TRESK adenovirus vector (pAdTrack-CMV-TRESK), negative adenovirus, PCR primer (Shanghai R&S Biotechnology Co., Ltd., Shanghai, China); the rabbit polyclonal anti-TRESK antibody, TRESK-conjugated anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA); the SP radio-immunity kit (the Second Army Medical College, Shanghai, China); the Superscript kit (Life Technologies, Gaithersburg, MD, USA); and the DNA engine Opticon 2 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) were used in this study.

DRGs were removed from young adult SD rats (6–9 weeks) and were dissociated into single isolated neurons by enzyme treatment of 0.125% collagenase for 90 min (twice), followed by 0.25% trypsin for 30 min at 37˚C and by trituration with fire-polished Pasteur pipettes of decreasing tipdiameter (11). The cells (2–3 DRG/well) were subsequently plated on polyethyleneimine and laminin-coated 96-well tissue-culture plates and incubated in DMEM containing 10% heat-inactivated horse serum, 1% penicillin/streptomycin and 200 mM glutamine. The cultures were maintained at 37˚C in a water-saturated atmosphere with 5% CO₂ for 5 days prior to the experiment. On the fifth day of culture, neurons exhibited globular cell bodies and extended axonal processes. Various non-neuronal cells, such as Schwann cells, fibroblasts and satellite cells, were also present as an oxford gray cellular background. Approval was received by the First People's Hospital of Foshan ethics committee.

DRG neurons were cultured in 54 wells and divided randomly into six groups, 9 holes per group, per well. Group C received no treatment. Each well in the S group was incubated for 10 min with fresh 300 nmol/l capsaicin culture medium. Each well in the NC group was administered 3×10⁷ negative adenovirus (Shanghai R&S Biotechnology Co., Ltd.). Each well in the NCS group was given 3×10⁷ negative adenovirus and then incubated for 10 min with fresh 300 nmol/l capsaicin culture medium 72 h later. Each well in the R group was given 3×10⁷ pAdTrack-CMV-TRESK (Shanghai R&S Biotechnology Co., Ltd.). Each well in the RS group was given 3×10⁷ pAdTrack-CMV-TRESK and then incubated for 10 min with fresh 300 nmol/l capsaicin culture medium 72 h later.

Total RNA harvested from 3 wells of each group by the acid guanidinium thiocyanate-phenol-chloroform extraction method was separately subjected to reverse transcription into cDNA using a Superscript kit (Life Technologies), according to the manufacturer's protocol. cDNA sample (2 μg) was used immediately in a real-time PCR assessment of TRESK mRNA levels with iQ SYBR-Green Supermix, and forward primer (5′-GGTGCCAACGATGATCT-3′) and reverse primer (5′-CTGCTGGGCTGTGGGTCTAG-3′) on a DNA engine Opticon 2 real-time PCR detection system (Bio-Rad). The thermal cycler parameters used were: denaturation of 1 cycle of 2 min at 95˚C, followed by 40 cycles of 20 sec at 95˚C, annealing for 15 sec at 57˚C and extension 20 sec at 72˚C. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control was run simultaneously with the same reaction recipe listed in the instructions manual for the iQ SYBR-Green Supermix. The 2^−ΔΔCt method was used to calculate the expression level of TRESK mRNA (12).

Three wells were removed from each group, and the cells were processed using western blotting. Primary antibodies were raised against TRESK (1:1,000 dilution; rabbit polyclonal TRESK antibodies; Santa Cruz) or β-actin. After washing, the membranes were further incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000 dilution; Santa Cruz Biotechnology) for 1 h at room temperature (22˚C). The proteins were then used for the detection of chemiluminescence, according to the manufacturer's instructions.

We measured the SP content of the culture medium (serum-free DMEM) in the last 3 wells from each group at 72 h following grouping. Cells were removed from the medium and were maintained for 10 min with 300 nmol/l capsaicin in groups S, NCS and RS. Subsequently, the medium from the cells in each group was collected and centrifuged at 1,200 × g for 5 min, followed by a radioimmunoassay with the SP radioimmunity kit (the Second Army Medical College, Shanghai, China) of the medium (13).

The data are presented as the means ± SD. Statistical analyses were performed by the multiple t-test with the Bonferroni correction following ANOVA. P<0.05 was considered to indicate a statistically significant difference.

In our previous studies, we successfully constructed the recombinant TRESK adenovirus vector (pAdTrack-CMV-TRESK). This vector was found to upregulate TRESK mRNA expression levels in the DRG neurons of rats (10).

Therefore, we investigated whether these mass duplicate pAdTrack-CMV-TRESK are affected in the enhancement of TRESK mRNA from cultured DRG neurons exposed to long-term (72 h) treatment with adenovirus vector. Fig. 1A shows that the TRESK mRNA from cultured DRG neurons was significantly enhanced when exposed to long-term (72 h) treatment with pAdTrack-CMV-TRESK in groups R and RS. However, no increase in the TRESK mRNA levels of the rat DRG neurons was observed following a 72-h exposure of negative adenovirus in groups NC and NCS, as was the case with groups C and S as controls (data not shown).

The protein levels of TRESK exposed to long-term (72 h) treatment with pAdTrack-CMV-TRESK or negative adenovirus vector were examined using western blotting. The increase in the protein levels of TRESK from cultured DRG induced by pAdTrack-CMV-TRESK was significantly enhanced when exposed to long-term (72 h) treatment in groups R and RS. However, no increase in the TRESK protein levels of the rat DRG neurons was observed following a 72-h exposure of negative adenovirus in groups NC, NCS, C and S as controls (Fig. 1B) (data not shown).

To investigate whether pre-treatment with upregulation of TRESK mRNA causes the attenuation of SP release evoked by capsaicin, the levels of capsaicin-induced SP release from cultured DRG neurons pre-treated with pAdTrack-CMV-TRESK or negative adenovirus vector were examined. Fig. 1C shows that the release of SP from cultured DRG neurons was significantly enhanced when incubated for 10 min with capsaicin in groups S, NCS and RS. Although the capsaicin-evoked SP release was significantly enhanced by pre-treatment with pAdTrack-CMV-TRESK in comparison to the control group which was not incubated with capsaicin, the capsaicin-evoked SP release from cultured DRG was significantly attenuated by exposure to long-term (72 h) treatment with pAdTrack-CMV-TRESK in the RS group compared to the NCS and S groups. There was no significant difference in the release of SP from cultured DRG neurons in the R, NC and C groups (data not shown).