温度敏感TRPV2离子通道的结构与药理学功能研究进展

瞬时受体电位离子通道香草素亚型2 (transient receptor potential vanilloid ion channel subtypes 2, TRPV2)是属于TRPV通道家族的一种钙渗透性非选择性阳离子通道。TRPV2表达于神经、血管和脾脏等器官组织,参与调节多种重要的生理功能,如伤害性感受、神经发育和免疫应答。本文综述了TRPV2通道的结构、调节剂的发现与结合位点以及生理功能几个方面的研究进展,为后续研究提供参考。     

高盐饮食增强内皮细胞TRPV4与cPLA2的空间偶联作用

目的探究内皮细胞中香草素受体4型瞬时感受器电位通道(TRPV4通道)与胞浆型磷脂酶A2(c PLA2)在空间上的偶联作用。方法通过设定梯度盐(培养基中外源加入20、40、80、160 mol·L~(-1)Na Cl)确定高盐处理内皮细胞的最适盐浓度;在细胞水平上,通过免疫荧光能量共振转移(immuno-FRET)技术检测人微血管内皮细胞(HMEC)和小鼠胸主动脉原代内皮细胞中TRPV4与c PLA2的空间偶联作用;在动物组织水平,通过同样的技术检测正常小鼠与高盐诱导的高血压小鼠的胸主动脉血管环上的内皮细胞中TRPV4与c PLA2的空间偶联情况。结果高盐诱导内皮细胞的最适盐浓度为40 mol·L~(-1)Na Cl;在细胞水平和动物组织水平上,高盐处理的HMEC、小鼠胸主动脉原代内皮细胞和高盐诱导的高血压小鼠胸主动脉血管环上的内皮细胞中,TRPV4与c PLA2的空间偶联作用均明显增强。结论在细胞和组织水平上,高盐饮食能增强内皮细胞中TRPV4与c PLA2的空间偶联作用,这种空间偶联可能进一步引起两者的功能偶联,为血管内皮功能紊乱的研究提供了新的思路。     

杜鹃花总黄酮对缺血/再灌注损伤模型大鼠脑基底动脉TRPV4的作用研究

目的探讨杜鹃花总黄酮(TFR)对缺血/再灌注损伤模型大鼠脑基底动脉(CBA)瞬时感受器电位通道香草酸受体亚型Ⅳ(TRPV4)的作用。方法以改良四血管阻断法(4-VO)建立大鼠缺血/再灌注模型(IR);运用加压灌注法和细胞膜电位记录法,离体观察TFR对KCl预收缩IR大鼠CBA的舒张作用和超极化反应及TRPV4阻断剂钌红(RR)对其的影响;采用实时荧光定量PCR法和蛋白质印迹法,在体观察TFR对IR大鼠脑血管内皮细胞TRPV4 mRNA和蛋白表达以及RR对其的影响。结果递增浓度的TFR可诱导IR大鼠离体CBA产生明显剂量依赖性的舒张效应和超极化反应。去除血管内皮细胞后,TFR仍能介导CBA产生较弱的舒张作用和超极化反应,与血管内皮完整组比较,差异有显著性(P<0.01);去除一氧化氮(NO)和前列环素(PGI2)舒张作用后,TFR仍能诱导IR大鼠CBA产生明显的舒张效应和超极化作用,此作用可被TRPV4通道阻断剂RR抑制;TFR可明显上调IR大鼠脑血管TRPV4 mRNA和蛋白表达,而阻断TRPV4可明显抑制TFR上调的TRPV4基因表达。结论 TFR能介导IR大鼠CBA产生较强的内皮依赖性和较弱的内皮非依赖性血管舒张反应和超极化作用,其内皮依赖性效应推测可能与TFR促使脑血管内皮激活,促进内皮细胞生成和释放内皮衍生性超极化因子(EDHF)增多,继而激活TRPV4,引发Ca~(2+)内流,导致血管平滑肌细胞膜超极化,产生血管舒张效应有关。     

TRPV1/SIRT1介导吴茱萸次碱抗AngⅡ诱导的血管平滑肌细胞衰老

目的探讨吴茱萸次碱(rutaecarpine,Rut)对长寿蛋白SIRT1表达及AngⅡ诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)衰老的影响。方法采用AngⅡ(1μmol·L^-1)孵育大鼠胸主动脉平滑肌细胞72 h,预先加入不同浓度的Rut(0.3、1、3μmol·L^-1),采用TRPV1拮抗剂CAPZ(10μmol·L^-1)和AMPK抑制剂Compound C(1μmol·L^-1)探讨TRPV1/AMPK是否介导Rut的保护效应。SA-β-Gal测定衰老细胞数目,DCFH-DA法测定细胞ROS水平。划痕愈合结合Transwell检测VSMCs迁移。Western blot检测VSMCs中长寿蛋白SIRT1和衰老相关蛋白p53、p21的表达以及p-AMPK水平。结果Rut明显地抑制AngⅡ诱导的VSMCs衰老和ROS生成,并抑制VSMCs迁移。预先给予TRPV1拮抗剂可取消Rut这一保护作用。AngⅡ可降低SIRT1的表达,给予Rut可剂量依赖性地恢复SIRT1的表达,且下调其下游衰老相关蛋白p53和p21的表达。AngⅡ可抑制p-AMPK,加入Rut能恢复p-AMPK水平。CAPZ和Compound C可消除Rut升高SIRT1表达的效应。结论Rut可上调SIRT1表达,抑制AngⅡ诱导的VSMCs衰老和迁移,其机制可能激活TRPV1/AMPK信号途径。     

基于荧光定量PCR评测热性中药成分对TRPV1通道功能的影响

目的深入研究热性中药的生物学功能和分子机制。方法该研究选择了临床常用的热性中药的主要活性成分,基于新型7900PCR仪尝试搭建背根神经节(DRG)神经元TRPV1通道温觉感知功能的检测评价系统,并对热性中药成分的TRPV1通道功能的调节作用进行了研究。结果TRPV1通道能够被温度升高所激活,且该激活过程可被TRPV1特异性阻断剂辣椒平抑制,所选大部分热性中药成分均具有上调TRPV1通道功能的作用。结论这些结果提示基于PCR仪建立的TRPV1通道功能检测体系适于药物调节TRPV1通道功能的研究;热性中药表征的热性属性可能与其所含的活性成分能够提高TRPV1通道感知温度的活性,从而增加机体的能量代谢有关。     

TRPV3通道蛋白的研究现状及进展

瞬时受体电位(transient receptor potential,TRP)通道是一种非电压依赖性的特殊阳离子通道,广泛表达于多种哺乳动物组织中,并参与多种重要的生理功能,包括钙的平衡、温度感觉等。TRPV3可以感受热刺激(32～39℃),并可被多种非热性刺激激活,在维持机体功能中发挥着重要     

慢性非细菌性前列腺炎小鼠前列腺组织TRPV5的表达

目的探讨钙通道-瞬时受体电位阳离子通道V5(TRPV5)在慢性非细菌性前列腺炎(NBCP)发病机制中的作用。方法建立NBCP小鼠模型,采用免疫组化SP法及实时荧光定量PCR法分别检测NBCP小鼠及正常对照组小鼠前列腺组织中TRPV5蛋白及其mRNA的表达。结果 NBCP小鼠前列腺组织TRPV5 mRNA的表达高于对照组[(0.815±0.062)vs.(0.587±0.080)](P<0.05)。结论钙通道TRPV5的mRNA水平升高在慢性前列腺炎发病机制中可能起一定作用。     

在LPS致大鼠发热过程中下丘脑TRPV4对体温及cAMP含量、[Ca~(2+)]i浓度的影响

目的探讨TRPV4在发热时是否参与体温调节过程。方法用脂多糖复制大鼠发热模型,结合应用钌红抑制TR-PV4的活性,观察发热时的下丘脑组织中TRPV4含量、钙离子浓度变化与cAMP含量的关系。结果单独给予钌红组体温降低;钌红+LPS组与LPS组相比,△T、cAMP与[Ca2+]i增高幅度均降低;钌红组与钌红+LPS组TRPV4表达明显低于对照组。结论①TRPV4通道可能参与正常体温的维持;②通过激活TRPV4通道,使钙离子内流增多,进而中枢发热介质cAMP表达增多,这可能是LPS致大鼠发热的重要途径。     

TRPV3通道研究进展

瞬时受体电位离子通道香草素亚家族3 (transient receptor potential vanilloid 3, TRPV3)是位于细胞膜上的一种非选择性阳离子通道蛋白,广泛表达于皮肤、大脑、背根神经节、心脏和结肠等器官。TRPV3参与感觉传导、皮肤屏障形成、毛发生长及血管舒张等生理过程,并被证明与瘙痒、皮肤炎症性疾病及癌症等病理进程密切相关。TRPV3能应答非伤害性热刺激(≥33℃)、内源性物质(如焦磷酸法尼酯)及外源性小分子化合物(如香芹酚、樟脑和2-APB等)。近年来,多种天然和合成的TRPV3抑制剂(如蛇床子素、74a和达克罗宁等)陆续被发现,并且在多种疾病动物模型中表现出一定疗效,说明TRPV3是具有潜力的药物开发靶点。本文综述了TRPV3通道蛋白结构、生理功能、相关疾病及调节剂的研究进展,为TRPV3的后续研究提供理论参考。     

香草素受体4型瞬时感受器电位通道及其抑制剂与相关呼吸系统疾病关系的研究进展

香草素受体4型瞬时感受器电位(TRPV4)通道是瞬时感受器电位通道家族成员之一,是一种对钙离子具有选择通透性的阳离子通道。该通道激活后可引起胞内钙离子浓度升高,进而参与调节机体多种生理或病理过程。近年来大量文献表明,TRPV4通道可能与多种呼吸系统疾病的发病机制有关,包括急性肺损伤、慢性阻塞性肺疾病、肺动脉高压、特发性肺纤维化和哮喘等,TRPV4抑制剂的应用可不同程度地缓解上述疾病进展。本文对TRPV4通道及其抑制剂与有关呼吸系统疾病关系的研究进展予以综述。     

Gadolinium and ruthenium red attenuate remote hind limb preconditioning-induced cardioprotection: possible role of TRP and especially TRPV channels

Remote ischemic preconditioning is a well reported therapeutic strategy that induces cardioprotective effects but the underlying intracellular mechanisms have not been widely explored. The current study was designed to investigate the involvement of TRP and especially TRPV channels in remote hind limb preconditioning-induced cardioprotection. Remote hind limb preconditioning stimulus (4 alternate cycles of inflation and deflation of 5 min each) was delivered using a blood pressure cuff tied on the hind limb of the anesthetized rat. Using Langendorff’s system, the heart was perfused and subjected to 30-min ischemia and 120-min reperfusion. The myocardial injury was assessed by measuring infarct size, lactate dehydrogenase (LDH), creatine kinase (CK), LVDP, +dp/dt_max, ?dp/dt_min, heart rate, and coronary flow rate. Gadolinium, TRP blocker, and ruthenium red, TRPV channel blocker, were employed as pharmacological tools. Remote hind limb preconditioning significantly reduced the infarct size, LDH release, CK release and improved coronary flow rate, hemodynamic parameters including LVDP, +dp/dt_max, ?dp/dt_min, and heart rate. However, gadolinium (7.5 and 15 mg kg^?1) and ruthenium red (4 and 8 mg kg^?1) significantly attenuated the cardioprotective effects suggesting the involvement of TRP especially TRPV channels in mediating remote hind limb preconditioning-induced cardioprotection. Remote hind limb preconditioning stimulus possibly activates TRPV channels on the heart or sensory nerve fibers innervating the heart to induce cardioprotective effects. Alternatively, remote hind limb preconditioning stimulus may also activate the mechanosensitive TRP and especially TRPV channels on the sensory nerve fibers innervating the skeletal muscles to trigger cardioprotective neurogenic signaling cascade. The cardioprotective effects of remote hind limb preconditioning may be mediated via activation of mechanosensitive TRP and especially TRPV channels.     

Nociceptive and pro-inflammatory effects of dimethylallyl pyrophosphate via TRPV4 activation

BACKGROUND AND PURPOSE

Sensory neuronal and epidermal transient receptor potential ion channels (TRPs) serve an important role as pain sensor molecules. While many natural and synthetic ligands for sensory TRPs have been identified, little is known about the endogenous activator for TRPV4. Recently, we reported that endogenous metabolites produced by the mevalonate pathway regulate the activities of sensory neuronal TRPs. Here, we show that dimethylallyl pyrophosphate (DMAPP), a substance produced by the same pathway is an activator of TRPV4.

EXPERIMENTAL APPROACH

We examined the effects of DMAPP on sensory TRPs using Ca²⁺ imaging and whole-cell electrophysiology experiments with a heterologous expression system (HEK293T cells transfected with individual TRP channels), cultured sensory neurons and keratinocytes. We then evaluated nociceptive behavioural and inflammatory changes upon DMAPP administration in mice in vivo.

KEY RESULTS

In the HEK cell heterologous expression system, cultured sensory neurons and keratinocytes, µM concentrations of DMAPP activated TRPV4. Agonistic and antagonistic potencies of DMAPP for other sensory TRP channels were examined and activation of TRPV3 by camphor was found to be inhibited by DMAPP. In vivo assays, intraplantar injection of DMAPP acutely elicited nociceptive flinches that were prevented by pretreatment with TRPV4 blockers, indicating that DMAPP is a novel pain-producing molecule through TRPV4 activation. Further, DMAPP induced acute inflammation and noxious mechanical hypersensitivities in a TRPV4-dependent manner.

CONCLUSIONS AND IMPLICATIONS

Overall, we found a novel sensory TRP acting metabolite and suggest that its use may help to elucidate the physiological role of TRPV4 in nociception and associated inflammation.     

Functional Expression of TRPV4 Cation Channels in Human Mast Cell Line (HMC-1)

Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, 23~25℃ to a moderately high temperature (MHT, 37~39℃ to activate TRPV3/4, a high temperature (HT, 44~46℃ to activate TRPV1, or a very high temperature (VHT, 53~55℃ to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV4 activator 4α-phorbol 12,13-didecanoate (4αPDD, 1µM) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The [Ca²⁺]_c of HMC-1 cells was also increased by MHT or by 4αPDD. In summary, our present study indicates that HMC-1 cells express Ca²⁺-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.     

TRPV4: New therapeutic target for inflammatory bowel diseases

The transient receptor potential vanilloid-4 (TRPV4) belongs to a family of ion channels and can be activated by warm temperature, hypotonicity, cell swelling or lipid mediators of the arachidonic cascade. The metabolites or events responsible for TRPV4 activation are associated with inflammation, arguing in favor of a role for this receptor in inflammatory diseases. The first studies have focused their attention on the role of TRPV4 in neurons and endothelial cells but TRPV4 cellular distribution is widespread, particularly in the gastrointestinal tract. Herein, we review a number of studies demonstrating the expression of TRPV4 in the gut, the regulation of its expression and functions by inflammatory mediators in that organ and the consequences of TRPV4 activation or inhibition in the intestine. We further discuss the relevance of considering this receptor as a potential target for therapeutic development in inflammatory bowel diseases.     

TRPV1 Channel: A Potential Drug Target for Treating Epilepsy

Epilepsy has 2-3% incidence worldwide. However, present antiepileptic drugs provide only partial control of seizures. Calcium ion accumulation in hippocampal neurons has long been known as a major contributor to the etiology of epilepsy. TRPV1 is a calcium-permeable channel and mediator of epilepsy in the hippocampus. TRPV1 is expressed in epileptic brain areas such as CA1 area and dentate gyrus of the hippocampus. Here the author reviews the patent literature on novel molecules targeting TRPV1 that are currently being investigated in the laboratory and are candidates for future clinical evaluation in the management of epilepsy. A limited number of recent reports have implicated TRPV1 in the induction or treatment of epilepsy suggesting that this may be new area for potential drugs targeting this debilitating disease. Thus activation of TRPV1 by oxidative stress, resiniferatoxin, cannabinoid receptor (CB1) activators (i.e. anandamide) or capsaicin induced epileptic effects, and these effects could be reduced by appropriate inhibitors, including capsazepine (CPZ), 5''-iodoresiniferatoxin (IRTX), resolvins, and CB1 antagonists. It has been also reported that CPZ and IRTX reduced spontaneous excitatory synaptic transmission through modulation of glutaminergic systems and desensitization of TRPV1 channels in the hippocampus of rats. Immunocytochemical studies indicated that TRPV1 channel expression increased in the hippocampus of mice and patients with temporal lobe epilepsyTaken together, findings in the current literature support a role for calcium ion accumulation through TRPV1 channels in the etiology of epileptic seizures, indicating that inhibition of TRPV1 in the hippocampus may possibly be a novel target for prevention of epileptic seizures.     

The functions of TRPA1 and TRPV1: moving away from sensory nerves

The transient receptor potential vanilloid 1 and ankyrin 1 (TRPV1 and TRPA1, respectively) channels are members of the TRP superfamily of structurally related, non-selective cation channels. It is rapidly becoming clear that the functions of TRPV1 and TRPA1 interlink with each other to a considerable extent. This is especially clear in relation to pain and neurogenic inflammation where TRPV1 is coexpressed on the vast majority of TRPA1-expressing sensory nerves and both integrate a variety of noxious stimuli. The more recent discovery that both TRPV1 and TRPA1 are expressed on a multitude of non-neuronal sites has led to a plethora of research into possible functions of these receptors. Non-neuronal cells on which TRPV1 and TRPA1 are expressed vary from vascular smooth muscle to keratinocytes and endothelium. This review will discuss the expression, functionality and roles of these non-neuronal TRP channels away from sensory nerves to demonstrate the diverse nature of TRPV1 and TRPA1 in addition to a direct role in pain and neurogenic inflammation.     

The pharmacological challenge to tame the transient receptor potential vanilloid-1 (TRPV1) nocisensor

The transient receptor potential vanilloid-1 (TRPV1) cation channel is a receptor that is activated by heat (>42 degrees C), acidosis (pH<6) and a variety of chemicals among which capsaicin is the best known. With these properties, TRPV1 has emerged as a polymodal nocisensor of nociceptive afferent neurones, although some non-neuronal cells and neurones in the brain also express TRPV1. The activity of TRPV1 is controlled by a multitude of regulatory mechanisms that either cause sensitization or desensitization of the channel. As many proalgesic pathways converge on TRPV1 and this nocisensor is upregulated and sensitized by inflammation and injury, TRPV1 is thought to be a central transducer of hyperalgesia and a prime target for the pharmacological control of pain. As a consequence, TRPV1 agonists causing defunctionalization of sensory neurones and a large number of TRPV1 blockers have been developed, some of which are in clinical trials. A major drawback of many TRPV1 antagonists is their potential to cause hyperthermia, and their long-term use may carry further risks because TRPV1 has important physiological functions in the peripheral and central nervous system. The challenge, therefore, is to pharmacologically differentiate between the physiological and pathological implications of TRPV1. There are several possibilities to focus therapy specifically on those TRPV1 channels that contribute to disease processes. These approaches include (i) site-specific TRPV1 antagonists, (ii) modality-specific TRPV1 antagonists, (iii) uncompetitive TRPV1 (open channel) blockers, (iv) drugs interfering with TRPV1 sensitization, (v) drugs interfering with intracellular trafficking of TRPV1 and (vi) TRPV1 agonists for local administration.     

Structure-function analysis of TRPV channels

In recent years many new members of the family of TRP ion channels have been identified. These channels are classified into several subgroups and participate in many sensory and physiological functions. TRPV channels are important for the perception of pain, temperature sensing, osmotic regulation, and maintenance of calcium homeostasis, and much recent research concerns the identification of protein domains involved in mediating specific channel functions. Recent literature on TRPV channel subunit composition, protein domains required for subunit assembly, trafficking, and regulation will be reviewed and discussed.     

TRPV channels' role in osmotransduction and mechanotransduction

In signal transduction of metazoan cells, transient receptor potential (TRP) ion channels have been identified that respond to diverse external and internal stimuli, among them osmotic and mechanical stimuli. This chapter will summarize findings on the TRPV subfamily, both its vertebrate and invertebrate members. Of the six mammalian TRPV channels, TRPV1, -V2, and -V4 were demonstrated to function in transduction of osmotic and/or mechanical stimuli. TRPV channels have been found to function in cellular as well as systemic osmotic homeostasis in vertebrates. Invertebrate TRPV channels, five in Caenorhabditis elegans and two in Drosophila, have been shown to play a role in mechanosensation, such as hearing and proprioception in Drosophila and nose touch in C. elegans, and in the response to osmotic stimuli in C. elegans. In a striking example of evolutionary conservation of function, mammalian TRPV4 has been found to rescue mechanosensory and osmosensory deficits of the TRPV mutant line osm-9 in C. elegans, despite no more than 26% orthology of the respective amino acid sequences.     

TRPV4

TRPV4 is a non-selective cation channel subunit expressed in a wide variety of tissues. TRP channels are formed by a tetrameric complex of channel subunits. The available evidence suggests that TRPV4 cannot form heteromultimers with other TRPV isoforms, and that TRPV4-containing channels are homotetramers. These channels have a characteristic outwardly rectifying current-voltage relation, and are 5-10 times more permeable for Ca2+ than for Na+. TRPV4 can be activated by a wide range of stimuli including physical (cell swelling, heat, mechanical stimulation) and chemical stimuli (endocannabinoids, arachidonic acid, and, surprisingly, 4alpha-phorbol esters). Activation by swelling and endocannabinoids involves cytochrome P450 epoxygenase-dependent arachidonic acid metabolism to the epoxyeicosatrienoic acids (EETs). Heat and 4alpha-phorbol esters also seem to share a common mechanism of activation, but the endogenous messenger involved in the response to heat has not yet been identified. Ca2+ acting from the intracellular side can have both potentiating and inhibitory effects on channel activity and is involved in channel activation and inactivation. Given its wide expression and the variety of activatory stimuli, TRPV4 is likely to play a number of physiological roles. Studies with TRPV4(-/-) mice suggest a role for the channel in the regulation of body osmolarity, mechanosensation, temperature sensing, vascular regulation and, possibly, hearing.