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Studies on the role of Ca²⁺ in the pancreatic acinar cell Ansah, Twum-Ampofo 1983

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STUDIES ON THE ROLE OF Ca*"" IN THE PANCREATIC ACINAR CELL 1  By TWUM-AMPOFO M.Sc.  The U n i v e r s i t y  A THESIS THE  SUBMITTED  of  British  IN PARTIAL  REQUIREMENTS DOCTOR  ANSAH Columbia,  FULFILMENT  F O R T H E D E G R E E OF  OF  PHILOSOPHY in  THE  FACULTY  (Faculty  of  We a c c e p t to  THE  OF G R A D U A T E  Pharmaceutical  this  thesis  the required  UNIVERSITY  as  ©Twum-Ampofo  Sciences)  conforming  standard  OF B R I T I S H  December  STUDIES  COLUMBIA  1983  Ansah,  1983  1980  OF  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t  the L i b r a r y s h a l l make  it  and  f r e e l y a v a i l a b l e for reference  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be  department o r by h i s o r her  granted by  the head of  representatives.  my  It i s  understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not  be  allowed without my  permission.  Department of Pharmaceutical Sciences The U n i v e r s i t y of B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  D a  7<n  te-  !  D  e  c  e  m  b  e  r  8,  19 83  Columbia  written  -  i i  -  ABSTRACT  Calcium secretion  ions  from  play  the  from  important  exocrine  secretagogue-induced release  an  rise  pancreatic  role  pancreas.  in  in It  cytoplasmic  acinar  the has  stimulation been  free  of  proposed  calcium  enzyme  that  triggers  a  enzyme  cells.  2+ The  mechanism  investigated  by w h i c h  using  membrane-enriched of  the  cells  of  trypan  role  secretion  was  calmodulin inhibited  was  the  of  on  a  secretin  Propranolol  amylase  the  did  the  possibility  release  regulated  step  from in  the  of  and  to  a  calmodulin the  selective  that  phenothiazines  stimulus-secretion  The  viability  the  exclusion  produced  in  (ED50 of  a  enzyme  10  both  significant  of  carbacholdegree  These  and  without  studies  calcium-mediated  acting  coupling  |jM).  release.  inhibit by  plasma  Trifluoperazine  inhibition.  cells  and  effects  mediated)  this  by  was  cells.  |jM i n h i b i t e d  (non-calcium  acinar  cells.  carbachol  acinar  release  10  cells  evaluated  process.  release  pancreatic  these  determining  amylase  amylase  show  the  secretion  acinar  95% a s  secretory  stimulated  not  from  protein,  by  concentration  pancreozymin-stimulated affecting  in  binding  carbachol-stimulated at  than  amylase  enzyme  pancreatic  pancreozymin  indirectly  antagonists  regulate  obtained  greater  calcium  studied  Chlorpromazine  indicate  intact  Secretin,  release  of  can  preparations  dye.  dose-dependent The  Isolated  isolated  blue  Ca  on a  calmodulin-  process. 24-  A high was  ATP  localized  Kinetic affinity  hydrolytic in  analysis (p  acinar  cell  revealed  < 0.05)  for  activity  C a  the  presence  of  either  Mg  preparations  enriched  in  plasma  membranes.  that 2  +  (K  the  d  of  in  2-  enzyme 1.73  had uM)  a  significantly  than Mg + 2  (Krf  of  or  higher 2.98  Ca  -  pM)  but  a  carried one  or  very  similar  out  in  more  order  to  enzymes.  A  similar  ATP  a n d GTP  both  maximal  profiles being  activities  the M g  most  of  activity.  These  due  enzyme  to  one  though,  revealed  addition, identify  when the  presence  of  data  tend  with  Ca  both  (M.W.  In  to  activity  by ADP.  addition,  were  represented  requirements  Ca^-stimulated  the  indicate  activation  revealed  activities  The  with  pH c u r v e s  combinations  in  the  the  for  degree ATPase  both  transition  gel  of  ( a p p r o x M.W.  2+  Mg "  of  for  saturating  of  maximal  activity  was  ATPase,  115,000) uM)  may  Arrhenius  temperatures.  the  =1.5-2  r  of  ions.  electrophoresis  intermediates  (endogenous  same  that  sites  polyacrylamide  alone  a n d Mg  130,000).  ,  Both  a  second  of  these  hydroxylamine-sensitive, of  and  studies  was  but  be  plots  In  used  to  one observed  in  the  upon  the  addition  intermediate  was  revealed  2+  Ca  presence  -  substrate  produced  difference  2+ of  Ca  intermediate  2+  of  ATPase  effectivefollowed  phosphoprotein  phosphoprotein  2 +  this  Further  2+ or  SDS  if  identical.  Mg  a  -  activity.  comparison  2+ concentrations  of  determine  for  the  were  rate  i i i  two  acyl  phosphoprotein phosphoproteins  indicating  that  phosphoproteins  they  would  were were  indicate  2+ intermediate  reaction  systems  for  the  Mg  acyl  phosphates.  possible  The  separate  24and  Ca  stimulated  ATPase  activity. Calmodulin  is  -transport  ATPases.  2+ Ca  known  to  regulate  The  effect  a number of  of  enzyme  exogenous  systems  calmodulin  on  including the  2+ Ca  -stimulated  investigated.  component In  the  pM.  Calmodulin  this  ATPase  presence  s t i m u l a t i o n by c a l m o d u l i n e f f e c t was d o s e - d e p e n d e n t 0.7  of  activity  2+  was  therefore  o f e n d o g e n o u s Mg , significant 2+ of Ca - A T P a s e a c t i v i t y was o b s e r v e d . This with a f o r c a l m o d u l i n of approximately  increased  the  2  Ca + - s e n s i t i v i t y  of  this  enzyme  -  system;  Mg  2+  stimulation  appeared  was  chlorpromazine  to  be  inhibited (ED50 of  required  by  55  iv  -  for  this  trifluoperazine  (jM)  and  compound  effect.  This  of  (ED50 48/80.  30  calmodulin  \M),  Using  an  125 I-labeled calmodulin dalton  calmodulin  binds  proteins  Further  in  gel  a Ca  present  studies  2 + -  technique,  dependent  in  revealed  overlay  the  that  fashion  plasma under  it  to  was  shown  133,000  and  membrane-enriched  conditions  that  that 230,000  fraction.  favour  2+ Ca  -dependent  of  a  30,000  stimulated  kinase  and by  a  activity,  19,000  acidic  calmodulin  dalton  protein.  enhanced The  Ca  2 +  the  phosphorylation  -ATPase  was  also  phospholipids.  2+ Ca  -flux  presence  of  measurements  two  in  processes:  plasma  there  membrane  were  vesicles  significantly  indicated  lower  the  levels  of  5 Ca  associated  with  'loaded'  in  the  system.  As  well,  absence a  was M g ^ - d e p e n d e n t In fluxes  order  degree  to  of  of  to the  ATPase  was  a  in  membranes.  may  be  ATP  (Mg  possible  and  This  externally-oriented.  function  remains  to  be  in 2 +  sealed  + Ca  2 +  an  site  of  provides The  the  when or  Mg  Ca  of  of  ATP  than  energy-dependent Ca  -uptake  those  efflux  system,  that  and  Ca  vesicles.  role  intact was  this  activity  in  enzyme  evidence  role  presence  )-ATPase  functional  observed  either  the  ATP-dependent  observed the  in  indicating  rapid  activity  with  'loaded'  ATP  catalytic  incubated broken  of  very  relate  observed  orientation  vesicles  the was  acinar  cell,  determined.  viable  acinar  similar  that  the  this  system  in  to  ATPase acinar  The  of  were  observed activity cell  elucidated. Signature  +  the  cells  that  2  Supervisor  -  v  -  TABLE OF CONTENTS Page ABSTRACT TABLE  i i  OF  CONTENTS  v  LIST  OF T A B L E S  v i i i  LIST  OF F I G U R E S  LIST  OF A B B R E V I A T I O N S  ix x i i  INTRODUCTION  1  LITERATURE 1.  2.  Structure  4.  2  and  Function  A.  Secretory  Products  B. C.  The S e c r e t o r y Secretagogues  Membrane  Regulation  C.  of  Ca  the  and  Exocrine  Cyclic  Nucleotides  MATERIALS I. II.  in 13 14 19 21 23  Turnover  24-  -ATPases  P l a s m a Membrane  24 Ca  24-  -ATPases  Role of Calmodulin Role of A c i d i c P h o s p h o l i p i d s , Long-Chain F a t t y A c i d s and C o n t r o l l e d P r o t e o l y s i s R e g u l a t i o n of the A c t i o n of C a l m o d u l i n and Other A c t i v a t o r s of Ca -ATPase A c t i v i t i e s  Objectives  of  AND M E T H O D S  the  2  3 7  Secretion  2 +  5.  Pancreas  2  Calcium C y c l i c GMP C y c l i c AMP Phosphatidylinositol  Plasma  A. B.  of  Pathway of Enzyme  The R o l e o f C a l c i u m Enzyme S e c r e t i o n A. B. C. D.  3.  REVIEW  Present  Study  30 30 34 36 40  42  MATERIALS  42  METHODS  45  -  vi  -  Page 1.  Preparation  of  Dispersed  2.  D e t e r m i n a t i o n of the Dispersed Pancreatic  Rat  Insulin Cells  3.  Cytochemical  Staining  4.  Measurement  of  Amylase  5.  Preparation  of  Pancreatic  6.  Treatment  7.  Measurement  of  of  Content  Pancreatic  Divalent  of  Cells..  Enzymatlcally  Cells  47  from Acinar  Cells  47  Plasma Membranes.....  EDTA  Cations  in  Membrane  9.  49  Determination Membrane Enzyme  of  Calmodulin  Content  of  Plasma  Preparations  49  Assays  50  a.  Measurement  of  Ca  -ATPase  b. c. d. e.  Measurement Measurement Measurement Measurement  of of of of  (Na + K )ATPase A c t i v i t y 5'-nucleotidase Activity Cytochrome C Oxidase A c t i v i t y Lactate Dehydrogenase A c t i v i t y +  Preparation  of  Phospholipid  11.  Measurement  of  Ca  2+  Activity  50  +  10.  Dispersion  -transport  Activities.....  Measurement of Calcium E f f l u x Membrane V e s i c l e P r e p a r a t i o n s  from  b.  Measurement of Ca -TJptake Vesicle Preparations  Plasma  2 +  Calmodulin  Gel  13.  Measurement  14.  Phosphorylation  of  Overlay  by  Plasma 53 Membrane  Technique  Endogenous  53  53  Acyl-Phosphoprotein of  50 50 51 52 52  a.  12.  15.  48 49  Preparations 8.  45  Acinar  Acinar  with  Acinar  46  Release  Membranes of  Pancreatic  54 Formation  Acinar  55  Membrane  Proteins  56  P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s and A u t o r a d i o g r a p h y of P h o s p h o r y l a t e d P l a s m a Membrane P r e p a r a t i o n s  56  a. b. c.  56 57 58  Gradient Gel Electrophoresis Acid-Polyacrylamide Gel Electrophoresis S t a i n i n g , D e s t a i n i n g and A u t o r a d i o g r a p h y . . . .  -  vii  -  Page 16.  Miscellaneous  Methods  59  a.  Protein  assay  59  b. c.  Statistical Analysis Determination of Free  Calcium  Concentrations..  RESULTS  59 59  60  1.  Determination of Plasma Membranes  Ca  2.  Determination of a ATPase A c t i v i t y to  2+  -ATPase  Activity  in  Pancreatic  60  2+  a. b.  Possible Relationship Amylase S e c r e t i o n  of  Ca  -  65  C h a r a c t e r i z a t i o n of Amylase S e c r e t i o n in Pancreatic Acinar Cells Determination of the E f f e c t s of Anticalmodulin  65  Agents  71  on A m y l a s e  Secretion  2+ 3. 4.  C h a r a c t e r i z a t i o n of Ca A c i n a r Plasma Membranes  -ATPase  activity  in  pancreatic 79  Regulation of Pancreatic Acinar Ca - A T P a s e by C a l m o d u l i n  Plasma  Regulation  Plasma  Membrane 91  +  5.  Ca  -ATPase  of by  Pancreatic  Acinar  Membrane  Phospholipids  117  2+ 6.  7.  D e t e r m i n a t i o n o f t h e P r e s e n c e o f a Ca -Transport P r o c e s s i n A c i n a r P l a s m a Membrane V e s i c l e Preparations  125  Determination of the O r i e n t a t i o n S i t e of the Ca -ATPase A c t i v i t y Membranes of A c i n a r C e l l s  135  2 +  of in  the the  Catalytic Plasma  DISCUSSION  143  CONCLUSIONS  157  BIBLIOGRAPHY  158  -  v i i i  -  LIST OF TABLES Table I  II  III  Page I n s u l i n content pancreatic cell acinar cells  V  VI  66  S p e c i f i c a c t i v i t y of C a - A T P a s e and marker enzymes i n c e l l homogenates and s u b c e l l u l a r fractions 2 +  K,. and V of diss max pancreatic acinar in  IV  of e n z y m a t i c a l l y dispersed homogenates and i s o l a t e d  the  presence  of  the  ATPase  plasma Ca  2  +  activity  membrane or  Mg  2  of  preparations 84  +  ATPase a c t i v i t y of p a n c r e a t i c a c i n a r plasma membrane p r e p a r a t i o n s i n t h e p r e s e n c e o f divalent cations  85  E f f e c t of monovalent c a t i o n s and d r u g s on the ATPase a c t i v i t y of p a n c r e a t i c acinar p l a s m a membrane p r e p a r a t i o n s  86  E f f e c t o f EGTA a n d EDTA o n c a l m o d u l i n s t i m u l a t e d Ca - A T P a s e a c t i v i t y  108  M g - A T P a s e a c t i v i t y o f EDTA t r e a t e d p l a s m a membrane p r e p a r a t i o n s of p a n c r e a t i c acinar cells  112  ATPase and l a c t a t e d e h y d r o g e n a s e activities of intact pancreatic acinar c e l l s and homogenates  140  The o r i e n t a t i o n p l a s m a membrane  142  +  VII  VIII  IX  80  2 +  of C a - A T P a s e a c t i v i t y vesicle preparations 2 +  in  -  ix  -  L I S T OP FIGURES Figure 1  2  3  4  5  6  7  8a  8b  9  Page R e a c t i o n sequence of (Ca + Mg " ) - A T P a s e p r o p o s e d by M u a l l e m and K a r l i s h (1980, 1981)  27  Ca or M g a c t i v a t i o n of ATPase activity i n p a n c r e a t i c p l a s m a membrane p r e p a r a t i o n s . . . .  61  E f f e c t o f ATP o n A T P a s e a c t i v i t y p l a s m a membrane p r e p a r a t i o n s  63  r  2  +  2 +  Light micrograph acinar cells Time course dissociated  of  isolated  in  pancreatic  pancreatic 67  o f a m y l a s e s e c r e t i o n by rat pancreatic acinar c e l l s .  69  C o n c e n t r a t i o n dependence of amylase secretion by d i s s o c i a t e d r a t p a n c r e a t i c a c i n a r c e l l s . . . .  72  E f f e c t o f t r i f l u o p e r a z i n e on a m y l a s e by d i s s o c i a t e d r a t p a n c r e a t i c a c i n a r  75  secretion cells....  E f f e c t o f c h l o r p r o m a z i n e on a m y l a s e secretion by d i s s o c i a t e d p a n c r e a t i c a c i n a r c e l l s  77  E f f e c t o f p r o p r a n o l o l on amylase s e c r e t i o n dissociated pancreatic acinar cells  77  The e f f e c t o f M g and C a i n plasma-membrane e n r i c h e d pancreatic acinar cells 2 +  2  +  on ATPase fractions  by  activity of 82  2+ 10  S u b s t r a t e r e q u i r e m e n t o f Ca -ATPase activity i n p l a s m a membrane p r e p a r a t i o n s o f a c i n a r cells  87  2+ 11  S u b s t r a t e r e q u i r e m e n t o f Mg ^ - A T P a s e activity i n p l a s m a membrane p r e p a r a t i o n s o f a c i n a r cells  89  12  E f f e c t of a d e n y l 3 , 5 ' - a d e n o s i n e (Ap5A) C a - A T P a s e a c t i v i t y i n p l a s m a membrane preparations of acinar c e l l s  on 92  E f f e c t o f pH on ATP h y d r o l y t i c a c t i v i t y p l a s m a membranes o f a c i n a r c e l l s  In  1  2 +  13  94  -  x  -  Figure 14  Page Arrhenius function  15  plots of  ATPase  activity  as  of  Ca  +  of  -ATPase  calmodulin activity  E f f e c t of p h e n o t h i a z i n e s on c a l m o d u l i n s t i m u l a t i o n of Ca -ATPase a c t i v i t y i n membranes of a c i n a r c e l l s 2 +  17  a 96  Concentration-dependence stimulation  16  of  temperature  99  plasma 101  E f f e c t o f Compound 4 8 / 8 0 on c a l m o d u l i n s t i m u l a t i o n o f Ca - A T P a s e a c t i v i t y i n p l a s m a membranes of a c i n a r c e l l s +  103  2+ 18  19  20  The e f f e c t o f e x o g e n o u s c a l m o d u l i n on Ca A T P a s e a c t i v i t y i n p l a s m a membrane e n r i c h e d preparations of pancreatic acinar c e l l s  106  (Ca + M g ) - A T P a s e a c t i v i t y i n EDTA p l a s m a membranes of a c i n a r c e l l s . . . . .  109  2 +  The e f f e c t o f c a l m o d u l i n calcium a c t i v a t i o n curve plasma  21  22  2 +  membranes  of  and M g o f EDTA  acinar  2  +  treated  on the treated  cells  113  C a l m o d u l i n g e l o v e r l a y and a u t o r a d i o g r a p h y of t a r g e t p r o t e i n s i n plasma membranes o f acinar cells Asolectin i n plasma acinar  115  s t i m u l a t i o n of Ca -ATPase activity membrane e n r i c h e d f r a c t i o n s of 2 +  cells  118  2+ 23  24  25  T h e e f f e c t o f e x o g e n o u s p h o s p h o l i p i d s o n Ca A T P a s e a c t i v i t y i n p l a s m a membrane e n r i c h e d f r a c t i o n s of a c i n a r c e l l s  120  SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s a n d autoradiography of a c y l phosphate intermediates of pancreatic acinar plasma membranes  123  SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and autoradiography of phosphorylated pancreatic acinar plasma membranes....  126  -  xi  -  Figure  Page  k5 26  27  28  29  30  Ca f l u x e s i n p r e l o a d e d p l a s m a membrane v e s i c l e s prepared from p a n c r e a t i c acinar cells  129  Ca c o n t e n t o f p r e l o a d e d p l a s m a membrane v e s i c l e s prepared from pancreatic acinar cells  131  2+ Time dependent Ca -uptake in pancreatic a c i n a r p l a s m a membrane v e s i c l e s  133  2+ A T P - d e p e n d e n t Ca -uptake as a f u n c t i o n o f Ca -concentration in pancreatic acinar membrane v e s i c l e s  136  ATPase a c t i v i t y of i n t a c t c e l l s and homogenates  138  pancreatic  acinar  -  xii  -  L I S T OF ABBREVIATIONS ADP  adenosine  5'-diphosphate  ATP  adenosine  5'-triphosphate  Ap5A  adenyl  GTP  guanosine  triphosphate  inorganic  phosphate  i  P  3',5'-adenosine  cyclic  AMP  cyclic  adenosine  5'-monophosphate  cyclic  GMP  cyclic  guanosine  5'-monophosphate  E  enzyme  EP  phosphorylated  ATPase  adenosine  (Ca  2 +  EDTA EGTA  +  Mg  2 +  enzyme  intermediate  triphosphatase  2+  )-ATPase Mg  dependent  calcium  ethylenediamine  stimulated  tetraacetate,  disodium  ethyleneglycol-bis-(p-aminoethyl N,N'-tetraacetic  SEM Tris  error  trichloroacetic  SDS PAGE  sodium  dodecyl  polyacrylamide  TFP  salt  ether)  acid  of  the  tris(Hydroxymethyl)  TCA  mean  aminomethane  acid sulfate gel  electrophoresis  nicotinamide  adenine  dinucleotide  dissociation  constant  trifluoperazine  CPZ  chlorpromazine  NADH  K  standard  ATPase  Ca + 2  of  the  (reduced)  enzyme  for  Ca  2+  -  x i i i  g  Gravitational  K,. diss  dissociation  Vmax  maximum  m  milli  u  micro  M  molar  M  -  Constant constant  velocity  molecular  weight  r mg  milligram  sec  second  min  minute  ml  m i l l i l i t e r  ul  microliter  mM  millimolar  concentration  uM  micromolar  concentration  nmoles  nanomoles  v/v  unit  of  volume  per  unit  of  volume  w/v  unit  of  weight  per  unit  of  volume  -  xiv  -  ACKNOWLEDGEMENTS  I advice,  wish  to express  encouragement I  advisory  would  like  and help  to extend  committee,  Dr. J . McNeill  criticism  and  Dr.  I  wish  Foundation I graduate  helpful  to g r a t e f u l l y  and L a b o r a t o r y  thanks  of  for his  this  t o members  D r . J . Diamond,  study.  o f my  D r . C.  for their  wish  constructive  a l l members  i n the faculty  Mahey  for their  the Canadian  of  this  and  study.  Cystic  the Faculty,  of Pharmaceutical  Fibrosis  Staff  Sciences  and  for  making  experience.  to express  colleagues,  during  Dr. A. Molla  assistance.  to thank  I wish  D r . D. J e f f e r y ,  contributions  acknowledge  a t UBC a n e n j o y a b l e  Finally  and R a j e s h  t o D r . M. B r i d g e s ,  for their  students  my p r o g r a m  my s p e c i a l  the course  and D r . B.D. R o u f o g a l i s  for financial  also  throughout  t o D r . S. K a t z  suggestions.  am g r a t e f u l  W. O v a l l e  gratitude  Dr. D.A. A p p l e g a r t h ,  Mcintosh,  I  my p r o f o u n d  my s i n c e r e  M r s . Loan Hoang, patience  and  gratitude Bruce  t o a l l my  Allen,  assistance.  Barry  friends Eibschutz  - 1INTRODUCTION  Cystic  fibrosis  characterized affected  (CF) i s  by a u n i v e r s a l  individuals.  mechanisms  of  that  secretions  mucus  properties. to  chronic  They  pancreatic calcium  content  Johansen either the  et  al  result  of  parotid  their  and  lower  the  acinar  control  secretion pancreas  in is  little  events  from  higher  exocrine one o f  the glands  information  involved  in  is  organ  reported of  (1981)  have  with  CF a r e  for  most  which  fully  amylase, is  available proteins.  of and  et^ a l 1 9 6 8 ;  secretions  could  macromolecules the duct  that  However, water  from  and  sodium  affected  of  the  and  isolated  cells  calcium  suggestive  a defect  in  cellular  macromolecular  Although by the  regarding  or  systems.  than  understood.  profoundly  (di  the protein  larger  electrolyte,  presently of  of  in  rise  hepatic  t h e amount  of  recognized  giving  demonstrated  patients.  are not  been  passages,  exocrine  and s e c r e t i o n  of  pathogenetic  complications in  i s  physicochemical  (Hadorn  these  the secretion  abnormal  been  of potassium  tissue  has long  in  concentrations  responsible  It  which  glands  and t h e  as e l e v a t i o n s  patients  of  defect  modifications  and D o n n e l l y  physiology  mechanisms  have  the exocrine  decreases  The a b n o r m a l i t i e s  concentrations cell  as w e l l  compositional  cells  and have  Large  disease  insufficiency,  and other  the synthesis  Mangos  acinar  controls  to  pancreatic  1976).  the juice  have  and obstruct  obstruction  1968).  be r e l a t e d  Recently,  very  of  in  unknown.  f r o m CF p a t i e n t s  produced  metabolic  molecular  s t i l l  disease,  and Davis juice  is  precipitate  intestinal  Sant'Agnese  dysfunction  The b a s i c  the disease  pulmonary  cirrhosis,  an i n h e r i t e d  the  the  disease, molecular  -  2  -  LITERATURE REVIEW  1.  Structure and Function of the Exocrine Pancreas The  tissue,  pancreas in  comprises  addition  to  exocrine  secreting  fluid  containing  electrolytes.  which  enters  duodenum v i a  endocrine (Kempen  tissue,  referred  acinus.  77%  of  The The the  major  and  neutralizing  of  cells  gastric  for  cell  the the  mass  a variety  secrete acid  a  in  duct of  4%  around  the  lumen  of  rat  secretes  a  juice The  in  the  rat  somatostatin  and  what  et_ a l  consists  are  (Kempen  digestive  exocrine  1978).  gland  lumen  cells et^ a l  enzymes  is  1975).  of  called  acinar  bicarbonate-rich the  (Case  a narrow  pancreatic  also  (Kimmel  pancreas  The  pancreatic  the  glucagon,  acinar  in  form  polypeptide  grouped  of  these  only  exocrine  of  hydrolases,  pancreatic  insulin,  the  function  cells  the  pancreatic  cells  secrete  Together  tissue.  to  form  an  centro-acinar which  1977)  while  comprise is  to  the  fluid  important  in  which  separates  proteins  intestine.  Secretory Products Using the  two-dimensional  first  dimension  second  dimension  showed  that  of  acinar  terminal  centro-acinar  in  unit  pancreatic  synthesize  A.  as  endocrine  digestive  accounts  secretes to  functional  pyramidal-shaped  cells.  which  et^ al^ 1 9 7 7 )  now b e i n g The  the  and  the  approximately  gel  according  according acinar 20  electrophoresis,  cell  enzymes  to  to  their  of and  the  their  isoelectric  molecular exocrine  zymogens.  weight,  pancreas These  point  and  Scheele secretes  include  in  the  (1975) a  mixture  proteases  - 3 -  (trypsinogen  and  chymotrypsinogen),  Chymotrypsinogen, appeared  B.  in  proelastase,  multiple  amylase,  lipase  and  procarboxypeptidase  A,  ribonucleases. and  lipase  forms.  The Secretory Pathway Pancreatic  (aggregations (RER).  From  enzymes, of  ribosomes)  their  site  transitional  elements.  transitional  elements  that  carry  their  proteins  residence  in with  gradually  bud  concentration eventual  of  process  of  electron (1967a  the  granule  (Case  1978).  secretory  microscopic b;  1971a  and and  the  proteins  become  route was  and  1977).  the  of  complex.  transporting  concave  rounded  up,  with  and  exportable fuse  with lumen  time  course  of  mainly  guinea  of  pig  the  increases.  acinar  studies  side  Gradually,  vacuole"  The  their  the  the  vesicles  their  obtained  Using  that  during  granules  into  so-called  Golgi  granule.  packed  autoradiographic b;  fusion  nearest  these  exocytosis  the  polysomes  reticulum  transporting  to  "condensing  the  is  modifications  storage  contents  into  next  small  saccules  stimuli,  on  as  on  endoplasmic  move  occur  material,  zymogen  external  they to  synthesized  rough  Following  their  of  and  in  are  proteins  extensive  immature  evidence  the  RER  ejecting  Experimental transport  the  stacks,  proteins  appropriate  plasmalemma,  off  secretory  an  the  to  appears  secretory as  is  joined  complex.  Golgi  off  result  of  proteins,  synthesis,  undergo  with  all  What  Golgi  the  f i l l  eventually  Under  the  of  bud  load  secretory  vesicles  like  from  The  proteins. the by  apical the  intracellular the  Jamieson pancreatic  classic and  Palade  slices  -  and  an  In  vitro  pulse-labeled occupied  by  'pulse-chase'  with  [ H]-leucine  first  After  non-radioactive  amino  acids,  maximum  Golgi  After  37  of  complex.  observed  on  the  grains  'chase', acinar  side  were  most  of  of  17 m i n  min the  -  technique,  RER.  many  the  4  of  'chase' labeling  complex  condensing  the  were  observed  cell  in  association  with  time  course  of  movement  pulse-labeled  same  in  prior  unstimulated  three-hour  1971b).  Bleger  (6-24h)  of  the  considerable this  of  pancreatic  stimulation et  al  (1976)  in_ v i t r o  extent  rat  the  rate  of  in  was  or  in  with  movement  that  of  found  and  of  the  be  and the  received  a  Palade stimulation  accelerated  pulse-labeled  and of  route to  had  continuous  caerulein  min  portion  that  (Jamieson  were  57  The  of  the  membrane  After  were  mixture  grains  granules.  cell  with  apical  apical  slices  the  complete  the  the  proteins  of  associated  proteins  found  pancreas  a  area  vacuoles.  carbachol  however  with  zymogen  slices  with  the  facing  with  mature  in  that  autoradiographic  associated grains  determined  appeared  'chase'  Golgi  they  to  a  proteins  along  pathway. Cell  fractionation  microsomes, separated  zymogen  by  techniques,  granules  differential  homogenization,  confirmed  Palade  It  1967a).  pulse-labeled was  neither  the  appearance  protein studied.  in  also  proteins  sharp  the These  of  nor  and  rate the  sedimentation  the  rough  that  the  supernatants  kinetics  significant  postmicrosomal have  quantities supernatant given  rise  was  movement  compartment  fraction to  of  of  another  were  (Jamieson  additionally  (5-20%)  smooth  tissue  findings  one m e m b r a n e - e n c l o s e d This  microsomes,  following  autoradiographic  unambiguous.  findings  which  post-microsomal  determined from  in  to  and of another  complicated  pulse-labeled at  all  school  time of  points thought  by  - 5 -  which pass  suggests through  that  the  (Rothman  1980).  indicated  that  leakage and  and  cell a.  under  cytoplasmic Recent  the  ambiguity  adsorption  all  times,  b.  of  proceeding  from  discharge  Studies  is  zymogens as  a  of  group  the by  a  individually  by  discharge).  The  pathway:  into  been  a  single  series  the  to  individual  was  is  (1978)  secondary  tissue  have to  homogenization  that:  segregated  therefore  is  within  excluded  vectorial  portion  from  the  of  the  and  cell  irreversible to  the  apical  interconnectable  membrane-enclosed  requiring,  normal  position  conducted  to  secretory  each  bovine  secretory space  determine are  conditions,  proteins  (Jamieson  of  for  and  seems  localization  pancreatic  to  Palade  favour  monospecific  enzymes and  discharge)  pathways  studies  proteins  the  stored  (parallel  secretory  five  whether  synthesized,  pathway  evidence  of  under  1975).  pancreas  available  that  of  and P a l a d e  Immunocytochemical  toward  with  are  extracellular  a multiplicity  indicated  and  proteins  basilar  30 m i n  exocrine  (1977)  studies  view  proteins  time-dependent  Scheele  have  held  co-workers  transport  and  approximately  1971b;  the  through  transport  early  proteins  intracellular  and  associated  compartments  secretory  compartments; c.  Scheele  in  The w i d e l y  secretory  their  secretory  space;  transport  portion  seen  conditions  during  of  artifacts  membrane-bound cytosolic  space  findings  fractionation. at  physiological  of  (non a  secreted or  parallel  single  secretory  Kraehenbuhl  antibodies  (trypsinogen,  and  et  directed  al  -  chymotrypsinogen  A,  deoxyribonuclease) a l l  bovine  carboxypeptidase reacted  pancreatic  provide  quantitative  granule  in  proteins Palade  each  (1975)  followed  the  and  four  found  that  constant these  up  workers while  of  the  were of  1976;  was  existence  of  pig  indicated  nonparallel of  upon Steer  to  1977;  of  incubation  and  the  an a l t e r n a t i v e  have  in  secretory  and  were  the  in  was be  vivo  end  of  by  Parallel  other However,  (Steer  studies when  on  the  rat  (Rinderknecht  viewed  supporting  which  that  stimulated  Though  pathway  and  found  changed. as  of  the  medium.  studies  subjects  in  proportions  1978).  unchanged  1975)  discharged  confirmed  vitro  time  the  co-workers in  human  could  the  into  been  their  and  at  and W i l k i n g  Steer  proportion  secretion  varying  were  discharge,  1983)  and  measured  tissue  and  and  zymogens  the  and  caerulein  and P a l a d e  zymogens  basal  or  (Scheele  Rothman  1979)  Scheele  medium at  Furthermore,  not  secretory  chymotrypsinogen  discharged  and  lobules,  lipase  in  zymogen  five  carbachol  do  They  stimulation  (Beaudoin  patterns  in  those  (1975)  enzymes  that  the  each  the  amylase,  point.  found to  of  trypsinogen,  time  and Manabe  compared  1978),  1978)  each  identical  Steer  pancreatic  granules  findings  that of  and  zymogen  these  indicate  enzymes,  and  all  a mixture  pig  into  enzymes  zymogens  and  they  stimulation.  and P a l a d e  proportion  (Dagorn  at  enzymes  (Glazer  discharge  al  each  Scheele  Glazer the  after  and  three  of  Although  concentrations  and B  2h  enzymes  discharge  A  ribonuclease,  content  contains  zymogens,  to  proportion  experiment  of  -  A,  cells.  guinea  optimal  procarboxypeptidases intervals  cell  Studying  used  the  information,  discharge  ribonuclease  with  acinar  acinar  tested.  6  involves  these  the  the  et  -  movement  of  secretory  1980)  is  also  it  responsible discharge  C.  proteins  possible  primarily  (Scheele  that  for  7  -  through one  basal  the  cytoplasmic  population  discharge  of  and  space  acinar  another  (Rothman  cells  might  be  for  stimulated  from  pancreatic  1980).  Secretagogues of Enzyme Secretion The  acini  physiological  are  the  stimulants  neurotransmitter  cholecystokinin-pancreozymin development  of  acini  and  to  capable  be  recognized  a.  in  vitro  dispersed of  acinar  secretion  acetylcholine  (CCK).  cells,  (ACh)  With  recent  such  as  many  enzyme  the  progress  slices  different  pancreatic  and  of  oligopeptide in  the  pancreas,  agents  have  secretion.  intact  been  The  found  generally,  are:  Muscarinic Cholinergic Agents  Acetylcholine  and  other  mobilization  of  secretion  pancreatic  blocked  enzyme  preparations  increasing  groups  of  by  benzilate  in  calcium acinar  atropine  (Gardner  ([ H]QNB),  Larose  3  demonstrated pancreatic  cellular  muscarinic  the  presence  broken  agonist-induced  cell  enzyme  preparations  (Larose  studies  [ H]QNB  et  well  cells  1979). et  of  as  al  Using  (1979)  muscarinic  secretion 1979;  as  agents  stimulation  (Gardner  preparations.  al  cholinergic  1979).  Ng e t  However,  could  not  Ng ^ t  al  be  the  actions  (1979)  receptors  are  have in  corresponding  measured  1979).  enzyme  quinuclidinyl  al  cholinergic  of  These  tritiated  and  cause  in  Recently,  these binding  q  with  performed  on  rat  pancreatic  dispersed  acini  with  - 8 -  concomitant  secretory  receptor  is  coupled  receptor  population  of  65  pM a n d  b.  studies  to  have  enzyme  secretion  consisted  approximately  of  2600  high  isolated contain  and 33  naturally gastrin  purified  amino  and  enzyme  1976;  different  peptides  et  chemical  al  is  Australian to  a  hylid  frog  seven  C-terminal  octapeptide  resides  the  fragment  is  have  same  The  the most  acid  C-terminal  the  C-terminal  efficacy  potent  are  caerulea  its of  but  CCK8  sequence  eight  CCK.  portion  and  acinar  to  with  was  a  al^ 1 9 8 1 ) .  found The  similar £t  of  al  its  (Gregory  and  1968)  and  they  calcium  several  1964).  skin  of  with  the  activity  The  of  CCK  smallest  active peptides  All  the  active  (Jensen  and  Gardner  more  the  subsequently  acids  biologic  times  are  1976)  Tracy  the  molecule.  CCK  3',5'-cyclic  in  from  (CCK4).  to  other  (Deschodt-Lanckman  naturally  amino  to  two  cellular  in  (10  originally  and  cells  CCK  potencies  caerulein  sites  1968).  release  intrinsic the  The  guanosine  isolated  tetrapeptide varying  that  (Christophe  C-terminal  of  1981).  et  intestine  (Anastasi  The  muscarinic  Peptides  occurs  similar  originally  Hyla of  the  dispersed  is  (Larose  cellular  cause  al  structurally ACh  Gastrin  and  decapeptide  share  in  and  amino  are  increase and  et  and J o r p e s  CCK a n d  1978).  f orras  carboxyl(C)-terminal Caerulein  GMP)  fragments  May  that  Like  secretion,  (cyclic  pancreatic  found  sites  small  (Mutt  the  affinity  a peptide  hog upper  residues  caerulein.  monophosphate  al  from  acid  occurring  stimulate  ejt  low  Cholecystoklnln-Gastrin-Caerulein is  that  (Larose  and  binding  Cholecystokinin-pancreozymin  from  established  potent  1981).  than  -  native  CCK).  high-affinity 1980;  with  binding  sites  Sankaran  appears cell  Studies  that  with  et  al  there  a K^  of  may  various  physiological  1979;  be  about  derivatives  al  2  5  of  Philpott  I-labeled  From  CCK33  the  cyclic  CCK33  GMP  responses  acinar  9000  and  and  al  et^  al  (1980),  per  it  acinar  caerulein.  competitively  with  These  et  sites  nM f o r  stimulation  (Jensen  Jensen  binding  0.3  1979).  revealed  cells  of  selectively  to  Petersen  have  studies  approximately  2nM f o r  and  -  on p a n c r e a t i c  1980).  Butyryl  et  1  9  inhibit  CCK p e p t i d e s  cyclic  GMP  (Peikin  derivatives  125 also  selectively  CCK33  to  the  suggested rather CCK  experiments  CCK by that  al  this  from the  et  1980).  may  al^ 1 9 8 3 ) .  that  cyclic  as  well  as  the  GMP  (Gardner  lack  the  actions  as  an It  the  of  control the  antagonist has  been  structurally  and  been  CCK,  certain  Therefore  are  of  has  inhibition  of  1983).  specifically  it  GMP a n d  for  acts  that  I-labeled  cyclic  mechanism  cyclic  compounds  nucleotides,  binding  the  of  Recently,  inconclusive.  receptor  benzotript,  binding  dibutyryl  However,  dibutyryl  the  al  be  interpretation  with  and  the  of  shown  very  competitively CCK  peptides  (Hahne  1981).  c.  Bombesln-llke Peptides  Another pancreatic comprises like  et  block  between  blockade,  remains  proglumide  (Jensen  interaction  interacting  antagonize  cells  (Miller make  view  different  et  the  competitively  receptor  activity  current  acinar  that  than  and  group enzyme  of  secretion  bombesin,  caerulein,  naturally  were  occurring  by  alytesin,  causing  release  ranatensin  originally  peptides  isolated  and from  of  that  cellular  litorin. the  can  skins  stimulate calcium  These of  peptides,  various  frogs  -  and  were  named a f t e r  isolated  (Erspamer  and M e l c h i o r r i activity the  of  bombesin  has  1973;  Erspamer  1978).  Since  and  same  tetradeca-peptide  activated  pancreatic  Deschodt-Lanckman  workers  direct  1979;  activation  high  5000  bombesin  bombesin  actions  1978;  on  was  Petersen  1975;  the  1976;  Jensen  demonstrated  directly  on  the  Philpott  1979)  units.  acinar  bombesin in  4 nM  [  1  2  guinea per  separate  I] pig  have  (Deschodt-Lanckman 1979;  from  peptides.  evoked of  rat  1978;  Philpott  presented and  with  clear  Other and and  with  have  acini.  a  pancreas  bombesin  studies  pancreatic  gut  only  cells.  CCK  Brown  the  acinar  Binding  cell  of  and  bombesin  Tyr^-bombesin  acinar  (Jensen  cells  5  CCK  bombesin  and P e t e r s e n  a  revealed There  are  dissociation  et_ a l ^ 1 9 7 8 ) .  While  been  in  the  mouse,  Iwatsuki  and  Petersen  et_ a l  observed  1976;  Jensen  et  al  of  Melchiorri  Rivier  fragments  have  and  CCK-gastrin  Iwatsuki  same  and  that  that  isolated  a property  Erspamer  via  biological  nonapeptide  et_ a l ^ 1 9 7 8 ;  thought  Erspamer  intrinsic is  were  native  gastrin  of  Philopott  the  they  1973;  peptides  as  al  coexistence  on a c i n a r  and  et  in  from which  C-terminal  indirectly  acted  and  CCK,  the  release  changes  sites  of  to  frog  related  originally  receptors  bombesin  pig  of  with  efficacy  cells  1978;  the  binding  for  guinea  it  showing  constant  and  and  able  bombesin  analogue  affinity  about  is  Petersen  sites  radiolabeled  potency  acinar  et_ a l  evidence  case  of  and M e l c h i o r r i  molecule;  et^ a l ^ ( 1 9 7 6 )  that  (Jensen  Petersen  the  calcium-flux  suggested  the  and M e l c h i o r r i  et^ a l ^ 1 9 7 6 )  and  Erspamer  structurally  of  bombesin  and  in  -  class  (Deschodt-Lanckman  (Konturek  secretion  1972;  As  portion  the  particular  et_ a l  1975).  C-terminal  bombesin  the  10  1978)  it  direct  appears  rat  that  -  dog  acinar  d.  cells  do n o t  respond  to  11  bombesin  work  peptides.  In  has  the  been  done  on  the  guinea  pig,  high  pancreatic  affinity  demonstrated  using  [  I]  physalaemin  appears  there  may  be  about  that  dissociation  constant  substance  P  like  CCK a n d  ACh,  (Jensen  cyclic  GMP,  by  other  e.  but  of  and  the  of  acid  Gardner  1981).  in  evoking  effects  1980).  and  sites  These  per  (Jensen  release smaller  been  with for  appear  to  and  an  than  It a  5 nM  and G a r d n e r  these  1981).  cell  and  of  have  Gardner  peptides  calcium much  actions  sites  physalaemin  are  secretagogues  that  increase  molecule,  the  et  al  Robberecht  et  al_ 1 9 7 6 ;  possess  secretin  two and  1976).  of  the  Binding Gardner  classes each  in  biological  portion  of  causing  and  release  activities  Robberecht  that  (Schulz  calcium  intrinsic  N-terminal  Intestinal  activities  sequences  biological  and  binding  2 riM f o r  and V a s o a c t i v e  intrinsic  cells  500  acinar  binding  (Jensen  about  maximal  biological  amino  peptides  the  et^ a l  act  increase  those  in  evoked  1981).  Secretin and Vasoactive I n t e s t i n a l Polypeptide  spectrum  in  of  bombesin  types  Secretin  their  (Bommelaer  Substance P - l l k e Peptides  Little  the  -  et  the  reflects Stolze from  studies al  of  cells  have  each in  of  a  similar  secretin et et  to  and  their  adenosine  have  of  and al al  the  the VIP  resides  1976; 1976;  demonstrated which  in  contrast  portion  (Christophe  increase  In  (Christophe  1979)  have  similarities  1980).  C-terminal  molecule  (VIP)  the  acinar  activity  receptors, an  Polypeptide  that  interact  acinar  with  3',5'-cyclic  VIP  -  monophosphate (K,j= 0 . 7 (K^ a  nM)  = 7 nM)  (cyclic which  and  relatively  135,000  high  has  interaction  with  cyclic  AMP,  it  of  the  VIP-preferring  be  noted  of  VIP  to  guinea  mouse the  and  The  but  mediate  may  in the  of  possess  receptor  receptors  in  that  cyclic  dog, is  VIP cat that  AMP  but  stimulation  may  of  lack  enzyme  cell  and  1979).  cause  an  increase  with  evoked  acinar  by  regard  to  the  from  must  ability in  et_ a l  dog,  that  VIP-preferring One  It  secretion  receptors  secretion.  in  occupation  (Robberecht cells  about  Although  secretion.  enzyme  mouse  the  are  acinar  enzyme  secretin-preferring  with  al  evokes or  nM)  et  AMP  mediates  (K^ = 0 . 3  per  can  receptor  secretin  There  (Gardner  specificity  VIP  hormone  = 80 n M ) .  d  release. in  affinity  the  receptors  species  not  high  for  VIP  explanation  cyclic  (K  increase  enzyme  rat,  pancreas  a  a  receptors  receptors  is  possible  increase  that  there  affinity  the  -  secretin  VIP  classes  only  stimulate  pig  1977).  is  for  secretin  both  is  affinity  affinity  high  affinity  There  low  affinity  9000  that  a  a high  low  approximately  AMP).  12  cat  and  mediate  receptors  finding  in  12 5 support  of  partially class  of  affinity  this  purified  plasma  is  sites  for  (Milutinovic  VIP  and VIP  pancreatic  concentration  (Case  available  with  ion-fluid  transport  mechanisms  that  had  control  duct  cells et  regard  al to  by  the  et  al  studies from  a high  et the  al  cat  secretion  pancreas for  the  of  binding  showed  secretin  a  to  single  and  bicarbonate-rich  cellular  However,  1981).  I-secretin  a  low  1976).  mechanism by nor  of  affinity  increasing  1980).  machinery  (Scratcherd  that  membranes  binding  Secretin from  possibility  there which  precise  cyclic is  no  cyclic  ion  fluid  AMP information  AMP  membrane  turns  on  transport  the  -  f. In  acinar  cells  enzyme  adenylate  cyclase  as  Singh  K  of  d  and  1982).  as  estimated  from  a  to  1 nM  guinea  secretion  choleragenoid  function  a  -  Cholera Toxin  increases  1979;  13  "B  also  have  g.  Insulin  The  finding  to  of  of  its  ability  of  the  and  a  cyclic cholera  cholera  21,000  and Rottam  high-  pancreas,  antagonist.  approximately  (Gardner  rat  cellular  subunit" bind  competitive  and  virtue  increase  The  can  by  pig  The  AMP  cholera  to  toxin  also  and  for  Rottam  referred  receptor  pancreatic  receptors  activate  (Gardner  toxin,  toxin  and  acinar  thereby  cell  cholera  to  is  toxin  with  1979).  low-affinity  binding  sites  using  125 biologically regulate Studies  specific  Kanno  that  1974) and  either  insulin  and  Saito  may n o t  potentiate  2.  the  may  1976).  the  perfused  synthesis The  directly of  and  not  stimulate hormonal  insulin  pancreas  or  glucose  (Korc  oxidation  of  may  pancreatic  secretion  relationship is  that  exocrine  regulate  coupling  effects  in  suggests  intact,  amylase  stimulus-secretion insulin  I-insulin  functions  employing  indicated Sehlin  active  has  It  amylase  secretion  1978). have  (Danielsson  been  and  and Unger  binding  clear.  secretagogues  et^ a l  fragments  (Soling  insulin  directly  to  suggested  but  (Williams  1972;  that  rather et^ al^ 1 9 8 1 ) .  The Role of Calcium and C y c l i c Nucleotides i n Enzyme Secretion Pancreatic  primary  acinar  function  of  cells  these  are  connected  "fused"  plasma  by  tight  membranes  junctions. is  to  While  prevent  the  -  leakage also  pancreatic  block  means and  of  by  off  the  apical  which  the  hormones  functional  interaction  An  therefore  the  of  reach  the  the  membrane  intracellular  cells  their  exists,  intracellular  -  target  with  and  mediator  from  the  spaces,  they  circulation,  organs.  Thus  the  structural  secretagogue-receptor  enzyme  release  (second  at  the  messenger)  for  apical secretion  is  required.  Calcium A number  addition  of  of a  fragments for  or  amylase al  such  as  calcium  carried  pancreas, (Hokin  have  1966;  Robberecht  terminals  pituitary  (Douglas  and  in  secretion  in  the  (Williams  and  the  Chandler  50% d e p r e s s i o n  of  of  1972;  an  Argent  et  (Katz  Poisner  immediate  Williams  a  alone  or  with  1973).  which  blockade  and  of  bethanechol-stimulated  Benz  However  a  in  and  dependence  was  clearly  stimulated  could  Chandler  1971;  1967)  secretion  calcium  requirement  Christophe  al  in  pancreatic  calcium  and M i l e d i  1964)  extracellular  1975).  either  incubated  and  secretagogue-induced  pancreas  absence  using  emphasized  on n e r v e  of  1972;  out  studies  calcium  al  removal,  ethyleneglycol-bis(p-aminoethylether)-  et  established,  a  which  (EGTA)  perfused  extracellular  only  in  Heisler  to  posterior  acid  secretion  1972;  contrast  studies  chelator  -N,N'tetraacetic  et  basal  into  region  polarization  at  membrane.  A.  secretions  14  not  (1975)  secretion  be  shown  observed  from  mouse  2+ pancreatic medium found  in a  fragments the  absence  complete  loss  after  30-90  min  of  preincubation  of  calcium  chelator.  of  the  response  ACh  In in  in  contrast,  pigeon  a  Ca  -free  Hokin  (1966)  pancreatic  slices  on  -  "presoaked" incubated  in  for  a Ca^-free  saline  15  -  solution  40 m i n  in  a Krebs-Ringer  significance  of  these  containing  bicarbonate  EDTA  medium  and  further  without  calcium. The lack  of  immediate  insufficient  effects  calcium  of  extracellular  calcium  or  provoke  missing  response.  short  by  exposure  of  to  A  Ca  However,  in  normally  causing  burst  secretion.  of  during  the  a Ca  first  extracellular which  the  2 +  -free  acini medium  -free  minutes  of  first  release  10 m i n ,  was  lost  Understanding and/or  appears of  was  examined  containing  but in  acini  how e n z y m e  extracellular  in  calcium  might  mouse  be  pancreatic  was  e v e n when EGTA was  secretion,  may  at  later  be  the  case  medium  release  was  ability  suspended release required  similar  in  can the  of  Ca  be  in  from  for  not  a  to  short  independent dependent from  on  studies  in  isolated calcium  It  observed  in  the  affected  normal was  the  two m e d i a  caerulein  24-free  of  intracellular  only  is  to  and  in  that for  stimulate  medium.  controlled  use  prolonged  stimulation,  comes  1980).  and  response  times  containing  (Williams  the  release  but  from  present.  ACh  resulted  amylase  since  fragments  intervals  EGTA,  assess  responsible  half-hour  release  thereafter  the  that  on a m y l a s e  EGTA  amylase  decrease  in  that  this  caerulein  eventually  secretion  secretion  That  result  complete  containing  enzyme  might  to  amylase  solution  solution  sustained It  that at  difficult  hand,  superfused  showed  24-free  caerulein-stimulated the  on  other  might  damage  stimulation  calcium.  effect  pancreatic Ca  2 +  study  1976)  ACh  a  calcium  is  omission  On t h e  nonspecific  and Ueda  pulses  calcium  removal.  removal  (Petersen  of  observations  by  intracellular  radioisotopes  to  -  monitor  calcium  fluxes.  Initial  16  -  observations  were  controversial.  In  45 pancreatic after  fragments  addition  (1973)  using  1973;  the  effect  Chandler  Similarly, increased while  "lanthanum  Ca e f f l u x  any  Ca  Ca  Ca  efflux al  was  from  (Gardner  et  1975;  could  attributed  be  influx  other  (Case  1974;  tissue,  observed  Ca p r e l o a d e d  enzymatically  influx  could  method",  from  on  intact  4-5  and W i l l i a m s  using 4 5  superfused  secretagogues  45  increased detect  of  and  tissue  and  Clausen  et  et  was  and  observed  al  1976).  not  tracer  The  Grondin  able  Matthews  to et^ a_l  1976).  acinar  in  and  not  al  Schulz  influx  observed  1973;  dispersed  by Kondo  cells  Heisler  and were  Deschodt-Lanckman  observed  Christophe  by  Ca  investigators  dissociated  preloaded  while  cells,  an  (1976a  and  other  studies  diversity  of  b),  results  2+ at  be  steady-state  to  and  whether  therefore  or  whether  the  Ca  exchange  movement  of  4 5  Ca  was  measured  was  an  2+ indication ** Ca f l u x  of  net  Ca  flux.  measurements  5  at  From  the  data  steady-state  obtained  conditions  by  unidirectional  (Kondo  and  Schulz  2+ 1976a al  and  1975;  calcium  b)  as  well  Renckens fluxes  There  release  Ca  into  a  et^ a l  during  biphasic. of  2  +  is  remained  mediate  Various  the  measurements  1978;  to  Stolze  i n i t i a l  of  and  "Ca  be  determined  calcium  calcium  approaches  release  transport  have  stores  from where  been  net  Schulz  Ca " ' ' - e x t r u s i o n  Intracellular  different  secretagogue-evoked could  by  secretagogue-stimulated  an  from  compartment  What  as  flux"  1980),  enzyme from  followed  calcium  has  (Gardner  it  appears  secretion  the by  cell a  were  the  site(s)  of  and  the  biochemical  that  are  due  to  reuptake  been  e_t  phase  released.  the mechanism  that  processes.  used  to  determine  the  site(s)  of  the  •  -  secretagogue-evoked  calcium  17  release.  -  Subcellular  fractionation  carried  45 out  on  cells  calcium b),  was  or  tissues  released  (Dormer  of  by  mimicked  released  from  produced  by  sites  action  of  al  Ca  1981).  inside  the  c a l c i u m was  lanthanum  In enzyme (Schulz  free  was  spite  of not  of  secretion 1980;  transport  a  from be  found  ruled  the  b)  that  using  the  an  a  cholinergic the  controls  a  was  requiring with  stimulation  The  to  not  that though  interfere  calcium  1 mM  but  possibility  membrane  trigger  of  (Wakasugi  membrane,  concluded  ATP  specific  concentration  therefore  plasma  metabolic  calcium  agents  plasma  membrane.  the  that  organelle  at  to  fluorescent  the  Lanthanum to  were  mitochondrial  using  bound  on  calcium  on  and  membrane  bound  concluded by  1975a  pool  with  located  out.  triggered  Williams  and  that  reticulum,  plasma  approach,  plasma  that  accumulating is  or  et^ a l ^ ( 1 9 8 1 )  acting  signal  mechanism(s)  calcium  response  Wakasugi  could  generation  elsewhere  Another  employed.  in  the  membrane  authors  suggested  endoplasmic  also  (1978a  Ca  and M e l d o l e s i  secretagogues  These  L a n t h a n u m was  released  in  mitochondria  been  of  observed  of  with  (Clement  perhaps  was  effect  the  release  cells.  and  It  signal.  has  loaded  consisting  and W i l l i a m s  the  either  been  Changes  mitochondrion.  abolished  the  1981).  Chandler  chlorotetracycline  that  complex  chlorotetracycline.  inhibitors  et  fraction  Golgi  and W i l l i a m s  monitored probe  the  have  from mitochondria  from a microsomal  elements  that  1980;  evidence by  is  increase  O'Doherty  required  concentration  an  suggesting  to  not  and  in  fully  worked  stimulation  cytosolic  Stark  critically  that  1982;  control out.  calcium  Dormer the  So  of  1983)  the  intracellular far,  -  electrophysiological in  the  ACh-evoked  sustained on  in  permeable 1982).  membrane  ACh-evoked  extracellular  increase  studies  to  At  sodium,  present,  identifying  active  membrane  (Petersen  intracellular  calcium  chloride  calcium  to  and  identify  depolarization  and Maruyama to  potassium  studies  transport  a  conductance  appears  and  biochemical  -  failed  potential  acinar  calcium  have  18  are  change is  up  being  although  the  ionic  and  pathways  Petersen  directed  in  dependent  Rather,  the  (Maruyama  component  acutely  1983).  open  processes  calcium  towards  various  cellular  24organelles. probably  An ATP-dependent  consisting  endoplasmic 1978; by  Ponnappa  et^ a l  associated  (Ponnappa et  reticulum  mitochondrial  was  al  1982)  were  or  Heisler  and  Senior  In  digitonin  both  reported  The  A  (Lucas  1976; 1980)  Ca  microsomes,  membranes jilt j a l  and  1975;  -transport  It  Lambert  and  using  almost  activity  has  the  activity  is  calcium  by  not in  Ca  to  with make  been  from whole  been  shown  was  Lucas not  saponin the  Ca  et  al  inhibited  other Le  that  Bel the  (Wakasugi  plasma  membrane  24-uptake  described  tissue  by  1978;  preparations  certain  the  has  Christophe  similar  equally  treated  non-mitochondrial  prepared  1977).  stimulated  obscure.  2 +  et^ a l ^ 1 9 8 3 )  and  24-ATPase  fractions  et^ a l  intracellular  plasma  Ca  cells  24-  ATPase  pancreatic  (Argent  active  acinar  mitochondrial  enriched  and  been  1981).  1981).  both  (Milutinovic  has  from  in  i n h i b i t o r s s u c h as a n t i m y c i n A, a z i d e or o l i g o m y c i n and 2+ 2+ a Mg - d e p e n d e n t , Ca - s t i m u l a t e d ATPase activity  identified.  membrane  vesicles  -transport  with  et^ a l  permeable,  of  Ca  in  systems plasma  horaogenates workers et  al  (Forget  1980;  ATPase  Martin  activity  is  24or  Mg  its  pancreatic  . role  Since in  acinar  the  the cell  cellular  regulation s t i l l  origin of  remains  of  -  Another  transport  mechanism  19  that  may p l a y  i of  intracellular  system as  a  uses  the  source  cardiac  of  calcium  the  Na  electrochemical  Na  energy  muscle  is  and  (DiPolo  has  and  -  role  in  the  regulation  24/Ca  countertransport  gradient  +  been w e l l  Beauge  a  1980;  across  the  characterized Mulins  system.  plasma  in  1979).  membrane  nerve  Using  This  axons  and  isolated  22 pancreatic (1979)  plasma  have  shown  membrane that  an  vesicles increase  loaded in  with  internal  Na, Ca  2  Schulz  and  increased  +  Heil  the  Na  24permeability  whereas  permeability. from  Petersen  is  to  to  be  studies  increase  the  an  important  that  membrane  external  one  decreased  system  major  sodium  Ca  since  effect  permeability  of  Na  it  is  known  cholinergic  (Maruyama  and  1982).  increase  secretagogues  cellular  Deschodt-Lanckman correlation  release  cyclic et  between  secretagogue  on  cellular broken  soluble  (Christophe  1)  May  et  cyclic  al  al  1978).  Calcium  forms  of  the  et  1980).  cyclic  can  findings  stimulate  (Christophe  enzyme  have  Calcium  et  been  and M g  2  Christophe  the  for  GMP b y  cellular  There  for  that  Three  of  1976;  curve  GMP a n d  1976).  cellular  release  et  dose-response  preparations  al  cause  (Albano  1976;  et^ a l  increase  that  GMP  cellular  calcium: cell  al the  (Christophe  secretagogues  and  appears  in  4-  C y c l i c GMP  Pancreatic  in  increase  electrophysiological  stimulation  B.  This  an  +  its  causing  1980).  can  et  al^ 1 9 7 6 ;  of on  a calcium  that  mobilization cyclase Both in  also  close  action  suggest  identified +  a  action  guanylate al  is  calcium  activity  particulate  the  substitute  of  pancreas  for  Mn  2  +  at  -  the  metal  activator  stimulated 1980). in  by  ii)  and  of  preparations  pancreas  1980).  reaching  The  continued  agonist  not  form. fatty  A23187  et^ a l  cause  increase  cyclic  levels  elevation  exposure  may  of  after the  result  particulate  form  acids  (Christophe  et^ al^  1976).  calcium  et^ a l  in  The  increases  (Christophe  maximum  transient  soluble  (Christophe  GMP d o  stimulant-evoked  transient,  the  ionophore,  cells  cyclic  of  on  -  polyunsaturated  calcium  acinar  derivatives  The  mono  The  dispersed  site  20  cellular and  efflux  i i i ) from  is  cyclic  GMP  Exogenous in  vitro  1976). GMP  1 to  2 min  cyclic from a  concentration  GMP  is  (Christophe  et  concentration  sequential  al  despite  stimulation  of  2+ guanylate  cyclase  and  phosphodiesterase.  a Ca  Indeed,  phosphodiesterase  have  Vandermeers  1977).  e_t a l  Vandermeers GMP a s  substrate  specifically  been  activity  of  by  and  The  GMP  175,000  than  role  of  cyclic  GMP a s  (1979)  reduction  in  that  a  medium  pancreas  that  (Terai  the  et  al  presence  1976;  of  cyclic  There  are  exhibit et  two  higher  al  forms  incubation  of  from  (P3)  is  the rat  affinity  for  1983).  mediator  secretion  enzyme  nucleotide  phosphodiesterases  containing  carbachol-stimulated  in  cyclic  these  potential  enzyme  of  phosphodiesterase  (Vandermeers  observed  calcium-free  of  nucleotide  AMP  and H a y m o v i t s in  one  nucleotide  forms  that  calmodulin.  cyclic  on p a n c r e a t i c  the  shown  116,000)  secretagogues  lobules  in  the  pancreas cyclic  found  have  cyclic  r  different  (1977)  stimulated  cyclic  three  et^ a l  calmodulin-dependent (M  -dependent  is of  of  s t i l l  the  unclear.  guinea  ImM E G T A secretion,  action  pig  resulted whereas  of Scheele  pancreatic in the  a  severe tissue  -  response  to  cyclic  depolarization  of  concentrations, without  the  in  elevation  Furthermore, compounds  GMP r e m a i n e d  the of  On t h e  hand,  of  a  GMP l e v e l s  slight  acinar  substantiated  C.  (Albano  1977).  others  increase  accompanied 1976;  calcium al  of et  al  However,  (Heisler  this  and  causing  12-0-tetradecanoyl with  There  no  are  Haymovits  1975;  elevation some  of  and  observation  Grondin  1980).  nitrosourea  derivatives  1976;  release  without  secretion  exogenous  that  KC1  enzyme  that  ester,  1979).  shown  Ueda  reports  cyclic  GMP  Scheele  could  not  et  al  secretin  or  of  on  1976;  be  1980).  C y c l i c AMP The  al  by  and J a m i e s o n  also  and H a y m o v i t s  dramatically  enzyme  was  caused  showed  a phorbol  effect  secretion  and J a c k s o n  levels  It  elevated  (Scheele  (1979)  marked  (Gunther  stimulatory  enzyme  Gardner  caused  by  atropine,  levels  and J a m i e s o n  other  intact.  membrane  of  GMP  GMP  phorbol-13-acetate, cyclic  cell  cyclic  -  largely  presence  cyclic  Gunther  elevated  secretion.  acinar  21  by  in  an  amylase  increase  Robberecht flux  1976).  or  et  al  cellular  Inhibitors  cyclic cyclic  well  on enzyme  secretion  1977)  both  membrane Svoboda  and  preparations et^ a l  1976;  secretin  Long  cyclic  Gardner GMP  et  nucleotide or  al  (Gardner  VIP  on  can  and  Gardner  levels  1979) et^ a l  with 1975;  cellular  acinar 1977).  et  cyclic al  no  cells  change  are  AMP  (Rutten  e_t in et_  known  levels  Gardner  cyclase  Furthermore,  is  Christophe  1975;  adenylate  VIP  (Christophe  phosphodiesterase  activate  pancreatic  by AMP  (Deschodt-Lanckman  peptides from  elicited  cellular  1976;  the  Jackson  of  in  augment as  action  of  secretion  to  as and  in  et^ a l  1972;  derivatives  of  -  cyclic  AMP  can  increase  (Haymovits  and  Scheele  enzyme of  secretion  cyclic  (Gardner The  finding  CCK a s  mediated  the  increase  has  of  that  same  to  CCK  activity  shown  broken Long of  the  cyclic  1975)  vitro  The  secretin  and  increase  in  derivatives  or  VIP  alone  1977)  may  in  intact  reflect  adenylate in  GTP  other and  1977;  analog  cells  was  shown  the  the  activate  al  that  1972;  cyclic  concentrations  amylase  acinar  et  cells  contamination  of  release (Long  the  the  observation  could  (Rutten  in  of  do  and  AMP CCK  not  Gardner  CCK w i t h  small  material.  cyclase  activation  systems  (Cassel  particularly stimulate Svoboda higher  activator  (Perkins  with  suggested  GMP a n d  AMP  concentrations  analogs  preparations  Gardner  AMP  together  cyclic  in  non-cholinergic,  plus  with  efflux,  et^ a l  recently  obtained  since  an u b i q u i t o u s  was  exogenous  VIP  However,  fluoride, animal  in  or  CCK.  [Gpp(NH)p],  GTP  pancreas 1977);  fragments  or V I P - l i k e  of  that  the  and J a c k s o n  increase  cell  and  cyclic  found  (DePont  of  in  secretin-  imidodiphosphate  It  could  C-terminal  findings  that  from  1977).  calcium  mechanism  been  effect  as  action  cellular  these  similar  the  1976;  increase  The  secretin  as  cyclase  et^ a l  amounts  by  that  well  Svoboda  1977),  Gardner  ( D e s c h o d t - L a n c k m a n jilt j i l  adenylate  that  is  1976;  -  secretion  produced  and J a c k s o n  pancreas that  AMP  enzyme  22  of  its  in  and  than  of  It  guanylyl  The  cyclase  stimulatory  secretin,  membrane-bound  is  1978):  adenylate  1978).  that  pancreas  Selinger  analog  pancreatic  et^ a l  the  CCK8  adenylate  or  cyclases  1973). that  non-adrenergic  electrical nerves  in  stimulation isolated  of  guinea-pig  pancreatic  -  segments marked  evokes  and  (Pearson  complex  et  al  concentration caerulein.  (Pearson  D.  ACh  response  be  on as  both well  preceded  cyclic cyclic  mimicked  the as  of  is  by  accompanied  nucleotide AMP  and  VIP,  theophylline  nerve-stimulationincrease  in  and  cyclic  by  cancentrations  cyclic  exogenous  inhibitor  the  and  GMP  but  had  not  by  a  VIP-evoked  AMP  concentration  1981).  the  first  pancreatic  not  causes  Michell of  preceed  calcium  agents  and  response) It  has  especially  CCK does been  in  Thus,  in  not  affect  recognized  of  1972;  PI  cellular  change  The  PI  diphosphoinositide  some (DPI)  the  which PI  In  of  time and  since  calcium  seems  that  is  in  in A23187  the and  Michell  may m e d i a t e thought  correlate  pancreas,  (Michell  and  ionophore  (Jones  to  in  confirmed  where  calcium  presence  1956)  et^ a l _ 1 9 8 0 )  secretin PI  the  been  turnover  the  in  (1955,  conditions  response  while  degradation  has  functions  in  process.  turnover  for  in  (PI)  Hokin  Calderon  under  tissues  the  secretory PI  and  Furthermore,  mobilization.  increase  Hokin  observation  other  agonists,  calcium-mediated  by  Cantraine  1975).  changes  various  This  degradation  1975).  actions  phosphatidylinositol  described  and  (Michell  accelerate  ionophore  of  slices.  (Bauduin  tissues  1975;  turnover  CCK was  pancreas  other does  increased or  pigeon  the  cellular  phophodiesterase  effect  which  pattern  could  -  Phoshatidyllnosltol Turnover The  of  in  The  changes  e£ al  secretion  changes  1981).  The  potentiating secretory  amylase  23  the  to with  cholinergic  (calcium-independent 1975).  phosphatidylinositols,  triphosphoinositide  (TPI)  have  a  -  high  affinity  possible that  for  calcium  activation  plasma  storage of  membranes of  calcium  (Hauser sites  muscarinic thus  directly  calcium  from  the  3.  Plasma Membrane Ca^-ATPases involved  including  (Porzig  terminals  (Katz  a  necessary The  1972)  and M i l e d i  calcium  10  There removing  Na the  /Ca  (Michell  transmitter of  in or,  cellular 1968),  of  membrane  from motor free  PI  1975).  of  (Douglas  nerve  cytosolic  Ca  is  regulation. cell  10  M in  to  the  much h i g h e r  10~ M)  in  3  cells  cytoplasm  is  typically  contrast the  extracellular  continuously  by  spaces.  passive  levels  Calcium  diffusion  of  calcium  therefore  down  its  gradient. are  various  calcium by  from  the  cellular the  cytosol:  reticulum.  system  and  the  apparent  that  Ca  Na /Ca +  responsible  endoplasmic  and  intracellular  It  are  calcium  low  mechanisms.  that  Intracellular  of  important now  mechanisms  mitochondria  maintenance exchange  is  release  calcium  the  into  sequestered long-term  metabolic  to  control  control  suggested  —7  (approximately  chemical  for  The  been of  a number  1973) ,  therefore  hydrolysis  fluid  of  are  has  in  and  penetrates  of  1967).  to  coupling  (Huxley  release  It  indirectly  regulation  and  concentration  —5 between  the  and  leads  extracellular  coupling  requirement  free  or  stimulus-secretion  excitation-contraction transport  in  1967)  membrane.  receptors  entry  functions  the  and  is  -  and Dawson  in  the  Calcium  24  2 +  free  -ATPase  exchange  in  is  calcium, the  not  can  be For  the  the  plasma  an  for  membrane  exclusive  are  -  feature axons in  of  and  excitable cardiac  nonexcitable  intestinal membranes 1979). cells and  muscle  cells  (Gmaj  such  et  as  cardiac have  axons  sarcolemma been  complimentary:  the  low  system  A Ca in  24-ATPase  erythrocyte  1969). wide  Since  variety  (DiPolo  1980) ,  liver  (Lotersztajn  Carafoli  1981),  (McDonald  et  and  1982)  Ca  2 +  Field  -ATPase  1982), and by  far  prototype  by w h i c h  therefore  be  suggest  that  the  (Ca  in  1981),  is  also  muscle  it  appears  a  low  present  1981).  ATPase,  Ca  24-transport  Ca  1966; 2 +  also  Heil  excitable  Beauge the  they  1979)  two  are  high-capacity a higher  was  affinity  has  macrophages  et_ a l  1981),  corpus  cells  (Gill  et  studied  such ATPases here.  2424-+Mg )-ATPase  al  but  luteum  al and  are  et  al  has  (Verma heart  Several  originally  The  been  judged.  and  lines  et  a  al  Stossel and  (Coroni  thyroid  human  regarded  Its  in  and  adipocytes  1982),  1983).  Vincenzi  described  (Lew  1981),  demonstrated  (Schneider  et_ al^ 1 9 8 1 ) ,  (Kotagal  (Shen  detail  et  and  been  granulocytes  (Lichtman  first  Schatzmann  -ATPase  include,  most  but  1982).  with  cells  in  and  affinity,  -pumping  and  Where  that  nerve  basolateral  (Schulz  (DiPolo  in  1982),  kidney  pancreas  only  1979),  Carafoli  1979),  islet  other  described  the  Ca  synaptosomes  bone  and  not  Mulins  Beauge  Lymphocytes brain  al  is  and  1980;  and C a r a f o l i  being  These  axons  1981),  cardiac  affinity  1979) ,  Penniston  -ATPase  (Schatzmann  a high  cells:  Beauge  exocrine  2 +  (Penniston  associated  then  demonstrated,  (Famulski  directly  the  membranes  of  Ca  (Caroni  and  been  and  and  exchanger  system,  -  and H i l d m a n  and  compared  transporting capacity  1979)  membrane  nerve  has  liver  (Murer  al  plasma  It  (DiPolo  like  epithelium  The  systems  cells.  25  (Kasai  erythrocyte as  the  properties of  described  will  evidence  by Dunham  and  -  Glynn  Ca  24-  (1961)  from  the  Recently,  enzyme  is  enzymatic  erythrocyte  this  and  has  cytoplasm  liposomes  mechanism by  of  its  Ca  (Carafoli  which  -  expression  the  Ca  al  from  -pump,  extruding  and V i n c e n z i  confirmed  24-transport et  energy  of  (Schatzmann  been u n e q u i v o c a l l y  demonstration  reconstituted The  the  26  by  1969).  purification  properties  of  the  in  1982). the  hydrolysis  of  ATP  is  not  is  utlized  24to  transport  Ca  understood.  against  In  elucidation  of  membranes.  The  intermediate  recent the  reaction  and  a  by  Garrahan  1975)  the  stimulation  Rega  2  +  2  in  Blostein  is  1975;  +  (Knauf  al  ATPase  Katz  a  dalton 1977)  and  Katz to  which  of  is  in  is  of  this  (Katz The  1975).  proposed  to  to  intermediate 1975;  and  et  Rega  is  and  dependence  Blostein  al  chemically  Blostein  the  of  1975;  phosphorylated  (Knauf  molecular  in  erythrocyte  sensitive  concentration  activity  which  b e e n made  -ATPase  and B l o s t e i n  the  protein  has  clearly  phosphorylated-protein  et^ a l ^ 1 9 7 8 ) .  phosphoenzyme  et^ a l _ 1 9 7 4 ;  Ca  Formation  1974;  Szasz  et^ a l  the  through  similar  gradient  progress  acyl-phosphate  the  150,000  Wolf  +  of  1975;  a  (Na +K )-ATPase  et  a manner  and G a r r a h a n  intermediate  proceeds  of  conditions.  (Knauf  +  considerable  reactions  labile  basic  enhanced  Ca  Ca  years  partial  [EP],  hydroxylamine  an e l e c t r o c h e m i c a l  weight  1974;  Katz  distinct  around  Whereas  the  and  from  100,000  the daltons  substrate  for  24the  Ca  1975;  pump w a s Schatzmann  proposed  that  at  Substrate  binding  E^.  K  The  d  for  initially 1977;  Richards  l o w ATP results the  et  al  the  formation  of  CaATP  formation E^  free  1978).  concentrations, in  be  (1-6  of uM)  ATP  (Rega  Graf  and  was  the  an is  and  Garrahan  Penniston  (1981)  substrate.  i n i t i a l similar  phosphoenzyme, to  the  K  d  -  for  ATP  hydrolysis  the  presence  of  Ca  (2-5 24-  uM)  (Richards  occurs  in Q  Garrahan is  1975).  accelerated  1978;  Enyedi  conversion Garrahan and  can  b y Mg  et  of  However,  al  E^P  and Rega undergo  the  a  rapid  al  dependent  The  second  1978).  et  absence  (Schatzmann  to  -  1978). of  Phosphorylation  a d d e d Mg  24-  (Rega  in  and  i  Ca  1980).  27  and  major  E P 2  hydrolysis  Biirgin  effect  state,  The  formation  E2P  1978;  of  Mg  (Rega  release  2  is  Pi  the  Rega  phosphoenzyme and  appears  +  and  intermediate to  of  to  Garrahan more  (Rega  Garrahan be  the  1975;  reactive  than  E^P  and  Garrahan  1978),  the  presence  of  24consistent  with  Mg  and B l o s t e i n  2  the be  +  (Katz  the  higher  turnover  1975;  sequence  is  the  regulated  by  calmodulin  of  Ca  Garrahan  conversion  of  E  (Muallem  Rega  to  2  ATPase  in  1978).  E^,  a  step  The  final  which  is  and K a r l i s h  1980).  The  membrane  thought  to  step  in  believed actual  24transport  of  Ca  across  conformational  changes  Biirgin  Figure Ca  1978).  2  +  in  the the  plasma  translocating  1 describes , ATP  the  full  is  protein  be  through  (Schatzmann  reaction  and  sequence.  Calmodulin  Figure  1.  Although ATP  in  the  R e a c t i o n sequence of fca + Muallem and K a r l i s h (1980,  there  2+  is  regulation  general of  the  agreement erythrocyte  on Ca  Mg )-ATPase 1981) 2 +  the  24-  role  of  -ATPase,  proposed  Ca  24, Mg  by  24-  considerable  and  to  -  discrepancy enzyme. both  exists  Analysis  a high  and  of  low  Rossi  1971;  these  activities and  the the  Ca  24-  1972).  was  however  as  activity The  1971;  -  to  activation  estimated  Rossi  (1973)  literature  affinity  Scharff  (Schatzmann Schatzmann  in  28  Ca to  2  +  be  Scharff  of  kinetic  the  (Horton  Ca  et  properties  24-ATPase  al  1970;  constant  4 uM a n d  uM,  100  Foder  that  the  the  revealed  (K )  and  for  d  respectively  1977). low  of  Schatzmann  dissociation  and  concluded  the  Scharff  affinity  (1972)  activity  and  was  24an a r t i f a c t use  of  due  chelating  preparation. et  al.  agents Ca  2 +  and  from  conversion  agents  such  who  used  Ca  hemolysing  2+  depend pump  Ca  EGTA a n d EDTA  in  at  the The  to  (Katz  the  variance  prepared  presence  of  the  in  in  calmodulin  presence  et^ al^ 1 9 7 9 ) .  or  affinity the  with  high  variability  on w h e t h e r  due  buffer  high  is  of  membranes  activities. may  as  conclusion  demonstrated  enzyme the  the  This  (1970),  -ATPase  the  to  and the is  results  absence low  of  Horton  chelating  affinities  associated of  of  affinity  apparent  absence  the  membrane  the  the  f o r m by  Ca  been  24-  of  or  dissociated  in  the  Also  it  has  observed  that  such  as  EGTA  and EDTA  in  -ATPase  (Sarkadi  et^ a l  the  24presence  of  Ca  medium w i l l Al-Jobore  chelating  T  affect  the  substances  kinetics  and R o u f o g a l i s  of  the  Ca  2 +  the  assay 1979;  1981a). 24-  Attempts have  yielded  to  determine  conflicting  the  results.  Schatzmann  (1973)  suggested  Roufogalis  (1975)  and  inhibition  of  Ca  inhibition  of  ATPase  24-  stoichiometry  a  transport  Schatzmann  calcium  Sarkadi  et by  activity.  of  al  to  Pi  (1977)  erythrocyte  and V i n c e n z i  ratio showed  l a n t h a n u m was They  the  of  one.  that  Ca  (1969) Quist  pump and and  maximum  a s s o c i a t e d w i t h o n l y 50% 34concluded that La -insensitive  -  ATPase  activity  subtracted  from  was  not  the  total  estimate  of  using  ion-selective  an  2 Ca  associated  ions  ATPase  pumped  29  -  with  Ca  activity  per  electrode  Pi  transport yielding  released.  method  for  resealed  red  a  and  be  stoichiometric  Larsen  rapid,  should  et  and  al  (1978a)  continuous  24assessment  of  Ca  efflux  outdated  blood  activity  and hence  from various  find  a  significant  reported  a  stoichiometry  the  clearly  pump  (Sarkadi  may  l i t t l e  properties  majority  can  the  point  vary  1980;  explain  of  of  information  other  high  membranes  plasma  1:1.Recently,  the  fact  on  that  the  et^ a l ^ 1 9 8 1 ;  differences  exists  from  corpus  1982)  do n o t  metal  ion  seem t o  chelator  rat al have  in  membrane  ATPases  ( L o t e r s z t a j n ^it  1981) a  Ca  reports  the  concentration  Akyempon  previously  and  of  Roufogalis  encountered  and  these  similar  but  require  was  (Verma  pancreatic  in  concerning  In 24-  contrast  Mg  ,  and  Penniston  islets  requirement.  trans-cyclohexane  membranes  literature  24-ATPases.  luteum  By  the  to  ATPases  use  the  of  the  1981),  rat  (Kotagal the  the  et of  al the  -1,2-diamine-N,N,N',N'-tetraacetic  a c i d (CDTA) i t was p o s s i b l e t o show an 2+ i n these systems, suggesting that from  the  that  Mg  the  to  of  depending  Larsen  affinity  liver  remove  c e l l "ghosts" prepared from 3424La - i n s e n s i t i v e Ca -ATPase  laboratories.  Very  plasma  of  used  1981) , w h i c h various  not  laboratories  stoichiometry effectors  did  from  apparent  requirement f o r added 24Mg was d i f f i c u l t to  endogenous  s t i l l  required  for  the  activity  of  enzyme. In  1981),  numerous  other  synaptosomes  systems  (Gill  et  such al  as  1981),  the  heart  squid  (Caroni  nerve  and  (DiPolo  Carafoli and  Beauge  -  1979) ,  adipocytes  (McDonald  et  al  30  -  1982)  and macrophages  (Lew  and  Stossel  2+ 1980)  the  presence  of  a  high  affinity  Ca  T  -ATPase  and  associated  2+ Ca  -transport  not  yet  4.  Regulation of Plasma Membrane Ca -ATPases  A.  Role of Calmodulin  been  have  been  observed.  demonstrated  In  the  (Lotersztajn  liver  such  a  correlation  has  et^ a l 1 9 8 1 ) .  2+  Bond present  and in  Clough  the  (1973)  hemolysates  first of  reported  that  a non-hemoglobin  human e r y t h r o c y t e s  increased  protein  the  2+ Ca  -ATPase  activity  purification Penniston the  by  1977,  purified  protein  a  1978)  brain  Calmodulin,  as  Ca  into  and V i n c e n z i  cellular et  al  (Hundain calmodulin Remtulla  it  was  are  has  (Whitfield  et  termed  the It  and  unique is  and  synaptosomes  1979)  and  into  and  cyclase  also  (Hinds  et  from  activator 1977). stimulated  al  1978;  Larsen  1978). of  in  Other  Jarrett  indistinguishable  and P e n n i s t o n  vesicles  insulin  be  adenylate  Green  Following  et^ a l 1 9 7 7 ;  et^ a l ^ ( 1 9 7 8 )  property,  transport  to  Jarrett  cell  involved  1979).  or  membranes.  (Luthra  shown  Cheung  red  induced  calcium  al  was  1977;  by  Maclntyre  Naftalin  1978)  factor  inside-out  calcium  and  this  laboratories  and V i n c e n z i  functions.  1979),  of  erythrocyte  phosphodiesterase  1979;  Calmodulin  isolated  number  (Gopinath  uptake  of  regulating  synaptic  release  and  cellular  fertilization  et of  variety  transmission intestinal  processes  sarcoplasmic  (Sobue  a  oocytes  regulated  cell by  (DeLorenzo secretion  reticulum  a l 1979),  of  (Katz  by and  multiplication  spermatozoa  -  (Jones  £t  al  number  of  enzymes  rase,  1978).  adenylate  kinase  and  Calmodulin  including  cyclase,  several  -  exerts  calcium  myosin  other  31  effects  transport  light  protein  its  chain  kinases  by  stimulating  ATPases,  kinase,  (Scharff  a  phosphodieste-  phosphorylase  B  1981).  2+ Calmodulin such  as  troponin  inducible group  of  C,  protein  (Stevens  cells  et^ a l  in  to  constitutes tissues,  is  2% o f  fractions  In al  the  (Smoake  et  lack  homologous  myosin  chains,  Calmodulin  Is  unique  species  tissue  tissues,  of  protein  distributed  or  al the  it  Calmodulin  and  the  in  most  in  brain  richest  eel  where  Siegel  soluble  binding  this  specificity  The  electric  D  among  highest  1977).  (Childers between  is  proteins  vitamin  of  Dedman e t  1974).  -binding  distributed  electroplax  al  light  Ca  ubiquitously  mammalian  total  is  is  1976;  the  calmodulin  it  shows  et  of  calsequestrin.  1976).  date  a group  that  and  (Watterson  reported  to  parvalbumin,  and  proteins  eucaryotic  testis  belongs  to  source calmodulin  1975).  and  and  In  all  particulate  particulate  2+ fractions  requires  (Vandermeers temperature  et_ a l and  Calmodulin weight 1980).  of  one-third  isolelectric tryptophan  is  the  as  characteristic  appears Binding  has is  stable  determined  amino  total  point  and  1978).  a heat  acidic  of  and  trypsin-sensitive  16,790,  The  Ca  acids,  amino  few the  tyrosine presence  is  occur  (Brostrom  from  its  amino and  residues, lacks  a  sites  reversible  acid  with  sequence  glutamic  acids  accounting  for  A  trimethyl  and  1981).  protein  cysteine,  residues. of  specific  and W o l f f  globular  aspartic  It  at  saturable,  acidic  acid  (3.5-4.3).  to  a  molecular  (Klee  et  constitute its  hydroxyproline  low and  distinguishing lysyl  residue  (Watterson  al  -  32  -  2i et  jil  such  1980). as  On t h e  troponin  internally  amino  1976),  sites  hand,  or  forming  et  like  and Hodges  sequences  acids  (Watterson  (Kretsinger binding  (Reid  homologous  appropriate structure  C  other  al  calmodulin  correspond  to  Ca  binding  calmodulin  domains,  each  of  Based  four  Ca  2  ions  +  domains.  It  four  which  contains  Ca  binding  2  +  on K r e t s i n g e r ' s  four  protein  exhibits  helix-loop-helix  binds  the  other  1980)  the  1980).  the  hypothesis  and has  the  the been  four  Ca  shown  2  +  that  of  24the  four  calcium fourth  Ca (K  site  1977).  A  divalent Direct mole 1 Mg 2  d  binding =0.2 has  of  uM)  cations  of  and  the  Ca  reported  are  regulation  systems  et  dependent  protein  al  K  of  for  Ca  (Katz  et  d  enzyme  2  (k  +  both C a  2  binding 4  d  affinity  = 140  and M g  +  conclusion  that  sites  on 24-  2  pM).  The  (Wolff  +  Mg  for  24-  et  and  by 4-  of  a_l  al  other  calmodulin.  m o l e s o f Mg o r Mn b i n d 94Ca ( W o l f f eit a l 1 9 7 7 ) .  2  per Also,  various  1979;  activity  Ca ^ - i n d e p e n d e n t  such  1982;  dephosphorylation The  that  Mg  high  B r o s t r o m ^t^ a l  has  generally  1977).  been  found  to  24-  (Greenlee  1980).  Ca  the 24-  exhibit  for  for  displaced  enzymes  although  in  the  indicated  21 require  for  sites  affinity  supports  apparent  calmodulin-dependent Calmodulin  low  data  studies  three  affinities  compete  calmodulin  increases  but  similar  variety  binding  sites,  (Yamaki  liver  mechanism  Bordetella  Kilhoffer  kinase by  as  of  calmodulin  et^ a l  pertusis 1983),  and H i d a k a  phosphoprotein  activation  in  effects  adenylate  pig 1980)  lung and  phosphatases  these  system  is  have  been  cyclase  cyclic  GMP  histone (Khandelwal not  fully  understood. Regulation  of  Ca  2+ -dependent  enzyme  (E)  by  calmodulin  (C)  is  et  ajL  - 33 -  believed  to  equations  proceed  by  (Brostrom  2 sequential,  and W o l f f  nCa  2 +  + C  fully  reversible  (Ca  s x\  2 +  )n  C  [(Ca^ )n  the  number  of  binding  an  activating  (1)  C]x  +  n equals  action  1981).  x  Where  mass  sites  E  (2)  active  which  must  be  occupied  by  2+ Ca  to  convert  equals  the  model,  Ca  calmodulin  to  unknown  number  of  first  interacts  (Ca  2 +  )n  form  calmodulin  for  the  enzyme  complexes.  In  and  x  this  2+ activating  the  inactivated 2).  enzyme  form  Saturation  change  of  involving  (Brostrom  of  (Eqn  the  with 1).  an  increase  and W o l f f  This  enzyme  calmodulin  1981).  calmodulin complex  forming with  in  Ca  the  Tanaka  to  2  an +  form a  then  associates  activated  results  a-helical  in  ternary a  (1980)  capable with  of  the  complex  (Eqn  conformational  content  and H i d a k a  complex  from have  28% t o shown  42%  that  the  2+ conformational high be  affinity  involved  change sites  in  produced  exposes  activating  in  calmodulin  hydrophobic  by  groups  binding which  calmodulin-dependent  in  enzymes.  of  Ca  turn  to  the  appear  Thus,  both  to the  24acidic  nature  properties enzymes. of t h e EP (Jeffery Even  membrane  of  calmodulin  seem t o  be  In the red complex of et  al  though  Ca  and  important  its in  Ca  -inducible  regulating  hydrophobic  calmodulin  dependent  blood c e l l , calmodulin s t i m u l a t e s the decomposition 2+ 24t h e Ca - A T P a s e , i n a m a n n e r s i m i l a r t o Mg  1981). calmodulin  24-ATPases  a  is  clear  known  to  regulate  demonstration  of  a  majority  this  effect  of  plasma  is  lacking  in  - 34 -  the  Ca  liver  -ATPases  (Lotersztajn  calmodulin Penniston  even 1981)  (Lotersztajn endogenous these  of  be  after or  luteum  washing  1981).  calmodulin  is  The  out.  of  enzyme  Penniston  did  membranes  the  such  the  Indeed  the  and  systems  possibility  bound  However  (Verma  These  of  purification  et_ a l  ruled  corpus  et^ a l ^ 1 9 8 1 ) .  treatments.  cannot  rat  not  with on  respond  EGTA  and  to  (Verma  rat  added  and  DEAE-cellulose  remains  though  that  it  could  not  existence  of  other  modes  Lotersztajn  1981)  be  that removed  of  et^ a l ^ ( 1 9 8 1 )  have  activated  an  by  regulation reported  that  24rat  liver  protein  plasma  membrane  activator  which  Ca  is  -ATPase  distinct  is  from  by  calmodulin  in  endogenous  a  number  of  24properties. by  both  contrast,  calmodulin  (Caroni  B.  In  et  al  and a  the  heart  cyclic  AMP-dependent  was  Roufogalis membrane  first  shown  (1981b)  by  and  by  that  detergents  phospholipids  Ca  -ATPase  is  phosphorylation  regulated process  1982).  Role of A c i d i c Phospholipids, Proteolysis It  sarcolemmal  by  Long-chain Fatty Acids and Controlled  Ronner ^t  erythrocyte could  be  al^ ( 1 9 7 7 ) Ca  -ATPase  activated  polyunsaturated  and  by  fatty  later  by A l - J o b o r e  dissolved  a variety  acids.  of  Niggli  from  and  the  acidic et^ al^ ( 1 9 7 9 )  24observed  that  erythrocyte  phosphatidylserine stimulated the an  by  purified eight-to  exhibited  calmodulin. enzyme  Ca  in  nine-fold  the  In  -ATPase high  purified  specific  contrast,  presence  stimulation  of  of  in  the  activity  presence  but  G i e t z e n ^it a l  could  (1980a)  phosphatidycholine  the  purified  enzyme  not  be  isolated  and by  of  reported  calmodulin.  -  It  soon  acids the it  became  could  mimic  purified is  apparent the  by  a  (phosphatidylserine, Unsaturated  effect.  On t h e  sphingomyelin Ca  2 +  -ATPase  significance much  lower  could  the of  Taverna limited  (DPI)  acidic  et  and  finding  a  proteolysis  Ca  of  2 +  and  linoleic  had It  -ATPase  on that  the  fact  no  membranes  (TPI)  a  on  are et  they  the  was  had  effect  phospholipids  al  phosphatidic  now b e e n  that  of  and  similar  (phosphatidylcholine,  has  Sarkadi £t  erythrocyte  done  indicated  acid)  (Carafoli  regulator  and  fatty  phospholipids  1981).  acidic  (1980)  studies  1981)  phospholipids  in  physiological  and Hanahan  est a l  triphosphoinositide  than  unsaturated  phosphatidylinositol  al  lies  and  Extensive  (Niggli  (oleic  reconstituted  the  as  of  neutral  (Niggli  concentration  function  -ATPase  acids  hand,  phospholipids  phosphatidylethanolamine)  activity  of  2 +  -  calmodulin.  cardiolipin,  fatty  diphosphoinositide activators  Ca  of  variety  other  and  acidic  effect  erythrocyte  stimulated  acid).  that  35  and  the  found also  al  that powerful  1982);  activate fatty  the  at  a  acids,  and  enzyme.  (1980)  observed  another  method  that of  24activation in  the  of  the  removal  Ca  from  ( S a r k a d i ^it  al  calmodulin,  Sarkadi  is  the  digestion  acidic  the  1980).  calmodulin  studies with c a p a c i t y was  -ATPase.  et  ATPase Since al  Maximum p r o t e o l y t i c of  a  phospholipids,  al  Mj. a b o u t  treatment  removed  (1980)  suggested  that  of  the  24p u r i f i e d Ca -ATPase l o s t upon removal of ^it  of  the  receptor  (Carafoli  fragment  1982).  fatty  acids,  enzyme  activation  the  molecule.  the  resulted  30,000  sensitivity  30,000 On t h e  M  r  to  fragment  contrary,  indicated that calmodulin binding a 1 0 , 0 0 0 Mj. p o l y p e p t i d e b y t r y p t i c The  and  possible  the  explanation  calmodulin-Ca  24-  is  that  complex  act  - 36 -  by  introducing  relatively result  in  into  hydrophobic a  Removing  enzyme  molecule  site.  Thus  C.  in  its  treatments  region  by the  a  case  of  active  such  that  treatment  (Tanaka Ca  et the  2-4-  the  enzyme  1982).  active  also  would  site  and H i d a k a  expose  be  will  becomes from the  required  1980)  a  This  polypeptide  would  Ca  of  al  inhibitory  calmodulin,  require  site  (Carafoli  hypothetical  proteolytic  not  the  change  hydrophobicity  would  of  environment  conformational  accessible.  increase  the  whereas  more  the  active to  other  2+  Regulation of the Action of Calmodulin and other A c t i v a t o r s of Ca -ATPase a c t i v i t i e s +  At  present,  control  calmodulin  inhibits has  relatively  Ca  mediated  -ATPase  2 +  generated  some  l i t t l e  known  functions.  activity  interest  is  in  due  rat to  Recent  the  activation  in  binding  calmodulin  to  cellular  in  the  literature.  calmodulin-binding binding the  name  to  protein  protein  calcineurin  from nerve 1978)  the  Wang a n d D e s a i  forms  tissue. of  erythrocytes, antagonized  was  to  CaM-BPs  have  from  to  the  (Sharma  been It  heat  was  activation  of  that  that  insulin  et  al  1982)  insulin  Evidence  may  for  direct  has  also  isolated  and  characterized  brain:  Klee labile  est a l  isolated  which  proteins  bovine  -dependent.  stable  respectively.  calmodulin  Ca  system.  (1977)  (CaM-BP)  refer  Heat  this  factors  (McDonald  possibility  calmodulin  various  the  reports  adipocytes  antagonize of  about  from  demonstrated erythrocyte  (1979)  CaM-BP and  bovine that Ca  2+  cited a  calmodulin  et^ a l  1978)  been  they heat  brain CaM-BP -ATPase  suggested isolated labile  (Au  and selectively and  -  Ca  -transport  function is  or  (Au  1978;  regulation  believed  that  Larsen £t  of  CaM-BPs  the may  37  al  levels  -  1978b). of  represent  Little  CaM-BPs a means  in of  is  known  cells.  about  the  However,  regulating  it  calmodulin  activity. The  demonstration  antipsychotic  drugs,  blocked  calmodulin  a major  advancement  systems.  Roufogalis  in  related  it  trifluoperazine affinity  (K  Ca  -ATPase et j i l of  (Levin  The  Ca  24-  -transport  1980b;  Raess  resulted  and Weiss  to  1979)  analysis  in  affinity  (K  in  antagonized  human  sites  and Weiss  two  types  = 1-1.5  d  1980;  calmodulin  revealed  affinity  also  and V i n c e n z i (Levin  high  low  activity  phenothiazines  and  phenothiazines,  uM)  1977)  is of and  are  24-  -independent,  Weiss  related  trifluoperazine  plot of  certain  calmodulin-regulated  24Ca  and  of  that  24-  sites,  that  phosphodiesterase  shown  Schatchard  = 5 uM).  (1977)  investigation  binding  binding  d  of  was  phenothiazines  and W e i s s  trifluoperazine  (Gietzen  The  calcium-dependent.  low  the  of  membranes 1981).  as  activation  activation  erythrocyte  Levin  such  Subsequently  calmodulin  and  by  1979).  A  chlorpromazine Since  the  activation  of  antagonize  the  agents,  in  contrast  similar (K  d  discovery enzyme action  sulfonamide  that  high of  Ca  sites  phenothiazines  local  affinity was  sites  found  (Levin  and  for  uM).  a variety  calmodulin.  antihypertensives, (W7),  130  systems, of  the  distribution  = 5 uM a n d  antidepressants,  alkaloids,  to  of  These  anesthetics,  inhibit  calmodulin-dependent  compounds agents  have  include  vincristine,  been  shown  to  antipsychotic rauwolfia  N-(6-aminohexyl)-5-chloro-l-naphthalene-  l-[bis(p-chlorophenyl)  methyl]-3-[2,4-dichloro-f?-  - 38 -  (2,4-dichlorobenzyloxyl) calmidazolium), (Gietzen  et_ a l  Triton  X-100 The  most  The  I50  for  inhibition  by  this  commonly  used  uM),  trifluoperazine  Levin and  drugs,  of  (Levin  10  =  and  the  that  are  among  the  or  48/80  to  date  is  activation  of  1981).  the  penfluridol  the  Of  (I50=  most  antianxiety  antidepressants, as  well  chlorpromazine  and Weiss  reported  uM ( B e l l e  uM),  (R24571  Compound  calmodulin  0.01  uM)  and  compound of  chloride  2.5  potent  1982).  noted  desipramine,  phenothiazines,  sulfoxide  (I50  (1976)  is  1982)  ( I 5 0 = 0.7  (Roufogalis  chlordiazepoxide, and  potent  compound  pimozide  drugs  and Weiss  protriptyline  (Roufogalis  .  phosphodiesterase  anticalmodulin  imidazolinium  1983)  calmidazolium.  and  phenethyl]  1979)  as  the  sulfoxide are  less  agents,  medazepam  amitryptyline, non-antipsychotic  and  analogs  trifluoperazine  potent  and  show  less  2+ Ca  -dependence  suggested  that  antipsychotic with of  this  there  is  a  activity.  of  Norman  of  calmodulin  correlation However,  agents  clinically,  yet  calmodulin-activated et  al  1979)  and  is  of  50-fold  has  a  It  are  the  more  similar  therefore  antagonism  not  potent or  class  than  lower  against  and  consistent  butyrophenone  phosphodiesterase  ineffective  was  calmodulin  findings  a member  is  it  action.  between  recent  Haloperidol,  antipsychotic  chlorpromazine  1979;  inhibitors  hypothesis:  clinical  antagonist  as  potency  (Levin  and  as  an  Weiss  calmodulin  O 1  activation Another  of  piece  erythrocyte  Ca  of  against  stereospecificity activity:  evidence of  Roufogalis  -ATPase  calmodulin (1981)  this  (Raess  and  Vincenzi  hypothesis  inhibition  demonstrated  in that  is  the  contrast all  of  lack  to a  1980a). of  antisychotic  series  of  - 39  -  c h l o r p r o m a z i n e a n a l o g s ; i n which the c h l o r i n e s u b s t i t u t i o n was from p o s i t i o n 1 to 4 on the A r i n g of the t r i c y c l i c n u c l e u s  varied  inhibited  24-  c a l m o d u l i n a c t i v a t i o n of e r y t h r o c y t e Ca  -ATPase a c t i v i t y , y e t o n l y the  compound w i t h c h l o r i n e s u b s t i t u t e d on p o s i t i o n 2 showed t r a n q u i l i z e r activity.  I n view of these d i s c r e p a n c i e s , the r e l a t i o n s h i p between the  antagonism  of c a l m o d u l i n and the c l i n i c a l e f f i c a c y of the a n t i p s y c h o t i c  agents seems u n l i k e l y . A c r i t i c a l study of the wide v a r i e t y of a n t i c a l m o d u l i n agents  by  Norman et a l (1979) r e v e a l e d t h a t h y d r o p h o b i c i t y i s a major f a c t o r c o n t r i b u t i n g to t h e i r a c t i o n . t h e s e workers  Indeed i n a d i v e r s e s e r i e s of d r u g s ,  found a l i n e a r r e l a t i o n s h i p between the l o g a r i t h m of the  potency of antagonism  of c a l m o d u l i n a c t i v a t i o n of p h o s p h o d i e s t e r a s e  the l o g o c t a n o l : water p a r t i t i o n c o e f f i c i e n t .  and  The p o t e n t c a l m o d u l i n  i n h i b i t o r s are a m p h i p a t h i c compounds t h a t c o n t a i n l a r g e r e g i o n s and c a r r y a p o s i t i v e charge a t n e u t r a l pH.  hydrophobic  The i n t e r a c t i o n s of  these drugs w i t h c a l m o d u l i n appear t o i n v o l v e two p r o c e s s e s :  There i s  the hydrophobic i n t e r a c t i o n between l i p o p h i l i c p o r t i o n s of the drug n o n - p o l a r r e g i o n s of c a l m o d u l i n and an e l e c t r o s t a t i c  and  interaction  o c c u r r i n g between a p o s i t i v e l y charged group on the drug and a n e g a t i v e l y charged a c i d i c r e s i d u e on c a l m o d u l i n (Weiss et^ a l ^ 1982). The  s p e c i f i c i t y of p h e n o t h i a z i n e s f o r i n h i b i t i o n of  c a l m o d u l i n - m e d i a t e d events has been a major concern of many investigators.  I t i s now known t h a t p h e n o t h i a z i n e s and o t h e r  a n t i c a l m o d u l i n agents a l s o e f f e c t i v e l y b l o c k the a c t i v a t i o n of p h o s p h o d i e s t e r a s e and e r y t h r o c y t e C a - A T P a s e by a c i d i c p h o s p h o l i p i d s 2+  -  and  unsaturated  been  shown  to  bind  Chlamydomonas 1981).  fatty  acids  to  some  flagella  blocking  activities,  activities studying  as  relationship  of  have  well  as  1982).  et^ a l  ^t  1982).  effects  al  proteins  isolated  and  rat  a-adrenergic  and  muscarinic  specific  of to  Phenothiazines  1980)  Thus  activation  these  -  "calmodulin-like"  also  (Roufogalis  calmodulin  (Gietzen  (Van E l d i k  Phenothiazines  40  dopamine  despite  various their  and  their  enzyme  mechanism  heart  (MacManus receptor  usefulness  of  from  serotonin  systems  as  in  action  have  blocking tools  vitro, in_ v i v o  in  the is  uncertain.  5.  Objectives of the Present Study Calcium  secretion  ions  from  play  the  enzyme  Williams  1980;  precise  rise  release  of  function  cell  plasma  has  been  free  shown  calcium  Carafoli  a  to  be  in  Stark  the  membrane  acinar Ca  responsible  concentrations  1981; McDonald  et  2+  cells  cell.  In and  a number associated of  and V i n c e n z i  that  (Schulz  critical  maintenance  enzyme a  concentration  1983).  is  of  proposed  calcium  calcium  (Schatzmann al  been  Dormer  -ATPase  for  has  stimulation  acinar  1982;  free  the  free  pancreatic  and  of  in It  cytosolic  cytosolic  physiological  role  pancreas.  from  O'Doherty  regulation  types,  important  exocrine  secretagogue-induced triggers  an  low  1980;  Therefore, for of Ca  the  the different  2+  -transport  intracellular  1969;  Caroni  and  1982).  2+ A  Ca  -ATPase  membrane-enriched (Milutinovic  et  al  activity fractions 1977).  has  been  described  prepared Various  from  in  plasma  pancreatic  investigators  tissue  (Forget  and  homogenates Heisler  -  1976;  Lambert  1980)  have  to  a  shown  similar  cellular since report  and  on  objectives  that  extent.  origin  the  Christophe  of  this It  the  1978;  enzyme  was  not  plasma  41  Le is  membrane  a multicellular  the  of  regulation  the  present  et  possible  is  of  Bel  of  study  al  stimulated  pancreas mode  -  by  though  fraction organ.  this  1980;  both  to in  Martin Ca  and  determine the  Senior Mg  the  exact  preparations  Moreover,  enzyme.  and  there  Therefore  is  used  no  the  are: 2+  1.  Determination activity  2.  in  and  characterization  pancreatic  Determination  of  investigating  the  the  acinar  cell  mechanism  effects  of  of  of  a  possible  plasma  -ATPase  membranes.  regulation  calmodulin,  Ca  and  of  this  enzyme  by  acidic  phospholipids. 243.  Characterization prepared  4.  24-  of  the  -transport to investigation  acinar  cells  Ca  acinar  possible  processes  in  vesicles  membranes.  relationship  of  Ca  24-ATPase  and  drugs)  on a m y l a s e  release  from  pancreatic  and,  identification the  -transport  a m y l a s e s e c r e t i o n by of the e f f e c t of phenothiazines  (anticalmodulin  b.  Ca  from pancreatic  Determination Ca a.  of  of  24-transport  the  orientation  process  in  the  of  the  intact  catalytic acinar  site  cell.  of  -  42  -  MATERIALS AND METHODS  I.  MATERIALS  The  chemicals  and/or  proteins  were  purchased  sources:  1.  Sigma  Chemical  Co.  Collagenase Carbamylcholine  chloride  (Carbachol)  inhibitor  Type  Pancreozymin Secretin Soybean  trypsin  Aprotinin Ascorbic  acid  EGTA EDTA Tris-ATP 2-Mercaptoethanol Cardiolipin Phosphatidylserine Phosphatidylinositol Phosphatidylcholine Oleic  acid  Compound  48/80  I-S  from  the  following  -  Tris  base  Tris  hydrochloride  Triton  43  -  X-100  Glucose Lithium  dodecyl  sulphate  Chlorpromazine Bovine  serum  Sodium  azide  albumin  Trichloroacetic Hydroxylamine Bromphenol  2.  acid monohydrochloride  blue  Amersham [Y 1 + 5  3 2  P]-ATP  (Specific  CaCl (Specific 2  3.  activity  activity  = 5-10  = 1.5  x  Calbiochem Calmodulin Adenyl  4.  Smith,  3 ,5'-adenosine 1  Kline  and  Trifluoperazine  5.  Grand  Island  blue  (a  gift)  Biological  Minimum E a g l e s Trypan  French  Co.  medium amino  acids  10  x 6  10  6  DPM/p.mole)  CPM/uraole)  -  6.  BDH  -  Biochemicals  Sodium  dodecylsulphate  Calcium  7.  44  chloride  Bio-Rad Acrylamide N,N'-methylene-bi Coomassie Ammonium  8.  Fisher  Brilliant  Blue  Scientific  Co.  Sodium  chloride  Sodium  phosphate  monobasic  Sodium  phosphate  dibasic  Glacial  MCB  (Na^PO^)  (Na2HP0i ) t  chloride  Hydrochloric  acid  acetic  (HC1)  acid  Manufacturing  Asolectin  10.  R-250  persulphate  Lanthanum  9.  sacrylamide  Boehringer-Mannheim Hyaluronidase  (CH C00H) 3  II.  1.  METHODS  Preparation of Dispersed Rat Pancreatic Acinar C e l l s The  as  cells  modified  killed cells  were  by  Chauvelot  by  a blow  were  done  equilibrated soybean  with  First  mesentery  to  and  the  using  trypsin  (a)  isolated ejt  neck.  and  2  inhibitor  and  of  tissue  1.5  1.2  by  means  of  distended  gland  and  and  the  of  Kondo  Pancreas steps  in  5% C 0  the  containing  2  and  was the  Schultz  obtained  from  isolation  (KRB)  (1976a)  of  rats,  the  solution  15 mM g l u c o s e  and  0.36  mg  ml. pancreas  was  enzyme m i x t u r e  trimmed  (0.9  mg h y a l u r o n i d a s e / m l  in  free  mg o f KRB  of  fat  and  crude  solution  containing  2+ and  siliconized  All  per  the  2+ mM C a  method  a Krebs-Ringer-bicarbonate  95% 0  10 m l  the  al_ ( 1 9 7 9 ) .  digestion:  collagenase/ml  0.1  by  100 m l  incubated  at  mM M g a  1 ml  ) was syringe  excess  for  with  enzyme  Erlenmeyer  37°C  inoculated a 25  solution  flask,  15 m i n .  into  the  gauge was  5/8  needle.  transferred  equilibrated  with  interstitium  agitation  with at  to  95% 0  2  of  the  5%  C0  The a and  108  oscillations/min. (b) was  Removal  decanted  20 m l  of  Divalent  KRB  and  and  replacement  of  the  tissue  incubated  solution  cations  were  then  without  Ca  2  +  subsequently  divalent  and M g added  2 +  cations:  twice and  back  for  the  supernatant  5 min.  containing  by  briefly  at  37°C  in  2 mM E D T A .  washing  the  2+ tissue mM M g  twice 2 +  with  20 m l  of  KRB  solution  containing  0.1  mM C a  and  1.2  .  2+ (c)  Second  digestion:  fresh  collagenase  (10  ml w i t h  0.1  mM C a  2  -  and  1.2  mM M g  incubated (d)  )  for  25 -  Final  dissociated  by  pipettes  with  isolated  cells  Cole-Parmer) with  albumin.  The  were  concentration 30 m i n .  at  dissociation sequential tips  cut  were  and  solution  at  0.1  mM C a  tubes  resuspended  2  were  in  two  (two  times  1.2  of  was  added  and  the  cells  at  2  +  nylon  over  x  1  filter  and  um,  columns  of  4% b o v i n e  KRB  serum  6 min.  The  counted  using  and  pellet  cells a  in  KRB  glucose,  10 m g / m l  of  bovine  and  kept  at  2.  Determination of the I n s u l i n Content of Enzymatlcally Dispersed Pancreatic C e l l s  a  urea. and  the  The  in  dispersed  mM C a  albumin,  pancreatic  centrifugation  2 0 mM T r i s - H C l  homogenized  serum  0.1  0.36  buffer  the  cellular  supernatant  same  step  (pH 7 . 5 ) buffer  homogenate assayed  for  cells  ,  1.2  mg/ml  described  containing  containing was  and  154  mM M g  ,  2 +  trypsin  I]radioimmunoassay  kit  (Becton  pellet  were  10 mM  inhibitor  using  Dickinson).  at an  3000  obtained  washed  mM N a C l  0.1% T r i t o n  centrifuged  insulin  the  above  125 [  2 +  was  temperature.  Enzymatlcally final  with  the  The  suspended  solution  centrifuged  (149  were  ml  finally  the  again  Eppendorf  10 m l  g for  solution  cells  mm r e s p e c t i v e l y .  containing  50  The  The  from  were  1.5  tubes  cells:  through  through  mM M g  same  the  hematocytometer.  room  cells  the  of  3 mm a n d  centrifuged  15 m l  mg/ml  times)  centrifuge  and  +  1.5  harvesting  diameters  in  -  37°C.  and  filtered  layered  of  passage  to  46  and  X-100 x  insulin  twice  then and  g for  5 M 30  min  in  - 47 -  3.  Cytochemlcal Staining of Pancreatic Acinar C e l l s The  piece of  pellet  of  at  muscle  Tagacant  sitting  in  -30°C  the  4% B S A  tissue.  The  gum a n d q u i c k l y a  flask  for  (Demon/IEC in  from  of  3 hours  tapwater  and  followed  by  then  tissue frozen  liquid  and  on  haematoxylin  dehydration  in  which  mounted  stained  was  for  through  was  sectioned  tissue  slides. min,  minute  alcohols,  disc  into by  a  means  (2-methylbutane)  frozen  3-6  embedded  cork  The  glass  another  was  on a  isopentane  it  for  step  mounted  nitrogen.  following  Division)  Cox-Lillie-Meyer  centrifugation  cleared  a  then  three  were  stained  times  1% e o s i n . in  kept  cryostat  Sections  washed with  in  was  xylene  in  This and  was then  mounted.  4.  Measurement of Amylase Release from Acinar C e l l s Cells  mM M g  ,  soybean final  were  resuspended  10 mM g l u c o s e , trypsin  volume  siliconized  of  at  2.5  The  Lil a l i q u o t s  and  mis.  were  taken  (Eppendorf  Centrifuge  50  transferred  ul  mg/ml  were  bovine  amylase  using  into Model to  buffer  the  bovine  cells at  containing  2.5  mM C a  serum albumin  and  0.12  10  cells/ml  were  37°C At  in  of  0.5  incubated the  and  of  Then  (Pharmacia)  ul  Kit.  and 45  sec.  100  Aliquots  consisting  dodecyl  sulphate  To  of  centrifuged  medium  aliquot  a  ml  incubation,  sodium  a 50  in  of  hypotonic  1 mg/ml  mg/ml  periods  rpm f o r  ul  1.2  absence  3200)  950  10  ,  or  tubes  10,000  a  6  2 +  presence  different  at  x in  microcentrifuge  pH 7 . 4 .  Phadebas  solution  concentration  drugs.  serum albumin  10 mM p h o s p h a t e  a  flask  other  KRB  10 m g / m l  inhibitor  Erlenmeyer  secretagogues  in  was  determine  of  total  1  (SDS)  assayed  of  in  for amylase,  -  50  ul  and  of  the  vortexed  Amylase  cell  suspension  vigorously.  released  was  Units % Release  where  5.  A  n  = time  at  t  Q  = zero  time  to  9.95  aliquot  expressed  as  a percentage  t  -Units  end  of  in  was  amylase  cell  —;  ml  of  then of  hypotonic  assayed total  medium  for  amylase.  amylase:  t x Dilution  lysate  Factor  x  100  to  the  incubation  sample  Preparation of Pancreatic Acinar Plasma Membranes Plasma  method  of  membranes Svoboda  were  et  homogenized  in  in  cold  (2-4°C)  a  sucrose,  buffer  glass-teflon  of  buffer,  filtered  centrifuged  at  180  for  again  at  15000  15 m l  of  was  layered  35%  and  x  g  g for  15 m i n  buffer  containing  on  of  top  38% ( w / w )  were  through  and  sucrose  and  (Model  aspirated,  with  layers  rev/min  with  at  was  of  25,000  SW 27  min M  inactivator diluted and  centrifuged was  of  1  0.3  further  this  resuspended suspension  gradient  rpm f o r  rotor.  sucrose-free  for  cheesecloth  was  density  cells  (pH 7 . 4 ) ,  pellet  Then 6 ml  sucrose  The  kallikrein  homogenate  resulting  centrifuged  diluted  900  supernatant  2 mM E D T A .  L5-50)  (500  two  The the  at  10 mM T r i s - H C l  The  a discontinuous  Beckman U l t r a c e n t r i f u g e fractions  of  according  modifications.  Aprotinin  10 m i n .  cells  homogenizer  2 mM M g C l 2 «  120 m l  acinar  slight  consisting  1 mM E D T A a n d  x  from  with  5 mM 2 - m e r c a p t o e t h a n o l ,  units/ml), with  a  prepared  (1976)  al  were  in  added  ul  ; amylase  t  -  50  amylase  = — „ Units  was  48  buffer  of  3 h  Various and  in  27%, a  - 49 -  centrifuged in  6.  at  70,000  sucrose-free  x g f o r 35 m i n .  buffer  and used  The f i n a l  immediately  or  pellet  stored  was  resuspended  frozen  membrane  volume  of  min  37°C.  at  20,000  buffer  preparation containing  After  x g for  cooling  20 m i n .  1 mM T r i s on i c e ,  The p e l l e t  EDTA  (pH 8 . 0 )  the suspension was washed  with  an  twice  membrane (TCA):  preparations  glacial  suspension  acetic  for  spectrophotometer or  at  285.2  identical EDTA;  8.  a solution  acid  mixture, 20,000  absorption nm, u s i n g  acetylene  concentrations  the standard  at  422.8  nm, u s i n g  at  and resuspended  curve  of  f r o m membrane  (1964).  with  atomic at  extracted  and Johnstone  was c e n t r i f u g e d  collected  30  in  buffer.  and magnesium were Sparrow  for  was c e n t r i f u g e d  Measurement of Divalent Cations i n Membrane Preparations  of  equal  and i n c u b a t e d  7.  method  of  was d i l u t e d  original  Calcium  volume  (1-2 mg/ml)  the  was  -80°C.  Treatment of Membranes with EDTA The  the  at  of  This  involved  treating  0.6 M t r i c h l o r o a c e t i c  x  g for  35 m i n a n d t h e  spectrometry nitrous  TCA: g l a c i a l from  in  a Techtron  The s t a n d a r d s acid  the  The  supernatant  oxide-acetylene  acetic  by  acid  1 mM E D T A a n d 0 . 5 mM L a C l 3 .  f o r magnesium.  was l i n e a r  preparations  mixture,  AA5  for  calcium  contained LaCl  3  and  0-20 uM.  Determination of Calmodulin Content of Plasma Membrane Preparations Plasma  presence  of  membrane  preparations  0 . 2 mM E G T A .  After  (2 mg/ml) cooling  were  on i c e ,  boiled calcium  for  5 min i n  was added  to  the  -  chelate min.  t h e EGTA a n d t h e s o l u t i o n  The s u p e r n a t a n t  was t h e n  [  I]radioimmunoassay  9.  Enzyme Assays a.  [Ethyleneglycolbis 2 mM N a N , 3  the desired  preincubation 25 u l o f  taking  Vincenzi  for  and/or  added  to  at  Ca  -ATPase  the assay  was m e a s u r e d  and Subbarow Under  preparation  concentration.  0.5 ml of  (1925)  the assay  a solution  presence  or  20 m i n i n c u b a t i o n defined  of  calmodulin  a c t i v i t y was pH 7 . 4 ,  (0.7 -  1.0  by the  was t e r m i n a t e d  after  3% ( w / v ) s o d i u m  0.1 mg/ml)  Calmodulin  was s t a r t e d  and/or  addition  15 m i n b y  dodecylsulfate  Pi  released  described  conditions,  0 . 0 6 mM  acid],  After  procedure  by an  by Raess  t h e enzyme  and  activity  was  ( N a + K )-ATPase +  7 5 mM T r i s - H C l  activity  as  Inorganic for Ca  2 +  Measurement of 5'-Nucleotidase A c t i v i t y : was d e t e r m i n e d  in a solution  containing  by  pH 7 . 4 ,  and i n  phosphate  -ATPase.  w h i c h was i n h i b i t e d  +  buffer  3 mM A T P , 0 . 1 mM E G T A  1 mM o u a b a i n .  was d e t e r m i n e d  as that  a  required.  by d e t e r m i n i n g  containing  2  absence  g f o r 30  30 m i n .  2 0 mM K C 1 , 3 mM M g C l ,  activity  2 +  +  in  using  4 8 mM T r i s - H C l  medium when  37°C,  into  activity  least  x  Nuclear).  Measurement of ( N a + K+j-ATPase A c t i v i t y :  assayed  c.  Mg  this  mM N a C l ,  was  calmodulin  0 . 0 7 m l o f membrane  15 m i n a t  (1980b).  at 70,000  (p-aminoethylether)-N,N'-tetraacetic  Ca  aliquots  Fiske  for  b.  free were  ATPase  automated  was  for  containing  5 mM A T P , a n d t h e r e a c t i o n  210 u l  (SDS).  linear  assayed  (New E n g l a n d  i n a 0 . 2 5 m l medium  phenothiazines  of  was c e n t r i f u g e d  2+  mM o u a b a i n , and  -  Measurement of Ca -ATPase A c t i v i t y :  determined EGTA  kit  50  (Na  the  released +  +  100  after  K )-ATPase +  ouabain.  5'-Nucleotidase 1 0 0 mM T r i s - H C l  (pH  -  8.5),  1 0 mM M g C l ,  activity  in  the  presence  5'-Nucleotidase phosphatase  d.  in  Wharton  and  potassium  acid  phosphate was  presence  Ten in  ul  by  absorbance  the  at  buffer,  of  5'-Nucleotidase  subtracted  5'-AMP  to  from  account  the  for  total nonspecific  Cytochrome  the  Specific  mg p r o t e i n  ul  1% f e r r o c y t o c h r o m e  c,  and  as  addition  at  of  an  monitored preparation  which  the  for  with  to  reaction  was  of  time  added  to  the  the  the  "blank" cuvettes  reaction  was  preparation  rate  determined  0.83  ascorbic  Both  cuvette.  3 minutes.  was  the  present.  membrane  "reaction" index  reduced  In  Activity  activity per  =  was  of  The  decrease  oxidation  Cytochrome using  minute.  (absorbance at time 0) min (absorbance at 1 minute)  the  c  as  nmole  of  cytochrome  c  in  of  Oxidase  following  _1  (concentration of cytochrome ( c o n c e n t r a t i o n of protein)  expressed  of  uM  equations:  k  per  used  a n d was  10  ferrocyanate  37°C  to  rate  specific  at  ug p r o t e i n ) nm w a s  of  c was  100  method  ul  its  cuvettes,  the  2  ferrocytochrome  ul  membrane  The  the  10  activity  order  to  of  c  the  prior  10 m i n u t e s  550  70  using  c  of  ferrocytochrome  oxidize  ferrocytochrome of  each  potassium  addition  5  To  pH 7 . 0 ,  The  0.1M  to  for  (approximately  first  was  mM K C 1 .  spectrophotometrically  (1967).  overnight  of  order  incubated  initiated  measured  added.  dialyzed  medium. cuvette  was  Tzagaloff  water and  were  3'-AMP  200  Measurement of Cytochrome c Oxidase A c t i v i t y : activity  of  the  of  and  -  activity.  oxidase  ml  10 mM 5 ' - A M P  2  51  c)  oxidized  -  e.  52  -  Measurement of Lactate Dehydrogenase A c t i v i t y :  suspensions  (10  and  thawing  were  rpm  for  45  sec  cells/ml)  6  or  centrifuged  and  the  cells  that  (Eppendorf  supernatant  had  been  disrupted  Centrifuge  assayed  for  Acinar  Model  lactate  cell  by  3200)  freezing at  10,000  dehydrogenase  activity. Lactate  dehydrogenase  using  the  method  added  0.1  ml  of  of  similarly  NADH. was  Both  buffer  treated  cuvettes  started  absorbance  Kornberg  by at  the 340  except  nm w a s  that  of  used  NADH a n d w a s  monitored  for  amount  which  causes  i n i t i a l  minute  using  an e x t i n c t i o n  expressed  10.  as  umoles  an  per  to  and  50 as  ul  3 min.  0.1  ml  of  One  coefficient of  capacity of  10 m i n cell  was at  The  37°C  the of  was  1.0  ml  'blank'  added  extract.  of  cuvette  2 uM N A D H ,  ml water.  unit  of  spectrophotometrically  buffer  index  rate  milligram  3-ml  ml  for  an  a  1.75  0.1  incubated  addition  measured  pyruvate,  (pH 7 . 4 )  were  was  (1955):  1 0 mM s o d i u m  1 0 0 mM p h o s p h a t e was  activity  cuvette  instead  of  and  the  reaction  The  decrease  rate  of  oxidation  enzyme  is  defined  oxidation  of  of  Specific  6.22.  of  1 umole  of  as  NADH  activity  in of that per was  protein.  Preparation of Phospholipid Dispersions Aliquots  dryness Tris-HCl  of  under (pH  chloroform  a  stream  7.4)  by  ultrasonicator  at  centrifuged  3000  large  at  aggregates  of  solutions nitrogen  sonication  of  x  g for  and  for  maximum p o w e r . 30 m i n  phospholipids.  of  the then  3 min  The to  in  lipids  evaporated  reconstituted  in  a Braunsonic  translucent remove  were  small  4 8 mM  1510  solution metal  to  obtained  particles  was and  -  11.  Measurement of Ca a.  method  of  instead  Schulz  membranes  prepared  (1979)  since  it  of  5 times  containing  Ca  hours.  2  concentration,  +  To d e t e r m i n e  mg/ml)  were  at  filtered  under  to  scintillation Blanks similar  in  vials  2  At  various  of  with  pancreatic  and any a c t i v i t y  buffer  ATP, the at  present  membranes  obtained  sucrose  aliquots  were  subtracted  (2-3 buffer  of  50  ul  (Millipore  were  of  Co.)  sucrose  then  determined  (New E n g l a n d  18  vesicles  filters  filters  desired  4°C f o r  two 5 ml p o r t i o n s  i n Aquasol  preloading,  2 0 mM T r i - H C l  the preloaded  The d r i e d  with  and  intervals,  and the r a d i o a c t i v i t y  of  For  and kept  0 . 4 5 urn m e m b r a n e  used  interfered  a non-radioactive  timed  washed  spectrophotometry  manner  50 u l  was  by t h e  hand homogenizer  5 mM T r i s  3  1.45 ml o f  3  a  0.3 M sucrose,  to  mM L a C l .  with  2 mM N a N ,  efflux;  +  mannitol  phosphate.  (10 uCi/mmol)  5  presoaked  1.2  the absence  experimental  * CaCl  and the f i l t e r s  containing  transferred  37°C.  through  vacuum,  buffer  2  transferred  incubated  were  Ca  t  preparations  0.3 M sucrose  that  inorganic  homogenized  a medium  that  was o b s e r v e d  determination were  f r o m membrane  except  7 . 4 ) , 1 0 0 mM K C 1 , 5 mM M g C l 2 ,  free  and  in  were  and H e i l  of mannitol  vesiculated (pH  -Transport A c t i v i t i e s  Vesicles  colorimetric  plasma  -  Measurement of Calcium E f f l u x from Plasma Membrane V e s i c l e  P r e p a r a t i o n s:  the  53  by  liquid  Nuclear).  determined from  in  a  the  values.  2+  b.  Measurement of Ca  P r e p a r a t i o n s: above.  Vesicles  The s e a l e d  containing  were  vesicles  2 0 mM T r i s  -Uptake by Plasma Membrane V e s i c l e prepared  were  HCl-buffer  then  i n 0.3 M sucrose suspended  as  i n a sucrose  described buffer  ( p H 7 . 4 ) , 1 0 0 mM K C 1 , 0 . 1 mM M g C l 2 ,  2  -  mM N a N ,  the desired  3  5  mM T r i s  ATP i n  non-specific  a  free  total  binding  of  Ca  54  -  concentration, ml.  (10 Ci/mmole)  CaCl2  volume  of  1.0  Because  calcium  to  t h e membrane  of  the  vesicles,  and  high the  reaction  45 at  37 C was s t a r t e d  ul  aliquots  by  the a d d i t i o n  were  transferred  mM E G T A  before  filtration.  described  above.  2  bound  12.  Ca  2+  resulted  calmodulin  modification  of  Ca  gel overlay  2+  that  steps  are  t h e EGTA  uptake  timed  sucrose  intervals,  buffer  similar  treatment  50  containing  to  those  which  removes  rates.  isopropanol  incubations  in  buffer  four  times  distilled  albumin,  5 0 mM T r i s - H C l ,  The  were  buffer,  buffer  A  Tris-HCl,  0.1  pH 7 . 5 ) .  the  the  pH 7 . 5 )  10 m g / m l  The g e l s  were  washed to  were  soaked  bovine  6 M  to  equilibration  soaked  The  two,  5  30 m i n i n  a  serum  24 h o u r s  in for  1 mM C a C l 2 ,  serum a l b u m i n , in  solution  guanidine-HCl.  prior  then  a  SDS.  for  least  acetate,  in  1 mM m a g n e s i u m  mg/ml with  of  Following  at  bovine  Following  remove  for  1 mM m a g n e s i u m  mM E D T A ,  acid  0.1  supplemented washed  were  glycerol,  mM E D T A ,  a  5-20% g r a d i e n t )  period.  gels  20% ( v / v )  using  et^ a l ^ ( 1 9 8 1 ) :  gels  a 2 hour  guanidine-HCl,  (0.15 M NaCl,  dithiothreitol,  in  0.1  continuously  without  by C a r l i n  water,  (0.15 M NaCl,  then  was p e r f o r m e d  a n d 10% ( v / v ) a c e t i c  1 mM d i t h i o t h r e i t o l ,  above  used  fractions,  acetate,  gels  technique  ( 2 0 0 \ig p r o t e i n / l a n e ,  membrane  was c h a n g e d  denaturing  in  Subsequent  higher  the procedure  plasma  25% ( v / v )  solution min  in  gel electrophoresis  pancreatic of  1 ml i c e - c o l d  was o b s e r v e d  At  CaCl2.  Calmodulin Gel Overlay Technique The  SDS  It  to  of  buffer  A  the 12  hours  1 mM  5 0 mM containing  -  1  2  5  I-labeled  calmodulin  hours.  Non-specifically  washing  for  24  hours  which  5 mM E G T A  dried  and  weights, rest  of  then the  the  Coomassie the  was  included  lanes,  to  A.  in  containing  Rp  values  10  -  k  cpm/  5  was  Control  the  final  were  used  50 m l / s l a b ) by  experiments wash  appropriate  following  pmol,  removed  autoradiography.  immediately  blue.  I0  -  calmodulin  buffer  subjected  gel  nM,  bound  in  calmodulin-labeled  13.  (240  55  assign  and  molecular  performed The  gels  in  were  molecular  were  electrophoresis to  were  determine  standards,  24  continuous  solutions. To  for  cut  from  stained weights  the  by to  bands.  Measurement of Acyl-Phosphoproteln Formation Membrane-ATPase  and B l o s t e i n final  phosphoprotein  (1975).  incubation  Pancreatic  volume)  were  containing  4 8 mM T r i s - H C l  (1000-3000  cpm/pmol),  60  (pH  was  measured  acinar  plasma  by  the  membranes  incubated  for  7.4),  0.1  mM o u a b a i n ,  the  presence  uM E G T A  in  method  15s  at  10°C 2  and  of  (130 in  ug/100  a  uM [ y -  Katz ul  medium P]  3 2  absence  ATP  of  0.4  mM  2+ magnesium  acetate.  concentration of  membranes  terminated mM T r i s  was  added  it  was  present  in  a  80  uM f r e e .  The  reaction  was  started  by  the  the  reaction  of to  by  the  phosphate  30% u r e a , tracking  0.5 dye.  formed,some sodium  When Ca  addition buffer  of  (pH  45  To  determine  (pH  were 5.2)  the  treated for  ul  6.5),  mM d i t h i o t h r e i t o l  samples  acetate  medium.  and  After of  'stop'  5% l i t h i u m 0.002%  stability with  10 m i n  15s  at  0.6 22°C  the  reaction  solution dodecyl  bromphenol of  the  addition was  consisting sulphate blue  as  of  the  in  the  0.8  addition  50  (LiDS),  phosphoprotein  M hydroxylamine before  final  of  M the  -  'stop'  solution.  The samples  acid-polyacrylamide  14.  (60 u l  -  per lane)  were  then  analyzed  by  electrohoresis.  Phosphorylation of Endogeneous Acinar Membrane Proteins Phosphorylation  30°C  gel  56  in a  100 u l  of  endogenous  reaction  mixture  substrate  proteins  containing  was c a r r i e d  130 ug membrane  out  at  proteins,  32 0.1  mM d i t h i o t h r e i t o l ,  (1000-3000  cpm/pmol),  absence  9 0 uM C a  of  incubation, 'stop' SDS,  30% u r e a , dye.  polyacrylaraide  15.  and  ,  2 . 2 mM E G T A  consisting  of  The samples  (pH 7.0)  by  in  the addition  phosphate  a n d 0.002%  (60 u l / l a n e )  gel  0 . 5 mM [y  were  After of  buffer  bromphenol  then  P] ATP  the presence  1 0 0 nM c a l m o d u l i n .  5 0 mM T r i s  0 . 5 mM d i t h i o t h r e i t o l  gradient  buffer  was t e r m i n a t e d  analyzed  or  5 min of  45 u l  of  ( p H 6 . 8 ) , 5% blue  as  the  b y SDS  electrophoresis.  of  Gradient G e l Electrophoresis: 1 . 5 mm t h i c k n e s s  Favre  (1973).  were  cast  (pH 8 . 8 ) , (TEMED)  1.35% g l y c e r o l ,  bisacrylamide  (30%: 0.8%) m i x t u r e ,  gradient  the method of  of  3.33 ml  (30%: 0.8%) m i x t u r e ,  0.1% SDS, 0.025%  gel contained  to  gel consisted  a n d 0 . 4 m l ammonium p e r s u l p h a t e  20% ' r e s o l v i n g '  Polyacrylamide  according  T h e 5% ' r e s o l v i n g '  acrylamide-methylene-bisacrylamide  The  or  acetate,  Polyacrylamide G e l Electrophoresis and Autoradiography of Phosphorylated Plasma Membrane Preparations a.  gels  2 0 mM T r i s - H C l  the r e a c t i o n  solution  tracking  2 +  1 0 mM m a g n e s i u m  (5-20%)  Laemmli of  3 7 5 mM T r i s - H C l  tetraethylmethylenediamine  (15 mg/ml)  13.33 ml of  in  a total  of  20 m l .  acrylamide-methylene-  3 7 5 mM T r i s - H C l  buffer  (pH 8 . 8 ) ,  - 57 -  5.75% (15  glycerol,  mg/ml)  solutions gradient the  gel  in  a  were  total  the  temperature. 'stacking'  of  0.05%  About gel  The  The  added  2 ml  was  TEMED a n d  20 m l .  immediately  former. and  The  0.1% SDS,  of  was  to  (15  (pH  6.8),  mg/ml)  in  was  a  gel  (5%)  gel.  gel  gel  the  Tris-Glycine SDS w a s  added  the  teflon  of  decanted  gel was  20%  with  carefully for  to  persulphate  'resolving'  chamber  prior  consisted  7.3  teflon  'comb'  to  (25  60  the  the  gel  aid  layered  12 h o u r s  of  on  at  addition  of a  for  molecular  12  top  a of  room  of  the  mixture,  After  mixing,  the  chamber  over  gel  was  then for  the  ml  least  samples  the into  2  The  syringe protein  daltons)  carbonic lysozyme  the and  the  anhydrase  cell,  plasma  membrane  Ca  by  created  by  gel  run  used  at  bovine  (31,000),  The  -ATPase  was  acyl  detected  25  for  (200,000),  2+ of  followed  wells the  0.1%  serum  albumin  soybean  (14,400).  Acld-Polyacrylamlde Gel Electrophoresis:  intermediate  stacking  containing  myosin  (92,500),  gel  polymerized  standards  were;  persulphate  hours.  pH 8 . 3  to  Tris-HCl  'stacking'  inserted at  3 1 5 mM  ammonium  electrophoresis  protein  (in  0.2  mM G l y c i n e )  phosjphorylase  and  of  and  hours.  (45,000),  (21,500)  0.8%)  100 m l H a m i l t o n  weights  (116,250),  inhibitor  the  ml  TEMED  the  of  1.25  (30%:  mM T r i s - 1 9 2  ul  of  polymerize  chambers  using  ovalbumin  ml.  into  both of  0.136%  poured  current)  p-galactosidase  b.  of  allowed  'comb'  mA ( c o n s t a n t  trypsin  A  to  application  (66,200),  total  buffer  the  estimation  0.1% SDS,  gently  'resolving' and  the  ammonium  gel.  'stacking'  solution  the  ml  polymerize  acrylamide-methylene-bisacrylamide buffer  5% a n d  buffer  allowed  buffer  to  0.3  by  phosphate  -  acid-polyacrylamide method 10.7  of  ml  Lichtner  ml.  a  total  of  constant  with  The  'resolving'  ml.  those  (40 used  (30%:  lithium  (5%)  contained  mixture,  Protein (pH mA)  for  for  0.3  ml  (15 ml  0.25% C o o m a s s i e  (60  ul)  for  procedure  in  and a  second  until  the  Prior (10:3:87)  30 m i n  were  protein  gel  methanol/acetic destaining was  to  the  drying,  for  30  dried  exposed  to X-ray  (Cronex  Lightning were  min,  in  (LiDS), total  of  40  at  (pH  (15  mg/ml)  in  a 0.2  M  4°C  under  the  standards  used  were  studies. The  followed  by  (45:9:46)  a  were  stained  acid/water first  for  methanol/acetic  gels  1 hour  destaining (3  changes)  acid/water  (4:1:15)  transparent. gels  were  vacuum  at  80°C  Min-R)  DuPont)  developed.  fixed  70% m e t h a n o l  (Kodak  Plus,  in  a  1 0 0 mM  buffer  run  methanol/acetic  acid/water  procedure  then  under film  Blue  room t e m p e r a t u r e ,  background  immediately  films  at  in  of  acrylaraide-methylene-  0.2% L i D S  The  gradient  Brilliant  sulphate  mg/ml)  of  the  mixture,  ammonium p e r s u l p h a t e  containing  7 hours.  the  0.8%)  to  consisted  1 2 5 mM T r i s - p h o s p h a t e  samples  7.0)  1.25  gel  dodecyl  ammonium p e r s u l p h a t e  0.8%)  buffer  current to  ml  0.2%  according  Staining, destaining and Autoradiography:  (5:1:4)  the  electrophoresis  (1979).  0 . 1 3 % TEMED a n d  7.4  Tris-phosphate  c.  gel  (pH 7 . 0 ) ,  gel  (30%:  0.2% L i D S ,  similar  0.8  'stacking'  bisacrylamide  in  and Wolf  buffer  TEMED a n d  The  6.5),  slab  -  acrylamide-methylene-bisacrylamide  Tris-phosphate 0.085%  (8%)  58  for  in for  for  along 7-16  acetic  acid/glycerol/water  20 m i n .  The  gels  2 hours.  The  dried  with days  an at  were gels  intensifying -80°C  following  were  screen which  -  16.  -  Miscellaneous Methods a.  Protein Assay:  ug p r o t e i n ) ml. a  59  To  ten  were  this  added  was  acid  aspirated,  for  using  albumin  was  room was  used  the as  at  and  variance  data  the  (S.E.M.)  mean  was  used  the  was  as  water  to  a  preparations final  2% d e o x y c h o l a t e  added  to  x  Lowry  0.5  ml  thirty  resuspended (1951)  of any  were  personal  constants  using  communication).  association adjusted  calculated  constant  for  of  EGTA,  temperature,  Students of  used were  first  to  8.615,  2.660,  2.000;  CaEGTA:  6.544,  3.945;  CaATP:  3.742,  5.987,  2.720,  2.009;  CaEDTA:  protein. the  in  and  water assay.  "t"  test  for  The  assayed  Bovine  unpaired,  significance.  a measure  of  serum  Standard  EDTA  and ATP  fourth  for  MgATP:  10.423;  the  error  of  Log  MgEGTA:  4.228,  MgEDTA:  and  (Goldstein,  magnesium,  association with  5.422,  2.240;  8.929,  calcium  apparent  association  5.190:  Free  CATIONS  calcium  strength.  proton  10.630, 1.890;  program  calculates  ionic  common  variation.  Fortran  program  pH a n d  1.5  protein.  the  The  of  Following  minutes,  protein  (10-150  cold  Determination of Free Calcium Concentrations:  c.  volume  solution.  precipitate  g for  pellet  a measure  used  membrane  temperature,  standard  as  a  3000  standard  the  of  S t a t i s t i c a l Analysis:  b.  values  at  centrifuged  acinar  distilled  ul  (24%)  supernatant protein  in  12.5  incubation  trichloroacetic suspension  suspended  was  minute  Pancreatic  EGTA: 3.810;  EDTA:  2.770.  9.235, HATP:  10.009,  -  60  -  RESULTS  1.  Determination of C a  -ATPase A c t i v i t y In Pancreatic Plasma  2 +  Membranes In  preliminary  membranes activity The Mg  2  +  from whole  was o b s e r v e d  activity  in  was s t i m u l a t e d  b y Ca  for Ca  that  the ATPase  activity  o r Mg  saturating  activity  2  +  21  in  in  plasma  activity,  suggesting  represent  the  same  of  that  Ca  activation  increased  membrane  of  with  of  o r Mg  Figure  3  a d d e d Mg  the  stimulated  indicates  hydrolytic  2-4-  .  Also  Figure  2  concentrations  A combination  24a n d Mg produced 2424-  plasma  preparations.  increasing  off.  in  ATP  the enzyme.  24-  t h e Ca  enzyme.  A high  the absence  u p t o 0 . 2 5 mM a n d l e v e l l e d  concentrations  was d e t e r m i n e d  homogenates.  the pancreatic  substitute  Ca  ATPase tissue  could  shows of  prepared  experiments  same  of  maximum  activities  t h e ATP d e p e n d e n c y  of  the  24ATPase  activity.  increased mM.  with  Higher  ATPase  activity  increasing  in  the presence  ATP c o n c e n t r a t i o n ,  concentrations  than  0 . 5 mM w e r e  of  either  reaching  Ca  24o r Mg  a maximum a t  0.35  inhibitory. 24-  Since activity  the  pancreas  observed  homogenates  could  in not  is  a heterocellular  plasma  membranes  be d i r e c t l y  organ,  prepared  related  to  t h e Ca  from  acinar  -ATPase  pancreatic cell  tissue  function.  24C h a r a c t e r i z a t i o n o f a Ca -ATPase a c t i v i t y i n p a n c r e a t i c acinar plasma membranes w o u l d r e q u i r e p r e p a r a t i o n o f hormone r e s p o n s i v e dissociated 24pancreatic processes  acinar that  cells,  could  be  and i d e n t i f i c a t i o n related  to  amylase  of  possible  secretion  in  Ca  -mediated  the acinar  cell.  - 61 -  Figure  2  Ca o r Mg " a c t i v a t i o n o f A T P a s e a c t i v i t y i n pancreatic p l a s m a membrane p r e p a r a t i o n s . A T P a s e a c t i v i t y was d e t e r m i n e d a s d e s c r i b e d i n M e t h o d s a t 0.5^ mM A T P i n t h e i  p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f Ca (• (O---o). R e s u l t s r e p r e s e n t t h e mean ± SEM o f different determinations. +  •) or five  Mg  -  Figure  3  Effect  of  ATP  63  on ATPase  membrane p r e p a r a t i o n s . d e s c r i b e d i n Methods at  -  activity  in  pancreatic  plasma  A T P a s e a c t i v i t y was d e t e r m i n e d v a r y i n g c o n c e n t r a t i o n s o f ATP  (0.01 - 1.0 mM) i n t h e p r e s e n c e o f 0.5 mM C a (• •) a n d 0.5 mM M g (o o). R e s u l t s r e p r e s e n t t h e mean ± SEM o f f i v e d i f f e r e n t determinations. 2  2  +  +  as  ATP cone (mM)  -  65- -  2.  Determination of a Possible Relationship of Ca "'"-ATPase A c t i v i t y to Amylase Secretion  a.  Characterization of Amylase Secretion i n Pancreatic Acinar The  from  i n i t i a l  rat  pancreas  population. of  cells  The  marker  there  was  cells  compared  the  and  a  was  used  35 -  insulin  to  p-cells.  To  to  verify  this,  were  was  of  30,  than  the  acinar of  45,  an  amylase 60,  75  in  and  was with  respect  has  the  The  acinar  size  of  the  by of  the  incubated  response  to  10  (Figure  5  5).  of  represent from Figure  cells.  with  cells  M carbachol  The  features  viability of  trypan  their  of  the  blue  cells dye.  dissociated  conditions was  4  Acinar  maintained  control  Amylase  acinar  appearance.  type  I  et_ al^ 1 9 7 8 ) ,  employed.  acinar  The  Table  with  might  secretion,  under -  were  exclusion  amylase  in  presence  (Korc  granular cell  The  contamination  the  configuration.  course  90 m i n  of  population  shown  the  preparation than  the  associated  cells  cell  cells.  As  reported  and one  whether  Since  acinar  acinar  acinar  methods  majority  evaluated  were  to  levels  rather  staining  their  the  insulin.  been  final  cells,  eosin  of  population.  cells in  of  insulin  cell  responsive  determine  presence in  hormone  purity  to  predominantly  time  cells  the  cytochemical  by  cells.  95% a s  determine  secretion  shows  exhibited  pancreatic  15,  4)  acinar  greater To  and  characterized  (Figure  and  mixed  acinar  cells  shape  the  measured  an h a e m a t o x y l i n  typical  approach  on a c i n a r  the  prepare  determine  was  shows  Figure  to  enriched  the  levels  bound  was  75% d e c r e a s e  receptors  insulin  to  first  obtained  cellular  insulin  objective  Cells  and  monitored  secretion  rat the at  increased  -  Insulin  66  -  Table  I  Content of E n z y m a t i c a l l y D i s p e r s e d P a n c r e a t i c Homogenates and I s o l a t e d A c i n a r Cells.  Insulin  Content(mU/mg  Cell  Tissue) Purification  Homogenate  Acinar  Cells  Preparation  1  5.66  3.71  1.5  fold  Preparation  2  5.94  1.66  3.5  fold  I n s u l i n was e x t r a c t e d f r o m c e l l s as d e s c r i b e d i n c o n t e n t was a s s a y e d u s i n g a r a d i o i m m u n o a s s a y k i t mU/mg o f c e l l u l a r protein.  Methods. Insulin and i s e x p r e s s e d as  Figure  4  L i g h t micrograph of i s o l a t e d Magnification: X 1019.  pancreatic  acinar  cells.  - 68-  - 69 -  Figure  5  Time c o u r s e o f a m y l a s e s e c r e t i o n by d i s s o c i a t e d rat pancreatic acinar cells. C e l l s were i n c u b a t e d i n s t a n d a r d i n c u b a t i o n s o l u t i o n as d e s c r i b e d i n M|thods the p r e s e n c e (• • ) o r a b s e n c e ( O - — O ) o f 10~ M carbachol. R e s u l t s s h o w n a r e t h e mean ± SEM f r o m separate experiments.  in  three  -"70-  T i m e (min)  -  linearly release  with  time  increased  studied.  Figure  up  about  secretin  pancreatic  acinar  pancreozymin,  maximal  at  secretin  10  as  the  rate  comparable (Gardner release termed the  and  the  of  to  higher  that  (10  of  thereafter. at  time  varying  on a m y l a s e  from  concentrations  respectively, was  points  concentrations  release  secretion  release  all  Amylase  and  then  dissociated  of  increased,  of  carbachol becoming  decreased.  submaximal  at  the  With  highest  M).  - 6  secretion  1977;  by  observed  other  Williams  carbachol  these  effect  amylase  U/ml  off  basal  increasing  amylase  "desensitization"  fact  of  reported  and J a c k s o n at  rate  amylase  those  the  With  agonist,  tested  over  pancreozymin  cells.  -  levelling  three-fold  M and 0.01  concentration The  60 m i n ,  6 illustrates  carbachol,  and  to  71  in  these  studies  is  workers  using  similar  preparations  et  al  1978).  and pancreozymin  (Williams  secretagogues  et  al  decreased  concentrations  1978).  produce  The  This  much h i g h e r  is  amylase  has  been  attributed  increases  to  in  2+ intracellular O  excess  Ca  levels  than  is  required  for  amylase  secretion.  The  I  Ca  levels  then  either  directly  or  indirectly  inhibit  amylase  release.  b.  Determination of the E f f e c t of Anticalmodulin Agents on Amylase Secretion Calmodulin  plays  a major  role  in  the  modulation  of  a wide  variety  of  24Ca  -dependent  presence and  of  Scheele  cellular  functions  calmodulin  in  1980),  role  its  the in  (Scharff  pancreas amylase  1981).  Based  (Vandermeers ^t secretion  from  al  on  the  1977;  dissociated  reported Bartelt rat  -  Figure  6  72  -  C o n c e n t r a t i o n d e p e n d e n c e o f a m y l a s e s e c r e t i o n by dissociated rat pancreatic acinar c e l l s . Amylase s e c r e t i o n s t i m u l a t e d by s e c r e t i n (o O) a n d p a n c r e o z y m i n ( A — A ) suspended i n standard i n c u b a t i o n Ca a n d 1 . 2 mM M g at 37°C f o r a r e mean ± SEM o f f i v e d i f f e r e n t 2  +  (• • ) , carbachol was m e a s u r e d i n cells m e d i u m c o n t a i n i n g 2 . 5 mM 45 m i n . Results shown experiments.  - u  - 74 -  acinar  cells  was  phenothiazines  studied  on  the  indirectly  secretory  by  determining  process.  These  the  effects  agents  are  of  known  to  bind  2+ to  calmodulin  mediated  in  events  a Ca  -dependent  (Levin  and W e i s s  phenothiazine  trifluoperazine  activity  dose-dependent  amylase was  in  a  release.  found  to  be  phenothiazine,  The 10  of  M inhibited  stimulated  maximal all  have  that  lower  release  release  At  any  8a  by  were on  (10~  calcium.  stabilizing These calcium on a  effects,  studies  mediated  calmodulin  induced  did  which  not  indicate  show  the  to  release  regulated  step  in  a  10~ and  CPZ by  5  CPZ  this  from the  (lO  at  a  produce  by  concentration  -  -  4  0.5  same a  that  pancreatic  (98% 10  CPZ  It  that  act  non-calcium  M)  did  not  by has  no  mediated  membrane  inhibition  (Figure  phenothiazines  stimulus-secretion  3  appears  concentration  acinar  -  of  amylase  non-specific  selective  x  degree.  inhibits  U/ml)  whereas  intact  amylase.  CPZ  secretin,  can  inhibition  induced  virtually  of  the  basal  pancreozymin-(0.01  pancreozymin) at  50%  on  another  similar  M),  the  effect  65% r e s p e c t i v e l y  s t i l l  possibility  amylase  and  release  -  6  intracellular  Propranolol,  was  basal  mobilizing  secretagogue.  and  for  release  concentrations  (carbachol  release  60  inhibited  the  concentrations  amylase  amylase  of  7,  stimulated  significant  effect  calmodulin  Figure  carbachol  required  M)  inhibit  in  determined.  release  secretagogues  on  on  about  CPZ  shown  no  TFP  the  was  by  effect  of  (CPZ)  higher  effect  with  carbachol-(10  stimulated  M)  5  both  secretagogues  though,  Figure  As  thereby  inhibited  manner,  secretagogues  response).  three  at  of  amylase  secretin-(10~  (TFP)  chlorpromazine  classes  10  In  and  1977).  concentration  uM.  various  manner  cells  8b).  inhibit by  coupling  acting  - 75 -  Figure  7  E f f e c t of t r i f l u o p e r a z i n e on a m y l a s e s e c r e t i o n f r o m dissociated pancreatic acinar cells. Enzyme secretion was measured i n t h e p r e s e n c e ( • — • ) and absence (O—-O) of 10 M carbachol. R e s u l t s r e p r e s e n t t h e mean ± S . E . M . of f i v e separate experiments. - 5  Amylase release (% of total)  -  Figure  8a  77  -  E f f e c t o f c h l o r p r o m a z i n e on a m y l a s e s e c r e t i o n by dissociated pancreatic acinar cells. Amylase secretion was measured i n t h e p r e s e n c e of 10~ M c a r b a c h o l (•—•). 5  0.01  U/ml  pancreozymin  (A  A)  and  10~  6  M  secretin  (0---0). C e l l s were suspended i n standard incubation m e d i u m c o n t a i n i n g 2 . 5 mM C a a n d 1 . 2 mM M g at 37°C f o r 45 m i n . R e s u l t s r e p r e s e n t t h e mean ± SEM o f three separate experiments. +  Figure  8b  2  E f f e c t o f p r o p r a n o l o l on a m y l a s e s e c r e t i o n pancreatic acinar cell|. Amylase s e c r e t i o n i n the presence of 10" M carbachol ( • — • ) secretin ( O — o ) . C e l l s were suspended i n i n c u b a t i o n s o l u t i o n c o n t a i n i n g 2 . 5 mM C a Mg a t 37°C f o r 45 m i n . Results represent ±SEM o f t h r e e s e p a r a t e experiments. 2  +  +  +  by d i s s o c i a t e d was m e a s u r e d and 1 0 " M standard a n d 1 . 2 mM t h e mean  -73 -  A m y l a s e r e l e a s e (% of m a x i m u m )  -  process. enzyme play  The mechanism  secretion  a role  plasma  in  is  the  membrane  Ca  by  not  which  2+  A of  -ATPase.  -  calmodulin  known.  regulation  79  would  regulate  calmodulin-dependent free  intracellular  Therefore  subsequent  pancreatic  enzyme  calcium studies  that  is  may  the  involved  2+ characterization from  3.  pancreatic  activity  in  plasma  membranes  obtained  cells.  preparations  contaminated  attempts cells  the  mainly  Methods  acinar  membranes Table  enzymes increase  to  cells (see  the  II  fractions from  characteristic +  and  This  illustrates  (Na  Isolation  the  of  of of  acinar  from  yielded  enzymes.  the  The  described these  pure  plasma  acinar  ATPase  There in  a  was  number  activity. of  marker  homogenates.  interface  in  cells.  by  cell  increase  as  from  distribution  acinar  cells,  stimulation  endogenous  membranes.  4-fold  to  Therefore  acinar  cells  could  endocrine  cells.  membranes  27%/35% s u c r o s e  plasma  homogenates the  from  digestive  plasma  of  from  centroacinar  sub-cellular  + K )-ATPase, +  cells  responsive  loss  obtained the  of  procedure  appreciable  collected  in  the  tissue  membranes  secretion  viable  above).  of  some  plasma  preparation  were  without  and  prepare with  from whole  membranes  duct  involved  then  obtained  plasma  concerned  among v a r i o u s fraction  made  first  and  agents  with  collecting  were  procedure  of  acinar  -ATPase  2+  pancreas,  The  a Ca  Characterization of Ca -ATPase A c t i v i t y In Pancreatic Acinar Plasma Membranes Membrane  be  of  was a  enzymes The  enriched  in  12-fold  5'-nucleotidase  and  Table  II  S p e c i f i c A c t i v i t y o f Ca -ATPase and M a r k e r Enzymes i n C e l l Homogenates and S u b c e l l u l a r Fractions.  Cytochrome (Na  + K )-ATPase  +  +  3  5'-Nucleotidase  3  c  Oxidase  Ca  2 +  -ATPase ' a  Homogenate  0.64  + 0.05  1.52  + 0.09  4.51  + 0.15  0.18  + 0.02  10-25%  3.62  + 0.31  2.45  + 0.16  0.34  + 0.08  0.32  + 0.01  27-35% s u c r o s e interface  7.89  + 0.12  5.51  + 0.10  0.67  + 0.05  0.80  + 0.03  Bottom  0.31  + 0.03  0.26  + 0.06  6.57  + 0.27  0.05  + 0.01  sucrose  interface  a  pellet  umoles  ^nmoles c  Ca  Pi/mg cyt  prot/hr.  c oxidized/mg  concentration  is  0.4  prot/min. uM  free.  C  - 8 1 -  4-fold  increase  oxidase  in  activity  mitochondrial differential membranes  Ca  was  2  stimulated  +  significantly  contamination degrees  may  be  ATPase  due  was  activities.  decreased  minimal  of  purification  to  the  in  of  different  Cytochrome  indicating  this  marker  that  fraction. enzymes  stabilities  of  c  The  for  the  plasma  enzymes  studied. A  high  enriched  ATP  hydrolytic  fraction.  with  increasing  free  ion  a  also  concentration. higher  maximum  activated  rate  (Table  and M g  produced  +  cations  though,  Similarly, maximal of  the  amylase  markedly  sodium  did  azide,  the  for  or  Mg  affect  the  2  S r of  from  V).  the  acinar  Whereas  enzyme The  Ca  2  ATPase.  comparison  A  activity  is  requirement  this  shown is  membrane increased  Ba  2  that (P  +  ATPase  enzyme  the  no  was  activity is  for  due was  on  10 a n d Ca  was  Ca  2  +  V).  produce  the  activity 1 mM  membrane  insensitive from  to  11. and  5 mM  the  requirement  2+  a  of  effect,  to  different  has  least  (Table  that  without  both  the  uM  Monovalent  effect  substrate  Figures  with  concentrations  of  88  activity  showing  +  of  system  < 0.05)  IV).  had  the  the  (Table  possibly  similar  a maximum  concentrations  enzyme  in  2  saturating  uM C P Z  of  plasma  activity  to  The  and  +  at  -ATPase  up  III).  activity, 2 +  ATPase  than M g  +  cells  10  the  indicated  activity  and pancreozymin  in  2-4-  Ca  with  +  the  (Table  that  substrate  2+  9,  analysis  indicating  phosphohydrolase  observed  same maximum a c t i v i t y  not  V).  2  Ca  combination  release  (Table  mitochondrial  for  A  (Table  of  activity and Z n  was  Figure  affinity  +  the  inhibited  disruption  2  IV).  carbachol  enzyme  in  Kinetic  of  by M n  activity 2  shown  concentrations  significantly  similar  As  activity  The Mg  for  profile  2+  the  - 82 -  Figure  9  The  effect  of  Mg  plasma-membrane  / : +  and  Ca  enriched  +  on A T P a s e  fractions  of  activity  in  pancreatic  acinar  cells. ATP h y d r o l y t i c a c t i v i t y i n t h e p r e s e n c e o f increasing Ca (•—•) and M g (O O) c o n c e n t r a t i o n s . Results r e p r e s e n t the mean ± S . E . M . o f f i v e different 2  +  determinations.  2  +  - 84"--  Table  Q1SS Acinar  III  and V „ „ of the ATPase A c t i v i t y of Pancreatic IUctX P l a s m a Membrane P r e p a r a t i o n s i n t h e P r e s e n c e Ca o r Mg .  of  +  K  (uM)  V  Ql SS  Ca  Mg  a  2  +  2 +  (5)  a  (5)  1.73  ± 0.25  2.98  ± 0.39  (umoles  Pi/mg  prot/hr)  TTlctX  b  ,  C  15.29  ±  0.76  16.32  ±  0.61  N u m b e r of p r e p a r a t i o n s .  ^Mean ± SEM  Significant  i n each case. (p < 0 . 0 5 ) when compared t o M g - s t i m u l a t e d 2 +  activity.  -  85  xable  -  IV  A T P a s e A c t i v i t y of P a n c r e a t i c A c i n a r P l a s m a Membrane P r e p a r a t i o n s i n the Presence of D i v a l e n t Cations.  Cation  Ca Mg Mn Zn S r Ba ' +  Specific  Activity  (umoles  2  +  13.42  + 0.69  2  +  13.45  + 0.99  2  +  10.32  + 0.46  2  +  8.69  + 0.32  2  +  1.40  + 0.05  2  +  1.02  + 0.07  13.56  + 0.08  Mg  2  +  Pi/mg  prot/hr)  A T P a s e a c t i v i t y w a s m e a s u r e d i n a m e d i u m c o n t a i n i n g 4 8 mM T r i s - H C l p H 7 . 4 , 0 . 5 mM A T P , 0 . 1 mM o u a b a i n , 2 mM N a N a n d 0 . 2 5 mM d i v a l e n t cation at 37°C. R e s u l t s r e p r e s e n t t h e mean ± S . E . M . o f f o u r separate determinations. 3  -  86  -  Table Effect  of  Monovalent Pancreatic  Cations Acinar  V  and Drugs  1 0 0 mM N a  +  ATPase  Activity  Preparations.  ATPase A c t i v i t y Ca  (umoles  2  Control  on t h e  P l a s m a Membrane  +  Pi/mg prot/hr) Mg 2  +  16.01  + 1.5  16.03  + 1.2  15.01  + 1.3  14.85  + 1.1  13.72  + 0.9  14.78  + 0.9  1 0 0 mM  K  10"  M  Carbachol  15.73  + 2.3  15.59  + 1.4  M  Chlorpromazine  14.04  + 1.2  16.59  + 2.0  M  Chlorpromazine  3.46  + 0.2  1.95  + 0.5  16.92  + 0.2  16.66  + 1.0  15.23  + 1.5  15.01  + 1.7  5  10 10  _3 0.01  +  U/ml  Pancreozymin  5 mM s o d i u m  azide  of  A T P a s e a c t i v i t y w a s m e a s u r e d i n a m e d i u m c o n t a i n i n g 4 8 mM T r i s - H C l p H 7 . 4 , 0 . 0 6 mM E G T A . 1 mM o u a b a i n , 2 mM N a N a n d 0 . 5 mM A T P a t 3 7 ° C . When either Ca o r Mg was a d d e d , i t was p r e s e n t a t a c o n c e n t r a t i o n o f 88 uM f r e e . R e s u l t s r e p r e s e n t t h e mean ± S . E . M . o f f i v e different experiments. 3  2  +  +  -  Figure  10  87  -  S u b s t r a t e r e q u i r e m e n t o f Ca -ATPase a c t i v i t y i n plasma membrane p r e p a r a t i o n s o f a c i n a r c e l l s . Enzyme activity was d e t e r m i n e d as d e s c r i b e d i n M e t h o d s a t varying c o n c e n t r a t i o n s ( 0 . 0 1 - 1 . 0 mM) o f A T P ( • • ) , GTP (A A) AD? ( O O ) a n d AMP (A A) i n the presence of 8 8 uM C a different  . The r e s u l t s r e p r e s e n t determinations.  the  m e a n ± SEM o f  five  -88-  Nucleotide cone. (mM)  -  Figure  11  89  -  S u b s t r a t e r e q u i r e m e n t o f Mg "'"-ATPase a c t i v i t y i n p l a s m a membrane p r e p a r a t i o n s of a c i n a r c e l l s . Enzyme activity was d e t e r m i n e d as d e s c r i b e d i n M e t h o d s a t varying c o n c e n t r a t i o n s ( 0 . 0 1 - 1 . 0 mM) o f A T P ( • • ) , GTP (A A ) , A D P (O O ) a n d AMP ( A A ) i n the presence  of  88  five  uM M g  different  2 +  .  The  results  determinations.  represent  the  m e a n ± SEM o f  Nucleotide cone. (mM)  -  stimulated  activities.  followed  by A D P .  presence  of  substrate,  The  produced  by  conversion  preparation.  activity  was  adenyl  that  systems  Rather,  stimulation  2  +  Figure or M g  2  dependency  +  activity Ca  2 +  of  -ATPase  of  ADP  the  a  by  of  enzyme,  activity  and  20°C  for  be  as  kinase  present  was  the  that  this  of  the As  adenylate shown  adenylate  in  was  observed  pH on  the  exhibited  pH o p t i m u m a r o u n d the  effect  transition 2 +  -ATPase  of  to  the  case,  ADPase  12,  Ap5A  activity  ADPase at  in  activity.  0.5  enzyme a  due  kinase  kinase  activity  on  a  in  Figure  inhibit  of  ADP w a s  not  not  Mg  used  non-specific  did  a  the  adenylate  activities  indicating  not  to  of  effect  obtained  a  due  effect  1975),  broad  possibly  substrates,  the  inhibit  the  ATPase  data  that  suitable  could  not  (Ap5A).  ADPase  relatively  plot  is  presence  et_ a l  the  stimulated  the  enzyme existed  of  13 i l l u s t r a t e s  with  an A r r h e n i u s  the  generally  (Feldhaus  most  activity  demonstrate  in  the  Pyrrophosphate  3',5'-Adenosine  various  Ca  To  determined  concentrations  Ap5A.  that  -  GTP w e r e  minimal  possibility  membrane  at  showed  suggesting  inhibitor,  and  5'-Nucleotidase.  phosphatase. ATP  AMP  ATP  91  and  1 mM  activity.  similar pH 8 .  pH  Figure  temperature  temperature  of  on 25°C  14 the  for  activity.  2+  4.  Regulation of Pancreatic Acinar Plasma Membrane Ca Calmodulin Having  secretion  demonstrated  in  a  Ca  that  the  24-dependent  phenothiazines  manner,  possibly  inhibit  through  -ATPase by amylase  calmodulin  24inhibition, also  it  regulated  was by  of  interest  calmodulin.  to  determine  whether  the  In  these  experiments  low  Ca Ca  -ATPase 2  +  is  is  -  Figure  12  92  -  E f f e c t of a d e n y l 3 ' , 5 ' - a d e n o s i n e (Ap5A) on C a ^ - A T P a s e a c t i v i t y i n p l a s m a membrane p r e p a r a t i o n s o f acinar cells. C a - A T P a s e a c t i v i t y w a s d e t e r m i n e d a t 8 8 uM C a a n d 0 . 5 mM A D P . D a t a r e p r e s e n t t h e m e a n ±SEM o f three separate experiments. 2 +  C a - A T P a s e Activity: 2+  gmoles Pi/mg prot/hr O)  — i —  > Ol >  o  o  3 O  o o o ro •  Ol  o  Ol  C-OJ  -  Figure  13  94  -  E f f e c t o f pH o n A T P h y d r o l y t i c a c t i membranes of a c i n a r c e l l s . ATPase d e t e r m i n e d a t 0 . 5 mM A T P a n d 8 8 uM Mg (O O). The r e a c t i o n medium 2 +  v i t y i n plasma a c t i v i t y was Ca (• • ) o r 88 was b u f f e r e d w i t h 2  +  T r i s - g l y c i n e - m a l e i c a c i d a n d a d j u s t e d b e t w e e n pH 6 9. R e s u l t s r e p r e s e n t t h e mean o f two d i f f e r e n t d e t e r m i n a t i o n s e a c h done i n d u p l i c a t e .  and  uM  - 96 -  Figure  14  A r r h e n i u s p l o t s of ATPase a c t i v i t y as a f u n c t i o n of temperature. A T P a s e a c t i v i t y was d e t e r m i n e d i n a c i n a r membrane p r e p a r a t i o n s a s d e s c r i b e d i n M e t h o d s i n t h e p r e s e n c e o f 8 8 uM C a ( • — • ) o r 8 8 uM M g (0....0). T h e p H w a s a d j u s t e d t o pH 7 . 4 a t e a c h t e m p e r a t u r e setting. R e s u l t s r e p r e s e n t t h e mean o f two d i f f e r e n t e x p e r i m e n t s e a c h done i n d u p l i c a t e . 2  +  2  +  - 97- -  1/Tx1000  -  concentrations found  were  used  to  be  only  presence  of  endogenous  as  marginal  98  -  calmodulin  at  Mg  higher (1.5  -  activation  of  the  Ca  Oa "*" c o n c e n t r a t i o n s . uM)  calmodulin  -ATPase  In  2  2  +  was  the  activation  of  the  Figure  exogenous  2+ Ca  stimulated  calmodulin manner.  activity  stimulated  At  increasing  Using  a  to  radioimmunoassay preparations  presence  of  enzyme  bound to  |jM f r e e Figure  Ca Ca 16  and  +  was  agents  found  shows  the  that  activity  enzyme  in  a  activity  showed the  in  ± 3.5  could  in  part  explain  calmodulin.  and  CPZ  Ca  TFP  would  -ATPase and  and  It  (up  ng/mg the  beyond of  of  280  175  1.7  uM.  the  plasma  poor  The  response  determined  was  if  of  the  stimulation  measured  nM  uM)  by  protein.  calmodulin  activity  to  increased  content  was  block  absence  CPZ  was  calmodulin  15,  dose-dependent  saturation  41.8  presence  while  shown  be  added  TFP  As  to  activity.  in  the  1 uM a n d  calmodulin  -ATPase 2  -ATPase  technique,  exogenously  'anticalmodulin' the  Ca  890% a t  membrane  of  the  observed.  2 8 0 nM c a l m o d u l i n ,  244%,  the  was  at  0.4  calmodulin. did  not  affect  2+ the  basal  Ca ^-ATPase  activity,  the  phenothiazines  inhibited  a dose-dependent  manner.  calmodulin  o_i_  stimulated maximal uM T F P  Ca  -ATPase  inhibition and  55  the  uM C P Z ,  was  reported  the  phenothiazines,  stimulated  of  by  Ca  activity  calmodulin  with  Gietzen is  in  maximal  inhibition  et^ a l _ ( 1 9 8 3 ) a more  -ATPases.  As  stimulated  that  specific shown  in  activity  observed  Compound  inhibitor Figure  17,  occurred  at  48/80,  of  The  125  half at  uM.  30 It  unrelated  to  calmodulin  compound  48/80  o x  inhibited  activation  calmodulin  (200  400  did  ug/ml,  nM) not  of in  have  acinar a  plasma  membrane  dose-dependent any  effect  on  Ca  fashion.  the  basal  -ATPase Compound  activity.  activity 48/80,  up  by to  - 99 -  Figure  15  C o n c e n t r a t i o n s dependence of c a l m o d u l i n s t i m u l a t i o n of Ca ATPase activity. ATPase a c t i v i t y i n the presence of 0 . 4 uM C a and endogenous M g ( m e a s u r e d t o be l - 2 u M ) . D a t a r e p r e s e n t the mean ± S . E . M . o f t h r e e separate determinations. 2 + -  2  +  2  +  -MOO  [Calmodulin^  •-  pM  -  Figure  16  101  -  E f f e c t of p h e n o t h i a z i n e s on c a l m o d u l i n s t i m u l a t i o n o f Ca A T P a s e a c t i v i t y i n p l a s m a membranes of acinar cells. R e a c t i o n m e d i u m c o n t a i n e d 0 . 4 uM C a , endogenous Mg ( 1 - 2 pM) a n d t r i f l u o p e r a z i n e ( A A) chlorpromazine ( 0 . . . . 0 ) o r 2 8 0 nM c a l m o d u l i n , t r i f l u o p e r a z i n e ( A — A ) and c h l o r p r o m a z i n e (• •). D a t a r e p r e s e n t t h e mean ± 2 + -  2  2 +  S.E.M.  of  four  separate  determinations.  +  ATPase ACTIVITY (%)  - 103 -  Figure  17  E f f e c t o f C o m p o u n d 48/80 o n c a l m o d u l i n s t i m u l a t i o n o f Ca -ATP ase a c t i v i t y i n p l a s m a membranes of acinar cells. A T P a s e a c t i v i t y w a s d e t e r m i n e d a t 0.4 uM C a , endogenous M g (1-2 pM) a n d i n t h e p r e s e n c e ( • — • ) and 2  2  absence mean o f  (o two  +  +  o) o f 280 nM c a l m o d u l i n . Data represent e x p e r i m e n t s e a c h done i n duplicates.  the  - 104 -  The Figure for  effect 18.  of  Calmodulin  calcium;  no  concentrations presence  calmodulin  of  significant (>0.4uM).  stimulation  to  be  in  occurred  105  the  Ca  increased  endogenous  spectrometry  on  -  effect  the  range  0.03  2+  activation  sensitivity  was  Calmodulin  calcium,  at  the  -  of  of  observed  at  stimulation  determined 6-7  uM f r e e  by  uM.  Ca  24-  curve the  in  system  Ca  observed  atomic  in  the  absorption  Maximal the  shown  enzyme  higher  was  , with  is  calmodulin degree  of  24stimulation  decreasing  with  presence  of  endogenous  Ca  activity  was  decreased  by  medium was  (Table  VI).  abolished.  the  This  24-  Ca  total), basal  Ca  In  endogenous  2 +  -ATPase to  calmodulin  Ca  concentrations.  stimulation  1 mM E G T A w a s  suggest  is  free  of  dependent  either  required  the to  the  Mg  24-  2 +  the  calmodulin  for  Ca  the  -ATPase  reaction  calmodulin  on e n d o g e n o u s  2t  Mg ^ i s  added  1 mM E D T A ,  that  of  In  stimulation  stimulation (1.5  -  2  expression  of  of  uM  the  activity.  further were  membranes  ,  presence  could  or  membranes  produce  the  activity  that  +  20% w h e n  -ATPase  order  plasma  In  2  increasing  establish  incubated  depleted  of  the  with  role  of  Mg  2  +  1 mM E D T A a t  divalent  cations  in 37°C  this  system,  for  30  min  to  (EDTA-treated  24raembranes). on Mg  (Ca 24-  2 +  Figure + Mg  )-ATPase  2 +  19  illustrates  )-ATPase  activity  activity  refers  to  the  in that  effect  of  EDTA-treated activity  exogenously  added  Mg  membranes.  (Ca  +  present  2 +  following  24s u b t r a c t i o n of t h e a c t i v i t y measured i n the a b s e n c e of added Ca and i n 2424t h e p r e s e n c e o f Mg . W h i l e 0 . 0 5 uM f r e e C a failed to stimulate the (Ca  24-  + Mg  addition This  24-  of  )-ATPase  0.5  activity  uM M g  was  activity  24-  of  resulted  further  EDTA-treated in  increased  membrane  stimulation by  1,  2,  and  of 5  the  preparations, ATPase  uM M g  24-  .  activity. The  - 106 -  Figure 18  The effect of exogenous calmodulin on Ca -ATPase a c t i v i t y i n plasma membrane enriched preparations of pancreatic acinar c e l l s . ATPase a c t i v i t y i n the presence of increasing Ca -concentrations ( 0 . . . . 0 ) ( M g estimated to be 1-2 uM), and a calmodulin concentration (CaM) of 280 nM (•—•) Data represent mean ±SEM of four different experiments. 2+  2+  ATPase ACTIVITY ( u m o l e s Pi • m g protT - h r . ) 1  1  -  108  Table Effect  of  -  VI  EGTA a n d EDTA on C a l m o d u l i n 2+ Ca -ATPase Activity.  Addition  Ca  2 +  -ATPase  Activity  Control  Calmodulin  + 1 mM E D T A  Calmodulin  +  1 mM E G T A  A T P a s e a c t i v i t y was a s s a y e d i n E G T A , 0 . 1 mM o u a b a i n , 2 mM N a N ( e s t i m a t e d t o b e 7 uM t o t a l ) . a t a c o n c e n t r a t i o n o f 280 n M . different determinations. free  calcium  (%  Control)  100  Calmodulin  Endogenous  Stimulated  1438 ±  70  100 ±  10  1154 ±  50  4 8 mM T r i s - H C l p H 7 . 4 , c o n t a i n i n g 0 . 0 6 mM , 0 . 5 mM A T P a n d e n d o g e n o u s Ca When c a l m o d u l i n was p r e s e n t i t was a d d e d D a t a r e p r e s e n t mean ± S . E . M . o f five 2  3  concentration  is  9.4  nM.  +  -  Figure  19  109  -  (Ca + Mg ) - A T P a s e a c t i v i t y i n E D T A t r e a t e d p l a s m a membranes of a c i n a r c e l l s . Calcium activation curve +  +  2+ d e t e r m i n e d i n t h e a b s e n c e o f Mg ( 0 ) and i n t h e p r e s e n o o f 0 . 5 uM ( • ) , 1 uM ( A ) , 2 uM ( • ) a n d 5 uM ( • ) Mg f ( C a + Mg )-ATPase a c t i v i t y r e f e r s to that a c t i v i t y present f o l l o w i n g s u b t r a c t i o n of the a c t i v i t y measured i n the a b s e n c e o f a d d e d c a l c i u m a n d i n t h e p r e s e n c e o f Mg . D a t a r e p r e s e n t t h e mean ± S . E . M . o f t h r e e separate determinations. +  (Ca  2 +  + M g ) - A T P a s e ACTIVITY 2+  (pmolesPimg protT - hr." ) 1  1  -  increase because total  in  activity  the  was  not  contribution  ATPase  activity  of  due Mg  (Table  2+  -  I l l  to  the  -ATPase  VII).  additive  effect  had  subtracted  been  Therefore  it  of  appears  Mg  "'"-ATPase  from  that  the  the  effect  2+ of  Mg It  the  is  to  increase  the  was  of  interest  to  calcium  indicates observed  that  with  the  expression  produce In  a  of  curve  to  of  Ca  effect  obtain  of  the  2+  the  presence conclusion  the  Mg  2  of  EDTA-treated  or  absence  that  affinity on  of  for  Mg  the  and  +  2  +  the  calmodulin  Figure  membranes  2+  of  Mg  is  required  Calmodulin  of  calcium.  membranes.  activity.  information  enzyme  effect  EDTA-treated  -ATPase  on  of  the  stimulation  previous the  similar  order  determine  calmodulin  irrespective  agreement the  activation  sensitivity  enzyme  mechanism by  .  This  for  20 was is  in  only  a n d Mg  2+  on  for seem  to  calcium.  which  calmodulin  2+ activates  the  Ca  -ATPase,  interacts  directly  with  the  it  is  important  ATPase  to  molecule.  know To  if  calmodulin  resolve  this  problem,  125 an  I-labeled  procedure target of  I O C  21). the  indicates  proteins I-labeled In  calmodulin the  present  to  autoradiogram,  overlay  ability on  calmodulin  contrast  gel  other  the to  of  calmodulin  gel. a  The  number  calmodulin  calmodulin  technique  binding  employed.  bind  autoradiogram  of  membrane  binding to  to  was  a  denatured  shows  proteins  proteins  133,000  to  and  that a  This  the  binding  (Figure appeared  230,000  dalton  2+ protein EGTA.  were  Ca  -dependent  as  no  binding  occurred  in  the  presence  on  of  -  112  Table Mg  2 +  -ATPase  Activity  Preparations  Mg  +  Cone.  uM  in  -  VII  EDTA T r e a t e d  from P a n c r e a t i c  Mg  -ATPase  Plasma  Acinar  Activity  Membrane  Cells.  (umoles  0.5  0.16  + 0.07  1.0  0.23  + 0.05  2.0  0.52  + 0.12  5.0  2.05  + 0.35  10.0  3.37  + 0.66  Pi/mg  prot/hr)  M e m b r a n e p r e p a r a t i o n s w e r e i n c u b a t e d w i t h 1 mM T r i s E D T A , pH 8 . 0 f o r 3 0 min at 37°C. The membranes were washed t w i c e and r e s u s p e n d e d in T r i s - H C l b u f f e r pH 7 . 4 . Mg -ATPase a c t i v i t y r e f e r s to the a c t i v i t y in t h e a b s e n c e o f a d d e d Ca and i n the p r e s e n c e of v a r i o u s concentrations of M g . 2 +  2 +  -  Figure  20  113  -  T h e e f f e c t o f c a l m o d u l i n a n d Mg on t h e c a l c i u m a c t i v a t i o n c u r v e o f EDTA t r e a t e d p l a s m a m e m b r a n e s o f acinar cells. M e m b r a n e s w e r e i n c u b a t e d w i t h 1 mM T r i s E D T A p H 8 . 0 f o r 30 m i n a t 3 7 ° C , w a s h e d f r e e o f E D T A a n d assayed f o r ATPase a c t i v i t y . The c a l c i u m activation c u r v e i n t h e a b s e n c e o f M g ( o ) , i n t h e p r e s e n c e o f 0 . 5 uM M g ( • ) , 2 8 0 nM C a l m o d u l i n ( A ) a n d M g plus calmodulin (A) i s s h o w n . D a t a r e p r e s e n t t h e mean ± S . E . M . o f three separate determinations. +  2  +  C a - A T P a s e Activity: 2+  prnoles Pi/mg prot/hr  00 r\ i  •-•.-I  o  <Q  I  o 0)  _ Ol  -  Figure  21  115  -  C a l m o d u l i n g e l o v e r l a y and a u t o r a d i o g r a p h y of target p r o t e i n s i n p l a s m a membranes of a c i n a r cells. [ I ] - C a l m o d u l i n b i n d i n g i n t h e p r e s e n c e o f 1 mM C a ( L a n e A ) and i n t h e a b s e n c e o f Ca ( 5 mM E G T A ; L a n e +  B).  -  5.  117  -  Regulation of Pancreatic Acinar Plasma Membrane CarJ-ATPase  by  Phospholipids It acids  has and  been  limited  membrane-bound Roufogalis Ca  -ATPase.  activated  (Gietzen or  Since  by  that  on  mimic  et j i l  the  this  the  was  effect  et  al  acinar  Figure  fatty  on  (Al-Jobore  plasma  22  unsaturated  calmodulin  1981)  investigated  system.  of  solubilized  (Niggli  pancreatic it  phospholipids,  1982),  purified  calmodulin,  effect  acidic  proteolysis  1981b)  24-  similar  reported  and  erythrocyte membrane  whether  Ca  24-  -ATPase  phospholipids  demonstrates  that  is  had  asolectin  a (a  2-4-  mixture plasma  of  membranes  require  the  observed was  phospholipids) by  approximately  presence  by A d u n y a h  reconstituted  asolectin  is  of et  in  the  acidic  al  composed  mainly  Ca  A  were  -ATPase This  similar  when  Thin-layer  of  activity  activation  activating  purified  of  did  effect  erythrocyte  chromatography  acinar  not was  Ca  2 +  -ATPase  revealed  that  phosphatidylcholine,  phosphatidylserine  phospholipids  phospholipids  (1982)  the  7-fold.  calcium.  asolectin.  phosphatidylinositol, When  stimulated  tested  and  their  separately,  cardiolipin,  it  lysoderivatives. was  observed  that  the  phosphatidylserine, 24-  andphosphatidyllnositol 23).  As  expected,  failed  to  found  that  stimulate  stimulating  the the  phospholipids solubilized The  reason  pure  the  ATPase  It  produce  this  activity  phospholipids  enzyme.  to  the  phosphatidylcholine  erythrocyte for  stimulated  is  were  required  Ca  which  not  2 +  -ATPase  apparent  is  not  as  23).  than  (Al-Jobore perhaps  the  as  10-fold  that  and  (Figure phospholipid  However,  effective  approximately  but  activity  a neutral  (Figure  maximum a c t i v a t i o n Ca  -ATPase  it  asolectin higher  determined  Roufogalis enzyme  was in  pure for  1981).  requires  a  -  Figure  22  118  -  A s o l e c t i n s t i m u l a t i o n o f Ca -ATPase a c t i v i t y i n plasma membrane e n r i c h e d f r a c t i o n s o f a c i n a r c e l l s . Asolectin s t i m u l a t i o n i n t h e p r e s e n c e ( • ) a n d a b s e n c e ( o ) o f 0 . 4 pM Ca . R e s u l t s r e p r e s e n t mean ± S . E . M . o f t h r e e separate determinations. +  -ATPase Activity: prnoles Pi/mg prot/hr  -  Figure  23  120  -  T h e e f f e c t o f e x o g e n o u s p h o s p h o l i p i d s on Ca -ATPase a c t i v i t y i n p l a s m a membrane e n r i c h e d f r a c t i o n s o f acinar cells. Enzyme a c t i v i t y was a s s a y e d i n t h e p r e s e n c e o f phosphatidylcholine (O-—o) , phosphatidylinositol (• — • ) , p h o s p h a t i d y l s e r i n e (*"• *) and c a r d i o l i p i n ( A ^ A ) . D a t a r e p r e s e n t t h e mean o f two d i f f e r e n t experiments each i n duplicates. —  -121 -  T °- r 75  I PHOSPHOLIPID I jjg/ml  -  milieu for  of  optimum Thus  Ca  2 +  different  phospholipids  122  -  rather  than  a  specific  phospholipid  activation.  far  -ATPase  it  has  has  a  been  demonstrated  number  of  that  properties  the  acinar  plasma  characteristic  of  membrane  the  2+ Ca  -transport  ATPase  in  other  systems.  A  common  feature  of  these  2+ ATPases  is  the  formation  intermediate.  In  the  a  Ca  An a u t o r a d i o g r a m  hydroxylamine 24.  of  sensitive  absence  phosphorylated  of  -dependent of  experiments  acyl-phosphate Ca  Q  I  intermediate  labile  (in  the  was  formed  phosphorylated  designed  intermediate  presence  of  (Figure  is  0.4 24  to  identify  shown  mM E G T A )  lane  A).  in  the  Figure  no Addition  of  2+ 80  uM C a  the O  resulted  region  of  115,000  the  formation  daltons  of  (Figure  a phosphorylated  24  lane  j  Mg  B).  In  intermediate  the  presence  in  of  oj  alone  (in  2+ Ca  in  the  presence  of  endogenous  two  phosphorylated  Ca  ),  or  in  the  presence  of  2+ a n d Mg  132,000  and  unstable It  ,  115,000  in  has  regulate  the  been known  by  cytoplasmic identify kinases  presence  certain  catalysed  Figure  cellular  protein protein  under  various  lane  sometime  that  functions. enzymes  proteins  In  were Both  (not  associated  conditions.  phosphorylation  were  (M  r  phosphoproteins  phosphorylation  phosphorylation with  experiment  phosphorylated  formed  were  shown).  membrane  Membrane  this  experimental  kinase  C and D ) .  hydroxylamine  kinases.  membrane  protein  of  kinase  any  measuring  for  24,  intermediates  by  the  were  endogenous  utilized,  can  membrane  attempts  Standard  may  or  by  made  to  protein  procedures such  be  as  a  for high  -  Figure  24  123  -  SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and a u t o r a d i o g r a p h y of a c y l phosphate i n t e r m e d i a t e s of p a n c r e a t i c a c i n a r plasma membranes. p-bound to p h o s p h o p r o t e i n s s e p a r a t e d o n 8% s l a b g e l . A lane: no added i o n s (presence of EGTA); B l a n e : Ca (80 uM); C lane: Mg (80 uM); D l a n e Ca + Mg ( 8 0 uM a n d 8 0 uM). 2  +  2 +  +  2  +  Mol. wt."  200116674530-  14-  x10 .  -  ATP  concentration  shown  in  and  a  longer  similar  phosphorylation  presence  and  absence  of  of  Ca " " p r o d u c e d 1  dalton  The  low  represent  a  greater  protein  bands  phosphorylation  2 +  degree  (Figure  molecular  Ca  25  at  of  .  of  gel  membrane  the  phosphorylation  of  platelet  and  red  membranes  kinase.  This  a  role  cell  plasma  suggests  in  the  that  regulation  calmodulin  front  (Figure  lipids.  As  observed in  of  a  24  and  Enyedi  by  cyclic  phosphorylation  of  min).  the  both  presence  19,000  and  a  C).  polyphosphoinosites  the  (5  were  phosphorylation  lane  the  time  patterns  However  reported  play  incubation  25,  the  30,000  mM)  -  Figure  in  2  (0.5  125  cellular  25)  e_ _1  in  (1983)  has  lymphocyte,  AMP-dependent of  may  membrane  protein  lipids  may  functions.  2+  6.  Determination of the Presence of a Ca Plasma Membrane V e s i c l e Preparations 2+ Many  Ca  affinity  Ca  -transport 2 +  -ATPase  processes  activity  are  -Transport Process In Acinar  mediated  (Penniston  by  1982).  a membrane Therefore  bound  it  high  was  2+ investigated plasma  membranes  studies, vesicle In  whether  plasma  an ATP-dependent  Ca  obtained  from  pancreatic  membranes  were  resuspended  -transport acinar in  0.3  process  cells.  existed  For  M sucrose  in  these to  favour  formation. view  of  the  uncertainty  of  the  'sidedness'  of  the  (Mg  +  2+ Ca in  )-ATPase at t h i s s t a g e of the t h e l i t e r a t u r e , i t was d e c i d e d  s t u d i e s and the c o n t r o v e r s y t h a t b o t h A T P - d e p e n d e n t Ca  existing efflux  2+ from pre-loaded measured.  vesicles  and ATP-dependent  Ca  -influx  should  be  -  Figure  25  126  -  SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and autoradiography of phosphorylated p a n c r e a t i c acinar plasma membranes. P-phosphorylated proteins separated on a g r a d i e n t (5-20%) g e l . A lane: no a d d e d i o n s (2.2 mM E G T A ) ; B l a n e : C a * (90 uM); C l a n e : Ca + C a l m o d u l i n ( 9 0 uM a n d 1 0 0 n M ) ; D l a n e ; c a l m o d u l i n + E G T A ( 1 0 0 nM a n d 2 . 2 m M ) ; E l a n e : no added i o n s . 2  2  2  +  -U7-  A B C D E  In  45 Ca  preliminary  2+  were  associated  loaded  in  suggested  that  involved.  A  efflux the  experiments,  with  the  perhaps  from preloaded  vesicles,  of  of  there  of  was  was  a  slow  was  but  -  observed  that  the  amount  was  consistently  lower  than  when ATP was  absent.  an A T P - d e p e n d e n t  experiments vesicles  by  it  ATP  an ATP-dependent  followed  128  vesicles  presence  series  presence  the  -  were  then  extrusion  steady  in  Figure  26  system  in  uptake.  when  vesicles  This  24-extrusion  performed  determined.  Ca  Ca  of  The  which  process '  t 5  Ca  illustrates the  obvious  membrane concern  at  24this  point  presence  was  or  extrusion  whether  absence  was  the  included  in  shown  Figure  in  presence  ATP  ATP-dependent  absence  was  incubation  was  existing  45  the  accumulation  ATP  Ca  content  T  similar,  as  process.  and  media.  Ca  content  much h i g h e r ,  measured.  of  ATP  only  However,  of  therefore  of  initial  of  would To  the  be  test  vesicles  expected  this,  in  if  Ca  oxalate  the 24-  was  24p r e l o a d i n g medium i n o r d e r t o p r e v e n t Ca -efflux. 242 7 , Ca - e f f l u x was g r e a t l y r e d u c e d u n d e r t h e s e  the  conditions.  the  45  these  Ca  and  vesicles  the  agents  observed  Ca  were were  was  preloaded  existence  dependent  vesicles  these  24Ca -uptake  The  ATP  studies  then  the  suggesting  process.  In  of  of  added  rapid,  the  an  2-4-uptake  formed  in  As  in  to  was the  the  reaching  a  24maximum i n  2 min  the  calcium  the  vesicles  (Figure  ionophore to  28).  A23187  a n EGTA  The (5  accumulated  uM).  containing  It  buffer  was  Ca  could  observed  prior  to  be  that  released  transferring  filtration  increased  24-  the  uptake  rates.  incorporated  in  The  EGTA w a s h i n g  subsequent  step  experiments.  to  remove  bound  Ca  by  T  was  -  Figure  26  Ca  fluxes  In  129  -  preloaded  plasma  membrane  vesicles  prepared from pancreatic acinar c e l l s . Calcium loaded v e s i c l e s in the presence ( • — • ) and absence ( O — O ) of 5 mM A T P a t 0 ° C w e r e r e s u s p e n d e d i n non-radioactive medium and t h e t r a n s p o r t a s s a y p e r f o r m e d a t 3 7 ° C . The r e s u l t i s t h e mean ± S . E . M . o f t h r e e determinations.  -  -!d"  130  -  t6 TIME (minutes)  6X»  -  Figure  27  131  -  Ca c o n t e n t of p r e l o a d e d p l a s m a membrane v e s i c l e s prepared from p a n c r e a t i c a c i n a r c e l l s . Oxalate f a c i l i t a t e d calcium loaded v e s i c l e s i n the presence (•) a n d a b s e n c e ( o ) o f 5 mM A T P a t 0 ° C w e r e r e s u s p e n d e d in n o n - r a d i o a c t i v e medium and t r a n s p o r t a s s a y p e r f o r m e d a t 37°C. R e s u l t s r e p r e s e n t t h e mean ± S . E . M . o f three determinations.  - 132 -  Time (min.)  - 133 -  Figure  28  T i m e - d e p e n d e n t Ca membrane v e s i c l e s . uM C a mM A T P . 2  +  in  the  -uptake i n pancreatic acinar plasma Ca - u p t a k e i n a medium c o n t a i n i n g  presence  (•  •)  and  absence  75  (0....0) o f  T h e v e s i c l e s w e r e p r e t r e a t e d w i t h 2 mM E G T A p r i o r t o t h e transport assay. R e s u l t s r e p r e s e n t t h e mean ± S . E . M . o f four different experiments.  5  -134---  -  Figure varying  29  free  increase  in  represents Ca  Ca  2  2-4-uptake  -  of  Mg  effect  concentrations.  +  2-4-uptake  the  135  activity  on Ca  +  Addition  of  indicating  -uptake  Mg  that  produced  2 +  Mg  activity  2+  is  a  marked  essential  the  Ca  7.  Determination of the Orientation of the C a t a l y t i c S i t e of the Ca  order  plasma  of  it  was  enzyme. was  cells  suspended  -ATPase  ATPases,  Ca  high 2  +  ATP  or M g  unchanged  intact  cells  cytoplasmic  An also  was  not  enzyme total)  investigation  carried  cytoplasm  activity 30).  out  of  than  VIII due  in  the  the  as  in  the  the  to  lactate  functional  of  the  a majority  would  be  the  by  in  the  or  2-4-  and  is  acinar site  acinar of of  the the  -transport  On t h e of  contrary, either  largely thawing  externally-oriented  ATPase  activity  damaged  dehydrogenase  the  site  presence  freezing  the  broken  Ca  remained  activity  that  active of  in  catalytic  activity  observed.  activity  cells  the  in  pancreatic  hydrolytic If  observed  role  of  dispersed  observed  90% o f  activity  orientation  ATP  ATPase  mainly  )-ATPase  medium.  indicates  these of  the  was  The  2 +  possible  activity  marker, in  a  incubation  greater Table  to  measure  disruption  that  VIII).  of  the  (Figure  (Table  to  the  + Ca  2 +  availabilty  hydrolytic  after  indicating  (0.005%  in  ATP  +  (Mg  determine  the  possible  hydrolytic 2  to  With  faces  no  the  preparations  important  it  a  relate  membrane  this  2_i  process.  to  cells  Ca  for  -ATPase A c t i v i t y i n the Plasma Membranes of Acinar C e l l s  In  cell  at  was  cells  observed  since  hardly  in  the  detectable  preparations. 'sidedness'  vesicle  of  preparation  the  (Mg  used  to  2 +  + Ca  2 +  )-ATPase  measure  was  - 136 -  Figure  29  ATP-dependent C a ^ - u p t a k e as a f u n c t i o n of Ca c o n c e n t r a t i o n i n p a n c r e a t i c a c i n a r membrane vesicles. Ca u p t a k e i n the presence ( • — • ) and absence (o-—O) o f 5 mM M g added t o the i n c u b a t i o n medium f o l l o w i n g vesicle sealing. T h e v e s i c l e s w e r e p r e t r e a t e d w i t h 2 mM EGTA p r i o r t o t h e t r a n s p o r t a s s a y . Data represent the mean ± S . E . M . o f f o u r d i f f e r e n t experiments. 2 + _  2 + -  2  +  -  137  -  400 ICa l free 2+  -  Figure  30  138  -  ATPase a c t i v i t y of i n t a c t p a n c r e a t i c a c i n a r c e l l s and homogenates. ATP h y d r o l y t i c a c t i v i t y i n c e l l suspension (10 c e l l s / m l ) i n the presence of C a (• • ) and M g 6  ,  +  ( A — A ) or c e l l homogenates i n the presence of (O O ) a n d Mg ( A — A ) . Data represent the S.E.M. of four d i f f e r e n t determinations.  2  Ca mean ± +  +  -•139 —  0  050 I ION I mM  tx>o  -  140  Table ATPase  and L a c t a t e  -  VIII  Dehydrogenase A c t i v i t i e s of I n t a c t A c i n a r C e l l s and Homogenates.  Specific Intact  Activity  Pancreatic  (umoles/mg  Cells  Cell  prot/hr)  Homogenates  Ca "'"-ATPase  4.26  ±0.6  4.16  ±  0.8  Mg  5.22  ±0.3  5.78  ±  0.5  1.81  ±  0.5  2 +  -ATPase  Lactate  dehydrogenase  0.01  ±  0.0  I n t a c t a c i n a r c e l l s were resuspended i n phosphate f r e e K r e b s - R i n g e r b i ^ a r b o n a t e s o l u t i o n c o n t a i n i n g 2 mM A T P , 5 mM N a N a n d 1 mM C a or Mg a t a c o n c e n t r a t i o n o f 10 c e l l s / m l f o r 15 m i n a t 3 7 ° C . A portion of t h e c e l l s u s p e n s i o n was s i m i l a r l y t r e a t e d e x c e p t t h a t i t was s u b j e c t e d t o f r e e z i n g and t h a w i n g b e f o r e enzyme a s s a y (cell homogenates). I n b o t h cases a l i q u o t s of the i n c u b a t i o n media were c e n t r i f u g e d a t 1 0 , 0 0 0 rpm f o r 45 s e c a n d t h e s u p e r n a t a n t a s s a y e d f o r i n o r g a n i c phosphate or l a c t a t e dehydrogenase a c t i v i t y . Specific a c t i v i t y i s e x p r e s s e d a s u m o l e s p e r mg c e l l p r o t e i n . Results represent mean ± S . E . M . o f f o u r d i f f e r e n t experiments. 2  3  +  -  ATP-dependent orientation exhibited that of  of  these  as  an  -transport  that  were  catalytic thawing  (see  in  (Mg "  membranes  vesicles  and  noted  accessible  broken  accessible freezing  Ca  the r  141  the  previous  intact  + Ca  (Table  -  )-ATPase  IX).  right-side  site,  neither  could  unmask  cells  out  This  section). was  indicates  deoxycholate latent  (0.04  the  vesicles  comparable that  Consistent  activity  similar  observed;  activity  (> 9 5 % ) .  A  -  (Table  the  majority  with  0.25 IX).  with  mM)  a  fully nor  -  142  Table The  -  IX  O r i e n t a t i o n of C a - A T P a s e A c t i v i t y Membrane V e s i c l e Preparations. 2 +  Treatment  Plasma  % Control  100  Control DOC  in  40  uM  101.3  80  uM  103.8  120  uM  98.7  160 uM  98.5  200  uM  91.0  240  uM  90.7  Freeze  thaw  lx  101.2  Freeze  thaw  2x  97.5  P l a s m a m e m b r a n e v e s i c l e p r e p a r a t i o n s w e r e i n c u b a t e d i n 2 0 mM T r i s buffer p H 7 . 4 c o n t a i n i n g 0 . 3 M s u c r o s e , 2 mM N a N , 0 . 5 mM A T P , 2 1 uM f r e e Ca and i n the p r e s e n c e of v a r i o u s c o n c e n t r a t i o n s of d e o x y c h o l a t e (DOC). D a t a r e p r e s e n t t h e mean o f two d i f f e r e n t e x p e r i m e n t s , e a c h d o n e i n duplicate. 2  3  +  -  143  -  DISCUSSION  A  Ca "'"-ATPase  enriched  activity  preparations parallels  ing  Ca "'"-ATPase  the  Various Lambert  and  workers  been  characterized  from pancreatic  distribution that  has  that  of  Christophe  plasma  activity  (Forget  and  1978;  acinar  Le  is  Bel  cells.  membrane  et  1976; al  plasma Its  marker  endogenous  Heisler  in  to  subcellular  enzymes,  the  plasma  Milutinovic  1980;  membrane  Martin  __  and  suggestmembrane.  al  1977;  Senior  1980)  2+ have  described  pancreatic  a  Ca  tissue  -ATPase  activity  homogenates.  in  Since  plasma  the  membranes  pancreas  prepared  is  a  be  directly  from  heterocellular  2+ organ, to  the  acinar  Ca cell  population decrease insulin cells  of  in  acinar  insulin  (Korc _ t  detect In  al  1978)  other  cell  contrast __  be  to  in  the  one  staining  previous  Senior  1980)  which  millimolar  range,  employed showed  from  homogeneous  a  35 -  contamination  cell  type  with  The  binding  than  by  75% residual  sites  on  p-cells.  features  u s e d was  related  a  preparation.  dislodged  studies  Lambert  in  final  not  acinar Cyto-  characteristics  general  enough  to  present.  1977;  and  study  technique  al  Martin  could  Radioimmunoassay  rather  type  observed  present  insulin  revealed The  Milutinovic  the  levels  cells.  any  The  cells.  may  analysis  acinar  activity  function.  detected  chemical of  -ATPase  and  (Forget  Christophe  reported  a high  and H e i s l e r  calcium  affinity  Ca  2 +  1978;  Le  1976; Bel  sensitivity  -ATPase  with  et_ a l of  a Kj  the for  2+ Ca  of  1.7  uM w a s  observed  in  these  plasma  membrane  preparations.  1980; ATPase  -  Since is  the  typically  regulation buffer  reveal  al  1981).  concentration  between  of  0.1  and  intracellular  system  to  could  free  and  the  the  high  These  explain  low  affinity  the  high  -  calcium  uM,  free  this  in  not  may  of  (0.5  the  Ca  employed  affinity  acinar play  cell a  concentrations.  concentrations  were  the  enzyme  calcium  component  conditions  why  of  10  ATP  144  mM)  by  component  used  not  in  the  Ca-EGTA  are  necessary  ( L o t e r s z t a j n et_  previous  was  role The  -ATPase  2 +  cytosol  workers  observed  and  in  their  studies. By this  the  Ca  use  24-  -ATPase  endogenous  or  requirement cells  from  et  1981)  al  cells  of  in  activity  tightly  of  this  corpus  requirements.  for  Ca  2+  24-  Mg 2  ( S h e n j^t a l  Increasing  apparent  plasma  have  24-  t h e Mg  ATP  et  to  satisfy  1981),  Ca  1982)  and  been  shown  to  hydrolytlc  the  2  +  -ATPase  of  (Lotersztajn  embryonic have  bone  similar  increases  rates  Mg  24-  liver  al  concentration  of  Presumably,  membrane  and P e n n i s t o n  1983)  requirement  demonstrated.  (Kotagal  reaching  an  sufficient  The  (Verma  islets  was  is  +  -ATPase.  luteum  eventually  membranes,  bound M g  pancreatic  culture  activity,  EDTA-treated  the  similar  to  Mg  2  +  ATPase that  24-  observed  in  that  the  activity  with  a  First,  the  presence  catalytic saturating  maximum a c t i v i t y . have  similar  substrate transition  of  measured site  that  Ca in  alone. the  has  pH o p t i m a .  requirement  both  Third,  profile.  temperatures  for  the  presence  different  concentrations Second,  Evidence  of Ca  Ca  2  +  of  and M g  -ATPase is  2  or  +  affinities  24-  there  Ca  obtained  a  2  +  a n d Mg  striking  Mg  Ca  and  2  +  is  +  the  one  the  -ATPase  same activities  similarity observed  activity  and  enzyme  cations:  24-  differences  +  for  2  produce  However, 2  Mg  indicates  the  in  their  in possible  -  presence the  of  basis  different  of  a  145  phosphoenzyme  single  -  intermediates  cannot  be  explained  on  enzyme.  2+ The  acinar  calmodulin; of  plasma  this  stimulation  trifluoperazine  trifluoperazine maximal those  (30  for  concentrations  of  those  for  Ca  2 +  and  required  -ATPase  of  and  the  other  was  -ATPase blocked  activity  by  calmodulin systems  activation 1981),  (55  (Roufogalis  of  high  uM)  (K^  of  produced  activity  1982).  by  concentrations  that  are  and  half  similar  Although  = 750 nM)  phosphodiesterase  much h i g h e r  stimulated  concentrations  stimulated  used were  was  micromolar  The  chlorpromazine  calmodulin  (Scharff  Ca  chlorpromazine.  uM)  inhibition  reported  membrane  to  the  compared  to  erythrocyte  concentrations  of  calmodulin  are  2+ required  for  membrane  (Kotagal  reticulum protein) explain  the  activation e_ _1  (Kirchberger that  this  remained decreased  calmodulin  used  calmodulin  in  (1.7  the  observed  In  the  presence  of  endogenous  estimated  20% o f  the  to  al  -ATPase  canine  membranes  to  the  is  only of  Calmodulin of  added  (7  The  In  ng/mg  isolation  could  concentration  total  of VI)  condition the  lost,  (42  (estimated  (Table  the  uM t o t a l ) . was  the  plasma  sarcoplasmic  during  of  cell  calmodulin  stimulation  under  islet  saturating  cells  calcium  activation  The  fraction  acinar  calcium;  9nM  a  of  ventricle  bound  responsiveness.  absence  calmodulin  and  Ca  1982).  1977).  be  the  and A n t o n e t z  cytoplasm  et  is  1982)  uM)  Vandermeers  of  amount  to  the  be  Ca  could of  presence  suggesting  be  the of  2 +  5  of uM;  -ATPase due  to  assay,  the  this  1 mM E G T A ,  that  of  perhaps  only not  2+ a l l  endogenous  further  Ca  increased  was to  chelated.  avoid  the  The  direct  EGTA effect  concentrations of  EGTA  were  on A T P a s e  not activity  -  observed Kotagal of  by  others  (Sarkadi  et^ al^ 1 9 8 3 ) .  1 mM E D T A  sensitivity  c a n be to  EDTA-treated  the  EDTA-treated  ATPase  performed  was  presence  Mg  lower  light  observations  of  the  upon  as  and R o u f o g a l i s  stimulation  that  Mg  shown by  2  in  confers  +  the Ca  experiments  1981a;  2  presence  +  with  19).  to  determine  The  consistently  fact  activity  both  alone.  Al-Jobore  calmodulin  the  revealed  observed  -  1979;  of  to  (Fig  membranes  stimulation  lack  related  membranes  Experiments  of  The  et_ j i l  146  complex  in  the  degree  addition of  the  of  effect  kinetics  absence of  of  uM C a  et^ j a l  calmodulin  (Fig.  added  stimulation  0.015  Kotagal  of  2+  (1983)  20).  Ca  was in  2+  Calmodulin  and  found  both  on  in  to  the  be  cases.  indicating  In  that  2+ Ca-EGTA  stimulates  possible on  the  that  the  ATPase  125  The  pancreatic Ca-EGTA  under  buffer  these  I-labeled  islet  plasma  used  may  membrane have  Ca  -ATPase,  produced  direct  it  is  effects  conditions.  calmodulin  gel  overlay  technique  revealed  a  2+ Ca  -dependent  weight Ca of  2 +  of  this  -ATPase,  a  protein  compares  dalton  identification  provides  to  reported  133,000  molecular  binding  of  weight  a  to  be  133,000  the  evidence  that  protein.  The  with  of  (Carafoli  binding  hydroxylamine in  dalton  favourably  138,000  calmodulin  observed  convincing  a  et  protein,  sensitive presence the  that al  molecular erythrocyte  1982).  coupled  The  to  the  phosphoprotein  of  2  Ca "  133,000  i r  a n d Mg  dalton  presence  of  2+  in  protein  similar this is  study  the  2i Ca  —ATPa s e  enzymes  it  Tawata  molecule, is  and  stimulated  and F i e l d  that by  (1982)  a  like direct  have  many  other  calmodulin  interaction  reported  the  with  presence  dependent  calmodulin. of  220,000  and  -  150,000  dalton  calmodulin  thyroid  plasma  membranes  calcium-dependent the  cytoskeleton  the  membranes  binding plasma  since  of  the  pancreas.  It  must  the  sarcolemma proteins  of  bind  to  _t  al  to  the  proteins in  plasma  appear  present Ca is  2  Ca  not  interact  treatment  of  acinar  in  other  the  exocrine  calcium-independent plasma  The  function been  It  has  (Carafoli  _t  al  large  acinar  1982)  shown and  proportion  column  can  be  of that  heart  of  the  eluted  with  2+ a  Ca  wash. In  calcium  intact  acinar  mediated  amylase  calmodulin-regulated This  corroborates  suggested pancreatic greater), intact amylase  that  cells  the  step  that  secretion  may as  shown  release, in  the  At  may h a v e  account indicated  for  that  possibly  by  phenothiazines acting  stimulus-secretion  may h a v e  secretion.  phenothiazines  cells  was  observations  calmodulin  enzyme  it  of a  Heisler  higher other the  on  inhibit  a  coupling  e_ a l  functional  (1981)  role  in  process.  who  (10  effects  function  by W i l l i a m s  _t  al  on  the  effects (1977)  have  exocrine  concentrations  inhibitory  of  calmodulin  with  known.  affinity  These  components  similar  pancreatic  a  be  pancreatic  several  -ATPase,  calmodulin  a  bovine  membranes.  to  exocytosis  in  and  after  and E G T A ) .  +  erythrocyte  in  may  that  liver  pellet  observed  though,  1982)  the  regulate  proteins of  rat  erythrocyte  protein  endogenous  binding  (Caroni  was  were  in  Interestingly,  This  noted,  purification  that  X-100.  proteins  (presence  calmodulin  during  be  in  remained  21).  -  proteins  binding  cytoskeleton  calmodulin-binding  these  not  = 230,000)  r  (Fig.  components  membranes,  they  Triton  (M  membranes  but  calmodulin  with  protein  binding  147  - l t  on and  M  or of  pancreatic Singh  -  (1980).  Phenothiazines  non-selectively possible  Phenothiazines  gradients  or  membrane-bound addition, blocking  the  receptors  isolated  tissue  Nevertheless,  of  (Douglas  needed  the  more  been  (Gietzen  cells  et  al  processes  which  be  invoke  cells  W7  may  agents  and  enzyme  may  affect  distribution 1982).  in  Tanaka  In  the on  caution. effects  calmodulin islets  et^ a l  1982)  of  in  the  (Gagliardino and mast  anticalmodulin  cells agents  preparations.  Some  the  are  literature  1982)  of  receptor  obtained  with  of  tissue  cited  the  inhibitory  specific  isolated  (Hidaka  data  pancreatic  more  which  complicate  role  (Meritt  with  and m u s c a r i n i c  the  the  sites,  (Roufogalis  interpreted  including  involving  affect  Therefore,  Clearly,  1981),  could  partition  -dependent  +  conditions, to  Ca  a-adrenergic  should  anticalmodulin  (Belle  turn  agents.  used  1982).  +  1982)  pituitary  studies  specific  the  of  anterior  calmidazolium  In  these  controlled  have  for  have  of  (Na +K )-ATPase  secretion  preparations  and Nemeth  are  48/80  of  a number  1980),  and  which  hydrophobic  antagonism  in  (Roufogalis  under  phenothiazines function  which  molecules  other  inhibit  phenothiazines  effects  and  than  can  tubulin,  activities  biological  et^ a l  other  -  hydrophobic  membranes  consequences  systems. ion  into  are  148  and  of  Compound  1983).  pancreas,  calmodulin  has  been  shown  to  stimulate  not  only  the  n i  plasma  membrane  nucleotide fact  that  release  -ATPase  activity  phosphodiesterase the  from  mechanism  Ca  by  role the  of  cyclic  exocrine  which  (Vandermeers nucleotides  pancreas  calmodulin  (see  is  above)  also  et^ al_ 1 9 8 3 ) . in  s t i l l  activation  but  of  In  cyclic view  calcium-mediated not  conclusive,  of  the  enzyme the  phosphodiesterase  regulates  -  pancreatic The  role  function shown  has  that  secretion protein also  enzyme  protein  been  increased  and the  proteins. amylase  dalton  proteins  are  and W i l l i a m s  recently.  CCK8  at  and  insulin did  not  soluble  in  23,000  which alter  Burnham  of  the  regulation  a M  that  = 32,000  r  proteins.  not  direct  involved  1982).  recently,  authors  in  the  a  amylase  agents soluble  stimulation  the  concluded of  have  particulate  20,500  of  process  (1982)  These  and  produce  pancreatic  produce  Mj. = 2 1 , 0 0 0  these  of  and W i l l i a m s  phosphorylation  specifically More  of  soluble  does  proteins,  the  concentrations  phosphorylation  Since  release  uncertain.  phosphorylation  a n d Mj. = 1 6 , 0 0 0 the  is  -  phosphorylation  examined  carbachol  altered  20,500  of  secretion  149  21,000 that  of  and  the  exocytosis  two (Burnham  phospholipid-sensitive  2+ Ca (M  -dependent  r  = 38,000,  pancreatic effective protein in  30,000,  acinar as  a  either  total  incubated 30°C,  30,000  in  (Fig.  dalton  and  for  studies  The  proteins  of  substrate  been  1983).  the  Wrenn  was  38,000  1983)  1982) with  in partially  substrate  phosphorylation  and W i l l i a m s  1982;  demonstrated  Calmodulin  of  proteins  or no  reactions  soluble reference  to  substrate. when Ca  2  +  pancreatic ,  Mg  2 +  formation  degree  was  have  examined  (Burnham  phosphoprotein 25).  15,000)  authors  the  presence  endogenous  (Wrenn  and W i l l i a m s of  its  phosphorylation  These  origin  the  and  extracts  particulate  present  covalent  proteins  cell  (Burnham  these  22,000  1983).  subcellular In  kinase  cofactor  (Wrenn  fractions the  protein  of  enhanced  acinar  and  0.5  was  observed  mM A T P  phosphorylaton by  plasma  calmodulin.  of  for  in the Of  membranes 5 min  at  a number  of  19,000  and  particular  are  -  interest to  the  upon  is  the  similarity  reported  to  The  effect as  calcium  pump  process  by  of  This of  of  target  (ATP, or  to  GTP) .  study), 1980)  result  dalton  32,000  with  membrane  protein  dalton  carbachol  provides proteins  as  in  the  similar  tissues. and  The  lack as  vanadate  diphosphohydrolase  of  a  phosphorylation  particulate  and  CCK8  possible  and  the  protein  (Burnham link  and  between  response  of  acinar  1976),  kidney  (Saffran  1976),  skeletal  oat  regulate  via or  the  in  this  present  can  hydrolyse  (ADP)  at  and  high  to  of  red,  Senior  1980)  ATPases.  A  tissues  Kinne  secretory  molecular  present  the  in  a  triphosphates  presence  of  cations  oligomycin  (Le  Bel  et  distinguishes  the  ATP  similar  mast  1979),  melanoma  the  cells Smooth  et^ a l , 1 9 8 2 )  and  in  certain  and monovalent  including  1974),  and M e i s s n e r  carcinoma  be  membrane  has  nucleoside  rates  azide  ruthenium  other  thus  phosphorylation  study  enzyme  and  can  the  high  enhance  diphosphohydrolase  (Anand-Srivastara cell  calcium,  plasma  ATP  a number  (Malouf  on a  the  (Martin  in  act  secretion  membrane.  sensitivity  from  could  observed,  described to  enzyme  cytoskeleton  plasma  ouabain,  characterized  muscles  the  protein  diphosphate  well  astrocytoma,  with  activity  on p a n c r e a t i c  intracellular  binding  proteins  The  and  cardiac  a  Calmodulin  regulate  interacting  ATPase  of  Mg  plasma  three-fold:  characteristics number  latter  30,000  of  cells  calmodulin  calmodulin  The  acinar  -  secretagogues.  envisaged  weight  of  1982).  phosphorylation cells  this  phosphorylation  stimulation  Williams  of  150  and  enzyme  has  liver  (Kwan  (Knowles,  +  al  been  and  1982)  et  al^  Stanworth and  sarcomas, jet  2  (this  (Garnett  (Cooper  mouse  Ca  human  al^ 1 9 8 3 ) .  The  -  function  of  this  enzymie s y s t e m 2+  shown from  that  rats.  In  or  Ca  largely  ATPase  smooth muscle  rat  -  unknown.  Kwan  (1982)  has  2+  t h e Mg  vascular  is  151  heart  was  activity  decreased  sarcolemma,  it  has  of  in  been  plasma  membranes  spontaneously proposed  that  isolated  hypertensive the  2+ Ca  -ATPase  component  (Anand-Srivastava A  feature  of  et  study  hydrolyse 30).  Ca is  to  o r Mg in  all  externally  surface  over  part  of  zymogen  granule  1980).  If  cytoplasmic plasma  the  of  the  the  this  and n a t i v e have  luminal  of the  the  calcium  channels  the  shown  membrane a  of  that  enzyme  Ca  the  Ca  be  2  2 +  cells  or M g  +  Senior  2  acinar  question the  1980).  s t i l l  and  on  of  fusion  could  be  derived  2 +  -ATPase  is  then  internally  exists  presence the  of  following oriented.  on  lead  indicates from al  the  fusion So  in  inner  (Beaudoin _t  situated  is  biochemical  The  exocytosis  this  remains  activity  outset  of  membrane  While  Both  at  -ATPase  presented  membrane  cells  the  (Fig  +  plasma  plasma  -ATPase  this  can  (1983)  acinar  granules,  would  Ca  in  ecto-enzyme.  with  Ca  al  of  cells  plasma membrane.  result  zymogen  the  associated to  an  an  acinar  of  and  as  is  intact  pancreatic  studies,  membranes  site  the  Hamlyn  expressed  surface  as  isolated  presence  (Beaudoin  granule  active  membrane,  is  activity  plasma  of  pancreatic  study  present  membranes  side  in  90-95% of  membranes  the  Using  a d d e d ATP  studies  zymogen  opening  diphosphohydrolases  that  ATPase  the  in  demonstrated  activity  the  ATP  site.  -ATPase  oriented  of  the  that  granule  deposits  of  suggest  with  involved  1982).  course  cytochemical  zymogen  that  was  the  agreement  whether  and  it  be  active  externally  During  evidence  al  some  externally-oriented present  may  with  far  the  this  -  has  not  been  mechanism  demonstrated  of  membrane and  unresolved.  Moreover,  penetrating destroyed plasma there the  origin  covalent  about  membranes may  be  a  fully  plasma  reagent, the  (Hamlyn  pool  externally  of  is  treatment  90% o f  -  cytochemically.  fusion  distribution  152  and  plasma  applied  probe.  Ca  intact  diazotized ATPase  Senior  of  understood  membrane of  total  Therefore, the  acid  could  that  of  the  slowly  (DSA) in  pancreatic  suggest  was  the  remain  with  present  This  ATPase  will  cells  sulphanilic activity  the  question  -ATPase  acinar  1983).  membrane  until  not  that  accessible  to  (Beaudoin  e_t  ni In al  addition  1980),  the  Ponnappa  associated Is  to  et  zymogen al  (1981)  with microsomal  possible  that  this  granule have  Ca  described  preparations  activity  -ATPase a Ca  obtained  represents  reported -ATPase  2 +  from  acinar  contamination  activity cells.  from  It  plasma  2-4membranes though by  may b e  Ponnappa  with Ca  present  the  in  this  present  as  the  e_t a l ^ ( 1 9 8 1 )  plasma  2-4-ATPase  indigenous  different  observed  was  An  characteristics  appear  membrane  activity  fraction.  not  in  the  stimulated  of  from  -ATPase  activity  the  activity  described  that  activity  associated  current by  Ca  work:  The  calmodulin  microsomal  (Williams,  2-4personal  communication)  membrane-related  and  activity  the  was  h i g h Mg  -stimulation  not  observed.  plasma  membrane  of  the  plasma  2-4Thus  in  addition  to  the  Ca  -ATPase  system 2_i_  described,  the  acinar  possibly  associated  activity  and  The  a  (Chambers  et  al  with  zymogen  presence  of  cell  may  the  also  endoplasmic  granule  a microsomal  reticulum  or  Ca  -ATPase  mitochondrial  system.  ecto-ATPases  1967),  possess  have  been  polymorphonuclear  reported  leucocytes  in  platelets  (De  Pierre  and  Karnovsky gland the  1974),  cells  (Cooper  and  (Carraway  _____  1980).  Despite  of  mouse  that  the  parotid  an  role  It  ATP is  ATP  signal  in  an e f f e c t  a  the  in  the  presence  which  can  secretagogue that  be  of  a  blocked work  fluxes  rat  mammary  occurrence,  clear.  It  has  mechanism  for  the  ATP  may be  acting  Gallacher  release  atropine,  amylase  calcium  not  Recently,  of  and  widespread  is  where  amylase  further  1976),  their  provide  1980).  stimulate  observation,  now k n o w n  may  processes  _____  can  Stanworth  ecto-ATPases  ecto-ATPases  ATP  glands  as  of  (Carraway  interesting of  the  external  propranolol, is  function  that  neurotransmitter showed  -  cells  suggested  termination  153  mast  physiological  been  -  as  (1982)  from  superfused  phentolamine  and  by  quinidine.  Although  is  required  establish  to  a  this the  secretion. during  secretagogue-stimulated 2+  enzyme the  secretion  cell  Case  and  1976  a  due  b)  pool  1973),  into  an  (Stolze  ATP-dependent  biphasic.  release  Clausen  and  trigger  to  are  calcium  from  Schulz  has  et  l i t t l e  known  is  an  been  stores  Ca  2 +  (Matthews (Kondo  the  mitochondria  recognized  -extrusion  different  Although  in  Ca  phase  compartment  1980).  about  i n i t i a l  a reuptake  sequestration  reticulum  1982)  by  intracellular and  is  intracellular  followed  endoplasmic al  There  (Ponnappa  -transport  e_ a l and  from  presence and  from 1973;  Schulz the  of  an  rough  et_ a l  1981;  processes  at  Wakasugi  the  level  2+ of  the  plasma  membrane.  In  view  of  the  bidirectional  Ca  fluxes  2+ reported vesicles  in  intact  b o t h A T P - d e p e n d e n t Ca e f f l u x from 2+ and A T P - d e p e n d e n t Ca - i n f l u x were measured i n these k  studies. release  The of  Ca  cells,  pre-loaded present  5 Ca  efflux  observed  sequestered  since  in La  preloaded (1.2  mM)  vesicles present  represented in  the  the  washing  -  medium would preloaded in  the  displace  vesicles  presence  considerable  bound  was  of  calcium.  considerably  oxalate.  degree  of  It  calcium  24-  ATP-dependent  Ca  observed  the  before  when  abolished was  in  sequestered  vesicles.  Moreover, reduced  was  Ca  efflux  +  when v e s i c l e s  recognized,  binding  influx.  Ca  vesicles  were  filtration.  the  -  which  though  had  the  from  were  that  preloaded  there  tendency  was  to  a  obscure  2-4-  T  Millipore  154  presence into  Kribben  of  though,  transferred  This  and  aj^ ( 1 9 8 3 )  to  was  ionophore  not  have  just  Ca  2 +  containing  -uptake  indicating  bound  also  consistently  a n EGTA  ATP-dependent  calcium  vesicles  et  -uptake  to  reported  the an  buffer  was  that  calcium  surface  of  the  ATP-dependent  2+ Ca  -uptake  in  observations 24Mg of  of  -dependent a d d e d Mg  these  Kribben  both microsomal  endoplasmic  level  However,  plasma  the  Ca  of  24-  preparations.  -uptake  uptake  the  than  reported  and  membrane  (1983),  much h i g h e r  et^ a l ^ ( 1 9 8 1 )  that, similar i n the acinar  some  29).  were  plasma  et^ a j l  although  (Fig.  studies  Ponnappa in  pancreatic  was  calcium  those  membrane  in  the  found  to  be  observed  in  the  concentrations  used  presence  was  As  used  by K r i b b e n j i t jal  of  Ca  -transport  preparations.  It  absence in  (1983).  processes  thus  appears 24-  t o c o n t r a c t i l e s y s t e m s , t h e r e g u l a t i o n o f c y t o s o l i c Ca c e l l may b e a f u n c t i o n o f b o t h a ' m i c r o s o m a l ' (possibly  reticulum)  Ca  -uptake  system  and  a  plasma-membrane  24-  ATP-dependent  Ca  extrusion  system. 24-  The sealed  great  right-side  accessible Intact  majority  from  cells.  out  the Based  of as  the  95% o r  outside, on  vesicles  the  used  in  the  Ca  -flux  more  of  the  ATPase  similar  to  the  orientation  presence  of  a Ca  2  +  activity  uptake  studies appeared  observed  system  in  in the  the  -  vesicle also are  preparations,  exist worth  down  in  intact  it  an e l e c t r o c h e m i c a l required.  tempting  cells.  considering.  normally  is  155  to  A number  Firstly,  Ca  gradient  so  Secondly,  the  2  -  +  speculate  that  such  a  of  problems  with  this  assumption  in  this  situation  an ATP-dependent acinar  membrane  system  would  uptake  be  moving  would  appears  to  process  does  may  not  be  be  2-4adequately appear  to  permeable be  uncertain, the  granules  to  granules  there  the  contain  (Heisler,  Ca  necessary.  though  cytosol  to  ATP  personal could  cell  .  Therefore  Thirdly, is  the  evidence  exterior  that  release  these  of  ATP  It  translocated is  known  communication)  to  exterior  that  and  exocytosis  the  not  extracellular  may b e  Therefore  effectors  the  1974).  personal  communication).  active  source  (Trans  (Grinstein,  an  is  from zymogen  calmodulin  of  of  ATP  zymogen  the  acinar  cell. Calcium inhibiting  influx the  outward  Carafoli  1982).  since  was  it  quinidine shown in  Co  of  hepatocytes  1975).  blood  calcium K  +  (Verecka  provision  isolated  Cockrell  This  red  transport  saturable,  and  that  Into  These  cells of  was  sensitive  respiratory  shown  and  demonstrated  vanadate to  be  Stratman  suggest  (Verecka  by  1974;  that,  in  and  verapamil,  Similarly,  supports  after  carrier-mediated  inhibited  1982).  energy  observations  by  and was  Carafoli  (Kleineke  been  calcium  influx  and  has  it  calcium  has  been  accumulation  Dubinsky  and  resting  cells,  a  2-4slow  cycling  phenomenon The  of  Ca  though,  regulatory  maintenance  of  low  across has role  not of  the been  plasma  membrane  demonstrated  calcium  concentrations  in of  in  cellular cytosolic  occurs.  This  pancreatic  acinar  cells.  functions  depends  on  calcium.  Studies  by  the  -  Schulz  and  may  responsible  be  cells.  Heil  It  is  (1979) at  known  have  indicated  least  that  156  in  the  part  -  that  for  secretory  the  Na /Ca +  calcium  extrusion  proteins  in  the  2+ are  accompanied  1980). during  The  by a  Ca  -secretory  maturation  extrusion may n o t  constant  the  secretory  mechanism,  the  time  major  component  2+ in  of  of  a n d Mg  vesicles.  the  of  pancreatic (Beaudoin  presumably Though a  such  calcium  acinar fluid  2+  Ca  association  course  from  system  an  event  takes  such  al  place  possible is  extrusion  e_  calcium  that  it  process.  2+  ecto-Mg  plasma  protein  of  be a  The  amount  exchange  +  or  membranes  Ca  -ATPase  represents  present  the  in  major  acinar  ATP  preparations  hydrolyzing  enriched  activity  in  the  2+ pancreas. important  Unlike role  In  concentrations, catalytic activity  site  other the  this does  plasma  membrane  maintenance enzyme  not  system  appear  characterized,  of  to  though,  Ca  intracellular  by  virtue  share  has  -ATPases  of  this  many  that  an  calcium  the  orientation  property.  similar  play  The  Ca  properties  to  of 2 +  its  -ATPase  other  2+ Ca  -transport  phospholipids, intermediate addition,  ATPases a Ca  and  this  including  stimulation  by  -dependent-hydroxylamine  an  affinity  activity  has  for  calcium  properties  in  sensitive  the  similar  calmodulin  acidic  phosphorylated  micromolar to  and  range.  In  other  2+ ATP-diphospholydrolases ADP of  as the  substrate cell  including  and  the  surface.  It  plasma  membrane  or  both a  high  low  and  if  h i g h Mg  presence is  this  not total  affinity  of  its  ectopic  nature  of  the  activity  system with Ca  catalytic  known w h e t h e r  2+ The  -sensitivity,  the  site enzyme  represents separate  the  utilization on  the  spans  of  outside the  presence  of  functions.  2+ or  Mg  -ATPase  activity  characterized  - 157 -  opens of  to  this  question enzyme.  the For  possible example,  mechanism(s) what  would  be  of  regulation  and  possible  source  of  significantly  higher  than  the  function  2+ calmodulin  and  since  extracellular  Ca  is  2+ intracellular  Ca  , what  would  be  the  function  of  a  high  affinity  2+ Ca -ATPase under these conditions? One p o s s i b i l i t y i s t h a t f u n c t i o n s as a s i g n a l t o o t h e r events f o l l o w i n g e x o c y t o s i s . present  in  zymogen  granules  and/or  released  by  exocytosis  this-, system Calmoduln  may b i n d  to  2+ Ca  -ATPase  sites  leading  to  a  localized  transport  that  may  effect  the  2+ next event. The Ca acinar c e l l s ) are not  l e v e l s i n the secretory ducts ( e x t r a c e l l u l a r to constant but are a l t e r e d at v a r i o u s stages i n the  2+ secretion  cycle.  depending  on  Therefore  the  activity  of  the  Ca  -ATPase  may  vary  2+ Of  the  environmental  significant  importance  Ca  .  is  the  if  this  exact  localization  of  this  2+ Ca  T  -ATPase:  basolateral this the  It or  is  luminal  information ATPase  with  not  will other  known  membrane be  of  events  of  value in  activity  the in  the  acinar  is  present  cell.  speculating  on  The the  stimulus-secretion  on  the  availability interaction  coupling  of of  process.  -  158  -  BIBLIOGRAPHY  Adunyah,  E.S.,  drugs  Niggli,  V.and  trifluoperazine  purified  erythrocyte  controlled  Carafoli,  a^d R 2 4 5 7 1 Ca  proteolysis.  +  -ATPase FEBS  E.  remove  (1982)  by a c i d i c  Lett.  143,  Akyempon, C.K. and R o u f o g a l i s , B.D. 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