Bradykinin stimulation of nitric oxide production is not sufficient for gamma-globin induction.

Abstract

Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases gamma-globin and fetal hemoglobin levels in human erythroid progenitors. In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase gamma-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 microM up to 30 minutes).This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/beta-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase gamma-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce gamma/beta globin ratio in erythroid progenitors in co-cultures with HUVEC. Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce gamma-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of gamma-globin gene expression.