CYCLIC LACTAM ANALOGS OF OVINE PITUITARY ADENYLATE-CYCLASE ACTIVATING POLYPEPTIDE (PACAP) - DISCOVERY OF POTENT TYPE-II RECEPTOR ANTAGONISTS

Binding of [I-125]PACAP-38 to rat liver membranes was investigated. It was rapid at 37 degrees C, reversible, and saturable, and it was time, concentration, and temperature dependent. Scatchard plots showed that [I-125]PACAP-38 bound to single noninteracting site(s), and [I-125]VIP bound to high- and low-affinity binding site(s). The order of potency of displacing [I-125]PACAP-38 from rat liver membranes was: PACAP-38 > PACAP-27 > VIP (IC50 = 5, 180, and 350 nM, respectively). Surprisingly, the order of potency of displacing [I-125]VIP was also the same (IC50 = 1, 8, and 52 nM, 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 nM, 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-terminal 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 receptors, and that the PACAP receptor(s) is probably similar, but not identical, to type I receptor(s) characteristic of the brain.