Studies of Plasma Membrane Domain-Specific Molecules in Isolated and Cultured Rat Hepatocytes

Pamela Leslie Zeitlin, Yale University.

This is an Open Access Thesis


Single rat hepatocytes, prepared by collagenase perfusion of liver, lost the three morphologically and functionally distinct plasma membrane domains that exist in situ (sinusoidal, bile canalicular, and lateral). Hepatocytes placed in monolayer culture re-established morphologically distinct plasma membrane regions analogous to the original three domains. The behavior of several domain-specific markers was studied in these cells. The asialoglycoprotein (ASGP) receptor, an integral membrane glycoprotein normally restricted at the surface to the sinusoidal domain in situ, was retained in both single hepatocytes (> 95%) and in cells after 24 hr in monolayer culture but at lower levels. Cytochemistry with asialo-oroso-mucoid-horseradish peroxidase revealed that the receptor was concentrated in pits and diffusely distributed along the entire cell surface of isolated cells. However, in 24 hr cultured cells, the tracer lined all plasma membrane surfaces except those of bile canalicular-like spaces. In both cell systems, the surface receptor continued to function in the receptor-mediated endocytosis of ('125)I-asialo-orosomucoid.Cell surface and internal pools of ASGP receptors were measured and localized by EM-ARG of fixed and permeabilized hepatocytes. Binding sites were concentrated in the plasma membrane, peripheral cytoplasm, Golgi complex, and cytoplasm within 1 (mu)m of the Golgi complex. Cell surface receptors accounted for 16% of total cellular receptors. The numbers of surface and total receptors were measured in cultured cells and found to both decrease by 80% after 72 hr in culture.The level of a bile canalicular domain antigen, HA4, was found by radioimmunoassay to remain at near normal levels for at least 72 hr in culture. By indirect immunofluorescence both HA4 and the ASGP receptor were found on all surfaces of the cultured cells, however the HA4 was concentrated in patches between cells at 24 hr.