Intracellular pH change does not accompany egg activation in the mouse

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pH_i). We measured pH_i, in germinal vesicle (GV)-intact mouse oocytes, ovulated eggs, and in vivo fertilized zygotes using the pH indicator dye, SNARF-1. The mean pH_i was 6.96 ± 0.004 (± SEM) in GV-intact oocytes, 7.00 ± 0.01 in ovulated, unfertilized eggs, and 7.02 ± 0.01 in fertilized zygotes, indicating no sustained changes in pH_i after germinal vesicle breakdown (GVBD) or fertilization. To examine whether transient changes in pH_i occur shortly after egg activation, mouse eggs were parthenogenetically activated by 7% ethanol in phosphate buffered saline (PBS); no significant change in pH_i followed ethanol activation. Since increased Na⁺/H⁺ antiporter activity is responsible for pH_i increase in the sea urchin, pH_i was measured in the absence of added bicarbonate or CO₂ la condition under which the antiporter would be the only major pH_i regulatory mechanism able to operate, since the others were bicarbonate- dependent) in GV-intact oocytes, ovulated eggs, and in vivo fertilized zygotes to determine whether a Na⁺/H⁺ antiporter was activated. There was no physiologically significant difference in pH_i after GVBD or fertilization, when pH_i was measured in bicarbonate-free medium, nor any change upon parthenogenetic activation. Thus, a change in pH_i is not a feature of egg activation in the mouse. © 1996 Wiley-Liss, Inc.     

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : I. Role of HCO3?

We studied the regulation of intracellular pH (pH_i) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH_i. Intrinsic buffering power (β_I) was 10.5 mM (pH unit)⁻¹ at pH_i 7.0, and decreased linearly with pH_i; the best-fit line to the data had a slope of −10.0 mM (pH unit)⁻². In the absence of HCO₃ ⁻, pH_i recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH_i 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH_i. Acid extrusion was inhibited 68% (pH_i 6.23) by substituting Li⁺ for Na⁺ in the bath solution. Switching from a CO₂/HCO₃ ⁻-free to a CO₂/HCO₃ ⁻-containing bath solution caused mean steady state pH_i to increase from 6.82 to 6.90, due to a Na⁺-driven HCO₃ ⁻ transporter. The HCO₃ ⁻-induced pH_i increase was unaffected by amiloride, but was inhibited 75% (pH_i 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pH_i 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO₂/HCO₃ ⁻-induced pH_i increase was blocked when external Na⁺ was replaced with N-methyl-d-glucammonium (NMDG⁺). In the presence of HCO₃ ⁻, the Na⁺-driven HCO₃ ⁻ transporter contributed to the pH_i recovery from an acid load. For example, HCO₃ ⁻ shifted the plot of acid-extrusion rate vs. pH_i by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO₃ ⁻ increased acid extrusion 3.8-fold (pH_i 6.20). When astrocytes were acid loaded in amiloride, with Li⁺ as the major cation, HCO₃ ⁻ failed to elicit a substantial increase in pH_i. Thus, Li⁺ does not appear to substitute well for Na⁺ on the HCO₃ ⁻ transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na⁺-driven HCO₃ ⁻ transporter are the predominant acid extruders in astrocytes.     

Comparison between drug-induced and K+-induced changes in molar acid extrusion fluxes (JH +) and in energy consumption rates in astrocytes

Neuronal excitation leads to an increase of the extracellular K⁺ concentration ([K⁺]_o) in brain. This increase has at least two energy-consuming consequences: (1) a depolarization-mediated change in intracellular pH (pH_i) in astrocytes due to depolarization-mediated increased activity of the acid-extruding Na⁺/bicarbonate transporter NBCe1 (driven by secondary active transport, supported by ion gradients established by the Na⁺, K⁺-ATPase); and (2) activation of cellular reuptake of K⁺ mediated by the Na⁺, K⁺-ATPase in both neurons and astrocytes. Astrocytic, but not neuronal increase in NBCe1 activity and pH_i is also seen after chronic treatment with either of the two anti-bipolar drugs carbamazepine or valproic acid. The third ‘classical’ anti-bipolar drug, ‘lithium’ increases astrocytic pH_i by a different mechanism (stimulation of the acid extruding Na⁺/H⁺ exchanger NHE1). The acid extruder fluxes, which depend upon the change in pH_i per time unit (ΔpH_i/Δt) and intracellular buffering power, have not been established in most of these situations. Therefore their stimulatory effects on energy metabolism has not been quantitated. This has been done in the present study in cultured mouse astrocytes. pH_i was determined using the fluorescent pH-sensitive indicator BCECF–AM and an Olympus IX71 live cell imaging fluorescence microscope. Molar acid extrusion fluxes (indicating transporter activity) were determined as pH_i changes/min during recovery after acid-loading with NH₃/NH₄ ⁺, NBCe1 mRNA and protein expression in the cultured cells by, respectively RT-PCR and Western blotting. Drug-induced up-regulation of acid extrusion flux was slow and less than physiologically seen after increase in K⁺ concentration. Energetically, K⁺ uptake is much costlier than NBCe1 activity.     

Comparative patterns of protein phosphorylation during activation of surf clam oocytes by different artificial agents

Oocytes from the surf clam Spisula solidissima are arrested at prophase I of meiotic maturation, until fertilization, We analyzed the patterns of phosphorylated proteins under procedures mimicking, to various degrees, the normal sperm-induced activation process. High K⁺-seawater, the phorbol ester TPA, serotonin, or a combination of these were used to analyze their effects on both germinal vesicle breakdown (GVBD) and protein phosphorylation. Oocytes were preloaded with ³⁶S-methionine or ³²P-phosphate, and the pattern of labeled proteins was analyzed by polyacrylamide gel electrophoresis followed by autoradiography. When comparing, in high K⁺-activated oocytes, the pattern of phosphorylated proteins with that of synthesized proteins, it appeared that these two processes were largely unrelated to one another. Activation induced by TPA was slower (60 min for GVBD) than that induced by high K⁺ or serotonin (12–15 min for GVBD), but was similarly sensitive to the protein phosphorylation inhibitor, 6-dimethylaminopurine, and resulted in a qualitatively similar pattern of phosphorylated proteins appearing with slower kinetics, reflecting slower GVBD. When both serotonin and TPA were added to oocytes, the kinetics of GVBD was intermediate (30 min), and so was the appearance of phosphorylated proteins. Finally, the kinetics of development of H1 kinase activities was evaluated in oocytes activated by serotonin, TPA, or both. Similar to the general pattern of phosphorylated proteins, increased histone H1 kinase activities developed to similar degrees but with kinetics reflecting those of GVBD in each case. In conclusion, activations by different artificial agents, utilizing different pathways, resulted in GVBD with different kinetics but similar overall patterns of phosphorylated proteins after a lag typical of the agent used. This suggests that diverse pathways may initially be used to activate oocytes, but that these different pathways eventually merge into a common one, resulting in a highly conserved and regulated sequence of phosphorylation processes. © 1996 Wiley-Liss, Inc.     

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pH _i ) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K⁺ and Na⁺ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pH _i depends strongly on the activity of a K⁺/H⁺ exchanger. In the absence of toxin, removal of K⁺ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pH _i equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pH _i . B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H⁺, K⁺ and Na⁺.     

Apical and basolateral Na+/H+ exchange in the rabbit outer medullary thin descending limb of henle: Role in intracellular pH regulation

Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH _i ) was measured using BCECF. Steady-state pH _i in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na⁺, pH _i decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH _i was significantly less, 0.73±0.18 pH/min. Steady-state pH _i decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na⁺ 140mm lumen and bath). When Na⁺ was removed from the basolateral side of the tubule, pH _i decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH _i was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na⁺ 140mm lumen and bath) caused steady-state pH _i to decrease significantly by 0.06 pH units. When pH _i was acutely decreased to 5.87±0.02 following NH₄Cl removal (lumen, bath), pH _i failed to recover in the absence of Na⁺ (lumen, bath). Addition of 140mm Na⁺ to the lumen caused pH _i to recover at a rate of 2.17±0.59 pH/min. The rate of pH _i recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na⁺ was added to the basolateral side, pH _i recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH _i , by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na⁺/N⁺ antiporters. In the steady state, the rate of Na⁺-dependent H⁺ flux across the apical antiporter exceeds the rate of Na⁺-dependent H⁺ flux via the basolateral antiporter. Recovery of pH _i following acute intracellular acidification is Na⁺ dependent and mediated primarily by the luminal antiporter.     

Intracellular pH plays a role in regulating protein synthesis in Xenopus oocytes

Full-grown Xenopus oocytes undergo meiotic maturation in response to progesterone stimulation. Using [¹⁴C]dimethyloxazolidine dione (DMO), we have measured a cytoplasmic alkalization in these oocytes starting at pH 7.14 ± 0.17 during the germinal vesicle (GV) stage, and increasing to 7.56 ± 0.14 at the time of germinal vesicle breakdown (GVBD). During this period, the rate of protein synthesis increases 2-fold from 18.9 ± 3.1 to 37.7 ± 8.8 ng/hr/oocyte. Artificial alkalization of GV stage oocytes to pH_i 7.68 ± 0.16, by exposure to the weak bases trimethylamine, methylamine, procaine, or imidazole, led to a 1.8-fold increase in the synthetic rate. Intracellular acidification from 7.5 back to 7.0 had no apparent effect on the elevated rate of protein synthesis following GVBD. Therefore, a cytoplasmic alkalization in the range of 7.5 to 7.6 seems to be one of the events that is necessary for initiating the increase in protein synthesis in maturing Xenopus oocytes; however, it does not appear that an elevated pH_i is necessary to maintain the increased synthetic rate following GVBD.     

Starfish oocyte maturation and fertilization: Intracellular pH is not involved in activation

Intracellular pH (pH_i) was assayed during the hormonally induced maturation of oocytes of the starfish Pisaster ochraceus. Cytoplasmic pH was measured by the DMO method, and concurrently, the initiation of maturation was determined by germinal vesicle breakdown (GVBD) and the increase in protein synthesis (percentage incorporation of amino acids). Our results indicate that (1) oocyte pH_i rises slightly after initiation of maturation; (2) GVBD is not inhibited by acidifying pH_i; and (3) amino acid incorporation can be affected by large changes in pH_i, but not by the small pH_i change promoted by maturation. Therefore, activation of GVBD and amino acid incorporation must proceed by mechanisms which do not include changing pH_i. These conclusions appear to be true of oocyte activation by fertilization as well, for the pH_i change following insemination is even smaller than during maturation. These results are discussed in terms of mechanisms by which dormancy is controlled.     

INHIBITION OF GERMINAL VESICLE BREAKDOWN AND POLAR BODY FORMATION BY NICOTINAMIDE IN STARFISH AND SURF CLAM OOCYTES*

Nicotinamide inhibited both germinal vesicle breakdown (GVBD) and polar body formation (PBF) in surf clam and starfish oocytes. In the surf clam nicotinamide at 0.3 mM completely blocked PBF in the fertilized oocytes. For blockage of GVBD higher concentration was required. In the starfish, nicotinamide (30 mM) prevented PBF but not GVBD, when added 7 min after the commencement of 1-methyladenine (1-MeAde) administration. These results suggest that PBF is blocked by nicotinamide independent of its effect on GVBD. In the case of starfish, NAD⁺was more effective than nicotinamide in inhibiting oocyte maturation. Nicotinamide also blocked GVBD induced by microinjection of the cytoplasm containing maturation-promoting factor (MPF) obtained from 1-MeAde-treatcd oocytes. These results suggest that nicotinamide prevents the action of MPF rather than inhibiting the interaction of 1-McAde with cell membrane or the induction of MPF.     

Hill Coefficient Analysis of Transmembrane Helix Dimerization

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Cytoplasmic pH (pH _i ) has been shown to be an important parameter in cell physiology, regulating namely cell metabolism and differentiation. However, membrane transport mechanisms involved in pH _i regulation mechanisms of Sertoli cells have not yet been elucidated. In this work, pH _i was determined using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Addition of weak acids resulted in rapid acidification of the intracellular milieu. Sertoli cells then recovered pH _i by a mechanism that was shown to be sensitive to external Na⁺. pH _i recovery was also greatly reduced in the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and amiloride. These results point toward the action of an Na⁺-driven HCO₃⁻/Cl⁻ exchanger and/or an Na⁺/HCO₃⁻ cotransporter and the action of the Na⁺/H⁺ exchanger on pH _i regulation in the experimental conditions used. pH _i recovery was only slightly affected by ouabain, suggesting that the inhibition of Na⁺/K⁺-ATPase affects recovery indirectly, possibly via the shift on the Na⁺ gradient. On the other hand, recovery from the acid load was independent of the presence of concanamycin A, a specific inhibitor of the V-type ATPases, suggesting that these pumps do not have a relevant action on pH _i regulation in bovine Sertoli cells.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Effect of Trace Levels of Nigericin on Intracellular pH and Acid-Base Transport in Rat Renal Mesangial Cells

Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pH _i ) changes in the next. Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pH _i in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH _i decrease elicited by removing extracellular Na⁺, (ii) an increase in acid extrusion during the pH _i increase caused by elevating extracellular [K⁺], and (iii) an acid shift in the pH _i dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pH _i dependence of Na-H exchange, computed by adding the pH _i dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment. Received: 14 October 1998/Revised: 2 March 1999     

Cytoplasmic pH Responses to Carbonic Anhydrase Inhibitors in Cultured Rabbit Nonpigmented Ciliary Epithelium

Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pH _i ) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pH _i was measured using the fluorescent dye BCECF and the pH _i responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid pH _i changes elicited by bicarbonate/CO₂ removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pH _i . We considered whether CA-IV might facilitate H⁺ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mm) reduced pH _i 0.52 ± 0.10 pH units. In the presence of DBI, the magnitude of pH _i reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 ± 0.09 pH units. ACTZ similarly reduced the magnitude of the pH _i reduction. DBI also reduced by ∼40% the rate of pH _i recovery in cells acidified by an ammonium chloride (20 mm) prepulse; a reduction in pH _i recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pH _i alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H⁺ and HCO⁻ ₃ with CO₂ in the unstirred layer outside the plasma membrane, preventing local accumulation of H⁺ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pH _i changes that might alter the function of other ion transporters and channels in the NPE. Received: 24 April 1997/Revised: 4 November 1997     

Regulation of Intracellular pH by p90Rsk-dependent Activation of an Na+/H+ Exchanger in Starfish Oocytes

Starfish oocytes arrest at metaphase of the first meiotic division (MI arrest) in the ovary and resume meiosis after spawning into seawater. MI arrest is maintained by lower intracellular pH (pH_i) and release from arrest by cellular alkalization. To elucidate pH_i regulation in oocytes, we cloned the starfish (Asterina pectinifera) Na⁺/H⁺ exchanger 3 (ApNHE3) expressed in the plasma membrane of oocytes. The cytoplasmic domain of ApNHE3 contains p90 ribosomal S6 kinase (p90Rsk) phosphorylation sites, and injection of a constitutively active p90Rsk and the upstream regulator Mos to immature oocytes, stimulated an increase in pH_i. This increase was blocked by 5-(N-ethyl-N-isopropyl)-amiloride, a NHE inhibitor, and SL0101, a specific Rsk inhibitor. The MAPK kinase (MEK) inhibitor U0126 blocked the Mos-induced, but not the p90Rsk-induced, pH_i increase, suggesting that the Mos-MEK-MAPK-p90Rsk pathway promotes ApNHE3 activation. In a cell-free extract, the Mos-MEK-MAPK-p90Rsk pathway phosphorylates ApNHE3 at Ser-590, -606, and -673. When p90Rsk-dependent ApNHE3 phosphorylation was blocked by a dominant-negative C-terminal fragment, or neutralizing antibody, the p90Rsk-induced pH_i increase was suppressed in immature oocytes. However, ApNHE3 is up-regulated via the upstream phosphatidylinositol 3-kinase pathway before MAPK activation and the active state is maintained until spawning, suggesting that the p90Rsk-dependent ApNHE3 phosphorylation is unlikely to be the primary regulatory mechanism involved in MI arrest exit. After meiosis is completed, unfertilized eggs maintain their elevated pH_i (∼7.4) until the onset of apoptosis. We suggest that the p90Rsk/ApNHE3-dependent elevation of pH_i increases fertilization success by delaying apoptosis initiation.     

Cytosolic acidification leads to Ca mobilization from intracellular stores in single and populational parietal cells and platelets

Regulatory relationship and gain control between cytosolic free Ca²⁺ concentration (Ca_i) and cytosolic pH (pH_i) were evaluated by two different cell types, gastric parietal cells, and blood platelets. Studies were carried out in both single cells and populations of cells, using Ca²⁺-indicative probe fura-2 (1-(2-(5′-carboxyoxazol-2′-yl)-6-aminobenzofuran-5-oxy)-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid) and pH-indicative probe BCECF (2′,7′-bis(carboxyethyl) carboxyfluorescein). Stimulation of single and populational parietal cells and platelets with gastrin and thrombin, respectively, resulted in an increase in Ca_i. In both populational cell types, an initial change in pH_i during agonist stimulation occurred almost simultaneously with the mobilization of Ca²⁺; an initial transient decrease in pH_i was followed by a slower increase in pH_i above the prestimulation level. When populational platelets were preloaded with the Ca²⁺ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′tetraacetic acid), the thrombin-induced initial large increase in Ca_i was apparently inhibited, whereas the pH_i decrease induced by thrombin was not altered. This suggests that the initial Ca_i change is not a prerequisite for the pH_i change. The effect of pH_i on Ca_i was examined next. In both single and populational cell types, application of the K⁺-H⁺ ionophore nigericin, which induced a transient decrease in pH_i, led to the release of Ca²⁺ from intracellular stores. In single parietal cells double-labeled with fura-2 and BCECF, a temporal decrease in pH_i preceded the rise in Ca_i after stimulation with nigericin. A decrease in pH_i, and an increase in Ca_i occurred at 1.5 and 4 s, respectively. In single parietal cells, replacement of medium Na⁺ with N-methyl- -glucamine (NMG⁺), which also induced a decrease in pH_i, resulted in repetitive Ca²⁺ spike oscillations. The source of Ca²⁺ utilized for the Ca²⁺ oscillation that was induced by NMG⁺ originated from the agonist-sensitive pool. Thus, several maneuvers, which were capable of decreasing pH_i, led to an increase in Ca_i. Cytosolic acidification may be a part of the trigger for Ca²⁺ mobilization from intracellular stores in both parietal cells and platelets.     

A role for the vacuole in auxin‐mediated control of cytosolic pH by Vicia mesophyll and guard cells

A role for cytosolic pH (pH_i) in hormonal signalling and transport control in plants has long been mooted. Yet, while changes in pH_i are a common consequence of hormonal stimuli in plant cells and contribute to hormonally evoked ion channel control, the origins of these changes remain unknown. To examine a possible role for the tonoplast and vacuolar compartment in these events, pH_i was measured in the presence of auxins and during cytosolic H⁺ loading with weak acid in vacuolate and evacuolate protoplasts, both from mesophyll and guard cells of Vicia faba L. Evacuolate protoplasts were obtained following ultracentrifugation on Percoll gradients, and pH_i of single protoplasts was recorded in both vacuolate and evacuolate preparations using fluorescence ratio microphotometry and the pH-sensitive dye BCECF. External pH measurements indicated a roughly twofold increase in the rate of net H⁺ secretion in evacuolate compared with vacuolate protoplasts, and showed that evacuolate protoplasts retained the characteristic stimulation of H⁺ secretion in the presence of auxin. BCECF fluorescence recording gave resting pH_i values near 7.5, and evacuolation had no significant effect on this parameter. Reversible decreases of 0.1–0.2 units in pH_i were evoked in vacuolate protoplasts by 10 μM concentrations of the auxins 1-naphthalene acetic acid and 3-indoyl-acetic acid, and not by the inactive (anti-auxin) analogue 2-naphthalene-acetic acid. However, auxin treatments failed to evoke a change in pH_i in all but one of 12 experiments with evacuolate protoplasts. Evacuolation also appeared to reduce the transient, dynamic H⁺ buffering capacity of the protoplasts in the face of acid pH_i loads imposed by adding Na⁺-butyrate to the bath. These results implicate the tonoplast or vacuolar compartment in short-term pH_i homeostasis and generation of hormonally evoked H⁺ signalling in plant cells; they also conform with the view that the decrease in pH_iper se is not a primary determinant in the stimulation of H⁺ secretion by auxin.     

Upregulation of Na/H exchanger by the AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O₂ deficiency glycolytic degradation of glucose leads to formation of lactate and H⁺, thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H⁺ export. The present study explored whether AMPK influences Na⁺/H⁺ exchanger (NHE) activity and/or Na⁺-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH_i) was estimated utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺-dependent re-alkalinization (ΔpH_i) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH_i and ΔpH_i in those cells were significantly increased by treatment with AMPK stimulator AICAR (1 mM) and significantly decreased by AMPK inhibitor compound C (10 μM). The effect of AICAR on pH_i and ΔpH_i was blunted in the presence of the Na⁺/H⁺ exchanger inhibitor cariporide (10 μM), but not by the H⁺ ATPase inhibitor bafilomycin (10 nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

Mechanism of aldosterone-induced increase of K+ conductance in early distal renal tubule cells of the frog

Summary Isolated early distal tubule cells (EDC) of frog kidney were incubated for 20–28 hr in the presence of aldosterone and then whole-cell K⁺ currents were measured at constant intracellular pH by the whole-cell voltage-clamp technique. Aldosterone increased barium-inhibitable whole-cell K⁺ conductance (gK⁺) threefold. This effect was reduced by amiloride and totally abolished by ouabain. However, aldosterone could still raisegK⁺ in ouabain-treated cells in the presence of furosemide.We tested whether changes in intracellular pH (pH _i ) could be a signal for cells to regulategK⁺. After removal of aldosterone, the increase ingK⁺ was preserved by subsequent incubation for 8 hr at pH 7.6 but abolished at pH 6.6. In the complete absence of aldosterone, incubation of cells at pH 8.0 for 20–28 hr raised pH _i and doubledgK⁺.Using the patch-clamp technique, three types of K⁺-selective channels were identified, which had conductances of 24, 45 and 59 pS.Aldosterone had no effect on the conductance or open probability (P _o) of any of the three types of channels. However, the incidence of observing type II channels was increased from 4 to 22%. Type II channels were also found to be pH sensitive,P _o was increased by raising pH.These results indicate that prolonged aldosterone treatment raises pH _i and increasesgK⁺ by promoting insertion of K⁺ channels into the cell membrane. Channel insertion is itself triggered by raising both pH _i and increasing the activity of the Na⁺/K⁺ pump in early distal cells of frog kidney. Present address: Department of Physiology, The University of Leeds, Leeds, LS2 9NQ, England     

Effects of extracellular potassium concentration on meiotic and cytoplasmic maturation in the porcine oocyte

Effects of extracellular potassium (K⁺) concentration in maturation media on the meiotic and cytoplasmic maturation of porcine oocytes were examined. Oocyte-cumulus cell complexes or cumulus cell denuded oocytes were cultured in Whitten's medium containing 0, 3, 6, 12 or 16 mM potassium. Absence of K⁺ in the media did not inhibit germinal vesicle breakdown (GVBD) in cumulus intact oocytes, but significantly decreased the frequency of meiotic maturation. In cumulus cell denuded oocytes, both GVBD and meiotic maturation were inhibited in K⁺-free medium. Millimole concentrations of K⁺ channel blockers, 4-aminopyridine or tetraethyl ammonium chloride inhibited GVBD and almost completely suppressed progression of meiotic maturation. The effect of varying the concentration of K⁺ on cytoplasmic maturation of pig oocytes was evaluated by the ability to form a male pronucleus after in vitro fertilisation. The percentage of sperm penetration or monospermic penetration was not different among treatments (P > 0.1). However, male pronuclear formation in oocytes in medium with 6 mM K⁺ was higher than in media with 12 and 16 mM K⁺. These results suggest that extracellular K⁺ is required for GVBD and meiotic maturation, and high concentrations (12 or 16 mM) of K⁺ in maturation media impair cytoplasmic maturation.