Evidence that P2X purinoceptors mediate the excitatory effects of αβmethylene-ADP in rat locus coeruleus neurones

Abstract

Extracellular and whole-cell patch clamp recordings were used to study the excitatory responses elicited by purine nucleotides in pontine slices of the rat brain containing the locus coeruleus (LC). The P2 purinoceptor agonists, α β-methyleneadenosine 5′-triphosphate ( α βmeATP) and adenosine 5′- O-(2-thiodiphosphate) (ADP βS), and a novel purinoceptor agonist, α β-methyleneadenosine 5′-diphosphate ( α βmeADP), elicited concentration-dependent increases in the spontaneous firing rate over the concentration range (1–300 μM). On vagus nerve or dorsal root preparations α βmeADP (100 μM) had no agonist activity. In the presence of both α βmeATP (300 μM), ADP βS (300 μM) elicited a further and significant increase in the firing rate of the LC neurones, whilst neither α βmeATP nor α βmeADP (of 300 μM) elicited a further response. The P2 purinoceptor antagonists, suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 30 μM), markedly attenuated responses to all three agonists. Whole-cell recording of membrane current showed that, at −60 mV, α βmeATP and α βmeADP (both 100 μM) elicited inward currents of a similar magnitude, whilst the inward currents elicited by a lower concentration of ADP βS (30 μM) were larger and faded in the presence of this agonist. In the presence of tetrodotoxin and a combination of other neurotransmission blockers, both α βmeATP and α βmeADP still produced inward currents. Based on the known selectivity of the agonists used in this study, there appear to be two distinct P2 purinoceptor types present on neurones in the LC, which correspond to the P2X and P2Y types. The responses elicited by α βmeADP appear to be mediated through a putative P2X purinoceptor, although further work is required to determine which P2X receptor subtype(s) are involved.