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Erythropoietin stimulates a rise in intracellular-free calcium concentration in single BFU-E derived erythroblasts at specific stages of differentiation.

Human cord blood progenitor-derived erythroblasts have recently been shown to respond to erythropoietin (Epo) or granulocyte-macrophage colony-stimulating factor (GM-CSF) with a transient increase in intracellular free calcium concentration [Cac]. However, the importance of [Cac] changes in mediating cell proliferation and/or differentiation is undefined. In the present study, the response of erythroid precursors at different stages of differentiation to Epo was examined. Erythroblasts were derived from adult blood erythroid progenitors (BFU-E) at day 7 or day 10 of culture. [Cac] was measured in individual Fura-2 loaded cells with fluorescence microscopy coupled digital video imaging. The dynamic range (Rmax/Rmin) of intracellular Fura-2 was similar to that measured in free solution, suggesting insignificant amounts of intracellular Ca insensitive forms of Fura-2. Baseline [Cac] of erythroid cells calculated with an in vitro calibration method was 44 +/- 4 nmol/L and with an in vivo method was 46 +/- 4 nmol/L. Treatment of day 7 BFU-E derived erythroblasts with Epo resulted in no significant increase in [Cac]. In contrast, in more mature erythroblasts (day 10 of culture), Epo stimulated a large increase in [Cac] from 49 +/- 11 nmol/L at baseline to 279 +/- 47 nmol/L. This [Cac] increase occurred in phosphate buffered saline (PBS) containing no added calcium. The increase in [Cac] persisted for 18 minutes and was dose dependent. Day 7 and day 10 control cells treated with either insulin or media showed no significant change in [Cac] during 18 minutes of observation. Our data demonstrate that early (day 7) and late (day 10) erythroblasts display different responses to Epo, at least in terms of intracellular Ca++ fluxes. The differential [Cac] response observed in early and late erythroid precursors to growth factor stimulation suggests that [Cac] may be an important signal in cell differentiation.