Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K+ from the cells. Hyperpolarization shows a peak at about 3 min and a net K+ efflux at 4-5 min. An additional transient hyperpolarization and net K+ efflux are found after 60-80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K+ efflux. At low pH, neither transient hyperpolarization nor a transient K+ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K+ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K+ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K+, whereas the same amount of Na+ does not reduce the hyperpolarization.     

Transport characteristics of L-glutamate in human jejunal brush-border membrane vesicles

Previous work using human jejunal brush-border membrane vesicles has demonstrated the existence of a distinct transport system in man for acidic amino acids. This system is energized by an inwardly directed Na+ gradient and an outwardly directed K+ gradient. These studies further characterize the transport of L-glutamate in the human jejunal brush-border membrane vesicles. Efflux studies were performed by loading the brush-border membrane vesicles with radiolabeled L-glutamate and sodium chloride. Extravesicular K+ accelerated the efflux of L-glutamate when compared to extravesicular Na+ or choline, indicating that potassium serves to recycle the carrier. Unlabeled extravesicular L-glutamate (but not D-glutamate) also enhanced the efflux of radiolabeled L-glutamate demonstrating that there is a bidirectional similarity to the transport system. The effect of pH on the transport system was also investigated by varying the intravesicular and extravesicular pH from 5.5 to 9. A pH environment of 6.5 produced the highest initial uptake rates as well as the greatest overshoots for transport of L-glutamate into brush-border membrane vesicles. The imposition of an inwardly directed pH gradient (5.5 outside, 7.5 inside) accelerated both the influx and efflux of L-glutamate. These results demonstrate that the L-glutamate carrier system in human jejunum appears to have similar energizing characteristics in either direction across the brush-border membrane. In addition, the system operates at an optimal pH of 6.5 and protonation of the system may enhance its mobility.     

Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells

Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells.     

Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles

The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium efflux and cycling across the synaptosomal plasma membrane.

Ca2+ efflux from intact synaptosomes is investigated. Net efflux can be induced by returning synaptosomes from media with elevated Ca2+ or high pH to a normal medium. Net Ca2+ efflux is accelerated when the Na+ electrochemical potential gradient is collapsed by veratridine plus ouabain. Under steady-state conditions at 30 degrees C, Ca2+ cycles across the plasma membrane at 0.38 nmol . min-1 . mg-1 of protein. Exchange is increased by 145% by veratridine plus ouabain, both influx and efflux being increased. Increased influx is probably due to activation of voltage-dependent Ca2+ channels, since it is abolished by verapamil. The results indicate that, at least under conditions of low Na+ electrochemical gradient, some pathway other than a Na+/Ca2+ exchange must operate in the plasma membrane to expel Ca2+.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells. Evidence for separate modes of operation of the transporter

(1) Unidirectional K+ (86Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K+ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 . 10(-3) M ouabain. (3) Measurements of net K+ loss and Na+ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K+ (Cl-) outward net flux uncoupled from net Na+ movement. Net K+ (Cl-) outward flux was half-maximally inhibited by 2 microM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K+ influx/efflux were essentially unchanged when compared to untreated controls.     

Activation of potassium ion transport in mitochondria by cadmium ion

Low levels of Cd2+ (1-5 microM) produce rapid swelling of mitochondria, which is respiration-dependent and uncoupler-sensitive. No cation requirement is apparent, since the swelling occurs in a medium containing only sucrose and the respiratory substrate. The swelling is inhibited by ruthenium red, suggesting that this effect of Cd2+ requires its entry into mitochondria. In medium containing 9 mM K+, addition of Cd2+ along with ruthenium red increases the rate of K+ influx threefold. In the presence of K+, Rb+ or Li+, but not of Na+, addition of Cd2+ produces first efflux of H+ into the medium followed by discharge of the pH gradient or uncoupling. Only the latter effect is inhibited by ruthenium red, showing that the efflux and influx of H+ are independent reactions. The H+ efflux appears to be an antiport response to the induced K+ entry. Its activation by Cd2+ is similar to the known effect of p-chloromercuriphenyl sulfonate. The H+ influx or uncoupling appears to result from binding of Cd2+ to some matrix-facing membrane site, perhaps the dithiol group on coupling factor B, and may relate to apparent permeability changes associated Cd2+-induced swelling.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.     

Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

Na+ for H+ exchange in rabbit erythrocytes

The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H+ efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 microM) and acetazolamide (1 mM). H+ efflux from acid loaded erythrocytes (pHi = 6.1) was measured in a K+ (145 mM) medium, pH0 = 8.0, in the presence and absence of 60 microM 5,N,N-dimethyl-amiloride (DMA). The H+ efflux rate in a K+-containing medium was 116.38 +/- 4.5 mmol/l cell X hr. Substitution of Nao+ for Ko+ strongly stimulated H+ efflux to 177.89 +/- 7.9 mmol/l cell X hr. The transtimulation of H+ efflux by Nao+ was completely abolished by DMA falling to values not different from controls with an ID50 of about 8.6 X 10(-7) M. The sequence of substrate selectivities for the external transport site were Na greater than greater than greater than Li greater than choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO3-. The DMA sensitive H+ efflux was a saturable function of [Na+]o, with an apparent Km and Vmax of about 14.75 +/- 1.99 mM and 85.37 +/- 7.68 mmol/l cell X hr, respectively. However, the Nao+-dependent and DMA-sensitive H+ efflux was sigmoidally activated by [H+]i, suggesting that Hi+ interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 +/- 3.0 mmol/l cell X hr) which was abolished when pHo was reduced to 6.0. The data is therefore consistent with the presence of a Na+/H+ exchange system in rabbit erythrocytes.     

Effect of Cd2+ on phosphate uptake by cadmium-resistant and cadmium-sensitive Staphylococcus aureus.

The effect of Cd2+ on phosphate (Pi) uptake was investigated in the growing cells of Cd(2+)-resistant Staphylococcus aureus 1781OR and Cd(2+)-sensitive S. aureus 17810S. Inhibitor and ionophore studies showed that 32Pi uptake in the two strains occurred via the Pi porter down pH gradient (delta pH) generated by the respiratory chain. Cd2+ inhibited 32Pi uptake in the cadmium-sensitive strain 1781OS at all concentrations used (10 microM-1 mM). In strain 1781OR, possessing the plasmid-coded Cd2+ efflux system, 10-100 microM Cd2+ did not inhibit 32Pi uptake. Even at 1 mM Cd2+, inhibition of 32Pi uptake in strain 1781OR was reversed when the external Cd2+ was chelated with cysteine and activity of Cd2+ efflux system was restored. Cd2+ efflux induced by cysteine was energized either by membrane potential (delta psi) or by delta pH, which indicated that electrochemical gradient of protons (delta mu H+) was required for this efflux.     

Mechanism of stimulation of Ca2+ uptake by miconazole and ethidium bromide in yeasts: role of vacuoles in Ca2+ detoxification

The antifungal antibiotic miconazole and the cationic dye ethidium bromide, both caused K+ efflux, membrane depolarization and intracellular acidification in the yeast Saccharomyces cerevisiae. Whereas miconazole inhibited the activity of the plasma membrane H+-ATPase, no such inhibition was observed using ethidium bromide at concentrations up to 600 microM. Low concentrations of both drugs caused marked stimulation of the energy dependent Ca2+ uptake. The extra Ca2+ taken up in the presence of the drugs was localized within the vacuoles, whereas K+ was lost mainly from the cytosolic pool. The ions Zn2+ and La3+ inhibited the effect of both drugs on the stimulation of Ca2+ uptake. The results indicated that both drugs caused an increase in the permeability of cell membranes to ions, leading to an increase in the influx of Ca2+ into the cytosol along its electrochemical gradient. Consequently, the concentration of Ca2+ within the cytosol increased and in turn led to the enhancement of Ca2+ uptake by the energy dependent vacuolar Ca2+ system, which functioned as a Ca2+ detoxification system.     

Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. Studies with L-carnosine and glycyl-L-proline

We examined the role of pH gradient and membrane potential in dipeptide transport in purified intestinal and renal brush-border membrane vesicles which were predominantly oriented right-side out. With an intravesicular pH of 7.5, changes in extravesicular pH significantly affected the transport of glycyl-L-proline and L-carnosine, and optimal dipeptide transport occurred at an extravesicular pH of 5.5-6.0 in both intestine and kidney. When the extravesicular pH was 5.5, glycyl-L-proline transport was accelerated 2-fold by the presence of an inward proton gradient. A valinomycin-induced K+ diffusion potential (interior-negative) stimulated glycyl-L-proline transport, and the stimulation was observed in the presence and absence of Na+. A carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced H+ diffusion potential (interior-positive) reduced dipeptide transport. It is suggested that glycyl-L-proline and proton(s) are cotransported in intestinal and renal brush-border membrane vesicles, and that the process results in a net transfer of positive charge.     

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO(3)(-) across their luminal membrane into a HCO(3)(-)-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO(3)(-) transport cannot be achieved by luminal Cl-/HCO(3)(-) exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO(3)(-) efflux, we have measured the intracellular potential (V(m)) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl- -25 mM HCO(3)(-) solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (approximately 5 mV) due to activation of electrogenic Na+-HCO(3)(-) cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately -50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO(3)(-) concentration to 125 mM caused a hyperpolarization (approximately 10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl- efflux across the luminal membrane is minimized by the depletion of intracellular Cl- and offset by the hyperpolarizing effects of Na+-HCO(3)(-) cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO(3)(-) that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO(3)(-) to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO(3)(-).     

Nigericin-induced death of an acidophilic bacterium.

At an external pH of 3.5, nigericin (which catalyses an electroneutral H+/K+ exchange) abolished the transmembrane proton gradient (delta pH) of Bacillus acidocaldarius, causing a rapid acidification of the cytoplasm from approximately pH 6.0 to pH 3.5. A pronounced loss of viability and fine-structural changes rapidly followed treatment with nigericin. A marked decline in respiration and an even more rapid decrease in cytoplasmic ATP were observed. Activity of at least one cytoplasmic enzyme decreased more slowly. There was no generalized loss in the integrity of the cytoplasmic membrane, as assayed by permeability to inulin or Na+ or by release of ultraviolet light-absorbing compounds. The loss of viability upon treatment with carbonyl cyanide m-chlorophenylhydrazone was similar to what observed with nigericin, so proton influx alone, rather than together with K+ efflux, was probably involved in the death of the organism. Moreover, acidification of the cytoplasm rather than abolition of the delta pH was the lethal event, since no loss of viability was observed when the delta pH was abolished by elevation of the external pH.     

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.     

Characteristics of tripeptide transport in human jejunal brush-border membrane vesicles

These studies are aimed at characterizing the transport of the tripeptide, glycylglycyl-L-proline (GlyGlyPro) across human jejunal brush-border membrane vesicles. GlyGlyPro (0.65 mM) was hydrolyzed by brush-border membrane vesicles with the extent of hydrolysis per mg protein being 23% at 0.5 min, 57% at 1 min and complete hydrolysis at 60 min. Treatment of the membrane vesicles with gel-complexed papain (to remove membrane peptidases) resulted in minimal hydrolysis of GlyGlyPro up to 10 min of incubation. Measurement of GlyGlyPro influx with papain-treated vesicles in the presence of increasing medium osmolarity showed that uptake occurred into an osmotically reactive intravesicular space. Transport of GlyGlyPro with normal and papain-treated membrane vesicles was similar in the presence of an inward Na+ or K+ gradient. No overshoot phenomenon was observed in the presence of an inward proton gradient (extravesicular pH 5.5; intravesicular pH 7.5). An interior negative membrane potential induced by a K+ diffusion potential in the presence of valinomycin stimulated the uptake of the peptide. The effect of increasing concentrations on initial rates of GlyGlyPro uptake revealed the presence of a saturable component as well as a diffusional component. Preloading the membrane vesicles with 20 mM glycylsarcosylsarcosine stimulated uptake by 4-fold. Uptake of GlyGlyPro was inhibited greater than 50% by dipeptides and tripeptides and less than 15% by free amino acids. These results indicate that GlyGlyPro uptake in jejunal brush-border membrane vesicles is not energized by a Na+ or proton gradient and that transport occurs by carrier-mediated and diffusional processes.     

A putative K(+)-selective channel in the plasma membrane of yeast that is blocked by micromolar concentrations of external divalent cations and is insensitive to tetraethylammonium

At pH 7, addition of glucose to an anaerobic suspension of non-metabolizing yeast cells causes a transient net efflux of K+ from the cells and a concomitant transient hyperpolarization of the plasma membrane (Van de Mortel, J.B.J., et al. (1988) Biochem. Biophys. Acta 936, 421-428). Both phenomena are effectively suppressed in the presence of low concentrations of polyvalent cations. The concentrations of Mn2+, Ca2+, Ba2+, Mg2+, Sr2+ and La3+ required for half-maximal suppression of the transient hyperpolarization are 10, 17, 20, 38, 47 and 5 microM, respectively. Subsequent addition of EDTA 90 s after that of Ca2+ immediately restores both K+ efflux and cellular uptake of the fluorescent membrane potential probe 2-(dimethylaminostyryl)-1-ethylpyridinium (DMP). This suggests that an interaction of polyvalent cations with an external binding site blocks the putative K(+)-selective channel. Opening of this channel is not blocked by 20 mM tetraethylammonium nor by 100 microM 3,4-diaminopyridine. It is argued that this glucose-induced K(+)-conductive pathway is not identical to the voltage-gated K+ channels identified until now in patch-clamp studies of the yeast plasma membrane.     

Amiloride-sensitive sodium transport in lamprey red blood cells: evidence for two distinct transport pathways

To determine Na+/H+ exchange in lamprey erythrocyte membranes, the cells were acidified to pH(i) 6.0 using the K+/H+ ionophore nigericin. Incubation of acidified erythrocytes in a NaCl medium at pH 8.0 caused a considerable rise in 22Na+ influx and H+ efflux during the first 1 min of exposure. In addition, exposure of acidified red cells to NaCl medium was associated with rapid elevation of intracellular Na+ content. The acid-induced changes in Na+ influx and H+ efflux were almost completely inhibited by amiloride and dimethylamiloride. In native lamprey erythrocytes, amiloride-sensitive Na+ influx progressively increased as the osmolality of incubation medium was increased by addition of 100, 200, or 300 mmol/l sucrose. Unexpectedly, the hypertonic stress induced a small, yet statistically significant decrease in intracellular Na+ content in these cells. The reduction in the cellular Na+ content increased with hypertonicity of the medium. The acid- and shrinkage-induced Na+ influxes were inhibited by both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in a dose-dependent manner. For both blockers, the half-maximal inhibitory values (IC50) were much greater for the shrinkage-induced (44 and 15 micromol/l for amiloride and EIPA, respectively) than for the acid-induced Na+ influx (5.1 and 3.3 micromol/l, respectively). The data obtained are the first demonstration of the presence of a Na+/H+ exchanger with high activity in acidified (pH(i) 6.0) lamprey red blood cells (on average, 512 +/- 56 mmol/l cells/h, n = 13). The amiloride-sensitive Na+ influxes produced by hypertonic cell shrinkage and acid load are likely to be mediated by distinct ion transporters in these cells.