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Extra-endothelial TRPV1 channels participate in alcohol and caffeine actions on cerebral artery diameter.

ABSTRACT: Alcohol (ethyl alcohol; ethanol) and caffeine are the two most widely used psychoactive substances in the world. Caffeine and ethanol have both been reported to constrict cerebral arteries in several species, including humans. We have recently shown that application of 10-?M caffeine mixed with 50 mM ethanol to in vitro pressurized cerebral arteries of rats reduced ethanol-induced constriction. This effect was dependent on the presence of nitric oxide (NO•) and could be observed in de-endothelialized arteries supplied with the NO donor sodium nitroprusside (SNP). The molecular target(s) of ethanol-caffeine interaction in cerebral arteries has remained unknown. In the present work, we used rat and mouse middle cerebral arteries (MCA) to identify the extra-endothelial effectors of NO-mediated, caffeine-induced protection against ethanol-evoked arterial constriction. Constriction of intact MCA of rat by either 50 mM ethanol or 10 ?M caffeine was ablated in the presence of a selective TRPV1 pharmacological blocker. TRPV1 pharmacological block, but not block of TRPA1, PKG, or BK channels, removed caffeine-induced protection against ethanol-evoked rat MCA constriction, whether evaluated in arteries with intact endothelium or in SNP-supplemented, de-endothelialized arteries. In mouse arteries, caffeine-induced protection against ethanol-induced MCA constriction was significantly amplified, resulting in actual vasodilation, upon pharmacological block of TRPV1, and in TRPV1 knock-out arteries. Despite some species-specific differences, our study unequivocally demonstrates the presence of functional, extra-endothelial TRPV1 that participates in both endothelium-independent MCA constriction by separate exposure to ethanol or caffeine and caffeine-induced protection against ethanol-evoked MCA constriction.

SUBMITTER: North KC

PROVIDER: S-EPMC6417832 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Extra-endothelial TRPV1 channels participate in alcohol and caffeine actions on cerebral artery diameter.

North Kelsey C KC Chang Jennifer J Bukiya Anna N AN Dopico Alex M AM

Alcohol (Fayetteville, N.Y.) 20180426

Alcohol (ethyl alcohol; ethanol) and caffeine are the two most widely used psychoactive substances in the world. Caffeine and ethanol have both been reported to constrict cerebral arteries in several species, including humans. We have recently shown that application of 10-μM caffeine mixed with 50 mM ethanol to in vitro pressurized cerebral arteries of rats reduced ethanol-induced constriction. This effect was dependent on the presence of nitric oxide (NO<sup>•</sup>) and could be observed in de ...[more]

PMID: 30268908

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Project description: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.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-?-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.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 ?1 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.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.

Project description:L-type voltage-dependent Ca(2+) channels (VDCCs) are essential for numerous processes in the cardiovascular and nervous systems. Alternative splicing modulates proteomic composition of Ca(v)1.2 to generate functional variation between channel isoforms. Here, we describe expression and function of Ca(v)1.2 channels containing alternatively spliced exon 9* in cerebral artery myocytes. RT-PCR showed expression of Ca(v)1.2 splice variants both containing (alpha(1)C(9/9*/10)) and lacking (alpha(1)C(9/10)) exon 9* in intact rabbit and human cerebral arteries. With the use of laser capture microdissection and RT-PCR, expression of mRNA for both alpha(1)C(9/9*/10) and alpha(1)C(9/10) was demonstrated in isolated cerebral artery myocytes. Quantitative real-time PCR revealed significantly greater alpha(1)C(9/9*/10) expression relative to alpha(1)C(9/10) in intact rabbit cerebral arteries compared with cardiac tissue and cerebral cortex. To demonstrate a functional role for alpha(1)C(9/9*/10), smooth muscle of intact cerebral arteries was treated with antisense oligonucleotides targeting alpha(1)C(9/9*/10) (alpha(1)C(9/9*/10)-AS) or exon 9 (alpha(1)C-AS), expressed in all Ca(v)1.2 splice variants, by reversible permeabilization and organ cultured for 1-4 days. Treatment with alpha(1)C(9/9*/10)-AS reduced maximal constriction induced by elevated extracellular K(+) ([K(+)](o)) by approximately 75% compared with alpha(1)C(9/9*/10-)sense-treated arteries. Maximal constriction in response to the Ca(2+) ionophore ionomycin and [K(+)](o) EC(50) values were not altered by antisense treatment. Decreases in maximal [K(+)](o)-induced constriction were similar between alpha(1)C(9/9*/10)-AS and alpha(1)C-AS groups (22.7 + or - 9% and 25.6 + or - 4% constriction, respectively). We conclude that although cerebral artery myocytes express both alpha(1)C(9/9*/10) and alpha(1)C(9/10) VDCC splice variants, alpha(1)C(9/9*/10) is functionally dominant in the control of cerebral artery diameter.

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Extra-endothelial TRPV1 channels participate in alcohol and caffeine actions on cerebral artery diameter.

Publications

Extra-endothelial TRPV1 channels participate in alcohol and caffeine actions on cerebral artery diameter.

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