application) The aims of this proposal are to reveal some of the mechanisms of depolarization and constriction of cerebral artery smooth muscle to physiological stimuli. The central hypothesis is that vasoconstrictor stimuli inhibit K+ channels and activate Cl- channels, resulting in smooth muscle depolarization and contraction. The goals of the following 3 specific aims are to demonstrate the involvement of specific K+ and Cl- ion channels in cerebral artery constrictor responses and to define the cellular signaling pathways which regulate these ion channels.
Specific Aim #1, will test the hypothesis that inhibition of calcium activated K+ channels is a mechanism of cerebral artery constriction. Calcium-activated potassium channel activity is increased by calcium. One source of this calcium is the sarcoplasmic reticulum which via ryanodine-sensitive channels releases calcium to localized regions near the plasmalemma. The calcium release channels as well as the calcium-activated potassium channel itself are sites where vasoconstrictor generated second messengers could inhibit calcium-activated potassium channels. These possibilities will be examined using activators and inhibitors of established signal transduction pathways.
Specific Aim #2 will test the hypothesis that inhibition of voltage-dependent potassium channels is another mechanism of agonist-induced vasoconstriction. Pharmacological inhibition of voltage-dependent potassium channels depolarizes and constricts isolated cerebral arteries. Voltage-dependent potassium channel currents contribute to resting membrane potential in vascular smooth muscle. In this aim the direct and indirect effects of vasoconstrictors on voltage-dependent potassium channels will be determined.
Specific Aim #3 will test the hypothesis that chloride channels contribute to regulation of membrane potential in cerebral arteries. Chloride channels are present in vascular smooth muscle. Chloride channel blockers hyperpolarized and dilate cerebral arteries contracted by agonists or pressure. In this aim, the properties and mechanisms of regulation and functional roles of cerebrovascular smooth muscle chloride channels will be evaluated. These studies will employ state-of-the-art techniques to study: 1) localized and global changes in intracellular calcium using conventional digital fluorescence imaging and confocal microscopy, 2) ion channel activity using the patch-clamp technique and 3) correlated electrical and contractile responses of pressurized cerebral resistance arteries. A more complete understanding of the mechanisms of contraction and relaxation of vascular smooth muscle is essential to the development of new strategies for treatment of pathophysiological states that involve excessive vasoconstrictor (hypertension, vasospasm and ischemia). The proposed studies should provide essential new information relevant to these issues.
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