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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 IN TELEOST FISH, WITH SPECIAL REFERENCE TO A CALCIUM-STIMULATED ATPase IN THE GILL PLASMA MEMBRANES by STEPHANIE W. Y. MA B.Sc. (Gen.), U n i v e r s i t y o f Hong Kong, 1970 M . S c , U n i v e r s i t y o f Hong Kong, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF PHYSIOLOGY FACULTY OF MEDICINE We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF^BRITISH COLUMBIA APRIL, 1976 0 S t e p h a n i e . Y . 1976 In present ing t h i s thes is in p a r t i a l fu l f i lment o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ib ra ry s h a l l make i t f r ee ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for s c h o l a r l y purposes may be granted by the Head of my Department or by his representa t ives . It is understood that copying or pub l i ca t ion o f th is thes is f o r f i n a n c i a l gain s h a l l not be allowed without my wri t ten permiss ion. Department of The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 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 a r e s t i l l p o o r l y u n d e r s t o o d , t h e r e i s 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 be i n v o l v e d . The g i l l , "being t h e r e s p i r a t o r y organ where b l o o d comes i n t o i n t i m a t e c o n t a c t w i t h t h e e x t e r n a l w a t e r , 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 . The o b j e c t i v e o f t h i s p r o j e c t was t o s t u d y the mechanisms o f c a l c i u m t r a n s -p o r t i n t e l e o s t f i s h , w i t h s p e c i a l r e f e r e n c e t o b r a n c h i a l 2+ c a l c i u m - u p t a k e , p r o p e r t i e s o f a g i l l Ca -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, c a l c i t o n i n 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 ) 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 . 2+ A method was d e v e l o p e d t o measure Ca t r a n s p o r t a c r o s s t h e i s o l a t e d and p e r f u s e d g i l l o f t h e A m e r i c a n e e l , A n g u i l l a r o s t r a t a . T h i s p r e p a r a t i o n d e m o n s t r a t e d a n e t 2+ Ca -uptake a c r o s s t h e g i l l e p i t h e l i u m a g a i n s t a c h e m i c a l and e l e c t r i c a l g r a d i e n t . T h i s uptake was i n h i b i t e d by 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 a c t i v e t r a n s p o r t . The b r a n -2 + c h i a l Ca t r a n s p o r t system was s e n s i t i v e t o changes 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 c o n -2+ c e n t r a t i o n s and pH. Ca -uptake was s t i m u l a t e d by salmon 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 o f S t a n n i u s . 2+ + + A Ca - 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 and 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 lasma membranes o f t h e American e e l . The enzyme was a l s o 2+ s t i m u l a t e d by Mg , b u t t h e V m o v and a f f i n i t y o f t h e enzyme were h i g h e r f o r C a 2 + t h a n f o r M g 2 + . C a 2 + and M g 2 + were f o u n d t o a c t on t h e same s i t e . ATP was 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. The pH optimum f o r enzyme a c t i -v a t i o n was 7.9-8.1. Salmon c a l c i t o n i n had no e f f e c t on 2+ t h e Ca -ATPase a c t i v i t y , however, t h e enzyme a c t i v i t y was m a r k e d l y i n h i b i t e d by e x t r a c t s o f t h e S t a n n i u s c o r p u s c l e s . 2+ The e x i s t e n c e o f a s i m i l a r Ca -ATPase was demon-s t r a t e d i n t h e g i l l plasma membranes o f a v a r i e t y o f t e l e o s t f i s h f r o m b o t h f r e s h w a t e r and ma r i n e h a b i t a t s . 2+ The g i l l Ca -ATPase a c t i v i t y was n o t a l t e r e d by s e a w a t e r a d a p t a t i o n 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 e l e v a t e d d u r i n g s e x u a l m a t u r a t i o n and spawning. T h i s 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 u p t a k e s y s t e m 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 Ca -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 S t a n n i u s " f a c t o r " 2+ were dem o n s t r a t e d 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 Ca t r a n s p o r t s y s t e m , s u g g e s t i n g t h e g i l l as an i m p o r t a n t s i t e o f hormonal a c t i o n i n t e l e o s t e a n c a l c i u m h o m e o s t a s i s . i v ACKNOWLEDGMENTS I w o u l d l i k e t o thank my r e s e a r c h s u p e r v i s o r , Dr. D. H. Copp, f o r h i s c o n t i n u o u s encouragement and s u p p o r t t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y , e s p e c i a l l y d u r i n g t h e p r e p a r a t i o n o f t h i s t h e s i s . I wo u l d a l s o l i k e t o thank Mrs . E l s p e t h W i l k i n s o n f o r h e r e x c e l l e n t t e c h n i c a l a s s i s t a n c e and g e n e r a l c a r i n g o f t h e f i s h l a b o r a t o r y . I am most g r a t e f u l t o Dr. H. H. Messer and Dr. Y. Shami 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 s u b j e c t and t h e u s e f u l a d v i c e s t h e y o f t e n gave. Members o f t h e r e s e a r c h team who had so w i l l i n g l y h e l p e d when-e v e r n e c e s s a r y a r e g r e a t l y a p p r e c i a t e d . I am p a r t i c u l a r l y i n d e b t e d t o Dr. D. G. B u t l e r , Department o f Z o o l o g y , U n i v e r s i t y o f T o r o n t o , f o r h i s k i n d n e s s i n a r r a n g i n g f o r t h e s u p p l y o f t h e c h i e f e x p e r i -m e n t a l a n i m a l , A n g u i l l a r o s t r a t a ; t o the F i s h e r i e s R e s e a r c h S t a t i o n , West Vancouver, and t h e Vancouver P u b l i c Aquarium f o r t h e i r g e n e r o s i t y i n p r o v i d i n g some o f t h e e x p e r i -m e n t a l f i s h . S p e c i a l t h a n k s a r e due t o Dr. W.K. O v a l l e , J r . , Department o f Anatomy, U n i v e r s i t y o f B r i t i s h C o l u m b i a , f o r t h e E l e c t r o n M i c r o s c o p i c work. My g r a t i t u d e i s a l s o e x t e n d e d t o Mr. K. Henze f o r p r e p a r i n g t h e t h e s i s i l l u s t r -a t i o n s . V TABLE OF CONTENTS PAGE GENERAL INTRODUCTION 1 DIVISION I CHAPTER I - CALCIUM TRANSPORT ACROSS THE ISOLATED GILL OF THE AMERICAN EEL, ANGUILLA ROSTRATA 11 INTRODUCTION 11 MATERIALS AND METHODS 15 RESULTS 31 DISCUSSION 44 DIVISION I I CHAPTER I I - CHARACTERIZATION OF CALCIUM-STIMULATED ATPase I N THE GILL PLASMA MEMBRANES OF THE AMERICAN EEL, ANGUILLA ROSTRATA .. 5^ INTRODUCTION 5^ MATERIALS AND METHODS 56 RESULTS 63 DISCUSSION 85 CHAPTER I I I - EFFECTS OF SALINITY ADAPTATION, SEXUAL MATURATION AND DIET ON GILL AND GUT MUCOSA PLASMA MEMBRANE CALCIUM-STIMU-LATED ATPase OF TELEOSTS 93 INTRODUCTION 93 v i PAGE MATERIALS AND METHODS 96 RESULTS 101 DISCUSSION 114 GENERAL DISCUSSION 123 LITERATURE CITED 12§ v i i LIST OF TABLES TABLE PAGE 1 Electrolyte composition, osmolarity and pH of eel plasma and perfusion f l u i d used in the isolated g i l l study . 20 2 Specific activities of marker enzymes i n different fractions 66 3 Specific effect of the corpuscles of Stan-nius on g i l l Ca 2 +-(Mg 2 +) ATPase 84 2+ 4 Ca -ATPase in g i l l plasma membrane of marine and freshwater teleosts 102 5 Electrolyte composition of the dechlorinated tap water and a r t i f i c i a l sea water i n the Salinity Adaptation Experiment 105 2+ 6 Effect of seawater adaptation on Ca -ATPase in g i l l plasma membranes of rainbow trout, S. gairdneri 106 7 Effect of sexual maturation and spawning on 2+ Ca -ATPase i n g i l l plasma membrane of rain-bow trout and migrating coho salmon ........ 107 2+ 8 Effect of starvation on Ca -ATPase i n g i l l plasma membrane of rainbow trout, £>.. gaird- neri I l l 2+ 9 Effect of starvation on Ca -ATPase i n gut plasma membrane of rainbow trout, S. gaird-neri 112 • • • V i l l LIST OF FIGURES FIGURE PAGE 1 Movement of -'Ca across the 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 32 2 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 perfused g i l l 3^  2+ 3, E f f e c t of external Ca concentration on 2+ Ca -uptake i n the i s o l a t e d and perfused g i l l 36 2+ k E f f e c t of i n t e r n a l Ca concentration on 2+ Ca -uptake i n the i s o l a t e d and perfused g i l l 37 2+ 5 E f f e c t of perfusate pH on Ca -uptake i n the i s o l a t e d and perfused g i l l 39 2+ 6 E f f e c t of incubation temperature on Ca -uptake i n the i s o l a t e d and perfused g i l l 40 2+ 7 E f f e c t of calcitonin- pn Ca -uptake i n the i s o l a t e d and perfused g i l l of the American e e l , A. ro s t r a t a 41 8 E f f e c t of corpuscles of Stannius extract 2+ on Ca -uptake i n the i s o l a t e d and per-fused g i l l of the American e e l , A., r o s t -r a t a 43 9 I s o l a t i o n of g i l l plasma membranes 58 10 A c t i v a t i o n of ATP hydrolysis by increasing concentrations of divalent cations 68 ix FIGURE PAGE 11 Lineweaver - Burk plots of ATPase activity 2+ 2+ at various concentrations of Ca , Mg and ATP 69 12 Effect of protein concentration on Pi release 71 13 Effect of Na + on enzyme activation by d i -valent cations 72 14 Enzyme activity with different nucleotides as substrate 73 15 Effect of pH on Ca2+-ATPase activity .... 75 16 Effect of inhibitors on Ca2+-ATPase a c t i -v i t y 76 2+ 17 Effect of incubation temperature on Ca -ATPase activity 78 18 Effect of storage at +4° C on Ca2+-ATPase act i v i t y 79 19 Effect of calcitonin on g i l l plasma mem-brane Ca 2 +-(Mg 2 +) ATPase 80 20 Effect of extract of the corpuscles of 2+ Stannius on g i l l plasma membrane Ca -(Mg 2 +) ATPase 82 21 Inhibitory effect of extract of the cor-2+ puscles of Stannius on g i l l Ca -ATPase activity 83 X FIGURE PAGE 2+ 22 G i l l plasma membrane Ca -ATPase of marine and freshwater teleosts 103 23 Effect 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 x i LIST OF PLATES PLATE PAGE 1 Bleaching and cannulation of the f i r s t g i l l arch of the American eel, A. rpstrata 23 2 General apparatus for isolated g i l l per-fusion study i n the American eel, A. rostrata 24 3 A schematic diagram i l l u s t r a t i n g the technique of g i l l transepithelial e l e c t r i -cal potential measurement 27a 4 The two encapsulated corpuscles of Stan-nius on the ventral surface of the kidneys in the American eel, A. rostrata 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 cellular functions such as excitation-secretion coupling at nerve endings (Eccles, 1964), excitation-contraction coupling in muscle (Bianchi, 1969)» maintenance of mem-brane integrity (Poste and Allison, 1973)» regulation of enzyme activity (Bianchi, 1968) and control of hormone secretion (Copp, 1970; Care et a l , , 1975). Calcium concentration i n the external environment can be very fluctuating, but vertebrates i n general are able to main-tain a constant plasma calcium level. Calcium regulation within the animal i s an important physiological function. In mammals, regulation of calcium metabolism i s mediated principally by the action of three hormonesi calcitonin, parathormone and 1, 25- dihydroxycholecalci-f e r o l (see Copp, 1972| 1975). Parathormone i s produced by the parathyroid glands which arise embryonically from the 3 and 4 branchial pouches. It i s a hypercalcemia hormone, having an important role i n combating hypocal-cemia (Talmage ejfc a l . , 1953* 1955). Calcitonin i s secreted by the parafollicular cells or 'C* cells (Foster ejt a l . . 1964* Bussolati and Pearse, 1967* Kalina et a l . , 1970) which, in most mammals, are structurally associated with 2 the thyroid tissue. More recently, i t has been shown that the calcitonin-secreting cells are of ultimobran-chial origin, derived from the neural crest (Pearse and Carvalheira, 19^7 * ^ e; Dourain and Le Lievre, 1972). Functionally, calcitonin i s a hypocalcemic hormone involved in controlling hypercalcemia (Copp, 1967* Copper ejt a l . , 1970* Hirsch et a i . f 1975). The steroid hormone, 1, 25 - dihydroxycholecalciferol i s the biolo-g i c a l l y active metabolite of vitamin D, produced in the kidney (DeLuca, 1972). It plays a significant role in intestinal absorption of calcium. It 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 to calcium homeostasis. Parathyroidectomy results in hypo-calcemia and treatment with exogenous parathormone induces hypercalcemia i n amphibians (see Cortelyou and McWhinnie, 1967)* reptiles (see Clark, 1967) and birds (see Urist, 1967). Since the recent finding of the ultimobranchial gland as the source of calcitonin i n a l l classes of jawed vertebrates (see Copp, 1972), there i s growing evidence that this endocrine tissue i s involved in calcium meta-bolism in non-mammalian species. In amphibiansfthe 3 u l t i m o b r a n c h i a l g l a n d s a ppear t o be i m p o r t a n t i n p r e -v e n t i n g e x c e s s i v e m o b i l i z a t i o n o f c a l c i u m f rom t h e p a r a -v 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 a g a i n s t h y p e r c a l c e m i a d u r i n g p e r i o d s o f h i g h d i e t a r y c a l c i u m i n t a k e (see R o b e r t s o n , 1 9 7 1 ) . I n b i r d s , h y p e r t r o p h y and h y p e r p l a s i a o f t h e u l t i m o b r a n c h i a l b o d i e s o c c u r i n t i m e s o f c a l c i u m s t r e s s , such as i n l a y i n g hens ( U r i s t , 1 9 6 7 ) 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 (Copp e t a l . , 1968; C i p e r a e t a l . , 1 9 7 0 ) . The a c t i o n o f t h e s t e r o i d hormone, 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 has a l s o been d e m o n s t r a t e d . A d m i n i s t r a t i o n o f v i t a m i n 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 t h e k i d n e y , 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 mucosa, s t i m u l a t i n g t h e 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, 1 9 7 5 ) . A l t h o u g h f i s h c o m p r i s e t h e l a r g e s t and most d i v e r s e group 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 known o f t h e e n d o c r i n e c o n t r o l o f t e l e o s t e a n c a l c i u m m e t a b o l i s m . F i s h l a c k f u n c t i o n a l p a r a t h y r o i d g l a n d s . A p a r t f rom t h e 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 have been shown t o be r i c h i n c a l c i t o n i n (Copp and P a r k e s , 1968; Pang e t a l . , 1971; Orimo e t a l . , 1972; Copp e t a l . , 1972), bony f i s h ( 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 unique hormonal system, t h e c o r p u s c l e s o f S t a n n i u s (see B e r n , 1967) w h i c h a r e shown t o be 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; Chan, 1969; Pang, 1973). I n a d d i t i o n , t h e h y p o p h y s i s a l s o appears t o a f f e c t c a l -cium m e t a b o l i s m i n t e l e o s t s . Pang and co-workers (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 h y p o c a l c e m i a i n k i l l i f i s h (Fundulus heteroclitu3) 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 d e f i c i e n t i n c a l c i u m . A d m i n i s t r -a t i o n o f p i t u i t a r y homogenate t o hy p o h y s e c t o m i z e d f i s h r e s t o r e d t h e plasma c a l c i u m t o n o r m a l l e v e l (Pang, 1973). B a s e d on t h e s e o b s e r v a t i o n s , Pang (1973) p r o p o s e d t h a t t h e p i t u i t a r y g l a n d has a p h y s i o l o g i c a l l y h y p e r c a l c e m i a f u n c t i o n i n t e l e o s t s . 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 organ 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 s y s t e m s , i t s s p e c i f i c r o l e i n c a l c i u m h o m e o s t a s i s r e m a i n s t o be d e f i n e d . A l t h o u g h v i t a m i n D i s p r e s e n t i n f i s h l i v e r i n l a r g e q u a n t i t i e s , i t s r o l e i n 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 has n o t been s t u d i e d . Whether t h e 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, 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 e x i s t s i n f i s h i s unknown. S i n c e t h e d i s c o v e r y o f u l t i m o b r a n c h i a l g l a n d s as a r i c h s o u r c e o f c a l c i t o n i n (Copp and P a r k e s , 1968), p u r i f i e d f i s h c a l c i t o n i n has been p r e p a r e d from salmon (0*Dor e t aJL., 1969a, b) and 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 and co-workers (1965)» t h e b i o l o g i c a l p o t e n c y o f b o t h salmon (5»000 MRCU/mg) and e e l (4,000 MRCU/mg) c a l c i t o n i n i s f o u n d t o be much 5 higher than that of porcine (200 MRC U/mg) and human (120 MRC U/mg). In the Japanese e e l (Anguilla .iauonica), removal of the ultimobranchial glands did not a l t e r the plasma calcium l e v e l , but muscle calcium was markedly reduced (Ma, 1973). In "the eels A. a n g u i l l a and A. .iaponica. hypercalcemia induced by the removal of the corpuscles of Stannius was accompanied by ultimobranchial hypertrophy and an increase i n i t s c e l l u l a r secretory a c t i v i t y (Lopez et a l . 1968j Chan, 1972). In the migrating salmon, the highest l e v e l of plasma c a l c i t o n i n was found i a the female at time of spawning when plasma calcium was elevated (Watts, 1973). While i t appears that the ultimobranchial glands are intimately involved i n f i s h calcium metabolism, response of te l e o s t species to c a l c i t o n i n has been incon-s i s t e n t . C a l c i t o n i n administration had no e f f e c t on the plasma calcium l e v e l of Fundulus h e t e r o c l i t u s . Oncorhynchus  kisu t c h (Pang, 1971a) and Salmo gairdneri (Watts, 1973). In contrast, a hypocalcemic response to c a l c i t o n i n was observed i n three freshwater-adapted An g u i l l a species (Chan et a l . , 1968; Pang, 1971a; Lopez and D e v i l l e , 1972; Ma, 1973). The corpuscles of Stannius arise i n the embryo from the pro- and meso-nephric ducts (Ford, 1959; de Smet, 1962). 6 Histologically and cytologically, 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 role of the corpuscles in the biosynthesis of adrenocortico-steroids in the teleost f i s h , increasing evidence now supports the view that this endocrine tissue i s non-steroidogenic (see Chester Jones et a l . . 1969; Colombo et a l . , 1971). Electron micro-scopic studies indicate that the secretion of the corpu-scles i s proteinaceous in nature (Ogawa, 1967; Tomasulo, 1968;, Cohen et a l . , 1975). The hypocalcemic role of the corpuscles of Stannius in teleostean calcium homeostasis i s well-established. Removal of the corpuscles resulted in a marked hyper-calcemia in a variety of teleosts (see Pang, 1971b). In the Japanese eel, a profound increase in muscle total calcium was also recorded (Chan and Ma, 1972; Ma, 1973). Administration of the corpuscle homogenate to "Stanni-ectomized" eels restored the disturbed plasma calcium to normal level (Fontaine,1964; Chan et a l . 1969; Lopez, 1970). Corpuscle extract was reported to e l i c i t a hypo-calcemic response in intact seawater-adapted k i l l i f i s h , but was ineffective in k i l l i f i s h adapted to fresh water (Pang, 7 1971b). Based on t h e o b s e r v a t i o n t h a t t h e c o r p u s c l e s were 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 , Pang (1973) p r o p o s e d t h a t t h e main p h y s i o l o g i c a l f u n c t i o n 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 i s t o m a i n t a i n serum c a l c i u m l e v e l i n h i g h c a l c i u m e n v i r o n m e n t s . However, t h e s i t e ( s ) and mech-a n i s m ( s ) o f a c t i o n o f t h i s hormonal system a r e n o t w e l l u n d e r s t o o d . 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 h e t e l e o s t may be q u i t e d i f f e r e n t f rom t h o s e i n t e r r e s t r i a l v e r t e b r a t e s , t h e r e a r e two p o i n t s o f s p e c i a l i n t e r e s t . F i r s t l y , 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 a c t i v e p a r t i n c a l c i u m h o m e o s t a s i s . I t i s a s i t e o f a c t i o n o f parathormone, c a l c i t o n i n and 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 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 p r e s e n t i n t h e a d u l t f i s h i s e i t h e r a c e l l -u l a r o r c e l l u l a r . Calcium - 4 5 s t u d i e s show t h a t o n l y t h e o utermost t h i n l a y e r o f t e l e o s t e a n bone i s exchangeable (see Simmons, 1971). S e c o n d l y , t h e i n t i m a t e r e l a t i o n s h i p o f t h e f i s h t o i t s a q u a t i c environment w i t h v a r i a b l e c a l -c i u m c o n c e n t r a t i o n p r e s e n t s a pro b l e m o f c a l c i u m r e g u l a t i o n d i f f e r e n t f r o m t h a t o f t h e t e r r e s t r i a l v e r t e b r a t e s . T e l e o s t a r e known t o have a w e l l - d e v e l o p e d c a p a -c i t y t o w i t h d r a w c a l c i u m f r o m t h e environment (see Simmons, 1971). 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 t o " S t a n n i e c t o r a y " i s dependent upon an e n v i r o n m e n t a l c a l c i u m c h a l l e n g e (Chan e t a l . , 1967; Pang, 1973; Pang e t a l . . 1973; Fenwick, 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 e n v i r o n m e n t a l c a l c i u m i n t e l e o s t e a n c a l c i u m h o m e o s t a s i s . I n t e l e o s t f i s h , t h r e e s i t e s a r e c o n s i d e r e d c a p a b l e 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 e n v i r o n m e n t s , namely t h e g i l l , t h e s k i n and the g u t . The s k i n has o n l y 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 c o n -s i d e r e d t o be c o n t i n u o u s l y impermeable t o b o t h w a t e r and i o n s . The gut p l a y s a minor p a r t due t o t h e f a c t t h a t f r e s h w a t e r f i s h do n o t d r i n k a p p r e c i a b l e q u a n t i t i e s o f w a t e r (Maetz and Skadhauge, 1968). The g i l l , b e i n g t h e r e s p i r a t o r y organ where b l o o d comes i n t o i n t i m a t e c o n t a c t 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 major s i t e f o r i o n t r a n s p o r t . The t e l e o s t e a n g i l l i s known t o have a major r o l e i n o s m o r e g u l a t i o n . The mechanisms u n d e r l y i n g b r a n c h i a l N a + 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 . How-e v e r , v e r y l i t t l e i s known c o n c e r n i n g t h e f u n c t i o n a l s i g n i -f i c a n c e o f t h e t e l e o s t e a n g i l l i n c a l c i u m h o m e o s t a s i s . 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 dat e has been d e r i v e d i n d i r e c t l y from 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 c a l c i u m f l u x e s i n i n t a c t a n i m a l s ( F o n t a i n e e t a l . , 1972; F l e m i n g e t a l . , 1973; Dacke, 1975)• A d i r e c t s t u d y on b r a n c h i a l 9 i n v o l v e m e n t 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 r e p o r t e d . The aim o f t h e p r e s e n t t h e s i s was t o i n v e s t i g a t e t h e r o l e o f t h e g i l l i n c a l c i u m h o m e o s t a s i s i n f r e s h -w a t e r t e l e o s t s , w i t h s p e c i a l r e f e r e n c e 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 f r om 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 the c o r p u s c l e s o f S t a n n i u s . I n t h e f i r s t s e r i e s o f e x p e r i m e n t s , c a l c i u m t r a n s -p o r t a c r o s s t h e i s o l a t e d and p e r f u s e d g i l l o f a t e l e o s t was s t u d i e d . The second s e r i e s o f e x p e r i m e n t s was c o n -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 ATPase system in t h e t e l e o s t e a n g i l l p l a s m a membranes. B i o c h e m i c a l a d a p t a t i o n o f t h e enzyme t o environmental and p h y s i o l o g i c a l changes was a l s o i n v e s t i -g a t e d . Based 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 , a mechanism o f c a l c i u m i o n uptake a c r o s s t h e g i l l e p i t h e l i u m was p o s t u l a t e d and 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 c a l c i u m t r a n s p o r t was d i s c u s s e d . DIVISION I CALCIUM TRANSPORT IN THE TELEOST GILL CHAPTER I CALCIUM TRANSPORT ACROSS THE ISOLATED GILL OF THE AMERICAN EEL, ANGUILLA ROSTRATA INTRODUCTION C a l c i u m r e q u i r e m e n t s 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 t h e i o n s f r o m t h e i r a q u a t i c e n v i r o n m e n t s , e i t h e r b y d i r e c t a b s o r p t i o n from w a t e r o r v i a d i e t a r y i n t a k e . The a b i l i t y o f t e l e o s t f i s h t o accumulate COn-L.1: s i d e r a b l e q u a n t i t i e s o f ^Ca from t h e s u r r o u n d i n g w a t e r was f i r s t d e m o n s t r a t e d b y Asano and co-workers (1957) and R o s e n t h a l (1957; I 9 6 0 ) . I n r e c e n t y e a r s , r e s e a r c h 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 t e l e o s t s . 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 r e m o v a l 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 r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n n e t c a l c i u m i n f l u x i n t h e Eu r o p e a n e e l . They p r o p o s e d t h a t t h e g i l l was l i k e l y 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 male k i l l i -f i s h e x h i b i t e d s e a s o n a l v a r i a t i o n s i n t h e r a t e o f enviroraental calcium uptake. Calcitonin injection stimu-l i ; lated calcium uptake and altered the -'Ca distribution between ca l c i f i e d and soft tissue compartments. In the European eel, calcitonin administration caused a marked reduction i n total calcium efflux (Dacke, 1975)• In both reports, the possible involvement of the branchial appar-atus was suggested. In a review on. Na + and Cl"* transport mechanisms i n the teleostean g i l l and amphibian skin, Motais and.Garcia-Romeu (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 in f i s h has been conducted mainly i n vivo". In teleost, the common experimental approach to calcium uptake study had been to expose the intact f i s h to -'Ca-labelled water i n an aquarium for various time periods and then either follow the appearance grid distribution of ^ C a £h the animal (Asano et a l . . 1956* Borough e£ a l . , 1956* Rosenthal, 1957* I960* Fleming et a l . . 1973) or measure the disappearance of ^ C a from the medium (Fontaine e£ a l . . 1972). In the determination of calcium efflux, the animal was f i r s t loaded with -'Ca by intaaperitoneal injection 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 overall uptake and e l i m i n a t i o n o f ^ C a by t h e f i s h , t h e y do n o t p e r m i t t h e s e p a r a t i o n o f g i l l f u n c t i o n from o t h e r p o s s i b l e a r e a s o f exchange. V a r i o u s o t h e r methods have been d e v e l o p e d i n an 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 t h e g i l l s i n c a l c i u m r e g u l a t i o n . They i n c l u d e p e r f u s i o n o f t h e a n t e r i o r r e g i o n v i a t h e v e n t r a l a o r t a ( S c h i f f m a n , 1961) and p e r f u s i o n o f s p e c i f i c r e g i o n s o f an i n t a c t f i s h i s o l a t e d b y t i g h t l y f i t t i n g impermeable s a c s (Mashiko and J o z u k a , 196I; 1964). However, a l l t h e s e i n v i v o p r e p a r a t i o n s t e n d t o s u f f e r from h a n d l i n g and cannu-l a t i o n e f f e c t s (Wood and R a n d a l l , 1971). p o s s i b l e i n t e r -v e n t i o n o f v a r i o u s f e e d b a c k mechanisms, l a c k o f f l e x i -b i l i t y i n t h e t y p e o f e x p e r i m e n t s w h i c h can be c a r r i e d out and t h e u n c e r t a i n t y ; w i t h w h i c h e x p e r i m e n t a l o b s e r -v a t i o n s can be 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 t h e b r a n c h i a l e p i t h e l i u m . R e c e n t l y , S h u t t l e w o r t h (1972) d e s c r i b e d a method + + • — f o r m e a s u r i n g Na , K , and C l f l u x e s a c r o s s an i s o l a t e d and p e r f u s e d g i l l . D a t a o b t a i n e d from s t u d i e s u s i n g t h i s t e c h n i q u e a r e 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 t h e a c c e p t e d r o l e o f t h e g i l l as deduced from f l u x s t u d i e s i n i n t a c t a n i m a l s . Hence, t h i s appears t o be a s u i t a b l e method f o r t h e s t u d y o f 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 ^ C a i n f l u x i n the "Stanniectomized" American e e l . They reported that , ,Stanniectomy , , resulted i n an increase i n g i l l calcium i n f l u x . However, there i s no available information on the nature of the branchial transport system and the possible mechanisms involved i n i t s regulation. This chapter describes a method f o r the study of calcium movements across an i s o l a t e d and perfused g i l l . Using t h i s technique, properties of the bran-c h i a l transport system of a freshwater t e l e o s t were investigated. The e f f e c t of two calcium-active hormones, c a l c i t o n i n from the ultimobranchial glands and extract of the corpuscles of Stannius on g i l l calcium uptake was also studied. 15 MATERIALS AND METHODS A. E x p e r i m e n t a l A n i m a l s 1. C h o i c e o f e x p e r i m e n t a l a n i m a l The e x p e r i m e n t a l a n i m a l chosen f o r t h e p r e s e n t s t u d y i s t h e s i l v e r A m e r i c a n 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 f o u n d a l o n g t h e A t l a n t i c c o a s t o f A m e r i c a , e x t e n d i n g f r o m t h e S t . Law-r e n c e R i v e r t o B r a z i l (see Ray and C i a m p i , 1956). The spawning season o f t h i s f i s h i s around l a t e September t o e a r l y November each y e a r . The e x a c t b l e e d i n g ground 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 i n t h e mid A t l a n t i c n e a r t h e S a r g a s s o Sea. When h a t c h e d , t h e l a r v a 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 where t h e y become t r a n s f o r m e d i n t o t i n y c y l i n d r i c a l e l v e r s w i t h i n a y e a r . The young e e l s may re m a i n i n t h e s h a l l o w c o a s t a l b r a c k i s h w a t e r s o r more o f t e n , t h e y m i g r a t e up r i v e r s where t h e y s t a y i n f r e s h w a t e r f o r s e v e r a l y e a r s . 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 and grow t o a c o n s i d e r a b l e s i z e . The f u l l y grown 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 as t h e y m i g r a t e downstream towards t h e s e a where t h e y t r a v e l t h e G u l f Stream t o t h e spawning grounds. 16 The 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-p e r i m e n t a l a n i m a l a r e t w o f o l d . F i r s t l y , t h e s i l v e r e e l s o b t a i n e d a r e i n a f a i r l y u n i f o r m and s t a b l e s t a t e o f c a l -c i u m m e t a b o l i s m . The a n i m a l s a r e n o t y e t f u l l y mature s e x u a l l y and t h e y a r e i n a f a s t i n g c o n d i t i o n . Hence, one w o u l d e x p e c t l e s s c o m p l i c a t i o n due t o v a r i a b l e d i e t a r y c a l -c i um i n t a k e and s e x u d i f f e r e n c e . The e x t r a c a l c i u m r e l e a s e d i n t o c i r c u l a t i o n d u r i n g d i g e s t i o n o f 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 t h e d a i l y l o s s o f c a l c i u m i n t h e u r i n e . S e c o n d l y , t h e genus A n g u i l l a has been e x t e n -s i v e l y used i n t h e s t u d i e s o f hormonal c o n t r o l o f c a l c i u m m e t a b o l i s m i n t e l e o s t s i n v i v o . Ample i n f o r m a t i o n i s a v a i l a b l e i n t h e l i t e r a t u r e on t h e p o s s i b l e mechanisms o f c a l c i u m r e g u l a t i o n i n t h e A n g u i l l a s p e c i e s . 2. K e e p i n g o f A n i m a l s S i l v e r A m e r i c a n e e l s o f body w e i g h t r a n g i n g f rom 800 -1,200 g were caught a l o n g th e S t . Lawrence R i v e r i n t h e P r o v i n c e s o f O n t a r i o and Quebec. They were s h i p p e d by a i r t o Vancouver and b r o u g h t back t o t h e l a b o r a t o r y unan-e s t h e t i z e d . I n t h e l a b o r a t o r y , t h e y were k e p t i n s e l f -c l e a n i n g , w e l l - a e r a t e d f i b r e g l a s s t a n k s o f r u n n i n g d e c h l -o r i n a t e d f r e s h w a t e r a t a t e m p e r a t u r e o f 1 2 + 2 ° C . To ensure complete r e c o v e r y o f t h e a n i m a l s from t h e trauma o f 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 u s e . O n l y h e a l t h y a n i m a l s f r e e f rom wounds and f u n g a l i n -f e c t i o n were chosen f o r e x p e r i m e n t s . B. Plasma E l e c t r o l y t e D e t e r m i n a t i o n I n 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 t o 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 , t h e b l o o d pH, p lasma o s m o l a r i t y and e l e c t r o l y t e c o m p o s i t i o n o f t h e A m e r i c a n e e l were d e t e r m i n e d . 1. B l o o d S a m p l i n g P r o c e d u r e B l o o d samples were o b t a i n e d from an u n a n e s t h e t i z e d f i s h u s i n g t h e c a u d a l v e i n s a m p l i n g t e c h n i q u e . The e e l was l a i d f l a t 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 and a h e p a r i n i z e d p l a s t i c 2 ml s y r i n g e w i t h a 21-G 1-1/2 i n c h n e e d l e was i n s e r t e d t h r o u g h t h e s k i n o f t h e mid-v e n t r a l a s p e c t o f t h e c a u d a l p e d u n c l e . The n e e d l e was d i r e c t e d f o r w a r d between t h e haemal s p i n e s i n t o t h e haemal c a n a l . U s i n g t h i s t e c h n i q u e , b l o o d was w i t h drawn i n t o t h e s y r i n g e f r o m t h e c a u d a l c i r c u l a t i o n w i t h l i t t l e i n j u r y t o t h e f i s h . F o r b l o o d pH measurement, b l o o d samples were c o l l e c t e d and c e n t r i f u g e d under o i l t o ensure a n a e r o b i c c o n d i t i o n s . 18 2. Chemical Analysis a. Blood pH was measured under anaerobic conditions, using a combination pH electrode (Sargent -Welch Scien-t i f i c Co., W. Germany) with a Research D i g i t a l pH/mV Meter (Orion, model 801). b. Plasma osmolarity was determined, using an Osmette Pr e c i s i o n Osmometer (Model 2007» Precision Systems, Massachusetts, U.S.A.). c. Plasma calcium and magnesium concentrations were determined by atomic absorption spectrophotometry ( J a r r e l l -Ash, Model 280 Atomsorb). To minimize inter-elementary interference i n the cation determination, 0.5 # lanthanum chloride was used as the diluent f o r both samples and standards. The readings were recorded on a s t r i p chart recorder (Sargent Recorderj Model SR). d. Sodium and potassium i n the plasma were analysed by flame photometry (Instrumentation Laboratory Inc., Model 143). A standard l i t h i u m s o l u t i o n (15 mEq. L i per l i t r e ) was used as d i l u e n t . e. Plasma chloride concentration was determined by automatic e l e c t r o - t i t r a t i o n , using a Buchler - Cotlove Ckloridometer (Buchler Instruments, Model 4-2008, Nuclear Chicago Corp.) A standard s o l u t i o n of sodium chloride was used f o r c a l i b r a t i o n . f . Plasma inorganic phosphorus was measured using a modification of the Technicon Auto-Analyzer N-^c Method (Alexander, 1968). This method i s based on the formation of phosphomolybdic a c i d , which i s then reduced by stannous chloride -hydrazine. C. Isolated G i l l Preparation Using the basic technique of Shuttleworth (1972) f o r measuring Na + fluxes, a constant-flow perfusion method was developed to study the transport of calcium ions across the i s o l a t e d and bleached t e l e o s t g i l l . 1. Materials a. Perfusion f l u i d t an E e l Saline, modified from the Cortland Saline (Wolf, 1963) to approximate the plasma e l e c t r o l y t e composition of the American e e l , was chosen as the standard perfusion f l u i d (Table 1). Poly-vinylpyrrolidone (1 $>) (P.V.P. with average molecular weight of 40,000; Sigma Chemical Co.) was added as a c o l l o i d substitute f o r plasma proteins and D-glucose (1 %) served as a substrate f o r energy supply to the c e l l s . P r i o r to each experiment, fresh-saline was prepared and the pH adjusted to 7.80. I t was saturated with oxygen and f i l t e r e d with a 0.22 jum disposable Table 1. Electrolyte composition, osmolarity and pH of eel plasma and perfusion f l u i d used i n the isolated g i l l study. Eel Plasma Perfusion Fluid Electrolyte (mM) Na + 140 133 K + 2.70 2.68 •Cl" 108 132 C a 2 + 1.50 1.50 Mg 2 + 1.20 0.93 HCC~ 8.00 8.00 Pi 0.69 1.50 — 0.93 Osmolarity (m.Osm) 263 263 a 2H 7.85+0.05 7.80 + 0.10 a With 0.1% glucose and 1# P.V.P. 40. 21 Millipore f i l t e r before use. b. Afferent and efferent cannulast a special type of leak-proof cannula was constructed for both afferent and efferent branchial arteries. Basically, i t consisted of a cut-off 23-G 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 effective "stop" to avoid possible leakage during perfusion. 2. Cannulation Procedure Prior to the experiment, a s i l v e r eel was removed from the water and transected behind the pectoral fins unanaesthetized. The f i r s t g i l l arch was carefully removed and placed immediately i n ice-cold heparinized saline j100.I.U. Heparin (sodium salt, Fisher Scientific Co.) per ml of saline \ bubbled with oxygen. The afferent cannula was introduced into the cut end of the afferent branchial artery to a depth of a few millimeters. A ligature was tied 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 ligature. The afferent cannula was connected to a motor-driven syringe pump with a variable speed f a c i l i t y (model 941, Harvard Apparatus Co., Maine, U.S.A.). H e p a r i n i z e d s a l i n e (200 I.U. Hep-a r i n p e r ml o f s a l i n e ) , b u b b l e d w i t h oxygen, was pumped t h r o u g h t h e i s o l a t e d g i l l a t a c o n s t a n t r a t e o f 0.6 ml p e r minute u n t i l t h e g i l l f i l a m e n t s were seen t o be c o m p l e t e l y f r e e o f b l o o d . The p e r f u s i o n f l u i d was t h e n changed t o a n o n - h e p a r i n i z e d s a l i n e . The e f f -e r e n t b r a n c h i a l a r t e r y was t h e n c a n n u l a t e d i n a s i m -i l a r manner. The r a t e o f p e r f u s i o n t h r o u g h t h e c a n -n u l a t e d g i l l ( P l a t e 1) was r e d u c e d t o 0.6 ml p e r h o u r . The g i l l p r e p a r a t i o n was t h e n r i n s e d t w i c e i n a e r a t e d d e c h l o r i n a t e d t a p w a t e r and f i n a l l y suspended i n a b a t h o f f i l t e r e d d e c h l o r i n a t e d w a t e r a t t h e d e s i r e d c a l c i u m c o n c e n t r a t i o n . The i n c u b a t i o n medium was 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 t h e e n t i r e c o u r s e o f t h e e x p e r i m e n t . The whole system was p l a c e d i n a c i r c u l a t i n g w a t e r b a t h a t a c o n s t a n t t e m p e r a t u r e ( P l a t e 2). 3« Sample C o l l e c t i o n and A n a l y s i s The i s o l a t e d g i l l p r e p a r a t i o n was a l l o w e d t o e q u i l i b r a t e a t a low p e r f u s i o n r a t e o f 0.6 ml p e r h o u r f o r 30 m i n u t e s . The e f f l u e n t was c o l -l e c t e d a t 15 minute i n t e r v a l s , u s i n g p i e c e s o f v o l u m e - s t a n d a r i z e d Clay-Adams PE 200 ( i . d . 1.397mm) p o l y e t h y l e n e t u b i n g . V a r i a t i o n i n t h e r a t e o f e f f e r e n t B l e a c h i n g and c a n n u l a t i o n o f t h e f i r s t g i l l a r c h 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 . 1. A f f e r e n t b r a n c h i a l c a n n u l a . 2. E f f e r e n t b r a n c h i a l c a n n u l a . 3 . B l e a c h e d g i l l f i l a m e n t s . G e n e r a l a p p a r a t u s f o r i s o l a t e d g i l l p e r f u s i s t u d y i n t h e Am 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 suspended i n i n c u b a t i o n medium. 2. A f f e r e n t b r a n c h i a l c a n n u l a . 3 . E f f e r e n t b r a n c h i a l c a n n u l a . 4. Sample c o l l e c t i n g t u b e . 5 . C o n s t a n t - f l o w 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. Haake c o n s t a n t - t e m p e r a t u r e i m m e r s i o n c i r c u l a t i n g pump. 25 flow within +. 3 % of the known afferent perfusion rate was accepted. G i l l preparations with efferent flow rate exceeding these limits were discarded. Calcium ion flux measurements began when -'Ca (as ^ C a C l 2 i n aqueous solution, Amersham -Searle Corp.) at a concentration of 7.4X10~^M (approximately 20 uCi per ml) was introduced into the system. For efflux studies, ^ C a 2 + was added to the perfusion f l u i d and 2+ the rate of outward Ca movement was measured by the appearance o f ^ C a 2 + in the incubation medium. For influx studies, ^ C a 2 * was added to the external solution and the rate of ^Ca -uptake was measured by the amount of ^ C a 2 + appearing i n the effluent. For ^ C a 2 + 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 in a Beckman liquid s c i n t i -l l a t i o n counter (model LS-233* Beckman Instruments Inc., California). At the termination of each experiment, the g i l l was removed from the incubation bath, rinsed and blotted l i g h t l y . The perfused g i l l filaments between the ligatures were cut off and placed in a preweighed aluminum dish. This was then l e f t to dry in an oven at 106° C for 24 hours. The dry weight of the filaments 26 was determined. The rate of Ca*^ flux was expressed 2+ as umoles Ca per 100 mg dry wt. per min. D. Studies of Calcium Transport Across the Isolated  and Perfused G i l l 1. Properties of the Branchial Calcium Transport  System The rate of *^Ca 2 + flux at 12° C, with an external 2+ water Ca concentration of 0 . 5 5 mM and a standard Eel 2+ Saline perfusate (Table 1) containing. 1 . 5 0 mM Ca at pH 7.80 was chosen as the control standard. The ionic calcium concentrations of both the incubation medium and the standard perfusate were directly measured, using a Beckman Select-Ion Calcium Electrode (model no. 39608, Beckman Instruments Inc., California) i n conjunction with a standard reference electrode and a Research Digital pH/mV Meter (Orion, model 801). The transepithelial e l e c t r i c a l potential of the isolated g i l l perfused under standard conditions was also determined. Potential difference between the glass micro-electrode inserted in the efferent end of the g i l l and the reference electrode situated in the incubation medium was recorded on an oscilloscope connected to an amplifier (Plate 3). For each series of experiments, a control level 2+ of Ca flux was always measured prior to changes i n the experimental parameters. At the end of the experi-ment, the rate of control flux was again determined to ensure that the preparation was s t i l l active. Each value was the mean of 2 - 5 samples and individual experiments were repeated at least three times. 2. Effect of Hormones on the Branchial Calcium  Transport System The action of two f i s h hormones which have "been shown to "be active i n calcium regulation i n the Anguilla species was investigated. They were calcitonin from the ultimobranchial glands and extract of the corpuscles of Stannius. a. Calcitonin t crystalline natural salmon calcitonin (Armour Pharmaceutical Co., No. AL 1025) was dissolved i n acetate - glycine buffer (1 % sodium acetate + 0.1# glycine, pH 4.3) to a f i n a l concentr-ation of 10 U/ml. Aliquots of 200 u l were lyophilized and stored at -20°C u n t i l use. The calcitonin activity of each batch was confirmed by rat bioassay, using a modification of the method developed by Kumar and his co-workers (1965)• 27a Recording Device Efferent cannula Glass micro-electrode inserted into efferent branchial artery Isolated,perfused gill preparation Amplifier nOscilloscoper~l Afferent cannula Reference electrode Bathing solution P l a t e 3. A s c h e m a t i c diagram i l l u s t r a t i n g t h e t e c h n i q u e o f 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. For perfusion studies, the lyophilized calcitonin was dissolved i n standard Eel Saline to 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 fluids were f i l t e r e d with 0.22 jum disposable Millipore f i l t e r before use. b. Extract of the corpuscles of Stanniust in the American eel, the corpuscles of Stannius are in the form of a pair of discrete, encapsulated glands, resting on the ventral surface of the kidneys (Plate 4) or more often, hidden on the sides of the posterior cardinal vein which passes between the two kidneys. Paired corpuscles of Stannius were collected from eels k i l l e d by decapitation. A slightly l a t e r a l incision was made i n the ventral body wall just opposite to the cloaca>. The kidneys were carefully exposed and the discrete corpuscles could be easily, be removed by a pair of curved forceps. They were either used immed-iately or stored intact at -20° C u n t i l use. For perfusion studies, the corpuscles were homo-genized i n ice-cold standard Eel Saline, using a Tri-R P l a t e 4 . The two e n c a p s u l a t e d c o r p u s c l e s o f S t a n n i u s 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 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. P a i r e d c o r p u s c l e s o f S t a n n i u s 2. 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 . 3. V e n t r a l body w a l l . 30 t i s s u e g r i n d e r w i t h T e f l o n p e s t l e . The homogenate was t h e n c e n t r i f u g e d a t 35tOOOXg f o r 30 minutes a t k° C, u s i n g a S o r y a l l s u p e r s p e e d c e n t r i f u g e (model RC 2-B). The c l e a r s u p e r n a t a n t was c a r e f u l l y removed and f i l t e r e d w i t h 0.22 um d i s p o s a b l e M i l l i p o r e f i l t e r . Subsequent d i l u t i o n t o t h e r e q u i r e d c o n c e n t r a t i o n s was made w i t h f i l t e r e d E e l S a l i n e w h i c h was u sed as t h e c o n t r o l p e r -f u s a t e . T o t a l p r o t e i n o f t h e f i l t e r e d c o r p u s c u l a r e x t r a c t was d e t e r m i n e d by t h e method o f Lowry and c o - w o r k e r s (1951), u s i n g b o v i n e a l b u m i n s t a n d a r d s . The c o n c e n t r a t i o n o f t h e e x t r a c t o f c o r p u s c l e s o f S t a n n i u s was e x p r e s s e d as mg p r o t e i n p e r ml o f p e r f u s i o n f l u i d . 31 RESULTS A. P r o p e r t i e s o f t h e 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 System 1. Ca G r a d i e n t A c r o s s t h e E p i t h e l i u m o f t h e  I s o l a t e d and P e r f u s e d G i l l The i o i c c a l c i u m c o n c e n t r a t i o n s were 0 .55 + 0.OlmM (average o f t h r e e measurements) 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+ s t a n d a r d E e l S a l i n e p e r f u s a t e . T h i s g i v e s a Ca g r a d i e n t o f 3 a c r o s s t h e e p i t h e l i u m o f t h e i s o l a t e d g i l l under s t a n d a r d p e r f u s i o n . 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 t h e E p i t h e l i u m o f t h e  I s o l a t e d and P e r f u s e d G i l l 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 app-r o x i m a t e l y + 30 t o +40 mV (30 measurements i n two g i l l p r e p a r a t i o n s ) w i t h r e s p e c t t o t h e e x t e r n a l i n c u b a t i o n medium. 2+ 3. Ca F l u x e s A c r o s s t h e I s o l a t e d G i l l P e r f u s e d  w i t h Normal E e l S a l i n e 2+ F i g u r e 1 i s a t y p i c a l t i m e - c o u r s e p r o f i l e o f Ca movements a c r o s s an i s o l a t e d and p e r f u s e d g i l l . D u r i n g i n f l u x measurements, ^ C a 2 + (7.4X10"^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. Ca i n f l u x , as i n d i c a t e d ILK p + b y t h e appearance o f -'Ca i n t h e e f f l u e n t , i n c r e a s e d 32 Figure 1. Movement of ^ C a 6 T across ah isolated and perfused g i l l of the American eel, A. rostrata. Incubation mixture was dechlorinated tap water with 0.55 mM Ca . Perfusion f l u i d was normal Eel Saline with 1.5 mM Ca 2 +, at pH 7.80. 33 progressively and reached a steady level 120 minutes after the addition of -'Ca . At this time, the average rate of ^ C a 2 + influx was (3.3^ + 0.32) X 10 3 CPM/100 mg dry wt./min. (mean+S.E., n«13) which i s equivalent to an inward movement of (3.34+ 0.32) XIO"2* ;umoles C a 2 + / 100 mg dry wt./min. During efflux measurements, ^ C a 2 + at a concentration of 7.4X10"^M was added to the standard Eel Saline per-2+ fusate. Ca efflux, as indicated by the appearance of ^ C a in the incubation medium, was small and i t reached a maximum level 75 minutes after the introduction of the 4*5 2+ isotope. At this 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 2+ rate of (8.48 +1.35) X10~ umoles Ca /100 mg dry wt./min. Net Ca influx was calculated to be approximately 3.26 X 10"^ jumoles /100 mg dry wt./min. 4. The Effect of Metabolic Inhibitor on Branchial Ca 2*- Uptake Figure 2 i s a typical time-course profile of the 2+ effect of metabolic inhibitor on branchial Ca -uptake. Administering the respiratory inhibitor, 2,4 dinitrophenol (ImM) to the perfused g i l l caused a rapid drop in the rate of net C a 2 + influx to only 45 + 2.0 % (mean + S .E., n » 3 ) that of the original control within 90 minutes. Addition of / ( I ) 2,4 DNP F i g u r e 2. E f f e c t o f m e t a b o l i c i n h i b i t o r on • *r-'Cafc" i n f l u x i n an i s o l a t e d and p e r f u s e d g i l l * © — ® Normal g i l l p r e p a r a t i o n . ( I ) 2,4 D N P » p e r f u s a t e w i t h 1 mM 2,4 d i n i t r o p h e n o l • ( I I ) 2,4 D N P J i n c u b a t i o n medium w i t h 1 mM 2,4 d i n i t r o p h e n o l . 0 — 0 G i l l p r e p a r a t i o n f r o m a d y i n g a n i m a l . 2,4 d i n i t r o p h e n o l (1 mM) 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 a f u r t h e r r e d u c t i o n i n t h e r a t e o f n e t Ca i n f l u x . A 76 +2 . 5 % (mean + S.D., n » 2 ) i n h i b i t i o n was obs e r v e d a t 165 minutes 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 i n t o t h e 2+ system. Net Ca -uptake i n t h e e n e r g y - d e p l e t e d g i l l i s o -l a t e d f r om a d y i n g a n i m a l was f o u n d t o be o n l y 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. The 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+ B r a n c h i a l Ca -Uptake 2+ -3 At e x t e r n a l Ca c o n c e n t r a t i o n s below 10 ^ M, n e t Ca i n f l u x was v e r y low ( l e s s t h a n 6.67X10" umoles /100 mg 2 + d r y wt./min.) ( F i g u r e 3 ) . Ca -uptake showed a r a p i d 2+ -3 i n c r e a s e 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 between 10 J M —2 —2 2+ and 10 M. A t 10 M, t h e r a t e o f n e t Ca -uptake was a p p r o x i m a t e l y 4.90X10""^ umoles / 1 0 0 mg d r y w t . / m i n . 2+ 6. The E f f e c t o f I n t e r n a l Ca C o n c e n t r a t i o n on .2+ B r a n c h i a l Ca2''"-Uptake F i g u r e 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 . I n c r e a s i n g 2+ t h e i n t e r n a l Ca fro m 0.75 mM t o 1.50mM (normal plasma 2+ l e v e l ) r e s u l t e d i n an e l e v a t e d r a t e o f n e t Ca i n f l u x . 2+ I t was s t a b l e a t Ca c o n c e n t r a t i o n s between 1.50mM and 1.75 mM, b u t was f u r t h e r enhanced a t a h i g h e r i n t e r n a l C a 2 + l e v e l of 2.25 mM. J 36 pCa in External Medium F i g u r e 3. E f f e c t o f e x t e r n a l C a ^ T c o n c e n t r a t i o n on 2+ Ca u p t a k e 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 . 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 d i f f e r e n t Ca c o n c e n t r a t i o n s . 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 C a 2 + , a t pH 7.80. V a l u e s a r e means + S.E. ( n » 9 ) . 37 ! X3 o> E O O \ OJ o E * O x x 2 + o o 5 r Perfusate [Ca 2 +] mM 2+ F i g u r e 4 . E f f e c t o f i n t e r n a l Ca c o n c e n t r a t i o n on 2+ Ca - u p t a k e 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 . 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 . P e r f u s i o n 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 C a 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 of Perfusate pH on Branchial Ca 2*- Uptake + 2+ The e f f e c t of H on g i l l Ca transport was deter-mined i n the pH range of 5»90-8.40. As indicated i n Figure 5» changes i n perfusate 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 stimulated net Ca i n f l u x . The rate of increase was more rapid "between pH 7.80-8.40 than between pH 7.80 -5.90. 8. The E f f e c t of Incubation Temperature on Branchial  Ca 2*-Uptake 2+ The e f f e c t of incubation temperature on g i l l Ca -uptake was studied and the r e s u l t s are presented i n Figure 6. Lowering the incubation temperature from the normal enviromental l e v e l of 12 °C to as low as 2 . 5 ° C did 2+ not induce any change i n the rate of net Ca i n f l u x . However, i f the incubation temperature was raised from 12° G to 17°G, a decrease of 35.% i n the rate of net 2+ 2+ Ca -uptake was observed. The rate of branchial Ca transport remained low at 20° C, B. The E f f e c t of Hormones on Branchial Ca2">"-Uptake I..The E f f e c t of C a l c i t o n i n 2+ The e f f e c t of salmon c a l c i t o n i n on g i l l Ca transport i s shown i n Figure 7. Using the vehicle 39 F i g u r e $. E f f e c t o f p e r f u s a t e pH on Ca*^- u p t a k e 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 . 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 2+ 0.55 mM 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 1.50 mM C a 2 + t a t d i f f e r e n t pH. V a l u e s a r e means o f 2 t o 5 samples i n 7 g i l l p r e p a r a t i o n s . 1 0 0 - i ^ 9 0 H CM o O 8 0 £ 7 0 o 6 0 H o JZ O • T" 5 I 10 15 —I 20 Temperature ( C ) 40 2+ F i g u r e 6. E f f e c t o f i n c u b a t i o n t e m p e r a t u r e on Ca -uptak e 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 . 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 0.55 mM C a 2 + . P e r f u s i o n f l u i d 2+ was n o r m a l E e l S a l i n e w i t h 1.50 mM Ca , a t pH 7 .80. V a l u e s a r e means + S.E. ( n « 3 ) T 1 1 1 1 1 100 200 300 Salmon Calcitonin (mil/ml) 2+ F i g u r e 7. E f f e c t o f c a l c i t o n i 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 g i l l 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 2+ 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 . 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 c o n c e n t r a t i o n s o f c a l c i t o n i n , a t pH 7.80. V a l u e s a r e means + S.E. (n = 3). CD c o O perfusate as the control, increasing doses of salmon calcitonin in the perfusion f l u i d (50 mU/ml and 120 mU/ml) resulted in a progressive stimulation of the rate of net C a 2 + influx. A higher dose of 240 mU/ml did not cause further activation. During the course of this study, i t was observed that a high dose of 300 mU/ml salmon calcitonin caused vasoconstriction, resulting i n a marked reduction i n the perfusion flow. 2. The Effect of Extract of the Corpuscles of  Stannius Figure 8 illustrates the effect of corpuscles of 2+ Stannius on g i l l Ca transport. Standard saline per-fusion was chosen as the control reference. Administr-ation of corpuscular extract at a low concentration of 0.1 mg protein /ml perfusate to the perfused g i l l 2+ caused a 42.5 # inhibition i n branchial Ca -uptake. The response was dose - dependent. Higher concentrations 2+ of extract resulted in further inhibition. Net Ca influx at extract concentration of 0.33 mg protein/ml perfusate was 44# that of the control. 100 0.0 Extract of Corpuscles of Stannius (mg Protein/ml) F i g u r e 8. E f f e c t o f c o r p u s c l e s o f S t a n n i u s e x t r a c t on Ca - u p t a k e 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 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 . P e r f u s i o n f l u i d 2+ was n o r m a l E e l S a l i n e w i t h 1.50 mM Ca and d i f f e r e n t c o n c e n t r a t i o n s o f c o r p u s c u l a r e x t r a c t , a t pH 7«80. V a l u e s a r e means + S.E ( n - 3 ) . ' 44 DISCUSSION A. P e r f u s i o n Technique The g i l l p e r f u s i o n t e c h n i q u e d e v e l o p e d f o r t h i s s t u d y has e f f e c t i v e l y p r e v e n t e d t h e problem o f l e a k a g e commonly e n c o u n t e r e d i n p e r f u s e d g i l l p r e p a r a t i o n s . P o s s i b l e l e a k a g e and b l o c k a g e o f t h e c a n n u l a t e d v e s s e l i s e l i m i n a t e d b y t h e i n t r o d u c t i o n o f a " s t o p " a t t h e t i p o f each c a n n u l a . Leaks as s m a l l as 1.50 j u l / m i n . c a n be r e a d i l y d e t e c t e d by p r e c i s e l y m o n i t o r i n g b o t h t h e 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 t h r o u g h o u t t h e e n t i r e e x p e r i m e n t . B. P r o p e r t i e s o f t h e 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 System C e r t a i n b i o l o g i c a l membranes such as 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 w a t e r . T r a n s e p i t h e l i a l p o t -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 om f r e e d i f f u s i o n o f 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 depends on 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 and 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 . I n s e a w a t e r t e l e o s t s , e l e c t r i c a l p o t e n t i a l mea-surements a c r o s s t h e a n i m a l i n d i c a t e t h a t t h e b l o o d i s p o s i t i v e w i t h r e s p e c t t o t h e e x t e r n a l w a t e r (Maetz and Camp a n i n i , 1966$ Evans, 1969? K i r s c h n e r g t al.,1974} P i c and Maetz, 1975). T h i s p o s i t i v e b l o o d p o t e n t i a l can be a t t r i b u t e d t o t h e f a c t t h a t t h e g i l l membrane i s ' more permeable t o c a t i o n s ( N a + and K + ) t h a n t o a n i o n s ( C l ~ ) ( K i r s c h n e r e t a l . , 1974} P i c and Maetz, 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 e l e c t r i c a l 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 . I n t h e European e e l k e p t i n f r e s h w a t e r , a n e g a t i v e b l o o d p o t e n t i a l (-21 mV) was o b s e r v e d (Maetz and Campanini, 1966). I n c o n t r a s t , a p o s i t i v e b l o o d p o t e n t i a l (+8.5 mV) was measured i n t h e ra 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 + 2+ c o n t a i n i n g 1 mM Na and Ca ( K e r s t e t t . e r et, a l . , 1970). I n t h e g o l d f i s h ( C a r a s s i u s a u r a t u s ) . t h e b l o o d p o t e n t i a l v a r i e d 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 (Maetz, 1974). I t was p o s i t i v e (+4.0 mV) i n f r e s h w a t e r , n e g a t i v e (-44 mV) 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 added. Measurements o f i o n i c membrane p e r m e a b i l i t i e s r e v e a l e d t h a t i n f r e s h w a t e r , P N a was alm o s t t h e same as P C 1 } i n d e i o n i z e d 2+ w a t e r , P N a was 8 X P C 1 and i n d e i o n i z e d w a t e r w i t h Ca , P N a was o n l y 0.5X P C 1 . 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 outward 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 in fresh water also contributed to the f i n a l potential difference. In the present study, the positive transepithelial potential (+30 to +40 mV) recorded i n the isolated per-fused g i l l preparation agrees qualitatively with the observations of Kerstetter and co-workers (1970) and Maetz (1974-). Quantitatively, the larger potential difference observed i n the American eel may be accounted for by species variations or difference in the experi-mental technique. Previous studies were a l l performed on intact animals. The e l e c t r i c a l potential in a caudal vein cannula or an intraperitoneal catheter was measured and assumed to be equivalent to that i n the branchial circulation. In contrast, the present data were obtained by a direct recording of potential difference across the isolated g i l l perfused with normal Eel Saline. 2+ Present results on Ca efflux revealed that the 2+ g i l l epithelium was f a i r l y impermeable to calcium. Ca influx against a concentration gradient was i n the order of 10 umoles/100 mg dry wt./min., while Ca efflux along a diffusion gradient was in the order of 10""^  units. 2+ The rates of Ca influx and efflux differed by a factor 2+ of 40, suggesting an efficient Ca -uptake system. G i l l C a ^ - u p t a k e was i n h i b i t e d by 2,4 d i n i t r o -p h e n o l w h i c h i s an u n c o u p l e r o f o x i d a t i v e 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 energy. Two c r i t e r i a a r e o f t e n used t o d e t e r m i n e d whether 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 a g a i n s t a c o n c e n t r a t i o n g r a d i e n t and ( o r ) an e l e c t r i c a l g r a d i e n t ; and a r e q u i r e m e n t f o r m e t a b o l i c e n e r g y (see G i e s e , 1968). B o t h c r i t e r i a have been s a t i s f i e d i n t h e p r e s e n t s t u d y , i n d i c a t i n g t h a t t h e 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 Ca -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 r e s embles t h a t 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 plasma membrane 2+ v e s i c l e s (Shami, 1974). I n b o t h c a s e s , Ca -uptake 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 below 10 ^M. Shami (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 by p l a c e n t a l plasma membrane v e s i c l e s was dependent 2+ on ATP h y d r o l y s i s by t h e membrane-bound Ca -ATPase. 2+ The s u b s t r a t e o f Ca -ATPase was a Ca-ATP complex n o t 2+ t a k e n up by t h e v e s i c l e s . O n l y f r e e Ca was t r a n s p o r t e d . Based on t h e s e f i n d i n g , i t was pr o p o s e d t h a t i n t h e p r e -sence o f ATP and a t C a 2 + c o n c e n t r a t i o n s below 10"-* M, 2+ l i t t l e f r e e Ca was a v a i l a b l e f o r membrane t r a n s p o r t . A s i m i l a r C a ^ - A T P a s e system may be r e s p o n s i b l e f o r a c t i v e 2+ 2+ b r a n c h i a l Ca - u p t a k e . I d e n t i f i c a t i o n o f a Ca -ATPase i n t h e g i l l p lasma membranes s u p p o r t s t h i s h y p o t h e s i s (see C h a p t e r 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+ by an 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 p h y s i o l o g i c a l l e v e l . I f b r a n c h i a l Ca t r a n s p o r t p l a y s 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 , 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 cause an i n h i b i t i o n i n g i l l Ca - u p t a k e . W h i l e t h e p r e s e n t r e s u l t appears t o c o n t r a d i c t t h e 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 p r e p a r -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 a b s e n t . The o b s e r v e d r e s p o n s e o n l y r e p r e s e n t s 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 t r a n s p o r t system w h i c h i s n o t 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 normal 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+ an e l e v a t e d b r a n c h i a l Ca t r a n s p o r t . The changes i n th e g i l l e p i t h e l i u m l e a d i n g t o t h i s phenomenon can be complex. I n t h e c o u r s e o f i n t e r p r e t i n g t h e d a t a , 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 . F i r s t l y , p r o l o n g e d exposure o f c e l l s t o abnormal pH r e s u l t s i n an 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 i n j u r y (see G i e s e , 1968). S e c o n d l y , changes i n pH may cause a 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 c e l l , t h u s a l t e r i n g i t s energy s t a t u s . Shami (1974) r e p o r t e d 2+ t h a t Ca - 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 plasma membranes was f a c i l i t a t e d by an a l k a l i n e pH. I t i s p o s s i b l e t h a t 2+ a s i m i l a r i n c r e a s e i n Ca - b i n d i n g a l s o o c c u r r e d i n t h e g i l l , p a r t l y a c c o u n t i n g f o r t h e more pronounced e l e v a t i o n 2+ i n Ca - t r a n s p o r t i n t h e a l k a l i n e pH r a n g e . 2+ B r a n c h i a l Ca -uptake was f o u n d t o be s e n s i t i v e t o changes i n i n c u b a t i o n t e m p e r a t u r e . I n p o i k i l o t h e r m i c organisms s u c h as f i s h , many m e t a b o l i c enzyme systems e x h i b i t a temperature-dependent v a r i a t i o n i n t h e enzyme-s u b s t r a t e a f f i n i t y . The enzyme a f f i n i t y o f t e n appears t o 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 a d a p t e d (see Hochachka and Somero, 1973). F o r t h e A m e r i c a n e e l , t h e n ormal range o f i t s h a b i t a t t e m p e r a t u r e i s between 4°-12°C. T h i s c o r r e s p o n d s t o t h e t e m p e r a t u r e range o f o p t i m a l b r a n c h i a l C a 2 + - u p t a k e a c t i v i t y (2°-12°C), sug-g e s t i n g p o s s i b l e t e m p e r a t u r e a d a p t a t i o n o f c e l l u l a r f u n c t i o n s . C The E f f e c t o f Hormones on B r a n c h i a l Ca 2*-Uptake A l t h o u g h t h e p r i m a r y p h y s i o l o g i c a l f u n c t i o n o f t h e u l t i m o b r a n c h i a l b o d i e s i n t e l e o s t s i s n o t y e t f u l l y 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 r e g u -l a t i o n . T e l e o s t e a n u l t i m o b r a n c h i a l g l a n d s a r e r i c h i n c a l c i t o n i n (Copp and P a r k e s , 1968; Copp, 1969). The h y p o c a l c e m i c e f f e c t o f 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). Whether c a l c i t o n i n a c t s t o e l i c i t a h y p o c a l c e m i c r e s p o n s e i n f i s h i s s t i l l d e b a t -a b l e . However, d a t a a v a i l a b l e t o dat 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 as 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 (Chan ejb a l . , 1968; Pang, 1971a; Lopez and D e v i l l e , 1972; Ma, 1973). The s i t e ( s ) 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 r e m a i n o b s c u r e . R e c e n t l y , F l e m i n g and co-workers (1973) demonstr-a t e d 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 r e s u l t e d i n a s t i m u l a t e d ^Ca-uptake from t h e e n o i o n m e n t a l w a t e r . F u r t h e r m o r e , t h e h o r m o n e - t r e a t e d 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 . A d d i t i o n a l e v i d e n c e by Dacke (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 r e d u c e d 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, a n g u i l l a ) . 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 g i l l p r e p a r a t i o n s , 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 mU/ml caused a s i g n i f i c a n t i n c r e a s e i n n e t b r a n c h i a l 2 + Ca - u p t a k e . Based on t h e s e r e s u l t s , i t seems l i k e l y 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 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 ppears t o he i n c o n s i s t e n t w i t h t h e h y p o c a l c e m i c e f f e c t o f c a l c i t o n i n o b s e r v e d 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 i n t o t h e s o f t t i s s u e compartment by t h e hormone ( F l e m i n g e t a l . , 1973) s h o u l d be c o n s i d e r e d . The f a c t t h a t i n A. .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 d e c l i n e i n muscle t o t a l c a l c i u m (Ma, 1973) p r o v i d e s a d d i t i o n a l e v i d e n c e i n s u p p o r t o f t h i s v i e w . I t i s now g e n e r a l l y a c c e p t e d t h a t t h e c o r p u s c l e s 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 f i s h (see Pang, 1973). However, t h e r e i s l i t t l e i n f o r m a t i o n c o n c e r n i n g t h e s i t e ( s ) and mechanism(s) o f a c t i o n o f t h i s e n d o c r i n e t i s s u e . F o n t a i n e and co-workers (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 f o l l o w e d by an r L 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 . They p r o p o s e d t h a t t h i s phenomenon may a c c o u n t , a t l e a s t p a r t l y , f o r t h e c o n c o m i t a n t r i s e i n plasma and muscle c a l c i u m l e v e l s n o r m a l l y o b s e r v e d . Fenwick and So (1974) p r o v i d e d t h e f i r s t p i e c e o f e v i d e n c e o f b r a n c h i a l i n v o l v e -ment i n c a l c i u m h o m e o s t a s i s . They demonstrated t h a t i n A n g u i l l a r o s t r a t a . " S t a n n i e c t o m y " r e s u l t e d i n a marked 52 e l e v a t i o n i n c a l c i u m i n f l u x a c r o s s t h e i s o l a t e d p e r f u s e d g i l l . Data o b t a i n e d from t h e p r e s e n t s t u d y i n d i c a t e t h a t 2+ b r a n c h i a l Ca -uptake i n A. r o s t r a t a was i n h i b i t e d by t h e c o r p u s c l e e x t r a c t and t h e r e s p o n s e was dose-depen-d e n t . These r e s u l t s s t r o n g l y s u ggest 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+ g i l l 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 an 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 absent i n f i s h a dapted t o a c a l c i u m - d e f i c i e n t e n v i r o n -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 . I n summary, t h i s s e r i e s o f s t u d i e s has demonstrated 2+ 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 Ca -uptake system i n t h e A m e r i c a n e e l . T h i s t r a n s p o r t system was s e n s i t i v e t o 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 c o n c e n t r a t i o n s , 2+ pH and e n v i r o m e n t a l t e m p e r a t u r e . B r a n c h i a l Ca -uptake was a f f e c t e d by c a l c i t o n i n 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 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 , sugg-e s t i n g t h e 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 t e l e o s t e a n c a l c i u m h o m e o s t a s i s . DIVISION I I CALCIUM - STIMULATED ATPase OF THE TELEOST GILL PLASMA MEMBRANES 5^ CHAPTER I I CHARACTERIZATION OF CALCIUM - STIMULATED ATPase IN THE GILL PLASMA MEMBRANES OF THE AMERICAN EEL, ANGUILLA ROSTRATA INTRODUCTION I n t h e f i r s t c h a p t e r , s t u d i e s on c a l c i u m t r a n s p o r t a c r o s s t h e i s o l a t e d g i l l o f t h e A m e r i c a n e e l have demon-s t r a t e d a n e t u p t a k e o f c a l c i u m i o n s f rom t h e e x t e r n a l 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 an e l e c t r i c a l -2+ c h e m i c a l g r a d i e n t . Such " u p h i l l " movement o f Ca i s dependent upon 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 energy. I n l i v i n g systems, most energy - u t i l i z i n g p r o c e s s e s a r e d r i v e n by t h e breakdown o f t h e e n e r g y - r i c h compound ATP ( D i x o n and Webb, 1964,.). To be e f f e c t i v e , t h e h y d r o l y s i s o f ATP must be e n z y m a t i c and c o u p l e d w i t h t h e energy -u t i l i z i n g p r o c e s s . The c o u p l i n g between t h e two e v e n t s (ATP h y d r o l y s i s and a c t i v e c a l c i u m t r a n s p o r t i n t h i s c a s e ) must t a k e p l a c e w i t h i n t h e membrane, so t h a t t h e enzyme h y d r o l y z i n g ATP i s f i x e d i n s p ace. Skou (1965) has e s t a b l i s h e d t h e r e l a t i o n s h i p between N a + , K + f l u x e s a c r o s s c e l l membranes and ATP 2+ + h y d r o l y s i s by t h e membrane - bound Mg -dependent, (Na -K ) - a c t i v a t e d ATPase. I n t e l e o s t f i s h , a c t i v e t r a n s p o r t o f sodium a c r o s s t h e g i l l e p i t h e l i u m has "been shown t o i n v o l v e a s i m i l a r Mg -dependent, (Na -K ) - a c t i v a t e d ATPase (see M o t a i s and Garcia-Romeu, 1972). Membrane-2 + 2+ bound Ca (Mg ) - a c t i v a t e d ATPases have been d e s c r i b e d i n s e v e r a l v e r t e b r a t e 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 c a l c i u m o c c u r s , s uch as t h e i n t e s t i n a l mucosa (Melancon and DeLuca, 1970), t h e r e n a l t u b u l e s ( P a r k i n s o n and Radde, 1971) and t h e p l a c e n t a (Shami and Radde, 1971). C a l c i u m -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 i n t i s s u e s 2+ where t h e l e v e l o f Ca i s c r i t i c a l f o r s p e c i f i c p h y s i o -l o g i c a l f u n c t i o n s , 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 r e t i c u l u m (see M a r t o n o s i , 1972) and t h e r e d b l o o d c e l l s (see S c h a t z -mann, 1975). 2+ I n t h i s c h a p t e r , t h e p r o p e r t i e s o f a Ca - s t i m u -l a t e d ATPase i n t h e g i l l plasma membranes o f t h e A m e r i c a n e e l . A n g u i l l a r o s t r a t a were examined and compared w i t h t h e p r o p e r t i e s o f o t h e r Ca -(Mg ) ATPases. 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 + 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 on t h e g i l l Ca -ATPase was a l s o i n v e s t i g a t e d . 56 MATERIALS AND METHODS A. Experimental Animals Silver American eel, Anguilla rostrata L. was chosen as the source of supply of teleost g i l l tissue (see Mater-i a l s and Methods, Chapter I ) . Silver eels weighing 800-1200 g were acclimated under laboratory conditions, in running dechlorinated water at 12*5 +1° C for at least two weeks before use. B. Isolation of G i l l Plasma Membranes The method of Post and Sen (I967) for the isolation of kidney cortical plasma membranes was adopted for 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 in i c e - c o l d 0,9% saline. The isolated g i l l tissue was then homogenised in 30 ml of a cold (4° C) solution cont-aining 87 g sucrose, 1.0 g NaCl, 1.86 g EDTA (disodium s a l t ) , 0.20 g MgCl 2»6H 20 and 0.68 g imidazole per l i t r e , using a T r i - R tissue grinder with Teflon pestle. The homogenised tissue^was then processed through a series of centrifugations at 35,000 Xg for 30 min i n different solutions at 4°C, using a Sorvall superspeed centrifuge (model RC2-B). The d e t a i l p r o c e d u r e i s o u t l i n e d i n F i g u r e 9. The i s o l a t e d plasma membranes were f i n a l l y suspended i n 5 mM T r i s -HC1 b u f f e r (pH 8.6) c o n t a i n i n g 0.5 mM i m i d a z o l e and s t o r e d a t 4° C u n t i l u s e . C. D e t e r m i n a t i o n o f Membrane C o n c e n t r a t i o n The p r o t e i n c o n c e n t r a t i o n o f t h e f i n a l p r e p a r a t i o n was t a k e n as an i n d i c a t o r o f plasma membrane c o n t e n t i n t h e i n c u b a t i o n medium. T o t a l p r o t e i n was d e t e r m i n e d by t h e method o f Lowry and c o - w o r k e r s (1951)» u s i n g b o v i n e a l b u m i n s t a n d a r d s . D. E v a l u a t i o n o f t h e 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 F i n a l  Membrane P r e p a r a t i o n 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 was f i x e d i n K a r n o s k y f i x a t i v e (50$) and c e n t r i f u g e d a t 35,000 X^g f o r 30 min. The p e l l e t was t h e n washed t h r e e t i m e s w i t h 0.1 M c a c o d y l a t e b u f f e r , r e suspended and c e n t r i f u g e d a t 45,000Xg f o r 30 min each t i m e . The p e l l e t was t h e n f i x e d w i t h phosphate - b u f f e r e d osmium t e t r a o x i d e (1$) and s t a i n e d w i t h s a t u r a t e d u r a n y l a c e t -a t e . The s t a i n e d membranes were f i n a l l y d e h y d r a t e d and 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 and 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 . 58 Homogenate centrifuged at 300 Xg for 15 min. Coarse residue (discarded) -Supernatant centrifuged at 35.000 Xg for 30 min, Mitochondrial and heavy plasma membrane fragments (discarded) Particle-free supernatant (discarded) —Microsomal fraction. This upper plasma membrane layer was scraped free and transferred to 25 ml of solution (A), homogenized and re-centrifuged at 35,000 Xg for 30 min. The same procedure was repeated 3X, in solutions (B), (C) and (D) to reduce progress-ively contamination by mitochon-dria. The f i n a l membrane prepar-ation was suspended i n (D). Figure 9. Isolation 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 Na2EDTA and 3 mM imadazole. Solution (D) contained 0.5 mM imada-zole and 5mM Tris-HCl (pH 8.0). 59 2. Enzyme Marker Assays After the upper phase membrane layer was scraped off, the remaining pellet from each centrifugation (35*000 Xg) was pooled. Both the f i n a l membrane pre-paration and the pooled residue were assayed for enzyme a c t i v i t i e s . Alkaline phosphatase was used as a marker for plasma membranes and succinic dehydrogenase was used as a marker for 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 for one hour at 12° C in 1.0 ml buffer - subst-rate mixture containing 0.1 M C0^2~-HC0^~ buffer (pH 9 * 5 ) » 10 mM MgCl 2 and 5.4 mM p-nitrophenyl phosphate (Sigma Chemical Co.). The reaction was terminated by addition of 5 ml 0 .05N NaOH. The p-nitrophenol released was measured as increase i n absorbance at 410 nm, using a Perkin -Elmer double beam spectrophotometer (model 124). Specific enzyme activity 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 prepar-ation, 0.1 ml 0 .5 M sodium succinate, 0 .3 ml 0 .33% INT 60 |2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetra-zolium chloride | (Eastman Kodak Co.) and 0 . 5 ml 0.1 M potassium phosphate buffer (pH 7.4). Incubation was terminated by adding 1.0 ml 10$ (w/v) trichloroacetic acid. The formazan produced was extracted with 4 ml ethyl acetate and measured as increase i n absorbance at 490 nm. Specific enzyme activity was expressed as absor-bance units per mg protein per hour. E. ATPase Activity of the G i l l Plasma Membranes To test the various enzymatic properties, 0.1 ml of membrane suspension (20-30 ug protein) was incubated with 1.0 ml of a solution containing 20 mM Tris-HCl buffer (pH 8.0), 70 mM Na* (as NaCl), divalent cations ( C a 2 + or 2+ Mg ) and Na 2 ATP (Sigma) at different concentrations as indicated. Divalent cations were omitted i n blanks. Tris-ATP (Sigma) was used instead of Na 2 ATP, and the reaction mixture contained no Na . Incubations were carried out in a water-bath at 1 2 . 5 + 1.0° C for one hour. The reaction was then stopped by quickly placing the tubes in an ice-bath and adding 1.0 ml 10$ (w/v) trichloroacetic acid. 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 activity 6 1 was e x p r e s s e d as umoles P i r e l e a s e d p e r mg p r o t e i n p e r h o u r . Each v a l u e was t h e mean of t r i p l i c a t e samples and 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 t i m e s . The f o l l o w i n g was a l i s t o f t h e s p e c i a l c h e m i c a l s u s e d i n v a r i o u s e x p e r i m e n t s » ( 1 ) N a 2 ATP {Adenosine 5 * - t r i p h o s p h a t e , d i s o d i u m s a l t } (Sigma No. A - 3 1 2 7 ) ; (2) T r i s - A T P J D i - T r i s ( h y d r o x y m e t h y l ) - a m i n o - methane s a l t } (Sigma No. A - 3 8 7 7 ) ; ( 3 ) GTP | Guanosine 5 * - t r i p h o s p h a t e , sodium s a l t \ (Sigma No. G - 5 7 5 6 ) ; ( 4 ) ITP { 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 } (Sigma No. 1-5000)i ( 5 ) ADP | Adenosine 5 ' - d i p h o s p h a t e , d i s o d i u m s a l t } (Sigma No. A - 0 1 2 7 ) ; ( 6 ) AMP { A d e n o s i n e 5*-monophosphoric a c i d , sodium s a l t } (Sigma No. A - 1 8 7 7 ) ; ( 7 ) Ouabain o c t a h y d r a t e (Sigma No. 0 - 3 1 2 5 ) ; ( 8 ) M e r s a l y l a c i d (Sigma No. M - 8 0 0 0 ) . F. E f f e c t o f Hormones on ATPase A c t i v i t y The e f f e c t o f two c a l c i u m - r e l a t e d hormones, namely c a l c i t o n i n 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 , on 2+ 2+ t h e g i l l plasma membrane Ca -(Mg ) ATPase a c t i v i t y was s t u d i e d . 62 1. E f f e c t o f Salmon C a l c i t o n i n N a t u r a l 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 Co., No. AL 1025) was p r e p a r e d as d e s c r i b e d i n M a t e r i a l s and Methods, C h a p t e r I . The l y o p h i l i z e d hormone-.was d i s s o l v e d i n 20 mM t r i s - H C l b u f f e r (pH 8.0) and i n t r o d u c e d i n t o t h e r e a c t i o n m i x t u r e i n 100 fil a l i q u o t s t o make up t h e r e q u i r e d c a l c i t o n i n c o n c e n t r a t i o n . An equal volume o f T r i s - H C l b u f f e r was added t o t h e c o n t r o l s a m p l e s . 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 C o r p u s c l e s ; o f S t a n n i u s were c o l l e c t e d f r o m s i l v e r A m e r i can e e l s and t h e e x t r a c t was p r e p a r e d as d e s c r i b e d i n M a t e r i a l s and Methods, C h a p t e r I , e x c e p t t h a t 20 mM T r i s -H C l b u f f e r (pH 8.0) was u sed f o r hormone e x t r a c t i o n . The c o r p u s c u l a r e x t r a c t was t h e n d i l u t e d and added t o t h e r e a c -t i o n m i x t u r e i n 100 u l a l i q u o t s . E q u a l volumes o f 20 mM T r i s - HC1 b u f f e r was used i n t h e c o n t r o l samples. T o t a l p r o t e i n o f t h e c o r p u s c u l a r e x t r a c t was d e t e r m i n e d by t h e method o f Lowry and co-workers (1951) and t h e hormone c o n c e n t r a t i o n was e x p r e s s e d as mg p r o t e i n p e r m l . To demonstrate th e s p e c i f i c a c t i o n 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 on g i l l Ca -(Mg ) ATPase, an e q u a l amount o f k i d n e y t i s s u e was t a k e n i n t h e v i c i n i t y o f t h e hormonal g l a n d s . I t was 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 t h e e f f e c t o f t h i s e x t r a c t on g i l l C a 2 + - ( M g 2 + ) ATPase a c t i v i t y was examined. 63 RESULTS A. Evaluation of the Isolation Method 1. Electron Microscopic Examination of the Final  Membrane Preparation Electron microscopy revealed that the f i n a l prepar-ation consisted mostly of plasma membranes (Plates 5 and 6 ) . No contamination with mitochondria could be observed. 2. Marker Enzymes Table 2 summarizes the distribution of marker enzymes. The specific activity of succinic dehydrogenase i n the " f i n a l membrane fraction" was very low, being only one-tenth of that i n the "pooled residue". This indicates that the f i n a l membrane preparation contained only very few mitochondria, confirming the electron microscopy observations. Ca -ATPase activity followed a distribution very similar to that of alkaline phosphatase, with almost the same act-2+ i v i t y ratio (1.66 for Ca -ATPase and 1.86 for alkaline phosphatase) between the " f i n a l membrane fraction" and the 2+ "pooled residue". This indicates that the Ca -ATPase under investigation i s located in the plasma membranes. The relatively high specific activity of the two phosphatases i n the "pooled residue" may be due to incomplete recovery of the heavier plasma membrane fragments. 64 Plate 5 . The f i n a l membrane preparation (X39,664). Plate 6. The f i n a l membrane preparation (X 82,075). 66 Table 2. Specific a c t i v i t i e s of marker enzymes i n different fractions. The pooled residue consisted of the remaining pellet after the upper membrane layer had been scraped off. It includes mitochondria and heavy plasma membrane fragments. Fraction Ca 2 +- Alkaline Succinic ATPase Phosphatase Dehydrogenase Final membrane fraction Pooled residue 13.9 a 0.93 b 8.4 0.50 0.086° 0.728 Final membrane fraction Pooled residue 1.66 1.86 0.12 a Activity in umoles Pi/mg protein/hr. b Activity i n umoles p-nitrophenol/rag protein/hr. c Activity i n A^ 0 Q ^ /mg protein/hr. 67 2 + B. P r o p e r t i e s o f t h e G i l l P lasma Membrane Ca -ATPase 1. A c t i v a t i o n o f t h e Enzyme by D i v a l e n t C a t i o n s A c t i v a t i o n o f t h e enzyme by c a l c i u m i o n s was a s s e s s e d by 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 c o n c e n t r a t i o n i n t h e i n c u b a t i o n medium from 0.1 t o 10 mM. 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 f o r 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 0.38+ 0.03 mM (mean + S.E.) ( F i g u r e 1 1 ) . The r a t e o f P i r e l e a s e a t peak a c t i v i t y w i t h 2 + 5 mM Ca was 24 . 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 t h a n 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 magnesium 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 f o r Mg 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 g i l l p r e p a r a t i o n s was 0.64+ 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 by Mg - s t i m u l a t i o n was 12.3+ Q.,4 ;uraoles P i p e r mg p r o t e i n p e r hour (mean + S.E., n = 8 ) , w h i c h 2+ was o n l y 50$ t h a t by Ca - s t i m u l a t i o n . E q u i m o l a r 2+ 2 + c o n c e n t r a t i o n s 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 between 2+ 2 + t h o s e w i t h Ca and Mg a l o n e ( F i g u r e 1 0 ) . Maximum 68 mM Divalent Cation Figure 10. Activation of ATP hydrolysis by increasing concentrations of divalent cations. 2+ • — • , activation by Ca ; © — © , activation by Mg24"; A A , activation by Ca 2 ++Mg 2 + (in equimolar concentration). Incubation mixtures contained 20 mM Tris-HCl (pH 8.0), 5mM ATP (disodium salt) and 70 mM Na + (as NaCl). 69 0.6 ~t Figure 11. Lineweaver - Buric plots of ATPase a c t i v i t y at 2 + 2 + various concentrations of Ca , Mg and. ATP. V i s expressed i n jumoles Pi/mg protein/hr.., [S] i n mM. • — • , 5 mM ATP + increasing 2 + concentration of Ca i © — © t 5 mM ATP + 2+ increasing concentration of Mg j • • 2+ 5 mM Ca + increasing concentration of ATP. Incubation mixtures contained 20 mM Tris-HCl (pH 8 .0) and 70 mM Na + (as NaCl). a c t i v i t y with C a 2 + plus Mg2* (1 « 1) was 71.4+ 1.7$ 2+ (mean + S.E., n«5)-that with Ca alone. 2. Effect of Protein Concentration on Pi Release The effect of protein concentration on ATP hydro-l y s i s i s shown i n Figure 12. Inorganic phosphate release was linear within the range of 20 to 240 mg protein per ml incubation mixture. 3. Sodium and Potassium Independence of the Enzyme 2+ 2+ Activation of the enzyme by Ca or Mg did not require the presence of Na + or K+. Incubation without Na + and using 5 mM Tris-A3?P resulted i n a slightly higher activation than that with 70 mM Na and 5 mM ATP (disodium salt) (Figure 13). 4. Effect of ATP Concentration and Substrate Specif- i c i t y The effect of ATP concentration on enzyme activity was examined by incubating the enzyme preparation with 5 mM Ca 2 +, 20 mM Tris-HCl (pH 8.0), 70 mM Na + and. various concentrations of ATP (disodium salt) from 0.1 2+ to 10 mM. Maximal Ca -activated ATP hydrolysis occurred at an ATP concentration of 5 mM (Figure 14). The apparent K m for ATP was 0.08 to 0.09 mM (Figure 11). 2 + The substrate specificity 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 + 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 T r i s - H C l (pH 8.0), 5 mM ATP ( d i s o d i u m s a l t ) and 70 mM N a + (as N a C l ) . Figure 13. Effect of Na on enzyme activation by 2+ divalent cations. • e , 5^M Ca j 2+ ©—© , 5 mM Mg . Incubation mixtures contained 20 mM Tris-HCl (pH 8.0) and 5 mM Tris-ATP. 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 n u c l e o t i d e s as s u b s t r a t e . • • , ATP; © — © , GTPj • A t I T P | A A o ADP; • • , AMP. 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 T r i s - H C l (pH 8.0) and 70 mM N a + (as N a C l ) . 74 ATPase was tested, using sodium salts of GTP, ITP, ADP and AMP to replace ATP (disodium salt) i n the incubation medium. The result (Figure 14) shows that the two t r i -phosphates (ITP and GTP) and diphosphate (ADP) a l l served as substrates, but with a V" „ only 70% (ADP and GTP) and 78% (ITP) that with ATP as substrate. Pi production with AMP as substrate was undetectable. 5. Effect of P H on Ca2*-ATPase Activity The effect of pH on enzyme activity was determined i n the range 4.0 to 10.0. In each instance, the pH was adjusted i n both the incubation mixture and the ATP solution and was determined before and after incubation. The pH curve i s presented i n Figure 15. The pH optimum of g i l l Ca -ATPase was between 7-9 and 8.1. At pH 6.8 and 9.4, only 50% activation by calcium ions was retained. 6. Effect of Inhibitors Figure 16 shows the effect of two inhibitors on 2+ Ca -ATPase activity. The presence of ouabain in the incubation medium at concentrations of 0.1 and 1.0 mM did not inhibit the enzyme. Mersalyl acid, when added to the reaction mixture at concentrations ranging from 0.1 to 10 mM, exhibited a progressive inhibitory effect on enzyme activity. At a mersalyl acid concentration of 2.1 mM, 50-% inhibition was observed. 0+V/-T-0 3 1 II pH F i g u r e 15. E f f e c t o f pH on C a 2 + - A T P a s e a c t i v i t y . 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 T r i s - H C l , 5 mM ATP ( d i s o d i u m s a l t ) and 70 mM N a + (as N a C l ) . 76 £ 20 H °i • 1 • 1 • 1 « r~ 1 ; 0 2 4 6 8 IO mM Inhibitor F i g u r e 16. E f f e c t o f i n h i b i t o r s on C a 2 + - A T F a s e a c t i v i t y . • • , e f f e c t o f o u a b a i n * © — 0 , e f f e c t o f m e r s a l y l a c i d . 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 C a 2 + , 20 mM T r i s - H C l (pH 8 . 0 ) , 5 mM ATP ( d i s o d i u m s a l t ) and 70 mM N a + (as N a C l ) . 7 7 7 . E f f e c t o f I n c u b a t i o n Temperature on Enzyme A c t i v i t y 2+ Ca -ATPase a c t i v i t y was d e t e r m i n e d i n t h e temper-a t u r e range 1-80°C. E n z y m a t i c h y d r o l y s i s o f ATP i n c r e a s e d p r o g r e s s i v e l y up t o 30° C ( F i g u r e 1 7 ) . 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 1 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 s h a r p l y and complete enzyme i n a c t i v a t i o n o c c u r r e d a t 7L° C. The Q 1 Q ( 5 - 1 5 ° C ) c a l c u l a t e d a c c o r d i n g t o t h e method o f G i e s e (1968) was 1.8. 8. S t a b i l i t y o f t h e Enzyme 2+ To d e t e r m i n e t h e 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 were 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 d e c l i n e d p r o g r e s s i v e l y w i t h i n c r e a s i n g t i m e o f s t o r a g e ( F i g u r e 1 8 ) . A 50 i> l o s s i n a c t i v i t y was o b s e r v e d i n samples s t o r e d a t 4-° C f o r 20 days. T h e r e f o r e , f r e s h p r e p a r a t i o n s were n o r m a l l y used w i t h i n a few days o f i s o l a t i o n . 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 Salmon C a l c i t o n i n 2+ 2+ 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 Ca -(Mg ) ATPase i s shown i n F i g u r e 19. Salmon c a l c i t o n i n a t v a r i o u s c o n c e n t r a t i o n s r a n g i n g f r o m 50 t o 520 mU p e r ml i n c u b a t i o n 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 o r Mg - s t i m u l a t e d ATP h y d r o l y s i s . 7 8 °~\— ' 1 ' 1 « 1 r~=- 1 0 20 40 60 80 Incubation Temperature ( °C) 2 + F i g u r e 1 7 . E f f e c t o f i n c u b a t i o n t e m p e r a t u r e on Ca -ATPase a c t i v i t y . The 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 C a 2 + , 2 0 mM T r i s - H C l (pH 8 . 0 ) , 5mM ATP ( d i s o d i u m s a l t ) and 7 0 mM N a + (as N a C l ) . 79 100-80H c o o 60 o X Ul o <u £ 4 0 H o >> > 5 2 0 o 5? T Stored at 4°C T - i 1 r— 4 8 12 Days after Original Enzyme Extraction i 16 20 F i g u r e 18. E f f e c t o f s t o r a g e a t +4 C on Ca -ATPase a c t i v i t y . 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 5mM C a 2 + , 20 mM T r i s - H C l (pH 8.0), 5 mM ATP ( d i s o d i u m s a l t ) and 70mM N a + (as N a C l ) 00 -D 1 0_ .E H « < o —. t_ • CL CM 10 en (0 o i — 0 5mM Mg 2 + _ T _ i r 200 400 Salmon Calcitonin (mU/ml) 60( F i g u r e 19* E f f e c t o f c a l c i t o n i n on g i l l p l a s m a membrane C a 2 + - ( M g 2 + ) ATPase. • — « , 5 mM C a 2 + i o — o , 5 mM M g 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 20 mM T r i s -H C l (pH 8 . 0 ) , 5 mM ATP ( d i s o d i u m s a l t ) and 70 mM N a + (as 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 e f f e c t o f 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 o f 2+ ?+ S t a n n i u s on g i l l Ca -(Mg ) ATPase i s p r e s e n t e d i n F i g u r e 20. C o r p u s c u l a r e x t r a c t , when added t o t h e i n c u b a t i o n m i x t u r e a t c o n c e n t r a t i o n s r a n g i n g f rom 0.03 t o 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 on b o t h Ca - s t i m u l a t e d and Mg -s t i m u l a t e d ATPase 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 -ATPase a c t i v i t y i s dose - r e l a t e d ( F i g u r e 2 1 ) . A t e x t r a c t c o n c e n t r a t i o n as low as 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 dose o f 0.24 mg p r o t e i n p e r ml r e s u l t e d i n a 30 % i n h i b i t i o n . T a b l e 3 shows t h e s p e c i f i c e f f e c t o f t h e c o r p u s -2+ c l e s o f S t a n n i u s on g i l l Ca -ATPase. C o r p u s c u l a r 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 ml i n c u b a t i o n m i x t u r e , caused a 2 4 - 2 5 % i n h i b i t i o n o f b o t h C a 2 + - a c t i v a t e d and M g 2 + - a c t i v a t e d ATP h y d r o l y s i s . A f t e r heat t r e a t m e n t of t h e same c o r p u s c u l a r e x t r a c t a t 100°C 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 r e d u c e d t o o n l y 8 - 9 %. 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 (0.122 mg p r o t e i n p e r ml i n c u b -a t i o n m i x t u r e ) r e s u l t e d i n a 5 - 7 % i n h i b i t i o n o f enzyme a c t i v i t y . 82 26 -j Extract of Corpuscles of Stannius ( mg Protein /ml) F i g u r e 20. E f f e c t o f e x t r a c t o f c o r p u s c l e s o f S t a n n i u s 2 + 2 + on g i l l p l asma membrane Ca -(Mg ) ATPase. • — • , 5 mM C a 2 + ; 0 O , 5 mM M g 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 20 mM T r i s -HC1 (pH 8.0), 5mM ATP ( d i s o d i u m s a l t ) and 70 mM N a + (as Na C l ) . 83 F i g u r e 2 1 . I n h i b i t o r y 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 2 + o f S t a n n i u s on g i l l Ca -ATPase a c t i v i t y . 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 t r i s - H C l (pH 8.0), 5mM ATP ( d i s o d i u m s a l t ) and 70 mM N a + (as Na C l ) Table 3. Specific effect of the corpuscles of Stannius on g i l l Cafc' (Mg 2 +) ATPase. Incubation mixtures contained 20 mM Tr i s -HCl (pH 8.0), 5 roM ATP (disodium salt) and ?0 mM Na* (as NaCl). Tissue extract was substituted by equal volumes of Tris - HC1 buffer in control samples. Treatment % Inhibition of Ca 2 +-(Mg 2 +) ATPase activity 5 mM C a 2 + 5 mM Mg 2 + Corpuscular extract (0.128 mg protein/ml) 24.6+1.4 23.8+0.8 Heat-treated corpuscular extract (100°C for 15 min) 8.6+1.8 9.0+1.6 Kidney extract (0.122 mg protein/ml) 7.2+1.3 4.9 +2.5 A l l values are given as means + S.E. (n« 3 ) DISCUSSION A. P r o p e r t i e s o f t h e Gij.1 Plasma Membrane Ca*'' - ATPase 2+ I n t h e p r e s e n t i n v e s t i g a t i o n , a Ca - a c t i v a t e d ATPase has been demonstrated i n t h e g i l l t i s s u e s o f t h e Am e r i c a n e e l . R e s u l t s f r om e l e c t r o n m i c r o s c o p i c exam-i n a t i o n and enzyme marker 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 -bound. 2+ 2+ The enzyme can be s t i m u l a t e d by e i t h e r Ca o r Mg 2+ a l o n e , b u t t h e v m a x and a f f i n i t y a r e h i g h e r f o r Ca t h a n 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 by 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 + (Shami and Radde, 1971). However, such Ca p r e f e r e n c e * 2+ 2+ i s n o t obser v e d i n o t h e r Ca -(Mg ) ATPases w h i c h v a r y c o n s i d e r a b l y i n t h e i r d i v a l e n t c a t i o n r e q u i r e m e n t s . 2+ B r a i n m i c r o s o m a l Ca -ATPase can be s t i m u l a t e d e q u a l l y 2+ 2 + w e l l by e i t h e r Ca o r Mg a l o n e (Nakamaru e t a l . , 1967; B e r l and P u s z k i n , 1970). I n t h e i n t e s t i n a l mucosa (Melancon and DeLuca, 1970) and t h e r e n a l t u b u l e s ( P a r k i n s o n and Radde, 1971). w h i l e C a 2 + and M g 2 + c a n s u b s t i t u t e f o r each 2+ 2+ o t h e r , a c t i v a t i o n by Mg i s g r e a t e r t h a n t h a t by Ca . I n t h e s a r c o p l a s m i c r e t i c u l u m (MacLennan, 1970) and t h e e r y t h r o c y t e (Schatzmann and R o s s i , 1971; W o l f , 1972), M g 2 + must be 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 c o n c e n t r a t i o n as ATP t o g i v e f u l l a c t i v i t y . 2+ 2 + E q u i m o l a r c o n c e n t r a t i o n s o f b o t h Ca and Mg 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 i t y l o w e r 2+ 2 + t h a n t h a t w i t h Ca a l o n e and h i g h e r t h a n t h a t w i t h Mgfc a l o n e , s u g g e s t i n g 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 t h e a c t i v e s i t e s . Shami and Radde (1972) have d e r i v e d an e q u a t i o n f o r d e t e r m i n i n g whether 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 . I n d i v i d u a l V , max 2+ 2 + and a p p a r e n t K m f o r Ca and Mg o b t a i n e d f r o m t h i s s t u d y 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 . The c a l c u l a t e d v a l u e 2+ 2+ o f maximum a c t i v i t y w i t h Ca p l u s Mg (1 i 1) was 78$ 2 + t h a t w i t h Ca a l o n e . T h i s d i f f e r s f r om t h e obse r v e d v a l u e (72 $) b y o n l y 6 $, i n d i c a t i n g t h a t C a 2 + and M g 2 + a c t i v a t e t h e g i l l plasma membrane ATPase 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 Ca -(Mg ) ATPases i s t h a t t h e y do n o t r e q u i r e Na or K f o r a c t i v a t i o n (Nakamaru e t a l . . 1967; Schatzmann and V i n c e n z i * 1969? MacLennan, 1970? Melancon and DeLuca, 1970? P a r k i n -s o n and Radde, 1971; Shami and Radde, 1971). T h i s i s a l s o t r u e f o r t h e g i l l C a 2 + - A T P a s e . 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. A l t h o u g h o t h e r n u c l e o t i d e t r i p h o s p h a t e s (ITP and GTP) and d i p h o s p h a t e (ADP) may a l s o be u t i l i z e d , t h e degree o f h y d r o -l y s i s i s much l o w e r t h a n t h a t o f ATP. AMP cannot s e r v e as s u b s t r a t e . U n l i k e t h e ( C a 2 + + M g 2 + ) -ATPase o f t h e r e d b l o o d c e l l (Cha e t a l . , 1971) w h i c h does n o t h y d r o l y s e o t h e r t r i p h o s p h a t e s 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 degree 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 o t h e r •» 2 + 2+ Ca -(Mg ) ATPases (Nakamaru e t a l . . 1967; MacLennan, 1970; Shami and Radde, 1971; D a v i e s and B r a g g , 1972). However, 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 s u b s t r a t e . T h i s i s t h e main 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 an ATPase, 2+ 2+ The r o l e o f d i v a l e n t c a t i o n s (Ca , Mg ) i n t h i s enzyme system i s t o produce a d i v a l e n t i o n -ATP complex w h i c h i s b e l i e v e d t o be 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 (Melanon and DeLuca, 1970; Hyde and R i m a i , 1971). I n t h e e r y t h r o c y t e , W o l f (1972) has demonstrated t h a t Mg2"*-ATP complex r a t h e r t h a n f r e e ATP s e r v e s as s u b s t r a t e f o r t h e enzyme. I n t h e g i l l , a l t h o u g h t h e same a p p a r e n t f o r ATP ( d i s o d i u m s a l t ) was o b t a i n e d by r e p l a c i n g 5 mM C a 2 + w i t h 5 mM M g 2 + , t h e V o f M g 2 + - s t i m u l a t e d ATPase ° ' max ° was o n l y 50% t h a t o f C a 2 + - s t i m u l a t e d ATPase. T h i s f i n d i n g 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 i s u b s t r a t e f o r t h e g i l l enzyme. 2 + The o p t i m a l pH f o r t h e g i l l Ca -ATPase was 7»9 -8.1. T h i s f a l l s w i t h i n t h e range (7.5-8.2) r e p o r t e d f o r t h e m a j o r i t y o f o t h e r C a 2 + - ( M g 2 + ) ATPases (MacLennan, 1970; Melancon and DeLuca, 1970; P a r k i n s o n and Radde, 1971; 88 Shami and Radde, 1971)» w i t h t h e e x c e p t i o n o f t h e b r a i n 2+ 2+ m i c r o s o m a l Ca -(Mg ) ATPase w h i c h has 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 ) ATPase w h i c h shows maximal a c t i v i t y o v e r a wide range o f pH ( P a r k i n s o n and Radde, 1971), t h e g i l l 2+ Ca -ATPase e x h i b i t s a b e l l - shaped pH c u r v e w i t h a d i s t i n c t pH optimum. T h i s f i n d i n g i s i n agreement w i t h t h o s e o f t h e s a r c o p l a s m i c r e t i c u l u m (MacLennan, 1970) and t h e p l a c e n t a (Shami and Radde, 1971). A l t h o u g h t h e enzyme was n o t i s o l a t e d and p u r i f i e d , t h e narrow 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 , under t h e a s s a y c o n d i t i o n s employed, 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 a l k a l i n e p hosphatase 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 o f 8.0. 2+ 2 + The s e a r c h f o r a s p e c i f i c i n h i b i t o r o f Ca -(Mg ) ATPase has been u n s u c c e s s f u l . Ouabain, a s p e c i f i c i n h i b i -t o r o f ( N a + - K + ) ATPase, has no e f f e c t on C a 2 + - ( M g 2 + ) 2+ 2 + ATPases. However, Ca -(Mg ) ATPase a c t i v i t y can 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 i n h i b i t o r s s u c h as e t h a c r y n i c a c i d ( V i n c e n z i , 1968; Shami and Radde, 1971) and m e r s a l y l a c i d ( H a s s e l b a c h and S e r a y d a r i a n , 1966; Schatzmann and V i n c e n z i , 1969; Shami and Radde, 1971). H a s s e l b a c h and S e r a y d a r i a n (1966) have demonstrated 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 a c t i v i t y 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 i n c u b a t e d i n t h e p r e s e n c e o f m e r s a l y l a c i d . S i m i l a r 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 b o t h 2 + t h e Ca -ATPase and t h e c a l c i u m pump a r e i n h i b i t e d by m e r s a l y l a c i d (Schatzmann and V i n c e n z i , 1969). B o t h e t h a c r y n i c a c i d and 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). I n h i b i t i o n 2 + 2 + o f Ca -(Mg ) ATPases by t h e s e i n h i b i t o r s suggests;: t h a t t h e r e a r e SH - groups 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. I n t h e p r e s e n t s t u d y , s i m i l a r i n h i b i t i o n 2+ 2 + o f g i l l Ca -(Mg ) ATPase by m e r s a l y l a c i d was o b s e r v e d , i n d i c a t i n g t h a t f r e e SH - groups 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 g i l l C a 2 + - A T P a s e i s r e l a t i v e l y heat s t a b l e . Marked 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 u n t i l t h e i n c u b a t i o n t e m p e r a t u r e was above 60°C. T h i s i s i n 2+ common w i t h t h e p l a c e n t a l Ca -ATPase (Shami, 1974). However, i n a d d i t i o n t o t h e f i r s t enzyme a c t i v i t y peak a t 30°C, t h e appearance o f a second peak a t 50° C i s 2+ 2 + r a t h e r u n u s u a l among t h e r e p o r t e d Ca -(Mg ) ATPases. The component c o n t r i b u t i n g t o t h i s second peak 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 r e -p a r a t i o n a t 4° C, s u g g e s t i n g t h e p o s s i b l e e x i s t e n c e o f a t e m p e r a t u r e - 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 C a 2 + - ( M g 2 + ) ATPase sys t e m . 9 0 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 a c t i v i t y + •* and 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 Ca -ATPase from t h e w e l l - known (Na -K ) ATPase r e p o r t e d i n t h e g i l l plasma; membranes o f t e l e o s t ( s e e M o t a i s and Garcia-Romeu, 1972). Mg^-dependent, (Na -K ) ATPase has been s t u d i e d i n g i l l t i s s u e p r e p a r -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; K i r s c h n e r , I9691 Zaugg and M c L a i n , 1971? P f e i l e r and K i r s c h n e r , 1972). I n a l l c a s e s , a h i g h 2+ " b a s e l i n e " Mg -ATPase a c t i v i t y , i n s e n s i t i v e t o o u a b a i n , was o b s e r v e d . On t h e b a s i s o f t h e p r e s e n t f i n d i n g s , i t 2+ i s s u g g e s t e d t h a t t h i s Mg - 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 be due t o a c t i v a t i o n o f t h e g i l l Ca -ATPase 2 + w h i c h can a l s o be s t i m u l a t e d by Mg t o a c o n s i d e r a b l e e x t e n t . S i n c e ( N a + - K + ) ATPase i n most s t u d i e s was a s s a y e d 2+ i n t h e pr e s e n c e o f 5 Mg , i t i s v e r y l i k e l y t h a t t h e 2+ Ca -ATPase system was a l s o a c t i v a t e d . One o f t h e pro p o s e d mechanisms f o r a c t i v e t r a n s -2+ p o r t o f Ca a c r o s s c e l l membranes i s v i a t h e a c t i o n o f a C a 2 + - ( M g 2 + ) ATPase. The i d e n t i f i c a t i o n o f a C a 2 + -s t i m u l a t e d ATPase i n t h e g i l l plasma membrane o f t h e A m e r i c a n e e l , w i t h e n z y m a t i c p r o p e r t i e s s i m i l a r t o t h o s e 2+ 2+ o f t h e r e p o r t e d Ca -(Mg ) ATPases, 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 t r a n s p o r t i n f r e s h w a t e r t e l e o s t . 91 B. The E f f e c t o f Hormones on G i l l C a ^ - A T P a s e I t has been demonstrated t h a t s e a w a t e r - a d a p t a t i o n and g i l l ( N a + - K + ) - A T P a s e a c t i v i t y i n t e l e o s t a r e under hormonal c o n t r o l . A d m i n i s t r a t i o n o f C o r t i s o l t o i n t a c t f r e s h w a t e r e e l s i n d u c e d a r i s e i n t h e s p e c i f i c a c t i v i t y o f t h e b r a n c h i a l ( N a + - K + ) - A T P a s e and an i n c r e a s e i n t h e a n i m a l s 1 a b i l i t y t o e x t r u d e N a + from t h e g i l l s ( E p s t e i n e t a l . , 1971; Kamiya, 1972; B u t l e r and C a r m i c h a e l , 1972). On t h e 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 h y p o p h y s e c t o m i z e d e e l s a c c e l e r a t e d t h e d e c r e a s e i n t h e b r a n c h i a l ( N a +-K +)-ATPase a c t i v i t y , and a marked r e d u -c t i o n i n t h e g i l l N a + e f f l u x was r e c o r d e d when t h e a n i m a l were t r a n s f e r r e d f r om s e a w a t e r t o f r e s h w a t e r (Kamiya, 1972). I n C h a p t e r I , c a l c i t o n i n 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 have been de m o n s t r a t e d t o have an 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 - u p t a k e . I t i s p o s s i b l e t h a t t h e s e two hormones a l s o i n f l u e n c e t h e 2+ b r a n c h i a l Ca -ATPase a c t i v i t y . P a r k i n s o n and Radde (1970) r e p o r t e d t h a t c a l c i t o n i n 2+ 2+ s t i m u l a t e d t h e a c t i v i t y o f a Ca -(Mg ) ATPase i n t h e r e d b l o o d c e l l . A s i m i l a r a c t i v a t i o n o f r e n a l plasma 2+ membrane Ca -ATPase by c a l c i t o n i n was o b s e r v e d by S t r e i f l e r and H a r e l l (1974). I n c o n t r a s t , d a t a o b t a i n e d 9 2 i n t h e p r e s e n t s t u d y 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 plasma membrane Ca -ATPase i n v i t r o . 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+ uptake by a mechanism n o t i n v o l v i n g t h e Ca -ATPase, i t i s a l s o p o s s i b l e t h a t hormonal c o n t r o l o f t h e enzyme i s m e d i a t e d 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 . 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 e x h i b i t e d 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 r e s p o n s e i s l o g - d o s e = r e l a t e d . T h i s f i n d i n g s u g g e s t s t h a t t h e c o r p u s c l e s o f S t a n n i u s 2+ may r e g u l a t e b r a n c h i a l Ca -uptake by a d i r e c t c o n t r o l 2+ on t h e membrane-bound g i l l Ca -ATPase. 93 CHAPTER I I I EFFECTS OF SALINITY ADAPTATION, SEXUAL MATURATION AND DIET ON GILL AND GUT MUCOSA PLASMA MEMBRANE CALCIUM - STIMULATED ATPase OF TELEOSTS INTRODUCTION I t has now been 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 e f f i c i e n t c o n t r o l o f t h e system i s t o be a t t a i n e d , t h e apparent K m o f t h e enzyme f o r N a + s h o u l d a p p r o x i m a t e t h e e f f e c t i v e N a + c o n c e n t r a t i o n range i n w h i c h t h e system n o r m a l l y f u n c t i o n s . Enzyme k i n e t i c s t u d i e s have r e v e a l e d t h e e x i s t e n c e o f t i s s u e - s p e c i f i c " f u n c t i o n a l v a r i a n t s " o f N a + - K + ATPase w h i c h appear t o be e l a b o r a t e d f o r p a r t i -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 o f an i n d i v i d u a l (see Hochachka and Somero, 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 + - ( M g 2 + ) ATPase w h i c h has been shown t o be a s s o c i a t e d w i t h a c t i v e t r a n s p o r t o f c a l c i u m i o n a c r o s s c e l l membranes (Malancon and DeLuca, 1970? see M a r t o n o z i , 1972* see Schatzmann, 1975). I n s p e c i f i c b i o l o g i c a l membranes such as the erythrocyte membrane and the sarco-2+ plasmic reticulum which regulate the intracellular Ca —7 —6 concentration (10"' -10 M), the apparent K m of the enzyme for C a 2 + i s in the range of 10"6 -10"^ B8 (Wolf, 1972; Martonosi and Feretos, 1964a, b). In other tissues such as the intestinal mucosa and the renal tubules 2+ where active transcellular Ca transport occurs, the 2+ apparent of the enzyme for Ca i s in a much higher range of 10""-* M (Melancon and DeLuca, 1970; Parkinson and Radde, 1971). + + The high degree of v e r s a t i l i t y in the Na -K ATPase system i s further demonstrated by the fact that more than one functional form of the enzyme is present in the g i l l of catadromous and anadromous fishes under different environmental conditions. In marine stenohaline f i s h and in 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. A high 24* + + level of Mg -dependent, Na -K ATPase activity 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 relatively impermeable g i l l epi-2+ + thelium occurs. Only low levels of Mg -dependent, Na -K + ATPase a c t i v i t y can be d e t e c t e d (Kamiya and U t i d a , 1969). I n s t e a d , t h e r e i s a h i g h l e v e l o f K + - i n d e p e n d e n t , 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 t e l e o s t g i l l p r e p a r a t i o n s ( P f e i l e r and K i r s c h n e r , 1972). I n C h a p t e r I I , a C a 2 + - s t i m u l a t e d ATPase, f o r t h e f i r s t t i m e , has been demonstrated i n g i l l plasma membr-anes o f a t e l e o s t . A l l t h e d a t a p r e s e n t e d a r e s t u d i e s o f a s i n g l e f r e s h w a t e r s p e c i e s , t h e American e e l ( A n g u i l l a  r o s t r a t a ) . 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 o c c u r r e n c e o f t h i s enzyme i n marine and f r e s h w a t e r t e l e o s t s was made. E x p e r i m e n t s were a l s o d e s i g n e d t o i n v e s t i g a t e 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 changes t h a t may o c c u r 2+ i n t h e g i l l Ca -ATPase when a n i m a l s a r e s u b j e c t e d t o d i f f e r e n t environmental c o n d i t i o n s w i t h a change i n e i t h e r t h e i n t e r n a l o r e x t e r n a l c a l c i u m c o n c e n t r a t i o n . 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 and spawning, and d i e t on 2+ t h e g i l l plasma membrane Ca -ATPase. MATERIALS AND METHODS A. Effect of Salinity Adaptation on G i l l Ca2*-ATPase Seven teleost species obtained from various marine and freshwater habitats were examined. Rockcod (Sebastodes paucispinis). Pacific herring (Clupea  pallasi) and striped sea perch (Taeniotoca lateralis ) were chosen as examples of marine f i s h . Rockcods (100-500 g) and striped sea perch (120-140 g) were obtained by diving i n Howe Sound, B.C. Pacific herrings (40-60 g) were caught by netting in the same coastal water. The animals were sampled immediately i n the f i e l d . Four freshwater teleost species were investigated. They were the s i l v e r American eel (Anguilla rostrata). the rainbow trout (Salmo gairdneri), the brown bullhead catfish (Ictalurus nebulosus) and the common carp (Cyprinus  carpio). Silver American eels (800-1,200 g) were caught in rivers along the Eastern coast of Canada (see Materials and Methods, Chapter I ) . Immature rainbow trout (80-120 g) were purchased locally from a commercial hatchery in Mission, B.C. Catfish (120-170 g) and carp (400-800 g) were caught in freshwater ponds i n the Lower Mainland, B.C. The animals were sampled i n the laboratory after an acclimation peroid of at least two weeks. L o c a l l y p u r c h a s e d f r e s h w a t e r r a i n b o w t r o u t (120-180 g) were adap t e d t o a r t i f i c i a l s e a w a t e r under l a b -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 Sea S a l t s (Aquarium Systems I n c . , Ohio, U.S.A.). E x p e r i -m e n t a l a n i m a l s were f i r s t h e l d f o r two weeks i n a 50 g a l l o n f i b r e g l a s s t a n k c o n t a i n i n g r u n n i n g d e c h l o r i n a t e d t a p w a t e r (12°C), t h e n g r a d u a l l y a d a p t e d t o f u l l s t r e n g t h s e a w a t e r o v e r a p e r i o d o f two weeks b e f o r e s a c r i f i c e . Sea w a t e r was f i l t e r e d c o n t i n u o u s l y t o remove n i t r o g e n o u s wastes and was changed t h r e e t i m e s a week. C o n t r o l a n i -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 w a t e r t h r o u g h o u t t h e e x p e r i m e n t . B o t h s e a w a t e r -adapted and c o n t r o l t r o u t were f e d d a i l y w i t h Co-op t r o u t p e l l e t s ( S u r r e y C o - o p e r a t i v e A s s o c i a t i o n , C l o v e r d a l e , B.C.) F o r enzyme a n a l y s i s , g i l l samples were always t a k e n f r o m 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 p lasma membranes were i s o l a t e d by t h e same p r o c e d u r e s as d e s c r i b e d i n M a t e r i a l s and Methods, C h a p t e r I I . Spec-2+ 2+ i f i c Ca - s t i m u l a t e d and Mg - s t i m u l a t e d ATPase a c t i v i -t i e s o f t h e f r e s h g i l l membrane,preparations were measured 2+ 2+ and t h e a p p a r e n t Kffl o f t h e enzyme f o r Ca and Mg were d e t e r m i n e d i n t h e same manner as d e s c r i b e d i n C h a p t e r 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 2*-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 rainbow t r o u t and t h e m i g r a t i n g coho salmon (Oncorhvnchus k i s u t c h ) . 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 c y c l e . 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 c o n d i t i o n s , i n r u n n i n g d e c h l o r i n a t e d w a t e r a t 1 0 + 2 ° C f o r 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 f r o m f r e s h l y s a c r i f i c e d a n i m a l s and g i l l p lasma 2+ membranes were i s o l a t e d . S p e c i f i c Ca - s t i m u l a t e d and M g 2 + - s t i m u l a t e d g i l l ATPase a c t i v i t i e s o f t h e f r e s h membrane p r e p a r a t i o n s were measured and t h e ap p a r e n t 2+ 2+ K m o f t h e enzyme f o r Ca and Mg were d e t e r m i n e d as d e s c r i b e d i n C h a p t e r I I . 1. Rainbow 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 i n M i s s i o n , B.C. F o u r groups o f t r o u t were sampled i a. Young, immature t r o u t w e i g h i n g 80 - 1 2 0 g. They were a p p r o x i m a t e l y 18 months o l d and t h e i r s e x e s were i n d i s t i n g u i s h a b l e a t t h i s s t a g e . b. A d u l t , m a t u r i n g 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 d e v e l o p e d . 99 c. Spawning male t r o u t w e i g h i n g 200 -500 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. d. Spawning fema l e t r o u t w e i g h i n g 600 -800 g. They were over 3 y e a r s o l d and were a t t h e peak o f spawning. 2. Coho Salmon M i g r a t i n g coho salmon were o b t a i n e d from 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 , West Vancouver, B.C. They were h a t c h e d 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 c r e e k a t t h e s t a t i o n . F o u r groups o f coho salmon were sampled i a. Young immature coho w e i g h i n g 8 0 - 1 0 0 g. They were one y e a r o l d f r e s h w a t e r f i s h and t h e i r sexes were s t i l l i n d i s t i n g u i s h a b l e . b. A d u l t m a t u r i n g coho w e i g h i n g 90-120 g. They were two y e a r s o l d seawater f i s h and t h e i r gonads were w e l l d e v e l o p e d a t t h i s s t a g e . c. Spawning male coho w e i g h i n g 500 -600 g. A f t e r 2 1/2 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. Spawning female coho w e i g h i n g 600 - 700 g. They were i n t h e same c o n d i t i o n as t h e spawning male coho i n group c. 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 groups o f l o c a l l y p u r c h a s e d mature rainbow t r o u t (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 o f r u n n i n g d e c h l o r -i n a t e d w a t e r f o r two weeks, and were f e d d a i l y w i t h Co-op t r o u t p e l l e t s . One group 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 weeks, under t h e same e x p e r i m e n t a l c o n d i -t i o n s . The o t h e r group was f e d t h r o u g h o u t t h e e n t i r e e x p e r i m e n t and s e r v e d as t h e c o n t r o l . On t e r m i n a t i o n 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 blow on t h e head. G i l l samples were t a k e n i m m e d i a t e l y . The g u t o f b o t h s t a r v e d and c o n t r o l a n i m a l s were c a r e f u l l y examined f o r m o r p h o l o g i c a l d i f f e r e n c e s and f o o d c o n t e n t . The e n t i r e i n t e s t i n e was removed from t h e f i s h and r i n s e d i n i c e - c o l d 0.9$ s a l i n e . I t was t h e n s l i t l e n g t h w i s e and t h e mucosa were h a r v e s t e d by s c r a p i n g w i t h a g l a s s k n i f e . G i l l and gut mucosa plasma membranes were t h e n i s o l a t e d by a p r o c e d u r e s i m i l a r t o t h a t d e s c r i b e d i n 2+ M a t e r i a l s and Methods, C h a p t e r I I . S p e c i f i c Ca - s t i m u -2+ l a t e d and 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 measured 2+ 2+ and t h e a p p a r e n t o f t h e enzymes f o r Ca and Mg were d e t e r m i n e d . 101 RESULTS A. E f f e c t of S a l i n i t y Adaptation on G i l l Ca 2*-ATPase A number of te l e o s t obtained from d i f f e r e n t marine and freshwater habitats were investigated. A s i m i l a r 2+ Ca -ATPase was i d e n t i f i e d i n the g i l l plasma membrane of a l l seven t e l e o s t species 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 of the 2+ seven t e l e o s t g i l l Ca -ATPases. The g i l l enzymes varied 2+ 2+ i n both t h e i r rate of Ca -stimulated and Mg -stimulated ATP hydrolysis and t h e i r a f f i n i t y f o r C a 2 + and Mg 2 +. The majority of s p e c i f i c enzyme a c t i v i t y f e l l within the range of 14 - 30 jumoles P i per mg protein per hour. An exceptionally high l e v e l of 125 units was observed i n the common carp, which was approximately 6X the average s p e c i f i c a c t i v i t y recorded. Apart from the c a t f i s h , v m a x was higher f o r C a 2 + than f o r Mg 2 + i n a l l the g i l l C a 2 + -2+ ATPases i d e n t i f i e d . In the c a t f i s h , v _ _ f o r Mg was 2+ about 20% higher than that f o r Ca . No d i s t i n c t 2+ difference i n the s p e c i f i c Ca -ATPase a c t i v i t y could be observed between the marine and the freshwater t e l e o s t However, the apparent 1^ f o r divalent cations appeared to be higher i n the marine t e l e o s t than i n most of the freshwater species. Table 4. Ca -ATPase in g i l l plasma membrane of marine and freshwater teleosts. Incubation mixtue contained 20 mM Tris-HCl (pH 8.0), 5 mM ATP (disodium salt) and 70 mM Na + (as NaCl). Species No. of Animals Sp. Activity 3 , (5mM Ca 2 +) Sp. Activity 3 , (5mM Mg 2 +) % Activity Mg 2 +/Ca 2 + App. K m(Ca2 +) (mM) App.Kffl(Mg2+) (mM) A. Marine Fish Rockcod 6 24 .0 + 3 . 2 1 9 .1 + 2 . 5 7 9 . 2 1.29+0.04 2.36 + 0.14 Herring 6 3 0 . 5 + 0 . 8 2 3 .9 + 0 . 4 7 8 . 2 0 . 8 9 + 0 . 0 2 1.9^ + 0 . 0 7 Sea Perch 2 18 .1 +1 .2 9 .0+0.6 49.9 1.18 2 . 0 0 6. Freshwater Fish Am. Eel 5 24 . 5 + 0 .8 12 . 0 + 0 . 4 49.7 0 . 3 8 + 0 . 0 3 0.64 + 0 . 0 7 Trout 10 1 4 . 6+ 1 . 0 ' 1 0 .6+0.7 72.8 0 .38 + 0 . 0 1 0.58 + 0 . 0 2 Catfish 9 1 8 . 4 + 0.6 22 . 9 + 0.6 119.1 0.64 + 0 .06 1 .34 + 0 . 0 5 Carp 11 125.0 + 3 . 9 107 . 0 + 3.7 8 5 . 0 1 .69 + 0.09 3 . 6 7 + 0.19 a Specific activity i s expressed as ;umoles Pi/mg protein/hr. Values are given as means+S.E. H o 140 - i 120 -100 -••-~ 80 -o < g 6 0 -o 0. < 40 -20 -0 -Cod rah Herring Perch Sea Water • 5 mM Ca 2 + Eel Fresh Water 5mM Mg 2* F i g u r e 22. G i l l plasma membrane C a 2 + - A T P a s e o f marine and f r e s h w a t e r t e l e o s t s . 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 as umoles P i / mg p r o t e i n / h r . Trout Catfish Carp •* The e l e c t r o l y t e c o m p o s i t i o n s o f d e c h l o r i n a t e d t a p w a t e r and a r t i f i c i a l s e a w a t e r i n t h e S a l i n i t y A d a p t a t i o n E x p e r i m e n t a r e p r e s e n t e d i n T a b l e 5* C a l c i u m 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 i n e x t r e m e l y low c o n c e n t r a t i o n s (10 -10 J mM). I n t h e a r t i f i c i a l s e a w a t e r , c a l c i u m l e v e l was 9.3 mM and magnesium c o n t e n t was 50 mM. A c c l i m a t i o n o f f r e s h w a t e r r a i n b o w t r o u t t o a r t i f i c i a l s e a w a t e r 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 plasma membrane Ca -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 se a w a t e r - adapt 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 f o r M g 2 + was 74$ 2+ t h a t f o r Ca i n t h e seawater - adapt e d a n i m a l s . I n t h e c o n t r o l f i s h , i t was 75$. 2+ B. E f f e c t o f M a t u r a t i o n and Spawning i n G i l l Ca -ATPase 2+ G i l l plasma membrane Ca -ATPases o f rainbow t r o u t and m i g r a t i n g coho salmon a t v a r i o u s s t a g e s o f s e x u a l development were examined and t h e r e s u l t s a r e summarized i n T a b l e 7. S p e c i f i c C a 2 + - s t i m u l a t e d and M g ^ - s t i m u l a t e d ATPase a c t i v i t i e s a r e i l l u s t r a t e d by h i s t o g r a m s ( F i g u r e 23). 2+ I n r a i n b o w t r o u t , s p e c i f i c Ca -ATPase a c t i v i t y i n t h e young immature a n i m a l s was f o u n d t o be o n l y 50$ t h a t o f t h e a d u l t mature f i s h . 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. E l e c t r o l y t e c o m p o s i t i o n o f t h e d e c h l o r i n a t e d t a p w a t e r and a r t i f i c i a l s e a w a t e r i n t h e S a l i n i t y A d a p t a t i o n E x p e r i m e n t . D e c h l o r i n a t e d Water A r t i f i c i a l Sea Water E l e c t r o l y t e (m.moles/l) (m.moles/l) N a + 5.45 444 K + 8.40 X10"* 1 9.5 C a 2 + 5 . 6 0 X 1 0 " 2 9.3 M g 2 + 7.00 X 1 0 " 3 50 P i 4.00 X 1 0 " 4 1.0 X 1 0 ~ 2 106 T a b l e 6. E f f e c t o f seawater a d a p t a t i o n on Ca -ATPase i n g i l l plasma membranes o f rai n b o w t r o u t , S. g a i r d n e r i . No. o f Sp. A c t i v i t y 2 1 Sp. A c t i v i t y a % A c t i v i t y C o n d i t i o n # 2+^ 2+/„ 2+ A n i m a l s (5mM C a 2 + ) (5 mM Mg^ ) Mg^ /Ca* C o n t r o l 5 30.5 +1.4 23.0 +1.0 75.4 Seawater- ? 31.9 +1.6 23.6 +1.2 74.0 adapted a 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 as 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 as means +S.E. Table 7. Effect of sexual maturation and spawning on Ca -ATPase i n g i l l plasma membrane of rainbow trout and migrating coho salmon. Incubation mixtures contained 20 mM Tris-HCl (pH 8.0), 5 mM ATP (disodium salt) and ?0 mM Na + (as NaCl). Mo. of Sp.Activity a Sp.Activity21 % Activity App.Km(Ca2+) App.Km(Mg2+) Animals (5mMCa*' ) (SmMMg* ) Mg* /Ca* (mM) (mM) Rainbow Trout Young immature, P.W. 10 14.6 +1.0*** 10.6 + 0.7*** 72.8 0.38 + 0.01 . 0 . 5 8 +0.02 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 Spawning °. , F.W. 5 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 0 , F.W. 4 18.6+1.2 11.4 + 0.7 60.9 0.67 1.11 aSpecific activity is expressed as jumoles Pi/mg protein/hr. Values are given as means +S.E. ( )*» ( )**» ( denote significant difference as compared with corresponding values of other groups within the same speciesj P being <0.05, < 0.005 and <0.001 respectively. F.W. -fresh water? S.W. . sea water. 30 20 H > o < co 10 o ft ft Young Adult * ? Spawn. Trout • 5 m M Ca 2 + Young Adult N ft ft Spawn. • 5 mM Mg Coho Salmon z* ,2+ F i g u r e 2 3 . 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 i n g i l l plasma membrane o f rainbow t r o u t and m i g r a t i n g coho salmon. 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 as jumoles Pi/mg p r o t e i n / h r . 109 2 + o b s e r v e d i n t h e r a t e o f Mg - s t i m u l a t e d ATP h y d r o l y s i s . 2+ S u b s e q u e n t l y , t h e r a t i o o f v m a x f o r Mg t o t h a t f o r 2+ Ca remained r e l a t i v e l y c o n s t a n t . Spawning had l i t t l e e f f e c t on t h e s p e c i f i c enzyme a c t i v i t y o f a d u l t mature a n i m a l s . F u r t h e r m o r e , no 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 2+ Ca -ATPase a c t i v i t y was o b s e r v e d between t h e spawning male and f e m a l e t r o u t . The a p p a r e n t o f t h e enzyme f o r d i v a l e n t c a t i o n s was f o u n d t o be h i g h e r i n t h e mature a d u l t s t h a n i n t h e young a n i m a l s . I n t h e spawning male, 2+ 2+ t h e a p p a r e n t f o r Mg was t h e same as t h a t f o r Ca . I n t h e 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+ i n s p e c i f i c g i l l Ca -ATPase a c t i v i t y w i t h m a t u r a t i o n 2+ and spawning was o b s e r v e d . The r a t e o f Ca - s t i m u l a t e d 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 t h e young immature a n i m a l s was 80 - 85 % t h a t o f t h e matured a d u l t s . No s i g n i f i c a n t d i f f e r e n c e i n s p e c i f i c enzyme a c t i v i t y was d e t e c t e d between t h e s e x u a l l y matured a d u l t s and t h e spawning a n i m a l s . The a p p a r e n t K m o f t h e enzyme f o r t h e 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 s e a -w a t e r a d u l t s t h a n i n t h e f r e s h w a t e r a n i m a l s . I n t h e 2+ spawning male salmon, t h e a p p a r e n t f o r Mg was 2+ a p p r o x i m a t e l y t h e same as t h a t f o r Ca . 2+ C. E f f e c t o f D i e t on G i l l and Gut Mucosa Ca -ATPase 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 s t i n e o f t h e c o n t r o l a n i m a l , t h e i n t e s t i n e o f t h e s t a r v e d f i s h was v e r y s m a l l and l i n e d w i t h o n l y 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 any o f t h e s t a r v e d t r o u t . The 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 plasma 2+ membrane Ca -ATPase o f r a i n b o w t r o u t i s summarized i n T a b l e 8. There was no 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 2+ 2+ s p e c i f i c g i l l Ca - s t i m u l a t e d and Mg - s t i m u l a t e d ATPase a c t i v i t y between t h e s t a r v e d and c o n t r o l a n i m a l s . S t a r v a t i o n f o r s i x weeks d i d n o t r e s u l t i n any marked a l t e r a t i o n i n t h e ap p a r e n t o f t h e enzyme f o r d i v a l e n t c a t i o n s . 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 Ca -ATPase o f r a i n b o w t r o u t was a l s o i n v e s t i -g a t e d and 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« S t a r -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 Ca -s t i m u l a t e d ATPase a c t i v i t y from 5*1 t o 7»5 ^moles P i 2+ p e r mg p r o t e i n p e r hour.- The s p e c i f i c Mg - s t i m u l a t e d ATPase a c t i v i t y , 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 about 7.4 u n i t s . I n c o n t r a s t t o t h e c o n t r o l 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 gut mucosa ATPase a c t i v i t y 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 by Ca -s t i m u l a t i o n , t h e gut enzyme o f t h e s t a r v e d t r o u t appeared t o r e s p o n d e q u a l l y w e l l t o s t i m u l a t i o n b y b o t h d i v a l e n t 2+ c a t i o n s . The a p p a r e n t o f t h e enzyme f o r Ca i n t h e Table 8. Effect of starvation on Ca2+-ATPase in g i l l plasma membrane of rainbow trout, S. gairdneri. No. of Sp. Activity 2 1 Sp. A c t i v i t y a % Activity App. K^Ca 2*) App. K^Mg2*) Condition o . 5 . P + O + Animals (5mM Ca* ) (5 mM Mg* ) Mg* /Ca* (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 a Specific activity 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 in gut mucosa plasma membrane of rainbow trout, S. gairdneri. No. of Sp. Activity a Sp. Activity 5 % Activity App. K (Ca2 ) App. K (Mg2 ) Condition 9 , 0. , m • m Animals (5 mM Ca**) (5mM Mg* ) Mg* /Ca* (mM) (mM) Control 8 5.14 + 0.56 7.43 + 0.68 144.6 0.38 + 0.02 1.40 +0.16 Starved 8 7«53±0.56** 7.36 + 0.44 97.4 0.93 + 0.02*** 1.18 +0.04 a Specific activity is 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 t h e c o n t r o l . No s i g n i f i c a n t change i n t h e a p p a r e n t K f o r M g 2 + was o b s e r v e d as a r e s u l t o f s t a r v a t i o n . 114 DISCUSSION I n t h e p r e s e n t s t u d y , t h e e x i s t e n c e o f a Ca f c'-ATPase has been demonstrated i n t h e g i l l plasma 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 f r om b o t h m a rine and 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 o c c u r r e n c e o f t h i s g i l l enzyme among t e l e o s t s . Some e n z y m a t i c p r o p e r t i e s s u c h as s p e c i f i c 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 n a t u r e o f t h e enzyme a c t i v e s i t e s a r e f u n d a m e n t a l l y s i m i l a r . I n a l l s p e c i e s , t h e g i l l enzyme can be s t i m u -l a t e d by e i t h e r Ca o r Mg a l o n e . Ca i s t h e p r e f e r -e n t i a l c a t i o n o f a c t i v a t i o n e x c e p t i n c a t f i s h . The 2+ enzyme a f f i n i t y f o r Ca i s c o n s i s t e n t l y h i g h e r t h a n 2+ t h a t f o r Mg , 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 a 2 + - a c t i v a t e d ATPase. A. S a l i n i t y A d a p t a t i o n U n l i k e t h e g i l l N a + - K + ATPase w h i c h i s found a t much h i g h e r l e v e l o f a c t i v i t y (2 - 5 X ) i n marine t e l e o s t s t h a n i n f r e s h w a t e r s p e c i e s (Kamiya and U t i d a , 1969)» t h e r e seems t o be no 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 between marine and 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 r e s p o n s e i n g i l l Ca -ATPase 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 ad a p t e d t o seawater under l a b o r a t o r y c o n d i t i o n s . However, 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 f r e s h w a t e r s p e c i e s t h a n i n marine t e l e o s t s . S i n c e t h e c a l c i u m 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 Ca a f f i n i t y i s i n acco r d a n c e w i t h a more e f f i c i e n t l y 2+ c o n t r o l l e d Ca -ATPase f u n c t i o n i n t h e g i l l s o f f r e s h -w a t e r t e l e o s t s . D u r i n g a d a p t a t i o n t o s e a w a t e r , a d i r e c t c o r r e l -a t i o n "between environmental s a l i n i t y and g i l l N a + - K + ATPase a c t i v i t y has been o b s e r v e d i n many e u r y h a l i n e t e l e o s t s s uch as t h e European e e l , A n g u i l l a a n g u i l l a ( 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 ( E p s t e i n e t a l . , 1967; B u t l e r and C a r m i c h a e l , 1972), t h e Japanese e e l , A. .iaponica ( U t i d a e t a l . , 1971)» t h e coho salmon, Oncorhynchus k i s u t c h (Zaugg and M c L a i n , 1970; 1971) and t h e r a i n b o w t r o u t , Salmo g a i r d n e r i ( P f e i l e r and K i r s c h n e r , 1972). Such a d a p t a t i v e i n c r e a s e i n g i l l Na -K ATPase a c t i v i t y d i s a p p e a r s when t h e s e a w a t e r - adap t e d a n i m a l s a r e r e t u r n e d t o f r e s h w a t e r . The h i g h e r l e v e l o f g i l l Na -K ATPase a c t i v i t y i n marine and seawater T 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 -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 b r a n c h i a l sodium e x c r e t i o n i n s e a w a t e r w h i c h i s v e r y h i g h i n N a + c o n t e n t (about 450 -500 mM). I n f r e s h w a t e r , l o w e r i n g 116 o f g i l l Na -K ATPase a c t i v i t y i s i n res 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 e x c r e t i o n t o o c c u r 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 problem o f c o n t i n u a l l o s s o f Na t o t h e e x t e r n a l w a t e r i n w h i c h t h e Na l e v e l i s v e r y low ( u s u a l l y l e s s t h a n 5 mM). I n c o n t r a s t , f r e s h w a t e r t e l e o s t s a r e c a p c a b l e o f a c t i v e u p t a k e o f N a + from t h e e n v i r o n m e n t a l w a t e r . The i n w a r d N a + pump i n th e g i l l s o f f r e s h w a t e r t e l e o s t i s t h o u g h t 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 f o r m o f N a + - 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, indepe n d e n t o f K + and 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 i n h i b i t o r ( M o t a i s , 1970; P f e i l e r and K i r s c h n e r , 1972). T h i s N a + a c t i v a t i o n i s p e c u l i a r t o f r e s h w a t e r a n i m a l s and 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 t o s e a w a t e r . 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 presumably would e n c o u n t e r t h e same problem i n 2 + + Ca r e g u l a t i o n as 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 d i f f e r e n t e n v i r o n m e n t a l s a l i n i t y . Assuming t h e r e i s 2+ a r o l e f o r t h e g i l l Ca -ATPase i n a c t i v e b r a n c h i a l 2+ Ca -up t a k e w h i c h has now been dem o n s t r a t e d i n t h e Amer-i c a n e e l (see C h a p t e r I ) , t h e l a c k o f e f f e c t o f e n v i r o n -m 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 t h a t b r a n c h i a l C a 2 + - u p t a k e i s p r o b a b l y u n a f f e c t e d . The 117 mechanism by 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 c a l c i u m l e v e l i n s e a w a t e r remains t o be e l u c i d a t e d . However, 2+ t h e p r e s e n t d a t a on g i l l Ca -ATPase a c t i v i t y a r e o b t a i n e d by k i n e t i c s t u d i e s on i s o l a t e d g i l l plasma - membrane p r e p a r a t i o n s i n w h i c h t h e v e c t o r i a l component o f t h e enzyme cannot be d i f f e r e n t i a t e d . I n view o f t h e f a c t t h a t i n f i s h g i l l t h e ATPase - r e l a t e d H a + pump can f u n c t i o n i n o p p o s i t e d i r e c t i o n s depending on t h e e x t e r n a l s a l i n i t y , i t i s p o s s i b l e t h a t d u r i n g 2+ s e a w a t e r - a d a p t a t i o n , 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 v i a a b i o c h e m i c a l m o d i f i -c a t i o n o f t h e v e c t o r a l component i n t h e enzyme. Our 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 t r a n s -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 does 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 -ATPase a c t i v i t y i n t h e g i l l s o f b o t h t h e rain b o w t r o u t and t h e coho 2+ salmon. . I n a d u l t m a t u r i n g f i s h , a h i g h e r plasma Ca l e v e l i s n o r m a l l y o b s e r v e d , w h i c h i s p r o b a b l y a s s o c i a t e d w i t h an i n c r e a s e d c a l c i u m s t o r a g e f o r t h e d e v e l o p i n g 2+ gonads. Assuming t h a t Ca -ATPase i s i n d e e d p a r t o f 2+ t h e b r a n c h i a l Ca t r a n s p o r t m achinery i n t h e e p i t h e l i a l membrane, t h i s increase i n capacity of the enzyme system may represent a biochemical adaptation i n response to a greater demand f o r calcium by the adult, maturing a n i -mals. There appears to be no s i g n i f i c a n t difference i n 2+ the branchial Ca -ATPase a c t i v i t y between the adult f i s h and the spawning animals. Hypercalcemia has been observed i n many female tel e o s t s during the breeding season (Oguri and Takada, 1967 j Woodhead, 19681 Fontaine et, a l . , I969; U r i s t et , 1972). The dramatic r i s e i n plasma calcium i s attributed to the increase i n endogenous secretion of female sex hormone. 2+ Based on the hypothesis that Ca -ATPase i s involved i n 2+ branchial Ca -uptake i n the t e l e o s t , the lack of observ-able response of the enzyme to spawning suggests that calcium source(s) other than environmental water c o n t r i -butes to the estrogen-induced increase i n plasma calcium of the spawning females. In b i r d s , i t i s evident that the long bones are involved i n the storage and mobilization of extra calcium during egg-production (see Simkiss, 1967). Whether the bone i s s i m i l a r l y involved i n teleosts i s s t i l l uncertain. However, during the experimentally induced sexual maturation of the European e e l pro-duced by chronic i n j e c t i o n of the carp p i t u i t a r y extract, Lopez and Bagot (1971) reported a strong stimulation of osteoclasis 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 showed a g r e a t l o s s i n m i n e r a l c o n s t i t u e n t . 2+ The p r e s e n t 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 t h e p o s s i b i l i t y t h a t d u r i n g s e x u a l m a t u r a t i o n o f t h e t e l e o s t , t h e r i s e i n body c a l c i u m c o n t e n t i s v 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 -uptake s y s t e m . 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 and i s s u b s e q u e n t l y m o b i l i z e d d u r i n g spawning f o r egg-p r o d u c t i o n . However, u n t i l more d i r e c t i n f o r m a t i o n 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 t r a n s -p o r t d u r i n g d i f f e r e n t d e v e l o p m e n t a l s t a g e s i n t h e 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 as s p e c u l a t i o n s . C. D i e t and 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 n o t e t h a t t h e 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 average enzyme l e v e l f o u n d i n seven o t h e r t e l e o s t s p e c i e s . T h i s p a r t i c u l a r l y h i g h l e v e l o f b r a n -2+ c h i a l Ca -ATPase a c t i v i t y may be r e l a t e d t o i t s h e r b i -v o r o u s h a b i t a t . Under n o r m a l c o n d i t i o n s , t h e p r i n c i p a l 2+ Ca i n t a k e i n t e l e o s t s i s i n t h e form o f f o o d w h i c h i s b e i n g a b s o r b e d t h r o u g h l o c a l i z e d r e g i o n s o f t h e gut d u r i n g f e e d i n g (see L o v e , 1970). S i n c e t h e l e v e l o f 120 c a l c i u m i n p l a n t m a t e r i a l s i s v e r y low, i t i s p o s s i b l e t h a t t h e c a r p has t o r e l y more on t h e e n v i r o n m e n t a l w a t e r f o r d i v a l e n t c a t i o n s u p p l y . As a p r e l i m i n a r y t e s t o f t h e above h y p o t h e s i s , t h e 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 Ca -ATPase was examined. S i n c e t e l e o s t f i s h a r e known t o p o s s e s s d i f f e r e n t s e t s o f gut d i g e s t i v e enzymes depending on t h e i r d i e t (see L o ve, 1970), t h e p o s s i b l e changes t h a t 2+ may o c c u r i n t h e gut mucosa Ca -ATPase w i t h s t a r v a t i o n 2+ were a l s o i n v e s t i g a t e d . I f t h e h i g h b r a n c h i a l Ca -ATPase a c t i v i t y i n t h e c a r p i s i n d e e d due t o t h e low l e v e l o f d i v a l e n t c a t i o n s a v a i l a b l e i n i t s d i e t , one w o u l d e x p e c t t h a t complete r e m o v a l o f t h e f o o d s o u r c e i n a n o r m a l f e e d i n g f i s h may i n d u c e an e l e v a t i o n i n t h e g i l l Ca -ATPase a c t i v i t y w i t h a p o s s i b l e conco-m i t a n t d e p r e s s i o n i n t h e g u t enzyme l e v e l . Data o b t a i n e d i n t h e p r e s e n t s t u d y i n d i c a t e t h a t s t a r v a t i o n f o r s i x weeks d i d n o t r e s u l t i n any o b s e r v a b l e changes i n t h e b r a n c h i a l C a 2 + - A T P a s e a c t i v i t y o f t h e r a i n b o w t r o u t . I n t h e gut mucosa, an i n c r e a s e i n t h e enzyme c a p a c i t y b y Ca - s t i m u l a t i o n was accompanied by a d e c r e a s e i n 2+ t h e enzyme a f f i n i t y f o r Ca . W h i l e t h e s e f i n d i n g s appear t o c o n t r a d i c t t h e p r o p o s e d h y p o t h e s i s , two o t h e r p o s s i b i l i t i e s s h o u l d be c o n s i d e r e d . F i r s t l y , t h e e v o l u t i o n o f a h i g h c a p a -2 + c i t y b r a n c h i a l Ca -ATPase system i s a l o n g - t e r m a d a p t -a t i v e event w h i c h cannot be d u p l i c a t e d i n a s h o r t - t e r m e x p e r i m e n t . S e c o n d l y , i t i s w e l l - e s t a b l i s h e d t h a t d u r i n g s t a r v a t i o n , t h e f i s h i s c a p a b l e o f d i g e s t i n g i t s own muscle i n o r d e r t o s u r v i v e . 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 c o n t e n t 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 b r e a k -down o f p a r i e t a l muscle f o r energy s u p p l y d u r i n g s t a r -v a t i o n w i l l c o n t i n u a l l y r e l e a s e e x t r a c a l c i u m i n t o t h e c i r c u l a t i o n . T h i s may w e l l compensate f o r t h e l o s s o f d i e t a r y c a l c i u m i n t a k e . As a r e s u l t , s h o r t - t e r m s t a r -v a t i o n i s n o t d i r e c t l y comparable t o a h e r b i v o r o u s d i e t i n w h i c h t h e e x t r a c a l c i u m s u p p l y f r o m muscle i s a b s e n t . 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 gut and g i l l enzymes o f h e r b i v o r o u s f i s h i s r e q u i r e d t o v e r i f y t h e proposed h y p o t h e s i s . 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 d e s c r i b e d i n t h i s c h a p t e r has c l e a r l y d emonstrated t h e g e n e r a l 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 o f t e l e o s t s . The enzyme system e x h i b i t s some degree 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 + ATPase syst e m , however, many o f t h e 2+ ob s e r v e d b i o c h e m i c a l v a r i a t i o n s i n t h e 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 1 2 2 e n v i r o n m e n t a l c o n d i t i o n s . Our l a c k o f knowledge on t h e k i n e t i c s o f a c t i v e 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 i n t e l e o s t under e n v i r o n m e n t a l i n f l u e n c e makes i t d i f f i c u l t t o i n t e r p r e t t h e c u r r e n t d a t a . U n t i l a b e t t e r u n d e r s t a n d i n g 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 , t h e f u n c t i o n a l s i g n i f i c a n c e o f t h e s e a d a p t a t i v e b i o c h e m i c a l changes i n t h e g i l l Ca -ATPase cannot be f u l l y a p p r e c i a t e d . 123 GENERAL DISCUSSION The objective of this thesis i s to study the role of the g i l l i n teleostean calcium regulation, with special 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 teleost, Anguilla rostrata. which enables the 2+ f i s h to ef f i c i e n t l y take up Ca from the environmental water. Whether this represents a primary or secondary 2+ Ca pump coupled with the transport of other ions has not 2+ been clearly defined. However, identification of a Ca -ATPase in the g i l l plasma membranes, with specific enzyme acti v i t y easily 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 Ca 2 +-(Mg 2 +) ATPase i n Ca 2 +-2+ uptake by the sarcoplasmic reticulum and in Ca -extrusion by the red blood cells i s well-demonstrated (Martonosi, 1972; Schatzmann, 1975). A role for Ca 2 +-(Mg 2 +) ATPase has also been proposed in the asymmetrical transcellular 2+ Ca transport that occurs in the intestinal mucosa (Melancon and DeLuca, 1970), the renal tubules (Parkinson 124 and Radde, 1971; R o r i v e and K l e i n z e l l e r , 1972) and t h e p l a c e n t a (Shami, 1974). The e x i s t e n c e o f a membrane-2+ bound g i l l Ca -ATPase, 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 ) ATPases, 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 be p a r t o f t h e b r a n c h i a l Ca t r a n s p o r t s y s t e m , s e r v i n g as a s o u r c e o f m e t a b l i c energy 2+ f o r t h e a c t i v e Ca -uptake p r o c e s s . C a l c i u m - u p t a k e i n t h e g i l l e p i t h e l i u m i n v o l v e s 2+ movement p f Ca from one c e l l s u r f a c e t o a n o t h e r c e l l 2+ s u r f a c e . 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+ t h e c o m b i n a t i o n o f a Ca pump i n one plasma membrane w i t h 2+ h i g h Ca p e r m e a b i l i t y i n t h e o p p o s i t e plasma membrane 2+ 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 o f Ca pump i n t h e b r a n c h i a l e p i t h e l i u m i s n o t e l u c i d a t e d i n t h e p r e s e n t s t u d y . By a n a l o g y w i t h t h e r e d b l o o d c e l l s (Schatzmann, 2+ 1975)» i t would seem l i k e l y t h a t t h e a c t i v e Ca t r a n s -p o r t o c c u r s 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 A m e r i c a n 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 hormonal c o n t r o l . C a l c i t o n i n f r om t h e 2+ u l t i m o b r a n c h i a l g l a n d s enhances t h e Ca -uptake p r o c e s s . However, t h e hormone has no e f f e c t on t h e a c t i v i t y o f 2+ Ca -ATPase i n g i l l plasma membrane p r e p a r a t i o n s . I n mammals, one o f t h e pro p o s e d mechanisms 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 by a c t i v a t i o n o f t h e 125 membrane-bound adenyl cyclase, leading to an increased intracellular level of cyclic-AMP which serves as a "second messenger" e l i c i t i n g the characteristic hormonal response (see Gray et aJL., 1 9 7 4 ) . It i s possible that calcitonin acts on the teleostean g i l l epithelial c e l l vi a a similar mechanismj hormonal regulation of branchial Ca 2 +-uptake i s achieved by an ultimate effect on the 2+ 2+ membrane Ca pump or the membrane permeability to Ca . Extract of the Stannius corpuscles has a potent 2+ inhibitory effect on both branchial Ca -uptake and g i l l 2+ Ca -ATPase ac 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 teleosts, the hypocalcemic effect of the corpuscles of Stannius observed i n vivo i s due, at 2+ least partly, to inhibition of branchial Ca -uptake by 2+ a direct action of the hormone on the membrane Ca pump. Although the physiological significance of the corpuscles of Stannius i n teleostean calcium metabolism i s now well-established, the chemical nature of the glan-dular secretion remains a subject of controversy. Bio-chemical characterisation of the active hypocalcemic principle(s) has not been possible due to the lack of a simple and sensitive bioassay for the corpuscle "hormone". Pang and co-workers ( 1 9 7 3 ) 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 intact k i l l i f i s h . The bioassay consists of 4 daily injections of the cor-puscle homogenate into k i l l i f i s h pre-adapted for six weeks to calcium-deficient sea water and a low-calcium diet. The hypocalcemic response i s determined two hours after the f i n a l injection. The routine application of this bioassay i s limited because i t i s both time-consuming and lacking i n sensitivity for quantitative analysis. As revealed i n the present study, the corpuscle extract exhibits a potent inhibition on the activity of the g i l l 2+ plasma membrane Ca -ATPase. The effect i s specific to corpuscles of Stannius (Table 3) and the response i s log-dose related (Figure 21)• The i n v i t r o enzyme assay i s quick (one hour incubation) and the experimental para-meters can be easily controlled. This relatively simple method provides a more efficient bioassay for elucidating the biochemical nature of the yet unidentified corpuscle hypocalcemic "hormone'*. The assay i s sensitive enough for quantitative analysis essential i n the purification of the hormone. The work described in this thesis provides infor-mation towards a new understanding of calcium regulation i n the teleost. In the freshwater American eel, bran-2+ chial 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 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 and t h e S t a n n i u s c o r p u s c l e " f a c t o r " have an e f f e c t on t h i s c a l c i u m t r a n s p o r t system, s u g g e s t i n g t h e g i l l as an i m p o r t a n t t a r g e t o r gan of hormonal a c t i o n i n t e l e o s t e a n c a l c i u m h o m e o s t a s i s . A d e t a i l e d i n v e s t i g a t i o n o f t h e k i n e t i c s 2+ o f b r a n c h i a l Ca t r a n s p o r t i n t e l e o s t s under v a r i o u s e n v i r o n m e n t a l and p h y s i o l o g i c a l c o n d i t i o n s w i l l p r o v e v a l u a b l e i n f u r t h e r e l u c i d a t i n g 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 f i s h . F u t u r e work s h o u l d a l s o be d i r e c t e d t o t h e p u r 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 t h e 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 c o r p u s c l e s o f S t a n n i u s , w h i c h w i l l c o n t r i b u t e t o a more complete u n d e r s t a n d i n g o f t h e p h y s i o l o g i c a l f u n c t i o n o f t h i s e n d o c r i n e t i s s u e . 128 LITERATURE CITED Alexander, E, (1968). Inorganic phosphorus methods manual. Vancouver Gen. Hosp., Vancouver, B.C. Asano, M., Ito, M. and Kumagai, T. (1956). 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