We investigated the effects of prostaglandin (EP) receptor subtype agonists on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes to elucidate their mechanisms of action. Maintained in short-term cultures (i.e. 3.5 h) in a serum-free, defined medium, hepatocyte parenchymal cells underwent DNA synthesis and proliferation in the presence of sulprostone (10 ؊6 M), PGE 2 (10 ؊6 M), and 17-phenyl-trinor-PGE 2 (10 ؊9 M) in a time-and dose-dependent manner. PGE 2 was less

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... nt than 17-phenyl-trinor-PGE 2 in stimulating hepatocyte mitogenesis. Sulprostone (10 ؊6 M) and 11-deoxy-PGE 1 (10 ؊6 M) showed weak and insignificant stimulation, respectively, for hepatocyte mitogenesis. These effects of PGE 2 , 17-phenyl-trinor-PGE 2 , and sulprostone were abolished by treatment with a specific EP 1 receptor antagonist, SC-51322, or the PLC inhibitor U-73122. The effects of these EP 1 receptor agonists were potentiated by ionomycin and blocked by verapamil. Hepatocyte mitogenesis was almost completely blocked by specific inhibitors of growth-related signal transducers, such as genistein, wortmannin, PD98059, and rapamycin. A monoclonal antibody against TGF-␣ dose-dependently inhibited PGE 2 -and 17-phenyltrinor-PGE 2 -induced hepatocyte mitogenesis. Treatment with the EP 1 receptor agonists significantly increased the secretion of TGF-␣, reaching a maximum within 5 min. The increase in TGF-␣ secretion was blocked by SC-51322, U-73122, somatostatin, and verapamil and potentiated by ionomycin. These results indicate that the proliferative mechanisms of action of EP 1 receptor agonists are mediated through an increase in the autocrine secretion of TGF-␣, which is dependent on the EP 1 receptor/G-protein involved in PLC regulation/PLC/Ca 2؉ system. The locally secreted TGF-␣, in turn, acts as a complete mitogen that stimulates the tyrosine kinase/MAPK pathway in these cells. (Endocrinology 142: 4428 -4440, 2001)