Pre- and postsynaptic actions of ATP on neurotransmission in rat submandibular ganglia
The pre- and postsynaptic actions of exogenously applied ATP were investigated in intact and dissociated parasympathetic neurones of rat submandibular ganglia. Nerve-evoked excitatory postsynaptic potentials (EPSPs) were not inhibited by the purinergic receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), or the desensitising agonist, α,β-methylene ATP. In contrast, EPSPs were abolished by the nicotinic acetylcholine receptor antagonists, hexamethonium and mecamylamine. Focal application of ATP (100 μM) had no effect on membrane potential of the postsynaptic neurone or on the amplitude of spontaneous EPSPs. Taken together, these results suggest the absence of functional purinergic (P2) receptors on the postganglionic neurone in situ. In contrast, focally applied ATP (100 μM) reversibly inhibited nerve-evoked EPSPs. Similarly, bath application of the non-hydrolysable analogue of ATP, ATPγS, reversibly depressed EPSPs amplitude. The inhibitory effects of ATP and ATPγS on nerve-evoked transmitter release were antagonised by bath application of either PPADS or suramin, suggesting ATP activates a presynaptic P2 purinoceptor to inhibit acetylcholine release from preganglionic nerves in the submandibular ganglia. In acutely dissociated postganglionic neurones from rat submandibular ganglia, focal application of ATP (100 μM) evoked an inward current and subsequent excitatory response and action potential firing, which was reversibly inhibited by PPADS (10 μM). The expression of P2X purinoceptors in wholemount and dissociated submandibular ganglion neurones was examined using polyclonal antibodies raised against the extracellular domain of six P2X purinoceptor subtypes (P2X1-6). In intact wholemount preparations, only the P2X5 purinoceptor subtype was found to be expressed in the submandibular ganglion neurones and no P2X immunoreactivity was detected in the nerve fibres innervating the ganglion. Surprisingly, in dissociated submandibular ganglion neurones, high levels of P2X2 and P2X4 purinoceptors immunoreactivity were found on the cell surface. This increase in expression of P2X2 and P2X4 purinoceptors in dissociated submandibular neurones could explain the increased responsiveness of the neurones to exogenous ATP. We conclude that disruption of ganglionic transmission in vivo by either nerve damage or synaptic blockade may up-regulate P2X expression or availability and alter neuronal excitability.