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 degrees C was characterized by a single class of binding site for the brain membrane with a dissociation constant (Kd) of 2.46 +/- 0.53 nM and a maximal binding capacity (Bmax) of 8.44 +/- 3.13 pmol/mg protein, but there were two classes of binding site for lung membranes with Kd of 1.02 +/- 0.51 nM and 5.19 +/- 0.99 nM, and Bmax 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.