Tóth Attila, Kedei Noémi, Wang Yun, Blumberg Peter M
Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bldg. 37, Rm. 4048, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-4255, USA.
Life Sci. 2003 Jun 13;73(4):487-98. doi: 10.1016/s0024-3205(03)00310-2.
The vanilloid receptor VR1 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner.
香草酸受体VR1是一种非特异性Ca(2+)通道，在外周神经系统的感觉神经元以及多个脑区中表达，被认为是多种引起疼痛的化学刺激（酸、香草酸类）和物理刺激（热）的重要分子整合器。最近，已鉴定出几种VR1的内源性配体，如花生四烯酸乙醇酰胺（花生四烯酸乙醇胺）和更强效的花生四烯酸多巴胺（AA-DO）。在此，我们进一步将AA-DO表征为在CHO和HEK293细胞中异源表达的大鼠VR1的配体。AA-DO抑制[3H]RTX与VR1的结合，K(d)值为5.49±0.68 microM，具有正协同性（p = 1.89±0.27），表明在该系统中AA-DO的效力比花生四烯酸乙醇胺高约5倍。AA-DO与脊髓膜结合的K(d)（9.7±3.3 microM）和p值（1.54±0.04）与CHO-VR1数据具有良好的相关性。AA-DO刺激CHO-VR1和HEK-VR1细胞摄取45Ca(2+)，EC(50)值分别为4.76±1.43和7.17±1.64 microM，希尔系数分别为1.28±0.11和1.13±0.13，与结合测量结果一致。与花生四烯酸乙醇胺不同，AA-DO诱导的45Ca(2+)摄取水平与辣椒素几乎相同（在表达VR1的CHO细胞中为90±6.6%，在表达VR1的HEK293细胞中为89.3±9.4%）。在激活后的时间依赖性方式下，AA-DO在45Ca(2+)摄取试验（IC(50) = 3.24±0.84 microM，抑制率为68.5±6.85%）以及使用CHO-VR1系统的Ca(2+)成像实验（抑制率为35.8±5.1%）中都使VR1部分脱敏。脱敏程度与辣椒素本身引起的相似。我们得出结论，AA-DO是VR1的完全激动剂，效力在低微摩尔范围内，并且能够以时间和剂量依赖性方式使细胞显著脱敏。
