Depolarization and Ca(2+) down regulate CB1 receptors and CB1-mediated signaling in cerebellar granule neurons.
Depolarization and Ca(2+) down regulate CB1 receptors and CB1-mediated signaling in cerebellar granule neurons.
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去极化和 Ca(2) 下调小脑颗粒神经元中 CB1 受体和 CB1 介导的信号传导。
DOI：
10.1016/j.neuropharm.2005.11.012
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发表时间：
2006
影响因子：
4.7
通讯作者：
Middleton,FrankA
中科院分区：
文献类型：
--
作者：
Vallano,MaryLou;Beaman-Hall,CarolM;Bui,CuongJ;Middleton,FrankA
小脑颗粒神经元突触前末端是大麻素的主要作用靶点，大麻素通过1型Gαi/o偶联大麻素受体（CB1）调节谷氨酸释放。为了研究CB1信号，研究人员使用颗粒神经元的原代培养物，通常在补充高KCl的培养基中生长，以提高长期存活率。在此，我们证明了CB1的表达和信号传导在这些条件下受到干扰。具体而言，免疫化学和RT-PCR分析表明，与在含有5mM KCl的生理培养基中培养相比，去极化生长条件降低了CB1蛋白、mRNA和CB1介导的腺苷酸环化酶抑制。CB1 mRNA的去极化依赖性下调，像生存一样，被l型VDCC拮抗剂而不是Na+通道拮抗剂河豚毒素减弱。比较5mM KCl和25mM KCl培养物的寡核苷酸微阵列证实，去极化减少了CB1 mRNA，但没有减少编码若干g蛋白亚基或腺苷酸环化酶的mRNA。然而，可能位于CB1下游的突触信号蛋白发生了显著变化，包括K+通道、α-神经毒素、cAMP-GEFII、Munc13-3、secretogranin和synaptotagmin。这些发现为采用5mM KCl培养物进行颗粒神经元中CB1信号传导的未来研究提供了令人信服的论据。此外，他们认为去极化和Ca2+依赖的信号通路抑制CB1基因转录。
Presynaptic terminals of cerebellar granule neurons are primary targets of cannabinoids, which act through type 1 Gαi/o-coupled cannabinoid receptors (CB1) to modulate glutamate release. To study CB1 signaling investigators use primary cultures of granule neurons, typically grown in medium supplemented with elevated KCl to improve long-term survival. Herein, we demonstrate that CB1 expression and signaling are perturbed under these conditions. Specifically, immunochemical and RT–PCR assays indicate that depolarizing growth conditions decrease CB1 protein, mRNA and CB1-mediated inhibition of adenylyl cyclase compared to cultures grown in physiologic medium containing 5mM KCl. Depolarization-dependent downregulation of CB1 mRNA, like survival, is attenuated by L-type VDCC antagonists but not the Na+-channel antagonist, tetrodotoxin. Comparison of oligonucleotide microarrays from cultures grown in 5mM versus 25mM KCl confirms that depolarization reduces CB1 mRNA, but not mRNAs encoding several G-protein subunits or adenylyl cyclases. However, significant alterations in synaptic signaling proteins that likely lie downstream of CB1 are observed, including K+channels, α-neurexins, cAMP-GEFII, Munc13-3, secretogranin and synaptotagmin. These findings make a compelling argument to adopt cultures grown in 5mM KCl for future study of CB1 signaling in granule neurons. Further, they suggest that a depolarization and Ca2+-dependent signaling pathway represses CB1 gene transcription.