Björn Wallmark

Plasma gastrin and gastric enterochromaffinlike cell activation and proliferation. Studies with omeprazole and ranitidine in intact and antrectomized rats.

Unoperated female rats were subjected to daily oral treatment with omeprazole (10 or 400 mumol/kg body wt), ranitidine (175 + 175 + 350 mumol/kg body wt), or vehicle and antrectomized rats were treated with omeprazole (400 mumol/kg body wt) or vehicle. After 10 wk of treatment, plasma gastrin levels were high in unoperated rats treated with the high omeprazole dose and with ranitidine, and low in antrectomized controls. Plasma gastrin levels were slightly higher in the low-dose omeprazole group than in the intact controls. In antrectomized rats treated with the high dose of omeprazole, the plasma gastrin level was in the same range as in intact control rats. A close correlation (r = 0.89, p less than 0.0001) was found between the plasma gastrin level and the oxyntic mucosal enterochromaffinlike cell density (as well as the tissue levels of histidine decarboxylase and histamine in the oxyntic mucosa) in all groups. The somatostatin cell density in the oxyntic mucosa was not altered by the various treatments. During a recovery period of 10 wk after the 10-wk treatment, the enterochromaffinlike cell density and histamine concentration decreased by 30%-40% in the rats treated with the high dose of omeprazole, whereas the corresponding values increased by 50% and 40%, respectively, in the control rats. The difference between the two groups, however, was still statistically significant. Plasma gastrin levels and gastric histidine decarboxylase activity returned to control values during recovery. The results suggest that the observed changes in enterochromaffinlike cell density are related to the plasma gastrin levels and that they are reversible. it is concluded that neither omeprazole nor ranitidine per se is likely to induce proliferation of enterochromaffinlike cells.

Inhibition of osteoclast proton transport by bafilomycin A1 abolishes bone resorption

Osteoclasts are the main bone resorbing cells with capacity to acidify their intimate contact area with bone. Recent studies have suggested that osteoclast acid secretion is carried out by an H(+)-ATPase. We demonstrate here, that specific inhibitor of vacuolar type H(+)-ATPases, bafilomycin A1, inhibits bone resorption in osteoclast cultures as well as blocks proton transport in isolated medullary bone derived microsomes containing a vacuolar type H(+)-ATPase. These results demonstrate an important role of vacuolar H(+)-ATPase in bone resorption.

Omeprazole and bafilomycin, two proton pump inhibitors: differentiation of their effects on gastric, kidney and bone H(+)-translocating ATPases.

The effects of omeprazole and bafilomycin on processes dependent on two different types of H(+)-translocating ATPases were compared. A H(+)-ATPase of the E1E2-type, the H+,K(+)-ATPase, was purified from gastric mucosa. Vacuolar type H(+)-ATPases were prepared both from kidney medulla and from osteoclast-containing medullary bone. H+,K(+)-ATPase-mediated proton transport in gastric vesicles was selectively inhibited by omeprazole with a high potency (inhibitory concentrations greater than or equal to 3 microM) and in time- and pH-dependent manner. This result is consistent with the mechanism of action of omeprazole, which is dependent on acid-induced transformation of the drug into an active inhibitor reacting with luminally accessible sulfhydryl groups of the enzyme. Accordingly, the presence of the membrane-impermeable mercaptane glutathione did not affect the inhibitory action of omeprazole on the H+,K(+)-ATPase. Proton transport in kidney- and bone-derived membrane vesicles was also inhibited by omeprazole, but with a lower potency (inhibitory concentrations greater than or equal to 100 microM). Furthermore, the presence of glutathione totally abolished this inhibition, indicating that cytosolic, rather than luminal, SH-groups of the respective vacuolar H(+)-ATPase were interacting with omeprazole at high concentrations. In line with these results, it was found that omeprazole was much more potent in inhibiting acid production in isolated gastric glands (IC50 approximately 0.25 microM) than in inhibiting osteoclast-mediated 45Ca-release in isolated mouse calvaria (IC50 approximately 200 microM). Bafilomycin, on the other hand, was much more effective in inhibiting proton transport mediated by the vacuolar H(+)-ATPases in the kidney- and bone-derived membrane vesicles (IC50 approximately 2 nM) than in inhibiting H+,K(+)-ATPase-mediated proton transport in gastric membrane vesicles (IC50 approximately 50 microM). Thus, approximately 10(4) times higher concentrations of bafilomycin were needed to inhibit the H+,K(+)-ATPase to the same extent as the vacuolar H(+)-ATPase. A similar difference in potency of bafilomycin was found when its inhibitory effect was determined in isolated mouse calvaria (IC50 approximately 2.5 nM) and in isolated gastric glands (IC50 approximately 5 microM). Hence, omeprazole was found to be a specific inhibitor of the H+,K(+)-ATPase under physiological conditions, i.e. in the presence of glutathione, while bafilomycin was found to be selective towards vacuolar H(+)-ATPases.

Time-Course of Development and Reversal of Gastric Endocrine Cell Hyperplasia After Inhibition of Acid Secretion: Studies With Omeprazole and Ranitidine in Intact and Antrectomized Rats

In intact rats plasma gastrin levels were increased during a 20-wk treatment course with either omeprazole or ranitidine. Although plasma gastrin levels were the same during treatment, the enterochromaffinlike (ECL) cell density increased approximately linearly with time at a rate correlated to the plasma gastrin level. Antrectomy prevented the ECL cell hyperplasia seen in omeprazole-treated rats, suggesting that it was not caused by omeprazole per se. Changes in ECL cell density roughly paralleled changes in oxyntic mucosal histidine carboxylase activity and histamine concentration. Treatment with omeprazole also raised stomach weight and antral gastrin and gastrin cell density, reduced antral somatostatin cell density, but did not affect enterochromaffin cell density. Within 19 days of cessation of a 10-wk treatment course, plasma gastrin levels, oxyntic mucosal histidine decarboxylase activity, and antral gastrin and somatostatin cell densities had returned to control levels. The stomach weight was normal within 5-10 wk, antral gastrin concentration within 10 wk, and oxyntic mucosal ECL cell density and histamine concentration within 20 wk. After renewed treatment with omeprazole for 10 wk starting 10 wk after completion of the first omeprazole treatment period, changes in all parameters were of similar magnitude in animals previously treated with omeprazole and those previously treated with vehicle. The results suggest that the effects described are reversible and that gastrin cells turn over more rapidly than ECL cells.

Evidence for acid-induced transformation of omeprazole into an active inhibitor of (H+ + K+)-ATPase within the parietal cell

The chemical reactions of omeprazole, leading to inhibition of gastric acid secretion, were investigated. In acid buffer solutions, omeprazole was found to be labile, whereas at physiological pH it was stable (t1/2 greater than 17 h at pH 7.4). The stability of omeprazole was also studied in isolated, acid producing, gastric glands under conditions where acid formation was either stimulated or inhibited. The rate of transformation of omeprazole was high (t1/2 approximately 3 min) under stimulation. Inhibition of acid formation in the gland greatly retarded the decomposition of omeprazole (t1/2 approximately 73 min). The time-course for inhibition of acid formation by omeprazole was parallel to that for decomposition. The major product formed from omeprazole was the reduced form, H 168/22. The inhibitory action of omeprazole was shown to depend on acid-induced transformation, since no inhibition was obtained when omeprazole was incubated under neutral conditions, both in the isolated gastric mucosal- and the (H+ + K+)-ATPase preparations. Despite the fact that H 168/22 was the major product formed in the glandular preparation, it was found to be virtually inactive in both the glandular- and (H+ + K+)-ATPase preparations. Therefore, a model is proposed in which the inhibition of acid formation by omeprazole is mediated by a compound formed during the reduction of omeprazole to H 168/22 within the acid compartments of the parietal cell. Furthermore, mercaptanes, such as beta-mercaptoethanol, were found to prevent as well as reverse inhibition by omeprazole in both the glandular- and (H+ + K+)-ATPase preparations. This indicates that -SH groups are most likely involved in the chemical reactions leading to inhibition of acid secretion.

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).

The mechanism for inhibition of gastric (H+ + K+)-ATPase by omeprazole

Omeprazole was found to inhibit the K+-stimulated ATPase activity of the gastric (H+ + K+)-ATPase in parallel with the K+-stimulated p-nitrophenylphosphatase activity and the phosphoenzyme formation. The degree of inhibition of ATPase activity was directly correlated to the amount inhibitor bound to the enzyme preparation down to about 15% of the control enzyme activity. The acid-decomposed form of omeprazole, i.e. the inhibitory form, was found to react with and bind to sulfhydryl groups within the (H+ + K+)-ATPase preparation with close to a 1:1 stoichiometry. beta-Mercaptoethanol, when added beforehand and in a 10-fold excess of omeprazole, completely prevented binding of the inhibitor and its inhibition of the enzyme. In the presence of beta-mercaptoethanol two different reaction products could be detected in addition to omeprazole; the reduced form of omeprazole (H 168/22), and a product formed between beta-mercaptoethanol and a decomposition product, generated from omeprazole. Under those conditions neither inhibition nor binding was obtained, indicating that none of these three compounds was the inhibitor. Rather, the compound generated from omeprazole and reacting rapidly with either beta-mercaptoethanol or the -SH groups of the enzyme was the likely inhibitor compound. In order to reverse already established inhibition higher concentrations of beta-mercaptoethanol were needed than for protection indicating two different reaction pathways for protection and reversal by beta-mercaptoethanol. The reversal reaction was explained by a two-step reaction; in the first step the bound inhibitor was exchanged for a beta-mercaptoethanol molecule resulting in formation of compound H 168/22 and a mixed disulfide between the enzyme and beta-mercaptoethanol. In the second step, attack of another beta-mercaptoethanol molecule results in liberation of active enzyme and generation of the disulfide form of beta-mercaptoethanol. This hypothesis was substantiated by the fact that when 1 mM beta-mercaptoethanol was added to inhibited enzyme the radiolabel was partially displaced, without any change in the concentration of modified -SH groups.

Mechanism of Action of Omeprazole

The inhibitory effect of omeprazole on gastric acid secretion in vivo and in vitro is presented. In the gastric fistula dog omeprazole was found to be about 10 times more potent than cimetidine. When omeprazole was administered in vivo, the inhibition of acid secretory rates was found to correlate with the degree of inhibition of the gastric H+K+ATPase purified from the omeprazole treated animals. The inhibitory action of omeprazole was found to depend on acid induced transformation of omeprazole into an active inhibitor of the gastric H+K+ATPase, as no inhibition was obtained when omeprazole was incubated under neutral conditions with either the isolated gastric mucosal or the H+K+ATPase preparations. A model is proposed in which the inhibition of acid formation is mediated by an inhibitory compound generated form omeprazole within the acid compartment of the parietal cell.

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

Abstract 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 .

Specific labelling of gastric H+,K+-ATPase by omeprazole

Abstract Acid secretion is conducted by the parietal cell of the gastric mucosa. The H + ,K + -ATPase has been shown to be specifically located to this cell and during recent years been recognized as the gastric proton pump. Omeprazole, a known inhibitor of acid secretion, administered in vivo was found to bind specifically to the H + ,K + -ATPase of the rabbit gastric mucosa. A stoichiometry of 2.1 mol radiolabel per mol phosphoenzyme was calculated at total inhibition of the H + ,K + -ATPase enzyme activity. In isolated gastric glands prepared from omeprazole-treated animals, the secretagogue-induced increase in oxygen consumption, related to acid secretion, was inhibited to the same level as the H + ,K + -ATPase activity. Both the degree of acid secretion inhibition induced by omeprazole and the amount of inhibitor bound to the H + ,K + -ATPase were found to be dependent on the stimulation state of the parietal cell. Inhibition of secretion by the H 2 -receptor blocker ranitidine prior to omeprazole treatment prevented both the inhibition of H + , K + -ATPase and oxygen consumption normally observed with omeprazole and, furthermore, reduced the binding levels of radiolabel to the enzyme. Inhibition of acid secretion by the H + , K + -ATPase inhibitor SCH 28080 totally prevented the binding of radiolabel to the H + , K + -ATPase. The inhibition by omeprazole could be fully reversed in gastric glands and H + ,K + ATPase isolated from omeprazole-treated animals by addition of β-mercaptoethanol. The major product formed during reactivation was the reduced form of omeprazole, compound H 168/22. Neutralization of the gastric glands in vitro with imidazole totally prevented the inhibitory action of omeprazole. These experiments demonstrate the necessity of acid for the inhibition of gastric acid secretion by omeprazole and the binding of the inhibitor to the H + ,K + -ATPase, both in vivo and in vitro , and also the specificity of omeprazole for the H + ,K + -ATPase.