Endothelin-1 release by erythropoietin involves calcium signaling in endothelial cells

We investigated the effects of recombinant human erythropoietin (rHuEPO) on intracellular calcium ([Ca²⁺ ]_j) and whether these changes regulate both endothelin-1 (ET-1) protein release and ET-1 messenger RNA (mRNA) production in bovine pulmonary arterial endothelial cells (BPAEC). rHuEPO (3.3 U/ml) induced [Ca²⁺ ]_j increases from a basal level of 54 ± 12.2 (SE) to 147 ± 21.1 nM (p < 0.001), in fura-2-loaded BPAEC. In the presence of nifedipine (10 μM), the increases in [Ca²⁺ ]_i) were significantly reduced. Furthermore, when extracellular calcium ([Ca²⁺ ]_Q) was reduced (200 μM), there was a significant reduction in [Ca²⁺ ]_i increase after stimulation with rHuEPO. Incubation of BPAEC with rHuEPO for 4 h increased ET-1 levels in the culture supernatant from 44.7 ± 5.3 to 85 ± 7.6 pg/ml (p < 0.001). However, when the cells were treated with rHuEPO and nifedipine, the ET-1 levels were decreased, as compared to levels resulting from treatment with rHuEPO alone (41 ± 6.1 vs. 85 ± 7.6 pg/ml, p < 0.001, respectively). rHuEPO also induced a fourfold increase in the level of the preproET-1 mRNA as compared with control. Pre-proET-1 mRNA was diminished in the presence of nifedipine and rHuEPO and rHuEPO can increase [Ca²⁺]_i in BPAEC, and this increase may be related to the stimulation of ET-1 synthesis and release.