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 hours 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 PCR, PACAP transcript levels increased progressively up to 48 hours in culture with no further increase after 72 hours. PACAP transcript levels were reduced by neurturin at 48 hours in culture but not after 24 or 72 hours in culture. In addition, neurturin partially suppressed the percentage of PACAP-IR neurons after 72 hours 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 CNTF, Il-1β, TNFα, bFGF, TGF βand NGF did not increase the percentage of PACAP-IR neurons after 24 hours 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 hour cultures, but not in 72 hour cultures. Neither treatment enhanced the number of VIP-IR neurons. The addition of the PAC 1 receptor antagonist, M65 (100 nM) suppressed the 20 nM PACAP-induced increase in percentage of PACAP-IR cells in 24 hour cultures indicating the effect of PACAP was mediated through the PAC 1 receptor. However, 100 nM M65 had no effect on the percentage of PACAP-IR cells in either 24 or 48 hour 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 can not be excluded. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content...

Neurturin suppresses injury‐induced neuronal activating transcription factor 3 expression in cultured guinea pig cardiac ganglia

“…For culture, the cardiac ganglia explants were dissected under sterile conditions and then maintained at 37°C in culture media consisting of DMEM-F12 (1:1) containing 10% horse serum, gentamicin (10 g/ml), amphotericin B (3.75 g/ml), penicillin (100 U/ml), and streptomycin (100 g/ml; Sigma, St. Louis, MO; Lynch et al, 1999;Girard et al, 2006Girard et al, , 2007. The preparations were placed on a wave platform shaker in a 37°C 5% CO 2 -95% air incubator and kept for 2, 4, 6, 24, 48, 72, or 96 hours, with the culture media replaced every 24 hours.…”

“…The lack of information about the response of cardiac neurons to injury is due in part to the limited access of cardiac ganglia in vivo to experimental manipulations. However, the guinea pig ICNS wholemount preparation can be maintained in explant culture for many days; thus, the cultured guinea pig ICNS makes a convenient model system in which to analyze injuryinduced changes in the cardiac neurons (Girard et al, 2006(Girard et al, , 2007. In recent studies, we used the cultured ICNS to investigate mechanisms responsible for an injuryinduced expression of neuropeptides such as cocaine-and amphetamine-transcript regulated peptide (CARTp) and pituitary adenylate cyclase-activating polypeptide (PACAP; Girard et al, 2006Girard et al, , 2007.…”

“…Cardiac ganglia whole mounts were immunolabeled using procedures described previously (Mawe et al, 1996;Braas et al, 1998;Calupca et al, 2000a,b;Parsons et al, 2006;Girard et al, 2006Girard et al, , 2007. Control and cultured preparations were fixed in 2% paraformaldehyde containing 0.2% picric acid for 2 hours at room temperature (Mawe et al, 1996;Braas et al, 1998;Lynch et al, 1999;Parsons et al, 2006;Girard et al, 2006Girard et al, , 2007.…”

“…Control and cultured preparations were fixed in 2% paraformaldehyde containing 0.2% picric acid for 2 hours at room temperature (Mawe et al, 1996;Braas et al, 1998;Lynch et al, 1999;Parsons et al, 2006;Girard et al, 2006Girard et al, , 2007. The fixed cardiac ganglia were washed in phosphate-buffered saline, permeabilized with 0.5% Triton X-100, and incubated at 4°C overnight with combinations of the primary antiserum.…”

Pituitary adenylate cyclase-activating peptide-27 (PACAP-27) is co-stored with galanin, substance P and corticotropin releasing factor (CRF) in intrapancreatic ganglia of the sheep

“…Since the discovery in 1989, PACAP has been an object of intense anatomical, physiological and pharmacological studies. Using immunohistochemical and radioimmunoassay techniques, the expression of PACAP and its receptors has been found in numerous mammalian tissues including neurons of the central and peripheral nervous system (Masuo et al 1991, Ghatei et al 1993, Csati et al 2012, Rytel et al 2014 as well as enteric Liu 2001, Miampamba et al 2002) and intramural ganglia of the pancreas (Hannibal and Fahrenkrug 2000) and heart (Girard et al 2007). At the central level, PACAP may exert a variety of functions like modulation of hypothalamic-pituitary hormones release (Kanasaki et al 2015), food consumption and water drinking (Puig de Parada et al 1995, Mounien et al 2009), neuronal differentiation, neuroprotection (Manecka et al 2013) and many more, whereas its role at the periphery is relatively less understood.…”