Co-release of ATP and ACh mediates hypoxic signalling at rat carotid body chemoreceptors

1. Using functional co-cultures of rat carotid body (CB) O-2 chemoreceptors and 'juxtaposed' petrosal neurones (JPNs), we tested whether ATP and ACh acted as co-transmitters. 2. Perforated-patch recordings from JPNs often revealed spontaneous and hypoxia-evoked (P-o2 similar to 5 mmHg) excitatory postsynaptic responses. The P2X purinoceptor blocker, suramin (50 mu M) or a nicotinic ACh receptor (nAChR) Meeker (hexamethonium, 100 mu M; mecamylamine, 1 mu M) only partially inhibited these responses, but together, blocked almost all activity. 3. Under voltage clamp (-60 mV), fast perfusion of 100 mu M ATP over hypoxia-responsive JPNs induced suramin-sensitive (IC50 = 73 mu M), slowly-desensitizing, inward currents (I-ATP) with time constant of activation tau(on) = 30.6 +/- 4.8 ms (n=7). I-ATP reversed at 0.33 +/- 3.7 mV (n = 4), and the dose-response curve was fitted by the Hill equation (EC50 = 2.7 mu M; Hill coefficient similar to 0.9). These purinoceptors contained immunoreactive P2X(2) subunits, but their activation by alpha,beta-methylene ATP (alpha,beta-meATP; EC50 = 2.1 mu M) suggests they are P2X(2)/P2X(3) heteromultimers. 4. Suramin and nAChR blockers inhibited the extracellular chemosensory discharge in the intact rat carotid body-sinus nerve preparation in vit ro. Further, P2X(2) immunoreactivity was widespread in rat petrosal ganglia in situ, and co-localized in neurones expressing the CB chemo-afferent marker, tyrosine hydroxylase (TH). P2X(2) labelling in the CB co-localized with nerve-terminal markers, and was intimately associated with TH-positive type 1 cells. 5. Thus ATP and ACh are co-transmitters during chemotransduction in the rat carotid body.