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058 INHIBITORY EFFECT OF PAR4 ACTIVATION IN NOCICEPTIVE MOUSE COLONIC DRG NEURONS R Karanjia1, ME Tsang1, N Vergnolle2, S Vanner1 Visceral pain expression in inflammatory states can result from a balance of nociceptive and anti-nociceptive factors. Activation of PAR4 receptors increases nociceptive thresholds in whole animals suggesting it may have a novel anti-nociceptive action. While it has been shown that activation of PAR4 on dorsal root ganglia (DRG) neurons can inhibit Ca2+ mobilization, the effect on excitability of colonic DRG neurons is unknown. Therefore, the aim of this study was to apply whole cell electrophysiological techniques to examine this question.
1GIDRU, Queen's University, Kingston, Ontario; 2Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta
METHODS: DRG neurons innervating the mouse colon were retrogradely labeled with Fast Blue injected into the colon. PAR-4 antibody (1:200; Santa Cruz) was used in immunohistochemical studies to identify PAR4 on colonic DRG's. Whole cell voltage clamp recording were made from acutely dissociated labeled small cells characteristic of nociceptors (<40 pF). The effects of PAR4 on neuron excitability were investigated using current clamp recordings by measuring the changes in membrane potential, rheobase, number of action potential fired at 2x rheobase and the input resistance following a brief application (3 min) of PAR4 Activating Peptide (AP: 100 µM), AYPGKF-NH2 or PAR4 inactive control peptide (ICP: 100 µM), YAPGKF-NH2. These values were normalized to the initial responses from the neurons pre-peptide application.
RESULTS: Immunohistochemical studies demonstrated labeled neurons co-localized PAR4 receptors. The application of AP significantly decreased the number of action potential fired at 2x rheobase 10 min post application (64% of initial). The average number of action potentials fired in response to 2x rheobase decreased from 4.64 ± 0.76 to 2.76 ± 0.54 over this time period (p=0.004). This decrease in excitability remained up to 20 min post AP application (p=0.001). Application of the ICP did not significantly alter the number of action potentials fired at 2x rheobase. The rheobase increased at all time points following the application of AP (228% of initial by 20 min) but did not reach statistical significance (p=0.08). Rheobase was not altered by ICP. There was no change in the resting membrane potential or input resistance post application of AP.
CONCLUSIONS: These findings directly demonstrate that PAR4 activation decreased the excitability of nociceptive DRG neurons innervating the colon and this effect was sustained for up to 20 min after washout of the AP. This anti-nociceptive effect may play a role counteracting nociceptive inflammatory mediators and provide novel targets for treatment.