Specific 125I Neuropeptide Y Binding to Intact Cultured Bovine Adrenal Medulla Capillary Endothelial Cells

Abstract

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.