Inhibition of H+K+ATPase by SCH 28080 and SCH 32651

The novel antiulcer agents, SCH 28080 and SCH 32651 were examined for their ability to inhibit the H+K+ ATPase enzyme activity in a preparation of microsomal membranes from rabbit fundic mucosa. SCH 28080 inhibited the isolated enzyme activity with a potency similar to omeprazole, IC50s of 2.5 and 4.0 microM respectively. SCH 32651 was less potent exhibiting an IC50 of 200.0 microM. Both compounds may therefore exert their antisecretory activity via a direct inhibition of the parietal cell H+K+ ATPase.     

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.     

Effects of newly synthesized benzimidazole derivatives on gastric H+/K+ ATPase

The effects of various synthetic benzimidazole derivatives on gastric H⁺/K⁺ ATPase activityin vitro were examined. The results showed that the effects of substituents on the benzimidazole ring were not significant. However, replacement of sulfoxide connecting two ring systems to sulfide resulted in a completely inactive compoundin vitro, suggesting the essential role of sulfoxide group in the inhibition. In addition, compounds with 5 or 6-membered oxacyclic substituents attached to the pyridine ring displayed the most effective inhibitory activity. Among these derivatives, AU-47 was the most potent, and detailed mechanistic studies with the compound were carried out. AU-47 inhibited gastric H⁺/K⁺ ATPase in a concentration and time dependent manner with 50% inhibition at 6 μM. The presence of sulfhydryl reducing agents or substrate analogue protected H⁺/K⁺ ATPase from the inactivation. The inhibition by AU-47 was potentiated by acid pretreatment of the compound, suggesting the structural conversion of AU-47 into a more active intermediate which was favored in acidic condition. Consistent within vitro results, AU-47 inhibitedin vivo gastric acid secretion. The results suggest that AU-47 is a relevant candidate for the development of new antiulcer agent.     

Inhibition of Mg2+-Na+-K+-dependent ATPase system from human gastric mucosa by prostaglandins E1 and E2

The Mg²⁺-dependent and Na⁺K⁺-dependent (Na⁺K⁺-activated or transport) ATPase (EC 3.6.1.3.) were prepared from human fundic gastric mucosa and the effects of prostaglandins E₁ and E₂ on their activity studied.Significant inhibitions of Mg²⁺-dependent, total (Mg²⁺-dependent and Na⁺K⁺-dependent) and Na⁺K⁺-dependent ATPase activities were caused by prostaglandins E₁ and E₂, in concentrations of 10^?9 to 10 ^?6 M.A possible role of Mg²⁺Na⁺K⁺-dependent ATPase system is discussed in the inhibition of gastric secretion by type E prostaglandins.     

Inhibition of H+/K+ ATPase in the gastroprotective effect of Baccharis illinita DC

Baccharis illinita DC (Compositae) is used in folk medicine to treat gastric disturbances. Preliminary studies with other extracts of B. Illinita showed gastric protection against ethanol-, indometacin- and stress-induced ulcers and the inhibition of gastric secretion. Based on these data, the aim of this study was to verify the pathways involved in the inhibition of gastric secretion. The chloroform extract (CE) of flowers from B. illinita (3, 10, 30 and 100 mg kg(-1) i.p.) tested on rats with pylorus ligature reduced the volume and the total acidity of gastric content by approximately 50% (ED50 = 69 mg kg(-1)). Treatment with CE (100 mg kg(-1) i.p.) reduced the gastric total acidity stimulated by histamine, bethanechol and pentagastrin to 42%, 27% and 57% of that in the stimulated control group, respectively. The CE (10, 30 and 100 microM) inhibited H+/K+ ATPase activity in-vitro, with an IC50 of 37 microM. The isolated flavonoid luteolin (1, 3, 10 and 30 microM) also inhibited H+/K+ ATPase activity by 50%, at a dose of 30 microM. Our results suggest that the reduction in gastric secretion occurs through inhibition of H+/K+ ATPase, which is the final step in acid secretion and therefore one of the most important steps.     

Inhibition of dog brain synaptosomal Na+ -K+ ATPase and K+-stimulated phosphatase activities by long chain n-alkyl-amine and -piperidine, and N'-alkylnicotinamide derivatives

The effects of piperidine, primary amine, and nicotinamide aliphatic derivatives on dog brain snyaptosomal Na⁺-K⁺ ATPase were investigated. These derivatives inhibited the enzyme activity in a manner dependent on alkyl chain length. Kinetic studies revealed that inhibition of Na⁺-K⁺ ATPase activity by long chain alkyl derivatives (C₁₂-C₁₈) was biphasic and non-competitive with respect to the inhibitor and substrate (ATP) concentrations respectively. These long alkyl derivatives caused changes in the Hill coefficient that suggest the occurrence of a possible conformational change in the enzyme molecule. Dual-inhibitor experiments showed that both saturated and unsaturated pentadecylpiperidine (C₁₅-pip) derivatives inhibited Na⁺-K⁺ ATPase by the same mechanism. Oleylamine (C_18:1-NH₂), and N-dodecylnicotinamide (C₁₂-NA⁺Cl^?) derivatives apparently inhibited the enzyme activity by a mechanism different from that of piperidine derivatives. Low concentrations of C_15:1-pip, C_18:1-NH₂, and C₁₂-NA⁺Cl^? inhibited dog brain Na⁺-K⁺ ATPase activity only, but at higher inhibitor concentrations K⁺-stimulated phosphatase activity was inhibited as much as Na⁺-K⁺ ATPase. It is concluded that long chain n-alkyl derivatives of piperidines, amines, and nicotinamide have a cationic detergent-like action on Na⁺-K⁺ ATPase, possibly by the disruption of protein-phospholipid interactions. The mechanism by which these compounds inhibit the overall Na⁺-K⁺ ATPase may involve two different inhibitory sites: one, a high affinity inhibitory site, binding to which inhibits the Na⁺-stimulated phosphorylation reaction, and the other, a low affinity inhibitory site, binding to which inhibits the K⁺-stimulated dephosphorylation reaction. These possible mechanisms may provide an explanation for the observed biphasic inhibition kinetics.     

Affinity and dose-dependent digoxin Na+K+ATPase dissociation by monoclonal digoxin-specific antibodies

The effect of three monoclonal digoxin-specific antibodies and of polyclonal Digidot^® as reference on digoxin dissociation from rat brain Na⁺K⁺ATPase microsomes was studied to determine the role of the affinity constant (Ka) and dose of the antibody on the rate of digoxin dissociation from Na⁺K⁺ATPase. Stoichiometrical doses of 1C10, 6C9, 9F5 IgG, and Digidot^® (Ka = 6 10⁹, 3.1 10⁸, 2.5 10⁷, and 8.5 10⁹ M⁻¹, respectively) resulted in digoxin dissociation related to Ka. When the IgG:digoxin molar ratio increased from 0.25 to 10, digoxin dissociation from Na⁺K⁺ATPase sites also increased according to the Hill equation, allowing comparative parameters among the three antibodies to be determined. 1C10 IgG was 2- and 10-fold more efficacious than 6C9 and 9F5, respectively. This in vitro model appears to be a useful predictive screening assay before in vivo experimentation.     

Inhibition of G protein-activated inwardly rectifying K+ channels by ifenprodil.

G protein-activated inwardly rectifying K+ channels (GIRK, also known as Kir3) are regulated by various G-protein-coupled receptors. Activation of GIRK channels plays an important role in reducing neuronal excitability in most brain regions and the heart rate. Ifenprodil, which is a clinically used cerebral vasodilator, interacts with several receptors, such as alpha1 adrenergic, N-methyl-D-aspartate, serotonin and sigma receptors. However, the molecular mechanisms underlying the various clinically related effects of ifenprodil remain to be clarified. Here, we examined the effects of ifenprodil on GIRK channels by using Xenopus oocyte expression assays. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, ifenprodil reversibly reduced inward currents through the basal GIRK activity. The inhibition was concentration-dependent, but voltage- and time-independent, suggesting that ifenprodil may not act as an open channel blocker of the channels. In contrast, Kir1.1 and Kir2.1 channels in other Kir channel subfamilies were insensitive to ifenprodil. Furthermore, GIRK current responses activated by the cloned kappa-opioid receptor were similarly inhibited by ifenprodil. The inhibitory effects of ifenprodil were not observed when ifenprodil was applied intracellularly, and were not affected by extracellular pH, which changed the proportion of the uncharged to protonated ifenprodil, suggesting its action from the extracellular side. The GIRK currents induced by ethanol were also attenuated in the presence of ifenprodil. Our results suggest that direct inhibition of GIRK channels by ifenprodil, at submicromolar concentrations or more, may contribute to some of its therapeutic effects and adverse side effects.     

Inhibition of gastric H+, K(+)-ATPase and acid secretion by ellagic acid.

The effects of ellagic acid on gastric H+, K(+)-ATPase, acid secretion, and the occurrence of gastric ulcers were studied. Ellagic acid inhibited hog gastric H+, K(+)-ATPase activity with a 50% inhibition at 2.1 x 10(-6)M; kinetic studies showed that the inhibition of H+, K(+)-ATPase by ellagic acid is competitive with respect to ATP and is noncompetitive with respect to K+. The effect on gastric ulcers was investigated by using a stress ulcer model. Intraperitoneal administration of ellagic acid at above 5 mg/kg markedly reduced the occurrence of gastric lesion. Ellagic acid significantly reduced acid secretion at the same doses. These results suggest that ellagic acid has a marked inhibitory effect on acid secretion and the occurrence of stress-induced gastric lesions, and these effects may be attributed to the inhibition of H+, K(+)-ATPase activity.     

Inhibition of voltage-gated K+ channels and Ca2+ channels by diphenidol

BackgroundAlthough diphenidol has long been deployed as an anti-emetic and anti-vertigo drug, its mechanism of action remains unclear. In particular, little is known as to how diphenidol affects neuronal ion channels. Recently, we showed that diphenidol blocked neuronal voltage-gated Na⁺ channels, causing spinal blockade of motor function, proprioception and nociception in rats. In this work, we investigated whether diphenidol could also affect voltage-gated K⁺ and Ca²⁺ channels.MethodsElectrophysiological experiments were performed to study ion channel activities in two neuronal cell lines, namely, neuroblastoma N2A cells and differentiated NG108-15 cells.ResultsDiphenidol inhibited voltage-gated K⁺ channels and Ca²⁺ channels, but did not affect store-operated Ca²⁺ channels.ConclusionDiphenidol is a non-specific inhibitor of voltage-gated ion channels in neuronal cells.     

(Na+ + K+)ATPase inhibition by Palythoa extracts--chemical nature of the inhibitor and kinetics of inhibition.

Crude toxic extracts obtained by ethanol extraction from the coelenterate Palythoa caribaeorum were shown to possess strong (Na⁺ + K⁺)ATPase inhibitory activity on enzyme preparations from the electroplax of Etectrophorus electricus. The toxic and inhibitory effects were foun to be separable. Chromatographie, spectrophotofluorimetric, electrophoretic and biological data demonstrate that the inhibitor is serotonin. It is a non-competitive inhibitor for Na⁺ and ATP but is a competitive inhibitor for K⁺. In enzyme preparations of a specific activity of 1.5 μM P_i/min, I₅₀ is of the order of 1 mM.     

Mechanism for potentiation of warfarin by phenylbutazone. Inhibition of vitamin K-dependent carboxylation and prothrombin synthesis by phenylbutazone in preparations from rat liver

Phenylbutazone potentiated the anticoagulant effects of racemic warfarin and of the individual enantiomers to similar extents in the rat. This indicates that the phenylbutazone did not act stereospecifically on the enantiomers, as it does in humans. Phenylbutazone doubled the turnover rate of warfarin in plasma, but it did not increase the amount of the anticoagulant in liver or the amount excreted in urine. The drug had no effect on plasma disappearance of [3H] or on hepatic levels of [3H] vitamin K1 or of its chief metabolite, [3H] vitamin K1 epoxide, after injection of [3H] vitamin K1. Phenylbutazone, however, at concentrations of 0.5 to 2.8 mM inhibited vitamin K-dependent carboxylation of a synthetic pentapeptide substrate in liver microsomes by 40-88 per cent. Vitamin K-dependent protein carboxylation was also inhibited by about 40 per cent in microsomes and post-mitochondrial supernatant fluid at drug concentrations of 2.8 to 4.8 mM. Most importantly, prothrombin synthesis was inhibited in post-mitochondrial supernatant fractions by 19 and 39 per cent at drug concentrations of 2.8 and 4.8 mM respectively. The inhibition of both carboxylation and prothrombin synthesis appears to have been of sufficient magnitude to account for the potentiation by phenylbutazone observed in vivo. The calculated hepatic level of phenylbutazone during potentiation was around 3 mM, a concentration that produced inhibition in vitro.     

Inhibition of mitochondrial Mg2+ ATPase activity in isolated perfused rat liver by kepone.

Hepatic mitochondria Mg²⁺ ATPase (MATPase) activity was determined in isolated perfused rat liver preparations. Perfusion for 1–4 hr with 30% rat blood did not affect either the native or DNP (10^?4 M)-stimulated MATPase activity. Mitochondrial preparations obtained from perfused livers were sensitive to added kepone: first, addition of kepone (10^?6M) resulted in total abolishment of DNP-stimulated MATPase activity; second, over and beyond the abolishment of DNP-stimulated activity, part of the native MATPase activity was also inhibited by kepone (10^?6M). Furthermore, a higher concentration of kepone (10^?5 M) inhibited the native MATPase activity in a dose-related manner. Liver perfusion with 10^?4 M kepone in the blood perfusate resulted in a similar abolishment of DNP-stimulated. as well as inhibition of native, MATPase activity. The requirement for a higher concentration of kepone in the perfusion system is because of the partial loss of intracellular kepone from the liver into the 9000 g supernatant fraction which would have been otherwise available for the inhibition of MATPase activity in the intact liver. These results suggest that interference of energy metabolism in the liver may bear a cause effect relationship to the previously reported kepone-induced impairment of biliary excretory function.     

Inhibition of H+ efflux and K+ uptake,and induction of K+ efflux in yeast by heavy metals

Energy and cell membrane ATPase-dependent H⁺ efflux from yeast cells was inhibited by heavy metals, which also inhibited energy-dependent K⁺ uptake by the cells. Heavy metals also induced K⁺ efflux from metabolizing cells in two phases: reversibly at low concentrations and irreversibly at high concentrations. The concentration dependence of all of these effects varied considerably between metals. The effect of in vivo inhibition of H⁺ transporting ATPases on cell membrane polarization and thus on cellular transport processes, including heavy metal uptake, are discussed. It is proposed that the inhibition of H⁺ efflux and the appearance of irreversible K⁺ efflux in yeast are potentially useful indicators of heavy metal toxicity toward yeast and possibly other fungi.