Multiple actions of a hybrid PACAP antagonist: Neuronal cell killing and inhibition of sperm motility

Pituitary stimulating adenylate cyclase (PACAP) is a major regulatory peptide with two active molecular forms: PACAP-27 and PACAP-38. Both molecular forms promote neuronal survival and protect against neurotoxicity. Based on our previous hybrid peptide strategy in designing vasoactive intestinal peptide (VIP) antagonists, novel PACAP analogues were synthesized (neurotensin_6-11 PACAP_7-27 and neurotensin_6-11 PACAP_7-38. In addition to the hybrid modification, the methionine in position 17 was replaced by norleucine (Nle). Treatment of rat cerebral cortical cultures for five days with the putative PACAP antagonists (1 nM) resulted in a 35-45% reduction in neuronal cell counts as compared to controls. Neuronal cell death was already obtained at picomolar concentrations for the neurotensin_6-11PACAP_7-27 antagonist with 70% death at 10^-8 M. Co-administration of the PACAP hybrid analogue with picomolar amounts of PACAP-27 or Nle¹⁷-PACAP-27 attenuated the reduction in neuronal cell counts. While the protective effects of both analogues exhibited a peak at 1 pM concentrations, the Nle-containing agonist displayed a broader range of active concentrations (10^-12 M-10^-9 M). The putative PACAP antagonist also inhibited sperm motility (golden hamster) in a dose-dependent manner as assessed in vitro. Complete inhibition was observed at 10 μM, suggesting a role for PACAP in sperm motility and sexual function. Thus, previous findings of a large number of PACAP and PACAP receptors in the nervous system and the reproductive system are now correlated with a function in neuronal survival and sperm motility. The structure-activity studies suggest that the methionine in position 17 and the first six amino acids are important in the determination of PACAP activity, knowledge that may facilitate PACAP-based drug design.