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J G Forte

Publications and source records attributed to J G Forte.

At least 73 records · Page 4Linked to original sources
PubMed1987

Protein phosphorylation associated with stimulation of rabbit gastric glands.

Changes in protein phosphorylation associated with stimulation of acid secretion were investigated using isolated rabbit gastric glands labeled with 32P. The glands were stimulated by 100 microM histamine plus either 10 microM forskolin or 50 microM isobutylmethylxanthine, homogenized, and fractionated into a series of pellets: 40 X g, 5 min; 4000 X g, 10 min; 14,500 X g, 10 min; 48,200 X g, 90 min (microsomes); and supernatant. Stimulation induced a redistribution of H+/K+-transporting ATPase among the membrane fractions, i.e., a reduction in activity of the microsomal fraction, and a compensatory increase in the 4000 X g fraction. Further subfractionation of the 4000 X g pellet by Ficoll density gradient produced an 18% Ficoll layer, greatly enriched in the H+/K+-ATPase, and which is thought to be rich in apical membranes of parietal cells. SDS-polyacrylamide gel electrophoresis showed that the amount of 94 kDa peptide (the molecular size of the H+/K+-ATPase) was increased in the 18% Ficoll layer and decreased in the microsomal fraction by stimulation. Analysis of autoradiograms of the gels revealed that apparent changes in level of phosphorylation occurred in the 120, 94 and 80 kDa regions of the 18% Ficoll layer, and in the 94 kDa region of the microsomal fraction. The phosphorylation changes in the 94 kDa region may not reflect changes in specific activity of a single peptide but may be due to the heterogeneity of proteins in this region, which was demonstrated by selective heat treatment of the samples as well as two-dimensional electrophoresis. Phosphorylation of 120 kDa protein in the 18% Ficoll layer was clearly increased by stimulation, and this appeared to be associated with protein distribution changes as well as phosphorylation. The 80 kDa protein in the 18% Ficoll layer showed marked increased phosphorylation by stimulation, with little change in protein distribution. This 80 kDa protein was focused on two-dimensional gels as several sequential spots, with the most radioactive peptide focused toward the acidic side; thus, we propose isomeric forms of an 80 kDa protein with sequential phosphorylation sites. The phosphorylation changes observed in this study are considered to be important to the process of gastric acid secretion because they occurred in the putative apical membrane fractions in which biochemical and functional changes with stimulation have been demonstrated.

Adenosine Triphosphatases↗
PubMed1987

Stimulation-associated redistribution of H+-K+-ATPase activity in isolated gastric glands.

The objective of this work is to establish a procedure to study the stimulation-dependent membrane redistribution and properties of H+-K+-ATPase in an in vitro model system, rabbit isolated gastric glands. Stimulated (10(-4) M histamine plus 10(-5) M forskolin) and resting (10(-4) M metiamide) glands were homogenized and fractionated into PO (40 g, 5 min), P1 (400 g, 10 min), P2 (14,500 g, 10 min), P3 (48,200 g, 90 min), and supernatant, S3. Significant changes occurred in the distribution of our marker for H+-K+-ATPase (K+-p-nitrophenyl phosphatase) activity: a reduction in activity of P3 and a compensatory increment in P1. P3 showed valinomycin (Val)-dependent vesicular H+ uptake, while H+ uptake in P1 was Val independent. Direct measurements of ATPase revealed that H+-K+-ATPase activity of P3 was Val dependent and decreased by stimulation; H+-K+-ATPase activity of P1 was Val independent and increased by stimulation. Further density gradient purification of P1 showed that membranes lighter than 17% Ficoll contained higher specific H+-K+-ATPase activity, and the observed increase in H+-K+-ATPase associated with stimulation was more pronounced. Also, the lighter fractions from stimulated P1 had much latent H+-K+-ATPase activity that was unmasked by n-octylglucoside. The properties of membrane fractions from isolated glands were consistent with results obtained in vivo: high H+-K+-ATPase activity of P3 from resting glands corresponds to cytoplasmic tubulovesicles lacking KCl transport pathways; high activity of P1 from stimulated glands corresponds to apical plasma membrane vesicles containing KCl transport in addition to the H+-K+-ATPase, and full competency for the generation of HCl.

Adenosine Triphosphatases↗
PubMed1987

Mechanisms of active Cl- secretion by frog gastric mucosa.

Net Cl- flux across the bullfrog gastric mucosa was examined to test the hypothesis that Cl-secretion (JClnet) can be driven by either of the two cation exchange pumps in the oxyntic cell. The effects on JClnet of ouabain, an Na+-K+ pump inhibitor, and omeprazole, an H+-K+ pump inhibitor were examined. Omeprazole abolished acid secretion (JH) and reduced JClnet in bullfrog gastric mucosa. For mucosae at open circuit the omeprazole-induced decrease in JH was not significantly different than the decrease in JClnet, and the transmucosal potential difference (PD) was increased. When short-circuited mucosae were treated with omeprazole, the decrease in JClnet was significantly less than the decrease in JH, and short-circuit current (SCC) was correspondingly increased. After treatment of short-circuited mucosae with ouabain, the omeprazole-induced decreases in JH and JClnet were not significantly different, and no change in SCC occurred. For open-circuited mucosae, pretreatment with ouabain resulted in a significantly smaller omeprazole-induced increase in the transmucosal PD than was seen without ouabain pretreatment. Our data 1) show that both the H+-K+ pump and the Na+-K+ pump can drive Cl- secretion and 2) suggest that inhibition of the H+-K+ pump with omeprazole stimulates the Na+-K+ pump.

Adenosine Triphosphatases↗
PubMed1986

Effect of ethanol on acid secretion by isolated gastric glands from rabbit.

Isolated gastric glands from rabbit, as well as basolateral and microsomal membranes derived therefrom, were used to examine the effect of ethanol on several parameters related to acid secretion. Low concentrations of ethanol, 0.2%-5% (vol/vol), had no effect on basal aminopyrine accumulation by isolated gastric glands but significantly potentiated aminopyrine accumulation stimulated by histamine. In contrast, this dose range of ethanol inhibited aminopyrine accumulation stimulated by forskolin or dibutyryl-cyclic adenosine monophosphate. This dose range of ethanol produced a similar effect on adenylate cyclase activity of basolateral membranes from isolated gastric glands, with potentiation of histamine stimulation and inhibition of forskolin stimulation. Low-dose ethanol was found to produce increased proton permeability of the apical membrane of the parietal cell but had no effect on hydrogen-potassium-stimulated adenosine triphosphatase activity. Ethanol (10%) significantly inhibited all parameters of acid secretion studied. Ethanol has a biphasic effect on acid secretion with potentiation of histamine-stimulated aminopyrine accumulation and adenylate cyclase activity at low doses and inhibition of all parameters of acid secretion at high doses.

Adenosine Triphosphatases↗
PubMed1986

Mechanism of inhibition of gastric acid secretion by SCN-: interrelation of SCN- flux and inhibition.

Effects of thiocyanate (SCN-) and imidazole on acid secretion and Cl- flux across the isolated bullfrog gastric mucosa have been examined. For open-circuited mucosae, with Cl- Ringer on the nutrient side and 110 mM Na isethionate on the secretory side, the addition of 15 mM SCN- to either the secretory or nutrient side gave equivalent reductions in acid secretion (JH) and nutrient-to-secretory Cl- flux (JnsCl) . JnsSCN, the nutrient-to-secretory flux of SCN- (0.2 mu eq . cm-2 . h-1), was much less than the decrease in JH (1.88 mu eq . cm-2 . h-1). Addition of imidazole, phosphate, or histidine to the secretory side reversed SCN- inhibition of JnsCl. Addition of imidazole to the nutrient side gave equivalent restorations of JnsCl and JH. The increase in JnsSCN, 0.07 mu eq . cm-2 . h-1, was much less than the increase in either JH, 0.77, or JnsCl, 0.86 mu eq . cm-2 . h-1. Similar results were obtained for mucosae bathed with NaCl on the secretory side. With 15 mM SCN- on both sides, the flux ratio for Cl- was significantly larger (1.0) than the flux ratio for SCN- (0.47). The addition of nutrient imidazole increased the flux ratios for Cl- and SCN- to 1.29 and 1.16, respectively. Both SCN- and Cl- showed exchange diffusion. Inhibition of acid secretion with nutrient SCN- at short circuit also inhibited JnsCl but did not alter the partial conductance of Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Active K+ absorption by the gastric mucosa: inhibition by omeprazole.

Unidirectional fluxes of K+ across the histamine-stimulated frog gastric mucosa bathed in Cl(-)-Ringer were measured. Comparison of nutrient-to-secretory and secretory-to-nutrient K+ fluxes determined using either 42K or 86Rb as radioisotopic tracers gave identical results, and thus either isotope could be used for K+ flux measurements; the majority of these experiments used 86Rb as the tracer. Short-circuited mucosas maintained a small net absorption of 0.07 +/- 0.03 mueq X cm-2 X h-1 (n = 9). Omeprazole decreased acid secretion from 4.98 +/- 0.58 to 0.08 +/- 0.02 mueq X cm-2 X h-1 and reversed the direction of net K+ flux giving a net secretion of 0.09 +/- 0.04 mueq X cm-2 X h-1. Ouabain increased the rate of K+ absorption to 0.32 +/- 0.08 mueq X cm-2 X h-1 with a flux ratio (JKn----s/JKs----n) of 0.31 +/- 0.09; subsequent addition of omeprazole significantly decreased the rate of K+ absorption and increased the flux ratio. Omeprazole did not alter the fraction of transmucosal current carried by either K+ or Cl-. For omeprazole inhibited-mucosas at open circuit increasing the secretory osmolarity by 100 mosM with sucrose decreased the transmucosal resistance by 14% and increased nutrient-to-secretory K+ flux by 31%. For actively secreting mucosas no significant changes were seen in either parameter. These results are shown to be consistent with an electroneutral apical H+-K+ exchange pump and diffusion from the gland lumen-to-the secretory fluid, being rate limiting for K+ flux.

Animals↗
PubMed1985

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.

Adenosine Triphosphatases↗
PubMed1985

Inhibition of the gastric (H+ + K+)-ATPase by fenoctimine.

The effects of fenoctimine, an inhibitor of gastric acid secretion, on the microsomal (H+ + K+)-ATPase were studied. In the micromolar concentration range, fenoctimine inhibited hydrolysis of ATP and p-nitrophenyl phosphate by the (H+ + K+)-ATPase. Inhibition was reversible and noncompetitive with substrate. The apparent Ki was dependent on the concentration of membranes, being increased by added liposomes or high microsomal membrane concentrations. Over the concentration range that (H+ + K+)-ATPase was inhibited, fenoctimine increased the turbidity of microsomal suspensions. The effects of fenoctimine were not specific for the gastric (H+ + K+)-ATPase, since the hydrolytic activities of the (Na+ + K+)-ATPase and mitochondrial ATPase were also inhibited by the drug. These results suggest that inhibition of hydrolysis may not be the direct result of an interaction between the (H+ + K+)-ATPase and fenoctimine but the secondary effect of a fenoctimine-induced perturbation of the microsomal membrane.

Adenosine Triphosphatases↗
PubMed1985

K+ and Cl- conductances in the apical membrane from secreting oxyntic cells are concurrently inhibited by divalent cations.

This study concerns the properties of rapid K+ and Cl transport pathways that are present in the (H+ + K+)-ATPase membrane from stimulated, and secreting, gastric oxyntic cells. Ion permeabilities in the isolated stimulation-associated vesicles were monitored via the rates of H+ efflux under conditions of exclusive H+/K+ counterflux or H+ - Cl co-efflux, as well as by comparison of equilibration rates for 86Rb and 36Cl under conditions of equilibrium exchange and unidirectional salt flux. These latter studies suggest that Rb+ and Cl pathways are conductive and independent. In spite of the functional independence of the ion pathways, several divalent cations inhibit Rb+ and Cl isotopic exchange as well as the H+ efflux that is dependent on either K+ or anion (Cl, SCN, NO2) fluxes. Zn2+ is the more potent inhibitor, reducing by 50% the sensitive component of K+, Cl, and NO2 fluxes at about 20 microM; Mn2+ has a similar effect at 200 microM. Ni2+ and Co2+ were roughly equipotent to Mn2+ while Mg2+ and Ca2+ had no inhibitory effect. These results suggest that the stimulation-induced permeabilities, while functioning independently, may be physically linked, i.e., residing within a single entity. In similar studies carried out in (H+ + K+)-ATPase vesicles obtained from nonstimulated cells, no vestiges of sensitivity to the inhibitory divalent cations could be detected. The implications of these findings for the physiology of the oxyntic cell in the context of a model for membrane fusion are discussed.

Animals↗
PubMed1984

Stimulation of oxyntic cell triggers K+ and Cl- conductances in apical H+-K+-ATPase membrane.

Vesicles isolated from the apical membrane of stimulated oxyntic cells [stimulation-associated (SA) vesicles] are highly permeable to KCl. The KCl flux is coupled to an electroneutral ATP-driven H+-K+ exchange (the H+-K+-ATPase) to produce net intravesicular HCl accumulation. In the past, we observed that rates of KCl transport were not accelerated by valinomycin and that dissipation of preformed H+ gradients in the presence of a protonophore (carbonyl cyanide, m-chlorophenylhydrazone, 10 microM) required the simultaneous presence of valinomycin. Consequently the fast KCl transport was attributed to an electroneutral cotransport system. Now we have been able to elicit fast H+ gradient dissipation in the absence of valinomycin by using the protonophore tetrachlorosalicylanilide. Experiments carried out in the absence of Cl- demonstrated the existence of a specific high-conductance pathway for K+. Experiments in K+-free medium demonstrated the existence of a high Cl- conductance. Parallel experiments in the equivalent H+-K+-ATPase-rich vesicles from nonsecreting oxyntic cells showed very little K+ and Cl- conductivity, suggesting that the appearance of large ionic conductance in the membrane is associated with the stimulation of the cell.

Adenosine Triphosphatases↗
PubMed1983

Actin and associated proteins in gastric epithelial cells.

A quantitative assessment of the distribution and state of microfilament-related proteins in the heterocellular fundic gastric epithelium was carried out. Actin content, as determined by the DNAase inhibition assay, ranged from 29 to 42 micrograms/mg of tissue protein, depending upon the tissue source. About 60% of the total actin existed in fresh tissue in the polymeric form (F-actin). The distribution of fluorescent-labelled phallicidin demonstrated that F-actin was concentrated predominantly in the acid-secreting oxyntic cells. The patterns of distribution corresponded to the location of the numerous elongated apical surface microvilli seen within oxyntic cell canaliculi. In the isolated apical membrane, actin represented about 10% of the total protein and was present entirely as F-actin. After mild treatment of membranes with Triton X-100, filaments could be readily visualized by negative staining. More extensive Triton X-100 extraction solubilized intrinsic membrane protein and yielded an insoluble residue highly enriched in actin and containing several additional polypeptides. Homogenization and fractionation of the gastric epithelium in low ionic strength media led to the depolymerization of a significant proportion of the tissue actin which was recovered in the homogenate supernatant. When purified by DNAase affinity chromatography, this gastric actin displayed structural and functional properties similar to muscle actin. Incubation of the homogenate supernatant in KCl-Mg2+ induced the formation of actin-rich gels. The gels contained myosin as well as several other peptides that may be actin-binding proteins.

Actins↗
PubMed1983

Anion exchange in oxyntic cell apical membrane: relationship to thiocyanate inhibition of acid secretion.

The effects of SCN- on H+-accumulation by inside-out gastric vesicles derived from the apical membrane of secreting oxyntic cells are reported. SCN- inhibited the formation of pH gradients in Cl- and isethionate media. In Cl-, the concentration of SCN- required to achieve a certain degree of inhibition of H+ uptake (or dissipation of performed gradients) was increased with the increase in Cl- concentration, indicating some competitive phenomena between these anions. Comparison of the rates of dissipation of similar pH gradients achieved in Cl- vs. isethionate suggested the existence of a fast Cl-/SCN- exchange. In addition, direct isotopic fluxes confirmed the existence of rapid anion exchange and K-salt transport for both Cl- and SCN-. The rates of anion-exchange and K-salt transport were of similar magnitude, and the rates for SCN- in either countertransport against Cl- or cotransport with K+ were twice as fast as the equivalent values for Cl-. These mediated pathways in the apical membrane provide the possible means for rapid access of SCN- to the acidic canalicular spaces of the oxyntic cell that is implicit in recent proposals to explain SCN- inhibition of gastric HCl secretion.

Adenosine Triphosphatases↗
PubMed1983

Correlation of parietal cell structure and function.

The apical surface of the gastric parietal cell is greatly expanded (5-10-fold) during maximal HCl secretion, as compared to the resting cell. The membrane recycling hypothesis has been proposed to account for the extensive, functionally related, rearrangement of cell membranes. Cytoplasmic membranes within the resting cell, the tubulovesicles, contain the H+/K+-ATPase. Fusion of tubulovesicles with the apical plasma membrane occurs when the cells are stimulated, thus providing the increased surface area and proper disposition of the H+ pump enzyme. Microfilaments, composed of actin and other regulatory proteins, serve to direct the reordering of the apical surface during stages of the secretory cycle. Cell fractionation of resting oxyntic mucosa reveals that virtually all of the H+/K+-ATPase activity is associated with light microsomal membrane vesicles, presumably derived from tubulovesicles. Although the enzyme from resting tissue is fully competent (e.g. ATP-driving pump, H+-K+ exchange), the microsomal vesicles lack an endogenous pathway to provide rapid access for K+ to its intravesicular activity site. In stimulated stomach, there is a redistribution of H+/K+-ATPase to a larger, denser membrane fraction, the so-called stimulation-associated vesicles. Morphological features and chemical content (e.g. microfilament proteins) suggest that the stimulation-associated vesicles are derived from the expanded apical surface of the stimulated oxyntic cell. A KCl cotransport system has been identified in the stimulation-associated membranes, which operates in parallel with the ATP-driven H+-K+ exchange pump. These two transport systems operate in concert within the apical membrane to provide the machinery for net HCl transport by the parietal cell.

Actins↗
PubMed1983

Action of thiocyanate on pH gradient formation by gastric microsomal vesicles.

The effects of thiocyanate anion (SCN-) on proton accumulation and ATP hydrolysis by hog gastric microsomal vesicles have been investigated. The addition of SCN- to vesicles in the presence of KC1 and valinomycin reduced ATP-dependent proton accumulation in a dose-dependent manner. Inhibition was most pronounced in the presence of internal SCN-, which was obtained by preincubation of vesicles with SCN- or by the addition of K+-valinomycin, which facilitates entry of SCN-. SCN- does not appear to act by inhibition of the vesicular H+ pump because 1) there were minimal effects of SCN- on the rate of ATP hydrolysis, and 2) the initial rate of pH gradient formation was greater with 20 mM SCN- than with 20 mM Cl-. In the presence of K+ and valinomycin, external SCN- inhibited ATP-dependent pH gradient formation by increasing the rate of proton efflux. Preformed pH gradients (acid interior) were rapidly dissipated by internal, but not external, SCN-. These results suggest that SCN- acts to increase the rate of passive proton loss from the vesicle interior and do not support a direct inhibition of ATP-dependent H+ translocation. The results are consistent with the formation of the permeant hydrothiocyanic acid within the vesicles, increasing the rate of proton loss. The data also lend support to the HSCN backflux hypothesis of Sanders et al. [Am. J. Physiol. 234 (Endocrinol. Metab. Gastrointest. Physiol. 3): E120-E128, 1978] for inhibition of gastric acid secretion by SCN-.

Adenosine Triphosphate↗
PubMed1983

Muscarinic receptors and guanylate cyclase in mammalian gastric glandular cells.

To investigate the involvement of guanosine 3',5'-cyclic monophosphate (cGMP) in the cholinergic activation of gastric acid and pepsinogen secretion, we studied the subcellular and cellular relation between particulate guanylate cyclase and muscarinic cholinergic receptor sites. Subcellular fractionation of homogenates from rabbit gastric glands showed that particulate guanylate cyclase and muscarinic receptors were distributed in similar patterns, which differed from the pattern found for Na+-K+-ATPase, a marker for basal-lateral plasma membranes. Assuming a basal-lateral membrane localization for particulate guanylate cyclase and cholinergic receptors, these results suggested a heterogeneity of glandular basal-lateral membranes. The distributions of these markers among fractions enriched in isolated canine parietal or chief cells were also followed. Na+-K+-ATPase correlated with parietal cell distribution (r = 0.86) and guanylate cyclase with chief cell distribution (r = 0.76). The distribution of quinuclidinyl benzilate (QNB) binding sites indicated association of muscarinic receptors with both cell types. The similar subcellular and cellular distributions of guanylate cyclase and QNB binding sites may reflect a functional relationship of these markers in muscarinic-activated pepsinogen secretion. As seen in most other tissues, gastric glandular guanylate cyclase was not stimulated by various gastric secretagogues. We found that small changes in Ca2+ concentration, within the micromolar range, can regulate glandular guanylate cyclase activity. These results are discussed in terms of the cholinergic activation of parietal and chief cell function.

Adenosine Triphosphatases↗
PubMed1982

The effects of microfilament disrupting agents on HCl secretion and ultrastructure of piglet gastric oxyntic cells.

A functionally responsive in vitro preparation of piglet gastric mucosa was used to investigate the involvement of microfilaments in the process of HCl secretion by oxyntic cells. A well-ordered array of microfilaments was observed in the short, stubby microvilli on the apical surface of nonsecreting oxyntic cells, as well as ii the longer microvilli of actively secreting cells. Treatment with cytochalasin B (10(-5)-10(-4) M) caused a dose-dependent inhibition of acid secretion and a concomitant gradient of morphologic alteration of oxyntic cells. Associated with slight (approximately 20%) inhibition of secretion was an initial collapse of the canalicular and glandular lumina and appearance of some pleomorphic-shaped microvilli. Maximum inhibition of secretion always produced a complete collapse of the oxyntic cell canalicular and glandular lumina, with a resultant apposition of apical surfaces. Microvilli were no longer readily distinguishable, and microfilaments were severely disorganized. Treatment with cytochalasin B (2-4 X 10(-5) M) before secretagogue stimulation also reduced the ability of the gastric mucosa to secrete acid; oxyntic cells retained the general appearance of nonsecreting cells. The correlation of disruption of microfilaments and the inhibition of acid secretion by cytochalasin B suggests an involvement of microfilaments in both the initiation and maintenance of high levels of acid secretion.

Animals↗