Nerve growth factor sensitises TRPV4 to induce bladder hyperreactivity in the mouse

Mai Khidir, Andy Grant. King’s College, London, United Kingdom.

Sensing bladder filling and regulation of bladder emptying involves a complex network of neural and non-neural mechanisms. The molecular identity of the mechanosensor(s) involved remains unclear, but recent studies implicated the ion channels transient receptor potential vanilloid 1 (TRPV1) and 4 (TRPV4). The argument for TRPV4 is particularly compelling, as it is also linked to mechanical nociception, is highly expressed in the urothelium and TRPV4 knockout mice show defective urination. Cystitis is characterised by decreased micturition threshold, frequent urination and pain during urination caused, in part, by increased sensitivity in the normal mechanosensory pathways. Nerve growth factor, NGF, is an inflammatory mediator that is believed to play a causative role in cystitis pathologies. Exposure of bladder sensory afferents to NGF produces hyperreactivity of the bladder similar to that seen in cystitis. NGF sensitises TRPV1 via tyrosine phosphorylation to increase its activity, at a residue conserved in TRPV4. We hypothesised that systemic NGF would sensitise TRPV4, increasing bladder sensitivity to filling.

Female C57BL/6 wild type and TRPV4 knockout mice (22-26g) were used in this study. Mice were anaesthetised with urethane (1.75 mg/g i.p.) and cannulae inserted into the bladder via the urethra. Following a laparotomy, the ureters were ligated and sectioned. Saline was instilled into the bladder (10 μl/min, 30 min) via the cannula, and bladder pressure monitored by a pressure transducer connected to a PowerLab. Voiding and non-voiding contractions of the bladder were measured as spikes of pressure. Cystometric recordings were repeated 1 and 3 hours following saline or NGF (1mg/kg i.v.) administration. Mice were euthanased, and bladders collected and processed for western blotting to measure TRPV4 expression.

Intravenous saline had no effect on micturition threshold in C57BL/6 mice (baseline: 177.9±36.8μl; 3 hours: 140.5±31.3μl), whereas it was significantly reduced 3 hours after NGF injection (Baseline: 166.3±37.5μl; 3 hours: 68.6±13.4μl; p<0.05, one-way RM ANOVA). There was no difference between baseline micturition threshold in TRPV4 wild type and knockout mice. A significant reduction in micturition threshold was observed at 1 and 3 hours following NGF injection in TRPV4 wild type mice (Baseline: 177.1±25.1μl; 3 hours: 108.9±15.1μl; p<0.05, one-way RM ANOVA), whereas no decrease was seen in knockout animals (Baseline: 180.8±28.7μl; 3 hours: 126.2±26.7μl). Expression of TRPV4 relative to the house-keeping protein GAPDH was unaffected by NGF injection.

We conclude that systemic injection of NGF sensitises TRPV4 signalling to induce bladder hyperreactivity, probably via phosphorylation of the channel protein. This mechanism may contribute to the bladder dysfunction that characterises cystitis.