engleski

Artifactual localization of a vacuolar H+-ATPase isoform in the apical cell membrane of the rat intestine

The renal vacuolar H+-ATPase is a multi-subunit protein that acidifies tubule fluid and the interior of various intracellular organelles. Two isoforms of the 56 kD H+-ATPase subunit have been detected in mammalian kidney which indicate possible functional differences of the pumps in the collecting duct intercalated cells (kidney isoform, 56 kDki) and in other tubule cells (brain isoform). We have observed that an antibody against the 56 kDki subunit labeled immunohistochemically the luminal membrane of intestinal absorptive cells in the rat. The aim of this paper is to elaborate this initial observation and to correlate it with an equivalent ATPase activity that may reside in these cell membranes. Material and methods: An antibody against the 56 kDki subunit was used to localize the antigen by indirect immunofluorescence in frozen sections of the fixed intestinal tissue and by immunoblotting in brush-border membrane vesicles (BBMV) isolated from the rat jejunum. Bafilomycin-sensitive ATPase activity was measured in intact and detergent-treated BBMV by the phosphate-liberation assay. Results: Immunofluorescence reaction was observed in the luminal membrane of small intestinal and colonic absorptive cells, and in the goblet cell cytoplasm. In detergent-pretreated tissue sections, staining of the luminal membrane was largely diminished, whereas staining in goblet cells was enhanced. In isolated jejunal BBMV we found neither a significant bafilomycin-sensitive ATPase activity nor a specific protein band labeled with the antibody. Conclusion: The immunoreaction of the luminal membrane in small intestinal and colonic absorptive cells with the antibody against the 56 kDki subunit may be an artifact ; the antibody may stick to the mucus produced by the goblet cells and mimic the presence of the specific antigen. A vacuolar H+-ATPase in the small intestinal BBMV could not be demonstrated.

small intestine; colon; brush-border membrane; vacuolar H+-ATPase

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