1. Rates of efflux of H+ and ammonia from a marine teleost and an elasmobranch were measured.

  2. Hypercapnia stimulated H+; efflux from both species, stimulated ammonia efflux from the elasmobranch, and inhibited ammonia efflux from the teleost.

  3. In both species the H+; and ammonia efflux were predominantly across the branchial epithelium. In Na+-free sea water, the H+ efflux from both species was completely abolished and the ammonia efflux was inhibited by approximately 50%.

  4. Injection of an acid load stimulated H+; efflux, which continued for 2-5 h until more than the injected acid load was excreted. It therefore appears that injection of an acid load also produces a metabolic acid load which must be excreted.

  5. The H+ efflux from mineral/metabolic acidotic fish is entirely branchial and dependent upon external Na+.

  6. The data support the conclusion that marine teleosts and elasmobranchs possess branchial Na+/NH4+ and Na+/H+; ionic exchange mechanisms and that Na+/H+; exchange plays a major role in the response to acidosis in both groups.

  7. The possible evolution of these ionic exchange systems is discussed.

In recent years it has become apparent that marine teleosts, and possibly marine elasmobranchs, excrete blood NH4+ in exchange for sea water Na+ (Evans, 1977, 1980a, b). In freshwater fish this branchial ionic exchange system provides for the uptake of Na+ required for existence in their hypo-osmotic environment, as well as for nitrogen (and presumably acid) excretion. However, in the marine environment the resulting Na+ influx is obviously maladaptive for ion regulation. We have no information on the magnitude of the net influx of Na+ presented by Na+/NH4+ exchange in the elasmobranchs, but in at least one teleost, Opsanus beta, it presents a Na+ load that is 10% of the total net influx of this ion, twice the load presented by oral ingestion (Evans, 1980 c).

Investigations of freshwater teleosts indicate that, in addition to Na+/NH4+ exchange, Na+/H+; exchange may also play a significant role in Na+ balance (Evans, 1980a, b). No direct evidence for Na+/H+ exchange exists for marine teleost fishes; however, its presence in the elasmobranchs is suggested by the finding that the Na+ influx into Scyliorhinus canicula can be stimulated by the injection of HC1 (Payan & Maetz, 1973) and inhibited by acidifying the external sea water (Bentley, Maetz & Payan, 1976). In addition, Evans, Kormanik & Krasny (1979) recently demonstrated that acid (but not ammonia) efflux from the skate, Raja erinacea, is dependent upon external Na+.

The importance of branchial extrusion pathways in acid/base regulation by both teleosts and elasmobranchs is presently under active investigation (see reviews by Cameron, 1978, and Heisler, 1980). Cameron & Wood (1976) found that compensation for the post-operative fall in blood pH of two Amazonian teleosts was made by an increased extrarenal efflux of acid; however, Wood & Caldwell (1976) showed that trout (Salmo gairdneri) responded to a mineral acid load with an increase in the renal excretion of acid which could account for the entire acid load within 72 h. More recently, Cameron (1980) has found that extrarenal mechanisms predominate in pH compensation in the catfish, Ictalurus punctatus, after the production of either hypercapnic or mineral acidosis. In addition, Kobayashi & Wood (1980) demonstrated that renal excretion could amount for only 2% of the lactate load and 6% of the proton load 72 h after the infusion of lactic acid into S. gairdneri. Branchial compensatory mechanisms also appear to be of major importance in elasmobranch pH regulation because Heisler, Weitz & Weitz (1976) found that hypercapnia stimulated extrarenal H+ efflux from 5. canicula, with less than 1 % of the acid load appearing in the urine (Heisler, 1980). The excretion of acid by this species after HC1 influsion or exercise is also approximately 99% via branchial pathways (Heisler, 1980). In addition King & Goldstein (1979) showed that only 7% of a mineral acid load injected into Squalus acanthias was excreted renally within the first 72 h after injection of the HC1. These data corroborate an earlier study (Cross et al. 1969) that found that an increased renal excretion of acid plays a vanishingly small (less than 1 %) role in the compensation of hypercapnic acidosis by S. acanthias.

While it appears that pH regulation in freshwater teleosts and marine elasmobranchs is branchial, there is no direct evidence that it is via Na+/NH4+ and/or Na+/H+; exchange. Both DeRenzis & Maetz (1973) and Cameron (1976) demonstrated that alteration of blood pH of freshwater fish (via injection of acid or base, or production of hypercapnia) was accompanied by alteration in the relative influxes of Na+ and Cl, presumably because they were linked to H+; (or NH4+) and HCO3 extrusion, respectively. However, alteration of blood pH could have had secondary effects on permeability or transepithelial electrical potentials which could have resulted in the observed alterations in isotopic influxes. In addition, Kerstetter & Mize (1976) found that acute metabolic acidosis in S. gairdneri did not affect Na+ or Clinfluxes measured over a short time-span (15 min). More recently Perry et al. (1981) have found that hypercapnia in the same species does not alter the uptake of either ion measured for 1h after production of hypercapnia for 24 h. Importantly, the role of branchial ionic exchange systems in pH regulation by marine teleost fishes has not been investigated, and it is possible that alternative pathways exist to avoid increased Na+ (and Cl) influxes secondary to pH regulation.

The purpose of the present investigation was to determine: (1) the rate of H+; and ammonia efflux from a marine teleost and elasmobranch, (2) the effect of either hypercapnia or mineral-acid loading on these effluxes, (3) the relative importance of branchial vs renal efflux, and (4) the role of external Na+ in these effluxes.

Toadfish (Opsanus beta) were collected by commercial shrimp fishermen in Biscayne Bay, Florida, and maintained in plastic aquaria containing filtered sea water (500 mM-Na+) at room temperature (23–25 °C). Fish (10–30 g) were used in experiments within 2 weeks of capture and were not fed. Dogfish (Squalus acanthias) ‘pups’ were removed from sacrificed females that had been caught by hook and line in Frenchman’s Bay, Maine, and maintained in running sea water (450 mM-Na+) at 13–17 °C in wooden aquaria. ‘Pups’ are near-term embryos at the end of an approximately 2-year gestation period (Woodhead, 1979). They are able to survive in sea water for periods over 41 days (Gilbert, 1958) and display the major hallmarks of elasmobranch osmoregulation (Kormanik & Evans, 1978; Evans & Mansberger, 1979; Evans & Oikari, 1980). The ‘pups’ (40–60 g) had large yolk sacs and were therefore not fed. Except for the initial normocapnic to hypercapnic experiments, the yolk sac was ligated and removed before all experiments.

Net H+ effluxes were measured using a technique modified from that of Heisler et al. (1976). Net H+; efflux is defined in the present study exclusive of ammonia effluxes, with the assumption that ammonia is excreted predominately as NH4+ and does not release a proton at the pH’s used in the present experiments. Total acid efflux would therefore be the sum of the net H+; efflux and the ammonia efflux. The net H+ efflux is best correlated with ‘titratable acidity’, but the latter term is not used here because back titrations were not performed. Individual fish were placed in aerated sea water, 100 ml for toadfish, 200 ml for dogfish, in a small plastic container. Experimental baths containing toadfish were maintained at room temperature (23–25 °C) while those containing shark ‘pups’ were placed in running sea-water troughs at 13–17 °C. At time zero, and at various time intervals thereafter, a 5 ml sample of the experimental bath was removed, bubbled with 1 % CO2 (in oxygen, from compressed gas cylinders, Air Products) and its pH recorded with a Radiometer PHM 62 pH meter attached to a Radiometer titragraph recorder. In this way the pH could be recorded to the nearest 0·002 units. The pH was noted only after it had been relatively stable (drift less than 0·02 pH units/h) for at least 2 min. The sample was then either frozen for ammonia analysis or returned to the experimental bath. As Heisler et al. (1976) have proposed, since the of the experimental sample is maintained constant during the recording of the pH, according to the Henderson-Hasselbalch equation, the net change in bath pH is dependent only upon acid or base addition. I have empirically determined that bubbling with 1 % CO2 immediately (less than 30 s) blows of any respiratory CO2 and results in a relatively stable pH of the sample (drift of 0·02 pH units/h in sea water). The pH of samples of the experimental baths was always compared with the pH of samples from a control bath, treated in the same manner as any experimental bath, but containing no fish. The pH differential between the control and experimental baths at any time can be converted to acid or base fluxes since the volume of the bath, the weight of the fish (determined to the nearest 0·1 g at the end of an experiment) and the buffering capacity of the solutions are known. Buffering capacity of the control was determined at the beginning of the experiment and that of experimental baths at the termination of the experiment. In both cases the buffering capacity was measured by the addition of known amounts of HC1 or NaOH (to a 5 ml sample) while recording the resulting change in pH. A linear regression of the data gives a slope of μM acid change per μM acid/base added. The final buffering capacity of the experimental solutions was determined over the pH range observed during the course of the experiment because the fish could change the buffering capacity, the control solution could drift spontaneously (probably due primarily to temperature changes, especially at MDIBL), and the buffering capacity depended on the pH range used. H+; fluxes were calculated by converting the pH differential between the control and the experimental bath to hydrogen ion concentration, and correcting for the average buffering capacity for that time period and pH range, the volume of the sample, and the weight of the fish. The calculation also took into account removal of any samples for ammonia analysis. Computation of the rate of H+; flux (μM 100 g−1 h−1) was performed on the University of Miami Univac 1100 computer using a program written by J. B. Claiborne. Ammonia concentrations of defrosted samples were determined using either the technique of Harwood & Kuhn (1970) or Solarzano (1969).

Experimental solutions were either bubbled with compressed air or 5 % CO2 (in air) produced by a Wostoff gas mixing pump connected to a tank of liquid CO2. To ascertain the role of renal/rectal pathways in acid and ammonia efflux, a washed rubber condom was placed over the tail and secured, anterior to the anus, with loops of cotton thread. The condom contained 10 ml of sea water, and at the end of 1 h the condom was removed and the pH of its contents and a sample of the experimental bath were determined as above. The sample was then frozen and saved for ammonia analysis. Mineral acidosis was produced in some experiments by injecting 50 μM 100 g−1 of HC1 under MS 222 anaesthesia (0·01 %). The acid was injected intraperitoneally as 0·5 μ 1 g−1 of 1 N-HCI, normally over 1–2 min. The role of external Na+ in H+; and ammonia efflux was determined by monitoring efflux in control sea water for a specific time period, rinsing the fish (for approximately 30 s) in Na+-free artificial sea water (formulated according to Evans and Cooper, 1976, with 2·5 mM-KHC03 and 7·5 mM-KCl, and choline chloride as the Na+ substitute) made to conform to the osmolarity of the ambient sea water (Miami v. MDIBL), and then transferred into Na+-free artificial sea water for 1 h. At the end of that experimental period the fish were transferred to another container of control sea water to test for reversibility of ‘Na+-free effect’.

In some experiments the transepithelial electrical potential (TEP) was measured as previously described (Evans & Cooper, 1976).

All data are expressed as + S.E. (N) and statistical significance between means was tested with Student’s t test (using paired data when appropriate).

Hypercapnic acidosis

(1) Normacapnic acid and ammonia efflux

Under normocapnic conditions both O. beta and S. acanthias excrete small amounts of ammonia and little or no net amounts of H+; (Table 1). The excretion rate of ammonia for 5. acanthias is similar to the value of 6–7 μM 100 g−1 h−1 found for another elasmobranch Raja erinacea (Evans, 1977). In the present study, the reason for the low rate of ammonia excretion by the dogfish ‘pups’ may have been that they were still manufacturing protein for rapid growth. Since both O. beta and S. acanthias excrete more ammonia than H+; under normocapnic conditions, it appears that protein catabolism provides the major pathway for the excretion of both nitrogen and protons under these conditions.

Table 1.

Effect of hypercapnia on H+ and ammonia efflux from the toadfish and dogfish

Effect of hypercapnia on H+ and ammonia efflux from the toadfish and dogfish
Effect of hypercapnia on H+ and ammonia efflux from the toadfish and dogfish

(2) Effect of hypercapnia

Production of hypercapnia by bubbling 5 % CO2 into the experimental medium produced a significant stimulation of net H+; excretion from both species (Table 1). The onset of compensatory H+; extrusion by both species was gradual (unpublished results) so the data presented in Table 1 were obtained 3–5 h after introduction of 5 % CO2. This delay in the onset of H+ excretion stimulated by hypercapnia has also been demonstrated in two freshwater teleosts (Cameron & Wood, 1976) and in the elasmobranch S. canicula (Heisler et al. 1976) and presumably parallels the time course of blood pH decline and activation of acid excretory mechanisms. Hypercapnia inhibited ammonia efflux from O. beta, and stimulated it slightly from S. acanthias. Whether the inhibition of ammonia efflux from the toadfish indicates competition between H+; and NH4+ for a common carrier remains to be seen, but it is clear that in both species compensation of hypercapnic acidosis is predominantly via H+;, rather than ammonia excretion. To ascertain the possible role of. changes of electrical potential in the stimulation of H+; efflux from hypercapnic fish the transepithelial electrical potential (TEP) was measured at least 2 h after the onset of hypercapnia in both species. Hypercapnic O. beta maintained a TEP of −8·5 ±1·8 mV (four fish, blood relative to medium), only 1–2 mV more negative than that described for normo-capnic individuals previously (Evans & Cooper, 1977; Evans, 1977). Hypercapnic acanthias maintained a TEP of −2·4 ±0·5 mV (4), no different from that of normocapnic individuals ( −2·7±0·6 mV, seven fish). Thus, it is clear that changes in the TEP cannot account for any of the changes in H+ (or ammonia) efflux in the present experiments. Since hypercapnia produced measurable H+; effluxes the subsequent studies of the role of branchial v. renal efflux and the role of external Na+ were made on fish which had been hypercapnic for at least 3 h.

(3) Role of branchial v. renal/rectal efflux

In both species, branchial pathways predominated over renal/rectal pathways for the extrusion of H+ from hypercapnic individuals (Table 2); indeed, both species appeared to excrete a net amount of base into the sea water surrounding the renal/ rectal openings. These data are the first demonstration of the importance of branchial H+ extrusion by a hypercapnic marine teleost and support the recent findings (see introduction) that branchial pathways are the major site of H+; extrusion by marine elasmobranchs and freshwater teleosts, and earlier studies (Smith, 1929; Fromm, 1963) which showed that ammonia was also excreted mostly via the gills in freshwater and marine fishes.

Table 2.

Branchial v. renal/rectal excretion of H+ and ammonia by the toadfish and dogfish

Branchial v. renal/rectal excretion of H+ and ammonia by the toadfish and dogfish
Branchial v. renal/rectal excretion of H+ and ammonia by the toadfish and dogfish

(4) Effect of Na+-free sea water

Transfer of hypercapnic toadfish and dogfish to Na+-free artificial sea water resulted in a cessation of H+; excretion and, in many cases, a net excretion of base (Table 3). Thus, it is clear that H+ extrusion by both species is completely dependent upon external Na+. The observation that many individuals started to excrete base in Na+-free sea water may indicate that Na+/H+; exchange normally masks a baseline base extrusion (linked to external Cl ?) ; more data are needed to support this interesting proposition. The ammonia extrusion by hypercapnic dogfish was reduced by some 40% in Na+-free sea water, supporting the previous finding that low Na+ (2·5 mM), K+-free artificial sea water reduces the ammonia efflux from O. beta by some 50 % (Table 3, data from Evans, 1977). Thus it appears that elasmobranchs, like teleosts (Evans, 1977), excrete a considerable portion of their ammonia via pathways other than Na/NH4 exchange. Indeed, the present data are the only direct demonstration of Na+/NH4+ exchange in an elasmobranch; it has been previously found that ammonia efflux from R. erinacea was not sensitive to external Na+ concentrations (Evans et al. 1979). One might propose that changes in the TEP could play a role in the Na+-free effect in these experiments. However, hypercapnia did not affect significantly the TEP of either species in sea water (see above) and the TEP of normocapnic individuals of both species only becomes 3–7 mV more negative (blood relative to sea water) in Na+-free (and K+-free) sea water, far less than that TEP change necessary to account for the reduction in efflux of either H+ or NH4+ shown in Table 3 (Evans & Cooper, 1976; Kormanik & Evans, 1978).

Table 3.

Effect of removal of external Na+ on excretion of and H+ ammonia by the toadfish and dogfish

Effect of removal of external Na+ on excretion of and H+ ammonia by the toadfish and dogfish
Effect of removal of external Na+ on excretion of and H+ ammonia by the toadfish and dogfish

Return to Na+-containing sea water restimulated both the H+; and ammonia efflux in O. beta, but only restored the acid efflux in S. acanthias. The reasons for the apparent irreversibility of Na+/NH4+ exchange in the latter species are unknown.

It is interesting to consider some reasons for the apparent discrepancy between my data and those of Perry et al. (1981). Their finding that production of hypercapnia in 5. gairdneri did not alter the influx of either Na+ or Cl supports the conclusion that alterations of Na+/H+;, Na+/NH4+ or C1/HCO3exchanges do not play a measurable role in hypercapnic-acidosis compensation in this species. Their data are especially surprising in light of their concurrent finding that addition of amiloride to the external bath inhibited Na+ uptake and resulted in a pronounced acidosis, while addition of SITS (4-acetamido-4′-iso-thiocyanatostilbene-2,2′ disulphonic acid) inhibited Cl uptake and resulted in a significant decrease in plasma H+; concentration. Thus, their work indicates that Na+/H+; or Na+/NH4+ and C1/HCO3 exchange systems are resident in the branchial epithelium S. gairdneri, can control blood pH, but do not seem to function in response to hypercapnic acidosis. However, they did find that addition of more Na+ (3 mM) to the normally very dilute Vancouver tap water (40 μM) resulted in less acidosis and a more rapid blood pH recovery after hypercapnia. In addition, similar increases in external Na+ concentration resulted in alkalosis under normocapnic conditions. It seems evident therefore that they did not see a change in the Na+ influx under hypercapnic conditions because the transport system rate of turnover was limited by exceedingly low levels of one of its substrates, i.e. Na+. It may be that under normal conditions the limitation of the Na+ influx dictates that the response to acidosis is shifts in intracellular v. extra-cellular buffers (bicarbonate and non-bicarbonate) so that cytoplasmic pH is carefully controlled despite the lack of fine regulation of blood pH. Such a system has been described for the freshwater, airbreathing teleost Synbranchus marmoratus (Heisler, 1980). This fish is found in South American fresh water with extremely low Na+ and Cl concentrations (3 μM) and responds to hypercapnia with an increased blood , but little increase in HCO3 concentration. The result is a fall in blood pH of some 0·6 units for at least 5 days. The usual pattern in other species (Heisler, 1980) is an initial fall in blood pH which is compensated for by an increased blood HCO3 concentration within a few hours (interestingly, it apparently takes some 50 h for this compensation in 5. gairdneri’, Janssen & Randall, 1975). Perry et al. (1981) found that hypercapnic 5. gairdneri in low-Na water also had an increased blood , but a relatively low blood pH (0·29 pH units below the control) secondary to only a slight rise in blood HCO3 concentration. When the fish were placed into 3 mM-Na+ solutions, the blood HCO3 concentrations were significantly increased and the blood pH was controlled at levels only slightly (but significantly) below the control (0·06 pH units) after 24 h. It therefore appears that fish species living in solutions of low Na+ and Cl content may have to rely on mechanisms other than branchial ionic exchanges to maintain blood pH relatively constant in the face of some environmental perturbations. This is obviously not a problem for marine species.

Mineral acidosis

(1) Time course

Injection of a small mineral acid load (50 μM-HC1 100 g−1) into O. beta and 5. acanthias resulted in a stimulation of the H+; efflux as outlined in Table 4. The efflux of ammonia was not measured in these experiments because preliminary studies showed that animals which had been anaesthetized with MS-222 excreted inordinately high, and variable, amounts of ammonia, presumably as a result of the metabolism of this amine-containing anaesthetic. The toadfish responded to an injected acid load within 30 min and continued to excrete H+ for 2 h after the injection. The dogfish did not begin to excrete acid until the second 30 min after the injection and continued to excrete acid until 4 or 5 h after the injection. This difference in time course presumably results from differences in buffering capacities, sensitivity to blood pH changes, and activation of extrusion mechanisms in the two species ; however, there are no data to allow a determination of the limiting factors involved. In both cases the amount of H+ finally excreted by the injected fish exceeded the injected acid load. Thus, it appears that, despite the use of an anaesthetic, the stress of mineral acid loading produced a mixed mineral/metabolic acidosis. In support of this, control injections of Ringer’s solution (under MS-222 anaesthesia) produced measurable H+; efflux in both species (unpublished results). Cameron (1980) found that the catfish, Ictalurus punctatus, responded to an injected acid load (100 μM-HCI 100 g−1) with an extrarenal efflux of acid; however, after 2 h only some 20–30% of the injected load had been excreted. Since the pH of the blood of I. punctatus had returned to normal levels after 2 h, Cameron proposed that the other 70–80 % of the injected acid load had been buffered in either intracellular or bone compartments. It is clear from the data in Table 4 that both O. beta and 5. acanthias are able to excrete the injected acid load within 2–4 h after the injection.

Table 4.

Effect of acid injection on excretion of H+ by the toodfish and dogfish

Effect of acid injection on excretion of H+ by the toodfish and dogfish
Effect of acid injection on excretion of H+ by the toodfish and dogfish

(2) Role of branchial v. renal/rectal efflux

Table 5 shows clearly that all of the H+; efflux subsequent to mineral acid loading is via branchial pathways. This dominance of branchial v. renal/rectal pathways corroborates most recent data on freshwater teleost species (see Introduction), but is the first description of the importance of branchial acid extrusion by a marine teleost made acidotic by an injection of a mineral acid load.

Table 5.

Effect of acid injection on excretion of H+ by the toadjish and dogfish: branchial v. renal/rectal excretion

Effect of acid injection on excretion of H+ by the toadjish and dogfish: branchial v. renal/rectal excretion
Effect of acid injection on excretion of H+ by the toadjish and dogfish: branchial v. renal/rectal excretion

(3) Effect of Na+-free sea water

When HCl-injected fish were transferred into Na+-free sea water the efflux of H+; fell to below zero, so the fish were actually excreting net amounts of base (Table 6). When the fish were subsequently transferred back into Na+-containing sea water the efflux of acid resumed and continued at a greater rate for a longer period than the control time-course in Table 4. This prolongation of the time of H+; excretion subsequent to HC1 injection was presumably because the experimental fish were unable to excrete acid (they actually excreted base) during the period in Na+-free sea water. Thus, it is clear that both species require external Na+ in order to excrete a mixed mineral/metabolic acid load across their branchial epithelium.

Table 6.

Effect of acid injection on excretion of H+ by the toadjish and dogfish: effect of Na+-free sea water

Effect of acid injection on excretion of H+ by the toadjish and dogfish: effect of Na+-free sea water
Effect of acid injection on excretion of H+ by the toadjish and dogfish: effect of Na+-free sea water

In summary, both the marine teleost Opsanus beta and the marine elasmobranch Squalus acanthias respond to either a hypercapnic or mineral/metabolic acidosis by excreting H+ via the branchial epithelium. We have no data on HC1 loaded fish but at ‘east in hypercapnic acidosis, excretion of ammonia plays an insignificant role in this esponse. The excretion of H+; by both species requires the presence of Na+ in the îxternal sea water -suggesting strongly that branchial Na+/H+; exchange plays a lominant role in this response to acidosis. This is the first direct evidence for branchial Na+/H+; exchange in either a marine teleost or elasmobranch, and supports the conclusion that marine fishes, like their freshwater ancestors, excrete unwanted H+; and ammonia across the branchial epithelium in exchange for external Na+. The role of Na+/H+ and Na+/NH4+ exchange in the Na41 balance of these (and other) marine fish remains to be determined. One might propose that Na+/H+; and Na+/NH4+ exchange in these marine forms is a remnant of freshwater ancestory (Evans, 1975); however, recent evidence (Evans, 1980d) indicates that H+ (and possibly ammonia) efflux from the agnathan hagfish Myxine glutinosa is also dependent upon external Na+. Since this line of agnathan evolution has apparently never entered fresh water (Hardisty, 1979) it appears that Na+/H+; and Na+/NH4+ exchange may have evolved before the vertebrates entered fresh water, as a vehicle for H+; and ammonia extrusion, rather than during entry into fresh water as a device for regulating blood Na+ concentrations at levels above those in fresh water.

This study was supported by NSF grants PCM 77-03914 and PCM 80-03866 to the author and by NSF PCM 77-2670 and NIH Bio-Medical Research Support Grant SO7 RR 05764 to the Mt Desert Island Biological Laboratory. The technical assistance of John Mendlein is greatly appreciated.

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