Exp Neurobiol 2012; 21(2): 75-82
Published online June 30, 2012
© The Korean Society for Brain and Neural Sciences
Department of Physiology and Biophysics, Inha University College of Medicine, Incheon 402-752, Korea
Correspondence to: *To whom correspondence should be addressed.
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Capsaicin, the pungent ingredient in hot pepper, activates nociceptors to produce pain and inflammation. However, prolonged exposures of capsaicin will cause desensitization to nociceptive stimuli. Hyperpolarization-activated cation currents (
Keywords: capsaicin, DRG neuron, hyperpolarization-activated cation current, rat
The hyperpolarization-activatedcurrent (
Capsaicin, the main pungent ingredient in chili peppers can excite nociceptive sensory neurons and produce transient pain in animals and humans. Capsaicin activates the transient receptor potential vanilloid subtype 1 (TRPV1), a nonselective cation channel with high Ca2+ permeability . The TRPV1 channel is expressed in subsets of primary sensory neurons and nerve terminals and plays an essential role in detecting noxious heat and several other nociceptive stimuli. Based on the reports that TRPV1knockout mice exhibit reduced inflammatory thermal hyperalgesia, TRPV1 appears to be essential for mediating thermal hyperalgesia induced by inflammation [15, 16]. Paradoxically, prolonged or repetitive exposure to capsaicincan desensitize nociceptive sensory neurons and results in long lasting pain relief . Ca2+-dependent desensitization of TRPV1 themselves likely contributes to the analgesic effects of capsaicin .
Neuronal excitability is due to the presence of voltage-sensitive ion channels in the plasma membrane . Thus, activation or inhibition of these ion channels may contribute to the excitability of the sensory neurons and capsaicin analgesia. Capsaicin blocks voltage-gated Na+ channels in trigeminal ganglion  and DRG neurons [20-22], Ca2+ channels in the sensory neurons [23-25] and K+ channels in the trigeminal ganglion neurons . Such inhibitions of voltage-sensitive channels by capsaicin may result in decrease oft he excitability of neurons and block the transmission of painful signal from peripheral tissues.
As stated above, inhibition of
Primary cultures of DRG neurons dissected from all levels of lower cervical, thoracic, and lumbar spinal cord of two-day-old neonatal rats were prepared. The dissected ganglia were collected in cold culture medium (4℃), which contained Dulbecco's modified Eagle medium/F-12 mixture (DMEM/F-12; Gibco, Invitrogen, Grand Island, NY, USA), 10% fetal bovine serum (FBS; Gibco, Invitrogen), 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The collected ganglia were washed with the culture medium and incubated at 37℃ for 30 min in 1 mg/ml collagenase (Type II; Worthington, Freehold, NJ, USA). The ganglia were then washed three times with Mg2+- and Ca2+-free Hank's balanced salt solution (HBSS; Gibco, Invitrogen) and incubated in 2.5 mg/ml trypsin (Gibco, Invitrogen) at 37℃ for 30 min. Subsequently, the ganglia were centrifuged at 1,000 rpm for 10 min, and the pellet was washed two or three times with the culture medium to inhibit the enzyme. The pellet in turn was suspended in the culture medium by gentle trituration with a Pasteur pipette, and the suspended cells were plated on square glass coverslips coated with poly-L-lysine (Sigma-Aldrich), which were placed in small Petri dishes. Then, 25 ng/ml nerve growth factor (Alomone Labs, Jerusalem, Israel) were added to each Petri dish. Cells were incubated at 37℃ in a 95% air -5% CO2 gas mixture and used 1~3 days after plating.
Whole-cell membrane currents were recorded from the somata of DRG neurons. Electrodes were made by pulling borosilicate glass capillaries (Harvard, Kent, UK). Tip resistances were 2~3 MΩ for whole-cell recordings. To record whole-cell current, cellmembrane was ruptured by gentle suction after the formation of a gigaohmseal. Capacitative transients were then cancelled. Whole-cell membrane currents were recorded at -60 mV using an Axopatch 200 Bamplifier (Molecular Devices, Union City, CA, USA). Membrane currents were low pass filtered at 1 kHz and sampled at 2.5 kHz with a Digidata 1,322 data acquisition system (Molecular Devices). Data were analyzed using the pClamp 8.0 software (Molecular Devices) and OriginPro 8.0 (OriginLab, Northampton, MA, USA). All electrophysiological experiments were performed at room temperature.
Normal Tyrode's (NT) solution contained (in mM) 140 NaCl, 3 KCl, 10 HEPES, 10 glucose, 2.5 CaCl2, 1 MgCl2 (pH was adjusted to 7.4 with NaOH). The recording electrodes were filled with apipette solution containing (in mM) 135 K gluconate, 1 MgCl2, 10 NaCl, 10 HEPES, 2 Mg-ATP, and 0.1 Na-GTPtitrated to pH 7.4 with KOH. For intracellular Ca2+ chelation, 10 mM BAPTA was added to the internal solution. The nominally Ca2+-free bath solution contained (in mM) 140 NaCl, 3 KCl, 10 HEPES, 10 glucose, 1 MgCl2,10 EGTA (pH was adjusted to 7.4 with NaOH). To isolate
Data are presented as means±standard error of the mean (SEM). Inhibitory effects capsaicin were given as % inhibition=100×(1-(
Hyperpolarization of DRG neurons voltage-clamped at -60 mV induced noninactivatinginward current consisting of an instantaneous and a slowly-activating component (Fig. 1A).
We found that in capsaicin-insensitive cells where capsaicin did not evoke membrane currents, capsaicin had no significant effect on the amplitudes of
Because TRPV1 is highly permeable to Ca2+ , intracellular Ca2+ concentration can be greatly increased when the channel is opened. To determine the role of Ca2+ influx in the inhibitory effect of capsaicin on
Rapid rise in intracellular Ca2+ levels may due to either Ca2+ influx or release of Ca2+ from an intracellular store. Thus, we investigated whether the inhibitory effect of capsaicin on
Because the increase in intracellular Ca2+ appeared to be required for the inhibitory effect of capsaicin on
Calcineurin (protein phosphatase 2B) is a Ca2+-sensitive protein phosphatase and can be activated by a rise in intracellular Ca2+. To determine whether capsaicin inhibited
In the present study, we demonstrate that capsaicin reversibly and voltage-independently inhibited hyperpolarization-activated cation current,
Because TRPV1, capsaicin receptor is highly permeable to Ca2+ , extracellular Ca2+ influx through the activated channel can influence on
As described in the result section, BAPTA, thapsigargin and cyclosporin A partly attenuated the inhibition of
Increase of intracellular Ca2+ concentration can trigger many intracellular events such as protein phosphorylation. Therefore, we were interested in the cellular events following the increase of intracellular Ca2+ level through activated TRPV1 in the modulation of
It has been reported that