e and m e c h a n i s m o f t h e a l t e r e d c a l c i u m s e n s i t i v i t y o b s e r v e d h e r e . I o n a n a l y s e s on t h e p l a s m a s a m p l e s d i d n o t r e v e a l much i n f o r m a t i o n , , as one w o u l d e x p e c t , b e c a u s e t h e h o m e o s t a t i c m e c h a n i s m s i n t h e body a r e c o n s t a n t l y a d j u s t i n g f o r m i n o r i m b a l a n c e s o f i o n s o r o t h e r c i r c u l a t i n g b l o o d f a c t o r s . T h e u r i n e i o n a n a l y s e s , on t h e o t h e r h a n d , were v e r y i n f o r m a t i v e . T h e r e was an i n c r e a s e i n Ca and Mg e x c r e t i o n by t h e a l c o h o l - t r e a t e d a n i m a l s when c o m p a r e d t o c o n t r o l v a l u e s . S i m i l a r i o n i c a b n o r m a l i t i e s h a v e been d o c u m e n t e d i n humans, and i n c r e a s e d r e n a l e x c r e t i o n o f M g + + was f o u n d t o be a s s o c i a t e d w i t h a l c o h o l - i n d u c e d r e n a l f a i l u r e ( 2 3 3 - 2 3 5 ) . The r e d u c e d - 110 -24 h r u r i n a r y o u t p u t and t h e low u r i n a r y s o d i u m c o n c e n t r a t i o n ( s e e R e s u l t s s e c t i o n 111F) s u g g e s t e d t h a t t h e r e m i g h t be v o l u m e e x p a n s i o n i n t h e a l c o h o l -t r e a t e d a n i m a l s . P l a s m a v o l u m e m e a s u r e m e n t s were t h e r e f o r e u n d e r t a k e n by i n j e c t i n g ^ C - m e t h y l - g a m m a g l o b u l i n s i n t o t h e j u g u l a r v e i n and s a m p l i n g b l o o d f r o m t h e f e m o r a l a r t e r i e s . T h e a d v a n t a g e o f g a m m a - g l o b u l i n s o v e r E v a n s B l u e dye i s t h a t t h e g l o b u l i n s a r e n o t c l e a r e d f r o m t h e c i r c u l a t i o n f o r a t l e a s t s e v e r a l h o u r s , m a k i n g e x t r a p o l a t i o n t o " z e r o " t i m e u n n e c e s s a r y ( 2 3 6 ) . A l s o t h e b l o o d s a m p l e s r e q u i r e d a r e much s m a l l e r , and s p e c t r o p h o t o -m e t r y c o n t a m i n a t i o n by b l o o d c o m p o n e n t s o f t e n s e e n i n E v a n s B l u e p r o c e d u r e s i s n o t a p r o b l e m . I o d i n e 1 2 5 - l a b e l l e d a l b u m i n i s s o m e t i m e s u s e d i n b l o o d v o l u m e m e a s u r e m e n t s b u t a l b u m i n a l s o e n t e r s i n t o c e r t a i n e x t r a v a s c u l a r s p a c e s s u c h as t h e l y m p h a t h i c s y s t e m , r e s u l t i n g i n o v e r e s t i m a t i o n o f p l a s m a v o l u m e . G a m m a - g l o b u l i n s do n o t d i s t r i b u t e i n t o e x t r a v a s c u l a r s p a c e s so t h a t t h e e s t i m a t e d v o l u m e i s c l o s e r t o t h e r e a l p l a s m a v o l u m e ( 2 3 6 ) . Our r e s u l t s showed t h a t t h e r e i s r o u g h l y a 2 4 % e x p a n s i o n i n t h e p l a s m a v o l u m e o f t h e a l c o h o l - t r e a t e d a n i m a l s when comp a r e d t o c o n t r o l s , i m p l i c a t i n g v o l u m e e x p a n -s i o n as one o f t h e m e c h a n i s m s r e s p o n s i b l e f o r t h e e l e v a t e d b l o o d p r e s s u r e o b s e r v e d i n t h e s e a n i m a l s ( s e e R e s u l t s s e c t i o n I V G ) . P a r a l l e l v o l u m e e x p a n -s i o n a s s o c i a t e d w i t h c h r o n i c e t h a n o l c o n s u m p t i o n was r e p o r t e d i n d o g s ( 2 3 7 , 238) and humans ( 2 3 9 , 2 4 0 ) . S i n c e c h r o n i c e t h a n o l c o n s u m p t i o n has been shown t o be a s s o c i a t e d w i t h e l e v a t e d p l a s m a a r g i n i n e v a s o p r e s s i n ( 2 4 0 ) and p l a s m a r e n i n a c t i v i t y ( 2 4 1 ) , t h e s e f a c t o r s may p l a y c o n t r i b u t e t o t h e v o l u m e e x p a n -s i o n o b s e r v e d i n o u r e t h a n o l - t r e a t e d a n i m a l s . I n v i t r o v a s c u l a r smooth m u s c l e r e s p o n s i v e n e s s was e x a m i n e d b u t no d i f f e r e n c e i n t h e r e s p o n s e o f a o r t i c s t r i p s f r o m t h e c o n t r o l and t h e a l c o h o l -t r e a t e d a n i m a l s was d e t e c t e d . T h e p o r t a l v e i n s t r i p s f r o m b o t h g r o u p s g a v e s i m i l a r r e s p o n s e s t o n o r a d r e n a l i n e a t n o r m a l (2.5 mM) e x t e r n a l c a l c i u m , b u t - Ill -t h e s t r i p s f r o m t h e a l c o h o l - t r e a t e d a n i m a l s a c t u a l l y g a v e l o w e r r e s p o n s e s t o t h e same a g o n i s t a t low (0.2 mM) e x t e r n a l c a l c i u m l e v e l s . T h e s e f i n d i n g s i m p l y t h a t i n c r e a s e d v a s c u l a r r e s p o n s i v e n e s s i s n o t a m e c h a n i s m r e s p o n s i b l e ~ f o r t h e b l o o d p r e s s u r e e l e v a t i o n i n t h e a l c o h o l - t r e a t e d a n i m a l s . T h e e r y t h r o c y t e membranes were e x t e n s i v e l y c h a r a c t e r i z e d i n t h e hope o f e l u c i d a t i n g t h e m e c h a n i s m u n d e r l y i n g t h e a l t e r e d c a l c i u m s e n s i t i v i t y f o u n d i n t h e i s o l a t e d p e r f u s e d , h e a r t s and p o r t a l v e i n s t r i p s f r o m t h e a l c o h o l -t r e a t e d a n i m a l s ( s e e R e s u l t s e c t i o n s I I I - E and H ) . T h e h i g h a f f i n i t y C a + + / M g + + A T P a s e was^ t h e o n l y membrane enzyme a c t i v i t y t h a t was e l e v a t e d i n t h e e r y t h r o c y t e membranes f r o m a l c o h o l - t r e a t e d a n i m a l s . S i n c e t h e e l e v a -t e d enzyme a c t i v i t y i s n o t a s s o c i a t e d w i t h o t h e r g r o s s s t r u c t u r a l o r compo-s i t i o n a l c h a n g e s i n t h e e r y t h r o c y t e membrane and t h e r e i s no i n c r e a s e i n p a s s i v e C a + + p e r m e a b i l i t y ( s e e R e s u l t s s e c t i o n 111 — I ) , we assume t h a t t h e C a + + / M g + + a c t i v i t y c h a n g e i s a r e f l e c t i o n o f v e r y s u b t l e a l t e r a t i o n s i n c a l c i u m h a n d l i n g n o t d e t e c t a b l e b y o u r p r e s e n t m e t h o d s . T h e o t h e r p o s s i b i l -i t y t h a t t h e s l i g h t a l t e r a t i o n i n membrane c h o l e s t e r o l c o n t e n t i n t h e c h r o n i c a l c o h o l i c r a t s r e s u l t s i n a l t e r e d enzyme a c t i v i t y s h o u l d a l s o be k e p t i n ++ ++ mind b e c a u s e t h e a c t i v i t y o f t h e Ca /Mg A T P a s e i s h i g h l y d e p e n d e n t o n membrane l i p i d c o m p o s i t i o n and f l u i d i t y ( 2 4 3 ) . VI/-II. S t r e s s and E t h a n o l C o n s u m p t i o n . P h y s i c a l and e m o t i o n a l s t r e s s h a s l o n g b e e n s u s p e c t e d t o be c a u s a l l y r e l a t e d t o h y p e r t e n s i o n and e x p e r i m e n t s i n a n i m a l s and humans p r o v i d e some e v i d e n c e s u p p o r t i n g t h i s s u s p i c i o n ( 2 4 - 2 6 ; s e e I n t r o d u c t i o n s e c t i o n I I f o r more d e t a i l s ) . A s w e l l , s t r e s s has g e n e r a l l y been r e g a r d e d as one o f t h e - 112 -c o n f o u n d i n g f a c t o r s r e s p o n s i b l e f o r t h e c a r d i o v a s c u l a r p a t h o l o g i e s f o u n d i n c h r o n i c a l c o h o l i c s ( 1 3 4 , 2 1 7 , 2 2 1 , 2 4 4 ) . We h a v e i n v e s t i g a t e d t h e p o s s i b l e i n t e r a c t i o n b etween s t r e s s and c h r o n i c e t h a n o l c o n s u m p t i o n b y d e v e l o p i n g and u s i n g a method o f h e a t s t r e s s i n c o n j u n c t i o n w i t h o u r model o f a l c o h o l i s m . T h e b l o o d p r e s s u r e r e s p o n s e s i n t h e 4 g r o u p s o f a n i m a l s were v e r y i n t e r -e s t i n g . A t week 4, s i g n i f i c a n t d i f f e r e n c e s i n s y s t o l i c p r e s s u r e s were o b s e r v e d b e t w e e n t h e two e t h a n o l - t r e a t e d g r o u p s and t h e two c o n t r o l g r o u p s . H e a t s t r e s s i n g , b e g i n n i n g a t Week 4, seemed t o e l e v a t e , t h e p r e s s u r e o f b o t h t h e c o n t r o l and e t h a n o l - t r e a t e d g r o u p s a t a s i m i l a r r a t e and t o a s i m i l a r e x t e n t , a b o v e t h a t o f t h e u n s t r e s s e d g r o u p s ( s e e F i g . 1 6 ) . T h i s o b s e r v a -t i o n s u g g e s t s t h a t s t r e s s b y i t s e l f c a n r a i s e t h e b l o o d p r e s s u r e i n b o t h t h e c o n t r o l and e t h a n o l - t r e a t e d a n i m a l s . T h e o b s e r v a t i o n t h a t t h e a l c o h o l i c s t r e s s e d a n i m a l s had t h e h i g h e r s y s t o l i c p r e s s u r e v a l u e when c o m p a r e d t o a l c o h o l i c u n s t r e s s e d a n i m a l s d e m o n s t r a t e d an a d d i t i v e e f f e c t o f s t r e s s t o e t h a n o l c o n s u m p t i o n i n e l e v a t i n g t h e b l o o d p r e s s u r e s o f o u r a n i m a l s . T h e m e c h a n i s m f o r t h e o b s e r v e d " a d d i t i v e " e f f e c t was e x a m i n e d and we f o u n d t h a t t h e b l o o d v o l u m e s o f t h e s t r e s s e d a n i m a l s were s i m i l a r t o t h e i r r e s p e c t i v e u n s t r e s s e d c o u n t e r p a r t s . T h i s i m p l i e s t h a t t h e a d d i t i o n a l e l e v a t i o n i n b l o o d p r e s s u r e s e e n i n b o t h s t r e s s e d g r o u p s o f a n i m a l i s p r o b a b l y n o t t h e r e s u l t o f p l a s m a v o l u m e e x p a n s i o n . P l a s m a c a t e c h o l a m i n e s were a s s a y e d u s i n g HPLC and we o b s e r v e d a s t r a t i f i e d d i s t r i b u t i o n i n t h e p l a s m a n o r a d r e n a l i n e l e v e l s . T he CU g r o u p had t h e l o w e s t n o r a d r e n a l i n e l e v e l s , t h e n t h e CS g r o u p had l e v e l s t h a t were m a r g i n a l l y e l e v a t e d when c o m p a r e d t o CU, t h e AU g r o u p had s i g n i f i c a n t l y h i g h e r l e v e l s , w i t h t h e AS g r o u p h a v i n g t h e h i g h e s t c i r c u -l a t i n g l e v e l s o f a l l . No d i f f e r e n c e s c o u l d be f o u n d i n p l a s m a a d r e n a l i n e l e v e l s b e t w e e n t h e f o u r g r o u p s . P l a s m a d o p a m i n e l e v e l s were n o t d i f f e r e n t b e t w e e n t h e CU, CS and AU g r o u p s b u t were s i g n i f i c a n t l y l o w e r i n t h e AS - 113 -g r o u p . T h e r e a s o n f o r t h e r e d u c e d d o p a m i n e l e v e l i s n o t i m m e d i a t e l y a p p a r -e n t b u t one c a n s p e c u l a t e on t h e b a s i s o f p r e v i o u s r e p o r t s . D a r d e n and Hunt as w e l l as M a j o r ' s g r o u p r e p o r t e d d e p r e s s e d d o p a m i n e r g i c a c t i v i t y i n t h e b r a i n s o f r o d e n t s (245) and man (246) d u r i n g w i t h d r a w a l f r o m c h r o n i c e t h a n o l i n g e s t i o n . M e t a b o l i s m o f o t h e r n e u r o t r a n s m i t t e r s was a l s o f o u n d t o be a l t e r e d a f t e r a c u t e and c h r o n i c e t h a n o l t r e a t m e n t s ( s e e r e v i e w by H u n t ; 2 4 7 ) . I t i s p o s s i b l e t h a t s t r e s s i n t e r a c t s w i t h c h r o n i c e t h a n o l c o n s u m p t i o n i n o u r AS a n i m a l s t o p r o d u c e w i t h d r a w a l - l i k e e f f e c t s . T h i s e x p l a n a t i o n i s deemed more p l a u s i b l e i f one c o n s i d e r s t h e o b s e r v a t i o n s t h a t t h e AS a n i m a l s a r e g e n e r a l l y more i r r i t a b l e and h a v e h i g h e r i n c i d e n c e o f s e i z u r e s and i n c r e a s e d m o r t a l i t y r a t e ( d u e t o c a r d i a c f a i l u r e ; s e e R e s u l t s s e c t i o n I V A ) . In c o n c l u s i o n , t h e d a t a o b t a i n e d t o d a t e s u g g e s t t h a t c h r o n i c e t h a n o l c o n s u m p t i o n i s a s s o c i a t e d w i t h b l o o d p r e s s u r e e l e v a t i o n i n o u r a n i m a l s . F u r t h e r e v i d e n c e s u p p o r t i n g t h i s s t a t e m e n t comes f r o m an o b s e r v a t i o n made i n o n e o f t h e s e r i e s o f e x p e r i m e n t s when we had t o d i s c o n t i n u e a t Week 6 due t o t h e d e v e l o p m e n t o f r e s p i r a t o r y i n f e c t i o n i n some o f t h e a n i m a l s . T h e b l o o d p r e s s u r e s o f b o t h t h e e t h a n o l - t r e a t e d and c o n t r o l a n i m a l s were m o n i t o r e d d e s p i t e d i s c o n t i n u a t i o n o f e t h a n o l f e e d i n g . I t was n o t e d t h a t t h e a l r e a d y -e l e v a t e d s y s t o l i c p r e s s u r e s o f t h e e t h a n o l - t r e a t e d a n i m a l s r e t u r n e d t o c o n t r o l p r e s s u r e l e v e l s w i t h i n 2 weeks o f t o t a l e t h a n o l w i t h d r a w a l . S i m i l a r o b s e r v a t i o n s were r e p o r t e d i n a l o n g - t e r m f o l l o w - u p s t u d y o f a g r o u p o f a l c o h o l i c p a t i e n t s where t h e mean b l o o d p r e s s u r e was shown t o be r e d u c e d by a b s t i n e n c e and s u b s e q u e n t l y e l e v a t e d i n t h o s e who r e s u m e d d r i n k i n g ( 2 4 8 ) . I n o u r e t h a n o l - t r e a t e d a n i m a l s , t h e b l o o d p r e s s u r e e l e v a t i o n seems t o be a s s o c i a t e d w i t h b o t h an e x p a n s i o n i n p l a s m a v o l u m e as w e l l as an e l e v a t i o n i n c i r c u l a t i n g l e v e l s o f n o r a d r e n a l i n e i n t h e p l a s m a . I n c r e a s e d v a s c u l a r r e s p o n s i v e n e s s was n o t f o u n d t o be a s s o c i a t e d w i t h t h e b l o o d p r e s s u r e e l e v a -- 114 -t i o n i n t h e e t h a n o l - t r e a t e d r a t s . T h e a d d i t i o n a l h y p e r t e n s i v e e f f e c t o f h e a t s t r e s s on t h e e t h a n o l - t r e a t e d and c o n t r o l a n i m a l s i s a s s o c i a t e a w i t h i n c r e a s e s i n t h e l e v e l s o f p l a s m a n o r a d r e n a l i n e and i s p r o b a b l y u n r e l a t e d t o p l a s m a v o l u m e e x p a n s i o n . I f one a c c e p t s t h e a s s u m p t i o n t h a t p l a s m a n o r -a d r e n a l i n e l e v e l s r e f l e c t s y m p a t h e t i c n e r v o u s a c t i v i t y , t h e n e t h a n o l and s t r e s s may i n t e r a c t t o e l e v a t e t h e s y m p a t h e t i c t o n e o f t h e a n i m a l s . S i n c e t h i s " s t r e s s - e t h a n o l " model f o r h y p e r t e n s i o n i s a v e r y r e c e n t d e v e l o p m e n t , more work i s n e e d e d t o d e l i n e a t e t h e u n d e r l y i n g m e c h a n i s m s r e s p o n s i b l e f o r t h e b l o o d p r e s s u r e e l e v a t i o n . R e s u l t s f r o m e x p e r i m e n t s on v a s c u l a r r e a c t i v i t y , e r y t h r o c y t e membrane p r o p e r t i e s , c e n t r a l and p e r i p h e r a l v a s o -p r e s s o r and v a s o d e p r e s s o r l e v e l s , and b l o o d p r e s s u r e i n t e r v e n t i o n u s i n g s y m p a t h o l y t i c and o t h e r p h a r m a c o l o g i c a l a g e n t s s h o u l d p r o v i d e more i n s i g h t i n t o t h e n a t u r e o f t h e v a r i o u s u n d e r l y i n g m e c h a n i s m s s u s t a i n i n g t h e e l e v a t e d b l o o d p r e s s u r e i n t h i s model o f h y p e r t e n s i o n . i I X . G e n e r a l C o n c l u s i o n s A f t e r a t t e m p t i n g t o e l u c i d a t e some o f t h e u n d e r l y i n g m e c h a n i s m s i n t h e v a r i o u s t y p e s o f h y p e r t e n s i o n d e s c r i b e d h e r e , one i s l e f t w i t h some i n s i g h t s b u t many more q u e s t i o n s . T h e r e i s m o u n t i n g e v i d e n c e t h a t a b n o r m a l i t i e s i n c a l c i u m h a n d l i n g a r e e t i o l o g i c a l l y r e l a t e d t o e s s e n t i a l h y p e r t e n s i o n . B l a u s t e i n p o s t u l a t e d t h a t s i n c e t h e r e i s e v i d e n c e f o r a r e d u c t i o n o f N a + / K + A T P a s e a c t i v i t y , h e n c e s o d i u m e x t r u s i o n , i n c e l l s o f h y p e r t e n -s i v e s , t h e r e s u l t a n t i n c r e a s e i n i n t r a c e l l u l a r s o d i u m w i l l t e n d t o s t i m u l a t e t h e Na /Ca e x c h a n g e m e c h a n i s m s and e f f e c t i v e l y i n c r e a s e i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n s ( 2 4 9 ) . We h a v e shown i n t h e SHR e r y t h r o c y t e t h a t i n c r e a s e d i n t r a c e l l u l a r c a l c i u m i s due t o i n c r e a s e d membrane p e r m e a b i l i t y t o t h e i o n i t s e l f and n e ed n o t be a s e c o n d a r y e f f e c t o f s o d i u m a b n o r m a l i t i e s . C a l c i u m a n t a g o n i s t s s u c h as v e r a p a m i l has b e e n shown t o s e l e c t i v e l y d i l a t e - 115 -t h e r e s i s t a n c e v e s s e l s i n t h e f o r e a r m s o f h y p e r t e n s i v e p a t i e n t s t o much g r e a t e r e x t e n t s t h a n i n n o r m o t e n s i v e c o n t r o l s w h i l e n i t r o p r u s s i d e d i l a t e s t h e v e s s e l s t o t h e same e x t e n t i n b o t h g r o u p s ( 2 5 0 ) . N i f e d i p i n e , a n o t h e r c a l c i u m a n t a g o n i s t , has been shown t o h a v e g r e a t e r b l o o d p r e s s u r e l o w e r i n g e f f e c t s i n p a t i e n t s w i t h e s s e n t i a l h y p e r t e n s i o n ( 2 5 1 ) . I f one a c c e p t s t h a t t h e SHR i s a r e a s o n a b l e model o f human e s s e n t i a l h y p e r t e n s i o n , t h e n o u r f i n d i n g s o f i n c r e a s e d c a l c i u m p e r m e a b i l i t y i n SHR e r y t h r o c y t e s and p o s s i b l y o t h e r t i s s u e s o f f e r a p l a u s i b l e e x p l a n a t i o n t o t h e a b o v e m e n t i o n e d c l i n i c a l o b s e r v a t i o n s . F u r t h e r e x p e r i m e n t a t i o n s u c h as g i v i n g c a l c i u m a n t a g o n i s t s t o p r e g n a n t a n d / o r n e o n a t a l SHR and m o n i t o r i n g t h e b l o o d p r e s s u r e d e v e l o p m e n t , may p r o v i d e more i n f o r m a t i o n on t h e p o s s i b i l i t y o f r e v e r s i n g t h e h y p e r t e n s i o n i n t h e s e a n i m a l s as w e l l as s u p p l y i n g a model w h e r e b y t h e b i o c h e m i c a l / b i o -p h y s i c a l c h a r a c t e r i z a t i o n s o f t h e membrane s u c h as t h o s e d e s c r i b e d h e r e c a n be p e r f o r m e d . A l s o , c o m p a r i s o n o f t h e m e m b r a n o l o g i c a l p r o p e r t i e s o f e r y t h r o c y t e s f r o m b o t h u n t r e a t e d h y p e r t e n s i v e p a t i e n t s and t h o s e p a t i e n t s on c a l c i u m a n t a g o n i s t t h e r a p y may p r o v i d e i n s i g h t i n t o t h e i n v o l o v e m e n t o f c a l c i u m i n e s s e n t i a l h y p e r t e n s i o n . I n a r e c e n t commentary, Page r e - s t a t e d h i s m o s a i c t h e o r y and l i s t e d t h e f o l l o w i n g f a c t o r s as i m p o r t a n t i n h y p e r t e n s i o n ( 2 5 2 ) . 1. G e n e t i c 5. N e u r a l 2. E n v i r o n m e n t a l 6. E n d o c r i n e 3. A n a t o m i c a l . 7. Humoral 1 4. A d a p t i v e . 8. Haemodynamic I f we a p p l y t h e s e f a c t o r s t o t h e a n i m a l m o d e l s o f h y p e r t e n s i o n s t u d i e d h e r e , s e v e r a l i n t e r e s t i n g o b s e r v a t i o n s emerge. I n t h e SHR, f a c t o r s s u c h as g e n e t i c , a n a t o m i c a l ( c h a n g e s i n v a s c u l a r a r c h i t e c t u r e and m e mbranes), a d a p -++ ++ t i v e ( i n c r e a s e d a c t i v i t y o f Ca /Mg A T P a s e s and c a r d i a c h y p e r t r o p h y ) , n e u r a l ( i n c r e a s e d s y m p a t h e t i c t o n e and b a r o r e c e p t o r r e s e t t i n g ) and haemody-- 116 -namic ( i n c r e a s e d h e a r t r a t e ) a r e p r o b a b l y e t i o l o g i c a l l y r e l a t e d t o t h e h y p e r t e n s i o n . I n t h e DOCA a n i m a l s , e n v i r o n m e n t a l ( s a l t i n t a k e ) , a n a t o m i c a l / / ( v a s c u l a r c h a n g e s ) , n e u r a l ( r e p u t e d i n c r e a s e i n s y m p a t h e t i c t o n e ) , e n d o c r i n e ( c o r t i c o s t e r o n e e x c e s s ) and haemodynamic ( b l o o d v o l u m e e x p a n s i o n ) a r e f a c t o r s t h a t a r e i m p o r t a n t . F i n a l l y i n t h e e t h a n o l / s t r e s s m o d e l , e n v i r o n -m e n t a l ( e t h a n o l c o n s u m p t i o n and h e a t s t r e s s ) , a d a p t i v e ( c a r d i a c h y p e r t r o p h y ) , n e u r a l ( e l e v a t e d p l a s m a n o r a d r e n a l i n e ) , e n d o c r i n e ( r e p u t e d i n c r e a s e i n p l a s m a r e n i n and a l d o s t e r o n e ) , and ~haemodynamic ( p l a s m a v o l u m e e x p a n s i o n ) a r e f a c t o r s t h a t c o n t r i b u t e t o h y p e r t e n s i o n i n t h i s m o d e l . I t i s o b v i o u s f r o m t h e f o r e g o i n g d i s c u s s i o n s t h a t any number o f P a g e ' s f a c t o r s c a n be i n v o l v e d i n a p a r t i c u l a r model o f h y p e r t e n s i o n . Our p r e s e n t u n d e r s t a n d i n g o f t h e s e m o d e l s seems t o s u g g e s t t h a t t h e r e i s no s e t r e q u i r e -ment f o r a p a r t i c u l a r c o m b i n a t i o n o f f a c t o r s t o be i n v o l v e d i n a l l o f t h e m o d e l s s t u d i e d . T h e r e i s , h o w e v e r , an o v e r l a p o f f a c t o r s i n a l l t h r e e m o d e l s b e c a u s e " n e u r a l " and "haemodynamic" a r e t h e f a c t o r s t h a t a r e common t o a l l o f them. T h i s m i g h t seem l i k e an o v e r s i m p l i f i c a t i o n b e c a u s e b o t h " n e u r a l " and "haemodynamic" f a c t o r s c a n encompass many s u b c l a s s e s o f a b n o r -m a l i t i e s . In o u r p r e s e n t d i s c u s s i o n , h o w e v e r , t h e s e f a c t o r s s e r v e d t h e p u r p o s e o f g i v i n g us t h e r i g h t p e r s p e c t i v e on v i e w i n g h y p e r t e n s i v e mechan-i s m s . A s o u r k n o w l e d g e o f h y p e r t e n s i o n i m p r o v e s , more f a c t o r s w i l l u n d o u b -t e d l y be i d e n t i f i e d as b e i n g i m p o r t a n t i n h y p e r t e n s i o n o f a l l c a u s e s . More i m p o r t a n t l y , i t may be f o u n d t h a t t h e i n t e r a c t i o n s b e t w e e n t h e f a c t o r s t h a t a r e i m p o r t a n t i n p r o d u c i n g h y p e r t e n s i o n . T h e a s c e r t a i n m e n t o f e t h a n o l c o n s u m p t i o n as a p o s s i b l e c a u s e o f h y p e r -t e n s i o n h as v e r y i m p o r t a n t c l i n i c a l i m p l i c a t i o n s . I n t h e g r o u p o f p a t i e n t s c u r r e n t l y c l a s s i f i e d as e s s e n t i a l h y p e r t e n s i v e s , e t h a n o l c o n s u m p t i o n h a b i t s s h o u l d be q u a n t i t a t e d . One may i d e n t i f y a s u b - p o p u l a t i o n o f h y p e r t e n s i v e - i n -p a t i e n t s whose b l o o d p r e s s u r e e l e v a t i o n , g i v e n t h e r i g h t c o m b i n a t i o n o f c o n f o u n d i n g f a c t o r s , i s i n d e e d e t h a n o l - r e l a t e d and n o t o f " u n k n o w n a e t i o l o g y " as p r e v i o u s l y t h o u g h t . A s more and more f a c t o r s t h a t a r e c a u s a l l y r e l a t e d t o h y p e r t e n s i o n a r e i d e n t i f i e d , p h y s i c i a n s may be l e f t ,with an e v e r d i m i n i s h -i n g p o p u l a t i o n o f t r u e e s s e n t i a l h y p e r t e n s i v e p a t i e n t s . - 118 -i R e f e r e n c e s 1. B u i l d and B l o o d P r e s s u r e S t u d y 1959, V o l s . I and I I . S o c i e t y o f A c t u a r i e s , C h i c a g o , 1959. 2. K a n n e l , W.B. R o l e o f b l o o d p r e s s u r e i n c a r d i o v a s c u l a r d i s e a s e : T h e F r a m i n g h a m S t u d y . 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I m p o r t a n c e o f h y p e r t e n s i o n as a m a j o r r i s k f a c t o r i n c a r d i o v a s c u l a r d i s e a s e . In: H y p e r t e n s i o n , E d s . G e n e s t , J . , Koiw E. and K u c h e l , 0. McGraw H i l l , N.Y., pp. 8 8 8 - 9 1 0 , 1977. 8. de C h a m p l a i n , J . T h e c o n t r i b u t i o n o f t h e s y m p a t h e t i c n e r v o u s s y s t e m t o a r t e r i a l h y p e r t e n s i o n . C a n a d . J . P h y s i o l . P h a r m a c o l . 5 6 ( 3 ) : 3 4 1 - 3 5 2 , 1978. 9. V e t e r a n s A d m i n i s t r a t i o n C o o p e r a t i v e S t u d y G r o u p on A n t i h y p e r t e n s i v e A g e n t s : E f f e c t s o f t r e a t m e n t on m o r b i d i t y i n h y p e r t e n s i o n : R e s u l t s i n p a t i e n t s w i t h d i a s t o l i c b l o o d p r e s s u r e s a v e r a g i n g 115 t h r o u g h 129 mm Hg. J . Am. Med. A s s n . 202: 1028-1034, 1967. - 119 -10. 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Q i n 25 ml g l a c i a l a c e t i c a c i d . b) W o r k i n g s o l u t i o n : D i l u t e 1.0 ml o f s t o c k s o l u t i o n i n t o 99.0 ml o f c o n c e n t r a t e d ^ S O ^ . 3. K r e b s ' - R i n g e r s o l u t i o n S o d i u m c h l o r i d e ' 112 mM P o t a s s i u m c h l o r i d e 4.5 mM M a g n e s i u m c h l o r i d e 1.2 mM C a l c i u m c h l o r i d e ' 2.5 mM S o d i u m p h o s p h a t e , m o n o b a s i c 1.2 mM S o d i u m b i c a r b o n a t e 26.2 mM G l u c o s e 11.1 mM EDTA ' 26 U M APPENDIX ic 1. ANS ( F i s k e - S u b b a R o w r e a g e n t ) v l - a m i n o - 2 - n a p h t h o l - 4 - s u l f o n i c a c i d S o d i u m s u l f i t e ( a n h y d r o u s ) S o d i u m b i s u l f i t e ( a n h y d r o u s ) D o u b l e d i s t i l l e d w a t e r 

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