Endothelial cell α-globin and its molecular chaperone α-hemoglobin–stabilizing protein regulate arteriolar contractility
Christophe Lechauve, Joshua T. Butcher, Abdullah Freiwan, Lauren A. Biwer, Julia M. Keith, Miranda E. Good, Hans Ackerman, Heather S. Tillman, Laurent Kiger, Brant E. Isakson, Mitchell J. Weiss
Christophe Lechauve, Joshua T. Butcher, Abdullah Freiwan, Lauren A. Biwer, Julia M. Keith, Miranda E. Good, Hans Ackerman, Heather S. Tillman, Laurent Kiger, Brant E. Isakson, Mitchell J. Weiss
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Research Article Hematology Vascular biology

Endothelial cell α-globin and its molecular chaperone α-hemoglobin–stabilizing protein regulate arteriolar contractility

Abstract

Arteriolar endothelial cell–expressed (EC-expressed) α-globin binds endothelial NOS (eNOS) and degrades its enzymatic product, NO, via dioxygenation, thereby lessening the vasodilatory effects of NO on nearby vascular smooth muscle. Although this reaction potentially affects vascular physiology, the mechanisms that regulate α-globin expression and dioxygenase activity in ECs are unknown. Without β-globin, α-globin is unstable and cytotoxic, particularly in its oxidized form, which is generated by dioxygenation and recycled via endogenous reductases. We show that the molecular chaperone α-hemoglobin–stabilizing protein (AHSP) promotes arteriolar α-globin expression in vivo and facilitates its reduction by eNOS. In Ahsp−/− mice, EC α-globin was decreased by 70%. Ahsp−/− and Hba1−/− mice exhibited similar evidence of increased vascular NO signaling, including arteriolar dilation, blunted α1-adrenergic vasoconstriction, and reduced blood pressure. Purified α-globin bound eNOS or AHSP, but not both together. In ECs in culture, eNOS or AHSP enhanced α-globin expression posttranscriptionally. However, only AHSP prevented oxidized α-globin precipitation in solution. Finally, eNOS reduced AHSP-bound α-globin approximately 6-fold faster than did the major erythrocyte hemoglobin reductases (cytochrome B5 reductase plus cytochrome B5). Our data support a model whereby redox-sensitive shuttling of EC α-globin between AHSP and eNOS regulates EC NO degradation and vascular tone.

Authors

Christophe Lechauve, Joshua T. Butcher, Abdullah Freiwan, Lauren A. Biwer, Julia M. Keith, Miranda E. Good, Hans Ackerman, Heather S. Tillman, Laurent Kiger, Brant E. Isakson, Mitchell J. Weiss

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Figure 7

AHSP stabilizes oxidized (Fe3+) α-globin and facilitates its reduction by eNOS.

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AHSP stabilizes oxidized (Fe3+) α-globin and facilitates its reduction b...
(A) Purified oxygenated (Oxy) (Fe2+) α-globin (15 μM) was incubated with AHSP and/or full-length recombinant eNOS for 60 minutes at 37°C. Control refers to (Oxy) (Fe2+) α-globin alone. Potassium ferricyanide was added at t0 (to a final concentration of 50 μM) to induce heme oxidation. Protein precipitation was monitored by light scattering at 700 nm. D, density. (B) Reduction of the(Fe3+) α-globin–AHSP complex by the indicated proteins (either 0.15 μM CyB5R3, 0.15 μM CyB5R3 plus 1 μM CyB5, or 1 μM full-length eNOS) with 50 to 100 μM NADH. Reactions were performed in PBS with 250 U/ml catalase at 25°C at a physiologically low O2 concentration (10 Torr) with CO (100 μM) to prevent the eventual partial reoxidation of reduced α-globin. All reactions were performed 3 times. ΔA, change in light absorbance.