Erythropoietin modulation of intracellular calcium: a role for tyrosine phosphorylation

We have reported that erythropoietin induces a dose-dependent increase in cytosolic calcium ([Ca i]) in single human peripheral blood BFU-E derived erythroblasts which is specific for stage of differentiation and that this increase is modulated by erythropoietin through an ion channel permeable to Ca 2+. Here, the role of protein phosphorylation in the increase in intracellular free calcium [Ca i] stimulated by erythropoietin was studied with digital video imaging. Preincubation of day 10 erythroblasts with a broad inhibitor of serine/threonine and tyrosine kinases, staurosporine (100 nM), blocked the increase in [Ca i] over 20 min following erythropoietin stimulation. However, erythropoietin-induced calcium influx was unaffected by preincubation of cells with specific inhibitions of protein kinase C (calphostin C) or the cAMP- or cGMP-dependent kinases (KT 5720, HA 1004), and [Ca i] did not increase following stimulation with phorbol 12-myristate 13-acetate (PMA) or dibutyryl cAMP. These results suggest that neither protein kinase C nor protein kinase A mediate the erythropoietin-induced [Ca i] increase. In contrast, preincubation with genistein, a tyrosine kinase inhibitor, blocked the erythropoietin induced increase in [Ca i]. To further study calcium entry in erythroblasts, we determined mastoparan, a peptide from wasp venom, induced a dose-dependent rise in [Ca i] in erythroblasts which required external calcium. Stimulation of erythroid precursors with 10 μM mastoparan resulted in an increase in [Ca i] from 52 ± 3 nM to 214 ± 36 nM which peaked at 20 min. The mastoparan-induced [Ca i] increase was also dependent on tyrosine phosphorylation since it was blocked by preincubation with genistein. These results demonstrate that both erythropoietin and mastoparan stimulate calcium entry by a mechanism which has a genistein sensitive step and suggest that tyrosine kinase activation is required for the rise in [Ca i] to occur.