Responses induced by acetylcholine and ATP in the rabbit petrosal ganglion

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Abstract

Acetylcholine and ATP appear to mediate excitatory transmission between receptor (glomus) cells and the petrosal ganglion (PG) neuron terminals in the carotid body. In most species these putative transmitters are excitatory, while inhibitory effects had been reported in the rabbit. We studied the effects of the application of acetylcholine and ATP to the PG on the carotid nerve activity in vitro. Acetylcholine and ATP applied to the PG increased the carotid nerve activity in a dose-dependent manner. Acetylcholine-induced responses were mimicked by nicotine, antagonized by hexamethonium, and enhanced by atropine. Bethanechol had no effect on basal activity, but reduced acetylcholine-induced responses. Suramin antagonized ATP-induced responses, and AMP had little effect on the carotid nerve activity. Our results suggest that rabbit PG neurons projecting through the carotid nerve are endowed with nicotinic acetylcholine and purinergic P2 receptors that increase the carotid nerve activity, while simultaneous activation of muscarinic cholinergic receptors reduce the maximal response evoked by nicotinic cholinergic receptor activation.

Introduction

The carotid body (CB) is the principal arterial chemoreceptor organ, constituted by specific parenchymal cells: the receptor (glomus, type-I) cells and the glial-like (sustentacular, type-II) cells. The glomus cells are innervated, through the carotid (sinus) nerve, by sensory neurons whose perikarya are located in the petrosal ganglion (PG). The most accepted paradigm of CB chemoreception states that, as a result of the transduction mechanism, the glomus cells release one or more transmitters that, acting on postsynaptic receptors located on the nerve terminals of PG neurons, generate or maintain the afferent activity (Eyzaguirre and Zapata, 1984, González et al., 1994, Prabhakar, 2000). There are several transmitter molecules present in the CB, but their exact participation in the generation of the chemo-afferent activity is still controversial. Part of the controversy arises from conflicting results obtained from different species and preparations.

Acetylcholine (ACh) was one of the first molecules postulated as a transmitter in the CB (see Heymans, 1955). ACh and its metabolic synthesis and degradation pathways are present in the CB of cats (Fidone et al., 1976, Wang et al., 1989), rabbits (Wang et al., 1989, Kim et al., 2004) and in the rat CB-PG reconstituted system in vitro (Nurse and Zhang, 1999). ACh is released from the cat CB during electrical (Eyzaguirre and Zapata, 1968) and hypoxic stimulation (Fitzgerald et al., 1999, Fitzgerald, 2000), but ACh release from the rabbit CB is reduced during hypoxic stimulation (Kim et al., 2004). The exogenous application of ACh to the cat CB increases the carotid nerve frequency of discharge (Eyzaguirre and Zapata, 1968), while its effect is mainly inhibitory in the rabbit CB (Docherty and McQueen, 1979, Monti-Bloch and Eyzaguirre, 1980). However, application of nicotine to the CB increases the carotid nerve frequency of discharge both in the cat (Eyzaguirre and Zapata, 1968, Reyes et al., 2007) and in the rabbit (Monti-Bloch and Eyzaguirre, 1980, Jonsson et al., 2004), effects blocked by nicotinic ACh receptor antagonists (Eyzaguirre and Zapata, 1968, Jonsson et al., 2004, Reyes et al., 2007). On the other hand, muscarinic ACh receptor subtypes M1 and M2 have been described in the cat CB (Shirahata et al., 2004). However, muscarinic antagonists have no effect on the cat chemosensory activity but depressed the carotid nerve frequency of discharge on the rabbit (Monti-Bloch and Eyzaguirre, 1980). In the rabbit CB, atropine antagonizes the inhibitory effects of muscarinic ACh receptor agonists on the carotid nerve frequency of discharge (Monti-Bloch and Eyzaguirre, 1980), increases basal ACh release and revert the reduction of ACh release induced by hypoxia to an increased release (Kim et al., 2004). Thus, the available data indicate that ACh appear to be excitatory in the cat CB, but may have both excitatory and inhibitory actions in the rabbit, mediated by nicotinic and muscarinic ACh receptors, respectively. However, the exogenous application of ACh to the CB may involve activation of both presynaptic and postsynaptic receptors (Kim et al., 2004, Shirahata et al., 2007).

Currently, is accepted that both ATP and adenosine (Buttigieg and Nurse, 2004, Conde and Monteiro, 2004, Conde and Monteiro, 2006a) are released from the rat CB by hypoxia (see Conde and Monteiro, 2006b). ATP increases the carotid nerve frequency of discharge when applied to the rat CB in situ and in vitro (McQueen and Ribeiro, 1981, Spergel and Lahiri, 1993), although the action of ATP through its metabolites AMP (McQueen and Ribeiro, 1983) or adenosine (Runold et al., 1990) has been suggested. In the rat CB preparation in vitro, adenosine appears to participate in the response to an hypoxic challenge, acting both on PG and CB cells trough A2A and A2B receptors, respectively (Conde et al., 2006). On the other hand, using the cat PG preparation in vitro we found that the application of ATP increases the carotid nerve frequency of discharge in a dose-dependent manner, effect that is only marginally mimicked by AMP (Alcayaga et al., 2000a, Alcayaga et al., 2000b). These data suggest that ATP increases the chemosensory activity in the rat and cat at the postsynaptic level in the PG neurons, but there is no information on the effects of ATP on the rabbit carotid chemosensory afferents.

Both ACh and ATP have been proposed to participate in the generation of the afferent chemosensory activity by acting on specific receptors located on the PG terminals in the CB of cats and rats (Prasad et al., 2001, Iturriaga and Alcayaga, 2004, Nurse, 2005). However, in the rabbit the effect of ACh is controversial and little or no information about the effect of ATP is available (see Alcayaga et al., 2006, Iturriaga et al., 2007). Thus, we studied the responses evoked by ACh and ATP on the rabbit PG neurons that project through the carotid nerve, using an isolated PG preparation (Alcayaga et al., 1998).

Section snippets

Animals

Experiments were performed on 24 male adult White New Zealand rabbits (2.30 ± 0.11 kg; mean ± SEM). The protocol was approved by the Ethical Committee of the Facultad de Ciencias of the Universidad de Chile, and meets the guidelines of the National Fund for Scientific and Technological Research (FONDECYT), Chile, and the Guiding Principles for the Care and Use of Animals of the American Physiological Society.

Surgical procedures and recording of nerve activity

The PGs were obtained from animals anaesthetized with an intramuscular injection of a

Electrical stimulation

Electrical stimulation of the PG elicited an antidromic compound action potential in both the carotid nerve and the glossopharyngeal branch of the glossopharyngeal nerve. Fig. 1 shows representative compound action potentials recorded from the peripheral branches of the glossopharyngeal nerve. The compound action potential recorded in the glossopharyngeal branch was dominated by components attributable to fast conducting fibers (Fig. 1A), while the compound action potential evoked in the

General

One main finding of this study is that application of ACh to the rabbit PG increases carotid nerve frequency of discharge, effect blocked by hexamethonium (10 μM) and mimicked by nicotine (0.1–1000 μg) but not by bethanechol (0.1–1000 μg). Acetylcholine-induced responses were transiently reduced by prior application of bethanechol (500–1000 μg), but were enhanced during atropine (10 μM) treatment. Similarly, ATP increases carotid nerve frequency of discharge, responses that were reversibly blocked

Acknowledgement

This work was supported by grants 1040638 and 1090157 from the National Fund for Scientific and Technological Development (FONDECYT) of Chile.

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    1

    Present address: Programa de Doctorado en Ciencias, mención Neurociencia, Universidad de Valparaiso, Gran Bretaña 1111 Playa Ancha, Valparaiso, Chile.

    2

    Present address: Programa de Doctorado en Ciencias Biológicas, mención Ciencias Fisiologicas, P. Universidad Católica de Chile, Facultad de Ciencias Biológicas, Portugal 49, Casilla 114-D, Santiago, Chile.

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