Studies both in vivo and in vitro have shown that substituted benzimidazoles inhibit the stimulation of acid secretion produced by dibutyryl cyclic AMP and histamine. Furthermore, the results differ from those produced by H2 antagonists and anticholinergic agents in that the inhibition is not competitive, and the site of action is intracellular and peripheral to that of dibutyryl cyclic AMP. To investigate the biochemical mechanism of action of substituted benzimidazoles, one such compound, H 149/94 (2-([2-(3-methyl)pyridyl-methyl]-sulphinyl)-5-methoxycarbonyl-6-methylbenzimidazol), has been tested either directly on an (H+ + K+)ATPase isolated from pig and human gastric mucosa or on the function of this enzyme in gastric glands isolated from rabbit and human gastric mucosa. (H+ + K+)ATPase, which has only been found at the secretory surface of the parietal cell, catalyses a one-to-one exchange of protons and potassium ions. It is possibly the proton pump within the gastric mucosa, and may thus be the terminal or one of the terminal steps of the acid secretory process. We show here that H 149/94 inhibits (H+ + K+)ATPase, which may explain its inhibitory action on acid secretion in vitro and in vivo. Because of the unique distribution and properties of the (H+ + K+)ATPase, the inhibitory action of H 149/94 on this enzyme may be a highly selective clinical means of suppressing the acid secretory process.
The gastric H+,K+ ATPase--the gastric acid pump--is the molecular target for the class of antisecretory drugs called the proton-pump inhibitors (PPIs). These compounds--omeprazole, lansoprazole, and pantoprazole--contain, as their core structure, 2-pyridyl methylsulfinyl benzimidazole. The H+,K+ ATPase is a heterodimer composed of a 1034-amino acid catalytic alpha peptide and a glycosylated 291-amino acid beta subunit. The alpha subunit probably contains 10 membrane-spanning sequences; the beta, a single transmembrane segment. The PPIs have a pKa of about 4.0; hence they accumulate only in the acidic secretory canaliculus of the stimulated parietal cell. Here they undergo conversion to a cationic sulfenamide, which then reacts with available cysteines on the extracytoplasmic face of the alpha subunit. Omeprazole reacts and forms disulfide bonds with cys813(822) and cys892; lansoprazole, with cys813(822), cys892, and cys321; and pantoprazole, with cys813 and -822. The antisecretory effect of the drugs reflects their short plasma half-life (approximately 60 min), the number of active pumps during that time, and the recovery of pumps following biosynthesis and reversal of inhibition. These drugs also show synergism with either amoxicillin or clari- thromycin in eradicating Helicobacter pylori, an organism shown to be important in duodenal and gastric ulcer disease. Their action is probably due to elevation of pH in the environment of the organism, rather than to any direct action.
Abstract. Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H+-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60-and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatmerit of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K+-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through vacuolar H+-ATPase.STEOCLASTS are multinucleated giant cells that are responsible for bone resorption. Like secreting epithelial cells, they are polarized when active in bone resorption, showing three distinct specialized cell membrane areas (for review see Vaes et al., 1988). In addition to the basolateral membrane, which is rich in Na+,K+-ATPase (Baron et al., 1986), resorbing osteoclast exhibits a clear zone that mediates the attachment of resorbing cells to the bone matrix. The cytoplasm in the vicinity of this clear zone contains specialized cytoskeletal structures (Holtrop et al., 1974;Lakkakorpi et al., 1989). The third specialized membrane area, the ruffled border, faces the actual bone resorption site on the bone surface. The resorption lacuna underneath the ruffled border membrane is acidic. This has been shown by acridine orange accumulation experiments (Anderson et al., 1986; Bar6n et al., 1985) and direct micropuncture measurements (Fallon, 1984). The acidic pH favors dissolution of the bone mineral. In addition, proteinases active at acid pH and capable of collagen degradation are present in osteoclasts (Vaes et al., 1988; Blair et al., 1986). Enzyme histochemistry suggests the presenc$ of ATPase activity in the plasma membrane of the osteoclast- (Akisaka et al., 1986). Baron et al. (1985) found at the ruffled:border of the osteoclast a 100-kD lysosomal membrane polypeptide that showed immunological similarity to gastric H+,K+-ATPase. Now we report that osteoclasts contain an ATP-dependent proton pump that is clearly different from the gastric proton pump and from the mitochondrial proton pump but shows considerable immunological similarities to the vacuolar type H+-ATPase of Neurospora crassa. Materials and Methods Preparation of Bone MicrosomesBone microsomes were prepared from medullary bone of regularly laying hens. Medullary bone from the tibia and femur was dissected out and immediately homogenized in a medium containing 5 mM Tris pH 7.4, 250 mM sucrose, 1 mM K2CO3, 1 mM DTT, and 1 mM EGTA in a glass-Teflun ...
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