Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume,intracellular pH and migration of human polymorphonuclear leucocytes

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pH_i) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pH_i and migration of fMLP stimulated PMNs.
2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling.
3. Inhibition of the Na⁺/H⁺ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling.
4. Step omission of extracellular Na⁺ caused intracellular acidification, which was accelerated by subsequent addition of gastric H⁺/K⁺ ATPase inhibitor SCH 28080, or by omission of extracellular K⁺ ions. In addition Na⁺ removal caused cell swelling, which was further enhanced by fMLP.
5. H⁺/K⁺ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV.
6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na⁺/H⁺ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited.
7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole.
8. Western analysis with a monoclonal anti gastric H⁺/K⁺ATPase β-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs.
9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na⁺/H⁺ exchanger and a K⁺-dependent H⁺-extruding mechanism, presumably an H⁺/K⁺ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

     

Inhibition of gastric K+/H+-ATPase by acid-activated 2-((2-pyridylmethyl)sulphinyl)benzimidazole products

The inhibitory effects of timoprazole- and omeprazole-derived metabolites were studied in different in vitro test systems in order to characterize the metabolites of substituted benzimidazoles originating from acid activation. Acidification of timoprazole and omeprazole to pH 1.0 markedly increased the inhibitory potency on gastric K+/H+-ATPase. The timoprazole-derived tetracyclic thiol and radical were found to be equally or more potent on the K+/H+-ATPase than the mother compounds dissolved at pH 1.0. Kinetic studies with omeprazole sulphide revealed a competitive inhibition of the K+/H+-ATPase with respect to K+. The mercaptan dithiothreitol reversed the inhibitory effect of omeprazole, acidified timoprazole and the timoprazole-derived radical in the parietal cell and K+/H+-ATPase preparation. In contrast, the inhibitory effect of omeprazole sulphide and the timoprazole-derived thiol could not be reversed by dithiothreitol. Wash-out experiments indicated that acidified timoprazole and the tetracyclic compounds interact irreversibly with the K+/H+-ATPase, which contrasts with the properties of timoprazole in the parietal cell preparation. It is concluded from these data that neither the tetracyclic compounds nor the sulphide act as the 'active principle' of substituted benzimidazoles in the parietal cell preparion.     

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.     

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.     

Influence of condensed benzimidazole derivatives on gastric secretion

Translated from Khimikofarmatsevticheskii Zhurnal, Vol. 24, No. 2, pp. 127–130, February, 1990.     

SCH 28080 is a more selective inhibitor than SCH 32651 at the K+ site of gastric K+/H+-ATPase

The anti-secretory agents SCH 32651 and SCH 28080 were compared for their potency to interact with the K+ site of guinea-pig parietal cell K+/H+-ATPase and dog kidney Na+/K+-ATPase. SCH 32651 and SCH 28080 had an inhibition constant of 9.0 and 0.02 mumol/l, respectively, for the K+/H+-ATPase. The Ki values for the Na+/K+-ATPase were 140 and 220 mumol/l. The data show that both drugs have a higher affinity to the K+ site of the K+/H+-ATPase than to that of the Na+/K+-ATPase and that the affinity ratio of SCH 28080 in favour of K+/H+-ATPase is much greater (11,000) than that of SCH 32651 (15).     

Bisabolangelone, a gastric H+/K+-ATPase inhibitor: homology modeling and docking study

The homology model of human gastric H⁺/K⁺-ATPase has been produced based on the template provided by pig gastric H⁺/K⁺-ATPase (PDB code: 3IXZ). After molecular mechanics optimization, induced-fit docking simulation between gastric H⁺/K⁺-ATPase and bisabolangelone that has significantly inhibition activity of H⁺/K⁺-ATPase was performed. The results of ligand docking showed that the binding pocket involves the amino acid residues Asp101, Asp102, Tyr105, Leu106, Val296, Phe297, Met299, Ala300, Tyr764, Tyr767, Leu774, Gly777, Cys778, Ile779, Gln889, Tyr893, and Ile952. The hydrogen bonds are formed between bisabolangelone and the amino acid residues Cys778, Gln889, and Tyr893.     

Substituted 2-[(2-benzimidazolylsulfinyl)methyl]anilines as potential inhibitors of H+/K+ ATPase

A series of substituted 2-[(2-benzimidazolylsulfinyl)methyl]anilines were synthesized as potential inhibitors of the acid secretory enzyme H+/K+ ATPase. Substitutions on the aniline nitrogen atom resulted in potent enzyme inhibition in vitro but weak activity in gastric fistula dogs. Electron-donating substituents on the aniline ring enhanced in vitro and in vivo potency relative to the unsubstituted analogue. The potency showed a correlation to the calculated pKa of the aniline nitrogen atom. Substitutions on the aniline and benzimidazole rings did not further enhance potency. Di- and trisubstituted aniline derivatives were potent inhibitors of the enzyme system. The preferred combination of substituents were a methoxy group on the benzimidazole ring and a single alkyl group on the aniline ring. One such compound, 76, was an effective inhibitor of acid secretion in the dog and was selected for further pharmacological study.     

Anti-ulcer activity of newly synthesized acylquinoline derivatives

Anti-ulcer activity of newly synthesized acylquinoline derivatives was investigated. For the in vitro screening, the effects of the compounds on gastric H+/K+ ATPase isolated from hog and rabbit were examined. Among them, AU-090, AU-091, AU-254, AU-413 and AU-466 exhibited good in vitro activity on both enzymes. To correlate the in vitro activity with in vivo action, the effects of the compounds on the basal gastric acid secretion were studied. Some derivatives showed considerable anti-secretory activities, and AU-413 was selected for further studies. AU-413 protected gastric damage induced by either ethanol or NaOH dose dependently when given orally. ED50 values of 12 mg/kg, p.o. (ethanol) and 41 mg/kg, p.o. (NaOH) were obtained. In addition, histamine-stimulated gastric secretion was reduced upon AU-413 administration. Taken together, newly synthesized acylquinoline derivatives, especially AU-413, is worthy of further investigation to be developed as an anti-ulcer agent.     

Alcohol Excites Cerebellar Golgi Cells by Inhibiting the Na+/K+ ATPase

Alcohol-induced alterations of cerebellar function cause motor coordination impairments that are responsible for millions of injuries and deaths worldwide. Cognitive deficits associated with alcoholism are also a consequence of cerebellar dysfunction. The mechanisms responsible for these effects of ethanol are poorly understood. Recent studies have identified neurons in the input layer of the cerebellar cortex as important ethanol targets. In this layer, granule cells (GrCs) receive the majority of sensory inputs to the cerebellum through the mossy fibers. Information flow at these neurons is gated by a specialized pacemaker interneuron known as the Golgi cell, which provides divergent GABAergic input to thousands of GrCs. In vivo electrophysiological experiments have previously shown that acute ethanol exposure abolishes GrC responsiveness to sensory inputs carried by mossy fibers. Slice electrophysiological studies suggest that ethanol causes this effect by potentiating GABAergic transmission at Golgi cell-to-GrC synapses through an increase in Golgi cell excitability. Using patch-clamp electrophysiological techniques in cerebellar slices and computer modeling, we show here that ethanol excites Golgi cells by inhibiting the Na⁺/K⁺ ATPase. Voltage-clamp recordings of Na⁺/K⁺ ATPase currents indicated that ethanol partially inhibits this pump and this effect could be mimicked by low concentrations of ouabain. Partial inhibition of Na⁺/K⁺ ATPase function in a computer model of the Golgi cell reproduced these experimental findings. These results establish a novel mechanism of action of ethanol on neuronal excitability, which likely has a role in ethanol-induced cerebellar dysfunction and may also contribute to neuronal functional alterations in other brain regions.     

Inhibition of gastric K+ ATPase by phenylbutazone and indomethacin.

Inhibition of gastric H⁺ secretion by phenylbutazone and indomethacin was investigated by examining the effects of these agents on a putative H⁺ transport enzyme, a K⁺ stimulated ATPase. unique to gastric mucosa. Phenylbutazone and indomethacin were found to inhibit both the K⁺ ATPase and the K⁺ pNPPase. K_is were 430 μM and 710 μM for the K⁺ ATPase and 330 μM and 670 μM for the K⁺ pNPPase for phenylbutazone and indomethacin respectively. Inhibition was not reversed by Mg²⁺, ATP, pNPP, or KCl and obeyed non-competitive kinetics. Inhibition of the pNPPase suggested that the mechanism of inhibition involved the K⁺ sensitive dephosphorylation of the phosphoenzyme. In the presence of 500 μM phenylbutazone dephosphorylation was significantly less at 3, 5, 7.5, 10 and 15 sec following KCl addition. These studies provide an alternate mechanism for inhibition of gastric H⁺ secretion by phenylbutazone and indomethacin.     

Activation of K+ channels and Na+/K+ ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K⁺ channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K⁺ channels and Na⁺/K⁺-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O₂⁻ production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K⁺-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K⁺-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K⁺ channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress.     

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.     

Role of Na+/K+ ATPase on the Relaxation of Rabbit Ear and Femoral Arteries

The role of Na⁺/K⁺ ATPase in vascular relaxation has been studied by determining its inhibitory effects on 2-mm segments from rabbit central ear and femoral arteries, mounted for isometric tension recording. Acetylcholine (10-⁸-10-⁴ M), the nitric oxide donor sodium nitroprusside (10-⁸-3 × 10-⁴ M), the potassium channel agonist cromakalim (10-⁸-3 × 10-⁵ M), histamine (10-⁸-10-⁴ M) in the presence of the H₁ antagonist chlorpheniramine (10-⁵ M), and papaverine (10-⁶-3 × 10-⁴ M) all produced arterial relaxation in ear and femoral arteries precontracted with endothelin 1. Addition of potassium (6 × 10-³- 1.2 × 10-² M) caused relaxation of the same arteries preincubated in potassium-free medium. Ouabain (10-⁵ M) an inhibitor of Na⁺/K⁺ ATPase, reduced the relaxation of ear arteries, but not of femoral arteries, in response to acetylcholine; it also reduced the response to sodium nitroprusside, cromakalim or histamine, and abolished the relaxation to potassium, but did not modify the response to papaverine, in both types of artery. These results suggest that Na⁺/K⁺ ATPase might play a role in the relaxation of ear and femoral arteries to nitrovasodilators, to potassium channel openers and to activation of histamine receptors, and that Na⁺/K⁺ ATPase might play a role in the cholinergic relaxation of ear, but not femoral arteries, suggesting that the mechanism of cholinergic relaxation might differ in each type of artery.     

Ligand docking in the gastric H+/K+-ATPase: homology modeling of reversible inhibitor binding sites

Using the recent high-resolution X-ray structures determined for the Ca2+-ATPase, we have generated two homology models of the gastric H+/K+-ATPase reflecting the E1 and E2 conformations adopted by P-type ATPases in their catalytic cycle. In regimes where the in situ solid-state NMR-determined structure for 1,2,3-trimethyl-8-(pentafluorophenylmethoxy)imidazo[1,2-a]pyridinium iodide (TMPFPIP), a reversible inhibitor of the gastric H+/K+-ATPase, was retained in its predefined conformation and was allowed full torsional flexibility in docking, the ligands localized to discrete binding volumes in the E1 model and to a single central binding space, together with secondary peripheral locations, in the E2 conformation. The results of these binding studies are in good agreement with current site-directed mutagenesis data and support the suggestion that the binding site is proximal to the loop between TM5 and TM6 and TM8, the transmembrane (TM) region considered important for cation translocation. Furthermore, the results of the simulation with the flexible ligand complement the solid-state NMR structural constraints of this inhibitor when bound in situ to the protein.     

Biochemical properties of a newly synthesized H(+)/K(+) ATPase inhibitor, 1-(2-methyl-4-methoxyphenyl)-4-

A new compound, 1-(2-methyl-4-methoxyphenyl)-4-[(3-hydroxypropyl)amino]-6-methyl-2,3-dihydropyrrolo[3,2-c]quinoline (DBM-819), inhibited gastric H(+)/K(+) ATPase in the rabbit (EC 3.6.1.3) with an IC(50) value of 5 microM. However, DBM-819 was a weak inhibitor of kidney Na(+)/K(+) ATPase in the dog, indicating that it has selectivity for the gastric H(+)/K(+) ATPase. The inhibition was reversible and non-competitive with respect to the activating cation K(+). The presence of dithiothreitol did not protect the H(+)/K(+) ATPase from inactivation. The inhibition by DBM-819 was potentiated by acid pretreatment of the compound, suggesting that DBM-819 is converted into a more active intermediate under acidic conditions. The results suggest that DBM-819 is a potent, selective and reversible inhibitor of gastric H(+)/K(+) ATPase, and that the essential cysteine residue may not be involved in the DBM-819-mediated inactivation of gastric H(+)/K(+) ATPase.     

Effect of stilbene derivatives on gastric H+, K(+)-ATPase.

The effect of naturally occurring hydroxystilbene, 3,3',4,5-tetrahydroxystilbene (piceatanol), and its derivatives on gastric H+, K(+)-ATPase was studied. Piceatanol inhibited H+, K(+)-ATPase in a dose-dependent manner. The 50% inhibition value was 4.3 x 10(-6) M. It was found from the kinetic study that the inhibition of the enzyme by piceatanol was competitive with respect to ATP and was noncompetitive with respect to K+. Piceatanol also effectively inhibited gastric acid secretion. However, methylation of phenolic hydroxy groups of piceatanol resulted in a complete loss of inhibition of the enzyme and acid secretion, suggesting the role of phenolic hydroxy groups in the inhibition. The study on hydroxystilbene derivatives also showed that phenolic hydroxy groups are important in the interaction with H+, K(+)-ATPase and that stilbenes with neighbouring hydroxy groups are the most effective inhibitors.     

Pharmacological properties of a newly synthesized H(+)/K(+) ATPase inhibitor, 1-(2-methyl-4-methoxyphenyl)-4-

A new compound, 1-(2-methyl-4-methoxyphenyl)-4-[(3-hydroxypropyl)amino]-6-methyl-2,3-dihydropyrrolo[3,2-c]quinoline (DBM-819), given intraduodenally in pylorus-ligated rats, inhibited basal acid secretion with an ED(50) value of 3.5 mg/kg. In addition, DBM-819 reduced histamine- and pentagastrin-stimulated gastric acid secretion with ED(50) values of 4.0 and 5.1 mg/kg, respectively. The duration of the anti-secretory effect was approximately 18 h when DBM-819 was administered orally to rats with a chronic gastric fistula. Oral administration of DBM-819 protected against gastric lesions induced by ethanol, NaOH, indomethacin and aspirin, and the duodenal ulcer induced by cysteamine, in a dose-dependent manner with ED(50) values of 7.0, 20, 3.1, 4.0 and 6.0 mg/kg, respectively. Taken together, these results suggest that DBM-819 acts as an effective oral anti-ulcer agent in vivo, and that DBM-819 could be developed as a new therapeutic agent for peptic ulcer disease.     

Antimicrobial screening of novel synthesized benzimidazole nucleus containing 4-oxo-thiazolidine derivatives

As a part of systematic investigation of synthesis, characterization and antimicrobial screening of some novel 3-(2-(6-methyl-1H-benzo[d]imidazol-2-yl)phenyl)-2-(aryl)thiazolidin-4-ones (3a–l), have been synthesized from Schiff bases of N-arylidene-2-(6-methyl-1H-benzo[d]imidazol-2-yl)anilines, (2a–l). The Schiff bases (2a–l) were prepared by condensation of different aldehydes with 2-(6-methyl-1H-benzo[d]imidazol-2-yl)aniline (1). The compound (1) was obtained from 2-amino benzoic acid and 4-methylbenzene-1,2-diamine. All the synthesized compounds were screened for in vitro antibacterial and antifungal activities on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus pyogenes, Candida albicans, Aspergillus niger, and Aspergillus clavatus. The structures of the compounds synthesized were elucidated by IR, ¹H-NMR, ¹³C-NMR, and mass spectra.     

Effects of omeprazole and famotidine on (H+-K+) ATPase and acid secretion in rabbit gastric glands

Effects of omeprazole, an anti-ulcer drug, on (H+-K+) ATPase activity and gastric acid secretion in a gastric mucosal gland preparation from rabbits were investigated. The mode of action of the substance was compared with famotidine, and H2 antagonist, by examining the effects of both drugs on the (H+-K+) ATPase of the rabbit gastric mucosa and on gastric acid secretion from the isolated rabbit gastric glands. Optimal assay conditions for (H+-K+) ATPase activity differed slightly from that reported for pig gastric mucosa, and they were pH 7.0, 2 mM of MgCl2 and 50 mM of KCl. Omeprazole dose-dependently inhibited the enzyme activity with an IC50 of 4.2 microM, whereas famotidine was not inhibitory even at the highest concentration of 100 microM. Acid secretion in the glands was determined by measuring accumulation of 14C-aminopyrine. Omeprazole and famotidine showed almost the same inhibitory effect against histamine-stimulated gastric secretion, and their IC50 values were 0.35 microM. Omeprazole inhibited dibutyryl cyclic AMP-stimulated gastric acid secretion, but famotidine was not inhibitory even at the highest concentration of 100 microM. The reason for this difference was that (H+-K+) ATPase activity is linked to the final step of acid secretion. From these results, omeprazole can be expected to be useful for the treatment of peptic ulcer disease.