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100 QUANTITATIVE CHANGES IN Nav 1.8 mRNA EXPRESSION IN NOCICEPTIVE DRG NEURONS INNERVATING THE INFLAMED MOUSE COLON DE King, RJ MacLeod, M Tsang, I Kosatka, S Vanner Nav 1.8 voltage-gated sodium channels provide the major current underlying generation of action potentials in nociceptive dorsal root ganglia (DRG) neurons. Whole cell voltage clamp studies in TNBS colitis demonstrate this current is increased and underlies hyperexcitability of these "pain sensing" neurons. This increase in Nav 1.8 current could be due to transcriptional increases in channel expression (ie increased mRNA) or post-translational modification. The aim of this study was to use quantitative PCR to examine changes in Nav 1.8 mRNA.
GIDRU, Queen's University, Kingston, Ontario
METHODS: Mouse colonic sensory neurons were identified with the retrograde label Fast blue injected into the distal colon wall and colitis induced by injection of TNBS at the same site. Animals were sacrificed on day 2 or day 4 and labeled neurons were harvested by laser-capture microdissection from dorsal root ganglia T9-T13 in TNBS and control animals. A control group was also obtained from TNBS animals by laser capturing neurons from ganglia which do not innervate the colon (T1-T3). Intron spanning primers were used to identify all Nav channel alpha subunits in DRG neurons, i.e. Nav 1.6, 1.7, 1.8, 1.9, and quantitative real-time PCR was used to determine mRNA transcription levels. Values were expressed as a ratio to the Alas I reference gene which exhibited constant expression during inflammation and had similar levels to the target Nav channels.
RESULTS: TNBS inflammation was confirmed by comparing macroscopic damage scores and tissue wt in inflamed and non-inflamed colons. Nav 1.8 and Nav 1.9 alpha subunit mRNA from neurons innervating the inflamed colon on day 2 was markedly decreased (6 fold; P<0.03, 0.01 respectively) whereas Nav 1.7, Nav1.6 mRNA levels were unaffected (n=3 separate experiments). None of the Nav channel alpha subunit mRNA levels, including Nav 1.8 and 1.9, were altered in ganglia which did not innervate the colon (i.e. segments T1-T3). In day 4 studies, inflammation was more severe and there was a 297 fold decrease in Nav 1.8 mRNA in neurons innervating the inflamed colon compared to controls.
CONCLUSION: These findings suggest that increased Nav 1.8 currents underlying neuronal hyperexcitability in TNBS colitis are due to post-translation modification of channels given the lack of increased mRNA. In fact, there is a marked decrease in mRNA that may suggest a feedback mechanism which alters mRNA stability and possibly acts to counteract the post-translational changes. Much less is known about the role of Nav 1.9 channels but it may also be modified in inflammation.