Oscillatory transepithelial H(+) flux regulates a rhythmic behavior in C. elegans

In C. elegans, rhythmic defecation is timed by oscillatory Ca(2+) signaling in the intestine [1-5]. Here, by using fluorescent biosensors in live, unrestrained worms, we show that intestinal pH also oscillates during defecation and that transepithelial proton movement is essential for defecation signaling. The intestinal cytoplasm is acidified by proton influx from the lumen during defecation. Acidification is predicted to trigger Na(+)/H(+) exchange activity and subsequent proton efflux. The Na(+)/H(+) exchanger NHX-7 (PBO-4) extrudes protons across the basolateral membrane and is necessary for both acute acidification of the pseudocoelom and for strong contractions of the posterior body wall muscles during defecation. This suggests that secreted protons transmit a signal between the intestine and muscle. NHX-2 is a second Na(+)/H(+) exchanger whose distribution is limited to the apical membranes facing the intestinal lumen. RNA interference of nhx-2 reduces the basal pH of the intestinal cells, reduces the rate of proton movement between the lumen and the cytoplasm during defecation, and extends the defecation period. Thus, the cell may integrate both pH and calcium signals to regulate defecation timing. Overall, these results establish the defecation cycle as a model system for studying transepithelial proton flux in tissues that maintain systemic acid-base balance.     

The role of the Na+/H+ exchange system in the regulation of the internal pH in cultured cardiac cells

The Na+/H+ exchange system is not the major mechanism that regulates the internal pH value (pHi) of chick cardiac cells in culture under normal physiological conditions in the absence of carbonate. In cardiac cells in which the internal pH has been lowered to 6.6-6.7, the Na+/H+ exchanger becomes the major mechanism to bring back pHi to normal values (pHi = 7.3). The blockade of the Na+/H+ exchange activity with an active amiloride derivative, ethylisopropylamiloride, prevents internal pH recovery. The internal pH dependence of the Na+/H+ exchanger activity has been carefully studied. The [H+]i-dependence is very cooperative. For an external pH of 7.4, the system is nearly completely inactive at pHi 7.8 and nearly completely active at pHi 6.9-7.0 with half-maximum activation at pHi = 7.35. The increased activity of the Na+/H+ exchange system which follows the acidification of the internal medium produces an activation of the (Na+,K+)-ATPase.     

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.     

The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock

The properties of the Na+/H+ exchange system in the glial cell lines C6 and NN were studied from 22Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethyl-carboxyfluorescein. In both cell types, the Na+/H+ exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride greater than amiloride greater than benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H+. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in C6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines. Increasing the osmolarity of the external medium with mannitol produces an activation of the exchanger in C6 cells, which leads to a cell alkalinization. Mannitol action on 22Na+ uptake and the pHi were not observed in the presence of amiloride derivatives. Mannitol produces a modification of the properties of interaction of the antiport with both internal and external H+. It shifts the pHi dependence of the system to the alkaline range and the external pH (pHo) dependence to the acidic range. It also suppresses the interdependence of pHi and pHo controls of the exchanger's activity. NN cells that possess an Na+/H+ exchange system with different properties do not respond to mannitol by an increased activity of the Na+/H+ exchanger. The action of mannitol on C6 cells is unlikely to be mediated by an activation of protein kinase C.     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. II. Regulation of H+ and HCO3- transporters by Ca2(+)-mobilizing agonists

Pancreatic acini loaded with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to examine the effect of Ca2(+)-mobilizing agonists on the activity of acid-base transporters in these cells. In the accompanying article (Muallen, S., and Loessberg, P. A. (1990) J. Biol. Chem. 265, 12813-12819) we showed that in 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-buffered medium the main pHi regulatory mechanism is the Na+/H+ exchanger, a while in HCO3(-)-buffered medium pHi is determined by the combined activities of a Na+/H+ exchanger, a Na(+)-HCO3- cotransporter and a Cl-/HCO3- exchanger. In this study we found that stimulation of acini with Ca2(+)-mobilizing agonists in HEPES or HCO3(-)-buffered media is followed by an initial acidification which is independent of any identified plasma membrane-located acid-base transporting mechanism, and thus may represent intracellularly produced acid. In HEPES-buffered medium there was a subsequent large alkalinization to pHi above that in resting cells, which could be attributed to the Na+/H+ exchanger. Measurements of the rate of recovery from acid load indicated that the Na+/H+ exchanger was stimulated by the agonists. In HCO3(-)-buffered medium the alkalinization observed after the initial acidification was greatly attenuated. Examination of the activity of each acid-base transporting mechanism in stimulated acini showed that in HCO3(-)-buffered medium: (a) recovery from acid load in the presence of H2-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS) (Na+/H+ exchange) was stimulated similar to that found in HEPES-buffered medium; (b) recovery from acid load in the presence of amiloride and acidification due to removal of external Na+ in the presence of amiloride (HCO3- influx and efflux, respectively, by Na(+)-HCO3- cotransport) were inhibited; and (c) HCO3- influx and efflux due to Cl-/HCO3- exchange, which was measured by changing the Cl- or HCO3- gradients across the plasma membrane, were stimulated. Furthermore, the rate of Cl-/HCO3- exchange in stimulated acini was higher than the sum of H+ efflux due to Na+/H+ exchange and HCO3- influx due to Na(+)-HCO3- cotransport. Use of H2DIDS showed that the latter accounted for the attenuated changes in pHi in HCO3(-)-buffered medium, as much as treating the acini with H2DIDS resulted in similar agonist-mediated pHi changes in HEPES- and HCO3(-)-buffered media. The effect of agonists on the various acid-base transporting mechanisms is discussed in terms of their possible role in transcellular NaCl transport, cell volume regulation, and cell proliferation in pancreatic acini.     

22Na+ fluxes in thymic lymphocytes. II. Amiloride-sensitive Na+/H+ exchange pathway; reversibility of transport and asymmetry of the modifier site

22Na+ flux and cytoplasmic pH (pHi) determinations were used to study the reversibility, symmetry, and mechanism of activation of the Na+/H+ exchange system in rat thymic lymphocytes. In acid-loaded cells, the antiport can be detected as an Na+-induced, amiloride-sensitive alkalinization. At pHi greater than or equal to 7.0, amiloride- sensitive net H+ fluxes are not detectable. To investigate whether at this pHi the transporter is operative in a different mode, e.g., Na+/Na+ exchange, 22Na+ uptake was measured as a function of pHi. The results indicate that the antiport is relatively inactive at pHi greater than or equal to 7.0. Comparison of the rates of H+ efflux (or equivalent OH- uptake) and Na+ uptake indicate that Na+/Na+ countertransport through this system is negligible at all values of pHi and that the Na+:H+ stoichiometry is 1:1. Measurements of pHi in Na+- loaded cells suspended in Na+-free medium revealed an amiloride- sensitive cytoplasmic acidification, which is indicative of exchange of internal Na+ for external H+. The symmetry of the system was analyzed by measuring the effect of extracellular pH (pHo) on Na+ efflux. Unlike cytoplasmic acidification, lowering pHo failed to activate the antiport. The results indicate that the amiloride-sensitive Na+/H+ exchanger is reversible but asymmetric. The system is virtually inactive at pHi greater than or equal to 7.0 but can be activated by protonation of a modifier site on the cytoplasmic surface. Activation can also occur by depletion of cellular Na+. It is proposed that Na+ may also interact with the modifier site, stabilizing the unprotonated (inactive) form.     

The role of HCO3- and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation

The intracellular pH (pHi) of a rat parotid acinar preparation was monitored using the pH-sensitive fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Under resting (unstimulated) conditions both Na+/H+ exchange and CO2/HCO3- buffering contribute to the regulation of pHi. Muscarinic stimulation (carbachol) of the acini produced a gradual rise in pHi (approximately 0.1 unit by 10 min) possibly due to activation of the Na+/H+ exchanger. When the exchanger was blocked by amiloride or sodium removal, carbachol induced a dramatic (atropine inhibitable) decrease in pHi (approximately 0.4 pH unit with t1/2 approximately 0.5 min at 1 mM carbachol). The rate of this acidification was reduced by removal of exogenous HCO3- and by the carbonic anhydrase inhibitor methazolamide. Also, acini stimulated with carbachol in Cl- -free solutions showed a more pronounced acidification than in the corresponding Cl- -replete media. Taken together, these data indicate that the carbachol-induced acidification of rat parotid acinar cells unmasked by inhibition of the Na+/H+ exchanger is due to a rapid loss of intracellular HCO3-. Carbachol induced acidification was inhibited by the Cl- channel blocker diphenylamine 2-carboxylate but not by 4-acetomido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Cl-/HCO3- exchange. In addition, this acidification could not be sustained in Ca2+-free media and was totally blocked by chelation of intracellular Ca2+. Interpreted in terms of HCO3- loss, these results closely parallel the pattern of carbachol-induced Cl- release from this same preparation and indicate that HCO3- is secreted in response to muscarinic stimulation via the same or a very similar exit pathway, presumably an apical anion channel. Under normal physiological conditions the intracellular acidification resulting from HCO3- secretion is buffered by the Na+/H+ exchanger.     

Na+-independent Cl(-)-HCO-3- exchange in Madin-Darby canine kidney cells. Role in intracellular pH regulation

The role of plasma membrane Cl(-)-HCO-3-exchange in regulating intracellular pH (pHi) was examined in Madin-Darby canine kidney cell monolayers. In cells bathed in 25 mM HCO-3, pH 7.4, steady state pHi was 7.10 +/- 0.03 (n = 14) measured with the fluorescent pH probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Following acute alkaline loading, pHi recovered exponentially in approximately 4 min. The recovery rate was significantly decreased by Cl- or HCO-3 removal and in the presence of 50 microM 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS). Na+ removal or 10(-3) M amiloride did not inhibit the pHi recovery rate after an acute alkaline load. Following acute intracellular acidification, the pHi recovery rate was significantly inhibited by 10(-3) M amiloride but was not altered by Cl- removal or 50 microM DIDS. At an extracellular pH (pHo) of 7.4, pHi remained unchanged when the cells were bathed in either Cl- free media, HCO-3 free media, or in the presence of 50 microM DIDS. As pHo was increased to 8.0, steady state pHi was significantly greater than control in Cl(-)-free media and in the presence of 50 microM DIDS. It is concluded that Madin-Darby canine kidney cells possess a Na+-independent Cl(-)-HCO-3 exchanger with a Km for external Cl- of approximately 6 mM. The exchanger plays an important role in pHi regulation following an elevation of pHi above approximately 7.1. Recovery of pHi following intracellular acidification is mediated by the Na+/H+ antiporter and not the anion exchanger.     

Effects of cyclosporin A on induced HIT cell alkalinization.

We studied whether therapeutic doses of cyclosporin A (CsA) modify the effects of nutrient and non-nutrient stimuli on pHi, in the insulin-secreting beta-cell line HIT-T15. Glucose caused a transient acidification, followed by alkalinization. CsA failed to block this alkalinization. PMA elicited a gradual alkalinization by a protein kinase C mediated mechanism which is not inhibited by CsA. The depolarization with high K+ was associated with a rise in pHi. CsA was able to completely block this increase in pHi. Ionomycin induced a rapid cytosolic alkalinization partially inhibited by CsA. We conclude that in HIT-T15 cells, therapeutical doses of CsA inhibit the Ca(2+)-dependent pathway of Na+/H+ antiport activation but not protein kinase C activation of this exchanger.     

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.     

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.     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. I. Characterization of H+ and HCO3- transporters

Rat pancreatic acini loaded with the pH sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to characterize intracellular pH (pHi) regulatory mechanisms in these cells. The acini were attached to cover slips and continuously perfused. In 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered solutions recovery from acid load (H+ efflux) required extracellular Na+ (Na+out) and was blocked by amiloride. Likewise, H+ influx initiated by removal of Na+out was blocked by amiloride. Hence, in HEPES-buffered medium the major operative pHi regulatory mechanism is a Na+/H+ exchange. In HCO3(-)-buffered medium, amiloride only partially blocked recovery from acid load and acidification due to Na+out removal. The remaining fraction required Na+out, was inhibited by H2-4,4'-diisothiocyanostilbene-2,2'-disulfunic acid (H2DIDS) and was independent of C1-. Hence, a transporter with characteristics of a Na(+)-HCO3- cotransport exists in pancreatic acini. Measurement of pHi changes due to Na(+)-HCO3- cotransport, suggests that the transporter contributes to HCO3- efflux under physiological conditions. Changing the Cl- gradient across the plasma membrane of acini maintained in HCO3(-)-buffered solutions reveals the presence of an H2DIDS-sensitive, Na(+)-independent, Cl(-)-dependent, HCO3- transporter with characteristics of a Cl-/HCO3- exchanger. In pancreatic acini the exchanger transports HCO3- but not OH- and under physiological conditions functions to remove HCO3- from the cytosol. In summary, only the Na+/H+ exchanger is functional in HEPES-buffered medium to maintain pHi at 7.28 +/- 0.03. In the presence of 25 mM HCO3- at pHo of 7.4, all the transporters operate simultaneously to maintain a steady-state pHi of 7.13 +/- 0.04.     

Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive

The mechanism by which human alpha-thrombin activates the Na+/H+ exchanger was studied in cultured neonatal rat aortic smooth muscle cells. Thrombin (0.4 unit/ml) caused a rapid cell acidification followed by a slow, amiloride-inhibitable alkalinization (0.10-0.14 delta pHi above base line). In protein kinase C down-regulated cells (exposed to phorbol 12-myristate 13-acetate for 24 or 72 h), the delta pHi induced by thrombin was only partially attenuated. This protein kinase C-independent activation of the Na+/H+ exchanger was blocked by pertussis toxin (islet activating protein (IAP)), reducing delta pHi by 50%. IAP did not directly inhibit Na+/H+ exchange activity as assessed by the response to intracellular acid loading. Thrombin also stimulated arachidonic acid release by 2.5 fold and inositol trisphosphate release by 6.2 fold. IAP inhibited both of these activities by 50-60%. Intracellular Ca2+ chelation with 120 microM quin2 prevented the thrombin-induced Ca2+ spike, inhibited thrombin-induced arachidonic acid release by 75%, and inhibited thrombin-induced activation of the Na+/H+ exchanger in protein kinase C-deficient cells by 65%. Increased intracellular [Ca2+] alone was not sufficient to activate the Na+/H+ exchanger, since ionomycin (0.3-1.5 microM) failed to elevate cell pH significantly. 10 microM indomethacin inhibited thrombin-induced delta pHi in both control and protein kinase C down-regulated cells by 30-50%. Thus, thrombin can activate the Na+/H+ exchanger in vascular smooth muscle cells by a Ca2+-dependent, pertussis toxin-sensitive pathway which does not involve protein kinase C.     

The action of low density lipoprotein on vascular smooth muscle cells involves increase in intracellular pH

The effect of low density lipoprotein (LDL) on the intracellular pH (pHi) of vascular smooth muscle cells (VSMC) was investigated using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). LDL and apoprotein B (apo-B), a binding protein for the LDL receptor, caused transient acidification followed by Na(+)-dependent and amiloride-sensitive alkalization of the cells due to stimulation of Na+/H+ exchanger. NH4Cl also caused intracellular alkalization, but independently of extracellular Na+. LDL, apo-B and NH4Cl all stimulated thymidine incorporation. These results indicate that the binding of LDL to its receptor stimulates Na+/H+ exchanger, resulting in alkalization of VSMC and suggest that this may function as a massage in stimulation of DNA synthesis evoked by LDL.     

Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia)

The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined.     

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.     

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.     

Kinetic dissection of two distinct proton binding sites in Na+/H+ exchangers by measurement of reverse mode reaction

We examined the effect of intracellular acidification on the reverse mode of Na+/H+ exchange by measuring 22Na+ efflux from 22Na+-loaded PS120 cells expressing the Na+/H+ exchanger (NHE) isoforms NHE1, NHE2, and NHE3. The 5-(N-ethyl-N-isopropyl)amiloride (EIPA)- or amiloride-sensitive fraction of 22Na+ efflux was dramatically accelerated by cytosolic acidification as opposed to thermodynamic prediction, supporting the concept that these NHE isoforms are activated by protonation of an internal binding site(s) distinct from the H+ transport site. Intracellular pH (pHi) dependence of 22 Na+ efflux roughly exhibited a bell-shaped profile; mild acidification from pHi 7.5 to 7 dramatically accelerated 22Na+ efflux, whereas acidification from pHi 6.6 gradually decreased it. Alkalinization above pHi 7.5 completely suppressed EIPA-sensitive 22Na+ efflux. Cell ATP depletion and mutation of NHE1 at Arg440 (R440D) caused a large acidic shift of the pHi profile for 22Na+ efflux, whereas mutation at Gly455 (G455Q) caused a significant alkaline shift. Because these mutations and ATP depletion cause correspondingly similar effects on the forward mode of Na+/H+ exchange, it is most likely that they alter exchange activity by modulating affinity of the internal modifier site for protons. The data provide substantial evidence that a proton modifier site(s) distinct from the transport site controls activities of at least three NHE isoforms through cooperative interaction with multiple protons.     

Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel.

The internal pH (pHi) of cytoplasts, derived from human neutrophils, falls 0.05 pH units upon activation of the superoxide-generating NADPH oxidase. The decrease in pHi is absent in diphenyleneiodonium-treated cytoplasts and therefore it is likely to arise directly from the activity of the oxidase. The addition of amiloride, to diminish the Na+/H+ exchanger, enhanced the extent of the internal acidification but not the initial rate. However the electroneutral Na+/H+ exchanger cannot be a contributor to H+ efflux to compensate for charge translocated by the oxidase. In the presence of Cd ions or valinomycin, phorbol-induced acidification of the cytosol was greatly increased, suggesting an inability to translocate the cytosolic H+ generated by an electrogenic oxidase. In the presence of both Cd and valinomycin the cytoplasts retained 0.8 H+ per O2-. generated. The rate of acidification of the external medium by stimulated cytoplasts is greatly reduced in the presence of Zn and valinomycin. Our results support the view that the plasma membrane of neutrophils contains Zn2+- or Cd2+-sensitive proton-conducting channels which maintain a stable membrane potential and pHi during the activity of the electrogenic NADPH oxidase.