Glucose-6-phosphatase distribution in isolated and cultured adult rat hepatocytes.
The cytochemical localization of glucose-6-phosphatase (G6Pase) and its biochemical quantification were studied in isolated and cultured adult rat parenchymal cells. Appropriate technical conditions were chosen to assume adequate ultrastructural preservation and retention of enzyme activity. Isolated hepatocytes separated by collagenase perfusion were shortly fixed in glutaraldehyde and entrapped in a pellet of fibrin. Frozen sections, 50 microns in thickness were incubated for cytochemical demonstration of G6Pase, in a slightly modified Wachstein-Meisel medium. Hepatocytes in culture, fixed for 1 min in glutaraldehyde, were impregnated in a 10% cryoprotective glycerol solution and quickly frozen in liquid nitrogen at -170 degrees C in order to induce penetration of the substrate. In these conditions, a homogeneous distribution of the enzyme was observed in both isolated and cultured cells. The cytochemical reaction appears continuous in the smooth and rough endoplasmic cisternae and in the nuclear envelope. Lead phosphate deposits, although evenly distributed, are reduced in intensity after 48 h culture. Biochemical determinations reveal the presence of a high specific enzymatic activity in isolated cells (108 nmolP/min/mg proteins), which decreases in culture, respectively to 70 and 50% of the original value, after 24 and 48 h culture. G6Pase induction by glucagon was obtained after 48 and 72 h in culture.
D000818:Animals; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005934:Glucagon / Q000494:pharmacology; D005952:Glucose-6-Phosphatase / Q000378:metabolism*; D006651:Histocytochemistry; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009685:Nuclear Envelope / Q000201:enzymology; D051381:Rats
D005934:Glucagon; D005952:Glucose-6-Phosphatase