MDMA glutamate receptor binding in spinal cords of mice lacking the adenosine A2A receptor

M.J. Hussey, S.M.O. Hourani and I. Kitchen, University of Surrey, Guildford, UK.

Adenosine is a neuromodulator with complex effects on pain pathways. Mice lacking the adenosine A2A receptor are hypoalgesic (Ledent et al., 1997), and have altered analgesic responses to opioid agonists (Bailey et al., 2002). The adenosine A2A receptor is absent from the spinal cord despite mRNA transcripts in dorsal root ganglion, indicating transport of receptors to the peripheral terminal only (Kaelin-Lang et al., 1998), and the A2A agonist CGS21680 causes hyperalgesia in the paw pressure test (Khasar et al., 1995). These findings suggest a role for the A2A receptor in sensitizing afferent fibres projecting to the spinal cord. As glutamate is a primary nociceptive transmitter we have used spinal cord binding of [ 3H]-MK801, an antagonist radioligand for the NMDA receptor, as an indirect measure of nociception in A2A receptor knockout mice, to investigate whether peripheral A2A receptors influence pain transmission.

Adult male wildtype and A2A receptor knockout mice (CD1) were killed and the spinal cords removed. Sections (20 µm) were cut and total binding determined by incubation with 70nM [3H]-MK801 for 1 hour. Adjacent sections were incubated in the additional presence of 1mM unlabelled MK801 to determine non-specific binding. Sections were apposed to [3H] sensitive film (Hyperfilm, Amersham) with microscale standards and developed after 3 weeks. Quantitative analysis was carried out using an MCID imaging system. Statistical analysis was carried out using 2-way ANOVA for factors genotype and region.

Table 1. Specific binding of [3H]-MK801 to NMDA glutamate receptors in spinal cords of wildtype and A2A knockout mice (mean ± S.E.M., n=3-5).

There was a substantial reduction in binding of [3H]-MK801 in all regions of the spinal cords of A2A knockout mice (P<0.001). The mean overall decrease was 61.3% throughout the spinal cord with the highest decreases seen in cervical and lumbar regions (Table 1.). The decrease in NMDA glutamate receptor binding could reflect reduced peripheral sensory input to the spinal cord and be related to the hypoalgesia in this genotype. These results support a key role for the adenosine A2A receptor in peripheral pain pathways.

Bailey A. et al (2002) J. Neurosci 22; 9210-9220.
Kaelin-Lang A. et al (1998) Neurosci lett. 246: 21-24.
Khasar S.G. et al (1995) Neurosci 67: 189-195.
Ledent C. et al (1997) Nature 388: 674-678.

This work was supported by a BBSRC CASE studentship with GlaxoSmithKline.