| Description |
Previous studies have shown mammalian hepatic adenylate cyclase is stimulated by several compounds including: fluoride ion, guanosine-5-trophosphate (GTP), glucagon, secretin and vasoactive intestinal polypeptide (VIP). In addition to these effectors, prostaglandins of the E series were found to stimulate the enzyme, and this response is entirely dependent upon the presence of GTP. Since liver is composed of several distinct cell types (including Kupffer cells and hepatocytes), these responses may be due to effects on different cell specific enzymes. The purpose of this work was to examine adenylate cyclases obtained from isolated homogeneous populations of liver cells. Previously developed methods for isolation of liver cells did not yield cell fractions free of cross-contamination. A procedure was developed to isolated homogenous fractions of hepatocytes and Kupffer cells from rat liver. Animals were given intravenous injections of particulate (3 um) carbonyl iron to "prelabel" the phagocytic Kupffer cells, livers were perfused with digestive enzymes which breakdown connective tissue of organ to be dispersed. Magnetic and centrifugal separation procedures were used to obtain 3 distinct cell fractions: hepatocytes, Kupffer cells and hepatocyte wash. Kupffer cell and hepatocyte fractions were determined by microscopic analysis to be 99% homogeneous; the hepatocyte wash fraction contained blood components, hepatocytes, nuclei, and cell debris. Isolated hepatocytes contain high specific activity glucose-6-phosphatase, some 5'-nucleotidase, and acid phosphatase activity. Kupffer cells contain high specific activities of 5'-nucleotidase, acid phosphatase, and an activity responsible for the conversion of isoproterenol to an unidentified product. Adenylate cyclase from isolated hepatocytes responds to all effectors as does enzyme obtained from whole liver. Glucagon stimulated enzyme is greatly enriched, and hepatocytes in this fraction account for most (70%) of the response measured in the intact organ. Kupffer cell enzyme has a high basal activity, and is stimulated by fluoride ion and GTP. An adenylate cyclase of high specific activity is present is the hepatocyte wash fraction; the enzyme responds to all effectors which stimulate the hepatocyte enzyme. At least 70% if whole liver prostaglandin sensitive adenylate cyclase is present in the hepatocyte wash. Blood components and nuclei contained in the wash cannot account for this activiaty. Stimulations caused by adding combinations of hormones to samples containing hepatocyte adenylate cyclase show the presence of separate receptors for glucagon and the gastrointestinal hormones. A model assuming mobile receptors is proposed to explain present in this cell type. |
