The increasing recognition of the role played by cerebral artery dysfunction in brain disorders has fueled the search for new cerebrovascular dilators. Celastrol, a natural triterpene undergoing clinical trials for treating obesity, exerts neuroprotection, which was linked to its antioxidant/anti-inflammatory activities. We previously showed that celastrol fitted pharmacophore criteria for activating calcium-and voltage-gated potassium channels of large conductance ("BK") made of subunits cloned from cerebrovascular smooth muscle (SM). These recombinant BKs expressed in a heterologous system were activated by celastrol. Activation of native SM BKs is well-known to evoke cerebral artery dilation. Current data demonstrate that celastrol (1-100 µM) dilates de-endothelialized, ex-vivo pressurized middle cerebral arteries (MCA) from rats with EC 50 =45 µM and E max =100 µM, with MCA diameter reaching a 10% increase over vehicle-containing, time-matched values (P<0.05). A similar vasodilatory efficacy is achieved when celastrol is probed on MCA segments with intact endothelium. Selective BK block with 1 M paxilline blunts celastrol vasodilation. Similar blunting is achieved with 0.8 mM 4-AP, which blocks voltage-gated K + channels other than BK. Using an in-vivo rat cranial window, we further demonstrate that intracarotid injections of 45 M celastrol onto pial arteries branching from MCA mimics celastrol ex-vivo action. MCA constriction by ethanol concentrations reached in blood during moderate-heavy alcohol drinking (50 mM), which involves SM BK inhibition, is both prevented and reverted by celastrol. We conclude that celastrol could be an effective cerebrovascular dilator and antagonist of alcohol-induced cerebrovascular constriction, its efficacy being uncompromised by conditions that disrupt endothelial and/or BK function.

Alcohol intake leading to blood ethanol concentrations (BEC) ≥ legal intoxication modifies brain blood flow with increases in some regions and decreases in others. Brain regions receive blood from the Willis' circle branches: anterior, middle (MCA) and posterior cerebral (PCA), and basilar (BA) arteries. Rats and mice have been used to identify the targets mediating ethanol-induced effects on cerebral arteries, with conclusions being freely interchanged, albeit data were obtained in different species/arterial branches. We tested whether ethanol action on cerebral arteries differed between male rat and mouse and/or across different brain regions, and identified the targets of alcohol action. In both species and all Willis' circle branches, ethanol evoked reversible and concentration-dependent constriction (EC50s≈37-86 mM; below lethal BEC in alcohol-naïve humans). While showing similar constriction to depolarization, both species displayed differential responses to ethanol: in mice, MCA constriction was highly sensitive to the presence/absence of the endothelium, while rat PCA was significantly more sensitive to ethanol than its mouse counterpart. In rat, but not mouse, BA was more ethanol-sensitive than other branches. Both interspecies and regional variability were ameliorated by endothelium. Selective BK channel block in de-endothelialized vessels demonstrated that these channels were the effectors of alcohol-induced cerebral artery constriction across regions and species. Variabilities in alcohol actions did not fully matched KCNMB1 expression across vessels. However, immunofluorescence data from KCNMB1-/- mouse arteries electroporated with KCNMB1-coding cDNA demonstrate that KCNMB1 proteins, which regulate SM BK channel function and vasodilation, regulate interspecies and regional variability of brain artery responses to alcohol.

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