Autocrine Expression and Ontogenetic Functions of the PACAP Ligand/Receptor System during Sympathetic Development

Abstract

The superior cervical ganglion (SCG) is a well-characterized model of neural development, in which several regulatory signals have been identified. Vasoactive intestinal peptide (VIP) has been found to regulate diverse ontogenetic processes in sympathetics, though functional requirements for high peptide concentrations suggest that other ligands are involved. We now describe expression and functions of pituitary adenylate cyclase-activating polypeptide (PACAP) during SCG ontogeny, suggesting that the peptide plays critical roles in neurogenesis. PACAP and PACAP receptor (PAC(1)) mRNA's were detected at embryonic days 14.5 (E14.5) through E17.5 in vivo and virtually all precursors exhibited ligand and receptor, indicating that the system is expressed as neuroblasts proliferate. Exposure of cultured precursors to PACAP peptides, containing 27 or 38 residues, increased mitogenic activity 4-fold. Significantly, PACAP was 1000-fold more potent than VIP and a highly potent and selective antagonist entirely blocked effects of micromolar VIP, consistent with both peptides acting via PAC(1) receptors. Moreover, PACAP potently enhanced precursor survival more than 2-fold, suggesting that previously defined VIP effects were mediated via PAC(1) receptors and that PACAP is the more significant developmental signal. In addition to neurogenesis, PACAP promoted neuronal differentiation, increasing neurite outgrowth 4-fold and enhancing expression of neurotrophin receptors trkC and trkA. Since PACAP potently activated cAMP and PI pathways and increased intracellular Ca(2+), the peptide may interact with other developmental signals. PACAP stimulation of precursor mitosis, survival, and trk receptor expression suggests that the signaling system plays a critical autocrine role during sympathetic neurogenesis.

Direct cAMP Signaling through G-Protein-Coupled Receptors Mediates Growth Cone Attraction Induced by Pituitary Adenylate Cyclase-Activating Polypeptide

“…In particular, PAC1 splice variants activate a number of signaling cascades, including the activation of adenylate cyclase, phosphatidyl inositol turnover, and L-type Ca 2ϩ channels (Pisegna and Wank, 1993;Spengler et al, 1993;Chatterjee et al, 1996). For instance, PAC1 hop splice variant activation increases PI turnover, protein kinase C localization, and intracellular Ca 2ϩ mobilization, whereas PAC1 short activation only stimulates adenylate cyclase (Lu et al, 1998;DiCicco-Bloom et al, 2000;Nicot and DiCicco-Bloom, 2001). Our current data demonstrate that PACAP-induced growth cone attraction was independent of Ca 2ϩ , PLC, and PI3 kinase pathways, implying that PAC1 short , not PAC1 hop , likely mediates the turning.…”

“…Different splice variants of PAC1 receptors elicit different intracellular signaling cascades besides the cAMP pathway, including PLC, PI-3 kinases, and L-type Ca 2ϩ channels (Pisegna and Wank, 1993;Spengler et al, 1993;DiCicco-Bloom et al, 2000;Nicot and DiCicco-Bloom, 2001). To determine whether PACAP elicits Ca 2ϩ responses in cultured Xenopus spinal neurons, we used fura-2 ratiometric imaging to measure intracellular Ca 2ϩ concentrations ([Ca 2ϩ ] i ).…”

Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression

“…E14 rat cortical cells (10) were plated on poly-D-lysine (0.1 mg/ml)͞laminin (20 g/ml)-coated glass coverslips at 4-8 ϫ 10 4 cells/cm 2 in defined medium [Neurobasal͞B27 (from BRL), 2 mM glutamine, penicillin (25 units/ml), streptomycin (25 g/ml), BSA (0.1%)] with basic fibroblast growth factor (bFGF) (10 ng/ml). E16 sympathetic neuroblasts were seeded at 3 ϫ 10 4 cells per well (24-multiwell) (12). Cultures were maintained in a humidified 5% CO 2 ͞air incubator at 37°C.…”

“…For example, in embryonic hindbrain (11) and cortical neuroblasts (10,17), which express the short variant, PACAP stimulates AC only and elicits mitotic inhibition. In contrast, in sympathetic neuroblasts, which express the hop variant, PACAP activates both AC and PLC pathways and stimulates precursor mitosis (12,17). Thus, alternative usage of the hop exon may determine receptor intracellular signaling and mitogenic response.…”

“…Ligands for G protein-coupled receptors (GPCR) may play significant roles in neurogenesis in a regionally or developmentally specific fashion (6)(7)(8)(9). The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide ) exhibits either positive or negative mitogenic effects depending on the neuroblast (10)(11)(12). The basis of these opposite effects is undefined, but may be due to differential expression of PACAP receptor isoforms and consequent activation of distinct signal transduction pathways.…”