Gastric H+, K(+)-ATPase inhibition by catechins.

Five catechins, (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin and (-)-epigallocatechin gallate, inhibited gastric H+, K(+)-ATPase activity with IC50 values ranging from 1.7 x 10(-4) to 6.9 x 10(-8) M, with (-)-epigallocatechin gallate as the most potent inhibitor. The intensity of inhibitor activity paralleled the number of phenolic hydroxy groups in the molecule. The inhibition of the enzyme by (-)-epicatechin was competitive with respect to ATP and noncompetitive with respect to K+. These findings suggest that the anti-secretory and anti-ulcerogenic effects of catechins previously reported, are due to their inhibitory activity on gastric H+, K(+)-ATPase.     

Novel techniques in the development of osteoporosis drug therapy: the osteoclast ruffled-border vacuolar H+-ATPase as an emerging target

Introduction: Bone loss occurs in many diseases, including osteoporosis, rheumatoid arthritis and periodontal disease. For osteoporosis alone, it is estimated that 75 million people are afflicted worldwide, with high risks of fractures and increased morbidity and mortality. The demand for treatment consumes an ever-increasing share of healthcare resources. Successive generations of antiresorptive bisphosphonate drugs have reduced side effects, minimized frequency of dosing, and increased efficacy in halting osteoporotic bone loss, but their shortcomings have remained significant to the extent that a monoclonal antibody antiresorptive has recently taken a significant market share. Yet this latter, paradigm-shifting approach has its own drawbacks.

Areas covered: This review summarizes recent literature on bone-remodeling cell and molecular biology and the background for existing approaches and emerging therapeutics and targets for treating osteoporosis. The authors discuss vacuolar H⁺-ATPase (V-ATPase) molecular biology and the recent advances in targeting the osteoclast ruffled-border V-ATPase (ORV) for the development of novel antiresorptive drugs. They also cover examples from the V-ATPase-targeted drug discovery literature, including conventional molecular biology methods, in silico drug discovery, and gene therapy in more detail as proofs of concept.

Expert opinion: Existing therapeutic options for osteoporosis have limitations and inherent drawbacks. Thus, the search for novel approaches to osteoporosis drug discovery remains relevant. Targeting the ORV may be one of the more selective means of regulating bone resorption. Furthermore, this approach may be effective without removing active osteoclasts from the finely balanced osteoclast–osteoblast coupling required for normal bone remodeling.     

Effects of prodigiosin 25-C on cultured cell lines: its similarity to monovalent polyether ionophores and vacuolar type H(+)-ATPase inhibitors.

Prodigiosin 25-C inhibited the proliferation of various cultured cell lines more strongly when concanavalin A (Con A) was added to the cultures. The increase in sensitivity was most evident in T lymphoma YAC-1 cells. The combination of prodigiosin 25-C and Con A induced characteristic morphological changes in these cells. In the presence of Con A, monovalent polyether ionophores and vacuolar type H(+)-ATPase inhibitors induced effects similar to those of prodigiosin 25-C on YAC-1 cells. Prodigiosin 25-C had neither K+ionophore activity nor inhibitory effect on vacuolar type H(+)-ATPase. A Golgi mannosidase II inhibitor, swainsonine, inhibited the proliferation of YAC-1 cells only when Con A was added. Prodigiosin 25-C and swainsonine increased Con A binding receptors on the surface of YAC-1 cells. These results suggest that prodigiosin 25-C affects the intracellular transport and/or processing of glycoproteins.     

Interaction of beta-eudesmol with Na+,K(+)-ATPase: inhibition of K(+)-pNPPase activity.

The effect of beta-eudesmol, one of the major components in So-jutsu (Atractylodis Lanceae Rhizoma), on K(+)-dependent p-nitrophenyl phosphatase (K(+)-pNPPase) activity was studied. It inhibited K(+)-pNPPase activity with an I50 value of 4.1 x 10(-4) M. The inhibition rate decreased as the K+ concentration was increased, whereas greater inhibition was observed with high concentrations of either Na+ or ATP. The Ki values for Na+ in the presence of 0, 0.1 and 1 mM ATP were 140, 260 and 310 mM, respectively, but with the addition of beta-eudesmol, these values decreased to 90 mM regardless of the ATP concentration. This study on K(+)-pNPPase activity supports the conclusion obtained from the study on Na+,K(+)-ATPase activity (Satoh K et al., Biochem Pharmacol 44: 373-378, 1992) that is, beta-eudesmol interacts with enzyme in the Na.E1 form and inhibits the reaction step Na.E1----Na.E1-P. Furthermore, in the study of the effects of K+ and beta-eudesmol on K(+)-pNPPase activity, it was confirmed that beta-eudesmol prevents the conformational change of Na.E1----K.E2.     

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.     

A novel inhibitor of vacuolar ATPase, FR167356, which can discriminate between osteoclast vacuolar ATPase and lysosomal vacuolar ATPase

1 Vacuolar ATPase (V-ATPase) has been proposed as a drug target in lytic bone diseases. Studies of bafilomycin derivatives suggest that the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is selective inhibition of osteoclast V-ATPase. Previous efforts to develop therapeutic inhibitors of osteoclast V-ATPase have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, we tried to find novel potent and specific V-ATPase inhibitors, which have new structural features and inhibition selectivity, from random screening using osteoclast microsomes. Finally, a novel V-ATPase inhibitor, FR167356, was obtained through chemical modification of a parental hit compound. 2 FR167356 inhibited not only H+ transport activity of osteoclast V-ATPase but also H+ extrusion from cytoplasm of osteoclasts, which depends on the V-ATPase activity. As expected, FR167356 remarkably inhibited bone resorption in vitro. 3 FR167356 also showed inhibitory effects on other V-ATPases, renal brush border V-ATPase, macrophage microsome V-ATPase and lysosomal V-ATPase. However, FR167356 was approximately seven-fold less potent in inhibiting lysosomal V-ATPase compared to osteoclast V-ATPase. Moreover, LDL metabolism in cells, which depends on acidification of lysosome, was blocked merely at higher concentration than bone resorption, suggesting that FR167356 inhibits V-ATPase of osteoclast ruffled border membrane still more selectively than lysosome at the cellular level. 4 These results from the experiments seem to indicate that osteoclast V-ATPase may be different from lysosomal V-ATPase in respect of their structure. 5 FR167356 had a novel chemical structural feature as well as inhibitory characteristics distinctly different from any previously known V-ATPase inhibitor family. Therefore, FR167356 is thought to be a useful tool for estimating the essential characteristics of V-ATPase inhibitors for drug development.     

Selective inhibition of K(+)-stimulation of Na,K-ATPase by bretylium.

1. The effects of bretylium were investigated on purified Na,K-ATPase from guinea-pig heart and on the Na/K pump in trout erythrocytes, with a view to further identifying the mechanism(s) associated with its antiarrhythmic effects. 2. Na,K-ATPase activity of the thiocyanate-dispersed enzyme was determined by the measurement of inorganic phosphate produced by ATP hydrolysis. 3. When the concentrations of each of the Na,K-ATPase activating components were varied in turn, bretylium (1-5 mmol l-1) exhibited competitive-type effects against K+ with a Ki of 1.4 mmol l-1 and noncompetitive-type effects against Na+, Mg2+ and ATP. 4. In K+ influx studies in trout erythrocytes with 86Rb+ used as the marker, the inhibition of total influx observed with bretylium (5 and 10 mmol l-1) was attributable to the bretylium cation selectively inhibiting the Na/K pump-mediated influx with the associated tosylate anion inhibiting Na/K cotransport. 5. The observed inhibition kinetics indicated that the bretylium cation (2-15 mmol l-1) competitively inhibited K+ stimulation of the Na/K pump at 6 and 1.25 mmol l-1 external K+ with a mean K1 of 2.3 mmol l-1. 6. The effects demonstrated on the functioning Na/K pump in erythrocytes confirmed the Na,K-ATPase findings, with bretylium selectively inhibiting K+ stimulation of the pump mechanism in both cases. 7. It is suggested that Na,K-ATPase inhibition may contribute to the antiarrhythmic and positive inotropic effects of bretylium with the cardiac accumulation of bretylium also possibly being a further important factor.     

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 gastric H+,K(+)-ATPase by the anti-ulcer agent, sofalcone.

Effects of the anti-ulcer agent, sofalcone, on gastric H+,K(+)-ATPase were studied as well as those of other chalcone derivatives, chalcone and sophoradin. These drugs inhibited pig gastric H+,K(+)-ATPase in a dose-dependent manner. They were 5-10-fold less inhibitory toward Na+,K(+)-ATPase than H+,K(+)-ATPase. The potencies of these drugs on the inhibition of enzymes were as follows: sophoradin greater than sofalcone greater than chalcone. Kinetic studies showed that the inhibition of H+,K(+)-ATPase by sofalcone was competitive with respect to ATP and was non-competitive with respect to K+. Sofalcone also inhibited H+,K(+)-ATPase mediated proton transport and reduced the phosphoenzyme level. These results suggest that sofalcone inhibits gastric H+,K(+)-ATPase competitively with ATP at the ATP site and thereby blocks the phosphorylation of the enzyme. This may be the cause of the anti-secretory activity of sofalcone.     

Vacuolar H(+)-ATPase: functional mechanisms and potential as a target for cancer chemotherapy

Tumor cells in vivo often exist in a hypoxic microenvironment with a lower extracellular pH than that surrounding normal cells. Ability to upregulate proton extrusion may be important for tumor cell survival. Such microenvironmental factors may be involved in the development of resistant subpopulations of tumor cells. In solid tumors, both intracellular and extracellular pH differ between drug-sensitive and -resistant cells, and pH appears critical to the therapeutic effectiveness of anticancer agents. Four major types of pH regulators have been identified in tumor cells: the sodium-proton antiporter, the bicarbonate transporter, the proton-lactate symporter and proton pumps. Understanding mechanisms regulating tumor acidity opens up novel opportunities for cancer chemotherapy. In this minireview, we describe the structure and function of certain proton pumps overexpressed in many tumors--vacuolar H(+)-ATPases--and consider their potential as targets for cancer chemotherapy.     

Specific H+/K(+)-ATPase inhibitors decreased contractile responses of isolated rat vas deferens.

The effect of H(+)/K(+)-ATPase inhibitors on rat vas deferens contractility was investigated in vitro. Omeprazole (100-300microM), lansoprazole (100-300microM) and SCH 28080 (10-100microM) (2-methyl-8-(phenylmethoxy)-imidazo[1,2-a]pyridine-3-acetonitrile) decreased contractile responses of vas deferens to electrical field stimulation, high K(+) (80mM) and phenylephrine in a reversible, reproducible and concentration-dependent manner. The inhibitory potency of lansoprazole on vas deferens contractility was increased in relatively acidic solution (pH 6.9), suggesting that the site of action may be related to H(+)/K(+)-ATPase. However, lansoprazole-induced inhibition on contractility was unaltered in K(+) free solution, indicating that the mechanism of action is independent from H(+)/K(+)-ATPase. Reversible nature of omeprazole and lansoprazole-induced inhibition on contractility also suggests that the effects are not due to inhibition of H(+)/K(+)-ATPase, since both compounds are irreversible inhibitors of the enzyme. Presence of ouabain (5microM) did not decrease lansoprazole-induced inhibition on contractility but potentiated the inhibitory effect of lansoprazole, suggesting that lansoprazole-induced inhibition is not mediated by the inhibition of Na(+)/K(+)-ATPase. Calcium-induced contractions in high K(+)-Ca(2+) free medium were completely antagonized by lansoprazole, implying that lansoprazole inhibits Ca(2+) entry through voltage-gated channels. In conclusion, three H(+)/K(+)-ATPase inhibitors decreased contractile responses of rat vas deferens to various stimulants in vitro. They may act on a common mechanism, which plays a crucial role in regulating rat vas deferens contractility and this mechanism is probably involved in the regulation of intracellular Ca(2+).     

Interaction of bretylium tosylate with guinea-pig myocardial Na(+)-K(+)-ATPase.

1. The effects of bretylium on myocardial Mg(2+)-dependent, Na(+)-K(+)-ATPase (EC 3.6.1.3) activity were compared with those of ouabain in guinea-pig heart preparations. 2. Both ouabain and bretylium inhibited microsomal Na(+)-K(+)-ATPase activity in a concentration-dependent fashion in the range of 0.01-100 and 1.0-4000 microM, respectively. The IC50 values were 1.93 +/- 0.27 microM for ouabain and 2.45 +/- 0.17 mM for bretylium. 3. In another set of experiments, the effects of bretylium on the enzyme activity were tested in combination with 2.5 or 5.0 microM ouabain. 4. The combined effects of the two drugs resulted in a net reduction in the total inhibitory activities of the individual drugs, which became more marked the higher the bretylium concentration. This trend seems to suggest a competitive mode of interaction of the two drugs in their inhibitory actions on Na(+)-K(+)-ATPase activity. 5. The results demonstrate therefore that bretylium is a potent inhibitor of ouabain-inhibited ATP hydrolysis by myocardial Na(+)-K(+)-ATPase. These actions may be pertinent with regard to some of its cardiac actions.     

Studies on the mechanism of action of the gastric H+,K(+)-ATPase inhibitor SPI-447

3-Amino-5-methyl-2(2-methyl-3-thienyl)- imidazo[1,2-a]thieno[3,2-c]pyridine, SPI-447, is a potent gastric H+,K(+)-ATPase inhibitor, but a detailed mechanism of the inhibition is unknown. This study was designed to investigate the mechanism by which SPI-447 inhibits gastric H+,K(+)-ATPase. For this purpose, the inhibitory action of SPI-447 on gastric H+,K(+)-ATPase from porcine gastric mucosa was compared with that of omeprazole (an irreversible inhibitor) and SCH28080 (a reversible inhibitor). All compounds produced dose-dependent inhibition of gastric H+,K(+)-ATPase, and the inhibitory intensities were increased under acidic conditions. The anti-H+,K(+)-ATPase actions of SPI-447 and SCH28080 were attenuated by dilution, but not influenced by glutathione pretreatment. In contrast, that of omeprazole was not influenced by dilution, but was suppressed by glutathione pretreatment. KCl addition reversed the inhibition of H+,K(+)-ATPase-mediated H(+)-transport by SPI-447 and SCH28080, but had no effect on that by omeprazole. The anti-gastric H+,K(+)-ATPase action of SPI-447 was additive with that of SCH28080. SPI-447 and SCH28080 had no effect on Na+,K(+)-ATPase activity. These findings indicated that the inhibitory mechanism of SPI-447 on gastric H+,K(+)-ATPase was similar to that of SCH28080, but different from that of omeprazole; i.e., 1) reversible, 2) SH-group independent, 3) K(+)-competitive, and 4) highly specific against gastric H+,K(+)-ATPase.     

Drug-induced parkinsonism: cinnarizine and flunarizine are potent uncouplers of the vacuolar H+-ATPase in catecholamine storage vesicles.

Cinnarizine (1-diphenylmethyl-4-(3-phenyl-2-propenyl)piperazine) and its di-fluorinated derivative flunarizine inhibit the MgATP-dependent generation of a transmembrane proton electrochemical gradient in chromaffin granule ghosts. The concentrations giving 50% inhibition (IC50) of the MgATP-dependent generation of the pH-gradient were 5.9+/-0.6 microM (n = 6) and 3.0+/-0.3 microM (n = 5) for cinnarizine and flunarizine, respectively. The IC50 values for inhibiting the generation of the membrane potential were even lower, i.e. 0.19+/-0.06 microM (n = 6) and 0.15+/-0.01 microM (n = 4) for cinnarizine and flunarizine, respectively. Cinnarizine (10 microM) also inhibited the energy-dependent vesicular uptake of [14C]-dopamine (50 microM) by 76%, i.e. from 2.1+/-0.9 to 0.5+/-0.6 nmol/mg protein/min (n = 5, P < 0.002). Cinnarizine (10 microM) increased the MgATPase activity of the granule ghosts by 47+/-26% (n = 4) compatible with an uncoupling of the vacuolar H+-ATPase activity. The IC50-values observed for the two compounds are in the same range as their reported therapeutic plasma concentrations in vivo, suggesting that cinnarizine and flunarizine may well inhibit proton pumping and catecholamine uptake in storage vesicles also in vivo. This mechanism of action may contribute to the drug-induced parkinsonism seen as a side-effect of the two drugs.     

Cibenzoline, an ATP-sensitive K(+) channel blocker, binds to the K(+)-binding site from the cytoplasmic side of gastric H(+),K(+)-ATPase.

1. Cibenzoline, (+/-)-2-(2,2-diphenylcyclopropyl-2-imidazoline succinate, has been clinically used as one of the Class I type antiarrhythmic agents and also reported to block ATP-sensitive K(+) channels in excised membranes from heart and pancreatic beta cells. In the present study, we investigated if this drug inhibited gastric H(+),K(+)-ATPase activity in vitro. 2. Cibenzoline inhibited H(+),K(+)-ATPase activity of permeabilized leaky hog gastric vesicles in a concentration-dependent manner (IC(50): 201 microM), whereas no effect was shown on Na(+),K(+)-ATPase activity of dog kidney (IC(50): >1000 microM). Similarly, cibenzoline inhibited H(+),K(+)-ATPase activity of HEK-293 cells (human embryonic kidney cell line) co-transfected with rabbit gastric H(+),K(+)-ATPase alpha- and beta-subunit cDNAs (IC(50): 183 microM). 3. In leaky gastric vesicles, inhibition of H(+),K(+)-ATPase activity by cibenzoline was attenuated by the addition of K(+) (0.5 - 5 mM) in a concentration-dependent manner. The Lineweaver-Burk plot of the H(+),K(+)-ATPase activity shows that cibenzoline increases K(m) value for K(+) without affecting V(max), indicating that this drug inhibits H(+),K(+)-ATPase activity competitively with respect to K(+). 4. The inhibitory effect of H(+),K(+)-ATPase activity by cibenzoline with normal tight gastric vesicles did not significantly differ from that with permeabilized leaky gastric vesicles, indicating that this drug reacted to the ATPase from the cytoplasmic side of the membrane. 5. These findings suggest that cibenzoline is an inhibitor of gastric H(+),K(+)-ATPase with a novel inhibition mechanism, which inhibits gastric H(+),K(+)-ATPase by binding its K(+)-recognition site from the cytoplasmic side.     

Substituted thieno[3,4-d]imidazoles, a novel group of H+/K(+)-ATPase inhibitors. Differentiation of their inhibition characteristics from those of omeprazole

The action of the H+/K(+)-ATPase inhibitors, Hoe 731 and S 4216, both thieno-imidazole derivatives, was compared with that of the benzimidazole derivative, omeprazole. In intact, gastric membrane vesicles under conditions shown to result in acidification of the vesicle interior. Hoe 731 and S 4216 inhibited H+/K(+)-ATPase activity with an IC50 value of about 1.0 microM. In the absence of a generated pH gradient the respective IC50 values were 5.5 and 2.1 microM. In contrast, omeprazole inhibited the enzyme only in the presence of proton accumulation (IC50: 0.7 microM). The inhibitory action of omeprazole on H+/K(+)-ATPase-mediated proton transport was prevented by the membrane permeable mercaptane, dithioerythritol, but not by the membrane impermeable, mercaptane glutathione, whereas both mercaptanes were able to prevent the effect of Hoe 731 and S 4216. These results indicate that the thienoimidazoles react with intravesicular (luminal) and extravesicular (cytosolic) SH groups of the H+/K(+)-ATPase, whereas omeprazole interacts uniquely with luminal SH groups of the enzyme. In isolated parietal cells all drugs caused a concentration-dependent inhibition of HCl production, as measured by [14C]aminopyrine uptake, during histamine and dibutyryl-cAMP stimulation. The IC50 value was 0.1 microM for Hoe 731 and omeprazole and 0.4 microM for S 4216 after 30-min incubation. The inhibitory action of Hoe 731 and S 4216 faded with increasing incubation time, whereas omeprazole caused an unchanged inhibition over the entire 120-min incubation period. We suggest that several factors, e.g. weaker chemical stability of the drugs or perturbation of cellular glutathione levels, may be responsible for the fading inhibitory action of thienoimidazoles in the parietal cell.     

Durable inhibition of rat cerebral capillary Na+/K(+)-ATPase after in vivo administration of mercuric chloride.

Intraperitoneal administration of a single dose (6 mg/kg body wt.) of mercuric chloride led to a rapid and irreversible inhibition of Na+/K(+)-ATPase activity in rat cerebral capillaries. The activity measured at 1 h, 18 h and 5 days after injection was, respectively, 53, 44 and 26% of the control. By contrast, Mg(2+)-ATPase activity in the capillaries remained uninhibited throughout the observation period. Mercuric chloride administration did not affect either of the two enzyme activities in nerve endings, which is consistent with the inability of the compound to penetrate the blood-brain barrier. The mercuric-chloride-induced impairment of the capillary sodium pump may contribute to disturbances of ion homeostasis in the brain and thus to the neurophysiological abnormalities accompanying this exposure. Direct treatment of the isolated cerebral capillary preparations with mercuric chloride evoked a stronger inhibitory effect on Mg(2+)-ATPase (IC50 = 0.25 microM) than on Na+/K(+)-ATPase (IC50 = 5.0 microM). This result indicates that the effect in vivo may not have resulted from direct interaction of the compound with the latter enzyme.     

Direct comparison between the ulcer-healing effects of two H(+)-K(+)-ATPase inhibitors, one M1-selective antimuscarinic and one H2 receptor antagonist in the rat

A direct comparison of the ulcer-healing effects of two H(+)-K(+)-ATPase inhibitors (pantoprazole and omeprazole), one M1 antimuscarinic (telenzepine) and one H2 receptor antagonist (cimetidine) was performed in the rat. Gastric and duodenal ulcers were induced by local application of acetic acid and thereafter treated over 10 days by the test drugs. Overall and on a molar basis, ulcer healing was comparably accelerated by pantoprazole, omeprazole and telenzepine and less so by cimetidine. The same rank order was found with respect to the inhibition of gastric acid secretion in the modified Shay rat.