It is well known that during and following resuscitation from hemorrhagic shock (HS), the vasculature becomes hyporeactive to vasoconstricting agents, and subtle changes in endothelial cell structure and function occur. Although nitric oxide (NO) clearly contributes to the genesis and maintenance of such vasodysregulation, the molecular and cellular basis of vascular pathology in HS remains unclear. The active or allosteric sites of most known bioregulatory targets for NO (and its secondary reaction products) contain thiols and/or iron. In this project, we suggest that in HS an alternative but critical target for NO is zinc sulfur clusters. After iron, zinc (Zc) is the most abundant intracellular metal. Zn metalloproteins comprise >1% of the entire human genome and are critical components of all classes of enzymes as well as many other proteins (e.g., transcription factors). Although Zn itself is redox, insert, its major intracellular binding partner metallothionein (MT) is readily nitrosylated by NO and thus we hypothesize that MT acts a molecular switch linking sensing of NO with a potential component of its transduction (labile zinc). Preliminary data regarding sensitivity of MT null mutant mice to HS and NO-dependent phenotypic changes in vasomoter regulation of resistance vessels of MT- /- mice underscores this hypothesis. According, the specific aims are to: I. Identify the role of MT in affecting the vascular response to hemorrhagic shock and vasomotor regulation of isolated mesenteric vessels in MT null mutant and transgenic mice. II. Study the physiology of zinc homeostasis in isolated cultured murine endothelial (and vascular smooth muscle) cells including use of a novel GTP-MT construct, FRET, and Zn-sensitive fluorophores. III. Determine the mechanism by which zinc contributes to: a) oxidant induced necrosis; or b) NO-mediated inhibition of LPS-induced apoptosis in cultured murine endothelial cells. IV. Determine the molecular mechanisms by which NO-mediated zinc release affects myogenic reflex in mouse mesenteric artery including calcium sparks and spontaneous transient outward currents.
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