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

Barbara Miller, J. Y. Cheung, D. L. Tillotson, S. M. Hope, Russell Scaduto

Research output: Contribution to journalArticle

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Abstract

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 [Ca(c)]. However, the importance of [Ca(c)] 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. [Ca(c)] was measured in individual Fura-2 loaded cells with fluorescence microscopy coupled digital video imaging. The dynamic range (R(max)/R(min)) of intracellular Fura-2 was similar to that measured in free solution, suggesting insignificant amounts of intracellular Ca++ insensitive forms of Fura-2. Baseline [Ca(c)] of erythroid cells calculated with an in vitro calibration method was 44 ± 4 nmol/L and with an vivo method was 46 ± 4 nmol/L. Treatment of day 7 BFU-E derived erythroblasts with Epo resulted in no significant increase in [Ca(c)]. In contrast, in more mature erythroblasts (day 10 of culture), Epo stimulated a large increase in [Ca(c)] from 49 ± 11 nmol/L at baseline to 279 ± 47 nmol/L. This [Ca(c)] increase occurred in phosphate buffered saline (PBS) containing no added calcium. The increase in [Ca(c)] 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 [Ca(c)] 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 [Ca(c)] response observed in early and late erythroid precursors to growth factor stimulation suggests that [Ca(c)] may be an important signal in cell differentiation.

Original languageEnglish (US)
Pages (from-to)1188-1194
Number of pages7
JournalBlood
Volume73
Issue number5
StatePublished - Jan 1 1989

Fingerprint

Erythroblasts
Erythroid Precursor Cells
Erythropoietin
Calcium
Fura-2
Cell Differentiation
Blood
Erythroid Cells
Fluorescence microscopy
Cell proliferation
Granulocyte-Macrophage Colony-Stimulating Factor
Fetal Blood
Fluorescence Microscopy
Calibration
Intercellular Signaling Peptides and Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

Cite this

Miller, Barbara ; Cheung, J. Y. ; Tillotson, D. L. ; Hope, S. M. ; Scaduto, Russell. / Erythropoietin stimulates a rise in intracellular-free calcium concentration in single BFU-E derived erythroblasts at specific stages of differentiation. In: Blood. 1989 ; Vol. 73, No. 5. pp. 1188-1194.
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abstract = "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 [Ca(c)]. However, the importance of [Ca(c)] 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. [Ca(c)] was measured in individual Fura-2 loaded cells with fluorescence microscopy coupled digital video imaging. The dynamic range (R(max)/R(min)) of intracellular Fura-2 was similar to that measured in free solution, suggesting insignificant amounts of intracellular Ca++ insensitive forms of Fura-2. Baseline [Ca(c)] of erythroid cells calculated with an in vitro calibration method was 44 ± 4 nmol/L and with an vivo method was 46 ± 4 nmol/L. Treatment of day 7 BFU-E derived erythroblasts with Epo resulted in no significant increase in [Ca(c)]. In contrast, in more mature erythroblasts (day 10 of culture), Epo stimulated a large increase in [Ca(c)] from 49 ± 11 nmol/L at baseline to 279 ± 47 nmol/L. This [Ca(c)] increase occurred in phosphate buffered saline (PBS) containing no added calcium. The increase in [Ca(c)] 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 [Ca(c)] 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 [Ca(c)] response observed in early and late erythroid precursors to growth factor stimulation suggests that [Ca(c)] may be an important signal in cell differentiation.",
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Erythropoietin stimulates a rise in intracellular-free calcium concentration in single BFU-E derived erythroblasts at specific stages of differentiation. / Miller, Barbara; Cheung, J. Y.; Tillotson, D. L.; Hope, S. M.; Scaduto, Russell.

In: Blood, Vol. 73, No. 5, 01.01.1989, p. 1188-1194.

Research output: Contribution to journalArticle

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T1 - Erythropoietin stimulates a rise in intracellular-free calcium concentration in single BFU-E derived erythroblasts at specific stages of differentiation

AU - Miller, Barbara

AU - Cheung, J. Y.

AU - Tillotson, D. L.

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N2 - 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 [Ca(c)]. However, the importance of [Ca(c)] 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. [Ca(c)] was measured in individual Fura-2 loaded cells with fluorescence microscopy coupled digital video imaging. The dynamic range (R(max)/R(min)) of intracellular Fura-2 was similar to that measured in free solution, suggesting insignificant amounts of intracellular Ca++ insensitive forms of Fura-2. Baseline [Ca(c)] of erythroid cells calculated with an in vitro calibration method was 44 ± 4 nmol/L and with an vivo method was 46 ± 4 nmol/L. Treatment of day 7 BFU-E derived erythroblasts with Epo resulted in no significant increase in [Ca(c)]. In contrast, in more mature erythroblasts (day 10 of culture), Epo stimulated a large increase in [Ca(c)] from 49 ± 11 nmol/L at baseline to 279 ± 47 nmol/L. This [Ca(c)] increase occurred in phosphate buffered saline (PBS) containing no added calcium. The increase in [Ca(c)] 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 [Ca(c)] 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 [Ca(c)] response observed in early and late erythroid precursors to growth factor stimulation suggests that [Ca(c)] may be an important signal in cell differentiation.

AB - 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 [Ca(c)]. However, the importance of [Ca(c)] 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. [Ca(c)] was measured in individual Fura-2 loaded cells with fluorescence microscopy coupled digital video imaging. The dynamic range (R(max)/R(min)) of intracellular Fura-2 was similar to that measured in free solution, suggesting insignificant amounts of intracellular Ca++ insensitive forms of Fura-2. Baseline [Ca(c)] of erythroid cells calculated with an in vitro calibration method was 44 ± 4 nmol/L and with an vivo method was 46 ± 4 nmol/L. Treatment of day 7 BFU-E derived erythroblasts with Epo resulted in no significant increase in [Ca(c)]. In contrast, in more mature erythroblasts (day 10 of culture), Epo stimulated a large increase in [Ca(c)] from 49 ± 11 nmol/L at baseline to 279 ± 47 nmol/L. This [Ca(c)] increase occurred in phosphate buffered saline (PBS) containing no added calcium. The increase in [Ca(c)] 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 [Ca(c)] 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 [Ca(c)] response observed in early and late erythroid precursors to growth factor stimulation suggests that [Ca(c)] may be an important signal in cell differentiation.

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