Characterization of Pituitary Adenylate Cyclase‐Activating Polypeptide 38 (PACAP38)‐, PACAP27‐, and Vasoactive Intestinal Peptide‐Stimulated Responses in N1E‐115 Neuroblastoma Cells

Abstract

In this study, the effects of three related peptides, pituitary adenylate cyclase‐activating polypeptide 38 (PACAP38), PACAP27, and vasoactive intestinal peptide (VIP), on cyclic AMP (cAMP) accumulation and intracellular Ca2+ concentration ([Ca2+]i) were compared in N1E‐115 cells. PACAP38 and PACAP27 stimulated cAMP accumulation up to 60‐fold with EC50 values of 0.54 and 0.067 nM, respectively. The effect of VIP on cAMP accumulation was less potent. The binding of 125I‐PACAP27 to intact cells was inhibited by PACAP38 and PACAP27 (IC50 values of 0.44 and 0.55 nM, respectively) but not by VIP. In fura‐2‐loaded cells, both PACAP38 and PACAP27 increased [Ca2+]i with EC50 values around 10 nM. The interactions of these three peptides with ionomycin, a Ca2+ ionophore, and 4β‐phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C, were also determined. Ionomycin increased the cAMP accumulation caused by all three peptides. With low concentrations of PACAP38 or PACAP27, the effect of PMA was inhibitory, whereas at higher concentrations of PACAP (>1 nM), the effect of PMA was stimulatory. Similar to other agents that elevate cAMP, PACAP38 was an effective stimulator of neurite outgrowth. These results show that (a) PACAP27 and PACAP38 stimulate cAMP accumulation and increase [Ca2+]i through the type I PACAP receptors in N1E‐115 cells, (b) ionomycin enhances cAMP accumulation by all three peptides, and (c) activation of protein kinase C has a dose‐dependent stimulatory or inhibitory effect on the PACAP38‐ or PACAP27‐stimulated cAMP accumulation.

PACAP Action in Nervous System Development, Regeneration, and Neuroblastoma Cell Proliferation

“…In 1994, Arimura and colleagues determined that PACAP immunoreactivity and PACAP‐preferring (PAC 1 ) binding sites could be detected in extracts of the developing rat brain on embryonic day (E) 14, a time of peak neurogenesis 1. Others had shown that PACAP can regulate the proliferation and differentiation of neuroblastoma cells in vitro 2–4. More recently, DiCicco‐Bloom and colleagues showed that cultured cortical neuroblasts isolated from the rat at E 13.5 respond to PACAP with decreased DNA synthesis, enhanced neurite outgrowth, and increased trkB expression 5.…”