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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 P I G SMOOTH MUSCLE by M. B.Pharm.,  CHANDRA SEKAR  B . I . T . S . , P i l a n i , I n d i a 1974; M . P h a r m . , P i l a n i , I n d i a 1976; M . S c , U . B . C , 1980  B.I.T.S,  A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS  FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Division  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 Pharmaceutical Sciences  We a c c e p t  this  to the  t h e s i s as  required  conforming  standard  THE UNIVERSITY OF B R I T I S H COLUMBIA December 0  M.  Chandra  1983  Sekar,  19 8 3  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  freely  a v a i l a b l e f o r r e f e r e n c e and study.  I further  agree t h a t p e r m i s s i o n for e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be granted by t h e head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . understood t h a t for financial  copying o r p u b l i c a t i o n o f t h i s  gain  Fkfayv^^C-JAs-kS>£^  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  UI>/{Lf  thesis  s h a l l n o t be allowed w i t h o u t my  permission.  Department o f  It i s  Columbia  £A^C£^  written  i i  Abstract Stimulation  of  various  enhanced  turnover  phenomenon,  referred  to  to  play  muscarinic  atria  a  and  hypothesis  to  has  was  density  is  observed  in rat  contrast  to  the  rat  left  these  receptors  These  results,  compared  receptor  suggested  that  muscarinic  receptors  are  receptors remaining probably cyclase.  termed  m,  coupled these  on  0.1  is  inositol  lipid  and C a 2 +  muscarinic  in  an  findings  to  analogy  support  i n t o PI  was By  into  to  of  a,  PI  density in  PI  manner  effect  the  of  breakdown, 15%)  of  turnover. adrenergic  termed m 2 , to  of  atria.  mobilization.  receptors,  inhibitory  an  (approximately  PI of  receptor atria,  lower  lack  by  turnover  ileum.  mM methoxamine  showing a  receptors,  rat  stimulation,  receptors  coupled  this  in  incorporation the  by  atrium.  receptor  by  population  atria  pig  right  despite  studies  response  guinea  in  the m u s c a r i n i c  with  in rat  population  While  to  study,  and r i g h t  muscarinic  atria,  suggested  mediated  incorporation  not  This  muscarinic  left  i n phosphate  a small  w h i c h enhance  is  in rat  stimulation  only  the  stimulation  and r i g h t  along  of  PI  an  stimulation  In t h i s  though  but of  increase  muscarinic  These  effect  been  receptor  by s t u d y i n g  even  atrium  (PI).  has  systems.  in phosphate  receptor  produced a t w o - f o l d both  that  left  produces  mobilization  smooth m u s c l e  p < 0.05)  alpha-adrenergic  calcium  be s i m i l a r  small  response, muscarinic  investigated  known t o (35%,  PI  receptor  observed  increase  the  in  other  receptors  phosphatidylinositol  as  role  been  surface  r e s p o n s e of  and l o n g i t u d i n a l It  in  of  be an o b l i g a t o r y  and  cell  The most  adenylate  postulate  that  PI  r e s p o n s e may accompany whose  response  results  also  population The  is  role  the  mediated  suggest  whose  only  the  PI  in l o n g i t u d i n a l  Consistent  with  stimulation  of  response.  effect  guinea  of  of  in  the  accumulation  Carbachol  accumulation Analysis  of  of  pig  occurred found that  in  i n the similar  primary  inositol  event  than  The putative and  than.  of  an  breakdown  by  a  PI  into PI.  The  lipids  was  which  inositol  of  by  in  lithium.  tissues,  [3H]inositol  while  fraction.  the  ion-exchange  many o t h e r the  causes  increase  i n the p r e s e n c e  receptor  ileum. receptor  inhibiting  20-fold  tissues  was  atropine-sensitive  lithium,  by  of  pig  inositol  unlike  20%  in other  muscarinic  guinea  accompanied  mM  a  polyphosphate  enhanced  of  of  monophosphate,  findings  i l e u m may be t h e rather  less  mobilization  phosphates  that,  turnover.  [3H]inositol  phosphate  inositol  inositol  to  was  10  phosphates  indicated  ileum  of  PI  the  receptor  muscarinic  produced  produced  inositol the  chromatography guinea  inositol  receptors  muscarinic  calcium  breakdown  presence  of  phosphatase.  in  ileum  mM)  on t h e  a  findings,  pig  (0.1  those  coupled to  [ 3 2 P ] p h o s p h a t e and  carbachol  studied  not  of  smooth m u s c l e  previous  Carbachol  incorporation  is  response  investigated  of  through calcium m o b i l i z a t i o n ,  presence  stimulation of  stimulation  the  label  remainder  This  in  was  indicated  reported  recently,  the  stimulation  in guinea  pig  of  polyphosphoinositides,  PI. effect  of  i n h i b i t o r of  phenylmethanesulfonylfluoride Pl-specific  potassium-stimulated  PI  phospholipase turnover  and  C , on  (PMSF),  a  carbachol  contraction  in  iv  longitudinal PMSF  smooth m u s c l e  almost  completely  incorporation stimulated stimuli  into PI,  inositol  produce  guinea  pig  while  it  had  no  responses"  PMSF p r o d u c e d a n o n s p e c i f i c ,  not  guinea  nonspecific the  result  suggestion turnover  of  its  Walenga e t  C  produced only inositol  a  was  indicating  that  fully  the  obtained involved  do n o t  transient  through  its  on  on  that  indirect of  the  accumulation  in  the  PMSF e f f e c t for  on  the  contradict  the  two In  PI  and  of  of  potassium. that  it  turnover.  is The  of  PI  Pl-specific  findings.  PMSF  (2 mM)  carbachol-stimulated presence  Pl-specific  possibility  PI  inhibition  of  inhibition  that  lithium,  phospholipase  incorporation.  in calcium mobilization.  the  PMSF i n h i b i t i o n  observed  inositol  that  suggests  inhibition  inhibition  account  carbachol-stimulated  (1980)  potassium-  carbachol-stimulated  on c o n t r a c t i o n  al.  inositol  mechanisms.  i l e u m by b o t h c a r b a c h o l  based  16%  phosphate  of  inhibitory effect  may be m e d i a t e d  phospholipase  cannot  pig  inhibition  PMSF e f f e c t  of  studied.  on  suggests  different  turnover,  The  specific  by  to  of  was  effect  This  contrast  contraction  ileum  inhibited carbachol-stimulated  incorporation.  "PI  its  of  The PI  C  by PMSF of results  breakdown  is  V  Table  of  Contents  Abstract L i s t of T a b l e s L i s t of F i g u r e s Acknowledgement L I S T OF ABBREVIATIONS  ii viii ix  xiii x  i  Chapter I INTRODUCTION 1 1.1 DISTRIBUTION OF PI 3 1.2 PI RESPONSE 4 1.2.1 Closed Cycle 4 1.2.2 The P r i m a r y E v e n t Of PI R e s p o n s e 6 1 . 2 . 3 A l t e r n a t e Mechanisms F o r 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 R e q u i r e m e n t Of PI R e s p o n s e 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 I o n o p h o r e 19 1 . 4 . 3 I s PI D e g r a d e d 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 G u a n o s i n e Monophosphate 29 1.8.2 P o t a s s i u m 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 R e c e p t o r And PI Response 32 1.8.4 A r e T h e r e 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 Receptors ? 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 M e c h a n i s m Of The A l p h a - And B e t a - A d r e n e r g i c Receptor-mediated P o s i t i v e Inotropic Effect 45 1.11 MUSCARINIC RECEPTORS IN LONGITUDINAL SMOOTH MUSCLE OF GUINEA PIG ILEUM 47 1.12 OBJECTIVE 50 C h a p t e r II METHODS 2.1 C h o i c e Of A n i m a l 2.2 K r e b s - b i c a r b o n a t e B u f f e r 2 . 3 P h o s p h a t e I n c o r p o r a t i o n In A t r i a 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 M u s c l e Of G u i n e a P i g Ileum 2 . 5 L a b e l - i n c o r p o r a t i o n In G u i n e a P i g Ileum 2 . 6 E x t r a c t i o n Of L i p i d 2.7 P h o s p h o l i p i d S e p a r a t i o n  51 51 52 52 53 53 54 55  —  vi  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 S p o t s 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 2.10 P h o s p h o r u s D e t e r m i n a t i o n 2.11 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 In The P r e s e n c e Of Lithium 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 2.13 2.14 2.15 2.16 2.17  S e p a r a t i o n Of I n o s i t o l P h o s p h a t e s Contraction Studies PI D e g r a d a t i o n S t u d i e s 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 Materials  56 56 57 58 59 59 60 60 61 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 V e r s u s Ventricle 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 R e c e p t o r 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 R e c e p t o r 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 S e p a r a t e L e f t And R i g h t 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 W i t h 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 Accumulation 70 3 . 3 . 4 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 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 [32P]Phosphate Incorporation 73 3.4.2 [ 3 H ] l n o s i t o l Incorporation 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 P h o s p h a t e s 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 T u r n o v e r 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 C h a p t e r 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 T e c h n i q u e 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 H e a r t . . . . 1 2 5  vii  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 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 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 Arachidonyl-Enriched 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 R i g h t Atria 132 4 . 2 . 4 E v i d e n c e F o r 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 133 4 . 3 ROLE OF PI TURNOVER IN MUSCARINIC RECEPTOR STIMULATED CALCIUM MOBILIZATION IN GUINEA PIG ILEUM 1 38 3 4.3.1 I n c o r p o r a t i o n Of [ H ] i n o s i t o l I n t o 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 R e s p o n s e 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 R e s p o n s e M e d i a t e d By I n h i b i t i o n Of P i - s p e c i f i c Phospholipase 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 G u i n e a P i g Ileum 143 4 . 3 . 5 A n a l y s i s Of I n o s i t o l P h o s p h a t e s A c c u m u l a t e d 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 R e s p o n s e In G u i n e a P i g I l e u m 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 Inositol Phosphate Accumulation 147 Chapter V SUMMARY  150  BIBLIOGRAPHY  154  vi i i  List  of  Tables  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  II.  E f f e c t of c a r b a c h o l of d i a c y l g l y c e r o l  stimulation  III.  E f f e c t of c a r b a c h o l phospholipid  on [ 3 H ] i n o s i t o l  IV.  Carbachol-stimulated phosphate  V.  C o m p o s i t i o n of i n o s i t o l guinea p i g ileum  VI.  E f f e c t of PMSF on t h e 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 in guinea p i g ileum.  on a r a c h i d o n y l  accumulation phosphates  content 101  incorporation  of  82  into 1 08  inositol  accumulated  109 in  116  1 23  ix  List  of  Figures  1 . PI  turnover  2.  PI  as  3.  Pirenzepine  .4.  Phospholipid separation  80  5.  Standard  83  6.  C o m p a r i s o n of t h e b a s a l 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 a t r i a and v e n t r i c l e s of r a t heart  85  7.  E f f e c t of c a r b a c h o l and methoxamine on [32P] i n c o r p o r a t i o n i n t o PI i n c o m b i n e d 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 atria  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 e p a r a t e l e f t and r i g h t a t r i a  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 p h o s p h a t i d y l i n o s i t o l of s e p a r a t e r a t l e f t and r i g h t atria  11.  .  5  a c a l c i u m gate  15  Structure  curve  for  36  phosphorus  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 [3 2 P ] p h o s p h a t e i n combined r a t a t r i a  prelabeled  rat 89 rat 91 into 93  with 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 with arachidonic a c i d in rat a t r i a  13.  Separation  14.  Gas chromatogram diacylglycerol  15.  F a t t y a c i d c o m p o s i t i o n of r a t a t r i a and v e n t r i c l e s  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 m u s c l e o f guinea p i g ileum 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 phosphate accumulation i n guinea p i g ileum  of  prelabeled  diacylglycerol of  fatty  acid the  97 99  methyl e s t e r s polar  lipid  of  fraction  102 of  inositol  104  110  X  18.  Elution  profile  of  standard  inositol  19.  E l u t i o n p r o f i l e of t h e in guinea p i g ileum  20.  Time c o u r s e of t h e 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 g u i n e a 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 p i g ileum  22.  E f f e c t of PMSF on c a r b a c h o l - and c o n t r a c t i o n of g u i n e a p i g i l e u m  accumulated  mono-phosphate.  inositol  112  phosphates 114  K+-stimulated  guinea 119 121  xi  List  of A b b r e v i a t i o n s  ACh  acetylcholine  nAChR  nicotinic  mAChr  muscarinic acetylcholine  ACS  aqueous c o u n t i n g s c i n t i l l a n t  cAMP  cyclic  adenosine  °C  degree  centigrade  Ci  Curie  mCi  millicurie  MCi  microcurie  cm  centimeter  cpm  counts  DAG  1-oleyl-2-acetyl-diacylglycerol  D-600  methoxy v e r a p a m i l  dpm  disintegrations  fmole  femto mole  cGMP  cyclic  GTP  guanosine  £  gravitational  g  gram  mg  milligram  Mg  microgram  cGMP  cyclic  GTP  guanosine  h  hour  IP  i n o s i t o l mono-phosphate  acetylcholine  receptor receptor  3',5'-monophosphate  per minute  per minute  guanosine  3',5'-monophosphate  triphosphate force  guanosine  35'-monophosphate  triphosphate  xii  IP2  inositol  di-phosphate  IPs  inositol  tri-phosphate  K  thousand  ml  milliliter  Ml  microliter  M  molar  mM  mi 1 1 i m o l a r  MM  micromolar  min  minute  nm  nanometer  QNB  quinuclidinyl  sec  second  msec  m i l l i second  TLC  thin  PI  phosphatidylinositol  PIP  phosphat i d y l i n o s i t o l  4-phosphate  PIP2  phosphatidylinositol  4,5-bisphosphate  PMSF  phenylmethanesulfonyl  PKC  protein  layer  benzilate  chromatography  kinase C  fluoride  xi i i  ACKNOWLEDGEMENT  I  wish  to  his  constant  this  work.  Drs,  J.H.  e x p r e s s my g r a t i u d e  to  encouragement  guidance d u r i n g  My s p e c i a l  thanks  McNeill,  S.Katz,  Schellenberg  for  their  and to  Dr.  B.D.  my r e s e a r c h D.  Godin,  genuine  Roufogalis the  course  for of  committee  members,  Diamond  and R . H .  J.  interest  and  valuable  suggest i o n s . I  will  programs,  Dr.  suggestions and  friends  memorable I by  to  thank  Christine  during for  the  making  Mr.  Nichol  c o u r s e of my s t a y  Ahmad F a w z i for  this  the  for  plotting  discussions  work and a l l  i n Vancouver  his  a very  and  my c o l l e a g u e s pleasant  and  one.  would l i k e  the  Heart  like  to  University  Foundation.  acknowledge the of  British  financial  C o l u m b i a and  the  support British  provided Columbia  xiv  To my parents  1  I. It  is  surface  now w e l l  enable  is  known a b o u t  of  the  to  the  the  (Sutherland According  membrane (Singer lipid the  cell  is  and N i c o l s o n ,  1960)  is  play  an  membrane  Sandermann,  1978),  membrane and a c t i v i t y suggestion in  that  of  appears q u i t e  (Levitzki  coupled  side and  of  receptor  and a d e n y l a t e  may  lipid  bilayer  the  major  cells.  Both  have  various  been  membrane  Raison,  1979; the  membrane bound e n z y m e s .  Therefore,  the  to  Helmreich,  i n v o l v e m e n t of  One  1970).  biological  chains  and  are  f l u i d i t y of  signals  of  assist  ions,  (including lipid)  across  a  adenylate  t h e membrane,  possible  and A x e l r o d ,  in  permeability  w h i c h employ cAMP as  be  cytoplasmic  acid  inside  the  may p l a y  bilogical  a  membrane  rational.  Receptors to  fatty  McMurchie  membrane components  transmitting  in a  various  role  1974;  s u c h as  the  in  less  language,  Phospholipids constitute  important  (Singer,  the  (Rasmussen,  "floating"  cell  but  which  into c e l l u l a r  p h o s p h o l i p i d head g r o u p and t h e to  the  to  messengers,  and N i c b l s o n m o d e l ,  1972).  at  transferred  and c a l c i u m  proteins  t h e plasma  receptors  incoming s i g n a l s ,  secondary  Singer  composed of  characteristics  shown  recognize  i n c o m i n g message  the  of  that  information  and R a i l ,  to  fraction  shown  role  to  Two i m p o r t a n t  transform  cAMP  established  how t h i s  cell.  INTRODUCTION  cyclase  have  been  on  the  present  t h r o u g h a GTP b i n d i n g  1979;  Rodbell,  phospholipid cyclase  s e c o n d messenger  has  in also  the  1980).  protein  Recently,  coupling  a  between  been p o s t u l a t e d  (Hirata  1980). ask  if  phospholipids  also  play  a  role  in  2  translating  the  employ cAMP as such et  a  role  al.,  1982  factor,  in  second is  ).  messenger.  PI c o n t a i n s head  is  One  with  and  rich  in  the  its  phosphate  phospholipid Galliard,  content  1973  concentration  of  derivatives. of  ;  Even  PI  is  total  phospholipid,  undergo  a very  rapid metabolic  pancreas  enhanced to  an In  different  tissues  labeled  similar  to  In  the  of  stimuli  be  the  that  were  labeled amylase  into  observed  s h o w i n g PI only  PI  acyl chain  acid,  in  of  (Rouser  its  a very  1953  al.,  1968;  tissues  the  that  al•,  into  PI,  (Hokin  by H o k i n and H o k i n  turnover.  Firstly,  w i t h those  ligands  in  an  addition Hokin,  stimuli  in  incorporation Michell,  (1953)  to e v o l v e  of  produced  and  other  1969;  1979).  stimulation  carbamylcholine  various  fraction  phospholipids  et  c a u s e an e n h a n c e d  a t r e n d began  total  small  (Griffin  1968,  PI,  phosphorylated  inositol  secretion  (Hokin,  rate-  the  et  most  phosphate  years  a  of  derivatives,  2-12%  than  three  turnover  found to  seventies,  associated  all  growth  phosphatidylinositol  in  greater  observed  following  phosphate  early  of  increased  1953).  of  and  by p a n c r e o z y m i n o r  incorporation  causing  about  Jones  prostaglandins.  and  tissues  1973),  the  pigeon  (PIP)  much  first  fatty  of  not for  1975;  an e s s e n t i a l  0  though they c o n s t i t u t e  H o k i n and H o k i n  (Michell,  phosphorylated  various  White,  w h i c h do  candidate  arachidonic  constitute  in  (Pi)  synthesis  two  (PIP2),  possible  the  in  phosphatidylinositol-4-phosphate 4,5-bis  in systems  myo - i n o s i t o l ,  group,  usually  precursor  along  information  phosphatidylinositol  the  mammalian PI limiting  incoming  in  i n the  1975),  pancreas. long  list  the  response  seemed  that  interact  with  to  cell  3  surface did  receptors  not  In the  and s e c o n d l y ,  employ cAMP as  past  decade,  possibility mediated  many  that  signal  their  PI  only  with  those  s e c o n d messenger  groups  have  turnover  may  been play  receptors (Michell,  that 1975).  investigating a  role  in  the  receptor-  transmission.  1 . 1 DISTRIBUTION OF PI If  inositol  transmission t h e major the  PI  is  must  be l o c a t e d  inositol  present  membrane,  in  various  and a s m a l l membrane  present  mostly  studies  to in  PI,  (Buckley  PIP  the  done on m y e l i n  membranes  quite  difficult.  studies 1978).  up a b o u t  outer  PI,  2-10%  of  The m a j o r i t y  intracellular  of  organelles,  m i t o c h o n d r i a l membrane  amount has  and  plasma  (Soukup  PIP2  been  The  most  are  membrane, et  al.,  and H a w t h o r n e ,  shown  to  be  Kirk  et  to  be  considered  b a s e d p r i n c i p a l l y on  1978)  1972).  and  In t h r e e  different  reported  values  Mmole o f  PIP2  gram o f  between  per the  of  their quantitative  reliable  regions  0.1  -  tissue.  receptor  and  erythrocyte  The l a b i l e  values  0.13  of  nature  brain,  al  Soukup  al  Mmole PIP and 0 . 2 9  inside  of  from  (Soukup e t  The p h o s p h o l i p i d a c t i n g the  of  estimation  were o b t a i n e d  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  .(1978)  link  membrane.  ( H o k i n - N e a v e r s o n , 1977;  t h e s e p o l y p h o s p h o i n o s i t i d e s makes  .,  makes  signal  1981). In c o n t r a s t  the  i n the p l a s m a  i n the c e l l .  reticulum,  the plasma  in receptor-mediated  lipid,  content  in  endoplasmic  and n u c l e a r present  have a r o l e  phospholipid  including  al. ,  they  component o f  total  this  lipids  the c e l l  et -  0.48 as  a  should  4  preferably  be  located  i n the  membrane  bilayer.  There  inositol  lipid  the  in  cytoplasmic  side,  ATP p o o l of  cytosol  PI,  suggests  cytoplasmic have  side  1.2  RESPONSE  PI  1.2.1  Closed The  observed occur  that  they  that  the  by an  that  tissue, for  PI  of  also  further  various  increase of  indicating  i n the  that  in  the  that  the  phosphorylation  be  located  on  of the  (1982)  PIP2  constitute  only  lipid  fraction,  the  inner  leaflet  of  the  5 mM.  in  head d i d not  labeled  receptors  phosphate  (PI  head g r o u p  .  p r o v i d e d by H o k i n and cortex  inositol of  enhanced  was  not  labeled  moiety  may  g r o u p was show  supporting  r e s p o n s e was  PI  cerebral  and  of  i n de novo s y n t h e s i s  the  first  diacylglycerol  content  suggest  Downes and M i c h e l l and  incorporation  incorporation  phosphoinositol PI  plasma  located  for  may  total  h i g h as  is  to  the  Cycle  phosphate  increased  the  c o u l d be as  mechanism was  labeled  PIP  concentration  turnover  found  phosphate lipids  of  evidence  evidence  membrane.  of  on s t i m u l a t i o n  increased latter  the  side  membrane  indirect  even when  increased  either  plasma  the  of  polyphosphoinositide bilayer  no d i r e c t  inositol  quantities  myelin  is  can p r o v i d e  that  calculated  trace  but  cytoplasmic  the  reconfirmed  in  mechanism.  i n many  PI  other  PI  could or  by  Evidence for  the  Hokin  (1958);  i n c o r p o r a t i o n of  This  by  the  an  indicated while  D i r e c t measurement  increase  above  of  reutilised  renewed.  an  response)  accompanied  glycerol. be  into  the of  stimulated  This tissues  mechanism by  the  5  Hokins,  including  (Hokin, gland  adrenal  (Hokin  other of  1968),  and  groups.  phosphate  been  shown  contents  sympathetic  Sherwin,  One w e l l  to  provoke  of  1957),  is a  the  cortex  and of  is  (Hokin,  et  two-fold  to  quite  above  increase PI,  mechanism  the  et  has  adrenal  PIP,  (Farese  from  by many  where ACTH in  phospholipid  different  salivary  verified  cortex,  diacylglycerol  pancreas  1958),  the  acid,  ACTH on t h e  1969),  al.,  and l a t e r  adrenal  phosphatidic  This effect  adrenal  (Hokin  known e x c e p t i o n  incorporation  phosphatidylglycerol 1980).  medulla  ganglia  PIP21  al.,  1979,  metabolism  of  classical  PI  the  response. The  general  mechanism  for  PI  turnover  is  shown i n  Receptor  stimulation  by  agonists  causes  the  specific  phospholipase  C,  resulting  i n the  breakdown  diacylglycerol phosphorylated reacts  with  and  inositol  by ATP t o cytidine  give  phosphate.  phosphatidic  triphosphate  and  Figure Reproduced  activation  PI-  PI  to  Diacylglycerol  is  inositol  1 - PI t u r n o v e r from B e r r i d g e , 1980  1.  of  acid,  PI .  Fig  of  which to  further  resynthesise  6  1.2.2  The P r i m a r y E v e n t Of PI Usually  measured  radioactivity later  that  rate  of  The  into  an  increase  phosphatidic  the p r i m a r y event breakdown  first  as  report  of  of  the  PI  of  (or  (1964)  of  mechanism f o r  decade  above later,  Neaverson, proven work  with  the  1974;  (Bansback parotid event  on  et  gland of  al.,  (Berridge response  phospholipase  C, g i v i n g  phosphate.  Studies  1978;  and  platelets  blowfly  et  al.,  was rise  et  et  1981)  have  than  PI  al.,  Imai  1983;  increased  derivatives).  the  the  and  and  PI  by  inositol al.,  1977,  recently  with  1980;  1983)  suggested  that  polyphosphoinositide  may  constitute  action.  be  primary  breakdown of  and h e p a t o c y t e s  the  to  brain  that  and M a j e r u s ,  former  Hokin-  pancreas  al.,  breakdown  a  1980),  ( A b d e l - L a t i f et  Bell  only  studied,  gland,  more  by  been  diacylglycerol  and  was  generality  et  (Downes and M i c h e l l , 1 9 8 2 ) , and t h e phosphodiesterase  an  Though s t i l l has  of  apparent  a l . (1969),  suggested  increased  1980)  was  apparent  Majerus,  salivary  Abdel-Latif,  (Agranoff  with  to  et  response and  But  became  (1974).  1982)  the  it  was  .  Durell  muscle  1982b;  rather  gland  iris  and L a p e t i n a , al.,  of  ,  concentration  response  (Bell  1974),  and PI  PI  salt  PI  incorporation  phosphorylated of  where a PI  platelets  PI  Akhtar  in avian  the  response  J o n e s and M i c h e l l  i n many t i s s u e s based  PI its  reports  in  acid  lowering  H o k i n and H o k i n the  Response  Billah (Michell  breakdown  primary  may be t h e  event  target  of  7  Activation  of  Phospholipase  breakdown  on a c e t y l c h o l i n e  proposed  by  were  inositol  as  stimulated have  the  been  by  accumulation  of  a  PI  and p a r o t i d  Alternate  by  described  briefly  into  PI c a n  has  been  1979)  and  substantially  followed  of  A second  When  inositol  present in  insect al.,  of  could  lithium,  was  PI  than  by  fatty  an  during  a  large  salivary  gland,  1982).  can o c c u r , PI  and has  been  They  are  response.  the  by r a p i d  acid,  action  rabbit  on PI  of  (Rittenhouse-Simmons,  (Schrey et  decrease  1981),  from p h o s p h o l i p a s e  C  label A2.  with arachidonic  (Rubin  can a l s o  of  phospholipase  cells  neutrophils  A2 action  incorporation  reacylation  in adrenocortical  c a n be d i s t i n g u i s h e d level  result  below.  be a f f e c t e d  observed  above  Incorporation  into  other  employing l a b e l e d  Phospholipase  the  label  mechanisms  Deacylation  (ACh)-  hydrolysis  et  conclusions  in acetylcholine  in  (Berridge  once  [3H]-  resulted  monophosphate  PI  only  phosphatase, it  of  was  Their detect  the  of  Mechanisms F o r L a b e l  reported  studies  that  result  mechanism pancreas  could  phosphatase.  gland  of  of  product  response,  inositol  Incorporation  In  a  inositol  of  1.2.3  as  a  (1975).  they  now a p p e a r s  inositol  of  stimulation  brain  It  obtained  inhibitor  that  al.,  water-soluble  tissue.  phosphate(s)  rat  finding  as  stimulation  H o k i n - N e a v e r s o n et  b a s e d on t h e  D  but  and  al.,  the the  action  Rubin, 1981).  PI  content  above by  acid  effect  measuring  lysophosphatidylinositol. mechanism of  increasing  label  i n t o PI  involves  the  8  increased certain  availability  tissues,  such  of  diacylglycerol.  as  platelets  Prescott  and M a j e r u s ,  1983)  observed  in  response  the  PI  Diacylglycerol  can  triacylglycerol  and o t h e r  the  PI c y c l e ,  PI.  Only  during  resulting  20% of  the  for  considered  to  arise  third  phosphatidic  unlike  acid  of  show  into  PI.  Secondly,  incorporation  an  the  sources  PI,  for  increased  to in  PI. a  This  number  classical  PI  acid label  will  into  other  inositol  incorporation D action  widespread  cells but  be  occurrence  of  is  1974). label  acyl  no  CoA,  mechanism  labeled  glycerol  is  a  de novo i n c r e a s e  al.,  of  Firstly,  this  by as  key in  the  increased in  ACTH  1980).  h i g h l y u n l i k e l y , way of  There  was  distinguished  acid  could  be  rest  al.,  phospholipids,  PI  obtained  ways.  response,  et  into  could  et  reflected  (Farese  into  on P I .  label  de novo s y n t h e s i s  i n c o r p o r a t i o n of  level  enter  increasing  of  phosphatidic  e.g.  then  w h i l e the  (Bansback  pathway  since  fourth possible,  the  can  pancreas,  breakdown of  increased  The  for  This  sources  in d i a c y l g l y c e r o l l e v e l ,  involves  on a d r e n o c o r t i c a l  phospholipase  breakdown.  from o t h e r  phospholipid metabolism,  of  PI  may be  also  phosphatidic  to  of  1981b;  in d i a c y l g l y c e r o l  i n c o r p o r a t i o n of  stimulation  turnover  shown i n  al•,  conversion  the m a j o r i t y  of  et  and f a t t y  c y c l i c PI  intermediate  due  been  from g l y c e r o l - 3 - p h o s p h a t e  will  action  increase  PI  increase  was  increased  from o t h e r  into  the  obtained  possible  subsequent  from t h e  in  has  (Lapetina  phospholipids.  by i n c r e a s e d  incorporation  with  the  be  pilocarpine  accounted  The  also  that  It  be  the  convincing  phospholipase  increasing result  of  evidence  D i n mammalian  9  tissue. In PI  spite  head  of  group  response.  In  (Berridge  and  occur,  of  limitations,  is  a  all  PI  label  tissues  (except  1979))  been  the  label first  is  the  step  the of  salivary  response into  into  indicator  blowfly  where PI  i n c o r p o r a t i o n of has  incorporation  c o n v e n i e n t and s e n s i t i v e  Fain,  enhanced  moiety  its  PI  gland  now known  to  phosphoinositol  in establishing  the  response. 1.2.4  Pi-specific  Phospholipase  C  As m e n t i o n e d  in a previous  section,  response  is  considered  and H o k i n , Jones  1964;  and  Akhtar  Durell  Michell,  derivatives,  to  PIP and  1982b;  action  Imai  of  C  1969;  (Abdel  1980; al.,  1983;  bond has  of been  pancreas  1961),  guinea  pig  1968),  among o t h e r s ,  plants  (for  as  a complete  characterized  (Dawson,  list  see  C exists  of  i n the  et  al.,  al., et  phosphorylated 1977,  1983; al.,  1981)  soluble  by  and the  the  Pi-specific  in various  liver  (Kemp  animal et  al.,  ( A t h e r t o n and H a w t h o r n e ,  certain  microorganisms  from w h i c h t h e  1982).  Billah  hydrolyses  be p r e s e n t  sources  1978;  polyposphoinositide  1959),  in  Shukla,  its  which  mucosa  as  et  or  phospholipid.  shown t o  well  1974;  or  the  intestinal  Hokin-Neaverson  Michell  C  PI  (Hokin  Latif  Agranoff  of  PI  1975)  respectively,  s u c h as  phospholipase  al.,  PIP2  tissues  been  et  phospholipase  glycerophosphate  e n h a n c e d breakdown of  Michell,  et  phosphodiesterase,  phospholipase  the  1974;  and A b d e l - L a t i f ,  Lapetina,  be  the primary event  Most of  form i n t h e  the  enzyme  and has  Pi-specific  cytoplasm  of  10  various  tissues,  (Allan  and M i c h e l l ,  1959; of  Kemp e t  the  and  al.,  enzyme,  that  and w h i c h d i f f e r e d described  by  (1973).  hydrolyses  1961;  Thompson,  from s o l u b l e et  Irvine  and  dehydrogenase  membrane bound a c t i v i t y Lapetina  cytoplasmic  and M i c h e l l ,  contamination  available  evidence  dependent  soluble  lysosomal The  phospholipase  showed PI.  (Atherton Latif  et  enzyme (Shukla,  some  The and  al., from  reflect  and k i d n e y ,  five  electrofocusing  and  was  Michell provided  marker,  (Friedel  of  plasma  membrane. presence  and that  et  accounted  and  C,  activation  be  from  C  in  al.,  for  by  Most of  the  of  a  Ca2+ -  a C a 2 + - independent  enzyme its  guinea  1968)  and  hydrolyzed  from  different  d e g r e e of  specificity  pig  iris  the  for  only  specificity  intestinal  mucosa  smooth m u s c l e  (Abdel-  different  forms  points,  (Hirasawa  et  of  have al.,  of  studies  the  the been  1982).  PIP2,  PI and n o t  the h y d r o l y s i s  in vivo s p e c i f i c i t y  isoelectric  in brain  the  turnover,  later  could  variation  Most of  not  the  Lapetina (1978)  enzyme  Dawson,  phospholipase  h y d r o l y s e d b o t h PIP and  optimal conditions  i n PI  a cytoplasmic  phospholipase  S.aureus  1982).  role  1973) the  calcium  C.  Hawthorne, 1980)  as  of  1968;  deoxycholate  indicates  enzyme  the  their  far  of  described  cytoplasmic  Pi-specific  sources towards  so  and  Dawson  lactate  1969;  important  al.(1969)  the the  presence  A membrane bound form  cytoplasmic  b a s e d on pH d e p e n d e n c e  of  1967).  c o u l d p l a y an  evidence, use  i n the  1 9 7 4 ; A t h e r t o n and H a w t h o r n e ,  Friedel  However,  PI  of  PI  the  PIP o r  PIP2  in v i t r o  employ  and t h e r e f o r e  enzyme. enzyme,  In r a t  by  do  liver  differing  separated This  whereas  in  column  heterogeneity  of  11  the  cytoplasmic  importance quite  enzyme  and f u n c t i o n of  possible  techniques could  that  used  have  others,  Hydrolysis of  and M a j e r u s ,  of  an  phospholipase  C,  and s h e e p s e m i n a l was  inhibited  proteins, to  fatty  forms  vesicular  glands  as  of  the  self-amplification  enzyme  over  when c o m p a r i n g  brain  PI  (Dawson e t  and  bound  enzyme a c t i v i t y . PI  al.,  1980)  charged  phospholipase  in  acid  C  unstimulated and  unsaturated  (produced  were  found to  be  This  provides  a  response  by  1982)  positively  PI  the  (Hofmann  Majerus,  acid  response),  by  hydrolysis  i n a b i l i t y of  the  is  enzyme  increased  1978).  arachidonic  mechanism f o r  this  deoxycholate  phosphatidic  PI  It  isolation  the  (Hofmann and  the  membrane  d u r i n g the  stimulating  is  phosphatidylcholine for  the  laboratories.  enzyme  from r a t  such  of  s h o u l d be e x e r c i s e d  and Dawson,  Unsaturated  acids  certain  obtained  hydrolyse  diacylglycerol of  Irvine  by  preparations.  of  this  w h i c h may a c c o u n t  rapidly  characterization  optimal concentration  1982;  enzyme.  for  from d i f f e r e n t by  of  and  caution  PI  forms  regarding  conditions  favoured  enzyme c h a r a c t e r i s t i c s  questions  assay  previously  selectively  new  these various  the  and t h e r e f o r e  presence  raises  from capable  possible  (Dawson e t  al.,  1980). The h y d r o l y s i s described PIP2 PI  as  early  phosphodiesterase  response a f t e r  hydrolysis, event  for  the  PIP2  as  1964  has  recent  rather PI  of  than  response  by  extracts  rat  (Thompson and Dawson,  become more  suggestions PI  of  important that  hydrolysis,  (Michell  et  with  brain  was  1964a),  but  respect  to  polyphosphoinositide may be t h e  al.,  1981;  triggering Downes  and  1 2  Michell,  1982).  and e r y t h r o c y t e one  Data  on t h e  this  any e f f e c t  conditions  requirement 1.3  its  and S h e r w i n , in  scope,  secretory early  1971;  had been PIP  occurrence  involvement  as  that  Hokin  PI  a  et  1972). PIP2  levels  calcium  the  PI  response  phospholipase  role.  1958)  was cells  .  for of  of  event  reevaluation  of  has  first  in exocrine was  In  the  be  potentials  1966;  and  Reinertsen,  (Hokin limited  occur  late  in  both  sixties  and  in  stimulated  H e n d r i c k s o n and  Reinertsen,  of  all  the  1969)  above  for  proposals  calcium  and t h e r e f o r e ,  phosphorylation-dephosphorylation  C  mediated  of the  PI  response  role  of  the  membrane.  breakdown  PI.  of  the  in  affinity  c a l c i u m bound t o  to  hypothesis  gland  found t o found to  led  polyphosphoinositides  action  (Dawson,  The  later  phosphorylation-dephosphorylation  to  C,  certain  two  a much h i g h e r  of  primary  PIP2,  and under  be  has  affect  the  activity,  The main f e a t u r e  by  amount  of  al.,  role  proposed  interconversion  that  with  phospholipase  to  its  response  (Hendrickson  the  its  in exocytosis  and p r o p a g a t i o n  Torda,  Pi-specific  seemed  regarding  1957;  was  consistent  1982).  seventies,  neurons  Like  and n o n - s e c r e t o r y  generation  are  TURNOVER  speculation  regarding  1981)  1964b)  h y d r o l y s i n g b o t h PIP and  calcium for  there  The w i d e s p r e a d much  PI.  (Irvine,  ROLE OF PI  activity  on  enzyme r e q u i r e d  assay  (Thompson and Dawson,  (Downes and M i c h e l l ,  phosphodiesterase  without  brain  of  PI,  than their might  Recognition rather  than  polyphosphoinositides,  was  i n many d i f f e r e n t Michell  in  1975  systems  led  (Michell,  1 3  1975),  after  concluded turnover has  that in  been  existing  literature  the  evidence  suggested a  available  "calcium gating". presented  Garcia-Sainz, and  r e v i e w i n g the  1980;  against  both  In in  Michell  the  past  favour  and K i r k ,  (Cockcroft,  1981;  on PI  eight  role  ;  for  years,  (Berridge, 1981  response, PI  evidence  1980;  Fain  and  Putney,1981,1982)  Hawthorne,  1982)  the  above  hypothesis. In have  been  will a  the  f o l l o w i n g pages  suggested  be a n a l y z e d .  source  of  which  a  initial  calcium  observations a)  of  across  kinase  PI  support  gate  was  (Michell,  Receptors  mediated b)  and t h a t  capable  message  protein  hypotheses PI  hypotheses,  role are  of  turnover  that  PI  turnover  is  turnover  is  a source  of  transmitting the  PI  which  cell  the  h o r m o n a l or  membrane  through  C (PKC).  PI AS A CALCIUM GATE The  as  of  acid  is  and two o t h e r  physiological  The o t h e r  neurotransmitter  1.4  the  arachidonic  diacylglycerol,  activation  for  this  whose  their  the  heavily  hypothesis  that  dependent  on  PI  functioned  the  following  1975):  stimulation  response  r e s p o n s e was  for  was  accompanied  by a PI  response  through calcium m o b i l i z a t i o n ;  independent  of  between  mediating  r e s p o n s e t h r o u g h c a l c i u m m o b i l i z a t i o n has  been  very  good  and u n i v e r s a l l y then  a)  it  (Michell,  1975,  producing  1979).  involved in receptor  s h o u l d be e v o k e d  in a l l  If  a  calcium.  The c o r r e l a t i o n their  stimuli  extracellular  PI  mediated cells  PI  response  breakdown calcium  and a l l  and  indeed is  only  gating,  tissues  where  1 4  receptor  stimulation  receptors should  which  1979).  cholinergic parotid  and  Berridge,  accompanies  a  list this  the  (Lakshmanan,  second messenger  is  in  al.,  but  the  response.  ionophore section), surface  where  enhance  receptor  1975,  Michell  of  pineal  growth  that  as  of  NGF  where  the  is  two in  greater  labelling et  et  al.,  PI  Huggins,  a  1981).  response  more One which  sympathetic preparations (1981)  have  the  role  of  as  is  still  not  clear  1980),  the  above  al•,  detail  Ca+2  Another receptor  in  a  exception mobilizes  which  produces  and  calcium  glucose  without  (Fain parotid  (for  and  stimulation  1981)  and  et  al.  nicotinic  stimuli,  in  (NGF)  reservation.  muscarinic  Only  (Michell  et  (Egawa  gland  al.,  gland  factor  Landreth with  et  be t h e  1953),  s y s t e m s where PI  mobilization  treated  lipid  cell  Ca2+  action  (described  (Gaut  However, M i c h e l l  the  deferens  receptors  and h e a r t  to  muscarinic  Hokin,  salivary  cAMP,  Michell,  as  and  vas  in blowfly  indicating  it  1974),  appears  nerve  cortex,  calcium PI  Michell  1978;  s h o u l d be  adrenal  by  1979).  arguments  et  (Hokin  those  s u c h as  1982;  such  many d i f f e r e n t  stimulation  1978,  al.,  receptors,  1974),  rule  to  et  alpha-adrenergic  few of  see  exposed  exception  (Jones  pancreas  and J o n e s ,  to  (Schubert  in  accompanied  comprehensive  provided  second messengers,  surface  1979b),  just is  ganglia  other  5-hydroxytryptamine  are  exception  c a l c i u m m o b i l i z a t i o n and b)  ( J o n e s and M i c h e l l ,  (Michell  response  to  breakdown  Cell  gland  1981),  1966)  PI  receptors  al.,  gland  employ  not produce  1975;  leads  the  acting  following at  a  cell  15  Figure  2 -  Reproduced  PI  as a c a l c i u m  from B e r r i d g e ,  gate 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 conformational 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 Pi-specific  Of PI  phospholipase  concentrations  for  activation  Pi-specific  of  breakdown, of  the  1979;  occurs  reported  studies  Trifaro,  1969b)  Conclusions on  such  Steinberg, indicate  1973)  or  by  recent  1981;  mechanism methods  for  the  usually  of  a)  of  calcium  experiment  is  calcium with  measure  (Griffin  al.,  to  whether  causes  the  cell.  Griffin  PI Many  et  al.,  incorporation  of  PI  requirement et  is  which  phosphate  response.  of  1979;  interpret,  PI  response  Lennon as  breakdown  they is  and do  of  PI  to  t h e whole p r o b l e m has  suggesting  that  and c a l c i u m i n d e p e n d e n t et  loss  al. , of  1982),  PI  to  addition  as  :  study of  an  the  not  calcium-  that  calcium  PI  breakdown  mobilization,  ionophore  as  an  s h o u l d not  1982a).  the cause  the  the  PI  PI of  increase  the the  b e h i n d s u c h an  cause  increase  of  agents.  increasing  The r a t i o n a l e is  Two  regulation  A23187,  thereby  et  additional  calcium chelating  s u c h as  both  (Egawa  c a l c i u m dependence  calcium,  concentration. if  well  been  may be  turnover  i o n o p h o r e and b)  Ionophores, to  PI  there  ( B i l l a h and L a p e t i n a ,  c a l c i u m by means of  the c e l l  cytosolic  of  a  calcium  reports  ionophore  permeability  effect  as  complexity  used  intracellular  Effect  C,  1975;  labeled  primary event  Farese  breakdown a r e : the  question  calcium  not.  calcium-dependent al. ,  al.,  difficult  A new d i m e n s i o n of added  The  micromolar  calcium enters  et  used  the  are the  after  and PI  studies  whether  dependent  or  (Oron  regarding  requires  phospholipase  have  acid  C  activity.  before  into phosphatidic  based  its  Response  rather in  than  cytosolic  breakdown.  In  1 7  many  tissues  produce  PI  (Jones  and  Berridge,  ionophore-mediated  breakdown. Michell,  1977a)  Some of  the  1975),  blowfly  and h e p a t o c y t e s  p a n c r e a s and p l a t e l e t s , (platelets  -  Billah  Neaverson  ,1977)  pancreas  -  breakdown  of  (Cockcroft  et  before  PI a  al. ,  phenomenon; concentration stimulation  must  and b) as  data are  Stimulation  maximal  nM),  w h i c h was  secretion  similar  for  and PI  100 and  was  accompanied  phosphatidic  acid  (Egawa  the  PI  a  neutrophils  breakdown  both  suggesting  for  Hokin1982;  leukocytes 1981)  a  requirements  calcium-mediated calcium  during by  receptor the  same  At p r e s e n t et  such al.,  fMet-Leu-Phe produced (half  breakdown.  and  -  al.,  maximal  and PI  at  stimulated  MM c a l c i u m to produce  range,  In  (Cockcroft  Neutrophils  PI  1979).  An i o n o p h o r e -  occur  response.  with  and  cytosolic  must  by i n c r e a s e d  and P I ,  as  in  loss  et  following  neutrophil secretion  calcium concentration  breakdown  PI  (Fain  ionophore-mediated  reported.  the  to  gland  and Nazawa,  range o b t a i n e d  calcium-dependent.  produced  been  neutrophils  i n both s e c r e t i o n  ionomycin,  deferens  the  for  only of  Ca2+  increase  observed  fMet-Leu-Phe r e q u i r e d  effects  into  the  gland  pancreas  - Imai  c a n be c o n s i d e r e d  in  parotid  presented  1982a;  a  satisfy  the  failed  polymorphonuclear  breakdown has  suggested  half  nM  and v a s  be  available  1981).  in  that  been  (platelets  to  are  salivary  Lapetina,  whereas  response a)  has  1980)  1981) PI  examples  al.,  breakdown has PI  mechanism  et  PI,  calcium-dependent mediated  and  increase  ( B i l l a h and M i c h e l l ,  evidence  and a g a i n s t  Farese  calcium  half  The  with 1 maximal  over  ionomycin-stimulated  that  the  0.3  ionophore,  breakdown  phosphate  a  a PI  incorporation PI  breakdown  18  was  at  least  However, of  a  the  partially  result  does not  receptor-mediated  neutrophils.  Recent  submaxillary et  al.,  calcium  independent  demonstration enough  calcium  found  to  eliminate PI  (Billah  and  Michell,  et  al.,  there  1982)  is  the  PI,  where  Michell,  1979;  the  Fain  breakdown  and e n h a n c e d by c a l c i u m  al., of  1981).  PIP2  observed  in  gland  (Weiss  parotid period  of  decreased not  a  few  observed  with  recently  presented  suggest  that  and that  in a tissue  receptor  is  mediated  to  hepatocytes et  al.,  gland)  and  1979a;  Jones  and  in  iris  medium ( A b d e l - L a t i f  et  al.,  in  this  1982b),  iris  smooth  (Lang  et  and b r a i n  was  the et  interesting  et  al.  1981)  (or  and  over  a  slightly  and was (1982b)  and c o n v i n c i n g d a t a  breakdown may be  et  breakdown  occurred  unaffected  Weiss  (Jolles  al.,  which  muscle  al.,1977) ,  rapid  by c a l c i u m d e p r i v a t i o n ,  polyphosphoinositide  tissues  abolished  (Michell  ionophore.  some v e r y  of  was  polyphosphoinositide,  1978)  was  breakdown  number  erythrocyte  exception  seconds  in parotid  a  (ACh) was  and H a w t h o r n e ,  The o n l y  of  ionophore  1978),  (Griffin  deferens  suggests  breakdown  of  in a calcium d e f i c i e n t  synaptosomes  turnover  large  by a c e t y l c h o l i n e  al.,  vas  rat  calcium-dependent  and B e r r i d g e ,  smooth m u s c l e  et  PI  in  tissues,  and r a b b i t  PI  in a  PIP2,  (Abdel-Latif  possibility breakdown  receptor-mediated  specifically  1978)  PI  two  both  occurence  independent  1979),  the  C.  breakdown.  to  be c a l c i u m  phospholipase  negate  in  calcium-dependent  contrast  to  that  receptor-mediated  independent  In  reports  1981),  of  completely  of  calcium-independent  gland,(Farese  (Egawa  not  through a c t i v a t i o n  also have to  responsible  19  for  calcium mobilization  in  the  phasic  response  of  parotid  breakdown of  inositol  cells. 1.4.2  Phosphatidic  Acid  Diacylglycerol, lipids is an  (Igarashi  obtained  and K o n d o ,  rapidly converted enzyme  also  Hawthorne,  including  located  during salt  in  pancreas  1979;  blood  the et  second  ionophore.  was  et  was  physical  ,  to  ask  too  whether  could act  phosphatidic  properties  was  Michell's  r e s p o n s e may be  response)  that  to  systems,  After PI  and  found  different  the  kinase,  (Lapetina  been  very n a t u r a l  d u r i n g PI  1977)  a  shown t o a c t (Tyson is  role  et  which (1977)  of  the  as  acid  membrane,  ( Keryer  et  undefined  for as  al.,  interesting  lines  was  of  acid  to  al., be  a calcium  acid.  of  as  w h i l e PI  and in  in  the (Serhan  and  red  a calcium tend  Phosphatidic  in liposomes  that  studies  evidence  ionophore  and  to note  proposed  functions  available  phosphatidic  1976)  first  b a s e d on t h e i r  phosphatidic  Many d i f f e r e n t  has  It  al.  that  such  1981).  many  hypothesis  hypothesis,  support  chamber  membrane  h y p o t h e t i c a l c a l c i u m gate  al.,  by M i c h e l l  cells,  been  by d i g l y c e r i d e  has  that  1982a),  use.  The rejected  1975) it  the  Lapetina,  and p l a t e l e t s .  (accumulated  may a l t e r  Michell  practical  (Michell,  The i n i t i a l  altering  plasma  response  in calcium gating,  thereby  the  and  acid  PI  involved  accumulation  Billah  acid  proposal  a calcium gate.  1980;  Phosphatidic  gland,  acid  d u r i n g the  in  initial  phosphatidic  Ionophore  to p h o s p h a t i d i c  1971).  accumulate  As A C a l c i u m  to acid  Pressman et  al.,  arachidonic  20  acid  failed  to  enhance  phosphatidic  acid  intermediates  obtained  uptake acid  into  the  acid  formation presence 1982)  of  In p a r o t i d  gland,  et  presented  to  (Putney  support et  the  increased  lipid  inhibit  with  for  phosphatidic an o r g a n i c  the  of  translocated Non-bilayer  ,  3 +  for  very well same  (Putney  amphiphilic  incorporation  of  nature  mimic  of  various  neomycin, C o 2 + ,  N i 2+ ,  the  inhibitory  during of  a 5  the  Ca  constants  inhibition  from  water  of  into  1980).  of  phosphatidic  exogenously of  a calcium  PI  Even  effect  a c i d as  been  during  difficult.  the  has  channel  added  i n d i c a t i n g that  acid  calcium  exogenously  somewhat  phosphatidic  by  with  al.,  al.,  uptake  substances  et  the  calcium  acid-induced partitioning  phase  in  ( V e r k l e i j et  constants  Tm3+,  the  acid  evidence  phosphatidic  Dissociation La  of  receptor-stimulated  calcium  obtained  cations  of  phosphatidic  1981).  of  calcium  following order  total  line  two  Phosphatidic  with a  al.,  divalent  role  (e.g.  correlated  to  et  a different  antagonists  response,  able  the  Liposomes  mole % of  acid,  1981).  ionophoric property  1980).  competing  studies  with  (Serhan  al.,  which  membrane  cations  1-5  the  1980).  The  al.,  c a l c i u m and o t h e r  al.,  Mg2+),  liposomes,  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 of  calcium  into  response,  et  >> Mg.  n o t magnesium  could explain  (Cullis  PI  (Serhan  divalent  Mn > Ca > Sr  transport  8,11,14-eicosatrienoic during  liposomes  concentration  c a l c i u m but  gate  and  translocated  selectivity;  calcium  acid  phosphatidic then,  there  makes acid  the into  have  been  some  added p h o s p h a t i d i c  acid  was  calcium-mobilizing agonists.  In  21  neuroblastoma  cells  little  1  as  production  of  treatment, of  (Ohsako and D e g u c h i ,  yg/ml  of  phosphatidic  phosphatidic  caused  an  1978)  smooth and  hand,  platelet  1.4.3  Is  release  being  of  (Putney,  degraded  determine variable  et  of  al.,  phospholipase  cGMP by  al.,  1978)  systems  (Gerrard  has or  C  activation  Other  1981).  acid  the  is  the  comes site  results.  in accepting lack  from of  of  the  et  On t h e no  al., other  effect  on  calcium-dependent  loss  of  In p a n c r e a s  site  reticulum. membranes  The  PI  appeared  and  salt  gland  in stimulated  be  the  Attempts  cells  have  ganglia, 1959;  PI to  given  b a s e d on  Gerber  e_t  was  endoplasmic  to  from p l a s m a  c o u l d not  that  1967;  stimulated  (Bennett  a calcium  ( H o k i n and H u e b n e r , found be  to  et  synaptosomes islet  membrane  located  al., in  be  from s e c r e t o r y  ( H o k i n - N e a v e r s o n , 1977)  neutrophils loss  membrane.  glucose-stimulated  a l . ,(1977),  suggest  (Redman and H o k i n ,  turnover  loss  1978)  (Clements et  stimulated  PI  in e l e c t r i c a l l y  Hawthorne,  stimulated  of  to  and s y m p a t h e t i c  and a u t o r a d i o g r a p h y  the  evidence  PI  al.,  1973)  PI breakdown as  plasma  centrifugation  of  or  1981).  differential  site  exogenously,  cyclase.  added p h o s p h a t i d i c ( G e r r a r d et  as  by  microsomes  (Harris  the problems  hypothesis  1965),  level  of  PI D e g r a d e d From Plasma Membrane?  One gating  platelet  synaptosomes  aggregation  potassium  i n the  addition  acid  in s i t u  guanylate  muscle,  exogeneously  acid  increase  (calcium-stimulated)  include  1981)  in  Hokin  vesicle  (Pickard of  and  Langerhans  acetylcholine-  and f M e t h - L e u - P h e -  1982).  By c o n t a s t ,  the  vasopressin-stimulated  22  hepatocytes of  and  this  PI among v a r i o u s If  the  was  subcellular  primary event  polyphosphoinositides, recently most  (Michell  probably  occur  at  the  shown t o 1.5  the  attributed  of  as  et  the  primary event  plasma  be p r e s e n t  membrane, only  PI  1981;  the  (Kirk  rapid et  response by  equilibration  al.,  is  1981).  the  Michell  breakdown of  and  Downes and M i c h e l l , of  as  i n the  inositol  lipid  coworkers 1982),  then  breakdown  does  polyphosphoinositides plasma  have  been  membrane.  DIACYLGLYCEROL AS AN ACTIVATOR OF PROTEIN KINASE C Phospholipid-dependent  (PKC), al., et  pools  suggested  al.,  to  w h i c h was  1979a)  al.,  (Ku e t other  has  turnover  in  when  was  it  smooth  1981),  tissues  al.,  protein  was acid  in  1979b).  the  /3  carbon  diacylglycerols  were  et for  al., the  et  al.,  An  (the  was  shift  found  PI  whereas to  be  Although  PKC a c t i v i t y ,  of  other  lymphocytes  first of  and many of  PI  postulated unsaturated  the  affinity  of  ( K i s h i m o t o e_t  in calcium  affinity  unsaturated is  et  (Helfman  1982)  MM range  containing  acid),  1980).  the  (S c a r b o n  (Takai  involvement  presence  This  with d i a c y l g l y c e r o l  fatty  essential  the  group  1982),  i n c u b a t i o n medium s h i f t e d  al.,  polyunsaturated  (Kishimoto  1981).  c a l c i u m from t h e mM t o et  al.,  C activation  that  kinase C  in neutrophils  (Tanigawa  al.,  kinase  i n the  Takai  greatest  et  occur  (Endo e t  pancreas  observed  enzyme f o r 1980;  rat  protein  by N i s h i z u k a ' s  shown t o muscle  (Minakuchi  diacylglycerol the  described  now been  1983), al.,  first  calcium-activated  also  fatty  enriched in  the  saturated  completely  ineffective  phosphatidylserine phospholipids  were  was capable  23  of  further  modulation  phosphatidylserine  of  PI  had  sphingomyelin et  al.,  evidence  was  a  PI  diacylglycerol  protein  was  platelets  al.,  (Limas, clearly  enzyme a c t i v i t y  PI  of  PI and  (Kaibuchi  activating caused  a  not  release,  activation  of  and  (40K)  of  al.,  it  diacylglycerol  was  however,  et  al.,  that  at  three  increase  The  release  same  PI  breakdown  or  PI  causing  1980). least  to  occur  response calcium  Recent in  by  (Mori  diacylglycerol  the m i c r o m o l a r  during  al the  diacylglycerol as  et  of  of  considered  that  40K  activation  (DAG) ( M o r i  by  in  phosphorylation  direct  PKC t o  responses without  Kishimoto  indicate,  formed  all  This phosphorylation  was p r o p o s e d of  with  (  1982b).  synthetic  affinity  platelets  and s e r o t o n i n  accompanied  PKC by the  i n the  physiological  1983).  al.,  transient  during  i n PKC et  factor  protein  therefore  Initially  shift  turnover (Ieyasu  Activation  1-oleyl-2-acetyldiacylglycerol  was  1980;  of  platelets  phosphorylated  by DAG  1982).  in  K a i b u c h i et  al.,  calcium,  mediate  1982;  weight  et  can c a u s e a of  PKC a c t i v i t y  intermediates  f o l l o w e d by a s i m u l t a n e o u s  Kaibuchi  40K p r o t e i n serotonin  recently  response),  also  with  1982;  direct  the  of  While  phosphatidylcholine  involvement  or p l a t e l e t  level,  al.,  the  al.,(1982b)•  a 40,000 m o l e c u l a r  (Ieyasu et  et  et  collagen  produced  for  provided  Kaibuchi  thrombin,  .,  other  and  presence  1981).  increased  (two  effect,  the  al.,  Mg/ml)  acid  in  1981) .  activation  of  no  et  markedly d i m i n i s h e d the  Further  1982;  (20  and p h o s p h a t i d i c  response)  activity  (Kaibuchi  phosphatidylethanolamine by 50%,  its  level could influx  experiments  platelets,  both  24  calcium for  mobilization  complete  (Kaibuchi  physiological  et  In  al.,  1982b,  model  phosphatidylserine the  phase  causing PI  and d i a c y l g l y c e r o l  diacylglycerol  curve  concentration  mediated  phase  possible  mechanism Most of  group,  and  that  turnover  1.6  PI PI  the  and  rate  logical  production the  it  of  al.,  in this are  concentration, In  separation 1981).  activation  involved  et to of  (Lapetina generate  et free  action  PI  acid  is  field  i n the  comparison,  at  the  same  Diacylglycerolmay  of  indicate  PKC (Ohki  is  consistent  from v a r i o u s  from  activation  et  a  al.,  Nishizuka's  w i t h the  hypothesis  of P K C .  al.,  postulate  al.,  in  van D o r p e t that  response 1981a;  that  the  PI  arachidonic  arachidonic of  established  factor 1964;  "free"  mammalian  tissues  (5,8,11,14-icosatetraenoic  now w e l l  physiological  direct  the  work done  may be  limiting  (Bergstrom is  10 M M c a l c i u m .  phosphatidylserine  observations  0 carbon  arachidonic  1973),  at  et  calcium  shifted  AS A SOURCE OF ARACHIDONIC ACID  The in  the  of  (from y e a s t )  lower  phase  (Ohki  for  the  release)  phosphatidylcholine/  a  o n l y 7%  separation  1981).  to  separation  calcium  required  1983)  mixtures,  produced  are  serotonin  containing  phase  from y e a s t  (i.e.  systems  separation 75%  response  formation  al.,  1964).  turnover  may be  acid,  through  its  M a r s h a l l et acid  phospholipase  by  acid;  arachidonic  formation  which  of  White, acid  is  Therefore, involved  can  then to  al.,  1982).  A 2 on PI  rich  eicosanoids  conversion  two  is  1980,  mechanisms:  it  in  the  mediate  eicosanoids  a)  PI by  (Hong and D e y k i n ,  can the  1981;  25  Rubin by  et  al.,  the  1981;  action  of  monoacylglycerol Majerus,  1983)  (Lapetina  et  Walsh et  lipase or  al.,  the  response, based  the  moiety acyl  was  turnover acid  was  composition  stimulation  levels)  indicated and  and  arachidonic  acid  PI  and b)  turnover  reacylation  the  not  PI  further  be  a  contained  of  fatty  of  be  the  before PI  to  of  acid  during  by  fatty  and  after  original amount  of  that  a)  thrombin-induced  resynthesized  produced  PI  arachidonate  suggested  1-stearoyl-2-arachidonyl  w h i c h may f i n a l l y  while  diacylglycerol  a greater  amount This  being  glycerol,  thrombin-stimulated  recovery  conserved  conserved  that  the  platelets  lower  not  the  is  Initial  labeled  Comparison  1981).  was  with  fatty a  the acid  deacylation-  cycle. of  arachidonyl-prelabeled  t h y r o t r o p i n produced  labeled  1981).  latter  being  of  acid  group  proposed.  platelets, during  and  on p h o s p h a t i d i c  renewal  human  and  of  Stimulation with  that  was  in  even a f t e r  Majerus,  characteristic composition,  (and  linoleate,  (Prescott  underwent  in  (Prescott  response concluded  conserved  PI  diacylglycerol;  w o u l d not  incorporation  At l e a s t  of  it  originally  and M a j e r u s ,  thrombin  oleate  moiety  not  (Prescott  the  response,  arachidonyl  then  d u r i n g PI  conserved.  chain  was  on  inositol  phosphoinositol  If  turnover, as  on  mooacylglycerol  1981b). PI  d u r i n g PI  lipase  A2 action  during  p h o s p h a t e and  on  b)  phospholipase  during  observations,  1981),  diacylglycerol  hydrolyzed PI  al.,  diacylglycerol proposed  (but  no  a  rapid  (Igarshi evidence  thyroid  and t r a n s i e n t and was  Kondo,  follicles  accumulation 1980).  presented),  It that  of was this  26  diacylglycerol present to  could  i n the  liberate  transient Marshall that  et  free  al.  was  only was was  was  possibility action  of  Schwartzman  probably  salivary  salivary  arachidonic  acid),  presented  i m p l i e d by  al.,  1981;  other  PI  gland  on  acid.  gland  necessarily  et  al.,  PI  simply  al.,  PI,  et the  decrease  1982). explained  by  its  and t h e  other  suggested synthesis that  An  it or  alternate  that  of  a  suggested  to  occur  direct  Hong and D e y k i n ,  (1982)  decrease  have  of  in  1981;  in  stimulation  by s a l i v a r y in  of  release  both  made  the  arachidonyl-  stimulation  in  The d e c r e a s e  the  carbachol  This  is  al.  result  accompanied  of  PI  1981).  5-Hydroxytryptamine  c a u s e d a 54%  (Litosch  that  during  indicate  result  5-hydroxytryptamine  should  during  authors.  1982a;  Litosch  assumption  lost  show  from d i a c y l g l y c e r o l  recently  Walsh et  hand,  (i.e  a similar  A 2 on P I ,  to  in prostaglandin  the  ( B i l l a h and L a p e t i n a , et  half  necessarily  turnover  give  Only  acid  in  pancreas,  residues  metabolites.  used  not  mouse evidence  phosphatidic  acid  acid  PI  1980)  resulting  In  release.  in  does  PI  which w i l l  phosphate-prelabeled  c a n be  but  as  incorrect  arachidonic  have  lipase  and K o n d o ,  thereby  from a r a c h i d o n y l  in arachidonic  during  the  prelabeled  acid,  amylase  phospholipase  many t i s s u e s  On  1982)  recovered  from P I ,  obtained  phosphatidic  (Igarshi  in arachidonyl-prelabeled  the  derived  by d i a c y l g l y c e r o l  prostaglandin.  derived  cause  found  that  of  (1980,  can  stimulation  upon  arachidonic  prostaglandins  radioactivity  acted  t h y r o i d p l a s m a membrane  synthesis  breakdown  half  be  blowfly of  of  blowfly  arachidonyl gland  free  or  secretion  arachidonyl-prelabeled  conversion  to  diacylglycerol,  27  as than  phosphate-prelabeled by t h e  that  arachidonic  during  gland,  authors  responses, with 1.7  of  free  acid  and  was  such  not  as  being  added  INHIBITORS OF PI  specific  see  in solving PI  N,N-diphenylcarbamate  and  specific the  of  least  1980;  inhibitors  platelets, collagen production  and  a  et  of al.,  above)  "puzzle"  such  that  secretion,  but  with  is  the  al.  as  lack  (1980)  dansyl  due  of  the  to  arachidonic 1980,  they  a  found  fluoride,  the  a  for  acid  added  (Igarashi  in  i.e.  and  esterase  prostaglandin  on  PI-  diacylglycerol  production  stimuli  et  esterase  decrease  Serine  the  had no e f f e c t  Walenga  i n h i b i t i o n of  in  of  inhibited  serine  1982).  product  exogeneously  acid.  of  substrate  various  of  2-nitro-4-carboxyphenyl-  inhibited  metabolic  papain;  PI  effect  partly  presence  obtained  suprising  gland  arachidonic  this  source  (mentioned  i n the  salivary  C , w h i c h would r e s u l t  Marshall  malondialdehyde,  blowfly  5-hydroxytryptamine  Walenga e t  of  diacylglycerol,  and a p r o p o s e d  Kondo,  be  released  in  p-nitrophenylanthranilate,  that  phospholipase  level  lipase  at  possible  and not  turnover  the  (PMSF),  mobilization  suggested was  is  acid.  inhibitors, fluoride  inhibitor  the  turnover.  phenylmethanesulfonyl  (1980)  It  would not of  arachidonic  protease  stimulus-induced  PI  it  any  acid.  rather  RESPONSE  i n h i b i t o r of  serine  case  same e x t e n t ,  conserved  c a l c i u m u p t a k e or  One main h u r d l e  al.  by the  arachidonic  in that  could  exogeneously  that  decreased  5-hydroxytryptamine-stimulated  salivary the  release  PI  of in  thrombin,  malondialdehyde  arachidonic  acid  28  (Walenga  et  al.,  1980).  N-N-diphenylcarbamate stimulated  PI,  and  phospholipase contrast to  to  C  from  the  well  observe  any  as  findings, effect  in  2-nitro-4-carboxyphenylcollagen-  arachidonic formation  platelets  above  or  acid  of  from  et  PMSF  on  and acid  Pi-specific  al.,  Downes and M i c h e l l  of  PI  phosphatidic  i n h i b i t i o n of  (Walenga  papain-  1980). (1981)  In failed  polyphosphoinositide  erythrocytes.  MUSCARINIC CHOLINERGIC RECEPTORS Cholinergic  major  receptors  categories,  cholinergic  and  short  Adams,  stimulation increase  the  muscarinic in  muscarine  of  greater or  et  membrane  can  by  molecule  al.,  by ACh ( N e l s o  detail)  (latency  1980),  al.,  of  and  msec)  review  see  (nAChR)  nonselective in been  binding  site  because the  ionic  containing  nAChR  1980).  stimulated  acetyl-|3-methacholine,  <  nAChR has  (mAChR)(which be  inhibited  resulting  (ACh)  bilayers et  a  cations,  acetylcholine  receptor  are  receptor  response  The s i n g l e  (Raftery  acetylcholine  onset  two  Nicotinic  (30-100 m s e c ) ( f o r  monovalent  both the  into  muscarinic.  fast  this  to  artificial  regulated  classified  acetylcholine  about  ionic channel  be  a very  Nicotinic  contain  and  response  depolarization.  permeability could  of  brings  suggested to and  and have  in permeability  membrane  l o n g been  can be m i m i c k e d by n i c o t i n e ,  duration  1981).  have  nicotinic  responses  by d - t u b o c u r a r i n e  with  as  of  the  showed a d o s e - d e p e n d e n t  phosphodiesterase 1.8  inhibited  mobilization  phosphatidylcholine, from  B o t h PMSF and  In  will  contrast, be  specifically  inhibited  by  dealt with the  29  classical  muscarinic  are  slow  in  (0.5  sec)  (for  and  onset  the  review  slow  -  onset  see  >  100 msec)  Heilbronn  of  Muscarinic and  longer  and B a r t f a i ,  the m u s c a r i n i c  f o l l o w i n g two h y p o t h e s e s :  Kehoe and M a r t y complexes  that  to  muscarinic  somehow  messenger  increase efflux  iii) All  to play 1.8.1  of  of  responses  in  duration  1978;  Wastek  of  formation  large  by  model p r o p o s e d  by  of  ACh the  model  the  PI  receptor  channel  can  hypothesizes  formation  turnover),  of  an  which  is  opening. responses  receptors levels  of  usually  i n whole  accompany  cells:  cGMP  PI  in various  i n the m u s c a r i n i c  Guanosine  in  to  biochemical  of  responses,  a role  increases  channel  muscarinic  turnover  Stimulation  very  the  messenger  cGMP,  explained  several  opening  leads  is  potassium  above  Cyclic  the  second  in i n t r a c e l l u l a r  increased the  for  that  before  (i.e.  following three  stimulation  ii)  the  response  a subunit  postulates  activation  responsible  The  First,  interact  Second,  intracellular  the  (1980),  have  take p l a c e .  to  (latency  atropine.  Yamamura,1981). The  i)  antagonist,  have  stimulus-response  been  suggested  coupling.  Monophosphate muscarinic  of  cGMP  systems,  cGMP levels  increase  muscarinic  stimulation  Nirenberg,  1975;  receptors  (for see  Richelson  list  Heilbronn  (200-fold) in  a  of  tissues  and B a r t f a i ,  the  level  neuroblastoma  cells  et  in  i n many t i s s u e s  al.,  1978)  of  leads  showing 1978). cGMP  (Matsuzawa  makes  this  A on and an  30  appropriate  system  muscarinic response 1977)  has  (El-Fakahany added cGMP  still the  shown t o  30 sec  that  highly  basal  (see  also  1978)  and  the  response.  guanylate  cyclase  However,  activation  Study  et  this  al., is  the  in  (Wastek  exogeneously  et  some o t h e r on  al.,  result,  et  rather  1981) Though  evidence  "cGMP i n  in  negative receptor-  requirement  systems  Murad  min)  stimulation.  calcium  in various  cGMP  2-3  cGMP i n t h e m u s c a r i n i c the  this  temperature-dependent  section  1978;  the  with  of  (Richelson,  In t h e s e c e l l s ,  along  for  cell,  level  obtained with muscarinic  mediated  that  to  1980).  this  aspects  r a p i d and t r a n s i e n t  al.,  suggested a r o l e  1978;  be  hyperpolarization  debatable,  literature  various  neuroblastoma  and r e t u r n s  ( R i c h e l s o n et  produced  inotropy")  the  and R i c h e l s o n ,  to  indicates  In  been  calcium-  similar  characterize  response.  (peaks at  and  to  for  ( R i c h e l s o n et  al., than  1979)  al  clearly  the  cause,  of  some g u a n y l a t e  cyclase  has  calcium mobilization. Mostly also  been shown  (Garbers with a  et  in soluble  to  al.,  be  cyclase  cyclase in  (Limbird exception  and is  to  similar  broken  cell  Lefkowitz, rat  M carbachol  or  form,  present  1978;  few e x c e p t i o n s ,  guanylate  10." 9  present  liver 10"7  in  Sulakhe  et  the  particulate  al.,  1976).  show a d i r e c t to  that  of  1975;  et  Murad membrane,  M ACh c a u s e d  cGMP,  w h i c h was a t r o p i n e - s e n s i t i v e  Unlike  cGMP r e s p o n s e s  in intact  of  mAChR  hormone s e n s i t i v e have  plasma  A l l attempts,  coupling  preparations  a  been  al.,  2-4  unsuccessful 1979).  fold  the  et  to  adenylate  One  where s t i m u l a t i o n  (deBecemberg  cells,  fraction  increase al.,  increase  with in  1982). in  cGMP  31  levels very  with  the  narrow  various  range  agents  activation  of  of  the g u a n y l a t e  generated 1.8.2  al.,  of  caused  are:  an  fatty  Spies  in  cell  free  al.,  1975;  et  which  al.,  (Goldberg et  to  activators,  could  note  that  fatty  (Marshall  al.,  et  acids  al.,  fatty  acid  1976;  Hidaka Zwiller  least in  two o f  platelets  and M a j e r u s , al.,  et  1978;  at  be  proteases  1980),  al.,  the Other  (Glass  et  1981b;. P r e s c o t t  by  activators.  acids  et  muscarinic  increased  and  rat  In t i s s u e s  receptors  efflux 1968),  parotid such  as  of  potential  and c a u s i n g  K+  1980,  1983) 1982)  cells heart,  in tissues from  cardiac  r a p i d h y p e r p o l a r i z a t i o n , thereby  action  only  Ca2+-dependent  interesting  ( B u r g e n and S p e r o , 1982),  cGMP,  explained  cyclase  Of  and are  turnover.  Stimulation  al.,  readily  a  in  George  activity,  1978;  in pancreas  Efflux  among o t h e r s .  the  is  Potassium  fragments  not  1982;  unsaturated  1981a,  d u r i n g PI  with K+ caused  et  It  cyclase  prostaglandins  increase  and l y s o l e c i t h i n ( S h i e r  1976)..  et  are  and p r o s t a g l a n d i n s  1977)  (Lapetina  al•,  over  concentration.  by c h o l i n e e s t e r s  enzyme  al.,  ACh  an  guanylate  1980), et  and  et  important,  al.,  hydroperoxides  al. ,  1974)  membrane was o b s e r v e d  cause  cyclase  this  Garbers  and A s a n o ,  can  other  physiologically  1977;  carbachol  which  of  (Lacombe e t  plasma  (deBecemberg  regulators  et  of  and M o n t a g u e ,  presence  liver  guanylate  preparations Howell  rat  preloaded  smooth  muscle  muscle c e l l s  (Galper  (Putney et enhanced  al.,  1980),  K+  efflux  r e d u c i n g the d u r a t i o n of  inhibition  of  the  slow  inward  32  calcium current for  K+  efflux  stimulation not  in  alters  considered  efflux  (Ten E i c k  in tissues  mobilization, 1.8.3  is  the K+  to  1976).  muscarinic  permeability  be a p a r t  of  not  (Hokin  of  and  t h e most 1975, diverse and  and t h e  tissues  Michell,  Trifaro,  1953).  widely studied  1979),  1969a,  response.  (as  the  present  of  guinea  calcium  the  receptor  that  stimulation  synaptosomes  stimulation whereas  produced  calcium  one  and PI  following  pigeon  pancreas  thing  PI  al.,  only  (Jafferji  not  i n many Lapetina  al.,  and  list  Michell,  see PI  1958;  Michell,  turnover. until  the  i n common; t h e y a r e  The  correlation so  good  intrinsic  1976).  that  to  since  (Griffin considered  et  all  between it  led  muscarinic  Presynaptic  exception,  response  m o b i l i z a t i o n was  1969; et  are  (Michell,  receptor  (Hokin  r e s p o n s e was  et  receptors  investigated  breakdown may be  the a  K+  made  w i t h enhanced  responses  least  (Michell  was  of  calcium  response  a comprehensive  mobilization.  PI  was  (Hokin,  ileum  be a s s o c i a t e d  stimulation  proposal  pig  (for  the m u s c a r i n i c  by  still  Response  muscarinic  1969b),  at  is  mediates  in  PI  medulla  have  muscarinic  of  to  adrenal  to  role  function  Muscarinic cholinergic  1974),  study  mediated  receptors  cortex  found  the  turnover  cerebral  are  a  How m u s c a r i n i c  channel  And PI  s u c h as  1975),  all  PI  stimulation  among many o t h e r s  K+  stimulation  with respect  1976a),  Almost  on  muscarinic  Hokin,  suggests  clear.  observation the  This  t h e mAChR) o r  where m u s c a r i n i c still  first  stimulation  of  al.,  M u s c a r i n i c A c e t y l c h o l i n e Receptor The  to  et  mAChR  muscarinic al.,  1979),  to mediate  the  33  receptor  response.  "mechanism i.e.  of  femoral  examples  negative artery  where  mobilization. al.,  1976)  The mAChR  (DeMey  receptor  test  accompanying  whether  it  accompanies  stimulations  which mediate  study  where  the  the  final  mobilization. Various  the  same  The c o n c e n t r a t i o n PI  response  of  i n most  higher  response  (Jafferji the  physiological observed  that  muscle  of  tissues  response  pattern  that  stimulation  for  (while  ileum,  carbachol. the  as  for  be  in d e t a i l  of was  several  orders  of  the et  physiological al., to  1976).  be of  Michell  response,  similar  of  of  spare  to spare  the  obtained smooth  receptor  receptors  receptors  no  (1976a)  mAChR i n l o n g i t u d i n a l  The l a c k  presence  receptors. the  and PI  the  produce  i n i t i a l l y considered  of  in  to  stimulation  other  1976a; M i c h e l l  curve  calcium  to  produce  Jafferji  tissue  response  mAChR  the  a  thesis.  required  to to  or  receptor  in  in  discusssed  appeared  dose-response  pig  turnover  agonist  required  unknown,  through  in t h i s  accompanying  and M i c h e l l ,  guinea curve  PI  intrinsic  muscarinic  mediated  been  response  r e s p o n s e was  the  carbachol  PI  of  e_t  by c a l c i u m m o b i l i z a t i o n ,  presented  significance.  during  occupation  is  calcium  (Michell  is  stimulation  not  cholinergic  than  PI  effect  is  have  general  magnitude  Therefore,  effect  PI  those  other  to  an  on  muscle  are  lead  is  function  muscarinic  receptors  sections.  follows  of  their  Such a s t u d y  of  whose  smooth  breakdown  stimulation  only  characteristics  stimulation earlier  effect  PI  section  1980)  does not  whether  receptor  the  Vanhoutte,  mAChR  of  to  and  (see  and v a s c u l a r  stimulation  One way t o  the  heart  inotropy")  characteristic  is  in  has  for been  34  shown et  for  al.,  from  the  1979),  receptor  may l i e  It are  response  suggested  occupation  closer  1976a). there  physiological  to  was  the  to  reported  receptors  response  physiological  al.,  1.8.4  (Birdsall, subtypes  (Ehlert  of  (Birdsall,  et  (Jafferji  and  in  leading  breakdown Michell,  neutrophils,  concentration  where  lysosomal  for  similar  the  PI  (Cockcroft  Of M u s c a r i n i c A c e t y l c h o l i n e  to  Strange  same et  al.,  muscarinic  receptors  the is  binding sites  1980; In  except  on  the  binding sites  exhibit  a  receptors  and  explained  having d i f f e r e n t  the  Rossini, solutions bind  to  membrane cells  have  been  similar  "heterogeneity" (Burgen  antagonist by  multiple  under  Yamamura and S n y d e r ,  agonist  single  in p i t u i t a r y  By c o n t r a s t ,  of  or  antagonists  sites  antagonist  1980).  1977;  1977)  been p r o p o s e d .  1977),  agonists  either  al.,  muscarinic  population  between  of  have  al.,  et  receptor  for  used,  W e l l s et Hammer  al.,  al.,  muscarinic the  1983;  of  sites et  criterion  strength,  W e l l s et  (Mukherjee  the  receptors  difference  classes  PI  r e s p o n s e were  1982;  population 1977;  conditions  of  al.,  ionic  two a f f i n i t y  reported  1974;  response,  fMet-Leu-Phe-induced  maximal  Venter,  muscarinic  uniform  binding  nature  1977;  physiological  where  events  ?  B a s e d on t h e  a  (Yoshida  1981) .  Receptors  of  half  Are There Subpopulations  1981)  c h a i n of  that  for  the  et  i n the  occupation  enzyme s e c r e t i o n , and t h e  contraction  physiological  receptor  recently  no s p a r e  that  i.e.  et  1974). binding  p r e s e n c e of  affinity  (super  in  al. , This to three high,  35  high  and low)  (Birdsall for  et  agonist  for  al.,  agonist 1980).  may not  electrophoresis  indicate  the  presence  from f i v e  others  postulated  that  sites  represent  the  same  et  of  different  receptor  somehow a l t e r  the  population  of  receptors  (Wei  Sulakhe,  of  and  the  cGMP  in  brain  to  1979;  a  of  et  and n e u r o b l a s t o m a  the  with cells  (Birdsall  however,  was  associated  sites  i n guinea  1.8.5  Subclassification Three  ileum  from The  IC50  factor  of  30  for or  i n the  showing a homogeneous  (Birdsall, cyclase PI  and M i c h e l l ,  muscarinic  pirenzepine  propylbenzilylcholine by a  of  binding the  1977), activity  response,  population  studies  pirenzepine  of  binding  1976a).  receptors  subclasses,  of  Binding  N-methyl scopolamine three  form  Occupation  b o t h h i g h and low a f f i n i t y  (Jafferji  of may  formation  1980b).  Based On A n t a g o n i s t  subclasses  suggested, 1980).  pig  to  al.,  states  affinity  the  in  et  binding  heterogeneous  1980).  GTP-stimulated adenylate  homogenates  agonist  low  al.,  have  nucleotides  and w i t h i n h i b i t i o n of cardiac  1982)  coupling  they convert  Rosenberger  analysis  Yamamura and  al.,  Guanine  form was c o r r e l a t e d  slices  et  predominantly  as  w i t h 80K m o l e c u l a r  affinities  as  states  macromolecules,  1983).  e n v i r o n m e n t a l or  environment,  low a f f i n i t y  Ehlert  macromolecule.  antagonist  inactivation  (Venter,  1980;  the  affinity  polypeptide  tissues  different  for  different  radiation  a single  al.,  the  affinity different  three  and  different  (Birdsall  equal  The t h r e e  represent  SDS-gel  weight  but  have (Hammer e t  inhibition binding  with a t r i a  low a f f i n i t y  been al., of  differed  and  receptors.  ileum  36  In IC  5 0  the  contrast,  of 5 x l 0 ~  B  presence  cerebral  M and 1.1x10"  muscarinic  exposure  canine  (QNB)  (0.1-1 binding  affinity Tris  .  apparent  mM)  r e s p e c t i v e l y , and  receptors  atrial  while  in their  absence.  was  type  reported  1982).  The  (10-100 mM)  or  in quinuclidinylbenzilate s i t e s maintained  to conclude in  showed  Another  (Sastre et a l . ,  the remaining  heterogeneity  had an  also  sites.  homogenates t o T r i s  l e d the authors a  binding  c a u s e d a 45% l o s s  sites,  This  unmasked  M,  and s u b l i n g u a l g l a n d  b a s e d on c h o l i n e / T r i s s e n s i t i v i t y  in myocardial  choline  7  of m u l t i p l e a f f i n i t y  of h e t e r o g e n e i t y ,  of  cortex  QNB  that  binding,  choline  their and  w h i c h was n o t  37  Three detected studies were  subpopulations in  cultured  (Galper  rapidly  insensitive second  lost  a  during  exposure  to  The  to c a r b a c h o l .  between  three  classified three  based  different  of  as  the  on  affinity  sites  for  of  is  the  which  lost  even  any  on  receptors after  relationship  muscarinic  receptors,  desensitization  agonist  A  desensitization  there  agonist-induced  and were  colchicine.  w h i c h were not  of  sites  receptors,  (30%)  subpopulations  been  receptor  with carbachol  of  Whether  have  desensitization  the  such  rest  population,  3 hour exposure these  44%  on  colchicine-sensitive  carbachol.  "stable"  26%  inhibitors of  receptors  based  incubation  consisted  slower,  cells  1980).  to m i c r o t u b u l e  underwent  muscarinic  heart  and S m i t h ,  subclass  comprised a  of  binding is  and  the  still  not  known. 1.8.6  Receptor-Effector Just  as  adrenergic, (e.g  receptor  can  receptors  adenylate be  in  heart  ionic of the  have  the m u s c a r i n i c effector  to  i n the  to  different  system  and  1982)  coupled  c o u p l i n g to a  to  that  effectors  Muscarinic  Weight  neurones et  al.,  types  of  subclassification  b a s e d on t h e  the  single  now b e i n g  two d i f f e r e n t  Possible  /3single  a  is  1981).  1981;  be c o u p l e d t o  can be made,  (e.g.  and s y m p a t h e t i c  Dodd,  same t i s s u e .  receptor  of  El-Fakahany,  Horn  receptors  the p o s s i b i l i t y  (Hartzell,  1980;  of  capable  cyclase)  and  been p r o p o s e d  channels  types  are  coupled  (Richelson  (Brown and Adams, 1979)  different  H2_histaminic)  effector  recognized  many  Coupling  receptors.  nature A  of large  38  proportion mediate  of  mAChR's b e l o n g  their  p r o d u c e a PI 1975,  response  response  1979).  proportion influx,  of  e.g  inhibition  of  that  Recently  but  has  (Murad e t the  al.,  level  1973).  activity  coupled  proposed  its  of  to  there  mAChR was is  system,  it  of is  adenylate  (Michell  al.,  1981).  have  of  and  thereby 1980;  suggested calcium  to  this  cyclase  decreasing  George  et  a1.,  affinity  and a d e n y l a t e  GTP  et  al.,  et  al.  cGMP. system,  Jones of  mAChR. PI  One  turnover  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 to mediate Unlike  known whether t h e is  of  cyclase  (Watanabe  t o be c o u p l e d t o  cyclase.  the  mAChR  adenylate  may be two p o p u l a t i o n s  cyclase  calcium  following calcium  both the  c o n v e r s i o n to  suggested  not  inhibiting et  of  of  belonging  1978),  cyclase  effects  smaller,  inhibit  step  may r e g u l a t e  suggested  adenylate still  not  and P a p p a n o ,  mediated  s e c o n d p o p u l a t i o n was inhibition  may  inhibition al.,  much  in heart  al.,(1979)  also  Michell,  presynaptic  the a l p h a - a d r e n e r g i c  that  response  by  receptors  adenylate  through i t s  analogy  population and  to  effect  et  some  (Beigon  by m o d u l a t i n g t h e  possibly  By (1982)  cAMP  which  (see  inhibition  phosphorylation  Cholinergic agonists  receptors  1978),  of  by  release  Watanabe e t  receptors  second,  inotropic  shown t o c a u s e  1962;  a  mediated  S t i m u l a t i o n of  been  of  stimulation  a  of  stimulation  Michelson  mAChR  rather  mobilization. class  are  negative  presynaptic  uptake,  receptor  transmitter  stimulation.  family  c a l c i u m m o b i l i z a t i o n and  responses  mAChRs the  a  through  on  The  to  its  response  the  PI  through  alpha-adrenergic  population  c o u p l e d to  the  of  mAChR  t u r n o v e r or  not  39  1.9  MECHANISM OF THE NEGATIVE INOTROPIC EFFECT Ligand  all  regions  In  rat  higher  binding studies of  and than  showed  the  rabbit, that  of  almost  observed  various  species  for  review  ventricle 1982) was  between  atria  observed two  i n the  (Furchgott see  et  Higgins  et  variable.The  and v e n t r i c l e  variation  i n the  is  guinea  these  two  al.,  1973),  1979;  pig  (Wei and  isolated Ravens  but  is  atrium  and  the  dog  force  and  (Ingerbretsen  et  of  Ziegler,  decrease  in  Sperelakis, al.,  1980)  mAChR  density  difference  in  the  not  to  account  response  and  regions  Josephson  enough  two-fold  for  t o mAChR s t i m u l a t i o n  the  in  these  in  basal  regions. The  direct  contractility)  negative  observed  sometimes v e n t r i c l e ) ,  inotropic  was  attributed  inward c a l c i u m c u r r e n t  and  Ziegler,  1980;  Ten E i c k  et  calcium  current  potential higher  c a u s e an  (Beigon  by  of  to  the  al.,  1976).  effect  force of  Ten E i c k  was  to  (and  of  of  the  et the  suggested  of  contraction slow  inward  the  action  of al.,  above to  the  Ravens  Concentrations  duration  ACh, in a d d i t i o n  mechanism a d i r e c t  atrium  inhibition  i n the  the 1980;  of  and F u r c h g o t t , 1 9 6 4 ;  inhibition  decreasing  and P a p p a n o ,  concentrations  indirect  indirect  (decrease  stimulation  (Grossman  ACh w h i c h p r o d u c e d a 30-40% d e c r e a s e shown t o  effect  on m u s c a r i n i c  slow  were  1960;  in  Sulakhe,1978).  contractile  mAChR i n  al.,  mAChRs  a t r i u m was  while  of  preparations  of  Wei and  i n the  basal  and Motomura,  and whole h e a r t and  in  upon s t i m u l a t i o n  (Endoh  small  density  density  presence  1980;  the v e n t r i c l e ,  A decrease  usually  the  (Hartzell,  mAChr  equal  Sulakhe,1978).  1980;  heart  suggest  be  1976).  At  mentioned involved  40  in  i n h i b i t i n g the  1976).  responsible  ACh,  between  et  because  al.,  of  the  al.,  i.e  1976;  et  Lee  in  includes  former a  the  inotropic and  was  action  effect  for  for  level  of  1973),  the  al.,  can  not  observed  and  the  1960;  Ten  negative  without producing a to c o n t r o l  for  et  i n the  level  al.,  1973;  et see  levels  but  the  cGMP  negative  1973).  inotropic  beating  atria  may  i n guinea  pig  mediate  its  the  kinase  both  an  level  of  inotropic  effect  correlation  evidence  effect  which  inotropy  of  8-bromo  (Nawrath,  1976),  d i b u t y r y l cGMP t o a n t a g o n i z e  cause  Severson,  i n the  Other  cyclic  Nawrath,  in ACh-mediated negative  dependent  cGMP  of  produced  showed a much b e t t e r  al.,  negative  Drummond and  cGMP and a d e c r e a s e  isoproterenol  to  ACh-mediated  ACh s t i m u l a t i o n  cGMP-dependent p r o t e i n  found  neutralize  the  review  stimulation  1975).  of  et  correlation  al.,  potential  (George  heart,  of of  good  et  alterations  spontaneously  ability  Besch,  activation which  the  - a concentration or  to  effect  potential  with epinephrine  rat  (George  role  very  (Furchgott  possible  1972;  al.,  ACh  cGMP i n p a c e d and  al.,  et  accompanying  suggested  of  a  action  ACh a l o n e  ACh  potential  inotropic  was  the  cAMP and cGMP  the  (George  the  of  was  action  negative  there  include  In p e r f u s e d  increase  the  (Ten E i c k  1960).  effects  nucleotides  with  of  of  p o s s i b l e mechanisms  inotropic  1979).  it  duration  et  Other  with  1976),  effect  (Furchgott  cAMP  the  while  inotropy  inotropic return  for  decreased duration  negative Eick  inward c a l c i u m c u r r e n t  The d e c r e a s e d d u r a t i o n  be s o l e l y with  slow  the  heart  positive (Watanabe  effect  (Gill  phosphorylation  et  al., of  through 1977), cardiac  41  troponin-1 protein  (Blumenthal  present  functional they  rat  under  (Blumenthal  et  al.,  1978;  certain  observed  (Diamond  concentrations  decrease al.,  in  et of  70K  (Wrenn  and  Kuo,  conditions  is  endogenous 1981).  The  and whether  still  Drummond and S e v e r s o n ,  1977).  guinea  (Endoh could  and  a  the  not  1979;  similar  in  both  clear  Wrenn and  was  atrial  any change  increase  also 1979).  tissue i n cGMP  in  (Diamond  and ACh  and B e s c h ,  1975)  was  Recent  and only  inotropic  with  dibutyryl  force  elevated  effect explain increasd  ventricle, in  not  observations  Sodium n i t r o p r u s s i d e  obtained  with  1977)  a negative  tension  a  al.,  tension.  atrium  At  showed  t h a n one mechanism may h e l p t o  canine  been  i n cGMP l e v e l s  (Diamond e t  that  has  the  but  was  the  atria.  A  cGMP  (Endoh and  1981).  stimulation  tension  in  between  and B r o o k e r ,  cat  (Watanabe  1981)  by a d e c r e a s e  When c o n t r a c t i l e  treatment  hand, atria  inotropy  Linden  iM),  above d i s c r e p a n c i e s .  result  Yamashita,  in cat  by more  dissociation  without  other  Yamashita,  levels  accompanied  ACh ( 0 . 0 5  decrease  be p r o d u c e d  some of  1977;  ventricles  by  a  negative  al.,  On t h e  pig  accompanied  and  twitch tension  sodium n i t r o p r u s s i d e  cGMP  a  conditions,  i n cGMP l e v e l  in  and  these phosphorylations  physiological  elevation  et  of  1978)  1981). Under  low  al., heart  significance  occur  Kuo,  i n the  et  or  was  isoproterenol  w i t h ACh p r o d u c e d a than  the  control  ("accentuated antagonism")  by  sympathetic  administration,  greater in  first  both  (Dempsey  fractional left  atria  and C o o p e r ,  subsequent decrease and  1969;  in  ventricle Levy  and  42  Zieske,  1969;  1975). in  Schwegler  The g r e a t e r  the  presence  negative of  catecholamine  was  and  1969).  Zieske,  observed such  only  as  agonists 1966;  referred As  responses  or  Most  its  of  t h r o u g h cAMP Rinaldi  et  protein,  (for  stimulated cause  (Josephson of  by  available  are  of  results  of  (Levy  w i t h ACh was  mediated  cAMP  ACh  level  antagonism"  by  cAMP,  but  not  with  (Levy  et  al.,  suggested  interfering  of  have  t h a t ACh  with  Drummond recently  leads  cAMP  increase  to  the  and S p e r a l a k i s ,  adenylate  on  GTP-dependent  receptor/adenylate  cardiac  by kinase  which  inward  thereby  system  al.,  calcium  about  et  current the  slow  level  al.,  of  inward  ACh-mediated  by  component  (Watanabe  1978)  the a c t i v a t i o n  r e d u c i n g the  brought  uptake.  isoproterenol-  (Keely et  may i n h i b i t  regulatory  1979).  sarcolemmal  A C h , by d e c r e a s i n g  was  the  mediated  calcium  membrane p r e p a r a t i o n s ,  cyclase  cyclase  are  Severson,  increased  slow  cAMP,  In c a r d i a c  of  an  1982).  protein kinase,  inhibition  weight  protein  in  and  that  shown t h a t c A M P - d e p e n d e n t  mechanism  cAMP-dependent  suggests  inotropic effects  a 23K m o l e c u l a r  a possible  calcium current.  evidence  positive  isoproterenol-stimulated  the  increased  H2 agonists,  independent  antagonism  (1981)  cAMP-dependent  responses  the  calciductin,  an  or  antagonism  and h i s t a m i n e are  obtained with  "accentuated  accentuated  a r e v i e w see  al.  may s u g g e s t  Watanabe and B e s c h ,  response. the  phosphorylation  tone  1975),  catecholamine-stimulated  may  t o as  Watanabe and B e s c h , accentuated  1976;  inotropic effect  w i t h a g e n t s whose  whose  generation  Jacob,  sympathetic  catecholamines  produced  This  and  an of  effect the  1978).  /3-  43  Other  proposed  inotropy calcium efflux 1.10  include: (Grossman  mechanisms a)  a  decrease  and F u r c h g o t t ,  (Prolopezuk  et  al.,  present  i n the  Brodde,  hearts  1978;  of  rat,  Schumann,  1980;  specificity  1982). be  Alpha,  exchangeable  increased  adrenoceptors list  of  detected  has  a  than phentolamine rabbit  Lefkowitz,  calcium  receptors  being  presence  of  sites  with a  a  Marinetii, binding  1977)  of  i n the post the  in d i s p l a c i n g and  cardiac labeled  a2  1980).  - a,  and  a, 2  (Exton,  considered membrane  postsynaptic  (McGrath,  receptors  1982; have  a  2  for been  1978)  20 t i m e s  less  potent  Brodde,  1979)  and r a t  suggesting  postsynaptic.  In  ( W i l l i a m s and  that  the  rat  alpha-  heart,  the  class  of  [3H]dihydroergocryptine binding  density  of  41  and 307  100 et  was  [3H]dihydroergocryptine-binding  homogenates, are  single  (Karliner  Benfey,  binding  of  be  (Wagner  and p r e s y n a p t i c  presence  to  and c a t  were p r e v i o u s l y  b l o c k i n g agent,  2  and  see  antagonist  postsynaptic  a  receptor  Lefkowitz,  review  shown  1982).  (Schumann 1978)  rabbit  shown i n many t i s s u e s  where  Exton,  Yohimbine,  now  been  tissues see  for  been  d i v i d e d i n t o two c a t e g o r i e s  located  but  now  pig,  and a 2 _ a d r e n o c e p t o r s  specifically  respectively,  and  p o o l of  and b)  have  guinea  b a s e d m a i n l y on t h e  in  1964)  receptors  are  a  i n the  negative  1981).  Alpha-adrenoceptors  to  ACh-mediated  ALPHA-ADRENERGIC RECEPTORS IN HEART Alpha-adrenergic  and  for  fmole/mg  fmole/mg of  fmole/mg p r o t e i n  al.,  1982)  has  protein  protein  (Ciaraldi  b a s e d on  been  (Williams  reported,  and  [3H]prazosin suggesting  44  the presence  of  a homogeneous  On t h e o t h e r h a n d ,  in  response  with  obtained  (Skomedal et a l . , guinea  pig  presence  of  Further,  a  single  (an a ,  et  class  stimulatory adrenoceptors  to  the  inhibitory  (Skomedal  inhibition  as  et  cyclase  interact or  interfere  stimulatory (Levitzki,  well  as  receptors) isolated  stimulation propranolol  the  considered to  catalytic  with  the  A decrease  i n the l e v e l  alpha1982). mediated  suggested to  protein, subunit  of  occur  which  of  between  cAMP  myocytes  and  Watanabe  perfused  rat  et  heart,  w i t h phenylephrine or a d r e n a l i n e beta al.,  blocker) 1977).  a c c o m p a n i e d by a n e g a t i v e  caused  on  effect.  rat  the  cyclase cardiac  (1977).  a  2  In  alpha-receptor  i n the presence  a decrease  In perfused  inotropic  al.  may  adenylate  adenylate  of  be  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  been r e p o r t e d by  cAMP (Watanabe e t  may  be  interaction  1982).  (a  there  and K a n n o ,  This is  rat  ( H a t t o r i and  of p o s t s y n a p t i c  Hattori  the  (both  by p h e n t o l a m i n e i n  populations  subunits  rat  blockade  that  cyclase.  and  i n o t r o p i c phase  suggestion  catalytic  has  1978)  receptors.  i n guinea p i g  1980;  muscle  argues against  complete  and  alpha-adrenergic  papillary  the p o s i t i v e  responses are  with  multiphasic  et a l . ,  by a c t i v a t i o n o f an i n h i b i t o r y - G T P b i n d i n g directly  a  alpha-adrenergic  and a  al•,  of a d e n y l a t e  rat  1981)  i n o t r o p i c response)  Alpha2-adrenergic by  of  (Osnes,  b l o c k a d e of  1980),  led  and  of  antagonist),  al.,  1982),  atria  alpha-adrenoceptors.  studies  ( H a t t o r i and K a n n o ,  p o s i t i v e and n e g a t i v e (Skomedal  stimulation rat  the s e l e c t i v e  by p r a z o s i n  Kanno,  pharmacological  1980),  atria  p o p u l a t i o n of  i n the l e v e l heart  this  of of was  45  A  monophasic  positive  transient  inhibitory  Osnes  al.,  et  receptors et  al.,  1980)  Brodde,  greatest  (Hattori  has  left  atria  Wenzel  (Shibata Su,  (Shibata  rat  et  left  al.,  Positive  Inotropic  Many d i f f e r e n t alpha  and  lines  1975;  and Y a m a s h i t a ,  inotropic  1979).  effects  Robinson  et  time  t o peak  al.,  1975;  contrast,  peak  tension  Among  and M c N e i l l , (Skomedal  workers  Shibata  Wagner  and  the m y o c a r d i a l  atria  showed  the to  Receptor,  1980;  clearly  induced p o s i t i v e  Skomedal e t  al.,  levels  tissues  of  cAMP  of  M a r t i n e z and M c N e i l l , relaxation  1975;  Skomedal  neither  1977),  by an  al.,  1982).  agree  that  the  Wagner  heart al.,  1977), time al.,  increase  al.,  positive  positive  nor by a d e c r e a s e  et  et  et  et  1982;  the  (Kelly  that  inotropic  (Endoh  /3-adrenergic-st imulated  various  al.,  indicate  mechanisms  and a s h o r t e n e d  accompanied  alpha  i n o t r o p i c response  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  tension Most  1965;  R a b i n o w i t z et  was  (Martinez  al.,  1977;  1980;  right  evidence  The in  al.,  positive  by d i f f e r e n t  a c c o m p a n i e d by i n c r e a s e d  effect  and  receptor  mediated  Schumann,  on s t i m u l a t i o n o f  And B e t a - A d r e n e r g i c  of  responses are  and  et  1982;  Effect  beta-adrenergic  Endoh  1981,  the  1980).  1.10.1 M e c h a n i s m Of The A l p h a mediated  Kanno,  1966).  phentolamine-sensitive  methoxamine  and  d e v o i d of  ( M a r t i n e z and M c N e i l l ,  and  studied,  response  been o b s e r v e d  and v e n t r i c l e s  1978;  preparations  effect  1978)  in rat  inotropic  1978;  decreased (Ledda  e_t  1982).  In  inotropic  i n cAMP  i n the  are  levels  time  to  alpha-adrenoceptor-stimulated  46  positive  inotropic  mobilization  effect  of c a l c i u m  Ledda e t a l . , 1980; calcium both  in  this  tissues  in  mediation and  1982).  the  t o D-600 response,  alpha  alpha  response  Endoh  transmembrane  enhanced  by  influx  calcium, carbachol,  and  were  able  cardiac al.  influx.  of c a l c i u m , of  the  to  (1978)  found  that  muscle,  alpha-adrenoceptor  calcium  current,  hypothesis. challenged  Some the  of  were  (Karliner  shown t o compete et a l . ,  1982).  by  the  of that  also  favoured  an  gradient  dinitrophenol,  (1981) and  for  Miura  rabbit  increased the  of  with  of p h e n y l e p h r i n e  +  e_t  papillary the  inward  Endoh e t a l .  (1975)  observations  have  recent hypothesis.  the  Verapamil  only  block  f o r the  alpha  receptor  may  explain  This  beta-  were  the  with  K -depolarized  this  the  action potential  affinity  more  more  increasing  conclusions  of c a l c i u m  supporting  the  sensitivity  of  methoxy d e r i v a t i v e (D-600) not but  than  effect  stimulation  of  was  plateau  in  the  validity  its  Inui et a l .  thereby  response  e l e v a t i o n of  alpha-adrenoceptors.  in various  l e d them t o c o n c l u d e  These  the  been  that  s u c h as  increase  general,  found  that c o n d i t i o n s which  increased efflux  of  have  responses  temperature  exerted  In  pools  antagonist)  above s t u d y  observation  shortening  all  Ca  1981;  source  debated.  inotropic  greater  mainly  calcium the  the  the  (1975)  channel  enhanced  I n u i et a l . ,  but  being  et a l .  positive  i n the  stimulation  supported  is s t i l l  (a c a l c i u m and  through  1978),  extracellular  alpha-receptor-mediated  receptor  et a l . ,  alpha-adrenergic-mediated  (Exton,  sensitive  mediated  (Handa e t a l . , 1982;  Miura  mitochondrial  implicated  is  calcium  and  channel  binding the  its  sites higher  47  sensitivity Endoh's pig  of  studies  slow  contractile  inward  depolarized rise  of  than  calcium  methoxamine  current  (Ledda  potential  was u s e d  of  the  the  supressing  the  the a l p h a beta  unaltered  increased  positive  involved,  what  its  of  Alpha  current  system, is  rate  of  the  inward  potential  could s t i l l  is  and  al.,  even  with i n an  compared  if  by  et  result  known about  produced,  smaller  probably  (Handa  In c o n c l u s i o n ,  little  K+-  stimulation  potential,  action  a  In  r e s p o n s e was much  1982).  the  calcium.  inotropy  maximum  a measure of  action  doseby  1980).  outward K+ c u r r e n t  calcium  source  as  produced a  al.,  in  guinea  accompanied  when t h e  al.,  the  duration  m o b i l i z a t i o n of  beta-adrenergic  mediated  of  time-dependent  inward  et  stimulated  (Handa e t  duration  The i n c r e a s e d  In K + - d e p o l a r i z e d  r e s p o n s e w h i c h was not  the a c t i o n  that  t o D-600 o b s e r v e d  stimulation  muscle,  increased  1.11  1975).  papillary  current,  1982).  al.,  responses  rabbit  calcium  the  (Endoh e t  v e n t r i c u l a r muscle,  dependent  an  alpha-adrenoceptor  to  how a l p h a calcium  is  is.  MUSCARINIC RECEPTORS IN LONGITUDINAL SMOOTH MUSCLE OF  GUINEA PIG ILEUM In  l o n g i t u d i n a l smooth m u s c l e  [ 3 H]QNB be  190  binding studies, fmol/mg t i s s u e  receptor  muscarinic  receptors  pig  ileum produced a b i p h a s i c component and a  Triggle,  1973;  guinea  density  of  i l e u m , b a s e d on  been e s t i m a t e d  1974).  contraction, tonic  consisting component  and R o u f o g a l i s ,  1981).  to  Stimulation  l o n g i t u d i n a l smooth m u s c l e  sustained  James-Kracke  pig  has  (Yamamura and S n y d e r ,  of  phasic  of  of of  guinea a  (Chang  rapid and  Both phases  48  of  contraction  pools  of  is  to  have  a  stimulation enhanced  suggested enhanced pig  i l e u m has  receptor  as  only  curve was  been  10% of  for  the  al.,  (Jafferji  to  of  shown t o  to  between  receptor  receptors  On  may o c c u r  role  calcium 1976a).  Honeyman,  1980)  the  step  al.,  of  evidence  are  Chang and  and  Triggle  free  calcium  contraction, in  the  observe  that  to  was  into  to  be  phosphate for This that  receptor  et  into  PI  occupancy additional  chain  of  events  response,  PI  and may p l a y Jafferji  cells  suggest  the  dose-response  provided  smooth m u s c l e  by  contraction  receptor  1975;  with  1979),  incorporation  occupation  an  guinea  al.,  physiological  to  also  of  occupied  the  i n the  (Michell,  and  agonists  a maximal  contrary,  provided  PI,  produced  r e s p o n s e may be  Yoshida  produce  and t h e  cultured  ileum  smooth m u s c l e  required  to  to  to muscarinic  1976a).  mobilization In  phases  used  pig  this  1975;  are  hypothesis  nearer  first  phosphate  the  stimulation  breakdown  Michell,  the  dose-response curve  support  of is  i n guinea  respond  et  Michell,  evidence  pool  Longitudinal  1979).  and  were  receptors  PI.  Chang  phase  the  1980;  extracellular  tonic  but  contraction.  oxotremorine  the  of  calcium  carbachol-stimulated  similar  in  a pool  (1976a)  (Taylor  agonist  et  1983).  al.,  primary  of  reserve  muscarinic (Yoshida  the  two  et  incorporation  breakdown  a  phasic  these  and S n e d d o n ,  bound  muscarinic  that  during  sustained  and M i c h e l l  of  calcium mobilization,  (Brading  that  w i t h the  and r a p i d  Jafferji  an  Rangachari  superficially  transient  of  mobilized  suggested  associated  while  result  be d i f f e r e n t  1973;  (1973)  the  calcium  considered Triggle,  are  that  (Salmon the  a  and and time  49  course  of  stimulated calcium Michell also  formation PI  turnover  of was  mobilization. (1976b)  produces  enhanced inhibition Rosenberger  phosphatidic  reported  rapid  In  a later  that  of  contraction  and T r i g g l e  K+-stimulation  of  mobilize  biphasic  [32P]phosphate by  various  (1979)  proposed  the  same p o o l  to  during play  communication,  K+-induced  a calcium-mediated  incorporation  enough  acid  of  carbachola  role  in  Jafferji  and  depolarization, contraction, into  PI.  calcium that  which  showed  B a s e d on  the  antagonists,  both c a r b a c h o l  calcium.  an  and  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  following  two  hypotheses:  a)  PI  breakdown  that  is  thesis  intrinsic  to  was  to  test  muscarinic  the  receptor  stimulation.  b)  that  the  PI  response  is  involved  in calcium m o b i l i z a t i o n .  These o b j e c t i v e s  were s o u g h t  1)  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  PI  response  2)  the  effect  of  in rat  primary  guinea  pig  3) t h e  effect  by s t u d y i n g : stimulation  on  heart. event  of  c a r b a c h o l - s t i m u l a t e d PI  response  in  ileum. of  the p u t a t i v e  on c a r b a c h o l - s t i m u l a t e d PI  phospholipase  response  C  inhibitor,  and c o n t r a c t i o n .  PMSF,  51  II.  2.1  C h o i c e Of Among  to  Sulakhe,  Animal  the  contain  METHODS  small  the  laboratory  largest  1978)  and  number  alpha  animals, of  both  adrenergic  (Schumann,  1980).  Pharmacological  stimulation  of  these  characterized et  al. ,  Ten E i c k guinea  both  i n the  1978; et  Ravens  al.,  pig  rat  and Z i e g l e r ,  1976).  i l e u m the  on c a l c i u m m o b i l i z a t i o n and  Triggle,  1979)  and M i c h e l l ,  1976a)  rather  than  male  results  with  Male W i s t a r were of  tissue.  heparin  in  min b e f o r e  has  guinea  In  pigs  Jafferji  in ether  the case  of  normal s a l i n e the  been  (300-350 g)  anaesthetized  rats  have  also  1980;  were  were u s e d  saturated  rats, was  0.4  anaesthetized.  Osnes  al.,  1980;  of  the  stimulation Rosenberger (Jafferji  reported.  Female,  t o compare  the  (1976a).  tank  injected  well  turnover  guinea  ml  et  muscle  in order  and M i c h e l l  and f e m a l e  1982;  1973;  previously  atria  following  receptor  Triggle,  and  been q u i t e  Skomedal smooth  shown  (Wei  in t h e i r  and S p e r e l a k i s ,  muscarinic  (Chang and  been  muscarinic  and p h o s p h a t i d y l i n o s i t o l  t h o s e of rats  of  has  receptors  In l o n g i t u d i n a l effect  rat  responses  receptors  (Josephson  the  of over  pigs  before 1600 5-10  (350-400 the  isolation  units/ml sec  g)  period,  of 10  52  2.2  Krebs-bicarbonate All  buffer 113,  Buffer  incubations of  the  potassium  dihydrogen  chloride -  -  -  25,  4.7,  1.2,  2.3  Incorporation  heart cold  buffer  chopped  into  studies)  or a f t e r  later atria Krebs final  Tissue #1  was  40  stopped  fragments  paper  lipid,  with  were  separation  radioactivity  of  6 ml.  with  them of  either the  left  i n 5.3  -  -  potassium  0.6,  sodium  or  After  of  the  isolated  b l o o d and p l a c e d (95/5).  together and ml of  to  ( i n mixed  atria  atria  (in  Krebs b u f f e r .  The  of  drugs  incubation,  20% t r i c h l o r o a c e t i c  i c e - c o l d Krebs b u f f e r . and in later  ice were  by vacuum f i l t r a t i o n on  described  in  orthophosphate,  60 min  rat  Atria  right  absence  [32P]sodium  phospholipid  were done as  2.5,  and t h e pH a d j u s t e d  02/C02  2 ml of  separated  and washed w i t h  11.5,  presence  u C i of  i n c u b a t i o n volume of  reaction  of  suspended i n the  sodium c h l o r i d e  sulphate  perfusion  with  fragments,  and  (mM) :  Atria  free  separation  incubated  buffer  to  Krebs-bicarbonate  (95/5).  In  saturated  smaller  studies) were  -  were removed a f t e r  with Krebs buffer Krebs  magnesium  with 0 2 / C 0 2  in  calcium chloride -  and d e x t r o s e  7.4  The a t r i a  out  composition  approximately Phosphate  carried  following  phosphate  bicarbonate  were  in in a the  acid. Whatman  E x t r a c t i o n of  determination sections.  of  53  2.4  Isolation For  guinea  each  pigs.  method of and  Of L o n g i t u d i n a l Smooth M u s c l e Of G u i n e a P i g  Rang  (1964).  in  longitudinal  with a muscles  (except  for  at  37°C),  2.5  cotton were  on  was  swab  a  in  of  a  manner  (Q-tip).  ileum  pieces  i n which case  preincubated  for  Krebs  buffer  pieces  were  further  under  an  by a d d i n g  described  in a  2 ml of  and later  extraction  2100x2  for  15  that  the  weight)  (95/5)  and  min,  buffer stored  was  for  in  atmosphere.  15  i n the  for  Lipid  was  organic  ml  The  was  tissue  extracted  phase  a  presence  The r e a c t i o n acid.  and  Individual  separately  4 ml.  with  phosphate  combined  [3H]inositol  s e p a r a t e d by  twice  i l e u m were  incorporation,  [3H]inositol  The l o w e r  washed  and  tangential  pig  were  incubated  volume of  lipid  rod  were  described  wet  washed w i t h b u f f e r .  of  in guinea  20% t r i c h l o r o a c e t i c  section.  contraction  longitudinal  they  [3H]inositol  1 MCi of  in a t o t a l  the  fragments  c o l d Krebs  For  02/C02  by  Ileum  30 MCi of  separated  agents,  separated  during  30 min w i t h  then  to  , mg  ice  female  (95/5).  incorporation  30 min p e r i o d w i t h  various  stopped  (15-25  in  2  glass  Isolated  studies,  atrium.  for  by c a r e f u l  stored  similar  in rat  37°C  usually  phosphate  cm l o n g  smooth  separated  1 or  obtained  2-3  4 ° C or at  and a e r a t e d w i t h 0 2 / C 0 2  incorporation  at  at  from  was  into  L a b e l - i n c o r p o r a t i o n In G u i n e a P i g  done  was  muscle  functional  Studies  of  buffer  was mounted  smooth  smooth  I l e u m was c u t  Krebs  Each piece  scraping  obtained  L o n g i t u d i n a l smooth m u s c l e  stored  studies.  e x p e r i m e n t , i l e u m was  Ileum  as  obtained  centrifugation  methanol/0.1  N  54  hydrochloric finally  acid/chloroform  removed by e v a p o r a t i o n .  chloroform  (2  aliquots  described  as  determined solvent  ml)  in  and Dyer  than  was  25 mg) 3  study  (1959),  were  all  mixed  of  added  sec.  Finally,  p h a s e was  lipids  in  i n two 75 ul  removal  was  of  the  Incorporation  were e x t r a c t e d  described  in  1.5  below. on i c e .  ml of  was  a percentage  of  and 1.8  vortex  homogenate  vortex  m i x t u r e was  separated  Tissue  2 M KCI  was  for  water 5  sec.  and  (usually  and  transferred  20  less  homogenized  sec  Chloroform  was  (a  After  by c e n t r i f u g a t i o n  and  15  was  ml  added  nonacidified to a v o i d  (1.8  on a v o r t e x  added  to a  v/v)  i n t e n t i o n a l l y used  tube mixed a g a i n  ml o f for  Homogenization  c h l o r o f o r m / m e t h a n o l (1:2  by  the  by t h e method of  P o t t e r - E l v e h j e m homogenizer.  polyphosphoinositides)  then  by  the  ml of  chloroform/methanol  mixed  as  suspended  5.2  tube  extraction  after  and e x p r e s s e d as  with a teflon-coated  tube,  and t h e  dissolved  The r a d i o a c t i v i t y  10 ml E c o n o f l u o r .  c a r r i e d out  homogenization,  capped  section.  solvent  Lipid  the  extraction  After  was  the  value.  Throughout  speed  of  and  was d e t e r m i n e d  portion,  dpm/jug p h o s p h o r u s  2 . 6 E x t r a c t i o n Of  at  later  remaining  v/v),  The r e s i d u e  phosphorus  in a  addition  as  the c o n t r o l  and  the  and  calculated  Bligh  (48:41:3  the  ml)  mixer  for  the was 20  suspension  20-30 m i n , t h e c h l o r o f o r m at  2100XC_ f o r  15  min  at  55  2.7  Phospholipid For  initial  used  for  good  separation,  plates  Separation  the  experiments  separation but  "stored"  precoated  TLC p l a t e s  mm,  were  good  separations.  separated plates  all  from  a  in later Merck  period silica  spots  were  gel  layer  previously  with  thickness  consistently plates  separated  mobilities  different  very  Therefore,  chromatography  relative  slightly  gave  time. 60,  were  obtained  w h i c h gave  layer  the  was  of  studies,  thin  but  New p l a t e s  separation  the p h o s p h o l i p i d s p o t s Supelco,  phospholipid  longer  from M e r c k ,  used  Redi Coat-2D p l a t e s  phospholipids.  unsatisfactory  for  0.25  of  Supelco  i n the  with  of  the  two t y p e s  of  plates. Plates four  equal  were a c t i v a t e d quadrants.  micropipette In  at  incorporation, spot. thin  only  nitrogen  filter  solvent  for  solvent, run  between of in  runs  i n the  ammonia was the  second  detected  paper  30 m i n .  by  were  completely  corners,  was  to  the  by  Chromatographic and p r e s a t u r a t e d  dried  for  After  iodine  edges. or the  two-dimensional tanks  with  each  were  the  lined  desired  with  60 ml  run.  Between  5 m i n , and f o r  10-15 min  and s e c o n d d i m e n s i o n , lost.  10 M1  d r y i n g of  was d e v e l o p e d  with  by  into  determination  for  was a c h i e v e d  d i m e n s i o n and r e m o v a l of e-xposure  spotted  2 cm from t h e  acid  used  was u s e d  air  first  20 min and d i v i d e d  sample was  Each p l a t e  and new s o l v e n t plates  for  arachidonic  chromatography.  system  30 ul  four  Phospholipid separation layer  each  the  involving  w i t h Whatman #1  of  105°C  A 20 t o  e a c h of  experiments  at  until  development the  vapor,  solvent, which  the  of  smell  the  plate  spots  were  gave a y e l l o w  56  colour. was  After  allowed  spots  were  methanol.  to  fade  The  the  silica  v/v)  aqueous phase was  plate  gel  allowed  discarded  Of  drop  of  tube  and  to  stand  and 2 ml of used to spots  for  the  lower  extract were  5 min.  acid The  acid  upper  saturated  phospholipids.  extracted  with  In  2  ml  Radioactivity  phospholipid (0.8-0.9 ml).  determination.  label  required  only.  The  fluid.  a test  was  initial  Determination  after  to  a  chloroform/methanol/water/acetic  experiments  portions  transferred  of  of  some of  2.9  addition  The  stain  Spots  phase was  methanol  after  was  chloroform/methanol the  the  by s t o r a g e o v e r n i g h t .  Of P h o s p h o l i p i d  mixture  (50:30:10:1  away  pencil,  extracted.  Extraction A  s p o t s were marked w i t h a  scraped off  phospholipids 2.8  the  removal ACS and  One  In a n o t h e r of  the  was  of  portion,  divided was  the  into  used  for  radioactivity  and a d d i t i o n  extract,  [3H]-label  was  portion  solvent  u s e d as  aqueous  determination  extract  was  two  equal  phosphorus determined  of  10 ml  scintillation  scintillation  fluid  for  while  Econofluor  i n nonaqueous  media.  was  phosphate used  for  57  2.10  Phosphorus D e t e r m i n a t i o n All  acid of  the  washed.  glassware  used  i n the phosphorus  P h o s p h o r u s was d e t e r m i n e d by a s l i g h t  t h e method o f  Duck-Chong  (1979).  30 M1 of  10% magnesium n i t r a t e  mixture  evaporated  85-l00°C  for  sample tip  for  of  5 to  blue  temperature,  by  2  to ml  reagent at  1 M  of  as  molybdate  malachite  green.  in  r e a c h e d room  was  added.  The  at  90-95°C  After  disposable  for  described  Just  mixture to give  before a final  be m e n t i o n e d h e r e freely,  M  that  malachite  tubes  15 m i n .  5 min t h e  were After  followed absorbance  cuvette.  With  to  a  each  tube  in  and  the  had been e v a p o r a t e d  above. 1  HCl with  use,  volume  Triton  while  of  3 volume o f X-100  c o n c e n t r a t i o n of  green  the  c u r v e was o b t a i n e d by a d d i n g a  nitrate  4.5  the  at  ml 1 M H C l was a d d e d ,  phosphorus  at  tubes  C was p r e p a r e d by m i x i n g  ammonium  dissolved  a  magnesium  was c a r r i e d o u t Reagent  in  and  sodium d i h y d r o g e n monophosphate  of  bath  After  flame.  HCl  C (see  a standard  i n a water  the  sec  and h e a t e d  620 nm  and  10-15  below).  30 til  should  the  flame  of  known sample  this  the  0.7  experiment  assay  cone o f  marbles  added  tube,  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  t h e t o p of  ml o f  it  modification  in a test  was  by h e a t i n g  room t e m p e r a t u r e ,  was measured  which  0.3  with  cooling  at  To a sample  i n methanol  to dryness  10 m i n .  15 sec  the  covered  d e t e r m i n a t i o n was  malachite  from o t h e r  4.2%  (w/v)  0.05%  (w/v)  was  added  to  0.075%  (w/v).  It  green  from  sources  BDH  did not.  58  2.11  Inositol In  values  preliminary c o u l d be  labeled  the  resin.  experiments  as  obtained  200  ul of  Dowex-1  of  [3H]inositol  saturated  with  Krebs-Ringer the presence lithium  i n 800  labeled  wherever  to  required,  0.1  b e g i n n i n g of  were c l o s e d  t i g h t l y and  hour.  Incubation  the  was  pieces  and  (1:2).  The t i s s u e s  described retained  of  alone  terminated them  phosphate  Krebs  the  buffer  ileum pieces volume of plastic  1  ml  tubes,  in  carbachol, of  and 6 . 4  were  10  mM  PMSF i n 1% uM a t r o p i n e (95/5)  Then t h e  was tubes  a p e r i o d of smooth  one  muscle  ml c h l o r o f o r m / m e t h a n o l  were h o m o g e n i z e d and t h e  inositol  mixed,  by r e m o v i n g t h e  The a q u e o u s p h a s e of  step.  was v o r t e x  the m i x t u r e .  1  anion-  contaminants  incubation 0 2 / C 0 2  into  the  30 MCi  incubation continued for  transferring  above. and  surface  of  with  added  r e q u i r e d amount  sulphate.  the  mM  blank  to  in a t o t a l  or d i m e t h y l s u l f o x i d e  above  pig  in disposable  60 mM K C I , t h e  introduced  was  14 ml of  Guinea  dimethylsulfoxide At t h e  that  removed d u r i n g t h i s  added  buffer  anionic  (50% w/v)  [3H]inositol  Lithium  treatment  The m i x t u r e  (95/5).  bicarbonate  chloride,  resin  supernatant  1 uCi of  observed  by p r i o r  to  ul w a t e r .  02/C02  of,  was  w h i c h were b e i n g  X8-formate  and t h e  incubated with  it  Of  from t h e m a n u f a c t u r e r ,  T h i s may be due  [3H]inositol,  centrifuged  A c c u m u l a t i o n In The P r e s e n c e  reduced d r a s t i c a l l y  inositol,  exchange in  Phosphate  lipids  extracted  extraction  d e t e r m i n e d as  follows.  as  medium was  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  Inositol  The method was  simplified  described (w/v) the  by  X8  times  with  eluted formate.  4 ml of  determination 2.13  Washed eluted  ml  of of  Separation  plus  0.1  0.1  t h e column s e p a r a t i o n  Standard  inositol  inositol  phosphate  mM  inositol  This  were  strengths  of  described or  after  was  washed  five were  1 M ammonium  used  for  the  addition  of  adsorbed  inositol  phosphates  tube  with  four  10 ml A C S .  5 mM b o r a x 0.4  i n the  or  in  a  an  no more r a d i o a c t i v i t y from t h e  column  ammonium  formate  1 ml f r a c t i o n s  the  and  extract  were c o l l e c t e d  eluted.  with  5  Inositol  increasing  and  of  through  column  formic  Dowex-  column.  was p a s s e d  with  5  formate  cm  aqueous  was  of  150 mM  2 ml of  1x4  tissue,  was  formate.  phosphates,  packed  plus  M ammonium  1 M ammonium  inositol was  2 ml p o r t i o n s  f o l l o w e d by w a s h i n g of  determined.  the  phosphates  was  to  Phosphates  accumulated  borax,  radioactivity  ml)  the  mono-phosphate,  eluted  above.  of  (50% w/v)  until  phosphates  (0.4  50%  after  was  inositol  M formic a c i d plus  In  resin  formic a c i d plus  M formic a c i d plus  resin  the  of  form was added  discarded  mM ammonium f o r m a t e ,  finally  column.  eluate  in a test  and  the  M  procedure  A 1 ml s l u r r y  was  of  assay  formate  After  containing  60  ammonium f o r m a t e ,  i n the  inositol,  Of I n o s i t o l  0.1  (1982).  mM  radioactivity  resin  1 X8-formate  al.  settle.  this  successively  borax  the  supernatant  to 5  0.8  Half  et  The  allowed  with  to  (100-200 mesh)  aqueous p h a s e . was  mM  Berridge  Dowex-1  resin  similar  Phosphate  ionic  acid,  as  phosphorous  60  2.14  Contraction Guinea  under  0.35  with  Studies  pig  ileum s t r i p s  g tension  02/C02  i n Krebs  (95/5).  isometrically  using  equilibration  period.  potassium-mediated dissolved  in  bicarbonate  force The  contraction  i n the  KCI  not  in  effect  tone  bath.  was  final  by  aerated monitored  a  by  30  min  adding  reduction  of of  and PMSF  concentration  The h y p e r o s m o l a r i t y  compensated  37°C  PMSF on c a r b a c h o l -  studied  to a  at  and  after  of  was  suspended  buffer  transducers  dimethylsulfoxide  was  l o o p e d and  Alteration  dimethylsulfoxide solution  were  of  1%  the  60 mM  the  NaCl  concentration. 2.15  PI D e g r a d a t i o n PI  degradation  [32P]phosphate pooled  and  small  fragments  orthophosphate  six  i n the  of  this  ml  of  labeled  2 h at  were  37°C  with  buffer.  w i t h Krebs  equal  incubation  period,  trichloroacetic  by f i l t r a t i o n .  phospholipids  further  and  and  for  they  of  tissue lipids,  rats  incubating  incubation the  divided  into  i n 5.9  ml of  37°C  was  5  After  was  mM c a r b a c h o l .  and  of  the  were  with  [32P]sodium  suspended  reaction  Extraction  of  end of  1 h at  0.1  acid  determination  mCi  by  filtration.  buffer,  portions  from  j_n v i t r o  0.2  by  prelabeled  Atria  At t h e  separated  p r e s e n c e o r a b s e n c e of  20%  separated  phospholipids  Further  incubation  fragments  follows.  were washed  buffer.  on a t r i a l as  i n 40 ml o f  approximately  Krebs out  for  fragments  fragments  studies  were p e r f o r m e d  were  period  Studies  carried  At t h e  stopped  end  with  fragments  were  separation  radioactivity  2  were done  of as  61  described In  i n the  earlier  degradation  acid,  whole  At  the  end of  Krebs for  acid,  atria  of  amount percent 2.16  were  was  PI  separated,  presence  or  37°C.  washed  with  2 ml of  incubated  absence  20%  w e i g h e d and l i p i d  i n PI p e r  2 h at  serum a l b u m i n and  section.  of  the  trichloroacetic and p h o s p h o l i p i d  Radioactivity  s p o t s was d e t e r m i n e d and r e s u l t s  Acid  in  calculated  as  expressed  as  diacylglycerol fractions  was  g of  tissue  and  fatty  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 acid composition  d e t e r m i n e d by methods (1974).  incubated (95/5)  In  i n 5.3  to  give  further  of  20%  a  similar  these  ml of  atmosphere.  a  final  in  in dry  -20°C,  After  fragments  buffer  15 min a  carbachol  and  the  tissues lipid  at  37°C,  stock  reaction Control  t h e a b s e n c e of  mixture,  extracted  weighed, as  from r a t  under  carbachol of  was  chopped in  were  was  After  with  were  and s t o r e d  02/C02  mM.  stopped  Tissues  described  an  et were  solution  0.1  tissues  carbachol.  were  by Bansback  whole a t r i a  concentration  acid.  ice/methylbutane  Frozen  those described  experiments,  Krebs  trichloroacetic  of  to  15 min i n c u b a t i o n ,  identically frozen  for  10 /uCi of  control.  The  added  were  i n an e a r l i e r  radioactivity  Fatty  al.  buffer,  with  arachidonic  incubated with  Krebs  i n the  stopped  [1"C]-labeled  were  1% b o v i n e  separated,  described  the  of  atria  15 min p e r i o d  The r e a c t i o n  each  i n 30 ml of  period,  drug.  as  employing 12 r a t s  containing  further  isolated  from  acid  this  buffer  a  studies  atria  [1"C]arachidonic  sections.  2 ml  treated rapidly  overnight  at  into  smaller  an  earlier  62  section.  Neutral  chromatography  lipids of  were  total  benzenerchloroform:methanol 0.01% by  dichlorofluorescein  comparison  extracted (9:1  with  from t h e  v/v)  acid  was  obtained  added  pentane  w i t h hexane was  further  ionization  in  (Hewlett  detector of  diethylene  glycol  and  under N 2 .  Hewlett  column  on  -  190°C;  injection  temperature  -  225°C;  carrier  ionization  sensitivity  for  detector all  the  fatty  10-15% b o r o n  extracted  with  chromatography to  a  extracted  twice  mixture  into  5830A)  equipped  with a  Packard  18850A  an  was  80/100 were  as  port  -  found acids  at  to  a  flame  on a  225°C;  column detector  56-58 m l / m i n .  examined.  10%  Supelcoport  follows:  have  gas  integrator.  achieved mesh  in  standard,  The m e t h y l e s t e r  gas-helium  was  with  injected  conditions  temperature  flame  with  were  layer  esters  were  potassium  and  methyl  succinate  acids  were e x t r a c t e d  from t h e p l a t e s ,  Packard  individual  The  acids  methyl e s t e r s  hexane  heneicosanoic  Fatty  corresponding  ul  ether/ethanol  and  portions  20  identified  15% m e t h a n o l i c  The m e t h y l e s t e r s  scraped  with  2-mercaptoethanol.  by t h i n  were  Separation  column.  their  and c o n c e n t r a t e d  chromatograph  (v/v)  purified  gel  resuspended  0.1%  standard.  in methanol.  stearate,  with d i e t h y l  internal  to  with  D i a c y l g l y c e r o l was  plates  The f a t t y  layer  and v i s u a l i z e d  under N 2  1 h.  thin  D i a c y l g l y c e r o l was  by h y d r o l y s i s w i t h  for  Silica  v/v),  to dryness  an  converted  and  benzene. methyl  taken as  70°C  and  trifluoride  gel  by  extract  1,2-dipalmitoylglycerol.  from l i p i d s  hydroxide at pentane  spray.  silica  was  lipid  (80:15:5  containing  Diacylglycerol  separated  a  The  similar Fatty  acid  63  quantities to  the  2.17  were e s t i m a t e d  internal  b a s e d on t h e a r e a  with  respect  standard.  Materials Carbamylcholine  monophosphate  chloride,  ester  Sigma  chemical  mixture  orthophosphate  and  (200  obtained  from  New  Phentolamine mesylate  was  Methoxamine  BDH  Amersham,  chemicals, Seattle,  chromatography Bellanfonte,  Wa.  hexane  Germany.  and a c e t o n e  inorganic  salts  analytical  grade.  N.C.  and  Precoated  Glass  used  in  the  acid  (55.8  Econofluor  were  Quebec. Dorval,  from B u r r o u g h s  Wellcome  resin  was  and magnesium n i t r a t e  were  Ontario.  molybdate  Redi-coat from  silica were  distilled  were p u r c h a s e d  from  from  Canada,  obtained  precoated  acid  [32P]sodium  Lachine, CIBA  ammonium  and b e n z e n e  fatty  Dowex-1 X 8 - f o r m a t e  42000)  were  and  chromatography p l a t e s Darmstadt,  from  Mississauga,  ACS  plates Pa.  Nuclear,  obtained  (C.I.  Mo.  C i / m m o l ) and  h y d r o c h l o r i d e was  green  inositols-  [1-C1"]-arachidonic  (15.8  T r i a n g l e Park,  Malachite  Louis,  England  from B i o - R a d l a b o r a t o r i e s ,  from  sulphate,  c h l o r i d e were o b t a i n e d  St.  mCi/mmol),  myo-[3Hj i n o s i t o l  Research  lithium  company,  mCi/mmol),  Quebec.  atropine  dl-monocyclohexylamine, m y o - i n o s i t o l ,  methyl  Co.,  ratio  gel  obtained  2D  preparation  of  thin  from layer  Supelco  Inc.,  60  layer  thin  from  chloroform, Caledon,  were  E.Merck, methanol,  Ontario. buffers  All  were of  64  . Ill.  3.1  SEPARATION OF PHOSPHOLIPIDS  3.1.1  Separation  And I d e n t i f i c a t i o n  Phospholipids chromatography on c o m p a r i s o n were  were  separated  on a s i l i c a with  standard  identified  -  phosphatidylserine,  identity  of  PI  incorporation  spot  distribution rat  atria  determine than  of is  the  the  was  of  four  shown  efficiency  determined  and t h e r e f o r e ,  shown  phospholipids  in Table  In a  similar  determined,  it  was  phosphatidylinositol present  per  spot  g tissue  f-  into  Table  As  spots  spot  well  where as  (one  the  specific relative  in studies  i n t o PI  g tissue  distribution  rather  was  not  of  the  determination,  by W h i t e  lipid  (1973).  phosphorus, 16.3  Mg  phosphatidylcholine of  to  phospholipids,  that of  on  was  phosphatidylinositol  total  uq  The  objective  all  reported  calculated 126.6  acid.  The  by p h o s p h o r u s  from t h a t  d-  the  (a).  b-  spot  phospholipids  percentage  estimated  .by  of  individual  the  weight  following  incorporated  determination  and  Based  determined  I.  layer  4.  phosphatidic  spot  phospholipids  of  the  confirmed  experiment, as  shown i n F i g  phosphatidylcholine,  and  I differs  phosphatidylcholine,  as  thin  phosphatidylinositol,  radioactivity  quantitative  major  ac-  in  specific  by two d i m e n s i o n a l  phospholipids,  [3H]inositol  the  Of PI  plate,  further  extraction  four  gel  spot  phosphatidylethanolamine  as  RESULTS  rat  heart  has  and were of are been  65 -  reported  to c o n t a i n  The r e p o r t e d 17.4  and  3.1.2  values  169.2  Phosphorus  is  was  in F i g  sensitive  5.  and  0.6  3.2  than  phosphorus  (White,  two p h o s p h o l i p i d s  determined of  a  respectively  the  in rat  (White,  curve  1973)). heart  are  1973).  line  in duplicate  0.01  absorbance  method  method  reproducible.  1 and c o r r e l a t i o n  than  Duck-Chong  by t h a t  determination was  The v a r i a t i o n  of  obtained  gave a s t r a i g h t  around  less  method  curve  phosphorus  phosphorus  were  by  standard  standard  0.99.  experiment  lipid  g tissue,  Mg, w i t h a s l o p e  greater each  these  This  the  absorbance versus to  Mg of  Determination  An example  shown  for  Mg p e r  Phosphorus  (1979).  470  was  A plot  between  of  0.1  Mg  coefficient  of  observations  in  unit.  PHOSPHATE INCORPORATION IN RAT HEART  3.2.1  [32P]Phosphate Incorporation Despite  contain  its  a  large  incorporated similar  incorporation  in into  is  labeled  than  the  of  phosphate-label atria  was  fraction  about  rat  of  heart  various  shown i n F i g  incorporated that  in c e l l s ,  into phospholipids  result  ventricles  low q u a n t i t y  6.  PI  the  has  total  (Table  I).  f o l d higher  into than  It  the  was  in  of  Ventricle found  We  to  label  obtained  rat  specific also  atria  and PI  activity  observed  that  phospholipids  label  a  phosphate  and v e n t r i c l e s  various  that  been  The b a s a l  Both i n a t r i a with a higher  Versus  phosphate  (Michell,1975).  phospholipids.  incorporation 5-6  Atria  phospholipids  phosphate  other  In Rat  of  incorporation  66  into  the  3.2.2  corresponding  Effect  Stimulation  and  in  been  by  to  verify  phosphate rat  mM  carbachol  the  pieces  effect  the  methoxamine,  result  an  [32P]phosphate adrenergic  into  receptor  a-adrenergic mM) PI  enhanced by  density  88% is  it  less  is  an  of  the  of  its  rat As not to  stimulation.  35%  rat  atrial  increase  (Fig  7).  In  particular  in order  labeled  i n combined  i n a b i l i t y to  measure  preparation, agonist,  on  combined r a t  atria  muscarinic  response,  the  than  in  interest  combined  receptor  (Fig  unlike  considered  was  a-adrenergic  to  contrast  but  responses.  stimulation  into that  In  (0.1  atria,  i n c o r p o r a t i o n of  of  investigated.  methoxamine  1980)  a  receptor  i n t o PI  the  (Schumann,  (p<0.05)  incorporation  of  (Wei  atria  caused  is  two r e c e p t o r s  in  [32P]phosphate  stimulation  Both m u s c a r i n i c  accompanies  d e g r e e of  incorporation  of  these  in rat  i n t o PI  small  was n o t  the  in rat  receptors  i n t o PI on m u s c a r i n i c  [32P]phosphate the  response  incorporation  that  atrial  of  stimulation  PI  usually  pharmacological  muscarinic  0.1  [32P]phosphate  be p r e s e n t  by c a l c i u m m o b i l i z a t i o n ,  whether of  i n t o PI  response.  stimulation  receptor  Stimulation pieces  shown t o  Receptor  In Combined A t r i a  alpha-adrenergic  opposite  be a c c o m p a n i e d  determine  and  tissues  produces  muscarinic  phosphate  i d e n t i f y i n g a PI 1978)  have  other  atria  to  i n c o r p o r a t i o n of  Sulakhe,  most  ventricles.  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  step  receptors  in  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  Enhanced first  phospholipids  of  rat  atria  was  with  incorporation 7),  even t h o u g h t h e  50% of  the  of a-  muscarinic  67  receptor by t h e  density.  This  a-adrenergic  similar  antagonist,  experiment,  stimulated  increase  PI  did  and  stimulation  it  was  phentolamine  further  in phosphate  not  occur  in  Stimulation Right  canine  us  to  so  that  (Fig  a  methoxamine-  was  specific  phospholipids,  to  including or  8). Receptor  In S e p a r a t e L e f t And  (1982)  receptor  reported  atrium,  showed  adrenergic  of  the  muscarinic left  atrium  incorporation  the  a similar  [32P]phosphate It  not  receptors  one of  preparations.  but  regions  whether  only  that  enhanced  muscarinic  test  small  canine  atria  found  receptor,  that  stimulation  Michell,  produced  a  incorporation 9).  other  Muscarinic  hand, PI  into  in rat  PI  in  left  receptor  produced left  110 and  been  shown  to  1978).  This  led  and  of  the  atrium,  in  was  the  atrial alpha-  response  (Jones  and  phosphate  respectively  by c a r b a c h o l ,  in phosphate but  PI  tissues  heart,  for  i n combined  increase  right  stimulation  (p<0.05),  in rat  shows a s m a l l e r  175%  a 35% i n c r e a s e  atrium  PI,  responsible  in other  right  into  exist  observed  which u s u a l l y  or  phosphate  stimulation  receptor  1975),  be  stimulation  left  have  might  may  incorporation  was  of  heart  situation  rat  or  (Wei and S u l a k h e ,  than m u s c a r i n i c  into  In  On [ 3 2 P I p h o s p h a t e I n c o r p o r a t i o n  though a l l  contain  (Fig  the  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  right  ventricle, even  yM).  Atria Quist  of  incorporation  blocked  phosphatidylcholine  phosphatidylethanolamine Effect  was  (20  shown t h a t  other  phosphatidylserine,  3.2.3  by methoxamine  on  the  incorporation  completely  without  68  effect  in  rat  incorporation (compare various  right  into  Fig  9  rat  d i d not results  muscarinic  receptors  turnover,  left 3.3  a  10),  magnitude  long  time  to  and  Hokin.,  blowfly  be  the  1964;  salivary that  accompanied increase receptor was  sensitivity  that  atria  do not  of  PI  while  variable  results  of  from  the  basal  to  agonists.  the  majority  of  be c o u p l e d  to  appear  muscarinic  basal  to  receptors  in  rat  turnover.  BREAKDOWN IN HEART p r i m a r y event  it  of  the  population  The  1979a)  quite  that  the  relative  a t r i u m was comparison  influence  in rat  The  the  suggested  small  10).  but  a t r i u m may be c o u p l e d t o PI  (Fig  and r i g h t  suggests  These  PI  left  and  experiments  incorporation  atrium  Michell,  gland,  it  has  by  rapid  to  on PI  were done b e f o r e  of  the  been  observed  inositol  the  least  effect  apparent).  i n one  tissue,  and  Berridge,  PI.  As  i n t o PI  of  was  only  during  not  small  muscarinic  muscarinic  response  was a  observed  atria  a  (Hokin  breakdown,  atria  i n PI  (Fain  for  lipid  lipid  i n combined r a t  differences  a t r i u m became  of  combined r a t  breakdown  inositol  At  incorporation  investigate  been c o n s i d e r e d  1975).  resynthesis  phosphate  stimulation  r e s p o n s e has  e n h a n c e d breakdown of  primary event,  decided  and r i g h t  PI  the  in  stimulation  of  (these  between  (Fig  7),  receptor studies rat  left  69  3.3.1  Breakdown Of P h o s p h o l i p i d s  Prelabeled  With  Radiolabel  Phosphate Muscarinic been  shown t o  labeled In t h e mM  cause  with  study  carbachol  3.3.2  an  for  past  few  that  arachidonic phospholipase turnover detect  breakdown  of  Prelabeled years,  A2  et of  the  al.,  1980,  effect  of  period  the  level  observed guinea  of  stimulation  level  for  the  PI  in pig  1979).  incubation  parotid ileum for  gland  (Jones 15  Similarly,  a  min  in  maximum  during acetylcholine  observed  after  a  15  min  through  or  of  activation  from P I ,  or  as  atria  in  was  chosen  stimulation period  as  the  examined. for  these  decrease  1975, after  ( B i l l a h and the  to  stimulation  stimulation  and  d u r i n g PI  well  A detectable  1979)  of  in order  receptor  15 min was  increase  incubation  PI  release  1982a)  hepatocytes  receptor  0.1  to  Therefore,  receptor  al.,  with  presented  cause  ( J o n e s and M i c h e l l , et  1974).  breakdown of  may  reasons.  a 30 min  previously  atria  been  i n combined r a t  following  after  has  acid  of  PI  has  11)  muscarinic  A  gland  Arachidonate  1982).  PI  experiments  (Fig  With  arachidonic  of  the  Lapetina,  on a r a c h i d o n y l - p r e l a b e l e d carbachol  enhance  either and  parotid  combined r a t  stimulation  (Billah  loss PI,  of  evidence  PI,  rat  ( J o n e s and M i c h e l l ,  significantly  from  (Marshall any  phosphate  receptor  acid  of  breakdown  45 min d i d not  phospholipids  the  suggest  enhanced  stimulation  Breakdown Of PI In  stimulation  radiolabeled  present  any o t h e r  receptor  has 1976)  in  been and  receptor Michell,  diacylglycerol of  pancreas  (Bansback  et  was al.,  70  1974).  As  conserved  the  d u r i n g the  prelabeled  PI  radioactivity specific  fraction 175 and  studies  present  for  of  unstimulated  breakdown  activation  of  have  of  group  increase  mediate  has  atria  were  of  weight,  PI  to  be  arachidonatethe  amount  of  than  as  with  0.1  the  PI  rather  stimulated  radioactivity  in  t i s s u e was  respectively stimulation  i n combined r a t  found to  (p>0.05)  d i d not  atria  A 2 or p h o s p h o l i p a s e  in  of  its  effect  either  be  (Fig  cause  a  through  C.  diacylglycerol  was  chromatography  shown i n F i g  13.  (Hokin  and  well  the  lipid  stimulation  i n combined r a t  Diacylglycerol  as  separated  as  formed  during  Hokin,  al.  the  of  an  content (1974).  effect  of  arachidonyl content  of  examined. phospholipids  in benzene/chloroform/methanol Phospholipids  PI  1953),  arachidonyl  breakdown,  from  al.,  where A C h - s t i m u l a t e d  et  was  et  activation  by Bansback  on t h e  atria  Kishimoto  through  In p a n c r e a s ,  been o b s e r v e d  inositol  receptor  1979b;  diacylglycerol  reported  has  al.,  that  diacylglycerol,  measure  et  PKC.  been  diacylglycerol,  muscarinic  as  as  tissue,  muscarinic  (Takai  suggested  may  turnover  layer  in  of  amount  tissue  phospholipid-dependent  a  results  expressed g  considered  D i a c y l g l y c e r o l Accumulation  turnover  As  is  and s t i m u l a t e d  phospholipase  Nishizuka's  of  per  15 m i n , t h e  suggested that  significant  PI  the  were  i n PI  194 cpm/mg wet  This  1980)  response,  arachidonyl-prelabeled  mM c a r b a c h o l  3.3.3  PI  moiety  radioactivity.  When  12).  diacylglycerol  remained at  the  by  thin  (80:15:5),  origin,  while  71  diacylglycerol  moved  with  was  present  mercaptoethanol acid  methyl e s t e r s  done  prior  standard Fatty  to  gas  methyl  chromatography.  the  similar  latter loss  was  very well  oleate  arachidonate  in  tissues  Fatty  Atria  And V e n t r i c l e s  origin In  Acid  of  its  composition  of  after  (Fig  the  the of  polar the  and v e n t r i c l e s  acid  of of  mixture  used,  with  3.04,  ;  9.88  and  composition  of  tissue  and  is  -untreated  diacylglycerol  and l i n o l e i c  acid,  14).  during  Lipid  Fraction  arachidonic  isolation,  fraction  diacylglycerol about  gas  result  linoleate  the  a b s e n c e of  lipid  by  palmitate  oleic  to  hexane.  the  -untreated  in  stearic,  degradation  separation  atria  acids  was  the absence  fatty  acid  and  fatty  arachidonate  not  carbachol-treated  absent  that  is  8.88;  fatty  fatty  in  methyl  -  C o m p o s i t i o n Of The P o l a r  to v e r i f y  result  both  4  a c i d was  3.3.4  acid  (min)  2-  benzene  column c o n d i t i o n s  arachidate  t h e y were p a l m i t i c ,  while arachidonic  the  the  Both  only  and  of  analyzed  The s t a n d a r d  The  II.  showed  In o r d e r  then  fraction  values  5.95;  When  corresponding  and t h e r e f o r e  in carbachol-treated  Table  fraction;  under  17.23.  diacylglycerol  not  were  m o b i l i t y i n benzene,  5.13;  Spots  in  removed and e x t r a c t e d  esters  retention  0.53.  chromatography  Both methyl s t e a r a t e  following  shown  layer  were  separated  of  a preliminary separation  d u r i n g TLC i n b e n z e n e .  stearate  value  i n the d i a c y l g l y c e r o l  its  the  R  chromatography.  methyl s t e a r a t e acid  have a  by t h i n  an  65% of  the  In  acid  the  remaining at was  was  fatty the  determined.  total  fatty  acid  72  content  was  saturated  unsaturated the  fatty  content  of  acids,  after  and  and  al.  a more d e t a i l e d  atria  content  linoleic  adrenergic  receptor  in rat  majority  the  of  carbachol  effect  possibility PI  turnover  It  is  therefore  muscarinic  by  of  the  in  differed  unsaturated  fatty  (Fig  similar  15).  conclusions  phospholipids  atria  is  receptor  enhanced  carbachol  PI  lipid  receptors  coupled to  absent used  or  so  small  in this  PI  in  rat  turnover.  The  is  a  that  it  the  atria  lack  rules  is  of  a  out  the  the p r i m a r y  step  accompanying  only  alpha-  turnover,  in rat  breakdown a l s o  atria  the  stimulation  the  that  in rat  while  may be s t i m u l a t i n g  without a c t i v a t i n g suggested  that  small  resynthesis. population  coupled to  breakdown a c c o m p a n y i n g m u s c a r i n i c  methods  but  acids  a r r i v e d at  was  d i d not d i f f e r  three  arachidonic  indicated  on i n o s i t o l  that  in  3.4  muscarinic  accompanied  either  remainder  acids,  all  investigation  heart  PI  fatty  of  (1983)  in  the  the  ventricles.  The s t u d i e s  not  and  and v e n t r i c l e s  saturated  the  Charnock et  acid  Atria  two  in  oleic,  Recently,  acid.  the  significantly  fatty  PI  receptor  turnover  stimulation  c o u l d not be d e t e c t e d  by  of and is the  investigation.  PI RESPONSE IN GUINEA PIG ILEUM Muscarinic  receptor  produces  a biphasic  through  calcium  Jafferji  and  longitudinal  c o n t r a c t i o n which mobilization  Michell smooth  stimulation  of  is  and  have  guinea  guinea  shown  (Chang  (l976a,b) muscle  of  pig  to  pig be  Triggle,  reported  ileum  mediated 1973). that  ileum s t i m u l a t i o n  in of  73  muscarinic  receptor  incorporation study,  the  of  radiolabeled-phosphate  PI  investigated  or K + - d e p o l a r i z a t i o n  response  in order  to  in  guinea  examine  its  produced into PI.  pig  an In t h e  i l e u m has  possible  enhanced present  been  further  in  calcium  role  mobilization. 3.4.1  [32P]Phosphate The  effect  Incorporation of  muscarinic  [32Pjphosphate  incorporation  carried  at  the  a  PI  to  out  detect  (Jafferji of  specific 3.4.2  [3H]lnositol  PI a l s o several  1975). (1980), the  which  PI,  and  increased  report  by  subsequent  was  observed label  radioactivity  into  was  stimulation 0.1 mM of  b l o c k e d by  MM) ( F i g  in  labeled  tissues  the  16).  inositol  examined  Schellenberg  into  inositol  t h e major  lipids.  incorporation studies, the  incorporation  see  and  in p r e l i m i n a r y experiments  incorporated  present  inositol  (1.6  report  incorporation  phosphate  i n c o r p o r a t i o n of  As i n a p r e v i o u s  fraction  ability  ileum with  the  increase  labeled  of  [3H]inositol  our  was  Incorporation  a list  was  to v e r i f y  pig  on  ileum  receptor  in  atropine  pig  w i t h an e a r l i e r  guinea  this  (for  it  study  increase  antagonist,  increased tissues  of  stimulation  guinea  muscarinic  muscle  100%  into  muscarinic  Agonists  in  a  this  in  In agreement  1976a),  smooth  caused  t32P]phosphate  into  response.  longitudinal  carbachol  i n t o PI  b e g i n n i n g of  and M i c h e l l ,  receptor  total  lipid  the  into  PI  Michell, Gillespie  that  98% of  phospholipid  Therefore,  in  all  incorporation  of  fraction  was  used  as  a  74  measure  of  inositol  lipids.  In  incorporation dpm/^g  the of  [3H]inositol blocked  (Table  by  was  III).  was  incorporation  16 uM a t r o p i n e  into  of  inositol-containing  carbachol increased  This  incorporated  period  from  represents  during  the  subtracted.  c a u s e d by (Table  (0.1  accumulation number  of  et of  al.  inositol  tissues.  accumulation  of  inositol  to  be o p t i m a l  In  the  there  was  a very  23.5  dpm/uq  small  total  of  lithium,  increase  in  the  of  of  of  was  lithium  response atropine  to  effect was  or  was  al.,  mM  receptor  30  min  increase was  in  completely  of  l i t h i u m causes  of  carbachol i n the were  of  0.1  on  a the  presence,  previously  mM c a r b a c h o l  radioactivity  alone  (Table  as  shown  that  the  The h a l f  In  a  i n the Fig  and the  20-fold to  IV).  b l o c k e d by t h e m u s c a r i n i c suggesting  (31  compared  (Table  in  alone  IV).  produced  by c a r b a c h o l  stimulation.  in  1982).  radioactivity,  phosphates  Phosphates  response  which  carbachol  lithium  17),  increase  PI  studied  respectively)  dose-dependent,  uM) ( F i g  from m u s c a r i n i c  the  accumulation  0.1  553  a 99%  that  during  10 mM l i t h i u m or  inositol  carbachol  (16  of  et  accumulation  carbachol  accumulation  shown  phosphates  phosphorus,  presence  effect  have  60 m i n , c o n d i t i o n s  presence  to  III).  study  (Berridge  the  368  The  carbachol  phosphate  In t h i s  10 mM l i t h i u m f o r  found  (1982)  mM),  initial  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 Berridge  of  [3H]inositol  radioactivity  preincubation  3.4.3  presence  phosphorus  when t h e  incorporation  the The  presence 17.  This  antagonist,  response maximal  results response  75  for  the  the  carbachol  carbachol using  Michell,  1976a).  from  an  et  al.  (1982)  that  lithium-amplified salivary the  Of I n o s i t o l  ion-exchange  electrophoresis  gland  previous  resin  (Fig  However,  accumulated carbachol  and  eluted that  resin,  the  presence  majority of  1981),  Based  resin 2/3  of  carbachol  phosphates. exchange  ionic  triphosphate.  and  brain,  parotid  1982),  plus 20% of of  in  19,  gland  standard  the  eluted  pig  from  the  formate which with  on a Dowex-  peak  corresponding  to  Table V ) .  The r e m a i n d e r  was  This  result  accumulated as  1982,  Downes  biphosphate  suggests in  inositol  elution characteristics  inositol  inositol  ileum  then adsorbed a  and As i n  radioactivity  guinea  solutions.  al.,  during  150 mM ammonium  radioactivity  et  voltage  accumulated  and l i t h i u m was p r e s e n t  t o be  high  be q u a n t i t a t i v e l y  strength  and  elution  al.,  eluted  (Berridge  on  et  than  the  (Jafferji  i n o s i t o l mono-phosphate.  (Fig  to  response  to  to  on t h e i r  appeared  in  and w h i c h was  was  similar  PI  based  radioactivity  incubation  monophosphate  by h i g h e r  reported,  the  less  lithium,  maximal  incorporation  5 mM b o r a x  following  1 X8-formate inositol  with  is  Phosphates  response  found  This  column  (Berridge  ion-exchange 18).  half  have  corresponds  was  10 uM.  resin  80% of  PI  report  mono-phosphate  for  [32P]phosphate  Identification Berridge  around  concentration  obtained  3.4.4  r e s p o n s e was  and  the poly-  from t h e and 1/3  ion  Michell, inositol  76  3.5  EFFECT OF PMSF ON PI TURNOVER AND CONTRACTION IN GUINEA P I G  ILEUM  3.5.1  Effect  Of PMSF On PI  Recently, esterase  Walenga  inhibitors  Turnover  et  may  al.  (1980)  also  be  reported  inhibitors  p h o s p h o l i p a s e C , b a s e d on t h e i r  studies  decided  serine esterase  as  to  i n v e s t i g a t e whether  PMSF, c a n  and  if  inhibit Pi-specific  they d i d ,  response  in ileum.  incorporation m u s c l e of  what t h e i r  into  guinea  30 m i n p r e i n c u b a t i o n incorporation time in but  for  its  was  absence.  Jafferji  and M i c h e l l guinea  c a l c i u m by o p e n i n g  The  [3H]inositol alone  of  almost  a  shown  on the  was  inhibitors,  such  and  2  (1976b) pig  studied,  mM  PMSF  remain  slight  is  completely  calcium  carbachol  shown i n F i g .  increase  and 21.  the  with  2 mM PMSF a n d  into  small PI.  shown  that  which m o b i l i z e s channel,  phosphate  in [3H]inositol  blocked  linear  PMSF c a u s e d a  i l e u m by p o t a s s i u m ,  on  initial  [3H]inositol  previously  labeled  smooth  A f t e r an  incorporation  voltage-dependent  mediated  [3H]inositol  PMSF, b a s a l  have  ileum,  longitudinal  shown t o  i n c o r p o r a t i o n of  the  basal  i n F i g 20.  in [3H]inositol  incorporation  caused it  a  an e n h a n c e d  effect  of  time p o i n t s  increase  produced  It  on t h e c a l c i u m  PMSF  serine  Pi-specific  platelets.  30 m i n , b o t h i n t h e p r e s e n c e o f  At a l l  of  was  i n the absence of  consistent  stimulation  but  ileum is  determined  a further  of  phospholipid  pig  of  phospholipase C in  effect  The e f f e c t  in  that  also  into  PI.  K+-stimulated PMSF  (2  mM)  incorporation,  carbachol-stimulated  77  increase  in  obtained slight  inositol  w i t h PMSF a l o n e  increase,  incorporation  not  into of  PI  nonspecific  stimulated  [3H]inositol  3.5.2  Both  and  effect  1973;  is  made  K+  is  above  Addition  to  0.4  mM  carbachol  that  and  occurring  R+-  through  relaxation  (Fig.  22a).  PMSF  added p r i o r  to  the  the  d e g r e e of  the  time  contracting  the  elapsed agent,  interval.  or K + ,  phasic  inhibition between  of  the  the  to  the affect  pig  ileum  strips,  transient  was  achieved  levels,  even  at  in 8 minutes.  When PMSF was  carbachol  K+,  to  phasic  was  or  both agents.  addition  the  PI  contraction.  relaxation  responses of  from  produced a r a p i d  of  and no i n h i b i t i o n  In c o n t r a s t  guinea  If  PMSF w i l l  returned to c o n t r o l  addition  initial  that  of  (Chang  1979).  mobilization,  concentration,  the  mobilization  K+-stimulated,  The peak  tension  contraction  Triggle,  2 mM PMSF t o  by c a r b a c h o l  highest  and  not  either  1 min and t h e  by C a + 2  calcium  contracted  time  first,  incorporation  a biphasic  one would e x p e c t  but  of  mediated  in  carbachol-stimulated,  inhibited  second,  produce  Rosenberger  involved  observations  the  and  inositol  a  [3H]inositol  suggested,  i n c o r p o r a t i o n may be  and  i l e u m , which  Triggle,  within  This  that  PMSF c a u s e d  in K+-stimulated  21b).  to  Of PMSF On C o n t r a c t i o n  carbachol  pig  turnover  In c o n t r a s t ,  187%,  (Fig  similar  mechanisms.  Effect  guinea  to  21a).  to a value  carbachol-stimulated  a  different  (Fig  from 167  PMSF i n h i b i t i o n is  incorporation,  it  also  However,  r e s p o n s e depended on of  PMSF  observed  transient  and  after  nature  of  a  the 10 min  the  PMSF  78  effect  on c a r b a c h o l - m e d i a t e d  its  inhibition  incorporation period,  of  i n the  suggesting  contraction  in  guinea  pig  carbachol-stimulated  same t i s s u e that  was  these  [3H]inositol  effective  two  ileum,  over  effects  of  a  30  PMSF  min  may be  unrelated. The specific  suggestion  that  phospholipase  evidence.  It  was  to  test  inhibition this,  is  capable  C d u r i n g PI  turnover  necessary  carbachol-stimulated due  PMSF  of  the  PI  to  verify  turnover  Pi-specific  effect  of  of is  whether  in guinea  inhibiting b a s e d on  indirect  PMSF i n h i b i t i o n pig  phospholipase  PMSF on i n o s i t o l  PI-  ileum is  C.  In  lipid  of  indeed  order  to  breakdown  was  invest igated. 3.5.3  Effect To  Of PMSF On I n o s i t o l - P h o s p h a t e  determine  the  effects  of  Accumulation  PMSF  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  was  as  used  a measure of  the  In p r e l i m i n a r y e x p e r i m e n t s decrease inositol  phosphates  than  been  shown  20% to  e x a m i n e d whether  indicated in  of be  the  inositol  r e s p o n s e of shown  to  [3H]inositol  accumulation  into  [3H]inositol  inositol  monophosphate  PMSF s p e c i f i c a l l y  PMSF c a u s e d  a c c u m u l a t i o n of  the  turnover. 18%  the  accumulated  lithium  PI  phosphates  an  of  cause  (Table  (Fig  VI).  As  phosphates  have  19),  it  was  i n h i b i t e d the a c c u m u l a t i o n  fraction.  mono- and p o l y - p h o s p h a t e that  was  inositol-lipid  i n the presence  i n o s i t o l mono-phosphate  inositol  PMSF  in carbachol-stimulated  less  the  primary  on  from  a similar inositol  Selective an (14.8 mono-  ion and and  e l u t i o n of exchange 16.4%)  of the  resin  decrease  poly-phosphate  79  pool, only of  but i n the  the  mediated  the  d e c r e a s e was  latter  PMSF  case.  inhibition  through  statistically  This of  inhibition  suggested  significant  that  only  carbachol-stimulated of  Pi-specific  a PI  (p<0.05)  small  part  turnover  phospholipase  C.  is  80  Figure  4 - Phospholipid  Two d i m e n s i o n a l t h i n  layer  atrial  in  Lipids  phospholipids were  described were  first  hydroxide plates  four  developed (17:7:1  were  chloroform/ v/v).  on  chromatographic  Merck  Lipids  corners  of  v/v).  acetone/  After  quadrants  of  from the  left four  in  the  TLC p l a t e .  t h e TLC p l a t e .  gel  or  right  gel  plate.  10.5  the  rat  plates. atria  Plates  M ammonium  ammonia by a i r  acid/  water  by e x p o s u r e b)  to  T r a c i n g of  as  samples  drying,  second dimension c o n s i s t i n g  methanol/ acetic  a  of  separate  silica  r e m o v a l of  P h o s p h o l i p i d s were d e t e c t e d of  rat  silica  in chloroform/ methanol/  developed  Photograph  separation  precoated  from a s i n g l e  i n the Methods.  spotted  were  a)  extracted  separation  of  (3:4:1:1:0.5 iodine  one o f  vapour, the  four  81  82  Table I label  Lipids  - 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 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  were  extracted  [32P]phosphate  for  from a t r i a 120  following  min.  determined  before  separation  of  the  phospholipid  once  with  2 ml of  extracted in  Methods),  eluant. in  and phosphorus  The amount  the four  and  spotting  Total  4210  of  cpm  samples  present  determined. to  contain  reported Spot  and  per  phosphorus four  Mg  per g tissue  lipid  of  atrial  phosphorus  (as  heart  (White,  was  described in  the  radioactivity were  the  2.5  amount  from  lipid  After  gel  determined  samples B)  total rat  was  and t o t a l  In  of  phosphorus  silica  phosphatidylcholine  470  40^Ci  medium  and r a d i o a c t i v i t y  A s one g wet w e i g h t 4 7 0 Mg o f  the  the e x t r a c t i n g  from  with  on t h e T L C p l a t e .  spots,  respectively.  phosphatidylinositol  lipid  the sample  total  phospholipids  incubation  16  1973),  of  atrial  phosphorus  has been  Mg  was  reported  results  are  weight.  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 i n o s i t o l  16.3 ± 0.9  17.4  Phosphatidylchol ine  126.6 ± 4.0  169.2  83  Figure The  standard  dihydrogen observations  5 - Standard  curve  for  phosphate. from  one  of  curve  phosphorus Points  are  several  such  for was the  phosphorus. obtained with mean  experiments.  of  sodium  duplicate  84  .6n  0  .2  .4  Microgram phosphorus  .6  85  Figure 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 . Incorporation one h o u r .  of  [32P]phosphate  Results  incorporated  are  per  Histograms  calculated  microgram  and H r e p r e s e n t s  experiment.  the  of  Bars  of  series  results  atria.  of  the  phosphorus.  were  (A), (E)  Histograms  phospholipids  mean  (±  S.E.M)  obtained  of  radioactivity  phospholipid  in rat  represent  Similar  amount  a period  phosphatidylcholine  (G)  same  the  over  phosphatidylserine  (C),  phosphatidylethanolamine  observations.  as  represent  phosphatidylinositol  ventricles.  was measured  in  and B,  D, F  from  rat  of  three  one  other  CPM/MICROGRRM PHOSPHORUS cn oi  87  Figure  7 - E f f e c t of c a r b a c h o l and methoxamine on i n c o r p o r a t i o n i n t o PI i n c o m b i n e d r a t a t r i a .  Combined  rat  atria  carbachol  (B),  with  methoxamine presence  plus  of  parentheses pooling  control.  20  into Bars  mM  data  statistically  (C) (D)  orthophosphate. in  represent the  methoxamine  phentolamine  uM  PI  represent  together  represents  0.1  incubated with buffer  [32P]sodium  incorporation phosphorus.  were  the  control  (A), with or  from  size.  three  significant  with  for  sample  Results separate increase  0.1 mM 0.1  60 min i n  The  t h e mean ± S . E . M .  sample  [32P]  mean was  The  863  mM the [32P]  cpm/Vg  numbers  in  were o b t a i n e d by experiments. (p<0.05)  * over  SPECIFIC RADIOACTIVITY (Z CONTROL) cn ca  _L_  ca ca i  cn at  _L_  Cl at  CD CD  n  89  Figure 8 - E f f e c t of methoxamine s t i m u l a t i o n on [32P]phosphate incorporation into various phospholipids combined r a t a t r i a . Combined  rat  methoxamine for  atria  or w i t h  were 0.1  incubated  of  (G),  phospholipids  from  represents  phosphatidylinositol  S.E.M.  and  four  represent  methoxamine-treated  phentolamine-treated of  H  respectively,  B,  plus  samples.  MM  phentolamine  orthophosphate.  phosphatidylcholine  and h i s t o g r a m s  the  D, F  20  0.1 mM  phosphatidylserine  (C),  phosphatidylethanolamine  without  54 y C i [ 3 2 P ] s o d i u m  represent  phosphatidylinositol  or  mM methoxamine and  75 min i n t h e p r e s e n c e  Histograms  with  in  sample.  (E)  in control  the  same  samples.  obtained Bars  (A), and  samples,  series  of  H i s t o g r a m DP  from  represent  methoxamine t h e mean ±  90  AB  CD DP E F  6 H  91  Figure 9 - E f f e c t o f 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 e p a r a t e r a t l e f t and r i g h t a t r i a . Left  and  right  [32P]phosphate for  60 m i n .  atria  in  the p r e s e n c e  Histograms  methoxamine-treated respectively. control. phosphorus. from  a  significant  Bars  A , B and  tissue  Results  Mean  were  are  of  single increase  of  buffer  C,  left  D  into  or  (p<0.05)  *  0.1  percent  left  control  of  a t r i u m was of  represents  over c o n t r o l .  with  mM methoxamine  and r i g h t  t h e mean ± S . E . M .  experiment.  separately  represent  (A,B)  e x p r e s s e d as  incorporation represent  incubated  and  (C,D)  atria,  left  atrium  3550  four  cpm/uq  samples,  statistically  SPECFIC RADIOACTIVITY CX CONT ROL) ro tn ca  JL_  CD  ca  tn ca _1_  ca ca _L_  —I  K>  tn ca  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 s e p a r a t e r a t l e f t right a t r i a . Left  and r i g h t  orthophosphate 60  min.  atria i n the  Histograms  carbachol-treated respectively. cpm/Vg  two  Mean  separate  significant  A,  B  of  buffer  and  C,  (A,B)  the  represent sample  increase  with  0.1  [32P]sodium  mM c a r b a c h o l  represent right  rat  (C,D)  left  *  Results  the  and  atria,  a t r i u m was  1612  Numbers  are  represents  for  control  t h e mean ± S . E . M .  size.  over  or  D  and  experiments.  (p<0.05)  separately  incorporation into Bars  indicate  incubated  presence  left  phosphorus.  parentheses  were  and  pooled  in  from  statistically  corresponding  control.  94  250 -.  o  (8) (9)  200-  o u  (8)  M  150-  cc cc ft:  (7)  4  U  C 50 in  B  95  Figure  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 with [ 3 2 P ] p h o s p h a t e i n combined rat a t r i a .  Combined  atria,  orthophosphate period  with  or  preincubated  for  2  without  h,  were  0.1  mM  radiolabeled-phosphate. phosphatidylserine, and  B,  phospholipids  D,  carbachol A,  juCi  F  and  H  in  a  in  the  C,  represent  carbachol-treated of  three  the  samples,  [32P]sodium  for  phosphatidylinositol,  from  t h e mean ± S . E . M .  40  incubated  Histograms  phosphatidylethanolamine  histograms  with  E  further  45 min  absence  and G  of  represent  phosphatidylcholine control the  same  samples. from a s i n g l e  sample,  and  series  of  Bars  represent  experiment.  CPM/MC IROGRP10M PHOSPHORUS  Ca ca  ca  _L_  Q  ca ca  JL.  ca ca ca  JL.  ca ca ca  Ji  n CD  J cn  rn  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 prelabeled with arachidonic Combined  atrial  [1"C]arachidonic 1% b o v i n e period  acid,  with  buffer  parentheses two s e p a r a t e  (A)  or  w i t h 0.1  represent  indicate  the  experiments.  mean  buffer  further  mM c a r b a c h o l  sample was  the  prelabeling  with Krebs  incubated  i n the c o n t r o l  Bars  after  were washed  serum a l b u m i n and  incorporation weight.  preparations,  on p h o s p h a t i d y l i n o s i t o l acid in rat atria.  ±  sample s i z e .  a  (B).  175 cpm/mg of  Results  containing  for  S.E.M. are  with  15  min  The mean wet  tissue  Numbers  in  pooled  from  98  99  Figure Thin fatty  layer  chromatographic  acids  (80:15:5).  13 - S e p a r a t i o n  and a,  b,  c  and  diacylglycerol.  separation  phospholipids  phosphatidylcholine, respect i v e l y .  of  d  in  of  from  benzene/chloroform/methanol  represents  myristic  diacylglycerol  acid  phosphatidylinositol, and  1,2-dioleate,  100  o  ©  a  b  101  Table  II  Incubation  - 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 c o n t e n t of d i a c y l g l y c e r o l .  of  composition expressed tissue)  tissues  is  described  i n uq/q were  size  arachidonic  tissue.  Results given  acid  in  was  determination  i n the Methods.  Fatty  from  are  the  fatty  e x p r e s s e d as  parentheses.  observed  i n one  A other  0.168 ± 0.029  fatty acid  n.s.  41.4  ±  Cl8:0  41.8  ± 7.6  Cl8:l  content  of  the  mean ± S . E . M of  the  similar  absence  9.16  ± 0.6  8.3 ± 1.8  ^20:4  nil  Not s i g n i f i c a n t l y  different  of  experiment.  Carbamylcholine ( 0 . 1 mM) (4)  5.2  Cl8:2  is  (ymoles/g  0.142  ± 0.025  Fatty acid composition of diacylglycerol (ug/g tissue) Cl6:0  acid  content  levels acid  the  Control  Diacylglycerol content (umoles/g tissue)  of  The d i a c y l g l y c e r o l  calculated  diacylgycerol. sample  and  on a r a c h i d o n y l  from control  28.4 ±  3.2  22.0 ± 3.9 8.6 ± 10.2  ±  nil (p >  0.05).  0.71 2.5  (5)n-s*  1 02  Figure  14 - Gas c h r o m a t o g r a m of f a t t y diacylglycerol.  A gas  c h r o m a t o g r a m of  from  a carbachol-treated  and d c o r r e s p o n d linoleate.  Peak  to e  fatty  a c i d methyl e s t e r s  rat  methyl is  represents  the p o s i t i o n  would have  eluted.  a c i d methyl esters  the  atria  is  shown.  palmitate, internal  where m e t h y l  of  diacylglycerol  The p e a k s a ,  stearate, standard  arachidonate  of  b,  oleate and if  point  c  and f  present,  103  1 04  The  Figure  15 - F a t t y a c i d f r a c t i o n of r a t  polar  lipid  layer  was  extracted  composition A2,  fraction  chromatography  v/v)  A3,  remaining  atria.  Histograms  from t h e  silica  by gas  of  represent different  fatty the  acids  mean  (p<0.05)  ±  the  origin after  V2, from  the  plate  fatty  acids  acids,  V3,  and  its  thin  (80:15:5 fatty  acid  Histograms  A1,  palmitic,  stearic,  respectively,  in  rat  V4 and V5 r e p r e s e n t  the  same  rat  S . E . M of  from t h e  gel  chromatography.  arachidonic V1,  at  lipid  benzene:chloroformrmethanol  A4 and A5 r e p r e s e n t  l i n o l e i c and  atria.  in  determined  oleic,  series  c o m p o s i t i o n of t h e p o l a r a t r i a and v e n t r i c l e s .  six  ventricle samples.  corresponding  fatty  samples. *,  Bars  significantly  a c i d content  in  105  50 -i  40-  30-  i  20-  10-  fll VI fl2 V2  A3 V3 fl4 V4 fl5 V5  1 06  Figure incorporation  Guinea with  0.1  atropine  pig  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 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 m u s c l e of g u i n e a p i g i l e u m . ileum  mM c a r b a c h o l (C)  orthophosphate. the  control  for  (B) 60  results  the were  were or  min  The mean  sample was  mean ± S . E . M of Similar  pieces  incubated  w i t h 0.1 in  the  sample  presence  obtained  of  i n one  and  indicated other  1.6  i n PI  Results in  (A), uM  [32P]sodium  incorporation  cpm/yg p h o s p h o r u s . size  carbachol  mM c a r b a c h o l  [32P]phosphate  1638  without  are  in the  parentheses.  experiment.  107  108  Table  Guinea  I I I - E f f e c t o f c a r b a c h o l on [ H ] i n o s i t o l incorporation into phospholipid. 3  p i g ileum  pieces  p r e s e n c e and a b s e n c e containing addition in  of  atropine,  Methods,  [ H]inositol  in  3  radioactivity subtraction  following  the  absence  mean  incorporation ileum  of pieces  were o b t a i n e d  Treatment  min  preparations  5 min b e f o r e t h e out as  described  preincubation  fraction  was c a l c u l a t e d a f t e r  during  %  increase  the i n i t i a l  in  30 min  [ H ] i n o s i t o l ( " b a s a l " ) from t h e 3  The  radioactivity ±  3  shown other  in  S.E.M.  represents  The number o f  parentheses.  Similar  experiments.  dpm/pg total phosphorus  % Increase  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)  with  The  [ H]inositol  i n four  In  drugs.  incorporated  is  3  was added  30  of  period.  [ H ] i n o s i t o l i n the  was c a r r i e d  a  of the t i s s u e with  60 min i n c u b a t i o n  with  carbachol.  i n the p h o s p h o l i p i d  total  results  mM  Incubation  of the counts  preincubation  guinea p i g  0.1  16 MM a t r o p i n e  of c a r b a c h o l .  the  were i n c u b a t e d  109  Table  IV - C a r b a c h o l - s t i m u l a t e d  accumulation  of  inositol  phosphate. Guinea  pig  containing presence  ileum  [3H]inositol, or  accumulated formic  as  the  shown  absence  radioactivity  was  plus  mean ± S . E . M .  of  mM  lithium  60 min i n a medium  eluted  (see  from t h e  phase.  The number o f  guinea  Similar  and  Methods).  resin  with  Phosphorus  organic  parentheses.  the  for  without carbachol,  1 M ammonium f o r m a t e .  in aliquots  other  incubated  w i t h or 10  in the  several  were  of  acid  determined  pieces  results  Total 0.1  are  experiments.  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)  (6)  M was  given  ileum pieces  were  492.0 ± 34.0  the  content  Results pig  in  obtained  is in  110  F i g u r e 17 - D o s e - r e s p o n s e c u r v e f o 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 accumulation i n guinea p i g i l e u m . The  dose-response  phosphate lithium,  curve  accumulation with  t h e mean of  (•)  or  duplicate  for  carbachol-stimulated  was d e t e r m i n e d  without  (•)  incubations  6.4  i n the  presence  MM a t r o p i n e .  from a s i n g l e  inositol of  10  Each p o i n t experiment.  mM is  111  •j—i 11 mm—i i iimi|—i i M I I M |  1  i i M I " 4 — « ' ' »"1  2 3 4 5 Log CorbomylcholIne (nM)  6  1 12  Figure  18 - E l u t i o n  200  Ail  of  salt  was  loaded  column. with plus  11  onto a  plus  60 mM ammonium f o r m a t e  by p h o s p h o r u s  20,  dl-monocyclohexylamine  Dowex-1 X 8 - f o r m a t e  mM ammonium f o r m a t e  uq p h o s p h o r u s and  i n o s i t o l mono-  ion  column was washed w i t h w a t e r ,  5 mM b o r a x  identified 14.2  1 mg/ml i n o s i t o l - 2 - p h o s p h a t e  The  150  p r o f i l e of s t a n d a r d phosphate .  mono-phosphate.  B.  determination  was e l u t e d  thereby  at  exchange  f o l l o w e d by e l u t i o n at  Inositol i n the  A and 5 mM  a complete  borax  monophosphate eluant  was  fractions.  from t h e column between  suggesting  resin  r e c o v e r y of  fractions inositol  Microgram Phosphorus 10 _I  to I  I  11 4  Figure  Inositol  19 - E l u t i o n p r o f i l e of t h e a c c u m u l a t e d phosphates i n guinea p i g i l e u m .  phosphates  accumulated  ileum  pieces  loaded  o n t o a Dowex-1  column with  was  with  at  determined  in  radioactivity  A,  B  .1  ml  eluted  phosphates.  In a  strength  and  presence  ion-exchange  M formic C,  acid  of  the  plus  pig  lithium  were  column.The successively  1  M  B  and  the  relative  amount  plus  The  solution of  two  was peak  C  were  inositol  control unstimulated  p e a k s on e l u t i o n w i t h  150  ammonium  Radioctivity  eluant.  solution  experiment  guinea  5 mM b o r a x  with  radioactivity  of  resin  respectively.  fractions  similar  solutions.  the  5 mM i n o s i t o l and e l u t e d  and 0.1  determine  no  i n c u b a t i o n of  60 mM ammonium f o r m a t e ,  compared t o  showed  in  X8-formate with  plus  mM ammonium f o r m a t e formate  carbachol  washed  5 mM b o r a x  during  inositol  increasing  tissue ionic  1 15  Frocrtlon Number  1 16  T a b l e V - C o m p o s i t i o n of  inositol  guinea Guinea  pig  ileum fragments  [3H]inositol min.  Inositol  described ± S.E.M. obtained  with  lithium  were (10  phosphates  i n the Methods. from  pig  four  i n one o t h e r  phosphates  incubated  i n a medium c o n t a i n i n g  mM) and c a r b a c h o l were  eluted are  samples.  (0.1  from  mM) f o r  60  resin  as  the  e x p r e s s e d as Similar  Percent r a d i o a c t i v i t y eluted (%Y  5 mM Borax + 60 mM ammonium formate  6.4 ± 0.4  5 mM Borax + 150 mM ammonium formate  16.9 ± 1.7  0.1 M Formic acid + 0.4 M ammonium formate  50.8 ± 0.9  0.1 M Formic acid + 1.0 M ammonium formate  24.3 ± 0.7  t h e mean  results  experiment.  Elution medium  in  ileum.  The r e s u l t s  tissue  accumulated  were  11 7  F i g u r e 20 - Time c o u r s e of t h e 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 into p h o s p h o l i p i d in guinea p i g ileum. Ileum  pieces  were  30 m i n .  Further  and  the  in  samples.  are  incubation  absence  Radioactivity results  preincubated  was  (•)  of  was  as  carried  2 mM PMSF,  determined  reported  w i t h 30  dpm/yg  in  the  lipid  uCi of out as  [3H]inositol  i n the shown  lipid  presence  i n the  fraction  phosphorus  for ( • )  figure. and  ± S . E . M of  the three  118  11 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 g u i n e a p i g ileum. a)  After  preincubation  ileum pieces  were  further  (A),  PMSF  (B),  plus b)  2  mM  2 mM PMSF  After  ileum  were  60 mM p o t a s s i u m  Bars  represent  in  the  was  phosphorus other  0.1  the  of  (B)  for  with [ 3 H ] i n o s i t o l  30 min w i t h no  mM c a r b a c h o l  (C)  pieces  incubated  and  21b).  experiments.  with  for  0.1  the  addition  mM c a r b a c h o l  The  The mean  during  the  phosphorus Similar  [3H]inositol  plus  numbers  2 mM PMSF in  [3H]inositol last  (Fig  results  the  30 min w i t h no a d d i t i o n  and 60 mM p o t a s s i u m  sample s i z e .  dprn/Vg l i p i d (Fig  ileum  t h e mean ± S . E . M .  c o n t r o l experiment  180  incubated  further  (A),  represent  ileum pieces  (D).  preincubation  pieces  of  30 min  21a)  and  were  (C).  parentheses incorporation  of  incubation  166 dpm/yg obtained  in  lipid two  (10)  2M-i  (11)  200-i (11)  I  (7)  150-  (5)  150-  O  (10)  (9)  100-  100-  I  50-  in o  fl  0  C  D  0-  A  B  C  121  Figure  Guinea  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 g u i n e a p i g i l e u m pig  i l e u m was c o n t r a c t e d  mM p o t a s s i u m . (a).  PMSF  transient tracing PMSF  with  2 mM PMSF was added as inhibition  nature  i n each  of set  addition.  this  of  the  effect  shows  is  Following  this,  t h e arrow and t i s s u e  later  K*.  stimulated added  1  Dose  phasic min  expressed compared t o  as the  dependent  response  before  is  indicated  phasic  by  the  contraction  shown  0.4  in  (b).  or  mM  The  PMSF  was  1,2  or  inhibition  by  PMSF  (c).  with  inhibition  PMSF-untreated t i s s u e .  of  PMSF  (0.4  60 mM K + . the  60  arrows and  restimulated  shown i n  stimulation  fractional  1MM c a r b a c h o l  the first  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  i n d i c a t e d at with  either  added 10 of  to as min K+-  mM)  was  Results  are  phasic  response  122  123  T a b l e VI - E f f e c t of PMSF on t h e 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 p h o s p h a t e s • i n guinea p i g i l e u m .  Incubation  with  phosphates  [3H]inositol  (TIP),  and d e t e r m i n a t i o n  i n o s i t o l mono-phosphate  (IP)  and t r i - p h o s p h a t e s  (IP2/IP3)  were d e t e r m i n e d  1OmM  described  i n the Methods.  lithium  concentration  as of  2 mM i n d i m e t h y l s u l f o x i d e .  PMSF c o n t a i n e d an e q u i v a l e n t Results  are  measured  i n one e x p e r i m e n t  another  experiment.  The number of  reported  is  of  are  and  dpm/ug l i p i d  used  The c o n t r o l  experiments.  i n the  as  were  without (1%). T I P was  measured  t h e mean ±  parentheses.  % Decrease  phosphorus  (3)  167.8 ± 11.6  (5)  18.5**  IP  22.3 ± 1.7  (7)  18.9 ± 0.8  (11)  14.8**  111.5 ± 6.5  (7)  93.2 ±  (11)  16.4*  p < 0.05 0.05 < P < 0.06  3.6  in  S.E.M.  206.1 ± 8.0  * **  of in a  TIP  IP2/IP3  bi-  presence  PMSF was  Carbachol + PMSF  Carbachol  inositol  and i n o s i t o l  i n the  IP3  reported  indicated  total  dimethylsulfoxide  two s e p a r a t e  and I P 2  Results  samples  Inositol phosphate  from  amount  of  -  124  IV.  4.1  IS  DISCUSSION  MUSCARINIC RECEPTOR STIMULATION IN THE HEART ACCOMPANIED  BY A PI RESPONSE?  4.1.1  Phospholipid Separation As  mentioned  increase  in  r e s p o n s e of valuable r a n g e of of  tissues.  inositol  turnover for  of  phosphate PI  Two-dimensional Luukkonen, by  many  1976), other  is  on p h o s p h a t e studied,  to  the  studies  the  the  first  a  very  in a  wide  primary by  step  enhanced  Therefore,  for  were  per  phosphate  method  to  study  effect  our  of  into  using 4.  PI  of  two d i m e n s i o n a l  thin  of  reported  in to  separation  The  muscarinic  and past  calculated  and  ability  (Renkonen i n the  the  microgram  content  incorporation verify  used  separation  incorporated  First,  one)  used s u c c e s s f u l l y  shown i n F i g  p h o s p h o l i p i d phosphorus control.  response  chromatography  been  was  such  incorporation  radioactivity  secondary  as  accompanied  as  a  served PI  PI.  chosen  layer  w h i c h has  chromatography  phosphate  thin  One  is  is  heart.  groups,  phospholipids. layer  was  of  (except  into  PI  has  occurrence  tissues  receptor-mediated  into  it  breakdown  in rat  though  cycle,  the  In a l l  into  response  even  incorporation  lipid  incorporation PI  PI  indicator  reincorporation  the  earlier,  phosphate the  And T e c h n i q u e V e r i f i c a t i o n  results as  the  the as  pig  o b s e r v e a PI  the  amount  of  individual percent  receptor  guinea  of  of  stimulation ileum  response.  was In  1 25  accordance  with a previous  carbachol-stimulated observed,  which  atropine  (Fig  phosphate  was  16).  The b a s a l  corresponds  to  value  to  (Jafferji  by  the  incorporation  previously  of  PI  1976a),  into  muscarinic  51 cpm/nmole of  reported  and M i c h e l l ,  incorporation  blocked  phosphorus the  report  cpm/^g of  is  (Jafferji  was  antagonist,  1638  and  PI  PI  comparable  and  Michell,  1976a,b). 4.1.2  Basal  [32P]phosphate Incorporation  Phospholipids 4).  By  were a l s o  comparison  with  phosphatidylinositol,  study,  only  it  phosphate  incorporated  incorporation  atria the  of  and  distribution  incorporated  into  the  50% of four  Brown  incorporation was  i n the  into  the  of  fraction  into  phospholipids  higher  that  of  than  ventricle.  The  cause  the for  have  was of  Fig  identified  as  into  6),  PI  was  in a t r i a  was  this  (Table  I). a  In b o t h  in that  various tissues,  radioactivity radioactivity 4-6  fold  corresponding  phospholipids  in  this  in basal  difference  at  60%  atria.  observed into  the  PI  radiolabeled-  i n mouse  total  However,  in  phospholipids  it  similar. the  Although  reported  radioactivity  phospholipids.  incorporated  total  phospholipids  (Fig  (see  measured  into various  two t i s s u e s  a large  acid.  the  (1982)  compared  profile  were  phospholipids  almost  atria  phosphatidylcholine,  phosphatidic  four  and  ventricle  incorporated  spots  radiolabeled-phosphate  phosphate  phospholipids PI  the  Brown  from r a t  standards,  and  incorporated  Similarly,  When t h e  6% of  Heart  phosphatidylserine,  phosphatidylethanolamine comprised  separated  In  least  phosphate  1 26  incorporation but  a  into atria  similar  ventricle  in to  rat  atria  Effect  Stimulation In  has  1982).  as  well  as  atria  The  greater  for  further  rat  receptor  phosphate  In Rat  atrial  pieces,  t o PI  of  consistent  with  response  in guinea  receptors et  density  1978),  to  led  and  pig  (1982)  receptor-mediated myocytes.  or c a t  They  incorporation  PI  alpha-  response  that  This  epinephrine  [32P]phosphate and  in v i t r o  1975).  /3  of et  The  rather  al.,  have o b s e r v e d  further  and c a l c i u m - i n d e p e n d e n t  of  The  lack  into  (Gaut of  and this  v e n t r i c l e u s i n g n o r e p i n e p h r i n e was  (Lapetina  phosphate  increase  stimulation.  observation  in vivo  Farkas,  richness  in v e n t r i c l e al.  both  alpha-  7).  i n d i c a t i n g that  incorporation  Kiss  the  and  (Fig  i n phosphate  receptor  the p r e v i o u s  increased  1966;  rat  phentolamine,  phosphatides,  attributed  receptor  and c o u l d be b l o c k e d by t h e  alpha-adrenergic  Huggins,  Uchida  (Fig  antagonist,  result  inositol  label  muscarinic  respectively  8),  an  of  s t i m u l a t i o n p r o d u c e d a 35% and 88%  incorporation into PI,  specific  caused  extent  and  Atria  was  was  in canine a t r i a  study.  enhancement  the  present,  (Wei and S u l a k h e ,  alpha-adrenergic-mediated  was  known a 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 R e c e p t o r  combined  adrenergic  not  the h i g h e r m u s c a r i n i c  On PI T u r n o v e r  adrenergic  is  been o b s e r v e d  compared t o v e n t r i c l e  t h e c h o i c e of  4.1.3  in  difference  (Quist,  incorporation,  and v e n t r i c l e  1976).  a similar  incorporation into characterized  (Uchida et  than  al.,  the  1982).  a-adrenergic  More  recently,  alpha-adrenergic PI  in  response  cultured to  be  a,  127  As t h e r e  was  incorporation a  receptor  almost  i n t o PI  density  density  smaller  muscarinic  PI  4.2.1  less  tissue,  effect  was  indicated  nearly  always  relationship  between is  study  effect  the  earlier,  than it  not  50%  phosphate  pieces, of  was c l e a r l y due t o an  in  which  the  have  muscarinic  evident  that  i n a b i l i t y to  the  detect  response.  still  (discussed  known.  it  muscarinic  testing  breakdown of  reported  parotid  (Egawa  lipid  for  lipid  resynthesis  breakdown,  breakdown and was  its  therefore  stimulation  two o t h e r  the  of  precise  subsequent decided  PI  to  breakdown,  probable  mechanisms  below).  Increased been  not  though compensatory  inositol  inositol  of  simultaneously  PI  even  accompanies  resynthesis  1979),  atrial  i n c o r p o r a t i o n d u r i n g a PI  Breakdown Of P r e l a b e l e d  while  enhancement  BREAKDOWN STUDIES  As  has  is  in this  enhanced phosphate  two-fold  i n combined r a t  which  receptor  4.2  a  et  in blowfly  gland  experiments,  combined  atria in  prelabeled  PI  Following  this  with  the  o r any  salivary  among  preliminary  enhancement  PI  0.1  loss of  it mM of the  several was  d u r i n g the  gland  ( J o n e s and M i c h e l l ,  a l . , , 1981),  rat  prelabeled  that  p r e l i m i n a r y experiment  Berridge, deferens In  stimulation  of  to  cause  an  [32P]phosphate-  phospholipids with  response  tissues.  failed  from e i t h e r  other  and vas  other  carbachol label  ( F a i n and  1975),  observed  PI  (Fig  11).  phosphate-prelabeled  phospholipids, -arachidonyl-prelabeled phospholipids  were  used,  1 28  for  the  following reason.  c a u s e an  increase  Nawrath,  1976;  guanylate  is  Diamond  et  prostaglandins could been  be  al.,  derived  shown t o  thrombin  (Billah  from  stimulation  1979),  investigated  it  was  occurred  during  guanylate If  PI  by  either  by  a  the  Stimulation  of  receptor  a possible  activators  (Glass  al.,  et  of  and  Asano,  1977)  acid  has  stimulation  by  and l i n o l e i c a c i d (Franson  of  al.,  1980)  arachidonic  release  during et  al.,  arachidonic  acid  combined r a t  mechanism f o r  d i d not (Fig  C A2  12)  level  of  the  atria,  activation  arachidonyl-prelabeled  activity  of  receptors  enhance  the  This  d i d not  . cause  or p h o s p h o l i p a s e  PI,  total  .  during  of  A 2 or p h o s p h o l i p a s e  i n the  muscarinic  stimulation  phospholipase  i n the  specific  PI  phospholipase  breakdown  decrease  mM c a r b a c h o l  prelabeled  of  stimulation  phospholipase  if  0.1  even  et  sarcolemma  whether a  i n the  would c a u s e a d e c r e a s e  PI,  acid  of  cyclase.  stimulation  reflected  canine  carbachol  which c o u l d then suggest of  of  1973;  cyclase  proposed  platelet  1982a)  al. , ,  activation  H i d a k a and  during  receptors  guanylate  Spies  As r e l e a s e  and L a p e t i n a ,  isoproterenol  for  1978;  et  the  Among s e v e r a l  al.,  PI  muscarinic  by  arachidonic  from P I .  occur  1977)  1978; et  of  (George  mechanism  two,  (Goldberg  heart  al.,  unknown.  cyclase,  Gabers  in  et  The  still  guanylate  1977;  cGMP  cyclase.  activation for  in  Stimulation  C occurred,  which would  radioactivity PI  remains  breakdown  of  indicated  that  A2.  muscarinic  The l a c k  be  present  atria  with  arachidonylmuscarinic  activation of  in  unchanged.  i n combined r a t  significant  then  it  activation  stimulation  was  of of not  1 29  surprising,  as  stimulation there  is  a  of  not  cardiac  A2  4.2.2  stimulation  (Franson  activation is  calcium i n the  that  the  isoproterenol  al. , ,  of  1979),  where  phospholipase  result  and t h e is  et  of  of  an  available  mobilized  A2,  increase  evidence  during  in  does  muscarinic  heart.  Arachidonyl-Enriched Diacylglycerol  the  be  phospholipase  formation  I e y a s u et to  except  sarcolemma  activation  that  Pi-specific to  cases,  calcium concentration  indicate  receptor  most  receptor-mediated  phospholipase cytosolic  in  al.,  of  1982;  diacylglycerol K a i b u c h i et  rapidly phosphorylated  phosphatidic  acid  diacylglycerol phospholipid quantities produced seconds  is  also  a  (Billah  and  increase,  during  carbachol  stimulation  still  longer  increased  (1983).  A c c u m u l a t i o n of  indicate  the  easily  existence  accessible  to  over of  of  of  As  found  On t h e  other  (Bansback  small  within  which  i n the m e t a b o l i c  and  hand,  a  observed et  al.,  over  by Homa e t  a longer  compartments  in  1980)  30 m i n , was  reported  other  stimulation  diacylglycerol  d i a c y l g l y c e r o l over  enzymes  1981).  and K o n d o ,  pancreas  r e c e n t . l y been  other  give  diacylglycerol  a p e r i o d of  cellular  to  Receptor of  considered  many  only  1982a).  mouse  is  kinase  for  is  (Igarshi  accumulation  p e r i o d has  which  Putney,  tissues.  Lapetina,  transient  and an  it  accumulation  slower  1974)  1982a;  intermediate  in both t h y r o i d f o l l i c l e s  platelets  ( B i l l a h and L a p e t i n a ,  1975;  pathways,  transient  leads  by d i a c y l g l y c e r o l  an  unstimulated  PI  al.,,1982),  (Michell,.  metabolic in  C - m e d i a t e d breakdown of  a  al.  p e r i o d may are  not  pathways.  1 30  Widespread  occurrence  dependent  protein  of  calcium-activated,  kinase  (PKC),  diacylglycerol,  has  diacylglycerol  d u r i n g breakdown  may c a u s e et  al.,  been and  the  increased  1974).  In  carbachol p>0.05) level  produce  (0.1  mM)  acid C,  a l . (1974).  of  production  of  al.,  muscarinic  response  phospholipid 1981;  Takai  stimulation  has  (Hokin-Neaverson,  that  (Bansback  1974)  et  i n combined r a t  only  a small  an  increase,  could acid  1973).  be  was  stimulated  combined  observation  that  combined r a t min  of  al., atria  (but  nonsignificant,  in  diacylglycerol  If  similar  detected  by in  atria  is  either  diacylglycerol, (Bansback  15-fold giving et  al.,  by  arachidonyl by  Bansback  arachidonyl  unstimulated-  or  of  small  increase of  PI  i n the  If  as  in  carbacholthe  initial  response  by 0.1  to a v a l u e 1974).  in  no  supported  increase rise  of  fraction  pancreas  rich  chromatography,  lack  may be o n l y a v e r y  a  observed  gas  is  degraded  i n the  nmole  further  of  being  that  0.5  The  Stimulation  produced  to  diacylglycerol  rat  tissue  increase  low as  tissue.  there  PI  an  detected  i n the  atria.  tissue  mammalian  one w o u l d e x p e c t  acid  nmole/g  PI  Even t h o u g h as  ester  of  that  or any o t h e r  observed  than  (White,  arachidonic  content  was  rather  by  diacylglycerol  caused  carbachol-stimulated  15  PI  regulation  PKC ( M i n a k u c h i e t  of  diacylglycerol,  arachidonic  for  of  its  2).  phospholipase  methyl  it  and  possiblity  b o t h a PI  j3-position  of  of  accumulation study  arachidonic  the  In mouse p a n c r e a s ,  this  (Table  content  .  decrease,  The  et  activation  1979b)  shown t o  raised  C  phospholipid-  in  mM c a r b a c h o l arachidonyl h i g h as a  290  similar  131  enhancement about  had  occurred  6 nmole of  arachidonic acid  t h e amount p r e s e n t detected  detection  fraction  of  atria  possibility  that  during  various  methyl  ester.  acid  ventricles contents  of  atria  1983). acid  of  all  but  as  was  completely  was the  polar  ventricle that  of  fatty  acids  a similar  of  this  difference  atria  and  reported  out  fatty  acid  saturated  fatty  from  was  i n some o f  observed  between  incorporation observed  the  destroyed  atria  and  in  the  observed  difference  (Fig in  (Charnock et  is  not  the  fraction  in unsaturated  ventricles  a role  as  lipid  i n the p h o s p h o l i p i d  been  phosphate  polar  difference  acids  has  could play  (1982).  fraction  fatty  study  of  w h i l e the  significant  unsaturated  compared t o a t r i a ,  Quist  that  lipid a  been  rules  i n the p r e p a r a t i o n  observed  similar,  between  lower  the  acid  t h e membrane c h a r a c t e r i s t i c s such  in  arachidonic  ventricles  it  acid  also  involved  have  14).  arachidonic  The s i g n i f i c a n c e  present,  should  15)  unsaturated  content  and i t  to  (approximately  (Fig  three  and  would c o r r e s p o n d  i n 20 mg t i s s u e  (Fig  In a more c o m p r e h e n s i v e  content of  were  it  and v e n t r i c l e  It of  heart,  incubation)  used  of  steps  contents  15).  i n each  by t h e methods  The  in rat  al., fatty  known  the d i f f e r e n c e s the  two  study  in  tissues,  into phospholipids in this  at  and  of in  1 32  4.2.3  [32P]Phosphate Incorporation All  of  the  evidence  muscarinic  either  no,  (1982)  or  receptors  that  response  on m u s c a r i n i c  or  or  right  interesting  unlike  the  in  far  canine  receptor  failed  independent  cells,  (Uchida et  in canine  incorporation  of  which  al.,  right  1982),  atrium  (as  calcium-dependent  (Quist,  1982).  only  possibility  that  Stimulation  of  stimulation an  incorporation It  was  also  radioactivity (compare  (35%)  right of  of  and  but  atrium  (Fig  alpha-adrenergic  approximately i n t o PI  observed into  rat  F i g 9 and  10).  that left  the  PI)  to  response. was  be  in  was  that effect  calcium  (p < rat In  .05) left  by 0.1  increase  left  and r i g h t  PI  raised  the  atria.  mM c a r b a c h o l  enhancement atrium,  in  but  to  no  this,  mM methoxamine in atria  phosphate (Fig  i n c o r p o r a t i o n of atrium  be  a  contrast  fold  right  of  in rat  by 0.1  PI  increased found to  atrium  atria  relative  and  atrium  inotropic  might e x i s t  receptors  in both rat  left  occurrence  10).  two  canine  shown  canine  significant PI  Quist  a t r i u m showed a PI  positive  into  right  into  time  observation  The  situation  left  incorporation  in rat  produced  part  similar  rat  produced a small  effect  a  one  that  d e t e r m i n e d by t h e  phosphate  in  was a c c o m p a n i e d by  the m u s c a r i n i c - s t i m u l a t e d  labeled  phosphate  his  Atria  stimulation  show a s i m i l a r  was  of  response  right  to  that  Around  stimulation,  characteristic  heart  atria  turnover.  while  And R i g h t  suggested  alpha-adrenergic-mediated  cultured rat  response  PI  ventricle,  Another  so  i n combined r a t  a very s m a l l ,  reported  left  obtained  In Rat L e f t  was  9). basal  variable  133  4.2.4  E v i d e n c e F o r Two S u b p o p u l a t i o n s  Results right  atrium,  suggested the  from t h e  the  as  to  receptors most  receptors  PI  stimulation  studied  muscarinic receptor  ,  may be c o u p l e d breakdown 11,  to  (Jones  cycle  gland caused a  that  the  media  a  four-fold the  enhancement level  of  PI,  In  alpha-adrenergic  the  receptor 1975). between  protein,  of  have  9 &  population atrium  inositol  lipid  of  atria  finding  (Fig  that  a  may be c o u p l e d t o  PI  reported  stimulation i n phosphate while  (Fig  in rat  stimulation the  Wei and Karliner  obtained  receptors  and  .relative  protein,  small  receptors  carbachol  turnover.  on  pmoles/g  The a b s e n c e  (1982)  not  muscarinic  efficiency  based  muscarinic  al  were  Michell,  responses  receptor  of  both a l p h a - a d r e n e r g i c  only  muscarinic  PI  and  may seem t o c o n t r a d i c t  of  decreasing  PI  and  (1982),  muscarinic  = 100 p m o l e s / g  turnover.  II)  for  of  to  coupling  220  relative  However, F a r e s e et  calcium-depleted  =  Quist  atrium  to  the  a t r i u m and  muscarinic  population  turnover.  without  PI  during  12 and T a b l e  small  tissue  left  w h i l e the m a j o r i t y  response  equal  by  population  than  adrenergic  of  PI  Receptors  separate  and r i g h t  small  (muscarinic  15%)  that  weaker  calculated  (approximately  left  the  an  and t h e  was  a  in rat  alpha  1982)) it  in rat  of  obtained  possibility  turnover  density  al.,  10)  PI  those  far,  same  receptors  Sualkhe,1978; et  so  assuming  and t h e  stimulation  a t r i u m may be c o u p l e d  usually  i n the  Nevertheless, receptor  left  is  stimulation  as  turnover,  in rat  tissues  well  interesting  muscarinic  coupled  carbachol  Of M u s c a r i n i c  of  that  in  submaxillary incorporation  in calcium-containing  1 34  media in  b o t h an  the  level  Therefore,  increase of  it  resulting  It  be  now  considered  to  (recognition  subunits  of  is  more  a  subunits)  can  be c o u p l e d t o  On t h e subunit  the  to  combination  receptor,  other  same t i s s u e  of  the  functional  can  one  recognition  a  receptors defines  and  subunit  receptors,  be  subunits,  Various if  of  these  than  of  that one  a  effector  e.g  which are  the  and E l - F a k a h a n y ,  a  has  been  single  systems receptors,  betacoupled  a,  may  and  physiological  norepinephrine  (Exton,  1982),  but  a,  is  suggested to  be m e d i a t e d  unit  a2,  in  even  only  in  very  coupled further  previously.been  The two  subclasses  have  similar  neurotransmitter,  they appear  (Garcia-Sainz  or  require  w h i c h have  the  systems  subunit  1981)  recognition  for  effector  recognition  recognized  b a s e d m a i n l y on b i n d i n g s t u d i e s . receptors,  same  effector  affinities  of  receptors  receptors  same e f f e c t o r  1982)  effector  alpha-adrenergic  response  breakdown  preparations.  subunit.  possiblity  (Richelson  (Hartzell,  different  different  that  muscarinic  most  both  H 2 , glucagon  hand,  subclassification  of  of the  Identification  classified  or  phosphate  cyclase.  tissues  recently.  and  appropriate,  may be c o u p l e d t o more  different  breakdown  two d i s t i n c t  and an e f f e c t o r  as  to adenylate  stimulation.  events  that  decrease  carbachol  population  recognized  receptor  adrenergic  PI  dissociable  be composed subunit  during  i n combined a t r i a l  well  recognition  i n c o r p o r a t i o n and a  that  such a s m a l l  be d e t e c t e d  is  observed  possible  may  from  not  was  is  incorporation  could  PI  in phosphate  to  be  and F a i n ,  coupled 1982).  by s t i m u l a t i o n  of  to The PI  1 35  turnover  w h i l e the  through  inhibition  1982;  of  systems  unit  has  hepatocytes. juvenile  rats,  mediated  only  adult  rats,  be  cyclase  coupled  provided  They  suggested  An i n t e r e s t i n g  can  been  is  adenylate  Levitzki,1982).  recognition  by  a2_receptor  by  observed  the  response  two  Morgan  et  of  was  its  effects  and  how  al.  Fain,  a  single  different  a,-receptor  stimulation  b o t h cAMP e l e v a t i o n  of  in hepatocytes  by c a l c i u m m o b i l i z a t i o n , but  c^-receptor  exert  (Garcia-Sainz  example to  that  to  effector  (1983)  in  derived  from  stimulation  is  in hepatocytes  shown t o be  from  accompanied  and c a l c i u m m o b i l i z a t i o n (Morgan e t  al.,  1983). Heterogeneity studies  has  Strange  et  al.,  Weight  channel  1977;  that  and s y m p a t h e t i c  of  neurones  et  al.,  may o r may n o t et  Quist  receptor.  al.  (1981)  muscarinic left  receptors  atrium  subtype  support is  which  (Quist, is  not  and  receptors  of  (Burgen  Snyder, in heart  have  1982)  et  binding  al.,  1974).  1974; Recent  (Hartzell,  1982)  Horn and  Dodd,  results,  proposed left  and  that right  may b e l o n g t o  c o u p l e d t o PI  type which  binding.  speculated  receptors  subclassification  in rat  on  heterogeneity,  recently  The p r e s e n t  further  1980;  by a n t a g o n i s t  muscarinic  a  based  may be c o u p l e d t o more t h a n one  be d i s t i n g u i s h e d  It  time  an e f f e c t o r - d e p e n d e n t  receptors.  (1982),  some  (Brown and Adams,  1979)  may be two s u b c l a s s e s adrenergic  for  receptors  Yamamura  muscarinic  indicates  Jones  muscarinic  been p o s t u l a t e d  suggestions  1981;  in  that  similar as  well  of  the  to as  there alpha-  those  of  muscarinic  the m a j o r i t y  of  atrium  canine  and  the m u s c a r i n i c  t u r n o v e r and  which  the  receptor will  be  1 36 called  m2.  receptors atrium  However,  i n mouse a t r i a (Quist,  receptors  1982)  in rat  receptors  in  reported)  many  of  levels  in cardiac may  (Garcia-Sainz to  protein  (Rodbell,  found  receptor  Fain,  1982),  cyclase  effect,  may  two  at  for  the  by  higher  effect.  types  of  of  requirements. response  heart Quist in  to  PI  muscarinic muscarinic  to  a,  of The  decrease Watanabe  adrenergic  2  been  turnover.  to  1973;  a  cAMP  et  al.,  receptors  receptors  inhibitory  multiple  may be  GTP  binding  of  and  them t o  alpha-adrenergic-stimulated  ACh  suggest  receptor  in  produced that  sites.  inotropic  to  reported  right PI  differ that  atrium response  a  there But  the  to  be  against  the  reponse  nature.  in  their  PI calcium  carbachol-stimulated  required in  They  negative  alpha-adrenergic-stimulated  appear  canine  Ach produced a  concentrations  nicotinic  receptor  al•,(1968).  or hexamethonium a r g u e d  or  (1982)  cholinergic  by B u c c i n o e t  cholinergic  atropine  muscarinicin  of  right  r e s p o n s e has  m2 m u s c a r i n i c  This led  responses being muscarinic  PI  to  ACh-mediated p o s i t i v e  either  responses  al.,  t h r o u g h an  were p r e s e n t e d  inotropic  The  coupled  et  canine  (analogous  stimulation  similar  suggestions  positive  blocked  is  m,  muscarinic  with  where a PI  w h i l e a low c o n c e n t r a t i o n  inotropic  inability  which  (along  of  1980).  in heart  that  be  subclass  the  that  adenylate  Previous subtypes  to  1982),  population  10)  tissues  (George  and  coupled  (Fig.  tissue  suggest  small  other  muscarinic  population  (Brown and Brown,  atrium  receptors)  ability  significant  and a v e r y  left  may b e l o n g  adrenergic  1978)  a  calcium,  cultured  rat  while heart  1 37 -  cells  was  f o u n d t o be c a l c i u m - i n d e p e n d e n t  Presently in  heart  with  the is  functional  not  their  possible  canine atrium), effect  this  on t h e  addition  of  the  subtype  such r e c e p t o r s , for  PI  along  response  positive  carbachol  note  (as  in  inotropic  following a prior  papillary  muscarinic  muscarinic  that  muscle  (Endoh  receptors  is  receptor  based  subunit  coupled  to  receptor  subtypes  may n o t  by b i n d i n g s t u d i e s . H o w e v e r , G i b s o n et  the m u s c a r i n i c as  of  effector  the  m, and c a u d a t e / p u t a m e n  al.  (1983)  have  in v e n t r i c u l a r  m 2 b a s e d on t h e i r  affinity  the  it  is  recently  muscle for  as  various  derivatives. It  reason two  is for  even  to  species 1979;  say  variation  regulation  by a PI  has  i n the  to  are  in  their  other  speculate  relative  subtypes  there  1980),  in these  Michell  response  receptor  that  Schumann,  role  more d i f f i c u l t  the d i f f e r e n c e s  muscarinic  except  a  of  subclassification  to  subclassified  of  small  in rabbit  be d i f f e r e n t i a t e d  interesting  the  1982).  1979).  subunit,  necessarily  QNB  for  al.,  t h e m, r e c e p t o r  calcium requirement  on  nature  recognition  of  the presence  w i t h methoxamine  and M o t o m u r a , As  but  may a c c o u n t  observed  stimulation  only  known,  significance  (Uchida et  in a t r i a receptors  of  various  which  of  the the  species, large  and V e r m a ,  differences  in  species.  previously predicted  that  if  PI  those  turnover receptors  m e d i a t e d by c a l c i u m m o b i l i z a t i o n w i l l  response  on  show a  (McNeill  representing  i n c a l c i u m m o b i l i z a t i o n , then only is  present  distribution  distribution  probably  at  (Michell,  1979).  The o b s e r v a t i o n  be  played whose  accompanied  in this  study  138  that  stimulation  of  positive  inotropic  coupled  to  receptors, not  PI  postulate.  first m2,  PI  effect  calcium  canine  evidence  to  has  left  been  to adenylate  mobilization,  was  consistent a PI  atrium, that  is  the  response,  response  (Quist,  was  muscarinic  with in  1982))  the m u s c a r i n i c  proposed cyclase,  of  pharmacological  absence of  suggest  which produced a  w h i l e the m a j o r i t y  turnover,  The c o m p l e t e  which  the  above  rat  right  provides  receptor  by  Jones et  al  .  not  simultaneously  did  the  subtype  (1982)  to  be  coupled  to  turnover.  4.3  ROLE OF PI TURNOVER IN MUSCARINIC RECEPTOR  CALCIUM MOBILIZATION Longitudinal homogeneous Snyder, guinea  pig  Triggle, al.,  PI  muscle  by  a  James-Kracke well  (Jafferji  as  mobilization.  of  guinea  agonists  an e n h a n c e d  role  (Yamamura  by  1981;  This pig  (Chang  Rangachari  PI  l e d to  ileum turnover  a  and of  potassium  i n c o r p o r a t i o n of  guinea of  or  contraction  and M i c h e l l , 1 9 7 6 a , b ) .  the  ileum contains  l o n g i t u d i n a l smooth m u s c l e  and R o u f o g a l i s ,  of  pig  receptors  biphasic  l o n g i t u d i n a l smooth m u s c l e  investigation  of  muscarinic  produces  as  of  muscarinic  Stimulation  ileum  1973;  1983)  into  smooth  1974).  STIMULATED  IN GUINEA PIG ILEUM  p o p u l a t i o n of  depolarisation  of  through  turnover,  PI  (and  coupled  receptors,  p r o d u c i n g an o p p o s i t e  enhance  atrium  alpha-adrenergic  and e_t  phosphate the  choice  for  further  in  calcium  139  4.3.1  Incorporation During  turnover,  of  complete  cycle  one  "new"  phosphate  into  PI.  produced  both phosphate  This  was  [3H]inositol  a  incorporated carbachol  Of  consistent  of  a two-fold 16)  with  and  the  produces  (Jafferji  and M i c h e l l ,  1976a).  suggested PI  inositol  both pools  undergoing  turnover.  carbachol-stimulated by  atropine,  of  a  of  inositol  suggesting  that  response  16,  to phosphate  this  tissue  of  response  both  Table  accessible  was  by  III).  in this  (Fig  equally  be  muscarinic  enhancement  incorporation  the  (Table  suggestion  are  will  incorporation  PI  i n t o PI  group  receptors  i n the  into  The e q u a l  Similar  group  muscarinic  PI  label  head  inositol  enhancement  incorporation  that  an  previous  stimulation  p h o s p h a t e and  and  inositol  receptor  PI  phosphoinositol  Stimulation  (Fig  Into  III)  to  the  incorporation, also  is  inhibited  muscarinic  in  nature. 4.3.2 And  Effect  And K + - s t i m u l a t e d  PI  Response  Contraction Even  three  though  decades,  significance and the  Of PMSF On C a r b a c h o l  Kirk PI  the  r e s p o n s e has  reason lack  pointed  of  out,  for  (Michell  Recently, serine  specific  and K i r k ,  Walenga protease  phospholipase  et  our  discovery step  been  known  ignorance  of  its  inhibitor. of  a specific  forward  for  over  functional As  Michell  inhibitor  in solving  the  of "PI  1981). al.  (1980)  inhibitors C.  now  a specific  r e s p o n s e w o u l d be a major  puzzle"  other  PI  one  is  have  the  It  was  reported  may a l s o decided  to  be  that  PMSF  inhibitors  investigate  of  and PI-  whether  1 40  PMSF i s  capable  of  turnover  in  carbachol  and K +  PI  guinea  in guinea  PMSF  (Fig.  by  of  PI  (1982)  C,  phospholipase  1981; to  F a r e s e et note  stimulated  If  stimulated mediated of  PI  found  to  (Michell,  PMSF  to  these  (which  contraction.  (Fig.  of  but  PMSF.  enhance  not  of  and  the  least  for  both  turnover  et  al.  in  (Egawa is  two  calciumet  al. ,  interesting  inositol  Therefore,  K+-  Pi-specific  at  it  they  Pi-specific  Hirasawa  forms  accumulation  1982).  inositol different  phosphate  calcium-independent, was  while  K+-  found  to  be  may  be  a  PMSF  effects.  played a role  turnover  and  be  of  and K + may  been p r e s e n t e d  phosphate  separating  carbachol-  carbachol,  In t h i s  b l o c k e d by  by  forms  substance P-stimulated  carbachol-stimulated  both  or  context  1982).  into  or a l t e r n a t i v e l y ,  al.,  turnover  mobilization,  carbachol  PI  inositol  PI  has  was  occurred  electrofocusing,  evidence  effect  PI  while both  incorporation  stimulation  pathways  the  K+-stimulated  found t h a t  calcium-independent  was  way o f  that  multiple  calcium-dependent useful  the  K+  separated  that  accumulation  and  pools  that  by  and  that  sensitive  C  was  carbachol  is  .tissues  dependent  the  by d i f f e r e n t  activity,  have  different  only  possible  so  It  and  labeled-inositol  different  phospholipase stimulated  ileum.  carbachol is  activate  carbachol-  suggesting  It  turnover  may  ileum,  21),  mechanisms.  pig  stimulated  pig  incorporation  the  inhibiting  in carbachol-stimulated  completely 2)),  should also  However, u n l i k e inositol  inhibits  its  K+-contracted  tissue  carbachol-  inhibit  selective  incorporation  , and  Ca2*  carbachol inhibition  PMSF  relaxed  i n h i b i t e d the  141  contraction to  the  effects  transient PMSF  produced  (Fig  on  correlated. HEPES  under  diminish under  pH  but  it  the  effects  similar  effects  PMSF.  inhibition  by  of  Harris  et  depolarized after a  calcium  a  transient 4.3.3  Is  Ca2+  15 sec  transient  al.  the  a  separate  cellular  uptake  period.  i n the  This  or  result  cellular  i n h i b i t i o n of  may a l s o  e f f e c t - of  Pi-specific  apparent  stimulated contraction in  calcium  however,  PI  dissociation  turnover  and  might appear  one must  of  assume t h a t  For the  such  hand, PI two  transient  interaction report K+ -  had no  effect  be c o n s i s t e n t  with  PMSF,  rather  than  its  C. PI  Response C ?  PMSF i n h i b i t i o n of  to argue a g a i n s t  rapidly  the  to  Phospholipase  Ca2+-dependent  mobilization.  for  but  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  The  PMSF  inhibited  phospholipase  M e d i a t e d By I n h i b i t i o n Of P i - s p e c i f i c  of  A recent  PMSF  10 sec  and  35 min  other  by n o n s p e c i f i c  first  be  i n h i b i t i o n of  mechanism  that  be  to  On t h e  components.  showed  to  rate  enough  of  Tris  to  degradation  were  effect  in  by PMSF may be due  accumulation  other  the  reported  fast  contrast  unlikely  PMSF gave p r o l o n g e d  (1983)  membrane  are  been  was  The r e l a x a t i o n  or  that  degradation  has  that  in  on c o n t r a c t i o n  PMSF on c o n t r a c t i o n .  suggests  t h e membrane  indicate  PMSF  conditions  conditions  This  of  for  appeared  of  effects  contraction  8 and 2 5 ° C  incubation  Furthermore,  the  results  and  life  at  turnover.  with  These  The h a l f  1978),  our  turnover,  turnover  buffer  (James,  on PI  22).  PI  by b o t h a g e n t s .  carbachol-  carbachol-stimulated a role  for  an argument  i n h i b i t i o n of  the  PI to  turnover be  valid,  carbachol-  142  simulated the  inositol  steps  breakdown  al.,  Michell,  inositol of  lipids  both  are  PMSF  may be an  i n h i b i t o r of  based p r i n c i p a l l y  could  occur  reported  collagen of  C  the  or  PI  PMSF was  also  the  mediated  its  phospholipase purified  PI  .,(1983)  have  depolarized  by  the  calcium  kinase  ,  arachidonic to  that  at  inhibition  by  or  both.  acid  A2.  In  the  with  phospholipase  C.  of  its  A2.  more  study,  its  from effect  However, PMSF  was  Pi-specific hydrolysis Harris  inhibition of  or  same  that  of  result  one  release  part  suggest  during  diacylglycerol  acid  least  Similarly,  release  C,  PMSF  the  platelets,  Pi-specific  i n h i b i t i o n of  PMSF-mediated  PMSF  phosphatidic  of  interacting  u p t a k e was  of  phospholipase to  that  G in  inhibition  provided  that  PI-  : PMSF i n h i b i t i o n of  arachidonic  by  et  accumulation  phospholipase  Pi-specific  assumed  calcium  proposed  findings  i n h i b i t i o n of  effect  C was  (1982)  phospholipase  suggesting  evidence  Serhan  inhibiting  by Walenga  phospholipase  by  inhibit  through  only d i r e c t  exerting  al.  Pi-specific  phosphatidylcholine,  of  in  and  would be  c o u l d be due  shown t o  capable  1982)  1980;  role  the  calcium mobilizing steps  following  specific  a  al.,  both  (1982),  or d i a c y l g l y c e r o l  following;  et  inhibits  al  PMSF i n h i b i t i o n of  lipase,  was  et  either  stimulation  play  proposed  Pi-specific  on t h e  (Putney  to  collagen-stimulated  phospholipase the  Walenga  1975;  indeed  these proposed  by PMSF.  acid  was  C , as  inhibited  or  acid  considered  If  of  (Michell,  phosphatidic  phospholipase  thrombin  by PMSF a l s o  of  mobilization.  then  PI  Presently,  1981)  specific  into  involved in calcium m o b i l i z a t i o n .  accumulation et  incorporation  et of  of al K+-  i n h i b i t i o n of  143  Pi-specific  phospholipase  diacylglycerol  present  As d i r e c t  accumulation  demonstrated,  the  phospholipase  C.  C  evidence during  results  during  argue  that  PI  inhibition  PI  turnover  PMSF  inhibits  turnover  against  this  needs  by  PMSF  has  not  of been  Pi-specific  verification.The  mechanism  in  guinea  pig  ileum. 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 A  sensitive  r e s p o n s e was response, been  method  found w i t h the  as  suggested  shown t o  inhibit  Blisner,  1976;  used t h i s  observation  PI  response.  et  to  three  of  al.,  develop  different  gland  and  turnover  i n the  presence  inositol  monophosphate that  use  blowfly of  lithium  et  primary  s t e p of  "amplify"  a l . ( 1 982).  PI  Lithium  has  1981).  et  Berridge  a n o v e l method tissues  et  (rat  al.,  act  brain  gland)  leads  for  and  al.,(1982)  studying  the  slices,  rat  stimulation  to  of  accumulation  1982).  They  as  an  PI  response  PI  the  (Allison  lithium  can  the  Ileum  monophosphatase  salivary  (Berridge  In G u i n e a P i g  l i t h i u m to  by B e r r i d g e  Sherman  In  measuring  inositol  parotid  therefore,  for  Response  PI of  suggested,  amplifier  of  the  PI  response. The use some  of  l i t h i u m to  interesting  stimulated  PI  conditions, period,  0.1  of  phase  IV).  into  in  which i n c l u d e d  accumulation (Table  insights  response  mM  study  t h e mechanism  guinea  pig  of  ileum.  has the  radioactive was  produced inositol  a  20-fold  with  carbachol-  i n the  the  optimum  incubation  increase  phosphate(s)  consistent  provided  Under  10 mM l i t h i u m and a 60 min  carbachol  This  the  in  the  aqueous  results  of  1 44  Berridge  et  al.  produced  only a  aqueous  phase.  et  al.  and t h a t  This  presence  of  that  one m o l e c u l e  cycle,  it  was  unstimulated  tissue  (Table  calculated.  T h i s was  inositol  into  other  o n l y about  inositol In dependent  atropine carbachol  than  10% of  (Fig  17).  was  of  1017  higher  of  PI  to  inositol  blocked  of  PI  hand,  the  carbachol  released  per  molecules  if  the  all  the  in  the  PI  turnover  per  hour  was  rate  obtained  As  inositol  it  occur  by  would appear into  cycle. produced  i n the  muscarinic  r e s p o n s e was  10 uM ( F i g  then  incorporation  turnover  the  medium  cycle,  could  cycle,  pool  60 min i n  studies.  phosphate  maximal  in  it  phosphate  1017  molecules  inositol  by  around  x  d u r i n g the  than  turnover  the  is  PI  If  inositol  phospholipid  phospholipids  lithium,  The h a l f  concentration  x  basal  due  2.52  other  accumulation  the  presence  this  28.5  1982).  turnover  turnover  occurred  small  inositol  PI  the  Berridge  incubation  phosphate  On t h e  III)  the  is  the  in  by  very  al.,  the  inositol  inositol  accumulation  and  IV).  et  to  that  undergo  10 t i m e s  phospholipid the  inositol  of  phosphate  incorporation mechanisms  rate  the  labelled  from t h e  into  (Table  turnover  added  calculated  incorporated  a  of  phospholipid tissue  of  lithium a l l  obtained  reported  to  activity  i n the  inositol  ileum  specific  10 mM l i t h i u m  radioactivity  (Berridge  inositol  unstimulated  that  tissue  tissue,  were  attributed  labelled  inositol  cycle,  was  is  of  observations  the  turnover  from  the  accumulated  assuming  of  accumulation  in unstimulated  same as  being  In u n s t i m u l a t e d  Similar  assumed t h a t  is  PI  small  (1982).  turnover is  (1982).  17),  a  dose-  guinea  pig  antagonist,  obtained similar  at to  a the  145  value  reported  stimulated suggests  by  Jafferji  phosphate that  4.3.5  incorporation  inositol  carbachol-stimulated  PI  phosphate  (1976a)  into  for  PI.  carbachol-  This  accumulation  further  occurred during  turnover.  A n a l y s i s Of I n o s i t o l  Amplification  and M i c h e l l  Phosphates  Accumulated During  Of C a r b a c h o l - s t i m u l a t e d  PI Response  Lithium  In G u i n e a P i g  I leum The  elution  profile  following  carbachol  exchange  resin  by  Berridge  of  stimulation,  et  al.  (1982)  eluted  ammonium f o r m a t e  (Fig  18),  radioactivity  radioactivity  eluted  event  of  receptor  breakdown 1976a),  of  the  breakdown o f  1982; al.,  PI  was  1983,  suggested  be  to  al.,  been  step  of  et  1982) al.,  the  only  reported Inositol  plus  160 mM  the  total  17% of  The  majority  inositol  considered  on PI  turnover  PIP2 o r of  1983;  1964;  of  the  biphosphate  and  that was  suggested that  primary event.  It  enhanced  and  Michell,  stimulation  is  not  is  the  t h a n of  and  Lapetina  (Billah  al.,  of  rather  b o t h PIP and P I P 2  Thomas e t  primary  i n a number  (PIP and P I P 2 ) alone  the  the  Jafferji  receptor  polyphosphoinositides  et  those  ion  Table V ) .  has  either  to  tissues.  19).  previously  it  primary  Rhodes to  19,  for  ( H o k i n and H o k i n ,  The breakdown of Weiss  (Fig  stimulation  more r e c e n t l y  tissues  PI.  it  (Fig  released  column by 5 mM b o r a x  corresponded  triphosphate  results  other  accounted  accumulated  Although  in  from t h e  phosphates,  from t h e Dowex-1 X8 f o r m a t e  c o l u m n , gave d i f f e r e n t  monophosphate,  inositol  inositol  (Agranoff  1983) known,  has  e_t  been  however,  1 46  whether  the  same  enzyme  polyphosphoinositides. phosphate  released  causes When  sympathetic  ganglia  it  found  while  during bi-  an e a r l y  that  period,  to  inositol  suggest  that  biphosphate  was  breakdown  time  inositol  stimulation  of  superior  was  was b e i n g  (IP2),  IP3——^>  by t h e  IP2  (Michell  an  respectively.  IP3  This  in to  rat be  brain,  inositol  attributed above from  radioactivity  to  rat  s e q u e n c e of  study  inositol et  al.  inositol,  via  P  more  than  et  to  al., of  the  80%  of  the  neurotransmitters  salivary  by l i t h i u m  in t h i s  e n h a n c e d breakdown of One p o s s i b l e in  involved inositol block  (Berridge  leading  of  =^5^Inositol  studies  events,  increased  f o l l o w i n g mechanism.  and b l o w f l y  inhibition  1983),  g l a n d was  1982).  the  last  release  shown  This step  of  in  was the  inositol  IP3. The f i n d i n g s  be,  to  P  monophosphate the  (IP3)  p r o d u c e d by v a r i o u s  parotid  al.,  led Michell  converted  -=^IP  lithium-amplification  accumulated  et  increase  P  In  these  of  triphosphate  there  of  course  studied  inositol  later  and m o n o - p h o s p h a t e ,  (1983)  the  during vasopressin  cervical was  the  explanation pig  i n the  sequence  for  the a c c u m u l a t i o n  i l e u m l i t h i u m may i n h i b i t  triphosphate perhaps  that  polyphosphoinositides,  guinea  one o r  suggest  of to  reactions  inositol.  several  inositol  in  carbachol  causes  and p r o b a b l y of all the  IP2 of  an  of  PI.  and I P 3  may  the  enzymes  conversion  of  A l t e r n a t i v e l y , l i t h i u m may phosphatases  responsible  147  for  the  inositol  direct  conversion  and p h o s p h a t e ,  as  of  inositol  shown  IP2  y  V  4.3.6  Effect  as  Inositol +P  + 2P  + 3P  polyphosphates,  IP  Inositol  lithium will  lead  observed  to  below:  IP3  Inositol  In b o t h c a s e s ,  polyphosphate(s)  to  t h e a c c u m u l a t i o n of  in t h i s  inositol  study.  Of PMSF On C a r b a c h o l - s t i m u l a t e d  Inositol  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 t o lipid  breakdown  step.  In  inhibition to  the  preliminary  of  inositol  small  its  (Table  significance.  at  monophosphate Pl-specific  this  it  effect  of  inositol not  polyphosphoinositide have been c o n s i s t e n t  the  the of  f i n d i n g of  PMSF on  caused but  an  less  be  without  both  due  smaller  (Fig  24),  of  much  t h a n 20% of to  selectively  Such  18%  significant  PMSF was much  to  this  probably  not  corresponded  C  inositol  incorporation  that  phosphodiesterase. with  of  PMSF may be  phospholipase  was  considered  phosphates  that  PMSF  accumulation,  observations  suggested  effect  difference  was  inositol  the  This  inhibition  Later  study  experiment,  VI).  first  accumulated  to  phosphate  sample s i z e  complete  and t h e r e f o r e  total  used  a  (0.05<p<.06) than  was  measure  the  inositol inhibiting inhibiting  a  result  Walenga  et  would al.  1 48  (1980)  and  however,  as  stimulated results  show  its  This the  PMSF  that  effect  steps  for  that  the  have  compared  and t h r o m b i n .  the  lack  of  a prolonged  of  polyphosphoinositides  possibility lipids  is  be  response  in t h i s  to  in  in  substance to  calcium  calcium P  or  know whether  of  more  one or  to  PMSF on  15-18%,  breakdown  these open.  during  breakdown  It plays  phasic  It  al.  PIP2  the  inositol is  (1982),  have  that  may  carbachol-  than  et  of  PMSF on  the  Weiss  the  of  block  gland.  during'  the  breakdown)  remains  suggest  PIP/PIP2  mobilization  effect  studies,  parotid  PI  Walenga  inhibits  b o t h of  that  as  stimulation  PPI  mobilization  in  kinase,  during  effect  inactivation  the  One p o s s i b l e  (PI  context  in  of  breakdown.  in calcium m o b i l i z a t i o n  note  smaller  one or more  completely  by  of  much  i n h i b i t i o n of  PMSF d i d not  convincing evidence  involved  interesting role  to  breakdown  cross-receptor  provided very may  the  involved  interesting using  that  Since  carbachol-  incorporation.  at  The s m a l l  calcium m o b i l i z i n g step  of  These  greater  acid  contraction.  or  PI  PMSF  stimulated PI  to  case,  17%.  breakdown  the  the  carbachol-  by  was  be d i g l y c e r i d e  that  the  inositol  for  phosphatidic  by c o l l a g e n  for  effect  lipid  account  shown  of  putative  the  not  breakdown  PMSF may be a c t i n g  PMSF i n h i b i t i o n m i g h t  platelets  the  that  inositol  which w i l l  VI)  polyphosphate  inhibits but  Such was  (Table  inositol  lipid,  suggests  accumulation  account  of  incorporation  al.,(1980)  the  decreased  PMSF  following  inositol  (1981).  on c a r b a c h o l - s t i m u l a t e d  response c y c l e ,  et  also  inositol  in turn  site  and M i c h e l l  accumulation  stimulated than  Downes  recently breakdown the  phasic,  would  be  a  similar  or  tonic  responses  in  guinea  pig  ileum.  1 50  V. The  effect  rat  heart  study,  of  muscarinic  and g u i n e a  in  order  pig  to  SUMMARY  receptor  stimulation  i l e u m have  understand  been  the  on PI  response  investigated  relationship  in  between  in  this the  PI  r e s p o n s e and c a l c i u m m o b i l i z a t i o n . 1.  C o m p a r i s o n of  into  various  that  in  phospholipids  both  tissues  radioactivity incorporation atria  was  of  phospholipids  two a r e a s 2. atria in  fold in  the  rate  of  a  incorporation  into  PI.  labeled  of  mM c a r b a c h o l  PI  diacylglycerol.  Similar  the  of the  This  in  corresponding  differences  heart.  amount  phospholipids  in  loss or  of by  This  receptors  smooth m u s c l e  may  have  been  represent  between  these  of  muscarinic  pig  i l e u m were  in  or  suggested  of  guinea  [32P]phosphate-  result  stimulation  muscarinic  receptors  of  0.05)  and  the  level  (p <  rat  atria  enhance  the  combined  increase  increase  d i d not  in  In c o n t r a s t ,  i n combined r a t  two-fold  Stimulation  by t h e  that  into PI.  receptors  by  measured  than  indicated  However,  various  mM) p r o d u c e d a s m a l l  in longitudinal  by 0.1  into  muscarinic  (0.1  accompanied  atria  higher  p h o s p h o l i p i d metabolism  incorporation  alpha-adrenergic  3.  a  phospholipids.  canine of  and v e n t r i c l e  incorporated  higher  in  incorporation  heart.  by c a r b a c h o l  receptors  atria  ventricle.  Stimulation  phosphate  in rat  radioactivity  i n the  of  [32P]phosphate  other  reported  differences  basal  PI  than  4-6  recently  the  [32P]phosphate  i n combined  breakdown of  PI  rat as  [1"C]arachidonyl-  arachidonyl-containing  that  muscarinic  receptor  151  stimulation rat  had  a very  small  Muscarinic  response  in rat  receptor  left  in rat  left  response  (35%,  p<0.05),  On t h e  other rat  hand,  left  incorporation consistent the  calcium  of  on PI  turnover  in  produced a d i f f e r e n t  PI  analogy and C a 2 +  of  in  to to  PI the  of  into  it  is  that  The  coupled  a  whose  right  r e s p o n s e may  relative  receptors are  mediated  a small in  remaining  population,  enhanced  by  population  manner  atria  receptors,  w h i c h enhance  the  are  densities  rat  receptors  inhibitory  enhanced  alpha-adrenergic  termed m,  i n an  in  accompany  receptor  and that  is  PI  atria.  findings  response  suggested  small  by an  These PI  muscarinic  receptors  accompanied  PI.  These  adrenergic  by  in rat  muscarinic  is  turnover. a,  was  muscarinic  mobilization.  probably  atria  of  of  alpha-adrenergic  Based on t h e  heart,  15%)  of  receptors  response  rat  Stimulation  accompanied  suggestion  mobilization.  (approximately  most  any,  had no e f f e c t  phosphate the  PI  but  right  atria. was  stimulation  stimulation  receptor  coupled  atria  and  with  and r e l a t i v e  stimulation  and r i g h t  receptors  only  if  atria. 4.  both  effect,  PI  by  turnover  termed to  is  m2,  adenylate  cyclase. 6.  In  [32Jphosphate longitudinal  addition into  to PI,  muscarinic  smooth m u s c l e  mM) p r o d u c e d an e n h a n c e d and a l s o  c a u s e d an  in  the  p r e s e n c e of  for  carbachol-stimulated  of  guinea  receptor pig  i n c o r p o r a t i o n of  increased lithium  accumulation (10  mM).  inositol  incorporation stimulation  i l e u m by c a r b a c h o l [3H]inositol of  inositol  into  in (0.1 PI,  phosphates  The h a l f - m a x i m a l  phosphate  of  response  accumulation  was  152  around  lOjxM,  similar  carbachol-stimulated binding  studies.  support  the  in  lipid  7.  ileum. PI  This  may  to  PMSF  contraction  of  guinea  probably  to  the  an e f f e c t would  of  their  for  receptor  evidence  to  receptor by e n h a n c e d  the  argue  PMSF  response,  receptor  of  a  and  of  mechanisms  on  against  non-specific and This  inhibition  contraction role  of  pig  and c o u l d  be  PMSF. on  inositol  and  transient  potassium-stimulated  Pi-specific  the  to  effects  effect of of  carbachol-stimulated of  in guinea  potassium-stimulated  inhibition  its  selectively stimulated  lipids  sensitivity  ileum.  inhibition of  PI  differential  pig  result  its  step)  accompany  different  nonspecific  the  of  further  accompanied  inositol  carbachol-  inhibition  lack  by  its  PMSF-mediated of  in  potassium-,  carbachol-  produced  both  the  not into  basis  of  than  and  muscarinic  is  i n h i b i t o r of  that  inhibition  result  ileum  but  occur  on t h e  incorporation,  rather  that  and may c l o s e l y  a putative  In c o n t r a s t  due  provide  previously  agonist.  suggested  differentiated 8.  findings  incorporation  response  reported  incorporation  pig  carbachol-,  [3H]inositol  value  suggestion  turnover  PMSF,  inhibited  These  guinea  o c c u p a n c y by t h e  the  phosphate  previous  stimulation inositol  to  PMSF  calcium PI PI  was  uptake  turnover.  If  turnover  was  phospholipase (a  PI  of  C,  the  calcium-mediated  turnover  in calcium  mobilization. 9.  A n a l y s i s of  the  carbachol-stimulated  PI  indicated  that  less  than  inositol turnover 20%  of  phosphates in the  the  accumulated presence  inositol  of  during lithium  phosphate  was  153  eluted the  i n the  rest  was  inositol to  turnover  in  at  higher  like  ionic strength,  some o t h e r  guinea  pig  may be t h e  i n o s i t o l mono-phosphate  This  ileum  the  during  breakdown of  corresponding  provided direct  tissues,  and to  evidence  primary event  muscarinic  of  PI  receptor  polyphosphoinositides  rather  PI. 10.  Accumulation  polyphosphates ileum  rather  i n the  inositol direct  or  a  it  that  lithium  phosphates  its  carbachol-stimulated  inositol  small  16%)  (approximately  PMSF of  phosphate Pi-specific  the  inositol following  observed  the  putative obtained breakdown  small  calcium do n o t is  inhibition  inhibition  C  by  effect  PMSF of  on t h e  the  step  enzymes to  inhibition  PMSF c a u s e d  only  inhibition  completely  possibility  a  by  account  carbachol-stimulated other  steps  As PMSF  i n o s i t o l - l i p i d breakdown, PI  of  carbachol-stimulated  PMSF may i n h i b i t  of  the  inositol.  i n o s i t o l - l i p i d breakdown.  mobilizing  contradict  cannot  three  pig  involved in  complete  of  inositol  triphosphate  i n d i c a t i n g that  Therefore,  s t e p of  to  incorporation,  phospholipase  initial  had o n l y a v e r y  almost  accumulation,  incorporation.  inositol  of  i n guinea  inhibited a l l  one o r more enzymes  inositol to  proportion  i n o s i t o l monophosphate  inhibited  In c o n t r a s t  inositol  large  s t e p w i s e c o n v e r s i o n of  c o n v e r s i o n of  11.  of  than  indicated either  involved  for  to  and t r i - p h o s p h a t e .  that  stimulation  corresponding  eluted  bi-  suggest  than  peak  turnover, that  involved in calcium m o b i l i z a t i o n .  the  a  results  inositol-lipid  1 54  BIBLIOGRAPHY 1.  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