Regulation of neuronal pituitary adenylate cyclase-activating polypeptide expression during culture of guinea-pig cardiac ganglia

Abstract

The trophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) increases in many different neuron types following injury; a response postulated to support cell survival and regeneration. In acutely isolated cardiac ganglia, approximately 1% of the cardiac neurons exhibited PACAP immunoreactivity whereas after 72 h in culture, ∼25% of the neurons were PACAP immunoreactive. In contrast, there was no increase in vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) cells. Using a combination of immunocytochemical and molecular techniques, we have quantified PACAP expression, during explant culture of guinea-pig cardiac ganglia. Using real time polymerase chain reaction, PACAP transcript levels increased progressively up to 48 h in culture with no further increase after 72 h. PACAP transcript levels were reduced by neurturin at 48 h in culture but not after 24 or 72 h in culture. In addition, neurturin partially suppressed the percentage of PACAP-IR neurons after 72 h in culture, an effect mediated by activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways. The addition of different known regulatory molecules, including ciliary neurotrophic factor (CNTF), interleukin-1 beta (Il-1β), tumor necrosis factor-alpha (TNFα), fibroblast growth factor basic (bFGF), transforming growth factor-beta (TGF-β) and nerve growth factor (NGF) did not increase the percentage of PACAP-IR neurons after 24 h in culture; a result indicating that the generation and secretion of these factors did not stimulate PACAP expression. The presence of 20 nM PACAP or 10 μM forskolin increased the percentage of PACAP-IR cardiac neurons in 24 h cultures, but not in 72 h cultures. Neither treatment enhanced the number of VIP-IR neurons. The addition of the PACAP selective receptor (PAC1) receptor antagonist, M65 (100 nM) suppressed the 20 nM PACAP-induced increase in percentage of PACAP-IR cells in 24 h cultures indicating the effect of PACAP was mediated through the PAC1 receptor. However, 100 nM M65 had no effect on the percentage of PACAP-IR cells in either 24 or 48 h cultures not treated with exogenous PACAP, suggesting that endogenous release of PACAP likely did not contribute to the enhanced peptide expression. We postulate that the enhanced PACAP expression, which occurs in response to injury is facilitated in the explant cultured cardiac ganglia by the loss of a target-derived inhibitory factor, very likely neurturin. In intact tissues the presence of neurturin would normally suppress PACAP expression. Lastly, our results indicate that many common trophic factors do not enhance PACAP expression in the cultured cardiac neurons. However, the stimulatory role of an, as yet, unidentified factor cannot be excluded.