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Alejandro M. Dopico - One of the best experts on this subject based on the ideXlab platform.
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bk β1 subunit dependent facilitation of ethanol inhibition of bk current and cerebral Artery Constriction is mediated by the β1 transmembrane domain 2
British Journal of Pharmacology, 2017Co-Authors: Guruprasad Kuntamallappanavar, Alejandro M. DopicoAbstract:Background and Purpose Ethanol at concentrations obtained in circulation during moderate-heavy episodic drinking (30-60 mM) causes cerebral Artery Constriction in several species, including humans. In rodents, ethanol-induced cerebral Artery Constriction results from ethanol inhibition of large conductance voltage/Ca2+i-gated potassium (BK) channels in cerebral Artery myocytes. Moreover, the smooth muscle-abundant BK β1 accessory subunit is required for ethanol to inhibit cerebral Artery myocyte BK channels under physiological Ca2+i and voltages, and thus constrict cerebral arteries. The molecular bases of these ethanol actions remain unknown. Here, we set to identify the BK β1 region(s) that mediates ethanol-induced inhibition of cerebral Artery myocyte BK channels and eventual arterial Constriction. Experimental Approach We used protein biochemistry, patch-clamp on engineered channel subunits, reversible cDNA permeabilization of KCNMB1 k/o mouse arteries, and Artery in vitro pressurization. Key Results Ethanol inhibition of BK current is facilitated by β1 but not β4 subunits. Furthermore, only BK complexes containing β chimeras with β1 TM domains on a β4 background or with a β1 TM2 domain on a β4 background displayed ethanol responses identical to those of BK complexes including wt β1. Moreover, β1 TM2 itself but not other β regions are necessary for ethanol-induced cerebral Artery Constriction. Conclusion and Implications BK β1 TM2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral Artery Constriction at physiological Ca2+ and voltages. Thus, novel agents that target β1 TM2 may be considered to counteract ethanol-induced cerebral Artery Constriction and associated cerebrovascular conditions.
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BK β1 subunit‐dependent facilitation of ethanol inhibition of BK current and cerebral Artery Constriction is mediated by the β1 transmembrane domain 2
British journal of pharmacology, 2017Co-Authors: Guruprasad Kuntamallappanavar, Alejandro M. DopicoAbstract:Background and Purpose Ethanol at concentrations obtained in circulation during moderate-heavy episodic drinking (30-60 mM) causes cerebral Artery Constriction in several species, including humans. In rodents, ethanol-induced cerebral Artery Constriction results from ethanol inhibition of large conductance voltage/Ca2+i-gated potassium (BK) channels in cerebral Artery myocytes. Moreover, the smooth muscle-abundant BK β1 accessory subunit is required for ethanol to inhibit cerebral Artery myocyte BK channels under physiological Ca2+i and voltages, and thus constrict cerebral arteries. The molecular bases of these ethanol actions remain unknown. Here, we set to identify the BK β1 region(s) that mediates ethanol-induced inhibition of cerebral Artery myocyte BK channels and eventual arterial Constriction. Experimental Approach We used protein biochemistry, patch-clamp on engineered channel subunits, reversible cDNA permeabilization of KCNMB1 k/o mouse arteries, and Artery in vitro pressurization. Key Results Ethanol inhibition of BK current is facilitated by β1 but not β4 subunits. Furthermore, only BK complexes containing β chimeras with β1 TM domains on a β4 background or with a β1 TM2 domain on a β4 background displayed ethanol responses identical to those of BK complexes including wt β1. Moreover, β1 TM2 itself but not other β regions are necessary for ethanol-induced cerebral Artery Constriction. Conclusion and Implications BK β1 TM2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral Artery Constriction at physiological Ca2+ and voltages. Thus, novel agents that target β1 TM2 may be considered to counteract ethanol-induced cerebral Artery Constriction and associated cerebrovascular conditions.
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Age-Dependent Susceptibility to Alcohol-Induced Cerebral Artery Constriction.
Journal of drug and alcohol research, 2016Co-Authors: Anna N. Bukiya, Olga Seleverstov, Shivantika Bisen, Alejandro M. DopicoAbstract:Background. Age has been recognized as an important contributor into susceptibility to alcohol-driven pathology. Purpose. We aimed at determining whether alcohol-induced Constriction of cerebral arteries was age-dependent. Study design. We used rat middle cerebral Artery (MCA) in vitro diameter monitoring, patch-clamping and fluorescence labeling of myocytes to study an age-dependent increase in the susceptibility to alcohol in 3 (50 g), 8 (250 g), and 15 (440 g) weeks-old rats. Results. An age-dependent increase in alcohol-induced Constriction of MCA could be observed in absence of endothelium, which is paralleled by an age-dependent increase in both protein level of the calcium-/voltage-gated potassium channel of large conductance (BK) accessory β1 subunit and basal BK channel activity. Ethanol-induced BK channel inhibition is increased with age. Conclusions. We demonstrate an increased susceptibility of MCA to ethanol-induced Constriction in a period equivalent to adolescence and early adulthood when compared to pre-adolescence. Our work suggests that BK β1 constitutes a significant contributor to age-dependent changes in the susceptibility of cerebral arteries to ethanol.
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dietary cholesterol protects against alcohol induced cerebral Artery Constriction
Alcoholism: Clinical and Experimental Research, 2014Co-Authors: Anna N. Bukiya, Alejandro M. Dopico, Charles W. Leffler, Alexander L. FedinecAbstract:Background Binge drinking represents the major form of excessive alcohol (ethanol [EtOH]) consumption in the United States. Episodic (such as binge) drinking results in blood alcohol levels (BAL) of 18 to 80 mM and leads to alcohol-induced cerebral Artery Constriction (AICAC). AICAC was shown to arise from EtOH-induced inhibition of large-conductance, calcium/voltage-gated potassium (BK) channels in the vascular smooth muscle. Factors that modulate BK channel-mediated AICAC remain largely unknown. Methods Male Sprague Dawley rats were placed on high-cholesterol (2% of cholesterol) diet for 18 to 23 weeks. Their littermates were placed on control iso-caloric diet. AICAC was evaluated both in vivo and in vitro, by means of pial arteriole diameter monitoring through a closed cranial window and diameter measurements of isolated, pressurized cerebral arteries. Cholesterol level in the cerebral Artery tissue was manipulated by methyl-β-cyclodextrin to reverse dietary-induced accumulation of cholesterol. BK channel surface presence on the plasma membrane of cerebral Artery myocytes was evaluated by immunofluorescence staining. BK channel function in pressurized cerebral Artery was assessed using selective BK channel blocker paxilline. Results Within 5 minutes of 50 mM EtOH injection into carotid Artery in vivo, arteriole diameter decreased by 20% in control group. Pial arteriole Constriction was significantly reduced in rats on high-cholesterol diet, resulting in only 10% reduction in diameter. BAL in both groups, however, remained the same. Significant reduction in AICAC in group on high-cholesterol diet compared to control was also observed after middle cerebral Artery dissection and in vitro pressurization at 60 mmHg, this reduction remaining after endothelium removal. Cholesterol level in de-endothelialized cerebral arteries was significantly increased in rats on high-cholesterol diet. Removal of excessive cholesterol content restored AICAC to the level observed in cerebral arteries of rats on normal diet. Immunofluorescence staining of BK channel-forming and accessory, smooth muscle-specific β1 subunit in freshly isolated cerebral Artery myocyte showed that high-cholesterol diet did not down-regulate surface presence of BK protein. However, paxilline-induced cerebral Artery Constriction was diminished in arteries from rats on high-cholesterol diet. Conclusions Our data indicate that dietary cholesterol protects against AICAC. This protection is caused by cholesterol buildup in the arterial tissue and diminished function (but not surface presence) of EtOH target—BK channel.
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Dietary Cholesterol Protects Against Alcohol‐Induced Cerebral Artery Constriction
Alcoholism clinical and experimental research, 2014Co-Authors: Anna N. Bukiya, Alejandro M. Dopico, Charles W. Leffler, Alexander L. FedinecAbstract:Background Binge drinking represents the major form of excessive alcohol (ethanol [EtOH]) consumption in the United States. Episodic (such as binge) drinking results in blood alcohol levels (BAL) of 18 to 80 mM and leads to alcohol-induced cerebral Artery Constriction (AICAC). AICAC was shown to arise from EtOH-induced inhibition of large-conductance, calcium/voltage-gated potassium (BK) channels in the vascular smooth muscle. Factors that modulate BK channel-mediated AICAC remain largely unknown. Methods Male Sprague Dawley rats were placed on high-cholesterol (2% of cholesterol) diet for 18 to 23 weeks. Their littermates were placed on control iso-caloric diet. AICAC was evaluated both in vivo and in vitro, by means of pial arteriole diameter monitoring through a closed cranial window and diameter measurements of isolated, pressurized cerebral arteries. Cholesterol level in the cerebral Artery tissue was manipulated by methyl-β-cyclodextrin to reverse dietary-induced accumulation of cholesterol. BK channel surface presence on the plasma membrane of cerebral Artery myocytes was evaluated by immunofluorescence staining. BK channel function in pressurized cerebral Artery was assessed using selective BK channel blocker paxilline. Results Within 5 minutes of 50 mM EtOH injection into carotid Artery in vivo, arteriole diameter decreased by 20% in control group. Pial arteriole Constriction was significantly reduced in rats on high-cholesterol diet, resulting in only 10% reduction in diameter. BAL in both groups, however, remained the same. Significant reduction in AICAC in group on high-cholesterol diet compared to control was also observed after middle cerebral Artery dissection and in vitro pressurization at 60 mmHg, this reduction remaining after endothelium removal. Cholesterol level in de-endothelialized cerebral arteries was significantly increased in rats on high-cholesterol diet. Removal of excessive cholesterol content restored AICAC to the level observed in cerebral arteries of rats on normal diet. Immunofluorescence staining of BK channel-forming and accessory, smooth muscle-specific β1 subunit in freshly isolated cerebral Artery myocyte showed that high-cholesterol diet did not down-regulate surface presence of BK protein. However, paxilline-induced cerebral Artery Constriction was diminished in arteries from rats on high-cholesterol diet. Conclusions Our data indicate that dietary cholesterol protects against AICAC. This protection is caused by cholesterol buildup in the arterial tissue and diminished function (but not surface presence) of EtOH target—BK channel.
Yu Tai - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial uncoupler BAM15 inhibits Artery Constriction and potently activates AMPK in vascular smooth muscle cells.
Acta pharmaceutica Sinica. B, 2018Co-Authors: Yu Tai, Xuan Peng, Jun-xue Zhu, Xi-hai Mao, Nan Qin, Rong Huo, Yunlong Bai, De-li DongAbstract:Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited Artery Constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced Constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC50 value of vasorelaxation in PE-induced Constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator.
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Mitochondrial uncoupler BAM15 inhibits Artery Constriction and potently activates AMPK in vascular smooth muscle cells
Elsevier, 2018Co-Authors: Yu Tai, Xuan Peng, Jun-xue Zhu, Xi-hai Mao, Nan Qin, Rong Huo, Yunlong Bai, De-li DongAbstract:Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited Artery Constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced Constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC50 value of vasorelaxation in PE-induced Constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator. KEY WORDS: BAM15, Mitochondrial uncoupling, AMPK, Smooth muscle cells, AICAR, Metformi
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Niclosamide ethanolamine inhibits Artery Constriction.
Pharmacological Research, 2017Co-Authors: Jie Yan, Chang-lin Zhen, Yan Qiu Zhang, Xin Shen, Ming-yu Liu, Jing Jin, Jin-lai Gao, Xiao-lin Xiao, Yu TaiAbstract:We previously demonstrated that the typical mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Artery Constriction, but CCCP was used only as a pharmacological tool. Niclosamide is an anthelmintic drug approved by FDA. Niclosamide ethanolamine (NEN) is a salt form of niclosamide and has been demonstrated to uncouple mitochondrial oxidative phosphorylation. The aim of the present study was to elucidate the vasoactivity of NEN and the potential mechanisms. Isometric tension of rat mesenteric Artery and thoracic aorta was recorded by using multi-wire myograph system. The protein levels were measured by using western blot techniques. Niclosamide ethanolamine (NEN) treatment relaxed phenylephrine (PE)- and high K+ (KPSS)-induced Constriction, and pre-treatment with NEN inhibited PE- and KPSS-induced Constriction of rat mesenteric arteries. In rat thoracic aorta, NEN also showed antagonism against PE- and KPSS-induced Constriction. NEN induced increase of cellular ADP/ATP ratio in vascular smooth muscle cells (A10) and activated AMP-activated protein kinase (AMPK) in A10 cells and rat thoracic aorta. NEN-induced aorta relaxation was attenuated in AMPKα1 knockout (-/-) mice. SERCA inhibitors cyclopiazonic acid and thapsigargin, but not KATP channel blockers glibenclamide and 5-hydroxydecanoic acid, attenuated NEN-induced vasorelaxation in rat mesenteric arteries. NEN treatment increased cytosolic [Ca2+]i and depolarized mitochondrial membrane potential in vascular smooth muscle cells (A10). Niclosamide in non-salt form showed the similar vasoactivity as NEN in rat mesenteric arteries. Niclosamide ethanolamine inhibits Artery Constriction, indicating that it would be promising to be developed as an anti-hypertensive drug or it would induce vasodilation-related side effects when absorbed in vivo.
Wolfgang Schaper - One of the best experts on this subject based on the ideXlab platform.
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in situ localization of transforming growth factor β1 in porcine heart enhanced expression after chronic coronary Artery Constriction
Journal of Molecular and Cellular Cardiology, 1991Co-Authors: Martin Wünsch, Hari S. Sharma, T. Markert, Robert J. Schott, Peter Kremer, Niels Bleese, Sabine Bernotatdanielowski, Wolfgang SchaperAbstract:We investigated the expression of transforming growth factor beta 1 (TGF-beta 1), a polypeptide differentiation factor probably associated with angiogenic properties in chronically hypoperfused heart tissue. A slowly swelling ameroid constrictor was implanted around the coronary circumflex Artery (CX) of young domestic pigs. Two to three weeks after, significant CX stenosis of more than 90% and coronary collateralization could be demonstrated angiographically. The CX dependent experimental myocardial tissue (E) was investigated, with the LAD dependent area of the same pig serving as a control (C). We found significantly enhanced TGF-beta 1 mRNA expression by northern blot hybridization in the experimental myocardium (E) of those pigs with demonstrable coronary collaterals in the absence of a major myocardial infarction. The presence of TGF-beta 1 protein could be demonstrated quantitatively in extracts of the experimental and the control area by immunoblot analysis. By in situ techniques, TGF-beta 1 mRNA and protein could be localized predominantly in cardiac myocytes. We conclude that one adaptive mechanism of the pig heart in chronic coronary Artery Constriction is the enhanced expression of TGF-beta 1. Cardiac myocytes are a major source of TGF-beta 1. The observed coronary collateralization could be mediated-at least in part-by the angiogenic properties of TGF-beta 1.
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In situ localization of transforming growth factor β1 in porcine heart: Enhanced expression after chronic coronary Artery Constriction
Journal of molecular and cellular cardiology, 1991Co-Authors: Martin Wünsch, Hari S. Sharma, T. Markert, Sabine Bernotat-danielowski, Robert J. Schott, Peter Kremer, Niels Bleese, Wolfgang SchaperAbstract:Abstract We investigated the expression of transforming growth factor beta 1 (TGF-β1), a polypeptide differentiation factor probably associated with angiogenic properties in chronically hypoperfused heart tissue. A slowly swelling ameroid constrictor was implanted around the coronary circumflex Artery (CX) of young domestic pigs. Two to three weeks after, significant CX stenosis of more than 90% and coronary collateralization could be demonstrated angiographically. The CX dependent experimental myocardial tissue (E) was investigated, with the LAD dependent area of the same pig serving as a control (C). We found significantly enhanced TGF-β1 mRNA expression by northern blot hybridization in the experimental myocardium (E) of those pigs with demonstrable coronary collaterals in the absence of a major myocardial infarction. The presence of TGF-β1 protein could be demonstrated quantitatively in extracts of the experimental and the control area by immunoblot analysis. By in situ techniques, TGF-β1 mRNA and protein could be localized predominantly in cardiac myocytes. We conclude that one adaptive mechanism of the pig heart in chronic coronary Artery Constriction is the enhanced expression of TGF-β1. Cardiac myocytes are a major source of TGF-β1. The observed coronary collateralization could be mediated — at least in part — by the angiogenic properties of TGF-β1.
De-li Dong - One of the best experts on this subject based on the ideXlab platform.
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Mitochondrial uncoupler BAM15 inhibits Artery Constriction and potently activates AMPK in vascular smooth muscle cells.
Acta pharmaceutica Sinica. B, 2018Co-Authors: Yu Tai, Xuan Peng, Jun-xue Zhu, Xi-hai Mao, Nan Qin, Rong Huo, Yunlong Bai, De-li DongAbstract:Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited Artery Constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced Constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC50 value of vasorelaxation in PE-induced Constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator.
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Mitochondrial uncoupler BAM15 inhibits Artery Constriction and potently activates AMPK in vascular smooth muscle cells
Elsevier, 2018Co-Authors: Yu Tai, Xuan Peng, Jun-xue Zhu, Xi-hai Mao, Nan Qin, Rong Huo, Yunlong Bai, De-li DongAbstract:Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited Artery Constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced Constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC50 value of vasorelaxation in PE-induced Constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator. KEY WORDS: BAM15, Mitochondrial uncoupling, AMPK, Smooth muscle cells, AICAR, Metformi
Guruprasad Kuntamallappanavar - One of the best experts on this subject based on the ideXlab platform.
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bk β1 subunit dependent facilitation of ethanol inhibition of bk current and cerebral Artery Constriction is mediated by the β1 transmembrane domain 2
British Journal of Pharmacology, 2017Co-Authors: Guruprasad Kuntamallappanavar, Alejandro M. DopicoAbstract:Background and Purpose Ethanol at concentrations obtained in circulation during moderate-heavy episodic drinking (30-60 mM) causes cerebral Artery Constriction in several species, including humans. In rodents, ethanol-induced cerebral Artery Constriction results from ethanol inhibition of large conductance voltage/Ca2+i-gated potassium (BK) channels in cerebral Artery myocytes. Moreover, the smooth muscle-abundant BK β1 accessory subunit is required for ethanol to inhibit cerebral Artery myocyte BK channels under physiological Ca2+i and voltages, and thus constrict cerebral arteries. The molecular bases of these ethanol actions remain unknown. Here, we set to identify the BK β1 region(s) that mediates ethanol-induced inhibition of cerebral Artery myocyte BK channels and eventual arterial Constriction. Experimental Approach We used protein biochemistry, patch-clamp on engineered channel subunits, reversible cDNA permeabilization of KCNMB1 k/o mouse arteries, and Artery in vitro pressurization. Key Results Ethanol inhibition of BK current is facilitated by β1 but not β4 subunits. Furthermore, only BK complexes containing β chimeras with β1 TM domains on a β4 background or with a β1 TM2 domain on a β4 background displayed ethanol responses identical to those of BK complexes including wt β1. Moreover, β1 TM2 itself but not other β regions are necessary for ethanol-induced cerebral Artery Constriction. Conclusion and Implications BK β1 TM2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral Artery Constriction at physiological Ca2+ and voltages. Thus, novel agents that target β1 TM2 may be considered to counteract ethanol-induced cerebral Artery Constriction and associated cerebrovascular conditions.
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BK β1 subunit‐dependent facilitation of ethanol inhibition of BK current and cerebral Artery Constriction is mediated by the β1 transmembrane domain 2
British journal of pharmacology, 2017Co-Authors: Guruprasad Kuntamallappanavar, Alejandro M. DopicoAbstract:Background and Purpose Ethanol at concentrations obtained in circulation during moderate-heavy episodic drinking (30-60 mM) causes cerebral Artery Constriction in several species, including humans. In rodents, ethanol-induced cerebral Artery Constriction results from ethanol inhibition of large conductance voltage/Ca2+i-gated potassium (BK) channels in cerebral Artery myocytes. Moreover, the smooth muscle-abundant BK β1 accessory subunit is required for ethanol to inhibit cerebral Artery myocyte BK channels under physiological Ca2+i and voltages, and thus constrict cerebral arteries. The molecular bases of these ethanol actions remain unknown. Here, we set to identify the BK β1 region(s) that mediates ethanol-induced inhibition of cerebral Artery myocyte BK channels and eventual arterial Constriction. Experimental Approach We used protein biochemistry, patch-clamp on engineered channel subunits, reversible cDNA permeabilization of KCNMB1 k/o mouse arteries, and Artery in vitro pressurization. Key Results Ethanol inhibition of BK current is facilitated by β1 but not β4 subunits. Furthermore, only BK complexes containing β chimeras with β1 TM domains on a β4 background or with a β1 TM2 domain on a β4 background displayed ethanol responses identical to those of BK complexes including wt β1. Moreover, β1 TM2 itself but not other β regions are necessary for ethanol-induced cerebral Artery Constriction. Conclusion and Implications BK β1 TM2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral Artery Constriction at physiological Ca2+ and voltages. Thus, novel agents that target β1 TM2 may be considered to counteract ethanol-induced cerebral Artery Constriction and associated cerebrovascular conditions.
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Endothelial nitric oxide mediates caffeine antagonism of alcohol-induced cerebral Artery Constriction
The Journal of pharmacology and experimental therapeutics, 2015Co-Authors: Jennifer Chang, Guruprasad Kuntamallappanavar, Alexander L. Fedinec, Charles W. Leffler, Anna N. Bukiya, Alex M. DopicoAbstract:Despite preventive education, the combined consumption of alcohol and caffeine (particularly from “energy drinks”) continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent Constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40–70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS−/−) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral Artery Constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular Constriction without significantly disrupting endothelium-independent, alcohol-induced cerebral Artery Constriction itself.
