Metallothionein, Nitric Oxide and Zinc Homeostasis in Vascular Endothelial Cells

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Abstract
Recent in vitro studies suggest that the oxidoreductive capacity of metal thiolate clusters in metallothionein (MT) contributes to intracellular zinc homeostasis. We used fluorescence-based techniques to address this hypothesis in intact endothelial cells, focusing on the contributory role of the important redox signaling molecule, nitric oxide. Microspectrofluorometry with Zinquin revealed that the exposure of cultured sheep pulmonary artery endothelial cells to S-nitrosocysteine resulted in the release of N,N,N′,N′-tetrakis(2 · pyridylmethyl)ethylendiamine (TPEN) chelatable zinc. Cultured sheep pulmonary artery endothelial cells were transfected with a plasmid expression vector suitable for fluorescence resonance energy transfer containing the cDNA of MT sandwiched between two mutant green fluorescent proteins. The exposure of cultured sheep pulmonary artery endothelial cells transfected with this chimera to nitric oxide donors or to agents that increased cytoplasmic Ca2+ via endogenously generated nitric oxide decreased the efficiency of fluorescence resonance energy transfer in a manner consistent with the release of metal (Zn) from MT. A physiological role for this interaction in intact tissue was supported by the lack of myogenic reflex in resistance arteries of MT knockout mice unless endogenous nitric oxide synthesis was blocked. These data suggest an important role for metal thiolate clusters of MT in nitric oxide signaling in the vascular wall.