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