Agonist-evoked calcium entry in vascular smooth muscle cells requires IP3 receptor-mediated activation of TRPC1

Transient receptor potential canonical (TRPC) proteins have been proposed to function as plasma membrane Ca2+ channels activated by store depletion and/or by receptor stimulation. However, their role in the increase in cytosolic Ca2+ activated by contractile agonists in vascular smooth muscle is not yet elucidated. The present study was designed to investigate the functional and molecular properties of the Ca2+ entry pathway activated by endothelin-1 in primary cultured aortic smooth muscle cells. Measurement of the Ca2+ signal in fura-2-loaded cells allowed to characterize endothelin-1-evoked Ca2+ entry, which was resistant to dihydropyridine, and was blocked by 2-aminoethoxydiphenylborate (2-APB) and micromolar concentration of Gd3+. It was not activated by store depletion, but was inhibited by the endothelin ETA receptor antagonist BQ-123, and by heparin. On the opposite, thapsigargin-induced store depletion activated a Ca2+ entry pathway that was not affected by 2-APB, BQ-123 or heparin, and was less sensitive to Gd3+ than was endothelin-1-evoked Ca2+ entry. Investigation of the gene expression of TRPC isoforms by real-time RT-PCR revealed that TRPC1 was the most abundant. In cells transfected with TRPC1 small interfering RNA sequence, TRPC1 mRNA and protein expression were decreased by 72+/-3% and 86+/-2%, respectively, while TRPC6 expression was unaffected. In TRPC1 knockdown cells, both endothelin-1-evoked Ca2+ entry and store-operated Ca2+ entry evoked by thapsigargin were blunted. These results indicate that in aortic smooth muscle cells, TRPC1 is not only involved in Ca2+ entry activated by store depletion but also in receptor-operated Ca2+ entry, which requires inositol (1,4,5) triphosphate receptor activation.     

The cytoskeleton plays a modulatory role in the association between STIM1 and the Ca channel subunits Orai1 and TRPC1

Store-operated Ca²⁺ entry (SOCE) is a major pathway for Ca²⁺ influx in non-excitable cells. Recent studies favour a conformational coupling mechanism between the endoplasmic reticulum (ER) Ca²⁺ sensor STIM1 and Ca²⁺ permeable channels in the plasma membrane to explain SOCE. Previous studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca²⁺ permeable channels is modulated by the actin or microtubular network. In HEK-293 cells, treatment with the microtubular disrupter colchicine enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of the microtubules by paclitaxel attenuated TG-evoked activation of SOCE and the interaction between STIM1 and the Ca²⁺ channels Orai1 and TRPC1, altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer results in inhibition of TG-evoked both association between STIM1, Orai1 and TRPC1 and SOCE. Interestingly, disruption of the actin filament network by cytochalasin D did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE. Finally, inhibition of calmodulin by calmidazolium enhances TG-evoked SOCE and disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. Thus, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between their main molecular components.     

Store-operated calcium entry in vascular smooth muscle

In non-excitable cells, activation of G-protein-coupled phospholipase C (PLC)-linked receptors causes the release of Ca(2+) from intracellular stores, which is followed by transmembrane Ca(2+) entry. This Ca(2+) entry underlies a small and sustained phase of the cellular [Ca(2+)](i) increases and is important for several cellular functions including gene expression, secretion and cell proliferation. This form of transmembrane Ca(2+) entry is supported by agonist-activated Ca(2+)-permeable ion channels that are activated by store depletion and is referred to as store-operated Ca(2+) entry (SOCE) and represents a major pathway for agonist-induced Ca(2+) entry. In excitable cells such as smooth muscle cells, Ca(2+) entry mechanisms responsible for sustained cellular activation are normally considered to be mediated via either voltage-operated or receptor-operated Ca(2+) channels. Although SOCE occurs following agonist activation of smooth muscle, this was thought to be more important in replenishing Ca(2+) stores rather than acting as a source of activator Ca(2+) for the contractile process. This review summarizes our current knowledge of SOCE as a regulator of vascular smooth muscle tone and discusses its possible role in the cardiovascular function and disease. We propose a possible hypothesis for its activation and suggest that SOCE may represent a novel target for pharmacological therapeutic intervention.     

Store-operated calcium channels and pro-inflammatory signals

In non-excitable cells such as T lymphocytes, hepatocytes, mast cells, endothelia and epithelia, the major pathway for calcium [Ca2+] entry is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, however, neither the gating mechanism nor the downstream targets of these channels has been clear established. Here, I review some of the proposed gating mechanisms of store-operated Ca2+ channels and the functional implications in regulating pro-inflammatory signals.     

Dual effects of tetrandrine on calcium-activated potassium channels in pulmonary artery smooth muscle cells

目的：研究粉防己碱（Ｔｅｔ）对大鼠肺动脉平滑肌细胞钙激活钾（ＫＣａ）通道的影响．方法：内面朝外膜片箝单通道记录法．结果：Ｔｅｔ７５和１５μｍｏｌ·Ｌ－１使ＫＣａ的开放概率由０２５１±００１２增加到０３４０±００１３和０４１５±００１１（Ｐ＜００１）．关闭时间由（６１±１５）ｍｓ缩短到（３３±１０）和（２８±１１）ｍｓ（Ｐ＜００１）．Ｔｅｔ３０μｍｏｌ·Ｌ－１使开放概率和开放时间分别降低到（０１１４±０００８）和（１４７±００９）ｍｓ（Ｐ＜０．０１）．结论：Ｔｅｔ对大鼠肺动脉平滑肌细胞ＫＣａ通道有双重作用．     

高血压发展过程中脑血管平滑肌细胞离子通道的变化

脑血管重构是高血压发展过程中最重要的病理生理改变,由此引起的脑卒中更日益危害人类的健康。在高血压发展过程中,脑血管平滑肌细胞上分布的多种离子通道,如钾钙、氯等均发生变化,导致细胞内离子浓度异常,在脑血管重构的发生发展过程中发挥了重要作用。     

The transient receptor potential channel antagonist SKF96365 is a potent blocker of low-voltage-activated T-type calcium channels

Background and purpose:

{"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (SKF), originally identified as a blocker of receptor-mediated calcium entry, is widely used diagnostically, as a blocker of transient receptor potential canonical type (TRPC) channels. While SKF has been used as a tool to define the functional roles of TRPC channels in various cell and tissue types, there are notable overlapping physiological and pathophysiological associations between TRPC channels and low-voltage-activated (LVA) T-type calcium channels. The activity of SKF against T-type Ca channels has not been previously explored, and here we systematically investigated the effects of SKF on recombinant and native voltage-gated Ca channel-mediated currents.

Experimental approach:

Effects of SKF on recombinant Ca channels were studied under whole-cell patch clamp conditions after expression in HEK293 cells. The effect of SKF on cerebellar Purkinje cells (PCs) expressing native T-type Ca channels was also assessed.

Key results:

SKF blocked recombinant Ca channels, representative of each of the three main molecular genetic classes (Ca_V1, Ca_V2 and Ca_V3) at concentrations typically utilized to assay TRPC function (10 µM). Particularly, human Ca_V3.1 T-type Ca channels were more potently inhibited by SKF (IC₅₀∼560 nM) in our experiments than previously reported for similarly expressed TRPC channels. SKF also inhibited native Ca_V3.1 T-type currents in a rat cerebellar PC slice preparation.

Conclusions and implications:

SKF was a potent blocker of LVA T-type Ca channels. We suggest caution in the interpretation of results using SKF alone as a diagnostic agent for TRPC activity in native tissues.     

基质相互作用蛋白分子1在肿瘤发生发展和 临床应用中的研究进展

基质相互作用蛋白分子1（STIM1）与肿瘤的发生发展密切相关,其参与多种癌症细胞凋亡、增殖、迁移和侵袭的调节过程。阻断或敲除STIM1可以显著抑制癌细胞的增殖和迁移。阐明STIM1在癌症细胞中的调节机制,将有助于新的治疗靶点的确定。本文对STIM1分子在不同肿瘤中的作用机制及临床应用前景作一综述。     

氨茶碱调节大鼠支气管平滑肌L型钙通道的活动性

目的 探讨氨茶碱对大鼠支气管平滑肌细胞L型钙通道(L-L_(Ca))的作用。方法 将20只大鼠随机分为2组,A组为正常对照组(n=10),B组为氨茶碱组(n=10),观测大鼠支气管平滑肌细胞L-L_(Ca)在正常时及给予氨茶碱治疗后的变化。结果 氨茶碱治疗后大鼠的L-L_(Ca)与正常对照组大鼠通道开放概率相比差异有显著性(P<0.05)。氨茶碱组通道开放时间常数(τ_(O1)和τ_(O2))不变,关闭时间常数(T_(c1)和T_(c2))延长,与正常对照组相比差异有显著性(P<0.05)。大鼠L-钙通道的活动经氨茶碱治疗后活动减弱,表现为通道开放时间常数不变,关闭时间常数延长。结论 氨茶碱通过缩短L-L_(Ca)的开放时间来抑制支气管平滑肌细胞的收缩,可显著抑制大鼠L-L_(Ca)的活动,促进大鼠支气管平滑肌细胞的舒张。     

Roles of TRP channels in the development of cardiac hypertrophy

Cardiac hypertrophy is induced by various stresses such as hypertension and myocardial infarction. It is believed that hypertrophy is adaptive in the early phase but becomes maladaptive in the late phase. Cardiac hypertrophy develops heart failure when the heart is exposed persistently to the stresses. The increase in intracellular Ca(2+) ([Ca(2+)](i)) plays an important role in the development of hypertrophy. It is generally thought that the increase in [Ca(2+)](i) for hypertrophy occurs via G(q)-stimulated production of inositol-1,4,5-trisphosphate (IP(3)) and IP(3)-mediated release of Ca(2+) from intracellular store. However, several groups recently reported that canonical transient receptor potential (TRPC) channels are responsible for the increase in [Ca(2+)](i). Among them, three TRPC subtypes (TRPC3/TRPC6/TRPC7) are activated by another G(q)-mediated second messenger, diacylglycerol. Although several groups independently demonstrated that TRPC channels mediate receptor-stimulated and pressure overload-induced hypertrophy, there is discrepancy of which subtypes of TRPC channels predominantly mediate hypertrophy. However, there is consensus that TRPC-mediated Ca(2+) influx is essential for hypertrophy. As TRPC channels participate in pathological hypertrophy, but not physiological contraction and the relaxation cycle, TPRC channels are a new target for the treatment of hypertrophy.     

家兔主动脉血管平滑肌细胞内钙动员及小檗胺作用的共聚焦研究(英文)

观察小檗胺 ( Ber)对高钾除极 ,Bay K8644,5-羟色胺 ( 5- HT)及咖啡因升高细胞内钙水平( [Ca2 + ]i)的影响。以 Fluo- 3/AM负载家兔培养的主动脉平滑肌细胞 ( VSMC) ,共聚焦显微术测定[Ca2 + ]i,结果以荧光强度 ( FI)表示 .结果 :( 1 )在细胞外钙为 1 .3mmol· L-1时 ,VSMC胞浆静息 FI明显高于核区 ,且不受 Ber的影响 . ( 2 ) Ber 1 0 -1 0 0 μmol·L-1预处理可抑制 KCl60 mmol·L-1或Bay K86441 0 0 μmol·L-1升高的 [Ca2 + ]i,抑制 5-HT 1μmol· L-1升高 [Ca2 + ]i 的持续相 ,但不影响[Ca2 + ]i 的一过性升高。维拉帕米 1 0 μmol· L-1具有相似作用 . ( 3)在无钙 Hanks液中 ,Ber预处理对咖啡因 1 0 0 mmol·L-1升高的 [Ca2 + ]i 无明显抑制作用。结果表明 ,Ber可阻断外钙内流 ,但不抑制内钙释放 ,这可能与 Ber阻断电压依赖性钙通道和受体依赖性钙通道的作用有关 .     

Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Cav1.2 calcium channels

Background and purpose:

As a calcium channel blocker, diltiazem acts mainly on the voltage-gated calcium channels, Ca_v1.2, for its beneficial effects in cardiovascular diseases such as hypertension, angina and/or supraventricular arrhythmias. However, the effects of diltiazem on different isoforms of Ca_v1.2 channels expressed in heart and vascular smooth muscles remain to be investigated. Here, we characterized the effects of diltiazem on the splice variants of Ca_v1.2 channels, predominant in cardiac and vascular smooth muscles.

Experimental approach:

Cardiac and smooth muscle isoforms of Ca_v1.2 channels were expressed in human embryonic kidney cells and their electrophysiological properties were characterized using whole-cell patch-clamp techniques.

Key results:

Under closed-channel and use-dependent block (0.03 Hz), cardiac splice variant Ca_v1.2CM was less sensitive to diltiazem than two major smooth muscle splice variants, Ca_v1.2SM and Ca_v1.2b. Ca_v1.2CM has a more positive half-inactivation potential than the smooth muscle channels, and diltiazem shifted it less to negative potential. Additionally, the current decay was slower in Ca_v1.2CM channels. When we modified alternatively spliced exons of cardiac Ca_v1.2CM channels into smooth muscle exons, we found that all three loci contribute to the different diltiazem sensitivity between cardiac and smooth muscle splice isoforms.

Conclusions and implications:

Alternative splicing of Ca_v1.2 channels modifies diltiazem sensitivity in the heart and blood vessels. Gating properties altered by diltiazem are different in the three channels.     

An increase in opening of BK(Ca) channels in smooth muscle cells in streptozotocin-induced diabetic mice

INTRODUCTION Diabetes mellitus (DM) is a kind of disease thatmetabolism decompensates with hyperglycemia and re-sults in multi-organ damage[1]. Thus, the risk of coro-nary disease, cerebrovascular disease, and other car-diovascular complications increase. These changes, atleastpartially, due todiabetes functionalchanges inbloodvessels including endothelial cell dysfunction. Simulta-neously, altered ion channelfunction in vascular smoothmuscle are also involved[2,3]. In vascular…     

T型钙通道在心肌肥厚大鼠心肌细胞钙内流中的作用

目的研究T型钙通道在心肌细胞钙离子内流中的作用及其对心脏兴奋收缩耦联的可能影响。方法测定选择性T型钙通道阻滞剂米贝拉地尔对培养的SD乳大鼠心室肌细胞和二肾一夹心肌肥厚大鼠心室肌细胞[Ca2+]i的影响。结果血管紧张素Ⅱ(AngⅡ)刺激使乳大鼠心室肌舒张期细胞[Ca2+]i增高,收缩期细胞[Ca2+]i降低,[Ca2+]i上升和下降的时间延长。米贝拉地尔1.25～5μmol·L-1浓度依赖性降低AngⅡ引起的细胞[Ca2+]i变化。在心肌肥厚模型大鼠,咖啡因刺激后,[Ca2+]i增幅和最高[Ca2+]i明显降低。而米贝拉地尔25mg·kg-1·d-1(灌胃给药7～9周)组加入咖啡因刺激后细胞内[Ca2+]i增幅和最高[Ca2+]i明显增高。结论T型钙通道异常开放可以引起心肌细胞内钙超载。阻断T型钙通道,可能通过改善肌浆网摄取及释放钙的功能而抑制心肌细胞钙超载。     

小鼠精母细胞钙通道特性及硝苯地平的作用

目的　为了建立一个合理、客观评价药物或毒物对生殖系统影响的细胞模型 ,研究了机械分离、未经酶消化的小鼠精母细胞Ca2 + 通道特性及硝苯地平对其的作用。方法　采用全细胞膜片钳技术。结果阻断K+ 电流后 ,当钳制电位 -90mV、指令电压 -80～ +1 0mV、步阶电压 1 0mV时 ,记录到Ca2 +电流 ,二价无机阳离子对Ca2 + 电流的抑制作用Ni2 +>Cd2 + ,而L型Ca2 + 通道激动剂BayK8644对电流无任何影响。分析小鼠精母细胞上记录的Ca2 + 电流为T型Ca2 + 通道开放产生。L型Ca2 + 通道阻断剂硝苯地平对精母细胞Ca2 + 电流有明显的抑制作用 ,半数最大抑制浓度为 0 .3 9μmol·L-1,而且细胞外液冲洗恢复缓慢 ,这支持了二氢吡啶类药物可用于男性避孕。结论　机械分离、未经酶消化的小鼠精母细胞模型适合进行药理学和毒理学研究     

Histamine-operated calcium channels in intestinal smooth muscle of the guinea-pig

The effects of Bay K 8644 and of nifedipine on histamine-induced mechanical and electrical responses were studied in the longitudinal smooth muscle of the ileum and in the taenia coli isolated from the guinea-pig. Nifedipine (10(-9)-10(-7) M) depressed the tonic and phasic components of histamine contraction. Phasic tension was less sensitive to nifedipine inhibition than was tonic tension (I50: 27 +/- 6 and 2.6 +/- 0.4 nM respectively). Bay K 8644 (10(-8)-10(-7) M) increased tension and rhythmic activity of intestinal smooth muscle and potentiated the histamine responses. The phasic tension evoked by histamine 10(-5) M and the phasic tension evoked by the KCl depolarizing solution showed the same sensitivity to nifedipine inhibition (I50: 28 +/- 5 nM) and were similarly potentiated by Bay K 8644. The tonic tension in response to the KCl-depolarizing solution was more sensitive to nifedipine inhibition than was the tonic tension in response to histamine and was not potentiated by Bay K 8644. These results indicate that different Ca entry pathways, dependent or not on modification of the membrane potential, are involved in the contractile response evoked by histamine in intestinal smooth muscle.     

家兔主动脉血管平滑肌细胞内钙动员及小檗胺作用的共聚焦研究

观察小檗胺 (Ber) 对高钾除极,Bay K8644,5-羟色胺 (5-HT) 及咖啡因升高细胞内钙水平 (［Ca²⁺］_i) 的影响。以Fluo-3/AM负载家兔培养的主动脉平滑肌细胞 (VSMC),共聚焦显微术测定［Ca²⁺］_i,结果以荧光强度(FI)表示. 结果：(1) 在细胞外钙为1.3 mmol·L^-1时, VSMC胞浆静息FI明显高于核区, 且不受Ber的影响. (2) Ber 10-100 μmol·L^-1预处理可抑制KCl 60 mmol·L^-1或Bay K8644 100 μmol·L^-1升高的［Ca²⁺］_i,抑制5- HT 1 μmol·L^-1升高［Ca²⁺］_i的持续相,但不影响［Ca²⁺］_i的一过性升高。维拉帕米10 μmol·L^-1具有相似作用. (3) 在无钙Hanks液中,Ber预处理对咖啡因100 mmol·L^-1升高的［Ca²⁺］_i无明显抑制作用。结果表明,Ber可阻断外钙内流,但不抑制内钙释放,这可能与Ber阻断电压依赖性钙通道和受体依赖性钙通道的作用有关.     

三七皂甙对血管平滑肌上受体操纵Ca~(2 )通道的特异性作用(英文)

三七皂甙能明显抑制狗肠系膜动脉及大隐静脉α肾上腺素能受体触发的收缩反应及~(45)Ca内流(从0.36±0.03降至0.14±0.05μmol·g~(-1)组织湿重),但对高K~ 引起的Ca~(2 )内流无影响。三七皂甙不影响Ca~(2 )释放及受体的亲和力。提示三七皂甙具有特异性阻断受体操纵Ca~(2 )通道的特性,对电位依赖性Ca~(2 )通道无作用。     

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