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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.