Stimulation of leucine transport by a phorbol ester through activation of protein kinase C and Na+/H+ exchanger

A synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) has been found to elevate the cytoplasmic pH and increase leucine uptake dose-dependently, when added to quiescent cultures of Chang liver cell. Addition of either a protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), or an Na+/H+ antiporter inhibitor, ethylisopropylamiloride (EIPA), abolished completely or incompletely the TPA-stimulated leucine uptake and TPA-induced cytoplasmic alkalinization. Therefore the stimulation of leucine uptake by OAG and TPA is proposed to be elicited at least partly through activation of Na+/H+ antiporter. We suggest that activation of protein kinase C by the phorbol ester is responsible for the stimulation of Na+/H+ exchange system and also leucine uptake in the cell.     

Reconstitution of a bacterial Na+/H+ antiporter

Membrane proteins from alkalophilic Bacillus firmus RAB were extracted with octylglucoside, reconstituted into liposomes made from alkalophile lipids. The proteoliposomes were loaded with 22Na+. Imposition of a valinomycin-mediated potassium diffusion potential, positive out, resulted in very rapid efflux of radioactive Na+ against its electrochemical gradient. That the Na+ efflux was mediated by the electrogenic Na+/H+ antiporter is indicated by the following characteristics that had been established for the porter in previous studies: dependence upon an electrical potential; pH sensitivity, with activity dependent upon an alkaline pH; inhibition by Li+; and an apparent concentration dependence upon Na+ that correlated well with measurements in cells and membrane vesicles.     

Inhibition by pertussis toxin of fibroblast growth factor-stimulated hexose transport in Swiss 3T3 cells

Addition of fibroblast growth factor to quiescent cultures of Swiss 3T3 cells stimulated the membrane transport of 2-deoxyglucose. Treatment of the cells with pertussis toxin (islet-activating protein) inhibited fibroblast growth factor-stimulated hexose transport. 5'-Guanylyl imidodiphosphate (p[NH]ppG), a non hydrolyzable analogue of GTP, increased the number of hexose carriers in the plasma membrane of saponin-permeabilized cells. These results suggest that guanine nucleotide binding protein may be involved in the regulation of hexose transport system by fibroblast growth factor in Swiss 3T3 cells.     

Effect of staurosporine on the phorbol ester-induced activation of the Na+/H+ antiporter in smooth muscle cells

Evidence is presented suggesting that the Na+/H+ antiporter activity of aortic smooth muscle cells is stimulated by protein kinase C activation. However, once the transporter has been activated, inhibitors of protein kinase C are not effective, supporting a model in which the Na+/H+ antiporter conserves memory of its activation by protein kinase C.     

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.     

Palytoxin acidifies chick cardiac cells and activates the Na+/H+ antiporter

The cardiotoxic action of palytoxin was investigated using embryonic chick ventricular cells. Under normal ionic conditions, palytoxin produced an intracellular acidification which is partially compensated for by the Na+/H+ antiporter thereby leading to an increased rate of ethylisopropylamiloride-sensitive 22Na+ uptake. Under depolarizing membrane conditions, palytoxin produced a cellular acidification, a cellular alkalinization or no change in intracellular pH depending on the value of the extracellular pH. We propose that palytoxin acidifies cardiac cells by opening preexisting H+ conducting pathways in the plasma membrane.     

Intracellular Ca2+ potentiates Na+/H+ exchange and cell differentiation induced by phorbol ester in U937 cells

The human cell line U937 differentiates to monocyte macrophage-like cells in response to tumour-promoting phorbol esters. This effect is attributed to activation of protein kinase C. We show here that U937 cell differentiation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) is associated with cytoplasmic alkalinization. Ethyl-isopropyl-amiloride (EIPA), a potent inhibitor of Na+/H+ exchange, blocked both cytoplasmic alkalinization and cell differentiation. Cell acidification by addition of 2-4 mM sodium propionate also blocked TPA-induced U937 cell differentiation. These results suggest that a sustained cell alkalinization mediated by activation of Na+/H+ exchange is essential for TPA-induced differentiation in U937 cells. The increase of cytoplasmic free calcium concentration ([Ca2+]i) by addition of the calcium ionophore ionomycin enhanced TPA-induced alkalinization by increasing the apparent affinity of the Na+/H+ antiporter for intracellular H+. Treatment with ionomycin also potentiated differentiation of U937 cells induced by TPA. This synergism suggests that [Ca2+]i either potentiates the activation of protein kinase C or triggers additional transducing mechanisms. The key events of this interaction occur during the first 30 min of treatment, even though cell differentiation manifests much later.     

Desensitization by external Na of the cyclic AMP-dependent Na+/H+ antiporter in trout red blood cells

The erythrocytes of the trout, Salmo gairdneri, react to beta-adrenergic stimulation by activating a cyclic AMP-dependent and amiloride-sensitive Na+/H+ antiporter (see Borgese, F., F. Garcia-Romeu, and R. Motais, Journal of General Physiology, 1986, 87:551-566). The present study traces the kinetic behavior of the unidirectional Na fluxes after stimulation by isoproterenol. A very considerable increase (100-fold) of the unidirectional Na influx (JNa(in)) follows the addition of isoproterenol to the erythrocyte suspension. After 1.5 min, JNa(in) falls suddenly, and asymptotically diminishes toward the nonstimulated flux level. The unidirectional Na efflux (JNa(out)) proceeds according to similar kinetics. The decrease of JNa(in) and JNa(out)is not linked to either a change in the driving forces of the transported ions or a decrease of the cyclic AMP concentration but to a desensitization of the Na+/H+ antiporter. This desensitization is dependent on the external Na concentration and is not controlled by internal Na, cell swelling, or external Ca.     

A stable Na+/H+ antiporter of thermophilic bacterium PS3

As a first step in the isolation of a stable Na⁺/H⁺ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na⁺, Li⁺, Mn²⁺, Cd²⁺, and Co²⁺, but not of choline⁺ or Ca²⁺, regardless of their counter anions.²²Na⁺ was taken up into the vesicles by NADH oxidation, and the²²Na⁺ uptake was inhibited by the addition of an uncoupler. H⁺ release was observed on addition of Na⁺ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na⁺/H⁺ antiport was affected by the pH of the medium (optimum pH=8.5). TheK _m 's of the antiporter for Na⁺ and Li⁺ were 2.5 and 0.1 mM, respectively, but theV _max values for the two ions were the same at pH 8.0. In the presence of Li⁺, no further decrease of fluorescence quenching was observed on addition of Na⁺ andvice versa. The Na⁺/H⁺ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H⁺ antiporters, this antiporter was not inhibited by a thiol reagent.Abbreviations Tricine N-tris(hydroxymethyl)methylglycine - MOPS morpholinopropane sulfonic acid - TMAHO tetramethylammonium hydroxide - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - H⁺ — ATPase proton-translocating adenosine triphosphatase - electrochemical proton gradient across membrane - electrochemical Na⁺ gradient across membrane - pH pH difference across membrane     

Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+

Growth factors (alpha-thrombin and insulin) activate a Na+/H+ antiport in G0/G1-arrested Chinese hamster lung fibroblasts (CCL39). In this report, we have examined the influence of intracellular pH on this exchange activity, measured by initial rates of amiloride-sensitive 22Na+ uptake, in the absence and presence of growth factors. Our results indicate the following. 1) In quiescent as in mitogen-stimulated cells, Na+/H+ antiport is regulated by internal H+ in an allosteric way, whereas, in contrast, interactions with external H+ and Na+ obey simple saturation kinetics. 2) The growth factor-induced activation of Na+/H+ exchange, which, under physiological conditions, is responsible for a sustained cytoplasmic alkalinization, is due to an increased affinity for internal H+ (the apparent pK is shifted by approximately 0.3 pH unit towards alkaline pH values). Therefore, we propose that growth factors promote a conformational change of the Na+/H+ antiporter, possibly at the level of an internal modifier site(s).     

Ha-ras activates the Na+/H+ antiporter by a protein kinase C-independent mechanism

In quiescent Ha-ras-transfected NIH 3T3 cells, addition of serum growth factors, bombesin or 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a dimethylamiloride-sensitive intracellular alkalinization which can be inhibited by staurosporine, a potent inhibitor of protein kinase C. Expression of the transforming Ha-ras gene causes a growth factor-independent increase in cytoplasmic pH. This Ha-ras-induced alkalinization is sensitive to dimethylamiloride but is not affected by staurosporine concentrations which prevent the pH response after addition of growth factors or TPA. Protein kinase C depletion by long term exposure to TPA eliminates the pH response to bombesin and phorbol ester but does not effect the Ha-ras-induced intracellular alkalinization. It is concluded that expression of Ha-ras causes an activation of the Na+/H+ antiporter by an as yet unknown protein kinase C-independent mechanism.     

Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption

We have examined the effects of inhibitors of proton transport systems on osteoclastic bone resorption using an in vitro bone slice assay, where osteoclasts (OCs) are free from the influence of other bone cells. Amiloride (AM) and dimethylamiloride (DMA), inhibitors of the Na+/H+ antiporter, were potent inhibitors of bone resorption (IC50 approximately 9 and 0.7 microM for AM and DMA, respectively). Omeprazole (OM), a potent inhibitor of parietal cell K+/H+(-)ATPase, was a poor inhibitor of OC bone resorption (IC50 approximately 100 microM). These results strongly suggest that the Na+/H+ antiporter is the primary proton system used by OCs during bone resorption.     

Multiple effects of the Na+/H+ antiporter inhibitor HMA on cancer cells

Amiloride derivatives are a class of new promising chemotherapeutic agents. A representative member of this family is the sodium–hydrogen antiporter inhibitor HMA (5-(N,N-hexamethylene amiloride), which has been demonstrated to induce cellular intracytosolic acidification and cell death through the apoptotic pathway(s). This work aims at characterizing drug response of human cancer cell lines to HMA. After a first screening revealing that HMA interferes with cancer cell survival, we focused our attention on SW613-B3 colon carcinoma cells, which are intrinsically resistant to a panel of drugs. Searching for the activation of canonical apoptosis, we found that this process was abortive, given that the final steps of this process, i.e. PARP-1 cleavage and DNA ladder, were not detectable. Thus, we addressed caspase-independent paradigms of cell death and we observed that HMA promotes the induction of the LEI/L-DNase II pathway as well as of parthanatos. Finally, we explored the possible impact of autophagy of cell response to HMA, providing the evidence that autophagy is activated in our experimental system. On the whole, our results defined the biochemical reactions triggered by HMA, and elucidated its multiple effects, thus adding further complexity to the intricate network leading to drug resistance.     

Inhibition of cardiac sarcolemmal Na+/H+ antiporter by opioids.

In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the opioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,488H, a selective kappa-opioid agonist, we characterized the nature of interaction between opioids and the Na+/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+ antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-50,488H with the Na+/H+ antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 +/- 0.2 to 5.0 +/- 0.3 mM, while the Vmax (52.0 +/- 5.0 nmol.mg-1.min-1) remained the same. These results suggest that U-50,488H and Na+ compete for the same site on the antiporter. When testing the effect of U-50,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na+/Ca2+ antiporter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride, we found that U-50,488H (IC50 = 100 +/- 15 microM) was threefold more potent than amiloride (IC50 = 300 +/- 20 microM) but it was three-fold less potent than the amiloride analog (IC50 = 30 +/- 10 microM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-50,488H is more potent than amiloride, the inhibitory characteristics of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter are similar to amiloride.     

Dimethylsulfoxide modifies the sensitivity of BALB/c-3T3 cells to the activation of Na+/H+ exchange by phorbol esters

In a companion paper (Gillies et al.: J. Cell. Physiol. 139:124-129, 1989) we show that phorbol esters (PEs) are unable to stimulate Na+/H+ exchange in BALB/c-3T3 cells under a wide variety of conditions. The Na+/H+ exchangers of a number other cell types are also not responsive to PEs yet have been rendered responsive by treatment with agents such as dimethylsulfoxide (DMSO). We undertook the present study to evaluate whether or not the treatment of BALB/c-3T3 cells with DMSO will induce modifications in the sensitivity of these cells to activation by PEs. The present study indicates that a 3-5 day exposure of BALB/c-3T3 cells to 1.25% DMSO leads to changes in the sensitivity of these cells to the activation of Na+/H+ exchanger by PEs. These changes in sensitivity were apparent at day 3 and maximal at day 5. Non-tumor-promoting analogues of PEs do not activate Na+/H+ exchange, suggesting that the effect is mediated through kinase C. Sphingosine prevents PE-, but not serum-induced alkalinization. However, the half-time of the intracellular pH (pHin) response to serum was increased by sphingosine, suggesting that kinase C participates in, but is not required for, the serum induced activation. Since DMSO does not induce any apparent morphological change, the change in sensitivity of Na+/H+ exchange to PEs is not likely to be related to differentiation, but may be associated with structural changes in the Na+/H+ exchanger and/or changes in isoforms of kinase C which recognize the exchanger as a substrate.     

Mitogen-stimulated activation of the Na+/H+ antiporter does not regulate S6 phosphorylation or protein synthesis in murine thymocytes or Swiss 3T3 fibroblasts

The activation of protein synthesis by mitogens in quiescent (G0) mammalian cells is obligatory for progression from G0 through G1 to DNA synthesis in S phase. When the activation of the Na+/H+ antiporter which occurs in mitogen-stimulated Swiss 3T3 fibroblasts or murine fibroblasts is completely blocked by dimethylamiloride, there is little or no effect on the phosphorylation of the ribosomal protein S6 or the activation of protein synthesis assayed by [35S]methionine incorporation. Furthermore, the accumulation of the protein product of the activated c-myc gene is unaffected by dimethylamiloride in 3T3 fibroblasts. The data show that there is no requirement for activation of the Na+/H+ antiporter for the activation of S6 phosphorylation or protein synthesis by mitogens but do not preclude the possibility that activation of the antiporter is necessary for some other response(s) obligatory for DNA synthesis. These data are contrasted with previous reports for Chinese hamster lung fibroblasts that the increase in intracellular pH which results from activation of the Na+/H+ antiporter in bicarbonate-free media is necessary for S6 phosphorylation, protein synthesis, and hence, for subsequent DNA synthesis (Pouyssegur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3935-3939; Chambard, J.C., and Pouyssegur, J. (1986) Exp. Cell Res. 164, 282-294).     

Biology of the 2Na+/1H+ antiporter in invertebrates

The functional expression of membrane transport proteins that are responsible for exchanging sodium and protons is a ubiquitous phenomenon. Among vertebrates the Na+/H+ antiporter occurs in plasma membranes of polarized epithelial cells and non-polarized cells such as red blood cells, muscle cells, and neurons, and in each cell type the transporter exchanges one sodium for one hydrogen ion, is inhibited by amiloride, and regulates intracellular pH and sodium concentration within tight limitations. In polarized epithelial cells this transporter occurs in two isoforms, each of which is restricted to either the brush border or basolateral cell membrane, and perform somewhat different tasks in the two locations. In prokaryotic cells, sodium/proton exchange occurs by an electrogenic 1Na+/2H+ antiporter that is coupled to a primary active proton pump and together these two proteins are capable of tightly regulating the intracellular concentrations of these cations in cells that may occur in environments of 4 M NaCl or pH 10-12. Invertebrate epithelial cells from the gills, gut, and kidney also exhibit electrogenic sodium/proton exchange, but in this instance the transport stoichiometry is 2Na+/1H+. As with vertebrate electroneutral Na+/H+ exchange, the invertebrate transporter is inhibited by amiloride, but because of the occurrence of two external monovalent cation binding sites, divalent cations are able to replace external sodium and also be transported by this system. As a result, both calcium and divalent heavy metals, such as zinc and cadmium, are transported across epithelial brush border membranes in these animals and subsequently undergo a variety of biological activities once accumulated within these cells. Absorbed epithelial calcium in the crustacean hepatopancreas may participate in organismic calcium balance during the molt cycle and accumulated heavy metals may undergo complexation reactions with intracellular anions as a detoxification mechanism. Therefore, while the basic process of sodium/proton exchange may occur in invertebrate cells, the presence of the electrogenic 2Na+/1H+ antiporter in these cells allows them to perform a wide array of functions without the need to develop and express additional specialized transport proteins. J. Exp. Zool. 289:232-244, 2001.     

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.     

The Na+ cycle of extreme alkalophiles: A secondary Na+/H+ antiporter and Na+/solute symporters

Extremely alkalophilic bacteria that grow optimally at pH 10.5 and above are generally aerobic bacilli that grow at mesophilic temperatures and moderate salt levels. The adaptations to alkalophily in these organisms may be distinguished from responses to combined challenges of high pH together with other stresses such as salinity or anaerobiosis. These alkalophiles all possess a simple and physiologically crucial Na⁺ cycle that accomplishes the key task of pH homeostasis. An electrogenic, secondary Na⁺/H⁺ antiporter is energized by the electrochemical proton gradient formed by the proton-pumping respiratory chain. The antiporter facilitates maintenance of a pH_in that is two or more pH units lower than pH_out at optimal pH values for growth. It also largely converts the initial electrochemical proton gradient formed by respiration into an electrochemical sodium gradient that energizes motility as well as a plethora of Na⁺/solute symporters. These symporters catalyze solute accumulation and, importantly, reentry of Na⁺. The extreme nonmarine alkalophiles exhibit no primary sodium pumping dependent upon either respiration or ATP. ATP synthesis is not part of their Na⁺ cycle. Rather, the specific details of oxidative phosphorylation in these organisms are an interesting analogue of the same process in mitochondria, and may utilize some common features to optimize energy transduction.