Savinainen Juha R, Saario Susanna M, Niemi Riku, Järvinen Tomi, Laitinen Jarmo T
Department of Physiology, University of Kuopio, PO Box 1627, Kuopio FIN-70211, Finland.
Br J Pharmacol. 2003 Dec;140(8):1451-9. doi: 10.1038/sj.bjp.0705577. Epub 2003 Nov 17.
At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB1 receptor. In the micromolar range, these compounds were shown to inhibit basal G-protein activity, and this is often interpreted to implicate constitutive activity of the CB1 receptors in native tissue. We show here, using [35S]GTPgammaS binding techniques, that micromolar concentrations of SR141716 and AM251 inhibit basal G-protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A1 receptor signaling is not eliminated. Unlike lipophilic A1 receptor antagonists (potency order DPCPX>>N-0840 approximately cirsimarin>caffeine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 receptor-dependent G-protein activity. Importantly, all antagonists reduced basal signal to the same extent (20%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N-0840. These data indicate that rat brain A1 receptors are not constitutively active, but that an ADA-resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. SR141716 and AM251, at concentrations fully effective in reversing CB1-mediated responses (10-6 m), did not reduce basal G-protein activity, indicating that CB1 receptors are not constitutively active in these preparations.4 At higher concentrations (1-2.5 x 10-5 m), both antagonists reduced basal G-protein activity in control and ADA-treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right-shifted A1 agonist dose-response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. When further optimizing G-protein activation assay for the labile endocannabinoid 2-arachidonoylglycerol (2-AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of 2-AG and concomitantly enhanced the potency of 2-AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB1 receptor.6 The findings establish an optimized method with improved signal-to-noise ratio to assess endocannabinoid-dependent G-protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of 2-AG are fully eliminated.
在纳摩尔浓度下，SR141716和AM251作为大麻素CB1受体的特异性和选择性拮抗剂。在微摩尔范围内，这些化合物被证明可抑制基础G蛋白活性，这通常被解释为意味着CB1受体在天然组织中具有组成性活性。我们在此使用[35S]GTPγS结合技术表明，微摩尔浓度的SR141716和AM251可抑制大鼠小脑膜中的基础G蛋白活性，但仅在紧张性腺苷A1受体信号未被消除的条件下。与亲脂性A1受体拮抗剂（效价顺序为DPCPX>>N - 0840≈cirsimarin>咖啡因）不同，腺苷脱氨酶（ADA）不能完全消除基础A1受体依赖性G蛋白活性。重要的是，所有拮抗剂将基础信号降低到相同程度（20%），并且反向激动剂DPCPX引起的反应不会被中性拮抗剂N - 0840逆转。这些数据表明大鼠脑A1受体不是组成性激活的，而是一个ADA抗性腺苷池负责脑膜中的紧张性A1受体活性。SR141716和AM251在完全有效逆转CB1介导反应的浓度（10 - 6 m）下，不会降低基础G蛋白活性，表明CB1受体在这些制剂中不是组成性激活的。在更高浓度（1 - 2.5×10 - 5 m）下，两种拮抗剂均可降低对照和ADA处理膜中的基础G蛋白活性，但在用DPCPX阻断A1受体信号时则无作用。此外，CB1拮抗剂使A1激动剂剂量 - 反应曲线右移而不影响最大反应，表明拮抗剂作用的竞争模式。CB1拮抗剂不影响毒蕈碱型乙酰胆碱或GABAB受体信号传导。当进一步优化针对不稳定内源性大麻素2 - 花生四烯酸甘油酯（2 - AG）的G蛋白激活测定时，我们通过使用高效液相色谱法表明，用甲基花生四烯酰基氟磷酸酯（MAFP）预处理小脑膜可完全防止2 - AG的酶促降解，并同时增强2 - AG的效力。与先前的说法相反，MAFP在CB1受体上没有表现出拮抗剂活性。这些发现建立了一种具有改善信噪比的优化方法，用于在完全消除基础腺苷能张力和2 - AG酶促降解的测定条件下评估脑膜中内源性大麻素依赖性G蛋白活性。
