The two-pore channel 2 (TPC2) is required for NAADP-evoked contraction in permeabilized mouse detrusor myocytes

Nezahat Tugba Durlu-Kandilci1, Alison F. Brading2, Antony Galione2. 1Hacettepe University Faculty of Pharmacy Department of Pharmacology, Ankara, Turkey, 2Oxford University Department of Pharmacology, Oxford, United Kingdom.

Ca2+ mobilizing pyridine nucleotide messengers cADPR and NAADP are involved in agonist induced contractions in smooth muscle1, the former acting on the sarcoplasmic reticulum (SR) and the latter on lysosome-related organelles,. Two-pore channels (TPCs) are members of the superfamily of voltage-gated cation channels, and have recently been shown to function as NAADP receptors2. This study investigated NAADP-mediated and agonist-induced Ca2+ release in detrusor muscle from wild-type and Tpc2 knockout mice. Strips of bladder detrusor were mounted in a chamber filled with Krebs’ solution under 100 mg tension. Isometric contractions were recorded and expressed as % of a control response to 80 mM K+. Strips were permeabilized with β-escin (100 μM) for 30 min and then the intracellular stores were loaded with Ca2+ before applying an agonist to measure contraction. NAADP (1-20 μM) showed a bell-shaped concentration-response relationship in wild-type mouse detrusor cells with a maximum of 11.5 ± 1.9 % (n = 11) at 10 μM, but no response in detrusor cells from Tpc2 knockout mice. NAADP (10 μM) contraction in wild-type mice was inhibited by bafilomycin (100 nM) (2.8 ± 0.7 %, n = 6, P<0.05), which disrupts lysosomal Ca2+ storage, and by high (desensitizing) concentrations of NAADP (100 μM) (3.9 ± 0.1 %, n = 4, P<0.05). IP3 (100 μM) contracted mouse detrusor muscle that is 12.9 ± 2.8 % (n = 11) in wild type and 4.6 ± 0.7 % (n = 13) in Tpc2 knockout mice and these contractions were both completely abolished by heparin (P<0.05). Carbachol (100 μM), in the presence of GTP (100 μM), contracted both wild-type and Tpc2 knockout mice detrusor that is 29.1 ± 6.0 % (n = 14) and 46.9 ± 2.3 % (n = 9), respectively, and these contractions were reduced by both heparin (5.2 ± 0.5 %, n = 6 in wild-type and 26.7 ± 3.1 %, n = 7 in Tpc2 knockout) (P<0.05) and ryanodine (100 μM) (17.4 ± 2.8 %, n = 6 in wild-type and 11.4 ± 2.5 %, n = 8 in Tpc2 knockout) (P<0.05). Bafilomycin (100 nM) and desensitizing concentrations of NAADP (100 μM) decreased carbachol-evoked contractions in wild-type mice detrusor to 15.0 ± 2.3 % (n = 6) and 15.4 ± 3.4 % (n = 10), in respectively (P<0.05) but not in Tpc2 knockout tissue (P>0.05). In detrusor myocytes from Tpc2 knockout mice, thapsigargin (5 μM), ryanodine and heparin which abrogate SR Ca2+ storage and release, completely abolished the carbachol response (2.3 ± 1.4 %, n = 5, P<0.05), whereas bafilomycin or NAADP (100 μM) were required in addition to block the residual contraction in myocytes from wild-type mice (n = 4, P<0.05). Our data is consistent with a requirement for TPC2 channels in mediating NAADP-evoked Ca2+ release from acidic stores in detrusor muscle, and also play a role in muscarinic receptor mediated responses in these cells.

1. Galione, A. and Churchill, G. C. (2002). Interactions between calcium release pathways:multiple messengers and multiple stores. Cell Calcium 32, 343-54.
2. Calcraft, P. J., Ruas, M., Pan, Z., Cheng, X., Arredouani, A., Hao, X., Tang, J., Rietdorf, K., Teboul, L., Chuang, K. T. et al. (2009). NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature 459, 596-600.

Supported by The Wellcome Trust.