Linden Anni-Maija, Schmitt Ulrich, Leppä Elli, Wulff Peer, Wisden William, Lüddens Hartmut, Korpi Esa R
Pharmacology, Institute of Biomedicine, University of Helsinki Helsinki, Finland.
Front Neurosci. 2011 Jan 20;5:3. doi: 10.3389/fnins.2011.00003. eCollection 2011.
Ethyl alcohol (ethanol) has many molecular targets in the nervous system, its potency at these sites being low compared to those of sedative drugs. This has made it difficult to discover ethanol's binding site(s). There are two putative binding sites at γ-aminobutyric acid (GABA) type A receptor subtypes for the proposed ethanol antagonist Ro 15-4513, the established γ2 subunit-dependent benzodiazepine site and the recently reported δ subunit-dependent Ro 15-4513/ethanol binding site. Here, we aimed at clarifying the in vivo role of Ro 15-4513 at these two sites. We found that the antagonism of ethanol actions by Ro 15-4513 in wildtype mice was dependent on the test: an open field test showed that light sedation induced by 1.5-1.8 g/kg ethanol was sensitive to Ro 15-4513, whereas several tests for ethanol-induced anxiolytic effects showed that the ethanol-induced effects were insensitive to Ro 15-4513. Antagonism of ethanol-induced sedation by Ro 15-4513 was unaffected in GABA(A) receptor δ subunit knockout mice. By contrast, when testing the GABA(A) receptor γ2 subunit F77I knock-in mouse line (γ2I77 mice) with its strongly reduced affinity of the benzodiazepine sites for Ro 15-4513, we found that the ethanol-induced sedation was no longer antagonized by Ro 15-4513. Indeed, γ2I77 mice had only a small proportion of high-affinity binding of [(3)H]Ro 15-4513 left as compared to wildtype mice, especially in the caudate-putamen and septal areas, but these residual sites are apparently not involved in ethanol antagonism. In conclusion, we found that Ro 15-4513 abolished the sedative effect of ethanol by an action on γ2 subunit-dependent benzodiazepine sites.
乙醇在神经系统中有许多分子靶点，与镇静药物相比，其在这些靶点的效力较低。这使得发现乙醇的结合位点变得困难。对于拟用的乙醇拮抗剂Ro 15-4513，在γ-氨基丁酸（GABA）A型受体亚型上有两个假定的结合位点，即已确定的γ2亚基依赖性苯二氮䓬位点和最近报道的δ亚基依赖性Ro 15-4513/乙醇结合位点。在此，我们旨在阐明Ro 15-4513在这两个位点的体内作用。我们发现，Ro 15-4513对野生型小鼠乙醇作用的拮抗作用取决于测试：旷场试验表明，1.5-1.8 g/kg乙醇诱导的轻度镇静对Ro 15-4513敏感，而几项乙醇诱导的抗焦虑作用测试表明，乙醇诱导的效应对Ro 15-4513不敏感。Ro 15-4513对乙醇诱导的镇静作用的拮抗在GABA(A)受体δ亚基敲除小鼠中不受影响。相比之下，当测试GABA(A)受体γ2亚基F77I敲入小鼠品系（γ2I77小鼠）时，其苯二氮䓬位点对Ro 15-4513的亲和力大幅降低，我们发现Ro 15-4513不再拮抗乙醇诱导的镇静作用。事实上，与野生型小鼠相比，γ2I77小鼠仅剩下一小部分[(3)H]Ro 15-4513的高亲和力结合，尤其是在尾状核-壳核和隔区，但这些残留位点显然不参与乙醇拮抗作用。总之，我们发现Ro 15-4513通过作用于γ2亚基依赖性苯二氮䓬位点消除了乙醇的镇静作用。
