Purinergic and cholinergic neuro-neuronal transmission underlying reflexes activated by mucosal stimulation in the isolated guinea-pig ileum.

We present evidence that adenosine triphosphate (ATP) plays a major role in excitatory neuro-neuronal transmission in ascending and descending reflex pathways to the longitudinal (LM) and circular muscle (CM). A partitioned bath was used for the pharmacological isolation of a segment of guinea-pig ileum (∼6 cm in length), allowing drugs to be selectively applied to an intermediate region between the region where mucosal stimulation was applied and that where mechanical recordings were made. Brush stroking the mucosa (3 strokes) elicited a synchronous contraction of the LM and CM both above (ascending excitation) and below (descending excitation) the site of stimulation. All reflexes were abolished when tetrodotoxin (1 μm) was applied to the intermediate chamber. Hexamethonium (300 μm) added to the intermediate chamber abolished the ascending contraction in 15 % of oral preparations (from 26 preparations, 18 animals) and the descending contraction in 13 % of anal preparations studied (from 53 preparations, 48 animals). In the remaining 85 % of oral preparations, hexamethonium usually attenuated the oral contraction of the LM and CM. However, in the remaining 87 % of anal preparations, hexamethonium had no effect on the anal contraction of the LM and CM. Oral and anal reflexes that were hexamethonium resistant were either abolished or attenuated by the further addition of the P2 purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 10 μm) or α,β-methylene ATP (50–100 μm) to the intermediate chamber. 1,1-Dimethyl-4-phenyl-piperazinium iodide (DMPP, 20 μm) or α,β-methylene ATP (50–100 μm) stimulated both ascending and descending excitatory pathways, when applied to the intermediate chamber. In conclusion, ascending and descending neuro-neuronal transmission in excitatory nervous pathways to the LM and CM is complex and clearly involves neurotransmitter(s) other than acetylcholine (ACh). We suggest mucosal stimulation releases ACh and ATP in both ascending and descending excitatory reflex pathways that synapse with excitatory motoneurons to the LM and CM. More is known about the enteric nervous system of the guinea-pig ileum than of any other species. It has developed as the model preparation for understanding how enteric neurons generate the peristaltic reflex. In this tissue, immunohistochemical and electrophysiological studies have resulted in a detailed understanding of the neural projections in the myenteric plexus (Costa et al. 1996). It is now known that there is only one major class of ascending interneuron in the guinea-pig ileum (Brookes et al. 1997) and that orally directed transmission in this tissue is markedly attenuated or abolished by hexamethonium (Smith & Furness, 1988; Holzer, 1989; Smith et al. 1990; Tonini & Costa, 1990; Johnson et al. 1996; Spencer et al. 1999b). In contrast, anally directed transmission in descending interneurons of the guinea-pig ileum is considerably more complex. There are at least four major classes of descending interneuron in this tissue, three of which are cholinergic (Costa et al. 1996). Surprisingly, it is generally believed that these descending interneurons largely utilize neurotransmitters other than ACh, since descending neuro-neuronal transmission in the ileum has consistently been reported to be resistant to hexamethonium (Smith et al. 1990; Johnson et al. 1996; Spencer et al. 1999b). Therefore, the identity of the neurotransmitter substance(s) involved in descending transmission from enteric interneurons has largely remained unclear. However, the neurotransmitter substance released from descending pathways would, like ACh in ascending interneurons, most probably have to be a fast-acting transmitter, operating through ligand-gated ion channels, since neurons in both reflex pathways communicate essentially via fast excitatory postsynaptic potentials (fEPSPs) (Hirst et al. 1975, 1976; Bornstein et al. 1991; Smith et al. 1992, 1999; Brookes et al. 1997). Although much evidence has been presented suggesting a role for ATP as a neurotransmitter at many neuromuscular synapses in autonomic smooth muscle (see reviews by Burnstock, 1972, 1997; Hirst & Edwards, 1989), little attention has been focused on a role for ATP as a neuro-neuronal transmitter in the enteric nervous system of mammals. There is increasing evidence that ATP may mediate fast synaptic transmission in some ganglia of the central nervous system (Pankratov et al. 1998). In fact, recent evidence has been presented that fast synaptic transmission in the enteric nervous system is likely to involve purinergic as well as nicotinic receptors (Galligan & Bertrand, 1994; Zhou & Galligan, 1996, 1998; Barajas-Lopez et al. 1998; Lepard & Galligan, 1999). However, a role for purinergic transmission during physiological reflexes has not been shown. Furthermore, it is unclear which classes of enteric neuron may involve such transmission. In the current study, we have used the partitioned organ bath technique to investigate the identity of the neurotransmitter substances released from ascending and descending interneurons involved in the transmission of the orally and anally directed reflex pathways. We present evidence that ACh and ATP both act as excitatory neurotransmitters, involved in ascending and descending neuro-neuronal transmission to motoneurons in the LM and CM.