Studying The Inhibitory Effect Of Capsaicin On VGCCs And Its Signal Pathway In Rat DRG Neurons

ObjectiveVoltage-gated Ca2+ channels (VGCCs) in sensory neurons play a critical role in transduction of pain. It has been found that application of capsaicin inhibits VGCCs and desensitizes nociceptors. The present study was to investigate the inhibitory effect of capsaicin on VGCCs 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 DRGs were dissected from the lumbar regions of male Sprague–Dawley rats (80 ~ 120 g) and rinsed in 0℃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℃for 30 min. After four washes in standard external solution, DRGs were triturated using progressively fine fire-polished Pasteur pipettes. Cell suspensions were then plated onto slide for 30 min until the cell adhered to the slide。Next, add the extracellular fluid in the culture dish。placed at 4℃refrigerator, studied within 6 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 Fura-2/AM at 37℃. The excitation wavelength of Fura-2/AM which bound to Ca2+ was 340 nm, while the excitation wavelength of Fura-2/AM which 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-gated calcium channel currents (VGCCs) and capsaicin-induced current 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 (109 ?) 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. VGCCs were elicited by depolarizing command pulses from ?70 mV to 0 mV for 200 ms. Capsaicin sensitivity was tested in DRG neurons by detecting inward current 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 means±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-sensitivity DRG neurons, application of capsaicin (1μmol/L) can induce an inward cation current (capsaicin current), which can inflow Ca2+, and trigger transient intracellular Ca2+ concentration rise . In capsaicin-insensitivity DRG neurons, application of capsaicin (1μmol/L) cannot induce any current or trigger intracellular Ca2+ concentration rise.2. In capsaicin-sensitivity DRG neurons, capsaicin (1μmol/L) resversibly inhibits VGCC currents, and also inhibits the high potassium (40 mmol/L)-mediated voltage-gated Ca2+ influx. The inhibitory effects of capsaicin on VGCCs are depended on intracellular Ca2+ concentration rise. In capsaicin-sensitivity DRG neurons, capsaicin has no obviously inhibitory effect on VGCC currents or VGCC-induced Ca2+ influx.3. The inhibitory effects of capsaicin on VGCCs can be significantly reduced by protein kinase C (PKC) selective inhibitor, Bisindolylmaleimide. But protein kinase A (PKA) selective inhibitor, H-89, do no significant change those inhibitory effects. It suggests that PKC may involve in the inhibition of capsaicin on VGCCs.ConclusionThe inhibitory effect of capsaicin on voltage-gated calcium channels in capsaicin-sensitive dorsal root ganglion neurons is via intracellular Ca2+ and protein kinase C pathway.