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.     

Diabetes alters intracellular calcium transients in cardiac endothelial cells

Diabetic cardiomyopathy (DCM) is a diabetic complication, which results in myocardial dysfunction independent of other etiological factors. Abnormal intracellular calcium ([Ca(2+)](i)) homeostasis has been implicated in DCM and may precede clinical manifestation. Studies in cardiomyocytes have shown that diabetes results in impaired [Ca(2+)](i) homeostasis due to altered sarcoplasmic reticulum Ca(2+) ATPase (SERCA) and sodium-calcium exchanger (NCX) activity. Importantly, altered calcium homeostasis may also be involved in diabetes-associated endothelial dysfunction, including impaired endothelium-dependent relaxation and a diminished capacity to generate nitric oxide (NO), elevated cell adhesion molecules, and decreased angiogenic growth factors. However, the effect of diabetes on Ca(2+) regulatory mechanisms in cardiac endothelial cells (CECs) remains unknown. The objective of this study was to determine the effect of diabetes on [Ca(2+)](i) homeostasis in CECs in the rat model (streptozotocin-induced) of DCM. DCM-associated cardiac fibrosis was confirmed using picrosirius red staining of the myocardium. CECs isolated from the myocardium of diabetic and wild-type rats were loaded with Fura-2, and UTP-evoked [Ca(2+)](i) transients were compared under various combinations of SERCA, sarcoplasmic reticulum Ca(2+) ATPase (PMCA) and NCX inhibitors. Diabetes resulted in significant alterations in SERCA and NCX activities in CECs during [Ca(2+)](i) sequestration and efflux, respectively, while no difference in PMCA activity between diabetic and wild-type cells was observed. These results improve our understanding of how diabetes affects calcium regulation in CECs, and may contribute to the development of new therapies for DCM treatment.     

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。     

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.     

Imaging of calcium transients in skeletal muscle fibers.

Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures.     

Delay of intracellular calcium transients using a calcium chelator: application to high-throughput screening of the capsaicin receptor ion channel and G-protein-coupled receptors.

Whole-cell functional assays are often used for high-throughput screening (HTS) of molecular targets such as ion channels and G-protein-coupled receptors. A common method for assaying the activity of these membrane proteins is to measure the change in intracellular calcium concentration upon receptor stimulation. These changes in calcium concentration are typically transient and therefore not readily adapted to high-density plate formats used in HTS instruments. We have demonstrated that an intracellular calcium chelator, BAPTA, was able to delay by 5- to 20-fold and extend for several minutes the observed calcium signals initiated by extracellular calcium influx or release of calcium from intracellular stores. As examples, we used cells expressing a calcium-permeable ion channel, vanilloid receptor type 1 (the capsaicin receptor), and two G-protein-coupled receptors. These receptor-mediated increases in intracellular calcium concentration were measured by both fluorescence-based and luminescence-based detection methods. The use of an intracellular calcium chelator to delay calcium signaling should have wide application since it allows the measurement of the functional activity of any cellular receptor that signals through calcium. With this procedure, calcium fluorescence and luminescence whole-cell functional assays may be performed with standard laboratory pipetting and detection systems.     

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.     

Modelling receptor-controlled intracellular calcium oscillators.

This paper presents mathematical models for the hepatocyte calcium oscillator which follow the concepts in a class of informal models developed to account for the striking dependence on the receptor type of several features of the calcium oscillations, in particular the shape and duration of the free calcium transients. The essence of these models is that the transients should be timed by a build-up of activated GTP-binding proteins, which, combined with positive feedback processes and perhaps with cooperative effects, leads to a sudden activation of phospholipase C (PLC), followed by negative feedback processes which switch off the calcium rise and lead to a fall in free calcium back to resting levels. These models predict pulsatile oscillations in inositol (1,4,5)P3 as well as in free calcium. We show that receptor-controlled intracellular calcium oscillators involving an unknown positive feedback pathway onto PLC and negative feedback from protein kinase C (PKC) onto G-proteins and receptors, or negative feedback by stimulation of GTPase activity can simulate many of the features of observed intracellular calcium oscillations. These oscillators exhibit a dependence of frequency on agonist concentration and a dependence of transient duration on receptor and G-protein type. We also show that a PLC-dependent GTPase activating factor (GAF) could provide explanations for some otherwise puzzling features of intracellular calcium oscillations.     

Repetitive calcium transients in hamster oocytes

Golden hamster oocytes show repetitive Ca2+ transients at fertilization: a propagating Ca2+ rise from the sperm attachment site in the first 2-3 responses and synchronous Ca2+ rise in the entire egg in the succeeding responses. Cyclic Ca2+ rises are produced in unfertilized eggs by an injection of GTP gamma S or continuous injection of inositol 1,4,5-trisphosphate (InsP3). Both InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR) are observed in hamster eggs, associated with a refractory period of 1-2 min after a Ca2+ release. In addition, external Ca2+ is a prerequisite for maintaining the repeated Ca2+ transients. The conditions that are expected to alter Ca2+ influx affect the frequency of Ca2+ transients with little effect on each response. The fertilizing sperm causes an increase in Ca2+ permeability of the egg plasma membrane and an increase in Ca2+ sensitivity of CICR. Feedback inhibition through protein kinase C is observed in G-protein-mediated Ca2+ transients but this inhibition seems to operate rather tonically. A model of Ca2+ oscillation is proposed: basically a second messenger-controlled oscillator model. InsP3 as the rigger of Ca2+ release is continuously supplied while an elevated basal [Ca2+]i level due to Ca2+ influx provides a favourable condition for IICR and CICR as well as for recharging the Ca2+ pools ready to release Ca2+ again.     

Polarity in intracellular calcium signaling.

The concentration of free calcium ions (Ca(2+)) in the cytosol is precisely regulated and can be rapidly increased in response to various types of stimuli. Since Ca(2+) can be used to control different processes in the same cell, the spatial organization of cytosolic Ca(2+) signals is of considerable importance. Polarized cells have advantages for Ca(2+) studies since localized signals can be related to particular organelles. The pancreatic acinar cell is well-characterized with a clearly polarized structure and function. Since the discovery of the intracellular Ca(2+)-releasing function of inositol 1,4,5-trisphosphate (IP(3)) in the pancreas in the early 1980s, this cell has become a popular study object and is now one of the best-characterized with regard to Ca(2+) signaling properties. Stimulation of pancreatic acinar cells with the neurotransmitter acetylcholine or the hormone cholecystokinin evokes Ca(2+) signals that are either local or global, depending on the agonist concentration and the length of the stimulation period. The nature of the Ca(2+) transport events across the basal and apical plasma membranes as well as the involvement of the endoplasmic reticulum (ER), the nucleus, the mitochondria, and the secretory granules in Ca(2+) signal generation and termination have become much clearer in recent years.     

Measurement of calcium transients and slow calcium current in myotubes

The purpose of this study was to characterize excitation-contraction (e- c) coupling in myotubes for comparison with e-c coupling of adult skeletal muscle. The whole cell configuration of the patch clamp technique was used in conjunction with the calcium indicator dye Fluo-3 to study the calcium transients and slow calcium currents elicited by voltage clamp pulses in cultured myotubes obtained from neonatal mice. Cells were held at -80 mV and stimulated with 15-20 ms test depolarizations preceded and followed by voltage steps designed to isolate the slow calcium current. The slow calcium current had a threshold for activation of about 0 mV; the peak amplitude of the current reached a maximum at 30 to 40 mV a and then declined for still stronger depolarizations. The calcium transient had a threshold of about -10 mV, and its amplitude increased as a sigmoidal function of test potential and did not decrease again even for test depolarizations sufficiently strong (> or = 50 mV) that the amplitude of the slow calcium current became very small. Thus, the slow calcium current in myotubes appears to have a negligible role in the process of depolarization-induced release of intracellular calcium and this process in myotubes is essentially like that in adult skeletal muscle. After repolarization, however, the decay of the calcium transient in myotubes was very slow (hundreds of ms) compared to adult muscle, particularly after strong depolarizations that triggered larger calcium transients. Moreover, when cells were repolarized after strong depolarizations, the transient typically continued to increase slowly for up to several tens of ms before the onset of decay. This continued increase after repolarization was abolished by the addition of 5 mM BAPTA to the patch pipette although the rapid depolarization-induced release was not, suggesting that the slow increase might be a regenerative response triggered by the depolarization-induced release of calcium. The addition of either 0.5 mM Cd2+ + 0.1 mM La3+ or the dihydropyridine (+)-PN 200-110 (1 microM) reduced the amplitude of the calcium transient by mechanisms that appeared to be unrelated to the block of current that these agents produce. In the majority of cells, the decay of the transient was accelerated by the addition of the heavy metals or the dihydropyridine, consistent with the idea that the removal system becomes saturated for large calcium releases and becomes more efficient when the size of the release is reduced.     

LY294002, but not wortmannin,increases intracellular calcium and inhibits calcium transients in bovine and human airway smooth muscle cells

To characterize the effect that a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002, has on cytosolic calcium concentrations ([Ca2+]i), bovine airway smooth muscle cells (BASMC) and cultured human bronchial smooth muscle cells (HBSMC) were loaded with fura 2-AM, imaged as single cells and [Ca2+]i measured ratiometrically. LY294002 (50 microM) increased [Ca2+]i by 294+/-76 nM (P<0.01, n=13) and 230+/-31 nM (P<0.001, n=10) in BASMC and HBSMC, respectively, and increases occurred in the absence of extracellular calcium. In contrast, after pre-treatment with thapsigargin, LY294002 no longer increased [Ca2+]i. This calcium mobilization by LY294002 was associated with a significant functional effect since LY294002 also inhibited calcium transients to carbachol (45+/-23 nM), caffeine (45+/-32 nM), and histamine (20+/-22 nM), with controls of 969+/-190, 946+/-156, and 490+/-28 nM, respectively. Wortmannin, a different PI3-kinase inhibitor, neither increased [Ca2+]i nor inhibited transients. Also, LY294002 increased [Ca2+]i in the presence of wortmannin, U-73122, and xestospongin C. We concluded that LY294002 increased [Ca2+]i, at least in part, by mobilizing intracellular calcium stores and inhibited calcium transients. The effects of LY294002 on [Ca2+]i were not dependent on wortmannin-sensitive PI3-kinases, phospholipase C, or inositol trisphosphate receptors (IP3R). For BASMC and HBSMC, LY294002 has effects on calcium regulation that could be important to recognize when studying PI3-kinases.     

Numerical methods to determine calcium release flux from calcium transients in muscle cells.

Several methods are currently in use to estimate the rate of depolarization-induced calcium release in muscle cells from measured calcium transients. One approach first characterizes calcium removal of the cell. This is done by determining parameters of a reaction scheme from a fit to the decay of elevated calcium after the depolarizing stimulus. In a second step, the release rate during depolarization is estimated based on the fitted model. Using simulated calcium transients with known underlying release rates, we tested the fidelity of this analysis in determining the time course of calcium release under different conditions. The analysis reproduced in a satisfactory way the characteristics of the input release rate, even when the assumption that release had ended before the start of the fitting interval was severely violated. Equally good reconstructions of the release rate time course could be obtained when the model used for the analysis differed in structure from the one used for simulating the data. We tested the application of a new strategy (multiple shooting) for fitting parameters in nonlinear differential equation systems. This procedure rendered the analysis less sensitive to ill-chosen initial guesses of the parameters and to noise. A locally adaptive kernel estimator for calculating numerical derivatives allowed good reconstructions of the original release rate time course from noisy calcium transients when other methods failed.     

Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators.

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.     

Characterizing the response of calcium signal transducers to generated calcium transients

Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological phenomena as diverse as muscle contraction, neurosecretion, and cell division. When Ca2+ is rapidly mixed with slow Ca2+ chelators, EGTA, or Mg2+/EDTA, artificial Ca2+ transients (ACTs) of varying duration (0.1-50 ms half-widths (hws)) and amplitude can be generated. We have exposed several Ca2+ indicators, Ca2+-binding proteins, and a Ca2+-dependent enzyme to ACTs of various durations and observed their transient binding of Ca2+, complex formation, and/or activation. A 0.1 ms hw ACT transiently occupied approximately 70% of the N-terminal regulatory sites of troponin C consistent with their rapid Ca2+ on-rate (8.7 +/- 2.0 x 10(7) M-1 s-1). A 1.1 ms hw ACT produced approximately 90% transient binding of the N-terminal of calmodulin (CaM) to the RS-20 peptide, but little binding of CaM's C-terminal to RS-20. A 0.6 ms hw ACT was sufficient for the N-terminal of CaM to transiently bind approximately 60% of myosin light chain kinase (MLCK), while a 1.8 ms hw ACT produced approximately 22% transient activation of the sarcoplasmic reticulum (SR) Ca2+/ATPase. In both cases, the ACT had fallen back to baseline approximately 10-30 ms before maximal binding of CaM to MLCK or SR Ca2+/ATPase activation occurred and binding and enzyme activation persisted long after the Ca transient had subsided. The use of ACTs has allowed us to visualize how the Ca2+-exchange rates of Ca2+-binding proteins dictate their Ca2+-induced conformational changes, Ca2+-induced protein/peptide and protein/protein interactions, and enzyme activation and inactivation, in response to Ca2+ transients of various amplitude and duration. By characterizing the response of these proteins to ACTs, we can predict with greater certainty how they would respond to natural Ca2+ transients to regulate cellular phenomena.     

Estradiol coupling to human monocyte nitric oxide release is dependent on intracellular calcium transients: evidence for an estrogen surface receptor.

We tested the hypothesis that estrogen acutely stimulates constitutive NO synthase (cNOS) activity in human peripheral monocytes by acting on an estrogen surface receptor. NO release was measured in real time with an amperometric probe. 17beta-estradiol exposure to monocytes stimulated NO release within seconds in a concentration-dependent manner, whereas 17alpha-estradiol had no effect. 17beta-estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17beta-estradiol and E2-BSA, whereas ICI 182,780, a selective inhibitor of the nuclear estrogen receptor, had no effect. We further showed, using a dual emission microfluorometry in a calcium-free medium, that the 17beta-estradiol-stimulated release of monocyte NO was dependent on the initial stimulation of intracellular calcium transients in a tamoxifen-sensitive process. Leeching out the intracellular calcium stores abolished the effect of 17beta-estradiol on NO release. RT-PCR analysis of RNA obtained from the cells revealed a strong estrogen receptor-alpha amplification signal and a weak beta signal. Taken together, a physiological dose of estrogen acutely stimulates NO release from human monocytes via the activation of an estrogen surface receptor that is coupled to increases in intracellular calcium.