The p7 viroporin of the hepatitis C virus contributes to liver inflammation by stimulating production of Interleukin-1ß.

Hepatitis C is one of the most widespread infectious diseases worldwide and hepatitis C virus (HCV)-induced chronic inflammation is highly associated with progredient liver damage. It was shown that HCV infection increases levels of pro-inflammatory cytokines via activation of NOD-like receptor (NLRP3) inflammasomes, yet the underlying mechanism is still under question. We propose modulation of intracellular pH by p7, a 63 residue ion channel produced by the hepatitis C virus as a possible pathomechanism for hepatitis C-associated inflammation. Recombinant constructs corresponding to HCV genotypes 1-4 were expressed in HEK 293 and RAW 264.7 cells and changes of intracellular pH were monitored using pH-sensitive fluorescent probes as well as production of inflammatory cytokines. Presence of p7 induced general loss of vesicular acidity as well as producing a significant increase in the levels of interleukin-1ß (IL-1ß). Effects showed a genotype-dependent pattern of IL-1ß production, in agreement with the pH-response profile of p7 channels corresponding to hepatitis C genotypes. Lowering the pH of the extracellular medium increased activity of p7 channels as well as production of IL-1ß for genotypes 1, 3, and 4, but less for genotype 2. Our data are in agreement with the hypothesis that p7 activity can trigger intracellular signaling cascades that are involved in HCV-associated cytopathy.

Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pump's constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henle's loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

The H+/K+-ATPase (proton) pump is expressed in human laryngeal submucosal glands.

OBJECTIVES/HYPOTHESIS: Diagnosis and treatment of gastroesophageal and laryngopharyngeal reflux disease has significantly increased over recent years. The larynx is highly sensitive to the effects of LPRD and is similarly responsive to proton pump inhibitor pharmacotherapy. The hypothesis of the study was that proton pump activity exists in the human larynx and plays a functional role in normal and/or pathological laryngeal tissue. STUDY DESIGN: Pathological investigation. METHODS: Two fresh human cadaveric larynges (one male and one female larynx) were obtained as part of an exempt protocol from the Human Subjects Committee and were formalin fixed and paraffin embedded. Banked human stomach tissue was also obtained for use as comparative positive and negative control specimens. Sections were immunostained with monoclonal antibodies reactive with both alpha and beta subunits of the H+/K+-ATPase (proton) pump. Specimens were reviewed for staining pattern and intensity. RESULTS: Stomach parietal cells (known to produce gastric acid) exhibited strongly positive staining for both the alpha and beta subunits of the proton pump. There was no staining in stomach cells that were not morphologically consistent with the parietal cell. In the human larynx there were strong focal and identical staining patterns in the serous cells and ducts of the minor seromucinous glands by both alpha and beta monoclonals to the proton pump. There was variable staining in the laryngeal epithelium that was thought to be consistent with artifact staining resulting from tissue processing. CONCLUSION: The H+/K+-ATPase (proton) pump is present in serous cells and ducts of submucosal glands in the human larynx. Proton pump inhibitor pharmacotherapy may have a site of action in seromucinous glands of the human larynx, with possible relevance for patients treated for chronic laryngitis with or without laryngopharyngeal reflux disease.

Neonatal injection of native proton pump antigens induces autoimmune gastritis in mice.

BACKGROUND & AIMS: Autoimmune gastritis is associated with gastric H+, K(+)-adenosine triphosphatase (ATPase)-specific autoantibodies (HKAb). The (auto) antigen that triggers disease and the pathogenic role of the autoantibodies are unknown. The aim of this study was to analyze when these autoantibodies are produced during autoimmune gastritis in neonatally thymectomized mice and whether a native H+, K(+)-ATPase antigen preparation can induce disease in mice. METHODS: Autoantibodies were characterized by a novel assay based on immunoprecipitation of a functional H+, K(+)-ATPase expressed in Xenopus oocytes. Normal mice were injected intraperitoneally with H+, K(+)-ATPase-enriched gastric membranes in the absence of adjuvant. RESULTS: Conformational autoantibodies recognizing both H+, K(+)-ATPase subunits appeared simultaneously with the gastric lesions 1 month after thymectomy. Immunization of neonates, but not adults, induced a persistent autoimmune gastritis in the body mucosa, characterized by lymphocytic infiltrations, loss of parietal and chief cells, metaplasia, and H+, K(+)-ATPase-specific autoantibodies. The histopathological lesions of this new model are similar to those in humans and thymectomized mice. CONCLUSIONS: The onset of gastritis and autoantibody production parallels the expression of the H+, K(+)-ATPase during ontogeny. Exposure of the neonatal immune system to organ-specific antigens expressed late after birth induces autoimmune gastritis in adult mice.

Acidification of vacuoles is required for autophagic degradation in the yeast, Saccharomyces cerevisiae.

Acidification inside vacuoles has been shown to play a key role in a number of physiologically important cellular events. We studied the role of vacuolar membrane H(+)-ATPase in the autophagic process of Saccharomyces cerevisiae. Mutants lacking VMA genes which encode their subunits of the vacuolar H(+)-ATPase accumulated autophagic bodies in vacuoles on starvation. vma mutants also had a defect in protein degradation induced by starvation. In vma mutants, the activities of vacuolar proteases were remarkably lower than those of the wild-type. Overexpression of vacuolar proteases did not overcome the defect in the disintegration of autophagic bodies in vma mutant, even the overexpressed proteinase A and proteinase B being substantially localized to the vacuolar compartment and undergoing proper proteolytic maturation. Our results showed that the acidification of vacuoles is not required for the formation and delivery of autophagosomes to vacuoles, but is essential for the disintegration of autophagic bodies.

Differential immunological cross-reactions with antisera against the V-ATPase of Kalanchoë daigremontiana reveal structural differences of V-ATPase subunits of different plant species.

Two antisera (ATP88 and ATP95) raised against the V-ATPase holoenzyme of Kalanchoë daigremontiana were tested for their cross-reactivity with subunits of V-ATPases from other plant species. V-ATPases from Kalanchoë blossfeldiana, Mesembryanthemum crystallinum, Nicotiana tabacum, Lycopersicon esculentum, Citrus limon, Lemna gibba, Hordeum vulgare and Zea mays were immunoprecipitated with an antiserum against the catalytic V-ATPase subunit A of M. crystallinum. As shown by silver staining and Western blot analysis with ATP88, subunits A, B, C, D and c were present in all immunoprecipitated V-ATPases. In contrast, ATP95 recognized the whole set of subunits only in K. blossfeldiana, M. crystallinum, H. vulgare and Z. mays. This differential cross reactivity of ATP95 indicates the presence of structural differences of certain V-ATPase subunits. Based on the Bafilomycin A1-sensitive ATPase activity of tonoplast enriched vesicles, and on the amount of V-ATPase solubilized and immunoprecipitated, the specific ATP-hydrolysis activity of the V-ATPases under test was determined. The structural differences correlate with the ability of V-ATPases from different species to hydrolyze ATP at one given assay condition for ATP-hydrolysis measurements. Interestingly V-ATPases showing cross-reactivity of subunits A, B, C, D and c with ATP95 showed higher rates of specific ATP hydrolysis compared to V-ATPases containing subunits which were not labeled by ATP95. Thus, V-ATPases with high turnover rates in our assay conditions may show common structural characteristics which separate them from ATPases with low turnover rates.

Interference with the endosomal acidification by a monoclonal antibody directed toward the 116 (100)-kD subunit of the vacuolar type proton pump.

A monoclonal antibody (OSW2) was prepared by using human osteosarcoma cells. OSW2 was found to be directed toward the 116 (also called 100)- kD protein that uniquely associates to the vacuolar-type proton pump. The antibody specifically localized acidic membrane compartments that could be visualized with acridine orange in many types of human cells. It also reacted with the surface and was internalized along the endosomal pathway. Monitoring the endosome pH by using FITC-dextran and acridine orange suggested that the antibody interfered with low pH. Cell-free experiments indicated that the ATP-dependent acidification was inhibited in endosomes associated with OSW2. In contrast, the antibody gave little effect on the ATPase activity of the solubilized H+ pump. The internalization of OSW2 reduced infectivity of certain enveloped viruses (influenza, SFV, VSV) by 50 to 80%. Inhibition of viral fusion was directly demonstrated by monitoring the fate of octadecylrhodamine-labeled influenza virus fluorescence. These results indicate that the 116 (100)-kD protein is necessary for the control of pH. The antibody represents a novel probe for understanding the role of the endosomal compartments in cellular physiology.

Vacuolar H(+)-ATPase of Dictyostelium discoideum. A monoclonal antibody study.

A Dictyostelium membrane fraction rich in vacuolar proton pumps, previously described by Nolta et al. (J. Biol. Chem. 266, 18,318-18,323, 1991), was used as the immunogen for production of monoclonal antibodies. We obtained antibodies that recognized polypeptides of 100 kDa and 68 kDa, corresponding to the two largest subunits of the vacuolar proton pump. In indirect immunofluorescence experiments, these two subunits were located on an interconnected collection of tubules and vacuoles. On frozen thin sections they were found principally on membranes of vacuoles and collections of small vesicles typically located just internal to the plasma membrane. These vesicles and vacuoles had electron-translucent lumens. No other structures in axenically grown Dictyostelium cells were labeled to a significant extent by these two antibodies. Using an affinity-purified antibody to calmodulin and a monospecific antibody to the B subunit of the chromaffin granule vacuolar ATPase, markers known to label the membranes of the contractile vacuole complex in Dictyostelium (Zhu and Clarke, J. Cell Biol. 118, 347-358, 1992; Heuser et al., J. Cell Biol. 121, 1311-1327, 1993), we showed that the 100 kDa and 68 kDa subunits had the same distribution as these two markers. Co-localization was seen in both interphase and mitotic cells. Thus, our results support the conclusion that vacuolar proton pumps are located principally on the membranes of the contractile vacuole complex in Dictyostelium. In addition, in indirect immunofluorescence experiments, these monoclonal antibodies provided improved images of the organization of the contractile vacuole system.

An immunocytochemical analysis of the vacuolar proton pump in Dictyostelium discoideum.

Antisera were generated in rabbits against the vacuolar proton pump (V-H(+)-ATPase) purified from Dictyostelium discoideum. The antisera inhibited V-H(+)-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H(+)-ATPase from cell homogenates. Immunocytochemical analysis of intact cells and subcellular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shapes, which corresponded to the acidosomes. The cytoplasmic surfaces of lysosomes, phagosomes and the tubular spongiome of the contractile vacuole also bore the pump antigen. The lumina of multivesicular bodies were often stained intensely; the internalized antigen may have been derived from acidosomes by autophagy. Antibodies against V-H(+)-ATPases from plant and animal cells cross-reacted with the proton pumps of Dictyostelium. Antisera directed against the V-H(+)-ATPase of Dictyostelium decorated a profusion of small vacuoles scattered throughout the cytoplasm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells. We conclude that the V-H(+)-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.

The osteoclast proton pump differs in its pharmacology and catalytic subunits from other vacuolar H(+)-ATPases.

Osteoclasts are multinucleated cells derived from the mononuclear phagocyte system in the hematopoietic bone marrow. Their function is to resorb bone during skeletal growth and remodeling. They perform this function by acidifying an enclosed extracellular space, the bone resorbing compartment. Analysis of proton transport by inside-out vesicles derived from highly purified chicken osteoclast membranes has revealed the presence of a novel type of multisubunit vacuolar-like H(+)-ATPase. Unlike H(+)-ATPases derived from any other cell type or organelle, proton transport and ATPase activity in osteoclast vesicles are sensitive to two classes of inhibitors, namely V-ATPase inhibitors [N-ethyl-maleimide (NEM) and bafilomycin A1] and vanadate (IC50 100 mumol l-1), an inhibitor previously found to affect only P-ATPases. The osteoclast V-ATPase morphologically resembles vacuolar proton pumps and contains several vacuolar-like subunits (115 x 10(3), 39 x 10(3) and 16 x 10(3)M(r)), demonstrated by Western blot analysis. Subunits A and B of the catalytic domain of the enzyme, however, differ from that of other V-ATPases. In osteoclasts, subunit A has an M(r) of 63 x 10(3) instead of 67 x 10(3)-70 x 10(3); in contrast, monocytes, macrophages and kidney microsomes, which contain a vanadate-insensitive H(+)-ATPase, express the classical subunit A (70 x 10(3)M(r)). Moreover, two types of 57 x 10(3)-60 x 10(3)M(r) B subunits are also found: they are differentially recognized by antibodies and one is expressed predominantly in osteoclasts and the other in bone marrow cells and in kidney microsomes. Preliminary cloning data have indicated that the B subunit expressed in osteoclasts may be similar to the brain isoform. The osteoclast proton pump may, therefore, constitute a novel class of V-ATPase, with a unique pharmacology and specific isoforms of two subunits in the catalytic portion of the enzyme.