Gastric Microsomes Research Articles

The effect of nimesulide on prostanoid formation.

Nimesulide (4-nitro-2-phenoxy-methansulfonanilide) is a weak acidic (pKa = 6.50) nonsteroidal anti-inflammatory drug (NSAID) belonging to a new chemical class, the sulfonanilide derivatives, and does not contain the carboxylic group. It exhibits potent anti-inflammatory, analgesic and antipyretic activities. Although nimesulide is not a cyclo-oxygenase inhibitor in gastric tissues or bovine seminal vesicle microsomes, it inhibits prostaglandin synthesis in zymosan-stimulated murine macrophages in inflammatory exudate. Nimesulide significantly antagonises the immune formation of thromboxane B2 in lung tissue. It neither induces gastrointestinal lesions nor affects renal function in animal models.

Limited utilization of exogenous arachidonic acid by the prostaglandin cyclooxygenase in gastric mucosa: The role of protein binding, glutathione peroxidase, and hydrogen peroxides

We investigated the utilization of exogenous 14C-labelled arachidonic acid by the cyclooxygenase system of the gastric mucosa and its alteration by cytosolic factors, protein binding, glutathione peroxidase (GSH-Px), and hydrogen peroxides. Total prostaglandin (PG) synthesis from gastric microsomes was reduced in a dose- dependent manner to 12% and 68% of controls by increasing amounts of the 105,000g supernatant or albumin (8mg protein/ml), respectively ( p<0.01). The inhibitory cytosolic factor was heat labile, protease sensitive, and was retained by a 300,000 Dalton ultrafiltration membrane. Thus, it was likely a protein. Other possible inhibitory mechanisms like protease- or heme-induced destabilization of the cyclooxygenase, haptoglobin-mediated inhibition, or self-inactivation by endogenous substrate were excluded. N-ethylmaleimide (NEM), an agent that alkylates sulfhydryl-groups thereby inhibiting GSH-Px, abolished the inhibitory effect of cytosol in a dose-dependent fashion. In contrast to their inhibition of prostaglandin synthesis, the binding of arachidonic acid by albumin or cytosolic proteins accounted to 75% and 19% under comparable conditions, respectively, however, cytosolic fatty acid binding was unaffected by NEM. Thus, it was concluded that the inhibitory effect of cytosol, in contrast to albumin, was mediated by a sulfhydryl-depending process, probably a GSH-Px. This conclusion was supported by a qualitatively comparable inhibition by a purified GSH-Px from bovine erythrocytes. The inhibitory action of cytosolic proteins was reduced significantly by increasing concentrations or repeated application of arachidonic acid; therefore, cytosolic GSH-Px was likely to affect substrate utilization by the microsomal PGH synthase through reduction of activating substrate peroxides. Similarly, the in vitro formation of cyclooxygenase products by mucosal homogenate or gastric microsomes in the absence of cytosol was limited at substrate concentrations below 80μM, despite sufficient nonesterified arachidonic acid remaining in the incubate. This limitation was mediated only partially by self-inactivation of the prostaglandin cyclooxygenase. Neither N-ethylmaleimide nor repeated application of hydrogen peroxides increased substrate utilization by isolated microsomes, excluding contamination by GSH-Px or simply a lack of hydrogen peroxides as possible mechanisms for the limited utilization. From these results, a special role of substrate-linked lipid peroxides in the activation of mucosal prostaglandin synthesis is proposed. The reduction of these peroxides by glutathione dependent or independent peroxidases, e.g. the PGH synthase-linked hydroperoxidase activity itself, could explain the reduced utilization of nonesterified arachidonic acid by the gastric mucosa.

Isolation of a gastric phosphoprotein which stimulates acid secretion.

Stimulation of gastric acid secretion is mediated by cAMP which regulates the proton pump through an A-kinase-dependent phosphoprotein. The purpose of this study was to isolate a stimulation-dependent gastric phosphoprotein capable of stimulating acid secretion. Gastric glands were prepared from rabbit gastric mucosa and acid secretion was stimulated with cAMP. A detergent extract of these stimulated gastric membranes was fractionated by gel chromatography and assayed for functional activity by measurement of [14C]-aminopyrine accumulation in permeabilized resting gastric glands or measurement of H(+)-K(+)-ATPase activity in inhibited gastric microsomes. We hereby report isolation of a membrane-bound, A-kinase-dependent phosphoprotein which enhances aminopyrine accumulation in digitonin-permeabilized gastric glands (32%) and stimulates H(+)-K(+)-ATPase activity in gastric microsomes to a level 55% of the maximal stimulation observed in the presence of valinomycin. Incubation of this phosphoprotein with [32P]ATP and the catalytic subunit of A-kinase resulted in [32P] incorporation into a protein which coincided with a single protein band on SDS-PAGE (17,500 Da).

Gastric H(+)-K(+)-ATPase activity is inhibited by reduction of disulfide bonds in beta-subunit.

H(+)-K(+)-ATPase activity of rabbit isolated gastric microsomes was irreversibly inactivated by reducing agents, such as 2-mercaptoethanol and dithiothreitol. Similar to what has been observed for Na(+)-K(+)-ATPase, high concentrations of reagents, at moderately elevated temperatures, were required to inactivate H(+)-K(+)-ATPase, suggesting relative inaccessibility of the responsible disulfide bonds. Resistance against inactivation was conferred by monovalent cation activators of K(+)-stimulated ATPase and p-nitro-phenylphosphatase. The effectiveness of K+ congeners in protecting the enzyme was similar in sequence (Tl+ greater than K+ greater than Rb+) and concentration to their respective affinities for stimulating enzymatic activity, suggesting that the K(+)-bound form of the enzyme is more resistant to reduction than the free enzyme. Furthermore, Na+ antagonized the protective effect of K+. Labeling studies using fluorescein-maleimide indicated that 60-70% of the cysteine residues in the beta-subunit are in the oxidized form. Coupled with primary sequence data, this suggests that three disulfide bonds are present in the native beta-subunit. In contrast, less than 10% of the cysteine residues in the alpha-subunit are in the oxidized form. Kinetic studies showed that the 2-mercaptoethanol-induced loss of H(+)-K(+)-ATPase activity was correlated with a reduction of disulfide groups in the beta-subunit, while there was no significant change in the alpha-subunit. We conclude that reduction of disulfide bonds irreversibly inhibits H(+)-K(+)-ATPase activity, binding of K+ to the enzyme confers a resistance to disulfide bond reduction, and the responsible disulfide bonds are present in the beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography.

We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized gastric H+/K(+)-ATPase complex.

Gastric H,K-ATPase topography: Amino acids 888–907 are cytoplasmic

Gastric acidification is mediated by H,K-ATPase, an integral protein of apical membranes of gastric parietal cells. Hydropathy analysis of H,K-ATPase alpha subunit primary structure predicts eight transmembrane (TM) domains, while omeprazole-binding data were interpreted in terms of ten TM domains (Mercier et al. (1991) FASEB J. 5, A749). In the present study, tryptic hydrolysis of gastric mucosal microsomes gave a set of peptides which bound the monoclonal antibody HK 12.18, a highly specific probe of the H,K-ATPase. An antiserum against the C-terminus of H,K-ATPase alpha subunit bound the same peptides, and one smaller peptide. The binding data suggested a putative epitope for HK 12.18, and a 20-mer peptide encompassing this site was synthesized. This peptide bound directly to HK 12.18, displaced HK 12.18 from microsomal H,K-ATPase, and blocked HK 12.18 immunostaining of gastric parietal cells. In addition, intact gastric microsomes competitively inhibited binding of HK 12.18 to peptide-BSA conjugate. Taken together, these data place the HK 12.18 epitope between amino acids 888-907 and identify this domain as cytosolic. This result specifically excludes a pair of TM domains between the sixth and seventh TM alpha helices of the H,K-ATPase and supports a secondary structure model with eight TM domains.

Effects of the enantiomers of lansoprazole (AG-1749) on (H + + K +)-ATPase activity in canine gastric microsomes and acid formation in isolated canine parietal cells

The effects of the enantiomers of 2-[[[3-methyl-4-(2,2,2-trifluoroethoxy) - 2 - pyridyl]methyl]sulfinyl] - 1 H - benzimidazole(lansoprazole, AG-1749) on acid formation in isolated canine parietal cells and (H + + K +)-ATPase activity in canine gastric microsomes were investigated. Both the (+)-and the (−)-enantiomer of lansoprazole inhibited the acid formation stimulated by dibutyryl cyclic AMP (db-cAMP) in isolated canine parietal cells in a concentration-dependent manner with IC 50 values of 59 and 82 nM, respectively. The enantiomers showed concentration-dependent inhibition of (H + + K +)-ATPase with IC 50 values of 4.2 and 5.2 μM, respectively. On the other hand, the IC 50 values of lansoprazole for db-cAMP-stimulated acid formation and (H + + K +)-ATPase were 59 nM and 2.1 μM, respectively. These results suggest that the two enantiomers of lansoprazole have antisecretory action due to inhibition of (H + + K +)-ATPase.

Effect of alcohols on gastric and small intestinal apical membrane integrity and fluidity.

Duodenal and jejunal brush border membrane vesicle integrity was studied after in vitro treatment of rabbit tissue with ethyl, benzyl or octyl alcohol. The effects of the alcohols on gastric parietal cell apical and microsomal membrane vesicle integrity was also studied. Membrane vesicle integrity was determined from the enclosed volume of the vesicle preparations, measured as [14C]glucose space at equilibrium. Exposure of vesicles to the three alcohols caused concentration dependent decreases in enclosed volume. The rank order of potency of the alcohol was octyl greater than benzyl greater than ethyl. Concentrations greater than or equal to 10 mM benzyl alcohol significantly reduced the enclosed volume of duodenal or jejunal vesicles; jejunal vesicles were disrupted by 625 mM ethanol, whereas 2 M ethanol was required to disrupt the duodenal vesicles. Gastric apical membrane integrity was reduced with 0.25 M ethanol, the vesicles being approximately an order of magnitude more sensitive to ethanol than gross estimates of gastric mucosal damage, but 1 M ethanol was required to significantly damage gastric microsomes. All concentrations of benzyl or octyl alcohol tested (greater than or equal to 5 mM) reduced the enclosed volume of both gastric apical membrane vesicles and gastric microsomes. As determined by shrink-swell techniques, benzyl alcohol permeated duodenal vesicles at a faster rate than NH4Cl (apparent rate constant of 9.89 (0.71) X 10(-3)s-1 compared with 4.48 (0.23) X 10(-3)s-1). Therefore, reductions in enclosed volume in response to alcohol treatment could not be explained by alcohol induced osmotic shrinkage. The enclosed volume of the vesicles after alcohol treatment was negatively correlated with membrane fluidity suggesting a common causal effect, the increased fluidity increasing membrane fragility. Duodenal vesicles were more resistant to disruption by the alcohols compared with gastric and jejunal vesicles.

Biosynthesis of platelet-activating factor in glandular gastric mucosa. Evidence for the involvement of the ‘de novo’ pathway and modulation by fatty acids

The biosynthesis of platelet-activating factor (PAF), a phospholipid autocoid with potent ulcerogenic properties that is produced in secretory exocrine glands by physiological secretagogues, was assessed in microsomal preparations of glandular gastric mucosa. For this purpose, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67); the enzymes of the 'de novo' pathway: 1-O-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP):acetyl-CoA acetyltransferase and 1-O-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-choline cholinephosphotransferase (EC 2.7.8.16); and some enzymes involved in the catabolism of PAF and lyso-PAF were assayed. Only the enzymes of the 'de novo' pathway and small amounts of PAF acetylhydrolase, phospholipase A2 and a lysophospholipase D acting on either lipids could be detected in the gastric preparations, whereas lyso-PAF:acetyl-CoA acetyltransferase activity was undetectable. The specific activity of alkyl-lyso-GP:acetyl-CoA acetyltransferase in the gastric mucosa was about one-tenth of that found in spleen microsomes and its apparent Km for acetyl-CoA was 454 microM compared with 277 microM in spleen microsomes. Glandular mucosa homogenates contained preformed PAF at a concentration of 2.7 +/- 0.7 ng equivalents of PAF (hexadecyl)/mg of protein. When gastric microsomes were incubated with micromolar concentrations of fatty acids (arachidonic, palmitic and oleic) prior to the assay of dithiothreitol (DTT)-insensitive cholinephosphotransferase, a dose-dependent reduction in the formation of PAF was observed, arachidonic acid being the most potent inhibitor, followed by linoleic acid (only tested on spleen microsomes) and oleic acid. By contrast, 1,2-diolein and phosphatidylcholine (dipalmitoyl) showed no or little effect. These results indicate that glandular gastric mucosa can produce PAF through the 'de novo' pathway, and that fatty acids, especially unsaturated, can reduce that synthesis by modulating the expression of DTT-insensitive cholinephosphotransferase.

Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes.

The (H+,K+)ATPase-enriched microsomal fraction prepared from hog gastric mucosa by sucrose density gradient centrifugation was effectively solubilized with Emulgen, with apparent preservation of the enzyme activity, and then the ATPase was highly purified by polyethylene glycol fractionation, and Blue Sepharose CL-6B and amino-hexyl Sepharose chromatographies. The purified enzyme showed a single band, with an apparent molecular mass of approximately 94 kDa, on SDS-PAGE, and exhibited both K+-ATPase and K+-stimulated-p-nitrophenyl phosphatase (pNPPase) activities. The optimum pH for the ATPase activity was 7.0. Amino acid analysis of the purified enzyme showed that it contains a large amount of hydrophobic amino acid (42%) and a small amount of glucosamine and galactosamine. The rabbit antibody monospecific for the ATPase, in the Ouchterlony double immunodiffusion and Western blotting tests, markedly inhibited both the K+-ATPase and K+-pNPPase activities.

Actions of antisecretory agents on proton transport in hog gastric microsomes

The properties of K +-stimulated ATP hydrolysis (K +-ATPase) and vesicular accumulation of H + (H + accumulation) in hog gastric microsomes were investigated. The microsomes consisted of smooth surfaced vesicular particles, 70–300 nm in diameter. Both the activities of ATPase and the vesicular accumulation of H + were stimulated by K + in the presence of Mg 2+, and enhanced by the K +-ionophore, valinomycin. However, there were differences in regulation of K +-ATPase and H + accumulation by K + ions, i.e. K + at concentrations higher than 10 mM decreased K +-ATPase activity but further enhanced H + transport. This observation suggests that the two reactions are partly independent. The H + accumulation was inhibited by omeprazole, fenoctimine, spennine, and NaSCN, but not by cimetidine, prostaglandin E 2, and atropine. The inhibitory effect of omeprazole on H + accumulation paralleled the inhibition of k +-ATPase, while fenoctimine, spermine, and NaSCN suppressed H + accumulation, without inhibiting K +-ATPase, under appropriate concentrations. In addition, the spontaneous diffusion of H + across the microsomal membrane was markedly enhanced by fenoctimine, but not by the other agents used. These results indicate that (1) omeprazole inhibits H + accumulation by inhibiting K +-ATPase, (2) fenoctimine suppresses H + accumulation mainly by increasing the loss of accumulated H + from the microsomal vesicles, (3) spermine and NaSCN reduce H + accumulation by inhibiting the transport of H + into microsomal vesicles.

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.

Thiophosphorylation of hog gastric (H + + K +)-ATPase membranes by endogenous protein kinases

(H + + K +)-ATPase-enriched membranes from hog stomachs were tested for their capacity to autophosphorylate using [γ- 32P]ATP or [γ- 35S]ATP[S] as phosphate donors. The radioactive polypeptides were characterized by SDS-PAGE. In the presence of Mg 2+ and 5 μM [γ- 32P]ATP, rapid and transient incorporation of 32P occurred at 0°C. Radioactivity was essentially found in the major polypeptide of the material, the 95 kDa subunit of (H + + K +)-ATPase. Under the same experimental conditions, thiophosphorylation was slower and reached a plateau within 1 h. Incorporation levels were higher with manganese than with magnesium. After one hour at 0°C, and in the presence of 10 mM manganese and 5 μM ATP[S], 0.58 ± 0.06 nmoles of thiophosphate were incorporated per mg of protein. Twenty seven percent of the thiophosphorylated amino acids were acylphosphates i.e. likely to be the ATPase thiophosphointermediate. The remaining thiophosphorylated amino acids (73%) were thought to be produced by protein kinases. This was supported by the autoradiographies of membrane SDS-PAGE which indicated that, in addition to the 95 kDa ATPase subunit, other polypeptides were thiophosphorylated especially at 108, 58, 47, 45 and 36–40 kDa. A previous study had provided strong evidence that chloride transport in gastric microsomes, is modulated by a protein kinase-dependent phosphorylation (Soumarmon, A., Abastado, M., Bonfils, S. and Lewin M.J.M. (1980) J. Biol. Chem. 255, 11682–11687). In the present work, we demonstrate that the peptidic inhibitor of cAMP-dependent protein kinases decreased thiophosphorylation of a 45 kDa polypeptide. We suggest that this polypeptide could be regarded as a candidate for the role of chloride transporter or chloride transport regulator.

NMR studies of pig gastric microsomal H+,K+-ATPase and phospholipid dynamics. Effects of ethanol perturbation.

The effects of ethanol on the gastric H+,K+-ATPase activity and the degree of mobility of various microsomal phospholipids were assessed using 31P and 1H NMR. This illuminated the role of lipid-protein association in the function of pig gastric microsomes. Treatment of gastric microsomes with 15% ethanol for 1 min at 37 degrees C inactivated the H+,K+-ATPase activity, which could largely be reconstituted by supplementation with phosphatidylcholine isolated from the gastric microsomes. Under similar conditions, the 1H NMR profile of the microsomal +N(CH3)3 choline moiety showed dramatic enhancement of peak intensity as well as a break point at 25 degrees C which was restored to the untreated control value after reconstitution. This break, together with the dramatic enhancement in the overall lipid profile, compared to the control and reconstituted microsomes, suggested a greater degree of freedom of movement of the microsomal lipids following ethanol perturbation. The data demonstrate the unique ability that a combined approach using 31P and 1H NMR holds as a noninvasive probe to study the structure-function relationship of biomembranes.

Mechanism of gastric antisecretory effect of thiocyanate: Further evidence for the thiocyanate-induced impediment in gastric H +,K +-ATPase function

Two hypotheses have recently been proposed for the thiocyanate inhibition of gastric acid secretion—a protonophore mechanism and an uncoupling model. The mechanistic aspects for the latter scheme have been examined on the following basis: capability of generating verifiable predictions, supporting evidence that is unambiguous, and compatibility with experimental realities. Gastric microsomes bind 5 nmol of SCN −/mg, and a “pure” and highly active fraction of H +,K +-ATPase prepared from gastric microsomes binds about 15 nmol of SCN −/mg. The affinity of SCN − binding to gastric microsomes changes from 10 to 25 mM in the presence of 20 mM K + suggesting competition between K + and SCN −. Potassium also displaces the bound SCN − from “pure” H +,K + ATPase with a K i of about 25 mM. Of the cations tested—Tl +, K +, Rb +, Cs +, NH 4 +, Na +, and Li +—Tl + was the most effective in displacing bound SCN − while Na + and Li + were without effect. The effects of anions such as Cl −, NO 3 −, and gluconate were found to be nonspecific and absolutely dependent on K + as cocation. Sulfate and OCN −, on the other hand, showed an ability to displace SCN − as both K + and Na + salts. For SO 4 −2 the K + form was much more effective than the Na + salt. Besides these antagonistic effects of K + and congeners with the H +,K +-ATPase-bound SCN −, a competition between K + and SCN − was also observed at the level of gastric K +-stimulated pNPPase reaction. The effects of SCN − and two other unrelated anions, F − and NO 2 −, on artificial ΔpH across the microsomal vesicles exhibited a lack of appreciable change up to 5 mM and a small (about 13%) reduction between 10 and 20 mM. However, a combination of CCCP and nigericin or valinomycin completely abolished the ΔpH under identical conditions. The present data in conjunction with other reports suggest that the proton impediment model best explains the gastric antisecretory effects of SCN −.

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.

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.

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.

Ion transport studies with H+-K+-ATPase-rich vesicles: implications for HCl secretion and parietal cell physiology

A summary of recent studies on relations between the properties of the membrane incorporating the H+-K+-ATPase, the H+ motive force in gastric acid secretion, and the secretory state of the parietal cell is presented. Depending on tissue secretory state, two distinct H+-K+-ATPase-rich membranes predominate in tissue homogenates, the gastric microsomes derived from the intracellular tubulovesicles of the resting cell and the stimulation-associated (SA) vesicle derived from the apical membrane of the acid-secreting cell. Structural and chemical differences between both vesicular types lend support to the notion that the formation of an expanded, elaborated apical membrane in the secreting parietal cell results from fusion of tubulovesicles containing the H+-K+-ATPase to an apical membrane of different chemical composition. Comparison of polypeptide composition of microsomes and SA membranes provides a way to identify and isolate membrane and cytoskeletal components putatively involved in the membrane interconversion process. Comparison of transport properties between gastric microsomes and SA vesicles demonstrates that stimulation triggers the appearance of rapid K+ and Cl- permeabilities in the H+-K+-ATPase membrane, allowing efficient acid accumulation in SA vesicles by the combination of rapid KCl influx followed by ATPase-driven H+ for K+ exchange, i.e., by K+ recycling. These stimulation-triggered conductances are functionally independent. Nevertheless, their concurrent inhibition by certain divalent cations (Mn2+,Zn2+) suggests their location within a single physical domain. The compatibility of the K+-recycling model for HCl accumulation in SA vesicles with gastric HCl secretion and selected electrophysiological observations and certain implications of the findings for cellular mechanisms of transport regulation in the context of a membrane fusion and recycling model are discussed.

Evidence for Distinct Ca2+‐ATPase Activities in Gastric Microsomes

Recently, a variant type of (Ca2+ + Mg2+)-ATPase stimulated by submicromolar Ca2+ has been reported in plasma membranes of erythrocyte,) Iiver,2 adipocyte,l corpus luteum,4 isolated bone cells,s and ascites hepatoma cells. The Ca2+ -stimulation of many of these A TPases2-5 was abolished by the addition of Mg2+ -a fact posing a major difficulty in demonstrating the high-affinity Ca2+ -activated ATPase. We previously reported? that purified dog gastric microsomes highly enriched in H+,K +ATPase activity also contain a high-affinity and a low-affinity Ca2+ -ATPase system. Our data suggested? the Ca2+ -ATPase activities to be a part of the Mg2+ -ATPase, which is always associated with the gastric microsomes. In this paper, we report complete separation of the H + ,K+ -ATPase from the Mg2 + -ATPase and the associated Ca2+ -ATPase activities, thus demonstrating that the latter activities are unrelated to the former.