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Studies on the mechanisms of branchial calcium transport in teleost fish, with special reference to a… Ma, Stephanie W. Y. 1976

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STUDIES ON THE MECHANISMS  OF BRANCHIAL CALCIUM TRANSPORT  I N TELEOST F I S H , WITH S P E C I A L REFERENCE TO A CALCIUM-STIMULATED A T P a s e I N THE G I L L PLASMA MEMBRANES by STEPHANIE W. Y. MA B.Sc.  ( G e n . ) , U n i v e r s i t y o f Hong K o n g ,  M . S c , U n i v e r s i t y o f Hong Kong,  1970  1973  A T H E S I S SUBMITTED I N P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY I N THE DEPARTMENT OF PHYSIOLOGY FACULTY OF MEDICINE  We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o the required  standard  THE U N I V E R S I T Y OF^BRITISH COLUMBIA APRIL,  0  Stephanie  1976  .Y.  1976  In p r e s e n t i n g t h i s  t h e s i s in p a r t i a l  an advanced degree at  the U n i v e r s i t y of  the L i b r a r y s h a l l make i t I  further  agree  fulfilment  freely  of  the  requirements  B r i t i s h Columbia, I agree  available  for  t h a t p e r m i s s i o n for e x t e n s i v e c o p y i n g o f  of  representatives.  this thesis for  It  financial  this  thesis  g a i n s h a l l not be allowed without my  of  The U n i v e r s i t y o f B r i t i s h Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1W5  or  i s understood that c o p y i n g o r p u b l i c a t i o n  written permission.  Department  that  r e f e r e n c e and study.  f o r s c h o l a r l y purposes may be granted by the Head o f my Department by h i s  for  i i ABSTRACT  A l t h o u g h mechanisms o f c a l c i u m r e g u l a t i o n i n t e l e o s t f i s h are s t i l l poorly understood, there  is i n -  2+  d i r e c t e v i d e n c e t h a t e n v i r o n m e n t a l Ca  may b e  involved.  The g i l l , "being t h e r e s p i r a t o r y o r g a n w h e r e b l o o d  comes  i n t o intimate contact with the external water, i s l i k e l y t o be a major s i t e f o r i o n t r a n s p o r t . t h i s p r o j e c t was t o s t u d y  The o b j e c t i v e o f  t h e mechanisms o f c a l c i u m  port i n teleost f i s h , with special reference 2+ calcium-uptake,  properties of a g i l l  Ca  trans-  to branchial  -ATPase and t h e  p o s s i b l e r o l e o f two c a l c i u m - a c t i v e hormones,  calcitonin  f r o m t h e u l t i m o b r a n c h i a l g l a n d s and t h e a c t i v e p r i n c i p l e ( s ) of the corpuscles across  of  Stannius.  2+ A m e t h o d was d e v e l o p e d t o m e a s u r e C a transport t h e i s o l a t e d and perfused g i l l o f t h e American e e l ,  Anguilla rostrata.  This  preparation  demonstrated a net  2+ Ca  -uptake a c r o s s  the g i l l  and  e l e c t r i c a l gradient.  epithelium against  a  chemical  T h i s u p t a k e was i n h i b i t e d  2,4 d i n i t r o p h e n o l , s u g g e s t i n g  active transport.  The  by bran-  2+ c h i a l Ca  t r a n s p o r t s y s t e m was s e n s i t i v e t o c h a n g e s i n 2+ e n v i r o n m e n t a l t e m p e r a t u r e , i n t e r n a l and e x t e r n a l Ca con2+ c e n t r a t i o n s a n d pH.  Ca  - u p t a k e was s t i m u l a t e d b y s a l m o n  c a l c i t o n i n , b u t i n h i b i t e d by e x t r a c t s o f t h e c o r p u s c l e s Stannius.  of  A Ca  2+  - s t i m u l a t e d ATPase,  i n d e p e n d e n t o f Na  +  and K  +  a n d i n s e n s i t i v e t o o u a b a i n , was i d e n t i f i e d i n t h e g i l l p l a s m a membranes o f t h e A m e r i c a n e e l . The enzyme was  also  2+  s t i m u l a t e d b y Mg were h i g h e r  , but the V  f o rCa  2 +  m  f o r t h e enzyme.  v a t i o n was 7 . 9 - 8 . 1 .  a n d a f f i n i t y o f t h e enzyme  v  than f o rM g  f o u n d t o a c t on t h e same s i t e . substrate  o  2 +  .  Ca  2 +  and M g  were  2 +  ATP was t h e p r e f e r e n t i a l  The pH optimum f o r enzyme  acti-  Salmon c a l c i t o n i n h a d no e f f e c t on  2+ the  Ca  -ATPase a c t i v i t y , however,  t h e enzyme a c t i v i t y  markedly i n h i b i t e d by e x t r a c t s of the Stannius 2+ The  existence  strated i n the g i l l  o f a s i m i l a r Ca  was  corpuscles.  - A T P a s e was  demon-  p l a s m a membranes o f a v a r i e t y o f  t e l e o s t f i s h from both freshwater  and marine h a b i t a t s .  2+ The g i l l  Ca  adaptation  -ATPase a c t i v i t y was n o t a l t e r e d b y s e a w a t e r o r b y s t a r v a t i o n , b u t was s i g n i f i c a n t l y  elevated  d u r i n g s e x u a l m a t u r a t i o n and spawning. This s e r i e s o f s t u d i e s has r e v e a l e d t h e e x i s t e n c e o f an a c t i v e b r a n c h i a l c a l c i u m uptake system i n t h e f r e s h 2+ w a t e r t e l e o s t , p o s s i b l y m e d i a t e d v i a a membrane-bound C a ATPase. C a l c i t o n i n and t h e c o r p u s c l e s o f Stannius " f a c t o r " 2+ were demonstrated t o p l a y a r e g u l a t o r y r o l e i n t h i s transport system, suggesting site  o f hormonal  the g i l l  Ca  as an i m p o r t a n t  action i n teleostean calcium homeostasis.  iv  ACKNOWLEDGMENTS I w o u l d l i k e t o t h a n k my r e s e a r c h Dr.  supervisor,  D. H. C o p p , f o r h i s c o n t i n u o u s e n c o u r a g e m e n t a n d  support throughout t h e course o f t h i s study, e s p e c i a l l y during  the preparation  l i k e t o thank Mrs.  Elspeth  technical assistance laboratory. and  of this thesis. Wilkinson  I would  also  for her excellent  and general c a r i n g o f t h e f i s h  I am m o s t g r a t e f u l t o D r . H. H. M e s s e r  D r . Y. S h a m i f o r t h e i n t e r e s t t h e y showed i n t h i s  subject  and t h e u s e f u l a d v i c e s they o f t e n gave.  Members  o f t h e r e s e a r c h t e a m who h a d s o w i l l i n g l y h e l p e d whenever necessary are greatly  appreciated.  I am p a r t i c u l a r l y i n d e b t e d t o D r . D. G. B u t l e r , Department o f Zoology, U n i v e r s i t y o f Toronto, f o r h i s kindness i narranging  f o r the supply of the chief  mental animal, A n g u i l l a r o s t r a t a ;  t o the Fisheries  S t a t i o n , West V a n c o u v e r , a n d t h e V a n c o u v e r P u b l i c for t h e i r generosity mental  i nproviding  experiResearch  Aquarium  some o f t h e e x p e r i -  fish. S p e c i a l t h a n k s a r e due t o D r . W.K. O v a l l e ,  Department o f Anatomy, U n i v e r s i t y o f B r i t i s h f o r theElectron Microscopic  work.  ations .  Columbia,  My g r a t i t u d e  e x t e n d e d t o M r . K. H e n z e f o r p r e p a r i n g  Jr.,  the thesis  i s also illustr-  V  TABLE  OF CONTENTS PAGE  GENERAL INTRODUCTION  1 DIVISION I  CHAPTER I - CALCIUM TRANSPORT ACROSS THE ISOLATED G I L L OF THE AMERICAN E E L , ANGUILLA ROSTRATA  11  INTRODUCTION  11  MATERIALS AND METHODS  15  RESULTS  31  DISCUSSION  44 DIVISION I I  CHAPTER I I - CHARACTERIZATION OF CALCIUM-STIMULATED A T P a s e I N THE G I L L PLASMA MEMBRANES OF THE AMERICAN E E L , ANGUILLA ROSTRATA ..  5^  INTRODUCTION  5^  MATERIALS AND METHODS  56  RESULTS  63  DISCUSSION  85  CHAPTER I I I - EFFECTS OF S A L I N I T Y ADAPTATION, SEXUAL MATURATION AND DIET ON G I L L AND  GUT  MUCOSA PLASMA MEMBRANE CALCIUM-STIMULATED A T P a s e OF TELEOSTS INTRODUCTION  93 93  vi PAGE MATERIALS AND METHODS  96  RESULTS  101  DISCUSSION  114  GENERAL DISCUSSION  123  LITERATURE CITED  12§  vii LIST OF TABLES TABLE 1  PAGE E l e c t r o l y t e composition, osmolarity and pH of e e l plasma and perfusion f l u i d used i n the i s o l a t e d g i l l study  2  .  S p e c i f i c a c t i v i t i e s of marker enzymes i n d i f f e r e n t fractions  3  5  66  S p e c i f i c e f f e c t of the corpuscles of Stannius on g i l l C a - ( M g ) ATPase 2+  4  20  2+  84  2+  Ca -ATPase i n g i l l plasma membrane of marine and freshwater teleosts  102  E l e c t r o l y t e composition of the dechlorinated tap water and a r t i f i c i a l sea water i n the S a l i n i t y Adaptation Experiment  105  2+  6  7  E f f e c t of seawater adaptation on Ca -ATPase i n g i l l plasma membranes of rainbow trout, S. gairdneri E f f e c t of sexual maturation and spawning on 2+  Ca -ATPase i n g i l l plasma membrane of r a i n bow trout and migrating coho salmon ........ 2+  8  106  107  E f f e c t of starvation on Ca -ATPase i n g i l l plasma membrane of rainbow trout, £>.. gairdneri I l l 2+  9  E f f e c t of starvation  on Ca  -ATPase i n gut  plasma membrane of rainbow trout, S. gairdneri  112  •  • •  Vill  LIST OF FIGURES FIGURE 1  PAGE Movement o f  -'Ca  a c r o s s the i s o l a t e d  and p e r f u s e d g i l l  o f the American e e l ,  A. r o s t r a t a 2  32  E f f e c t of metabolic  i n h i b i t o r on ^ C a  2 +  i n f l u x i n the i s o l a t e d and p e r f u s e d g i l l  3^  2+ 3,  E f f e c t o f e x t e r n a l Ca c o n c e n t r a t i o n on 2+ Ca -uptake i n t h e i s o l a t e d and p e r f u s e d gill  36  2+ k  E f f e c t o f i n t e r n a l Ca  2+  c o n c e n t r a t i o n on  Ca -uptake i n t h e i s o l a t e d and p e r f u s e d gill  37  2+ 5  E f f e c t o f p e r f u s a t e pH on Ca  -uptake i n  the i s o l a t e d and p e r f u s e d g i l l  39  2+ 6  E f f e c t o f i n c u b a t i o n temperature on Ca  -  uptake i n t h e i s o l a t e d and p e r f u s e d g i l l  40  2+ 7  E f f e c t o f c a l c i t o n i n - pn Ca  -uptake i n  the i s o l a t e d and p e r f u s e d g i l l 8  o f the  American e e l , A. r o s t r a t a E f f e c t o f c o r p u s c l e s o f Stannius e x t r a c t  41  2+ on Ca  -uptake i n the i s o l a t e d and p e r -  fused g i l l 9 10  o f t h e American e e l , A., r o s t -  rata I s o l a t i o n o f g i l l plasma membranes Activation  43 58  o f ATP h y d r o l y s i s by i n c r e a s i n g  concentrations of divalent cations  68  ix FIGURE 11  PAGE Lineweaver - Burk plots of ATPase a c t i v i t y 2+  at various concentrations of Ca and ATP 12  2+  , Mg  69  E f f e c t of protein concentration on P i 71  release 13  E f f e c t of Na  +  on enzyme a c t i v a t i o n by d i 72  valent cations 14  Enzyme a c t i v i t y with d i f f e r e n t nucleotides 73  as substrate 15  E f f e c t of pH on Ca -ATPase a c t i v i t y ....  75  16  E f f e c t of i n h i b i t o r s on Ca -ATPase a c t i vity  76  2+  2+  2+  17 18  E f f e c t of incubation temperature on Ca ATPase a c t i v i t y  -  E f f e c t of storage at +4° C on Ca -ATPase 2+  79  activity 19  E f f e c t of c a l c i t o n i n on g i l l plasma membrane C a - ( M g ) ATPase 2+  20  78  80  2+  E f f e c t of extract of the corpuscles of 2+  Stannius on g i l l plasma membrane Ca (Mg ) ATPase  -  2+  21  Inhibitory  e f f e c t of extract of the cor2+  puscles of Stannius on g i l l Ca activity  82  -ATPase 83  X  FIGURE  PAGE 2+  22 23  G i l l plasma membrane Ca -ATPase of marine and freshwater teleosts  103  E f f e c t of sexual maturation and spawning 2+  on Ca -ATPase i n g i l l plasma membranes of rainbow trout and migrating coho salmon  108  xi LIST OF PLATES PLATE 1  PAGE Bleaching and cannulation of the f i r s t g i l l arch of the American e e l , A. r p s t r a t a  2  General apparatus f o r i s o l a t e d g i l l  23  per-  fusion study i n the American e e l , A. rostrata 3  24  A schematic diagram i l l u s t r a t i n g the technique of g i l l t r a n s e p i t h e l i a l e l e c t r i c a l p o t e n t i a l measurement  4  27a  The two encapsulated corpuscles of Stannius on the v e n t r a l surface of the kidneys i n the American e e l , A. r o s t r a t a  29  5  The f i n a l membrane preparation (X 39»664)  64  6  The f i n a l membrane preparation (X 82,075)  65  1  GENERAL INTRODUCTION  Calcium ion plays a v i t a l role i n regulating c e l l u l a r functions such as excitation-secretion coupling at nerve endings (Eccles, 1964), excitation-contraction coupling i n muscle (Bianchi, 1969)» maintenance of membrane i n t e g r i t y (Poste and A l l i s o n , 1973)» regulation of enzyme a c t i v i t y (Bianchi, 1968) and control of hormone secretion (Copp, 1970; concentration  Care et a l , , 1975).  Calcium  i n the external environment can be very  f l u c t u a t i n g , but vertebrates  i n general are able to main-  t a i n a constant plasma calcium l e v e l .  Calcium regulation  within the animal i s an important p h y s i o l o g i c a l function. In mammals, regulation of calcium metabolism i s mediated p r i n c i p a l l y by the action of three hormonesi c a l c i t o n i n , parathormone and 1, 2 5 - dihydroxycholecalcif e r o l (see Copp, 1972| 1975).  Parathormone i s produced  by the parathyroid glands which a r i s e embryonically from the 3  and 4  branchial pouches.  I t i s a hypercalcemia  hormone, having an important r o l e i n combating hypocalcemia (Talmage ejfc a l . , 1953* 1955).  C a l c i t o n i n i s secreted  by the p a r a f o l l i c u l a r c e l l s or 'C* c e l l s (Foster ejt a l . . 1964*  B u s s o l a t i and Pearse, 1967*  Kalina et a l . , 1970)  which, i n most mammals, are s t r u c t u r a l l y associated with  2 the thyroid t i s s u e .  More recently, i t has been shown  that the c a l c i t o n i n - s e c r e t i n g c e l l s are of ultimobranc h i a l o r i g i n , derived from the neural crest (Pearse and Carvalheira, 19^7 * ^e; Dourain and Le L i e v r e , 1972). Functionally, c a l c i t o n i n i s a hypocalcemic hormone involved i n c o n t r o l l i n g hypercalcemia Copper ejt a l . , 1970*  Hirsch et a i .  f  (Copp, 1967* 1975).  The s t e r o i d  hormone, 1, 2 5 - dihydroxycholecalciferol i s the b i o l o g i c a l l y active metabolite of vitamin D, produced i n the kidney  (DeLuca, 1972).  I t plays a s i g n i f i c a n t r o l e i n  i n t e s t i n a l absorption of calcium.  I t i s also involved  i n renal-tubular reabsorption of calcium (Taylor and Wasserman, 1971) and causes mobilization of calcium from old bone (Omdahl and DeLuca, 1973)• In other tetrapods, parathyroid glands have long been recognised as an endocrine tissue important t o calcium homeostasis.  Parathyroidectomy  results i n hypo-  calcemia and treatment with exogenous parathormone induces hypercalcemia  i n amphibians (see Cortelyou and McWhinnie,  1967)* r e p t i l e s (see Clark, 1967) and b i r d s (see U r i s t , 1967). Since the recent f i n d i n g of the ultimobranchial gland as the source of c a l c i t o n i n i n a l l classes of jawed vertebrates (see Copp,  1972), there i s growing evidence  that t h i s endocrine tissue i s involved i n calcium metabolism i n non-mammalian species.  In amphibians the f  3 u l t i m o b r a n c h i a l glands  a p p e a r t o be i m p o r t a n t  i n pre-  v e n t i n g excessive m o b i l i z a t i o n of calcium from the parav e r t e b r a l l i m e s a c s and i n p r o t e c t i n g t h e a n i m a l hypercalcemia intake  against  during periods of high d i e t a r y calcium 1971).  (see Robertson,  I n b i r d s , hypertrophy  and  hyperplasia of the u l t i m o b r a n c h i a l bodies  occur  of c a l c i u m s t r e s s , such as i n l a y i n g hens  (Urist, 1967)  and  i n c h i c k s f e d on h i g h c a l c i u m d i e t  1968;  Cipera et a l . , 1970).  i n times  (Copp e t a l . ,  The a c t i o n o f t h e s t e r o i d  h o r m o n e , 1, 25 - d i h y d r o x y c h o l e c a l c i f e r o l i n c h i c k s h a s a l s o been demonstrated.  Administration of vitamin D  l e a d s t o t h e f o r m a t i o n o f t h e hormone  i n the kidney,  w h i c h a c t s on t h e c h i c k i n t e s t i n a l m u c o s a , s t i m u l a t i n g the s y n t h e s i s o f a c a l c i u m - b i n d i n g p r o t e i n and c a l c i u m a b s o r p t i o n i s a c t i v a t e d (Lawson and Emtage, Although  1975).  f i s h c o m p r i s e t h e l a r g e s t a n d most  diverse  g r o u p o f v e r t e b r a t e s , u n t i l r e c e n t l y v e r y l i t t l e was k n o w n of the endocrine  c o n t r o l of teleostean calcium metabolism.  Fish lack f u n c t i o n a l parathyroid glands.  Apart  from the  p a i r e d u l t i m o b r a n c h i a l b o d i e s w h i c h h a v e b e e n shown t o be r i c h i n c a l c i t o n i n 1971;  (Copp a n d P a r k e s ,  O r i m o e t a l . , 1972;  1968;  Copp e t a l . ,  ( t e l e o s t e a n s and Amia) p o s s e s s a n o t h e r system, the corpuscles of Stannius  Pang e t a l . ,  1972), b o n y  fish  unique hormonal  (see Bern,  1967)  w h i c h a r e shown t o b e i n t i m a t e l y i n v o l v e d i n c a l c i u m  4 h o m e o s t a s i s ( F o n t a i n e , 1964; C h a n , 1969; In  P a n g , 1973).  a d d i t i o n , t h e hypophysis a l s o appears t o a f f e c t  cium metabolism i n t e l e o s t s .  Pang a n d co-workers  cal-  (1971)  r e p o r t e d t h a t r e m o v a l o f t h e p i t u i t a r y i n d u c e d extreme hypocalcemia i n k i l l i f i s h to  artificial  ( F u n d u l u s heteroclitu3) a d a p t e d  sea water d e f i c i e n t i ncalcium.  Administr-  a t i o n o f p i t u i t a r y homogenate t o h y p o h y s e c t o m i z e d r e s t o r e d t h e plasma c a l c i u m t o normal l e v e l  ( P a n g , 1973).  B a s e d o n t h e s e o b s e r v a t i o n s , P a n g (1973) p r o p o s e d the  fish  that  p i t u i t a r y gland has a p h y s i o l o g i c a l l y hypercalcemia  function i n teleosts.  However, s i n c e p i t u i t a r y i s a master  e n d o c r i n e o r g a n i n v o l v e d i n t h e r e g u l a t i o n o f many o t h e r hormonal systems, i t s s p e c i f i c r o l e i n c a l c i u m homeostasis r e m a i n s t o be d e f i n e d . fish  Although vitamin D i s present i n  l i v e r i nlarge q u a n t i t i e s , i t s r o l e i n calcium  r e g u l a t i o n i n t e l e o s t has n o t been s t u d i e d . b i o l o g i c a l l y a c t i v e m e t a b o l i t e , 1, calciferol  e x i s t s i nf i s h  Whether t h e  25 - d i h y d r o x y c h o l e -  i s unknown.  Since t h e discovery o f u l t i m o b r a n c h i a l glands as a r i c h source o f c a l c i t o n i n purified fish  (Copp a n d P a r k e s , 1968),  c a l c i t o n i n has been prepared from  salmon  (0*Dor e t aJL., 1969a, b ) a n d e e l ( O t a n i e t a i . , 1975). U s i n g t h e r a t b i o a s s a y o f Kumar a n d c o - w o r k e r s (1965)» the  b i o l o g i c a l potency o f b o t h salmon  a n d e e l (4,000 M R C U / m g )  (5»000 M R C U / m g )  c a l c i t o n i n i s f o u n d t o b e much  5 higher than that o f porcine  (200 MRC U/mg) and human  (120 MRC U/mg). In t h e Japanese e e l ( A n g u i l l a .iauonica), removal o f the u l t i m o b r a n c h i a l glands d i d n o t a l t e r t h e plasma c a l c i u m l e v e l , but muscle c a l c i u m was markedly reduced (Ma, 1973).  I n "the e e l s A. a n g u i l l a and A. .iaponica.  h y p e r c a l c e m i a induced by t h e removal o f t h e c o r p u s c l e s o f S t a n n i u s was accompanied by u l t i m o b r a n c h i a l hypertrophy and an i n c r e a s e i n i t s c e l l u l a r s e c r e t o r y a c t i v i t y (Lopez et a l .  1968j  Chan, 1972).  I n t h e m i g r a t i n g salmon, the  h i g h e s t l e v e l o f plasma c a l c i t o n i n was found i a t h e female a t time o f spawning when plasma c a l c i u m was e l e v a t e d (Watts, 1973).  While i t appears t h a t the u l t i m o b r a n c h i a l  glands a r e i n t i m a t e l y i n v o l v e d i n f i s h c a l c i u m metabolism, response of t e l e o s t s p e c i e s t o c a l c i t o n i n has been i n c o n sistent.  C a l c i t o n i n a d m i n i s t r a t i o n had no e f f e c t on t h e  plasma c a l c i u m l e v e l o f Fundulus h e t e r o c l i t u s . k i s u t c h (Pang, 1971a) and Salmo g a i r d n e r i  Oncorhynchus  (Watts, 1973).  I n c o n t r a s t , a hypocalcemic response t o c a l c i t o n i n was observed i n t h r e e f r e s h w a t e r - a d a p t e d A n g u i l l a s p e c i e s (Chan e t a l . , 1972;  1968;  Pang, 1971a;  Lopez and D e v i l l e ,  Ma, 1973). The c o r p u s c l e s o f S t a n n i u s a r i s e i n the embryo from  the p r o - and meso-nephric d u c t s (Ford, 1959;  de Smet, 1962).  6  H i s t o l o g i c a l l y and c y t o l o g i c a l l y , the corpuscles answer to the description of an active endocrine tissue (Hanke et a l . , 1969J  Lopez, 1969;  Heyl, 1970;  Chan, 1972),  but the exact biochemical nature of the "hormone(s)" secreted i s s t i l l unknown.  Although considerable contro-  versy s t i l l exists as to the r o l e of the corpuscles i n the biosynthesis of adrenocortico-steroids i n the teleost f i s h , increasing evidence now  supports the view that t h i s  endocrine tissue i s non-steroidogenic et a l . . 1969;  (see Chester Jones  Colombo et a l . , 1971).  Electron micro-  scopic studies indicate that the secretion of the s c l e s i s proteinaceous  i n nature  (Ogawa, 1967;  corpu-  Tomasulo,  1968;, Cohen et a l . , 1975). The hypocalcemic role of the corpuscles of Stannius i n teleostean calcium homeostasis i s well-established. Removal of the corpuscles resulted i n a marked hypercalcemia i n a v a r i e t y of t e l e o s t s (see Pang, 1971b).  In  the Japanese e e l , a profound increase i n muscle t o t a l calcium was  also recorded  (Chan and Ma,  1972;  Ma,  1973).  Administration of the corpuscle homogenate to "Stanniectomized" eels restored the disturbed plasma calcium to normal l e v e l (Fontaine,1964;  Chan et a l . 1969;  1970).  reported to e l i c i t a hypo-  Corpuscle extract was  Lopez,  calcemic response i n intact seawater-adapted k i l l i f i s h , but was i n e f f e c t i v e i n k i l l i f i s h adapted to fresh water (Pang,  7 1971b).  B a s e d on t h e  observation  t h a t the  corpuscles  w e r e more a c t i v e i n c a l c i u m - r i c h e n v i r o n m e n t s , P a n g  (1973)  proposed t h a t the main p h y s i o l o g i c a l f u n c t i o n of the  cor-  puscles high  of Stannius  calcium  i s to maintain  environments.  serum c a l c i u m l e v e l  H o w e v e r , t h e s i t e ( s ) and  anism(s) of a c t i o n of t h i s hormonal system are not  in  mech-  well  understood.  W h i l e t h e mechanisms o f c a l c i u m r e g u l a t i o n i n t e l e o s t may  be  q u i t e d i f f e r e n t from those i n  v e r t e b r a t e s , t h e r e a r e two Firstly,  terrestrial  points of s p e c i a l i n t e r e s t .  i n t e t r a p o d s , bone e v i d e n t l y p l a y s an  part i n calcium homeostasis. p a r a t h o r m o n e , c a l c i t o n i n and ferol.  the  It i s a site  active  of a c t i o n  of  1, 25 - d i h y d r o x y c h o l e c a l c i -  I n t e l e o s t f i s h , l o n g bones a r e a b s e n t . The  osseous t i s s u e present u l a r or c e l l u l a r .  i n the a d u l t f i s h i s e i t h e r a c e l l -  C a l c i u m - 4 5 s t u d i e s show t h a t o n l y  the  o u t e r m o s t t h i n l a y e r o f t e l e o s t e a n bone i s e x c h a n g e a b l e ( s e e Simmons, 1971). of the  Secondly, the  f i s h to i t s aquatic  cium concentration presents  intimate relationship  environment w i t h v a r i a b l e a problem of calcium  d i f f e r e n t from that of the t e r r e s t r i a l Teleost city  1971).  The  regulation  vertebrates.  a r e known t o h a v e a w e l l - d e v e l o p e d  to withdraw calcium  from the  environment  f a c t that the hypercalcemia  cal-  capa-  (see  response  to  Simmons,  "Stanniectoray" challenge 1973;  i s dependent upon an e n v i r o n m e n t a l  ( C h a n e t a l . , 1967;  P a n g , 1973;  calcium  Pang e t a l . .  F e n w i c k , 197^) s u g g e s t s a p h y s i o l o g i c a l r o l e o f  environmental calcium  i n teleostean calcium  In t e l e o s t f i s h , three  homeostasis.  s i t e s are considered  capable  o f i o n exchange between t h e a n i m a l ' s i n t e r n a l and e x t e r n a l environments, namely t h e g i l l ,  t h e s k i n and t h e g u t .  The  s k i n has only a n e g l i g i b l e r o l e s i n c e i t i s g e n e r a l l y cons i d e r e d t o be c o n t i n u o u s l y ions.  The g u t p l a y s a m i n o r p a r t due t o t h e f a c t t h a t  freshwater water  impermeable t o b o t h w a t e r and  f i s h do n o t d r i n k a p p r e c i a b l e  ( M a e t z a n d S k a d h a u g e , 1968).  r e s p i r a t o r y organ where b l o o d  quantities of  The g i l l ,  being the  comes i n t o i n t i m a t e  contact  w i t h t h e e x t e r n a l e n v i r o n m e n t , i s l i k e l y t o be t h e m a j o r site f o r ion transport. The in Na  teleostean g i l l  osmoregulation. +  i s known t o h a v e a m a j o r r o l e  The m e c h a n i s m s u n d e r l y i n g  branchial  and C l ~ t r a n s p o r t have been e x t e n s i v e l y s t u d i e d .  ever, very l i t t l e  i s known c o n c e r n i n g  ficance of the teleostean g i l l  the functional s i g n i -  i n calcium  homeostasis.  Most o f t h e i n f o r m a t i o n a v a i l a b l e t o date has been i n d i r e c t l y f r o m s t u d i e s on e n d o c r i n e c o n t r o l Qf f l u x e s i n i n t a c t animals e t a l . , 1973;  (Fontaine  D a c k e , 1975)•  How-  e t a l . , 1972;  A d i r e c t study  derived  calcium Fleming  on b r a n c h i a l  9  involvement i n f i s h c a l c i u m r e g u l a t i o n has n o t been reported.  The a i m o f t h e p r e s e n t the  r o l e of the g i l l  t h e s i s was t o i n v e s t i g a t e  i n calcium homeostasis i n f r e s h -  water t e l e o s t s , with s p e c i a l reference  t o t h e mechanism  o f b r a n c h i a l c a l c i u m t r a n s p o r t and t h e a c t i o n o f two c a l c i u m - a c t i v e hormones, c a l c i t o n i n from t h e u l t i m o b r a n c h i a l g l a n d s and t h e e x t r a c t o f t h e c o r p u s c l e s  of  Stannius. In the f i r s t port across  s e r i e s of experiments, calcium  t h e i s o l a t e d and p e r f u s e d  was s t u d i e d .  gill  trans-  of a t e l e o s t  The s e c o n d s e r i e s o f e x p e r i m e n t s was  con-  c e r n e d w i t h t h e i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f a 2+ Ca  - s t i m u l a t e d A T P a s e s y s t e m in t h e t e l e o s t e a n  p l a s m a membranes.  Biochemical  adaptation  gill  o f t h e enzyme  t o e n v i r o n m e n t a l a n d p h y s i o l o g i c a l c h a n g e s was a l s o gated.  B a s e d on t h e r e s u l t s o f t h e s e e x p e r i m e n t s ,  mechanism o f c a l c i u m  i o n uptake across  the g i l l  investia  epithelium  was p o s t u l a t e d a n d t h e p o s s i b l e r o l e o f hormones i n b r a n c h i a l calcium  t r a n s p o r t was  discussed.  DIVISION I  CALCIUM TRANSPORT IN THE TELEOST GILL  CHAPTER  I  CALCIUM TRANSPORT ACROSS THE ISOLATED OF THE AMERICAN E E L , ANGUILLA  GILL  ROSTRATA  INTRODUCTION  Calcium requirements o f f i s h a r e s a t i s f i e d by a c q u i s i t i o n o f the ions from t h e i r aquatic  environments,  e i t h e r by d i r e c t a b s o r p t i o n from water o r v i a d i e t a r y intake. The a b i l i t y o f t e l e o s t f i s h t o a c c u m u l a t e C O n L.1:  siderable quantities of was f i r s t  ^Ca f r o m t h e s u r r o u n d i n g w a t e r  demonstrated b y Asano and co-workers  a n d R o s e n t h a l (1957; I 9 6 0 ) .  (1957)  In recent years, research  has been u n d e r t a k e n t o s t u d y t h e e f f e c t o f e n d o c r i n e systems  on e n v i r o n m e n t a l c a l c i u m exchange i n f r e s h w a t e r  teleosts.  F o n t a i n e and h i s co-workers  (1972) r e p o r t e d  t h a t removal of t h e corpuscles o f Stannius r e s u l t e d i n a significant increase i n net calcium influx i n the European e e l .  T h e y p r o p o s e d t h a t t h e g i l l was  likely  t o be t h e most i m p o r t a n t s i t e f o r c a l c i u m u p t a k e . F l e m i n g and co-workers  (1973) o b s e r v e d t h a t m a l e  f i s h exhibited seasonal variations i n the rate of  killi-  enviroraental calcium uptake.  C a l c i t o n i n i n j e c t i o n stimuli;  l a t e d calcium uptake and altered the  -'Ca d i s t r i b u t i o n  between c a l c i f i e d and soft tissue compartments.  In the  European e e l , c a l c i t o n i n administration caused a marked reduction i n t o t a l calcium e f f l u x (Dacke, 1975)•  In both  reports, the possible involvement of the branchial apparatus was  suggested.  In a review on. Na  +  and Cl"* transport mechanisms i n  the teleostean g i l l and amphibian skin, Motais and.GarciaRomeu (1972) commentedi "the. g i l l has proved to be a much more d i f f i c u l t organ to study i n v i t r o : research into the transport mechanisms i n f i s h has been conducted mainly i n vivo ". to  In t e l e o s t , the common experimental approach  calcium uptake study had been to expose the i n t a c t  f i s h to  -'Ca-labelled water i n an aquarium f o r various  time periods and then either follow the appearance grid d i s t r i b u t i o n of ^ C a £h the animal (Asano et a l . . 1956* Borough e£ a l . , et  al..  1956*  Rosenthal, 1957* I960*  1973) or measure the disappearance of ^ C a  the medium (Fontaine e£ a l . . of  Fleming  1972).  from  In the determination  calcium e f f l u x , the animal was f i r s t loaded with  -'Ca  by intaaperitoneal i n j e c t i o n and the appearance of the isotope in. an enclosed medium was then followed with time (Fontaine et a l . ,  1972*  Dacke, 1975).  While these  methods provide an understanding of the o v e r a l l uptake  and  e l i m i n a t i o n of ^ C a  the  separation  areas of  of g i l l  by  the  f i s h , t h e y do n o t  f u n c t i o n from other  permit  possible  exchange.  Various  o t h e r methods have been d e v e l o p e d i n  a t t e m p t t o d e f i n e more s p e c i f i c a l l y t h e a c t i o n o f gills  i n calcium regulation.  and  the  They i n c l u d e p e r f u s i o n  the a n t e r i o r region v i a the v e n t r a l a o r t a 1961)  p e r f u s i o n of s p e c i f i c regions  J o z u k a , 196I;  preparations  1964).  tend  o f an  intact  t o s u f f e r from h a n d l i n g Randall,  and  cannu-  1971). p o s s i b l e  v e n t i o n o f v a r i o u s feedback mechanisms, l a c k of  out  and  vations  i n the  (Mashiko  However, a l l t h e s e i n v i v o  l a t i o n e f f e c t s (Wood and  bility  of  (Schiffman,  f i s h i s o l a t e d by t i g h t l y f i t t i n g impermeable sacs and  an  inter-  flexi-  t y p e o f e x p e r i m e n t s w h i c h c a n be c a r r i e d  the u n c e r t a i n t y ; w i t h which experimental  obser-  can be  branchial  a s s o c i a t e d s p e c i f i c a l l y w i t h the  epithelium. Recently, + for  m e a s u r i n g Na  and  perfused  technique  +  , K  gill.  •  , and  d e s c r i b e d a method  —  Cl  fluxes across  Data obtained  an  isolated  from s t u d i e s u s i n g  are q u a l i t a t i v e l y i n c l o s e agreement w i t h  accepted r o l e of the g i l l in  (1972)  Shuttleworth  intact animals.  method f o r t h e  study  as d e d u c e d f r o m f l u x  H e n c e , t h i s a p p e a r s t o be  this the  studies a suitable  of b r a n c h i a l i o n i c r e g u l a t i o n .  14  Fenwick and So (1974) adopted t h i s method f o r measuring ^Ca  i n f l u x i n the "Stanniectomized"  reported that  ,,  Stanniectomy  g i l l calcium i n f l u x .  ,,  American e e l .  They  r e s u l t e d i n an i n c r e a s e i n  However, t h e r e i s no a v a i l a b l e  i n f o r m a t i o n on the nature of the b r a n c h i a l t r a n s p o r t system and the p o s s i b l e mechanisms i n v o l v e d i n i t s regulation. T h i s chapter  d e s c r i b e s a method f o r the study  o f c a l c i u m movements a c r o s s an i s o l a t e d and perfused gill.  Using t h i s technique,  p r o p e r t i e s of the b r a n -  c h i a l t r a n s p o r t system o f a freshwater investigated.  t e l e o s t were  The e f f e c t o f two c a l c i u m - a c t i v e hormones,  c a l c i t o n i n from the u l t i m o b r a n c h i a l glands and e x t r a c t o f the c o r p u s c l e s was a l s o s t u d i e d .  o f Stannius  on g i l l c a l c i u m uptake  15  MATERIALS AND METHODS  A.  Experimental  Animals  1. C h o i c e o f e x p e r i m e n t a l The  animal  experimental animal chosen f o r t h e present  study i s t h e s i l v e r American  e e l , A n g u i l l a r o s t r a t a L.  I t i s a m i g r a t i n g e u r y h a l i n e t e l e o s t found along t h e A t l a n t i c coast o f America, rence R i v e r t o B r a z i l The  e x t e n d i n g f r o m t h e S t . Law-  ( s e e R a y a n d C i a m p i , 1956).  spawning season  o f t h i s f i s h i s around  S e p t e m b e r t o e a r l y November e a c h y e a r . ground  larvae  The e x a c t b l e e d i n g  i s n o t c l e a r l y i d e n t i f i e d , b u t presumably  mid A t l a n t i c near t h e Sargasso  Sea.  late  i n the  When h a t c h e d , t h e  ( g l a s s e e l s ) a r e c a r r i e d by c u r r e n t s towards t h e  c o a s t o f A m e r i c a w h e r e t h e y become t r a n s f o r m e d i n t o c y l i n d r i c a l elvers within a year. remain  tiny  T h e y o u n g e e l s may  i n t h e shallow c o a s t a l b r a c k i s h waters  o r more  o f t e n , t h e y m i g r a t e u p r i v e r s where t h e y s t a y i n f r e s h water f o r s e v e r a l years.  T h i s i s t h e y e l l o w phase d u r i n g  w h i c h t h e e e l s f e e d v i g o r o u s l y a n d grow t o a c o n s i d e r a b l e size.  The f u l l y g r o w n e e l s u l t i m a t e l y e n t e r i n t o t h e  s i l v e r phase and cease f e e d i n g .  They b e g i n t o mature  s e x u a l l y a s t h e y m i g r a t e downstream towards t h e y t r a v e l t h e G u l f Stream t o t h e spawning  t h e s e a where grounds.  16  The perimental obtained  r e a s o n s f o r c h o o s i n g t h i s t e l e o s t as t h e  ex-  animal are  eels  are  twofold.  Firstly,  i n a f a i r l y u n i f o r m and  cium metabolism. s e x u a l l y and  The  cium intake  and  Secondly, the  s i v e l y used i n the  calcium  Hence,  one  extra calcium  cal-  released  d a i l y l o s s of calcium  genus A n g u i l l a has  studies  l i t e r a t u r e on  calcium  Ample i n f o r m a t i o n the  in  been e x t e n -  of hormonal c o n t r o l of  r e g u l a t i o n i n the A n g u i l l a  2. K e e p i n g o f  f u l l y mature  to variable dietary  The  the  metabolism i n t e l e o s t s i n v i v o . a v a i l a b l e i n the  cal-  d i g e s t i o n of p a r i e t a l muscle f o r  e n e r g y s u p p l y i s b a l a n c e d by urine.  due  sexudifference.  into c i r c u l a t i o n during  the  yet  i n a fasting condition.  would expect l e s s complication  silver  s t a b l e s t a t e of  animals are not  they are  the  is  p o s s i b l e mechanisms  of  species.  Animals  S i l v e r A m e r i c a n e e l s of body w e i g h t r a n g i n g from 800 - 1 , 2 0 0 g w e r e c a u g h t a l o n g t h e the  Provinces  of Ontario  a i r t o V a n c o u v e r and esthetized.  In the  To the  Quebec.  laboratory,  in  They were s h i p p e d  brought back t o the  cleaning, well-aerated orinated  and  S t . Lawrence R i v e r  laboratory  t h e y were k e p t i n  f i b r e g l a s s tanks of running  f r e s h water at a temperature of ensure complete r e c o v e r y of the  unanselfdechl-  12+2°C. animals  by  from  trauma of t r a n s p o r t a t i o n , p u r c h a s e d e e l s were always  17 a l l o w e d t o a c c l i m a t e d f o r a t l e a s t two weeks b e f o r e  use.  O n l y h e a l t h y a n i m a l s f r e e f r o m wounds a n d f u n g a l i n f e c t i o n were chosen f o r  B.  Plasma E l e c t r o l y t e In  to  experiments.  Determination  o r d e r t o choose a g i l l  p e r f u s i o n f l u i d as c l o s e  p h y s i o l o g i c a l c o n d i t i o n s as p o s s i b l e , the b l o o d  p l a s m a o s m o l a r i t y and A m e r i c a n e e l were 1.  e l e c t r o l y t e composition of the  determined.  Blood Sampling  Procedure  B l o o d samples were o b t a i n e d f r o m an  unanesthetized  f i s h u s i n g the caudal v e i n sampling technique. flat  pH,  The  was  laid  and  a h e p a r i n i z e d p l a s t i c 2 m l s y r i n g e w i t h a 21-G  eel  on i t s d o r s a l s i d e on t h e o p e r a t i o n t a b l e  i n c h n e e d l e was  i n s e r t e d through the s k i n of the  v e n t r a l aspect of the caudal peduncle.  The  1-1/2 mid-  needle  was  d i r e c t e d f o r w a r d between the haemal s p i n e s i n t o the haemal canal.  U s i n g t h i s t e c h n i q u e , b l o o d was  withdrawn  into  the syringe from the caudal c i r c u l a t i o n w i t h l i t t l e to  the  fish. F o r b l o o d pH m e a s u r e m e n t , b l o o d s a m p l e s w e r e  and  injury  c e n t r i f u g e d under o i l t o ensure  collected  anaerobic c o n d i t i o n s .  18 2. Chemical A n a l y s i s a. B l o o d pH was u s i n g a combination t i f i c Co., W.  measured under anaerobic c o n d i t i o n s ,  pH e l e c t r o d e (Sargent -Welch S c i e n -  Germany) w i t h a Research  Digital  pH/mV  Meter ( O r i o n , model 801). b . Plasma o s m o l a r i t y was  determined,  u s i n g an  Osmette P r e c i s i o n Osmometer (Model 2007» P r e c i s i o n Systems, Massachusetts,  U.S.A.).  c. Plasma c a l c i u m and magnesium c o n c e n t r a t i o n s were determined  by atomic a b s o r p t i o n spectrophotometry  Ash, Model 280 Atomsorb).  To minimize  inter-elementary  i n t e r f e r e n c e i n the c a t i o n d e t e r m i n a t i o n , 0.5 # c h l o r i d e was standards. recorder  (Jarrell-  lanthanum  used as the d i l u e n t f o r both samples and The r e a d i n g s were r e c o r d e d on a s t r i p c h a r t  (Sargent Recorderj Model SR).  d. Sodium and potassium  i n the plasma were a n a l y s e d  by flame photometry ( I n s t r u m e n t a t i o n L a b o r a t o r y Inc., Model 143). l i t r e ) was  A s t a n d a r d l i t h i u m s o l u t i o n (15 mEq. used as  diluent.  e. Plasma c h l o r i d e c o n c e n t r a t i o n was by automatic  electro - titration,  Ckloridometer Chicago Corp.)  L i per  determined  u s i n g a Buchler - Cotlove  (Buchler Instruments,  Model 4-2008, N u c l e a r  A s t a n d a r d s o l u t i o n o f sodium c h l o r i d e  was  used f o r c a l i b r a t i o n . f . Plasma i n o r g a n i c phosphorus was  measured u s i n g  a m o d i f i c a t i o n of the Technicon Auto-Analyzer Method (Alexander, 1968).  N-^c  T h i s method i s based on the  f o r m a t i o n of phosphomolybdic a c i d , which i s then  reduced  by stannous c h l o r i d e - h y d r a z i n e .  C. I s o l a t e d G i l l P r e p a r a t i o n U s i n g the b a s i c technique of S h u t t l e w o r t h f o r measuring N a method was  +  (1972)  f l u x e s , a constant-flow perfusion  developed t o study the t r a n s p o r t of c a l c i u m  i o n s a c r o s s the i s o l a t e d and b l e a c h e d t e l e o s t  gill.  1. M a t e r i a l s a. P e r f u s i o n f l u i d t  an E e l S a l i n e , m o d i f i e d from  t h e C o r t l a n d S a l i n e (Wolf, 1963)  t o approximate the  plasma e l e c t r o l y t e composition o f the American e e l , was chosen as the s t a n d a r d p e r f u s i o n f l u i d v i n y l p y r r o l i d o n e (1 $>)  (Table 1 ) .  (P.V.P. w i t h average m o l e c u l a r  weight o f 40,000; Sigma Chemical Co.) was c o l l o i d s u b s t i t u t e f o r plasma p r o t e i n s and (1 %) cells.  Poly-  added as a D-glucose  served as a s u b s t r a t e f o r energy s u p p l y t o the P r i o r t o each experiment,  fresh-saline  prepared and the pH a d j u s t e d t o 7.80. w i t h oxygen and f i l t e r e d w i t h a 0.22  I t was  was saturated  jum d i s p o s a b l e  Table 1. E l e c t r o l y t e composition, osmolarity and pH of e e l plasma and perfusion f l u i d used i n the i s o l a t e d g i l l study.  E e l Plasma  Perfusion F l u i d  140  133  2.70  2.68  •Cl"  108  132  Ca  2 +  1.50  1.50  Mg  2+  1.20  0.93  HCC~  8.00  8.00  Pi  0.69  1.50  Electrolyte Na K  (mM)  +  +  —  Osmolarity (m.Osm) 2H a  263  7.85+0.05  0.93 263  a  7.80 + 0.10  With 0.1% glucose and 1# P.V.P. 40.  21 M i l l i p o r e f i l t e r before use. b. Afferent and efferent cannulast a s p e c i a l type of leak-proof cannula was constructed f o r both afferent and efferent branchial a r t e r i e s . of a cut-off 23-G  B a s i c a l l y , i t consisted  6mm hypodermic needle connected to a  length of Clay-Adams PE 50 ( i . d . 0.584mm) polyethylene tubing at one end.  The other end of the needle was  attached to a small section (1mm)  of PE 50 tubing which  served as an e f f e c t i v e "stop" to avoid possible leakage during perfusion. 2. Cannulation Procedure P r i o r to the experiment, a s i l v e r e e l was removed from the water and transected behind the pectoral f i n s unanaesthetized.  The f i r s t g i l l arch was  carefully  removed and placed immediately i n i c e - c o l d heparinized s a l i n e j100.I.U. Heparin (sodium s a l t , Fisher S c i e n t i f i c Co.) per ml of saline \ bubbled with oxygen.  The afferent  cannula was introduced into the cut end of the afferent branchial a r t e r y to a depth of a few millimeters.  A  l i g a t u r e was t i e d around the whole g i l l arch including the artery, and the cannula was then withdrawn u n t i l the "stop" caught  on the l i g a t u r e .  The afferent cannula  was connected to a motor-driven syringe pump with a v a r i a b l e speed f a c i l i t y (model 941, Harvard Apparatus  Co.,  Maine, U.S.A.).  Heparinized  s a l i n e (200  a r i n p e r ml  of s a l i n e ) , bubbled with  through the  isolated g i l l  completely  The  e r e n t b r a n c h i a l a r t e r y was  nulated g i l l The  gill  The  r a t e o f 0.6  perfusion f l u i d  t h e n changed t o a n o n - h e p a r i n i z e d  saline.  r a t e of p e r f u s i o n through the  can-  p r e p a r a t i o n was  r e d u c e d t o 0.6  ml  then r i n s e d twice  per  in  concentration.  The  course of the experiment.  aerated  desired  i n c u b a t i o n medium  The  was  entire  w h o l e s y s t e m was  a c i r c u l a t i n g water bath at a constant  3«  hour.  f i n a l l y suspended i n a  c o n t i n u a l l y b u b b l e d w i t h oxygen d u r i n g the  (Plate  effsim-  b a t h of f i l t e r e d d e c h l o r i n a t e d water at the  in  be  in a  ( P l a t e 1) was  ml  was  The  then cannulated  d e c h l o r i n a t e d t a p w a t e r and  calcium  Hep-  pumped  f i l a m e n t s were s e e n t o  f r e e of b l o o d .  i l a r manner.  o x y g e n , was  at a constant  per minute u n t i l the g i l l  I.U.  placed  temperature  2). Sample C o l l e c t i o n and The  isolated g i l l  t o e q u i l i b r a t e a t a low p e r h o u r f o r 30 l e c t e d a t 15  minutes.  p r e p a r a t i o n was  allowed  p e r f u s i o n r a t e o f 0.6 The  e f f l u e n t was  minute i n t e r v a l s , u s i n g pieces  volume-standarized polyethylene  Analysis  C l a y - A d a m s PE  tubing.  200  (i.d.  ml  colof 1.397mm)  V a r i a t i o n i n the r a t e of e f f e r e n t  B l e a c h i n g and  cannulation  of the f i r s t  a r c h o f t h e A m e r i c a n e e l , A. 1.  rostrata.  Afferent branchial  cannula.  2. E f f e r e n t b r a n c h i a l  cannula.  3.  Bleached g i l l  filaments.  gill  General apparatus f o r i s o l a t e d g i l l  perfusi  s t u d y i n t h e A m e r i c a n e e l , A. r o s t r a t a . 1. I s o l a t e d g i l l medium.  suspended  i n incubation  2. A f f e r e n t b r a n c h i a l  cannula.  3. E f f e r e n t b r a n c h i a l  cannula.  4. S a m p l e c o l l e c t i n g  tube.  5. Constant-flow p e r f u s i o n  pump.  6. O x y g e n - b u b b l i n g d e v i c e . 7. H a a k e c o n s t a n t - t e m p e r a t u r e c i r c u l a t i n g pump.  immersion  25  flow within +. 3 % was  accepted.  of the known afferent perfusion rate  G i l l preparations with efferent flow rate  exceeding these l i m i t s were discarded. Calcium ion f l u x measurements began when (as ^ C a C l  2  i n aqueous s o l u t i o n , Amersham -Searle Corp.)  at a concentration of 7.4X10~^M per ml) was  -'Ca  (approximately  introduced i n t o the system.  studies, ^ C a  2 +  For e f f l u x  was added to the perfusion f l u i d and 2+  the rate of outward Ca appearance o f ^ C a  2 +  movement was measured by the  i n the incubation medium.  i n f l u x studies, ^ C a * was 2  s o l u t i o n and the rate of by the amount of ^ C a For ^ C a  2 +  2 +  For  added to the external ^Ca  -uptake was measured  appearing i n the e f f l u e n t .  analysis, 100 p i samples were added  to 10 ml s c i n t i l l a t i o n f l u i d (aquasol, New  England  Nuclear) and then counted i n a Beckman l i q u i d l l a t i o n counter  20 uCi  scinti-  (model LS-233* Beckman Instruments  Inc., C a l i f o r n i a ) . At the termination of each experiment, the was  gill  removed from the incubation bath, rinsed and blotted  lightly.  The perfused g i l l filaments between the  l i g a t u r e s were cut o f f and placed i n a preweighed aluminum dish.  This was  at 106° C f o r 24 hours.  then l e f t to dry i n an oven The dry weight of the filaments  26 was  determined. The rate of Ca*^ f l u x was expressed 2+ as umoles Ca per 100 mg dry wt. per min. D.  Studies of Calcium Transport Across the Isolated and Perfused  Gill  1. Properties of the Branchial Calcium  Transport  System The rate of * ^ C a  2+  f l u x at 12° C, with an external  2+ water Ca  concentration of 0 . 5 5 mM  and a standard E e l 2+  Saline perfusate (Table 1) containing. 1 . 5 0 mM pH 7.80 was  Ca  at  chosen as the control standard.  The i o n i c calcium concentrations of both the incubation medium and the standard perfusate were d i r e c t l y measured, using a Beckman Select-Ion Calcium Electrode (model no. 39608, Beckman Instruments Inc., C a l i f o r n i a ) i n conjunction with a standard reference electrode and a Research D i g i t a l pH/mV Meter (Orion, model 801). The t r a n s e p i t h e l i a l e l e c t r i c a l potential of the i s o l a t e d g i l l perfused under standard conditions was  also determined.  P o t e n t i a l difference between the  glass micro-electrode inserted i n the efferent end of the g i l l and the reference electrode situated i n the incubation medium was recorded on an oscilloscope  connected to an amplifier (Plate 3 ) . For each series of experiments, a control l e v e l 2+ of Ca  f l u x was  always measured p r i o r to changes i n  the experimental parameters.  At the end of the  ment, the rate of control f l u x was ensure that the preparation was value was  experi-  again determined to  s t i l l active.  Each  the mean of 2 - 5 samples and i n d i v i d u a l  experiments were repeated at l e a s t three times. 2. E f f e c t of Hormones on the Branchial Calcium Transport System The action of two  f i s h hormones which have "been  shown to "be a c t i v e i n calcium regulation i n the A n g u i l l a species was  investigated.  They were c a l c i t o n i n from  the ultimobranchial glands and extract of the  corpuscles  of Stannius. a. C a l c i t o n i n t  c r y s t a l l i n e natural salmon  c a l c i t o n i n (Armour Pharmaceutical Co., was  No. AL  1025)  dissolved i n acetate - glycine buffer (1 % sodium  acetate + 0.1#  glycine, pH 4.3) to a f i n a l concentr-  a t i o n of 10 U/ml.  Aliquots of 200 u l were l y o p h i l i z e d  and stored at - 2 0 ° C u n t i l use. of each batch was  The  calcitonin activity  confirmed by rat bioassay, using a  modification of the method developed by Kumar and his co-workers (1965)•  27a  Recording Device Amplifier nOscilloscoper~l  Afferent cannula  Efferent cannula  Reference electrode  Glass micro-electrode inserted into efferent branchial artery Isolated,perfused gill preparation  Plate  3.  Bathing solution  A schematic diagram of  gill  illustrating  transepithelial  measurement.  the technique  electrical  potential  For perfusion studies, the l y o p h i l i z e d c a l c i t o n i n was dissolved i n standard E e l Saline t o the required concentration.  Control perfusate contained 200 u l of  acetate-glycine buffer (pH 4.3) i n the same volume of Eel Saline.  The f i n a l pH of these perfusates was  measured to ensure that they were within the normal pH range of 7.80 + 0.10.  A l l perfusion f l u i d s were  f i l t e r e d with 0.22 jum disposable M i l l i p o r e f i l t e r before use. b. Extract of the corpuscles of Stanniust  i n the  American e e l , the corpuscles of Stannius are i n the form of a p a i r of d i s c r e t e , encapsulated  glands, r e s t i n g on  the v e n t r a l surface of the kidneys (Plate 4) or more often, hidden on the sides of the posterior c a r d i n a l v e i n which passes between the two kidneys. Paired corpuscles of Stannius were c o l l e c t e d from eels k i l l e d by decapitation.  A slightly lateral  i n c i s i o n was made i n the v e n t r a l body wall just to the cloaca>.  opposite  The kidneys were c a r e f u l l y exposed and  the discrete corpuscles could be easily, be removed by a p a i r of curved forceps.  They were either used immed-  i a t e l y or stored intact at -20° C u n t i l use. For perfusion studies, the corpuscles were homogenized i n i c e - c o l d standard E e l S a l i n e , using a Tri-R  Plate 4.  The  two  encapsulated  corpuscles of  Stannius  on t h e v e n t r a l s u r f a c e o f t h e k i d n e y s A m e r i c a n e e l , A.  rostrata.  1. P a i r e d c o r p u s c l e s o f 2.  Stannius  V e n t r a l surface of the  3. V e n t r a l b o d y w a l l .  kidneys.  i n the  30  tissue grinder with Teflon pestle. t h e n c e n t r i f u g e d a t 35tOOOXg  The  homogenate  was  f o r 30 m i n u t e s a t k°  C,  u s i n g a S o r y a l l superspeed c e n t r i f u g e The  c l e a r s u p e r n a t a n t was  w i t h 0.22  um  disposable  ( m o d e l RC  c a r e f u l l y removed and  Millipore filter.  d i l u t i o n t o the r e q u i r e d concentrations f i l t e r e d E e l S a l i n e w h i c h was fusate.  co-workers The  d e t e r m i n e d by  Stannius fluid.  was  of the  Subsequent  was  made w i t h  corpuscular  t h e method o f Lowry  (1951), u s i n g b o v i n e a l b u m i n  concentration  and  standards.  e x t r a c t of corpuscles  e x p r e s s e d a s mg  filtered  used as the c o n t r o l p e r -  T o t a l p r o t e i n of the f i l t e r e d  e x t r a c t was  2-B).  p r o t e i n per ml  of  of perfusion  31 RESULTS  A.  P r o p e r t i e s of the B r a n c h i a l Calcium Transport 1. C a  Gradient Across  the Epithelium of the  I s o l a t e d and Perfused The i o i c  System  Gill  c a l c i u m c o n c e n t r a t i o n s w e r e 0 . 5 5 + 0.OlmM  ( a v e r a g e o f t h r e e m e a s u r e m e n t s ) f o r t h e i n c u b a t i o n medium and  1.39+0.02 mM  (average o f t h r e e measurements) f o r t h e 2+  standard E e l Saline perfusate.  T h i s g i v e s a Ca  of 3 across t h e epithelium o f the i s o l a t e d g i l l standard  gradient under  perfusion.  2. E l e c t r i c a l G r a d i e n t A c r o s s I s o l a t e d and Perfused  theEpithelium of the  Gill  The e l e c t r i c a l p o t e n t i a l i n s i d e t h e g i l l was a p p roximately  + 30 t o + 4 0 mV (30 m e a s u r e m e n t s i n t w o g i l l  preparations) with respect t o the external incubation medium. 2+ 3. C a  Fluxes Across  the Isolated G i l l  Perfused  w i t h Normal E e l S a l i n e 2+ Figure 1 i s a t y p i c a l time-course  profile  movements a c r o s s a n i s o l a t e d a n d p e r f u s e d g i l l . i n f l u x measurements, ^ C a  o f Ca During  ( 7 . 4 X 1 0 " ^ M ) was i n t r o d u c e d 2+ i n t o t h e i n c u b a t i o n medium. C a i n f l u x , as i n d i c a t e d ILK  by  t h e appearance o f  2  +  p+  -'Ca  i nthe effluent,  increased  32  Figure 1.  Movement of ^ C a  6 T  across ah  i s o l a t e d and  perfused g i l l of the American e e l , A. r o s t r a t a . Incubation mixture was dechlorinated tap water with 0.55 mM  Ca  .  Perfusion f l u i d was normal  E e l Saline with 1.5 mM  C a , at pH 7.80. 2 +  33  progressively and reached a steady l e v e l 120 minutes a f t e r the addition of rate of ^ C a  2 +  -'Ca  i n f l u x was  .  At t h i s time, the average  (3.3^ + 0.32)  dry wt./min. (mean+S.E., n « 1 3 )  X 10  3  CPM/100 mg  which i s equivalent  an inward movement of (3.34+ 0.32) XIO" * ;umoles C a 2  2 +  to /  100 mg dry wt./min. During e f f l u x measurements, ^ C a of 7.4X10"^M was  2 +  at a  concentration  added to the standard E e l Saline per-  2+ fusate. ^Ca  Ca  e f f l u x , as indicated by the appearance of  i n the incubation medium, was  small and i t reached a  maximum l e v e l 75 minutes a f t e r the introduction of the 4*5 2+ isotope. At t h i s time, the measured rate of ^Ca efflux was 84.8+13.5 CPM/lOO mg dry wt./min. (mean+S.D., n«=2) 2+ which i s equivalent to an outward movement of Ca at a 6  rate of (8.48 +1.35) X10~ Net Ca  i n f l u x was  10"^ jumoles /100  2+  umoles Ca  /100  mg dry wt./min.  calculated to be approximately 3.26 X  mg dry wt./min.  4. The E f f e c t of Metabolic  I n h i b i t o r on Branchial Ca *2  Uptake Figure 2 i s a t y p i c a l time-course p r o f i l e of the 2+ e f f e c t of metabolic i n h i b i t o r on branchial Ca Administering (ImM)  the r e s p i r a t o r y i n h i b i t o r , 2,4  -uptake. dinitrophenol  to the perfused g i l l caused a rapid drop i n the rate  of net C a  2 +  i n f l u x to only 45 + 2.0 % (mean + S .E., n » 3 )  of the o r i g i n a l control within 90 minutes.  Addition of  that  /  (I) 2,4 DNP  F i g u r e 2.  E f f e c t of metabolic i n f l u x i n an © — ®  Normal g i l l  2,4  2,4  D N P J  preparation. 2,4  r  ( I )  fc  gill*  2,4  D N P »  dinitrophenol•  i n c u b a t i o n medium w i t h 1 mM  d i n i t r o p h e n o l . 0—0  from a dying  o n • * -'Ca "  i s o l a t e d and p e r f u s e d  p e r f u s a t e w i t h 1 mM ( I I )  inhibitor  animal.  Gill  preparation  2,4 d i n i t r o p h e n o l  (1 mM)  i n a further reduction  t o t h e i n c u b a t i o n medium r e s u l t e d  i n the rate o f net Ca  influx.  A  76 + 2 . 5 % (mean + S.D., n » 2 ) i n h i b i t i o n w a s o b s e r v e d a t 165 m i n u t e s a f t e r t h e i n h i b i t o r was i n t r o d u c e d  into the  2+ system.  Net Ca -uptake i n t h e energy-depleted g i l l  l a t e d from a dying  a n i m a l was f o u n d t o b e o n l y  iso-  3«9+1.4#  (mean + S.E., n = 3) t h a t o f t h e a c t i v e c o n t r o l . 2+ 5. T h e E f f e c t o f E x t e r n a l C a C o n c e n t r a t i o n o n 2+ B r a n c h i a l C a -Uptake 2+ -3 A t e x t e r n a l C a c o n c e n t r a t i o n s b e l o w 10 ^ M ,  net  i n f l u x was v e r y l o w ( l e s s t h a n 6.67X10" u m o l e s / 1 0 0 mg 2+ d r y w t . / m i n . ) ( F i g u r e 3 ) . C a - u p t a k e showed a r a p i d 2+ -3 increase a t e x t e r n a l Ca c o n c e n t r a t i o n s b e t w e e n 10 M —2 —2 2+ a n d 10 M. A t 10 M, t h e r a t e o f n e t C a - u p t a k e was  Ca  J  a p p r o x i m a t e l y 4.90X10""^ u m o l e s / 1 0 0 mg d r y w t . / m i n .  2+  6. T h e E f f e c t o f I n t e r n a l C a Branchial Figure  Concentration on  Ca ''"-Uptake 2  .2+ 4 i l l u s t r a t e s t h e e f f e c t o f i n t e r n a l Ca*  2+ c o n c e n t r a t i o n on b r a n c h i a l Ca t r a n s p o r t . Increasing 2+ the i n t e r n a l Ca f r o m 0.75 mM t o 1.50mM ( n o r m a l p l a s m a 2+ l e v e l ) r e s u l t e d i n an elevated r a t e o fn e t Ca influx. 2+ I t was s t a b l e a t C a 1.75 mM, Ca  2 +  concentrations  b e t w e e n 1.50mM a n d  b u t was f u r t h e r e n h a n c e d a t a h i g h e r  l e v e l o f 2.25 mM.  internal  J  36  pCa in External Medium  Figure  3.  Effect  o f e x t e r n a l C a ^ c o n c e n t r a t i o n on T  2+ Ca  uptake i n t h e i s o l a t e d and p e r f u s e d  gill.  I n c u b a t i o n medium w a s d e c h l o r i n a t e d 2+  tap water w i t h d i f f e r e n t Ca  concentrations.  P e r f u s i o n f l u i d was n o r m a l E e l S a l i n e w i t h 1.50 mM S.E.  Ca  2 +  , a t pH 7 . 8 0 . V a l u e s a r e means +  (n»9).  37  !5r X3  o> E O O \ OJ  o E * O x x  2  +  o o  Perfusate [ C a ] mM 2+  2+ Figure 4.  Effect  o f i n t e r n a l Ca  concentration  on  2+ Ca - u p t a k e i n t h e i s o l a t e d a n d p e r f u s e d gill. I n c u b a t i o n medium was d e c h l o r i n a t e d 2+  t a p w a t e r w i t h 0.55 mM  Ca  .  Perfusion  f l u i d was E e l S a l i n e w i t h d i f f e r e n t  Ca  2 +  c o n c e n t r a t i o n s , a t pH 7»80. V a l u e s a r e means + S.E. ( n . 9 ) .  38  7. The E f f e c t o f P e r f u s a t e  pH on B r a n c h i a l  Ca *2  Uptake + The e f f e c t o f H  2+ on g i l l Ca  t r a n s p o r t was  mined i n the pH range o f 5 » 9 0 - 8 . 4 0 . F i g u r e 5» changes i n p e r f u s a t e  deter-  As i n d i c a t e d i n  pH from pH 7»80 (normal  plasma pH) i n e i t h e r d i r e c t i o n r e s u l t e d i n s t i m u l a t e d n e t Ca  influx.  The r a t e of i n c r e a s e was more r a p i d  "between pH 7.80-8.40 than between pH 7.80 - 5 . 9 0 . 8. The E f f e c t of I n c u b a t i o n Temperature  on B r a n c h i a l  Ca *-Uptake 2  2+ The e f f e c t o f i n c u b a t i o n temperature on g i l l Ca uptake was  s t u d i e d and the r e s u l t s are p r e s e n t e d i n  F i g u r e 6.  Lowering the i n c u b a t i o n temperature from the  -  normal e n v i r o m e n t a l l e v e l of 12 °C t o as low as 2 . 5 ° C d i d 2+ n o t induce any change i n the r a t e o f n e t Ca influx. However, i f the i n c u b a t i o n temperature was r a i s e d from 12° G  to 17°G,  a decrease of 35.%  i n the r a t e of n e t  2+ Ca  2+ -uptake was  observed.  The r a t e o f b r a n c h i a l Ca  t r a n s p o r t remained low a t 20° C, B.  The E f f e c t o f Hormones on B r a n c h i a l Ca " "-Uptake 2  >  I..The E f f e c t of C a l c i t o n i n The e f f e c t o f salmon c a l c i t o n i n on g i l l t r a n s p o r t i s shown i n F i g u r e 7.  2+ Ca  U s i n g the v e h i c l e  39  Figure  $.  Effect the  of perfusate  pH o n Ca*^- u p t a k e i n  i s o l a t e d and perfused  gill.  Incubation  medium was d e c h l o r i n a t e d t a p w a t e r  with  2+ 0.55 mM Eel  Ca  .  P e r f u s i o n f l u i d was n o r m a l  S a l i n e w i t h 1.50 mM  Ca  2 + t  at different  V a l u e s a r e means o f 2 t o 5 s a m p l e s i n 7 preparations.  pH.  gill  40  100-i  ^  9 0H  CM  o O  £ o  80  70  60 o JZ  H •  O  T" 5  —I  I  10  20  15  Temperature ( C )  Figure  6.  Effect  2+ o f i n c u b a t i o n temperature on Ca -  uptake i n t h e i s o l a t e d and perfused Incubation  gill.  medium was d e c h l o r i n a t e d t a p  w a t e r w i t h 0.55 mM  Ca  2 +  .  Perfusion  fluid 2+  was n o r m a l E e l S a l i n e w i t h 1.50 mM a t pH 7 . 8 0 .  Ca  ,  V a l u e s a r e means + S.E. ( n « 3 )  CD  c o  T  O  1  1  100  1  1  200  1  300  Salmon Calcitonin (mil/ml)  2+ Figure  7.  Effect  o f c a l c i t o n i n on Ca  i s o l a t e d and p e r f u s e d e e l , A. r o s t r a t a *  gill  -uptake i n t h e of the American  I n c u b a t i o n medium w a s  d e c h l o r i n a t e d t a p w a t e r w i t h 0.55 mM  2+  Ca  .  P e r f u s i o n f l u i d was n o r m a l E e l S a l i n e w i t h 2+ 1.50 mM  Ca  and d i f f e r e n t  o f c a l c i t o n i n , a t pH 7.80. means + S.E. ( n = 3 ) .  concentrations Values are  perfusate as the c o n t r o l , increasing doses of salmon c a l c i t o n i n i n the perfusion f l u i d (50 mU/ml and 120 mU/ml) resulted i n a progressive stimulation of the rate of net C a  2 +  influx.  A higher dose of 240 mU/ml did not  cause further a c t i v a t i o n .  During the course of t h i s  study, i t was observed that a high dose of 300 mU/ml salmon c a l c i t o n i n caused vasoconstriction, r e s u l t i n g i n a marked reduction i n the perfusion flow. 2. The E f f e c t of Extract of the Corpuscles of Stannius Figure 8 i l l u s t r a t e s the e f f e c t of corpuscles of 2+  Stannius on g i l l Ca  transport.  Standard s a l i n e per-  fusion was chosen as the control reference.  Administr-  a t i o n o f corpuscular extract at a low concentration of 0.1 mg protein /ml perfusate to the perfused  gill  2+  caused a 42.5 # i n h i b i t i o n i n branchial Ca -uptake. The response was dose - dependent. Higher concentrations 2+  of extract resulted i n further i n h i b i t i o n .  Net Ca  i n f l u x at extract concentration of 0.33 mg p r o t e i n / m l perfusate was 44# that of the c o n t r o l .  100  0.0 Extract of Corpuscles of Stannius (mg Protein/ml)  F i g u r e 8.  Effect  o f corpuscles o f Stannius  on C a  -uptake i n the i s o l a t e d and perfused  gill  extract  o f t h e A m e r i c a n e e l , A. r o s t r a t a .  I n c u b a t i o n medium was d e c h l o r i n a t e d t a p w a t e r w i t h 0 . 5 5 mM  Ca  .  Perfusion  fluid 2+  w a s n o r m a l E e l S a l i n e w i t h 1.50 mM and  different  extract, (n-3). '  Ca  concentrations of corpuscular  a t pH 7«80.  V a l u e s a r e means + S.E  44 DISCUSSION  A.  P e r f u s i o n Technique The  gill  p e r f u s i o n technique developed  study has e f f e c t i v e l y prevented commonly e n c o u n t e r e d  tip  of leakage  i n perfused g i l l preparations.  P o s s i b l e leakage and blockage is  t h e problem  f o r this  of the cannulated v e s s e l  eliminated by the introduction of a "stop" a t the o f each c a n n u l a .  L e a k s a s s m a l l a s 1.50  jul/min.  c a n be r e a d i l y d e t e c t e d b y p r e c i s e l y m o n i t o r i n g the a f f e r e n t and e f f e r e n t p e r f u s i o n r a t e s the e n t i r e  B.  both  throughout  experiment.  Properties of the B r a n c h i a l Calcium Transport  System  C e r t a i n b i o l o g i c a l membranes s u c h a s t h e g i l l a r e o n l y p a r t i a l l y permeable t o water.  Transepithelial  e n t i a l s a c r o s s t h e membranes r e s u l t f r o m f r e e of  pot-  diffusion  p e n e t r a t i n g i o n s a l o n g t h e i r c h e m i c a l g r a d i e n t s and  t h e a c t i v i t y o f t h e i o n pumps l o c a t e d i n t h e membranes. The  d i f f u s i o n p o t e n t i a l d e p e n d s o n t h e s i z e o f t h e chem-  i c a l g r a d i e n t s a n d t h e r e l a t i v e membrane p e r m e a b i l i t y t o c a t i o n s and a n i o n s . In  seawater  teleosts, electrical potential  mea-  surements a c r o s s t h e animal i n d i c a t e t h a t t h e b l o o d i s  positive with respect t o the external C a m p a n i n i , 1966$  E v a n s , 1969?  P i c a n d M a e t z , 1975). can  be a t t r i b u t e d  (Kirschner  K i r s c h n e r g t al.,1974}  This positive blood  t o the fact that  more p e r m e a b l e t o c a t i o n s (Cl~)  w a t e r (Maetz and  the g i l l  potential membrane i s '  ( N a and K ) than t o anions +  e t a l . , 1974}  +  P i c a n d M a e t z , 1975).  I n f r e s h w a t e r t e l e o s t s , r e p o r t s on b l o o d  electrical  p o t e n t i a l measurements a r e few and i n c o n s i s t e n t . the  In  European e e l kept i n f r e s h water, a negative  potential 1966).  (-21 mV) was o b s e r v e d  I n contrast,  blood  (Maetz and Campanini,  a positive blood potential  (+8.5  mV) was m e a s u r e d i n t h e r a i n b o w t r o u t k e p t i n a s o l u t i o n +  containing  1 mM  In the goldfish varied  Na  2+  and Ca  (Kerstett.er  et, a l . , 1970).  (Carassius auratus). the blood  potential  w i t h t h e i o n i c c o n t e n t o f t h e w a t e r ( M a e t z , 1974).  I t was p o s i t i v e (-44 mV)  (+4.0 mV)  i n fresh water, negative  i n d e i o n i z e d w a t e r and p o s i t i v e  (+7.4 mV) i n  2+  d e i o n i z e d w a t e r w i t h 1.5 mM  Ca  o f i o n i c membrane p e r m e a b i l i t i e s water, P  N a  added.  Measurements  revealed that  was a l m o s t t h e same a s P  C 1  i n fresh  } i n deionized 2+  water, P was 8 X P was o n l y 0.5X N a  N a  P P  C 1 C 1  and i n d e i o n i z e d w a t e r w i t h Ca , . I t was c o n c l u d e d t h a t a p o s i t i v e  ?+  b l o o d p o t e n t i a l r e c o r d e d i n t e l e o s t k e p t i n Ca - r i c h f r e s h w a t e r was due t o o u t w a r d d i f f u s i o n o f C l ~ a t a + + r a t e f a s t e r t h a n t h a t o f Na . A c t i v e b r a n c h i a l Na -  46 uptake i n fresh water  also contributed to the f i n a l  potential difference. In the present study, the p o s i t i v e t r a n s e p i t h e l i a l p o t e n t i a l (+30 to +40 mV)  recorded i n the i s o l a t e d per-  fused g i l l preparation agrees q u a l i t a t i v e l y with the observations of K e r s t e t t e r and co-workers (1970) and Maetz (1974-).  Quantitatively, the larger p o t e n t i a l  difference observed i n the American e e l may be for  accounted  by species v a r i a t i o n s or difference i n the experi-  mental technique. on i n t a c t animals.  Previous studies were a l l performed The e l e c t r i c a l p o t e n t i a l i n a caudal  v e i n cannula or an i n t r a p e r i t o n e a l catheter was measured and assumed to be equivalent to that i n the branchial circulation.  In contrast, the present data were obtained  by a d i r e c t recording of p o t e n t i a l difference across the i s o l a t e d g i l l perfused with normal E e l Saline. 2+  Present r e s u l t s on Ca  e f f l u x revealed that the 2+  g i l l epithelium was f a i r l y impermeable to calcium. Ca i n f l u x against a concentration gradient was i n the order of  10  umoles/100 mg dry wt./min., while Ca  along a d i f f u s i o n gradient was  efflux  i n the order of 10""^ u n i t s .  2+  The rates of Ca  i n f l u x and e f f l u x d i f f e r e d by a f a c t o r 2+  of  40, suggesting an e f f i c i e n t Ca  -uptake system.  G i l l Ca^-uptake  was i n h i b i t e d b y 2,4  phenol which i s an u n c o u p l e r o f o x i d a t i v e  dinitro-  phosphory-  2+ l a t i o n , i n d i c a t i n g t h a t b r a n c h i a l Ca  t r a n s p o r t depended  on t h e a v a i l a b i l i t y o f m e t a b o l i c e n e r g y . Two c r i t e r i a a r e o f t e n u s e d t o d e t e r m i n e d w h e t h e r a s u b s t a n c e moves a c r o s s a b i o l o g i c a l membrane b y a c t i v e t r a n s p o r t , namely moving and  against a concentration gradient  ( o r ) an e l e c t r i c a l g r a d i e n t ;  metabolic energy been s a t i s f i e d  and a requirement f o r  ( s e e G i e s e , 1968).  Both c r i t e r i a  have  i n the present study, indicating that the  2+ b r a n c h i a l Ca  -uptake i s an a c t i v e p r o c e s s . 2+  The b r a n c h i a l C a  -uptake p r o f i l e as a f u n c t i o n  2+ o f e x t e r n a l Ca  c o n c e n t r a t i o n c l o s e l y resembles  that  r e p o r t e d f o r t h e g u i n e a p i g p l a c e n t a l p l a s m a membrane 2+ v e s i c l e s ( S h a m i , 1974). I n b o t h c a s e s , C a - u p t a k e was 2+ -3 i n s i g n i f i c a n t a t e x t e r n a l Ca c o n c e n t r a t i o n s b e l o w 10 ^M. S h a m i (1974) d e m o n s t r a t e d t h a t a c t i v e a c c u m u l a t i o n o f 2+ Ca b y p l a c e n t a l p l a s m a membrane v e s i c l e s was d e p e n d e n t 2+ o n ATP h y d r o l y s i s b y t h e membrane-bound C a -ATPase. 2+ The s u b s t r a t e o f C a - A T P a s e was a Ca-ATP c o m p l e x n o t 2+ t a k e n up by t h e v e s i c l e s .  Only f r e e Ca  was t r a n s p o r t e d .  B a s e d o n t h e s e f i n d i n g , i t was p r o p o s e d t h a t i n t h e p r e s e n c e o f ATP a n d a t C a little  2 +  c o n c e n t r a t i o n s b e l o w 10"-* M,  2+ f r e e Ca was a v a i l a b l e f o r membrane  transport.  A s i m i l a r C a ^ - A T P a s e s y s t e m may b r a n c h i a l Ca in (see  the g i l l  2+  -uptake.  be r e s p o n s i b l e f o r a c t i v e  I d e n t i f i c a t i o n o f a Ca  2+  -ATPase  p l a s m a membranes s u p p o r t s t h i s h y p o t h e s i s  Chapter I I ) . 2+ G i l l Ca  t r a n s p o r t was p r o g r e s s i v e l y s t i m u l a t e d 2+ b y a n i n c r e a s e i n i n t e r n a l Ca c o n c e n t r a t i o n up t o t w i c e 2+ the physiological level. I f b r a n c h i a l Ca transport p l a y s a r o l e i n c a l c i u m homeostasis, h y p e r c a l c e m i a would 2+ be e x p e c t e d t o c a u s e a n i n h i b i t i o n i n g i l l  Ca  -uptake.  While the present r e s u l t appears t o c o n t r a d i c t  the  p h y s i o l o g i c a l r e q u i r e m e n t i n v i v o , one s h o u l d b e a r i n mind t h a t i n an i s o l a t e d and s a l i n e p e r f u s e d g i l l  prepar-  a t i o n , f u n c t i o n a l r e g u l a t o r s s u c h as hormones a r e  comple-  t e l y absent.  The  observed response o n l y represents a 2+ 2+ d i r e c t a c t i o n o f i n t e r n a l Ca on t h e g i l l Ca transport system which i s not under hormonal c o n t r o l i n v i t r o . A l t e r a t i o n i n p e r f u s a t e pH f r o m t h e n o r m a l e e l b l o o d pH  o f 7.80  i n e i t h e r d i r e c t i o n always r e s u l t e d i n 2+  a n e l e v a t e d b r a n c h i a l Ca the  gill  complex.  transport.  The  changes i n  e p i t h e l i u m l e a d i n g t o t h i s phenomenon c a n  be  In the course of i n t e r p r e t i n g the data,  two  p o s s i b l e f a c t o r s s h o u l d be t a k e n i n t o a c c o u n t . prolonged exposure  of c e l l s t o abnormal  Firstly,  pH r e s u l t s i n a n  i n c r e a s e d p e r m e a b i l i t y o f t h e c e l l membrane due t o  injury  (see  G i e s e , 1968).  S e c o n d l y , changes  i n pH may  cause  change i n t h e o v e r a l l m e t a b o l i s m o f t h e e p i t h e l i a l thus a l t e r i n g i t s energy s t a t u s .  a  cell,  S h a m i (1974) r e p o r t e d  2+ t h a t Ca was  - b i n d i n g t o g u i n e a p i g p l a c e n t a l p l a s m a membranes  facilitated  b y a n a l k a l i n e pH.  a s i m i l a r i n c r e a s e i n Ca gill, i n Ca  2+  I t i s possible  -binding a l s o occurred i n the  p a r t l y a c c o u n t i n g f o r t h e more p r o n o u n c e d  2+  that  - t r a n s p o r t i n t h e a l k a l i n e pH  elevation  range.  2+ B r a n c h i a l Ca changes  - u p t a k e was  i n incubation temperature.  organisms s u c h as  substrate a f f i n i t y .  The  systems  v a r i a t i o n i n t h e enzyme-  enzyme a f f i n i t y  often  appears  be h i g h e r a t t h e t e m p e r a t u r e t o w h i c h t h e f i s h i s  adapted eel,  ( s e e H o c h a c h k a a n d S o m e r o , 1973).  For the  American  t h e normal range o f i t s h a b i t a t temperature i s between  4°-12°C.  T h i s corresponds t o the temperature range  optimal branchial Ca -uptake 2 +  activity  (2°-12°C),  g e s t i n g p o s s i b l e temperature a d a p t a t i o n of  of sug-  cellular  functions. C  The E f f e c t o f Hormones on B r a n c h i a l  Ca *-Uptake 2  Although the primary p h y s i o l o g i c a l f u n c t i o n of the  to  In poikilothermic  f i s h , many m e t a b o l i c enzyme  e x h i b i t a temperature-dependent  to  f o u n d t o be s e n s i t i v e  u l t i m o b r a n c h i a l bodies i n t e l e o s t s i s not yet  fully  50  e s t a b l i s h e d , t h e y appear t o be i n v o l v e d i n c a l c i u m lation.  regu-  Teleostean ultimobranchial glands are r i c h i n  calcitonin  (Copp a n d P a r k e s , 1968;  C o p p , 1969).  The  hypocalcemic e f f e c t of f i s h c a l c i t o n i n i s w e l l - e s t a b l i s h e d i n mammals (Copp e t a l . . 1970). to e l i c i t able.  Whether c a l c i t o n i n  acts  a hypocalcemic response i n f i s h i s s t i l l  debat-  However, d a t a a v a i l a b l e t o d a t e i n d i c a t e t h a t t h e  hormone may f u n c t i o n a s a h y p o c a l c e m i c f a c t o r , a t l e a s t i n t h e f r e s h w a t e r - a d a p t e d A n g u i l l a ( C h a n ejb a l . , 1968; P a n g , 1971a; site(s) remain  L o p e z a n d D e v i l l e , 1972;  Ma, 1973).  The  and mechanism(s) o f a c t i o n o f c a l c i t o n i n i n f i s h obscure. R e c e n t l y , Fleming and co-workers  (1973)  demonstr-  ated t h a t a d m i n i s t r a t i o n o f c a l c i t o n i n t o i n t a c t Fundulus kansae  resulted i n a stimulated  enoionmental water.  ^Ca-uptake  from t h e  Furthermore, t h e hormone-treated  a n i m a l s were found t o have a h i g h e r p e r c e n t a g e o f  ^Ca  i n t h e s o f t t i s s u e compartment and l o w e r p e r c e n t a g e i n bone t h a n t h e s o l v e n t - t r e a t e d c o n t r o l s .  Additional  e v i d e n c e b y D a c k e (1975) i n d i c a t e d t h a t t o t a l ^ C a e f f l u x was  a l s o reduced i n c a l c i t o n i n - t r e a t e d i n t a c t e e l s (A,  anguilla). gill  I n t h e p r e s e n t s t u d y on i s o l a t e d p e r f u s e d  preparations,  c a l c i t o n i n p e r f u s i o n i n a low dose o f  50 m U / m l c a u s e d a s i g n i f i c a n t i n c r e a s e  i nnet branchial  Ca  2 + -uptake.  Based  on t h e s e r e s u l t s , i t seems  t h a t c a l c i t o n i n a c t s on t h e g i l l  likely  t o enhance e n v i r o n m e n t a l  2+ Ca - u p t a k e . W h i l e t h i s a p p e a r s t o he i n c o n s i s t e n t w i t h the hypocalcemic e f f e c t of c a l c i t o n i n observed i n v i v o . 2+ a p o s s i b l e s i m u l t a n e o u s s t i m u l a t i o n o f Ca  -uptake  into  t h e s o f t t i s s u e c o m p a r t m e n t b y t h e hormone ( F l e m i n g et a l . , A.  1973)  s h o u l d be c o n s i d e r e d .  The  fact that i n  .iaponica. u l t i m o b r a n c h i a l e c t o m y caused a marked  i n muscle  t o t a l c a l c i u m (Ma, 1973)  evidence i n support of t h i s I t i s now  provides additional  view.  g e n e r a l l y accepted that the corpuscles  o f S t a n n i u s have a h y p o c a l c e m i c r o l e i n t e l e o s t ( s e e P a n g , 1973).  However, t h e r e i s l i t t l e  fish  information  c o n c e r n i n g t h e s i t e ( s ) and m e c h a n i s m ( s ) o f a c t i o n t h i s endocrine  decline  of  tissue.  F o n t a i n e and c o - w o r k e r s  (1972) r e p o r t e d t h a t i n  A n g u i l l a a n g u i l l a . " S t a n n i e c t o m y " was rL  f o l l o w e d by 2+  i n c r e a s e i n n e t i n w a r d f l u x o f e n v i r o m e n t a l Ca p r o p o s e d t h a t t h i s phenomenon may p a r t l y , f o r the concomitant r i s e calcium l e v e l s normally observed. provided the f i r s t  .  They  account, at l e a s t i n p l a s m a and Fenwick  muscle  a n d So  piece of evidence of branchial  ment i n c a l c i u m h o m e o s t a s i s .  an  They demonstrated  (1974) involve-  that i n  A n g u i l l a r o s t r a t a . "Stanniectomy" r e s u l t e d i n a marked  52 elevation i n calcium i n f l u x across the isolated perfused gill.  Data o b t a i n e d from t h e p r e s e n t study i n d i c a t e  that  2+ b r a n c h i a l Ca the  - u p t a k e i n A. r o s t r a t a was i n h i b i t e d b y  c o r p u s c l e e x t r a c t a n d t h e r e s p o n s e was d o s e - d e p e n -  dent.  These r e s u l t s s t r o n g l y suggest t h a t i n t e l e o s t  f i s h , t h e c o r p u s c l e s o f S t a n n i u s may p l a y a r o l e i n c a l c i u m h o m e o s t a s i s v i a i t s r e g u l a t o r y a c t i o n on t h e 2+ gill  Ca  t r a n s p o r t system.  The h y p e r c a l c e m i a d e v e l o p e d  f o l l o w i n g " S t a n n i e c t o m y " c o u l d be due t o a n i n c r e a s e d 2+ b r a n c h i a l Ca  -uptake i n t h e absence o f t h i s h y p o c a l -  cemic hormone.  The f a c t t h a t t h e h y p e r c a l c e m i a r e s p o n s e  was a b s e n t i n f i s h a d a p t e d t o a c a l c i u m - d e f i c i e n t  environ-  ment f u r t h e r s u p p o r t s t h i s h y p o t h e s i s . In  summary, t h i s s e r i e s o f s t u d i e s h a s d e m o n s t r a t e d 2+  the  e x i s t e n c e o f an a c t i v e b r a n c h i a l Ca  -uptake  in  the American e e l .  T h i s t r a n s p o r t s y s t e m was  to  changes i n e x t e r n a l and i n t e r n a l c a l c i u m  pH a n d e n v i r o m e n t a l t e m p e r a t u r e . was  system sensitive  concentrations, 2+  B r a n c h i a l Ca  -uptake  a f f e c t e d by c a l c i t o n i n from the u l t i m o b r a n c h i a l  g l a n d s and e x t r a c t o f t h e c o r p u s c l e s o f S t a n n i u s , suggesting the g i l l  as an i m p o r t a n t t a r g e t organ o f hormonal  a c t i o n i n teleostean calcium homeostasis.  DIVISION I I  CALCIUM - STIMULATED A T P a s e OF THE TELEOST G I L L PLASMA MEMBRANES  5^ CHAPTER  CHARACTERIZATION OF I N THE OF  THE  II  CALCIUM - STIMULATED A T P a s e  G I L L PLASMA MEMBRANES  AMERICAN E E L ,  ANGUILLA ROSTRATA  INTRODUCTION  I n the f i r s t across  the  chapter,  isolated g i l l  s t u d i e s on c a l c i u m  o f t h e A m e r i c a n e e l h a v e demon-  s t r a t e d a net uptake of calcium  ions from the  w a t e r i n t o t h e i n t e r n a l medium, a g a i n s t a n chemical  gradient.  dependent upon the  transport  Such " u p h i l l " availability  external  electrical2+  movement o f Ca  of metabolic  energy.  l i v i n g s y s t e m s , most energy - u t i l i z i n g p r o c e s s e s d r i v e n by t h e breakdown o f t h e (Dixon  a n d Webb, 1964,.).  o f ATP  must be  To  e n z y m a t i c and  energy-rich  be  w i t h the  energy -  The  (ATP  active calcium transport i n t h i s  must t a k e  c o u p l i n g b e t w e e n t h e two  p l a c e w i t h i n t h e membrane, s o t h a t t h e  h y d r o l y z i n g ATP  +  K  +  events case)  enzyme  i s f i x e d i n space.  S k o u (1965) h a s between Na ,  ATP  hydrolysis  u t i l i z i n g process. h y d r o l y s i s and  In  are  compound  e f f e c t i v e , the  coupled  is  e s t a b l i s h e d the r e l a t i o n s h i p  fluxes across  c e l l membranes and  h y d r o l y s i s b y t h e membrane - b o u n d Mg  2+  ATP  - d e p e n d e n t , (Na  +  -  K ) - a c t i v a t e d ATPase.  In teleost fish, active  o f sodium a c r o s s t h e g i l l i n v o l v e a s i m i l a r Mg ATPase  bound Ca  and  - d e p e n d e n t , (Na -K ) - a c t i v a t e d Membrane-  2+ (Mg  ) - a c t i v a t e d ATPases have been  several vertebrate  calcium  h a s "been shown t o  ( s e e M o t a i s a n d G a r c i a - R o m e u , 1972). 2+  in  epithelium  transport  occurs,  described  t i s s u e s where a c t i v e t r a n s p o r t o f  s u c h a s t h e i n t e s t i n a l mucosa (Melancon  D e L u c a , 1970), t h e r e n a l t u b u l e s  1971) a n d t h e p l a c e n t a  (Parkinson  ( S h a m i a n d R a d d e , 1971).  s e n s i t i v e ATPases have a l s o been c h a r a c t e r i z e d 2+ where t h e l e v e l  and Radde,  o f Ca  i s critical  Calcium i n tissues  f o rspecific  physio-  logical functions,  such as t h e b r a i n and n e r v e t i s s u e  ( B e r l and P u s z k i n ,  1970), t h e s a r c o p l a s m i c  (see  reticulum  M a r t o n o s i , 1972) a n d t h e r e d b l o o d c e l l s  (see Schatz-  mann, 1975).  2+ In t h i s chapter, the properties l a t e d ATPase i n t h e g i l l eel. the  o f a Ca  -stimu-  p l a s m a membranes o f t h e A m e r i c a n  A n g u i l l a r o s t r a t a were examined and compared properties  o f o t h e r Ca  -(Mg  ) ATPases.  with  The a c t i o n  o f two c a l c i u m - r e l a t e d hormones, c a l c i t o n i n and e x t r a c t 2+ of the corpuscles was a l s o  o f S t a n n i u s on t h e g i l l  investigated.  Ca  -ATPase  56  MATERIALS AND METHODS  A.  Experimental Animals S i l v e r American e e l , Anguilla r o s t r a t a L. was chosen  as the source of supply of teleost g i l l tissue (see Materi a l s and Methods, Chapter I ) . S i l v e r eels weighing 8001200 g were acclimated under laboratory conditions, i n running dechlorinated water at 12*5 +1° C f o r at least two weeks before use.  B.  I s o l a t i o n of G i l l Plasma Membranes The method of Post and Sen (I967) f o r the i s o l a t i o n  of kidney c o r t i c a l plasma membranes was adopted f o r the present study of g i l l plasma membranes. Experimental animals were k i l l e d by decapitation. G i l l filaments were freed from the g i l l arches and rinsed i n i c e - c o l d 0,9%  saline.  The i s o l a t e d g i l l tissue was  then homogenised i n 30 ml of a cold (4° C) solution containing 87 g sucrose, 1.0 g NaCl, 1.86 g EDTA (disodium s a l t ) , 0.20 g MgCl »6H 0 and 0.68 g imidazole per l i t r e , 2  2  using a T r i - R t i s s u e grinder with Teflon pestle.  The  homogenised tissue^was then processed through a series of centrifugations at 35,000 X g f o r 30 min i n d i f f e r e n t solutions at 4 ° C , using a S o r v a l l superspeed centrifuge  (model RC2-B). 9. in  The 5 mM  d e t a i l procedure i s o u t l i n e d i n Figure  i s o l a t e d p l a s m a membranes w e r e f i n a l l y T r i s -HC1  i m i d a z o l e and  C.  The  s t o r e d a t 4° C  Determination The  b u f f e r (pH 8.6)  taken  the  i n c u b a t i o n medium.  Concentration final  preparation  as a n i n d i c a t o r o f p l a s m a membrane c o n t e n t T o t a l p r o t e i n was  t h e method o f L o w r y and  D.  determined  c o - w o r k e r s (1951)» u s i n g  by  bovine  I s o l a t i o n Method  1. E l e c t r o n M i c r o s c o p i c E x a m i n a t i o n o f t h e Membrane An  a l i q u o t o f t h e f i n a l membrane p r e p a r a t i o n (50$)  35,000 X^g f o r 30 m i n .  p e l l e t was  w i t h 0.1 M  The  cacodylate  and  t h e n washed  each time.  p e l l e t was  then f i x e d w i t h phosphate - b u f f e r e d  tetraoxide  (1$)  and  three  and The osmium  stained with saturated uranyl  s t a i n e d membranes w e r e f i n a l l y  examination.  acet-  dehydrated  embedded i n R e s i n , r e a d y f o r s e c t i o n i n g a n d microscopic  was  centrifuged at  b u f f e r , resuspended  c e n t r i f u g e d a t 4 5 , 0 0 0 X g f o r 30 m i n  The  Final  Preparation  f i x e d i n Karnosky f i x a t i v e  ate.  in  standards.  E v a l u a t i o n of the  times  mM  use.  p r o t e i n c o n c e n t r a t i o n of the  was  albumin  c o n t a i n i n g 0.5  until  o f Membrane  suspended  electron  and  58  Homogenate centrifuged at 300 X g f o r 15 min.  -Supernatant Coarse  centrifuged at 35.000 X g f o r 30 min,  residue (discarded)  Mitochondrial and heavy plasma membrane fragments (discarded)  Figure 9.  P a r t i c l e - f r e e supernatant (discarded) — M i c r o s o m a l f r a c t i o n . This upper plasma membrane layer was scraped free and transferred to 25 ml of solution (A), homogenized and re-centrifuged at 35,000 X g f o r 30 min. The same procedure was repeated 3X, i n solutions (B), (C) and (D) to reduce progressi v e l y contamination by mitochond r i a . The f i n a l membrane prepara t i o n was suspended i n (D).  I s o l a t i o n of g i l l plasma membranes. Solution (A) contained 0.25 M NaCl, 1.0 M MgClg, 5 mM Na^EDTA and 10 mM imadazole. Solution (B) contained 0.25 M sucrose, 0.1 mM MgClg, 2 mM NagEDTA, 4 mM imadazole and 0.02% (w/v) Na-Haparin. Solution (C) contained 15 mM NaCl, 1.0 mM Na EDTA and 3 mM imadazole. Solution (D) contained 0.5 mM imadazole and 5mM Tris-HCl (pH 8.0). 2  59  2. Enzyme Marker Assays A f t e r the upper phase membrane layer was scraped o f f , the remaining p e l l e t from each centrifugation (35*000 Xg) was pooled.  Both the f i n a l membrane pre-  paration and the pooled residue were assayed f o r enzyme activities.  Alkaline phosphatase was used as a marker  f o r plasma membranes and succinic dehydrogenase was used as a marker f o r mitochondria (Dixon and Webb, 1964). Alkaline phosphatase was assayed by the method of Linhardt and Walter (1965).  Aliquots (100 Ail) of the  f i n a l membrane preparation and the pooled residue were incubated f o r one hour at 12° C i n 1.0 ml buffer - substrate mixture containing 0.1 M C0^ ~-HC0^~ buffer (pH 9 * 5 ) » 2  10 mM MgCl  2  and 5.4 mM p-nitrophenyl phosphate (Sigma  Chemical Co.).  The reaction was terminated by addition  of 5 ml 0 . 0 5 N NaOH.  The p-nitrophenol released was  measured as increase i n absorbance at 410 nm,  using a  Perkin -Elmer double beam spectrophotometer (model 124). S p e c i f i c enzyme a c t i v i t y was expressed as yx mole  p-nitro-  phenol released per mg protein per hour. Succinic dehydrogenase was assayed by the method of Pennington (1961).  The reaction mixture contained, i n  a f i n a l volume of 1.0 ml, 0.1 ml of the enzyme preparation, 0.1 ml 0.5 M sodium succinate, 0.3 ml 0.33%  INT  60 |2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride | (Eastman Kodak Co.) and 0 . 5 ml 0.1 M potassium phosphate buffer (pH 7 . 4 ) . terminated by adding 1.0 ml 10$ acid.  Incubation  (w/v)  was  trichloroacetic  The formazan produced was extracted with 4 ml  e t h y l acetate and measured as increase i n absorbance at 490 nm.  S p e c i f i c enzyme a c t i v i t y was  expressed as absor-  bance u n i t s per mg protein per hour.  E.  ATPase A c t i v i t y of the G i l l Plasma Membranes To t e s t the various enzymatic properties, 0.1  of membrane suspension  (20-30 ug protein) was  with 1.0 ml of a solution containing 20 mM (pH 8.0), 70 mM  Na*  ml  incubated  Tris-HCl buffer  (as NaCl), divalent cations ( C a  2 +  or  2+ Mg  ) and Na ATP (Sigma) at d i f f e r e n t concentrations as 2  indicated.  Divalent cations were omitted i n blanks.  Tris-ATP (Sigma) was used instead of Na reaction mixture contained no Na .  ATP,  and the  Incubations were  c a r r i e d out i n a water-bath at 1 2 . 5 + hour.  2  1.0° C f o r one  The reaction was then stopped by quickly placing  the tubes i n an i c e - b a t h and adding 1.0 ml 10$ trichloroacetic acid.  (w/v)  ATP hydrolysis was measured by  the release of inorganic phosphate from the samples, as determined by using a modification of the Technicon Auto Analyzer N-4c  Method (Alexander, 1968).  Specific a c t i v i t y  61  was  e x p r e s s e d as umoles  hour.  E a c h v a l u e was  P i r e l e a s e d p e r mg  protein  t h e mean o f t r i p l i c a t e  per  samples  i n d i v i d u a l e x p e r i m e n t s were r e p e a t e d a t l e a s t t h r e e The  f o l l o w i n g was  a list  and times.  of the s p e c i a l chemicals  used i n various experiments » (1)  Na  2  {Adenosine 5 * - t r i p h o s p h a t e , disodium s a l t }  ATP  A-3127);  ( S i g m a No.  (2)  Tris-ATP  JDi-Tris salt }  (3)  G-5756);  { Adenosine 5*-monophosphoric  AMP  (8)  Mersalyl acid  The  ( S i g m a No.  Activity namely  of the corpuscles of Stannius, 2+  studied.  0-3125);  o f two c a l c i u m - r e l a t e d h o r m o n e s ,  c a l c i t o n i n and e x t r a c t  gill  salt}  M-8000).  o f Hormones on A T P a s e effect  a c i d , sodium  A-1877);  O u a b a i n o c t a h y d r a t e ( S i g m a No.  Effect  (Sigma  A-0127);  (7)  the  (Sigma  1-5000)i  ( S i g m a No.  F.  (Sigma  | Adenosine 5 ' - d i p h o s p h a t e , disodium s a l t }  ADP  No.  (6)  A-3877);  { I n o s i n e 5 * - t r i p h o s p h a t e , sodium s a l t }  ITP  No. (5)  ( S i g m a No.  | Guanosine 5 * - t r i p h o s p h a t e , sodium s a l t \  GTP  No.  (4)  ( h y d r o x y m e t h y l ) - a m i n o - methane  p l a s m a membrane Ca  on  2+ -(Mg  ) ATPase a c t i v i t y  was  62 1.  E f f e c t o f Salmon C a l c i t o n i n Natural  No.  AL  1025)  salmon c a l c i t o n i n (Armour P h a r m a c e u t i c a l  was  p r e p a r e d as  Methods, Chapter I . in  20 mM  The  i n Materials  and  l y o p h i l i z e d hormone-.was d i s s o l v e d  t r i s - H C l buffer  r e a c t i o n m i x t u r e i n 100  described  (pH  8.0)  and  introduced  fil a l i q u o t s t o make up  into  the  added t o the 2.  Corpuscles; A m e r i c a n e e l s and  HCl  and  buffer  the  8.0)  was  was  described  k i d n e y t i s s u e was I t was  Tris-  reac-  used i n the  added t o the  E q u a l v o l u m e s o f 20 c o n t r o l samples.  e x t r a c t was  the  p r o t e i n per  ) A T P a s e , an  ml. corpuscles  e q u a l amount  taken i n the v i c i n i t y of the  Ca  2 +  -(Mg  2 +  of  hormonal  t h e n p r o c e s s e d i n a s i m i l a r manner and  e f f e c t o f t h i s e x t r a c t on g i l l  the  hormone  s p e c i f i c a c t i o n of the  -(Mg  mM  Total  d e t e r m i n e d by  c o - w o r k e r s (1951) and  Ca  in  t h e n d i l u t e d and  demonstrate the  examined.  silver  The  e x p r e s s e d as mg  o f S t a n n i u s on g i l l  was  Stannius  u s e d f o r hormone e x t r a c t i o n .  corpuscular  m e t h o d o f L o w r y and  glands.  p r e p a r e d as  u l aliquots.  b u f f e r was  p r o t e i n of the  To  e x t r a c t was  e x t r a c t was  concentration  Corpuscles of  o f S t a n n i u s were c o l l e c t e d f r o m  t i o n m i x t u r e i n 100 T r i s - HC1  of the  M e t h o d s , C h a p t e r I , e x c e p t t h a t 20 mM  (pH  corpuscular  buffer  c o n t r o l samples.  E f f e c t of E x t r a c t  Materials  the  required  c a l c i t o n i n c o n c e n t r a t i o n . An e q u a l v o l u m e o f T r i s - H C l was  Co.,  ) ATPase  the  activity  63 RESULTS  A.  Evaluation of the I s o l a t i o n Method 1. Electron Microscopic Examination of the F i n a l Membrane Preparation Electron microscopy revealed that the f i n a l  prepar-  a t i o n consisted mostly of plasma membranes (Plates 5 and 6 ) . No contamination with mitochondria could be observed. 2. Marker Enzymes Table 2 summarizes the d i s t r i b u t i o n of marker enzymes. The s p e c i f i c a c t i v i t y of succinic dehydrogenase i n the " f i n a l membrane f r a c t i o n " was very low, being only onetenth of that i n the "pooled residue".  This indicates that  the f i n a l membrane preparation contained only very few mitochondria, Ca  confirming the electron microscopy observations.  -ATPase a c t i v i t y followed a d i s t r i b u t i o n very s i m i l a r  to that of a l k a l i n e phosphatase, with almost the same act2+  i v i t y r a t i o (1.66 f o r Ca -ATPase and 1.86 f o r alkaline phosphatase) between the " f i n a l membrane f r a c t i o n " and the 2+ "pooled residue".  This indicates that the Ca  -ATPase under  i n v e s t i g a t i o n i s located i n the plasma membranes. The relatively  high s p e c i f i c a c t i v i t y of the two phosphatases  i n the "pooled residue" may be due to incomplete of the heavier plasma membrane fragments.  recovery  64  Plate 5 .  The f i n a l membrane preparation (X39,664).  Plate  6.  The  f i n a l membrane p r e p a r a t i o n  (X  82,075).  66  Table 2 . S p e c i f i c a c t i v i t i e s of marker enzymes i n different fractions.  The pooled residue  consisted of the remaining p e l l e t after the upper membrane layer had been scraped o f f . I t includes mitochondria and heavy plasma membrane fragments.  Ca ATPase 2 +  Fraction  Alkaline Succinic Phosphatase Dehydrogenase 0.086°  F i n a l membrane fraction  13.9  Pooled residue  8.4  0.50  0.728  F i n a l membrane f r a c t i o n Pooled residue  1.66  1.86  0.12  a  0.93  a  A c t i v i t y i n umoles Pi/mg protein/hr.  b  A c t i v i t y i n umoles p-nitrophenol/rag  c  Activity i n A^  0 Q  ^/mg  protein/hr.  b  protein/hr.  67  B.  P r o p e r t i e s o f t h e G i l l P l a s m a Membrane C a 1. A c t i v a t i o n o f t h e Enzyme b y D i v a l e n t  2+ -ATPase  Cations  A c t i v a t i o n o f t h e enzyme b y c a l c i u m i o n s was a s s e s s e d b y ATP h y d r o l y s i s w i t h i n c r e a s i n g c a l c i u m concentration 10 mM.  i n t h e i n c u b a t i o n medium f r o m 0.1 t o  Maximum enzyme a c t i v i t y was r e a c h e d a t c a l c i u m  l e v e l s o f 3 t o 5 mM  ( F i g u r e 1 0 ) . The a p p a r e n t  for  2+ Ca d e t e r m i n e d f r o m f i v e d i f f e r e n t f r e s h g i l l membrane p r e p a r a t i o n s was 0 . 3 8 + 0.03 mM (mean + S.E.) ( F i g u r e 11).  The r a t e o f P i r e l e a s e a t p e a k a c t i v i t y  with  2+ 5 mM C a  was 2 4 . 5 + 0.82 jumoles P i p e r mg p r o t e i n p e r  h o u r (mean+  S.E., n=5)<  Magnesium i o n a l o n e a l s o a c t i v a t e d t h e enzyme, though l e s s e f f e c t i v e l y than c a l c i u m i o n .  Maximum  enzyme  a c t i v i t y was o b s e r v e d a t a m a g n e s i u m c o n c e n t r a t i o n o f 2+ 5 mM  ( F i g u r e 1 0 ) . The a p p a r e n t  from f i v e d i f f e r e n t g i l l  f o r Mg  determined  p r e p a r a t i o n s was 0 . 6 4 + 0.07  mM  (mean+  S.E.) ( F i g u r e 1 1 ) . The maximum r a t e o f ATP 2+ h y d r o l y s i s b y Mg - s t i m u l a t i o n was 1 2 . 3 + Q.,4 ;uraoles P i p e r mg p r o t e i n p e r h o u r (mean + S.E., n = 8 ) , w h i c h 2+ was o n l y 50$ t h a t b y C a - s t i m u l a t i o n . E q u i m o l a r 2+ 2+ concentrations  o f Mgf'  and Ca  i n t h e i n c u b a t i o n medium  r e s u l t e d i n enzyme a c t i v a t i o n i n t e r m e d i a t e b e t w e e n 2+ 2+ t h o s e w i t h Ca a n d Mg a l o n e ( F i g u r e 1 0 ) . Maximum  68  mM Divalent Cation  Figure 10.  A c t i v a t i o n of ATP hydrolysis by increasing concentrations  of divalent cations. 2+  •—•  , a c t i v a t i o n by Ca  by Mg "; A 24  ; © — © , activation  A , a c t i v a t i o n by C a  ( i n equimolar concentration). mixtures contained  2 +  +Mg  2 +  Incubation  20 mM T r i s - H C l (pH 8.0),  5mM ATP (disodium s a l t ) and 70 mM N a (as +  NaCl).  69  0.6  Figure 11.  ~t  Lineweaver - Buric plots of ATPase a c t i v i t y at 2+  various concentrations of Ca  2+  , Mg  and. ATP.  V i s expressed i n jumoles Pi/mg protein/hr.., [S] i n mM.  • — • , 5 mM  ATP + i n c r e a s i n g  2+  concentration of Ca  i © — ©  t  increasing concentration of Mg 2+  5 mM  Ca  5 mM  2+  ATP +  j •  •  + increasing concentration of ATP.  Incubation mixtures contained 20 mM (pH 8 . 0 ) and 70 mM  Na  +  (as NaCl).  Tris-HCl  a c t i v i t y with C a  2 +  plus Mg * (1 « 1) was 71.4+ 2  1.7$  2+  (mean + S.E., n « 5 ) - t h a t  with Ca  alone.  2. E f f e c t of Protein Concentration on P i Release The e f f e c t of protein concentration on ATP hydrol y s i s i s shown i n Figure 12.  Inorganic phosphate release  was l i n e a r within the range of 20 to 240 mg protein per ml incubation mixture. 3. Sodium and Potassium Independence of the Enzyme 2+ 2+ A c t i v a t i o n of the enzyme by Ca require the presence of Na Na  +  +  or K . +  or Mg  did not  Incubation without  and using 5 mM Tris-A3?P resulted i n a s l i g h t l y  higher a c t i v a t i o n than that with 70 mM Na  and 5 mM  ATP (disodium s a l t ) (Figure 1 3 ) . 4. E f f e c t of ATP Concentration and Substrate Specificity The e f f e c t of ATP concentration on enzyme a c t i v i t y was examined by incubating the enzyme preparation with 5 mM C a , 20 mM T r i s - H C l (pH 8.0), 70 mM Na and. 2 +  +  various concentrations of ATP (disodium s a l t ) from 0.1 2+ t o 10 mM.  Maximal Ca  -activated ATP hydrolysis occurred  at an ATP concentration of 5 mM (Figure 14). K  m  The apparent  f o r ATP was 0.08 to 0.09 mM (Figure 11). 2+  The substrate s p e c i f i c i t y of the Ca -activated  71  Protein Concentration (/jg/ml)  F i g u r e 12.  E f f e c t o f p r o t e i n c o n c e n t r a t i o n on P i r e l e a s e . 2+  c o n t a i n e d 5 mM C a  Incubation mixtures  20 mM T r i s - H C l ( p H 8 . 0 ) , s a l t ) a n d 70 mM N a  +  (as  ,  5 mM A T P ( d i s o d i u m NaCl).  Figure 13.  E f f e c t of Na  on enzyme a c t i v a t i o n by 2+ divalent cations. • e , 5^M Ca j 2+  ©—© , 5 mM  Mg  contained 20 mM 5 mM  Tris-ATP.  .  Incubation mixtures  T r i s - H C l (pH 8.0)  and  73  F i g u r e 14.  Enzyme a c t i v i t y w i t h d i f f e r e n t as •  substrate. A  t  ITP|  A  •  • , ATP; A  o  ADP;  nucleotides  ©—©  •  • ,  , GTPj AMP. 2+  Incubation mixtures contained 20 mM NaCl).  T r i s - H C l (pH  8.0)  and  5 mM  70 mM  Ca Na  +  , (as  74  ATPase was and AMP  tested, using sodium s a l t s of GTP, ITP,  to replace ATP  medium.  ADP  (disodium s a l t ) i n the incubation  The r e s u l t (Figure 14) shows that the two  phosphates (ITP and GTP)  and diphosphate (ADP) a l l served  as substrates, but with a V"  „ only 7 0 %  and 78% (ITP) that with ATP as substrate. with AMP  tri-  as substrate was  (ADP and  GTP)  P i production  undetectable.  5. E f f e c t of P H on Ca *-ATPase A c t i v i t y 2  The effect of pH on enzyme a c t i v i t y was i n the range 4.0 to 10.0.  determined  In each instance, the pH  adjusted i n both the incubation mixture and the solution and was  and 9.4,  ATP  determined before and a f t e r incubation.  The pH curve i s presented i n Figure 15. of g i l l Ca  was  The pH optimum  -ATPase was between 7-9 and 8.1.  only 5 0 %  At pH  6.8  a c t i v a t i o n by calcium ions was retained.  6. E f f e c t of Inhibitors Figure 16 shows the e f f e c t of two i n h i b i t o r s on 2+  Ca  -ATPase a c t i v i t y .  The presence of ouabain i n the  incubation medium at concentrations of 0.1 and 1.0 did not i n h i b i t the enzyme.  mM  Mersalyl acid, when added  to the reaction mixture at concentrations ranging from 0.1 to 10 mM,  exhibited a progressive i n h i b i t o r y e f f e c t  on enzyme a c t i v i t y . of 2.1 mM,  50-%  At a mersalyl acid concentration  i n h i b i t i o n was  observed.  0+V/-T0  1 II  3  pH  F i g u r e 15.  Effect  o f pH o n C a - A T P a s e 2 +  activity. 2+  I n c u b a t i o n m i x t u r e s c o n t a i n e d 5 mM  Ca  20 mM  salt)  and  Tris-HCl, 70 mM  Na  +  5 mM  ATP  (as NaCl).  (disodium  ,  76  £  20 H  °i  •  0  F i g u r e 16.  1  2  Effect •  •  1  4  mM Inhibitor  1  6  r~  «  8  1  o f i n h i b i t o r s on C a - A T F a s e 2 +  • , effect  mersalyl acid. 5 mM  •  Ca  2 +  , 20 mM  of ouabain*  © — 0  ( d i s o d i u m s a l t ) a n d 70 mM  Na  +  IO  activity.  , effect  Incubation mixtures T r i s - H C l (pH 8 . 0 ) ,  ;  of  contained 5 mM  (as NaCl).  ATP  77  7.  E f f e c t of Incubation  T e m p e r a t u r e on Enzyme A c t i v i t y  2+ Ca ature  - A T P a s e a c t i v i t y was  r a n g e 1-80°C.  p r o g r e s s i v e l y up  determined i n the  temper-  E n z y m a t i c h y d r o l y s i s o f ATP (Figure 1 7 ) .  t o 30° C  increased  A second peak  w i t h enzyme a c t i v i t y 10 fo l o w e r t h a n t h a t o f t h e f i r s t o was o b s e r v e d a t 50 C. ATP h y d r o l y s i s t h e n d e c l i n e d 1  s h a r p l y and  c o m p l e t e enzyme i n a c t i v a t i o n o c c u r r e d  7°  Q  L  C.  The  1 Q  method of G i e s e 8.  (5-15°C)  calculated according  (1968) was  S t a b i l i t y of the  at  to  the  1.8.  Enzyme 2+  To  determine the  s t a b i l i t y o f Ca  -ATPase, a l i q u o t s  o f enzyme p r e p a r a t i o n w e r e t e s t e d f o r a c t i v i t y a f t e r s t o r age  a t 4-° C  f o r up  t o 20  days.  Enzyme a c t i v i t y  p r o g r e s s i v e l y with i n c r e a s i n g time of storage A 50 i> l o s s i n a c t i v i t y was a t 4-° C  f o r 20  normally  days.  u s e d w i t h i n a few  (Figure  observed i n samples  Therefore,  declined  stored  fresh preparations  days o f  18).  were  isolation.  2+ C.  E f f e c t o f Hormones on Ca  -ATPase A c t i v i t y  1. E f f e c t o f S a l m o n C a l c i t o n i n 2+ The  e f f e c t o f s a l m o n c a l c i t o n i n on g i l l  A T P a s e i s shown i n F i g u r e  19.  ranging  medium d i d n o t  a l t e r t h e r a t e o f e i t h e r Ca  - s t i m u l a t e d ATP  -(Mg  Salmon c a l c i t o n i n a t  concentrations  o r Mg  f r o m 50  Ca  t o 520  hydrolysis.  mU  per ml  2+ )  various  incubation  -stimulated  78  °~\—  '  1  0  '  1  20  «  40  1  r~=-  60  1 80  Incubation Temperature ( °C)  2+  Figure 1 7 . Effect  o f i n c u b a t i o n temperature on Ca  ATPase a c t i v i t y . contained 5mM  5 mM  Ca  ATP ( d i s o d i u m  NaCl).  The i n c u b a t i o n 2 +  , 2 0 mM  -  mixtures  T r i s - H C l (pH 8 . 0 ) ,  s a l t ) a n d 7 0 mM  Na  +  (as  79  100-  80H  Stored at 4 ° C  c  o o 60 o X  Ul  o <u £  40 H  o >> >  5  20  o 5? T  -i  1  r—  T  i  8 12 Days after Original Enzyme Extraction 4  Figure 18.  Effect  o f s t o r a g e a t +4  activity. 5mM ATP  Ca  2 +  C  on Ca  Incubation mixtures , 20 mM  Tris-HCl  20  16  -ATPase  contained  (pH 8.0),  ( d i s o d i u m s a l t ) a n d 70mM N a  +  5 mM (as NaCl)  00 D 1  0_ .E  H  5mM Mg  «  2+  < o —. t_  •  CL  CM  en  10  (0  o i —  0  i  _ _ T  r  200  60(  400 Salmon Calcitonin (mU/ml)  Figure  19*  Effect Ca  2 +  o f c a l c i t o n i n o n g i l l p l a s m a membrane  -(Mg  • — « ,  2 +  )  5 mM  ATPase. Ca  2 +  i  Incubation mixtures HCl 70 mM  (pH 8 . 0 ) , Na  +  5 mM  o—o,  5 mM  Mg  c o n t a i n e d 20 mM  2 +  . Tris-  ATP ( d i s o d i u m s a l t ) a n d  ( a s Na C l ) .  2. E f f e c t o f E x t r a c t o f t h e C o r p u s c l e s o f S t a n n i u s The  effect of the extract of the corpuscles of 2+  S t a n n i u s on g i l l Figure 20.  Ca  ?+ -(Mg  ) ATPase i s p r e s e n t e d i n  C o r p u s c u l a r e x t r a c t , when a d d e d t o t h e  i n c u b a t i o n mixture a t c o n c e n t r a t i o n s r a n g i n g from  0.03  to  0.24 mg p r o t e i n p e r m l , e x h i b i t e d a p r o g r e s s i v e 2+ 2+ i n h i b i t o r y e f f e c t o n b o t h C a - s t i m u l a t e d a n d Mg s t i m u l a t e d A T P a s e a c t i v i t y . The i n h i b i t o r y e f f e c t o f 2+  t h e e x t r a c t on Ca (Figure 21).  -ATPase a c t i v i t y i s dose - r e l a t e d  A t e x t r a c t c o n c e n t r a t i o n a s l o w a s 0.05  mg p r o t e i n p e r m l , a 1 2 % i n h i b i t i o n o f enzyme a c t i v i t y was o b s e r v e d .  A h i g h e r d o s e o f 0.24 mg p r o t e i n p e r m l  r e s u l t e d i n a 30 % Table  inhibition.  3 shows t h e s p e c i f i c  e f f e c t o f the corpus-  2+ c l e s o f S t a n n i u s on g i l l  Ca  -ATPase.  Corpuscular  e x t r a c t a t a c o n c e n t r a t i o n o f 0.128 mg p r o t e i n p e r m l i n c u b a t i o n mixture, caused a 2 4 - 2 5 % both Ca  2 +  - a c t i v a t e d and M g  A f t e r heat treatment at  100°C  to  o n l y 8 - 9 %.  2 +  inhibition of  - a c t i v a t e d ATP h y d r o l y s i s .  o f t h e same c o r p u s c u l a r e x t r a c t  f o r 15 m i n u t e s ,  t h e % i n h i b i t i o n was  reduced  A d d i t i o n o f k i d n e y e x t r a c t a t about  t h e same c o n c e n t r a t i o n ( 0 . 1 2 2 mg p r o t e i n p e r m l i n c u b ation mixture) resulted i n a 5 - 7 % activity.  i n h i b i t i o n o f enzyme  82 26 -j  Extract of Corpuscles of Stannius ( mg Protein /ml) F i g u r e 20.  Effect  of extract  on g i l l • — •  of corpuscles o f Stannius 2+ 2+ p l a s m a membrane C a - ( M g ) A T P a s e .  , 5 mM  Ca  2 +  ;  0  O  , 5 mM  I n c u b a t i o n m i x t u r e s c o n t a i n e d 20 mM HC1  ( p H 8.0),  70 mM N a  +  5mM  Mg  2 +  .  Tris -  ATP ( d i s o d i u m s a l t ) a n d  (as Na C l ) .  83  Figure 21.  Inhibitory  effect  of extract  of the corpuscles  2 + of  S t a n n i u s on g i l l  Incubation mixtures (pH 8.0), Na  +  5mM  ( a s Na C l )  ATP  Ca  -ATPase  c o n t a i n e d 5 mM  activity. tris-HCl  ( d i s o d i u m s a l t ) and  70  mM  Table 3.  S p e c i f i c effect of the corpuscles of Stannius on g i l l Ca ' fc  (Mg ) ATPase. 2+  Incubation mixtures contained 20 mM T r i s -  HCl (pH 8.0), 5 roM ATP (disodium s a l t ) and ?0 mM Na* (as NaCl).  Tissue extract was substituted by equal volumes of  T r i s - HC1 buffer i n control samples.  % Inhibition of C a - ( M g ) ATPase a c t i v i t y 2+  Treatment  5 mM C a  2 +  2+  5 mM M g  2+  24.6+1.4  23.8+0.8  Heat-treated corpuscular extract (100°C f o r 15 min)  8.6+1.8  9.0+1.6  Kidney extract  7.2+1.3  4.9 +2.5  Corpuscular extract (0.128 mg protein/ml)  (0.122 mg protein/ml) A l l values are given as means + S.E. ( n « 3 )  DISCUSSION  A.  Properties  o f t h e Gij.1 P l a s m a Membrane Ca*'' - A T P a s e 2+  I n t h e present i n v e s t i g a t i o n , a Ca ATPase h a s been demonstrated i n t h e g i l l American e e l .  Results  -activated  tissues of the  from e l e c t r o n microscopic  exam-  i n a t i o n a n d enzyme m a r k e r s t u d y i n d i c a t e t h a t t h e enzyme i s l o c a l i z e d i n t h e m i c r o s o m a l f r a c t i o n and appears t o be membrane - b o u n d . 2+ 2+ enzyme c a n b e s t i m u l a t e d b y e i t h e r C a o r Mg 2+ but the and a f f i n i t y a r e h i g h e r f o r Ca than The  alone,  v  m  a  x  2+  f o r Mg . T h i s p r e f e r e n t i a l a c t i v a t i o n o f t h e enzyme b y 2+ 2+ Ca i s s i m i l a r t o t h a t d e s c r i b e d f o r p l a c e n t a l Ca -ATPase 2+ ( S h a m i a n d R a d d e , 1971). H o w e v e r , s u c h C a preference * 2+ 2+ i s n o t o b s e r v e d i n o t h e r C a -(Mg ) A T P a s e s w h i c h v a r y considerably  i n t h e i r divalent cation requirements.  2+ B r a i n m i c r o s o m a l Ca -ATPase c a n be s t i m u l a t e d 2+  w e l l by e i t h e r Ca B e r l and Puszkin, and  o r Mg 1970).  alone  ( N a k a m a r u e t a l . , 1967;  I n t h e i n t e s t i n a l mucosa  D e L u c a , 1970) a n d t h e r e n a l t u b u l e s  R a d d e , 1971). w h i l e other,  Ca  2 +  a c t i v a t i o n b y Mg  In the sarcoplasmic erythrocyte  equally  2+  2+  and M g  (Melancon  (Parkinson and  c a n s u b s t i t u t e f o r each 2+ i s g r e a t e r than t h a t by Ca .  reticulum  2 +  ( M a c L e n n a n , 1970) a n d t h e  ( S c h a t z m a n n a n d R o s s i , 1971;  must b e p r e s e n t i n a t l e a s t t h e same m o l a r  W o l f , 1972),  Mg  concentration  2 +  a s ATP t o g i v e f u l l  activity. 2+  Equimolar concentrations the  o f b o t h Ca  a n d Mg  i n c u b a t i o n medium r e s u l t e d i n enzyme a c t i v i t y 2+  t h a n t h a t w i t h Ca alone,  suggesting  alone  equation  (1972)  fc  2+ f o r Ca  have d e r i v e d an  w h e t h e r two i o n s a c t i v a t e t h e  same s i t e w h i l e t h e enzyme i s s a t u r a t e d .  m  lower 2+  a n d h i g h e r t h a n t h a t w i t h Mg  S h a m i a n d Radde  f o r determining  apparent K  in  c o m p e t i t i o n between t h e two c a t i o n s f o r  the a c t i v e s i t e s .  and  2+  Individual V  , max  2+ a n d Mg  were i n t r o d u c e d i n t o t h e e q u a t i o n . 2+ o f maximum a c t i v i t y w i t h C a  obtained  from t h i s  study  The c a l c u l a t e d v a l u e 2+  p l u s Mg  (1 i 1) was  78$  2+ t h a t w i t h Ca  alone.  This d i f f e r s from the observed  (72 $) b y o n l y 6 $, i n d i c a t i n g t h a t C a the g i l l  2 +  and M g  2 +  value  activate  p l a s m a membrane A T P a s e a t t h e same s i t e . 2+  One o f t h e common p r o p e r t i e s o f t h e v a r i o u s C a (Mg  ) A T P a s e s i s t h a t t h e y do n o t r e q u i r e Na  activation  1969?  ( N a k a m a r u e t a l . . 1967;  1970? M e l a n c o n a n d D e L u c a , 1970? 1971; S h a m i a n d R a d d e , 1971). T h i s  true f o r the g i l l  for  Schatzmann and V i n c e n z i *  MacLennan,  son and Radde,  or K  -  Parkini s also  Ca -ATPase. 2 +  ATP i s t h e p r e f e r e n t i a l s u b s t r a t e f o r t h e enzyme. Although  other nucleotide triphosphates  diphosphate lysis  ( I T P a n d GTP) a n d  (ADP) may a l s o b e u t i l i z e d , t h e d e g r e e o f h y d r o -  i s much l o w e r  t h a n t h a t o f ATP.  AMP  cannot serve  as  substrate. blood  cell  Unlike the ( C a  +Mg  2 +  2 +  ) -ATPase  of the red  ( C h a e t a l . , 1971) w h i c h d o e s n o t h y d r o l y s e  other triphosphates  t o any a p p r e c i a b l e e x t e n t , t h i s low  d e g r e e o f s u b s t r a t e s p e c i f i c i t y i s common t o many •» 2 + Ca  2+ -(Mg  1970;  ) ATPases  (Nakamaru e t a l . . 1967;  S h a m i a n d R a d d e , 1971;  MacLennan,  D a v i e s a n d B r a g g , 1972).  H o w e v e r , i n a l l c a s e s , ATP i s t h e p r e f e r e n t i a l This  other  substrate.  i s t h e m a i n j u s t i f i c a t i o n f o r c a l l i n g t h e enzyme a n  ATPase, 2+ The r o l e  of divalent cations  (Ca  2+ , Mg  ) i n this  enzyme s y s t e m i s t o p r o d u c e a d i v a l e n t i o n - A T P  complex  w h i c h i s b e l i e v e d t o b e t h e a c t u a l s u b s t r a t e f o r t h e enzyme ( M e l a n o n a n d D e L u c a , 1970;  Hyde a n d R i m a i ,  1971).  In  t h e e r y t h r o c y t e , W o l f (1972) h a s d e m o n s t r a t e d t h a t Mg "*2  ATP c o m p l e x r a t h e r t h a n f r e e ATP s e r v e s a s s u b s t r a t e f o r t h e enzyme.  In the g i l l ,  f o r ATP ( d i s o d i u m Ca  2 +  w i t h 5 mM  t h e same a p p a r e n t  s a l t ) was o b t a i n e d  Mg  2 +  , the V  ° was o n l y 5 0 %  although  '  b y r e p l a c i n g 5 mM  of Mg -stimulated  ATPase  2 +  max  °  that of Ca -stimulated 2 +  ATPase.  This  finding  2+ i n d i c a t e s t h a t Ca  -ATP complex i s t h e p r e f e r e n t i a l  substrate f o r the g i l l  i  enzyme. 2+  The o p t i m a l pH f o r t h e g i l l 8.1.  Ca  -ATPase was 7»9 -  T h i s f a l l s w i t h i n t h e r a n g e (7.5-8.2) r e p o r t e d f o r  the m a j o r i t y of other C a  2 +  M e l a n c o n a n d D e L u c a , 1970;  -(Mg  2 +  ) ATPases  Parkinson  (MacLennan,  and Radde,  1971;  1970;  88 S h a m i a n d R a d d e , 1971)» w i t h t h e e x c e p t i o n  2+ m i c r o s o m a l Ca  of the b r a i n  2+ -(Mg  ) A T P a s e w h i c h h a s a pH optimum o f  9.0 (Nakamaru e£ a i . , 1967). U n l i k e t h e r e n a l c o r t i c a l 2+ 2+ Mg - ( C a ) A T P a s e w h i c h shows m a x i m a l a c t i v i t y o v e r a w i d e r a n g e o f pH  ( P a r k i n s o n a n d R a d d e , 1971), t h e g i l l  2+ Ca  - A T P a s e e x h i b i t s a b e l l - s h a p e d pH c u r v e w i t h  d i s t i n c t pH o p t i m u m .  T h i s f i n d i n g i s i n agreement w i t h  those of the sarcoplasmic the placenta  a  r e t i c u l u m ( M a c L e n n a n , 1970)  ( S h a m i a n d R a d d e , 1971).  and  A l t h o u g h t h e enzyme  was n o t i s o l a t e d a n d p u r i f i e d , t h e n a r r o w pH c u r v e w i t h  a  s h a r p pH optimum s u g g e s t s t h a t , u n d e r t h e a s s a y c o n d i t i o n s e m p l o y e d , i t i s u n l i k e l y t h a t more t h a n one enzyme i s c o n t r i b u t i n g s i g n i f i c a n t l y t o ATP h y d r o l y s i s .  An  alkaline  p h o s p h a t a s e i s u n l i k e l y t o be a c t i v a t e d a t t h e a s s a y pH of  8.0.  2+ The s e a r c h  f o r a specific  ATPase has been u n s u c c e s s f u l .  i n h i b i t o r o f Ca  +  2+ ATPases.  However, Ca  2 +  -(Mg  )  inhibi2 +  )  2+ -(Mg  ) ATPase a c t i v i t y c a n be  i n h i b i t e d b y a number o f n o n - s p e c i f i c enzyme s u c h a s e t h a c r y n i c a c i d ( V i n c e n z i , 1968; 1971)  -(Mg  Ouabain, a s p e c i f i c  t o r o f ( N a - K ) ATPase, has no e f f e c t on C a +  2+  and m e r s a l y l a c i d (Hasselbach  S c h a t z m a n n a n d V i n c e n z i , 1969; H a s s e l b a c h and S e r a y d a r i a n  inhibitors  Shami and Radde,  and S e r a y d a r i a n ,  1966;  S h a m i a n d R a d d e , 1971).  (1966) h a v e d e m o n s t r a t e d t h a t  b o t h c a l c i u m t r a n s p o r t and C a  2 +  - a c t i v a t e d ATPase  activity  a r e a b o l i s h e d when s a r c o p l a s m i c r e t i c u l u m membranes a r e incubated i n the presence  of mersalyl acid.  Similar  r e s u l t s a r e r e p o r t e d i n t h e r e d b l o o d c e l l s where  both  2+ t h e Ca  - A T P a s e a n d t h e c a l c i u m pump a r e i n h i b i t e d b y  mersalyl acid  ( S c h a t z m a n n a n d V i n c e n z i , 1969).  Both  e t h a c r y n i c a c i d a n d m e r s a l y l a c i d a r e b e l i e v e d t o be SH - b l o c k e r s ( S l a t e r , 1967; D a v i s , 1970). Inhibition 2+ 2+ o f C a -(Mg ) A T P a s e s b y t h e s e i n h i b i t o r s s u g g e s t s ; : t h a t t h e r e a r e SH - g r o u p s i n o r n e a r t h e a c t i v e c e n t r e o f t h e enzymes. 2+ of g i l l  Ca  I n the present  study, s i m i l a r  inhibition  2+ -(Mg  ) A T P a s e b y m e r s a l y l a c i d was  observed,  i n d i c a t i n g t h a t f r e e SH - g r o u p s a r e e s s e n t i a l f o r f u l l e x p r e s s i o n o f enzyme a c t i v i t y . The  gill  Ca  2 +  - A T P a s e i s r e l a t i v e l y heat  M a r k e d d e c l i n e i n enzyme a c t i v i t y d i d n o t o c c u r the i n c u b a t i o n temperature  was a b o v e 6 0 ° C .  stable. until  This i s i n  2+ common w i t h t h e p l a c e n t a l C a  - A T P a s e ( S h a m i , 1974).  However, i n a d d i t i o n t o t h e f i r s t at  30°C,  enzyme a c t i v i t y  peak  t h e a p p e a r a n c e o f a s e c o n d p e a k a t 50° C i s 2+ 2+  rather unusual  among t h e r e p o r t e d C a  -(Mg  ) ATPases.  The  component c o n t r i b u t i n g t o t h i s s e c o n d p e a k i s u n s t a b l e  and  d i s a p p e a r s w i t h b r i e f s t o r a g e o f t h e membrane  p a r a t i o n a t 4 ° C, a temperature Ca  2 +  -(Mg  2 +  pre-  suggesting the possible existence of  - d i f f e r e n t i a t e d iso-enzyme i n t h e g i l l  ) ATPase  system.  90  The  i n a b i l i t y o f o u a b a i n t o i n h i b i t enzyme +  and  activity  •*  t h e l a c k o f r e q u i r e m e n t f o r Na and K c l e a r l y d i s t i n 2+ + + g u i s h t h i s C a - A T P a s e f r o m t h e w e l l - known (Na -K ) ATPase r e p o r t e d  i nthe g i l l  p l a s m a ; membranes o f t e l e o s t  ( s e e M o t a i s a n d G a r c i a - R o m e u , 1972). (Na  Mg^-dependent,  -K ) A T P a s e h a s b e e n s t u d i e d i n g i l l  tissue  prepar-  a t i o n s o f a l a r g e v a r i e t y o f t e l e o s t (Kamiya and U t i d a ,  1968} 1969;  Kirschner,  I9691  P f e i l e r a n d K i r s c h n e r , 1972).  Z a u g g a n d M c L a i n , 1971? I n a l l cases,  a high  2+ " b a s e l i n e " Mg  -ATPase a c t i v i t y ,  was  On t h e b a s i s o f t h e p r e s e n t  observed.  i n s e n s i t i v e t o ouabain, findings, i t  2+ - s t i m u l a t e d ATP h y d r o l y s i s 2+ may i n f a c t b e due t o a c t i v a t i o n o f t h e g i l l C a - A T P a s e 2+ w h i c h c a n a l s o b e s t i m u l a t e d b y Mg t o a considerable is  s u g g e s t e d t h a t t h i s Mg  extent.  Since  ( N a - K ) A T P a s e i n m o s t s t u d i e s was a s s a y e d +  +  2+ in Ca  t h e presence o f 5  Mg  , i t i svery l i k e l y that the  2+ - A T P a s e s y s t e m was a l s o a c t i v a t e d . One  o f t h e p r o p o s e d mechanisms f o r a c t i v e t r a n s -  2+ port a Ca  o f Ca 2 +  -(Mg  across 2 +  c e l l membranes i s v i a t h e a c t i o n o f  ) ATPase.  The i d e n t i f i c a t i o n o f a C a  s t i m u l a t e d ATPase i n t h e g i l l  2 +  -  p l a s m a membrane o f t h e  American e e l , w i t h enzymatic p r o p e r t i e s s i m i l a r t o those 2+ 2+ o f t h e r e p o r t e d Ca -(Mg ) A T P a s e s , i s p r e s u m p t i v e 2+ e v i d e n c e f o r i t s r o l e i n a c t i v e b r a n c h i a l Ca in  freshwater  teleost.  transport  91 B.  The  E f f e c t o f Hormones on G i l l I t has  and  gill  been demonstrated t h a t  seawater-adaptation  (Na -K )-ATPase a c t i v i t y i n t e l e o s t are +  +  hormonal c o n t r o l .  Administration  freshwater eels induced a r i s e of the b r a n c h i a l animals  Ca^-ATPase  i n the  specific activity an  +  a b i l i t y to extrude Na  1  of C o r t i s o l to i n t a c t  ( N a - K ) - A T P a s e and +  increase  from the  +  under  K a m i y a , 1972;  1972).  o t h e r hand, p r o l a c t i n i n j e c t i o n i n t o  the  hypophysectomized e e l s accelerated branchial  the  +  c t i o n i n the  +  gill  Na  +  e f f l u x was  Carmichael,  decrease i n  ( N a - K ) - A T P a s e a c t i v i t y , and  the  (Epstein  e t a l . , 1971; On  B u t l e r and  gills  in  the  a marked r e d u -  r e c o r d e d when t h e  were t r a n s f e r r e d from sea w a t e r t o f r e s h water  animal  (Kamiya,  1972). I n C h a p t e r I , c a l c i t o n i n and puscles  e x t r a c t of the  o f S t a n n i u s have been demonstrated t o have  coran  2+ e f f e c t on t h e  a c t i v e b r a n c h i a l Ca  p o s s i b l e t h a t t h e s e two  -uptake.  It is  hormones a l s o i n f l u e n c e  the  2+ b r a n c h i a l Ca  -ATPase  P a r k i n s o n and  activity. Radde (1970) r e p o r t e d 2+  stimulated  the  a c t i v i t y o f a Ca  red blood c e l l .  that c a l c i t o n i n  2+ -(Mg  ) ATPase i n  the  A s i m i l a r a c t i v a t i o n of r e n a l plasma  2+ membrane Ca S t r e i f l e r and  -ATPase b y  c a l c i t o n i n was  H a r e l l (1974).  observed  In contrast,  data  by obtained  92  in  t h e present study i n d i c a t e t h a t salmon c a l c i t o n i n 2+  had  no d i r e c t e f f e c t on t h e g i l l  ATPase i n v i t r o .  p l a s m a membrane C a  -  W h i l e t h e l a c k o f enzyme r e s p o n s e 2+  s u g g e s t s t h a t c a l c i t o n i n may r e g u l a t e  b r a n c h i a l Ca 2+  u p t a k e b y a mechanism n o t i n v o l v i n g t h e Ca  -  -ATPase, i t  is  a l s o p o s s i b l e t h a t h o r m o n a l c o n t r o l o f t h e enzyme  is  mediated v i a i n t r a c e l l u l a r messengers i n i n t a c t c e l l s . Extract  of the corpuscles  of Stannius  exhibited  2+ a marked i n h i b i t o r y e f f e c t on t h e g i l l  Ca  -ATPase  a c t i v i t y i n v i t r o and t h e response i s l o g - d o s e = r e l a t e d . This  f i n d i n g suggests that the corpuscles of Stannius 2+ may r e g u l a t e b r a n c h i a l C a - u p t a k e b y a d i r e c t c o n t r o l 2+ o n t h e membrane-bound g i l l C a -ATPase.  93 CHAPTER  III  E F F E C T S OF S A L I N I T Y ADAPTATION, SEXUAL MATURATION AND DIET ON G I L L AND GUT MUCOSA PLASMA MEMBRANE CALCIUM - STIMULATED A T P a s e OF TELEOSTS  INTRODUCTION  I t h a s now b e e n w e l l e s t a b l i s h e d t h a t t h e N a - K +  +  s t i m u l a t e d ATPase i s r e s p o n s i b l e f o r a c t i v e sodium i o n movement a c r o s s  b i o l o g i c a l membranes.  I f efficient  c o n t r o l o f t h e s y s t e m i s t o be a t t a i n e d , t h e a p p a r e n t K  o f t h e enzyme f o r N a  m  Na  +  +  should  approximate the e f f e c t i v e  c o n c e n t r a t i o n range i n which t h e system  functions. existence Na -K +  +  normally  Enzyme k i n e t i c s t u d i e s h a v e r e v e a l e d t h e of tissue - specific  "functional variants" of  ATPase w h i c h appear t o be e l a b o r a t e d  for parti-  c u l a r p h y s i o l o g i c a l f u n c t i o n s w i t h i n d i f f e r e n t organs of an i n d i v i d u a l  ( s e e H o c h a c h k a a n d S o m e r o , 1973).  A  s i m i l a r e v o l u t i o n a r y t r e n d i s f o u n d i n t h e membrane bound C a  2 +  -(Mg  2 +  ) A T P a s e w h i c h h a s b e e n shown t o b e  associated with active transport of calcium  i o n across  c e l l membranes ( M a l a n c o n a n d D e L u c a ,  see M a r t o n o z i ,  1972*  see Schatzmann,  1975).  1970?  In specific  biological  membranes such as the erythrocyte membrane and the sarco2+ plasmic reticulum which regulate the i n t r a c e l l u l a r Ca —7 —6 concentration (10"' -10 enzyme f o r C a 1972;  2 +  M),  the apparent K  i s i n the range of 10"  m  of the  -10"^ B8  6  Martonosi and Feretos, 1964a, b ) .  (Wolf,  In other tissues  such as the i n t e s t i n a l mucosa and the renal tubules 2+ where active t r a n s c e l l u l a r Ca transport occurs, the 2+  apparent  of the enzyme f o r Ca  range of 10""-* M  i s i n a much higher  (Melancon and DeLuca, 1970;  Parkinson  and Radde, 1971).  + The high degree of v e r s a t i l i t y i n the Na  + -K  ATPase system i s further demonstrated by the fact that more than one functional form of the enzyme i s present i n the g i l l of catadromous and anadromous fishes under d i f f e r e n t environmental conditions.  In marine stenohaline  f i s h and i n euryhaline f i s h adapted to sea water, there i s a continual "downhill" inflow of Na  +  from the sea  water across the permeable g i l l epithelium. 24* + + l e v e l of Mg  -dependent, Na -K  A high  ATPase a c t i v i t y i s  generally observed, which i s believed to be associated with active branchial sodium excretion (see Motais and Garcia-Romeu, 1972).  In freshwater stenohaline f i s h  and i n euryhaline f i s h adapted to fresh water, active uptake of Na  across a r e l a t i v e l y impermeable g i l l e p i 2+ + thelium occurs. Only low l e v e l s of Mg -dependent, Na +  K  A T P a s e a c t i v i t y c a n be d e t e c t e d  +  1969).  (Kamiya and U t i d a ,  Instead, there i s a high l e v e l of K -independent, +  N a - a c t i v a t e d ATPase a c t i v i t y p r e s e n t i n f r e s h w a t e r +  teleost g i l l In first  preparations  Chapter  I I , a Ca  ( P f e i l e r a n d K i r s c h n e r , 1972). 2 +  - s t i m u l a t e d ATPase, f o r t h e  time, has been demonstrated i n g i l l  anes o f a t e l e o s t .  A l l the data presented  p l a s m a membrare studies  of a s i n g l e freshwater s p e c i e s , t h e American e e l ( A n g u i l l a rostrata).  I n t h i s c h a p t e r , a s u r v e y on t h e g e n e r a l  occurrence  o f t h i s enzyme i n m a r i n e a n d f r e s h w a t e r  was made.  Experiments  were a l s o designed  teleosts  toinvestigate  t h e p o s s i b l e a d a p t i v e b i o c h e m i c a l c h a n g e s t h a t may  occur  2+ in  the g i l l  Ca  - A T P a s e when a n i m a l s  d i f f e r e n t environmental  are subjected t o  c o n d i t i o n s w i t h a change i n e i t h e r  the i n t e r n a l o r e x t e r n a l calcium concentration.  Three  a s p e c t s were examined, i n c l u d i n g t h e e f f e c t s o f s a l i n i t y a d a p t a t i o n , s e x u a l m a t u r a t i o n a n d s p a w n i n g , a n d d i e t on 2+ t h e g i l l p l a s m a membrane C a - A T P a s e .  MATERIALS AND METHODS  A.  E f f e c t of S a l i n i t y Adaptation on G i l l Ca *-ATPase 2  Seven teleost species obtained from various marine and freshwater habitats were examined. Rockcod (Sebastodes p a u c i s p i n i s ) . P a c i f i c herring (Clupea p a l l a s i ) and s t r i p e d sea perch (Taeniotoca l a t e r a l i s ) were chosen as examples of marine f i s h .  Rockcods (100-  500 g) and striped sea perch (120-140 g) were obtained by d i v i n g i n Howe Sound, B.C. P a c i f i c herrings ( 4 0 60 g) were caught by netting i n the same coastal water. The animals were sampled immediately i n the f i e l d . freshwater teleost species were investigated.  Four  They were  the s i l v e r American e e l (Anguilla r o s t r a t a ) . the rainbow trout (Salmo g a i r d n e r i ) , the brown bullhead c a t f i s h (Ictalurus nebulosus) and the common carp (Cyprinus carpio).  S i l v e r American eels (800-1,200 g) were  caught i n r i v e r s along the Eastern coast of Canada (see Materials and Methods, Chapter I ) .  Immature  rainbow trout (80-120 g) were purchased l o c a l l y from a commercial hatchery i n Mission, B.C. Catfish (120170 g) and carp  (400-800 g) were caught i n freshwater  ponds i n the Lower Mainland, B.C. The animals were sampled i n the laboratory a f t e r an acclimation peroid of at l e a s t two weeks.  L o c a l l y purchased f r e s h w a t e r rainbow t r o u t (120180 g ) w e r e a d a p t e d t o a r t i f i c i a l s e a w a t e r u n d e r o r a t o r y c o n d i t i o n , u s i n g I n s t a n t Ocean S y n t h e t i c Salts  (Aquarium Systems I n c . , Ohio, U.S.A.).  labSea  Experi-  m e n t a l a n i m a l s w e r e f i r s t h e l d f o r t w o weeks i n a  50  g a l l o n f i b r e g l a s s tank containing running dechlorinated tap water  (12°C),  then g r a d u a l l y adapted t o f u l l  s e a w a t e r o v e r a p e r i o d o f t w o weeks b e f o r e S e a w a t e r was f i l t e r e d  continuously  w a s t e s a n d was c h a n g e d t h r e e  strength  sacrifice.  t o remove n i t r o g e n o u s  t i m e s a week.  Control ani-  mals were h e l d i n a s i m i l a r t a n k i n n o r m a l r u n n i n g d e c h l o r i n a t e d water throughout the experiment.  Both  seawater-  a d a p t e d a n d c o n t r o l t r o u t w e r e f e d d a i l y w i t h Co-op t r o u t pellets  (Surrey Co-operative A s s o c i a t i o n ,  Cloverdale,  B.C.) For  enzyme a n a l y s i s , g i l l  samples were  t a k e n from f r e s h l y k i l l e d a n i m a l s and t h e g i l l  always plasma  membranes w e r e i s o l a t e d b y t h e same p r o c e d u r e s a s described  i n M a t e r i a l s and Methods, C h a p t e r I I .  2+ ific ties  Spec-  2+  Ca - s t i m u l a t e d a n d Mg - s t i m u l a t e d ATPase a c t i v i o f t h e f r e s h g i l l membrane,preparations were measured 2+  and t h e a p p a r e n t K  ffl  o f t h e enzyme f o r Ca  d e t e r m i n e d i n t h e same manner a s d e s c r i b e d  2+ a n d Mg  were  i n Chapter I I .  98 B.  E f f e c t o f M a t u r a t i o n and Spawning on G i l l  Ca *-ATPase  Two s p e c i e s o f t e l e o s t f i s h , t h e r a i n b o w and t h e m i g r a t i n g coho salmon (Oncorhvnchus  2  trout  kisutch).  were i n v e s t i g a t e d a t v a r i o u s s t a g e s i n t h e i r l i f e  cycle.  E x p e r i m e n t a l a n i m a l s were a c c l i m a t e d under l a b o r a t o r y conditions, i n running d e c h l o r i n a t e dwater a t 1 0 + 2 ° C for  a t l e a s t two weeks b e f o r e u s e . G i l l  samples were  t a k e n from f r e s h l y s a c r i f i c e d animals and g i l l  plasma  2+ membranes w e r e i s o l a t e d . Mg -stimulated g i l l 2 +  S p e c i f i c Ca  ATPase a c t i v i t i e s  - s t i m u l a t e d and of the fresh  membrane p r e p a r a t i o n s w e r e m e a s u r e d a n d t h e a p p a r e n t 2+ 2+ K  m  o f t h e enzyme f o r C a  a n d Mg  were determined as  described i n Chapter I I . 1. R a i n b o w T r o u t F r e s h w a t e r rainbow t r o u t were p u r c h a s e d f r o m a l o c a l c o m m e r c i a l h a t c h e r y , t h e Sun V a l l e y T r o u t Farm in  M i s s i o n , B.C.  Four groups  o f t r o u t were sampled i  a . Y o u n g , i m m a t u r e t r o u t w e i g h i n g 80 - 1 2 0 g . T h e y w e r e a p p r o x i m a t e l y 18 months o l d a n d t h e i r sexes were i n d i s t i n g u i s h a b l e a t t h i s stage. b. A d u l t , maturing t r o u t w e i g h i n g 1 2 0 - 1 8 0 g. They were 2 - 3 y e a r s o l d and t h e i r gonads were w e l l  developed.  99 c. S p a w n i n g m a l e t r o u t w e i g h i n g 200 -500  g.  They  w e r e o v e r 3 y e a r s o l d a n d were a t t h e p e a k o f spawning. d . S p a w n i n g f e m a l e t r o u t w e i g h i n g 600 - 8 0 0  g.  They were o v e r 3 y e a r s o l d and were a t t h e peak o f spawning. 2. Coho S a l m o n M i g r a t i n g coho s a l m o n were o b t a i n e d f r o m t h e F i s h e r i e s R e s e a r c h S t a t i o n , W e s t V a n c o u v e r , B.C.  They were  hatched  i n t h e Samish h a t c h e r y and r a i s e d i n t h e a r t i f i c i a l at the s t a t i o n .  creek  F o u r g r o u p s o f coho s a l m o n were sampled i  a. Young immature coho w e i g h i n g 8 0 - 1 0 0 g.  They  w e r e one y e a r o l d f r e s h w a t e r f i s h a n d t h e i r s e x e s were s t i l l  indistinguishable.  b. A d u l t m a t u r i n g coho w e i g h i n g 9 0 - 1 2 0 g.  They  w e r e two y e a r s o l d s e a w a t e r f i s h a n d t h e i r gonads were w e l l developed a t t h i s s t a g e . c. S p a w n i n g m a l e c o h o w e i g h i n g 500 - 6 0 0 g . 2 1/2  After  y e a r s i n s e a w a t e r , t h e y were back i n  f r e s h w a t e r , r e a d y t o spawn. d . S p a w n i n g f e m a l e c o h o w e i g h i n g 6 0 0 - 700 w e r e i n t h e same c o n d i t i o n coho i n g r o u p c.  g.  They  as t h e spawning male  100  C.  E f f e c t o f D i e t on G i l l  and Gut Mucosa C a ^ - A T P a s e  Two g r o u p s o f l o c a l l y trout  purchased mature rainbow  (120 - 1 8 0 g) were a c c l i m a t e d t o l a b o r a t o r y c o n d i -  t i o n s i n 50 g a l l o n f i b r e g l a s s t a n k s  of running  dechlor-  i n a t e d w a t e r f o r t w o w e e k s , a n d w e r e f e d d a i l y w i t h Co-op trout pellets.  One g r o u p o f t r o u t was s t a r v e d f o r a  p e r i o d o f s i x w e e k s , u n d e r t h e same e x p e r i m e n t a l tions.  condi-  The o t h e r g r o u p was f e d t h r o u g h o u t t h e e n t i r e  experiment and served  as t h e c o n t r o l .  On  termination  o f t h e e x p e r i m e n t , t h e f i s h were k i l l e d b y a b l o w on the head. gut  Gill  samples were t a k e n  immediately.  o f b o t h s t a r v e d and c o n t r o l animals  examined f o r m o r p h o l o g i c a l  were  The  carefully  d i f f e r e n c e s and food  content.  The e n t i r e i n t e s t i n e was r e m o v e d f r o m t h e f i s h a n d r i n s e d in ice-cold and  0.9$  saline.  I t was t h e n s l i t  t h e mucosa were h a r v e s t e d  knife.  Gill  lengthwise  by scraping w i t h a g l a s s  a n d g u t m u c o s a p l a s m a membranes w e r e t h e n  i s o l a t e d by a procedure s i m i l a r t o that described i n 2+ M a t e r i a l s and Methods, Chapter I I .  S p e c i f i c Ca  -stimu-  2+ l a t e d a n d Mg - s t i m u l a t e d g i l l and g u t mucosa ATPase a c t i v i t y o f t h e f r e s h membrane p r e p a r a t i o n s was m e a s u r e d 2+ 2+ and  the apparent  determined.  o f t h e enzymes f o r C a  a n d Mg  were  101 RESULTS  A.  Effect  o f S a l i n i t y A d a p t a t i o n on G i l l Ca *-ATPase 2  A number o f t e l e o s t  o b t a i n e d from d i f f e r e n t  and f r e s h w a t e r h a b i t a t s were i n v e s t i g a t e d .  marine  A similar  2+ Ca  -ATPase was i d e n t i f i e d i n t h e g i l l plasma membrane  o f a l l seven t e l e o s t  s p e c i e s examined (Table 4 ) .  Figure  22 i s a histogram comparing the s p e c i f i c a c t i v i t y o f t h e 2+ seven t e l e o s t g i l l Ca -ATPases. The g i l l enzymes v a r i e d 2+ 2+ i n both t h e i r r a t e o f Ca - s t i m u l a t e d and Mg - s t i m u l a t e d ATP h y d r o l y s i s and t h e i r a f f i n i t y f o r C a and M g . 2 +  2 +  The m a j o r i t y o f s p e c i f i c enzyme a c t i v i t y f e l l w i t h i n t h e range o f 14 - 30 jumoles P i p e r mg p r o t e i n p e r hour.  An  e x c e p t i o n a l l y h i g h l e v e l o f 125 u n i t s was observed i n the common c a r p , which was approximately 6X the average s p e c i f i c a c t i v i t y recorded. was  higher f o r C a  Apart from t h e c a t f i s h ,  m  a  x  i n a l l the g i l l C a 2+ ATPases i d e n t i f i e d . I n the c a t f i s h , v _ _ f o r Mg was 2+ about 2 0 % h i g h e r than t h a t f o r Ca . No d i s t i n c t 2+ 2 +  than f o r M g  v  d i f f e r e n c e i n t h e s p e c i f i c Ca  2 +  2 +  -ATPase a c t i v i t y c o u l d  be observed between t h e marine and t h e freshwater However, t h e apparent  1 ^ f o r d i v a l e n t c a t i o n s appeared  t o be h i g h e r i n t h e marine t e l e o s t freshwater species.  teleost  than i n most o f t h e  Table 4.  Ca  -ATPase i n g i l l plasma membrane of marine and freshwater t e l e o s t s .  Incubation mixtue contained 20 mM Tris-HCl (pH 8.0), 5 mM ATP (disodium s a l t ) and 70 mM Na  Species  No. of  +  (as NaCl).  Sp. A c t i v i t y  Animals (5mM  Ca ) 2 +  3,  Sp. A c t i v i t y  3,  (5mM Mg ) 2+  % Activity Mg /Ca 2+  2+  App.  K ( C a ) App.K (Mg ) (mM) (mM) 2+  2 +  m  ffl  A. Marine F i s h 19  .1 + 2 . 5  79.2  1.29+0.04  2.36 + 0.14  23  .9 + 0 . 4  78.2  0.89+0.02  1.9^ + 0 . 0 7  18.1 + 1 . 2  9 .0+0.6  49.9  1.18  2.00  0.8  12 . 0 + 0 . 4  49.7  0.38+0.03  0.64 + 0 . 0 7  Rockcod  6  24.0  Herring  6  30.5+0.8  Sea Perch  2  5  24.5  +  3.2  6. Freshwater F i s h Am. E e l Trout Catfish Carp a  +  14.6+1.0'  10  .6+0.7  72.8  0.38 +0.01  0.58 + 0 . 0 2  9  1 8 . 4 + 0.6  22 . 9 + 0 . 6  119.1  0.64 + 0.06  1.34 + 0 . 0 5  11  125.0 + 3 . 9  85.0  1 . 6 9 + 0.09  3.67 + 0.19  10  107 . 0  +  3.7  S p e c i f i c a c t i v i t y i s expressed as ;umoles Pi/mg protein/hr. Values are given as means+S.E.  H  o  140 - i 120 100 ••-  ~ o  80 -  g  60-  < o  0.  <  40 rah 20 0 -  Herring  Cod  Eel  Perch  Catfish  Carp •*  Fresh Water  Sea Water • F i g u r e 22.  Trout  5 mM Ca  2 +  5mM Mg * 2  G i l l p l a s m a membrane C a - A T P a s e 2 +  teleosts.  Specific  mg p r o t e i n / h r .  o f marine and freshwater  a c t i v i t y i s expressed  as umoles P i /  The tap  e l e c t r o l y t e compositions of dechlorinated  water and a r t i f i c i a l  A d a p t a t i o n Experiment  sea water  i n the S a l i n i t y  a r e p r e s e n t e d i n T a b l e 5*  Calcium  and magnesium were p r e s e n t i n d e c h l o r i n a t e d t a p w a t e r in  e x t r e m e l y l o w c o n c e n t r a t i o n s (10  the a r t i f i c i a l  J  mM).  In  s e a w a t e r , c a l c i u m l e v e l was 9.3 mM a n d  m a g n e s i u m c o n t e n t was 50 mM. rainbow  -10  trout to a r t i f i c i a l  Acclimation of freshwater s e a water under l a b o r a t o r y  c o n d i t i o n s f o r two weeks d i d n o t i n d u c e any s i g n i f i c a n t 2+ changes i n t h e g i l l  p l a s m a membrane C a  -ATPase ( T a b l e 6 ) .  2+ S p e c i f i c Ca -ATPase a c t i v i t y i n t h e s e a w a t e r - a d a p t e d t r o u t was 31.9 + 1.6 u n i t s , w h i l e t h a t i n t h e c o n t r o l a n i m a l s was 30.5 + 1.4 u n i t s .  V  m o v  for Mg  2 +  was  74$  2+ t h a t f o r Ca  i n t h e seawater - adapted  control fish,  i t was  animals.  Inthe  75$. 2+  B.  E f f e c t o f M a t u r a t i o n and Spawning i n G i l l Ca  -ATPase  2+ G i l l p l a s m a membrane C a  -ATPases o f rainbow  trout  and m i g r a t i n g coho salmon a t v a r i o u s s t a g e s o f s e x u a l development were examined and t h e r e s u l t s a r e summarized in  T a b l e 7.  Specific Ca  2 +  - s t i m u l a t e d and M g ^ - s t i m u l a t e d  ATPase a c t i v i t i e s a r e i l l u s t r a t e d b y h i s t o g r a m s 2+ In  rainbow  t r o u t , s p e c i f i c Ca  -ATPase a c t i v i t y i n t h e  y o u n g i m m a t u r e a n i m a l s was f o u n d t o be o n l y 5 0 $ of  t h e a d u l t mature f i s h .  ( F i g u r e 23).  that  A s i m i l a r d i f f e r e n c e was  105  T a b l e 5.  Electrolyte  composition of the  t a p w a t e r and a r t i f i c i a l  dechlorinated  sea water i n the  S a l i n i t y Adaptation Experiment.  D e c h l o r i n a t e d Water Electrolyte  (m.moles/l)  Na  +  5.45  K  +  Sea Water  (m.moles/l)  444  8.40 X 1 0 " *  1  9.5  2  9.3  Ca  2 +  5.60X10"  Mg  2 +  7.00 X 1 0 "  3  4.00 X 1 0 "  4  Pi  Artificial  50 1.0  X10~  2  106  T a b l e 6.  Effect in g i l l S.  o f s e a w a t e r a d a p t a t i o n on Ca p l a s m a membranes o f r a i n b o w  Animals  Control Seawateradapted  a  trout,  gairdneri.  No. o f Condition  -ATPase  5  ?  Sp. A c t i v i t y (5mM  Ca  2 +  2 1  )  30.5 +1.4 31.9 +1.6  Sp. A c t i v i t y 2+^ (5 mM Mg^ ) #  23.0 +1.0 23.6 +1.2  a  %  Activity 2+/„ 2+ Mg^ / C a *  75.4 74.0  S p e c i f i c a c t i v i t y i s e x p r e s s e d a s jumoles/mg p r o t e i n / h r . V a l u e s a r e g i v e n a s means + S . E .  Table 7.  E f f e c t of sexual maturation and spawning on Ca  -ATPase i n g i l l plasma membrane  Incubation mixtures contained 20 mM  of rainbow trout and migrating coho salmon.  Tris-HCl (pH 8 . 0 ) , 5 mM ATP (disodium s a l t ) and ?0 mM Na  Mo. of Sp.Activity Animals  a  Sp.Activity  21  +  % Activity Mg* /Ca*  (as NaCl).  App.K (Ca ) 2+  m  (mM)  App.K (Mg ) 2+  m  (5mMCa*' )  (SmMMg* )  (mM)  14.6 +1.0***  10.6 + 0.7***  72.8  0.38 + 0.01  . 0 . 5 8 +0.02  Rainbow Trout Young immature, P.W.  10  Adult maturing, F.W.  8  29.6 + 1.6  24.8 + 1.1  84.4  0.61 + 0.07  0.82 + 0.04  Spawning J , F.W.  7  28.7 + 1.6  22.5 + 0.7  79-2  0.87  0.87  5  Spawning °. , F.W.  27.6 + 0.7  21.4 + 0.7  77.4  0.63  0.?4  Coho Salmon Young immature, F.W.  9  14.9 + 0.7*  9.^ + 0.5**  63.2  0.57 + 0.01  0.81 + 0.01  Adult maturing, S.W.  9  17.2 + 1.7  11.9 + 0.5  69.6  0.92 + 0.14  1.13 + 0.13  Spawning </ , F.W.  4  17.2 + 1.7  11.4 + 1.1  66.1  0.67  O.63  Spawning a  0  , F.W.  4  18.6+1.2  11.4 + 0.7  60.9  0.67  1.11  Specific activity i s expressed as jumoles Pi/mg protein/hr. Values are given as ( )*» ( )**» ( other groups within F.W. -fresh water?  means +S.E. denote significant difference as compared with corresponding values of the same speciesj P being <0.05, < 0.005 and <0.001 respectively. S.W. . sea water.  30 ft  20  ft  H ft  >  ft  o  < co 10 o  Young  *  Adult  Spawn.  ?  Young  Coho Salmon  Trout •  Figure 23.  Spawn.  Adult  N  5 m M Ca  2+  •  5 mM  Mg  z*  ,2+ E f f e c t o f s e x u a l m a t u r a t i o n and spawning on C a ^ - A T P a s e in g i l l  p l a s m a membrane o f r a i n b o w t r o u t a n d m i g r a t i n g  coho s a l m o n .  S p e c i f i c a c t i v i t y i s e x p r e s s e d a s jumoles  Pi/mg p r o t e i n / h r .  109 o b s e r v e d i n t h e r a t e o f Mg Subsequently, the  2 + - s t i m u l a t e d ATP 2+  r a t i o of  v m  a  x  f o r Mg  hydrolysis.  to that  for  2+ Ca  remained r e l a t i v e l y constant.  effect  on t h e  animals.  specific  Spawning had  little  enzyme a c t i v i t y o f a d u l t m a t u r e  F u r t h e r m o r e , no  significant  difference i n  the  2+ Ca  -ATPase a c t i v i t y was  m a l e and for  observed between the  female t r o u t .  The  d i v a l e n t c a t i o n s was  apparent  spawning  of the  f o u n d t o be h i g h e r  enzyme  i n the  mature  a d u l t s than i n the young animals. In the spawning male, 2+ 2+ the apparent f o r Mg was t h e same as t h a t f o r Ca . I n the m i g r a t i n g coho salmon, a s i m i l a r e l e v a t i o n 2+ in specific and  g i l l Ca  s p a w n i n g was  -ATPase a c t i v i t y w i t h  observed.  The  maturation 2+  r a t e o f Ca  -stimulated  2+ and  Mg  - s t i m u l a t e d ATP  h y d r o l y s i s i n the young immature  a n i m a l s was  80 - 85 %  significant  difference i n specific  detected  between the  spawning animals. the  t h a t of the matured a d u l t s .  No  enzyme a c t i v i t y  s e x u a l l y m a t u r e d a d u l t s and  The  apparent K  m  d i v a l e n t c a t i o n s a p p e a r e d t o be  water a d u l t s than i n the  freshwater  of the higher  was  the  enzyme f o r i n the  animals.  In  seathe  2+ spawning male salmon, the apparent approximately the C.  same as  f o r Mg 2+ t h a t f o r Ca .  was  2+ I n c o n t r a s t t o t h e f u l l y d i s t e n d e d i n t E f f e c t o f D i e t on G i l l and Gut M u c o s a Ca e s-tAiTnPea s eo f  the  control animal, the intestine of the starved  was v e r y s m a l l  and l i n e d w i t h  only a t h i n mucosal l a y e r .  No f o o d c o n t e n t was f o u n d i n a n y o f t h e s t a r v e d The  fish  e f f e c t o f s t a r v a t i o n on t h e g i l l  trout.  plasma  2+ membrane C a - A T P a s e o f r a i n b o w t r o u t i s s u m m a r i z e d i n T a b l e 8. T h e r e was n o s i g n i f i c a n t d i f f e r e n c e i n t h e specific g i l l  Ca  2+  -stimulated  a c t i v i t y between t h e s t a r v e d Starvation  a n d Mg  2+  -stimulated  and c o n t r o l  ATPase  animals.  f o r s i x weeks d i d n o t r e s u l t i n a n y marked  a l t e r a t i o n i n t h e apparent  o f t h e enzyme f o r  divalent  cations. The  e f f e c t o f s t a r v a t i o n on g u t mucosa plasma 2+  membrane C a  - A T P a s e o f r a i n b o w t r o u t was a l s o  investi-  g a t e d a n d t h e r e s u l t s a r e p r e s e n t e d i n T a b l e 9«  Star-  2+  v a t i o n f o r s i x weeks l e d t o a r i s e i n s p e c i f i c C a s t i m u l a t e d A T P a s e a c t i v i t y f r o m 5*1 t o 7»5 ^ m o l e s P i  2+ per  mg p r o t e i n p e r h o u r . -  ATPase a c t i v i t y ,  The s p e c i f i c Mg  -stimulated  on t h e o t h e r hand, remained a t a c o n s t a n t  l e v e l o f a b o u t 7.4 u n i t s .  In contrast  t o the control  a n i m a l s i n w h i c h t h e s p e c i f i c g u t mucosa ATPase  activity  2+  2+  b y Mg - s t i m u l a t i o n was 45 % h i g h e r t h a n t h a t b y C a s t i m u l a t i o n , t h e g u t enzyme o f t h e s t a r v e d t r o u t a p p e a r e d t o respond equally w e l l t o s t i m u l a t i o n by both cations.  The a p p a r e n t  divalent  2+  o f t h e enzyme f o r C a  i n the  Table 8.  E f f e c t of starvation on Ca -ATPase i n g i l l plasma membrane of rainbow 2+  trout,  S. gairdneri.  No. of Condition  a  Animals  Sp. A c t i v i t y o. (5mM Ca* )  21  Sp. A c t i v i t y 5. (5 mM Mg* )  % Activity P O Mg* /Ca*  App. K ^ C a * )  a  +  2  App. K^Mg *) 2  +  (mM)  (mM)  Control  8  29.6 +1.6  24.8+1.1  84.4  0.61 + 0.07  0.82 +0.04  Starved  8  31.3 + 1.8  24.6 +1.2  79.0  0.80 + 0.03  1.00 + 0.02  S p e c i f i c a c t i v i t y i s expressed as jumoles Pi/rag protein/hr. Values are given as means + S.E.  H  Table 9. Effect of starvation on Ca -ATPase i n gut mucosa plasma membrane of rainbow trout, S. gairdneri.  Condition  a  No. of  Sp. A c t i v i t y  Animals  (5 mM Ca**)  a  9  Sp. A c t i v i t y , (5mM Mg* )  5  % Activity . , Mg* /Ca* 0  Control  8  5.14 + 0.56  7.43 + 0.68  144.6  Starved  8  7«53±0.56**  7.36 + 0.44  97.4  App. K (Ca ) App. K (Mg ) • (mM) (mM) 2  m  0.38 + 0.02  2  m  1.40 +0.16  0.93 + 0.02*** 1.18 +0.04  Specific activity i s expressed as jumoles Pi/mg protein/hr. Values are given as means +S.E. ( )** and ( )*** denote significant difference as compared with corresponding values of control, P being <0.01 and <0.001 respectively.  s t a r v e d f i s h was  f o u n d t o be a p p r o x i m a t e l y t w i c e t h a t  i n the c o n t r o l .  No  K  for Mg  2 +  was  s i g n i f i c a n t change i n t h e  o b s e r v e d as a r e s u l t  of  apparent  starvation.  114  DISCUSSION  In t h e present  study, t h e e x i s t e n c e o f a Ca 'fc  ATPase has been demonstrated i n t h e g i l l  p l a s m a membrane  p r e p a r a t i o n s o f a v a r i e t y o f t e l e o s t f i s h e s from  both  m a r i n e a n d f r e s h w a t e r h a b i t a t s , s u g g e s t i n g a common occurrence enzymatic  of this g i l l  enzyme among t e l e o s t s .  p r o p e r t i e s such as s p e c i f i c  Some  a c t i v i t y and t h e  a f f i n i t y c o n s t a n t s v a r y between s p e c i e s , b u t t h e g e n e r a l nature  o f t h e enzyme a c t i v e s i t e s  similar.  I n a l l species, the g i l l  l a t e d b y e i t h e r Ca ential  are fundamentally  o r Mg  alone.  c a t i o n o f a c t i v a t i o n except  enzyme c a n b e s t i m u Ca  i sthe prefer-  i ncatfish.  The  2+ enzyme a f f i n i t y f o r C a  i s consistently higher  than  2+ t h a t f o r Mg a Ca A.  2 +  , i n d i c a t i n g t h a t t h e enzyme i s p r i m a r i l y  - a c t i v a t e d ATPase.  Salinity  Adaptation  Unlike the g i l l much h i g h e r l e v e l  Na -K +  +  of a c t i v i t y  than i n freshwater species  ATPase w h i c h i s found a t (2 - 5 X ) i n m a r i n e  teleosts  ( K a m i y a a n d U t i d a , 1969)»  t h e r e seems t o b e n o d i s t i n c t d i f f e r e n c e i n t h e g i l l 2+ Ca  -ATPase a c t i v i t y b e t w e e n m a r i n e a n d f r e s h w a t e r s p e c i e s .  T h i s o b s e r v a t i o n i s i n agreement w i t h t h e l a c k o f response in  gill  Ca  - A T P a s e a c t i v i t y when f r e s h w a t e r  rainbow  t r o u t a r e adapted t o seawater under l a b o r a t o r y c o n d i t i o n s .  H o w e v e r , i t i s o f i n t e r e s t t o n o t e t h a t t h e enzyme 2+ a p p e a r s t o have a h i g h e r Ca  a f f i n i t y i n freshwater  s p e c i e s t h a n i n marine t e l e o s t s .  Since the calcium  c o n c e n t r a t i o n o f f r e s h w a t e r i s much l o w e r t h a n t h a t 2+ o f s e a w a t e r , t h e e v o l u t i o n o f a h i g h e r enzyme C a a f f i n i t y i s i n a c c o r d a n c e w i t h a more  efficiently  2+ c o n t r o l l e d Ca water  -ATPase f u n c t i o n i n t h e g i l l s  of fresh-  teleosts. During adaptation t o sea water, a d i r e c t  a t i o n "between e n v i r o n m e n t a l s a l i n i t y a n d g i l l A T P a s e a c t i v i t y h a s b e e n o b s e r v e d i n many t e l e o s t s such as t h e European  Na -K +  a l . , 1967;  Japanese  eel, Anguilla  anguilla  e e l , A . . i a p o n i c a ( U t i d a e t a l . , 1971)» t h e kisutch  (Zaugg and M c L a i n ,  1970; 1971) a n d t h e r a i n b o w t r o u t , S a l m o  in  gill  a n d K i r s c h n e r , 1972). Na -K  ATPase a c t i v i t y  gairdneri  Such a d a p t a t i v e  higher l e v e l of g i l l  Na -K  increase  d i s a p p e a r s when t h e s e a  water - adapted animals a r e r e t u r n e d t o f r e s h The  (Epstein  B u t l e r a n d C a r m i c h a e l , 1972), t h e  coho salmon, Oncorhynchus  (Pfeiler  +  euryhaline  ( M o t a i s , 1970), t h e A m e r i c a n e e l , A. r o s t r a t a et  correl-  water.  ATPase a c t i v i t y  i n marine  a n d s e a w a t e r a d a p t e d t e l e o s t s i s b e l i e v e d t o be a s s o T  c i a t e d w i t h t h e i n c r e a s e d demand f o r a c t i v e sodium content  branchial  e x c r e t i o n i n sea water which i s v e r y high i n N a ( a b o u t 450 -500 mM).  I n fresh water,  lowering  +  116 of  gill  Na -K  ATPase a c t i v i t y i s i n r e s p o n s e t o t h e  m i n i m a l n e c e s s i t y f o r a c t i v e sodium  excretion t o occur  s i n c e t h e a n i m a l now e n c o u n t e r s t h e p r o b l e m o f c o n t i n u a l l o s s o f Na is  t o t h e e x t e r n a l water i n w h i c h t h e Na  v e r y l o w ( u s u a l l y l e s s t h a n 5 mM).  level  In contrast,  freshwater t e l e o s t s a r e capcable o f a c t i v e uptake o f N a from t h e environmental water. the  gills  The i n w a r d N a  +  pump i n  +  o f f r e s h w a t e r t e l e o s t i s thought t o be a s s o -  c i a t e d w i t h a second f u n c t i o n a l form o f Na -K +  +  ATPase  w h i c h i s a s p e c i f i c N a - a c t i v a t e d enzyme, i n d e p e n d e n t +  of K  +  a n d i n s e n s i t i v e t o a c t i n o m y c i n . D , a RNA s y n t h e s i s  inhibitor This N a  +  ( M o t a i s , 1970;  P f e i l e r a n d K i r s c h n e r , 1972).  a c t i v a t i o n i s p e c u l i a r t o freshwater animals  a n d seems t o d i s a p p e a r when t h e f i s h a r e t r a n s f e r r e d to  sea water. The c a l c i u m l e v e l i n s e a w a t e r i s a p p r o x i m a t e l y  150-200 t i m e s h i g h e r t h a n t h a t i n f r e s h w a t e r .  The  a n i m a l p r e s u m a b l y w o u l d e n c o u n t e r t h e same p r o b l e m i n 2+ Ca  + r e g u l a t i o n a s i n Na  r e g u l a t i o n when a d a p t i n g t o  a different environmental s a l i n i t y .  Assuming t h e r e i s  2+ a role f o rthe g i l l  Ca  -ATPase i n a c t i v e  branchial  2+ Ca  - u p t a k e w h i c h h a s now b e e n d e m o n s t r a t e d i n t h e Amer-  i c a n e e l (see Chapter I ) , t h e l a c k o f e f f e c t o f environm e n t a l s a l i n i t y on t h e g i l l  enzyme a c t i v i t y s u g g e s t s  that branchial Ca -uptake i sprobably unaffected. 2 +  The  117  mechanism b y w h i c h t h e a n i m a l h a n d l e s i t s e x t r a l e v e l i n s e a water remains t o be e l u c i d a t e d .  calcium  However,  2+ the  p r e s e n t d a t a on g i l l  obtained  Ca  by k i n e t i c studies  - membrane p r e p a r a t i o n s  -ATPase a c t i v i t y a r e on i s o l a t e d g i l l  i nwhich the vectorial  o f t h e enzyme c a n n o t b e d i f f e r e n t i a t e d . the  fact that i n f i s h g i l l  external salinity,  component  I n view o f  t h e ATPase - r e l a t e d H a  pump c a n f u n c t i o n i n o p p o s i t e the  plasma  +  d i r e c t i o n s depending on  i t i spossible that  during  2+ seawater - adaptation,  t h e d i r e c t i o n o f t h e g i l l Ca  pump i s s i m i l a r l y r e v e r s e d cation of the vectoral  v i a a biochemical  modifi-  c o m p o n e n t i n t h e enzyme. 2+  l a c k o f knowledge on t h e k i n e t i c s o f g i l l  Ca  p o r t under d i f f e r e n t environmental c o n d i t i o n s  Our  transdoes n o t  p e r m i t f u r t h e r s p e c u l a t i o n on t h e s u b j e c t . B. Growth and S e x u a l M a t u r a t i o n Growth and s e x u a l m a t u r a t i o n r e s u l t i n s i g n i 2+ f i c a n t e l e v a t i o n o f t h e s p e c i f i c Ca in  the g i l l s  -ATPase  activity  o f b o t h t h e r a i n b o w t r o u t and t h e coho 2+  salmon. . I n a d u l t maturing f i s h ,  a higher  plasma Ca  l e v e l i s normally  observed, which i s probably  w i t h an increased  calcium  associated  storage f o r t h e developing 2+ g o n a d s . A s s u m i n g t h a t Ca -ATPase i s i n d e e d p a r t o f 2+ the b r a n c h i a l Ca t r a n s p o r t machinery i n t h e e p i t h e l i a l  membrane, t h i s i n c r e a s e i n c a p a c i t y of the enzyme system may  r e p r e s e n t a b i o c h e m i c a l a d a p t a t i o n i n response t o  a g r e a t e r demand f o r c a l c i u m by the a d u l t , maturing a n i mals.  There appears t o be no  significant difference i n  2+ the b r a n c h i a l Ca  -ATPase a c t i v i t y between the a d u l t  f i s h and the spawning Hypercalcemia  animals. has been observed  i n many female  t e l e o s t s d u r i n g the b r e e d i n g season (Oguri and Takada, 1967 j Woodhead, 19681 The  Fontaine  et, a l . , I969;  U r i s t et  , 1972).  dramatic r i s e i n plasma c a l c i u m i s a t t r i b u t e d t o the  i n c r e a s e i n endogenous s e c r e t i o n of female sex hormone. 2+ Based on the h y p o t h e s i s t h a t Ca  -ATPase i s i n v o l v e d i n  2+ b r a n c h i a l Ca  -uptake i n the t e l e o s t , the l a c k of  a b l e response o f the enzyme t o spawning suggests c a l c i u m s o u r c e ( s ) other than environmental butes t o the estrogen-induced o f the spawning females.  observthat  water c o n t r i -  i n c r e a s e i n plasma c a l c i u m  I n b i r d s , i t i s e v i d e n t t h a t the  l o n g bones are i n v o l v e d i n the storage and m o b i l i z a t i o n of e x t r a calcium d u r i n g egg-production 1967).  (see S i m k i s s ,  Whether the bone i s s i m i l a r l y i n v o l v e d i n t e l e o s t s  i s s t i l l uncertain.  However, d u r i n g the  induced s e x u a l maturation  experimentally  of the European e e l p r o -  duced by c h r o n i c i n j e c t i o n of the carp  pituitary  e x t r a c t , Lopez and Bagot (1971) r e p o r t e d a s t r o n g s t i m u l a t i o n of o s t e o c l a s i s and o s t e o l y s i s .  At  the  same t i m e , t h e i n t e r c e l l u l a r s u b s t a n c e s h o w e d a g r e a t loss i nmineral  constituent. 2+  The  present  d a t a on t h e changes i n g i l l Ca  -  ATPase a c t i v i t y w i t h development i n d i c a t e i n d i r e c t l y the p o s s i b i l i t y that during sexual maturation t e l e o s t , t h e r i s e i n body c a l c i u m content  i sv i a an 2+  i n c r e a s e i n t h e c a p a c i t y o f t h e b r a n c h i a l Ca system. is  of the  -uptake  The e x t r a c a l c i u m i s s t o r e d i n t h e a n i m a l a n d  subsequently  production.  m o b i l i z e d d u r i n g spawning f o r egg-  H o w e v e r , u n t i l more d i r e c t  information 2+  i s a v a i l a b l e on t h e k i n e t i c s o f b r a n c h i a l Ca port during d i f f e r e n t developmental stages  trans-  i nthe l i f e  c y c l e o f t h e t e l e o s t , t h e above p r o p o s a l remains a s speculations. C. D i e t a n d S t a r v a t i o n I t i s o f s p e c i a l i n t e r e s t t o note that the s p e c i f i c 2+ Ca  -ATPase a c t i v i t y i n t h e g i l l s  o f common c a r p i s a p p r o -  x i m a t e l y 6X t h e a v e r a g e enzyme l e v e l f o u n d i n s e v e n teleost species.  other  This p a r t i c u l a r l y high l e v e l of bran-  2+ c h i a l Ca  - A T P a s e a c t i v i t y may b e r e l a t e d t o i t s h e r b i -  vorous h a b i t a t .  Under normal c o n d i t i o n s , t h e p r i n c i p a l  2+ Ca being  intake i n t e l e o s t s i s i n t h e form o f food which i s absorbed through l o c a l i z e d regions  during feeding  ( s e e L o v e , 1970).  of the gut  Since the l e v e l of  120 calcium that  i n plant materials  the  c a r p has  t o r e l y more on t h e  water f o r divalent cation As the  i s v e r y low,  a preliminary  t e s t of the  of gut  t h e i r d i e t (see  environmental  above  gill  Since t e l e o s t f i s h are  different sets  possible  supply.  e f f e c t o f s t a r v a t i o n on t h e  examined.  i t is  Ca  hypothesis, -ATPase  known t o  was  possess  d i g e s t i v e enzymes d e p e n d i n g  L o v e , 1970), t h e  possible  on  changes  that  2+ may  occur i n the  gut  m u c o s a Ca  were a l s o i n v e s t i g a t e d . ATPase a c t i v i t y level  i n the  -ATPase w i t h  I f t h e h i g h b r a n c h i a l Ca c a r p i s i n d e e d due  of d i v a l e n t cations  the  a normal feeding gill  Ca  low  available i n i t s diet,  one  f i s h may  in  the  i n d u c e an  -ATPase a c t i v i t y w i t h  m i t a n t depression i n the  gut  r e s u l t i n any  food source  elevation  a possible  enzyme l e v e l .  present study i n d i c a t e that  weeks d i d not  -  t o the  would expect t h a t complete removal of the in  starvation 2+  in  conco-  Data  obtained  starvation for six  o b s e r v a b l e changes i n  the  branchial Ca -ATPase a c t i v i t y  of the  rainbow t r o u t .  In the  gut  i n the  enzyme  by  - s t i m u l a t i o n was  2 +  the  Ca  m u c o s a , an  increase  accompanied by 2+ enzyme a f f i n i t y f o r Ca .  capacity  a decrease i n  While these f i n d i n g s appear to c o n t r a d i c t proposed hypothesis,  two  other p o s s i b i l i t i e s  the  should  be  considered.  F i r s t l y , the evolution of a high 2+  c i t y b r a n c h i a l Ca  capa-  -ATPase s y s t e m i s a l o n g - t e r m  adapt-  a t i v e e v e n t w h i c h c a n n o t be d u p l i c a t e d i n a s h o r t - t e r m experiment.  Secondly,  i ti s well-established that  during s t a r v a t i o n , the f i s h i s capable own m u s c l e i n o r d e r t o s u r v i v e .  of digesting i t s  P a r i e t a l muscle i n  t e l e o s t s c o n t a i n a r e l a t i v e l y h i g h c a l c i u m content as compared t o t h a t o f t h e t e t r a p o d s  (Ma, 197£)•  The  down o f p a r i e t a l m u s c l e f o r e n e r g y s u p p l y d u r i n g vation w i l l  break-  star-  continually release extra calcium into the  circulation.  T h i s may w e l l c o m p e n s a t e f o r t h e l o s s o f  dietary calcium intake.  As a r e s u l t , short-term  star-  v a t i o n i s n o t d i r e c t l y comparable t o a herbivorous  diet  i n which t h e e x t r a calcium supply from muscle i s absent. A d d i t i o n a l i n f o r m a t i o n on t h e g u t and g i l l herbivorous  f i s h i s required to verify  enzymes o f  the proposed  hypothesis. v_ - V  i n summary, t h e s e r i e s o f e x p e r i m e n t s  described  i n t h i s chapter has c l e a r l y demonstrated the general 2+ e x i s t e n c e o f a Ca  -ATPase i n t h e g i l l s  of teleosts.  The enzyme s y s t e m e x h i b i t s some d e g r e e o f v e r s a t i l i t y . U n l i k e t h e N a - K A T P a s e s y s t e m , h o w e v e r , many o f t h e 2+ +  observed  +  biochemical variations i n the g i l l  Ca  -ATPase  cannot be c o r r e l a t e d d i r e c t l y t o t h e changes i n t h e  122  environmental kinetics  conditions.  O u r l a c k o f k n o w l e d g e on t h e  of a c t i v e b r a n c h i a l calcium transport i n t e l e o s t  under environmental  i n f l u e n c e makes i t d i f f i c u l t  interpret the current data.  Until a better  o f t h e b r a n c h i a l c a l c i u m pump i s a c h e i v e d , significance  of these  the g i l l  - A T P a s e c a n n o t be f u l l y  Ca  to  understanding the f u n c t i o n a l  a d a p t a t i v e b i o c h e m i c a l changes i n appreciated.  123 GENERAL DISCUSSION  The objective of t h i s thesis i s to study the role of the g i l l i n teleostean calcium regulation, with s p e c i a l 2+  reference to the mechanisms of branchial Ca  transport  and the action of hormones. From the data presented, i t i s evident that an 2+  active branchial Ca  transport system i s present i n the  freshwater t e l e o s t , A n g u i l l a r o s t r a t a . which enables the 2+  f i s h to e f f i c i e n t l y take up Ca water.  from the environmental  Whether t h i s represents a primary or secondary  2+  Ca  pump coupled with the transport of other ions has not 2+  been c l e a r l y defined.  However, i d e n t i f i c a t i o n of a Ca -  ATPase i n the g i l l plasma membranes, with s p e c i f i c enzyme a c t i v i t y e a s i l y measured at the physiological  temperature 2+  of  the f i s h , strongly suggests that the branchial Ca  pump may be a primary one. The involvement of C a - ( M g ) ATPase i n C a 2+  2+  2 +  2+  uptake by the sarcoplasmic reticulum and i n Ca -extrusion by the red blood c e l l s i s well-demonstrated 1972;  Schatzmann, 1975).  (Martonosi,  A role f o r C a - ( M g ) ATPase 2+  has also been proposed i n the asymmetrical  2+  transcellular  2+  Ca  transport that occurs i n the i n t e s t i n a l mucosa  (Melancon and DeLuca, 1970), the renal tubules (Parkinson  124 and  R a d d e , 1971; R o r i v e a n d K l e i n z e l l e r , 1972) a n d t h e  placenta  ( S h a m i , 1974).  The e x i s t e n c e o f a membrane-  2+ b o u n d g i l l C a - A T P a s e , w i t h enzyme p r o p e r t i e s s i m i l a r 2+ 2+ t o t h o s e o f o t h e r Ca -(Mg ) A T P a s e s , i s p r e s u m p t i v e 2+ e v i d e n c e f o r t h e enzyme t o b e p a r t o f t h e b r a n c h i a l C a t r a n s p o r t system, s e r v i n g as a source for  2+ t h e a c t i v e Ca -uptake  of metablic  energy  process.  Calcium-uptake i n t h e g i l l  epithelium involves  2+ movement p f C a f r o m one c e l l surface.  surface t o another c e l l 2+ T h i s t r a n s c e l l u l a r Ca t r a n s p o r t may r e f l e c t 2+  the combination  o f a Ca  pump i n o n e p l a s m a membrane w i t h  2+ h i g h Ca permeability i n the opposite w i t h i n an e p i t h e l i a l c e l l .  The s i t e  p l a s m a membrane 2+  o f Ca  pump i n t h e  b r a n c h i a l epithelium i s n o t e l u c i d a t e d i n the present study.  By analogy with t h e r e dblood  cells  (Schatzmann, 2+  1975)» i t w o u l d seem l i k e l y t h a t t h e a c t i v e C a port occurs  trans-  f r o m c e l l t o i n t e r n a l medium.  2+ I n t h e f r e s h w a t e r American e e l , a c t i v e b r a n c h i a l Ca t r a n s p o r t is,,,under h o r m o n a l c o n t r o l . C a l c i t o n i n from t h e 2+ u l t i m o b r a n c h i a l glands enhances t h e Ca -uptake p r o c e s s . H o w e v e r , t h e hormone h a s n o e f f e c t o n t h e a c t i v i t y o f 2+ Ca  -ATPase i n g i l l  p l a s m a membrane p r e p a r a t i o n s .  In  mammals, one o f t h e p r o p o s e d m e c h a n i s m s o f a c t i o n o f c a l c i t o n i n on t a r g e t c e l l s i s b y a c t i v a t i o n o f t h e  125  membrane-bound adenyl cyclase, leading to an increased i n t r a c e l l u l a r l e v e l of cyclic-AMP which serves as a "second messenger" e l i c i t i n g the c h a r a c t e r i s t i c hormonal response (see Gray et aJL.,  1974).  I t i s possible that  c a l c i t o n i n acts on the teleostean g i l l e p i t h e l i a l c e l l via  a s i m i l a r mechanismj  hormonal regulation of branchial  Ca -uptake i s achieved by an ultimate e f f e c t on the 2+ 2+ membrane Ca pump or the membrane permeability to Ca . 2+  Extract of the Stannius corpuscles has a potent 2+ i n h i b i t o r y e f f e c t on both branchial Ca -uptake and g i l l 2+  Ca  -ATPase a c t i v i t y £n v i t r o .  Based on the present  data,  i t i s proposed that i n t e l e o s t s , the hypocalcemic e f f e c t of the corpuscles of Stannius observed i n vivo i s due, at 2+ least p a r t l y , t o i n h i b i t i o n of branchial Ca -uptake by 2+ a d i r e c t action of the hormone on the membrane Ca pump. Although the p h y s i o l o g i c a l s i g n i f i c a n c e of the corpuscles of Stannius i n teleostean calcium metabolism i s now well-established, the chemical nature of the glandular secretion remains a subject of controversy.  Bio-  chemical characterisation of the active hypocalcemic p r i n c i p l e ( s ) has not been possible due to the lack of a simple and s e n s i t i v e bioassay f o r the corpuscle "hormone". Pang and co-workers  (1973)  described a bioassay based on  the hypocalcemic response e l i c i t e d by administration of  /  126 the Stannius corpuscle homogenate to i n t a c t k i l l i f i s h . The bioassay consists of 4 d a i l y i n j e c t i o n s of the corpuscle homogenate into k i l l i f i s h pre-adapted f o r s i x weeks t o calcium-deficient sea water and a low-calcium diet.  The hypocalcemic response i s determined two hours  a f t e r the f i n a l i n j e c t i o n .  The routine application of  t h i s bioassay i s l i m i t e d because i t i s both time-consuming and l a c k i n g i n s e n s i t i v i t y f o r quantitative a n a l y s i s . As revealed i n the present study, the corpuscle extract exhibits a potent i n h i b i t i o n on the a c t i v i t y of the g i l l 2+  plasma membrane Ca  -ATPase. The e f f e c t i s s p e c i f i c to  corpuscles of Stannius (Table 3 ) and the response i s l o g dose r e l a t e d (Figure 21)• The i n v i t r o enzyme assay i s quick (one hour incubation) and the experimental parameters can be e a s i l y c o n t r o l l e d .  This r e l a t i v e l y simple  method provides a more e f f i c i e n t bioassay f o r e l u c i d a t i n g the biochemical nature of the yet u n i d e n t i f i e d corpuscle hypocalcemic "hormone'*. for  The assay i s sensitive enough  quantitative analysis e s s e n t i a l i n the p u r i f i c a t i o n  of the hormone. The work described i n t h i s thesis provides i n f o r mation towards a new understanding of calcium regulation i n the t e l e o s t .  In the freshwater American e e l , bran-  2+  c h i a l Ca  -uptake i s by an active process, possibly  m e d i a t e d v i a a membrane-bound C a ^ - A T P a s e .  The  calcium-active  Stannius  h o r m o n e s , c a l c i t o n i n and  c o r p u s c l e " f a c t o r " have an transport target  e f f e c t on  system, s u g g e s t i n g the  organ of hormonal a c t i o n  homeostasis.  the  this  gill  as  two  calcium an  important  i n teleostean  calcium  A d e t a i l e d i n v e s t i g a t i o n of the  kinetics  2+ of branchial  Ca  e n v i r o n m e n t a l and  transport  i n t e l e o s t s under v a r i o u s  physiological conditions  valuable i n further  e l u c i d a t i n g the  calcium regulation  in fish.  be  p u r i f i c a t i o n and  d i r e c t e d to the  of the  will  mechanisms  Future work should  understanding of the endocrine  tissue.  contribute  of also  characterization  h y p o c a l c e m i c "hormone" p r e s e n t i n t h e  of Stannius, which w i l l  prove  corpuscles  t o a more c o m p l e t e  physiological function  of  this  128  LITERATURE CITED  Alexander, E, (1968). manual. 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