TRPM8 Channel Activation Reduces the Spontaneous Contractions in Human Distal Colon
Antonella Amato, Simona Terzo, Laura Lentini, Pierenrico Marchesa, Flavia Mulè
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.