PEROXYNITRITE MEDIATED MECHANISMS IN CEREBRAL ISCHEMIA

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The long-term goal of this project is to advance our understanding of the mechanisms that are involved in stroke. Stroke is a leading cause of brain disease in the United States, and involves abnormal regulation of vascular tone during ischemia and subsequent reperfusion. It is known that free radicals and reactive oxidants, including nitric oxide (NO) and superoxide (O2-), are produced during ischemia/reperfusion. Peroxynitrite (ONOO-) is a highly reactive oxidant formed by the reaction of NO and O2-. Preliminary evidence from our own laboratory and from others indicates that ONOO- may be a key modulator of vascular tone. In the proposed experiments, we will use freshly isolated cells and vessels from the rat brain Circle of Willis arteries. Cell-imaging amd quantitative videomicroscopy will be used to define the contraction responses of single cells and small arteries to ONOO. Ion channels are major determinants of vascular tone through their influence on resting membrane potential in endothelial and vascular smooth muscle cells. Our preliminary data indicate that ONOO-activates a cation current in vascular endothelial cells and inhibits calcium-activated K+ current in cerebrovascular smooth muscle cells. The patch-clamp technique will be used to (1) identify the endothelial cell ion channels that are activated by ONOO- and that are responsible for membrane depolarization, and (2) define the effect of ONOO- on K+ channels in vascular smooth muscle cells. Fluxes of O2 and NO will be varied and titrated against each other to define the underlying chemical basis for the electrical and contractile responses to ONOO. We will test whether the modulation of channel activity and cell contraction by ONOO- is thiol-dependent and involves glutathione. By bringing together free radical chemistry, ion channel electrophysiology and whole-vessel contractile responses, these studies are expected to reveal important mechanistic information regarding the effects of NO, O2- and ONOO on membrane potential, cellular-ionic signaling and vascular tone within the cerebral vasculature. This information will provide new insight into the regulation of vascular homeostasis in the brain and could help identify new measures to reduce the impact of stroke.