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Biomedical subjects

G Sachs

Publications and source records attributed to G Sachs.

At least 217 records · Page 12Linked to original sources
PubMed1988

Inhibitory effects of cations on the gastric H+, K+ -ATPase. A potential-sensitive step in the K+ limb of the pump cycle.

The presence of a cation inhibitory site on the dephosphoform of the H+, K+ -ATPase was confirmed by comparing the effects of K+ and NH4+ on overall activity and on phosphorylation and dephosphorylation. Inhibition of ATPase activity was pronounced at high cation/ATP ratios, but NH4+ was much less effective. At 60 mM cation, although the ATPase activity was greater in the presence of NH4+ (17.1 mumol/mg.h) as compared to K+ (5.1 mumol/mg.h), dephosphorylation of preformed phosphoenzyme was faster with K+ (2101 min-1) than with NH4+ (1401 min-1). Increasing K+ concentrations at the cytosolic face of the enzyme, at constant ATP, decreased the rate of phosphorylation from 1343 to 360 min-1 at 25 mM K+. Increasing ATP concentrations in the presence of constant K+ concentrations accelerated ATPase activity and increased the steady-state phosphoenzyme level. Therefore, inhibition by cations was due to cation stabilization of a dephospho form of the enzyme at a cytosolically accessible cation-binding site. ATP promoted cation dissociation from this site. In ion-permeable vesicles, increasing K+ concentrations, at constant ATP, activated and then inhibited ATPase activity, with a K0.5(I) of 22 mM. In intact, ion-impermeable inside-out vesicles, in the presence of valinomycin, ATPase activity increased up to 175 mM K+. Collapse of this potential by the addition of the electrogenic protonophore 3,3',4', 5-tetrachlorosalicylanilide restored the K+ inhibition of ATPase activity. Thus, the cation inhibition of the ATPase activity appears to be voltage-sensitive; and hence, its connection to the voltage sensitivity of acid secretion demonstrated in intact gastric mucosa is discussed.

Adenosine Triphosphatases↗
PubMed1988

Inactivation of H+,K+-ATPase by a K+-competitive photoaffinity inhibitor.

A light-sensitive derivative, 2,3-dimethyl-8-[(4-azidophenyl)methoxy]imidazo[1,2-a]pyridine (DAZIP), of the drug 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine (SCH 28080) has been synthesized and shown to be a K+-competitive inhibitor of gastric H+,K+-ATPase in the dark. The apparent dissociation constants calculated for DAZIP at pH 6.4 and 7.4 were 1.8 +/- 0.2 and 4.7 +/- 1.2 microM, respectively. Inhibition required binding of DAZIP to a luminal-facing site on the enzyme. Irradiation in the presence of DAZIP and 2 mM Mg2+ resulted in irreversible loss of ATPase activity that was more than 2-fold greater at pH 6.4 than at pH 7.4, showing the enhanced efficiency of covalent incorporation at the lower pH. Further photolyses were conducted at pH 6.4 in the presence of either 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), ATP and CDTA, or MgATP. The specificity of light-dependent, covalent insertion of DAZIP for the site of reversible inhibition was shown both by protection against photoinactivation given by K+ (the competing ligand) and by the observation that the amount of K+-protectable photoinactivation approached a maximum limiting value as a function of DAZIP concentration. The effectiveness of K+ in protecting against photoinactivation was 100-fold greater in the presence of ATP and CDTA than in the presence of either Mg2+ or CDTA and suggests the formation of a ternary complex of the apoenzyme with ATP and tightly bound K+. The dissociation constant for DAZIP (2 microM) calculated from photolyses in the presence of MgATP without added K+ agreed with the kinetic experiments and suggests that DAZIP inhibits turnover by binding to E.MgATP.

Adenosine Triphosphatases↗
PubMed1988

Limitations on psychiatrists' discretionary civil commitment authority by the Stone and dangerousness criteria.

The dangerousness-oriented commitment criteria of Massachusetts were compared with the paternalistic Stone model of commitment in an emergency setting. In a sample of 1062 patients, the Stone criteria were more restrictive than the dangerousness-oriented criteria; 91 patients were committable by the Stone model compared with 218 under the present statute. A second comparison of only those patients who refused treatment yielded similar findings--52 patients were committable according to the Stone criteria compared with 80 under the Massachusetts statute. The greater restrictiveness of the Stone criteria in each comparison was in large part the result of the requirement that the treatment decisions of the patient be incompetent; in the broader sample, the criterion of major patient distress had a comparable impact. A comparison of patients committable under both standards with those patients currently committable who would be excluded under the Stone standard revealed significant differences. The dually committable patients were more likely to be psychotic, to meet statutory criteria for grave disability, and to be diagnosed as manic. The implications of the findings are discussed.

Adult↗
PubMed1988

Passive and active transport in the parietal cell.

1. Models are presented for (a) HK ATPase acting in the presence of K and Cl conductances; (b) a pH regulatory system where Na/H exchange is regulated directly by second messenger and the anion exchanger is activated secondarily to the rise in cell pH; (c) vesicle fusion and K and Cl conductances activation in the gastric parietal cell. 2. It is suggested that H transport involves protonation and deprotonation of histidine groups as well as the motion of these groups relative to the membrane barrier. 3. The HK ATPase would have a voltage generating and voltage sensitive step in the forward direction. 4. Given net electroneutrality the K transport reaction would also be charge translocating and voltage sensitive.

Animals↗
PubMed1988

Gastric H+-K+-ATPase in situ: relation to secretory state.

Isolated gastric glands from rabbit were used to measure the gastric H+-K+-adenosinetriphosphatase (ATPase) and its partial reaction, a K+ p-nitrophenyl phosphatase (pNPPase), in situ. Measurement of the enzyme activities required permeabilization of the cells with digitonin and the use of several ATPase inhibitors to reduce nonspecific activity. The enzyme activities were identified as the H+-K+-ATPase according to the following criteria: dependence on K+, association with parietal cells, insensitivity to ouabain and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and inhibition by the specific inhibitors omeprazole and Sch 28080. K+-stimulated ATPase, but not K+-pNPPase, was enhanced by K+ ionophores, valinomycin and nigericin, with nigericin resulting in the greatest activity. Comparison of tissues that were preincubated to establish resting and stimulated states showed that prestimulation results in an increase in K+-stimulated ATPase activity with no change in the total activity. With the use of a two-step assay procedure, it could be shown that stimulation also results in an increase in the omeprazole-sensitive maximal ATPase activity. These results indicate that the major effects of stimulation are to enhance KCl activation of the enzyme and to increase the number of active enzyme molecules.

4-Nitrophenylphosphatase↗
PubMed1988

Monoclonal antibodies to the H+-K+ ATPase of gastric mucosa selectively stain the non-pigmented cells of the rabbit ciliary body epithelium.

Monoclonal antibodies developed against the transport enzyme H+-K+ ATPase of gastric mucosa selectively bind to the nonpigmented cell layer in both the pars plana and pars plicata of the rabbit ciliary body epithelium. The most specific of these antibodies (HK 12.18) reacts with the nonpigmented cells but with no other cell type in ciliary body, retina or iris. Antibody reactivity is evinced by a distinct particulate staining which is greater on the surfaces of the nonpigmented cells than within the cytoplasm and seems most intense on the apical membrane between the pigmented and nonpigmented cell layers. This pattern of staining was present in 2-3-day-old pups and in young adults, in both pigmented and albino animals. Similar staining was not observed in control sections treated with preimmune serum, nonspecific mouse monoclonal antibody, or HK 12.18 preabsorbed to gastric microsomes. In addition to staining intact ciliary body, the antibody HK 12.18 can be shown to react with a population of cells lacking pigment granules in suspensions of dissociated ciliary body epithelium. This antibody may therefore be useful as a marker for the identification of nonpigmented cells in tissue culture.

Animals↗
PubMed1987

The lysosomal H+ pump: 8-azido-ATP inhibition and the role of chloride in H+ transport.

Lysosomes (tritosomes) were purified from the livers of rats injected with Triton WR 1339. The lysosomes developed an Mg2+-ATP-dependent pH gradient as measured by Acridine orange accumulation. H+ transport was supported by chloride, but not sulfate, and was independent of the cation used. H+ transport and Mg2+-stimulated ATPase was inhibited by diethylstilbesterol (K0.5 = 2 microM). N-Ethylmaleimide inhibited H+ transport (K0.5 = 30 microM). At low concentrations of N-ethylmaleimide, ATP partially protected H+ transport from inhibition with N-ethylmaleimide. Photolysis with 8-azido-ATP inhibited H+ transport and Mg2+-stimulated ATPase activity. Under these same conditions, 8-azido-[alpha-32P]ATP reacted with a number of polypeptides of the intact lysosome and lysosomal membranes. Pump-dependent potentials were measured using the fluorescent potential-sensitive dye, DiSC3(5) (3,3'-dipropylthiocarbocyanine) and ATP-dependent potential generation was inhibited by diethylstilbesterol. Chloride, but not sulfate reduced the magnitude of the ATP-dependent membrane potential, as measured using merocyanine 540. The chloride conductance, independent of ATP, was of sufficient magnitude to generate a H+ gradient driven by external chloride in the presence of tetrachlorosalicylanilide. In Cl- free media, ATP-dependent H+ transport was restored to control levels by outwardly directed K+ gradients in the presence of valinomycin. The role of cell Cl- is to provide the necessary conductance for supporting lysosomal acidification by the electrogenic proton pump.

Acridine Orange↗
PubMed1987

Gastric antisecretory activity of cycloheximide due to inhibition of protein synthesis.

Treatment of rats with cycloheximide 1 h before carbachol dose-dependently reduced the secretagogue-stimulated gastric acid secretion in pylorus ligated rats, and partially blocked carbachol- or histamine-induced activation of rat gastric (H+ + K+)-ATPase which includes translocation of reserve intracellular (H+ + K+)-ATPase into the apical membrane of the parietal cells and induction of a KCl pathway. Time-course studies showed that the drug was effective only when administered at least 30 min before the secretagogues. Puromycin showed the same effect as cycloheximide. Pulse labelling studies with [35S]methionine led to identification of two most actively synthesized polypeptides in rat gastric mucosa; the proteins of 38,000 and 14,000 molecular weight. The larger polypeptide was identified as rat pepsinogen. The identity of the smaller protein is not known yet. We suggest that synthesis of nascent polypeptide(s) is required for certain steps of the acid secretory process leading to the activation of the acid pump.

Adenosine Triphosphatases↗
PubMed1987

Manoalide, a natural sesterterpenoid that inhibits calcium channels.

Manoalide is a marine natural product that has anti-inflammatory and anti-proliferative activities and is an irreversible inhibitor of phospholipase A2 and phospholipase C. It is now shown that the compound is a potent inhibitor of Ca2+ mobilization in several cell types. In A431 cells the increase in epidermal growth factor receptor-mediated Ca2+ entry and release from intracellular Ca2+ stores were blocked by manoalide in a time-dependent manner with an IC50 of 0.4 microM. The effect of manoalide on phosphoinositide metabolism, namely the production of inositol monophosphate, did not coincide with its effect on the epidermal growth factor response. In GH# cells, manoalide blocked the thyrotropin-releasing hormone-dependent release of Ca2+ from intracellular stores without inhibition of the formation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate. Manoalide also blocked the K+ depolarization-activated Ca2+ channel in these cells as well as the activation of the channel by Bay K8644 with an IC50 of 1 microM. In addition, manoalide also inhibited the Ca2+ influx induced by concanavalin A in mouse spleen cells in a time- and temperature-sensitive manner with an IC50 of 0.07 microM. However, neither forskolin-activated adenylate cyclase in A431 cells nor the distribution of the potential sensitive dye, 3,3'-dipropylthiodicarbocyanide iodide in GH3 cells was affected by manoalide. Thus, manoalide acts as a Ca2+ channel inhibitor in all cells examined. This action may account for its effects on inflammation and proliferation and may be independent of its effect on phospholipases.

Animals↗
PubMed1987

Inhibition of gastric H+,K+-ATPase and acid secretion by SCH 28080, a substituted pyridyl(1,2a)imidazole.

A hydrophobic amine, SCH 28080, 2-methyl-8-(phenylmethoxy)imidazo(1,2a)pyridine-3-acetonitrile, previously shown to inhibit gastric acid secretion in vivo and in vitro, was also shown to inhibit basal and stimulated aminopyrine accumulation in isolated gastric glands when histamine, high K+ concentrations, or dibutyryl cAMP were used as secretagogues. Stimulated, but not basal, oxygen consumption was also inhibited. Neutralization of the acid space of the parietal cell by high concentrations of the weak base, imidazole, reduced the potency of the drug, suggesting that SCH 28080 was active when protonated. Studies on the isolated H+,K+-ATPase showed that the compound inhibited the enzyme competitively with K+, whether ATP or p-nitrophenyl phosphate were used as substrates. In contrast, the inhibition was mixed with respect to p-nitrophenyl phosphate and uncompetitive with respect to ATP. The drug reduced the steady state level of the phosphoenzyme but not the observed rate constant for phosphoenzyme formation in the absence of K+ nor the quantity of phosphoenzyme reacting with K+. The drug quenched the fluorescence of fluorescein isothiocyanate-modified enzyme and also inhibited the ATP-independent K+ exchange reaction of the H+,K+-ATPase. Its action on gastric acid secretion can be explained by inhibition of the H+,K+-ATPase by reversible complexation of the enzyme. This class of compound, therefore, acts as a reversible inhibitor of gastric acid secretion.

Adenosine Triphosphatases↗
PubMed1987

Inhibition of (H+ + K+)-ATPase by omeprazole in isolated gastric vesicles requires proton transport.

Omeprazole was found to inhibit the (H+ + K+)-ATPase activity in isolated gastric vesicles only when acid was accumulated in the vesicle lumen. The ATPase activity was time- and dose-dependently inhibited in the presence of K+ and valinomycin. Under conditions in which no pH-gradient was generated, i.e., in the presence of K+ alone or NH4+, no effect of omeprazole was found. The degree of inhibition was directly correlated to the amount of inhibitor bound to the preparation. A stoichiometry of 2 mol radiolabelled inhibitor bound per mol phosphoenzyme was found on total inhibition of the K+ plus valinomycin-stimulated activity. This inhibitory action of omeprazole on the ATPase activity could be fully reversed by addition of beta-mercaptoethanol. The inhibition of the proton transport in the (H+ + K+)-ATPase-containing vesicles by omeprazole was also strictly correlated to the amount of bound inhibitor. The stoichiometry of binding at total inhibition of this reaction was found to be 1.4 mol per mol phosphoenzyme. The K+-stimulated p-nitrophenylphosphatase activity was inhibited in parallel with the ATPase activity, whereas the phosphoenzyme levels were affected to a lesser extent by omeprazole. Gel electrophoresis of an omeprazole-inhibited vesicle preparation showed that the radiolabel was mainly found at 94 kDa, the molecular weight of the (H+ + K+)-ATPase catalytic subunit(s).

4-Nitrophenylphosphatase↗
PubMed1987

Regulation of K+ channels in the basolateral membrane of Necturus oxyntic cells.

Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands from Necturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67 +/- 21 pS in 24% of the patches and channels of smaller conductance, 33 +/- 6 pS in 56% of the patches. Channels in both classes were highly selective for K+ over Na+ and Cl-, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca2+ in the range between 5 and 500 nM, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca2+. The presence of K+ channels in the basolateral membrane which are regulated by these known "second messengers" can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.

Animals↗