Expression and physiological actions of neuropeptide Y in guinea pig parasympathetic cardiac ganglia

doi:10.1016/S0165-1838(98)00072-1

Journal of the Autonomic Nervous System

Volume 71, Issues 2–3, 15 July 1998, Pages 190-195

Journal of the Auton...

https://doi.org/10.1016/S0165-1838(98)00072-1 Get rights and content

Abstract

Guinea pig atrial whole mount preparations containing the parasympathetic cardiac ganglia were used to establish the expression, distribution and actions of neuropeptide Y (NPY) in atrial tissues. NPY-immunoreactive fibers densely innervated the atrial myocardium and blood vessels. Fibers containing NPY also innervated intrinsic parasympathetic cardiac neurons. Four percent of the cardiac neurons, identified using microtubule associated protein-2 antiserum, were NPY-positive. An endogenous source of NPY was confirmed with reverse transcription PCR which demonstrated the presence of proNPY mRNA. Sixty percent of the parasympathetic cardiac neurons were hyperpolarized by local application of NPY. NPY also decreased the amplitude and duration of the action potential after hyperpolarization in 60% of the neurons and decreased the fast excitatory postsynaptic potential in about 50% of the cells. These observations indicate that NPY is anatomically positioned to directly alter the output of the parasympathetic cardiac ganglia either by hyperpolarizing the cardiac neurons or by decreasing the fast synaptic input which drives individual neurons.

Introduction

Cardiac function is regulated not only by the classical neurotransmitters acetylcholine (ACh) and norepinephrine (NE) from autonomic parasympathetic and sympathetic postganglionic nerve fibers, respectively, but also by the co-release of neuropeptides from the same terminals (Lundberg, 1996). Most of the extrinsic NE containing postganglionic sympathetic fibers innervating the heart also co-store the α-amidated 36 amino acid neuropeptide Y (neuropeptide tyrosine, NPY; Dalsgaard et al., 1986; Gu et al., 1984; Lundberg, 1996; Sosunov et al., 1997; Sternini and Brecha, 1985). The dense pattern of NPY-immunoreactive (-IR) fiber innervation of cardiac tissues coincides with the patterns of catecholamine biosynthetic enzymes. Also, NPY and NE are co-released upon stimulation of the sympathetic postganglionic fibers innervating the heart (Lundberg, 1996). It has been postulated that NPY release from sympathetic postganglionic fibers reduces vagal induced bradycardia (Potter, 1987; Revington and McCloskey, 1990; Warner and Levy, 1989a, Warner and Levy, 1989b). This may represent actions of NPY released from sympathetic nerves onto adjacent parasympathetic postganglionic nerve fiber terminals innervating cardiac muscle. Other studies have provided indirect evidence that NPY may act within the parasympathetic cardiac ganglia, as well as on parasympathetic postganglionic nerve terminals, to reduce the vagal cardiac inhibition (Pardini et al., 1992). In accord with these physiological studies, staining of cardiac tissue sections have demonstrated NPY-IR fibers enveloping intrinsic neurons of the cardiac ganglia (Dalsgaard et al., 1986; Sosunov et al., 1997; Sternini and Brecha, 1985).

Several important issues of NPY innervation of the parasympathetic cardiac ganglia remain to be resolved. Some intrinsic parasympathetic neurons have been shown to contain NPY (Hassall and Burnstock, 1984; Sosunov et al., 1997; Steele et al., 1994). However, previous studies were done under conditions that may have promoted atypic neuropeptide expression such as dissociated neurons in culture or tissue explantation with colchicine treatment (Hassall and Burnstock, 1984; Steele et al., 1994). Furthermore, the extent of NPY-IR in cardiac ganglia has not been evaluated and thus the prevalence of NPY innervation of intrinsic neurons in the cardiac ganglia has not been determined. Consequently, estimates of neurons expressing NPY may not have accurately reflected the in vivo condition. Also, the direct actions of NPY on neurons within the parasympathetic cardiac ganglia have not been examined.

The aims of the present study were to: (a) establish the distribution of NPY-IR fibers in atrial whole mount preparations containing cardiac ganglia; (b) establish the relationship between NPY-IR fibers and parasympathetic cardiac neurons; (c) establish that intrinsic cardiac ganglia neurons express proNPY mRNA and therefore the ability to endogenously synthesize NPY; (d) determine the fraction of intrinsic cardiac neurons expressing NPY; and (e) test whether NPY has direct actions on individual cardiac neurons and/or alters fast ganglionic transmission.

Section snippets

Materials and methods

Experiments utilized tissues from adult guinea pigs (250–300 g of either sex), which were anesthetized with isoflurane and exsanguinated; methods were approved by the University of Vermont Animal Care and Use Committee. Atrial whole mount preparations containing the cardiac ganglia were prepared as described previously (Hardwick et al., 1995, Hardwick et al., 1997; Mawe et al., 1996). For electrophysiological recordings, the atrial whole mount preparations were maintained in a standard Krebs

Results and discussion

We have previously demonstrated that with the atrial whole mount preparation, it is possible to assess intrinsic cardiac neuron distribution and neurotransmitter phenotype and analyze the functional relationships between intrinsic cardiac neurons and the innervating fibers (Hardwick et al., 1995, Hardwick et al., 1997; Mawe et al., 1996). In the present study, we have employed the same techniques to establish the distribution of NPY-IR fibers in guinea pig cardiac atrial tissue (Fig. 1). Atrial

Acknowledgements

Supported by NIH grants NS 23978 (RLP) and NS 26995 and DK 45410 (GMM) and AHA Grant In Aid #94615540 (KMB). We thank Dr Victor May for critical reading of the manuscript and help in designing the NPY primer templates.

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The mammalian parasympathetic cardiac ganglia form a complex intrinsic cardiac nervous system presumed to contain multiple neuron types and are innervated by extrinsic fibers from multiple sources, each containing specific neurotransmitters and neuropeptides. In the guinea pig, the preganglionic parasympathetic cholinergic fibers contain the neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), and essentially all cardiac neurons express the PACAP selective PAC₁ receptor. Application of exogenous PACAP depolarizes and enhances the excitability of guinea pig cardiac neurons. The mechanism by which PACAP enhances excitability is not established. However, Ca²⁺ influx through PACAP-activated nonselective cation channels is required for the PACAP-induced increase in excitability of guinea pig cardiac neurons. In addition, a PACAP-induced shift in the voltage dependence of activation of the cyclic nucleotidegated, hyperpolarization-activated current, I_h, most likely participates in the peptide-induced increase in neuronal excitability. The release of endogenous PACAP by repetitive stimulation of preganglionic fibers in vitro contributes to the generation of a slow excitatory postsynaptic potential and also can enhance cardiac neuron excitability. We hypothesize that PACAP released during ongoing vagal activity may regulate cardiac neuron excitability in vivo. [Tzu Chi Med J 2008;20(1):11–18]
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Quantification of PACAP-IR parasympathetic neurons present in the cardiac ganglia followed procedures similar to those published previously (Lynch et al., 1999; Braas et al., 2004; Parsons et al., 2006). The number of cardiac neurons in each preparation was determined by counting ChAT-IR or PGP-9.5-IR cells (Mawe et al., 1996; Braas et al., 1998, 2004; Kennedy et al., 1998; Lynch et al., 1999; Calupca et al., 2000; Parsons et al., 2006). The percentage of cardiac ganglia neurons that expressed PACAP was then determined as a fraction of the total number of neurons that were ChAT or PGP-95 IR: [(PACAP-IR neurons/ChAT-IR or PGP-95-IR neurons)×100=%].
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Short communicationExpression and physiological actions of neuropeptide Y in guinea pig parasympathetic cardiac ganglia

Abstract

Introduction

Section snippets

Materials and methods

Results and discussion

Acknowledgements

J. Auton. Nerv. Syst.

Neurosci. Lett.

Neurosci. Lett.

Neuroscience

Pituitary adenylate cyclase activating polypeptide, PACAP38 and PACAP27, regulation of sympathetic neuron catecholamine and neuropeptide Y expression through activation of type I PACAP/VIP receptor isoforms

Ann. New York Acad. Sci.

Identification of endogenous sympathetic neuron pituitary adenylate cyclase activating polypeptide (PACAP): Depolarization regulates production and secretion through induction of multiple propeptide transcripts

J. Neurosci.

Distribution and origin of substance P- and neuropeptide Y-immunoreactive nerves in the guinea-pig heart

Cell Tissue Res.

High concentrations of a novel peptide, neuropeptide Y, in the innervation of mouse and rat heart

J. Histochem. Cytochem.

Tachykinin-induced activation of non-specific cation conductance via NK3 neurokinin receptors in guinea-pig intracardiac neurones

J. Physiol.

Sources and actions of neuropeptide Y (NPY) in the guinea pig cardiac ganglia

Soc. Neurosci. Abstr.

Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric acid

Pharmacol. Rev.

Short communication
Expression and physiological actions of neuropeptide Y in guinea pig parasympathetic cardiac ganglia