| ObjectiveCalcium influx through voltage-activated Ca2+ channels (VACCs) plays a critical role in neurotransmission. It has been found that capsaicin application inhibits VACCs and desensitizes nociceptors. The present study was to investigate the inhibitory effect of capsaicin on VACCs and its intracellular signaling mechanisms in acutely-isolated rat dorsal root ganglion (DRG) neurons with patch clamp and calcium imaging techniques.Methods1. Isolation of DRG neurons. Briefly, DRGs were dissected from the lumbar regions of male Sprague–Dawley rats (80 ? 120 g) and rinsed in 0°C DMEM solution, which is oxygenated with 95% O2 and 5% CO2. The ganglia were treated with 1 mg/ml collagenase (type IA) and 0.5 mg/ml trypsin (type I) at 37°C for 40 min. After four washes in standard external solution, DRGs were triturated using progressively fine fire-polished Pasteur pipettes. Cell suspensions were then plated onto culture dishes and studied within 10 hrs.2. Calcium imaging. Calcium imaging can monitor the change of intracellular concentration ([Ca2+]i). [Ca2+]i was measured using a Ca2+-sensitive dye Fura-2 acetoxymethyl ester (Fura-2/AM). Isolated DRG neurons were incubated for 30 min in a bath solution containing 2μmol/L Fura-2/AM at 37°C. The excitation wavelength of Fura-2/AM which bound to Ca2+ was 340 nm, while the excitation wavelength of Fura-2/AM which was free was 380 nm, the emission wavelength of both was 510 nm. Fluorescent image intensities were expressed as the ratio F340/F380 to allow quantitative estimates of changes in [Ca2+]i. Thus the changes of [Ca2+]i could be evaluated immediately by use of TILLvision software which could display real-time rations.3. Whole-cell patch clamp records. Voltage-activated calcium channel currents and capsaicin-induced currents were recorded by patch clamp technique. The patch electrodes were pulled with a Sutter electrode puller and then were fire polished with a microforge. When filled with the intracellular pipette solution, the patch electrodes had a resistance of 3–5 M?. After a giga-ohm seal was obtained and the membrane under the pipette tip was ruptured, the whole cell mode was established. The holding potential was clamped at ?70 mV. VACC currents were elicited by depolarizing command pulses to 0 mV for 200 ms. Capsaicin sensitivity was tested in DRG neurons by measuring inward currents induced by the application of capsaicin (1μmol/L) to cells. Whole-cell patch clamp recordings were performed at room temperature.4. Statistical analysis. Data were analyzed with Igor Pro software (USA). Statistical data are presented as mean±SEM. Comparisons between means were performed using Student's unpaired t-test. Differences were considered to be significant when p < 0.05.Results1. In capsaicin-sensitive DRG neurons, where capsaicin could evoke inward currents, capsaicin clearly produced an inhibitory effect on VACC currents. In contrast, in capsaicin-insensitive DRG neurons, where capsaicin could not evoke any currents, the inhibitory effect on VACC currents produced by capsaicin was not observed.2. High K+ (40 mmol/L) led to an elevation of [Ca2+]i via activating VACCs. The high K+-evoked [Ca2+]i enhancement was almost completely inhibited by capsaicin in capsaicin-sensitive DRG neurons where capsaicin (1μmol/L) evoked [Ca2+]i transient enhancement. In capsaicin-insensitive DRG neurons, where capsaicin (1μmol/L) hardly induced [Ca2+]i transient enhancement, capsaicin had no effect on high K+-induced elevation of [Ca2+]i.3. Bisindolylmaleimide, a selective inhibitor of PKC, significantly attenuated the inhibitory effect of capsaicin on voltage-gated calcium influx.ConclusionThe inhibitory effect of capsaicin on voltage-activated calcium channels in capsaicin-sensitive dorsal root ganglion neurons is via intracellular Ca2+ and in part regulated by protein kinase C.
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