The activation of cAMP-dependent protein kinase is directly linked to the inhibition of osteoblast proliferation (UMR-106) by parathyroid hormone-related protein

The present study was performed to compare the effect of parathyroid hormone-related protein (PTHrP) on the proliferation of osteoblastic osteosarcoma cells (UMR-106) with that of PTH and characterize the direct involvement of cAMP in the change of osteoblast proliferation by PTHrP. Human(h)PTHrP-(1-34) (10(-11)-10(-7)M) dose-dependently inhibited [3H]thymidine incorporation (TdR) in the same manner as hPTH-(1-34). The simultaneous addition of PTHrP and PTH at a maximal effective dose of 10(-7) M did not cause additive suppressive effect on cell proliferation. Rp-cAMPs, which has been recently shown to act directly as antagonist in the activation of cAMP-dependent protein kinase (PKA), dose-dependently (10(-6)-10(-4)M) antagonized PTHrP-induced suppression of TdR in the same manner as PTH. Present study indicated that PTHrP has the same effect on osteoblast proliferation as PTH and that the activation of PKA is directly linked to the change of osteoblast proliferation by PTHrP.     

Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs

Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.01-0.04 units (correlated with the rise of [Ca2+]i measured with quin2) followed after 10-15 s by a sustained rise of 0.1-0.15 units pHi. The fall of pHi and [Ca2+]i mobilization was reduced by early (5 s) addition of hirudin, but the later elevated pHi was not reversed by hirudin added after 30 s, although this strips thrombin from receptors and rapidly returns [Ca2+]i to basal levels. In Na+-free medium, or in presence of the Na+/H+ antiport inhibitors, 5-(N,N-dimethyl)amiloride (DMA) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thrombin caused a greater fall of pHi (0.22-0.26 units) that was sustained. DMA or EIPA could also reverse the alkalinization response to thrombin. Ca2+ ionophores (ionomycin, A23187) decreased platelet pHi by 0.02-0.15 units, but without an increase of pHi comparable to that following thrombin; DMA and EIPA enhanced the fall of pHi (0.14-0.33 units). Cytoplasmic acidification produced by nigericin (K+/H+ ionophore) was followed by return towards normal that was abolished by Na+/H+ antiport inhibitors. The phorbol diester phorbol 12-myristate 13-acetate had little effect on resting pHi but increased the rate of recovery 2-3-fold after cytoplasmic acidification by nigericin, ionomycin, or sodium propionate. These results indicate that elevation of [Ca2+]i by thrombin enhances H+ production, but the subsequent alkalinization is independent of receptor occupancy or elevated [Ca2+]i and stimulation of the Na+/H+ antiporter by thrombin probably involves some mechanism apart from regulation by H+ and protein kinase C.     

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.     

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.     

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

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

The activation of cAMP-dependent protein kinase is directly linked to the regulation of osteoblast proliferation (UMR-106) by parathyroid hormone.

In order to characterize the direct involvement of cAMP in the change of osteoblast proliferation by parathyroid hormone (PTH), we employed the diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, Sp-cAMPS and Rp-cAMPS, which have been recently shown to act directly as agonist and antagonist, respectively in the activation of cAMP-dependent protein kinase (PKA). Dibutyryl cAMP (dbcAMP) and cholera toxin as well as human(h) PTH-(1-34) significantly inhibited [3H]thymidine incorporation (TdR) in osteoblastic osteosarcoma cells, UMR-106. Sp-cAMPS (10(-6)-10(-4) M) inhibited TdR in a dose-dependent manner. Although Rp-cAMPS (10(-6)-10(-4) M) itself did not affect TdR, it significantly blocked dbcAMP-, cholera toxin- and Sp-cAMPS-induced suppression of TdR. Moreover, Rp-cAMPS (10(-6)-10(-4) M) dose-dependently antagonized hPTH-induced suppression of TdR. Present studies first indicated that the activation of PKA is directly linked to the change of osteoblast proliferation by PTH.     

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

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

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.     

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.     

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.     

Kinetic characteristics of 22Na transport in human and rat erythrocytes during cytoplasm acidification and cell compression

Under normal conditions (pH0 = 7.4, pHi = 7.1-7.2) amiloride, a Na+/H+ exchange inhibitor, does not influence Na+ intake by human and rat erythrocytes. Acidification of the cytoplasm (pHi approximately 6.4) is accompanied by the acceleration of 22Na intake, which is decreased after addition of 1 mM amiloride (by 50 and 80%, respectively). The Ki value of amiloride for human and rat erythrocytes is 30 and 250 microM, respectively. In rat erythrocytes the dependence of the rate of the delta pH-induced incorporation of 22Na on Na+ concentration is described by a saturation curve (K0.5 for Na0+ is approximately 40 mM), whereas in human erythrocytes it obeys the diffusion kinetics. These results suggest that the Na+/H+ exchange takes place in rat erythrocytes, but is absent in human erythrocytes. In rat erythrocytes the Na+/H+ exchange can be induced by cell compression which can be caused either by decreasing the KCl content (after addition of valinomycin) or by increasing the osmolarity of the medium (in the presence of sucrose). The rate of Na+/H+ exchange induced by cell compression is increased by 60-70% after addition of protein kinases A and C activators. No effect of intracellular Ca2+ on the rate of the Na+/H+ exchange in rat erythrocytes is observed.     

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.     

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.     

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.     

Evidence for a Na+/H+ antiport in stomach smooth muscle cells

Single smooth muscle cells were isolated from circular muscle of the canine gastric corpus by collagenase incubation. Cytoplasmic pH (pHi) of these cells was measured fluorometrically using the trapped dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Cells were examined for their Na+/H+ exchange activity after intracellular acidification. Cells acid-loaded by propionate exposure, the NH4+ prepulse technique or suspension in a Na+-depleted medium regained almost normal pHi upon exposure to a Na+ medium. The Na+-dependent alkalinization was amiloride sensitive. As well, addition of amiloride to cells suspended in a Na+ medium caused a concurrent decrease in pHi. The study indicates that a Na+/H+ antiport is present in these smooth muscle cells.     

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.     

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent,but can couple to an H+ conductance

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT- PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time- course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH- sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.     

Ethyl isopropylamiloride downregulates Na,K-ATPase gene expression which confers cytotoxicity in primary proximal tubule cell cultures

Our original attempt was to examine whether inhibition of Na/H exchange in proximal tubule would affect the expression of basolateral membrane protein Na,K-ATPase. Three amiloride analogues were tested within the range of 10(-6) M to 10(-4) M in primary cultures of proximal tubule cells. Only ethylisopropyl amiloride (EIPA) dose-dependently downregulated Na,K-ATPase activity in cultured proximal tubule cells. The time course study revealed that EIPA (10(-4) M) significantly decreased Na,K-ATPase alpha- and alpha-mRNA abundance within 4 hr and suppressed Na,K-ATPase alpha- and beta-mRNA levels by 76.3 +/- 4.5% and 85.5 +/- 5.8%, respectively, within 24 hr. The decrease in Na,K-ATPase mRNA was followed by a decrease in Na,K-ATPase activity by 22.5 +/- 10.8% and 48.8 +/- 5.9% within 12 and 24 hr, respectively, which could be reflected by a coordinate decrease in levels of both alpha- and mature beta-protein. The cell viability was not affected until 20 hr of EIPA treatment, when an increase in LDH release and cell detachment was observed. Because EIPA rapidly decreased intracellular pH (pHi) to 6.7 within 2 hr and raising pHi to 6.6 by metabolic acidosis could not elicit changes in Na,K-ATPase activity, EIPA-induced downregulation of Na,K-ATPase should not be mediated through H+. In view of the time course of EIPA effects on Na,K-ATPase subunit mRNA, protein, activity and cell toxicity, the cytotoxic effect is likely resulted from a decrease in Na,K-ATPase activity. Take together, we conclude that EIPA induces downregulation of Na,K-ATPase expression via both pre- and post-translational mechanisms, which confers cytotoxic effects on proximal tubule cells.     

The effect of endothelin-1 on Na+/H+ metabolism in vascular smooth muscle cells]

The effects of endothelin on intracellular pH (pHi) were examined in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent probe BCECF. Endothelin induced biphasic changes in pHi: initial decrease followed by a subsequent increase above the basal level due to activation of the Na+/H+ exchange. The elevation of pHi was slow and sustained, but depended on the dose of endothelin: IC50 was about 3 x 10(-8) M. Na+/H+ exchange inhibition by EIPA (10(-7) M) or by equimolar replacement of external Na+ by choline abolished the pHi increase by enhancing the first phase of cytoplasm acidification. Effects of endothelin were compared with the action of protein kinase C (PK-C) activator phorbol 12-13 myristate ester (PMA). PMA induced a monophasic slow and sustained increase in pHi. The treatments of VSMC with H-7 and staurosporine (PK-C) inhibitors prevented the pHi response to endothelin and PMA. These results suggest that protein kinase C may play an important role in mediating the effects of endothelin on Na+/H+ exchange in VSMC.