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
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.