Although hypoxic pulmonary vasoconstriction (HPV) has been reported to affect all segments of the pulmonary circulation relatively little is known regarding vasoconstriction of small (<50um) intra-acinar arterioles. Our preliminary data suggest that hypoxic induced increases in NO biosynthesis result in enhanced intracellular free zinc that in turn may contribute to vasoconstriction of microvessels in the intact isolated perfused lung (IPL). Since this anatomic site is composed primarily of endothelial cells with solitary or discontinuous smooth muscle like cells (e.g. pericytes) in their wall, the nature of contractile events within the microcirculation are likely to be distinct from vasoregulation of proximal pulmonary vessels. We propose to use contemporary optical microscopy and fluorescent reporter molecules, including a green fluorescent protein (GFP) chimera of the zinc-binding protein metallothionein (MT) suitable for fluorescence resonance energy transfer (FRET) analysis of the metallation status of MT, to facilitate our first insights into the role of intracellular zinc in pulmonary vasoregulation.
The specific aims of this amended proposal are to: I. Determine the role of NO-Zn signaling in mediating the intrinsic capacity of isolated pulmonary microvascular endothelial and smooth muscle cells to constrict in hypoxia by pharmacological or genetic inhibition of the distinct components of the pathway (NOS inhibition, TPEN mediated Zn chelation, siRNA inhibition of MT expression). II. Assess the role of NO-Zn signaling in mediating HPV in the intact rat lung by monitoring perfusion pressure responses to inhibition of the known vasodilatory limbs of NO mediated effects on HPV and manipulation of hypoxia-induced, NO-mediated increases in labile Zn. We will examine the role of NO- MT-Zn on constriction of the microcirculation via intravital microscopy of subpleural vessels of IPLs from TIE- 2-GFP and MT and eNOS null mice. Furthermore, we will use endothelial cell directed non-viral mediated somatic gene transfer of FRET reporter constructs, and zinc-sensitive fluorophores followed by spectral laser scanning confocal microscopy to reveal elements of hypoxia-NO-MT-Zn pathway. III. Investigate a pathway by which NO-induced changes in labile zinc modulate HPV via activation of PKC using live cell and intravital imaging approaches to visualize PKC activation, alterations in Zn homeostasis, and vascular reactivity against a background of PKC inhibition in MT +/+ and MT -/- mice. ? ? ?
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