Painful crises in sickle cell disease (SCD) are associated with increased plasma cytokines levels, including endothelin-1 (ET-1). Reduced red cell magnesium content, mediated in part by increased Na + /Mg 2+ exchanger (NME) activity, contributes to erythrocyte K + loss, dehydration and sickling in SCD. However, the relationship between ET-1 and the NME in SCD has remained unexamined. We observed increased NME activity in sickle red cells incubated in the presence of 500 nM ET-1. Deoxygenation of sickle red cells, in contrast, led to decreased red cell NME activity and cellular dehydration that was reversed by the NME inhibitor, imipramine. Increased NME activity in sickle red cells was significantly blocked by pre-incubation with 100 nM BQ788, a selective blocker of ET-1 type B receptors. These results suggest an important role for ET-1 and for cellular magnesium homeostasis in SCD. Consistent with these results, we observed increased NME activity in sickle red cells of three mouse models of sickle cell disease greater than that in red cells of C57BL/J6 mice. In vivo treatment of BERK sickle transgenic mice with ET-1 receptor antagonists reduced red cell NME activity.Among patients with sickle cell disease (SCD), 5% experience 3-10 potentially lethal vaso-occlusive painful crises annually. 1,2 Vaso-occlusive crises are initiated, in part, by red cell deoxygenation and by elevated plasma levels of cytokines such as endothelin-1 (ET-1), leading to increased adhesive interactions among sickle red cells, between sickle red cells and endothelium and involving plasma factors, through mechanisms that remain incompletely understood. [3][4][5][6] ET-1 plays important roles in the disordered erythrocyte homeostasis, inflammation, vaso-occlusion, tissue injury, and pain common to the pathophysiology of SCD. 7,8 ET-1 levels are increased in SCD, 9,10 and in vivo treatment with ET-1 receptor antagonists improve hematological parameters and reduced oxidant stress in mouse models of SCD. 11 However, the mechanisms by which ET-1 receptor antagonists mediate their beneficial effects in SCD remain unclear.Dysregulated Mg 2+ levels have been reported in SCD and in patients with Type 2 diabetes, 12,13 hypertension, and stroke, all diseases with an important inflammatory component. [14][15][16] Low Mg 2+ levels are accompanied by oxidant stress, impaired vascular function and increased inflammation, all associated with the pathophysiology of SCD. 17,18 Low cellular Mg 2+ levels stimulate cytokine production in and release from endothelial cells and modulate activity of NF-κB, a master regulator of cytokine production. 19,20 Intracellular Mg 2+ regulates multiple additional activities linked to SCD, including casein kinase II (CK2), 21 PKC [22][23][24] and hypoxia-inducible factor-1 (HIF-1). 25 Thus, regulation of Mg 2+ levels has been proposed as a therapeutic target in SCD. [26][27][28] A preliminary study reported that the activity of the Na + /Mg 2+ exchanger (NME) in sickle erythrocytes was significantly reduced after 6 months of...

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.