ERYTHROPOIETIN RECEPTOR-OPERATED CA2- ACTIVATION BY PHOSPHOLIPASE C-GAMMA-1( CHANNELS )

Erythropoietin (EPO) increases Ca2+ influx in vascular smooth muscle c ells and acts both as a direct vasoconstrictor and vascular growth fac tor (that is, angiogenesis). However, the mechanism by which EPO promo tes extracellular Ca2+ entry in contractile cells has not been elucida ted. In hematopoietic cells, EPO induces tyrosine kinase (TK)-dependen t activation of phospholipase C (PLC)-gamma 1 and Ca2+ influx via a vo ltage-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-gamma 1 activity. Therefore, we e xamined cultured rat glomerular mesangial cells after timed exposure t o recombinant human EPO (20 U/ml). Erythropoietin increased the tyrosi ne phosphorylation of PLC-gamma 1, promoted membrane complex formation between PLC-gamma 1 and the EPO receptor itself, and raised the level s 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 p atches, out was dramatically increased when EPO was present in the pat ch pipetre. Tyrosine kinase inhibition with 100 mu M genistein or 1 mu M PP1 (Src; selective tyrosine kinase inhibitor) prevented all of the se EPO-induced responses. We conclude that: (1) EPO-induced stimulatio n of 1 pS Ca2+ channels is mediated via a cytosolic Src TK in glomerul ar mesangial cells. (2) Stimulation of this Ca2+-activated, Ca2+-perme able channel is dependent on the tyrosine phosphorylation/activation o f PLC-gamma 1. (3) This cascade provides a possible mechanism for the vasoconstriction and hypertension observed with clinical EPO use for t he treatment of chronic anemias.