Modulation of TRPC5 cation channels by halothane, chloroform and propofol

BACKGROUND AND PURPOSE: TRPC5 is a mammalian homologue of the Drosophila Transient Receptor Potential (TRP) channel and has expression and functions in the cardiovascular and nervous systems. It forms a calcium-permeable cation channel that can be activated by a variety of signals including carbachol (acting at muscarinic receptors), lanthanides (e.g. Gd3+) and phospholipids (e.g. lysophosphatidylcholine: LPC). Here we report the effects of inhalational (halothane and chloroform) and intravenous (propofol) general anaesthetics upon TRPC5. EXPERIMENTAL APPROACH: Human TRPC5 channels were expressed in HEK 293 cells and studied using fura-2 and patch-clamp recording to measure intracellular calcium and membrane currents respectively at room temperature. Human TRPM2 channels were studied for comparison. KEY RESULTS: TRPC5 activation by carbachol, Gd3+ or LPC was inhibited by halothane and chloroform at > or =0.1 and 0.2 mM respectively. Neither agent inhibited TRPM2. Propofol had an initial stimulatory effect on TRPC5 (evident in patch-clamp recordings only) and an inhibitory effect at > or =10 microM. TRPM2 was not affected by propofol. Propofol inhibited activation of TRPC5 by Gd3+ but not LPC, suggesting the effect was not directly on the channel. Propofol's anti-oxidant property was not necessary for its inhibitory effect because di-isopropyl benzene, a propofol analogue that lacks the hydroxyl group, also inhibited TRPC5.Conclusions and implications:The data show the sensitivity of TRPC5 channel to general anaesthetics and suggest that some of the effects could have clinical relevance. The effects may be explained in part by the sensitivity of the channel to biophysical properties of the lipid bilayer.     

Riluzole activates TRPC5 channels independently of PLC activity

BACKGROUND AND PURPOSE

The transient receptor potential channel C5 (TRPC5) is a Ca²⁺-permeable cation channel, which is predominantly expressed in the brain. TRPC5 is activated in a PLC-dependent manner by, as yet, unidentified endogenous messengers. Recently, modulators of TRPC5, like Ca²⁺, pH and phospholipids, have been identified. However, the role of TRPC5 in vivo is only poorly understood. Novel specific modulators of TRPC5 might help to elucidate its function.

EXPERIMENTAL APPROACH

Novel modulators of TRPC5 were identified in a compound screening of approved drugs and natural compounds. The potency and selectivity of TRPC5-activating compounds were determined by fluorometric calcium imaging. The biophysical properties of channel activation by these compounds were analysed using electrophysiological measurements.

KEY RESULTS

Riluzole was identified as a novel activator of TRPC5 (EC₅₀ 9.2 ± 0.5 μM) and its mechanism of action was shown to be independent of G protein signalling and PLC activity. Riluzole-induced TRPC5 currents were potentiated by La³⁺ and, utilizing TRPC5 mutants that lack La³⁺ binding sites, it was confirmed that riluzole and La³⁺ activate TRPC5 by different mechanisms. Recordings of excised inside-out patches revealed a relatively direct effect of riluzole on TRPC5.

CONCLUSIONS AND IMPLICATIONS

Riluzole can activate TRPC5 heterologously expressed in HEK293 cells as well as those endogenously expressed in the U-87 glioblastoma cell line. Riluzole does not activate any other member of the TRPC family and could, therefore, despite its action on other ion channels, be a useful pharmacological tool for identifying TRPC5-specific currents in immortalized cell lines or in acutely isolated primary cells.     

Stereo-selective inhibition of transient receptor potential TRPC5 cation channels by neuroactive steroids

BACKGROUND AND PURPOSE

Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids.

EXPERIMENTAL APPROACH

Human TRPC5 channels were conditionally expressed in HEK293 cells and studied using intracellular Ca²⁺ measurement, whole-cell voltage-clamp and excised patch techniques. For comparison, control experiments were performed with cells lacking TRPC5 channels or expressing another TRP channel, TRPM2. Native TRPC channel activity was recorded from vascular smooth muscle cells.

KEY RESULTS

Extracellular application of pregnenolone sulphate, pregnanolone sulphate, pregnanolone, progesterone or dihydrotestosterone inhibited TRPC5 activity within 1–2 min. Dehydroepiandrosterone sulphate or 17β-oestradiol had weak inhibitory effects. Pregnenolone, and allopregnanolone, a progesterone metabolite and stereo-isomer of pregnanolone, all had no effects. Progesterone was the most potent of the steroids, especially against TRPC5 channel activity evoked by sphingosine-1-phosphate. In outside-out patch recordings, bath-applied progesterone and dihydrotestosterone had strong and reversible effects, suggesting relatively direct mechanisms of action. Progesterone inhibited native TRPC5-containing channel activity, evoked by oxidized phospholipid.

CONCLUSIONS AND IMPLICATIONS

Our data suggest that TRPC5 channels are susceptible to relatively direct and rapid stereo-selective steroid modulation, leading to channel inhibition. The study adds to growing appreciation of TRP channels as non-genomic steroid sensors.     

蛋白质酪氨酸磷酸化和氯通道参与瞬时受体电位蛋白介导钙池耗竭引起的钙内流

目的　探讨蛋白质酪氨酸磷酸化与Cl-通道对瞬时受体电位 (TRP)蛋白参与的Ca2 + 池耗竭引起的Ca2 + 内流(SOC)的调控作用。方法　采用脂质体转染和Fura 2 /AM荧光光度法 ,测定胞浆游离Ca2 +浓度 ( [Ca2 + ]i) ,比较转染人源性TRP1 (hTRP1 )和人源性TRP3 (hTRP3 )cDNA对毒胡罗卜素 (TG)引起的Ca2 + 内流的作用 ,并观察酪氨酸激酶抑制剂染料木黄酮、Cl-通道阻断剂呋塞米和 4,4 二异硫氰基芪 2 ,2 二磺酸 (DIDS)对其的影响。结果　HEK2 93细胞转染hTRP1cDNA后 ,TG引起的Ca2 + 内流显著增加 ;转染hTRP3cDNA则无明显影响。 5～ 3 0 μmol·L-1染料木黄酮、1～ 8μmol·L-1呋塞米、0 .5～ 1μmol·L-1DIDS对转染hTRP1cDNA细胞的TG诱发的Ca2 + 内流均有抑制作用。结论　hTRP1蛋白可能是HEK2 93细胞SOC的物质基础 ;酪氨酸激酶和Cl-通道均参与HEK2 93细胞SOC的调控 ,而且酪氨酸激酶可能直接作用于TRP1蛋白     

Production of a specific extracellular inhibitor of TRPM3 channels

Background and purpose:

Isoform-specific ion channel blockers are useful for target validation in drug discovery and can provide the basis for new therapeutic agents and aid in determination of physiological functions of ion channels. The aim of this study was to generate a specific blocker of human TRPM3 channels as a tool to help investigations of this member of the TRP cationic channel family.

Experimental approach:

A polyclonal antibody (TM3E3) was made to a conserved peptide of the third extracellular (E3) loop of TRPM3 and tested for binding and functional effect. Studies of channel activity were made by whole-cell planar patch-clamp and fura-2 intracellular Ca²⁺ measurement.

Key results:

Ionic current mediated by TRPM3 was inhibited partially by TM3E3 over a period of 5–10 min. Ca²⁺ entry in TRPM3-expressing cells was also partially inhibited by TM3E3 in a peptide-specific manner and independently of the type of agonist used to activate TRPM3. TM3E3 had no effect on TRPC5, TRPV4, TRPM2 or an endogenous ATP response.

Conclusions and implications:

The data show the successful development of a specific TRPM3 inhibitor and give further confidence in E3 targeting as an approach to producing isoform-specific ion channel blockers.     

Emerging functions of 10 types of TRP cationic channel in vascular smooth muscle

1. The influx of Ca2+, Mg2+ and Na+ and the efflux of K+ have central importance for the function and survival of vascular smooth muscle cells, but progress in understanding the influx/efflux pathways has been restricted by a lack of identification of the genes underlying many of the non-voltage-gated cationic channels. 2. The present review highlights evidence suggesting the genes are mammalian homologues of the Transient Receptor Potential (TRP) gene of the fruit-fly Drosophila. The weight of evidence supports roles for TRPC1, TRPP2/1 and TRPC6, but recent studies point also to TRPC3, TRPC4/5, TRPV2, TRPM4 and TRPM7. 3. Activity of these TRP channels is suggested to modulate contraction and sense changes in intracellular Ca2+ storage, G-protein-coupled receptor activation and osmotic stress. Roles in relation to myogenic tone, actions of vasoconstrictors substances, Mg2+ homeostasis and the vascular injury response are suggested. 4. Knowledge that TRP channels are relevant to vascular smooth muscle cells in both their contractile and proliferative phenotypes should pave the way for a better understanding of vascular biology and provide the basis for the discovery of a new set of therapeutic agents targeted to vascular disease.     

Voltage-gated cation channel modulators for the treatment of stroke

Neuronal voltage-gated cation channels regulate the transmembrane flux of calcium, sodium and potassium. Neuronal ischaemia occurring during acute ischaemic stroke results in the breakdown in the normal function of these ion channels, contributing to a series of pathological events leading to cell death. A dramatic increase in the intracellular concentration of calcium during neuronal ischaemia plays a particularly important role in the neurotoxic cascade resulting in stroke-related acute neurodegeneration. One approach to provide therapeutic benefit following ischaemic stroke has been to target neuronal voltage-gated cation channels, and particularly blockers of calcium and sodium channels, for post-stroke neuroprotection. A recent development has been the identification of openers of large-conductance calcium- and voltage-dependent potassium channels (maxi-K channels), which hyperpolarise ischaemic neurons, reduce excitatory amino acid release, and reduce ischaemic calcium entry. Thus far, targeting these voltage-gated cation channels has not yet yielded significant clinical benefit. The reasons for this may involve the lack of small-molecule blockers of many neuronal members of these ion channel families and the design of preclinical stroke models, which do not adequately emulate the clinical condition and hence lack sufficient rigor to predict efficacy in human stroke. Furthermore, there may be a need for changes in clinical trial designs to optimise the selection of patients and the course of drug treatment to protect neurons during all periods of potential neuronal sensitivity to neuro-protectants. Clinical trials may also have to be powered to detect small effect sizes or be focused on patients more likely to respond to a particular therapy. The development of future solutions to these problems should result in an improved probability of success for the treatment of stroke.     

大豆异黄酮体外对大鼠淋巴细胞激活和天然杀伤细胞活性的作用(英文)

目的 :研究大豆异黄酮对大鼠免疫细胞的体外作用。方法 :取大鼠抗凝血 ,密度梯度离心法分离外周血单个核细胞 ,以伴刀豆球蛋白A(ConA)和体内所能达到的不同浓度的金雀异黄素或大豆苷元共同孵育 6 8h后 ,四唑蓝还原法 (MTT)测定体外ConA诱导的淋巴细胞增殖 ;另取外周血单核细胞为效应细胞 ,YAC 1细胞为靶细胞 ,以乳酸脱氢酶法测定天然杀伤细胞 (NK )活性。结果 :在 0 .0 1～ 1μmol·L- 1浓度范围内 ,大豆苷元可剂量依赖性的增强体外ConA诱导的淋巴细胞增殖 (P <0 .0 1) ,1μmol·L- 1的大豆苷元可明显增强NK细胞活性 (P<0 .0 1)。而同样浓度范围内的金雀异黄素对淋巴细胞增殖及NK细胞活性均无明显作用。结论 :大豆苷元体外能剂量依赖性的刺激ConA诱导的淋巴细胞转化 ,增强NK细胞活性 ,这种增强的免疫功能可能是大豆制品抗肿瘤作用的机制之一     

Structural requirements of steroidal agonists of transient receptor potential melastatin 3 (TRPM3) cation channels

Background and Purpose

Transient receptor potential melastatin 3 (TRPM3) proteins form non-selective but calcium-permeable membrane channels, rapidly activated by extracellular application of the steroid pregnenolone sulphate and the dihydropyridine nifedipine. Our aim was to characterize the steroid binding site by analysing the structural chemical requirements for TRPM3 activation.

Experimental Approach

Whole-cell patch-clamp recordings and measurements of intracellular calcium concentrations were performed on HEK293 cells transfected with TRPM3 (or untransfected controls) during superfusion with pharmacological substances.

Key Results

Pregnenolone sulphate and nifedipine activated TRPM3 channels supra-additively over a wide concentration range. Other dihydropyridines inhibited TRPM3 channels. The natural enantiomer of pregnenolone sulphate was more efficient in activating TRPM3 channels than its synthetic mirror image. However, both enantiomers exerted very similar inhibitory effects on proton-activated outwardly rectifying anion channels. Epiallopregnanolone sulphate activated TRPM3 almost equally as well as pregnenolone sulphate. Exchanging the sulphate for other chemical moieties showed that a negative charge at this position is required for activating TRPM3 channels.

Conclusions and Implications

Our data demonstrate that nifedipine and pregnenolone sulphate act at different binding sites when activating TRPM3. The latter activates TRPM3 by binding to a chiral and thus proteinaceous binding site, as inferred from the differential effects of the enantiomers. The double bond between position C5 and C6 of pregnenolone sulphate is not strictly necessary for the activation of TRPM3 channels, but a negative charge at position C3 of the steroid is highly important. These results provide a solid basis for understanding mechanistically the rapid chemical activation of TRPM3 channels.     

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.     

Post-transcriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca entry in A7r5 vascular smooth muscle cells

This study investigates functional consequences of TRPC1 ion channel downregulation observed in aging rat aorta by employing RNA interference in cultured vascular smooth muscle cells. For this purpose, A7r5 aortic smooth muscle cells were used in quantitative gene and protein expression as well as in functional analyses. According to quantitative RT-PCR results, TRPC3, TRPC4 and TRPC5 mRNAs were not at detectable levels. In siTRPC1-transfected cells, TRPC1 mRNA and protein levels were decreased by 40% and 64%; however, those of TRPC6 were drastically increased by 100% and 200%, respectively. In fura-2-loaded TRPC1 knockdown cells, despite the decreased TRPC1 levels, cyclopiazonic acid-induced Ca²⁺ entry and store-operated Ca²⁺ entry following Ca²⁺ addition were elevated by 77% and 135%, respectively. Results suggest that decrease in TRPC1 may be compensated by upregulated TRPC6 that possibly takes part in store-operated Ca²⁺ entry in vascular smooth muscle cells.     

Dual signaling pathways of arterial constriction by extracellular uridine 5'-triphosphate in the rat

We investigated actions of uridine 5'-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 μM) elicited an inward-rectifying current strongly reminiscent of activation of P2X(1) receptor, and a similar current was also induced by α,β-methylene adenosine 5'-triphosphate (ATP) (≥100 nM). UTP desensitized α,β-methylene ATP-evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X(1)-receptor antibody. Both UTP (1 mM) and α,β-methylene ATP (10 μM) elicited similar conductance single channel activities. UTP (≥10 μM) provoked a dose-dependent contraction of de-endothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca(2+) or bath-applied 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 μM) or nifedipine (10 μM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 μM). Similar biphasic rises in [Ca(2+)](i) were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X(1) receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X(1)-like and P2Y receptors, but not TRPC3.     

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 TRPC2 ion channel and pheromone sensing in the accessory olfactory system

The mammalian vomeronasal organ (VNO) has emerged as an excellent model to investigate the signaling mechanisms, mode of activation, biological function, and molecular evolution of transient receptor potential (TRP) channels in real neurons and real physiological systems. TRPC2, a member of the canonical TRPC subfamily, is highly localized to the dendritic tip of vomeronasal sensory neurons. Targeted deletion of the TRPC2 gene has established that TRPC2 plays a fundamental role in the detection of pheromonal signals by the VNO. TRPC2-deficient mice exhibit striking behavioral defects in the regulation of sexual and social behaviors. A novel Ca²⁺-permeable, diacylglycerol-activated cation channel found at the dendritic tip of vomeronasal neurons is severely defective in TRPC2 mutants, providing the first clear example of native diacylglycerol-gated cation channels in the mammalian nervous system. The TRPC2 gene has become an important marker for the evolution of VNO-dependent pheromone signaling in primates.     

辣椒素受体-5在头孢曲松结石大鼠模型肾脏中的表达及其意义

目的：通过构建头孢曲松钠结石大鼠模型,探讨TRPV5（辣椒素受体-5）在结石模型组大鼠肾脏中和正常对照组大鼠肾脏中的表达差异,分析其表达量与头孢曲松钠应用时间的关系,为研究头孢曲松钠相关肾结石的发生机制提供新的理论依据。方法：30只雄性SD大鼠随机分为3组,A组给予120 mg·kg^-1·d^-1纯化水灌胃4周,B组、C组分别以120 mg·kg^-1·d^-1头孢曲松钠灌胃2周和4周;试剂盒检测各组血、尿生化指标;用相差显微镜观察各组结石结晶形成情况;采用免疫组化、双抗体夹心法分别定性、定量大鼠肾组织TRPV5的表达。结果：3组血钙浓度无统计学差异（P>0.05）,C组与A组、B组比较血肌酐明显升高[（96.29±21.81）μmol·L^-1 vs（34.72±10.49）μmol·L^-1、（59.98±20.45）μmol·L^-1,P<0.05],C组尿钙浓度相比较A组明显升高[（0.72±0.25）mmol·L^-1 vs（0.46±0.23）mmol·L^-1,P<0.01],C组与A组、B组比较尿肌酐降低[（2 488.28±435.75）μmol·L^-1 vs（3 463.57±221.76）μmol·L^-1、（2 971.37±319.27）μmol·L^-1,P<0.01]。光镜下C组可见有大量结石结晶形成。TRPV5在3组血液中表达无明显差异（P>0.05）,C组中TRPV5在肾脏中表达明显低于A组、B组[（217.79±48.31）ng·L^-1 vs（395.66±74.69）ng·L^-1、（343.08±74.08）ng·L^-1,P<0.01]。结论：TRPV5在头孢曲松钠干预4周组大鼠肾组织中的表达降低,表达量且与头孢曲松钠干预时间呈负相关,可能由于头孢曲松钠抑制了钙离子通道蛋白TRPV5在肾脏的表达,增加肾小管尿钙排泄从而参与了头孢曲松结石的形成。     

Functional characterization and physiological relevance of the TRPC3/6/7 subfamily of cation channels

The mammalian transient receptor potential (TRP) superfamily of cation channels can be divided into six major families. Among them, the classical or canonical TRPC family is most closely related to Drosophila TRP, the founding member of the superfamily. All seven channels of this family designated TRPC1-7 share the common property of activation through phospholipase C (PLC)-coupled receptors, but their gating by receptor- or store-operated mechanisms is still controversial. The TRPC3, 6, and 7 channels are 75% identical and are also gated by direct exposure to diacylglycerols (DAG). TRPC3, 6, and 7 interact physically and, upon coexpression, coassemble to form functional tetrameric channels. This review will focus on the TRPC3/6/7 subfamily and describe their functional properties and regulation as homomers obtained from overexpression studies in cell lines. It will also summarize their heteromultimerization potential in vitro and in vivo and will present preliminary data concerning their physiological functions analyzed in isolated tissues with downregulated channel activity and gene-deficient mouse models.     

Activation of two types of Ca2+-permeable nonselective cation channel by endothelin-1 in A7r5 cells

1. In A7r5 cells loaded with the Ca²⁺ indicator fura-2, we examined the effect of a Ca²⁺ channel blocker SK&F 96365 on increases in intracellular free Ca²⁺ concentrations ([Ca²⁺]i) and Mn²⁺ quenching of fura-2 fluorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed.
2. Higher concentrations (⩾10 nM) of ET-1 (higher [ET-1]) evoked a transient peak and a subsequent sustained elevation in [Ca²⁺]i: removal of extracellular Ca²⁺ abolished only the latter. A blocker of L-type voltage-operated Ca²⁺ channel (VOC) nifedipine at 1 μM reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 μM).
3. Lower [ET-1] (⩽1 nM) evoked only a sustained elevation in [Ca²⁺]i which depends on extracellular Ca²⁺. The elevation was partly sensitive to nifedipine but not SK&F 96365.
4. In the presence of 1 μM nifedipine, higher [ET-1] increased the rate of Mn²⁺ quenching but lower [ET-1] had little effect.
5. In whole-cell recordings, both lower and higher [ET-1] induced inward currents at a holding potential of −60 mV with linear I-V relationships and reversal potentials close to 0 mV. The current at lower [ET-1] was resistant to SK&F 96365 but was abolished by replacement of Ca²⁺ in the bath solution with Mn²⁺. The current at higher [ET-1] was abolished by the replacement plus SK&F 96365.
6. In a bath solution containing only Ca²⁺ as a movable cation, ET-1 evoked currents: the current at lower [ET-1] was sensitive to Mn²⁺, whereas that at higher [ET-1] was partly sensitive to SK&F 96365.
7. These results indicate that in addition to VOC, ET-1 activates two types of Ca²⁺-permeable nonselective cation channel depending on its concentrations which differ in terms of sensitivity to SK&F 96365 and permeability to Mn²⁺.

     

Stim, ORAI and TRPC channels in the control of calcium entry signals in smooth muscle

Ca2+ entry signals are crucial in the control of smooth muscle contraction. Smooth muscle cells are unusual in containing plasma membrane (PM) Ca2+ entry channels that respond to voltage changes, receptor activation and Ca2+ store depletion. Activation of these channel subtypes is highly coordinated. The TRPC6 channel, widely expressed in most smooth muscle cell types, is largely non-selective to cations and is activated by diacylglycerol arising from receptor-induced phosholipase C activation. Receptor activation results largely in Na+ ion movement through TRPC6 channels, depolarization and subsequent activation of voltage-dependent L-type Ca2+ channels. The TRPC6 channels also appear to be activated by mechanical stretch, resulting again in depolarization and L-type Ca2+ channel activation. Such a coupling may be crucial in mediating the myogenic tone response in vascular smooth muscle. The emptying of stores mediated by inositol 1,4,5-trisphosphate receptors triggers the endoplasmic reticulum (ER) Ca2+ sensing protein stromal-interacting molecule (STIM) 1 to translocate into defined ER-PM junctional areas in which coupling occurs to Orai proteins, which serve as highly Ca2+-selective low-conductance Ca2+ entry channels. These ER-PM junctional domains may serve as crucial sites of interaction and integration between the function of store-operated, receptor-operated and voltage-operated Ca2+ channels. The STIM, Orai and TRPC channels represent highly promising new pharmacological targets through which such control may be induced.     

L-type calcium channel blockers enhance 5-HTP-induced antinociception in mice

INTRODUCTIONAlthough the numerous studies have shown theimportance of the peripheral and central serotonin (5-hydroxytryptamine, 5-HT) in modulating nociceptivetransmission, several modes of its actions requireclarification. 5-HT is generally described as prono-ciceptive substance in peripheral tissues, but as anti-nociceptive substance in the central nervous systems(CNS), including spinal and supraspinal levels. How-ever, there exist several lines of evidence which are inconflict wit…