Canalicular ATPase

The mechanism of action of drug induced hepatocellular tumors is quite similar to epidermal carcinogenesis. There are a series of biochemical changes such as enzyme induction that are parallel in liver and skin. Hyperplastic nodules result from promoter treatment and these nodules regress if the promoting stimulus is removed up to a critical point, after which the nodule is committed to develop into a hepatoma. The preneoplasic nodules contain enzyme altered foci for y-glutamyltranspeptidase, canalicular ATPase, glu-cose-6-phosphatase and iron deficient foci (Pitot et al., 1982). It is not clear whether or not all of the enzyme alTered foci result in hepatomas but they are certainly direct evidence of enhanced clonal growth or cell section and proliferation of presumably initiated cells. The promotional model can be modified by hormonal and dietary factors and this model has potential use for extrapolation to the human population. 

In 1991 bile-acid secretion was shown to be energy driven by a 110-kDa glycoprotein that was dependent on ATP. This protein was subsequently characterised as liver ecto-ATPase by Sippel and co-workers. However, while further work with COS cells showed that expression of ecto-ATPase enhanced secretion of bile acids purified canalicular membranes lacking this enzyme efficiently exported bile acids showing that at least one other bile-acid transporter existed. ... 

The parietal cell contains receptors for gastrin, histamine (H2), and acetylcholine (muscarinic, M3) (Figure 63-1). When acetylcholine or gastrin bind to the parietal cell receptors, they cause an increase in cytosolic calcium, which in turn stimulates protein kinases that stimulate acid secretion from a H+/K+ ATPase (the proton pump) on the canalicular surface. 

As a weak base (pKa = 3.9), pantoprazole is highly ionized at low pH and readily accumulates in the highly acidic canalicular lumen of the stimulated parietal cell. In this acidic environment, pantoprazole is rapidly converted to the active species, a cationic cyclic sulfonamide, which binds covalently to cysteine residues on the luminal (acidic) surface of H+, K+-ATPase to form a mixed disulphide, thereby causing irreversible inhibition of gastric proton pump function. As H+, K+-ATPase represents the final step in the secretary process, inhibition of this enzyme suppresses gastric acid secretion regardless of the primary stimulus [1],... 

The limited distribution of the H+, K+-ATPase, which is found mainly in the tubulovesicular and canalicular membranes of the gastric parietal cell. This is in contrast to, for example, the more widely distributed H2 and cholinergic receptors. 

Mg -ATPase activity can likewise be inhibited in the canalicular side of the membrane by cholestatic factors (in particular bile acids). This metabolic pump transports bicarbonate and chloride into the canaliculi and is probably closely associated with the function of the microfilaments. 

The H /K -ATPase enzyme, present in tubu-lovesicular and canalicular membranes of the gastric parietal cell, consists of two subunits, a 114-kDa a-subunit and a 34-kDa jS-subunit. The a-subunit has been shown to contain 10 transmembrane helices, with the j3-subunit possessing only a single transmembrane helix (113). The a-subunit carries out the catalytic and transport functions of the enzyme because it contains both ATP and cation binding sites it also contains the sequences responsible for apical membrane localization. When... 

The last mediator of gastric secretion in the parietal cell is an H+,K+-ATPase (proton or acid pump) which is a member of the phosphorylating class of ion transport ATPases. Hydrolysis of ATP results in ion transport. This chemical reaction induces a conformational change in the protein that allows an electroneutral exchange of cytoplasmic H+ for K+. The pump is activated when associated with a potassium chloride pathway in the canalicular membrane which allows potassium chloride efflux into the extracytoplasmic space, and thus results in secretion of hydrochloric acid at the expense of ATP breakdown. The activity of the pump is determined by the access of K+ on this surface on the pump. In the absence of K+, the cycle stops at the level of the phosphoenzyme [137]. 

Enzymes, such as 7-GTP, glucose-6-phosphate (G-6-Pase), and canalicular adenosine triphosphatase (ATPase) have been generally used as markers for assaying initiated cell populations in liver carcinogenesis [15]. In order to confirm the essential similarity of foci induced by DEN plus ABA to those observed after DEN alone, a comparison of histochemical characteristics was made. The foci induced by DEN plus PH, typically showing positive for 7-GTP and periodic acid Schiff stain and negative for G-6-Pase and ATPase. 

The transport reactions of the gastric H,K ATPase. As a function of binding MgATP and H or hydronium (HsO ) ion, the export of protons or hydronium ions occurs after phosphorylation. In the presence of K extracellularly enabled by the presence of a KCI channel in the canalicular membrane, K binds to the outward conformation of the phosphorylated pump. The K is then transported inwardly during the dephosphorylation step. In the absence of K, the pump stops in the E2P conformation. 

In the parietal cell, the Na,K ATPase Is sorted to the basolateral surface and the H,K ATPase to the cytoplasmic tubules destined for Insertion Into the canalicular membrane with stimulation of secretion. The basis for this selective sorting Is not well defined. Part of the sorting signal may be due to the 6 subunit. Recent work has shown that mutation of Tyr 19 of the B subunit In transgenic mice results In retention of the ATPase In the canalicular membrane, suggesting that perhaps phosphorylation of this residue Is Important In the stimulatory cycle of the enzyme. However, this event Is subsequent to the cellular sorting of the two pumps. 

Activity of the H,K ATPase results In a primary secretion of 160 mM of HCl Into the secretory canaliculus. Because the H,K ATPase Is electroneutral. It Is necessary that the KCl permeability pathway assodated with the canaliculus transfer a minimum of 160 mmol of KCl for each liter of acidic fluid secreted. This Is true whether the KCl pathway consists of conductive or electroneutral transporters. It Is likely. In fact, that the KCl pathway allows transfer of a slight excess of KCl over the minimum required for the production of HCl. This Is suggested both by the observation that gastric secretions contain a low but significant concentration of KCl and by the likelihood that the H,K ATPase Is not fully efficient at recovering K from the canalicular fluid. In the absence of other mechanisms, the combined activity of the transporters at the apical pole of the parietal cell would lead to alkallnizatlon of the cell and depletion of cellular Cl" and K. The potential disturbances In electrolyte balance are prevented by the activity of transporters at the basolateral membrane. These Include an anion exchanger (AE, HCOj /Cl"), a sodlum/proton exchanger (NHE), and the Na,K ATPase. 

General reaction mechanism of the PPIs with the H,K ATPase in the membrane of the parietal cell canaliculus, showing passive diffusion across the canalicular membrane, accumulation of the protonated form, conversion to the sulfenamide, and reaction with one or more cysteines in the catalytic subunit of the H,K ATPase. The outline of the pump structure illustrates the vestibule of the pump on its outside surface where binding of the PPIs results in inhibition of acid secretion correlated with inhibition of ATPase activity. 

Figure 2. This illustrates the general mechanism of acid secretion catalyzed by H,K ATPase. It secretes acid only when present in the canalicular membrane. There is activation of a KVCl pathway that enables efflux of KCl and H2O from the cytoplasm of the parietal cell. The K secreted into the lumen of the secretory canaliculus of the parietal cell is transported inward in exchange for by the cycle of phosphorylation/dephosphorylation on the catalytic subunit of the H,K ATPase, leaving HCl in the canalicular lumen.

The nature and origin of the permeability which is associated with the H,K ATPase in the canaliculus remain undefined. It has been proposed that the canalicular membrane contains parallel conductances for and CT or alternatively that there is a KCl cotransporter. Further, the proteins that interact with the enzyme in the cytoplasmic membranes that enable stimulus-dependent conversion to the canalicular form remain elusive, although ezrin and actin are clearly associated with the enzyme in its stimulated form. 

Evidence that the H, K -ATPase was the target enzyme for omeprazole came from autoradiographic studies in which the tubulovesicular and canalicular membranes of the parietal cell were selectively labeled from radiolabeled omeprazole. Other studies including monoclonal antibodies showed that these membrane structures harbor the H, K -ATPase [9-11]. Direct proof that omeprazole inhibited the H, K -ATPase under in vivo conditions came from studies in the rat in which a linear relationship was found between inhibition of the H, K -ATPase and acid secretion [12, 13]. Thus, both direct binding studies and measurements of H, K -ATPase activity provided strong evidence that omeprazole selectively inhibited acid secretion under in vivo conditions. 

By and large, the events leading to inhibition of the H, K -ATPase by the various PPIs can be described in identical terms After intestinal absorption, the drugs are distributed by the bloodstream and gain diffusional access to the canalicular system of the parietal cell. In this acid compartment, they are trapped and acid-activated to form short-lived sulfenamides ready to bind to sulfhydryl groups of the H, K -ATPase. Elimination depends on hepatic metabolism, followed by renal and faecal excretion. Along this way, a number of differences between the various PPIs can be identified. 

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