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Studies on the role of Ca²⁺ in the pancreatic acinar cell Ansah, Twum-Ampofo 1980

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STUDIES ON CA -TRANSPORT PROCESSES 2+  IN CYSTIC FIBROSIS PATIENTS AND CONTROLS  by TWUM-AMPOFO(ANSAH  B.Sc,  The U n i v e r s i t y  of S c i e n c e and Technology, 1975  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE  THE FACULTY OF GRADUATE STUDIES D i v i s i o n o f Pharmacology and T o x i c o l o g y of t h e F a c u l t y We accept  of Pharmaceutical  t h i s t h e s i s as  to the r e q u i r e d  Sciences  conforming  standard  THE UNIVERSITY OF BRITISH COLUMBIA January,  1980  (^) Twum-Ampofo Ansah, 1980  In p r e s e n t i n g  this thesis in p a r t i a l  an advanced degree at the U n i v e r s i t y  f u l f i l m e n t of the requirements f o r of B r i t i s h Columbia, I agree  the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  that  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g 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 by h i s r e p r e s e n t a t i v e s .  be  granted by  the Head of my  I t i s understood t h a t c o p y i n g or  of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not written  permission.  Department of  Pharmacology and  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 Date  February  12,  1980.  Department  Toxicology  Columbia  be  or  publication  allowed without  my  -ii-  ABSTRACT Plasma membrane-enriched preparations obtained from cultured human skin f i b r o b l a s t s by d i f f e r e n t i a l centrifugation and sucrose density 2+ centrifugation techniques were found to contain a Ca -stimulated ATPase 2+ a c t i v i t y . This enzyme was Mg -dependent and was stimulated by calmodulin 2+ 2+ and thus was similar to the (Mg + Ca )-ATPase a c t i v i t y observed i n other 2+ 2+ tissues. The s p e c i f i c a c t i v i t y of the (Mg + Ca )-ATPase present was 4-5 fold higher than that present i n crude membrane preparations and 802+ 100 f o l d higher than that present i n homogenates.  2+  The (Mg + Ca  )-ATPase  a c t i v i t y of both crude membrane and plasma membrane-enriched preparations of cultured f i b r o b l a s t s from Cystic F i b r o s i s ( C F . ) patients was s i g n i f i cantly reduced when compared to that a c t i v i t y observed i n age-matched controls (p^O.Ol  i n the crude membrane preparations; p<.0.05 i n the  p u r i f i e d plasma membrane preparations).  Reciprocal plots indicated that  i t i s the maximal a c t i v a t i o n of the enzyme ( c 2+) and not the a f f i n i t y v  a  of the enzyme system (K^igg) f o r calcium that i s altered i n C F . strains. 2+ 2+ In order to determine i f t h i s decrease i n (Mg + Ca  )-ATPase a c t i v i t y  in C F . was related to a decrease i n the a b i l i t y of these c e l l s to transport calcium, inside-out (10) v e s i c l e s were prepared from red c e l l s 2+ obtained from C F . patients and age-matched controls.  Ca  -uptake a c t i v i t y  was found to be s i g n i f i c a n t l y reduced i n the preparations derived from CF.  patients.  This reduction was apparent  at a l l time points s t u d i e d  (10 seconds - 120 minutes) and at a l l free calcium concentrations used (10-150 um).  Reciprocal plots of the data revealed that the K  d i s s  for  calcium of the calcium pump, was similar i n control and C F . samples but  -iii-  t h a t t h e £ 2 + was s i g n i f i c a n t l y r e d u c e d (p ( 0.001) i n t h e C F . V  &  preparations.  C a l m o d u l i n p r e p a r e d from r e d c e l l hemolysates o f c o n t r o l s was found t o s t i m u l a t e Ca  2+ -transport a c t i v i t y t o a s i m i l a r extent 2+  samples; i t d i d n o t , though, r e t u r n Ca tions to control levels. CF.  i n b o t h C F . and c o n t r o l  -uptake a c t i v i t y i n C F . p r e p a r a -  An a l t e r a t i o n i n c a l c i u m t r a n s p o r t a c t i v i t y i n  may have a number o f i m p l i c a t i o n s t h a t may e x p l a i n some o f t h e  manifestations  of the disease.  -iv-  TABLE OF CONTENTS PAGE ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  viii  LIST OF ABBREVIATIONS  viii  INTRODUCTION  1  Pathophysiology of c y s t i c f i b r o s i s  1  Transport mechanisms  4  Factors related to c y s t i c f i b r o s i s  5  2+ 2+ 2+ -Transport and (Mg + Ca )-ATPase a c t i v i t y  7  2+ 2+ RBC activator of the (Mg + Ca )-ATPase a c t i v i t y  12  Ca  AIMS OF THE PRESENT STUDY  15  MATERIALS AND METHODS  17  Establishment of f i b r o b l a s t cultures  17  Preparation of plasma membrane-enriched fractions of human skin f i b r o b l a s t s  18  Preparation of red blood c e l l inside-out v e s i c l e s  19  Enzyme assays  20 2+  A. Measurement of (Mg  2+  + Ca  )-ATPase a c t i v i t y  20  B. 5'-Nucleotidase assay  21  C. Adenylate cyclase assay  21  D. Acetylcholinesterase assay  22  E. Glyceraldehyde 3-phosphate dehydrogenase assay  22  -vPAGE Measurement of calcium transport a c t i v i t y  23  Preparation of calmodulin  23  Preparation of lymphoblast plasma membranes  24  Protein assay  24  Statistics  24  Materials  24  RESULTS  25  P a r t i a l p u r i f i c a t i o n of plasma membranes from cultured skin fibroblasts  25  2+ 2+ P a r t i a l characterization of the (Mg + Ca )-ATPase a c t i v i t y i n f i b r o b l a s t membrane preparations 2+ 2+ Comparison of (Mg + Ca )-ATPase a c t i v i t y i n cultured f i b r o b l a s t s derived from C F . patients and controls  29  Studies on inside-out v e s i c l e preparations red c e l l s of controls and C F . patients  44  25  derived from  2+ Comparison of Ca -uptake a c t i v i t y i n 10 v e s i c l e preparation obtained from C F . patients and controls 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane preparations of human cultured lymphoblasts  45 52  DISCUSSION  56  BIBLIOGRAPHY  63  -vi-  LIST OF TABLES  TABLE 1.  5'-Nucleotidase and adenylate cyclase a c t i v i t i e s of f i b r o b l a s t membrane preparations  *  K  3.  K  2  diss ca ^ 8 Ca )-ATPase of f i b r o b l a s t membrane preparations a  d  v  2+  o f  t h e  M  2 + +  2+  2+  diss * Ca °^ * ^ ~ P ^ system i n control and C F . preparations of 10 v e s i c l e s a n c  2+ 4.  n  V  2 +  t  ie  a  u  t a  e  2+  (Mg + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane preparations of human cultured lymphoblasts  -vii-  LIST  OF FIGURES  PAGE FIGURE 1.  The f u l l  2.  2+ Mg ^dependency in  fibroblast 2+  3.  4.  5. 6.  7.  8.  9.  r e a c t i o n sequence  o f t h e (Mg  2+ 2+ + Ca )-ATPase  2+ o f t h e Ca -stimulated  ATPase  11  activity  membrane p r e p a r a t i o n s  28  2+  (Mg + C a )-ATPase a c t i v i t y i n crude p u r i f i e d p l a s m a membrane p r e p a r a t i o n s b l a s t s i n t h e p r e s e n c e and absence o f 2+ 2+ (Mg + C a )-ATPase a c t i v i t y i n crude p u r i f i e d p l a s m a membrane p r e p a r a t i o n s b l a s t s i n t h e p r e s e n c e and absence o f 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y i n c r u d e tions of cultured fibroblasts  membrane a n d i n of cultured fibroadded c a l m o d u l i n  31  membrane a n d i n of c u l t u r e d f i b r o added c a l m o d u l i n  33  membrane  prepara35  Kj .and V 2+ o f t h e ( M g + C a ) - A T P a s e a c t i v i t y o r c r u d e membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o b l a s t s  38  2+ 2+ (Mg + C a )-ATPase a c t i v i t y i n p u r i f i e d preparations of cultured fibroblasts  40  2 +  i s g  2 +  Q  plasma  membrane  K a n d V 2+ o f the (Mg + Ca )-ATPase a c t i v i t y of p u r i f i e d p l a s m a membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o blasts 2 +  d  i  s  s  2 +  Cg  2+ T i m e - c o u r s e o f C a - u p t a k e a c t i v i t y i n 10 v e s i c l e p r e p a r a t i o n s from c o n t r o l and C F . samples  442  47  2+ 10.  11.  I n i t i a l r a t e s o f Ca and C F . samples  - u p t a k e i n 10 v e s i c l e s  from c o n t r o l  2+ Ca - u p t a k e a c t i v i t y i n t h e p r e s e n c e and a b s e n c e o f added c a l m o d u l i n i n 10 v e s i c l e p r e p a r a t i o n s f r o m C F . p a t i e n t s and c o n t r o l s  49  51  -viii-  LIST OF ABBREVIATIONS  AChE  acetylcholinesterase  ADP  adenosine 5'-diphosphate  AMP  adenosine 5'-monophosphate  ATP  adenosine  5'-triphosphate  ATPase  adenosine  5'-triphosphatase  Ca  t o t a l calcium 2+  Ca  free calcium ion  c y c l i c AMP  c y c l i c 3', 5'-adenosine monophosphate  DEAE  diethylaminoethyl  DTNB  5, 5'-dithiobis-(2-nitrobenzoic acid)  E  enzyme  EDTA  ethylene diamine tetraacetate, disodium  EGTA  ethyleneglycolbis-( -aminoethyl)  E^P  phosphorylated enzyme intermediate  G3-PD  glyceraldehyde 3-phosphate dehydrogenase  GTP  guanosine triphosphate  10  inside-out  K^£  s s  MBP /3-NAD  , N'-tetracetate  dissociation constant modulator binding protein fb -nicotinamide adenine dinucleotide  Pi  inorganic phosphate  RBC  red blood c e l l ,  salt  -ix-  S.E.M.  standard e r r o r  TCA  trichloroacetic  Tris  tris  V,-, 2+  maximum  o f the mean acid  (hydroxymethyl) velocity  aminomethane  ACKNOWLEDGEMENTS  I am deeply g r a t e f u l t o Dr. S. K a t z f o r h i s guidance and encouragement throughout t h i s study.  I would  l i k e t o thank D r s .  J.H. M c N e i l l , D.A. A p p l e g a r t h and B.D. R o u f o g a l i s f o r t h e i r c o n s t r u c t i v e c r i t i c i s m s o f t h i s work. the  I a l s o w i s h t o acknowledge  e x c e l l e n t c o - o p e r a t i o n o f Dr. A.G.F. Davidson and Dr.  Lawrence Wongtand t h e s t a f f and p a t i e n t s o f t h e H e a l t h C e n t r e f o r C h i l d r e n C F . c l i n i c , Vancouver,  B.C. Canada.  -1-  INTRODUCTION C y s t i c f i b r o s i s o f the pancreas ( C F . )  i s now  r e c o g n i z e d as the most  common g e n e t i c d i s e a s e a f f e c t i n g p e o p l e of C a u c a s i a n o r i g i n . r e p o r t e d by F a n c o n i  (1936) i n S w i t z e r l a n d , but i t was  I t was  first  not r e c o g n i z e d as a  s e p a r a t e and d i s t i n c t d i s o r d e r u n t i l Andersen (1938) gave the f i r s t d e t a i l e d p a t h o l o g i c a l d e s c r i p t i o n o f the  disease.  C y s t i c f i b r o s i s i s an i n b o r n e r r o r o f m e t a b o l i s m which i n v o l v e s e x o c r i n e glands  and i s t r a n s m i t t e d as an autosomal r e c e s s i v e t r a i t  Sant' Agnese and D a v i s  1976).  newborns i s a f f e c t e d by C F . (Danks et a l 1965; Pathophysiology  I t i s estimated and t h a t one  (Lobeck 1972;  that approximately  di  one i n  i n 20 i n d i v i d u a l s i s a h e a l t h y  1600 carrier  Lobeck 1972).  of C y s t i c F i b r o s i s  A s t r i k i n g i n c r e a s e i n the l e v e l of sodium, c h l o r i d e and, potassium i s present  to a l e s s e r extent,  i n the sweat of v i r t u a l l y a l l homozygote p a t i e n t s w i t h  and i s the most c o n s i s t e n t and e a s i l y r e c o g n i z a b l e c h e m i c a l a b n o r m a l i t y disease  ( d i Sant' Agnese et a l 1953).  present  from b i r t h and  The  CF.  in this  sweat e l e c t r o l y t e a b n o r m a l i t y  is  throughout l i f e and i s u n r e l a t e d e i t h e r to s e v e r i t y of  the u n d e r l y i n g d i s e a s e or to which o t h e r e x o c r i n e organ i s i n v o l v e d ( d i Sant' Agnese and Talamo 1967).  The  "sweat t e s t " i s  '•' - t h e r e f o r e g e n e r a l l y  accepted  as the s i m p l e s t and most r e l i a b l e l a b o r a t o r y p r o c e d u r e i n the d i a g n o s i s of CF.  ( d i Sant' Agnese and V i d a u r r e t a 1960 ; Shwachman'. and Antonowicz 1962).  M o r p h o l o g i c a l l y , no d i f f e r e n c e has been seen between the e c c r i n e sweat of p a t i e n t s w i t h C F .  glands  and those of c o n t r o l s u b j e c t s e i t h e r by l i g h t microscopy  or by e l e c t r o n - m i c r o s c o p y  techniques  (Munger et a l 1961).  In s k i n biopsies,  Munger and co-workers (19.61) d i d not f i n d s i g n i f i c a n t d i f f e r e n c e s between the  -2-  two  groups i n the f i n e s t r u c t u r e of t h e c e l l s o f the duct of sweat glands  b e f o r e and a f t e r sweating.  both  U s i n g i n - v i t r o m i c r o p e r f u s i o n , Mangos (1973)  demonstrated t h a t t h e duct o f t h e sweat gland i n C F . behaved n o r m a l l y when p e r f u s e d w i t h normal sweat, whereas both normal and C F . sweat gland ducts behaved abnormally CF.  when p e r f u s e d w i t h C F . sweat.  e c c r i n e sweat a b n o r m a l i t y  I t i s h i g h l y p r o b a b l e t h a t the  i s due to an e f f e c t of a sodium r e a b s o r p t i o n i n -  h i b i t i n g f a c t o r e x e r t e d i n t r a l u m i n a l l y by t h e sweat of a f f e c t e d persons.  The  f a c t o r i s so l a b i l e t h a t i t has been i m p o s s i b l e so f a r to i s o l a t e and c h a r a c t e r i z e it.  Wiesmann et a l (1972) demonstrated t h a t i t i s s p e c i f i c a l l y  the o r a l submucosal minor s a l i v a r y creased i n p a t i e n t s with C F .  the s a l i v a  from  glands i n which sodium i s c o n s i s t e n t l y i n -  Thus, t h i s i s the o n l y o t h e r e x o c r i n e gland system,  b e s i d e s t h e e c c r i n e sweat glands to m a n i f e s t t h i s sodium a b n o r m a l i t y  consistently  i n the d i s e a s e . In c o n t r a s t t o the s e r o u s , h y p o t o n i c s e c r e t i o n s o f p a t i e n t s w i t h C F . ( v i z , sweat and s a l i v a ) which show e l e v a t e d sodium and c h l o r i d e l e v e l s  ( d i Sant'  Agnese et a l 1953; Wiesmann e t a l 1972), the more p r o t e i n - r i c h o r mucoid, i s o t o n i c secretions  ( v i z , from the pancreas  and  t r a c h e o b r o n c h i a l mucus glands) a r e found  to have sodium c o n c e n t r a t i o n s near normal o r even somewhat decreased Agnese and Talamo 1967). water content  However these s e c r e t i o n s have a s i g n i f i c a n t l y  ( d i Sant' Agnese and Talamo 1967).  hepatic  reduced  and o b s t r u c t organ  thus g i v i n g r i s e t o c h r o n i c pulmonary d i s e a s e , p a n c r e a t i c i n s u f f i c i e n c y , c i r r h o s i s , i n t e s t i n a l o b s t r u c t i o n and other c o m p l i c a t i o n s .  E l e v a t e d c a l c i u m c o n c e n t r a t i o n s have been noted et a l 1971) and p a r o t i d s a l i v a 1971)  1  P h y s i c o c h e m i c a l l y , the mucus  s e c r e t i o n s behave i n an abnormal way; they p r e c i p i t a t e , passages,  ( d i Sant  of C F . p a t i e n t s .  i n submaxillary s a l i v a  (Gibson  ( d i Sant' Agnese and Talamo 1967; Wotman e t a l  Gibson e t a l demonstrated e x p e r i m e n t a l l y t h a t C F .  -3-  mucus d e r i v e d and  from s u b m a x i l l a r y  s a l i v a was  electrolytes  a l s o showed that normal mucus became hyperpermeable when c a l c i u m  added.  They proposed t h a t h y p e r s e c r e t i o n  i s a primary d e f e c t i n the d i s e a s e , water and  thus p r o d u c i n g  calcium concentrations  (Chernick  and  Boat and  rendering  the e x o c r i n e  the mucus  ( K o p i t o 1964)  and  was glands  hyperpermeable to Elevated  have a l s o been observed i n f e c a l m a t e r i a l  Barbero 1963;  tracheobronchial  (Johansen  secretions  P o t t e r et a l 1963).  co-workers (1974) s t u d i e d a c a l c i u m p r e c i p t a b l e p r o t e i n  p r e v i o u s l y shown by  Gugler (1967) to be  t u r b i d i t y i n submaxillary  gland  one  of two  f r a c t i o n s which cause  s a l i v a of p a t i e n t s w i t h  shown to be a p h o s p h o g l y c o p r o t e i n  daltons, with  of c a l c i u m by  the o t h e r e l e c t r o l y t e a b n o r m a l i t i e s .  1963), duodenal contents  was  permeable to water and  with  a molecular  CF.  This p r o t e i n  weight o f 12,000  f i v e phosphate groups per m o l e c u l e , which may  aggregate  i n the presence o f c a l c i u m owing to r e d u c t i o n of e l e c t r o s t a t i c  charge.  B e t t e l h e i m (1971) u s i n g m a t e r i a l f u r n i s h e d by Boat, proposed t h a t  aggregation  may  be  sites  for  non-covalent bonding.  associated with  patients with  CF.  a c i d composition  and  conformational  change exposing hydrophobic  Calcium-precipitable protein i s identical i n  c o n t r o l s i n many chemical  (Boat et a l 1964)'.  Calcium-precipitable protein i s  found i n comparable q u a n t i t i e s i n s a l i v a I t thus appears to be the c a l c i u m t h a t produces a g g r e g a t i o n .  tration  ( F o r s t n e r and  from p a t i e n t s and normal c o n t r o l s .  concentration  i n s a l i v a of C F .  Though the p r o t e i n i t s e l f  b r o n c h i a l or duodenal f l u i d , have decreased s o l u b i l i t y  f e a t u r e s , i n c l u d i n g amino  i s not  patients  detectable  in  i t has-been shown t h a t r a t i n t e s t i n a l mucins  i n the presence of i n c r e a s e d  Forstner  1976).  calcium  concen-  -4-  Transport  Mechanisms  E l e v a t i o n s of sweat sodium and normal c o n c e n t r a t i o n s 1969)  of these  chloride concentrations  substances i n p r e c u r s o r  i n the face of  fluid  (Schultz  suggest t h a t plasma membrane t r a n s p o r t of e l e c t r o l y t e s by  e p i t h e l i u m i s abnormal.  CF.  e r y t h r o c y t e s have been used as a  model to d e l i n e a t e t h i s a b n o r m a l i t y . e r y t h r o c y t e sodium t r a n s p o r t i n C F . workers concluded the sweat and  duct simple  B a l f e et a l (1968) r e p o r t e d and  abnormal  o b l i g a t e heterozygotes.  These  t h a t i f the p o s t u l a t e d d e f e c t of sodium t r a n s p o r t i n  salivary  ducts  in CF.  i n t h i s d i s e a s e d s t a t e , a study  represents  a fundamental  of t h i s abnormality  h e l p to determine the pathogenic  defect.  Lapey and  abnormality  i n erythrocytes  may  Gardner (1971) observed  t h a t o n l y the o u a b a i n - i n s e n s i t i v e , e t h a c r y n i c a c i d - s e n s i t i v e component of sodium e f f l u x was females w i t h C F .  diminished  i n erythrocytes  S i m i l a r l y , Cole and  Dirks  from males and  post-puberty  (1972) found t h a t whereas  the o u a b a i n - s e n s i t i v e component of e r y t h r o c y t e ATPase a c t i v i t y was changed i n C F .  p a t i e n t s , the o u a b a i n - i n s e n s i t i v e component was  c a n t l y decreased.  T h i s i m p l i e d t h a t an ATPase a c t i v i t y o t h e r  i n t i m a t e l y r e l a t e d to N a - t r a n s p o r t  was  signifi-  than the  one  a f f e c t e d by the d i s e a s e s t a t e .  Subsequently, Horton et a l (19 70) measured the Mg activity  un-  2+  -dependent Ca  of - e r y t h r o c y t e membranes from-both normals and  2+  -ATPase  C F . .patients  and  2+ found a decreased.Ca -ATPase a c t i v i t y i n the C F . of enzyme a c t i v i t y  d e p r e s s i o n was  erythrocytes.  r e l a t e d to the s e v e r i t y of the  The  extent  disease.  Other workers "(McEvoy et a l 1974; not observe any CF.  patients.  F e i g e t a l 1974; Duffy et a l 1974) d i d 2+ a l t e r a t i o n i n the^Ca ^ATPase a c t i v i t y i n e r y t h r o c y t e s from 2+ Recent s t u d i e s  (Katz 1978  a) have i n d i c a t e d t h a t the  2+ Ca  )-ATPase ^ a c t i v i t y of e r y t h r o c y t e s  obtained  from C F .  patients i s  (Mg  +  -5-  significantly  decreased  when compared to the a c t i v i t y noted  T h i s a l t e r a t i o n i n a c t i v i t y was maximal a c t i v a t i o n of the  (Mg  found  2+  + Ca  of the enzyme system f o r c a l c i u m . xn  to be due  -2+  i n controls.  to an a l t e r a t i o n i n the  )-ATPase and not i n the  I t was  a l s o found  that t h i s  affinity decrease  . 2+ 2-k (Mg - + Ca ) - A T P a s e , a c t i v i t y i s not a p a r t of a g e n e r a l i z e d membrane  or membrane-bound enzyme a l t e r a t i o n i n C F .  s i n c e s t u d i e s on the Na ,  K-  +  +  2+  ATPase and Mg  - ATPase i n e r y t h r o c y t e membranes i n d i c a t e d no  i n the a c t i v i t y of these enzymes.  S t u d i e s done u s i n g f i b r o b l a s t membrane 2+  p r e p a r a t i o n s a l s o i n d i c a t e d a decrease in C F .  strains  Shapiro  (1979  fibroblast The  i n (Mg  compared to normal s t r a i n s  a) r e p o r t e d an e n l a r g e d  ( F e i g a l and  Shapiro  1979  a).  )-ATPase a c t i v i t y  (Katz 1978  intracellular  d e r i v e d from s u b j e c t s w i t h C F .  that t h i s abnormality  2+ + Ca  and  b) .  Feigal  and  calcium pool i n skin  obligate  o b s e r v a t i o n of an a l t e r a t i o n i n f i b r o b l a s t s  suggested  alteration  from  heterozygotes  obligate'heterozygotes  i s r e l a t e d to the b a s i c gene d e f e c t i n  Subsequently, i t was  shown t h a t  CF.  mitochondrial  f r a c t i o n s i s o l a t e d from s k i n f i b r o b l a s t s of s u b j e c t s w i t h C F .  accumulated  more c a l c i u m than those from c o n t r o l c e l l s  1979  ( F e i g a l and S h a p i r o  b).  F a c t o r s R e l a t e d to C y s t i c F i b r o s i s Spock et a l (1967) observed parents  caused n o r m a l l y  synchronous c i l i a r y  e x p l a n t s to become i r r e g u l a r and used by Bowman and and  t h a t s e r a from C F .  dyskinetic.  Oyster  s a l i v a from C F .  genotypes.  These workers  energy exchange.  A similar factor  their  in rabbit tracheal g i l l p r e p a r a t i o n s were  co-workers (1969) to d e t e c t a c i l i a r y  t h a t the f a c t o r which a f f e c t e d c i l i a t e d and  activity  p a t i e n t s and  i n h i b i t o r i n sera  (Bowman et a l 1970)  suggested  t i s s u e might a f f e c t membrane t r a n s p o r t (or f a c t o r s ) has  s i n c e been demonstrated  -6-  i n u r i n e , c u l t u r e d s k i n f i b r o b l a s t s ( B e r a t i s et a l 1973) (Conover et a l 1973) The  ciliary  within 19 75)  from C F .  inhibitor  a c t i v i t y was  as w e l l as i n o b l i g a t e h e t e r o z y g o t e s .  to 10,000 d a l t o n s  i s presumed to be  in a basic fraction  lymphoblasts  from serum i s a s m a l l m o l e c u l a r weight  the range of 1,000 and  subjects,  and  (Conover et a l 1973;  c a t i o n i c s i n c e i t i s e l u t e d on  (Bowman et a l 1973).  polypeptide  Recently,  demonstrated i n a l l s e r a from C F .  Bowman et a l  chromatography  ciliary  dyskinesia  homozygotes and  hetero-  zygotes as w e l l as i n s e r a from p a t i e n t s w i t h b r o n c h i a l asthma. isoelectric  f o c u s i n g showed t h a t the presence of C F .  w i t h t h a t of d y s k i n e s i a  activity  samples from the asthma p a t i e n t s speculated a CF.  that C F .  other  from C F .  dyskinesia and  factor.  The  et a l 1978;  disturbance  Although no  serum f a c t o r a c t i v i t y 1979)  active  These workers  that patients with b r o n c h i a l  t h a t d i f f e r s from C F .  p r e s e n c e of C F .  and  transport  across  sera  been confirmed by  -  other  like  when added to the  culture  found between the amount of  the c l i n i c a l s t a t u s of C F . ciliary  specific  Bogart et a l (1977) has  i n r a b b i t t r a c h e a l explants c o r r e l a t i o n was  -  p r o t e i n i n the  ionophore A23187 produces a C F .  significant  to  autoimmune d i s o r d e r s harbor a substance  T u l l y et a l 1979).  i t i s p o s s i b l e that C F .  a c t i v e calcium  et a l 1977).  f a c t o r and  d y s k i n e s i a but  demonstrated t h a t c a l c i u m  medium.  (Wilson  o b l i g a t e h e t e r o z y g o t e i n d i v i d u a l s has  workers (Scholey  mucociliary  i n a l l s e r a t e s t e d except f o r the  dyskinesia  r e s p i r a t o r y and  t h a t can produce c i l i a r y ciliary  corresponded  p r o t e i n i s r e l a t e d s t r u c t u r a l l y or m e t a b o l i c a l l y  - specific ciliary  asthma and  protein  However,  patients  (Nagy et a l  f a c t o r can modify p a s s i v e  and/or  plasma membranes ( V i n c e n z i ; 1979).  -72~T~  O j  Ca  O |  - T r a n s p o r t and (Mg + Ca ) - ATPase A c t i v i t y C a l c i u m i s i n v o l v e d i n the r e g u l a t i o n of a number of c e l l u l a r  including stimulus-secretion coupling coupling  (Rubin 1974), e x c i t a t i o n - c o n t r a c t i o n  (Huxley 1973), c o n t r o l of membrane t r a n s p o r t  r e l e a s e of t r a n s m i t t e r from motor nerve t e r m i n a l s Many r e s e a r c h e r s for studying calcium  x 10  Long  per  s u r f a c e of the membrane.  of about 10  concentration  cells  hemolysis.  of RBC  M  contained  Long and  remaining 10% was  The  low  Romero and  cytoplasmic  of the plasma membrane of f r e s h red  Ca  1975), ( i i ) by  c h e l a t i o n of  or  ( i i i ) by a c t i v e c a l c i u m  Whittam 1971).  calcium (Weed  extrusion  Schatzmann  Maintenance of low  (i)  blood  to h i g h a f f i n i t y membrane b i n d i n g s i t e s  Mouat 1971)  the  b e l i e v e d to  intracellular  the plasma membrane-bound c a l c i u m pump (Schatzmann 1966; V i n c e n z i 1969;  bound to  and  c e l l s i s thought to be m a i n t a i n e d by  F e r r e i r a and Lew  to i n t r a c e l l u l a r anions and  calcium  These workers ( H a r r i s o n  s m a l l molecules l e a v i n g a  i n normal red b l o o d  ( P o r z i g 1972;  The  (Schatzmann 1973).  calcium permeability  et a l 1969;  cellular  cellular  suggested t h a t most of the c e l l u l a r c a l c i u m was  be bound mostly to p r o t e i n s and  the low  as a model  EDTA or s i m i l a r non-  c h e l a t i n g agents c o u l d remove about 90%  extracellular  f r e e Ca  (RBC)  (1968) found t h a t the RBC  l i t e r of packed c e l l s .  i n i s o t o n i c medium w i t h o u t c a u s i n g Long 1968)  cell  2+ moles Ca  penetrating  and  and  (Katz and M i l e d i 1965).  have used the human r e d b l o o d  Harrison  -5 1.58  ( P o r z i g 1972)  the r o l e of the plasma membrane i n the r e g u l a t i o n of  content.  functions  by  and  intracellular  2+ Ca An  has  been shown to be  e s s e n t i a l f o r normal r e d c e l l shape and 2+  i n c r e a s e i n i n t r a c e l l u l a r Ca  chinocyte  shape t r a n s f o r m a t i o n s  was  reported  to cause  (Weed et a l 1969;  function.  discoid-spheroe-  P a l e k et a l 1974),  an  + increase i n K ability  permeability  (Gardos 1958;  Lew  1971), decreased deform-  (Weed et a l 1969), i n c r e a s e d v i s c o s i t y  h i b i t i o n o f Na"^ K  - transport  +  I n s p i t e of the f a c t  (La C e l l e 1973)  and i n -  (Dunn 1974) .  t h a t RBCs have r e l a t i v e l y low  permeability  2+ to Ca  ( P o r z i g 1972;  presence of "AmM  - s t a r v e d RBCs was and o  Ca  Lew  F e r r e i r a and Lew 1975), i t was shown t h a t i n the 2+ 2+ , the maximum p a s s i v e l e a k of Ca i n t o ATP  e x t e r n a l Ca  1977).  about 6 umoles per l i t e r  of c e l l s per hour  T h i s suggested t h a t i n o r d e r  I  _ ^  i n the f a c e of a p p r o x i m a t e l y 10  O  to m a i n t a i n  low  (Ferreira intracellular  j  M Ca  i n the e x t r a c e l l u l a r  fluid,  2+ the RBC reported  must have assystem f o r removing Ca t r a n s p o r t of c a l c i u m  loaded w i t h  c a l c i u m and ATP.  t h a t c a l c i u m was chemical  r e q u i r e d Mg  RBC  "ghosts" ATP  against  and  "ghosts" (1969) showed an  electro-  temperature dependent,  2+ and  from r e s e a l e d RBC and  Schatzmann (1966)  L a t e r , Schatzmann and V i n c e n z i  T h i s t r a n s p o r t , which was  2+  In 1966,  from r e v e r s i b l y - h e m o l y z e d  extruded from r e s e a l e d RBC  gradient.  .  Shin 1969;  Ca  i n s i d e the c e l l .  "ghosts"  a c t i v e t r a n s p o r t of  calcium  confirmed by a number of l a b o r a t o r i e s  (Lee  1969;:Qulst and R o u f o g a l i s 1975). 2+ The requirements f o r ATP and Mg l e d Schatzmann and V i n c e n z i (1969) 2+ 2+ to propose t h a t the Mg - dependent - Ca a c t i v a t e d adenosine t r i p h o s p h a t a s e 2+ 2+ (ATP Phosphohydrolase E C 3.6.1.3; (Mg + Ca ) - ATPase) a c t i v i t y , f i r s t r e p o r t e d i n RBC membranes by Dunham and Glynn (1961) and l a t e r by Wins and Schoffeniels process. mann 1969;  O l s o n and  was  The  (1966), was  There now  Cazort  the b i o c h e m i c a l  expression  of t h i s Ca  e x i s t s ample evidence to support  2+  transport  t h i s hypothesis  (Schat-  V i n c e n z i and Hinds 19*76) .  The mechanism by which energy from the h y d r o l y s i s of ATP  is utilized  2+ to t r a n s p o r t Ca  against  an e l e c t r o c h e m i c a l . . g r a d i e n t  i s not  clearly  under-  -9-  stood.  I n r e c e n t y e a r s c o n s i d e r a b l e p r o g r e s s has been made i n the e l u c i -  d a t i o n of the p a r t i a l r e a c t i o n s of the (Mg 2+ c e l l membranes.  The  (Mg  2+  + Ca  2+  )-ATPase  i n red  2+ + Ca  )-ATPase  phosphorylated-protein intermediate.  r e a c t i o n proceeds.through  a  Formation of t h i s i n t e r m e d i a t e i s  2+  enhanced by Ca  (Knauf e t a l 1974; Katz and B l o s t e i n 1975; Rega and  Garrahan 19 75) i n a manner s i m i l a r to the c o n c e n t r a t i o n dependence  of  2+ Ca  s t i m u l a t i o n of the ATPase a c t i v i t y  and Garrahan 1975; Szasz et a l 1978).  (Katz and B l o s t e i n 1975; Rega The p h o s p h o r y l a t e d  intermediate i s  a 150,000 d a l t o n p r o t e i n (Knauf et a l 1974; Katz and B l o s t e i n 1975; et a l 1977) which i s c h e m i c a l l y d i s t i n c t  from the Na  , K  Wolf  - ATPase  phosphoenzyme of m o l e c u l a r weight around 100,000 d a l t o n s (Knauf et a l 1974; Katz and B l o s t e i n 1975). I t has been proposed t h a t the s u b s t r a t e f o r the ATPase i s f r e e ATP (Rega and Garrahan 1975; Schatzmann 1977; Richards et a l 1978). E-P.  S u b s t r a t e b i n d i n g r e s u l t s i n the f o r m a t i o n of an i n i t i a l The K  m  f o r the f o r m a t i o n of E-P  ATP h y d r o l y s i s - ( 2 - 5 uM) presence of Ca 1975).  2+  ( R i c h a r d s et a l 1978).  i s s i m i l a r to the K  2+  m  for  P h o s p h o r y l a t i o n i n the  occurs i n the absence of added Mg  The major e f f e c t of Mg  second s t a t e , E-P  (1-6 uM) "  phosphoenzyme,  2+  (Rega and Garrahan  appears to be the c o n v e r s i o n of E-P to a  (Rega and Garrahan 1975; Garrahan and Rega 19.78).  The  t  E-P i n t e r m e d i a t e i s more r e a c t i v e than E-P and can undergo r a p i d h y d r o l y s i s 2+ (Rega and Garrahan 1978), c o n s i s t e n t w i t h the h i g h e r t u r n o v e r o f (Mg_ + Ca  2+  )-ATPase  Rega 1978)  i n the presence of Mg  2+  (Katz and B l o s t e i n 1975; Garrahan and  and the h i g h e r s t e a d y - s t a t e l e v e l s of phosphoenzyme i n t e r m e d i a t e  (Garrahan and Rega 1978; Schatzmann  and B u r g i n 1978).  The f i n a l step i n the  sequence i s the c o n v e r s i o n of E' to E, the r e g u l a t i o n o f which remains unknown.  The a c t u a l t r a n s l o c a t i o n of Ca  2+  a c r o s s the plasma membrane i s  -10-  th ought t o be through c o n f o r m a t i o n a l protein  changes i n the t r a n s l o c a t i n g  (Schatzmann and B u r g i n 1978).  Figure 1 describes the f u l l  r e a c t i o n sequence. Attempts t o determine t h e number o f c a l c i u m ions pumped p e r ATP h y d r o l y z e d have y i e l d e d c o n f l i c t i n g r e p o r t s . (1969) and Schatzmann (1973) suggested Q u i s t and R o u f o g a l i s  Schatzmann and V i n c e n z i  a c a l c i u m t o P i r a t i o of one.  (1975) and S a r k a d i e t a l (1977) showed t h a t maximum  2+ i n h i b i t i o n of Ca t r a n s p o r t by lanthanum was a s s o c i a t e d w i t h o n l y a 50% 3+ i n h i b i t i o n o f ATPase a c t i v i t y . They concluded t h a t L a - i n s e n s i t i v e ATPase 2+ a c t i v i t y was n o t a s s o c i a t e d w i t h Ca  t r a n s p o r t and s h o u l d be deducted from  the t o t a l ATPase a c t i v i t y y i e l d i n g a s t o i c h i o m e t r y e s t i m a t e i o n s pumped p e r P i r e l e a s e d .  Recently, Larsen  of 2 calcium  e t a l (1978 a) u s i n g an i o n -  s e l e c t i v e e l e c t r o d e method f o r r a p i d , continuous assessment o f C a efflux from r e s e a l e d RBC " g h o s t s " r e p o r t e d a s t o i c h i o m e t r y o f one c a l c i u m i o n 2+ pumped p e r ATP h y d r o l y z e d . T h i s may i n d i c a t e t h e r e f o r e t h a t t h e Ca 2 +  pump has more than one mode of o p e r a t i o n . 2+ 2+ 2+ K i n e t i c a n a l y s i s of the Ca a c t i v a t i o n o f (Mg + Ca )-ATPase a c t i v i t y 2+ 2+ i n i s o l a t e d RBC membranes r e v e a l e d t h e presence o f two (Mg + Ca )-ATPase 2+ 2+ activities. These a r e r e f e r r e d t o as h i g h and low a f f i n i t y (Mg + Ca ) ATPase t o r e f l e c t  their different  affinities  Schatzmann and R o s s i 1971; S c h a r f f 1972).  2+ f o r Ca (Horton  e t a l 1970;  2+ The Ca dissociation  ( K ( j ^ ) f ° t h e h i g h a f f i n i t y enzyme was e s t i m a t e d r  ss  constant  t o be between 1 and 4  2+ uM Ca (Schatzmann and R o s s i 1971; S c h a r f f 1972; Schatzmann 1973). 2+ low  affinity  enzyme was r e p o r t e d to have a  K(ji 'r s s  f o r Ca  The  o f 46-100 uM  (Schatzmann and R o s s i 1971; S c h a r f f 1976; S c h a r f f and Foder 1977).  It i s  -11-  E + ATP  Ca + 2  Mg  7PT  2+  E-vP  Mg  E-xP  H 0 9  E~P  + ADP  E'~P  E  + Pi  2+ F i g u r e 1.  The f u l l r e a c t i o n sequence of (Mg  2+ + Ca  )-ATPase  -12-  still  not c l e a r whether t h e r e a r e two d i s t i n c t 2+  two s t a t e s o f a s i n g l e  (Mg  (Mg  2+ 2+ + Ca )-ATPase or  2+  + Ca  )-ATPase r e g u l a t e d by o t h e r p r o t e i n s  i n the membrane. 2+ I t has now been demonstrated t h a t plasma-membrane bound a c t i v e Ca 2+ t r a n s p o r t and/or (Mg  2+  + Ca  i n c l u d i n g those o f l i v e r Lindsay Borowitz  1976), k i d n e y  )-ATPase e x i s t s i n a number o f mammalian  (Lamb and L i n d s a y 1971; Garnett and Kemp 1975;  (Moore e t a l 1974), a d r e n a l medulla  1975), b r a i n (Robinson  and u t e r u s  1974), h e a r t  RBC A c t i v a t o r o f the (Mg  ( S t . L o u i s and Sulakhe 1976)  2+ + Ca  )-ATPase A c t i v i t y  RBC a c t i v a t o r or c a l m o d u l i n was f i r s t  X19 73) who noted  membranes.  r e p o r t e d by Bond and Clough  t h a t a non-hemoglobin p r o t e i n p r e s e n t i n the hemolysate o f 2+ 2+  human RBCs i n c r e a s e d the (Mg  + Ca  )-ATPase a c t i v i t y o f i s o l a t e d  P a r t i a l p u r i f i c a t i o n was accomplished  The presence  of calmodulin  c a l m o d u l i n have been o b t a i n e d Calmodulin  approximately  4.0.  RBC  by L u t h r a e t a l (1976 a ) .  i n t h e RBCs of s e v e r a l mammalian s p e c i e s has  been r e p o r t e d ( L u t h r a e t a l 1976 b ) .  1978).  ( L e s l i e and  ( J a n i s e t a l 1977; J a n i s and D a n i e l 1977; Kroeger e t a l 1977). 2+  The  cells,  R e c e n t l y , more p u r i f i e d  ( L u t h r a et a l 1977; J a r r e t t  f r a c t i o n s of  and P e n n i s t o n  i s an a c i d i c p r o t e i n w i t h an i s o e l e c t r i c p o i n t o f I t has a m o l e c u l a r weight o f about  has no i n h e r e n t ATPase a c t i v i t y  (Jung 1978) .  to the i n n e r s u r f a c e of RBC membranes 2+ was dependent on Ca  16,500 d a l t o n s and  Calmodulin was shown t o b i n d  ( F a r r a n c e and V i n c e n z i 1977 a ) :  ( F a r r a n c e and V i n c e n z i 1977 b ) .  binding  The requirement f o r  2+ Ca  becomes important  i n t h e l i g h t o f the f a c t t h a t c a l m o d u l i n was  to be p r e s e n t i n the RBC a t a much h i g h e r c o n c e n t r a t i o n than was  found  necessary  to achieve maximum a c t i v a t i o n of the (Mg has been observed  t h a t low a f f i n i t y - l i k e 2+  resemble  2+ 2+ + Ca )-ATPase a c t i v i t y .  high a f f i n i t y - l i k e  (Mg  (Mg  2+  + Ca  2+  It  )-ATPase - membranes  2+  + Ca  )-ATPase - membranes i f i n c u b a t e d  i n the presence of added c a l m o d u l i n ( F a r r a n c e and V i n c e n z i 1977 b ) . i t was suggested  t h a t t h e presence  o r absence o f c a l m o d u l i n may  f o r the h i g h and low a f f i n i t y b e h a v i o r r e s p e c t i v e l y ,  Thus,  account  i n i s o l a t e d RBC  membrane p r e p a r a t i o n s (Schatzmann and B u r g i n 1978). Calmodulin  i s o l a t e d from RBCs shares many :physicochemical p r o p e r t i e s  with a p r o t e i n p u r i f i e d from b o v i n e b r a i n and h e a r t shown t o a c t i v a t e AMP p h o s p h o d i e s t e r a s e  cyclic  ( K a k i u c h i et a l 1972) and a d e n y l a t e c y c l a s e (Brostrom  et a l 1975) a c t i v i t i e s .  The f i n d i n g t h a t t h i s l a t t e r p r o t e i n 2+ 2+  s e l e c t i v e a c t i v a t i o n of RBC membrane  (Mg  + Ca  produced  )-ATPase (Gopinath and  V i n c e n z i 1977; J a r r e t t and P e n n i s t o n 1977) and t h a t RBC c a l m o d u l i n a c t i v a t e d cyclic-AMP p h o s p h o d i e s t e r a s e  activity  ( J a r r e t t and P e n n i s t o n 1977)  i n d i c a t e d t h a t t h e two p r o t e i n s c o u l d be i d e n t i c a l .  Recent  shown t h a t the mode o f s t i m u l a t i o n of human RBC membrane  s t u d i e s have 2+ 2+  (Mg  + Ca  )-ATPase  by c a l m o d u l i n i s s i m i l a r t o the mode of s t i m u l a t i o n by c a l m o d u l i n o f a d e n y l a t e c y c l a s e and cyclic-AMP  phosphodiesterase  (Lynch and Cheung 1979).  In 1977, Wang and D e s a i (1977) d e s c r i b e d the presence o f a p r o t e i n b o v i n e b r a i n named modulator  binding protein  (MBP).  from  This p r o t e i n could  a n t a g o n i z e the a c t i v a t i o n o f p h o s p h o d i e s t e r a s e by b i n d i n g the c a l m o d u l i n 2+ Ca  complex.  Thus MBP and p h o s p h o d i e s t e r a s e  compete f o r t h e c a l m o d u l i n -  complex.  L a r s e n e t a l (1978 b) found t h a t MBP would a l s o 2+ 2+  2+ Ca  the a c t i v a t i o n o f RBC membrane (Mg  + Ca  antagonize  )-ATPase a c t i v i t y by p u r i f i e d RBC  c a l m o d u l i n , p r o v i d i n g f u r t h e r e v i d e n c e f o r the s i m i l a r i t y o f these two  -14-  proteins.  Though i t s r o l e i s not known, MBP  regulatory  protein.  c o u l d be  another  cellular  -15Aims of the P r e s e n t  Study  Although C F . a f f e c t s a number o f t i s s u e s , t h e s e a r c h  continues f o r  a p o s s i b l e common denominator t h a t w i l l e x p l a i n t h e cause o f t h e d i s e a s e . Many of the problems a s s o c i a t e d w i t h mucoid s e c r e t i o n o f the e x o c r i n e An  the d i s e a s e s t a t e i n v o l v e the v i s c o u s  glands  i n c r e a s e i n t h e c a l c i u m content  ( d i Sant' Agnese and Talamo 1967).  o f many o f the g l y c o p r o t e i n - r i c h  s e c r e t i o n s i n C F . p a t i e n t s has been r e p o r t e d  (Johansen 1963; P o t t e r e t a l  1963;  T h i s suggests t h a t a major  Gibson e t a l 1971; Wotman et a l 1971).  d e f e c t i n C F . might be a s s o c i a t e d w i t h  a calcium-dependent process  such  as s e c r e t i o n or t r a n s p o r t . 2+ 2+ s t u d i e s have r e v e a l e d a s p e c i f i c decrease i n t h e (Mg + Ca ) -  Previous  ATPase a c t i v i t y o f e r y t h r o c y t e membrane p r e p a r a t i o n s s t u d i e s r e v e a l e d a decrease i n (Mg  2+ if  the (Mg  Further  2+ 2+ + Ca )-ATPase a c t i v i t y i n crude membrane  homogenates of c u l t u r e d f i b r o b l a s t s to age-matched c o n t r o l s .  (Katz 1978 a ) .  (Katz  1978.»•'- from .CF.  The o b j e c t i v e of t h i s p r e s e n t  p a t i e n t s compared  study was t o determine  2+  + Ca  )-ATPase a c t i v i t y noted i n t h e crude membrane homogenates  of c u l t u r e d f i b r o b l a s t s was i n d i g i n o u s t o the plasma membrane and f u n c t i o n e d , as i n t h e r e d b l o o d c e l l , t o m a i n t a i n low i n t r a c e l l u l a r f r e e c a l c lum i concentrations.  T h i s r e q u i r e d the d e t e r m i n a t i o n  a c t i v i t y present  i n p u r i f i e d plasma membrane p r e p a r a t i o n s  of whether the (Mg  o f the (Mg  2+ 2+ + Ca )-ATPase  and an e v a l u a t i o n  2+ 2+ + Ca )-ATPase a c t i v i t y was a l t e r e d i n samples d e r i v e d  from C F . p a t i e n t s . 2+ The  (Mg  2+  + Ca  )-ATPase i s g e n e r a l l y accepted  as t h e b i o c h e m i c a l  basis  of the c a l c i u m t r a n s p o r t system i n a number o f t i s s u e s (Katz e t a l 1975; Schatzmann 1973). pump a c t i v i t y  As a more d i r e c t approach to the e v a l u a t i o n of the c a l c i u m  i n C F . , t h i s study  sought t o e s t a b l i s h whether  calcium  -16-  t r a n s p o r t i n c e l l membranes d e r i v e d from C F . p a t i e n t s was d e f e c t i v e . Thus, i n t h i s p r e s e n t  2+  work, t h e Ca  out v e s i c l e p r e p a r a t i o n s age-matched c o n t r o l s was  - t r a n s p o r t a c t i v i t y of i n s i d e -  d e r i v e d from r e d c e l l s of C F . p a t i e n t s and evaluated.  Parameters of t h e c a l c i u m pump have been shown t o be a f f e c t e d by 2+ calmodulin  ( S a r k a d i e t a l 1978; L a r s e n  and V i n c e n z i 1979).  T h i s Ca  b i n d i n g p r o t e i n appears t o p l a y an important r o l e i n t h e r e g u l a t i o n o f the c a l c i u m pump. was in  I t ' remained t o be determined whether c a l m o d u l i n  altered i n C F . tissue. this  T h i s p o s s i b i l i t y was t h e r e f o r e i n v e s t i g a t e d  study. 2+  F i n a l l y , to a s c e r t a i n t h e g e n e r a l i z e d n a t u r e o f the (Mg a c t i v i t y i n C F . , another c e l l  2+ (Mg  2+ + Ca  were o b t a i n e d and  2+ + Ca  preparations  )-ATPase a c t i v i t y o f C F . samples compared t o c o n t r o l evaluated.  )-ATPase  type d e r i v e d from C F . p a t i e n t s was i n -  v e s t i g a t e d ; lymphoblast plasma membrane p r e p a r a t i o n s  the  function  -17-  M a t e r i a l s and Methods Establishment The  cell  of f i b r o b l a s t  cultures .  s t r a i n s used i n t h e experiments d e s c r i b e d were d e r i v e d  s k i n b i o p s i e s o f C F . c h i l d r e n and age-matched c o n t r o l s o b t a i n e d  from  from t h e  f i b r o b l a s t bank o f t h e C h i l d r e n ' s H o s p i t a l , Vancouver, through the c o o p e r a t i o n o f Dr. D.A. A p p l e g a r t h Medical  and from Dr. M. Buchwald, Department o f  G e n e t i c s , H o s p i t a l f o r S i c k C h i l d r e n , Toronto.  p a t i e n t s had h i g h  A l l the C F .  c h l o r i d e l e v e l s i n t h e i r sweat and v a r y i n g degrees o f  pulmonary and p a n c r e a t i c  involvement.  from s k i n b i o p s i e s of c h i l d r e n w i t h 4 C F . and 3 c o n t r o l f i b r o b l a s t v e n t i o n a l procedures  (Krooth  The c o n t r o l s t r a i n s were d e r i v e d  diseases  unrelated  to C F .  In a l l ,  c e l l c u l t u r e s were e s t a b l i s h e d by con-  1969).  When c o n f l u e n t , t h e c e l l s were sub-  c u l t u r e d 1:4 u s i n g 0.25% t r y p s i n (Flow L a b o r a t o r i e s ) ' i n Hank's Balanced 2 S a l t s o l u t i o n and grown i n F a l c o n F l a s k s days w i t h  f e e d i n g every t h r e e days.  (75 cm , Corning  The F l a s k s were m a i n t a i n e d a t 37° i n  an i n c u b a t i o n chamber ( F i s h e r Isotemp I n c u b a t o r ) . in  c u l t u r e medium w i t h  nitrogen.  10% d i m e t h y l  Co.) f o r 10-14  C e l l s t r a i n s were f r o z e n  s u l f o x i d e (DMSO) and s t o r e d i n l i q u i d  When needed, c u l t u r e s t r a i n s were r e c o n s t i t u t e d w i t h  medium c o n t a i n i n g  20% (v/v) F e t a l Bovine Serum.  Dulbecco's M o d i f i e d  E a g l e Medium  culture  The growth medium  contained  (MEM, Grand I s l a n d B i o l o g i c a l Co.) w i t h  a lower than normal amount of sodium b i c a r b o n a t e  t o prevent  alkalinity,  w i t h heat i n a c t i v a t e d 15% (v/v) F e t a l Bovine Serum (Flow  Laboratories)  and  units/ml,  an a n t i b i o t i c - a n t i m y c o t i c mixture  fungizone  25 mcg/ml and s t r e p t o m y c i n  ( p e n i c i l l i n 10,000  10,000 mcg/ml, Grand I s l a n d  l o g i c a l Co.; 10 ml per l i t e r of p r e p a r a t i o n ) .  Bio-  A l l the f i b r o b l a s t s t r a i n s  -18-  were s u b c u l t u r e d  s e v e r a l times then t r a n s f e r r e d to 1 l i t e r r o l l e r b o t t l e s  continually rotated as p r e v i o u s .  (Wheaten Co.  Rotator  apparatus) and  f e d and  maintained  Growing i n r o l l e r b o t t l e s i n c r e a s e d the t i t e r of c e l l s to  l e v e l r e q u i r e d f o r p r e p a r a t i o n of plasma membrane-enriched f r a c t i o n s .  the The  experiments d e s c r i b e d were performed on f i b r o b l a s t s of passages 7-and 8, P r e p a r a t i o n of plasma-membrane-enriched f r a c t i o n s of human s k i n f i b r o b l a s t s Plasma-membrane-enriched f r a c t i o n s of f i b r o b l a s t s d e r i v e d from and  c o n t r o l s u b j e c t s were prepared  T y p i c a l l y , two  by the method of K a r t n e r  r o l l e r b o t t l e s c o n t a i n i n g f i b r o b l a s t s , one  c o n t r o l s t r a i n , were i n i t i a l l y washed 6 times w i t h S a l t s o l u t i o n (0,15  M NaGl, 0.01  M"NaH^P0 and 4  CF.  et a l (1977).  CF.  and  one  25 ml Phosphate Balanced  11'mM  K H P 0 , pH.7.4), 2  4  The washed c e l l s were s w o l l e n by slow r o t a t i o n . o f b o t t l e s c o n t a i n i n g - " 25 ml of 1 mM step was  sodium b i c a r b o n a t e  repeated  twice.  The  from the b o t t l e s u r f a c e and c o n t a i n i n g 25 ml of 1 mM rupted  suspension  minutes.  The  was  sodium b i c a r b o n a t e  a d d i t i o n of EDTA was  discontinuous  s o l u t i o n (pH  mM  necessary  to prevent  '(w/v) sucrose  and  d e n s i t y g r a d i e n t of 30%,  (pH 7.4)  by  The  aggregation  designated,  48%  and  60%  sucrose  M a t e r i a l banding a t the 10/30  c e n t r i f u g a t i o n at  dis-  33,000 xg  The the  and pellet  crude  then l a y e r e d on a  the crude membrane f r a c t i o n were washed f r e e of s u c r o s e bicarbonate  7.4).  EDTA'at 27,000 xg f o r 20'  A p a r t of t h i s p r e p a r a t i o n was  76,000 xg f o r 2 hours.  of the b o t t l e  of p a r t i c u l a t e m a t e r i a l p r e s e n t .  resuspended i n 10 ml of 10%  This  c e l l s c o u l d then be removed e a s i l y  c e n t r i f u g e d i n 0.5  membrane p r e p a r a t i o n .  at  swollen  d i s r u p t e d by manual shaking  clumping of the v a r i o u s types was  s o l u t i o n (pH 7.4)v','for 1 minute.  V  and  centrifuged  i n t e r f a c e and i n 1 mM  sodium  f o r 20 minutes.  The  -19-  p e l l e t s were resuspended i n 1 mM for  5'-nucleotidase,  Preparation CF. Health  a d e n y l a t e c y c l a s e and (Mg  of r e d b l o o d  blood  sodium b i c a r b o n a t e  cell  2+  (pH 7.4) and assayed  + Ca  2+  )-ATPase a c t i v i t i e s .  inside-out vesicles  samples were o b t a i n e d  from p a t i e n t s a t t e n d i n g  the Vancouver  Center C y s t i c F i b r o s i s C l i n i c through the c o - o p e r a t i o n  o f Dr. A.G.F.  Davidson, Dr. Lawrence Wong and s t a f f .  Approximately'8 ml b l o o d was drawn  from each p a t i e n t d i r e c t l y i n t o h e p a r i n . from student v o l u n t e e r s  C o n t r o l samples were  obtained  a t the same time and under e x a c t l y the same c o n d i t i o n s .  The  age range of the C F . donors was 12-29 y e a r s w i t h a mean age of 20 years  and  t h a t o f the c o n t r o l s u b j e c t s was 22-34 y e a r s w i t h a mean age of 27 y e a r s .  The  patients  chosen were a t t e n d i n g  the C F . c l i n i c  f o r routine  follow-up.  A l l had been i d e n t i f i e d as h a v i n g C F . on the b a s i s of e l e v a t e d chloride.  The p a t i e n t s had v a r y i n g  problems.  Some of the p a t i e n t s were r e c e i v i n g p a n c r e a t i c  sweat  degrees of r e s p i r a t o r y and d i g e s t i v e enzymes and some  were r e c e i v i n g a n t i b i o t i c s . Preparation  of r e d c e l l  hours of o b t a i n i n g  the blood  inside-out samples.  (10) v e s i c l e s was s t a r t e d w i t h i n 2 C F . and c o n t r o l r e d b l o o d  cells  were washed 3 times i n i s o t o n i c s a l i n e to remove the b u f f y coat and were l y s e d i n 35 volumes of i c e - c o l d 5 mM Na-phosphate b u f f e r  (pH 8.0).  The  l y s e d c e l l were c e h t r i f u g e d a t 12,000 xg f o r 10 minutes i n a Beckman J2-21 c e n t r i f u g e u s i n g a JA 20 r o t o r .  The p e l l e t s were washed and  two more times i n the same b u f f e r .  centrifuged  The r e s u l t i n g white membrane p e l l e t s  were suspended i n 35 volumes o f i c e - c o l d 0.5 mM Na-phosphate b u f f e r for  30 minutes and c e n t r i f u g e d a a t  27,000 xg f o r 30 minutes.  (pH 8.0)  The membranes  -20-  were resuspended i n 17.5 and  volumes of 0.5  s t o r e d o v e r n i g h t on i c e .  The  mM  next morning, the suspensions  c e n t r i f u g e d a t 27,000 xg f o r 30 minutes. an equal .volume of the 0.5 through a 1 i n c h No.  were  f i v e times.  and were  passed  The membranes were  t r i s - g l y c y l g l y c i n e b u f f e r (pH 7.1)  MgCl2 and once i n 20 mM  8.5)  The p e l l e t s were d i s p e r s e d i n  Na-phosphate b u f f e r (pH 8.0)  27 hypodermic needle  then washed once i n 10 mM OV025 mM  mM  Na-phosphate b u f f e r (pH  containing  t r i s - g l y c y g l y c i n e b u f f e r (pH 7,1)  con-  t a i n i n g 0.05 mM MgCl2 by c e n t r i f u g a t i o n a t 31,000 xg f o r 35 m i n u t e s . membrane-vesicle p e l l e t s were then s t o r e d i n 20 mM (pH 7.1)  The  tris-glycylglycine buffer  at a p r o t e i n c o n c e n t r a t i o n of 4 to 5 mg/ml and used w i t h i n s i x hours.  Enzyme Assays 2+ A.  Measurement of (Mg 2+ (Mg  2+ + Ca  )-ATPase a c t i v i t y  2+ + Ca  )-ATPase a c t i v i t y was  by Katz and B l o s t e i n (1975). 50 mM  Tris-HCL  (pH 7.4),  3 mM  The Y-  determined e s s e n t i a l l y as  r e a c t i o n mixture 3 2  p  (ATP)  contained  5 mM  described MgS04,  (20,000 cpm/sample) and 0.75  of membranes (0.5-0.8 mg/ml of the crude membrane p r e p a r a t i o n or 0.1-0.3 mg/ml of the p r e p a r a t i o n e n r i c h e d i n plasma membranes) i n the presence or absence of c a l m o d u l i n and/or the d e s i r e d f r e e c a l c i u m c o n c e n t r a t i o n . When c a l c i u m was  present  i n the i n c u b a t i o n medium i t was  added  together  w i t h EGTA i n order to o b t a i n the d e s i r e d f r e e c a l c i u m c o n c e n t r a t i o n . The  f r e e c a l c i u m c o n c e n t r a t i o n was  constant equations  then determined by the a s s o c i a t i o n 2+ f o r the i n t e r a c t i o n of EGTA w i t h Ca at pH 7.4, u s i n g the of Katz et a l (1970) t a k i n g i n t o c o n s i d e r a t i o n the ATP  and  2+ Mg  c o n c e n t r a t i o n s used.  preparations  Ouabain, 0.1  mM,  was  added to the membrane  d u r i n g p r e - i n c u b a t i o n to i n h i b i t Na , +  K -ATPase +  (ATP  ml  -21-  phosphohydrolase EC the by and  The  a d d i t i o n of the membrane p r e p a r a t i o n the  a d d i t i o n of 5%  2 mM  KR^PO^.  sample-) was f o r one  assay at 37  and  then added to each sample and  hour w i t h i n t e r m i t t e n t  m i x i n g and,  started  by  - TCA  containing (1.5  g/10  5 mM ml;  ATP  0.45  ml/  the samples i n c u b a t e d a t following  0°  centrifugation,  c l e a r supernatant counted f o r r a d i o a c t i v i t y .  5' - N u c l e o t i d a s e assay The  method of M i c h e l l and  Hawthorne (1965) was  v o l v e d the c o l o r i m e t r i c measurement of i n o r g a n i c the h y d r o l y s i s  of AMP.  The  37°  i n a medium c o n t a i n i n g  (pH  7.4),  was  stopped w i t h 1 ml of  was  d i l u t e d to 2 ml  a 1:1 at 660 C.  was  t e r m i n a t e d a f t e r 20 minutes  t r i c h l o r o a c e t i c a c i d (TCA)  A s u s p e n s i o n of c h a r c o a l  an a l i q u o t of the B.  3.L6.3) a c t i v i t y .  5 MM  AMP  m i x t u r e of 1% nm  using  and  and  and  in-  phosphate-produced  by  membranes were i n c u b a t e d f o r 15 minutes at 100  10 mM 25% the  followed  mM  KC1,  10 mM  M g C l , 50 mM 2  sodium potassium t a r t r a t e . '(w/v) TCA.  An  aliquot*  a Beckman Model 25  1%  The  of the  c o l o r developed f o l l o w i n g  ammonium molybdate and  Tris-HCl  buffer  reaction  supernatant  the a d d i t i o n  a s c o r b i c a c i d and  of  measured  spectrophotometer.  A d e n y l a t e C y c l a s e Assay For  the measurement of c y c l i c AMP  p r e p a r a t i o n s were i n c u b a t e d w i t h 50 mM threitol,  1 mM  EDTA, 5 mM  phosphoenolpyruvate and 2.5  mM  T r i s ATP  volume of 0.25  theophylline,  the r e a c t i o n was  Following  s t a r t e d by  t r i s - H C I ! ( p H 7.4), 10 mM  KC1,  5 mM  mg/ml p y r u v a t e k i n a s e , 0.01  0.35  i n the p r e s e n c e and ml.  a c c u m u l a t i o n , membrane  absence of 10 mM  NaF  a 10 minute p r e - i n c u b a t i o n  the  a d d i t i o n of ATP  and  1 mM  Dithio-  M g C l , 20  mM  2  mM  GTP,  in a  and  final  period  at  proceeded f o r  37°, 15  -2-2minutes.  The r e a c t i o n was  t e r m i n a t e d by p l a c i n g the samples i n a b o i l i n g  water bath.  The samples were then c e n t r i f u g e d a t 3,000 xg and  of  supernatant assayed  the c l e a r  Gilman p r o t e i n b i n d i n g assay D.  Acetylcholinesterase  f o r the presence of c y c l i c AMP  (Gilman  1970).  determined by a m o d i f i c a t i o n of the method of  Ellman et a l (1961) as d e s c r i b e d by Steck and Kant  for in  u s i n g the  (AChE) assay  AChE-- a c t i v i t y was  assay, 0.05  aliquots  (1974).  In t h i s  ml o f the r e d c e l l v e s i c l e p r e p a r a t i o n was p r e - i n c u b a t e d  5 minutes  w i t h an e q u a l volume of 20 mM  the presence o r absence  imidazole buffer  (pH 7.1;  of 0.2% 9 mM)  tris-glycylglycine(pH  T r i t o n X-100  was  (v/v).  added to produce  7.1),  Histidine-  a final  volume of  3.0 ml.DTNB and a c e t y l t h i o c h o l i n e c h l o r i d e 0.1 ml each were then added and the r e a c t i o n a t room temperature 25 r e c o r d i n g spectrophotometer E.  was  f o l l o w e d u s i n g a Beckman model  a t a wavelength  G l y c e r a l d e h y d e 3-phosphate Dehydrogenase E s s e n t i a l l y , the assay of  0.05  20 mM or  nm.  (G3-PD) assay  ( C o r i e t a l 1948)  involved pre-incubation  ml o f the red c e l l v e s i c l e p r e p a r a t i o n w i t h an equal volume of  tris-glycylglycine buffer  absence  0.3 ml)  o f 412  of 0.2%  T r i t o n X-100  and c y s t i n e - I I C l (4 mM;  sodium a r s e n a t e (0.4 M;  0.09  (pH. 7.4) (v/v). 1.75  f o r 5 minutes,  i n the presence of  Sodium pyrophosphate  (30  mM;  ml) were then added f o l l o w e d by  ml) and 20 mM ^>-NAD (20 mM;  0.16  ml).  s t a r t the r e a c t i o n , 0.1 mM g l y c e r a l d e h y d e 3-Phosphate (0.03 ml). was The r e a c t i o n at room temperature was 25 r e c o r d i n g spectrophotometer, at  To added. •'  then f o l l o w e d u s i n g a Beckman Model  340  nm.  -23-  Measuremetit of c a l c i u m t r a n s p o r t  activity  C a - u p t a k e experiments were performed a t 30°.  The  2 +  preparations  10  vesicle  (90 ug/ml f i n a l i n c u b a t i o n volume) were p r e - i n c u b a t e d  minutes i n a medium c o n t a i n i n g 9 mM  h i s t i d i n e - i m i d a z o l e b u f f e r (pH  0.6  40 mM  mM  tris-glycylglycine  (pH  7.1),  NaCl, 7.5  mM  CaCl2 ( c o n t a i n i n g 5 x 10~* cpm/samples of ^ 5 c a C l ) •  The  2  c o n c e n t r a t i o n was  maintained  concentrations present  by  KC1,  7.1),  MgCl  and  2  desired free calcium  the a d d i t i o n of EGTA and  determined by the e q u a t i o n s  3 mM  f o r 30  the f r e e c a l c i u m  of Katz et a l (1970)  2+ t a k i n g i n t o c o n s i d e r a t i o n the Mg c a l m o d u l i n was The  added, i t was  r e a c t i o n was  present  c o n c e n t r a t i o n s used.  in a final  s t a r t e d by the a d d i t i o n of 3 mM  i n t e r v a l s a l i q u o t s of 100 c o l d 40 mM  and ATP  V e s i c l e s were trapped  c o n c e n t r a t i o n of 0.8 ATP.  At s p e c i f i c  u l were then taken and quenched i n 1.0  t r i s - g l y c y l g l y c i n e b u f f e r (pH on a 0.45  once w i t h the same b u f f e r and  um  7.1)  filter  When  c o n t a i n i n g 0.1  mM  ml  ug/ml.  time of  MgCl . 2  ( M i l l i p o r e Co.), the f i l t e r s washed  counted f o r r a d i o a c t i v i t y i n Aquasol  (New  England N u c l e a r ) . P r e p a r a t i o n of  calmodulin  Calmodulin  was  prepared  from r e d c e l l s  of outdated  a c c o r d i n g to the method of Jung (1978) by h e m o l y s i s i s o l a t i o n of the c a l m o d u l i n The  human b l o o d  i n hypotonic  essentially  medium and  p r o t e i n by DEAE-Sephadex column chromatography.  a c t i v e f r a c t i o n s were e l u t e d by a b u f f e r e d NaCl g r a d i e n t s o l u t i o n  concentrated f r a c t i o n s was  by a M i l l i p o r e s e p a r a t o r technique. done on Sephadex G-15  of the a c t i v i t y  and  G-25  D e s a l t i n g of the a c t i v e  columns.  of the c a l m o d u l i n p r e p a r a t i o n s was  the s t i m u l a t i o n of  (Mg  2+  + Ca  2+  )-ATPase a c t i v i t y  and  A concentration  curve  o b t a i n e d by the assay  of  of an EDTA-washed membrane  the  -24-  p r e p a r a t i o n from c o n t r o l e r y t h r o c y t e s . (by  The maximal a c t i v a t o r  concentration  volume) o b t a i n e d by t h i s procedure was then used i n subsequent  ments.  experi-  The c a l m o d u l i n p r e p a r a t i o n s were then s t o r e d at -80° and used w i t h i n  3 weeks. P r e p a r a t i o n o f lymphoblast plasma membranes P r e p a r a t i o n was done by Dr. Riordan's group, Research I n s t i t u t e , C h i l d r e n ' s H o s p i t a l , Toronto.  Sick  The p r e p a r a t i o n was s t o r e d a t -80° and used  w i t h i n 3 weeks. P r o t e i n assay P r o t e i n c o n c e n t r a t i o n s were measured by the method of Lowry et a l (1951) u s i n g b o v i n e serum albumin as a standard.. Statistics Student's " t " t e s t f o r u n p a i r e d , common v a r i a n c e data was used as a measure of s i g n i f i c a n c e .  Standard e r r o r of the mean (S.E.M.) was used as  a measure of v a r i a t i o n . Materials A l l chemicals used were a n a l y t i c a l grade.  ATP, DEAE-Sephadex,  EDTA and Trizma base were purchased from Sigma Chemical Company.  EGTA,  \/ 3 2 • -P  45 (ATP) (10-20 Ci/mmole and  CaCl  2  (lOCCi/mmole) were purchased from  Amersham Company.Charcoal ( N o r i t ' A ) was purchased from F i s h e r and Aquasol s c i n t i l l a t i o n  Scientific  f l u i d from New England N u c l e a r Company.  -25Results P a r t i a l p u r i f i c a t i o n o f plasma membranes from c u l t u r e d s k i n Table 1 i l l u s t r a t e s  t h e r e s u l t s o f assays  of 5 ' - n u c l e o t i d a s e and a d e n y l a t e  done t o determine the presence  c y c l a s e a c t i v i t y i n t h e crude plasma mem-  brane f r a c t i o n and i n the "10/30" f r a c t i o n o b t a i n e d density gradient c e n t r i f u g a t i o n .  fibroblasts  f o l l o w i n g sucrose  When t h e crude plasma membrane  fraction,  o b t a i n e d by d i f f e r e n t i a l c e n t r i f u g a t i o n , was s e p a r a t e d on a sucrose density and  g r a d i e n t t h e r e was a 5 - f o l d i n c r e a s e i n the a c t i v i t y o f 5 ' - n u c l e o t i d a s e adenylate  c y c l a s e i n the f r a c t i o n p r e s e n t  These enzymes a r e known t o be p r e s e n t  a t the "10/30" i n t e r f a c e .  i n t h e plasma membrane o f f i b r o b l a s t s  (Solymon and Trams 1972; K a r t n e r e t a l 1977).  The enhancement i n a c t i v i t y  of these enzymes i n the "10/30" f r a c t i o n t h e r e f o r e suggests i s e n r i c h e d i n plasma membranes.  that t h i s  fraction  T h i s f r a c t i o n o f f i b r o b l a s t membranes was  t h e r e f o r e u t i l i z e d as t h e plasma membrane-enriched p r e p a r a t i o n i n f u r t h e r experiments. 2+ P a r t i a l c h a r a c t e r i z a t i o n o f the (Mg membrane p r e p a r a t i o n s  + Ca  2+ )-ATPase a c t i v i t y  A h i g h degree of t h e ATP h y d r o l y t i c a c t i v i t y noted  i n fibroblast  i n both  the crude  2+ membrane and plasma membrane-enriched p r e p a r a t i o n s of f i b r o b l a s t s was Ca 2+ 2+ dependent. F i g u r e 2 i l l u s t r a t e s the Mg -dependency o f the Ca stimulated component o f t h e ATPase a c t i v i t y i n t h e crude membrane and plasma membrane -enriched f r a c t i o n .  The r e s u l t s a r e expressed  mg p r o t e i n p e r minute. present  as nmoles  2+ 2+ (Mg + Ca )-ATPase a c t i v i t y  32 P i released per  r e f e r s to that a c t i v i t y  f o l l o w i n g s u b t r a c t i o n o f the a c t i v i t y measured i n the absence o f  added c a l c i u m and i n t h e presence  o f 0.1 mM EGTA.  The Ca  2+  stimulated  component of t h i s a c t i v i t y was markedly i n c r e a s e d i n t h e presence 0.5-5.0 mM Mg 20.0  2+  mM MgS0 . 4  of f r om  w i t h maximal s t i m u l a t i o n i n both p r e p a r a t i o n s o b t a i n e d a t  -26-  T a b l e 1.  5 ' - N u c l e o t i d a s e and a d e n y l a t e c y c l a s e a c t i v i t i e s of f i b r o b l a s t membrane p r e p a r a t i o n s  . Adenylate-Cyclase Fraction  5'-Nucleotidase  Basal  Crude  5.11+1.20  3.35+0.75  6.75+1.20  10/30  26.36 + 2.80  14.00 + 2.50  76.00 + 5.20  Assays were performed  as i n Methods.  i s expressed as pinoles.  of 5'-nucleotidase  P i r e l e a s e d p e r mg p r o t e i n per hour.  Adenylate c y c l a s e a c t i v i t y p r o t e i n per minute.  The a c t i v i t y  NaF  i s expressed as pmoles c y c l i c AMP p e r mg  -27-  F i g u r e 2.  Mg  -dependency o f the Ca  - s t i m u l a t e d ATPase  activity  i n f i b r o b l a s t membrane p r e p a r a t i o n s . ATPase a c t i v i t y  i n t h e presence of 200 uM Ca  2+  f r e e was  determined as d e s c r i b e d i n Methods i n t h e absence 2+ (0.1 mM EGTA) and presence o f v a r y i n g Mg tions and  (0.5-50 mM)  concentra-  i n crude plasma membrane ( O  plasma membrane-enriched p r e p a r a t i o n s  of c u l t u r e d f i b r o b l a s t s .  The r e s u l t shown i s a  t y p i c a l experiment o f d u p l i c a t e  determinations.  O ) ©)  -28-  -292+  2+  In order to f u r t h e r c h a r a c t e r i z e the (Mg + Ca )-ATPase a c t i v i t y p r e s e n t i n the f i b r o b l a s t membrane p r e p a r a t i o n s , t h e e f f e c t of c a l m o d u l i n was i n v e s t i g a t e d .  T h i s p r o t e i n has p r e v i o u s l y been shown to s p e c i f i c a l l y  2+ 2+ s t i m u l a t e r e d c e l l membrane (Mg + Ca )-ATPase a c t i v i t y 1973;  L u t h r a e t a l 1976 a&b).  (Bond and Clough  F i g u r e 3 and 4 i l l u s t r a t e t h a t  calmodulin 2+  obtained  from r e d c e l l hemolysates s i g n i f i c a n t l y  s t i m u l a t e d the (Mg  +  2+ Ca )-ATPase a c t i v i t y p r e s e n t i n these f r a c t i o n s . Two d i f f e r e n t c a l m o d u l i n p r e p a r a t i o n s were used i n t h i s study: One o f t h e c a l m o d u l i n p r e p a r a t i o n s 2+ u t i l i ' z e d produced a much h i g h e r s t i m u l a t i o n o f t h e (Mg activity  + Ca  )-ATPase  i n t h e membrane f r a c t i o n s than the o t h e r ; t h e extent of  a c t i v a t i o n over t h a t a c t i v i t y noted 150%  2+  i n t h e absence o f added c a l m o d u l i n was  and 296% i n the crude and t h e plasma membrane-enriched  respectively  fractions,  ( F i g u r e 4) as compared to 83% and 114% s t i m u l a t i o n when t h e  l e s s a c t i v e c a l m o d u l i n p r e p a r a t i o n was used  ( F i g u r e 3 ) . The amount o f  c a l m o d u l i n p r e p a r a t i o n ( F i g u r e 4) n e c e s s a r y  t o produce  half-maximal  a c t i v a t i o n was 0.63 ug i n the crude membrane and 1.52 ug i n t h e plasma membrane-enriched  fraction. 2+  2+  Comparison of (Mg + Ca )-ATPase a c t i v i t y i n c u l t u r e d f i b r o b l a s t s d e r i v e d from C F . p a t i e n t s and c o n t r o l s In a l l , 4 C F . and 3 c o n t r o l s t r a i n s were used i n t h i s study. 2+ 2+ F i g u r e 5 i l l u s t r a t e s a t y p i c a l experiment o f the measurement o f (Mg + Ca )• ATPase a c t i v i t y  i n crude membrane p r e p a r a t i o n s of C F . and c o n t r o l  blast  (Mg  strains.  fibro-  2+ 2+ + Ca )-ATPase a c t i v i t y was i n c r e a s e d w i t h i n c r e a s i n g  f r e e c a l c i u m c o n c e n t r a t i o n i n both C F . and c o n t r o l s t r a i n s t o a maximum at  200 uM Ca  2+  free.  The (Mg  2+ 2+ + Ca )-ATPase a c t i v i t y noted  i n the c o n t r o l  -30-  F i g u r e 3.  (Mg  + Ca  purified  )-ATPase a c t i v i t y i n crude membrane and i n  plasma membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o -  b l a s t s i n t h e presence 2+ (Mg  and absence o f added  calmodulin.  2+  + Ca  )-ATPase a c t i v i t y i n t h e presence  o f 200 uM  2+ Ca  f r e e was determined  absence and presence  as d e s c r i b e d i n Methods i n the  o f varying c a l m o d u l i n  concentrations  (3.25-9. 75 ug) i n the crude plasma membrane ( O plasma membrane-enriched p r e p a r a t i o n s cultured fibroblasts.  The r e s u l t  (@  O ) and  ® ) of  shown i s a t y p i c a l  experiment of d u p l i c a t e d e t e r m i n a t i o n s .  -31-  -32-  F i g u r e 4.  (Mg  + Ca  purified  )-ATPase a c t i v i t y i n crude membrane and i n  plasma membrane p r e p a r a t i o n s of c u l t u r e d f i b r o -  b l a s t s i n t h e presence 2+ (Mg  and absence o f added  calmodulin.  2+  + Ca  )-ATPase a c t i v i t y i n t h e presence  o f 200 pM  2+ Ca  f r e e was determined  absence and presence  as d e s c r i b e d i n Methods i n t h e  of varying calmodulin  (0.85-^7-6.4 ug) i n the crude and  plasma membrane ( O  plasma membrane-enriched p r e p a r a t i o n s  cultured fibroblasts.  concentrations  The r e s u l t  (<@  —-O) @) o f  shown i s a t y p i c a l  experiment o f d u p l i c a t e d e t e r m i n a t i o n s .  -33-  -34-  F i g u r e 5.  (Mg  + Ca  of c u l t u r e d (Mg  )-ATPase a c t i v i t y i n crude membrane  preparations  fibroblasts.  2+ 2+ + Ca )-ATPase a c t i v i t y was measured as d e s c r i b e d i n  Methods i n the absence (0.1 mM EGTA) and presence (50-600 2+ uM Ca  f r e e ) o f added c a l c i u m i n crude membrane  t i o n s d e r i v e d from c o n t r o l ( O fibroblast  strains.  O)  a  n  prepara-  d C F . (©  The r e s u l t s shown a r e a t y p i c a l  experiment of d u p l i c a t e  determinations.  @)  -35-  nmoles Pi released mg min32  -1  -36-  s t r a i n s was c o n s i s t e n t l y h i g h e r than t h a t noted free calcium concentrations tested. data i n d i c a t i n g t h a t t h e ^Q 2+  a  n  a  i n the C F . s t r a i n s a t a l l  F i g u r e 6 i s a r e c i p r o c a l p l o t of t h i s  d not the K ^ i s s o f the (Mg  2+ 2+ + Ca )-ATPase  for calcium i s altered i n the C F . s t r a i n s . 2+ (Mg  2+  + Ca  )-ATPase a c t i v i t y  t i o n s was g r e a t l y i n c r e a s e d over preparations. of the (Mg  i n the plasma membrane-enriched t h a t observed  prepara-  i n the crude membrane  F i g u r e 7 i l l u s t r a t e s a t y p i c a l experiment of the d e t e r m i n a t i o n  2+ 2+ + Ca )-ATPase a c t i v i t y  i n plasma membrane-enriched p r e p a r a t i o n s 2+ 2+ of both C F . and c o n t r o l s t r a i n s . A g a i n , the (Mg + Ca )-ATPase a c t i v i t y of t h i s p r e p a r a t i o n i n c r e a s e d w i t h i n c r e a s i n g f r e e c a l c i u m c o n c e n t r a t i o n 2+  2+  to a maximum a t 200 uM Ca  free.(Mg  s t r a i n s was c o n s i s t e n t l y h i g h e r than  2+  + Ca  )-ATPase a c t i v i t y  i n the c o n t r o l  t h a t i n the C F . s t r a i n s .  Figure 8 i s  a r e c i p r o c a l p l o t of t h i s d a t a i n d i c a t i n g a g a i n , t h a t i t i s t h e V^ 2+ and 2+ 2+ a  not the K ^ g g of the (Mg CF.  + Ca  )-ATPase f o r c a l c i u m t h a t i s a l t e r e d i n t h e  strains. T a b l e 2 shows a comparison o f the crude membrane and plasma membrane-  e n r i c h e d p r e p a r a t i o n s used-in- t h i s study.  P u r i f i c a t i o n o f t h e crude membrane 2+ 2+  p r e p a r a t i o n i n c r e a s e d t h e s p e c i f i c a c t i v i t y o f t h e (Mg approximately  4-5 f o l d .  + Ca  )-ATPase  T h i s i s an 80-100 f o l d i n c r e a s e over the a c t i v i t y  noted p r e v i o u s l y i n f i b r o b l a s t membrane homogenate p r e p a r a t i o n s 1978 b; not shown).  The v^ 2+ was s i g n i f i c a n t l y a  decreased  (Katz  i n both t h e  crude membrane (p<0.01) and t h e plasma membrane-enriched p r e p a r a t i o n s (p^ 0.05) o b t a i n e d obtained  from C F . f i b r o b l a s t s t r a i n s  from c o n t r o l s t r a i n s .  compared t o p r e p a r a t i o n s  The K ^ g g f o r c a l c i u m was i n c r e a s e d  -37-  F i g u r e 6.  K,.  and V  2+ f the (Mg^+ C a ^ ) - A T P a s e a c t i v i t y  r  D  of crude membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o b l a s t s . 2+ R e c i p r o c a l p l o t s of a t y p i c a l d e t e r m i n a t i o n of (Mg  +  2+ Ca )-ATPase a c t i v i t y o f crude membrane p r e p a r a t i o n s d e r i v e d from c o n t r o l ( O — - O ) and C F . ( ® «®) fibroblast  strains.  i n nmoles mg"*" min "^". -  -  The K ^ g g i s expressed  i n pM and V^ 2+ &  -38-  -39-  Figure  7.  (Mg  + Ca  )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane  preparations (Mg  of c u l t u r e d  fibroblasts.  2+ 2+ + Ca )-ATPase a c t i v i t y was measured as d e s c r i b e d  i n Methods i n the absence (0.1 mM EGTA) and presence 2+ (50-600 uM Ca  f r e e ) of added c a l c i u m i n p u r i f i e d  plasma membrane p r e p a r a t i o n s (O-  O)  The r e s u l t s  d e r i v e d from  and C F . («@ shown a r e a t y p i c a l  determinations.  @)  control  fibroblast  strains.  experiment o f - d u p l i c a t e  - 4 1 -  F i g u r e 8.  K  d l s g  and ^ 2+  purified  Qg  o f t h e (Mg  + Ca  )-ATPase a c t i v i t y o f  plasma membrane p r e p a r a t i o n s  of c u l t u r e d  fibroblasts.  2+ Reciprocal p l o t s of a t y p i c a l determination  of (Mg  +  2+ Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane p r e p a r a t i o n s d e r i v e d from c o n t r o l ( O O ) and C F . (<g)  @) f i b r o b l a s t  i n uM and t h e V  strains.  The K ^ d  2 + in~ nmoles mg"'" min \ -  F  s g  i s expressed  -42-  -43-  T a b l e 2.  K,.  Q l S S  and V  p  L<cL  2+ o f the (Mg  + Ca  )-ATPase of f i b r o b l a s t  membrane p r e p a r a t i o n s .  K  diss  XpM) V 2 + c &  (NMOLES MG  MIN - )  1  1  CRUDE PLASMA MEMBRANES: CONTROL STRAINS C F . STRAINS  39.1 + 5.3 41.5 + 2.2  48.9 + 3.8 27.8 + 2.1  a  PURIFIED PLASMA MEMBRANES: CONTROL STRAINS C F . STRAINS  113.0 + 6.1 103.4 + 6.6  207.0 + 39.4 102.7 + 10. 8  b  ( M g + C a ) - A T P a s e a c t i v i t y o f f i b r o b l a s t s membrane p r e p a r a t i o n s was determined as d e s c r i b e d i n Methods i n the presence o f v a r y i n g Calcium' c o n c e n t r a t i o n s (50-600 uM Ca +) f r e e ) . R e c i p r o c a l p l o t s of d u p l i c a t e s o f i n d i v i d u a l experiments were then drawn and the i n t e r c e p t s determined by r e g r e s s i o n a n a l y s i s . The r e s u l t s a r e the mean + S.E.M. o f 3 c o n t r o l s t r a i n s and 4 C F . s t r a i n s . 2+  2+  2  statistically (P < 0.01)  s i g n i f i c a n t compared t o t h e c o n t r o l s  b statistically (P< 0.05)  s i g n i f i c a n t compared t o the c o n t r o l s  a  a p p r o x i m a t e l y 2 1/2  f o l d i n the plasma membrane-enriched  compared to the crude membrane f r a c t i o n . though, i n the K ^ i s s f ° control  calcium  r  No  i n the C F .  d i f f e r e n c e was  In t h i s p r e s e n t  s t r a i n s compared to  the  whether the a l t e r a t i o n i n (Mg samples c o u l d s i g n i f i c a n t l y accomplish t h i s ,  d e r i v e d from red c e l l s  work, subsequent s t u d i e s were d i r e c t e d at 2+  of  determining  2+ + Ca  )-ATPase a c t i v i t y  a f f e c t calcium  observed i n  transport i n C F .  red c e l l v e s i c l e p r e p a r a t i o n s  obtained  CF.  cells.  from C F .  To  patients  c o n t r o l s were used. The  r e l a t i v e proportion  of i n s i d e - o u t , r i g h t - s i d e - o u t and  membranes i n the red c e l l v e s i c l e p r e p a r a t i o n s  was  estimated  the a c t i v i t y of the enzymes l o c a t e d on the o u t e r  surface  inner surface  The  (G3-PD) of the red c e l l membrane.  a c t i v i t y measured i n the absence and was  observed,  strains.  S t u d i e s on i n s i d e - o u t v e s i c l e p r e p a r a t i o n s c o n t r o l s and C F . p a t i e n t s  and  fraction  by  unsealed determining  (AChE) and  the  r a t i o of the enzyme  i n the p r e s e n c e of 0.1%  Triton  X-100  used as a measure of the percentage a c c e s s i b i l i t y of the marker enzymes  .{Cohen and  Solomari 1976).  Only 20-25% of the t o t a l AChE was  w h i l s t 55-60% of the t o t a l G3-PD was  accessible.  15-25% unaccounted f o r , c o u l d be  to the presence of membrane f r a g -  ments.  Thus, the p r e p a r a t i o n s  10, v e s i c l e s . and  due  remaining  activity,  were found to c o n s i s t e n t l y c o n t a i n 55-60%  10 v e s i c l e s were not  broken membranes due  The  accessible  separated  from r i g h t - s i d e - o u t v e s i c l e s  to the s i g n i f i c a n t l o s s i n Ca  produced by the s e p a r a t i o n procedures a v a i l a b l e .  -uptake a c t i v i t y  -45-  2+ Comparison of Ca -uptake a c t i v i t y i n 10 v e s i c l e p r e p a r a t i o n o b t a i n e d from C F . p a t i e n t s and c o n t r o l s 2+ F i g u r e 9 compares the Ca -uptake a c t i v i t y of f i v e d i f f e r e n t c o n t r o l and  six different C F .  10 v e s i c l e p r e p a r a t i o n s , each o b t a i n e d from a 2+  different per mg  subject.  The r e s u l t s are expressed as Ca  -uptake  i n nmoles  p r o t e i n f o l l o w i n g s u b t r a c t i o n o f t h a t a c t i v i t y noted i n the  of ATP.  I t can be observed t h a t c a l c i u m a c c u m u l a t i o n was  and v a r i e d l i t t l e  from p r e p a r a t i o n to p r e p a r a t i o n .  p r e p a r e d from c o n t r o l b l o o d samples have a s i m i l a r Ca  absence  time-dependent  The 10 red c e l l  vesicles  i n t h i s p r e s e n t study were found to  2+ . - t r a n s p o r t a c t i v i t y t o those prepared by o t h e r workers  ( S a r k a d i et a l 1978;  L a r s e n and V i n c e n z i 1979).  I t can be seen t h a t the  2+ amount of Ca  -uptake observed i n the c o n t r o l p r e p a r a t i o n s was  h i g h e r than t h a t observed i n the C F . studied.  considerably  p r e p a r a t i o n s at a l l time p o i n t s  Experiments were then c a r r i e d out to determine whether t h i s  d i f f e r e n c e was  apparent at the i n i t i a l times of i n c u b a t i o n . F i g u r e 10 2+ i l l u s t r a t e s the Ca -uptake a c t i v i t y of both C F . and c o n t r o l p r e p a r a t i o n s at sample times between 10 seconds and 10 minutes. In both C F . and c o n t r o l 2+ p r e p a r a t i o n s , t h e r e was a l i n e a r r e l a t i o n s h i p between the Ca -uptake 2+ a c t i v i t y and the time of i n c u b a t i o n . Ca -uptake a c t i v i t y was found to be c o n s i d e r a b l y reduced i n the C F . and at a l l subsequent  p r e p a r a t i o n s at the e a r l i e s t time of a s s a y  assay t i m e s .  2+ Ca  -uptake a c t i v i t y i n 10 v e s i c l e p r e p a r a t i o n s d e r i v e d from C F .  c o n t r o l red c e l l s was (10-150 uM Ca  2+  free).  and  then determined at v a r i o u s f r e e c a l c i u m c o n c e n t r a t i o n s F i g u r e 11a i l l u s t r a t e s  i n c r e a s e d w i t h i n c r e a s i n g f r e e Ca  2+  t h a t Ca  2+ -uptake  activity  c o n c e n t r a t i o n to a maximum a t 100  uM  -46-  F i g u r e 9.  Time-Course preparations Ca  of Ca  -Uptake A c t i v i t y i n 10 v e s i c l e  from C o n t r o l and C F . samples.  2+ -uptake was determined as d e s c r i b e d i n Methods 2+  i n the presence of 150 pM Ca (O  -O)  free i n control  and C F . p r e p a r a t i o n s  Results represent  (®  the mean + S.E.M. of f i v e  ® ) . control  and s i x C F . p r e p a r a t i o n s each from a d i f f e r e n t  donor.  -47-  ujajojd Bui/«»|ouiu :»])04dn +  +  03  -48-  F i g u r e 10.  I n i t i a l r a t e s o f Ca  -uptake i n 10 v e s i c l e s from C o n t r o l  and C F . samples. Ca  2+ -uptake was determined as d e s c r i b e d i n Methods i n t h e 2+  presence o f 150 pM Ca CF.  preparations  Results represent  (@  free i n control ( O  O ) and  ®) .  t h e mean + S.E.M. o f f i v e c o n t r o l and  six C F . preparations  each from a d i f f e r e n t  donor.  -49-  C  'tt)  Time (min)  -50-  F i g u r e 11.  Ca  -uptake a c t i v i t y  added c a l m o d u l i n  i n the presence and  absence of  i n 10 v e s i c l e p r e p a r a t i o n s from  CF.  p a t i e n t s and c o n t r o l s . 2+ a) Ca  -uptake was  measured as d e s c r i b e d i n Methods 2+  i n the presence of v a r i o u s f r e e Ca (10-150 uM) CF.  in five control ( O  preparations  donor and  (©  @)  concentrations O)  and s i x  each from a d i f f e r e n t  i n the presence of added c a l m o d u l i n  (0.8  pg/ml f i n a l i n c u b a t i o n volume) i n these c o n t r o l (A  A)  and  CF.  (^  A)  preparations.  b)' R e c i p r o c a l p l o t s of data shown i n F i g u r e  11a.  -52-  Ca  2+  2+ free i n both C F . and control preparation; Ca -uptake a c t i v i t y was  consistently reduced i n the C F . preparations at a l l free calcium concentrations studied. Calmodulin has been shown to be present i n a number of c e l l types and to regulate the a c t i v i t y of the calcium pump system i n red c e l l preparations (Luthra et a l a&b; Larsen and Vincenzi i n disease states i s not yet known.  1979), I t s r o l e or possible a l t e r a t i o n  Figure 11a i l l u s t r a t e s that when calmo-  dulin from red c e l l hemolysates was added to the incubation medium i t 2+ stimulated Ca -uptake a c t i v i t y i n both C F . and control preparations to approximately  the same extent (35-39%).  Calmodulin, though, did not  2+ restore the Ca  -uptake a c t i v i t y of the C F . samples to levels  observed  i n the control preparations. 2+ Figure l i b and Table 3 i l l u s t r a t e the K ^ d  g g  for Ca  and the V^ 2+ a  2+ of the Ca -transport system i n the 10 v e s i c l e preparations of the control and C F . preparations i n the presence and absence of added calmodulin. 2+ It can be noted that there was l i t t l e a l t e r a t i o n i n the K ^ g g for Ca i n the C F . samples compared to the controls. Calmodulin had l i t t l e e f f e c t 2+ on the K ^ g g f o r Ca  i n either preparation.  I t can_>- also be noted that v  the V^ 2+ was s i g n i f i c a n t l y decreased i n C F . samples (p ^ 0.001) compared a  to the controls.  Calmodulin s i g n i f i c a n t l y increased the V^ 2+ i n both groups &  (p<0.01 i n the C F . group and p ^0.001 i n the control group) but did not restore the V 2+ of the C F . preparations to control values. Ca  2+ 2+ (Mg + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane preparations of human cultured lymphoblasts Lymphoblasts obtained from C F . patients have been reported to be  -53-  Table  3.  K c  j  i  s  s  and V 2 + - u p t a k e system i n c o n t r o l Ca  and C F . p r e p a r a t i o n s  of 10 v e s i c l e s .  Kdiss^) Control  (5) a  CF. (6)  a  +  calmodulin calmodulin  22.17 + 1.54 24.30 + 1.83  - calmodulin + calmodulin  20.75 + 1.54 18.56 + 2.19  Ca"'-uptake was assayed  as d e s c r i b e d i n Methods.  V  t  Ca  (nmoles/mg)  2 +  61.23 + 1.63 85.24 + 1.82c 28.47 + 1.48 38.50 + 2.25°' c  When c a l m o d u l i n was  added i t was p r e s e n t i n a c o n c e n t r a t i o n o f 0.8 pg/ml f i n a l i n c u b a t i o n volume. a  Number of p r e p a r a t i o n s i n each group, each from a d i f f e r e n t  subject,  b Mean + S.E.M. i n each case. c  d  S i g n i f i c a n t (P K 0.001) when compared t o c o n t r o l added c a l m o d u l i n .  i n t h e absence o f  S i g n i f i c a n t (P< 0.01) when compared to C F . i n t h e absence o f added c a l m o d u l i n .  d  -54-  abnormal w i t h r e s p e c t t o the a c t i v i t y of oC.- L - f u c o s i d a s e (Maler and Riordan  1979).  Other membrane-bound enzyme a c t i v i t i e s may a l s o be a l t e r e d  in this  cell-type.  I t was t h e r e f o r e i n v e s t i g a t e d whether (Mg  a c t i v i t y was p r e s e n t i n lymphoblast was a l t e r e d i n C F . samples. ATPase a c t i v i t y of p u r i f i e d  membrane p r e p a r a t i o n s and i f t h i s  T a b l e 4 i s a comparison o f t h e (Mg  b l a s t s d e r i v e d from C F . p a t i e n t s and c o n t r o l s . observed  than t h a t noted fibroblasts 2+ (Mg  + Ca  activity  2+ 2+ + Ca ) -  plasma membrane p r e p a r a t i o n s o f c u l t u r e d lympho2+  activity  2+ 2+ + Ca )-ATPase  i n the lymphoblast  (Mg  + Ca  2+ )-ATPase  membrane p r e p a r a t i o n s was much  lower  i n t h e p u r i f i e d plasma membrane p r e p a r a t i o n s of c u l t u r e d  (see T a b l e 2 ) . No s i g n i f i c a n t  d i f f e r e n c e was d e t e c t e d i n the  2+ )-ATPase a c t i v i t y p r e s e n t  compared to t h a t a c t i v i t y p r e s e n t  i n the C F . plasma membrane samples  i n the c o n t r o l s .  -55-  T a b l e 4.  (Mg + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane p r e p a r a t i o n s of human c u l t u r e d lymphoblasts.  nmoles P-; r e l e a s e d mg  min'  CONTROL PREPARATIONS PM 23 PM 17 PM 19  36.0 33.7 38.4 + S.E.M.  36.0 + 1.3  C y s t i c F i b r o s i s PREPARATIONS PM 22 PM 18 PM 16  46.0 39.8 30.5 + S.E.M.  38.8 + 4.5  a  P u r i f i e d plasma membranes from c u l t u r e d lymphoblasts were prepared by Tom Maler and Dr. J . R i o r d a n , H o s p i t a l f o r S i c k C h i l d r e n , These p r e p a r a t i o n s  Toronto.  were f r o z e n and thawed twice p r i o r t o use and i n -  cubated a t 37° f o r 30 minutes i n t h e presence of 5 mM MgCl2,50 mM T r i s - H C l , pH 7.4, 3 mM Y =  3 2  P(ATP) and 0.10 mM f r e e Ca  was t e r m i n a t e d w i t h 5% t r i c h l o r o a c e t i c a c i d as d e s c r i b e d  .  The r e a c t i o n  previously.  r e s u l t s shown a r e d u p l i c a t e s f o l l o w i n g the s u b t r a c t i o n o f the ATPase a c t i v i t y measured i n the absence of added f r e e Ca a  not s t a t i s t i c a l l y  2+  s i g n i f i c a n t when compared t o the c o n t r o l s .  The  -56-  Discussion  I n t h e p r e s e n t study, t h e presence  2+ 2+ o f a (Mg + Ca )-ATPase a c t i v i t y  i n p u r i f i e d plasma membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o b l a s t s was demonstrated.  The s i g n i f i c a n t decrease  i n (Mg  2+ 2+ + 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 f i b r o b l a s t s and r e d c e l l s  observed  (Katz 1978 a)  2+ d e r i v e d from C F . s t r a i n s activity  observed  t h i s present  c o u l d be r e l a t e d  to the a l t e r e d  Ca  -uptake  i n 10 v e s i c l e p r e p a r a t i o n s from C F . r e d b l o o d c e l l s i n  study.  The p r e p a r a t i o n of plasma membrane-enriched f r a c t i o n s from c u l t u r e d f i b r o b l a s t s i n v o l v e d mechanical hypotonic  conditions.  agents, It  c e l l s under  T h i s has advantages over many of the e x i s t i n g methods  (Warren et a l 1966; B r u n e t t e proteases  d i s r u p t i o n o f the h a r v e s t e d  and T i l l  1971),in  t h a t i t a v o i d s the use o f  f o r h a r v e s t i n g o f c e l l s and the use of d e n a t u r i n g and c r o s s l i n k i n g  l i k e heavy metals  or aldehydes,  which i n f l u e n c e c e l l f u n c t i o n .  appears e v i d e n t from these  s t u d i e s t h a t the plasma membrane-enriched 2+ 2+ p r e p a r a t i o n s of c u l t u r e d f i b r o b l a s t s c o n t a i n a h i g h degree of (Mg + Ca ) ATPase a c t i v i t y . P u r i f i c a t i o n o f the membrane p r e p a r a t i o n s by sucrose 2+ d e n s i t y g r a d i e n t c e n t r i f u g a t i o n r e v e a l e d a 4-5 f o l d i n c r e a s e i n (Mg ATPase a c t i v i t y which i s 80-100 f o l d h i g h e r than t h a t p r e v i o u s l y i n homogenates (Katz 1978 b ) .  + Ca  )-  observed  A l s o a 4-5 f o l d i n c r e a s e i n 5 ' - n u c l e o t i d a s e  and a d e n y l a t e c y c l a s e a c t i v i t i e s was observed The  2+  i n the "10/30" f r a c t i o n .  f a c t t h a t these two enzymes, b e l i e v e d to be i n d i g i n o u s t o the plasma 2+ 2+  membrane, " c o - p u r i f y " w i t h t h i s f r a c t i o n i n d i c a t e s t h a t the (Mg ATPase a c t i v i t y  + Ca  )-  i s i n d i g i n o u s t o the plasma membrane and p o s s i b l y f u n c t i o n s  to r e g u l a t e t h e i n t r a c e l l u l a r  f r e e c a l c i u m c o n c e n t r a t i o n , s i m i l a r to i t s  r o l e i n the red b l o o d membranes was 2+ (Mg  cell.  The  (Mg  2+  + Ca  2+  )-ATPase of f i b r o b l a s t plasma  found to share many of the same c h a r a c t e r i s t i c s as  the  2+ + Ca  )-ATPase from other  tissues  ( V i n c e n z i and Hinds 1976).  This 2+  i n c l u d e s a c t i v a t i o n by micromolar c o n c e n t r a t i o n s -dependency.  T h i s source of the enzyme i s a l s o s t i m u l a t e d  p r e p a r e d from r e d c e l l hemolysates. p u r i f i e d and blasts.  of c a l c i u m and  Recently,  characterized calmodulin  T h e i r p r e p a r a t i o n was  by  a  calmodulin  E l d i k and Watterson  from transformed c h i c k e n  g i z z a r d and b r a i n and  c h e m i c a l and  functional properties.  (1979)  embryo  i n d i s t i n g u i s h a b l e from c a l m o d u l i n  from c h i c k e n  Mg  fibro  prepared  bovine b r a i n i n terms of p h y s i c a l ,  2+ 2+  I t was  observed t h a t w h i l s t the degree of s t i m u l a t i o n of the  (Mg  +  Ca )-ATPase a c t i v i t y by c a l m o d u l i n i n c r e a s e d markedly i n p u r i f i e d plasma membranes compared to crude p r e p a r a t i o n s , the apparent K ^ i s s of c a l m o d u l i n for  stimulation.'increased. 2+  VQ 2+ of the a  (Mg  S i m i l a r l y , i t was  + Ca  )-ATPase i n c r e a s e d  i n t h i s p r e p a r a t i o n was  affinity- calmodulin-sensitive  lower.  arations  Moore and  (Mg  s i g n i f i c a n t l y as the f i b r o b l a s t  calcium a f f i n i t y  2+  blast organelle.  + Ca  of the enzyme system  T h i s i m p l i e s t h a t there  is a  )-ATPase.system i n .another  t r a n s p o r t system i n c r e a s e d up thus appears t h a t at l e a s t two  They a l s o observed  to 8 f o l d as a f u n c t i o n of c e l l energy-dependent c a l c i u m  e x i s t i n c u l t u r e d f i b r o b l a s t s ; one the o t h e r  fibro-  Paston (1977) have shown t h a t microsomal prep-  from f i b r o b l a s t s a c t i v e l y accumulate c a l c i u m .  f r a c t i o n and  higher  2+  t h a t i n growing c u l t u r e s the s p e c i f i c a c t i v i t y of the microsomal  may  the  2+  plasma membranes were p u r i f i e d , the present  observed t h a t a l t h o u g h  calcium  density.  transport  systems  a s s o c i a t e d w i t h the microsomal  a s s o c i a t e d w i t h the plasma membrane.  The  It  former  -58-  may  be  involved  (Moore and  i n the m o d u l a t i o n o f c e l l u l a r movement and  Paston 1977)  a t the l e v e l of the  c o n t r o l o f the i n t r a c e l l u l a r f r e e c a l c i u m  concentration  2+ I t was  demonstrated t h a t the  (Mg  plasma membrane-enriched p r e p a r a t i o n s duced i n C F . was  strains.  s i m i l a r i n the C F .  cytoplasm and  proliferation the l a t t e r i n the  (Kartner  et a l 1977).  2+ + Ca  )-ATPase a c t i v i t y present  of f i b r o b l a s t s was  in  significantly  K i n e t i c a n a l y s i s i n d i c a t e d t h a t the K ^ g g  for  recalcium  and  c o n t r o l s t r a i n s but t h a t t h e r e was a s i g n i f i c a n t 2+ 2+ decrease i n the maximal a c t i v a t i o n of the (Mg + Ca )-ATPase i n the CF.  fibroblast strains. F i b r o b l a s t s represent might be and  abnormal i n C.F.;  release  (Matalon and  as w e l l as an i n c r e a s e d blue  (Danes and  DNA  a c e l l - t y p e t h a t many s t u d i e s have i n d i c a t e d abnormally h i g h  Dorfman 1968)  r a t e s of g l y c o s a m i n o g l y c a n  have been observed i n C F .  o c c u r r e n c e of c y t o p l a s m i c  B e a m 1969)  (Barranco et a l 19 76).  and  synthesis  fibroblasts  metachromasia w i t h t o l u i d i n e  a decreased i n c o r p o r a t i o n of thymidine i n t o  There are  few  reports  i n the l i t e r a t u r e  possible alterations i n ion-transport  in CF.  and  f i b r o b l a s t s t r a i n s found no a l t e r a t i o n  L i n (1972) i n a study of two  i n Na , +  K -ATPase a c t i v i t y and +  i n 3 -ouabain b i n d i n g H  F e i g a l and  in CF.  Shapiro  CF.  Q u i s s e l and  fibroblast strains;  regarding  Pitot  Fletcher  (1974) observed no  difference  fibroblasts.  (1979a) have r e c e n t l y r e p o r t e d  an e n l a r g e d  intra-  2+ c e l l u l a r Ca  pool  i n s k i n f i b r o b l a s t s from s u b j e c t s with C F . and o b l i g a t e 2+ h e t e r o z y g o t e s and i n d i c a t e d t h a t the a l t e r e d Ca p o o l represented a change 2+ inomitochondfia'l Ca content. These workers ( F e i g a l and S h a p i r o 1979b) have a l s o shown t h a t m i t o c h o n d r i a from C F .  c e l l s accumulate more Ca  2+  than  those from c o n t r o l c e l l s .  These s t u d i e s a r e i n agreement w i t h  our f i n d i n g s  s i n c e a l e s s e f f e c t i v e plasma membrane c a l c i u m pump would l e a d t o the accumulation  of i n t e r n a l c e l l c a l c i u m and suggests  t h a t the i n t r a c e l l u l a r  p o o l of c a l c i u m t h a t i s enhanced e x i s t s i n the m i t o c h o n d r i a . Ca  2+ - t r a n s p o r t s t u d i e s were done i n 10 v e s i c l e p r e p a r a t i o n s from r e d  b l o o d c e l l s because i n such an i n v e r t e d system the c o n d i t i o n s a t t h e ATP, 2+ Mg  2+ and Ca  -binding sites  c o u l d be easily, c o n t r o l l e d .  Although  the 10  v e s i c l e p r e p a r a t i o n s were n o t p u r i f i e d by e l i m i n a t i o n o f r i g h t - s i d e - o u t v e s i c l e s and broken fragments they were found results.  to y i e l d  extremely  reproducible  The s p e c i f i c a c t i v i t y o f these p r e p a r a t i o n s was a l s o found  to be  e q u a l t o o r h i g h e r than t h a t o f s i m i l a r p r e p a r a t i o n s used by o t h e r workers ( L a r s e n and V i n c e n z i 1979; S a r k a d i et a l 1978) when the p r o p o r t i o n o f 10 v e s i c l e present  T h i s study  to t o t a l p r o t e i n i s taken i n t o c o n s i d e r a t i o n . 2+ i n d i c a t e d t h a t Ca  -uptake a c t i v i t y i n ;IQ v e s i c l e  a r a t i o n s o b t a i n e d from C F . p a t i e n t s was s i g n i f i c a n t l y reduced  prep-  when compared  to c o n t r o l s ; t h i s r e d u c t i o n i s observed  a t a l l t i m e - p o i n t s and a t a l l 2+ f r e e c a l c i u m c o n c e n t r a t i o n s t e s t e d . That t h e Ca -uptake a c t i v i t y i n the C F . p r e p a r a t i o n s i s reduced a t the i n i t i a l times s t u d i e d i n d i c a t e s t h e p h y s i o l o g i c a l relevance of t h i s f i n d i n g . Ca  2+ -uptake a c t i v i t y  is also  i s noted  The f a c t  at r e l a t i v e l y  that t h i s d i f f e r e n c e i n  low f r e e c a l c i u m  concentrations  significant.  Calmodulin  prepared  from r e d c e l l hemolysates o f normal v o l u n t e e r s was  2+ found  to s t i m u l a t e Ca  - t r a n s p o r t i n both  c o n t r o l and C F . p r e p a r a t i o n s ;  the degree of c a l m o d u l i n s t i m u l a t i o n appeared to be s i m i l a r i n both  groups.  -60The  low  degree of c a l m o d u l i n  s t i m u l a t i o n observed compared to the t h a t  found by o t h e r workers i n s i m i l a r ; p r e p a r a t i o n s c o u l d e i t h e r be due presence of i n d i g i n o u s i n h i b i t o r s i n the c a l m o d u l i n possibility  t h a t some c a l m o d u l i n was  not be removed d u r i n g  associated with  the p r e p a r a t i o n .  t h a t a d d i t i o n of c a l m o d u l i n  to  the  p r e p a r a t i o n or to  the  the v e s i c l e s and  I t i s important  (0.8 ug/ml f i n a l  could  to note, though,  i n c u b a t i o n volume) d i d not  2+ r e t u r n the Ca present  -uptake a c t i v i t y  i n the C F .  i n the c o n t r o l p r e p a r a t i o n s .  preparations  to those  levels  T h i s suggests t h a t r e g u l a t i o n of  the  2+  Ca  -transport a c t i v i t y by,calmodulin  disease s t a t e .  The  i n C F . red c e l l s i s unaffected by the  p o s s i b i l i t y e x i s t s though, t h a t e i t h e r C F .  i s a l t e r e d i n some way  compared to c a l m o d u l i n  t h a t r e d c e l l membranes from C F .  calmodulin  from c o n t r o l p r e p a r a t i o n s  p a t i e n t s respond d i f f e r e n t l y  to  or  CF.  calmodulin. S t u d i e s to date i n d i c a t e t h a t no plasma membrane s t r u c t u r e , w i t h and  lipid  content,  in CF.  ( F l e t c h e r and L i n 1973)  significant differences exist i n  r e s p e c t to t o t a l p r o t e i n ,  red c e l l s  (McEvoy et a l 1974),  carbohydrate fibroblasts  or lymphoblasts  (Maler and R i o r d a n 1978). A 2+ 2+ s i g n i f i c a n t decrease i n the V 2 + of the (Mg + Ca )-ATPase a c t i v i t y of 2+ f i b r o b l a s t membrane p r e p a r a t i o n s and Ca -uptake a c t i v i t y of 10 v e s i c l e C a  preparations K  diss  ^  o r  c  a  d e r i v e d from C F . l  c  of the enzyme.  i  u  m  m a  y  p a t i e n t s without  t h e r e f o r e be due  There may  an a l t e r a t i o n i n the  to an e f f e c t on the a c t i v e s i t e  be l e s s enzyme per mg  p r o t e i n , a d e f e c t i n the  mechanisms t h a t r e g u l a t e c a l c i u m t r a n s p o r t or the enzyme may a t a s i t e other than the c a t a l y t i c  site  be a l t e r e d  (an a l l o s t e r i c s i t e ) which u l t i m a t e l y  i n f l u e n c e s the c a t a l y t i c a c t i v i t y of the enzyme.  -61-  2+  2+  A d e c r e a s e d (Mg + Ca )-ATPase a c t i v i t y i n C F . c e l l s may r e p r e s e n t a b a s i c a l t e r a t i o n i n the a b i l i t y of these c e l l s t o t r a n s p o r t c a l c i u m . T h i s i s i n d i c a t e d by t h e experiments conducted u s i n g preparations, in  and may be a g e n e r a l i z e d  t h e 10 v e s i c l e  phenomenon i n C F .  No a l t e r a t i o n  2+ 2+ (Mg + Ca )-ATPase a c t i v i t y was observed, though, i n lymphoblast  membrane p r e p a r a t i o n s  d e r i v e d from C F . p a t i e n t s .  Lymphoblasts a r e t r a n s -  2+ formed c e l l s and t h i s l o s t during  c h a r a c t e r i s t i c o f low Ca  transformation.  -transport  a c t i v i t y may be  I t i s noted t h a t i n n o n - d i s e a s e d samples,  d i f f e r e n c e s e x i s t i n Na , K -ATPase a c t i v i t y between transformed and nont r a n s f o r m e d c u l t u r e d f i b r o b l a s t s (Kimelberg and Mayhew 1976). In these s t u d i e s no attempt was made to c o r r e l a t e t h e r e s u l t s w i t h t h e s e v e r i t y of t h e d i s e a s e s i z e o f each group  small  sample  studied.  Mangos (1978) using CF.  s t a t e due t o t h e r e l a t i v e l y  obtained  i s o l a t e d rat  p a r o t i d a c i n a r c e l l s observed t h a t  s a l i v a produced a s i g n i f i c a n t i n c r e a s e  comparison w i t h c e l l s i n c u b a t e d  i n saliva  p o s s i b l e t h a t the a l t e r e d c a l c i u m  i n i n t r a c e l l u l a r calcium i n  from c o n t r o l s u b j e c t s .  It is  pump a c t i v i t y noted i n C F . c e l l - t y p e s  may be a response to a c i r c u l a t i n g f a c t o r which a c t s as a modulator o f calcium  pump a c t i v i t y .  An a l t e r a t i o n i n c a l c i u m  pump a c t i v i t y  inCF.  may have a number o f p o s s i b l e i m p l i c a t i o n s t h a t may e x p l a i n some of the 2+ manifestations  of the disease;  Ca  i s i n v o l v e d i n many s e c r e t o r y  i n c l u d i n g macromolecular s e c r e t i o n (Douglas 1968).  processes  An a l t e r a t i o n i n c a l c i u m  pump a c t i v i t y would thus a f f e c t t h e s e c r e t i o n of g l y c o p r o t e i n s .  A number of  workers have a l s o shown t h a t a l t e r e d e x t r a c e l l u l a r c a l c i u m  levels w i l l affect  glycoprotein f l u i d i t y  ( B e t t e l h e i m 1971;  ( v i s c o s i t y ) and thus mucus c l e a r a n c e  Boat et a l 1974; F o r s t n e r and F o r s t n e r 1976), an important and problem i n C F .  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