State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
Int J Mol Sci. 2021 Dec 21;23(1):42. doi: 10.3390/ijms23010042.
Benzodiazepines (BZDs) produce versatile pharmacological actions through positive modulation of GABA receptors (GABARs). A previous study has demonstrated that high concentrations of diazepam potentiate GABA currents on the αβγ and αβ GABARs in a flumazenil-insensitive manner. In this study, the high-concentration effects of BZDs and their sensitivity to flumazenil were determined on synaptic (αβγ, αβγ, αβγ) and extra-synaptic (αβδ) GABARs using the voltage-clamp electrophysiology technique. The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice via the loss of righting reflex (LORR) test. Diazepam induced biphasic potentiation on the αβγ, αβγ and αβγ GABARs, but did not affect the αβδ receptor. In contrast to the nanomolar component of potentiation, the second potentiation elicited by micromolar diazepam was insensitive to flumazenil. Midazolam, clonazepam, and lorazepam at 200 µM exhibited similar flumazenil-insensitive effects on the αβγ, αβγ and αβγ receptors, whereas the potentiation induced by 200 µM zolpidem or triazolam was abolished by flumazenil. Both the GABAR antagonist pentylenetetrazol and Fa173, a proposed transmembrane site antagonist, abolished the potentiation induced by 200 µM diazepam. Consistent with the in vitro results, flumazenil antagonized the zolpidem-induced LORR, but not that induced by diazepam or midazolam. Pentylenetetrazol and Fa173 antagonized the diazepam-induced LORR. These findings support the existence of non-classical BZD binding sites on certain GABAR subtypes and indicate that the flumazenil-insensitive effects depend on the chemical structures of BZD ligands.
苯二氮䓬类药物（BZDs）通过正向调节 GABA 受体（GABARs）产生多种药理学作用。先前的研究表明，高浓度的地西泮以氟马西尼不敏感的方式增强 αβγ 和 αβ GABARs 上的 GABA 电流。在这项研究中，使用电压钳电生理学技术确定了 BZD 的高浓度作用及其对氟马西尼的敏感性在突触（αβγ、αβγ、αβγ）和 extrasynaptic（αβδ）GABARs 上的作用。通过翻正反射（LORR）试验在小鼠体内评估了氟马西尼不敏感的 BZD 作用。地西泮在 αβγ、αβγ 和 αβγ GABARs 上诱导双相增强作用，但不影响 αβδ 受体。与增强的纳摩尔成分相反，微摩尔地西泮引起的第二次增强对氟马西尼不敏感。在 200µM 时，咪达唑仑、氯硝西泮和劳拉西泮表现出对 αβγ、αβγ 和 αβγ 受体的类似氟马西尼不敏感作用，而 200µM 唑吡坦或三唑仑引起的增强作用被氟马西尼消除。GABAR 拮抗剂戊四唑和 Fa173（一种拟议的跨膜位点拮抗剂）都消除了 200µM 地西泮引起的增强作用。与体外结果一致，氟马西尼拮抗唑吡坦引起的 LORR，但不拮抗地西泮或咪达唑仑引起的 LORR。戊四唑和 Fa173 拮抗地西泮引起的 LORR。这些发现支持某些 GABAR 亚型上存在非经典 BZD 结合位点，并表明氟马西尼不敏感的作用取决于 BZD 配体的化学结构。
