Differentiation of human promyelocytic HL 60 cells by retinoic acid is accompanied by an increase in the intracellular pH. The role of the Na+/H+ exchange system

Retinoic acid, which induces the differentiation of HL 60 cells to granulocytes, produces a cell alkalinization from pHi = 7.03 to pHi = 7.37. The half-maximum effect of retinoic acid is observed at 10 nM. The effect of retinoic acid on the pHi develops slowly, and it precedes the differentiation of the cells. A cell alkalinization is also observed after differentiation of the cells by dimethyl sulfoxide. It is not observed using etretinate, a synthetic retinoid that does not promote the differentiation of HL 60 cells. Two pHi regulating mechanisms coexist in HL 60 cells. The Na+/H+ exchange system is the major mechanism that allows HL 60 cells to recover from an intracellular acidosis. A second mechanism is a Na-HCO3 cotransport system. During differentiation of the cells by retinoic acid, a 2-fold increase in the activity of the Na+/H+ exchange system is observed, while the activity of the NaHCO3 cotransport remains constant. The properties of interaction of the Na+/H+ exchanger with internal H+, external Na+, and Li+ as well as with amiloride and its derivatives are defined. The Na+/H+ exchanger of HL 60 cells is characterized by unusually low affinities for alkali cations and a high affinity for amiloride and its derivatives. The pHi dependence of the exchanger is not modified after differentiation by retinoic acid. It is concluded that the mechanism of activation of the Na+/H+ exchanger by retinoic acid is distinct from the short-term effect produced by mitogens and phorbol esters which change the pHi dependence of the system.     

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by approximately 12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by approximately 13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100-200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.     

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.     

Mechanisms regulating intracellular pH in sea urchin eggs

Intracellular pH (pHi) of sea urchin eggs (Paracentrotus lividus) was determined using DMO (dimethyloxazolidinedione) and a rapid filtration technique (P. Payan, J.P. Girard, R. Christen and C. Sardet (1981). Exp. Cell Res. 134, 339-344). Transfer of unfertilized or fertilized eggs from normal sea water into Na+-free artificial sea water leads to a progressive acidification and fall of intracellular Na+ content. A step rise in external Na+ to 10 meq causes a rapid but transient Na+ entry coupled to an excretion of H+, giving rise to a pHi increase. It is shown that the plasma membrane of unfertilized eggs contains a permanent and reversible Na+/H+ exchanger which contributes to the regulation of pHi. This exchange occurs with a 1:1 stoichiometry and is independent of metabolic energy. Proton excretion and sodium entry follow saturable kinetics with respect to external Na+ and are completely inhibited by amiloride. At fertilization, pHi increases from 7.38 to 7.64 and is maintained at this level by two separate mechanisms: (1) a Na+/H+ exchange with the same characteristics as in unfertilized eggs; (2) a H+-excreting system that is dependent on external Na+, amiloride sensitive, and requiring metabolic energy. The relationship between the permanent Na+/H+ exchange involved in pHi regulation and the transient Na+/H+ exchange occurring at fertilization is discussed.     

Regulation of intracellular pH in sheep cardiac Purkinje fibre: interactions among Na+, H+, and Ca2+1

Experiments were performed on sheep cardiac Purkinje fibres using pH- and sodium-selective microelectrodes, while simultaneously measuring tension, to determine if the fall in intracellular pH (pHi) following a rise in intracellular Na+ activity (aiNa) is caused by inhibition or reversal of acid extrusion on Na+-H+ exchange. A rise in aiNa was induced either by using the cardioactive steroid strophanthidin to inhibit the sarcolemmal Na+-K+ pump or by increasing the frequency of stimulation (0-4 Hz). Both of these manoeuvres led to an increase in aiNa and a decrease in pHi. Following exposure to strophanthidin, amiloride (an inhibitor of sarcolemmal Na+-H+ exchange) produced a decrease in both pHi and aiNa. These effects of amiloride increased with decreasing pHi, indicating that acid extrusion on Na+-H+ exchange is stimulated by the fall in pHi. The changes in intracellular Na+ and H+ caused by amiloride were quantitatively consistent with an electroneutral stoichiometry. The fall in pHi during strophanthidin exposure is therefore not caused by inhibition or reversal of acid extrusion Na+-H+ exchange. It is likely that the fall in pHi during a rate increase is also independent of Na+-H+ exchange. This is because (i) it has been shown previously to occur in the presence of amiloride and (ii) the calcium antagonist D600 completely abolished the stimulation-dependent fall in pHi. It is concluded that the intracellular acidosis following inhibition of the sarcolemmal Na+-K+ pump or following an increase in the rate of stimulation is secondary to a rise in intracellular calcium.     

Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils

Pretreatment of isolated human neutrophils (resting pHi congruent to 7.25 at pHo 7.40) with 30 mM NH4Cl for 30 min leads to an intracellular acidification (pHi congruen to 6.60) when the NH4Cl prepulse is removed. Thereafter, in 140 mM Na+ medium, pHi recovers exponentially with time (initial rate, approximately 0.12 pH/min) to reach the normal resting pHi by approximately 20 min, a process that is accomplished mainly, if not exclusively, though an exchange of internal H+ for external Na+. This Na+/H+ countertransport is stimulated by external Na+ (Km congruent to 21 mM) and by external Li+ (Km congruent to 14 mM), though the maximal transport rate for Na+ is about twice that for Li+. Both Na+ and Li+ compete as substrates for the same translocation sites on the exchange carrier. Other alkali metal cations, such as K+, Rb+, or Cs+, do not promote pHi recovery, owing to an apparent lack of affinity for the carrier. The exchange system is unaffected by ouabain or furosemide, but can be competitively inhibited by the diuretic amiloride (Ki congruent to 8 microM). The influx of Na+ or Li+ is accompanied by an equivalent counter-reflux of H+, indicating a 1:1 stoichiometry for the exchange reaction, a finding consistent with the lack of voltage sensitivity (i.e., electroneutrality) of pHi recovery. These studies indicate that the predominant mechanism in human neutrophils for pHi regulation after intracellular acidification is an amiloride-sensitive alkali metal cation/H+ exchange that shares a number of important features with similar recovery processes in a variety of other mammalian cell types.     

Role of calcium in regulating intracellular pH following the stepwise release of the metabolic blocks at first-meiotic prophase and second-meiotic metaphase in amphibian oocytes

31P-NMR has been used to monitor changes in intracellular pH following the sequential release of the block at first-meiotic prophase by hormones and the block at second-meiotic metaphase by fertilization in Rana eggs and oocytes. The broad phosphoprotein signal was eliminated by a combination of spin-echo and deconvolution techniques. pHi was determined from the pH-dependent separation of intracellular Pi and phosphocreatine resonances. Agents that release the prophase block (progesterone, insulin, D-600, La3+) increased pHi from 7.38 to 7.7-7.8 within 1-3 h. Noninducers such as 17 beta-estradiol were without effect. By second-metaphase arrest (ovulated, unfertilized) the pHi had fallen to 7.1-7.2. pHi underwent a transient increase to about 7.7 within the first 30 min at fertilization, with a slow 0.1-0.2 pH unit oscillation during early cleavage. The progesterone-induced elevation of intracellular pH is not blocked by amiloride and occurs in Na+-free medium. A transient rise in pHi occurs when the prophase-arrested oocyte is transferred to Ca2+-free medium or when ionophore A23187 is added to the Ca2+-containing medium. Agents that inhibit the resumption of the first meiotic division either block the rise in pHi (procaine, PMSF) or shorten the time-course of the rise in pHi (ionophore A23187). Conditions that elevate intracellular Ca2+ levels and/or increase Ca2+ exchange produce an increase in pHi, whereas those conditions that decrease intracellular Ca2+ levels and/or exchange produce a fall in pHi within 1 h. The time-course of the increase in pHi both following release of the prophase block and at fertilization coincide with a fall in intracellular cAMP and release of surface and/or intracellular Ca2+. These results suggest that: (1) pHi is a function of cytosolic free Ca2+ levels and/or Ca2+ exchange across the oocyte plasma membrane, and (2) meiotic agonists (progesterone, insulin, D-600) and mitogens (sperm, ionophore A23187) modulate intracellular and/or membrane Ca2+ with the resulting changes in pHi and cAMP and resumption of the meiotic divisions.     

Na+-H+ exchange in the process of glucose-induced insulin release from the pancreatic B-cell. Effects of amiloride on 86Rb, 45Ca fluxes and insulin release

The effect of amiloride, an inhibitor of Na+-H+ exchange, on intracellular pH (pHi), 86Rb outflow, 45Ca outflow and insulin release from pancreatic rat islets was examined. In the 0.1-1 mM range, amiloride transiently reduced pHi of glucose-deprived islets and allowed glucose to induce a sustained decrease in pHi of the islet cells. Amiloride reproduced the effect of glucose to decrease 86Rb and 45Ca outflow. In the presence of glucose (5.6 mM or more), amiloride (100 microM) acted synergistically with the sugar to reduce K+ outflow, and to stimulate 40Ca inflow and insulin release from perifused islets. These results add strong support to the view that the generation of protons through the metabolism of glucose represents an important step in the process of glucose-induced release. The stimulation by glucose of Na+-H+ exchange apparently masks and even overcomes the glucose-induced decrease in pHi otherwise expected from the increase in catabolic fluxes.     

Na+-H+ exchange at the apical membrane of Necturus gallbladder. Extracellular and intracellular pH studies

The mechanism of luminal solution acidification was studied in Necturus gallbladder by measurement of mucosal solution and intracellular pH with glass electrodes. When the gallbladder was bathed by a Na-Ringer's solution it acidified the luminal side by a Na+-dependent, amiloride- inhibitable process. In the presence of ouabain, acidification was reduced but could be stimulated to a rate greater than that under control conditions by the imposition of an inwardly directed Na+ gradient. These results suggest that luminal acidification results from Na+-H+ exchange at the apical membrane and not by diffusion of metabolic CO2. Li+ can substitute for Na+ but K+, Rb+, Cs+, and tetramethylammonium (TMA+) cannot. The maximal rate of exchange was about five times greater for Na+ than for Li+. Intracellular pH (pHi) was measured with recessed-tip glass microelectrodes; with the tissue bathed in Na-Ringer's solution (pH 7.75), pHi was 7.51 +/- 0.04. After inhibition of Na+-H+ exchange by mucosal perfusion with amiloride (1 mM) or by complete Na+ replacement with TMA+, phi fell reversibly by 0.15 and 0.22 pH units, respectively. These results support the conclusion that Na+-H+ exchange at the apical membrane is the mechanism of luminal acidification and is involved in the maintenance of steady state pHi.     

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.     

The Na+-dependent regulation of the internal pH in chick skeletal muscle cells. The role of the Na+/H+ exchange system and its dependence on internal pH.

The internal pH (pHi) of chick muscle cells is determined by the transmembrane Na+ gradient. Li+, but not K+, Rb+ or Cs+, can substitute for Na+ for regulating the internal pH of chick muscle cells. Pharmacological evidence using amiloride and amiloride analogs has shown that the Na+/H+ exchange system is the membrane mechanism that couples the pHi to the transmembrane Na+ gradient. The pHi dependence of the amiloride-sensitive Na+/H+ exchange mechanism was defined. Internal H+ interacts cooperatively with the Na+/H+ exchange system, in contrast with external H+, thus indicating an asymmetrical behaviour of this exchanger. The half-maximum effect for the activation by the internal H+ of the Na+ transporting activity of the amiloride-sensitive Na+/H+ exchange was observed at pH 7.4. The Hill coefficient of the H+ concentration dependence is higher than 3. Insulin was shown to have no effect on the pHi of chick muscle cells.     

Na+/H+ exchange activation mediates the lipopolysaccharide-induced proliferation of human B lymphocytes and is impaired in malignant B-chronic lymphocytic leukemia lymphocytes

In various mammalian cell types the stimulation of the plasma membrane amiloride-sensitive Na+/H+ exchange and the resulting increase of intracellular pH (pHi) play a key role in the initiation of cell proliferation. In the present work we have investigated whether Na+/H+ exchange is involved in normal human B cell proliferation and whether it is also operating in malignant B-chronic lymphocytic leukemia (B-CLL) lymphocytes. Our results show that: 1) normal human B cells contain an operating Na+/H+ exchanger, as inferred by their ability to recover pHi after acid-loading in a HCO3- -free medium and by evidences that LPS and phorbol ester PMA elicit a pHi rise inhibitable by either 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or a Na+-free medium; 2) LPS-induced proliferation of normal human B cells is strongly inhibited when the amiloride analog EIPA (5 microM) is present in the culture medium (after 72 h the proportion of B cells incorporation bromodeoxyuridine falls from 13.9 +/- 3.9% to 2.8 +/- 1.1%); 3) EIPA does not affect BdR incorporation when B cells proliferation is induced by the co-mitogenic activity of IL-4 and low m.w. B cell growth factor (BCGF); 4) B-CLL cells, which proliferate in response to IL-4/BCGF but not to LPS, fail to increase pHi above their pHi resting levels when challenged with LPS or PMA and pHi recovery after acid-loading is highly impaired. These results lead to conclude that Na+/H+ exchange operation is necessary for LPS-(but not for IL-4/BCGF)-induced proliferation of human normal B lymphocytes and that Na+/H+ exchange activation is impaired in malignant B-CLL lymphocytes.     

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.     

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.     

Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder

The hypothesis of Cl-/HCO3- exchange across the apical membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pHo), intracellular pH (pHi), and Cl- activity (alpha Cli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na+/H+ exchange inhibitor amiloride (10(-3) M) to the superfusate, alkalinization was observed. During prolonged (10 min) exposure to amiloride, no significant pHo change occurred. Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na+ in the superfusate (TMA+ substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of Cl- (cyclamate or sulfate substitution) caused a short-lived increase in the rate of luminal acidification, followed by a return to control values (10-30 min). Upon re-exposure to Cl-, there was a transient reduction of luminal acidification. The initial increase in acidification produced by Cl- removal was partially inhibited by SITS (0.5 mM). The pHi increased rapidly and reversibly when the Cl- concentration of the mucosal bathing solution was reduced to nominally 0 mM. The pHi changes were larger in 10 mM HCO3-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO3- is transported in exchange for Cl-. In both HEPES- and HCO3-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of alpha Cli. These results support the hypothesis of apical membrane Cl-/HCO3- exchange, which can be dissociated from Na+/H+ exchange and operates under control conditions. The coexistence at the apical membrane of Na+/H+ and Cl-/HCO3- antiports suggests that NaCl entry can occur through these transporters.     

Basolateral Na-H exchange in the rabbit cortical collecting tubule

We used the intracellular absorbance spectrum of the dye 4',5'-dimethyl-5- (and -6-) carboxyfluorescein (Me2CF) to measure intracellular pH (pHi) in the isolated, perfused cortical collecting tubule (CCT) of the rabbit nephron. The incident spot of light was generally 10 micron in diameter, large enough to illuminate from two to six cells. No attempt was made to distinguish principal from intercalated cells. All experiments were carried out in HCO3- -free Ringer to minimize HCO3- transport. When cells were acid-loaded by briefly exposing them to Ringer containing NH+4 and then withdrawing the NH+4, pHi spontaneously recovered from the acid load. The pHi recovery was best fit by the sum of two exponentials. When the acid loading was performed in the absence of Na+, the more rapid of the two phases of pHi recovery was absent. The remaining slow phase never returned pHi to normal and was sometimes absent. Returning Na+ to the lumen had only a slight effect on the pHi recovery. However, when Na+ was returned to the basolateral (i.e., blood-side) solution, pHi recovered rapidly and completely. The apparent Km for basolateral Na+ was 27.3 +/- 4.5 mM. The basolateral Na-dependent pHi recovery was reversibly inhibited by amiloride. We conclude that the mechanism responsible for the rapid phase of pHi recovery is an Na-H exchanger confined primarily, if not exclusively, to the basolateral membrane of the CCT.     

Intravesicular pH changes in submitochondrial particles induced by monovalent cations: relationship to the Na+/H+ and K+/H+ antiporters

The fluorescence of internalized fluorescein isothiocyanate dextran has been used to monitor the intravesicular pH of submitochondrial particles (SMP). Respiring SMP maintain a steady-state delta pH (interior acid) that results from the inwardly directed H+ flux of respiration and an opposing passive H+ leak. Addition of K+, Na+, or Li+ to SMP results in a shift to a more alkaline interior pH (pHi) in both respiring and nonrespiring SMP. The K+-dependent change in pHi, like the K+/H+ antiport in intact mitochondria, is inhibited by quinine and by dicyclohexylcarbodiimide. The Na+-dependent reaction is only partially inhibited by these reagents. Both the Na+- and the K+-dependent pH changes are sensitive to amiloride derivatives. The Km for both Na+ and K+ is near 20 mM whereas that for Li+ is closer to 10 mM. The K+/H+ exchange reaction is only slightly inhibited by added Mg2+, but abolished when A23187 is added with Mg2+. The passive exchange is optimal at pHi 6.5 with either Na+ or K+, and cannot be detected above pHi of 7.2. Both the Na+/H+ and the K+/H+ exchange reactions are optimal at an external pH of 7.8 in respiring SMP (pHi 7.1). Valinomycin stimulates the K+-dependent pH change in nonrespiring SMP, as does nigericin. It is concluded that SMP show K+/H+ antiport activity with properties distinct from those of Na+/H+ antiport. However, the properties of the K+/H+ exchange do not correspond in all respects to those of the antiport in intact mitochondria. Donnan equilibria and parallel uniport pathways for H+ and cations appear to contribute to cation-dependent pH changes in SMP.     

Increased intracellular pH is not necessary for ribosomal protein s6 phosphorylation, increased protein synthesis, or germinal vesicle breakdown in Xenopus oocytes

An increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation during Xenopus oocyte maturation has been reported by several laboratories. In this paper, the question of whether the pHi increase is necessary to induce S6 phosphorylation, an increase in protein synthesis, or germinal vesicle breakdown (GVBD) was assessed using sodium-free medium and the putative Na/H exchange blocker amiloride. Sodium-free medium decreased basal pHi by 0.3 unit and prevented increases in pHi in response to both insulin and progesterone, but S6 phosphorylation occurred normally with both hormones. GVBD occurred normally in sodium-free medium in response to progesterone, but the effect of insulin was reduced by 60%. In sodium-containing medium, amiloride inhibited GVBD and prevented insulin or progesterone-induced increases in pHi but the hormone-induced increase in S6 phosphorylation was unaffected. In the absence of sodium, amiloride inhibited GVBD but did not affect pHi, indicating that amiloride inhibits GVBD by a pHi-independent mechanism. Both progesterone and insulin increased protein synthesis in oocytes by 35%, and amiloride inhibited basal protein synthesis but not the increase with hormone. In the presence of cholera toxin, protein synthesis increases with insulin were inhibited but increased S6 phosphorylation was unaffected. Priming of animals with pregnant mare's serum gonadotropin prior to oocyte isolation reduced the time required for progesterone-induced GVBD, and increased the synchrony of GVBD of the population. Priming also increased oocyte basal pHi and basal protein synthesis as well as the magnitude of the increase in protein synthesis with progesterone but had no effect on S6 phosphorylation. The results indicate that in Xenopus oocytes increased pHi is not necessary for increased S6 phosphorylation, increased protein synthesis, or GVBD in response to insulin or progesterone nor is increased S6 phosphorylation sufficient for GVBD or increased protein synthesis.     

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 regulation in the renal proximal tubule of the salamander. Na-H exchange

Using pH-sensitive microelectrodes to measure intracellular pH (pHi) in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum, we have found that when cells are acid-loaded by pretreatment with NH+4 in a nominally HCO3--free Ringer, pHi spontaneously recovers with an exponential time course. This pHi recovery, which is indicative of active (i.e., uphill) transport, is blocked by removal of Na+ from both the luminal and basolateral (i.e., bath) solutions. Re-addition of Na+ to either the lumen or the bath results in a full pHi recovery, but at a lower-than-normal rate; the maximal rate is achieved only with Na+ in both solutions. The diuretic amiloride reversibly inhibits the pHi recovery when present on either the luminal or basolateral sides, and has its maximal effect when present in both solutions. The pHi recovery is insensitive to stilbene derivatives and to Cl- removal. A transient rise of intracellular Na+ activity accompanies the pHi recovery; there is no change of intracellular Cl- activity. These data suggest that these proximal tubule cells have Na-H exchangers in both the luminal and basolateral membranes.