pH regulation of an egg cortex tyrosine kinase

Fertilization of the echinoderm egg is known to result in the phosphorylation, on tyrosine, of a high-molecular-weight cortical protein (HMWCP) localized in the egg cortex. Studies using various parthenogenic agents indicate that this phosphorylation event occurs in response to the alkaline shift in cytoplasmic pHi which normally occurs 1 to 2 min after fertilization. In the present study, the purified egg cell surface complex was used as in vitro system to determine whether a small alkaline shift in pH, such as occurs upon fertilization, could stimulate the activity of the egg cortex-associated tyrosine kinase toward endogenous protein substrates. The results demonstrated that the cell surface complex is highly enriched in a tyrosine kinase activity which accounts for the majority of the protein kinase activity in this preparation. The activity of this tyrosine kinase toward the HMWCP and other cortical proteins was highly dependent on pH over the range pH 6.8 to 7.3. This indicates that the fertilization-associated change in cytoplasmic pH would be sufficient to trigger increased tyrosine phosphorylation of the high-molecular-weight cortical protein in vivo. The regulation of tyrosine phosphorylation by small changes in pH represents a novel control mechanism in which a tyrosine protein kinase may act as a pH-sensitive transducer.     

22Na+ fluxes in thymic lymphocytes. II. Amiloride-sensitive Na+/H+ exchange pathway; reversibility of transport and asymmetry of the modifier site

22Na+ flux and cytoplasmic pH (pHi) determinations were used to study the reversibility, symmetry, and mechanism of activation of the Na+/H+ exchange system in rat thymic lymphocytes. In acid-loaded cells, the antiport can be detected as an Na+-induced, amiloride-sensitive alkalinization. At pHi greater than or equal to 7.0, amiloride- sensitive net H+ fluxes are not detectable. To investigate whether at this pHi the transporter is operative in a different mode, e.g., Na+/Na+ exchange, 22Na+ uptake was measured as a function of pHi. The results indicate that the antiport is relatively inactive at pHi greater than or equal to 7.0. Comparison of the rates of H+ efflux (or equivalent OH- uptake) and Na+ uptake indicate that Na+/Na+ countertransport through this system is negligible at all values of pHi and that the Na+:H+ stoichiometry is 1:1. Measurements of pHi in Na+- loaded cells suspended in Na+-free medium revealed an amiloride- sensitive cytoplasmic acidification, which is indicative of exchange of internal Na+ for external H+. The symmetry of the system was analyzed by measuring the effect of extracellular pH (pHo) on Na+ efflux. Unlike cytoplasmic acidification, lowering pHo failed to activate the antiport. The results indicate that the amiloride-sensitive Na+/H+ exchanger is reversible but asymmetric. The system is virtually inactive at pHi greater than or equal to 7.0 but can be activated by protonation of a modifier site on the cytoplasmic surface. Activation can also occur by depletion of cellular Na+. It is proposed that Na+ may also interact with the modifier site, stabilizing the unprotonated (inactive) form.     

Ligand-affected shift of Na+/H+ exchange pHi set point in human blood platelets, rapidly revealed by a novel approach.

The Na+/H+ exchange time-course of BCECF-loaded human platelets, suspended in isotonic media containing NaCl and sodium propionate and activated by intracellular acidification, was measured spectrofluorimetrically. Sequential alkalinization rates decline exponentially as a function of the changing intracellular pH (pHi) and its linear expression (log rate vs. pHi) extrapolates reproducibly to the pHi set point for the Na+/H+ exchange activation. The set point of control platelets (7.28 +/- 0.01) is shifted rapidly (discernibly less than or equal to 30 s) and markedly to alkaline pHi (7.62 +/- 0.03) by PMA, that activates protein kinase C and is shifted to acidic pHi (7.05 +/- 0.01) by staurosporine, which inhibits protein kinases. The addition of 5-N-(3-aminophenyl)amiloride reveals that the alkalinization measured is predominantly Na+/H+ exchange with only a minute contribution (delta pHi = 0.012 +/- 0.002 in 1 min) of an acid loading component, at pHi greater than 7.2. The results support recent studies concluding that the set point indeed reflects the phosphorylation state of the Na+/H+ exchanger.     

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.     

Okadaic acid, a phosphatase inhibitor, induces activation and phosphorylation of the Na+/H+ antiport.

We determined the effect of okadaic acid (OA), a potent phosphoprotein phosphatase inhibitor, on the intracellular pH (pHi) of rat thymic lymphocytes and human bladder carcinoma cells. OA induced a rapid and sustained cytosolic alkalinization. This pHi increase was Na(+)-dependent and was inhibited by 5,N-disubstituted analogs of amiloride, indicating mediation by the Na+/H+ antiport. As described for other stimulants, such as mitogens and hypertonic challenge, activation of the antiport by OA is attributable to an upward shift in its pHi dependence. Accordingly, the alkalinization produced by the phosphatase inhibitor was not additive with that induced osmotically. Activation of the antiport by OA was accompanied by a marked increase in phosphoprotein accumulation, revealing the presence of active protein kinases in otherwise unstimulated cells. We considered the possibility that phosphorylation of the antiport itself or of an ancillary protein is responsible for activation of Na+/H+ exchange. Consistent with this notion, the alkalinization induced by OA was absent in ATP depleted cells. More importantly, immunoprecipitation experiments demonstrated increased phosphorylation of the antiport following treatment with OA. We conclude that, upon inhibition of phosphoprotein phosphatase activity, constitutively active kinases induce the activation of Na+/H+ exchange, possibly by direct phosphorylation of the antiport.     

The Na+/H+ antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process

The properties of the Na+/H+ exchange system have been studied with 22Na+ uptake techniques at two stages of muscle development: proliferating myoblasts and differentiated myotubes. The characteristics of the interactions of the exchanger with external H+, with external Na+, and with amiloride or its more potent analogs are the same at both stages of development. Differences between the two stages of development concern: (i) the internal pH (pHi) dependence of the Na+/H+ exchanger, and (ii) the activation of the Na+/H+ exchanger by serum and phorbol ester which is observed in myoblasts but not in myotubes. Properties of the Na+/H+ exchanger in myoblasts after serum activation seem to be identical to those observed in myotubes with or without serum as if myotube formation stabilized a fully activated state of the exchanger. The activation of the myoblast Na+/H+ exchange system by serum is due to a shift of the pHi dependence towards alkaline pHi values and to an increase in the maximal activity of the Na+/H+ exchange system at acidic pH. Phorbol esters which are well-known activators of protein kinase C can only partially mimic the effects of serum on the Na+/H+ exchanger: they produce a shift of the pH dependence, but they do not increase the maximal activity at acidic pH.     

Cytoplasmic [Ca2+] and intracellular pH in lymphocytes. Role of membrane potential and volume-activated Na+/H+ exchange

The effect of elevating cytoplasmic Ca2+ [( Ca2+]i) on the intracellular pH (pHi) of thymic lymphocytes was investigated. In Na+-containing media, treatment of the cells with ionomycin, a divalent cation ionophore, induced a moderate cytoplasmic alkalinization. In the presence of amiloride or in Na+-free media, an acidification was observed. This acidification is at least partly due to H+ (equivalent) uptake in response to membrane hyperpolarization since: it was enhanced by pretreatment with conductive protonophores, it could be mimicked by valinomycin, and it was decreased by depolarization with K+ or gramicidin. In addition, activation of metabolic H+ production also contributes to the acidification. The alkalinization is due to Na+/H+ exchange inasmuch as it is Na+ dependent, amiloride sensitive, and accompanied by H+ efflux and net Na+ gain. A shift in the pHi dependence underlies the activation of the antiport. The effect of [Ca2+]i on Na+/H+ exchange was not associated with redistribution of protein kinase C and was also observed in cells previously depleted of this enzyme. Treatment with ionomycin induced significant cell shrinking. Prevention of shrinking largely eliminated the activation of the antiport. Moreover, a comparable shrinking produced by hypertonic media also activated the antiport. It is concluded that stimulation of Na+/H+ exchange by elevation of [Ca2+]i is due, at least in part, to cell shrinking and does not require stimulation of protein kinase C.     

Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes

The activity of the Na+/H+ exchange system of rat thymic lymphocytes was determined by means of intracellular (pHi) and extracellular pH (pH0) measurements. In isotonic media, the antiport is virtually quiescent at physiological pHi (7.0-7.1), but is greatly activated by cytoplasmic acidification. At normal pHi, the antiport can also be activated by osmotic shrinking. Osmotic activation occurs after a delay of 20-30 s and is reversed several minutes after iso-osmolarity is restored. The mechanism of activation was analyzed by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. The affinities of the external substrate site for Na+ and H+ are not altered in shrunken cells. In contrast, the Hi+ sensitivity of the antiport (which is largely dictated by an allosteric modifier site) was increased, which accounted for the activation. The concentration of free cytoplasmic Ca2+ [( Ca2+]i) increased after osmotic shrinking. This increase was dependent on the presence of extracellular Ca2+ and Na+ and was blocked by inhibitors of Na+/H+ exchange, which suggests that it is a consequence, rather than the cause, of the activation of the antiport. It is concluded that the shift in the pHi dependence of the modifier site of the Na+/H+ antiport is the primary event underlying the regulatory volume increase that follows osmotic shrinkage.     

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.     

Photosensitized irreversible binding of estrone to protein via a hydroperoxide intermediate: an explanation of (photo-) allergic side-effects of estrogens

Recent studies have established that polypeptide growth factors cause an elevation of the cytoplasmic pH (pHi) in cultured mammalian cells by stimulating Na+/H+ exchange. We show that vanadate, previously found to act as a mitogen for a number of cells, reversibly activates Na+/H+ exchange at micromolar concentrations in A431 cells, leading to a large increase of pHi. The stimulation of Na+/H+ exchange by vanadate is not due to inhibition of the Na+/K+ ATPase and is unrelated to possible effects of vanadate on cAMP levels. Elevation of pHi by vanadate and by epidermal growth factor (EGF) both display similar kinetics, and both EGF and vanadate stimulate the rate of pHi recovery following an acute acid load, suggesting that vanadate stimulates Na+/H+ exchange by a mechanism similar to that of polypeptide growth factor stimulation. Thus, stimulation of Na+/H+ exchange may be a common property not only of polypeptide growth factors but also of other, chemically unrelated mitogens.     

Modulation of Na+/H+ exchange and intracellular pH by protein kinase C and protein phosphatase in blood platelets.

Phosphorylation of the Na+/H+ exchanger in human platelets is apparently controlled by the balancing activities of protein kinase C (PKC) and protein phosphatase (PP). To explore cellular expressions of these activities, we have examined the impact of modulation of PKC and PP on Na+/H+ exchange activity, its pHi set point and intracellular pH (pHi). These parameters were followed spectrofluorimetrically in BCECF-loaded platelets. Phorbol 12-myristate 13-acetate (PMA) and dihexanoylglycerol (DHG), which stimulate PKC, and okadaic acid, which inhibits PP 1 and 2A, elevate the measured parameters in concert, while staurosporine, which inhibits protein kinases, had opposite effects. The stimulatory and inhibitory effects are similarly very rapid, being discerned within seconds. It is concluded that: (a) phosphorylation of the Na+/H+ exchanger is the common origin of the diverse effects of PMA, DHG, okadaic acid and staurosporine, (b) Na+/H+ exchange properties are tightly regulated by phosphorylation and dephosphorylation, and (c) the exchanger plays a major role in pHi regulation in platelets.     

Cytoplasmic pH regulation in thymic lymphocytes by an amiloride- sensitive Na+/H+ antiport

The mechanisms underlying cytoplasmic pH (pHi) regulation in rat thymic lymphocytes were studied using trapped fluorescein derivatives as pHi indicators. Cells that were acid-loaded with nigericin in choline+ media recovered normal pHi upon addition of extracellular Na+ (Nao+). The cytoplasmic alkalinization was accompanied by medium acidification and an increase in cellular Na+ content and was probably mediated by a Nao+/Hi+ antiport. At normal [Na+]i, Nao+/Hi+ exchange was undetectable at pHi greater than or equal to 6.9 but was markedly stimulated by internal acidification. Absolute rates of H+ efflux could be calculated from the Nao+-induced delta pHi using a buffering capacity of 25 mmol X liter-1 X pH-1, measured by titration of intact cells with NH4+. At pHi = 6.3, pHo = 7.2, and [Na+]o = 140 mM, H+ extrusion reached 10 mmol X liter-1 X min-1. Nao+/Hi+ exchange was stimulated by internal Na+ depletion and inhibited by lowering pHo and by addition of amiloride (apparent Ki = 2.5 microM). Inhibition by amiloride was competitive with respect to Nao+. Hi+ could also exchange for Lio+, but not for K+, Rb+, Cs+, or choline+. Nao+/Hi+ countertransport has an apparent 1:1 stoichiometry and is electrically silent. However, a small secondary hyperpolarization follows recovery from acid-loading in Na+ media. This hyperpolarization is amiloride- and ouabain-sensitive and probably reflects activation of the electrogenic Na+-K+ pump. At normal Nai+ values, the Nao+/Hi+ antiport of thymocytes is ideally suited for the regulation of pHi. The system can also restore [Na+]i in Na+-depleted cells. In this instance the exchanger, in combination with the considerable cytoplasmic buffering power, will operate as a [Na+]i- regulatory mechanism.     

The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock

The properties of the Na+/H+ exchange system in the glial cell lines C6 and NN were studied from 22Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethyl-carboxyfluorescein. In both cell types, the Na+/H+ exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride greater than amiloride greater than benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H+. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in C6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines. Increasing the osmolarity of the external medium with mannitol produces an activation of the exchanger in C6 cells, which leads to a cell alkalinization. Mannitol action on 22Na+ uptake and the pHi were not observed in the presence of amiloride derivatives. Mannitol produces a modification of the properties of interaction of the antiport with both internal and external H+. It shifts the pHi dependence of the system to the alkaline range and the external pH (pHo) dependence to the acidic range. It also suppresses the interdependence of pHi and pHo controls of the exchanger's activity. NN cells that possess an Na+/H+ exchange system with different properties do not respond to mannitol by an increased activity of the Na+/H+ exchanger. The action of mannitol on C6 cells is unlikely to be mediated by an activation of protein kinase C.     

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.     

The Na+/H+ exchanger is constitutively activated in P19 embryonal carcinoma cells, but not in a differentiated derivative. Responsiveness to growth factors and other stimuli

We have examined the functional properties and growth factor responsiveness of the plasma membrane Na+/H+ exchanger in pluripotent P19 embryonal carcinoma (EC) cells and in a differentiated mesodermal derivative (MES-1) by analyzing the recovery of cytoplasmic pH (pHi) from an acute acid load under bicarbonate-free conditions. In the absence of exogenous growth factors, the mean steady-state pHi of undifferentiated P19 cells (7.49 +/- 0.03) is 0.55 unit higher than the value of differentiated MES-1 cells (6.94 +/- 0.01). In both cell types, recovery of pHi from an NH+4-induced acid load follows an exponential time course and is entirely mediated by the amiloride-sensitive Na+/H+ exchanger in the plasma membrane. Kinetic analysis indicates that the higher steady-state pHi in P19 EC cells is due to an alkaline shift in the pHi sensitivity of the Na+/H+ exchange rate, as compared to that in MES-1 cells. The Na+/H+ exchanger of MES-1 cells is responsive to epidermal growth factor, platelet-derived growth factor, serum, phorbol esters, and diacylglycerol, as shown by a rapid amiloride-sensitive rise in pHi of 0.15-0.35 unit. This mitogen-induced alkalinization is attributable to an alteration in the pHi sensitivity of the exchanger. In contrast, the Na+/H+ exchanger of P19 EC cells fails to respond to any of these stimuli. Similarly, hypertonic medium rapidly activates the Na+/H+ exchanger in MES-1, but not in P19 EC cells. We conclude that the Na+/H+ exchanger in undifferentiated P19 EC stem cells is maintained in a fully activated state which is unaffected by extracellular stimuli, as if signal pathways normally involved in growth factor action are constitutively operative.     

Mitogen-independent activation of Na+/H+ exchange in human epidermoid carcinoma A431 cells: regulation by medium osmolarity

Previous studies have documented the activation of Na+/H+ exchange in A431 cells by the addition of epidermal growth factor or serum (Rothenberg et al., 1983b). Here we show that exposure of A4 31 cells to medium of increased osmolarity also leads to activation of Na+/H+ exchange and to an increase in intracellular pH (pHi), which under a variety of conditions displays similar kinetics to that observed upon addition of mitogens to the cells. Measurements of cell volume using the 3-0-methylglucose equilibration technique clearly show that mitogens do not activate Na+/H+ exchange by an osmotic mechanism (i.e., a decrease in cell volume). In fact, mitogens can induce further intracellular alkalinization if added to cells which have been shrunken in hypertonic medium. Activation of the Na+/H+ antiport does not lead to an obligatory change in pHi. Addition of epidermal growth factor of hypertonic solution to A431 cells in bicarbonate buffer activates Na+/H+ exchange without a concomitant increase in pHi. Under these conditions the increased proton efflux via Na+/H+ exchange must therefore be compensated by other mechanisms that control cytoplasmic pH.     

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.     

Na+/H+ exchange and the regulation of intracellular pH in polymorphonuclear leukocytes

1. Regulation of the cytoplasmic pH(pHi) was studied in quiescent and activated human neutrophils. Acid-loaded unstimulated cells regulate pHi by activating an electroneutral Na+/H+ exchange. 2. When activated, neutrophils undergo a biphasic change in pHi: an acidification followed by an alkalinization. The latter is due to stimulation of the Na+/H+ antiport. 3. The acidification, which is magnified in Na+-free or amiloride-containing media, is associated with net H+ efflux from the cells. 4. A good correlation exists between cytoplasmic acidification and superoxide generation: inhibition of the latter by adenosine, deoxyglucose or pertussis toxin also inhibits the pHi changes. 5. Moreover, acidification is absent in chronic granulomatous disease patients, which cannot generate superoxide. 6. Regulation of pHi is essential for neutrophil function. The oxygen dependent bactericidal activity is inhibited upon cytoplasmic acidification. This can result from impairment of Na+/H+ exchange, or from influx of exogenous acid equivalents. 7. The latter mechanism may account for the inability of neutrophils to resolve bacterial infections in abscesses, which are generally made acidic by accumulation of organic acids that are by-products of bacterial anaerobic metabolism.     

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.     

The role of the Na+/H+ exchange system in the regulation of the internal pH in cultured cardiac cells

The Na+/H+ exchange system is not the major mechanism that regulates the internal pH value (pHi) of chick cardiac cells in culture under normal physiological conditions in the absence of carbonate. In cardiac cells in which the internal pH has been lowered to 6.6-6.7, the Na+/H+ exchanger becomes the major mechanism to bring back pHi to normal values (pHi = 7.3). The blockade of the Na+/H+ exchange activity with an active amiloride derivative, ethylisopropylamiloride, prevents internal pH recovery. The internal pH dependence of the Na+/H+ exchanger activity has been carefully studied. The [H+]i-dependence is very cooperative. For an external pH of 7.4, the system is nearly completely inactive at pHi 7.8 and nearly completely active at pHi 6.9-7.0 with half-maximum activation at pHi = 7.35. The increased activity of the Na+/H+ exchange system which follows the acidification of the internal medium produces an activation of the (Na+,K+)-ATPase.