Observation of cytochemical alkaline phosphatase activity on the plasma membrane of cultured rat hepatocytes by backscattered electron imaging.

Abstract

Distribution of alkaline phosphatase (ALPase) activity on the plasma membrane of rat hepatocytes in primary monolayer culture was observed using the backscattered electron image (BEI) mode. Apparent reaction product was seen in hepatocytes cultured for 48 or 72 hours in a concentration of 8 X 10(5) cells per ml, and those cultured for 72 or 96 hours in a concentration of 6 or 4 X 10(5) cells per ml. The reaction product was observed as scattered fine dots, as spots, and as bands along the cell edge, with high contrast in flattened polyhedral hepatocytes forming cell trabeculae. The deposition of the reaction product was generally more abundant at the periphery of the cell trabeculae. X-ray microanalysis revealed that the reaction product contains both lead and phosphorus as real ALPase reaction product. In transmission electron microscopy, the reaction product was exclusively localized on the external surface of the plasma membrane. However, the plasma membrane adjoining the culture substratum was devoid of the reaction product. Further, the highest biochemical ALPase activity appeared in earlier culture stages when the density of cultured cells was larger. Thus, during a few days of culture, ALPase activity increases on the plasma membrane predominantly at the periphery of the hepatocyte trabeculae. This increase in the activity might be related to the mechanism of preserving the cell shape.