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Erythropoietin stimulates a rise in intracellular-free calcium
concentration in single BFU-E derived erythroblasts at specific stages of
differentiation
BA Miller, JY Cheung, DL Tillotson, SM Hope and RC Scaduto
Department of Pediatrics, Milton S. Hershey Medical Center, PA 17033.
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.
Volume 73,
Issue 5,
pp. 1188-1194,
04/01/1989
Copyright © 1989 by The American Society of Hematology
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