Episodic, moderate-to-heavy alcohol intake, such as in binge drinking, represents the primary form of alcohol consumption in the US and other industrialized countries. Alcohol abuse is one of the three most common risk factors among stroke patients, with binge drinking being associated with an increased risk for cerebrovascular ischemia and stroke. Cerebrovascular ischemia results from impaired vasodilation and/or enhanced constriction of cerebral arteries. The activity of large-conductance, calcium- and voltage-gated (BK) channels critically limits vascular myocyte contraction, favoring cerebrovascular dilation. Ethanol at levels reached in circulation after moderate-heavy episodic drinking (50 mM) inhibits the activity of cerebral artery myocyte BK channels via their21 subunits, leading to endothelium-independent cerebrovascular constriction. Apart from ethanol, high cholesterol levels in myocyte membrane also inhibit BK currents, decrease vascular compliance, favoring vasoconstriction. Cholesterol is proposed to amplify ethanol-driven BK channel inhibition, thus, ethanol and high cholesterol level would have a profound negative impact on vascular compliance. Based on preliminary findings in artificial lipid bilayers, we hypothesize that BK 21 subunit facilitates cholesterol amplification of ethanol-induced BK channel inhibition and thus, vasoconstriction. In the present study we are aiming to use patch-clamp electrophysiology to record ethanol-induced decrease in BK channel activity in myocytes freshly isolated from wild type (C57BL/6) and KCNMB1 knock-out mouse cerebral arteries. While wild type animals have arterial myocyte BK channels with functional 21 subunits, KCNMB1 knock-outs lack this subunit. Experiments will be performed with intact cholesterol in the cell membranes, as well as after depletion of cholesterol, and cholesterol enrichment. Patch-clamp recording of BK currents under conditions desrcribed above will allow us to compare degree of ethanol-cholesterol synergistic inhibition of BK channel with of without 21 subunits. We anticipate to prove, that BK 21 subunit serves as a facilitator for cholesterol to amplify ethanol-induced inhibition of arterial myocyte BK channel. Results obtained on isolated myocytes will be matched with experiments on in vitro pressurized cerebral arteries from wild type and KCNMB1 knock-out mice. Our studies will bolster a novel concept in alcohol research and cerebrovascular pathophysiology: ethanol concentrations obtained in circulation after moderate to heavy episodic drinking and cholesterol, two well-known risk factors for stroke, impair cerebral artery dilation by inhibiting 21-containing BK channels in vascular smooth muscle itself.

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Alcohol and cholesterol are two well-known risk factors for stroke and impaired cerebral artery relaxation. We are aiming to show that BK channel 21 subunits facilitate cholesterol amplification of alcohol-induced cerebral vasospasm via inhibition of 21 subunit-containing BK channels. This study will bolster a novel concept in alcohol research and will open a new venue for therapeutic intervention on alcohol-driven cerebrovascular disease.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Small Research Grants (R03)
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Special Emphasis Panel (ZRG1-IFCN-L (50))
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Noronha, Antonio
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University of Tennessee Health Science Center
Schools of Medicine
United States
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Bukiya, Anna N; McMillan, Jacob; Liu, Jianxi et al. (2014) Activation of calcium- and voltage-gated potassium channels of large conductance by leukotriene B4. J Biol Chem 289:35314-25
Bukiya, Anna; Dopico, Alejandro M; Leffler, Charles W et al. (2014) Dietary cholesterol protects against alcohol-induced cerebral artery constriction. Alcohol Clin Exp Res 38:1216-26
Dopico, Alejandro M; Bukiya, Anna N; Singh, Aditya K (2012) Large conductance, calcium- and voltage-gated potassium (BK) channels: regulation by cholesterol. Pharmacol Ther 135:133-50
Bukiya, Anna N; Vaithianathan, Thirumalini; Kuntamallappanavar, Guruprasad et al. (2011) Smooth muscle cholesterol enables BK ?1 subunit-mediated channel inhibition and subsequent vasoconstriction evoked by alcohol. Arterioscler Thromb Vasc Biol 31:2410-23