Demonstration of specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytes

Abstract

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 microM, 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 structurally 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.