ISOLATION AND ACTION OF ADULT RAT HEPATOCYTE MITOGENS

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Monoclonal antibodies (MCA) and primary adult rat liver cell cultures (ARL) will be used to isolate a new hepatocyte serum protein growth factor (HGF) and to study its mechanism of action. Either partially (5700-fold) or further purified (cation exchange/Cibacron Blue Dye/HPLC) HGF will be used to generate mouse hybridoma cell lines whose fluids will be screened for anti-HGF activity by standard enzyme-linked immunoadsorbant assays and by ARL bioassays. Anti-HGF-MCA obtained will be used to immunoaffinity purify HGF, which will then be characterized by conventional and advanced semi-micro protein chemistry technology. The hypothesis that HGF and other peptide mitogens (EGF, insulin and glucagon) activate monovalent cation fluxes which are necessary for growth will be tested critically with a new panel of 9 mouse anti-rat (Na+, K+)-ATPase "pump" MCA's constructed in this laboratory. Starting with "9B1" (one of the new MCA's that binds to bile canalicular membranes, and inhibits ouabain-sensitive [Na+, K+]-ATPase as well as cultured hepatocyte DNA synthesis), detailed experiments (requiring large amounts of pure well-characterized MCA, that will be disseminated to other laboratories working on unrelated problems) will be conducted to determine if (from ARL and dose-response bioassays), when (from interval/duration and proliferation kinetic studies), and how (from measurements of ouabain-sensitive ATPase, 86Rb+ influx and Na+ and K+ content; and from electron microscopic immunocytochemical localization studies) anti-pump MCA's influence hepatocyte proliferative transitions under chemically defined conditions. This research will provide new insights into fundamental problems of how mitogens control animal cell proliferation. In particular, this work will lead to new approaches towards isolating blood borne factors that might relationships between the effects of growth factors and the eventual initiation of DNA synthesis; and to new information about the structure, function and location of the liver sodium pump.