Cyclic lactam analogues of ovine pituitary adenylate cyclase activating polypeptide (PACAP): Discovery of potent type II receptor antagonists

Binding of [ 125I]PACAP-38 to rat liver membranes was investigated. It was rapid at 37°C, reversible, and saturable, and it was time, concentration, and temperature dependent. Scatchard plots showed that [ 125I]PACAP-38 bound to single noninteracting site(s), and [ 125I]VIP bound to high- and low-affinity binding site(s). The order of potency of displacing [ 125I]PACAP-38 from rat liver membranes was: PACAP-38 > PACAP-27 > VIP ( IC 50 = 5 , 180, and 350 n M, respectively). Surprisingly, the order of potency of displacing [ 125I]VIP was also the same ( IC 50 = 1 , 8, and 52 n M, respectively). The order of potency of stimulating adenylate cyclase to release cyclic AMP was: PACAP-27 > VIP>PACAP-38 ( EC 50 = 0.06 , 1, and 6 n M, respectively). Modification of PACAP-27 or PACAP-38 structures either through deletions, substitutions, or cyclization involving amino acid residues, Asp 3, Asp 8, Lys 15, Lys 20, or Lys 21 indicated that the N- termial region of the molecule is important for both binding and transduction. Of the various lactam analogues synthesized, cyclo[Asp 3,Lys 15]PACAP-38 and cyclo[Asp 8,Lys 15]PACAP-38 appear to be competitive receptor antagonists of the release of cAMP by PACAP-38. The results presented suggest that liver membranes possess distinct PACAP and VIP recceptors, and that the PACAP receptor(s) is probably similar, but not identical, to type I receptor(s) characteristic of the brain.