Proceedings of The Physiological Society

University of Edinburgh (2011) Proc Physiol Soc 25, C01 and PC01

Oral Communications

NAADP and two-pore channels are important for the contraction of airway smooth muscle cells

K. Rietdorf1,3, P. K. Aley1, C. Lim1, M. D. Bootman2, M. J. Sanderson3, J. Parrington1, G. C. Churchill1, A. Galione1

1. Department of Pharmacology, Oxford University, Oxford, United Kingdom. 2. Laboratory of Molecular Signalling, The Babraham Institute, Cambridge, United Kingdom. 3. Department of Microbiology and Physiology Systems, University of Massachusetts Medical School, Worcester, Massachusetts, United States.

Three second messengers that activate intracellular Ca2+ release are established so far: IP3, cADPR, and NAADP. Of these, NAADP is the most recently identified and the most potent. It has been shown to act on acidic organelles, but there is also evidence that it might act on the RyR. We have recently identified a member of the two-pore channel (TPC) family as a major component of the NAADP-receptor complex, and as a NAADP-gated Ca2+-channel (Calcraft et al., 2009; Pitt et al., 2010). A role for NAADP and TPCs has been shown to be important for Ca2+ release and/or contraction in various smooth muscle cells systems (Durlu-Kandilci et al., 2010, Aley et al., 2010, Kinnear et al., 2004). Since TPCs and NAADP-mediated Ca2+-signalling has been shown to be of importance in smooth muscle cells, we were interested whether it could be involved in asthma. Here we present data showing the intracellular localization of TPCs on the endolysosomal system using immunofluorescence staining (n=3). Using RT-PCR, we show the presence of TPC1 and TPC2 in mouse trachea. To investigate whether TPCs and NAADP are important for the contraction of airway smooth muscle cells, we established dose-response curves to cholinergic stimulation using carbachol (CCh; 0.3 nM - 300 µM) in trachea from WT and TPC2 KO mice. In addition, we tested the effect of pre-incubation of the tissue with 10 µM Ned-19, a selective inhibitor of NAADP receptors. Pre-incubation with Ned-19 caused an increase in the EC50 of CCh-stimulated Ca2+ elevation from 0.3 ± 0.04 µM (n=3) to 1 ± 0.5 µM (n=4) in trachea from WT mice, and an increase from 0.2 ± 0.04 µM (n=4) to 0.5 ± 0.2 µM (n=3) in trachea from TPC2 KO mice. All values are given as mean ± S.E.M. Besides this change in the EC50, we also found differences in the maximum contractility: trachea from TPC2 KO mice showed a significantly higher maximum contractility (11.9 ± 0.5 mN/mg tissue, n=4) compared to WT mice (5.2 ± 0.2 mN/mg, n=4; p<0.0001, T-test). Consistent with the finding in TPC2 KO mice, we also observed an increased maximum contractility after pre-incubation with Ned-19 in trachea from WT mice (9.1 ± 0.7 mN/mg, n=3). These results indicate that Ca2+ release via NAADP and TPCs acts in an inhibitory manner on the contractile response of airway smooth muscle cells. One yet to be tested hypothesis to explain this inhibition is that NAADP causes localised Ca2+ release near the plasma membrane, thus activating BKCa channels and causing a hyperpolarisation of the plasma membrane. This hyperpolarization could in turn effectively dampen the CCh response. This inhibition is missing after pre-treatment with Ned-19, or in the absence of TPC2, explaining the higher maximum contraction observed.

Where applicable, experiments conform with Society ethical requirements