Showing papers on "Electrochemical gradient published in 1994"

Energy Transduction by Cytochrome Complexes in Mitochondrial and Bacterial Respiration: The Enzymology of Coupling Electron Transfer Reactions to Transmembrane Proton Translocation

Abstract: INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676 THE PROTONMOTIVE ENZYME COMPLEXES OF RESPIRATION ..... . . . 676 A COMPARISON OF MITOCHONDRIAL AND BACTERIAL RESPIRATORY SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 677 PROTON TRANSLOCATION SCHEMES . . . . . . . . . . . . . . . . . . . . . . . . . . 679 Cytochrome bd: Formation of a Proton Gradient by Substrate Protons without a Transmembrane Channel . . . . . . . . . . . . . . . . . . . . . . . . . . 679 Cytochrome bo: Proton Translocation by Substrate Protons Plus a Transmembrane Channel 680 Cytochrome c Oxidase: A Proton Pump . . . . . . . . . . . . . . . . . . . . . . . . . . 681 Cytochrome bCI Complex: Proton Translocation by Substrate Protons . . . . . . . 681

Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) pH decrease

Abstract: The formation of zeaxanthin (Zea) from violaxanthin (Vio) in chloroplasts of leaves and algae upon strong illumination is currently suggested to play a role in the photoprotection of plants. Properties and location of the enzyme Vio de-epoxidase, which is responsible for the transformation of Vio to Zea, were studied using thylakoid membrane vesicles isolated from leaves of Spinacia oleracea L. Without using detergents a repeated freeze-thaw treatment of thylakoid vesicles was sufficient to release the enzyme into the medium. With the same procedure the mobile electron carrier plastocyanin, known to occur in the thylakoid lumen, was also released. The enzyme was demonstrated by its activity in the supernatant of the pelleted thylakoid vesicles in the presence of the added substrates Vio and ascorbic acid, as well as by staining of the released proteins after polyacrylamide gel electrophoresis. The release of the deepoxidase from the vesicles was pH-dependent, declined below pH 6.5 and ceased in the pH range around 5, which corresponds to the pH optimum of the enzyme activity. By using thylakoid vesicles isolated from pre-illuminated and therefore Zea-containing leaves the release by freeze-thaw cycles of both the de-epoxidase and plastocyanin was diminished compared with the dark control. However, the reason for this effect was not the Zea content but an unknown effect of the illumination on the thylakoid membrane properties. The de-epoxidase collected at pH 7 was able to re-bind to thylakoid membranes at pH 5.5 and to transform intrinsic Vio to Zea in the presence of ascorbate. The isolated de-epoxidase, as well as the endogenous membrane-bound de-epoxidase, was inhibited by dithiothreitol. From these results it is concluded that Vio de-epoxidase, like plastocyanin, is mobile within the thylakoid lumen at neutral pH values which occur under in-vivo conditions in the dark. However, upon strong illumination, when the lumen pH drops (pH < 6.5) due to the formation of a proton gradient, the properties of the de-epoxidase are altered and the enzyme becomes tightly bound to the membrane (in contrast to plastocyanin) thus gaining access to its substrate Vio. These findings corroborate the assumption of a transmembrane opposite location of the two enzymes of the xanthophyll cycle, the ascorbate-dependent Vio deepoxidase at the lumenal side and the NADPH-dependent Zea epoxidase at the stromal side. Indications in favour of a location of Vio within the lipid bilayer of the thylakoid membrane and of a binding of the active deepoxidase to these areas are discussed.

Transport of Ascorbic and Dehydroascorbic Acids across Protoplast and Vacuole Membranes Isolated from Barley (Hordeum vulgare L. cv Gerbel) Leaves.

Abstract: Protoplasts, vacuoles, and chloroplasts were isolated from leaves of 8-d-old barley (Hordeum vulgare L. cv Gerbel) seedlings. Transport of ascorbate and dehydroascorbate into protoplasts and vacuoles was investigated. Contents of ascorbic acid, glutathione, and [alpha]-tocopherol and ascorbate peroxidase activity and glutathione reductase activity were analyzed in protoplasts, vacuoles, and chloroplasts. Uptake of ascorbate and dehydroascorbate by protoplasts showed saturation kinetics (Km = 90 [mu]M reduced ascorbic acid, 20 [mu]M dyhydroascorbic acid). Effects of various membrane transport inhibitors suggested that transport was carrier mediated and driven by a proton electrochemical gradient. Translocation of ascorbate and dehydroascorbate into vacuoles did not show saturation kinetics. Neither was it influenced by effectors or by ATP but only by Mg2+, suggesting that translocation did not occur by carrier. Ascorbic acid was predominantly localized in the cytosol. Contents in the chloroplasts and vacuoles were low. The results are consistent with the view that ascorbate is synthesized in the cytosol and released to chloroplasts, apoplast, and vacuole following a concentration gradient. Translocation from the apoplast into the cytosol is against a steep gradient and appears to control the concentration of ascorbic acid in the apoplast. In its function as an antioxidant, ascorbate in the apoplast may be oxidized to dehydroascorbate, which can be efficiently transported back into the cytosol for regeneration to ascorbate.

Transcellular transport of benzoic acid across Caco-2 cells by a pH-dependent and carrier-mediated transport mechanism.

Abstract: The pH-dependent transcellular transport of [14 C]benzoic acid across a Caco-2 cell monolayer is shown to be mediated by a monocarboxylic acid-specific carrier-mediated transport system, localized on the apical membrane. Evidence for the carrier-mediated transport of benzoic acid includes (a) the significant temperature and concentration dependence, (b) the metabolic energy dependence, (c) the inhibition by unlabeled benzoic acid and other monocarboxylic acids, (d) countertransport effects on the uptake of [14C]benzoic acid, and (e) effects of a proteinase (papain) and amino acid-modifying reagents. Furthermore, since carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin significantly inhibited the transport of [14C] benzoic acid, the direct driving force for benzoic acid transport is suggested to be the inwardly directed proton gradient. From these results, together with previous observations using intestinal brush border membrane vesicles, the pH dependence of the transcellular transport of certain organic weak acids across Caco-2 cells is considered to result mainly from a proton gradient-dependent, carrier-mediated transport mechanism, rather than passive diffusion according to the pH-partition theory.

Characterization of Sulfate Transport in Chlamydomonas reinhardtii during Sulfur-Limited and Sulfur-Sufficient Growth.

Abstract: We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.

Experimental discrimination between proton leak and redox slip during mitochondrial electron transport

Abstract: By measuring the relationship between protonmotive force and the increment in oxygen consumption by mitochondria treated with submaximal amounts of uncoupler, we have experimentally tested four different models of imperfect coupling of oxidative phosphorylation. The results show that the increased rate of oxygen consumption at high protonmotive force is explained entirely by the dependence on protonmotive force of the passive proton leak conductance of the mitochondrial inner membrane. There is no measurable contribution from redox-slip reactions in the proton pumps caused by high protonmotive force. Neither is there any contribution from increased proton conductance of the membrane or increased redox slip in the respiratory chain caused by high turnover rates of the complexes.

Activity of the plasma membrane H+-ATPase is a key physiological determinant of thermotolerance in Saccharomyces cerevisiae

Abstract: The role of membrane integrity and the membrane ATPase in the mechanism of thermotolerance in Saccharomyces cerevisiae was investigated. The resistance to lethal heat of a mutant strain with reduced expression of the membrane ATPase was significantly less than that of the wild-type parent. However, prior exposure to sub-lethal temperatures resulted in the induction of similar levels of thermotolerance in the mutant compared to the parent strain, suggesting that the mechanism of sub-lethal heat-induced thermotolerance is independent of ATPase activity. Supporting this, exposure to sub-lethal heat stress did not result in increased levels of glucose-induced acid efflux at lethal temperatures and there was little correlation between levels of acid efflux and levels of heat resistance. ATPase activity in crude membrane preparations from sub-lethally heat-stressed cells was similar to that in preparations from unstressed cells. Study of net acid flux during heating revealed that pre-stressed cells were able to protect the proton gradient for longer. This may confer an ‘advantage’ to these cells that results in increased thermotolerance. This was supported by the observation that prior exposure to sub-lethal heat resulted in a transient protection against the large increase in membrane permeability that occurs at lethal temperatures. However, no protection against the large drop in intracellular pH was detected. Sub-lethal heat-induced protection of membrane integrity also occurred to the same extent in the reduced-expression membrane ATPase mutant, further implying that the mechanism of induced thermotolerance is independent of ATPase activity. To conclude, although the membrane ATPase is essential for basal heat resistance, thermotolerance induced by prior exposure to stress is largely conferred by a mechanism that is independent of the enzyme.

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Abstract: Methanosarcina mazei Go1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Go1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.

The effect of extracellular pH and lactic acid on pH homeostasis inLactococcus lactis andStreptococcus bovis

Abstract: WhenStreptococcus bovis JB1 andLactococcus lactis ML3 were grown with an excess of glucose, lactic acid accumulation caused a decrease in extracellular pH;S. bovis grew at extracellular pH values as low as 4.9, butL. lactis was unable to grow below pH 5.3. Because both bacteria maintained a low ΔpH across the cell membrane, it appeared that intracellular pH was controlling their pH sensitivities.S. bovis glycolyzed glucose and maintained high concentrations of ATP at intracellular pH values as low as 5.4.L. lactis could not glycolyze glucose when the intracellular pH was less than 5.6, and ATP declined.L. lactis cells that were washed and incubated in buffers with an excess glucose had higher ΔpH values than growing cells. Lactic acid addition, however, prevented the interconversion of membrane potential (Δψ) and chemical gradient of protons (ZΔpH).

Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos.

Abstract: The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (delta psi) and a pH gradient (delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate. Membrane vesicles of L. oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport. Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive. A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles. Kinetic analysis of delta pH-driven citrate uptake over a range of external pHs suggests that the monovalent anionic species (H2cit-) is the transported particle. Together, the data show that the transport of citrate is an electrogenic process in which H2cit- is translocated across the membrane via a uniport mechanism. Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found. It is concluded that the generation of metabolic energy by citrate utilization in L. oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H(+)-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient. The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).

Mechanism of electron transfer in the cytochrome b/f complex of algae: evidence for a semiquinone cycle.

Abstract: The most widely accepted mechanism of electron and proton transfer within the cytochrome (Cyt) b/f complex derives from the Q-cycle hypothesis originally proposed for the mitochondrial Cyt b/c1 complex by Mitchell [Mitchell, P. (1975) FEBS Lett. 57, 135-137]. In chloroplasts, the Cyt b/f complex catalyzes the oxidation of a plastoquinol at a site, Qo (the plastoquinol binding site), close to the inner aqueous phase and the reduction of a quinone at a site, Qi (the plastoquinone binding site), close to the stromal side of the membrane. In an alternative model, the semiquinone cycle [Wikstrom, M. & Krab, K. (1986) J. Bioenerg. Biomembr. 18, 181-193], a charged semiquinone formed at site Qo is transferred to site Qi where it is reduced into quinol. Flash-induced kinetics of the redox changes of Cyt b and of the formation of a transmembrane potential have been measured in Chlorella sorokiniana cells incubated in reducing conditions that induce a full reduction of the plastoquinone pool. The experiments were performed in the presence of an uncoupler that collapses the permanent electrochemical proton gradient and thus accelerates the rate of the electrogenic processes. The results show that the electrogenic reaction driven by the Cyt b/f complex precedes the processes of reduction or oxidation of the b-hemes. This electrogenic process is probably due to a transmembrane movement of a charged semiquinone, in agreement with the semiquinone-cycle hypothesis. This mechanism may represent an adaptation to reducing conditions when no oxidized quinone is available at the Qi site.

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625

Abstract: The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.

Interactions of Ca2+ and NaCI stress on the ion relations and intracellular pH of Sorghum bicolor root tips: An in vivo 31P-NMR study

Abstract: In vivo 31 P-NMR measurements showed that supplemental Ca 2+ (5.0 mM CaSO 4 ) decreased the magnitude of the NaCl-induced reduction of the pH gradient across the tonoplast (ΔpH tonoplast ) in Sorghum bicolor root tips exposed to 200 mM NaCl. The reduced ΔpH tonoplast in root tips exposed to 200 mM NaCl was primarily due to vacuolar alkalization rather than cytoplasmic acidification. Maintenance of the ΔpH tonoplast may be important for salinity tolerance since the trans-tonoplast H + electrochemical gradient is the putative driving force for Na + transport from the cytoplasm into the vacuole via a Na + /H + antiport

Developmental cell-specific regulation of Na(+)-K(+)-ATPase alpha 1-, alpha 2-, and alpha 3-isoform gene expression.

Abstract: Na(+)-K(+)-activated adenosine triphosphatase (Na(+)-K(+)-ATPase) is the integral membrane protein that maintains the Na(+)-K(+) electrochemical gradient across the plasma membrane. Because of the ...

Na(+)-H+ antiport detected through hydrogen ion currents in rat alveolar epithelial cells and human neutrophils.

Abstract: Voltage-activated H(+)-selective currents were studied in cultured adult rat alveolar epithelial cells and in human neutrophils using the whole-cell configuration of the patch-clamp technique. The H+ conductance, gH, although highly selective for protons, was modulated by monovalent cations. In Na+ and to a smaller extent in Li+ solutions, H+ currents were depressed substantially and the voltage dependence of activation of the gH shifted to more positive potentials, when compared with the "inert" cation tetramethylammonium (TMA+). The reversal potential of the gH, Vrev, was more positive in Na+ solutions than in inert ion solutions. Amiloride at 100 microM inhibited H+ currents in the presence of all cations studied except Li+ and Na+, in which it increased H+ currents and shifted their voltage-dependence and Vrev to more negative potentials. The more specific Na(+)-H+ exchange inhibitor dimethylamiloride (DMA) at 10 microM similarly reversed most of the suppression of the gH by Na+ and Li+. Neither 500 microM amiloride nor 200 microM DMA added internally via the pipette solution were effective. Distinct inhibition of the gH was observed with 1% [Na+]o, indicating a mechanism with high sensitivity. Finally, the effects of Na+ and their reversal by amiloride were large when the proton gradient was outward (pHo parallel pHi 7 parallel 5.5), smaller when the proton gradient was abolished (pH 7 parallel 7), and absent when the proton gradient was inward (pH 6 parallel 7). We propose that the effects of Na+ and Li+ are due to their transport by the Na(+)-H+ antiporter, which is present in both cell types studied. Electrically silent H+ efflux through the antiporter would increase pHi and possibly decrease local pHo, both of which modulate the gH in a similar manner: reducing the H+ currents at a given potential and shifting their voltage-dependence to more positive potentials. A simple diffusion model suggests that Na(+)-H+ antiport could deplete intracellular protonated buffer to the extent observed. Evidently the Na(+)-H+ antiporter functions in perfused cells, and its operation results in pH changes which can be detected using the gH as a physiological sensor. Thus, the properties of the gH can be exploited to study Na(+)-H+ antiport in single cells under controlled conditions.

L-alanine absorption in human intestinal Caco-2 cells driven by the proton electrochemical gradient

Abstract: In human Caco-2 intestinal epithelial layers, xxxl-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na+-free media, is associated with a saturable net transepithelial absorption of xxxl-alanine. xxxl-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. xxxl-Alanine transport in Na+-free media is rheogenic, stimulating an inward short-circuit current in voltageclamped epithelial monolayers. By measurement of rapid xxxl-alanine influx across the apical membrane, xxxl-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate xxxl-alanine/proton stoichiometry to be 1∶0.62 ±0.25 (xxxsd) (short-circuit current) or 1∶0.73 ±0.19 (intracellular acidification). From competition studies, it is likely that xxxl-proline, α-aminoisobutyric acid, and β-alanine, but not xxxl-valine and xxxl-serine, are substrates for protonlinked, substrate transport in the brush border of Caco-2 cells.

Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.

Abstract: The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium has been genetically transferred into the inner mitochondrial membrane (IM) of the eukaryotic cell Schizosaccharomyces pombe, where the archaebacterial proton pump replaces or increases the proton gradient usually formed by the respiratory chain. For targeting and integration, as well as for the correct orientation of bR in the IM, the bacterioopsin gene (bop) was fused to signal sequences of IM proteins. Northern and Western blot analysis proved that all hybrid gene constructs containing the bop gene and a mitochondrial signal sequence were expressed and processed to mature bR. Fast transient absorption spectroscopy showed photocycle activity of bR integrated in the IM by formation of the M intermediate. Experiments with the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein revealed bR-mediated proton pumping from the mitochondrial matrix into the intermembrane space. Glucose uptake measurements under anaerobic conditions showed that yeast cells containing photoactive mitochondria need less sugar under illumination. In summary, our experiments demonstrate the functional genetic transfer of a light energy converter to a naturally nonphotoactive eukaryotic organism.

The role of monovalent cation/proton antiporters in Na(+)-resistance and pH homeostasis in Bacillus: an alkaliphile versus a neutralophile.

Abstract: Both neutralophilic Bacillus subtilis and alkaliphilic Bacillus firmus OF4 depend upon electrogenic Na+/H+ antiporters, which are energized by the gradients established by respiration-coupled proton extrusion, to achieve Na(+)-resistance and pH homeostasis when the external pH is very alkaline. The interplay of proton and sodium cycles is discussed. In B. subtilis, pH homeostasis, up to pH9, can be achieved using K+ when Na+ is unavailable or when the gene encoding the Na+/H+ antiporter that is involved in Na(+)-dependent pH homeostasis is disrupted. That gene is a member of the tetracycline efflux family of genes. A second gene, encoding a Na+/H+ antiporter that functions in Na(+)-resistance, has been identified, and candidates for the K+/H+ antiporter genes are under investigation. Aggregate Na+/H+ antiport activity in B. subtilis is as much as 10 times lower than in the alkaliphile, and the neutralophile cannot regulate its internal pH upon a shift to pH 10.5. Upon such a shift, there is a pronounced reduction in the generation of a primary electrochemical proton gradient. The alkaliphile, by contrast, maintains substantial driving forces and regulates its internal pH in an exclusively Na(+)-coupled manner upon shifts to either pH 8.7 or 10.5. One gene locus has been identified and a second locus has been inferred as encoding relevant antiporter activities.

Acute reductions in PO2 depolarize pulmonary artery endothelial cells and decrease [Ca2+]i

Abstract: Whereas pulmonary artery endothelial cells (PAECs) are sensitive to oxygen, neither the effect of an acute reduction in PO2 on PAEC membrane potential nor its effect on intracellular free Ca2+ ([Ca2+]i) is known. We hypothesized that in confluent primary cultures of PAECs, an acute decrease in PO2 would depolarize the cell membrane, inhibit Ca2+ influx, and reduce [Ca2+]i. To test this hypothesis, the membrane-sensitive fluorophore bis (1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4, 1 microM) and [Ca2+]i-sensitive probe fura 2 (3 microM) were used. A decrease in PO2 from 125 to 35 mmHg caused membrane depolarization and a 60 +/- 8% (data are means +/- SE) reduction in Ca2+ influx, estimated by manganese quenching of fura 2 fluorescence. While basal [Ca2+]i was 79 +/- 5 nM in normoxic cells, it decreased to 31 +/- 2 nM after 15 min of hypoxia. Decreasing the electrochemical gradient for Ca2+ entry with either low extracellular Ca2+, the K+ channel blockers tetraethylammonium or charybdotoxin, or blockade of Ca2+ entry with lanthanum decreased [Ca2+]i by 54-71% of that observed during an acute reduction in PO2. These results demonstrate that an acute reduction in PO2 1) depolarizes PAECs, 2) reduces Ca2+ influx, and 3) decreases [Ca2+]i, and that a similar reduction in [Ca2+]i was observed with interventions designed to reduce the electrochemical driving force for Ca2+ entry.