| Abstract No.: | A-D1142 |
| Country: | Canada |
| | |
| Title: | ACIDOSIS INDUCES RAPID MOBILIZATION OF THE NERVE GROWTH FACTOR (NGF) RECEPTOR TROPOMYOSIN-RELATED KINASE A (TRKA) TO THE CELL MEMBRANE OF ADULT SENSORY NEURONS |
| | |
| Authors/Affiliations: | 1 Geoffrey E. Bray*; 1 Valerie M.K. Verge;
1 Univeristy of Saskatchewan, Saskatoon, SK, Canada
|
| | |
| Content: | Inflammation is a ubiquitous response of the body to cellular damage and injury. It not only leads to increased production of inflammatory molecules, including NGF that sensitizes sensory neurons, but is also associated with acidosis due to decrease in extracellular pH. The addition of protons results in increased membrane conductance and depolarization in sensory neurons involving the acid-sensing ion channels (ASICs) and the capsaicin-sensitive transient receptor potential vanilloid type 1 receptor (TRPV1). As NGF-associated trkA expression and activation are critically linked to inflammation-associated nociceptor sensitization, we hypothesized that decreased pH contributes to this process by depolarizing the neuron and mobilizing more trkA to the cell membrane. This hypothesis is premised by research demonstrating that increased neuronal activity mobilizes more of the neurotrophin receptor trkB to the membrane (Meyer-Franke et al.,Neuron 1998 21:681-693). Objectives: (i) to determine whether trkA can be mobilized in sensory neurons in response to decreased pH; (ii) to assess whether increased mobilization of trkA to the membrane results in greater trkA activation in response to NGF challenge; and (iii) to assess the contribution of proton-sensitive channels activity in this response Methods: Adult male Wistar rat sensory neurons were isolated and grown in the presence of 10 ng/ml NGF for two days prior to removal of NGF and exposure to MES-buffered media at a pH of 7.4 or 6.5 +/- anti-BDNF for 15 minutes to two hours. To assess impact of pH on trkA mobilization to the membrane, cultures were processed for trkA immunohistochemistry under non-permeabilizing conditions and average fluorescence levels were quantified using Northern Eclipse software. To assess response to NGF, the cells were washed twice with DMEM containing anti-NGF for 5 mins and once without anti-NGF for 5 mins prior to the shift in pH. Cells were then challenged with 50 ng/ml NGF for 15 mins and processed for phospho trkA IHC under permeabilizing conditions. To assess the role of proton sensitive channels, channel blockers for ASIC (250M amiloride) and TRPV1 (10M capsazepine), were added to the media for the duration of the pH challenge, followed by trkA IHC under non-permeabilizing conditions. Results: Decreased pH resulted in a dramatic increase in mobilization of trkA to the membranes of the cell bodies and processes, detectable at 15 mins but maximal 30 mins following pH shift. Similarly, an increase in phospho trkA was also detected when cells were challenged with NGF after a pH shift to 6.5. Treatment with anti-BDNF at the time of pH shift did not alter the level of trk phosphorylation observed. When the ASIC and TRPV1 channels were blocked by amiloride and capsazepine, the mobilization of trkA to the neuronal membrane was attenuated as detected by IHC under non-permeablizing conditions. Conclusions: These findings suggest that acidosis can rapidly alter the responsiveness of sensory neurons to NGF a critical signaling molecule in inflammatory-mediated pain responses and that proton sensitive channel activity |
| | |
| Back |
|
