Na+/H+ exchange in volume regulation and cytoplasmic pH homeostasis in lymphocytes

Osmotic shrinking activates an amiloride-sensitive Na+/H+ exchange in the membrane of blood and thymic lymphocytes. The exchange, which is virtually quiescent in isotonic conditions, can also be activated by lowering the cytoplasmic pH (pHi). Activation by pHi is largely caused by an allosteric interaction of H+ with a kinetic modifier site, different from the internal substrate site. The set point or threshold pHi for activation of the exchanger is dictated by the protonation of the modifier. Evidence is presented that indicates that cell shrinking alters the pHi sensitivity of the modifier, shifting the set point to more alkaline levels. In the presence of HCO3- and Cl- a volume increase will accompany the change in pHi. Volume changes can also be produced in isotonic solutions if the exchange is activated by acidification of the cytoplasm, e.g., by addition of propionate to the medium. The latter phenomenon provides a simple method for the detection of the Na+/H+ antiport by electronic cell sizing.     

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.     

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.     

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.     

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange

Summary Suspensions of LLC-PK₁ cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na⁺ and K⁺ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05±0.01,n=5) of cells which have recovered in (pH 7.4) Na⁺-containing medium is not affected over several minutes by addition of 100 M amiloride or removal of extracellular Na⁺ (Na _o ⁺ /H _i ⁺ and Na _i ⁺ /H _o ⁺ exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na _o ⁺ /H _i ⁺ exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H⁺ efflux or net Na⁺ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a set point of Na⁺/H⁺ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.     

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.     

Na+/H+ exchange and the regulation of intracellular pH in polymorphonuclear leukocytes

1. Regulation of the cytoplasmic pH(pHi) was studied in quiescent and activated human neutrophils. Acid-loaded unstimulated cells regulate pHi by activating an electroneutral Na+/H+ exchange. 2. When activated, neutrophils undergo a biphasic change in pHi: an acidification followed by an alkalinization. The latter is due to stimulation of the Na+/H+ antiport. 3. The acidification, which is magnified in Na+-free or amiloride-containing media, is associated with net H+ efflux from the cells. 4. A good correlation exists between cytoplasmic acidification and superoxide generation: inhibition of the latter by adenosine, deoxyglucose or pertussis toxin also inhibits the pHi changes. 5. Moreover, acidification is absent in chronic granulomatous disease patients, which cannot generate superoxide. 6. Regulation of pHi is essential for neutrophil function. The oxygen dependent bactericidal activity is inhibited upon cytoplasmic acidification. This can result from impairment of Na+/H+ exchange, or from influx of exogenous acid equivalents. 7. The latter mechanism may account for the inability of neutrophils to resolve bacterial infections in abscesses, which are generally made acidic by accumulation of organic acids that are by-products of bacterial anaerobic metabolism.     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Modulation of Na+/H+ exchange activity by Cl-

Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl- ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl- sensitivity of the individual isoforms has not been established. To explore their Cl- sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl- with nitrate or thiocyanate inhibited the activity of all isoforms. Cl- depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl- removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl- into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl- is critical for activity. Thus interaction of intracellular Cl- with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.     

Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification

After swelling in hypotonic solutions, peripheral blood mononuclear cells (PBM) shrink toward their original volumes. Upon restoration of isotonicity, the cells initially shrink but then regain near-normal size again. This regulatory volume increase (RVI) is abolished by removal of Na+o or Cl-o or by addition of amiloride. RVI is unaffected by removal of K+o or by ouabain and is only partially inhibited by 1 mM furosemide. As a result of increased influx, the cells gain both Na+ and K+ during reswelling. In contrast, only Na+ content increases in the presence of ouabain. Amiloride largely eliminates the changes in the content of both cations. Using diS-C3-(5), no significant membrane potential changes were detected during RVI, which suggests that the fluxes are electroneutral. The cytoplasmic pH of volume-static cells was measured with 5,6-dicarboxyfluorescein. After acid loading, the addition of extracellular Na+ induced an amiloride-inhibitable alkalinization, which is consistent with Na+/H+ exchange. Cytoplasmic pH was not affected by cell shrinkage itself, but an internal alkalinization, which was also amiloride sensitive and Na+ dependent, developed during reswelling. In isotonic lightly buffered solutions without HCO-3, an amiloride-sensitive acidification of the medium was measurable when Na+ was added to shrunken PBM. K+ was unable to mimic this effect. The observations are compatible with the model proposed by Cala (J. Gen. Physiol. 1980. 76:683-708), whereby an electroneutral Na+o/H+i exchange is activated by osmotic shrinking. Cellular volume gain occurs as Cl-o simultaneously exchanges for either HCO-3i or OH-i. Na+i is secondarily replaced by K+ through the pump, but this step is not essential for RVI.     

Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells

Summary The present studies were designed to test our previous suggestion that Na⁺/H⁺ exchange was activated by muscarinic stimulation of rat parotid acinar cells. Consistent with this hypothesis, we demonstrate here that intact rat parotid acini stimulated with the muscarinic agonist carbachol in HCO ₃ ^– -free medium show an enhanced recovery from an acute acid load as compared to similarly challenged untreated preparations. Amiloride-sensitive²²Na uptake, due to Na⁺/H⁺ exchange, was also studied in plasma membrane vesicles prepared from rat parotid acini pretreated with carbachol. This uptake was stimulated twofold relative to that observed in vesicles from control (untreated) acini. This stimulation was time dependent, requiring 15 min of acinar incubation with carbachol to reach completion, and ws blocked by the presence of the muscarinic antagonist atropine (2×10^–5 m) in the pretreatment medium. The effect of carbachol was dose dependent withK _0.53×10^–6 m. Stimulation of the exchanger was also seen in vesicles prepared from acini pretreated with the -adrenergic agonist epinephrine, but not with the -adrenergic agonist isoproterenol, or with substance P. Kinetic analysis indicated that the stimulation induced by carbachol was due to an alkaline shift in the pH responsiveness of the exchanger in addition to an increasedapparent transport capacity. Taken together with previous results from this and other laboratories, these results strongly suggest that the Na⁺/H⁺ exchanger and its regulation are intimately involved in the fluidsecretory response of the rat parotid.     

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells

Using thepH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF),we examined the effect of hyperosmolar solutions, which presumablycaused cell shrinkage, on intracellular pH(pH_i) regulation in mesangialcells (single cells or populations) cultured from the rat kidney. Thecalibration of BCECF is identical in shrunken and unshrunken mesangialcells if the extracellular K⁺concentration ([K⁺])is adjusted to match the predicted intracellular[K⁺]. ForpH_i values between ~6.7 and~7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH₂O) is threefold larger than in unshrunken cells (~300mosmol/kgH₂O). In the nominalabsence ofCO₂/HCO₃,exposing cell populations to a HEPES-buffered solution supplementedwith ~300 mM mannitol (600 mosmol/kgH₂O) causes steady-statepH_i to increase by ~0.4. The pH_i increase is due to activationofNa⁺/H⁺exchange because, in single cells, it is blocked in the absence ofexternal Na⁺ or in the presence of50 µM ethylisopropylamiloride (EIPA). Preincubating cells in aCl-free solution for atleast 14 min inhibits the shrinkage-induced pH_i increase by 80%. Wecalculated the pH_i dependence oftheNa⁺/H⁺exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) thepH_i dependence of the totalacid-extrusion rate and 2) thepH_i dependence of theEIPA-insensitive acid-loading rate. Shrinkage alkali shifts thepH_i dependence ofNa⁺/H⁺exchange by ~0.7 pH units.     

Volume changes in activated human neutrophils: The role of Na+/H+ exchange

The apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12-O-tetradecanoylphorbol 13, acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle-van't Hoff plots, and with an increase in Na⁺ content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na⁺ with K⁺ or N-methyl-D-glucamine⁺, or by addition of amiloride. Swelling was also inhibited by the 5-N-substituted analogs of amiloride, which are potent specific inhibitors of the Na⁺/H⁺ antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na⁺/H⁺ exchange, determined as a Na⁺-dependent and amiloride-sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na⁺/H⁺ exchange can lead to significant cell swelling in neutrophils.     

Thyrotropin-releasing hormone activates Na+/H+ exchange in rat pituitary cells.

The effects of thyrotropin-releasing hormone (TRH) and 12-O-tetradecanoylphorbol 13-acetate (TPA) on cytosolic pH (pHi) were studied on GH4C1 pituitary cells loaded with the fluorescent pH indicator bis(carboxyethyl)carboxyfluorescein (BCECF) and the fluorescent Ca2+ indicator quin2. TRH, which was minimally effective at around 10(-9) M, and TPA, 100 nM, produced very small elevations in pHi of about 0.05 pH units from the normal basal resting pHi of GH4C1 cells of around 7.05. The effects were more marked after acid-loading the cells using 1 micrograms of nigericin/ml. Preincubation with amiloride or replacing the extracellular Na+ with choline+ completely blocked the elevations stimulated by TRH or TPA, consistent with an activation of the Na+/H+ antiport mechanism. The effects were completely independent of the cytoplasmic free calcium concentration ([Ca2+]i). The calcium ionophore ionomycin produced an elevation in [Ca2+]i with no concomitant effect on pHi, and amiloride, although completely inhibiting the pH change stimulated by TRH, failed to affect the initial stimulated [Ca2+]i transient. Although the data are consistent with an elevation in pHi by TRH which is caused by stimulation of a protein kinase C and subsequent activation of the antiporter, the rapidity of the onset of the pHi response to TRH could not be mimicked by a combination of TPA and ionomycin. These results, together with previous findings which show that secretion can be mimicked by TPA and ionomycin, suggest that TRH-stimulated Na+/H+ exchange plays no part in the acute stimulation of secretion, but that TRH increases the pH-sensitivity of the antiport system during increased synthesis of prolactin and growth hormone.     

Na+-dependent elevation of the acidic cell surface pH (microclimate pH) of rat jejunal villus cells induced by cyclic nucleotides and phorbol ester: possible mediators of the regulation of the Na+/H+ antiporter

The effects of cyclic nucleotides and phorbol ester on the acidic cell surface pH of rat jejunal villi were studied by using single-barrelled pH-sensitive microelectrodes. Addition of dibutyryl cAMP (1 mM) to the mucosal bathing solution caused an elevation of the cell surface pH from 6.19 +/- 0.04 (n = 12 measurements from three animals) to 6.53 +/- 0.03 (12) in the presence of Na+ in the medium. However, dibutyryl cAMP had no significant effect in the absence of Na+ and presence of 1 mM amiloride. Dibutyryl cGMP (1 mM) also had an Na+-dependent inhibitory effect on the cell surface pH. A phorbol ester, phorbol 12-myristate 13-acetate, caused an elevation of the cell surface pH only in the presence of Na+ from 6.14 +/- 0.07 (12) to 6.46 +/- 0.08 (12). Phorbol and phorbol 13-acetate, which do not stimulate protein kinase C, were without significant effects. These results suggest that increased levels of the intracellular cyclic nucleotides and activation of protein kinase C raise the acidic cell surface pH by inhibiting the activity of the brush-border Na+/H+ antiporter in the rat jejunal villus cells.