Binding sites of a novel neuropeptide pituitary‐adenylate‐cyclase‐activating polypeptide in the rat brain and lung

Pituitary‐adenylate‐cyclase‐activating polypeptide (PACAP) is a novel 38‐amino‐acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27‐amino‐acid N‐terminal derivative of PACAP [PACAP(1–27)] labelled with ¹²⁵I. Particularly high specific binding sites of ¹²⁵I‐PACAP(1–27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of ¹²⁵I‐PACAP(1–27)‐binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of ¹²⁵I‐PACAP(1–27) was observed at pH 7.4. Interaction of ¹²⁵I‐PACAP(1–27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1–27) is more potent than PACAP in displacing the binding of ¹²⁵I‐PACAP(1–27) with brain membranes [concentration that inhibits 50% of the binding (IC₅₀) = 7.45 ± 1.52 nM and 11.45 ± 3.65 nM, respectively); mean ± SEM. n= 4] and lung membranes (IC₅₀= 4.41 ± 0.87 nM and 10.68 ± 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of ¹²⁵I‐PACAP(1–27) in lung membrane (IC₅₀= 16.88 ± 5.14 nM) but not in brain membranes. The equilibrium binding of ¹²⁵I‐PACAP(1–27) at 4°C was characterized by a single class of binding site for the brain membrane with a dissciation constant (K_d) of 2.46 ± 0.53 nM and a maximal binding capacity (B_max) of 8.44 ± 3.13 pmol/mg protein, but there were two classes of binding site for lung menbranes with K_d of 1.02 ± 0.51 nM and 5.19 ± 0.99 nM, and B_max of 2.84 ± 0.72 pmol/mg protein and 9.13 ± 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP‐binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung.