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.     

Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+ antiport. Evidence for two distinct roles for Na+/H+ antiport activation.

Thapsigargin stimulates an increase of cytosolic free Ca2+ concentration [( Ca2+]c) in, and 45Ca2+ efflux from, a clone of GH4C1 pituitary cells. This increase in [Ca2+]c was followed by a lower sustained elevation of [Ca2+]c, which required the presence of extracellular Ca2+, and was not inhibited by a Ca2(+)-channel blocker, nimodipine. Thapsigargin had no effect on inositol phosphate generation. We used thyrotropin-releasing hormone (TRH) to mobilize Ca2+ from an InsP3-sensitive store. Pretreatment with thapsigargin blocked the ability of TRH to cause a transient increase in both [Ca2+]c and 45Ca2+ efflux. The block of TRH-induced Ca2+ mobilization was not caused by a block at the receptor level, because TRH stimulation of InsP3 was not affected by thapsigargin. Rundown of the TRH-releasable store by Ca2(+)-induced Ca2+ release does not appear to account for the action of thapsigargin on the TRH-induced spike in [Ca2+]c, because BAY K 8644, which causes a sustained rise in [Ca2+]c, did not block Ca2+ release caused by TRH. In addition, caffeine, which releases Ca2+ from intracellular stores in other cell types, caused an increase in [Ca2+]c in GH4C1 cells, but had no effect on a subsequent spike in [Ca2+]c induced by TRH or thapsigargin. TRH caused a substantial decrease in the amount of intracellular Ca2+ released by thapsigargin. We conclude that in GH4C1 cells thapsigargin actively discharges an InsP3-releasable pool of Ca2+ and that this mechanism alone causes the block of the TRH-induced increase in [Ca2+]c.     

Calcium mobilization and Na+/H+ antiport activation by endothelin in human skin fibroblasts

Endothelin (ET-1) has been shown to exert vasoconstrictor activity in vivo and mobilize Ca2+ in vascular smooth muscle cells in culture. In this paper we show that the human skin fibroblast exhibits specific receptors to ET-1 and that activation of these receptors results in increased intracellular Ca2+ (Ca2+i) and accelerated Na+/H+ antiport activity. ET-1 raised Ca2+i in a dose-response manner; the peak Ca2+i rise was from basal levels of 112.2 +/- 21.9 to 299.2 +/- 49.7 nM at 300 nM ET-1. This rise was attenuated by removal of extracellular Ca2+i0. Although ET-1 did not alter basal intracellular pH, it enhanced Na+/H+ antiport activity of acidified cells. Fibroblasts demonstrated 156 +/- 18 (mean +/- SE) ET-1 receptors per unit cell and an equilibrium dissociation constant of 203.4 +/- 35.6 pM. Inasmuch as ET-1 plays a role in the metabolism of cells such as the undifferentiated fibroblast, an important action of this peptide may be to act as a growth factor.     

The amiloride-sensitive Na+/H+ antiport in 3T3 fibroblasts

BALB/c 3T3 fibroblasts have an amiloride-sensitive Na+ uptake mechanism which is hardly detectable under normal physiological conditions. The activity of this Na+ transport system can be increased to a large extent by treatments that decrease the internal pH such as loss of intracellular NH4+ as NH3 or incubation with nigericin in the presence of a low external K+ concentration. These treatments have made possible an analysis of the interaction of the Na+/H+ antiport with amiloride and of the external pH dependence of the system. The addition of fetal bovine serum to quiescent 3T3 cells stimulates the initial rate of the amiloride-sensitive 22Na+ uptake by only 50%. However, after treatment of the cells with ammonia or nigericin, serum produces a 40-fold stimulation of the rate of the amiloride-sensitive 22Na+ uptake. Control experiments show that serum does not stimulate the activity of the Na+/H+ antiport by an indirect mechanism involving a depolarization of the membrane or a modification of the internal Ca2+ concentration. It is suggested that some serum component directly interacts with the Na+/H+ exchanger to modify its catalytic properties.     

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.     

Activation of the Na+/H+ antiport is not required for lectin-induced proliferation of human T lymphocytes

Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes.     

Sphingosine inhibits phorbol 12-myristate 13-acetate-, but not serum-induced, activation of Na+/H+ exchange in mammalian cells

Addition of serum to quiescent mammalian cells in culture initiates a series of events which culminates in DNA replication and cell division. One of the earliest events in this sequence of events is activation of Na+/H+ exchange, which can result in an increase in intracellular pH (pHin). The regulation of this change in activity is not known. Since treatment of 3T3 cells with activators of protein kinase C (kinase C) can result in an increased pHin, it has been hypothesized that serum stimulation of kinase C is responsible for activation of Na+/H+ exchange. Recently, sphingolipids have been discovered to inhibit kinase C both in vitro and in vivo. Therefore, we undertook the present study to ask whether or not inhibition of kinase C using sphingolipids prevents mitogen-induced alkalinization in 3T3 cells. Our results indicate that activators of kinase C stimulate Na+/H+ exchange in normal human fibroblasts (BoGi), but not in mouse embryo (3T3) cells. Addition of serum to BoGi cells, on top of saturating doses of phorbol 12-myristate 13-acetate (PMA), results in a further cytoplasmic alkalinization. Furthermore, sphingosine prevents the PMA-induced increase in pHin in BoGi cells, and phosphorylation of an 80 kDa protein in 3T3 cells, but not the serum-induced alkalinization in either BoGi or 3T3 cells. These data indicate that activation of kinase C does not participate in the physiological activation of Na+/H+ exchange in human fibroblasts or mouse embryo cells by serum.     

Lactoferrin activates plasma membrane oxidase and Na+/H+ antiport activity.

Lactoferrin is a growth stimulant. The basis for this effect is not clear since it is not thought to be involved in iron uptake through endocytosis. Ferric lactoferrin supports external ferrous chelate formation by K562 and HeLa cells, and ferric lactoferrin stimulates the reduction of external ferric iron by cells. Ferric lactoferrin also stimulates NADH oxidase activity in isolated rat liver plasma membranes and stimulates amiloride sensitive proton release from K562 cells. The evidence that ferric lactoferrin can participate in oxidoreduction reactions at the plasma membrane leading to activation of Na+/H+ exchange provides an alternative explanation for the proliferative effect.     

The Na+/Ca2+ antiporter in aortic smooth muscle cells. Characterization and demonstration of an activation by phorbol esters

The Na+/Ca2+ antiporter is present in aortic smooth muscle cells of the A7r5 cell line. Imposing an outward Na+ gradient to the cells promoted a 45Ca2+ uptake component which was sensitive to amiloride derivatives and insensitive to blockers of the voltage-dependent Ca2+ channel. The Ca2+ uptake system was dependent on intracellular Na+ concentration; it was inactive when Li+ replaced intracellular Na+ and it was electrogenic. Flow cytometric analysis of cells that had been loaded with the Ca2+ indicator indo-1 showed that all conditions that promoted Ca2+ influx led to corresponding increases in the free cytoplasmic Ca2+ concentration. Treatment of the A7r5 cells with phorbol myristate acetate, a known activator of protein kinase C (Ca2+/phospholipid-dependent enzyme), led to a two-fold activation of the system and to larger intracellular Ca2+ transients when cells were shifted to Na+-free solutions. Activation was observed at all intracellular Na+ concentrations. Changing the activity of the Na+/Ca2+ system did not affect the size and duration of intracellular Ca2+ transients elicited by the Ca2+ mobilizing hormone vasopressin. It is concluded that the Na+/Ca2+ antiporter in smooth muscle cells is a target for protein kinase C but that the system is not involved in the regulation of Ca2+ transients induced by vasopressin.     

Proton motive force and Na+/H+ antiport in a moderate halophile.

The influence of pH on the proton motive force of Vibrio costicola was determined by measuring the distributions of triphenylmethylphosphonium cation (membrane potential, delta psi) and either dimethyloxazolidinedione or methylamine (osmotic component, delta pH). As the pH of the medium was adjusted from 5.7 to 9.0, the proton motive force steadily decreased from about 170 to 100 mV. This decline occurred, despite a large increase in the membrane potential to its maximum value at pH 9.0, because of the loss of the pH gradient (inside alkaline). The cytoplasm and medium were of equal pH at 7.5; membrane permeability properties were lost at the pH extremes of 5.0 and 9.5. Protonophores and monensin prevented the net efflux of protons normally found when an oxygen pulse was given to an anaerobic cell suspension. A Na+/H+ antiport activity was measured for both Na+ influx and efflux and was shown to be dissipated by protonophores and monensin. These results strongly favor the concept that respiratory energy is used for proton efflux and that the resulting proton motive force may be converted to a sodium motive force through Na+/H+ antiport (driven by delta psi). A role for antiport activity in pH regulation of the cytosol can also explain the broad pH range for optimal growth, extending to the alkaline extreme of pH 9.0.     

Tetracycline/H+ antiport and Na+/H+ antiport catalyzed by the Bacillus subtilis TetA(L) transporter expressed in Escherichia coli.

The properties of TetA(L)-dependent tetracycline/proton and Na+/proton antiport were studied in energized everted vesicles of Escherichia coli transformed with a cloned tetA(L) gene (pJTA1) from Bacillus subtilis. Inhibition patterns by valinomycin and nigericin indicated that both antiports were electrogenic, in contrast to the tetracycline/proton antiport encoded by gram-negative plasmid tet genes. Tetracycline uptake in the everted system was dependent upon a divalent cation, with cobalt being the preferred one. The apparent Km for tetracycline was markedly increased at pH 8.5 versus pH 7.5, whereas the Vmax was unchanged. The much higher apparent Km for Na+ decreased at pH 8.5 relative to that at pH 7.5, as did the Vmax. Na+ did not affect tetracycline uptake, nor did Co2+ and/or tetracycline affect Na+ uptake; complex patterns of inhibition by amiloride and analogs thereof were observed.     

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.     

Extracellular Na+, but not Na+/H+ exchange, is necessary for receptor-mediated arachidonate release in platelets.

The effect of extracellular Na+ removal and replacement with other cations on receptor-mediated arachidonate release in platelets was studied to investigate the role of Na+/H+ exchange in this process. Replacement with choline+, K+, N-methylglucamine+ (which abolished the thrombin-induced pHi rise) or Li+ (which allowed a normal thrombin-induced pHi rise) significantly decreased arachidonate release in response to all concentrations (threshold to supra-maximal) of thrombin and collagen. This inhibition was not reversed by NH4Cl (10 mM) addition, which raised the pHi in the absence of Na+, but, on the contrary, NH4Cl addition further decreased the extent of thrombin- and collagen-induced arachidonate release, as well as decreasing 'weak'-agonist (ADP, adrenaline)-induced release and granule secretion in platelet-rich plasma. No detectable pHi rises were seen with collagen (1-20 micrograms/ml) and ADP (10 microM) in bis-(carboxyethyl)carboxyfluorescein-loaded platelets. Inhibition of thrombin-induced pHi rises was seen with 0.5-5 microM-5-NN-ethylisopropylamiloride (EIPA), but at these concentrations EIPA had little effect on thrombin-induced arachidonate release. At higher concentrations such as those used in previous studies (20-50 microM), EIPA inhibited aggregation/release induced by collagen and ADP in Na+ buffer as well as in choline+ buffer (where there was no detectable exchanger activity), suggesting that these concentrations of EIPA exert 'non-specific' effects at the membrane level. The results suggest that (i) Na+/H+ exchange and pHi elevations are not only necessary, but are probably inhibitory, to receptor-mediated arachidonate release in platelets, (ii) inhibition of receptor-mediated release in the absence of Na+ is most likely due to the absent Na+ ion itself, and (iii) caution should be exercised in the use of compounds such as EIPA, which, apart from inhibiting the Na+/H+ exchanger, have other undesirable and misleading effects in platelets.     

Protein phosphorylation during activation of Na+/H+ exchange by phorbol esters and by osmotic shrinking. Possible relation to cell pH and volume regulation

In lymphocytes, the Na+/H+ antiport can be stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and by osmotic shrinking. Since TPA acts by stimulating protein kinase C, we undertook experiments to determine if protein phosphorylation also underlies the osmotic stimulation of the antiport. We found that at least one of the membrane polypeptides labeled in cells treated with TPA is also phosphorylated by hypertonic shrinking. In both instances phosphorylation is alkali labile and associated with serine and threonine residues. We tested the possibility that shrinking activates phospholipase C, thereby stimulating protein kinase C through release of diacylglycerol. No decrease in phosphatidylinositol 4,5-bisphosphate levels was detected in hypertonically treated cells. Moreover, the concentrations of inositol phosphates, including inositol trisphosphate, were not altered in shrunken cells. Thus, shrinking does not appear to activate phospholipase C. Whereas TPA induced intracellular redistribution of soluble protein kinase C, no such effect was detected in osmotically activated cells. It was concluded that osmotic stimulation of the Na+/H+ antiport is associated with activation of protein phosphorylation by a kinase that is similar, but not identical to protein kinase C. Experiments in Na+-free or amiloride-containing media indicate that phosphorylation is not a consequence of activation of the antiport.     

Properties of two different Na+/H+ antiport systems in alkaliphilic Bacillus sp. strain C-125.

Na+/H+ antiport was studied in alkaliphilic Bacillus sp. strain C-125, its alkali-sensitive mutant 38154, and a transformant (pALK2) with recovered alkaliphily. The transformed was able to maintain an intracellular pH (pHin) that was lower than that of external milieu and contained an electrogenic Na+/H+ antiporter driven only by delta psi (membrane potential, interior negative). The activity of this delta psi-dependent Na+/H+ antiporter was highly dependent on pHin, increasing with increasing pHin, and was found only in cells grown at alkaline pH. On the other hand, the alkali-sensitive mutant, which had lost the ability to grow above pH 9.5, lacked the delta psi-dependent Na+/H+ antiporter and showed defective regulation of pHin at the alkaline pH range. However, this mutant, like the parent strain, still required sodium ions for growth and for an amino acid transport system. Moreover, another Na+/H+ antiporter, driven by the imposed delta pH (pHin > extracellular pHout), was active in this mutant strain, showing that the previously reported delta pH-dependent antiport activity is probably separate from delta psi-dependent antiporter activity. The delta pH-dependent Na+/H+ antiporter was found in cells grown at either pH 7 or pH 9. This latter antiporter was reconstituted into liposomes by using a dilution method. When a transmembrane pH gradient was applied, downhill sodium efflux was accelerated, showing that the antiporter can be reconstituted into liposomes and still retain its activity.     

The amiloride sensitive Na+/H+ antiport in guinea pig pancreatic acini. Characterization and stimulation by caerulein

Amiloride and analogs decrease the initial rate of 22Na+ uptake by dispersed acini from guinea pig pancreas in a dose-dependent manner. The initial rate of amiloride-sensitive 22Na+ uptake depends on external Na+ and H+ concentrations and on internal pH. These results provide evidence for the existence of a Na+/H+ antiport in pancreatic acinar cells. Caerulein, a cholecystokinin analog, stimulates the activity of the Na+/H+ antiport.     

Control of cytoplasmic pH by Na+/H+ exchange in rat peritoneal macrophages activated with phorbol ester

The mechanisms underlying cytoplasmic pH (pHi) regulation in elicited rat peritoneal macrophages were investigated by electronic sizing and fluorescence determinations. Acid-loaded cells rapidly regained normal pHi by means of an amiloride-sensitive Na+/H+ exchange. When stimulated by 12-O-tetradecanoyl phorbol 13-acetate, macrophages displayed a biphasic pHi change: a marginal acidification followed by an alkalinization. The latter results from activation of Na+/H+ exchange, since it is Na+-dependent and prevented by amiloride. When the antiport is inhibited, the full magnitude of the initial acidification can be appreciated. This acidification is independent of the nature of the ionic composition of the medium and probably reflects accumulation of protons generated during the metabolic burst. Under physiological conditions, these protons are rapidly extruded by the Na+/H+ antiport.     

Cytotoxic T lymphocyte (CTL)-mediated cytolysis proceeds in the absence of Na+/H+ antiport activity: regulation of cytosolic pH by the Na+/H+ antiport in a cloned CTL.

Cytotoxic T lymphocyte (CTL)-mediated cytolysis of specifically bound target cells (TC) is thought to be triggered by cross-linking the T-cell antigen receptor (TcR). Biochemical events associated with TcR cross-linking include increased intracellular calcium levels [Ca2+]i, hydrolysis of phosphatidylinositol (PI), and an increase in intracellular pH [pH]i. Whereas CTL-mediated cytolysis of some TC is calcium-dependent, and PI hydrolysis is speculated to trigger the CTL lethal hit via activation of PKC, little is known about changes in [pH]i relating to activation of the lethal hit stage. We report regulation of [pH]i in a cloned CTL by the electroneutral Na+/H+ antiport during activation with PMA and specific antigen-bearing TC. Furthermore, using 5-(N-methyl-N-isobutyl) amiloride (MIBA), a potent antiport inhibitor, we demonstrate that Na+/H+ exchange is not required for activation of CTL cytolytic activity.     

Insulin stimulation of Na/H antiport in L-6 cells: A different mechanism in myoblasts and myotubes

Insulin modulation of the Na/H antiport of L-6 cells, from rat skeletal muscle was studied in both myoblasts and myotubes using the fluorescent, pH sensitive, intracellular probe 2′,7′ bis (carboxyethyl)-5(6)-carboxyfluorescein. Insulin stimulated the Na/H antiport activity in L-6 cells, showing a bell-shaped dose response typical of other insulin responses: a maximum at 10 nM (ΔpH of 0.132 ± 0.007 and 0.160 ± 0.040 over basal value, for myoblasts and myotubes, respectively; means ± SD, n = 6–8) and smaller effects at higher and lower concentrations. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated the antiport in myoblasts but not in myotubes. Surprisingly the rapid increase in intracellular pH was not observed when insulin and PMA were added simultaneously to myoblasts; apparently these two activators mutually excluded each other. Downregulation of protein kinase C, obtained by preincubation of cells with PMA for 20 hr, totally abolished both hormone and PMA effects in myoblasts, whereas in myotubes insulin stimulation was not affected. Inhibitors of tyrosine kinase activity, such as erbstatin analog and genistein abolished insulin effect on the Na/H antiport, both in myoblasts and in myotubes. Different sensitivity to pertussis toxin in the two cell types suggests that the differentiation process leads to a change in the signal pathways involved in the physiological response to insulin. J. Cell. Physiol. 171:235–242, 1997. © 1997 Wiley-Liss, Inc.     

Rapid activation of Na+/H+ exchange by EPEC is PKC mediated

Enteropathogenic Escherichia coli (EPEC) increases sodium/hydrogen exchanger 2 (NHE2)-mediated sodium uptake by intestinal epithelial cells in a type III secretion-dependent manner. However, the mechanism(s) underlying these changes are not known. This study examines the role of a number of known secreted effector molecules and bacterial adhesins as well as the signaling pathways involved in this process. Deletion of the bacterial adhesins Tir and intimin had no effect on the increase in sodium/hydrogen exchanger (NHE) activity promoted by EPEC infection; however, there was a significant decrease upon deletion of the bundle-forming pili. Bacterial supernatant also failed to alter NHE activity, suggesting that direct interaction with bacteria is necessary. Analysis of the signal transduction cascades responsible for the increased NHE2 activity during EPEC infection showed that PLC increased Ca2+, as well as PKCalpha and PKCepsilon were involved in increasing NHE activity. The activation of PKCepsilon by EPEC has not been previously described nor has its role in regulating NHE2 activity. Because EPEC markedly increases NHE2 activity, this pathogen provides an exceptional opportunity to improve our understanding of this less-characterized NHE isoform.