Tushar K. Ray

Modulation of gastric H+, K+-transporting ATPase function by sodium

Gastric H+, K+‐ATPase activity is not affected by Na+ at pH 7.0 but is significantly stimulated by Na+ at pH 8.5. For the stimulation at the latter pH, the presence of both Na+ and K+ were essential. Contrary the H+, K+‐ATPase, the associated K+‐pNPPase was inhibited by Na+ at both pH values. Sodium competes with K+ for the K+‐pNPPase reaction. Also, unlike the H+, K+‐ATPase activity the ATPase‐mediated transport of H+ within the gastric microsomal vesicles was inhibited by Na+. For the latter event only the extravesicular and not the intravesicular Na+ was effective. The data suggest that the K+‐pNPPase activity does not represent the phosphatase step of the H+, K+‐ATPase reaction. In addition, the observed inhibition of vesicular H+ uptake by Na+ appears to be due to the displacement by Na+ of a cytosolic (extravesicular) H+ site responsible for the vectorial translocation of H+

Bilayer orientation of membrane-bound NH2 groups across microsomal vesicles and their role in the function of gastric K+-stimulated adenosine triphosphatase

The transversal distribution of the free NH2 groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K+-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH2 groups was studied after modification with the probes under various conditions and relating the inhibition of the K+-stimulated ATPase to the ATPase-dependent H+ accumulation by the gastric microsomal vesicles. TNBS (2 mm) inhibits nearly completely the K+-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K+. Both the K+-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 mm ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly (P < 0.05) reduced the labeling of protein-bound NH2 groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH2 groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH2 appeared to be associated with the K+-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH2 groups of the K+-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K+-stimulated ATPase and dye uptake.

Mechanism of action of gastric secretory inhibitors: Effects of SCN−, OCN−, NO2−, and NH4+ on (H+ + K+)-ATPase-mediated transport of H+ inside gastric microsomal vesicles☆

Abstract The mechanisms of action of the known inhibitors of gastric acid secretion such as SCN − , OCN − , NO 2 − , and NH 4 + ( M. E. LeFevre, E. J. Gohmann, Jr. and W. S. Rehm, 1964, Amer. J. Physiol. 207 , 613–618 ) were investigated using isolated pig gastric microsomal vesicles as a model system. The gastric microsomal vesicles enriched in (H + + K + )-ATPase have previously been demonstrated to accumulate H + in exchange for K + . The vesicular accumulation of acridine orange, which is a measure of H + uptake, shows sigmoidal kinetics in the presence of increasing K + with a Hill coefficient of 2.27 and a S 50 of 19.05 m m . None of those agents affects the microsomal (H + + K + )-ATPase activity, although they inhibit vesicular H + transport in a dose-dependent manner; the order of efficacy being NH 4 + > SCN − > OCN − > NO 2 − . The inhibitory effects of NH 4 + on vesicular H + transport appear to be due to neutralization of the transported H + by freely permeable NH 3 generated from the dissociation of NH 4 + in the bulk medium. SCN − , OCN − , and NO 2 − appear to work by a different mechanism. These agents do not act as protonophores. Our data demonstrate that the presence of SCN − , OCN − , and NO 2 − within the vesicle interior are essential for exerting their inhibitory effects. Furthermore, the inhibitory effects of SCN − and OCN − on vesicular H + transport could be reversed by an elevation of intravesicular K + . Our data strongly suggest that the effects of SCN − , OCN − , and NO 2 − are exerted by interfering with a low-affinity K + site (S 50 = 19.05 m m ) within the domain of the gastric ATPase complex. This low-affinity K + site is accessible only from the vesicle interior and appears to be essential for the vectorial transport of H + by the gastric microsomal (H + + K + )-ATPase system.

Control of the potassium ion-stimulated adenosine triphosphatase of pig gastric microsomes: effects of lipid environment and the endogenous activator.

Abstract The K + -stimulated ATPase activity associated with the purified gastric microsomes from the pig gastric mucosa can be completely inactivated by treatment with 15% ethanol for 60 s at 37 °C but not at 25 °C. Sequential exposure of the microsomes to 15% ethanol at 25 and 37 °C caused the release of 2.9 and 4.3% of the total membrane phospholipids, respectively, consisting entirely of phosphatidyl choline and phosphatidyl ethanolamine. The ethanol-treated (37 °C) membrane had high basal (with Mg 2+ as the only cation in the assay mixture) activity, which was further enhanced during reconstitution with phosphatidyl choline or phosphatidyl ethanolamine. The high basal activities could be reduced to the normal control level by assaying the enzyme in presence of the “activator protein,” partially purified from the soluble supernatant of the pig gastric cells. Phosphatidyl choline was somewhat more effective than phosphatidyl ethanolamine in the restoration of the activity of the ethanol-treated enzyme while phosphatidyl serine, phosphatidyl inositol, and sphingomyelin were without any effect. Synthetic phosphatidyl choline with various fatty acid substitutions were tested for their effectiveness in the restoration of the ethanol-inactivated enzyme. The distearoyl (18:0), dioleoyl (18:1), and dilinoleoyl (18:2) derivatives of phosphatidyl choline were almost equally effective while dipalmitoyl (16:0) phosphatidyl choline was somewhat less effective in the reconstitution process. Cholesterol appeared to interfere with phosphatidyl choline in the restoration of the activity of ethanol-treated enzyme. The fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine extracted by 15% ethanol at 37 °C was clearly different than those of the total microsome. Our data suggest that the phospholipids extracted by 15% ethanol at 37 °C are derived primarily from the immediate lipid environment of the enzyme and ATP together with Mg 2+ and K + help the partially delipidated enzyme to retain the appropriate conformation for the subsequent reconstitution. Furthermore, ethanol appears to either release or inactivate the membrane-associated activator protein, demonstrated to be essential for the K + -stimulated activity of the pig gastric ATPase.

Studies of gastric Ca2+-stimulated adenosine triphosphatase I. characterization and general properties

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in significant (2-3 fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmasking of latent Mg2+-dependent Ca2+-stimulation ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+, and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 microM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 x 10(-4) and 10(-7) M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.

Characterization of gastric mucosal membranes: II. Lipid compositions of purified gastric mitochondria from pig, rabbit and frog☆

1. The lipid composition of purified mitochondrial fractions from the fundic mucosa of pig, rabbit and frog were determined. 2. The total lipids expressed as mg lipid per 100 mg mitochondrial protein were approx. the same in pig and rabbit (13.4 and 15.5, respectively) and much higher than in frog (8.5). 3. The levels of phospholipids were about the same in pig and frog (approx. 61% of the total lipid) and lower than rabbit (78%). However, the levels of cholesterol were significantly different in the three species and constituted 22, 9 and 18.2% of the total lipids in pig, rabbit and frog mitochondria, respectively. 4. The glycolipid content in the mitochondrial lipids from pig, rabbit and frog were 7, 5.6 and 10.5%, respectively. 5. Cardiolipin contributed from 5.6 to 7.5% of the total phospholipids in the various species. Phosphatidylethanolamine and phosphatidylcholine together accounted for 80 90% of the total phospholipids in the various species; the contribution of phosphatidylcholine being always higher than that of phosphatidylethanolamine. Small but significant amounts of phosphatidylinositol were present in all species. 6. Generally, the predominant saturated fatty acid in the phospholipids was 16:0 from all species (except in phosphatidylethanolamine from pig and frog), and 18:1 and 18:2 were the predominant unsaturated fatty acids from all species. Sphingomyelin contained the highest amount of saturated fatty acids (over 80%) in both the species (pig and rabbit) studied.

Oral administration of spermine inhibits gastric acid secretion in rats

Administration of spermine by oral route inhibits the histamine-stimulated gastric acid secretion in pylorous ligated rats. The extent of both the acid and volume of secretion is dependent on the dose of sperimen. The polyamine is completely ineffective when injected by the intravenous route. A single oral dose of spermine suppresses acid secretion for at least 6 hr. An inverse relationship between the efficacy of oral spermine and age of the animals was observed. These data suggest a therapeutic potential for the naturally occurring polyamine compounds.

Purification and Characterization of a Cytosolic Activator Protein for the Gastric H+, K+-ATPase System from Dog Fundic Mucosa

An endogenous protein activator (AF) responsible for the activation of the gastric H+,K+-ATPase system, identified recently as the biochemical mechanism for the transport of H+, has been purified to homogeneity and partially characterized. The purification procedure (at 0-4 degrees C) involves simultaneous concentration and dialysis of the cytosolic fraction from dog fundic cells under negative pressure, pH 4.8 precipitation and two consecutive gel filtration steps on sephacryl S-200 columns. The highly purified and active AF is a protein of 80 Kd consisting of two identical subunits of 40 Kd each. The AF not only stimulates the gastric H+,K+-ATPase activity but also greatly enhances the rate of ATPase dependent proton pumping inside gastric microsomal vesicles. The data clearly suggest an important regulatory role of the cytosolic AF in the gastric HCl secretory process.

Effects of a Naturally Occurring Polyamine on Acid Secretion by Isolated Gastric Mucosa

Abstract Determination of the effects of spermine on acid secretion by isolated rabbit gastric mucosa shows paradoxical responses at neutral luminal pH. Initial inhibition of acid secretion was followed by a return to near basal rates. However, measurement of mucosal and serosal rates of CO2 release indicated that spermine causes prolonged inhibition of acid secretion. Similar prolonged inhibition is seen with mucosa exposed to an acidic luminal pH. The inhibitory effect of spermine is reversed by the addition of K+ to the mucosal side, suggesting spermine interferes with a K+ site at the secretory membrane. Serosal addition of spermine is without effect. The apparent acid secretory rebound phenomenon observed after the addition of spermine is most likely related to formation of H+ in the luminal bathing solution rather than proton secretion by the mucosa.

Trypsinization unmasks a Ca+2- stimulated AtPase activity from purified pig gastric microsomes

Abstract Trypsinization of gastric microsomal K+- stimulated ATPase in absence of ATP nearly abolished the K+- stimulated component of the enzyme activity without any significant effect on the basal (with Mg+2 alone) activity. The K+- stimulated component, however, was completely restored by the ‘activator protein” partially purified form the soluble supernatant fraction of the pig gastric cells. On the other hand, trypsinization of the microsomes in presence of ATP significantly increased (2–3 fold) the basal rate with virtual elimination of the K+- stimulated component. Assay of the trypsinized microsomes in presence of the activator protein not only demonstrated complete restoration of the K+- stimulated ATPase but also revealed an additional activity which has been characterized as a Ca+2- stimulated ATPase. Tryptic digestion has recently been used as a tool to understand the mechanism of action of various transport enzymes such as Na+, K+- ATPase (1), Ca+2- ATPase (2,3) and gastric H+, K+- ATPase (4). Controlled tryptic digestion of purified enzymes under various conditions of ligand binding may provide us with many valuable informations regarding the molecular architecture of the enzyme protein. However, when dealing with a membrane system containing a host of many different intrinsic and extrinsic proteins one must be cautious about the interpretation of the trypsin effects. In the present paper we report the effects of trypsin digestion of the purified pig gastric microsomes on the microsomal K+- stimulated ATPase activity. Our studies demonstrated that digestion of the microsomes with trypsin in absence of ATP inactivated the K+- stimulated ATPase but the activity could be fully restored by the addition of partially purified activator protein (5). Microsomes treated with trypsin in presence of ATP responded to the activator protein to the same extent as that without ATP but in addition demonstrated the manifestation of another enzymatic activity which has been characterized as a Ca+2- stimulated ATPase. This is a preliminary report dealing primarily with the unmasking of a new ATPase after trypsin treatment. Detailed reports on the characterization and mechanism of action of the gastric Ca+2- stimulated ATPase will be published elsewhere.