Inhibiting fatty acid amide hydrolase normalizes endotoxin-induced enhanced gastrointestinal motility in mice

Abstract

Background and purpose: Gastrointestinal (GI) motility is regulated in part by fatty acid ethanolamides (FAEs), including the endocannabinoid (EC) anandamide (AEA). The actions of FAEs are terminated by fatty acid amide hydrolase (FAAH). We investigated the actions of the novel FAAH inhibitor AM3506 on normal and enhanced GI motility.

Experimental approach: We examined the effect of AM3506 on electrically-evoked contractility in vitro and GI transit and colonic faecal output in vivo, in normal and FAAH-deficient mice treated with saline or LPS (100 µg·kg(-1), i.p.), in the presence and absence of cannabinoid (CB) receptor antagonists. mRNA expression was measured by quantitative real time-PCR, EC levels by liquid chromatography-MS and FAAH activity by the conversion of [(3)H]-AEA to [(3)H]-ethanolamine in intestinal extracts. FAAH expression was examined by immunohistochemistry.

Key results: FAAH was dominantly expressed in the enteric nervous system; its mRNA levels were higher in the ileum than the colon. LPS enhanced ileal contractility in the absence of overt inflammation. AM3506 reversed the enhanced electrically-evoked contractions of the ileum through CB(1) and CB(2) receptors. LPS increased the rate of upper GI transit and faecal output. AM3506 normalized the enhanced GI transit through CB(1) and CB(2) receptors and faecal output through CB(1) receptors. LPS did not increase GI transit in FAAH-deficient mice.

Conclusions and implications: Inhibiting FAAH normalizes various parameters of GI dysmotility in intestinal pathophysiology. Inhibition of FAAH represents a new approach to the treatment of disordered intestinal motility.

Figure 1

(A) Chemical structure of AM3506. AM3506 is a structural analogue of phenylmethylsulphonyl fluoride. (B) MPO activity; (C) TNF; (D) FAAH mRNA; (E) CB₁ receptor; and (F) CB₂ receptor expression in the ileum and distal colon of CD1 mice treated with saline (control) or LPS (100 µg·kg⁻¹, i.p.). There were no significant changes in MPO activity after LPS treatment, although TNF mRNA was significantly increased in both ileum and distal colon. LPS treatment had no effect on FAAH mRNA, which was significantly higher in the ileum than the colon. CB₁ receptor mRNA was increased in the distal colon, and CB₂ receptor mRNA was reduced in both regions of the gut by LPS treatment. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control in (E, C and F) and compared with ileum in (D); n = 4–6 per group.

Figure 2

FAAH immunoreactivity in full-wall thickness sections (A–D) and whole mount preparations of the myenteric plexus (E–H) of the ileum and distal colon from animals treated with saline and LPS. LPS (100 µg·kg⁻¹, i.p.) treatment did not alter the distribution of FAAH (B, D, F and H). Scale bars: 100 µm (A–D) and 50 µm (E–H).

Figure 3

FAAH activity in the ileum (A) and the distal colon (B) of CD1 mice. Note that FAAH activity in the colon of control group was significantly lower than in the ileum (P < 0.05). LPS (100 µg·kg⁻¹, i.p.) treatment or incubation with AM3506 (100 nM) did not inhibit FAAH activity in the ileum and the distal colon. After incubation with AM3506 (100 nM), a significant reduction in the FAAH activity of the ileum of LPS-treated mice was observed (A). *P < 0.01 compared with control; n = 6–10 per group.

Figure 4

FAE and 2-AG levels in the ileum and distal colon of saline-treated (control) or LPS (100 µg·kg⁻¹, i.p.)-treated mice pretreated with AM3506 (0.5 mg·kg⁻¹, i.p.) or its vehicle. AM3506 treatment enhanced AEA levels in the ileum (A), but not the colon (B) of LPS-treated mice. There were no significant differences in the levels of PEA (C and D), OEA (E and F) or 2-AG (G and H) in the ileum (C, E and G) or colon (D, F and H). *P < 0.05 compared with control; n = 3–10 per group.

Figure 5

Effect of LPS (100 µg·kg⁻¹, i.p.) and AM3506 (100 nM) on EFS-evoked contractions (4 Hz in C and D) of mouse ileum (A and C) and distal colon (B and D) and bethanechol(300 nM–10 µM)-induced contractions of mouse ileum and distal colon (E and F). Note that LPS enhanced contractility in the ileum (A) but not the distal colon (B) of the CD1 mice at all frequencies. (C) AM3506 reduced ileal contractions in LPS-treated CD1 mice but not in control mice. The trace represents the effect of AM3506 on ileal EFS contractility in a LPS-treated mouse. The arrow shows the time that tissue was exposed to AM3506 (100 nM). (D) AM3506 had no effect on colonic electrical contractions in control and LPS-treated animals. The trace represents the effect of AM3506 on colonic EFS contractility in a LPS-treated mouse. The arrow shows the time that tissue was exposed to AM3506 (100 nM). (E and F) Contractilities induced by bethanechol were not changed after LPS treatment. *P < 0.05 compared with control; n = 4–8 per group in all cases.

Figure 6

Effect of AM3506 (100 nM) on ileal contractions in gene-deficient mice and in the presence of CB receptor antagonists. (A) AM3506 did not change EFS contractility in control or LPS(100 µg·kg⁻¹, i.p.)-treated FAAH^−/− mice. (B) AM251 (100 nM) and AM630 (300 nM) blocked the effect of AM3506 on ileal contractility in LPS treated CD1 mice. (C) AM3506 had no effect on ileal contractions of vehicle or LPS-treated CB₁^−/− mice; however, it decreased EFS contractility in C57BL/6N wild-type mice treated with LPS (100 µg·kg⁻¹, i.p.). *P < 0.05 compared with AM3506 in (B) and compared with vehicle in (C); n = 4–8 per group in all cases.

Figure 7

The effect of WIN55,212-2 (10–100 nM) and JWH133 (100 nM–10 µM) in the ileum and distal colon of CD1 mice after treatment with saline (control) or LPS (100 µg·kg⁻¹, i.p.). (A) WIN55,212-2 significantly reduced contractility of the ileum in control and LPS-treated mice to about the same extent and this effect was fully reversed by the CB₁ receptor antagonist AM251 (100 nM) but not the CB₂ receptor antagonist AM630 (300 nM). (B) WIN55,212-2 reduced contractility of the colon in control mice, but this was only significant after LPS treatment. AM251 did not fully reverse the effects of WIN55,212-2 (100 nM) after LPS treatment in the distal colon. (C) JWH133 concentration-dependently reduced contractions evoked by EFS in the ileum. This effect was abolished by AM630 (300 nM). (D) JWH133 had no effect on evoked contractions in the distal colon. *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle; n = 3–6 per group for all panels.

Figure 8

Effect of AM3506 (0.5 mg·kg⁻¹, i.p.) on upper GI transit of a charcoal marker (A) and total stool weight (B) of control and LPS (100 µg·kg⁻¹, i.p.)-treated mice. (A) AM3506 reversed the enhanced transit produced by LPS while having no effect alone. The effect of AM3506 was reversed by AM251 and the higher dose of AM630. (B) AM3506 reversed the enhanced stool output produced by LPS while having no effect alone. The effect of AM3506 was reversed by AM251, but not by AM630. (C) Effect of LPS (100 µg·kg⁻¹, i.p.) and AM3506 (0.5 mg·kg⁻¹, i.p.) on ambulatory motor activity of mice; AM3506 did not decrease ambulatory motor activity. LPS decreased ambulatory activity to the same extent in AM3506- or vehicle-pretreated mice. WIN55,212-2 (5 mg·kg⁻¹) significantly decreased ambulatory activity. *P < 0.05, **P < 0.01, ***P < 0.001, bar indicates significant differences between the groups. n = 4–14 per group.

Figure 9

GI transit in FAAH-deficient (FAAH^−/−) and wild-type (WT) mice. (A) Whole-gut transit was identical in both groups. (B) Upper GI transit was identical in both groups under control conditions; however, LPS (100 µg·kg⁻¹, i.p.) increased transit in wild-type but not the FAAH^−/− mice. *P < 0.05; n = 4–8 per group.