Divergent changes to muscarinic and serotonergic signalling following colitis

Abstract

Background: The altered motility of the inflamed intestine derives in part from changes to the contractility of the intestinal smooth muscle cell. While modifications to the muscarinic receptor system are identified, changes to 5-hydroxytryptamine (5-HT; serotonin) receptors that also mediate contraction are less well studied.

Methods: In the trinitrobenzene sulphonic acid model of rat colitis, we used receptor antagonists to identify changes in receptor utilisation that accompany the selective reversal of the impaired contractile response to acetylcholine (ACh) and 5-HT during colitis (day 4 (D4)) and following resolution of inflammation (day 36 (D36)).

Results: In isolated circular smooth muscle cells, challenged with ACh, the muscarinic 3 receptor (M3R) antagonists 4-DAMP and pF-HSD each showed a 50% decrease in antagonism on D4 while the M2R antagonist methoctramine more than doubled its potency, showing a decreased role of M3R and an increased role of M2R, respectively. These changes were fully reversed by D36. In contrast, the 5-HT2 receptor (5-HT2R) antagonist ketanserin was sharply decreased in effectiveness on D4, with a further decrease by D36, when the contribution of 5-HT(2A)R was only 22% of control. There were no changes in response to the 5-HT4R antagonist SDZ-205-557 at any time. Western blotting identified decreased expression of 5-HT(2A)R on D36 versus controls, further supporting the conclusion that the persistence of the impaired response to 5-HT was due to decreased expression of the excitatory 5-HT(2A)R.

Conclusions: Thus the lasting decrease in receptor expression and resulting impairment of the contractile response will compromise the capacity for an appropriate response to 5-HT, which may contribute to the intestinal dysfunction seen in post-enteritis syndromes.

Figure 1

Inflammation reversibly impairs acetylcholine (ACh) induced contraction of circular smooth muscle cells (CSMC) from the rat colon. CSMC were enzymatically isolated on day 0 (DO, control), day 4 (D4), and day 36 (D36) post colitis, and contraction assessed under direct microscopic visualisation at 37°C. (A–C) Micrographs showing contraction of an individual D0 CSMC following application of 1 μM ACh by micropipette, seen on the right side of each image (arrowhead). (A) Control; (B) +750 ms; and (C) +3500 ms after ACh application. Scale bar 100 μm. (D) Time course of mean per cent contraction of cells from D0, D4, and D36 animals in response to ACh (n = 3 animals per category at 10 cells/animal). Arrows indicate per cent contraction of the cell illustrated in (A–C). Per cent contraction for D4 was significantly less than that for D0 and D36 at all time points ⩾500 ms (p⩽0.05).

Figure 2

Inflammation alters utilisation of muscarinic receptors in acetylcholine (ACh) induced contraction of colonic circular smooth muscle cells isolated from rats on day 0 (D0, control), day 4 (D4), and day 36 (D36) post colitis. ACh was applied in the presence of the selective muscarinic M₂ (methoctramine) and M₃ (4-DAMP or F-HSD) receptor antagonists. Data are presented as per cent inhibition of maximal contraction under each condition (that is, per cent of the contraction of cells receiving no antagonist). Each population of cells served as their own control for maximal contraction before treatment, thus normalising for the differences in contraction that occur during inflammation. During inflammation (D4), the M₃R antagonists pF-HSD (A) and 4-DAMP (B) became significantly less effective at blocking contraction but this decrease was reversed following resolution of the inflammation (D36). In contrast, the M₂R antagonist methoctramine (C) showed increased effectiveness in blocking contraction during inflammation, which was also reversed following colitis. n = 4–6 animals, with 10 cells/animal. *p⩽0.05 versus D0.

Figure 3

Alterations in expression of muscarinic M₃ receptors (M₃R) in colitis. Photomicrographs of immunofluorescence detecting M₃R in sections of normal and inflamed colonic circular smooth muscle from (A) control (day 0), (B) day 4, and (C) day 36 tissue. Day 0 and day 36 tissue demonstrated a similar diffuse staining pattern that was membrane associated. In contrast, M₃R localisation on day 4 smooth muscle was largely perinuclear (arrows), suggesting internalisation of the M₃R. Scale bar 20 μm.

Figure 4

Colitis causes lasting impairment to 5-hydroxytryptamine (serotonin; 5-HT) induced contraction of isolated circular smooth muscle cells (CSMC). Time course of contraction of CSMC isolated from day 4 (D4) or day 36 (D36) animals showed a significant decrease in 5-HT induced response by D4 and a further significant decrease by D36 relative to control (D0). Data points represent the mean per cent contraction (n = 3 animals per condition at 10 cells/animal). Per cent contraction for D4 and D36 was significantly less than for D0 at all time points ⩾500 ms. Per cent contraction for D36 was significantly less than that for D4 at all time points ⩾2000 mc (p⩽0.05).

Figure 5

The excitatory 5-hydroxytryptamine receptor 2A (serotonin; 5-HT_2AR) is expressed in rat colonic smooth muscle. (A) Immunocytochemistry of a section of rat mid-descending colon showing localisation of 5-HT_2AR labelling (red) to smooth muscle cell junctions, with nuclear staining (blue) using Hoechst 33342. (B) Confocal microscopy showing 5-HT_2AR (green) on the surface of an isolated circular smooth muscle cell, with nuclear staining using propidium iodide (red). Staining for the 5-HT_2AR was localised in linear arrays on the cell surface. Scale bars 25 μm (A) and 10 μm (B).

Figure 6

Inflammation alters the utilisation of the 5-hydroxytryptamine (serotonin; 5-HT) receptor in contraction of circular smooth muscle cells. 5-HT was applied in the presence of the 5-HT receptor 2A (5-HT_2AR) antagonist ketanserin (A) or the 5-HT₄R antagonist SDZ-205-557 (B), with data presented as per cent inhibition of contraction relative to contraction of cells receiving no antagonist. Each population of cells served as their own controls for maximal contraction, thus normalising for the differences in contraction during inflammation. Ketanserin was less effective at blocking contraction during inflammation and there was no recovery of this impairment following resolution of the colitis. No changes were observed in the effectiveness of SDZ-205-557, showing no alteration in the contribution of 5-HT₄R to contraction. n = 3 animals per time point, with 10 cells/animal per condition; *p⩽0.05 versus day 0 (D0). D4, D36, days 4 and 36, respectively.

Figure 7

Western blot analysis showing decreased expression of 5-hydroxytryptamine receptor 2A (serotonin; 5-HT_2AR) during colitis. Inset, representative blots for 5-HT_2AR in control (day 0, D0), day 4 (D4), and day 36 (D36) animals identifying the presence of the receptor in colonic smooth muscle. Graph shows outcome of image analysis of immunoblots for 5-HT_2AR, with integrated optical density (IOD) normalised against β-actin and expressed relative to control. By D4, there was a significant decrease in the amount of 5-HT_2AR present in smooth muscle, and this remained decreased on D36. n = 5–6 experiments using n = 3–4 rats; *p⩽0.05 versus D0.