Mechanism of intracellular calcium transients.

Calcium ions mediate extracellular signals on intracellular processes. The signalling system based on transient rises or oscillations of the cytoplasmic calcium concentration has potential advantages. The relevant mechanisms of intracellular concentration changes include calcium-induced calcium release and calcium dependent inactivation of calcium release. A model has been devised based on these processes to generate repetitive transients of the cytoplasmic calcium concentration.     

Morphine and anandamide stimulate intracellular calcium transients in human arterial endothelial cells: coupling to nitric oxide release.

Both morphine and anandamide significantly stimulated cultured endothelial intracellular calcium level increases in a concentration-dependent manner in cells pre-loaded with fura 2/AM. Morphine is more potent than anandamide (approximately 275 vs. 135 nM [Ca]i), and the [Ca]i for both ligands was blocked by prior exposure of the cells to their respective receptor antagonist, i.e., naloxone and SR 171416A. Various opioid peptides did not exhibit this ability, indicating a morphine-mu3-mediated process. In comparing the sequence of events concerning morphine's and anandamide's action in stimulating both [Ca]i and nitric oxide production in endothelial cells, we found that the first event precedes the second by 40+/-8 sec. The opiate and cannabinoid stimulation of [Ca]i was attenuated in cells leeched of calcium, strongly suggesting that intracellular calcium levels regulate cNOS activity.     

Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells

Tunicamycin is anucleoside antibiotic that inhibits protein glycosylation andpalmitoylation. The therapeutic use of tunicamycin is limited inanimals because of its toxic effects, particularly in cerebralvasculature. Tunicamycin decreases palmitoylation of the endothelialisoform of nitric oxide synthase, stimulates nitric oxide synthesis,and increases the concentration of intracellular calcium([Ca²⁺]_i)in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton.FASEB J. 11: A110, 1997). In the present study,we investigated the mechanism by which tunicamycin alters[Ca²⁺]_iusing the Ca²⁺-sensitive dye fura2. We found that tunicamycin increased[Ca²⁺]_iwithout increasing levels of inositol phosphates. When cells wereincubated in the absence of extracellularCa²⁺,[Ca²⁺]_irapidly rose in response to tunicamycin, although a full response wasnot achieved. The pool of intracellularCa²⁺ mobilized by tunicamycinoverlapped with that mobilized by thapsigargin. Extracellular nickelblocked a full response to tunicamycin when cells were incubated in thepresence of extracellular Ca²⁺.The effects of tunicamycin on[Ca²⁺]_iwere partially reversed by washing out the drug, and the remainder ofthe response was inhibited by removing extracellularCa²⁺. These results indicate thattunicamycin mobilizes Ca²⁺ fromintracellular stores in a manner independent of phospholipase Cactivation and increases the influx ofCa²⁺ across the plasma membrane.

     

Ion channels and regulation of intracellular calcium in vascular endothelial cells

Endothelial cells in vivo form an interface between flowing blood and vascular tissue, responding to humoral and physical stimuli to secrete relaxing and contracting factors that contribute to vascular homeostasis and tone. The activation of endothelial cell-surface receptors by vasoactive agents is coupled to an elevation in cytosolic Ca2+, which is caused by Ca2+ entry via ion channels in the plasma membrane and by Ca2+ release from intracellular stores. Ca2+ entry may occur via four different mechanisms: 1) a receptor-mediated channel coupled to second messengers; 2) a Ca2+ leak channel dependent on the electrochemical gradient for Ca2+; 3) a stretch-activated nonselective cation channel; and 4) internal Na+-dependent Ca2+ entry (Na+-Ca2+ exchange). The rate of Ca2+ entry through these ion pathways can be modulated by the resting membrane potential. Membrane potential may be regulated by at least two types of K channels: inwardly rectifying K channels activated upon hyperpolarization or shear stress; and a Ca2+-activated K channel activated upon depolarization, which may function to repolarize the agonist-stimulated endothelial cell. After agonist stimulation, cytosolic Ca2+ increases in a biphasic manner, with an initial peak due to inositol 1,4,5-trisphosphate-mediated Ca2+ release from intracellular stores, followed by a sustained plateau that is dependent on the presence of [Ca2+]o and on membrane potential. The delay in agonist-activated Ca2+ influx is consistent with the coupling of receptor activation to Ca2+ entry via a second messenger. Oscillations in [Ca2+]i, which may involve both Ca2+ entry and release, have been observed in isolated and confluent endothelial cell monolayers stimulated by histamine and bradykinin. Receptor-mediated Ca2+ entry, release, and refilling of intracellular stores follows a cycle that involves the plasma membrane.     

Laser exposure of gold nanorods can induce intracellular calcium transients

Uncoated and poly(styrene sulphonate) (PSS)‐coated gold nanorods were taken up by NG108‐15 neuronal cells. Exposure to 780 nm laser light at the plasmon resonance wavelength of the gold nanorods was found to induce intracellular Ca²⁺ transients. The higher Ca²⁺ peaks were observed at lower laser doses, with the highest levels obtained at a radiant exposure of 0.33 J/cm². In contrast, the cells without nanoparticles showed a consistently small response, independent of the laser dose. These initial results open up new opportunities for peripheral nerve regeneration treatments and for more efficient optical stimulation techniques. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Regulation of stretch-activated intracellular calcium transients by actin filaments.

Stretch activation of cation-permeable channels may be an important proximal sensory mechanism in mechanotransduction. As actin filaments may mediate cellular responses to changes of the mechanical properties of the substrate and regulate stretch-induced calcium transients, we examined the role of actin filaments and substrate flexibility in modulating the amplitude of stretch-activated intracellular calcium transients. Human gingival fibroblasts were subjected to mechanical stretch through integrins by magnetic force acting on collagen-coated ferric oxide beads. Intracellular calcium concentration was measured in fura-2-loaded cells by ratio fluorimetry. Cytochalasin D-treatment greatly increased (3-fold) the amplitude of stretch-activated calcium transients in well-spread cells grown on glass coverslips while phalloidin, colchicine or taxol exerted no signficant effects, indicating that actin filaments but not microtubules modulate stretch-activated calcium transients. In freshly plated cells with rounded shapes and poorly developed cortical actin filaments, stretch-induced calcium transients were of 3-fold higher amplitude than well-spread cells plated for 6-24 hrs and with well developed actin filaments. Cells plated on soft collagen-polyacrylamide gels showed round morphology but exhibited <50% of the response to stretch of well-spread cells on inflexible gels. Notably, cells on soft gels showed very heavy phalloidin staining for cortical actin filaments compared with cells on more inflexible surfaces which showed only light staining for cortical actin. While cell shape may have some effect on responsiveness to mechanical stretch, the rigidity of the cell membrane mediated by the extensive cortical actin network appears to be a central determinant in the regulation of stretch-induced calcium signals.     

Testosterone inhibits bradykinin-induced intracellular calcium kinetics in rat aortic endothelial cells in culture

Sex steroids have been associated with cardiovascular diseases and the modification of the risk of coronary artery disease (CAD). We cultured aortic endothelial cells from young adult male rats and loaded them with Fura 2 in order to evaluate the direct effects of testosterone on endothelial cells and the probable regulation of bradykinin-induced effects on intracellular calcium ([Ca(2+)](i)) kinetics, effects that are mediated through an increase in intracellular [IP(3)], which in turn stimulates the rapid release of Ca(2+) from ER stores. Our results show that testosterone had no direct effects on [Ca(2+)](i) kinetics, but did block bradykinin-induced increases in intracellular calcium concentration in endothelial cells. This effect was concentration-dependent; the steroid was applied only 30 s before bradykinin application and thus, the effect can be considered nongenomic in origin. Membrane localization of a putative androgen receptor in endothelial cells could be responsible for this effect. In summary, testosterone can modulate the effects induced by activation of membrane-bound bradykinin receptors.     

Mechanisms through which PDGF alters intracellular calcium levels in U-1242 MG human glioma cells

PDGF-BB induces a rapid, sustained increase in intracellular calcium levels in U-1242 MG cells. We used several calcium channel blockers to identify the types of channels involved. L channel blockers (verapamil, nimodipine, nicardipine, nitrendipine and taicatoxin) had no effect on PDGF-BB induced alterations in intracellular calcium. Blockers of P, Q and N channels (ω-agatoxin-IVA, ω-conotoxin MVIIC and ω-conotoxin GVIA) also had no effect. This indicates that these channels play an insignificant role in supplying the Ca²⁺ necessary for PDGF stimulated events in U-1242 MG cells. However, a T channel blocker (NDGA) and the non-specific (NS) calcium channel blockers (FFA and SK&F 9365) abolished PDGF-induced increases in intracellular calcium. This indicates that PDGF causes calcium influx through both non-specific cationic channels and T channels. To study the participation of intracellular calcium stores in this process, we used thapsigargin, caffeine and ryanodine, all of which cause depletion of intracellular calcium stores. The PDGF effect was abolished using both thapsigargin and caffeine but not ryanodine. Collectively, these data indicate that in these human glioma cells PDGF-BB induces release of intracellular calcium from caffeine- and thapsigargin-sensitive calcium stores which in turn lead to further calcium influx through both NS and T channels.     

Aggregating platelets increase intracellular calcium in endothelial cells through release of adenine nucleotides

Aggregating platelets relax isolated coronary arteries through the release of endothelium-derived relaxing factor (EDRF). Since release of EDRF may be calcium dependent, we tested if and how aggregating platelets stimulated a calcium response in cultured endothelial cells. Aggregating platelets caused a transient increase in intracellular calcium in endothelial cells loaded with the fluorescent calcium indicator fura-2. The adenine nucleotides ADP and ATP, but not other platelet-derived mediators, mimicked the platelet-induced calcium response, and inhibition of adenine nucleotides impaired the response to aggregating platelets. Thus, aggregating platelets release adenine nucleotides and stimulate a rise in intracellular calcium in cultured endothelial cells. This calcium response may represent the intracellular transduction mechanism by which aggregating platelets induce endothelial release of EDRF and subsequent relaxation of coronary arteries.     

白介素-2对心肌细胞[Ca~(2 )]_i的作用及其信号转导途径

为研究白介素 2 (interleukin 2 ,IL 2 )对心肌细胞内钙浓度 ([Ca2 ]i)的影响及其信号转导途径 ,实验采用酶解法分离成年大鼠心室肌细胞 ,以Fura 2 /AM为钙探针 ,用细胞内双波长钙荧光系统检测细胞 [Ca2 ]i 的变化。结果发现 :(1)IL 2 (0 5～ 2 0 0U/ml)浓度依赖性地降低单个心室肌细胞内钙瞬态 ,IL 2 (2 0 0U/ml)对咖啡因诱导的肌浆网内储钙的释放无影响 ;(2 )纳洛酮 (naloxone ,Nal) (10 -8mol/L)和nor binaltorphimine (nor BNI,10 -8mol/L)可阻断IL 2对心肌细胞钙瞬态的作用 ,而纳曲吲哚 (naltrindole ,NTI) (10 -6mol/L)不能阻断此作用 ;(3)κ阿片受体激动剂U5 0 488H (10 -6mol/L)降低心肌细胞钙瞬态 ,nor BNI (10 -8mol/L)可阻断此作用 ;(4 ) 5mg/L百日咳毒素 (PTX)预处理可取消IL 2降低心肌细胞钙瞬态的作用 ,而酪氨酸激酶抑制剂genistein (10 -4 mol/L)不能取消IL 2的作用 ;(5 )U7312 2预处理可阻断IL 2的作用。研究结果表明 ,IL 2降低心肌细胞钙瞬态的作用 ,是通过心肌细胞上κ阿片受体介导的 ,其下游途径包括PTX敏感的G蛋白和磷脂酶C。     

白介素—2对心肌细胞[Ca^2＋]i的作用及其信号转导途径

为研究白介素－2（interleukin-2,IL-2）对心肌细胞内钙浓度（[Ca^2 ]i）的影响及其信号转导途径,实验采用酶解法分离成年大鼠心室肌细胞,以Fura-2/AM为钙探针,用细胞内双波长钙荧光系统检测细胞[Ca^2 ]i的变化。结果发现：（1）IL－2（0.5-200U/ml）浓度依赖性地降低单个心室肌细胞内钙态,IL－2（200U／ml）对咖啡因诱导的肌浆网内储钙的释放无影响;（2）纳洛酮(naloxone,Nal)(10^-8mol/L）和nor-binaltorphimine(nor-BNI,10^-8mol/L）可阻断IL－2对心肌细胞钙瞬态的作用,而纳曲吲哚（naltrindole,NTI）（10^-6mol/L）不能阻断此作用;（3）κ阿片受体激动剂U50488H（10^-6mol/L）降低心肌细胞钙瞬态,nor-BNI(10^-8mol/L）可阻断此作用;（4）5mg/L百日咳毒素（PTX）预处理可取消IL－2降低心肌细胞钙瞬态的作用,而酪氨酸激酶抑制剂genistein(10^-4mol/L）不能取消IL－2的作用;（5）U73122预处理可阻断IL－2的作用。研究结果表明,IL－2降低心肌细胞钙瞬态的作用,是通过心肌细胞上κ阿片受体介导的,其下游途径包括PTX敏感的G蛋白和磷脂酶C。     

Effect of hypoxia upon intracellular calcium concentration of human endothelial cells.

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2. We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (beta-hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia-induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i. These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation.     

Release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells

Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.     

Scaffold topography alters intracellular calcium dynamics in cultured cardiomyocyte networks

Structural and functional changes ensue in cardiac cell networks when cells are guided by three-dimensional scaffold topography. We report enhanced synchronous pacemaking activity in association with slow diastolic rise in intracellular Ca2+ concentration ([Ca2+]i) in cell networks grown on microgrooved scaffolds. Topography-driven changes in cardiac electromechanics were characterized by the frequency dependence of [Ca2+]i in syncytial structures formed of ventricular myocytes cultured on microgrooved elastic scaffolds (G). Cells were electrically paced at 0.5-5 Hz, and [Ca2+]i was determined using microscale ratiometric (fura 2) fluorescence. Compared with flat (F) controls, the G networks exhibited elevated diastolic [Ca2+]i at higher frequencies, increased systolic [Ca2+]i across the entire frequency range, and steeper restitution of Ca2+ transient half-width (n = 15 and 7 for G and F, respectively, P < 0.02). Significant differences in the frequency response of force-related parameters were also found, e.g., overall larger total area under the Ca2+ transients and faster adaptation of relaxation time to pacing rate (P < 0.02). Altered [Ca2+]i dynamics were paralleled by higher occurrence of spontaneous Ca2+ release and increased sarcoplasmic reticulum load (P < 0.02), indirectly assessed by caffeine-triggered release. Electromechanical instabilities, i.e., Ca2+ and voltage alternans, were more often observed in G samples. Taken together, these findings 1) represent some of the first functional electromechanical data for this in vitro system and 2) demonstrate direct influence of the microstructure on cardiac function and susceptibility to arrhythmias via Ca(2+)-dependent mechanisms. Overall, our results substantiate the idea of guiding cellular phenotype by cellular microenvironment, e.g., scaffold design in the context of tissue engineering.     

Criticality in intracellular calcium signaling in cardiac myocytes

Calcium (Ca) is a ubiquitous second messenger that regulates many biological functions. The elementary events of local Ca signaling are Ca sparks, which occur randomly in time and space, and integrate to produce global signaling events such as intra- and intercellular Ca waves and whole-cell Ca oscillations. Despite extensive experimental characterization in many systems, the transition from local random to global synchronous events is still poorly understood. Here we show that criticality, a ubiquitous dynamical phenomenon in nature, is responsible for the transition from local to global Ca signaling. We demonstrate this first in a computational model of Ca signaling in a cardiac myocyte and then experimentally in mouse ventricular myocytes, complemented by a theoretical agent-based model to delineate the underlying dynamics. We show that the interaction between the Ca release units via Ca-induced Ca release causes self-organization of Ca spark clusters. When the coupling between Ca release units is weak, the cluster-size distribution is exponential. As the interactions become strong, the cluster-size distribution changes to a power-law distribution, which is characteristic of criticality in thermodynamic and complex nonlinear systems, and facilitates the formation and propagation of Ca waves and whole-cell Ca oscillations. Our findings illustrate how criticality is harnessed by a biological cell to regulate Ca signaling via self-organization of random subcellular events into cellular-scale oscillations, and provide a general theoretical framework for the transition from local Ca signaling to global Ca signaling in biological cells.     

Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes

Abstract

Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15?Hz, 50?Hz, 75?Hz and 100?Hz) and at a flux density of 2?mT. Intracellular calcium concentration ([Ca²⁺]_i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10?mM) was carried out to verify the function of the cardiac Na⁺/Ca²⁺ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca²⁺-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca²⁺]_i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.