Magnesium homeostasis in deoxygenated sickle erythrocytes is modulated by endothelin-1 via Na+ /Mg2+ exchange

Abstract

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⁺ /Mg²⁺ 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. Our results suggest that ET-1 receptor blockade may be a promising therapeutic approach to control erythrocyte volume and magnesium homeostasis in SCD and may thus attenuate or retard the associated chronic inflammatory and vascular complications of SCD.

SCHEME 1

In vivo protocol for ETRA testing in sickle cell mouse model, BERK.

FIGURE 1

Mg²⁺ efflux in Na⁺‐containing and Choline‐containing media. Normal and sickle erythrocytes were Mg‐loaded as in Methods, and Mg²⁺ efflux was measured in the absence (open bars) or presence of extracellular Na⁺ (gray bars). Post‐loading [Mg]_i was 11.86 ± 0.49 mmol/Kg Hb. Open circles represent individual triplicate experiments. Bars represent means ± SEM of >6 triplicate experiments. *p < .01; **p < .002; ***p < .0001 vs. corresponding choline data. Wilcoxon rank test for paired matched data, with Holm‐Sidak correction for multiple comparisons.

FIGURE 2

ET‐1 stimulated Na⁺/Mg²⁺ exchange (NME) activity in normal and sickle erythrocytes. Mg²⁺ efflux from AA (open bars) and SS red cells (gray bars) was measured as described above, and Na⁺/Mg²⁺ exchange activity was estimated from the difference between Mg flux in the presence and absence of extracellular Na⁺. Fluxes were normalized to MCV in each experiment. Data are means ± SEM of triplicate determinations. *p = .007; ^# p = .04, ^## p = .031, each vs. corresponding control data (CTL). Mann–Whitney test.

FIGURE 3

Cyclic deoxygenation decreases ET‐1‐stimulated Mg²⁺ transport sensitive to BQ788 and to imipramine in ex vivo human sickle erythrocytes. Sickle red cell suspensions were exposed to cyclic deoxygenation for 3 h. Mg²⁺ efflux was then measured in Mg²⁺‐unloaded red cells. ET‐1, 500 nM; BQ788, 1 μM; Imipramine (IMP), 100 μM. (A) Mg²⁺ efflux in the absence or presence of extracellular Na⁺. *p = .015, **p = .0001 vs. CTL in each condition; ^# p = .0003 compared with CTL in Na media; Wilcoxon test for matched data. (B) Na⁺‐dependent Mg²⁺ efflux. Data are means ± SEM from ≥7 independent triplicate experiments. *p = .021 for ET‐1 vs. ET‐1 + BQ788; **p = .0025 for ET‐1 vs. ET‐1 + IMP; ***p = .0005 for ET‐1 vs. CTL. Mann–Whitney test for unpaired data. CTL vs. ET‐1 + BQ788 and CTL vs. IMP comparisons were not statistically significant.

FIGURE 4

Red cell density distribution in normal (A) and in sickle erythrocytes (B). Density distribution profiles were measured at baseline (black, in‐room air) and subsequently after 3 h cyclic deoxygenation in the absence (red squares), CTL) or presence of 500 nM ET‐1 (blue triangles), 100 μM Imipramine (green inverted triangles, IMP) or 1 μM BQ788 (purple diamonds) (see Methods). Data are means ± SEM of 5 triplicate experiments. D₅₀ values (see Table 1) were calculated from curves fit to sigmoid equations (GraphPad PRISM 9.0).

FIGURE 5

Na⁺/Mg²⁺ exchange activity in red cells from several mouse models of hemoglobinopathies. (A) Na⁺‐dependent Mg²⁺ efflux was measured in red cells of mouse hemoglobinopathy models as described in Methods. These included Wild‐type mice (WT: C57Bl/6j), SAD, BERK and NY1DD mouse models of sickle cell disease, and human HbC‐expressing mice. Values represent means ± SEM for ≥3 quadruplicate experiments. *p = .02; **,p = .015 vs. WT. (B) Na⁺‐independent Mg²⁺ efflux was measured in Choline Cl media. Values represent means ± SEM of ≥3 quadruplicate experiments. ^# p = .008 BERK vs. SAD. (C) Post‐loading Mg²⁺ content in red cells from the indicated mouse hemoglobinopathy models. Values represent means ± SEM of 3 quadruplicate experiments. *p = .039 vs. WT. Mann–Whitney test.

FIGURE 6

The selective ETB receptor antagonist, BQ788, inhibits ET‐1‐stimulated NME activity in sickle mouse erythrocytes. (A) In vitro Na⁺‐dependent Mg²⁺ efflux was measured in Mg‐loaded red cells from BERK mice, in the presence or absence of 500 nM ET‐1 and 1 μM BQ788. Bars represent means ± S.E.M of ≥3 triplicate experiments. *p < .028 for Control vs. ET‐1); ^# p < .03 for ET‐1 vs. ET_1 + BQ788. Mann–Whitney test. ET‐1 + BQ788 did not differ from Control. (B) BERK mice of ~4 months of age were injected i.p. for 14 days with an ETR antagonists cocktail containing a 1:1 mix of an ETRB antagonist BQ788 and ETRA antagonist BQ123, as described in Methods. Mg²⁺ efflux was measured in the extracellular presence of choline (white bars) or Na⁺ (gray bars). *p = .035; **p = .0003 vs. choline Cl media, by Wilcoxon rank test.

FIGURE 7

Cytokine regulation of NME in human AA and SS erythrocytes. Mg²⁺‐loaded AA (white bars) and SS red cells (gray bars) were subjected to measurement of NME at 37°C in the absence or presence of 100 nM platelet‐activating factor (PAF), 100 nM prostaglandin E2 (PGE2) or 40 ng/ml RANTES. (A) Na⁺‐dependent Mg²⁺ efflux. ^# p < .04 (CTL vs. each cytokine) in AA cells; *p < .04 (control vs. each cytokine in SS cells). (B) Na⁺‐independent Mg²⁺ efflux. *p = .0357 (SS + PGE2 vs. AA + PGE2). Means ± SEM of >3 triplicate determinations. Mann–Whitney test. Differences between CTL and each cytokine did not achieve significance in AA or SS red cells.