Pico145 inhibits TRPC4-mediated mI_CATand postprandial small intestinal motility

Abstract Background & Aims 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 Ca 2+ signalling in mouse ileal myocytes, and on intestinal motility. Methods 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 Ca 2+ 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. Results Carbachol (50 µM)-induced mI CAT was strongly inhibited by Pico145 starting from 1 pM. The IC 50 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 Ca 2+ rises were inhibited with IC 50 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. Conclusions 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.