NEUROANATOMICAL AND PHYSIOLOGICAL EVIDENCE FOR THE INVOLVEMENT OF PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE IN THE REGULATION OF THE DISTAL LOBE OF THE FROG PITUITARY

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 ami no-acid peptide which belongs to the glucagon/secretin/vasoactive inte stinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino-a cid from mammalian PACAP. The aim of the present study was to investig ate the presence of PACAP in the hypothalamo-pituitary complex of the frog Rana ridibunda and to determine the biological activity of frog P ACAP on homologous pituitary cells. The distribution of PACAP-containi ng neurons and fibers was examined by the indirect immunofluorescence method using an antiserum raised against the N-terminal region of the peptide. In the hypothalamus, PACAP-immunoreactive perikarya were loca lized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral in fundibular nucleus to the external vascular layer of the median eminen ce. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointerme diate lobe and the distal lobe of the pituitary were devoid of immunor eactive elements. The amount of PACAP-like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent c oncentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 an d PACAP27 induced a similar dose-dependent stimulation of cAMP product ion in isolated frog distal lobe pituitary fragments (ED50 = 2 x 10(-8 ) M). At the maximum dose tested (5 x 10(-6) M), both frog PACAP38 and PACAP27 produced a 4-fold increase in cAMP production. In contrast, t he truncated form [Des-His1]frog PACAP38 did not affect adenylate cycl ase activity demonstrating therefore that the N-terminal histidyl resi due is essential for the biological activity of the peptide. [Des-His1 ]frog PACAP38 did not antagonize the stimulatory effect of frog PACAP3 8 or PACAP27 on cAMP production. Taken together, these data support th e concept that, in amphibians as in mammals, PACAP may act as a hypoph ysiotropic neuropeptide.