Effects of TNFα on 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, Queen’s Medical Centre, Nottingham NG7 2UH, UK

Tumour necrosis factor-α (TNFα) is a pro-inflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain conditions. The transient receptor potential V1 ligand gated channel (TRPV1), which is activated by noxious heat and capsaicin, is involved in mediating inflammatory hyperalgesia (Davis et al., 2000) and may be modulated by inflammatory mediators such as TNFα . Here we have investigated the effects of TNFα on capsaicin-evoked changes in intracellular calcium [Ca2+]i in adult rat dorsal root ganglion (DRG) neurones, an in vitro model of sensory nerves.
Spinal columns were removed and DRGs dissected from male Sprague Dawley rats (180-200g). DRG neurones were loaded with the calcium sensitive dye Fura 2AM. Capsaicin and TNFα -evoked changes in [Ca2+]i were measured in single DRG neurones using an Improvision system.
DRG neurones were suprafused with capsaicin (1nM-1μM) or with TNFα (10, 30, 50, 100, and 200 ng/ml). In separate experiments, DRG neurones were pre-exposed to 50ng/ml TNFα for 2 minutes before suprafusion with 50nM capsaicin for 1 minute. [Ca2+]i was estimated as the 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). Experiments were repeated at least three times. Capsaicin evoked a concentration-related increase in [Ca2+]i in DRG neurones (pEC50 = -7.5; Emax = 86.0% of KCl response).

Fig.1 TNFα-evoked Increases in [ Ca2+] i in DRG neurones.

Application of TNFα increased levels of [ Ca2+]i in DRG neurones ( pEC50 = -7.3; Emax=39.3% of the KCl response) (Fig. 1). Following pretreatment of DRG neurones with 50ng/ml TNFα, capsaicin (50nM)-evoked increases in [ Ca2+]i were significantly greater than capsaicin alone (TNFα + capsaicin: 58 ± 2% KCl; capsaicin alone 46 ± 5% % KCl; p< 0.05, unpaired t-test). Pre-treatment with TNFα increased the percentage of neurones responding to capsaicin ( 35%) compared to capsaicin alone (27%).

We have demonstrated that both capsaicin and TNFα produce excitatory effects on DRG neurones in vitro and that pre-treatment with TNFα facilitates capsaicin-evoked calcium responses. Further work will determine whether the responses are independent and simply additive or co-operative via as yet to be determined mechanisms.

Davis, J.B., Gray J., (2000) Nature, 405, 183-7.