Regulation of Na⁺/Mg²⁺ Exchange in Sickle Erythrocytes By Endothelin-1

Excess levels of endothelin-1 (ET-1), erythrocyte sickling and chronic inflammation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have shown that ET-1 receptor antagonists improve hematological parameters by reducing Gardos channel activity in two transgenic mouse models of SCD while reducing oxidant stress by decreasing circulating levels of protein disulfide isomerase. Magnesium (Mg²⁺) deficiency, mediated in part via increased erythrocyte Na⁺/Mg²⁺ exchanger activity, has been demonstrated to contribute to erythrocyte dehydration, K⁺ loss and sickling in SCD. However, the relationship between ET-1 and the Na⁺/Mg²⁺ exchanger in SCD remains unclear. We measured Na⁺/Mg²⁺ exchange activity in ex vivo red cells and observed increased activity following in vitro incubation of human (2.2 ± 0.2 to 3.2 ± 0.1 mmol/10¹³ cell x h, P<0.03, n=5) and mouse red blood cells with ET-1 (P<0.001, n=5); events that were significantly blocked by pre-incubation of cells with 1 μM BQ788, a selective inhibitor of ET-1 type B receptors. In addition, in vitro deoxygenation of sickle red cells led to increased exchanger activity that was inhibited by impramine, a Na⁺/Mg²⁺ exchange inhibitor, and associated with reduced deoxygenation-stimulated sickle cell dehydration. These results suggest an important role for ET-1 and cellular magnesium homeostasis in sickle cell disease. To this end, we studied Na⁺/Mg²⁺ exchange activity in ex vivo erythrocytes from three transgenic sickle mouse models and observed increased activity in these cells when compared to red cells from either Hb A transgenic or C57BL/J6 wild-type mice (P<0.03, n=4). We then tested the in vivo effects of ET-1 receptor antagonists on erythrocyte Na⁺/Mg²⁺ exchange activity in the BERK mouse, a transgenic model of SCD. We blocked ET-1 receptors type A and B by in vivo treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed lower erythrocyte exchanger activity when compared to cells from vehicle treated BERK mice (P<0.02, n=6). Thus our results suggest that ET-1 receptor blockade represents an important therapeutic approach to control erythrocyte volume and magnesium homeostasis that may lead to improved inflammatory and vascular complications observed in SCD. Supported by NIH R01HL090632 to AR.