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

Publisher Website
Google Scholar
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.