Differential Contribution of TRPA1, TRPV4 and TRPM8 to Colonic Nociception in Mice

Abstract

Background: Various transient receptor potential (TRP) channels in sensory neurons contribute to the transduction of mechanical stimuli in the colon. Recently, even the cold-sensing menthol receptor TRPM(melastatin)8 was suggested to be involved in murine colonic mechano-nociception.

Methods: To analyze the roles of TRPM8, TRPA1 and TRPV4 in distension-induced colonic nociception and pain, TRP-deficient mice and selective pharmacological blockers in wild-type mice (WT) were used. Visceromotor responses (VMR) to colorectal distension (CRD) in vivo were recorded and distension/pressure-induced CGRP release from the isolated murine colon ex vivo was measured by EIA.

Results: Distension-induced colonic CGRP release was markedly reduced in TRPA1-/- and TRPV4-/- mice at 90/150 mmHg compared to WT. In TRPM8-deficient mice the reduction was only distinct at 150 mmHg. Exposure to selective pharmacological antagonists (HC030031, 100 μM; RN1734, 10 μM; AMTB, 10 μM) showed corresponding effects. The unselective TRP blocker ruthenium red (RR, 10 μM) was as efficient in inhibiting distension-induced CGRP release as the unselective antagonists of mechanogated DEG/ENaC (amiloride, 100 μM) and stretch-activated channels (gadolinium, 50 μM). VMR to CRD revealed prominent deficits over the whole pressure range (up to 90 mmHg) in TRPA1-/- and TRPV4-/- but not TRPM8-/- mice; the drug effects of the TRP antagonists were again highly consistent with the results from mice lacking the respective TRP receptor gene.

Conclusions: TRPA1 and TRPV4 mediate colonic distension pain and CGRP release and appear to govern a wide and congruent dynamic range of distensions. The role of TRPM8 seems to be confined to signaling extreme noxious distension, at least in the healthy colon.

Fig 1. Normalized pressure/distension (30 mmHg—150 mmHg)-induced CGRP release from isolated colon of wild-type C57BL/6 mice (ΔCGRP release in pg/ml).

Pressure-dependent increased CGRP release shows a plateau in the middle pressure range. “*” indicates significance level at p < 0.05, “##” at p < 0.01 (Mann-Whitney U-test) versus 30 mmHg-induced colonic CGRP release. Each data point is representative for 6–10 experiments with different animals.

Fig 2

(A,C) Time course of 90 and 150 mmHg-induced CGRP release from the isolated colon of wild-type (WT) mice as compared to TRPA1, TRPV4 and TRPM8 knockout mice. (B,D) Normalized 90 and 150 mmHg-induced CGRP release from the isolated colon of WT as compared to TRPA1, TRPV4 and TRPM8 knockout mice. TRPA1^-/- and TRPV4^-/- mice show reduced pressure-induced colonic CGRP release compared to WTs at both pressure levels. In TRPM8-deficient mice pressure-induced CGRP release was only reduced at the high (150 mmHg) pressure level compared to WT. “+”, “#” and “*” indicate significance levels at p < 0.05 (Mann-Whitney U-test) versus WT mice. Each data point n = 6–10.

Fig 3

(A) Exposure of the isolated colon of wild-type (WT) mice to selective antagonists of TRPA1 (HC030031, HC, 100 μM) and TRPV4 (RN1734, RN, 10 μM) inhibited 90 mmHg-induced CGRP release. The TRPM8 antagonist AMTB (10 μM) had no effect at this pressure level. The unselective TRP-channel blocker ruthenium red (RR, 10 μM), the DEG/ENaC antagonist amiloride (Amil, 100 μM) and the SAC blocker gadolinium (Gadol, 50 μM) were about equally effective as HC and RN. (B) Analogeous results at 150 mmHg with the same pharmacological blockers, however, the TRPM8 antagonist AMTB distinctly reduced 150 mmHg-induced CGRP release. “*” indicates significance level at p < 0.05 and “**” at p < 0.01 (Mann-Whitney U-test) versus WT. Each data point is representative for 6–10 experiments.

Fig 4. Representative pictures of murine visceromotor responses (VMR) to 90 mmHg-induced colorectal distension.

Original EMG (in mV) recorded from the abdominal muscle in the different mouse strains during the 25 s of pressure/distension stimulation.

Fig 5. Representative pictures of murine visceromotor responses (VMR) to 90 mmHg-induced colorectal distension.

Original abdominal EMG (in mV) in mice treated with the respective TRP receptor antagonist compared to wild-type mice.

Fig 6

(A) Visceromotor responses to colorectal distension in WT compared to TRPM8, TRPA1 and TRPV4 knockout mice. Visceromotor responses (VMR) are presented as integrated EMG (iEMG in μVxs). TRPA1 and TRPV4 but not TRPM8 knockouts demonstrate reduced pain behavior at all tested pressure levels. Baseline EMG activity was about equal in all mouse strains. (B) Accordingly, the respective pharmacological blockers for TRPA1 and TRPV4 but not TRPM8 reduced VMRs at all pressure levels at the given dosage (HC 10 mg/kg, RN 1 mg/kg, AMTB 10 mg/kg). “+”indicates significance for TRPA1^-/- or HC vs. WT, “#” for TRPV4^-/- or RN vs. WT (p < 0.05; 2-way ANOVA followed by post hoc LSD test). Each data point is representative for 6–10 experiments.