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Metabolic acidosis is thought to exacerbate chronic kidney disease in part by stimulating the release of potentially injurious substances. To define the genes whose expression is affected by exposure to an acidic milieus, we examined the effect of exposure of MDCK cells to pH 7.4 and pH 7.0 for 24 h on gene expression using a canine derived microarray. Exposure to this pH stress for 24 h led to increased expression of 278 genes (2.2% of the transcriptome) by at least 2-fold and 60 of these (21%) were upregulated by >3-fold. On the other hand, 186 genes (1.5% of the transcriptome) were downregulated by at least 2-fold and 16 of these (9%) were downregulated by 3-fold or more. Ten percent of the genes upregulated by at least threefold encode proinflammatory cytokine proteins, including colony stimulating factor 2, chemokine ligand 7, chemokine ligand 20, chemokine ligand 8, and interleukin-1α. Two others encode metallopeptidases. The most highly upregulated gene encodes a protein, lubricin, shown to be important in preventing cartilage damage and in tissue injury or repair. Upregulation of four genes was confirmed by quantitative PCR. Housekeeping genes were not increased. To examine the effect of decreasing medium pH, we measured intracellular pH (pHi) using 2,7-bis (2-carboxyethyl)5-carboxyfluorescein. With extracellular pH (pHo) of 7.0, pHifell and remained depressed. These findings suggest that a pH stress alone can increase renal expression of proinflammatory and other genes that contribute to renal injury.

Inhibition of the gastric H,K-ATPase by the potassium-competitive acid blocker (P-CAB) 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine (TAK-438), is strictly K(+)-competitive with a K(i) of 10 nM at pH 7. In contrast to previous P-CABs, this structure has a point positive charge (pK(a) 9.06) allowing for greater accumulation in parietal cells compared with previous P-CABs [e.g., (8-benzyloxy-2-methyl-imidazo(1,2-a)pyridin-3-yl)acetonitrile (SCH28080), pK(a) 5.6]. The dissociation rate of the compound from the isolated ATPase is slower than other P-CABs, with the t(1/2) being 7.5 h in 20 mM KCl at pH 7. The stoichiometry of binding of TAK-438 to the H,K-ATPase is 2.2 nmol/mg in the presence of Mg-ATP, vanadate, or MgP(i). However, TAK-438 also binds enzyme at 1.3 nmol/mg in the absence of Mg(2+). Modeling of the H,K-ATPase to the homologous Na,K-ATPase predicts a close approach and hydrogen bonding between the positively charged N-methylamino group and the negatively charged Glu795 in the K(+)-binding site in contrast to the planar diffuse positive charge of previous P-CABs. This probably accounts for the slow dissociation and high affinity. The model also predicts hydrogen bonding between the hydroxyl of Tyr799 and the oxygens of the sulfonyl group of TAK-438. A Tyr799Phe mutation resulted in a 3-fold increase of the dissociation rate, showing that this hydrogen bonding also contributes to the slow dissociation rate. Hence, this K(+)-competitive inhibitor of the gastric H,K-ATPase should provide longer-lasting inhibition of gastric acid secretion compared with previous drugs of this class.

Proton pump inhibitors (PPIs) are acid-activated prodrugs that covalently bind to the gastric H,K-ATPase on its luminal surface. Only active pumps can be inhibited. The short plasma residence time of current PPIs prevents inhibition of pumps synthesized or activated after the PPI has disappeared, limiting the degree of acid inhibition even with twice-daily administration. PPIs with a longer residence time should improve acid control. Various K+competitive inhibitors of the pump are being developed (acid pump antagonists or potassium-competitive acid blockers), with the advantage of complete inhibition of acid secretion independent of pump activity. Early data on these suggest that twice daily administration would improve acid control compared with PPIs.

Polarized distribution of plasma membrane transporters and receptors in epithelia is essential for vectorial functions of epithelia. This polarity is maintained by sorting of membrane proteins into apical or basolateral transport containers in the trans-Golgi network and/or endosomes followed by their delivery to the appropriate plasma membrane domains. Sorting depends on the recognition of sorting signals in proteins by specific sorting machinery. In the present review, we summarize experimental evidence for and against the hypothesis that N-glycans attached to the membrane proteins can act as apical sorting signals. Furthermore, we discuss the roles of N-glycans in the apical sorting event per se and their contribution to folding and quality control of glycoproteins in the endoplasmic reticulum or retention of glycoproteins in the plasma membrane. Finally, we review existing hypotheses on the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36 and galectin-3, as putative apical sorting receptors.

Gastric H,K-ATPase is an electroneutral transmembrane pump that moves protons from the cytoplasm of the parietal cell into the gastric lumen in exchange for potassium ions. The mechanism of transport against the established electrochemical gradients includes intermediate conformations in which the transferred ions are trapped (occluded) within the membrane domain of the pump. The pump cycle involves switching between the E1 and E2P states. Molecular dynamics simulations on homology models of the E2P and E1 states were performed to investigate the mechanism of K+movement in this enzyme. We performed separate E2P simulations with one K+in the luminal channel, one K+ion in the occlusion site, two K+ions in the occlusion site, and targeted molecular dynamics from E2P to E1 with two K+ions in the occlusion site. The models were inserted into a lipid bilayer system and were stable over the time course of the simulations, and K+ions in the channel moved to a consistent location near the center of the membrane domain, thus defining the occlusion site. The backbone carbonyl oxygen from residues 337 through 342 on the nonhelical turn of M4, as well as side-chain oxygen from E343, E795, and E820, participated in the ion occlusion. A single water molecule was stably bound between the two K+ions in the occlusion site, providing an additional ligand and partial shielding the positive charges from one another. Targeted molecular dynamics was used to transform the protein from the E2P to the E1 state (two K+ions to the cytoplasm). This simulation identified the separation of the water column in the entry channel as the likely gating mechanism on the luminal side. A hydrated exit channel also formed on the cytoplasmic side of the occlusion site during this simulation. Hence, water molecules became available to hydrate the ions. The movement of the M1M2 transmembrane segments, and the displacement of residues Q159, E160, Q110, and T152 during the conformational change, as well as the motions of E343 and L346, acted as the cytoplasmic-side gate.

The recent progress in therapy if acid disease has relied heavily on the performance of drugs targeted against the H,K ATPase of the stomach and the H2 receptor antagonists. It has become apparent in the last decade that the proton pump is the target that has the likelihood of being the most sustainable area of therapeutic application in the regulation of acid suppression. The process of activation of acid secretion requires a change in location of the ATPase from cytoplasmic tubules into the microvilli of the secretory canaliculus of the parietal cell. Stimulation of the resting parietal cell, with involvement of F-actin and ezrin does not use significant numbers of SNARE proteins, because their message is depleted in the pure parietal cell transcriptome. The cell morphology and gene expression suggest a tubule fusion-eversion event. As the active H,K ATPase requires efflux of KCl for activity we have, using the transcriptome derived from 99 pure parietal cells and immunocytochemistry, provided evidence that the KCl pathway is mediated by a KCQ1KCNE2 complex for supplying Kand CLIC6 for supplying the accompanying Cl−. The pump has been modeled on the basis of the structures of different conformations of the sr Ca ATPase related to the catalytic cycle. These models use the effects of site directed mutations and identification of the binding domain of the K competitive acid pump antagonists or the defined site of binding for the covalent class of proton pump inhibitors. The pump undergoes conformational changes associated with phosphorylation to allow the ion binding site to change exposure from cytoplasmic to luminal exposure. We have been able to postulate that the very low gastric pH is achieved by lysine 791 motion extruding the hydronium ion bound to carboxylates in the middle of the membrane domain. These models also allow description of the Kentry to form the Kliganded form of the enzyme and the reformation of the ion site inward conformation thus relating the catalytic cycle of the pump to conformational models. The mechanism of action of the proton pump inhibitor class of drug is discussed along with the cysteines covalently bound with these inhibitors. The review concludes with a discussion of the mechanism of action and binding regions of a possible new class of drug for acid control, the Kcompetitive acid pump antagonists.

There is a continuing increase in gastroesophageal reflux disease (GERD), its diverse manifestations and complications worldwide. Indeed the substantial increase in Asia remains is a major epidemiologic and a developing health care issue. This manifesto provides a contemporary review of the current status of areas of uncertainty and in particular highlights the lack of knowledge and unmet needs in the field of GERD biology and especially management. There seems to be no widely accepted rationale for the increasing incidence and prevalence of GERD globally. Although consideration has been given to the decline in Helicobacter pyloriinfection, others consider the steadily growing problem of obesity as the most likely etiologic factor. What actually comprises the disease entity of GERD continues to confound physicians, and the steady increase in new definitions suggests that a clear picture is still emerging. Of note, however, is the rising awareness of its protean systemic manifestations and the fact that while erosive reflux disease is easily recognizable endoscopically, nonerosive disease (NERD) comprises the majority of presentations in many populations. In view of this, GERD-specific questionnaires and other evaluative tools have been designed to capture the multidimensional nature of the symptom complexes in GERD patients who for the most part may have no endoscopic evidence of disease. Such tools have obvious utility in the evaluation of changes with treatment, especially because there is no endoscopic abnormality detectable. The recognition that NERD is becoming a major clinical entity that requires substantial further investigation is now clearly apparent. At this time, however, little is known of the cellular barrier mechanisms of the esophagus and what is responsible for their reconstitution after injury. Similarly, the neural basis of esophageal pain appreciation, or its relation to the visceral sensory mechanisms that may link NERD with aspects of functional bowel disease are as yet ill understood. Apart from the clinical concern with symptom-focused issues, it is the specter of Barrett esophagus, the most-feared complication of GERD that mostly continues to drive endoscopic practice in GERD. At this time, however, current surveillance strategies remain severely limited in their ability to adequately detect or prevent most esophageal adenocarcinoma. Although considerable effort has been directed at defining the neural circuitry of the esophagus in terms of both motility and sensation, the topography and function of the esophageal-brain-gut axis in respect of the generation of esophageal symptoms remains terra incognita. At this time therefore acid suppression remains the mainstay of therapy in terms of treating the GERDNERD disease process. Despite the efficacy of the currently available proton pump inhibitor class of drugs, there are clearly unmet needs in this area and agents with a more rapid onset of action and prolonged effect, particularly at night remain important goals in the future advance of pharmacotherapy.

The gastric entero-chromaffin-like (ECL) cell plays a key regulatory role in peripheral regulation of acid secretion due to the release of histamine that stimulates acid secretion by the parietal cell. Studies in intact animals, gastric glands, and isolated cells after short-term culture have shown expression of stimulatory CCK2 and PAC1 and inhibitory SST2 and Gal1 receptors as well as histidine decarboxylase. However, the pattern of its gene expression as a neuroendocrine cell has not been explored. Comparison of gene expression by 95% pure ECL cells obtained by density gradient, elutriation, and fluorescence-assisted cell sorting with isolates of the intact fundic gastric epithelium (i.e., “subtractive hybridization”) identified a variety of additional expressed gene families characteristic of this neuroendocrine cell. These include genes 1) involved in neuropeptide synthesis and secretory vesicle exocytosis, 2) involved in control of inflammation, 3) implicated in healing of the epithelium, 4) encoding inhibitory Giprotein-coupled receptors, 5) playing a role in neuroendocrine regulation of food intake, and 6) encoding proteins likely involved in maintenance of circadian rhythm, in addition to the ECL cell-specific genes histidine decarboxylase and monoamine transporter. Particularly, the inhibitory apelin receptor gene, APJ, was highly expressed in the ECL cell preparation. Because parietal cells express apelin, immunohistochemical and functional studies showed that there is an inhibitory feed back loop between the parietal and ECL cell during gastrin stimulation, providing evidence for a novel pathway of downregulation of acid secretion due to interaction between these two cell types.

The gastric entero-chromaffin-like (ECL) cell plays a key regulatory role in peripheral regulation of acid secretion due to the release of histamine that stimulates acid secretion by the parietal cell. Studies in intact animals, gastric glands, and isolated cells after short-term culture have shown expression of stimulatory CCK2 and PAC1 and inhibitory SST2 and Gal1 receptors as well as histidine decarboxylase. However, the pattern of its gene expression as a neuroendocrine cell has not been explored. Comparison of gene expression by 95% pure ECL cells obtained by density gradient, elutriation, and fluorescence-assisted cell sorting with isolates of the intact fundic gastric epithelium (i.e., "subtractive hybridization") identified a variety of additional expressed gene families characteristic of this neuroendocrine cell. These include genes 1) involved in neuropeptide synthesis and secretory vesicle exocytosis, 2) involved in control of inflammation, 3) implicated in healing of the epithelium, 4) encoding inhibitory Gi protein-coupled receptors, 5) playing a role in neuroendocrine regulation of food intake, and 6) encoding proteins likely involved in maintenance of circadian rhythm, in addition to the ECL cell-specific genes histidine decarboxylase and monoamine transporter. Particularly, the inhibitory apelin receptor gene, APJ, was highly expressed in the ECL cell preparation. Because parietal cells express apelin, immunohistochemical and functional studies showed that there is an inhibitory feed back loop between the parietal and ECL cell during gastrin stimulation, providing evidence for a novel pathway of downregulation of acid secretion due to interaction between these two cell types.

Helicobacter pyloriis a Gram-negative neutralophile associated with peptic ulcers and gastric cancer. It has a unique ability to colonize the human stomach by acid acclimation. It uses the pH-gated urea channel, UreI, to enhance urea access to intrabacterial urease and a membrane-anchored periplasmic carbonic anhydrase to regulate periplasmic pH to ~6.1 in acidic media, whereas other neutralophiles cannot regulate periplasmic pH and thus only transit the stomach.

The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K+ and H+ ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E1 and P-E2, are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and ≤4.5 in the E1 conformation; 6.7 and ≤2 in the P-E2 conformation. The equilibrium dissociation constants for K+ binding on the cytoplasmic side were 11 and 16mM. The respective equilibrium dissociation constants on the luminal side were obtained via K+ concentration dependence of the enzyme activity and determined to be 0.11mM for both luminal binding sites.

Genomic microarray analysis of genes specifically expressed in a pure cell isolate from a heterocellular organ identified the likely K efflux channel associated with the gastric H-K-ATPase. The function of this channel is to supply K to the luminal surface of the pump to allow H for K exchange. KCNQ1-KCNE2 was the most highly expressed and significantly enriched member of the large variety of K channels expressed in the gastric epithelium. The function of this K channel in acid secretion was then shown by inhibition of secretion in isolated gastric glands with specific KCNQ inhibitors and by colocalization of the channel with the H-K-ATPase in the secretory canaliculus of the parietal cell. KCNQ1-KCNE2 appears to be the K efflux channel that is essential for gastric acid secretion.

Reciprocal interactions between Helicobacter pylori and cells of the gastric epithelium to which it adheres may affect colonization. Changes in gene expression of H. pylori induced by adhesion to AGS gastric cancer cells by coculture were compared to changes in gene expression of H. pylori cultured without AGS cells by using cDNA filter macroarrays. Adhesion was quantitatively verified by confocal microscopy of green fluorescent protein-expressing bacteria. Four experiments showed that 22 and 21 H. pylori genes were consistently up- and down-regulated, respectively. The up-regulated genes included pathogenicity island, motility, outer membrane protein, and translational genes. The sigma(28) factor antagonist flgM, flgG, the stress response gene, flaA, omp11, and the superoxide dismutase gene (sodB) were down-regulated. The up-regulation of cag3, flgB, tonB, rho, and deaD was confirmed by quantitative PCR, and the up-regulation of lpxD, omp6, secG, fabH, HP1285, HP0222, and HP0836 was confirmed by reverse transcription (RT)-PCR. The down-regulation of flaA, sodB, and HP0874 was confirmed by quantitative PCR, and the down-regulation of omp11 was confirmed by RT-PCR. The alteration of gene expression in H. pylori after adhesion to gastric cells in vitro suggests that changes in motility, outer membrane composition, and stress responses, among other changes, may be involved in gastric colonization.

ABSTRACTReciprocal interactions between Helicobacter pyloriand cells of the gastric epithelium to which it adheres may affect colonization. Changes in gene expression of H. pyloriinduced by adhesion to AGS gastric cancer cells by coculture were compared to changes in gene expression of H. pyloricultured without AGS cells by using cDNA filter macroarrays. Adhesion was quantitatively verified by confocal microscopy of green fluorescent protein-expressing bacteria. Four experiments showed that 22 and 21 H. pylorigenes were consistently up- and down-regulated, respectively. The up-regulated genes included pathogenicity island, motility, outer membrane protein, and translational genes. The σ28factor antagonist flgM, flgG, the stress response gene, flaA, omp11, and the superoxide dismutase gene (sodB) were down-regulated. The up-regulation of cag3, flgB, tonB, rho, and deaDwas confirmed by quantitative PCR, and the up-regulation of lpxD, omp6, secG, fabH, HP1285, HP0222, and HP0836 was confirmed by reverse transcription (RT)-PCR. The down-regulation of flaA, sodB, and HP0874 was confirmed by quantitative PCR, and the down-regulation of omp11was confirmed by RT-PCR. The alteration of gene expression in H. pyloriafter adhesion to gastric cells in vitro suggests that changes in motility, outer membrane composition, and stress responses, among other changes, may be involved in gastric colonization.

Helicobacter pylori is the only neutralophile that has been able to colonize the human stomach by using a variety of acid-adaptive mechanisms. One of the adaptive mechanisms is increased buffering due to expression of an acid-activated inner membrane urea channel, UreI, and a neutral pH-optimum intrabacterial urease. To delineate other possible adaptive mechanisms, changes in gene expression in response to acid exposure were examined using genomic microarrays of H. pylori exposed to different levels of external pH (7.4, 6.2, 5.5, and 4.5) for 30 min in the absence and presence of 5 mM urea. Gene expression was correlated with intrabacterial pH measured using 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein and compared to that observed with exposure to 42 degrees C for 30 min. Microarrays containing the 1,534 open reading frames of H. pylori strain 26695 were hybridized with cDNAs from control (pH 7.4; labeled with Cy3) and acidic (labeled with Cy5) conditions. The intrabacterial pH was 8.1 at pH 7.4, fell to 5.3 at pH 4.5, and rose to 6.2 with urea. About 200 genes were up-regulated and approximately 100 genes were down-regulated at pH 4.5 in the absence of urea, and about half that number changed in the presence of urea. These genes included pH-homeostatic, transcriptional regulatory, motility, cell envelope, and pathogenicity genes. The up-regulation of some pH-homeostatic genes was confirmed by real-time PCR. There was little overlap with the genes induced by temperature stress. These results suggest that H. pylori has evolved multifaceted acid-adaptive mechanisms enabling it to colonize the stomach that may be novel targets for eliminating infection.

ABSTRACTHelicobacter pyloriis the only neutralophile that has been able to colonize the human stomach by using a variety of acid-adaptive mechanisms. One of the adaptive mechanisms is increased buffering due to expression of an acid-activated inner membrane urea channel, UreI, and a neutral pH-optimum intrabacterial urease. To delineate other possible adaptive mechanisms, changes in gene expression in response to acid exposure were examined using genomic microarrays of H. pyloriexposed to different levels of external pH (7.4, 6.2, 5.5, and 4.5) for 30 min in the absence and presence of 5 mM urea. Gene expression was correlated with intrabacterial pH measured using 2′,7′-bis-(2-carboxyethyl)-5-carboxyfluorescein and compared to that observed with exposure to 42°C for 30 min. Microarrays containing the 1,534 open reading frames of H. pyloristrain 26695 were hybridized with cDNAs from control (pH 7.4; labeled with Cy3) and acidic (labeled with Cy5) conditions. The intrabacterial pH was 8.1 at pH 7.4, fell to 5.3 at pH 4.5, and rose to 6.2 with urea. About 200 genes were up-regulated and ∼100 genes were down-regulated at pH 4.5 in the absence of urea, and about half that number changed in the presence of urea. These genes included pH-homeostatic, transcriptional regulatory, motility, cell envelope, and pathogenicity genes. The up-regulation of some pH-homeostatic genes was confirmed by real-time PCR. There was little overlap with the genes induced by temperature stress. These results suggest that H. pylorihas evolved multifaceted acid-adaptive mechanisms enabling it to colonize the stomach that may be novel targets for eliminating infection.

The gastric H,K-ATPase and the Na,K-ATPase both are stimulated by luminal K, but differ in sensitivity to K-competitive inhibitors (ouabain and SCH28080), which implies a difference in structure near the luminal ion pathways in these two pumps. Knowledge of the amino acids in the H,K-ATPase that affect the mode of inhibition by SCH28080 and inhibitor affinity should provide insight into the regions of the membrane domain influencing the inhibitor selectivity and the luminal route to the ion transport site. Mutational scans in M4, 5, 6, and 8 have shown that amino acid residues affecting ion affinity (E343, K791, E795, E820, D824, E936) with either no or a lesser effect on the inhibitor affinity are located in the middle of the membrane domain. The residues significantly reducing inhibitor affinity, but not ion affinity (L809, P810, L811, T813, I816, Y925, T929), are located in the exoplasmic 5-6 loop and the luminal ends of M6 and M8. This suggests that the binding domain for SCH28080 contains the surface between L809 in the 5-6 loop and C813 at the luminal end of M6, ?two helical turns out from the ion binding region, where it blocks an ion access pathway. The mutations that change inhibitor kinetics are on the opposing faces of M6 and M8 and apparently modify the normal ion pathway or, perhaps, create an alternate ion pathway.

We have used homology molecular modeling based on the srCaATPase E2conformation, pdb1kju, to predict side chains involved in docking the Kcompetitive inhibitor, SCH28080, to the H,K-ATPase. A model for SCH28080 binding between residues L809 and A335 in the same space utilized by omeprazole is proposed. We also describe modeling MgATP binding to the E1structure of the srATPase, pdb1eul, as a paradigm for the Na,K- and H,K-ATPases. The resulting model, E1·MgATP, visualizes a conformation not yet available by crystallization and successfully predicts a range of published results, including backbone cleavages near V440 (N domain) and V712 (P domain) mediated by FeATP in the Na,K-ATPase. A separate model for MgATP docked to E2(pdb1kju) shows that access of the ? phosphate to D351 is blocked by the A domain. The E2· MgATP model explains FeATP-mediated cleavages of the Na,K-ATPase near V440 and E214 (A domain) and homologous results in the H,K-ATPase.

Background & Aims:Helicobacter pylori, a neutralophile, uses acid neutralization by urease to combat gastric acidity, allowing gastric colonization. Both acute and chronic acid resistance mechanisms are present. Acute mechanisms of acid adaptation could be due to surface urease, increased inner-membrane urea permeability via UreI, or both. Slower mechanisms may involve increased nickel insertion into apoenzyme, posttranscriptional regulation, or increased enzyme synthesis. The aim of this study was to further define regulation of urease under acidic conditions. Methods:Surface-bound urease was analyzed by measurement of free and bound urease after centrifugation through a step gradient and by quantitative urease immunostaining of intact and fixed bacteria. Changes in urease synthesis or assembly were determined by incubation of the organisms at pH 5.5 or 7.0 in the absence and presence of chloramphenicol, urea, or nickel chelator and in ureI-positive and -negative organisms. Results:The amount of surface urease was below detection limits with either centrifugation washing or immunostaining. Total bacterial urease activity was increased 3–5-fold by incubation at pH 5.5 in the presence of chloramphenicol but not in nickel-free medium or in ureIknockout organisms. There was also a 3-fold increase in survival of acid shock in acid-adapted organisms. Conclusions:Surface-bound urease is too low to contribute to acid resistance. Acidic medium pH induces UreI-dependent nickel incorporation into apoenzyme. This augmentation of urease activity increases survival in acid and is part of the gastric colonization strategy of the organism.

In the presence of ascorbate/H2O2, Fe2+ions or the ATP-Fe2+complex catalyze selective cleavage of the α subunit of gastric H+,K+-ATPase. The electrophoretic mobilities of the fragments and dependence of the cleavage patterns on E1and E2conformational states are essentially identical to those described previously for renal Na+,K+-ATPase. The cleavage pattern of H+,K+-ATPase by Fe2+ions is consistent with the existence of two Fe2+sites: site 1 within highly conserved sequences in the P and A domains, and site 2 at the cytoplasmic entrance to trans-membrane segments M3 and M1. The change in the pattern of cleavage catalyzed by Fe2+or the ATP-Fe2+complex induced by different ligands provides evidence for large conformational movements of the N, P, and A cytoplasmic domains of the enzyme. The results are consistent with the Ca2+-ATPase crystal structure (Protein Data Bank identification code 1EUL; Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature405, 647–655), anE1Ca2+conformation, and a theoretical model of Ca2+-ATPase in anE2conformation (Protein Data Bank identification code 1FQU). Thus, it can be presumed that the movements of N, P, and A cytoplasmic domains, associated with theE1↔ E2transitions, are similar in all P-type ATPases. Fe2+-catalyzed cleavage patterns also reveal sequences involved in phosphate, Mg2+, and ATP binding, which have not yet been shown in crystal structures, as well as changes which occur in E1↔E2transitions, and subconformations induced by H+,K+-ATPase-specific ligands such as SCH28080.

An H+-K+-ATPase contributes to hydrogen secretion and potassium reabsorption by the rat and rabbit collecting ducts. Transport of these ions appears to be accomplished by one or both of two isoforms of the H+-K+-ATPase, HKα1and HKα2,because both isoforms are found in the collecting ducts and transport of hydrogen and potassium is attenuated by exposure to inhibitors of these transport proteins. To evaluate whether an H+-K+-ATPase is present in the human kidney, immunohistochemical studies were performed using normal human renal tissue probed with antibodies directed against epitopes of three of the known isoforms of the H+-K+-ATPase, HKα1, HKα2, and HKα4, and the V-type H+-ATPase. Cortical and medullary tissue probed with antibodies against HKα1showed cytoplasmic staining of intercalated cells that was less intense than that observed in the parietal cells of normal rat stomach stained with the same antibody. Also, weak immunoreactivity was detected in principal cells of the human collecting ducts. Cortical and medullary tissue probed with antibodies directed against HKα4revealed weak, diffuse staining of intercalated cells of the collecting ducts and occasional light staining of principal cells. Cortical and medullary tissue probed with antibodies directed against the H+-ATPase revealed staining of intercalated cells of the collecting ducts and some cells of the proximal convoluted tubules. By contrast, no discernible staining was noted with the use of the antibody against HKα2. These data indicate that HKα1and HKα4are present in the collecting ducts of the human kidney. In this location, these isoforms might contribute to hydrogen and potassium transport by the kidney.

LEP503is a novel gene product isolated from lens epithelial cells by a subtractive cDNA cloning strategy. It is highly conserved in different vertebrate species and developmentally regulated in postnatal rat lens, suggesting that LEP503may be an important lens epithelium gene involved in the processes of lens epithelial cell differentiation. The expression of LEP503is highly restricted to lens epithelial cells invivo. To investigate the molecular mechanisms regulating the promoter of the human LEP503, we cloned and characterized the promoter of the human LEP503gene. The transcription start site was localized to a nucleotide C 22 base pairs (bp) 5′ of the initiation methionine codon. By reporter gene transfection experiments, we found that ∼2.5-kb of LEP5035′-flanking sequence directed high level luciferase activity in human lens epithelial cells; further deletion analysis revealed positive regulatory element between bp −401 and +22. Mutation analysis in each of the seven potential binding sites for transcription factors within the region between −401 and +22 showed that the AP-1 element at −131 and the Sp1 element at −48 are the most important sites for the tissue-specific expression of LEP503. Consistent with lens epithelial cell-restricted expression of LEP503mRNA, we found that the ∼2.5-kb 5′-flanking sequence directed high-level promoter activity in lens epithelial cells but not in other cell types. Understanding the LEP503promoter will allow us to investigate lens epithelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to lens epithelial cells. The LEP503gene is mapped to human chromosome 1q22, the same location to which zonular pulverulent cataract was previously mapped.

We previously reported that PAC1is expressed on ECL cells resulting in stimulation of [Ca2]i, histamine and acid secretion. The study reported here characterized the signaling by PAC1mon ECL cells; determined the effects of PACAP on the gastric acid secretion in vivo, and determined the effects of chronic administration of PACAP-27 on ECL cell proliferation. PACAP-27 dose dependently stimulated ECL cell Ca2and AC with detectable stimulation at 1 nM and maximal stimulation at 100 nM (six-fold). In rats PACAP-27 administration (10 pmolkgh) increased the rate of gastric acid secretion when an antisomatostatin antibody was co-administered. Chronic administration of PACAP (10 pmolh for seven days) via osmotic pump resulted in a more than twofold increase in BrdU incorporation into ECL cells. PACAP acting at the PAC1results in dual signaling responses to both [Ca2]i. AC in ECL cells stimulates gastric acid secretion via the actions of histamine acting at the parietal cell and in whole animals leads to proliferation of ECL cells when administered chronically.

The gastric H,K-ATPase is covalently inhibited by substituted pyridyl-methylsulfinyl-benzimidazoles, such as omeprazole, that convert to thiophilic probes of luminally accessible cysteines in the acid space. The K+competitive inhibitor, SCH28080, prevented inhibition of acid transport by omeprazole. In stably expressing HEK293 cells, the benzimidazole-reactive cysteines, Cys-321 (transmembrane helix (TM) 3), Cys-813 and Cys-822 (TM5/6), and Cys-892 (TM7/8) were mutated to the amino acids found in the SCH28080-resistant Na,K-ATPase and kinetic parameters of H,K-ATPase activity analyzed. Mutations of Cys-822 and Cys-892 had insignificant effects on the Ki(app),Km(app)or Vmax, but mutations of Cys-813 to threonine and Cys-321 to alanine decreased the affinity for SCH28080. Mutation of Cys-321 to alanine produced mixed kinetics of inhibition, still with higher affinity for the cation-free form of phosphoenzyme. Since the phenylmethoxy ring of the imidazo-pyridine inhibitors binds to TM1/2, as shown by earlier photoaffinity studies, and the mutations in TM6 (Cys-813 → Thr) as well as the end of TM3 (Cys-321 → Ala) decrease the affinity for SCH28080, the TM1/2, TM3, and TM6 helices lie within ∼ 16 Å of each other based on the size of the active, extended conformation of SCH28080.

ABSTRACTureIencodes an integral cytoplasmic membrane protein. It is present in the urease gene cluster of Helicobacter pyloriand is essential for infection and acid survival, but its role is unknown. To determine the function of UreI protein, we producedH. pylori ureIdeletion mutants and measured the pH dependence of urease activity of intact and lysed bacteria and the effect of urea on the membrane potential. We also determinedureIexpression, urease activity, and the effect of urea on membrane potential of several gastric and nongastricHelicobacterspecies. ureIwas found to be present in the genome of the gastric Helicobacterspecies and absent in the nongastric Helicobacterspecies studied, as determined by PCR. Likewise, Western blot analysis confirmed that UreI was expressed only in the gastric Helicobacterspecies. When UreI is present, acidic medium pH activation of cytoplasmic urease is found, and urea addition increases membrane potential at acidic pH. The addition of a low concentration of detergent raised urease activity of intact bacteria at neutral pH to that of their homogenates, showing that urease activity was membrane limited. No acidic pH activation or urea induced membrane potential changes were found in the nongastric Helicobacterspecies. The ureIgene product is probably a pH activated urea transporter or perhaps regulates such a transporter as a function of periplasmic pH.

The development of the lens is dependent on the proliferation of lens epithelial cells and their differentiation into fiber cells near the lens bow/equator. Identification of genes specifically expressed in the lens epithelial cells and their functions may provide insight into molecular events that regulate the processes of lens epithelial cell differentiation. In this study, a novel lens epithelium gene product, LEP503, identified from rat by a subtractive cDNA cloning strategy was investigated in the genome organization, mRNA expression and protein localization. The genomic sequences for LEP503 isolated from rat, mouse and human span 1754 bp, 1694 bp and 1895 bp regions encompassing the 5′-flanking region, two exons, one intron and 3′-flanking region. All exon-intron junction sequences conform to the GT/AG rule. Both mouse and human LEP503 genes show very high identity (93% for mouse and 79% for human) to rat LEP503 gene in the exon 1 that contains an open reading frame coding for a protein of 61 amino acid residues with a leucine-rich domain. The deduced protein sequences also show high identity (91% between mouse and rat and 77% between human and rat). Western blot shows that LEP503 is present as a specific ∼6.9 kDa band in the water-insoluble-urea-soluble fraction of lens cortex where lens epithelium is included. Immuno-staining shows that LEP503 is localized in the epithelial cells along the entire anterior surface of rat lens. Developmentally, LEP503 is expressed at a low level at newborn, and then the expression level increases by about ten-fold around postnatal day 14 and remains at this high level for about 25 days before it drops back to the low level by postnatal day 84. These data suggest that the LEP503 may be an important lens epithelial cell gene involving the processes of epithelial cell differentiation.

The current study was undertaken to determine the presence and distribution of PAC1-Rs within the gastric mucosa. Polyclonal antibodies to the carboxyl terminus of the rat PAC1-R were generated and shown to be specific against the PAC1-R expressed in NIH 3T3 cells. Western blot analysis using isolated (?85 pure) ECL cell membranes identified a 48 kD protein consistent with the calculated molecular mass of the cloned PAC1-R. RTPCR performed using specific primers for the PAC1-R confirmed the presence of splice variants of the rat PAC1-R, but not VPAC1-R or VPAC2-R. These data provide the first direct evidence for the existence of functional PACAP Type I receptors on ECL cells of the gastric mucosa and suggest a potential role for PACAP in the stimulation of gastric acid secretion and in the regulation of the growth of ECL cells.

Binding of vascular endothelial growth factor (VEGF) to its receptor, VEGFR-2 (Flk-1/KDR), induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for signaling proteins that relay the signals for cell proliferation, migration, and permeability enhancement. We explored the VEGF/receptor signaling pathway by performing a two-hybrid screen of a rat lung cDNA library in yeast using the intracellular domain of rat VEGFR-2 as bait. Two clones encoding lipocortin V were isolated. Subsequent studies with the yeast two-hybrid assay showed that the complete intracellular domain of VEGFR-2 was required for the interaction. Co-immunoprecipitation of translated proteins confirmed the interaction between the VEGF receptor and lipocortin V. VEGF induced a rapid tyrosine phosphorylation of lipocortin V in human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with antisense oligodeoxyribonucleotide (ODN) for lipocortin V significantly inhibited VEGF-induced cell proliferation, which was accompanied by a decrease in protein synthesis and tyrosine phosphorylation of lipocortin V. Our results indicate that lipocortin V may function as a signaling protein for VEGFR-2 by directly interacting with the intracellular domain of the receptor and appears to be involved in regulation of vascular endothelial cell proliferation mediated by VEGFR-2.

The integral membrane protein, the gastric H,K-ATPase, is an α-β heterodimer, with 10 putative transmembrane segments in the α-subunit and one such segment in the β-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na,K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995)Proc. Natl. Acad. Sci.U. S. A. 92, 7936–7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

Summary Uptake of a-aminoisobutyric acid (AIB) was examined in Ehrlich ascites tumor cells treated with the cation-exchange ionophore nigericin (20 µg/ml). Membrane voltages were measured using the voltage-sensitive dye diethyloxadicarbocyanine (DOCC). In normal phosphate-buffered media, nigericin changed the distribution ratios of Na+ and K+ (the ratio of intra- to extracellular concentrations) nearly to unity, but AIB was still accumulated to a distribution ratio of ~9.0. When all but 40mm Na+ in the medium was replaced by choline, nigericin resulted in K+ loss and Na+ gain and both cation distribution ratios approached 2.8–3.4, as would be expected if both ions were distributing near electrochemical equilibrium with a membrane voltage in the range of -28 to -33 mV. This conclusion was supported by the observation that the addition of 5×10-7m valinomycin to the nigericin-treated cell suspension produced no change in DOCC absorbance. In spite of the apparent zero electrochemical potential gradients for Na+ and K+, AIB was accumulated to a distribution ratio of 5.4 in the low-Na+ medium. Addition of 0.1mm oubain or 50 µm vanadate did not alter the extent of AIB accumulation as would have been expected if a large component of the membrane voltage were due to electrogenic operation of the (Na++K+)-ATPase. Addition of lactate, pyruvate or glucose increased the AIB distribution ratios to 11.9, 9.4 and 15.3, respectively. The effect of glucose could be explained, at least in part, by an enhanced Na+ electrochemical potential gradient. However, neither lactate nor pyruvate produced any change either in membrane voltage or the intracellular Na+ concentration. Therefore, these results confirm the existence of a metabolic energy source which is coupled to AIB accumulation and operates in addition to the Na+ co-transport mechanism, and which is augmented by metabolic substrates such as lactate and pyruvate.

Summary The addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H+ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H+ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10mm glucose an outward-directed H+ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H+ reverses the direction of the H+ concentration gradient. Changes in absorbance of the voltagesensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H+ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.

Background & Aims:Galanin, a 29–amino acid neuropeptide found in the gastric mucosa, inhibits basal and pentagastrin-stimulated acid secretion. Its cellular target is unknown. The aim of this study was to determine whether galanin inhibits Ca2+signaling and histamine release in enterochromaffin-like (ECL) cells. Methods:Isolated rat ECL cells were purified to 85% homogeneity by a combination of elutriation, density gradient centrifugation, and 48-hour culture. Intracellular calcium concentration ([Ca2+]i) was determined using video imaging with Fura-2 in a 37°C superfusion chamber. Histamine was measured by radioimmunoassay. Results:Reverse-transciption polymerase chain reaction of the ECL cell RNA showed a galanin type I receptor subtype. Galanin inhibited gastrin, Bay K8644, and K+depolarization–induced calcium mobilization and entry as well as reduced basal calcium levels. Pretreatment with pertussis toxin decreased the effect of galanin. Galanin inhibited basal and gastrin-stimulated histamine release by approximately 60% with a median effective concentration of 1.10−10mol/L. The inhibitory actions of galanin on histamine release and Ca2+influx could be reduced by a galanin antagonist, galantide. Conclusions:Galanin's inhibition of acid secretion can be explained in part by inhibition of calcium signaling and histamine release from the ECL cells due to activation of a Gi,oprotein–coupled receptor.

Waldenstrom's macroglobulinemia is a malignant lymphoproliferative disease associated with a monoclonal macroglobulinemia. Most explanations of the cochlear and vestibular symptoms in this disease have dealt with vascular thrombotic or hemorrhagic phenomena. Five patients currently being treated for macroglobulinemia have been reviewed. Three patients gave a history of periodic dizziness and underwent extensive neuro-otologic examination. The results suggested an alternate mechanism for the pathophysiology of the vestibular disturbance in macroglobulinemia. Vestibular examination by use of electronystagmography provided information which suggests that the cupulae may be altered by the macroglobulins to act as gravity receptors.

Summary Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa ofNecturus maculosa. In cell-attached patches with pipette solution containing 100mm KCl, four major classes of K+ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec fora, and 8 msec forb). Some channels showed subconductance levels. The maximal inward conductancegmax was 31±5 pS (n=13) and the reversal potentialErev was atVp=-34±6 mV (n=9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels withgmax=40 ±18 pS (n=3) andErev was atVp=-31±5 mV (n=3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1mm) to the bath solution in cell-attached patches increased the open probabilityPo of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels withgmax=21±3 pS (n=4) andErev was atVp=-24±9 mV (n=4). These channels exhibited very high open probability (Po=0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26±2 pS (n=3) andErev atVp=-33±3 mV (n=3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K+ in the pipette solution replaced by Na+, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K+ over Na+ and Cl-. A variety of K+ channels contributes to the basolateral K+ conductance of resting oxyntic cells.

Summary: Intracellular calcium [Ca2+] i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca2+] i transient followed by a steady-state increase as the characteristic [Ca2+] i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca2+] i signal in single cells. The distribution of [Ca2+] i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca2+] i present in the subplasmalemmal space and in one cell pole. [Ca2+] i gradients within these regions were not constant but showed temporal changes in the form of [Ca2+] i oscillations and spatial changes in the form of [Ca2+] i waves. [Ca2+] i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca2+] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca2+], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca2+] i waves was also independent of influx of extracellular Ca2+. [Ca2+] i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 mgrm per sec (n = 6). Increases of [Ca2+] i induced by different agonists were encoded into changes of baseline [Ca2+] i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca2+] i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca2+] i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca2+] i oscillations.