Elsevier

Neuropharmacology

Volume 39, Issue 2, February 2000, Pages 172-180
Neuropharmacology

Pharmacological comparison of P2X receptors on rat coeliac, mouse coeliac and mouse pelvic ganglion neurons

https://doi.org/10.1016/S0028-3908(99)00145-8Get rights and content

Abstract

Characteristics of P2X receptors on neurons of the rat coeliac, mouse coeliac and mouse pelvic ganglia have been studied using the whole cell voltage-clamp technique. Fast application of ATP (100 μM) on to isolated neurons voltage clamped at −70 mV induced a slowly desensitising inward current in 96% of the cells tested. Concentration–response curves for ATP yielded EC50 values of 86 μM, 64 μM and 123 μM, for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively, while α,β-methylene ATP was inactive. The response to ATP was antagonised by suramin, Cibacron blue and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS). The potency of ATP was increased by extracellular acidification and by co-application of micromolar concentrations of Zn2+, while raising pH decreased it. On rat coeliac ganglion neurons, the EC50 values for ATP were 35 μM and 253 μM at pH 6.8 and 8.0, respectively. On mouse coeliac and pelvic ganglion neurons, altering the pH produced comparable changes. In conclusion, our results indicate that, in contrast to the guinea-pig coeliac ganglion, the characteristics of the P2X receptors present on rat coeliac, mouse coeliac and mouse pelvic ganglia are all identical to those present on rat pelvic ganglion, i.e. they are homomeric P2X2 receptors, or heteromultimers with P2X2 being the dominant subunit.

Introduction

ATP acts as a fast excitatory neurotransmitter (for review, see Surprenant et al., 1995, Burnstock, 1997), where it activates a class of ligand-gated ion channels, the P2X receptors. To date, seven P2X receptor subunits have been cloned (P2X1–7), all of which have been reported to form functional homo-oligomeric receptors with different though overlapping biophysical and pharmacological properties, including agonist profiles, desensitisation and sensitivities to antagonists (Brake et al., 1994, Valera et al., 1994, Bo et al., 1995, Chen et al., 1995, Collo et al., 1996, Surprenant et al., 1996). In addition, these P2X receptor subtypes are differentially modulated by pH (King et al., 1996, Stoop et al., 1997) and Zn2+ (Wildman et al., 1998, Wildman et al., 1999). The pharmacological profiles of the recombinant P2X receptors do not always match those of the endogenous P2X receptors; thus, it is plausible that some native P2X receptors are hetero-multimeric channels composed of different P2X subunits (Lewis et al., 1995, Lê et al., 1998).

In autonomic and sensory neurons, two broad groups of native P2X receptors can be distinguished by their sensitivity to α,β-methylene ATP (α,β-meATP) (Evans and Surprenant, 1996). Thus, neurons from rat dorsal root (DRG) (Robertson et al., 1996, Rae et al., 1998), nodose (Lewis et al., 1995) and trigeminal ganglion neurons (Cook et al., 1997) are activated by α,β-meATP. The involvement of P2X3 and P2X2/3 receptors has been suggested. In another group of neurons, such as those of the rat superior cervical ganglion (SCG) (Nakazawa, 1994) and rat pelvic ganglion (Zhong et al., 1998), α,β-meATP is inactive or very weak as an agonist. The molecular and pharmacological properties of P2X receptors on those neurons suggest them to be of the P2X2 subtype. This seems to suggest that in the rat, sensory neurons demonstrate α,β-meATP sensitivity, while the autonomic neurons demonstrate α,β-meATP insensitivity. However, neurons from guinea-pig SCG (Reekie and Burnstock, 1994) and guinea-pig coeliac ganglion (Khakh et al., 1995) respond to α,β-meATP and the receptors present on guinea-pig coeliac ganglion neurons resemble those of rat nodose ganglion (Khakh et al., 1995).

Therefore, in this study, we sought to determine the pharmacological properties of the P2X receptors on rat coeliac ganglion neurons. We were interested in finding whether P2X receptors on rat coeliac (sympathetic) ganglion neurons are similar to those on rat SCG (also sympathetic) and pelvic neurons, or whether they exhibit similar properties to those on guinea-pig coeliac ganglion neurons, i.e. are similar to those on rat nodose (sensory) ganglion neurons. We have also characterised the P2X receptors on mouse coeliac and pelvic ganglia, to explore further the existence of inter-species and inter-ganglion variation, and to provide background information for studies of “knock-out” mice lacking specific P2X subunits. A preliminary report on part of the work has appeared in the form of an abstract (Zhong et al., 1999).

Section snippets

Isolation of neurons

Single neurons of the coeliac ganglion from 17-day-old male Sprague-Dawley rats and coeliac and pelvic ganglia from adult male mice were enzymatically isolated as described previously (Zhong et al., 1998). The animals were killed by a rising concentration of CO2 and death was confirmed by cervical dislocation. The coeliac and pelvic ganglia were rapidly dissected out and placed in Leibovitz's L-15 medium (Life Technologies, Paisley, UK). The ganglia were desheathed and three to four deep cuts

Response to agonists

Fast application of ATP (100 μM) to isolated rat coeliac, mouse coeliac and mouse pelvic ganglion neurons voltage-clamped at −70 mV induced a rapidly activating inward current in 96% of cells tested (Fig. 1). The peak amplitudes (means±SD) of response to ATP (100 μM) were 0.73±0.89 nA (n=94; range 0.025–5.6 nA) for rat coeliac ganglion neurons, 0.66±0.63 nA (n=70; range 0.02–3.2 nA) for mouse coeliac ganglion neurons, and 0.71±0.60 nA (n=61; range 0.025–3.2 nA) for mouse pelvic ganglion

Discussion

In the present study, the pharmacological properties of the P2X receptors on neurons from rat coeliac, mouse coeliac and mouse pelvic ganglia were compared.

Acknowledgements

The authors are grateful to E.W. Moules for excellent technical support, to R. Jordan for help in the preparation of the manuscript. YZ and PMD were supported by Roche Bioscience.

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