Ionic exchange mechanisms in fish gills

Comp. Biochem. Physiol., 1975, Vol. 51A, pp. 491 to 495. Pergamon Press. Printed in Great Britain

REVIEW ARTICLE IONIC EXCHANGE MECHANISMS IN FISH GILLS DAVID H. EVANS Department of Biology, University of Miami, Coral Gables, Florida 33124, U.S.A. (Received 3 April 1974)

Abstract--l. The evidence for the current model for ionic exchange mechanisms across the outer surface of the fresh water acclimated fish gill is reviewed. 2. It is suggested that recent evidence indicates that the uptake of NaCl for ionic regulation is secondary to the extrusion of HCOa-, H + and NH~ + and that the exchange systems may be functioning in sea water acclimated fish that already face a NaCl load. 3. It is further suggested that all marine fish may already have the NaCl uptake mechanisms needed for ionic regulation in fresh water but are restricted to the marine environment because of a low efficiency NaCI uptake mechanism and a relatively high ionic permeability. This ionic permeability may be under the control of intrinsic hormones (prolactin) and external calcium concentration. KROGH (1939) first suggested that, because they are hyperosmotic, fresh water fish extract sodium and chloride independently from the environment and, in an attempt to maintain approximate electroneutrality, the sodium ions are exchanged for blood ammonium ions and the chloride ions are changed for blood bicarbonate ions. Maetz & Garcia Romeu (1964) corroborated these propositions by showing that, in the goldfish Carassius auratus, raising the external ammonium ion concentration inhibited sodium uptake while the injection of ammonium ions stimulated sodium uptake. In addition, they found that raising the external bicarbonate concentration inhibited chloride uptake while the injection of bicarbonate ions stimulated chloride uptake. These data suggested quite strongly that ionic coupling mechanisms exist. While the model of CI-/HCO3- exchange has remained unchallenged, more recent work on various species of fish has shown that revision of the model for N a + / N H , + exchange is necessary. De Vooys (1968) found that NI-I, + effiux continued even when Cyprinus carpio (carp) was placed into distilled water where N a + uptake was nearly zero. In addition, Kerstetter et al. (1970) showed that, in the trout Salmo gairdneri, if the rate o f N a + uptake is varied by presenting various external N a + concentrations to the experimental fish, the rate of N H , + excretion did not change while the rate o f H + excretion varied directly (but not stoichiometrically) with the variation in the rate o f N a + uptake. More recently, Maetz (1973) has reexamined the exchange system in C. auratus and found that N a + uptake is best correlated with the sum of NI-I,+ and H + effiux. It appears therefore that Fig. 1 best describes the ionic exchange mechanisms taking place across a fish 17

Cell in t e rio r

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~"

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Fig. 1. Current model for the ionic exchange mechanisms at the apex (outer surface) of the transporting cells of the fresh water acclimated teleost fish gill. See text for details and supporting evidence.

acclimated to fresh water. On examining fig. 1, one is immediately struck by the idea that these exchange mechanisms serve much more than merely the NaCI balance of the organism. Excretion of H C O s provides for the net loss o f respiratory CO,, excretion of H + provides for the net loss of metabolic acid while the excretion o f N H , + provides for the net loss o f both acid and nitrogenous waste since fish are predominantly ammoniotelic (Forster & Goldstein, 1969). It appears therefore that the fish gill has

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D . H . EVANS

ingeneously provided for four metabolic needs via the acquisition o f two ionic exchange systems. These metabolic needs are divided between the kidney and lung in terrestrial vertebrates. One might ask what is/are the primary mechanism(s), i.e. what are the primary needs of the organism: NaC1 balance, carbon dioxide excretion, acid/base regulation or nitrogenous waste excretion ? One can start to answer this question in an intuitive manner. NaC1 uptake is "necessary" only in fresh water while the other parameters are probably important to both fresh water and marine fish. It seems unlikely that these four parameters would be coupled in fresh water but uncoupled in the marine environment when the fish is already facing a net gain of NaCl due to diffusion and ingestion (see Maetz, 1971, for a review of fish osmoregulation in sea water). One might argue that the metabolic needs o f the fish might change with salinity so that the excretion of C O s , H + or NH4 + is reduced in the marine environment. This is unlikely but there are no data to substantiate a firm statement. One might also argue that in marine fish excretion of COs, H + or NH4 + might take place at non-branchial sites such as the kidney or gut. Again, no information exists that this might be the case but it seems unlikely that major pathway differences exist. It is obvious that information is needed on the relative roles of the gills, kidney and gut in CO2, H + and NH~ + excretion and changes in these roles in the fresh vs. the marine environment. Experimental evidence that NaCI uptake is not the primary control mechanism for these ionic exchanges does exist. In two cases it has been shown that the ionic exchange mechanisms exist in fish that are acclimated to sea water. Evans (1973) showed that when the euryhaline molly, Poecilia latipinna, is acclimated to sea water, N a + influx is still via a saturable, and therefore presumably carriermediated, system. More recent evidence (Evans, 1975) on the same species indicates that the N a + uptake by sea water acclimated individuals can be partially inhibited by external H +, NH4 + and the antibiotic amiloride. (Kirschner et al. (1973) have shown that amiloride is a potent inhibitor of Na+/H + exchanges in S. gairdneri, a crayfish, Procambarus and the frog, Rana pipiens.) In addition, Payan & Maetz (1973) found that, in the cat shark, Scyliorhinus canicula, the injection of H + or NH~ + stimulated sodium influx. They also found that injection of the carbonic anhydrase inhibitor acetazolamide (Diamox) reduced N a + uptake. It has been proposed that inhibition o f carbonic anhydrase reduces the production o f H + and HCO3- and thereby reduces the rate o f exchange of Na+/H + (Kerstetter et al., 1970), N a + / N H , + (Maetz & Garcia Romeu, 1964) and C I - / H C O s - (Maetz & Garcia Romeu, 1964). It is interesting to note that injection of acetazolamide has no effect on CI- uptake by S. gairdneri (Kerstetter & Kirschner, 1972) or N a + uptake by

P. latipinna acclimated to sea water (Evans, unpublished). It may be that in these two species, part of the ionic exchange mechanisms are not limited by carbonic anhydrase activity. Since both P. latipinna and S. cannicula are apparently exchanging N a + for either H + or NH4 + when they are in sea water and therefore already facing a salt-load, one can only conclude that the physiological necessity for excretion of H + and NH4 + outweighs the problems of adding to the N a + imbalance. De Renzis & Maetz (1973) have recently provided more direct evidence that NaCI uptake is secondary to excretion of unwanted ions. They found that when C. auratus is maintained in choline chloride solutions both the blood N a + and CI- concentrations fell while the blood p H fell slightly. If fish were maintained in sodium sulfate solutions the blood N a + and CI- fell while the blood p H rose. They also monitored ionic uptake in fish maintained in these solutions and found that after maintenance in choline chloride solutions N a + uptake was increased more than CIuptake while the reverse was true after the fish had been maintained in sodium sulfate solutions, i.e. CIuptake predominated. Since the blood concentration of both ions fell in these choline chloride and sodium sulfate solutions they concluded that "under the present experimental conditions it would certainly seem that the stimulus pH-shift (author's italics) takes precedence over the stimulus internal Na and CI concentration drop in the feedback modulating the relative intensities of the branchial absorptions of sodium and chloride and thus regulating their internal concentrations." (De Renzis & Maetz, 1973). While all of these data are preliminary and are studies of single fish species, it appears that they provide strong evidence that NaCI uptake by both cartilagenous and bony fish is secondary but coupled to the excretion of metabolically produced carbon dioxide, acid and ammonium ions. Therefore, could it be true that the raison d'etre of NaCI uptake in fish is not for NaCI regulation but merely to provide ions as exchangers for other ions that are being excreted ? To extend this line of reasoning even further, one could then theorize that carriermediated NaC1 uptake was present in the marine precursors of the first vertebrates (who presumably also had to rid their bodies of unwanted carbon dioxide, acid and ammonium ions) and that they were therefore at least partially "preadapted" to life in fresh water with regard to NaCI regulation. And, indeed, do all modern fish, fresh water and marine, bony and cartilagenous have the metabolic machinery for NaCI uptake in fresh water ? Obviously, the answer to the latter question can only be given after an extensive systematic survey of fish groups. If, indeed, it can be shown that marine fish do have the metabolic machinery for NaC1 uptake in fresh water then what is limiting their

Ionic exchange mechanisms in fish gills entrance into fresh water? To put it another way, why are all fish not euryhaline ? We are now back to the primary question underlying studies in fish osmoregulation--what are the mechanisms controlling euryhaliniW ? Entrance into fresh water (or any hypo-osmotic salinity for that matter) depends on five interrelated phenomena: (1) Cessation of oral ingestion of the medium. (2) Increase in urine flow rate to rid the body of excess water. (3) Reduction of urine ionic concentration to conserve needed ions. (4) Reduction in ionic and osmotic permeability to decrease the effects of the osmotic and ionic gradients. (5) Active extraction of NaCI from the environment. Let us examine each of these phenomena with regard to their importance in and control of euryhalinity. (1) Cessation of oral ingestion of the medium has never been systematically examined but it seems to be a minor limitation since it has been shown that the euryhaline killifish, Fundulus heteroclitus actually continues to ingest the medium after acclimation to fresh water (Potts & Evans, 1967). In addition it is possible that urine flows could be increased to offset this addition to the osmotic uptake of water already present in fresh water. (2) Increase in urine flow rates does not seem to be a major problem since it involves a quantitative change in the functioning of structures in the kidney that most marine fish already possess. Aglomerular fish might be expected to be severely limited but at least one aglomerular fish, Microphis boaja, has been described in fresh water (Hickman & Trump, 1969) and the aglomerular toadfish, Opsanus tau, can survive in fresh water despite a very low free water clearance (Lahlou et al., 1969). (3) Reduction of urine ionic concentrations may appear to be a severe limitation because it is generally assumed that the hyperosmotic reabsorption from the urine is dependent on the addition of a new distal segment to the marine kidney (Hickman & Trump, 1969). However, some species of euryhaline fish (Fundulus heteroclitus and Gasterosteus aculeatus) can survive in fresh water without this additional kidney segment (Hickman & Trump, 1969), and O. tau produces a slightly hypo-osmotic urine in fresh water without the presence of a distal segment (Lahlou et al., 1969). It is interesting to note that elasmobranchs have a distal kidney segment despite the fact that they are generally restricted to sea water (Hickman & Trump, 1969). It therefore appears that there is no clear dependence on the possession of a distal renal segment for acclimation to fresh water. (4) It is clear that euryhaline fish do actually decrease their ionic permeability in hypoosmotic environments (Evans, 1967, 1969a; Potts & Evans, 1967) but that osmotic permeability may actually increase (Evans, 1967, 1969a, b; Potts et al., 1967; Motais et al., 1969). These permeability adjustments seem to be under the control of the pituitary hormone prolactin (Potts & Evans, 1966; Maetz et al., 1967; Lahlou & Giordan, 1970; Potts &

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Fleming, 1970). The importance of these permeability changes in limiting euryhalinity is unstudied, but it is obvious that these adjustments are interrelated with the other parameters, i.e. total impermeability is unlikely so that other regulatory mechanisms still must be present. (5) It is clear that even if parameters (1)-(3) are satisfied, unless the fish is absolutely ion impermeable in fresh water some means of NaCI extraction must be present in order for fresh water survival. Net ionic gain via feeding is poss~le, and has not been systematically examined with regard to fish, but is generally assumed not to be sufficient to allow for fresh water survival due to the relatively high ionic permeability of fish. The efficiency of the NaCl uptake mechanisms must be keyed to the efficiency of the other parameters. In other words, the greater the magnitude of ion loss due to urinary losses and net diffusional effiux the greater thedemand for efficient NaCI uptake mechanisms. It appears, therefore, that the most strict requirement for life in fresh water is the presence of a NaCI uptake mechanism. But I have just argued that all fish may have this uptake mechanism functioning all the time secondary to excretion of unwanted ions which have been produced metabolically. Then, what is the limiting factor to survival in fresh water? It seems most reasonable to propose that most marine fish are restricted to sea water, not because they lack the NaCI uptake mechanism, but because this mechanism is not efficient enough to balance the NaCI loss caused by net diff~ional loss and/or possibly kidney insufficiency with regard to production of a hypo-osmotic urine. Since I have already pointed out that renal insufficiency probably does not play a major role in some euryhaline fish it is most appropriate to concentrate on the relationship between net diffusional ion loss and active extraction. In fact there is evidence that the relation between these two parameters may be limiting fresh water survival of some species of teleosts. Breder (1934) and Neill (1957) showed that, on Andros Island, Bahamas, and in Florida, one could find fish in fresh water that were normally considered to be stenohaline marine species. In Lake Forsyth, Andros (approximately 20mM NaC1/l., l mM Ca/L), Breder found specimens of needlefish (Strongylura), halfbeak (Chriodorus), jack (Caranx), snapper (Lutianus), mojarra (Eucionostomus) and puffer (Spheroides). Further, Breder (1934) found that the addition of calcium to New York City tap water prolonged the survival of the queen angelfish (Holacanthus ciliaris) and the beaugregory (Eupomacentrus leucostictus) in fresh water. A single specimen of schoolmaster snapper (Lutianus apodus) survived for 20 days in high calcium tap water. More recently, Hulet et al. (1967) have shown that the sergeant major, Abudefduf saxatilis, can survive in fresh water only if relatively high (5-25 mM Ca/1.)

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concentrations of calcium are present. The fresh water survival of the pinfish, Lagodon rhomboides, is also dependent on the presence of at least 5 m M Ca/l. (Carrier, unpublished). What is the role of calcium in promoting the survival in fresh water o f these supposedly stenohaline marine fish? Studies on known euryhaline fish can give us some clues. Potts & Fleming (1971) found that removal of Ca t+ from the environment produces a substantial increase in N a + permeability of the kiUifish, Fundulus kansae, whether the animal is acclimated to fresh water or sea water. They also found that the osmotic permeability of this species was increased after the removal of Ca 2+ (Potts & Fleming, 1970). It has also been shown that the removal of external Cas+ ions doubles the N a + influx into sea water acclimated eels, Anguilla anguilla (Bornancin et al., 1972). On the other hand, the N a + permeability o f the goldfish, C. auratus, is little affected by external Cat+ concentration (Cuthbert & Maetz, 1972). Nevertheless, it appears that the ionic and osmotic permeability of at least two species of euryhaline fish is affected by external Ca~+ concentration. Does Cat+ have the same effect on the ionic permeability o f those marine fish whose survival in fresh water is dependent on external Cas+ concentrations? Carrier (unpublished) has found that the N a e ~ u x from L. rhomboides is significantly reduced by the addition of Cas+ to the medium. However, the reduction of ionic permeability is not complete and L. rhomboides still faces a net loss of N a + into fresh water. One would therefore propose that some sort of N a + uptake mechanisms must exist in this species and, in fact, Carrier has found a saturable N a + uptake mechanism in L. rhomboides whether this species is acclimated to sea water or high Ca~+ fresh water. The uptake mechanism has a relatively low affinity for N a + ( K m greater than 10 m M Na+/l.) and apparently is able to maintain L. rhomboides in positive N a + balance in low salinities only as long as the N a + loss is restricted by external Ca~+ ions. (Note added in proof: Carrier has also found that the sodium uptake by sea water acclimated L. rhomboides can beinhibited by external ammonium or hydrogen ions and by amiloride. (Unpublished PhD. dissertation, 1974.) ) One can better understand this interplay between Ca z+ maintained permeability and a low-Na +affinity uptake mechanism by examining Fig. 2 which diagrams this relationship for a hypothetical fish (or any aquatic organism for that matter) facing lowered N a + concentrations. As Shaw (1959) fLrst described, this hypothetical species will only be in positive N a ÷ balance at concentrations above S since this is the point where the line for efflux E crosses the uptake curve. A t any concentrations below S the uptake will be below the etIlux E and the animal will be in negative N a + balance. The addition of Ca 2+ to the external medium will reduce the Na + effiux to E ' so that now the two lines meet

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Fig. 2. Relationship between external Na concentration, Na + uptake rate and Na + loss rate for a hypothetical fish. E refers to the loss rate in calcium-free solutions while E' refers to the loss rate in calcium-enriched solutions. The fish is in positive sodium balance at external Na concentrations where the uptake rate is equal to or above the loss rate (S in calcium-free solutions and S' in calcium enriched solutions). It is clear that the addition of calcium will allow the fish to survive in solutions with a substantially lower Na + concentration.

at a point S ' which is a new minimal survival salinity below that of the fish without the addition of external Ca t+. If S ' is approximately the N a + concentration of fresh water then the addition of external Ca ~+ has allowed the survival in fresh water of a fish that normally could not live in that low salinity. Whether Fig. 2 diagrams the situation for L. rhomboides or any other fish remains to be seen. It should be obvious that the minimal survival salinity can also be affected by changes in the uptake curve, i.e. an increase in the affinity of the carrier system for N a + would result in a shift of the uptake curve in Fig. 2 to the left and in this case the minimal survival salinity S" may be approached without the intervention of external Ca 2+. There is some evidence that the fish pituitary octapeptide arginine vasotocin may play such a role since Maetz et al. (1964) found that the injection of this hormone stimulated N a + uptake by C. auratus, but these data have not been extended to other species. In summary, then, I propose that the limiting factor in euryhalinity of marine fish is not the absence of the necessary ionic uptake mechanisms--they are always present as secondary couplers to needed ionic extrusion (HCO3-, H +, NH4+). The entrance into fresh water is limited by the relation between these uptake mechanisms and the ionic permeability of a given species. Proper levels of intrinsic hormones may affect either the ionic permeability (prolactin) or the efficiency of the carrier-mediated ionic uptake mechanisms (arginine vasotocin). Some species with low efficiency uptake mechanisms and relatively high ionic permeability can survive in fresh water if the external calcium concentration is

Ionic exchange mechanisms in fish gills raised. It remains to be seen if most or all marine fish can be m a d e to live in fresh water by the proper levels o f prolactin, arginine vasotocin and external calcium. Acknowledgements--The research from the author's laboratory quoted in this review was supported by funds from N S F grants GB 16839 and GB 36423. REFERENCF~ BORNANCINM., CUTHBERTA. W. & MAIETZJ. (1972) The effects of calcium on branchial sodium fluxes in the seawater adapted eel, Anguilla anguilla, L. J. PhysioL, Land. 222, 487-496. BREDER C. M., JR. (1934) Ecology of an oceanic fresh water lake, Andros Island, Bahamas, with special reference to its fishes. Zoologica 18, 57-80. Ctrrl-mERT A. W. & MAETZJ. (1972) The effects of calcium and magnesium on sodium fluxes through gills of Carassius auratus, L. J. Physiol., Lond. 221, 633-643. EVANS D. H. (1967) Sodium, chloride and water balance of the intertidal teleost, Xiphister atropurpureus---III. The roles of simple diffusion, exchange diffusion, osmosis and active transport. J. exp. Biol. 47, 525--534. EvANs D. H. (1969a) Sodium, chloride and water balance of the intertidal teleost, Pholis gunnellus. J. exp. Biol. 50, 179-190. EVANSD. H. (1969b) Studies on the permeability to water of selected marine, freshwater and euryhaline teleosts. J. exp. Biol. 50, 689-703. EVANS D. H. (1973) Sodium uptake by the sailfin molly, Poecilia latipinna: kinetic analysis of a carrier system present in both fresh-water-acclimated and sea-wateracclimated individuals. Comp. Biochem. Physiol. 45A, 843-850. EVANS D. H. (1975) The effects of various external cations and sodium transport inhibitors on sodium uptake by the sailfin molly, Poecilia latipinna, acclimated to sea water. J. comp. Physiol. (In press). FORSTER R. P. & GOLDSTEIN L. (1969) Formation of excretory products. In Fish Physiology (Edited by HOAR W. S. & RANDALLD. J.), Voi. 1, pp. 313-350. Academic Press, New York. HICKMAN C, P., JR. & TRUMP B. F. (1969) The kidney. In Fish Physiology (Edited by HOAR W. S. & RANDALL D. J.), Vol. 1, pp. 91-239. Academic Press, New York. HULLerW. H., MASEL S. J., JODREYL. H. & WEHR R. G. (1967) The role of calcium in the survival of marine teleosts in dilute sea water. Bull. mar. Sci. 17, 677-685. KERs~evt~R T. H. & KmSeHNER L. B. (1972) Active chloride transport by the gills of rainbow trout (Salmo gairdneri). J. exp. Biol. 56, 263-272. KERSTETIr.a T. H., KIRSCHNER L. B. & RArUSE D. D. (1970) On the mechanisms of sodium ion transport by the irrigated gills of rainbow trout (Salmo gairdneri). J. gen. Physiol. 56, 342-359. KIRSCHNER L. B., GREENWALDL. & KERSTETTERT. H. (1973) Effect of amiloride on sodium transport across body surfaces of fresh water animals. Am. J. Physiol. 224, 832-837. KROGH A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press.

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L~m.ou R., HENDERSON I. W. & SAWYER W. H. (1969) Renal adaptations by Opsanus tau, a euryhaline aglomerular teleost, to dilute media. Am. d. Physiol. 216, 1266-1272. LAHLOU B. & GIORDANA. (1970) Le controle hormonal des 6~hanges et de la balance de l'eau chez le t~t~osteen d'eau douce Carassius auratua, intact et hypophysectomis~. Gen. & compar. Endocr. 14, 491-509. MAETZ J. (1971) Fish gills: mechanisms of salt transfer in fresh water and sea water. Phil. Trans. R. Soc. Lond. B 262, 209-249. MAETZ J. (1973) Na+/NH, +, Na+/H + exchanges and NHs movement across the gill of Carassius auratus, d. exp. BioL 58, 255--275.

MAETZ J. & GimClA ROMEU F. 0964) The mechanism of sodium and chlorideuptake by the gillsof a fresh-water fish,Carassius auratus--II. Evidence for N H d N a and HCOa/CI exchanges, d. gen.PhysioL 50, 391--422. MAETZ J., SAWYERW. H., PICKVCmoG. E. & MAYERN. (1967) Evolution de la balance min6rale du sodium chez Fundulus heteroclitus an cours du transfer d'eau de mer en eau douce: effets de l'hypophysectomie et de la prolactine. Gen. & compar, endoer. 8, 163-176. MOTAIS R., ISAIA J., RANKIN J. C. & MAETZ J. (1969) Adaptive changes of the water permeability of the teleostean gill epithelium in relation to external salinity. J. exp. Biol. 51,529-546. NEJLL W. T. (1957) Historical biogeography of present day Florida. Bull. Florida State Mus. 2, 175-220. PAYAN P. & MAETZ J. (1973) Branchial sodium transport mechanisms in Seylliorhinus canicula: evidence for Na+/NH4 + and Na+/H + exchanges and for a role of carbonic anhydrase. J. exp. BioL 58, 487-502. PoTrs W. T. W. & EVANS D. H. (1966) The effects of hypophysectomy and bovine prolactin on salt fluxes in fresh-water-adapted Fundulus heteroclitus. BioL Bull. mar. biol. Lab., Woods Hole 131,362-368. POTTS W. T. W. & EVANS n . H. (1967) Sodium and chloride balance in the killifish Fundulus heteroclitus. BioL Bull. mar. biol. Lab., Woods Hole 133, 411-425. POTIS W. T. W. & FLEMINGW. R. (1970) The effects of prolactin and divalent ions on the permeability to water of Fundulus kansae. J. exp. Biol. 53, 317-327. POTTS W. T. W. & FLEMING W. R. (1971) The effect of environmental calcium and ovine prolactin on sodium balance in Fundulus kansae, d. exp. Biol. 54, 63-75. POTTS W. T. W., FOSTER M. A., RUDY P. P. & PARRY HOWELLS G. (1967) Sodium and water balance in the cichlid teleost, Tilapia mossambica. J. exp. Biol. 47, 461-470. DE RENZIS G. & MAETZ J. (1973) Studies on the mechanisms of chloride absorption by the goldfish gill: relation with acid-base regulation. J. exp. Biol. 59, 339-358. SHAWJ. (1959) Salt and water balance in the East African freshwater crab Potamon niloticus (M. Edw.). J. exp. Biol. 36, 157-176. DE VOOYS G. G. N. (1968) Formation and excretion of ammonia in teleostei--I. Excretion of ammonia through the gills. Archs int. Physiol. Biochem. 76, 268-272. Key Word Index--Ionic exchange; fish gills; prolactin; uptake of NaCI.