Cytoplasmic pH regulation in normal and abnormal neutrophils. Role of superoxide generation and Na+/H+ exchange

The cytoplasmic pH of human neutrophils was determined fluorometrically using carboxylated fluorescein derivatives. When normal neutrophils were activated by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) in Na+-containing medium, the cytoplasmic pH initially decreased but then returned to near normal values. In Na+-free media or in Na+ medium containing amiloride, TPA induced a marked monophasic intracellular acidification. The cytoplasmic acidification is associated with net H+ equivalent efflux, suggesting metabolic acid generation. The metabolic pathways responsible for the acidification were investigated by comparing normal to chronic granulomatous disease neutrophils. These cells are unable to oxidize NADPH and generate superoxide. When treated with TPA in Na+-free or amiloride-containing media, chronic granulomatous disease cells did not display a cytoplasmic acidification. This suggests that in normal cells NADPH oxidation and/or the accompanying activation of the hexose monophosphate shunt are linked to the acidification. Unlike normal neutrophils, chronic granulomatous disease cells treated with TPA in Na+-containing medium displayed a significant cytoplasmic alkalinization. The alkalinization was Na+-dependent and amiloride-sensitive, indicating activation of Na+/H+ exchange. Thus, the Na+/H+ antiport, which can be indirectly stimulated by the metabolic cytoplasmic acidification, is also directly activated by the phorbol ester.     

Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes

The Na+/H+ antiport of rat thymic lymphocytes is activated when protein kinase C is stimulated by phorbol esters. A similar activation of the antiport is obtained when the cells are treated with hypertonic solutions. We tested the possibility that protein kinase C also mediates the osmotic activation of Na+/H+ exchange. Protein kinase C was depleted by preincubation of thymocytes for 24 hr in the presence of high concentrations of phorbol ester. Disappearance of the enzyme was assessed by direct measurement of phosphotransferase activity, and by the loss of biological responses to phorbol esters. The Na+/H+ antiport in protein kinase C-depleted cells was not stimulated by addition of phorbol ester, but responded normally to hypertonic treatment. The results indicate that the osmotic activation of countertransport does not require stimulation of protein kinase C.     

Dependence of Na+/H+ antiport activation in cultured rat aortic smooth muscle on calmodulin, calcium, and ATP. Evidence for the involvement of calmodulin-dependent kinases

The role of Ca2+/calmodulin-dependent processes in the activation of the Na+/H+ antiport of primary cultures of rat aortic smooth muscle was studied using 22Na+ uptake and measurement of intracellular pH (pHi) with the fluorescent pH dye 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein. Antiport activation following exposure to serum and by the induction of an intracellular acidosis could be markedly attenuated by calmodulin antagonists. Ionomycin also transiently elevated pHi and 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ influx, effects consistent with activation of the antiport; these effects were abolished in cells exposed to calmodulin antagonists or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. Activation of the antiport following intracellular acidosis was markedly affected by cellular ATP depletion. A comparison of the abilities of control and 2-deoxy-D-glucose-treated cells to increase 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ influx in response to graded acidifications indicated that attenuation of Na+/H+ antiport activity was due to both a shift of its pHi dependence and to a reduction in maximal activity. The results suggest that the Na+/H+ antiport of rat aortic smooth muscle is dependent on Ca2+/calmodulin-dependent processes, presumably phosphorylation, which influences its activity by modulating (i) an intracellular proton dependent regulatory mechanism (allosteric site) and (ii) the maximum activity of the 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.     

Estimation of the number and turnover rate of Na+/H+ exchangers in lymphocytes. Effect of phorbol ester and osmotic shrinking

Rat thymic lymphocytes possess an amiloride-sensitive Na+/H+ exchanger in their plasma membrane. Kinetic studies revealed that 5-(N-methyl-N-isobutyl)amiloride (MIA) was a more potent inhibitor of the antiport than amiloride (cf. apparent Ki of 174 nM and 6 microM, respectively). Inhibition by MIA was rapid (less than 5 s) and readily reversible. [3H]MIA binding to whole cells was assayed by rapid centrifugation following short (5 s) incubations to minimize nonspecific binding. A saturable binding component (Kd approximately equal to 170 nM) which was displaced by amiloride was detected. In contrast, there was no significant amiloride-displaceable binding to human erythrocytes, which have comparatively little Na+/H+ exchange activity. In lymphocytes, the ability of amiloride and several of its analogs to displace [3H]MIA correlated with their potency as inhibitors of the antiport. Both kinetic and binding studies revealed that extracellular H+, but not Na+, inhibited the interaction of MIA with its receptor(s). Taken together, these data suggest that [3H]MIA binds to the Na+/H+ exchanger. Scatchard analysis revealed that [3H]MIA bound to a maximum of 8000 high affinity sites/cell. Activation of Na+/H+ exchange by osmotic shrinking or by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate was not accompanied by a significant change in [3H]MIA binding. Given an upper limit of 8000 functional sites/thymocyte, we estimate that the turnover number of each maximally activated exchanger is at least 2000 cycles/s.     

Cyclosporine A inhibits Ca2+-dependent stimulation of the Na+/H+ antiport in human T cells

The cyclic undecapeptide cyclosporine A (CsA) is a potent immunosuppressive agent that inhibits the initial activation of T lymphocytes. This agent appears to be most effective in blocking the action of mitogens such as concanavalin A and the calcium ionophore A23187, which cause an influx of Ca2+, but not those that may act by alternate mechanisms. These observations suggest that CsA may block a Ca2+-dependent step in T cell activation. We have shown that stimulation of the T3-T cell receptor complex-associated Ca2+ transporter activates the Na+/H+ antiport (Rosoff, P. M., and L. C. Cantley, 1985, J. Biol. Chem., 260: 14053-14059). The tumor-promoting phorbol esters, which are co-mitogenic for T cells, activate the exchanger by a separate pathway which is mediated by protein kinase C. Both the rise in intracellular Ca2+ and intracellular pH may be necessary for the successful triggering of cellular activation. In this report we show that CsA blocks the T3-T cell receptor-stimulated, Ca2+ influx-dependent activation of Na+/H+ exchange, but not the phorbol ester-mediated pathway in a transformed human T cell line. CsA inhibited mitogen-stimulation of interleukin-2 production in a separate cell line. CsA also inhibited vasopressin stimulation of the antiporter in normal rat kidney fibroblasts, but had no effect on serum or 12-O-tetradecanoyl phorbol 13-acetate stimulation. CsA did not affect serum or vasopressin or serum stimulation of normal rat kidney cell proliferation. CsA also had no effect on lipopolysaccharide or phorbol ester stimulation of Na+/H+ exchange activity or induction of differentiation in 70Z/3 pre-B lymphocytes in which these events are initiated by the protein kinase C pathway. These data suggest that mechanisms of activation of Na+/H+ exchange that involve an elevation in cytosolic Ca2+ are blocked by CsA but that C kinase-mediated regulation is unaffected. The importance of the Na+/H+ antiport in the regulation of growth and differentiation of T cells is discussed.     

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.     

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.     

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

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

1,2-Diacylglycerols mimic phorbol 12-myristate 13-acetate activation of the sea urchin egg

Phorbol diesters have been reported to stimulate the Na+/H+ antiport of a variety of cells including sea urchin eggs. Since stimulation of the Na+/H+ antiport is necessary for metabolic derepression during fertilization and protein kinase C is a target of phorbol diesters, enhanced Na+/H+ exchange during fertilization may be a result of protein kinase C activity. Protein kinase C is probably physiologically activated by diacylglycerols, which are derived from hydrolysis of phosphatidylinositol. Treatment of sea urchin eggs with 1,2-diacylglycerols was found to stimulate the Na+/H+ antiport. The 1,3-isomers were without effect. Further, the effects of 1,2-diacylglycerol and phorbol diester are not additive with respect to Na+/H+ exchange. While a direct participation of protein kinase C activity during fertilization remains to be demonstrated, these data support the hypothesis that protein kinase C activity plays a role in fertilization. However, the cytotoxic effect of protein kinase C activators suggests effects associated with their pleiotropic nature.     

The relationship between mitogen-induced changes in the cytoplasmic pH and free Ca2+ concentration in rat thymocytes

The relationship between pHi and [Ca]i signals generated in rat thymocytes by the mitogen Con A has been investigated. It is shown that the mitogen-induced [Ca]i rise is dependent on Na+/H+ exchange or some other Na(+)-sensitive process. This conclusion is based on the following findings: (i) [Ca]i response to Con A weakens upon decreasing the concentration of extracellular Na+, or inhibiting Na+/H+ exchange; (ii) agents that alkalinize the cytoplasm (the phorbol ester TPA, the Na+/H+ ionophore monensin and NH4Cl) cause an increase in [Ca]i (Klip, A., Rothstein, A. and Mack, E. (1984) Biochem. Biophys. Res. Commun. 124, 14-22; Grinstein, S. and Goetz, J.D. (1985) Biochim. Biophys. Acta 819, 267-270); (iii) The effects of Con A, TPA and monensin on [Ca]i are not additive. The last observation suggests that all these agents activate the same Na+/H+ (Na+ and/or H+)-dependent system of Ca2+ transport. It is found that the pH i and [Ca]i responses in rat thymocytes are sensitive to changes in the intracellular levels of cyclic nucleotides, ATP and in temperature. These regulatory effects on the ionic signals are different for Con A, TPA and monensin. In particular, both the stimulation of Na+/H+ antiport and the [Ca]i rise brought about by Con A or TPA are inhibited upon elevating the cellular cAMP. In contrast, the monensin-induced [Ca]i signal is almost independent of cAMP but is highly sensitive to changes in cGMP and temperature. Reducing the ATP level eliminates both the pHi and [Ca]i responses to Con A but not to monensin. These different characteristics of [Ca]i signals elicited by the mitogen and the Na+/H+ ionophore indicate that these agents use different mechanisms to activate the Na+/H(+)-dependent Ca2+ transporting system. A [Ca]i response to monensin has been obtained in some other cell types, namely, in lymphoblastoid Raji cells, Ehrlich ascites tumor cells and also in platelets.     

Amiloride-sensitive Na+/H+ exchange in human neutrophils: mechanism of activation by chemotactic factors

The cytoplasmic pH (pHi) of human blood neutrophils was measured using trapped carboxyfluorescein derivatives. Cells were acid-loaded using propionate or by pretreatment with NH4+. Acid-loaded cells were found to regain near-normal pHi by means of a Na+-dependent process. A concomitant Na+ uptake was recorded as a change in cell volume. Both events were amiloride-sensitive, indicating involvement of a Na+/H+ antiport. Activation of Na+/H+ exchange was also observed with chemotactic factors. Studies of the pHi-dependence of the H+ extrusion rate indicate that chemotactic factors increase the [H+i] sensitivity of the antiport.     

Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+H+ exchange?

The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport.     

Calcium-dependent intracellular acidification dominates the pH response to mitogen in human T cells

The effects of a phorol ester and a mitogenic lectin on the intracellular pH (pHi) of human T lymphocytes was investigated. In contrast to the cytoplasmic alkalinization induced by 12-0-tetradecanoylphorbol-13-acetate, an acidification was recorded in cells treated with phytohemagglutinin. This decrease in pHi was magnified in Na+-free medium or in the presence of amiloride analogues, suggesting that activation of Na+/H+ exchange partially counteracts the phytohemagglutinin-induced acidification. The decrease in pHi was dependent on a sustained increase in cytosolic free Ca2+ and could be mimicked by addition of the divalent cation ionophore, ionomycin. The elevation of cytosolic free Ca2+ leads to metabolic H+ (equivalent) generation with consequent cytoplasmic acidification, which in human T cells predominates over the concurrent activation of the Na+/H+ antiport. These findings argue against the notion that activation of Na+/H+ exchange is a signal for the initiation of proliferation.     

Differences in nucleotide effects on intracellular pH, Na+/H+ antiport activity, and ATP-binding proteins in endothelial cells

Summary Bovine (BPAEC) and human (HPAEC) pulmonary artery endothelial cell monolayers were incubated with either ATP, ATP analogues, or UTP, followed by measurement of intracellular pH (pHi) and the rate of recovery from acidosis. ATP increased baseline pHi and the rate of acid recovery in BPAEC. This response was inhibited by the amiloride analogue, methyisobutylamiloride, demonstrating that activation of the Na⁺/H⁺ antiport was responsible for the increase in baseline pHi and the recovery from acidosis. This response had the features of both a P_2Y and P_2U purinergic receptor, based on the responses to a series of ATP analogues and UTP. In contrast, none of the nucleotides had any significant effect on pHi and Na⁺/H⁺ antiport activity in HPAEC. This difference in the response to extracellular nucleotides was not due to a difference in ATP metabolism between cell types, since the ectonucleotidase-resistant analogue, ATPγS, also had no effect on HPAEC. Analogues of cAMP had no effect on pHi or acid recovery in either cell type. Incubation of BPAEC and HPAEC with the photoaffinity ligand [³²P] 8-AzATP indicated that both BPAEC and HPAEC possess an ATP-binding protein of 48 kDa. However, BPAEC exhibited an additional binding protein of 87 kDa. Thus, the contrasting response to extracellular ATP between bovine and human pulmonary artery endothelial cells may be related to differences in the signal transduction pathway leading to antiport activation, including different ATP-binding sites on the cell membrane.     

Changes in pH in the cytoplasm of rat thymocytes after treatment with the mitogen concanavalin A and protein kinase C activator phorbol ester

The intracellular pH (pHi) of rat thymocytes has been measured with the fluorescent probe 2', 7'-bis(carboxyethyl)-5,6-carboxyfluorescein, both in the resting cells and under mitogenic stimulation. Concanavalin A (Con A) has been found to increase pHi from 7.16 +/- 0.02 to 7.30 +/- 0.02 during the first minutes after addition; the phorbol ester TPA raised pHi to 7.25 +/- 0.02. The Con A- and TPA-induced rise of pHi is due to activation of Na+/H+ exchange since it was abolished by amiloride, an inhibitor of Na+/H+ antiport, or in a low-Na+ medium. The elevation of intracellular cAMP level, decrease of cellular ATP, or the lowering of the temperature from 37 degrees down to 25 degrees C inhibited the pHi rise induced by Con A or TPA.     

Biochemical properties of the Na+/H+ exchange system in rat brain synaptosomes. Interdependence of internal and external pH control of the exchange activity

Properties of the Na+/H+ exchange system in synaptosomes have been studied primarily by using acridine orange fluorescence to follow H+ efflux. Results obtained from 22Na+ uptake experiments and [3H]ethylpropylamiloride binding experiments are also presented for comparison. The basal properties of the Na+/H+ antiport in synaptosomes are similar to those found in other systems; (i) the stoichiometry of Na+/H+ exchange is 1:1; (ii) Li+ can be successfully substituted for Na+; its affinity for the exchanger (KLi+ = 3 mM) is higher than that of Na+ (KNa+ = 12 mM), but the maximal rate of H+ efflux in the presence of Li+ is about 3 times lower than the maximal rate of H+ efflux in the presence of Na+; and (iii) the Na+/H+ antiport is inhibited by amiloride derivatives with the rank order:ethylisopropylamiloride greater than ethylpropylamiloride greater than amiloride greater than benzamil. The most important finding of this paper is that the external pH dependence of the synaptosomal Na+/H+ antiport is controlled by the value of internal pH and vice versa. For example apparent pHo values for half-maximum activation of the Na+/H+ exchanger are pHo = 7.12 when pHi = 6.4 and pHo = 7.95 when pHi = 7.3. Therefore, a 0.9 pH unit increase in internal pH produces a shift of at least a 0.83 pH unit in the external pH dependence. In addition, changing pHo from 7.75 to 8.50 also shifts the half-maximum pHi value for activation of the Na+/H+ antiport from 6.67 to 7.54.     

Interleukin-3-stimulated haemopoietic stem cell proliferation. Evidence for activation of protein kinase C and Na+/H+ exchange without inositol lipid hydrolysis.

Interleukin 3 (IL-3) is an important regulator of haemopoietic stem cell proliferation both in vivo and in vitro. Little is known about the possible mechanisms whereby this growth factor acts on stem cells to stimulate cell survival and proliferation. Here we have investigated the role of intracellular pH and the Na+/H+ antiport in stem cell proliferation using the multipotential IL-3-dependent stem cell line, FDCP-Mix 1. Evidence is presented that IL-3 can stimulate the activation of an amiloride-sensitive Na+/H+ exchange via protein kinase C activation. IL-3-mediated activation of the Na+/H+ exchange is not observed in FDCP-Mix 1 cells where protein kinase C levels have been down-modulated by treatment with phorbol esters. Also the protein kinase C inhibitor H7 can inhibit IL-3-mediated increases in intracellular pH. This activation of Na+/H+ exchange via protein kinase C has been shown to occur with no measurable effects of IL-3 on inositol lipid hydrolysis or on cytosolic Ca2+ levels. Evidence is also presented that this IL-3-stimulated alkalinization acts as a signal for cellular proliferation in stem cells.     

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).     

Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes

The activity of the Na+/H+ exchange system of rat thymic lymphocytes was determined by means of intracellular (pHi) and extracellular pH (pH0) measurements. In isotonic media, the antiport is virtually quiescent at physiological pHi (7.0-7.1), but is greatly activated by cytoplasmic acidification. At normal pHi, the antiport can also be activated by osmotic shrinking. Osmotic activation occurs after a delay of 20-30 s and is reversed several minutes after iso-osmolarity is restored. The mechanism of activation was analyzed by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. The affinities of the external substrate site for Na+ and H+ are not altered in shrunken cells. In contrast, the Hi+ sensitivity of the antiport (which is largely dictated by an allosteric modifier site) was increased, which accounted for the activation. The concentration of free cytoplasmic Ca2+ [( Ca2+]i) increased after osmotic shrinking. This increase was dependent on the presence of extracellular Ca2+ and Na+ and was blocked by inhibitors of Na+/H+ exchange, which suggests that it is a consequence, rather than the cause, of the activation of the antiport. It is concluded that the shift in the pHi dependence of the modifier site of the Na+/H+ antiport is the primary event underlying the regulatory volume increase that follows osmotic shrinkage.