Metallothionein, nitric oxide and zinc homeostasis in vascular endothelialcells

Recent in vitro studies suggest that the oxidoreductive capacity of metal t hiolate clusters in metallothionein (MT) contributes to intracellular zinc homeostasis. We used fluorescence-based techniques to address this hypothes is in intact endothelial cells, focusing on the contributory role of the im portant redox signaling molecule, nitric oxide. Microspectrofluorometry wit h Zinquin revealed that the exposure of cultured sheep pulmonary artery end othelial cells to S-nitrosocysteine resulted in the release of N,N,N',N'-te trakis(2.pyridylmethyl) ethylendiamine (TPEN) chelatable zinc. Cultured she ep pulmonary artery endothelial cells were transfected with a plasmid expre ssion vector suitable for fluorescence resonance energy transfer containing the cDNA of MT sandwiched between two mutant green fluorescent proteins. T he exposure of cultured sheep pulmonary artery endothelial cells transfecte d with this chimera to nitric oxide donors or to agents that increased cyto plasmic Ca2+ via endogenously generated nitric oxide decreased the efficien cy of fluorescence resonance energy transfer in a manner consistent with th e 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 o f MT in nitric oxide signaling in the vascular wall.