Endothelial-derived superoxide anions in pig coronary arteries: Evidence from lucigenin chemiluminescence and histochemical techniques

1. The generation of superoxide anions (O-2(-)) 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 O-2(-) 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 NET 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 O-2(-) generation by 184 +/- 55 % (n = 10; P < 0.01). Stimulation of protein kinase C with phorbol 12-myristate 13-acetate (5 mu M) enhanced endothelium-dependent O-2(-) generation by 136 +/- 20 % (n = 19; P < 0.01). Neither stimulation with bradykinin or substance P, nor inhibition with N-G-nitro-L-arginine methyl ester of endothelial nitric oxide synthase had a significant effect on O-2(-) generation. In contrast, the inhibition of flavoproteins with diphenyliodonium decreased concentration-dependent O-2(-) generation (IC50, 1.85 +/- 5.33 mu M). Inhibition of tetrahydrobiopterin synthesis with 2,4-diamino-6-hydroxy-pyrimidine resulted in a reduced generation of O-2(-) by about 55 %. 3. The addition of 100 mu M NADH and 100 mu M NADPH resulted in an excessive generation of O-2(-) at a rate of 0.68 +/- 0.03 and 0.26 +/- 0.01 nmol O-2(-) 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 O-2(-) under basal conditions and that the endothelial layer significantly contributes to this phenomenon. This generation of O-2(-) is greatly influenced by intrinsic SOD activity. It is suggested that basal vascular O-2(-) generation is mainly due to membrane-bound NAD(P)H oxidase activity and/or tetrahydrobiopterin-dependent processes.