Chiao-Wang Sun1, Jiangning Yang2, Andrei L Kleschyov3,4, Zhengbing Zhuge3, Mattias Carlström3, John Pernow2, Nadeem Wajih5, T Scott Isbell6, Joo-Yeun Oh7,8, Pedro Cabrales9, Amy G Tsai9, Tim Townes1, Daniel B Kim-Shapiro5, Rakesh P Patel7,8, Jon O Lundberg3. 1. Department of Biochemistry (C.W.S., T.T.), University of Alabama at Birmingham. 2. Department of Medicine, Division of Cardiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden (J.Y., J.P.). 3. Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (A.L.K., Z.Z., M.C., J.O.L.). 4. Freiberg Instruments GmbH, Freiberg, Germany (A.L.K.). 5. Department of Physics, Wake Forest University, Winston-Salem, NC (N.W., D.B.K.-S.). 6. Department of Pathology, Saint Louis University, MO (T.S.I.). 7. Department of Pathology (J.-Y.O., R.P.P.), University of Alabama at Birmingham. 8. Center for Free Radical Biology (J.-Y.O., R.P.P.), University of Alabama at Birmingham. 9. Department of Bioengineering, University of California San Diego (P.C., A.G.T.).
Abstract
BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin β chain (β93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the β93 cysteine had been replaced with alanine (β93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (β93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with β93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in β93C and β93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between β93C and β93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in β93C and β93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the β93 cysteine of Hb.
BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin β chain (β93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the β93 cysteine had been replaced with alanine (β93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (β93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with β93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in β93C and β93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between β93C and β93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in β93C and β93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the β93 cysteine of Hb.
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