Volume-dependent regulation of ion carriers in human and rat erythrocytes: role of cytoskeleton and protein phosphorylation

This study examines the effect of heat-induced cytoskeleton transitions and phosphoprotein phosphatase inhibitors on the activity of shrinkage-induced Na+, K+, 2Cl- cotransport and Na+/H+ exchange in rat erythrocytes and swelling-induced K+, Cl- cotransport in human and rat blood cells. Preincubation of human and rat erythrocytes at 49 degrees C drastically activated K+, Cl- cotransport and completely (rat) or partly (human) abolished its volume-dependent regulation. The same procedure did not affect basal activity of Na+, K+, 2Cl- cotransport but completely abolished its activation by shrinkage thus suggesting the involvement of a thermosensitive element of cytoskeleton network in the volume-dependent regulation of cotransporters. Both the shrinkage- and electrochemical proton gradient-induced Na+/H+ exchange was inhibited by the heat treatment to the same extent (50-70%), thus indicating the different signaling pathways involved in the activation of Na+, K+, 2Cl- cotransport and Na+/H+ exchange by cell shrinkage. This suggestion is in accordance with data on the different kinetics of volume-dependent activation and inactivation of these carriers as well as on their sensitivity to medium osmolality. Both swelling- and heat-induced increments of K+, Cl- cotransport activity were diminished by inhibitors of phosphoprotein phosphatases (okadaic acid and calyculin). In rat erythrocytes these compounds potentiate shrinkage-induced Na+/H+ exchange. On the contrary, neither basal nor shrinkage-induced Na+, K+, 2Cl- cotransport was affected by these compounds. Our results indicate a key role of cytoskeleton network in volume-dependent activation of K+, Cl- and Na+, K+, 2Cl- cotransport and the involvement of protein phosphorylation-dephosphorylation cycle in regulation of the activity of K+, Cl- cotransport and Na+/H+ exchange.     

Kinetic characteristics of 22Na transport in human and rat erythrocytes during cytoplasm acidification and cell compression

Under normal conditions (pH0 = 7.4, pHi = 7.1-7.2) amiloride, a Na+/H+ exchange inhibitor, does not influence Na+ intake by human and rat erythrocytes. Acidification of the cytoplasm (pHi approximately 6.4) is accompanied by the acceleration of 22Na intake, which is decreased after addition of 1 mM amiloride (by 50 and 80%, respectively). The Ki value of amiloride for human and rat erythrocytes is 30 and 250 microM, respectively. In rat erythrocytes the dependence of the rate of the delta pH-induced incorporation of 22Na on Na+ concentration is described by a saturation curve (K0.5 for Na0+ is approximately 40 mM), whereas in human erythrocytes it obeys the diffusion kinetics. These results suggest that the Na+/H+ exchange takes place in rat erythrocytes, but is absent in human erythrocytes. In rat erythrocytes the Na+/H+ exchange can be induced by cell compression which can be caused either by decreasing the KCl content (after addition of valinomycin) or by increasing the osmolarity of the medium (in the presence of sucrose). The rate of Na+/H+ exchange induced by cell compression is increased by 60-70% after addition of protein kinases A and C activators. No effect of intracellular Ca2+ on the rate of the Na+/H+ exchange in rat erythrocytes is observed.     

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 U/ml thrombin, whereas increasing pHi by NH4Cl enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2.     

A phorbol ester and 1-oleoyl-2-acetylglycerol induce Na+/H+ exchange in human platelets

This study aimed at investigating the mechanisms by which stimulation of human platelets results in activation of Na+/H+ exchange. Platelets were suspended in a slightly buffered medium and the stimulus-induced, amiloride-sensitive H+ release, reflecting Na+/H+ exchange, was estimated from changes in the medium pH. H+ release could be evoked by thrombin and by activators of protein kinase C such as 1-oleoyl-2-acetylglycerol (OAG) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the thrombin-and the OAG-induced Na+/H+ exchange could be blocked by trifluoperazine, a protein kinase C inhibitor. The thrombin-induced H+ release was also sensitive to increased intracellular cAMP levels, probably due to inhibition of phospholipase C activation, whereas the OAG-induced activation of Na+/H+ exchange was unaffected. Our data suggest that activation of Na+/H+ exchange is mediated by protein kinase C.     

Kinetic properties of sodium transport pathways in the river lamprey Lampetra fluviatilis erythrocytes

To activate Na+/H+ exchange, intracellular pH (pHi) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 to 8 using nigericin. The Na+/H+ exchanger activity was estimated from the values of amiloride-sensitive components of Na+ (22Na) inflow or of H+ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na+ and H+ transport on pHi value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9-10 mmol/l cells/min, and the H+ concentration producing the exchanger 50% activation amounted to 0.6-1.0 microM. Stimulation of H+ outcome from acidified erythrocytes (pHi 5.9) with increase of H+ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration of Na+: for the semimaximal stimulation of H+ outcome amounted to 19 mM. The obtained results indicate the presence in lamprey erythrocytes of only one binding site for H+ from the cytoplasm side and the presence of positive cooperativity in Na+ binding from the extracellular side of the Na+/H+ exchanger. Its efflux from cells in the Na+ -free medium did not change at a 10-fold increase of H+ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na+/H+ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na+ inflow on its concentration in the medium is described by Hill equation with n 1.5. The Na+ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm the concept of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Na+/H+ exchange in erythrocytes of spontaneously hypertensive rats: a study in F2 SHR x WKY hybrids.

The rate of proton gradient-induced Na+/H+ exchange in the erythrocytes of SHR was increased by 50-60% as compared to WKY animals. No significant correlation between Na+/H+ exchange and blood pressure was revealed in F2 hybrids of SHR and WKY rats. Na+/H+ exchange rate in the erythrocytes of F2 SHR x WKY hybrids was twice as high as in SHR and three times higher than in WKY rats.     

Vasopressin elevation of Na+/H+ exchange is inhibited by genistein in human blood platelets.

The regulation of intracellular Na+ and pHi in human blood platelets is known to be controlled by the function of the Na+/H+ exchanger. The phosphorylation state of the Na+/H+ exchanger which determines the exchanger activity in human blood platelets is regulated by the activities of protein kinases and protein phosphatases. Observations in this study indicate that arginine vasopressin (AVP) that interacts with a V1 receptor, activates the Na+/H+ exchange in human blood platelets through a genistein-inhibited mechanism. The AVP-activated Na+/H+ exchange is probably not regulated by protein kinase C (PKC), since this activation is not inhibited by staurosporine. The multiple ways in which platelet Na+/H+ exchange can be modulated may indicate the critical role played by this exchanger in the homeostasis control of pHi in human blood platelets.     

Calcium-dependent intracellular acidification dominates the pH response to mitogen in human T cells

The effects of a phorol ester and a mitogenic lectin on the intracellular pH (pHi) of human T lymphocytes was investigated. In contrast to the cytoplasmic alkalinization induced by 12-0-tetradecanoylphorbol-13-acetate, an acidification was recorded in cells treated with phytohemagglutinin. This decrease in pHi was magnified in Na+-free medium or in the presence of amiloride analogues, suggesting that activation of Na+/H+ exchange partially counteracts the phytohemagglutinin-induced acidification. The decrease in pHi was dependent on a sustained increase in cytosolic free Ca2+ and could be mimicked by addition of the divalent cation ionophore, ionomycin. The elevation of cytosolic free Ca2+ leads to metabolic H+ (equivalent) generation with consequent cytoplasmic acidification, which in human T cells predominates over the concurrent activation of the Na+/H+ antiport. These findings argue against the notion that activation of Na+/H+ exchange is a signal for the initiation of proliferation.     

Effects of ultraviolet irradiation on the Na/H exchange rate in erythrocytes of healthy subjects and patients with atherosclerosis of lower limb arteries]

Na+/H+ exchange rate in erythrocytes of patients with atherosclerosis of lower limb arteries is less than in erythrocytes of healthy subjects. Ultraviolet exposure of the patients blood in vitro activates Na/H exchange in erythrocytes.     

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.     

Thrombin-induced platelet aggregation is affected by external Na+ independently of the Na+/H+ exchange

Thrombin affects blood platelets by activation of Na+/H+ exchange and induction of aggregation, but the relationship between these effects is under debate. The present study attempts to clarify whether the activation of the exchanger activity is required for platelet aggregation. In apparent support of such a requirement, thrombin-induced aggregation is higher in Na+ medium than in N-methylglucamine+ medium and is inhibited by sphingosine, an inhibitor of protein kinase C known to regulate the Na+/H+ exchanger. However, the inhibition of aggregation by sphingosine occurs in both Na+-containing and Na+-free media, the aggregation is identical in Na+ and K+-containing media, and is not inhibited by 5-N-(3-aminophenyl)amiloride, at a concentration 10-fold higher than its Ki for platelet Na+/H+ exchange. Furthermore, at low concentration (0.005 U/ml) thrombin induces aggregation but does not activate the exchange. It is concluded that the activation of Na+/H+ exchange is not required for thrombin-induced platelet aggregation and that the apparent augmentation of aggregation by Na+ is due to an inhibitory effect of N-methylglucamine+.     

Elevated Na+/H+ exchange in erythrocytes of patients with hypertension

The kinetics of proton efflux from erythrocytes with acidified cytoplasm (pHi 6.4) in a medium with pH 8.0 has been studied. The participation of the anion exchanger in this process was blocked by a stilbene disulfonic acid derivative. It was shown that the rate of Na+/H+ exchange (amiloride-inhibited component of proton efflux) is increased 2 fold. The addition of protein kinase C activator (1 microM of TPA) results in the increase of the rate of Na+/H+ exchange by 4 fold.     

Modulation of the voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa through membrane potential changes induced by the egg peptide speract

Sea urchin sperm motility can be activated by alkalinization of the internal pH, and previous studies have shown that the internal pH can be regulated by a voltage-sensitive Na+/H+ exchanger present in the flagellar plasma membrane. In this study, the effects of speract, a peptide purified from egg conditioned media, on the Na+/H+ exchange were investigated. Evidence presented indicates that speract activates K+ channels in the flagellar membrane and modulates the Na+/H+ exchange activity through resultant changes in membrane potential. In the presence of tetraphenylphosphonium, a lipophilic ion, or high external Na+, the isolated flagella were depolarized, and Na+/H+ exchanger was inhibited. Speract and valinomycin, a K+ ionophore, were able to reactivate 22Na+ uptake, H+ efflux, and alkalinization of intraflagellar pH under either of the depolarizing conditions. Membrane potential measurements using 3,3'-dipropylthiodicarbocyanide iodide indicated repolarization by either speract or valinomycin. The speract-induced voltage changes did not require Na+ but were sensitive to [K+]. Thus, speract induced a slight depolarization in Na+-free seawater with 10 mM K+ but a hyperpolarization with 2 mM K+. Further support for the activation of K+ channels in the flagella was the 2-5-fold stimulation of K+ efflux induced by speract as measured with a K+ electrode. The ionic selectivity of the speract-activated channel assessed by voltage measurements was K+ greater than Rb+ greater than Cs+. The half-maximally effective concentration of speract was about 0.2 nM. That the H+ and K+ efflux in response to peptide was receptor-mediated was confirmed by the use of speract or resact on intact sea urchin spermatozoa, where the peptides were found to stimulate K+ efflux and to reverse the tetraphenylphosphonium inhibition on H+ efflux only in the homologous spermatozoa. Modulation of the voltage-sensitive Na+/H+ exchange by egg peptides, therefore, appears to be indirect and is coupled through its action on membrane potential.     

Catecholamine-induced transport systems in trout erythrocyte. Na+/H+ countertransport or NaCl cotransport?

It has previously been shown (Baroin, A., F. Garcia-Romeu, T. Lamarre, and R. Motais. 1984a, b. Journal of Physiology. 350:137, 356:21; Mahé, 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 cAMP-dependent, 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 Cl- could therefore result from the parallel and simultaneous exchange of Na+out for H+in (via the cAMP-dependent Na+/H+ antiporter) and Cl- out for HCO3- in (via the anion exchange system located in band 3 protein). However, in view of the following arguments, it had been proposed that NaCl uptake does not proceed by the double-exchanger system but via an NaCl cotransport: (a) Na+ entry requires Cl- as anion (in NO3- medium, the Na uptake is strongly inhibited, whereas NO3- is an extremely effective substitute for Cl- in the anion exchange system); (b) Na uptake is not significantly affected by the presence of HCO3- in the suspension medium despite the fact that in red cells, Cl-/HCO3- exchange occurs more readily than the exchanges of Cl- for basic equivalents in a theoretically CO2-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, NO3- 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 CO2-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.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a role of NA+/H+ exchange in platelets activated with calcium-ionophore A 23187

We have investigated the release of protons from human platelets and platelet aggregation induced by the calcium ionophore, A 23187. Addition of the ionophore to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.2 mM amiloride, a potent inhibitor of Na+/H+ countertransport, the amount of protons liberated was decreased by 50% and was further reduced to about 10% by 1 mM amiloride. Similar inhibition of H+-release was observed after decreasing Na+ in the incubation medium. Both results suggest that increasing internal Ca2+ by the ionophore induces Na+/H+ exchange in human platelets. Platelet aggregation could be induced by adding the ionophore to the platelet suspension. This aggregation was inhibited by amiloride, at least when induced by low ionophore concentrations. The results suggest that stimulation of Na+/H+ exchange, and the concomitant increase in intraplatelet pH, are important mechanisms in platelet activation.     

Control of the sodium-proton antiporter in human placental microvillous membranes by transport substrates

The microvillous membrane of the human placental syncytiotrophoblast contains an amiloride-inhibitable, electroneutral, Na+/H+ antiporter. The kinetic characteristics of this antiporter have been investigated to determine its response to alterations in intracellular and extracellular H+ and Na+ concentrations. Antiporter activity was measured using a pH-sensitive fluorescent probe entrapped in placental microvillous vesicles. We report here on the kinetic characterization of the antiporter, a transporter which displays simple, saturable kinetics for the external site but complex kinetics at the internal site. Measurement of the external Na+ and H+ dependences demonstrated that Na+ and H+ compete for binding to a single external binding site which displays saturation kinetics. The external Km determined for Na+ was 8.2 +/- 4.0 mM, while the external pK was 7.29 +/- 0.02. The Vmax calculated from these experiments was 0.57 +/- 0.10 nequiv./s per mg membrane protein. By contrast, the internal dependences for both Na+ and H+ showed significant deviations from simple linear kinetics. Decreasing internal pH to 6.0 stimulated Na+/H+ exchange to a greater degree than predicted for a single-site saturable binding model, in a manner which suggested allosteric activation. At the other extreme, Na+/H+ exchange ceased above an internal pH of 7.1, despite the existence of an inwardly-directed Na+ gradient. Increasing intracellular Na+ caused inhibition of Na+/H+ exchange but the intracellular Na+ dependence showed that the effect is due to a mechanism more complex than simple, competitive inhibition between Na+ and H+. These results show that the microvillous Na+/H+ antiporter is insensitive to changes in extracellular Na+ and H+ concentrations in the physiological range. Changes in intracellular Na+ and H+ however are likely to cause marked changes in antiporter activity. These characteristics suggest that cellular Na+ and H+ concentrations are tightly controlled in the placental syncytiotrophoblast and that the Na+/H+ antiporter may play a significant role in their regulation.     

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.     

Effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TTPA) upon membrane ionic exchanges in sea urchin eggs

The effect of TPA (12-O-tetradecanoylphorbol-13-acetate) upon ionic exchanges was investigated in eggs of the sea urchin Arbacia lixula. Ouabain-sensitive 86Rb uptake and amiloride-sensitive 24Na influx were dramatically stimulated after TPA addition, indicating an enhancement of total ionic permeabilities. Stimulation by TPA of both Na+/H+ and Na+/K+ exchanges was canceled by amiloride, suggesting that activation of protein kinase C elicits, via Na+/H+ activity, stimulation of the sodium pump. However, TPA did not stimulate sodium pump activity and Na+/H+ exchange at the same rate as fertilization, probably because of an absence of calcium-dependent events. Further fertilization of TPA-pretreated eggs triggered an enhancement of sodium pump activity when the TPA treatment duration did not exceed 10 min. It is suggested that TPA activates preexisting transporting mechanisms in plasma membranes of unfertilized eggs (Na+ pump, Na+/H+ exchange) without eliciting corresponding regulatory mechanisms (Na+ stat, pH stat).     

Low density lipoproteins inhibit the Na+/H+ antiport in human platelets via activation of p38MAP kinase

Low density lipoproteins (LDL) inhibit the Na+/H+ antiport and thereby sensitize platelet towards agonist. However, mechanisms underlying the suppressing effect of LDL on Na+/H+ exchange are unclear. We here show that the lowering of intracellular pH and the suppression of the sodium propionate-induced Na+/H+ exchange in the presence of LDL are abolished by SKF86002, a selective inhibitor of p38MAP kinase (p38MAPK). The inhibitory effect of LDL on Na+/H+ exchange was mimicked by H2O2, which directly activates p38MAPK. Exposure of platelets to LDL or H2O2 led to phosphorylation of p38MAPK, its upstream regulator MAP kinase kinase 3/6 (MKK 3/6), and its downstream target heat shock protein 27 (HSP27), and this effect was abrogated in SKF86002-pretreated platelets. In addition, both LDL and H2O2 produced the SKF86002-sensitive phosphorylation of an oligopeptide encompassing p38MAPK phosphorylation sequence derived from NHE-1, a major Na+/H+ exchanger in platelets. We further show that the sensitizing effects of LDL on the thrombin-induced platelet activation, as reflected by aggregation and granule secretion, are abolished in cells pretreated with SKF86002. We conclude that activation of p38MAPK is required for the inhibitory effect of LDL on Na+/H+ antiport and thereby for LDL-dependent sensitization in human platelets.     

The Na+-dependent regulation of the internal pH in chick skeletal muscle cells. The role of the Na+/H+ exchange system and its dependence on internal pH.

The internal pH (pHi) of chick muscle cells is determined by the transmembrane Na+ gradient. Li+, but not K+, Rb+ or Cs+, can substitute for Na+ for regulating the internal pH of chick muscle cells. Pharmacological evidence using amiloride and amiloride analogs has shown that the Na+/H+ exchange system is the membrane mechanism that couples the pHi to the transmembrane Na+ gradient. The pHi dependence of the amiloride-sensitive Na+/H+ exchange mechanism was defined. Internal H+ interacts cooperatively with the Na+/H+ exchange system, in contrast with external H+, thus indicating an asymmetrical behaviour of this exchanger. The half-maximum effect for the activation by the internal H+ of the Na+ transporting activity of the amiloride-sensitive Na+/H+ exchange was observed at pH 7.4. The Hill coefficient of the H+ concentration dependence is higher than 3. Insulin was shown to have no effect on the pHi of chick muscle cells.