The effects of prolonged epinephrine infusion on the physiology of the rainbow trout, Salmo gairdneri: II. Branchial solute fluxes

In freshwater fishes, internal ionic regulation is dependent upon the active branchial uptake of Na⁺ and Cl⁻ ions from the external environment. The branchial ionic uptake mechanisms involved have been studied in great detail (see reviews by Maetz, 1971; Evans, 1982, 1984; Payan, Girard & Mayer-Gostan, 1984) and there is strong evidence that Na⁺ uptake is linked to the excretion of acidic equivalents (H⁺ and/or NH₄⁺) while Cl⁻ uptake is coupled to the excretion of basic equivalents (HCO₃⁻ and/or OH⁻). For these reasons, ionic regulation and acid-base regulation are intimately related. Indeed, it has been demonstrated that perturbations of the branchial ionic exchange processes, either by altering the ionic composition of the external environment (DeRenzis & Maetz, 1973) or by selective pharmacological inhibition (Perry, Haswell, Randall & Farrell, 1981), cause predictable changes in blood acid-base status. Evidence is also accruing to suggest that the branchial ionic exchange mechanisms are dynamically manipulated in an appropriate fashion to regulate internal acid—base status during acid—base disturbances (Cameron, 1976; Wood, Wheatly & Hōbe, 1984; Claiborne & Heisler, 1984; S. F. Perry, S. Malone & D. Ewing, in preparation). Few studies, however, have addressed the problem of how such adjustments of branchial ionic fluxes are achieved. In this study we examine the hypothesis that epinephrine is involved in the control of gill ionic exchange mechanisms for the purposes of acid-base regulation. This hypothesis is based upon observations of elevated plasma epinephrine levels during a variety of acid—base disturbances induced by intravascular acid infusion (Boutilier, Iwama & Randall, 1986), external hypercapnia (S. F. Perry, S. Malone & D. Ewing, in preparation), exhaustive exercise (Opdyke, Carrol & Keller, 1982; Primmett, Randall, Mazeaud & Boutilier, 1986) and acute hypoxia (Fievet, Motais & Thomas, 1986), as well as the results of gill perfusion experiments that indicate adrenergic control of branchial ionic uptake (Payan, Matty & Maetz, 1975; Payan, 1978; Perry, Payan & Girard, 1984a). As in the previous paper (Perry & Vermette, 1986), experiments were performed in vivo during continuous infusion of epinephrine to elevate its level to that measured during periods of extracellular acidosis.

Experimental conditions and the treatment of animals prior to and during experimentation were as outlined in Perry & Vermette (1986). Branchial solute fluxes were determined on fish fitted with indwelling dorsal aortic cannulae and urinary catheters. Continuous urine collection permitted separation of branchial and renal fluxes. Skin and gut contributions were considered to be negligible. Fish were free swimming but restrained in opaque Perspex boxes (volume = 3 litres) similar in design to those used by McDonald (1983). The flux boxes were partially immersed in a cooling bath to maintain constant temperature and vigorously aerated to provide adequate water convection.

Fish were subjected to 4 h of pre-experimental saline (for ion concentrations, see Perry & Vermette, 1986) infusion, then a 3-h period of saline infusion followed by a 24-h interval of either saline (control group; N= 14) or 2×10^−Smoll⁻¹ L-epinephrine (experimental group; N= 19) infusion. Both groups were subjected finally to a further 12h of saline infusion. Fish were infused at a flow rate of 0·6 ml h⁻¹ to achieve an estimated blood epinephrine concentration of 5×10⁻⁸ moll⁻¹. Actual epinephrine levels were determined by high pressure liquid chromaStography, as described previously (Perry & Vermette, 1986).

Ammonia accumulation, water acid-base disturbances and isotopic backflux problems were minimized by determining solute fluxes over 3-h intervals only. Additionally, the boxes were flushed for at least 10 min (0·7–0·81H₂O min⁻¹) between successive flux periods.

Branchial unidirectional Na⁺ and Cl⁻ fluxes were determined on separate groups of fish by monitoring the disappearance of ²²Na (as NaCl; Amersham) or ³⁶C1 (as HCl; ICN). Approximately 1–1·25 μCi of isotope (the amount added was increased gradually at each flux period in an attempt to maintain a constant ratio of external to internal specific activities) was added to each box and allowed to mix for 15 min. An initial 10-ml water sample was removed following the mixing period and another after 3 h. Activity of ²²Na or ³⁶C1 was determined immediately on 5·0-ml samples while the remaining sample was frozen for subsequent ionic analysis (Na⁺, Cl⁻, K⁺ and NH₄⁺). Eight time periods were identified as being most interesting (3–6h saline; 0–3h, 3–6h, 9–12h and 21–24h epinephrine; 0–3 h, 3–6h and 9–12h final saline) and were selected for flux determinations. Otherwise, fish were supplied with continuous water flow.

Branchial net acid fluxes were determined on a separate group of fish (N = 5) from measurements of titratable alkalinity and ammonia concentrations (see below) on initial and final water samples.

²²Na and ³⁶C1 activities were determined on 5-ml water samples by liquid scintillation counter (LKB 1211 Rackbeta). Water concentrations of Na⁺ and K⁺ were determined by flame photometry (EEL Flame Photometer) ; concentrations of Cl⁻ were measured by amperometric titration (Buchler-Cotlove Chloridometer). Total ammonia concentrations were determined using the method of Verdouw, vanEchteld & Dekkers (1978). Net fluxes (J_nct) for these electrolytes and unidirectional fluxes (i.e. J_in, J_out) for Na⁺ and Cl⁻ were determined according to Maetz (1956). Accordingly, influxes (J_in) and net gains by the animal have positive signs while effluxes (J_out) and net losses have negative signs.

Titratable alkalinity was determined on 10-ml water samples by titrating to pH4·00 with 0·02moll⁻¹ HC1 as described by McDonald & Wood (1981). The net branchial acid flux was calculated as the sum of the titratable acidity flux (JTA; i.e. negative titratable alkalinity) and ammonia flux (J_Amm)-

Data shown in Figures are means ± S.E. Where appropriate, paired or unpaired Student’s Z-tests were used to compare sample means and 5 % was taken as the fiducial limit of significance.

Occasionally significant differences were noted between the control and experimental groups during the initial period of saline infusion (Figs IB, 2A, 3A). As a consequence, the results have been statistically analysed in two ways: (1) by using a paired approach in which all values were compared to the initial saline infusion values, and (2) by comparing control and experimental values at identical time periods using a non-paired design. Saline infusion (control group) had no effect on any measured variable.

Infusion of epinephrine caused a reduction in branchial net fluxes of Na⁺ and Cl⁻ (i.e. becoming more negative) indicating an increase in net NaCl loss from the animal into the external environment. However, there were significant differences between the pattern of and adjustment during epinephrine infusion. Most importantly, the changes in were more rapid and of a greater magnitude than the changes in -The reductions in and both tended to persist upon return to saline infusion (Fig. 1A,B). Branchial was slightly negative in both groups and was unaffected by epinephrine infusion (Fig. 1C).

The reduction in was due solely to an inhibition of sodium influx Fig. 2A) whereas the larger reduction in was a result of inhibition of Cl⁻ influx (Fig. 2B), combined with increased Cl⁻ efflux ⁠, Fig. 3B). Changes in and were most pronounced following 9–12 h of epinephrine infusion. was restored to pre-epinephrine levels after 21–24 h while remained slightly depressed (Fig. 2). Neither nor were significantly different from preepinephrine values during the final saline infusion period. remained elevated until 3–6 h of the final saline infusion (Fig. 3).

As a consequence of the differential effects of epinephrine on the branchial unidirectional fluxes of Na⁺ and Cl⁻ (see above), a physiologically significant pattern emerged with respect to the difference between and Fig. 4). was elevated rapidly during epinephrine infusion, peaked between 3 and 12 h of infusion and was returning towards pre-epinephrine levels by 21–24 h (Fig. 4). was unaffected by further saline infusion. Based on empirical evidence (Wood et al. 1984), the magnitude of is the prime determinant of branchial net acid excretion Indeed, in the present study, a positive relationship was observed between and net acid excretion (Fig. 5). Although the relationship was somewhat obscured by a high degree of variability, it is clear that branchial extrusion of acidic equivalents was greatest (i.e. becoming less positive) when was also elevated (i.e. during the first 12h of epinephrine infusion) (Fig. 5). Two variables contribute to the calculation of J net these are titratable alkalinity flux (JTA) and ammonia flux (J_Amm)-In the present investigation, the elevation of net acid extrusion (Fig. 6C) was due entirely to a reduction in JTA (Fig. 6A). The minor changes in J_TA during the final saline infusion were offset by similar non-significant changes in J_Amm such that remained constant.

The effects of catecholamines on branchial solute fluxes in freshwater fishes have been investigated previously in some detail using isolated, saline-perfused gill preparations (Payan et al. 1975; Payan, 1978; Payan, Mayer-Gostan & Pang, 1981 ; Girard & Payan, 1977, 1980; Perry et al. 1984a). The conclusions based on such perfusion experiments have been criticized for a variety of reasons, including the use of abnormally high levels of catecholamines in order to elicit effects, difficulty in distinguishing between specific effects of catecholamines and secondary effects caused by haemodynamic adjustments, and non-physiological conditions of perfusion (e.g. lack of dorsal aortic pressure). In the present investigation, we have evaluated the effects of epinephrine on branchial solute fluxes in the intact fish using levels of this catecholamine consistent with those measured during a variety of stressful conditions (6·2×l0⁻⁸moll⁻¹ after 4h of continuous intra-arterial infusion; see Perry & Vermette, 1986). A comparison between the present results and those of previous perfusion studies reveals some common features but also considerable differences. Unlike the results of perfused trout head experiments, which show a marked stimulatory effect of epinephrine on Na⁺/NH₄⁺ exchange (Payan et al. 1975; Payan, 1978; Girard & Payan, 1977), in vivo infusion actually caused significant inhibition of Na⁺ uptake that was not associated with lowered ammonia efflux. However, inhibition of Cl⁻ uptake (Fig. 2) is consistent with the α-receptor-mediated inhibition of branchial in the perfused trout head preparation (Perry et al. 1984a). The reasons for these discrepancies are unclear, although we speculate that branchial ionic uptake in some earlier perfusion studies may have been limited by the diffusive properties (such as surface area and thickness of the diffusion barrier) of the gill epithelium. If this were so, epinephrine may have stimulated in previous studies simply by increasing the diffusive conductance of the gill via lamellar recruitment (Booth, 1979) in a similar manner as for oxygen transfer (Pettersson, 1983; Perry, Daxboeck & Dobson, 1985). It is unlikely that active ionic uptake is diffusion-limited in the intact animal.

The results presented here suggest an important role for epinephrine in acid-base regulation and are consistent with observations of elevated levels of circulating catecholamines during conditions of internal acidosis induced by intravascular acid infusion (Boutilieret al. 1986), external hypercapnia (S. F. Perry, S. Malone & D. Ewing, in preparation), exhaustive exercise (Opdyke et al. 1982; Primmett et al. 1986; see also Wood & Perry, 1985) or acute hypoxia (Fievet et al. 1986). Based on those observations and the stimulatory effect of elevated plasma [epinephrine] on branchial excretion of acidic equivalents reported here, it is tempting to assign a role to epinephrine in the compensation of extracellular acidoses as speculated by S. F.

Perry, S. Malone & D. Ewing (in preparation) during regulation of hypercapnic acidosis. Certainly, a role for epinephrine in acute erythrocyte acid—base balance has already been established (Nikinmaa, Cech & McEnroe, 1984; Boutilier et al. 1986; Primmett et al. 1986; Perry & Vermette, 1986). However, the elevations of plasma epinephrine levels during persistent acid-base disturbances have been shown to be transient (Boutilier et al. 1986; S. F. Perry, S. Malone & D. Ewing, in preparation), so other factors are probably involved in the regulation of chronic acid-base disturbances.

Based on strong ion difference theory (i.e. blood pH is determined by the difference between strong cations and anions; Stewart, 1981) and empirical data (Wood et al. 1984), it is apparent that the magnitude of net branchial acid excretion ultimately depends upon the arithmetic difference between and across the gill. Typically, the regulation of depressed extracellular pH caused by respiratory disturbances is characterized by an increase of branchial (Cameron, 1976; Wood et al. 1984; Claiborne & Heisler, 1984; S. F. Perry, S. Malone & D. Ewing, in preparation) that is believed to result from dynamic manipulation of the influx and/or efflux rates of these ions. The relationship between and gill acid excretion apparently stems from the relationship between the exchanges of Na⁺ and CP for acidic and basic equivalents, respectively. Epinephrine infusion, in the absence of any external acid—base disturbance, caused similar increases in and net H⁺ excretion as has been observed during actual acid-base perturbations. It is clear, however, that the changes in did not result from simple modifications in the rates of Na⁺ and Cl⁻ influx but from complex and differential adjustments to both influx and efflux. Indeed, the apparent stimulation of Cl⁻ efflux in the absence of any effect on Na⁺ efflux or K⁺ net flux suggests independent control of the passive outward movement of these ions. Such a scheme, however, is difficult to explain given that salt loss in freshwater fishes is thought to be governed by gill diffusive properties. Regardless of the mechanisms involved, there is little doubt that epinephrine enhances the extrusion of acid equivalents into the external environment which should be reflected by elevated levels of bicarbonate in the plasma. In the preceding paper (Perry & Vermette, 1986), it was demonstrated that plasma [HCO3 ] increased by approximately 1·9 mmol I⁻¹ between 1·5 and 10·5 h of epinephrine infusion over and above the increase attributable to non-bicarbonate buffering. Assuming that plasma HCO₃⁻ is in equilibrium with the extracellular fluid (ECF) compartment and an ECF volume of 274 ml kg⁻¹ body mass (Hōbe, Wood & Wheatly, 1984), approximately 525μmolkg⁻¹ of additional acidic equivalents must have been excreted across the gill. Our calculations indicate that additional branchial H⁺ excretion over this particular period could account for the removal of about 1800μmol kg⁻¹, an amount well in excess of the calculated increase in ECF [HCO₃⁻]. Such a discrepancy may have resulted from the transfer of HCO₃⁻ between extracellular and intracellular compartments.

Although the results obtained in the present study suggest a distinct role for epinephrine in the regulation of branchial acid excretion, the possibility of interactive factors being involved cannot be rule out. One such factor is the acidosis induced by epinephrine infusion. To our knowledge, the direct effects of reduced blood pH on branchial acid excretion have not been evaluated; however, the transient and relatively minor extent of the acidification indicates that direct pH effects probably cannot account for all the various physiological responses observed in our study. Certainly, branchial net H⁺ excretion remained elevated and other branchial effects also persisted even after plasma pH had been restored to pre-infusion values. Specific effects of reduced pH on branchial ionic fluxes have rarely been investigated, although Payan (1978) reported that Na⁺ uptake was inhibited in the perfused trout head preparation following acidification of the perfusion fluid. In a similar preparation, Perry et al. (1984b) noted that hypercapnic acidosis of the internal perfusate caused a stimulation of Cl⁻ uptake. It is difficult to reach a conclusion on the possibility of direct pH modulation of branchial ion uptake processes given the paucity of available data. Other experiments designed to investigate the relationships oetween internal acid-base status and gill ionic uptake have been performed on intact animals in which secondary events such as catecholamine mobilization could not be controlled. Certainly, this is an area of research that warrants further investigation.

This study was supported by an NSERC operating grant to SFP. We wish to thank Drs T. W. Moon and J. C. Fenwick for helpful communication and comments and Dr D. J. Randall for access to unpublished manuscripts. Valuable technical assistance was provided by D. Ewing and S. Malone.