Endothelial-derived superoxide anions in pig coronary arteries: evidence from lucigenin chemiluminescence and histochemical techniques.
1. The generation of superoxide anions (O2-) by intact pig coronary artery rings was measured using a lucigenin-enhanced chemiluminescence technique and a histochemical technique with Nitroblue Tetrazolium (NBT) staining. 2. Isolated arteries with intact endothelium generated O2- at a rate of 9.0 +/- 0.8 pmol min-1 (mg dry weight)-1; this rate was diminished by about 24% when the endothelium was removed. The NBT staining of arterial ring preparations showed formazan precipitation mainly in the intima. Arterial rings were pretreated with diethylthiocarbamate in order to inhibit Cu-Zn superoxide dismutase (SOD) activity which increased the O2- generation by 184 +/- 55% (n = 10; P < 0.01). Stimulation of protein kinase C with phorbol 12-myristate 13-acetate (5 microM) enhanced endothelium-dependent O2- generation by 136 +/- 20% (n = 19; P < 0.01). Neither stimulation with bradykinin or substance P, nor inhibition with NG-nitro-L-arginine methyl ester of endothelial nitric oxide synthase had a significant effect on O2- generation. In contrast, the inhibition of flavoproteins with diphenyliodonium decreased concentration-dependent O2- generation (IC50, 1.85 +/- 5.33 microM). Inhibition of tetrahydrobiopterin synthesis with 2,4-diamino-6-hydroxy-pyrimidine resulted in a reduced generation of O2- by about 55%. 3. The addition of 100 microM NADH and 100 microM NADPH resulted in an excessive generation of O2- at a rate of 0.68 +/- 0.03 and 0.26 +/- 0.01 nmol O2- min-1 (mg protein)-1, respectively, in the membrane fraction, but not in the cytosolic fraction, of homogenates obtained from arteries. 4. The results suggest that intact coronary arteries do generate O2- under basal conditions and that the endothelial layer significantly contributes to this phenomenon. This generation of O2- is greatly influenced by intrinsic SOD activity. It is suggested that basal vascular O2- generation is mainly due to membrane-bound NAD(P)H oxidase activity and/or tetrahydrobiopterin-dependent processes.
Brandes
R P
RP
Division of Cardiology, Hannover Medical School, Germany.
Barton
M
M
Philippens
K M
KM
Schweitzer
G
G
Mügge
A
A
D000166:Acridines; D000818:Animals; D000838:Anions / Q000032:analysis* / Q000378:metabolism; D001158:Arteries / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D002474:Cell-Free System; D003331:Coronary Vessels / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D004730:Endothelium, Vascular / Q000737:chemistry / Q000378:metabolism*; D006651:Histocytochemistry; D008163:Luminescent Measurements; D009580:Nitroblue Tetrazolium; D010100:Oxygen / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000032:analysis* / Q000378:metabolism; D013552:Swine
D000166:Acridines; D000838:Anions; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; D009580:Nitroblue Tetrazolium; D013482:Superoxide Dismutase; D010100:Oxygen