Tubulovesicles

In whole-body autoradiography studies in mice, the radiolabel originating from uC-labeled omeprazole was found to be confined to the gastric mucosa (Fig. 2.6). With a similar technique, and using both fight and electron microscopic evaluation, omeprazole was found to label only the tubulovesicles and secretory membranes of the parietal cell, which contain the H+, K+-ATPase [3]. Electrophoretic analyses of the proteins from such membranes, purified after systemic administration of... 

Trypsinization of H,K-ATPase-enriched gastric microsomal vesicles. Tubulovesicles (-100 pg of protein) were treated with trypsin (5 pg) in Tris.HCl (20 mM, pH 7.5) at 37°C for 30 min. The vesicles were next centrifuged at 100,000 X g on a TLIOO table top centrifuge for 1 hr at 4°C. The supernatant was carefully separated from the pellet. The supernatant was next boiled for 5 min and stored at -20°C until further analysis. 

Figure 3. MALDI mass spectrum of the total supernatant from the tryptic digest of the H,K-ATPase-enriched tubulovesicles. The H,K-ATPase was digested with tiypsin and the vesicles centrifuged to separate supernatant from the pellet. An aliquot of the supernatant was analyzed by MALDl/MS in the reflectron ion mode using a-cyano 4-hydroxy cinnamic acid as a matrix. The signals are denoted by numbers and were assigned to a-subunit peptides (Table 1).

After the stomach has reached a sufficiently low pH to denature food proteins and to allow the full activity of pepsin, the proton pumps are retrieved from the plasma membrane. This retrieval occurs in the same manner as the procesis of endocytosis, except that membrane-bound proteins rather than the soluble contents in the interior of the vesicles are being retrieved, in short, parts of the plasma membrane bud off, reforming the tubulovesicles. 

Ion Exchange and Transport Systems of the Tubulovesicles and Plasma Membrane... 

When the resting parietal cell is stimulated by acid secretagogues, the tubulovesicles are transformed into the secretory canaliculus. The parietal cell has the largest mitochondrial content of any mammalian cell (—34% of cell volume) and the ATP generated by this is mainly used for acid secretion. Hydrolysis of ATP results in a conformational change in the protein that mediates the electroneutral exchange of intracellular and extracellular K+. The pump is activated only when it is associated with a potassium chloride pathway in the canalicular membrane (Fig. 3.6). This allows potassium chloride efflux into the extra-cytoplasmic space and thus results in the secretion of HCl at the expense of ATP... 

Schematic representation of the resting (left side) and stimulated (right side) state of the parietal cell. Basolateral membrane contains three major receptor classes gastrin (G), acetylcholine (ACh), and histamine (H). Their actions are mediated by cAMP responses, Ca changes, or both. In addition, there are a number of ion transport pathways. In the stimulated state, the apical membrane acquires H", K -ATPase contained in the tubulovesicles (tv) as well as the property of K+ and CI conductance, both of which are essential in the secretion of HCl. A change in cytoskeletal arrangement is also associated with stimulation. CaM = calmodulin SC -secretory canaliculus mf = microfilaments. [Reproduced with permission from D. H. Malinowska and G. Sachs, Cellular mechanisms of acid secretion, Clin. Gastroenterol. 13, 322 (1984).]...

Gerbino, A. Hofer, A. M. Mckay, B. Lau, B. W. Soybel, D. I. Divalent cations regulate acidity within the lumen and tubulovesicle compartment of gastric parietal cells. Gastroenterology 2004,126, 182-195. 

Electron micrographs of a resting and stimulated parietal cell showing the conversion of the cytoplasmic tubulovesicles to the microvilli of the secretory canaliculus. 

A model depicting turnover and biosynthesis of the H,K ATPase is shown in the figure. The two subunits of the ATPase are synthesized and coassembled in the endoplasmic reticulum and proceed to the trans-Golgi. From there, tubules are budded off. The turnover of the protein in the cytoplasmic tubules is relatively slow compared to when the protein is present in the membrane of the secretory canaliculus, where it is subject to endocytosis. On the right is shown the synthesis of the two subunits followed by processing in the Golgi. The mature pump subunits are inserted into the tubulovesicles. Stimulation of add secretion by histamine or acetylcholine results in rapid movement to the secretory canaliculus (t 2 = 5 minutes). Return from the canaliculus to the tubulovesicles has a t 2 of 60 minutes. The half-life of the pump protein is approximately 50 hours. 

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