Forman S A, Raines D E
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Boston 02114, USA.
Anesthesiology. 1998 Jun;88(6):1535-48. doi: 10.1097/00000542-199806000-00018.
Nonanesthetic volatile compounds fail to inhibit movement in response to noxious stimulation at concentrations predicted to induce anesthesia from their oil-water partitioning. Thus they represent tools to determine whether molecular models behave like the targets that mediate in vivo anesthetic actions. The effects of volatile anesthetics and nonanesthetics were examined in two experimental models in which anesthetics interact directly with proteins: the pore of the nicotinic acetylcholine receptor and human serum albumin.
Wild-type mouse muscle nicotinic receptors and receptors containing pore mutations (alphaS252I + betaT263I) were studied electrophysiologically in membrane patches from Xenopus oocytes. Patch currents evoked by brief pulses of acetylcholine were measured in the presence of enflurane and two nonanesthetics, 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane. Nonanesthetic interactions with human serum album were assessed by quenching of intrinsic protein fluorescence.
Both anesthetic and nonanesthetic volatile compounds inhibited wild-type and alphaS252I + betaT263I mutant nicotinic channels but displayed different selectivity for open versus resting receptor states. Median inhibitory concentrations (IC50s) in wild-type nicotinic receptors were 870+/-20 microM for enflurane, 37+/-3 microM for 1,2-dichlorohexafluorocylcobutane, and 11.3+/-5.6 microM for 2,3-dichlorooctafluorobutane. For all three drugs, ratios of wild-type IC50s to mutant IC50mut ranged from 7-10, and ratios of wild-type IC50s to predicted anesthetic median effective concentrations (EC50s) ranged from 1.8-2.3. 1,2-Dichlorohexafluorocyclobutane quenched human serum albumin with an apparent dissociation constant (Kd) of 160+/-11 microM. The ratios of dissociation constants to predicted EC50s for the nonanesthetics were within a factor of two of the dissociation constant:EC50 ratios calculated for halothane and chloroform from previous published results.
In two models in which anesthetics bind to protein sites, both anesthetic and nonanesthetic volatile drugs cause similar steady state effects with potencies that are predicted by hydrophobicity. These protein sites do not sterically discriminate between anesthetic and nonanesthetic drugs. However, differential state-selective actions on ion channel targets may underlie the distinct in vivo effects of anesthetics and nonanesthetics.
非麻醉性挥发性化合物在根据其油水分配系数预测可诱导麻醉的浓度下，无法抑制对有害刺激的反应性运动。因此，它们是用于确定分子模型是否表现得像介导体内麻醉作用的靶点的工具。在麻醉剂直接与蛋白质相互作用的两个实验模型中，研究了挥发性麻醉剂和非麻醉剂的作用：烟碱型乙酰胆碱受体的孔道和人血清白蛋白。
采用膜片钳技术，在非洲爪蟾卵母细胞的膜片上，对野生型小鼠肌肉烟碱型受体以及含有孔道突变（αS252I + βT263I）的受体进行电生理研究。在恩氟烷以及两种非麻醉剂1,2 - 二氯六氟环丁烷和2,3 - 二氯八氟丁烷存在的情况下，测量由乙酰胆碱短暂脉冲诱发的膜片电流。通过内在蛋白质荧光猝灭评估非麻醉剂与人血清白蛋白的相互作用。
麻醉性和非麻醉性挥发性化合物均抑制野生型和αS252I + βT263I突变型烟碱型通道，但对开放与静息受体状态表现出不同的选择性。在野生型烟碱型受体中，恩氟烷的半数抑制浓度（IC50）为870±20 μM，1,2 - 二氯六氟环丁烷为37±3 μM，2,3 - 二氯八氟丁烷为11.3±5.6 μM。对于所有这三种药物，野生型IC50与突变型IC50mut的比值范围为7 - 10，野生型IC50与预测的麻醉剂半数有效浓度（EC50）的比值范围为1.8 - 2.3。1,2 - 二氯六氟环丁烷猝灭人血清白蛋白的表观解离常数（Kd）为160±11 μM。非麻醉剂的解离常数与预测的EC50的比值，与根据先前发表结果计算的氟烷和氯仿的解离常数：EC50比值相差不超过两倍。
在麻醉剂与蛋白质位点结合的两个模型中，麻醉性和非麻醉性挥发性药物均产生相似的稳态效应，其效能可由疏水性预测。这些蛋白质位点在空间上不会区分麻醉剂和非麻醉剂药物。然而，对离子通道靶点的不同状态选择性作用可能是麻醉剂和非麻醉剂在体内产生不同效应的基础。
