INVOLVEMENT OF TRPV1, via p38 MITOGEN ACTIVATED PROTEIN KINASE IN TNFα FACILITATED CAPSAICIN-EVOKED CALCIUM RESPONSES IN ADULT RAT DORSAL ROOT GANGLION NEURONES

Leonie Norris, David Kendall, Victoria Chapman
Institute of Neuroscience, School of Biomedical Sciences, University of Nottingham, Queens Medical Centre, Nottingham, NG7 2UH, United Kingdom

Tumour necrosis factor-α (TNFα) is a pro-inflammatory cytokine involved in the development and maintenance of inflammatory pain. The transient receptor potential V1 ligand gated channel (TRPV1) is activated by noxious heat and capsaicin and is involved in mediating inflammatory hyperalgesia (Davis et al., 2000). Capsaicin and TNFα increase intracellular calcium [Ca2+]i in dorsal root ganglion (DRG) neurones in vitro and TNFα pre-treatment facilitates capsaicin-evoked responses (Norris et al, 2006). TNFα activates p38 mitogen-activated kinase (p38) in cultured DRG neurones ((Pollock et al., 2002) and activation of p38 up-regulates TRPV1, without changes in mRNA levels (Ji et al., 2002). The aim of the present study was to investigate whether TNFα activation of p38 contributes to the facilitation of capsaicin-evoked calcium responses by TNFα through increased expression of TRPV1 in adult rat DRG neurones.

DRG neurones were dissected from male Sprague Dawley rats (180-200g) and loaded with the calcium sensitive dye Fura 2AM. Capsaicin and TNFα-evoked changes in [Ca2+]i were measured in single neurones using an Improvision imaging system. [Ca2+]i was estimated as ratios of peak fluorescence intensities (measured at 500 nm) at excitation wavelengths of 340 and 380 nm respectively and expressed as a percentage of the response to KCl (50mM). DRG neurones were pre-exposed to the selective p38 inhibitor SB706504 (1μM) for 4 minutes, then suprafused with capsaicin (50nM) or TNFα (50ng/ml). In separate experiments, DRG neurones were suprafused with SB706504 for 4 minutes, then exposed to 50ng/ml TNFα for 3 minutes before suprafusion with 50nM capsaicin for 1 minute. Western blot analysis was performed on whole DRG and membrane extracts. Cells were left untreated or treated with TNFα (50ng/ml) for 4minutes. Phospho-p38 and TRPV1 specific antibodies were used to determine any change in p38 activity and TRPV1 expression

Pre-exposure to the p38 inhibitor SB706504 had no effect on responses to TNFα (SB706504 + TNFα =11±4%; TNFα alone 14±3%) or capsaicin alone (SB706504 + capsaicin =49±3%; capsaicin alone 51±5%). SB706504 pre-exposure did not alter the proportion of neurones responsive to capsaicin or TNFα. Pre-exposure to SB706504 did, however, significantly reduce the proportion of neurones responding to the combination of TNFα and capsaicin (11±3% compared with 55±9% in the absence of SB706504, p<0.05, unpaired t-test). TNFα treatment of DRG neurones increased phosphorylation of p38 (0.91 ±0.1% compaired with 0.38±0.05% for control treated, p<0.005,unpaired t-test), but the total p38 content was unaltered. TNFα treatment did not later TRPV1 expression in membrane fractions, but caused a significant increase in TRPV1 expression in whole cell extracts (0.71 ±0.06% compared with 0.37±0.05% for control treated, p<0.05,unpaired t-test).

These data suggest that TNFα facilitates capsaicin-evoked calcium responses perhaps via p38 MAPK-mediated phosphorylation and sensitisation of TRPV1 receptors. Inhibition of this pathway may prevent sensitization of sensory neurones to some inflammatory mediators.

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Norris et al (2006) www.pA2online.org/abstracts/Vol4Issue2abst170P
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