The Role Of Nav1.9 In The Activation of Visceral Afferent Fibres By Bradykinin

George Boundouki1, James Hockley1, Mark Baker2, Charles Knowles1, David Bulmer1. 1Centre for Digestive Diseases, Barts & The London School of Medicine and Dentistry, Queen Mary, University of London, London, UK, 2Neuroscience Group, Barts & The London School of Medicine and Dentistry, Queen Mary, University of London, London, UK.

Behavioural responses to somatic inflammatory pain are significantly attenuated in Nav1.9 knock-out (-/-) mice consistent with its expression in small diameter IB4 positive DRGs and the increase in persistent sodium current observed in DRGs following application of inflammatory mediators. By contrast, the role of Nav1.9 in visceral pain is ambiguous with enhanced or attenuated behavioural responses in Nav1.9 -/- mice.

To help clarify the role of Nav1.9 in visceral pain, the aim of this study was to examine the effect of the algogenic mediator bradykinin on visceral afferent fibre activity in Nav1.9 -/- mice.

Experiments were performed using male and female (12-35 weeks) Nav1.9 -/-, heterozygous (+/-) (bred on a C57/B6 background) and C57/B6 mice. The mice were euthanased by exposure to rising CO2 concentration and the intestine/colon removed and placed in a tissue bath superfused with carbogenated Krebs solution supplemented with 3µM indomethacin, 10µM nifedipine and 10µM atropine as appropriate. Small intestine or descending colon was canulated and luminally perfused with buffer (7ml/minute, 32-34 °C) and the mesenteric or lumbar splanchnic nerve bundles were dissected free and whole nerve activity recorded using a suction electrode. Following a 30 minute stabilisation period, tissue was superfused with a 20ml volume of 3µM bradykinin in intestinal preparations. Colonic preparations had consecutive administration of 0.3, 1 and 3µM (60 minute interval between concentrations) bradykinin. Peak nerve activity before and after application of bradykinin was determined in spikes/second for intestinal preparations and spikes/20 seconds for colonic preparations and the change in activity expressed as a mean ± sem. For intestinal preparations, peak changes in nerve activity were statistically compared using a one-way ANOVA with Dunnett’s post hoc comparisons versus the C57/B6 group. In colonic preparations, peak changes in nerve activity were statistically compared between Nav1.9 +/- and Nav1.9 -/- mice using a two-way ANOVA.

In intestinal preparations, bradykinin produced a robust increase in nerve activity in C57/B6 mice (72.2Hz ± 7.4, n = 5). A comparable increase was seen in Nav1.9 +/- mice (84.0Hz ± 10.8, n = 5), however responses to bradykinin were greatly attenuated in Nav1.9 -/- mice (22.0Hz ± 5.0, n = 5, p<0.05). In colonic preparations, bradykinin similarly produced a robust increase in nerve activity in Nav1.9 +/- mice (0.3µM: 96.0Hz ± 21.6, 1µM: 78.8Hz ± 16.9, 3µM: 73.5Hz ± 40.5, n = 2-4). This response was virtually abolished in Nav1.9 -/- mice (0.3µM: 2.0Hz ± 1.0, 1µM: 4.3Hz ± 4.3, 3µM: 0Hz ± 0, n = 3, p<0.0001).

The data generated suggest that Nav1.9 has an important role in the response of visceral afferent fibres to stimulation by bradykinin.