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 a nd that this increase is modulated by erythropoietin through an ion ch annel permeable to Ca2+. Here, the role of protein phosphorylation in the increase in intracellular free calcium [Ca-i] stimulated by erythr opoietin was studied with digital video imaging. Preincubation of day 10 erythroblasts with a broad inhibitor of serine/threonine and tyrosi ne kinases, staurosporine (100 nM), blocked the increase in [Ca-i] ove r 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- o r cGMP-dependent kinases (KT 5720, HA 1004), and [Ca-i] did not increa se following stimulation with phorbol 12-myrisate 13-acetate (PMA) or dibutyryl cAMP. These results suggest that neither protein kinase C no r protein kinase A mediate the eryuthropoietin-induced [Ca-i] increase . In contrast, preincubation with genistein, a tyrosine kinase inhibit or, blocked the erythropoietin induced increase in [Ca-i]. To further study calcium entry in erythroblasts, we determined mastoparan, a pept ide from wasp venom, induced a dose-dependent rise in [Ca-i] in erythr oblasts which required external calcium. Stimulation of erythroid prec ursors with 10 mu M mastoparan resulted in an increase in [Ca-i] from 52 +/- 3 nM to 214 +/- 36 nM which peaked at 20 min. The mastoparan-in duced [Ca-i] increase was also dependent on tyrosine phosphorylation s ince it was blocked by preincubation with genistein. These results dem onstrate that both erythropoietin and mastoparan stimulate calcium ent ry by a mechanism which has a genistein sensitive step and suggest tha t tyrosine kinase activation is required for the rise in [Ca-i] to occ ur.