Role Of Nav1.9 In The Excitation Of Visceral Afferent Fibres By ATP

James Hockley1, George Boundouki1, Charles Knowles1, Mark Baker2, Wendy Winchester3, David Bulmer1. 1Wingate Institute of Neurogastroenterology, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AJ, UK, 2Neuroscience Group, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AJ, UK, 3Pfizer Research & Development, Cambridge, CB21 6GP, UK.

ATP is an important algogenic mediator that is released from the epithelium of hollow visceral organs in response to distension and inflammation, leading to the activation of sensory nerves via P2X2 and P2X2/3 receptors [1]. Recent work [2] has demonstrated that ATP enhances the persistent current of the voltage gated sodium channel subtype 1.9 (NaV1.9), found in small diameter DRGs, suggesting that NaV1.9 may also play a significant role in the excitation of sensory nerves by ATP. In order to explore this further we examined the effects of ATP on visceral afferent activity recorded from isolated mouse intestine or colon in wild-type and NaV1.9 knock-out mice.

In separate experiments, either the small intestine or distal colon and associated mesentery from male and female (12-16 weeks) C57B6, NaV1.9 +/+ or NaV1.9 -/- mice (NaV1.9 mice bred on a C57B6 background) was removed following euthanasia with rising concentration of CO2. Tissue was placed in a recording chamber and superfused with carbogenated Krebs buffer (7ml/min; 32-34°C), supplemented with nifedipine (10µM), atropine (10µM) and indomethacin (3µM). Respective intestinal mesenteric or lumbar splanchnic nerve bundles were dissected free and whole nerve activity was recorded using a suction electrode. The tissue was luminally perfused with buffer (0.1mL/min) allowing for ramp distensions (0 to 80mmHg in 3 mins) to confirm the quality of the recording. ATP made up as a 300mM stock solution in buffer and diluted in buffer to the test concentration in a 20mL volume. Peak firing data are expressed as mean ± S.E.M. Responses were compared by Student’s t test and 2-way ANOVA, where appropriate.

In intestinal preparations, administration of 1mM ATP produced a robust increase (change in peak firing rate 65.97Hz ± 7.60Hz; n = 7) in visceral afferent fibre activity in C57B6 preparations, which was greatly reduced in NaV1.9 -/- mice (26.22Hz ± 6.57Hz; n = 6; p<0.0025: unpaired t test). In colonic preparations, consecutive administration of ATP (0.1mM, 1mM, 3mM and 10mM, 60min intervals) produced concentration dependent increases in visceral afferent fibre activity in NaV1.9 +/+ animals (3.56Hz ± 0.67Hz, 9.08 Hz ± 0.92Hz, 18.93Hz ± 1.08Hz and 60.59 ± 7.18Hz, respectively; n = 3). These responses were significantly reduced (1.40Hz ± 0.48Hz, 1.58Hz ± 0.32Hz, 5.43Hz ± 0.57Hz, 32.14Hz ± 6.72Hz, respectively; n = 4; p<0.0001: 2-way ANOVA) in the NaV1.9 -/- animals. In addition the peak firing response to distension pressures of 80mmHg was also greatly attenuated in the NaV1.9 -/- animals (12.06Hz ± 2.99Hz; n = 4) compared to NaV1.9 +/+ (41.33Hz ± 10.39Hz; n = 3; p<0.05).

This data demonstrates that NaV1.9 plays a critical role in the excitatory activation of visceral afferent fibres by ATP and luminal distension.

1. Burnstock, G., Purinergic mechanosensory transduction and visceral pain. Mol Pain, 2009. 5: p. 69.

2. Baker, M.D., Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones. J Physiol, 2005. 567(Pt 3): p. 851-67.