Regulation of gastric acid secretion via modulation of a chloride conductance

When gastric microsomes were purified from resting and stimulated rabbit mucosae, they were found to be generally similar in (H+ + K+)-ATPase activity, peptide composition in single-dimension sodium dodecyl sulfate-gel electrophoresis, and in size. In the stimulated vesicles, optimal proton transport activity was found at pH 7.4, 20-50 mM KCl, and 1 mM ATP-Mg. However, in the case of resting vesicles, the presence of valinomycin and an inward Cl-gradient was also necessary for Mg-ATP-dependent proton transport. Measurement of K+ and Cl-diffusion potentials using 3,3-dipropylthiadicarboxocyanine iodide as a potential sensitive dye showed that both resting and stimulated vesicles developed K+ gradient-dependent potentials in the presence of an impermeant anion, but that Cl- gradient-dependent potentials were observed only in the stimulated preparation. 86Rb+ self-exchange was found in both types of vesicles, but Cl- self-exchange was confined to vesicles derived from stimulated mucosae. Putative inhibitors of anion conductance such as furosemide and anthracene 9-carboxylic acid blocked proton transport, Cl- conductance, 36Cl- uptake, and Cl- exchange. The inhibition of proton transport was overcome by valinomycin. ATPase activity in the presence of nigericin, an H+:K+ exchanger, was unaffected by these inhibitors. K+ conductance, Rb+ uptake, and Rb+ exchange were insensitive to these inhibitors. Thus, activation of acid secretion by the stimulated parietal cell appears to involve at least the appearance of a discrete Cl- conductance in the pump-associated membrane.     

Proton transport by gastric membrane vesicles.

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H+ uptake into an osmotically sensitive space. The apparent Km for ATP was 1.7-10(-4) M in the absence of a K+ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ and inhibited by ATPase inhibitors such as N,N'-dicylclohexyl-carbodiimide. Maximal H+ uptake occurs with an outward K+ gradient but the minimal apparent KA is found in the absence of a K+ gradient. The pH optimum for H+ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphroylation of the enzyme, but is considerably less than the pH maximum of the K+ dependent dephosphorylation. In the presence of an inward K+ gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H+ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K+ conductance but a measurable H+ conductance, hence a K+ gradient can produce an H+ gradient in the presence of valinomycin. The uptake and spontaneous leak of H+ are temperature sensitive with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H+ uptake by these vesicles is probably due to a dimeric (H+ + K+)-ATPase and is probably non-electrogenic.     

Modulation of ATP-dependent Ca2+ transport in rat parotid basolateral membrane vesicles by K+ + Cl- flux

In basolateral membrane vesicles (BLMV) isolated from rat parotid glands, the initial rate of ATP-dependent Ca2+ transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N-methyl-D-glucamine (NMDG)-gluconate. Only NH4+, Rb+, or Br- could effectively substitute for K+ or Cl-, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca2+ transport, while equilibration of the vesicles with KCl (greater than 100 mM) increased transport 15-20%. In presence of Cl-, 86Rb+ uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb+ flux by approx. 60% in a Cl- medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca2+ transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 45Ca2+ transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had a higher (approx. 25%) rate of Ca2+ transport than mannitol loaded vesicles. Addition of valinomycin (5 microM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca2+ transport rate. These results suggest that during ATP dependent Ca2+ transport in parotid BLMV, K+ can be recycled by the concerted activities of a K+ and Cl- coupled flux and a K+ conductance.     

Potassium channels in basolateral membrane vesicles from pars convoluta of rabbit proximal tubule

The characteristics of 86Rb+ fluxes through conductive channels in basolateral-membrane vesicles isolated from pars convoluta of rabbit proximal tubule were investigated. In KCl loaded vesicles a transient accumulation of 86Rb+ was observed which was inhibited by BaCl2. The accumulation was driven by an electrical diffusion potential, as shown in experiments using membrane vesicles loaded with Li2SO4 and an outwardly directed Li+ gradient established with a Li(+)-ionophore. The vesicles containing the channel showed a cation selectivity with the order K+ = Rb+ much greater than Li+ greater than or equal to Na+ greater than choline+. The 86Rb+ flux was dependent on intravesicular Ca2+. Increasing concentrations of Ca2+ gradually decreased the 86Rb+ uptake.     

Ba2+-sensitive K+ channels in luminal-membrane vesicles from pars convoluta of rabbit proximal tubule

This paper describes properties of 86Rb+ fluxes through a novel K+ channel in luminal-membrane vesicles isolated from pars convoluta of rabbit proximal tubule. The uptake of 86Rb+ into potassium salt loaded vesicles was specifically inhibited by Ba2+. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using these membrane vesicles loaded with anions of different membrane permeability and was as follows: gluconate greater than SO4(2-) greater than Cl-. Furthermore, the vesicles containing the channels show a cation selectivity with the order K+ greater than Rb+ greater than Li+ greater than Na+ = choline+.     

Solubilization and reconstitution of the gastric H,K-ATPase

Proteoliposomes containing the hog gastric H+,K+-ATPase were prepared from cholate and n-octyl glucoside extracts of native microsomes. Experiments were presented which show reconstitution-dependent selective purification of a 94-kDa peptide capable of Rb+/Rb+ exchange and active H+ transport. The absence of selective enrichment of residual protein contamination in this material suggests but does not prove that those transport reactions are attributable only to the 94-kDa peptide. Transport demonstrated inhibitor sensitivity and cation specificity comparable to the microsomal gastric ATPase. In K2SO4 media the H+ transport reaction was protonophore insensitive and correlated with MgATP-dependent 86Rb+ extrusion. This and other evidence suggested that active transport occurs via electroneutral H+in for K+out exchange. 86Rb+ exchange (uptake) in the proteoliposomes demonstrated both saturable and nonsaturable components. At a K0.5 = 1.5 mM, saturable 86Rb+ uptake accounted for about 90% of Rb+ influx. The vanadate-sensitive cation exchange indicated that the ATPase was reconstituted asymmetrically into the proteoliposomes (70% cis-/30% trans-vanadate site). 86Rb+ exchange was inhibited by ATP and stimulated about 2-fold by low Mg2+ and 5 mM phosphate. These ligand effects and the demonstration of comparable rates of passive exchange and active Rb+ efflux suggest that passive K+ exchange is not severely limited by a K+-occluded enzyme form in the H,K-ATPase. A model compatible with this hypothesis is suggested.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

Identification and reconstitution of a Na+/K+/Cl- cotransporter and K+ channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na+/K+/Cl- cotransport system and a K+ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na+/K+/Cl- cotransporter and K+ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of 86Rb+ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two 86Rb+ fluxes. (A) A furosemide-inhibited 86Rb+ flux in the absence of Na+ (K+-K+ exchange). This flux is stimulated by an inward Na+ gradient (Na+/K+ cotransport) and is inhibited also by bumetanide. (B) A Ba2+-inhibited 86Rb+ flux, through the K+ channel. Luminal membranes containing the Na+/K+/Cl- cotransporter and K+ channels, and basolateral membranes containing the Na+/K+ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na+/K+/Cl- cotransporter and K+ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Finding of a KCl-independent, electrogenic, and ATP-driven H+-pumping activity in rat light gastric membranes and its effect on the membrane K+ transport activity

Resting rat light gastric membranes prepared through 2H2O and Percoll gradient centrifugations were enriched not only with (H+-K+)-ATPase and K+ transport activity (Im, W. B., Blakeman, D. P., and Davis, J. P. (1985) J. Biol. Chem. 260, 9452-9460), but also with a K+-independent, ATP-dependent H+-pumping activity. This intravesicular acidification has been ascribed to an oligomycin-insensitive H+-ATPase which differed from (H+-K+)-ATPase in several respects. The H+-ATPase is electrogenic, apparently of lower capacity, required a lower optimal ATP concentration (4 microM for the H+-ATPase and 500 microM for (H+-K+)-ATPase), of lower sensitivity to vanadate and sulfhydryl agents such as p-chloromercuribenzoate and N-ethylmaleimide, and insensitive to SCH 28,080, a known competitive inhibitor of (H+-K+)-ATPase with respect to K+. Operation of the H+-ATPase, however, appeared to interfere with the K+ transport activity in the light gastric membranes, probably through development of intravesicular positive membrane potential; for example, micromolar levels of Mg2+-ATP fully inhibited K+ uptake and stimulated K+ efflux as measured with 86Rb+. Involvement of (H+-K+)-ATPase in the K+ transport is not likely, since the inhibitory effect of Mg2+-ATP continued even after removal of the nucleotide with an ATP-scavenging system. Moreover, nigericin, an electroneutral H+/K+ exchanger, could bypass the inhibitory effect of Mg2+-ATP and equilibrate the membrane vesicles with 86Rb+ while valinomycin, an electrogenic K+ ionophore, could not. Finally, the H+-ATPase could possibly be involved in the acid secretory process, since its H+-pumping activity was removed from the light gastric membrane fraction upon carbachol treatment, along with the K+ transport and (H+-K+)-ATPase activities. We have speculated that the H+-ATPase is responsible for maintaining the K+-permeable intracellular membrane vesicles acidic and K+ free during the resting state of acid secretion and may contribute to basal acid secretion.     

Na+, K+, H+ and Cl- permeability properties of rabbit skeletal muscle sarcolemmal vesicles

The ion permeability properties of rabbit skeletal muscle sarcolemmal vesicles were investigated by means of radioisotope flux, membrane potential, and light-scattering measurements. An enriched sarcolemmal fraction was obtained from the 22-27% region of sucrose gradients after isopycnic centrifugation. The presence of contaminating sarcoplasmic reticulum was assessed with the use of a purified sarcoplasmic reticulum vesicle fraction. 22Na+, 86Rb+, 36Cl-, and [3H]sucrose flux measurements indicated that the sarcolemmal fraction possessed isotope spaces ranging between 1.5 and 4 microliters/mg protein. Membrane potential measurements using the voltage-sensitive fluorescent probe 3,3'-dipentyl-2,2'-oxadicarbocyanine iodide (diO-C5-(3)) indicated that sarcolemmal vesicles were impermeable to H+ and Na+ but that 10-15% of the vesicles were permeable to K+. Light-scattering measurements indicated a small fraction of sarcolemmal vesicles were permeable to both K+ and Cl-. Whether the low permeability of sarcolemmal vesicles to Na+, K+, and Cl- is the result of a low concentration of ion channels or the inactivation of these channels during isolation is at present uncertain.     

Stimulation of cation transport in mitochondria by gramicidin and truncated derivatives

Gramicidin and the truncated derivatives desformylgramicidin (desfor) and des(formylvalyl)gramicidin (desval) stimulate monovalent cation transport in rat liver mitochondria. Cation fluxes were compared indirectly from the effect of cations on the membrane potential at steady state (state 4) or from the associated stimulation of electron transport. Rb+ transport was measured directly from the uptake of 86Rb. The truncated gramicidins show enhanced selectivity for K+ and Rb+ when compared to gramicidin. Moreover, the pattern of selectivity within the alkali cation series is altered, i.e., Rb+ greater than K+ greater than Cs+ greater than Na+ greater than Li+ for desfor and desval as compared to Cs+ greater than Rb+ greater than K+ = Na+ greater than Li+ for gramicidin. The cation fluxes through the truncated derivatives are more strongly dependent on the cation concentration. The presence of high concentrations of permeating cation enhances the transport of other cations through the truncated derivative channels, suggesting that cations are required for stabilizing the channel structure. In high concentrations of KCl, desfor and desval are nearly as effective as gramicidin in collapsing the mitochondrial membrane potential, and, consequently, in the uncoupling of oxidative phosphorylation and enhancement of ATP hydrolysis. Preliminary experiments with liposomes show that 86Rb exchange is stimulated by desfor and desval almost to the same extent as gramicidin. These results strongly suggest that the truncated gramicidins form a novel conducting channel which differs from the gramicidin head-to-head, single-stranded beta 6.3-helical dimer ("channel") in its conductance characteristic and its structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disulfide cross-linking of H,K-ATPase opens Cl- conductance, triggering proton uptake in gastric vesicles. Studies with specific inhibitors

An S-S cross-linking reagent, Cu2+-o-phenanthroline, increased the 36Cl-/Cl- exchange rate across the hog gastric vesicle membrane, which contains H,K-ATPase, but did not affect the 86Rb+/Rb+ exchange rate. The results show that closed Cl- conductance can be opened by S-S cross-linking. Gastric vesicles with opened Cl- conductance could take up H+ upon addition of MgATP without prolonged preincubation in a solution containing K+. Preincubation of gastric vesicles with picoprazole, which is a specific inhibitor of H,K-ATPase and binds to 100-kDa polypeptides of the enzyme, dose dependently inhibited opening of the Cl- conductance by Cu2+-o-phenanthroline, indicating that the Cl- conductance is part of the function of the H,K-ATPase. The effect of picoprazole was greater at alkaline pH than at acidic pH. Another H,K-ATPase inhibitor, 2-[2-(3,5-dimethyl-4-methoxy)-pyridylmethylsulfinyl] (5-methoxycarbonyl-6-methyl)-benzimidazole (H compound), had a similar but stronger effect on the Cl- conductance than that of picoprazole. A pungent ingredient of curry, allylisothiocyanate, caused similar pH-dependent inhibition to that of picoprazole. However, another substituted benzimidazole, omeprazole, did not inhibit Cl- conductance. Substituted benzimidazoles, such as picoprazole, H compound, and omeprazole, inhibited the H,K-ATPase activity progressively with a decrease in pH of the medium. This pH dependence was the reverse of that in inhibition of Cl- conductance, suggesting that the inhibitory site of Cl- conductance is different from that of the H,K-ATPase activity and that the conformational states of the two sites change in different ways with change in pH of the medium.     

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.     

Effects of lidocaine on transport properties of renal brush-border membranes

The effect of lidocaine was examined in membrane vesicles from rabbit renal brush borders. Changes in the ionic permeability and the kinetics of Na+-dependent metabolite transport were observed at different concentrations of anesthetic. Lidocaine was found to alter the membrane permeability of all inorganic cations examined (Li+, Rb+, K+, and Na+). At low lidocaine concentrations, there was a saturable decrease in permeability, whereas at higher concentrations (greater than 0.2 mM) there was a non-saturable general increase in cation permeability. Lidocaine (1.0 mM) inhibited Na+-coupled transport of all ten substrates examined (sugars, amino acids and Krebs cycle intermediates). The affinity for the substrate decreased in the presence of the anesthetic.     

Non-selective cation channels in basolateral-membrane vesicles from pars recta of rabbit kidney proximal tubule.

The characteristics of 86Rb+ fluxes through conductive channels in basolateral-membrane vesicles isolated from pars recta of rabbit kidney proximal tubule were investigated. In RbCl-, KCl- and NaCl-loaded vesicles a transient and almost equal accumulation of 86Rb+ was observed. The uptakes of 86Rb+ were inhibited to the same extent by 10 mM-BaCl2 in all loadings. The accumulation was driven by an electrical diffusion potential. The 86Rb+ flux was dependent on intravesicular Ca2+. Increasing concentrations of Ca2+ gradually decreased the 86Rb+ uptake. At 10 microM-Ca2+ the radioisotope flux was below 20% of control. The vesicles containing the channel showed very low selectivity among the univalent cations K+, Rb+, Li+, Na+ and choline+.     

Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase.

We have studied the accumulation of dibenzyldimethyl-ammonium ion (DDA+) by respiring membrane vesicles of Escherichia coli, as an index of the generation of an electrical gradient during respiration. Nonrespiring vesicles accumulated DDA+ when K+ efflux was induced by valinomycin or monactin. By various criteria this was shown to be the exchange of one cation for another, independent of metabolism and coupled entirely by electrical forces. Uptake of DDA+ by respiring vesicles was inhibited by ionophores that translocate electrical charge and by reagents that block the respiratory chain. Oxamate and p-chloromercuribenzoate inhibited accumulation of DDA+ but did not dissipate a preformed pool; the reason appears to be that these reagents are less inhibitory to transport after lactate oxidation has begun than they are in resting vesicles. Uptake does not appear to involve a biological carrier, but requires trace amounts of a lipid-soluble anion such as tetraphenylboron, which has a catalytic role in DDA+ translocation. Respiring K+ vesicles accumulated substantially less DDA+ than did Na+ vesicles. Na+ was expelled from the vesicles concurrently with DDA+ uptake, whereas Rb+ and K+ were not. Thus, DDA+ uptake may be limited in the latter case by the availability of anionic groups. This explanation was supported by the finding that the addition of nigericin doubled the capacity of K+ vesicles to take up DDA+, presumably by providing a route for K+ to exit in exchange for H+. Parallel experiments on the valinomycin-dependent accumulation of Rb+ by respiring vesicles indicate that this process is analogous to the uptake of DDA+. Ionophores that elicit electrogenic K+ movement also induced respiration-linked transport. Proton-conducting ionophores and several inhibitors of respiration blocked Rb+ uptake and dissipated a preformed gradient. Preincubation of the vesicles with oxamate or p-chloromrecuribenzoate inhibited Rb+ uptake, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ be, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ compete for uptake when present simultaneously. We conclude that the accumulation of both DDA+ and Rb+ occurs in response to an electrical gradient, vesicle interior negative, produced by respiration.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Evidence for tripeptide/H+ co-transport in rabbit renal brush-border membrane vesicles.

L-Phe-L-Pro-L-Ala is a tripeptide which is hydrolysable almost exclusively by dipeptidyl peptidase IV in rabbit renal brush-border membrane vesicles. In order to delineate the mechanism of the transport of an intact tripeptide across the brush-border membrane, we studied the characteristics of the uptake of [3H]Phe-Pro-Ala in membrane vesicles in which the activity of dipeptidylpeptidase IV was completely inhibited by treatment with di-isopropyl fluorophosphate. In these vesicles, uptake of radiolabel from the tripeptide was found to be Na(+)-independent, but was greatly stimulated by an inwardly directed H+ gradient. The H(+)-gradient-dependent radiolabel uptake appeared to be an active process, because the time course of uptake exhibited an overshoot phenomenon. The process was also electrogenic, being stimulated by an inside-negative membrane potential. Under the uptake-measurement conditions there was no detectable hydrolysis of [3H]Phe-Pro-Ala in the incubation medium when di-isopropyl fluorophosphate-treated membrane vesicles were used. Analysis of intravesicular contents revealed that the radiolabel inside the vesicles was predominantly (greater than 90%) in the form of intact tripeptide. These data indicate that the uptake of radiolabel from [3H]Phe-Pro-Ala in the presence of an inwardly directed H+ gradient represents almost exclusively uptake of intact tripeptide. Uphill transport of the tripeptide was also demonstrable in the presence of an inwardly directed Na+ or K+ gradient, but only if nigericin was added to the medium. Under these conditions, nigericin, an ionophore for Na+, K+ and H+, was expected to generate a transmembrane H+ gradient. Uptake of Phe-Pro-Ala in the presence of a H+ gradient was inhibited by di- and tri-peptides, but not by free amino acids. It is concluded that tripeptide/H+ co-transport is the mechanism of Phe-Pro-Ala uptake in rabbit renal brush-border membrane vesicles.