In the nervous system purinergic excitatory synapses use ATP to mediate fast synaptic transmission via activation of P2X receptor cation channels. In the carotid body ATP is co-released with ACh during hypoxia, which activates P2X2 and P2X3 receptors on petrosal neurons to initiate corrective changes in ventilation (Prasad et al. 2001). However, the role of P2X receptors in this response is unclear. This study examined the response of cloned rat P2X2 receptors to hypoxia.
Whole-cell currents were recorded from P2X2 receptors stably expressed in HEK293 cells (Evans et al. 1996). Pipettes were filled with (mM) 10 NaCl, 117 KCl, 2 MgSO4, 1 CaCl2, 11 EGTA, 2 Na-ATP, 11 Hepes (pH 7.2), and cells were continuously perfused with 135 NaCl, 5 KCl, 1.2 MgCl2, 2.5 CaCl2, 5 Hepes and 10 glucose (pH 7.4). Data are given as means ± S.E.M. and statistical analysis was peformed using Student’s paired t test with P < 0.05 regarded as significant.
In the presence of external Ca2+ and Mg2+ perfusion of ATP elicited an inwardly rectifying current with an EC50 of 26 ± 0.1 µM. At a potential of -70 mV, 5 µM ATP induced an inward current that showed little desensitization during repeated exposures under normoxic conditions (-50.5 ± 16.7 pA pF-1 to -47.1 ± 18.4 pA pF-1, mean of first three and last three exposures respectively, n = 3). Exposure to an hypoxic ATP (5 µM) solution (PO2 25 mm Hg, EC solution bubbled with 100 % N2) reduced the whole-cell current from -58.0 ± 11.2 pA pF-1 to -31.1 ± 8.1 pA pF-1 (P < 0.01, n = 4). Removal of external Ca2+ and Mg2+ plus buffering with 1 mM EGTA shifted the EC50 to 0.2 ± 0.3 µM but did not prevent the reduction in current following a hypoxic challenge. Hypoxia reduced the inward current induced by ATP (0.1 µM) from -19.3 ± 4.4 pA pF-1 to -13.7 ± 3.5 pA pF-1 (P < 0.01, n = 5).
In summary, this is the first report indicating that hypoxia modulates the response of P2X receptors to ATP and suggests that P2X receptors are involved in mediating the response to hypoxia in the nervous system.
This work was funded by the Welcome Trust 065887/Z/01/Z.