Effects of Tiaomaiyin and Its Disassembled Prescription on Expression of L-type Calcium Channel β2 Subunit in Rat Model of Tachyarrhythmia

[Objectives] To study the effects of Tiaomaiyin and its disassembled prescription on expression of L-type calcium channel β2 subunit in rat model of tachyarrhythmia. [Methods] Sixty Wistar rats were randomly divided into model group,Tiaomaiyin prescription group( whole prescription group),main efficacy group of removing heat to cool blood( blood cooling group),and auxiliary drug efficacy group of benefiting qi and nourishing heart( qi benefiting group),auxiliary efficacy group of promoting flow of qi and blood circulation( qi flow promoting group),and amiodarone group( western medicine group). Aconitine was given 7 d after the intragastric administration of the corresponding drugs,and the time of occurrence of arrhythmia in each group was observed. The left ventricular myocardium was subjected to reverse transcription-polymerase chain reaction and Western blotting. [Results] The ventricular premature beats( VPB) time in the whole prescription group and western medicine group was significantly longer than that in the model group. Ventricular tachycardia( VT),ventricular fibrillation( VF),and cardiac arrest( CA) were longer in the whole prescription group,blood cooling group,and western medicine group. The mRNA and protein expression of L-type calcium channel β2 subunit in the whole prescription group,blood cooling group and western medicine group were significantly decreased. [Conclusions] Tiaomaiyin whole prescription group and blood cooling group can reduce the occurrence time of tachyarrhythmia and reduce the expression of LTCC β2 in myocardium.

Types of voltage-dependent calcium channels involved in high potassium depolarization-induced amylase secretion in the exocrine pancreatic tumour cell line AR4-2J

In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted large amount of amylase. High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation. High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel antagonists with an order of potency as follows: nifedipine > ω-agatoxin IVA > ω-conotoxin GVIA. In contrast, the L-type calcium channel antagonist nifedipine almost completely inhibited potassium-induced amylase secretion, whereas the N-type channel antagonist ω-conotoxin GVIA was without effect. The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect, but this inhibition was not significant at the level of amylase secretion. In conclusion, the AR4-2J cell line possesses different voltage-dependent calcium channels (L, P,N) with the L-type predominantly involved in depolarization induced amylase secretion.

Altered expression of stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs)in cancer:will they become a new battlefield for oncotherapy?

The stromal interaction molecule(STIM)-calcium release-activated calcium channel protein(ORAI) and inositol1,4,5-trisphosphate receptors(IP_3Rs) play pivotal roles in the modulation of Ca^(2+)-regulated pathways from gene transcription to cell apoptosis by driving calcium-dependent signaling processes.Increasing evidence has implicated the dysregulation of STIM-ORAI and IP_3Rs in tumorigenesis and tumor progression.By controlling the activities,structure,and/or expression levels of these Ca^(2+)-transporting proteins,malignant cancer cells can hijack them to drive essential biological functions for tumor development.However,the molecular mechanisms underlying the participation of STIM-ORAI and IP_3Rs in the biological behavior of cancer remain elusive.In this review,we summarize recent advances regarding STIM-ORAI and IP_3Rs and discuss how they promote cell proliferation,apoptosis evasion,and cell migration through temporal and spatial rearrangements in certain types of malignant cells.An understanding of the essential roles of STIM-ORAI and IP_3Rs may provide new pharmacologic targets that achieve a better therapeutic effect by inhibiting their actions in key intracellular signaling pathways.

Effects of Ca^(2+) channel blockers on store-operated Ca^(2+) channel currents of Kupffer cells after hepatic ischemia/reperfusion injury in rats

AIM： To study the effects of hepatic ischemia/ reperfusion （I/R） injury on store-operated calcium channel （SOC） currents （Isoc） in freshly isolated rat Kupffer cells, and the effects of Ca^2＋ channel blockers, 2-aminoethoxydiphenyl borate （2-APB）, SK＆F96365, econazole and miconazole, on Isoc in isolated rat Kupffer cells after hepatic I/R injury.METHODS： The model of rat hepatic I/R injury was established. Whole-cell patch-clamp techniques were performed to investigate the effects of 2-APB, SK＆F96365, econazole and miconazole on Isoc in isolated rat Kupffer cells after hepatic I/R injury.RESULTS： I/R injury significantly increased Isoc from -80.4±25.2pA to -159.5±34.5pA （^bp 〈 0.01, n = 30）. 2-APB （20, 40, 60, 80, 100 pmol/L）, SK＆F96365 （5, 10, 20, 40, 50 pmol/L）, econazole （0.1, 0.3, 1, 3, 10 μmol/L） and miconazole （0.1, 0.3, 1, 3, 10 μmol/L） inhibited Isoc in a concentration-dependent manner with IC50 of 37.41 μmol/L （n = 8）, 5.89 μmol/L （n = 11）, 0.21 μmol/L （n = 13）, and 0.28 μmol/L （n = 10）. The peak value of Isoc in the I-V relationship was decreased by the blockers in different concentrations, but the reverse potential of Isoc was not transformed. CONCLUSION： SOC is the main channel for the influx of Ca^2＋ during hepatic I/R injuries. Calcium channel blockers, 2-APB, SK＆F96365, econazole and miconazole,have obviously protective effects on I/R injury, probably by inhibiting Isoc in Kupffer cells and preventing the activation of Kupffer cells.

Polydatin attenuated food allergy via store-operated calcium channels in mast cell

AIM: To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and antiallergic activity. METHODS: After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwent PD treatment for 4 d, all the rats were stimulated by 100 mg/mL OVA for24 h and then sacrificed for the following experiments. The small intestines from all the groups were prepared for morphology examination by hematoxylin and eosin staining. We also used a smooth muscle organ bath to evaluate the motility of the small intestines. The OVA-specific immunoglobulin E (IgE) production and interleu-kin-4 (IL-4) levels in serum or supernatant of intestinal mucosa homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA). Using toluidine blue stain, the activation and degranulation of isolated rat peritoneal mast cells (RPMCs) were analyzed. Release of histamine from RPMCs was measured by ELISA, and regulation of PD on intracellular Ca 2+ mobilization was investigated by probing intracellular Ca 2+ with fluo-4 fluo-rescent dye, with the signal recorded and analyzed. RESULTS: We found that intragastric treatment with PD significantly reduced loss of mucosal barrier integrity in the small intestine. However, OVA-sensitization caused significant hyperactivity in the small intestine of allergic rats, which was attenuated by PD administration by 42% (1.26 ± 0.13 g vs OVA 2.18 ± 0.21 g, P < 0.01). PD therapy also inhibited IgE production (3.95 ± 0.53 ng/mL vs OVA 4.53 ± 0.52 ng/mL, P < 0.05) by suppressing the secretion of Th2-type cytokine, IL-4, by 34% (38.58 ± 4.41 pg/mLvs OVA 58.15 ± 6.24 pg/mL, P < 0.01). The ratio of degranulated mast cells, as indicated by vehicles (at least five) around the cells, dramatically increased in the OVA group by 5.5 fold (63.50% ± 15.51% vs phosphate-buffered saline 11.15% ± 8.26%, P < 0.001) and fell by 65% after PD treatment (21.95% ± 4.37% vs OVA 63.50% ± 15.51%, P < 0.001). PD mediated attenuation of mast cell degranulation was further confirmed by decreased histamine levels in both serum (5.98 ± 0.17 vs OVA 6.67 ± 0.12, P < 0.05) and intestinal mucosa homogenates (5.83 ± 0.91 vs OVA 7.35 ± 0.97, P < 0.05). Furthermore, we demonstrated that administration with PD significantly decreased mast cell degranulation due to reduced Ca 2+ influx through store-operated calcium channels (SOCs) (2.35 ± 0.39vs OVA 3.51 ± 0.38,P < 0.01).CONCLUSION: Taken together, our data indicate that PD stabilizes mast cells by suppressing intracellular Ca 2+ mobilization, mainly through inhibiting Ca 2+ entry via SOCs, thus exerting a protective role against OVA-sensitized food allergy.

EFFECT OF ELECTROACUPUNCTURE AND CALCIUM-CHANNEL INHIBITORS ON CYTOPLASMIC FREE CALCIUM CONCENTRATION OF MOUSE BRAIN CELLS

Objective: To study the effect of electroacupuncture (EA) and Verapamil and Nifedipine (calcium channel inhibitors) on free calcium concentrations of cells and intrasynaptosomes in hypothalamus (HT), periaqueductual grey matter (PAG) and hippocampus (HIP) of mice. Methods: The female ICR mice were randomly divided into control, EA, CaCl2 and CaCl2+EA groups (n=8 in each group). Pain threshold was detected by using radiation-heat irradiation-induced tail flick method. EA (8 Hz, a suitable stimulating strength, dense-sparse waves and duration of 30 min) was applied to“Shuigou” (水沟 GV 26) and “Chengjiang” (承浆CV 24). CaCl2 (10 μL, 0.2 μmol/L) was injected into the lateral cerebral ventricle of mice after EA. The concentrations of cytosolic free calcium ([Ca 2+]i) in HIP, PAG, HT cell suspension specimen and hippocampal intrasynaptosome suspension of mice were determined by the fluorescent calcium indicator Fura-2-AM and a spectrofluorometer. Results: During EA analgesia, the intracellular free [Ca 2+]i in HT and PAG specimens and intrsynaptosomal [Ca 2+]i of the 3 cerebral regions decreased considerably (P<0.05～0.01), but that in hippocampal cell suspension increased significantly (P<0.01) in comparison with control group. The concentrations of hippocampal intrasynaptosomal free [Ca 2+]i decreased significantly after adding Verapamil and Nifedipine to the extracted hippocampal intrasynaptosomal specimen. Microinjection of CaCl2 into lateral ventricle had no apparent influence on degree of analgesia (DA)% and intracellular and intrasynapsotomal [Ca 2+]i, but significantly lower DA% and reduce changes of cytosolic and intrasynaptosomal [Ca 2+]i induced by EA stimulation. Conclusion: Calcium ion in the neurons and intrasynaptosome of HT, PAG and HIP is involved in electroacupuncture analgesia.

Caribbean maitotoxin elevates [Ca^(2+)]i and activates non-selective cation channels in HIT-T15 cells

AIM:To investigate the cytotoxic mechanism of caribbean maitotoxin(MTX-C) in mammalian cells.METHODS:We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells,which is a system where the effects of MTX have been observed.HIT-T15 cells stably express L-type calcium current,making it a suitable model for this study.Using the fluorescence calcium indicator Indo-1 AM,we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C.RESULTS:About 3 min after perfusion of MTX-C,a gradual increase in free calcium concentration was observed.This elevation was sustained throughout the entire recording period.Application of MTX-C did not elicit the L-type calcium current,but large cationiccurrents appeared after applying MTX-C to the extracellular solution.The current-voltage relationship of the cation current is approximately linear within the voltage range from-60 to 50 mV,but flattened at voltages at-80 and-100 mV.These results indicate that MTX-C induces a non-voltage activated,inward current under normal physiological conditions,which by itself or through a secondary mechanism results in a large amount of cationic influx.The biophysical mechanism of MTX-C is different to its isoform,pacific maitotoxin(MTX-P),when the extracellular calcium is removed.CONCLUSION:We conclude that MTX-C causes the opening of non-selective,non-voltage-activated ion channels,which elevates level of intracellular calcium concentration and leads to cellular toxicities.

Effects of Arecoline on Calcium Channel Currents and Caffeine-induced Calcium Release in Isolated Single Ventricular

The effects of Arecoline (Are) on calcium m obilization were investigated. In isolated single ventricular m yocyte of guinea pig,patch clamp whole cell recording techniques were used to record the current of L - type calcium channel and cytosolic Ca2 + level ([Ca2 + ]i) labeled with fluo- rescence probe Fluo- 3/ AM was m easured under a laser scanning confocal microscope.Results re- vealed that Are(3- 10 0 μm ol/ L) could inhibit L- type calcium current in a concentration- depen- dent manner and the value of IC50 was33.73μm ol/ L (n=5 ) .In the absence of extracellular calci- um,the resting levels of[Ca2 + ]i was not affected by Are(n=6 ,P>0 .0 5 ) ,but pretreatment with Are(30 μmol/ L) could significantly inhibit the[Ca2 + ]i elevation induced by caffeine(10 m mol/ L,n=6 ,P<0 .0 1) .It was concluded that Are could inhibit not only calcium influx through L- type calcium channel but also calcium release from sarcoplasm ic reticulum.

CLCA2的表达与宫颈癌临床病理特征及预后的关系

目的探究宫颈癌组织中钙激活性氯离子通道2(CLCA2)的表达与患者临床病理特征及预后的关系。方法收集97例宫颈癌患者临床资料,分析其癌组织及癌旁组织CLCA2表达情况,以及癌组织中CLCA2表达与临床病理特征的关系;随访3年,采用生存分析、Cox回归分析法探究CLCA2表达与预后的关系。结果宫颈癌组织中CLCA2阴性表达率高于癌旁组织(P<0.05);CLCA2阴性组患者BMI≥30 kg/m^(2)、淋巴结转移、低分化程度及>1/2肌层浸润程度患者占比高于CLCA2阳性组(P<0.05);CLCA2阴性、阳性患者3年累计存活率差异存在显著性(P<0.05);Cox分析显示,宫颈癌TNMⅢ期、淋巴结转移、>1/2肌层浸润及CLCA2阴性是影响宫颈癌预后的独立危险因素(P<0.05)。结论宫颈癌组织中CLCA2下调,CLCA2表达与BMI、淋巴结转移、浸润程度及预后情况均相关,且高表达患者预后更好。

MicroRNA-1在心肌肥大中对L-型钙通道β_2亚基的负性调控作用

目的:研究微小RNA-1(microRNA-1,miR-1)在心肌细胞肥大中对L-型钙通道β2亚基(Cavβ2)的负性调控作用及机制。方法:应用异丙肾上腺素(ISO)诱导心肌细胞肥大;采用HJ2000通用图像分析系统测定心肌细胞表面积;应用数据库microCosm预测miR-1的靶基因;构建含Cavβ23’UTR报告基因质粒和miR-1瞬时共转染HEK293细胞,验证Cavβ2为miR-1靶基因;应用qRT-PCR或Western blot方法检测心房钠尿肽(ANP)、β-肌球蛋白重链(β-MHC)、miR-1和Cavβ2mRNA和蛋白表达水平;转染miR-1模拟物上调miR-1或应用Cavβ2RNAi干扰Cavβ2蛋白的表达,观察对心肌细胞肥大的影响。结果:①在ISO诱导的心肌细胞肥大中,miR-1表达显著下降;应用miR-1 mimic转染心肌细胞使miR-1表达上调,心肌细胞表面积、ANP和β-MHC mRNA表达均显著低于ISO组(P<0.05)。②网络数据库预测显示Cavβ2为miR-1的潜在靶点;将miR-1和含Cavβ23’UTR报告基因质粒共转染HEK293细胞,其萤光值显著降低(P<0.01)。转染miR-1 mimic使心肌细胞miR-1表达上调,可以明显抑制Cavβ2蛋白的表达。③在ISO诱导心肌细胞肥大中Cavβ2表达较对照组显著增加;应用RNAi技术下调Cavβ2表达可明显抑制心肌细胞表面积、ANP和β-MHC mRNA表达的增加。结论:预测并验证L-型钙通道β2亚基为miR-1的靶基因。miR-1可能通过抑制其靶基因Cavβ2蛋白的表达,降低细胞内钙离子浓度,抑制心肌细胞肥大。

高压氧对脑缺血及再灌注时海马游离Ca^（2＋）及钙通道的作用

应用新型钙离子荧光指示剂Ｆｕｒａ－２／ＡＭ测定海马突触体内游离Ｃａ￣（２＋）浓度，观察在脑缺血时及不同压力高压氧治疗后的变化规律，并应用［￣３Ｈ］ＰＮ２００－１１０作为放射性配基，用放射配体结合法测定海马组织Ｌ－型钙通道生物学特性和缺血及高压氧治疗后的变化。结果表明：脑缺血及再灌注后海马脑区突触体内游离Ｃａ￣（２＋）浓度显著增加，其Ｌ－型钙通道的Ｂｍａｘ和Ｋｄ值均显著上升，但经吸入高压氧后，可降低胞浆内游离Ｃａ￣（２＋）浓度，其中以２５３．２５ｋＰａ高压氧作用明显，并可降低钙通道的Ｂｍａｘ与Ｋｄ值。说明高压氧部分地通过减少细胞内游离Ｃａ￣（２＋）而起作用，Ｌ－型钙通道参与了高压氧降低脑缺血后胞浆内游离Ｃａ￣（２＋）浓度的作用，高压氧可使其开放的通道数量减少。

ERK1/2通路在低氧上调大鼠PASMCs钙激活性氯离子通道表达的作用

目的:探讨钙激活性氯离子通道(CLCA2)在大鼠低氧性肺动脉平滑肌细胞(PASMCs)中m RNA和蛋白表达的变化及其与ERK1/2信号通路的关系。方法:PASMCs随机分为:常氧组(N组),低氧组(H组),DMSO对照组(D组),U0126干预组(U组),Staurosporine aglycone干预组(SA组),采用免疫印迹法检测CLCA2蛋白的表达;选用半定量逆转录-聚合酶链反应(RT-PCR)技术测定CLCA2 m RNA水平的表达。结果:PASMCs中CLCA2m RNA和蛋白的表达量,H组较N组明显上调(P<0.01);U组较D组明显上调(P<0.01);SA组较D组m RNA的表达显著下调(P<0.01),蛋白的表达轻微下调。结论:低氧可上调CLCA2中m RNA和蛋白在PASMCs的表达;ERK1/2通路激活剂-Staurosporine aglycone能下调CLCA2在PASMCs中m RNA和蛋白的表达量;ERK1/2通路抑制剂-U0126可上调CLCA2在PASMCs中m RNA和蛋白的表达量。

重叠PCR法构建人心房肌SK2(KCNN2)基因表达质粒及鉴定和序列分析

目的:小电导钙激活钾通道亚型2(SK2)在心房肌功能活动中起重要作用,但是由于其表达密度低,直接进行RT-PCR一步法无法得到该基因(KCNN2)的编码区全长序列,本研究旨在采用Overlapping PCR(重叠PCR)法进行基因全长序列的扩增和表达质粒的构建,探讨其在长片段基因扩增的应用。方法:收集人心房肌标本,采用提取总RNA之后逆转录为cDNA,分三段设计KCNN2基因(AY258141)引物进行分段扩增,同时进行分段测序,然后采用Overlapping PCR得到KCNN2基因编码区全长序列,通过限制性酶切位点定向克隆到表达载体pIRES-hrGFP上。采用酶切法和测序法进行鉴定。结果:三段KCNN2基因扩增产物大小与预测值一致,最后得到的表达质粒测序结果与基因库数据基本一致。结论:成功构建人心房肌SK通道基因表达质粒pIRES-hrGFP-SK2,Overlapping PCR能够很好的用于长片段基因扩增。

三叉神经痛大鼠中钙通道α2δ-1亚单位表达的变化

目的研究大鼠眶下神经结扎后钙通道α2δ-1亚单位(Cavα2δ-1)表达的变化,探讨Cavα2δ-1与三叉神经痛的关系。方法将12只SD大鼠随机分为神经结扎组和假手术对照组,每组6只。用铬肠线轻松结扎神经结扎组大鼠的眶下神经,建立三叉神经的神经病理性疼痛动物模型;假手术对照组仅暴露神经,不结扎。术前1 d,术后第3、6、9、12、15天测定机械刺激反应阈值。术后第15天收集三叉神经节(TG)和三叉神经脊束核尾侧亚核及颈段第一、二脊髓背侧角(Vc/C2)组织,采用Western印迹杂交法测定Cavα2δ-1蛋白浓度。结果神经结扎组大鼠术侧机械刺激反应阈值在术后逐渐降低,与术前比较,术后第9、12、15天机械刺激反应阈值差异有统计学意义(P<0.05)。术后第15天神经结扎组术侧TG和Vc/C2中Cavα2δ-1表达明显上调(P<0.01)。结论 Cavα2δ-1参与了大鼠三叉神经痛的发生发展,为进一步探讨三叉神经痛发病机制提供实验基础。

AMPK调控Ca2＋内流对高糖诱导内皮细胞凋亡的作用及其机制研究

目的观察AMP依赖的蛋白激酶(AMPK)对高糖刺激内皮细胞凋亡的抑制作用,并初步探讨其机制。方法体外培养MS-1内皮细胞株,分别用AMPK激动剂、AMPK抑制剂、钙库依赖性钙离子通道(SOCC)抑制剂2-APB和(或)高糖处理,另设对照组(未经任何方式干预)。采用TUNEL法检测细胞凋亡情况,激光共聚焦显微镜检测细胞内钙离子(Ca2+)内流,Western blotting检测SOCC蛋白Stim1和Orai1的表达。结果与对照组比较,高糖能够明显诱导内皮细胞凋亡,增加Stim1和Orai1蛋白表达(P<0.05)。与高糖组比较,AMPK抑制剂+高糖能够明显增强高糖诱导的内皮细胞的凋亡(P<0.05),而AMPK激动剂+高糖能够明显抑制高糖诱导的内皮细胞凋亡,并降低Stim1和Orai1蛋白表达(P<0.05)。与对照组比较,高糖能够明显诱导内皮细胞Ca2+内流;与高糖组比较,2-APB+高糖能够明显抑制高糖诱导的内皮细胞Ca2+内流,并阻断高糖对内皮细胞凋亡的诱导作用,而AMPK激动剂能够明显抑制高糖诱导的内皮细胞Ca2+内流。结论 AMPK能够通过降低Stim1和Orai1蛋白的表达,抑制SOCC介导的Ca2+内流,进而阻断高糖刺激的内皮细胞凋亡,对内皮细胞功能起重要的保护作用。

动物CACNA2D1基因研究进展

CACNA2D1基因是编码动物L-型钙离子通道2αδ1亚基的基因。有研究表明,CACNA2D1基因可影响钙离子通道钙流的性质,其单核苷酸变异与某些疾病有关。综述了CACNA2D1基因在生物学特性、染色体上的位置、基本功能、表达调控及其单核苷酸多态性与疾病的关系等方面的最新研究成果。

用Fura-2测定突触体内游离Ca^(2+)浓度及Ca^(2+)通道激动剂和阻断剂的影响

用Fura-2测定大鼠突触体内游离Ca^(2+)浓度,探讨Ca^(2+)通道激动剂和阻断剂对突触体内钙浓度的影响。测得突触体内Ca^(2+)浓度为200～400 nmol/L。观察了不同浓度KCl,CaCl_2,NMDA和谷氨酸对突触体内Ca^(2+)增加的影响以及维拉帕米及MgCl_2对KCl和NMDA引起钙内流的阻断作用。本实验提供一个研究突触体内Ca^(2+)变化的准确而稳定的方法,并对测定中几个影响因素加以讨论。

miR-1和miR-133a对大鼠肥大心肌细胞L-型钙通道Cavβ_2和α1C亚基的调控作用

目的研究miR-1和miR-133a在大鼠心肌细胞肥大中对L-型钙通道β2亚基(Cavβ2)和α1C亚基的调控作用。方法异丙肾上腺素(ISO)诱导大鼠心肌细胞肥大;在线数据库micro Cosm和Targetscan预测miR-1和miR-133a的靶基因;分别构建含有Cavβ23'UTR或α1C 3'UTR的重组质粒,与miR-1或miR-133a共转染HEK293细胞,验证Cavβ2亚基是miR-1的靶基因,α1C亚基是miR-133a的靶基因;用Western blot法检测心肌细胞内Cavβ2和α1C蛋白表达;用siRNA干扰Cavβ2和α1C表达,明确Cavβ2和α1C在心肌细胞肥大中的作用。结果 1)Cavβ2为miR-1的潜在靶基因,α1C为miR-133a的潜在靶基因。2)分别将miR-1和Cavβ23'UTR,miR-133a和α1C 3'UTR共转染HEK293细胞,荧光素酶荧光值均显著降低(P<0.05,P<0.01)。3)分别转染miR-1 mimic、miR-133a mimic上调miR-1、miR-133a的表达后,心肌细胞内Cavβ2和α1C蛋白表达均明显下降(P<0.01,P<0.05)。4)用RNAi下调Cavβ2和α1C表达可明显抑制心肌细胞表面积(P<0.01),ANP和β-MHC mRNA表达增加(P<0.05)。结论Cavβ2亚基是miR-1的靶基因,α1C亚基是miR-133a的靶基因。miR-1和miR-133a可能通过负性调控L-型钙通道Cavβ2和α1C蛋白表达,抑制心肌细胞肥大。

心肌SarcK_(ATP)通道kir6.2亚基在运动预适应心肌保护效应中变化的研究

目的:探讨心肌SarcKATP通道kir6.2在运动预适应心肌保护效应中的变化以及与PKC的关系。方法:SD大鼠分对照组(C组)、力竭运动组(EE组)、运动预适应组(EP组)、运动预适应+力竭运动组(EP+EE组)、PKC阻断剂+运动预适应组(CHE+EP)和PKC阻断剂+运动预适应+力竭运动组(CHE+EP+EE组)。一次大强度间歇跑台运动建立运动预适应模型,力竭跑台运动致大鼠心肌损伤。用原位杂交和实时荧光定量PCR法检测kir6.2mRNA变化,用免疫荧光和免疫印迹法检测kir6.2蛋白变化。结果:与C组比,EE组和EP组kir6.2mRNA无明显变化,而EE组kir6.2蛋白明显升高,EP组kir6.2蛋白明显下降。与EE组比,EP+EE组kir6.2mRNA无明显变化,kir6.2蛋白明显下降。与EP组比,CHE+EP组kir6.2mRNA明显降低,kir6.2蛋白明显升高。与EP+EE组比,CHE+EP+EE组kir6.2mRNA和kir6.2蛋白无明显变化。结论:在EP诱导的保护效应中,心肌SarcKATP通道kir6.2mRNA水平未发生明显变化,而kir6.2蛋白水平明显降低,提示,心肌SarcKATP通道通过kir6.2蛋白水平的降低介导EP诱导的心肌保护效应。PKC对心肌SarcKATP通道的表达具有调控作用。

T－2毒素对心肌细胞三型钙通道的阻滞作用

用膜片钳连细胞电压钳法，在培养的Ｗｉｓｔａｒ大鼠单个心肌细胞上记录了Ｔ－２毒素对Ｂ、Ｌ和Ｔ三型Ｃａ２＋通道单通道电活动的影响．结果表明，Ｔ－２毒素浓度为１０ｍｇ／Ｌ时，心肌细胞Ｂ、Ｌ和Ｔ三型Ｃａ２＋通道均受到明显的阻滞，其阻滞作用表现为使Ｃａ２＋通道的开放概率减小，开放时间缩短，关闭时间延长，而对流过Ｃａ２＋通道的Ｂａ２＋流幅值无影响．