A confocal study of mechanism of 5-hydroxytryptamino induced intracellular calcium dynamics in cultured rat stomach fundus smooth muscle cells with a new Ca~(2+) indicator STDIn-AM

A new fluorescent Ca2+ indicator STDIn-AM for detecting [Ca2+]i transients in cultured smooth muscle cells is presented. By making a comparison, the difference between STDIn and fluo-3 is discussed in detail. Using the new Ca2+ indicator, the mechanism of 5-hydroxytryptamino (5-HT) induced intracellular calcium dynamics in stomach fundus smooth muscle cells (SFSMC) of rats is investigated. It is shown that in contrast with fluo-3, STDIn is uniformly distributed in the cytosolic compartment but excluded from the nucleus, when it is transfected into cells. This feature makes it a real cytosol Ca2+ indicator and can reflect changes of cytosol [Ca2+] more accurately than that of fluo-3. In addition, STDIn responds to the [Ca2+]i transients more sensitive and faster than fluo-3. The results also show that, the L-type Ca2+channel inhibitor Mn9202 and the PLC inhibitor Compound 48/80 can significantly inhibit the [Ca2+]i elevation induced by 5-HT, while the PKC inhibitor D-Sphingosine can enhance the effect of     

A confocal study of mechanism of 5-hydroxytryptamino induced intracellular calcium dynamics in cultured rat stomach fundus smooth muscle cells with a new Ca2+ indicator STDln-AM

A new fluorescent Ca²⁺ indicator STDln-AM for detecting [Ca²⁺], transients in cultured smooth muscle cells is presented. By making a comparison, the difference between STDln and fluo-3 is discussed in detail. Using the new Ca²⁺ indicator, the mechanism of 5-hydroxytryptamino (5-HT) induced intracellular calcium dynamics in stomach fundus smooth muscle cells (SFSMC) of rats is investigated. It is shown that in contrast with fluo-3, STDln is uniformly distributed in the cytosolic compartment but excluded from the nucleus, when it is transfected into cells. This feature makes it a real cytosol Ca²⁺ indicator and can reflect changes of cytosol [Ca²⁺] more accurately than that of fluo-3. In addition, STDln responds to the [Ca²⁺], transients more sensitive and faster than fluo-3. The results also show that, the L-type Ca²⁺ channel inhibitor Mn9202 and the PLC inhibitor Compound 48/80 can significantly inhibit the [Ca²⁺], elevation induced by 5-HT, while the PKC inhibitor D-Sphingosine can enhance the effect of 5-HT. The results suggest that 5-HT acts by the way of 5-HT₂ receptors on SFSMC, then through 5-HT₂ receptors coupled IP₃/Ca²⁺ and GC/PKC double signal transduction pathways to make Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx possibly through L-type calcium channels.     

A confocal study of mechanism of 5-hydroxytryptamino induced intracellular calcium dynamics in cultured rat stomach fundus smooth muscle cells with a new Ca2+ indicator STDln-AM

A new fluorescent Ca²⁺ indicator STDln-AM for detecting [Ca²⁺], transients in cultured smooth muscle cells is presented. By making a comparison, the difference between STDln and fluo-3 is discussed in detail. Using the new Ca²⁺ indicator, the mechanism of 5-hydroxytryptamino (5-HT) induced intracellular calcium dynamics in stomach fundus smooth muscle cells (SFSMC) of rats is investigated. It is shown that in contrast with fluo-3, STDln is uniformly distributed in the cytosolic compartment but excluded from the nucleus, when it is transfected into cells. This feature makes it a real cytosol Ca²⁺ indicator and can reflect changes of cytosol [Ca²⁺] more accurately than that of fluo-3. In addition, STDln responds to the [Ca²⁺], transients more sensitive and faster than fluo-3. The results also show that, the L-type Ca²⁺ channel inhibitor Mn9202 and the PLC inhibitor Compound 48/80 can significantly inhibit the [Ca²⁺], elevation induced by 5-HT, while the PKC inhibitor D-Sphingosine can enhance the effect of 5-HT. The results suggest that 5-HT acts by the way of 5-HT₂ receptors on SFSMC, then through 5-HT₂ receptors coupled IP₃/Ca²⁺ and GC/PKC double signal transduction pathways to make Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx possibly through L-type calcium channels.     

The influence of dihydropyridines calcium antagonists on 5-HT-induced intracellular calcium signal

Confocal laser scanning microscopy (CLSM) was applied to detect the intracellular [Ca2 ] variety of fluorescent intension, with Fluo-3/AM fluorescence loaded in SFSMC. The results show that 10 μmol/L Lacidipine can reduce the frequence which 10 μmol/L 5-HT induced [Ca2 ] spark in SFSMC of calcium over loading to 50%, and amplitude to 50% or so. We can draw a conclusion that dihydropyridines cal-cium antagonists lacidipine can antagonize the release of intracellular [Ca2 ] which 5-HT-induced in dose dependent manner.     

Vascular dilatory action of the Chinese crude drug. II. Effects of scoparone on calcium mobilization

Further experiments were conducted to examine the effect of scoparone (6,7-dimethoxycoumarin), a coumarin derivative found in the Chinese crude drug "Capillaris Flos," on calcium mobilization. Scoparone does not affect Ca2+ influx through the voltage-dependent channel due to membrane depolarization. Its inhibitory action may be dependent not only on the inhibition of Ca2+ release from sarcoplasmic reticulum but also on the inhibition of extracellular Ca2+ influx through the receptor-operated channel.     

Effect of Pinellia ternata Lectin on Membrane Currents of Mouse Motor Nerve Terminals

Pinellia ternata lectin (PTL) extracted from the fresh juice of rhizome of pinellia ternata used as a traditional Chinese medicine facilitated the quantal release of acetylcholine (ACh) in the mouse motor nerve terminals and formed cation channels in artificial lipid bilayer. Here we report the action of PTL on presynaptic membrane currents of motor nerve terminals.The experiments were performed on the intercostal nerve triangularis sterni muscle preparations. By means of the perineurial recording, the effects of PTL on the sodium current in the preterminal part , three potassium currents and two calcium currents generated from the nerve terminals were investigated. The results show that PTL increases voltage-dependent fast Ca2+ current (ICa,f), Na+ current (INa) and Ca2+-acti-vated K+ current (IK,Ca) without action on either the voltage-dependent fast K+ current (IK,f) or the slow K+ current (IK,S). These effects are irreversible, but can be reversed by mannan, the specific binding sugar for PTL.The to     

二氢吡啶类新衍生物的合成

二氢吡啶类化合物是一类重要的钙通道阻滞剂 ,广泛用于治疗心绞痛 ,室上心律失常 ,高血压和外周血管性疾病[1] 。近年来 ,对二氢吡啶新衍生物的研究中 ,在提高其药效的前提下 ,更注重开发其长效性 ,提高生物利用度以及功效扩增 ,如兼备抗血栓[2 ] ,抗心律失常等作用。本着这样的目的 ,设计合成了系列二氢吡啶新衍生物。以硝苯吡啶作阳性对照 ,对化合物 ( 1 )～ ( 5 ) (表 1 ) ,应用跨膜流动技术[3 ] ,研究其对细胞膜Ｃａ2 + 通道的作用 ,结果表明 5个化合物对电压依赖性钙离子通道均有显著阻滞作用 (表 2 ) ,其它药理考查尚在进行中。化合物…     

Research Note: Ultraviolet Irradiation (UVB) Interrupts Calcium Cell Signaling in Lens Epithelial Cells

A preliminary study was undertaken to establish whether low-dose UV irradiation (UVB) affects calcium cell signaling in rabbit lens epithelia. In a suspension of lens epithelial cells (line NN1003A), changes in intracellular Ca2+ were measured by Fura-2 fluorescence in response to exogenously added ATP. The cellular response to ATP, referred to as the calcium signal, is characterized by a brief increase and subsequent decrease in cytosolic Ca2+ levels. Ultraviolet B irradiation (1.8-9 mJ/cm2) was found to reduce the magnitude of the Ca2+ signal in a dose-dependent manner. A 5 min UVB exposure (9 mJ/cm2) completely altered the biphasic nature of the calcium signal, causing only an immediate and steady rise in cytosol Ca2+ levels. Lower fluences of UVB irradiation (2 min exposure times or 3.6 mJ/cm2) induced a 50% reduction in the calcium signal. When irradiated cells were returned to culture for 3 h after irradiation, calcium signals induced by ATP were normal. In view of the photooxidative nature of UVB irradiation, the oxidative state of cells was assessed by measuring glutathione (GSH) levels. Ultraviolet B irradiation caused a rapid 20% decline in GSH levels that returned to near-control values after a 3 h postirradiation incubation. The results of this study indicate that fluences lower than previously found to be cataractogenic can perturb calcium cell signaling in cultured lens epithelial cells.     

Quantitative study on La~(3+) influx mediated by sodium-calcium exchanger in human lymphocytes

Whether La3+ can enter human peripheral blood lymphocytes by the Na+/Ca2+ exchanger or not and the effect of La3+on the Na+/Ca2+ exchanger activity are examined by fura-2 technique. And that whether La3+ is sequestered by intracellular organelles (mainly endoplasmic reticulum and mitochondria) is studied by this method. La3+uptake is obviously stimulated by pre-treating the cells with ouabain and by removing extracellular Na+, and intracellular La3+concentration ([La3+]i) is directly proportional to its extracellular concentration ([La3+]o). But when [La3+]o exceeds 0.4 mmol/L, the 340/380 nm ratio of fluorescence is no longer varied and the maximum [La3+], is 1.5×10-12 mol · L-1. The higher concentration of La3+ (0.1 mmol/L) increases Na+/Ca2+ exchange-mediated calcium influx, but lower concentration (10 μmol/L) appears to block calcium influx. The results also suggest that cytosolic La3+ is transported by the ATP-dependent Ca2+ pump. Intracellular Ca2+ stores are depleted by ionomycin, and then ion     

Na^+／Ca^2+交换介导的Nd^3+跨淋巴细胞膜的行为研究

利用Fura-2荧光浓度指示剂法对Na^+／Ca^2+交换介导的Nd^3+跨入外赂血淋巴 细胞膜行为进行了一系列研究。结果表明：当细胞形成向外的Na^+梯度时Nd^3+能 跨膜进入细胞,电压依赖性L-型Ca^2+通道对Nd^3+进入无贡献,提出了Na^+／ Ca^2+交换系统是Nd^3+进入细胞的主要途径;在安全浓度范围内进入胞内的游 Nd^3+浓度成正比,计算表明进入胞内的最大游离Nd^3+浓度为（3.67±0.32）× 10^-14mol·L^-1;当胞外pH值降低时进入胞内的游离Nd^3+浓度减小,胞内游离 Ca^2+浓度减小时进入的游离Nd^3+浓度略微增大,胞外Nd^3+和Ca^2+竞争Na^+／ Ca^2+交换位点;结果进一步推测进入胞内的Nd^3+可被质膜钙泵泵出胞外,初步实 验表明进入胞浆中的Nd^3+会在内质网中进一步累积,而在线粒体中不累积。     

Na+-Ca2+ exchanger modulates Ca2+ content in intracellular Ca2+ stores in rat osteoblasts

Na+-Ca2+ exchanger (NCX) transports Ca2+ coupled with Na+ across the plasma membrane in a bi-directional mode. Ca2+ flux via NCX mediates osteogenic processes, such as formation of extracellular matrix proteins and bone nodules. However, it is not clearly understood how the NCX regulates cellular Ca2+ movements in osteogenic processes. In this study, the role of NCX in modulating Ca2+ content of intracellular stores ([Ca2+]ER) was investigated by measuring intracellular Ca2+ activity in isolated rat osteoblasts. Removal of extracellular Na+ elicited a transient increase of intracellular Ca2+ concentration ([Ca2+]i). Pretreatment of antisense oligodeoxynucleotide (AS) against NCX depressed this transient Ca2+ rise and raised the basal level of [Ca2+]i. In AS-pretreated cells, the expression and activity of alkaline phosphatase (ALP), an osteogenic marker, were decreased. However, the cell viability was not affected by AS-pretreatment. Suppression of NCX activity by the AS-pretreatment decreased ATP-activated Ca2+ release from intracellular stores and significantly enhanced Ca2+ influx via store operated calcium influx (SOCI), compared to those of S-pretreated or control cells. These results strongly suggest that NCX has a regulatory role in cellular Ca2+ pathways in osteoblasts by modulating intracellular Ca2+ content.     

ATP released from beta-amyloid-stimulated microglia induces reactive oxygen species production in an autocrine fashion

Present study demonstrated that fibrillar beta-amyloid peptide (fAbeta1-42) induced ATP release, which in turn activated NADPH oxidase via the P2X7 receptor (P2X7R). Reactive oxygen species (ROS) production in fAbeta1-42- treated microglia appeared to require Ca2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca2+. Considering previous observation of superoxide generation by Ca2+ influx through P2X7R in microglia, we hypothesized that ROS production in fAbeta-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAbeta1-42-induced Ca2+ influx was mediated through P2X7R activation. In serial experiments, we found that microglial pretreatment with the P2X7R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'- disulfonate (100 microM) or oxidized ATP (100 microM) inhibited fAbeta-induced Ca2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAbeta1-42- stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAbeta-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.     

The effect of staurosporine on Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells

We examined the effect of staurosporine on cytosolic calcium response in rat basophilic leukemia (RBL-2H3) cells using fura-2 as a fluorescent indicator of calcium ion. Staurosporine at a dose of 30 nM inhibited antigen-stimulated Ca2(+)-influx into the cells from the extracellular environment. In contrast, the drug at this concentration inhibited neither the mobilization of Ca2+ from intracellular stores nor inositol 1,4,5-trisphosphate (IP3) formation. At a high concentration (300 nM), however, staurosporine completely inhibited the cytosolic calcium responses as well as IP3 formation. These results indicate that staurosporine, if used at an appropriate concentration, can be used to discriminate Ca2(+)-influx from extracellular environment from mobilization of the ion from intracellular stores. These results also suggest that protein kinases, possibly protein kinase C, are involved in the calcium influx of RBL-2H3 cells from the extracellular environment. Serotonin release was strongly inhibited by the drug at 30 nM staurosporine. Since the inhibition of serotonin release and suppression of cytosolic calcium increase in response to the antigen were in parallel, we concluded that the inhibition of serotonin release from RBL-2H3 cells caused by the drug was elicited by the suppression of Ca2(+)-influx into the cells.     

Co-activation of Gi and Gq proteins exerts synergistic effect on human platelet aggregation through activation of phospholipase C and Ca2+ signalling pathways

Our previous studies have shown that subthreshold concentrations of two platelet agonists exert synergistic effects on platelet aggregation. Here we studied the mechanism of synergistic interaction of 5-hydroxytryptamine (5-HT) and epinephrine mediated platelet aggregation. We show that 5-HT had no or little effect on aggregation but it did potentiate the aggregation response of epinephrine. The synergistic interaction of 5-HT (1-5 microM) and epinephrine (0.5-2 microM) was inhibited by alpha2-adrenoceptor blocker (yohimbine; IC50= 0.4 microM), calcium channel blockers (verapamil and diltiazem with IC50 of 10 and 48 mM, respectively), PLC inhibitor (U73122; IC50=6 microM) and nitric oxide (NO) donor, SNAP (IC50=1.6 microM)). The data suggest that synergistic effects of platelet agonists are receptor-mediated and occur through multiple signalling pathways including the activation PLC/Ca2+ signalling cascades.     

Effects of low power laser irradiation on intracellular calcium and histamine release in RBL-2H3 mast cells

Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca2+]i) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o-phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca2+]i from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for the biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing.     

Lead interferes with calcium entry through membrane pores

Calcium is an important intracellular messenger in all cells, represented here by nerve cells and osteoblast-like (OBL) cells. In neurons the intracellular calcium signal is related, e.g., to bioelectric phenomena. In OBL cells the intracellular calcium concentration ([Ca²⁺]_i) plays a role in the intercellular communication via gap junction channels. [Ca²⁺]_i might be affected by lead (Pb²⁺). In the nervous system even low Pb²⁺ concentrations impair learning and memory functions. Considering long-term potentiation (LTP) as a model for learning and memory it has been proven that the generation and maintenance of LTP is reduced by Pb²⁺ (1–10 μM). As the induction of LTP depends on a rise of [Ca²⁺]_i, we examined the effects of Pb²⁺ on [Ca²⁺]_i and on currents through calcium permeable membrane pores in dorsal-root ganglion (DRG) neurons, using calcium measurements (Fura-2/ AM) and whole cell patch clamp techniques. To study the effects of Pb²⁺ on intercellular communication via gap junctions we used rat OBL cells investigating interactions of Pb²⁺ with electric cell coupling. Furthermore, we examined calcium release activated channel currents (CRACCs) of these cells. Lead (1–10 μM) reduced the stimulated increase of [Ca²⁺]_i in a concentration dependent manner, by reducing both voltage-activated calcium channels (VACCs) and N-methyl-D-aspartate activated calcium channels (NACCs) in neurons. Voltage-activated calcium channel currents (VACCCs) were reduced by Pb²⁺ with an IC₅₀ of 0.46 μM. The effect was quite specific as voltage activated sodium and potassium channel currents were not significantly altered in the same concentration and voltage range. Furthermore, this effect was not voltage dependent and only partly reversible. A 100-fold higher concentration of Pb²⁺ (IC₅₀ of 46 μM) was found for the reduction of NACC currents. A small portion of this effect was not reversible. Other agonist activated channel currents (kainate and quisqualate) are not affected. In OBL cells, the calcium entry through calcium release activated channels (CRACs) was reduced in a concentration dependent manner by extracellular Pb²⁺, the concentrations were between 2 and 20 μM. Surprisingly the electric coupling through gap junction channels in OBL cells was not reduced by either extracellular or intracellular Pb²⁺ (5–25 μM). Received: 1 August 1997 / Revised: 24 October 1997 / Accepted: 30 October 1997     

Molecular identification of Ca2+ channels in human sperm

The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+) channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+) channels has been limited. Here we identified Ca(2+) channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non- L-type, and T-type Ca(2+) channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H sequences. RT-PCR using specific primers repeatedly detected alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H mRNAs, and additionally alpha1I mRNA. But alpha1A and alpha1D messages were not detected. Relative expression levels of the detected Ca(2+) channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H alpha1G alpha1E alpha1B alpha1C alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+) channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.     

Development of intracellular calcium measurement by time-resolved photon-counting fluorescence

Calcium green I, a ratiometric probe based on fluorescence lifetime measurements, was used to monitor intracellular calcium activity ([Ca2+]i) in RINm5F cells using a time-resolved fluorescence confocal microscope. The probe affinity constant has been recalibrated in single cells using ionomycin as a calcium ionophore and ethylenebis(oxyethylenenitrilo)tetraacetic acid as a calcium buffer; Kd was found to equal 150 nmol/L. The kinetics of ionomycin equilibration showed that the calcium release from calcium stores occurs before equilibration with extracellular calcium. The response to the muscarinic agonist carbachol, measured on 17 cells receiving three consecutive applications was characterized both by a [Ca2+]i peak lasting 50 s without any trailing plateau and by desensitization with a 30% decrease in the response. The dose-dependent response was obtained for a carbachol concentration from 5 mumol/L to 0.5 mmol/L. The ability of our set-up to obtain a value every 10 ms enabled us to record asynchronous spikes of [Ca2+]i in the RINm5F cells. The spikes, lasting less than 1 s, are significantly bigger than the noise, and they are not observed in the colonic HT29 cells.     

Design, synthesis, and structure-activity relationships of 3,4-dihydropyridopyrimidin-2(1H)-one derivatives as a novel class of sodium/calcium exchanger inhibitor

Design, synthesis, and structure-activity relationships for 3,4-dihydropyridopyrimidin-2(1H)-one derivatives, which are aza-3,4-dihydro-2(1H)-quinazolinone derivatives, as the sodium/calcium (Na+/Ca2+) exchanger inhibitors are discussed. These studies based on 3,4-dihydro-2(1H)-quinazolinone derivatives led to the discovery of a structurally novel and potent Na+/Ca2+ exchanger inhibitor, 3,4-dihydropyridopyrimidin-2(1H)-one derivative (26), with an IC30 value of 0.02 microM. Compound 26 directly inhibited the Na+-dependent Ca2+ influx via the Na+/Ca2+ exchanger after Na+-free treatment in cardiomyocytes.     

Calcium-induced phospholipid ordering depends on surface pressure

The effect of sodium and calcium ions on zwitterionic and anionic phospholipids monolayers is investigated using vibrational sum-frequency generation in conjunction with surface pressure measurements and fluorescence microscopy. Sodium ions only subtly affect the monolayer structure, while the effect of calcium is large and depends strongly on the surface pressure. At low surface pressures (approximately 5 mN/m), the presence on Ca2+ results in the unexpected appearance of ordered domains. For pressures between approximately 5 and approximately 25 mN/m, Ca2+ ions induce disorder in the monolayer. For pressures exceeding 25 mN/m, calcium cations expand the monolayer, while simultaneously ordering the lipid chains. Interestingly, effects are similar for both zwitterionic lipids and negatively charged lipids. In both vibrational sum-frequency generation and surface tension measurements, the molecular signature of the association of Ca2+ with the lipids is evident from Ca2+-induced changes in the signals corresponding to area changes of 4 A2/lipid-precisely the surface area of a Ca2+ ion, with evidence for a change in lipid Ca2+ complexation at high pressures.