孕烯醇酮是一种调节神经胶质生长和分化的神经类固醇,神经元放电,和几个大脑功能,这些影响归因于孕烯醇酮对神经元和神经胶质细胞本身的作用。尽管脑循环对脑功能的重要作用,而且孕烯醇酮是一种血管活性剂,孕烯醇酮对脑动脉的作用尚不清楚。这里,我们获得了麻醉的雄性和雌性C57BL/6J小鼠对孕烯醇酮在大脑中动脉(MCA)直径上的体内浓度反应曲线。在雄性和雌性动物中,孕烯醇酮(1nM-100μM)以浓度依赖性方式收缩MCA,其最大效果达到~22-35%的直径减少。孕烯醇酮的作用在完整和去内皮化的情况下被复制,体外加压的MCA节段与孕烯醇酮在完整和去内皮化的MCA中引起类似的收缩。1μM帕索林消除了神经类固醇作用,大电导(BK)的Ca2-和电压门控K通道的选择性阻断剂。细胞附着,在从小鼠MCAs中新鲜分离的平滑肌细胞上的膜片钳记录表明,在体外和体内(10μM)收缩MCAs的浓度下,孕烯醇酮降低BK活性(NPo),NPo的平均跌幅达到24.2%。脑动脉的孕烯醇酮收缩的浓度依赖性和完整细胞中BK活性的抑制与无细胞中获得的数据平行,由内而外的补丁,在10μM孕烯醇酮时达到最大抑制。MCA平滑肌BKs包括通道形成α(slo1蛋白)和调节β1亚基,分别由KCNMA1和KCNMB1编码。然而,在KCNMB1-/-小鼠的MCA心肌细胞中,孕烯醇酮驱动的NPo降低仍然很明显。将slo1通道重建为人造通道后,二元磷脂双层,10μM孕烯醇酮引起slo1NPo抑制,与天然膜中看到的相似。最后,孕烯醇酮未能抑制KCNMA1-/-小鼠的MCA。总之,孕烯醇酮独立于神经元收缩MCA,胶质,内皮和循环因子,以及细胞完整性,细胞器,复杂的膜细胞结构,和胞质信号的持续存在。相反,这种作用涉及直接抑制SMBK通道,它不需要β1亚基,但通过通道形成α亚基对神经类固醇的直接感知来介导。
Pregnenolone is a neurosteroid that modulates glial growth and differentiation, neuronal firing, and several brain functions, these effects being attributed to pregnenolone actions on the neurons and glial cells themselves. Despite the vital role of the cerebral circulation for brain function and the fact that pregnenolone is a vasoactive agent, pregnenolone action on brain arteries remain unknown. Here, we obtained in vivo concentration response curves to pregnenolone on middle cerebral artery (MCA) diameter in anesthetized male and female C57BL/6J mice. In both male and female animals, pregnenolone (1 nM-100 μM) constricted MCA in a concentration-dependent manner, its maximal effect reaching ~22-35% decrease in diameter. Pregnenolone action was replicated in intact and de-endothelialized, in vitro pressurized MCA segments with pregnenolone evoking similar constriction in intact and de-endothelialized MCA. Neurosteroid action was abolished by 1 μM paxilline, a selective blocker of Ca2+ - and voltage-gated K+ channels of large conductance (BK). Cell-attached, patch-clamp recordings on freshly isolated smooth muscle cells from mouse MCAs demonstrated that pregnenolone at concentrations that constricted MCAs in vitro and in vivo (10 μM), reduced BK activity (NPo), with an average decrease in NPo reaching 24.2%. The concentration-dependence of pregnenolone constriction of brain arteries and inhibition of BK activity in intact cells were paralleled by data obtained in cell-free, inside-out patches, with maximal inhibition reached at 10 μM pregnenolone. MCA smooth muscle BKs include channel-forming α (slo1 proteins) and regulatory β1 subunits, encoded by KCNMA1 and KCNMB1, respectively. However, pregnenolone-driven decrease in NPo was still evident in MCA myocytes from KCNMB1-/- mice. Following reconstitution of slo1 channels into artificial, binary phospholipid bilayers, 10 μM pregnenolone evoked slo1 NPo inhibition which was similar to that seen in native membranes. Lastly, pregnenolone failed to constrict MCA from KCNMA1-/- mice. In conclusion, pregnenolone constricts MCA independently of neuronal, glial, endothelial and circulating factors, as well as of cell integrity, organelles, complex membrane cytoarchitecture, and the continuous presence of cytosolic signals. Rather, this action involves direct inhibition of SM BK channels, which does not require β1 subunits but is mediated through direct sensing of the neurosteroid by the channel-forming α subunit.