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. 2020 Jul 29;21(15):5403.
doi: 10.3390/ijms21155403.

TRPM8 Channel Activation Reduces the Spontaneous Contractions in Human Distal Colon

Antonella Amato  1 , Simona Terzo  1   2 , Laura Lentini  1 , Pierenrico Marchesa  3 , Flavia Mulè  1
Affiliations

TRPM8 Channel Activation Reduces the Spontaneous Contractions in Human Distal Colon

Antonella Amato et al. Int J Mol Sci. .

Abstract

The transient receptor potential-melastatin 8 (TRPM8) is a non-selective Ca2+-permeable channel, activated by cold, membrane depolarization, and different cooling compounds. TRPM8 expression has been found in gut mucosal, submucosal, and muscular nerve endings. Although TRPM8 plays a role in pathological conditions, being involved in visceral pain and inflammation, the physiological functions in the digestive system remain unclear as yet. The aims of the present study were: (i) to verify the TRPM8 expression in human distal colon; (ii) to examine the effects of TRPM8 activation on colonic contractility; (iii) to characterize the mechanism of action. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting were used to analyze TRPM8 expression. The responses of human colon circular strips to different TRPM8 agonists [1-[Dialkyl-phosphinoyl]-alkane (DAPA) 2-5, 1-[Diisopropyl-phosphinoyl]-alkane (DIPA) 1-7, DIPA 1-8, DIPA 1-9, DIPA 1-10, and DIPA 1-12) were recorded using a vertical organ bath. The biomolecular analysis revealed gene and protein expression of TRPM8 in both mucosal and smooth muscle layers. All the agonists tested, except-DIPA 1-12, produced a concentration-dependent decrease in spontaneous contraction amplitude. The effect was significantly antagonized by 5-benzyloxytryptamine, a TRPM8 antagonist. The DIPA 1-8 agonist resulted in the most efficacious and potent activation among the tested molecules. The DIPA 1-8 effects were not affected by tetrodotoxin, a neural blocker, but they were significantly reduced by tetraethylammonium chloride, a non-selective blocker of K+ channels. Moreover, iberiotoxin, a blocker of the large-conductance Ca2+-dependent K+-channels, but not apamin, a blocker of small-conductance Ca2+-dependent K+ channels, significantly reduced the inhibitory DIPA 1-8 actions. The results of the present study demonstrated that TRPM8 receptors are also expressed in human distal colon in healthy conditions and that ligand-dependent TRPM8 activation is able to reduce the colonic spontaneous motility, probably by the opening of the large-conductance Ca2+-dependent K+-channels.

Keywords: 1-[Diisopropyl-phosphinoyl]-alkane (DIPA); IBS; TRPM-8; human colon contractility.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
TRPM8 mRNA and protein expression in human distal colon. (A) Representative images of the reverse transcriptase-polymerase chain reaction (RT-PCR) results. A product of 621 bp corresponding to TRPM8 was detected in mucosa, smooth muscle, and HeLa cells used as positive control. The expression of β-actin (396 bp) was used as loading control. Negative control was obtained without addition of cDNA. (B) Western blot detecting protein levels for TRPM8 from colon mucosa and muscle. (C) Densitometric analysis of blots was performed using the NIH Image J 1.40 analysis program. HeLa cells were used as positive control. Human tubulin was used as loading control. Negative control was obtained by using blocking peptide added to TRPM8 Ab. n = 6; * p < 0.05 when compared to TRPM8 mucosa expression.
Figure 2
Figure 2
Typical recordings showing the inhibitory effects of increasing concentrations of DAPA 2–5 (1 μM–1 mM) (A), DIPA 1–7 (1 nM–1 mM) (B), DIPA 1–8 (1 nM–100 μM) (C), DIPA 1–9 (1 nM–100 μM) (D), DIPA 1–10 (1 nM–1 mM) (E), and DIPA 1–12 (10 nM–1 mM) (F) on the spontaneous contractions of human colon circular muscle. C = spontaneous contractions in control conditions. W = spontaneous contractions after washing out. Dotted line indicates the basal tone of the preparation.
Figure 3
Figure 3
Concentration–response curves showing the inhibitory effects of increasing concentrations of DAPA 2–5 (1 μM–1 mM) (A), DIPA 1–7 (1 nM–1 mM) (B), DIPA 1–8 (1 nM–100 μM) (C), DIPA 1–9 (1 nM–100 μM) (D), and DIPA 1–10 (1 nM–1 mM) (E) on the spontaneous contractions of human colon circular muscle, in the presence or in the absence of 5-BT (1 μM). Data are means S.E.M. (n = 6 for each experimental conditions) and are expressed as percentage of inhibition of the spontaneous contractions. * p < 0.05 compared with the respective control conditions.
Figure 4
Figure 4
Concentration–response curves for the inhibitory effects induced by DIPA 1–8 (1 nM–100 μM) before and after TTX (1 μM) (A) and TEA (10 mM) (B). All values are means ± S.E.M (n = 6) and are expressed as percentage of inhibition of the spontaneous contractions. * p <0.05 compared with the respective control conditions.
Figure 5
Figure 5
Concentration–response curves for the inhibitory effects induced by DIPA 1–8 (1 nM–100 μM) before and after apamin (100 nM) (A) and IbTX (10 µM) (B). All values are means ± S.E.M (n = 6) and are expressed as percentage of inhibition of the spontaneous contractions. * p <0.05 compared with the respective control conditions.
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