Nitric-oxide-mediated zinc release contributes to hypoxic regulation of pulmonary vascular tone.

The metal binding protein metallothionein (MT) is a target for nitric oxide (NO), causing release of bound zinc that affects myogenic reflex in systemic resistance vessels. Here, we investigate a role for NO-induced zinc release in pulmonary vasoregulation. We show that acute hypoxia causes reversible constriction of intraacinar arteries (<50 microm/L) in isolated perfused mouse lung (IPL). We further demonstrate that isolated pulmonary (but not aortic) endothelial cells constrict in hypoxia. Hypoxia also causes NO-dependent increases in labile zinc in mouse lung endothelial cells and endothelium of IPL. The latter observation is dependent on MT because it is not apparent in IPL of MT(-/-) mice. Data from NO-sensitive fluorescence resonance energy transfer-based reporters support hypoxia-induced NO production in pulmonary endothelium. Furthermore, hypoxic constriction is blunted in IPL of MT(-/-) mice and in wild-type mice, or rats, treated with the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), suggesting a role for chelatable zinc in modulating HPV. Finally, the NO donor DETAnonoate causes further vasoconstriction in hypoxic IPL in which NO vasodilatory pathways are inhibited. Collectively, these data suggest that zinc thiolate signaling is a component of the effects of acute hypoxia-mediated NO biosynthesis and that this pathway may contribute to constriction in the pulmonary vasculature.

Intravital fluorescence microscopy in pulmonary research.

Over the last several years, microscopy as a scientific tool has reinvented itself evolving from a group of principally descriptive methodologies to encompass a wide range of primary tools and techniques to investigate the molecular organization of organs, tissues and cells. Advances in microscope and camera design, fluorescent dye technology, the development of fluorescent proteins as well as the advent of inexpensive powerful computers, has led to the feasibility of simultaneous sub micron resolution and quantitation of multiple concurrent molecular markers for both protein and DNA. Confocal microscopy has allowed optical sectioning and reconstruction of tissues in three dimensions. Finally, the development of multiphoton methodologies as an extension of optical sectioning microscopy has further improved the potential utility of this technology when examining living or light scattering tissues such as the lung. In order to illustrate the utility of two-photon methods in pulmonary biology, we present the application of this approach to the study of cellular trafficking in situ and to the study of pulmonary vasoregulation in an ex vivo rodent model.

Nitric oxide-induced modification of protein thiolate clusters as determined by spectral fluorescence resonance energy transfer in live endothelial cells.

Low-molecular-weight S-nitrosothiols are found in many tissues and affect a diverse array of signaling pathways via decomposition to *NO or exchange of their -NO function with thiol-containing proteins (transnitrosation). We used spectral laser scanning confocal imaging to visualize the effects of D- and L-stereoisomers of S-nitrosocysteine ethyl ester (SNCEE) on fluorescence resonance energy transfer (FRET)-based reporters that are targets for the following NO-related modifications: (a) S-nitrosation, via the cysteine-rich protein metallothionein (FRET-MT), and (b) nitrosyl-heme-Fe, via guanosine 3',5'-cyclic monophosphate (cygnet-2). Conformational changes consistent with S-nitrosation of FRET-MT were specific to l-SNCEE. In addition, they were reversed by dithiothreitol (DTT) but unaffected by exogenous oxyhemoglobin. In contrast, d- and l-SNCEE had comparable effects on cygnet-2, likely via activation of soluble guanylyl cyclase (sGC) by *NO as they were sensitive to the sGC inhibitor 1H-[1,2,4]-oxadiazolo[4,3-alpha] quinoxalin-1-one and exogenous oxyhemoglobin. These data demonstrate the utility of spectral laser scanning confocal imaging in revealing subtle aspects of NO signal transduction in live cells. Stereoselective transnitrosation of MT emphasizes the specificity of posttranslational modification as a component of NO signaling.