Pico145 inhibits TRPC4-mediated mICATand postprandial small intestinal motility

Abstract

AbstractBackground & AimsIn intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mICAT), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mICATand Ca2+signalling in mouse ileal myocytes, and on intestinal motility.MethodsIleal 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 standardin vitrotensiometry,ex vivovideo recordings andin vivopostprandial intestinal transit measurements using carmine red.ResultsCarbachol (50 µM)-induced mICATwas strongly inhibited by Pico145 starting from 1 pM. The IC50value 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 IC50of 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.ConclusionsPico145 is a potent GI-active small-molecule which completely inhibits mICATat picomolar concentrations and which is as effective astrpc4gene deficiency inin vivointestinal motility tests.