Sanabria P, Silva W I
Department of Physiology, Universidad Central del Caribe, School of Medicine, Bayamón, Puerto Rico 00960-6032.
Microcirculation. 1994 Dec;1(4):267-73. doi: 10.3109/10739689409146753.
This study involved the pharmacological detection and characterization of binding sites for the neuromodulator neuropeptide Y (NPY) in an in vitro preparation of capillary endothelial cells derived from bovine adrenal medulla.
Equilibrium binding assays were conducted on intact cells with 125I Bolton-Hunter labeled NPY (125I-BH-NPY). The specificity of the high-affinity binding site was evaluated in competition experiments with cold NPY, (Leu31, Pro34)NPY (a Y1 receptor ligand, Y1RL), NPY13-36 (a Y2 receptor ligand, Y2RL), and two other members of the pancreatic polypeptide-fold (PP-fold) family: peptide YY (PYY) and avian pancreatic polypeptide (APP). Forskolin-stimulated adenylate cyclase activity was assessed to detect the participation of this second messenger pathway in the neuromodulator action at the studied cell preparation.
Nonlinear regression analysis of the binding data indicated the existence of high-affinity binding sites with an equilibrium dissociation constant (Kd) value of 39.00 +/- 12.84 nM and a maximal binding (Bmax) of 489.89 +/- 155.49 fmol/10(6) cells (mean +/- SE, n = 6). NPY, Y1RL, and PYY displayed a concentration that inhibits the specific binding by 50% IC50 (nM) values of 4.06 +/- 1.66 (n = 4), 2.94 +/- 0.75 (n = 5), and 18.36 +/- 10.36 (n = 3), respectively. APP and Y2RL were unable to compete with 125I-NPY in the concentration range 0.001-1 microM. Further evaluation of second messenger pathways suggested that NPY binding sites in this model are coupled to the inhibition of adenylate cyclase. NPY significantly inhibited the forskolin-stimulated adenosine cyclic 3',5'-(hydrogen phosphate) (cAMP) accumulation with a maximal effect of 37.03 +/- 6.28%, n = 5 and an IC50 of 5.96 +/- 1.87 nM. The Y1RL produced a comparable response (IC50 = 5.35 +/- 1.39 nM, n = 4; maximal inhibition of 61.05 +/- 13.03%) and Y2LR had no detectable effect at a similar concentration range.
The results demonstrate the existence of a Y1 receptor in the adrenal medulla capillary endothelial cells, which may be relevant to the postjunctional effect of NPY on this gland.
本研究涉及对牛肾上腺髓质来源的毛细血管内皮细胞体外制剂中神经调质神经肽Y（NPY）结合位点的药理学检测和特性分析。
用125I博尔顿-亨特标记的NPY（125I-BH-NPY）对完整细胞进行平衡结合试验。在与冷NPY、（亮氨酸31，脯氨酸34）NPY（一种Y1受体配体，Y1RL）、NPY13-36（一种Y2受体配体，Y2RL）以及胰多肽折叠（PP折叠）家族的其他两个成员：肽YY（PYY）和禽胰多肽（APP）的竞争实验中，评估高亲和力结合位点的特异性。评估福斯可林刺激的腺苷酸环化酶活性，以检测该第二信使途径在研究的细胞制剂中神经调质作用的参与情况。
结合数据的非线性回归分析表明存在高亲和力结合位点，其平衡解离常数（Kd）值为39.00±12.84 nM，最大结合量（Bmax）为489.89±155.49 fmol/10(6)个细胞（平均值±标准误，n = 6）。NPY、Y1RL和PYY分别显示出抑制特异性结合50%（IC50）时的浓度值（nM）为4.06±1.66（n = 4）、2.94±0.75（n = 5）和18.36±10.36（n = 3）。在0.001 - 1 microM浓度范围内，APP和Y2RL无法与125I-NPY竞争。对第二信使途径的进一步评估表明，该模型中的NPY结合位点与腺苷酸环化酶的抑制相关联。NPY显著抑制福斯可林刺激的腺苷环化3',5'-（磷酸氢）（cAMP）积累，最大效应为37.03±6.28%，n = 5，IC50为5.96±1.87 nM。Y1RL产生了类似的反应（IC50 = 5.35±1.39 nM，n = 4；最大抑制率为61.05±13.03%），而Y2LR在类似浓度范围内没有可检测到的作用。
结果表明肾上腺髓质毛细血管内皮细胞中存在Y1受体，这可能与NPY对该腺体的节后效应相关。
