Temporal Requirement for the Protective Effect of Dietary Cholesterol against Alcohol-Induced Vasoconstriction.

Moderate-to-heavy episodic alcohol drinking resulting in 30-80 mM of ethanol in blood constricts cerebral arteries and constitutes a risk factor for cerebrovascular disease. Alcohol-induced constriction of cerebral arteries in vivo and ex vivo has been shown to be blunted by dietary cholesterol (CLR) in a rat model of a high-CLR diet. Such protection has been proposed to arise from the high-CLR diet-driven increase in blood CLR levels and accompanying buildup of CLR within the cerebral artery smooth muscle. Here we used a rat model of high-CLR feeding in vivo and pressurized cerebral arteries ex vivo to examine whether the degree and time-course of alcohol-induced constriction are related to blood CLR levels. We demonstrate that subjecting young (3 weeks-old, 50 g) male Sprague-Dawley rats to a high- CLR feeding up to 41 weeks, resulted in an age-dependent increase in total blood CLR levels, when compared to those of age-matched rats on isocaloric (control) chow. This increase was paralleled by a high-CLR diet-driven elevation of blood low-density lipoproteins whereas high-density lipoprotein levels matched those of age-matched, chow-fed controls. Alcohol-induced constriction was only blunted by high-CLR dietary intake when high-CLR chow was taken for up to 8-12 and 18-23 weeks. However, alcohol-constriciton was not blunted when high-CLR chow intake lasted for longer times, such as 28-32 and 38-41 weeks. Thus, alcohol-induced constriction of rat middle cerebral arteries did not critically depend on the total blood CLR levels. Alcohol-induced constriction seemed unrelated to the natural, progressive elevation of the total blood CLR level in control- or high-CLR-fed animals over time. Thus, neither the exogenously nor endogenously driven increases in blood CLR could predict cerebral artery susceptibility to alcohol-induced constriction. However, we identified a temporal requirement for the protective effect of dietary CLR against alcohol, that could be governed by the young age of the high- CLR chow recipients (3 weeks of age) and/or the short duration of high-CLR chow feeding lasting for up to 23 weeks.

Maternal alcohol exposure during mid-pregnancy dilates fetal cerebral arteries via endocannabinoid receptors.

Prenatal alcohol exposure often results in fetal alcohol syndrome and fetal alcohol spectrum disorders. Mechanisms of fetal brain damage by alcohol remain unclear. We used baboons (Papio spp.) to study alcohol-driven changes in the fetal cerebral artery endocannabinoid system. Pregnant baboons were subjected to binge alcohol exposure via gastric infusion three times during a period equivalent to the second trimester of human pregnancy. A control group was infused with orange-flavored drink that was isocaloric to the alcohol-containing solution. Cesarean sections were performed at a time equivalent to the end of the second trimester of human pregnancy. Fetal cerebral arteries were harvested and subjected to in vitro pressurization followed by pharmacological profiling. During each alcohol-infusion episode, maternal blood alcohol concentrations (BAC) reached 80 mg/dL, that is, equivalent to the BAC considered legal intoxication in humans. Circulating anandamide (AEA) and 2-arachidonoylglycerol (2-AG) remained unchanged. Ultrasound studies on pregnant mothers revealed that fetal alcohol exposure decreased peak systolic blood velocity in middle cerebral arteries when compared to pre-alcohol levels. Moreover, ethanol-induced dilation was observed in fetal cerebral arteries pressurized in vitro. This dilation was abolished by the mixture of AM251 and AM630, which block cannabinoid receptors 1 and 2, respectively. In the presence of AM251, the cannabinoid receptor agonist AEA evoked a higher, concentration-dependent dilation of cerebral arteries from alcohol-exposed fetuses. The difference in AEA-induced cerebral artery dilation vanished in the presence of AM630. CB1 and CB2 receptor mRNA and protein levels were similar in cerebral arteries from alcohol-exposed and control-exposed fetuses. In summary, alcohol exposure dilates fetal cerebral arteries via endocannabinoid receptors and results in an increased function of CB2.

Distinct mechanisms underlying cholesterol protection against alcohol-induced BK channel inhibition and resulting vasoconstriction.

Alcohol (ethanol) at concentrations reached in blood following moderate to heavy drinking (30-80mM) reduces cerebral artery diameter via inhibition of voltage- and calcium-gated potassium channels of large conductance (BK) in cerebral artery smooth muscle. These channels consist of channel-forming α and regulatory ß1 subunits. A high-cholesterol diet protects against ethanol-induced constriction via accumulation of cholesterol within the vasculature. The molecular mechanisms of this protection remain unknown. In the present work, we demonstrate that in vitro cholesterol enrichment of rat middle cerebral arteries significantly increased cholesterol within arterial tissues and blunted constriction by 50mM of ethanol. Ethanol-induced BK channel inhibition in inside-out patches excised from freshly isolated cerebral artery myocytes was also abolished by cholesterol enrichment. Enrichment of arteries with enantiomeric cholesterol (ent-cholesterol) also blunted BK channel inhibition and cerebral artery constriction in response to ethanol. The similar protection of cholesterol and ent-cholesterol against ethanol action indicates that this protection does not require protein site(s) that specifically sense natural cholesterol. Cholesterol-driven protection against ethanol-induced BK channel inhibition and vasoconstriction was replicated in myocytes and middle cerebral arteries of C57BL/6 mice. BK ß1 subunits are known to regulate vascular diameter and its modification by ethanol. However, blunting of an ethanol effect by in vitro cholesterol enrichment was observed in arteries and myocyte membrane patches from BK ß1 (KCNMB1) knockout mice. Thus, BK ß1 subunits are not needed for cholesterol protection against ethanol effect on BK channel function and cerebral artery diameter.

Age-Dependent Susceptibility to Alcohol-Induced Cerebral Artery Constriction.

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 ß1 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 ß1 constitutes a significant contributor to age-dependent changes in the susceptibility of cerebral arteries to ethanol.