DEMONSTRATION OF SPECIFIC BINDING-SITES FOR PITUITARY ADENYLATE-CYCLASE ACTIVATING POLYPEPTIDE (PACAP) IN RAT ASTROCYTES

The high and low affinity binding sites for PACAP were identified in rat astrocytes using [125I]PACAP27 as the labeled ligand. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites, with the dissociation constant (Kd) = 1.22±0.4 nM, the binding maximal capacity (Bmax) = 821±218 fmols/mg protein for the high affinity binding site, and Kd=0.59±0.06 μM, Bmax=563±12 pmols/mg protein for the low affinity binding site, respectively. The specificity of [125I]PACAP27 binding was tested using PACAP38 and peptides structually related to PACAP, such as VIP, GHRF, PHI, secretin and glucagon. PACAP38 completely displaced the binding of [125I]PACAP27 and Scatchard analysis also indicated the presence of two classes of binding sites with similar Kd and Bmax to those for PACAP27. VIP and GHRF competed with [125I]PACAP27, but to a much lesser extent than unlabeled PACAP27 in binding. Other peptides tested did not displace the binding of [125I]PACAP27 at 10-6 M. © 1990.