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Biomedical subjects

G Sachs

Publications and source records attributed to G Sachs.

At least 271 records · Page 15Linked to original sources
PubMed1982

ATP/ADP exchange activity of gastric (H+ +K+)-ATPase.

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.

Adenosine Diphosphate↗
PubMed1982

The gastric [H,K]ATPase:H+/ATP stoichiometry.

An H+/ATP ratio of 2 for H+ transport was determined from initial rate measurements at pH 6.1 in a purified gastric microsomal fraction containing the [H,K]ATPase. This ratio was independent of external KCl, though the apparent K0.5 for ATP was increased from 10.78 +/- 0.51 (n = 3) to 64.6 +/- 11.9 (n = 3) microM ATP and from 5.13 +/- 0.64 (n = 3) to 65.2 +/- 0.64 (n = 3) microM ATP for H+ transport and the K+-stimulated ATPase, respectively, as K+external was increased from 12 to 150 mM. The H+/ATP ratio was also relatively independent of ATP concentration. Maximum initial rates obtained in KCl-equilibrated vesicles were independent of added valinomycin, though net H+ transport was increased 29.3 +/- 1.03% (n = 6) by the addition of ionophore. Maximum net H+ transport in this vesicle preparation was 185 +/- 2.1 (n = 14) nmol mg-1 of protein. Initial rate measurements of ATPase represent a burst of K+-dependent activity of approximately 10-15 s duration. The H+/ATP stoichiometry was calculated based on the K+-stimulated component of hydrolysis. Under most conditions, the Mg2+-dependent component of hydrolysis was less than 10% of the (Mg2+ + K+) component of hydrolysis.

Adenosine Triphosphatases↗
PubMed1982

Ion pathways in renal brush border membranes.

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na+/H+ or Li+/H+ exchange was demonstrated by diluting Na2SO4 or Li2SO4 loaded vesicles into Na+-or Li+-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K+ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na+ conductance smaller than electroneutral Na+/H+ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na+ conductance greater than the H+ conductance was suggested. K+ gradients also induced changes of pH, at about 10% of the Na+ or Li+ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K+ was a low affinity substrate for the Na+/H+ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K+ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles has a low K+ conductance. Inward Cl- gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl- conductance greater than the H+ conductance and a Cl-/OH- exchange were present. The rate of Na+/H+ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent Km for Na+ or Li+ of about 10 mM and an EA of 14.3 kcal per mol. A 61-fold Na2SO4 gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower delta pH even in the presence of gramicidin showing that the H+ and Cl- pathways could alter the effect of the Na+/H+ exchange.

Acridine Orange↗
PubMed1982

Quantitation of corneal endothelial potentials using a carbocyanine dye.

The carbocyanine dye, diS-C3-(5) was used to quantitate the plasma membrane potential of the bullfrog corneal endothelium. It was shown that valinomycin hyperpolarized the endothelial cell and that in the presence of the ionophore the membrane potential largely reflected the K+ equilibrium potential. Using calibration curves constructed by changing medium K+ concentration in the presence of valinomycin, and nigericin and ouabain to abolish ion gradients and electrogenic pump activity, the cell membrane potential was calculated to be 28.6 +/- 4.2 mV. The major source of this potential was a K+ diffusion potential, and the membrane Na+ conductance reduced the cell potential to less than the apparent K+ equilibrium potential of 51.5 +/- 5.1 mV. About 20% of the cell potential could be ascribed to the rheogenic (Na+ + K+)-ATPase.

Animals↗
PubMed1982

Heterozygote detection of type I Gaucher disease using blood platelets.

This report describes a reliable and reproducible method for the identification of carriers of Type I Gaucher disease using blood platelets as the source of beta-glucosidase and 4-methylumbelliferyl-beta-D-glucoside as substrate. Platelet lysates have at least two identifiable beta-glucosidase activities with the synthetic substrate. One is maximally active at pH 5.0 in the absence of sodium taurocholate and the other at pH 5.6 in the presence of taurocholate. In platelets of Gaucher homozygotes and heterozygotes, the beta-glucosidase activity at pH 5.6 with the bile salt is reduced whereas the activity at pH 5.0 is the same in non-carriers, carriers and affected patients. In addition to differences in specific activity, the ratio of beta-hexosaminidase to beta-glucosidase activities is a useful parameter in the evaluation of the carrier state. Since carriers have normal activity of hexosaminidase and a reduced activity of beta-glucosidase, their mean activity ratio is about 70% higher than in non-carriers. Therefore we propose that the specific activity of beta-glucosidase at pH 5.6 in the presence of sodium taurocholate with the ratio of beta-hexosaminidase to beta-glucosidase serve as useful and reliable indices in the evaluation of the carrier state for Gaucher disease.

Adult↗
PubMed1982

Proton/hydroxyl transport in gastric and intestinal epithelia.

Proton transport across the plasma membrane of the gastrointestinal epithelium occurs by various pathways. There is the permeability of H+ across the lipid components of the membranes, probably of minor significance at physiological pH, but at the pH of the secretory surface of the parietal cell a factor that cannot be neglected. Transport of H+ dependent on the protein components of the plasma membrane involves various mechanisms. For example Na+ :H+ or Cl- :HCO-3 antiport (exchange) are generally electroneutral mechanisms (i.e., neither affected by potential gradients nor affecting membrane conductance) that are widely distributed throughout the body. Plasma membranes may contain proton or bicarbonate conductances (i.e., gradients of either ion may be determined by the potential across the membrane). This type of pathway is often of minor significance, hence the electrical component of hydrogen ion gradients across the plasma membrane can often be neglected. In the case of the gastric parietal cell, proton transport depends on the activity of a specific ATPase. This ATPase may be present elsewhere in the intestinal tract. This review will consider many of these proton pathways. In the case of brush border pathways, some of the data presented on Na+ :H+ antiport wil be derived from studies done on renal brush border rather than those of the small intestine, on the assumption that the properties of the antiporter are similar in the two tissues.

Adenosine Triphosphatases↗
PubMed1982

Glucose-induced proton uptake in secretory granules of beta-cells in monolayer culture.

The weak base acridine orange (AO) has been shown to be accumulated by the insulin-containing secretory granules of cultured beta-cells in response to high glucose. Various lines of evidence indicate that this accumulation is due to a pH gradient. Thus ionophores such as monensin and nigericin abolish the glucose-induced accumulation, and a high concentration of the weak base, benzylamine, results in swelling of the granules. In the absence of glucose, ATP addition to digitonin-permeabilized cells also results in dye uptake. These data also suggest that a primary or secondary active accumulation mechanism for hydrogen ions exists across the granule membrane.

Acridine Orange↗
PubMed1982

Pepsinogen release from isolated gastric glands.

The in vitro release of pepsinogen was studied using a preparation of isolated gastric glands from rabbits. The pepsinogen content of the glands was estimated to be about 700 U/mg dry wt. Spontaneous release of pepsinogen was found to be less than 1% of the total per hour and relatively constant for at least 2 h. Pepsinogen release was stimulated in a dose-dependent manner by both carbachol and isoproterenol. The cholinergic and beta-adrenergic stimulation was selectively inhibited by atropine and propranolol, respectively. Removal of external calcium inhibited the responses to both isoproterenol (partially) and carbachol (completely). Several agents, including histamine, prostaglandin (E2), and synthetic secretin, were found not to stimulate pepsinogen release. However, a crude secretin preparation (Boots) was found to produce significant stimulation. Dibutyryl cAMP increased pepsinogen release in a dose-dependent manner. Isoproterenol was found to increase the cAMP content of gastric glands and to stimulate adenylyl cyclase activity in homogenates. The beta-adrenergic stimulation of adenylyl cyclase was found to be selective for a population of gastric cells that was relatively depleted of parietal cells and distinct from the histamine-stimulated adenylyl cyclase activity. The results indicate that pepsinogen secretion by the gastric chief cell is regulated, in part, by separate cholinergic and beta-adrenergic mechanisms and that both calcium and cAMP play a role in this regulation.

Animals↗
PubMed1982

Interrelationships of ion transport in rat submaxillary duct epithelium.

The transport of Na+, K+, Cl-, and HCO3(-) across the epithelium of the rat submaxillary salivary duct is postulated to be due to the coupling of the basolateral Na+-K+-ATPase with various ion transport systems in the luminal and basolateral membranes. Na+ reabsorption depends on the presence of a rheogenic (Na+ conductance) and an electroneutral (Na+:H exchange) pathway, both of which are sensitive to amiloride. K+ secretion is postulated to be mediated by a K+: H+ antiport, coupling between Na+ reabsorption and K+ secretion, thus depending on local H+ ion concentration. The ratio between electroneutral Na+ influx and K+ efflux, therefore, determines the rate of HCO3(-) secretion. In the absence of Na+ influx, although K+ efflux falls, HCO3(-) secretion rises to a value equal to that of K+ secretion. The maintenance of K+ secretion in the absence of luminal Na+ requires an additional Na+-entry step across the basolateral membrane, also postulated to be due to Na+:H+ exchange.

Animals↗
PubMed1982

[Diet triggered depression as well as its treatment: a behaviorally oriented group therapy concept].

Preliminary results of a behavior-oriented therapeutic concept for adipose females, who can be called a risk group because of their psychic instability, are presented. 6 adipose females, who at the beginning of the therapy showed a partial endomorphic cyclothymic axial syndrome according to Berner, participated in group therapy. Half of the patients were additionally supported with antidepressants. The results show that those patients with additional medicamental treatment did profit far more from the therapeutic supply than did the others in all examined parameters: they were better motivated to stay with the group, became socially more competent, and lost more weight.

Adult↗
PubMed1981

Reactivity of gastric (H+ + K+)-ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.

The K+-dependent ATPase and p-nitrophenyl phosphatase activity of, and formation of phosphoenzyme by, hog parietal cell membranes were inhibited in a time- and concentration-dependent manner by the carboxyl-activating reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The kinetics of inactivation was pseudo first order and was similar to the EEDQ-catalyzed incorporation of [14C]glycine ethyl ester. The most likely mechanism is the EEDQ-dependent formation of inter- and intramolecular amide bonds. Cross-linking between the subunits of the ATPase occurs with EEDQ treatment. The presence of K+ on the luminal face of the enzyme is able to prevent EEDQ inhibition of K+ ATPase activity (but not intermolecular cross-linking), whereas ATP enhanced the rate of inactivation. EEDQ reaction with the enzyme therefore allows investigation of K+- and ATP-dependent states of the enzyme.

Adenosine Triphosphatases↗
PubMed1981

Target molecular weight of the gastric (H+ + K+)-ATPase functional and structural molecular size.

The state of assembly of the (H+ + K+)-ATPase in purified hog gastric mucosa membranes was studied by target size analysis applied to radiation-induced enzyme inactivation and polypeptide degradation data. Radiation inactivated the Mg2+-ATPase, K+-stimulated ATPase, and p-nitrophenyl phosphatase activities of the membrane preparation with a dose dependence characteristic of a target size of 270,000-daltons. Radiation also bleached the major 100,000-dalton sodium dodecyl sulfate-gel electrophoresis band of this preparation, indicating a radiation-induced degradation. This apparent polypeptide degradation exhibited a dose dependency corresponding to a target size of 250,000 daltons in situ. It is suggested that the gastric ATPase is a trimeric assembly of the 100,000-dalton polypeptides.

Adenosine Triphosphatases↗
PubMed1981

The interaction of H+ and K+ with the partial reactions of gastric (H+ + K+)-ATPase.

The influence of H+ and K+ on the partial reactions and transport of gastric (H+ + K+)-ATPase was studied. Using transient kinetics, the effects and sidedness of effects of H+ and K+ on formation and breakdown of phosphoenzyme were determined in intact and lyophilized reconstituted vesicles in the absence and presence of gramicidin. Whereas increasing H+ concentrations on the ATP-binding face of the vesicles accelerates phosphorylation, increasing K+ concentrations inhibits phosphorylation. Increasing H+ on this side reduces K+ inhibition of the phosphorylation rate. At low ATP/K+ ratios, the phosphorylation step can become rate-limiting for steady state hydrolysis. Decreasing H+ accelerates dephosphorylation in the absence of K+. K+ on the internal or luminal face of the vesicles accelerates dephosphorylation, and this rate is reduced with increasing H+ concentrations. At low internal pH, K+-dependent dephosphorylation may become rate-limiting. H+ transport measurements using fluorescence quenching of acridine orange show that whereas internal K+ is required for H+ transport, external K+ inhibits the rate of formation of a pH gradient, and the inhibition is reduced by decreasing medium pH. The pH optimum for ATPase activity and transport correlated in the vesicles, and the K0.5 of K+ for transport correlated with data for intact parietal cells.

Adenosine Triphosphatases↗
PubMed1981

Substituted benzimidazoles inhibit gastric acid secretion by blocking (H+ + K+)ATPase.

Studies both in vivo and in vitro have shown that substituted benzimidazoles inhibit the stimulation of acid secretion produced by dibutyryl cyclic AMP and histamine. Furthermore, the results differ from those produced by H2 antagonists and anticholinergic agents in that the inhibition is not competitive, and the site of action is intracellular and peripheral to that of dibutyryl cyclic AMP. To investigate the biochemical mechanism of action of substituted benzimidazoles, one such compound, H 149/94 (2-([2-(3-methyl)pyridyl-methyl]-sulphinyl)-5-methoxycarbonyl-6-methylbenzimidazol), has been tested either directly on an (H+ + K+)ATPase isolated from pig and human gastric mucosa or on the function of this enzyme in gastric glands isolated from rabbit and human gastric mucosa. (H+ + K+)ATPase, which has only been found at the secretory surface of the parietal cell, catalyses a one-to-one exchange of protons and potassium ions. It is possibly the proton pump within the gastric mucosa, and may thus be the terminal or one of the terminal steps of the acid secretory process. We show here that H 149/94 inhibits (H+ + K+)ATPase, which may explain its inhibitory action on acid secretion in vitro and in vivo. Because of the unique distribution and properties of the (H+ + K+)ATPase, the inhibitory action of H 149/94 on this enzyme may be a highly selective clinical means of suppressing the acid secretory process.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1981

Thallium interaction with the gastric (K, H)-ATPase.

The gastric (K, H)-ATPase has been shown to catalyze an electroneutral H+ for K+ exchange. Tl+ is able to substitute for K+ as an activating cation in the hydrolytic reaction with an apparent dissociation constant of 90 microM as compared to about 870 microM for K+. The ability of Tl+ to participate in transport is shown by the development of pH gradients in the presence of Tl+ following addition of ATP to gastric vesicles and by the ATP-dependent efflux of Tl+ from gastric vesicles. Inhibition of hydrolysis is observed at pH 7.4 with external Tl+ concentrations above 3.0 mM. This inhibition of hydrolysis is correlated with inhibition of pH-gradient formation. The inhibition of transport activity is partially relieved by a decrease in medium pH. This inhibitory effect is attributed to Tl+ binding at an external, low affinity cation site. In contrast to rubidium chloride, at high Tl+ concentrations, following the initial Tl+ efflux, there is reuptake of the cation. This rapid uptake is attributed to lipid-dependent Tl+ entry pathways. The vesicles exhibit a high permeability to thallium nitrate demonstrating a half-time (t1/2) for uptake of about 1.0 min in contrast to 46 min for rubidium chloride. In both gastric vesicles or liposomes, external Tl+ concentrations in excess of 1 to 4 mM are able to dissipate intravesicular proton gradients. Thus, although Tl+ is able to activate the gastric ATPase by mimicking K+, the permeability of this cation in lipid bilayers tends to uncouple H+ transport at concentrations high enough to generate detectable proton gradients.

Adenosine Triphosphatases↗
PubMed1981

Amino Acid Transport and stimulation by substrates in the absence of a Na2+ electrochemical potential gradient.

Uptake of alpha-aminoisobutyric acid (AIB) was examined in Ehrlich ascites tumor cells treated with the cation-exchange ionophore nigericin (20 microgram/ml). Membrane voltages were measured using the voltage-sensitive dye diethyloxadicarbocyanine (DOCC). In normal phosphate-buffered media, nigericin changed the distribution ratios of Na+ and K+ (the ratio of intra- to extracellular concentrations) nearly to unity, but AIB was still accumulated to a distribution ratio of approximately 9.0. When all but 40 mM Na+ in the medium was replaced by choline, nigericin resulted in K+ loss and Na+ gain and both cation distribution ratios approached 2.8-3.4, as would be expected if both ions were distributing near electrochemical equilibrium with a membrane voltage in the range of -28 to -33 mV. This conclusion was supported by the observation that the addition of 5 X 10(-7) M valinomycin to the nigericin-treated cell suspension produced no change in DOCC absorbance. In spite of the apparent zero electrochemical potential gradients for Na+ and K+, AIB was accumulated to a distribution ratio of 5.4 in the low-Na+ medium. Addition of 0.1 mM oubain or 50 microM vanadate did not alter the extent of AIB accumulation as would have been expected if a large component of the membrane voltage were due to electrogenic operation of the (Na+ + K+)-ATPase. Addition of lactate, pyruvate or glucose increased the AIB distribution ratios to 11.9, 9.4 and 15.3, respectively. The effect of glucose could be explained, at least in part, by an enhanced Na+ electrochemical potential gradient. However, neither lactate nor pyruvate produced any change either in membrane voltage or the intracellular Na+ concentration. Therefore, these results confirm the existence of a metabolic energy source which is coupled to AIB accumulation and operates in addition to the Na+ co-transport mechanism, and which is augmented by metabolic substrates such as lactate and pyruvate.

Amino Acids↗