6533b7d6fe1ef96bd12665b3

RESEARCH PRODUCT

Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

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Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize a2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized a2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and fluorography. a2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5 - 11 days after the isolation of the cells, increasing amounts of a2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled a2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. a2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled a2-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 a2-macroglobulin. a2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where a2-macroglobulin was synthesized in Ito cells, no synthesis of al-macroglobulin, al-inhibitor 3, a,-proteinase inhibitor, al-acid glycoprotein, al-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that a2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases. Ito cells of the liver (fat-storing cells, lipocytes, stellate cells) are located in the perisinusoidal space underneath the endothelial cells and in tight contact with hepatocytes. In the rat they represent 8 - 13% of the non-parenchymal cells [l,