Expression of plasma membrane Ca2+-ATPase on hepatic sinusoidal endothelial fenestrae: modification of the one-step method.

Abstract

The intracytoplasmic free calcium ion (Ca2+) concentration is maintained at a low level in mammalian tissues by extruding Ca2+ against a high extracellular Ca2+ concentration, mainly through the activity of the plasma membrane Ca2+-ATPase pump. The objective of the present study was to localize the plasma membrane Ca2+-ATPase activity on hepatic sinusoidal endothelial cells (SECs) by electron microscopic cytochemistry. The ultrastructural localization of Ca2+-ATPase activity on ultrathin sections of liver tissue and cultured SEC monolayer was examined by the electron microscopic cytochemical method of Ando (method A: original method) and by our modified method (method B: shortened fixation method). By method A, scanty cytochemical reaction products of Ca2+-ATPase were found in the SECs. By method B, Ca2+-ATPase activity was clearly localized on the outer surface of the plasma membrane of sinusoidal endothelial fenestrae (SEF). Our modification of Ando's method by shortening the incubation time of liver tissue or isolated SEC sections in the substrate allowed clear demonstration of Ca2+-ATPase activity on the SEF membrane. Use of tangential sections of primary cultures of SEC provided excellent localization results. The cytochemically reactive Ca2+-ATPase expressed on the SEF plasma membrane may be involved in regulation of the intracytoplasmic Ca2+ concentration.