The alkaloid, nicotine, the primary component of tobacco that drives its habitual use, is a major health hazard with proven deleterious effects on the cerebral circulation, Epidemiological evidence indicates that smoking elevates risk of stroke by promoting development of atherosclerosis, formation of multiple cerebral aneurysms, and vasospasm following subarachnoid hemorrhage. Ca dysregulation in vascular smooth muscle may play an important role in nicotine-induced cerebral vasculopathy by predisposing to elevated intracellular CA, leading to Ca-mediated cell injury and cell death. We have made novel observations on effects of nicotine in patch clamp studies of cerebrovascular smooth muscle cells: 1) we found that prolonged exposure (3 hr) of vessels to nicotine causes a significant increase in Ca channel availability in smooth muscle cells subsequently isolated from the vessels; 2) we found that prolonged exposure of vessels to nicotine abrogates normal downregulation of Ca channel availability by nitric oxide (NO), but does not affect normal upregulation of Ca activated K (BK) channel activity by NO. These and other findings have led us to hypothesize that nicotine may exert its deleterious effects by blocking a protein phosphatase that may be important in regulating Ca channel availability, and that this mechanism may not involve a classical nicotinic receptor. The purpose of this grant is to 1) elucidate mechanism(s) for the effects of nicotine on Ca channel availability and on NO-signalling that we observed in smooth muscle cells following prolonged and chronic exposure of intact vessels to nicotine, and 20 to elucidate normal regulation of Ca and BK channels in cerebrovascular smooth muscle by tyrosine kinases and phosphatases, the balance between which may be altered by nicotine. We will use patch clamp techniques, supplemented with antibody/immunofluorescence techniques, Western blots and electron microscopy, to study Ca and BK channel regulation in freshly isolated smooth muscle cells from rats after prolonged (> 3 hr) and chronic (6 months) exposure to nicotine and other pharmacological agents. The proposed studies will constitute the first physiological demonstration of effects of nicotine on cerebrovascular smooth muscle CA and BK channels, and will be the first to address cellular mechanism of nicotine-induced CA channel dysregulation in smooth muscle cells from cerebral arterioles. The importance of this work lies in 1) elucidating a new physiological mechanism related to NO -induced downregulation of Ca channels in cerebrovascular smooth muscle cells, and 2) elucidating a new pathophysiological mechanism related to nicotine-induced changes in cerebral blood vessels that may predispose to stroke.
