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The Celluar Signal Transduction of Capsaicin-induced Inhibition of Voltage-activated Calcium Channels in Rat Sensory Neurons

Author: LiuYueYan
Tutor: KongDeHu；WangLieCheng
School: Anhui Medical University,
Course: Physiology
Keywords: Capsaicin Capsaicin receptor Dorsal root ganglion Voltage-gated calcium channel Protein kinase C
CLC: R33
Type: Master's thesis
Year: 2009
Downloads: 104
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Abstract

Purpose of Ca2 into the cells by the voltage-gated calcium channels (voltage-activated calcium channels, VACCs) play an important role in the process of information transmission neurons, studies have shown that capsaicin can inhibit VACCs, make the nociceptor desensitization. In this study, patch-clamp and calcium imaging technology research capsaicin (capsaicin, Cap) in acutely isolated rat dorsal root ganglia (dorsal root ganglion, DRG) of neurons VACCs the inhibitory effect and to explore the cell signal transduction pathways. Method 1. The DRG neurons separation male SD rats, weighing 80 - 120 g, clean grade. Decapitated, taken thoracic spine, in the mixed gas (95% O2 5% CO2) saturated 0 ° C in DMEM solution, exposed spinal separation the DRG, and immediately placed containing pancreatic protease (0.5 mg / ml, I type) and collagenase (1 mg / ml, IA type) in DMEM solution, and digested for 30 minutes in a heated water bath (37 ℃). Digestion is completed, repeated washing with DMEM solution four times, and then dispersed into single cells to form a cell suspension DRG neurons with a different caliber of straw wind and percussion pipette cell suspension drops in a small glass slide and allowed to stand for 30 minutes, until the cells adherent plus extracellular fluid in a Petri dish, the cells can be used in the experiment within 10 hours. Calcium imaging calcium imaging techniques to monitor the intracellular calcium concentration ([Ca2] i) changes. [Ca2] i measurement using a Ca2-sensitivity fluorescent dye Fura-2 acetoxymethyl ester (by Fura 2/AM). The outer liquid containing 2μmol / L Fura-2/AM incubated at 37 ° C with DRG neurons, combined with Ca2 of Fura-2/AM excitation wavelength is 340 nm, the Fura-2/AM excitation of unbound Ca2 optical wavelength is 380 nm and the emission wavelength was 510 nm. So that you can use the ratio of 340 nm and 380 nm, two fluorescence quantitative [Ca2] i changes. The use of calcium ion imaging analysis software TILLvision software, real-time detection [Ca2] i changes. 3 whole-cell patch-clamp recording VACC current as well as capsaicin-induced currents recorded by patch-clamp technique. Clamp electrode drawn from by microelectrode drawing instrument, then polished instrument electrode tip polishing. After electrode fillable inner solution so that the electrode tip into the water resistance of 3 to 5 m? The electrode with the cell membrane reached Gao impedance (G?) Sealing clamp electrode tip the film sucked broken, whole-cell modeling. Clamped at a membrane potential? 70 mV, 200 ms in time depolarized to 0 mV, neurons this depolarization pulse can induce VACC current. Perfusion of neurons with capsaicin (1μmol / L), by observing whether inward currents appear to detect the sensitivity of DRG neurons to capsaicin. The entire experiment is conducted at room temperature. Statistical analysis using Igor Pro software for statistical analysis. Statistics as mean ± standard error (mean ± SEM), said the group mean comparison with the unpaired t test, p lt; 0.05 Differences were considered significant. Results in capsaicin-sensitive DRG neurons, capsaicin (1μmol / L) can be induced inward current, and has a significant inhibitory effect of capsaicin on VACC current. DRG neurons in capsaicin insensitivity to detect less than the current evoked by capsaicin was not observed capsaicin VACC current inhibitory effect. 2 high potassium (40 mmol / L), so by activating VACC [Ca2] i rise. In capsaicin-sensitive DRG neurons, capsaicin (1μmol / L) so that [Ca2] i rise, high potassium so that [Ca2] i rise effect almost capsaicin completely inhibited. Insensitivity DRG neurons in capsaicin, capsaicin can not make [Ca2] i was significantly elevated in the high potassium [Ca2] i rise effect from capsaicin's impact. 3. Capsaicin inhibition the hyperkalemic induced voltage-gated Ca2 flow effects can be protein kinase C (PKC) the selective inhibitor Bisindolylmaleimide significantly weakened. Conclusion capsaicin-sensitive DRG neurons, capsaicin can inhibit VACCs, this inhibitory effect depends on intracellular calcium signaling and regulation by PKC.

The Celluar Signal Transduction of Capsaicin-induced Inhibition of Voltage-activated Calcium Channels in Rat Sensory Neurons

Abstract

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