Erythropoietin receptor-operated Ca²⁺ channels: Activation by phospholipase C-γ1

Erythropoietin (EPO) increases Ca²⁺ influx in vascular smooth muscle cells and acts both as a direct vasoconstrictor and vascular growth factor (that is, angiogenesis). However, the mechanism by which EPO promotes extracellular Ca²⁺ entry in contractile cells has not been elucidated. In hematopoietic cells, EPO induces tyrosine kinase (TK)-dependent activation of phospholipase C (PLC)-γ1 and Ca²⁺ influx via a voltage-independent Ca²⁺ conductance. In contractile mesangial cells, we have recently characterized a voltage-independent, 1 pS Ca²⁺ channel that is dependent on both TK and PLC-γ1 activity. Therefore, we examined cultured rat glomerular mesangial cells after timed exposure to recombinant human EPO (20 U/ml). Erythropoietin increased the tyrosine phosphorylation of PLC-γ1, promoted membrane complex formation between PLC-γ1 and the EPO receptor itself, and raised the levels of intracellular inositol 1,4,5-trisphosphate and intracellular Ca²⁺. Consistent with our previous studies, 1 pS Ca²⁺ channel activity was extremely low under basal, unstimulated conditions in cell-attached patches, but was dramatically increased when EPO was present in the patch pipette. Tyrosine kinase inhibition with 100 μM genistein or 1 μM PP1 (Src; selective tyrosine kinase inhibitor) prevented all of these EPO-induced responses. We conclude that: (1) EPO-induced stimulation of l pS Ca²⁺ channels is mediated via a cytosolic Src TK in glomerular mesangial cells. (2) Stimulation of this Ca²⁺-activated, Ca²⁺-permeable channel is dependent on the tyrosine phosphorylation/activation of PLC-γ1. (3) This cascade provides a possible mechanism for the vasoconstriction and hypertension observed with clinical EPO use for the treatment of chronic anemias.