Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, in the ovine hypothalamus.

We recently reported isolation, characterization and synthesis of a novel ovine hypothalamic peptide with 38 residues which stimulates accumulation of cAMP in rat anterior pituitary cell cultures. The peptide was named PACAP38 (pituitary adenylate cyclase-activating polypeptide with 38 residues). The presence of another peptide corresponding to the N-terminal 1-27 residues (PACAP27) was also demonstrated. Both PACAP38 and PACAP27 have an amidated C-terminus. Antisera against synthetic PACAP27 were generated in rabbits. These antisera were tested for titer and specificity in enzyme-linked immunosorbent assay. One of the antisera (no. 88121-3) exhibited a high titer of antibody, which was specific to PACAP27 and PACAP38 with exception of slight cross-reactivity with ovine CRF (oCRF). Therefore, the antibodies against oCRF were removed from the antiserum using a solid phase method. Removal of oCRF antibodies was confirmed by enzyme-linked immunosorbent assay. A dense immunoreactive fiber network was found in both external and internal zones of the median eminence and pituitary stalk. The fibers were demonstrated to be in close contact with the hypophysial portal capillaries. The preabsorption of antiserum with vasoactive intestinal polypeptide or with the mixture containing TRH, LHRH, oCRF, ovine GH-releasing factor, somatostatin, and bovine thyroglobulin did not affect the immunostaining. On the other hand, the preabsorption of antiserum with an excess of PACAP27 or PACAP38 abolished the immunostaining. Therefore, the staining is considered specific for PACAP27 and PACAP38. Stained fibers were also present in the posterior pituitary. A dense fiber network was observed and the lateral hypothalamus the fibers appeared to cling to unstained neuronal cell bodies and their dendrites. In the lateral septum the fibers surrounded some blood vessels. Immunolabeled cell bodies were found in the paraventricular and supraoptic nuclei. These findings support the view that PACAP may play a multifunctional role, including that of a hypophysiotropic hormone, neurotransmitter, neuromodulator, and vasoregulator.