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Detection of ouabain-insensitive H(+)-transporting, K(+)-stimulated p- nitrophenylphosphatase activity in rat gastric glands by cerium-based cytochemistry

T Kobayashi and H Seguchi

Department of Anatomy, Kochi Medical School, Japan.

We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p- nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain- insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p- nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron- dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).

Volume 38, Issue 12, pp. 1895-1905, 12/01/1990
Copyright © 1990 by The Histochemical Society


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V. S. Zinchuk, T. Kobayashi, E. G. del Saz, and H. Seguchi
Biochemical Properties and Cytochemical Localization of Ouabain-insensitive, Potassium-dependent p -Nitrophenylphosphatase Activity in Rat Atrial Myocytes
J. Histochem. Cytochem., February 1, 1997; 45(2): 177 - 188.
[Abstract] [Full Text] [PDF]



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