Tóth Fanni, Horváth Gyöngyi, Szikszay Margit, Farkas Judit, Tóth Géza, Borsodi Anna, Benyhe Sándor
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary.
Regul Pept. 2004 Oct 15;122(2):139-46. doi: 10.1016/j.regpep.2004.06.017.
Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [(35)S]GTPgammaS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [(3)H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing delta-, kappa-or micro-opioid receptors. The highest affinity for [(3)H]DADN binding was observed in membranes from CHO cells transfected with micro-opioid receptors confirming the micro-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of micro-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the delta-selective naltrindole (NTI) and the kappa-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [(35)S]GTPgammaS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.
酪氨酰-D-丙氨酰-甘氨酰-苯丙氨酰-D-亮氨酰-精氨酰-苯丙氨酸（DADN）是内源性甲硫氨酸脑啡肽-精氨酰-苯丙氨酸（酪氨酰-甘氨酰-甘氨酰-苯丙氨酰-甲硫氨酸-精氨酰-苯丙氨酸；MERF）的合成类似物，在放射性配体结合试验、[³⁵S]GTPγS刺激实验以及体内痛觉测定试验中进行了研究。在大鼠脊髓组织的粗膜部分以及选择性表达δ、κ或μ阿片受体的中国仓鼠卵巢（CHO）细胞匀浆中测量了[³H]DADN的结合特性。在用μ阿片受体转染的CHO细胞膜中观察到[³H]DADN结合的最高亲和力，证实了该肽的μ选择性。还通过测量大鼠脑膜部分中阿片受体介导的G蛋白激活，在功能性生化实验中研究了未标记的DADN。该肽以浓度依赖性方式刺激调节性G蛋白的活性，并且在存在μ受体特异性拮抗剂配体的情况下刺激被有效抑制，进一步支持了DADN的选择性特征。在大鼠热水甩尾试验中，鞘内注射DADN产生剂量相关的、纳洛酮可逆的镇痛作用。在所测试的选择性阿片拮抗剂中，与纳洛酮相比，δ选择性纳曲吲哚（NTI）和κ特异性去甲二氢吗啡酮（norBNI）仅显示出轻微的阻断作用。在体外激动剂刺激的[³⁵S]GTPγS结合试验中获得的结果与体内疼痛试验中观察到的阿片激动剂效应高度一致。
