Pico145 inhibits TRPC4-mediated mICAT and postprandial small intestinal motility

Abstract

In intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mI_CAT), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mI_CAT and Ca²⁺ signalling in mouse ileal myocytes, and on intestinal motility. Ileal myocytes freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents and for intracellular Ca²⁺ imaging using Fura-2. Functional assessment of Pico145's effects was carried out by standard in vitro tensiometry, ex vivo video recordings and in vivo postprandial intestinal transit measurements using carmine red. Carbachol (50 µM)-induced mI_CAT was strongly inhibited by Pico145 starting from 1 pM. The IC₅₀ value for the inhibitory effect of Pico145 on this current evoked by intracellularly applied GTPγS (200 µM), and thus lacking desensitisation, was found to be 3.1 pM, while carbachol-induced intracellular Ca²⁺ rises were inhibited with IC₅₀ of 2.7 pM. In contrast, the current activated by direct TRPC4 agonist (-)-englerin A was less sensitive to the action of Pico145 that caused only ∼43 % current inhibition at 100 pM. The inhibitory effect developed rather slowly and it was potentiated by membrane depolarisation. In functional assays, Pico145 produced concentration-dependent suppression of both spontaneous and carbachol-evoked intestinal smooth muscle contractions and delayed postprandial intestinal transit. Thus, Pico145 is a potent GI-active small-molecule which completely inhibits mI_CAT at picomolar concentrations and which is as effective as trpc4 gene deficiency in in vivo intestinal motility tests.

Keywords: Intestinal motility; Intestinal smooth muscles; Pico145; TRPC4 channels.