LOCATIONS AND MOLECULAR-FORMS OF PACAP AND SITES AND CHARACTERISTICS OF PACAP RECEPTORS IN CANINE ILEUM

In canine ileum we investigated the distribution of pituitary adenylat e cyclase-activating peptide (PACAP), using immunofluorescence and rad ioimmunoassay and the binding of I-125-PACAP-27 to membranes. Nerve pr ofiles immunoreactive to PACAP-27, and often to vasoactive intestinal polypeptide (VIP) as well, were found in all plexi, but PACAP was pres ent in similar to 100-fold lesser amounts than VIP. High-performance l iquid chromatography analysis of deep muscular plexus (DMP) synaptosom es suggested the presence of PACAP-38, PACAP-27, and a third unidentif ied molecular form. High- and low-affinity I-125-PACAP-27 binding site s were found in DMP synaptosomes and circular smooth muscle (CMI plasm a membranes. In competition studies with DMP membranes, high (H)- and low (L)-affinity dissociation constants (K-d) and maximal binding capa cities (B-max) were as follows: K-dH = 66.9 pM, B-maxH = 101 fmol/mg; K-dL = 2.18 nM, B-maxL = 580 fmol/mg protein. The binding of I-125-PAC AP-27 was fast. Dissociation was slow and incomplete in the pres ence of unlabeled PACAP-27 but accelerated by pretreatment with guanosine 5 '-O-(3-thiotriphosphate) (GTP gamma S). GTP gamma S or cholera toxin t reatment eliminated high-affinity binding in both membranes. VIP had s imilar to 100-fold lower affinity than PACAP-27 in both membranes. Cro ss-linking studies identified an similar to 70-kDa PACAP receptor in e ach membrane. Thus PACAP coexists with VIP in ileal enteric nerves and acts on PACAP-preferring, possibly G(s)-coupled, receptors in DMP syn aptosomes and CM membranes.