P2X receptor activation elicits transporter-mediated noradrenaline release from rat hippocampal slices

This study was designed to test the hypothesis of whether activation of presynaptic P2X receptor-gated ion channels elicits noradrenaline release from central catecholaminergic terminals. ATP, alpha,beta- methylene-adenosine 5'-triphosphate (alpha,beta-methyleneATP), and ADP elicited concentration-dependent [H-3] noradrenaline outflow from superfused rat hippocampal slices with the following rank order of agonist potency: alpha,beta-methyleneATP > ATP > ADP. Among P2 receptor antagonists, pyridoxal-phosphate-6-azophenyl-2 ', 4'-disulphonic acid (30 muM), 4,4', 4", 4'''-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))] tetrakis-1,3-benzenedisulfonic acid (100 nM), and 8,8'-[ carbonybis(imino-3,1-phenylenecarbonylimino)] bis1,3,5-naphthalenetrisulphonic acid ( 10 muM) significantly inhibited the outflow of [ 3H] noradrenaline, evoked by ATP, whereas Brilliant Blue G (100 nM), 2'-deoxy-N-6-methyladenosine 3', 5'-bisphosphate tetraammonium (10 muM), the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (250 nM), and the A(2A) receptor antagonist 3,7-dimethyl-1-propargylxanthine ( 250 nM) were ineffective. Pretreatment with the G(i) protein inhibitor pertussis toxin (2.5 mug/ml) did not change the effect of ATP on [H-3] noradrenaline outflow. In contrast, a decrease in extracellular pH from 7.4 to 6.6 significantly attenuated the response by ATP. When extracellular Na+ was replaced by choline chloride and in the presence of the noradrenaline uptake inhibitor desipramine (10 muM), the ATP-evoked [H-3] noradrenaline outflow was almost completely abolished, indicating that its underlying mechanism is the sodium-dependent reversal of the noradrenaline transporter. Reverse transcription-polymerase chain reaction analysis revealed that mRNA encoding P2X(1), P2X(2), P2X(3), P2X(4), P2X(6), P2X(7), and P2Y(1) receptor subunits were expressed in the brainstem containing catecholaminergic nuclei projecting to the hippocampus, whereas mRNA encoding P2X(5), P2Y(2), P2Y(4), and P2Y(6) receptors were absent. Taken together, these results indicate that noradrenergic terminals of the rat hippocampus are equipped with presynaptic facilitatory P2X receptors, displaying a pharmacological profile similar to homomeric P2X(1) and P2X(3) receptors.