To study the existence of the erythropoietin receptor (Epo-R) mRNA in brain capillary endothelial cells, the reverse transcription (RT) PCR was performed using total RNAs from rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines. Southern analysis of the RT-PCR products indicated that both RBECs and MBEC4 expressed an authentic form of Epo-R mRNA as a minor form and an intron-5-inserted form of Epo-R mRNA, thus a soluble form of Epo-R mRNA, as a major form. Furthermore, the effect of recombinant human erythropoietin (rHuEpo) on the DNA synthesis in RBECs was analyzed. rHuEpo showed a dose-dependent mitogenic action on RBECs as a competence factor. Radioiodinated rHuEpo was bound specifically to RBECs with time, cell number and dose dependencies. Binding studies with "'I-rHuEpo showed that RBECs had a single class of receptors with low-affinity (K,, = 860 pM) and that the number of siteskell (10300) was abundant. These results suggest that brain capillary endothelial cells express not only an authentic form of Epo-R but also a soluble form of Epo-R and that erythropoietin acts directly on brain capillary endotheha1 cells as a competence factor.Keywords: erythropoietin ; recombinant human erythropoietin ; erythropoietin receptor; brain capillary endothelial cells.Hypoxemia resulting from lung and heart diseases, anemia and high-altitude residence induces a series of modifications in the mammals. As an adaptation mechanism to hypoxia, mammals increase the number of the capillaries/tissue mass [I] and the erythrocytes in the blood (21 to maintain an adequate 0, delivery.Erythropoietin (Epo) is a serum glycoprotein hormone required for survival, proliferation, and differentiation of committed erythroid progenitor cells and its production is accelerated in the kidney by hypoxia [3-51. Recently, i t has been reported that Epo may act on such non-erythroid cells as endothelial cells [6-91, smooth Chemistry, Osaka Prefecture University, Sakai, Osaka, Japan 593 [9]. Although these results suggest that Epo may function as an angiogenic factor for adapting to hypoxia, after embryogenesis, angiogenesis proceeds by the growth of new capillary vessels from an established microvasculature following stimulation by various physiological or pathological processes [18, 191. Furthermore, the mean microvessel density increases in brain [20] and no new capillaries develop in muscle [21] under hypoxic conditions such as exposure to high altitudes, implying that capillary endothelial cells from various tissues each has specific characteristics.HUVECs express Epo-R mRNA [7]. In the present study, we use rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines [22], to analyze the existence of Epo-R mRNA in brain capillary endothelial cells, which have been thought to be associated with angiogenesis under hypoxic conditions, and provide evidence that both RBECs and MBEC4 express ...

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