Functional characteristics of pyruvate transport in Phycomyces blakesleeanus

A saturable and accumulative transport system for pyruvate has been detected in Phycomyces blakesleeanus NRRL 1555(-) mycelium. It was strongly inhibited by alpha-cyano-4-hydroxycinnamate. l-Lactate and acetate were competitive inhibitors of pyruvate transport. The initial pyruvate uptake velocity and accumulation ratio was dependent on the external pH. The Vmax of transport greatly decreased with increasing pH, whereas the affinity of the carrier for pyruvate was not affected. The pyruvate transport system mediated its homologous exchange, which was essentially pH independent, and efflux, which increased with increasing external pH. The uptake of pyruvate was energy dependent and was strongly inhibited by inhibitors of oxidative phosphorylation and of the formation of proton gradients. Glucose counteracted the inhibitory effect of the pyruvate transport produced by inhibitors of mitochondrial ATP synthesis. Our results are consistent with a pyruvate/proton cotransport in P. blakesleeanus probably driven by an electrochemical gradient of H+ generated by a plasma membrane H+-ATPase.     

Energy transduction in the thermophilic anaerobic bacterium Clostridium fervidus is exclusively coupled to sodium ions

The thermophilic, peptidolytic, anaerobic bacterium Clostridium fervidus is unable to generate a pH gradient in the range of 5.5-8.0, which limits growth of the organism to a narrow pH range (6.3-7.7). A significant membrane potential (delta psi approximately -60 mV) and chemical gradient of Na+ (-Z delta pNa approximately -60 mV) are formed in the presence of metabolizable substrates. Energy-dependent Na+ efflux is inhibited by the Na+/H+ ionophore monensin but is stimulated by uncouplers, suggesting that the Na+ gradient is formed by a primary pumping mechanism rather than by secondary Na+/H+ antiport. This primary sodium pump was found to be an ATPase that has been characterized in inside-out membrane vesicles and in proteoliposomes in which solubilized ATPase was reconstituted. The enzyme is stimulated by Na+, resistant to vanadate, and sensitive to nitrate, which is indicative of an F/V-type Na(+)-ATPase. In the proteoliposomes Na+ accumulation depends on the presence of ATP, is inhibited by the ATPase inhibitor nitrate, and is completely prevented by the ionophore monensin but is stimulated by protonophores and valinomycin. These and previous observations, which indicated that secondary amino acid transport uses solely Na+ as coupling ion, demonstrate that energy transduction at the membrane in C. fervidus is exclusively dependent on a Na+ cycle.     

ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin. 2. Relationships between proton motive force and ATP synthesis

The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to to 200 nmol ATP x min(-1) x mg protein (-1) were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal deltapH attainable at 40 degrees C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40 degrees C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of delta pH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96-->Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by delta pH/delta psi transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.     

Acid, protons and Helicobacter pylori

The anti-ulcer drugs that act as covalent inhibitors of the gastric acid pump are targeted to the gastric H+/K+ ATPase by virtue of accumulation in acid and conversion to the active sulfenamide. This results in extremely effective inhibition of acid secretion. Appropriate dosage is able to optimize acid control therapy for reflux and peptic ulcer disease as compared to H2 receptor antagonists. However, clinical data on recurrence show that Helicobacter pylori eradication should accompany treatment of the lesion. These drugs have been found to synergize with many antibiotics for eradication. The survival of aerobes depends on their ability to maintain a driving force for protons across their inner membrane, the sum of a pH and potential difference gradient, the protonmotive force (pmf). The transmembrane flux of protons across the F1F0 ATPase, driven by the pmf, is coupled to the synthesis of ATP. The internal pH of H. pylori was measured using the fluorescent dye probe, BCECF, and the membrane potential defined by the uptake of the carbocyanine dye, DiSC3 [5] at different pHs to mimic the gastric environment. The protonmotive force at pH 7.0 was composed of a delta pH of 1.4 (-84mV) and a delta potential difference of -131mV, to give a pmf of -215 mV. The effect of variations in external pH on survival of the bacteria in the absence of urea correlated with the effect of external pH on the ability of the bacteria to maintain a pmf. The effect of the addition of 5 mM urea on the pmf was measured at different medium pH values. Urea restored the pmf at pH 3.0 or 3.5, but abolished the pmf at pH 7.0 or higher, due the production of the alkalinizing cation, NH3. Hence H. pylori is an acid-tolerant neutrophile due to urease activity, but urease activity also limits its survival to an acidic environment. These data help explain the occupation of the stomach by the organism and its distribution between fundus and antrum. This distribution and its alteration by proton pump inhibitors also explains the synergism of proton pump inhibition and antibiotics such as amoxicillin and clarithromycin in H. pylori eradication.     

Effects of Surface-Bound Fe2+ on Nitrate Reduction and Transformation of Iron Oxide(s) in Zero-Valent Iron Systems at Near-Neutral pH

Nitrate reduction in an iron/nitrate/water system with or without an organic buffer was investigated using multiple batch reactors under strict anoxic conditions. Nitrate reduction was very limited (<10%) at near-neutral pH in the absence of the organic buffer. However, nitrate reduction was greatly enhanced if the system: (1) had a low initial pH ( ～ 2–3); (2) was primed with adequate aqueous Fe2+; or (3) was in the presence of the organic buffer. In Cases (1) and (3), nitrate reduction usually was involved in three stages. The first stage was quick, and H+ ions directly participated in the corrosion of iron grains. The second stage was very slow due to the formation of amorphous oxides on the surface of iron grains, while the third stage was characterized by a rapid nitrate reduction concurrent with the disappearance of aqueous Fe2+. Results indicate that reduction of nitrate by Fe0 will form magnetite; Fe2+ (aq.) can accelerate reduction of nitrate and will be substoichiometrically consumed. Once nitrate is exhausted in the system, no more Fe2+ will be consumed. In the presence of nitrate, Fe2+ (aq) will be adsorbed onto the surface of iron grains or iron oxides; the surface-complexed Fe(II) (extracted by acetate with pH = 4.1) might be oxidized and become structural Fe(III), resulting in a steadily increasing ratio of Fe(III)/Fe(II) in the oxides formed. The transformation of nonstoichiometric amorphous iron oxides into crystalline magnetite, a nonpassive oxide, triggers the rapid nitrate removal thereafter.     

pH dependence of corneal oxygen consumption

PURPOSE: To determine whether corneal acidosis, which occurs during contact lens wear, alters corneal O2 consumption (QO2) and if so, whether increased ion transport activity could contribute to altered QO2 during acidosis. METHODS: PO2 was measured, using the phosphorescence quenching of Pd-meso-tetra-(4-carboxyphenyl) porphine, in an airtight chamber that held a trephined rabbit cornea. The rate of change in chamber PO2 was used as a measure of QO2. QO2 was measured at pH 7.5 and then at either pH 6.7, 7.1, or 7.3. Measurements of QO2 at pHs 7.5 and 6.7 were repeated in the presence of 0.5 mM amiloride and 0.5 mM ouabain. RESULTS: When pH was changed from 7.5 to 6.7, 7.1, or 7.3, O2 consumption increased by a factor of 1.80+/-0.11 (+/-SE), 1.65+/-0.12, and 1.44+/-0.06, respectively. Amiloride (0.5 mM) and ouabain (0.5 mM) inhibited 50% and 65%, respectively, of the increase in QO2 at pH 6.7. CONCLUSIONS: Corneal acidosis leads to increased QO2 in a dose-dependent manner. The increased QO2 is in part secondary to the activation of pH regulatory mechanisms, including Na+/H+ exchange, which then stimulates Na+/ K+-ATPase activity. These findings indicate that contact lens-induced acidosis can exacerbate corneal hypoxia and related complications.     

Effect of H+ extracellular and intracellular buffering capacity on the activity Ca2+/H+ exchange in the lymphocyte plasma membrane

Formation of delta pH is registered when studying Ca2+ passive transport through lymphocytes' plasma membrane (PM). The pHi values strongly depended on pH0. Changes of pH0 lead to unidirectional changes of pHi and affect Ca2+ concentration in cytoplasm of the intact cells. The presence of Ca(2+)-channels antagonists does not affect this phenomenon. Ca2+/H+ exchange is supposed to exist in PM. It is also of great interest that cytoplasmic Ca2+ and H+ activities are some equal in physiological range. Besides, H(+)-buffering as well as Ca(2+)-buffering systems are present in the cell and have their maximal capacity about 7.2 in the intact cells. The spectrofluorimetric study of internal lymphocytes' H(+)-buffering capacity with titration technique using weak base, acid or other buffer addition has demonstrated maximal value of 9.0-1.1 mM depending on the substance to be added.     

Rapid computerized analysis of Na+/H+ exchange flux

The regulation of intracellular pH (pHi) is mediated by membrane transporters whose activity is directly controlled by pHi. Therefore, transport rates must be compared at identical pHi values in functional studies of these transporters. This is conventionally performed using scatter plots showing initial rates of proton flux versus intracellular pH. We present justification for determining proton flux over a wide range of pHi, by digitally smoothing a pHi trace and then directly taking the first derivative versus time of smoothed data. Compared to conventional least-squares analysis of initial rates, the derivative method generates much more information per experiment. Compared to other methods which fit pHi traces to a defined equation prior to rate calculation, the new method does not require that the pHi trace be well fit by any given mathematical function. The derivative technique is illustrated in an analysis of Na+/H+ exchange in Caco-2 cells. Intracellular pH is measured fluorometrically in cells loaded with BCECF (2',7'-bis[2-carboxyethyl]-5-(and-6)carboxyfluorescein). To validate the analysis of Na+/H+ exchange over an extended time range, we demonstrate that cellular acidification with NH4Cl does not change steady state Na+ content. We find that proton flux rates analyzed by the derivative method are equivalent to initial rates measured by least-squares analysis.     

Glass fragility and the stability of pharmaceutical preparations--excipient selection

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K(m) of 39.2 +/- 4.8 mM and a Jmax of 8.9 +/- 0.7 nmoles mg protein-1 sec-1. A very small conductive pathway for L-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H(+)-lactate cotransporter, whereas in the apical membrane both H(+)-lactate and Na(+)-lactate cotransporters are present, even if they exhibit a low transport rate.     

Can we use erythrocytes for the study of the activity of the ubiquitous Na+/H+ exchanger (NHE-1) in essential hypertension?

Both Na+/Li+ countertransport and electrochemical proton gradient (delta mu(H+))-induced Na+ and H+ fluxes are increased in erythrocytes from patients with essential hypertension. It was assumed that these abnormalities are related to ubiquitous (housekeeping) forms of the Na+/H+ exchanger (NHE-1). To examine this hypothesis, we compared kinetic and regulatory properties of erythrocyte Na+/Li+ countertransport and delta mu(H+)-induced Na+ and H+ fluxes with data obtained for cloned isoforms of the Na+/H+ exchanger. In human erythrocytes, Na+/Li+ countertransport exhibited a hyperbolic dependence on [Na+]0 with a K0.5 of approximately 30 to 40 mmol/L. The activity of this carrier was increased by two-fold in the fraction of erythrocytes enriched with the old cells, was inhibited by 0.1 mmol/L phloretin, and was insensitive to both 1 mmol/L amiloride and ATP depletion. In contrast, delta mu(H+)-induced 22Na influx was exponentially increased at [Na+]0 > 60 mmol/L, was insensitive to phloretin, was partly decreased by both 1 mmol/L amiloride and ATP depletion, and was the same in total erythrocytes and in the old cells. The values of Na+/Li+ countertransport and delta mu(H+)-induced Na+ influx in erythrocytes from different species were not correlating and their ratio in human, rat, and rabbit erythrocytes was 10:1:170 and 1:5:1 for Na+/ Li+ countertransport and delta mu(H+)-induced Na+ influx, respectively. In contrast to the majority of nonepithelial cells and cells transfected with an ubiquitous isoform of Na+/H+ exchanger, both delta mu(H+)-induced Na+ influx and Na+/Li+ countertransport in human erythrocytes were completely insensitive to ethylisopropyl amiloride (20 micromol/L) and cell shrinkage. Thus, our data strongly suggest that human erythrocyte Na+/Li+ countertransport and delta mu(H+)-induced Na+/H+ exchange are mediated by the distinct transporters. Moreover, because the properties of these erythrocyte transporters and NHE-1 are different, it complicates the use of erythrocytes for the identification of the mechanism for activating the ubiquitous form of Na+/H+ exchanger in primary hypertension.     

Effects of Selected Good’s pH Buffers on Nitrate Reduction by Iron Powder

The effects of three selected Good’s pH buffers on the performance of an Fe0/nitrate/H2O system were evaluated. The Good’s pH buffer itself did not reduce nitrate directly. Nitrate reduction by iron powder at near-neutral pH was negligible in an unbuffered system, but it was greatly enhanced with the presence of the buffer. A significant amount of aqueous Fe2+ (or Fe3+) was released after adding the Good’s pH buffer into the Fe0/H2O system with or without nitrate. In general, the pH of the buffered solution increased from the initial pH ( = ～ 4.6–5.3, depending on buffer’s pKa) to near-neutral pH. After the initial pH hiking, the pH in the system was more or less stable for a period of time ( ～ 5–10?h, usually concurrent with a fairly stable aqueous Fe2+). The pH then drifted to ～ 7.1 to 8.6, depending on the buffer’s initial concentration, the buffer’s pKa, and the consumption of Fe2+ concurrent with nitrate reduction. While a common assumption made by researchers is that Good’s pH buffers do not directly participate in reaction processes involved in contaminant remediation, this study shows that as side effects, the Good’s pH buffer may react with iron powder.     

Evaluation of a new semiautomatic electrode system for simultaneous measurement of ionized calcium and pH

The instrument (ICA 1 Radiometer, Copenhagen) measures the activity of calcium ion and hydrogen ion and displays the estimated concentration of free calcium ion (cCa2+) and pH at 37 degrees C in 110 microliter of whole blood or serum. The cCa2+ at pH 7.40 is calculated by means of a fixed relationship between cCa2+ and pH. The results are indicated on a display approximately 1 min after sample introduction. The measuring cycle plus automatic rinsing takes 3 min. The apparatus is equipped with a number of control functions. The sensitivity of the calcium electrode showed a fall from 100% to 92% over 20 weeks. The cell potential of the calcium cell showed a slow drift of--0.02 mV per day over a 20-week period while the pH cell which employs the same reference electrode showed no drift. Interference of Na+, K+, Li+, Mg2+ and H+ on the calcium measurement in whole blood is estimated to be at most +0.1%. The effect of erythrocytes on the static liquid junction potential is minimized by a special extrapolation procedure. The analytical standard deviation for cCa2+ in serum was 0.008 mmol/l within series, 0.017 mmol/l between days. The mean value for cCa2+ (at pH 7.4) in serum for 53 healthy adults was 1.249 mmol/l with a standard deviation of 0.036 mmol/l. cCa2+ in capillary blood at the actual pH from 20 healthy volunteers gave a mean value of 1.215 +/- 0.047 mmol/l (+/- 2SD) with pH 7.428 +/- 0.031 (+/- 2 SD). The slopes delta lgcCa2+/delta pH measured after equilibration at two different PCO2 values in venous blood (n = 20) and in serum (n = 104) gave a mean value of -0.221 +/- 0.040 (+/- 2 SD). The semi-automatic combined Ca2+ - pH electrode system makes the measurement of ionized calcium for clinical use a reliable and accurate analysis.     

K+/NH4+ antiporter: a unique ammonium carrying transporter in the kidney inner medulla

The mechanism of NH4+ transport in inner medulla is not known. The purpose of these experiments was to study the process that is involved in ammonium (NH4+) transport in cultured inner medullary collecting duct (mIMCD-3) cells. Cells grown on coverslips were exposed to NH4+ and monitored for pHi changes by the use of the pH-sensitive dye BCECF. The rate of cell acidification following the initial cell alkalinization was measured as an index of NH4+ transport. The rate of NH4+ transport was the same in the presence or absence of sodium in the media (0.052 +/- 0.003 vs 0.048 +/- 0.004 pH/min. P > 0.05), indicating that NH4+ entry into the cells was independent of sodium. The presence of ouabain, bumetanide, amiloride, barium, or 4,4'-di-isothiocyanostilbene-2-2'-disulfonic acid (DIDS) did not block the NH4(+)-induced cell acidification, indicating lack of involvement of Na+:K(+)-ATPase, Na+:K+:2Cl- transport, Na+:H+ exchange, K+ channel, or Cl-/base exchange, respectively, in NH4+ transport. The NH4(+)-induced cell acidification was significantly inhibited in the presence of high external [K+] as compared to low external [K+] (0.018 +/- 0.001 vs. 0.049 +/- 0.003 pH/min for 140 mM K+ vs. 1.8 mM K+ in the media, respectively, P < 0.001). Inducing K+ efflux by imposing an outward K+ gradient caused intracellular acidification by approximately 0.3 pH unit in the presence but not the absence of NH4+. This K+ efflux-induced NH4+ entry increased by extracellular NH4+ in a saturable manner with a Km of approximately 5 mM, blocked by increasing extracellular K+ and was not inhibited by barium. The K+ efflux-coupled NH4+ entry was electroneutral as monitored by the use of cell membrane potential probe 3,3'-dipropylthiadicarbocyanine. These results are consistent with the exchange of internal K+ with external NH4+ in a 1:1 ratio. The K(+)-NH4+ antiporter was inhibited by verapamil and Schering 28080 in a dose-dependent manner, was able to work in reverse mode, and did not show any affinity for H+ as a substrate, indicating that it is distinct from other NH4(+)-carrying transporters. We conclude that a unique transporter, a potassium-ammonium (K+/NH4+) antiport, is responsible for NH4+ transport in renal inner medullary collecting duct cells. This antiporter is sensitive to verapamil and Schering 28080, is electroneutral, and is selective for NH4+ and K+ as substrates. The K+/NH4+ antiporter may play a significant role in acid-base regulation by excretion of ammonium and elimination of acid.     

Molecular characterization and in situ localization of a mouse retinal taurine transporter

Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high-affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na+- and Cl(-)-dependent [3H]taurine transport activity in Xenopus laevis oocytes that saturated with an average Km of 13.2 microM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl- was varied, a single saturation process with an average apparent equilibrium constant (K(Cl-)) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na+ was varied (apparent equilibrium constant, K(Na+) approximately 54.8 mM). Analyses of the Na+- and Cl(-)-concentration dependence data suggest that at least two Na+ and one Cl- are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > beta-alanine > L-diaminopropionic acid > guanidinoethane sulfonate > beta-guanidinopropionic acid > chloroquine > gamma-aminobutyric acid > 3-amino-1-propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4alpha-phorbol 12,13-didecanoate but was inhibited significantly by the active phorbol ester phorbol 12-myristate 13-acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.     

Disruption of the transmembrane pH gradient--a possible mechanism for the antibacterial action of azelaic acid in Propionibacterium acnes and Staphylococcus epidermidis

The effect of the topical acne treatment azelaic acid on the transmembrane proton gradient (delta pH) of Propionibacterium acnes and Staphylococcus epidermidis was studied in vitro at external pH values found on human skin (pH 4.0-6.0). Bacteria were grown in defined media using continuous culture and delta pH was estimated by measuring the accumulation of [14C] benzoic by the cells using flow dialysis. In both P. acnes and S. epidermidis the addition of 30 mM azelaic acid and the membrane active inhibitors nigericin (150 microM) and CCCP (150 microM) resulted in a rapid release of [14C] label into the dialysate indicating the dissipation of delta pH between external pH values of 4.0-6.0. The addition of 60 mM NaCl as an iso-osmotic control and 150 microM valinomycin did not induce the release of [14C] label. The addition of 30 mM azelaic acid reduced the delta pH of P. acnes by 44% at external pH 4.0 and 28% at external pH 6.0. In S. epidermidis 30 mM azelaic acid reduced delta pH by 88% at external pH 5.0 and 20% at external pH 6.0. Rapid loss of viability occurred in suspensions of P. acnes and S. epidermidis containing 30 mM azelaic acid at pH 4.0 with no viable cells recovered after 60 min incubation. At pH 6.0 little change in viable numbers of P. acnes and S. epidermidis were observed over a 2 h incubation period. The results indicate that the antibacterial activity of azelaic acid is associated with the perturbation of intracellular pH.     

Uptake of 4-toluene sulfonate by Comamonas testosteroni T-2

The mechanism of transport of the xenobiotic 4-toluene sulfonate (TS) in Comamonas testosteroni T-2 was investigated. Rapid uptake of TS was observed only in cells grown with TS or 4-methylbenzoate as a carbon and energy source. Initial uptake rates under aerobic conditions showed substrate saturation kinetics, with an apparent affinity constant (Kt) of 88 microM and a maximal velocity (Vmax) of 26.5 nmol/min/mg of protein. Uptake of TS was inhibited completely by uncouplers and only marginally by ATPase inhibitors and the phosphate analogs arsenate and vanadate. TS uptake was also studied under anaerobic conditions, which prevented intracellular TS metabolism. TS was accumulated under anaerobic conditions in TS-grown cells upon imposition of an artificial transmembrane pH gradient (delta pH, inside alkaline). Uptake of TS was inhibited by structurally related methylated and chlorinated benzenesulfonates and benzoates. The results provide evidence that the first step in the degradation of TS by C. testosteroni T-2 is uptake by an inducible secondary proton symport system.     

Proton secretion in the upper part of the descending limb of long-looped nephron from hamsters

The proton transport processes in the upper part of the descending limb of the long-looped nephron (LDLu) from hamsters were studied using a fluorescent dye, 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF) in microperfused single nephron preparations. Intracellular pH (pHi), as assessed by the measurement of the fluorescence of BCECF trapped in the cytoplasm, was 7.23 +/- 0.05 (n = 18) under nominally HCO3--free conditions. Ouabain, when added to the bath, decreased pHi by 0.22 units. After an NH4Cl prepulse, the initial proton extrusion rate was 1.23 +/- 0.26 (n = 9) pH units/min, and was retarded in the presence of 1 mM amiloride either in the bath or in the lumen. pHi failed to recover when Na+ was eliminated from ambient solutions. These observations suggest that Na+/H+ antiporters exist both in the apical and basolateral cell membranes. By measuring tubular fluid pH (pHt) under stopped flow conditions, we examined whether the hamster LDLu has the capacity to generate and maintain a transmural H+ gradient. After the tubular outflow was obstructed, the luminal fluid was rapidly acidified, reaching a steady-state pH of 6.84 +/- 0.09 (n = 7). The steady-state pH was influenced by bath pH. Tubular fluid acidification was not observed in the absence of Na+ and was prevented by ouabain. We conclude that the hamster LDLu has the capability to generate and maintain a transmural proton gradient by proton secretion via a luminal Na+/H+ antiporter which is secondarily driven by the Na+-K+ ATPase in the basolateral membrane.     

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.     

Structure-function study on gastric proton pump

H+, K(+)-ATPase is a proton pump responsible for gastric acid secretion. It actively transport proton and K+ coupled with the hydrolysis of ATP, resulting in the formulation of a 10(6) fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K(+)-ATPase) and the Ca2+ pump (Ca(2+)-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K(+)-ATPase . One of the antibodies, HK2032 inhibited the H+, K(+)-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K(+)-ATPase molecule, and that the H+, K(+)-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K(+)-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K(+)-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K(+)-ATPase. This compound inhibited the H+, K(+)-ATPase activity by stabilizing the K(+)-form of the enzyme. Third we studied the conformational changes of the H+, K(+)-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K(+)-ATPase did not utilize acetylphosphate instead the ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K(+)-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.     

Characterization of the catecholamine extraneuronal uptake2 carrier in human glioma cell lines SK-MG-1 and SKI-1 in relation to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) selective cytotoxicity

Transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) and (-)-norepinephrine was investigated in SarCNU-sensitive SK-MG-1 and -resistant SKI-1 human glioma cell lines. [3H]SarCNU influx was inhibited by SarCNU, sarcosinamide, and (+/-)-epinephrine in SK-MG-1 cells with competitive inhibition observed by (+/-)-epinephrine (Ki = 140 +/- 12 microM) and (+/-)-norepinephrine (Ki = 255 +/- 41 microM). No effect on influx was detected in SKI-1 cells. [3H](-)-Norepinephrine influx was linear to 15 sec in both cell lines and temperature dependent only in SK-MG-1 cells. Influx of [3H](-)-norepinephrine was found to be saturable in SK-MG-1 (K(m) = 148 +/- 28 microM, Vmax = 1.23 +/- 0.18 pmol/microL intracellular water/sec) but not in SKI-1 cells. In SK-MG-1 cells, [3H](-)-norepinephrine influx was found to be inhibited competitively by (-)-epinephrine (Ki = 111 +/- 7 microM) and SarCNU (Ki = 1.48 +/- 0.22 mM). Ouabain and KCl were able to inhibit the [3H](-)-norepinephrine influx in SK-MG-1 cells, consistent with influx being driven by membrane potential. Several catecholamine uptake2 inhibitors were able to reduce significantly the influx of [3H](-)-norepinephrine and [3H]SarCNU with no inhibition by a catecholamine uptake1 inhibitor. These findings suggest that increased sensitivity of SK-MG-1 to SarCNU is secondary to enhanced accumulation of SarCNU mediated via the catecholamine extraneuronal uptake2 transporter, which is not detectable in SKI-1 cells. The introduction of SarCNU into clinical trials will confirm if increased uptake via the catecholamine extraneuronal uptake2 transporter will result in increased antitumor activity.