Biochemical Properties and Cytochemical Localization of Ouabain-insensitive, Potassium-dependentp-Nitrophenylphosphatase Activity in Rat Atrial Myocytes

Abstract

Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3, Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).

Identification and regulation of the gastric H+/K+‐ATPase in the rat heart

“…We thus anticipated that there might be another mechanism than the Na + /K + ‐ATPase and Na + /K + /2Cl − co‐transporter that could translocate K + into the cardiac myocytes. In previous studies, Northern blot and cytochemical analysis had indicated presence of the gastric H + /K + ‐ATPase in rat myocardial tissue (Crowson & Shull 1992) and atrial myocytes (Zinchuk 1992, Zinchuk et al. 1997), but there was no other report of similar findings at both RNA, protein and functional levels, in the left ventricle.…”

“…This observation is compatible with our finding of a relatively low level of mRNA expression, using competitive RT‐PCR. Zinchuk (1992) and Zinchuk et al. (1997) showed enzyme activity that represented ouabain‐insensitive, potassium‐dependent p ‐nitrophenylphosphatase ( p ‐NPPase) in rat atrial myocytes and the p ‐NPPase activity detected was considered to be a H + /K + ‐ATPase.…”

“…In a series of Northern blot hybridization experiments using probes derived from the gastric H + /K + -ATPase cDNA, trace amounts of the gastric isoform of H + /K + -ATPase were detected in the rat heart (Crowson & Shull 1992). Possible biochemical and physiological evidence for a H + /K + -ATPase in the myocardium have also been reported (Zinchuk et al 1997, Nagashima et al 1999, but molecular, biochemical and functional evidence for a H + /K + -ATPase in ventricular cardiac myocytes has not been reported.…”

Placebokontrollierte, doppelblinde Crossover-Studie zur Untersuchung der Wirkung einer parenteralen Gabe von Pantoprazol auf die Kontraktilität des Herzen

“…Oral applizierte PPI gelten in ihrer Anwendung allgemein als gut verträglich und arm an Nebenwirkungen (Robinson 2001).Darüber hinaus ist die Expression des Zielenzyms der PPI, die H + /K + -ATPase, ebenfalls nicht nur auf den Magen beschränkt. Bereits vor einiger Zeit wurde die H + /K + -ATPase in Vorhofmyozyten der Ratte(Shin et al 1993, Zinchuk et al 1997),…”