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Recently, we observed that recombinant human erythropoietin (rHuEPO) inhibits the interleukin (IL)-1β induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMC). The mechanisms of these inhibitory effects of rHuEPO were evaluated.Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify a specific erythropoietin receptor (EpoR). Tyrosine phosphorylation of phospholipase C (PLC) was analyzed by combination of immunoprecipitation and Western blotting. Protein kinase C (PKC) activities were analyzed by phosphorylation assay of myelin basic protein (MBP4-14). VSMC were incubated with test agents for 24 h and nitrite as a stable NO metabolite was measured. iNOS mRNA and protein expression was analyzed by Northern and Western blotting, respectively.RT-PCR analysis revealed that EpoR m-RNA was expressed; furthermore, it might be alternatively spliced in VSMC. rHuEPO induced tyrosine phosphorylation of PLC-γ1 and activation of PKC. rHuEPO inhibited not only IL-1β induced nitrite production, but also the expression of iNOS mRNA and protein. These inhibitory effects of rHuEPO were reversed in the presence of PKC inhibitors, calphostin C (1 μmol/l) orstaurosporine (10 nmol=l). PKC activation by phorbol myristate acetate inhibited nitrite production. The inhibitory effect of rHuEPO on IL-1β induced nitrite production was also eliminated in PKC depleted cells or in the existence of anti-EpoR antibody.rHuEPO inhibits IL-β induced NO production by suppressing iNOS mRNA and protein expressions through EpoR, and the PLC-γ1 and PKC pathway may be involved.