HYPOPHYSIOTROPIC ACTION OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) IN THE GOLDFISH - IMMUNOHISTOCHEMICAL DEMONSTRATION OF PACAP IN THE PITUITARY, PACAP STIMULATION OF GROWTH-HORMONE RELEASE FROM PITUITARY-CELLS, AND MOLECULAR-CLONING OF PITUITARY TYPE-I PACAP RECEPTOR

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon/secretin peptide family, and its molecular structure is highly conserved in vertebrates. In this study, the functional role of PACAP in regulating GH release in the goldfish was investigated. U sing immunohistochemical staining, nerve fibers with PACAP immunoreact ivity were identified in the vicinity of goldfish somatotrophs, sugges ting that this neuropeptide may influence GH release in the goldfish. The direct regulatory action of PACAP on GH secretion was demonstrated in vitro in perifused goldfish pituitary cells. PACAPs (0.01 nM to 1 mu M) from different species, including ovine PACAP(27), ovine PACAP(3 8), frog PACAP(38), zebra fish PACAP(27), and zebra fish PACAP(38), we re all effective in stimulating GH release with ED50 values of 8.9 +/- 3.5, 3.3 +/- 1.6, 14.4 +/- 3.5, 15.4 +/- 4.1, and 1.4 +/- 0.2 nM, res pectively. Similar concentrations of vasoactive intestinal polypeptide (VIP), a peptide related to PACAP, was not effective in this respect. In addition, the GH-releasing action of ovine PACAP(38) (10 nM) was i nhibited by the PACAP antagonist PACAP(6-39) (10 mu M), but not by the VIP antagonist [4-Cl-D-Phe(6),Leu(17)]VIP (10 mu M). The pharmacology of these GH responses is consistent with the mammalian type I PACAP r eceptors, suggesting that a similar receptor subtype is present in the goldfish pituitary and mediates the GH-releasing action of PACAP. To establish the structural identity of this goldfish PACAP receptor, a c omplementary DNA (cDNA) clone sharing a high degree of sequence homolo gy with mammalian type I PACAP receptors was isolated from a goldfish pituitary cDNA library. This cDNA was 5.2 kb in size with a 1.4-kb ope n reading frame and encoded a 465-amino acid protein with the typical structure of a 7-transmembrane domain-containing, G protein-coupled re ceptor. Functional expression of this cDNA in COS-7 cells revealed tha t this fish type I PACAP receptor could be activated by ovine PACAP(27 ) and PACAP(38) to increase cAMP synthesis with ED50 values of 2.4 +/- 0.8 and 4.2 +/- 1.2 nM, respectively. Other structurally related pept ides, including VIP (100 nM), GH-releasing hormone (100 nM), glucagon (100 nM), secretin (100 nM), gastric inhibitory polypeptide (100 nM), and PTH (100 nM), were not effective in altering cAMP production. Usin g Northern blot and RT-PCR, messenger RNA transcripts of this PACAP re ceptor were identified in the brain, heart, and pituitary of the goldf ish. These results, taken together, support the hypothesis that PACAP functions as a novel OH-releasing factor in the goldfish through activ ation of type I PACAP receptors.