Is the Intracellular Calcium-Mediated Pathway Involved in Erythropoietin-Induced Hypertension?

Kiyoshi Morikawa, Department of Clinical and Laboratory Science, Fukui Medical School, Matsuoka, Yoshida-gun, Fukui 910-11 (Japan) phore, ionomycin (Wako Pure Pharmaceutical, Tokyo, Japan), with a chemotactic pep-tide, FMLP (Peptide Institute, Osaka, Japan), or with epoetin-α (EPO, Kirin Beer Co. Ltd., Tokyo, Japan). In a short coculture study, at a concentration of 10,000 mlU/ml of rhEPO, morphological changes in ECs and SMCs Dear Sir, Hypertension is one of the adverse effects associated with recombinant human erythropoietin (rhEPO) therapy for anemia in hemo-dialysis patients. The incidence of hypertension is reported to be 10-15% [1,2], sometimes requiring a reduction in dosage or discontinuation of rhEPO therapy [2]. The exact mechanism of rhEPO-induced hypertension has not yet been fully elucidated, although several theories have been advanced. Of these, an attractive possibility is a direct vasopressor effect of rhEPO as suggested by two recent reports [3, 4]. We examined whether rhEPO directly affects the major components of the vessel wall, the endothelial cells (ECs) and smooth muscle cells (SMCs), through the intracellular calciummediated pathway which plays an important role in vascular contraction induced by angiotensin II and vaso-pressin [5]. ECs were prepared from an endothelial cell line derived from the human umbilical cord (Endocell, Kurabo Industry Ltd., Osaka, Japan). SMCs were from a smooth muscle cell line of the rat thoracic aorta, A10 (Dainip-pon Pharmaceutical Co. Ltd., Osaka, Japan). Both cell types were cultured with Dulbecco’s minimum essential medium (Flow Laboratories Inc., Va., USA) for 7 days. The confluent cells were trypsinized to disperse, and then loaded with 2 μM fura 2-AM (Dojin Chemical, Kumamoto, Japan) for 15 min at 37 °C and resuspended at 1 × lOVml in a HEPES buffer. Intracellular free calcium ([Ca2+]i) levels were determined according to the method of Grynkiewicz et al. [6], using a two-wave spectrofluorophotometer, RF-5000 (Shimazu Co. Ltd., Kyoto, Japan). Changes in the [Ca2+]i levels of these cells were examined by stimulation with a calcium iono[Qê*]\ nM 300 – ionomycin (50nM) 250 – 200 v^1⁄8i 10 min