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Effect of muscarinic receptor stimulation on phosphatidylinositol turnover in rat heart and guinea pig… Chandra Sekar, M. 1983

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EFFECT OF MUSCARINIC RECEPTOR STIMULATION ON PHOSPHATIDYLINOSITOL TURNOVER IN RAT HEART AND GUINEA PIG SMOOTH MUSCLE by M . CHANDRA SEKAR B . P h a r m . , B . I . T . S . , P i l a n i , I n d i a 1974; M . P h a r m . , B . I . T . S , P i l a n i , I n d i a 1976; M . S c , U . B . C , 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES D i v i s i o n Of P h a r m a c e u t i c a l C h e m i s t r y Of The F a c u l t y Of P h a r m a c e u t i c a l S c i e n c e s 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 December 1983 0 M. C h a n d r a S e k a r , 19 8 3 In presenting t h i s thesis 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 University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s t h e s i s fo r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publ i c a t i o n of t h i s t h e s i s for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Fkfayv^^C-JAs-kS>£^ £A^C£^ The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date UI>/{Lf i i A b s t r a c t S t i m u l a t i o n of v a r i o u s c e l l s u r f a c e r e c e p t o r s produces an enhanced t u r n o v e r of p h o s p h a t i d y l i n o s i t o l ( P I ) . T h i s phenomenon, r e f e r r e d to as the PI re sponse , has been suggested to be an o b l i g a t o r y response of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n and to p l a y a r o l e i n c a l c i u m m o b i l i z a t i o n mediated by m u s c a r i n i c and other r e c e p t o r systems. In t h i s s t u d y , t h i s h y p o t h e s i s has been i n v e s t i g a t e d by s t u d y i n g PI response i n r a t a t r i a and l o n g i t u d i n a l smooth muscle of the gu inea p i g i l e u m . I t was observed that even though m u s c a r i n i c r e c e p t o r d e n s i t y i s known to be s i m i l a r i n r a t l e f t and r i g h t a t r i a , an i n c r e a s e (35%, p < 0.05) i n phosphate i n c o r p o r a t i o n i n t o PI was observed i n r a t l e f t a t r i u m but not i n r i g h t a t r i u m . By c o n t r a s t to the sma l l e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n , a l p h a - a d r e n e r g i c r e c e p t o r s t i m u l a t i o n by 0.1 mM methoxamine produced a t w o - f o l d i n c r e a s e i n phosphate i n c o r p o r a t i o n i n t o PI i n both r a t l e f t and r i g h t a t r i a , d e s p i t e the lower d e n s i t y of these r e c e p t o r s compared to the m u s c a r i n i c r e c e p t o r s i n a t r i a . These r e s u l t s , a l o n g wi th s t u d i e s showing a l a ck of e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n on i n o s i t o l l i p i d breakdown, suggested tha t o n l y a smal l p o p u l a t i o n ( approx imate ly 15%) of m u s c a r i n i c r e c e p t o r s i n ra t a t r i a i s coup led to PI t u r n o v e r . These are termed m, r e c e p t o r s , by analogy to a, a d r e n e r g i c r e c e p t o r s which enhance PI t u r n o v e r and C a 2 + m o b i l i z a t i o n . The remain ing p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s , termed m 2 , most p r o b a b l y i s c o u p l e d i n an i n h i b i t o r y manner to a d e n y l a t e c y c l a s e . W h i l e these f i n d i n g s support the p o s t u l a t e t h a t PI response may accompany on ly the s t i m u l a t i o n of those r e c e p t o r s whose response i s mediated through c a l c i u m m o b i l i z a t i o n , the r e s u l t s a l s o suggest the presence of a m u s c a r i n i c r e c e p t o r p o p u l a t i o n whose s t i m u l a t i o n i s not c o u p l e d to PI t u r n o v e r . The r o l e of PI response i n c a l c i u m m o b i l i z a t i o n was i n v e s t i g a t e d i n l o n g i t u d i n a l smooth muscle of guinea p i g i l e u m . C o n s i s t e n t w i t h p r e v i o u s f i n d i n g s , m u s c a r i n i c r e c e p t o r s t i m u l a t i o n of guinea p i g i l eum was accompanied by a PI re sponse . C a r b a c h o l (0.1 mM) produced an a t r o p i n e - s e n s i t i v e i n c o r p o r a t i o n of [ 3 2 P ] p h o s p h a t e and [ 3 H ] i n o s i t o l i n t o P I . The e f f e c t of c a r b a c h o l on the breakdown of i n o s i t o l l i p i d s was s t u d i e d i n the presence of 10 mM l i t h i u m , which causes a c c u m u l a t i o n of i n o s i t o l phosphate by i n h i b i t i n g i n o s i t o l phosphatase . C a r b a c h o l produced a 2 0 - f o l d i n c r e a s e i n the a c c u m u l a t i o n of i n o s i t o l phosphates i n the presence of l i t h i u m . A n a l y s i s of the i n o s i t o l phosphates by ion-exchange chromatography i n d i c a t e d t h a t , u n l i k e many o t h e r t i s s u e s , i n guinea p i g i l eum l e s s than. 20% of the [ 3 H ] i n o s i t o l l a b e l o c c u r r e d i n i n o s i t o l monophosphate, w h i l e the remainder was found i n the i n o s i t o l po lyphosphate f r a c t i o n . T h i s i n d i c a t e d tha t s i m i l a r to f i n d i n g s i n o ther t i s s u e s r e p o r t e d r e c e n t l y , the pr imary event of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n guinea p i g i l eum may be the enhanced breakdown of p o l y p h o s p h o i n o s i t i d e s , r a t h e r than P I . The e f f e c t of p h e n y l m e t h a n e s u l f o n y l f l u o r i d e (PMSF), a p u t a t i v e i n h i b i t o r of P l - s p e c i f i c p h o s p h o l i p a s e C , on c a r b a c h o l and p o t a s s i u m - s t i m u l a t e d PI t u r n o v e r and c o n t r a c t i o n i n i v l o n g i t u d i n a l smooth muscle of guinea p i g i l eum was s t u d i e d . PMSF almost c o m p l e t e l y i n h i b i t e d c a r b a c h o l - s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n i n t o P I , w h i l e i t had no e f f e c t on pota s s ium-s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n . T h i s suggests t h a t the two s t i m u l i produce "PI re sponses " by d i f f e r e n t mechanisms. In c o n t r a s t to i t s s p e c i f i c i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d PI t u r n o v e r , PMSF produced a n o n s p e c i f i c , t r a n s i e n t i n h i b i t i o n of c o n t r a c t i o n of guinea p i g i l eum by both c a r b a c h o l and po ta s s ium. The n o n s p e c i f i c PMSF e f f e c t on c o n t r a c t i o n suggests tha t i t i s not the r e s u l t of i t s i n h i b i t o r y e f f e c t on PI t u r n o v e r . The s u g g e s t i o n of Walenga et a l . (1980) tha t PMSF i n h i b i t i o n of PI t u r n o v e r may be mediated through i t s i n h i b i t i o n of P l - s p e c i f i c p h o s p h o l i p a s e C was based on i n d i r e c t f i n d i n g s . PMSF (2 mM) produced o n l y a 16% i n h i b i t i o n of the c a r b a c h o l - s t i m u l a t e d i n o s i t o l phosphate a c c u m u l a t i o n i n the presence of l i t h i u m , i n d i c a t i n g that the PMSF e f f e c t on P l - s p e c i f i c p h o s p h o l i p a s e C cannot f u l l y account for the observed i n h i b i t i o n by PMSF of c a r b a c h o l - s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n . The r e s u l t s o b t a i n e d do not c o n t r a d i c t the p o s s i b i l i t y tha t PI breakdown i s i n v o l v e d i n c a l c i u m m o b i l i z a t i o n . V Tab le of Content s A b s t r a c t i i L i s t of T a b l e s v i i i L i s t of F i g u r e s ix Acknowledgement x i i i LIST OF ABBREVIATIONS x i Chapter I INTRODUCTION 1 1.1 DISTRIBUTION OF PI 3 1.2 PI RESPONSE 4 1.2.1 C l o s e d C y c l e 4 1.2.2 The Pr imary Event Of PI Response 6 1.2.3 A l t e r n a t e Mechanisms For L a b e l I n c o r p o r a t i o n 7 1.2.4 P i - s p e c i f i c P h o s p h o l i p a s e C 9 1 .3 ROLE OF PI TURNOVER 12 1.4 PI AS A CALCIUM GATE 13 1.4.1 C a l c i u m Requirement Of PI Response 16 1.4.2 P h o s p h a t i d i c A c i d As A C a l c i u m Ionophore 19 1.4.3 Is PI Degraded From Plasma Membrane? 21 1.5 DIACYLGLYCEROL AS AN ACTIVATOR OF PROTEIN KINASE C 22 1.6 PI AS A SOURCE OF ARACHIDONIC ACID 24 1.7 INHIBITORS OF PI RESPONSE 27 1.8 MUSCARINIC CHOLINERGIC RECEPTORS 28 1.8.1 C y c l i c Guanosine Monophosphate 29 1.8.2 Potass ium E f f l u x 31 1.8.3 M u s c a r i n i c A c e t y l c h o l i n e Receptor And PI Response 32 1.8.4 Are There S u b p o p u l a t i o n s Of M u s c a r i n i c A c e t y l c h o l i n e Receptor s ? 34 1.8.5 S u b c l a s s i f i c a t i o n Based On A n t a g o n i s t B i n d i n g . . . 3 5 1.8.6 R e c e p t o r - E f f e c t o r C o u p l i n g 37 1.9 MECHANISM OF THE NEGATIVE INOTROPIC EFFECT 39 1.10 ALPHA-ADRENERGIC RECEPTORS IN HEART 43 1.10.1 Mechanism Of The A l p h a - And B e t a - A d r e n e r g i c R e c e p t o r - m e d i a t e d P o s i t i v e I n o t r o p i c E f f e c t 45 1.11 MUSCARINIC RECEPTORS IN LONGITUDINAL SMOOTH MUSCLE OF GUINEA PIG ILEUM 47 1.12 OBJECTIVE 50 Chapter II METHODS 51 2.1 C h o i c e Of Animal 51 2.2 K r e b s - b i c a r b o n a t e Bu f f e r 52 2.3 Phosphate I n c o r p o r a t i o n In A t r i a 52 2.4 I s o l a t i o n Of L o n g i t u d i n a l Smooth Muscle Of Guinea P i g I leum 53 2.5 L a b e l - i n c o r p o r a t i o n In Guinea P i g Ileum 53 2.6 E x t r a c t i o n Of L i p i d 54 2.7 P h o s p h o l i p i d S e p a r a t i o n 55 — v i 2.8 E x t r a c t i o n Of P h o s p h o l i p i d Spots 56 2 .9 D e t e r m i n a t i o n Of R a d i o a c t i v i t y 56 2.10 Phosphorus D e t e r m i n a t i o n 57 2.11 I n o s i t o l Phosphate A c c u m u l a t i o n In The Presence Of L i t h i u m 58 2.12 D e t e r m i n a t i o n Of T o t a l L a b e l e d I n o s i t o l Phosphate 59 2.13 S e p a r a t i o n Of I n o s i t o l Phosphates 59 2.14 C o n t r a c t i o n S t u d i e s 60 2.15 PI D e g r a d a t i o n S t u d i e s 60 2.16 F a t t y A c i d Compos i t ion Of D i a c y l g l y c e r o l 61 2.17 M a t e r i a l s 63 Chapter III RESULTS 64 3.1 SEPARATION OF PHOSPHOLIPIDS 64 3.1.1 S e p a r a t i o n And I d e n t i f i c a t i o n Of PI 64 3 .1 .2 Phosphorus D e t e r m i n a t i o n 65 3.2 PHOSPHATE INCORPORATION IN RAT HEART 65 3.2.1 [ 3 2 P ] P h o s p h a t e I n c o r p o r a t i o n In Rat A t r i a Ver sus V e n t r i c l e 65 3 .2 .2 E f f e c t Of M u s c a r i n i c And A l p h a - a d r e n e r g i c Receptor S t i m u l a t i o n On [ 3 2 P ] p h o s p h a t e I n c o r p o r a t i o n In Combined A t r i a 66 3 .2 .3 E f f e c t Of M u s c a r i n i c And A l p h a - a d r e n e r g i c Receptor S t i m u l a t i o n On [ 3 2 P ] p h o s p h a t e I n c o r p o r a t i o n In Separate L e f t And R ight A t r i a 67 3.3 PI BREAKDOWN IN HEART 68 3.3.1 Breakdown Of P h o s p h o l i p i d s P r e l a b e l e d W i t h R a d i o l a b e l Phosphate 69 3 .3 .2 Breakdown Of PI P r e l a b e l e d With A r a c h i d o n a t e . . . . 6 9 3 .3 .3 D i a c y l g l y c e r o l Accumula t ion 70 3 .3 .4 F a t t y A c i d Compos i t ion Of The P o l a r L i p i d F r a c t i o n In A t r i a And V e n t r i c l e s 71 3.4 PI RESPONSE IN GUINEA PIG ILEUM 72 3.4.1 [ 3 2 P ] P h o s p h a t e I n c o r p o r a t i o n 73 3 .4 .2 [ 3 H ] l n o s i t o l I n c o r p o r a t i o n 73 3 .4 .3 L i t h i u m - a m p l i f i e d A c c u m u l a t i o n Of I n o s i t o l Phosphates 74 3 .4 .4 I d e n t i f i c a t i o n Of I n o s i t o l Phosphates 75 3.5 EFFECT OF PMSF ON PI TURNOVER AND CONTRACTION IN GUINEA PIG ILEUM 76 3.5.1 E f f e c t Of PMSF On PI Turnover 76 3 .5 .2 E f f e c t Of PMSF On C o n t r a c t i o n 77 3 .5 .3 E f f e c t Of PMSF On I n o s i t o l - P h o s p h a t e A c c u m u l a t i o n 78 Chapter IV DISCUSSION 124 4.1 IS MUSCARINIC RECEPTOR STIMULATION IN THE HEART ACCOMPANIED BY A PI RESPONSE? 124 4.1 .1 P h o s p h o l i p i d S e p a r a t i o n And Technique V e r i f i c a t i o n 124 4 . 1 . 2 B a s a l [ 3 2 P ] p h o s p h a t e I n c o r p o r a t i o n In Heart . . . . 1 2 5 v i i 4 . 1 . 3 E f f e c t Of M u s c a r i n i c And A l p h a - A d r e n e r g i c Receptor S t i m u l a t i o n On PI Turnover In Rat A t r i a 126 4.2 PI BREAKDOWN STUDIES 127 4.2 .1 Breakdown Of P r e l a b e l e d PI 127 4 . 2 . 2 A r a c h i d o n y l - E n r i c h e d D i a c y l g l y c e r o l 129 4 . 2 . 3 [ 3 2 P ] P h o s p h a t e I n c o r p o r a t i o n In Rat L e f t And Right A t r i a 132 4 . 2 . 4 E v i d e n c e For Two Subpopu la t ions Of M u s c a r i n i c Receptor s 133 4.3 ROLE OF PI TURNOVER IN MUSCARINIC RECEPTOR STIMULATED CALCIUM MOBILIZATION IN GUINEA PIG ILEUM 1 38 4 .3 .1 I n c o r p o r a t i o n Of [ 3 H ] i n o s i t o l Into PI .139 4 . 3 . 2 E f f e c t Of PMSF On C a r b a c h o l And K + - s t i m u l a t e d PI Response And C o n t r a c t i o n 139 4 . 3 . 3 Is PMSF I n h i b i t i o n Of C a r b a c h o l - s t i m u l a t e d PI Response Media ted By I n h i b i t i o n Of P i - s p e c i f i c P h o s p h o l i p a s e C ? 141 4 . 3 . 4 L i t h i u m - A m p l i f i c a t i o n Of PI Response In Guinea P i g I leum 143 4 . 3 . 5 A n a l y s i s Of I n o s i t o l Phosphates Accumulated D u r i n g L i t h i u m A m p l i f i c a t i o n Of C a r b a c h o l - s t i m u l a t e d PI Response In Guinea P i g Ileum 145 4 . 3 . 6 E f f e c t Of PMSF On C a r b a c h o l - s t i m u l a t e d I n o s i t o l Phosphate Accumula t ion 147 Chapter V SUMMARY 150 BIBLIOGRAPHY 154 v i i i L i s t of T a b l e s I . P h o s p h o l i p i d d i s t r i b u t i o n and [ 3 2 P ] p h o s p h a t e l a b e l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d s of r a t a t r i a 82 I I . E f f e c t of c a r b a c h o l s t i m u l a t i o n on a r a c h i d o n y l conten t of d i a c y l g l y c e r o l 101 I I I . E f f e c t of c a r b a c h o l on [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d 1 08 I V . C a r b a c h o l - s t i m u l a t e d a c c u m u l a t i o n of i n o s i t o l phosphate 109 V . Compos i t ion of i n o s i t o l phosphates accumulated i n guinea p i g i l eum 116 V I . E f f e c t of PMSF on the a c c u m u l a t i o n of c a r b a c h o l -s t i m u l a t e d i n o s i t o l phosphates i n gu inea p i g i l e u m . 1 23 ix L i s t of F i g u r e s 1 . PI t u r n o v e r . 5 2. PI as a c a l c i u m gate 15 3. P i r e n z e p i n e S t r u c t u r e 36 .4. P h o s p h o l i p i d s e p a r a t i o n 80 5. S tandard curve for phosphorus 83 6. Comparison of the b a s a l phosphate i n c o r p o r a t i o n i n t o v a r i o u s p h o s p h o l i p i d s i n a t r i a and v e n t r i c l e s of r a t h e a r t 85 7. E f f e c t of c a r b a c h o l and methoxamine on [ 3 2 P ] i n c o r p o r a t i o n i n t o PI i n combined r a t a t r i a . 87 8. E f f e c t of methoxamine s t i m u l a t i o n on [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o v a r i o u s p h o s p h o l i p i d s i n combined ra t a t r i a 89 9. E f f e c t of methoxamine s t i m u l a t i o n on [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l i n o s i t o l of s epara te ra t l e f t and r i g h t a t r i a 91 10. E f f e c t of c a r b a c h o l on [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l i n o s i t o l of separa te r a t l e f t and r i g h t a t r i a 93 11. E f f e c t of c a r b a c h o l on p h o s p h o l i p i d s p r e l a b e l e d w i t h [ 3 2 P]phosphate i n combined r a t a t r i a 95 12. E f f e c t of c a r b a c h o l on p h o s p h a t i d y l i n o s i t o l p r e l a b e l e d w i t h a r a c h i d o n i c a c i d i n r a t a t r i a 97 13. S e p a r a t i o n of d i a c y l g l y c e r o l 99 14. Gas chromatogram of f a t t y a c i d methyl e s t e r s of d i a c y l g l y c e r o l 102 15. F a t t y a c i d c o m p o s i t i o n of the p o l a r l i p i d f r a c t i o n of r a t a t r i a and v e n t r i c l e s 104 16. E f f e c t of c a r b a c h o l on [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l i n o s i t o l of l o n g i t u d i n a l smooth muscle of gu inea p i g i l eum 106 17. Dose-response curve for c a r b a c h o l - s t i m u l a t e d i n o s i t o l phosphate a c c u m u l a t i o n i n guinea p i g i l eum 110 X 18. E l u t i o n p r o f i l e of s t andard i n o s i t o l mono-phosphate. 112 19. E l u t i o n p r o f i l e of the accumulated i n o s i t o l phosphates i n gu inea p i g i l eum 114 20. Time cour se of the e f f e c t of PMSF on b a s a l [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d i n guinea p i g i l e u m . 117 21. E f f e c t of PMSF on c a r b a c h o l - and K + - s t i m u l a t e d [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d s of guinea p i g i l eum 119 22. E f f e c t of PMSF on c a r b a c h o l - and K + - s t i m u l a t e d c o n t r a c t i o n of guinea p i g i l eum 121 x i L i s t of A b b r e v i a t i o n s ACh a c e t y l c h o l i n e nAChR n i c o t i n i c a c e t y l c h o l i n e r e c e p t o r mAChr m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r ACS aqueous c o u n t i n g s c i n t i l l a n t cAMP c y c l i c adenos ine 3 ' , 5 ' -monophosphate ° C degree c e n t i g r a d e C i C u r i e mCi m i l l i c u r i e MCi m i c r o c u r i e cm c e n t i m e t e r cpm counts per minute DAG 1 - o l e y l - 2 - a c e t y l - d i a c y l g l y c e r o l D-600 methoxy v e r a p a m i l dpm d i s i n t e g r a t i o n s per minute fmole femto mole cGMP c y c l i c guanos ine 3 ' , 5 ' -monophosphate GTP guanosine t r i p h o s p h a t e £ g r a v i t a t i o n a l f o r c e g gram mg m i l l i g r a m Mg microgram cGMP c y c l i c guanosine 3 5 ' - m o n o p h o s p h a t e GTP guanosine t r i p h o s p h a t e h hour IP i n o s i t o l mono-phosphate x i i I P 2 i n o s i t o l d i - p h o s p h a t e IPs i n o s i t o l t r i - p h o s p h a t e K thousand ml m i l l i l i t e r Ml m i c r o l i t e r M molar mM mi 11 imolar MM micromolar min minute nm nanometer QNB q u i n u c l i d i n y l b e n z i l a t e sec second msec m i l l i second TLC t h i n l a y e r chromatography PI p h o s p h a t i d y l i n o s i t o l PIP phosphat i d y l i n o s i t o l 4-phosphate P I P 2 p h o s p h a t i d y l i n o s i t o l 4,5-bisphosphate PMSF p h e n y l m e t h a n e s u l f o n y l f l u o r i d e PKC p r o t e i n k inase C xi i i ACKNOWLEDGEMENT I wish to express my g r a t i u d e to D r . B . D . R o u f o g a l i s for h i s c o n s t a n t encouragement and guidance d u r i n g the course of t h i s work. My s p e c i a l thanks to my r e s e a r c h committee members, D r s , J . H . M c N e i l l , S . K a t z , D. G o d i n , J . Diamond and R . H . S c h e l l e n b e r g for t h e i r genuine i n t e r e s t and v a l u a b l e suggest i o n s . I w i l l l i k e to thank M r . Ahmad Fawzi fo r h i s p l o t t i n g programs, D r . C h r i s t i n e N i c h o l for the d i s c u s s i o n s and s u g g e s t i o n s d u r i n g the course of t h i s work and a l l my c o l l e a g u e s and f r i e n d s for making my s tay i n Vancouver a v e r y p l e a s a n t and memorable one. I would l i k e to acknowledge the f i n a n c i a l support p r o v i d e d by the U n i v e r s i t y of B r i t i s h Columbia and the B r i t i s h Columbia Heart F o u n d a t i o n . x i v To my parents 1 I . INTRODUCTION I t i s now w e l l e s t a b l i s h e d that r e c e p t o r s a t the c e l l su r f ace enable the c e l l to r e c o g n i z e incoming s i g n a l s , but l e s s i s known about how t h i s i n f o r m a t i o n i s t r a n s f e r r e d to the i n s i d e of the c e l l . Two important secondary messengers , which a s s i s t to t r a n s f o r m the incoming message i n t o c e l l u l a r language , are cAMP ( S u t h e r l a n d and R a i l , 1960) and c a l c i u m (Rasmussen, 1970). A c c o r d i n g to the S inger and N i c b l s o n model , the b i o l o g i c a l membrane i s composed of p r o t e i n s " f l o a t i n g " i n a l i p i d b i l a y e r (S inger and N i c o l s o n , 1972). P h o s p h o l i p i d s c o n s t i t u t e the major l i p i d f r a c t i o n of the plasma membrane i n v a r i o u s c e l l s . Both the p h o s p h o l i p i d head group and the f a t t y a c i d c h a i n s have been shown to p l a y an important r o l e i n v a r i o u s membrane c h a r a c t e r i s t i c s ( S i n g e r , 1974; McMurchie and R a i s o n , 1979; Sandermann, 1978), such as p e r m e a b i l i t y of i o n s , f l u i d i t y of the membrane and a c t i v i t y of membrane bound enzymes. T h e r e f o r e , the sugge s t ion tha t membrane components ( i n c l u d i n g l i p i d ) may p l a y a r o l e i n t r a n s m i t t i n g s i g n a l s a c r o s s the b i l o g i c a l membrane appears q u i t e r a t i o n a l . Receptor s which employ cAMP as a second messenger have been shown to be c o u p l e d to a d e n y l a t e c y c l a s e pre sent on the c y t o p l a s m i c s i d e of the membrane, through a GTP b i n d i n g p r o t e i n ( L e v i t z k i and H e l m r e i c h , 1979; R o d b e l l , 1980). R e c e n t l y , a p o s s i b l e involvement of p h o s p h o l i p i d i n the c o u p l i n g between r e c e p t o r and a d e n y l a t e c y c l a s e has a l s o been p o s t u l a t e d ( H i r a t a and A x e l r o d , 1980). One may ask i f p h o s p h o l i p i d s a l s o p l a y a r o l e i n 2 t r a n s l a t i n g the incoming i n f o r m a t i o n in systems which do not employ cAMP as second messenger. One p o s s i b l e c a n d i d a t e for such a r o l e i s p h o s p h a t i d y l i n o s i t o l (P i ) ( M i c h e l l , 1975; Jones et a l . , 1982 ) . PI c o n t a i n s myo - i n o s i t o l , an e s s e n t i a l growth f a c t o r , i n the head group , and the 0 f a t t y a c y l c h a i n of mammalian PI i s u s u a l l y r i c h i n a r a c h i d o n i c a c i d , a r a t e -l i m i t i n g p r e c u r s o r i n the s y n t h e s i s of p r o s t a g l a n d i n s . P I , a l o n g w i t h i t s two p h o s p h o r y l a t e d d e r i v a t i v e s , p h o s p h a t i d y l i n o s i t o l - 4 - p h o s p h a t e (PIP) and p h o s p h a t i d y l i n o s i t o l 4 , 5 - b i s phosphate ( P I P 2 ) , c o n s t i t u t e about 2-12% of the t o t a l p h o s p h o l i p i d content i n v a r i o u s t i s s u e s (Rouser et a l . , 1968; G a l l i a r d , 1973 ; W h i t e , 1973), and i n most t i s s u e s the c o n c e n t r a t i o n of PI i s much g r e a t e r than i t s p h o s p h o r y l a t e d d e r i v a t i v e s . Even though they c o n s t i t u t e a very s m a l l f r a c t i o n of the t o t a l p h o s p h o l i p i d , a l l three i n o s i t o l p h o s p h o l i p i d s undergo a very r a p i d m e t a b o l i c t u r n o v e r ( G r i f f i n et a l • , 1979). Hokin and Hokin f i r s t observed i n 1953 t h a t s t i m u l a t i o n of p igeon pancreas by pancreozymin or c a r b a m y l c h o l i n e produced an enhanced i n c o r p o r a t i o n of l a b e l e d phosphate i n t o P I , i n a d d i t i o n to c a u s i n g an i n c r e a s e d amylase s e c r e t i o n (Hokin and H o k i n , 1953). In the f o l l o w i n g year s v a r i o u s o t h e r s t i m u l i i n d i f f e r e n t t i s s u e s were found to cause an enhanced i n c o r p o r a t i o n of l a b e l e d phosphate i n t o PI ( H o k i n , 1968, 1969; M i c h e l l , 1975), s i m i l a r to tha t observed by Hokin and Hokin (1953) i n p a n c r e a s . In the e a r l y s e v e n t i e s , a t r e n d began to e v o l v e i n the long l i s t of s t i m u l i showing PI t u r n o v e r . F i r s t l y , the response seemed to be a s s o c i a t e d o n l y w i t h those l i g a n d s tha t i n t e r a c t w i t h c e l l 3 sur face r e c e p t o r s and s e c o n d l y , o n l y w i t h those r e c e p t o r s tha t d i d not employ cAMP as t h e i r second messenger ( M i c h e l l , 1975). In the past decade, many groups have been i n v e s t i g a t i n g the p o s s i b i l i t y t h a t PI tu rnover may p l a y a r o l e i n r e c e p t o r -mediated s i g n a l t r a n s m i s s i o n . 1 . 1 DISTRIBUTION OF PI I f i n o s i t o l l i p i d s have a r o l e i n r e c e p t o r - m e d i a t e d s i g n a l t r a n s m i s s i o n they must be l o c a t e d i n the plasma membrane. P I , the major component of i n o s i t o l l i p i d , makes up about 2-10% of the t o t a l p h o s p h o l i p i d content i n the c e l l . The m a j o r i t y of t h i s PI i s p re sent i n v a r i o u s i n t r a c e l l u l a r o r g a n e l l e s , i n c l u d i n g endoplasmic r e t i c u l u m , outer m i t o c h o n d r i a l membrane and n u c l e a r membrane, and a s m a l l amount has been shown to be present i n the plasma membrane (Hok in-Neaver son , 1977; K i r k et a l . , 1981). In c o n t r a s t to P I , PIP and P I P 2 are c o n s i d e r e d to be present mos t ly i n the plasma membrane, based p r i n c i p a l l y on s t u d i e s done on m y e l i n (Soukup et a l . , 1978) and e r y t h r o c y t e membranes (Buckley and Hawthorne, 1972). The l a b i l e nature of these p o l y p h o s p h o i n o s i t i d e s makes t h e i r q u a n t i t a t i v e e s t i m a t i o n q u i t e d i f f i c u l t . The most r e l i a b l e va lue s were o b t a i n e d from the s t u d i e s u s i n g microwave i r r a d i a t i o n t e c h n i q u e s (Soukup et a l . , 1978). In t h r e e d i f f e r e n t r e g i o n s of b r a i n , Soukup et a l . (1978) r e p o r t e d v a l u e s of 0.1 - 0.13 Mmole PIP and 0.29 - 0.48 Mmole of P I P 2 per gram of t i s s u e . The p h o s p h o l i p i d a c t i n g as a l i n k between the r e c e p t o r and the i n s i d e of the c e l l s h o u l d 4 p r e f e r a b l y be l o c a t e d i n the c y t o p l a s m i c s i d e of the plasma membrane b i l a y e r . There i s no d i r e c t ev idence to suggest tha t i n o s i t o l l i p i d i n the plasma membrane i s l o c a t e d in the c y t o p l a s m i c s i d e , but the i n d i r e c t ev idence i n d i c a t i n g that the ATP p o o l of c y t o s o l can p r o v i d e phosphate for p h o s p h o r y l a t i o n of P I , suggests that i n o s i t o l l i p i d s may be l o c a t e d on the c y t o p l a s m i c s i d e of the membrane. Downes and M i c h e l l (1982) have c a l c u l a t e d that even when PIP and P I P 2 c o n s t i t u t e o n l y t r a c e q u a n t i t i e s of the t o t a l l i p i d f r a c t i o n , the p o l y p h o s p h o i n o s i t i d e c o n c e n t r a t i o n i n the inner l e a f l e t of the m y e l i n b i l a y e r c o u l d be as h i g h as 5 mM. 1.2 PI RESPONSE 1.2.1 C l o s e d C y c l e The i n c r e a s e d i n c o r p o r a t i o n of l a b e l e d phosphate i n t o PI observed on s t i m u l a t i o n of v a r i o u s r e c e p t o r s (PI response) c o u l d occur e i t h e r by an i n c r e a s e i n de novo s y n t h e s i s of PI or by i n c r e a s e d t u r n o v e r of the PI head group . Ev idence fo r the l a t t e r mechanism was f i r s t p r o v i d e d by Hokin and Hokin (1958) ; they found tha t in c e r e b r a l c o r t e x enhanced i n c o r p o r a t i o n of l a b e l e d phosphate and i n o s i t o l was not accompanied by an i n c r e a s e d i n c o r p o r a t i o n of l a b e l e d g l y c e r o l . T h i s i n d i c a t e d tha t the d i a c y l g l y c e r o l moiety may be r e u t i l i s e d w h i l e the p h o s p h o i n o s i t o l head group was renewed. D i r e c t measurement of PI c o n t e n t a l s o d i d not show an i n c r e a s e i n the s t i m u l a t e d t i s s u e , f u r t h e r s u p p o r t i n g the above mechanism. T h i s mechanism for PI response was r e c o n f i r m e d i n many other t i s s u e s by the 5 H o k i n s , i n c l u d i n g sympathet ic g a n g l i a ( H o k i n , 1969), pancreas ( H o k i n , 1968), a d r e n a l medul la (Hokin et a l . , 1958), s a l i v a r y g l and (Hokin and Sherwin , 1957), and l a t e r v e r i f i e d by many o ther g roups . One w e l l known e x c e p t i o n to the above mechanism of phosphate i n c o r p o r a t i o n i s the a d r e n a l c o r t e x , where ACTH has been shown to provoke a t w o - f o l d i n c r e a s e i n the a d r e n a l c o n t e n t s of p h o s p h a t i d i c a c i d , P I , P IP , P I P 2 1 p h o s p h a t i d y l g l y c e r o l and d i a c y l g l y c e r o l (Farese et a l . , 1979, 1980). T h i s e f f e c t of ACTH on the p h o s p h o l i p i d metabol i sm of a d r e n a l c o r t e x i s q u i t e d i f f e r e n t from the c l a s s i c a l PI re sponse . The g e n e r a l mechanism for PI t u r n o v e r i s shown i n F i g 1. Receptor s t i m u l a t i o n by a g o n i s t s causes the a c t i v a t i o n of P I -s p e c i f i c p h o s p h o l i p a s e C , r e s u l t i n g i n the breakdown of PI to d i a c y l g l y c e r o l and i n o s i t o l phosphate . D i a c y l g l y c e r o l i s p h o s p h o r y l a t e d by ATP to g ive p h o s p h a t i d i c a c i d , which f u r t h e r r e a c t s w i t h c y t i d i n e t r i p h o s p h a t e and i n o s i t o l to r e s y n t h e s i s e PI . F i g u r e 1 - PI tu rnover Reproduced from B e r r i d g e , 1980 6 1.2.2 The Pr imary Event Of PI Response U s u a l l y measured as an i n c r e a s e i n the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o p h o s p h a t i d i c a c i d and PI , i t was apparent l a t e r tha t the pr imary event of PI response was an i n c r e a s e d r a t e of breakdown of PI (or i t s p h o s p h o r y l a t e d d e r i v a t i v e s ) . The f i r s t r e p o r t of the l o w e r i n g of PI c o n c e n t r a t i o n was by Hokin and Hokin (1964) i n a v i a n s a l t g l and . But the g e n e r a l i t y of the above mechanism for PI response became apparent only a decade l a t e r , w i t h the r e p o r t s of D u r e l l et a l . (1969) , H o k i n -Neaverson , 1974; Jones and M i c h e l l (1974) . Though s t i l l to be proven i n many t i s s u e s where a PI response has been s t u d i e d , work based on p l a t e l e t s ( B e l l and M a j e r u s , 1980), pancreas (Bansback et a l . , 1974), b l o w f l y s a l i v a r y g l a n d , b r a i n and p a r o t i d g l and ( B e r r i d g e et a l . , 1982) suggested that the pr imary event of PI response was the i n c r e a s e d breakdown of PI by p h o s p h o l i p a s e C , g i v i n g r i s e to d i a c y l g l y c e r o l and i n o s i t o l phosphate . S t u d i e s w i t h i r i s muscle ( A b d e l - L a t i f et a l . , 1977, 1978; Akhtar and A b d e l - L a t i f , 1980) and more r e c e n t l y w i t h p l a t e l e t s (Agranof f et a l . , 1983; B e l l and M a j e r u s , 1980; B i l l a h and L a p e t i n a , 1982b; Imai et a l . , 1983) and h e p a t o c y t e s ( M i c h e l l et a l . , 1981) have suggested that p o l y p h o s p h o i n o s i t i d e breakdown r a t h e r than PI breakdown may c o n s t i t u t e the p r imary event (Downes and M i c h e l l , 1 9 8 2 ) , and the former may be the t a r g e t of p h o s p h o d i e s t e r a s e a c t i o n . 7 A c t i v a t i o n of Phospho l ipa se D as a mechanism of PI breakdown on a c e t y l c h o l i n e s t i m u l a t i o n of pancreas was once proposed by Hokin-Neaverson et a l . , (1975) . T h e i r c o n c l u s i o n s were based on the f i n d i n g tha t they c o u l d d e t e c t o n l y [ 3 H ] -i n o s i t o l as the w a t e r - s o l u b l e product i n a c e t y l c h o l i n e (ACh)-s t i m u l a t e d t i s s u e . I t now appears tha t the above r e s u l t c o u l d have been o b t a i n e d as a r e s u l t of h y d r o l y s i s of i n o s i t o l phosphate( s ) by i n o s i t o l phosphatase . When l i t h i u m , an i n h i b i t o r of i n o s i t o l phosphatase , was p re sent d u r i n g s t i m u l a t i o n of a PI re sponse , i t r e s u l t e d i n a l a r g e a c c u m u l a t i o n of i n o s i t o l monophosphate i n i n s e c t s a l i v a r y g l a n d , r a t b r a i n and p a r o t i d g l a n d ( B e r r i d g e et a l . , 1982). 1.2.3 A l t e r n a t e Mechanisms For L a b e l I n c o r p o r a t i o n I n c o r p o r a t i o n of l a b e l i n t o PI can o c c u r , and has been r e p o r t e d by mechanisms o ther than PI r e sponse . They are d e s c r i b e d b r i e f l y below. In s t u d i e s employing l a b e l e d f a t t y a c i d , i n c o r p o r a t i o n of l a b e l i n t o PI can be a f f e c t e d by the a c t i o n of p h o s p h o l i p a s e A 2 . D e a c y l a t i o n f o l l o w e d by r a p i d r e a c y l a t i o n w i t h a r a c h i d o n i c a c i d has been observed i n a d r e n o c o r t i c a l c e l l s (Schrey and R u b i n , 1979) and r a b b i t n e u t r o p h i l s (Rubin et a l . , 1981). Phospho l ipa se A 2 a c t i o n on PI can a l s o decrease the PI content s u b s t a n t i a l l y (Ri t tenhouse-Simmons, 1981), but the above e f f e c t can be d i s t i n g u i s h e d from p h o s p h o l i p a s e C a c t i o n by measuring the l e v e l of l y s o p h o s p h a t i d y l i n o s i t o l . A second mechanism of i n c r e a s i n g l a b e l i n t o PI i n v o l v e s the 8 i n c r e a s e d a v a i l a b i l i t y of d i a c y l g l y c e r o l . I t has been shown i n c e r t a i n t i s s u e s , such as p l a t e l e t s ( L a p e t i n a et a l • , 1981b; P r e s c o t t and Ma jerus , 1983) that the i n c r e a s e in d i a c y l g l y c e r o l observed i n the PI response was due to PI breakdown. D i a c y l g l y c e r o l can a l s o be o b t a i n e d from other sources e . g . t r i a c y l g l y c e r o l and other p h o s p h o l i p i d s . T h i s can then en te r the PI c y c l e , r e s u l t i n g i n i n c r e a s e d i n c o r p o r a t i o n of l a b e l i n t o P I . Only 20% of the i n c r e a s e i n d i a c y l g l y c e r o l l e v e l , o b t a i n e d d u r i n g the p i l o c a r p i n e s t i m u l a t i o n of p a n c r e a s , c o u l d be accounted for by i n c r e a s e d breakdown of P I , w h i l e the r e s t was c o n s i d e r e d to a r i s e from o ther sources (Bansback et a l . , 1974). The t h i r d p o s s i b l e pathway for i n c r e a s i n g l a b e l i n c o r p o r a t i o n i n t o PI i n v o l v e s i n c r e a s e d de novo s y n t h e s i s of p h o s p h a t i d i c a c i d from g l y c e r o l - 3 - p h o s p h a t e and f a t t y a c y l CoA, w i t h subsequent c o n v e r s i o n to P I . T h i s may be d i s t i n g u i s h e d from the c y c l i c PI tu rnover i n a number of ways. F i r s t l y , u n l i k e the m a j o r i t y of the c l a s s i c a l PI r e sponse , t h i s mechanism w i l l a l s o show an i n c r e a s e d i n c o r p o r a t i o n of l a b e l e d g l y c e r o l i n t o P I . S e c o n d l y , s i n c e p h o s p h a t i d i c a c i d i s a key i n t e r m e d i a t e of p h o s p h o l i p i d metabo l i sm, de novo i n c r e a s e i n the p h o s p h a t i d i c a c i d l e v e l w i l l be r e f l e c t e d by i n c r e a s e d i n c o r p o r a t i o n of l a b e l i n t o o ther p h o s p h o l i p i d s , as i n ACTH a c t i o n on a d r e n o c o r t i c a l c e l l s (Farese et a l . , 1980). The f o u r t h p o s s i b l e , but h i g h l y u n l i k e l y , way of i n c r e a s i n g i n o s i t o l i n c o r p o r a t i o n i n t o PI c o u l d be the r e s u l t of p h o s p h o l i p a s e D a c t i o n on P I . There i s no c o n v i n c i n g ev idence fo r the widespread o c c u r r e n c e of p h o s p h o l i p a s e D i n mammalian 9 t i s s u e . In s p i t e of i t s l i m i t a t i o n s , l a b e l i n c o r p o r a t i o n i n t o the PI head group i s a conven ient and s e n s i t i v e i n d i c a t o r of PI re sponse . In a l l t i s s u e s (except b l o w f l y s a l i v a r y g l and ( B e r r i d g e and F a i n , 1979)) where PI response i s now known to o c c u r , enhanced i n c o r p o r a t i o n of l a b e l i n t o the p h o s p h o i n o s i t o l moiety of PI has been the f i r s t s tep i n e s t a b l i s h i n g the re sponse . 1.2.4 P i - s p e c i f i c Phospho l ipa se C As mentioned i n a p r e v i o u s s e c t i o n , the pr imary event of PI response i s c o n s i d e r e d to be the enhanced breakdown of PI (Hokin and H o k i n , 1964; D u r e l l et a l . , 1969; Hokin-Neaverson 1974; Jones and M i c h e l l , 1974; M i c h e l l , 1975) or i t s p h o s p h o r y l a t e d d e r i v a t i v e s , PIP and P I P 2 (Abdel L a t i f et a l . , 1977, 1978; Akhtar and A b d e l - L a t i f , 1980; Agranof f et a l . , 1983; B i l l a h and L a p e t i n a , 1982b; Imai et a l . , 1983; M i c h e l l et a l . , 1981) by the a c t i o n of phospho l ipa se C or p o l y p o s p h o i n o s i t i d e p h o s p h o d i e s t e r a s e , r e s p e c t i v e l y , which h y d r o l y s e s the g l y c e r o p h o s p h a t e bond of the p h o s p h o l i p i d . P i - s p e c i f i c p h o s p h o l i p a s e C has been shown to be p re sen t i n v a r i o u s an imal t i s s u e s such as pancreas (Dawson, 1959), l i v e r (Kemp et a l . , 1961), guinea p i g i n t e s t i n a l mucosa ( A t h e r t o n and Hawthorne, 1968), among o t h e r s , as w e l l as i n c e r t a i n microorgani sms and p l a n t s ( for a complete l i s t of sources from which the enzyme has been c h a r a c t e r i z e d see S h u k l a , 1982). Most of the P i - s p e c i f i c p h o s p h o l i p a s e C e x i s t s i n the s o l u b l e form i n the c y t o p l a s m of 10 v a r i o u s t i s s u e s , and h y d r o l y s e s PI i n the presence of c a l c i u m ( A l l a n and M i c h e l l , 1974 ;Ather ton and Hawthorne, 1968; Dawson, 1959; Kemp et a l . , 1961; Thompson, 1967). A membrane bound form of the enzyme, that c o u l d p l a y an important r o l e i n PI t u r n o v e r , and which d i f f e r e d from s o l u b l e c y t o p l a s m i c p h o s p h o l i p a s e C , was d e s c r i b e d by F r i e d e l et a l . ( 1 9 6 9 ) and L a p e t i n a and M i c h e l l (1973) . However, I r v i n e and Dawson (1978) l a t e r p r o v i d e d e v i d e n c e , based on pH dependence of deoxycho la te a c t i v a t i o n and the use of l a c t a t e dehydrogenase as a c y t o p l a s m i c marker , tha t the membrane bound a c t i v i t y d e s c r i b e d i n b r a i n ( F r i e d e l et a l . , 1969; L a p e t i n a and M i c h e l l , 1973) c o u l d be accounted fo r by c y t o p l a s m i c c o n t a m i n a t i o n of the plasma membrane. Most of the a v a i l a b l e ev idence so far i n d i c a t e s the presence of a C a 2 + -dependent s o l u b l e c y t o p l a s m i c enzyme and a C a 2 + - independent ly so somal p h o s p h o l i p a s e C . The P i - s p e c i f i c p h o s p h o l i p a s e C enzyme from d i f f e r e n t sources showed some v a r i a t i o n i n i t s degree of s p e c i f i c i t y towards P I . The enzyme from guinea p i g i n t e s t i n a l mucosa (Ather ton and Hawthorne, 1968) and i r i s smooth muscle ( A b d e l -L a t i f et a l . , 1980) h y d r o l y s e d both PIP and P I P 2 , whereas the enzyme from S .aureus h y d r o l y z e d o n l y PI and not PIP or P I P 2 ( S h u k l a , 1982). Most of the s p e c i f i c i t y s t u d i e s i n v i t r o employ the o p t i m a l c o n d i t i o n s for the h y d r o l y s i s of PI and t h e r e f o r e do not r e f l e c t the i n v i v o s p e c i f i c i t y of the enzyme. In r a t l i v e r and k i d n e y , f i v e d i f f e r e n t forms of the enzyme, d i f f e r i n g i n t h e i r i s o e l e c t r i c p o i n t s , have been separa ted by column e l e c t r o f o c u s i n g (Hirasawa et a l . , 1982). T h i s h e t e r o g e n e i t y of 11 the c y t o p l a s m i c enzyme r a i s e s new q u e s t i o n s r e g a r d i n g the importance and f u n c t i o n of these v a r i o u s forms of enzyme. I t i s q u i t e p o s s i b l e that the assay c o n d i t i o n s and i s o l a t i o n t e c h n i q u e s used p r e v i o u s l y for c h a r a c t e r i z a t i o n of t h i s enzyme c o u l d have s e l e c t i v e l y favoured c e r t a i n forms of the enzyme over o t h e r s , and t h e r e f o r e c a u t i o n s h o u l d be e x e r c i s e d when comparing enzyme c h a r a c t e r i s t i c s from d i f f e r e n t l a b o r a t o r i e s . H y d r o l y s i s of PI by t h i s enzyme i s i n c r e a s e d by the presence of an o p t i m a l c o n c e n t r a t i o n of d e o x y c h o l a t e (Hofmann and M a j e r u s , 1982; I r v i n e and Dawson, 1978). PI h y d r o l y s i s by p h o s p h o l i p a s e C , o b t a i n e d from r a t b r a i n (Dawson et a l . , 1980) and sheep semina l v e s i c u l a r g l ands (Hofmann and M a j e r u s , 1982) was i n h i b i t e d by p h o s p h a t i d y l c h o l i n e and p o s i t i v e l y charged p r o t e i n s , which may account fo r the i n a b i l i t y of p h o s p h o l i p a s e C to r a p i d l y h y d r o l y s e membrane bound PI i n u n s t i m u l a t e d p r e p a r a t i o n s . U n s a t u r a t e d p h o s p h a t i d i c a c i d and u n s a t u r a t e d f a t t y a c i d s such as a r a c h i d o n i c a c i d (produced from d i a c y l g l y c e r o l d u r i n g the PI r e s p o n s e ) , were found to be capab le of s t i m u l a t i n g the enzyme a c t i v i t y . T h i s p r o v i d e s a p o s s i b l e s e l f - a m p l i f i c a t i o n mechanism fo r the PI response (Dawson et a l . , 1980). The h y d r o l y s i s of P I P 2 by e x t r a c t s of r a t b r a i n was d e s c r i b e d as e a r l y as 1964 (Thompson and Dawson, 1964a), but P I P 2 p h o s p h o d i e s t e r a s e has become more important w i t h r e s p e c t to PI response a f t e r recent s u g g e s t i o n s that p o l y p h o s p h o i n o s i t i d e h y d r o l y s i s , r a t h e r than PI h y d r o l y s i s , may be the t r i g g e r i n g event fo r the PI response ( M i c h e l l et a l . , 1981; Downes and 1 2 M i c h e l l , 1982). Data on the b r a i n (Thompson and Dawson, 1964b) and e r y t h r o c y t e (Downes and M i c h e l l , 1981) are c o n s i s t e n t w i t h one p h o s p h o d i e s t e r a s e a c t i v i t y h y d r o l y s i n g both PIP and P I P 2 , wi thout any e f f e c t on P I . L i k e P i - s p e c i f i c p h o s p h o l i p a s e C , t h i s enzyme r e q u i r e d c a l c i u m for i t s a c t i v i t y , and under c e r t a i n assay c o n d i t i o n s there seemed to be two l e v e l s of c a l c i u m requirement ( I r v i n e , 1982). 1.3 ROLE OF PI TURNOVER The widespread o c c u r r e n c e of the PI response has l e d to much s p e c u l a t i o n r e g a r d i n g i t s r o l e . The f i r s t h y p o t h e s i s r e g a r d i n g i t s involvement in e x o c y t o s i s i n e x o c r i n e g l and (Hokin and Sherwin , 1957; Hokin et a l . , 1958) was found to be l i m i t e d i n scope , as PI response was l a t e r found to occur i n both s e c r e t o r y and n o n - s e c r e t o r y c e l l s . In the l a t e s i x t i e s and e a r l y s e v e n t i e s , a r o l e for p o l y p h o s p h o i n o s i t i d e s i n the g e n e r a t i o n and p r o p a g a t i o n of a c t i o n p o t e n t i a l s i n s t i m u l a t e d neurons was proposed (Dawson, 1966; H e n d r i c k s o n and R e i n e r t s e n , 1971; T o r d a , 1972). The main f e a t u r e of a l l the above p r o p o s a l s had been tha t P I P 2 has a much h i g h e r a f f i n i t y for c a l c i u m than PIP (Hendr ickson and R e i n e r t s e n , 1969) and t h e r e f o r e , t h e i r i n t e r c o n v e r s i o n by p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n might a f f e c t the amount of c a l c i u m bound to the membrane. R e c o g n i t i o n tha t p h o s p h o l i p a s e C mediated breakdown of P I , r a t h e r than p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n of p o l y p h o s p h o i n o s i t i d e s , was the pr imary event of PI response i n many d i f f e r e n t systems l e d to r e e v a l u a t i o n of the r o l e of P I . M i c h e l l i n 1975 ( M i c h e l l , 1 3 1975), a f t e r r e v i e w i n g the e x i s t i n g l i t e r a t u r e on PI re sponse , c o n c l u d e d tha t the a v a i l a b l e ev idence suggested a r o l e fo r PI t u r n o v e r i n " c a l c i u m g a t i n g " . In the past e i g h t y e a r s , ev idence has been p r e s e n t e d both i n favour ( B e r r i d g e , 1980; F a i n and G a r c i a - S a i n z , 1980; M i c h e l l and K i r k , 1981 ; Putney ,1981 ,1982) and a g a i n s t ( C o c k c r o f t , 1981; Hawthorne, 1982) the above h y p o t h e s i s . In the f o l l o w i n g pages t h i s and two other h y p o t h e s e s , which have been suggested for the p h y s i o l o g i c a l r o l e of PI t u r n o v e r w i l l be a n a l y z e d . The o ther hypotheses are tha t PI t u r n o v e r i s a source of a r a c h i d o n i c a c i d and tha t PI t u r n o v e r i s a source of d i a c y l g l y c e r o l , which i s capab le of t r a n s m i t t i n g the hormonal or n e u r o t r a n s m i t t e r message a c r o s s the c e l l membrane through a c t i v a t i o n of p r o t e i n k inase C (PKC) . 1.4 PI AS A CALCIUM GATE The i n i t i a l support for the h y p o t h e s i s tha t PI f u n c t i o n e d as a c a l c i u m gate was h e a v i l y dependent on the f o l l o w i n g o b s e r v a t i o n s ( M i c h e l l , 1975): a) Receptor s whose s t i m u l a t i o n was accompanied by a PI response mediated t h e i r response through c a l c i u m m o b i l i z a t i o n ; b) PI response was independent of e x t r a c e l l u l a r c a l c i u m . The c o r r e l a t i o n between s t i m u l i p r o d u c i n g a PI response and m e d i a t i n g t h e i r response through c a l c i u m m o b i l i z a t i o n has indeed been ve ry good ( M i c h e l l , 1975, 1979). I f PI breakdown i s on ly and u n i v e r s a l l y i n v o l v e d i n r e c e p t o r mediated c a l c i u m g a t i n g , then a) i t shou ld be evoked i n a l l c e l l s and a l l t i s s u e s where 1 4 r e c e p t o r s t i m u l a t i o n leads to c a l c i u m m o b i l i z a t i o n and b) those r e c e p t o r s which employ o ther second messengers , such as cAMP, s h o u l d not produce PI breakdown (Jones et a l . , 1982; M i c h e l l , 1975; 1979). C e l l su r f ace r e c e p t o r s , such as m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s i n pancreas (Hokin and H o k i n , 1953), p a r o t i d g l and (Jones and M i c h e l l , 1974), vas de fe rens (Egawa et a l . , 1981), 5 -hydroxyt ryptamine i n b l o w f l y s a l i v a r y g l a n d ( F a i n and B e r r i d g e , 1979b), a l p h a - a d r e n e r g i c r e c e p t o r s i n p a r o t i d g l and ( M i c h e l l and Jones , 1974), and hear t (Gaut and Hugg ins , 1966) are j u s t a few of many d i f f e r e n t c e l l systems where PI response i s accompanied by C a 2 + m o b i l i z a t i o n ( fo r a more comprehensive l i s t see M i c h e l l 1975, M i c h e l l et a l . , 1981). One e x c e p t i o n to t h i s r u l e appears to be the PI response which accompanies the s t i m u l a t i o n of p i n e a l g land and sympathet ic g a n g l i a exposed to nerve growth f a c t o r (NGF) p r e p a r a t i o n s (Lakshmanan, 1978, 1979). However, M i c h e l l et a l . (1981) have p r o v i d e d arguments i n d i c a t i n g tha t as the r o l e of C a + 2 as a second messenger i n the a c t i o n of NGF i s s t i l l not c l e a r (Schubert et a l . , 1978; L a n d r e t h et a l • , 1980), the above e x c e p t i o n shou ld be t r e a t e d w i t h r e s e r v a t i o n . Another e x c e p t i o n i s a d r e n a l c o r t e x , where the n i c o t i n i c r e c e p t o r m o b i l i z e s c a l c i u m but where i t i s m u s c a r i n i c s t i m u l a t i o n which produces the PI r e sponse . Only two s t i m u l i , g lucose and c a l c i u m ionophore ( d e s c r i b e d i n g r e a t e r d e t a i l i n the f o l l o w i n g s e c t i o n ) , enhance l i p i d l a b e l l i n g wi thout a c t i n g at a c e l l s u r f a c e r e c e p t o r ( M i c h e l l et a l . , 1981) 15 F i g u r e 2 - PI as a c a l c i u m gate Reproduced from B e r r i d g e , 1980 Three possible models for linking the PI response to calcium gating. The main components of the system (shown on the left) are a receptor (R), phospholipase C (PhC), a calcium gale and phosphatidylinositol (PI). The diagrams on the right illustrate three ways in which agonists might act to enhance calcium permeability (a) The agonist occupies the receptor (R) which somehow activates phospholipase C (Ph. C) to hydrolyse PI to diglyceride (DC) and the gate opens, (b) The activated receptor acts directly on the gate. As the gale opens there are confor-mational changes in the surrounding bilayer which alio wsPh C'. to hydrolyse PI as part of a closing mechanism .(c) The activated receptor stimulates Ph.Cto hydrolyse PI to DC The latter is rapidly phosphorylaled to phosphatidic acid (PA) which functions as a calcium ionophore. 16 1.4.1 C a l c i u m Requirement Of PI Response P i - s p e c i f i c p h o s p h o l i p a s e C r e q u i r e s micromolar c a l c i u m c o n c e n t r a t i o n s for i t s a c t i v i t y . The q u e s t i o n i s whether a c t i v a t i o n of P i - s p e c i f i c p h o s p h o l i p a s e C , which causes PI breakdown, o c c u r s before or a f t e r c a l c i u m e n t e r s the c e l l . Many of the r e p o r t e d s t u d i e s (Oron et a l . , 1975; G r i f f i n et a l . , 1979; T r i f a r o , 1969b) have used l a b e l e d phosphate i n c o r p o r a t i o n i n t o p h o s p h a t i d i c a c i d and PI as a measure of PI r e sponse . C o n c l u s i o n s r e g a r d i n g the c a l c i u m requirement of PI response based on such s t u d i e s ( G r i f f i n et a l . , 1979; Lennon and S t e i n b e r g , 1973) are d i f f i c u l t to i n t e r p r e t , as they do not i n d i c a t e whether the pr imary event of PI breakdown i s c a l c i u m -dependent or n o t . A new d imens ion of c o m p l e x i t y to the whole problem has been added by recent r e p o r t s s u g g e s t i n g that t h e r e may be both ca l c ium-dependent and c a l c i u m independent PI t u r n o v e r (Egawa et a l . , 1981; Fare se et a l . , 1982), as w e l l as an a d d i t i o n a l mechanism for the l o s s of PI ( B i l l a h and L a p e t i n a , 1982a). Two methods u s u a l l y used to s tudy the c a l c i u m dependence of PI breakdown a r e : a) a d d i t i o n of an ionophore and b) r e g u l a t i o n of the i n t r a c e l l u l a r c a l c i u m by means of c a l c i u m c h e l a t i n g a g e n t s . E f f e c t of ionophore : Ionophores , such as A23187, i n c r e a s e the p e r m e a b i l i t y of the c e l l to c a l c i u m , thereby i n c r e a s i n g the c y t o s o l i c c a l c i u m c o n c e n t r a t i o n . The r a t i o n a l e behind such an exper iment i s t h a t i f PI breakdown i s the cause r a t h e r than e f f e c t of c a l c i u m m o b i l i z a t i o n , the i n c r e a s e i n c y t o s o l i c c a l c i u m w i t h ionophore s h o u l d not cause the PI breakdown. In 1 7 many t i s s u e s ionophore-media ted c a l c i u m i n c r e a s e f a i l e d to produce PI breakdown. Some of the examples are p a r o t i d g l and (Jones and M i c h e l l , 1975), b l o w f l y s a l i v a r y g l and ( F a i n and B e r r i d g e , 1977a) and hepa tocy te s ( B i l l a h and M i c h e l l , 1979). In pancreas and p l a t e l e t s , ev idence has been p r e s e n t e d both for ( p l a t e l e t s - B i l l a h and L a p e t i n a , 1982a; pancreas - H o k i n -Neaverson ,1977) and a g a i n s t ( p l a t e l e t s - Imai and Nazawa, 1982; pancreas - Farese et a l . , 1980) a C a 2 + i onophore-media ted breakdown of P I , whereas i n po lymorphonuclear l e u k o c y t e s ( C o c k c r o f t et a l . , 1981) and vas de fe rens (Egawa et a l . , 1981) a ca l c ium-dependent PI breakdown has been r e p o r t e d . An ionophore-mediated PI breakdown has to s a t i s f y the f o l l o w i n g requi rements before a PI response can be c o n s i d e r e d as a c a l c i u m - m e d i a t e d phenomenon; a) tha t the i n c r e a s e i n c y t o s o l i c c a l c i u m c o n c e n t r a t i o n must be i n the range o b t a i n e d d u r i n g r e c e p t o r s t i m u l a t i o n and b) the observed PI l o s s must occur by the same mechanism as suggested for the PI re sponse . At p re sen t such data are a v a i l a b l e o n l y for n e u t r o p h i l s ( C o c k c r o f t et a l . , 1981). S t i m u l a t i o n of n e u t r o p h i l s w i t h fMet-Leu-Phe produced a h a l f maximal s e c r e t i o n and PI breakdown ( h a l f maximal at 0.3 nM), which was c a l c i u m - d e p e n d e n t . N e u t r o p h i l s s t i m u l a t e d w i t h 1 nM fMet-Leu-Phe r e q u i r e d 100 MM c a l c i u m to produce h a l f maximal e f f e c t s i n both s e c r e t i o n and PI breakdown. The i o n o p h o r e , i o n o m y c i n , produced n e u t r o p h i l s e c r e t i o n and PI breakdown over a s i m i l a r c a l c i u m c o n c e n t r a t i o n range , and i o n o m y c i n - s t i m u l a t e d PI breakdown was accompanied by i n c r e a s e d phosphate i n c o r p o r a t i o n i n t o p h o s p h a t i d i c a c i d and P I , s u g g e s t i n g that the PI breakdown 18 was at l e a s t p a r t i a l l y t h r o u g h a c t i v a t i o n of p h o s p h o l i p a s e C . However, the r e s u l t does not c o m p l e t e l y negate the p o s s i b i l i t y of a r e c e p t o r - m e d i a t e d c a l c i u m - i n d e p e n d e n t PI breakdown i n n e u t r o p h i l s . Recent r e p o r t s tha t i n two t i s s u e s , r a t s u b m a x i l l a r y g l a n d , ( F a r e s e et a l . , 1982) and r a b b i t vas de fe rens (Egawa et a l . , 1981), t h e r e i s both ca l c ium-dependent and c a l c i u m independent r e c e p t o r - m e d i a t e d PI t u r n o v e r suggests tha t demons t ra t ion of ca l c ium-dependent PI breakdown i n a t i s s u e i s not enough to e l i m i n a t e the occurence of a r e c e p t o r mediated c a l c i u m independent PI breakdown. In c o n t r a s t to P I , where r e c e p t o r - m e d i a t e d breakdown was found to be c a l c i u m independent i n a l a r g e number of t i s s u e s ( B i l l a h and M i c h e l l , 1979; F a i n and B e r r i d g e , 1979a; Jones and M i c h e l l , 1979), the breakdown of p o l y p h o s p h o i n o s i t i d e , s p e c i f i c a l l y P I P 2 , by a c e t y l c h o l i n e (ACh) was a b o l i s h e d i n i r i s smooth muscle i n a c a l c i u m d e f i c i e n t medium ( A b d e l - L a t i f et a l . , 1978) and enhanced by c a l c i u m ionophore i n i r i s smooth muscle ( A b d e l - L a t i f et a l . , 1978), e r y t h r o c y t e (Lang et a l . , 1 9 7 7 ) , synaptosomes ( G r i f f i n and Hawthorne, 1978) and b r a i n ( J o l l e s et a l . , 1981). The o n l y e x c e p t i o n to t h i s was the r a p i d breakdown of P I P 2 observed i n h e p a t o c y t e s ( M i c h e l l et a l . , 1981) and p a r o t i d g l a n d (Weiss et a l . , 1982b), which o c c u r r e d over a p e r i o d of a few seconds and was u n a f f e c t e d (or s l i g h t l y decrea sed i n p a r o t i d g land) by c a l c i u m d e p r i v a t i o n , and was a l s o not observed w i t h i o n o p h o r e . Weiss et a l . (1982b) have r e c e n t l y p r e s e n t e d some v e ry i n t e r e s t i n g and c o n v i n c i n g data to suggest tha t p o l y p h o s p h o i n o s i t i d e breakdown may be r e s p o n s i b l e 19 for c a l c i u m m o b i l i z a t i o n i n the p h a s i c response of p a r o t i d c e l l s . 1.4.2 P h o s p h a t i d i c A c i d As A C a l c i u m Ionophore D i a c y l g l y c e r o l , o b t a i n e d d u r i n g the breakdown of i n o s i t o l l i p i d s ( I g a r a s h i and Kondo, 1980; B i l l a h and L a p e t i n a , 1982a), i s r a p i d l y c o n v e r t e d to p h o s p h a t i d i c a c i d by d i g l y c e r i d e k i n a s e , an enzyme a l s o l o c a t e d i n the plasma membrane ( L a p e t i n a and Hawthorne, 1971). P h o s p h a t i d i c a c i d has been found to accumulate d u r i n g PI response i n many d i f f e r e n t systems, i n c l u d i n g s a l t g l a n d , pancreas and p l a t e l e t s . A f t e r M i c h e l l ' s i n i t i a l p r o p o s a l ( M i c h e l l , 1975) that the PI response may be i n v o l v e d i n c a l c i u m g a t i n g , i t was very n a t u r a l to ask whether p h o s p h a t i d i c a c i d (accumulated d u r i n g PI response) c o u l d ac t as a c a l c i u m g a t e . The i n i t i a l h y p o t h e s i s tha t p h o s p h a t i d i c a c i d a c c u m u l a t i o n may a l t e r the p h y s i c a l p r o p e r t i e s of the membrane, thereby a l t e r i n g the h y p o t h e t i c a l c a l c i u m gate ( Keryer et a l . , 1979; M i c h e l l et a l . , 1977) , was too unde f ined to be of p r a c t i c a l u se . The second h y p o t h e s i s , which was f i r s t proposed and r e j e c t e d by M i c h e l l et a l . (1977) based on t h e i r s t u d i e s i n red b lood c e l l s , was that p h o s p h a t i d i c a c i d f u n c t i o n s as a c a l c i u m i o n o p h o r e . Many d i f f e r e n t l i n e s of a v a i l a b l e ev idence t end to support such a r o l e fo r p h o s p h a t i d i c a c i d . P h o s p h a t i d i c a c i d has been shown to ac t as a c a l c i u m ionophore i n the Pressman chamber (Tyson et a l . , 1976) and i n l iposomes (Serhan et a l . , 1981). I t i s i n t e r e s t i n g to note t h a t w h i l e PI and a r a c h i d o n i c 20 a c i d f a i l e d to enhance c a l c i u m t r a n s p o r t i n t o l i po somes , p h o s p h a t i d i c a c i d and 8 , 1 1 , 1 4 - e i c o s a t r i e n o i c a c i d , two i n t e r m e d i a t e s o b t a i n e d d u r i n g PI re sponse , i n c r e a s e d c a l c i u m uptake i n t o the l iposomes (Serhan et a l . , 1981). P h o s p h a t i d i c a c i d t r a n s l o c a t e d d i v a l e n t c a t i o n s w i t h the f o l l o w i n g order of s e l e c t i v i t y ; Mn > Ca > Sr >> Mg. Liposomes wi th a p h o s p h a t i d i c a c i d c o n c e n t r a t i o n of 1-5 mole % of t o t a l l i p i d t r a n s l o c a t e d c a l c i u m but not magnesium (Serhan et a l . , 1981). N o n - b i l a y e r fo rmat ion i n model membrane c o n t a i n i n g p h o s p h a t i d i c a c i d i n the presence of c a l c i u m and o ther d i v a l e n t c a t i o n s ( V e r k l e i j et a l . , 1982) c o u l d e x p l a i n the i o n o p h o r i c p r o p e r t y of p h o s p h a t i d i c a c i d ( C u l l i s et a l . , 1980). In p a r o t i d g l a n d , a d i f f e r e n t l i n e of ev idence has been p r e s e n t e d to support the r o l e of p h o s p h a t i d i c a c i d as a c a l c i u m gate (Putney et a l . , 1980). D i s s o c i a t i o n c o n s t a n t s of v a r i o u s c a l c i u m a n t a g o n i s t s ( e . g . L a 3 + , T m 3 + , neomycin, C o 2 + , N i 2 + , M g 2 + ) , which i n h i b i t r e c e p t o r - s t i m u l a t e d c a l c i u m uptake by compet ing wi th c a l c i u m for the c a l c i u m channe l d u r i n g PI re sponse , c o r r e l a t e d very w e l l w i t h the i n h i b i t o r y c o n s t a n t s o b t a i n e d for the same substances d u r i n g the i n h i b i t i o n of p h o s p h a t i d i c a c i d - i n d u c e d p a r t i t i o n i n g of a 5 C a from water i n t o an o r g a n i c phase (Putney et a l . , 1980). The a m p h i p h i l i c nature of p h o s p h a t i d i c a c i d makes the i n c o r p o r a t i o n of exogenously added p h o s p h a t i d i c a c i d i n t o membrane somewhat d i f f i c u l t . Even t h e n , there have been some s t u d i e s i n d i c a t i n g that exogenously added p h o s p h a t i d i c a c i d was a b l e to mimic the e f f e c t of c a l c i u m - m o b i l i z i n g a g o n i s t s . In 21 neuroblastoma c e l l s (Ohsako and D e g u c h i , 1981) a d d i t i o n of as l i t t l e as 1 yg/ml of p h o s p h a t i d i c a c i d exogenous ly , or p r o d u c t i o n of p h o s p h a t i d i c a c i d in s i t u by p h o s p h o l i p a s e C t r e a t m e n t , caused an i n c r e a s e i n the l e v e l of cGMP by a c t i v a t i o n of ( c a l c i u m - s t i m u l a t e d ) guany la te c y c l a s e . Other systems i n c l u d e smooth musc le , p l a t e l e t microsomes ( G e r r a r d et a l . , 1978) and synaptosomes ( H a r r i s et a l . , 1981). On the o ther hand, exogeneously added p h o s p h a t i d i c a c i d has no e f f e c t on p l a t e l e t a g g r e g a t i o n ( G e r r a r d et a l . , 1978) or ca l c ium-dependent pota s s ium r e l e a s e (Putney, 1981). 1.4.3 Is PI Degraded From Plasma Membrane? One of the problems i n a c c e p t i n g PI breakdown as a c a l c i u m g a t i n g h y p o t h e s i s i s the l a c k of ev idence to suggest tha t the PI b e i n g degraded comes from the plasma membrane. Attempts to determine the s i t e of l o s s of PI i n s t i m u l a t e d c e l l s have g i v e n v a r i a b l e r e s u l t s . In pancreas and sympathet ic g a n g l i a , based on d i f f e r e n t i a l c e n t r i f u g a t i o n (Redman and H o k i n , 1959; Gerber e_t a l . , 1973) and a u t o r a d i o g r a p h y (Hokin and Huebner , 1967; Hokin 1965), the s i t e of PI t u r n o v e r was found to be endoplasmic r e t i c u l u m . The l o s s appeared to be from s e c r e t o r y v e s i c l e membranes i n e l e c t r i c a l l y s t i m u l a t e d synaptosomes ( P i c k a r d and Hawthorne, 1978) and g l u c o s e - s t i m u l a t e d i s l e t of Langerhans (Clements et a l . , ( 1977 ) , from plasma membrane i n a c e t y l c h o l i n e -s t i m u l a t e d s a l t g l and (Hok in-Neaver son , 1977) and fMeth-Leu-Phe-s t i m u l a t e d n e u t r o p h i l s (Bennett et a l . , 1982). By c o n t a s t , the s i t e of PI l o s s c o u l d not be l o c a t e d i n v a s o p r e s s i n - s t i m u l a t e d 22 hepa tocy te s and t h i s was a t t r i b u t e d to the r a p i d e q u i l i b r a t i o n of PI among v a r i o u s s u b c e l l u l a r p o o l s ( K i r k et a l . , 1981). I f the pr imary event of the PI response i s the breakdown of p o l y p h o s p h o i n o s i t i d e s , as suggested by M i c h e l l and coworkers r e c e n t l y ( M i c h e l l et a l . , 1981; Downes and M i c h e l l , 1982), then most p r o b a b l y the pr imary event of i n o s i t o l l i p i d breakdown does occur at the plasma membrane, as p o l y p h o s p h o i n o s i t i d e s have been shown to be p r e s e n t o n l y i n the plasma membrane. 1.5 DIACYLGLYCEROL AS AN ACTIVATOR OF PROTEIN KINASE C P h o s p h o l i p i d - d e p e n d e n t c a l c i u m - a c t i v a t e d p r o t e i n k inase C (PKC) , which was f i r s t d e s c r i b e d by N i s h i z u k a ' s group (Taka i et a l . , 1979a) has now been shown to occur i n n e u t r o p h i l s (Helfman et a l . , 1983), smooth muscle (Endo et a l . , 1982), lymphocytes (Ku et a l . , 1981), r a t pancreas (Tanigawa et a l . , 1982) and many o ther t i s s u e s (Minakuchi et a l . , 1981). An involvement of PI t u r n o v e r i n p r o t e i n k inase C a c t i v a t i o n was f i r s t p o s t u l a t e d when i t was observed tha t the presence of u n s a t u r a t e d d i a c y l g l y c e r o l i n the i n c u b a t i o n medium s h i f t e d the a f f i n i t y of the enzyme fo r c a l c i u m from the mM to the MM range (K i sh imoto e_t a l . , 1980; T a k a i et a l . , 1979b). T h i s s h i f t i n c a l c i u m a f f i n i t y was g r e a t e s t w i t h d i a c y l g l y c e r o l c o n t a i n i n g u n s a t u r a t e d f a t t y a c i d i n the /3 carbon (the (S carbon of PI i s a l s o e n r i c h e d i n p o l y u n s a t u r a t e d f a t t y a c i d ) , whereas the s a t u r a t e d d i a c y l g l y c e r o l s were found to be c o m p l e t e l y i n e f f e c t i v e (K i sh imoto et a l . , 1980). A l t h o u g h p h o s p h a t i d y l s e r i n e was e s s e n t i a l f o r the PKC a c t i v i t y , o ther p h o s p h o l i p i d s were capab le 23 of f u r t h e r modula t ion of i t s a c t i v i t y i n the presence of p h o s p h a t i d y l s e r i n e ( K a i b u c h i et a l . , 1981). Whi le p h o s p h a t i d y l e t h a n o l a m i n e (20 Mg/ml) i n c r e a s e d the PKC a c t i v i t y by 50%, PI and p h o s p h a t i d i c a c i d (two other i n t e r m e d i a t e s of PI response) had no e f f e c t , and 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 markedly d i m i n i s h e d the enzyme a c t i v i t y ( K a i b u c h i et a l . , 1981) . F u r t h e r ev idence for the involvement of PI t u r n o v e r i n PKC a c t i v a t i o n was p r o v i d e d r e c e n t l y i n p l a t e l e t s ( Ieyasu et a l . , 1982; K a i b u c h i et a l . , ( 1 9 8 2 b ) • A c t i v a t i o n of p l a t e l e t s w i t h t h r o m b i n , c o l l a g e n or p l a t e l e t a c t i v a t i n g f a c t o r ( a l l three produced a PI r e s p o n s e ) , caused a t r a n s i e n t i n c r e a s e i n d i a c y l g l y c e r o l l e v e l , f o l l o w e d by a s imul taneous p h o s p h o r y l a t i o n of a 40,000 m o l e c u l a r weight (40K) p r o t e i n and s e r o t o n i n r e l e a s e ( Ieyasu et a l . , 1982; K a i b u c h i et a l . , 1982b). The same 40K p r o t e i n was a l s o p h o s p h o r y l a t e d d u r i n g d i r e c t a c t i v a t i o n of p l a t e l e t s w i t h 1 - o l e y l - 2 - a c e t y l d i a c y l g l y c e r o l (DAG) (Mori et a l  . , 1982; K a i b u c h i et a l . , 1983). T h i s p h o s p h o r y l a t i o n of the 40K p r o t e i n by DAG was not accompanied by PI breakdown or s e r o t o n i n r e l e a s e , and t h e r e f o r e was c o n s i d e r e d to occur by d i r e c t a c t i v a t i o n of PKC by the s y n t h e t i c d i a c y l g l y c e r o l (Mori et a l . , 1982). I n i t i a l l y i t was proposed that as d i a c y l g l y c e r o l can cause a s h i f t i n the a f f i n i t y of PKC to the micromolar l e v e l of c a l c i u m , d i a c y l g l y c e r o l formed d u r i n g PI response c o u l d mediate p h y s i o l o g i c a l responses wi thout c a u s i n g c a l c i u m i n f l u x (L imas , 1980; K i s h i m o t o et a l . , 1980). Recent exper iments c l e a r l y i n d i c a t e , however, tha t at l e a s t i n p l a t e l e t s , both 24 c a l c i u m m o b i l i z a t i o n and d i a c y l g l y c e r o l fo rmat ion are r e q u i r e d for complete p h y s i o l o g i c a l response ( i . e . s e r o t o n i n r e l e a s e ) ( K a i b u c h i et a l . , 1982b, 1983) In model systems c o n t a i n i n g p h o s p h a t i d y l c h o l i n e / p h o s p h a t i d y l s e r i n e m i x t u r e s , d i a c y l g l y c e r o l (from yea s t ) s h i f t e d the phase s e p a r a t i o n curve to a lower c a l c i u m c o n c e n t r a t i o n , c a u s i n g 75% phase s e p a r a t i o n at 10 MM c a l c i u m . In compar i son , PI from yeas t produced o n l y 7% phase s e p a r a t i o n at the same c a l c i u m c o n c e n t r a t i o n (Ohki et a l . , 1981). D i a c y l g l y c e r o l -mediated phase s e p a r a t i o n of p h o s p h a t i d y l s e r i n e may i n d i c a t e a p o s s i b l e mechanism for the a c t i v a t i o n of PKC (Ohki et a l . , 1981). Most of the work done i n t h i s f i e l d i s from N i s h i z u k a ' s group , and the o b s e r v a t i o n s are c o n s i s t e n t w i t h the h y p o t h e s i s tha t PI t u r n o v e r may be i n v o l v e d i n the a c t i v a t i o n of PKC. 1.6 PI AS A SOURCE OF ARACHIDONIC ACID The 0 carbon of PI from v a r i o u s mammalian t i s s u e s i s r i c h i n a r a c h i d o n i c a c i d ( 5 , 8 , 1 1 , 1 4 - i c o s a t e t r a e n o i c a c i d ; W h i t e , 1973), and i t i s now w e l l e s t a b l i s h e d that a r a c h i d o n i c a c i d i s the r a t e l i m i t i n g f a c t o r i n the fo rmat ion of e i c o s a n o i d s (Bergstrom et a l . , 1964; van Dorp et a l . , 1964). T h e r e f o r e , i t i s l o g i c a l to p o s t u l a t e that PI t u r n o v e r may be i n v o l v e d i n the p r o d u c t i o n of " f r e e " a r a c h i d o n i c a c i d , which can then mediate the p h y s i o l o g i c a l response through i t s c o n v e r s i o n to e i c o s a n o i d s ( L a p e t i n a et a l . , 1981a; M a r s h a l l et a l . , 1980, 1982). PI can generate f ree a r a c h i d o n i c a c i d by two mechanisms: a) by the d i r e c t a c t i o n of phospho l ipa se A 2 on PI (Hong and D e y k i n , 1981; 25 Rubin et a l . , 1981; Walsh et a l . , 1981), b) d u r i n g PI re sponse , by the a c t i o n of d i a c y l g l y c e r o l l i p a s e on d i a c y l g l y c e r o l ; m o n o a c y l g l y c e r o l l i p a s e on m o o a c y l g l y c e r o l ( P r e s c o t t and Ma je rus , 1983) or p h o s p h o l i p a s e A 2 a c t i o n on p h o s p h a t i d i c a c i d ( L a p e t i n a et a l . , 1981b). I f the a r a c h i d o n y l group i s h y d r o l y z e d d u r i n g PI t u r n o v e r , then i t would not be conserved d u r i n g PI re sponse , as was o r i g i n a l l y p roposed . I n i t i a l o b s e r v a t i o n s , based on the i n c o r p o r a t i o n of l a b e l e d g l y c e r o l , phosphate and i n o s i t o l d u r i n g PI response c o n c l u d e d t h a t w h i l e the p h o s p h o i n o s i t o l moiety underwent renewal the d i a c y l g l y c e r o l moiety was c o n s e r v e d . At l e a s t i n p l a t e l e t s , the f a t t y a c i d a c y l c h a i n was not conse rved d u r i n g t h r o m b i n - s t i m u l a t e d PI t u r n o v e r ( P r e s c o t t and M a j e r u s , 1981). Comparison of the f a t t y a c i d c o m p o s i t i o n of PI i n human p l a t e l e t s be fore and a f t e r thrombin s t i m u l a t i o n (and even a f t e r r e c o v e r y of PI to o r i g i n a l l e v e l s ) i n d i c a t e d that the l a t t e r c o n t a i n e d a g r e a t e r amount of o l e a t e and l i n o l e a t e , and a lower amount of a r a c h i d o n a t e ( P r e s c o t t and M a j e r u s , 1981). T h i s suggested tha t a) a r a c h i d o n i c a c i d was not b e i n g conse rved d u r i n g t h r o m b i n - i n d u c e d PI t u r n o v e r and b) PI was not b e i n g r e s y n t h e s i z e d w i t h the c h a r a c t e r i s t i c of 1 - s t e a r o y l - 2 - a r a c h i d o n y l f a t t y a c i d c o m p o s i t i o n , which may f i n a l l y be produced by a d e a c y l a t i o n -r e a c y l a t i o n c y c l e . S t i m u l a t i o n of a r a c h i d o n y l - p r e l a b e l e d t h y r o i d f o l l i c l e s w i t h t h y r o t r o p i n produced a r a p i d and t r a n s i e n t a c c u m u l a t i o n of l a b e l e d d i a c y l g l y c e r o l ( I g a r s h i and Kondo, 1980). I t was f u r t h e r proposed (but no ev idence was p r e s e n t e d ) , t h a t t h i s 26 d i a c y l g l y c e r o l c o u l d be ac ted upon by d i a c y l g l y c e r o l l i p a s e present i n the t h y r o i d plasma membrane ( I g a r s h i and Kondo, 1980) to l i b e r a t e f ree a r a c h i d o n i c a c i d , thereby r e s u l t i n g i n t r a n s i e n t s y n t h e s i s of p r o s t a g l a n d i n . In mouse p a n c r e a s , M a r s h a l l et a l . (1980, 1982) have p re sen ted ev idence to show that p r o s t a g l a n d i n s d e r i v e d from a r a c h i d o n y l r e s i d u e s d u r i n g PI breakdown can cause amylase r e l e a s e . Only h a l f of the r a d i o a c t i v i t y i n a r a c h i d o n y l - p r e l a b e l e d PI l o s t d u r i n g c a r b a c h o l s t i m u l a t i o n was r e c o v e r e d i n p h o s p h a t i d i c a c i d and the o ther h a l f was found i n a r a c h i d o n i c a c i d m e t a b o l i t e s . T h i s suggested on ly that the a r a c h i d o n i c a c i d used i n p r o s t a g l a n d i n s y n t h e s i s was d e r i v e d from P I , but does not n e c e s s a r i l y i n d i c a t e t h a t i t was o b t a i n e d d u r i n g PI tu rnover ( i . e from d i a c y l g l y c e r o l or p h o s p h a t i d i c a c i d ) , as i m p l i e d by the a u t h o r s . An a l t e r n a t e p o s s i b i l i t y which w i l l g i v e a s i m i l a r r e s u l t i s tha t of a d i r e c t a c t i o n of p h o s p h o l i p a s e A 2 on P I , r e c e n t l y suggested to occur i n many t i s s u e s ( B i l l a h and L a p e t i n a , 1982a; Hong and D e y k i n , 1981; Schwartzman et a l . , 1981; Walsh et a l . , 1981). On the o ther hand, L i t o s c h et a l . (1982) have made the p r o b a b l y i n c o r r e c t assumption that the decrease of a r a c h i d o n y l -p r e l a b e l e d PI on 5-hydroxytryptamine s t i m u l a t i o n of b l o w f l y s a l i v a r y g l a n d s h o u l d n e c e s s a r i l y r e s u l t i n r e l e a s e of f ree a r a c h i d o n i c a c i d . 5-Hydroxytryptamine s t i m u l a t i o n of b l o w f l y s a l i v a r y g l a n d caused a 54% decrease i n both a r a c h i d o n y l or p h o s p h a t e - p r e l a b e l e d P I , accompanied by s a l i v a r y g l and s e c r e t i o n ( L i t o s c h et a l . , 1982). The decrease i n a r a c h i d o n y l - p r e l a b e l e d PI can be s imply e x p l a i n e d by i t s c o n v e r s i o n to d i a c y l g l y c e r o l , 27 as p h o s p h a t e - p r e l a b e l e d PI decreased by the same e x t e n t , r a t h e r than by the r e l e a s e of f r ee a r a c h i d o n i c a c i d . I t i s p o s s i b l e that a r a c h i d o n i c a c i d was be ing conserved and not r e l e a s e d d u r i n g 5 - h y d r o x y t r y p t a m i n e - s t i m u l a t e d PI t u r n o v e r i n b l o w f l y s a l i v a r y g l a n d , and i n tha t case i t would not be s u p r i s i n g that the au thor s c o u l d not see any of the 5 -hydroxyt ryptamine re sponses , such as c a l c i u m uptake or s a l i v a r y g l and s e c r e t i o n , w i t h exogeneously added a r a c h i d o n i c a c i d . 1.7 INHIBITORS OF PI RESPONSE One main h u r d l e i n s o l v i n g the PI " p u z z l e " i s the l a ck of a s p e c i f i c i n h i b i t o r of PI t u r n o v e r . Walenga et a l . (1980) found that s e r i n e pro tea se i n h i b i t o r s , such as d a n s y l f l u o r i d e , p h e n y l m e t h a n e s u l f o n y l f l u o r i d e (PMSF), 2 - n i t r o - 4 - c a r b o x y p h e n y l -N ,N-d ipheny lca rbamate and p - n i t r o p h e n y l a n t h r a n i l a t e , i n h i b i t e d s t i m u l u s - i n d u c e d m o b i l i z a t i o n of a r a c h i d o n i c a c i d . Walenga et a l . (1980) suggested t h a t t h i s e f f e c t of s e r i n e e s t e r a s e i n h i b i t o r was at l e a s t p a r t l y due to the i n h i b i t i o n of P I -s p e c i f i c p h o s p h o l i p a s e C , which would r e s u l t i n a decrease i n the l e v e l of d i a c y l g l y c e r o l , the s u b s t r a t e for d i a c y l g l y c e r o l l i p a s e and a proposed source of a r a c h i d o n i c a c i d ( I g a r a s h i and Kondo, 1980; M a r s h a l l et a l . , 1980, 1982). S e r i n e e s t e ra se i n h i b i t o r s (mentioned above) i n h i b i t e d the p r o d u c t i o n of m a l o n d i a l d e h y d e , a m e t a b o l i c product of p r o s t a g l a n d i n i n p l a t e l e t s , i n the presence of v a r i o u s s t i m u l i i . e . th rombin , c o l l a g e n and p a p a i n ; but they had no e f f e c t on malondia ldehyde p r o d u c t i o n o b t a i n e d w i t h exogeneous ly added a r a c h i d o n i c a c i d 28 (Walenga et a l . , 1980). Both PMSF and 2 - n i t r o - 4 - c a r b o x y p h e n y l -N-N-diphenylcarbamate i n h i b i t e d the c o l l a g e n - or p a p a i n -s t i m u l a t e d m o b i l i z a t i o n of a r a c h i d o n i c a c i d from PI and p h o s p h a t i d y l c h o l i n e , as w e l l as fo rmat ion of p h o s p h a t i d i c a c i d from P I , and showed a dose-dependent i n h i b i t i o n of P i - s p e c i f i c p h o s p h o l i p a s e C from p l a t e l e t s (Walenga et a l . , 1980). In c o n t r a s t to the above f i n d i n g s , Downes and M i c h e l l (1981) f a i l e d to observe any e f f e c t of PMSF on p o l y p h o s p h o i n o s i t i d e phosphod ie s t e ra se i n e r y t h r o c y t e s . 1.8 MUSCARINIC CHOLINERGIC RECEPTORS C h o l i n e r g i c r e c e p t o r s have long been c l a s s i f i e d i n t o two major c a t e g o r i e s , n i c o t i n i c and m u s c a r i n i c . N i c o t i n i c c h o l i n e r g i c responses can be mimicked by n i c o t i n e , are i n h i b i t e d by d - t u b o c u r a r i n e and have a very f a s t onset ( l a t e n c y < msec) and shor t d u r a t i o n of response (30-100 msec ) ( fo r review see Adams, 1981). N i c o t i n i c a c e t y l c h o l i n e r e c e p t o r (nAChR) s t i m u l a t i o n b r i n g s about t h i s response by a n o n s e l e c t i v e i n c r e a s e i n p e r m e a b i l i t y to monovalent c a t i o n s , r e s u l t i n g i n membrane d e p o l a r i z a t i o n . The s i n g l e molecu le of nAChR has been suggested to c o n t a i n both the a c e t y l c h o l i n e (ACh) b i n d i n g s i t e and the i o n i c c h a n n e l (Raf tery et a l . , 1980), because the i o n i c p e r m e a b i l i t y of a r t i f i c i a l membrane b i l a y e r s c o n t a i n i n g nAChR c o u l d be r e g u l a t e d by ACh (Nelso et a l . , 1980). In c o n t r a s t , m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r (mAChR)(which w i l l be d e a l t w i t h i n g r e a t e r d e t a i l ) can be s t i m u l a t e d s p e c i f i c a l l y wi th muscar ine or a c e t y l - | 3 - m e t h a c h o l i n e , and i n h i b i t e d by the 29 c l a s s i c a l m u s c a r i n i c a n t a g o n i s t , a t r o p i n e . M u s c a r i n i c responses are slow i n onset ( l a t e n c y > 100 msec) and longer i n d u r a t i o n (0.5 sec) ( fo r review - see H e i l b r o n n and B a r t f a i , 1978; Wastek and Yamamura,1981). The slow onset of the m u s c a r i n i c response i s e x p l a i n e d by the f o l l o w i n g two hypothese s : F i r s t , a subun i t model proposed by Kehoe and Marty (1980), p o s t u l a t e s that s e v e r a l ACh r e c e p t o r complexes have to i n t e r a c t be fore opening of the channe l can take p l a c e . Second, the second messenger model h y p o t h e s i z e s tha t m u s c a r i n i c a c t i v a t i o n l eads to the fo rmat ion of an i n t r a c e l l u l a r messenger ( i . e . cGMP, PI t u r n o v e r ) , which i s somehow r e s p o n s i b l e for the channe l o p e n i n g . The f o l l o w i n g three b i o c h e m i c a l responses u s u a l l y accompany the s t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n whole c e l l s : i ) i n c r e a s e i n i n t r a c e l l u l a r l e v e l s of cGMP i i ) e f f l u x of potas s ium i i i ) i n c r e a s e d t u r n o v e r of PI A l l the above re sponses , i n v a r i o u s systems, have been suggested to p l a y a r o l e i n the m u s c a r i n i c s t i m u l u s - r e s p o n s e c o u p l i n g . 1.8.1 C y c l i c Guanosine Monophosphate S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n many t i s s u e s l eads to the fo rmat ion of cGMP ( fo r a l i s t of t i s s u e s showing i n c r e a s e s i n cGMP l e v e l s see H e i l b r o n n and B a r t f a i , 1978). A very l a r g e i n c r e a s e (200- fo ld ) i n the l e v e l of cGMP on m u s c a r i n i c s t i m u l a t i o n i n neuroblas toma c e l l s (Matsuzawa and N i r e n b e r g , 1975; R i c h e l s o n et a l . , 1978) makes t h i s an 30 a p p r o p r i a t e system to c h a r a c t e r i z e v a r i o u s a s p e c t s of t h i s m u s c a r i n i c r e sponse . In the neuroblastoma c e l l , the cGMP response has been shown to be r a p i d and t r a n s i e n t ( R i c h e l s o n , 1977) (peaks at 30 sec and r e t u r n s to b a s a l l e v e l i n 2-3 min) and c a l c i u m - ( R i c h e l s o n et a l . , 1978) and temperature-dependent (E l -Fakahany and R i c h e l s o n , 1980). In these c e l l s , exogeneously added cGMP produced h y p e r p o l a r i z a t i o n (Wastek et a l . , 1981) s i m i l a r to t h a t o b t a i n e d w i t h m u s c a r i n i c s t i m u l a t i o n . Though s t i l l h i g h l y d e b a t a b l e , t h i s a l o n g w i t h some o ther ev idence in the l i t e r a t u r e (see a l s o the s e c t i o n on "cGMP i n n e g a t i v e i n o t r o p y " ) suggested a r o l e for cGMP in the m u s c a r i n i c r e c e p t o r -mediated r e sponse . However, the c a l c i u m requirement for guany la te c y c l a s e a c t i v a t i o n i n v a r i o u s systems ( R i c h e l s o n et a l 1978; Study et a l . , 1978; Murad et a l . , 1979) c l e a r l y i n d i c a t e s tha t t h i s i s the r e s u l t , r a t h e r than the cause , of c a l c i u m m o b i l i z a t i o n . M o s t l y p re sen t i n s o l u b l e form, some guany la te c y c l a s e has a l s o been shown to be pre sent i n the p a r t i c u l a t e f r a c t i o n (Garbers et a l . , 1978; Sulakhe et a l . , 1976). A l l a t t empt s , w i t h a few e x c e p t i o n s , to show a d i r e c t c o u p l i n g of mAChR to guany la te c y c l a s e s i m i l a r to tha t of hormone s e n s i t i v e a d e n y l a t e c y c l a s e i n broken c e l l p r e p a r a t i o n s have been u n s u c c e s s f u l ( L i m b i r d and L e f k o w i t z , 1975; Murad et a l . , 1979). One e x c e p t i o n i s r a t l i v e r plasma membrane, where s t i m u l a t i o n wi th 10."9 M c a r b a c h o l or 10" 7 M ACh caused a 2-4 f o l d i n c r e a s e i n cGMP, which was a t r o p i n e - s e n s i t i v e (deBecemberg et a l . , 1982). U n l i k e cGMP responses i n i n t a c t c e l l s , the i n c r e a s e i n cGMP 31 l e v e l s w i t h the ra t l i v e r plasma membrane was observed over a very narrow range of c a r b a c h o l and ACh c o n c e n t r a t i o n . Of v a r i o u s agents which can cause an i n c r e a s e i n cGMP, on ly a c t i v a t i o n of guany la te c y c l a s e by c h o l i n e e s t e r s i n c e l l f ree p r e p a r a t i o n s (deBecemberg et a l • , 1982; George et a l . , 1975; Howel l and Montague, 1974) are not r e a d i l y e x p l a i n e d by the presence of o ther guany la te c y c l a s e a c t i v a t o r s . Other r e g u l a t o r s of t h i s enzyme a c t i v i t y , which c o u l d be p h y s i o l o g i c a l l y i m p o r t a n t , a r e : C a 2 + - d e p e n d e n t p r o t e a s e s (Lacombe et a l . , 1980), u n s a t u r a t e d f a t t y a c i d s (Glas s et a l . , 1977; Garbers et a l . , 1978; Sp ie s et a l . , 1980), f a t t y a c i d h y d r o p e r o x i d e s and p r o s t a g l a n d i n s (Goldberg et a l . , 1978; Hidaka and Asano, 1977) and l y s o l e c i t h i n ( Sh ie r et a l . , 1976; Z w i l l e r et a l . , 1976).. I t i s i n t e r e s t i n g to note tha t at l e a s t two of the guany la te c y c l a s e a c t i v a t o r s , f a t t y a c i d s i n p l a t e l e t s ( L a p e t i n a et a l . , 1981a, 1981b;. P r e s c o t t and M a j e r u s , 1983) and p r o s t a g l a n d i n s i n pancreas ( M a r s h a l l et a l . , 1980, 1982) are generated d u r i n g PI t u r n o v e r . 1.8.2 Potass ium E f f l u x S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n t i s s u e s p r e l o a d e d w i t h K + caused an i n c r e a s e d e f f l u x of K + from smooth muscle fragments (Burgen and Spero , 1968), c a r d i a c muscle c e l l s (Galper et a l . , 1982), and r a t p a r o t i d c e l l s (Putney et a l . , 1980), among o t h e r s . In t i s s u e s such as h e a r t , enhanced K + e f f l u x caused r a p i d h y p e r p o l a r i z a t i o n , thereby r e d u c i n g the d u r a t i o n of the a c t i o n p o t e n t i a l and c a u s i n g i n h i b i t i o n of the slow inward 32 c a l c i u m c u r r e n t (Ten E i c k et a l . , 1976). T h i s suggests a r o l e for K + e f f l u x in the m u s c a r i n i c re sponse . How m u s c a r i n i c s t i m u l a t i o n a l t e r s K + p e r m e a b i l i t y (as the K + c h a n n e l i s s t i l l not c o n s i d e r e d to be a pa r t of the mAChR) or the f u n c t i o n of K + e f f l u x i n t i s s u e s where m u s c a r i n i c s t i m u l a t i o n mediates c a l c i u m m o b i l i z a t i o n , i s s t i l l not c l e a r . 1.8.3 M u s c a r i n i c A c e t y l c h o l i n e Receptor And PI Response The f i r s t o b s e r v a t i o n on PI turnover was made f o l l o w i n g s t i m u l a t i o n of the m u s c a r i n i c r e c e p t o r s i n p igeon pancreas (Hokin and H o k i n , 1953). M u s c a r i n i c c h o l i n e r g i c r e c e p t o r s are the most w i d e l y s t u d i e d w i t h r e s p e c t to PI response ( M i c h e l l , 1975, 1979), and the s t i m u l a t i o n of m u s c a r i n i c r e c e p t o r i n many d i v e r s e t i s s u e s such as c e r e b r a l c o r t e x ( H o k i n , 1969; L a p e t i n a and M i c h e l l , 1974), a d r e n a l medul la (Hokin et a l . , 1958; T r i f a r o , 1969a, 1969b), guinea p i g i l eum ( J a f f e r j i and M i c h e l l , 1976a), among many o t h e r s ( for a comprehensive l i s t see M i c h e l l , 1975), are found to be a s s o c i a t e d w i t h enhanced PI t u r n o v e r . Almost a l l of the m u s c a r i n i c responses i n v e s t i g a t e d u n t i l the p re sen t s tudy have at l e a s t one t h i n g in common; they are a l l mediated by c a l c i u m m o b i l i z a t i o n . The c o r r e l a t i o n between m u s c a r i n i c s t i m u l a t i o n and PI response was so good that i t l e d to the p r o p o s a l tha t PI breakdown may be i n t r i n s i c to m u s c a r i n i c r e c e p t o r s t i m u l a t i o n ( M i c h e l l et a l . , 1976). P r e s y n a p t i c mAChR of synaptosomes was the o n l y e x c e p t i o n , s i n c e m u s c a r i n i c s t i m u l a t i o n produced a PI response ( G r i f f i n et a l . , 1979), whereas c a l c i u m m o b i l i z a t i o n was not c o n s i d e r e d to mediate the 33 r e c e p t o r r e sponse . The mAChR i n hear t (see the s e c t i o n on "mechanism of nega t ive i n o t r o p y " ) and v a s c u l a r smooth muscle i . e . femoral a r t e r y (DeMey and V a n h o u t t e , 1980) are o ther examples where r e c e p t o r s t i m u l a t i o n does not l e a d to c a l c i u m m o b i l i z a t i o n . One way to t e s t whether PI breakdown ( M i c h e l l e_t a l . , 1976) accompanying mAChR s t i m u l a t i o n i s an i n t r i n s i c c h a r a c t e r i s t i c of the r e c e p t o r whose f u n c t i o n i s unknown, or whether i t accompanies o n l y those m u s c a r i n i c r e c e p t o r s t i m u l a t i o n s which mediate t h e i r e f f e c t by c a l c i u m m o b i l i z a t i o n , i s to s tudy the e f f e c t of m u s c a r i n i c s t i m u l a t i o n i n a t i s s u e where the f i n a l e f f e c t i s not mediated through c a l c i u m m o b i l i z a t i o n . Such a s tudy i s p r e s e n t e d i n t h i s t h e s i s . V a r i o u s c h a r a c t e r i s t i c s of PI t u r n o v e r i n response to s t i m u l a t i o n of r e c e p t o r s have been d i s c u s s s e d i n d e t a i l i n the e a r l i e r s e c t i o n s . PI response accompanying mAChR s t i m u l a t i o n f o l l o w s the same g e n e r a l p a t t e r n as for the o t h e r r e c e p t o r s . The c o n c e n t r a t i o n of c h o l i n e r g i c a g o n i s t r e q u i r e d to produce the PI response i n most t i s s u e s appeared to be s e v e r a l o r d e r s of magnitude h i g h e r than that r e q u i r e d to produce the p h y s i o l o g i c a l response ( J a f f e r j i and M i c h e l l , 1976a; M i c h e l l et a l . , 1976). T h e r e f o r e , the PI response was i n i t i a l l y c o n s i d e r e d to be of no 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 . J a f f e r j i and M i c h e l l (1976a) observed t h a t the dose-response curve of PI r e s p o n s e , o b t a i n e d d u r i n g c a r b a c h o l s t i m u l a t i o n of mAChR i n l o n g i t u d i n a l smooth muscle of gu inea p i g i l e u m , was s i m i l a r to the r e c e p t o r o c c u p a t i o n curve for c a r b a c h o l . The l a c k of spare r e c e p t o r s for PI response (whi le the presence of spare r e c e p t o r s has been 34 shown for the p h y s i o l o g i c a l response i . e . c o n t r a c t i o n (Yoshida et a l . , 1979), suggested tha t i n the c h a i n of events l e a d i n g from r e c e p t o r o c c u p a t i o n to p h y s i o l o g i c a l re sponse , PI breakdown may l i e c l o s e r to the r e c e p t o r o c c u p a t i o n ( J a f f e r j i and M i c h e l l , 1976a). I t was r e c e n t l y r e p o r t e d that i n n e u t r o p h i l s , where there are no spare r e c e p t o r s for f M e t - L e u - P h e - i n d u c e d ly sosomal enzyme s e c r e t i o n , the h a l f maximal c o n c e n t r a t i o n for the PI response and the p h y s i o l o g i c a l response were s i m i l a r ( C o c k c r o f t et a l . , 1981) . 1.8.4 Are There S u b p o p u l a t i o n s Of M u s c a r i n i c A c e t y l c h o l i n e  Receptor s ? Based on the nature of the c r i t e r i o n used , e i t h e r s i n g l e ( B i r d s a l l , 1977; V e n t e r , 1983; W e l l s et a l . , 1977) or m u l t i p l e subtypes ( E h l e r t et a l . , 1982; Hammer et a l . , 1980; R o s s i n i , 1981) of m u s c a r i n i c r e c e p t o r s have been p r o p o s e d . In s o l u t i o n s of p h y s i o l o g i c a l i o n i c s t r e n g t h , m u s c a r i n i c a n t a g o n i s t s b i n d to a u n i f o r m p o p u l a t i o n of r e c e p t o r s i t e s on the membrane ( B i r d s a l l , 1977; W e l l s et a l . , 1977), except i n p i t u i t a r y c e l l s where two a f f i n i t y s i t e s for a n t a g o n i s t b i n d i n g s i t e s have been r e p o r t e d (Mukherjee et a l . , 1980). By c o n t r a s t , under s i m i l a r c o n d i t i o n s m u s c a r i n i c a g o n i s t s e x h i b i t a " h e t e r o g e n e i t y " i n b i n d i n g to the same p o p u l a t i o n of r e c e p t o r s (Burgen et a l . , 1974; S trange et a l . , 1977; Yamamura and Snyder , 1974). T h i s d i f f e r e n c e between the a g o n i s t and a n t a g o n i s t b i n d i n g to m u s c a r i n i c r e c e p t o r s i s e x p l a i n e d by the presence of three c l a s s e s of b i n d i n g s i t e s hav ing d i f f e r e n t a f f i n i t y (super h i g h , 35 h i g h and low) for a g o n i s t but equa l a f f i n i t y for the a n t a g o n i s t ( B i r d s a l l et a l . , 1980). The three d i f f e r e n t a f f i n i t y s t a t e s for a g o n i s t may not r e p r e s e n t t h r e e d i f f e r e n t macromolecu les , as SDS-gel e l e c t r o p h o r e s i s and r a d i a t i o n i n a c t i v a t i o n a n a l y s i s i n d i c a t e the presence of a s i n g l e p o l y p e p t i d e w i t h 80K m o l e c u l a r weight from f i v e d i f f e r e n t t i s s u e s (Vente r , 1983). Yamamura and o t h e r s ( B i r d s a l l et a l . , 1980; E h l e r t et a l . , 1982) have p o s t u l a t e d that the d i f f e r e n t a f f i n i t i e s of a g o n i s t b i n d i n g s i t e s r e p r e s e n t d i f f e r e n t e n v i r o n m e n t a l or c o u p l i n g s t a t e s of the same r e c e p t o r macromolecu le . Guanine n u c l e o t i d e s may somehow a l t e r the env i ronment , as they conver t the heterogeneous p o p u l a t i o n of r e c e p t o r s to a p r e d o m i n a n t l y low a f f i n i t y form (Wei and Su lakhe , 1979; Rosenberger et a l . , 1980). O c c u p a t i o n of the low a f f i n i t y form was c o r r e l a t e d w i t h the fo rmat ion of cGMP i n b r a i n s l i c e s and neuroblastoma c e l l s ( B i r d s a l l , 1977), and w i t h i n h i b i t i o n of G T P - s t i m u l a t e d adeny la te c y c l a s e a c t i v i t y i n c a r d i a c homogenates ( B i r d s a l l et a l . , 1980b). PI r e sponse , however, was a s s o c i a t e d to both h i g h and low a f f i n i t y b i n d i n g s i t e s i n guinea p i g i l eum ( J a f f e r j i and M i c h e l l , 1976a). 1.8.5 S u b c l a s s i f i c a t i o n Based On A n t a g o n i s t B i n d i n g Three s u b c l a s s e s of m u s c a r i n i c r e c e p t o r s have been sugges ted , from p i r e n z e p i n e b i n d i n g s t u d i e s (Hammer et a l . , 1980). The I C 5 0 for the p i r e n z e p i n e i n h i b i t i o n of p r o p y l b e n z i l y l c h o l i n e or N-methyl scopolamine b i n d i n g d i f f e r e d by a f a c t o r of 30 i n the three s u b c l a s s e s , w i t h a t r i a and i l eum showing a homogeneous p o p u l a t i o n of low a f f i n i t y r e c e p t o r s . 36 In c o n t r a s t , c e r e b r a l cortex and s u b l i n g u a l gland had an I C 5 0 of 5 x l 0 ~ B M and 1.1x10" 7 M, r e s p e c t i v e l y , and a l s o showed the presence of m u l t i p l e a f f i n i t y b i n d i n g s i t e s . Another type of h e t e r o g e n e i t y , based on c h o l i n e / T r i s s e n s i t i v i t y was repo r t e d in myocardial muscarinic r e c e p t o r s ( S a s t r e et a l . , 1982). The exposure of canine a t r i a l homogenates to T r i s (10-100 mM) or c h o l i n e (0.1-1 mM) caused a 45% l o s s i n q u i n u c l i d i n y l b e n z i l a t e (QNB) b i n d i n g s i t e s , while the remaining s i t e s maintained t h e i r a f f i n i t y . T h i s l e d the authors to conclude that c h o l i n e and T r i s unmasked a h e t e r o g e n e i t y i n QNB b i n d i n g , which was not apparent i n t h e i r absence. 37 Three s u b p o p u l a t i o n s of m u s c a r i n i c r e c e p t o r s have been d e t e c t e d i n c u l t u r e d hear t c e l l s based on d e s e n s i t i z a t i o n s t u d i e s (Galper and Smi th , 1980). 26% of the r e c e p t o r s i t e s were r a p i d l y l o s t d u r i n g i n c u b a t i o n w i t h c a r b a c h o l and were i n s e n s i t i v e to m i c r o t u b u l e i n h i b i t o r s such as c o l c h i c i n e . A second s u b c l a s s c o n s i s t e d of 44% of the r e c e p t o r s , which underwent a s l ower , c o l c h i c i n e - s e n s i t i v e d e s e n s i t i z a t i o n on exposure to c a r b a c h o l . The r e s t (30%) of the r e c e p t o r s compr i sed a " s t a b l e " p o p u l a t i o n , which were not l o s t even a f t e r 3 hour exposure to c a r b a c h o l . Whether there i s any r e l a t i o n s h i p between these three s u b p o p u l a t i o n s of m u s c a r i n i c r e c e p t o r s , c l a s s i f i e d based on a g o n i s t - i n d u c e d d e s e n s i t i z a t i o n and the three d i f f e r e n t a f f i n i t y s i t e s for a g o n i s t b i n d i n g i s s t i l l not known. 1.8.6 R e c e p t o r - E f f e c t o r C o u p l i n g Ju s t as many d i f f e r e n t types of r e c e p t o r s ( e . g . /3-a d r e n e r g i c , H 2 _ h i s t a m i n i c ) are capab le of c o u p l i n g to a s i n g l e e f f e c t o r ( e .g a d e n y l a t e c y c l a s e ) the p o s s i b i l i t y that a s i n g l e r e c e p t o r can be c o u p l e d to d i f f e r e n t e f f e c t o r s i s now b e i n g r e c o g n i z e d ( R i c h e l s o n and E l - F a k a h a n y , 1981). M u s c a r i n i c r e c e p t o r s i n h e a r t ( H a r t z e l l , 1982) and sympathet ic neurones (Brown and Adams, 1980; Horn and Dodd, 1981; Weight et a l . , 1979) have been proposed to be c o u p l e d to two d i f f e r e n t types of i o n i c channe l s i n the same t i s s u e . P o s s i b l e s u b c l a s s i f i c a t i o n of the m u s c a r i n i c r e c e p t o r can be made, based on the nature of the e f f e c t o r system c o u p l e d to the r e c e p t o r s . A l a r g e 38 p r o p o r t i o n of mAChR's be long to a f a m i l y of r e c e p t o r s which mediate t h e i r response through c a l c i u m m o b i l i z a t i o n and a l s o produce a PI response on r e c e p t o r s t i m u l a t i o n (see M i c h e l l , 1975, 1979). The responses of a second, much s m a l l e r , p r o p o r t i o n of mAChRs are mediated by i n h i b i t i o n of c a l c i u m i n f l u x , e . g the nega t ive i n o t r o p i c e f f e c t i n hear t and the i n h i b i t i o n of t r a n s m i t t e r r e l e a s e by p r e s y n a p t i c mAChR s t i m u l a t i o n . R e c e n t l y M i c h e l s o n et a l . , ( 1 9 7 9 ) have suggested tha t p r e s y n a p t i c mAChR s t i m u l a t i o n may not i n h i b i t c a l c i u m uptake , but r a t h e r a p h o s p h o r y l a t i o n s tep f o l l o w i n g c a l c i u m m o b i l i z a t i o n . S t i m u l a t i o n of some r e c e p t o r s b e l o n g i n g to t h i s c l a s s has been shown to cause i n h i b i t i o n of a d e n y l a t e c y c l a s e (Murad et a l . , 1962; Watanabe et a l . , 1978), thereby d e c r e a s i n g the l e v e l of cAMP (Beigon and Pappano, 1980; George et a 1 . , 1973). C h o l i n e r g i c a g o n i s t s may r e g u l a t e both the a f f i n i t y of r e c e p t o r s c o u p l e d to a d e n y l a t e c y c l a s e and a d e n y l a t e c y c l a s e a c t i v i t y by modula t ing the e f f e c t s of GTP (Watanabe et a l . , 1978), p o s s i b l y through i t s c o n v e r s i o n to cGMP. By ana logy to the a l p h a - a d r e n e r g i c system, Jones et a l . (1982) proposed that there may be two p o p u l a t i o n s of mAChR. One p o p u l a t i o n of mAChR was suggested to be c o u p l e d to PI tu rnover and i t s response i s mediated by c a l c i u m m o b i l i z a t i o n , w h i l e the second p o p u l a t i o n was suggested to mediate i t s response through i n h i b i t i o n of adeny la te c y c l a s e . U n l i k e the a l p h a - a d r e n e r g i c system, i t i s s t i l l not known whether the p o p u l a t i o n of mAChR i n h i b i t i n g a d e n y l a t e c y c l a s e i s c o u p l e d to PI t u r n o v e r or not ( M i c h e l l et a l . , 1981). 39 1.9 MECHANISM OF THE NEGATIVE INOTROPIC EFFECT L i g a n d b i n d i n g s t u d i e s suggest the presence of mAChRs i n a l l r e g i o n s of the hear t ( H a r t z e l l , 1980; Wei and S u l a k h e , 1 9 7 8 ) . In ra t and r a b b i t , mAChr d e n s i t y i n the a t r i u m was t w o - f o l d h i g h e r than tha t of the v e n t r i c l e , w h i l e guinea p i g and dog showed almost equa l d e n s i t y i n these two r e g i o n s (Wei and S u l a k h e , 1 9 7 8 ) . A decrease i n the b a s a l c o n t r a c t i l e f o r c e i s u s u a l l y observed upon s t i m u l a t i o n of mAChR i n i s o l a t e d a t r i u m of v a r i o u s s p e c i e s (Furchgot t et a l . , 1960; Ravens and Z i e g l e r , 1980; for review see H i g g i n s et a l . , 1973), but the decrease i n v e n t r i c l e (Endoh and Motomura, 1979; Josephson and S p e r e l a k i s , 1982) and whole hear t p r e p a r a t i o n s ( I n g e r b r e t s e n et a l . , 1980) was sma l l and v a r i a b l e . T h e d i f f e r e n c e i n the mAChR d e n s i t y between a t r i a and v e n t r i c l e i s not enough to account for the observed v a r i a t i o n i n the response to mAChR s t i m u l a t i o n i n these two r e g i o n s . The d i r e c t n e g a t i v e i n o t r o p i c e f f e c t (decrease i n b a s a l c o n t r a c t i l i t y ) observed on m u s c a r i n i c s t i m u l a t i o n of a t r i u m (and sometimes v e n t r i c l e ) , was a t t r i b u t e d to the i n h i b i t i o n of the slow inward c a l c i u m c u r r e n t (Grossman and F u r c h g o t t , 1 9 6 4 ; Ravens and Z i e g l e r , 1980; Ten E i c k et a l . , 1976). C o n c e n t r a t i o n s of ACh which produced a 30-40% decrease i n the fo rce of c o n t r a c t i o n were shown to cause an i n d i r e c t i n h i b i t i o n of the slow inward c a l c i u m c u r r e n t by d e c r e a s i n g the d u r a t i o n of the a c t i o n p o t e n t i a l (Beigon and Pappano, 1980; Ten E i c k et a l . , 1976). At h i g h e r c o n c e n t r a t i o n s of ACh, i n a d d i t i o n to the above mentioned i n d i r e c t mechanism a d i r e c t e f f e c t was suggested to be i n v o l v e d 40 i n i n h i b i t i n g the slow inward c a l c i u m c u r r e n t (Ten E i c k et a l . , 1976). The decrea sed d u r a t i o n of the a c t i o n p o t e n t i a l can not be s o l e l y r e s p o n s i b l e for the n e g a t i v e i n o t r o p i c e f f e c t observed wi th ACh, because w h i l e there was a very good c o r r e l a t i o n between decrea sed d u r a t i o n of the a c t i o n p o t e n t i a l and the n e g a t i v e i n o t r o p y wi th ACh a lone ( F u r c h g o t t et a l . , 1960; Ten E i c k et a l . , 1976), i t was p o s s i b l e to n e u t r a l i z e the n e g a t i v e i n o t r o p i c e f f e c t of ACh w i t h e p i n e p h r i n e wi thout p r o d u c i n g a r e t u r n of the d u r a t i o n of the a c t i o n p o t e n t i a l to c o n t r o l l e v e l s (Furchgot t et a l . , 1960). Other p o s s i b l e mechanisms for the ACh-mediated n e g a t i v e i n o t r o p i c e f f e c t s i n c l u d e a l t e r a t i o n s i n the l e v e l of c y c l i c n u c l e o t i d e s i . e cAMP and cGMP (George et a l . , 1973; Nawrath, 1976; Lee et a l . , 1972; for review see Drummond and Sever son , 1979). In per fu sed ra t h e a r t , ACh s t i m u l a t i o n produced both an i n c r e a s e i n the l e v e l of cGMP and a decrease i n the l e v e l of cAMP (George et a l . , 1973), but the n e g a t i v e i n o t r o p i c e f f e c t accompanying ACh s t i m u l a t i o n showed a much b e t t e r c o r r e l a t i o n wi th the former (George et a l . , 1973). Other ev idence which suggested a r o l e for cGMP i n ACh-mediated n e g a t i v e i n o t r o p y i n c l u d e s - a c o n c e n t r a t i o n dependent i n o t r o p i c e f f e c t of 8-bromo cGMP i n paced or spontaneous ly b e a t i n g a t r i a (Nawrath, 1976), and the a b i l i t y of d i b u t y r y l cGMP to a n t a g o n i z e the p o s i t i v e i n o t r o p i c e f f e c t of i s o p r o t e r e n o l i n guinea p i g hear t (Watanabe and Besch , 1975). cGMP may mediate i t s e f f e c t through a c t i v a t i o n of cGMP-dependent p r o t e i n k inase ( G i l l et a l . , 1977), which was found to cause the p h o s p h o r y l a t i o n of c a r d i a c 41 t r o p o n i n - 1 (Blumenthal et a l . , 1978) and a 70K endogenous p r o t e i n pre sent i n the r a t hear t (Wrenn and Kuo, 1981). The f u n c t i o n a l s i g n i f i c a n c e of these p h o s p h o r y l a t i o n s and whether they occur under p h y s i o l o g i c a l c o n d i t i o n s i s s t i l l not c l e a r (B lumenthal et a l . , 1978; Drummond and Sever son , 1979; Wrenn and Kuo, 1981). Under c e r t a i n c o n d i t i o n s , a d i s s o c i a t i o n between the e l e v a t i o n i n cGMP l e v e l and n e g a t i v e i n o t r o p y has a l s o been observed (Diamond et a l . , 1977; L inden and B r o o k e r , 1979). At low c o n c e n t r a t i o n s of ACh (0.05 i M ) , ca t a t r i a l t i s s u e showed a decrease i n t w i t c h t e n s i o n wi thout any change i n cGMP (Diamond et a l . , 1977). On the o ther hand, i n c r e a s e i n cGMP l e v e l s w i t h sodium n i t r o p r u s s i d e i n c a t a t r i a (Diamond et a l . , 1977) and ACh i n gu inea p i g v e n t r i c l e s (Watanabe and Besch , 1975) was not accompanied by a decrease i n t e n s i o n . Recent o b s e r v a t i o n s (Endoh and Yamash i t a , 1981) that a n e g a t i v e i n o t r o p i c e f f e c t c o u l d be produced by more than one mechanism may h e l p to e x p l a i n some of the above d i s c r e p a n c i e s . Sodium n i t r o p r u s s i d e i n c r e a s d cGMP l e v e l s i n both c a n i n e a t r i u m and v e n t r i c l e , but was accompanied by a decrease i n t e n s i o n o n l y i n the a t r i a . A s i m i l a r r e s u l t was o b t a i n e d w i t h d i b u t y r y l cGMP (Endoh and Yamash i t a , 1981). When c o n t r a c t i l e f o r c e was e l e v a t e d f i r s t by sympathet ic s t i m u l a t i o n or i s o p r o t e r e n o l a d m i n i s t r a t i o n , subsequent t reatment w i t h ACh produced a g r e a t e r f r a c t i o n a l decrease i n t e n s i o n than the c o n t r o l i n both l e f t a t r i a and v e n t r i c l e ( " a ccentua ted antagonism") (Dempsey and Cooper , 1969; Levy and 42 Z i e s k e , 1969; Schwegler and J acob , 1976; Watanabe and Besch , 1975). The g r e a t e r nega t ive i n o t r o p i c e f f e c t o b t a i n e d w i t h ACh i n the presence of sympathet ic tone or i n c r e a s e d l e v e l of c a techo lamine was r e f e r r e d to as " a c c e n t u a t e d antagonism" (Levy and Z i e s k e , 1969). As a c c e n t u a t e d antagonism w i t h ACh was observed o n l y w i t h agents whose responses are mediated by cAMP, such as c a t e c h o l a m i n e s and h i s t a m i n e H 2 a g o n i s t s , but not w i t h a g o n i s t s whose responses are independent of cAMP (Levy et a l . , 1966; Watanabe and Besch , 1975), the r e s u l t s suggested that ACh produced a c c e n t u a t e d antagonism by i n t e r f e r i n g w i t h cAMP g e n e r a t i o n or i t s re sponse . Most of the a v a i l a b l e ev idence suggests that the c a t e c h o l a m i n e - s t i m u l a t e d p o s i t i v e i n o t r o p i c e f f e c t s are mediated through cAMP ( fo r a review see Drummond and Sever son , 1979). R i n a l d i et a l . (1981) have r e c e n t l y shown that cAMP-dependent p h o s p h o r y l a t i o n of a 23K m o l e c u l a r weight c a r d i a c sarcolemmal p r o t e i n , c a l c i d u c t i n , l eads to an i n c r e a s e d c a l c i u m uptake . T h i s may suggest a p o s s i b l e mechanism by which i s o p r o t e r e n o l -s t i m u l a t e d cAMP-dependent p r o t e i n k inase (Keely et a l . , 1978) may cause an i n c r e a s e i n the slow inward c a l c i u m c u r r e n t (Josephson and S p e r a l a k i s , 1982). ACh, by d e c r e a s i n g the l e v e l of i s o p r o t e r e n o l - s t i m u l a t e d cAMP, may i n h i b i t the a c t i v a t i o n of cAMP-dependent p r o t e i n k i n a s e , thereby r e d u c i n g the slow inward c a l c i u m c u r r e n t . In c a r d i a c membrane p r e p a r a t i o n s , ACh-mediated i n h i b i t i o n of a d e n y l a t e c y c l a s e was brought about by an e f f e c t on the GTP-dependent r e g u l a t o r y component of the /3-r e c e p t o r / a d e n y l a t e c y c l a s e system (Watanabe et a l . , 1978). 43 Other proposed mechanisms for ACh-mediated nega t ive i n o t r o p y i n c l u d e : a) a decrease i n the p o o l of exchangeable c a l c i u m (Grossman and F u r c h g o t t , 1964) and b) i n c r e a s e d c a l c i u m e f f l u x (Pro lopezuk et a l . , 1981). 1.10 ALPHA-ADRENERGIC RECEPTORS IN HEART A l p h a - a d r e n e r g i c r e c e p t o r s have now been shown to be p r e s e n t i n the h e a r t s of r a t , guinea p i g , r a b b i t and ca t (Wagner and Brodde, 1978; Schumann, 1980; for review see Benfey , 1980). A l p h a - a d r e n o c e p t o r s are d i v i d e d i n t o two c a t e g o r i e s - a, and a2, based ma in ly on the s p e c i f i c i t y of a n t a g o n i s t b i n d i n g (Ex ton , 1982). A l p h a , and a 2 _ a d r e n o c e p t o r s were p r e v i o u s l y c o n s i d e r e d to be s p e c i f i c a l l y l o c a t e d i n the post and p r e s y n a p t i c membrane r e s p e c t i v e l y , but now the presence of p o s t s y n a p t i c a2 a d r e n o c e p t o r s has been shown i n many t i s s u e s (McGrath, 1982; for a l i s t of t i s s u e s where p o s t s y n a p t i c a 2 r e c e p t o r s have been d e t e c t e d see E x t o n , 1982). Yohimbine , a a2 b l o c k i n g agent , was 20 t imes l e s s potent than phento lamine i n d i s p l a c i n g [ 3 H ] d i h y d r o e r g o c r y p t i n e - b i n d i n g i n r a b b i t (Schumann and Brodde, 1979) and r a t ( W i l l i a m s and L e f k o w i t z , 1978) c a r d i a c homogenates, s u g g e s t i n g tha t the a l p h a -r e c e p t o r s b e i n g l a b e l e d are p o s t s y n a p t i c . In r a t h e a r t , the presence of a s i n g l e c l a s s of [ 3 H ] d i h y d r o e r g o c r y p t i n e b i n d i n g s i t e s w i t h a r e c e p t o r d e n s i t y of 41 fmole/mg p r o t e i n ( W i l l i a m s and L e f k o w i t z , 1978) and 307 fmole/mg of p r o t e i n ( C i a r a l d i and M a r i n e t i i , 1977) and 100 fmole/mg p r o t e i n based on [ 3 H ] p r a z o s i n b i n d i n g ( K a r l i n e r et a l . , 1982) has been r e p o r t e d , sugge s t ing 44 t h e p r e s e n c e of a homogeneous p o p u l a t i o n of a l p h a - a d r e n o c e p t o r s . On t h e o t h e r h a n d , i n p h a r m a c o l o g i c a l s t u d i e s a m u l t i p h a s i c r e s p o n s e o b t a i n e d w i t h s t i m u l a t i o n of r a t p a p i l l a r y musc le (Skomedal e t a l . , 1980) , r a t a t r i a (Osnes , e t a l . , 1978) and g u i n e a p i g a t r i a ( H a t t o r i and Kanno , 1981) a r g u e s a g a i n s t the p r e s e n c e of a s i n g l e c l a s s of a l p h a - a d r e n e r g i c r e c e p t o r s . F u r t h e r , t h e s e l e c t i v e b l o c k a d e of t h e p o s i t i v e i n o t r o p i c phase by p r a z o s i n (an a , a n t a g o n i s t ) , and a c o m p l e t e b l o c k a d e ( b o t h p o s i t i v e and n e g a t i v e i n o t r o p i c r e s p o n s e ) by p h e n t o l a m i n e i n r a t (Skomedal e t a l . , 1980) , as w e l l as i n g u i n e a p i g ( H a t t o r i and Kanno , 1 9 8 2 ) , l e d t o the s u g g e s t i o n t h a t t h e r e may be s t i m u l a t o r y and i n h i b i t o r y p o p u l a t i o n s of p o s t s y n a p t i c a l p h a -a d r e n o c e p t o r s (Skomedal e t a l • , 1980; H a t t o r i and K a n n o , 1 9 8 2 ) . A l p h a 2 - a d r e n e r g i c r e s p o n s e s a r e c o n s i d e r e d t o be m e d i a t e d by i n h i b i t i o n of a d e n y l a t e c y c l a s e . T h i s i s s u g g e s t e d t o o c c u r by a c t i v a t i o n of an i n h i b i t o r y - G T P b i n d i n g p r o t e i n , w h i c h may d i r e c t l y i n t e r a c t w i t h the c a t a l y t i c s u b u n i t of a d e n y l a t e c y c l a s e or i n t e r f e r e w i t h t h e i n t e r a c t i o n between t h e s t i m u l a t o r y and c a t a l y t i c s u b u n i t s of a d e n y l a t e c y c l a s e ( L e v i t z k i , 1982) . A d e c r e a s e i n t h e l e v e l of cAMP on c a r d i a c a l p h a - a d r e n e r g i c s t i m u l a t i o n ( p r o b a b l y t h r o u g h a c t i v a t i o n of a2 r e c e p t o r s ) has been r e p o r t e d by Watanabe et a l . ( 1 9 7 7 ) . In i s o l a t e d r a t myocytes and p e r f u s e d r a t h e a r t , a l p h a - r e c e p t o r s t i m u l a t i o n w i t h p h e n y l e p h r i n e 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 of p r o p r a n o l o l (a b e t a b l o c k e r ) c a u s e d a d e c r e a s e i n the l e v e l of cAMP (Watanabe e t a l . , 1977 ) . I n p e r f u s e d r a t h e a r t t h i s was accompanied by a n e g a t i v e i n o t r o p i c e f f e c t . 45 A monophasic p o s i t i v e i n o t r o p i c response d e v o i d of the t r a n s i e n t i n h i b i t o r y e f f e c t ( H a t t o r i and Kanno, 1981, 1982; Osnes et a l . , 1978) has been observed on s t i m u l a t i o n of a lpha r e c e p t o r s i n r a t l e f t a t r i a (Mar t inez and M c N e i l l , 1977; S h i b a t a et a l . , 1980) and v e n t r i c l e s ( Sh iba ta et a l . , 1980; Wagner and Brodde , 1978; Wenzel and Su, 1966). Among the m y o c a r d i a l p r e p a r a t i o n s s t u d i e d , r a t l e f t and r i g h t a t r i a showed the g r e a t e s t p h e n t o l a m i n e - s e n s i t i v e p o s i t i v e i n o t r o p i c response to methoxamine ( Sh iba ta et a l . , 1980). 1.10.1 Mechanism Of The A l p h a - And B e t a - A d r e n e r g i c R e c e p t o r - mediated P o s i t i v e I n o t r o p i c E f f e c t , Many d i f f e r e n t l i n e s of ev idence c l e a r l y i n d i c a t e t h a t a lpha and b e t a - a d r e n e r g i c r e c e p t o r induced p o s i t i v e i n o t r o p i c responses are mediated by d i f f e r e n t mechanisms (Endoh et a l . , 1975; Endoh and Yamashi ta , 1980; Skomedal et a l . , 1982; Wagner and Schumann, 1979). The /3-adrenerg ic- s t imula ted p o s i t i v e i n o t r o p i c e f f e c t s i n v a r i o u s t i s s u e s of the hear t are accompanied by i n c r e a s e d l e v e l s of cAMP ( K e l l y et a l . , 1978; Robinson et a l . , 1965; M a r t i n e z and M c N e i l l , 1977), decrea sed time to peak t e n s i o n and a shor tened r e l a x a t i o n time (Ledda e_t a l . , 1975; Rab inowi tz et a l . , 1975; Skomedal et a l . , 1982). In c o n t r a s t , the a l p h a - a d r e n o c e p t o r - s t i m u l a t e d p o s i t i v e i n o t r o p i c e f f e c t was accompanied n e i t h e r by an i n c r e a s e i n cAMP l e v e l s ( Mar t inez and M c N e i l l , 1977), nor by a decrease i n the time to peak t e n s i o n (Skomedal et a l . , 1982). Most workers agree tha t the a l p h a - a d r e n o c e p t o r - s t i m u l a t e d 46 p o s i t i v e i n o t r o p i c e f f e c t i s mediated through enhanced m o b i l i z a t i o n of c a l c i u m (Handa et a l . , 1982; Inui et a l . , 1981; Ledda et a l . , 1980; Miura et a l . , 1978), but the source of c a l c i u m i n t h i s mediation i s s t i l l being debated. In g e n e r a l , both m i t o c h o n d r i a l and e x t r a c e l l u l a r Ca pools have been i m p l i c a t e d i n alpha-adrenergic-mediated responses i n v a r i o u s t i s s u e s (Exton, 1982). Endoh et a l . (1975) found that the alpha-receptor-mediated p o s i t i v e i n o t r o p i c response was more s e n s i t i v e to D-600 (a c a l c i u m channel a n t a g o n i s t ) than the beta-r e c e p t o r response, and the g r e a t e r temperature s e n s i t i v i t y of the alpha response i n the above study l e d them to conclude that alpha s t i m u l a t i o n mainly e x e r t e d i t s e f f e c t by i n c r e a s i n g transmembrane c a l c i u m i n f l u x . These c o n c l u s i o n s were a l s o supported by the o b s e r v a t i o n that c o n d i t i o n s which favoured an enhanced i n f l u x of c a l c i u m , such as e l e v a t i o n of the g r a d i e n t of c a l c i u m , s h o r t e n i n g of the p l a t e a u of the a c t i o n p o t e n t i a l with c a r b a c h o l , and i n c r e a s e d e f f l u x of c a l c i u m with d i n i t r o p h e n o l , were a l l a b l e to i n c r e a s e the a f f i n i t y of phenylephrine f o r c a r d i a c alpha-adrenoceptors. Inui et a l . (1981) and Miura e_t a l . (1978) found that i n K + - d e p o l a r i z e d r a b b i t p a p i l l a r y muscle, alpha-adrenoceptor s t i m u l a t i o n i n c r e a s e d the inward c a l c i u m c u r r e n t , thereby s u p p o r t i n g the Endoh et a l . (1975) h y p o t h e s i s . Some of the more recent o b s e r v a t i o n s have c h a l l e n g e d the v a l i d i t y of t h i s h y p o t h e s i s . Verapamil and i t s methoxy d e r i v a t i v e (D-600) not only block the c a l c i u m channel but were shown to compete f o r the alpha r e c e p t o r b i n d i n g s i t e s ( K a r l i n e r et a l . , 1982). T h i s may e x p l a i n the higher 47 s e n s i t i v i t y of a l p h a - a d r e n o c e p t o r responses to D-600 observed in Endoh ' s s t u d i e s (Endoh et a l . , 1975). In K + - d e p o l a r i z e d guinea p i g v e n t r i c u l a r musc le , methoxamine s t i m u l a t i o n produced a dose-dependent c o n t r a c t i l e response which was not accompanied by a slow inward c a l c i u m c u r r e n t (Ledda et a l . , 1980). In K + -d e p o l a r i z e d r a b b i t p a p i l l a r y m u s c l e , when the maximum r a t e of r i s e of the a c t i o n p o t e n t i a l was used as a measure of the inward c a l c i u m c u r r e n t , the a lpha s t i m u l a t e d response was much s m a l l e r than tha t of the beta (Handa et a l . , 1982). A lpha s t i m u l a t i o n i n c r e a s e d the d u r a t i o n of the a c t i o n p o t e n t i a l , p r o b a b l y by s u p r e s s i n g the t ime-dependent outward K + c u r r e n t (Handa et a l . , 1982). The i n c r e a s e d d u r a t i o n of the a c t i o n p o t e n t i a l even w i t h an u n a l t e r e d inward c a l c i u m c u r r e n t c o u l d s t i l l r e s u l t i n an i n c r e a s e d m o b i l i z a t i o n of c a l c i u m . In c o n c l u s i o n , compared to the b e t a - a d r e n e r g i c system, l i t t l e i s known about how a l p h a -mediated p o s i t i v e i n o t r o p y i s p roduced , and i f c a l c i u m i s i n v o l v e d , what i t s source i s . 1.11 MUSCARINIC RECEPTORS IN LONGITUDINAL SMOOTH MUSCLE OF  GUINEA PIG ILEUM In l o n g i t u d i n a l smooth muscle of guinea p i g i l e u m , based on [ 3H]QNB b i n d i n g s t u d i e s , r e c e p t o r d e n s i t y has been e s t i m a t e d to be 190 fmol/mg t i s s u e (Yamamura and Snyder , 1974). S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s of l o n g i t u d i n a l smooth muscle of guinea p i g i l eum produced a b i p h a s i c c o n t r a c t i o n , c o n s i s t i n g of a r a p i d p h a s i c component and a s u s t a i n e d t o n i c component (Chang and T r i g g l e , 1973; James-Kracke and R o u f o g a l i s , 1981). Both phases 48 of c o n t r a c t i o n are the r e s u l t of c a l c i u m m o b i l i z a t i o n , but the p o o l s of c a l c i u m m o b i l i z e d d u r i n g these two phases are c o n s i d e r e d to be d i f f e r e n t (Brad ing and Sneddon, 1980; Chang and T r i g g l e , 1973; Rangachar i et a l . , 1983). Chang and T r i g g l e (1973) have suggested that a p o o l of e x t r a c e l l u l a r f ree c a l c i u m i s a s s o c i a t e d w i t h the s u s t a i n e d t o n i c phase of c o n t r a c t i o n , w h i l e a s u p e r f i c i a l l y bound c a l c i u m p o o l i s used i n the t r a n s i e n t and r a p i d p h a s i c c o n t r a c t i o n . J a f f e r j i and M i c h e l l (1976a) were the f i r s t to observe t h a t s t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n guinea p i g i l eum produced an enhanced i n c o r p o r a t i o n of phosphate i n t o P I , and a l s o suggested that the pr imary s t ep of t h i s response may be an enhanced breakdown of P I . L o n g i t u d i n a l smooth muscle of gu inea p i g i l eum has been shown to respond to m u s c a r i n i c a g o n i s t s w i t h a r e c e p t o r r e s e r v e ( T a y l o r et a l . , 1975; Yosh ida et a l . , 1979), as o n l y 10% of the r e c e p t o r s are r e q u i r e d to be o c c u p i e d by the m u s c a r i n i c a g o n i s t oxotremor ine to produce a maximal c o n t r a c t i o n (Yoshida et a l . , 1979). On the c o n t r a r y , the dose-response curve for c a r b a c h o l - s t i m u l a t e d phosphate i n c o r p o r a t i o n i n t o PI was s i m i l a r to the dose-response curve for r e c e p t o r occupancy ( J a f f e r j i and M i c h e l l , 1976a). T h i s p r o v i d e d a d d i t i o n a l ev idence to support the h y p o t h e s i s tha t i n the c h a i n of events between r e c e p t o r s t i m u l a t i o n and the p h y s i o l o g i c a l re sponse , PI breakdown may occur nearer to r e c e p t o r o c c u p a t i o n and may p l a y a r o l e i n c a l c i u m m o b i l i z a t i o n ( M i c h e l l , 1975; J a f f e r j i and M i c h e l l , 1976a). In c u l t u r e d smooth muscle c e l l s (Salmon and Honeyman, 1980) ev idence was p r o v i d e d to suggest that the t ime 49 course of fo rmat ion of p h o s p h a t i d i c a c i d d u r i n g c a r b a c h o l -s t i m u l a t e d PI t u r n o v e r was r a p i d enough to p l a y a r o l e i n c a l c i u m m o b i l i z a t i o n . In a l a t e r communicat ion , J a f f e r j i and M i c h e l l (1976b) r e p o r t e d that K + - i n d u c e d d e p o l a r i z a t i o n , which a l s o produces a c a l c i u m - m e d i a t e d b i p h a s i c c o n t r a c t i o n , showed an enhanced i n c o r p o r a t i o n of [ 3 2 P ] p h o s p h a t e i n t o P I . Based on the i n h i b i t i o n of c o n t r a c t i o n by v a r i o u s c a l c i u m a n t a g o n i s t s , Rosenberger and T r i g g l e (1979) proposed tha t both c a r b a c h o l and K + - s t i m u l a t i o n m o b i l i z e the same p o o l of c a l c i u m . 50 1.12 OBJECTIVE The o v e r a l l o b j e c t i v e of the p r e s e n t t h e s i s was to t e s t the f o l l o w i n g two hypothese s : a) t h a t PI breakdown i s i n t r i n s i c to m u s c a r i n i c r e c e p t o r s t i m u l a t i o n . b) tha t the PI response i s i n v o l v e d i n c a l c i u m m o b i l i z a t i o n . These o b j e c t i v e s were sought by s t u d y i n g : 1) the e f f e c t of m u s c a r i n i c and a l p h a - a d r e n e r g i c s t i m u l a t i o n on PI response i n r a t h e a r t . 2) the pr imary event of c a r b a c h o l - s t i m u l a t e d PI response i n gu inea p i g i l e u m . 3) the e f f e c t of the p u t a t i v e p h o s p h o l i p a s e C i n h i b i t o r , PMSF, on c a r b a c h o l - s t i m u l a t e d PI response and c o n t r a c t i o n . 51 I I . METHODS 2.1 C h o i c e Of Animal Among the s m a l l l a b o r a t o r y a n i m a l s , the r a t has been shown to c o n t a i n the l a r g e s t number of both m u s c a r i n i c (Wei and S u l a k h e , 1978) and a lpha a d r e n e r g i c r e c e p t o r s i n t h e i r a t r i a (Schumann, 1980). P h a r m a c o l o g i c a l responses f o l l o w i n g s t i m u l a t i o n of both these r e c e p t o r s have a l s o been q u i t e w e l l c h a r a c t e r i z e d i n the r a t (Josephson and S p e r e l a k i s , 1982; Osnes et a l . , 1978; Ravens and Z i e g l e r , 1980; Skomedal et a l . , 1980; Ten E i c k et a l . , 1976). In l o n g i t u d i n a l smooth muscle of the gu inea p i g i l eum the e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n on c a l c i u m m o b i l i z a t i o n (Chang and T r i g g l e , 1973; Rosenberger and T r i g g l e , 1979) and p h o s p h a t i d y l i n o s i t o l t u r n o v e r ( J a f f e r j i and M i c h e l l , 1976a) has been p r e v i o u s l y r e p o r t e d . Female , r a t h e r than male guinea p i g s were used i n order to compare the r e s u l t s w i t h those of J a f f e r j i and M i c h e l l (1976a) . Male W i s t a r r a t s (300-350 g) and female guinea p i g s (350-400 g) were a n a e s t h e t i z e d in e ther s a t u r a t e d tank be fore the i s o l a t i o n of t i s s u e . In the case of r a t s , 0.4 ml of 1600 u n i t s / m l of h e p a r i n i n normal s a l i n e was i n j e c t e d over 5-10 sec p e r i o d , 10 min be fore the r a t s were a n a e s t h e t i z e d . 52 2.2 K r e b s - b i c a r b o n a t e Bu f f e r A l l i n c u b a t i o n s were c a r r i e d out i n K r e b s - b i c a r b o n a t e b u f f e r of the f o l l o w i n g c o m p o s i t i o n (mM) : sodium c h l o r i d e -113, po ta s s ium c h l o r i d e - 4 . 7 , c a l c i u m c h l o r i d e - 2 . 5 , potas s ium d ihydrogen phosphate - 1.2, magnesium su lpha te - 0 . 6 , sodium b i c a r b o n a t e - 25, and dex t ro se - 11 .5 , and the pH a d j u s t e d to a p p r o x i m a t e l y 7.4 w i t h 0 2 / C 0 2 ( 9 5 / 5 ) . 2.3 Phosphate I n c o r p o r a t i o n In A t r i a The a t r i a were removed a f t e r p e r f u s i o n of the i s o l a t e d ra t hear t w i t h Krebs b u f f e r to f r ee them of b l o o d and p l a c e d i n i c e c o l d Krebs b u f f e r s a t u r a t e d w i t h 0 2 / C 0 2 ( 9 5 / 5 ) . A t r i a were chopped i n t o sma l l e r f ragments , e i t h e r toge ther ( i n mixed a t r i a s t u d i e s ) or a f t e r s e p a r a t i o n of the l e f t and r i g h t a t r i a ( i n l a t e r s t u d i e s ) and suspended i n 5.3 ml of Krebs b u f f e r . The a t r i a were i n c u b a t e d i n the presence or absence of drugs i n Krebs b u f f e r w i t h 40 uCi of [ 3 2 P ] s o d i u m o r t h o p h o s p h a t e , i n a f i n a l i n c u b a t i o n volume of 6 m l . A f t e r 60 min i n c u b a t i o n , the r e a c t i o n was s topped w i t h 2 ml of 20% t r i c h l o r o a c e t i c a c i d . T i s s u e fragments were s epara ted by vacuum f i l t r a t i o n on Whatman #1 paper and washed w i t h i c e - c o l d Krebs b u f f e r . E x t r a c t i o n of l i p i d , s e p a r a t i o n of p h o s p h o l i p i d and d e t e r m i n a t i o n of r a d i o a c t i v i t y were done as d e s c r i b e d i n l a t e r s e c t i o n s . 53 2.4 I s o l a t i o n Of L o n g i t u d i n a l Smooth Muscle Of Guinea P i g Ileum For each e x p e r i m e n t , i l e u m was o b t a i n e d from 1 or 2 female guinea p i g s . L o n g i t u d i n a l smooth muscle was o b t a i n e d by the method of Rang (1964) . I leum was cut i n t o 2-3 cm long fragments and s t o r e d i n Krebs b u f f e r at 4 ° C or at 3 7 ° C for c o n t r a c t i o n s t u d i e s . Each p i e c e was mounted on a smooth g l a s s rod and l o n g i t u d i n a l smooth muscle was s epara ted by c a r e f u l t a n g e n t i a l s c r a p i n g w i t h a c o t t o n swab ( Q - t i p ) . I s o l a t e d l o n g i t u d i n a l smooth muscles were u s u a l l y s t o r e d in i c e c o l d Krebs b u f f e r (except fo r f u n c t i o n a l s t u d i e s , i n which case they were s t o r e d at 3 7 ° C ) , and a e r a t e d w i t h 0 2 / C 0 2 ( 9 5 / 5 ) . 2.5 L a b e l - i n c o r p o r a t i o n In Guinea P i g Ileum S t u d i e s of phosphate i n c o r p o r a t i o n i n guinea p i g i l eum were done i n a manner s i m i l a r to tha t d e s c r i b e d for phosphate i n c o r p o r a t i o n i n r a t a t r i u m . For [ 3 H ] i n o s i t o l i n c o r p o r a t i o n , i l eum p i e c e s (15-25 , mg wet weight) were combined and p r e i n c u b a t e d for 30 min w i t h 30 MCi of [ 3 H ] i n o s i t o l i n 15 ml Krebs b u f f e r under an 0 2 / C 0 2 (95/5) atmosphere. I n d i v i d u a l p i e c e s were then separa ted and i n c u b a t e d s e p a r a t e l y fo r a f u r t h e r 30 min p e r i o d w i t h 1 MCi of [ 3 H ] i n o s i t o l i n the presence of v a r i o u s a g e n t s , i n a t o t a l volume of 4 m l . The r e a c t i o n was s topped by adding 2 ml of 20% t r i c h l o r o a c e t i c a c i d . The t i s s u e was s epara ted and washed w i t h b u f f e r . L i p i d was e x t r a c t e d as d e s c r i b e d i n a l a t e r s e c t i o n . The lower o r g a n i c phase o b t a i n e d d u r i n g e x t r a c t i o n of the l i p i d was s epara ted by c e n t r i f u g a t i o n at 2100x2 fo r 15 min , washed twice w i t h methanol /0 .1 N 54 h y d r o c h l o r i c a c i d / c h l o r o f o r m (48 :41 :3 v / v ) , and the s o l v e n t f i n a l l y removed by e v a p o r a t i o n . The r e s i d u e was d i s s o l v e d i n c h l o r o f o r m (2 ml) and phosphorus was de termined i n two 75 ul a l i q u o t s as d e s c r i b e d i n a l a t e r s e c t i o n . The r a d i o a c t i v i t y was determined i n the remain ing p o r t i o n , a f t e r removal of the s o l v e n t and a d d i t i o n of 10 ml E c o n o f l u o r . I n c o r p o r a t i o n was c a l c u l a t e d as dpm/jug phosphorus and expres sed as a percentage of the c o n t r o l v a l u e . 2 . 6 E x t r a c t i o n Of L i p i d Throughout the s tudy l i p i d s were e x t r a c t e d by the method of B l i g h and Dyer (1959) , as d e s c r i b e d below. Homogenizat ion and e x t r a c t i o n were a l l c a r r i e d out on i c e . T i s s u e ( u s u a l l y l e s s than 25 mg) was suspended i n 1.5 ml of 2 M KCI and homogenized at speed 3 w i t h a t e f l o n - c o a t e d P o t t e r - E l v e h j e m homogenizer . A f t e r h o m o g e n i z a t i o n , the homogenate was t r a n s f e r r e d to a 15 ml capped tube , 5.2 ml of c h l o r o f o r m / m e t h a n o l (1:2 v /v ) was added and the tube mixed by v o r t e x for 20 sec (a n o n a c i d i f i e d c h l o r o f o r m / m e t h a n o l mix ture was i n t e n t i o n a l l y used to a v o i d the e x t r a c t i o n of p o l y p h o s p h o i n o s i t i d e s ) C h l o r o f o r m (1.8 ml) was then added and the tube mixed a g a i n on a v o r t e x mixer fo r 20 s e c . F i n a l l y , 1.8 ml of water was added and the suspens ion mixed by v o r t e x for 5 s e c . A f t e r 20-30 m i n , the c h l o r o f o r m phase was s epara ted by c e n t r i f u g a t i o n at 2100XC_ fo r 15 min at 55 2.7 P h o s p h o l i p i d S e p a r a t i o n For i n i t i a l exper iments Supelco Redi Coat-2D p l a t e s were used for the s e p a r a t i o n of p h o s p h o l i p i d s . New p l a t e s gave very good s e p a r a t i o n , but u n s a t i s f a c t o r y s e p a r a t i o n was o b t a i n e d wi th p l a t e s " s t o r e d " for a longer p e r i o d of t i m e . T h e r e f o r e , p r e c o a t e d TLC p l a t e s from Merck , s i l i c a g e l 60, l a y e r t h i c k n e s s 0.25 mm, were used i n l a t e r s t u d i e s , which gave c o n s i s t e n t l y good s e p a r a t i o n s . Merck t h i n l a y e r chromatography p l a t e s s epara ted a l l the p h o s p h o l i p i d spots p r e v i o u s l y s epara ted w i t h p l a t e s from S u p e l c o , but the r e l a t i v e m o b i l i t i e s of the p h o s p h o l i p i d spots were s l i g h t l y d i f f e r e n t i n the two types of p l a t e s . P l a t e s were a c t i v a t e d at 1 0 5 ° C for 20 min and d i v i d e d i n t o four equa l q u a d r a n t s . A 20 to 30 ul sample was s p o t t e d by 10 M1 m i c r o p i p e t t e at each of the four c o r n e r s , 2 cm from the edges . In exper iments i n v o l v i n g a r a c h i d o n i c a c i d d e t e r m i n a t i o n or i n c o r p o r a t i o n , on ly n i t r o g e n was used fo r the d r y i n g of the s p o t . P h o s p h o l i p i d s e p a r a t i o n was a c h i e v e d by two-d imens iona l t h i n l a y e r chromatography. Chromatographic tanks were l i n e d w i t h Whatman #1 f i l t e r paper and p r e s a t u r a t e d w i t h the d e s i r e d s o l v e n t system for 30 m i n . Each p l a t e was deve loped w i t h 60 ml of s o l v e n t , and new s o l v e n t was used w i t h each r u n . Between each run p l a t e s were a i r d r i e d for 5 m i n , and fo r 10-15 min between runs i n the f i r s t and second d i m e n s i o n , u n t i l the sme l l of ammonia was c o m p l e t e l y l o s t . A f t e r development of the p l a t e i n the second d imens ion and removal of the s o l v e n t , spots were d e t e c t e d by e-xposure to i o d i n e vapor , which gave a y e l l o w 56 c o l o u r . A f t e r the spots were marked w i t h a p e n c i l , the s t a i n was a l l o w e d to fade away by s torage o v e r n i g h t . The r e q u i r e d spots were s c raped o f f the p l a t e a f t e r a d d i t i o n of a drop of m e t h a n o l . The s i l i c a g e l was t r a n s f e r r e d to a t e s t tube and p h o s p h o l i p i d s e x t r a c t e d . 2.8 E x t r a c t i o n Of P h o s p h o l i p i d Spots A mix ture of c h l o r o f o r m / m e t h a n o l / w a t e r / a c e t i c a c i d (50 :30 :10 :1 v / v ) was a l l o w e d to s tand for 5 min . The upper aqueous phase was d i s c a r d e d and 2 ml of the lower a c i d s a t u r a t e d c h l o r o f o r m / m e t h a n o l phase was used to e x t r a c t p h o s p h o l i p i d s . In some of the i n i t i a l exper iments spots were e x t r a c t e d w i t h 2 ml methanol o n l y . 2.9 D e t e r m i n a t i o n Of R a d i o a c t i v i t y The p h o s p h o l i p i d e x t r a c t was d i v i d e d i n t o two equa l p o r t i o n s ( 0 . 8 - 0 . 9 m l ) . One p o r t i o n was used for phosphorus d e t e r m i n a t i o n . In another p o r t i o n , r a d i o a c t i v i t y was de termined a f t e r removal of the s o l v e n t and a d d i t i o n of 10 ml s c i n t i l l a t i o n f l u i d . ACS was used as the s c i n t i l l a t i o n f l u i d for phosphate l a b e l and aqueous e x t r a c t , w h i l e E c o n o f l u o r was used fo r d e t e r m i n a t i o n of [ 3 H ] - l a b e l i n nonaqueous media . 57 2.10 Phosphorus D e t e r m i n a t i o n A l l the g lassware used i n the phosphorus d e t e r m i n a t i o n was a c i d washed. Phosphorus was de termined by a s l i g h t m o d i f i c a t i o n of the method of Duck-Chong (1979) . To a sample i n a t e s t tube , 30 M1 of 10% magnesium n i t r a t e i n methanol was added and the mix ture evapora ted to dryness by h e a t i n g i t i n a water bath at 8 5 - l 0 0 ° C for 5 to 10 min . D i g e s t i o n was a c h i e v e d by h e a t i n g the sample for 15 sec at the top of the flame and 10-15 sec at the t i p of the b lue cone of the f lame. A f t e r tubes reached room tempera ture , 0.3 ml of 1 M HCl was added. The tubes were c o v e r e d wi th marbles and heated at 9 0 - 9 5 ° C for 15 min . A f t e r c o o l i n g to room tempera ture , 0.7 ml 1 M HCl was added, f o l l o w e d by 2 ml of reagent C (see b e l o w ) . A f t e r 5 min the absorbance was measured at 620 nm i n a d i s p o s a b l e c u v e t t e . With each experiment a s t andard phosphorus curve was o b t a i n e d by adding a known sample of sodium d i h y d r o g e n monophosphate to a tube i n which 30 til of magnesium n i t r a t e had been evapora ted and the assay was c a r r i e d out as d e s c r i b e d above. Reagent C was p repared by mix ing 1 volume of 4.2% (w/v) ammonium molybdate i n 4.5 M HCl w i t h 3 volume of 0.05% (w/v) m a l a c h i t e g r e e n . Ju s t be fore use , T r i t o n X-100 was added to t h i s mixture to g i v e a f i n a l c o n c e n t r a t i o n of 0.075% (w/v ) . I t shou ld be mentioned here tha t w h i l e m a l a c h i t e green from BDH d i s s o l v e d f r e e l y , m a l a c h i t e green from other sources d i d n o t . 58 2.11 I n o s i t o l Phosphate A c c u m u l a t i o n In The Presence Of L i t h i u m In p r e l i m i n a r y exper iments i t was observed that b lank v a l u e s c o u l d be reduced d r a s t i c a l l y by p r i o r t reatment of the l a b e l e d i n o s i t o l , as o b t a i n e d from the manufac turer , w i t h a n i o n -exchange r e s i n . T h i s may be due to l a b e l e d a n i o n i c contaminants i n the [ 3 H ] i n o s i t o l , which were be ing removed d u r i n g t h i s s t e p . 200 ul of Dowex-1 X8-formate r e s i n (50% w/v) was added to 30 MCi of [ 3 H ] i n o s i t o l i n 800 ul water . The mix ture was v o r t e x mixed , c e n t r i f u g e d and the supernatant added to 14 ml of Krebs b u f f e r s a t u r a t e d w i t h 0 2 / C 0 2 ( 9 5 / 5 ) . Guinea p i g i l eum p i e c e s were i n c u b a t e d w i t h 1 uCi of [ 3 H ] i n o s i t o l i n a t o t a l volume of 1 ml K r e b s - R i n g e r b i c a r b o n a t e b u f f e r i n d i s p o s a b l e p l a s t i c tubes , i n the presence o f , wherever r e q u i r e d , 0.1 mM c a r b a c h o l , 10 mM l i t h i u m c h l o r i d e , 60 mM K C I , the r e q u i r e d amount of PMSF i n 1% d i m e t h y l s u l f o x i d e or d i m e t h y l s u l f o x i d e a lone and 6.4 uM a t r o p i n e s u l p h a t e . At the b e g i n n i n g of the i n c u b a t i o n 0 2 / C 0 2 (95/5) was i n t r o d u c e d above the s u r f a c e of the m i x t u r e . Then the tubes were c l o s e d t i g h t l y and i n c u b a t i o n c o n t i n u e d for a p e r i o d of one h o u r . I n c u b a t i o n was t e r m i n a t e d by removing the smooth muscle p i e c e s and t r a n s f e r r i n g them i n t o 1 ml c h l o r o f o r m / m e t h a n o l ( 1 : 2 ) . The t i s s u e s were homogenized and the l i p i d s e x t r a c t e d as d e s c r i b e d above . The aqueous phase of the e x t r a c t i o n medium was r e t a i n e d and i n o s i t o l phosphate de termined as f o l l o w s . 59 2.12 D e t e r m i n a t i o n Of T o t a l L a b e l e d I n o s i t o l Phosphate The method was s i m i l a r to the s i m p l i f i e d assay procedure d e s c r i b e d by B e r r i d g e et a l . (1982) . A 1 ml s l u r r y of 50% (w/v) Dowex-1 X8 (100-200 mesh) i n the formate form was added to the aqueous phase . The supernatant was d i s c a r d e d a f t e r the r e s i n was a l l o w e d to s e t t l e . A f t e r the r e s i n was washed f i v e t imes w i t h 4 ml of 5 mM i n o s i t o l , i n o s i t o l phosphates were e l u t e d w i t h 0.8 ml of 0.1 M formic a c i d p l u s 1 M ammonium formate . H a l f of t h i s e l u a t e (0 .4 ml) was used for the d e t e r m i n a t i o n of r a d i o a c t i v i t y a f t e r the a d d i t i o n of 10 ml ACS. 2.13 S e p a r a t i o n Of I n o s i t o l Phosphates Washed r e s i n c o n t a i n i n g adsorbed i n o s i t o l phosphates was e l u t e d s u c c e s s i v e l y i n a t e s t tube w i t h four 2 ml p o r t i o n s of 5 mM borax p l u s 60 mM ammonium formate , 5 mM borax p l u s 150 mM ammonium formate , 0.1 M formic a c i d p l u s 0.4 M ammonium formate and f i n a l l y 0.1 M formic a c i d p l u s 1 M ammonium formate . In the column s e p a r a t i o n of i n o s i t o l phosphates , 2 ml of Dowex-1 X8-formate r e s i n (50% w/v) was packed i n a 1x4 cm co lumn. S tandard i n o s i t o l mono-phosphate, or an aqueous e x t r a c t of i n o s i t o l phosphate accumulated i n the t i s s u e , was passed through the co lumn. T h i s was f o l l o w e d by washing of the column w i t h 5 mM i n o s i t o l u n t i l no more r a d i o a c t i v i t y was e l u t e d . I n o s i t o l phosphates were e l u t e d from the column wi th i n c r e a s i n g i o n i c s t r e n g t h s of borax , ammonium formate and formic a c i d , as d e s c r i b e d above . 1 ml f r a c t i o n s were c o l l e c t e d and phosphorous or r a d i o a c t i v i t y d e t e r m i n e d . 60 2.14 C o n t r a c t i o n S t u d i e s Guinea p i g i l eum s t r i p s were looped and suspended at 3 7 ° C under 0.35 g t e n s i o n i n Krebs b i c a r b o n a t e b u f f e r and a e r a t e d w i t h 0 2 / C 0 2 ( 9 5 / 5 ) . A l t e r a t i o n in tone was moni tored i s o m e t r i c a l l y u s i n g f o r c e t r a n s d u c e r s a f t e r a 30 min e q u i l i b r a t i o n p e r i o d . The e f f e c t of PMSF on c a r b a c h o l - and potas s ium-media ted c o n t r a c t i o n was s t u d i e d by adding PMSF d i s s o l v e d i n d i m e t h y l s u l f o x i d e to a f i n a l c o n c e n t r a t i o n of 1% d i m e t h y l s u l f o x i d e i n the b a t h . The h y p e r o s m o l a r i t y of the 60 mM KCI s o l u t i o n was not compensated by r e d u c t i o n of the NaCl c o n c e n t r a t i o n . 2.15 PI Degrada t ion S t u d i e s PI d e g r a d a t i o n s t u d i e s on a t r i a l fragments p r e l a b e l e d w i t h [ 3 2 P ] p h o s p h a t e were performed as f o l l o w s . A t r i a from 5 r a t s were p o o l e d and p h o s p h o l i p i d s l a b e l e d j_n v i t r o by i n c u b a t i n g s m a l l fragments for 2 h at 3 7 ° C w i t h 0.2 mCi of [ 3 2 P ] s o d i u m or thophosphate i n 40 ml of b u f f e r . At the end of the i n c u b a t i o n p e r i o d fragments were s epara ted by f i l t r a t i o n . A f t e r the fragments were washed w i t h Krebs b u f f e r , they were d i v i d e d i n t o s i x a p p r o x i m a t e l y equa l p o r t i o n s and suspended i n 5.9 ml of Krebs b u f f e r . F u r t h e r i n c u b a t i o n fo r 1 h at 3 7 ° C was c a r r i e d out i n the presence or absence of 0.1 mM c a r b a c h o l . At the end of t h i s i n c u b a t i o n p e r i o d , f u r t h e r r e a c t i o n was s topped w i t h 2 ml of 20% t r i c h l o r o a c e t i c a c i d and t i s s u e fragments were s epara ted by f i l t r a t i o n . E x t r a c t i o n of l i p i d s , s e p a r a t i o n of p h o s p h o l i p i d s and d e t e r m i n a t i o n of r a d i o a c t i v i t y were done as 61 d e s c r i b e d i n the e a r l i e r s e c t i o n s . In d e g r a d a t i o n s t u d i e s employing [ 1 " C ] - l a b e l e d a r a c h i d o n i c a c i d , whole a t r i a from 12 r a t s were incuba ted w i t h 10 /uCi of [ 1 " C ] a r a c h i d o n i c a c i d i n 30 ml of Krebs b u f f e r , for 2 h at 3 7 ° C . At the end of t h i s p e r i o d , a t r i a were s e p a r a t e d , washed w i t h Krebs b u f f e r c o n t a i n i n g 1% bovine serum albumin and incuba ted for a f u r t h e r 15 min p e r i o d i n the presence or absence of the d r u g . The r e a c t i o n was s topped w i t h 2 ml of 20% t r i c h l o r o a c e t i c a c i d , a t r i a were s e p a r a t e d , weighed and l i p i d and p h o s p h o l i p i d i s o l a t e d as d e s c r i b e d i n an e a r l i e r s e c t i o n . R a d i o a c t i v i t y i n each of the PI spots was de te rmined and r e s u l t s c a l c u l a t e d as amount of r a d i o a c t i v i t y i n PI per g of t i s s u e and expres sed as p e r c e n t c o n t r o l . 2.16 F a t t y A c i d Compos i t ion Of D i a c y l g l y c e r o l The f a t t y a c i d c o m p o s i t i o n of d i a c y l g l y c e r o l f r a c t i o n s was de termined by methods s i m i l a r to those d e s c r i b e d by Bansback et a l . (1974) . In these e x p e r i m e n t s , whole a t r i a from r a t were i n c u b a t e d i n 5.3 ml of Krebs b u f f e r at 3 7 ° C , under an 0 2 / C 0 2 (95/5) a tmosphere . A f t e r 15 min a s tock c a r b a c h o l s o l u t i o n was added to g i v e a f i n a l c a r b a c h o l c o n c e n t r a t i o n of 0.1 mM. A f t e r a f u r t h e r 15 min i n c u b a t i o n , the r e a c t i o n was s topped w i t h 2 ml of 20% t r i c h l o r o a c e t i c a c i d . C o n t r o l t i s s u e s were t r e a t e d i d e n t i c a l l y i n the absence of c a r b a c h o l . T i s s u e s were r a p i d l y f rozen i n dry i c e / m e t h y l b u t a n e m i x t u r e , and s t o r e d o v e r n i g h t at - 2 0 ° C , F r o z e n t i s s u e s were weighed, chopped i n t o s m a l l e r fragments and l i p i d e x t r a c t e d as d e s c r i b e d i n an e a r l i e r 62 s e c t i o n . N e u t r a l l i p i d s were separa ted by t h i n l a y e r chromatography of t o t a l l i p i d e x t r a c t wi th b e n z e n e r c h l o r o f o r m : m e t h a n o l (80 :15 :5 v / v ) , and v i s u a l i z e d w i t h 0.01% d i c h l o r o f l u o r e s c e i n s p r a y . D i a c y l g l y c e r o l was i d e n t i f i e d by compari son w i t h 1 , 2 - d i p a l m i t o y l g l y c e r o l . D i a c y l g l y c e r o l was e x t r a c t e d from the s i l i c a g e l p l a t e s w i t h d i e t h y l e t h e r / e t h a n o l (9:1 v / v ) c o n t a i n i n g 0.1% (v/v) 2 - m e r c a p t o e t h a n o l . D i a c y l g l y c e r o l was taken to dryness under N 2 and h e n e i c o s a n o i c a c i d was added as an i n t e r n a l s t a n d a r d . F a t t y a c i d s were o b t a i n e d from l i p i d s by h y d r o l y s i s w i t h 15% m e t h a n o l i c potas s ium h y d r o x i d e at 7 0 ° C for 1 h . The f a t t y a c i d s were e x t r a c t e d w i t h pentane and c o n v e r t e d to t h e i r methyl e s t e r s w i t h 10-15% boron t r i f l u o r i d e i n methano l . The methyl e s t e r s were e x t r a c t e d w i t h pentane and f u r t h e r p u r i f i e d by t h i n l a y e r chromatography i n benzene. S i l i c a g e l p o r t i o n s c o r r e s p o n d i n g to a s t a n d a r d , methyl s t e a r a t e , were scraped from the p l a t e s , e x t r a c t e d twice w i t h hexane and c o n c e n t r a t e d under N 2 . The methyl e s t e r mixture was resuspended i n 20 ul hexane and i n j e c t e d i n t o a gas chromatograph (Hewlett Packard 5830A) equipped w i t h a flame i o n i z a t i o n d e t e c t o r and Hewlet t Packard 18850A i n t e g r a t o r . S e p a r a t i o n of i n d i v i d u a l methyl e s t e r s was a c h i e v e d on a 10% d i e t h y l e n e g l y c o l s u c c i n a t e on an 80/100 mesh S u p e l c o p o r t co lumn. The column c o n d i t i o n s were as f o l l o w s : column temperature - 1 9 0 ° C ; i n j e c t i o n p o r t - 2 2 5 ° C ; d e t e c t o r temperature - 2 2 5 ° C ; c a r r i e r g a s - h e l i u m at 56-58 m l / m i n . The flame i o n i z a t i o n d e t e c t o r was found to have a s i m i l a r s e n s i t i v i t y fo r a l l the f a t t y a c i d s examined. F a t t y a c i d 63 q u a n t i t i e s were e s t i m a t e d based on the area r a t i o w i t h re spec t to the i n t e r n a l s t a n d a r d . 2.17 M a t e r i a l s C a r b a m y l c h o l i n e c h l o r i d e , a t r o p i n e s u l p h a t e , i n o s i t o l s -monophosphate d l - m o n o c y c l o h e x y l a m i n e , m y o - i n o s i t o l , f a t t y a c i d methyl e s t e r mixture and l i t h i u m c h l o r i d e were o b t a i n e d from Sigma c h e m i c a l company, S t . L o u i s , Mo. [ 3 2 P ] s o d i u m or thophosphate (200 mCi/mmol) , [ 1 - C 1 " ] - a r a c h i d o n i c a c i d (55.8 mCi/mmol) , m y o - [ 3 H j i n o s i t o l (15.8 Ci/mmol) and E c o n o f l u o r were o b t a i n e d from New Eng land N u c l e a r , L a c h i n e , Quebec. Phentolamine mesy la te was o b t a i n e d from CIBA Canada, D o r v a l , Quebec. Methoxamine h y d r o c h l o r i d e was from Burroughs Wellcome C o . , Research T r i a n g l e Park , N . C . Dowex-1 X8-formate r e s i n was from B io-Rad l a b o r a t o r i e s , M i s s i s s a u g a , O n t a r i o . M a l a c h i t e green ( C . I . 42000) and magnesium n i t r a t e were from BDH c h e m i c a l s , ACS and ammonium molybdate were from Amersham, S e a t t l e , Wa. P r e c o a t e d R e d i - c o a t 2D t h i n l a y e r chromatography p l a t e s were o b t a i n e d from Supe lco I n c . , B e l l a n f o n t e , P a . and p r e c o a t e d s i l i c a g e l 60 t h i n l a y e r chromatography p l a t e s and benzene were o b t a i n e d from E . M e r c k , Darmstadt , Germany. G l a s s d i s t i l l e d c h l o r o f o r m , methanol , hexane and acetone were purchased from C a l e d o n , O n t a r i o . A l l i n o r g a n i c s a l t s used i n the p r e p a r a t i o n of b u f f e r s were of a n a l y t i c a l g r a d e . 64 . I l l . RESULTS 3.1 SEPARATION OF PHOSPHOLIPIDS 3.1.1 S e p a r a t i o n And I d e n t i f i c a t i o n Of PI P h o s p h o l i p i d s were s epara ted by two d i m e n s i o n a l t h i n l a y e r chromatography on a s i l i c a g e l p l a t e , as shown i n F i g 4. Based on compari son w i t h s t andard p h o s p h o l i p i d s , the f o l l o w i n g spots were i d e n t i f i e d - spot a- p h o s p h a t i d y l i n o s i t o l , spot b-p h o s p h a t i d y l s e r i n e , spot c - p h o s p h a t i d y l c h o l i n e , spot d-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 spot f- p h o s p h a t i d i c a c i d . The i d e n t i t y of PI was f u r t h e r conf i rmed .by the s p e c i f i c i n c o r p o r a t i o n of [ 3 H ] i n o s i t o l i n t o spot ( a ) . The r e l a t i v e d i s t r i b u t i o n of the four p h o s p h o l i p i d s determined i n s t u d i e s on ra t a t r i a i s shown i n T a b l e I . As the o b j e c t i v e was to determine the s p e c i f i c r a d i o a c t i v i t y i n c o r p o r a t e d i n t o PI r a t h e r than the q u a n t i t a t i v e d e t e r m i n a t i o n of a l l p h o s p h o l i p i d s , e x t r a c t i o n e f f i c i e n c y of i n d i v i d u a l p h o s p h o l i p i d s was not de termined and t h e r e f o r e , the percentage d i s t r i b u t i o n of the four major p h o s p h o l i p i d s e s t i m a t e d by phosphorus d e t e r m i n a t i o n , as shown i n T a b l e I d i f f e r s from tha t r e p o r t e d by White (1973). In a s i m i l a r exper iment , where p h o s p h a t i d y l i n o s i t o l and p h o s p h a t i d y l c h o l i n e , as w e l l as t o t a l l i p i d phosphorus , were d e t e r m i n e d , i t was c a l c u l a t e d that 16.3 Mg of p h o s p h a t i d y l i n o s i t o l and 126.6 uq of p h o s p h a t i d y l c h o l i n e are pre sent per g t i s s u e weight (one g t i s s u e of r a t h e a r t has been 65 -r e p o r t e d to c o n t a i n 470 Mg of l i p i d phosphorus (White , 1973)) . The r e p o r t e d v a l u e s for these two p h o s p h o l i p i d s i n r a t hear t are 17.4 and 169.2 Mg per g t i s s u e , r e s p e c t i v e l y (White , 1973). 3 .1 .2 Phosphorus D e t e r m i n a t i o n Phosphorus was determined by the method of Duck-Chong (1979) . An example of a s t andard curve o b t a i n e d by that method i s shown i n F i g 5. T h i s phosphorus d e t e r m i n a t i o n method was s e n s i t i v e and the s t andard curve was r e p r o d u c i b l e . A p l o t of absorbance ve r su s phosphorus gave a s t r a i g h t l i n e between 0.1 Mg to 0.6 Mg, w i t h a s lope around 1 and c o r r e l a t i o n c o e f f i c i e n t of g r e a t e r than 0 . 9 9 . The v a r i a t i o n i n d u p l i c a t e o b s e r v a t i o n s i n each experiment were l e s s than 0.01 absorbance u n i t . 3.2 PHOSPHATE INCORPORATION IN RAT HEART 3.2.1 [ 3 2 P ] P h o s p h a t e I n c o r p o r a t i o n In Rat A t r i a Ver sus V e n t r i c l e D e s p i t e i t s low q u a n t i t y i n c e l l s , PI has been found to c o n t a i n a l a r g e f r a c t i o n of the t o t a l phosphate l a b e l i n c o r p o r a t e d i n t o p h o s p h o l i p i d s ( M i c h e l l , 1 9 7 5 ) . We o b t a i n e d a s i m i l a r r e s u l t i n ra t hear t (Table I ) . The b a s a l phosphate i n c o r p o r a t i o n i n t o v a r i o u s p h o s p h o l i p i d s i n r a t a t r i a and v e n t r i c l e s i s shown i n F i g 6. Both i n a t r i a and v e n t r i c l e s PI i n c o r p o r a t e d l a b e l e d phosphate w i t h a h i g h e r s p e c i f i c a c t i v i t y than tha t of the o ther p h o s p h o l i p i d s . I t was a l s o observed tha t p h o s p h a t e - l a b e l i n c o r p o r a t i o n i n t o the v a r i o u s p h o s p h o l i p i d s of a t r i a was about 5-6 f o l d h i g h e r than tha t of l a b e l i n c o r p o r a t i o n 66 i n t o the c o r r e s p o n d i n g p h o s p h o l i p i d s i n v e n t r i c l e s . 3 .2 .2 E f f e c t Of M u s c a r i n i c And A l p h a - a d r e n e r g i c Receptor  S t i m u l a t i o n On [ 3 2 P J p h o s p h a t e I n c o r p o r a t i o n In Combined A t r i a Enhanced i n c o r p o r a t i o n of phosphate i n t o PI i s u s u a l l y the f i r s t s t ep i n i d e n t i f y i n g a PI r e sponse . Both m u s c a r i n i c (Wei and S u l a k h e , 1978) and a l p h a - a d r e n e r g i c (Schumann, 1980) r e c e p t o r s have been shown to be pre sent i n ra t a t r i a , but u n l i k e most o ther t i s s u e s s t i m u l a t i o n of these two r e c e p t o r s i n ra t a t r i a produces o p p o s i t e p h a r m a c o l o g i c a l r e sponse s . As m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n r a t a t r i a i s c o n s i d e r e d not to be accompanied by c a l c i u m m o b i l i z a t i o n , i t was of i n t e r e s t to determine whether PI response accompanies i t s s t i m u l a t i o n . S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n combined ra t a t r i a l p i e c e s by 0.1 mM c a r b a c h o l caused a 35% i n c r e a s e i n [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o PI (p<0.05) ( F i g 7 ) . In order to v e r i f y tha t the s m a l l degree of i n c o r p o r a t i o n of l a b e l e d phosphate i n t o PI on m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n combined r a t a t r i a l p i e c e s was not the r e s u l t of an i n a b i l i t y to measure [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o tha t p a r t i c u l a r p r e p a r a t i o n , the e f f e c t of methoxamine, an a - a d r e n e r g i c a g o n i s t , on [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o PI of combined r a t a t r i a was i n v e s t i g a t e d . In c o n t r a s t to the m u s c a r i n i c re sponse , s t i m u l a t i o n of the a - a d r e n e r g i c r e c e p t o r of r a t a t r i a w i t h methoxamine (0.1 mM) enhanced the i n c o r p o r a t i o n of [ 3 2 P ] p h o s p h a t e i n t o PI by 88% ( F i g 7 ) , even though the a-a d r e n e r g i c r e c e p t o r d e n s i t y i s l e s s than 50% of the m u s c a r i n i c 67 r e c e p t o r d e n s i t y . T h i s s t i m u l a t i o n by methoxamine was b l o c k e d by the a - a d r e n e r g i c a n t a g o n i s t , phentolamine (20 yM). In a s i m i l a r exper iment , i t was f u r t h e r shown tha t the methoxamine-s t i m u l a t e d i n c r e a s e in phosphate i n c o r p o r a t i o n was s p e c i f i c to PI and d i d not occur i n o ther p h o s p h o l i p i d s , i n c l u d i n g p h o s p h a t i d y l s e r i n e , p h o s p h a t i d y l c h o l i n e or p h o s p h a t i d y l e t h a n o l a m i n e ( F i g 8 ) . 3 .2 .3 E f f e c t Of M u s c a r i n i c And A l p h a - a d r e n e r g i c Receptor  S t i m u l a t i o n On [ 3 2 P Iphospha te I n c o r p o r a t i o n In Separate L e f t And  R ight A t r i a Quist (1982) r e p o r t e d that m u s c a r i n i c r e c e p t o r s t i m u l a t i o n of can ine r i g h t a t r i u m , but not l e f t a t r i u m or l e f t or r i g h t v e n t r i c l e , showed enhanced i n c o r p o r a t i o n of phosphate i n t o P I , even though a l l r e g i o n s of the can ine hear t have been shown to c o n t a i n m u s c a r i n i c r e c e p t o r s (Wei and Su l akhe , 1978). T h i s l e d us to t e s t whether a s i m i l a r s i t u a t i o n might e x i s t i n r a t h e a r t , so that o n l y one of the r a t a t r i a may be r e s p o n s i b l e for the s m a l l [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n observed i n combined a t r i a l p r e p a r a t i o n s . I t was found tha t s t i m u l a t i o n of the a l p h a -a d r e n e r g i c r e c e p t o r , which u s u a l l y shows a s m a l l e r PI response than m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n o ther t i s s u e s (Jones and M i c h e l l , 1975), produced a 110 and 175% i n c r e a s e i n phosphate i n c o r p o r a t i o n i n t o PI i n l e f t and r i g h t a t r i u m , r e s p e c t i v e l y ( F i g 9 ) . M u s c a r i n i c r e c e p t o r s t i m u l a t i o n by c a r b a c h o l , on the o ther hand, produced a 35% i n c r e a s e i n phosphate i n c o r p o r a t i o n i n t o PI i n r a t l e f t a t r i u m (p<0.05), but was c o m p l e t e l y wi thout 68 e f f e c t i n r a t r i g h t a t r i u m ( F i g 10) . The r e l a t i v e b a s a l i n c o r p o r a t i o n i n t o r a t l e f t and r i g h t a t r i u m was q u i t e v a r i a b l e (compare F i g 9 and 10), but the comparison of r e s u l t s from v a r i o u s exper iments suggests that the magnitude of the b a s a l i n c o r p o r a t i o n d i d not i n f l u e n c e the s e n s i t i v i t y to a g o n i s t s . These r e s u l t s suggested t h a t w h i l e the m a j o r i t y of m u s c a r i n i c r e c e p t o r s i n r a t a t r i a do not appear to be c o u p l e d to PI t u r n o v e r , a s m a l l p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s i n ra t l e f t a t r i u m may be c o u p l e d to PI t u r n o v e r . 3.3 PI BREAKDOWN IN HEART The pr imary event of PI response has been c o n s i d e r e d for a long time to be the enhanced breakdown of i n o s i t o l l i p i d (Hokin and Hokin., 1964; M i c h e l l , 1975). At l e a s t i n one t i s s u e , b l o w f l y s a l i v a r y g l a n d , i t has been observed ( F a i n and B e r r i d g e , 1979a) that the pr imary e v e n t , i n o s i t o l l i p i d breakdown, was not accompanied by r a p i d r e s y n t h e s i s of P I . As o n l y a s m a l l i n c r e a s e i n phosphate i n c o r p o r a t i o n i n t o PI d u r i n g m u s c a r i n i c r e c e p t o r s t i m u l a t i o n of combined r a t a t r i a was observed ( F i g 7 ) , i t was d e c i d e d to i n v e s t i g a t e the e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n on PI breakdown i n combined ra t a t r i a ( these s t u d i e s were done be fore the d i f f e r e n c e s i n PI response between r a t l e f t and r i g h t a t r i u m became a p p a r e n t ) . 69 3.3.1 Breakdown Of P h o s p h o l i p i d s P r e l a b e l e d With R a d i o l a b e l  Phosphate M u s c a r i n i c r e c e p t o r s t i m u l a t i o n of r a t p a r o t i d g l and has been shown to cause an enhanced breakdown of PI p r e v i o u s l y l a b e l e d w i t h r a d i o l a b e l e d phosphate (Jones and M i c h e l l , 1974). In the p re sen t s tudy s t i m u l a t i o n of combined r a t a t r i a w i t h 0.1 mM c a r b a c h o l for 45 min d i d not enhance the breakdown of PI or any o ther p h o s p h o l i p i d s s i g n i f i c a n t l y ( F i g 11) 3 .3 .2 Breakdown Of PI P r e l a b e l e d With A r a c h i d o n a t e In the pas t few y e a r s , ev idence has been p r e s e n t e d to suggest that r e c e p t o r s t i m u l a t i o n may cause r e l e a s e of a r a c h i d o n i c a c i d from P I , e i t h e r through a c t i v a t i o n of p h o s p h o l i p a s e A 2 ( B i l l a h and L a p e t i n a , 1982a) or d u r i n g PI t u r n o v e r ( M a r s h a l l et a l . , 1980, 1982). T h e r e f o r e , i n o rder to d e t e c t any l o s s of a r a c h i d o n i c a c i d from P I , as w e l l as the breakdown of P I , the e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n on a r a c h i d o n y l - p r e l a b e l e d PI i n combined r a t a t r i a was examined. A c a r b a c h o l i n c u b a t i o n p e r i o d of 15 min was chosen fo r these exper iments for the f o l l o w i n g r e a s o n s . A d e t e c t a b l e decrease i n the l e v e l of PI a f t e r a 30 min r e c e p t o r s t i m u l a t i o n has been observed i n p a r o t i d g l and (Jones and M i c h e l l , 1975, 1976) and guinea p i g i l eum (Jones et a l . , 1979) and a f t e r r e c e p t o r s t i m u l a t i o n for 15 min i n hepa tocy te s ( B i l l a h and M i c h e l l , 1979). S i m i l a r l y , a maximum i n c r e a s e i n the d i a c y l g l y c e r o l l e v e l d u r i n g a c e t y l c h o l i n e r e c e p t o r s t i m u l a t i o n of pancreas was observed a f t e r a 15 min i n c u b a t i o n p e r i o d (Bansback et a l . , 70 1974). As the d i a c y l g l y c e r o l moiety i s c o n s i d e r e d to be conserved d u r i n g the PI re sponse , the r e s u l t s i n a r a c h i d o n a t e -p r e l a b e l e d PI s t u d i e s were expres sed as the amount of r a d i o a c t i v i t y p re sent in PI per g of t i s s u e , r a t h e r than as s p e c i f i c r a d i o a c t i v i t y . When a r a c h i d o n y l - p r e l a b e l e d a t r i a were s t i m u l a t e d w i t h 0.1 mM c a r b a c h o l fo r 15 min , the amount of r a d i o a c t i v i t y i n the PI f r a c t i o n of u n s t i m u l a t e d and s t i m u l a t e d t i s s u e was found to be 175 and 194 cpm/mg wet t i s s u e we ight , r e s p e c t i v e l y (p>0.05) ( F i g 12) . T h i s suggested tha t m u s c a r i n i c s t i m u l a t i o n d i d not cause a s i g n i f i c a n t breakdown of PI i n combined r a t a t r i a e i t h e r through a c t i v a t i o n of p h o s p h o l i p a s e A 2 or p h o s p h o l i p a s e C . 3 .3 .3 D i a c y l g l y c e r o l Accumula t ion N i s h i z u k a ' s group (Takai et a l . , 1979b; K i s h i m o t o et a l . , 1980) have suggested tha t d i a c y l g l y c e r o l formed d u r i n g PI t u r n o v e r may mediate i t s e f f e c t through a c t i v a t i o n of p h o s p h o l i p i d - d e p e n d e n t PKC. In p a n c r e a s , where A C h - s t i m u l a t e d PI t u r n o v e r has been r e p o r t e d (Hokin and H o k i n , 1953), an i n c r e a s e i n d i a c y l g l y c e r o l , as w e l l as the a r a c h i d o n y l c o n t e n t of d i a c y l g l y c e r o l , has been observed by Bansback et a l . (1974) . As a measure of i n o s i t o l l i p i d breakdown, the e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n on the a r a c h i d o n y l conten t of d i a c y l g l y c e r o l i n combined ra t a t r i a was examined. D i a c y l g l y c e r o l was s epara ted from p h o s p h o l i p i d s by t h i n l a y e r chromatography i n b e n z e n e / c h l o r o f o r m / m e t h a n o l ( 8 0 : 1 5 : 5 ) , as shown i n F i g 13. P h o s p h o l i p i d s remained at the o r i g i n , w h i l e 71 d i a c y l g l y c e r o l moved w i t h an R va lue of 0 . 5 3 . When 2-mercaptoe thano l was pre sent a p r e l i m i n a r y s e p a r a t i o n of f a t t y a c i d methyl e s t e r s by t h i n l a y e r chromatography i n benzene was done p r i o r to gas chromatography. Spots c o r r e s p o n d i n g to s t a n d a r d methyl s t e a r a t e were removed and e x t r a c t e d i n hexane. F a t t y a c i d methyl e s t e r s were then a n a l y z e d by gas chromatography. Both methyl s t e a r a t e and methyl a r a c h i d o n a t e have a s i m i l a r m o b i l i t y i n benzene, and t h e r e f o r e the absence of the l a t t e r i n the d i a c y l g l y c e r o l f r a c t i o n i s not the r e s u l t of i t s l o s s d u r i n g TLC i n benzene. The s t andard f a t t y a c i d mixture was very w e l l s epara ted under the column c o n d i t i o n s used , w i t h the f o l l o w i n g r e t e n t i o n v a l u e s (min) - p a l m i t a t e 3 .04 , ; s t e a r a t e 5 .13 ; o l e a t e 5 .95 ; a r a c h i d a t e 8 .88 ; l i n o l e a t e 9.88 and a r a c h i d o n a t e 17 .23. The f a t t y a c i d c o m p o s i t i o n of d i a c y l g l y c e r o l i n c a r b a c h o l - t r e a t e d and - u n t r e a t e d t i s s u e i s shown i n T a b l e I I . Both c a r b a c h o l - t r e a t e d and - u n t r e a t e d t i s s u e s showed o n l y 4 f a t t y a c i d s i n the d i a c y l g l y c e r o l f r a c t i o n ; they were p a l m i t i c , s t e a r i c , o l e i c and l i n o l e i c a c i d , w h i l e a r a c h i d o n i c a c i d was absent ( F i g 14) . 3 .3 .4 F a t t y A c i d Compos i t ion Of The P o l a r L i p i d F r a c t i o n In  A t r i a And V e n t r i c l e s In order to v e r i f y tha t the absence of a r a c h i d o n i c a c i d was not the r e s u l t of i t s d e g r a d a t i o n d u r i n g i s o l a t i o n , the f a t t y a c i d c o m p o s i t i o n of the p o l a r l i p i d f r a c t i o n remain ing at the o r i g i n a f t e r s e p a r a t i o n of the d i a c y l g l y c e r o l was d e t e r m i n e d . In both a t r i a and v e n t r i c l e s about 65% of the t o t a l f a t t y a c i d 72 conten t was s a t u r a t e d f a t t y a c i d and the remainder was u n s a t u r a t e d f a t t y a c i d . A t r i a and v e n t r i c l e s d i d not d i f f e r i n the content of the two s a t u r a t e d f a t t y a c i d s , but d i f f e r e d s i g n i f i c a n t l y i n the content of a l l three u n s a t u r a t e d f a t t y a c i d s , o l e i c , l i n o l e i c and a r a c h i d o n i c a c i d s ( F i g 15) . R e c e n t l y , Charnock et a l . (1983) a r r i v e d at s i m i l a r c o n c l u s i o n s a f t e r a more d e t a i l e d i n v e s t i g a t i o n of the p h o s p h o l i p i d s i n ra t a t r i a and v e n t r i c l e s . The s t u d i e s i n hear t i n d i c a t e d that w h i l e the a l p h a -a d r e n e r g i c r e c e p t o r in ra t a t r i a i s c o u p l e d to PI t u r n o v e r , the m a j o r i t y of the m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n ra t a t r i a i s not accompanied by enhanced PI t u r n o v e r . The l a c k of a c a r b a c h o l e f f e c t on i n o s i t o l l i p i d breakdown a l s o r u l e s out the p o s s i b i l i t y tha t c a r b a c h o l may be s t i m u l a t i n g the pr imary s tep i n PI t u r n o v e r wi thout a c t i v a t i n g the accompanying r e s y n t h e s i s . I t i s t h e r e f o r e suggested t h a t o n l y a s m a l l p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s i n ra t a t r i a i s c o u p l e d to PI t u r n o v e r and the PI breakdown accompanying m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i s e i t h e r absent or so sma l l tha t i t c o u l d not be d e t e c t e d by the methods used i n t h i s i n v e s t i g a t i o n . 3.4 PI RESPONSE IN GUINEA PIG ILEUM M u s c a r i n i c r e c e p t o r s t i m u l a t i o n of guinea p i g i l eum produces a b i p h a s i c c o n t r a c t i o n which i s shown to be mediated through c a l c i u m m o b i l i z a t i o n (Chang and T r i g g l e , 1973). J a f f e r j i and M i c h e l l ( l 976a ,b ) have r e p o r t e d tha t i n l o n g i t u d i n a l smooth muscle of guinea p i g i l eum s t i m u l a t i o n of 73 m u s c a r i n i c r e c e p t o r or K + - d e p o l a r i z a t i o n produced an enhanced i n c o r p o r a t i o n of r a d i o l a b e l e d - p h o s p h a t e i n t o P I . In the pre sent s t u d y , the PI response i n guinea p i g i l eum has been f u r t h e r i n v e s t i g a t e d i n order to examine i t s p o s s i b l e r o l e i n c a l c i u m m o b i l i z a t i o n . 3.4.1 [ 3 2 P ] P h o s p h a t e I n c o r p o r a t i o n The e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n on [ 3 2 P j p h o s p h a t e i n c o r p o r a t i o n i n t o PI i n guinea p i g i l eum was c a r r i e d out at the b e g i n n i n g of t h i s s tudy to v e r i f y our a b i l i t y to de tec t a PI re sponse . In agreement w i t h an e a r l i e r r e p o r t ( J a f f e r j i and M i c h e l l , 1976a), m u s c a r i n i c r e c e p t o r s t i m u l a t i o n of l o n g i t u d i n a l smooth muscle of guinea p i g i l eum w i t h 0.1 mM c a r b a c h o l caused a 100% i n c r e a s e i n the i n c o r p o r a t i o n of t 3 2 P ] p h o s p h a t e i n t o P I , and t h i s i n c r e a s e was b l o c k e d by the s p e c i f i c m u s c a r i n i c a n t a g o n i s t , a t r o p i n e (1 .6 MM) ( F i g 16) . 3 .4 .2 [ 3 H ] l n o s i t o l I n c o r p o r a t i o n A g o n i s t s which i n c r e a s e d l a b e l e d phosphate i n c o r p o r a t i o n i n t o PI a l s o i n c r e a s e d i n c o r p o r a t i o n of l a b e l e d i n o s i t o l i n t o PI i n s e v e r a l t i s s u e s ( for a l i s t of t i s s u e s examined see M i c h e l l , 1975). As i n a p r e v i o u s r e p o r t by S c h e l l e n b e r g and G i l l e s p i e (1980) , i t was observed i n p r e l i m i n a r y exper iments t h a t 98% of the [ 3 H ] i n o s i t o l l a b e l i n c o r p o r a t e d i n t o the major p h o s p h o l i p i d f r a c t i o n was pre sent i n i n o s i t o l l i p i d s . T h e r e f o r e , i n a l l subsequent i n o s i t o l i n c o r p o r a t i o n s t u d i e s , the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o the t o t a l l i p i d f r a c t i o n was used as a 74 measure of i n o s i t o l i n c o r p o r a t i o n i n t o i n o s i t o l - c o n t a i n i n g l i p i d s . In the presence of c a r b a c h o l (0.1 mM), the i n c o r p o r a t i o n of [ 3 H ] i n o s i t o l was i n c r e a s e d from 368 to 553 dpm/^g phosphorus (Table I I I ) . T h i s r e p r e s e n t s a 99% i n c r e a s e when the r a d i o a c t i v i t y i n c o r p o r a t e d d u r i n g the i n i t i a l 30 min p r e i n c u b a t i o n p e r i o d was s u b t r a c t e d . The i n c r e a s e i n [ 3 H ] i n o s i t o l i n c o r p o r a t i o n caused by c a r b a c h o l was c o m p l e t e l y b l o c k e d by 16 uM a t r o p i n e (Table I I I ) . 3 .4 .3 L i t h i u m - a m p l i f i e d A c c u m u l a t i o n Of I n o s i t o l Phosphates B e r r i d g e et a l . (1982) have shown tha t l i t h i u m causes a c c u m u l a t i o n of i n o s i t o l phosphate d u r i n g PI response i n a number of t i s s u e s . In t h i s study the e f f e c t of c a r b a c h o l on the a c c u m u l a t i o n of i n o s i t o l phosphates was s t u d i e d i n the presence , of 10 mM l i t h i u m for 60 min , c o n d i t i o n s which were p r e v i o u s l y found to be o p t i m a l ( B e r r i d g e et a l . , 1982). In the presence of 10 mM l i t h i u m or 0.1 mM c a r b a c h o l a lone there was a very s m a l l a c c u m u l a t i o n of r a d i o a c t i v i t y (31 and 23.5 dpm/uq t o t a l phosphorus , r e s p e c t i v e l y ) (Table I V ) . In the presence of l i t h i u m , 0.1 mM c a r b a c h o l produced a 2 0 - f o l d i n c r e a s e i n the a c c u m u l a t i o n of r a d i o a c t i v i t y , compared to the e f f e c t of c a r b a c h o l or l i t h i u m a lone (Table I V ) . The a c c u m u l a t i o n of i n o s i t o l phosphates by c a r b a c h o l i n the presence of l i t h i u m was dose-dependent , as shown i n F i g 17. T h i s response to c a r b a c h o l was b l o c k e d by the m u s c a r i n i c a n t a g o n i s t , a t r o p i n e (16 uM) ( F i g 17) , s u g g e s t i n g tha t the response r e s u l t s from m u s c a r i n i c r e c e p t o r s t i m u l a t i o n . The h a l f maximal response 75 for the c a r b a c h o l response was around 10 uM. T h i s i s s i m i l a r to the c a r b a c h o l c o n c e n t r a t i o n for h a l f maximal PI response o b t a i n e d u s i n g [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n ( J a f f e r j i and M i c h e l l , 1976a). 3 .4 .4 I d e n t i f i c a t i o n Of I n o s i t o l Phosphates B e r r i d g e et a l . (1982) have r e p o r t e d , based on e l u t i o n from an ion-exchange r e s i n column and h i g h v o l t a g e e l e c t r o p h o r e s i s tha t 80% of the r a d i o a c t i v i t y accumulated d u r i n g l i t h i u m - a m p l i f i e d PI response i n b r a i n , p a r o t i d g l a n d and s a l i v a r y g l a n d corre sponds to i n o s i t o l mono-phosphate. As i n the p r e v i o u s r e p o r t (Ber r idge et a l . , 1982), s t andard i n o s i t o l mono-phosphate was found to be q u a n t i t a t i v e l y e l u t e d from the ion-exchange r e s i n w i t h 5 mM borax p l u s 150 mM ammonium formate ( F i g 18) . However, l e s s than 20% of the r a d i o a c t i v i t y which accumulated f o l l o w i n g i n c u b a t i o n of guinea p i g i l eum w i t h c a r b a c h o l and l i t h i u m , and which was then adsorbed on a Dowex-1 X8-formate r e s i n , was e l u t e d i n a peak c o r r e s p o n d i n g to i n o s i t o l monophosphate ( F i g 19, Tab le V ) . The remainder was e l u t e d by h i g h e r i o n i c s t r e n g t h s o l u t i o n s . T h i s r e s u l t suggests tha t the m a j o r i t y of the r a d i o a c t i v i t y accumulated i n the presence of c a r b a c h o l and l i t h i u m was present as i n o s i t o l p o l y -phosphate s . Based on t h e i r e l u t i o n c h a r a c t e r i s t i c s from the ion exchange r e s i n ( B e r r i d g e et a l . , 1982, Downes and M i c h e l l , 1981), 2/3 appeared to be i n o s i t o l b iphosphate and 1/3 i n o s i t o l t r i p h o s p h a t e . 76 3.5 EFFECT OF PMSF ON PI TURNOVER AND CONTRACTION IN GUINEA PIG  ILEUM 3 . 5 . 1 E f f e c t Of PMSF On PI T u r n o v e r R e c e n t l y , Walenga e t a l . (1980) r e p o r t e d t h a t s e r i n e e s t e r a s e i n h i b i t o r s may a l s o be i n h i b i t o r s of P i - s p e c i f i c p h o s p h o l i p a s e C , based on t h e i r s t u d i e s i n p l a t e l e t s . I t was d e c i d e d t o i n v e s t i g a t e whether s e r i n e e s t e r a s e i n h i b i t o r s , such as PMSF, can i n h i b i t P i - s p e c i f i c p h o s p h o l i p a s e C i n the i l e u m , and i f t h e y d i d , what t h e i r e f f e c t was on t h e c a l c i u m m e d i a t e d r e s p o n s e i n i l e u m . The e f f e c t of PMSF on b a s a l [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d of the l o n g i t u d i n a l smooth musc le of g u i n e a p i g i l e u m i s shown i n F i g 2 0 . A f t e r an i n i t i a l 30 min p r e i n c u b a t i o n i n t h e absence of PMSF, b a s a l [ 3 H ] i n o s i t o l i n c o r p o r a t i o n was d e t e r m i n e d and shown t o r ema in l i n e a r w i t h t i m e f o r a f u r t h e r 30 m i n , b o t h i n t h e p r e s e n c e of 2 mM PMSF and i n i t s a b s e n c e . A t a l l t i m e p o i n t s s t u d i e d , PMSF caused a s m a l l but c o n s i s t e n t i n c r e a s e i n [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o P I . J a f f e r j i and M i c h e l l (1976b) have p r e v i o u s l y shown t h a t s t i m u l a t i o n of g u i n e a p i g i l e u m by p o t a s s i u m , w h i c h m o b i l i z e s c a l c i u m by o p e n i n g a v o l t a g e - d e p e n d e n t c a l c i u m c h a n n e l , a l s o p r o d u c e d an enhanced i n c o r p o r a t i o n o f l a b e l e d phospha te i n t o P I . The e f f e c t of 2 mM PMSF on c a r b a c h o l and K + - s t i m u l a t e d [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i s shown i n F i g . 2 1 . PMSF (2 mM) a l o n e c a u s e d a s l i g h t i n c r e a s e i n [ 3 H ] i n o s i t o l i n c o r p o r a t i o n , but i t a l m o s t c o m p l e t e l y b l o c k e d t h e c a r b a c h o l - s t i m u l a t e d 77 i n c r e a s e i n i n o s i t o l i n c o r p o r a t i o n , to a va lue s i m i l a r to that o b t a i n e d w i t h PMSF a lone ( F i g 21a) . In c o n t r a s t , PMSF caused a s l i g h t i n c r e a s e , from 167 to 187%, i n K + - s t i m u l a t e d [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o PI ( F i g 21b) . T h i s sugges ted , f i r s t , tha t PMSF i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n i s not a n o n s p e c i f i c e f f e c t and second, c a r b a c h o l and R + -s t i m u l a t e d [ 3 H ] i n o s i t o l i n c o r p o r a t i o n may be o c c u r r i n g through d i f f e r e n t mechanisms. 3 .5 .2 E f f e c t Of PMSF On C o n t r a c t i o n Both c a r b a c h o l and K + produce a b i p h a s i c c o n t r a c t i o n of guinea p i g i l e u m , which i s mediated by C a + 2 m o b i l i z a t i o n (Chang and T r i g g l e , 1973; Rosenberger and T r i g g l e , 1979). I f PI t u r n o v e r i s i n v o l v e d i n c a l c i u m m o b i l i z a t i o n , from the o b s e r v a t i o n s made above one would expect tha t PMSF w i l l a f f e c t c a r b a c h o l - s t i m u l a t e d , but not K + - s t i m u l a t e d , c o n t r a c t i o n . A d d i t i o n of 0.4 mM to 2 mM PMSF to guinea p i g i l eum s t r i p s , c o n t r a c t e d e i t h e r by c a r b a c h o l or K + , produced a r a p i d t r a n s i e n t r e l a x a t i o n ( F i g . 22a) . The peak of r e l a x a t i o n was a c h i e v e d w i t h i n 1 min and the t e n s i o n r e t u r n e d to c o n t r o l l e v e l s , even at the h i g h e s t PMSF c o n c e n t r a t i o n , i n 8 m i n u t e s . When PMSF was added p r i o r to the a d d i t i o n of c a r b a c h o l or K + , i t a l s o i n h i b i t e d the i n i t i a l p h a s i c responses to both a g e n t s . However, the degree of the i n h i b i t i o n of the p h a s i c response depended on the t ime e l a p s e d between the a d d i t i o n of PMSF and the c o n t r a c t i n g agent , and no i n h i b i t i o n was observed a f t e r a 10 min time i n t e r v a l . In c o n t r a s t to the t r a n s i e n t nature of the PMSF 78 e f f e c t on c a r b a c h o l - m e d i a t e d c o n t r a c t i o n i n guinea p i g i l e u m , i t s i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n the same t i s s u e was e f f e c t i v e over a 30 min p e r i o d , sugge s t ing that these two e f f e c t s of PMSF may be u n r e l a t e d . The sugge s t ion that PMSF i s capable of i n h i b i t i n g P I -s p e c i f i c p h o s p h o l i p a s e C d u r i n g PI t u r n o v e r i s based on i n d i r e c t e v i d e n c e . I t was necessary to v e r i f y whether PMSF i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d PI t u r n o v e r i n guinea p i g i l eum i s indeed due to i n h i b i t i o n of P i - s p e c i f i c p h o s p h o l i p a s e C . In order to t e s t t h i s , the e f f e c t of PMSF on i n o s i t o l l i p i d breakdown was i n v e s t i g a t e d . 3 .5 .3 E f f e c t Of PMSF On I n o s i t o l - P h o s p h a t e A c c u m u l a t i o n To determine the e f f e c t s of PMSF on i n o s i t o l - l i p i d breakdown, l i t h i u m - a m p l i f i e d a c c u m u l a t i o n of i n o s i t o l phosphates was used as a measure of the pr imary response of PI t u r n o v e r . In p r e l i m i n a r y exper iments PMSF was shown to cause an 18% decrease i n c a r b a c h o l - s t i m u l a t e d [ 3 H ] i n o s i t o l a c c u m u l a t i o n i n t o i n o s i t o l phosphates i n the presence of l i t h i u m (Table V I ) . As l e s s than 20% of the accumulated [ 3 H ] i n o s i t o l phosphates have been shown to be i n o s i t o l monophosphate ( F i g 19), i t was examined whether PMSF s p e c i f i c a l l y i n h i b i t e d the a c c u m u l a t i o n of the i n o s i t o l mono-phosphate f r a c t i o n . S e l e c t i v e e l u t i o n of the i n o s i t o l mono- and po ly -phospha te from an ion exchange r e s i n i n d i c a t e d t h a t PMSF caused a s i m i l a r (14.8 and 16.4%) decrease i n the a c c u m u l a t i o n of the i n o s i t o l mono- and po ly -phospha te 79 p o o l , but the decrease was s t a t i s t i c a l l y s i g n i f i c a n t (p<0.05) o n l y i n the l a t t e r c a s e . T h i s suggested tha t o n l y a sma l l pa r t of the PMSF i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d PI t u r n o v e r i s mediated through i n h i b i t i o n of P i - s p e c i f i c p h o s p h o l i p a s e C . 80 F i g u r e 4 - P h o s p h o l i p i d s e p a r a t i o n Two d i m e n s i o n a l t h i n l a y e r chromatographic s e p a r a t i o n of the ra t a t r i a l p h o s p h o l i p i d s i n Merck p r e c o a t e d s i l i c a g e l p l a t e s . L i p i d s were e x t r a c t e d from a s i n g l e r a t l e f t or r i g h t a t r i a as d e s c r i b e d i n the Methods . L i p i d s from four separa te samples were s p o t t e d on four c o r n e r s of the s i l i c a g e l p l a t e . P l a t e s were f i r s t deve loped i n c h l o r o f o r m / methano l / 10.5 M ammonium h y d r o x i d e (17:7:1 v / v ) . A f t e r removal of ammonia by a i r d r y i n g , p l a t e s were deve loped i n the second d imens ion c o n s i s t i n g of c h l o r o f o r m / a c e t o n e / m e t h a n o l / a c e t i c a c i d / water ( 3 : 4 : 1 : 1 : 0 . 5 v / v ) . P h o s p h o l i p i d s were d e t e c t e d by exposure to i o d i n e vapour , a) Photograph of a TLC p l a t e . b) T r a c i n g of one of the four quadrants of the TLC p l a t e . 81 82 T a b l e I - P h o s p h o l i p i d d i s t r i b u t i o n a n d [ 3 2 P ] p h o s p h a t e l a b e l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d s o f r a t a t r i a L i p i d s w e r e e x t r a c t e d f r o m a t r i a f o l l o w i n g i n c u b a t i o n w i t h 4 0 ^ C i [ 3 2 P ] p h o s p h a t e f o r 120 m i n . T o t a l l i p i d p h o s p h o r u s was d e t e r m i n e d b e f o r e s p o t t i n g t h e s a m p l e on t h e T L C p l a t e . A f t e r s e p a r a t i o n o f t h e p h o s p h o l i p i d s p o t s , t h e s i l i c a g e l was e x t r a c t e d o n c e w i t h 2 m l o f t h e e x t r a c t i n g m e d i u m ( a s d e s c r i b e d i n M e t h o d s ) , a n d p h o s p h o r u s a n d r a d i o a c t i v i t y d e t e r m i n e d i n t h e e l u a n t . The amount o f t o t a l p h o s p h o r u s a n d t o t a l r a d i o a c t i v i t y i n t h e f o u r p h o s p h o l i p i d s f r o m f o u r a t r i a l s a m p l e s w e r e 2 . 5 Mg a n d 4210 cpm r e s p e c t i v e l y . I n B) t h e amount o f p h o s p h a t i d y l i n o s i t o l a n d p h o s p h a t i d y l c h o l i n e f r o m 16 a t r i a l s a m p l e s p r e s e n t p e r 470 Mg o f t o t a l l i p i d p h o s p h o r u s was d e t e r m i n e d . A s one g wet w e i g h t o f r a t h e a r t h a s b e e n r e p o r t e d t o c o n t a i n 470 Mg o f l i p i d p h o s p h o r u s ( W h i t e , 1 9 7 3 ) , r e s u l t s a r e r e p o r t e d p e r g t i s s u e w e i g h t . Spot Phospholipid % phosphorus % f 3 2 p i ] incorporation a Phosphatidyl inositol 6.3 ± 1.2 50.0 ± 3.0 b Phosphati dylseri ne 4.2 ± 0.6 2 .9 ± 0.3 c Phosphatidylchol ine 46.1 ± 1.1 35 .7 ± 0.8 d Phosphati dylethanol ami ne 40.0 ± 2.2 10 .5 ± 1.6 Phospholipid ug Phosphorus per 470 pg total l i p i d phosphorus Reported White 1973 Phosphatidyl inositol 16.3 ± 0.9 17.4 Phosphatidylchol ine 126.6 ± 4.0 169.2 83 F i g u r e 5 - Standard curve f o r phosphorus. The standard curve f o r phosphorus was obtained with sodium dihydrogen phosphate. P o i n t s are the mean of d u p l i c a t e o b s e r v a t i o n s from one of s e v e r a l such experiments. 84 .6n 0 .2 .4 .6 Microgram phosphorus 85 F i g u r e 6 - Comparison of the b a s a l phosphate i n c o r p o r a t i o n i n t o v a r i o u s p h o s p h o l i p i d s i n a t r i a and v e n t r i c l e s of r a t h e a r t . I n c o r p o r a t i o n of [ 3 2 P ] p h o s p h a t e was measured over a p e r i o d of one h o u r . R e s u l t s are c a l c u l a t e d as the amount of r a d i o a c t i v i t y i n c o r p o r a t e d per microgram of p h o s p h o l i p i d phosphorus . Hi s tograms r e p r e s e n t p h o s p h a t i d y l s e r i n e ( A ) , p h o s p h a t i d y l i n o s i t o l ( C ) , p h o s p h a t i d y l c h o l i n e (E) and p h o s p h a t i d y l e t h a n o l a m i n e (G) i n ra t a t r i a . His tograms B, D, F and H r e p r e s e n t s the same s e r i e s of p h o s p h o l i p i d s from ra t v e n t r i c l e s . Bars r e p r e s e n t the mean ( ± S . E . M ) of three o b s e r v a t i o n s . S i m i l a r r e s u l t s were o b t a i n e d i n one other exper iment . CPM/MICROGRRM PHOSPHORUS cn oi 87 F i g u r e 7 - E f f e c t of c a r b a c h o l and methoxamine on [ 3 2 P ] i n c o r p o r a t i o n i n t o PI i n combined r a t a t r i a . Combined r a t a t r i a were i n c u b a t e d w i t h b u f f e r ( A ) , w i t h 0.1 mM c a r b a c h o l (B ) , w i t h 0.1 mM methoxamine (C) or w i t h 0.1 mM methoxamine p l u s 20 uM phento lamine (D) for 60 min i n the presence of [ 3 2 P ] s o d i u m o r t h o p h o s p h a t e . The mean [ 3 2 P ] i n c o r p o r a t i o n i n t o PI i n the c o n t r o l sample was 863 cpm/Vg phosphorus . Bars r epre sent the mean ± S . E . M . The numbers i n parentheses r e p r e s e n t the sample s i z e . R e s u l t s were o b t a i n e d by p o o l i n g toge ther data from t h r e e separate e x p e r i m e n t s . * r e p r e s e n t 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 i n c r e a s e (p<0.05) over c o n t r o l . SPECIFIC RADIOACTIVITY (Z CONTROL) cn ca _L_ ca ca i cn at _L_ C l at n CD CD 89 F i g u r e 8 - E f f e c t of methoxamine s t i m u l a t i o n on [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o v a r i o u s p h o s p h o l i p i d s i n combined ra t a t r i a . Combined r a t a t r i a were i n c u b a t e d wi th or wi thout 0.1 mM methoxamine or w i t h 0.1 mM methoxamine and 20 MM phentolamine fo r 75 min i n the presence of 54 y C i [ 3 2 P ] s o d i u m o r t h o p h o s p h a t e . His tograms repre sent p h o s p h a t i d y l s e r i n e ( A ) , p h o s p h a t i d y l i n o s i t o l ( C ) , p h o s p h a t i d y l c h o l i n e (E) and p h o s p h a t i d y l e t h a n o l a m i n e (G) , r e s p e c t i v e l y , i n c o n t r o l samples , and h i s tograms B, D, F and H r e p r e s e n t the same s e r i e s of p h o s p h o l i p i d s from methoxamine-trea ted samples . Hi s togram DP r e p r e s e n t s the p h o s p h a t i d y l i n o s i t o l o b t a i n e d from methoxamine p l u s p h e n t o l a m i n e - t r e a t e d sample. Bars r e p r e s e n t the mean ± S . E . M . of four samples . 90 AB CD DP EF 6 H 91 F i g u r e 9 - E f f e c t of methoxamine s t i m u l a t i o n on [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l i n o s i t o l of separa te r a t l e f t and r i g h t a t r i a . L e f t and r i g h t a t r i a were incuba ted s e p a r a t e l y w i t h [ 3 2 P ] p h o s p h a t e i n the presence of bu f f e r or 0.1 mM methoxamine for 60 m i n . His tograms A , B and C , D r e p r e s e n t c o n t r o l and methoxamine- t rea ted t i s s u e of l e f t (A,B) and r i g h t (C,D) a t r i a , r e s p e c t i v e l y . R e s u l t s are expres sed as percent of l e f t a t r i u m c o n t r o l . Mean i n c o r p o r a t i o n i n t o l e f t a t r i u m was 3550 cpm/uq phosphorus . Bars r e p r e s e n t the mean ± S . E . M . of four samples , from a s i n g l e exper iment . * r e p r e s e n t 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 i n c r e a s e (p<0.05) over c o n t r o l . SPECFIC RADIOACTIVITY CX CONTROL) tn ca JL_ ca tn ca _1_ ro ca ca _L_ K> tn ca CD — I 93 F i g u r e 10 - E f f e c t of c a r b a c h o l on [ 3 2 P diphosphate i n c o r p o r a t i o n i n t o p h o s p h a t i d y l i n o s i t o l of separa te r a t l e f t and r i g h t a t r i a . L e f t and r i g h t a t r i a were i n c u b a t e d s e p a r a t e l y w i t h [ 3 2 P ] s o d i u m orthophosphate i n the presence of b u f f e r or 0.1 mM c a r b a c h o l fo r 60 min . His tograms A , B and C, D r e p r e s e n t c o n t r o l and c a r b a c h o l - t r e a t e d l e f t (A,B) and r i g h t (C,D) a t r i a , r e s p e c t i v e l y . Mean i n c o r p o r a t i o n i n t o r a t l e f t a t r i u m was 1612 cpm/Vg phosphorus . Bars r e p r e s e n t the mean ± S . E . M . Numbers i n parentheses i n d i c a t e the sample s i z e . R e s u l t s are p o o l e d from two separate e x p e r i m e n t s . * r e p r e s e n t 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 (p<0.05) i n c r e a s e over the c o r r e s p o n d i n g c o n t r o l . 94 250 -. o 200-o u M 150-cc cc ft: U C 50 in (8) (9) (8) (7) 4 B 95 F i g u r e 11 - E f f e c t of c a r b a c h o l on p h o s p h o l i p i d s p r e l a b e l e d w i t h [ 3 2 P ] p h o s p h a t e i n combined ra t a t r i a . Combined a t r i a , p r e i n c u b a t e d w i t h 40 juCi [ 3 2 P ] s o d i u m or thophosphate for 2 h , were i n c u b a t e d for a f u r t h e r 45 min p e r i o d w i t h or wi thout 0.1 mM c a r b a c h o l i n the absence of r a d i o l a b e l e d - p h o s p h a t e . His tograms A , C , E and G r e p r e s e n t p h o s p h a t i d y l s e r i n e , p h o s p h a t i d y l i n o s i t o l , p h o s p h a t i d y l c h o l i n e and p h o s p h a t i d y l e t h a n o l a m i n e i n the c o n t r o l sample, and h i s tograms B, D, F and H r e p r e s e n t the same s e r i e s of p h o s p h o l i p i d s from c a r b a c h o l - t r e a t e d samples . Bars r e p r e s e n t the mean ± S . E . M . of three samples , from a s i n g l e exper iment . CPM/MICROGRPM PHOSPHORUS Ca c a ca _L_ 10 Q c a c a J L . c a c a c a J L . c a c a c a J i CD n J cn r n H cn II 97 F i g u r e 12 - E f f e c t of c a r b a c h o l on p h o s p h a t i d y l i n o s i t o l p r e l a b e l e d w i t h a r a c h i d o n i c a c i d i n r a t a t r i a . Combined a t r i a l p r e p a r a t i o n s , a f t e r p r e l a b e l i n g w i t h [ 1 " C ] a r a c h i d o n i c a c i d , were washed wi th Krebs b u f f e r c o n t a i n i n g 1% bovine serum albumin and i n c u b a t e d f u r t h e r for a 15 min p e r i o d w i t h b u f f e r (A) or w i t h 0.1 mM c a r b a c h o l (B ) . The mean i n c o r p o r a t i o n i n the c o n t r o l sample was 175 cpm/mg of wet t i s s u e w e i g h t . Bars r epre sent the mean ± S . E . M . Numbers i n parentheses i n d i c a t e the sample s i z e . R e s u l t s are p o o l e d from two separa te e x p e r i m e n t s . 98 99 F i g u r e 13 - S e p a r a t i o n of d i a c y l g l y c e r o l . T h i n l a y e r chromatographic s e p a r a t i o n of d i a c y l g l y c e r o l from f a t t y a c i d s and p h o s p h o l i p i d s i n b e n z e n e / c h l o r o f o r m / m e t h a n o l ( 8 0 : 1 5 : 5 ) . a , b, c and d r e p r e s e n t s p h o s p h a t i d y l i n o s i t o l , p h o s p h a t i d y l c h o l i n e , m y r i s t i c a c i d and 1 , 2 - d i o l e a t e , r e spec t i v e l y . 100 o © a b 101 Tab le II - E f f e c t of c a r b a c h o l s t i m u l a t i o n on a r a c h i d o n y l content of d i a c y l g l y c e r o l . I n c u b a t i o n of t i s s u e s and d e t e r m i n a t i o n of f a t t y a c i d c o m p o s i t i o n i s d e s c r i b e d i n the Methods . F a t t y a c i d c o n t e n t i s expressed in uq/q t i s s u e . The d i a c y l g l y c e r o l l e v e l s (ymoles /g t i s s u e ) were c a l c u l a t e d from the f a t t y a c i d conten t of the d i a c y l g y c e r o l . R e s u l t s are expres sed as the mean ± S . E . M of the sample s i z e g i v e n i n p a r e n t h e s e s . A s i m i l a r absence of a r a c h i d o n i c a c i d was observed i n one o ther exper iment . Control Carbamylcholine ( 0 . 1 mM) Diacylglycerol content 0 . 1 6 8 ± 0 . 0 2 9 ( 4 ) 0 . 1 4 2 ± 0 . 0 2 5 ( 5 ) n - s * (umoles/g tissue) Fatty acid composition of diacylglycerol (ug/g tissue) C l 6 : 0 4 1 . 4 ± 5 . 2 2 8 . 4 ± 3 . 2 C l 8 : 0 4 1 . 8 ± 7 . 6 2 2 . 0 ± 3 . 9 C l 8 : l 9 . 1 6 ± 0 . 6 8 . 6 ± 0 . 7 1 C l 8 : 2 8 . 3 ± 1 . 8 1 0 . 2 ± 2 . 5 ^ 2 0 : 4 ni l nil n.s. Not significantly different from control (p > 0 . 0 5 ) . 1 02 F i g u r e 14 - Gas chromatogram of f a t t y a c i d methyl e s t e r s of d i a c y l g l y c e r o l . A gas chromatogram of f a t t y a c i d methyl e s t e r s of d i a c y l g l y c e r o l from a c a r b a c h o l - t r e a t e d ra t a t r i a i s shown. The peaks a , b , c and d c o r r e s p o n d to methyl p a l m i t a t e , s t e a r a t e , o l e a t e and l i n o l e a t e . Peak e i s the i n t e r n a l s t andard and p o i n t f r e p r e s e n t s the p o s i t i o n where methyl a r a c h i d o n a t e i f p r e s e n t , would have e l u t e d . 1 0 3 1 04 F i g u r e 15 - F a t t y a c i d c o m p o s i t i o n of the p o l a r l i p i d f r a c t i o n of r a t a t r i a and v e n t r i c l e s . The p o l a r l i p i d f r a c t i o n remain ing at the o r i g i n a f t e r t h i n l a y e r chromatography i n b e n z e n e : c h l o r o f o r m r m e t h a n o l (80 :15 :5 v / v ) was e x t r a c t e d from the s i l i c a g e l p l a t e and i t s f a t t y a c i d c o m p o s i t i o n de termined by gas chromatography. His tograms A 1 , A2, A3 , A4 and A5 r e p r e s e n t the f a t t y a c i d s p a l m i t i c , s t e a r i c , o l e i c , l i n o l e i c and a r a c h i d o n i c a c i d s , r e s p e c t i v e l y , i n r a t a t r i a . Hi s tograms V 1 , V2 , V 3 , V4 and V5 r e p r e s e n t the same s e r i e s of f a t t y a c i d s from r a t v e n t r i c l e samples . Bars r e p r e s e n t the mean ± S . E . M of s i x samples . * , s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from the c o r r e s p o n d i n g f a t t y a c i d content i n a t r i a . 105 50 -i 40-30-20- i 10-fll VI fl2 V2 A3 V3 fl4 V4 fl5 V5 1 06 F i g u r e 16 - E f f e c t of c a r b a c h o l on [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o p h o s p h a t i d y l i n o s i t o l of l o n g i t u d i n a l smooth muscle of gu inea p i g i l e u m . Guinea p i g i l eum p i e c e s were i n c u b a t e d wi thout c a r b a c h o l ( A ) , w i t h 0.1 mM c a r b a c h o l (B) or w i t h 0.1 mM c a r b a c h o l and 1.6 uM a t r o p i n e (C) for 60 min i n the presence of [ 3 2 P ] s o d i u m o r t h o p h o s p h a t e . The mean [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n PI i n the c o n t r o l sample was 1638 cpm/yg phosphorus . R e s u l t s are the mean ± S . E . M of the sample s i z e i n d i c a t e d i n p a r e n t h e s e s . S i m i l a r r e s u l t s were o b t a i n e d i n one other exper iment . 107 108 Table III - E f f e c t of car b a c h o l on [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d . Guinea p i g ileum p i e c e s were incubated with [ 3 H ] i n o s i t o l i n the presence and absence of 0.1 mM c a r b a c h o l . In p r e p a r a t i o n s c o n t a i n i n g a t r o p i n e , 16 MM a t r o p i n e was added 5 min before the a d d i t i o n of c a r b a c h o l . Incubation was c a r r i e d out as d e s c r i b e d in the Methods, f o l l o w i n g a 30 min p r e i n c u b a t i o n with [ 3 H ] i n o s i t o l i n the absence of drugs. The % i n c r e a s e i n r a d i o a c t i v i t y i n the p h o s p h o l i p i d f r a c t i o n was c a l c u l a t e d a f t e r s u b t r a c t i o n of the counts i n c o r p o r a t e d during the i n i t i a l 30 min p r e i n c u b a t i o n of the t i s s u e with [ 3 H ] i n o s i t o l ("basal") from the t o t a l 60 min i n c u b a t i o n p e r i o d . The r a d i o a c t i v i t y r e p r e s e n t s mean i n c o r p o r a t i o n of [ 3 H ] i n o s i t o l ± S.E.M. The number of guinea p i g ileum p i e c e s i s shown i n parentheses. S i m i l a r r e s u l t s were obtained i n four other experiments. Treatment dpm/pg total % Increase phosphorus Basal 182 ± 27 (4) no addition 368 ± 32 (5) 100 Carbachol (0.1 mM) 553 ± 29 (6) 199 Carbachol (0.1 mM) 386 ± 34 (4) 110 + atropine (16 yM) 109 Tab le IV - C a r b a c h o l - s t i m u l a t e d a c c u m u l a t i o n of i n o s i t o l phosphate . Guinea p i g i l eum p i e c e s were i n c u b a t e d for 60 min i n a medium c o n t a i n i n g [ 3 H ] i n o s i t o l , w i t h or wi thout c a r b a c h o l , and i n the presence or absence of 10 mM l i t h i u m (see Methods ) . T o t a l accumulated r a d i o a c t i v i t y was e l u t e d from the r e s i n w i t h 0.1 M formic a c i d p l u s 1 M ammonium formate . Phosphorus content was determined i n a l i q u o t s of the o r g a n i c phase . R e s u l t s are g i v e n as the mean ± S . E . M . The number of guinea p i g i l eum p i e c e s i s shown in the p a r e n t h e s e s . S i m i l a r r e s u l t s were o b t a i n e d i n s e v e r a l o ther e x p e r i m e n t s . Treatment dpm/ug total phosphorus No addition 3.21 ± 1.9 (4) Lithium (10 mM) 31.0 ± 2.6 (4) Carbachol (0.1 mM) 23.5 ± 2.1 (4) Carbachol (0.1 mM) + Lithium (lOmM) 492.0 ± 34.0 (6) 110 F i g u r e 17 - Dose-response curve fo r c a r b a c h o l - s t i m u l a t e d i n o s i t o l phosphate a c c u m u l a t i o n i n guinea p i g i l e u m . The dose-response curve for c a r b a c h o l - s t i m u l a t e d i n o s i t o l phosphate a c c u m u l a t i o n was determined i n the presence of 10 mM l i t h i u m , w i t h (•) or wi thout (•) 6.4 M M a t r o p i n e . Each p o i n t i s the mean of d u p l i c a t e i n c u b a t i o n s from a s i n g l e exper iment . 111 •j—i 11 mm—i i iimi|—i i M I I M | i i M I " 4 — « ' ' »"1 1 2 3 4 5 6 Log CorbomylcholIne (nM) 1 12 F i g u r e 18 - E l u t i o n p r o f i l e of s t andard i n o s i t o l mono-phosphate . 200 Ail of 1 mg/ml i n o s i t o l - 2 - p h o s p h a t e d l -monocyc lohexy lamine s a l t was loaded onto a Dowex-1 X8-formate ion exchange r e s i n column. The column was washed w i t h water , f o l l o w e d by e l u t i o n w i t h 5 mM borax p l u s 60 mM ammonium formate at A and 5 mM borax p l u s 150 mM ammonium formate at B. I n o s i t o l monophosphate was i d e n t i f i e d by phosphorus d e t e r m i n a t i o n i n the e l u a n t f r a c t i o n s . 14.2 uq phosphorus was e l u t e d from the column between f r a c t i o n s 11 and 20, thereby sugges t ing a complete r e c o v e r y of i n o s i t o l mono-phosphate. Microgram Phosphorus 10 to _I I I 1 1 4 F i g u r e 19 - E l u t i o n p r o f i l e of the accumulated i n o s i t o l phosphates i n gu inea p i g i l e u m . I n o s i t o l phosphates accumulated d u r i n g i n c u b a t i o n of guinea p i g i leum p i e c e s w i t h c a r b a c h o l i n the presence of l i t h i u m were loaded onto a Dowex-1 X8-formate ion-exchange r e s i n column.The column was washed w i t h 5 mM i n o s i t o l and e l u t e d s u c c e s s i v e l y w i t h 5 mM borax p l u s 60 mM ammonium formate , 5 mM borax p l u s 150 mM ammonium formate and 0.1 M formic a c i d p l u s 1 M ammonium formate at A , B and C , r e s p e c t i v e l y . R a d i o c t i v i t y was de termined i n .1 ml f r a c t i o n s of the e l u a n t . The peak r a d i o a c t i v i t y e l u t e d w i t h s o l u t i o n B and s o l u t i o n C were compared to determine the r e l a t i v e amount of two i n o s i t o l phosphate s . In a s i m i l a r exper iment c o n t r o l u n s t i m u l a t e d t i s s u e showed no r a d i o a c t i v i t y peaks on e l u t i o n w i t h i n c r e a s i n g i o n i c s t r e n g t h s o l u t i o n s . 1 15 Frocrtlon Number 1 16 T a b l e V - Compos i t ion of i n o s i t o l phosphates accumulated i n gu inea p i g i l e u m . Guinea p i g i l eum fragments were i n c u b a t e d i n a medium c o n t a i n i n g [ 3 H ] i n o s i t o l w i t h l i t h i u m (10 mM) and c a r b a c h o l (0.1 mM) fo r 60 m i n . I n o s i t o l phosphates were e l u t e d from the r e s i n as d e s c r i b e d i n the Methods . The r e s u l t s are expres sed as the mean ± S . E . M . from four t i s s u e samples . S i m i l a r r e s u l t s were o b t a i n e d i n one o ther exper iment . Elution medium Percent radioactivity eluted (%Y 5 mM Borax + 60 mM ammonium 6.4 ± 0.4 formate 5 mM Borax + 150 mM ammonium 16.9 ± 1.7 formate 0.1 M Formic acid + 0.4 M ammonium 50.8 ± 0.9 formate 0.1 M Formic acid + 1.0 M ammonium 24.3 ± 0.7 formate 1 1 7 F i g u r e 20 - Time course of the e f f e c t of PMSF on b a s a l [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d i n gu inea p i g i l e u m . Ileum p i e c e s were p r e i n c u b a t e d w i t h 30 uCi of [ 3 H ] i n o s i t o l for 30 m i n . F u r t h e r i n c u b a t i o n was c a r r i e d out i n the presence ( • ) and i n the absence (•) of 2 mM PMSF, as shown i n the f i g u r e . R a d i o a c t i v i t y was determined i n the l i p i d f r a c t i o n and the r e s u l t s are r e p o r t e d as dpm/yg l i p i d phosphorus ± S . E . M of three samples . 118 1 1 9 F i g u r e 21 - E f f e c t of PMSF on c a r b a c h o l - and K + - s t i m u l a t e d [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o p h o s p h o l i p i d s of guinea p i g i l e u m . a) A f t e r p r e i n c u b a t i o n of i l eum p i e c e s w i t h [ 3 H ] i n o s i t o l the i l eum p i e c e s were f u r t h e r i n c u b a t e d for 30 min w i t h no a d d i t i o n ( A ) , 2 mM PMSF (B) , 0.1 mM c a r b a c h o l (C) and 0.1 mM c a r b a c h o l p l u s 2 mM PMSF (D) . b) A f t e r p r e i n c u b a t i o n of i l eum p i e c e s w i t h [ 3 H ] i n o s i t o l the i l eum p i e c e s were f u r t h e r i n c u b a t e d for 30 min w i t h no a d d i t i o n ( A ) , 60 mM potas s ium (B) and 60 mM potas s ium p l u s 2 mM PMSF ( C ) . Bars r e p r e s e n t the mean ± S . E . M . The numbers i n parenthese s r e p r e s e n t the sample s i z e . The mean [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n the c o n t r o l experiment d u r i n g the l a s t 30 min of i n c u b a t i o n was 180 dprn/Vg l i p i d phosphorus ( F i g 21a) and 166 dpm/yg l i p i d phosphorus ( F i g 21b) . S i m i l a r r e s u l t s were o b t a i n e d i n two o ther exper iment s . (10) 2M-i I 150-100-in o (5) (9) 200-i (7) 150-100-50-fl 0 C D 0-(11) (11) (10) I A B C O 121 F i g u r e 22 - E f f e c t of PMSF on c a r b a c h o l - and K + - s t i m u l a t e d c o n t r a c t i o n of guinea p i g i l eum Guinea p i g i l eum was c o n t r a c t e d w i t h e i t h e r 1MM c a r b a c h o l or 60 mM p o t a s s i u m . 2 mM PMSF was added as i n d i c a t e d by the arrows ( a ) . PMSF i n h i b i t i o n of the p h a s i c c o n t r a c t i o n and the t r a n s i e n t na ture of t h i s e f f e c t i s shown i n ( b ) . The f i r s t t r a c i n g i n each set shows the K + - s t i m u l a t e d c o n t r a c t i o n p r i o r to PMSF a d d i t i o n . F o l l o w i n g t h i s , 0.4 mM PMSF was added as i n d i c a t e d at the arrow and t i s s u e r e s t i m u l a t e d 1 , 2 or 10 min l a t e r w i t h K * . Dose dependent i n h i b i t i o n by PMSF of K + -s t i m u l a t e d p h a s i c response i s shown i n ( c ) . PMSF (0.4 mM) was added 1 min be fore s t i m u l a t i o n w i t h 60 mM K + . R e s u l t s are expres sed as f r a c t i o n a l i n h i b i t i o n of the p h a s i c response compared to the PMSF-untreated t i s s u e . 122 123 Tab le VI - E f f e c t of PMSF on the accumula t ion of c a r b a c h o l -s t i m u l a t e d i n o s i t o l p h o s p h a t e s • i n guinea p i g i l e u m . I n c u b a t i o n w i t h [ 3 H ] i n o s i t o l and d e t e r m i n a t i o n of t o t a l i n o s i t o l phosphates ( T I P ) , i n o s i t o l mono-phosphate (IP) and i n o s i t o l b i -and t r i - p h o s p h a t e s ( I P 2 / I P 3 ) were determined i n the presence of 1OmM l i t h i u m as d e s c r i b e d i n the Methods . PMSF was used i n a c o n c e n t r a t i o n of 2 mM i n d i m e t h y l s u l f o x i d e . The c o n t r o l wi thout PMSF c o n t a i n e d an e q u i v a l e n t amount of d i m e t h y l s u l f o x i d e (1%). R e s u l t s are r e p o r t e d from two separa te e x p e r i m e n t s . TIP was measured i n one experiment and I P 2 and I P 3 were measured i n another exper iment . R e s u l t s are r e p o r t e d as the mean ± S . E . M . The number of samples i s i n d i c a t e d i n the p a r e n t h e s e s . Inositol Carbachol % phosphate Carbachol + PMSF Decrease dpm/ug l i p i d phosphorus TIP 206.1 ± 8.0 (3) 167.8 ± 11.6 (5) 18.5** IP 22.3 ± 1.7 (7) 18.9 ± 0.8 (11) 14.8** I P 2 / I P 3 111.5 ± 6.5 (7) 93.2 ± 3 .6 (11) 16.4* * p < 0.05 * * 0.05 < P < 0.06 - 124 IV . DISCUSSION 4.1 IS MUSCARINIC RECEPTOR STIMULATION IN THE HEART ACCOMPANIED  BY A PI RESPONSE? 4.1 .1 P h o s p h o l i p i d S e p a r a t i o n And Technique V e r i f i c a t i o n As ment ioned e a r l i e r , even though r e c e p t o r - m e d i a t e d i n c r e a s e i n phosphate i n c o r p o r a t i o n i n t o PI i s a secondary response of the PI t u r n o v e r c y c l e , i t has served as a very v a l u a b l e i n d i c a t o r for the o c c u r r e n c e of PI response i n a wide range of t i s s u e s . In a l l t i s s u e s (except one) the pr imary s tep of i n o s i t o l l i p i d breakdown i s accompanied by enhanced r e i n c o r p o r a t i o n of phosphate i n t o P I . T h e r e f o r e , phosphate i n c o r p o r a t i o n i n t o PI was chosen as the f i r s t method to s tudy the PI response i n r a t h e a r t . Two-d imens iona l t h i n l a y e r chromatography (Renkonen and Luukkonen, 1976), which has been used s u c c e s s f u l l y i n the pas t by many o t h e r g roups , was used fo r the s e p a r a t i o n of p h o s p h o l i p i d s . One such s e p a r a t i o n u s ing two d i m e n s i o n a l t h i n l a y e r chromatography i s shown i n F i g 4. The r e s u l t s of the phosphate i n c o r p o r a t i o n s t u d i e s were c a l c u l a t e d as the amount of r a d i o a c t i v i t y i n c o r p o r a t e d per microgram of the i n d i v i d u a l p h o s p h o l i p i d phosphorus content and r e p o r t e d as percent of c o n t r o l . F i r s t , the e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n on phosphate i n c o r p o r a t i o n i n t o PI i n guinea p i g i l eum was s t u d i e d , t o v e r i f y our a b i l i t y to observe a PI re sponse . In 1 25 accordance w i t h a p r e v i o u s r e p o r t ( J a f f e r j i and M i c h e l l , 1976a), c a r b a c h o l - s t i m u l a t e d phosphate i n c o r p o r a t i o n i n t o PI was ob se rved , which was b l o c k e d by the m u s c a r i n i c a n t a g o n i s t , a t r o p i n e ( F i g 16) . The b a s a l i n c o r p o r a t i o n of 1638 cpm/^g of PI phosphorus cor re sponds to 51 cpm/nmole of PI and i s comparable to the v a l u e r e p o r t e d p r e v i o u s l y ( J a f f e r j i and M i c h e l l , 1976a ,b ) . 4 . 1 . 2 B a s a l [ 3 2 P ] p h o s p h a t e I n c o r p o r a t i o n In Heart P h o s p h o l i p i d s were a l s o s epara ted from ra t a t r i a (see F i g 4 ) . By compari son w i t h s t a n d a r d s , spots were i d e n t i f i e d as p h o s p h a t i d y l i n o s i t o l , p h o s p h a t i d y l s e r i n e , p h o s p h a t i d y l c h o l i n e , 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 i c a c i d . A l t h o u g h PI compr i sed o n l y 6% of the four p h o s p h o l i p i d s measured i n t h i s s t u d y , i t i n c o r p o r a t e d a lmost 50% of the t o t a l r a d i o l a b e l e d -phosphate i n c o r p o r a t e d i n t o the four p h o s p h o l i p i d s (Table I ) . S i m i l a r l y , Brown and Brown (1982) have r e p o r t e d a 60% i n c o r p o r a t i o n of r a d i o l a b e l e d - p h o s p h a t e i n t o PI i n mouse a t r i a . When the phosphate i n c o r p o r a t i o n i n t o v a r i o u s p h o s p h o l i p i d s i n a t r i a and v e n t r i c l e was compared ( F i g 6 ) , i t was observed t h a t the d i s t r i b u t i o n p r o f i l e of r a d i o a c t i v i t y i n t o v a r i o u s p h o s p h o l i p i d s i n the two t i s s u e s was s i m i l a r . In both t i s s u e s , PI i n c o r p o r a t e d a l a r g e f r a c t i o n of the t o t a l r a d i o a c t i v i t y i n c o r p o r a t e d i n t o the p h o s p h o l i p i d s . However, the r a d i o a c t i v i t y i n c o r p o r a t e d i n t o p h o s p h o l i p i d s i n a t r i a was at l e a s t 4-6 f o l d h i g h e r than that of the c o r r e s p o n d i n g p h o s p h o l i p i d s i n v e n t r i c l e . The cause fo r t h i s d i f f e r e n c e i n b a s a l phosphate 1 26 i n c o r p o r a t i o n i n t o a t r i a and v e n t r i c l e i s not known at p r e s e n t , but a s i m i l a r d i f f e r e n c e has been observed i n can ine a t r i a and v e n t r i c l e (Qui s t , 1982). The g r e a t e r extent of l a b e l i n c o r p o r a t i o n , as w e l l as the h i g h e r m u s c a r i n i c r e c e p t o r d e n s i t y i n r a t a t r i a compared to v e n t r i c l e (Wei and Su l akhe , 1978), l e d to the c h o i c e of a t r i a for f u r t h e r s t u d y . 4 . 1 . 3 E f f e c t Of M u s c a r i n i c And A l p h a - A d r e n e r g i c Receptor  S t i m u l a t i o n On PI Turnover In Rat A t r i a In combined ra t a t r i a l p i e c e s , m u s c a r i n i c and a l p h a -a d r e n e r g i c r e c e p t o r s t i m u l a t i o n produced a 35% and 88% i n c r e a s e i n phosphate i n c o r p o r a t i o n i n t o P I , r e s p e c t i v e l y ( F i g 7 ) . The a l p h a - a d r e n e r g i c - m e d i a t e d enhancement i n phosphate i n c o r p o r a t i o n was s p e c i f i c to PI ( F i g 8 ) , and c o u l d be b l o c k e d by the a l p h a -a d r e n e r g i c a n t a g o n i s t , phento l amine , i n d i c a t i n g t h a t PI response was the r e s u l t of a l p h a - a d r e n e r g i c r e c e p t o r s t i m u l a t i o n . T h i s was c o n s i s t e n t w i t h the p r e v i o u s o b s e r v a t i o n tha t e p i n e p h r i n e caused an i n c r e a s e d i n c o r p o r a t i o n of [ 3 2 P ] p h o s p h a t e i n t o i n o s i t o l p h o s p h a t i d e s , both i n v i v o and i n v i t r o (Gaut and Hugg ins , 1966; K i s s and F a r k a s , 1975). The l a c k of t h i s response i n guinea p i g or ca t v e n t r i c l e u s ing n o r e p i n e p h r i n e was a t t r i b u t e d to the r i c h n e s s of /3 r a t h e r than a - a d r e n e r g i c r e c e p t o r s i n v e n t r i c l e ( L a p e t i n a et a l . , 1976). More r e c e n t l y , Uchida et a l . (1982) have observed a s i m i l a r a l p h a - a d r e n e r g i c r e c e p t o r - m e d i a t e d phosphate i n c o r p o r a t i o n i n t o PI i n c u l t u r e d ra t myocytes . They f u r t h e r c h a r a c t e r i z e d the response to be a , and c a l c i u m - i n d e p e n d e n t (Uchida et a l . , 1982). 127 As there was almost a t w o - f o l d enhancement i n phosphate i n c o r p o r a t i o n i n t o PI i n combined r a t a t r i a l p i e c e s , which have a r e c e p t o r d e n s i t y which i s l e s s than 50% of the m u s c a r i n i c r e c e p t o r d e n s i t y i n t h i s t i s s u e , i t was c l e a r l y e v i d e n t that the s m a l l e r m u s c a r i n i c e f f e c t was not due to an i n a b i l i t y to d e t e c t enhanced phosphate i n c o r p o r a t i o n d u r i n g a PI r e sponse . 4.2 PI BREAKDOWN STUDIES 4.2 .1 Breakdown Of P r e l a b e l e d PI As i n d i c a t e d e a r l i e r , even though compensatory r e s y n t h e s i s n e a r l y always accompanies i n o s i t o l l i p i d breakdown, the p r e c i s e r e l a t i o n s h i p between i n o s i t o l l i p i d breakdown and i t s subsequent r e s y n t h e s i s i s s t i l l not known. i t was t h e r e f o r e d e c i d e d to s tudy the e f f e c t of m u s c a r i n i c s t i m u l a t i o n of PI breakdown, w h i l e s i m u l t a n e o u s l y t e s t i n g for two o ther p r o b a b l e mechanisms ( d i s c u s s e d b e l o w ) . Increa sed breakdown of p r e l a b e l e d PI d u r i n g the PI response has been r e p o r t e d i n b l o w f l y s a l i v a r y g l and ( F a i n and B e r r i d g e , 1979), p a r o t i d g l and (Jones and M i c h e l l , 1975), and vas de ferens (Egawa et a l . , , 1981), among s e v e r a l o ther t i s s u e s . In p r e l i m i n a r y exper iment s , i t was observed t h a t s t i m u l a t i o n of combined r a t a t r i a w i t h 0.1 mM c a r b a c h o l f a i l e d to cause an enhancement i n the l o s s of l a b e l from e i t h e r [ 3 2 P ] p h o s p h a t e -p r e l a b e l e d PI or any of the o ther p h o s p h o l i p i d s ( F i g 11) . F o l l o w i n g t h i s p r e l i m i n a r y experiment w i t h p h o s p h a t e - p r e l a b e l e d p h o s p h o l i p i d s , - a r a c h i d o n y l - p r e l a b e l e d p h o s p h o l i p i d s were used , 1 28 for the f o l l o w i n g r e a s o n . S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s cause an i n c r e a s e i n cGMP i n h e a r t (George et a l . , , 1973; Nawrath, 1976; Diamond et a l . , 1977) by the a c t i v a t i o n of guany la te c y c l a s e . The mechanism for guany la te c y c l a s e a c t i v a t i o n i s s t i l l unknown. Among s e v e r a l proposed a c t i v a t o r s for guany la te c y c l a s e , two, a r a c h i d o n i c a c i d (G la s s et a l . , 1977; Gabers et a l . , 1978; Sp ie s et a l . , 1980) and p r o s t a g l a n d i n s (Goldberg et a l . , 1978; Hidaka and Asano , 1977) c o u l d be d e r i v e d from P I . As r e l e a s e of a r a c h i d o n i c a c i d has been shown to occur from PI d u r i n g p l a t e l e t s t i m u l a t i o n by thrombin ( B i l l a h and L a p e t i n a , 1982a) and l i n o l e i c a c i d d u r i n g i s o p r o t e r e n o l s t i m u l a t i o n of c an ine sarcolemma (Franson et a l . , 1979), i t was i n v e s t i g a t e d whether a r e l e a s e of a r a c h i d o n i c a c i d o c c u r r e d d u r i n g c a r b a c h o l s t i m u l a t i o n of combined r a t a t r i a , which c o u l d then suggest a p o s s i b l e mechanism for the a c t i v a t i o n of guany la te c y c l a s e . I f s t i m u l a t i o n i n the breakdown of a r a c h i d o n y l - p r e l a b e l e d PI by e i t h e r p h o s p h o l i p a s e A 2 or p h o s p h o l i p a s e C o c c u r r e d , i t would cause a decrease i n the l e v e l of P I , which would then be r e f l e c t e d by a decrease i n the t o t a l r a d i o a c t i v i t y p re sen t i n P I , even i f the s p e c i f i c a c t i v i t y of PI remains unchanged. S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n combined r a t a t r i a w i t h 0.1 mM c a r b a c h o l d i d not enhance the breakdown of a r a c h i d o n y l -p r e l a b e l e d PI ( F i g 12) . T h i s i n d i c a t e d tha t m u s c a r i n i c r e c e p t o r s t i m u l a t i o n d i d not . cause s i g n i f i c a n t a c t i v a t i o n of p h o s p h o l i p a s e C or p h o s p h o l i p a s e A 2 . The l a ck of a c t i v a t i o n of p h o s p h o l i p a s e A 2 d u r i n g m u s c a r i n i c s t i m u l a t i o n was not 1 29 s u r p r i s i n g , as i n most c a s e s , except that of i s o p r o t e r e n o l s t i m u l a t i o n of c a r d i a c sarcolemma (Franson et a l . , , 1979), where there i s a r e c e p t o r - m e d i a t e d a c t i v a t i o n of p h o s p h o l i p a s e A 2 , p h o s p h o l i p a s e A 2 a c t i v a t i o n i s the r e s u l t of an i n c r e a s e i n c y t o s o l i c c a l c i u m c o n c e n t r a t i o n and the a v a i l a b l e ev idence does not i n d i c a t e that c a l c i u m i s m o b i l i z e d d u r i n g m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n the h e a r t . 4 . 2 . 2 A r a c h i d o n y l - E n r i c h e d D i a c y l g l y c e r o l P i - s p e c i f i c p h o s p h o l i p a s e C-mediated breakdown of PI l eads to the fo rmat ion of d i a c y l g l y c e r o l ( B i l l a h and L a p e t i n a , 1982a; Ieyasu et a l . , 1982; K a i b u c h i et a l . , , 1 9 8 2 ) , which i s c o n s i d e r e d to be r a p i d l y p h o s p h o r y l a t e d by d i a c y l g l y c e r o l k inase to g i v e p h o s p h a t i d i c a c i d ( M i c h e l l , . 1975; Putney , 1981). As d i a c y l g l y c e r o l i s a l s o an i n t e r m e d i a t e for many other p h o s p h o l i p i d m e t a b o l i c pathways, i t i s o n l y found i n sma l l q u a n t i t i e s i n u n s t i m u l a t e d t i s s u e s . Receptor s t i m u l a t i o n produced a t r a n s i e n t a c c u m u l a t i o n of d i a c y l g l y c e r o l w i t h i n seconds i n both t h y r o i d f o l l i c l e s ( I g a r s h i and Kondo, 1980) and p l a t e l e t s ( B i l l a h and L a p e t i n a , 1982a). On the o ther hand, a s lower t r a n s i e n t i n c r e a s e , over a p e r i o d of 30 min , was observed d u r i n g c a r b a c h o l s t i m u l a t i o n of mouse pancreas (Bansback et a l . , 1974) and an i n c r e a s e d a c c u m u l a t i o n of d i a c y l g l y c e r o l over a s t i l l l onger p e r i o d has recent . ly been r e p o r t e d by Homa et a l . (1983) . A c c u m u l a t i o n of d i a c y l g l y c e r o l over a l onger p e r i o d may i n d i c a t e the e x i s t e n c e of c e l l u l a r compartments which are not e a s i l y a c c e s s i b l e to o ther enzymes i n the m e t a b o l i c pathways. 1 30 Widespread o c c u r r e n c e of c a l c i u m - a c t i v a t e d , p h o s p h o l i p i d -dependent p r o t e i n k inase C (PKC) , and i t s r e g u l a t i o n by d i a c y l g l y c e r o l , has r a i s e d the p o s s i b l i t y tha t p r o d u c t i o n of d i a c y l g l y c e r o l d u r i n g breakdown of PI or any o ther p h o s p h o l i p i d may cause the a c t i v a t i o n of PKC (Minakuchi et a l . , 1981; T a k a i et a l . , 1979b) . In mouse p a n c r e a s , m u s c a r i n i c s t i m u l a t i o n has been shown to produce both a PI response (Hok in-Neaver son , 1974) and i n c r e a s e d a c c u m u l a t i o n of d i a c y l g l y c e r o l (Bansback et a l . , 1974). In t h i s study i t was observed that i n combined r a t a t r i a c a r b a c h o l (0.1 mM) caused o n l y a sma l l (but n o n s i g n i f i c a n t , p>0.05) d e c r e a s e , r a t h e r than an i n c r e a s e , i n d i a c y l g l y c e r o l l e v e l (Table 2 ) . The j3 -pos i t ion of PI of mammalian t i s s u e i s r i c h i n a r a c h i d o n i c a c i d (White , 1973). I f PI i s b e i n g degraded by p h o s p h o l i p a s e C , one would expect an i n c r e a s e i n the a r a c h i d o n y l conten t of d i a c y l g l y c e r o l , s i m i l a r to that observed by Bansback et a l . (1974) . Even though as low as 0.5 nmole of a r a c h i d o n y l methyl e s t e r c o u l d be d e t e c t e d by gas chromatography, no a r a c h i d o n i c a c i d was d e t e c t e d i n e i t h e r u n s t i m u l a t e d - or c a r b a c h o l - s t i m u l a t e d t i s s u e . The l a ck of i n c r e a s e i n a r a c h i d o n i c a c i d i n the d i a c y l g l y c e r o l f r a c t i o n of c a r b a c h o l -s t i m u l a t e d combined r a t a t r i a f u r t h e r suppor ted the i n i t i a l o b s e r v a t i o n tha t there may be o n l y a very s m a l l PI response i n combined r a t a t r i a . S t i m u l a t i o n of pancreas by 0.1 mM c a r b a c h o l fo r 15 min produced a 1 5 - f o l d i n c r e a s e i n the a r a c h i d o n y l conten t of d i a c y l g l y c e r o l , g i v i n g r i s e to a v a l u e as h i g h as 290 nmole/g t i s s u e (Bansback et a l . , 1974). I f a s i m i l a r 131 enhancement had o c c u r r e d i n ra t h e a r t , i t would c o r r e s p o n d to about 6 nmole of a r a c h i d o n i c a c i d i n 20 mg t i s s u e ( approx imate ly the amount p re sen t i n each i n c u b a t i o n ) and i t s h o u l d have been d e t e c t e d by the methods used ( F i g 14) . The d e t e c t i o n of a r a c h i d o n i c a c i d i n the p o l a r l i p i d f r a c t i o n of a t r i a and v e n t r i c l e ( F i g 15) a l s o r u l e s out the p o s s i b i l i t y t h a t a r a c h i d o n i c a c i d was c o m p l e t e l y d e s t r o y e d d u r i n g v a r i o u s s teps i n v o l v e d i n the p r e p a r a t i o n of f a t t y a c i d methyl e s t e r . I t was observed tha t w h i l e the s a t u r a t e d f a t t y a c i d c o n t e n t s of the p o l a r l i p i d f r a c t i o n from a t r i a and v e n t r i c l e s were s i m i l a r , a s i g n i f i c a n t d i f f e r e n c e i n the c o n t e n t s of a l l three u n s a t u r a t e d f a t t y a c i d s was observed ( F i g 15) . In a more comprehensive s tudy a s i m i l a r d i f f e r e n c e i n the content of u n s a t u r a t e d f a t t y a c i d s i n the p h o s p h o l i p i d f r a c t i o n of a t r i a and v e n t r i c l e s has been r e p o r t e d (Charnock et a l . , 1983). The s i g n i f i c a n c e of t h i s d i f f e r e n c e i n u n s a t u r a t e d f a t t y a c i d conten t between a t r i a and v e n t r i c l e s i s not known at p r e s e n t , but i t c o u l d p l a y a r o l e i n some of the d i f f e r e n c e s i n the membrane c h a r a c t e r i s t i c s observed between the two t i s s u e s , such as lower phosphate i n c o r p o r a t i o n i n t o p h o s p h o l i p i d s of v e n t r i c l e compared to a t r i a , as observed i n t h i s s tudy and i n tha t of Quis t (1982) . 1 32 4 . 2 . 3 [ 3 2 P ] P h o s p h a t e I n c o r p o r a t i o n In Rat L e f t And Right A t r i a A l l the ev idence o b t a i n e d so far suggested tha t s t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n combined r a t a t r i a was accompanied by e i t h e r no, or a very s m a l l , PI t u r n o v e r . Around that t ime Quis t (1982) r e p o r t e d tha t w h i l e c a n i n e r i g h t a t r i u m showed a PI response on m u s c a r i n i c r e c e p t o r s t i m u l a t i o n , c a n i n e l e f t a t r i u m or l e f t or r i g h t v e n t r i c l e , f a i l e d to show a s i m i l a r r e sponse . Another i n t e r e s t i n g c h a r a c t e r i s t i c of h i s o b s e r v a t i o n was tha t u n l i k e the a l p h a - a d r e n e r g i c - m e d i a t e d p o s i t i v e i n o t r o p i c e f f e c t i n c u l t u r e d r a t hear t c e l l s , which was shown to be c a l c i u m independent (Uchida et a l . , 1982), the m u s c a r i n i c - s t i m u l a t e d PI response i n c a n i n e r i g h t a t r i u m (as determined by the i n c r e a s e d i n c o r p o r a t i o n of l a b e l e d phosphate i n t o PI) was found to be ca l c ium-dependent (Qui s t , 1982). The o c c u r r e n c e of a PI response i n o n l y one p a r t of c an ine a t r i u m r a i s e d the p o s s i b i l i t y tha t a s i m i l a r s i t u a t i o n might e x i s t i n ra t a t r i a . S t i m u l a t i o n of r a t l e f t and r i g h t a t r i a by 0.1 mM c a r b a c h o l produced a s m a l l (35%) but s i g n i f i c a n t (p < .05) enhancement i n phosphate i n c o r p o r a t i o n i n t o PI i n ra t l e f t a t r i u m , but no e f f e c t i n r a t r i g h t a t r i u m ( F i g 10) . In c o n t r a s t to t h i s , s t i m u l a t i o n of a l p h a - a d r e n e r g i c r e c e p t o r s by 0.1 mM methoxamine produced an a p p r o x i m a t e l y two f o l d i n c r e a s e i n phosphate i n c o r p o r a t i o n i n t o PI i n both r a t l e f t and r i g h t a t r i a ( F i g 9 ) . I t was a l s o observed tha t the r e l a t i v e i n c o r p o r a t i o n of b a s a l r a d i o a c t i v i t y i n t o r a t l e f t and r i g h t a t r i u m was v a r i a b l e (compare F i g 9 and 10) . 133 4 . 2 . 4 E v i d e n c e For Two S u b p o p u l a t i o n s Of M u s c a r i n i c Receptors R e s u l t s from the c a r b a c h o l s t i m u l a t i o n of separa te l e f t and r i g h t a t r i u m , as w e l l as those o b t a i n e d by Quis t (1982), sugges ted the i n t e r e s t i n g p o s s i b i l i t y that w h i l e the m a j o r i t y of the m u s c a r i n i c r e c e p t o r s i n ra t l e f t and r i g h t a t r i u m were not c o u p l e d to PI t u r n o v e r , a s m a l l p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s i n ra t l e f t a t r i u m may be coup led to PI t u r n o v e r . In most t i s s u e s s t u d i e d so f a r , the PI response to a l p h a - a d r e n e r g i c s t i m u l a t i o n i s u s u a l l y weaker than the m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n the same t i s s u e (Jones and M i c h e l l , 1975). N e v e r t h e l e s s , assuming an equa l c o u p l i n g e f f i c i e n c y between r e c e p t o r and the PI t u r n o v e r c y c l e for both a l p h a - a d r e n e r g i c and m u s c a r i n i c r e c e p t o r s i n r a t a t r i u m and based on the . r e l a t i v e r e c e p t o r d e n s i t y ( m u s c a r i n i c = 220 pmoles /g p r o t e i n , Wei and Sua lkhe ,1978 ; a lpha a d r e n e r g i c = 100 pmoles /g p r o t e i n , K a r l i n e r et a l . , 1982)) and the r e l a t i v e PI responses o b t a i n e d ( F i g 9 & 10) , i t was c a l c u l a t e d tha t o n l y a s m a l l p o p u l a t i o n ( approx imate ly 15%) of the m u s c a r i n i c r e c e p t o r s i n r a t a t r i u m may be c o u p l e d to PI t u r n o v e r . The absence of i n o s i t o l l i p i d breakdown d u r i n g m u s c a r i n i c r e c e p t o r s t i m u l a t i o n of a t r i a ( F i g 11, 12 and T a b l e II ) may seem to c o n t r a d i c t the f i n d i n g tha t a s m a l l p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s may be c o u p l e d to PI t u r n o v e r . However, Farese et a l (1982) have r e p o r t e d tha t i n c a l c i u m - d e p l e t e d media c a r b a c h o l s t i m u l a t i o n of s u b m a x i l l a r y g l a n d caused a f o u r - f o l d enhancement i n phosphate i n c o r p o r a t i o n without d e c r e a s i n g the l e v e l of P I , whi le i n c a l c i u m - c o n t a i n i n g 1 34 media both an i n c r e a s e in phosphate i n c o r p o r a t i o n and a decrease i n the l e v e l of PI was observed d u r i n g c a r b a c h o l s t i m u l a t i o n . T h e r e f o r e , i t i s p o s s i b l e tha t PI breakdown and phosphate i n c o r p o r a t i o n may be d i s s o c i a b l e events or that breakdown r e s u l t i n g from such a s m a l l p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s c o u l d not be d e t e c t e d i n combined a t r i a l p r e p a r a t i o n s . I t i s now w e l l r e c o g n i z e d that most r e c e p t o r s can be c o n s i d e r e d to be composed of two d i s t i n c t f u n c t i o n a l s u b u n i t s , a r e c o g n i t i o n subun i t and an e f f e c t o r s u b u n i t . V a r i o u s r e c e p t o r s ( r e c o g n i t i o n s u b u n i t s i s more a p p r o p r i a t e , i f one d e f i n e s a r e c e p t o r as a combina t ion of both r e c o g n i t i o n and e f f e c t o r s u b u n i t s ) can be c o u p l e d to the same e f f e c t o r subun i t e . g b e t a -a d r e n e r g i c r e c e p t o r , H 2 , g lucagon r e c e p t o r s , which are c o u p l e d to a d e n y l a t e c y c l a s e . On the o t h e r hand, the p o s s i b l i t y tha t the same r e c o g n i t i o n subuni t may be c o u p l e d to more than one e f f e c t o r subun i t i n d i f f e r e n t t i s s u e s ( R i c h e l s o n and E l - F a k a h a n y , 1981) or even i n the same t i s s u e ( H a r t z e l l , 1982) has been r e c o g n i z e d o n l y ve r y r e c e n t l y . I d e n t i f i c a t i o n of a s i n g l e r e c o g n i t i o n u n i t c o u p l e d to d i f f e r e n t e f f e c t o r systems may r e q u i r e f u r t h e r s u b c l a s s i f i c a t i o n of these r e c e p t o r s , which have p r e v i o u s l y . b e e n c l a s s i f i e d based main ly on b i n d i n g s t u d i e s . The two s u b c l a s s e s of a l p h a - a d r e n e r g i c r e c e p t o r s , a , and a 2 , have s i m i l a r a f f i n i t i e s f o r the p h y s i o l o g i c a l n e u r o t r a n s m i t t e r , n o r e p i n e p h r i n e (Ex ton , 1982), but they appear to be c o u p l e d to d i f f e r e n t e f f e c t o r systems ( G a r c i a - S a i n z and F a i n , 1982). The response of a , i s suggested to be mediated by s t i m u l a t i o n of PI 1 35 t u r n o v e r w h i l e the a 2 _ r e c e p t o r i s suggested to exer t i t s e f f e c t s through i n h i b i t i o n of adeny la te c y c l a s e ( G a r c i a - S a i n z and F a i n , 1982; L e v i t z k i , 1 9 8 2 ) . An i n t e r e s t i n g example of how a s i n g l e r e c o g n i t i o n u n i t can be c o u p l e d to two d i f f e r e n t e f f e c t o r systems has been p r o v i d e d by Morgan et a l . (1983) i n h e p a t o c y t e s . They observed tha t i n hepa tocy te s d e r i v e d from j u v e n i l e r a t s , the response of a , - r e c e p t o r s t i m u l a t i o n i s mediated o n l y by c a l c i u m m o b i l i z a t i o n , but i n h e p a t o c y t e s from a d u l t r a t s , c ^ - r e c e p t o r s t i m u l a t i o n was shown to be accompanied by both cAMP e l e v a t i o n and c a l c i u m m o b i l i z a t i o n (Morgan et a l . , 1983). H e t e r o g e n e i t y i n m u s c a r i n i c r e c e p t o r s based on b i n d i n g s t u d i e s has been p o s t u l a t e d for some time (Burgen et a l . , 1974; S t range et a l . , 1977; Yamamura and Snyder , 1974). Recent s u g g e s t i o n s tha t m u s c a r i n i c r e c e p t o r s i n hear t ( H a r t z e l l , 1982) and sympathet ic neurones (Brown and Adams, 1980; Horn and Dodd, 1981; Weight et a l . , 1979) may be c o u p l e d to more than one type of channe l i n d i c a t e s an e f f e c t o r - d e p e n d e n t h e t e r o g e n e i t y , which may or may not be d i s t i n g u i s h e d by a n t a g o n i s t b i n d i n g . Jones et a l . (1981) have r e c e n t l y s p e c u l a t e d tha t there may be two s u b c l a s s e s of m u s c a r i n i c r e c e p t o r s s i m i l a r to a l p h a -a d r e n e r g i c r e c e p t o r s . The pre sent r e s u l t s , as w e l l as those of Quis t (1982) , support a s u b c l a s s i f i c a t i o n of the m u s c a r i n i c r e c e p t o r . I t i s f u r t h e r proposed tha t the m a j o r i t y of the m u s c a r i n i c r e c e p t o r s i n r a t l e f t and r i g h t a t r i u m and c a n i n e l e f t a t r i u m ( Q u i s t , 1982) may be long to the m u s c a r i n i c r e c e p t o r subtype which i s not coup led to PI t u r n o v e r and which w i l l be 1 36 c a l l e d m 2 . However, a s i g n i f i c a n t p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s i n mouse a t r i a (Brown and Brown, 1982), c an ine r i g h t a t r i u m ( Q u i s t , 1982) and a very s m a l l p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s i n r a t l e f t a t r i u m ( F i g . 10) (a long w i t h m u s c a r i n i c r e c e p t o r s i n many other t i s s u e s where a PI response has been r e p o r t e d ) may be long to the s u b c l a s s m, (analogous to a, of a d r e n e r g i c r e c e p t o r s ) which i s coup led to PI t u r n o v e r . The a b i l i t y of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n to decrease cAMP l e v e l s i n c a r d i a c t i s s u e (George et a l . , 1973; Watanabe et a l . , 1978) may suggest that s i m i l a r to a2 a d r e n e r g i c r e c e p t o r s ( G a r c i a - S a i n z and F a i n , 1982), m2 m u s c a r i n i c r e c e p t o r s may be c o u p l e d to a d e n y l a t e c y c l a s e through an i n h i b i t o r y GTP b i n d i n g p r o t e i n ( R o d b e l l , 1980). P r e v i o u s sugges t ions fo r m u l t i p l e c h o l i n e r g i c r e c e p t o r subtypes i n hear t were p r e s e n t e d by Bucc ino et a l • , ( 1 9 6 8 ) . They found that w h i l e a low c o n c e n t r a t i o n of Ach produced a n e g a t i v e i n o t r o p i c e f f e c t , at h i g h e r c o n c e n t r a t i o n s ACh produced a p o s i t i v e i n o t r o p i c e f f e c t . T h i s l e d them to suggest tha t there may be two types of c h o l i n e r g i c r e c e p t o r s i t e s . But the i n a b i l i t y of the ACh-mediated p o s i t i v e i n o t r o p i c reponse to be b l o c k e d by e i t h e r a t r o p i n e or hexamethonium argued a g a i n s t the responses be ing m u s c a r i n i c or n i c o t i n i c i n n a t u r e . The m u s c a r i n i c - and a l p h a - a d r e n e r g i c - s t i m u l a t e d PI responses i n hear t appear to d i f f e r i n t h e i r c a l c i u m r e q u i r e m e n t s . Quis t (1982) r e p o r t e d that c a r b a c h o l - s t i m u l a t e d PI response i n can ine r i g h t a t r i u m r e q u i r e d c a l c i u m , w h i l e a l p h a - a d r e n e r g i c - s t i m u l a t e d PI response i n c u l t u r e d r a t hear t 1 37 -c e l l s was found to be c a l c i u m - i n d e p e n d e n t (Uchida et a l . , 1982). P r e s e n t l y the f u n c t i o n a l s i g n i f i c a n c e of the m, r e c e p t o r subtype in hear t i s not known, but the presence of such r e c e p t o r s , a long w i t h t h e i r p o s s i b l e c a l c i u m requirement for PI response (as i n can ine a t r i u m ) , may account for the sma l l p o s i t i v e i n o t r o p i c e f f e c t observed on a d d i t i o n of c a r b a c h o l f o l l o w i n g a p r i o r s t i m u l a t i o n w i t h methoxamine i n r a b b i t p a p i l l a r y muscle (Endoh and Motomura, 1979). As t h i s s u b c l a s s i f i c a t i o n of m u s c a r i n i c r e c e p t o r s i s based on ly on the na ture of the e f f e c t o r subuni t c o u p l e d to the r e c o g n i t i o n s u b u n i t , the m u s c a r i n i c r e c e p t o r subtypes may not n e c e s s a r i l y be d i f f e r e n t i a t e d by b i n d i n g s t u d i e s . H o w e v e r , i t i s i n t e r e s t i n g to note tha t G ibson et a l . (1983) have r e c e n t l y s u b c l a s s i f i e d the m u s c a r i n i c r e c e p t o r i n v e n t r i c u l a r muscle as m, and caudate/putamen as m2 based on t h e i r a f f i n i t y for v a r i o u s QNB d e r i v a t i v e s . I t i s even more d i f f i c u l t to s p e c u l a t e at pre sent on the reason for the d i f f e r e n c e s i n the r e l a t i v e d i s t r i b u t i o n of the two m u s c a r i n i c r e c e p t o r subtypes i n a t r i a of v a r i o u s s p e c i e s , except to say t h a t there are o ther r e c e p t o r s which show a l a r g e s p e c i e s v a r i a t i o n i n t h e i r d i s t r i b u t i o n ( M c N e i l l and Verma, 1979; Schumann, 1980), p r o b a b l y r e p r e s e n t i n g d i f f e r e n c e s i n r e g u l a t i o n i n these s p e c i e s . M i c h e l l has p r e v i o u s l y p r e d i c t e d that i f PI t u r n o v e r p l a y e d a r o l e i n c a l c i u m m o b i l i z a t i o n , then o n l y those r e c e p t o r s whose response i s mediated by c a l c i u m m o b i l i z a t i o n w i l l be accompanied by a PI response ( M i c h e l l , 1979). The o b s e r v a t i o n i n t h i s s tudy 138 tha t s t i m u l a t i o n of a l p h a - a d r e n e r g i c r e c e p t o r s , which produced a p o s i t i v e i n o t r o p i c e f f e c t through c a l c i u m m o b i l i z a t i o n , was c o u p l e d to PI t u r n o v e r , w h i l e the m a j o r i t y of the m u s c a r i n i c r e c e p t o r s , p r o d u c i n g an o p p o s i t e p h a r m a c o l o g i c a l re sponse , d i d not enhance PI t u r n o v e r , was c o n s i s t e n t w i t h the above p o s t u l a t e . The complete absence of a PI response i n ra t r i g h t a t r i u m (and c a n i n e l e f t a t r i u m , (Qui s t , 1982)) p r o v i d e s the f i r s t ev idence to suggest that the m u s c a r i n i c r e c e p t o r subtype m 2 , which has been proposed by Jones et a l . (1982) to be c o u p l e d to a d e n y l a t e c y c l a s e , i s not s i m u l t a n e o u s l y c o u p l e d to PI t u r n o v e r . 4.3 ROLE OF PI TURNOVER IN MUSCARINIC RECEPTOR STIMULATED  CALCIUM MOBILIZATION IN GUINEA PIG ILEUM L o n g i t u d i n a l smooth muscle of guinea p i g i l eum c o n t a i n s a homogeneous p o p u l a t i o n of m u s c a r i n i c r e c e p t o r s (Yamamura and Snyder , 1974). S t i m u l a t i o n of l o n g i t u d i n a l smooth muscle of guinea p i g i l eum by m u s c a r i n i c a g o n i s t s or by pota s s ium d e p o l a r i s a t i o n produces a b i p h a s i c c o n t r a c t i o n (Chang and T r i g g l e , 1973; James-Kracke and R o u f o g a l i s , 1981; Rangachar i e_t a l . , 1983) as w e l l as an enhanced i n c o r p o r a t i o n of phosphate i n t o PI ( J a f f e r j i and M i c h e l l , 1 9 7 6 a , b ) . T h i s l e d to the c h o i c e of l o n g i t u d i n a l smooth muscle of gu inea p i g i l e u m for f u r t h e r i n v e s t i g a t i o n of the r o l e of PI t u r n o v e r i n c a l c i u m m o b i l i z a t i o n . 139 4.3 .1 I n c o r p o r a t i o n Of [ 3 H ] i n o s i t o l Into PI D u r i n g a complete c y c l e of p h o s p h o i n o s i t o l head group t u r n o v e r , one "new" phosphate and an i n o s i t o l group w i l l be i n c o r p o r a t e d i n t o P I . S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s by c a r b a c h o l produced a t w o - f o l d enhancement i n the i n c o r p o r a t i o n of both phosphate ( F i g 16) and i n o s i t o l i n t o PI (Table I I I ) . T h i s was c o n s i s t e n t w i t h the p r e v i o u s sugge s t ion t h a t m u s c a r i n i c r e c e p t o r s t i m u l a t i o n produces a PI response i n t h i s t i s s u e ( J a f f e r j i and M i c h e l l , 1976a). The equa l enhancement of both phosphate and i n o s i t o l i n c o r p o r a t i o n i n t o PI ( F i g 16, Tab le I I I ) suggested tha t both p o o l s of l a b e l are e q u a l l y a c c e s s i b l e to the PI undergoing t u r n o v e r . S i m i l a r to phosphate i n c o r p o r a t i o n , c a r b a c h o l - s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n was a l s o i n h i b i t e d by a t r o p i n e , s u g g e s t i n g the t h i s response i s m u s c a r i n i c i n n a t u r e . 4 .3 .2 E f f e c t Of PMSF On C a r b a c h o l And K + - s t i m u l a t e d PI Response  And C o n t r a c t i o n Even though the PI response has now been known for over t h r e e decades , one reason for our ignorance of i t s f u n c t i o n a l s i g n i f i c a n c e i s the l a c k of a s p e c i f i c i n h i b i t o r . As M i c h e l l and K i r k have p o i n t e d o u t , d i s c o v e r y of a s p e c i f i c i n h i b i t o r of the PI response would be a major s t e p forward i n s o l v i n g the "PI p u z z l e " ( M i c h e l l and K i r k , 1981). R e c e n t l y , Walenga et a l . (1980) r e p o r t e d t h a t PMSF and o ther s e r i n e pro tea se i n h i b i t o r s may a l s o be i n h i b i t o r s of P I -s p e c i f i c p h o s p h o l i p a s e C . I t was d e c i d e d to i n v e s t i g a t e whether 1 40 PMSF i s capab le of i n h i b i t i n g c a r b a c h o l - and K + - s t i m u l a t e d PI t u r n o v e r i n guinea p i g i l e u m . I t was found that w h i l e both c a r b a c h o l and K + s t i m u l a t e d l a b e l e d - i n o s i t o l i n c o r p o r a t i o n i n t o PI i n guinea p i g i l e u m , o n l y the c a r b a c h o l e f f e c t was b l o c k e d by PMSF ( F i g . 21) , sugges t ing tha t the s t i m u l a t i o n of i n o s i t o l i n c o r p o r a t i o n by c a r b a c h o l and K + o c c u r r e d by d i f f e r e n t mechanisms. I t i s p o s s i b l e tha t c a r b a c h o l and K + may enhance the t u r n o v e r of PI by d i f f e r e n t pathways or a l t e r n a t i v e l y , they may a c t i v a t e d i f f e r e n t p o o l s or forms of P i - s p e c i f i c p h o s p h o l i p a s e C , so tha t the c a r b a c h o l , but not the K + -s t i m u l a t e d a c t i v i t y , i s s e n s i t i v e to PMSF. Hirasawa et a l . (1982) have separa ted m u l t i p l e forms of P i - s p e c i f i c p h o s p h o l i p a s e C by e l e c t r o f o c u s i n g , and at l e a s t i n two d i f f e r e n t . t i s sues ev idence has been p r e s e n t e d for both c a l c i u m -dependent and c a l c i u m - i n d e p e n d e n t PI t u r n o v e r (Egawa et a l . , 1981; Fare se et a l . , 1982). In t h i s contex t i t i s i n t e r e s t i n g to note t h a t substance P - s t i m u l a t e d i n o s i t o l phosphate a c c u m u l a t i o n was found to be c a l c i u m - i n d e p e n d e n t , w h i l e K + -s t i m u l a t e d i n o s i t o l phosphate a c c u m u l a t i o n was found to be ca l c ium-dependent ( M i c h e l l , 1982). T h e r e f o r e , PMSF may be a u s e f u l way of s e p a r a t i n g these e f f e c t s . I f PI t u r n o v e r p l a y e d a r o l e i n c a r b a c h o l - s t i m u l a t e d C a 2 * m o b i l i z a t i o n , PMSF (which c o m p l e t e l y i n h i b i t s c a r b a c h o l -s t i m u l a t e d PI t u r n o v e r ( F i g . 2 ) ) , shou ld a l s o i n h i b i t c a r b a c h o l mediated c o n t r a c t i o n . However, u n l i k e i t s s e l e c t i v e i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n , PMSF r e l a x e d both c a r b a c h o l - and K + - c o n t r a c t e d t i s s u e and i n h i b i t e d the 141 c o n t r a c t i o n produced by both a g e n t s . F u r t h e r m o r e , i n c o n t r a s t to the e f f e c t s on PI t u r n o v e r , the e f f e c t s on c o n t r a c t i o n were t r a n s i e n t ( F i g 22 ) . These r e s u l t s i n d i c a t e t h a t the e f f e c t of PMSF on PI t u r n o v e r and c o n t r a c t i o n are u n l i k e l y to be c o r r e l a t e d . The h a l f l i f e for PMSF d e g r a d a t i o n i n T r i s and HEPES b u f f e r at pH 8 and 2 5 ° C has been r e p o r t e d to be 35 min (James, 1978), but i t appeared tha t the d e g r a d a t i o n r a t e of PMSF under our i n c u b a t i o n c o n d i t i o n s was f a s t enough to r a p i d l y d i m i n i s h the e f f e c t s of PMSF on c o n t r a c t i o n . On the o ther hand, under s i m i l a r c o n d i t i o n s PMSF gave pro longed i n h i b i t i o n of PI t u r n o v e r . T h i s suggests a separa te mechanism for the two e f f e c t s of PMSF. The r e l a x a t i o n by PMSF may be due to t r a n s i e n t i n h i b i t i o n of c a l c i u m a c c u m u l a t i o n by n o n s p e c i f i c i n t e r a c t i o n w i t h the membrane or o ther c e l l u l a r components. A recent r e p o r t by H a r r i s et a l . (1983) showed tha t PMSF i n h i b i t e d K + -d e p o l a r i z e d C a 2 + uptake i n the f i r s t 10 sec but had no e f f e c t a f t e r a 15 sec p e r i o d . T h i s r e s u l t may a l s o be c o n s i s t e n t w i t h a t r a n s i e n t membrane or c e l l u l a r e f fec t - of PMSF, r a t h e r than i t s t r a n s i e n t i n h i b i t i o n of P i - s p e c i f i c p h o s p h o l i p a s e C . 4 . 3 . 3 Is PMSF I n h i b i t i o n Of C a r b a c h o l - s t i m u l a t e d PI Response  Media ted By I n h i b i t i o n Of P i - s p e c i f i c P h o s p h o l i p a s e C ? The apparent d i s s o c i a t i o n of PMSF i n h i b i t i o n of c a r b a c h o l -s t i m u l a t e d PI t u r n o v e r and C a 2 + - d e p e n d e n t c a r b a c h o l - s t i m u l a t e d c o n t r a c t i o n might appear to argue a g a i n s t a r o l e for PI t u r n o v e r i n c a l c i u m m o b i l i z a t i o n . For such an argument to be v a l i d , however, one must assume that the i n h i b i t i o n of the c a r b a c h o l -142 s i m u l a t e d i n o s i t o l i n c o r p o r a t i o n i n t o PI by PMSF a l s o i n h i b i t s the s teps i n v o l v e d i n c a l c i u m m o b i l i z a t i o n . P r e s e n t l y , both the breakdown of i n o s i t o l l i p i d s ( M i c h e l l , 1975; M i c h e l l , 1982) and a c c u m u l a t i o n of p h o s p h a t i d i c a c i d (Putney et a l . , 1980; Serhan et a l . , 1981) are c o n s i d e r e d to p l a y a r o l e i n c a l c i u m m o b i l i z a t i o n . I f PMSF was indeed capab le of i n h i b i t i n g P I -s p e c i f i c p h o s p h o l i p a s e C , as proposed by Walenga et a l (1982), then both of these proposed c a l c i u m m o b i l i z i n g s teps would be i n h i b i t e d by PMSF. Walenga et a l . (1982) proposed t h a t PMSF may be an i n h i b i t o r of P i - s p e c i f i c p h o s p h o l i p a s e G i n p l a t e l e t s , based p r i n c i p a l l y on the f o l l o w i n g f i n d i n g s : PMSF i n h i b i t i o n of thrombin or c o l l a g e n - s t i m u l a t e d a c c u m u l a t i o n of p h o s p h a t i d i c a c i d c o u l d occur e i t h e r by i n h i b i t i o n of P i - s p e c i f i c p h o s p h o l i p a s e C or d i a c y l g l y c e r o l k inase , or b o t h . S i m i l a r l y , the r e p o r t e d PMSF i n h i b i t i o n of a r a c h i d o n i c a c i d r e l e a s e d u r i n g c o l l a g e n s t i m u l a t i o n c o u l d be due to i n h i b i t i o n of one or more of the f o l l o w i n g ; P i - s p e c i f i c p h o s p h o l i p a s e C , d i a c y l g l y c e r o l l i p a s e , or PI s p e c i f i c p h o s p h o l i p a s e A 2 . In the same s tudy , PMSF was a l s o shown to i n h i b i t a r a c h i d o n i c a c i d r e l e a s e from p h o s p h a t i d y l c h o l i n e , s u g g e s t i n g tha t at l e a s t p a r t of i t s e f f e c t was mediated through i n h i b i t i o n of p h o s p h o l i p a s e A 2 . However, the o n l y d i r e c t ev idence p r o v i d e d to suggest tha t PMSF was e x e r t i n g i t s e f f e c t by i n t e r a c t i n g w i t h P i - s p e c i f i c p h o s p h o l i p a s e C was the i n h i b i t i o n by PMSF of h y d r o l y s i s of p u r i f i e d PI by P i - s p e c i f i c p h o s p h o l i p a s e C . H a r r i s et a l . , (1983) have assumed t h a t PMSF-mediated i n h i b i t i o n of K + -d e p o l a r i z e d c a l c i u m uptake was the r e s u l t of i t s i n h i b i t i o n of 143 P i - s p e c i f i c p h o s p h o l i p a s e C . As d i r e c t i n h i b i t i o n by PMSF of d i a c y l g l y c e r o l a c c u m u l a t i o n d u r i n g PI t u r n o v e r has not been demonst ra ted , the ev idence tha t PMSF i n h i b i t s P i - s p e c i f i c p h o s p h o l i p a s e C d u r i n g PI t u r n o v e r needs v e r i f i c a t i o n . T h e pre sent r e s u l t s argue a g a i n s t t h i s mechanism i n guinea p i g i l e u m . 4 . 3 . 4 L i t h i u m - A m p l i f i c a t i o n Of PI Response In Guinea P i g Ileum A s e n s i t i v e method for measuring the pr imary s tep of PI response was found w i t h the use of l i t h i u m to " a m p l i f y " the PI re sponse , as suggested by B e r r i d g e et a l . ( 1 982) . L i t h i u m has been shown to i n h i b i t i n o s i t o l monophosphatase ( A l l i s o n and B l i s n e r , 1976; Sherman et a l . , 1981). B e r r i d g e et a l . , ( 1 9 8 2 ) used t h i s o b s e r v a t i o n to d e v e l o p a n o v e l method for s t u d y i n g the PI r e sponse . In three d i f f e r e n t t i s s u e s ( r a t b r a i n s l i c e s , r a t p a r o t i d g l and and b l o w f l y s a l i v a r y g land) s t i m u l a t i o n of PI turnover i n the presence of l i t h i u m leads to a c c u m u l a t i o n of i n o s i t o l monophosphate ( B e r r i d g e et a l . , 1982). They sugges ted , t h e r e f o r e , t h a t l i t h i u m can ac t as an a m p l i f i e r of the PI re sponse . The use of l i t h i u m to s tudy the PI response has p r o v i d e d some i n t e r e s t i n g i n s i g h t s i n t o the mechanism of the c a r b a c h o l -s t i m u l a t e d PI response i n guinea p i g i l e u m . Under optimum c o n d i t i o n s , which i n c l u d e d 10 mM l i t h i u m and a 60 min i n c u b a t i o n p e r i o d , 0.1 mM c a r b a c h o l produced a 2 0 - f o l d i n c r e a s e i n the a c c u m u l a t i o n of r a d i o a c t i v e i n o s i t o l phosphate( s ) i n the aqueous phase (Table I V ) . T h i s was c o n s i s t e n t w i t h the r e s u l t s of 1 44 B e r r i d g e et a l . (1982) . In u n s t i m u l a t e d t i s s u e , 10 mM l i t h i u m produced o n l y a smal l a c c u m u l a t i o n of r a d i o a c t i v i t y i n the aqueous phase . 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 by B e r r i d g e et a l . (1982) . T h i s was a t t r i b u t e d to the very s m a l l PI t u r n o v e r i n u n s t i m u l a t e d t i s s u e ( B e r r i d g e et a l . , 1982). I f i t i s assumed tha t the s p e c i f i c a c t i v i t y of l a b e l l e d i n o s i t o l p o o l i s same as the l a b e l l e d i n o s i t o l added to the i n c u b a t i o n medium and tha t i n the presence of l i t h i u m a l l the i n o s i t o l phosphate be ing accumulated i s o b t a i n e d from the PI t u r n o v e r c y c l e , then assuming tha t one molecule of i n o s i t o l phosphate i s r e l e a s e d per PI t u r n o v e r c y c l e , i t was c a l c u l a t e d tha t 2.52 x 1 0 1 7 mo lecu le s of i n o s i t o l p h o s p h o l i p i d undergo t u r n o v e r i n 60 min i n the u n s t i m u l a t e d t i s s u e (Table I V ) . On the o ther hand, i f a l l the i n o s i t o l i n c o r p o r a t e d i n t o i n o s i t o l p h o s p h o l i p i d i n the u n s t i m u l a t e d t i s s u e (Table I I I ) o c c u r r e d d u r i n g the PI t u r n o v e r c y c l e , a t u r n o v e r r a t e of 28.5 x 1 0 1 7 m o l e c u l e s per hour was c a l c u l a t e d . T h i s was 10 t imes h i g h e r than the r a te o b t a i n e d from i n o s i t o l phosphate a c c u m u l a t i o n s t u d i e s . As i n o s i t o l i n c o r p o r a t i o n i n t o i n o s i t o l p h o s p h o l i p i d s c o u l d occur by mechanisms o t h e r than the PI t u r n o v e r c y c l e , i t would appear tha t o n l y about 10% of the b a s a l i n o s i t o l i n c o r p o r a t i o n i n t o i n o s i t o l p h o s p h o l i p i d i s due to the PI t u r n o v e r c y c l e . In the presence of l i t h i u m , c a r b a c h o l produced a dose-dependent a c c u m u l a t i o n of i n o s i t o l phosphate i n the gu inea p i g i l eum and t h i s was b l o c k e d by the m u s c a r i n i c a n t a g o n i s t , a t r o p i n e ( F i g 17). The h a l f maximal response was o b t a i n e d at a c a r b a c h o l c o n c e n t r a t i o n of around 10 uM ( F i g 17), s i m i l a r to the 145 va lue r e p o r t e d by J a f f e r j i and M i c h e l l (1976a) for c a r b a c h o l -s t i m u l a t e d phosphate i n c o r p o r a t i o n i n t o P I . T h i s f u r t h e r suggests t h a t i n o s i t o l phosphate a c c u m u l a t i o n o c c u r r e d d u r i n g c a r b a c h o l - s t i m u l a t e d PI t u r n o v e r . 4 . 3 . 5 A n a l y s i s Of I n o s i t o l Phosphates Accumulated D u r i n g L i t h i u m  A m p l i f i c a t i o n Of C a r b a c h o l - s t i m u l a t e d PI Response In Guinea P i g  I leum The e l u t i o n p r o f i l e of i n o s i t o l phosphate s , r e l e a s e d f o l l o w i n g c a r b a c h o l s t i m u l a t i o n , from the Dowex-1 X8 formate ion exchange r e s i n column, gave d i f f e r e n t r e s u l t s to those r e p o r t e d by B e r r i d g e et a l . (1982) i n o ther t i s s u e s . I n o s i t o l monophosphate, e l u t e d from the column by 5 mM borax p l u s 160 mM ammonium formate ( F i g 18), accounted for o n l y 17% of the t o t a l accumulated r a d i o a c t i v i t y ( F i g 19) . The m a j o r i t y of the r a d i o a c t i v i t y e l u t e d cor re sponded to i n o s i t o l b iphosphate and i n o s i t o l t r i p h o s p h a t e ( F i g 19, Tab le V ) . A l t h o u g h i t was p r e v i o u s l y c o n s i d e r e d tha t the pr imary event of r e c e p t o r s t i m u l a t i o n on PI t u r n o v e r was the enhanced breakdown of PI (Hokin and H o k i n , 1964; J a f f e r j i and M i c h e l l , 1976a), more r e c e n t l y i t has been suggested tha t i n a number of t i s s u e s the pr imary s t ep of r e c e p t o r s t i m u l a t i o n i s the breakdown of p o l y p h o s p h o i n o s i t i d e s (PIP and P I P 2 ) r a t h e r than of P I . The breakdown of e i t h e r P I P 2 a lone ( B i l l a h and L a p e t i n a 1982; Weiss et a l . , 1982) or of both PIP and P I P 2 (Agranof f e_t a l . , 1983, Rhodes et a l . , 1983; Thomas et a l . , 1983) has been suggested to be the pr imary e v e n t . I t i s not known, however, 1 46 whether the same enzyme causes the breakdown of these p o l y p h o s p h o i n o s i t i d e s . When the time course of i n o s i t o l phosphate r e l e a s e d d u r i n g v a s o p r e s s i n s t i m u l a t i o n of s u p e r i o r c e r v i c a l sympathet ic g a n g l i a was s t u d i e d ( M i c h e l l et a l . , 1983), i t was found that w h i l e i n o s i t o l t r i p h o s p h a t e ( I P 3 ) i n c r e a s e d d u r i n g an e a r l y p e r i o d , l a t e r there was an i n c r e a s e of i n o s i t o l b i - and mono-phosphate, r e s p e c t i v e l y . T h i s l e d M i c h e l l et a l . (1983) to suggest that I P 3 was be ing c o n v e r t e d to i n o s i t o l , v i a i n o s i t o l b iphosphate ( I P 2 ) , by the f o l l o w i n g mechanism. I P 3 — — ^ > I P 2 - = ^ I P = ^ 5 ^ I n o s i t o l P P P In l i t h i u m - a m p l i f i c a t i o n s t u d i e s more than 80% of the accumulated r a d i o a c t i v i t y produced by v a r i o u s n e u r o t r a n s m i t t e r s i n r a t b r a i n , r a t p a r o t i d and b l o w f l y s a l i v a r y g l a n d was shown to be i n o s i t o l monophosphate ( B e r r i d g e et a l . , 1982). T h i s was a t t r i b u t e d to the i n h i b i t i o n by l i t h i u m of the l a s t s tep i n the above sequence of e v e n t s , l e a d i n g to the r e l e a s e of i n o s i t o l from I P 3 . The f i n d i n g s i n t h i s study suggest that c a r b a c h o l causes an enhanced breakdown of p o l y p h o s p h o i n o s i t i d e s , and p r o b a b l y of P I . One p o s s i b l e e x p l a n a t i o n fo r the a c c u m u l a t i o n of I P 2 and I P 3 may be, i n gu inea p i g i l eum l i t h i u m may i n h i b i t a l l of the enzymes i n v o l v e d i n the sequence of r e a c t i o n s i n the c o n v e r s i o n of i n o s i t o l t r i p h o s p h a t e to i n o s i t o l . A l t e r n a t i v e l y , l i t h i u m may b l o c k one or perhaps s e v e r a l i n o s i t o l phosphatases r e s p o n s i b l e 147 for the d i r e c t c o n v e r s i o n of i n o s i t o l p o l y p h o s p h a t e ( s ) to i n o s i t o l and phosphate , as shown below: I P 3 y I n o s i t o l + 3P I P 2 V I n o s i t o l + 2P IP I n o s i t o l + P In both c a s e s , l i t h i u m w i l l l e a d to the a c c u m u l a t i o n of i n o s i t o l p o l y p h o s p h a t e s , as observed i n t h i s s tudy . 4 . 3 . 6 E f f e c t Of PMSF On C a r b a c h o l - s t i m u l a t e d I n o s i t o l Phosphate  Accumulat ion The l i t h i u m - a m p l i f i c a t i o n method to measure the i n o s i t o l l i p i d breakdown was used to s tudy the e f f e c t of PMSF on t h i s s t e p . In a p r e l i m i n a r y exper iment , PMSF caused an 18% i n h i b i t i o n of i n o s i t o l phosphate a c c u m u l a t i o n , but p r o b a b l y due t o the s m a l l sample s i z e t h i s d i f f e r e n c e was not s i g n i f i c a n t (0.05<p<.06) (Table V I ) . T h i s e f f e c t of PMSF was much s m a l l e r than i t s complete i n h i b i t i o n of i n o s i t o l i n c o r p o r a t i o n ( F i g 24) , and t h e r e f o r e at f i r s t i t was not c o n s i d e r e d to be of much s i g n i f i c a n c e . L a t e r o b s e r v a t i o n s that l e s s than 20% of the t o t a l accumulated i n o s i t o l phosphates cor re sponded to i n o s i t o l monophosphate suggested that PMSF may be s e l e c t i v e l y i n h i b i t i n g P l - s p e c i f i c p h o s p h o l i p a s e C wi thout i n h i b i t i n g p o l y p h o s p h o i n o s i t i d e p h o s p h o d i e s t e r a s e . Such a r e s u l t would have been c o n s i s t e n t w i t h the f i n d i n g of both Walenga et a l . 1 48 (1980) and Downes and M i c h e l l (1981). Such was not the ca se , however, as PMSF a l s o decreased (Table VI) the c a r b a c h o l -s t i m u l a t e d a c c u m u l a t i o n of i n o s i t o l po lyphosphate by 17%. These r e s u l t s show t h a t PMSF i n h i b i t s the breakdown of c a r b a c h o l -s t i m u l a t e d i n o s i t o l l i p i d , but that the e f f e c t was much s m a l l e r than i t s e f f e c t on c a r b a c h o l - s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n . T h i s i n t u r n suggests that PMSF may be a c t i n g at one or more of the s teps f o l l o w i n g the i n o s i t o l l i p i d breakdown i n the PI response c y c l e , which w i l l account for the g r e a t e r i n h i b i t i o n of i n o s i t o l i n c o r p o r a t i o n compared to PI breakdown. One p o s s i b l e s i t e for PMSF i n h i b i t i o n might be d i g l y c e r i d e k i n a s e , as Walenga et a l . , ( 1 9 8 0 ) have shown t h a t PMSF c o m p l e t e l y i n h i b i t s the accumula t ion of p h o s p h a t i d i c a c i d d u r i n g s t i m u l a t i o n of p l a t e l e t s by c o l l a g e n and t h r o m b i n . The s m a l l e f f e c t of PMSF on the p u t a t i v e c a l c i u m m o b i l i z i n g s tep (PI or PPI breakdown) may account for the l a c k of a p r o l o n g e d e f f e c t of PMSF on c a r b a c h o l -s t i m u l a t e d c o n t r a c t i o n . S i n c e PMSF d i d not b l o c k the breakdown of PI or p o l y p h o s p h o i n o s i t i d e s by more than 15-18%, the p o s s i b i l i t y tha t the breakdown of one or both of these i n o s i t o l l i p i d s i s i n v o l v e d i n c a l c i u m m o b i l i z a t i o n remains open. I t i s i n t e r e s t i n g to note i n t h i s c o n t e x t tha t Weiss et a l . (1982) , u s i n g c r o s s - r e c e p t o r i n a c t i v a t i o n s t u d i e s , have r e c e n t l y p r o v i d e d ve ry c o n v i n c i n g ev idence to suggest t h a t P I P 2 breakdown may be i n v o l v e d i n c a l c i u m m o b i l i z a t i o n d u r i n g the phas i c , response to substance P i n p a r o t i d g l a n d . I t would be i n t e r e s t i n g t o know whether P I P / P I P 2 breakdown p l a y s a s i m i l a r r o l e i n c a l c i u m m o b i l i z a t i o n d u r i n g ' the p h a s i c or t o n i c responses i n gu inea p i g i l e u m . 1 50 V . SUMMARY The e f f e c t of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n on PI response i n r a t hear t and guinea p i g i l eum have been i n v e s t i g a t e d i n t h i s s t u d y , i n order to unders tand the r e l a t i o n s h i p between the PI response and c a l c i u m m o b i l i z a t i o n . 1. Comparison of the b a s a l [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o v a r i o u s p h o s p h o l i p i d s i n r a t a t r i a and v e n t r i c l e i n d i c a t e d t h a t i n both t i s s u e s PI i n c o r p o r a t e d a h i g h e r amount of r a d i o a c t i v i t y than o ther p h o s p h o l i p i d s . However, the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o v a r i o u s p h o s p h o l i p i d s i n a t r i a was 4-6 f o l d h i g h e r than tha t i n the c o r r e s p o n d i n g p h o s p h o l i p i d s i n v e n t r i c l e . S i m i l a r d i f f e r e n c e s have been r e c e n t l y r e p o r t e d i n c a n i n e h e a r t . T h i s may repre sent d i f f e r e n c e s i n the r a t e of p h o s p h o l i p i d metabol i sm between these two areas of the h e a r t . 2. S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n combined ra t a t r i a by c a r b a c h o l (0.1 mM) produced a s m a l l i n c r e a s e (p < 0.05) i n phosphate i n c o r p o r a t i o n i n t o P I . In c o n t r a s t , s t i m u l a t i o n of a l p h a - a d r e n e r g i c r e c e p t o r s i n combined r a t a t r i a and m u s c a r i n i c r e c e p t o r s i n l o n g i t u d i n a l smooth muscle of guinea p i g i l eum were accompanied by a t w o - f o l d i n c r e a s e i n [ 3 2 P ] p h o s p h a t e i n c o r p o r a t i o n i n t o P I . 3. S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n combined r a t a t r i a by 0.1 mM c a r b a c h o l d i d not enhance the breakdown of PI as measured by the l o s s of [ 3 2 P ] p h o s p h a t e - or [ 1 " C ] a r a c h i d o n y l -l a b e l e d PI or by the l e v e l of a r a c h i d o n y l - c o n t a i n i n g d i a c y l g l y c e r o l . T h i s r e s u l t suggested tha t m u s c a r i n i c r e c e p t o r 151 s t i m u l a t i o n had a very sma l l e f f e c t , i f any, on PI turnover in ra t a t r i a . 4. M u s c a r i n i c r e c e p t o r s t i m u l a t i o n produced a d i f f e r e n t PI response i n r a t l e f t and r i g h t a t r i a . S t i m u l a t i o n of m u s c a r i n i c r e c e p t o r s i n r a t l e f t a t r i a was accompanied by a sma l l PI response (35%, p<0.05) , but had no e f f e c t i n ra t r i g h t a t r i a . On the o ther hand, s t i m u l a t i o n of a l p h a - a d r e n e r g i c r e c e p t o r s i n both r a t l e f t and r i g h t a t r i a was accompanied by an enhanced i n c o r p o r a t i o n of phosphate i n t o P I . These f i n d i n g s are c o n s i s t e n t w i t h the s u g g e s t i o n tha t PI response may accompany o n l y the s t i m u l a t i o n of r e c e p t o r s whose response i s mediated by c a l c i u m m o b i l i z a t i o n . Based on the r e l a t i v e r e c e p t o r d e n s i t i e s and r e l a t i v e PI response of m u s c a r i n i c and a l p h a - a d r e n e r g i c r e c e p t o r i n r a t h e a r t , i t i s suggested that a s m a l l p o p u l a t i o n ( approx imate ly 15%) of m u s c a r i n i c r e c e p t o r s i n r a t a t r i a i s c o u p l e d to PI t u r n o v e r . These are termed m, r e c e p t o r s , by analogy to the a , a d r e n e r g i c r e c e p t o r s which enhance PI t u r n o v e r and C a 2 + m o b i l i z a t i o n . The remain ing p o p u l a t i o n , termed m 2 , most p r o b a b l y i s c o u p l e d i n an i n h i b i t o r y manner to a d e n y l a t e c y c l a s e . 6. In a d d i t i o n to the enhanced i n c o r p o r a t i o n of [ 3 2 J p h o s p h a t e i n t o P I , m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n l o n g i t u d i n a l smooth muscle of gu inea p i g i l eum by c a r b a c h o l (0.1 mM) produced an enhanced i n c o r p o r a t i o n of [ 3 H ] i n o s i t o l i n t o P I , and a l s o caused an i n c r e a s e d a c c u m u l a t i o n of i n o s i t o l phosphates i n the pre sence of l i t h i u m (10 mM). The h a l f - m a x i m a l response for c a r b a c h o l - s t i m u l a t e d i n o s i t o l phosphate accumula t ion was 152 around lOjxM, s i m i l a r to the va lue r e p o r t e d p r e v i o u s l y for c a r b a c h o l - s t i m u l a t e d phosphate i n c o r p o r a t i o n and i n r e c e p t o r b i n d i n g s t u d i e s . These f i n d i n g s p r o v i d e f u r t h e r ev idence to support the p r e v i o u s sugge s t ion that m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n guinea p i g i l eum i s accompanied by enhanced i n o s i t o l l i p i d t u r n o v e r and may c l o s e l y accompany the r e c e p t o r occupancy by the a g o n i s t . 7. PMSF, a p u t a t i v e i n h i b i t o r of PI r e sponse , s e l e c t i v e l y i n h i b i t e d c a r b a c h o l - , but not p o t a s s i u m - , s t i m u l a t e d [ 3 H ] i n o s i t o l i n c o r p o r a t i o n i n t o i n o s i t o l l i p i d s i n guinea p i g i l e u m . T h i s suggested tha t c a r b a c h o l - and p o t a s s i u m - s t i m u l a t e d PI response may occur by d i f f e r e n t mechanisms and c o u l d be d i f f e r e n t i a t e d on the b a s i s of t h e i r s e n s i t i v i t y to PMSF. 8. In c o n t r a s t to i t s d i f f e r e n t i a l e f f e c t s on i n o s i t o l i n c o r p o r a t i o n , PMSF produced a n o n - s p e c i f i c and t r a n s i e n t i n h i b i t i o n of both c a r b a c h o l - and p o t a s s i u m - s t i m u l a t e d c o n t r a c t i o n of guinea p i g i l e u m . T h i s e f f e c t of PMSF was p r o b a b l y due to the n o n s p e c i f i c i n h i b i t i o n of c a l c i u m uptake r a t h e r than the r e s u l t of i t s i n h i b i t i o n of PI t u r n o v e r . I f PMSF-mediated i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d PI tu rnover was the r e s u l t of i t s i n h i b i t i o n of P i - s p e c i f i c p h o s p h o l i p a s e C , the l a c k of an e f f e c t of PMSF on c o n t r a c t i o n (a c a l c i u m - m e d i a t e d s tep) would argue a g a i n s t the r o l e of PI t u r n o v e r i n c a l c i u m m o b i l i z a t i o n . 9. A n a l y s i s of the i n o s i t o l phosphates accumulated d u r i n g c a r b a c h o l - s t i m u l a t e d PI tu rnover i n the presence of l i t h i u m i n d i c a t e d t h a t l e s s than 20% of the i n o s i t o l phosphate was 153 e l u t e d i n the peak c o r r e s p o n d i n g to i n o s i t o l mono-phosphate and the r e s t was e l u t e d at h i g h e r i o n i c s t r e n g t h , c o r r e s p o n d i n g to i n o s i t o l b i - and t r i - p h o s p h a t e . T h i s p r o v i d e d d i r e c t ev idence to suggest tha t l i k e some o ther t i s s u e s , the pr imary event of PI t u r n o v e r i n guinea p i g i l eum d u r i n g m u s c a r i n i c r e c e p t o r s t i m u l a t i o n may be the breakdown of p o l y p h o s p h o i n o s i t i d e s r a t h e r than P I . 10. A c c u m u l a t i o n of a l a r g e p r o p o r t i o n of i n o s i t o l po lyphosphate s r a t h e r than i n o s i t o l monophosphate i n guinea p i g i l eum i n d i c a t e d e i t h e r t h a t l i t h i u m i n h i b i t e d a l l t h r e e enzymes i n v o l v e d i n the s tepwise c o n v e r s i o n of i n o s i t o l t r i p h o s p h a t e to i n o s i t o l or i t i n h i b i t e d one or more enzymes i n v o l v e d i n the d i r e c t c o n v e r s i o n of i n o s i t o l phosphates to i n o s i t o l . 11. In c o n t r a s t to i t s a lmost complete i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n , PMSF caused o n l y a s m a l l ( approx imate ly 16%) i n h i b i t i o n of c a r b a c h o l - s t i m u l a t e d i n o s i t o l phosphate a c c u m u l a t i o n , i n d i c a t i n g tha t i n h i b i t i o n by PMSF of P i - s p e c i f i c p h o s p h o l i p a s e C cannot c o m p l e t e l y account fo r the observed i n h i b i t i o n by PMSF of c a r b a c h o l - s t i m u l a t e d i n o s i t o l i n c o r p o r a t i o n . T h e r e f o r e , PMSF may i n h i b i t o ther s teps f o l l o w i n g the i n i t i a l s t ep of i n o s i t o l - l i p i d breakdown. 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