Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA311

Poster Communications

Muscarinic receptor-induced contractions of the detrusor are mediated by activation of TRPC4 channels

C. Griffin1, M. Hollywood1, K. D. Thornbury1, N. G. McHale1, G. P. Sergeant1

1. DKIT, Dundalk, Ireland.

Activation of M3 muscarinic receptors (M3Rs) on detrusor myocytes by acetycholine (ACh), is the predominant mechanism responsible for contraction of the bladder (Andersson, 1993). Muscarinic receptor-mediated contractions of the detrusor rely on Ca2+ influx through voltage-gated Ca2+ channels, but the mechanism that links stimulation of M3Rs to activation of voltage-dependent Ca2+ channels has not been established. Transient receptor potential channel 4 (TRPC4) are receptor operated cation channels that couple muscarinic receptor activation to depolarisation of intestinal smooth muscle cells, voltage-activated Ca2+ influx and contraction (Tsvilovskyy, 2009). The purpose of this study was to investigate if TRPC4 channels are involved in cholinergic-mediated contractions of the detrusor. Isometric tension recordings were made from strips of murine detrusor and intracellular Ca2+ measurements were made from isolated detrusor myocytes using confocal microscopy. Transcriptional expression of TRPC and IP3R isoforms in intact detrusor strips and isolated detrusor myocytes was assessed using RT-PCR. All procedures were carried out in accordance with current EU legislation (EU Directive 2010/63/EU) and with approval from Dundalk Institute of Technology Animal Use and Care Committee. Cholinergic stimulation of the detrusor, induced by electric field stimulation (EFS) or via exogenous application of carbachol (300 nM), or neostigmine (1 μM), evoked two types of contraction; i) a transient, plus tonic response, that was blocked by the TRPC4 blocker, ML204 (10 μM) and ii) a phasic oscillatory response that was blocked by the IP3R inhibitor, 2-APB (100 μM). ML204 reduced the overall contraction amplitude (expressed as the area under the contraction) in response to 4 Hz EFS from 704.6 ± 127.7 to 400.6 ± 109.9 mN.s, and 2-APB reduced the residual contraction to 15.3 ± 4.8 mN.s (p<0.05, n=7). Similarly carbachol-evoked contractions were reduced from 6220 ± 724 to 1777 ± 232 mN.s, and 2-APB further inhibited the remaining response to 102.2 ± 40.7 mN.s (p<0.01, n=6). Carbachol induced reproducible Ca2+ responses in isolated murine detrusor myocytes. ML-204 inhibited the initial component of the response, whereas 2-APB reduced the oscillatory component. For example, ML204 reduced the initial response from 2.85 ± 0.33 to 0.37 ± 0.16 Δ(F/F0) (p<0.001, n=8). RT-PCR experiments showed that TRPC4β, TRPC6 and IP3R1 were selectively expressed in isolated detrusor myocytes. Control experiments showed that ML204 did not affect L-type Ca2+ or BK current amplitude, caffeine-induced Ca2+ transients or KCl-induced contractions of the detrusor. These data show that muscarinic receptor-mediated contractions of the murine detrusor involve activation of TRPC4β channels.

Where applicable, experiments conform with Society ethical requirements