UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Isolation and phosphorylation of guinea pig cardiac sarcolemma Hui, Chi Wai 1976

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

Item Metadata

Download

Media
831-UBC_1976_A1 H83.pdf [ 4.49MB ]
Metadata
JSON: 831-1.0093871.json
JSON-LD: 831-1.0093871-ld.json
RDF/XML (Pretty): 831-1.0093871-rdf.xml
RDF/JSON: 831-1.0093871-rdf.json
Turtle: 831-1.0093871-turtle.txt
N-Triples: 831-1.0093871-rdf-ntriples.txt
Original Record: 831-1.0093871-source.json
Full Text
831-1.0093871-fulltext.txt
Citation
831-1.0093871.ris

Full Text

ISOLATION AND PHOSPHORYLATION OF GUINEA PIG CARDIAC  SARCOLEMMA  by Hui C h i Wai A.B.,  U n i v e r s i t y o f C a l i f o r n i a , L.A., 1970  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  i n the Department of PHARMACOLOGY  We accept t h i s t h e s i s as conforming required  THE  t o the  standard  UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1976 © Hui Chi Wai, 1 9 7 6  In  presenting  an  advanced degree  the I  Library  further  for  shall  agree  thesis  in p a r t i a l  fulfilment  of  at  the  University  of  Columbia,  make  it  this  w r i t ten  that permission  representatives. thesis  for  for  financial  of  The U n i v e r s i t y  of  British  2075 Wesbrook P l a c e Vancouver, Canada V 6 T 1W5  by  the  is understood gain  Columbia  for  extensive  be g r a n t e d  It  British  available  pe rm i s s i on .  Department  Date  freely  s c h o l a r l y p u r p o s e s may  by h i s of  this  shall  requirements  reference copying of  Head o f  that  not  the  I  agree  and  be a l l o w e d  that  study.  this  thesis  my D e p a r t m e n t  copying or  for  or  publication  without  my  i  ABSTRACT Plasma membranes were prepared from guinea p i g h e a r t by homogenizing the t i s s u e i n a P o l y t r o n followed  by f o u r c y c l e s o f washing and d i f f e r e n t i a l c e n t r i -  f u g a t i o n u s i n g low g f o r c e s minutes). with KC1  ific  (380 x g/10 minutes t o 120  The crude p a r t i c l e s  thus obtained  x  g/10  were e x t r a c t e d  (1.25 M) , f o l l o w e d by i s o p y c n i c c e n t r i f u g a t i o n i n  a discontinuous purified  homogenizer,  10-15  sucrose g r a d i e n t .  Adenylate c y c l a s e  was  f o l d over the whole homogenate w i t h a spec-  a c t i v i t y o f 3.6±0.72 nmoles/mg/minute.  Ouabain-sensitive  Mg -dependent Na +K -ATPase, another plasma membrane s p e c i f ++  +  i c enzyme, was  +  enriched  by 4 f o l d , with a s p e c i f i c  of 107+8.2 nmoles/mg/minute. predominantly o f m i t o c h o n d r i a l yield.  activity  Cytochrome C oxidase, o r i g i n , was  an enzyme  recovered  i n low  These membrane "marker" enzyme s t u d i e s i n d i c a t e d t h a t  the i s o l a t e d membrane p r e p a r a t i o n  consisted of highly  purified  plasma membranes. Functional i n d i c a t e d t h a t an as C a - d e p e n d e n t ++  kinase  s t u d i e s w i t h the c a r d i a c  ATP-dependent  Ca  + +  binding  ATPases were present.  sarcolemma system as w e l l  Intrinsic  a c t i v i t i e s and membrane-bound s u b s t r a t e s  protein  f o r phosphor-  y l a t i o n were a l s o found t o be a s s o c i a t e d w i t h these membranes, which were phosphorylated by endogenous  or added p r o t e i n  kinase.  Membrane p h o s p h o r y l a t i o n was and  was  r e v e r s e d by  indicating  s t i m u l a t e d by  the presence  y l a t e d , were c a p a b l e  orated rease  of a  (1 jjM) phosphatase,  phosphorylation-dephosphorylation P l a s m a membranes, when p h o s p h o r -  of accumulating  preparations.  twice  as much C a  + +  as:  F o r e a c h nanomole o f p h o s p h a t e i n c o r p -  i n t o t h e membrane, t h e r e were 15 nanomoles o f n e t in Ca  possibility the  AMP  a membrane-bound p h o s p h o p r o t e i n  s y s t e m i n c a r d i a c sarcolemma.  control  cyclic  + +  uptake.  that cyclic  cardiac c e l l v i a a  mechanism.  These d a t a are c o n s i s t e n t w i t h AMP  may  facilitate Ca  + +  inc-  the  movement  phosphorylation-dephosphorylation  into  iii TABLE OF CONTENTS Page PART I :  CARDIAC SARCOLEMMA - ISOLATION CHARACTERIZATION A.  Introduction  B.  Experimental a. b.  C.  2.  PART I I :  1 Procedures  6  Materials Methods  6 6  1. Adenylate c y c l a s e assay 2. Enzyme assays - ATPase assay 3. Other enzyme assays  6 8 8  Results 1.  D.  AND  9  Membrane P r e p a r a t i o n s from guinea pig. h e a r t C h a r a c t e r i z a t i o n o f c a r d i a c sarcolemma by "marker" enzymes  Discussion  9 22 29  CARDIAC SARCOLEMMA - PHOSPHORYLATION AND CALCIUM UPTAKE A.  Introduction  B.  Experimental a. b.  C.  49  Materials Methods  49  1. 2. 3.  49 50 50  I s o l a t i o n o f c a r d i a c sarcolemma Calcium b i n d i n g and uptake P h o s p h o r y l a t i o n of sarcolemma  53  1.  53  3. 4. 5. 6.  REFERENCES  Procedures  Results  2.  D.  34  Calcium b i n d i n g and uptake i n c a r d i a c sarcolemma Ca - s t i m u l a t e d ATPase o f c a r d i a c sarcolemma P h o s p h o r y l a t i o n o f sarcolemma by exogenous p r o t e i n kinase Endogenous p r o t e i n kinase and s e l f p h o s p h o r y l a t i o n o f c a r d i a c sarcolemma Ca accumulation by phosphorylated c a r d i a c sarcolemma R e v e r s i b i l i t y of membrane phosphoryla t i o n and d i s t r i b u t i o n o f phosphop r o t e i n phosphatase  53 56 59  + +  Discussion  64 72 79 86  iv  LIST OF TABLES Page I II  III  IV  V  VI VII VIII  E f f e c t i v e n e s s of v a r i o u s s a l t s i n i s o l a t i o n o f c a r d i a c sarcolemma  16  E f f e c t s of d i t h i o t h r e i t o l (DTT) on the a c t i v i t y and y i e l d o f adenylate c y c l a s e d u r i n g i s o l a t i o n o f c a r d i a c sarcolemma  18  E f f e c t s of d i f f e r e n t b u f f e r s and the number of washes on removal of p r o t e i n from heart homogenate  20  D i s t r i b u t i o n and a c t i v i t i e s of marker enzymes i n v a r i o u s f r a c t i o n s obtained d u r i n g i s o l a t i o n of c a r d i a c sarcolemma  24  D i s t r i b u t i o n and a c t i v i t i e s o f 5' N u c l e o t i d a s e i n f r a c t i o n s o b t a i n e d d u r i n g i s o l a t i o n of c a r d i a c sarcolemma  28  Calcium b i n d i n g and uptake a c t i v i t i e s of c a r d i a c sarcolemma  54  Ca— -stimulated sarcolemma  55  +  ATPase a c t i v i t i e s o f c a r d i a c  E f f e c t of d i f f e r e n t preincubation conditions on C a uptake by c a r d i a c sarcolemma  65  D i s t r i b u t i o n of phosphoprotein phosphatase i n f r a c t i o n s obtained during i s o l a t i o n o f c a r d i a c sarcolemma  78  + +  IX  V  L I S T OF FIGURES Page  1.  Scheme o f i s o l a t i o n o f p l a s m a membranes from guinea p i g hearts  13  2.  D i s t r i b u t i o n o f enzyme a c t i v i t i e s i n s u c r o s e gradient fractions  27  3.  P h o s p h o r y l a t i o n o f c a r d i a c s a r c o l e m m a by purified protein kinase  58  4.  H i s t o n e p h o s p h o r y l a t i o n by c a r d i a c  61  5.  Self-phosphorylation of cardiac by e n d o g e n o u s p r o t e i n k i n a s e  6.  E f f e c t o f c y c l i c AMP a n d p r o t e i n k i n a s e o n c a l c i u m u p t a k e by c a r d i a c s a r c o l e m m a  68  Effect of CaC^.on phosphorylation of cardiac s a r c o l e m m a by e x o g e n o u s p r o t e i n k i n a s e  71  R e l a t i o n s h i p between p h o s p h o r y l a t i o n and s t i m u l a t i o n o f c a l c i u m u p t a k e by c a r d i a c sarcolemma  74  P h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n c a r d i a c sarcolemma  76  7. 8.  9.  sarcolemma  sarcolemma  63  of  vi  ABBREVIATIONS  ATP  adenosine  cyclic  adenosine  1  5'-cyclic  3',  monophosphate  adenosine  5'-monophosphate  3'-AMP  adenosine  3 *-monophosphate  EDTA  ethylenediamine  EGTA  ethylene  5'  AMP  AMP  5 -triphosphate  ether)  glycol N,  tetraacetate bis  (B-amino  ethyl  N'-tetraacetate  Tris  tris  (hydroxymethyl)  TCA  trichloroacetic  DTT  dithiothreitol  acid  aminomethane  vii  ACKNOWLEDGEMENTS  I w i s h t o t h a n k P r o f e s s o r G e o r g e I . Drummond f o r h i s guidance during the course o f t h i s work. I am p a r t i c u l a r l y g r a t e f u l t o h i m f o r e n c o u r a g i n g me t o engage i n a c o m b i n e d medi c a l and g r a d u a t e s t u d i e s p r o g r a m . I w i s h t o t h a n k my w i f e f o r h e r p a t i e n c e and s u p p o r t d u r i n g my l o n g y e a r s o f U n i v e r s i t y e d u c a t i o n , and f o r t y p i n g t h e o r i g i n a l manuscript of this thesis. The f i n a n c i a l a s s i s t a n c e by t h e C a n a d i a n Heart Foundation i n t h e form o f a M e d i c a l S c i e n t i s t F e l l o w s h i p i s hereby acknowledged.  PART I CARDIAC SARCOLEMMA - ISOLATION AND  CHARACTERIZATION  INTRODUCTION  P l a s m a membranes o f mammalian an  assembly o f d i v e r s e , but s p e c i f i c a l l y  ated ing  f u n c t i o n a l systems a r r a y e d intricate  cognized (1)  and c r u c i a l  mones,  antibodies  and o t h e r  (1,2);  In order forms these  lar  cell  a s w e l l as  since these contents.  cell  surface  receptors  agents;  and  no n u c l e u s tensively  f o r hor-  (3) v i a acting  t h e p l a s m a membrane must f i r s t  be  per-  isolated. of a p a r t i c u -  t h a t component i n . i t s most  native  n o t p o s s i b l e i n t h e c a s e o f p l a s m a memb-  must f i r s t be d i s r u p t e d  A preparation  "ghosts"  en-  intracellularly.  complex f u n c t i o n s , t h e y  i s clearly  small  c o m m u n i c a t i o n between  regulatory molecules  t o u n d e r s t a n d how  component i s t o o b t a i n  form, t h i s  now r e -  include:  t h e a i m o f most p r o c e d u r e s f o r t h e i s o l a t i o n  cell  ranes  Functions  and t r a n s p o r t o f  (2)  regulatory  enzymes, s y n t h e s i z e  both e x t r a c e l l u l a r l y  perform-  substances i n v o l v i n g s p e c i f i c  and i t s e n v i r o n m e n t by c e l l  membrane-bound  While  roles.  permeability  and numerous b i o - o r g a n i c  cell  periphery  t o be m e d i a t e d by t h e p l a s m a membrane  zymes and " c a r r i e r mechanisms" the  c a n be v i e w e d a s  and g e n e t i c a l l y r e g u l -  a t the c e l l  biological  regulation of cellular  ions  the  cells  obtained  representing  i n order  t o remove  i n t a c t membranes i s  from e r y t h r o c y t e s  (3).  This  cell  has  and so one d o e s n o t have t o d i s r u p t t h e membrane ext o remove i n t r a c e l l u l a r  organelles.  Furthermore, i t  - 2 -  i s devoid  of endoplasmic r e t i c u l u m , which f r e q u e n t l y  compli^  c a t e s t h e p r e p a r a t i o n o f s u r f a c e membranes f r o m more c o m p l e x cells.  Although  red c e l l  t h e r e are undoubted advantages i n u s i n g  "ghosts"  f o r s t u d y i n g membrane f u n c t i o n s , i t s u f f e r s  the d i s t i n c t disadvantage d e r i v e d from a c e l l The malian  simplified  a hypotonic  sucrose  for liver cells  i s o l a t i o n o f a mam-  The  (6) has  t i s s u e was The  procedure  liver  homogenate  N e v i l l e method, l a t e r m o d i f i e d  (7), kidney  ( 9 ) , t h y r o i d ( 1 0 ) , He  La c e l l s  anterior pituitary  i s o l a t i o n of  (8), i n t e s t i n a l  plasma  epithe-  ( 1 1 , 12) , b o v i n e mammary  be  a d a p t e d t o a number o f  s o f t t i s s u e s , i t s a p p l i c a t i o n t o s t r i a t e d m u s c l e s has  not  Muscles are h i g h l y s p e c i a l i z e d t i s s u e s w i t h  major p r o p o r t i o n of the apparatus.  by  (14).  W h i l e N e v i l l e ' s m e t h o d may  successful.  that  isopycnic centrifugation i n  been adapted f o r the  membranes f r o m h e p a t o m a s  ( 1 3 ) , and  (4, 5 ) .  c e n t r i f u g a t i o n s of the  b u f f e r f o l l o w e d by  gradients.  Emmelot e t a l  gland  for specialized functions.  p l a s m a membrane f r a c t i o n f r o m a s o l i d  employed d i f f e r e n t i a l  lium  a membrane p r e p a r a t i o n  f i r s t u s e f u l method f o r t h e  d e s c r i b e d by N e v i l l e  in  of being  c e l l mass made up o f t h e  M y o f i b r i l s a r e bound t o g e t h e r  r e t i c u l u m and  of the  complex i n t e r l o c k i n g networks  by w h a t was  defined  fibrils  c a n be  Z membrane s y s t e m w h i c h  (15).  and  the form  E l i m i n a t i o n o f t h e myo-  a c c o m p l i s h e d by b r e a k i n g  various chemical  a  contractile  by P e t e r s a s a " c y t o s k e l e t o n " l o c a t e d w i t h i n e l e m e n t s o f sarcoplasmic  been  this cytoskeleton  p h y s i c a l techniques.  Kono and  with  Colowick  (16)  - 3-  were f i r s t t o d e s c r i b e a procedure f o r the i s o l a t i o n o f sarcolemma from r a t s k e l e t a l muscle.  The method i n v o l v e d e x t r a c t i o n  of c e l l fragments w i t h 1.0 M KC1 s o l u t i o n t o remove the  contrac-  t i l e p r o t e i n s , and  from  other KBr  the t u b u l a r membranes were separated  c e l l p a r t i c l e s by d i f f e r e n t i a l c e n t r i f u g a t i o n i n a 25%  solution.  T h i s method has  been m o d i f i e d  by Severson e t a l  (17) who used 0.4 M L i B r i n s t e a d o f 1.0 M KC1 t o e x t r a c t contr a c t i l e proteins. t i o n s and  Membranes thus i s o l a t e d contained  appeared as empty, t r a n s p a r e n t  no s t r i a -  sac-like structures.  Adenylate c y c l a s e , g e n e r a l l y accepted as the best plasma membrane "marker" enzyme, was e n r i c h e d  approximately 1 5 - f o l d over  the whole homogenate w i t h a y i e l d o f a c t i v i t y o f about Other procedures r e p o r t e d  30%.  i n the l i t e r a t u r e f o r p u r i f i c a t i o n  of s k e l e t a l sarcolemma employed one  o r s e v e r a l o f the  following:  "ageing" o f the muscle c e l l segments, washing i n v a r i o u s solutions i n concentrations  ranging  salt  from m i l l i m o l a r to molar  l e v e l s , o r i n c u b a t i o n a t 37°C w i t h i n a l i m i t e d pH range, f o l l o w e d by d i f f e r e n t i a l c e n t r i f u g a t i o n t o y i e l d tubules  o r sheets  (18-21).  sarcolemmal  More r e c e n t l y , techniques were des-  c r i b e d t o o b t a i n , i n s t e a d o f s a c - l i k e sarcolemma, plasma membranes i n the  form of v e s i c l e s .  ated from m y o f i b r i l s by strong The  Plasma membranes were separ-  s a l t s o l u t i o n s o r by f i l t r a t i o n .  f i n a l p u r i f i c a t i o n was obtained  density gradients  by c e n t r i f u g a t i o n on sucrose  (22-24).  Attempts t o i s o l a t e plasma membranes from c a r d i a c muscle are l e s s numerous.  P o r t i u s and Repke (25) were the  first  -  4 -  t o apply the methods developed f o r the i s o l a t i o n o f sarcolemma from s k e l e t a l muscle the myocardium. a c t i v e Mg  (16, 18) t o prepare plasma membranes from  The sarcolemmal p r e p a r a t i o n o b t a i n e d had an  2+ -dependent  + .+. Na +K -ATPase, but e l e c t r o n microscopy  r e v e a l e d s i g n i f i c a n t amounts o f extraneous m a t e r i a l . al  Stam e t  (26) d e s c r i b e d a c a r d i a c plasma membrane p r e p a r a t i o n i n  which the crude homogenate was potassium ..iodide.  e x t r a c t e d up t o 16 hours i n , 1 M  Although s t r i a t i o n s were a p p a r e n t l y r e -  moved from these c a r d i a c f i b e r s a f t e r such treatment, i t was c l e a r t h a t the use o f such powerful s t r u c t u r e - d i s r u p t i n g had d e s t r o y e d many important membrane f u n c t i o n s al  (27).  salts  Katz e t  (2 7) and Kidwai e t a l (28) t r i e d t o a v o i d u s i n g h i g h con-  c e n t r a t i o n s o f s a l t , and employed  density gradient  ultracent-  r i f u g a t i o n to s u b f r a c t i o n a t e a microsomal p r e p a r a t i o n o f the h e a r t i n t o s a r c o p l a s m i c r e t i c u l u m and plasma membrane f r a c t i o n s , but the y i e l d o f plasma membrane was extremely low and c r o s s contamination by o t h e r i n t r a c e l l u l a r membranes was s i g n i f i c a n t . More r e c e n t l y , Tada e t a l (2 9) attempted t o i s o l a t e  plasma  membranes from guinea p i g h e a r t by exposing i s o l a t e d c e l l segments t o osmotic shock, f o l l o w e d by e x t r a c t i o n o f actomyosin i n 1 M KC1.  These p r e p a r a t i o n s c o n t a i n e d as much as 15% o f t h e  p r o t e i n i n the o r i g i n a l homogenate, and when examined by phase c o n t r a s t microscopy, many s t r i a t i o n s were seen. both a d e n y l a t e c y c l a s e and Mg  2+  -dependent  Furthermore,  + + Na' + K -ATPase . were  e n r i c h e d only 2 - 3 - f o l d over the o r i g n a l homogenate, i n d i c a t i n g t h a t the p u r i f i c a t i o n was f a r from complete.  - 5 -  It i s clear that o membranes, t h e  f o l l o w i n g must be  contractile proteins, c y t o p l a s m as  branes of  cited  a b u n d a n t and  separation  intracellular  functional  achieved:  integrity  plasma  themselves f i r m l y i n (2)  removal  d e e p l y embedded  in  of the  of the  o f p l a s m a membranes f r o m o t h e r mem-  origin;  of  cardiac  (1) e x t r a c t i o n  a complex i n t e r l o c k i n g n e t w o r k ;  (3)  ;;  to i s o l a t e  which organize  m i t o c h o n d r i a , which are myofibrils;,  i n order  the  and  (4) p r e s e r v a t i o n  p l a s m a membrane.  Most  of  attempts  above e i t h e r u s e d h a r s h p r o c e d u r e s t o a c h i e v e  extraction  of c o n t r a c t i l e proteins  at the  expense of  p l a s m a membrane, o r t o o  gentle  methods y i e l d i n g s a r c o l e m m a l  preparations materials.  c o n t a m i n a t e d by As  a result,  rane preparations limited the  to  procedure.  order  to  study  cally  meaningful.  preparation the  The  would a l l o w  a highly  c a r r y out,  t h i s p r o j e c t was  isolation  of  cardiac  the  i n a manner t h a t  example, one  not  an  ideal  only  to  or  and  survived to  sarcolemma  develop in  i s physiologi-  cardiac  sarcolemmal  i n v e s t i g a t e how  also to  subsequently take place  t o changes o f p e r m e a b i l i t y  ferential  to  of  p l a s m a membrane, b u t  e v e n t s t h a t may  trace  the  hormones  metabolic  which u l t i m a t e l y  i o n i c movements.  In  the  lead first  t h e s i s , a p r o c e d u r e i s p r e s e n t e d which employs  c e n t r i f u g a t i o n and purified  the  intracellular  aim  i t s function For  of  f u n c t i o n a l s t u d i e s w i t h t h e s e memb-  were e i t h e r d i f f i c u l t  a b e t t e r method f o r t h e  part of  amounts o f  integrity  i s o l a t e d a s p e c t s o f membrane a c t i v i t y w h i c h  isolation  activate  large  the  the  isopycnic  centrifugation to  p l a s m a membrane f r a c t i o n f r o m g u i n e a  dif-  yield  pig  - 6 -  ventricles.  Some r e s u l t s i n d i c a t i n g the l i m i t a t i o n s of v a r i o u s  s a l t s and agents used i n i s o l a t i o n o f plasma membranes w i l l be presented, and the concept o f plasma membrane "marker" zyme w i l l be b r i e f l y  also  en-  discussed.  EXPERIMENTAL PROCEDURES  A.  Materials  C y c l i c [H]AMP 3  (22 C i per mmole) ,  4 5  CaCl  2  (20 mCi per  14 r T mg)  and u n i f o r m l y l a b e l l e d  t a i n e d from New  [Cj ATP  England N u c l e a r .  (400mCi per mmole) were ob-  E t h a n o l was  removed from the  s o l u t i o n under vacuum and the r a d i o a c t i v e m a t e r i a l s were d i l u t e d w i t h water t o the d e s i r e d c o n c e n t r a t i o n and s t o r e d a t -20°C. U n l a b e l l e d n u c l e o t i d e s were o b t a i n e d from Calbiochem and Sigma. Pyruvate Kinase (from r a b b i t muscle) and 2-phosphoenol pyruvate ( t r i s o d i u m s a l t ) , cytochrome C, d i t h i o t h r e i t o l and p r o t e i n k i n a s e i n h i b i t o r were o b t a i n e d from Sigma. was  Ouabain  ( s t r o p h a n t h i n G)  purchased from Calbiochem, and sucrose ( d e n s i t y g r a d i e n t grade  - r i b o n u c l e a s e f r e e ) was o b t a i n e d from Schwarz/Mann. s a l t ) was B.  ATP  (tris  purchased from Sigma.  Methods  Adenylate c y c l a s e - Assay A;  Membrane f r a c t i o n s  (150-  400 ug p r o t e i n ) were i n c u b a t e d i n a medium ( t o t a l volume 150 u l ) c o n t a i n i n g 40 mM 6 mM  KC1,  15 mM  T r i s - H C l , pH 7.5, MgSO., 20 mM  8 mM  t h e o p h y l l i n e , 8 mM  phosphoenol p y r u v a t e , 21.7  ug  NaF,  - 7 -  [c]ATP  pyruvate k i n a s e , and 0.3 mM  (20 u C i per pmole).  Assay  tubes were e q u i l i b r a t e d a t 37°C and the r e a c t i o n s t a r t e d by the a d d i t i o n of membrane p r o t e i n .  Incubations were c a r r i e d out f o r  10 minutes and were terminated by p l a c i n g the tubes i n a b o i l i n g water bath f o r 3 minutes. of membranes.  C o n t r o l tubes c o n t a i n e d water  instead  The tubes were c e n t r i f u g e d a t 8,000 x g f o r 10  minutes t o remove denatured p r o t e i n , and 100 u l of the supernatant was  s t r e a k e d over 2 cm on Whatman 3 MM  chromatography  Descending paper chromatograms were developed f o r 18-22  paper.  hours a t  room temperature u s i n g 1 M ammonium a c e t a t e : 95% e t h a n o l (15:35) as s o l v e n t . was  A f t e r d r y i n g , the area c o r r e s p o n d i n g t o c y c l i c  AMP  v i s u a l i z e d under u l t r a v i o l e t l i g h t , cut out and p l a c e d i n  18 ml of s c i n t i l l a t i o n f l u i d  (4 g of 2,5-diphenyloxazole and  50  mg of 1,4-bis-2-(5-phenyloxazole)-benzene d i s s o l v e d i n 1 l i t e r of toluene) and the r a d i o a c t i v i t y was measured i n a Nuclear Chicago S c i n t i l l a t i o n spectrometer. c y c l i c AMP the  formed was  c a l c u l a t e d from the s p e c i f i c a c t i v i t y of  14 r T LCJATP s u b s t r a t e a f t e r c o r r e c t i o n f o r r a d i o a c t i v i t y present  i n the c y c l i c AMP c y c l a s e was 14 r  The amount of r a d i o a c t i v e  area of the c o n t r o l .  Assay B:  f o r adenylate  i d e n t i c a l to assay A except t h a t i n s t e a d of 0.3  mM  T  |_CjATP, 1 mM l i c AMP  u n l a b e l l e d ATP  formed was  to assay B.  used and the c y c -  determined a c c o r d i n g t o the s p e c i f i c b i n d i n g  p r o t e i n method of Gilman a l l c y c l i c AMP  ( t r i s - s a l t ) was  (30).  Except f o r r e s u l t s i n Table I,  d e t e r m i n a t i o n s i n t h i s t h e s i s were done a c c o r d i n g  - 8 -  A T P a s e s - Mg  - d e p e n d e n t A T P a s e a c t i v i t y was  deter-  m i n e d by i n c u b a t i n g membrane p r o t e i n (150-200 ug) i n a medium ( f i n a l v o l u m e 0.2 ml) c o n t a i n i n g 50 mM T r i s - H C l pH 7.5, 5 mM M g C l , 5 mM N a N , 0.5 mM EGTA a n d 3 mM T r i s ATP. 2  3  Mg -depend2 +  e n t N a + K - A T P a s e was m e a s u r e d by t h e i n c l u s i o n o f 100 mM +  and  +  10 mM KC1.  NaCl  I n t h e p r e s e n c e o f 0.1 mM o u a b a i n , p a r t o f t h e  2+  +  +  Mg - d e p e n d e n t Na +K - A T P a s e a c t i v i t y was i n h i b i t e d . The d i f f e r e n c e i n t h e ATPase a c t i v i t y i n t h e p r e s e n c e and absence o f ouabain i s taken  + + a s t h e o u a b a i n - s e n s i t i v e Na +K - A T P a s e .  Ca  2+  s t i m u l a t e d MgATPase was d e t e r m i n e d i n t h e same medium a s f o r MgATPase, e x c e p t t h a t EGTA was o m i t t e d cluded.  To m e a s u r e C a A T P a s e , t h e o n l y i o n i c  t h e medium was 5 mM C a C l EGTA.  a n d 50 uM C a C l  2  2  was i n -  species present i n  a n d 0.5 mM EDTA was s u b s t i t u t e d f o r  The r e a c t i o n was s t a r t e d by a d d i t i o n o f s u b s t r a t e a n d  a f t e r i n c u b a t i o n f o r 5 m i n u t e s a t 37°C, t e r m i n a t e d t i o n o f 0.2 m l o f c o l d 1 2 % t r i c h l o r o a c e t i c a c i d .  by t h e a d d i The p r e c i p i -  t a t e d p r o t e i n was r e m o v e d b y c e n t r i f u g a t i o n and t h e i n o r g a n i c phosphate content  of the supernatant  was d e t e r m i n e d b y t h e method  o f Ames ( 3 1 ) . U n d e r a l l c o n d i t i o n s u s e d , n o t more t h a n 30% o f the  s u b s t r a t e was  hydrolyzed.  Other assays:  5 -nucleotidase 1  was a s s a y e d b y i n c u b a t -  i n g membrane p r o t e i n  (100-150 jug) i n a medium  ml)  2 - a m i n o - 2 - m e t h y l - l , 3 - p r o p a n e d i o l , pH 9.0,  c o n t a i n i n g 50 mM  2.5 mM M g C l , 100 mM KC1 a n d 8 mM 2  5'-AMP.  ( f i n a l volume  Appropriate  0.2  controls  w e r e p e r f o r m e d i n w h i c h 5'-AMP was r e p l a c e d w i t h 3'-AMP o r w a t e r . The  r e a c t i o n was s t a r t e d b y a d d i t i o n o f s u b s t r a t e a n d a f t e r i n -  c u b a t i o n a t 37°C f o r 10 m i n u t e s , t e r m i n a t e d  b y a d d i t i o n o f 0.2  -  9 -  ml c o l d 12% t r i c h l o r o a c e t i c a c i d .  The p r e c i p i t a t e d p r o t e i n was  removed by c e n t r i f u g a t i o n and the i n o r g a n i c phosphate content of the supernatant chrome C oxidase  was determined by the method o f Ames (31).  Cyto-  a c t i v i t y was determined as d e s c r i b e d by Cooper-  s t e i n and Lazarow (33).  P r o t e i n determinations  were made by the  method o f Lowry e t a l (34), u s i n g bovine serum albumin as standard.  RESULTS  1.  Membrane p r e p a r a t i o n s  from guinea p i g h e a r t  Numerous attempts were made t o o b t a i n a p r e p a r a t i o n o f c a r d i a c sarcolemma f u l f i l l i n g  a l l the c r i t e r i a l i s t e d above.  f i n a l method s e t t l e d upon w i l l be d e s c r i b e d f i r s t ,  The  f o l l o w e d by  more d e t a i l e d e x p l a n a t i o n o f each step and o f attempts which proved u n s a t i s f a c t o r y . P r e p a r a t i o n o f washed p a r t i c l e s - Guinea p i g s were { k i l l e d by a sharp blow t o t h e head, b l e d from t h e neck f o r 30 seconds t o 1 minute, and the h e a r t s were promptly e x c i s e d and p e r f u s e d w i t h 10 ml o f warm (30°-35°C) Krebs-Ringer s o l u t i o n d e l i v e r e d by s y r i n g e . performed a t 4°C.  bicarbonate  A l l the f o l l o w i n g procedures were  A t r i a l t i s s u e , f a t and l a r g e v e s s e l s were  removed and d i s c a r d e d ; v e n t r i c u l a r t i s s u e was s l i c e d t o 4-5 p i e c e s with s c i s s o r s and weighed.  U s u a l l y 2.0-2.5 g o f t i s s u e  from 2 t o 3 guinea p i g s was used. The t i s s u e was suspended i n 5 volumes  ( a l l volumes were based on t h e o r i g i n a l wet weight  - 10 -  of the t i s s u e ) of hyptonic 10 mM  T r i s - H C l , pH  genizer  7.5,  b u f f e r c o n t a i n i n g 2 mM  and homogenized i n a P o l y t r o n PT 10 Homo-  f o r 15 seconds at one  t h i r d maximal v e l o c i t y , f o l l o w e d  by 2 seconds a t maximal v e l o c i t y .  The  homogenate was  adding 5 volumes of the same b u f f e r , and 250  dithiothreitol,  d i l u t e d by  a f t e r p a s s i n g through a  ym nylon mesh under m i l d s u c t i o n , c e n t r i f u g e d at 620  10 minutes.  The  supernatant f l u i d was  removed and  d i s t i n c t l a y e r , predominantly of mitochondria  and  x g for  discarded. capillaries  as determined by phase c o n t r a s t microscopy, sedimented on upper s u r f a c e of the p e l l e t . separated  T h i s l a y e r c o u l d be  the  loosened  and  from the p e l l e t by the a d d i t i o n of 1 ml of b u f f e r  lowed by r o c k i n g the c e n t r i f u g e tube g e n t l y ; the s l u r r y was removed w i t h a pasteur  pipette.  the p e l l e t undisturbed, volumes of b u f f e r and  which was  120  x g, and  120  folthen  exercised to  leave  washed by re-suspending i n 10  c e n t r i f u g e d at 380  T h i s washing procedure was g a t i o n at d e c r e a s i n g  Great care was  A  repeated  x g f o r 10 minutes.  3 more times with  g r a v i t a t i o n a l forces  centrifu-  (successively:  196  x g, each f o r 10 minutes), t o y i e l d the washed  particles. KCl-extracted  p a r t i c l e s - Washed p a r t i c l e s were sus-  pended i n 10 volumes o f i s o t o n i c b u f f e r c o n t a i n i n g 2 50 mM rose,  2 mM  d i t h i o t h r e i t o l and  homogenized i n a P o l y t r o n PT maximal v e l o c i t y .  10 mM  T r i s - H C l , pH  7.5  suc-  and were  10 Homogenizer f o r 30 seconds at  These p a r t i c l e s were then subjected  to  salt  e x t r a c t i o n by adding dropwise an equal volume of a s o l u t i o n c o n t a i n i n g 2.5 10 mM KC1  M KC1,  T r i s - H C l , pH  was  1.25  M.  x g,  250  7.5,  The  mM  sucrose,  2 mM  d i t h i o t h r e i t o l in  so t h a t the f i n a l c o n c e n t r a t i o n  s o l u t i o n was  of  g e n t l y s t i r r e d f o r 5 minutes  - 11 -  and then c e n t r i f u g e d a t 100,000 x g f o r 30 minutes.  The p e l l e t e d  p a r t i c l e s were thoroughly washed w i t h 15 volumes o f i s o t o n i c  buf-  f e r and c e n t r i f u g e d a t 37,000 x g f o r 15 minutes. Sucrose g r a d i e n t c e n t r i f u g a t i o n - Discontinuous  sucrose  g r a d i e n t s c o n t a i n i n g l a y e r s o f 5 ml each o f 50%, 55%, 60% and 65% sucrose i n 2 mM d i t h i o t h r e i t o l , 10 mM T r i s - H C l , pH 8.2 were prepared 4 hours b e f o r e use and s t o r e d a t 4°C.  KCl-extracted p a r t i c l e s  o b t a i n e d from the p r e v i o u s step were homogenized i n 10 ml o f a s o l u t i o n c o n t a i n i n g 250 mM sucrose, 2 mM d i t h i o t h r e i t o l i n 10 mM HCl, pH 8.2, u s i n g a P o t t e r - E l v e h j e m  Tris-  (10 s t r o k e s w i t h a l o o s e - f i t -  t i n g p e s t l e , and 10 s t r o k e s with a t i g h t - f i t t i n g p e s t l e ) .  The sus-  pended p a r t i c l e s were l a y e r e d on the sucrose g r a d i e n t s and c e n t r i fuged a t 40,000 x g f o r 1 hour i n a SW 25.1 swinging bucket The m a t e r i a l sedimenting  rotor.  a t the d i f f e r e n t i n t e r f a c e s was s e p a r a t e l y  c o l l e c t e d w i t h Pasteur p i p e t t e s and d i l u t e d 3 - f o l d with a b u f f e r c o n t a i n i n g 2 mM d i t h i o t h r e i t o l i n 10 mM T r i s - H C l , pH 7.5 b e f o r e being c e n t r i f u g e d a t 100,000 x g f o r 1 hour.  The f i n a l p e l l e t s were  suspended i n 250 mM sucrose, 2 mM d i t h i o t h r e i t o l i n 10 mM T r i s - H C l , pH 7.5, and were used f o r v a r i o u s s t u d i e s w i t h i n 2 hours. of the procedure  A scheme  i s p r o v i d e d i n F i g u r e 1.  Before we a r r i v e d a t the f i n a l procedure d e s c r i b e d above, our i n i t i a l approach was t o apply the techniques by Severson  and Drummond  f o r the i s o l a t i o n of s k e l e t a l  emma (17) t o c a r d i a c t i s s u e s .  Adenylate  developed sarcol-  c y c l a s e was used as  plasma membrane "marker" enzyme, and i n each step o f i s o l a t i o n , phase c o n t r a s t microscopy  was used t o check the presence or  FIGURE 1  Scheme o f i s o l a t i o n o f p l a s m a membranes from guinea p i g h e a r t s . D e t a i l s of the procedure a r e d e s c r i b e d i n t e x t under Results i n Part I of this thesis.  -  13 -  J  | G U I N E A PIG VENTRICLES Homogenization Polytron PTIO Max. Sp., 2 sec.  CRUDE H O M O G E N A T E Hypotonic W a s h i n g s (5x)  SUPERNATANT containing mito. capillaries etc. DISCARDED  Centrifuge with decreasing g forces 6 0 0 g to I20g/I0min.  W A S H E D PARTICLES Re-homogenization Polytron PTIO Max.Sp., 30 sec.  R E - H O M O G E N I Z E D PARTICLES KCI (1.25M) Extraction (5min.) Centrifuge at I O O , O O O g / 3 0 min.  H  KCI SUPERNATANT containing solubilized protein.DISCARDED.  EXTRACTED PARTICLES Sucrose G r a d i e n t SUCROSE GRADIENT FRACTIONS SAMPLE' 50% 55% 60% 65%  Fl 40,000 g  F2  I HOUR  F3 F4  removal  of s t r i a t i o n s .  Some of these i n i t i a l attempts  and  the  r e s u l t s o b t a i n e d are b r i e f l y d e s c r i b e d below. Heart s l i c e s were homogenized f o r 30 seconds T r i s - H G l , pH 7.5 w i t h a S o r v a l l Omnimixer The homogenate was 2 1 mm  s t r a i n e d through a n y l o n s e i v e (pore s i z e  The  1,000  x g p e l l e t was  pending i n 15 volumes of 10 mM i n g a t 1,000  x g  washed twice by sus-  T r i s - H C l , pH 7.5  and  centrifug-  x g> f o r 10 minutes t o y i e l d the washed p a r t i c l e s .  P r e p a r a t i o n of lithium-bromide icles:  mM  ( r h e o s t a t s e t a t 120).  ) to remove c o n n e c t i v e t i s s u e and c e n t r i f u g e d a t 1,000  f o r 10 minutes.  (LiBr) e x t r a c t e d p a r t -  Washed p a r t i c l e s obtained from above were suspended i n  15 volumes of 10 mM was  i n 15  added dropwise  T r i s - H C l , pH 7.5  and L i B r  (4.0 M s o l u t i o n )  to a f i n a l c o n c e n t r a t i o n of 0.4  t i o n , g e n t l y s t i r r e d f o r 45 minutes w i t h a magnetic  M.  The  solu-  stirrer,  d i l u t e d w i t h an equal volume of 10 mM  T r i s - H C l , pH 7.5  r i f u g e d a t 2,000 x g f o r 10 minutes.  The e x t r a c t e d p a r t i c l e s  were washed twice by suspending i n 10 mM  T r i s - H C l , pH  f o l l o w e d by c e n t r i f u g a t i o n a t 2,000 x g f o r 10  was  and cent-  7.5  minutes.  I n i t i a l r e s u l t s with L i B r e x t r a c t i o n were encouraging. As shown i n Table I, 75% of the p r o t e i n i n the o r i g i n a l homogenate was  removed a f t e r L i B r e x t r a c t i o n .  a c t i v i t y was  e n r i c h e d about  2.7  Adenylate c y c l a s e  f o l d over the o r i g i n a l homoge-  nate w i t h a y i e l d of 70% of enzyme a c t i v i t y .  Cross  striations  were l a r g e l y removed, but much r e f r a c t i l e m a t e r i a l arranged l o n g i t u d i n a l l y along the l o n g a x i s of the muscle f i b e r s c o u l d s t i l l be seen.  We  t h e r e f o r e attempted  t o e x t r a c t f u r t h e r the  -  LiBr-extracted particles. c e n t r a t i o n s o f KCI N a C l G , 25% 4  KBr  (0.6  and  1 mM  The  15  -  agents t e s t e d i n c l u d e d v a r i o u s  to 3.5  M),  0.5  M NaSCN, 1 M L i B r , 0.5  ATP.  The  L i B r - e x t r a c t e d p a r t i c l e s were  thoroughly suspended f o r about 1 minute i n 15 volumes of 10 T r i s - H C l pH agents. f e r and  7.5  The  s o l u t i o n c o n t a i n i n g one  suspension was  of the  M  mM  above-mentioned  immediately d i l u t e d 3 - f o l d w i t h buf-  c e n t r i f u g e d a t 7,000 x g f o r 20 minutes.  thus obtained  con-  were washed by suspending i n 10 mM  The  membranes  T r i s - H C l pH  7.5  f o l l o w e d by c e n t r i f u g a t i o n at 2,000 x g f o r 10 minutes to y i e l d the f i n a l plasma membrane f r a c t i o n . R e s u l t s of f u r t h e r treatment of L i B r - e x t r a c t e d  part-  i c l e s by the v a r i o u s agents mentioned above were g e n e r a l l y poor. As  shown i n Table I, although the p r o t e i n content o f the  membrane f r a c t i o n was  reduced to 6-8%,  there was  enrichment o f adenylate c y c l a s e a c t i v i t y and enzyme a c t i v i t y was p a r t i c l e s w i t h KCI r a t i o n of KCI was  20%  or l e s s .  Treatment of  from 0.6  o f the f i n a l membrane f r a c t i o n was t h i s was  accompanied by an i n c r e a s e  c y c l a s e t o a maximum of 5.3  to 3.5  M,  of  LiBr-extracted As  the  concent-  p r o t e i n content  p r o g r e s s i v e l y reduced,  and  i n enrichment o f adenylate  f o l d w i t h a y i e l d of 28%.  r e s u l t s l e d us t o b e l i e v e t h a t w h i l e L i B r was s o l u b i l i z i n g agent, and was  concomitant  the f i n a l y i e l d  seemed t o be most promising. increased  no  final  These  a good p r o t e i n -  i t s e l f not d i r e c t l y damaging to ade-  n y l a t e c y c l a s e a c t i v i t y , i t d i s r u p t e d membrane s t r u c t u r e i n such a manner t h a t any to e x t e n s i v e  f u r t h e r treatment of the membrane would l e a d  damage.  Attempts were made to s u b s t i t u t e L i B r  TABLE  I  E f f e c t i v e n e s s o f v a r i o u s s a l t s i n t h e i s o l a t i o n o f c a r d i a c sarcolemma as determined by a d e n y l a t e c y c l a s e a c t i v i t y i n t h e v a r i o u s s t a g e s o f i s o l a tion. A d e n y l a t e c y c l a s e a c t i v i t y was measured a c c o r d i n g t o Assay A (see Methods f o r d e t a i l ) and i n t h e presence o f f l u o r i d e . S p e c i f i c a c t i v i t y and r e c o v e r y o f a d e n y l a t e c y c l a s e and r e c o v e r y o f p r o t e i n were e x p r e s s e d i n percentage r e l a t i v e t o t h e homogenate (taken as 100) t o f a c i l i t a t e comparison o f d i f f e r e n t agents used t o t r e a t L i B r - e x t r a c t e d p a r t i c l e s . Numbers were a t l e a s t d u p l i c a t e p r e p a r a t i o n s i n each c a s e .  Membrane F r a c t i o n s  Adenylate Cyclase Specific ActiRecovery i n v i t y i n percent percent  Protein Recovery i n percent  (a) Crude Homogenate  100  100  100  (b) Washed  131  81  59  268  70  26  (d) 0.6 M KC1  290  39  14  (e) 2.0 M KC1  330  22  6  (f) 3.5 M KC1  534  28  5  .(g) 0.5 M NaSCN  335  20  6  (h) 1.0 M L i B r  255  20  8  ( i ) 0.5 M NaCIO,, 4  270  19  8  (j) 25% KBr  240  16  7  (k) 1.0 mM ATP  112  11  11  Particles  (c) L i B r - e x t r a c t e d P a r t i c l e s F i n a l Membrane a f t e r extraction of LiBr-extracted p a r t i c l e s with  w i t h s a l t s such as Nal or K l which had been employed by e a r l ier  workers (26, 35).  Unfortunately, results  with these  salts  were e q u a l l y , i f not more d i s a p p o i n t i n g w i t h r e s p e c t t o adenyl a t e c y c l a s e a c t i v i t y , both i n terms of s p e c i f i c a c t i v i t y and t o t a l recovery. I t was logy and i n t e r n a l  soon apparent  t h a t due t o the unique morpho-  o r g a n i z a t i o n of the m y o c a r d i a l c e l l ,  extrac-  t i o n of i n t r a c e l l u l a r contents, p a r t i c u l a r l y mitochondria contractile  p r o t e i n s , was  and  dependent not o n l y on the c h o i c e of  chemical agents, but a l s o on the method of homogenization. t h i s r e s p e c t , both the S o r v a l l  In  Omnimixer and the Waring Blender  were found t o be u n s u i t a b l e f o r the c a r d i a c t i s s u e  because i n  o r d e r to achieve the degree of d i s r u p t i o n necessary f o r e f f i c i ent e x t r a c t i o n , r e l a t i v e l y long p e r i o d s of homogenization remely  (1 minute or longer)  were r e q u i r e d and t h i s was  found t o be e x t -  damaging t o the i n t e g r i t y of the plasma membrane as  i n d i c a t e d by the low recovery of "marker" enzymes. t r o n PT 10 Homogenizer, i t was was  With a P o l y  found t h a t , p r o v i d e d the  tissue  thoroughly d i s p e r s e d by p r e l i m i n a r y low-speed homogeniza-  t i o n , a p e r i o d o f homogenization cient  of e x a c t l y 2 seconds was  to e f f e c t i v e l y d i s r u p t the f i b e r s .  Dithiothreitol  suffi (2  mM)  was  found t o have a " p r o t e c t i v e " e f f e c t on the membranes s i n c e  it  improved both the s p e c i f i c a c t i v i t y and the r e c o v e r y of ade-  nylate cyclase a c t i v i t y .  The e f f e c t of t h i s agent on  c y c l a s e i s shown i n Table I I .  When 2 mM  omitted i n the i n i t i a l homogenization,  adenylate  dithiothreitol  was  the d i f f e r e n c e i n the  TABLE I I E f f e c t s o f d i t h i o t h r e i t o l (DTT) on the a c t i v i t y and y i e l d o f adeny l a t e c y c l a s e d u r i n g i s o l a t i o n o f c a r d i a c sarcolemma. R e s u l t s were the means w i t h S.E.M. o f 3 membrane p r e p a r a t i o n s .  ••  Fractions  Protein (%)  -DTT Adenylate  +DTT (2 mM) Cyclase  nmoles/mg/min  Crude Homogenate  100  .17 + .02  Protein (%)  %recovery  100  Adenylate nmoles/mg/min  100  .23 + .03  Cyclase %  recovery  100  Washed Particles  42 '+_ 2  .67 + .08  159 + 15  41 + 3  1.1 + .12  164 + 13  KCl-extracted Membranes  15 + 1  .54 + .12  31 + 16  13 + 1  2.1 + .38  89 + 10  - 19 -  s p e c i f i c a c t i v i t y of the whole homogenate was (compare 0.23  nmoles/mg/minute w i t h DTT  without DTT).  In the washed p a r t i c l e s ,  d i t h i o t h r e i t o l was  "soticeable  to 0.17  nmoles/mg/minute  the p r o t e c t i v e e f f e c t  even more s i g n i f i c a n t ,  as evidenced by the a l -  most 2 - f o l d d i f f e r e n c e i n adenylate c y c l a s e a c t i v i t y . t h a t the presence of t h i s agent was homogenization, and was procedures.  particularly  I t appeared  critical  during  l e s s c r u c i a l i n the subsequent washing  I t s presence was  again of c r i t i c a l  importance when  the washed p a r t i c l e s were homogenized before e x t r a c t i o n w i t h as w e l l as d u r i n g  of  extraction.  KC1  As seen i n Table I I , adenylate  c y c l a s e a c t i v i t y i n the K C l - e x t r a c t e d  p a r t i c l e s p r e v i o u s l y homo-  genized i n the presence of d i t h i o t h r e i t o l was  4 times higher than  those homogenized i n the absence of t h i s agent.  As w i l l be  seen  2+ l a t e r , d i t h i o t h r e i t o l d i d not appear to " p r o t e c t " the Mg endent Na +K -ATPase whose a c t i v i t y was +  +  e x t r a c t i o n w i t h KC1. iothreitol. interfere  d r a s t i c a l l y reduced  P r o t e i n r e c o v e r y was  At the c o n c e n t r a t i o n s  -dep-  not a f f e c t e d by  used, t h i s reagent d i d  The  binding  AMP.  main purpose f o r s u b j e c t i n g the crude homogenate  to s e v e r a l c y c l e s of washings and  c e n t r i f u g a t i o n s was  s o l u b l e p r o t e i n s , endoplasmic r e t i c u l u m , p r o t e i n s and  dithnot  w i t h the adenylate c y c l a s e assay or the p r o t e i n  assay f o r c y c l i c  during  m i t o c h o n d r i a e t c . , and  "cytoskeleton".  Both hypotonic and  employed by e a r l i e r workers.  "exposed"  to remove  contractile  to f u r t h e r break up isotonic  Results  s o l u t i o n s have been  shown i n Table I I I  ate t h a t the e f f e c t i v e n e s s of e i t h e r 10 mM  the  T r i s - H C l , pH  indic7.5  or  - '20 -  TABLE  III  E f f e c t o f d i f f e r e n t b u f f e r s and the number o f washes on removal o f p r o t e i n s from h e a r t homogenate. Results are means o f d u p l i c a t e p r e p a r a t i o n s and the numbers are p e r c e n t a g e s o f p r o t e i n r e m a i n i n g i n the p e l l e t r e l a t i v e t o the homogenate taken as 100.  Number o f Washes Buffers Homogenate  I  II  III  IV  V  VI  10 mM T r i s - H C l pH 7.5  100  67  53  47  42  39  37  10 mM T r i s - H C l 250 mM s u c r o s e pH 7.5  100  67  51  45  43  35  33  0.1 M NaCIO lOmM T r i s - H C l pH 7.5  100  53  46  38  34  31  30  - 21 -  250 mM  sucrose i n 10 mM  T r i s - H C l , pH 7.5 to remove p r o t e i n  the whole homogenate were i d e n t i c a l .  from  Hypotonic medium had the  advantage o f l y s i n g r e s i d u a l red blood c e l l s t h a t were trapped i n the m y o c a r d i a l c a p i l l a r y beds.  NaC10  (0.1 M) was  4  most e f f i c i e n t agent i n removing p r o t e i n .  by f a r the  A f t e r the f i r s t  cycle  of washing and c e n t r i f u g a t i o n w i t h t h i s agent, c l o s e to 50% of the p r o t e i n was  removed.  But t h i s agent was  not used i n our  f i n a l procedure due to i t s d e l e t e r i o u s e f f e c t s on c e r t a i n enzyme activities.  Regardless of the b u f f e r s used, p r o t e i n removal tended  to l e v e l o f f a f t e r 4 washes and f u r t h e r washing tended t o decrease the y i e l d o f "marker" enzymes without f u r t h e r removing We  therefore  tions.  r o u t i n e l y used 4 c y c l e s o f washings  centrifuga-  The c e n t r i f u g a l f o r c e s chosen f o r the f i n a l procedure  decreased from 620 x g/10 it  and  protein.  minutes to 120, x g/10  minutes.  While  i s g e n e r a l l y accepted t h a t m i t o c h o n d r i a w i l l not sediment at 5  a g r a v i t a t i o n a l f o r c e l e s s than 10 g-minute  (time i n t e g r a t e d  f o r c e ) , c a r d i a c m i t o c h o n d r i a are l a r g e r and denser than those from o t h e r c e l l types and the d e c r e a s i n g g f o r c e was  necessary t o p r e -  vent the sedimentation of these p a r t i c l e s . The f i n a l step i n the procedure i n v o l v e d the use of d i s c o n t i n u o u s sucrose d e n s i t y l e t e the s e p a r a t i o n  gradients.  The aim here was  t o comp-  o f plasma membranes from any contaminating  s u b c e l l u l a r components which escaped the p r e v i o u s e x t r a c t i o n procedures.  One o f the major problems  encountered was  the  tendency o f the membrane bands to o v e r l a p i n the g r a d i e n t .  In  -  the i n i t i a l attempts,  2 2/  sucrose g r a d i e n t s were prepared i n 10 mM  T r i s - H C l , pH 7.5 and 3 bands of membranous m a t e r i a l were r e covered from the g r a d i e n t .  The bands were c l o s e t o g e t h e r and  adenylate c y c l a s e and cytochrome C oxidase a c t i v i t i e s were t r i b u t e d almost evenly i n a l l 3 bands.  dis-  With the a d d i t i o n of 1  mM EDTA there was a dramatic improvement i n the r e s o l u t i o n of g r a d i e n t bands both i n terms of t h e i r p h y s i c a l s e p a r a t i o n on the g r a d i e n t as w e l l as d i s t r i b u t i o n of "marker" enzymes.  The  best r e s o l u t i o n of g r a d i e n t bands was found when the sucrose was prepared i n 10 mM T r i s - H C l a t pH 8.2 i n s t e a d o f pH  7.5.  By simply a l t e r i n g the pH of the g r a d i e n t , 4 d i s t i n c t bands were r e s o l v e d and as w i l l be seen i n the f o l l o w i n g s e c t i o n , one o f the bands was h i g h l y e n r i c h e d i n plasma membranes.  2.  C h a r a c t e r i z a t i o n o f c a r d i a c sarcolemma by "marker" enzymes The washed p a r t i c l e s , a f t e r 4 c y c l e s o f washing and  c e n t r i f u g a t i o n s , were found t o have as l i t t l e t e i n i n the o r i g i n a l homogenate.  as 40% o f the pro-  The remaining 60% o f the pro-  t e i n was r e l e a s e d i n the supernatant which, i n the main, cont a i n e d c a p i l l a r i e s , r e s i d u a l r e d blood c e l l s , endoplasmic culum, s o l u b l e p r o t e i n s and m i t o c h o n d r i a .  reti-  As much as 50% o f  the cytochrome C oxidase a c t i v i t y , a m i t o c h o n d r i a l "marker" enzyme, was r e l e a s e d i n t o the supernatant by t h i s procedure.  On  the other hand, adenylate c y c l a s e and o u a b a i n - s e n s i t i v e Na +K +  ATPase a c t i v i t i e s , enzymes c o n s i d e r e d t o be predominantly l o c a l i z e d i n plasma membranes  (36) , were e n r i c h e d i n the washed  +  23 -  p a r t i c l e s , as i n d i c a t e d by the 4 - f o l d and 2 - f o l d i n c r e a s e i n their respective specific a c t i v i t i e s  (Table I V ) . When viewed  under the phase c o n t r a s t microscope, these washed p a r t i c l e s were seen t o c o n t a i n some s t r i a t i o n s , i n d i c a t i n g the incomplete r e moval of c o n t r a c t i l e p r o t e i n s .  A f t e r homogenization and e x t r a c -  t i o n with K C l , another 30% o f t h e p r o t e i n was removed. was a f u r t h e r r e d u c t i o n o f cytochrome C oxidase Adenylate c y c l a s e was e n r i c h e d to 8 - f o l d , having  There  a c t i v i t y by 30%.  i n these e x t r a c t e d membranes up  a s p e c i f i c a c t i v i t y o f 2 nmoles/mg/minute, 2+  but a s i g n i f i c a n t p o r t i o n , as much as 65% of the Mg + + Na +K -ATPase was l o s t .  -dependent  The s p e c i f i c a c t i v i t y . o f t h i s  enzyme  was reduced from 56 nmoles/mg/minute i n the washed p a r t i c l e s t o 41 nmoles/mg/minute a f t e r e x t r a c t i o n .  T h i s l o s s was  due t o i n a c t i v a t i o n o f the enzyme by the high s a l t s i n c e repeated  attempts t o recover  supernatant were not s u c c e s s f u l .  probably  concentration  the a c t i v i t y from the K C l The K C l - e x t r a c t e d  particles,  c o n t a i n i n g 10% o f the p r o t e i n o f the o r i g i n a l homogenate, was r e s o l v e d by sucrose  density gradient centrifugation into 4 d i s t -  i n c t bands (Figure 1 ) .  The d i s t r i b u t i o n of enzyme a c t i v i t i e s i n  these f r a c t i o n s i s shown i n F i g u r e 2.  The m a t e r i a l i n Band 1 on  the top of the g r a d i e n t was h i g h l y e n r i c h e d i n cytochrome C oxidase IV).  ( 5 - f o l d i n c r e a s e i n s p e c i f i c a c t i v i t y as shown i n Table  I t contained  60% o f the t o t a l cytochrome C oxidase  acti2+  v i t y and only 10% each o f the t o t a l adenylate  c y c l a s e and Mg  -dependent Na +K -ATPase  from the g r a d i e n t ,  +  +  a c t i v i t i e s recovered  i n d i c a t i n g t h a t the m a t e r i a l was predominantly of m i t o c h o n d r i a l  TABLE IV Distribution sarcolemma.  and a c t i v i t i e s o f "marker" enzymes i n v a r i o u s f r a c t i o n s o b t a i n e d d u r i n g i s o l a t i o n o f h e a r t A l l v a l u e s were t h e means w i t h S.E.M. o f 6 d i f f e r e n t c a r d i a c membrane p r e p a r a t i o n s  Protein  Adenylate Cyclase  Ouabain-sensitive ++ (Na +K )-ATPase Mg  Cytochrome C Oxidase  nmoles ATP hydrolysed mg/minute  percent recovery  nmoles/mg/ minute  26 + 1.6  100  +  Fractions %  100  Homogenate  nmoles cAMP p e r c e n t formed/mg/ r e c o v e r y minute  .26 + .03  100  166 ± 17  percent . . . recovery  100  40 ± 2.8  1.1 + .12 164 ± 13  56 + 3.4  87 ± 6.0  196 ± 5.7  48 ± 10  12 ± 1.7  2.1 + .38  89 ± 10  41 + 3.7  19 ± 2.2  308 ± 27  21 ± 3.0  2.3 ± .16  1.3 + .11  11 ± 88  48 + 7.3  3.1±  829 ± 124  11 ± 1.4  2.1 ± .18  2.8 + .48  22 ± 2.3  98 + 13  5.6 ± 1.5  318 ± 32  4.3 ± .26  G r a d i e n t Band 3  2.6 ± .45  3.6 + .72  33 ± 4.0  107 + 8.2  7.5 ± 2.0  164 ± 21  2.6 ± .52  G r a d i e n t Band 4  5.1 ± .27  2.2 + .27  44 ± 4.6  60 + 8.9  7.8 ± 1.1  93 ± 14  2.5 ± .35  Washed  Particles  KCl-extracted Particles G r a d i e n t Band 1 G r a d i e n t Band  2'.  .55  origin.  The m a t e r i a l i n Band 2 which sedimented  a t the  between 50-60% sucrose r e p r e s e n t e d a p a r t i a l enrichment  interface o f memb-  ranes o f sarcolemmal o r i g i n as i n d i c a t e d by the i n c r e a s e i n spe2+ + + c i f i c a c t i v i t i e s o f adenylate c y c l a s e and Mg -dependent Na*+K -ATPase.  But t h i s f r a c t i o n was a l s o s i g n i f i c a n t l y  contaminated  by mitochondria s i n c e 20% o f the cytochrome C oxidase recovered from the g r a d i e n t was l o c a t e d here.  activity  Material sedi-  mented i n Band 3 a t the i n t e r f a c e between 55-60% sucrose and i n Band 4 a t the i n t e r f a c e between 60-65% sucrose c o n t a i n e d membranes  t h a t were h i g h l y e n r i c h e d i n sarcolemma.  Adenylate  cyc-  l a s e s p e c i f i c a c t i v i t y was i n c r e a s e d 1 4 - f o l d i n Band 3 w i t h a s p e c i f i c a c t i v i t y o f 3.6 nmoles/mg/minute, and even the p a r t i 2+ + + a l l y i n a c t i v a t e d Mg -dependent Na +K -ATPase was e n r i c h e d over 4-fold.  Together,  Bands 3 and 4 c o n t a i n e d over 65% o f the  total  adenylate c y c l a s e and 60% o f the t o t a l Mg2+ -dependent Na++K+ATPase a c t i v i t i e s r e c o v e r e d from the g r a d i e n t . t i o n s were m i n i m a l l y contaminated  These two f r a c -  by m i t o c h o n d r i a .  Specific  a c t i v i t y f o r cytochrome C oxidase a s s o c i a t e d w i t h these membranes were low,  164 and 93 nmoles/mg/minute r e s p e c t i v e l y f o r  Bands 3 and 4.  Only 2% o f the t o t a l cytochrome C oxidase  acti-  v i t y i n the whole homogenate were recovered from each o f these fractions.  5' N u c l e o t i d a s e , commonly used as a plasma memb-  rane marker, was found not t o be s i g n i f i c a n t l y e n r i c h e d f o l d ) i n sucrose g r a d i e n t Bands 3 and 4. Table V, the enzyme appeared  (1.3-  In f a c t , as shown i n  t o a s s o c i a t e w i t h a l l the  fractions  o b t a i n e d d u r i n g the i s o l a t i o n o f the sarcolemma, i n d i c a t i n g t h a t  FIGURE 2  D i s t r i b u t i o n o f enzyme a c t i v i t i e s i n s u c r o s e gradient fractions. F o r e a c h enzyme, t h e t o t a l a c t i v i t y from t h e g r a d i e n t i s t a k e n as 100%, and t h e r e c o v e r y from each g r a d i e n t f r a c t i o n i s expressed as p e r c e n t o f the t o t a l .  60  < O  50  r-  (A) Mg (Na -K ) ATPase + +  o Sf  +  +  (B)  (C)  (D)  Adenylate Cyclase  5' Nucleotidase  Cytochrome C Oxidase  II  I  40  >-  >  t— U <  30  h-  20  h-  10  h"  mmJ •  t—  o  I  II  III  IV  I  III  IV  II  III  GRADIENT FRACTIONS  IV  I  II  III  IV  -  -  28 -  TABLE V  D i s t r i b u t i o n and a c t i v i t i e s o f 5 ' N u c l e o t i d a s e i n f r a c t i o n s o b t a i n e d d u r i n g i s o l a t i o n o f c a r d i a c sarcolemma. P r o t e i n y i e l d and r e c o v e r y o f enzyme a c t i v i t i e s were expressed as p e r c e n t r e l a t i v e t o the homogenate. A l l v a l u e s were means w i t h S.E.M. o f 6 d i f f e r e n t membrane p r e p a r a t i o n s .  Protein  Fractions  Homogenate  . Recovery ^ '': i n Percent  100  5'Nucleotidase  nmoles 5'AMP hydrolyzed/ mg/minute  18 + .50  Recovery in Percent  100  Washed P a r t i c l e s  40 + 2.8  21 + .95  47 + 2.2  KCl-extracted P a r t i c l e s  12 + 1.7  18 + 1.3  11 + .80  G r a d i e n t Band 1  2.3 + .16  18 + 3.1  2.4 + .21  G r a d i e n t Band 2  2.1 + .18  19 + 1.6  2.3 + .19  G r a d i e n t Band 3  2.6 +. .45  23 + 2.9  3.1 + .33  G r a d i e n t Band 4  5.1 + .27  23 + 2.8  6.4 ± .44  -  29  -  the plasma membrane i s not the e x c l u s i v e source of the enzyme, at l e a s t i n the guinea p i g myocardium. bined Bands 3 and  We  have r o u t i n e l y com-  4 as our f i n a l sarcolemmal p r e p a r a t i o n .  Each  gram of v e n t r i c u l a r t i s s u e (wet weight) y i e l d e d approximately 7-8  mg  of membrane p r o t e i n , p r o v i d i n g adequate m a t e r i a l f o r  v a r i o u s enzymatic s t u d i e s .  DISCUSSION Although we  have used phase c o n t r a s t microscopy to  examine v a r i o u s membrane f r a c t i o n s d u r i n g the i s o l a t i o n procedure, the technique was  of l i m i t e d use  i n view of the f a c t t h a t  the membranes assumed v e s i c u l a r forms a f t e r e x t e n s i v e i z a t i o n s and  thus l o s t t h e i r p h y s i c a l i d e n t i t y .  based our c r i t e r i a o f membrane p u r i t y on t h i s r e s p e c t , we  had  We  therefore  "marker" enzymes.  to r e l y h e a v i l y on data p u b l i s h e d  o t h e r t i s s u e s to choose the a p p r o p r i a t e  homogen-  In  for  "marker" enzymes s i n c e  o n l y very l i t t l e data were a v a i l a b l e r e l a t e d s p e c i f i c a l l y  to  c a r d i a c sarcolemma. In r a t l i v e r , adenylate a s s o c i a t e with  c y c l a s e has been shown to  the plasma membrane to the e x c l u s i o n of a l l  other s u b c e l l u l a r components  (37).  M a r i n e t t i e t a l (38)  p o r t e d p u r i f i c a t i o n of the enzyme up to 100-fold plasma membranes.  Wolff  from bovine t h y r o i d and  and  in rat liver  Jones prepared plasma membranes  found t h a t the enzyme was  80 to 150-fold over the whole homogenate (39). adenylate  re-  enriched  from  Enrichment i n  c y c l a s e a c t i v i t y t o v a r i o u s degrees has a l s o been  demonstrated adrenal c e l l s  i n plasma membranes i s o l a t e d from e r y t h r o c y t e s (40), (41), a n t e r i o r p i t u i t a r y  (14) and f a t c e l l s (47).  I f no enzyme has y e t been proven t o be an e x c l u s i v e and u n i v e r s a l enzyme marker o f plasma membranes, these d a t a i n d i c a t e t h a t adenylate  c y c l a s e appears t o be the c l o s e s t p o s s i b l e candidate  for this q u a l i f i c a t i o n .  R e s u l t s i n Table IV show t h a t  this  enzyme a c t i v i t y was e n r i c h e d more than 1 0 - f o l d i n the p r e s e n t c a r d i a c sarcolemmal  preparation.  The s m a l l amount o f adenylate  c y c l a s e a c t i v i t y t h a t was found t o a s s o c i a t e w i t h g r a d i e n t bands 1 and 2 might r e p r e s e n t contaminating plasma membranes i n these f r a c t i o n s , o r they c o u l d be due t o i n t r i n s i c adenylate c y c l a s e a c t i v i t y a s s o c i a t e d with membranes not o f sarcolemmal Sulakhe  origin.  e t a i (43) and Katz e t al (44) have r e c e n t l y p r o v i d e d  some i n d i r e c t evidence t h a t a minor p o r t i o n o f the adenylate c y c l a s e a c t i v i t y i n the myocardium may be a s s o c i a t e d w i t h the microsomal  fraction. As mentioned above, 2 mM d i t h i o t h r e i t o l was necessary  t o p r e s e r v e the adenylate c y c l a s e a c t i v i t y i n the course o f the i s o l a t i o n o f sarcolemma.  T h i s enzyme has been r e p o r t e d t o be  associated.with c e r t a i n e s s e n t i a l s u l f h y d r y l group(s).  In pur-  i f i e d bovine t h y r o i d plasma membranes, Wolff e t a_l (39) found t h a t both TSH- and f l u o r i d e - s t i m u l a t e d adenylate c y c l a s e a c t i v i t y were e q u a l l y s e n s i t i v e t o s u l f h y d r y l reagents such as N-ethyl-maleimide  and p-chloromercuribenzoate.  Similar  findings  were r e p o r t e d by Schramm e t a l (45) who s t u d i e d the adenylate c y c l a s e o f r a t p a r o t i d gland.  Strong evidence was a l s o p r o v i d e d  - '31 -  by  studies with the soluble adenylate  coccus s a l i v a r i u s . and  c y c l a s e from  T h i s enzyme h a s b e e n p u r i f i e d  Strepto3200-fold  h a s b e e n shown t o be s t r o n g l y i n h i b i t e d by s u l f h y d r y l r e -  agents.  The i n h i b i t i o n c a n be r e v e r s e d  by a g e n t s such as  cysteine, mercaptoethanol o r d i t h i o t h r e i t o l  (46). I t i s there-  fore quite probable that e s s e n t i a l s u l f h y d r y l s are a of a l l adenylate  cyclases.  feature  I n t h e i n t a c t t i s s u e , components o f  t h e c e l l membrane may p r o t e c t t h e s e g r o u p s t o some e x t e n t , b u t i n t h e c o u r s e o f t i s s u e f r a c t i o n a t i o n , l a b i l i z a t i o n o f membrane s t r u c t u r e s can presumably l e a d t o exposure o f s u l f h y d r y l groups which, unless ol,  p r o t e c t e d by r e d u c i n g  agents such as d i t h i o t h r e i t -  c a n e a s i l y be o x i d i z e d . The  p u r i t y of the present  sarcolemmal preparation i s  a l s o s u p p o r t e d by t h e 4 - f o l d e n r i c h m e n t o f t h e o u a b a i n - s e n s i t i v e Na +K -ATPase a c t i v i t y . +  +  specifically muscle  T h i s enzyme h a s b e e n shown t o be  a s s o c i a t e d w i t h p l a s m a membranes f r o m  ( 1 7 , 2 2 , 2 3 , 47) a s w e l l a s c a r d i a c m u s c l e  skeletal (28, 4 8 ) . 2+  Although c o - p u r i f i c a t i o nof adenylate ent Na +K -ATPase has been r e p o r t e d +  +  c y c l a s e a n d Mg  -depend-  i n i s o l a t e d p l a s m a memb-  r a n e f r a g m e n t s f r o m a number o f t i s s u e s , t h e d e g r e e o f p u r i f i c a t i o n f o r t h e two enzymes was s e l d o m t h e same. e n c e s h a v e b e e n o b s e r v e d i n membrane p r e p a r a t i o n s thyroid ary  ( 3 9 ) , HeLa c e l l s  (49), l i v e r  ( 1 4 ) . Tada e t a l (29) r e p o r t e d +  cyclase a c t i v i t i e s  differ-  from bovine  (50) a n d a n t e r i o r p i t u i t 2+  a 2-fold increase  dependent Na +K -ATPase and a 4 - f o l d i n c r e a s e +  Such  in-Mg  i n adenylate  i n their p a r t i a l l y purified cardiac  sarccv-  -  - 32 -  lemma.  T h a t t h e s e t w o enzymes w e r e s i m u l t a n e o u s l y e n r i c h e d t o  a much g r e a t e r e x t e n t i n t h e p r e s e n t s a r c o l e m m a l  preparation 2+  ( 1 0 - f o l d f o r a d e n y l a t e c y c l a s e a n d 4 - f o l d f o r Mg  -dependent  Na +K -ATPase) emphasized i t s h i g h degree o f p u r i t y . +  +  Contamination  by m i t o c h o n d r i a was m i n i m a l ;  t h i s was  i n d i c a t e d by t h e low recovery o f cytochrome C oxidase in the f i n a l  sarcolemmal  activity  p r e p a r a t i o n , and a s w i l l be seen i n a  l a t e r s e c t i o n , t h e a z i d e - i n s e n s i t i v e Ca  uptake  b y t h e memb-  ranes. In for  t h e absence o f any a c c e p t e d  s p e c i f i c m a r k e r enzyme  s a r c o p l a s m i c r e t i c u l u m , i t was n o t p o s s i b l e t o d e t e r m i n e t h e  distribution of this subcellular  fraction.  But i n view o f t h e  s e r i e s o f washes a n d l o w speed c e n t r i f u g a t i o n s employed, contami n a t i o n by microsomes i s n o t l i k e l y  t o be s i g n i f i c a n t .  s t u d i e s w i t h s k e l e t a l sarcolemma, Sulakhe demonstrated  a n d Drummond  the effectiveness of d i f f e r e n t i a l  o f m i c r o s o m e s was m i x e d w i t h i s o l a t e d  individually  20,000 g - m i n u t e .  i d e n t i f i e d a f t e r a one-step I n the present procedure,  r a n e s w e r e d e r i v e d f r o m a 1,200 g - m i n u t e In for  c o n c l u s i o n , by c a r e f u l l y  sarcolemma.  sarcolemma,  t h e s e t w o d i f f e r e n t membrane f r a c t i o n s w e r e c o m p l e t e l y and  (32) h a v e  centrifugation at  low g r a v i t a t i o n a l f o r c e s i n s e p a r a t i n g microsomes from When a n e x c e s s  In previous  separated  centrifugation at t h e sarcolemmal  memb-  pellet.  selecting the conditions  p h y s i c a l d i s r u p t i o n and chemical e x t r a c t i o n o f c a r d i a c  muscle f i b e r s ,  and by combining  differential  and i s o p y c n i c c e n t -  r i f u g a t i o n t e c h n i q u e s , a membrane f r a c t i o n h i g h l y e n r i c h e d i n  s a r c o l e m m a was contaminated  isolated.  The p r e p a r a t i o n was  by o t h e r c e l l u l a r  t a i n e d a good y i e l d o f a d e n y l a t e  of a l l ,  the p r e p a r a t i o n con-  c y c l a s e a n d , a s w i l l be s e e n  i m p o r t a n t b i o c h e m i c a l p r o c e s s were p r e s e n t  also,  i n g t h a t t h e f u n c t i o n a l i n t e g r i t y o f t h e s a r c o l e m m a was preserved.  un-  o r g a n e l l e s a s i n d i c a t e d by m a r k e r  enzyme s t u d i e s ; a n d m o s t i m p o r t a n t  later,  relatively  indicatlargely  PART I I :  CARDIAC SARCOLEMMA:  PHOSPHORYLATION AND CALCIUM UPTAKE  INTRODUCTION Adenosine 3', 5' monophosphate ( c y c l i c AMP) was o r i g i n a l l y d i s c o v e r e d as the i n t r a c e l l u l a r mediator genolytic effect  o f e p i n e p h r i n e and glucagon  o f the g l y c o -  i n the l i v e r ; i t  has s i n c e been r e c o g n i z e d as a 'second messenger"' mediating a v a r i e t y o f hormonal e f f e c t s effects  o f catecholamines  crease c o n t r a c t i l i t y ,  (51). One o f the most notable  on t h e h e a r t i s t h e i r  a b i l i t y to i n -  and much r e s e a r c h has been devoted  i n the  past decade t o determine the p r e c i s e r e l a t i o n s h i p between c y c l i c AMP and the hormone-induced i n o t r o p i c  response.  Sutherland and h i s a s s o c i a t e s proposed f o u r c r i t e r i a which, i f s a t i s f i e d , would c o n s t i t u t e s u f f i c i e n t  evidence t o  i m p l i c a t e c y c l i c AMP as an i n t e r m e d i a r y i n a given hormone-end organ system:  (1) adenylate c y c l a s e , t h e enzyme t h a t c a t a l y z e s  the formation o f c y c l i c AMP from ATP, should be s t i m u l a t e d by the hormone i n i n t a c t  tissue;  (2) a s i m i l a r  effect  should be ob-  served i n broken c e l l p r e p a r a t i o n s of the same t i s s u e ; hanced end organ a c t i v i t y should be observed inhibitors  (3) en-  i n the presence of  of 3',5' c y c l i c n u c l e o t i d e phosphodiesterase, the  enzyme r e s p o n s i b l e f o r the breakdown o f c y c l i c AMP, and (4) c y c l i c AMP s h o u l d have the a b i l i t y t o produce the end organ directly.  response  In the case o f hormone-induced i n o t r o p i s m , i t may be  - 35 -  added t h a t  t h e r e s h o u l d be a p r o p e r  ween i n t r a c e l l u l a r response  levels  temporal  of cyclic  AMP  first  as a m e d i a t o r  and s e c o n d  criteria  i n the i n o t r o p i c response  catecholamines  and t h e m e c h a n i c a l  perfused hearts  (52-54) as w e l l  i n tissue  phrine preceded  of cardiac  levels  of cyclic  AMP  levels  AMP  the i n o t r o p i c  heart s l i c e s  AMP  tissue to  shown t h a t  the administ-  i n isolated,  i n response  the increase i n c o n t r a c t i l e  i s o p r o t e r e n o l , which prevented  cyclic  as i n h e a r t s i n v i v o  B e t a - a d r e n e r g i c b l o c k i n g agents  abolished  to implicate  were s a t i s f i e d when i t was  ration of epinephrine increased c y c l i c  in  bet-  of the heart. The  creases  relationship  force  (55).  In-  to epine(52, 56, 57).  such as p r o n e t h a l o l o r d i c h l o r o the increase i n c y c l i c  response  (52, 55).  (58, 59) were a l s o  Cyclic  increased.by  AMP,  AMP  also  levels  incubation with  r catecholamines, The  observations i n i s o l a t e d  similar cat  and t h e i n c r e a s e was p r e v e n t e d  studies using particulate  (61) and d o g  agents  intact  prevented  (62, 63) h e a r t s .  t i s s u e s were c o n f i r m e d by p r e p a r a t i o n s from  c y c l a s e by c a t e -  cholamines  i n a l l these  stimulated  adenylate c y c l a s e i n these p a r t i c u l a t e  quantitatively  r a t (60),  Beta-adrenergic blocking  the a c t i v a t i o n of adenylate tissues.  by p r o p a n o l o l (59).  Furthermore,  i n the order of t h e i r  inotropic  catecholamines cell  potency  fractions i n vivo  (60-62). Positive c y c l i c AMP by  Rail  support  as a m e d i a t o r  and West who  f o r the t h i r d  criteria  i n the i n o t r o p i c  noted  a greatly  implicating  response  was  enhanced i n o t r o p i c  provided effect  - 36 -  of norepinephrine diesterase  i n the presence  inhibitor  that c y c l i c  AMP  response  was  subsequently  well  after  of phosphodiesterase,  c y c l i c AMP  mediation  The  t h e o p h y l l i n e treatment  ficult  and  last  inotropic  to e s t a b l i s h  to c y c l i c  AMP.  criteria  response  was  s u c c e s s i n t h e use  who  observed  an  intracardiac  igation  hibited  a positive  inotropic  propanolol.  cyclic  AMP,  K u k o v e t z and  as w e l l  dif-  o f exogenous c y c l i c  AMP  limited  t o one  r e p o r t by  output  a c t i o n of AMP  and  on  itself  on  (70)  the AMP  allowed the  cardiac  that  isolated  invest-  contractility. cyclic cat  AMP  showed t h a t  as i t s d i h e x a n o y l d e r i v a t i v e ,  ex-  papillary  these responses were not  Poch  of  phosphodiesterase  that dibutyryl  effect and  i n t r o d u c t i o n of  an a n a l o g o f c y c l i c  agent  demonstrated  muscle d r i v e n e l e c t r i c a l l y , by  AMP,  s o l u b l e than c y c l i c  (69)  AMP  AMP  The  to the degrading  of the e f f e c t of t h i s  Skelton et a l  t o be more  a d m i n i s t r a t i o n o f h i g h doses  d i b u t y r y l d e r i v a t i v e of c y c l i c  and more l i p i d  found  increased cardiac  i n u n a n e s t h e t i z e d dogs.  i s more r e f r a c t o r y  i n order  of c y c l i c  (68)  AMP  for  between c y c l i c  the d i b u t y r y l d e r i v a t i v e  Levine et a l  after  potent  low m y o c a r d i a l p e r m e a b i l i t y  t h e a c t i o n o f hormone was  cyclic  imidazole' , a  t o .be s a t i s f i e d  to reproduce  rate  (65,  provided further evidence  because of the  Until  became a v a i l a b l e ,  heart  by  a cause-and-effect relationship  the p o s i t i v e  this  (67) .  fourth  to e s t a b l i s h  demonstrated  correlated with  Antagonism o f the methylxanthines  stimulant  and  I n was  accumulation  augmented p o s i t i v e 66).  (64).  o f t h e o p h y l l i n e , a phospho-  altered  dibutyryl produced  - 37 -  i n c r e a s e s i n r a t e and amplitude o f c o n t r a c t i o n s measured i n h e a r t s of r a t s , guinea p i g s and r a b b i t s . (71)  a l s o provided  Drummond and Hemmings  evidence t h a t t h i s c y c l i c AMP analog has  a c t i o n s on the b e a t i n g r a t heart q u a n t i t a t i v e l y s i m i l a r t o those o f a d r e n e r g i c  amines.  T h e i r data a l s o i n d i c a t e d t h a t  cyc-  l i c AMP, formed by a d e a c y l a t i o n r e a c t i o n , was i n f a c t the a c t ive  component i n the a c t i o n o f d i b u t y r y l c y c l i c  AMP.  These s t u d i e s , which have s a t i s f i e d a l l the f o u r c r i t e r i a , p r o v i d e very  strong evidence f o r a cause-and-effect r e -  l a t i o n s h i p between c y c l i c AMP and the i n o t r o p i c a c t i o n of c a t e cholamines.  However, s e v e r a l experiments r e p o r t e d  i n the l i t -  e r a t u r e have i n d i c a t e d t h a t such an a s s o c i a t i o n i s not i n d i s p u t a b l e . F o r example, Benfey and h i s a s s o c i a t e s  (72, 73) observed  t h a t while dopamine, phenylephrine and norepinephrine contractility, and  only norepinephrine  i n c r e a s e d c y c l i c AMP l e v e l s .  stimulated  adenylate  increased cyclase  More r e c e n t l y , the same authors  demonstrated t h a t phenoxybenzamine prevented the i n o t r o p i c e f fect of  5-hydroxytryptamine, but d i d not i n h i b i t the r i s e i n  c y c l i c AMP accumulation.  A single i n j e c t i o n of reserpine  24 hours b e f o r e t h e experiment d i d not i n h i b i t the i n o t r o p i c e f f e c t o f 5-hydroxytryptamine but prevented the r i s e i n c y c l i c AMP formation  (74).  These authors concluded t h a t c a r d i a c  cont-  r a c t i l i t y may be i n c r e a s e d without a r i s e i n c y c l i c AMP forma t i o n and, c o n v e r s e l y ,  c y c l i c AMP accumulation need not be  accompanied by a r i s e i n c o n t r a c t i l i t y . u s i n g i s o l a t e d perfused  Shanfeld  e_t a l (75) ,  r a t h e a r t s , r e p o r t e d t h a t a t low doses  of catecholamines, s i g n i f i c a n t  increases i n c o n t r a c t i l e tension  - 38 -  c o u l d be demonstrated  without measurable  levels.  they demonstrated  Furthermore,  isopropylmethoxamine  increase i n c y c l i c t h a t the b l o c k i n g  AMP  agent  a b o l i s h e d the r i s e i n c y c l i c AMP without  a f f e c t i n g the i n c r e a s e d c o n t r a c t i l i t y produced by n o r e p i n e p h r i n e . I t was concluded t h a t under proper c o n d i t i o n s , i n c r e a s e d c a r d i a c c o n t r a c t i l i t y produced by catecholamines c o u l d take p l a c e w i t h out changes i n c y c l i c AMP.  In c o n t r a s t , W a s t i l a e t a l (76) found  t h a t a s i m i l a r b l o c k i n g agent,  N-tertiary-butyl-methoxamine,  when i n f u s e d i n t o open c h e s t dogs, produced  a dose dependent  decrease i n n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i l i t y , c y c l i c  AMP  l e v e l s and phosphorylase a l e v e l s , i n d i c a t i n g t h a t i n c r e a s e i n c y c l i c AMP l e v e l s c o u l d not be d i s s o c i a t e d from the c o n t r a c t i l e response.  S i m i l a r l y , the support gained from the use o f methyl-  xanthines has been s e r i o u s l y c h a l l e n g e d because  these agents  have t h e i r own i n t r i n s i c a c t i o n s on c a r d i a c t i s s u e which may account f o r e f f e c t s a t t r i b u t e d t o c y c l i c AMP.  For example,  t h e o p h y l l i n e i s known t o a l t e r i n t r a c e l l u l a r c a l c i u m b i n d i n g by the s a r c o p l a s m i c r e t i c u l u m calcium  (77) , m i t o c h o n d r i a l accumulation o f  (78) as w e l l as membrane t r a n s p o r t of c a l c i u m (79).  Under these circumstances, the i n c r e a s e d c o n t r a c t i l i t y  observed  may be due t o e l e v a t i o n of f r e e i n t r a c e l l u l a r c a l c i u m l e v e l s r a t h e r than a l t e r a t i o n s i n c a r d i a c c y c l i c AMP l e v e l .  The i s s u e  i s f u r t h e r complicated by the f a c t t h a t methylxanthines have been shown t o r e l e a s e catecholamines from both the b r a i n (80) and, h e a r t  (81).  T h e o p h y l l i n e has a l s o been observed t o e x e r t a  positive inotropic e f f e c t at concentrations i n s u f f i c i e n t to  i n h i b i t phosphodiesterase  (82).  Thus, e f f e c t s o f these  agents  which appear t o mimic a c t i o n s o f catecholamines might be due t o catecholamine  l i b e r a t i o n o r a l t e r a t i o n o f c a l c i u m l e v e l s or  some o t h e r y e t u n s p e c i f i e d mechanisms r a t h e r than of  facilitation  i n t r a c e l l u l a r accumulation o f c y c l i c AMP v i a i n h i b i t i o n of  phosphodiesterase. In have adopted  view o f these c o n s i d e r a t i o n s , s e v e r a l d i f f e r e n t approaches  investigators  whereby i n t r a c e l l u l a r  cyclic  AMP may be e l e v a t e d by mechanisms t h a t bypass the a d r e n e r g i c r e ceptor.  D i m e t h y l s u l f o x i d e (DMSO) i s an agent t h a t  facilitates  the d i f f u s i o n of. a wide v a r i e t y o f compounds i n t o c e l l s . et  Kjekshus  a l (83) s t u d i e d changes i n c a r d i a c c o n t r a c t i l i t y d u r i n g per-  f u s i o n o f i s o l a t e d h e a r t s w i t h c y c l i c AMP i n the presence of DMSO.  T r a n s f o r m a t i o n o f phosphorylase b to phosphorylase  used as an index o f the e n t r y o f c y c l i c AMP.  a was  Under these c o n d i t -  i o n s , both c y c l i c AMP and i t s d i b u t y r y l analog were e f f e c t i v e i n t r a n s f o r m i n g phosphorylase b t o a.  However, there was no change  i n m y o c a r d i a l c o n t r a c t i l i t y d e s p i t e the f a c t t h a t the h e a r t s r e t a i n e d normal i n o t r o p i c response t o e p i n e p h r i n e .  Thus t h i s  i n d i c a t e d t h a t although c y c l i c AMP e n t e r s t h e c e l l ,  study  i t does not  i n f l u e n c e c a r d i a c mechanics under those circumstances.  Another  study c a r r i e d by L a n g l e t and 0 y e (84) showed t h a t n u c l e o t i d e entrance i n t o m y o c a r d i a l c e l l s i s f a c i l i t a t e d by p e r f u s i n g r a t h e a r t s a t low temperatures. AMP e n t e r the c e l l .  A t 16°C, both e p i n e p h r i n e and c y c l i c  However, o n l y e p i n e p h r i n e e x h i b i t s  positive  i n o t r o p i c and c h r o n o t r o p i c e f f e c t s , s u g g e s t i n g t h a t when c y c l i c AMP e n t e r s the c e l l  and achieves a c o n c e n t r a t i o n s u f f i c i e n t t o  - 40 -  t r a n s f o r m phosphorylase  b t o phosphoylase a, i t does not augment  c o n t r a c t i l i t y i n p r e p a r a t i o n s capable o f responding  mechanically  as w e l l as m e t a b o l i c a l l y t o e p i n e p h r i n e . While an all-encompassing  e x p l a n a t i o n f o r these c o n t r o -  v e r s i a l data i s not y e t a v a i l a b l e , i t has been suggested authors  by some  (85) t h a t t h e r e might e x i s t i n the c e l l a l o c a l i z e d and  critical  i n t r a c e l l u l a r p o o l o f c y c l i c AMP which i n f l u e n c e s cont-  ractility  when i t i s augmented under p h y s i o l o g i c a l  i n response  t o catecholamines.  circumstances  Recently, some very e x c i t i n g s t u d i e s  have been r e p o r t e d by T s i e n and h i s c o l l e a g u e s  (86) who examined the  e l e c t r i c a l e f f e c t s o f i o n t o p h o r e t i c i n j e c t i o n o f c y c l i c AMP ( c o n c e n t r a t i o n used estimated t o be 0.6 mM) active Purkinje fibers.  i n spontaneously  Compared t o c o n t r o l experiments,  i n j e c t e d w i t h c y c l i c AMP showed i n c r e a s e d frequency  fibers  o f spontaneous  a c t i v i t y as a r e s u l t o f a steeper pacemaker d e p o l a r i z a t i o n and a shortened of  p l a t e a u phase i n the a c t i o n p o t e n t i a l .  These e f f e c t s  c y c l i c AMP were r e p r o d u c i b l e and f u l l y r e v e r s i b l e .  On the  other hand, i o n t o p h o r e t i c i n j e c t i o n o f 5'-AMP produced no change i n the a c t i o n p o t e n t i a l o r pacemaker d e p o l a r i z a t i o n . s u l t s p r o v i d e d i r e c t support  f o r the involvement  of c y c l i c AMP i n  the e l e c t r i c a l e f f e c t s o f epinephrine i n the h e a r t . Brooker  These r e -  Recently  (87) s t u d i e d the c o n c e n t r a t i o n o f c y c l i c AMP w i t h i n each  myocardial  c o n t r a c t i o n c y c l e and showed t h a t myocardial  c y c l i c AMP  -  concentrations centrations tension. ence o f  oscillate  of c y c l i c  during  AMP  concentrations raction.  of  which  the  by  cyclic  70%  and  cardiac  GMP  peak d e v e l o p m e n t o f  increased  (150)  both d i a s t o l i c  cycle corresponding  by  to the  v e n t r i c l e s had  the  preceding  the  nucleotides  AMP as  as  AMP.  during  latency  the  cyclic  cyclic  90%  e a r l y phase of m e c h a n i c a l s y s t o l e .  and  levels. cyclic  and  pres-  systolic  force of  At  phase of the  levels  a period  contin  rose of  the  contraction  time j u s t a f t e r  begun t o r e l a x , b o t h n u c l e o t i d e s  diastolic  systolic  conducted s i m i l a r studies  and  cardial  as w e l l  observed that  levels f e l l  con-  i s a l t e r e d i n the  nucleotide  Wollenberger et a l v e n t r i c l e s and  e a c h c o n t r a c t i o n w i t h peak  oscillation  cyclic  frog heart  -  preceding  Furthermore, t h i s epinephrine  41  returned  to  T h e s e t r a n s i e n t c h a n g e s i n myo-  GMP  suggest a p o t e n t i a l r o l e  beat-to-beat regulators  of myocardial  for  contract-  ility. A l t h o u g h , c y c l i c AMP  "has  b e e n p r o v e d o r presumed  m e d i a t e a l a r g e number o f p h y s i o l o g i c a l p r o c e s s e s , little  i s known a b o u t t h e  Evidence i s accumulating activation The  t h a t c y c l i c AMP  a n o t h e r enzyme: - t h e  enzymes grew o u t  associates  in their  of  the  i t s action  protein kinase(s)  i n the  muscle.  T h e s e a u t h o r s showed t h a t  the  cascade of  r e g u l a t i o n of glycogenolysis  s t i m u l a t i o n of  adenylate cyclase  as  acts. via  (88-91).  a class  f i n d i n g s l a r g e l y of Krebs  e l u c i d a t i o n of  involved  of  exerts  relatively  nucleotide  c o n c e p t o f c y c l i c AMP-dependent p r o t e i n k i n a s e  regulatory his  of yet  mechanism whereby t h i s  to  cyclic by  AMP,  and  events  by.epinephrine f o r m e d as  epinephrine,  of  in  a result  accelerated  - 42 -  the r a t e of p h o s p h o r y l a t i o n and a c t i v a t i o n of phosphorylase k i n a s e . The l a t t e r enzyme then c a t a l y z e d the c o n v e r s i o n of phosphorylase b t o phosphorylase a, and i n the presence of c a l c i u m , l e d to g l y c o gen breakdown. r e a c t i o n was was  The nature of the phosphorylase k i n a s e a c t i v a t i o n  not c l e a r u n t i l these authors found t h a t c y c l i c  AMP  i n t e r a c t i n g with another enzyme, a phosphorylase k i n a s e k i n a s e  (92, 93).  T h i s k i n a s e was  i s o l a t e d from s k e l e t a l muscle and  found  to c a t a l y z e the ATP-dependent p h o s p h o r y l a t i o n and a c t i v a t i o n o f phosphorylase b k i n a s e i n the presence of extremely low c o n c e n t r a t i o n s of c y c l i c AMP.  S i n c e the k i n a s e was  a l s o capable of phosphor-  y l a t i n g o t h e r p r o t e i n s such as c a s e i n and protamine, to as p r o t e i n k i n a s e (92).  referred  Based on s t u d i e s c a r r i e d out u s i n g beef  h e a r t p r o t e i n k i n a s e (94), i t was of a r e g u l a t o r y  i t was  found t h a t the enzyme i s composed  (R) and a c a t a l y t i c  (C) s u b u n i t .  The b i n d i n g of  the r e g u l a t o r y subunit to the c a t a l y t i c component r e s u l t s i n an e s s e n t i a l l y i n a c t i v e holoenzyme  (RC).  C y c l i c AMP  i a t i o n to y i e l d a r e g u l a t o r y s u b u n i t - c y c l i c AMP  promotes d i s s o c -  complex and a f r e e  enzymatically a c t i v e , c a t a l y t i c subunit:  RC (inactive)  +  c y c l i c AMP  .  i R-cAMP  +  C (active)  The r o l e of c y c l i c AMP-dependent p r o t e i n k i n a s e i n the m e d i a t i o n of hormonal a c t i o n was by Kuo  and Greengard  expanded by o b s e r v a t i o n s made  (95) of the widespread d i s t r i b u t i o n of t h i s  enzyme i n v a r i o u s mammalian t i s s u e s and among d i f f e r e n t s p e c i e s i n c l u d i n g the bacterium E s c h e r i c h i a C o l l  (96).  T h i s l e d t o the  - 43 -  hypothesis AMP  (96, 97)  are due  t h a t a l l p h y s i o l o g i c a l e f f e c t s of c y c l i c  to tne a c t i o n o f t h i s enzyme.  Sutherland's  T h i s concept  o r i g i n a l second messenger h y p o t h e s i s , and  embodied extended  the c h a i n of events to i n c l u d e a p r o t e i n p h o s p h o r y l a t i o n step i n a sequence as f o l l o w s :  Hormonal or s i m i l a r s i g n a l  » c y c l i c AMP  > p r o t e i n kinase a c t i v a t i o n functional protein(s)  -» p h o s p h o r y l a t i o n of  > a c t i v a t i o n or  of f u n c t i o n a l p r o t e i n ( s )  production  inactivation  > physiological  The p r o t e i n ( s ) t h a t i s phosphorylated  events  c o u l d be an enzyme, a nuc-  l e a r p r o t e i n , a membrane component or any o t h e r . p r o t e i n i n v o l v e d at a c r i t i c a l c o n t r o l s i t e . and Schlender  For example, S o d e r l i n g e t aJL  e t a l (99) observed  t h a t c y c l i c AMP-dependent p r o t -  e i n k i n a s e c a t a l y z e d the c o n v e r s i o n of glycogen D form.  (98)  S i m i l a r l y , C o r b i n e t a l (100)  synthetase I to the  and Huttenen e t a l  e s t a b l i s h e d t h a t hormonal r e g u l a t i o n of l i p o l y s i s . i n  (101)  adipocytes  c o u l d be a s c r i b e d to a c y c l i c AMP-dependent, p r o t e i n k i n a s e mediated a c t i v a t i o n of t r i g l y c e r i d e l i p a s e .  P h o s p h o r y l a t i o n of  b a s i c n u c l e a r p r o t e i n s by a c y c l i c AMP-dependent p r o t e i n kinase was  demonstrated by Langan (102) who  subsequently  showed, i n a  h i g h l y s i g n i f i c a n t study, t h a t h i s t o n e p h o s p h o r y l a t i o n occurs i n v i v o i n response events  t o c y c l i c AMP  stimulation.  Other p h y s i o l o g i c a l  i n v o l v i n g c y c l i c AMP-dependent p r o t e i n p h o s p h o r y l a t i o n  i n c l u d e the f a c i l i t o r y e f f e c t of protamine p h o s p h o r y l a t i o n upon  - 44 -  i t s t r a n s p o r t from t h e c y t o p l a s m i c ribosomes t o . chromatin (103); t h e i n s u l i n - a n d p r o l a c t i n - d e p e n d e n t  -•  phosphorylation of  n u c l e a r p l a s m a membrane a n d r i b o s o m a l p r o t e i n s d u r i n g mammary gland d i f f e r e n t i a t i o n i n v i t r o  (104, 1 0 5 ) ; t h e p h o s p h o r y l a t i o n  o f r e t i c u l o c y t e r i b o s o m e s and i t s r o l e i n t r a n s l a t i o n a l r e g u l a t o r y mechanisms (106); t h e p h o s p h o r y l a t i o n o f b o v i n e  adrenal prot-  e i n s and r e g u l a t i o n o f s t e r o i d o g e n e s i s (107); r e g u l a t i o n o f neuronal  f u n c t i o n s through  p h o s p h o r y l a t i o n o f membrane f r a g m e n t s  isolated  f r o m o x (89) a n d r a t (90) b r a i n s ; t h e p o s s i b l e r o l e o f  c a t i o n t r a n s p o r t i n t h e p h o s p h o r y l a t i o n o f e r y t h r o c y t e membrane (91, 1 0 9 ) ; p h o s p h o r y l a t i o n o f b r a i n t u b u l i n and i t s r o l e i n t h e regulation of neurosecretion  (110); and t h e p h o s p h o r y l a t i o n o f  t r o p o n i n a n d i t s p o s s i b l e e f f e c t on m u s c l e c o n t r a c t i o n ( 1 1 1 ) . The c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e o r i g i n a l l y  found  i n r a b b i t m u s c l e i s a s o l u b l e enzyme a n d i s l o c a t e d p r i m a r i l y i n t h e c y t o s o l ( 9 2 ) . A s i m i l a r d i s t r i b u t i o n was f o u n d muscle  (94) a n d i n t h e l a c t a t i n g mammary g l a n d  liver,  Chen a n d W a l s h  (113) f o u n d  located i n the cytosol, and  (112).  heart  In r a t  t h a t 8 9 % o f t h e enzyme i s  6% i n t h e n u c l e i , 4% i n t h e m i c r o s o m e s  l e s s t h a n .1% i n t h e m i t o c h o n d r i a .  been found  i n beef  I n c o n t r a s t t o what has  i n s k e l e t a l muscle and l i v e r ,  Maeno e t a l f o u n d  that  t h e c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e f r o m b r a i n was b o t h uble and p a r t i c u l a t e  (88). P a r t i c u l a t e  protein kinase of high s p e c i f i c a c t i v i t y  sol-  f r a c t i o n s from which c o u l d be s o l u b i l i z e d  i n c l u d e d t h e microsomes, synaptosomes, s y n a p t i c v e s i c l e s and s y n a p t i c membranes.  Furthermore,.the  subcellular distribution of  - 45  protein kinase c y c l a s e and  a c t i v i t y was  -  c l o s e l y s i m i l a r to that of  phosphodiesterase.  adenylate  B o t h l a t t e r enzymes h a v e b e e n  f o u n d t o be r i c h i n f r a c t i o n s c o n t a i n i n g m i c r o s o m e s and endings.  These s t u d i e s i n d i c a t e t h a t c y c l i c AMP-dependent p r o t -  ein kinase  a c t i v i t y may  the p o s s i b i l i t y  be  an  i n t e g r a l p a r t o f membranes and  t h a t p h y s i o l o g i c a l phenomenon s u c h as  p e r m e a b i l i t y , s e c r e t o r y p r o c e s s e s and be  c o n t r o l l e d by  zymes f o r t h e are  a h i g h l y compartmentalized system i n which  en-  degradation  of the c y c l i c  nucleotide  f o u n d i n c l o s e p r o x i m i t y t o t h e enzyme r e s p o n s i b l e  been r e p o r t e d  (115),  o f membrane s u b s t r a t e s  t o o c c u r i n s y n a p t i c v e s i c l e s (88-90),  ghosts (91, 109),  adenohypophyseal granules  r e n a l medullary  plasmic  reticulum  muscle  (119).  p l a s m a membranes  (117,  I t i s now  118)  b e e n d e m o n s t r a t e d by (120-122).  l e v e l s o f c y c l i c AMP concurrent  (116),  and  and  + +  membranes  cardiac  sarcoskeletal  influx;.the latter  phenomenon  a number o f e l e c t r o p h y s i o l o g i c a l and Thus, i n c r e a s e  in.the.intracellular  the transmembrane C a  + +  i n f l u x appear  e f f e c t s o f c a t e c h o l a m i n e s on m y o c a r d i a l  cumulation  movement r e m a i n s c o n t r o v e r s i a l .  + +  to  cells.  o f t h e r e l a t i o n s h i p b e t w e e n c y c l i c AMP  Ca  in  levels i n cardiac-tissue, also  However, t h e n a t u r e and  since  .erythrocyte  generally accepted that catecholamines,  the transmembrane C a  tracer studies  (114)  regulat-  have  as w e l l as s a r c o l e m m a f r o m  a d d i t i o n . t o i n c r e a s i n g c y c l i c AMP increases  for i t s  Membrane-associated p r o t e i n kinases  i n g endogenous p h o s p h o r y l a t i o n  be  cellular may  s y n t h e s i s and  synaptic  raise  transmission  biological activity.  has  nerve  Two  ac-  different  - 46 -  models have been proposed:  i n the f i r s t model, s t i m u l a t i o n of  b e t a - a d r e n e r g i c . r e c e p t o r s by catecholamines c o u l d s i m u l t a n e o u s l y but independently cause an a c t i v a t i o n of adenylate c y c l a s e  and  an a l t e r a t i o n . o f sarcolemmal  cyclic  AMP  and C a  + +  p e r m e a b i l i t y to C a  both acted as second messengers.  + +  , so t h a t  In the second  model, s t i m u l a t i o n o f the b e t a - a d r e n e r g i c r e c e p t o r l e d t o format i o n o f c y c l i c AMP,  which i n t u r n caused a change i n C a  a b i l i t y of the membrane. c y c l i c AMP  In t h i s second type of i n t e r a c t i o n ,  would f u n c t i o n as the second messenger and C a  serve as the t h i r d messenger.  in C a  + +  accumulation by a  + +  f r a c t i o n of canine myocardium..  each of these agents produced  would  + +  Entman e t aJL (123) s t u d i e d the  e f f e c t s o f e p i n e p h r i n e and glucagon on C a microsomal  perme-  + +  They r e p o r t e d t h a t  a concentration-dependent i n c r e a s e  accumulation, and t h e i r e f f e c t s c o u l d be mimicked by u s i n g  exogenous c y c l i c AMP.  Furthermore,  p r e s e n t i n the microsomal  since, adenylate c y c l a s e  p r e p a r a t i o n used, they observed  that  p r o p a n o l o l not o n l y a b o l i s h e d the p r o d u c t i o n . o f c y c l i c AMP a l s o the accumulation of C a  + +  .  Bloom and Sweat  c o v a r i a n c e of m y o c a r d i a l c y c l i c AMP  and C a  + +  (151)  was  but  s t u d i e d the  during beta-adrenergic  s t i m u l a t i o n i n v i v o and noted a p a r a l l e l i n c r e a s e i n m y o c a r d i a l Ca  + +  and c y c l i c AMP  nol i n rats. was  a f t e r i n t r a p e r i t o n e a l i n j e c t i o n , of i s o p r o t e r e -  The maximum response f o r both C a  observed a f t e r two minutes  f o r the two responses was  + +  and c y c l i c  and the c o r r e l a t i o n  highly s i g n i f i c a n t  AMP  coefficient  (0.974).  ings are c o n s i s t e n t w i t h the concept t h a t the i n o t r o p i c  These f i n d effects  of catecholamines occur as a r e s u l t of an accumulation of C a  + +  - 47  m e d i a t e d by  c y c l i c AMP.  -  R e c e n t l y Watanabe and  s t u d i e d the r e l a t i o n s h i p between c e l l u l a r and  the  slow inward  pig hearts  Ca  + +  i n which the  Besch  (124)  l e v e l s of c y c l i c  current i n i s o l a t e d perfused  guinea  f a s t Na  +  channels  e i t h e r by d e p o l a r i z a t i o n w i t h K  +  or blockade w i t h tetro.dotoxin.  They f o u n d t h a t e x c i t a b i l i t y and to these  contractility  been i n a c t i v a t e d  c o u l d be  l e v e l s o f c y c l i c AMP.  A r i s e i n c y c l i c AMP  observed to precede r e s t o r a t i o n of e x c i t a b i l i t y  i z e d h e a r t s , and h e a r t s was  levels  in  depolar-  t h e m a g n i t u d e o f t e n s i o n d e v e l o p e d by  restored  d i r e c t l y r e l a t e d to the e x t e r n a l C a  as w e l l as t h e  + +  concentration  c o n c e n t r a t i o n of the i n o t r o p i c agent used.  m o r e , a h i g h l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n was + +  o f c y c l i c AMP  i n these  colemmal C a  c h a n n e l s w i t h Dggg o r v e r a p a m i l . ,  + +  hearts.  t h e t e n s i o n . d e v e l o p m e n t by  not attenuated.  could a c t i v a t e slow C a  w i t h those  o f T s i e n who  the  s p e c i f i c blockade of  r e s t o r e d h e a r t s was  t h o u g h t h e r i s e i n c y c l i c AMP u l a t i o n was  Using  i t was  level sar-  shown t h a t  abolished, a l -  l e v e l s i n r e s p o n s e t o hormone s t i m These f i n d i n g s s u g g e s t e d t h a t  + +  Further-  found between  t h e m a g n i t u d e o f C a - d e p e n d e n t t e n s i o n d e v e l o p m e n t and  AMP  restored  hearts w i t h a v a r i e t y of i n o t r o p i c agents which a l s o  r a i s e d the was  had  AMP  c h a n n e l s i n t h e h e a r t and  cyclic  agreed  showed t h a t t h e i o n f l u x e s i n t h e  plateau  phase o f the a c t i o n p o t e n t i a l i n c a r d i a c p u r k i n j e f i b e r s were s e n s i t i v e t o c y c l i c AMP who  (125)  and  those  of Meinertz. et a l  d e m o n s t r a t e d t h a t d i b u t y r y l c y c l i c AMP,  increased contractile atria.  f o r c e and  These o b s e r v a t i o n s  Ca  + +  like  epinephrine,  u p t a k e by t h e b e a t i n g  suggest s t r o n g l y a causative  a c t i o n b e t w e e n a u g m e n t a t i o n o f c y c l i c AMP  (126)  l e v e l s and  rat  inter-  increase  - 48  i n transarcolemmal C a cellular Ca  + +  + +  -  i n f l u x , r e s u l t i n g i n a net  l e v e l s which i n turn leads  increase  in  to a p o s i t i v e .inotropic  response. I n view of the  e v i d e n c e t h a t c y c l i c AMP  c o n t r o l s many  p h y s i o l o g i c a l processes v i a the a c t i v a t i o n of p r o t e i n k i n a s e , i t i s conceivable AMP  may  t h a t the  s t i m u l a t i o n of C a  a l s o be m e d i a t e d by membrane p h o s p h o r y l a t i o n .  Katz et a l  (127,  140,  147-149) showed t h a t c a r d i a c  c o n s i s t i n g p r i m a r i l y of sarcoplasmic phorylated  by p r o t e i n k i n a s e  epinephrine.  net  + +  u p t a k e and  s t i m u l a t i o n of C a  + +  phosphoprotein formation. phorylation occurred weight  and  Morkin  u p t a k e by by  an  phos-  p r e s e n c e o f c y c l i c AMP  Ca -activated  or  uptake p a r a l l e l e d the I t was  w h i c h was  (118)  the  cardiac  reticulum.  + +  trans-  regulate LaRaia  a l s o succeeded i n demonstrating increased  a cardiac microsomal preparation  t h a t c a t e c h o l a m i n e s can  may  i n o t r o p i c a g e n t s on  a t the  l e v e l of the  i n o t r o p i c r e s p o n s e has  cardiac tissue. been c l e a r l y  + +  + +  strongly  movements  sarcoplasmic  e f f e c t s of  But  Ca  phosphorylation  These d a t a s u g g e s t v e r y  a c c o u n t f o r some o f t h e the  after  influence intracellular C a  v i a membrane p h o s p h o r y l a t i o n and  phos-  molecular  from the C a  sarcoplasmic  and  amount o f  f u r t h e r shown t h a t t h e  separable  en-  ATPase a c t i v i t y ,  + +  endogenous p r o t e i n k i n a s e .  reticulum,  c o u l d be  ( 1 0 0 , 0 0 0 ) , b u t when p h o s p h o r y l a t e d c o u l d  t r a n s p o r t by  + +  Recently,  microsomes,  a t a membrane component o f l o w  (22,000 d a l t o n )  port protein Ca  i n the  reticulum,  cyclic  Membranes t h u s p h o s p h o r y l a t e d showed a m a r k e d  hancement o f C a the  movements by  + +  these  s i n c e the p o s i t i v e  shown t o be  r e l a t e d to  an  i n c r e a s e of Ga  i n f l u x across the plasma membrane  (the slow i n -  ward c u r r e n t ) , i t i s our purpose to i n v e s t i g a t e whether membrane p h o s p h o r y l a t i o n c a t a l y z e d by c y c l i c AMP-dependent p r o t e i n k i n a s e a l s o take p l a c e i n the plasma membrane.  In t h i s  connection,  Sulakhe and Drummond (128), u s i n g a h i g h l y p u r i f i e d s k e l e t a l s a r colemmal p r e p a r a t i o n , showed t h a t the membranes c o u l d be phosp h o r y l a t e d by p r o t e i n k i n a s e and t h a t the phosphorylated membranes c o u l d accumulate more C a  + +  than c o n t r o l p r e p a r a t i o n s .  In  the f o l l o w i n g s e c t i o n , data w i l l be presented to demonstrate t h a t t h e r e i s an i n t e r a c t i o n between c y c l i c AMP  and C a  + +  via  p r o t e i n kinase-dependent p h o s p h o r y l a t i o n a t the l e v e l of the c a r d i a c sarcolemma, and these i n t e r a c t i o n s may  p r o v i d e a mechan-  ism f o r the p o s i t i v e i n o t r o p i c e f f e c t s of catecholamines, agon and other s i m i l a r  gluc-  agents.  EXPERIMENTAL PROCEDURES  A.  Materials £ Y ~  from New tone  32  Pj  England  ATP  (10 to 20 C i per mmole) was  Nuclear.  P r o t e i n kinase  ( c a l f thymus IIA) were purchased  obtained  (beef heart) and h i s -  from Sigma.  Other chemi-  c a l s were o b t a i n e d from s i m i l a r sources as i n d i c a t e d i n the previous section.  B.  Methods I s o l a t i o n of c a r d i a c sarcolemma  c r i b e d i n P a r t I of t h i s t h e s i s .  -  was  c a r r i e d as des-  Unless i n d i c a t e d o t h e r -  - 50 -  wise, membranes were used f o r v a r i o u s . s t u d i e s w i t h i n 2 hours of  isolation. Calcium b i n d i n g and uptake -  was incubated  membrane p r o t e i n (200-300 ug)  i n a medium ( f i n a l volume 1.0 ml) c o n t a i n i n g 50  mM T r i s - m a l e a t e , pH 6.0, 5 mM M g C l , 2 mM Tris-ATP, and 100 uM 2  CaCl  (8,000-12,000 cpm/nmole).  2  The r e a c t i o n was s t a r t e d by  a d d i t i o n o f ATP, and a f t e r i n c u b a t i o n f o r v a r i o u s times a t 30°, terminated filter  by f i l t e r i n g a 0.5 ml a l i q u o t through a M i l l i p o r e  (0.45 u, 25 mm)  under l i g h t s u c t i o n .  The f i l t e r  was  washed w i t h 5 ml o f 100 mM T r i s - m a l e a t e , pH 6.0, d r i e d a t 70° i n a scintillation vial,  and r a d i o a c t i v i t y was determined by l i q u i d  s c i n t i l l a t i o n spectrometry. ATP,  no M g C l  2  Appropriate  c o n t r o l s c o n t a i n i n g no  or no p r o t e i n were i n c l u d e d .  Ca  b i n d i n g was 45  c a l c u l a t e d from the s p e c i f i c a c t i v i t y o f the added  C a C l , and  the r a d i o a c t i v i t y r e t a i n e d by t h e membrane p r o t e i n .  In the ab-  2  sence of membrane p r o t e i n , r e t e n t i o n of r a d i o a c t i v i t y by the f i l t e r was n e g l i g i b l e  (32). Measurement o f C a . u p t a k e was sim-  i l a r t o the above, except  ++  t h a t 5 mM potassium  o x a l a t e was i n -  cluded i n the medium. P h o s p h o r y l a t i o n o f sarcolemma - membrane p r o t e i n (100-200 ug) was incubated  i n a medium  ( f i n a l . v o l u m e 0.2 ml) c o n t a i n i n g  50 mM sodium a c e t a t e , pH 6.0, 10 mM M g C l , 10 mM NaF, 2 mM 2  p h y l l i n e , 0.5 mM EGTA, 25 uM [ Y picomole),  3 2  P]ATP  1 uM c y c l i c AMP, with o r without  theo-  (500-2,000 cpm per bovine  heart p r o t e i n  -  kinase  (10-100 u g ) .  51  -  P h o s p h o r y l a t i o n of sarcolemma i n the  absence  o f a d d e d p r o t e i n k i n a s e i s d u e . t o a u t o p h o s p h o r y l a t i o n by ous  membrane-bound p r o t e i n k i n a s e .  tracted  from.those obtained.in  r e a c t i o n was for  s t a r t e d by  various  cold  10%  the  t i m e s a t 30°,  the  and  after  a d d i t i o n of  solution)  in  i c e f o r a t l e a s t 10 m i n u t e s b e f o r e c e n t r i f u g a t i o n .  with  10%  solved  carefully aspirated  i n 0.1  ml  of  d e t e r m i n e d by  Preparation  was  32  of  or  0.5  mg  of h e a t - t r e a t e d  incubated at  30°  containing  EGTA, 5 mM  r e a c t i o n was  s t o p p e d by  - 3.34 (100°  mg  1 uM  2 5 uM  s t a r t e d by  a d d i t i o n of  6 ml  ml  20%  and  substrates (calf  6.0,  (final 10 mM  mCi  volume  MgC^,  c y c l i c AMP, (500  0.5  240 per  ml  ug  mmole). and  trichloroacetic acid.  NaOH f o l l o w e d  thymus  sarcolemma  was  IN  times  spectrometry.  a t 7,000 x g f o r 15 m i n u t e s and  i n 0.2  a  an  of Aquasbr^,  tubes were c e n t r i f u g e d redissolved  of  dis-  water,  of histone  ATP  of  super-  washed 3  a d d i t i o n o f r a d i o a c t i v e ATP of  ml  The  f o r 15 m i n u t e s )  theophylline,  ml  t u b e s were k e p t  32 B - s a r c o l e m m a . as  pH  p u r i f i e d protein kinase, The  0.2  i n 20 ml  sodium a c e t a t e ,  2 mM  4.0  f i n a l p e l l e t was  f o r 60 m i n u t e s i n a medium  50 mM  NaF,  The  scintillation  P - h i s t o n e and  phosphoprotein phosphatase  1 ml) mM  dissolved  The  p e l l e t was  1 N NaOH, d i l u t e d w i t h  s a m p l e was  r a d i o a c t i v i t y was  IIA)  the  cold trichloroacetic acid.  a l i q u o t of the  for  and  carrier.  The  incubation  B o v i n e s e r u m a l b u m i n . (0.2  a protein  sub-  a d d e d enzyme.  1%  n a t a n t was  a d d e d as  presence of  t e r m i n a t e d by  t r i c h l o r o a c e t i c acid.. was  These v a l u e s were always  a d d i t i o n o f ATP  was  endogen-  the  The pellet  by p r e c i p i t a t i o n  - 52 -  w i t h 4 ml of,20% t r i e h l o r o a c e t i c acid-and c e n t r i f u g a t i o n a t 7,000 x. g f o r 15 minutes.  T h i s washing procedure was repeated  twice and t h e r e s i d u e was suspended a g a i n s t water  i n water and d i a l y s e d  (histone) or 10 mM T r i s - H C l ,  f o r 24 hours a t room  temperature.  Assay o f Phosphoprotein Phosphatase ions  (sarcolemma) pH 7.5,  - Various.muscle  fract-  (40-60 ug p r o t e i n ) were i n c u b a t e d a t 30° i n a medium  (final  volume 0.2 ml) c o n t a i n i n g 50 mM T r i s - H C l , pH 7.5, 1 mM d i t h i o t h r e i t o l and [ was  3 2  P ] - l a b e l e d h i s t o n e o r sarcolemma.  The r e a c t i o n  s t a r t e d by the a d d i t i o n o f substrate.and. stopped a f t e r 10  minutes  by t h e a d d i t i o n o f 4 ml o f 20% c o l d t r i c h l o r o a c e t i c  acid.  To each tube, 0.2 ml o f 1% albumin s o l u t i o n was added and the tubes were kept a t 4° f o r 15 minutes b e f o r e b e i n g c e n t r i f u g e d at 7,000 x g f o r 15 minutes.  The p e l l e t was washed once by d i s -  s o l v i n g i n 0.1 ml o f 1 N NaOH, f o l l o w e d by p r e c i p i t a t i o n w i t h c o l d t r i c h l o r o a c e t i c a c i d and c e n t r i f u g a t i o n . was  The f i n a l  pellet  d i s s o l v e d i n 0.1 ml o f 1 N NaOH, 0.2 ml water added and  r a d i o a c t i v i t y of a 2 00 p i a l i q u o t was determined Aquasol** s c i n t i l l a t i o n s o l u t i o n . denatured  (100°C f o r 30 minutes)  Phosphoprotein phosphatase  i n 15 ml o f  C o n t r o l s were c a r r i e d w i t h heate x t r a c t s or without added p r o t e i n .  a c t i v i t y was c a l c u l a t e d from the d i f -  f e r e n c e i n r a d i o a c t i v i t y between the experimentals and c o n t r o l s .  RESULTS  1.  Ca  b i n d i n g and uptake i n c a r d i a c  + +  sarcolemma  In view of the s i g n i f i c a n t r o l e of the plasma membrane i n the r e g u l a t i o n of i o n t r a n s p o r t , we s t u d i e d the C a and uptake a c t i v i t i e s o f c a r d i a c sarcolemma. d e f i n e d as the amount of C a  + +  binding  + +  Calcium uptake i s  accumulated by membranes i n the  presence o f o x a l a t e , a C a - p r e c i p i t a t i n g agent t h a t d i f f u s e s i n + +  and out o f membrane v e s i c l e s , whereas c a l c i u m b i n d i n g i s the amount of C a  accumulated i n the absence o f o x a l a t e .  + +  As shown  i n Table VI, these membranes.bound a s m a l l amount o f C a  i n the  + +  absence of ATP, but b i n d i n g was s t i m u l a t e d 5 - f o l d when ATP was added.  mM)  The b i n d i n g process appeared to be a r a p i d one, s i n c e  maximum b i n d i n g was reached a t 1 minute. Ca  (2  The maximal amount of  bound .remained r a t h e r c o n s t a n t f o r up.to 5 minutes.  presence o f o x a l a t e , C a  + +  uptake was almost l i n e a r up to 5 minutes  of i n c u b a t i o n .  Since muscle m i t o c h o n t r i a a l s o possess C a  take a c t i v i t i e s  (129) which are s e n s i t i v e to m e t a b o l i c  such as a z i d e  In the  + +  up-  inhibitors  (130), we s t u d i e d the e f f e c t of t h i s agent on C a  uptake by sarcolemma. had no e f f e c t on C a  + +  As shown i n Table VI, 5 mM  + +  sodium a z i d e  uptake by sarcolemma.  ++ 2.  Ca  - s t i m u l a t e d ATPase of c a r d i a c Since both C a  + +  sarcolemma  b i n d i n g and uptake are dependent on the  presence of ATP, it.was o f i n t e r e s t t o know whether the membranes c o n t a i n e d ATPase a c t i v i t i e s .  As shown i n Table V I I , i n the  - 54 -  TABLE V I  Calcium  b i n d i n g and uptake a c t i v i t i e s o f c a r d i a c  sarcolemma.  C a l c i u m b i n d i n g and uptake were measured as d e s c r i b e d under "Methods" w i t h t h e v a r i o u s a d d i t i o n s i n d i c a t e d . When added, ATP ( t r i s s a l t ) was 2 mM; potassium o x a l a t e , 5 mM; sodium a z i d e , 5 mM. V a l u e s a r e t h e mean w i t h S.E.M. o f 4 s e p a r a t e membrane p r e p a r a t i o n s .  Calcium  B i n d i n g and Uptake 1 min  Additions  3 min  5 min  nmoles/mg -ATP  .67 + .28  .80 + .16  .75 + .13  +ATP  3.5  + .57  3.9  + .41  3.2  + .67  +ATP + o x a l a t e  8.8  + 2.0  18  + 1.4  23  + 1.6  +ATP + o x a l a t e azide  9.1  + 1.9  19  + 1.2  25  + 1.4  - 55 -  TABLE V I I  Ca  - s t i m u l a t e d ATPase a c t i v i t i e s o f c a r d i a c sarcolemma.  ATPase a c t i v i t i e s were measured as d e s c r i b e d under "Methods" w i t h 2 mM ATP ( t r i s s a l t ) . V a l u e s a r e t h e mean w i t h S.E.M. o f 4 s e p a r a t e membrane p r e p a r a t i o n s .  ATPase a c t i v i t i e s  Additions  nmoles/mg/min  No a d d i t i o n 5 mM Mg 5 mM Mg 5 mM Ca  8 + 1.1  2+ 2+  109+7.3  50  + uM Ca  2  +  148 + 8.2 186 + 14  a b s e n c e o f Mg""", ATP h y d r o l y s i s was n e g l i g i b l e . 1  M g - d e p e n d e n t A T P a s e was f o u n d t o be p r e s e n t  i n t h e sarcolemma,  + +  and  t h i s a c t i v i t y was.further  of C a  + +  ( 5 0 uM).  s t i m u l a t e d by l o w c o n c e n t r a t i o n s  I n a d d i t i o n , t h e sarcolemma p o s s e s s e d a CaATPase  w h i c h was n o t d e p e n d e n t on M g previous  W i t h a d d e d Mg' ' ,  1  r e p o r t s t h a t Mg  + +  .  Our o b s e r v a t i o n s  - d e p e n d e n t a n d Mg  confirmed  - i n d e p e n d e n t CaATPase  a r e a c t i v e c o m p o n e n t s o f p l a s m a membranes f r o m c a r d i a c a n d s k e l e t a l tissues  3.  ( 1 7 , 3 2 , 3 5 , 47, 1 3 1 ) .  Phosphorylation  o f sarcolemma by exogenous p r o t e i n  When c a r d i a c s a r c o l e m m a were, i n c u b a t e d and  purified.bovine protein kinase,  w i t h [T -  t h e r e was a r a p i d  kinase 3 2  P]  ATP  incorpora-  t i o n o f t h e t e r m i n a l p h o s p h a t e o f ATP i n t o t h e t r i c h l o r o a c e t i c acid-precipitable residue  ( F i g u r e 3A)..  I n t h e p r e s e n c e o f 1 uM  c y c l i c AMP, t h e amount o f p h o s p h a t e i n c o r p o r a t e d doubled. plateau  was more t h a n  The r e a c t i o n was n o t l i n e a r w i t h t i m e a n d b e g a n t o a f t e r 3 minutes, probably  t h e ATPase and a d e n y l a t e  due t o h y d r o l y s i s o f ATP by  c y c l a s e t h a t t h e s e membranes p o s s e s s e d .  U n d e r t h e s e i n c u b a t i o n c o n d i t i o n s i n w h i c h r e a c t i o n was t e r m i n ated a t 5 minutes, phosphorylation l i n e a r with respect F i g u r e 3B.  o f c a r d i a c s a r c o l e m m a was  t o protein kinase  up t o 1 0 0 u g , a s shown i n  Figure 3 (A) Time c o u r s e o f p h o s p h o r y l a t i o n o f c a r d i a c s a r c o l e m m a by p u r i f i e d p r o t e i n k i n a s e i n t h e p r e s e n c e (•) and a b s e n c e (A) o f 1 pM c y c l i c AMP. C a r d i a c s a r c o l e m m a (* 135 p g ) , p r o t e i n k i n a s e (10 p g ) . Membrane p h o s p h o r y l a t i o n by e n d o g e n ous p r o t e i n k i n a s e h a s b e e n c o r r e c t e d f o r . (B) E f f e c t o f v a r i o u s amounts o f p r o t e i n k i n a s e on p h o s p h o r y l a t i o n o f c a r d i a c s a r c o l e m m a (~100 pg) i n t h e p r e s e n c e (•) a n d a b s e n c e (A) o f 1 pM c y c l i c AMP ( i n c u b a t i o n t i m e was 5 m i n u t e s ) .  n M O L E S 32p  INCORPORATED/MG  PROTEIN  o  o  o  o  o  —•  rO  CO  K  Ol  Ui °  VI 01  — b  —' to  to  - 85 -  - 59 -  4.  Endogenous p r o t e i n k i n a s e a n d a u t o p h o s p h o r y l a t i o n c a r d i a c sarcolemma  of  Recent f i n d i n g s of p r o t e i n k i n a s e ( s ) a s s o c i a t e d w i t h plasma membranes from v a r i o u s t i s s u e s 132-133) prompted us to determinate  (88-91, 114-117, 109,  whether c a r d i a c sarcolemma  contained endogenous p r o t e i n k i n a s e . were i n c u b a t e d i n the presence  119,  of [ Y -  F r e s h l y prepared P ] ATP  3 2  membranes  and h i s t o n e , and  shown i n F i g u r e 4A,  there was  a t i o n which reached  a p l a t e a u a f t e r 2 minutes of i n c u b a t i o n .  added c y c l i c AMP  (1 pM),  a low l e v e l of h i s t o n e  h i s t o n e p h o s p h o r y l a t i o n was  as  phosphorylWith  stimulated  5 to 7 - f o l d and d i d not reach a p l a t e a u a t the end of 5 minutes. Since c a r d i a c sarcolemma c o n t a i n e d both p r o t e i n k i n a s e ( s ) as w e l l as s u b s t r a t e ( s ) f o r p h o s p h o r y l a t i o n , . i t was see whether these membranes c o u l d e f f e c t When sarcolemma was  incubated w i t h  [ Y -  3  2  of i n t e r e s t to  autophosphorylation. P  ~J  ATP  without  p r o t e i n k i n a s e or h i s t o n e , phosphate i n c o r p o r a t i o n was  added detected  as e a r l y as 15 seconds and the r e a c t i o n proceeded r a p i d l y to a plateau. was  In the presence  s t i m u l a t e d moderately  o f added c y c l i c AMP,  autophosphorylation  (25% to 40%), but c o n s i s t e n t l y i n 6 sep-  a r a t e membrane p r e p a r a t i o n s s t u d i e d . experiment are shown i n F i g u r e  The r e s u l t s of a t y p i c a l  5A.  Since membrane-associated p r o t e i n kinase a c t i v i t y  ob-  served i n these p r e p a r a t i o n s represented only about 1% of the p r o t e i n kinase a c t i v i t y of the s t a r t i n g homogenate, the e x i s t e d t h a t the a c t i v i t y was  total  possibility  due to a d s o r p t i o n of s o l u b l e enzyme  Figure 4 (A) Time c o u r s e o f h i s t o n e p h o s p h o r y l a t i o n (100 ug c a l f thymus I I A h i s t o n e ) by c a r d i a c sarcolemma ( " 1 3 0 pg) i n t h e p r e s e n c e (•) and a b s e n c e (A) o f 1 uM c y c l i c AMP. I n a l l numbers g i v e n f o r . h i s t o n e p h o s p h o r y l a t i o n , v a l u e s due t o membrane a u t o p h o s p h o r y l a t i o n had b e e n s u b t r a c t e d . (B) A s s a y c o n d i t i o n s were t h e same a s i n ( A ) , e x c e p t t h a t c a r d i a c sarcolemma was washed w i t h a b u f f e r c o n t a i n i n g 250 mM s u c r o s e , 2 mM d i t h i o t h r e i t o l , 10 mM T r i s - H C L , pH 7.5 and c e n t r i f u g e d a t 37,000 x g f o r 15 m i n u t e s . The w a s h i n g p r o c e d u r e was r e p e a t e d 5 t i m e s and t h e washed sarcolemma r e s u s p e n d e d by a g r o u n d - g l a s s h o m o g e n i z e r b e f o r e u s e . (0) d e n o t e s p r e s e n c e and (A) a b s e n c e o f 1 uM cAMP i n the incubation.  MINUTES  MINUTES  Figure 5 (A) Time course o f a u t o p h o s p h o r y l a t i o n o f c a r d i a c sarcolemma (~ 130 ug) by endogenous p r o t e i n k i n a s e i n the presence ( • ) and absence ( A ) of 1 uM c y c l i c AMP. (B) Assay c o n d i t i o n s were the same as i n (A), except t h a t c a r d i a c sarcolemma was washed 5 times bef o r e use. Washing procedure i s as d e s c r i b e d i n legend t o F i g . 4B. ( O ) denotes presence and ( A ) absence o f 1 uM c y c l i c AMP i n the i n c u b a t i o n medium.  - 64  t o t h e membranes.  -  To r u l e o u t t h i s p o s s i b i l i t y ,  2.0-3.0  o f s a r c o l e m m a was  w a s h e d 5 t i m e s w i t h 25 m l o f i s o t o n i c  c o n t a i n i n g 250 mM  s u c r o s e , 2 mM  H C l , pH  7.5,  dithiothreitol  and  buffer  10 mM  e a c h t i m e c e n t r i f u g e d a t 37,000 x g f o r 15  Approximately  15% t o 25% membrane p r o t e i n was  Trisminutes.  r e m o v e d by  p r o c e d u r e , b u t t h e a b i l i t y o f t h e s e "washed s a r c o l e m m a " e f f e c t a u t o p h o s p h o r y l a t i o n ( F i g u r e 5B)  mg  this to  and h i s t o n e p h o s p h o r y l a -  tion  ( F i g u r e 4B)  was u n a l t e r e d .  5.  C a l c i u m a c c u m u l a t i o n by p h o s p h o r y l a t e d c a r d i a c s a r c o l e m m a I n v i e w o f t h e r e c e n t f i n d i n g s t h a t membranes d e r i v e d  f r o m c a r d i a c s a r c o p l a s m i c r e t i c u l u m (127, 140) sarcolemma  (128) w e r e c a p a b l e o f i n c r e a s e d C a  and  from  uptake  + +  skeletal when  p h o s p h o r y l a t e d by c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e , i t was of importance  to determine whether C a  a c c u m u l a t i o n by c a r d i a c  + +  s a r c o l e m m a c o u l d a l s o be a f f e c t e d as a r e s u l t o f p h o s p h o r y l a t i o n by e i t h e r e x o g e n o u s o r e n d o g e n o u s p r o t e i n k i n a s e . 5 separate experiments  a r e shown i n T a b l e V I I I .  p a r e d s a r c o l e m m a w e r e i n c u b a t e d a t 30° Ca  + +  Results  Freshly pre-  f o r 5 minutes  u p t a k e medium ( s e e M e t h o d s ) u n d e r a v a r i e t y o f  t i o n c o n d i t i o n s p r i o r t o measurement o f C a  + +  from  i n the phosphoryla-  uptake.  At  the  45 end o f t h e p r i o r i n c u b a t i o n p e r i o d , ATP a d d e d t o g e t h e r and 5 minutes prior  t h e amount o f C a  of i n c u b a t i o n .  + +  (2 mM)  and  u p t a k e was  When 25 uM ATP  was  CaCl  measured  2  were after  present during the  i n c u b a t i o n p e r i o d , ( a l l o w i n g endogenous p h o s p h o r y l a t i o n  t o t a k e p l a c e ) , t h e r e was increase i n C a  + +  uptake  a slight,  but c o n s i s t e n t l y  over the c o n t r o l .  observed  A further increase  - 65 -  TABLE V I I I  E f f e c t o f d i f f e r e n t p r e i n c u b a t i o n c o n d i t i o n s on Ca^ uptakeby c a r d i a c sarcolemma. F r e s h l y p r e p a r e d sarcolemma (100130 jig) was i n c u b a t e d f o r f i v e minutes under v a r i o u s a d d i ^ tions. When added, ATP ( t r i s s a l t ) was 25 uM; c y c l i c AMP, 1 uM; p r o t e i n k i n a s e , 100 ug. A t t h e end o f t h e p r i o r i n c u b a t i o n p e r i o d , 2 mM ATP and 100 pM C a C l 2 was added, and c a l c i u m uptake was measured f o r 5 minutes as d e s c r i b e d under "Methods-. R e s u l t s from 5 s e p a r a t e sarcolemmal p r e p a r a t i o n s were shown. V a l u e s a r e averages o f t r i p l i c a t e determinations. 4 5  Additions i n p r i o r tion  incuba-  Calcium Exp.  1  Exp.  2  uptake Exp.  3  Exp.  4  Exp.  ninoles/mg p r o t e i n / 5 minute No a d d i t i o n  21.9  14.2  16.2  14.5  17.2  ATP  23.4  15.2  17.3  15.5  18.5  ATP  + cAMP  25.5  16.9  18.2  17.0  20.7  ATP  + P r o t e i n Kinase  41.9  22.7  35.2  30.6  36.9  ATP  + cAMP + P r o t e i n K i n a s e  42.8  25.0  37.0  29.5  38.5  5  -  was noted, when c y c l i c AMP  66 -  (1 uM) was added.  This  indicated  t h a t as a u t o p h o s p h o r y l a t i o n o f the membrane was.allowed t o take p l a c e i n the p r i o r  i n c u b a t i o n p e r i o d by the a d d i t i o n o f ATP  (25 pM), the sarcolemma was capable o f accumulating  more C a  + +  ,  and as a u t o p h o s p h o r y l a t i o n was s t i m u l a t e d by the presence of added c y c l i c AMP, t h e r e was a g r e a t e r i n c r e a s e i n C a With p r o t e i n k i n a s e added t o the p r i o r 2-fold stimulation of C a  + +  uptake.  + +  i n c u b a t i o n mixture, a  uptake was observed.  When present  t o g e t h e r , c y c l i c AMP and p r o t e i n k i n a s e produced the g r e a t e s t i n c r e a s e above c o n t r o l . hancement o f C a small  + +  I t should be p o i n t e d out t h a t the en-  uptake due t o a u t o p h o s p h o r y l a t i o n was q u i t e  (7% and 20% i n the absence and presence  r e s p e c t i v e l y , ) and C a  + +  b e f o r e any s i g n i f i c a n t increase i n C a  + +  uptake had t o proceed  of c y c l i c  up t o 5 minutes  d i f f e r e n c e c o u l d be observed.  In c o n t r a s t ,  uptake due t o exogenous p r o t e i n kinase was s u f -  f i c i e n t l y l a r g e and the time course c o u l d be e a s i l y The  AMP  followed.  r e s u l t s o f a t y p i c a l experiment are shown i n F i g u r e 6A.  P r o t e i n k i n a s e i n c r e a s e d both the r a t e and the extent o f C a take even i n the absence o f added c y c l i c AMP.  + +  up-  In the presence o f  1 uM c y c l i c AMP, there was o n l y a s l i g h t increment.  T h i s may be  a t t r i b u t e d to the f a c t t h a t these membranes contained an a c t i v e adenylate c y c l a s e and produced c y c l i c AMP c l o s e to micromolar levels  under these c o n d i t i o n s .  Since adenylate c y c l a s e i s a  l a b i l e enzyme and i t s a c t i v i t y can be s i g n i f i c a n t l y storage a t 4° o v e r n i g h t , the e f f e c t demonstrated more c l e a r l y  reduced by  o f added c y c l i c AMP might be  by u s i n g such an "aged" p r e p a r a t i o n .  Figure  6  (A) Time c o u r s e o f c a l c i u m u p t a k e by c a r d i a c sarcolemma. F r e s h l y p r e p a r e d membranes (<~135 pg) was i n c u b a t e d f o r 5 m i n u t e s i n t h e p r e s e n c e o f 25 pM ATP w i t h t h e f o l l o w i n g c o n d i t i o n s : (• ) c o n t r o l ; (A ) p r e s e n c e o f 1 pM cAMP; (• ) 100 u g p r o t e i n k i n a s e ; (<• ) p r e s e n c e o f 100 p g p r o t e i n k i n a s e a n d 1 pM cAMP. A t t h e end o f 5 m i n u t e s , 2 mM ATP a n d 100 pM CaCl2 w e r e a d d e d a n d c a l c i u m u p t a k e was m e a s u r e d f o r the next 5 minutes. 4 5  (B) C o n d i t i o n s and l e g e n d s were s i m i l a r t o ( A ) , e x c e p t t h a t c a r d i a c s a r c o l e m m a was s t o r e d a t 4° f o r 20 h o u r s b e f o r e u s e .  MINUTES  MINUTES  - 69 -  R e s u l t s of such an experiment are shown i n F i g u r e 6B. the values f o r C a prepared  + +  sarcolemma  Although  uptake were lower than t h a t of f r e s h l y (since C a  f u n c t i o n ) , added c y c l i c AMP  + +  uptake i t s e l f  caused  i s also a l a b i l e  a larger increase i n C a  take, both i n the absence and presence  + +  up-  of p r o t e i n k i n a s e .  In the p r e v i o u s experiments s t u d y i n g the e f f e c t of phosphorylation i n C a  + +  uptake, p r i o r i n c u b a t i o n of sarcolemma w i t h  p r o t e i n k i n a s e took p l a c e i n a mixture  optimal f o r C a  + +  uptake,  but not f o r p h o s p h o r y l a t i o n , i t seemed necessary t o e s t a b l i s h t h a t under the c o n d i t i o n s o f the p r i o r i n c u b a t i o n , membranes were a c t u a l l y phosphorylated,  and t h a t d u r i n g the C a  + +  uptake phase,  an adequate l e v e l of membrane p h o s p h o r y l a t i o n was  maintained.  R e s u l t s i n F i g u r e 7 show t h a t when sarcolemma were incubated with p r o t e i n k i n a s e r  32  [V -  n P J ATP  2  (1 pM), and  ++ (25 uM) i n a Ga  phosphorylated, CaCl  (10 t o 200 p g ) , c y c l i c AMP  uptake medium, they were r a p i d l y  r e a c h i n g a maximum l e v e l i n 3 t o 4 minutes. When  ( f i n a l c o n c e n t r a t i o n 0.1 mM)  was added a t the end of 5  minutes and the l e v e l o f p h o s p h o r y l a t i o n determined  at different  time i n t e r v a l s up t o 10 minutes, i t was found t h a t , d e s p i t e a small gradual d e c l i n e , the maximal l e v e l of p h o s p h o r y l a t i o n was maintained  a t the end of 10 minutes.  Since p h o s p h o r y l a t i o n o f membranes would y i e l d p o l a r phosphate groups, the i n c r e a s e d C a  + +  uptake observed  r e f l e c t e l e c t r o s t a t i c i n t e r a c t i o n between p o s i t i v e l y c a l c i u m ions and the n e g a t i v e l y charged the membrane.  might simply charged  s i t e s on the s u r f a c e o f  I f t h i s were the case, t h e r e would be a simple  Figure 7 E f f e c t of CaCl2 on p h o s p h o r y l a t i o n of c a r d i a c sarcolemma by v a r i o u s amounts of p u r i f i e d p r o t e i n k i n a s e . C a r d i a c Sarcolemma ("IOO ug) was phosphorylated i n a C a uptake medium i n the presence of 2 5 uM T r i s - A T P and 1 uM c y c l i c AMP. At the end of 5 minutes, 100 uM C a C l was added and the l e v e l of membrane phosphorylat i o n was f o l l o w e d f o r the next 5 minutes. Numbers on each curve r e f e r to the amount o f p r o t e i n k i n a s e added i n ug. + +  2  MINUTES  -  72  -  s t o i c h i o m e t r i c r e l a t i o n s h i p b e t w e e n t h e amount o f p h o s p h a t e i n corporated As  and  t h e n e t amount o f Ca  shown i n F i g u r e  t a k e was  8,  t h i s was  not  b o u n d due the case.  phosphorylation.  Increased  Ca  15 n m o l e s o f n e t i n c r e a s e i n C a  t h a t the i n c r e a s e i n C a  + +  u p t a k e was  a c t i o n on t h e membrane s u r f a c e . o n l y a f f e c t e d t h e Ca  (up t o 1.5 uptake.  + +  n o t due  and  nmoles), This  suggested  to simple i o n i c  I n f a c t , membrane  u p t a k e s y s t e m and  up-  + +  l i n e a r w i t h t h e amount o f p h o s p h a t e i n c o r p o r a t e d ,  f o r e v e r y nmole o f p h o s p h a t e i n c o r p o r a t e d , t h e r e was  to  n o t Ca  inter-  phosphorylation binding.  e x a c t m e c h a n i s m w h e r e b y membrane p h o s p h o r y l a t i o n  The  leads to C a  + +  up-  t a k e r e m a i n s t o be e l u c i d a t e d .  6.  R e v e r s i b i l i t y o f membrane p h o s p h o r y l a t i o n of phosphoprotein phosphatase  F o r membrane p h o s p h o r y l a t i o n o l o g i c a l r e g u l a t i o n of C a  and  distribution  t o have i m p o r t a n c e i n p h y s i -  t r a n s p o r t i n t o the c a r d i a c c e l l ,  + +  m u s t a l s o be a m e c h a n i s m f o r r a p i d d e p h o s p h o r y l a t i o n . attempt to demonstrate dephosphorylation, phosphorylated  In  an  c a r d i a c sarcolemma  by e x o g e n o u s p r o t e i n k i n a s e , a n d when p l a t e a u  o f p h o s p h o r y l a t i o n was  reached  there  a f t e r 5 minutes,excess  was level  unlabeled  32 ATP  (3 mM)  was  added t o s t o p f u r t h e r  l e v e l o f p h o s p h o r y l a t i o n was  measured a t d i f f e r e n t time  d u r i n g t h e f o l l o w i n g 15 m i n u t e s . v a l e n t v o l u m e o f w a t e r was  added.  a f t e r the a d d i t i o n of excess  P i n c o r p o r a t i o n , and  In c o n t r o l experiments, As  shown i n F i g u r e  u n l a b e l e d ATP,  9,  the  intervals an  equi-  immediately  t h e l e v e l o f membrane  p h o s p h o r y l a t i o n began t o d e c l i n e w h i l e c o n t r o l l e v e l s  remained  Figure 8  R e l a t i o n s h i p between p h o s p h o r y l a t i o n and s t i m u l a t i o n of c a l c i u m uptake by c a r d i a c sarcolemma. Sarcolemmal membranes (125 ug) were phosphorylated by v a r i o u s amounts of p r o t e i n k i n a s e (20 pg t o 200 pg) i n the same medium used f o r c a l c i u m uptake. The amount o f p h o s p h o r y l a t i o n ( c o r r e c t e d f o r endogenous a c t i v i t i e s ) i s p l o t t e d a g a i n s t the net amount of c a l c i u m uptake s t i m u l a t e d by p h o s p h o r y l a t i o n .  NET INCREASE IN nMOLES  3 2  PHOSPHORYLATION  P I N C O R P . / M G PROTEIN/5  MIN.  Figure 9 P h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n of c a r d i a c sarcolemma. F r e s h l y prepared membranes ( 1 mg) was phosphorylated i n the presence ( c i r c l e s ) and absence ( t r i a n g l e s ) of 1 pM cAMP, 25 pM [ Y - P ] ATP by exogenous p r o t e i n k i n a s e (175 pg) i n a f i n a l volume of 2 ml. F l u o r i d e was excluded from the medium. P h o s p h o r y l a t i o n was measured a t v a r i o u s time, i n t e r v a l s up t o f i v e minutes by removing 200 p i a l i q u o t s (see Methods f o r d e t a i l s ) . At the end of 5 minutes, u n l a b e l e d ATP was added ( f i n a l c o n c e n t r a t i o n 2 mM) and dephosphorylat i o n was f o l l o w e d f o r the next 15 minutes (open symbols) again by removing 200 p i a l i q u o t s . In c o n t r o l tubes, an equal amount of water was added (closed symbols). 3 2  MINUTES  - 77 -  unaltered.  Although d e p h o s p h o r y l a t i o n was  not complete i n 15  32  minutes,  as much as 50% of  P was  removed from membranes phos-  p h o r y l a t e d i n the presence or absence of c y c l i c AMP.  This i s  h i g h l y s u g g e s t i v e t h a t phosphoprotein  be a s s o c i a t e d ,  phosphatase may  w i t h c a r d i a c sarcolemma as p a r t of a r e g u l a t o r y complex membrane p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n . f u r t h e r examined the presence  and d i s t r i b u t i o n  We,  controlling  therefore,  of t h i s phospho-  p r o t e i n phosphatase a c t i v i t y  i n sarcolemma and i n v a r i o u s f r a c t i o n s  o b t a i n e d d u r i n g the i s o l a t i o n  of the sarcolemma, u s i n g phosphoryl-  ated sarcolemma and h i s t o n e s as s u b s t r a t e s . at the stage of "washed p a r t i c l e s " phosphoprotein  largely  removed.  More than 75% of  recovered from the supernatant, which c o n t a i n e d  s o l u b l e p r o t e i n and the microsomal f r a c t i o n . .  the washed p a r t i c l e s  E x t r a c t i o n of  with KCI removed most of the remaining  so t h a t i n the f i n a l sarcolemmal membranes, o n l y 1% of the a c t i v i t y remained.  IX,  (see F i g u r e 1 ) , 80% of the  phosphatase a c t i v i t y was  t h i s a c t i v i t y was  As shown i n Table  activity total  Because of the s m a l l recovery of enzyme a c t i v i t y ,  i n order to e s t a b l i s h t h a t the enzyme i s i n t r i n s i c to the membrane, we washed sarcolemmal membranes thoroughly  (5 times) as  we  d i d p r e v i o u s l y i n the s t u d i e s of membrane bound p r o t e i n k i n a s e . As shown i n Table IV, d e s p i t e a r e d u c t i o n by the washing 0.5% iated  of enzyme a c t i v i t y  s u r v i v e d such treatment  w i t h the sarcolemma.  procedure,  and remained assoc-  - 78 -  TABLE IX  D i s t r i b u t i o n o f p h o s p h o p r o t e i n phosphatase i n f r a c t i o n s o b t a i n e d d u r i n g i s o l a t i o n o f sarcolemma. Measurement o f p h o s p h o p r o t e i n phosphatase a c t i v i t y , and i s o l a t i o n o f v a r i o u s f r a c t i o n s from g u i n e a p i g h e a r t were d e s c r i b e d under "Methods". "Supernatant" was o b t a i n e d by p o o l i n g a l l s u p e r n a t a n t f r a c t i o n s from the 5 washings d e s c r i b e d i n the i s o l a t i o n p r o c e d u r e ; "Washed sarcolemma" r e f e r r e d t o sarcolemma s u b j e c t e d t o 5 more washings w i t h b u f f e r c o n t a i n i n g 250 mM Sucrose, 2 mM d i t h i o t h r e i t o l , 10 mM T r i s - H C l pH 7.5 and c e n t r i f u g e d a t 37,000 x g f o r 15 minu t e s a f t e r each wash. V a l u e s g i v e n on the t a b l e were the means w i t h S.E.M. o f 4 d i f f e r e n t membrane p r e p a r a t i o n s .  Phosphoprotein Fractions  Specific Activity , (pmoles "^P/mg/min^.: ute) '"  KCl-extracted Particles  8:5  +  7.8  +  5.0  +  3.1  Phosphatase  '• T b t a ' l l a c t i v i t y } ; (Percent)  100  .98 52.2  +  5.4  .12  17.1  +  1.8  +  .70  3.2  +  .57  2.8  +  .47  .90 +  .06  1.5  +  .12  .51 +  .03  1.2  - 79 -  DISCUSSION  S t u d i e s w i t h i s o l a t e d c a r d i a c sarcolemma p r o v i d e e v i d ence f o r the presence o f an ATP-dependent C a w e l l as C a  + +  dependent ATPase  + +  b i n d i n g system as  (Tables VI and V I I ) .  These a c t i v -  i t i e s are not l i k e l y t o be due t o contamination by m i t o c h o n d r i a l or microsomal fragments because of (1) t h e i r l a c k of s e n s i t i v i t y to a z i d e  (we have observed, as o t h e r s  mitochondrial C a  + +  (130) t h a t 65% t o 85% of  accumulation i s a b o l i s h e d by 5. mM  (2) t h e i r r e l a t i v e l y low s p e c i f i c a c t i v i t y  NaN^),  ( f r e s h l y prepared c a r d -  i a c microsomal p r e p a r a t i o n s accumulate at l e a s t 6 t o 10 times more c a l c i u m under s i m i l a r c o n d i t i o n s ) , and ity  (3) t h e i r r e l a t i v e  stabil-  ( c a r d i a c microsomes l o s e 60%-80% of t h e i r a b i l i t y to accumu-  late C a  + +  3 hours a f t e r i s o l a t i o n whereas c a r d i a c sarcolemma can  be s t o r e d a t 4° o v e r n i g h t w i t h 90% of the Ca""" accumulation 1  activity intact).  I t i s a l s o u n l i k e l y t h a t these a c t i v i t i e s are  due to contamination by m y o f i b r i l s , centration  1  i n view of the h i g h s a l t con-  (1.25 M KCI) used to e x t r a c t the  membranes.  Further-  more, o x a l a t e i s not known to e x e r t any e f f e c t on m y o f i b r i l l a r Ca  binding.  Ca  - s t i m u l a t e d ATPase a c t i v i t i e s have been r e p o r t e d  i n plasma membrane f r a c t i o n s from dog cardium.  Energy-dependent C a  + +  (146) and r a t (134) myo-  t r a n s p o r t mechanisms have  been d e s c r i b e d i n plasma membranes from red c e l l s blasts  (137), smooth muscle  (135,136), f i b r o -  (138) and s k e l e t a l muscle  r e p o r t s have argued f o r an a c t i v e C a  + +  also  (32).  These  t r a n s p o r t at the plasma  membrane analogous t o the t r a n s p o r t of N a  +  and K . +  The present  f i n d i n g i n the c a r d i a c sarcolemma p r o v i d e s f u r t h e r support t o t h i s  - 80 -  concept and suggests a r o l e f o r the sarcolemma i n C a  T T  mobiliza-  t i o n and e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n the heart. Our  s t u d i e s show t h a t c a r d i a c sarcolemma possess memb-  rane-bound, c y c l i c AMP-dependent p r o t e i n kinase w e l l as endogenous s u b s t r a t e s  a c t i v i t i e s as  f o r auto-phosphorylation.  procedure t o i s o l a t e sarcolemma i n v o l v e d vigorous repeated  washings w i t h hypotonic  The  homogenization,  and i s o t o n i c b u f f e r s and ex-  t r a c t i o n w i t h high' c o n c e n t r a t i o n o f KCl.  T h i s makes i t u n l i k e l y  t h a t the p r o t e i n kinase a c t i v i t i e s observed were due t o the ads o r p t i o n o f s o l u b l e enzymes to the membranes. f a c t that auto-phosphorylation were washed 5 times w i t h convincing  was u n a l t e r e d a f t e r the sarcolemma  l a r g e volumes of b u f f e r makes i t r a t h e r  t h a t both the p r o t e i n k i n a s e ( s )  s t r a t e (s) were indeed  Furthermore, the  and the membrane  sub-  i n t r i n s i c components of the membrane.  Phosphoprotein phosphatase a c t i v i t y was a l s o found t o be associ a t e d w i t h the sarcolemma.  Maeno and Greengard  (139) have shown  t h a t i n r a t c e r e b r a l c o r t e x , more than 50% of the t o t a l phosphoprotein  phosphatase a c t i v i t y was found i n the p a r t i c u l a t e  f r a c t i o n s and t h a t the s p e c i f i c a c t i v i t y was h i g h e s t i n s u b t r a c t i o n s r i c h i n s y n a p t i c membranes.  The present  data  c a r d i a c sarcolemma was able t o r e v e r s e membrane s t i m u l a t e d by r e l a t i v e l y h i g h c o n c e n t r a t i o n s kinase.  shows t h a t  phosphorylation  of p u r i f i e d p r o t e i n  T h i s makes i t h i g h l y probable t h a t a t l e a s t p a r t o f the  phosphoprotein phosphatase a c t i v i t y i s a s s o c i a t e d with the plasma membranes, c o n f e r r i n g r e v e r s i b i l i t y t o membrane  phosphorylation.  -  Auto-phosphorylation slightly  stimulated  t i o n o f h i s t o n e by e n h a n c e d by  81  o f sarcolemma  (25%-40%) by the  -  ( F i g u r e 5A)  c y c l i c AMP,  whereas  of c y c l i c AMP-stimulation  ( F i g u r e 4A).  (133).  greatly  S i m i l a r low  have been o b s e r v e d i n the  p h o r y l a t i o n o f e r y t h r o c y t e membranes  m i n o r c o m p o n e n t was  c y c l i c AMP. membranes  Similarly,  unequivocally  In these  shown t o be  i n the p h o s p h o r y l a t i o n  levels  auto-phosmembranes,  o f t h e t h r e e p r o t e i n components t h a t were p h o s p h o r y l a t e d , one  only  phosphoryla-  same membrane p r e p a r a t i o n s was  this cyclic nucleotide  was  only  stimulated  of nerve  by  synaptic  ( 9 ) , Johnson e t a l f o u n d t h a t o n l y 2 p r o t e i n c o m p o n e n t s  showed i n c r e a s e d p h o s p h o r y l a t i o n  i n the presence of c y c l i c  AMP.  A t l e a s t s i x o t h e r membrane c o m p o n e n t s w e r e p h o s p h o r y l a t e d ,  but  their  l e v e l of phosphorylation  These o b s e r v a t i o n s o n l y one  o r a few  c y c l i c AMP  was  unaffected  s u g g e s t e d t h a t i n membrane  whose e f f e c t c a n  the  Although  we  have not  s i t e phosphorylated,  i n c a r d i a c s a r c o l e m m a may The sent  s i t u a t i o n was  auto-phosphorylation,  e a s i l y be o b s c u r e d by h i g h  the r e s u l t of a s i m i l a r  f u r t h e r complicated  T h i s e x p l a i n s why,  of  by  the  to p a r t l y  of  observation phenomenon.  f a c t t h a t the  pre-  active  c y c l a s e so t h a t s u f f i c i e n t l y h i g h l e v e l s o f c y c l i c  were a l r e a d y p r e s e n t  kinase  levels  attempted to determine the nature  i t i s q u i t e l i k e l y t h a t our be  by  o f o t h e r membrane compon-  s a r c o l e m m a l p r e p a r a t i o n p o s s e s s e d an e x t r e m e l y  adenylate  AMP.  possible substrate(s) i s subject to control  c y c l i c AMP-independent p h o s p h o r y l a t i o n ents.  by c y c l i c  stimulate protein kinase  AMP  activities.  even i n the p r e s e n c e o f added s o l u b l e p r o t e i n  ( F i g u r e 3 A ) , membrane p h o s p h o r y l a t i o n . w a s s t i m u l a t e d  no  -  more t h a n  82 -  3 - f o l d by a d d e d c y c l i c  AMP,  whereas h i s t o n e  phosphoryla-  t i o n by t h e same p r o t e i n k i n a s e was s t i m u l a t e d more t h a n by  cyclic  AMP. Our  finding of a phosphorylation  s y s t e m i n t h e c a r d i a c sarcolemma d i f f e r s Wray e t a l ( 1 1 7 ) , 147)  mainly  authors  and  from the o b s e r v a t i o n s o f  i n the l o c a l i z a t i o n  of these  e t a l (127, 140,  enzyme s y s t e m s .  used c a r d i a c microsomes which c o n s i s t  indicated  dephosphorylation  L a R a i a e t a l (118) and K a t z  mented e n d o p l a s m i c r e t i c u l u m .  largely  t h a t t h e s e membranes were p h o s p h o r y l a t e d  the s t i m u l a t i o n o f the r a t e o f C a  a functional  r e l a t i o n s h i p between p h o s p h o r y l a t i o n  reported tion  exist  reported  reticulum.  recently that sites i n the skeletal  of fragstudies  by c y c l i c  uptake,  + +  and C a  S u l a k h e and Drummond  of phosphorylation  and  Recently,  phosphorylation  (12 8)  p r e p a r a t i o n f r o m p i g myocardium..  together with  our s t u d i e s , suggest  phosphorylation-dephosphorylation  Although  membrane  a number o f t i s s u e s ,  have  dephosphoryla-  i n sar-  K r a u s e e t a l (141)  s y s t e m i n a p l a s m a memb-  rane-enriched  level  trans-  + +  sarcolemma; Andrew e t a l (110) a l s o  colemma d e r i v e d f r o m s k e l e t a l m u s c l e .  t h e p l a s m a membrane  cor-  suggesting  t h e e x i s t e n c e o f a membrane-bound p r o t e i n k i n a s e  demonstrated a r e v e r s i b l e  AMP-  of phosphoprotein  related with  i n sarcoplasmic  These  As m e n t i o n e d a b o v e , t h e i r  d e p e n d e n t p r o t e i n k i n a s e , and t h e f o r m a t i o n  port  10-fold  These  that a similar  observations,  system o f auto-  i n v o l v i n g c y c l i c AMP  e x i s t s at.  o f t h e myocardium. p h o s p h o r y l a t i o n has been o b s e r v e d i n  the p h y s i o l o g i c a l  importance of t h i s  phenomenon  - 83 -  remains obscure.  Membrane p e r m e a b i l i t y to i o n s has been p o s t u l -  ated t o be r e g u l a t e d by t h i s p r o c e s s i n s y n a p t i c membranes 90,  142),  toad-bladder  (88,  membranes (143), r e n a l medullary memb-  ranes  (116), l i v e r plasma membranes (108), c a r d i a c microsomes  (118,  127,  140)  and  s k e l e t a l sarcolemma  (128).  shows t h a t c a r d i a c sarcolemma, phosphorylated ous or exogenous p r o t e i n k i n a s e , was than c o n t r o l p r e p a r a t i o n s . t i o n of C a  + +  e i t h e r by endogen-  a b l e t o accumulate more Ca  As shown i n F i g u r e 8, the net s t i m u l a -  suggesting  t h a t the enhanced C a  indeed the r e s u l t of membrane p h o s p h o r y l a t i o n .  + +  transport i s  While  relatively  i s known about the exact mechanism whereby p h o s p h o r y l a t i o n  controls C a Siegel  + +  (144)  t r a n s p o r t , i t i s of i n t e r e s t to note t h a t Wolff i s o l a t e d from p i g b r a i n a C a  + +  activity,  suggesting a r o l e f o r  i n r e g u l a t i o n of C a 147,  148)  + +  binding.  and  b i n d i n g phospho-  p r o t e i n which when t r e a t e d with phosphatase, l o s t i t s C a  (145,  study  uptake p a r a l l e l e d the net i n c r e a s e i n k i n a s e - c a t a l y z e d  phosphorylation,  little  T h i s present  + +  binding  phosphorylation-dephosphorylation In recent s t u d i e s , Tada e t a l  showed t h a t c y c l i c AMP-dependent p r o t e i n kinase  c a t a l y z e d the p h o s p h o r y l a t i o n of a 22,000-dalton p r o t e i n i n c a r d i a c microsomes which was i n t e r m e d i a t e o f the C a  + +  d i s t i n c t from the  t r a n s p o r t ATPase.  phosphoprotein  I t was  f u r t h e r shown  t h a t b r i e f d i g e s t i o n with t r y p s i n i n the presence o f 1 M d i d not s i g n i f i c a n t l y a f f e c t microsomal C a but prevented and  + +  sucrose  transport a c t i v i t y ,  both p h o s p h o r y l a t i o n of the 22,000-dalton p r o t e i n  s t i m u l a t i o n of C a  + +  uptake by c y c l i c AMP-dependent p r o t e i n  k i n a s e , s u g g e s t i n g t h a t t h i s p r o t e i n i s a modulator of the pump.  Ca  + +  -  84  -  From a p h y s i o l o g i c a l p o i n t f u n c t i o n of ractility,  catecholamines i s the  contractile proteins.  of  c o n t r a c t i l e process  the  The  variations in C a  release  of C a  the  sarcolemma and  Ca  the  sarcolemma i s a s s o c i a t e d  influx  current cepted  that that  increasing for at  across  takes place  agonists.  The  of  have shown t h a t  basis  cardiac  lemma n o t  only  contain  action of  the  protein(s)  leading  relevance  sarcolemma c o n t a i n  the  microscopy  the  interpret  f o r the  the  nucleotide,  + +  + +  provid-  the  We  adenylate  s y s t e m s as  well  cardiac of  of c e r t a i n  transport.  + +  these data i s f u r t h e r  accumulation  B-adrenergic  sarcocyclic bio-  a c t i v a t i o n o f memb-  phosphorylation Ca  by  account  c o n c e p t by  active  ac-  content  machinery f o r the  i . e . , the  sarcolemma were done by Ca  Ca  production  regulator  The  physio-  s t r e n g t h e n e d by  electron  i s o l a t e d sarcolemmal  c o n s i s t e d m a i n l y o f v e s i c l e s and  cardiac  generally  Thus t h e  a l s o the  s t u d i e s w h i c h showed t h a t  preparation of  an  activity.  apparatus  and  inward  mediation of these events.  t o enhancement o f of  w i t h a slow  t h i s e f f e c t may  t h e s i s supports t h i s  f o r the  rane-bound p r o t e i n k i n a s e  logical  and  two  reticulum.  p o s i t i v e inotropic action of  transport  + +  delivery  + +  sarcoplasmic  s y s t o l e ; i t i s now  i n r e s p o n s e t o hormones, b u t  logical  the  auto-phosphorylation-dephosphorylation  as A T P - d e p e n d e n t C a  AMP  the  data i n t h i s  ing a biochemical  cyclase,  during  slow inward c u r r e n t ,  least part  cont-  for activation  + +  c a t e c h o l a m i n e s augment i n t r a c e l l u l a r this  important  i s c o n t r o l l e d p r e d o m i n a n t l y by  membrane s y s t e m s : + +  most  m o d u l a t i o n o f myocardial,  which u l t i m a t e l y i n v o l v e s  to the  o f view, the  Dr.  G.  open s a c s .  (EM  I . Drummond).  i n t h e s e v e s i c l e s and  the  studies We  enhancement  -  of  this  activity  analogous Ca  by c y c l i c AMP-dependent p h o s p h o r y l a t i o n t o be  t o an i n v i v o s i t u a t i o n  t r a n s p o r t i n response  + +  logical  data  indicates  intracellular  from which C a  subsequent d e p o l a r i z a t i o n  ile  proteins or a c t indirectly  somes s u g g e s t e d  Ca  + +  first  + +  some  c a n be r e l e a s e d d u r -  + +  by r e l e a s i n g C a  + +  on c o n t r a c t -  from  other  sites  e t a l on c a r d i a c m i c r o -  the existence of a s i m i l a r These a u t h o r s  Physio-  fill  to act either d i r e c t l y  The f i n d i n g s o f K a t z  reticulum.  transarcolemmal  stimulation.  that the inflowing C a  storage s i t e s  within the c e l l .  of increased  t o B-adrenergic  ing  plasmic  85 -  system i n the s a r c o -  p o s t u l a t e d that the increased  u p t a k e by t h e s a r c o p l a s m i c r e t i c u l u m f o l l o w i n g p h o s p h o r y l a -  in t i o n by p r o t e i n k i n a s e h a s t h e e f f e c t some o f t h e C a  + +  which would otherwise  This increased C a available beats, It  + +  storage  fordelivery  thus  can then  to.assume t h a t both  reticulum are activated  ylation  i n the m o b i l i z a t i o n of C a  plore  during  apparatus  f u t u r e work w i l l  importance  of these  + +  i n subsequent  contractility.  AMP-dependent  i n response  be n e c e s s a r y two c o n t r o l  AMP m e d i a t e s t h e i n o t r o p i c  to  phosphor-  B-adrenergic  not only t o assess the  sites,  o t h e r mechanisms, e . g . , r e l e a s e o f C a  whereby c y c l i c  diastole.  t h e sarcolemma and t h e s a r c o -  by c y c l i c + +  the c e l l  add t o t h e amount o f C a  t o the c o n t r a c t i l e  plasmic  relative  be l o s t  p r o v i d i n g augmentation of myocardial  i s reasonable  stimulation;  of retaining with  + +  but a l s o t o ex-  from  response.  mitochondria,  - 86 -  REFERENCES' 1.  SNELL, F., WOLKEN, J . , IVERSON, G., a n d LAM, J . ( e d s ) P h y s i c a l P r i n c i p l e s i n B i o l o g i c a l Membranes (1970) G o r d o n & B r e a c h , N. Y.  2.  HOPE, A. B . , I o n T r a n s p o r t a n d w o r t h & Co.  3.  DODGE, J . T.,  !  MITCHELL, C ,  Biochem. B i o p h y s .  100,  Membranes  (1971) L o n d o n ,  a n d HANAHAN, D.  Butter-  (1962) A r c h .  119.  4.  N E V I L L E , D. M. , J r . (1960) J . B i o p h y s . B i o c h e m . C y t o l .  5.  N E V I L L E , D. M.,  6.  EMMELOT, P., BOS, C. J . , BENEDETTI, E. L., a n d RUMKE, P. L. (1964) B i o c h i m . B i o p h y s . A c t a . 90, 126 EMMELOT, P., V I S S E R , A., a n d BENEDETTI, E. L. ( 1 9 6 8 ) . B i o c h i m . B i o p h y s . A c t a . 150-y 364.  7.  J r . (1968) B i o c h i m . B i o p h y s . A c t a . 154,  8.  F I N E A N , J . B., COLEMAN, B., and GREEN, W. o f N..Y. A c a d . S c i . V o l . 137 (#2) 414  9.  COLEMAN, R., 125,  8,  a n d F I N E A N , J . B.  A.  413  540.  (1966) i n A n n a l s  (1966) B i o c h i m . B i o p h y s .  Acta  197  10.  TURKINGTON, R. W.  11.  HOLLAND, J . J . (1962) V i r o l o g y  12.  HOLLAND, J . J . , a n d HOYER, B. H. (1962) C o l d S p r i n g H a r b o r Symp. Q u a n t . B i o l . 27^, 101 KEENAN, T. W., MORRE, D. J . , OLSON, D. E., YUNGHAUS, W. N., and PATTON, S. (1970) J . C e l l . B i o l . _4_4, 80  13. 14.  P O I R I E R , G.,  (1962) B i o c h i m . B i o p h y s . A c t a (55, 386  DeLEAN, A.,  L A R R I E , F.  16,  163  P E L L E T I E R , G.,  (1974) J . B i o l .  LEMAY, A.,  Chem. 249,  316  15.  PETERS, R. A.,  16.  KONO, T., a n d KOLOWICK, S. P. (1961) A r c h . B i o c h e m . B i o p h y s . 93, 520 SEVERSON, D. L., DRUMMOND, G. I . , a n d SULAKHE, P. V. (1971) J . B i o l . Chem. 247, 2949  17. 18.  (1956) N a t u r e 177,  and  McCOLLESTER, D. L.  426  (1962) B i o c h i m . B i o p h y s . A c t a 57,  427  ROSENTHAL, S. L . , EDELMAN, P. M., a n d SCHWARTZ, I . (1965) B i o c h i m B i o p h y s . A c t a . 10 9, 512 WESTORT, C , and HULTIN, H. 0. (1966) A n a l . Biochem. 16, 314 HULTIN, H. 0., and WESTORT, C. (1969) J . Food S c i . _3_4, 165 BOEGMAN, R. J . , MANERY, J . F., and PINTERIC, L. (1970) Biochim. Biophys. A c t a . 203, 506 KIDWAI, A. M., R A D C L I F F E , A., L E E , E . Y., a n d DANIEL, E . E . (1973) B i o c h i m . B i o p h y s . A c t a 2 9 8 , 593 SCHAPIRA, G., DOBOCZ, I . , P I A U , J . P., a n d D E L A I N , E . (1974) B i o c h i m . B i o p h y s . A c t a . 3 4 5 , 34 8 PORTIUS, H. J . , a n d REPKE, K.R.H. M e d i c a G e r m a n i c a 19_, 876  (1967) A c t a  Biologica  • STAM, A. C , J r . , WEGLICKI, W. B., J r . , FELDMAN, D., SHELBURNE J . C , a n d SONNENBLICK, E. H. (1970) J . M o l . C e l l . C a r d i o l 1, 117 KATZ, A. M., REPKE, D. I . , UPSHAW, J . E . , a n d POLASCIK, M. A. (1970) B i o c h i m . B i o p h y s . A c t a 2 0 5 , 473 KIDWAI, A. M., R A D C L I F F E , M. A., DUCHON, G., a n d DANIEL, E. E. (1971) B i o c h e m . B i o p h y s . R e s . Comm. 45_, 9 0 1 TADA, M., FINNEY, J . 0., J r . , SWARTZ, M. H., a n d KATZ, A. M. (1972) J . M o l . C e l l C a r d . A, 417 GILMAN, A. (1970) P r o c . N a t . A c a d . S c i . U.S.A. 6_7, 305 AMES, B. N., (1966) i n M e t h o d s i n E n z y m a l o g y (COLOWICK, S. P., a n d KAPLAN, N. 0. e d s ) V o l X V I I I p. 1 1 5 , A c a d e m i c P r e s s , N. Y. SULAKHE, P. V., DRUMMOND, G. I . , a n d NG, D. C. (1973) J . B i o l . Chem. 2 4 7 , 4150 COOPERSTEIN, S. J . , a n d LAZAROW, A. (1951) J . B i o l . 189, 665  Chem.  LOWRY, 0. H., ROSEBROUGH, N. J . , FARR, A. L . , a n d RANDALL, R. (1951) J . B i o l . Chem. 1 9 3 , 265 D I E T Z , G., a n d HEPP, K. C. (1972) B i o c h e m . B i o p h y s . R e s . Comm. 4 6 , 269  - 88 -  36.  SOLYOM, A., a n d TRAMS, E. G. (1972) Enzymes: 1 3 , 329  37.  SUTHERLAND, E. W. , RALL, T. W., a n d MENON, T. (1962) J . B i o l . Chem. 2 3 7 , 1220  38.  MARINETTI, G. V., RAY, T. K., a n d TOMASI, V. (1969) B i o c h e m . B i o p h y s . R e s . Comm. 3_6, 185  39.  WOLFF, J . , a n d JONES, A. B. (1971) J . B i o l .  40.  ROSEN, 0. M., a n d ROSEN, S. M. (1969) A r c h . B i o c h e m . B i o p h y s . 1 3 1 , 449 LEFKOWITZ, R. J . , ROTH, J . , PRICER, W., a n d PASTAN, I .  41.  Chem. 2 4 6 , 3939  (1970) P r o c . N a t . A c a d . S c i . U.S.A. 65, 745 42.  RODBELL, M.  43.  SULAKHE, P. V., a n d DHALLA, N. S. (1973) B i o c h i m . B i o p h y s . A c t a 2 9 3 , 379 KATZ, A. M., TADA, M., P E P K E , D. I . , IORIO, J.A.M., a n d  44.  (1967) J . B i o l .  Chem. 2 4 2 , 5744  KIRCHBERGER, M. A. (1974) J . M o l . C e l l .  Card.  6, 73  45.  SCHRAMM, M., a n d NAIM, E. (1970) J . B i o l .  46.  KHANDELWAL, R. L . , a n d HAMILTON, I . R. (1971) J . B i o l . Chem. 2 4 6 , 3297 McNAMARA, D. B., SULAKHE, P. V., a n d DHALLA, N. S. (1971) Biochem. J . 125, 525.  47.  Chem. 2 4 5 , 3225  48.  STAM, A. C , J r . , SHELBURNE, J . W., FELDMAN, D., a n d SONNENBLICK, E . H. (1969) B i o c h i m . B i o p h y s . A c t a 1 8 9 , 304  49.  BOSMANN, H. B., HAGOPIAN, A., a n d ELYAR, E. H. (1968) A r c h . B i o c h e m . B i o p h y s . 1 2 8 , 51  50.  WATTIAUX-DE CONINCK, S., a n d WATTIAUX, R. (1969) B i o p h y s . A c t a 1 8 3 , 118  51.  SUTHERLAND, E . W., ROBISON, G. A., a n d BUTCHER, R. W. (1968) C i r c u l a t i o n 3 7 , 279  52.  ROBINSON, G. A., BUTCHER, R. W., 0 Y E , I . , MORGAN, H. E . , a n d SUTHERLAND, E. W.  Biochim.  (1965) M o l . P h a r m a c o l . 1, 168  53.  WILLIAMS, B. J . , a n d MAYER, S. E. (1966) M o l . P h a r m a c o l . 2, 454  54.  DRUMMOND, G. I . , DUNCAN, L . , a n d HERTZMAN, E. J . (1965) J . B i o l . Chem. 2 4 1 , 5899 NAMM, D. H., a n d MAYER, S. E. (1968) M o l . P h a r m a c o l . 4, 61  55.  - 89 -  56.  CHEUNG, W. Y., a n d WILLIAMSON, J . R. (1965) N a t u r e 2 0 7 , 979  57.  DRUMMOND, G. I . , a n d HEMMING. S. J . (1973) i n M y o c a r d i a l M e t a b o l i s m V o l . 3 ( D h a l l a e d . ) p . 213  58.  LaRAIA, P. J . , a n d REDDY, W. J . (1969) B i o c h i m . A c t a 1 7 7 , 189  59.  L E E , T. P., KUO, J . F., a n d GREENGARD, P. (1971) B i o c h i m . B i o p h y s . R e s . Comm. 45_,  Biophys.  991  60.  MURAD, F., VAUGHN, M. (1969) B i o c h e m . P h a r m a c o l . 1 8 , 1053  61.  LEVEY, G. S., a n d EPSTEIN  62.  MURAD, F., C H I , Y. M., RALL, T. W., a n d SUTHERLAND, E. W. (1962) J . B i o l . Chem. 2 3 7 , 1233 SUTHERLAND, E . W., a n d RALL, T.W. (1958) J . B i o l . Chem. 232, 1077  63.  S. E. (1969) C i r c . R e s . 2 4 , 1 5 1  64.  RALL, T. W., a n d WEST, T. C. (1963) J . P h a r m a c o l . E x p . T h e r . 1 3 9 , 269  65.  L a R A I A , P. J . , a n d SONNENBLICK, E. H. (1969) Amer. J . C a r d i o l . 23_, 123  66.  SKELTON, C. L . , LEVEY, G. D., a n d E P S T E I N , S. E. (1969) C i r c u l a t i o n 4£ ( I I I ) 188  67.  BUTCHER, R. W., a n d SUTHERLAND, E . W. Chem. 2 3 7 , 1244  68.  L E V I N E , R. A., a n d VOGEL, J . A. (1966) J . P h a r m a c o l . E x p . T h e r . 1 5 1 , 262  69.  SKELTON, C. L . , LEVEY, G. S., a n d E P S T E I N , S. E. (1970) C i r c . R e s . 26_, 35  70.  KUKOVETZ, W. R., a n d POCH, G. (1970) A r c h i v s P h a r m a k o l . 2 6 6 , 236  71.  DRUMMOND, G. I . , a n d HEMMING. S. J . (1972) i n A d v a n c e s i n C y c l i c N u c l e o t i d e R e s e a r c h V o l . 1, p. 3 0 7 . Raven P r e s s , N.Y.  72.  BENFEY, B. G. (1971) B r i t .  73.  BENFEY, B. G., a n d CAROLIN T. (1971) C a n . J . P h y s i o l . P h a r m a c o l . 4 9 , 508  (1962) J . B i o l .  Naunyn-Sehmiedegergs  J . P h a r m a c o l . 4 3 , 757  -  90  -  74.  BENFEY, B. G., COHEN, J . , KUNOS, G., a n d VERNES-KUNOS, I . (1974) B r i t . J . P h a r m a c o l . 50_, 581  75.  SHANFELD, J . , FRAZER, A., and HESS, M. E. E x p . T h e r . 16 9, 315  76.  WASTILA, W. B., SU, J . Y., FRIEDMAN, W. F., a n d MAYER, S. E.  (1969) J . P h a r m a c o l .  (1972) J . P h a r m a c o l . E x p . T h e r . 1 8 1 , 126 77.  WEBER, A.  78.  NAYLER, W. G.  79.  DEGUBAREFF, T., a n d A L E A T O R , W., J r . (1965) J . P h a r m a c o l . E x p . T h e r . 1 4 8 , 202 BERKOWITZ, B. A., TARVER, J . H., SPECTOR, S. (1969) F e d . P r o c . 28^, 415  80.  (1968) J . Gen. P h y s i o l .  5 2 , 760  (1967) Am. H e a r t . J . 73_, 379  81.  WESTFALL, D. P., a n d FLEMING, W. W. E x p . T h e r . 1 5 9 , 98  82.  MCNEILL, J . H., NASSAR, M., a n d BRODY, T. M. P h a r m a c o l . E x p . T h e r . 1 6 5 , 234  83.  KJEKSHUS, J . K., HENRY, P. D., a n d SOBEL, B. E. Circ.  (1968) J . P h a r m a c o l . (1969) J . (1971)  R e s . 2 9 , 468  84.  LANGSLET, A., a n d 0 Y E , I . (1970) E u r . J . P h a r m a c o l . 12,  85.  SOBEL, B. E., a n d MAYER, S. E.  86.  T S I E N , R. W.  87.  BROOKER, G.  88.  MAENO, H., JOHNSON, E. M., a n d GREENGARD, P. Biol.  (1973) C i r c .  R e s . 3_2, 407  (1973) N a t u r e ( N e w . B i o l o g y ) 2 4 5 , 120 (1973) S c i e n c e 1 8 2 , 933 (1971) J .  Chem. 2 4 6 , 134  89.  WELLER, M., a n d RODNIGHT, R.  90.  JOHNSON, E. M., MAENO, H., a n d GREENGARD, P. ( I y 7 1 ) J . B i o l . Chem. 2 4 6 , 7731 RUBIN, C. S., ERLICHMAN, J . , a n d ROSEN, 0. M. (1972) J . B i o l . Chem. 24 7, 6135 WALSH, D. A., P E R K I N S , J . P., a n d KREBS, E. G. (1968) J . B i o l . Chem. 2 4 3 , 3763  91. 92. 93.  (1970) N a t u r e 2 5 5 , 187  REIMANN, E. M., WALSH, D. A., a n d KREBS, E. G. J . B i o l . Chem. 2 4 6 , 1986  (1971)  137  - 91 -  94.  BROSTROM, M. A., REIMANN, E. M., WALSH, D. A., a n d KREBS, E . G. (1970) A d v . E n z . R e g u l . 8, 1 9 1  95.  KUO, J . F., a n d GREENGARD, P. (1969) P r o c . N a t . A c a d . S c i . U.S.A. 6 4 ,  1349  96.  KUO, J . F . , a n d GREENGARD, P. (1969) J . B i o l .  97.  GREENGARD, P., a n d KUO, J . F. (1970) A d v . B i o c h e m . P s y c h o p h a r m a c o l . 3_, 287 SODERLING, T. R., HICKENBOTTOM, J . P., REIMANN, E. M., HUNKELER, F. L . , WALSH, D. A., a n d KREBS, E. G. (1970) J . B i o l . Chem. 2 4 5 , 6317  98.  99.  Chem. 2 4 4 , 3417  SCHLENDER, K. K., WEI, S. H., a n d V I L L A R - P A L A S I , C. (1969) B i o c h i m . B i o p h y s . A c t a 1 9 1 , 272  100.  CORBIN, J . D., a n d KREBS, E. G. (1969) B i o c h e m . Res. Comm. 3_6, 328  Biophys.  101.  HUTTUNEN, J . K., STEINBERG, D., a n d MAYER, S. E. (1970) P r o c . N a t . A c a d . S c i . U.S.A. 67_, 290  102.  LANGAN, T. A. (1968) S c i e n c e  103.  I N G L E S , C. J . , a n d DIXON, G. H. (1967) P r o c . N a t . A c a d . S c i . U.S.A. 5_8, 1 0 1 1 MARZLUFF, W. F., J r . , McCARTY, K. S., a n d TURKINGTON, R. W. (1969) B i o c h i m . B i o p h y s . A c t a 1 9 0 , 517  104. 105.  1 6 2 , 579  MAJUMDER, G. C., a n d TURKINGTON, R..W.  (1972) J . B i o l .  Chem. 2 4 7 , 7207 106.  RABAT, D. (1970) B i o c h e m i s t r y 9_, 4160  107.  WALTON, G. M., B I L L , G. N., ABRASS, I . B., a n d GARDEN, L. D. (1971) P r o c . N a t . A c a d . S c i . U.S.A. 6 8 , 880 SCHLATZ, L . , a n d MARINETTI, G. V. (1971) B i o c h e m . B i o p h y s . Res. Comm. 45_, 51  108. 109.  GUTHROW, C. E . , J r . , A L L E N , J . E., a n d RASMUSSEN, H. (1972) J . B i o l . Chem. 2 4 7 , 8145  110.  GOODMANN, D.B.P., RASMUSSEN, H., D I B E L L A , P., a n d GUTHROW, C. E . , J r . (1970) P r o c . N a t . A c a d . S c i . U.S.A. 6 7 , 652  111.  STULL, J . T., BROSTROM, C. 0., a n d KREBS, E . G. (1972) J . B i o l . Chem. 2 4 7 , 5272  -  112.  92 -  MAJUMDER, G. C. , and TURKINGTON, Chem.  R. W.  (1971) J . B i o l .  246, 2650  113.  CHEN, L . J . , a n d WALSH, D. A.  114.  LABRIE, F., LEMAIRE, S., POIRIER, G., P E L L E T I E R , G., and BOUCHER, R. (1971) J . B i o l . Chem. 246, 7411 LEMAY, A., DESCHENES, M., LEMAIRE, S., POIRIER, G., POULIN, L . , and LABRIE, F. (1974) J . B i o l . Chem. 249, 323"  115.  (1971) B i o c h e m i s t r y  116.  DOUSA, H. P., SANDS, 91, 757  117.  WRAY, H. L . , GRAY, R. R., and OLSSON, R. A. Biol.  Chem.  248,  H., a n d HECHTER, 0.  10,  3614  (1972) E n d o c r i n . (1973) J .  1496  118.  LaRAIA, P. L . , a n d MORKIN, E . (1974) C i r c .  119.  ANDREW, C. G., ROSES, A. D., and ALMON, R. R., and APPEL, S. H.  (1973) S c i e n c e ,  R e s . _3_5, 298  182_, 927  120.  REUTER, H. , a n d S E I T Z , N.  121.  LANGER, G. A.  122.  GROSSMAN, A., FURCHGOTT, R. F. (1964) J . P h a r m a c o l . Exp. T h e r . 145, 162 ENTMAN, M. L . , LEVEY, G. S., and EPSTEIN, S. E . (1969) C i r c . R e s . 25_, 429 WATANABE, A. M., a n d BESCH, H. R., J r . (1974) C i r c . R e s . _3_5, 316  123. 124.  (1968) J . PHysiol.':. 19J5, 451  (1968) P h y s i o l .  Rev. 48,  708  125.  T S I E N , R. W., GILES, W., a n d GREENGARD, P. (New B i o l o g y ) 240, 181  (1972) N a t u r e  126.  MEINERTZ, T., NAWRATH, H., and SCHOLZ, H. S c h m i e d e b e r g A r c h P h a r m a c o l . 277, 107  127.  KIRCHBERGER, M. A., TADA, M., REPKE, D. I . , and KATZ, A. M. (1972) J . M o l . C e l l . C a r d i o l . ± 673  12b.  SULAKHE, P. V., and DRUMMOND, G. I . (1974) A r c h . . B i o p h y s . 161, 44 8  129.  CARAFOLI, E . , and LEHNINGER, A. L . 122, 681  130.  WEBER, A. (1966) i n C u r r e n t T o p i c s i n B i o e n e r g e t i c s D. R. ed) V o l . 1, P. 203, A c a d e m i c P r e s s , N.Y.  (1973) Naunyn  Biochem.  (1971) B i o c h e m . J . (SANADI,  -  93 -  131.  FERDMAN, D. L . , HIMMELREICH, N. G. , a n d DYADYUSHA, G. P. (1970) B i o c h i m . B i o p h y s . A c t a 2 1 9 , 372  132.  ROSES, A. D., a n d APPEL, S. H. (1973) J . B i o l .  Chem.  248, 140 8 133.  RUBIN, C. S., a n d ROSEN, 0. M. (1973) B i o c h e m . B i o p h y s . Res. Comm. 5 0 , 421  134.  DIETZ, G., a n d HEPP, K. D. (1971) B i o c h e m . B i o p h y s . Rec.  Comm. 4£,  1041  135.  WEINER, M. L . , a n d L E E , K. S. (1972) J . Gen. P h y s i o l .  136.  SCHATZMANN, H. J . , a n d VINCENTI, F . F. (1969) J . P h y s i o l . (London) 2 0 1 ,  59,  462  369  137.  PURDUE, J . F . (1971) J . B i o l .  138.  HURWITZ, L . , FITZPATRICK, D. F . , LANDON, E . J . , a n d DEBBAS, G. (1972) F e d . P r o c . 31,  Chem. 2 4 6 , 6750  587  139.  MAENO, H., a n d GREENGARD, P. (1972) J . B i o l .  140.  KIRCHBERGER, M. A., TADA, M., a n d KATZ, A. M. (1974) J . B i o l . Chem. 2 4 9 , 6166 KRAUSE, E . G., a n d WOLLENBERGER, A. U 9 7 5 ) i n A d v a n c e s i n C y c l i c N u c l e o t i d e R e s e a r c h (DRUMMOND, G. I . , a n d GREENGARD, P. e d s . ) V o l . V, Raven P r e s s , N.Y. UEDA, T., MAENO, H., a n d GREENGARD, P. (1973) J . B i o l . Chem. 24_8, 8295 DeLORENZO, R. J . , WALTON, K. G., CURRAN, P. F., a n d GREENGARD, P. (1973) P r o c . N a t . A c a d . S c i . U.S.A.  141. 142. 143.  Chem. 2 4 7 , 3269  70, 880  144.  WOLFF, D., a n d SIEGEL, F. (1972) J . B i o l .  Chem. 2 4 7 ,  4180 145.  TADA, M., KIRCHBERGER, M. A., IORIO, J.A.M., a n d KATZ, A.M. (1974) F e d . P r o c . 3_3,  479  146.  SULAKHE, P. V., and DHALLA, N. S. (1971) L i f e  147.  TADA, M., KIRCHBERGER, M. A., REPKE, D. I . , a n d KATZ, A. M. (1974) J . B i o l . Chem. 2A3_, 6174 TADA, M., KIRCHBERGER, M. A., a n d KATZ, A. M. (1975) J . B i o l . Chem. 2 5 0 , 2640  148.  S c i . 10,  185  -  94 -  149.  KATZ, A. M., (1975) New England J o u r n a l o f Medicine 24 3, 1184  150.  WOLLENBERGER, A., BABSKII, E . G., KRAUSE, E.-G., GENZ, S., BLOHM, D., and BOGDANOVA, E . V. (1973) Biochem. Biophys. Res. Comm. 5_5, 446  151.  BLOOM, S., and SWEAT, F. W. (1974) Res. Comm. i n Chem. Path, and Pharm. 8, 505.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0093871/manifest

Comment

Related Items