Effect of ethanol on the kinetics of rat brain (Na+ + K+) ATPase and K+-dependent phosphatase with different alkali ions.

(Na⁺ + K⁺)-dependent ATPase [(Na + K)-ATPase] and K⁺-dependent p-nitrophenyl phosphatase [pNPPase] activities in rat brain heavy microsomal fractions were studied in the presence of 120 mM Na⁺ and varied concentrations of K⁺, Rb⁺, Cs⁺, Li⁺ or NH₄⁺. Scatchard and Hill plots indicated non-hyperbolicity (cooperativity) with all except Li⁺, which supported a considerably lower activity than any of the other ions tested. Addition of 0.22 M ethanol to the incubation mixtures produced a formally competitive inhibition of ATPase activity with K⁺, Rb⁺ and Cs⁺, a non-competitive inhibition with Li⁺, and a mixed inhibition with NH₄⁺. The changes in pNPPase activity generally followed a similar but less clear-cut pattern. The values of the Hill constants were not changed for either enzyme activity. The findings are interpreted as evidence that ethanol inhibits ATPase activity by inducing conformational changes which alter the consequences of ion binding to the various receptor sites.     

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.     

Biochemical and pharmacological properties of a new proton pump inhibitor, 2-amino-4,5-dihydropyrido[1,2-a]thiazolo [5,4-g] benzimidazole (YJA20379-5)

This study was designed to determine biochemical and pharmacological properties of a newly synthesized benzimidazole derivative, 2-amino-4,5-dihydropyrido [1,2-a] thiazolo [5,4-g] benzimidazole (YJA20379-5)in vitro andin vivo. In the leaky membrane vesicles of pig gastric mucosa, YJA20379-5 inhibited the K⁺-stimulated H⁺,K⁺-ATPase activity in a concentration- and time-dependent manner, with IC₅₀ values being 43 μM and 31 μM at pH 6.4 and 7.4, respectively. YJA20379-5, given intraduodenally, had a potent inhibitory effect on the gastric acid secretion in pylorus-ligated rats. The ED₅₀ value for acid secretion was 15.4 mg/kg. YJA20379-5, administered orally, also suppressed gastric damages induced by water-immersion stress, indomethacin and ethanol, and duodenal damage induced by mepirizole in rats; the ED₅₀ values were 17.6, 4.7, 3.0 and 18.7 mg/kg, respectively. Furthermore, repeated oral administration of YJA20379-5 accelerated the spontaneous healing of acetic acid-induced gastric ulcers in rats. It is concluded that the antisecretory activity of YJA20379-5 appears to be associated with inhibition of H⁺,K⁺-ATPase, while its antigastric and antiduodenal lesion activities are primarily related to the antisecretory effect.     

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 guinea pig aortic sarcolemmal Ca2+-Mg2+ ATPase and cAMP phosphodiesterase activity by milrinone

Milrinone is a positive inotrope/vasodilator that inhibits cardiovascular low K_m cAMP phosphodiesterase (PDE) and not Na⁺?K⁺ ATPase activity. To explore other possible mechanisms of action, we quantitated the effects of milrinone on Ca²⁺-stimulated Mg²⁺ ATPase activity in guinea pig aortic smooth muscle plasma membranes. Milrinone inhibited Ca²⁺- stimulated activity, but not basal activity, in aortic microsomes. Maximum inhibition (70%) occurred at 1 μM, which coincided with the inflection of a parabolic dose-response curve. In a sarcolemmal-enriched (F1) aortic preparation, 1 μM cAMP, 1 μM Cl-930 (another low K_m cAMP PDE inhibitor), and 100 μM W-7 (a calmodulin antagonist) all inhibited Ca² -stimulated Mg²⁺ ATPase activity. This F1 preparation contained cAMP PDE activity which was inhibited by 1 μM milrinone (26%) and 1 μM Cl-930 (40%) but not by 100 μM W-7. The inhibition of F1 Ca²⁺ -Mg²⁺ ATPase activity by 1 μM milrinone could be diminished by increasing the concentration of CaCl₂ in reaction mixtures. In sum, these studies show that milrinone can inhibit vascular sarcolemmal Ca²⁺ -stimulated Mg²⁺ ATPase activity. However, inhibition may be direct or direct or may be secondary to cAMP PDE inhibition in vascular sarcolemma, since inhibition also occurs with cAMP and another low-K_m cAMP PDE inhibitor, Cl-930.     

Gastric antisecretory activity of an acid stable H+/K+ ATPase inhibitor,WY-26,769

WY-26,769 (2-3-dihydro-2-(2-pyridinyl) thiazolo-[3,2-a]benzimidazole) is a gastric antisecretory agent that acts by inhibiting the parietal cell proton pump. In vitro, WY-26,769 was active against dbcAMP-stimulated aminopyrine uptake in isolated parietal cells. Preincubating the drug with liver microsomal enzymes increased its potency 50 times, to an IC₅₀ of 6.8 μM. Wy-26,769 and its oxidative metabolite Wy-26,876 each inhibited the H⁺/K⁺ ATPase (IC₅₀ = 42 μM and 18 μM, respectively). In whole animal studies, Wy-26,769 inhibited histaminestimulated gastric acid secretion (ID₅₀ = 3 mg/kg) irrespective of whether Wy-26,769 was administered orally or intravenously. Wy-26,769 was not degraded in acid and also inhibited gastric acid secretion in the pylorus-ligated rat. Wy-26,769 was equiactive after oral or intravenous administration demonstrating that it is an acid stable proton pump inhibitor.     

Effects of antirheumatic drugs on sponge-induced granulation tissue, rheumatoid synovial tissue, matrix-free tendon cells and fibroblast plasma membranes in vitro.

Slices of sponge-induced, mature granulation tissue were incubated in the presence of antirheumatic drugs (sodium acetylsalicylate, indomethacin and phenylbutazone at concentrations of 10^?7–10^?2M) and labelled precursors of nucleic acids, proteins and acid mucopolysaccharides. Analogous experiments were made with sliced human synovial tissue from rheumatoid patients and with matrix-free embryonic chick tendon cells. The synthetic functions were suppressed by 10^?4–10^?3M indomethacin and phenylbutazone but only by 10^?2m acetylsalicylic acid. At low concentrations (10^?5–10^?6M) there was an increase in the radioactivity in proteins during the early period of the incubation but this was reversed later. Indomethacin and phenylbutazone affected the production of collagen at two points: (i) the synthesis of the peptide chain and. more notably, (ii) the secretion of collagen to the extracellular space. Indomethacin affected the final content of cellular collagen only slightly. Preliminary experiments on the effect of indomethacin on the activities of enzymes in plasma membranes prepared from granulation tissue showed some effect on leucyl-β-naphthylamidase and Na⁺, K⁺ -activated Mg²⁺- adenosine triphosphatase. The use of such systems for the screening of new connective tissue-active drugs is discussed. The secretion of collagen from matrix-free embryonic tendon cells is the most sensitive target for indomethacin in vitro.     

Effects of adrenochrome on rat heart sarcolemmal ATPase activities

Effects of adrenochrome on rat heart sarcolemmal ATPase, calcium binding and adenylate cyclase activities were studied _vitro. Adrenochrome (1–100 μg/ml) did not affect calcium binding activity. Significant decreases in adenylate cyclase and Ca²⁺ ATPase activities were seen only at a concentration of 100 μg/ml adrenochrome. Adrenochrome depressed Na⁺?K⁺ ATPase activity in a dose-dependent manner. The inhibitory effect on Na⁺?K⁺ ATPase of adrenochrome was also observed in sarcolemmal membranes washed with a buffer after treatment with adrenochrome. The percentage inhibition of the enzyme activity was independent over a wide range of pH (6.6–7.8) and concentrations of NaCl/KCl (40/4–100/10,mM/mM). A study on the combined effects of adrenochrome and either ouabain or CaCl₂ showed that, unlike ouabain, calcium produced additive inhibition with adrenochrome. Depression of Na⁺?K⁺ ATPase activity was also observed in sarcolemma isolated from the heart perfused with adrenochrome. Since adrenochrome has been demonstrated to produce myocardial cell damage and contractile failure in the perfused heart, the present experiments provide a possible explanation for the genesis of adrenochrome-induced cardiotoxicity.     

Patent Evaluation: Novel 2,2′-alkylenediindoles as anti-ulcer agents

Summary

Novelty: Novel 2,2′-alkylenediindole derivatives are claimed to be H⁺/K⁺ ATPase inhibitors. They are potentially useful as anti-ulcer agents.

Biology: An H⁺/K⁺ ATPase inhibitory activity assay was performed in vitro using a pig stomach preparation. Inhibition was determined colorimetrically. Results are given for twelve compounds. ATPase inhibition was 5-100% (dose 5–20 μg/ml).

Chemistry: A total of twenty-three compounds are disclosed. Syntheses are given in five examples. Characterization is by ¹H nmr and ms. None of the compounds are specifically claimed. 3-(2-Phenylethylaminoethyl)-2,2′-tetramethylenediindole is typical of the claim.     

Inhibition by uranyl nitrate of adenosine triphosphatases derived from animal and human tissues

Inhibition of adenosine triphosphatase (ATPase) by uranyl nitrate (UO₂²⁺ or U⁶⁺) was studied in microsomal fractions and tissue homogenates of several organs and species. U⁶⁺ inhibited ouabain-sensitive (Na⁺ + K⁺-dependent) ATPase and ouabain-insensitive (Mg²⁺-dependent) ATPase with I50 of 2 × 10^?5 to 2 × 10^?4m. Higher concentrations of U⁶⁺ were required to inhibit the enzyme in homogenates than in microsomal fractions. Mg²⁺ ATPase was somewhat more sensitive to U⁶⁺ than was Na⁺ + K⁺ ATPase when data were corrected for protein content of enzyme preparations. The inhibition of Na⁺ + K⁺ ATPase, but not Mg²⁺ ATPase, was markedly antagonized by Na⁺. This suggests that U⁶⁺ may inhibit Na⁺ + K⁺ ATPase at the Na⁺ site on the enzyme, whereas ouabain inhibits at the K⁺ site. ATP decreased and Mg²⁺ increased the inhibition of both enzymes. K⁺ had no effect. The remaining studies were done with Na⁺ + K⁺ ATPase. Increasing pH enhanced inhibition. The enzyme was protected by bovine serum albumin and citric acid. Ascorbic acid increased inhibition possibly by reducing U⁶⁺ to U⁴⁺, thus rendering the new ionic species reactive with sulfhydryl groups in addition to organic anions.     

Ammonia and gastric acid secretion: a key to understanding activity and regulation of the H+,K+-ATPase

Ammonia is a cytotoxic substance liberated during Helicobacter pylori infection that may be responsible, in part, for the significant reduction in gastric acid secretion in human patients. However, it is not clear how ammonia blocks acid secretion. Here, we investigate several potential pathways for ammonia blockade in gastric oxyntic cells.___TAGSTART___BR___TAGEND___ Methods: Stomachs from the bullfrog, Rana catesbeiana, were stripped and mounted in Ussing chambers. Four possible pathways of blockade were investigated: (1) blockade of basolateral K⁺-channel activity, (2) blockade of ion transport activity, (3) neutralization of secreted H⁺ or (4) ATP depletion.___TAGSTART___BR___TAGEND___ Results: Addition of nutrient 10 mM NH₄Cl at pH 7.4, yielding 92.5 M NH₃ and 9.91 mM NH₄ ⁺, abolished acid secretion within 30 min. Inhibition of acid secretion did not occur by blockade of basolateral K⁺-channel activity or ion transport activity, nor did NH₄ ⁺ enter cells by substituting for Na⁺ or K⁺ on individual ion transporters. Furthermore, neutralization of the luminal solution by NH₃ and/or ATP depletion cannot account for the total reduction in acid secretion. We demonstrate that NH₄Cl acts specifically on stimulated tissues.___TAGSTART___BR___TAGEND___ Conclusions: We show that small concentrations of ammonia completely block gastric acid secretion. We propose that inhibition of acid secretion occurs by blockade of an apical K⁺-channel, specifically inwardly rectifying K⁺-channels. Our data suggest that apical K⁺-channel activity may be essential for the regulation of acid secretion and could be a new therapeutic target for acid inhibitory drugs.     

Stimulation of Na+,K+-ATPase of isolated smooth muscle membranes by the Ca2+ channel inhibitors,nimodipine and nitrendipine

Nimodipine (0.015 to 1.5 μM) increased Na⁺, K⁺-ATPase activity by 70–120% in isolated smooth muscle membranes. At 0.015 μM, nitrendipine, but not nifedipine, verapamil or diltiazem, also activated this enzyme. Nimodipine stimulated this Na⁺, K⁺ATPase three times more than nitrendipine at 15 nM. Marked stimulation of Na⁺,K⁺-ATPase by nimopidine was seen in membranes from rat and guinea pig aorta and rat vas deferens, but not in membranes from guinea pig heart or brain. Although it is not known whether these results are applicable to intact cells, the results are consistent with the hypothesis that vasodilation produced by nimodipine and nitrendipine may be due not only to inhibition of Ca²⁺ entry but also to the stimulation of the Na^? pump.     

Biochemical and Pharmacological Characteristics of 3-Butyryl-8-methoxy-4-[(2-thiophenyl)amino]quinoline,a New Proton-pump Inhibitor,in Rabbit Gastric Microsomes and in Rats

We have investigated the properties of the newly synthesized proton-pump inhibitor, 3-butyryl-8-methoxy-4-[(2-thiophenyl)amino]quinoline (YJA20379–6), on gastric mucosal proton-pump (H⁺/K⁺-ATPase) activity, gastric acid secretion and gastroduodenal lesions in experimental rats. YJA20379–6 markedly inhibited H⁺/K⁺-ATPase activity in rabbit isolated gastric mucosal microsomes, confirming its classification as a proton-pump inhibitor. The inhibitory efficacy of YJA20379-6 on the proton pump was approximately 14-times higher than that of omeprazole at pH 7.4. YJA20379–6 given intraduodenally had a potent inhibitory effect on gastric secretion in pylorus-ligated rats (ED50 22.9 mg kg^?1) but was less active than omeprazole. Pretreatment of rats with YJA20379-6 dose-dependently protected the gastric mucosa from damage induced by water-immersion stress, indomethacin and absolute ethanol, and the duodenal mucosa from damage induced by mepirizole. Repeated administration of YJA20379-6 also dose-dependently accelerated the spontaneous healing of acetic acid-induced gastric ulcers. These results suggest that YJA20379-6 has potent anti-secretory and anti-ulcer effects which are exerted by suppression of H⁺/K⁺-ATPase activity in gastric parietal cells. YJA20379–6 might be useful for the clinical treatment of peptic ulcer diseases.     

Secretagogue-induced transport of H+ and K+ by in vitro amphibian gastric mucosa

The effects of histamine, pentagastrin (PG), urecholinc (UC) amd dibutyryl cyclic AMP (cAMP) on the chambered fundic mucosa from Rana catesbeiana were studied to determine the H⁺ and K⁺ transport characteristics of the in vitro preparation. The three secretagogues (histamine, PG and UC) elicited very similar responses—a sustained steady-state secretion of H⁺ within 15—30 min and a transitory efflux of K⁺ that returned to control level within 60 min. Inhibition of H⁺ transport of the secretagogue-stimulated mucosa by 2.5 × 10^?4 Mp-chloromercuribenzene sulfonic acid (pCMBS) caused an increased efflux of K⁺. Replacement of pCMBS by β-mercaptoethanol (2 × 10^?3 M) rapidly restored the H⁺ transport, with a concomitant decrease of K⁺ efflux, to the pre-stimulated control level. The effects of cAMP were qualitatively similar to those of other secretagogues except that there was a delayed peak response for both H⁺ and K⁺. Our data are compatible with a K⁺ conductive pathway and a membrane-recycyling mechanism for K⁺in exchange for H⁺ at the secretory membrane of the gastric cells. Thiocyanate inhibited H⁺ transport, and the effect appeared to be due to inhibition of the transport and coupling of K⁺ to the $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange mechanism.     

Influence of TMB-8 on secretion of catecholamines from the perfused rat adrenal glands

An attempt was made to investigate the effect of TMB-8 [3,4,5-trimethoxybenzoate-8 (N,N-diethylamino) octyl ester], which is known to be an inhibitor of intracellular Ca²⁺ release, on catecholamines (CA) secretion evoked by Ach, excess K⁺, DMPP, McN-A-343 and caffeine from the isolated perfused rat adrenal glands and to clearify its mechanism of action. The pretreatment with a low dose of TMB-8 (10 μM) for 20 min led to marked inhibition in CA secretion evoked by Ach (5.32 mM), excess K⁺ (56 mM), DMPP (100 μM), McN-A-343 (100 μM) and BAY-K 8644 (10^?5M). Caffeine-induced CA secretion was similar to that of control only during the first periods (0–3 min) but thereafter marked inhibition in CA secretion evoked by caffeine was observed during the rest periods up to 30 min. The increased moderate concentration of TMB-8 (30 μM) caused the result similar to that of 10 μM TMB-8. However, in adrenal glands preloaded with a high dose of TMB-8 (100 μM), CA releases evoked by Ach, excess K⁺, DMPP, McN-A-343 and caffeine were almost completely blocked by the drug. These experimental data demonstrate that TMB-8 may inhibit cholinergic receptor-mediated and also depolarization-dependent CA secretion, suggesting that these TMB-8 effects seem to be mediated through inhibiting influx of extracellular calcium into the rat adrenal medullary chromaffin cells as well as reducing the release of calcium from intracellular sources.     

Lack of effect of diphenylhydantoin administration on cortex potassium activated p-nitrophenylphosphatase

Potassium-activated p-nitrophenylphosphatase (K⁺-activated phosphatase) activity was assayed in rat cerebral cortex following diphenylhydantoin (DPH) administration. No effect was found at any of 5K⁺ concentrations ranging from 1 to 20 mM in contrast to the stimulation of sodium-potassium-activated ATPase (NaKATPase) by DPH which has been reported under certain ionic conditions. In addition, the inhibition of K⁺-activated phosphatase by Na⁺ was not influenced by DPH. Diphenylhydantoin administration had no effect on the ouabain inhibition of K⁺-activated phosphatase, although at 10^?4m ouabain a slight increase in residual NaKATPase activity was found. As K⁺-activated phosphatase activity is thought to reflect the K⁺-activated dephosphorylation step of the NaKATPase reaction, DPH may act primarily on the Na⁺-dependent phosphorylation step.     

Epigallocatechin-3-gallate is an inhibitor of Na,K-ATPase by favoring the E1 conformation

Four catechins, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, and epicatechin, inhibited activity of the Na⁺,K⁺-ATPase. The two galloyl-type catechins were more potent inhibitors, with IC₅₀ values of about 1 μM, than were the other two catechins. Inhibition by epigallocatechin-3-gallate was noncompetitive with respect to ATP. Epigallocatechin-3-gallate reduced the affinity of vanadate, shifted the equilibrium of E₁P and E₂P toward E₁P, and reduced the rate of the E₁P to E₂P transition. Epigallocatechin-3-gallate potently inhibited membrane-embedded P-type ATPases (gastric H⁺,K⁺-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase) as well as the Na⁺,K⁺-ATPase, whereas soluble ATPases (bacterial F₁-ATPase and myosin ATPase) were weakly inhibited. Solubilization of the Na⁺,K⁺-ATPase with a nonionic detergent reduced sensitivity to epigallocatechin-3-gallate with an elevation of IC₅₀ to 10 μM. These results suggest that epigallocatechin-3-gallate exerts its inhibitory effect through interaction with plasma membrane phospholipid.     

Effects of aldehydes on sodium plus potassium ion-stimulated adenosine triphosphatase of mouse brain.

The biogenic aldehydes, 3,4-dihydroxyphenylglycolaldehyde and 3,4-dihydroxyphenylacetal-dehyde, derived from norepinephrine and dopamine, respectively, as well as acetaldehyde, porpionalde-hyde, benzaldehyde and phenylacetaldehyde, inhibited both Na⁺ + K⁺-activated ATPase and Mg²⁺-ATPase. In addition, K⁺ ion-dependent p-nitrophenylphosphatase activity was inhibited by these compounds. The Na⁺ + K⁺-ATPase was much more sensitive than Mg²⁺-ATPase of K⁺-activated phosphatase to inhibition by various aldehydes. The inhibition of Na⁺ + K⁺-ATPase by aldehydes was reversible and was non-competitive with ATP or K⁺ as the variable substrate or activator respectively. Addition of cysteine or mercaptoethanol protected the enzymes from inhibition by aldehydes. The concentrations of aldehydes which produced marked inhibition of Na⁺ + K⁺-ATPase ranged from 2 × 10^?2 M to 6 × 10^?6 M for acetaldehyde and 3,4-dihydroxyphenylglycoladehyde respectively. All aldehydes, including acetaldehyde, were more potent inhibitors of Na⁺ + K⁺-ATPase activity than was ethanol.     

Structure—activity relationships of omeprazole analogues and their mechanism of action

Omeprazole represents a new class of gastric acid secretion inhibitors, the sulphinylbemimidazoles. Per Lindberg and colleagues describe this first example of a clinically useful anti-ulcer agent in this group whose mechanism involves highly specific inhibition of H⁺/K⁺-ATPase, the gastric proton pump. Omeprazole is activated only at low pH, as occurs in the parietal cell and is thus highly selective. The active intermediate formed, a sulphenamide, reacts with a mercapto group of H⁺/K⁺-ATPase to form a disulphide inhibitor complex. The quantity of this sulphenamide on the luminal side of the parietal cell is related to the H⁺/K⁺-ATPase inhibitory effect.     

Proton Pump Inhibitors and Acid-Related Diseases

Proton pump inhibitors (PPIs) are targeted to the gastric acid pump, H⁺,K⁺-adenosine triphosphatase (ATPase). The drugs accumulate in the acid space of the parietal cell and convert to active sulfenamide by an acid-catalyzed reaction. Consequent covalent inhibition of H⁺,K⁺-ATPase blocks the final step of acid secretion, hence the PPIs omeprazole, lansoprazole, and pantoprazole are more effective than histamine₂-receptor antagonists (H₂RAs) in controlling acid secretion. Preclinical short- and long-term clinical surveillance data show these drugs to be well tolerated and safe. The PPIs heal the lesions of gastroesophageal reflux disease and lessen symptoms more effectively and more quickly than the H₂RAs, and are effective' and faster acting for peptic ulcer disease. Helicobacter pylori is causally implicated in the majority of peptic ulcers and in atrophic gastritis. Since PPIs, but not H₂RAs, are synergistic with antibiotics in eradicating H. pylori, their use is appropriate in all acid-related diseases since all patients who are H. pylori positive require eradication as well as healing.