title: Scavenging of nitric oxide by hemoglobin in the tunica media of porcine coronary arteries.
abstract: Scavenging of nitric oxide (NO) often interferes with studies on NO signaling in cell-free preparations. We observed that formation of cGMP by NO-stimulated purified soluble guanylate cyclase (sGC) was virtually abolished in the presence of cytosolic preparations of porcine coronary arteries, with the scavenging activity localized in the tunica media (smooth muscle layer). Electrochemical measurement of NO release from a donor compound and light absorbance spectroscopy showed that cytosolic preparations contained a reduced heme protein that scavenged NO. This protein, which reacted with anti-human hemoglobin antibodies, was efficiently removed from the preparations by haptoglobin affinity chromatography. The cleared cytosols showed only minor scavenging of NO according to electrochemical measurements and did not decrease cGMP formation by NO-stimulated sGC. In contrast, the column flow-through caused a nearly 2-fold increase of maximal sGC activity (from 33.1 ± 1.6 to 54.9 ± 2.2 μmol × min(-1) × mg(-1)). The proteins retained on the affinity column were identified as hemoglobin α and β subunits. The results indicate that hemoglobin, presumably derived from vasa vasorum erythrocytes, is present and scavenges NO in preparations of porcine coronary artery smooth muscle. Selective removal of hemoglobin-mediated scavenging unmasked stimulation of maximal NO-stimulated sGC activity by a soluble factor expressed in vascular tissue.
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kollau
authors.affiliation: Department of Pharmacology and Toxicology, Ruhr University Bochum, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Russwurm
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria.
authors.forename: Andrea
authors.initials: A
authors.lastname: Neubauer
authors.affiliation: Institute of Molecular Biosciences, NAWI Graz, University of Graz, Austria; Omics-Center, BioTechMed-Graz, Austria.
authors.forename: Gerald
authors.initials: G
authors.lastname: Rechberger
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria.
authors.forename: Kurt
authors.initials: K
authors.lastname: Schmidt
authors.affiliation: Department of Pharmacology and Toxicology, Ruhr University Bochum, Germany.
authors.forename: Doris
authors.initials: D
authors.lastname: Koesling
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria.
authors.forename: John
authors.initials: J
authors.lastname: Fassett
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria.
authors.forename: Astrid
authors.initials: A
authors.lastname: Schrammel
authors.affiliation: Institute of Pharmaceutical Sciences, Department of Pharmacology and Toxicology, University of Graz, Austria. Electronic address: mayer@uni-graz.at.
authors.forename: Bernd
authors.initials: B
authors.lastname: Mayer
mesh_terms: D000818:Animals; D002417:Cattle; D003331:Coronary Vessels / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D000078842:Cytoglobin; D005914:Globins / Q000378:metabolism; D006242:Haptoglobins / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase / Q000378:metabolism; D013552:Swine; D017540:Tunica Media / Q000378:metabolism*
chemical_list: D000078842:Cytoglobin; D006242:Haptoglobins; D006454:Hemoglobins; D009569:Nitric Oxide; D005914:Globins; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP
keywords: Affinity chromatography; Haptoglobin; Heme protein; Nitric oxide scavenging; Selective removal; cGMP