Seabrook Guy R, Sutton Kathy G, Jarolimek Wolfgang, Hollingworth Gregory J, Teague Simon, Webb Janine, Clark Natalie, Boyce Susan, Kerby Julie, Ali Zahid, Chou Margaret, Middleton Richard, Kaczorowski Gregory, Jones A Brian
The Neuroscience Research Centre, Merck Sharp and Dohme, Harlow, Essex, United Kingdom.
J Pharmacol Exp Ther. 2002 Dec;303(3):1052-60. doi: 10.1124/jpet.102.040394.
We have synthesized iodinated resiniferatoxin bearing a 4-hydroxy-5-iodo-3-methoxyphenylacetate ester (I-RTX) and have characterized its activity on rat and human TRPV1 (VR1) receptors, as well as in behavioral assays of nociception. In whole cell patch-clamp recordings from transfected cells the functional activity of I-RTX was determined. Currents activated by capsaicin exhibited characteristic outward rectification and were antagonized by capsazepine and I-RTX. On rat TRPV1 the affinity of I-RTX was 800-fold higher than that of capsazepine (IC50 = 0.7 and 562 nM, respectively) and 10-fold higher on rat versus human receptors (IC50 = 0.7 and 5.4 nM, respectively). The same difference was observed when comparing the inhibition of [3H]RTX binding to rat and human TRPV1 membranes for both RTX and I-RTX. Additional pharmacological differences were revealed using protons as the stimulus. Under these conditions capsazepine only partly blocked currents through rat TRPV1 receptors (by 70 to 80% block), yet was a full antagonist on human receptors. In contrast, I-RTX completely blocked proton-induced currents in both species and that activated by noxious heat. I-RTX also blocked capsaicin-induced firing of C-fibers in a rat in vitro skin-nerve assay. Despite this activity and the high affinity of I-RTX for rat TRPV1, only capsazepine proved to be an effective antagonist of capsaicin-induced paw flinching in rats. Thus, although I-RTX has limited utility for in vivo behavioral studies it is a high-affinity TRPV1 receptor antagonist that will be useful to characterize the functional properties of cloned and native vanilloid receptor subtypes in vitro.
我们合成了带有4-羟基-5-碘-3-甲氧基苯乙酸酯的碘化树脂毒素（I-RTX），并对其在大鼠和人类TRPV1（VR1）受体上的活性以及伤害感受行为试验进行了表征。在转染细胞的全细胞膜片钳记录中测定了I-RTX的功能活性。辣椒素激活的电流表现出典型的外向整流特性，并被辣椒素拮抗剂和I-RTX拮抗。在大鼠TRPV1上，I-RTX的亲和力比辣椒素拮抗剂高800倍（IC50分别为0.7和562 nM），在大鼠受体上比人类受体高10倍（IC50分别为0.7和5.4 nM）。在比较RTX和I-RTX对大鼠和人类TRPV1膜上[3H]RTX结合的抑制作用时，也观察到了相同的差异。使用质子作为刺激物揭示了其他药理学差异。在这些条件下，辣椒素拮抗剂仅部分阻断通过大鼠TRPV1受体的电流（阻断70%至80%），但对人类受体是完全拮抗剂。相比之下，I-RTX完全阻断了两种物种中质子诱导的电流以及有害热激活的电流。在大鼠体外皮肤-神经试验中，I-RTX还阻断了辣椒素诱导的C纤维放电。尽管I-RTX具有这种活性且对大鼠TRPV1具有高亲和力，但只有辣椒素拮抗剂被证明是大鼠中辣椒素诱导的爪部退缩的有效拮抗剂。因此，尽管I-RTX在体内行为研究中的用途有限，但它是一种高亲和力的TRPV1受体拮抗剂，将有助于在体外表征克隆的和天然香草酸受体亚型的功能特性。
