TPA induces cytoplasmic alkalinization in human monoblastic U-937 cells without activation of Na+/H+ exchange

The effect of the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on cytoplasmic pH (pHi) and H+ extrusion was studied in the human monoblastic cell line U-937. About 2 min after addition of TPA, pHi started to increase and reached a steady state 10-15 min later. The resulting alkalinization corresponded to 0.03 and 0.09 pH units at 10(-10) and 10(-7) M TPA, respectively. The TPA-induced increase in pHi was independent of the presence of extracellular Na+. Moreover, TPA did not affect the H+ extrusion from the U-937 cells. Together these observations indicate the presence of a novel mechanism for TPA-induced cytoplasmic alkalinization. This mechanism is independent of Na+/H+ exchange across the plasma membrane, but may involve organelle sequestration of H+.     

Evidence that Na+/H+ exchange regulates angiotensin II-stimulated diacylglycerol accumulation in vascular smooth muscle cells

Angiotensin II stimulation of vascular smooth muscle cells results in initial, rapid diacylglycerol (DG) formation from the polyphosphoinositides accompanied by intracellular acidification, as well as a more sustained DG accumulation which is accompanied by a prolonged intracellular alkalinization. To determine whether intracellular pH (pHi) modulates DG accumulation, NH4Cl and potassium acetate were used to alter pHi and DG formation was measured. NH4Cl (10 mM) increased pHi from 7.15 +/- 0.05 to 7.34 +/- 0.02 pH units and markedly enhanced the sustained (5 min), but not the initial (15 s), phase of DG formation in response to 100 nM angiotensin II (65 +/- 13% increase). Conversely, intracellular acidification with Na+-free buffer and potassium acetate (20 mM) decreased pHi to 6.93 +/- 0.08 and reduced subsequent angiotensin II-induced sustained DG formation by 82 +/- 9%. In intact cells, inhibition of angiotensin II-stimulated alkalinization by incubation in Na+-free buffer or by addition of the Na+/H+ exchange inhibitor dimethylamiloride (10 microM) decreased the ability of the cell to sustain DG formation, suggesting that active Na+/H+ exchange is necessary for continued DG formation. Thus, it seems that sustained, angiotensin II-induced diacylglycerol accumulation is regulated by intracellular alkalinization secondary to Na+/H+ exchange in cultured vascular smooth muscle cells.     

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.     

Characterization of Na+/H+ exchange in FRTL-5 thyroid cells. Evidence for dependence on activation of protein kinase C.

Na+/H+ exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pHi) was 7.13 +/- 0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity beta i was determined using the NH4Cl-pulse method, yielding a beta i value of 85 +/- 12 mM/pH unit. The relationship between extracellular Na+ and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent Km value of 44 +/- 26 mM, and an Vmax value of 0.3 +/- 0.01 pH unit/min. The agonist-induced activation of Na+/H+ exchange was investigated in cells acidified with nigericin. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 microM amiloride, by substituting extracellular Na+ with equimolar concentrations of choline+, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca2+, or depleating intracellular Ca2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na+/H+ exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.     

Cytoplasmic pH in cultured porcine thyroid cells: alkalinization by epidermal growth factor

We studied the effects of epidermal growth factor (EGF), thyroid-stimulating hormone (TSH) and amiloride on cytoplasmic pH (pHi) in cultured porcine thyroid cells. We used 2',7'-bis(2-carboxyethyl)-5- (and 6-)carboxyfluorescein (BCECF), an internalized fluorescent pH indicator, to measure pHi. EGF stimulated thyroid cell alkalinization and proliferation, which were blocked by amiloride. EGF-stimulated thyroid cell alkalinization depended on extracellular Na+ concentrations. EGF stimulation resulted in an activation of Na+/H+ exchange, which alkalinized the cells. The results indicated that Na+/H+ exchange or cell alkalinization might function as a transmembrane signal transducer in the action of EGF. In the present system, TSH did not stimulate alkalinization or proliferation.     

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.     

Na/H exchange in cultured chick heart cells. pHi regulation

The purpose of this study was to establish the existence of Na/H exchange in cardiac muscle and to evaluate the contribution of Na/H exchange to pHi regulation. The kinetics of pHi changes in cultured chick heart cells were monitored microfluorometrically with 6-carboxyfluorescein and correlated with Nai content changes analyzed by atomic absorption spectrophotometry; transmembrane H+ movements were evaluated under pH stat conditions. After induction of an intracellular acid load by pretreatment with NH4Cl, a regulatory cytoplasmic alkalinization occurred with a t1/2 of 2.9 min. pHi regulation required external Na+ and was concomitant with transmembrane H+ extrusion as well as a rapid rise in Nai content in an Na/H ratio of 1:1. Microelectrode recordings of membrane potential demonstrated directly the electroneutral character of pHi regulation. Acid-induced net Na+ uptake could be either stimulated by further decreasing pHi or inhibited by decreasing pHo; Na+ uptake was unaffected by tetrodotoxin (10 micrograms/ml), quinidine (10(-3) M), DIDS (10(-4) M), Clo-free solution, or HCO3-free solution. Amiloride (10(-3) M) maximally inhibited both pHi regulation and Na+ uptake; the ID50 for amiloride inhibition of Na+ uptake was 3 microM. Nao-dependent H+ extrusion showed half-maximal activation at 15 mM Nao; Li+, but not K+ or choline+, could substitute for Na+ to support H+ extrusion. Cao-free solution also stimulated acid-induced Na+ uptake. We conclude that pHi regulation following an acid load in cardiac muscle cells is by an amiloride-sensitive, electroneutral Na/H exchange. Stimulation of Na/H exchange up to 54 pmol/cm2 X s indicates the rapidity of this exchange across cardiac cell membranes. Na/H exchange may also participate in steady state maintenance of pHi.     

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes

The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+.     

Priming effect of hyperosmotic stress on TRH-induced activation of Na+/H+ exchange in GH4C1 rat pituitary cells

The effect of mild hyperosmotic stress on cytosolic pH (pHi) alone, and in combination with thyrotropin-releasing hormone (TRH) or the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was investigated in GH4C1 cells at resting pHi. Hyperosmotic stress induced by addition of 50 mM choline was without an effect on pHi. In cells stimulated with either TRH or TPA after choline, pHi increased 0.15 +/- 0.05 and 0.14 +/- 0.03 pH units, respectively (mean +/- SD). A similar response was obtained if TRH or TPA was added prior to choline. The effect was abolished by replacing extracellular Na+ with choline+, and by pretreatment of the cells with amiloride, indicating that the change in pHi probably was dependent on activation of Na+/H+ exchange. The results thus indicate that, in GH4C1 cells, hyperosmotic stress in combination with TRH or TPA can activate Na+/H+ exchange at resting pHi levels.     

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.     

Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell

Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in the cytosol. The ability of this cell type to regulate cytosolic pH (pHi) was examined as a function of stimulation of acid secretion by histamine or forskolin. The pHi was estimated from the change of fluorescence of the trapped dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-bis-carboxyethylcarbo xy fluorescein in a purified cell suspension of rabbit parietal cells. Stimulation of the cell suspension raised pHi by an average of 0.13 +/- 0.038 pH units. The H+,K+-ATPase inhibitor, SCH28080 (2-methyl-8-[phenyl-methoxy]-imidazo-(1,2)-pyridine-3-acetonitrile) had only a small effect on the increase of pHi, therefore, was largely independent of H+,K+-ATPase activity. In Na+-free medium, where Na+/H+ exchange would be absent, the rise of pHi was only 0.03 pH units. This increase was blocked by SCH28080, showing that this small increment was the result of acid secretion. In Na+-containing medium, 90% of the increase was inhibited by an inhibitor of Na+/H+ exchange, dimethyl amiloride (DMA). This compound also blocked changes in pHi due to changes in extracellular Na+. Accordingly, most of the change in pHi upon stimulation of acid secretion by histamine and forskolin is due to activation of Na+/H+ exchange in the parietal cell basal-lateral membrane. The addition of DMA to stimulated, but not resting cells, gave a rapid acidification that was blocked by inhibition of anion exchange by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), showing that anion exchange was also activated by stimulation. In single cell recording, canalicular and cytosolic pH were monitored simultaneously using 9-amino acridine and dimethyl carboxyfluorescein, respectively. Cytosolic alkalinization correlated with acid accumulation in the secretory canaliculus until a set point was reached. Thereafter, acidification continued without further change in pHi. To determine the role of Na+/H+ and Cl-/HCO3- exchange in acid secretion, Cl(-)-depleted cells were suspended in medium containing 40 mM Cl-. DMA and DIDS each blocked acid secretion by about 40%, but in combination, acid secretion was blocked by more than 90%. Thus, basal-lateral Na+/H+ and Cl-/HCO3- exchange activities are necessary for acid secretion across the apical membrane of the parietal cell.     

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.1 units/ml) increased [Ca2+]i with no detectable lag period to maximum levels within 13 s followed by a slow return to resting levels. There was a transient decrease in pHi within 9 s that was immediately followed by an alkalinization response, attributable to activation of Na+/H+ exchange, that raised pHi above resting levels within 22 s. At 10-15 degrees C, thrombin-induced changes in [Ca2+]i and pHi were delayed and therefore better resolved, although no differences in the magnitude of changes in [Ca2+]i and pHi were observed. However, the increase in [Ca2+]i had peaked or was declining before the alkalinization response was detected, suggesting that Ca2+ mobilization occurs before activation of Na+/H+ exchange. In platelets preincubated with 5-(N-ethyl-N-isopropyl)amiloride or gel-filtered in Na+-free buffer (Na+ replaced with N-methyl-D-glutamine) to inhibit Na+/H+ exchange, thrombin stimulation caused a rapid, sustained decrease in pHi. Under these conditions there was complete inhibition of the alkalinization response, whereas Ca2+ mobilization was only partially inhibited. Nigericin (a K+/H+ ionophore) caused a rapid acidification of more than 0.3 pH unit that was sustained in the presence of 5-(N-ethyl-N-isopropyl)amiloride. Subsequent stimulation with thrombin resulted in slight inhibition of Ca2+ mobilization. These data show that, in human platelets stimulated with high or low concentrations of thrombin, Ca2+ mobilization can occur without a functional Na+/H+ exchanger and in an acidified cytoplasm. We conclude that Ca2+ mobilization does not require activation of Na+/H+ exchange or preliminary cytoplasmic alkalinization.     

Regulation of cytoplasmic pH in resident and activated peritoneal macrophages

Cytoplasmic pH (pHi) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pHi and/or its regulation existed between activated and resident macrophages (RES MOs) which might explain the increased NADPH oxidase activity observed in the former. The pHi of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pHi did not differ between resident (RES) and elicited (ELI) MOs (7.16 +/- 0.05 and 7.20 +/- 0.05, respectively). pHi recovery after intracellular acid loading was partially dependent on the presence of Na+ in the extracellular medium, and was partially inhibited by the Na+/H+ antiport inhibitor, amiloride. At comparable pHi, the rate of acid extrusion during recovery was not different in RES and ELI MOs (1.48 +/- 0.12 and 1.53 +/- 0.06 mM/min, respectively). In both RES and ELI MOs, approx. 40% of total pHi recovery was insensitive to amiloride and independent of extracellular Na+. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pHi response: an initial acidification followed by a sustained alkalinization to a new steady-state pHi. This alkalinization was Na(+)-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na+/H+ antiport activity. The new steady-state pHi attained after TPA stimulation was equivalent in RES and ELI MOs (7.28 +/- 0.04 and 7.31 +/- 0.06, respectively), indicating comparable stimulated Na+/H+ antiport activity. However, the initial acidification induced by TPA was greater in ELI than in RES MOs (0.18 +/- 0.02 vs. 0.06 +/- 0.02 pH unit, respectively, P less than 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pHi reduction by only about 50%. This suggested that the TPA-induced pHi reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.     

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.     

Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium

The regulation of intracellular pH (pHi) was monitored in a virus-transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate-free media using the pH-sensitive absorbance of 5- (and 6-) carboxy-4',5'-dimethylfluorescein. Under resting conditions, pHi averaged 6.98 +/- 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and then withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ -depleted cells led to an alkalinization, which was sensitive to amiloride, with an IC50 of about 20 microM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.     

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.     

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

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

Cytoplasmic pH in the action of epidermal growth factor (EGF) in cultured porcine thyroid cells

We demonstrate measurement of cytoplasmic pH (pHi), using 2',7'-bis(2-carboxyethyl)-5 (and 6-) carboxyfluorescein (BCECF), and internalized fluorescent pHi indicator, in thyroid cells. Using cultured porcine thyroid cells, we studied the effects of epidermal growth factor (EGF) on pHi and [3H] thymidine incorporation; 10 nM EGF alkalinizes thyroid cells and stimulates thymidine incorporation. The results indicate that Na+/H+ exchange or cell alkalinization may function as a transmembrane signal transducer in the action of EGF in the thyroid cells.     

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.     

Alpha 2-adrenergic receptors accelerate Na+/H+ exchange in neuroblastoma X glioma cells

The regulation of cytoplasmic pH (pHi) was examined in neuroblastoma X glioma hybrid cell-line cells (NG108-15 cells) using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein. The pHi of NG108-15 cells suspended in nominally HCO-3-free, Na+-containing buffer could be reduced by the external application of acetate. The recovery of pHi to its resting value was blocked by the removal of extracellular Na+, by the addition of extra-cellular H+, and by the addition of analogs of amiloride selective for inhibition of Na+/H+ exchange. The rate of recovery of pHi from acid load exhibited an ionic selectivity of Na+ greater than Li+ much greater than K+, and no recovery was observed in N-methyl-D-glucamine+. Tetrodotoxin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on early pHi recovery. These data suggest that Na+/H+ exchange accounts primarily for the recovery of pHi in NG108-15 cells under our experimental conditions. Na+/H+ exchange in NG108-15 cells was accelerated by alpha 2-adrenergic receptors. Thus, (-)epinephrine, but not (+)epinephrine, elicited an intracellular alkalinization which was blocked by the alpha 2-adrenergic receptor selective antagonist yohimbine but not by the alpha 1-adrenergic receptor antagonist, prazosin, nor the beta-adrenergic antagonist, propranolol. Norepinephrine, clonidine, and the clonidine analog, UK-14304, also caused alkalinization of NG108-15 cells, whereas isoproterenol, a beta-adrenergic receptor agonist, and phenylephrine, a selective alpha 1-adrenergic receptor agonist, did not. Manipulations that blocked Na+/H+ exchange blocked the ability of alpha 2-adrenergic agonists to alkalinize the interior of NG108-15 cells without blocking the ability of these agonists to attenuate cAMP accumulation. These findings provide the first direct evidence of modulation of Na+/H+ exchange activity by a receptor linked to inhibition of adenylate cyclase and offer a possible mechanism whereby alpha 2-adrenergic receptors might influence cellular activity apart from changes in cyclic nucleotide metabolism.