MiR-29a靶向调控瞬时受体电位通道家族4在睾丸缺血再灌注损伤中对GC-1细胞增殖及凋亡的影响及作用机制

目的:研究miR-29a通过靶向调控瞬时受体电位通道家族4(TRPV4)在睾丸缺血再灌注损伤(IRI)中对GC-1细胞增殖和凋亡的影响及作用机制。方法:建立睾丸GC-1细胞缺氧复氧模型,RT-PCR检测不同复氧损伤时间点miR-29a和TRPV4的表达水平;分别采用MTT实验、流式细胞术检测转染miR-29a对GC-1...     

钙离子通道蛋白TRPV5小激活RNA慢病毒载体及其稳定转染细胞株的构建

目的:构建钙离子通道蛋白TRPV5小激活RNA(SaRNA)慢病毒载体,建立稳定激活TRPV5表达的大鼠肾小管上皮细胞株(NRK细胞株)。方法:针对大鼠TRPV5基因的启动子区域,设计并合成5条特异性SaRNA序列,通过脂质体转染和Western blot筛选出激活效应最强的SaRNA序列,连入慢病毒表达载体中构建重组质粒LV3-TRPV5-SaRNA,对重组质粒进行测序鉴定后进行慢病毒包装;利用生产的慢病毒颗粒感染NRK细胞,经嘌呤霉素筛选得到阳性克隆后进行扩增培养,通过RT-PCR和Western blot检测扩增后的阳性克隆细胞中TRPV5的mRNA和蛋白表达水平。结果:Western blot的检测结果显示SaRNA-320可以明显激活即上调TRPV5的表达水平;测序结果显示目的序列SaRNA-320正确插入LV3表达载体中;SaRNA-320慢病毒颗粒稳定感染的NRK细胞中TRPV5的mRNA和蛋白表达水平均明显升高。结论:成功构建了大鼠TRPV5基因的SaRNA慢病毒重组载体并获得TRPV5稳定过表达的NRK细胞株,为进一步利用小激活RNA技术以及研究钙离子通道在含钙结石的病因及防治中的作用及机制提供了扎实的实验基础。     

静水压对人膀胱平滑肌细胞内钙离子浓度及香草型瞬时受体电位表达的影响

目的研究静水压作用下人膀胱平滑肌细胞(human bladder smooth muscle cells,hb-SMCs)内游离钙离子浓度(intracellular free calcium concentration,[Ca2+]i)的变化及香草型瞬时受体电位(transient receptor potential vanilloid,TRPV)基因的表达情况,初步探讨机械应力在介导hb-SMCs增殖中的分子机制。方法取第6～7代细胞进行实验,采用新型Ca2+荧光染色剂Fluo-3/AM负载hb-SMCs,应用倒置激光扫描共聚焦显微镜分别测定静水压加压0 cm H2O(1 cm H2O=0.098 kPa)(A组)、200 cm H2O作用30 min(B组)及撤销200 cm H2O静水压(C组)后hb-SMCs内[Ca2+]i。采用RT-PCR技术测定200 cm H2O静水压作用0、2、6、12、24 h时TRPV1、TRPV2及TRPV4基因mRNA的表达量。结果 A、B、C组hb-SMCs内[Ca2+]i分别为(100.808±1.724)、(122.008±1.575)、(99.918±0.887)U,B组[Ca2+]i明显高于A、C组(P<0.001),C组撤销200 cm H2O静水压力后[Ca2+]i下降至A组基线水平(t=0.919,P=0.394)。RT-PCR检测示,200 cm H2O静水压加压0 h组、2 h组、6 h组、12 h组及24 h组TRPV1、TRPV2及TRPV4在hb-SMCs中均有表达,随时间推移表达无明显变化;各组间TRPV1、TRPV2、TRPV4 mRNA表达量比较差异均无统计学意义(P>0.05)。结论高水平静水压作用下hb-SMCs内[Ca2+]i明显升高。作为可能表达为机械敏感性阳离子通道的TRPV1、TRPV2及TRPV4基因在hb-SMCs中均有表达,机械应力可能通过调节其开放量而非上调其表达量使hb-SMCs内[Ca2+]i升高。     

瞬时感受器电位香草酸受体1与局部麻醉药物神经毒性

背景 瞬时感受器电位香草酸受体1 (transient receptor potential vanilloid 1,TRPV1)是一种非选择性阳离子通道,主要分布于感觉神经纤维,介导伤害性感受的传入.同时,它能够将外来刺激进行整合影响神经递质的释放以及激活细胞内蛋白酶,从而调节突触传递和细胞功能(如细胞凋亡).局部麻醉药物可以激活TRPV1,且在高浓度时具有神经毒性,表现为病理性疼痛和痛觉过敏,可能与神经细胞的损伤有关.目的 综述TRPV1受体在局部麻醉药物外周神经毒性中的作用机制的研究进展.内容 TRPV1受体的活化能够升高神经纤维局部麻醉药物的浓度而增加其阻断效能,同时增加细胞内钙离子浓度,释放谷氨酸及激活N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR),最终通过钙超载、氧化应激损伤及细胞凋亡途径的活化导致神经元凋亡,介导局部麻醉药物物神经毒性反应.趋向 鉴于TRPV1受体在局部麻醉药物神经毒性的发生机制中所起的作用,靶向调节TRPV1受体的功能是否可以防治局部麻醉药物的神经毒性值得研究.     

TRPV4在骨关节炎与正常软骨中的表达差异及意义

目的探索瞬时受体电位香草素受体4型通道蛋白(transient receptor potential vanilloid receptor 4,TRPV4)在骨关节炎(osteoarthritis,OA)软骨与正常软骨中的表达差异,为进一步研究其在OA防治中的作用提供理论依据。方法取膝关节OA患者软骨组织(OA组)与股骨颈骨折患者正常软骨组织(对照组)。其中,OA组男6例,女9例;年龄55~78岁,平均69岁;Kellgren-Lawrence(K-L)评分为(3.0±0.8)分。对照组男5例,女10例;年龄57~91岁,平均71岁。两组患者性别及年龄比较差异无统计学意义(P>0.05)。采用Western blot、实时荧光定量PCR、Masson染色以及免疫组织化学染色,观察并比较两组软骨组织中TRPV4蛋白及mRNA表达差异,以及软骨组织退变程度;分析OA组患者K-L评分与TRPV4阳性细胞率的相关性。结果OA组TRPV4蛋白及mRNA相对表达量分别为0.454±0.199、2.951±1.200,高于对照组的0.165±0.074、1.437±0.682,其中蛋白相对表达量组间差异有统计学意义(t=2.718,P=0.026)。组织染色显示,OA组软骨细胞排列紊乱,细胞外基质失去正常结构,局部软骨缺损可达深层,退变组织中可见较多TRPV4阳性细胞,阳性细胞率为37.353%±13.496%;对照组软骨细胞层次分明,阳性细胞率仅为9.642%±3.284%;两组阳性细胞率比较差异有统计学意义(t=7.491,P=0.000)。OA组TRPV4阳性细胞率与OA的K-L评分成正相关(r=0.775,P=0.001)。结论TRPV4在OA软骨组织中表达升高,且其表达升高与OA进展有相关性。     

新型钙离子通道TRPV5在遗传性高钙尿结石大鼠肾组织中的表达

目的 探讨新型钙离子通道(TRPV5)在遗传性高钙尿结石(GHS)大鼠肾组织的表达水平及其在尿石形成中的作用. 方法 取3月龄清洁级雄性GHS大鼠模型4只,体质量250～300 g,另取月龄和体质量相仿的正常雄性SD大鼠4只作为对照组,处死后取新鲜肾脏组织,Western blot检测TRPV5蛋白表达水平,实时荧光定量PCR检测其基因表达水平. 结果 GHS大鼠TRPV5蛋白表达水平0.30±0.05,低于对照组大鼠0.59±0.08;对照组大鼠TRPV5 mRNA基因表达水平为GHS组大鼠的12.5倍(分别为1.00和0.08),组间差异均有统计学意义(P＜0.05). 结论 TRPV5可能是尿钙重吸收重要的调控分子,其表达水平下降可能是特发性高钙尿症及尿石形成的重要发病机制.     

瞬时感受器电位离子通道香草素受体4在睾丸缺血再灌注损伤中对GC-1细胞增殖及凋亡的作用及其机制

目的:探讨瞬时感受器电位离子通道香草素受体4(TRPV4)在睾丸缺血再灌注损伤(IRI)中对GC-1细胞增殖和凋亡的作用及其机制。方法:建立睾丸GC-1细胞缺氧复氧模型,采用蛋白质印迹法(Western blot)检测不同复氧损伤时间点TRPV4的表达变化;分别采用噻唑蓝(MTT)实验、流式细胞术检测转染TRPV4对G...     

TRPV5与钙调节及其影响因素研究进展

TRPV5(transient receptor potential cation channel,subfamily V,member 5)作为瞬时性受体电位通道(TRP) vanilloid受体亚类成员之一,是介导远曲小管和集合管对钙主动重吸收的主要蛋白.钙离子、PH和激素等因素参与其表达水平或活性的调控.鉴于TRPV5在调节尿钙水平中的重要作用,我们对TRPV5与钙调节及其影响因素的相关研究作一综述.     

TRPV5/TRPV6调节骨代谢作用的研究进展

骨质疏松是一种严重的老年性疾病。全球骨质疏松的患者约2亿人。在中国,骨质疏松的患者超过9 000万,约占总人口的7.1%。骨质疏松症及其引起的骨折位居常见疾病的第7位,严重威胁着中、老年人的健康,成为全球公共卫生问题之一。骨质疏松与成骨细胞/破骨细胞功能障碍有关。影响成骨细胞/破骨细胞增殖、分化及功能的各种因素是骨质疏松发病的重要原因。在上皮组织中高度表达的钙离子通道TRPV5/TRPV6,对全身钙离子代谢有着非常重要的作用。认识骨组织代谢调控因子TRPV5/TRPV6在骨质疏松发病机制中的作用,     

辣椒素激活5637细胞的TRPV1通道提高吡柔比星疗效

膀胱癌术后是否进行化疗是影响其复发的主要因素,之前的研究表明瞬时受体电位香草酸亚型1(TRPV1)是诱导膀胱癌细胞凋亡的肿瘤抑制基因。吡柔比星(THP)术后灌注为膀胱癌的主要化疗手段之一,激活 TRPV1对 THP 是否有协同作用有待确定。     

TRPV4 ion channel as important cell sensors

This review provides a summary of the physiological significance of the TRPV4 ion channel. Although TRPV4 was initially characterized as an osmosensor, we found that TRPV4 can also act as a thermosensor or a mechanosensor in brain neurons or epithelial cells in the urinary bladder. Here, we summarize the newly characterized functions of TRPV4, including the research progress that has been made toward our understanding of TRPV4 physiology, and discuss other recent data pertaining to TRPV4. It is thought that TRPV4 may be an important drug target based on its broad expression patterns and important physiological functions. Possible associations between diseases and TRPV4 are also discussed.     

TRPV5: an ingeniously controlled calcium channel

Body Ca(2+) homeostasis is tightly controlled and slight disturbances in renal Ca(2+) reabsorption can lead to excessive urine Ca(2+) excretion and promote kidney stone formation. The epithelial Ca(2+) channel TRPV5 constitutes the rate-limiting step of active Ca(2+) reabsorption in the kidney. Elucidation of the molecular pathways controlling TRPV5 function provides important information for our understanding of renal Ca(2+) handling, since active Ca(2+) reabsorption fine-tunes the final amount of Ca(2+) excreted into the urine. Over the last years, the molecular regulation of TRPV5 has been dismantled in detail. Various calciotropic hormones, known to alter renal Ca(2+) reabsorption, affect the expression of TRPV5. Others stimulate the trafficking of TRPV5 to the plasma membrane, while a number of associated proteins and ions control channel activity at the plasma membrane. Dynamic cell surface presence of TRPV5 is largely mediated by endosomal recycling processes allowing internalized channels to reappear at the plasma membrane. We present recently identified factors shown to modulate TRPV5 activity by diverse mechanisms to ultimately control renal Ca(2+) handling. The selected factors include klotho, tissue kallikrein, pH, Ca(2+), Mg(2+), PIP(2) and WNK4. This review covers the distinctive properties and regulation of the highly Ca(2+)-selective TRPV5 channel and highlights the implications for our understanding of the process of Ca(2+) reabsorption.     

Regulation of the epithelial calcium channel TRPV5 by extracellular factors

PURPOSE OF REVIEW: Recent studies have greatly increased our knowledge concerning the regulation of renal calcium handling. This review focuses on newly identified calciotropic factors present in the pro-urine and the mechanisms by which they control the transient receptor potential channel vanilloid subtype 5 (TRPV5) which forms the gatekeeper of active renal calcium reabsorption. RECENT FINDINGS: The antiaging hormone klotho regulates TRPV5 activity via a novel mechanism modifying its glycosylation status, thereby entrapping the channel at the cell surface. Functional characterization of tissue kallikrein knockout mice revealed that these animals exhibit a pronounced hypercalciuria, comparable to the calcium leak observed in TRPV5 knockout mice. Recently, it has been demonstrated that tissue kallikrein stimulates active calcium reabsorption via the bradykinin receptor type 2 pathway involving protein kinase C-dependent activation of TRPV5. Finally, the extracellular pH appears to act as a dynamic switch controlling cell surface expression of TRPV5. SUMMARY: Unraveling the molecular mechanisms of TRPV5 channel regulation by the antiaging hormone klotho, tissue kallikrein and extracellular pH demonstrated the existence of novel regulatory mechanisms of active calcium reabsorption acting from the tubular lumen.     

The role of the transient receptor potential (TRP) superfamily of cation-selective channels in the management of the overactive bladder

? The pathophysiology of lower urinary tract symptoms (LUTS), detrusor overactivity (DO), and the overactive bladder (OAB) syndrome is multifactorial and remains poorly understood. ? The transient receptor potential (TRP) channel superfamily has been shown to be involved in nociception and mechanosensory transduction in various organ systems, and studies of the LUT have indicated that several TRP channels, including TRPV1, TRPV2, TRPV4, TRPM8, and TRPA1, are expressed in the bladder, and may act as sensors of stretch and/or chemical irritation. ? However, the roles of these individual channels for normal LUT function and in LUTS/DO/OAB, have not been established. ? TRPV1 is the channel best investigated. It is widely distributed in LUT structures, but despite extensive information on morphology and function in animal models, the role of this channel in normal human bladder function is still controversial. Conversely, its role in the pathophysiology and treatment of particularly neurogenic DO is well established. ? TRPV1 is co-expressed with TRPA1, and TRPA1 is known to be present on capsaicin-sensitive primary sensory neurones. Activation of this channel can induce DO in animal models. ? TRPV4 is a Ca(2+)-permeable stretch-activated cation channel, involved in stretch-induced ATP release, and TRPV4-deficient mice exhibit abnormal frequencies of voiding and non-voiding contractions in cystometric experiments. ? TRPM8 is a cool receptor expressed in the urothelium and suburothelial sensory fibres. It has been implicated in the bladder-cooling reflex and in idiopathic DO. ? The occurrence of other members of the TRP superfamily in the LUT has been reported, but information on their effects on LUT functions is scarce. There seem to be several links between activation of different members of the TRP superfamily and LUTS/DO/OAB, and further exploration of the involvement of these channels in LUT function, normally and in dysfunction, may be rewarding.     

Regulation of calcium-permeable TRPV2 channel by insulin in pancreatic beta-cells

OBJECTIVE—Calcium-permeable cation channel TRPV2 is expressed in pancreatic β-cells. We investigated regulation and function of TRPV2 in β-cells.RESEARCH DESIGN AND METHODS—Translocation of TRPV2 was assessed in MIN6 cells and cultured mouse β-cells by transfecting TRPV2 fused to green fluorescent protein or TRPV2 containing c-Myc tag in the extracellular domain. Calcium entry was assessed by monitoring fura-2 fluorescence.RESULTS—In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition. Addition of exogenous insulin induced translocation and insertion of TRPV2 to the plasma membrane. Consistent with these observations, insulin increased calcium entry, which was inhibited by tranilast, an inhibitor of TRPV2, or by knockdown of TRPV2 using shRNA. A high concentration of glucose also induced translocation of TRPV2, which was blocked by nefedipine, diazoxide, and somatostatin, agents blocking glucose-induced insulin secretion. Knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2. Similarly, the effect of insulin on TRPV2 translocation was not observed in a β-cell line derived from islets obtained from a β-cell–specific insulin receptor knockout mouse. Knockdown of TRPV2 or addition of tranilast significantly inhibited insulin secretion induced by a high concentration of glucose. Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2. Finally, insulin-induced translocation of TRPV2 was observed in cultured mouse β-cells, and knockdown of TRPV2 reduced insulin secretion induced by glucose.CONCLUSIONS—TRPV2 is regulated by insulin and is involved in the autocrine action of this hormone on β-cells.Insulin elicits pleiotropic actions in a variety of target cells and plays a pivotal role in regulating nutrient metabolism. Recent studies have revealed that the insulin signal is necessary to maintain the normal function of pancreatic β-cells. Thus, deletion of the insulin receptor (IR) in β-cells impairs insulin secretion and results in glucose intolerance (1). In β-cells of βIRKO mice, glucose-induced insulin secretion is reduced, which is accompanied by reduction of the expression of GLUT2 and glucokinase (1). However, insulin secretion induced by glyceraldehyde and KCl is also reduced in islets obtained from a βIRKO mouse (2), which cannot be explained simply by reduction of GLUT2 and/or glucokinase expression. Because addition of anti-insulin antibody immediately reduces insulin secretion from islets (3), it is likely that insulin modifies a molecule(s) involved in insulin secretion by a nongenomic mechanism. In accordance with these observations, knockdown of IR attenuates glucose-induced insulin secretion in MIN6 cells (4). In addition, postnatal β-cell growth is impaired in βIRKO mice. Consequently, the mechanism by which insulin maintains β-cell function is not totally known at present. It is thought that there must be a target molecule(s) of insulin that regulates secretion and possibly growth of β-cells.Transient receptor potential (TRP) (5) is a calcium-permeable channel expressed in Drosophila. A large number of mammalian homologues have been identified, and they regulate various cellular functions (6,7). Among them, calcium-permeable cation channels resembling the vanilloid receptor channel (TRPV1) (8) have common features and now are classified as members of the TRPV subfamily (9). The TRPV subfamily consists of six members, which function as cellular sensors responding to changes in the temperature, osmolarity, and mechanical stresses, and they are also regulated by various ligands (9).TRPV2 is regulated by heat, growth factors, and other ligands (10–13). An intriguing feature of TRPV2 is that its intracellular localization is changed by various stimulations. For example, IGF-I induces translocation of TRPV2 from an intracellular compartment to the plasma membrane (11). Regarding its expression, TRPV2 is abundantly expressed in neurons, neuroendocrine cells in the gastrointestinal tract, and blood cells such as macrophages (14). In the pancreas, TRPV2 is expressed in β-cells and ductal cells. In this regard, we previously reported that TRPV2 is expressed in an insulinoma cell line MIN6 (11). When MIN6 cells are cultured in a serum-free condition, immunoreactivity of TRPV2 is localized in an intracellular compartment. Addition of serum induces translocation of TRPV2 to the plasma membrane (11).In the present study, we further investigated regulation of TRPV2 in β-cells. Because these cells secrete insulin, and the mode of action of insulin resembles that of IGF-I, special attention was paid to the effect of insulin and insulin secretagogues on the localization of TRPV2. The results indicate that TRPV2 is regulated by insulin in an autocrine manner in β-cells. TRPV2 functions as an insulin-mediated regulator of calcium entry.     

TRPV1 is involved in stretch-evoked contractile changes in the rat autonomous bladder model: a study with piperine, a new TRPV1 agonist

AIM: Vanilloids like capsaicin and resiniferatoxin (RTX) have been used for more than a decade in the treatment of neurogenic detrusor overactivity. Recently, the vanilloid molecule piperine (PIP) has been shown to have similar pharmacological properties as these drugs. In this study, we looked at PIP-effects on autonomous bladder contractile activity, with particular interest for its selectivity for the transient receptor potential channel 1 (TRPV1) receptor. Additionally, we studied the role of TRPV1 in volume-induced contractile changes using selective and non-selective TRPV1 antagonists. METHODS: The acute and prolonged effects of PIP were studied on rat bladders. Each bladder was excised and placed in a heated organ bath, where intravesical pressures were measured. In acute experiments, PIP was added directly to the bathing solution. For prolonged effects, animals were pre-treated intravesically with vehicle (ethanol 5%) or PIP (10(-4) M) and sacrificed 72 hr later. The effects of selective (capsazepine (CZP)) and non-selective (ruthenium red (RR)) TRPV1 antagonists on volume-evoked contractile parameters were also studied. RESULTS: Acute administration of PIP 10(-4) M significantly increased amplitude of bladder contractions (P < 0.05). These effects were significantly antagonized (P < 0.05) by the TRPV1-selective antagonist CZP (10(-5) M) and the non-selective TRP-antagonist RR (10(-5) M). Intravesical pre-treatment with PIP induced shorter contractions with more periods of non-activity (P < 0.05) compared to controls. Inhibition of TRPV1 with CZP and RR significantly reduced the volume-evoked rise in contractile amplitude in isolated bladders (P < 0.05). CONCLUSION: We found evidence for acute and prolonged effects of PIP on bladder contractility, which seem to be mediated through TRPV1. Furthermore, we found evidence for involvement of TRPV1 in afferent signaling of mechanical stimuli.     

SD大鼠前列腺上皮新型钙通道TRPV5的实验研究

目的探讨新型钙通道TRPV5在前列腺疾病发病机制中的作用。方法年轻和老年的雄性大鼠各30只,先取出前列腺,使用光学显微镜观察其HE切片的组织病理学特点,应用SP法对其进行免疫组化染色,分别检测TRPV5钙通道在SD大鼠前列腺上皮细胞中的表达。结果30例老年DS大鼠前列腺标本中TRPV5钙通道强表达的有6例,中表达的11例,低表达的13例;30例年轻SD大鼠前列腺标本中TRPV5钙通道强表达的有19例,中表达的8例,低表达的3例。两组相比,差异有统计学意义(P<0.05)。结论TRPV5钙通道在SD大鼠前列腺上皮细胞中表达差异有统计学意义,新型钙通道TRPV5的改变可能在前列腺疾病的发病中起一定作用。