A Valve for Nitric Oxide

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Science Signaling  20 Nov 2012:
Vol. 5, Issue 251, pp. ec297
DOI: 10.1126/scisignal.2003790

Nitric oxide (NO) signaling mediates vasodilation. In blood vessels, the internal elastic lamina separates endothelial cells (ECs) from smooth muscle cells (SMCs), but cells project through gaps in the lamina to form myoendothelial junctions (MEJs), which allow the gaseous NO produced by endothelial NO synthase (eNOS) in ECs to diffuse into SMCs and stimulate guanylyl cyclase (see commentary by Gladwin and Kim-Shapiro). Using vascular cell cocultures (VCCCs) grown in transwells and methods for separating ECs, SMCs, and the MEJs of cocultured ECs and SMCs, Straub et al. identified hemoglobin α (Hb α) as an abundant protein in lysates of MEJs but not lysates from ECs or SMCs (either isolated from VCCCs or cultured separately). Hb α localized to MEJs in mouse thoracodorsal arteries (TDAs). There was a decrease in the vasoconstriction elicited by phenylephrine, which is an α-adrenoreceptor agonist that causes smooth muscle contraction, or an increase in vasodilation elicited by acetylcholine, which stimulates NOS in isolated canulated TDAs in which Hb α was knocked down in the endothelium by silencing RNAs, compared to the responses of control TDAs. Knockdown of Hb α also increased the amount of NO detected outside isolated TDA vessel walls perfused with NO-containing medium or on the SMC side of the VCCCs after adding NO-containing medium to the EC side. The iron (Fe) in the heme group of Hb α exists in two oxidation states: the reduced state Fe2+, which reacts quickly with NO, converting it to nitrate, or the oxidized state Fe3+, which reacts much more slowly with NO. Both Fe2+ and Fe3+ were present in TDAs and VCCCs, and treatment with carbon monoxide, which competes with NO for Fe2+ heme, increased the amount of NO that diffused outside isolated TDAs or through VCCCs. Cytochrome b5 reductase 3 (CYB5R3), which maintains hemoglobins in an Fe2+ state, colocalized with Hb α at MEJs of TDAs and coimmunoprecipitated with Hb α in TDA cell lysates and lysates of MEJs isolated from VCCCs. Consistent with the knockdown of Hb α, knockdown or chemical inhibition of CYB5R3 increased the amount of NO that diffused outside isolated TDAs or through VCCCs and decreased the vasoconstriction elicited by phenylephrine or increased vasodilation elicited by acetylcholine in TDAs. The oxidation state of Hb α thus controls the amount of NO signal that can propagate through blood vessels.

A. C. Straub, A. W. Lohman, M. Billaud, S. R. Johnstone, S. T. Dwyer, M. Y. Lee, P. S. Bortz, A. K. Best, L. Columbus, B. Gaston, B. E. Isakson, Endothelial cell expression of haemoglobin α regulates nitric oxide signalling. Nature 491, 473–477 (2012). [PubMed]

M. T. Gladwin, D. B. Kim-Shapiro, Nitric oxide caught in traffic. Nature 491, 344–345 (2012). [PubMed]

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