A B S T R A C T It has previously been shown that addition of catecholamines to a suspension of trout erythrocytes induces an enlargement of the cells owing to an uptake of NaC! mediated by a cAMP-dependent, amiloride-sensitive Na+/ H + exchange. In this article, we show that the change in cell volume induced by catecholamines is much greater when the erythrocytes are incubated in N2 than when they are in 02. This difference is explained by an inhibition of the cAMP-dependent Na+/H + exchange by 02. The inhibition is not reversed in cells incubated in O~ but poisoned with cyanide. It cannot be explained by a difference in the content of cAMP in O~ and in N~. In a CO atmosphere, in which the cells are anoxic, swelling and Na permeability are not increased as they are in N~: in CO, the cells behave as they do in O~. Moreover, cells previously exposed to CO and then put in an N~ atmosphere do not show the expected increase in Na+/H + exchange. This strongly indicates that the binding of CO to hemoglobin, which persists during the subsequent exposure to N~, is the primary event responsible for the inhibition. As CO substitutes for O~ in binding to hemoglobin, the effect of O~ in the control of Na+/H + exchange is probably explained by this interaction with heine. (Allen and McManus [1968.

SUMMARY1. An osmolality reduction of the suspending medium leads to osmotic swelling of trout erythrocytes, which is followed by a volume readjustment towards the original level. The regulatory volume decrease (RVD) was not complete after 1 h.2. During RVD the cells lost K+ and Cl-but gained Nat. This entry of Na+, which is about half the K+ loss, explains the incomplete volume recovery (it was complete when Na+ was replaced by impermeant N-methyl-D-glucamine). The cells also lose large quantities of taurine, which accounts for about 53 % of the volume recovery. In addition RVD is accompanied by the activation of a pathway allowing some large organic cations which are normally impermeant, such as choline or tetramethylammonium, to rapidly penetrate the cells.3. The swelling-activated K+ loss is not significantly affected by replacement of Cl-by N03-, indicating that K+ moves through a Cl--independent K+ pathway.Furosemide, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and niflumic acid inhibit the K+ loss. From experiments performed in high-K+-containing media, it appears that these compounds block the K+ flux, not by inhibiting Cl-movements but by interfering with the K+ pathway. 4. All the volume-activated pathways (K+, Na+, taurine, choline) are fully inhibited by furosemide and by inhibitors of the anion exchanger such as DIDS and niflumic acid. The concentration required for 50 % inhibition (IC50) of both inorganic cations and taurine appears to be similar. It is proposed that DIDS interacts with a unique target which controls all the volume-sensitive transport systems.

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SUMMARY1. The addition of isoprenaline to an isotonic suspension of red blood cells of rainbow trout induces an amiloride-sensitive Na+ transport which is independent of Cl-and insensitive to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) and furosemide.2. Na+ uptake is accompanied by amiloride-sensitive H+ release. The H+ efflux is dependent upon the external Na+ concentration, the Ko.5 value for Na+ being 16 mm.3. In the presence of DIDS, when the coupled NaCl entry (NaCl co-transport) induced by catecholamine is blocked, the results provide evidence for a linked movement of Na+ and H+, with a stoicheiometry of 1 :1.4. Exchange of H+ for Na+ induces osmotic swelling of the cells which is due to the replacement of a bound proton by an osmotically active Na+ cation.5. In the absence of DIDS when the bulk of the Na+ uptake is the result of a coupled entry of Na+ and Cl-, H+ extrusion still occurs and the magnitude of acid excretion is identical to that found in DIDS-treated cells. This suggests that Na+-H+ exchange remains active.6. Addition of isoprenaline first stimulates the Na+-H+ exchange but only transiently. This is followed by a more permanent stimulation ofthe NaCl co-transport.

It has previously been shown (Baroin, A,, F . Garcia-Romeu, T . Lamarre, and R . Motais . 1984a, b . Journal of Physiology . 350 :137, 356 :21 ; Mahe, Y ., F . Garcia-Romeu, and R . Motais . 1985 . European Journal of Pharmacology . 116 :199) that the addition of catecholamines to an isotonic suspension of nucleated red blood cells of the rainbow trout first stimulates a cAMPdependent, amiloride-sensitive Na'/H' exchange . This stimulation seems to be transient . It is followed by a more permanent activation of a coupled entry of Na' and Cl -, which is inhibited by amiloride but also by inhibitors of band 3 protein (DIDS, furosemide, niflumic acid) . The coupled entry of Na' and Clcould therefore result from the parallel and simultaneous exchange of Na .' for H (via the cAMP-dependent Na'/H' antiporter) and Clo-, for HCO3-; (via the anion exchange system located in band 3 protein) . However, in view of the following arguments, it had been proposed that NaCI uptake does not proceed by the double-exchanger system but via an NaCI cotransport : (a) Na' entry requires Cl -as anion (in NO-3 medium, the Na uptake is strongly inhibited, whereas NO-3 is an extremely effective substitute for Cl-in the anion exchange system) ; (b) Na uptake is not significantly affected by the presence of HCO-3 in the suspension medium despite the fact that in red cells, CI-/HCO-exchange occurs more readily than the exchanges of Cl -for basic equivalents in a theoretically C0 2-free medium (the so-called Cl -/OH-exchanges) . The purpose of the present paper was a reassessment of the two models by using monensin, an ionophore allowing Na'/H' exchange . From this study, it appears that NaCl entry results from the simultaneous functioning of the Na'/H' antiporter and the anion exchange system . The apparent Cl dependence is explained by the fact that, in these erythrocytes, NO-clearly inhibits the turnover rate of the Na'/H' antiporter . As Na'/H' exchange is the driving component in the salt uptake process, this inhibition explains the Cl requirement for Na entry . The lack of stimulation of cell swelling by bicarbonate is explained by the fact that the rate of anion exchange in a C0 2-free medium (Cl-/OH -exchange) is roughly equivalent to that of Na'/H' exchange and thus in practice is not limiting to the net influx of NaCl through the two exchangers . The acidification J . GEN . PHYSIOL .

SUMMARY1. Kinetic and electrophysiological studies were carried out in the in vitro Rana esculenta skin, bathed in dilute sodium solution, to characterize the proton pump and coupling between sodium absorption (JNa+) and proton excretion (JH+).2. JNa+ and JH+ were both dependent on transepithelial potential (V'ms); hyperpolarizing the skin decreased JNa+ and increased JnH+; depolarization produced the opposite effects. These changes were accompanied by depolarization of the apical membrane potentialVfmc from -47 to -25 mV and an increase in apical membrane resistance of 30 % ; no significant effects on basolateral membrane potential (Vfc) and resistance (Rb) nor on shunt resistance (Rj) were observed. The proton pump appears to be localized at the apical membrane. 6. The proton pump was also inhibited by deoxygenation, oligomycin, dicyclohexylcarbodiimide and vanadate (100, 78, 83 and 100 % inhibition respectively). The variations ofJH+ and ofthe measured electrical currents were significantly correlated. These findings are supportive evidence of a primary active proton pump, electrogenic and strictly linked to aerobic metabolism.7. The current-voltage (I-V) relation of the proton pump was obtained as the difference in the I-V curves of the apical membrane extracted before and after proton-pump inhibition by ethoxzolamide during amiloride block ofsodium transport.The proton-pump current (Ip) was best described by a saturable exponential function of Vfmc. Maximal pump current (IWmX) was calculated to be 200 nequiv h-1 cm-2 at a Vmc of + 50 mV and the pump reversal potential ER was -130 mV. The effect of ethoxzolamide to depolarize Vfmc was dependent on the relation between Vfmc and ER. Maximal induced depolarization occurred at a Vmc of + 50 mV whereas ethoxzolamide exerted minimal effect on Vfmc when the ER was approached either by voltage clamping the apical membrane or by the addition of amiloride.8. We show that electroneutral sodium-proton countertransport is not the mechanism of active proton excretion in frog skin but that it is the proton excretion J. EHRENFELD, F. GARCIA-ROMEU AND B. J. HARVEY which provides a favourable electrical driving force for passive apical sodium entry. The apparent coupling between net proton and sodium fluxes depends on the relation between V1mc and ER and the effect of the sodium-electrode-like behaviour of the apical membrane on this relation.

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