Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein

Abstract

We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn 2+, plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn 2+by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20–30% increases in fluorescence of the Zn 2+-specific fluorophore Zinquin that were rapidly reversed by the Zn 2+chelator N,N,N′, N′-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn 2+in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in the regulation of Zn 2+homeostasis by NO. Additional data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn 2+-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn 2+that were reversed by Zn 2+supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn 2+release from thiolate clusters. This effect was restored by Zn 2+supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn 2+and suggest that the ratio of MT to T can regulate Zn 2+homeostasis in response to nitrosative stress.