Recent work has implicated nicotine specifically as an important factor causing vessel wall injury in smokers, and emerging data suggest that the major pathophysiological mechanism by which nicotine exerts its effects on vessels is by causing oxidant stress. The main body of evidence supporting the hypothesis for involvement of oxidant stress is comprised of observations showing that effects of nicotine are reversed by co-treatment with anti-oxidants. However, no study to date has determined the precise cellular or molecular mechanism by which nicotine induces oxidant stress in the vessel wall. In this project, we will pursue 3 Specific Aims intended to elucidate the functional role of nicotinic acetylcholine receptors (nAChR) in cerebral blood vessels, and to elucidate the mechanisms by which these receptors produce oxidant stress in cerebral blood vessels. Our preliminary data show that basilar artery endothelial cells in situ express alpha3 and alpha7 nAChR subunits, suggesting the presence of functional nAChR.
In aim 1, we will expand on these observations by completing our survey to identify all nAChR subunits expressed in cerebral arteries, and to delineate their cellular location (endothelium vs. vascular smooth muscle). Our preliminary data show that freshly isolated endothelial cells from basilar artery demonstrate inward currents with complex deactivation kinetics in response to application of nicotine, consistent with expression of functional nAChR.
In aim 2, we will pursue these observations to expand on functional characterization of nAChR in freshly isolated cerebral endothelial cells using patch clamp and Ca imaging. Our preliminary data show that application of nicotine to freshly isolated endothelial cells causes activation of eNOS, as measured in DAF-2 imaging for NO. Also, our preliminary data suggest that in response to chronic nicotine infusion in vivo, markers of oxidant stress, nitrotyrosine and SOD-l, are increased in endothelial layers in situ.
In aim 3, we will pursue these observations to elucidate the molecular mechanism involving eNOS activation that results in production of oxidant stress in vivo with nicotine.
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| Simard, J Marc; Gerzanich, Volodymyr (2006) Sphingolipids and transient receptor potential channels: evolutionarily ancient families now joined. Circ Res 98:1347-8 |
