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Biomedical subjects

G Sachs

Publications and source records attributed to G Sachs.

At least 253 records · Page 14Linked to original sources
PubMed1984

Regulation of gastric acid secretion via modulation of a chloride conductance.

When gastric microsomes were purified from resting and stimulated rabbit mucosae, they were found to be generally similar in (H+ + K+)-ATPase activity, peptide composition in single-dimension sodium dodecyl sulfate-gel electrophoresis, and in size. In the stimulated vesicles, optimal proton transport activity was found at pH 7.4, 20-50 mM KCl, and 1 mM ATP-Mg. However, in the case of resting vesicles, the presence of valinomycin and an inward Cl-gradient was also necessary for Mg-ATP-dependent proton transport. Measurement of K+ and Cl-diffusion potentials using 3,3-dipropylthiadicarboxocyanine iodide as a potential sensitive dye showed that both resting and stimulated vesicles developed K+ gradient-dependent potentials in the presence of an impermeant anion, but that Cl- gradient-dependent potentials were observed only in the stimulated preparation. 86Rb+ self-exchange was found in both types of vesicles, but Cl- self-exchange was confined to vesicles derived from stimulated mucosae. Putative inhibitors of anion conductance such as furosemide and anthracene 9-carboxylic acid blocked proton transport, Cl- conductance, 36Cl- uptake, and Cl- exchange. The inhibition of proton transport was overcome by valinomycin. ATPase activity in the presence of nigericin, an H+:K+ exchanger, was unaffected by these inhibitors. K+ conductance, Rb+ uptake, and Rb+ exchange were insensitive to these inhibitors. Thus, activation of acid secretion by the stimulated parietal cell appears to involve at least the appearance of a discrete Cl- conductance in the pump-associated membrane.

Adenosine Triphosphatases↗
PubMed1984

Changes in cytosolic free Ca2+ in isolated parietal cells. Differential effects of secretagogues.

The relationship of free cytosolic Ca2+ to secretagogue-dependent activation of acid secretion by the mammalian parietal cell was studied using quin 2 as an intracellular Ca2+ probe. The resting [Ca2+]in of isolated dog parietal cells was found to be 134 +/- 11 nM. Carbachol produced a steady-state increase of [Ca2+]in and its effect was blocked by atropine and Ca2+ -channel blocking agents. Gastrin transiently elevated [Ca2+]in and this was not affected by Ca2+ -channel blocking agents. Neither histamine nor dbcAMP changed resting [Ca2+]in in rabbit parietal cells.

Aminoquinolines↗
PubMed1984

Isolation of pepsinogen granules from rabbit gastric mucosa.

Pepsinogen granules were isolated from rabbit stomachs by a combination of differential and isopycnic gradient centrifugation. The isolation procedure utilized 1 M sucrose and alkaline pH to stabilize the granules. The isolated granules were shown to be 8.4-fold enriched in pepsinogen and free of mitochondria and microsome enzyme markers. In addition to pepsinogen, a cation-insensitive but anion-sensitive Mg2+-ATPase co-purified with the zymogen. The enzyme was unaffected by aurovertin, oligomycin, and ouabain, but inhibited by high concentrations of vanadate, N,N'-dicyclohexylcarbodiimide, and azide. The enzyme activity was stimulated by tetrachlorosalicylanilide and the combination of valinomycin and nigericin in K+-containing media. The similarities between this enzyme and other secretory granule ATPases are discussed.

Adenosine Triphosphatases↗
PubMed1984

Inhibition of (H+ + K+)-ATPase and H+ accumulation in hog gastric membranes by trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate.

The mechanism of gastric antisecretory action for trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) has been studied utilizing isolated hog gastric membranes enriched with (H+ + K+)-ATPase. The drugs inhibited the gastric ATPase due to their apparent competition with K+ for the luminal high-affinity K+-site of the ATPase. The dose to inhibit 50% (ID50) of the ATPase in the membranes rendered freely permeable to K+ (20 mM) was 50 microM for trifluoperazine and 1.5 mM for verapamil and TMB-8. In intact hog gastric membranes which develop a pH gradient in the presence of valinomycin, ATP and KCl, however, trifluoperazine at 4 microM, verapamil and TMB-8 at 15 microM inhibited 40 and 30% of the valinomycin-stimulated ATPase activity, respectively, and also blocked the ionophore-dependent intravesicular acidification as measured by aminopyrine accumulation. The enhanced potency of the drugs to inhibit the ATPase in the intact membrane vesicles may be attributed to the accumulation of the drugs as a weak base within the vesicles, where the luminal K+-site of the ATPase is accessible. Calmodulin and Ca2+ had no effect on the extent of H+-accumulation as measured by aminopyrine accumulation in the membrane vesicles which were prepared in the presence of 1 mM EGTA. Since the drugs showed similar potency in interfering with H+ movements either in the membrane vesicles or isolated rabbit gastric glands stimulated by dibutyryl cAMP, it is reasonable to suggest the inhibitory effect of the drugs on (H+ + K+)-ATPase as a primary cause for such interferences in both cases. A trifluoperazine analog and other lipophilic amine drugs similarly inhibited (H+ + K+)-ATPase and H+ accumulation in the membrane vesicles or in the glands. We have concluded that a tertiary amine, the only common functional group among these drugs, is primarily responsible for their ability to interact with the high-affinity K+ site of the gastric ATPase.

Adenosine Triphosphatases↗
PubMed1984

Cellular mechanisms of acid secretion.

Current ideas about the parietal cell are presented in the model shown in Figure 6; the cell is illustrated both in resting and stimulated states. The basal-lateral surface contains three major receptor classes mediated by either cyclic AMP responses or Ca2+ changes, Ca2+ channels and a Ca2+ pump, and a variety of ion transport pathways such as Na+:H+ and Cl-:HCO-3 exchange, a K+ conductance, a postulated NaKCl2 cotransport and the (Na+ + K+)ATPase. At rest, the tubulovesicles contain the (H+ + K+)ATPase. In the stimulated state, the apical (secretory canalicular) membrane contains a K+ and Cl- conductance as well as the (H+ + K+)ATPase. There is also a change in cytoskeletal arrangement associated with stimulation.

Animals↗
PubMed1983

Vanadate binding to the gastric H,K-ATPase and inhibition of the enzyme's catalytic and transport activities.

Vanadate inhibition of the catalytic and transport activities of the gastric magnesium-dependent, hydrogen ion transporting, and potassium-stimulated adenosinetriphosphatase (EC 3.6.1.3) (H,K-ATPase) has been studied. The principal experiment observations are the following: (1) Inhibition of adenosine 5'-triphosphate (ATP) hydrolysis is biphasic. Vanadate binding with a stoichiometry of 1.5 nmol mg-1 approximately halves K+-stimulated ATPase activity at physiological temperature. The remaining activity is inhibited by binding an additional 1.5 nmol mg-1 vanadate with lower apparent ions bind specifically to gastric vesicles with two affinities. Vanadate binding in the presence of nucleotide is compatible with competition for the kinetically defined high-affinity and low-affinity ATP sites. (3) Vanadate inhibits phosphoenzyme formation and the K+-stimulated p-nitrophenyl phosphatase activity of the enzyme monophasically. A maximum of 1.5 nmol mg-1 acid-stable phosphoenzyme is formed. The half-time for vanadate dissociation from the site that inhibits p-nitrophenyl phosphate hydrolysis is 5 min (4) At most, 3 nmol mg-1 vanadate is required to inhibit proton transport. The simplest interpretation of the data is that vanadate inhibits the H,K-ATPase by binding competitively with ATP at two catalytic sites. Different catalytic mechanisms at the high-affinity and low-affinity sites are suggested by the different stoichiometries found for vanadate binding and phosphoenzyme formation.

Adenosine Triphosphatases↗
PubMed1983

Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase.

Fluorescein isothiocyanate was used to covalently label the gastric (H+ + K+)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated p-nitrophenylphosphatase activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of pI 5.6 was also noted. Fluorescence was quenched by K+, Rb+ and Tl+ in a dose-dependent manner, and the K0.5 values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg2+-dependent fluorescent quench. Ca2+ reversed the K+-dependent loss of fluorescence and inhibited it when added prior to K+. This may relate to the slow phosphorylation in the presence of ATP found when Ca2+ was substituted for Mg2+ and the absence of K+-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K+ binding to the enzyme.

Adenosine Triphosphatases↗
PubMed1983

Inhibition of gastric (H+ + K+)-ATPase by the substituted benzimidazole, picoprazole.

The substituted benzimidazole, picoprazole, inhibited the gastric (H+ + K+)-ATPase in a concentration-and time-dependent manner. Half-maximal inhibition of the (H+ + K+)-ATPase activity was obtained at about 2 . 10(-6)M under standard conditions. In addition to the inhibition of ATPase activity, parallel inhibition of phosphoenzyme formation and the proton transport activity were achieved. Radiolabelled picoprazole was found to bind to 100 kDa peptide; this peptide was shown by phosphorylation experiments to contain the catalytic centre of the (H+ + K+)-ATPase. Studies on the (Na+ + K+)-ATPase indicated that this enzyme was unaffected by picoprazole. From the data presented and from other pharmacological studies, it is proposed that this compound inhibits acid secretion at the level of the parietal cell by its ability to inhibit the gastric proton pump, the (H+ + K+)-ATPase.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1983

Changes of serum hexosaminidase for the presumptive diagnosis of type I Gaucher disease in Tay-Sachs carrier screening.

Although reduced acid beta-glucosidase activity appears to be the primary enzyme defect in type I Gaucher disease, patients with this disorder also have marked elevation of serum acid phosphatase and beta-hexosaminidase activities but with a normal level of lactic dehydrogenase activity. Moreover, there is a characteristic alteration in the hexosaminidase isozyme distribution with a striking increase in hexosaminidase B. Since these changes appear to be consistent and unlike those associated with other disorders or the hormonally induced alterations associated with pregnancy, routine serum testing for the Tay-Sachs carrier state may offer a useful approach for the presumptive diagnosis and screening for Gaucher disease. Unlike the changes in affected homozygotes, there are no characteristic alterations of acid phosphatase or hexosaminidase in heterozygotes for Gaucher disease.

Acid Phosphatase↗
PubMed1983

Cl- requirement of acid secretion in isolated gastric glands.

The dependence of acid formation, as measured by aminopyrine (AP) accumulation, on medium and intracellular Cl- was investigated in resting (10(-4) M cimetidine) and stimulated (10(-3) M dibutyryl cAMP) gastric glands isolated from the rabbit stomach. Intracellular Cl- concentrations (Cli-) were measured by the steady-state distribution of 36Cl-. In Cl- -free conditions AP accumulation was absent. Medium Cl- induced AP accumulation (AP ratio = 125) in stimulated glands with a K0.5 of 10.4 +/- 1.1 mM, equivalent to 18.0 +/- 1.2 mM Cli-, and had a small effect on resting glands (AP ratio = 6). With normal Nai+ and Ki+ maintained, similar results were obtained in stimulated glands treated with 10(-5) M amphotericin (ampho) and 10(-4) M ouabain (ouab), where the basal-lateral membrane was confirmed to be short-circuited with respect to Cl- pathways, i.e., medium Cl- and Cli- were equal. The K0.5 for Cl-i was 17.5 +/- 2.5 mM. In resting glands treated with ampho and ouab, AP accumulation increased linearly (no saturation was observed) with increasing Cl- (AP ratio = 35). These results suggest that stimulation activates a Cl- component in the secretory membrane and not in the basal-lateral membrane of the parietal cell. The K+ requirement of AP accumulation at a physiological Cl-i of 60 mM was also investigated in ampho- and ouab-treated glands. On stimulation the K0.5 for Ki+ decreased from 19.5 to 12 mM, coupled with a large increase in AP accumulation, indicating that stimulation also activates a K+ component in the secretory membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1983

Monoclonal antibodies against gastric H+ + K+ ATPase.

Monoclonal antibodies were prepared against a purified membrane fraction from hog gastric mucosa containing the H+ + K+ ATPase. On sodium dodecyl sulfate gels the molecular weight of this fraction corresponds to a single band of about 95,000. In contrast, on isoelectric focusing gels three groups of peptides are resolved with isoelectric points of 5.7, 6.2, and 8.5. One of the monoclonal antibodies (HK111) was shown to react selectively with the acidic peptide, whereas another antibody (HK113) reacted with the alkaline peptide, showing that the three peptides were antigenically distinct. Both monoclonal antibodies selectively labeled the parietal cell, and antibody HK111 labeled the tubulovesicles of the resting parietal cell and the microvilli of the secretory canaliculus of the secreting cell. This finding suggests translocation of membrane from the tubulovesicles to the secretory surface on stimulation.

Adenosine Triphosphatases↗
PubMed1983

Proton secretion by the gastric parietal cell.

The parietal cell occupies a unique niche among eukaryotic cells in that it develops a proton gradient of more than 4 million-fold across the membrane of the secretory canaliculus. At rest, the cell is still able to develop a proton gradient across intracellular membranes, such that the acid compartment has a pH of less than 4. Acidification depends on the simultaneous presence of ATP, K+ and Cl- as demonstrated in permeabilized cells. With acidification of the luminal side of the proton pump, there is a corresponding alkalinization of the cytosolic face as revealed by carboxyfluorescein fluorescence enhancement. Disposal of the resultant alkali depends on carbonic anhydrase activity and the functioning of a coupled Na+:H+ and Cl-:OH-antiport across the basal lateral membrane. Accordingly, with secretion there is an increased cellular Cl- level, which is exported across the apical membrane in association with K+. The Na+ pump dependent secretion of KCl across this membrane is one of the major sites of the gastric ATPase. Membranes isolated from secreting tissue contain a KCl permeation pathway largely absent from membranes isolated from resting tissue. The pump itself acts as an H+ for K+ exchange ATPase which is most probably composed of at least two peptides of 100 000 Mr. That catalytic cycle consists of formation and breakdown of a covalent aspartyl phosphate. Formation of the intermediate depends on loss of K+ from cytosolic binding sites, and breakdown of the intermediate depends on K+ binding to the luminal face of the enzyme. During breakdown, an acid labile E . P is formed, and, at high ATP concentrations, loss of this form of the enzyme is probably the rate limiting step.

Adenosine Triphosphatases↗
PubMed1983

Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis.

The tumorigenic roles of specific types of cells that emerge during chemical hepatocarcinogenesis in rats could potentially be determined if the specific types of cells could be purified adequately. Type II cells, defined (J. M. Jacobs, T. P. Pretlow, N. Fausto, A. M. Pitts, and T. G. Pretlow, J. Natl. Cancer Inst., 66: 967-973, 1981) as small, slowly sedimenting cells with histochemically demonstrable gamma-glutamyl transpeptidase, have oval nuclei similar in size to those of lymphocytes. Adult male F344 rats were fed a choline-deficient diet containing 0.05% ethionine for 4 weeks. Liver cells were obtained in suspension by in situ perfusion of collagenase. A two-step process, i.e., sedimentation in an isokinetic gradient followed by free-flow electrophoresis, was used to purify type II cells. We obtained preparations of cells with 32.3 +/- 5.0% (S.D.) type II cells, 13.4 +/- 2.0% erythrocytes, 52.9 +/- 3.3% small nucleated cells, and only 1.4 +/- 0.3% hepatocytes. The small nucleated cells were Kupffer cells and cells smaller than Kupffer cells that included many lymphocytes and unidentified cells similar in size to lymphocytes. Because both the electrophoretic mobilities and the rates of sedimentation of type II cells and hepatocytes are different, there is some advantage to be obtained from the sequential use of these techniques that exploit independent differences in the physical properties of these cells.

Acyltransferases↗
PubMed1982

Identification of gastric cyclic AMP binding proteins.

The photoaffinity ligand 8-azidoadenosine 3',5-monophosphate was employed to label cAMP binding proteins in both fractionated and freeze-thawed rabbit gastric glands. Fractionated glands incorporated the azido-cAMP label primarily into two cytosolic proteins with apparent molecular weights of 58 000 and 48 000. No enrichment of label was found in fractions containing basolateral or apical membranes. DEAE-cellulose chromatography of the cytosolic proteins resulted in the separation of two cAMP-dependent protein kinase peaks. Azido cAMP labelling of each peak suggested the initial peak contained type I cAMP-dependent protein kinase while the second peak contained the type II kinase. Labelling of 'resting' gastric glands resulted in radioactive proteins of apparent molecular weights of 58 000 and 48 000. When gastric glands were stimulated to produce acid by the addition of 10(-4) M histamine or 1 mM dibutyryl cAMP there was 32-44% dimunition of ligand incorporation compared to control glands. The results strongly suggest that histamine-mediated stimulus-secretion coupling in gastric glands involves activation of parietal cell cAMP-dependent protein kinases.

1-Methyl-3-isobutylxanthine↗