SECRETION OF INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-1 FROM INDIVIDUAL HEPATOCYTES

The reverse hemolytic plaque assay (RHPA) uses complement-mediated red blood cell lysis to detect peptide secretion by individual cells. Ini tially, the RHPA was used to study the function of neurons and B lymph ocytes. More recently, the RHPA has been adapted to measure hormone re lease from individual pituitary, parathyroid, luteal, and pancreatic i slet cells. We have applied this technique to detect insulin-like grow th factor binding protein-1 (IGFBP-1) secretion by a human hepatoma ce ll line (HepG2). We proposed that the technique of RHPA could be used to study peptide release from single hepatocytes in various defined co nditions. Our goal was the study of the kinetics of IGFBP-1 secretion from hepatoma cells and rat hepatocytes and to determine the heterogen eity of the cell population regarding the secretion of IGFBP-1. To eva luate the optimal conditions of IGFBP-1 secretion by hepatoma cells an d rat hepatocytes and to evaluate the influence of cell dispersion on hepatocyte's behavior, we evaluated three techniques of cell dispersio n: trypsin digestion, collagenase digestion, and mechanical dispersion . We tested cell viability, determined the percentage of secreting cel ls versus non-secreting cells, and measured mean plaque area which is a function of the amount of IGFBP-1 secreted by an individual cell. We determined the optimal IGFBP-1 antibody dilution for the detection of secreted IGFBP-1 by hepatocytes, evaluated the initiation of IGFBP-1 secretion from cultured cells, and quantified time-dependent IGFBP-1 s ecretion. In addition to demonstrating the feasibility of measuring IG FBP-1 from a cultured cell line, we measured IGFBP-1 release from fres hly dispersed rat hepatocytes.