Effects of Nitric Oxide Donor Supplementation on Copper Deficient Embryos and Nitric Oxide-Mediated Downstream Signaling

Yang, Soo Jin

Abstract

One suggested mechanism underlying copper (Cu) deficiency teratogenicity is a low availability of nitric oxide (NO), a signaling molecule which is essential in developmental processes. Increased superoxide anions secondary to decreased activities of Cu-zinc superoxide dismutase (Cu-Zn SOD) in Cu deficiency can interact with NO to form peroxynitrite, which can nitrate proteins at tyrosine residues. In addition, peroxynitrite formation can limit NO bioavailability. We previously reported low NO availability and increased protein nitration in Cu deficient (Cu-) embryos. In the current study, we tested whether Cu deficiency alters downstream signaling of NO by assessing cyclic GMP (cGMP) and phosphorylated vasodilator-stimulating phosphoprotein (VASP) levels, and whether NO supplementation can affect these targets as well as protein nitration. Gestation day 8.5 embryos from Cu adequate (Cu+) or Cu- dams were collected and cultured in either Cu+ or Cu- media for 48 hr. A subset of embryos was cultured in Cu- media supplemented with a NO donor (DETA/NONOate; 20 micrometer) and/or Cu-Zn SOD. Cu-/Cu- embryos showed a higher incidence of embryonic and yolk sac abnormalities, low NO availability, blunted dose-response in NO concentrations to increasing doses of acetylcholine, low mRNA expression of endothelial nitric oxide synthase (eNOS), increased levels of 3-nitrotyrosine (3-NT) compared to Cu+/Cu+ controls. cGMP concentrations tended to be low in Cu-/Cu- embryos, and they were significantly lower in Cu-/Cu- yolk sacs than in controls. Levels of phosphorylated VASP at serine 239 (P-VASP) were similar in all groups. NO donor supplementation to the Cu- media ameliorated embryonic and yolk sac abnormalities, and resulted in increased levels of cGMP without altering levels of P-VASP and 3-NT. Taken together, these data support the concept that Cu deficiency limits NO availability and alters NO/cGMP-dependent signaling in Cu- embryos and yolk sacs, which contributes to Cu deficiency-induced abnormal development.