Descending inhibitory reflexes involve P2X receptor-mediated transmission from interneurons to motor neurons in guinea-pig ileum

Abstract

The role of P2X receptors in descending inhibitory reflexes evoked by distension or mucosal distortion in the guinea-pig ileum was studied using intracellular recording from the circular muscle in a two-chambered organ bath. This allowed separate superfusion of the sites of reflex stimulation and recording, thereby allowing drugs to be selectively applied to different parts of the reflex pathway. Inhibitory junction potentials (IJPs) evoked by electrical field stimulation (EFS) in the recording chamber were compared with those evoked during reflexes to control for effects of P2 receptor antagonists on neuromuscular transmission. The P2 receptor antagonists suramin (100 microM) and pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (10 and 60 microM; PPADS), when added to the recording chamber, depressed reflexly evoked IJPs significantly more than those evoked by EFS. In particular, 10 microM PPADS depressed IJPs evoked by distension or mucosal distortion by about 50 %, but had little effect on IJPs evoked by EFS. Blockade of synaptic transmission in the stimulation chamber with a low Ca2+-high Mg2+ solution depressed, but did not abolish, IJPs evoked by distension. The residual reflex IJPs were unaffected by PPADS (10 microM), hyoscine (1 microM), hyoscine plus hexamethonium (200 microM), or hysocine plus hexamethonium plus PPADS in the recording chamber. We conclude that P2X receptors are important for synaptic transmission from descending interneurons to inhibitory motor neurons in descending inhibitory reflex pathways of guinea-pig ileum. Transmission from anally directed axons of distension-sensitive intrinsic sensory neurons to inhibitory motor neurons is unlikely to involve P2X, muscarinic or nicotinic receptors.

Figure 1. Schematic diagram illustrating the experimental design

A, organ bath (cross-hatching) divided by a plastic wall () into an oral stimulation chamber and an anal recording chamber. A balloon (white) was embedded in the stimulation chamber to distend the intestinal wall and a pair of silver wires () was place above and below the sheet of intestine in the recording chamber to allow electrical stimulation of the enteric nerves. An opened segment of ileum (black) was pinned mucosa up in the divided organ bath. The anal end of the segment was folded over to allow microelectrodes to be inserted through the longitudinal muscle into circular muscle cells. B, the descending neural circuit inferred from anatomical and physiological studies. All intrinsic sensory neurons excite neighbouring descending interneurons, and some have long descending axons that can directly excite inhibitory motor neurons. EFS excites both local inhibitory motor neurons and the pathways that impinge on them.

Figure 2. Effects of suramin (100 μm) on IJPs evoked by distension and EFS in guinea-pig ileum

A, IJPs recorded from circular muscle in control (top) and IJPs evoked in the same circular muscle cells when suramin was present in the recording chamber (bottom). The traces on the left show IJPs evoked by EFS and on the right are IJPs evoked by distension. The strength of each stimulus was adjusted so that the control responses were about 12 mV. B, pooled data from 8 experiments showing the relative amplitudes (expressed as a percentage of the controls) of IJPs evoked by EFS and distension with suramin in the recording chamber. Error bars show the s.e.m. in this and all subsequent figures. IJPs evoked by EFS and distension were both reduced by suramin (P < 0·05, Student’s paired t test). IJPs evoked by distension were significantly more depressed than IJPs evoked by EFS (P < 0·01, Wilcoxon signed rank test).

Figure 3. Effect of PPADS (10 μm) or PPADS (10 μm) plus hexamethonium (200 μm) on IJPs evoked by distension and EFS

A, IJPs recorded from a circular muscle cell in control (top) and when PPADS (middle) or PPADS and hexamethonium (Hex) (bottom) were present in the recording chamber. The left column shows IJPs evoked by EFS and the right IJPs evoked by distension. B, the relative amplitudes of IJPs evoked by EFS and distension with PPADS () or PPADS plus hexamethonium (▪) in the recording chamber. IJPs evoked by EFS and distension were reduced by PPADS (P < 0·05, Student’s paired t test, n = 12). IJPs evoked by distension were significantly more depressed than IJPs evoked by EFS (P < 0·01, Wilcoxon signed rank test, n = 12). Addition of hexamethonium to the PPADS in the recording chamber significantly reduced the IJPs evoked by EFS, but did not depress IJPs evoked by distension more than PPADS alone (one-way ANOVA, n = 6).

Figure 4. Effect of PPADS (10 μm) on IJPs evoked by EFS and mucosal compression

A, IJPs recorded from a circular muscle cell in control (top), with PPADS in the recording chamber (middle) and after 20 min washout of the PPADS (bottom). B, relative amplitudes of IJPs evoked by EFS (left) and compression (right) with PPADS in the recording chamber. IJPs evoked by EFS and compression were both reduced by PPADS (P < 0·05, n = 7). IJPs evoked by compression were significantly more depressed than those evoked by EFS (P < 0·01, Wilcoxon signed rank test).

Figure 5. Effect of PPADS (10 μm) in the recording chamber on IJPs evoked by EFS and distension when synaptic transmission in the stimulation chamber was blocked

A, IJPs recorded from a circular muscle cell in control (top) and when PPADS was present in the recording chamber (bottom). B, relative amplitudes of IJPs evoked by EFS (left) and distension (right) with PPADS in the recording chamber (n = 6). There were no significant changes in the responses to either stimulus (P > 0·05, n = 6). Synaptic transmission in the stimulation chamber was blocked with a low Ca²⁺-high Mg²⁺ solution.

Figure 6. Lack of effect of muscarinic and nicotinic antagonists in the recording chamber, when synaptic transmission is blocked in the stimulation chamber

A, IJPs recorded from a circular muscle cell in control (a), in hyoscine (1 μm; b), in hyoscine (1 μm) plus hexamethonium (200 μm; c) and in hyoscine (1 μm) plus hexamethonium (200 μm) and PPADS (10 μm; d). B, the relative amplitudes of IJPs evoked by EFS and distension in hyoscine (□), in hyoscine plus hexamethonium () and in hyoscine plus hexamethonium and PPADS (▪). Hyoscine plus hexamethonium in the recording chamber significantly reduced the IJPs evoked by EFS (P < 0·01, one-way ANOVA; n = 6), but not the IJPs evoked by distension. Hyoscine plus hexamethonium and PPADS did not significantly change the IJPs evoked by either EFS or distension (P > 0·05, one-way ANOVA, n = 5).