PMID: 2496770May 1, 1989Paper

The effect of recombinant erythropoietin on intracellular free calcium in erythropoietin-responsive cells.

Blood
S ImagawaH F Bunn

Abstract

We have investigated whether recombinant erythropoietin (r-Epo) elicits a change in intracellular free calcium (IFC) in purified Epo-responsive cells in spleens of mice treated with phenylhydrazine. Colony-forming units (CFU-E) were prepared by negative selection through immunologic panning. Anti-Forssman, Mac-1, Ia, and HSA antibodies were used to eliminate nonhematopoietic progenitors. After two pannings, 29 +/- 1.5% (mean +/- 1 SD) of the recovered cells were CFU-E. IFC was measured by labeling cells with the fluorescent dye Indo-1 and analyzing them on a flow cytometer from 15 seconds to 30 minutes after the addition of agonist. At each step of the panning procedure, there was no effect of r-Epo (0 to 10 U/mL) on IFC even in the larger cells that are predominantly CFU-E. As a positive control, calcium ionophore (A23187) significantly increased IFC in greater than 90% of the spleen cells enriched in CFU-E. During growth of CFU-E in methylcellulose, the calcium ionophore did not affect the r-Epo-dependent formation of erythroid colonies. EGTA inhibited the formation of erythroid colonies. This inhibition appeared to be the result of a toxic effect of the chelator because the colony growth could not be restored when Ca2+ was a...Continue Reading

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