Cultured Ito cells of rat liver express the alpha2-macroglobulin gene

Abstract

Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize .alpha.2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized .alpha.2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. .alpha.2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5-11 days after the isolation of cells, increasing amounts of .alpha.2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled .alpha.2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. .alpha.2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled .alpha.2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with .alpha.-macroglobulin. .alpha.2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where .alpha.2-macroglobulin was synthesized in Ito cells, no synthesis of .alpha.1-macroglobulin, .alpha.1-inhibitor 3, .alpha.1-proteinasee inhibitor, .alpha.1-acid glycoprotein, .alpha.1-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that .alpha.2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases.