Gastric Membrane Vesicles Research Articles

Helicobacter pylori augments the acid inhibitory effect of omeprazole on parietal cells and gastric H+/K+-ATPase

BACKGROUNDIn duodenal ulcer patients, intragastric acidity during omeprazole treatment is significantly lower before Helicobacter pylori eradication than after cure.AIMSTo determine ifH pylori enhances the acid inhibitory potency of omeprazole in...

Differential effects of GTP gamma S on acid and pepsinogen secretion by permeable gastric glands.

Gastric glands isolated from rabbit stomach were permeabilized with Staphylococcus aureus alpha-toxin. Acid secretion by parietal cells, as measured by the accumulation of weak base, was inhibited by incubation with alpha-toxin but could be restored by addition of exogenous ATP (1 mM). The permeable glands were found to retain acid secretory responses to receptor-linked secretagogues, histamine and carbachol, as well as to intracellular mediators, forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate, indicating the presence of intact, functional intracellular coupling mechanisms. Both basal and stimulated acid secretion by the permeable glands were blocked by the Mg2+ chelator, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA; 5 mM), whereas CDTA had no effect on nonpermeabilized glands. These results are interpreted to show that alpha-toxin permeabilizes parietal cells to moderate sized molecules without causing a loss of critical intracellular components. The acid secretory responses to histamine and carbachol persisted in media containing low ( < 50 nM) levels of free Ca2+ buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (0.5 mM), indicating that changes in bulk Ca2+ are not required for these responses. Inclusion of the nonhydrolyzable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM), resulted in inhibition of spontaneous acid secretion, blocked responses to all agents tested, and inhibited stimulated acid secretion. GTP gamma S had no effect on nonpermeabilized glands. No effects on acid secretion by either permeable or nonpermeable glands were observed with GTP, guanosine diphosphate, or guanosine 5'-O-(2-thiodiphosphate). GTP gamma S had no effect on H+ gradient formation by gastric membrane vesicles, showing that it does not inhibit the gastric H(+)-K(+)-adenosinetriphosphatase directly. These results are interpreted to show that GTP gamma S interacts at a postreceptor site to inhibit or reverse a critical step in stimulus-secretion coupling in parietal cells. In contrast to the effect on parietal cells, GTP gamma S was found to stimulate pepsinogen secretion by alpha-toxin-permeabilized chief cells. The differential effects of GTP gamma S on acid and pepsinogen secretions suggest unique roles for GTP binding proteins in these two secretory processes. The use of alpha-toxin-permeabilized gastric glands should prove useful in defining the stimulus-secretion coupling mechanisms involved in both acid and pepsinogen secretions.

Interaction of Helicobacter pylori and its fatty acids with parietal cells and gastric H+/K(+)-ATPase.

Helicobacter pylori and the fatty acids produced by this organism were compared for their acid inhibitory activity in isolated parietal cells and their interaction with gastric H+/K(+)-ATPase. H pylori (intact organisms, sonicates, methanolic extracts, and extracts from culture medium) and the fatty acids cis 9,10-methyleneoctadecanoic acid and tetradecanoic acid inhibited at fairly high concentrations histamine- and dibutyryl cyclic adenosine monophosphate stimulated acid production in isolated parietal cells, dissipated (with a slow onset) the H+/K(+)-ATPase created H+ gradient in gastric membrane vesicles, and inhibited H+/K(+)-ATPase activity in a concentration dependent manner. The inhibitory potency of H pylori and the fatty acids in relation to H+/K(+)-ATPase depended on the amount of membrane protein. Bovine serum albumin prevented enzyme inhibition and proton dissipation from gastric vesicles. The data indicate that H pylori establishes its antisecretory action in parietal cells by blocking H+/K(+)-ATPase activity and also by a detergent action at the apical parietal cell membrane. The fatty acids cis 9,10-methyleneoctadecanoic acid and tetradecanoic acid are probably the acid inhibitory factors secreted by H pylori.

Effect of proton pump inhibitor, omeprazole, on expression of rat parietal cell specific carbohydrate antigen, type 2 chain N-acetyllactosamine.

The effect of a proton pump inhibitor, omeprazole, on the rat gastric mucosal expression of carbohydrate antigens was studied. Type 2 chain N-acetyllactosamine was detected specifically on the apicocanalicular cell membranes of parietal cells. Pretreatment of rats with omeprazole profoundly suppressed the antigen expression, which followed the inhibition of gastric acid secretion. When omeprazole was discontinued, the antigen was reexpressed, which preceded the restoration of acid secretion. The antigen-negative tissues became antigen-positive when they were desialylated. Gastric membrane vesicles from the normal and omeprazole-treated rats were antigen-positive and -negative, respectively. SDS-PAGE revealed that a glycoprotein with an apparent molecular weight of 64-78 kDa carried type 2 chain N-acetyllactosamine. In the omeprazole-treated rats, the same molecular weight glycoprotein was positively immunostained only after desialylation. We concluded that: (1) the expression of type 2 chain N-acetyllactosamine was closely correlated with gastric acid secretion, and (2) the inhibition of acid secretion was accompanied by the sialylation of the parietal cell membrane glycoprotein.

Inhibition of Acid Secretion in Gastric Parietal Cells by the Ca 2+/Calmodulin-Dependent Protein Kinase II Inhibitor KN-93

A novel Ca 2+/calmodulin-dependent protein kinase II(CaM Kinase II) inhibitor, KN-93 potently inhibits gastric acid secretion from parietal cells. As previously reported (1), treatment of parietal cells with a selective inhibitor of CaM kinase II, KN-62 resulted in the inhibition of cholinergic-stimulated rabbit parietal cell secretion , whereas it failed to inhibit the histamine and forskolin response. In contrast effects of carbachol, histamine and forskolin were significantly inhibited by KN-93 with an IC 50 of 0.15, 0.3 and 1 μM, respectively; these effects occurred without any changes in intracellular cyclic AMP and Ca 2+ levels. In the present study we investigated the mechanism by which KN-93 acts upon the acid-secreting machinery of gastric parietal cells. Neither redistribution of the proton pump activity nor the morphological transformation were affected by KN-93. The drug only weakly inhibited the H +, K +-ATPase activity but strongly dissipated the proton gradient formed in the gastric membrane vesicles and reduced the volume of luminal space. Thus KN-93 acts at pH gradient formation whereas KN-62 acts only at CaM Kinase II.

Pantoprazole: a novel H +/K +-ATPase inhibitor with an improved pH stability

The action of the H +/K +-ATPase inhibitors pantoprazole and omeprazole was compared in different in vitro test systems. In gastric membrane vesicles under conditions shown to result in acidification of the vesicle interior, pantoprazole and omeprazole inhibited H +/K +-ATPase activity with IC 50 values of 6.8 and 2.4 μM, respectively. When intravesicular acidification was reduced by inclusion of imidazole (5 mM), a membrane permeable weak base, the inhibitory action of omeprazole was partially lost (IC 50 30 μM) and that of pantoprazole almost completely lost. After incubation for 40 min with pumping membrane vesicles, a half-maximal reduction in intravesicular H + concentration occurred at pantoprazole and omeprazole concentrations of 1.1 and 0.6 μM, respectively. Again, when the intravesicular H + concentration was reduced by inclusion of imidazole (2.5 mM), pantoprazole (20 and 60 μM) did not reduce the remaining intravesicular proton concentration, whereas omeprazole (10 and 30 μM) did. Both drugs inhibited, with similar potency, papain activity at pH 3.0 and inactivated the enzyme in a similar time-dependent manner; at pH 5.0 omeprazole (IC 50 17 μM) was more potent than pantoprazole (IC 50 37 μM) and enzyme inhibition was faster than with pantoprazole. These results indicate that pantoprazole is a potent inhibitor of H +/K +-ATPase under highly acidic conditions and that it is more stable than omeprazole at a slightly acidic pH such as pH 5.0.

Interaction of the non-steroidal anti-inflammatory drug flufenamic acid with gastric acid secretion and H+/K(+)-ATPase.

1. The effects of the non-steroidal anti-inflammatory drug (NSAID) flufenamic acid on H+ production in isolated and enriched guinea-pig parietal cells and on H+/K(+)-ATPase activity in ion-tight inside-out membrane vesicles from pig gastric mucosa were studied. 2. At low concentrations (0.1 and 1.0 mumol/L), flufenamic acid increased the secretory response of parietal cells to dibutyryl cyclic AMP (dbcAMP). At higher concentrations (10 and 100 mumol/L) it progressively inhibited basal and dbcAMP-stimulated acid production. 3. Flufenamic acid (10 mumol/L) increased K+ (0.5-10.0 mmol/L) and K+ (0.5-1.0 mmol/L) plus gramicidin-stimulated ATPase activity in gastric membrane vesicles. The Km value for K+ (1.6 and 1.0 mmol/L in the absence and presence of gramicidin, respectively) was decreased to 0.8 and 0.5 mmol/L, respectively. At higher concentrations (greater than or equal to 50 mumol/L), flufenamic acid inhibited K+ plus gramicidin-stimulated ATPase activity (inhibited concentration at 50% [IC50] = 186 mumol/L) and reduced the proton concentration (IC50 = 50 mumol/L). 4. It is concluded that flufenamic acid-induced enhancement of dibutyryl cyclic AMP-stimulated H+ production in the parietal cell reflects the stimulation of H+/K(+)-ATPase. We suggest that activation of the enzyme involves increased affinity of K+ towards the K(+)-binding site of the enzyme and/or increased KCl permeability at the vesicle membrane. The inhibitory action of the drug on H+ production in parietal cells results from a detergent and/or protonophoric-like action at the apical parietal cell membrane, and from inhibition of H+/K(+)-ATPase activity.

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles.

Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (kH+) was determined by addition of vesicles (pHi = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 x 10(-3) (gastric parietal cell stimulation-associated vesicles; SAV) and 5 x 10(-3) (duodenal brush-border membrane vesicles; dBBMV) to 11 x 10(-3) sec-1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mM, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in kH+ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na+/H+ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pHi less than pHo) 22Na+ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K(+)-diffusion potential ([K+]i much much greater than [K+]o). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K(+)-ionophore valinomycin, indicating that the native K+ permeability is rate limiting. In the presence of valinomycin, the K(+)-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K+ permeability. Valinomycin was without effect, but the K+/H+ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Omeprazole and bafilomycin, two proton pump inhibitors: Differentiation of their effects on gastric, kidney and bone H +-translocating ATPases

The effects of omeprazole and bafilomycin on processes dependent on two different types of H +-translocating ATPases were compared. A H +-ATPase of the E 1E 2-type, the H +,K +-ATPase, was purified from gastric mucosa. Vacuolar type H +-ATPases were prepared both from kidney medulla and from osteociast-containing medullary bone. H +,K +-ATPase-mediated proton transport in gastric vesicles was selectively inhibited by omeprazole with a high potency (inhibitory concentrations ≥ 3 μM) and in a time- and pH-dependent manner. This result is consistent with the mechanism of action of omeprazole, which is dependent on acid-induced transformation of the drug into an active inhibitor reacting with luminally accessible sulfhydryl groups of the enzyme. Accordingly, the presence of the membrane-impermeable mercaptane glutathione did not affect the inhibitory action of omeprazole on the H +, K +-ATPase. Proton transport in kidney- and bone-derived membrane vesicles was also inhibited by omeprazole, but with a lower potency (inhibitory concentrations ≥ 100 μM). Furthermore, the presence of glutathione totally abolished this inhibition, indicating that cytosolic, rather than luminal, SH-groups of the respective vascuolar H +-ATPase were interacting with omeprazole at high concentrations. In line with these results, it was found that omeprazole was much more potent in inhibiting acid production in isolated gastric glands (IC 50 ≈ 0.25 μM) than in inhibiting osteoclast-mediated 45Ca-release in isolated mouse calvaria (IC 50 ≈ 200 μM). Bafilomycin, on the other hand, was much more effective in inhibiting proton transport mediated by the vacuolar H +-ATPases in the kidney- and bone-derived membrane vesicles (IC 50 ≈ 2 nM) than in inhibiting H +,K +-ATPase-mediated proton transport in gastric membrane vesicles (IC 50 ≈ 50 μM). Thus, approximately 10 4 times higher concentrations of bafilomycin were needed to inhibit the H +, K +-ATPase to the same extent as the vacuolar H +-ATPase. A similar difference in potency of befilomycin was found when its inhibitory effect was determined in isolated mouse calvaria (IC 50 ≈ 2.5 nM) and in isolated gastric glands (IC 50 ≈ 5 μM). Hence, omeprazole was found to be a specific inhibitor of the H +,K +-ATPase under physiological conditions, i.e. in the presence of glutathione, while bafilomycin was found to be selective towards vacuolar H +-ATPases.

Interaction of the anti-inflammatory seleno-organic compound ebselen with acid secretion in isolated parietal cells and gastric H +/K +-ATPase

The effects of the anti-inflammatory seleno-organic compound ebselen on gastric H +/K +-ATPase, H +/K +-ATPase-mediated proton transport and on parietal cell HCl production was studied. Ebselen inhibited K +-stimulated ATPase activity in leaky gastric membranes ( ic 50:0.15 μM) and H +/ K +-ATPase-mediated proton transport in intact gastric membrane vesicles ( ic 50:0.7μM). Histamine- and dibutyryl-cAMP-stimulated HCl production in isolated and enriched guinea-pig parietal cells was inhibited with an ic 50 value of 12 μM. The mercaptan dithioerythritol and the nucleotide ATP prevents the H +/K +-ATPase against inactivation and dithioerythritol was found to restore already inhibited enzyme activity and ATPase mediated H + transport. Furthermore, dithioerythritol could prevent ebselen-induced inhibition of HCl production in the parietal cell preparation. It is concluded that ebselen inhibits acid secretion in the parietal cell by interference with SH groups of the gastric proton pump, the H +/K +-ATPase. Therefore ebselen can be regarded as an anti-inflammatory drug for which in vitro anti-secretory properties can be demonstrated.

Substituted thieno[3,4-d]imidazoles, a novel group of H +/K +-ATPase inhibitors. Differentiation of their inhibition characteristics from those of omeprazole

The action of the H +/K +-ATPase inhibitors, Hoe 731 and S 4216, both thieno-imidazole derivatives, was compared with that of the benzimidazole derivative, omeprazole. In intact, gastric membrane vesicles under conditions shown to result in acidification of the vesicle interior, Hoe 731 and S 4216 inhibited H +/K +-ATPase activity with an IC 50 value of about 1.0 μM. In the absence of a generated pH gradient the respective IC 50 values were 5.5 and 2.1 μM. In contrast, omeprazole inhibited the enzyme only in the presence of proton accumulation (IC 50: 0.7 μM). The inhibitory action of omeprazole on H +/K +-ATPase-mediated proton transport was prevented by the membrane permeable mercaptane, dithioerythritol, but not by the membrane impermeable, mercaptane glutathione, whereas both mercaptanes were able to prevent the effect of Hoe 731 and S 4216. These results indicate that the thienoimidazoles react with intravesicular (luminal) and extravesicular (cytosolic) SH groups of the H +/K +-ATPase, whereas omeprazole interacts uniquely with luminal SH groups of the enzyme. In isolated parietal cells all drugs caused a concentration-dependent inhibition of HCl production, as measured by [ 14C]aminopyrine uptake, during histamine and dibutyryl-cAMP stimulation. The IC 50 value was 0.1 μM for Hoe 731 and omeprazole and 0.4 μM for S 4216 after 30-min incubation. The inhibitory action of Hoe 731 and S 4216 faded with increasing incubation time, whereas omeprazole caused an unchanged inhibition over the entire 120-min incubation period. We suggest that several factors, e.g. weaker chemical stability of the drugs or perturbation of cellular glutathione levels, may be responsible for the fading inhibitory action of thienoimidazoles in the parietal cell.

Interaction of Calcium Channel Antagonists with Parietal Cell Acid Production, Adenylate Cyclase, Intracellular-Free Ca&lt;sup&gt;2+&lt;/sup&gt; and H&lt;sup&gt;+&lt;/sup&gt;/K&lt;sup&gt;+&lt;/sup&gt;-ATPase

The calcium channel antagonists verapamil, gallopamil and nifedipine were tested for their effects on acid secretion stimulated by histamine and dibutyryl-cyclic AMP in isolated and enriched guinea pig parietal cells, on adenylate cyclase activity mediated by histamine H2 receptors, on histamine-stimulated increase in cytosolic-free Ca2+ concentration [Ca2+], on gastric H+/K(+)-ATPase activity and on H+/K(+)-ATPase-mediated proton uptake in intact gastric membrane vesicles. Verapamil and gallopamil impaired all cellular and enzymatic test systems studied. Both drugs affected with highest potency the acid secretion in the parietal cell preparation (IC50: 1-2 mumol/l) and the H+/K(+)-ATPase-mediated H+ uptake in gastric membrane vesicles, whereas their inhibitory action was less pronounced on adenylate cyclase and on histamine-induced increase in cytosolic-free [Ca2+]. The type of interaction found in the gastric membrane vesicle preparation indicates that both drugs act as protonophores. Nifedipine was less effective as an inhibitor of acid secretion in the parietal cell preparation and in reducing proton concentration in isolated gastric membrane vesicles. The drug failed to block adenylate cyclase and H+/K+-ATPase activity. Since nifedipine is a more effective calcium channel blocking agent but a less lipophilic drug than verapamil and gallopamil, we conclude that the antisecretory activity of calcium channel antagonists in vitro is mediated by a nonspecific, i.e. a protonophoric, action. We suggest that verapamil exhibits its antisecretory activity in vivo partially by its protonophoric action at the secretory membrane of the parietal cell, whereas the decrease in acid secretion by nifedipine is not mediated by this mechanism.

Pig Gastric (H+ + K+)-ATPase: Lys-497 Conserved in Cation Transporting ATPases is Modified with Pyridoxal 5′-Phosphate

Abstract 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.

Modification of gastric (H+ + K+)-ATPase with pyridoxal 5'-phosphate.

Pig gastric membrane vesicles enriched in (H+ + K+)-ATPase were covalently modified with pyridoxal 5'-phosphate (PLP). The modification resulted in inhibition of K+-dependent ATP hydrolysis, formation of phosphoenzyme and ATP-driven H+-uptake catalyzed by (H+ + K+)-ATPase. ATP, ADP, and adenyl-5'-yl imidodiphosphate were protective ligands, whereas Mg2+ and K+ were not. Specific PLP-binding of about 4.5 nmol/mg membrane protein was necessary for complete inhibition of the enzyme activity, indicating that the stoichiometry of PLP-binding to the enzyme was about 1:1. Limited proteolysis of the enzyme modified with [3H]PLP by trypsin suggests that PLP specifically modifies the lysine residue located in the 16-kDa fragment of the enzyme cleaved by trypsin. These results suggested that PLP binds to a specific lysine residue in the nucleotide-binding site or a region in its vicinity and inhibits the substrate binding or phosphorylation step of (H+ + K+)-ATPase.

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 × 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 limitingstep 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

A protein phosphatase associated with rat heavy gastric membranes enriched with (H+-K+)-ATPase influences membrane K+ transport activity.

Rat stimulated heavy gastric membranes enriched with (H+-K+)-ATPase, a marker for the apical membrane of the parietal cell, displayed a 32P-histone-dephosphorylating activity which appeared to be physically copurified with, but functionally independent of, the ATPase. The protein phosphatase activity was optimal at pH 7.5 and was inhibited by fluoride (50 mM), inorganic phosphate (50 mM), and p-chloromercuribenzoate (0.1 mM), but was insensitive to vanadate (1 mM). The 32P-phosphoproteins in the heavy gastric membranes were also dephosphorylated, apparently by their own membrane-bound phosphatase in the presence of Mg2+ at millimolar concentrations, which is likely to enhance membrane-membrane interaction. Heavy gastric membrane vesicles incubated with Mg2+ (2 mM) exhibited no alterations in K+-dependent ATP-hydrolyzing activity, Cl permeability, and protein and lipid compositions, but irreversibly lost the ATP, K+-dependent H+-pumping activity. Since valinomycin, a K+-specific ionophore, restored the intravesicular acidifying activity and an inhibitor of the protein phosphatase, inorganic phosphate, largely blocked the Mg2+-induced change in the membrane transport function, it is reasonable to propose that the phosphatase action on certain membrane proteins, possibly the putative K+ transporter or regulatory proteins, selectively decreases K+-conductance in the apical membranes of gastric parietal cells.

Inhibitory action of omeprazole on acid formation in gastric glands and on H+,K+-ATPase isolated from human gastric mucosa.

The inhibitory effect of omeprazole on acid formation has been studied in vitro in gastric glands and partly purified H+,K+-ATPase, prepared from mucosa obtained either from healthy subjects by gastroscopic biopsy or from gastric ulcer patients during antrectomy. The effect of omeprazole was compared with the inhibitory pattern of the H2-antagonist cimetidine. Acid production in the glands was determined by measuring the accumulation of 14C-aminopyrine. In glands isolated from patients, omeprazole inhibited acid production maximally stimulated by histamine, db-cAMP, and potassium in a dose-dependent manner, with an IC50 value of about 50 nM irrespective of the agonist used. In contrast, cimetidine inhibited only histamine-induced aminopyrine accumulation, with an IC50 of about 30 micron. The inhibitory effect of omeprazole in db-cAMP-stimulated glands from healthy volunteers was of the same magnitude as seen in glands from gastric ulcer patients. Basal aminopyrine accumulation in glands from both patients and healthy volunteers was almost totally inhibited by omeprazole, whereas cimetidine was without effect. Omeprazole also concentration-dependently inhibited the H+,K+-ATPase activity in isolated gastric membrane vesicles. The estimated IC50 value was 4 micron.

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.

The H +/ATP transport ratio of the (K + + H +)-ATPase of pig gastric membrane vesicles

Various values have been reported for the H +/ATP transport ratio of the (K + + H +)-ATPase of the gastric parietal cell: 4, 2 and 1. We have, therefore, reinvestigated this matter with a vesicle preparation isolated from pig gastric mucosa. The vesicles are suspended in glycylglycine buffer (pH 6.11) at 22°C, and incubated until equalization of the K + concentration inside and outside (75 mM). After addition of ATP, the initial rates of H + uptake and ATP hydrolysis are then measured. Proton uptake is inhibited in the absence of K + or in the presence of nigericin. The K 0.5 value for proton transport is 154 μM and the K m value for ATP hydrolysis is 61 μM. The Lineweaver-Burk plot for ATP hydrolysis vs. ATP concentration is linear with a V max of 5.5 nmol/mg protein per s, but that for H + uptake is not. Thus with increasing ATP concentration (6.7 to 1670 μM) the transport ratio increases from 0.3 to 1.8. Extrapolation to infinite ATP concentration gives a value of 1.89. (S.E. 0.13, N= 5) and a Hill coefficient of n = 1.21 (S.E. 0.06, N = 5) implying that the true transport ratio is 2 H +/ATP with positive cooperativity between the protons.

An acid transporting enzyme in human gastric mucosa.

Isolation of a microsomal fraction from human gastric mucosa followed by density gradient centrifugation yielded a vesicular membrane preparation free of mitochondrial markers, containing a K+-activated, ouabain-insensitive ATPase with an activity of 20.7 mumol P1 released/mg protein per h. Sodium dodecyl sulfate gel electrophoresis showed that the human gastric membrane vesicles contained a major polypeptide of 110,000 daltons, which accounted for approximately or equal to 30% of the total protein stained and was phosphorylated by [gamma-32P]ATP and dephosphorylated in the presence of K+. Electron microscopy revealed the presence of vesicles with an average size of 0.13 micrometer in diameter. Addition of 0.65 microM ATP to this vesicular preparation resulted in the uptake of 17 nmol H+/mg protein which was dependent on the presence of K+. The gradient was dissipated by a combination of valinomycin and protonophore after consumption of the ATP. Incubation of fixed human fundic sections or human gastric biopsy with monospecific hog gastric membrane antibody followed by fluorescein-conjugated goat anti-rabbit gamma-globulin, showed fluorescent staining in the middle portion of the gastric glands. These data indicate that human stomach contains a H+ transport ATPase with characteristics similar to those established for lower species.