INTERACTION OF OVINE PITUITARY ADENYLATE CYCLASE-ACTIVATING PEPTIDE (PACAP-38) WITH RAT LUNG MEMBRANES

The binding of ovine pituitary adenylate cyclase-activating peptide (PACAP-38) to rat lung membranes was investigated using [I-125]PACAP-38 as radioligand. Binding was rapid at 37-degrees-C, reversible, saturable, and time, concentration, and temperature dependent. Kinetic parameters derived from saturation experiments revealed a K(d) = 100 +/- 15 pM, B(max) = 310 +/- 36 fmol/mg protein, and a Hill slope factor (n(H)) of 1.17 +/- 0.12. Various chemically synthesized analogues of PACAP-38, as well as related peptides, were tested for their ability to displace [I-125]PACAP-38. Of those that had an IC50 < 0.2 muM, the following order of potency was determined: PACAP-38 (IC50 = 25 nM) greater-than-or-equal-to [Ile2]PACAP-38 (IC50 = 31 nM) > PACAP-27 (IC50 = 54 nM) > [Tyr1]PACAP-38 (IC50 = 104 nM) > GHRH(1-29)NH2 (IC50 = 108 nM) > PHI (IC50 = 181 nM) > [Ser2]PACAP(2-38) (IC50 = 198 nM). Glucagon, PHM, secretin, and GIP exhibited little affinity in the same binding assay. Vasoactive intestinal peptide (VIP) had an IC50 in excess of 1 muM. When [I-125]VIP was used as radioligand, PACAP-27 had an IC50 = 0.2 nM > PACAP-38 (IC50 = 0.5 nM) > VIP (IC50 = 16 nM). A novel analog of PACAP-38, [4-Cl-D-Phe6,Leu17]PACAP-38, was able to displace [I-125]-VIP very efficiently (IC50 = 1 nM), but had little potency in displacing [I-125]PACAP-38 (IC50 = 320 nM). The results presented suggest that PACAP-38 binds to single, noninteracting binding sites on rat lung membranes that are distinct from those for VIP, but could be shared with PACAP-27; PACAP-38 can also efficiently bind to the VIP receptor(s), but VIP has much less potency toward the PACAP-38 receptor. Moreover, modification of the PACAP-38 molecule, either through chain length or amino acid residue substitutions, results in apparent decreased binding.