ENDOTHELIAL-DERIVED SUPEROXIDE ANIONS IN PIG CORONARY-ARTERIES - EVIDENCE FROM LUCIGENIN CHEMILUMINESCENCE AND HISTOCHEMICAL TECHNIQUES

Citation
Rp. Brandes et al., ENDOTHELIAL-DERIVED SUPEROXIDE ANIONS IN PIG CORONARY-ARTERIES - EVIDENCE FROM LUCIGENIN CHEMILUMINESCENCE AND HISTOCHEMICAL TECHNIQUES, Journal of physiology, 500(2), 1997, pp. 331-342
Citations number
38
Categorie Soggetti
Physiology
Journal title
ISSN journal
00223751
Volume
500
Issue
2
Year of publication
1997
Pages
331 - 342
Database
ISI
SICI code
0022-3751(1997)500:2<331:ESAIPC>2.0.ZU;2-Y
Abstract
1. The generation of superoxide anions (O-2(-)) by intact pig coronary artery rings was measured using a lucigenin-enhanced chemiluminescenc e 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. Th e NET staining of arterial ring preparations showed formazan precipita tion mainly in the intima. Arterial rings were pretreated with diethyl thiocarbamate in order to inhibit Cu-Zn superoxide dismutase (SOD) act ivity 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 o r substance P, nor inhibition with N-G-nitro-L-arginine methyl ester o f endothelial nitric oxide synthase had a significant effect on O-2(-) generation. In contrast, the inhibition of flavoproteins with dipheny liodonium 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 NADP H 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), respectiv ely, in the membrane fraction, but not in the cytosolic fraction, of h omogenates obtained from arteries. 4. The results suggest that intact coronary arteries do generate O-2(-) under basal conditions and that t he endothelial layer significantly contributes to this phenomenon. Thi s 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.