The induced synthesis of NO in response to cytokine stimulation contrasts with its constitutive release in picomolar (pM) quantities for a few seconds as an endothelium dependent mediator of vascular relaxation. Stimulated by inflammatory mediators such as E. coli lipopolysaccharide (LPS), tumor necrosis factor (TNFa), and interferon gamma (IFNg), an inducible NOS (iNOS) present in vascular smooth muscle and endothelium releases NO in uM quantities for up to 48 hours. Cytokine induced production of NO may have exaggerated effects on vascular reactivity and may be cytotoxic. The induced production of NO in the lung may have pathophysiologic relevance to acute lung injury and adult respiratory distress syndrome (ARDS). Preliminary studies indicate that a mixture of LPS and cytokines, TNFa and IFNg in particular, stimulate rat pulmonary artery smooth muscle (RPASM) cells in culture to increase NO production in a time and tetrahydrobiopter (BH4) dependent manner, with increased mRNA levels for iNOS and GTP cyclohydrolase (GTP-CH), the rate limiting enzyme in the production of BH4. NO, either released from an NO generating compound or from stimulated RPASM in coculture, appears to be cytotoxic for endothelial target cells. The applicants' findings led to the overall hypothesis that the inappropriate vascular lability and endothelial dysfunction seen in lung during sepsis is in part the result of NO release by vascular smooth muscle and endothelium. The up regulation of iNOS and GTP-CH results from enzyme induction by inflammatory cytokines, principally TNFa and IFNg. Accordingly, aim (1) is to define the mechanisms underlying TNFa and IFNg induced NO production by RPASM and endothelium (RPAE) in cell culture.
Aim 2 is to determine the effects of cytokine induced, smooth muscle and endothelial derived NO on (a) the reactivity of pulmonary vascular rings; and (b) the maintenance of oxidant sensitive endothelial functions. The relationship between iNOS expression and cytotoxicity will be established by antisense inhibition to iNOS and GTP-CH expression.
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