Purinergic neuromuscular transmission is selectively attenuated in ulcerated regions of inflamed guinea pig distal colon

Abstract

This study was undertaken to investigate neuromuscular transmission in regions of the inflamed colon in which motility is disrupted. Propulsive motility was evaluated in segments of control guinea pigs and those treated 6 days previously with trinitrobenzene sulfonic acid. Intracellular recordings were then obtained from circular muscle cells to examine excitatory and inhibitory junction potentials (EJPs and IJPs). In inflamed preparations, propulsion of fecal pellets was temporarily halted or obstructed at sites of mucosal damage, whereas the propulsive motility was linear in control colons. The amplitudes of evoked and spontaneous IJPs were significantly reduced in ulcerated regions of inflamed preparations, but EJPs were comparable to controls. Pharmacological dissection of the IJP revealed that the purinergic component was reduced, while the nitrergic IJP was slightly increased. Furthermore, the reduction in the purinergic IJP in inflamed preparations persisted in the presence of hexamethonium, suggesting that the deficit involved the inhibitory motor neuron and/or smooth muscle. Nerve fibre density was not altered in the circular muscle, and pre-contracted rings of inflamed colon relaxed normally to ATP, suggesting that the deficit involves altered ATP release and/or degradation. The P2Y(1) receptor antagonist MRS2179 slowed propulsive motility indicating that decreased purinergic neuromuscular transmission could contribute to the inflammation-induced motor deficit. We conclude that purinergic inhibitory neuronal input to the circular muscle is selectively reduced in regions of the colon in experimental colitis where the mucosa is damaged, and this is likely to contribute to altered motility in colitis by diminishing downstream relaxation during the peristaltic reflex.

Figure 1. Propulsive motility is linear in colons from non-treated animals, but is disrupted in ulcerated regions 6 days following TNBS administration

A, data from a normal colon illustrating the progress of a fecal pellet over time in 3 consecutive trials. B, graph illustrating data from a TNBS-treated animal in which propulsive motility was temporarily halted in ulcerated regions. This pattern was observed in 51 of 73 (70%) animals tested. C, graph illustrating data from a TNBS-treated animal in which propulsive motility was completely obstructed at the site of an ulcer. This pattern was observed in 15 of 73 (21%) animals tested.

Figure 2. Evoked inhibitory junction potentials (IJPs) are reduced at an ulcer, but excitatory junction potentials (EJPs) are unchanged

Junction potentials were evoked by transmural electrical field stimulation 2–10 mm oral (IJPs) or aboral (EJPs) from the impaled circular muscle cell, which in TNBS-treated preparations, was at the site of an ulcer. A, graph showing IJP amplitude in ulcerated regions versus control with transmural stimulation at distances of 10 mm and 2 mm oral to the recording electrode. B, representative traces for IJPs at 10 mm (top traces) and 2 mm (bottom traces). C, graph illustrating EJP amplitude in ulcerated regions versus control with transmural stimulation at distances of 10 mm and 2 mm oral to the recording electrode. D, representative traces for EJPs at 10 mm (top traces) and 2 mm (bottom traces).

Figure 3. The frequencies of spontaneous IJPs and EJPs, and the amplitudes of spontaneous IJPs were reduced in TNBS-inflamed preparations

A and B, quantification of spontaneous IJP frequency (A) and amplitude (B) in ulcerated regions versus control. C, representative traces illustrating spontaneous IJPs. D and E, quantification of spontaneous EJP frequency (D) and amplitude (E) in ulcerated regions versus control. F, representative traces illustrating spontaneous EJPs.

Figure 4. The purinergic component of the IJP is reduced in TNBS-inflamed preparations, but the nitrergic component of the IJP is unchanged

A, graph illustrating evoked purinergic IJP amplitudes in the presence of l-NNA. B, representative recordings of IJPs in the presence of l-NNA. C, graph of evoked nitrergic IJPs in the presence of apamin. D, representative traces of IJPs in the presence of apamin. *P < 0.0001.

Figure 5. The frequency and amplitude of spontaneous purinergic IJPs were reduced in TNBS-inflamed preparations

A and B, quantification of spontaneous purinergic IJP frequency (A) and amplitude (B) in ulcerated regions versus control. C, representative traces illustrating spontaneous purinergic IJPs.

Figure 6. The attenuation of the IJP in the ulcerated region does not appear to involve TNBS-induced AH neuron hyperexcitability or facilitation of interneuronal synaptic activity

A, graph of data from TNBS-treated preparations illustrating that the amplitude of the IJP is not increased by the HCN channel blocker, ZD7288 (10 μm), which should increase the afterhyperpolarization in AH neurons and decrease their excitability. B, graph illustrating that the TNBS colitis-induced attenuation of the evoked IJP is not reversed by inhibition of interneuronal synaptic transmission with 300 μm hexamethonium. *P < 0.002.

Figure 7

At a 56 day post-TNBS treatment time point, which is 4 weeks after inflammation can be detected, the amplitude of the evoked IJP is comparable to that detected in weight-matched control preparations

Figure 8. The concentration–effect curves for ATP-induced relaxations of colonic rings that were pre-contracted with bethanechol were comparable

No significant differences were detected between control and TNBS treated colons at any given ATP concentration (ANOVA; n= 4 per group).

Figure 9. The density of nerve fibres in the circular muscle layer is comparable in the normal colon as compared to the ulcerated region of the TNBS-inflamed colon

A, graph illustrating measurements of nerve fibres immunostained with antisera directed against NOS and choline acetyltransferase. Immunostaining for both antigens were observed together in circular muscle fibre bundles. B, micrographs of NOS immunoreactivity in sections of normal and inflamed distal colon.

Figure 10. Inhibition of P2Y₁ receptors decreases propulsive motility in the distal colon

The P2Y₁ receptor is known to mediate inhibitory purinergic neurotransmission in colonic smooth muscle. To test the effects of blockade of this receptor, motility experiments were performed on colons from non-treated animals (n= 10) with bath application of the selective P2Y₁ receptor antagonist, MRS-2179 (3 μm). Graph demonstrates a significant decrease in propulsive motility. *P < 0.002, paired t test.