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.     

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.     

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.     

Differentiation of human promyelocytic HL 60 cells by retinoic acid is accompanied by an increase in the intracellular pH. The role of the Na+/H+ exchange system

Retinoic acid, which induces the differentiation of HL 60 cells to granulocytes, produces a cell alkalinization from pHi = 7.03 to pHi = 7.37. The half-maximum effect of retinoic acid is observed at 10 nM. The effect of retinoic acid on the pHi develops slowly, and it precedes the differentiation of the cells. A cell alkalinization is also observed after differentiation of the cells by dimethyl sulfoxide. It is not observed using etretinate, a synthetic retinoid that does not promote the differentiation of HL 60 cells. Two pHi regulating mechanisms coexist in HL 60 cells. The Na+/H+ exchange system is the major mechanism that allows HL 60 cells to recover from an intracellular acidosis. A second mechanism is a Na-HCO3 cotransport system. During differentiation of the cells by retinoic acid, a 2-fold increase in the activity of the Na+/H+ exchange system is observed, while the activity of the NaHCO3 cotransport remains constant. The properties of interaction of the Na+/H+ exchanger with internal H+, external Na+, and Li+ as well as with amiloride and its derivatives are defined. The Na+/H+ exchanger of HL 60 cells is characterized by unusually low affinities for alkali cations and a high affinity for amiloride and its derivatives. The pHi dependence of the exchanger is not modified after differentiation by retinoic acid. It is concluded that the mechanism of activation of the Na+/H+ exchanger by retinoic acid is distinct from the short-term effect produced by mitogens and phorbol esters which change the pHi dependence of the system.     

Biochemical properties of the Na+/H+ exchange system in rat brain synaptosomes. Interdependence of internal and external pH control of the exchange activity

Properties of the Na+/H+ exchange system in synaptosomes have been studied primarily by using acridine orange fluorescence to follow H+ efflux. Results obtained from 22Na+ uptake experiments and [3H]ethylpropylamiloride binding experiments are also presented for comparison. The basal properties of the Na+/H+ antiport in synaptosomes are similar to those found in other systems; (i) the stoichiometry of Na+/H+ exchange is 1:1; (ii) Li+ can be successfully substituted for Na+; its affinity for the exchanger (KLi+ = 3 mM) is higher than that of Na+ (KNa+ = 12 mM), but the maximal rate of H+ efflux in the presence of Li+ is about 3 times lower than the maximal rate of H+ efflux in the presence of Na+; and (iii) the Na+/H+ antiport is inhibited by amiloride derivatives with the rank order:ethylisopropylamiloride greater than ethylpropylamiloride greater than amiloride greater than benzamil. The most important finding of this paper is that the external pH dependence of the synaptosomal Na+/H+ antiport is controlled by the value of internal pH and vice versa. For example apparent pHo values for half-maximum activation of the Na+/H+ exchanger are pHo = 7.12 when pHi = 6.4 and pHo = 7.95 when pHi = 7.3. Therefore, a 0.9 pH unit increase in internal pH produces a shift of at least a 0.83 pH unit in the external pH dependence. In addition, changing pHo from 7.75 to 8.50 also shifts the half-maximum pHi value for activation of the Na+/H+ antiport from 6.67 to 7.54.     

Role of Na+/H+ exchange in the control of intracellular pH and cell membrane conductances in frog skin epithelium

Ion-sensitive microelectrodes and current-voltage analysis were used to study intracellular pH (pHi) regulation and its effects on ionic conductances in the isolated epithelium of frog skin. We show that pHi recovery after an acid load is dependent on the operation of an amiloride-sensitive Na+/H+ exchanger localized at the basolateral cell membranes. The antiporter is not quiescent at physiological pHi (7.1-7.4) and, thus, contributes to the maintenance of steady state pHi. Moreover, intracellular sodium ion activity is also controlled in part by Na+ uptake via the exchanger. Intracellular acidification decreased transepithelial Na+ transport rate, apical Na+ permeability (PNa) and Na+ and K+ conductances. The recovery of these transport parameters after the removal of the acid load was found to be dependent on pHi regulation via Na+/H+ exchange. Conversely, variations in Na+ transport were accompanied by changes in pHi. Inhibition of Na+/K+ ATPase by ouabain produced covariant decreases in pHi and PNa, whereas increases in Na+ transport, occurring spontaneously or after aldosterone treatment, were highly correlated with intracellular alkalinization. We conclude that cytoplasmic H+ activity is regulated by a basolateral Na+/H+ exchanger and that transcellular coupling of ion flows at opposing cell membranes can be modulated by the pHi-regulating mechanism.     

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.     

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.     

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.     

Kinetic dissection of two distinct proton binding sites in Na+/H+ exchangers by measurement of reverse mode reaction

We examined the effect of intracellular acidification on the reverse mode of Na+/H+ exchange by measuring 22Na+ efflux from 22Na+-loaded PS120 cells expressing the Na+/H+ exchanger (NHE) isoforms NHE1, NHE2, and NHE3. The 5-(N-ethyl-N-isopropyl)amiloride (EIPA)- or amiloride-sensitive fraction of 22Na+ efflux was dramatically accelerated by cytosolic acidification as opposed to thermodynamic prediction, supporting the concept that these NHE isoforms are activated by protonation of an internal binding site(s) distinct from the H+ transport site. Intracellular pH (pHi) dependence of 22 Na+ efflux roughly exhibited a bell-shaped profile; mild acidification from pHi 7.5 to 7 dramatically accelerated 22Na+ efflux, whereas acidification from pHi 6.6 gradually decreased it. Alkalinization above pHi 7.5 completely suppressed EIPA-sensitive 22Na+ efflux. Cell ATP depletion and mutation of NHE1 at Arg440 (R440D) caused a large acidic shift of the pHi profile for 22Na+ efflux, whereas mutation at Gly455 (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na+/H+ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.     

Alpha 2-adrenergic receptors and the Na+/H+ exchanger in the intestinal epithelial cell line, HT-29

alpha 2-Adrenergic receptors (alpha 2-AR) are negatively coupled to adenylyl cyclase via the GTP-binding protein Gi. However, inhibition of adenylylcyclase does not account for many effector cell responses to alpha 2-AR agonists, suggesting that the receptor can couple to other signal transduction pathways. One potential pathway may be the stimulation of Na+/H+ exchange elicited by alpha 2-AR activation in renal proximal tubule cells, platelets, and the NG-10815 cell line. To determine whether the various receptor-effector coupling mechanisms operate in a tissue-specific manner, we studied the effect of alpha 2-AR activation on basal and stimulated Na+/H+ exchange in epithelial cells isolated from human colon (HT-29 adenocarcinoma cells). Na+/H+ exchange was measured by quantitation of intracellular hydrogen ion concentration (acetoxymethyl ester 2,7-biscarboxyethyl-5(6)carboxyfluorescein) and 22Na+ uptake. HT-29 cells expressed an amiloride-sensitive Na+/H+ exchanger that was activated by reduction of intracellular pH (pHi) to 6.0 but was quiescent at a physiological pHi. The rapid alkalinization observed after acid loading (0.57 +/- 0.07 pH units/min/10(4) cells) was dependent on external sodium and was blocked by amiloride (Ki approximately 2.1 microM). Although epinephrine and the selective alpha 2-AR agonists clonidine and UK-14304 inhibited forskolin-activated adenylylcyclase, these compounds did not alter basal Na+/H+ exchange. Stimulated Na+/H+ exchange was similarly unaffected by epinephrine. In contrast, stimulated Na+/H+ exchanger activity was completely inhibited by the selective alpha 2-agonists clonidine, UK-14304, and guanabenz. This inhibitory effect was not blocked by the alpha 2-AR antagonist rauwolscine, and it is likely due to a direct interaction with the exchanger molecule itself. Structure/activity studies indicated that the compounds inhibiting exchanger activity possess either an imidazoline or guanidinium moiety. Although these molecules bear structural similarity to amiloride, they did not inhibit the amiloride-sensitive epithelial sodium channel in toad urinary bladder, suggesting that these compounds may be useful as "amiloride-like" ligands selective for the Na+/H+ exchanger. These data indicate that in the HT-29 intestinal cell line, in contrast to observations in other tissues, alpha 2-adrenergic receptors are not coupled to the Na+/H+ exchanger, suggesting that the cell-signaling mechanisms utilized by the alpha 2-AR are tissue specific.     

Mechanisms regulating intracellular pH in sea urchin eggs

Intracellular pH (pHi) of sea urchin eggs (Paracentrotus lividus) was determined using DMO (dimethyloxazolidinedione) and a rapid filtration technique (P. Payan, J.P. Girard, R. Christen and C. Sardet (1981). Exp. Cell Res. 134, 339-344). Transfer of unfertilized or fertilized eggs from normal sea water into Na+-free artificial sea water leads to a progressive acidification and fall of intracellular Na+ content. A step rise in external Na+ to 10 meq causes a rapid but transient Na+ entry coupled to an excretion of H+, giving rise to a pHi increase. It is shown that the plasma membrane of unfertilized eggs contains a permanent and reversible Na+/H+ exchanger which contributes to the regulation of pHi. This exchange occurs with a 1:1 stoichiometry and is independent of metabolic energy. Proton excretion and sodium entry follow saturable kinetics with respect to external Na+ and are completely inhibited by amiloride. At fertilization, pHi increases from 7.38 to 7.64 and is maintained at this level by two separate mechanisms: (1) a Na+/H+ exchange with the same characteristics as in unfertilized eggs; (2) a H+-excreting system that is dependent on external Na+, amiloride sensitive, and requiring metabolic energy. The relationship between the permanent Na+/H+ exchange involved in pHi regulation and the transient Na+/H+ exchange occurring at fertilization is discussed.     

Activation of Na+ /H+ exchange on rat preadipocyte plasma membrane and its role in cell proliferation and differentiation

An in vitro cultured rat perirenal preadipocyte (PA) was established as a model system to investigate the role of the intracellular pH (pHi) and of the Na~ /H~ exchanger during PA proliferation and differentiation, pH sensitive probe, 2' ,7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), was employed to measure the pHi of PA and to determine the Na~ /H~ exchange activity. The results showed that there was Na~ /H~ exchange activity in the plasma membrane of PA, FCS stimulated DNA synthesis measured by ~3H-TdR incorporation, and the activation of Na~ /H~ exchanger resulted in phi increase (nearly 0.2 pH unit) within 2 min. Ethyl-isopropyl-amiloride (EIPA), a specific Na~ /H~ exchange inhibitor, inhibited Na~ /H~ exchange activity and DNA synthesis. In the absence of serum insulin did not stimulate DNA synthesis but did induce PA differentiation characterized by the appearance of adiposome in the cell and the enhancement of glycerol-3-phosphate dehydrogenase (G_3PDHase) activity. Meantime, insu     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.     

The voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa flagellar membrane vesicles studied with an entrapped pH probe

Flagellar plasma membrane vesicles were isolated from sea urchin sperm using osmotic lysis. A membrane impermeant fluorescence pH indicator, pyranine, was incorporated into the vesicles as they resealed after lysis and was used to measure the intravesicular pH (pHi). Addition of Na+ rapidly alkalinized the pHi of vesicles prepared with an internal acidic pH gradient. The pHi increase showed ionic selectivity in the order of Na+ greater than Li+ much greater than K+ approximately equal to Cs+ approximately equal to O. Complete removal of monovalent anions such as Cl- and HCO3- did not affect the exchange, thus ruling out the participation of an anion carrier in the process. The optimal operation of the exchanger, however, required the presence of a transmembrane potential, which could be generated by the diffusion potential of either K+, a naturally permeant ion, or Cs+ which was artificially made permeant by the ionophore valinomycin. Depolarization inhibited the exchange in both the forward and the reverse directions, which is consistent with the voltage-gated electroneutral exchange mechanism proposed previously for this exchanger (Lee, H. C. J. Biol. Chem. (1984) 259, 15315-15319). The voltage sensitivity of the Na+/H+ exchanger was found to be modulated by the presence of Mg2+. A model involving the screening of the internal surface potential was proposed to account for the Mg2+ effect. The vesicle preparation used in this study allows complete control of the internal contents and represents a major simplification of the system as compared with the intact sperm and the isolated flagella used previously.     

Effects of glucocorticoids on Na+/H+ exchange and growth in cultured vascular smooth muscle cells

We have examined the effects of hydrocortisone on growth and Na+/H+ exchange in cultured rat aortic vascular smooth muscle cells (VSMC). Hydrocortisone (2 microM) treatment of growth-arrested VSMC significantly decreased VSMC growth in response to 10% calf serum assayed by 3H-thymidine incorporation and cell number at confluence. This effect was associated with the appearance of an altered cell phenotype characterized by large, flat VSMC that did not form typical "hillocks." Na+/H+ exchange was also altered in hydrocortisone-treated cells assayed by dimethylamiloride-sensitive 22Na+ influx into acid-loaded cells or by intracellular pH (pHi) change using the fluorescent dye BCECF. Resting pHi was 7.25 +/- 0.04 and 7.15 +/- 0.05 in control and hydrocortisone-treated cells, respectively (0.1 less than P less than 0.05). Following intracellular acidification in the absence of external Na+, pHi recovery upon addition of Na+ was increased 89% in hydrocortisone-treated cells relative to control. This was due to an increase in the Vmax for the Na+/H+ exchanger from 17.5 +/- 2.4 to 25.9 +/- 2.0 nmol Na+/mg protein x min (P less than 0.01) without a significant change in Km. Treatment of VSMC with actinomycin D (1 microgram/ml) or cycloheximide (10 microM) completely inhibited the hydrocortisone-mediated increase in Na+/H+ exchange, indicating a requirement for both RNA and protein synthesis. Because hydrocortisone altered the Vmax for Na+/H+ exchange, in contrast to agonists such as serum or angiotensin II which alter the Km for intracellular H+ or extracellular Na+, respectively, we studied the effect of hydrocortisone on activation of Na+/H+ exchange by these agonists. In cells maintained at physiological pHi (7.2), the initial rate (2 min) of angiotensin II-stimulated alkalinization was increased 66 +/- 39% in hydrocortisone-treated compared with control cells. Hydrocortisone caused no change in angiotensin II-stimulated phospholipase C activity assayed by measurement of changes in intracellular Ca2+ or diacylglycerol formation. However, angiotensin II and serum stimulated only small increases in Na+/H+ exchange in acid-loaded (pHi = 6.8) hydrocortisone-treated cells. These findings suggest that hydrocortisone-mediated increases in VSMC Na+/H+ exchange occur in association with a nonproliferating phenotype that has altered regulation of Na+/H+ exchange activation. We propose that hydrocortisone-mediated growth inhibition may be a useful model for studying the role of Na+/H+ exchange in cell growth responsiveness.     

Sodium fluoride prevents receptor- and protein kinase C-mediated activation of the human platelet Na+/H+ exchanger without inhibiting its basic pHi-regulating activity

When human platelets are stimulated with thrombin or activators of protein kinase C, cytosolic pH (pHi) increases due to activation of Na+/H+ exchange. In order to further elucidate the molecular mechanisms that regulate the exchanger, we used sodium fluoride, which is a known activator of guanine nucleotide-binding proteins (G proteins) in platelets. Although NaF induced the mobilization of Ca2+ from intracellular storage sites in fura2-loaded platelets, it failed to raise pHi as determined from the fluorescence of 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded platelets. Furthermore, when thrombin (0.1 unit/ml) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) had raised pHi from 7.13 +/- 0.05 to 7.35 +/- 0.07 (n = 30), addition of NaF (2.5-10 mM) rapidly restored pHi to values found before stimulation. Conversely, preincubation of platelets with low concentrations of NaF (2.5 mM) completely prevented alkalinization in response to thrombin or TPA. Unlike ethylisopropylamiloride, which completely blocked Na+/H+ exchange, NaF did not prevent the recovery of pHi from an artificial acid load. Hence, the inhibitory action of NaF is restricted to receptor-mediated activation of the antiport. In order to investigate whether the NaF effect was attributable to a G protein, platelets were preincubated with N-ethylmaleimide (50 microM), which is known to inhibit the adenylyl cyclase-inhibitory G protein. N-Ethylmaleimide treatment not only prevented inhibition of adenylyl cyclase by epinephrine but also completely reversed the inhibitory effect of NaF on the Na+/H+ exchanger. Our data suggest the existence of a novel G protein which is activated by fluoride and functions as a negative regulator of the Na+/H+ exchanger in platelets.     

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.     

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.     

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.