Effect of prolonged incubation with copper on endothelium-dependent relaxation in rat isolated aorta

1 We investigated the effects of prolonged exposure to copper (Cu2+) on vascular functioning of isolated rat aorta. 2 Aortic rings were exposed to CuSO4 (3-24 h) in Dulbecco's modified Eagle medium with or without 10% foetal bovine serum (FBS) and then challenged with vasoconstrictors or vasodilators in the absence Of Cu2+. 3 Exposure to 2 pm Cu2+ in the absence of FBS did not modify the response to phenylephrine (PE) or acetylcholine (ACh) in aortic rings incubated for 24 h. Identical exposure in the presence of FBS increased the contractile response to 1 muM PE by 30% (P<0.05) and impaired the relaxant response to 3 muM ACh or 1 muM A23187 (ACh, from 65.7 +/- 7.1 to 6.2 +/- 1.1%, n = 8; A23187, from 74.6 +/- 8.2 to 12.0 +/- 0.8%, n = 6; P<0.01 for both). Cu2+ exposure did not affect the relaxant response to NO-donors. 4 Impairment of vasorelaxation appeared 3 h after incubation with 2 muM Cu2+ and required 12 h ;to attain a steady state. Vasorelaxation to ACh was partially restored by 1 mm tiron (intracellular scavenger of superoxide ions; maximum relaxation 34.2 +/- 6.4%, n = 10, P<0.01 VS Cu2+ alone), whereas catalase, superoxide dismutase or cycloheximide were ineffective. 5 Twenty-four hour-exposure to 2 muM Cu2+ did not affect endothelium integrity or eNOS expression, and increased the Cu content in arterial rings from 6.8 +/- 1.1 to 18.9 +/- 2.9 ng mg(-1) wet weight, n = 8; P<0.01. 6 Our results show that, in the presence of FBS, prolonged exposure to submicromolar concentrations Of Cu2+ impaired endothelium-dependent vasorelaxation in aortic rings, probably through an intracellular generation of superoxide ions.