Szallasi A, Acs G, Cravotto G, Blumberg P M, Lundberg J M, Appendino G
Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden.
Eur J Pharmacol. 1996 Mar 28;299(1-3):221-8. doi: 10.1016/0014-2999(95)00864-0.
Capsaicin binds to a specific recognition site, referred to as the vanilloid receptor, which it shares with the natural, ultrapotent agonist resiniferatoxin and with the competitive antagonist capsazepine. Upon binding to its receptor, capsaicin opens a cation channel leading to Ca2+ influx. The binding of capsaicin or resiniferatoxin by the vanilloid receptor follows a sigmoidal saturation curve, indicative of positive cooperativity. The biological significance of this positive cooperative behaviour is unknown, as is the mechanism responsible for it. We have developed a novel ligand, phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), which binds to cultured rat sensory neurons (with a Ki of 3.1 +/- 0.4 microM), and induces Ca2+ uptake by them (with an ED50 of 1.8 +/- 0.3 microM) with similar affinities and in a non-cooperative manner (Hill coefficients are 0.99 and 1.06 for binding and Ca2+ uptake, respectively). The behaviour of PPAHV thus contrasts with resiniferatoxin or capsaicin not only in the lack of cooperativity but also in the relative potencies for resiniferatoxin binding versus Ca2+ uptake (resiniferatoxin is less potent and capsaicin is more potent for induction of Ca2+ uptake than for binding). In further experiments in which the concentration of [3H]resiniferatoxin was varied, 1 microM PPAHV likewise reduced the cooperativity index that characterizes resiniferatoxin binding to rat spinal cord membranes from 2.3 +/- 0.1 to 1.1 +/- 0.2; in parallel experiments, neither capsaicin nor capsazepine (both at a concentration of 2 microM) affected binding cooperativity. Moreover, PPAHV (1 microM) turned the bi-phasic dissociation curve of resiniferatoxin into a monophasic curve, eliminating the second, slow-dissociation phase. The present results suggest that positive cooperativity is a ligand-induced feature rather than an inherent property of vanilloid receptors. A comparison of the spectrum of biological activity of ligands which bind to vanilloid receptors with different degrees of cooperativity may provide an approach to explore the functional significance of this binding behaviour.
辣椒素与一个特定的识别位点结合，该位点被称为香草酸受体，它与天然的超高效激动剂树脂毒素以及竞争性拮抗剂辣椒平共享。与受体结合后，辣椒素会打开一个阳离子通道，导致钙离子内流。香草酸受体对辣椒素或树脂毒素的结合遵循S形饱和曲线，表明存在正协同性。这种正协同行为的生物学意义尚不清楚，其产生机制也未知。我们开发了一种新型配体，佛波醇12-苯乙酸酯13-乙酸酯20-高香草酸酯（PPAHV），它与培养的大鼠感觉神经元结合（解离常数Ki为3.1±0.4微摩尔），并以相似的亲和力且以非协同方式诱导这些神经元摄取钙离子（半数有效浓度ED50为1.8±0.3微摩尔）（结合和钙离子摄取的希尔系数分别为0.99和1.06）。因此，PPAHV的行为不仅在缺乏协同性方面，而且在树脂毒素结合与钙离子摄取的相对效力方面（树脂毒素诱导钙离子摄取的效力低于其结合效力，而辣椒素诱导钙离子摄取的效力高于其结合效力）与树脂毒素或辣椒素形成对比。在进一步的实验中，改变[3H]树脂毒素的浓度，1微摩尔的PPAHV同样将表征树脂毒素与大鼠脊髓膜结合的协同指数从2.3±0.1降低到1.1±0.2；在平行实验中，辣椒素和辣椒平（浓度均为2微摩尔）均不影响结合协同性。此外，PPAHV（1微摩尔）将树脂毒素的双相解离曲线转变为单相曲线，消除了第二个缓慢解离阶段。目前的结果表明，正协同性是一种配体诱导的特征，而非香草酸受体的固有属性。比较以不同协同程度与香草酸受体结合的配体的生物活性谱，可能为探索这种结合行为的功能意义提供一种方法。
