Deletion of the ion channel TRPV4 increases detrusor contractility in the mouse

Antonio Ramos-Filho2, Edson Antunes2, Andy Grant1. 1King’s College, London, UK, 2University of Campinas, Campinas, Brazil.

The mechanosensitive Ca2+ permeable cation channel TRPV4 is a member of the Transient Receptor Potential Vanilloid family of channel proteins. Immunohistochemical and functional studies have identified TRPV4 expression in the urothelium and detrusor smooth muscle (DSM) of rodent bladders (Birder et al., 2007; Thorneloe et al., 2008). Urothelial stretch during bladder filling activates TRPV4 and produces a Ca2+-dependent release of ATP (Mochizuki et al., 2009). This ATP release activates P2X3 receptors on sensory nerves to produce a sensation of bladder filling (Vlaskovska et al., 2001). Activation of TRPV4 in the DSM by the selective agonist GSK1016790A (10-100nM) produces a slow and sustained contraction, which can be inhibited by nifedipine (Thorneloe et al., 2008). We hypothesised that deletion of the TRPV4 channel would reduce the contractility of isolated DSM strips by reducing the intracellular [Ca2+].

Male C57BL/6 wild type (+/+) and TRPV4 knockout (-/-) mice (22-30g) were used in this study. Mice were euthanased with an overdose of pentobarbital (1mg/g) and the bladder removed. The bladder dome was removed above the level of the ureters, and cut into 2 longitudinal strips. These were suspended in a 10ml organ bath containing Krebs’ solution (117mM NaCl, 4.7mM KCl, 2.5mM CaCl2, 1.2mM MgSO4, 1.2mM KH2PO4, 24.8mM NaHCO3 and 11mM glucose, pH7.4) at 37ºC and bubbled with a mixture of 95% O2 and 5% CO2. Changes in isometric force were recorded using a Power Lab v.4 system (AD Instruments, UK). Following 60 min equilibration, the resting tension was adjusted to 0.5 g at the beginning of the experiments. Concentration-response curves to the muscarinic agonist carbachol, KCl (both cumulative) and the P2X receptor agonist α,β-methylene ATP (non-cumulative) were constructed. Following pre-contraction of the tissue strips with KCl, a concentration-response curve was obtained for relaxation to the β-adrenergic agonist isoprenaline. All drugs were dissolved and administered in Krebs’ solution. Frequency-response curves to electrical field stimulation (EFS: 10s; 80V; pulse width 1ms) were also obtained. Emax and pEC50 values (stated as mean ± S.E.M) were compared by unpaired t-tests.

Maximal contractions to carbachol were significantly increased in TRPV4 -/- strips (1nM-30μM; +/+ = 0.029±0.004 vs -/- = 0.077±0.011g/mg, p<0.05, n = 5) with no effect on pEC50 values (+/+ = 5.92±0.17 vs -/- = 6.01±0.14). Contractions to EFS (4,8,16,32Hz) were also increased in TRPV4 -/- strips compared to +/+ strips. Conversely, the pEC50 of KCl contractions was decreased in TRPV4 -/- strips (1mM-1M; +/+ = 1.6±0.2 vs -/- = 1.2±0.1), p<0.05, n = 4) with no effect on maximal contractions (+/+ = 0.068±0.011 vs -/- = 0.070±0.007g/mg). Maximal relaxation to isoprenaline was significantly decreased in TRPV4 -/- strips (1nM-30µM; +/+ = 61.6±8.3 vs -/- = 41.8±3.8 % of KCl contraction, p<0.05, n = 5) with no effect on pEC50 values (+/+ = 7.13±0.20 vs -/- = 6.97±0.20). Contractions to α,β-methylene ATP (1-10μM, n = 4) were unaltered by loss of TRPV4.

These data suggest that muscarinic and β-adrenergic receptors activate a TRPV4-dependent mechanism, probably in urothelial cells, that inhibits DSM contraction. In contrast, DSM TRPV4 receptors contribute to the Ca2+ entry and contraction following exposure to KCl. Activation of P2X receptors is sufficient to cause DSM contraction without the contribution of TRPV4.

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Mochizuki, T., Sokabe, T., Araki, I., Fujishita, K., Shibasaki, K., Uchida, K., Naruse, K., Koizumi, S., Takeda, M., Tominaga, M. (2009) The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures. J. Biol. Chem. 284(32), 21257-64.

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