Erythropoietin stimulates a rise in intracellular free calcium concentration in single early human erythroid precursors.

Abstract

Erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the differentiation and proliferation of erythroid cells. To determine the cellular mechanism of action of these growth factors, we measured changes in intracellular free calcium concentration (jCaJ) in single human erythroid precursors in response to recombinant erythropoietin and GM-CSF. [CaJ in immature erythroblasts derived from cultured human cord blood erythroid progenitors was measured with fluorescence microscopy digital video imaging.When stimulated with erythropoietin, [CaJ in the majority of erythroblasts increased within 3 min, peaked at 5 min, and returned toward baseline at 10 min. The percentage of cells that responded to erythropoietin stimulation increased in a dose-dependent manner. Additional stimulation with GM-CSF in cells previously exposed to erythropoietin resulted in a second [Ca¢J increase. Immature erythroblasts treated with GM-CSF followed by erythropoietin responded similarly to each factor with a rise in [Ck]. The source of transient calcium is intracellular since erythroblasts were incubated in medium devoid of extracellular calcium. Our observations suggest that changes in [CaJ may be an intracellular signal that mediates the proliferative/differentiating effect of hematopoietic growth factors.

Post Transplant Erythrocytosis in Hypercalcemic Renal Transplant Recipients

“…Several in vitro reports have proposed that Ca 2+ flux and intracellular Ca 2+ concentration are important determinants of erythroid proliferation and differentiation. Erythropoietin has been shown to induce increases in intracellular Ca 2+ concentration in late human erythroblasts through an EPO-regulated Ca 2+ channel (17)(18)(19). The EPO-induced increase in intracellular free Ca 2+ is dependent on the extracellular Ca 2+ concentration and can be blocked by nifedipine (18).…”

Rap1 Is Activated by Erythropoietin or Interleukin-3 and Is Involved in Regulation of β1 Integrin-mediated Hematopoietic Cell Adhesion

“…It should be also mentioned that an intracellular calcium chelator, BAPTA-AM, showed no inhibitory effect on Epo-or IL-3-induced activation of Rap1 in 32D cells, 2 which is in contrast to previous reports on PLC-dependent Rap1 activation observed rapidly and transiently following thrombin stimulation in platelets or that observed in lymphocytes stimulated though the B cell antigen receptor (20,21). It is thus possible that, although Epo stimulation induces an increase in intracellular calcium (45), it may not play a critical role in Rap1 activation. Further studies are required to elucidate the downstream signaling pathways mediating the PLC-␥-dependent Rap1 activation in cytokinestimulated hematopoietic cells.…”

Erythropoietin Modulates Calcium Influx through TRPC2

“…At 5 h, an equal volume of Dulbecco's modified Eagle's medium with 20% FCS was added, and 18 h later this medium was replaced with Dulbecco's modified Eagle's medium with 10% FCS. Successful transfection of CHO cells with Epo-R and TRPC was verified by detection of GFP (excitation, 478 nm; emission, 535 nm) and BFP (excitation, 380 nm; emission, 435 nm), respectively, in the cells with digital video imaging (4,5,12,13,23). The optimal time for expression of pTracer CMV Epo-R and pQBI50 TRPC2 was 48 -72 h after transfection, and this time interval was selected to examine the response of transfected CHO cells to Epo.…”

“…Imaging-A fluorescence microscopy-coupled digital video imaging system was used to measure [Ca] i (4,5,12,13,23). To study changes in [Ca] i in transfected cells, we were not able to use Fura-2 as the detection fluorophore because its excitation and emission wavelengths overlap with those of GFP.…”

“…The mechanism of regulation of [Ca] i by erythropoietin has previously been examined at the single cell level using fluorescence microscopy coupled to digital video imaging (4,5,12,13,23). In this report, we used RT-PCR to determine the expression pattern of TRPC on murine erythroid cell lines HCD-57 and Ba/F3 Epo-R, in which Epo stimulates a rise in [Ca] i (23).…”