The effect of ooplasmic pH regulation on the formation of yolk spheres in the telotrophic ovariole of Dysdercus intermedius

Vitellogenic oocytes of Dysdercus intermedius (Heteroptera: Pyrrhocoridae) were treated with the proton ionophore monensin in order to load the ooplasm with protons along the electrochemical gradient. Additionally, changes in the ooplasmic pH (DeltapH(OOC)) were recorded during exposure the oocytes to potassium-free medium (K(+)(MED)=0mM; choline for K(+)) or sodium-free medium (Na(+)(MED)=0mM; 40mM of choline for 40mM of Na(+)). The following observations were made: 1) The average ooplasmic pH (pH(OOC)) recorded during immersion in physiological saline solution (PSS) was pH(OOC(PSS))=7.40. 2) K(+)(MED) had no effect on pH(OOC) (pH(OOC(K-FREE)) congruent with pH(OOC(PSS))). 3) In sodium-free medium the pH(OOC) decreased by H(+) influx in the magnitude of DeltapH(OOC(Na-FREE))=pH(OOC(Na-FREE))-pH(OOC(PSS))=-0.05 pH units. 4) The decreased pH(OOC) observed in sodium-free medium returned to initial values (7.40) by pumping out H(+) when 40mM of choline were replaced by 40mM of Na(+). 5) Addition of monensin (10&mgr;M; under the condition of Na(+)(MED)=0mM) reduced pH(OOC) in the magnitude of DeltapH(OOC(MON))=pH(OOC(MON))-pH(OOC(PSS))=-0.14. 6) Monensin induced ooplasmic proton loading was reversible when 40mM choline were replaced by 40mM Na(+).VITELLOGENESIS WAS DEMONSTRATED BY THE ACCUMULATION OF FLUORESCENCE LABELLED HEMOLYMPH PROTEINS IN YOLK SPHERES IN THE CORTEX OF THE OOCYTE: 1) Yolk formation continued in potassium-free medium. 2) The formation of yolk spheres came to a halt in sodium-free medium and, additionally, in the presence of monensin (10&mgr;M; Na(+)(MED)=0mM). 3) Breaks in yolk formation under the condition of Na(+)(MED)=0mM or during monensin treatment were stopped by replacing 40mM of choline with 40mM of Na(+). The results obtained using proton-specific microelectrodes and the in vitro assay to detect the formation of yolk spheres indicate that both the ooplasmic pH regulation and the acidification of vesicles during vitellogenesis are under control of a H(+)/Na(+) antiporter.     

Granulosa cells regulate oocyte intracellular pH against acidosis in preantral follicles by multiple mechanisms

Mammalian oocytes grow within ovarian follicles in which the oocyte is coupled to surrounding granulosa cells by gap junctions. We report here that growing oocytes isolated from mouse preantral follicles are incapable of recovering from an experimentally induced acidosis, and that oocytes acquire the ability to manage acid loads by activating Na(+)/H(+) exchange during growth. By contrast, granulosa cells from similar preantral follicles possess substantial Na(+)/H(+) exchange capacity, which is attributable to the simultaneous action of two Na(+)/H(+) exchanger isoforms: NHE1 and NHE3. Granulosa cells were also found to possess a V-type H(+)-ATPase that drives partial acidosis recovery when Na(+)/H(+) exchange is inactivated. By monitoring intracellular pH (pH(i)) in small follicle-enclosed oocytes, we found that the oocyte has access to each of these acidosis-correcting activities, such that small follicle-enclosed oocytes readily recover from acidosis in a manner resembling granulosa cells. However, follicle-enclosed oocytes are unable to access these activities if gap-junction communication within the follicle is inhibited. Together, these experiments identify the NHE isoforms involved in regulating oocyte pH(i), indicate that gap junctions allow granulosa cells to exogenously regulate oocyte pH(i) against acidosis until the oocyte has acquired endogenous pH(i) regulation, and reveal that granulosa cells possess multiple mechanisms for carrying out this function.     

Measuring ion transport activities in Xenopus oocytes using the ion-trap technique

The ion-trap technique is an experimental approach allowing measurement of changes in ionic concentrations within a restricted space (the trap) comprised of a large-diameter ion-selective electrode apposed to a voltage-clamped Xenopus laevis oocyte. The technique is demonstrated with oocytes expressing the Na(+)/glucose cotransporter (SGLT1) using Na(+)- and H(+)-selective electrodes and with the electroneutral H(+)/monocarboxylate transporter (MCT1). In SGLT1-expressing oocytes, bath substrate diffused into the trap within 20 s, stimulating Na(+)/glucose influx, which generated a measurable decrease in the trap Na(+) concentration ([Na(+)](T)) by 0.080 +/- 0.009 mM. Membrane hyperpolarization produced a further decrease in [Na(+)](T), which was proportional to the increased cotransport current. In a Na(+)-free, weakly buffered solution (pH 5.5), H(+) drives glucose transport through SGLT1, and this was monitored with a H(+)-selective electrode. Proton movements can also be clearly detected on adding lactate to an oocyte expressing MCT1 (pH 6.5). For SGLT1, time-dependent changes in [Na(+)](T) or [H(+)](T) were also detected during a membrane potential pulse (150 ms) in the presence of substrate. In the absence of substrate, hyperpolarization triggered rapid reorientation of SGLT1 cation binding sites, accompanied by cation capture from the trap. The resulting change in [Na(+)](T) or [H(+)](T) is proportional to the pre-steady-state charge movement. The ion-trap technique can thus be used to measure steady-state and pre-steady-state transport activities and provides new opportunities for studying electrogenic and electroneutral ion transport mechanisms.     

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.     

Na(+)/H(+) exchange in mosquito Malpighian tubules

Fluid secretion and intracellular pH were measured in isolated mosquito Malpighian tubules to determine the presence of Na(+)/H(+) exchange. Rates of fluid secretion by individual Malpighian tubules in vitro were inhibited by 78% of control in the presence of 100 microM 5-(N-ethyl-n-isopropyl)-amiloride (EIPA), a specific inhibitor of Na(+)/H(+) exchange. Steady-state intracellular pH was measured microfluorometrically by using 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in individual Malpighian tubules. Bathing the Malpighian tubules in 0 mM extracellular Na(+) or in the presence of 100 microM EIPA reduced the steady-state intracellular pH by 0.5 pH units. Stimulation of the Na(+)/H(+) exchanger by using the NH(4)Cl pulse technique resulted in a rate of recovery from the NH(4)Cl-induced acute acid load of 8.7 +/- 1.0 x 10(-3) pH/s. The rates of recovery of intracellular pH after the acute acid load in the absence of extracellular Na(+) or in the presence of 100 microM EIPA were 0.7 +/- 0.6 and -0.3 +/- 0.3 x 10(-3) pH/s, respectively. These results indicate that mosquito Malpighian tubules possess a Na(+)/H(+) exchanger.     

Detection of apical Na(+)/H(+) exchanger activity inhibition in proximal tubules induced by acute hypertension

We previously showed that acute arterial hypertension induces an inhibition of fluid and NaCl reabsorption in proximal tubules of Sprague-Dawley rats, which is associated with a rapid reversible internalization of apical Na(+)/H(+) exchanger in brush border. To determine whether there is a corresponding inhibition of apical Na(+)/H(+) exchanger activity in proximal tubules to account for the reduced tubular reabsorption, an instrument capable of measuring intracellular pH (pH(i)) ratiometrically and repeatedly on the surface of kidney with high temporal resolution is required. We report the design and validation of such a fluorimetric system based on two ultraviolet nitrogen-pulsed lasers and a photomultiplier. pH(i) of proximal tubules in situ was measured with pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein at 5 Hz. Using the initial rate of change of pH(i) (dpH(i)/dt) during luminal Na(+) removal as an index of apical Na(+)/H(+) exchanger activity, the exchanger activity was found to be reduced by 52 +/- 11% (n = 14, P < 0.05) compared with the baseline after 20 min of induced acute hypertension. The inhibition of Na(+)/H(+) exchange activity was alleviated when the blood pressure was returned to prehypertensive level. These observations indicate that acute changes in arterial pressure can reversibly inhibit apical Na(+)/H(+) exchanger activity, which might contribute to pressure natriuresis in proximal tubule.     

A possible role of the Na(+)/K(+)-ATPase for O(2) production from H(2)O(2)

We are attempting to supply a new insight on interaction between Na(+)/K(+)-ATPase and H(2)O(2). We demonstrate that in vitro the Na(+)/K(+)-ATPase, a non heme-protein, is able to disproportionate H(2)O(2) catalatically into dioxygen and water, as well as C(40) catalase. By polarography, we quantify O(2) production and by Raman spectroscopy H(2)O(2) consumption. A comparative analysis of kinetics parameters relative to O(2) production shows that for Na(+)/K(+)-ATPase the affinity of the catalytic site able to transform H(2)O(2) into O(2) is twice weaker than that for C(40) catalase. It also shows that the molar activity for O(2) production is 300-fold weaker for ATPase than for catalase. Inhibitors, pH and GSH studies highlight the differences between the heme- and nonheme-proteins. Indeed, for C(40), NaN(3) is strongly inhibiting, but much less for ATPase. The pH range for the catalatic activity of ATPase is wide (6.5 to 8.5), while it is not for C(40) catalase (optimum at pH 8). The Na(+)/K(+)-ATPase catalatic activity is reduced in presence of glutathione, while it is not the case with C(40) catalase.     

Mechanism of intracellular pH increase during parthenogenetic activation of In vitro matured porcine oocytes

Parthenogenetic activation of porcine oocytes by using 7% ethanol, 50 or 100 microM A23187 results in an increase in intracellular pH as does prolonged exposure to thimerosal. We attempt to specify which transporters or mechanisms are involved in the observed increase in intracellular pH during oocyte activation. Experiments were performed in the absence of sodium; the presence of 2.5 mM amiloride, a potent inhibitor of the Na(+)/H(+) antiport; in the absence of bicarbonate; and in the presence of 4, 4'-diisothiocyanatodihydrostilbene-2,2'-di-sulfonic acid, disodium salt (H(2)DIDS) for all three activation methods. These treatments had no effect on the increase in intracellular pH induced by the calcium ionophore or thimerosal, but all reduced the increase in pH (P < 0.001) in the 7% ethanol group. This suggests that the Na(+)/H(+) antiport and the HCO(3)(-)/Cl(-) exchangers are not playing a role during treatment with calcium ionophore or thimerosal, and the pH increase observed during treatment with 7% ethanol may be dependent upon a sodium or bicarbonate flux (or both) into the oocyte. Bafilomycin A1 (500 nm), an inhibitor of vacuolar-type H(+) ATPases, had no effect on 7% ethanol or thimerosal treatments, but significantly reduced the increase in intracellular pH observed during calcium ionophore treatment. This may be the result of an initial local increase in intracellular free calcium levels.     

Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na(+)/H(+) exchanger

The present study was aimed at evaluating the role of D(1)- and D(2)-like receptors and investigating whether inhibition of Na(+) transepithelial flux by dopamine is primarily dependent on inhibition of the apical Na(+)/H(+) exchanger, inhibition of the basolateral Na(+)-K(+)-ATPase, or both. The data presented here show that opossum kidney cells are endowed with D(1)- and D(2)-like receptors, the activation of the former, but not the latter, accompanied by stimulation of adenylyl cyclase (EC(50) = 220 +/- 2 nM), marked intracellular acidification (IC(50) = 58 +/- 2 nM), and attenuation of amphotericin B-induced decreases in short-circuit current (28.6 +/- 4.5% reduction) without affecting intracellular pH recovery after CO(2) removal. These results agree with the view that dopamine, through the activation of D(1)- but not D(2)-like receptors, inhibits both the Na(+)/H(+) exchanger (0.001933 +/- 0.000121 vs. 0.000887 +/- 0.000073 pH unit/s) and Na(+)-K(+)-ATPase without interfering with the Na(+)-independent HCO transporter. It is concluded that dopamine, through the action of D(1)-like receptors, inhibits both the Na(+)/H(+) exchanger and Na(+)-K(+)-ATPase, but its marked acidifying effects result from inhibition of the Na(+)/H(+) exchanger only, without interfering with the Na(+)-independent HCO transporter and Na(+)-K(+)-ATPase.     

H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1-liposomes

The H(+)/ATP ratio and the standard Gibbs free energy of ATP synthesis were determined with a new method using a chemiosmotic model system. The purified H(+)-translocating ATP synthase from chloroplasts was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. During reconstitution, the internal phase was equilibrated with the reconstitution medium, and thereby the pH of the internal liposomal phase, pH(in), could be measured with a conventional glass electrode. The rates of ATP synthesis and hydrolysis were measured with the luciferin/luciferase assay after an acid-base transition at different [ATP]/([ADP][P(i)]) ratios as a function of deltapH, analysing the range from the ATP synthesis to the ATP hydrolysis direction and the deltapH at equilibrium, deltapH (eq) (zero net rate), was determined. The analysis of the [ATP]/([ADP][P(i)]) ratio as a function of deltapH (eq) and of the transmembrane electrochemical potential difference, delta micro approximately (H)(+) (eq), resulted in H(+)/ATP ratios of 3.9 +/- 0.2 at pH 8.45 and 4.0 +/- 0.3 at pH 8.05. The standard Gibbs free energies of ATP synthesis were determined to be 37 +/- 2 kJ/mol at pH 8.45 and 36 +/- 3 kJ/mol at pH 8.05.     

Pancreatic bicarbonate secretion involves two proton pumps

Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium express H(+)/HCO(3)(-) transporters, which depend on gradients created by the Na(+)/K(+)-ATPase. However, the model cannot fully account for high-bicarbonate concentrations, and other active transporters, i.e. pumps, have not been explored. Here we show that pancreatic ducts express functional gastric and non-gastric H(+)-K(+)-ATPases. We measured intracellular pH and secretion in small ducts isolated from rat pancreas and showed their sensitivity to H(+)-K(+) pump inhibitors and ion substitutions. Gastric and non-gastric H(+)-K(+) pumps were demonstrated on RNA and protein levels, and pumps were localized to the plasma membranes of pancreatic ducts. Quantitative analysis of H(+)/HCO(3)(-) and fluid transport shows that the H(+)-K(+) pumps can contribute to pancreatic secretion in several species. Our results call for revision of the bicarbonate transport physiology in pancreas, and most likely other epithelia. Furthermore, because pancreatic ducts play a central role in several pancreatic diseases, it is of high relevance to understand the role of H(+)-K(+) pumps in pathophysiology.     

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.     

Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (～7.6) and ΔpH(m) (～0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (～5 mm, pH 7.8) compared with the cytosol (～20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification.     

Development of the follicle complex and oocyte staging in red drum,sciaenops ocellatus linnaeus, 1776 (perciformes,sciaenidae)

Pelagic egg development in red drum, Sciaenops ocellatus, is described using tiered staging. Based on mitosis and meiosis, there are five periods: Mitosis of Oogonia, Active Meiosis I, Arrested Meiosis I, Active Meiosis II, and Arrested Meiosis II. The Periods are divided into six stages: Mitotic Division of Oogonia, Chromatin Nucleolus, Primary Growth, Secondary Growth, Oocyte Maturation and Ovulation. The Chromatin Nucleolus Stage is divided into four steps: Leptotene, Zygotene, Pachytene, and Early Diplotene. Oocytes in the last step possess one nucleolus, dispersed chromatin with forming lampbrush chromosomes and lack basophilic ooplasm. The Primary Growth Stage, characterized by basophilic ooplasm and absence of yolk in oocytes, is divided into five steps: One‐Nucleolus, Multiple Nucleoli, Perinucleolar, Oil Droplets, and Cortical Alveolar. During primary growth, the Balbiani body develops from nuage, enlarges and disperses throughout the ooplasm as both endoplasmic reticulum and Golgi develop within it. Secondary growth or vitellogenesis has three steps: Early Secondary Growth, Late Secondary Growth and Full‐Grown. The Oocyte Maturation Stage, including ooplasmic and germinal vesicle maturation, has four steps: Eccentric Germinal Vesicle, Germinal Vesicle Migration, Germinal Vesicle Breakdown and Resumption of Meiosis when complete yolk hydration occurs. The period is Arrested Meiosis II. When folliculogenesis is completed, the ovarian follicle, an oocyte and encompassing follicle cells, is surrounded by a basement membrane and developing theca, all forming a follicle complex. After ovulation, a newly defined postovulatory follicle complex remains attached to the germinal epithelium. It is composed of a basement membrane that separates the postovulatory follicle from the postovulatory theca. Arrested Meiosis I encompasses primary and secondary growth (vitellogenesis) and includes most of oocyte maturation until the resumption of meiosis (Active Meiosis II). The last stage, Ovulation, is the emergence of the oocyte from the follicle when it becomes an egg or ovum. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Spinotrapezius muscle microcirculatory function: effects of surgical exteriorization

Intravital microscopy facilitates insights into muscle microcirculatory structural and functional control, provided that surgical exteriorization does not impact vascular function. We utilized a novel combination of phosphorescence quenching, microvascular oxygen pressure (microvascular PO(2)), and microsphere (blood flow) techniques to evaluate static and dynamic behavior within the exposed intact (I) and exteriorized (EX) rat spinotrapezius muscle. I and EX muscles were studied under control, metabolic blockade with 2,4-dinitrophenol (DNP), and electrically stimulated conditions with 1-Hz contractions, and across switches from 21 to 100% and 10% inspired O(2). Surgical preparation did not alter spinotrapezius muscle blood flow in either I or EX muscle. DNP elevated muscle blood flow approximately 120% (P < 0.05) in both I and EX muscles (P > 0.05 between I and EX). Contractions reduced microvascular PO(2) from 30.4 +/- 4.3 to 21.8 +/- 4.8 mmHg in I muscle and from 33.2 +/- 3.0 to 25.9 +/- 2.8 mmHg in EX muscles with no difference between I and EX. In each O(2) condition, there was no difference (each P > 0.05) in microvascular PO(2) between I and EX muscles (21% O(2): I = 37 +/- 1; EX = 36 +/- 1; 100%: I = 62 +/- 5; EX = 51 +/- 9; 10%: I = 20 +/- 1; EX = 17 +/- 2 mmHg). Similarly, the dynamic behavior of microvascular PO(2) to altered inspired O(2) was unaffected by the EX procedure [half-time (t(1/2)) to 100% O(2): I = 23 +/- 5; EX = 23 +/- 4; t(1/2) to 10%: I = 14 +/- 2; EX = 16 +/- 2 s, both P > 0.05]. These results demonstrate that the spinotrapezius muscle can be EX without significant alteration of microvascular integrity and responsiveness under the conditions assessed.     

Effect of azathioprine on Na(+)/H(+) exchanger activity in dendritic cells

Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug.     

Plasmalemmal V-H(+)-ATPases regulate intracellular pH in human lung microvascular endothelial cells

The lung endothelium layer is exposed to continuous CO(2) transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na(+)/H(+) exchanger and HCO(3)(-)-dependent H(+)-transporting mechanisms regulate intracellular pH (pH(cyt)) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H(+)-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na(+)/H(+) exchanger and HCO(3)(-)-based H(+)-transporting mechanisms, to maintain pH(cyt) homeostasis. Immunocytochemical studies revealed V-H(+)-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na(+) and HCO(3)(-) that were similar to those observed in the presence of either Na(+), or Na(+) and HCO(3)(-). The Na(+)- and HCO(3)(-)-independent pH(cyt) recovery was inhibited by bafilomycin A(1), a V-H(+)-ATPase inhibitor. These studies show a Na(+)- and HCO(3)(-)-independent pH(cyt) regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases.     

Proton uptake upon anaerobic reduction of the Paracoccus denitrificans cytochrome c oxidase: a kinetic investigation of the K354M and D124N mutants

The kinetics and stoichiometry of the redox-linked protonation of the soluble Paracoccus denitrificans cytochrome c oxidase were investigated at pH = 7.2-7.5 by multiwavelength stopped-flow spectroscopy, using the pH indicator phenol red. We compared the wild-type enzyme with the K354M and the D124N subunit I mutants, in which the K- and D-proton-conducting pathways are impaired, respectively. Upon anaerobic reduction by Ru-II hexamine, the wild-type enzyme binds 3.3 +/- 0.6 H(+)/aa(3), i.e., approximately 1 H(+) in excess over beef heart oxidase under similar conditions and the D124N mutant 3.2 +/- 0.5 H(+)/aa(3). In contrast, in the K354M mutant, in which the reduction of heme a(3)-Cu(B) is severely impaired, approximately 0.8 H(+) is promptly bound synchronously with the reduction of heme a, followed by a much slower protonation associated with the retarded reduction of the heme a(3)-Cu(B) site. These results indicate that complete reduction of heme a (and Cu(A)) is coupled to the uptake of approximately 0.8 H(+), which is independent of both H(+)-pathways, whereas the subsequent reduction of the heme a(3)-Cu(B) site is associated with the uptake of approximately 2.5 H(+) transferred (at least partially) through the K-pathway. On the basis of these results, the possible involvement of the D-pathway in the redox-linked protonation of cytochrome c oxidase is discussed.     

Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth

Oocytes grow within ovarian follicles in which the oocyte is coupled to the surrounding granulosa cells by gap junctions. It was previously found that small growing oocytes isolated from juvenile mice and freed of their surrounding granulosa cells (denuded) lacked the ability to regulate their intracellular pH (pH(i)), did not exhibit the pH(i)-regulatory HCO(3)(-)/Cl(-) and Na(+)/H(+) exchange activities found in fully-grown oocytes, and had low pH(i). However, both exchangers became active as oocytes grew near to full size, and, simultaneously, oocyte pH(i) increased by approximately 0.25 pH units. Here, we show that, in the more physiological setting of the intact follicle, oocyte pH(i) is instead maintained at approximately 7.2 throughout oocyte development, and the growing oocyte exhibits HCO(3)(-)/Cl(-) exchange, which it lacks when denuded. This activity in the oocyte requires functional gap junctions, as gap junction inhibitors eliminated HCO(3)(-)/Cl(-) exchange activity from follicle-enclosed growing oocytes and substantially impeded the recovery of the oocyte from an induced alkalosis, implying that oocyte pH(i) may be regulated by pH-regulatory exchangers in granulosa cells via gap junctions. This would require robust HCO(3)(-)/Cl(-) exchange activity in the granulosa cells, which was confirmed using oocytectomized (OOX) cumulus-oocyte complexes. Moreover, in cumulus-oocyte complexes with granulosa cells coupled to fully-grown oocytes, HCO(3)(-)/Cl(-) exchange activity was identical in both compartments and faster than in denuded oocytes. Taken together, these results indicate that growing oocyte pH(i) is controlled by pH-regulatory mechanisms residing in the granulosa cells until the oocyte reaches a developmental stage where it becomes capable of carrying out its own homeostasis.