Inside Alkaline Research Articles

The kinetics of transport of lactate and pyruvate into rat hepatocytes. Evidence for the presence of a specific carrier similar to that in erythrocytes.

Time courses of L-lactate and pyruvate uptake into isolated rat hepatocytes were measured in a citrate-based medium to generate a pH gradient (alkaline inside), by using the silicone-oil-filtration technique at 0 degrees C to minimize metabolism. At low concentrations of lactate and pyruvate (0.5 mM), transport was inhibited by over 95% by 5 mM-alpha-cyano-4-hydroxycinnamate, whereas at higher concentrations (greater than 10 mM) a significant proportion of transport could not be inhibited. The rate of this non-inhibitable transport was linearly related to the substrate concentration, was less with pyruvate than with L-lactate, and appeared to be due to diffusion of undissociated acid. Uptake of D-lactate was not inhibited by alpha-cyano-4-hydroxycinnamate and occurred only by diffusion. Kinetic parameters for the carrier-mediated transport process were obtained after correction of the initial rates of uptake of lactate and pyruvate in the absence of 5 mM-alpha-cyano-4-hydroxycinnamate by that in the presence of inhibitor. Under the conditions used, the Km values for L-lactate and pyruvate were 2.4 and 0.6 mM respectively and the Ki for alpha-cyano-4-hydroxycinnamate as a competitive inhibitor was 0.11 mM. Km values for the transport of L-lactate and pyruvate into rat erythrocytes under similar conditions were 3.0 and 0.96 mM. The Vmax. of lactate and pyruvate transport into hepatocytes at 0 degrees C was 3 nmol/min per mg of protein. Carrier-mediated transport of 0.5 mM-L-lactate was inhibited by 0.2 mM-p-chloromercuribenzenesulphonate (greater than 90%), 0.5 mM-quercetin (80%), 0.6 mM-isobutylcarbonyl-lactyl anhydride (70%) and 0.5 mM-4,4'-di-isothiocyanostilbene-2,2'-disulphonate (50%). A similar pattern of inhibition of lactate transport is seen in erythrocytes. It is suggested that the same or a similar carrier protein exists in both tissues. The results also show that L-lactate transport into rat hepatocytes is very rapid at physiological temperatures and is unlikely to restrict the rate of its metabolism. Differences between our results and those of Fafournoux, Demigne & Remesy [(1985) J. Biol. Chem. 260, 292-299] are discussed.

A variable stoichiometry model for pH homeostasis in bacteria

The composition of the proton-motive force of a hypothetical bacterial cell of wide pH tolerance is analyzed according to a model whereby the electron transport chain and various proton-linked sodium and potassium ion transporting modes are responsible for the development of the membrane potential and the chemical potentials of the three cations. Simultaneous use of two or more modes employing the same metal cation, but at a different stoichiometric ratio with respect to protons, produces nonintegral stoichiometry; the modes could represent either different devices or different states of a single device. Cycling of the cation, driven by proton-motive force, results. The relative conductances of the various modes are postulated to be pH-dependent. The pattern of potentials that results is qualitatively in accord with current knowledge and may reflect the mechanism of pH homeostasis in bacteria. The membrane potential is outwardly directed (positive inside) at extremely acid pH, becoming inwardly directed as the pH increases; the pH gradient across the membrane is large and inwardly directed (alkaline inside) at acid pH, becoming smaller and eventually inverting at alkaline pH values; the transmembrane potassium gradient is outwardly directed (high concentration inside) at all pH values; the transmembrane sodium gradient is inwardly directed at all pH values, following the pH gradient from acid through neutral pH, but then diverging at alkaline pH.

Calcium-induced opacification is dependent upon lens pH

The intracellular pH of a normal lens is 6.8 in the cortex and remains unchanged during culture in media buffered at pH 7.2. Incubation of rabbit lenses in calcium enriched media, either at 24 degrees C or 37 degrees C, results in lens opacification provided that the lens pH remains slightly acidic. Opacities are prevented in cultured lenses with an alkaline interior (pH 7.1-7.3) despite the accumulation of calcium (1.3 mM). The mechanism by which an intracellular pH shift from 6.8 to 7.1 prevents opacification in the presence of excess calcium is not known, but does not appear to depend upon the total level of bound calcium. This study provides the first data that opacification caused by calcium is associated with lens pH.

Mechanism of action of Pseudomonas syringae phytotoxin, syringomycin. Interaction with the plasma membrane of wild-type and respiratory-deficient strains of Saccharomyces cerevisiae.

The effects of the phytotoxin, syringomycin, produced by Pseudomonas syringae pv. syringae, were examined on cells of a wild-type and a respiratory-deficient (rho0) mutant of Saccharomyces cerevisiae. The growth of both strains in liquid culture was inhibited by 0.5 micrograms syringomycin per ml and higher. Uptake rates of tetraphenylphosphonium and dimethyloxazolidine ions in cell suspensions of both strains increased when 1.5 micrograms per ml syringomycin was added. These responses were kinetically and quantitatively similar in the two strains and indicated increases in electrical potential (cell interior negative) and pH differences (cell interior alkaline) across the plasma membrane. Glucose (0.1 M) enhanced the effect on the electrical potential, was required for the pH changes, and increased the cellular ATP levels. These results show that the effects of syringomycin are energy-dependent and are due to alterations of plasma membrane and not to mitochondrial function.

Effects of triorganotin-mediated anion-hydroxide exchange upon reconstituted cytochrome c oxidase proteoliposomes.

Proteoliposomes containing cytochrome c oxidase and an internally trapped fluorescent pH probe (pyranine) were used to monitor respiration-dependent internal alkalinization and membrane potential formation. A maximum steady-state pH gradient of about 0.4 pH unit (vesicle interior alkaline) was obtained during active respiration in presence of reducing substrates and cytochrome c. This pH gradient was abolished by the triorganotin compounds tripropyl-, tributyl-, and triphenyl-tin chloride. At the same time, the membrane potential, measured by carbocyanine dye uptake, was slightly increased in value. Valinomycin, which abolishes the membrane potential, restores the value of delta pH at low trialkyltin concentrations. The organotin compounds acted as electroneutral ionophores which exchanged intravesicular OH- ions with external SCN-, I-, and CI- ions, but not NO3- or SO4(2-) ions. Abolition of delta pH is accompanied by an increase in respiration rate, but full respiratory stimulation only occurs when both delta psi and delta pH are abolished by addition of both triorganotin and valinomycin. The triorganotin-valinomycin combination leads to active KCl accumulation by the respiring proteoliposome, and it is necessary to postulate an electrically neutral KCl efflux process to explain the continued steady respiration of the proteoliposomes in the presence of this ionophore combination.

Oxalate transport by anion exchange across rabbit ileal brush border.

This study demonstrates the presence of oxalate transporters on the brush border membrane of rabbit ileum. We found that an inside alkaline (pH = 8.5 inside, 6.5 outside) pH gradient stimulated [14C]oxalate uptake 10-fold at 1 min with a fourfold accumulation above equilibrated uptake at 5 min. 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (disodium salt; DIDS) profoundly inhibited the pH-gradient stimulated oxalate uptake. Using an inwardly directed K+ gradient and valinomycin, we found no evidence for potential sensitive oxalate uptake. In contrast to Cl:HCO3 exchange, HCO3 did not stimulate oxalate uptake more than was seen with a pH gradient in the absence of HCO3. An outwardly directed Cl gradient (50 mM inside, 5 mM outside) stimulated oxalate uptake 10-fold at 1 min with a fivefold accumulation above equilibrated uptake. Cl-stimulated oxalate uptake was largely inhibited by DIDS. Addition of K+ and nigericin only slightly decreased the Cl gradient-stimulated oxalate uptake, which indicates that this stimulation was not primarily due to the Cl gradient generating an inside alkaline pH gradient via Cl:OH exchange. Further, an outwardly directed oxalate gradient stimulated 36Cl uptake. These results suggested that both oxalate:OH and oxalate:Cl exchange occur on the brush border membrane. To determine if one or both of these exchanges were on contaminating basolateral membrane, the vesicle preparation was further fractionated into a brush border and basolateral component using sucrose density gradient centrifugation. Both exchangers localized to the brush border component. A number of organic anions were examined (outwardly directed gradient) to determine if they could stimulate oxalate and Cl uptake. Only formate and oxaloacetate were found to stimulate oxalate and Cl uptake. An inwardly directed Na gradient only slightly stimulated oxalate uptake, which was inhibited by DIDS.

Mechanism of action of Pseudomonas syringae phytotoxin, syringomycin. Interaction with the plasma membrane of wild-type and respiratory-deficient strains of Saccharomyces cerevisiae

The effects of the phytotoxin, syringomycin, produced by Pseudomonas syringae pv. syringae, were examined on cells of a wild-type and a respiratory-deficient ( rho 0 ) mutant of Saccharomyces cerevisiae. The growth of both strains in liquid culture was inhibited by 0.5 μg syringomycin per ml and higher. Uptake rates of tetraphenylphosphonium and dimethyloxazolidine ions in cell suspensions of both strains increased when 1.5 μg per ml syringomycin was added. These responses were kinetically and quantitatively similar in the two strains and indicated increases in electrical potential (cell interior negative) and pH differences (cell interior alkaline) across the plasma membrane. Glucose (0.1 M) enhanced the effect on the electrical potential, was required for the pH changes, and increased the cellular ATP levels. These results show that the effects of syringomycin are energy-dependent and are due to alterations of plasma membrane and not to mitochondrial function.

Chloride/formate exchange with formic acid recycling: a mechanism of active chloride transport across epithelial membranes.

The pathways for transport of Cl- and formate in microvillus membrane vesicles isolated from rabbit renal cortex were evaluated. An outward formate gradient stimulated the uptake of Cl-, and an outward Cl- gradient stimulated the uptake of formate, indicating Cl-/formate exchange. In addition, an inside alkaline pH gradient induced the accumulation of formate, consistent with nonionic diffusion of formic acid. Although an inward Na+ gradient also stimulated uphill formate accumulation, suggesting Na+/formate cotransport, this effect was abolished when ionophores were used to prevent the generation of a transmembrane pH gradient, indicating an indirect coupling of formic acid transport to Na+/H+ exchange. An inside alkaline pH gradient only minimally stimulated the uptake of 82Br-, used as tracer for Cl-, confirming the absence of appreciable Cl-/OH- exchange. However, the same pH gradient in the presence of a physiologic formate concentration (0.2 mM) markedly stimulated 82Br- influx. These data suggest that Cl-/formate exchange with recycling of formic acid by nonionic diffusion is a potential mechanism for active Cl- absorption across the luminal membrane in the proximal tubule and perhaps in other epithelia.

A polyvinylchloride-membrane based anion selective electrode for continuous registration of ΔpH (interior alkaline) with salicylate as the indicator probe

An anion sensitive electrode has been constructed with the use of the lipid soluble cation benzyl-dimethyl-hexadecylammonium analogous to the procedure described for tetraphenylphosphonium-sensitive electrodes [Shino, T., Kamo, N., Kurihara, K. and Kobatake Y. (1978) Arch. Biochem. Biophys. 187, 414–422]. The anion electrode responds to salicylate concentrations above 400 μM with a Nernstian sensitivity. Less lipid soluble anions like chloride and phosphate do not interfere. Below 400 μM salicylate the response of the electrode decreases gradually so that the sensitivity of the electrode is less than 10 mV per decade change at concentrations of the anion of 50 μM. A computer program has been developed to fit the electrode response curve with a polynomal function of the fourth power. Additional software allows calculation of changes in the concentration of the salicylate anion, also under conditions where the sensitivity of the electrode for the anion is not constant. In this way the electrode can be used to measure changes in salicylate concentration that occur in a suspension of bacteria when, upon energization, a pH gradient is generated. 31P nuclear magnetic resonance measurements demonstrated that the pH gradient measured with the salicylate-sensitive electrode in the phototrophic bacterium Rhodopseudomonas sphaeroides is quantitatively correct. The response time of the electrode decreases from 1 min at 20 μM salicylate to 10 s at concentrations ⩾ 200 μM.

Methanogenesis and the K+ transport system are activated by divalent cations in ammonia-treated cells of Methanospirillum hungatei.

We describe a K+ transport system in Methanospirillum hungatei cells depleted of cytoplasmic K+ via an ammonia/K+ exchange reaction (Sprott, G. D., Shaw, K. M., and Jarrell, K. F. (1984) J. Biol. Chem. 259, 12602-12608). Ammonia-treated cells contained low concentrations of ATP and were unable to make CH4 or to transport 86Rb+. All of these properties were restored by CaCl2, MgCl2, or MnCl2, and not by CoCl2 or NiCl2. The Rb+ transport system had a Km of 0.42 and Vmax of 29 nmol/min X mg; K+ inhibited competitively. Both H2 and CO2 were required for appreciable transport, whereas air, valinomycin, or nigericin were potent inhibitors. The influx of Rb+ was electrogenic and associated with proton efflux, producing a delta pH (alkaline inside) in acidic media. In the absence of K+ (or Rb+), the activation of CH4 synthesis by Mg2+ produced little change in the cytoplasmic pH, showing that methanogenesis did not elicit a net efflux of protons. The pH optimum for transport was in the range 6.0-7.3 where the transmembrane pH gradient would contribute minimally to the proton motive force. Protonophores at pH 6.3 caused a partial decline in CH4 synthesis and the ATP content and dramatically collapsed Rb+ transport. These and other inhibitor experiments, coupled with the fact that the Rb+ gradient was too large to be in equilibrium with the proton motive force alone, suggest a role for both ATP and the proton motive force in Rb+ transport. Also, a role for K+ in osmoregulation is indicated.

Effect of trypsin treatment of bacteriorhodopsin on its orientation in reconstituted vesicles

The light-induced proton pump bacteriorhodopsin (bR) can be used to generate an electrochemical proton gradient (Ap) across the membrane of reconstituted vesicles, bR can be incorporated into membrane vesicles derived from the lactic acid bacterium Streptococcus eremoris by fusion of these bacterial membrane vesicles with bR-containing proteoliposomes (Driessen et al., 1985). For studies on the relationship between the Ap and solute transport (e.g. amino acids) in such model systems, a Ap, inside alkaline and negative, is required. Only one method has been described for the reconstitution of bR proteoliposomes that show light-dependent proton extrusion (Happe et al., 1977; Hellingwerf, 1982) and consequently generate a rightside-out-oriented Ap. These rightsideout bR proteoliposomes can be obtained by co-sonication of purple membranes together with phospholipid vesicles, containing acidic phospholipids using a short acidic stage (pH 2.4--3.0) during sonication. Although these preparations show light-dependent proton extrusion, a considerable part of the vesicles contains bR with an inside-out orientation. It has been suggested (Hellingwerf, 1982) that the distribution of charges over the two sides of bR is an important factor in determining the orientation of the protein in the reconstituted membrane. We therefore studied the effect of proteolytic treatment ofbR on the orientation of the protein in the reconstituted membrane. Under fully native conditions only the C-terminal part of the protein is accessible to proteolytic enzymes (e.g. trypsine). A peptide fragment is removed containing five carboxylic acid groups, negatively charged at neutral pH. Removal of this peptide results in a decrease of the negative surface charge density of the C-side (cytoplasmic side) of bR (Arrio et al., 1984) resulting in an extensive aggregation of the purple membrane sheets. Trypsin-treated bR was reconstituted into phospholipid vesicles, containing the negatively charged phospholipid cardiolipin, by the use of the rightside-out reconstitution procedure (Happe et al., 1977; Hellingwerf, 1982) at different pH values during the acid stage. An inversion of the direction of proton transloeation upon illumination can be observed when the pH of the acid stage of the reconstitution procedure is varied. This inflection point is around pH 3.0 for non-treated bR and about 3.5 for trypsin-treated bR. Trypsin-treated bR can be reconstituted in a rightside-out orientation with a higher efficiency, compared to non-treated bR. This is concluded from the following observations: (i) the extent of proton extrusion by trypsin-treated bR proteoliposomes is considerably higher than by non-treated bR, (ii) the proton extrusion extent is stimulated 1.2-2-fold by valinomycin and this is not observed in non-treated bR, (iii) tetraphenylphosphonium (TPP § is accumulated and tetraphenylboron (TPB) is extruded by proteoliposomes prepared with trypsintreated bR. Proteoliposomes prepared with native bR show some TPB- uptake, indicating the presence of proteoliposomes generating a Ap, interior positive and acid. These results support the mechanism proposed by Hellingwerf (1982) for the reconstitution of bR with an inside-out orientation in which the charge distribution over the two sides of bR plays an important role. The use of bR proteoliposomes with an inside-out orientation may be an important factor for a successful incorporation of bR into bacterial membrane vesicles, such that a Ap, inside negative and alkaline, is generated upon illumination.

Solubilization and reconstitution of the Na +-motive NADH oxidase activity from the marine bacterium Vibrio alginolyticus

The Na +-motive NADH oxidase activity from Vibrio alginolyticus was extracted with octylglucoside and reconstituted into liposomes by dilution. On the addition of NADH, the reconstituted proteoliposomes generated Δψ (inside positive) and ΔpH (inside alkaline) in the presence of a proton conductor CCCP, and accumulated Na + in the presence of valinomycin. These results indicate that the NADH oxidase activity, reconstituted in opposite orientation, leads to the generation of an eletrochemical potential of Na + by the influx of Na +.

Kinetic properties of sodium-proton antiport in Escherichia coli membrane vesicles: effects of imposed electrical potential, proton gradient, and internal pH

Modifications of the kinetic properties of the Escherichia coli (RA11) Na(+) - H(+) antiport system by imposed pH gradients (deltapH, interior alkaline) and membrane potential(delta(psi), interior negative) were studied by looking at the accelerating effects of deltapH and delta on downhill Na(+) efflux from membrane vesicles incubated at different external pHs. First,variations of the Na(+) efflux rate ( VNa) as a function of imposed delta pH appear to be strongly dependent on the external pH value.The individual VN, vs. deltapH relationships observed between pH 5.5 and pH 6.6 are all nonlinear and indicate the existence of a threshold deltapH above which V(Na) increases steeply as the deltapH magnitude increases; threshold deltapH values progressively decrease as the pH is raised from 5.5 to 6.6. In contrast, at or above neutrality, V(Na) acceleration is linearly related to deltapH amplitude. Strikingly, it is shown that the deltapH-dependent variations in the Na(+) efflux rate measured in vesicles incubated at different external pHs can be accounted for by variations of internal pH; the observed relationship suggests that a high internal H(+) concentration inhibits the Na(+) -H(+) antiport activity.This inhibition results from a drastic increase in the apparent K(m), of the Na(+) efflux reaction as the internal H(+) concentration increases. On the other hand, imposed Δ increases the Na(+) efflux rate linearly by a selective modification of the V(max) value of the Na(+) efflux. Together, these data indicate that the internal H(+) concentration controls the Na(+)-H(+) antiport activity and that the chemical and electrical proton gradients affect two different kinetic steps of the Na(+)-H(+) exchange reaction.

Cytochrome o type oxidase from Escherichia coli. Characterization of the enzyme and mechanism of electrochemical proton gradient generation.

Cytochrome o type oxidase purified from the membrane of Escherichia coli consists of four polypeptides (Mr 66000, 35000, 22000, and 17000), and the monomeric form predominates in octyl beta-D-glucopyranoside. The oxidase complex contains two b-type cytochromes (b-558 and b-563) and 2 mol of heme/mol of enzyme. Cytochrome o utilizes ubiquinol-1 and a number of other artificial electron donors as substrates but does not oxidize reduced cytochrome c or ferrocyanide. Activity is highly dependent upon exogenous phospholipids and/or Tween 20, and the quinone analogues 2-heptyl-4-hydroxyquinoline N-oxide and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole are potent inhibitors. Proteoliposomes were formed by detergent dilution or dialysis in the presence of the oxidase and phospholipids, followed by freeze-thaw/sonication. Vesicles formed by this means are unilamellar and contain a random distribution of 85-90-A intramembranous particles on the convex and concave fracture surfaces. During oxidase turnover, the reconstituted system generates a proton electrochemical gradient (interior negative and alkaline) of -115 to -140 mV; however, respiratory control is minimal (i.e., respiratory control ratios of about 1.5 are observed). By using a glass electrode to measure changes in external pH and the fluorescence of entrapped 8-hydroxy-1,3,6-pyrenetrisulfonate to measure changes in internal pH, it is apparent that during ubiquinol oxidation, protons are released on the external surface of the membrane and consumed on the internal surface. In contrast, with N,N,N',-N'-tetramethyl-p-phenylenediamine, an electron donor that carries few protons at neutral pH, little change in external pH is observed until the protonophore carbonyl cyanide m-chlorophenylhydrazone is added, at which point the medium becomes alkaline. The results taken as a whole are consistent with the concept that oxidase turnover generates an electrical potential (interior negative) due to vectorial electron flow from the outer to the inner surface of the membrane. The pH gradient (interior alkaline), on the other hand, appears to result from scalar (i.e., nonvectorial) reactions that consume and release protons at the inner and/or outer surfaces of the membrane, respectively. In other words, cytochrome o oxidase from Escherichia coli does not appear to catalyze vectorial proton translocation.

Intracellular pH distribution and transmembrane pH profile of yeast cells

The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 μm. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0–7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15–20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5–7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20–30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the ΔpH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.

Transport by reconstituted lactose carrier from parental and mutant strains of Escherichia coli.

The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lower Km but similar Vmax compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.

Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles.

The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen ion cotransport by the renal brush border glutamate transporter.

Sodium ion dependent glutamate transport into rabbit renal brush border membrane vesicles is stimulated by low external pH and inhibited by low internal pH. Imposition of a pH difference (delta pH) (interior alkaline) across the vesicle membrane drives glutamate accumulation in the absence of other driving forces. This process requires Na+ but is not due to generation of an Na+ gradient in response to delta pH. Internal K+ stimulates both the rate and extent of glutamate accumulation, although K+ is not absolutely required for transport. Internal H+ inhibits the rate of glutamate accumulation by decreasing this K+ stimulation. Conversely, external K+ inhibits glutamate influx, and this inhibition can be overcome by lowering the external pH. These results indicate that H+ is cotransported with glutamate and suggest the possibility that, in the absence of internal K+, H+ can also fulfill the requirement for a countertransported ion.

Proton motive force and Na+/H+ antiport in a moderate halophile.

The influence of pH on the proton motive force of Vibrio costicola was determined by measuring the distributions of triphenylmethylphosphonium cation (membrane potential, delta psi) and either dimethyloxazolidinedione or methylamine (osmotic component, delta pH). As the pH of the medium was adjusted from 5.7 to 9.0, the proton motive force steadily decreased from about 170 to 100 mV. This decline occurred, despite a large increase in the membrane potential to its maximum value at pH 9.0, because of the loss of the pH gradient (inside alkaline). The cytoplasm and medium were of equal pH at 7.5; membrane permeability properties were lost at the pH extremes of 5.0 and 9.5. Protonophores and monensin prevented the net efflux of protons normally found when an oxygen pulse was given to an anaerobic cell suspension. A Na+/H+ antiport activity was measured for both Na+ influx and efflux and was shown to be dissipated by protonophores and monensin. These results strongly favor the concept that respiratory energy is used for proton efflux and that the resulting proton motive force may be converted to a sodium motive force through Na+/H+ antiport (driven by delta psi). A role for antiport activity in pH regulation of the cytosol can also explain the broad pH range for optimal growth, extending to the alkaline extreme of pH 9.0.

Proton electrochemical potential of the inner mitochondrial membrane in isolated perfused rat hearts, as measured by exogenous probes

The membrane potential (Δψ) and ΔpH of the inner mitochondrial membrane were studied in isolated perfused rat hearts using exogenous labelled probes and tissue fractionation in non-aqueous media. The mitochondrial Δψ, measured by means of the subcellular distribution of [ 3H]triphenylmethylphosphonium (TPMP +), was 125 ± 7 mV (negative inside) in hearts beating at 5 Hz and 150 ± 3 mV (negative inside) in hearts beating at 1.5 Hz. The mitochondrial membrane ΔpH, measured by means of the subcellular distribution of low concentrations of [1- 14C]propionate, was 0.63 ± 0.06 pH units (alkaline inside) in hearts beating at 5 Hz and 0.53 ± 0.12 pH units (alkaline inside) in hearts beating at 1.5 Hz. The implication of proton and electron gradients in the regulation of cellular respiration is discussed. In combination with previous evidence on adenylate distribution in the isolated perfused rat heart, the results indicate that the mitochondrial electrogenic adenylate translocator is in near equilibrium with Δψ.