Changjin Liu

Changes in osmolality modulate voltage-gated calcium channels in trigeminal ganglion neurons.

Voltage-gated calcium channels (VGCCs) participate in many important physiological functions. However whether VGCCs are modulated by changes of osmolarity and involved in anisotonicity-induced nociception is still unknown. For this reason by using whole-cell patch clamp techniques in rat and mouse trigeminal ganglion (TG) neurons we tested the effects of hypo- and hypertonicity on VGCCs. We found that high-voltage-gated calcium current (I(HVA)) was inhibited by both hypo- and hypertonicity. In rat TG neurons, the inhibition by hypotonicity was mimicked by Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator but hypotonicity did not exhibit inhibition in TRPV4(-/-) mice TG neurons. Concerning the downstream signaling pathways, antagonism of PKG pathway selectively reduced the hypotonicity-induced inhibition, whereas inhibition of PLC- and PI3K-mediated pathways selectively reduced the inhibition produced by hypertonicity. In summary, although the effects of hypo- and hypertonicity show similar phenotype, receptor and intracellular signaling pathways were selective for hypo- versus hypertonicity-induced inhibition of I(HVA).

Osmolality-induced tuning of action potentials in trigeminal ganglion neurons

The present study explored the effect of anisotonicity on action potential (AP) in cultured trigeminal ganglion (TG) neurons. We demonstrate that the number of evoked APs was increased by both hypo- and hypertonic treatment. Transient Receptor Potential Vanilloid 4 receptor (TRPV4) activator increased the number of APs, but only hypotonic-response was markedly blocked in TRPV4-/- mice. Additionally, inhibition of PKC attenuated hypotonicity-induced increase, whereas antagonism of PKA attenuated hypertonicity-response. We conclude that anisotonicity increases excitability of nociceptors, which might be involved in anisotonicity-induced nociception. The increase of APs by hypo- and hypertonicity is mediated through different receptor and intracellular signaling pathways.

Effects of capsaicin on VGSCs in TRPV1−/− mice

Two different mechanisms by which capsaicin blocks voltage-gated sodium channels (VGSCs) were found by using knockout mice for the transient receptor potential V1 (TRPV1(-/-)). Similar with cultured rat trigeminal ganglion (TG) neurons, the amplitude of tetrodotoxin-resistant (TTX-R) sodium current was reduced 85% by 1 muM capsaicin in capsaicin sensitive neurons, while only 6% was blocked in capsaicin insensitive neurons of TRPV1(+/+) mice. The selective effect of low concentration capsaicin on VGSCs was reversed in TRPV1(-/-) mice, which suggested that this effect was dependent on TRPV1 receptor. The blockage effect of high concentration capsaicin on VGSCs in TRPV1(-/-) mice was the same as that in capsaicin insensitive neurons of rats and TRPV1(+/+) mice. It is noted that non-selective effect of capsaicin on VGSCs shares many similarities with local anesthetics. That is, firstly, both blockages are concentration-dependent and revisable. Secondly, being accompanied with the reduction of amplitude, voltage-dependent inactivation curve shifts to hyperpolarizing direction without a shift of activation curve. Thirdly, use-dependent blocks are induced at high stimulus frequency.

Changes in osmolality modulate voltage-gated sodium channels in trigeminal ganglion neurons.

Voltage-gated sodium channels (VGSCs) are important channels which participate in many physiological functions. Whether VGSCs can be modulated by changes in osmolality in trigeminal ganglion (TG) neurons remains unknown. In this study, by using whole-cell patch clamp techniques, we tested the effects of hypo- and hypertonicity on VGSCs in cultured TG neurons. Our data show that tetrodotoxin-resistant sodium current (TTX-R current) was inhibited in the presence of hypo- and hypertonic solutions. In hypertonic solutions both voltage-dependent activation and inactivation curves shifted to the hyperpolarizing direction, while in hypotonic solutions only inactivation curve shifted to the hyperpolarizing direction. Transient Receptor Potential Vanilloid 4 (TRPV4) receptor activator mimicked the inhibition of TTX-R current by hypotonicity and the inhibition by hypotonicity was markedly attenuated by TRPV4 receptor blocker and in TRPV4(-/-) mice TG neurons. We also demonstrate that the inhibition of PKA selectively attenuated hypotonicity-induced inhibition, whereas antagonism of PLC and PI3K selectively attenuated hypertonicity-induced inhibition. We conclude that although hypo- and hypertonicity have similar effect on VGSCs, receptor and intracellular signaling pathways are different for hypo- versus hypertonicity-induced inhibition of TTX-R current.

The modulation of voltage-gated potassium channels by anisotonicity in trigeminal ganglion neurons

Voltage-gated potassium channels (VGPCs) play an important role in many physiological functions by controlling the electrical properties and excitability of cells. Changes in tonicity in the peripheral nervous system can activate nociceptors and produce pain. Here, using whole cell patch clamp techniques, we explore how hypo- and hypertonicity modulate VGPCs in cultured rat and mouse trigeminal ganglion (TG) neurons. We found that hypo- and hypertonicity had different effects on slow-inactivating K+ current (IK) and fast-inactivating K+ current (IA): hypotonicity increased IK but had no effect on IA while hypertonicity depressed both IK and IA. The increase of IK by hypotonicity was mimicked by transient receptor potential vanilloid 4 (TRPV4) receptor activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) but hypotonicity did not exhibit increase in TRPV4-/- mice TG neurons, suggesting that TRPV4 receptor was involved in hypotonicity-induced response. We also found that inactivation of PKC selectively reversed the increase of IK by hypotonicity, whereas antagonism of G-protein selectively rescued the inhibitions of IK and IA by hypertonicity, indicating that different intracellular signaling pathways were required for the modulation by hypo- and hypertonicity. In summary, changes in osmolality have various effects on IK and IA and different receptors and second messenger systems are selective for the modulation of VGPCs induced by hypo- versus hypertonicity.

Action of aluminum on high voltage‐dependent calcium current and its modulation by ginkgolide B

AbstractAim:To investigate the effect of aluminum (Al) on high voltage-dependent calcium current (IHVA) and its modulation by ginkgolide B (Gin B).Methods:The whole-cell, patch-clamp technique was used to record IHVA from acutely isolated hippocampal CA1 pyramydal neurons in rats.Results:Al 0.1 mmol/L (low concentration) reduced IHVA; Al 0.75 and 1.0 mmol/L (high concentrations) increased IHVA, and Al decreased and increased IHVA at intermediate concentrations of 0.25 and 0.5 mmol/L. The increase of IHVA by Al 1.0 mmol/L was enhanced by the adenylyl cyclase (AC) agonist forskolin and was partly abolished by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) antagonist H-89, whereas the decrease observed with Al 0.1 mmol/L was neither reversed by forskolin nor affected by H-89. Gin B had no effect on IHVA in normal neurons, but canceled the increase in IHVA by 1.0 mmol/L Al.Conclusion:The results indicate that the mechanism of Al affecting IHVA differs at different concentrations, and this may be attributed to its complex actions. Gin B could prevent neurons from injury by inhibiting calcium influx.

Hypertonic stimulation inhibits synaptic transmission in hippocampal slices through decreasing pre-synaptic voltage-gated calcium current

Acute changes in the cerebrospinal fluid osmotic pressure modulate the brain excitability. The present study investigated the effect of hypertonic stimulation on the synaptic transmission in hippocampal slices. It was found that the slope of excitatory postsynaptic potential (EPSP) in hippocampal CA1 area was inhibited after the hypertonic treatment. Accompanied with the inhibition in EPSP slope, the paired-pulse facilitation (PPF) was increased by hypertonicity. Transient receptor potential vanilloid 4 (TRPV4 receptor) antagonists did not block hypertonicity-action. High voltage-gated calcium current (I(HVA)) in hippocampal CA3 neurons was decreased by hypertonicity, whereas the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-induced current in hippocampal CA1 neurons was unaffected. Additionally, inhibition of phosphatidylinositol 3-kinase (PI3K) or protein kinase A (PKA) markedly attenuated hypertonicity-induced decrease of I(HVA), whereas antagonism of phosphorylated ERK1/2 mitogen-activated protein kinase (pERK1/2) had no effect. We conclude that hypertonic stimulation inhibits synaptic transmission in hippocampal slices through decreasing pre-synaptic voltage-gated calcium current.

Regulatory effects of anandamide on intracellular Ca(2+) concentration increase in trigeminal ganglion neurons.

Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neurotransmission by decreasing Ca2+ influx through high voltage-gated Ca2+ channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca2+ influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca2+ concentration by the cannabinoid receptor type 1 agonist anandamide, and its underlying mechanisms. Using whole cell voltage-clamp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca2+ influx in a dose-dependent manner, which then triggered an increase of intracellular Ca2+ concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca2+ concentration by anandamide. This result showed that anandamide increased intracellular Ca2+ concentration and inhibited high voltage-gated Ca2+ channels through different signal transduction pathways.

An optimized recording method to characterize biophysical and pharmacological properties of acid-sensing ion channel

ObjectiveTo re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a (hASIC1a) current in HEK293 cells with a modified perfusion methods.MethodsWith cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASIC1a currents evoked by low pH external solution.ResultsUsing cell floating method, the amplitude of hASIC1a currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is (21±5) ms and (270±25) ms, respectively. Inactivation time constants are (496±23) ms and (2284±120) ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC1a [IC50 is (3.4±1.1) μmol/L and (2.4±0.9) μmol/L, respectively]. Both recording methods have similar pH activation EC50 (6.6±0.6, 6.6±0.7, respectively).ConclusionASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC1a current was re-confirmed and the biophysical and pharmacological properties of hASIC1a channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.摘要目的用一种改良的灌流方法研究和再次证实HEK 293细胞内源性酸敏感性离子通道的生物物理和药理学特性。方法使用全细胞膜片钳技术, 将记录细胞与玻片分离, 并使细胞位于输液맜前, 呈漂浮状态, 以记录在低pH 值外液中, HEK293 细胞的酸敏感性1a 型离子通道的电流。结果使用细胞漂浮方法, pH 5.0 的细胞外液诱发的酸敏感性1a 型离子通道电流是传统细胞附着法诱发的两倍。在两种不同的方法下, 通道电流达到峰值的时间分别是 (21±5) ms和 (270±25) ms, 失活的时间常数分别是(496±23) ms 和(2284±120) ms。细胞漂流法也明显增强amiloride 对酸敏感性1a型离子通道的阻断效能。两种方法具有相似的pH激活的EC50 (分别为6.6±0.6, 6.6±0.7)。结论酸敏感性1a 型离子通道的激活需要细胞外液快速的交换。通过细胞漂浮的方法, 我们再次证明了酸敏感性1a型离子通道电流的存在, 更精确地分析了HEK 293 细胞内源性酸敏感型离子通道的生物物理和药理学特性。这一改良的方法能够用于研究所有的酸敏感型离子通道和需要快速细胞外液交换的配体门控通道。