Facilitated Transport of Lactate by Rat Jejunal Enterocyte

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 J _max 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. Received: 22 October 1996/Revised: 11 March 1997     

Characterization of H+/OH− currents in phospholipid vesicles

In pure phospholipid vesicles, the conductivity of H⁺/OH^– ions exceeds that for other simple inorganic ions. Protons achieve electrochemical equilibrium across egg phosphatidylcholine vesicles within tens of minutes. When pH gradients are established across vesicles, transmembrane potentials develop. Conversely, the establishment of transmembrane potentials leads to the formation of pH gradients. When the phenomenological permeability of H⁺/OH^– ions in vesicles is estimated, values are obtained that are much greater (six orders of magnitude larger) than those for Na⁺ or K⁺. A wide range in the values for this permeability has been reported; however, much of the discrepancy can be attributed to differences in the vesicle systems and experimental conditions. The H⁺/OH^– current appears to be modulated by changes in membrane dielectric constant. However, the dependence of this current on the pH gradient and on the membrane voltage argues against simple diffusion mechanisms as the source of the H⁺/OH^– current. In addition, in vesicle systems the H⁺/OH^– current shows a surprising invariance to changes in the membrane dipole potential, an observation that argues against the role of simple carriers for H⁺ and OH^– ions.     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.     

The effects of pH and calcium concentrations on gill potentials in the Brown Trout,Salmo trutta

Summary Measurements of the transepithelial potential (V_int-V_ext) across the gills of Brown Trout,Salmo trutta, were made in solutions of a range of pH and calcium concentrations. The potential was strongly dependent on external pH, being negative in neutral solutions but positive in acid solutions. The addition of calcium to the external medium produced a positive shift in potential in all but very acid media (pH 4.0–3.5), where very little change was seen. The gill membrane appears to act as a hydrogen electrode having a very high permeability to H⁺ ions, and the potential behaves as a diffusion potential. The presence of calcium reduced the permeability to both H⁺ and Na⁺ ions but even at a calcium concentration of 8.0 mM/l the permeability ratio H⁺/Na⁺ was still more than 900. The transepithelial potential is shown to be diffusional in origin and is discussed in terms of the relative permeability of the gill to H⁺, Na⁺ and Cl^– ions. Sodium fluxes across the gills were measured and provide the basis for a theoretical consideration of Na⁺, Cl^– and H⁺ fluxes across the gills in neutral and acid solutions. The positive potential at low pH largely accounts for the increased loss of sodium from fish in these conditions.     

Sodium transport measured in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits

Right-side-out plasma membrane vesicles were isolated from wheat roots using an aqueous polymer two-phase system. The purity and orientation of the vesicles were confirmed by marker enzyme analysis. Membrane potential (Ψ)-dependent ²²Na⁺ influx and sodium/proton (Na⁺/ H⁺) antiport-mediated efflux across the plasma membrane were studied using these vesicles. Membrane potentials were imposed on the vesicles using either K⁺ gradients in the presence of valinomycin or H⁺ gradients. The ΔΨ was quantified by the uptake of the lipophilic cation tetraphenylphosphonium. Uptake of Na⁺ into the vesicles was stimulated by a negative ΔΨ and had a K_m for extrav-esicular Na⁺ of 34.8 ± 5.9 mol m³. The ΔΨ-dependent uptake of Na⁺ was similar in vesicles from roots of hexaploid (cv. Troy) and tetraploid (cv. Langdon) wheat differing in a K⁺/Na⁺ discrimination trait, and was also unaffected by growth in 50 mol m^?3 NaCl. Inhibition of ΔΨ-dependent Na⁺ uptake by Ca²⁺ was greater in the hexaploid than in the tetraploid. Sodium/proton antiport was measured as Na⁺-dependent, amiloride-inhibited pH gradient formation in the vesicles. Acidification of the vesicle interior was measured by the uptake of ¹⁴C-methylamine. The Na⁺/H⁺ antiport had a K_m, for intravesicular Na⁺ of between 13 and 19 mol m^?3. In the hexaploid, Na⁺/H⁺ antiport activity was greater when roots were grown in the presence of 50 mol m^?3NaCl, and was also greater than the activity in salt-grown tetraploid wheat roots. Antiport activity was not increased in a Langdon 4D chromosome substitution line which carries a trait for K⁺/Na⁺ discrimination. It is concluded that neither of the transport processes measured is responsible for the Na⁺/K⁺ discrimination trait located on the 4D chromosome of wheat.     

Effect of pH on the transport of Krebs cycle intermediates in renal brush border membranes

Lowering extravesicular pH stimulated Na⁺-dependent citrate transport in renal brush border membrane vesicles: e.g., at pH_out = 5.5, the initial rate of citrate uptake was increased 10-fold compared to parallel control experiments at pH 7.5. The same experimental conditions had little effect on succinate uptake. The influence of pH on citrate transport is a product of the extravesicular H⁺ concentration; pH gradients did not potentiate the effects nor were proton gradients capable of driving transport in the absence of Na⁺. The effect of pH is adequately explained if only the mono- and divalent species of citrate (Cit^1?, Cit^2?) are considered acceptable substrates for transport. The stimulatory influence of pH on transport correlated quite well with pH-related increases in the concentrations of Cit^1? and Cit^2?, and over the same pH range [Cit^3?] was inversely related to citrate uptake. A model of the Na⁺-dependent dicarboxylate transport system is discussed in which three sodium ions are translocated per molecule of dicarboxylic acid.     

Thallium interaction with the gastric (K,H)-ATPase

Summary The gastric (K,H)-ATPase has been shown to catalyze an electroneutral H⁺ for K⁺ exchange. Tl⁺ is able to substitute for K⁺ as an activating cation in the hydrolytic reaction with an apparent dissociation constant of 90 m as compared to about 870 m for K⁺. The ability of Tl⁺ to participate in transport is shown by the development of pH gradients in the presence of Tl⁺ following addition of ATP to gastric vesicles and by the ATP-dependent efflux of Tl⁺ from gastric vesicles. Inhibition of hydrolysis is observed at pH 7.4 with external Tl⁺ concentrations above 3.0mm. This inhibition of hydrolysis is correlated with inhibition of pH-gradient formation. The inhibition of transport activity is partially relieved by a decrease in medium pH. This inhibitory effect is attributed to Tl⁺ binding at an external, low affinity cation site. In contrast to rubidium chloride, at high Tl⁺ concentrations, following the initial Tl⁺ efflux, there is reuptake of the cation. This rapid uptake is attributed to lipid-dependent Tl⁺ entry pathways. The vesicles exhibit a high permeability to thallium nitrate demonstrating a half-time (t _1/2) for uptake of about 1.0 min in contrast to 46 min for rubidium chloride. In both gastric vesicles or liposomes, external Tl⁺ concentrations in excess of 1 to 4mm are able to dissipate intravesicular proton gradients by an electrically coupled H⁺ for Tl⁺ exchange. Thus, although Tl⁺ is able to activate the gastric ATPase by mimicking K⁺, the permeability of this cation in lipid bilayers tends to uncouple H⁺ transport at concentrations high enough to generate detectable proton gradients.     

Effect of nickel on certain physiological and biochemical behaviors of an acid tolerantChlorella vulgaris

This study concerns the inhibitory effects of acid pH and nickel on growth, nutrient (NO₃ ^- and NH₄ ⁺) uptake, carbon fixation, O₂ evolution, electron transport chain and enzyme (nitrate reductase and ATPase) activities of acid tolerant and wild-type strains of Chlorella vulgaris. Though a general reduction in all these variables was noticed with decreasing pH, the tolerant strain was found to be metabolically more active than the wild-type. A reduced cation (NH₄ ⁺, Na⁺, K⁺ and Ca²⁺) uptake, coupled with a facilitated influx of anions (NH₄ ⁺, PO₄ ^3- and HCO₃ ^-), suggested the development of a positive membrane potential in acid tolerant Chlorella. Nevertheless, a tremendous increase in ATPase activity at decreasing pH revealed the involvement of superactive ATPase in exporting H⁺ ions and keeping the internal pH neutral. A difference in Na⁺ and K⁺ efflux of the two strains at decreasing pH suggests there is a difference in membrane permeability. The low toxicity of Ni in the acid tolerant strain may be due to the low Ni uptake brought about by a change in membrane potential as well as in permeability. Hence, the development of superactive ATPase and a change in both membrane potential and permeability not only offers protection against acidity, but also co-tolerance to metals.     

Diclofop-methyl increases the proton permeability of isolated oat-root tonoplast

Diclofop-methyl (methyl ester of 2-[4-(2′,4′-dichlorophenoxy)phenoxy]propionate; 100 micromolar) and diclofop (100 micromolar) inhibited both ATP- and PPi-dependent formation of H⁺ gradients by tonoplast vesicles isolated from oat (Avena sativa L., cv Dal) roots. Diclofop-methyl (1 micromolar) significantly reduced the steady-state H⁺ gradient generated in the presence of ATP. The ester (diclofop-methyl) was more inhibitory than the free acid (diclofop) at pH 7.4, but this relative activity was reversed at pH 5.7. Neither compound affected the rate of ATP or PPi hydrolysis by the proton-pumping enzymes. Diclofop-methyl (50, 100 micromolar), but not diclofop (100 micromolar), accelerated the decay of nonmetabolic H⁺ gradients established across vesicle membranes. Diclofop-methyl (100 micromolar) did not collapse K⁺ gradients across vesicle membranes. Both the (+)- and (−)-enantiomers of diclofop-methyl dissipated nonmetabolic H⁺ gradients established across vesicle membranes. Diclofop-methyl, but not diclofop (each 100 micromolar), accelerated the decay of H⁺ gradients imposed across liposomal membranes. These results show that diclofop-methyl causes a specific increase in the H⁺ permeability of tonoplast.     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum

Summary The ion permeability of sarcoplasmic reticulum vesicles from skeletal and heart muscle has been characterized by radioisotope flux, osmotic and membrane potential measurements, and by incorporating vesicles into planar phospholipid bilayers. The sarcoplasmic reticulum membrane is uniquely permeable to various biologically relevant monovalent ions. At least two and possibly three separate passive permeation systems for monovalent ions have been identified: 1) a K⁺, Na⁺ channel, 2) an anion channel, and 3) a H⁺ (OH^–) permeable pathway which may or may not be synonymous with the anion channel. A possible physiological function of these monovalent ion permeation systems is to permit rapid movement of K⁺, Na⁺, H⁺ and Cl across the membrane to counter electrogenic Ca²⁺ fluxes during Ca²⁺ release and uptake by sacroplasmic reticulum.     

A stable Na+/H+ antiporter of thermophilic bacterium PS3

As a first step in the isolation of a stable Na⁺/H⁺ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na⁺, Li⁺, Mn²⁺, Cd²⁺, and Co²⁺, but not of choline⁺ or Ca²⁺, regardless of their counter anions.²²Na⁺ was taken up into the vesicles by NADH oxidation, and the²²Na⁺ uptake was inhibited by the addition of an uncoupler. H⁺ release was observed on addition of Na⁺ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na⁺/H⁺ antiport was affected by the pH of the medium (optimum pH=8.5). TheK _m 's of the antiporter for Na⁺ and Li⁺ were 2.5 and 0.1 mM, respectively, but theV _max values for the two ions were the same at pH 8.0. In the presence of Li⁺, no further decrease of fluorescence quenching was observed on addition of Na⁺ andvice versa. The Na⁺/H⁺ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H⁺ antiporters, this antiporter was not inhibited by a thiol reagent.Abbreviations Tricine N-tris(hydroxymethyl)methylglycine - MOPS morpholinopropane sulfonic acid - TMAHO tetramethylammonium hydroxide - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - H⁺ — ATPase proton-translocating adenosine triphosphatase - electrochemical proton gradient across membrane - electrochemical Na⁺ gradient across membrane - pH pH difference across membrane     

ATP-dependent and ionophore-induced proton translocation in pea stem microsomal vesicles

The presence of an electrogenic pump in pea stem microsomal vesicles has already been demonstrated, but no evidence on the nature of the electrogenic ion has been presented (Rasi-Caldogno, F., De Michelis, M.I. and Pugliarello, M.C. (1981) Biochim. Biophys. Acta 642, 37–45). In this work we tested the usefulness of the ΔpH probe Acridine orange to monitor both ATP-dependent and ionophore-induced H⁺ fluxes in pea stem microsomal vesicles. The H⁺/K⁺ exchanger nigericin causes a marked uptake of protons into the vesicles that can be followed, with similar results, both as Acridine orange absorbance changes and pH changes of the external medium. ATP induces an uptake of Acridine orange into the vesicles which is reversed by FCCP and abolished by the presence of Triton X-100 in the incubation medium, thus indicating an inward, ATP-driven, H⁺ translocation. The ATP-dependent acridine orange uptake is Mg²⁺-requiring and KCl-stimulated. Such activity is inhibited by two specific ATPase inhibitors, dicyclohexylcarbodiimide and diethylstilbestrol, while it is unaffected by oligomycin and Na₃VO₄. These results show that Acridine orange is a useful probe to measure pH gradients in our membrane system and are consistent with the hypothesis that an ATPase of plasmalemma may act as a proton pump.     

Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions: Implications for the Use of Acridine Orange as a pH Probe

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I⁻, ClO₃⁻, NO₃⁻, Br⁻, or SCN⁻, acridine orange reported lower pH gradients than either quinacrine or [¹⁴C]methylamine. Acridine orange, but not quinacrine, reduced [¹⁴C]methylamine accumulation when NO₃⁻ was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO₃⁻ increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24°C than at 12°C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H⁺, possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO₃⁻/H⁺ symport involved in the export of NO₃⁻ from the vacuole is probably an artefact caused by acridine orange.     

Passive H+/OH− permeability in epithelial brush border membranes

Passive H⁺/OH^– permeability across epithelial cell membranes is rapid and leads to partial dissipation of H⁺/OH^– gradients produced by H⁺ pumps and ion gradient-coupled H⁺/OH^– transporters. A heterogeneous set of H⁺/OH^– transport mechanisms exist in biological membranes: lipid solubility/diffusion, protein-mediated transport by specific proteins or by slippage through ion-coupled H⁺/OH^– transporters, and transport at the protein/lipid interface or through protein-dependent defects in the lipid structure. A variety of methods are available to study protein transport mechanisms accurately in cells and biomembrane vesicles including pH electrode recordings, pH-sensitive fluorescent and magnetic resonance probes, and potentiometric probes. In brush border vesicles from the renal proximal tubule, the characteristics of passive H⁺/OH^– permeability are quite similar to those reported for passive H⁺/OH^– permeability through pure lipid bilayers; slippage of protons through the brush border Na⁺/H⁺ antiporter or through brush border water channels is minimal. In contrast, passive H⁺/OH^– permeability in brush border vesicles from human placenta is mediated in part by a stilbene-sensitive membrane protein. To demonstrate the physiological significance of passive renal brush border H⁺/OH^– transport, proximal tubule acidification and cell pH regulation mechanisms are modeled mathematically for states of normal and altered H⁺/OH^– permeabilities.     

Properties of Ca2+ uptake and release by Golgi membrane vesicles from rat intestine

A previous study of energy-independent in vitro Ca²⁺ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH)₂D₃ repletion (R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca²⁺ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg²⁺ and Sr²⁺, but not by Na⁺ or K⁺. Lowering the external [H⁺] or raising the internal [H⁺] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H⁺] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl₂ or SrCl₂ but not CaCl₂, NaCl, or KCl. Vesicles preloaded with K₂SO₄ failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl₂ was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca²⁺ uptake maintained at equilibrium. Therefore, the remainder of the Ca²⁺ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10^?6m was found for this binding. The bound Ca²⁺ could be rapidly released by external Mg²⁺ or Sr²⁺, but not Ca²⁺, Na⁺, or K⁺. Release of bound Ca²⁺ could also be induced by raising the [H⁺] of the external medium. Failure of external Ca²⁺ to release bound Ca²⁺ suggested that the release induced by external Mg²⁺, Sr²⁺, or H⁺ was not due to competitive displacement of Ca²⁺ from its binding sites. These results indicated that Ca²⁺ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca²⁺ to the vesicle. The effects of H⁺, Mg²⁺, and Sr²⁺ on Ca²⁺ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles.     

An estimation of the proton conductance of the inner membrane of turnip (Brassica napus L.) mitochondria

The permeability of the inner membrane of turnip mitochondria to H⁺ and OH^- ions has been investigated using an acid pulse technique. The rate of decay of a H⁺ pulse across the inner membrane is exponential having first-order kinetics and gives t 1/2 values of approx 54 s at neutral pH and at 25° C. Valinomycin or 1799 alone have little effect on t 1/2 values, whereas in combination, values of <15 s are observed. Nigericin produces a similar effect. The effective proton conductance of the inner membrane near pH 7 at 25° C is 0.27 nmol H⁺ min^-1 mg protein^-1 mV^-1. The results suggest that at neutral pH, the inner membrane of plant mitochondria is relatively impermeable to H⁺ and OH^- ions.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.     

Faster-than-anticipated Na/Cl diffusion across lipid bilayers in vesicles

Maintenance of electrochemical potential gradients across lipid membranes is critical for signal transduction and energy generation in biological systems. However, because ions with widely varying membrane permeabilities all contribute to the electrostatic potential, it can be difficult to measure the influence of diffusion of a single ion type across the bilayer. To understand the electrodiffusion of H⁺ across lipid bilayers, we used a pH-sensitive fluorophore to monitor the lumenal pH in vesicles after a stepwise change in the bulk pH. In vesicles containing the ion channel gramicidin, the lumenal pH rapidly approached the external pH. In contrast, the lumen of intact vesicles showed a two stage pH response: an initial rapid change occurred over ~ 1 min, followed by a much slower change over ~ 24 h. We provide a quantitative interpretation of these results based on the Goldman–Hodgkin–Katz ion fluxes discharging the electrical capacitance of the bilayer membrane. This interpretation provides an estimate of the permeability of the membranes to Na⁺ and Cl⁻ ions of ~ 10^− 8 cm/s, which is ~ 3 orders of magnitude faster than previous reports. We discuss possible mechanisms to account for this considerably higher permeability in vesicle membranes.     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.