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J G Forte

Publications and source records attributed to J G Forte.

At least 91 records · Page 5Linked to original sources
PubMed1981

Proton gradients in renal cortex brush-border membrane vesicles. Demonstration of a rheogenic proton flux with acridine orange.

The fluorescence quenching of acridine orange has been used to study the formation and dissipation of acid interior pH gradients in brush-border membrane vesicles from rabbit renal cortex. Acidic interior pH gradients were produced by 1) outwardly directed gradients of Na+ or K+, and 2) the addition of vesicles equilibrated at pH 6.0 to 7.5 buffer. The rate of pH gradient dissipation was stimulated 6.3-fold by the replacement of tetramethylammonium gluconate by tetramethylammonium chloride. A further increase, of 2-fold, was seen upon the addition of carbonyl cyanide-m-chlorophenylhydrazine, demonstrating the existence of a Cl- conductance pathway. In the presence of valinomycin, the replacement of tetramethylammonium gluconate by K gluconate increased the rate of delta pH dissipation by 11-fold, demonstrating the existence of a conductive pathway for protons. This pathway for protons was also shown by the formation of an acidic interior space by an outwardly directed K gradient in the presence of valinomycin. The parallel conductive pathways for H+ and Cl- may dissipate pH and chloride gradients across the luminal membrane of the proximal tubule.

Acridine Orange↗
PubMed1981

Specific modification of gastric K+-stimulated ATPase activity by thimerosal.

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K+-ATPase and the K+-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K+-stimulated ATPase, while K+-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K+-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K+-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K+-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; Km for p-nitrophenylphosphate unchanged for Ka for K+ increased. ATP, which is a potent inhibitor of K+-p-nitrophenylphosphatase activity in native membranes (KI approximately 200 microM). These data suggest that there are multiple SH groups which differentially influence the gastric K+-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K+-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H+-transport reaction.

4-Nitrophenylphosphatase↗
PubMed1981

An enriched preparation of basal-lateral plasma membranes from gastric glandular cells.

A procedure is described for the preparation of a membrane fraction enriched in basal-lateral plasma membranes from gastric mucosa. Gastric glands isolated from rabbit were employed as starting material, greatly reducing contamination from non-glandular cell types. The distribution of cellular components during the fractionation procedure was monitored with specific marker enzymes. (Na+ + K+)-ATPase, ouabain-sensitive K+-stimulated p-nitrophenyl-phosphatase and histamine-stimulated adenylate cyclase were used as markers for basal-lateral membranes. These three markers were similarly distributed during both differential and equilibrium density gradient centrifugation. The enriched membrane fraction contained more than 30% of the total initial activities of the three basal-lateral membrane markers which were purified better than 11-fold with respect to protein. (Na+ + K+)-ATPase activity was resolved from the activities of acid phosphatase, pepsin, Mg2+-ATPase, cytochrome c oxidase, NADPH-cytochrome c reductase, glucose-6-phosphatase, (K+ + H+)-ATPase, DNA and RNA.

Animals↗
PubMed1981

Changes in the membrane environment of the (K+ + H+)-ATPase following stimulation of the gastric oxyntic cell.

Biochemical evidence is presented for changes in the membrane environment of the (K+ + H+)-dependent ATPase enzyme of the oxyntic cell following in vivo gastric stimulation of young New Zealand rabbits. The changes are inferred from the marked differences in the sedimentation properties of the (K+ + H+)-ATPase when obtained from homogenates of either stimulated or nonstimulated (resting) fundic gastric epithelium. Stimulation resulted in a redistribution of K+-ATPase activity that was reduced to less than half in the microsomal pellet and concomitantly increased in the membrane fractions normally associated with nuclei and mitochondria. Density gradient fractionation of the mitochondrial pellet yield a preparation rich in (K+ + H+)-ATPase. Our studies indicated that the membranes in this preparation are far larger and apparently denser than the microsomal vesicles associated with the nonstimulated state of the cell. The specific nature of the relationship between stimulation and the observed changes is suggested by the lack of change in the distribution of enzymatic activities unrelated to the apical pole of the oxyntic cell. Preliminary, tentative information aimed at identifying the processes responsible for the observed changes is presented.

Adenosine Triphosphatases↗
PubMed1981

H+/ATP stoichiometry for the gastric (K+ + H+)-ATPase.

The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150 mM KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639 +/- 84 and 619 +/- 65 nmol/min x mg protein, respectively, giving an H+/ATP ratio of 1.03 +/- .07. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (less than 10 nmol/min x mg protein) was exhibited by vesicles in 159 mM NaCl at pH 6.1 despite ATP hydrolysis of 187 +/- 46 nmol/min x mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H+/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K+ greater than 10 mM. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H+/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K+ + H+)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.

Adenosine Triphosphatases↗
PubMed1981

Ultrastructural changes related to functional activity in gastric oxyntic cells.

When stimulated to secrete HCl the gastric oxyntic cell undergoes profound morphological change. The identifiable apical cell surface is greatly expanded in the stimulated oxyntic cell as compared with nonsecreting ones. To account for this change, one hypothesis proposes that the expanded surface is derived from the fusion of cytoplasmic tubulovesicular membranes with the existing limited apical membrane surface. An alternative hypothesis suggests that the tubulovesicular compartment is actually confluent with the apical surface at all times and that the morphological appearance follows the expansion of this supercollapsed compartment as HCl secretion commences. A variety of morphological evidence is reviewed here including transmission electron microscopy during various stages of secretion and inhibition, analysis of freeze-fracture replicas, penetration of macromolecular tracers, and membrane surface-staining characteristics. It is concluded that the weight of evidence favors a membrane fusion process. Moreover, recent comparative studies of membrane fractions from resting and secreting stomachs show different morphological and functional properties that are also consistent with a fusion hypothesis as a fundamental event in the membrane transformation of the oxyntic cell.

Animals↗
PubMed1981

Inhibition of HCl secretion and the effects on ultrastructure and electrical resistance in isolated piglet gastric mucosa.

Rates of H+ secretion and transepithelial resistance were measured for gastric mucosas from neonatal piglets before and after the addition of histamine and with the deployment of conditions that inhibit HCl secretion. At designated times tissue biopsy specimens were taken and prepared for electron microscopy. Oxyntic cell morphology was evaluated by classification of the cells into one of four categories: resting, partially stimulated, stimulated, or returning. Morphologic profiles for each experimental condition were thus established. In order to assess this method of quantitation, detailed morphometric analyses of oxyntic cells from resting and stimulated preparations were undertaken. The observed differences in tubulovesicular and microvillar surface densities were similar to those of previous investigators and correlated well with the morphologic profiles for these conditions. Actively secreting gastric preparations subjected to 15 mM NaSCN, substrate deprivation, or removal of histamine showed an inhibition of HCl secretion coupled with an increase in tissue resistance. However, only the morphologic profile of cells from the latter condition was different from that observed for control stimulated cells; histamine removal caused cells to be predominantly of a resting or returning morphology. Treatment of resting tissue with SCN- or removal of substrate inhibited subsequent histamine-induced secretion though tissue resistance decreased somewhat (approximately 15%). The morphologic profile of cells from these conditions was similar to that of control stimulated cells, despite the lack of acid secretion. These data support the hypothesis that oxyntic cell morphology does not absolutely dictate the secretory state of the tissue and that the resistance of gastric mucosa cannot be accounted for solely by the ultrastructure (i.e., surface area) of oxyntic cells.

Animals↗
PubMed1980

A novel method for measurement of intravesicular pH using fluorescent probes.

A method for the measurement of intravesicular pH of phospholipid vesicles and gastric microsomes is described. The present method makes use of the well characterized pH-dependent shift of the emission maximum of two fluorescent amines, quinine and acridine. As the probes distribute into the intravesicular space, according to the existing pH gradient, they respond to the acidic environment and emit fluorescence at a longer wavelength than the external probes which sense the more alkaline medium. By measuring both the decrease in the alkaline fluorescence peak and the enhancement of the acidic peak, direct determination of the internal pH can be obtained. This method has the advantages of giving a positive signal (enhancement of fluorescence instead of quenching), measuring intravesicular pH directly without the need of independent measurement of the volume of the H+ space, and also enabling the possible use of a much lower probe concentration than that required by the fluorescence-quenching method. The accuracy of the present method was quantitatively verified using phospholipid vesicles as a well defined model system. Application to biological systems was demonstrated using gastric microsomes which actively transport H+ into the microsomal space via an H+-K+ exchange ATPase system. The reasoning of the present method is also extended to the monitoring of internal alkaline pH gradient by using a fluorescent weak acid, o-hydroxycinnamic acid. Some general criteria for the future search for better Ph gradient probes are presented.

Animals↗
PubMed1980

Chemical modification of gastric microsomal potassium-stimulated ATPase.

Selective chemical modification was used to examine amino acid residues that might be critical for the operation of the gastric K+-stimulated ATPase. Modification of amino groups with the fluorigenic reagent 2-methoxy-2,4-diphenyl-3-dihydrofuranone resulted in selective inhibition of the K+-stimulated ATPase and H+-transporting activities of the gastric microsomes, while the Mg2+-atpase was not affected. Half-maximal inhibition occurred at about 3 microgram 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent Km for ATP protection was about 50 microM. Nucleotide selectivity for protection was ATP greater than ADP greater than ITP greater than GTP greater than CTP greater than AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the Mr 100 000 peptide band, a very small peptide, and aminolipids. In the presence of ATP there was about 75% reduction in the fluorescent label on the Mr 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg2+-ATPase and K+-ATPase activities, with the former being much less reactive. Similar to 2-methoxy-2,4-diphenyl-3-dihydrofuranone, ATP provided complete protection from butanedione treatment. It is concluded that amino and guanidino groups are critical to the function of the K+-ATPase and may be actually at the ATP binding site.

Animals↗
PubMed1980

Structure of oxyntic cell membranes during conditions of rest and secretion of HCl as revealed by freeze-fracture.

The density and distribution of membrane associated particles of piglet oxyntic cell tubulovesicular and apical surface membranes were investigated during resting (nonsecreting) and secreting conditions. For the resting oxyntic cell, the abundant tubulovesicles showed a highly asymmetrical distribution of particles between fracture faces, with the P face heavily studded by particles and the E face particle deficient. The apical surface, however, had a relatively symmetrical distribution of particles on both membrane fracture faces. In contrast to the resting state, the apical surface of the stimulated oxyntic cell showed a marked asymmetry of membrane particles; the P face had a high density of particles, while there was a scarcity of particles on the E face. The observed changes in apical surface membrane particle distribution support the hypothesis that, following the initiation of acid secretion, the tubulovesicles fuse with and become an integral part of the apical surface. Thus, the apical membrane P face of the stimulated cell is enriched and the E face is diluted by the incorporation of tubulovesicular membranes.

Animals↗
PubMed1980

Substrate dependency for HCl secretion by isolated piglet gastric mucosa.

Gastric mucosa was isolated from newborn piglets and bathed with balanced salt solutions. In the presence of glucose (ca. 0.01 M), this gastric preparation has been shown to be responsive to histamine by relatively prompt and vigorous H+ secretory rates. Secretion is dependent on glucose concentration in the serosal bathing solution, showing saturation kinetics with an apparent Km of about 2 mM glucose. Acetate and pyruvate were about as effective as glucose in sustaining H+ secretory rates. Short-chain fatty acids supported secretory rates that were significantly lower than rates measured with glucose. The order of effectiveness was butyrate greater than valerate greater than hexanoate greater than propionate. The results show absolute dependence of H+ secretion by piglet gastric mucosa on exogenous substrate and the preferential utilization of carbohydrate sources as substrates for secretion. They suggest that it is unlikely for any specialized and essential involvement of fatty acids in the primary H+ secretory mechanism as had been previously proposed.

Acetates↗
PubMed1979

Asymmetric labeling of amino lipids in liposomes.

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H+ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin-K+ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents. When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching. These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surface, monitor vesicular fusion, and assess liposomal structure.

Amines↗
PubMed1979

Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles.

The Mg2+-dependent, K+-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H+ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth. Valinomycin stimulated both dye uptake and K+-ATPase (valinomycin-stimulated K+-ATPase); stimulation by valinomycin was due to increased K+ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K+-ATPase and H+ accumulation as an index, the sequence for anion permeation was NO-3 greater than Br- greater than Cl- greater than I- greater than acetate approximately isethionate. When permeability to both K+ and H+ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K+-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an 'uncoupling' of ATP hydrolysis from H+ accumulation. Thiocyanate interacts with valinomycin inhibiting the typical action of the K+ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN- to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K+-ATPase, it appears that SCN- is a very permeant anion which can be placed before NO-3 in the sequence of permeation. Valinomycin-stimulated ATPase and H+ uptake showed similar dependent correlations, including: dependence on [ATP] and [K+], pH optima, temperature activation, and selective inhibition by SH- or NH2-group reagents. These results are consistent with a pump-leak model for the gastric microsomal K+-ATPase which was simulated using Nernst-Planck conditions for passive pathways and simple kinetics for the pump. The pump is a K+/H+ exchange pump requiring K+ at an internal site. Rate of K+ entry would depend on permeability to K+ as well as the counterion, either (1) the anion to accompany K+ or (2) the H+ efflux path as an exchange ion. The former leads to net accumulation of H+ and anion, while the latter results in non-productive stimulation of ATP hydrolysis.

Adenosine Triphosphatases↗