Lipid Modulators of Pulmonary Vascular Tone

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DESCRIPTION (provided by applicant): Although greatly enhanced 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis and co hydroxylase activity in pregnant rabbit lung was reported in the late 1970s, the physiological role of 20-HETE in the lung remains elusive. Our laboratory has identified 20-HETE synthesis and one cytochrome P450 (CYP) 4 isoform that converts arachidonic acid into 20-HETE in pulmonary artery endothelial cells. Expression of CYP4 in endothelial cells appears to be unique to the lung, and may be related to opposite effects of 20-HETE on the tone of systemic compared to pulmonary arteries. Endothelial derived nitric oxide (NO) is a critical pulmonary vasodilator. Several lines of our evidence suggest that CYP4/20-HETE modulate eNOS activity, thereby regulating pulmonary vascular tone. Relaxation of pulmonary arteries to 20-HETE is blunted by eNOS inhibitors, and 20-HETE evokes slow, sustained increases in intracellular free calcium with NO release from pulmonary artery endothelial cells (PAECs). 20-HETE increases phosphorylation of eNOS and association of Akt to eNOS in PAECs- both modifications known to promote activation of eNOS and enhance NO release. CYP4 and eNOS co-precipitate from PAEC lysates- and therefore are likely in very close proximity in these cells. Vascular endothelial growth factor (VEGF) is a potent, NO-dependent dilator found abundantly in the lung, where it plays a critical role in maintenance of pulmonary vascular function. Our preliminary data demonstrate that VEGF-dependent relaxation of pulmonary arteries is blocked by an inhibitor of 20-HETE synthesis; again consistent with the hypothesis that 20-HETE couples VEGF receptor binding to NO release in PAECs. This proposal will examine the mechanisms by which 20-HETE stimulates NO release from PAECs, and the contribution of 20-HETE to VEGF-stimulated eNOS activation and decreased PA tone. The specific aims are: (1) To determine the contribution of CYP4/20-HETE to VEGF induced changes in pulmonary artery tone (2) To define the contribution of CYP4/20-HETE to VEGF induced activation of eNOS and stimulated NO release and (3) To investigate cellular mechanisms by which 20- HETE enhances NO release by measuring 20-HETE or CYP4-dependent, VEGF- induced changes in (a) [Ca2+]i, IP-3 activation, (b) binding of calmodulin, hsp-90, and Akt to NOS, and phosphorylation of Akt and eNOS, and (c) co-localization of CYP4 and eNOS from PAECs. We speculate that enhanced release of 20-HETE evoked by VEGF may modulate pulmonary vascular tone locally through NO release.