The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia.
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...
Cultured guinea pig atrial whole mounts containing the intrinsic cardiac ganglia were used as an in vitro model to investigate the induction of the stress/injury marker activating transcription factor 3 (ATF-3). ATF-3 expression was quantified by using immunocytochemical labeling and real-time PCR. In freshly isolated ganglia, no neuronal or Schwann cell nuclei exhibited ATF-3 immunoreactivity. In 2-hour cultures, the induction of ATF-3 expression was evident in many Schwann cell nuclei, whereas no neuronal nuclei were ATF-3 immunoreactive. Beginning at 4 hours, the percentage of neurons with ATF-3-immunoreactive nuclei increased progressively, and, by 48 hours in culture, approximately 95% of the cardiac neurons had ATF-3-immunoreactive nuclei. Neurturin significantly suppressed ATF-3 expression in 48-hour-cultured neurons without effect on ATF-3 expression in Schwann cell nuclei. Neuturin also could reverse neuronal ATF-3 expression after its induction. The suppression of ATF-3 induction by neurturin was mediated by activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Glial-derived neurotrophic factor (GDNF) also suppressed neuronal ATF-3 induction during culture. However, culture in serum-free media, presence of nerve growth factor, or addition of pituitary adenylate cyclase-activating polypeptide had no effect on ATF-3 induction in the 48-hour-cultured cardiac neurons. By 4 hours in culture, there was a significant increase in ATF-3 transcript levels, and neurturin partially suppressed ATF-3 transcript levels in 48-hour cultures. It is proposed that the loss of target-derived neurturin is a potential mechanism stimulating injury-induced expression of ATF-3 in cardiac neurons.
The major pelvic ganglia (MPG) contain both parasympathetic and sympathetic postganglionic neurons and provide much of the autonomic innervation to urogenital organs and components of the lower bowel. Whereas many parasympathetic neurons were found to express vasoactive intestinal polypeptide (VIP), no MPG neurons exhibited immunoreactivity for pituitary adenylate cyclase activating polypeptide (PACAP). However, in 3-day cultured MPGs, numerous PACAP-IR cells and nerve fibers were present and transcript levels for PACAP increase As the MPG contains both sympathetic and parasympathetic postganglionic neurons, d significantly. In 3-day cultured MPGs, PACAP immunoreactivity was seen in cells that were also immunoreactive for VIP or neuronal nitric oxide synthase (nNOS), but not tyrosine hydroxylase (TH), indicating that PACAP expression occurred preferentially in MPG parasympathetic postganglionic neurons. Transcript levels for the VPAC2, but not VPAC1 or PAC1 receptor, also increased significantly following 3 days in culture. Transcript levels of activating transcription factor 3 (ATF-3), a marker of cellular injury, were increased 64-fold in 3-day explants and ATF-3-IR nuclei were evident in both TH-IR and nNOS-IR neurons as well as in non-neuronal cells. In sum, these results demonstrate that although only the parasympathetic neurons in explant cultured MPGs increase expression of PACAP, both sympathetic and parasympathetic postganglionic neurons in the cultured MPG whole mount increase expression of ATF-3.
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