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

Abstract

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: $\text{PACAP-38 > PACAP-27 > VIP}$ ($\text{IC}{}_{50}\text{= 5}$, 180, and 350 nM, respectively). Surprisingly, the order of potency of displacing [¹²⁵I]VIP was also the same ($\text{IC}{}_{50}\text{= 1}$, 8, and 52 nM, respectively). The order of potency of stimulating adenylate cyclase to release cyclic AMP was: $\text{PACAP-27 > VIP>PACAP-38}$ ($\text{EC}{}_{50}\text{= 0.06}$, 1, and 6 nM, respectively). Modification of PACAP-27 or PACAP-38 structures either through deletions, substitutions, or cyclization involving amino acid residues, Asp³, Asp⁸, Lys¹⁵, Lys²⁰, or Lys²¹ indicated that the $\text{N-}\text{termial}$ region of the molecule is important for both binding and transduction. Of the various lactam analogues synthesized, cyclo[Asp³,Lys¹⁵]PACAP-38 and cyclo[Asp⁸,Lys¹⁵]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.