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)     

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.     

Species dependence of mitochondrial calcium transients during excitation-contraction coupling in isolated cardiomyocytes.

Whether mitochondrial Ca(2+) transport is rapid enough to respond to changes in cytosolic [Ca(2+)] ([Ca(2+)](c)) which occur during excitation-contraction coupling in the heart is controversial; different results wereobtained with different techniques and different species. In this study mitochondrial [Ca(2+)] ([Ca(2+)](m)) was measured in indo-1/AM-loaded myocytes from rat and guinea-pig hearts where the cytosolic indo-1 had been removed by extended incubation of cells at 37 degrees C ("heat treatment"). The mitochondrial origin of the remaining fluorescence was confirmed by sensitivity of the indo-1 signal to ruthenium red. In resting rat myocytes, [Ca(2+)](m) was lower than [Ca(2+)](c), whereas in guinea-pig cells [Ca(2+)](m) was higher than [Ca(2+)](c). Upon electrical stimulation of cells, no change occurred in [Ca(2+)](m) in rat myocytes. However, in guinea-pig cells mitochondrial Ca(2+) transients were clearly visible with a mean indo-1 ratio amplitude of 0.153 +/- 0.2 (n = 20), compared with 0.306 +/- 0.02 (n = 25), p < 0.001, prior to heat treatment. These observations suggest significant differences in mitochondrial Ca(2+) transport in cardiomyocytes from different species.     

Developmental changes of calcium transients and contractility during the cultivation of rat neonatal cardiomyocytes

In the normal myocardium matrix metalloproteinases (MMP) are present in the latent form. To examine whether MMP are activated following infarction or idiopathic dilated cardiomyopathy (DCM), we extracted and measured MMP activity in tissue derived from 7 explanted, failing human hearts due to either previous myocardial infarction (MI) or DCM. MMP activity in infarcted left ventricle (LV), noninfarcted IV and right ventricle (RV) from MI patients, as well as tissue from either ventricle of DCM patients, were compared to the activity of donor heart tissue. SDS-PAGE and dye-binding assays were used to determine total protein concentration, while collagenase activity was measured by SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Accuracy of the zymographic technique was shown for tissue samples as small as 0.05 mg and was comparable to results obtained by a spectrophotometric method.. After normalization for total protein concentration, we found 3 ± 1 % collagenase activity in normal atrial tissue which could be activated to 80–90% by trypsin or plasmin, indicating that collagenase is normally inactive or in a latent form in human heart. In endo- and epimyocardium of infarcted LV on the other hand, collagenase activity was 85–95% and 10–20%, respectively, while 5–10% and 3–5%, respectively, in noninfarcted LV In DCM, collagenolytic activity in the endo and epimyocardium was 75 ± 5 and 35 ± 5% in the LV and 35 ± 7 and 20 ± 5% in the RV, respectively. Thus, in dilated failing human hearts secondary to previous MI or DCM, MMP activity is increased. This is particularly the case within the endomyocardium of the infarcted and noninfarcted portions of either ventricle with MI and in both ventricles in DCM. This suggests that an activation of collagenase throughout the myocardium may contribute to its remodeling that includes ventricular dilatation and wall thinning.This work was supported in part by NIH grant GM-48595 and by a Grant-In-Aid from the American Heart Association, Missouri Affiliate (92-10517).     

Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes

Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A₂ (PLA₂) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca²⁺ concentration([Ca²⁺]_i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca²⁺]_i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca²⁺]_i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca²⁺]_i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA₂.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA₂/AA pathway in mediating thecontractile effects of TNF-.

     

Three-dimensional simulation of calcium waves and contraction in cardiomyocytes using the finite element method

To investigate the characteristics and underlying mechanisms of Ca²⁺ wave propagation, we developed a three-dimensional (3-D) simulator of cardiac myocytes, in which the sarcolemma, myofibril, and Z-line structure with Ca²⁺ release sites were modeled as separate structures using the finite element method. Similarly to previous studies, we assumed that Ca²⁺ diffusion from one release site to another and Ca²⁺-induced Ca²⁺ release were the basic mechanisms, but use of the finite element method enabled us to simulate not only the wave propagation in 3-D space but also the active shortening of the myocytes. Therefore, in addition to the dependence of the Ca²⁺ wave propagation velocity on the sarcoplasmic reticulum Ca²⁺ content and affinity of troponin C for Ca²⁺, we were able to evaluate the influence of active shortening on the propagation velocity. Furthermore, if the initial Ca²⁺ release took place in the proximity of the nucleus, spiral Ca²⁺ waves evolved and spread in a complex manner, suggesting that this phenomenon has the potential for arrhythmogenicity. The present 3-D simulator, with its ability to study the interaction between Ca²⁺ waves and contraction, will serve as a useful tool for studying the mechanism of this complex phenomenon. cardiac muscle cell; excitation-contraction coupling; mechanoelectrical feedback; spiral wave; arrhythmia     

Calcium transients and calcium binding to troponin at the contraction threshold in skeletal muscle.

Antipyrylazo III calcium transients from voltage-clamped, cut skeletal muscle fibers of the frog were recorded, and the calcium binding to the regulatory sites of troponin C was calculated. The strength-duration curve for the contraction threshold was determined. It was found that the increase in myoplasmic calcium concentration necessary to produce the same level of contractile activation, i.e., the just visible movement, was approximately 60% higher at more positive membrane potentials resulting from short depolarizing pulses than at rheobase. However, using biochemical data for the kON and kOFF rate coefficients of the binding sites, the calculated maximums of the calcium binding curves were about the same at different voltages, and the time to maximum saturation was roughly equal to the latency of the contractions. To characterize the calcium binding in intact fibers more accurately, those values of the kON and kOFF rate coefficients that gave equal peak saturations during threshold movement at different membrane potentials were determined.     

Purified endogenous inhibitor of the Na/Ca exchanger can enhance the cardiomyocytes contractility and calcium transients

Previous studies have shown that the newly found endogenous inhibitor (NCX(IF)) of the cardiac Na/Ca exchanger (NCX1) is capable of regulating the muscle strip's contractility and relaxation. Here, the effects of purified NCX(IF) were tested on single cell shortening-lengthening (by using the IR CCD camera coupled with the two-edge video-detector) and [Ca]i-transients (by monitoring the changes in fluo-3 fluorescence). A perfusion of isolated cardiomyocytes (paced at 0.5-1.0 Hz) with NCX(IF) results in 4-6-fold enhancement in the amplitude of cell shortening-lengthening reaching the steady-state levels within 5-8 min (n=20, p<0.009). Simultaneous recordings of cell shortening-lengthening and [Ca]i-transients from the same cell show that the amplitude enhancement is associated with accelerated decay of both signals. Therefore, the NCX(IF)-dependent modulation of the single cell contractility is primarily governed by Ca-related mechanisms. The observed data are consistent with a proposal suggesting that the inhibition of NCX1 by NCX(IF) results in Ca-dependent activation of SERCA (SR Ca ATPase), yielding the accelerated decay of the [Ca]i-transients. The subsequent increase in the SR Ca content may result in enhanced Ca-release reflecting the manifested promotion of [Ca]i-transients. More systematic study is required for confirming this working hypothesis.     

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.     

Rabbit tendon cells produce MMP-3 in response to fluid flow without significant calcium transients

Forces applied to tendon during movement cause cellular deformation, as well as fluid movement. The goal of this study was to test the hypothesis that rabbit tendon fibroblasts detect and respond to fluid-induced shear stress. Cells were isolated from the paratenon of the rabbit Achilles tendon and then subjected to fluid flow at 1 dyn/cm(2) for 6h in a specially designed multi-slide flow device. The application of fluid flow led to an increased expression of the collagenase-1 (MMP-1), stromelysin-1 (MMP-3), cyclooxygenase II (COX-2) and interleukin-1beta (IL-1beta) genes. The release of proMMP-3 into the medium exhibited a dose-response with the level of fluid shear stress. However, not all cells aligned in the direction of flow. In other experiments, the same cells were incubated with the calcium-reactive dye FURA-2 AM, then subjected to laminar fluid flow in a parallel plate flow chamber. The cells did not significantly increase intracellular calcium concentration when exposed to fluid shear stress levels of up to 25 dyn/cm(2). These results show that gene expression in rabbit tendon cells is sensitive to fluid flow, but that signal transduction is not dependent on intracellular calcium transients. The upregulation of the MMP-1, MMP-3 and COX-2 genes shows that fluid flow could be an important mechanical stimulus for tendon remodelling or injury.     

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.     

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.     

Use of recombinant aequorin to study calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria

We investigated the possibility of Ca(2+) signaling in cyanobacteria (blue-green algae) by measuring intracellular free Ca(2+) levels ([Ca(2+)](i)) in a recombinant strain of the nitrogen fixing cyanobacterium Anabaena strain sp. PCC7120, which constitutively expresses the Ca(2+)-binding photoprotein apoaequorin. The homeostasis of intracellular Ca(2+) in response to increasing external Ca(2+) has been studied in this strain. The resting level of free Ca(2+) in Anabaena was found to be between 100 and 200 nM. Additions of increasing concentrations of external Ca(2+) gave a transient burst of [Ca(2+)](i) followed by a very quick decline, reaching a plateau within seconds that brought the level of [Ca(2+)](i) back to the resting value. These results indicate that Anabaena strain sp. PCC7120 is able to regulate its internal Ca(2+) levels. We also monitored Ca(2+) transients in our recombinant strain in response to heat and cold shock. The cell's response to both stresses was dependent on the way they were induced. The use of inhibitors suggests that heat shock mobilizes cytosolic Ca(2+) from both intracellular and extracellular sources, while the Ca(2+) source for cold shock signaling is mostly extracellular.     

Salvia miltiorrhiza attenuates the changes in contraction and intracellular calcium induced by anoxia and reoxygenation in rat cardiomyocytes

The aim of the present study is to investigate the effect of Salvia miltiorrhiza (SM) on contraction and the intracellular calcium of isolated ventricular myocytes during normoxia or anoxia and reoxygenation using a video tracking system and spectrofluorometry. Cardiac ventricular myocytes were isolated enzymatically by collagenase and exposed to 5 min of anoxia followed by 10 min of reoxygenation. SM (1-9 g/L) depressed both contraction and the [Ca(2+)](i) transient in a dose-dependent manner. SM did not affect the diastolic calcium level and the sarcolemmal Ca(2+) channel of myocytes but decreased the caffeine-induced calcium release. During anoxia, the +/-dL/dtmax, amplitudes of contraction (dL) of cell contraction and [Ca(2+)](i) transients were decreased, while the diastolic calcium level was increased. None of the parameters returned to the pre-anoxia level during reoxygenaton. However, SM (3 g/L) did attenuate the changes in cell contraction and intracellular calcium induced by anoxia and reoxygenation. It is concluded that SM has different effects on normoxic and anoxic cardiomyocytes. The SM-induced reduction of changes in contraction and intracellular calcium induced by anoxia/reoxygenation indicates that SM may be beneficial for cardiac tissue in recovery of mechanical function and intracellular calcium homeostasis.     

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.     

牛磺酸对心肌细胞钙离子的调节作用

牛磺酸占心肌游离氨基酸的５０％以上,它对心肌细胞许多依赖于Ｃａ＾２＋的生理两全是现象具有明显的调节作用。牛磺酸能增加高亲和力Ｃａ＾２＋转驼系统转运Ｃａ＾２＋的量和速度,对要和力Ｃａ＾２＋转运系统的调节作用依胞外Ｃａ＾２＋浓度的不同而不同,具有稳态调节细胞内的Ｃａ＾２＋作用;另外,牛磺酸尚能抑制各种原因经起的胞内Ｃａ＾２＋超负荷,显示其具有细胞保护作用。     

Histamine activates H1-receptors to induce cytosolic free calcium transients in cultured vascular smooth muscle cells from rat aorta

Using an intracellularly trapped dye, quin 2, the effects of histamine on cytosolic free calcium concentrations in rat aortic vascular smooth muscle cells in primary culture were recorded, microfluorometrically. When the cells were exposed to histamine, both in the presence and the absence of extracellular Ca2+, there was a rapid, transient and dose-dependent elevation of cytosolic Ca2+ concentrations, with a similar time course. This elevation of cytosolic Ca2+ was dose-dependently inhibited by mepyramine, but not by cimetidine. Thus, histamine activates H1- but not H2- receptors to mediate a release of Ca2+ from the store sites, and there is a rapid and transient elevation of cytosolic Ca2+.