Bedecs K, Langel U, Bartfai T, Wiesenfeld-Hallin Z
Department of Biochemistry, Stockholm University, Sweden.
Acta Physiol Scand. 1992 Mar;144(3):213-20. doi: 10.1111/j.1748-1716.1992.tb09289.x.
The ligand binding properties of galanin receptors were examined in crude synaptosomal fraction preparations of lumbar dorsal spinal cord, using chloramin-T mono-iodinated porcine Tyr26 galanin as ligand. The equilibrium binding of [125I]galanin showed the presence of a single population of high-affinity binding sites with KD = 0.6 +/- 0.2 nM in a concentration of 55 +/- 15 fmol mg-1 protein (Bmax). The N-terminal fragments galanin (1-16) and galanin (1-12) fully displaced specific [125I]galanin binding from membranes with IC50 values 6 nM and 4 microM, respectively. The C-terminal fragment galanin (17-29) did not displace [125I]galanin when applied in the concentration range 10(-11)-10(-4) M. GTP inhibited the specific binding of [125I] galanin in a concentration dependent manner, with 54% inhibition at 1 mM, suggesting that the galanin receptor found in lumbar dorsal spinal cord is G-protein coupled. Second messenger systems, through which the galanin receptor in lumbar dorsal spinal cord may exert its effect, were also studied. A galanin (10 microM) produced inhibition (58%) of the depolarization induced cGMP increase was found, whereas galanin (10 microM) did not inhibit the noradrenalin (100 microM) activated cAMP synthesis or phosphoinositide turnover in tissue slices of the spinal cord. Bilateral transection of the sciatic nerve at midthigh level 14 days prior to the binding experiment was performed, a treatment which is known to cause a dramatic increase of galanin-like immunoreactivity in the superficial layers of the dorsal spinal cord, dorsal root ganglion and in galanin mRNA levels, but no significant effect on Bmax or KD of the galanin receptor was found.
使用氯胺 - T 单碘化猪 Tyr26 甘丙肽作为配体，在腰段背侧脊髓的粗制突触体部分制剂中检测甘丙肽受体的配体结合特性。[125I]甘丙肽的平衡结合显示存在单一群体的高亲和力结合位点，KD = 0.6±0.2 nM，浓度为 55±15 fmol mg-1 蛋白质（Bmax）。N 端片段甘丙肽（1 - 16）和甘丙肽（1 - 12）分别以 IC50 值 6 nM 和 4 μM 完全取代膜上特异性[125I]甘丙肽结合。C 端片段甘丙肽（17 - 29）在 10(-11)-10(-4) M 浓度范围内应用时不能取代[125I]甘丙肽。GTP 以浓度依赖性方式抑制[125I]甘丙肽的特异性结合，在 1 mM 时抑制率为 54%，表明在腰段背侧脊髓中发现的甘丙肽受体是 G 蛋白偶联的。还研究了腰段背侧脊髓中的甘丙肽受体可能发挥作用的第二信使系统。发现甘丙肽（10 μM）对去极化诱导的 cGMP 增加有抑制作用（58%），而甘丙肽（10 μM）不抑制脊髓组织切片中去甲肾上腺素（100 μM）激活的 cAMP 合成或磷酸肌醇周转。在结合实验前 14 天，在大腿中部水平对坐骨神经进行双侧横断，已知该处理会导致背侧脊髓浅层、背根神经节中甘丙肽样免疫反应性以及甘丙肽 mRNA 水平显著增加，但未发现对甘丙肽受体的 Bmax 或 KD 有显著影响。
