Abstract
We have previously shown that: a) iNOS or chemically derived (S-nitroso-N-acetylpenicillamine (SNAP)) NO is associated with resistant phenotype to LPS induced apoptosis in a zinc dependent fashion; and b) NO mediated elevations in intracellular zinc ([Zn]i) are critically dependent upon MT in cultured pulmonary mouse fibroblasts and endothelial cells. In the current study, we used RNA interference (siRNA) to couple these observations and reveal a central role for NO→MT→ [Zn]i↑ in affecting sensitivity of SPAEC to LPS-induced apoptosis. We exposed SPAEC to a combination of siRNA to sheep MT-Ia, b, c and MT-II and noted a lack of change in [Zn]i (as detected microspectrofluorometrically with the zinc indicator, FluoZin-3) compared to a huge increase (6X increase in FluoZin-3 fluorescence) after SNAP in wildtype SPAEC after a brief exposure to large concentrations of SNAP. Pretreatment of SPAEC with SNAP (250μM, 6h) made them resistant to LPS (1 ug/ml) -induced apoptosis. Importantly, the anti-apoptotic effect of SNAP was abrogated in SPAEC treated with non-toxic (1 uM) doses of zinc chelator, TPEN. Collectively, these data confirm that: a) increases in labile [Zn]i are an important component of NO mediated resistance to LPS induced aopotosis; and b) s-nitrosation of MT (with subsequent zinc release) may be a critical source of such NO mediated increases in labile zinc.
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