酸敏感离子通道在非神经组织中的功能研究

酸敏感离子通道(ASICs)是一类由细胞外酸化所激活的阳离子通道,属于钠通道超家族的新成员。近来发现,ASICs不但在神经系统具有重要的生物学功能,对神经系统以外的组织(如:味蕾、心血管、骨等)的生理和病理过程中也具有重要的作用。该文对有关ASICs在神经以外其他组织中研究的最新进展作一综述,以增进对ASICs生物学功能和病理作用的了解。     

酸敏感离子通道1a在酸诱导的大鼠肝星状细胞活化中的作用

目的：探讨酸敏感离子通道1a（ASIC1a）在酸诱导的大鼠肝星状细胞活化中的作用。方法：体外培养肝星状细胞株（HSC-T6）经处理后,乳酸脱氢酶（LDH）检测细胞毒性变化,RT—PCR检测ASIC1a mRNA表达,Western Blot和RT—PCR分别检测Calpain、Calcineurin蛋白和mRNA表达变化,免疫细胞化学法检测α-平滑肌肌动蛋白（α—SMA）的变化。结果：细胞酸化处理后ASIC1a含量明显上升;随着胞外pH值的下降,LDH释放量逐渐升高,ASIC1a阻滞剂可抑制酸化诱导的肝星状细胞LDH的释放量;酸化组肝星状细胞Calpain、Calcineurin mRNA和蛋白表达水平均明显增高,ASIC1a阻断剂组Calpain、Calcineurin mRNA和蛋白表达水平较酸化组明显降低;与酸化组相比,阻断ASIC1a可以降低HSC—T6中α—SMA的表达。结论：胞外酸化环境下可诱导肝星状细胞活化,阻断ASIC1a能明显降低酸化诱导的细胞活化。     

酸敏感离子通道1a在自身免疫性疾病发病机制中的研究进展

酸敏感离子通道(acid-sensing ion channels,ASICs)是在外周和中枢神经系统中广泛表达的质子门控的阳离子通道,ASIC1a作为其重要的组成部分在许多生理和病理过程中起重要作用。作为细胞外质子的关键受体,ASIC1a参与涉及组织酸中毒的多种病理生理过程,如疼痛、炎症、癫痫发作、多发性硬化等。自身免疫疾病是机体在应对自身抗原发生免疫反应时,过度活化的T、B细胞导致机体多组织器官受损的慢性炎症性疾病。近年来研究发现,ASIC1a在多种自身免疫性疾病发生中发挥着重要作用,该文就ASIC1a的生物学特征作简要综述,重点探讨ASIC1a在自身免疫性疾病发病机制中的研究进展。     

Current perspectives on acid-sensing ion channels: new advances and therapeutic implications

Acid-sensing ion channels (ASICs) form a family of voltage-independent cation channels that predominantly conduct Na⁺ ions, and were identified at the molecular level a little more than a decade ago. ASICs are activated by extracellular acidification within the physiological range, and they form effective proton sensors in both central and peripheral sensory neurons. A combination of genetic and pharmacologic approaches has revealed their implication in an increasing number of physiological and pathophysiological processes – most of them associated with extracellular pH fluctuations, ranging from synaptic plasticity, learning, memory, fear, depression, seizure termination and neuronal degeneration to nociception and mechanosensation. ASICs, therefore, emerge as new potential therapeutic targets in the management of psychiatric disorders, stroke, neurodegenerative diseases and pain.     

酸敏感离子通道1a在酸诱导的大鼠关节软骨细胞自噬的作用及其机制研究

目的研究酸敏感离子通道1a(acid-sensing ion chan-nel 1a)在体外培养的酸诱导的大鼠关节软骨细胞自噬的作用及其可能机制。方法Ⅱ型胶原酶消化法分离大鼠关节软骨细胞,鉴定后传代培养,观察在不同pH条件下诱导的细胞自噬情况以确定胞外酸化条件;将软骨细胞分为pH7.4环境下培养的正常组、pH 6.0的酸化组、以及经ASIC1a非特异性阻断剂Amiloride和特异性阻断剂PcTX1处理的酸化组,RT-PCR法检测细胞自噬基因Beclin-1 mRNA的表达,Western blot法检测自噬蛋白LC3及ERK1/2、p38MAPK磷酸化蛋白的表达,透射电子显微镜法(TEM)观察自噬小体数量的变化;通过使用ERK1/2、p38MAPK磷酸化抑制剂(PD98059、SB203580),采用RT-PCR和Western blot法观察ERK1/2及p38MAPK对酸诱导的软骨细胞自噬的影响。结果 pH 5.5和pH 6.0胞外酸化刺激均能明显升高软骨细胞自噬水平(P<0.01);与pH 7.4组比较,酸化组软骨细胞Beclin-1 mRNA及LC3、磷酸化的ERK1/2和p38MAPK蛋白表达水平均明显升高(P<0.01),且自噬小体数量增多,ASIC1a阻断剂组自噬水平及ERK1/2、p38磷酸化蛋白表达水平较酸化组明显降低(P<0.01),且自噬体数量减少;与pH 6.0组相比,ERK1/2磷酸化抑制剂处理后,Beclin-1 mR-NA和LC3蛋白的表达均下调(P<0.05,P<0.01),而p38磷酸化抑制剂组自噬水平则无明显变化。结论胞外酸化环境下能诱发软骨细胞自噬,阻断ASIC1a能明显减弱酸化诱导的软骨细胞自噬,其机制可能与抑制ERK1/2磷酸化有关。     

Functional role of T-type calcium channels in tumour growth and progression: prospective in cancer therapy

T-type Ca²⁺ channels represent a specific channel family overexpressed in different types of tumours. Their involvement in controlling the proliferation, angiogenesis and invasion of tumour cells, has been partially clarified. The article by Zhang et al. in this issue of BJP provides the first evidence of anti-tumoural effects of endostatin (ES) in U87 glioma cells. He demonstrated that ES or mibefradil (a L/T-type calcium channel blocker), reduces the proliferation and migration of U87 glioma cells in a T-type Ca²⁺ channel-dependent manner. However, the difference in the blocking effect of mibefradil on T-type calcium channel expression as compared with its ability to inhibit proliferation and migration, supports the idea of a broader T/L-type-independent effect of the mibefradil blocker. Overall, these findings provide new insights for the future development of a novel class of anti-T-type calcium channel blockers in the therapy of glioblastoma.

LINKED ARTICLE

This article is a commentary on Zhang et al., pp. 1247–1260 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2012.01852.x     

大脑中动脉栓塞模型大鼠的中枢电压依赖性钾通道mRNA表达的改变

目的观察几种代表性电压依赖性钾通道亚型在脑缺血不同时间大鼠海马和皮层mRNA表达水平的变化。方法采用大脑中动脉栓塞模型致大鼠脑缺血损伤，应用RT-PCR方法检测Kv1.4，Kv1.5，Kv2.1和Kv4.2 mRNA表达水平在海马和皮层中的改变。结果大脑中动脉栓塞模型大鼠出现明显的神经损伤症状。缺血2 h时，海马组织的Kv1.4，Kv2.1和Kv4.2 mRNA表达水平分别增加了50%，67%和90%，在缺血24 h时Kv1.4和Kv4.2 mRNA仍保持高水平表达。大鼠皮层组织在缺血2 h后，Kv1.4，Kv1.5，Kv2.1和Kv4.2 mRNA水平均无明显改变，缺血24 h后，Kv2.1和Kv4.2 mRNA水平分别增加了70%和62%。结论大脑中动脉栓塞模型大鼠的海马和皮层组织中电压依赖性钾通道亚型的mRNA表达发生明显上调。     

Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain

Background and purpose:

T-cells may play a role in the evolution of ischaemic damage and repair, but the ability to image these cells in the living brain after a stroke has been limited. We aim to extend the technique of real-time in situ brain imaging of T-cells, previously shown in models of immunological diseases, to models of experimental stroke.

Experimental approach:

Male C57BL6 mice (6–8 weeks) (n= 3) received a total of 2–5 × 10⁶ carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled lymphocytes from donor C57BL6 mice via i.v. injection by adoptive transfer. Twenty-four hours later, recipient mice underwent permanent left distal middle cerebral artery occlusion (MCAO) by electrocoagulation or by sham surgery under isoflurane anaesthesia. Female hCD2-green fluorescent protein (GFP) transgenic mice that exhibit GFP-labelled T-cells underwent MCAO. At 24 or 48 h post-MCAO, a sagittal brain slice (1500 µm thick) containing cortical branches of the occluded middle cerebral artery (MCA) was dissected and used for multiphoton laser scanning microscopy (MPLSM).

Key results:

Our results provide direct observations for the first time of dynamic T-cell behaviour in living brain tissue in real time and herein proved the feasibility of MPLSM for ex vivo live imaging of immune response after experimental stroke.

Conclusions and Implications:

It is hoped that these advances in the imaging of immune cells will provide information that can be harnessed to a therapeutic advantage.This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x     

Chloroquine impairs visual transduction via modulation of acid sensing ion channel 1a

Acid-sensing ion channels (ASICs) are extracellular pH sensors activated by protons, which influence retinal activity and phototransduction. Among all ASICs, ASIC1a is abundantly expressed in the retina and involved in normal retinal activity. Chloroquine, which has been used in the treatment of malaria, rheumatoid arthritis and systemic lupus erythematosus, has been shown to be toxic to the retina. However, the underlying mechanisms remain unclear. In this study, we investigated the role of chloroquine in phototransduction by measuring the electroretinogram (ERG). The effect of chloroquine on acid-evoked currents in either isolated rat retinal ganglion neurons (RGNs) or Chinese hamster ovary (CHO) cells transfected with ASIC1a were assessed using a whole-cell patch-clamp technique. Chloroquine reduced the b-wave of scotopic 0.01 and photopic 3.0 and amplitudes of oscillatory potentials (OPs), an effect which was almost completely reversed by PcTx1, an ASIC1a-specific channel blocker. Further, patch-clamp experiments demonstrated that chloroquine reduced the peak current amplitude and prolonged the activation and desensitization of ASIC1a currents. These chloroquine-induced effects on the kinetics of ASIC 1a were dose-, pH- and Ca²⁺-dependent. Taken together, these results demonstrate that chloroquine affects vision conduction by directly modifying the kinetics of ASIC1a. Such a mechanism, may, in part, explain the retinal toxicity of chloroquine.     

The pharmacology and therapeutic potential of small molecule inhibitors of acid-sensing ion channels in stroke intervention

In the nervous system, a decrease in extracellular pH is a common feature of various physiological and pathological processes, including synaptic transmission, cerebral ischemia, epilepsy, brain trauma, and tissue inflammation. Acid-sensing ion channels (ASICs) are proton-gated cation channels that are distributed throughout the central and peripheral nervous systems. Following the recent identification of ASICs as critical acid-sensing extracellular proton receptors, growing evidence has suggested that the activation of ASICs plays important roles in physiological processes such as nociception, mechanosensation, synaptic plasticity, learning and memory. However, the over-activation of ASICs is also linked to adverse outcomes for certain pathological processes, such as brain ischemia and multiple sclerosis. Based on the well-demonstrated role of ASIC1a activation in acidosis-mediated brain injury, small molecule inhibitors of ASIC1a may represent novel therapeutic agents for the treatment of neurological disorders, such as stroke.     

Angelica gigas root ameliorates ischaemic stroke-induced brain injury in mice by activating the PI3K/AKT/mTOR and MAPK pathways

ContextTraditionally, the root of Angelica gigas Nakai (Umbelliferae), has long been used to treat ischaemic diseases and is considered safe in humans.ObjectiveTo investigate the neuroprotective effects of a methanol extract of A. gigas root (AGmex) on the middle cerebral artery occlusion (MCAO)-induced brain injury in mice, and the underlying mechanisms.Materials and methodsTwo hours of transient MCAO (tMCAO) was induced in C57BL/6 mice (MCAO control group and AGmex groups), AGmex was administered to the AGmex group at 300-3,000 mg/kg bw at 1, 1, and 24 h before tMCAO or at 1000 mg/kg bw at 1 h before and after tMCAO. Infarction volumes, tissue staining, and western blotting were used to investigate the mechanism underlying the neuroprotective effects of AGmex.ResultsThe median effective dose (ED₅₀) could not be measured because the AGmex treatment did not reduce the infarction volume caused by 2 h of tMCAO to within 50%; however, pre-treatment with AGmex twice at 1,000 mg/kg bw before tMCAO significantly reduced the infarction volumes. The proteins related to cell growth, differentiation, and death were upregulated by this treatment, and the major recovery mechanisms appeared to involve the attenuation of the mitochondrial function of Bcl-2/Bax and activation of the PI3K/AKT/mTOR and MAPK signalling pathways in ischaemic neurons.ConclusionsThis study provides evidence supporting the use of A. gigas root against ischaemic stroke and suggests a novel developmental starting point for the treatment of ischaemic stroke.     

Mechanosensitive ion channels in skeletal muscle: a link in the membrane pathology of muscular dystrophy

1. Mechanosensitive (MS) channels are expressed abundantly in skeletal muscle at all stages of development. In recordings from membrane patches, MS channels are constitutively active at the resting potential. The channels are selective for cations and have a large single-channel conductance (approximately 25 pS in physiological saline) and a high Ca2+ permeability (relative permeability of Ca2+ to K+ (PCa/PK) = 7). 2. Mechanosensitive channel activity recorded from the surface of myotubes from dystrophic mdx mice was substantially greater than the activity recorded from wild-type myotubes. Increased channel activity in the mutant results from the induction in a subpopulation of channels of a novel MS gating mode characterized by markedly prolonged channel openings and inactivation in response to membrane stretch. 3. Membrane stretch or a strong depolarization causes an irreversible switch to the stretch-inactivated gating mode in mdx myotubes. A stretch-induced shift in MS channel gating mode may contribute to stretch-induced elevations in [Ca2+]i during the early stages of disease pathogenesis. 4. Abnormalities of MS channel behaviour are also detected in recordings from patches on flexor digitorum brevis fibres acutely isolated from mdx mice. Mechanosensitive channel opening probability is higher in mdx fibres at all developmental stages. In addition, channel numbers are persistently elevated during postnatal development, failing to undergo a normal process of downregulation during the first 3 postnatal weeks. 5. Two distinct mechanisms may contribute to elevations of [Ca2+]i in dystrophin-deficient skeletal muscle: (i) a membrane stress-dependent switch of MS channels into to a prolonged opening mode; and (ii) a loss of developmental downregulation leading to persistent MS channel expression during postnatal muscle development.     

Oxytocin inhibits the activity of acid-sensing ion channels through the vasopressin,V1A receptor in primary sensory neurons

BACKGROUND AND PURPOSE

A growing number of studies have demonstrated that oxytocin (OT) plays an analgesic role in modulation of nociception and pain. Most work to date has focused on the central mechanisms of OT analgesia, but little is known about whether peripheral mechanisms are also involved. Acid-sensing ion channels (ASICs) are distributed in peripheral sensory neurons and participate in nociception. Here, we investigated the effects of OT on the activity of ASICs in dorsal root ganglion (DRG) neurons.

EXPERIMENTAL APPROACH

Electrophysiological experiments were performed on neurons from rat DRG. Nociceptive behaviour was induced by acetic acid in rats and mice lacking vasopressin, V_1A receptors.

KEY RESULTS

OT inhibited the functional activity of native ASICs. Firstly, OT dose-dependently decreased the amplitude of ASIC currents in DRG neurons. Secondly, OT inhibition of ASIC currents was mimicked by arginine vasopressin (AVP) and completely blocked by the V_1A receptor antagonist SR49059, but not by the OT receptor antagonist L-368899. Thirdly, OT altered acidosis-evoked membrane excitability of DRG neurons and significantly decreased the amplitude of the depolarization and number of action potentials induced by acid stimuli. Finally, peripherally administered OT or AVP inhibited nociceptive responses to intraplantar injection of acetic acid in rats. Both OT and AVP also induced an analgesic effect on acidosis-evoked pain in wild-type mice, but not in V_1A receptor knockout mice.

CONCLUSIONS AND IMPLICATIONS

These results reveal a novel peripheral mechanism for the analgesic effect of OT involving the modulation of native ASICs in primary sensory neurons mediated by V_1A receptors.     

Multiple structural elements contribute to voltage-dependent facilitation of neuronal α1C (CaV1.2) L-type calcium channels

Voltage- and frequency-dependent facilitation of calcium channel activity has been implicated in a number of key physiological processes. Various mechanisms have been proposed to mediate these regulations, including a switch between channel gating modes, voltage-dependent phosphorylation, and a voltage-dependent deinhibition of G-protein block. Studying such modulation on recombinant Ca channels expressed in oocytes, we previously reported that α_1C L-type calcium channel contrast with non-L type Ca channels by its ability to exhibit facilitation by pre-depolarization (Voltage-dependent facilitation of a neuronal α_IC L-type calcium channel, E. Bourinet et al., EMBO Journal, 1994; 13, 5032–5039). To further analyze this effect, we have investigated the molecular determinants which mediate the differences in voltage-dependent facilitation between «facilitable» α_1C and «non facilitable» α_1E calcium channels. We used a series of chimeras which combine the four transmembrane domains of the two channels. Results show that the four domains of α_1C contribute to facilitation, with domain I being most critical. This domain is required but not sufficient alone to generate facilitation. The minimal requirement to observe the effect is the presence of domain I plus one of the three others. We conclude that similarly to activation gating, voltage-dependent facilitation of α_1C is a complex process which involves multiple structural elements were domains I and III play the major role.     

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca_v channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca_v channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca_v channels, and failed to modulate LVA Ca_v channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca_v channel currents. The concentration–response curve was biphasic in GH4 neurons and the subnanomolar IC₅₀ values were at least 1000-fold lower than in GH3 cells. L-type Ca_v channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I_Na(v) or I_K(v) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca_v channels.     

Electrical resonance with voltage-gated ion channels: perspectives from biophysical mechanisms and neural electrophysiology

Electrical resonance, providing selective signal amplification at preferred frequencies, is a unique phenomenon of excitable membranes, which has been observed in the nervous system at the cellular, circuit and system levels. The mechanisms underlying electrical resonance have not been fully elucidated. Prevailing hypotheses attribute the resonance to voltage-gated ion channels on the membrane of single neurons. In this review, we follow this line of thinking to summarize and analyze the biophysical/molecular mechanisms, and also the physiological relevance of channel-mediated electrical resonance.     

Myrtenol improves brain damage and promotes angiogenesis in rats with cerebral infarction by activating the ERK1/2 signalling pathway

ContextCerebral ischaemia/reperfusion (I/R) injury has a high disability and fatality worldwide. Myrtenol has protective effects on myocardial I/R injury through antioxidant and anti-apoptotic effects.ObjectiveThis study investigated the effect of myrtenol on cerebral ischaemia/reperfusion (I/R) injury and the underlying mechanism.Materials and methodsCerebral I/R injury was induced in adult Sprague-Dawley rats by middle cerebral artery occlusion (MCAO) for 90 min. MCAO rats were treated with or without myrtenol (10, 30, or 50 mg/kg/day) or/and U0126 (10 μL) intraperitoneally for 7 days.ResultsIn the present study, myrtenol had no toxicity at concentrations up to 1.3 g/kg. Myrtenol treatment improved neurological function of MCAO rats, with significantly (p < 0.05) improved neurological deficits (4.31 ± 1.29 vs. 0.00) and reduced brain edoema (78.95 ± 2.27% vs. 85.48 ± 1.24%). Myrtenol extenuated brain tissue injury and neuronal apoptosis, with increased Bcl-2 expression (0.48-fold) and decreased Bax expression (2.02-fold) and caspase-3 activity (1.36-fold). Myrtenol promoted angiogenesis in the brain tissues of MCAO rats, which was reflected by increased VEGF (0.86-fold) and FGF2 (0.51-fold). Myrtenol promoted the phosphorylation of MEK1/2 (0.80-fold) and ERK1/2 (0.97-fold) in MCAO rats. U0126, the inhibitor of ERK1/2 pathway, reversed the protective effects of myrtenol on brain tissue damage and angiogenesis in MCAO rats.Discussion and conclusionsMyrtenol reduced brain damage and angiogenesis through activating the ERK1/2 signalling pathway, which may provide a novel alternative strategy for preventing cerebral I/R injury. Further in vitro work detailing its mechanism-of-action for improving ischaemic cerebral infarction is needed.     

An assessment of the present and future roles of non-ligand gated ion channel modulators as CNS therapeutics

Few approved drugs have, as their primary known mechanism of action, modulation of non-ligand gated ion channels. However, these proteins are important regulators of neuronal function through their control of sodium, potassium, calcium and chloride flux, and are ideal candidates as drug discovery targets. Recent progress in the molecular biology and pharmacology of ion channels suggests that many will be associated with specific pharmacological profiles that will include both activators and inhibitors. Ion channels, through their regulation by G-proteins, are a major component of the final common pathway of many drugs acting at classical neuronal receptors. Thus, targeting of the ion channels themselves may confer different profiles of efficacy and specificity to drug action in the brain and spinal cord. Three areas for drug discovery are profiled that the authors consider prime targets for ion channel based therapies, anticonvulsant drugs, cognition enhancing drugs and drugs for improving neurone survival following ischaemia.     

Effects of berberine on L-and T-type calcium channels in guinea pig ventricular myocytes

目的：研究小檗碱（Ｂｅｒ）对心室肌细胞钙通道的影响．方法：全细胞膜片箝技术．结果：Ｂｅｒ（１０，３０μｍｏｌ·Ｌ－１）使豚鼠心室肌细胞Ｌ型钙流由１４００±２４７ｐＡ分别减至９７８±２０４ｐＡ及６１７±２３ｐＡ（ｎ＝５，Ｐ＜００５），抑制效应呈浓度依赖及非频率依赖，其电流－电压曲线的峰值下降．Ｂｅｒ（１０μｍｏｌ·Ｌ－１）使Ｌ型钙流失活曲线的最大半激活电压由－２７８ｍＶ变为－３４２ｍＶ，斜率因子由９２２变为１３０３，对激活曲线无影响．Ｂｅｒ（１０，３０μｍｏｌ·Ｌ－１）使Ｔ型钙流峰值由加药前的１５４±８０ｐＡ降至１０１±７８ｐＡ及４８±４５ｐＡ（ｎ＝８，Ｐ＜００５）．结论：Ｂｅｒ对Ｌ和Ｔ型钙通道均有抑制作用．