Role and mechanism of Glut-1 and H+/K+-ATPase expression in pepsin-induced development of vocal cord leukoplakia.

PURPOSE: We investigated the role of Glut-1 and H+/K+-ATPase expression in pepsin-induced development of human vocal cord leukoplakia cells (HVCLCs). Next, we analyzed the relationship between Glut-1 and H+/K+-ATPase expression with the clinicopathological features of laryngeal carcinoma. METHODS: Glut-1 and H+/K+-ATPase expression levels in HVCLCs were determined after treatment with artificial gastric juice containing pepsin and laryngeal carcinoma tissues. RESULTS: Exposure to pepsin-containing artificial gastric juice significantly enhanced the migration and proliferation of VSCLCs in a time-dependent manner. The apoptotic rate of VSCLCs decreased over time after exposure to pepsin and reached a nadir on day 7 (p < 0.01). With increasing duration of exposure to pepsin, the proportion of VSCLCs in G0/G1 phase decreased and the proportions in the S and G2/M phases significantly increased (p < 0.05). After treatment with pepsin-containing artificial gastric juice, RT-PCR and Western blotting showed that the expression of Glut-1 and H+/K+-ATPase α, ß significantly increased in HVCLCs compared to in the absence of pepsin (p < 0.05). The expression of Glut-1 and H+/K+-ATPase α, ß gradually increased from vocal cord leukoplakia (VLC) to laryngeal carcinoma (p < 0.05). Lentivirus-mediated inhibition of Glut-1 expression in VCL significantly inhibited the cells' migration and proliferation (p < 0.05) but enhanced their apoptosis (p < 0.05). Also, inhibition of Glut-1 expression resulted in an increased proportion of cells in G0/G1 phase and a significantly decreased proportion in G2/M phase (p < 0.05). CONCLUSIONS: Elevated Glut-1 expression may promote the development of VCL by upregulating laryngeal H+/K+-ATPase expression to reactivate absorbed pepsin, thus damaging the laryngeal mucosa.

H+/K+ATPase Expression in the Larynx of Laryngopharyngeal Reflux and Laryngeal Cancer Patients.

OBJECTIVES: The gastric H+/K+ ATPase proton pump has previously been shown to be expressed in the human larynx, however its contribution to laryngopharyngeal reflux (LPR) signs, symptoms and associated diseases such as laryngeal cancer is unknown. Proton pump expression in the larynx of patients with LPR and laryngeal cancer was investigated herein. A human hypopharyngeal cell line expressing the proton pump was generated to investigate its effects. STUDY DESIGN: In-vitro translational. METHODS: Laryngeal biopsies were obtained from three LPR and eight LSCC patients. ATP4A, ATP4B and HRPT1 were assayed via qPCR. Human hypopharyngeal FaDu cell lines stably expressing proton pump were created using lentiviral transduction and examined via transmission electron microscopy and qPCR for genes associated with inflammation or laryngeal cancer. RESULTS: Expression of ATP4A and ATP4B was detected in 3/3 LPR, 4/8 LSCC-tumor and 3/8 LSCC-adjacent specimens. Expression of ATP4A and ATP4B in FaDu elicited mitochondrial damage and expression of IL1B, PTGS2, and TNFA (P < .0001); expression of ATP4B alone did not. CONCLUSIONS: Gastric proton pump subunits are expressed in the larynx of LPR and LSCC patients. Mitochondrial damage and changes in gene expression observed in cells expressing the full proton pump, absent in those expressing a single subunit, suggest that acid secretion by functional proton pumps expressed in upper airway mucosa may elicit local cell and molecular changes associated with inflammation and cancer. LEVEL OF EVIDENCE: NA Laryngoscope, 131:130-135, 2021.

Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System.

P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed proteins can be isolated. Insect cells are a perfect system to study P-type ATPases as they have little or no endogenous Na,K-ATPase activity and other ATPase activities can be inhibited easily. Here we describe in detail the expression and isolation of Na,K-ATPase and H,K-ATPase isoforms with the baculovirus expression system.

Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion.

The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05) and 53% (P<0.001) respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H+] of the gastric contents was significantly (P<0.025) lower by 9.4 fold (89%) in the ClC-2-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84-95%, P<0.005) in ClC-2-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion.

Benzimidazole derivative, BMT-1, induces apoptosis in multiple myeloma cells via a mitochondrial-mediated pathway involving H+/K+-ATPase inhibition.

2-(1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-ol (BMT-1), a bicyclic compound, belongs to the benzimidazole group and consists of the fusion of benzene and imidazole. The objective of the present study was to assess the effect of BMT-1 on the proliferation of multiple myeloma (MM) cells and identify whether BMT-1 induces apoptosis in MM cells. Our results showed a dose- and time-dependent decrease in the proliferation of MM cells treated with BMT-1. Further studies revealed that the antiproliferative effects of BMT-1 were caused by induction of apoptosis with activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in MM cells. In addition, BMT-1 induced the loss of mitochondrial membrane potential resulting in the activation of caspase-8 and -9. Furthermore, the MM cells treated with BMT-1 showed a more acidic intracellular pH (pHi) as indicated by a lower FL1/FL2 ratio caused by inhibition of H+/K+-ATPase. Collectively, these findings demonstrated that a decrease in pHi, caused by H+/K+-ATPase inhibition induced by BMT-1, triggered the dysfunction of the mitochondria resulting in the apoptosis of MM cells. Therefore, BMT-1 may be used as a lead compound for the design and development of new agents with which to treat MM and other forms of cancer.

H+ /K+ ATPase expression in human parietal cells and gastric acid secretion in elderly individuals.

OBJECTIVE: To investigate whether the ultrastructure and hydrogen potassium adenosine triphosphate (H+ /K+ ATPase) expression of human parietal cells were associated with aging. METHODS: In all, 50 participants who underwent gastroscopy due to dyspepsia were divided into two age groups, with 19 in the younger group (YG, aged 20-59 years) and 31 in the elder group (EG, aged ≥60 years). The ultrastructure of their parietal cell was determined by electron microscopy (EM), and the expressions of H+ /K+ ATPase α-subunit mRNA and ß-unit protein were detected. Furthermore, 24-h esophageal pH monitoring was performed in the two groups. RESULTS: EM images showed no distinct difference in the morphology and distribution of parietal cells or the acid secretion-related organelle between the two groups. There were no differences between YG and EG in the proportion of mitochondria and the tubulovesicular system area. The expressions of H+ /K+ ATPase α-subunit mRNA and ß-subunit protein showed no age-related alteration between YG and EG. The expression of H+ /K+ ATPase α-subunit mRNA in EG was higher than that in YG, whereas the expression of ß-subunit protein was significantly higher in those aged ≥80 years than in the YG. No significant difference was found in the 24-h esophageal pH monitoring between YG and EG. CONCLUSION: Acid secretion-related organelles in parietal cells do not degenerate with aging, the expression of H+ /K+ ATPase even shows a trend to increase, indicating the existence of intact molecular biological basis for acid secretion in healthy elderly individuals.

Isoform specificity of cardiac glycosides binding to human Na+,K+-ATPase alpha1beta1, alpha2beta1 and alpha3beta1.

Cardiac glycosides inhibit the Na(+),K(+)-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na(+),K(+)-ATPase are expressed: alpha(1)beta(1), alpha(2)beta(1) and alpha(3)beta(1). It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na(+),K(+)-ATPase isoforms alpha(1)beta(1), alpha(2)beta(1) and alpha(3)beta(1) were expressed in yeast which has no endogenous Na(+),K(+)-ATPase. Isoform specific affinities of digoxin, digitoxin, beta-acetyldigoxin, methyldigoxin and ouabain were assessed in [(3)H]-ouabain binding assays in the absence or presence of K(+) (each n=5). The subcellular localizations of the Na(+),K(+)-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K(+), methyldigoxin (alpha(1)>alpha(3)>alpha(2)) and ouabain (alpha(1)=alpha(3)>alpha(2)) showed distinct isoform specific affinities, while for digoxin, digitoxin and beta-acetyldigoxin no differences were found. In the presence of K(+), also digoxin (alpha(2)=alpha(3)>alpha(1)) and beta-acetyldigoxin (alpha(1)>alpha(3)) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only alpha(1)beta(1) and alpha(2)beta(1) are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na(+),K(+)-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance.

Expression of H(+),K(+)-ATPase and glycopattern analysis in the gastric glands of Rana esculenta.

A multidisciplinary study involving lectin histochemistry, IHC, immuno-lectin blotting, and immunogold was carried out to determine the distribution of sugar residues in the glycoproteins of Rana esculenta oxynticopeptic cells. We considered animals in two experimental conditions, fasting and fed. It is known that, in mammals, the tubulovesicular membranes are rich in proteins with several functions. The proton pump H(+),K(+)-ATPase, a heterodimeric complex with a catalytic alpha-subunit and a heavily glycosylated beta-subunit, responsible for acid secretion, is the most abundant. No data have been published regarding the localization and the structures of H(+),K(+)-ATPase in amphibians. In the water frog, the luminal membrane and tubulovesicular system of oxynticopeptic cells, which differ in morphology according to their functional stage, reacted with the primary gold-conjugated antibody against the H(+),K(+)-ATPase alpha-subunit. By lectin histochemistry and immunoblotting, in the oxynticopeptic cells of R. esculenta we detected the presence of N-linked glycans having fucosylated (poly)lactosamine chains, which could correspond to the oligosaccharide chains of the beta subunit. The latter are somewhat different from those described in mammals, and this is probably because of an adaptation to the different microenvironmental conditions in which the oxynticopeptic cells find themselves, in terms of their different habits and phylogeny.

Cyclo-oxygenase-1 inhibition increases acid secretion by modulating H+,K+-ATPase expression and activation in rabbit parietal cells.

1. In the present study, we evaluated the role of cyclo-oxygenase (COX)-1 and COX-2 on gastric acid secretion in rabbit isolated parietal cells and gastric glands by examining [(14)C]-aminopyrine uptake, prostaglandin (PG) E(2) synthesis and COX-1, COX-2 and proton pump expression at baseline and after treatment with various concentrations of specific COX-1 (SC-560), COX-2 (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methyl-sulphonyl)phenyl-2 (5H)-furanone; DFU) and non-specific COX (indomethacin) inhibitors. 2. In parietal cells, SC-560 and indomethacin, over the concentration range 10(-8) to 10(-4) mol/L, dose-dependently increased basal and 10(-4) mol/L histamine-stimulated aminopyrine uptake and inhibited PGE(2) synthesis, whereas DFU (10(-8) to 10(-5) mol/L) had no effect. However, at 10(-4) mol/L, DFU augmented histamine-stimulated aminopyrine uptake by 135% and inhibited PGE(2) synthesis by 39%, indicating an inhibition of COX-1 at this higher concentration. 3. The SC-560-, DFU- and indomethacin-induced augmentation of histamine-stimulated aminopyrine uptake was reduced to basal levels after 10(-5) mol/L lansoprazole treatment in parietal cells and gastric glands, whereas 10(-4) mol/L ranitidine only partially inhibited such augmentation. 4. Only COX-1 was detected in parietal cells. However, both COX-1 and COX-2 were expressed in gastric glands, with relative protein density of COX-1 being sixfold higher than that of COX-2. Protein levels of COX-1 in parietal cells and those of COX-1 and COX-2 in gastric glands remained unchanged, regardless of inhibitor treatment, either alone or with histamine. 5. Parietal cell proton pump expression was significantly enhanced by 10(-5) mol/L SC-560 and 10(-4) mol/L indomethacin (by 29 and 31%, respectively) and pump activity was enhanced by 61 and 65%, respectively. In contrast, 10(-5) mol/L DFU had no effect. 6. In conclusion, the data indicate that inhibition of COX-1- but not COX-2-derived PGE(2) synthesis is involved in augmentation of non-steroidal anti-inflammatory drug-induced gastric acid secretion in parietal cells by enhancing expression and activation of the proton pump.

[The effects of H. pylori and its crude extracted proteins on isolated rabbit parietal cells acid secretion].

OBJECTIVE: To explore the effects of H. pylori and crude extracted proteins secreted by H. pylori (broth culture filtrate protein, BCF-P) on acid secretion from isolated rabbit parietal cells. METHODS: Parietal cells from rabbit gastric mucosa were isolated and enriched with digestion and elutriation. H. pylori (NCTC 11637, CagA+ VacA+) were grown in liquid broth culture and BCF-P was precipitated with ammonium sulfate. The vacuolation activity of BCF-P was evaluated with neutral red dye uptake test in HeLa cell. Isolated parietal cells were incubated with H. pylori (bacteria/cell = 100:1) for 2 h and 16 h,or BCF-P (100 microLg/ml) for 1 h and 12 h. Acid secretion from parietal cells was studied using 14C-aminopyrine (14C-AP) accumulation indirectly and H+-K+ ATPase alpha subunit mRNA expression was assessed using RT-PCR. RESULTS: (1) BCF-P containing vacuolating cytotoxin (VacA) with vacuolation activity on HeLa cells had positive result on neutral red uptake test. (2) The basal expression of H+-K+ ATPase alpha subunit mRNA could be detected in isolated parietal cells and 14C-AP accumulation was significantly increased in response to the stimulation of histamine with different concentrations for 30 min (P < 0.05). These results indicated that the isolated parietal cells retain relative intact acid secretion function. (3)The histamine (1.0 x 10(-4) mol/L) stimulated acid secretion was inhibited sustainedly in response to H. pylori by 81% at 2 h and by 94% at 16 h (P < 0.05). However, H+-K+ ATPase alpha subunit mRNA expression was up-regulated in the acute period (2 h) and was down-regulated in the chronic period (16 h) by H. pylori (P < 0.05). (4) BCF-P significantly inhibited the histamine-stimulated acid secretion by 24% at 1 h and by 58% at 12 h (P < 0.05), and this inhibition was accompanied by the down-regulated expression of H+-K+ ATPase alpha subunit mRNA. Conclusions Intact H. pylori and VacA secreted by H. pylori could directly inhibit histamine-stimulated acid secretion from parietal cells and this inhibition may be mediated by the down-regulated H+-K+ ATPase expression.

Systematic expression profiling of the gastric H+/K+ ATPase in human tissue.

OBJECTIVE: The potassium-competitive acid blockers (P-CABs), comprise a new, innovative group of competitive and reversible inhibitors of the gastric H+/K+ ATPase. Our aim was to identify sites of expression of the H+/K+ ATPase that are potential targets of these compounds by examining the expression profile of the gastric H+/K+ ATPase in the human body from a broad range of tissues. MATERIAL AND METHODS: Expression profiling was done by quantitative mRNA analysis (TaqMan PCR). Tissues that were mRNA-positive for the alpha subunit were investigated further by Western blot and immunohistochemistry (IHC) for the presence of gastric H+/K+ ATPase protein. RESULTS: In addition to the very high expression levels in the stomach, the adrenal gland, cerebellum and pancreas gave unexpectedly positive mRNA signals for the alpha subunit of gastric H +/K+ ATPase. However, they were negative for mRNA of the beta subunit, and Western blot and IHC were negative for alpha and beta subunit protein. Another group of tissues with low alpha subunit mRNA expression including the frontal cortex, cortex grey matter, testis, thymus and larynx submucosa were also found negative for both alpha and beta subunit protein. In contrast to mouse kidney, no gastric H+/K+ ATPase could be detected in human kidney. CONCLUSIONS: We therefore conclude that the only organ in humans expressing significant levels of the P-CAB target gastric H+/K+ ATPase is the stomach.

IL-1beta modulation of H,K-ATPase alpha-subunit gene transcription in Helicobacter pylori infection.

Helicobacter pylori infection of the human gastric body induces hypochlorhydria by perturbing acid secretion. H. pylori inhibits parietal cell H,K-ATPase alpha-subunit (HKalpha) gene and protein expression, providing a mechanistic basis for clinical hypochlorhydria. Given that H. pylori infection increases gastric mucosal IL-1beta, an acid secretory inhibitor, we investigated the role of IL-1beta in H. pylori-mediated inhibition of HKalpha transcription. Human gastric adenocarcinoma (AGS) cells were transfected with promoter-reporter constructs containing human HKalpha 5'-flanking sequence deletions. IL-1beta (10 ng/ml) had no effect on the transcriptional activity of six progressively shorter deletion constructs of the HKalpha promoter (HKalpha2179-HKalpha340) and significantly stimulated the activity of HKalpha206, HKalpha177, HKalpha165, and HKalpha102 deletion constructs (80%, 100%, 46%, and 35%, respectively). H. pylori inhibited the transcriptional activity of HKalpha2179, HKalpha206, HKalpha177, and HKalpha165; IL-1beta relieved the H. pylori inhibition of HKalpha2179 and HKalpha206 activity but not HKalpha177 and HKalpha165 activity. AGS cell pretreatment with a MEK1/2 inhibitor prevented the IL-1beta-mediated stimulation, but p38 and JNK pathway inhibitors did not. IL-1beta mRNA levels in AGS cells were low and unaffected by H. pylori, and ELISAs of H. pylori-conditioned AGS culture media showed no measurable IL-1beta secretion. These data indicate that an IL-1beta-dependent cis-response element lies downstream of -206 nt in the HKalpha promoter and that IL-1beta-mediated upregulation of HKalpha transcription is affected by an ERK1/2 kinase signal pathway. We conclude that an IL-1beta-responsive HKalpha cis element positively regulates HKalpha gene transcription in shortened deletion constructs and that H. pylori-induced inhibition of HKalpha transcription is not mediated by IL-1beta.

Helicobacter pylori eradication induces marked increase in H+/K+-adenosine triphosphatase expression without altering parietal cell number in human gastric mucosa.

BACKGROUND AND AIMS: Gastric acid secretion is downregulated by Helicobacter pylori infection and upregulated after its eradication, but the mechanisms are still unclear. We examined the effects of H pylori eradication on the number of parietal cells and on expression of molecules functioning in acid secretion in the human gastric mucosa. METHODS: We enrolled 111 consecutive men with chronic gastritis induced by H pylori. Biopsy specimens were endoscopically obtained before and 12 weeks after successful eradication of H pylori and parietal cell numbers were counted. mRNA expression levels of H+/K+-adenosine triphosphatase (H+/K+-ATPase), anion exchanger 2, M3 muscarinic receptor, intrinsic factor, and interleukin 1beta were determined with a real time reverse transcriptase-polymerase chain reaction method. The severity of gastric atrophy was evaluated using the serum pepsinogen I/II ratio. RESULTS: No significant difference was observed in parietal cell numbers before and after H pylori eradication. Median mRNA expression levels of H+/K+-ATPase in the gastric mucosa increased 250-fold after H pylori eradication accompanied by attenuation of interleukin 1beta. A large increase in H+/K+-ATPase expression was observed even in patients with severe atrophic gastritis. In contrast, fold increases in mRNA expression levels, including intrinsic factor, anion exchanger 2, and M3 muscarinic receptor, after eradication therapy, were limited to 1.4, 2.3, and 2.5 times, respectively. CONCLUSIONS: In the absence of alteration of parietal cell number, gastric H+/K+-ATPase mRNA expression was markedly restored after successful H pylori eradication, suggesting a central role for the restoration of H+/K+-ATPase expression in gastric acid secretion recovery after H pylori eradication.

Eye regeneration assay reveals an invariant functional left-right asymmetry in the early bilaterian, Dugesia japonica.

Consistent visceral asymmetry in vertebrates raises fascinating questions about the developmental mechanisms and evolutionary origin of fixed chirality of the left-right axis. One persistent controversy is whether consistently biased asymmetry is a later innovation imposed on a bilaterally symmetrical primitive body-plan, or whether asymmetry is a fundamental property predating the bilateria. The morphology of planaria suggests proximity to the origin of the bilateral body-plan, and they are commonly thought to be left-right symmetrical, as no consistent anatomical asymmetries have been described despite over a century of study of regeneration. Here, we show that D. japonica possess a consistent functional asymmetry in eye patterning defects caused by inhibition of H+/K+-ATPase activity (an ion flux mechanism recently shown to be an important early step in the asymmetry of several vertebrate embryos). Moreover, an endogenous transcript of the non-gastric H+/K+-ATPase subunit alpha is expressed in the head blastema shortly after amputation. Taken together, these data suggest that (1) left-right asymmetry is at least as old as planaria, (2) subtle functional asymmetries should be sought in other more primitive model systems that are believed to be symmetrical, and (3) symmetrical paired structures may in fact contain information about their position on the L or R side.

Thymic expression of a gastritogenic epitope results in positive selection of self-reactive pathogenic T cells.

Intrathymic expression of tissue-specific self-Ags can mediate tolerance of self-reactive T cells. However, in this study we define circumstances by which thymic expression of a tissue-specific autoepitope enhances positive selection of disease-causing, self-reactive T cells. An immunodominant gastritogenic epitope, namely the gastric H/K ATPase beta subunit(253-277) (H/Kbeta(253-277)), was attached to the C terminus of the invariant chain (Ii) and the hybrid Ii (Ii-H/Kbeta(253-277)) expressed in mice under control of the Ii promoter. The Ii-H/Kbeta(253-277) fusion protein was localized to MHC class II-expressing cells in the thymus and periphery of Ii-H/Kbeta(253-277) transgenic mice. In one transgenic line the level of presentation in the periphery (spleen) was insufficient to activate naive, low affinity H/Kbeta(253-277)-specific transgenic T cells (1E4-TCR), whereas thymic presentation of H/Kbeta(253-277) enhanced positive selection of 1E4-TCR cells in Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice. Furthermore, Ii-H/Kbeta(253-277)/1E4-TCR double-transgenic mice had an increased incidence of autoimmune gastritis compared with 1E4-TCR single-transgenic mice, demonstrating that the 1E4 T cells that seeded the periphery of Ii-H/Kbeta(253-277) mice were pathogenic. Therefore, low levels of tissue-specific Ags in the thymus can result in positive selection of low avidity, self-reactive T cells. These findings also suggest that the precise level of tissue-specific Ags in the thymus may be an important consideration in protection against autoimmune disease and that perturbation of the levels of self-Ags may be detrimental.

Lipopolysaccharide-induced changes in rat gastric H/K-ATPase expression.

OBJECTIVE: To evaluate lipopolysaccharide (LPS)-induced inhibition of gastric acid secretion. SUMMARY BACKGROUND DATA: Endotoxemia from LPS inhibits gastric acid secretion by an unknown mechanism. Bacterial overgrowth in the stomach caused by decreased acid secretion could be responsible for nosocomial pneumonia developing in critically ill intensive care unit patients. Because acid secretion is via the H/K-ATPase and the effects of LPS on this enzyme are unknown, we hypothesized that LPS causes inhibition of gastric acid secretion by down-regulating the H/K-ATPase. METHODS: A rat model to study gastric acid secretion was created. Saline or LPS (0.05-20 mg/kg IP) was given for 1 hour, after which basal acid secretion was determined for 1 hour. Pentagastrin (PG; 10 microg/kg IV) or saline was then given and gastric acid output collected for another 2 hours. RESULTS: LPS dose dependently inhibited basal and PG stimulated acid secretion. LPS increased alpha- and beta-H/K-ATPase subunit mRNA expression (Northern blot) in the absence of PG compared with saline. In the presence of PG, LPS did not have this effect. Western blot analysis did not show any difference in alpha- or beta-subunit immunoreactivity. Immunofluorescence analysis demonstrated that PG increased staining in the secretory membranes for H/K-ATPase subunits whereas in all LPS-treated rats, it appeared that H/K-ATPase subunits remained within the tubulovesicles. Furthermore, changes in H/K-ATPase mRNA expression may not be related to changes in NF-kappaB activity. CONCLUSIONS: These data suggest that inhibition of gastric acid secretion by LPS is due to inhibition of H/K-ATPase enzymatic function or changes in cytoskeletal rearrangements in H/K-ATPase subunits rather than by down-regulation of transcriptional or translational events.

Spontaneous organ-specific Th2-mediated autoimmunity in TCR transgenic mice.

CD4(+) T cells that lead to autoimmune gastritis (AIG) in BALB/c mice are either Th1 or Th2 cells. To test whether the phenotype of disease is related to the particular TCR expressed by the pathogenic cell, we have generated several lines of TCR transgenic mice using receptors cloned from pathogenic Th1 or Th2 cells. We previously described spontaneous inflammatory AIG in A23 mice, caused by the transgenic expression of the TCR from a Th1 clone, TXA23. In this study we describe the generation of A51 mouse lines, transgenic for the TCR of a CD4(+) self-reactive Th2 clone, TXA51. A proportion of A51 mice spontaneously develop AIG by 10 wk of age, with a disease characterized by eosinophilic infiltration of the gastric mucosa and Th2 differentiation of transgenic T cells in the gastric lymph node. The Th2 phenotype of this autoimmune response seems to be related to a low availability of MHC class II-self peptide complexes. This in vivo model of spontaneous Th2-mediated, organ-specific autoimmunity provides a unique example in which the clonotypic TCR conveys the Th2 disease phenotype.

Molecular and cellular regulation of the gastric proton pump.

The gastric H+, K+-ATPase is a proton pump that is responsible for gastric acid secretion and that actively transports protons and K+ ions in opposite directions to generate in excess of a million-fold gradient across the membrane under physiological conditions. This pump is also a target molecule of proton pump inhibitors which are used for the clinical treatment of hyperacidity. In this review, we wish to summarize the molecular regulation of this pump based on mutational studies, particularly those used for the identification of binding sites for cations and specific inhibitors. Recent reports by Toyoshima et al (2000, 2002) presented precise three-dimensional (3-D) structures of the sarcoplasmic reticulum (SR) Ca2+-ATPase, which belongs to the same family as the gastric H+, K+-ATPase. We have studied the structure-function relationships for the gastric H+, K+-ATPase using 3-D structures constructed by homology modeling of the related SR Ca2+-ATPase, which was used as a template molecule. We also discuss in this review, the regulation of cell surface expression and synthesis control of the gastric proton pump.

Identification of the beta-subunit for nongastric H-K-ATPase in rat anterior prostate.

The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase alpha-subunit (alpha(ng)) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to alpha(1)-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase beta-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of beta-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three beta-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is beta(1) > beta(3) >> beta(2). Immunohistochemical experiments demonstrate that beta(1) is present predominantly in apical membranes, coinciding with alpha(ng), whereas beta(3) is localized in the basolateral compartment, coinciding with alpha(1). This is the first direct demonstration of the alpha(ng)-beta(1) colocalization in situ indicating that, in rat AP, alpha(ng) associates only with beta(1). The existence of alpha(ng-)beta(1) complex has been confirmed by immunoprecipitation experiments. These results indicate that beta(1)-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.

Stable expression of gastric proton pump activity at the cell surface.

Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the alpha+beta-expressing cell line, K(+)-stimulated ATPase (K(+)-ATPase) activity and the acylphosphorylation of the alpha-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K(+)-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the alpha-subunit was retained in the intracellular compartment and was unstable in the absence of the beta-subunit, but it was stabilized and reached the cell surface in the presence of the beta-subunit. On the other hand, the beta-subunit was stable and able to travel to the cell surface in the absence of the alpha-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.