The effect of erythropoietin on interleukin-1 beta mediated increase in nitric oxide synthesis in vascular smooth muscle cells

Objective Recently, we observed that recombinant human erythropoietin (rHuE PO) inhibits the interleukin (IL)-1 beta induced nitric oxide (NO) producti on and inducible NO synthase (iNOS) expression in cultured rat vascular smo oth muscle cells (VSMC), The mechanisms of these inhibitory effects of rHuE PO were evaluated, Methods Reverse transcription-polymerase chain reaction (RT-PCR) was perfor med to identify a specific erythropoietin receptor (EpoR), Tyrosine phospho rylation of phospholipase C (PLC) was analyzed by combination of immunoprec ipitation and Western blotting. Protein kinase C (PKC) activities were anal yzed 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 w as measured. iNOS mRNA and protein expression was analyzed by Northern and Western blotting, respectively. Results RT-PCR analysis revealed that EpoR m-RNA was expressed; furthermore , it might be alternatively spliced in VSMC, rHuEPO induced tyrosine phosph orylation of PLC-gamma 1 and activation of PKC, rHuEPO inhibited not only I L-1 beta induced nitrite production, but also the expression of iNOS mRNA a nd protein. These inhibitory effects of rHuEPO were reversed in the presenc e of PKC inhibitors, calphostin C (1 mu mol/l) or staurosporine (10 nmol/l) , PKC activation by phorbol myristate acetate inhibited nitrite production. The inhibitory effect of rHuEPO on IL-1 beta induced nitrite production wa s also eliminated in PKC depleted cells or in the existence of anti-EpoR an tibody. Conclusion rHuEPO inhibits IL-1 beta induced NO production by suppressing i NOS mRNA and protein expressions through EpoR, and the PLC-gamma 1 and PKC pathway may be involved. I Hypertens 1999, 17:1249-1256 (C) Lippincott Will iams & Wilkins.