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.     

Interferon-gamma and interleukin-1 alpha induce transient translocation of protein kinase C activity to membranes in a B lymphoid cell line. Evidence for a protein kinase C-independent pathway in lymphokine-induced cytoplasmic alkalinization

We have previously shown that recombinant murine interferon-gamma, rIFN-gamma, and recombinant human interleukin-1 alpha, rIL-1 alpha, induce differentiation of murine pre-B-like cell line 70Z/3, a finding associated with stimulation of Na+/H+ exchange across the plasma membrane. The present study was designed to test whether the enhanced Na+/H+ exchange is mediated by Ca2+/phospholipid-dependent protein kinase C. The results show that two structurally different peptides, rIFN-gamma and rIL-1 alpha, induce identical patterns of transient translocation of protein kinase C from the cytosol to the membranes. The increase in membrane-associated protein kinase C activity was first detected 20 min after exposure to the lymphokines. This activity peaked at 30 min and was back to baseline by 2 h. At each time point, the increase in membrane-associated protein kinase C activity corresponded to a decrease in the activity of protein kinase C in the cytoplasmic fraction. The total cellular activity (cytosol + membrane) remained the same. Two series of experiments were carried out to test the role of protein kinase C in mediating the lymphokine-stimulated Na+/H+ exchange. In the first, the effects of rIFN-gamma and rIL-1 alpha on cytoplasmic pH were measured in the presence of a protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, H-7. In the second, rIFN-gamma- and rIL-1 alpha-induced cytoplasmic alkalinization was determined in cells containing decreased protein kinase C activity. Under both experimental conditions, lymphokine-induced cytoplasmic alkalinization was not attenuated. These results indicate that, although both rIFN-gamma and rIL-1 alpha cause association of protein kinase C with membranes, activation of protein kinase C is not required for rIFN-gamma or rIL-1 alpha to stimulate Na+/H+ exchange across the plasma membrane.     

Interferon-gamma-induced astrocyte class II major histocompatibility complex gene expression is associated with both protein kinase C activation and Na+ entry

Astrocytes can be induced by interferon-gamma (IFN-gamma) to express class II major histocompatibility complex (MHC) antigens. This study was undertaken to elucidate the intracellular signaling pathways involved in IFN-gamma induction of class II MHC. We examined the effects of Na+/H+ antiporter and protein kinase C (PKC) inhibitors on class II expression and Na+ influx in astrocytes. We found that amiloride and ethyl isopropylamiloride, inhibitors of Na+/H+ exchange, blocked IFN-gamma-induced class II gene expression. IFN-gamma stimulated Na+ influx, and this increased influx was inhibited by amiloride. Treatment of astrocytes with the PKC inhibitor H7 also blocked the increase in Na+ uptake induced by IFN-gamma, indicating that IFN-gamma-induced PKC activation is required for subsequent Na+ influx. IFN-gamma treatment produced an increase of total PKC activity, which was associated with a rapid translocation of PKC activity from cytosolic to particulate fraction. H7 and another PKC inhibitor, staurosporine, inhibited IFN-gamma-induced class II gene expression. However, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, a potent PKC activator, did not affect class II expression. Taken together, our data indicate that both IFN-gamma-induced PKC activation and Na+ influx are required for class II MHC expression in astrocytes but that activation of PKC alone is not sufficient for ultimate expression of this gene.     

Amiloride inhibits phorbol ester-stimulated Na+/H+ exchange and protein kinase C. An amiloride analog selectively inhibits Na+/H+ exchange

The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by phorbol esters in HL-60 cells is the stimulation of Na+-dependent H+ efflux. In efforts to determine whether stimulation of Na+/H+ exchange by phorbol esters is coupled to induction of cellular differentiation, we found that 1) amiloride, a frequently used inhibitor of Na+/H+ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which amiloride inhibits Na+/H+ exchange; 2) an amiloride analog, dimethylamiloride, is a far more potent inhibitor of Na+/H+ exchange than is amiloride, while being no more potent than amiloride in inhibiting phorbol ester/protein kinase C-mediated phosphorylation; and 3) at concentrations sufficient to completely inhibit Na+/H+ exchange, amiloride blocked phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) did not. Thus, dimethylamiloride represents a potential tool for distinguishing protein kinase C-coupled from Na+/H+ exchange-coupled events in phorbol ester-stimulated cells.     

Osmotic and phorbol ester-induced activation of Na+/H+ exchange: possible role of protein phosphorylation in lymphocyte volume regulation

The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1-oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.     

Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways

Angiotensin II, a potent vasoconstrictor, is known to stimulate Ca2+ mobilization and Na+ influx in vascular smooth muscle cells (VSMC). The fact that the Na+/H+ exchange inhibitor, amiloride, blocks angiotensin II-stimulated Na+ influx and is itself a vasodilator suggests that Na+/H+ exchange may play a role in the angiotensin II-mediated effects on VSMC. We have used a pH-sensitive fluorescent dye to study Na+/H+ exchange in cultured rat aortic VSMC. Basal intracellular pH was 7.08 in physiological saline buffer. Angiotensin II stimulation caused an initial transient acidification, followed by a Na+-dependent alkalinization. Angiotensin II increased the rate of alkalinization with apparent threshold, half-maximal, and maximal effect of 0.01, 3, and 100 nM, respectively. Angiotensin II stimulation appeared to be mediated by a shift in the Km of the Na+/H+ exchanger for extracellular Na+. Since angiotensin II activates phospholipase C in VSMC, we tested the possibility that angiotensin II increased Na+/H+ exchange by activation of protein kinase C via stimulation of diacylglycerol formation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated Na+/H+ exchange in VSMC cultured for 24 h in serum-free medium, and the subsequent angiotensin II response was inhibited. However, VSMC grown in serum and treated for 24 h with TPA to decrease protein kinase C activity showed no inhibition of angiotensin II-stimulated Na+/H+ exchange. TPA caused no intracellular alkalinization of VSMC grown in serum, while the angiotensin II response was actually enhanced compared to VSMC deprived of serum for 24 h. We conclude that angiotensin II stimulates an amiloride-sensitive Na+/H+ exchange system in cultured VSMC which is mediated by protein kinase C-dependent and -independent mechanisms. Angiotensin II-mediated Na+ influx and intracellular alkalinization may play a role in excitation-response coupling in vascular smooth muscle.     

WS-1 human fibroblasts contain distinct calcium and protein kinase C-mediated pathways for activation of Na+/H+ exchange: contrasting effects of thrombin and PMA

PMA and thrombin were examined for their ability to activate Na+/H+ exchange in growth-arrested WS-1 human fibroblasts. PMA or thrombin caused a cytoplasmic alkalinization that required extracellular sodium and was sensitive to 1 mM amiloride, suggesting that the rise in pH was mediated by the Na+/H+ exchanger. However, PMA and thrombin activated Na+/H+ exchange by distinctly different mechanisms. The rate of cytoplasmic alkalinization caused by 30 nM PMA was slower than 10 nM thrombin. The PMA-induced pH change was sensitive to the protein kinase inhibitors staurosporine (50 nM) and H-7 (100 microM). No increase in intracellular calcium was observed after PMA treatment and the cytoplasmic alkalinization caused by PMA was not sensitive to the drug TMB8 (200 microM) or the intracellular calcium-chelator BAPTA. In contrast, the thrombin-induced rise in cytoplasmic pH was insensitive to 50 nM staurosporine and only partially reduced with 100 microM H-7. The thrombin-induced activation of Na+/H+ exchange was inhibited by 200 microM TMB8 or pretreatment with BAPTA. PMA caused translocation of PKC activity from a cytoplasmic to membrane fraction whereas thrombin did not. Pretreatment with 50 nM staurosporine significantly reduced measurable PKC activity with or without PMA treatment. PMA and thrombin were also examined for their ability to induce DNA synthesis in growth-arrested WS-1 human fibroblasts. Unlike thrombin, PMA did not stimulate [3H]-thymidine incorporation in cells serum-deprived for 48 hours. In addition, PMA inhibited thrombin-induced DNA synthesis when added at the same time or as late as 10 hours after thrombin addition. Therefore, thrombin and PMA activate Na+/H+ exchange by distinct pathways, but only the thrombin-induced pathway correlates with a mitogenic response.     

Sphingosine differentially inhibits activation of the Na+/H+ exchanger by phorbol esters and growth factors

The role of protein kinase C in activation of the plasma membrane Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. The basic lipid, sphingosine, was used to block enzymatic activity of protein kinase C. Na+/H+ exchange was activated by phorbol 12-myristate 13-acetate (PMA), diacylglycerols, platelet-derived growth factor (PDGF), thrombin, or by osmotically-induced cell shrinkage. Intracellular pH and Na+/H+ exchange activity were measured using the intracellular pH indicator, 2',7'-bis(carboxyethyl)-5(6) carboxyfluorescein. Acting alone, both crude sphingosine and pure, synthetic C18 D-(+)-erythro-sphingosine raised pHi in a dose-dependent manner (from 6.95 +/- 0.02 to 7.19 +/- 0.09 over 10 min for 10 microM sphingosine). This alkalinization was not due to Na+/H+ exchange as it was not altered by t-butylamiloride (50 microM) nor by replacement of the assay medium with a Na(+)-free solution. Sphingosine-induced alkalinization did not require protein kinase C activity, since it was fully intact in protein kinase C-depleted cells. It was also not due to a detergent action of sphingosine on the cell membrane, since both ionic and non-ionic detergents caused cell acidification. Rather, alkalinization induced by sphingosine appeared to be due to cellular uptake of NH3 groups since N-acetylsphingosine showed no alkalinization. After the initial cell alkalinization, cellular uptake of [3H]sphingosine continued slowly for up to 24 h. The ability of PMA or dioctanoylglycerol to activate Na+/H+ exchange fell to 20% of control after 24 h of sphingosine exposure. At all times, C11 and N-acetylsphingosine failed to block PMA-induced activation of the exchanger. Activation of the Na+/H+ exchanger by sucrose, which does not depend on protein kinase C activity, was unaffected by sphingosine. Activation of Na+/H+ exchange by thrombin and PDGF was partially inhibited by 30 and 20%, respectively. These data indicate that both thrombin and PDGF activate Na+/H+ exchange by pathway(s) that are primarily independent of protein kinase C.     

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent,but can couple to an H+ conductance

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT- PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time- course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH- sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.     

Separate regulatory control of apical and basolateral Na+/H+ exchange in renal epithelial cells

Epithelial layers of LLC-PK1/PKE20 cells, a renal epithelial cell line which expresses Na+/H+ exchange activities in the apical as well as basolateral membrane domains, are examined in the single cell mode by microspectrofluorometry. We provide evidence that basolateral Na+/H+ exchange is more sensitive to amiloride inhibition than is apical Na+/H+ exchange. Furthermore, we demonstrate that the two exchange activities differ in their regulatory control: kinase A activation (forskolin, 8-Br-cAMP) leads to inhibition of both exchange activities, whereas kinase C activation (phorbol ester) stimulates basolateral and inhibits apical Na+/H+ exchange. Thus, renal epithelial cells may contain two Na+/H+ exchange activities: an apical ("epithelial") and basolateral ("housekeeping") which may serve different cellular functions and are under separate regulatory controls.     

Tumor promoter phorbol 12-myristate 13-acetate inhibits mitogen- stimulated Na+/H+ exchange in human epidermoid carcinoma A431 cells

Addition of polypeptide growth factors to cultured cells results in a rapid stimulation of Na+/H+ exchange, which leads to cytoplasmic alkalinization. We studied the effects of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on the Na+/H+ exchange system of A431 cells. Stimulation of Na+/H+ exchange by epidermal growth factor (EGF) and serum as well as by vanadate ions is strongly inhibited after treatment of cells with nanomolar concentrations of PMA. Phorbol esters that have no activity as tumor promoters also do not modulate the activation of Na+/H+ exchange. By contrast, the stimulation of Na+/H+ exchange that is produced upon exposure of cells to hypertonic solution is only slightly inhibited by PMA treatment, indicating that PMA treatment does not directly block the activity of the Na+/H+ antiporter. Furthermore, incubation of cells with PMA causes a weak stimulation of Na+/H+ exchange, although this effect is mostly observed at relatively high PMA concentrations and appears to require external Ca2+. The inhibition BY PMA of EGF-promoted Na+/H+ exchange is not due to inhibition of EGF-binding to the EGF receptor. Since PMA activates protein kinase C, our observations are consistent with the hypothesis that protein kinase C functions to attenuate the stimulation of Na+/H+ exchange by polypeptide growth factors.     

Mechanism of activation of lymphocyte Na+/H+ exchange by concanavalin A. A calcium- and protein kinase C-independent pathway

Treatment of thymic lymphocytes with the mitogenic lectin concanavalin A (ConA) increases the intracellular free Ca2+ concentration and stimulates phosphoinositide turnover. ConA also induced a rapid, amiloride-sensitive, Na+-dependent increase in cytosolic pH of 0.13 +/- 0.01, indicative of stimulation of the Na+/H+ antiport. To investigate the mechanism underlying activation of Na+/H+ exchange by ConA, the intracellular free Ca2+ concentration changes induced by this lectin were precluded by loading the cells with Ca2+-buffering agents and suspension in Ca2+-free media. Under these conditions, the ConA-induced cytoplasmic alkalinization proceeded normally. Two approaches were used to assess the role of protein kinase C. First, this enzyme was inhibited by the addition of 1-(5-isoquinolinysulfonyl)-2-methylpiperazine. In the presence of this potent antagonist, stimulation of the antiport by 12-O-tetradecanoylphorbol-13-acetate was greatly inhibited. In contrast, stimulation by ConA was unaffected. Second, protein kinase C was depleted by overnight incubation with phorbol esters. Following this treatment, Na+/H+ exchange was no longer activated by 12-O-tetradecanoyl-13-acetate, but was still stimulated by ConA. These data suggest that a Ca2+- and protein kinase C-independent mechanisms mediates the activation of Na+/H+ exchange by ConA. The possible role of GTP-binding proteins in the activation was also studied. The antiport was not stimulated by either fluoroaluminate or vanadate. Moreover, pretreatment with pertussis toxin failed to inhibit the ConA-induced cytoplasmic alkalinization. In contrast, preincubation with cholera toxin partially inhibited activation. Under these conditions, cholera toxin significantly elevated intracellular cAMP levels. Inhibition was also observed in cells treated with forskolin at concentrations that increased [cAMP]. The data suggest that a novel cAMP-sensitive signaling mechanism not involving Ca2+ and protein kinase C is involved in the stimulation of Na+/H+ exchange by mitogens in T lymphocytes.     

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+.     

The effect of endothelin-1 on Na+/H+ metabolism in vascular smooth muscle cells]

The effects of endothelin on intracellular pH (pHi) were examined in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent probe BCECF. Endothelin induced biphasic changes in pHi: initial decrease followed by a subsequent increase above the basal level due to activation of the Na+/H+ exchange. The elevation of pHi was slow and sustained, but depended on the dose of endothelin: IC50 was about 3 x 10(-8) M. Na+/H+ exchange inhibition by EIPA (10(-7) M) or by equimolar replacement of external Na+ by choline abolished the pHi increase by enhancing the first phase of cytoplasm acidification. Effects of endothelin were compared with the action of protein kinase C (PK-C) activator phorbol 12-13 myristate ester (PMA). PMA induced a monophasic slow and sustained increase in pHi. The treatments of VSMC with H-7 and staurosporine (PK-C) inhibitors prevented the pHi response to endothelin and PMA. These results suggest that protein kinase C may play an important role in mediating the effects of endothelin on Na+/H+ exchange in VSMC.     

Recombinant murine interferon-gamma-induced differentiation of pre-B lymphocytes is associated with Na+/H+ exchange-dependent and -independent cytoplasmic alkalinization

We have previously shown that in a murine pre-B lymphocyte cell line, 70Z/3, interleukin-1-induced IgM expression (differentiation) was associated with Na+/H+-mediated cytoplasmic alkalinization. Because interferon-gamma also induces 70Z/3 differentiation, in this study we examined the effects of recombinant murine interferon-gamma (rIFN-gamma) on cell pH. We found that rIFN-gamma (50 units/ml) induced a sustained increase in cell pH, averaging 0.09 pH units above the base line at 30 min and 0.08 at 4 h. Because rIFN-gamma also induced increases in cell Na+ concentration, the data suggests stimulation of Na+/H+ exchange across the cell membrane. Amiloride inhibited the rIFN-gamma-mediated pH rise by only 31% at 30 min, but at 4 h the inhibition was more complete (89%). In contrast to pHi, amiloride totally blocked the Na+ rise at both 30 min and 4 h. This indicates that at the earlier time point the pH rise had a Na+/H+ exchange-dependent and -independent component while at the later time most of the pH rise was due to the Na+/H+ exchanger. The presence of a Na+ independent amiloride-insensitive component was further confirmed by the 0.05 pH rise induced by rIFN-gamma in Na+-free media. Failure to block the Na+-independent rIFN-gamma-mediated pHi rise by anion exchange inhibitors or removal of Ca2+ indicate that this component was not mediated by Cl-/OH- or Ca2+/H+ exchange. The nature of the Na+-independent cell alkalinization process or its role in rIFN-gamma-mediated 70Z/3 differentiation remains to be determined. Because amiloride did not have an effect on rIFN-gamma-induced surface Ig expression or the rIFN-gamma-mediated increase in new kappa light chain-specific mRNA the results indicate that rIFN-gamma-stimulated Na+/H+ is not required for 70Z/3 differentiation.     

Interferon gamma induces in human neutrophils and macrophages expression of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I or CD64)

Immune interferon (IFN-gamma) induces in human neutrophils accumulation of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I, CD64) with a mechanism that is independent from de novo protein synthesis and from activation of the Na+/H+ antiporter. Monocyte-derived macrophages can also be induced to express high levels of Fc gamma R-I mRNA by IFN-gamma, without requirement of protein synthesis. Unlike what is observed in neutrophils, induction by IFN-gamma of macrophage Fc gamma R-I mRNA was significantly depressed by the Na+/H+ antiporter inhibitor amiloride. These results indicate that phagocytes' Fc gamma R-I mRNA induction by IFN-gamma is regulated by different mechanisms depending on the target cells.