TRANSFER OF RETINOL-BINDING PROTEIN FROM HEPG2 HUMAN HEPATOMA-CELLS TO COCULTURED RAT STELLATE CELLS

Rat liver stellate cells were cocultured with HepG2 human hepatoma cel ls, which are known to synthesize and secrete retinol-binding protein (RBP). Transfer of human RBP from HepG2 cells to stellate cells was st udied by cryoimmunoelectron microscopy. In stellate cells, human RBP w as found on the cell surface and within endosomes. The transfer of hum an RBP from HepG2 cells to stellate cells was blocked by addition of R BP antibodies to the culture medium. Very little uptake of RBP was obs erved when fibroblasts were cocultured with HepG2 cells. In a series o f experiments, RBP was bound to its putative cell surface receptor at 4-degrees-C, and the stellate cells were washed and then incubated at 37-degrees-C in order to allow them to internalize a pulse of RBP. Abo ut 50% of the RBP was internalized after 6 min of incubation. The RBP- positive vesicles were initially (after 1-2 min) located close to the cell surface and later were found deeper in the cytoplasm. During the first 10 min, RBP was mainly observed in close association with membra nes. After 2 hr, however, most RBP was localized in intracellular vesi cles at a distance from the vesicular membranes, suggesting that R-BP had been released from its receptor. Saturable binding of RBP to liver cells was demonstrated when cells were incubated with I-125-RBP at 4- degrees-C and cell-associated radioactivity was determined. The calcul ated dissociation constant for the specific binding was 12.7 +/- 3.2 n M. A binding assay was also developed for determination of solubilized RBP receptor. Solubilized proteins from the nonparenchymal liver cell s bound about 30 times more I-125-labeled RBP than did parenchymal cel ls (based on mass of cell protein). These data suggest that RBP mediat es the paracrine transfer of retinol from hepatocytes to perisinusoida l stellate cells in liver and that stellate cells bind and internalize RBP by receptor-mediated endocytosis.