Intravesicular Acidification Research Articles

Characteristics of the proton pump in rat renal cortical endocytotic vesicles

The characteristics of the H+ pump in isolated rat renal endocytotic vesicles were studied by the delta pH-sensitive dye acridine orange, the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide, and by a coupled optical ATPase assay. Intravesicular acidification depended on ATP and Mg2+ concentrations with half-maximal activations at 73 and 77 microM, respectively. CTP, GTP, UTP, and ITP partially supported acidification, but ADP and AMP did not. Ouabain, ethoxzolamide, levamisole, and vanadate did not inhibit H+ uptake into endocytotic vesicles. Oligomycin inhibited partially. Depending on concentration and preincubation time, Dio-9, filipin, N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD) inhibited H+ uptake completely. Filipin and, partially, DCCD acted nonspecifically by dissipating pH gradients. A specific cation was not required for the H+ pump; Zn2+ inhibited. Compared with mannitol, ATP-driven H+ uptake was stimulated by SCN- greater than Cl- greater than Br- greater than I- much greater than HPO4(2-) = gluconate = HCO3- = F-, but not by SO4(2-), NO3-, CH3COO-, S2O3(2-), and S4O6(2-). Chloride stimulated H+ uptake from the outside of the vesicles with an apparent Km of 27 mM. In the absence of Cl-, ATP-driven proton uptake was increased by intravesicular K+ and valinomycin, suggesting that the pump is electrogenic. The electrogenicity, however, could not be demonstrated with voltage-sensitive dyes. The vesicle membrane contains no significant K+ and Cl- conductances; only a conductance for H+ was found. The vesicles exhibited an ouabain-, oligomycin-, and vanadate-insensitive ATPase activity that was inhibited by DCCD and NEM. Our data indicate the presence of an electrogenic H+ pump in endocytotic vesicles from rat renal proximal tubules with similar characteristics as H+ pumps present in various intracellular (nonmitochondrial) membranes.

Leucine-proton cotransport system in Chang liver cell.

The stimulatory effect of an inward H+ gradient on the Na+-independent L-leucine uptake by the plasma membrane vesicles from Chang liver cells (Mohri, T., Mitsumoto, Y., and Ohyashiki, T. (1983) Biochem. Int. 7, 159-167) has been shown to be due to the increase of the Km value without changing the Vmax value in the transport kinetics. The uptake of leucine by the vesicles is accompanied by intravesicular acidification, and a stimulated uptake of leucine by the countertransport with a high concentration of leucine in the vesicles enhances the acidification. All of these uptakes of leucine and proton and their stimulations are amplified by imposing an inward proton gradient. These results suggest appreciably different affinities of proton for the leucine transport carrier in the inner and outer sides of the plasma membrane. A rapid decrease in the cytoplasmic pH was observed only in the first minute of incubation of intact cells with leucine in Na+-containing medium. But the leucine-dependent decrease of the cytoplasmic pH persisted longer when either Na+ in the medium was replaced by choline or amiloride was present along with Na+. Addition of amiloride to Na+-containing medium was inhibitory on the leucine uptake of cells, without effect on the early phase of glycine uptake. We conclude that Chang liver cells are provided in their plasma membrane with an amino acid-H+ cotransport system, and this is coupled to the amiloride-sensitive Na+/H+ exchange system.

Potassium/proton exchange in brush-border membrane of rat ileum.

These experiments were designed to determine whether proton-driven 86Rb uptake was present in apical membrane vesicles prepared from rat ileum. The uptake of 86Rb was approximately 300 to 350% greater in the presence of a 100-fold H+ gradient than in its absence and was greater at 1, 2 and 5 minutes ("overshoot") than that at 90 minutes. Proton-driven 86Rb uptake was decreased by 20% in TMA-nitrate compared to that in TMA-gluconate. 0.3 mM amiloride did not significantly inhibit proton-driven 86Rb uptake; in contrast, proton-driven 22Na uptake was significantly inhibited by 0.3 mM amiloride by 34%. Similarly, 25 mM KCl inhibited proton-driven 86Rb uptake more than that of 22Na, while the inhibition of proton-driven 22Na uptake by 25 mM NaCl was greater than that of 86Rb. In additional studies intravesicular acidification measured by acridine orange fluorescence was demonstrated in the presence of an outwardly directed K gradient. These studies demonstrate that a proton gradient stimulates 86Rb uptake and a K gradient induces intravesicular acidification; and that these fluxes are mediated by a K/H exchange distinct from Na/H exchange which is also present in this membrane. We conclude that a specific exchange process for K/H is located in ileal apical membrane vesicles.

Fusicoccin stimulates the H +-ATPase of plasmalemma in isolated membrane vesicles from radish

The effect of fusicoccin on the plasmalemma H +-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H +-transport is mediated only by the plasmalemma H +-ATPase. Fusicoccin stimulated the plasmalemma H +-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of Δψ and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H +-ATPase was evident at pH 6.6.

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles. Neutral red as a probe for external surface charge rather than internal protonation

The occurrence of short-range proton displacements in Photosystem I (PS I) -enriched subchloroplast vesicles was studied under single-turnover conditions, using the pH indicators Cresol red and Neutral red. Bulk proton translocation was not detectable with Cresol red. Neutral red (externally added) was adsorbed to the vesicles in a salt-reversible way. It showed a flash-induced absorbance transient, recorded between 545 and 550 nm, consisting of a fast decrease (halftime < 5 ms) followed by an increase (halftime 20–40 ms). On the basis of the effects of uncoupler, ionophores, buffers and pH, it was concluded that the fast decrease represents Neutral red reduction and the following increase mainly reflects Neutral red protonation (and includes its reoxidation). The results further demonstrate that Neutral red is adsorbed to external membrane sites that have different accessibility towards various buffers. Bovine serum albumin, in a concentration at which it hardly contributed to external bulk buffering, stimulated the Neutral red response in parallel with the electric potential, indicating carotenoid response. This was presumably due to a general stabilizing effect. Our results strongly suggest that in these Photosystem I-enriched vesicles Neutral red senses flash-induced changes of external membrane surface charge, rather than intravesicular acidification, due to proton displacements across the membrane. This behavior is in many respects similar to that of the aminoacridine probes.

Electrolyte transport across the basolateral membrane of the parietal cells.

The ion-transport properties of the basal lateral membranes of intact isolated parietal cells were studied at the cellular and subcellular level. The presence of an amiloride-sensitive Na+:H+ exchange was demonstrated in cells by proton gradient-driven Na+ uptake and by changes in cell pH as monitored by dimethylcarboxylfluorescein fluorescence both in a fluorimeter and on single isolated cells using a fluorescence microscope and an attached intensified photodiode array spectrophotometer. The presence of the Na+:H+ antiport in vesicles was shown both by intravesicular acidification monitored by acridine orange fluorescent quenching and by proton gradient-dependent Na+ uptake. The presence of Cl-:HCO-3 exchange was determined in intact cells by monitoring changes in cell pH due to Cl- uptake and was shown to be 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid- and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid-sensitive. In vesicles, Cl-:HCO-3 exchange was demonstrated by Cl- flux measurement. The apparent affinities for both Cl- and HCO-3 on either side of the membrane were determined to be Km Cli = 20 mM, Km Clout = 17.5 mM, Km HCO-3in = 2.5 mM, and Km HCO-3out = 7.5 mM. A K+ conductance in cells and vesicles was demonstrated by monitoring K+ gradient-dependent 86Rb uptake. No evidence was found for the presence of a Cl- conductance in either cells or vesicles but a H+ conductance was found to be present in vesicles but not in intact cells. In the latter, by determining the effect of either Na+ or Cl- gradients on cell pH and by flux calculations it was concluded that the Cl-:HCO-3 exchange was the major passive flux mechanism for pH regulation in this cell type.

Electrogenic Transport of Protons Driven by the Plasma Membrane ATPase in Membrane Vesicles from Radish

Mg:ATP-dependent H(+) pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Delta psi). DeltapH was measured as the decrease of absorbance of Acridine orange and Delta psi as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Delta pH and Delta psi generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Delta pH generation is not inhibited by NO(3) (-). These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H(+)-ATPases. Both intravesicular acidification and Delta psi generation are influenced by anions: Delta pH increases and Delta psi decreases following the sequence SO(4) (2-), Cl(-), Br(-), NO(3) (-). ATP-dependent H(+) pumping strictly requires Mg(2+). It is very specific for ATP (apparent K(m) 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Delta pH generation is inhibited by CuSO(4) and diethylstilbestrol as well as vanadate. Delta pH generation is specificially stimulated by K(+) (+ 80%) and to a lesser extent by Na(+) and choline (+28% and +14%, respectively). The characteristics of H(+) pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.

Na/H- and Cl/OH-exchange in rat jejunal and rat proximal tubular brush border membrane vesicles. Studies with acridine orange.

The quenching of the acridine orange fluorescence was used to monitor the formation and/or dissipation of a delta pH in brush border vesicles isolated from rat kidney cortex or rat jejunum. Similar findings were obtained with both brush border membrane vesicle preparations. Acridine orange fluorescence was quenched by a preset delta pH (intravesicular acid) or by the ionophore (valinomycin/CCCP) dependent development of a delta pH (intravesicular acid) under conditions of potassium efflux. Under sodium efflux conditions, an acidification of the intravesicular space occurred: a) due to indirect (electrical) coupling of sodium and proton fluxes; b) due to directly coupled sodium/proton exchange. The initial rate of the dissipation of a preset delta pH was accelerated by pulse injections of sodium in a saturable manner; lithium partially replaced sodium. The sodium dependent acceleration in the rate of dissipation of a preset delta pH was not altered by replacing gluconate with chloride. Amiloride was an inhibitor of directly coupled sodium/proton exchange. An inwardly directed chloride gradient did not induce intravesicular acidification. The initial rate of the dissipative proton fluxes (preset delta pH) was slightly accelerated by an outwardly directed chloride gradient. Sodium/proton exchange dependent acidification of the intravesicular space was not altered by replacing gluconate with chloride. These results clearly document the existence of sodium/proton exchange in both renal and intestinal brush border membrane vesicles. In contrast, Cl/OH exchange--under our experimental conditions--must have a much smaller rate than Na/H exchange.

Inhibition of ( [formula omitted] and H + accumulation in hog gastric membranes by trifluoperazine, verapamil and [formula omitted

The mechanism of gastric antisecretory action for trifluoperazine, verapamil and 8-(N,N- diethylamino)octyl-3,4,5- trimethoxybenzoate (TMB-8) has been studied utilizing isolated hog gastric membranes enriched with ( H + + K +)- ATPase . The drugs inhibited the gastric ATPase due to their apparent competition with K + for the luminal high-affinity K +-site of the ATPase. The dose to inhibit 50% (ID 50) of the ATPase in the membranes rendered freely permeable to K + (20mM) was 50 μM for trifluoperazine and 1.5 mM for verapamil and TMB-8. In intact hog gastric membranes which develop a pH gradient in the presence of valinomycin, ATP and KCl, however, trifuoperazine at 4 μM, verapamil and TMB-8 at 15 μM inhibited 40 and 30% of the valinomycin-stimulated ATPase activity, respectively, and also blocked the ionophore-dependent intravesicular acidification as measured by aminopyrine accumulation. The enhanced potency of the drugs to inhibit the ATPase in the intact membrane vesicles may be attributed to the accumulation of the drugs as a weak base within the vesicles, where the luminal K +-site of the ATPase is accessible. Calmodulin and Ca 2+ had no effect on the extent of H +-accumulation as measured by aminopyrine accumulation in the membrane vesicles which were prepared in the presence of 1 mM EGTA. Since the drugs showed similar potency in interfering with H + movements either in the membrane vesicles or isolated rabbit gastric glands stimulated by dibutylryl cAMP, it is reasonable to suggest the inhibitory effect of the drugs on ( H + + K +)- ATPase as a primary cause for such interferences in both cases. A trifluoperazine analog and other lipophilic amine drugs similarly inhibited ( H + + K +- ATPase and H + accumulation in the membrane vesicles or in the glands. We have concluded that a tertiary amine, the only common functional group among these drugs, is primarily responsible for their ability to interact with the high-affinity K + site of the gastric ATPase.

Proton pathways in rat renal brush-border and basolateral membranes

The quenching of acridine orange fluorescence was used to monitor the formation and dissipation of pH gradients in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. The fluorescence changes of acridine orange were shown to be sensitive exclusively to transmembrane ΔpH and not to membrane potential difference. In brush-border membrane vesicles, an Na + (Li +)H + exchange was confirmed. At physiological Na + concentrations, 40–70% of Na +H + exchange was mediated by the electroneutral Na +H + antiporter; the remainder consisted of Na + and H + movements through parallel conductive pathways. Both modes of Na +H + exchange were saturable, with half-maximal rates at about 13 and 24 mM Na +, respectively. Besides a Na + gradient, a K + gradient was also able to produce an intravesicular acidification, demonstrating conductance pathways for H + and K + in brush-border membranes. Experiments with Cl − or SO 2− 4 gradients failed to demonstrate measurable Cl −OH − or SO 2− 4OH − exchange by an electroneutral antiporter in brush-border membrane vesicles; only Cl − conductance was found. In basolateral membrane vesicles, neither Na +(Li +)H + exchange nor Na + K + conductances were found. However, in the presence of valinomycin-induced K + diffusion potential, H + conductance of basolateral membranes was demonstrated, which was unaffected by ethoxzolamide and 4,4′-diisothiocyanostilbene-2,2-disulfonic acid. A Cl − conductance of the membranes was also found, but antiporter-mediated electroneutral Cl −OH − or SO 2− 4OH − exchange could not be detected by the dye method. The restriction of the electroneutral Na +H + exchanger to the luminal membrane can explain net secretion of protons in the mammalian proximal tubule which leads to the reabsorption of bicarbonate.

Kinetic properties of the KCl transport at the secreting apical membrane of the oxyntic cell

We have previously reported marked structural and functional differencces between (H+/K+)-ATPase-rich vesicles obtained from resting fundic gastric mucosa and those obtained from secreting tissue. The functional differences seem to arise from activation of a permeability pathway for K+ salts in the vesicles originating from stimulated tissue. By providing the intravesicular K+ needed for the ATPase-mediated H+/K+ exchange, this pathway facilitates formation of large pH gradients (J.M. Wolosin & J.G. Forte.FEBS Lett.125:208–212, 1981). We have now expanded our observations on the characteristics of the K-salt transport. The dependence of intravesicular acidification on the concentration of various anions or of K+ was monitored using the rate of fluorescence quenching of the pH probe, acridine orange. Rates of acid accumulation followed the series I−>Br−>Cl−>NO3−>CH3SO3−>isethionate>gluconate. The effects of exogenous ionophores on pH gradients suggested that K+-salt transport is electroneutral. On the basis of the known properties of the H+/K+ pump, we devised a method by which the rates of unidirectional K+ salt influx can be monitored via the resulting rates of H+ accumulation. Accordingly, the dependence of these rates on the concentration of K+ and Cl− was studied and subjected to a kinetic analysis. It was found that the dependence follows that expected for an electroneutral K+ and Cl− transport system with apparent Michaelis constants equal to 15±2mm for K+ and 46±5mm for Cl−. It is concluded that intravesicular acid accumulation, believed to represent HCl secretion in whole tissue, results from the combined action of a KCl symport and the well known (H++K+)-ATPase. Though the calculated maximal transport capacity of the symport exceeds by several times the maximal transport capacity of the pump, at physiological concentrations of the ions both transport systems are expected to transport K+ in opposite directions at similar rates. Implications of these observations to HCl secretion by intact tissue are discussed.

Apparent inhibition of Na +/H + exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na + gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na + gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K + gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I 50) of Na +/H + exchange and K + gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I 50 for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na +/H + exchange by these drugs.

Properties of an ATP-Fueled, Cl--Dependent Proton Pump Localized in Membranes of Microsomal Vesicles from Maize Coleoptiles

Abstract Vesicles prepared from the microsomal membrane fraction of maize coleoptiles possess an ATP-fueled H+-transport into the vesicles. It is highly possible that the vesicles from the microsomal membrane fraction actually are unchanged, native vesicles (originating from the ER or Golgi-apparatus) that can fuse with the plasmalemma or the vacuole. Therefore, they can reflect properties of the vacuole or the plasmalemma. The energy dependent acidification within the vesicles, which can be completely reverted through the addition of CCCP, was determined on the basis of photometric difference spectra using NR (Hager et al., Z. Naturforsch. 35 c, 794-804 1980). The proton pumps possess a very high substrate specificity; only ATP (+ Mg2+) can be used as substrate, while GTP, ITP, UTP and CTP or other nucleoside tri- or diphosphates cannot be used. DCCD and DES inhibit H+-ATPase completely, oligomycin has only a slight, orthovanadate no inhibitory effect at all. The energy dependent transport of H+ across the membrane takes place only in the presence of Cl- or Br- (not as well in the presence of I-). Other anions (F-, NO- 3, SO2- 4, SCN-, IDA-, H2BO- 3 cannot cause an intravesicular acidification through ATP if chloride (or Br-) is not present. In the presence of chloride, however, some of these anions inibit the H+/Cl--symport (J-, NO- 3, SO2- 4, SCN-, H2BO- 3). They obviously are in competitive interaction with Cl- ions for Cl--binding sites on a carrier or channel without being able to be transported themselves. Pi, which renders the acidification of the vesicles at a low rate possible without Cl-, is the only tested anion which can augment the Cl- dependent acidification. This supports the idea that either Pi functions as a positive effector on the Cl-transport or that a Cl-/Pi-antiporter exists which reduces Cl- accumulation and therefore facilitates the Cl- coupled H+ transport into the vesicles also. The anion transport inhibitor, DIDS, blocks the ATP-dependent H+-transport. This again supports the idea of a relatively tight coupling between H+ and Cl- transport in a possibly electroneutral system. The presence of monovalent cations, such as K+, Na+, Li+ and choline+, are not important for H+ transport. The dependence of the ATP-fueled acidification of the vesicles on the same anion pattern which seems to regulate elongation growth and stomata aperture speaks for the eminent importance of the H+-pump and vesicles described in this report for growth and turgor of plant cells.

Energy-linked Changes of Hydrogen Ion Concentration in Submitochondrial Particles

Abstract 1. Submitochondrial particles supplemented with bromthymol blue, found in independent experiments to be bound to the particles, exhibit characteristic decreases of absorbance upon activation of electron transport by addition of substrate or oxygen to the anaerobic material or by addition of adenosine 5'-triphosphate to the sulfide-inhibited material. The responses are reversible upon exhaustion of substrate or oxygen or upon utilization of adenosine 5'-triphosphate. 2. Inhibitors of electron transport (sulfide or antimycin A) reverse the bromthymol blue responses activated by substrate and oxygen. 3. Uncoupling agents, for example, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, rapidly reverse the bromthymol blue responses upon addition of substrate, oxygen, or adenosine 5'-triphosphate. 4. Glass electrode measurements external to the submitochondrial particles confirm an alkalinization outside the particles on activation of electron transport. On the basis of these observations, the decolorization of bromthymol blue upon activation of electron transport is attributed to an intravesicular acidification. 5. The total amount of hydroxyl ion production exterior to the vesicles is 50 to 60 mµmoles of hydroxyl ions per mg of protein, a value roughly 10 times in excess of the maximal content of respiratory carriers of the particles, and several fold greater than the measured calcium content, but considerably less than the magnesium content. 6. In view of the observed cation content of the submitochondrial particles employed and the similarities in the bromthymol blue responses caused by activation of electron transport to those caused by the addition of acetate or ethylene glycol 2-(2-aminoethyl)-tetraacetate, the intravesicular acidification is attributed to the energy-linked movement of bound cations from the inside of the vesicle to the outside medium. 7. The response of bromthymol blue affords a highly sensitive measure of energy conservation; a response to the oxidation of 1 nmole of reduced β-diphosphopyridine nucleotide is readily measurable. 8. The kinetics of activation of the bromthymol blue response and of its inactivation by inhibitors of electron transport and by uncouplers as well suggests that the cation movement is a reversible phenomenon and that a circulation of cations through the vesicular membrane occurs. 9. Much experimental evidence is consistent with a chemical mechanism for cation movements below 0.2 mg of protein per ml, suggesting that the bromthymol blue response is due to a dissociable component of the vesicular membranes, presumably a cation such as calcium. Also, ethylene glycol 2-(2-aminoethyl)-tetraacetic acid and acetate give bromthymol blue responses similar to those induced by activation of electron transport or adenosine 5'-triphosphate. The slow initial rate of hydrogen ion formation is consistent with the chemical mechanism. 10. The data of this paper do not afford support for compulsory movements of electrons in the respiratory chain and of hydrogen ions through the vesicular membrane. The initial rate of hydrogen ion formation is over 1000-fold slower than the initial rate of oxidation of reduced quinone and flavoprotein. The magnitude of the hydrogen ion change is too small by a factor of about 10 to represent a jump due to the oxidation of reduced quinones and flavin. The collapse of the pH gradient across the vesicular membrane occurs nearly as rapidly upon inhibition of electron transport by antimycin A as it does upon addition of an uncoupling agent. 11. A structural model for the movement of cations in a circulating pathway through the vesicular membrane operating according to the chemical mechanism has been proposed and is consistent with the available experimental data. 12. It is concluded that the chemical mechanism for oxidative phosphorylation is worthy of detailed experimental investigation.