Selley D E, Stark S, Sim L J, Childers S R
Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Winston-Salem, NC 27157, USA.
Life Sci. 1996;59(8):659-68. doi: 10.1016/0024-3205(96)00347-5.
Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [35S]GTP gamma S binding in rat brain membranes. Assays were conducted with 0.05 nM [35S]GTP gamma S, incubated with rat cerebellar membranes, 1-30 microM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [35S]GTP gamma S binding increased with increasing concentrations of GDP, with 10-30 microM GDP providing approximately 150-200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [35S]GTP gamma S binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [35S]GTP gamma S binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [35S]GTP gamma S binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [35S]GTP gamma S autoradiography. These results demonstrate that [35S]GTP gamma S binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.
大麻素受体属于抑制腺苷酸环化酶的G蛋白偶联受体类别。受体与G蛋白的偶联可通过激动剂在过量GDP存在下刺激鸟苷-5'-O-(3-[35S]硫代)三磷酸([35S]GTPγS)结合的能力来评估。本研究检测了大麻素激动剂对大鼠脑膜中[35S]GTPγS结合的影响。实验采用0.05 nM [35S]GTPγS，与大鼠小脑膜、1-30 μM GDP以及大麻素激动剂WIN 55212-2一起孵育。结果显示，WIN 55212-2刺激[35S]GTPγS结合的能力随GDP浓度增加而增强，10-30 μM GDP时大麻素激动剂提供约150-200%的刺激。大麻素激动剂刺激[35S]GTPγS结合的药理学与先前报道的受体结合和腺苷酸环化酶检测结果相似，且大麻素拮抗剂SR141716A可阻断激动剂对[35S]GTPγS结合的刺激。脑区研究显示，WIN 55212-2在多个脑区广泛刺激[35S]GTPγS结合，这与体外[35S]GTPγS放射自显影结果一致。这些结果表明，在过量GDP存在下的[35S]GTPγS结合是衡量大麻素受体与脑膜中G蛋白偶联的有效指标。
