Cell volume regulation: the role of taurine loss in maintaining membrane potential and cell pH.

1. In response to a hypo-osmotic stress cells undergo a regulatory volume decrease (RVD) by losing osmotically active solutes and obliged water. During RVD, trout red cells lost taurine, K+ and Cl- but gained Na+ and Cl-. Over the full time course of RVD the chloride concentration in the cell water remained remarkably constant. Thus membrane potential and cell pH, which depends on the ratio of internal to external chloride concentration ([Cl-]i:[Cl-]o), remained fixed. 2. When cell volume decreases it is only possible to keep the chloride concentration in the cell water constant if an equal percentage of the cell chloride pool and of the cell water pool are lost simultaneously. Quantitative analysis of our data showed that this requirement was fulfilled because, over the full time course of RVD, cells lost osmotically active solutes with a constant stoichiometry: 1 Cl-:1 positive charge:2.35 taurine. Any change in taurine permeability, by modifying the stoichiometric relationship, would affect the amount of water lost and consequently cell chloride concentration. 3. Experiments carried out with different cations as substitutes for external Na+ suggest that the constancy of the chloride concentration is not finely tuned by some mechanism able to modulate the channel transport capacity, but results in part from the fact that the swelling-dependent channel constitutively possesses an adequately fixed relative permeability for cations and taurine. However, as a significant fraction of K+ and Cl- loss occurs via a KCl cotransporter, the contribution of the cotransport to the stoichiometric relationship remains to be defined. 4. The large amount of taurine released during RVD (50 % of all solutes) was shown to be transported as an electroneutral zwitterion and not as an anion. How the channel can accommodate the zwitterionic form of taurine, which possesses a high electrical dipole, is considered.

Regulation of Na+/H+ antiporter in trout red blood cells.

The trout red blood cell Na+/H+ antiporter (beta NHE) plays two interesting properties: it is the only NHE own to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself. Cloning and expression of beta NHE have provided inificant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter. Expression of beta NHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge a common kinase. Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses. NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a "NHE1 transmembrane domain/beta NHE cytoplasmic domain' chimera is fully activated by cyclic AMP. In red cells, activation of beta NHE is the result of phosphorylation by PKA of at least two independent sites. Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites. Furthermore, Calyculin A (CIA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter. It is suggested that CIA may be able to revive "sequestered' antiporters. We propose that the molecular events underlying beta NHE desensitisation could be similar to those involved in rhodopsin desensitisation. Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger. Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.

Association of the band 3 protein with a volume-activated, anion and amino acid channel: a molecular approach.

In response to swelling, cells recover their initial volume by releasing intracellular solutes via volume-sensitive pathways. There is increasing evidence that structurally dissimilar organic osmolytes (amino acids, polyols, methyl amines), which are lost from cells in response to swelling, share a single pathway having the characteristics of an anion channel. However, the molecular identity of this pathway remains to be established. It has been suggested that the erythrocyte anion exchanger (AE1) or some AE1-related proteins could be involved. A direct evaluation of this possibility has been made by comparing the functional properties of two AE1s when expressed in Xenopus laevis oocytes: tAE1 is from a fish erythrocyte which releases taurine when swollen, and mAE1 is from a mammalian erythrocyte which does not regulate its volume when swollen. While mAE1 performs exclusively Cl-/Cl- exchange, tAE1 behaves as a bifunctional protein with both anion exchange and Cl-/taurine channel functions. Construction of diverse tAE1/mAE1 chimaeras allows the identification of protein domains associated with this channel activity. Thus, some AE1 isoforms could act as a swelling-activated osmolyte channel, a result having a potentially important implication in malaria. This review also discusses the possibility that several different proteins might function as swelling-activated osmolyte channels.

A role for the anion exchanger AE1 (band 3 protein) in cell volume regulation.

In response to swelling cells recover their volume by releasing ions (mainly K+, Cl-) and different organic solutes (e.g. taurine) via volume-sensitive pathways. Depending on the cause of swelling (net uptake of electrolytes or decrease in external osmolality) cells use specifically some of these pathways. Previous data indicate that the anion exchanger (AE1) is involved in the choice of the regulatory pattern the cells adopt. Molecular cloning and functional expression of AE1 from the trout erythrocyte shows that this anion exchanger can function as a channel mediating taurine fluxes. In the erythrocyte, the channel activation depends on the conditions as the cell is swollen: when swelling is caused by an accumulation of electrolytes (resulting in an increase of the intracellular ionic strength) the channel is not activated and the regulatory volume decrease occurs exclusively by a release of K and Cl via a KCl cotransporter. When swelling is caused by hypotonic shock (resulting in a decrease in intracellular ionic strength) the KCl cotransporter is then mainly inactivated or even silent; conversely the channel is activated and allows volume recovery by mediating the release of both taurine and probably K and Cl. The possibility that AEs function as volume-activated taurine channels in other cell types and as a malaria-induced channel in malaria-infected human red cells is considered.

The erythrocyte Na+/H+ exchangers of eel (Anguilla anguilla) and rainbow trout (Oncorhynchus mykiss): a comparative study

Trout and eel red blood cell Na+/H+ exchangers show widely different regulatory properties. Catecholamines, cyclic AMP and phorbol esters, which activate the trout red cell antiporter, do not affect the eel exchanger. Unlike the trout red cell exchanger, the eel red cell exchanger is strongly activated by cell shrinkage, allowing a remarkable cell volume recovery. These different regulatory properties probably indicate the existence of different isoforms of the exchangers in nucleated erythrocytes, since sensitivity to catecholamines is known to be dependent upon the presence of protein kinase A consensus sites on the cytoplasmic domain of the antiporter. After shrinkage of eel erythrocytes, the Na+/H+ exchange rate gradually increases to reach a maximum value after about 10 min. The magnitude of activation is a graded function of cell shrinkage. Deactivation, like activation, is induced by a volume change and occurs after some delay (lag time). The response of the trout antiporter (betaNHE) to cell shrinkage is much reduced compared with that of the eel antiporter. In addition, the antiporter is deactivated prior to restoration of the normal control volume, leaving cell volume regulation notably defective. The trout red cell antiporter, which is desensitized and enters a refractory state following hormonal activation, is only deactivated (it can be reversibly reactivated) after shrinkage-induced activation. This dual control may occur by both phosphorylation-dependent and phosphorylation-independent mechanisms. In view of the similarities in the regulatory properties of eel and salamander (Amphiuma sp.) Na+/H+ exchangers, the expression of a putative K+/H+ exchange mediated by the N+/H+ exchanger was sought in eel erythrocytes. However, neither osmotic swelling nor calyculin-A-dependent phosphorylation revealed such a K+/H+ exchange.

Regulation of Na+/H+ exchange activity by recruitment of new Na+/H+ antiporters: effect of calyculin A.

The Na+/H+ antiporter of trout red blood cells, beta-NHE, is activated by agonists of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) and by those of protein kinase C (PKC). beta-NHE, once activated, shifts into a refractory state, accounting for its desensitization. It had previously been shown that desensitization is blocked and reversed by the protein phosphatase inhibitor okadaic acid (OA). In this study we examined the effect of another protein phosphatase inhibitor, calyculin A (CIA). CIA was at least 10 times more potent than OA in blocking beta-NHE desensitization, suggesting that desensitization is controlled by phosphatase-1. Furthermore, CIA alone induced a large Na+/H+ exchange in unstimulated red blood cells, a property not shared by OA. The characteristics of ClA-induced Na+/H+ exchange are very different from those of the exchange triggered by activation of beta-NHE by PKA or PKC agonists, i.e., a flat pH dependence and total insensitivity to PKA and PKC inhibitors. Simultaneous addition of maximal concentrations of ClA and catecholamine produced an additive stimulation of the Na+/H+ exchange, consistent with the interpretation that these agents act on two distinct pools of exchangers. Screening of different cDNA libraries suggested that only one isoform of antiporter exists in the trout red blood cell; it therefore seems likely that regulation of the Na+/H+ antiporter beta-NHE involves a recycling mechanism. The reasons why intracellular beta-NHE show different properties from membrane beta-NHE are discussed.

Role of protein phosphorylation and dephosphorylation in activation and desensitization of the cAMP-dependent Na+/H+ antiport.

The Na+/H+ antiporter of trout erythrocytes is activated by agents raising intracellular cAMP, whereas other Na+/H+ exchangers are insensitive to or inhibited by cAMP. Cloning of the beta agonist-activated exchanger (beta NHE) reveals the presence of two consensus sites for phosphorylation by the cAMP-dependent protein kinase A (cAMP-PKA) on the cytoplasmic loop. Transfected to fibroblasts, beta NHE can no longer be activated by cAMP when these consensus sites are removed, indicating regulation through cAMP-PKA. Moreover, it has been shown that activation of the exchanger is rapidly followed by its desensitization. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase and phosphatase inhibitors on the antiporter activation and desensitization in trout red cells. Na+/H+ exchange was not induced by strong acidification, indicating that beta NHE is normally in a nonfunctional state, whereas cAMP did activate the system by forcing beta NHE into a functional conformation; preincubation of cells with the kinase inhibitor H89 blocked cAMP-activation, confirming the role of cAMP-PKA in the activation process. The protein phosphatase inhibitor okadaic acid (OA) neither activated the exchange when added on unstimulated cells nor prevented deactivation of beta agonist-activated beta NHE by propranolol. Hence, the cAMP-dependent phosphorylation involved in the activating process is controlled by an OA-insensitive phosphatase. beta NHE activated by beta agonist or cAMP shifts rapidly into a refractory state, accounting for the previously described desensitization. Desensitization was blocked and reversed by OA, indicating a control by an OA-sensitive phosphatase of the phosphorylation level of a site critical for the desensitizing process. Phosphorylation of this (site 2) and of the activating site (site 1) is mediated by cAMP-PKA, as demonstrated by the effects of both intracellular cAMP concentration and kinase inhibitor H89 on the Na+/H+ exchange activity. Based on these data, we proposed that beta NHE can exist in three different states (inactive I, activated A, and desensitized D). Conversion of I to A needs the simultaneous phosphorylation by cAMP-PKA of sites 1 and 2. These two sites might constitute the two neighboring cAMP-PKA sites located on the cytoplasmic loop as deduced from the oligonucleotide sequence. Dephosphorylation of site 2 and subsequent binding of an arrestin-like protein are assumed to account for desensitization of the antiport.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a K(+)-H+ exchange in trout red blood cells.

1. Exposure of trout red blood cells to beta-adrenergic agonist isoprenaline activates a cAMP-dependent Na(+)-H+ antiport, the movements of protons being compensated by a Cl(-)-OH- (or HCO3-) exchange mediated by band 3 protein. The absorption of water osmotically linked to sodium and chloride induces cell swelling. 2. In the presence of acetazolamide, anionic exchange is inhibited and activation of cationic exchange resulted in the first 2 min in a strong external acidification and a large internal alkalinization leading to a reversal of the transmembrane pH gradient. Then, for at least 1 h and despite the inhibition of Cl- entry, a net Na+ uptake occurred which was balanced by an equivalent K+ loss, with the result that cell volume and pH gradient remained unchanged. 3. In such conditions, the inactivation of the Na(+)-H+ exchanger by a beta-antagonist, propranolol, blocked Na+ entry while K+ continued to be lost. This volume-independent K+ efflux, which is thus independent of the Na(+)-H+ exchanger, was not accompanied by a Cl- efflux but was associated with large internal and external pH changes consistent with K(+)-H+ exchange. 4. The K+ loss and the related pH changes are inhibited by compounds which are known to inhibit the K(+)-anion co-transporter in trout red cells, i.e. 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (Dids) and niflumic acid.

Volume-activated Cl(-)-independent and Cl(-)-dependent K+ pathways in trout red blood cells.

1. Swelling of trout erythrocytes can be induced either by addition of catecholamine to the cell suspension, thus promoting NaCl uptake via beta-adrenergic-stimulated Na(+)-H+ exchange (isotonic swelling) or by suspending red blood cells in a hypotonic medium (hypotonic swelling). In both cases cells tend to regulate their volume by losing K+, but the characteristics of the volume-activated K+ pathways are different: after hormonally induced swelling the K+ loss is strictly Cl- dependent; after hypotonic swelling the K+ loss is essentially Cl- independent. 2. In order to determine the nature of these volume regulatory pathways (i.e. whether the net K+ loss was conductive or was by electroneutral K(+)-H+ exchange or KCl co-transport), studies were performed to analyse ion fluxes and associated electrical phenomena. The cell membrane potential and intracellular ionic activities of volume-regulating and volume-static cells were measured by impalement with conventional microelectrodes and double-barrelled ion-sensitive microelectrodes. 3. The information gained from the electrical and ion flux studies leads to the conclusion that both Cl(-)-independent and Cl(-)-dependent K+ loss proceed via electrically silent pathways. 4. Experiments were designed to distinguish between electroneutral K(+)-H+ exchange or KCl co-transport. These were based upon the inhibition of Cl(-)-OH- exchange to evaluate the degree of coupling between K+ and Cl- (KCl stoichiometry, pH change). The experimental observations are consistent with the fact that both Cl(-)-independent and Cl(-)-dependent K+ loss are mediated by coupled K(+)-anion co-transport and not by K(+)-H+ exchange. 5. On the basis of previous data, we suggest that only one type of K(+)-anion co-transport exists in the cell membrane, for which the selectivity for anions varies according to the change in cellular ionic strength induced by swelling.

Regulation of Na+/H+ exchange and pH in erythrocytes of fish.

1. The function of trout RBC Na+/H+ antiport is unrelated to cell volume or cell pH regulation. Its role is to improve oxygen transport capacity when the supply of oxygen becomes limited. 2. Antiport activation, mediated by cAMP, promotes complex changes in blood pH which have been analyzed in vivo and in vitro. 3. The regulation of antiport (activation, desensitization, control by molecular oxygen and by a newly discovered cytosolic protein, arrestin) is presented. 4. Molecular cloning of the antiport shows that two typical site motifs of phosphorylation by cAMP-dependent protein kinase are localized on the cytoplasmic region.

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.

A volume increase of trout erythrocytes can be induced either by beta-adrenergic stimulation of a Na+/H+ antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K+, Na+, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K+ loss was mediated essentially via a 'Cl(-)-independent' pathway. After isotonic swelling, the taurine, choline and Na+ pathways were practically not activated and the K+ loss was strictly 'Cl(-)-dependent'. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K+ pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl(-)-dependent, DIDS-sensitive K+ pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O2 or CO to the heme.

Regulation of Cl-dependent K transport by oxy-deoxyhemoglobin transitions in trout red cells.

The oxygenation of trout red cells opens a Cl-dependent K pathway inhibited by furosemide, and by inhibitors of the erythrocyte anion exchanger such as DIDS and niflumic acid. The trigger is the deoxy-oxy conformational change of hemoglobin. The binding of carbon monoxide to heme, which induces a similar conformational change, mimics the effect of oxygen. The possible mechanisms enabling molecular oxygen to control the transport protein are discussed. This oxygenation-activated K transport appears to play a regulatory role in the control of the extracellular K concentration.

Cell volume regulation by trout erythrocytes: characteristics of the transport systems activated by hypotonic swelling.

1. 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 Na+. 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 tetramethyl-ammonium, to rapidly penetrate the cells. 3. The swelling-activated K+ loss is not significantly affected by replacement of Cl- by NO3-, 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.

Effects of anions on the Na(+)-H+ exchange of trout red blood cells.

1. Replacement of chloride by foreign anions in the suspending medium of trout erythrocytes can affect in a complex manner both the activation by catecholamines of the latent Na(+)-H+ exchanger and its subsequent desensitization. These changes are discussed in relation to other cellular modifications (distribution of permeant anions and accumulation of cyclic AMP) induced by foreign anions. 2. The transfer of trout erythrocytes from a chloride-containing medium to media containing lyophilic permeable anions, NO3- or SCN-, immediately induces a decrease of distribution ratios of permeable anions across the red cell membrane (i.e. Donnan ratios). It is probable that the binding of lyophilic anions to haemoglobin, by altering the amount of negative fixed charges, results in changes of distribution of permeant anions across the membrane. 3. The effectiveness of anions in decreasing both the activation of the Na(+)-H+ exchanger and the Donnan ratio follows the same sequence in both cases, i.e., SCN- greater than NO3- greater than Cl- = propionate. It was demonstrated that a change in Donnan ratio affects antiport activity possibly through a shift in intracellular pH; such a mechanism however cannot account for all the effects of foreign anions on antiport activity. 4. The present results show that lyophilic anions do not modify the affinity of the antiporter for sodium ions but greatly decrease the transport capacity of the exchange system. This is interpreted as indicating that the binding of lyophilic anions to some component of the transport system prevents antiporters from establishing their activated configuration once stimulated. Since the inhibitory effect of anions on Na(+)-H+ exchange has been demonstrated in all erythrocytes studied but in no other cell, the crucial substance involved in this inhibition could well be haemoglobin, which appears to control antiport activity in erythrocytes. 5. Some anions affect desensitization of the exchanger. This effect is not related to the lyophilic character of the anion and is not mediated by a change in intracellular cyclic AMP. 6. Propionate and acetate drastically reduce the intracellular level of cyclic AMP and seem to facilitate the activated configuration of the exchanger.

Immunological detection of arrestin, a phototransduction regulatory protein, in the cytosol of nucleated erythrocytes.

Cytosolic extracts of trout and turkey erythrocytes were tested for their immunoreactivity with polyclonal and monoclonal antibodies to retinal arrestin (S-antigen), a cytosolic protein of photoreceptor cells involved in the desensitization of rhodopsin. After adsorption or immunoaffinity chromatography of the extracts, these antibodies specifically recognized a protein having a molecular weight similar to that of retinal arrestin. Because the G-protein-mediated transduction systems, such as visual and beta-adrenergic systems, display a high degree of structural and functional homology, the presence of arrestin-like proteins in non-photosensitive cells suggests that these proteins are involved in the transduction of chemical signals, with a possible role in receptor desensitization.

Glutaraldehyde fixation of the cAMP-dependent Na+/H+ exchanger in trout red cells.

It has been shown that the addition of a beta-adrenergic catecholamine to a trout red blood cell suspension induces a 60-100-fold increase of sodium permeability resulting from the activation of a cAMP-dependent Na+/H+ antiport. Subsequent addition of propranolol almost instantaneously reduces the intracellular cAMP concentration, and thus the Na permeability, to their basal values (Mahé et al., 1985). If glutaraldehyde (0.06-0.1%) is added when the Na+/H+ exchanger is activated after hormonal stimulation, addition of propranolol no longer inhibits Na permeability: once activated and fixed by glutaraldehyde, the cAMP dependence disappears. Glutaraldehyde alone causes a rapid decrease in the cellular cAMP concentration. In its fixed state the antiporter is fully amiloride sensitive. The switching on of the Na+/H+ exchange by cAMP is rapidly (2 min) followed by acute but progressive desensitization of the exchanger (Garcia-Romeu et al., 1988). The desensitization depends on the concentration of external sodium, being maximal at a normal Na concentration (145 mM) and nonexistent at a low Na concentration (20 mM). If glutaraldehyde is added after activation in nondesensitizing conditions (20 mM Na), transfer to a Na-rich medium induces only a very slight desensitization: thus the fixative can "freeze" the exchanger in the nondesensitizing conformation. NO3- inhibits the activity of the cAMP-dependent Na+/H+ antiporter of the trout red blood cell (Borgese et al., 1986). If glutaraldehyde is added when the cells are activated by cAMP in a chloride-containing medium, the activity of the exchanger is no longer inhibited when Cl- is replaced by NO3-. Conversely, after fixation in NO3- medium replacement of NO3- by Cl- has very little stimulatory effect. This indicates that the anion dependence is not a specific requirement for the exchange process but that the anion environment is critical for the switching on of the Na+/H+ exchanger and for the maintenance of its activated configuration.

Na+-H+ exchange and pH regulation in red blood cells: role of uncatalyzed H2CO3 dehydration.

Erythrocytes of rainbow trout respond to adrenergic stimulation by activation of a Na+-H+ exchange. When red blood cells are suspended in their own plasma and equilibrated with a convenient gas mixture in a tonometer, the extrusion of H+ induces a fast, very strong acidification of the blood (by 0.5-0.7 pH units), explained as follows. Excretion of H+ into a medium containing HCO3- causes the formation of H2CO3. The uncatalyzed dehydration of H2CO3 is slow so that H+ accumulates above the level that would prevail at equilibrium, promoting a strong acid disequilibrium pH. Then the blood pH progressively returns to a value close to its initial value because of the slow uncatalyzed dehydration of H2CO3 and washout of the CO2 so produced. The period of acid disequilibrium pH, however, is lengthened because part of the CO2 generated by the spontaneous dehydration is not washed out by tonometry but diffuses into the red cells where it is rapidly converted into HCO3- and H+ by carbonic anhydrase and then excreted by Na+-H+ and Cl-HCO3- exchangers. This recycling process "refuels" the ionic reaction, increasing the time needed to reach equilibrium. The anion exchanger does not sense this strong acid disequilibrium pH, since the external HCO3- concentration is practically unchanged at that time. During the extracellular pH (pHe) recovery period, simultaneously extracellular HCO3- content decreases and intracellular Cl- content increases. Thus intracellular pH and pHe appear to be uncoupled. This overall interpretation is confirmed by experiments using carbonic anhydrase and drugs such as propranolol and amiloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Desensitization by external Na of the cyclic AMP-dependent Na+/H+ antiporter in trout red blood cells.

The erythrocytes of the trout, Salmo gairdneri, react to beta-adrenergic stimulation by activating a cyclic AMP-dependent and amiloride-sensitive Na+/H+ antiporter (see Borgese, F., F. Garcia-Romeu, and R. Motais, Journal of General Physiology, 1986, 87:551-566). The present study traces the kinetic behavior of the unidirectional Na fluxes after stimulation by isoproterenol. A very considerable increase (100-fold) of the unidirectional Na influx (JNa(in)) follows the addition of isoproterenol to the erythrocyte suspension. After 1.5 min, JNa(in) falls suddenly, and asymptotically diminishes toward the nonstimulated flux level. The unidirectional Na efflux (JNa(out)) proceeds according to similar kinetics. The decrease of JNa(in) and JNa(out)is not linked to either a change in the driving forces of the transported ions or a decrease of the cyclic AMP concentration but to a desensitization of the Na+/H+ antiporter. This desensitization is dependent on the external Na concentration and is not controlled by internal Na, cell swelling, or external Ca.

The control of Na+/H+ exchange by molecular oxygen in trout erythrocytes. A possible role of hemoglobin as a transducer.

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 NaCl 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 O2. This difference is explained by an inhibition of the cAMP-dependent Na+/H+ exchange by O2. The inhibition is not reversed in cells incubated in O2 but poisoned with cyanide. It cannot be explained by a difference in the content of cAMP in O2 and in N2. In a CO atmosphere, in which the cells are anoxic, swelling and Na permeability are not increased as they are in N2: in CO, the cells behave as they do in O2. Moreover, cells previously exposed to CO and then put in an N2 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 N2, is the primary event responsible for the inhibition. As CO substitutes for O2 in binding to hemoglobin, the effect of O2 in the control of Na+/H+ exchange is probably explained by this interaction with heme. (Allen and McManus [1968. Biophysical Journal. 8:125a] previously described a similar effect of CO on passive Na permeability in duck red cells.) It is proposed that the hemoglobin, by interacting differently, according to its degree of oxygenation, with the cytoplasmic segment of band 3 protein, may influence some transport function, such as Na+/H+ exchange. The physiological significance of a control of Na+/H+ exchange by molecular O2 is discussed.

Ion movements and volume changes induced by catecholamines in erythrocytes of rainbow trout: effect of pH.

1. Trout red cells suspended in an isotonic medium containing catecholamines or adenosine 3',5'-phosphate (cyclic AMP) enlarge rapidly to reach a new steady-state volume which is maintained as long as hormone is present. 2. The present investigation demonstrates that the maximum swelling reached by the cells is strongly pH dependent. At pH 7.55 the cells enlarge more rapidly than at pH 7.95 and they reach a maximal volume which is much greater. It is explained by a differential effect of pH on two pathways controlling the movements of cations: K+ loss decreases as pH becomes more acidic in a roughly linear manner. On the contrary Na+ uptake increases as pH becomes more acidic with a maximum around pH 7.30 and then decreases. From this pH dependence it can be expected that the maximum enlargement occurs at about pH 7.30. 3. The complex relationship describing the change in the activity of the Na+-H+ exchanger as a function of pH (bell-shaped curve) is explained by the predominant influence of internal H+ on the antiporter in the alkaline range of pH and by the predominant influence of external H+ on the transporter in the acidic range.