John G. Forte

K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells

Abstract Differential centrifugation of oxyntic cell homogenates yielded microsomal fractions which contained large amounts of mitochondrial membrane. The presence of marker enzymes (succinate dehydrogenase and cytochrome c oxidase) indicated that mitochondrial contamination of crude microsomes ranged from 20 to 60% in different preparations. A discontinuous sucrose density gradient procedure was developed for the routine preparation of purified oxyntic cell microsomes. A K + -stimulated, Mg 2+ -requiring ATPase was localized in these purified membranes and coincided with the presence of a K + -stimulated p -nitrophenylphosphatase. Na + and ouabain had no effect on the K + stimulation of the microsomal ATPase. The apparent activation constant for K + was approximately 1 mM at pH 7.5, the optimal pH for stimulation. An anion-sensitive ATPase has been widely studied in gastric microsomal preparations. We found that the basal microsomal ATPase ( i.e. without K + ) and the mitochondrial ATPase were inhibited by SCN − and enhanced by HCO 3 − , however, the K + -stimulated component of the microsomal ATPase was virtually unaffected by these anions.

A study of H+ transport in gastric microsomal vesicles using fluorescent probes

Fluorescent amines, 9-aminoacridine, acridine orange and quinacrine, were used as probes for a pH gradient (deltapH) across gastric microsomal vesicles. Analysis of probe uptake data indicates that 9-aminoacridine distributes across the membrane as a weak base in accordance with the deltapH. On the other hand, acridine orange and quinacrine show characteristics of binding to membrane sites in addition to the accumulation in response to deltapH. A discussion of the advantages and limitations of the probes is presented. Application of these probes to pig gastric microsomal vesicles indicates that that K+-stimulated ATPase is responsible for the transport of H+ into the vesicles and thus develops a deltapH across the membrane. The deltapH generated by the K+-ATPase has a definite requirement for internal K+. The proton gradient can be discharged slowly after ATP depletion or rapidly either by detergent disruption of the vesicles or by increasing their leakiness using both H+ and K+ ionophores. On the other hand, the sole use of the K+ ionophore, valinomycin, stimulates the ATP-induced formation of deltapH by increasing the availability of K+ to internal sites. This stimulation by valinomycin requires the presence of permeable anions like Cl-. Analysis of the Cl- requirement indicates that in the presence of valinomycin the net effect is the accumulation of HCl inside the gastric vesicles. With an external pH of 7.0, the ATP-generated deltapH was calculated to be from 4 to 4.5 pH units. The results are consistent with the hypothesis that the K+-stimulated ATPase drives a K+/H+ exchange across the gastric vesicles. Since other lines of evidence suggest that these gastric microsomes are derived from the tubulovesicular system of the oxyntic cell, the participation of the ATP-driven transport processes in gastric HCl secretion is of interest.

The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli.

Stimulation of gastric acid secretion in parietal cells involves the translocation of the proton pump (H,K‐ATPase) from cytoplasmic tubulovesicles to the apical membrane to form long, F‐actin‐containing, microvilli. Following secretion, the pump is endocytosed back into tubulovesicles. The parietal cell therefore offers a system for the study of regulated membrane recycling, with temporally separated endocytic and exocytic steps. During cAMP‐mediated stimulation, an 80 kDa peripheral membrane protein becomes phosphorylated on serine residues. This protein is a major component, together with actin and the pump, of the isolated apical membrane from stimulated cells, but not the resting tubulovesicular membrane. Here we show that the gastric 80 kDa phosphoprotein is closely related or identical to ezrin, a protein whose phosphorylation on serine and tyrosine residues was recently implicated in the induction by growth factors of cell surface structures on cultured cells [Bretscher, A. (1989) J. Cell Biol., 108, 921–930]. Light and electron microscopy reveal that ezrin is associated with the actin filaments of the microvilli of stimulated cells, but not with the filaments in the terminal web. In addition, a significant amount of ezrin is present in the basolateral membrane infoldings of both resting and stimulated cells. Extraction studies show that ezrin is a cytoskeletal protein in unstimulated and stimulated cells, and its association with the cytoskeleton is more stable in stimulated cells. These studies indicate that ezrin is a membrane cytoskeletal linker that may play a key role in the control of the assembly of secretory apical microvilli in parietal cells and ultimately in the regulation of acid secretion. Taken together with the earlier studies, we suggest that ezrin might be a general substrate for kinases involved in the regulation of actin‐containing cell surface structures.

The membrane-recruitment-and-recycling hypothesis of gastric HCl secretion

In the unstimulated oxyntic (or parietal) cell, the primary pump for gastric HCl secretion, the H+/K+-ATPase, is retained within the cytoplasm in a membranous compartment of tubulovesicles. Neural or hormonal stimulation of acid secretion induces extensive membrane transformations consistent with a fusion and recruitment of tubulovesicles to the apical plasma membrane. The consequent placement of H+/K+-ATPase in parallel with K(+) and Cl(-) channels provides the necessary ionic flow and ATP-driven exchange for net HCl secretion. Current evidence is consistent with a recruitment and recycling of membrane transporters, such as H+/K+-ATPase, through docking/fusion machinery analogous to that in many other systems.

Signal transduction and activation of acid secretion in the parietal cell.

Gastric acid secretion has long fascinated physiologists, cell biologists and biochemists because of many attractive riddles in the field. Pure gastric juice, secreted by the parietal cell, contains highly concentrated hydrochloric acid, with pH as low as 0.8, that is, the established proton gradient is greater than 4 million times that in the blood stream. The origin and mechanism of how this gigantic electrochemical potential can be formed and maintained has been extensively studied. Also the hormonal and neural regulation of acid secretion, especially the role of histamine as a final common mediator, had been focus of much debate (Black, 1993). In this review we will concentrate on the intracellular events that couple the stimulus to activation of the parietal cell. The parietal cell is considered to have at least three types of activating receptors on its basolateral membrane, i.e., histamine H 2, acetylcholine M3, and gastrin CCKB, although much of the action of acetylcholine or gastrin is mediated by the release of endogenous histamine. It is believed that the H 2 receptor couples to Gs to activate adenylate cyclase (ACase) producing adenosine 38,58-cyclic monophosphate (cAMP) and subsequent activation of cAMP-dependent protein kinase (PKA), whereas both M3and CCKB-type receptors couple to non-Gs/Gi systems, probably Gq, to activate phospholipase C (PLC) producing inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, with the former releasing Ca 2+

Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells and impairs acid secretion in the stomach

Slc26a9 is a recently identified anion transporter that is abundantly expressed in gastric epithelial cells. To study its role in stomach physiology, gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant (Slc26a9−/−) mice appeared healthy and displayed normal growth. Slc26a9 deletion resulted in the loss of gastric acid secretion and a moderate reduction in the number of parietal cells in mutant mice at 5 weeks of age. Immunofluorescence labeling detected the H-K-ATPase exclusively on the apical pole of gastric parietal cells in Slc26a9−/− mice, in contrast to the predominant cytoplasmic localization in Slc26a9+/+ mice. Light microscopy indicated that gastric glands were dilated, and electron micrographs displayed a distinct and striking absence of tubulovesicles in parietal cells and reductions in the numbers of parietal and zymogen cells in Slc26a9−/− stomach. Expression studies indicated that Slc26a9 can function as a chloride conductive pathway in oocytes as well as a Cl−/HCO3− exchanger in cultured cells, and localization studies in parietal cells detected its presence in tubulovesicles. We propose that Slc26a9 plays an essential role in gastric acid secretion via effects on the viability of tubulovesicles/secretory canaliculi and by regulating chloride secretion in parietal cells.

Vesicular trafficking machinery, the actin cytoskeleton, and H+-K+-ATPase recycling in the gastric parietal cell.

Gastric HCl secretion by the parietal cell involves the secretagogue‐regulated re‐cycling of the H+–K+‐ATPase at the apical membrane. The trafficking of the H+–K+‐ATPase and the remodelling of the apical membrane during this process are likely to involve the co‐ordination of the function of vesicular trafficking machinery and the cytoskeleton. This review summarizes the progress made in the identification and characterization of components of the vesicular trafficking machinery that are associated with the H+–K+‐ATPase and of components of the actin‐based cytoskeleton that are associated with the apical membrane of the parietal cell. Since many of these proteins are also expressed at the apical pole of other epithelial cells, the parietal cell may represent a model system to characterize the protein‐ protein interactions that regulate apical membrane trafficking in many other epithelial cells.

Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H+-K+-ATPase.

Stimulation of the gastric parietal cell results in a massive redistribution of H+-K+-ATPase from cytoplasmic tubulovesicles to the apical plasma membrane. Previous studies have implicated the small GTPase rab11 in this process. Using matrix-assisted laser desorption mass spectrometry, we confirmed that rab11 is associated with H+-K+-ATPase-enriched gastric microsomes. A stoichiometry of one rab11 per six copies of H+-K+-ATPase was estimated. Furthermore, rab11 exists in at least three forms on rabbit gastric microsomes: the two most prominent resemble rab11a, whereas the third resembles rab11b. Using an adenoviral expression system, we expressed the dominant negative mutant rab11a N124I in primary cultures of rabbit parietal cells under the control of the tetracycline transactivator protein (tTA). The mutant was well expressed with a distribution similar to that of the H+-K+-ATPase. Stimulation of these cultures with histamine and IBMX was assessed by measuring the aminopyrine (AP) uptake relative to resting cells (AP index). In experiments on six culture preparations, stimulated uninfected cells gave an AP index of 10.0 ± 2.9, whereas parallel cultures expressing rab11a N124I were poorly responsive to stimulation, with a mean AP index of 3.2 ± 0.9. Control cultures expressing tTA alone or tTA plus actin responded equally well to stimulation, giving AP index values of 9.0 ± 3.1 and 9.6 ± 0.9, respectively. Thus inhibition by rab11a N124I is not simply due to adenoviral infection. The AP uptake data were confirmed by immunocytochemistry. In uninfected cells, H+-K+-ATPase demonstrated a broad cytoplasmic distribution, but it was cleared from the cytoplasm and associated with apically derived membranes on stimulation. In cells expressing rab11a N124I, H+-K+-ATPase maintained its resting localization on stimulation. Furthermore, this effect could be alleviated by culturing infected cells in the presence of tetracycline, which prevents expression of the mutant rab11. We therefore conclude that rab11a is the prominent GTPase associated with gastric microsomes and that it plays a role in parietal cell activation.

Isolation and characterization of gastric microsomal glycoproteins. evidence for a glycosylated β-subunit of the H+/K−-ATPase

Abstract Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60–80 (two glycoproteins sharing this molecular mass); 125–150; and 190–210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K+-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60–80 kDa glycoprotein. Characterization of the 60–80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated β-subunit of the Na+/K+-ATPase, this 60–80 kDa gastric microsomal glycoprotein is suggested to be a β-subunit of the H+/K+-ATPase.

The Effects of Microfilament Disrupting Agents on HCl Secretion and Ultrastructure of Piglet Gastric Oxyntic Cells

A functionally responsive in vitro preparation of piglet gastric mucosa was used to investigate the involvement of microfilaments in the process of HCl secretion by oxyntic cells. A well-ordered array of microfilaments was observed in the short, stubby microvilli on the apical surface of nonsecreting oxyntic cells, as well as ii the longer microvilli of actively secreting cells. Treatment with cytochalasin B (10(-5)-10(-4) M) caused a dose-dependent inhibition of acid secretion and a concomitant gradient of morphologic alteration of oxyntic cells. Associated with slight (approximately 20%) inhibition of secretion was an initial collapse of the canalicular and glandular lumina and appearance of some pleomorphic-shaped microvilli. Maximum inhibition of secretion always produced a complete collapse of the oxyntic cell canalicular and glandular lumina, with a resultant apposition of apical surfaces. Microvilli were no longer readily distinguishable, and microfilaments were severely disorganized. Treatment with cytochalasin B (2-4 X 10(-5) M) before secretagogue stimulation also reduced the ability of the gastric mucosa to secrete acid; oxyntic cells retained the general appearance of nonsecreting cells. The correlation of disruption of microfilaments and the inhibition of acid secretion by cytochalasin B suggests an involvement of microfilaments in both the initiation and maintenance of high levels of acid secretion.