Thompson C L, Razzini G, Pollard S, Stephenson F A
Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, England.
J Neurochem. 2000 Mar;74(3):920-31. doi: 10.1046/j.1471-4159.2000.0740920.x.
Exposure of rat cerebellar granule cells cultured to maturity in vitro to forskolin, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP), and 3-isobutyl-1-methylxanthine (IBMX) down-regulated GABA(A) receptor alpha6 and beta3 subunits but up-regulated alpha1 and beta2 subunits with respect to vehicle-treated controls. Dideoxyforskolin had no effect on subunit expression. Protein kinase A inhibitors, H-89 and Rp-adenosine 3',5'cyclic monophosphothioate, prevented these effects on alpha1 but not alpha6 subunit expression. Flunitrazepam-sensitive [3H]Ro 15-4513 binding sites were increased by 144 +/- 20% following forskolin treatment. [3H]Ro 15-4513 photoaffinity labelling showed that the GABA(A) receptor alpha1 subunit was the principal locus of the increased flunitrazepam-sensitive [3H]Ro 15-4513 binding. Forskolin decreased flunitrazepam-insensitive [3H]Ro 15-4513 binding sites by 25 +/- 8% and resulted in a 20% decrease in the irreversible incorporation of radioactivity in the alpha6 subunit. Steady-state levels of GABA(A) receptor subunit mRNAs were determined by semiquantitative RT-PCR in forskolin-treated cultures. Forskolin, Bt2cAMP, and IBMX down-regulated GABA(A) receptor alpha6 subunit mRNA expression; alpha1 and beta3 mRNA levels were unaffected, whereas beta2 subunit mRNA was up-regulated. Dideoxyforskolin had no significant effect on alpha1, alpha6, beta2, and beta3 mRNA levels. Thus, in mature cerebellar granule cells, GABA(A) receptor expression can be regulated by intracellular cyclic AMP levels. This occurs at the level of gene transcription and/or translation by mechanisms that are only partially governed by protein kinase A.
将体外培养至成熟的大鼠小脑颗粒细胞暴露于福司可林、N6,2'-O-二丁酰腺苷 3',5'-环磷酸 (Bt2cAMP) 和 3-异丁基-1-甲基黄嘌呤 (IBMX) 中，相对于用载体处理的对照，GABA(A) 受体α6和β3亚基表达下调，但α1和β2亚基表达上调。双脱氧福司可林对亚基表达无影响。蛋白激酶A抑制剂H-89和Rp-腺苷3',5'-环磷酸硫代酯可阻止这些对α1亚基表达的影响，但不能阻止对α6亚基表达的影响。福司可林处理后，氟硝西泮敏感的[3H]Ro 15-4513结合位点增加了144±20%。[3H]Ro 15-4513光亲和标记显示，GABA(A) 受体α1亚基是氟硝西泮敏感的[3H]Ro 15-4513结合增加的主要位点。福司可林使氟硝西泮不敏感的[3H]Ro 15-4513结合位点减少25±8%，并导致α6亚基中放射性的不可逆掺入减少20%。通过半定量RT-PCR测定福司可林处理的培养物中GABA(A) 受体亚基mRNA的稳态水平。福司可林、Bt2cAMP和IBMX下调GABA(A) 受体α6亚基mRNA表达；α1和β3 mRNA水平未受影响，而β2亚基mRNA上调。双脱氧福司可林对α1、α6、β2和β3 mRNA水平无显著影响。因此，在成熟的小脑颗粒细胞中，GABA(A) 受体表达可受细胞内环磷酸腺苷水平的调节。这发生在基因转录和/或翻译水平，其机制仅部分受蛋白激酶A控制。
