Effects of Phorbol-12,13-dibuterate on Sodium Currents and Potassium Currents in Rat Trigeminal Ganglion Neurons

The effects of phorbol-12,13-dibuterate （PDBu） on total sodium current （INa-total）, tetrodotoxin-resistant sodium current （INa-TFXr）, 4-AP-sensitive potassium current （IA） and TEA-sensitive potassium current （IK） in trigeminal ganglion （TG） neurons were investigated. Whole-cell patch clamp techniques were used to record ion currents in cultured TG neurons of rats. Results revealed that 0.5μmol/L PDBu reduced the amplitude of INa-total by （38.3±4.5）% （n=6, P〈0.05）, but neither the G-V curve （control： V0.5 =-17.1±4.3 mV, k=7.4±1.3; PDBu： V0.5=-15.9±5.9 mV, k=5.9±1.4; n=6, P〉0.05） nor the inactivation rate constant （control： 3.6±0.9 ms; PDBu： 3.6±0.8 ms; n=6, P〉0.05） was altered. 0.5 μmol/L PDBu could significantly increase the amplitude of INa-TFXr by （37.2± 3.2）% （n=9, P〈0.05） without affecting the G-V curve （control： V0.5=-14.7±6.0 mV, k=6.9± 1.4; PDBu： V0.5=- 11.1±5.3 mV, k=8.1± 1.5; n=5, P〉0.05 ） or the inactivation rate constant （control： 4.6±0.6 ms; PDBu： 4.2±0.5 ms; n=5, P〉0.05）. 0.5 μmol/L PDBu inhibited IK by （15.6±5.0） % （n=16, P〈0.05）, and V0.5 was significantly altered from - 4.7±1.4 mV to -7.9 ±1.8 mV （n=16, P〈0.05）. IA was not significantly affected by PDBu, 0.5μmol/L PDBu decreased IA by only （0.3±3.2）% （n=5, P〉0.05）. It was concluded that PDBu inhibited INa-total ：.but enhanced INa-TFXr, and inhibited IK without affecting IA. These data suggested that the activation of PKC pathway could exert the actions.

Chlorogenic acid alters the voltage-gated potassium channel currents of trigeminal ganglion neurons

Chlorogenic acid（5-caffeoylquinic acid, CGA） is a phenolic compound that is found ubiquitously in plants, fruits and vegetables and is formed via the esterification of caffeic acid and quinic acid. In addition to its notable biological functions against cardiovascular diseases, type-2 diabetes and inflammatory conditions, CGA was recently hypothesized to be an alternative for the treatment of neurological diseases such as Alzheimer＇s disease and neuropathic pain disorders. However, its mechanism of action is unclear.Voltage-gated potassium channel（Kv） is a crucial factor in the electro-physiological processes of sensory neurons. Kv has also been identified as a potential therapeutic target for inflammation and neuropathic pain disorders. In this study, we analysed the effects of CGA on the two main subtypes of Kv in trigeminal ganglion neurons, namely, the IK,Aand IK,Vchannels. Trigeminal ganglion（TRG）neurons were acutely disassociated from the rat TRG, and two different doses of CGA（0.2 and 1 mmol·L21） were applied to the cells.Whole-cell patch-clamp recordings were performed to observe alterations in the activation and inactivation properties of the IK,Aand IK,Vchannels. The results demonstrated that 0.2 mmol·L21CGA decreased the peak current density of IK,A. Both 0.2 mmol·L21and1 mmol·L21CGA also caused a significant reduction in the activation and inactivation thresholds of IK,Aand IK,V. CGA exhibited a strong effect on the activation and inactivation velocities of IK,Aand IK,V. These findings provide novel evidence explaining the biological effects of CGA, especially regarding its neurological effects.

Effect of Interleukin-1β on I_A and I_K Currents in Cultured Murine Trigeminal Ganglion Neurons

To investigate the effect of intedeukin-1β （IL-1β） on IA and IK currents in cultured murine trigeminal ganglion （TG） neurons, whole-cell patch clamp technique was used to record the IA and IK currents before and after 20 ng/mL IL-1β perfusion. Our results showed that 20 ng/mL IL-1β inhibited IA currents （18.3±10.7）% （n=6, P〈0.05）. IL-1β at 20 ng/mL had no effect on G-V curve of IA but moved the H-infinity curve V0.5 from -36.6±6. 1 mV to-42.4±5.2 mV （n=5, P〈0.01）. However, 20 ng/mL IL-1β had effect on neither the amplitude nor the G-V curve of IK. IL-1β was found to selectively inhibit IA current in TG neurons and the effect may contribute to hyperalgesia under various inflammatory conditions.

Inhibitory effects of synthetic cannabinoid WIN55,212-2 on nicotine-activated currents in rat trigeminal ganglion neurons

Cannabinoid and nicotinic acetylcholine receptors are strongly associated with algesia. Previous studies in our laboratory have reported inhibitory effects of synthetic cannabinoid WIN55, 212-2 on nicotine-activated currents （Inic）, but the underlying mechanisms remain poorly understood. The present study used whole-cell patch clamp techniques to investigate the modulatory effects of synthetic cannabinoid WIN55, 212-2 on Inic in cultured rat trigeminal ganglion neurons. The results revealed several major findings： WIN55, 212-2 inhibited Inic in rat trigeminal ganglion neurons. In addition, when WIN55, 212-2 （3 μmol/L） was applied simultaneously with nicotine （100 μmol/L）, the inhibition of WIN55, 212-2 on Inic was reversible, concentration-dependent and voltage-independent This effect was not mediated by CB1, CB2 or VR1 receptors; neither the selective CB1 receptor antagonist AM281, CB2 receptor antagonist AM630 nor VR1 receptor antagonist capsazepine reduced the inhibitory effect of WIN55, 212-2. Further, the inhibition of nicotinic responses by WIN55, 212-2 was not sensitive to the membrane permeable cyclic adenosine monophosphate （cAMP） analog 8-Br-cAMP. The G-protein inhibitor GDP-I3-S （1 mmol/L） did not block the inhibitory effects of WIN55, 212-2 on/n^c, excluding the involvement of G-protein mediation. The results suggested that WIN55, 212-2 inhibits/n^o directly via the neuronal nicotinic acetylcholine receptor, and that this inhibition is non-competitive. WIN55, 212-2 did not act as an open channel blocker of the neuronal nicotinic acetylcholine receptor, and did not affect the desensitization of Into. The results suggest that nicotine receptors may be physically plugged from outside the membrane by drugs containing WIN55, 212-2.

Inhibition of 5-HT_3 Receptors-activated Currents by Cannabinoids in Rat Trigeminal Ganglion Neurons

This study investigated the modulatory effect of synthetic cannabinoids WIN55,212-2 on 5-HT3 receptor-activated currents (I5-HT3) in cultured rat trigeminal ganglion (TG) neurons using whole-cell patch clamp technique. The results showed that: (1) The majority of examined neurons (78.70%) were sensitive to 5-HT (3–300 μmol/L). 5-HT induced inward currents in a concentration-dependent manner and the currents were blocked by ICS 205-930 (1 μmol/L), a selective antagonist of the 5-HT3 receptor; (2) Pre-application of WIN55,212-2 (0.01–1 μmol/L) significantly inhibited I5-HT3 reversibly in concentration-dependent and voltage-independent manners. The concentra-tion-response curve of 5-HT3 receptor was shifted downward by WIN55,212-2 without any change of the threshold value. The EC50 values of two curves were very close (17.5±4.5) mmol/L vs. (15.2±4.5) mmol/L and WIN55,212-2 decreased the maximal amplitude of I5-HT3 by (48.65±4.15)%; (3) Neither AM281, a selective CB1 receptor antagonist, nor AM630, a selective CB2 receptor antagonist reversed the inhibition of I5-HT3 by WIN55,212-2; (4) When WIN55,212-2 was given from 15 to 120 s before 5-HT application, inhibitory effect was gradually increased and the maximal inhibition took place at 90 s, and the inhibition remained at the same level after 90 s. We are led to concluded that-WIN55,212-2 inhibited I5-HT3 significantly and neither CB1 receptor antagonist nor CB2 receptor antagonist could reverse the inhibition of I5-HT3 by WIN55,212-2. Moreover, WIN55,212-2 is not an open channel blocker (OCB) of 5-HT3 receptor. WIN55,212-2 significantly inhibited 5-HT-activated currents in a non-competitive manner. The inhibition of I5-HT3 by WIN55,212-2 is probably new one of peripheral analgesic mechanisms of WIN55,212-2, but the mechanism by which WIN55,212-2 inhibits I5-HT3 warrants further investigation.

Effects of WIN 55,212-2 on I_K Current in Cultured Trigeminal Ganglion Neurons of Rat

Summary: To investigate the effects of WIN 55,212-2 on I K in cultured rat trigeminal ganglion (TG) neurons, whole-cell patch clamp techniques were used to record the I K before and after WIN 55,212-2 perfusion at different concentrations. 30 μmol/L WIN 55,212-2 markedly (35 7 %± 7 3 %, P<0.01, n=8) inhibited I K currents, and the currents were partially recovered after washing. 30 μmol/L WIN 55,212-2 also induced a significant depolarizing shift in conductance-voltage parameters (control: V 0 5=10 43 ± 4.25 mV, k=16 27±3 86; WIN 55,212-2: V 0.5=24.71±3.91 mV, k =16.69±2.75; n = 8, P<0.01 for V 0.5). 0.01 μmol/L WIN 55,212-2 slightly (27.0 %± 7.9 %, P<0.05, n=7) increased I K currents, but had no significant change in conductance–voltage parameters (control: V 0.5=10.74±5.27 mV, k=17.33±2.96; WIN 55,212-2: V 0.5=11.06±2.05 mV, k=19.69±6.60; n=7, P>0.05 for V 0.5 and k). These results suggested that WIN 55,212-2 has dual action, which might be through different receptors.

Flunarizine inhibits sensory neuron excitability by blocking voltage-gated Na＋ and Ca2＋ currents in trigeminal ganglion neurons

Background Although flunarizine has been widely used for migraine prophylaxis with clear success, the mechanisms of its actions in migraine prophylaxis are not completely understood. The aim of this study was to investigate the effects of flunarizine on tetrodotoxin-resistant Na＋ channels and high-voltage activated Ca2＋ channels of acutely isolated mouse trigeminal ganglion neurons. Methods Sodium currents and calcium currents in trigeminal ganglion neurons were monitored using whole-cell patch-clamp recordings. Paired Student＇s t test was used as appropriate to evaluate the statistical significance of differences between two group means. Results Both tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were blocked by flunarizine in a concentration-dependent manner with the concentration producing half-maximal current block values of 2.89 μmol/L and 2.73 μmol/L, respectively. The steady-state inactivation curves of tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were shifted towards more hyperpolarizing potentials after exposure to flunarizine. Furthermore, the actions of flunarizine in blocking tetrodotoxin-resistant sodium currents and high-voltage activated calcium currents were use-dependent, with effects enhanced at higher rates of channel activation. Conclusion Blockades of these currents might help explain the peripheral mechanism underlying the preventive effect of flunarizine on migraine attacks.

Lipopolysaccharide-induced Trigeminal Ganglion Nerve Fiber Damage is Associated with Autophagy Inhibition

Objective This study aimed to determine whether lipopolysaccharide(LPS)induces the loss of corneal nerve fibers in cultured trigeminal ganglion(TG)cells,and the underlying mechanism of LPS-induced TG neurite damage.Methods TG neurons were isolated from C57BL/6 mice,and the cell viability and purity were maintained for up to 7 days.Then,they were treated with LPS(1µg/mL)or the autophagy regulator(autophibib and rapamycin)alone or in combination for 48 h,and the length of neurites in TG cells was examined by the immunofluorescence staining of the neuron-specific proteinβ3-tubulin.Afterwards,the molecular mechanisms by which LPS induces TG neuron damage were explored.Results The immunofluorescence staining revealed that the average length of neurites in TG cells significantly decreased after LPS treatment.Importantly,LPS induced the impairment of autophagic flux in TG cells,which was evidenced by the increase in the accumulation of LC3 and p62 proteins.The pharmacological inhibition of autophagy by autophinib dramatically reduced the length of TG neurites.However,the rapamycin-induced activation of autophagy significantly lessened the effect of LPS on the degeneration of TG neurites.Conclusion LPS-induced autophagy inhibition contributes to the loss of TG neurites.