cDNA cloning and sequence determination of pig gastric (H+ + K+)-ATPase

Complementary DNA to pig gastric mRNA encoding (H+ + K+)-ATPase was cloned, and its amino acid sequence was deduced from the nucleotide sequence. The enzyme contained 1034 amino acid residues (Mr. 114,285) including the initiation methionine. The sequence of pig (H+ + K+)-ATPase was highly homologous with that of the corresponding enzyme from rat, but had high degree of synonymous codon changes. Potential sites of phosphorylation by cAMP-dependent protein kinase and N-linked glycosylation sites were identified. The amino terminal region contained a lysine-rich sequence similar to that of the alpha subunit of (Na+ + K+)-ATPase, although a cluster of glycine residues was inserted into the sequence of the (H+ + K+)-ATPase. As the pig enzyme is advantageous for biochemical studies, the information of the primary structure is useful for further detailed studies.     

Preparation of rat gastric heavy and light microsomal membranes enriched in (H+-K+)-ATPase using 2H2O and Percoll gradients

Gastric heavy microsomal membranes highly enriched in (H+-K+)-ATPase were obtained from cimetidine- or carbachol-treated rats through 2H2O and Percoll gradient centrifugations. Both the resting (cimetidine-treated) and the stimulated (carbachol-treated) heavy membranes which presumably represent the apical membrane of gastric parietal cells were enriched with the polypeptides of 81,000 and 45,000 besides that of 93,000 representing (H+-K+)-ATPase. No apparent differences could be detected between the resting and the stimulated heavy membranes in their polypeptide profiles or their specific activity of (H+-K+)-ATPase. Nevertheless, the level of 86RbCl uptake was greater in the stimulated than the resting heavy microsomal membrane vesicles. The light gastric microsomes which abound in intracellular tubulovesicles containing reserve (H+-K+)-ATPase as isolated from cimetidine-treated rats were similarly purified with respect to (H+-K+)-ATPase. The purified light gastric membranes were largely devoid of the polypeptides of 81,000 and 45,000 found in the heavy gastric membranes. These observations further support the current hypothesis that secretagogues bring about changes in the environment of (H+-K+)-ATPase and induce KCl permeability in the apical membrane of the parietal cells, although at present we have been unable to identify the polypeptide(s) responsible for the KCl pathway.     

Inhibition of gastric (H+ + K+)-ATPase by the substituted benzimidazole, picoprazole

The substituted benzimidazole, picoprazole, inhibited the gastric (H+ + K+)-ATPase in a concentration-and time-dependent manner. Half-maximal inhibition of the (H+ + K+)-ATPase activity was obtained at about 2 . 10(-6)M under standard conditions. In addition to the inhibition of ATPase activity, parallel inhibition of phosphoenzyme formation and the proton transport activity were achieved. Radiolabelled picoprazole was found to bind to 100 kDa peptide; this peptide was shown by phosphorylation experiments to contain the catalytic centre of the (H+ + K+)-ATPase. Studies on the (Na+ + K+)-ATPase indicated that this enzyme was unaffected by picoprazole. From the data presented and from other pharmacological studies, it is proposed that this compound inhibits acid secretion at the level of the parietal cell by its ability to inhibit the gastric proton pump, the (H+ + K+)-ATPase.     

胃(H~(+)+K~(+))-ATPase与急性胃粘膜病变的关系的研究

 本文对急性胃粘膜病变与胃粘膜(H~(+)+K~(+))-ATP_ase的关系作了初步探讨,实验结果说明以消炎痛为诱因引起大白鼠急性胃粘膜病变时胃粘膜上的胃酸分泌的质子泵(H~(+)+K~(+))-ATP_ase活力受到抑制。在离体实验中低浓度的消炎痛<2μmol,大白鼠胃粘膜(H~(+)+K~(+))-ATP_ase的活力便受到显著的抑制,在整体实验中维酶素对消炎痛引起的胃粘膜(H~(+)+K~(+))-ATP_ase的抑制具有保护作用。     

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.     

Redistribution and characterization of (H+ + K+)-ATPase membranes from resting and stimulated gastric parietal cells.

When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+)-ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation.     

Effect of lysophosphatidylcholine on K+ transport in rat heavy gastric membranes enriched with (H+-K+)-ATPase

K+- and ATP-dependent H+-accumulation in rat heavy gastric membrane vesicles enriched with (H+-K+)-ATPase was markedly stimulated by amphiphiles like lysophosphatidylcholine and Zwittergent 3-14 at concentrations of 10(-5) M. Their stimulatory effect was dependent on K+-concentration in the medium and was abolished by SCH 28,080, a specific inhibitor of (H+-K+)-ATPase. Lysophosphatidylcholine at the optimal dose (3 X 10(-5) M) showed dual effects on K+-dependent membrane functions; it stimulated the rate of K+-uptake by nearly 60%, but partially inhibited SCH 28,080-sensitive and K+-dependent ATP-hydrolysis (about 20% reduction). These data indicate that H+-pumping through (H+-K+)-ATPase in the inside-out gastric membrane vesicles was facilitated by the stimulatory effect of lysophosphatidylcholine on membrane K+-transport in spite of its partial inhibition of ATP-hydrolysis. It appears that the rate limiting step for operation of the ATPase is the availability of K+ ions in the luminal side of the pump. We propose that ionic amphiphiles may modulate K+-transport in rat heavy gastric membranes through specific interactions with the putative K+-transporter.     

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.     

Inhibition of gastric (H+ + K+)-ATPase by unsaturated long-chain fatty acids

Arachidonic acid and unsaturated C18 fatty acids at concentrations near 10(-5) M markedly inhibited (H+ + K+)-ATPase in hog or rat gastric membranes. Arachidonic acid was a more potent inhibitor than unsaturated C18 fatty acids, but the involvement of the metabolites of arachidonic acid cascade was ruled out. Linolenic acid inhibited the formation of phosphoenzyme and the K+ -dependent p-nitrophenylphosphatase activity of the hog ATPase. Treatment with fatty acid-free bovine serum albumin abolished only the inhibitory effect of the fatty acid on the phosphatase activity without restoring the overall ATPase action. These data suggest the existence of at least two groups of hydrophobic binding sites in the gastric ATPase for unsaturated long-chain fatty acids which affect differentially the catalytic reactions of the ATPase. (H+ + K+)-ATPase in rat gastric membranes was found more susceptible to the fatty acid inhibition and also more unstable than the ATPase in hog gastric membranes. The presence of a millimolar level of lanthanum chloride or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid stabilized the rat ATPase probably via the inhibition of Ca2+ -dependent phospholipases in the gastric membranes.     

Characteristics of a pure endogenous activator of the gastric H+,K+-ATPase system. Evaluation of the role as a possible intracellular regulator

An endogenous activator capable of stimulating the gastric H+,K+-ATPase activity has been purified to homogeneity from dog and pig gastric cells and found to be a dimer of two identical 40-kDa subunits in the active state. Identical nature of the activator monomers was revealed by the detection of lysine as the sole N-terminal amino acid. The activator from one species can stimulate the H+,K+-ATPase from another species and vice versa. Such cross-activation is consistent with the striking similarities in the amino acid composition between the two species, suggesting considerable homology in the activator molecules from different species. The activator exhibited several unique features during modulation of the H+,K+-ATPase reaction. It appreciably enhances affinity of the H+,K+-ATPase for K+, known to increase turnover of the enzyme. To complement this K+ affinity, the activator also enhances ability of the H+,K+-ATPase to generate more transition state (E*.ATP) complex by increasing the entropy of activation (delta S++) of the system as revealed from an Arrhenius plot of the data on temperature activation. In addition, the activator shows both positive cooperativity and strong inhibition, depending on its concentration. Thus, up to the ratio of the H+,K+-ATPase and activator of about 1:2 (on the protein basis), the activator shows sigmoidal activation (Hill coefficient = 4.5), but beyond such concentration a strong inhibition was observed. Finally, Ca2+ at low (2-4 microM) concentration strongly inhibits the activator-stimulated H+,K+-ATPase. It is proposed that the activator may be acting as a link in the signal transducing cascade system between the intracellular second messenger (Ca2+) and the physiological response (gastric H+ transport).     

The stomach divalent ion-sensing receptor scar is a modulator of gastric acid secretion

Divalent cation receptors have recently been identified in a wide variety of tissues and organs, yet their exact function remains controversial. We have previously identified a member of this receptor family in the stomach and have demonstrated that it is localized to the parietal cell, the acid secretory cell of the gastric gland. The activation of acid secretion has been classically defined as being regulated by two pathways: a neuronal pathway (mediated by acetylcholine) and an endocrine pathway (mediated by gastrin and histamine). Here, we identified a novel pathway modulating gastric acid secretion through the stomach calcium-sensing receptor (SCAR) located on the basolateral membrane of gastric parietal cells. Activation of SCAR in the intact rat gastric gland by divalent cations (Ca(2+) or Mg(2+)) or by the potent stimulator gadolinium (Gd(3+)) led to an increase in the rate of acid secretion through the apical H+,K+ -ATPase. Gd(3+) was able to activate acid secretion through the omeprazole-sensitive H+,K+ -ATPase even in the absence of the classical stimulator histamine. In contrast, inhibition of SCAR by reduction of extracellular cations abolished the stimulatory effect of histamine on gastric acid secretion, providing evidence for the regulation of the proton secretory transport protein by the receptor. These studies present the first example of a member of the divalent cation receptors modulating a plasma membrane transport protein and may lead to new insights into the regulation of gastric acid secretion.     

Effects of phenothiazines on acid secretion in the toad gastric mucosa

The effect of phenothiazine antipsychotic drugs on acid secretion was investigated in the isolated toad gastric mucosa. The acid secretory responses induced by maximal doses of histamine, carbachol and theophylline were all inhibited in a similar fashion by chlorpromazine. The ID50 was between 300 and 600 microM. Histamine-stimulated H+ secretion was also inhibited by trifluoperazine. Soluble cyclic AMP-dependent protein kinase activity was not significantly affected by 300 microM chlorpromazine. Microsomal (H+ + K+)-ATPase activity was significantly reduced by chlorpromazine. The results indicate that phenothiazines can inhibit acid secretion in the toad gastric mucosa and that inhibition of the gastric (H+ + K+)-ATPase may be involved in the mechanism of action.     

Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric (H+-K+)-ATPase

Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent. The NH2-terminal sequence of the 27-kDa polypeptide corresponded exactly to that beginning at Asn-369 of the cDNA-deduced primary structure of the rat ATPase. The presence of the phosphorylation site, Asp-385, and FITC-labeled Lys-517, which is known to be a part of the ATP-binding site, indicates that the 27-kDa polypeptide contains a major cytoplasmic portion of (H+-K+)-ATPase. Interestingly, the polypeptide was stained with periodate-Schiff's base, indicating its glycoprotein nature. The carbohydrate group attached to the polypeptide seems to include at least an N-linked high-mannose moiety, since the polypeptide showed Con A binding activity as detected with a Con A-biotin/avidin-peroxidase assay on nitrocellulose transblots. Also, its Con A binding activity was inhibited by excess methyl alpha-D-mannopyranoside and disappeared upon treatment of the polypeptide with endoglycosidase H and N-glycanase. Further tryptic action converted the 27-kDa polypeptide to 2 smaller FITC-labeled polypeptides of 25 and 15 kDa, which lost 18 and 96 amino acid residues, respectively, from the NH2 terminus of the parent polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Target molecular weight of the gastric (H+ + K+)-ATPase functional and structural molecular size

The state of assembly of the (H+ + K+)-ATPase in purified hog gastric mucosa membranes was studied by target size analysis applied to radiation-induced enzyme inactivation and polypeptide degradation data. Radiation inactivated the Mg2+-ATPase, K+-stimulated ATPase, and p-nitrophenyl phosphatase activities of the membrane preparation with a dose dependence characteristic of a target size of 270,000-daltons. Radiation also bleached the major 100,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation, indicating a radiation-induced degradation. This apparent polypeptide degradation exhibited a dose dependency corresponding to a target size of 250,000 daltons in situ. It is suggested that the gastric ATPase is a trimeric assembly of the 100,000-dalton polypeptides.     

Sulphatide content in a membrane fraction isolated from rabbit gastric mucosal: its possible role in the enzyme involved in H+ pumping

The sulphatide content of vesicular membrane fraction from rabbit mucosal gastric microsomes was analyzed. This vesicular membrane fraction, in addition to a high sulphatide content, was enriched in an ouabain-insensitive (H+ + K+)-ATPase, a (Mg+2 + K+)-activated phosphatase, and a H+ pumping activity. The enzyme system involved in the process of acid secretion and the translocation of K+ was studied in these membrane preparations treated with arylsulphatase A, an enzyme that specifically hydrolyzes sulphatide. The results indicate that the breakdown of sulphatides of the vesicular membrane fraction inactivated both the (H+ + K+)-ATPase activity and the H+ pumping. Both activities were partially restored by the sole addition of sulphatide. The K+-stimulated ouabain-insensitive phosphatase activity, suggested as a partial reaction of the (H+ + K+)-ATPase sequence, was unaffected by arylsulphatase. These results suggest that sulphatides may play a function in the high activity binding site for K+ of the enzyme involved in H+ pumping.     

Pig gastric (H+ + K+)-ATPase. Lys-497 conserved in cation transporting ATPases is modified with pyridoxal 5'-phosphate

Pig gastric (H+ + K+)-ATPase can be covalently modified with pyridoxal 5'-phosphate (PLP) (about 1 mol/mol enzyme), and this modification is not observed in the presence of ATP, suggesting that PLP binds to a specific Lys residue in the ATP binding site or the region in its vicinity (Maeda, M., Tagaya, M., and Futai, M. (1988) J. Biol. Chem. 263, 3652-3656). The peptides labeled with radioactive PLP could be released from the gastric membrane vesicles quantitatively by chymotrypsin treatment, and two peptides were purified by high performance liquid chromatographies. These peptides were not obtained from vesicles incubated with PLP in the presence of ATP. The sequences of the two peptides were NH2-Asn-Ser-Thr-Asn-Lys-Phe-COOH and NH2-Ser-Thr-Asn-Lys-Phe-COOH, exactly corresponding to residues 493-498 and 494-498, respectively, of pig gastric (H+ + K+)-ATPase sequenced recently (Maeda, M., Ishizaki, J., and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157, 203-209). Lys-497 was concluded to be the binding site of PLP, as pyridoxyl-Lys was identified at the corresponding position. This Lys residue is conserved in (Na+ + K+)- and Ca2+-ATPases. The possible amino acid residues in the catalytic site of gastric (H+ + K+)-ATPase are discussed.     

The ontogeny of rat gastric H+/K+-ATPase

The ontogeny of rat H+/K+-ATPase was studied between foetal day 18 and neonatal day 18, using a specific monoclonal antibody (95-111 mAb). The H+/K+-ATPase content of gastric subcellular membranes was assayed and the ATPase subunits were characterized by Western blot. The epithelium density in parietal cells was measured by immunohistochemistry. H+/K+-ATPase was present in the 18-day-old foetuses and parietal cells were detected on foetal day 19. The H+/K+-ATPase concentration remained stable from foetal day 18 to neonatal day 1, while the parietal cell density increased 2.5-fold. The H+/K+-ATPase concentration increased by 2.5-fold on day 6, then remained constant up to day 18. The parietal cell density remained unchanged during this period, suggesting that the concentration increase on day 6 was due to an increase in parietal cell ATPase content. The 95-111 mAb recognized a 95 kDa single band on foetal day 18 and a doublet at all the other stages of development. Previous studies had demonstrated that acid secretion drops critically at day 12 post partum in the rat and that H+/K+-ATPase activity is lost. The present study demonstrates that the H+/K+-ATPase is, however, present on day 12.     

Molecular cloning of the rat stomach (H+ + K+)-ATPase

We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N-terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic.     

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.