Erythropoietin depresses nitric oxide synthase expression by human endothelial cells

We have recently shown that erythropoietin (EPO)-induced hypertension is unrelated to the rise in hematocrit and is marked by elevated cytosolic [Ca+2] and nitric oxide (NO) resistance. The present study was done to determine the effect of EPO on NO production and endothelial NO synthase (eNOS) expression by endothelial cells. Human coronary artery endothelial cells were cultured to subconfluence and then were incubated for 24 hours in the presence of either EPO (0, 5, and 20 U/mL) alone or EPO plus the calcium channel blocker felodipine. The experiments were carried out with quiescent (0.5% FCS) and proliferating (5% FCS) cells. Total nitrate and nitrite, eNOS protein, DNA synthesis (thymidine incorporation), and cell proliferation (cell count) were determined. In addition, NO production in response to acetylcholine stimulation was tested. EPO resulted in a dose-dependent inhibition of basal and acetylcholine-stimulated NO production and eNOS protein expression and also led to a significant dose-dependent stimulation of DNA synthesis in endothelial cells. The inhibitory effects of EPO on NO production and eNOS expression were reversed by felodipine. Thus, EPO downregulates basal and acetylcholine-stimulated NO production, depresses eNOS expression, and stimulates proliferation in isolated human endothelial cells. The suppressive effects of EPO on NO production and on eNOS expression are reversed by calcium channel blockade.