Comparative analysis of methods and models for predicting biochemical methane potential of various organic substrates.

Biochemical methane potential (BMP) corresponds to the maximum methane production at anaerobic digestion infinite time and is a key parameter to evaluate the suitability of substrates to obtain biogas. The main objective of this work is to explore the data available in the literature for ten categories of substrates to compare and develop new methods and mathematical models able to predict BMP. Indeed, experimental procedure is time-consuming, laborious and costly, and the development of methods or models based on properties easily assessed may be very helpful at industrial scale. In this study, three substrates (banana waste, tomato waste and winery wastewater) were tested and compared with >150 results from the literature. The analysis involved four methods (Met_I to Met_IV) and five models developed by multivariate regression (Mod_I to Mod_V). Met_I is related to elemental analysis; Met_II with the organic fraction composition; Met_III is associated with chemical oxygen demand (COD); Met_IV is based on NIR spectra. Regression models are combinations by grouping single variables: C, H, O, N (Mod_I); hemicellulose, lignin (LG), acid detergent fibre (ADF) (Mod_II); volatile solids (VS), COD (Mod_III); proteins (PT), carbohydrates (CRB), lipids (LP) (Mod_IV); and CRB, LP, PT, LG, ADF (Mod_V). The results showed that no significant correlation can be found between BMP and single common properties (e.g. VS or C/N ratio). However, good results may be achieved with models developed by multivariate regression (R2 from 0.93 to 0.98, and R2adj from 0.91 to 0.96). The prediction of BMP based on Met_IV, which is based on NIR spectroscopy combined with a multivariate regression model, revealed to be a promising method for both data from literature as well as for substrates analyzed in the present work.

Interleukin 1beta induces gastric epithelial cell matrix metalloproteinase secretion and activation during Helicobacter pylori infection.

BACKGROUND: and aims: Matrix metalloproteinases (MMPs) are endopeptidases with roles in extracellular matrix remodelling, cell proliferation, and inflammatory processes. We showed previously that Helicobacter pylori infection of human gastric adenocarcinoma (AGS) cells increased epithelial secretion of epithelial MMP-1 and MMP-3 and bacterial secretion of MMP-3-like activity. In the present study, we sought to characterise the role of interleukin (IL)-1beta in H pylori induced secretion of epithelial MMPs. METHODS AND RESULTS: AGS cells were treated with H pylori and/or IL-1beta. Comparable IL-8 secretory responses (approximately 1700 ng/ml) measured by ELISA were induced by 2.0 ng/ml IL-1beta and by H pylori at a multiplicity of infection (MOI) of 50. The same IL-1beta and H pylori concentrations induced comparable increases in AGS cell caseinolytic activity at 60 kDa. MMP-3 monoclonal antibody immunoblots of AGS cell conditioned media detected immunoreactive bands at 71 kDa and 56 kDa. H pylori (MOI=50-100) induced dose dependent increases in both bands whereas IL-1beta (0.2-2 ng/ml) induced dose dependent increases only in the 71 kDa band, which was identified as a MMP-3/TIMP-3 (tissue inhibitor of metalloproteinases 3) heterodimer. AGS/H pylori conditioned media expressed 24 times more MMP-3 activity than AGS/IL-1beta conditioned media. There was a strong interaction between IL-1beta and H pylori on MMP-3 secretion. CONCLUSIONS: We conclude that IL-1beta induces gastric epithelial cell MMP-3 secretion, contributing to epithelial tissue destruction during H pylori infection. However, other bacterial/host factors are needed to mediate the full gastric epithelial cell MMP-3 secretory response induced by H pylori infection.

Epithelial and bacterial metalloproteinases and their inhibitors in H. pylori infection of human gastric cells.

To test the hypothesis that Helicobacter pylori regulates gastric cell secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), culture media from infected and uninfected human gastric adenocarcinoma (AGS) cells were analyzed by zymography, MMP activity assays, and immunoblotting. AGS cells secreted gelatinolytic (prominently 90 kDa) and caseinolytic (110 kDa) activity together with MMP-1, MMP-3, and TIMP-1, TIMP-2, and TIMP-3 isoforms. H. pylori secreted caseinolytic activity (60 kDa), MMP-3-like enzyme activity, and TIMP-3 immunoreactivity. H. pylori infection increased the 110-kDa caseinolytic activity and induced new gelatinolytic (~35 kDa) and caseinolytic (22 kDa) activities. Infection also increased both basal secretion and activation of MMP-1 and MMP-3, enhanced TIMP-3 secretion, and increased the formation of MMP-3/TIMP-3 complexes. TIMP-1 and TIMP-2 secretion were unchanged. Normal AGS cells showed a pancellular distribution of TIMP-3, with redistribution of immunoreactivity toward sites of bacterial attachment after H. pylori infection. The data indicate that MMP and TIMP secretion by AGS cells is modulated by H. pylori infection and that host MMP-3 and a TIMP-3 homolog expressed by H. pylori mediate at least part of the host cell response to infection.

Location of a cytoplasmic epitope for monoclonal antibody HK 12.18 on H,K-ATPase alpha subunit.

The enzyme responsible for gastric acidification is a heterodimeric (alpha and beta subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes electroneutral exchange of exoplasmic K(+) for cytoplasmic H(3)O(+). The molecular mechanisms of the catalytic exchange reaction are imperfectly understood, and await clarification of the precise topology of the enzyme with respect to the secretory membrane. Antibodies directed against H,K-ATPase subunits have been useful in confirming hydropathy plot predictions of HKalpha and HKbeta secondary structure. The monoclonal antibody HK 12.18, which labels gastric mucosal parietal cells by immunocytochemistry, and which binds to a single M(r) approximately 94,000 polypeptide by SDS-PAGE immunoblot of gastric microsomes, has been widely used as a specific marker of parietal cells in clinical and cell biological studies of acid secretion, and as a specific HKalpha probe in biochemical studies. However, the uncertain location of the HK 12.18 epitope has limited the antibody's usefulness as a topology probe. In this study, HK 12. 18 immune reactivity with native H,K-ATPase tryptic peptides, HKalpha cDNA fragments expressed in bacteria, and overlapping synthetic HKalpha tridecapeptides, was used to identify the HK 12.18 epitope as seven consecutive amino acids (Asp(682)-Met-Asp-Pro-Ser-Glu-Leu(688)) in the cytoplasmic middle third of HKalpha.

Inhibition of human gastric H(+)-K(+)-ATPase alpha-subunit gene expression by Helicobacter pylori.

Clinical studies and in vitro data from isolated parietal cells suggest that acute Helicobacter pylori infection inhibits acid secretion. Gastric acidification is mediated by H(+)-K(+)-ATPase, an integral protein of parietal cell apical membranes. To test the hypothesis that H. pylori downregulates H(+)-K(+)-ATPase alpha-subunit (HKalpha) gene expression and to identify potential intracellular signaling pathways mediating such regulation, we transfected human gastric adenocarcinoma (AGS) cells with human and rat HKalpha 5'-flanking DNA fused to a luciferase reporter plasmid. Histamine caused dose-dependent, cimetidine-sensitive (10(-4) M) increases in cAMP, free intracellular Ca(2+), and HKalpha promoter activation in AGS cells. H. pylori infection of transfected AGS cells dose dependently inhibited basal and histamine-stimulated HKalpha promoter activity by 80% and 66%, respectively. H. pylori dose dependently inhibited phorbol myristate acetate-induced (10(-7) M) and staurosporine- (10(-7) M) and calphostin C-sensitive (5 x 10(-8) M) activation of HKalpha promoter. Also, H. pylori inhibited epidermal growth factor (EGF) (10(-8) M), genistein-sensitive (5 x 10(-5) M) activation of HKalpha promoter, reducing activity to 60% of basal level. These data suggest that H. pylori inhibits HKalpha gene expression via intracellular pathways involving protein kinases A and C and protein tyrosine kinase, AGS cells have functional histamine H(2) and EGF receptors, and transiently transfected AGS cells are a useful model for studying regulation of HKalpha gene expression.

H,K-ATPase alpha subunit C-terminal membrane topology: epitope tags in the insect cell expression system.

The H,K-ATPase responsible for gastric acidification is a heterodimeric (alpha and beta subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes the electroneutral exchange of K+ for protons, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1, 2-alpha]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of the catalytic alpha subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP binding and phosphorylation sites, and a C-terminal region with four or six putative transmembrane domains. Several lines of evidence implicate the C-terminal region of P-type ATPases in cation-binding and occlusion, conformational changes, and interactions with the beta subunit (HKbeta), making the definition of topology a prerequisite for understanding the structural basis of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag) were inserted in predicted hydrophilic segments of the alpha subunit (HKalpha) to establish the membrane orientation of two amino acids with different predicted topologies in the C-terminal four- and six-transmembrane models. Wild-type and mutated HKalpha and HKbeta cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HKalpha- and HKbeta-specific and flu-tag-specific antibodies. Functional assays showed K+-stimulated, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags was determined by microsomal protease protection assays in Sf9 cells and immunolabelling of HKalpha and HKbeta in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasmically oriented HKalpha residues. The results indicated cytoplasmic exposure of Leu844 and Phe996, and luminal exposure of Pro898, leading to a revised secondary structure model of the C-terminal third of HKalpha.

The Na(+)-K(+)-ATPase beta 1 subunit is associated with the HK alpha 2 protein in the rat kidney.

The Na-K-ATPase beta 1 subunit acts as the beta subunit for the HK alpha 2 protein in the rat kidney. The colonic H(+)-K(+)-ATPase is a member of the P-type ATPases, and has been shown to contribute to potassium transport by the mammalian kidney and colon. The P-type ATPases often consist of an alpha subunit that contains the catalytic site and a beta subunit that participates in regulation of enzyme activity and targeting of the enzyme to the plasma membrane. The cDNA of the alpha subunit (HK alpha 2) has been cloned and the HK alpha 2 protein has been isolated from the rat kidney and colon. However, a unique beta subunit for the colonic H(+)-K(+)-ATPase has not been described. To determine if one of the known beta subunits present in the kidney might act as the beta subunit for the colonic H(+)-K(+)-ATPase, microsomes enriched in the colonic H(+)-K(+)-ATPase were isolated using an HK alpha 2-specific antibody (AS 31.7) and the Minimac magnetic separation system. Immunoblots of rat kidney microsomal protein isolated with antibody AS 31.7 were probed with antibodies directed against the gastric HK beta subunit, Na(+)-K(+)-ATPase alpha 1, and Na(+)-K(+)-ATPase beta 1 subunits. A band of the appropriate size was detected with Na(+)-K(+)-ATPase beta 1-specific antibodies, but not those directed against HK beta 1. These data suggest that Na(+)-K(+)-ATPase beta 1 could be the beta subunit for the colonic H(+)-K(+)-ATPase in the kidney.

Effect of hypokalemia on the abundance of HK alpha 1 and HK alpha 2 protein in the rat kidney.

An H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) contributes to potassium reabsorption by the collecting ducts of the rat kidney. mRNAs for two isoforms of the H(+)-K(+)-ATPase, HK alpha 1 and HK alpha 2, have been found in the rat kidney. To evaluate whether the HK alpha 1 and HK alpha 2 proteins are present in the rat kidney, microsomes enriched in HK alpha 1 or HK alpha 2 were isolated using the MiniMac magnetic separation system with antibodies directed against either HK alpha 1 (HK 12.18) or HK alpha 2 (AS 31.7). Immunoblots of rat kidney microsomal protein isolated with HK 12.18 revealed a band approximately 94 kDa in size that comigrated with the G1 fraction of the stomach. Immunoblots of rat kidney microsomal protein isolated with AS 31.7 revealed a band slightly greater than 94 kDa that comigrated with a band obtained from rat colonic microsomal protein. To examine the effect of perturbations in potassium metabolism, the abundance of the HK alpha 1 and HK alpha 2 isoforms was compared in rats fed a normal or potassium-deficient diet. A low-potassium diet increased the abundance of HK alpha 2, whereas that of HK alpha 1 was not altered. These data suggest that HK alpha 2 might be the isoform responsible for potassium conservation by the kidney.

Parietal cells in the duodenal bulb and their relation to Helicobacter pylori infection.

AIM: To investigate the prevalence, and relation to Helicobacter pylori, of parietal cells in the duodenal bulb using a monoclonal antibody directed against H+,K(+)-ATPase (HK12.18). METHODS: Twenty six patients with duodenal ulcer disease and 16 healthy controls were studied. H pylori status was determined by gastric histology and culture and by the 13C-urea breath test. Four biopsy specimens were taken from the duodenal bulb and stained with HK12.18. The presence/absence and number of parietal cells in the duodenal bulb were assessed blindly by a histopathologist. RESULTS: The overall prevalence of parietal cells in the duodenal bulb was 31% (13/42) and was similar in patients with duodenal ulcer and in controls, and in H pylori positive and negative subjects. The median (range) number of parietal cells in the duodenal bulb was 7.5 (4-20) parietal cells/subject, and was similar in all four groups. CONCLUSIONS: The prevalence of parietal cells in the duodenal bulb (31%) is notably higher than previously reported in endoscopic studies, and is in keeping with reports from studies on necropsy/operative specimens. There was no difference in the prevalence or number of parietal cells in the duodenal bulb between patients with duodenal ulcer and controls, regardless of H pylori status. These findings suggest that parietal cells in the duodenal bulb do not contribute to the pathogenesis of duodenal ulcer.

Epithelial response of the rat gastric mucosa to chronic superficial injury.

UNLABELLED: Chronic injury to the healthy gastric mucosa with noxious agents such as aspirin or alcohol induces a progressive strengthening of the stomach wall against these insults. The present study examined the histologic response of the rat gastric mucosa to chronic destruction of the superficial mucosa for one month with hypertonic saline. The number, position and morphology of proliferating, parietal, G and D cells were followed during mucosal injury and one month of recovery. The results showed that chronic injury reduced parietal cell numbers by about 30 percent, particularly in the middle of the mucosal thickness where a clear zone was formed by hypertrophy of mucous neck-like cells. G cells were also reduced by about 50 percent, but there were no changes in D cells. Chronic injury induced a marked increase in the number of antral (+112 percent) and fundic (+250 percent) proliferating cells. CONCLUSION: The rat gastric mucosa responds to chronic superficial injury by down-regulation of acid secretory cells and gastrin secreting cells and an up-regulation of proliferating cells. The appearance of a prominent layer of mucous neck-like cells may indicate a new secretory function for these cells.

Identification of parietal cells in gastric body mucosa with HMFG-2 monoclonal antibody.

AIMS: To identify parietal cells in the upper gastrointestinal tract by an immunoperoxidase method, using commercially available monoclonal antibodies. METHODS: Routine surgical biopsy specimens of gastric body mucosa were examined using the avidin-biotin peroxidase method with the monoclonal antibodies HMFG-1 and HMFG-2 to identify parietal cells. Double immunoperoxidase labelling with HK12.18, a well characterised monoclonal antibody directed against an epitope on the alpha (catalytic) subunit of H+ translocating, K+ stimulated adenosine triphosphatase (H,K-ATPase), was used to confirm that HMFG-1 and -2 stained parietal cells. RESULTS: HMFG-1 and HMFG-2 showed consistent parietal cell staining patterns in the gastric body mucosa. HMFG-2 gave a more intense staining pattern of the secretory canaliculi. This was confirmed by double immunolabelling with HK12.18. CONCLUSIONS: HMFG monoclonal antibodies are recommended as highly specific markers of human gastric parietal cells.

Function and structure of H-K-ATPase in the kidney.

The present review summarizes recent functional and structural evidence indicating that the kidney possesses at least one and probably more than one isoform of a proton- and potassium-activated adenosinetriphosphatase (H-K-ATPase). Functional studies have examined in detail the mechanism of luminal acidification and K/Rb absorption by the outer medullary collecting duct (OMCD) from the inner stripe, a high-capacity distal site of urinary acidification. These studies indicate that the mechanism of proton secretion in this segment is similar to a model proposed for gastric acid secretion. Specifically, the profound effect of H-K-ATPase inhibitors or luminal K removal on net bicarbonate (HCO3) absorption indicates a major role for an H-K pump in luminal acidification by the OMCD. The importance of an H-K-ATPase is further supported by the finding that nanomolar concentrations of bafilomycin A1, which specifically inhibit vacuolar-type H-ATPase, have significantly smaller effects on net HCO3 absorption than do H-K-ATPase inhibitors. Studies on the perfused inner medullary collecting duct (IMCD) and cultured IMCD cells also suggest a significant role for H-K-ATPase in luminal acidification by the IMCD. Evidence has accrued from studies in the cortical CD and OMCD that the mechanism of H-K-ATPase-mediated luminal proton secretion differs under K-replete and K-restricted conditions. In K repletion, luminal K ions transported by the pump recycle back into the lumen by a Ba-sensitive mechanism. However, in K restriction, the mechanism of the H-K-ATPase involves luminal proton secretion and K absorption that is insensitive to luminal Ba and, by inference, apical K recycling. Moreover, in K restriction, K/Rb absorption is inhibited by basolateral Ba, indicating that the pump operates to reabsorb K/Rb across the epithelium. The structural evidence reviewed here indicates the presence of mRNA within the mammalian kidney that is either identical or highly homologous to mRNAs for gastric and putative colonic H-K-ATPase alpha-subunits and gastric H-K-ATPase beta-subunit. Localization of these transcripts by in situ hybridization demonstrates gastric alpha- and beta-subunit mRNAs in intercalated cells of both the cortical and medullary CD, principal cells of the CD, and IMCD cells. Additional studies in transgenic mice indicate that regulatory sequences upstream to the H-K-ATPase beta-subunit gene direct transcription in both gastric parietal cells and the renal CD.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural interactions between alpha- and beta-subunits of the gastric H,K-ATPase.

Structural and functional interactions between alpha- and beta-subunits of the H,K-ATPase were explored. The sensitivity to trypsinolysis of alpha-subunit was monitored by SDS-PAGE in control H,K-ATPase-enriched microsomes and in microsomes in which disulfide bonds of the beta-subunit were reduced using 2-mercaptoethanol (2-ME). Reduction of beta-subunit disulfide bonds increased the susceptibility of the alpha-subunit to tryptic digestion. Kinetics of trypsinolysis were also carried out in the presence of ligands known to bind with H,K-ATPase and favor a particular conformer state in the native enzyme. The time-course for release of tryptic peptides was monitored in protein stained gels and Western blots probed with monoclonal antibody alpha-H,K,12.18. In control preparations, where beta-subunit disulfides remained intact, trypsinolysis in the presence of ATP or K+ produced distinctive patterns of tryptic fragments, each characteristic of the conformational states induced by the respective ligand. For 2-ME-treated microsomes the altered alpha-subunit was unable to undergo ligand-induced conformational changes. The increased susceptibility of the alpha-subunit to trypsinization, the change in accessibility of tryptic cleavage sites and the inability of the alpha-subunit to undergo ligand-induced conformational changes after reduction of the beta-subunit disulfides suggest that the interactions between alpha- and beta-subunits are important for the conformational stability of the functional holoenzyme. A model localizing the most susceptible tryptic cleavage sites in control and 2-ME-reduced states is presented.

Renal expression of the gene encoding the gastric H(+)-K(+)-ATPase beta-subunit.

The gastric mucosal parietal cells and cells of the renal collecting duct both possess H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) activities. In the stomach, the H(+)-K(+)-ATPase (EC 3.6.1.3) is responsible for acidification of luminal contents. The kidney H(+)-K(+)-ATPase protein(s) contribute to potassium reabsorption and secretion of hydrogen ions to maintain potassium and acid-base homeostasis. The stomach H(+)-K(+)-ATPase is well defined and consists of an alpha-catalytic subunit of apparent molecular mass of 95 kDa and a highly glycosylated beta-subunit of 60-90 kDa. The molecular identity of the protein that mediates the H(+)-K(+)-ATPase activity in the kidney has been addressed in this paper. A combination of RNA hybridizations, polymerase chain reaction analysis of kidney RNA, and sequence analysis of cDNAs indicated that gastric H(+)-K(+)-ATPase beta-subunit mRNA is present in kidney. Immunoblotting with antibodies specific for the gastric H(+)-K(+)-ATPase beta-subunit detected proteins, which, after deglycosylation, had the same molecular mass as the gastric beta-subunit in membrane protein preparations from rabbit, pig, rat, and mouse kidneys. Furthermore, we have used transgenic mice to demonstrate that the gastric H(+)-K(+)-ATPase beta-subunit gene contains cis-acting regulatory sequences that are active in both gastric parietal cells and the renal collecting ducts. Overall, these data indicate that the gastric H(+)-K(+)-ATPase beta-subunit is found in the kidney and probably associates with the gastric H(+)-K(+)-ATPase alpha-subunit and/or other P-type ATPase alpha-subunits, thus contributing to acid-base and potassium homeostasis.

Physiological and immunocytochemical evidence for a putative H-K-ATPase in elasmobranch renal acid secretion.

The mechanism of renal acid secretion in marine fish is largely unknown. We explored whether H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) is present and functional in acid secretion in the kidney of the elasmobranch spiny dogfish shark, Squalus acanthias. In whole animal studies, a specific inhibitor of mammalian H(+)-K(+)-ATPase, Sch-28080, abolished greater than 87% of basal (62 mg/kg) and 75% of imidazole-stimulated titratable acid excretion (5 and 62 mg/kg). Antibodies directed against the COOH-terminus hog gastric H(+)-K(+)-ATPase alpha-subunit stained specific subdivisions of the neck, early and late proximal tubule, late intermediate tubule, both segments of the distal tubule, and the early collecting duct of the renal tubule of these fish. These findings are consistent with a major role for a protein similar to the mammalian gastric H(+)-K(+)-ATPase in elasmobranch renal acid secretion.

Identification of gastric H,K-ATPase in an early vertebrate, the Atlantic stingray Dasyatis sabina.

Virtually all vertebrates acidify their gastric contents to a pH between 0.8 and 2.0. In mammals, acid secretion is mediated by a K-stimulated proton-translocating adenosine triphosphatase (H,K-ATPase), which establishes a million-fold gradient of protons across the apical membrane of the gastric parietal cell. The earliest phylogenetic appearance of gastric acid secretion is in cartilaginous fish, and we sought to verify in this class (Chondrichthyes) the presence and distribution of H,K-ATPase in gastric epithelial cells. An antibody against a synthetic peptide based on the C-terminus of pig H,K-ATPase alpha-subunit was localized in the gastric glands of the Atlantic stingray Dasyatis sabina. The C-terminal antibody stained all cells with tubulovesicles and the apical membrane domain of mucous neck cells. In proximal stomach, gastric glands showed the strongest immunoreactivity in cells close to the isthmus; in the distal stomach, strongest immunoreactivity was found in cells at the base of the glands. Oxyntic cells were more intensely immunoreactive than oxynticopeptic cells. This antibody labeled a single band of M(r) 100,600 on immunoblots of D. sabina gastric microsomes. These results show the earliest phylogenetic occurrence of a gastric ATPase in putative acid-secreting cells and suggest that this enzyme shares structural features with mammalian H,K-ATPase.

Characterization of membrane and cytoskeletal compartments in cultured parietal cells: immunofluorescence and confocal microscopic examination.

Primary cultures of rabbit gastric parietal cells respond to various gastric secretagogues as evidenced by morphological alterations and [14C]aminopyrine uptake. The availability of cultures of > 95% purity has allowed us to utilize immunofluorescence and confocal microscopy to observe the direct effect of histamine upon the distribution of membrane and cytoskeletal proteins in parietal cells. Cells cultured for 3 days were incubated for 45 min with or without 10(-4) M histamine, washed, and fixed with 3% paraformaldehyde. Immunofluorescence was performed with antibodies against H+/K(+)-ATPase, Na+/K(+)-ATPase, ezrin, and beta-tubulin, as well as with Bodipy-phallacidin. Anti-H+/K(+)-ATPase antibody stained resting cells in a vesicular cytoplasmic pattern. Stimulation with histamine resulted in the development of a well-defined linear pattern, outlining the expanded secretory canaliculi. The Na+/K(+)-ATPase was restricted to predominantly the lateral surface in both the resting and stimulated cells, suggesting that the cultured parietal cells retain membrane polarity. Ezrin was visualized outlining the intracellular canaliculi in the resting state, and surrounding the large secretory canaliculi in the stimulated cell. Phallacidin labeling of F-actin localized to an area tightly surrounding the intracellular canaliculi in the resting cell, and was comparable with the staining observed with ezrin. In the stimulated cells this fluorescence pattern became more diffuse and surrounded the expanded secretory surface. In both the resting and stimulated cells, antibodies to beta-tubulin revealed a microtubular pattern located predominantly in the basal portion of the cell. These results demonstrate that the cells are capable of translocating the H+/K(+)-ATPase-containing tubulovesicles to a secretory surface, and that they exhibit organization and maintenance of basolateral and canalicular membrane domains. Furthermore, these studies demonstrate the directed movement of membrane and cytoskeletal proteins upon stimulation of the cultured parietal cells.

A study of autoimmune gastritis in the postpartum period and at a 5-year follow-up.

The presence of autoimmune gastritis was investigated in 54 women with postpartum thyroiditis. Parietal cell antibodies (PCA) specific against H+, K(+)-adenosine triphosphatase (EC 3.6.1.36) were found in 18 women during pregnancy; in 10 of them, a 2-9-fold increase in the PCA level was observed in the postpartum period. At a 5-year follow-up, the initially PCA-positive women still had elevated antibody levels. Hypergastrinemia and low pepsinogen levels were noted in 4 women. In 2 of these women low serum vitamin B12 levels had developed. In 6 of 9 PCA-positive women examined by gastroscopy, biopsy specimens from the gastric body mucosa contained mononuclear cells, mainly T lymphocytes (CD3+) and macrophages (Leu-M3+) combined with an aberrant epithelial expression of HLA-DR. In four patients with chronic gastritis, all parietal cells, as defined by a specific monoclonal antibody, were found to have immunoglobulin G (IgG) deposits by a double-immunostaining method. Three of them had microscopic evidence of atrophy, whereas in 1 patient the body mucosa was intact. In 1 further patient with intact glands at histological examination, the basolateral membrane of some oxyntic glands was coated with IgG. The selective in situ deposition of antibodies associated with histologically intact parietal cells may support the concept that specific autoantibodies participate in the early pathogenesis of parietal cell destruction.

Site-directed antibodies as topographical probes of the gastric H,K-ATPase alpha-subunit.

Gastric acid is secreted by an ATP-driven H+ and K+ exchanger (H,K-ATPase), an integral apical membrane protein of parietal cells. Although the primary structure of the enzyme is known, its higher order structure is uncertain. In order to acquire topographical probes of native, microsomal H,K-ATPase, synthetic peptides corresponding to the 17 amino-terminal (N-peptide) and 16 carboxyl-terminal (C-peptide) residues of pig gastric H,K-ATPase alpha-subunit were coupled to keyhole limpet hemocyanin (KLH). Rabbits were immunized with peptide-KLH conjugates and their sera were tested for specificity by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunocytochemistry. All sera showed high ELISA reactivities with synthetic peptides, peptide-BSA conjugates, and microsomal H,K-ATPase adsorbed to microtiter wells (some titers greater than 1:10(4)). Immunoblots of H,K-ATPase resolved by SDS-PAGE showed both N-peptide and C-peptide antibodies reacting with a single 94 kDa band. All sera selectively stained parietal cells in pig gastric mucosal sections. Preimmune sera gave negative or weak signals in all assays. In competition ELISAs, N-peptide antibodies, but not C-peptide antibodies, were displaced from the corresponding bound synthetic peptides by added microsomal H,K-ATPase. One of the N-peptide antibodies inhibited H,K-ATPase activity by more than 50%; binding of this antibody was decreased when ATP or K+ were bound to the enzyme. These results indicate a cytoplasmically-oriented alpha-subunit N-terminus which may participate conformationally in the H,K-ATPase catalytic cycle, and suggest that antibodies against synthetic H,K-ATPase peptides are potentially useful probes of native microsomal H,K-ATPase topography.

Gastric H,K-ATPase topography: amino acids 888-907 are cytoplasmic.

Gastric acidification is mediated by H,K-ATPase, an integral protein of apical membranes of gastric parietal cells. Hydropathy analysis of H,K-ATPase alpha subunit primary structure predicts eight transmembrane (TM) domains, while omeprazole-binding data were interpreted in terms of ten TM domains (Mercier et al. (1991) FASEB J. 5, A749). In the present study, tryptic hydrolysis of gastric mucosal microsomes gave a set of peptides which bound the monoclonal antibody HK 12.18, a highly specific probe of the H,K-ATPase. An antiserum against the C-terminus of H,K-ATPase alpha subunit bound the same peptides, and one smaller peptide. The binding data suggested a putative epitope for HK 12.18, and a 20-mer peptide encompassing this site was synthesized. This peptide bound directly to HK 12.18, displaced HK 12.18 from microsomal H,K-ATPase, and blocked HK 12.18 immunostaining of gastric parietal cells. In addition, intact gastric microsomes competitively inhibited binding of HK 12.18 to peptide-BSA conjugate. Taken together, these data place the HK 12.18 epitope between amino acids 888-907 and identify this domain as cytosolic. This result specifically excludes a pair of TM domains between the sixth and seventh TM alpha helices of the H,K-ATPase and supports a secondary structure model with eight TM domains.