Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein.
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.
St  Croix
Claudette M
CM
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA. cls13+@pitt.edu
Wasserloos
K J
KJ
Dineley
K E
KE
Reynolds
I J
IJ
Levitan
E S
ES
Pitt
B R
BR
D000818:Animals; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003545:Cysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004880:Ergothioneine / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes; D015870:Gene Expression / Q000502:physiology; D006706:Homeostasis / Q000502:physiology*; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D011651:Pulmonary Artery / Q000166:cytology; D015363:Quinolones; D026403:S-Nitrosothiols / Q000494:pharmacology; D012756:Sheep; D013050:Spectrometry, Fluorescence; D014105:Tosyl Compounds; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology
D002614:Chelating Agents; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D020030:Nitric Oxide Donors; D015363:Quinolones; D026403:S-Nitrosothiols; D014105:Tosyl Compounds; D009569:Nitric Oxide; D008668:Metallothionein; C021315:S-nitrosocysteine; D004880:Ergothioneine; D015032:Zinc; D003545:Cysteine; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; C084731:zinquin