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Relationship between the arterial blood acid-base status and ventilation in the rainbow trout, Salmo… Janssen, Robert Gerrit 1973

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THE RELATIONSHIP BETWEEN THE ARTERIAL BLOOD ACID - BASE STATUS AND VENTILATION IN THE RAINBOW TROUT, Salmo g a i r d n e r i by ROBERT G. JANSSEN B.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF • THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1973 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a llowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT Studies were carr i e d out to determine the influence of the change i n the acid-base status of the blood on regulation of pH i n r e l a t i o n to control of v e n t i l a t i o n i n the rainbow trout. By placing trout i n v e n t i l a t i o n (V^) boxes d i r e c t measurement of v e n t i l a t i o n volume and rate could be made. A r t e r i a l blood was c o l l e c t e d via c h r o n i c a l l y implanted catheters i n the dorsal aorta; these catheters also allowing administration of the various acids and bases. The f i r s t s eries of experiments were designed to determine v e n t i l a t o r y responses to high ambient PCO^ l e v e l s (hypercapnia) and the e f f e c t on regulation of a r t e r i a l blood pH. Both short-term (up to 8 hours) and long-term (up to 72 hours) exposures were studied. ^a®2 l e v e l s remained saturated, or nearly so, throughout these experiments. The general response to high PCO^ " l e v e l s i s an increase i n the v e n t i l a t o r y stroke volume, t h i s being mainly due to an increase i n rather than VR. Compensation of v e n t i l a t i o n during the sustained hypercapnia i s slow, taking up to 3 days. A r t e r i a l H + l e v e l s increased during CO^ exposure, increasing from a control l e v e l of 11.8 + 0.5 to 41.0 + 3.5 nM/L within 5 minutes. There i s a gradual decrease i n a r t e r i a l H + concentration such that at 72 hours i t i s near normal. The time course of compensation f o r both V and pHa coincide. The hypercapnia experiments indicate that i n the face of an increase i n ambient PCO^ trout do not adjust the PCO, difference ( APCOJ between a r t e r i a l blood and water. i i PaC0 o changes i n proportion to the change i n P CO such that 2 - j 2 PaCO^ i s always about 2 mm Hg above ambient, demonstrating that APCO^ i s not affected by changes i n v e n t i l a t i o n . The change i n a r t e r i a l blood pH i s shown to be related to the transfer of CO^ rather than by a transfer of H + ions from water to blood. A r t e r i a l blood pH i s regulated via adjustment of blood HCO^ l e v e l s , adjustment being i n the order of 2 - 3 days. HCO~ can be regulated, or adjusted by either the kidney or the g i l l s . The r o l e of the kidney was shown to be minor i n the adjustment of a NaHCO^ induced a l k a l o s i s . Uptake of HCO^ i s shown to occur when f i s h are placed i n NaHCO^ containing water, demonstrating the ro l e of the g i l l s i n the amelioration of a r t e r i a l blood pH. These observations are discussed i n r e l a t i o n to a riCO-/Cl~ exchange. 3 The v e n t i l a t i o n volume i s dependent on an increase i n PaCO? and/or P_CO? and not to pHa or pH'. A decrease i n pHj, although causing a f a l l i n pHa, has only a delayed effect on V_. 9 The response i n V Q i s transient. I t i s postulated that receptors are either adapting or are not located i n the blood or water but i n another compartment whose contents or properties change i n proportion to v e n t i l a t i o n . I t i s hypothesized that a chemosens.itive area may exist on the ventrolateral surface of the medulla as i n mammals. Perfusion of the cran i a l cavity of trout with mock CSF, i n which CO^ -HGO^  was altered, did not e l i c i t r e s piratory responses. These experiments do not preclude the existence i i i of medullary chemoreceptors. These r e s u l t s are c o n s i s t e n t w i t h the hypothesis that v e n t i l a t i o n i n t r o u t i s dependent on the CO,, t e n s i o n w i t h i n the body or elsewhere and that blood pH l e v e l s are re g u l a t e d v i a i o n i c exchange mechanisms at the g i l l s u r f a c e , r a t h e r than by i o n exchange at the kidney or by d i f f u s i v e washout of gaseous CO v i a v e n t i l a t i o n . TABLE OF CONTENT o Page ABSTRACT i TABLE OF'CONTENTS. i v LIST OF FIGURES v LIST OF TABLES. .... v i i ACKNOWLEDGEMENT «.... v i i i INTRODUCTION •. 1 ACID-BASE DISTURBANCES AND THEIR DEFINITION.. ... 7 GENERAL MATERIALS AND METHODS 8" OPERATING PROCEDURE 9 EXPERIMENTAL SERIES . 18 DATA RECORDING..... 24 SYMBOLS AND ABBREVIATIONS 26 RESULTS: EXPERIMENTAL SERIES........ f..............' 27 X * H Y P JiRC APT*! IA • ©•*©•••©««**©}••••©•©©•••••* ^7 I I . METABOLIC ACID-BASE DISTURBANCES....... 41 XXX* P 3? ER.PU S101^ « © » o » « • • « • . a * © o e * e « » o © * c * ' > e '^3 IV. RADIOTRACER STUDIES. 46 GhiNERAXj DXSGUSSXQN a 6 « . * » « « © « « « « * « « ' e 0 » f l * o * e e © © « « © » * » * < > .60 SUI^Jvlji. H Y 6 Q » « « * « « 0 c « o « e « © © « A o a « © « o 6 « 0 « « 6 * © o e o a 9 O 4 9 e o o o 7~ R. EP IJ]R.E!N G H>3 » * « 0 * - © e » e © » © » e » c * » > a » o © © © * i » e « » © o * e © © e » © © « c i o 73 APPEI'J.OXX Q A « « « > v e * f > « f > * * « « o » o o o * « « a o « o © < i - « o © 0 6 0 © » e © © a o e o 7^ ' V LIST OF FIGURES Facing Page 1 Detail of the dorsal aorta cannula i n t r o u t . 12 2 Detail of a. urinary bladder cannula. 13 3 Details of i n t r a - c r a n i a l cavity cannulae. 14 4 Experimental setup used f o r the hypercapnia experiments. 15 5 Experimental setup used f o r the radiotracer study. 16 6 Effect of hypercapnia on v e n t i l a t o r y and a r t e r i a l parameters: short-term. 30 7 Effect of hypercapnia: long-term. 31 8 Effect of. hypercapnia on a r t e r i a l CO^ tensions: short-term. 33 9 Hypercapnia and i t s effect on a r t e r i a l COp tensions during compensation: long-term. 34 10 pH-HC0~ diagram with isopleths of PaCO^. 35 11 Low pH^ . and the effect on v e n t i l a t i o n and a r t e r i a l pH. 36 12 Responses of trout to HC1 induced metabolic acidosis. 47 13 Effect of NaHCO-^  induced a l k a l o s i s on v e n t i l a t i o n and a r t e r i a l pH. 48 14 NaOH induced a l k a l o s i s and the effect on a r t e r i a l pH and. v e n t i l a t i o n . 49 15 Sham i n j e c t i o n of NaCl and the effect on a r t e r i a l pH and v e n t i l a t i o n . 50 16 Relationship between [H +]a and VR i n trout 51 v i 17 R e l a t i o n s h i p between [ H + ] a and i n t r o u t . 52 18 L i n e a r r e g r e s s i o n on the r e l a t i o n s h i p between V Q and [ H + ] a d u r i n g hypercapnia. 53 19 R e l a t i o n s h i p between and [H ]a accord i n g t o l i n e a r r e g r e s s i o n d u r i n g HC1 a c i d o s i s . 54 20 R e l a t i o n s h i p between Vsv and V_ i n t r o u t . 55 21 Excretory pathways of carbon-14 v i a the g i l l and kidney. 58 22 Cumulative CO^ e x c r e t i o n p a t t e r n . 59 23 Carbon-l4 quench curve. 83 24 E f f e c t of s e l f - a b s o r p t i o n on the count r a t e of B a l 4 C 0 3 . 84 v i i LIST OF TABLES V e n t i l a t i o n r a t e , volume and a r t e r i a l {U J Facing Page du r i n g long-term exposure "to high P^CO^. 32 I I Maintenance of f i s h i n V boxes f o r G extended periods of time. 37 I I I The e f f e c t of NaHCO^ buf f e r e d water on the responses of f i s h to high CO,, l e v e l s . ' 38 v i i i ACKNOWLEDGEMENTS To my supervisor, Dr. David Randall, I wish to express my sincere thanks for his stimulating guidance throughout the course of t h i s i n v e s t i g a t i o n and f o r his assistance i n preparing t h i s manuscript, • I am also indebted to Mrs. Judy Porter who helped i n the i n i t i a l stages of the experiments, and to Dr. Bren Gannon f o r h i s p r o f i t a b l e discussion of my problems, and also to Dr. John Davis for use of his f a c i l i t i e s at the P a c i f i c Environmental I n s t i t u t e . The radioisotope experiments were made possible through usage of apparatus supplied by Drs. J.E. P h i l l i p s and B. Tregunna. I am grateful to the Fisheries Research Board of 'Canada f o r providing f i n a n c i a l support through a studentship and to the National Research Council of Canada for providing support for t h i s research through grants to Dr. Randall. 1 INTRODUCTION B o d i l y f u n c t i o n i s dependent on a r e g u l a t e d a r t e r i a l pH. S l i g h t changes i n a r t e r i a l pH d r a m a t i c a l l y a f f e c t the d i s s o c i a t i o n s t a t e of p r o t e i n m o i e t i e s , thereby a f f e c t i n g such f u n c t i o n s as enzyme a c t i v i t y and gas t r a n s p o r t i n the animal. For t h i s reason the r e g u l a t i o n of the blood plasma H + i o n c o n c e n t r a t i o n i s one of the most important aspects of homeostasis. T h i s manuscript deals with the chemical r e a c t i o n s and the p h y s i o l o g i c a l responses when a r t e r i a l pH and PC0 2 l e v e l s are a l t e r e d by e i t h e r i n c r e a s i n g ambient C0 2 l e v e l s or by the i n j e c t i o n of a c i d s and bases. Since a c i d s and bases are substances capable of d e l i v e r i n g or a c c e p t i n g H + i o n s , the c o n c e n t r a t i o n of H + depends p r i m a r i l y on the amount of the v a r i o u s a c i d s present i n the body f l u i d s . The most important of these i s carbonic a c i d which i s d e r i v e d from C0 2, one of the major end products of metabolism. Hence, the e q u i l i b r i u m equations of H2CO^ and i t s r e a c t i o n s with other b u f f e r substances i n the blood, f o r example haemoglobin, form the chemical b a s i s of C0 2 t r a n s p o r t and acid-base balance ( A l b e r s , 1970). C0 2 and carbonic a c i d are r e l a t e d as f o l l o w s : C0 2 + H £0 H 2C0 3 H + + HCO^ The c l a s s i c a l d e s c r i p t i o n of the acid-base s t a t u s of the blood s t a r t s with the Henderson-Hassel.balch equation: n v 1 n [HCOo] pH = pK + l o g _ L — • " °<C02 • PaC0 2 where pK"^  i s the negative l o g a r i t h m of the apparent f i r s t 2 d i s s o c i a t i o n constant of carbonic a c i d , ©< CO^ i s the s o l u b i l i t y c o e f f i c i e n t of GO i n plasma, and PaCO ? i s the a r t e r i a l CQ^ t e n s i o n . According to t h i s equation, pH i s a f u n c t i o n of the PCO? and HCO"! c o n c e n t r a t i o n . From a chemical p o i n t of view the PCO^ and the [HCO^] are not mutually independent v a r i a b l e s s i n c e as the POO^ r i s e s the i n c r e a s e i n carbonic a c i d i s b u f f e r e d by the nonbicarbonate b u f f e r s and thus the [HCO^l in c r e a s e s a c c o r d i n g l y (Astrup et a l , 1 9 6 6 ) . In r e s p i r a t o r y physiology the blood i s considered as a b u f f e r i n e q u i l i b r i u m w i t h a gas phase. The pH of the blood can thus be a l t e r e d i n two ways: by changing e i t h e r the PCO2 or the blood plasma c o n c e n t r a t i o n of n o n v o l a t i l e a c i d or base. The c o n c e n t r a t i o n of CO,, and the chemical composition of the blood are t h e r e f o r e the p r i n c i p a l f a c t o r s governing acid-base balance. The former can be c o n t r o l l e d by v e n t i l a t i o n , the l a t t e r by the a c t i o n of v a r i o u s e x c r e t o r y mechanism. I t has been e s t a b l i s h e d t h a t p o i k i l o t h e r m animals r e g u l a t e a constant degree of a l k a l i n i t y , or [OH""]/[H+] r a t i o , i n r e l a t i o n to the n e u t r a l p o i n t of water (Robin, 1962, Rahn, 1967, Rahn and Baumgardner, 1972, Reeves, 1969, 1972, and Howell et a l , 1970). Thus as temperature i n c r e a s e s , a r t e r i a l pH decreases. T h i s preserves a constant r a t i o between 0H°° and H1" ions even though t h e i r absolute concentrations w i l l vary g r e a t l y . I t was p o s t u l a t e d t h a t t h i s apparent constancy i s necessary to c o n t r o l the d i s s o c i a t i o n s t a t e of c e r t a i n p r o t e i n m o i e t i e s , p a r t i c u l a r l y the i m i d a z o l e groups of h i s t i d i n e , s i n c e t h e i r d i s s o c i a t i o n remains the same 3 when pH v a r i e s i n the observed manner. In other words, a r t e r i a l dpH/dT p a r a l l e l s dpK/dT f o r p r o t e i n systems i n po i k i l o t h e r m s . Homeotherms possess a blood pH which i s s i t u a t e d on the pH-temperature continuum c h a r a c t e r i s t i c of the p o i k i l o t h e r m . Cross et a l (1969) , Randall and Cameron (1972) , Truchot (1973) , and Howell et a l (1973) have shown t h a t d o g f i s h , t r o u t , shore crabs, and aqua t i c i n v e r t e b r a t e s , r e s p e c t i v e l y , have a c o n s i d e r a b l y lower PCO^ than a i r b r e a t h e r s at any temperature, as p o s t u l a t e d e a r l i e r by Rahn and West ( 1 9 6 3 ) , and Rahn (1966) . The P a C 0 2 of waterbreathers cannot t h e o r e t i c a l l y r i s e higher than 5.0 mm Hg above ambient. The reason f o r t h i s l i m i t a t i o n i s to be found i n the d i f f e r e n t s o l u b i l i t i e s of 0£ and C0£ i n water and the r e s u l t a n t enormous v e n t i l a t i o n r e q u i r e d t o achieve saturated a r t e r i a l O2 t e n s i o n s . According t o Rahn (1966) the CO^ t e n s i o n i n the g i l l w i l l be 1 . 8 mm Hg and stands i n c o n t r a s t to the PCO^ of 50 mm Hg i n the a l v e o l u s w i t h .the same 0£ t e n s i o n of 100 mm Hg, assuming equal exchange between 0^ and .CO./. i . e . R = "^002^ ^0£ = loO* F i s h which are o b l i g a t o r y a i r b r e a t h e r s have PCO,, values t h a t are approximately s i x times those found i n waterbreathing f i s h (Garey and Rahn, 1970). S i m i l a r d i f f e r e n c e s i n CO2 t e n s i o n s are found between the s t r i c t l y aquatic tadpole and the a i r b r e a t h i n g a d u l t amphibian (Erasmus et a l , 1970) . A r t e r i a l pH can e i t h e r be re g u l a t e d by adjustment of the HCO^ con c e n t r a t i o n , or by manipulation of the a r t e r i a l CO^ t e n s i o n i n face of a r i s e i n temperature. T h i s i s 4 because the kpK 1/dT f o r the HCO - H_CO„ system i s small 3 3 compared with t h a t f o r a r t e r i a l blood pH (R a n d a l l and Cameron, 1 9 7 2 ) , hence PaC0 2 • <=< CO^  or plasma [HCOp must adjust with temperature (Rahn, 1967 , and Howell et a l , 1970) , In various a i r b r e a t h i n g p o i k i l o t h e r m s , as shown by Reeves (1969) and Jackson ( 1 9 7 3 ) , the PaC0 ? v a r i e s d i r e c t l y w i t h temperature, as a r e s u l t of a r e l a t i v e h y p e r v e n t i l a t i o n at lower body temperatures. Hence, the observed dependence of pH on temperature i n a i r b r e a t h i n g p o i k i l o t h e r m s i s r e g u l a t e d p h y s i o l o g i c a l l y by v e n t i l a t o r y c o n t r o l of P C 0 2 (Rahn,1967, Jackson, 1973) . F i s h , however, cannot compromise 0 2 t r a n s f e r by an inc r e a s e i n v e n t i l a t i o n or r e d u c t i o n i n the V Q / V Q Q 2 r a t i o i n order t o r e g u l a t e PaC0 2, and hence a r t e r i a l pH (Ra n d a l l and Cameron, 1 9 7 2 ). A r e d u c t i o n i n the V Q / V Q O ^ S accompanied by a r e d u c t i o n i n Pa0 2 and an impairment of 0 2 d e l i v e r y t o the t i s s u e s . F i s h r e g u l a t e pHa i n the face of a temperature r i s e by adjustment of plasma HC0~ and pH i s independent of v e n t i l a t i o n volume and 0 2 t r a n s f e r r a t e s . Thereby the [0H~]/[H +] r a t i o remains constant. In both p o i k i l o t h e r m i c and homeothermic a i r b r e a t h e r s the d i f f e r e n c e between ambient a i r and a r t e r i a l PC0 2 l e v e l s can be adjusted by a l t e r i n g v e n t i l a t i o n . F i s h appear unable to r e g u l a t e PaC0 2, and hence the PaCO^ - FjCO^ g r a d i e n t , i n face of a r i s e i n ambient PCO by a l t e r a t i o n of v e n t i l a t i o n . 2 Increases i n v e n t i l a t i o n volume above the r e s t i n g l e v e l does not cause any change i n a r t e r i a l blood pH or t o t a l C0 2 content (Randall and Cameron, 1 9 7 2 ) . The PaCO„ - P_CO , or APCO difference, and CO output i n f i s h has been shown to be affected by changes i n the i o n i c composition of the inspired water (Dejours et a l , 1968, and.Dejours, 1969) . This f i n d i n g i s best explained by the existence of an obligatory exchange of HCO^ f o r CI at the g i l l surface. When a freshwater f i s h (goldfish) i s switched from a high to a low CI solution, the excretion of CO^ drops off sharply (Dejours, 1 9 6 9 ). In addition, when trout are subjected to high l e v e l s of CO,,, Lloyd and White (1967) observed a concomitant decrease i n plasma C l " concentration. These findings suggest then that i n freshwater f i s h , Cl i n f l u x i s l i n k e d to HCO~ e f f l u x , thereby maintaining e l e c t r o n e u t r a l i t y . i n the plasma. Evidence of t h i s ion exchange system has been presented by Maetz and Garcia-Romeu (1964) i n the goldfish and Kerstetter and Kirschner (1972) i n trout. Regulation of pH i n freshwater f i s h can thus be postulated to be by anion exchange rather than by adjustment of PaCO^, as i n mammals. As airbreathers increase t h e i r v e n t i l a t i o n i n response to an increase i n PaCO^, the question a r i s e s : what occurs i n a waterbreather, i n which the HCO^ concentration rather than the a r t e r i a l 00^ tension i s adjusted to regulate a r t e r i a l blood pH? Is v e n t i l a t i o n related to changes i n a r t e r i a l FCO^ l e v e l s or to a r t e r i a l blood pH levels? V.hat i s the time course i n adjustment of pH during sustained exposure to high ambient C0^ levels? To test these problems rainbow trout were subjected to various acid-base disorders, namely metabolic.acidosis and a l k a l o s i s and re s p i r a t o r y 6 a c i d o s i s . From r e s u l t s obtained i t i s hoped that a better understanding of acid-base regulation and i t s r e l a t i o n to v e n t i l a t i o n i s obtained i n view of the animal kingdom as a whole. 7 ACID-BASE DISTURBANCES AND THEIR DEFINITIONS Metabolic acidosis: the reduction of pH associated with accumulation of nonvolatile acid i n the body, or a decrease i n the bicarbonate f r a c t i o n . Respiratory acidosis: the reduction of pH caused by an increase i n carbonic acid r e l a t i v e to bicarbonate. Metabolic a l k a l o s i s : occurs when there i s an increase i n the bicarbonate f r a c t i o n , with either no change or a r e l a t i v e l y smaller, change i n the carbonic acid f r a c t i o n . There i s an increase i n pH. Respiratory al k a l o s i s: , an increase i n pH as a r e s u l t of a decrease i n the carbonic acid f r a c t i o n v/ith no corresponding change i n bicarbonate. After: Winters, R.W., E. Engel and R.B.Dell (1969). Acid Base Physiology i n Medicine, 2nd Ed., London Company, Cleveland, 290 p . 8 GENERAL MATERIALS AND METHODS In order t o t e s t the r e l a t i o n s h i p s between the a r t e r i a l acid-base s t a t u s and v e n t i l a t i o n i n the rainbow t r o u t , f o u r s e r i e s of experiments were performed. The f i r s t s e r i e s was designed t o determine a f i s h ' s v e n t i l a t o r y response t o high ambient CO^ l e v e l s (hypercapnia). Both short-term (up t o 8 hours) and long-term (up t o 72 hours) exposure were s t u d i e d , measuring a r t e r i a l P G O 2 , pH, PO^ l e v e l s and v e n t i l a t o r y r a t e s and volumes. The second s e r i e s of experiments were executed t o f u r t h e r i l l u m i n a t e the r e l a t i o n -ship between v e n t i l a t i o n and acid-base s t a t u s of blood as observed during the hypercapnic experiments. Both metabolic a c i d o s i s and a l k a l o s i s were induced through i n j e c t i o n of ac i d s and bases (H C 1 , NaOH, NaHCO^) v i a the d o r s a l aorta., R e s p i r a t o r y responses and a r t e r i a l acid-base parameters were fo l l o w e d under t h i s experimental regime. One of the compensatory mechanisms t h a t p r o t e c t an animal from acute change i n e x t r a c e l l u l a r pH with i n j e c t i o n of NaHGO^ i s e i t h e r by e x c r e t i o n of HCO or by C 0 ? e l i m i n a t i o n . Since f i s h possess both r e n a l and e x t r a r e n a l ( g i l l s ) pathways, both expired water t o t a l C O 2 and uri n e t o t a l C O 2 were monitored f o l l o w i n g i n j e c t i o n of l a b e l l e d NaH^^CO . R a d i o a c t i v i t y i n e i t h e r expired water or u r i n e and t h e i r r e l a t i v e r a t i o s would demonstrate the route of C O 2 e x c r e t i o n i n the t r o u t . The p e r f u s i o n of the c r a n i a l c a v i t y w i t h mock c e r e b r o s p i n a l f l u i d (CSF) comprises the f o u r t h s e r i e s of experiments. These t e s t s were devised i n l i g h t of i n f o r m a t i o n made a v a i l a b l e from observations made on mammals. The p e r f u s i o n of mock GSF, with a l t e r e d CO / HCO r a t i o s near C0 o, H 2 3 * s e n s i t i v e areas on the v e n t r a l surface of the medulla i n mammals evoke responses i n v e n t i l a t i o n . The p o s s i b i l i t y of t h i s c o n t r o l system e x i s t i n g i n the t r o u t was, t h e r e f o r e , t e s t e d . A l l experimental work was c a r r i e d out on a d u l t rainbow t r o u t , Salmo g a i r d n e r i , obtained from the Sun V a l l e y Trout Farm i n P o r t Coquitlam, B. C. The f i s h were maintained i n l a r g e , c i r c u l a r h o l d i n g tanks at the U n i v e r s i t y of B r i t i s h Columbia, s u p p l i e d with d e c h l o r i n a t e d f r e s h water at seasonal temperatures. Feeding was c a r r i e d out on a r e g u l a r b a s i s on a mixed d i e t of t r o u t p e l l e t s (J.R. C l a r k Co.) and canned crab meat. Tests were conducted from e a r l y May t o December, 1972, and March to J u l y , 1973. OPERATING PROCEDURE Experiments i n t h i s study i n v o l v e d prolonged surgery. To accomplish such surgery an o p e r a t i n g t a b l e s i m i l a r t o t h a t described by Smith and B e l l (1964) was used. F i s h were netted and immersed i n a s o l u t i o n of 1:10,000 t r i c a i n e methane s u l f o n a t e (M.S. 222, Sandoz). A f t e r swimming and opercular movements had ceased, the animal was then placed v e n t r a l side up on the o p e r a t i n g t a b l e and water, c o n t a i n i n g a n a e s t h e t i c , perfused c o n t i n u o u s l y over the g i l l s . At the end of surgery, the f i s h were t r a n s f e r r e d t o v e n t i l a t i o n boxes and w i t h i n 2 t o 5 minutes r e g u l a r breathing movements commenced. A c t u a l experimentation d i d not commence u n t i l a recovery p e r i o d of 20-40 hours had passed. Such recovery i s necessary as these s u r g i c a l procedures lead- t o c o n s i d e r a b l e 10 trauma, including acidosis (Wedemeyer, 1970). Dorsal Aortic Cannulation The dorsal aorta was cannulated i n the midline at i t s point of i n t e r s e c t i o n with the second efferent branchial a r t e r i e s as described by Smith and B e l l (1964). Intramedic (Clay Adams, Inc.) PE 60 tubing, tipped with a #21 Huber point needle and f i l l e d with heparinized (10 I.U./ml) Cortland saline (Wolf, 1963), was passed through a hole punched between the nares of the snout, the hole being l i n e d with a short length of heat f l a r e d PE 190 tubing ( F i g . 1). This cannulation allowed both sampling of blood f o r determination of a r t e r i a l P0p, PC0 , pH, and i n j e c t i o n of the various acids and bases. Such a cannulation proved quite secure and dependable, allowing blood sampling f o r up to 4 weeks. Urinary Cannulation A urinary cannula was constructed by glueing with epoxy r e s i n , approximately 1.5 cm of PE 190 around an 80 cm length of PE 60 about 1.5 cm from the t i p of the l a t t e r (Wood, 1971, Wood & Randall, 1973). Numerous small holes were punched i n the proximal 0.5 cm of PE 60 with a heated #22 needle ( F i g . 2). Successful cannulation was f a c i l i t a t e d by having a variety of preconstructed catheters on hand, representing a range of f l a r e widths, jacket lengths, and proximal t i p lengths. A cannula could then be f i t t e d to the p a r t i c u l a r urogenital morphology of a s p e c i f i c trout. This l a t t e r structure varies greatly between i n d i v i d u a l t r o u t . Cannulation was performed by opening the aperture of 11 the u r o g e n i t a l p a p i l l a with a p a i r of f i n e f o r c e p s , and i n s e r t i n g the proximal t i p of the c a t h e t e r d o r s a l u n t i l the proximal f l a n g e of the PE 2C0 j a c k e t was j u s t i n s i d e the p a p i l l a . Immediately f o l l o w i n g i n s e r t i o n , 0.5 ml of s a l i n e was i n f u s e d i n t o the u r i n a r y system and the cannula plugged with a p i n . The dead space of the cannula, at 80 cm, equals 0 . 3 5 ml (4.4 pl/cm, PE 60) . T h i s o p e r a t i o n was c a r r i e d out 14 only on those f i s h subjected t o r a d i o a c t i v e NaH CO^. C r a n i a l C a v i t y Cannulation The c r a n i a l c a v i t y , or e x t r a d u r a l space (Davson, 196?) was perfused u s i n g a mock s p i n a l f l u i d ( M i t c h e l l et a l , 1963) modified f o r rainbow t r o u t . -#20G short bevel needles, f i t t e d with PE 50 t u b i n g , were passed i n t o the foramen magnum from the d o r s a l surface of the head ( a t the margin at which s c a l e development commences) and d o r s o l a t e r a l l y passed through the p t e r o t i c bone s t r u c t u r e on both s i d e s of the cranium ( F i g . 3). The former c a n n u l a t i o n perfused the area d i r e c t l y d o r s a l to the medulla oblongata and the p t e r o t i c c a n n u l a t i o n allowed p e r f u s i o n l a t e r a l l y i n the area between the o p t i c lobe and the cerebellum on e i t h e r s i d e of the b r a i n . Mock s p i n a l f l u i d could be i n j e c t e d v i a any of the three cannulae; thus one cannula would serve as an i n l e t , the other .two as o u t l e t s of p e r f u s a t e . 12 F i g u r e 1 D e t a i l of the d o r s a l a o r t i c cannula i n rainbow t r o u t . The d o r s a l a o r t a cannula provides sampling of a r t e r i a l blood and o f f e r s a pathway f o r the i n j e c t i o n of the various a c i d s and bases used i n t h i s study. -PE 60 TUBING -PE 200 JACKET HEAT FLARED TIP no.21 HUBER POINT NEEDLE IN DORSAL AORTA STITCH 13 F igure 2 D e t a i l of a cannula implanted i n t o the urinary-bladder of a. rainbow t r o u t . T h i s cannula allowed c o l l e c t i o n of u r i n e . Placement of the l i g a t u r e s was such t h a t movement a n t e r i o r l y and p o s t e r i o r l y was reduced to a minimum, thereby reducing chance of damage done to the d e l i c a t e u r o g e n i t a l p a p i l l a . The diagram i s a f t e r Wood, 1971. a P E 190 J A C K E T ^ V s \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ | DISTAL F L A N G E H E A T P O L I S H E D TIP P R O X I M A L F L A N G E UROGENITAL P A P L L S T I C H IN V E N T R A L BODY W A L L PELVIC FIN A N T E R I O R 14 Figure 3 Implantation of cannulae i n t o the c r a n i a l c a v i t y or e x t r a - d u r a l space of rainbow t r o u t a l l o w i n g p e r f u s i o n and evacuation of f l u i d . Dorsal and l a t e r a l views. 2 cannulae were implanted d o r s o l a t e r a l l y on both side s of the head. The d o r s o l a t e r a l cannulae pass through the p t e r o t i c bone s t r u c t u r e , the d o r s a l cannula i n t o the foramen magnum. f 15 F i g u r e 4 D e t a i l of experimental setup used f o r most of t h i s study. The f i r s t stage header tank r e c e i v e d d e c h l o r i n a t e d water from a tap. The two header tanks were necessary to maintain a steady waterflow t o the v e n t i l a t i o n box. A double gas r e g u l a t o r system was r e q u i r e d t o s t a b i l i z e gas f l o w from the compressed CO^ i n a i r tank. The v e n t i l a t i o n box allowed d i r e c t measurement of v e n t i l a t i o n volume v i a an o r a l membrane s t i t c h e d around the margin of the mouth t o separate i n s p i r e d from expired water. Movement of the f i s h e i t h e r forward or backward i s prevented by a l i g a t u r e attached to the snout of the f i s h thereby p r e v e n t i n g the f i s h from t e a r i n g l o o s e . The h o l d i n g tube i s covered i n black p l a s t i c . 1 STAGE HEADER TANK P 2 n a STAGE TANK 16 Figure 5 This experimental setup was used f o r the radiotracer study. A dechlorinator, consisting of 1 A an activated charcoal f i l t e r , was used. NaH CO^ was injected via the dorsal a o r t i c cannula. Urine and expired water were analyzed f o r r a d i o a c t i v i t y . The flow was adjusted at a rate of 200 ml/min. 50 ml of expired water was collected i n a f l a s k below a layer of hexane. AIR — W A T E R L E V E L HEAD TANK L O V E R F L O W CANNULAE DORSAL \ A O R T A ^ ^ / U R I N A R Y n DECHLORINATOR RESPIROMETER BOX 3J— F R O M TAP EXPIRED WATER 17 Oral Membrane Attachment A technique which enables d i r e c t measurement of v e n t i l a t i o n volume (V ) , i n v o l v i n g attachment of a p o r t i o n G of a rubber surgeon's glove to the mouth, was developed by Davis and Cameron (1970). In t h i s technique, a 12 cm c i r c l e , which i n c l u d e s the thumb, was cut from a number 8t? l a t e x glove. The t i p was cut to f i t the f i s h head so t h a t i t covered the d o r s a l a o r t i c cannula at i t s p o i n t of emergence but l e f t the eyes uncovered. The membrane was s t i t c h e d around the margin of the f i s h ' s mouth. When p r o p e r l y cut and p o s i t i o n e d the membrane exerted l i t t l e t e n s i o n on the head or jaws. F i s h w i t h i l l - f i t t i n g membranes, or those unable to execute proper v e n t i l a t o r y movements were r e j e c t e d . Some f i s h were t e s t e d f o r p o s s i b l e v e n t i l a t o r y impairment. No such impairment was found as the d o r s a l a o r t a blood remained oxygen s a t u r a t e d , or n e a r l y so. F i s h with attached o r a l membranes were placed i n a l u c i t e box ( F i g . 4) d i v i d e d i n t o 2 connecting chambers. Once the membrane was attached t o the p a r t i t i o n between the 2 chambers, i t served as a b a r r i e r , s e p a r a t i n g i n s p i r e d and expired water. The top and s i d e s of the h o l d i n g tube was covered with black p l a s t i c to prevent f i s h r e c e i v i n g v i s u a l s t i m u l i . F o l l o w i n g the o p e r a t i o n the d r a i n at the ends of the box could be set at various l e v e l s by s l i d i n g i t up or down through a rubber stopper. P o s i t i v e pressure i n the buccal c a v i t y was provided by r a i s i n g the d r a i n at the head end of the box. T h i s was used to f a c i l i t a t e v e n t i l a t i o n during 18 recovery from the operation. After the 20-40 hour recovery period following surgery the drains i n the box were l e v e l l e d and the f i s h were allowed to adjust for at l e a s t an hour i n order to establi s h a normal breathing pattern. V e n t i l a t i o n rate ( VR) , v e n t i l a t i o n volume (V ), inspired oxygen and carbon dioxide tension ^1^2' ^1^2^ ' e x P ^ r e c ^ o x y g e n tension (PgOp>) > and blood parameters (pHa, PaCO^) were then measured at i n t e r v a l s . V e n t i l a t i o n volume was measured by c o l l e c t i n g the water s p i l l i n g over the rear drain as the f i s h breathed. Water was collected f or 1 minute and v e n t i l a t i o n rate was simultaneously determined by counting mouth movements. EXPERIMENTAL SERIES I. Hypercapnia Fish successfully operated upon were placed i n V_ boxes. Before subjecting the f i s h to an increase i n ambient PjCO^, the animals were allowed to acclimate to the adjusted water l e v e l s i n the boxes for at l e a s t 1 - 2 hours. Any f i s h which f a i l e d to demonstrate proper respiratory movements were rejected. Fish were supplied dechlorinated water d i r e c t l y from the tap system during non-experimental periods. To ensure s t a b i l i t y of hypercapnic waters during the actual experiment, double systems of both gas and water were constructed. The former consisted of a primary and secondary gas regulator system, the l a t t e r of a f i r s t and second stage header tank. Accurate flows could be maintained during the entirety of the experiments, which i n some cases las t e d up to 72 hours (Fig.. 4) . 19 Once the l e v e l of GO i n the secondary header tank 2 ' had s t a b i l i z e d at 5 mm Hg PCO^, "the water i n l e t s were switched such t h a t water c o n t a i n i n g the high CO^ l e v e l passed d i r e c t l y i n t o the head end of the V_ box. In order t o reduce J G the response time of the f i s h subjected to t h i s high CO^ water, the volume of the head end of the V box was reduced 0 by p l a c i n g a one l i t e r p l a s t i c b o t t l e i n i t . I n t r o d u c t i o n of high C0 2 water was taken as time zero. A r t e r i a l blood, obtained by a p p l y i n g s l i g h t s u c t i o n onto the cannula, was c o l l e c t e d at predetermined times during the course of the experiment. Blood was analyzed almost immediately f o l l o w i n g exsanguination and analyzed f o r pH every sampling p e r i o d and f o r PaOp, PaCO^ every few sampling p e r i o d s . T h i s sampling technique prevented undue hemodilution ( l o w e r i n g of haematocrit) d u r i n g the experiment. Water samples f o r P^COp were taken roughly every 15 minutes. 0^ l e v e l s were monitored at approximately the same time i n t e r v a l s . In a l l experiments 0^ l e v e l s were kept between 70 - 100% s a t u r a t i o n . At the end of the experiment, cannulae and o r a l membranes were removed, and the f i s h weighed. Oral membranes were c l o s e l y inspected f o r t e a r s and holes and r e s u l t s of those w i t h damaged membranes were r e j e c t e d . I I . M etabolic acid-base disturbances In t h i s experimental s e r i e s , metabolic a c i d o s i s and a l k a l o s i s was induced v i a i n j e c t i o n s of a c i d s and bases i n t o the d o r s a l a orta cannula. In order t o reduce the degree of hemodilution, a l l compounds i n j e c t e d i n t o the f i s h were done 20 so i n 1 ml volumes. To produce the two acid-base disorders, the following concentrations of acids and bases were used: 0.025 mM HCl/ml of saline 0.025 mM NaOH/ml of saline 1.000 mM NaHCO /ml of saline 3 and as a control 0.025 mM NaCI/ml of saline These concentrations were adequate to induce the two metabolic acid-base disorders. As i n the hypercapnic experiments, f i s h were allowed to acclimate to the adjusted water l e v e l s i n the V boxes f o r G up to 2 hours. Fish had already recovered from the operation over a 20 - AO hour period. Control values f o r V VR, pHa G and PaOg were taken at the end of t h i s time. Fish were maintained on dechlorinated tap water during the course of the experimental and control periods. To reduce unwanted stimulation of f i s h a perfusion rate of 1 ml/minute was used i n the i n j e c t i o n of the compounds. Commencement of administration was taken as time zero for the experiments. At the end of each run f i s h were weighed and oral membranes inspected f o r possible leaks. I l l . CSF perfusion In t h i s t h i r d experimental series a mock spinal f l u i d modified for rainbow trout, after M i t c h e l l et a l ( 1 9 6 3 ) , v/as used as a perfusate f o r the c r a n i a l cavity of the f i s h . The composition i s as follows: NaCI 125 mM/liter NaHCO 10(5,25) 11 " KC1 3.5 " " 21 1.3 rnM/liter 1.14 tt NaHoP0, 0 .51 tt glucose 0 . 5 6 The C0 9/H + responsiveness of the sensitive areas was tested tt tt by a l t e r i n g the PCO^ or [HCO~] of the mock spinal f l u i d . This was achieved by bubbling either 1% or 5% C0 ? (a PCO^ of 7.5 and 35.0 mm Hg, respectively) or by a l t e r i n g the [HCO^] of the perfusate. Injection was at a very slow rate of approximately 1 ml per 3 to 5 minutes and continued u n t i l a t o t a l of 5 to 10 ml of mock spinal f l u i d was perfused. operated upon were placed i n a black p l a s t i c box, s i m i l a r to the one used i n F i g . 5, and allowed to recover 2 0 - 4 0 hours. A l l f i s h were maintained on dechlorinated tap water. One of the cran i a l cavity cannulae, f i l l e d with mock CSF, was attached to a 5 ml syringe via a 3-way stopcock. As stated, the experimental procedure consisted of slow perfusion of mock CSF with d i f f e r e n t COg/HCO^ r a t i o s into the c r a n i a l cavity via either one of the 3 cannulae ( F i g . 3) » Recordings were made of the v e n t i l a t i o n rate by manually counting respiratory movements. In these experiments oral membranes were not attached thus no v e n t i l a t i o n volumes could be measured. IV. Radiotracer studies Fish that were successfully operated upon were placed i n a watertight, l u c i t e v e n t i l a t i o n chamber. A l l sides were covered with black p l a s t i c .to reduce visual- stimulation, As i n the previous experiments f i s h successfully 22 since a g i t a t i o n of f i s h r e s u l t s i n an increa s e d v e n t i l a t i o n r a t e and a d i u r e s i s , r e s u l t i n g i n anomalous r a d i o a c t i v i t y -r eadings. The v e n t i l a t i o n box was su p p l i e d with aerated, d e c h l o r i n a t e d f r e s h tap water fronr a system c o n s i s t i n g of a head tank and an a c t i v a t e d charcoal f i l t e r ( F i g . 5)• The temperature and P T 0 ? of the system never v a r i e d more than 6.0 1 0.5 °C and 146 i 2 mm Hg, r e s p e c t i v e l y . A steady f l o w r a t e of 200 ml/min was maintained by adjustment of a stopcock on the e x i t end of the v e n t i l a t i o n box. 1 L. NaH C0^ (Amershaw, S e a r l e , Inc.) was prepared such th a t a 1 ml sample of s a l i n e contained 1 mM NaHCO wit h a 3 s p e c i f i c a c t i v i t y of 5 ( see Appendix A) . T h i s amount provided adequate NaHCO^ t o induce metabolic a l k a l o s i s and enough a c t i v i t y f o r d e t e c t i o n c o n s i d e r i n g the water f l o w r a t e of the system and the time taken f o r the f i s h t o compensate f o r the metabolic d i s o r d e r . The t o t a l time f o r i n j e c t i o n approximated 1 minute. S t a r t of a d m i n i s t r a t i o n was taken as time zero. Both urine and expired water samples were analyzed by a technique r e s u l t i n g i n a BaCO^ p r e c i p i t a t e , as described i n Appendix B. The p r i n c i p l e of t h i s technique i s c o l l e c t i o n of expired CO^ , or bicarbonate i n an a l k a l i n e t r a p and. subsequent p r e c i p i t a t i o n of the bicarbonate-carbonate with a BaCl^-NH^Cl s o l u t i o n . The p r e c i p i t a t e , v/eighing from 1 to 3 mg was f i l t e r e d u s i n g a m i l l i p o r e system. M i l l i p o r e f i l t e r s proved very s u i t a b l e since accurate and reproducable 14 readings of beta r a d i a t i o n ( C) are p o s s i b l e due to the o r i e n t a t i o n of the p r e c i p i t a t e on the f i l t e r surface 23 ( M i l l i p o r e B u l l e t i n , 1 9 7 0 ) . Surface r e t e n t i o n of the BaCO^ prevents masking of the low energy beta. Since the r e f r a c t i v e index of the m i l l i p o r e f i l t e r ( c e l l u l o s e ester) i s 1 .50 + 0 . 1 0 , i t i s t o t a l l y transparent i n a toluene based s c i n t i l l a t i o n f l u i d , r e s u l t i n g i n t o t a l geometry s c i n t i l l a t i o n counting. S p e c t r a f l u o r P P 0 - P 0 P 0 P , a concentrate l i q u i d s c i n t i l l a t o r , was used as a b a s i s of the s c i n t i l l a t i o n " c o c k t a i l s " , at a co n c e n t r a t i o n of 4 g POPOP, 50 mg PPO i n toluene. Benakis (1970) found t h i s t o be a s u f f i c i e n t l y i L e f f i c i e n t counting f l u i d f o r Ba CO^. A blank, c o n s i s t i n g of u n l a b e l l e d BaCO^ ( 1 . 5 mg) on a m i l l i p o r e f i l t e r was used f o r background count determination. The average background count v a r i e d 35 * 2 cpm. I t was i n i t i a l l y intended t o c a l c u l a t e the s p e c i f i c a c t i v i t y ( i . e . the "^CO / 1 2 C 0 „ r a t i o ) from the weight of BaCO p r e c i p i t a t e d . T h i s proved t o be f r u i t l e s s : no 3 c o r r e l a t i o n was obtained between the weight of BaCO^ and the amount of r a d i o a c t i v i t y and t h e r e f o r e .this a n a l y t i c a l approach was abandoned. Urine sampling proved f a i r l y i n c o n s i s t e n t , p r o v i d i n g 2 ml of f l u i d one moment, none at another time during the experiment. The best sampling technique was as f o l l o w s : i f the f i s h d id not c o n t r i b u t e any u r i n e , some s a l i n e was fl u s h e d i n t o the bladder. The r e s u l t a n t s a l i n e / u r i n e sample was then drawn i n t o a NaOH c o n t a i n i n g (about 0 . 0 2 ml of 5 M NaOH) syringe.. The u r i n e was then i n j e c t e d below the hexane l a y e r i n t o 50 ml of water, and subsequently t r e a t e d with NaOH and the BaCl--NH,Cl mixture. 24 DATA RECORDING Gas t e n s i o n s i n water and blood were measured u s i n g a Radiometer e l e c t r o d e system. PO determinations were made 2 with Radiometer type E 5046 oxygen e l e c t r o d e , PCO^ w i t h a Radiometer type E 5036 carbon d i o x i d e e l e c t r o d e . For pH measurements, a c a p i l l a r y e l e c t r o d e , type G297/G2 was used. A l l e l e c t r o d e s were contained i n thermostatted c e l l s and maintained at the same temperature as.the experimental animal. C a l i b r a t i o n of both COg and 0 e l e c t r o d e s was c a r r i e d out by exposure t o moist gas samples of known te n s i o n s from a gas mixing pump system (Wosthoff, Germany) and a compressed CO,, c y l i n d e r f o r C0£ e q u i l i b r i u m and from a compressed N-c y l i n d e r and water saturated a i r f o r 0^ e q u i l i b r a t i o n . The pH e l e c t r o d e was e q u i l i b r a t e d using Radiometer p r e c i s i o n b u f f e r s . The oxygen and pH e l e c t r o d e s were hooked up t o a Radiometer PH3V) 71 Acid-Base Analyzer. The l a t t e r , a l a t e r model of meter, f e a t u r e s a more s t a b l e a m p l i f i e r , important i n measuring the low p a r t i a l pressures of COp encountered. Blood was exsanguinated a n a e r o b i c a l l y by a p p l y i n g s u c t i o n onto the d o r s a l aorta cannula and drawing the blood i n t o a 1 ml s y r i n g e . The i n i t i a l 0.0 5 ml of blood was r e j e c t e d . Although blood pH i s p r a c t i c a l l y unaffected by small d i l u t i o n s , the PCO,, (and bicarbonate concentration) of plasma f a l l s i n d i r e c t p r o p o r t i o n t o , or s l i g h t l y more than the d i l u t i o n (Siggaard Anderson, 1 9 6 1 ) . To provide c o n s i s t e n t r e s u l t s , p r i o r t o i n t r o d u c t i o n of a blood or water sample, the e l e c t r o d e s were a i r - d r i e d . 25 The sample was then introduced i n t o the cuvettes and readings taken a f t e r a set time. 4 minutes was r e q u i r e d f o r s t a b i l i -z a t i o n t o occur i n the case of CO^ measurements, 3 minutes f o r 0^. pH values were read when the needle s t a b i l i z e d . Quench curve determination and sample a n a l y s i s f o r r a d i o a c t i v i t y were monitored i n a l i q u i d s c i n t i l l a t i o n system c o n s i s t i n g of a nuclear-Chicago U n i l u x IIA., as discussed i n Appendices C and D. Since i t i s not mathematically c o r r e c t to take a r i t h m e t i c a l means of pH values (Davonport, 1969) and th a t pH p l o t t e d on a l i n e a r s c a l e d i s t o r t s the p h y s i o l o g i c a l r e a l i t y , a l l a r t e r i a l pH values were converted to corresponding hydrogen i o n c o n c e n t r a t i o n s . S t a t i s t i c s were executed on the l a t t e r , and reconverted back t o pH values f o r comparison purposes. Standard e r r o r s shown thus apply o n l y t o hydrogen i o n c o n c e n t r a t i o n s , not pH values. Plasma HC0 o concentrations were c a l c u l a t e d from the 3 Henderson-Hasselbalch equation, u s i n g measured values of pHa and PaCO^. Values of pK are those from, human plasma since t r o u t and human plasma have a s i m i l a r i o n i c strength ( A l b e r s , 1970). <^C0p i s assumed the same as COp s o l u b i l i t y i n 150 mM MaCl/L, and i s expressed i n mM/ ml/mm Hg ( t r o u t plasma = 130 mM NaCl/L). 26 SYMBOLS AND ABBREVIATIONS USED IN THIS STUDY V^ , -volume of water passed over the g i l l s / m i n u t e ( v e n t i l a t i o n volume) VR -mouth or opercular closures/minute ( v e n t i l a t i o n rate) Vsv -volume of water passed over the g i l l s / b r e a t h i n g c y c l e ( v e n t i l a t o r y stroke volume) V - - i n a i r b r e a t h e r s , t o t a l amount of new a i r i n t o the r e s p i r a t o r y passages/minute ( r e s p i r a t o r y minute volume) P,p - p a r t i a l pressure of gas i n mm Hg A P G 0 2 -PaCO^ - PjCO ? d i f f e r e n c e or gradient ©< - s o l u b i l i t y c o e f f i c i e n t f o r gas i n water or plasma [ H + ] -hydrogen i o n co n c e n t r a t i o n , i n n a n o m o l s / l i t e r dpm - r a d i o a c t i v e decay r a t e , d i s i n t e g r a t i o n s / m i n u t e dps - r a d i o a c t i v e decay r a t e , d i s i n t e g r a t i o n s / s e c o n d 4 _JUC -microCurie, 3 .700 X 10 dps; based on r a t e of nuclear d i s i n t e g r a t i o n S.E,, -standard e r r o r S u b s c r i p t s a - a r t e r i a l p i -plasma I - i n s p i r e d E -expired 27 RESULTS: EXPERIMENTAL SERIES I. THE EFFECT OF INCREASED C0 2 TENSION ON ARTERIAL pH, PC0 2 AND ON VENTILATION. Hypercapnia was induced and maintained by subjecting 13 f i s h , weighing 243.9 + 10.5 g, to water at 5.20 + 0 .33 mm Hg PC02„ The data presented i n Figures 6 and 7 are s i m i l a r , the former showing d e t a i l of short-term exposure, the l a t t e r the-effect of long-term exposure. Because of the large i n d i v i d u a l v a r i a t i o n i n both v e n t i l a t i o n rate and volume (e s p e c i a l l y i n the former) and small, sample size (n="5) , data i n F i g . 7 represent that of 1 f i s h only (no. 41) at both 48 and 72 hours. Values of a l l 5 f i s h are portrayed i n Table I . The general response to high CO i s an increase i n 2 both VR and"V„ and a gradual return to normocapnic l e v e l s i n u 0 2 or 3 days. However, the increase i n V i s "much more 0 pronounced than that of VR, changing from a r e s t i n g l e v e l of 47.1 + 2 .7 ml/min to a maximum of 254,9 ± 29.3 ml/min at 5 minutes. This represents a 5-fold' increase"in v e n t i l a t i o n volume, compared with only a s l i g h t increase i n VR. This i s re f l e c t e d i n F i g . 20, where a l i n e a r r e l a t i o n s h i p exists between v e n t i l a t i o n volume and v e n t i l a t o r y stroke volume showing that the increase i n V i s c h i e f l y due to an increase i n Vsv. At 72 hours of exposure, both VR and V are generally G back to control l e v e l s , demonstrating that compensation has occured. The a r t e r i a l hydrogen ion concentration increased from a mean control value of 11.8 + 0 .5 to 41.0 + 3 .5 nM/L 28 (a pH of 7 . 9 3 "to 7.39) w i t h i n 5 minutes, thereby producing acute r e s p i r a t o r y a c i d o s i s ( F i g s , 6 , 7 ) . With time, during c o n t i n u a l exposure t o high CO,,, there i s a gradual decrease i n a r t e r i a l [ H + ] such t h a t at 7 2 hours i t i s near normal. There i s suggestion of a r e l a t i o n s h i p e x i s t i n g between [H + ] a or PaCO^ and v e n t i l a t i o n because of the synchronous trends e x h i b i t e d . A r t e r i a l P^O^ l e v e l s were measured r e l a t i v e t o the PCO^ of i n s p i r e d water i n 2 f i s h (no. 5 8 , no, 5 9 ) : s h o r t -term ( 8 hours) and long-term ( 7 2 h o u r s ) , r e s p e c t i v e l y . Both f i s h were without attached o r a l membranes. I t i s shown that f i s h cannot reduce t h e i r PaCO,, w i t h respect to the p r e v a i l i n g i n s p i r e d CO^ t e n s i o n and t h a t the PaCO^ - ^ j ^ ° 2 d i f f e r e n c e (APCO^) i s r e - e s t a b l i s h e d i n l j j t o 2 hours ( F i g s . 8 , 9 ) . At 5 min of exposure there e x i s t s an apparent negative APCO^ of -0,4 and -0.1 mm Hg f o r f i s h 58 and 5 9 , r e s p e c t i v e l y . I t i s to be kept i n mind t h a t these P C O 2 values are from the d o r s a l aorta and i s thus probably not i n d i c a t i v e of CO,, t e n s i o n s a c t u a l l y e x i s t i n g at the g i l l . Although the v e n t i l a t i o n r a t e s are lower f o r f i s h having no attached o r a l membranes, these 2 f i s h show the same general trend i n response t o hypercapnia. There i s an i n i t i a l h y p e r v e n t i l a t i o n which may l a s t up to 24 hours f o l l o w e d by a slow r e t u r n t o basal v e n t i l a t o r y l e v e l s at 7 2 hours. However, the i n i t i a l peak and f o l l o w i n g decrease i n YR obtained i n "membraned" f i s h i s not seen, showing p o s s i b l e i n t e r f e r e n c e of the v e n t i l a t o r y mechanism by the o r a l membrane. The p a t t e r n i n a r t e r i a l [H 4 -] l e v e l s are 29 a l s o s i m i l a r between the 2 groups of f i s h . Since the c a p a c i t y of t r o u t t o reduce the PaCO^ - PjCO^ d i f f e r e n c e i s small as i n d i c a t e d , how does the animal adjust i t s pH t o normal l e v e l s i n face of maintained hypercapnia? To answer t h i s a pH - HCO^ diagram ( F i g . 10) was constructed to i l l u m i n a t e the r e l a t i o n between pH, PCO^ and HC0~ from the Henderson-Hasselbalch equation. Since PaC02 and ^COg are h e l d constant (both temperature and i o n i c strength d i d not change i n the animal), HCO^ must change i n order to adjust pH. A r t e r i a l PCO^ and pH values of 4 d i f f e r e n t f i s h (no. 56 to 59)were p l o t t e d f o l l o w i n g the time course d u r i n g hypercapnia. The l i n e A - C, connecting these p o i n t s , i s assumed to represent the blood b u f f e r l i n e . T h i s l i n e i s an approximation only s i n c e i t should show the c h a r a c t e r i s t i c upward slope to the l e f t , where there i s a s l i g h t i n c r e a s e i n [HC0'~] and a l a r g e i n c r e a s e i n the [ H + ] (Refsum, 1971) . The PaCO^ values probably do not represent the a c t u a l a r t e r i a l l e v e l s . T h i s could be the r e s u l t of a time l a g i n the HC0~ - C0 2 system. A f i s h subjected to P j C 0 2 - l 6 " ^ 6 - 1 - 3 °^ 5.0 mm Hg w i l l f o l l o w t h i s f i t t e d l i n e (A - C) i n response t o the r a i s e d C0 2 l e v e l s . T h i s i s the immediate response up t o approximately 1-hour ( a t C) . A f t e r t h i s p e r i o d , as P a C°2 ^ a s adjusted t-° about 7.0 mm Hg, an i n c r e a s e i n [HCO^] i s seen. T h i s i n c r e a s e f o l l o w s the 7.0 mm Hg i s o p l e t h . Thus p o i n t G to B i n F i g . 10 describes the animal's compensation to c h r o n i c , or sustained hypercapnia u n t i l pH i s adjusted. 30 Figure 6 The e f f e c t of hypercapnia on the v e n t i l a t i o n r a t e , v e n t i l a t i o n volume and a r t e r i a l hydrogen c o n c e n t r a t i o n . Hypercapnia was induced by s u b j e c t i n g 13 f i s h t o a PCO^ of 5.20 + 0.33 mm Hg i n water. The f i s h weighed 243.9 + 10.5 g (8.9 + 0.1 °C). Values are means + 1 standard e r r o r . Time zero designates the s t a r t of the experiment. The values t o the l e f t of the dashed l i n e designate c o n t r o l v a l u e s . 31 F i g u r e 7 The e f f e c t of hypercapnia on v e n t i l a t i o n r a t e and volume, and a r t e r i a l hydrogen c o n c e n t r a t i o n . 13 f i s h (243.9 + 10.5 g, 8.9 + 0.1 °C) were subjected t o a P O O g of 5.22 + 0.33 mm Hg. A s i n g l e f i s h ' s values are p l o t t e d a f t e r the v e r t i c a l dotted l i n e ; i . e . at k& and 72 hours. Values are means + 1 standard e r r o r . T h i s f i g u r e i s s i m i l a r t o t h a t of F i g u r e 6, up t o 5 hours. T I M E (minutes) 32 Table I V e n t i l a t i o n r a t e s and volumes, and a r t e r i a l hydrogen i o n c o n c e n t r a t i o n during long-term exposure to high P TCO . Values of f i s h no. 41 have been p l o t t e d i n the previous diagram, F i g . 7. Time F i s h 0 \ VR Vsv pHa P I C 0 2 no „ ml/min no./min ml/breath nM/L mm Hg I . 48 hours of 41 75 70.6 1.07 7.85 14.1 4.8 exposure' 63 76.4 59 0.83 7.82 1 5 . 1 5 .1 60 76 63.5 1.20 7.89 12.9 5.1 61 64 94.5 0.68 5.1 62 84 70.6 1 .19 7 . 8 1 15.5 5.1 X + S.E. 72.4 + 3.5 75.1 + 5.9 14.5 + 0.8 5.00 + o.o: I I . 72 hours of 41 . 59 71.4 0.83 7.99 10.2 5.0 exposure 59 55 73.6 0.75 7.87 13.5 5.1 60 48 64.5 0.74 7.94 11.5 5.1 61 54 87.6 0.62 4.2 62 102 70.2 1.45 7.99 10.2 4.2 X + S.E. 63.6 + 10.9 73.5 +4.3 11.4 + 0.9 4.7 + -.2 33 Figure 8 The e f f e c t of hypercapnia (5°0 mm Hg PC0 ?) on a r t e r i a l CO^ t e n s i o n s . F i s h 58 ( 2 2 8 g, 9 °C) i s shown. This f i s h d i d not have an attached o r a l membrane. Induction of hypercapnia i s at time zero. The values of PaC0 o - P_C0„ ( APCO ) are shown 2 1 2 2 immediately to the r i g h t of the v e r t i c a l l i n e s connecting a r t e r i a l and i n s p i r e d C00 t e n s i o n s . T I M E (hours) 3 4 Figure 9 Hypercapnia and i t s e f f e c t , o n a r t e r i a l CO^ tensions during compensation of r e s p i r a t o r y a c i d o s i s . F i s h 59 (257 g, 9 °C) without an attached o r a l membrane. Values of A PCO^ are shown adjacent t o the l i n e s connecting a r t e r i a l and i n s p i r e d C0 9 t e n s i o n s . T I M E (hours) 35 Figure 10 A r t e r i a l pH - plasma HCO^ concentration coordinates showing the isopleths f o r the two PaC02 values tested. The f i g u r e takes into account the changes i n pK of carbonic acid as a function of temperature and blood pH. The s o l u b i l i t y of GO2 remains constant because temperature and i o n i c strength did'not change. ^^^2 va^-nes a r e ^n r n m ^g. A r t e r i a l PCO2, pH values of 4 f i s h (no. 56 to no. 59, inclusive) are included, as designated by the ind vidual points. Letter A represents values of normocapnic f i s h , B those of f i s h which have compensated for the sustained hypercapnia. 36 Figure 11 The e f f e c t of low pH of water on the a r t e r i a l pH and v e n t i l a t i o n r a t e and volume. One f i s h , no. 65 (241 g, 1 2 + 1 °C) was subjected t o a pH of 5-0 maintained by a d d i t i o n of HC1 t o a r e c i r c u l a t i n g tank system (volume 75 L ) • Exposure i s at time zero. O 1 2 3 4 T I M E (hours) 37 T a b l e I I Maintenance of f i s h i n v e n t i l a t i o n boxes f o r extended p e r i o d s of t i m e and t h e e f f e c t on v e n t i l a t i o n r a t e and volume and a r t e r i a l p H . Time . F i s h Ya VR h o u r s no. ml/min no./min 4 A 34 71.9 B 41 77.9 8 A 38 72.3 B 40 79.5 24 A Lp 71.9 B 39 76.9 48 A 42 73.2 B 45 80.0 sv * a a 2 ml/breath mm Hg 0.47 7.94 92 0.52 7.98 102 0.52 7.97 101 0.50 8.02 114 0.58 7.95 98 0 o 50 8.00 109 0.58 7.97 105 0.57 7.99 115 Table I I I The e f f e c t of NaHCO b u f f e r i n g of water on 3 the responses of f i s h t o high C0o l e v e l s . F i s h no. 15 ( 2 7 7 g, T ~ 1 3 . 0 °C) was exposed t o a 1% NaHCO^ bu f f e r system and the FjCO^ r a i s e d as i n F i g s . 6 t o 9 . The responses are noted i n P a r t I . F i s h no. 17 ( 2 5 1 g, T = 1 3 . 0 °C) was subjected t o aerated 1% NaHCO. alone, and responses t a b u l a t e d i n P a r t I I . T I M E T E M P . \ VR Vsv ' pHa [H+ ] a P I C 0 2 min °C ml/min breath/min ml/min nM/L mm Hg I . HIGH PCO £ c o n t r o l 12.0 46 75.9 0.61 8.01 9.8 0 e 6 + 11 304 82.8 , 3.67 7.81 15.6 4.8 1% NaHCO^ • 15 tt 212 90.2 2.35 7.88 13.3 4.8 i n v/ater 30 II 194 87.6 2.22 4.7 ( f i s h no.15) 60 it 206 92.3 2.23 7.83 14.7 4.9 120 12.5 246 93.0 2.64 7.86 13.8 4.9 180 1? 228 • 96.0 2.32 7.89 12.9 • 5.1 240 it 224 96.8 2.31 7.92 125.0' 5o0 300 1! 216 92.3 2.34 7.95 11.4 5.3 I I . 1% NaHCO^ ' c o n t r o l 13.0 48 92.3 0.52 8.00 10.0 aerated 5 II 54 97.6 0.55 8.08 8.3 ( f i s h no.17) 60 t? 70 103.4 0.68 8.11 7.7 120 It 60 105.3 0.57 8.20 6.3 39 As an example, f i s h no, 5 9 , under normocapnic c o n d i t i o n s (PaCO £ = 2.3 mm Hg) , would have a plasma [HC0~] of 10.0 mM/L at a pH of 7 - 9 8 ( p o i n t A) . P o i n t B (on the 7.0 mm Hg PaCO^ i s o p l e t h ) d e s c r i b e s i t s compensation t o the maintained high C 0 £ l e v e l s a f t e r 72 hours, g a i n i n g 21.0 mM/L of HC0~ t o adjust i t s pH t o 7 . 9 7 at a PaCOg o f m m H g * T h u s r e g u l a t i o n of pH i n fa c e of a r i s e i n CO^ i s by adjustment of the plasma [HCO^] du r i n g hypercapnia. I n c r e a s i n g the p a r t i a l pressure of CO^ i n water causes a f a l l i n the pH of water. To t e s t the e f f e c t of lov; water pH, f i s h no. 65 (241 g, 1 2 + 1 °C) was exposed o and maintained at a pH^ . of 5«00. A r t e r i a l pH, VR and V^ were observed ( F i g . 11)„ The response noted i s t y p i c a l of s e v e r a l f i s h t e s t e d at lov/ pH^. Water, at a PCO^ of 5 mm Hg, never f e l l t h a t low i n pH, u s u a l l y ranging from a pH of 6 .3 to 6 . 6 during the hypercapnia experiments. There was a s l i g h t decrease i n a r t e r i a l pH, from a p r e - s t r e s s value of 7 . 9 5 t o a low of 7 . 8 6 at 30 min, and a gradual increase to a pH of 7 . 8 9 at 3 hours. Upon r e t u r n to normal water of pH of 7.30, the pHa returned t o c o n t r o l values, i . e . between 7 . 9 8 and 8,00. A pH of 5.00 represents an H i o n gradient 100 times t h a t normally present i n water. These data i n d i c a t e t h a t d u r i n g hypercapnia the a r t e r i a l a c i d o s i s observed i s due t o C0„ and net. a lov/ pH_. 2 I I t i s p o s s i b l e t h a t the observed, r e t a r d e d ' i n c r e a s e i n V^ and VR at lov; pH could be due t o mucous accumulation at the * 1 g i l l surface l e a d i n g t o an increase i n 0^ d i f f u s i o n d i stance, 40 THE EFFECT OF NaHCO^ BUFFERED WATER ON RESPONSE OF FISH. Dejours et a l (1968) and Dejours (1969).observed t h a t the e f f e c t of a change i n the. i o n i c composition of i n s p i r e d water a f f e c t e d the APCO^and CO,, output i n g o l d f i s h . To t e s t i f these e f f e c t s e x i s t e d i n rainbow t r o u t , s e v e r a l f i s h were subjected t o waters c o n t a i n i n g 1% NaHCO^ i n a r e c i r c u l a t i n g system of 75 l i t e r s . Short-term responses t o r a i s e d l e v e l s of PjCO^ were observed (Table I I I ) . The f i s h (no. 15), whose response i s portrayed, t y p i f i e s the responses under these experimental c o n d i t i o n s . The degree of a r t e r i a l blood a c i d o s i s i s much l e s s pronounced i n the NaHCO^ buffered system (Table I I I , p a r t I) than i n tap water system ( F i g s . 6,7) , even though PjCC^ l e v e l s are s i m i l a r . T h i s i m p l i e s t h a t HC0~ must be a c t i v e l y taken up from the water. F i s h exposed t o 1% NaHCO^ i n water alone provide evidence t h a t HC0~ i s indeed a c t i v e l y 3 a s s i m i l a t e d s i n c e the a r t e r i a l blood pH in c r e a s e s from a c o n t r o l of 8.00 t o 8.20 i n 2 hours ( f i s h no. 17, Table I I I , part II) . The general t r e n d of v e n t i l a t i o n volume under these c o n d i t i o n s of high POO^ along ( F i g s . 6,7), g i v i n g evidence © t h a t V i s r e l a t i v e l y independent of the a r t e r i a l bipod pH G but dependent on a r t e r i a l PCO l e v e l s . 2 In order to determine the e f f e c t of m a i n t a i n i n g a o f i s h , with attached o r a l membrane, i n a V box f o r extended G periods of time, f i s h were placed i n boxes f o r extended periods of time and observed. The r e s u l t s are i n Table I I . 0 s a t u r a t i o n o f blood remained normal d u r i n g the l e n g t h of 41 the experiment, as d i d a r t e r i a l pH l e v e l s . There appeared 9 to be a s l i g h t i n c r e a s e i n VR and V , but these were w i t h i n G the range of experimental e r r o r obtained i n c o n t r o l values of f i s h subjected t o the various acid-base d i s o r d e r s . I I . INDUCED METABOLIC ACIDOSIS WITH HC1 INJECTIONS AND ITS EFFECT ON VENTILATION AND ARTERIAL pH. Si x f i s h (264.3 + 19.2 g at 11.8 + 0.4 °C) were used i n t h i s experiment. I n j e c t i o n of 0.025 mM HC1 i n s a l i n e r e s u l t e d i n acute metabolic a c i d o s i s , a f f e c t i n g both V and G VR, and a r t e r i a l pH. From a c o n t r o l value of 10.9 + 0.5 nM/L, the [H 4 -] at 90 minutes equalled 12.2 + 2.0 nM/L, demonstrating the l a r g e v a r i a t i o n e x i s t i n g between i n d i v i d u a l f i s h . Thus some f i s h had readjusted t h e i r pHa, some had not ( F i g . 12). An a c i d l o a d i s d e a l t w i t h i n two ways; i t i s buffered by both the non-bicarbonate and the bicarbonate b u f f e r system as f o l l o w s : 1) nonbicarbonate component H + + Buf" HBuf I 2) bicarbonate component H + + HCO" **HoC0o ^H o0 + ' C0 9 | 3 2 ) 2 <c The former c o n s i s t s of plasma p r o t e i n s , mainly haemoglobin. B u f f e r i n g by Buf i s immediate. The added a c i d r e a c t s with haemoglobin, plasma p r o t e i n s and HC0~ of blood, but some of the added H remains i n the i o n i z e d form, and thus r a i s e s the [ H + ] a . The H + i o n , i n combining w i t h KCO^, causes an increase i n carbonic a c i d c o n c e n t r a t i o n , hence there i s a 42 r i s e i n CO,, i n the blood. As the blood passes through the g i l l s , some of the CO,, i s blown o f f . In response to the H + load, both v e n t i l a t i o n rate and volume increased: VR from a control l e v e l of 64.7 ± 1.2 to 79.0 +3.8 breath/min, and V from $ 0 . 5 + 3.6 to 187.5 G ~" + 2 0 . 3 ml/min at 2 min. Near normal l e v e l s are attained i n 90 min, although there i s considerable v a r i a b i l i t y i n the ve n t i l a t i o n rate, i . e . 68.1 + 4.0 breath/min. Very l i t t l e v a r i a b i l i t y existed at t h i s time for V^. THE EFFECT OF NaHCO INJECTION ON VENTILATION AND ARTERIAL pH IN TROUT I n t r a - a r t e r i a l i n j e c t i o n of 1 mM NaHCO i n saline was completed on 7 f i s h (245.6 + 10.8 g at 10.7 + 0.8 °C), re s u l t i n g i n acute metabolic a l k a l o s i s ( F i g . 13) . Both VR and VQ were affected. There was an i n i t i a l hyperventilation upon completion of i n j e c t i o n . Normal v e n t i l a t i o n rates were attained i n 2 to 3 hours. V^ reached a peak volume of 130,4 + 23.1 ml/min at 15 min after i n j e c t i o n . • This compares to a pre-i n j e c t i o n control .level of 45.0 + 4.5 ml/min, shov/ing approximately a 3-fold increase i n V^. Vsv, i n accordance, rose l i n e a r l y to 1.42 + C . 2 3 ml/breath from 0,54 + 0.04 ml/breath. Injection of • NaHCO causes- an a l k a l o s i s i n the b l o o d ' 3 as follows: 1) HCO" + HBuf -Buf" + H oC0 o -H_0 + C0 9 3 2 3 2 ^ 2) KCO^ j + H + " H 2 C 0 3 " H 2 ° + C 02 causing a f a l l i n [H +]a, hence pH increases'. This was 43 + observed, the [H ]a f a l l i n g from a c o n t r o l of 10.9 +0.9 to 4.9 +0.9 nM/L i n 5 minutes a f t e r i n j e c t i o n . T h i s i s a change of 6.0 nM/L. A gradual r e t u r n t o near normal l e v e l s i s observed i n 3 hours, the mean pHa being 9.9 + 0.6 nM/L. The NaHCO and HC1 data give f u r t h e r evidence t h a t V G i s not l i n e a r l y r e l a t e d t o a r t e r i a l pH l e v e l s alone, since both an incr e a s e and decrease i n pHa r e s u l t e d i n a r i s e i n V Q . However, since both NaHCO and HC1 i n j e c t i o n s cause an incr e a s e i n PaCOp i n i t i a l l y , V Q may be l i n e a r l y r e l a t e d t o a r t e r i a l CO^ t e n s i o n s . INDUCED METABOLIC ALKALOSIS WITH NaOH AND THE EFFECT ON VENTILATION AND pHa LEVELS. NaOH, l i k e NaHCO^, produces a metabolic a l k a l o s i s , but u n l i k e NaHCO^ i s n o n v o l a t i l e . Seven f i s h , weighing 2$9.7 + 20.2 g, ( a t 12.0 + 0.4 °C) were i n j e c t e d with 0.025 mM NaOH i n t o the d o r s a l a o r t a ( F i g . 1 4 ) . NaOH i s b u f f e r e d as f o l l o w s : 1) OH" + HBuf <-Buf~ + H £0 2) OH" +. HpCO^ -H £0 + KC0~ C0 2 + HO by the nonbicarbonate and bicarbonate system, r e s p e c t i v e l y , r e s u l t i n g i n a r i s e i n [HCO^] and hence an in c r e a s e i n pHa. COp i s consumed i n t h i s r e a c t i o n i n i t i a l l y , i n the formation of carbonic a c i d . There was a quicker r e t u r n of a r t e r i a l [H 1 -] t o normal values with NaOH a l k a l o s i s i n comparison to tha t w i t h 44 NaHCO^ i n j e c t i o n , even though [H +]a values achieved by either i n j e c t i o n were s i m i l a r . V e n t i l a t i o n volume increased to a maximum of 98.0 + 1 5 . 4 ml/min from a r e s t i n g , control value of 53.0 + 4.2 ml/min. This -is s l i g h t l y l e s s than a 2-fold increase i n V compared to a 3-fold increase obtained G o with NaHC0„ a l k a l o s i s . That the stimulation to V i s not an 3 <J i o n i c e f f e c t , i . e . Na + e f f e c t , i s shown i n F i g . 15. NaCI, injected at equimolar amounts as NaOH, caused no s i g n i f i c a n t change i n V_, VR, nor pHa. These data ( i . e . HC1, NaHCOQ and NaOH) indicate that both a r i s e and f a l l i n a r t e r i a l pH re s u l t i n an increase i n V Q , but that the system c o n t r o l l i n g v e n t i l a t i o n i s predominantly CO^ s e n s i t i v e . RELATIONSHIPS BETWEEN ARTERIAL H+ ION LEVELS AND VENTILATION. In F i g . 16, mean values of VR are plotted against corresponding H + ion concentrations. The plotted values represent progressive stages of the various acid-base disorders with time during each of the experiments. Normal values represent controls f o r each experimental group. Due to the considerable v a r i a b i l i t y , no re l a t i o n s h i p appears to exist between.arterial [ H + J and VR. On the other hand, a relat i o n s h i p does exist between V Q and [H']a ( F i g . 1 7 ) . The 3 acid-base disorders are depicted. Values are means taken from Figs. 6 ,7 , 11,12 and 13. Metabolic a l k a l o s i s , and both respiratory and metabolic acidosis cause increases i n v e n t i l a t i o n volume. A comparison of dV^/dTH ] between VJ hypercapnia and I-IC1 acidosis i s not v a l i d since d i f f e r e n t degrees of a r t e r i a l acidosis were induced. A clearer picture i s obtained when a l l experimental values of V r and [H ]a are 45 plotted as a scatter diagram with a regression l i n e drawn through these points. The equations obtained are: Y = 11.6219 + 6.1085 X Y = -25.6636 + 9.5517 X for hypercapnia and HC1 acidosis, respectively ( F i g . 18,19). These relationships should not be considered v a l i d f o r s t a t i s t i c a l treatment since i t contains, f o r one, the variable time. In addition, the functional r e l a t i o n s h i p between V & and [H +] i s f a r more complex than i t appears. The l i n e a r regressions are thus only rough approximations. NaHCO i s more of a stimulus f o r V Q than NaOH i s , even though both bases caused i d e n t i c a l a r t e r i a l [H +] changes. A l l these data suggest then, that although CO^ appears to be the main stimulus of v e n t i l a t i o n , the actual functional r e l a t i o n s h i p i s more complex than t h i s , as demonstrated by NaOH i n j e c t i o n . I t does not appear to be by a simple i o n i c e f f e c t , -i.e. by Na ions, since NaCl i n j e c t i o n does not stimulate v e n t i l a t i o n . Adjustment of v e n t i l a t i o n i t s e l f i s dominated by a change i n V Q rather than V R , since the v e n t i l a t o r y stroke volume (Vsv) rose i n a l i n e a r fashion bet-ween 40 - 240 ml/min, r e f l e c t i n g the r i s i n g V with l i t t l e or no s i g n i f i c a n t change i n VR ( F i g . 20). Adjustment i n mammals i s s i m i l a r , where the V „ or respiratory minute volume increases rather than E * J respiratory rate. . I I I . PERFUSION OF MOCK CSF INTO.THE CRANIAL CAVITY AND THE - EFFECT ON VENTILATION Cerebrospinal f l u i d , modified after M i t c h e l l , et a l 46 ( 1 9 6 3 ) , was perfused i n t o the c r a n i a l c a v i t y to t e s t CO^ /H4" ion s e n s i t i v e areas shown t o be at the v e n t r a l surface of the medulla oblongata, or pons i n mammals. Slow p e r f u s i o n of mock CSF with e i t h e r the NaHCO^ c o n c e n t r a t i o n or CO^  t e n s i o n changed caused no p e r c e i v a b l e a l t e r a t i o n i n v e n t i l a t i o n r a t e during the time course of approximately 1 hour. T h i s suggests t h a t e i t h e r the perfusate was not c i r c u l a t i n g near the CO s e n s i t i v e areas or t h a t the s e n s i t i v e area i s not - 2 l o c a l i z e d s u p e r f i c i a l l y as i n mammals. The l a t t e r i s probably more c o r r e c t s i n c e t o p i c a l or l o c a l i z e d a p p l i c a t i o n of mock CSF with high PCCv, on the medulla d i d not a l t e r v e n t i l a t i o n (Jones, personal communication). P e r f u s i o n of the b r a i n i t s e l f i s not f e a s i b l e i n t r o u t , mainly because of s i z e . IV. INDUCED ALKALOSIS WITH RADIOACTIVE NaH l 4CO^ AND PATHS OF EXCRETION OF CARBON-14. R e s u l t s from 6 f i s h ( 2 9 6 . 2 + 2 7 . 4 g at 5 . 4 + 0 . 4 °C) are presented i n F i g . 2 1 . The f l u x of carbon-14 i s p l o t t e d versus time. The data obtained i n the 6 experiments are i n d i v i d u a l l y shown. The curve was f i t t e d by eye. The values of expired water equals the amount of r a d i o a c t i v i t y , as carbon - 1 4 , passed from g i l l s to ex p i r e d water i n a 1 min pe r i o d at time of sampling. Because of the small amounts of u r i n e one i s able to c o l l e c t at any one time, each value of r a d i o a c t i v i t y i n u r i n e p l o t t e d represents the t o t a l amount of u r i n e passed from kidney t o bladder from one sampling p e r i o d t o another. 47 F i g u r e 12 Responses of rainbow t r o u t t o induced metabolic a c i d o s i s by the i n j e c t i o n of 0.025 mM HG1 i n s a l i n e . 6 f i s h (264.3 + 19.2 g, 11.8 + 0,4 °C) were t e s t e d , and the v e n t i l a t i o n r a t e and volume and a r t e r i a l hydrogen concent r a t i o n recorded. I n j e c t i o n i s at time zero. The values are means + 1 standard e r r o r . il cx/ x° CL o o o o o o o o ^ LO o K 0 3 c>0 o r< p< r< i—cao cr> 8 Q o 48 F i g u r e 13 The e f f e c t of induced metabolic a l k a l o s i s on v e n t i l a t i o n r a t e , volume and a r t e r i a l pH. A l k a l o s i s was induced by i n j e c t i o n of 1 mM NaHCO_ i n C o r t l a n d s a l i n e i n 7 f i s h (24$.6 + 10.8 g, 10.7 ± 0.8 °C). Values shown are means + 1 standard e r r o r . O 1 2 3 T I M E (hours) 49 Figure 14 Induction of metabolic a l k a l o s i s through NaOH i n j e c t i o n and i t s e f f e c t on a r t e r i a l pH and v e n t i l a t i o n r a t e and volume. A l k a l o s i s was produced by i n j e c t i o n of 0.02$ mM NaOH i n s a l i n e . 7 f i s h , weighing 2 $9.7 + 20.2 g, at a temperature of 12.0 + 0.4 °C, were t e s t e d . Values are means + 1 standard e r r o r . 50 Figure 15 Sham i n j e c t i o n of NaCI i n saline and the effect on a r t e r i a l pH, v e n t i l a t i o n rate and volume. 0.025 mM NaCI i n Cortland saline was used as a control i n j e c t i o n i n 7 f i s h (278»0 + 23.0 g, 10.1 + 0„9 °G). Values are presented as means ± 1 standard error. T I M E (hours) 51 Figure 16 Relationship between a r t e r i a l hydrogen concentration and v e n t i l a t i o n rate i n rainbow t r o u t . Values represent progressive stages of the various acid-base disorders and t h e i r compensation. Thus the points plotted for elevated PCO^ f o r example, are mean values at 5 min, 15 min, etc., up to 72 hours of exposure. Normal refers to control values. 4 0 3 0 [ H i a nM./l-2 0 ® normal © elevated pCQ~ o NaHCOQ 2 B NaOH J ° HCI 740 7.50 a 7.60 7 7 0 IO 65 7 0 • • 9 00 o 1 1 I u o 7.80 7 9 0 8.00 8.10 8.20 8.30 75 8 0 VR no./min. 85 9 0 52 F i g u r e 17 R e l a t i o n s h i p between a r t e r i a l hydrogen co n c e n t r a t i o n and v e n t i l a t i o n volume. Values represent p r o g r e s s i v e stages of the v a r i o u s acid-base d i s o r d e r s and t h e i r compensation. 4 0 i 3 0 [ H i nM./l 2 0 to ® normal • elevated pCCu o N a H C O o " N a O H 3 a HCI o O \ 0 o © B 0 ^ f c c o ^As 1 0 0 2 0 0 mis./min. 7.40 7.50 PH G 7.60 7 7 0 7.80 7.90 8.00 8.10 8.20 8.30 3 0 0 53 Figure 18 Linear regression on the r e l a t i o n s h i p betv;een V Q and a r t e r i a l [ H ] during hypercapnia. A l l experi-mental values during the time course of the experiment have been plotted by computer. The regression l i n e i s described by the equation: Y = 1 1 .6219 + 6 . 1 0 8 5 X 53^ VG ml min 400 • .Vli« 3C0-375-550 v 335 > 300-x75». 150'. 100'.. 75»_! 50-35. Y = 1CQE>X N = ±15 X X X x/ X X x ^ r ; x V x x - x xx ,X^/X x X x ^ x x x ^ X > r - X v v ^ -J •"• - / X s A x x< %x<x" X 1 0 -X X X Xx n b ° :30-nM/l 54 F i g u r e 19 R e l a t i o n s h i p between V and a r t e r i a l [U J G according t o l i n e a r r e g r e s s i o n during HC1 a c i d o s i s . A l l values during the time of the experiments have been p l o t t e d by computer. The r e l a t i o n s h i p i s described by the equation: Y = - 2 5 . 8 6 3 6 + 9 . 5 5 1 7 X 55 Figure 20 The r e l a t i o n s h i p between v e n t i l a t o r y stroke volume and v e n t i l a t i o n volume i n rainbow t r o u t . Ea po i n t represents p r o g r e s s i v e stages of the v a r i o u s acid-base d i s o r d e r s and t h e i r compensation. 3 0 0 2 0 0 VG mJs./min. 1 0 0 ® normal ® elevated pC00 o N a H C O - d • N a O H J • HCI ©a c o © © 9, 9 o • o o t o 2.0 3.0 STROKE \/OLUME mis./breath 4 0 56 A plot of the percent of recovery of injected dose against time i s expressed i n F i g . 22, giving cumulative excretion of carbon~ l 4 . 50% excretion of the injected dose i s obtained at 75 minutes after administration. The blood has circ u l a t e d and passed through the g i l l s approximately 75 times according to Davis' (1970) data on c i r c u l a t i o n time i n the rainbow trout ( i . e . 64.»1 -i- 16.4 sec (n = 9) f o r f i s h of similar weight). Assuming that cumulative excretion of radioactive carbon i n 4 hours i s 9 0 % , as Gould, et a l ( 1948) observed 14 i n NaH C0^ i n j e c t i o n studies i n the r a t , then the percent excreted via the g i l l or kidneys can be calculated. The t o t a l urine a c t i v i t y i s divided by the t o t a l a c t i v i t y i njected. On. 2 f i s h , from which complete sampling was made during the 4 hour run, the percent excreted v i a the kidney was calculated to be 0.40 and 0 .03% respectively. This indicates that the kidneys play only a minor r o l e i n the elimination of a HCO^ load. The NaHCO^ load e l i c i t e d a net l o s s of CO^ and/or HCO^ through the g i l l s , whereas the excretion rate by the kidneys remained v i r t u a l l y unchanged. There are three possible ways to restore the aci d -base balance following a NaHCO0 a l k a l o s i s : the passive intervention of blood buffers, or an increased HCO^ excretion by the active intervention of either kidney or g i l l . Of course, the load can also be blown off as C0 2. That the kidney plays only a minor part i n acid-base balance i s supported by Smith ( 1 9 3 9 ), Hodler et a l , and Murdaugh and Robin ( 1 9 6 7 ). They found that urine pH remains constant 57 despite important variations i n blood .pH produced by-inje c t i o n s of acids, a l k a l i or acetazolamide; neither did exposure to high CO i n the elasmobranch aff e c t the urine pH (Cross et a l , 1969) « Thus the g i l l i s obviously the main route of a l k a l i - a c i d excretion thereby maintaining the inte r n a l pH (Payan and Maetz, 1973). As Maetz (1971) states: i n freshwater f i s h the kidney has the task of removing excess water and also excreting some of the organic/inorganic acids as shown, by a renal Na"h and CI" l o s s . Unfortunately there i s no a n a l y t i c a l technique available at present i n separating the expired carbon-14 as either CO " , • 2 or as HCO'^ . However, evidence suggests that the NaHCO^ load i s probably mainly excreted as gaseous CO^ rather than ioni c HCO^ since g i l l tissue carbonic anhydrase l e v e l s are r e l a t i v e l y low (R. Milne, personal communication). Therefore, the NaHx CO^ experiment adds to the general theory that the teleostean g i l l i s a multi-purpose organ, specialized for ' respiratory gas exchanges, clearance of waste products of nitrogen metabolism and maintenance of acid-base and mineral balances (Maetz, 1971) . Figure 21 Excretory pathways of carbon-14 as CO and/ 14 or HCO^ after i n j e c t i o n of NaH CO^ i n 6 f i s h (296.2 + 27.4 g, 5.4 + 0.4 °C). The l i n e s have been f i t t e d by eye. Values of expired water equals t o t a l amount of r a d i o a c t i v i t y excreted by the f i s h i n a 1 minute period. Each urine value represents the t o t a l amount of urine collected between 2 sampling periods; i . e . value at 90 min represents a l l urine excreted by the animal i n the time i n t e r v a l from 60 to 90 min. 30 60 90 120 ' 150 180 210 240 * 290 MINUTES AFTER INJECTION 59 Figure 22 14 Cumulative CO excretion pattern from f i s h 2 14 injected with NaH CO^ plotted against time. The cumulative excretion rate v/as calculated by the "cut and weigh" technique from the curve i n Figure 21. C U M U L A T I V E E X C R E T I O N O F 1 4 C O O o o O —r™ CO O ~T~ 2 6 o (°/o O F I N J E C T E D D O S E ) 60 GENERAL DISCUSSION Although there i s an abundance of l i t e r a t u r e regarding pH regulation i n mammals, very l i t t l e i s known about waterbreathers. I t i s only through the work of Rahn and colleagues that an i n t e r e s t has been focused i n t h i s l a t t e r group of animals. However, they have only concentrated on pH regulation per se, s p e c i f i c a l l y i n conjunction with change i n temperature and the evolution of water to a i r -breathing,, I t i s only i n the l a s t few years that the functional r e l a t i o n s h i p between the a r t e r i a l acid-base status and v e n t i l a t i o n i n waterbreathers has been studied, s p e c i f i c a l l y through the work of Randall and Cameron. I. Regulation of a r t e r i a l blood pH v i a bicarbonate. The hypercapnia experiments indicate that i n the face of an increase i n ambient PCOg trout do not adjust the PCO^ difference (APCO^) between a r t e r i a l blood and water. PaCO^, as e a r l i e r shown by Cameron and Randall (1972), i n f a c t increases i n proportion to the change i n PjCOp such that PaCOp i s always about 2 mm Hg above ambient. That i s , the A P C O 2 i s not affected by changes i n v e n t i l a t i o n . The COp gradient between a r t e r i a l blood and water may r e s u l t from either a l i m i t a t i o n i n the rate of formation of COp i n the blood (Maren, 1967, as c i t e d from Randall, 1970) or from a d i f f u s i o n resistance between blood and water. With the increase i n PaCOp there i s a concomitant and s i g n i f i c a n t f a l l i n a r t e r i a l blood pH at the ambient PCO^ l e v e l s studied, producing respiratory a c i d o s i s . A sim i l a r response exists i n airbreathing animals, a r i s e i n 61 PaCC>2 associated with a f a l l i n pHa (Nichols, 1958) . However, airbreathers are capable of reducing the difference between ambient and a r t e r i a l CO^ tensions during sustained hypercapnia by hyperventilation as shown by Manfred! (1962). He found that experimental subjects, during maintained high CO^ l e v e l s , could reduce t h e i r TaCO through hyperventilation to l e v e l s below 40 mm Hg. A PaCO^ of 40 mm Hg i s considered normal i n man when ambient CO^ tensions are nearly zero. Thus airbreathers, unlike waterbreathers, can adjust blood pH and PaCO via v e n t i l a t i o n during hypercapnia. In trout, 2 the increase i n PaCO^ i s i n proportion to the increase i n P T C 0 o and.thus v e n t i l a t i o n has l i t t l e e f fect on APCO?. 1 2 ^ In trout i t i s shown that there i s a delay i n the increase of PaC'O^  r e l a t i v e to the ambient PCOp. One would expect equilibrium to be established within minutes v/ith the commencement of high ambient COp l e v e l s . However, i f one considers movements of CO,, within the f i s h ' s body during hypercapnia i n r e l a t i o n to CO,, production by the tis s u e s , the observed time l a g of PaCO,, r e l a t i v e to PjCOp may be explained. I n i t i a l l y , CO,, l e v e l s within body compartments w i l l be raised due to COp entry across the g i l l s and by tissu e C O 2 production i t s e l f . In addition, the reaction v e l o c i t y of COp to HCO^ w i l l affect both C 0 o and HCO_, l e v e l s within each compartment. 2 J Eventually equilibrium w i l l be achieved, COp uptake across the g i l l s w i l l stop and CO,, l e v e l s i n each compartment w i l l r i s e above ambient. A new steady state w i l l thus be established. The time of t h i s response w i l l be dependent on the rate of exchange and magnitude of the COp/HCOo stores within the body 62 compartments. Nothing i s known about many of the.time constants involved. The change i n a r t e r i a l blood pH i s related to the change i n PaCCr rather than the i n f l u x of H + ions as a resuT of the decrease i n pHj associated with an increase i n ambient PCO,,. Support for t h i s comes from experiments i n which the pH of water was altered. Although the decrease i n pHj was much greater than that encountered when PjCO^ was increased to 5 mm Hg, the s h i f t i n a r t e r i a l blood pH was s i g n i f i c a n t l y smaller. Therefore, the f a l l i n pHa i s the re s u l t of an increase i n PaCO^ and not a r e s u l t of the transfer of H + from water to blood. In order to regulate a r t e r i a l blood pH i n the face of a r i s e i n ambient PCO , blood bicarbonate l e v e l s were 2 adjusted. That the time course of HCO-^  adjustment i s slow during hypercapnia was also noted by Lloyd and White (196?). In trout, complete compensation to sustained hypercapnia required up to 3 days to complete. Mammals, i n general, display a simi l a r long-term mechanism during prolonged hypercapnia i n the regulation of pHa. The period of hypercapnia, before compensatory mechanisms have begun, r e f l e c t s the effect of blood b u f f e r i n alone. In t h i s s i t u a t i o n , as observed i n trout, reduction of PjCO^ t o z e r o leads to rapid restoration of the o r i g i n a l , normal acid-base balance, thus r e f l e c t i n g a back t i t r a t i o n along the blood buffer curve ( l i n e C-A, F i g . 10). This i s a observed i n mammals, the time course being similar (Refsum, 1971) . Prolonged- elevation of PaC0 ? leads to an increase i n 63 pHa via HCO~ along the actual TaCO^ isopleth ( l i n e C-B, Fig.10). In t h i s s i t u a t i o n , rapid reduction of FCO^ ( a s done i n several fish) l e d to r e l a t i v e l y marked increase i n pHa above the i n i t i a l value, frequently above 8.20, demonstrating the increase i n the plasma [HCO-^]. KCO^ can either be regulated v i a the kidney or the g i l l s . The kidney plays a major r o l e i n HCO^ regulation i n mammals, either by reabsorption or excretion of HCO^ (Refsum, 1 9 7 1 ) . In f i s h , evidence points to the contrary, the kidney playing only a minor rol e i n both the dogfish and trou t . The renal response to NaHCO^ induced a l k a l o s i s i s neg l i g i b l e as shown by the labelled--NaHCO experiments. This i s also supported by Murdaugh and Robin ( 1 9 6 7 ) . They found neither a l k a l i z a t i o n nor a c i d i f i c a t i o n of urine occured i n the dogfish following NaHCO^ and HC1 administration. Evidence supports that HC0~ i s regulated v i a the g i l l s . Fish subjected to NaHCO^ buffered water i n which PjCO^ was raised showed a much le s s severe change i n a r t e r i a l blood pH than f i s h i n nonbuffered water, even though PjCOp l e v e l s were s i m i l a r . This implies that HCO^ must have been taken up from the water by the g i l l s . Fish placed i n high bicarbonate water show a r i s e i n pHa. Lloyd and White ( 1 9 6 7 ) add further support to the role of the g i l l s i n HCO-j regulation: they describe increases i n blood plasma HCO^ following acclimation to high l e v e l s of CO^. They r i g h t l y conclude that the purpose of the observed increase i n HC0~ i s to maintain pH. A concomitant decrease i n plasma C l " was also observed by these authors, the changes i n 64 HCO~ and Cl being of approximately the same magnitude and reversed when the animals were restored to water of low PCO,> Maetz and Garcia-Romeu (1964) were the f i r s t to present data supporting a HC0~/C1~ exchange i n f i s h . By a l t e r i n g the CO,, l e v e l i n the water they were able to vary C l " movement across the g i l l s . Dejours (1969) demonstrated that f i s h transferred to water of very low C l " content shov/ed a marked reduction i n CO,, excretion immediately following tr a n s f e r . Thus i n freshwater, indications are that f i s h C l " i n f l u x i s l i n k e d to-HCO^ e f f l u x . The mechanism of pH adjustment i n trout may involve regulation of a HCO^/Cl" exchange. In addition, there i s evidence for a Na /H exchange and dir e c t H + excretion (Kerstetter et a l . , 1 9 7 0 , Maetz, 1 9 7 1 , 1973 , Payan and Maetz, 1 9 7 3 ) . However, i t has not been demonstrated that the rate of HCO^/Cl" exchange i s modified to adjust plasma HCO^ l e v e l s i n order to regulate pHa. This could equally be achieved by regulation of the rate of Na /H exchange or by H excretion (Randall and Cameron, 1973) . COp could be excreted by the trout either as gaseous COp or as HC0~. Because of some lo s s of ions by the kidney ( i . e . Na +, C l " ; Maetz, 1 9 7 1 ) , HC0~ ef f l u x could thus be link e d to a C l " i n f l u x thereby maintaining plasma el e c t r o n e u t r a l i t y . However, the extent of t h i s HCO^/Cl™ exchange may vary from species to species. In go l d f i s h , an extensive re l a t i o n s h i p exists between HCO^ and Cl since COp excretion seems very dependent on the external Cl concentration. In salmonids perhaps t h i s HC0^/C1" exchange 65 has a very small c a p a c i t y as shown by the 2 to 3 day p e r i o d before pHa was adjusted v i a bicarbonate. T h i s time course i s supported by R. Milne (unpublished data) i n h i s study i n t r a n s f e r of t r o u t , and t h e i r adjustment, from f r e s h to s a l t -water. In a d d i t i o n , he has demonstrated very low carbonic anhydrase l e v e l s i n the t r o u t g i l l e p i t h e l i u m versus high l e v e l s i n the g o l d f i s h g i l l e p i t h e l i u m . Carbonic anhydrase i s r e q u i r e d i n the f a s t conversion of COp t o bicarbonate (Maren, 1 9 6 7 ) . However, a l t e r a t i o n of bicarbonate l e v e l s i n the water aided pH r e g u l a t i o n i n t r o u t i n these s t u d i e s . This was not confirmed by Mi l n e , pHa not being a f f e c t e d by a s a l i n i t y change. I I . The r e l a t i o n s h i p between v e n t i l a t i o n volume and a r t e r i a l  CO^ l e v e l s . In t r o u t , the in c r e a s e i n the v e n t i l a t o r y s t roke volume, as a r e s u l t of an a l t e r a t i o n of the acid-base s t a t u s of the blood i s mainly due to an in c r e a s e i n v e n t i l a t i o n volume, ra t h e r than r a t e . In a i r b r e a t h e r s a s i m i l a r response p r e v a i l s , there being a greater i n c r e a s e . i n the r e s p i r a t o r y minute volume r a t h e r than breathing frequency (Schaefer et a l . , 1963) . The v e n t i l a t i o n volume i n t r o u t i s dependent on an increase i n ?aCO^ and/or F^CC^ and not to pHa or pHj as i n d i c a t e d . A decreased ambient pH l e v e l , although causing a f a l l i n pHa, has only a delayed e f f e c t on V ^ . Hoglund and Hardig (1969) have a l s o concluded t h a t behavioural r e a c t i o n s ( i . e . r i s e i n V R ) provoked by ambient pH/PCOp changes i s e s s e n t i a l l y due to an inc r e a s e i n P-rCO and not 66 to an inc r e a s e i n ambient pH. IvS The a r t e r i a l a c i d o s i s ma^ t- not be due t o a l a c t a t e accumulation (Hoglund and Borjeson, 1971) as observed when A t l a n t i c salmon were subjected t o a low pH^. The observed « increase i n V i s probably r e l a t e d t o c o n d i t i o n s l e a d i n g to G hypoxia. I t i s w e l l documented th a t f i s h exposed t o low ambient pH show a decreased a b i l i t y t o e x t r a c t 0^ from the water, t h i s p o s s i b l y r e l a t i n g to the observed i n c r e a s e i n mucous production at the g i l l surface (Jones, 1964, Town send-and Cheyne, 1944; as c i t e d from Packer and Dunson, 1970) . Mucous accumulation at the g i l l may cause an inc r e a s e i n the 0^ d i f f u s i o n d i s t a n c e . However, nothing i s known about the r a t e s of d i f f u s i o n of gases through mucous produced by the g i l l s ( R a n d a l l , 1970). In a d d i t i o n a f a l l i n a r t e r i a l blood pH i s as s o c i a t e d with a decrease i n the 0^ c a r r y i n g c a p a c i t y of the blood (Bohr-Root e f f e c t ) ( R a n d a l l , 1970). These r e s u l t s are c o n s i s t e n t w i t h the hypothesis t h a t v e n t i l a t i o n i s dependent on the C0£ t e n s i o n v/ithin the body or elsewhere and t h a t blood pH l e v e l s are r e g u l a t e d v i a i o n i c exchange mechanisms at the g i l l s u r f a c e , r a t h e r than by i o n exchange at the kidney or by d i f f u s i v e washout of gaseous CO,, v i a v e n t i l a t i o n . ' Receptors and the r e g u l a t i o n of v e n t i l a t i o n . The response i n i s r a p i d and t r a n s i e n t and i s shown to respond to PGOp changes at the surface or w i t h i n the f i s h . Although the change i n V Q i s t r a n s i e n t , PaCO^ i s held constant r e l a t i v e to P C0~. Therefore, r e c e p t o r s are I <~ e i t h e r adapting or the rec e p t o r s are not l o c a t e d i n the blood 67 or water but i n another compartment whose contents or properties change i n proportion to V . From the rapid G response noted to acid-base changes i n v e n t i l a t i o n , receptors are most l i k e l y a r t e r i a l ! y located, or possibly c e n t r a l l y i n the brain, since blood c i r c u l a t i o n time i n trout i s of the order of minutes rather than seconds (Davis, 1 9 7 0 ; Jones et a l . , 1970) . However, t h i s does not preclude oral cavity or external g i l l recepters. Since COp s t i l l increases breathing following denervation of peripheral chemoreceptors i n mammals, central chemosensitivity i s or obvious importance i n regulating v e n t i l a t i o n (Hornbein, I965) . Leusen (1954) reported that v e n t i l a t i o n could be changed by a l t e r i n g the PCOg-fH*] composition of f l u i d s perfusing the v e n t r i c l e s i n dogs. This led to the delineation of an area on the s u p e r f i c i a l layers of the ventrolateral surface of the medulla as the chemosensitive area for v e n t i l a t i o n ( M i t c h e l l et a l . , 1963)• Both an increase i n cerebrospinal f l u i d PCO^ and decrease i n CSF pH causes an increase i n v e n t i l a t i o n ; a f a l l i n CSF PCO and an increase i n pH causes hypoventilation i n mammals. 2 The CSF i s a HCO"^  containing f l u i d with a very low protein content ( M i t c h e l l et a l . , . 1963) and i t has, therefore, a low buffer capacity toward CO.,, The ba r r i e r interposed between 2 plasma and the CSF (blood-brain barrier) i s r e l a t i v e l y impermeable to H + ions and HC0~ ions but highly permeable to C0 2 (Messeter, 1971) . The pH of the CSF i s extremely well regulated i n • mammals (Loeschke, 1971). This regulation i s exerted 68 by signals from the sensitive areas on the medulla (Schaefke et al_., 1 9 7 0 ) , the signals depending on the l o c a l pH around these structures. These signals are responsible for the ven t i l a t o r y drives by both CSF PCO^ and pH.in mammals. Local receptor pH i s determined by blood FCO^ and blood pH, the l a t t e r by way of r e d i s t r i b u t i o n of HCO^ (Loeschke, 1 9 7 1 ) . The regulator acts by increasing v e n t i l a t i o n as soon as l o c a l e x t r a c e l l u l a r pH drops, the drops being counteracted by the elimination of CO^ i n mammals. This suggests that i n trout, central receptor a c t i v i t y , i f e x i s t i n g , and hence, ac t i v a t i o n of v e n t i l a t i o n , could be the r e s u l t of f a s t entry of CC> versus slow entry of HCO~ or H + across the blood-brain 2 J 3 b a r r i e r , thereby causing a f a l l i n CSF pH. The observed transient nature of the increase i n V Q could be explained by the adjustment of CSF pH to normal l e v e l s during sustained hypercapnia. Thus V Q could be dependent upon CSF pH via PaCO^ i n f i s h , the CSF pH af f e c t i n g receptor a c t i v i t y of the respiratory mechanism. Adjustment of CSF pH i s probably by HCO^ since CSF i s e s s e n t i a l l y a CO^-HCO^ buffer system. Maren ( 1 9 7 2 ) demonstrated that as the PCO^ rose i n the blood of dogfish an immediate r i s e i n CSF PCC^ V 7 a s noted, as observed i n mammals. As a r e s u l t , CSF pH dropped. This drop i n CSF pH i s associated with an increase i n v e n t i l a t i o n i n the dogfish (D.J. Randall, personal communication) . CSF pH was regulated i n the dogfish within 3 hours as a re s u l t of HCO^ formation (Maren, 1 9 7 2 ) during respiratory acidosis. This i s simi l a r to the time course of the Vp change as observed by Randall. The rate data of 69 bicarbonate formation and the carbonic anhydrase i n h i b i t i o n suggests that HCO~ reaches the CSF by hydroxylation of gaseous CO^ at the choroid plexus and probably at the g l i a i n the dogfish. I f respiratory responses of trout to changes i n the acid-base status of the blood are mediated by changes i n CSF PCC^-pH as i n mammals and as suggested i n dogfish, then perfusion of the cr a n i a l cavity with mock CSF with altered PC0 2~[H +] should affect v e n t i l a t i o n . Although t h i s was attempted i n trout, no response could be e l i c i t e d by the perfusion technique used. D.R. Jones (personal communication) did not obtain any response i n trout with t o p i c a l application of mock CSF1 on the medulla. This suggests that the M i t c h e l l CO,p-[H+] sensitive area may not exis t super-f i c i a l l y on the medulla as implied. This i s supported by McCarthy and Borison (1972) who suggest that surface receptors are an addendum used to explain the r a p i d i t y of response to t o p i c a l l y applied agents i n a system where d i f f u s i o n i s the sole mechanism of penetration. They believe there exists a rapid access route capable of carrying substances from the surface of the medulla, to more deeply l y i n g structures. The receptors may be located anatomically more c e n t r a l l y i n the trout brain, possibly within the v e n t r i c l e s themselves. However, one should consider the anatomical differences of the brain e x i s t i n g between f i s h and mammals. The f l u i d surrounding the brain i n mammals i s CSF, the cranial cavity being the subarachnoid space. Because teleosts (and a l l lower vertebrates) have no true dura a true 70 subarachnoid space does not e x i s t . The area s u r r o u n d i n g the b r a i n i s r a t h e r a perimeningeal l a y e r c o n t a i n i n g not CSF but plasma exudate or d i a l y s a t e (Davson, 1967). When l a b e l l e d serum albumen was i n j e c t e d i n t o the v e n t r i c l e s of sharks, K l a t z o and S t e i n w a l l , 1965 (as c i t e d from Davson, 1967) found no r a d i o a c t i v i t y on the b r a i n s u r f a c e or su r r o u n d i n g f l u i d s . The CSF i s thus c o n f i n e d o n l y t o the v e n t r i c l e s . Thus t o e l i c i t v e n t i l a t o r y responses i n t r o u t , one p o s s i b l y needs t o p e r f u s e the v e n t r i c l e s d i r e c t l y , i f c e n t r a l r e c e p t o r s are l o c a t e d t h e r e . Even i f c e n t r a l chemoreceptors were the main r e g u l a t o r of v e n t i l a t i o n as i n mammals, p e r i p h e r a l r e c e p t o r s , e x i s t i n g e i t h e r w i t h i n or o u t s i d e the f i s h ' s body, cannot be p r e c l u d e d . In mammals, the a c t i v i t y of the m e d u l l a r y r e s p i r a t o r y center i s mediated by the a c t i v i t y of s e v e r a l r e c e p t o r s . These r e c e p t o r s i n c l u d e the a o r t i c and c a r o t i d body chemoreceptors, mechanoreceptors i n the l u n g s and c e n t r a l H + r e c e p t o r s w i t h i n the medulla i t s e l f (Hornbein, 1965). Analogous s t r u c t u r e s may e x i s t i n the t r o u t , m o n i t o r i n g blood or water P0£, p c ° 2 ( a n d t o a l e s s e r degree, pH) and m a i n t a i n i n g r e s p i r a t o r y r h y t h m i c i t y . 71 SUMMARY 1) The e f f e c t of a l t e r i n g the acid-base s t a t u s of blood on pH r e g u l a t i o n and v e n t i l a t i o n was s t u d i e d . In a d d i t i o n , p e r f u s i o n of the c r a n i a l c a v i t y of t r o u t was attempted w i t h the hypothesis t h a t v e n t i l a t i o n could be a f f e c t e d by a l t e r a t i o n of CSF RCO -[H 4"]. 3 2) In the face of an inc r e a s e i n ambient PCO^ the a r t e r i a l CO^ t e n s i o n r o s e . Trout are not capable of a d j u s t i n g the blood-to-water gradient of PC0 2 ( APCOp) v i a V Q, the A P C O ' r e m a i n i n g at 2 mm Hg above ambient duri n g both normo-and-hypercapnic c o n d i t i o n s . 3) Hypercapnia produced a severe a r t e r i a l a c i d o s i s . The increase i n a r t e r i a l blood pH Is the r e s u l t of the increase i n ambient PCO- r a t h e r than a r e s u l t of an i n f l u x 2 of H + ions from water t o blood. 4) A r t e r i a l blood pH r e g u l a t i o n i s slow, t a k i n g up to 3 days. pHa i s r e g u l a t e d by adjustment of plasma HCO^ level's during sustained hypercapnia along the PaCO^ i s o p l e t h . 5) HCO^ can e i t h e r be re g u l a t e d v i a the kidneys or the g i l l s . Evidence i s presented showing d i r e c t uptake of bicarbonate from KaHCO^ enriched waters by the g i l l s . The r e n a l response i s shown t o be n e g l i g i b l e In a m e l i o r a t i n g the NaHCO^ induced a l k a l o s i s . 6) CO i s shown to be the main stimulus to v e n t i l a t i o n . 2 However, the r e l a t i o n s h i p i s probably more complex. The increase i n v e n t i l a t i o n i s mainly due t o an in c r e a s e i n volume r a t h e r than r a t e . 7) P e r f u s i o n of the c r a n i a l c a v i t y w i t h mock CSF of a l t e r e d CO^-HCO^ composition d i d not e l i c i t v e n t i l a t o r y responses. These r e s u l t s do not preclude the presence of c e n t r a l l y l o c a t e d chemoreceptors, 8) The r e s u l t s are c o n s i s t e n t w i t h the hypothesis t h a t v e n t i l a t i o n i s dependent on the PCO^ of the blood or elsewhere, and t h a t blood pH l e v e l s are r e g u l a t e d v i a i o n i c exchange mechanisms at the g i l l s urface r a t h e r than by i o n exchange at the kidney or by d i f f u s i v e washout of CO^ v i a v e n t i l a t i o n . 73 REFERENCES A l b e r s , C. ( 1 9 7 0 ) . Acid-base balance. In F i s h P h y s i o l o g y ( ed. W.S. Hoar and D.J. Randall) AcaaTjrac~Pre"ss, New York. IV: 1 7 3 - 2 0 8 . Astrup, P., K. Engel, K. J^rgensen and 0. Siggaard-Anderson. (1966). D e f i n i t i o n s and terminology i n blood a c i d -base chemistry. Ann. N.Y. Acad..Sci. 133: 59-65. Benakis, A. ( 1 9 7 1 ) . A new g e l i f y i n g agent i n l i q u i d s c i n t i l l a t i o n counting. In Organic S c i n t i l l a t o r s  and L i q u i d S c i n t i l l a t i o n Counting. ( ed'. D.L. Horrocks and C.f. Peng). Academic 'Press, pp . 70 5-74 5. Cameron, J.N. and D.J. R a n d a l l . ( 1 9 7 2 ) . The e f f e c t of increased ambient CO,-, on a r t e r i a l C 0 ? t e n s i o n , C0 9 content and pH i n rainbow t r o u t . J . Exp. B i o l . 57: 673-680. Cross, C.E., B.S. Packer, J.M. L i n t a , H.V. Murdaugh, J r . and E.D. Robin. (196 9 ). H + b u f f e r i n g and e x c r e t i o n i n response t o acute hypercapnia i n the d o g f i s h , Squalus a c a n t h i a s . Am. J . P h y s i o l . 2 1 6 : 4 4 0 - 4 5 2 . Davis, J.C. ( 1 9 7 0 ) . E s t i m a t i o n of c i r c u l a t i o n time i n rainbow t r o u t , Salmo. g a i r d n e r i . J . 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Normal a r t e r i a l gas tensi o n s and pH and the br e a t h i n g frequency of the e l e c t r i c e e l . Resp. P h y s i o l . 9:141-150. 74 Gould, R.G., F.M. Sinex, I.N. Rosenberg, A.K. Solomon and A.B. Hastings, (1948). E x c r e t i o n of r a d i o a c t i v e C0~ by r a t s a f t e r a d m i n i s t r a t i o n of i s o t o p i c bi6arbonate, acetate and s u c c i n a t e . J . B i o l . Chem. 177:295-301. Hodler, J . , H.O. Heinemann, A.P. Fishman and H.W. Smith. (1955). Urine pH and carbonic anhydrase a c t i v i t y i n the marine d o g f i s h . Am. J . P h y s i o l . 183:155-162. Hoglund, L.B. and H. Borjeson. (1971). A c i d i t y and l a c t a t e content i n the blood of young A t l a n t i c salmon exposed to high PCOp. I n s t . Freshwater Res., Drottingholm, F i s h e r y Bd. Sweden. Hoglund, L.B. and J . Hardig. (1969). Reactions of young salmonids t o sudden changes of pH, PC0£ and 0, content. I n s t . Freshwater Res., Drottingholm, F i s h e r y Bd. Sweden. Hornbein, T.F. (1965). The chemical r e g u l a t i o n of v e n t i l a t i o n . I n Physiology and B i o p h y s i c s . (Ed. T.C, Ruch and H.D7~Patton). W.B. Saunders, P h i l a d e l p h i a , pp. 803-819. Howell, B.J., F.W. Baumgarten, K. Bondi and H. Rahn. (1970). A c i d base balance i n cold-blooded v e r t e b r a t e s as a f u n c t i o n of body temperature. Am. J . P h y s i o l . 218: 600-606. Howell, B.J., H. Rahn, D. Goodfellow and C. H e r r e i d . (1973). A c i d base r e g u l a t i o n and temperature i n s e l e c t e d i n v e r t e b r a t e s as a f u n c t i o n of temperature. Amer. Z o o l . 13:557-563. Jackson, D.C, (1973). V e n t i l a t i o n and a c i d base balance of t u r t l e s a t various temperatures. In press. Jones, D,R.S D.J. Randall and G.M. Jarman, (1970). A gr a p h i c a l a n a l y s i s of 0 o t r a n s f e r i n f i s h , Resp. P h y s i o l . 10:28 5-298. c K e r s t e t t e r , T.H. and L.B. K i r s c h n e r . (1972). A c t i v e c h l o r i d e t r a n s p o r t bv the g i l l s of rainbov; t r o u t . J . Exp. B i o l . 56:263^72. K e r s t e t t e r , T.H., L.B. K i r s c h n e r and D. Rafuse. (1970), On the mechanism of sodium i o n t r a n s p o r t by the i r r i g a t e d g i l l s of rainbow t r o u t (Salmo g a i r d n e r i ) . J . Gen. P h y s i o l . 56:342-359. ' '"~ " Leusen, I.R. (1954). C h e m o s e n s i t i v i t y of the r e s p i r a t o r y center: i n f l u e n c e of C0 9 i n the c e r e b r a l v e n t r i c l e on r e s p i r a t i o n . Am. J . ^ P h y s i o l . 176:39-44. 75 L l o y d , R. and W.R. White.' (1967) . E f f e c t of high concentration of C0 ? on the i o n i c composition of t r o u t blood. Nature 216:1341-1342. Loeschke, H.H. ( 1 9 7 1 ) . The a c i d base s t a t u s of c e r e b r o s p i n a l f l u i d and the r e g u l a t i o n of br e a t h i n g . Proc. I n t e r n . P h y s i o l . Sciences. V I I I : 2 3 1 - 2 3 3 . McCarthy, L.E. and H.L. B o r i s o n . ( 1 9 7 2 ) . Separation of c e n t r a l e f f e c t s of COp and n i c o t i n e on v e n t i l a t i o n and blood pressure. Kesp. P h y s i o l . 1 5 : 3 2 1 - 3 3 0 . Maetz, J . ( 1 9 7 1 ) . F i s h g i l l s : mechanisms of s a l t t r a n s f e r i n freshwater and seawater. P h i l . Trans. Roy. Soc. Lond. B. 262:209 - 2 4 9 . Maetz, J . (1973). Na+/NH"|, N"a+/H exchanges and NH~ movements across trie g i l l of Car a s s i u s auratds. J . Exp. B i o l . 58:255-275. " ~~ ' Maetz, J . and F. Garcia-Romeu. (1964). I I . Evidence f o r NHt/Na + and HCO^/Cl"" exchanges. J . Gen. P h y s i o l . 48+1209-1227. -> Maren, T.H. ( I 9 6 7 ) . Carbonic anhydrase: chemistry, physiology and i n h i b i t i o n . P h y s i o l . Rev. 47:595-781. Maren, T.H. (1972). Bicarbonate formation i n c e r e b r o s p i n a l f l u i d : r o l e of sodium t r a n s p o r t and pH r e g u l a t i o n . Amer. J . P h y s i o l . 222:885-899. M i t c h e l l , R.A., H.H. Loeschke, W.H. Massion and J.W. Severinghaus. (1963). R e s p i r a t o r y responses mediated through s u p e r f i c i a l chemosensitive areas on the medulla. J . Appl. P h y s i o l . 18: 523-533» Murdaugh, H.V. and E.D. Robin. (1967). A c i d base metabolism i n the dog f i s h shark. In Sharks, Skates and Rays, (ed. P.W. G i l b e r t , R.F. Mathews on and "D". FT"ltaTlT. John Hopkins P r e s s , B a l t i m o r e , pp. 249-264. N i c h o l s , G. (1958). S e r i a l changes i n t i s s u e COp content during acute r e s p i r a t o r y a c i d o s i s . J . C l i n . I n v e s t . 37:1111-1122. Packer, R.K. and W.A. Dunson. (1970). E f f e c t of low environmental pH on blood pH and sodium balance of brook t r o u t , j . Exp. Zool'. 174:65-72. Pappenheimer, J.R., V. F e n c l , S.R. Heisey and D. Held. (1965). Role of c e r e b r a l f l u i d s i n c o n t r o l of r e s p i r a t i o n as studi e d i n unaesthetized goats. Am. J . P h y s i o l . 208:436-450. 76 Payan, P. and J . Maetz. (1973). B r a n c h i a l Na t r a n s p o r t mechanisms i n S c v j i o r h i n u s c a n i c u l a : evidence f o r Na+/NH^ and lla:r/tt' exchanges and f o r a r o l e of carbonic anhydrase. J . Exp. B i o l . 58:487-502. Rahn, H. (1966). Aquatic gas exchange: theory. Resp. P h y s i o l . 1:1-12. Rahn, H. (1967). Gas t r a n s p o r t from the e x t e r n a l environment t o the c e l l . In Development of the Lung, (ed. A.V.S. deReuck and R. P o r t e r ) . J . & A. C h u r c h i l l L t d . , London. 408 p. Rahn, H. and F.W. Baumgardner. (1972). Temperature and acid-base r e g u l a t i o n i n f i s h . Resp. P h y s i o l . 14: 171-182. Rahn, H. and J.B. West. (1963). Aquatic gas exchange p r e d i c t i o n of a r t e r i a l gas te n s i o n s and " v e n t i l a t i o n " of g i l l s . P h y s i o l o g i s t 6:259. ( A b s t r a c t ) . R a n d a l l , D.J. (1970). Gas exchange i n f i s h . In F i s h P h ysiolog y, (ed. W.S. Hoar and D.J. RandalXTT" Icademic Press, New York. IV:253-292. R a n d a l l , D.J. and J.N. Cameron. (1973). R e s p i r a t o r y c o n t r o l of a r t e r i a l pH as temperature changes i n rainbow t r o u t , Salmo g a i r d n e r i . Am. J . P h y s i o l . In press. Reeves, R.B. (1969). Role of body temperature i n d e t e r -mining the acid-base s t a t e i n v e r t e b r a t e s . Fed. Proc. 28:1204-1208. Reeves, R.B. (1972). An imidaz o l e a l p h a s t a t hypothesis f o r v e r t e b r a t e acid-base r e g u l a t i o n : t i s s u e C0£ content and. body temperature i n b u l l f r o g s . Resp. P h y s i o l . 14:219-236. Refsum, H.E. (1971). P a t t e r n s of a r t e r i a l acid-base balance i n r e s p i r a t o r y and metabolic acid-base dist u r b a n c e s . Scand. J . C l i n . Lab. I n v e s t . 118:8-11. Robin, E.D. (1962). R e l a t i o n s h i p between temperature and plasma p] 249-251". P H and PC0 ? i n the t u r t l e . Nature 195: Schaefer, K.E., B.J. Hastings, C.R. Carey and G. N i c h o l s , J r . (1963). R e s p i r a t o r y a c c l i m a t i z a t i o n t o COp. J . Appl. Physiol.'18:1071-1078. Schlaefke, M.E., W.R. See and H.H. Loeschke. (1970). V e n t i l a t o r y response t o a l t e r a t i o n s of hydrogen i o n concentration i n small areas of the v e n t r a l medullary sur f a c e . Resp. P h y s i o l . 10:198-212. 77 Siggaard-Anderson, 0. (1961),. Sampling and s t o r i n g of blood f o r determination of acid-base s t a t u s . Scand, J . C l i n . Lab. In v e s t . IJ:196-204. Sinex, F.M., J . P l a z i n , D. Clareus, W. B e r n s t e i n , D.D. van Slyke and R. Chase. (1955). Determination of t o t a l carbon and i t s r a d i o a c t i v i t y . J . B i o l . Chem. 213: 673-680. Singer, R.B., R.C. Deering and J . K. C l a r k . (1956). The acute e f f e c t s i n man of a r a p i d intravenous i n f u s i o n of hypertonic NaHCO., s o l u t i o n . I I . Changes i n r e s p i r a t i o n and output of COp. J . C l i n . I n v e s t . 35:245-253. Smith, L.S. (1966). Blood volume of three salmonids. J . F i s h . Res. Bd. Canada. 23:1439-1446. Smith, L.S. and G.R. B e l l . (1964). A technique f o r prolonged blood sampling i n f reeswirnming salmon. J . F i s h . Res. Bd. Canada. 21:711-717. Smith, W.W. ( 1939). The e x c r e t i o n of phosphate i n the dog f i s h Squalus a c a n t h i a s . J . C e l l . Comp. P h y s i o l , 14:95-102. ~ Truchot, J.P. (1973). Temperature and acid.base r e g u l a t i o n i n the shore crab. Resp, P h y s i o l . 17:11-20. Wang, C H . and D.L. W i l l i s . (1965). R a d i o t r a c e r Methodology i n B i o l o g i c a l ScienceT P r e n t i c e - H a l l , Inc.' 382 pages'. " Wedemeyer, G. (1970). S t r e s s of anaesthesia w i t h MS-222 and benzocaine i n rainbow t r o u t , Salmo g a i r d n e r i . J . F i s h . Res. Bd. Canada. 27:909-91^7" Wolf, K. (1963). P h y s i o l o g i c a l s a l i n e s f o r freshwater t e l e o s t s . Progr. F i s h C u l t u r i s t . 25:135-140. Wood, CM. (1971 MS), The i n f l u e n c e of swimming a c t i v i t y on sodium and water balance i n the rainbow t r o u t (Salmo g a i r d n e r i ) . M.Sc. T h e s i s . U n i v e r s i t y of B r i t i s h CJoTumbia, Vancouver, B.C. Wood, CM. and D.J. R a n d a l l . ( 1973) . The i n f l u e n c e of swimming a c t i v i t y on sodium balance i n the rainbow t r o u t , Salmo g a i r d n e r i . J . Comp. P h y s i o l . 8 2 : 2 0 7 - 2 3 3 . ~ ~ 78 14 APPENDIX A. ASSUMPTIONS AND CALCULATIONS FOR NaH CO^ INJECTION AND l 4C0 2/H l 4CO~ RECOVERY In order t o formulate the amount of HCO^ t o i n j e c t i n t o the animal t o induce a l k a l o s i s , the f o l l o v / i n g c r i t e r i a were considered. Knowing that the plasma bicarbonate c o n c e n t r a t i o n of rainbow t r o u t averages 7 mM/L, a 200 g f i s h , c o n t a i n i n g approximately 10 ml of blood (Smith, 1966), and-with a haematocrit of 30% (packed c e l l volume) , would have about 7 ml of plasma. The c a l c u l a t e d plasma HCO^ c o n c e n t r a t i o n would be 49 juM/ml or 350 jaM f o r a 200 g f i s h . Thus an i n j e c t i o n of 1 mM NaHCO was considered adequate 3 i n e l i c i t i n g the d e s i r e d response. 14 A d m i n i s t r a t i o n of 5 JiO of NaH CO^ was c a l c u l a t e d t o be adequate f o r the d e t e c t i o n of r a d i o a c t i v i t y i n the expired water." T h i s value was approximated as f o l l o w s . Knowing t h a t the f l o w r a t e of the system ( F i g . 4) i s 200 ml/min ( l e s s could l e a d t o a n o x i a ) , and that the background r a t e i s 30 cpm, the 50 ml of water sample (15 sec of sampling) should show a net count r a t e at l e a s t 10 times the background. Then an a c t i v i t y of 6 cpm/ml of water i s r e q u i r e d . I f 1 sampling i s f o r a p e r i o d of 4 hours , a r a t e of approximately 300,000 cpm i s , t h e r e f o r e , r e q u i r e d . I f the o v e r a l l d e t e c t i o n e f f i c i e n c y f o r the beta p a r t i c l e of carbon-14 i s approximately 25 t o 30% ( a d m i t t e d l y low) i n the s c i n t i l l a t i o n system used, the f o r e g o i n g counting r a t e i s equivalent t o 1 T h i s time p e r i o d i s assumed t o be the time f o r the f i s h t o take i n order t o compensate f o r the induced metabolic a l k a l o s i s , thus having excreted most of the l a b e l l e d bicarbonate. about 1,000,000 cpm. T h i s , then, i s the a c t i v i t y r e q u i r e d i n the a d m i n i s t r a t i o n of NaH^CO^, or 0,5 of carbon-14 to be used. Thus i n j e c t i o n of 5 JiC of NaH^ +C0 should be adequate f o r e x c e l l e n t d e t e c t i o n . APPENDIX B. C0 o/HC0" COLLECTION AND METHOD OF ANALYSIS Both urine and expired water samples were analyzed by the f o l l o w i n g technique. Standard stocks of a 5M NaOH and 1 M BaCl£ - NH^Cl s o l u t i o n were prepared and kept i n capped p l a s t i c c o n t a i n e r s . A high c o n c e n t r a t i o n of NaOH keeps the CO,, co n c e n t r a t i o n t o a minimum. Expired water was c o l l e c t e d below a l a y e r of hexane i n a 250 ml volumetric f l a s k . NaOH was p i p e t t e d i n t o the f l a s k , f o l l o w e d by the BaCl - NH CI s o l u t i o n , r e s u l t i n g i n a white p r e c i p i t a t e . ( L b . The NH^Cl was added to n e u t r a l i z e the r a t h e r basic s o l u t i o n . P r e c i p i t a t i o n i s v i a the f o l l o w i n g equation: B a + + + 20H~ + C0 2 BaCO^ + HpO The s o l u b i l i t y product of BaCO^ i s 1 . 6 x 10 ^ , and i s , t h e r e f o r e very i n s o l u b l e (Sinex, et a l , 19 55)' . F i l t r a t i o n was completed w i t h a M i l l i p o r e f i l t e r system. The f i l t e r s were allowed t o dry overn i g h t . The next day each f i l t e r was weighed and prepared f o r s c i n t i l l a t i o n counting. 81 APPENDIX C. QUENCH CURVE DETERMINATION A s e r i e s of 6 quenched samples (Nuclear-Chicago, Inc.) with a d i s i n t e g r a t i o n r a t e of 2 3 8 , 0 0 0 cpm were used and monitored i n a l i q u i d s c i n t i l l a t i o n system c o n s i s t i n g of a Nuclear-Chicago U n i l u x IIA. In the procedure of channels r a t i o technique, one channel i s used to monitor the e n t i r e carbon-14 spectrum ( channel B) and the second channel ( A ) " i s used t o monitor the low energy p o r t i o n of the spectrum. This arrangement provides the r a t i o of lower energy channel A to the t o t a l energy channel B. F i r s t of a l l the balance p o i n t attenuator s e t t i n g was determined on channel B i n a 20 to 5 dynamic range window f o r the l e a s t quenched, carbon-14 standard. This procedure c o n s i s t s of a d j u s t i n g the a m p l i f i e r attenuator on channel B so t h a t the count r a t e i s maximized f o r a given lower and upper l e v e l 2 d i s c r i m i n a t o r s e t t i n g . That i s , the r a t i o S /B i s maximized by t h i s technique. Then a s e r i e s of counts are made on channel A, a d j u s t i n g the attenuator u n t i l the count r a t e s on.both channels are approximately equal. In accordance w i t h the quenching phenomena, the net count r a t e r a t i o f o r the quenched standards w i l l i n c r e a s e , as the concentration, of the quenching agent i n the samples increases•and as the counting e f f i c i e n c y decreases (Wang & W i l l i s , 1965). The p l o t of counting e f f i c i e n c y versus the channels net count r a t i o thus provides one with a standard quench c o r r e c t i o n curve ( F i g . 23) where:-82 cpm (channel B) - background count % efficiency = = X 100 DPM of standard where background count = 30 cpm, and DPM of standard = 2 3 ^ , 0 0 0 and the net count ratio is CPM (channel A) . This ratio is CTM~T"cTiannel BT used to locate the intersect on the curve and the percentage can be read on the vertical scale for the particular sample. The actual disintegration rate is calculated as follows: count (channel B) _ b a c k g r o u n d count = t l m e X 100 % efficiency from curve 63 Figure 23 Carbon-l4 quench curve, a l l c w i n g determination of the percent e f f i c i e n c y of a p a r t i c u l a r system once the channels r a t i o i s c a l c u l a t e d . g4 Figure 24 The e f f e c t of s e l f - a b s o r p t i o n on the count Ba l 4CO , wher< 3 zero sample thickness, 1  r a t e of Ba 00^, where 100% maximum e f f i c i e n c y i s 85 APPENDIX D. EFFECT OF SELF-ABSORPTION ON THE COUNT RATE 14 OF Ba C0 3 In order t o see the e f f e c t of s e l f - a b s o r p t i o n ^on the count r a t e of Ba 1 / fCO_, known amounts of NaH l 4CO ( a t a DPM 3 3 of 500,000) were i n j e c t e d i n t o volumetric f l a s k s c o n t a i n i n g water of d i f f e r e n t t o t a l CO^ and the maximum e f f i c i e n c y p l o t t e d versus the weight of Ba^CO . The r e s u l t s are presented i n F i g . 24, and e x t r a p o l a t e d to 100% maximum e f f i c i e n c y . T h i s p l o t , of course, i s analogous t o the quench curve, as presented i n F i g . 23. S e l f - a b s o r p t i o n c o r r e c t i o n i s p r i m a r i l y a problem i n the assay of low energy beta e m i t t e r s , such as carbon-14 (Wang and W i l l i s , 1965) v/ith the usual sample t h i c k n e s s e s 14 encountered. T h i s treatment of Ba CO^ thus makes i t p o s s i b l e t o compare a s e r i e s of counting samples of the same composition but v a r i e d t h i c k n e s s e s . 

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