Rhythmic contraction and intracellular Ca2 + oscillatory rhythm in spontaneously beating cultured cardiac myocytes

Cultured cardiac myocytes from neonatal rats show spontaneous and rhythmic contractions. The intracellular concentration of free Ca^2 + also changes rhythmically, associated with the rhythmic contraction of myocytes (Ca^2 + oscillation). This study aims to elucidate whether spontaneous rhythmic contraction affects the dynamics of intracellular Ca^2 + oscillation in cultured cardiac myocytes. In cultures at four days in vitro (4 DIV), spontaneous Ca^2 + oscillation was synchronized among myocytes. Treatment of cultures with an uncoupler of E - C coupling resulted in a cessation of the spontaneous contraction of cardiac myocytes, but did not abolish the Ca^2 + oscillation. The intercellular synchronization of intracellular Ca^2 + oscillation persisted, and both the intervals and the fluctuation of the oscillation tended to increase after the termination of rhythmic contraction. The present study demonstrated that mechanical factors associated with rhythmic contraction did not affect the intercellular synchronization of intracellular Ca^2 + oscillation, but possibly contributed to the stability of the oscillatory rhythm.     

Fluctuations in the concentration of extracellular ATP synchronized with intracellular Ca(2+) oscillatory rhythm in cultured cardiac myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca²⁺ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca²⁺ oscillation). Both the contraction and Ca²⁺ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP-purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca²⁺ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca²⁺ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca²⁺ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca²⁺ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca²⁺ oscillation. In addition, power spectrum and cross-correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca²⁺ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca²⁺ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca²⁺, contributing to the intercellular synchronization of Ca²⁺ oscillation among cultured cardiac myocytes.     

Simulation analysis of intracellular Na+ and Cl- homeostasis during beta 1-adrenergic stimulation of cardiac myocyte

To quantitatively understand intracellular Na⁺ and Cl⁻ homeostasis as well as roles of Na⁺/K⁺ pump and cystic fibrosis transmembrane conductance regulator Cl⁻ channel (I_CFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na⁺, K⁺, Ca²⁺ and Cl⁻), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca²⁺ transient and cell shortening, and the decreases in both Cl⁻ concentration and cell volume. These results are consistent with experimental data. Increasing the density of I_CFTR shortened APD and augmented the peak amplitudes of the L-type Ca²⁺ current (I_CaL) and the Ca²⁺ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of I_CaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na⁺ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na⁺ affinity of Na⁺/K⁺ pump by protein kinase A. However it was predicted that Na⁺ increases at higher beating rate because of larger Na⁺ influx through forward Na⁺/Ca²⁺ exchange. It was demonstrated that dynamic changes in Na⁺ and Cl⁻ fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.     

Modelling of calcium handling in airway myocytes

Airway myocytes are the primary effectors of airway reactivity which modulates airway resistance and hence ventilation. Stimulation of airway myocytes results in an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the subsequent activation of the contractile apparatus. Many contractile agonists, including acetylcholine, induce [Ca²⁺]_i increase via Ca²⁺ release from the sarcoplasmic reticulum through InsP₃ receptors. Several models have been developed to explain the characteristics of InsP₃-induced [Ca²⁺]_i responses, in particular Ca²⁺ oscillations. The article reviews the modelling of the major structures implicated in intracellular Ca²⁺ handling, i.e., InsP₃ receptors, SERCAs, mitochondria and Ca²⁺-binding cytosolic proteins. We developed theoretical models specifically dedicated to the airway myocyte which include the major mechanisms responsible for intracellular Ca²⁺ handling identified in these cells. These biocomputations pointed out the importance of the relative proportion of InsP₃ receptor isoforms and the respective role of the different mechanisms responsible for cytosolic Ca²⁺ clearance in the pattern of [Ca²⁺]_i variations. We have developed a theoretical model of membrane conductances that predicts the variations in membrane potential and extracellular Ca²⁺ influx. Stimulation of this model by simulated increase in [Ca²⁺]_i predicts membrane depolarisation, but not great enough to trigger a significant opening of voltage-dependant Ca²⁺ channels. This may explain why airway contraction induced by cholinergic stimulation does not greatly depend on extracellular calcium. The development of such models of airway myocytes is important for the understanding of the cellular mechanisms of airway reactivity and their possible modulation by pharmacological agents.     

Arrhythmogenic Ca(2+) release from cardiac myofilaments

We investigated the initiation of Ca²⁺waves underlying triggered propagated contractions (TPCs) occurring in rat cardiac trabeculae under conditions that simulate the functional non-uniformity caused by mechanical or ischemic local damage of the myocardium. A mechanical discontinuity along the trabeculae was created by exposing the preparation to a small constant flow jet of solution with a composition that reduces excitation–contraction coupling in myocytes within that segment. Force was measured and sarcomere length as well as [Ca²⁺]_i were measured regionally. When the jet-contained Caffeine, BDM or Low-[Ca²⁺], muscle-twitch force decreased and the sarcomeres in the exposed segment were stretched by shortening of the normal regions outside the jet. During relaxation the sarcomeres in the exposed segment shortened rapidly. Short trains of stimulation at 2.5 Hz reproducibly caused Ca²⁺-waves to rise from the borders exposed to the jet. Ca²⁺-waves started during force relaxation of the last stimulated twitch and propagated into segments both inside and outside of the jet. Arrhythmias, in the form of non-driven rhythmic activity, were triggered when the amplitude of the Ca²⁺-wave increased by raising [Ca²⁺]_o. The arrhythmias disappeared when the muscle uniformity was restored by turning the jet off. We have used the four state model of the cardiac cross bridge (Xb) with feedback of force development to Ca²⁺ binding by Troponin-C (TnC) and observed that the force–Ca²⁺ relationship as well as the force–sarcomere length relationship and the time course of the force and Ca²⁺ transients in cardiac muscle can be reproduced faithfully by a single effect of force on deformation of the TnC·Ca complex and thereby on the dissociation rate of Ca²⁺. Importantly, this feedback predicts that rapid decline of force in the activated sarcomere causes release of Ca²⁺ from TnC.Ca²⁺,which is sufficient to initiate arrhythmogenic Ca²⁺ release from the sarcoplasmic reticulum. These results show that non-uniform contraction can cause Ca²⁺-waves underlying TPCs, and suggest that Ca²⁺ dissociated from myofilaments plays an important role in the initiation of arrhythmogenic Ca²⁺-waves.     

'Ca(2+)-induced Ca(2+) entry' or how the L-type Ca(2+) channel remodels its own signalling pathway in cardiac cells

The adjustment of Ca²⁺ entry in cardiac cells is critical to the generation of the force necessary for the myocardium to meet the physiological needs of the body. In this review, we present the concept that Ca²⁺ can promote its own entry through Ca²⁺ channels by different mechanisms. We refer to it under the general term of ‘Ca²⁺-induced Ca²⁺ entry’ (CICE). We review short-term mechanisms (usually termed facilitation) that involve a stimulating effect of Ca²⁺ on the L-type Ca²⁺ current (I_Ca-L) amplitude (positive staircase) or a lessening of Ca²⁺-dependent inactivation of I_Ca-L. This latter effect is related to the amount of Ca²⁺ released by ryanodine receptors (RyR2) of the sarcoplasmic reticulum (SR). Both effects are involved in the control of action potential (AP) duration. We also describe a long-term mechanism based on Ca²⁺-dependent down-regulation of the Kv4.2 gene controlling functional expression of the repolarizing transient outward K⁺ current (I_to) and, thereby, AP duration. This mechanism, which might occur very early during the onset of hypertrophy, enhances Ca²⁺ entry by maintaining Ca²⁺ channel activation during prolonged AP. Both Ca²⁺-dependent facilitation and Ca²⁺-dependent down-regulation of I_to expression favour AP prolongation and, thereby, promote sustained voltage-gated Ca²⁺ entry used to enhance excitation–contraction (EC) coupling (with no change in the density of Ca²⁺ channels per se). These self-maintaining mechanisms of Ca²⁺ entry have significant functions in remodelling Ca²⁺ signalling during the cardiac AP. They might support a prominent role of Ca²⁺ channels in the establishment and progression of abnormal Ca²⁺ signalling during cardiac hypertrophy and congestive heart failure.     

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca²⁺ channels in the surface/transverse tubular membrane and ryanodine receptor Ca²⁺ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca²⁺ release through ryanodine receptors in cardiac muscle is a Ca²⁺ influx through the L-type Ca²⁺ channel. The Ca²⁺ entering through the surface membrane Ca²⁺ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.     

Calcium ion control of platelet thrombosthenin ATPase activity

This study demonstrates that Ca²⁺ regulates thrombosthenin ATPase activity, likening the control of platelet contraction to that of cardiac and skeletal muscle. Thrombosthenin, the platelet contractile protein, was isolated by repeated low ionic strength and isoelectric precipitation. Thrombosthenin superprecipitation and ATPase activity were measured in 10⁻⁴ M CaCl₂ (high ionized Ca²⁺) and 0.25 mM ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) (low ionized Ca²⁺). In both high and low Ca²⁺, superprecipitation, measured as an increase in turbidity, ocurred shortly after addition of ATP. ATP hydrolysis by thrombosthenin, which proceeded linearly for several hours, was greater in high Ca²⁺ (approx. 2.3 nmoles·mg⁻¹·min⁻¹) than in low Ca²⁺ (approx. 1.8 nmoles·mg⁻¹·min⁻¹). This difference, when analyzed by the Student's t-test for paired samples was highly significant (P < 0.001). Thrombosthenin ATPase activity was not significantly altered by azide, an inhibitor of mitochondrial ATPase, nor by ouabain, an inhibitor of (Na⁺ + K⁺)-activated ATPase. The dependence of thrombosthenin activation on ionized Ca²⁺, measured with the use of CaEGTA buffers, was studied. The Ca²⁺-dependent portion of thrombosthenin ATPase was half maximal at 4.5·10⁻⁷ M Ca²⁺. This corresponds to an apparent binding constant of 2.2·10⁶ M⁻¹, a value that is comparable to that of skeletal and cardiac muscle. These data suggest that a Ca²⁺ control mechanism similar to that of the troponin-tropomyosin complex of muscle exists in the platelet.     

Actions of emigrated neutrophils on Na(+) and K(+) currents in rat ventricular myocytes

Interactions between neutrophils and the ventricular myocardium can contribute to tissue injury, contractile dysfunction and generation of arrhythmias in acute cardiac inflammation. Many of the molecular events responsible for neutrophil adhesion to ventricular myocytes are well defined; in contrast, the resulting electrophysiological effects and changes in excitation–contraction coupling have not been studied in detail. In the present experiments, rat ventricular myocytes were superfused with either circulating or emigrated neutrophils and whole-cell currents and action potential waveforms were recorded using the nystatin-perforated patch method. Almost immediately after adhering to ventricular myocytes, emigrated neutrophils caused a depolarization of the resting membrane potential and a marked prolongation of myocyte action potential. Voltage clamp experiments demonstrated that following neutrophil adhesion, there was (i) a slowing of the inactivation of a TTX-sensitive Na⁺ current, and (ii) a decrease in an inwardly rectifying K⁺ current.

One cytotoxic effect of neutrophils appears to be initiated by enhanced Na⁺ entry into the myocytes. Thus, manoeuvres that precluded activation of Na⁺ channels, for example holding the membrane potential at −80 mV, significantly increased the time to cell death or prevented contracture entirely. A mathematical model for the action potential of rat ventricular myocytes has been modified and then utilized to integrate these findings. These simulations demonstrate the marked effects of (50-fold) slowing of the inactivation of 2–4% of the available Na⁺ channels on action potential duration and the corresponding intracellular Ca²⁺ transient. In ongoing studies using this combination of approaches, are providing significant new insights into some of the fundamental processes that modulate myocyte damage in acute inflammation.     

A significant fraction of calcium transients in intact Guinea Pig ventricular myocytes is mediated by Na-Ca exchange

Ca²⁺ mobilization elicited by simulation with brief pulses of high K + were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca²⁺ changes across the cell, suggesting the presence of significant spatial Ca²⁺ gradients. Treatment with 20 μM ryanodine, an inhibitor of Ca²⁺ release from the SR, and 10 μM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca²⁺]_i elicited by membrane depolarization. The overall spatial distribution of [Ca²⁺]_i changes appeared unchanged. Ca²⁺ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 μM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the exchange mechanism. Conversely, when the reversal potential of the exchange was shifted to negative potentials by lowering [Na⁺]₀ or by increasing [Na⁺]_i by treatment with 20 μM monensin, the amplitude of these Ca²⁺ transients increased. Ca²⁺ transients elicited by membrane depolarization and largely mediated by reverse operation of Na⁺-Ca²⁺ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These findings suggest that in intact guinea pig cardiac cells, Ca²⁺ influx through the exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling.     

A model of the guinea-pig ventricular cardiac myocyte incorporating a transverse-axial tubular system

A model of the guinea-pig cardiac ventricular myocyte has been developed that includes a representation of the transverse–axial tubular system (TATS), including heterogeneous distribution of ion flux pathways between the surface and tubular membranes. The model reproduces frequency-dependent changes of action potential shape and intracellular ion concentrations and can replicate experimental data showing ion diffusion between the tubular lumen and external solution in guinea-pig myocytes. The model is stable at rest and during activity and returns to rested state after perturbation. Theoretical analysis and model simulations show that, due to tight electrical coupling, tubular and surface membranes behave as a homogeneous whole during voltage and current clamp (maximum difference 0.9 mV at peak tubular I_Na of −38 nA). However, during action potentials, restricted diffusion and ionic currents in TATS cause depletion of tubular Ca²⁺ and accumulation of tubular K⁺ (up to −19.8% and +3.4%, respectively, of bulk extracellular values, at 6 Hz). These changes, in turn, decrease ion fluxes across the TATS membrane and decrease sarcoplasmic reticulum (SR) Ca²⁺ load. Thus, the TATS plays a potentially important role in modulating the function of guinea-pig ventricular myocyte in physiological conditions.     

Altered function and regulation of cardiac ryanodine receptors in cardiac disease

In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum (SR) releases the calcium required for muscle contraction. The magnitude of Ca²⁺ release by RyR2, which is subject to regulation by several physiological mediators, determines cardiac contractility. In heart failure, chronic stimulation of the β-adrenergic signaling pathway leads to hyperphosphorylation of RyR2 by protein kinase A, which dissociates calstabin2 (FKBP12.6) from the receptor. Calstabin2-depleted channels display altered channel gating and can cause diastolic Ca²⁺ release from the SR. This release depletes the SR Ca²⁺ stores, leading to reduced myocardial contractility. Mutant RyR2, found in patients with catecholaminergic polymorphic ventricular tachycardia, has decreased calstabin2 binding affinity, which can trigger ventricular arrhythmias and sudden cardiac death after stress and exercise. Thus, defects in RyR2 have been linked to heart failure and exercise-induced sudden cardiac death and might provide novel therapeutic targets for the treatment of these common diseases of the heart.     

Physiological modulation of voltage-dependent inactivation in the cardiac muscle L-type calcium channel: a modelling study

The inactivation of the L-type Ca²⁺ current is composed of voltage-dependent and calcium-dependent mechanisms. The relative contribution of these processes is still under dispute and the idea that the voltage-dependent inactivation could be subject to further modulation by other physiological processes had been ignored. This study sought to model physiological modulation of inactivation of the current in cardiac ventricular myocytes, based upon the recent detailed experimental data that separated total and voltage-dependent inactivation (VDI) by replacing extracellular Ca²⁺ with Mg²⁺ and monitoring L-type Ca²⁺ channel behaviour by outward K⁺ current flowing through the channel in the absence of inward current flow. Calcium-dependent inactivation (CDI) was based upon Ca²⁺ influx and formulated from data that was recorded during β-adrenergic stimulation of the myocytes. Ca²⁺ influx and its competition with non-selective monovalent cation permeation were also incorporated into channel permeation in the model. The constructed model could closely reproduce the experimental Ba²⁺ and Ca²⁺ current results under basal condition where no β-stimulation was added after a slight reduction of the development of fast voltage-dependent inactivation with depolarization. The model also predicted that under β-adrenergic stimulation voltage-dependent inactivation is lost and calcium-dependent inactivation largely compensates it. The developed model thus will be useful to estimate the respective roles of VDI and CDI of L-type Ca²⁺ channels in various physiological and pathological conditions of the heart which would otherwise be difficult to show experimentally.     

Two bombesin analogues discriminate between neuromedin B- and bombesin-induced calcium flux in a lung cancer cell line

We examined the profile of two bombesin (BN) antagonists, (CH₃)₂CHCO-His-Trp-Ala-Val- -Ala-His-Leu-NHCH₃] (ICI 216140) and [ -Phe⁶,des-Met¹⁴]BN(6–14)ethylamide (DPDM-BN EA), against neuromedin B-induced Ca²⁺ mobilization in the small cell lung cancer (SCLC) line NCI-H345. Neuromedin B (NMB), a BN-like peptide sharing sequence homology with ranatensin, elicited a concentration-dependent Ca²⁺ release (in part) from intracellular stores. Sequential addition of NMB attenuated Ca²⁺ mobilization. Desensitization occurred between BN and NMB; depletion of intracellular Ca²⁺ is a likely mechanism because thapsigargin stimulated Ca²⁺ release after a maximally desensitizing dose of NMB. ICI 216140 and DPDM-BN EA competitively inhibited BN-induced Ca²⁺ transients. In contrast, these compounds antagonized NMB-stimulated Ca²⁺ transients in a noncompetitive manner. The pharmacological profiles obtained support receptor heterogeneity for BN-like peptides on this SCLC line, underscoring the need for thorough examination of dose-response relationships when investigating effects of BN analogues on intact cells.     

Effect of Ca channel blockers on glycerol levels in Dunaliella tertiolecta under hypoosmotic stress

The role of Ca²⁺ in glycerol dissimilation under hypoosmotic stress in the halotolerant alga Dunaliella tertiolecta was investigated using a pharmacological approach. A stretch-activated Ca²⁺ channel blocker, GdCl₃, inhibited glycerol dissimilation under hypoosmotic stress. However, addition of voltage-dependent Ca²⁺ channel blockers and inhibitors of mitochondrial and endoplasmic reticulum Ca²⁺ channels did not affect the glycerol dissimilation under hypoosmotic stress. The results of the present study suggest that the influx of Ca²⁺ from the extracellular space via the stretch-activated Ca²⁺ channels localized in the plasma membrane is required for the transduction of osmotic signal of D. tertiolecta.     

Influence of hyperthyroidism on the superprecipitation response and Ca-sensitivity of natural actomyosin in cardiac and skeletal muscle

In cardiac natural actomyosin prepared from hyperthyroid rabbits, the time of onset of the superprecipitation response was shortened by 58% and the rate of response was increased 4-fold compared with euthyroid animals. However, Ca²⁺-sensitivity of cardiac natural actomyosin prepared from either euthyroid or hyperthyroid rabbits was not changed over the range of 10⁻⁷ to 6.6·10⁻⁵ mM free Ca²⁺ concentrations. Skeletal natural actomyosin prepared from either euthyroid or hyperthyroid rabbits showed a far higher Ca²⁺-sensitivity than cardiac natural actomyosin, but there was no difference in either time of onset or rate of superprecipitation response.     

神经元钙信号系统异常与阿尔茨海默病的“钙假说”

钙信号是细胞调节各项生命活动的重要机制。神经元通过胞外钙离子(calcium ion, Ca²⁺)内流、内质网Ca²⁺释放以及Ca²⁺释放介导的Ca²⁺内流等方式产生具有时空特异性的钙信号,用于调控多种生物学过程,例如动作电位的调节、神经递质的释放、轴突的生长以及突触可塑性等。神经元胞内Ca²⁺浓度因受到细胞精确调控而处于动态平衡之中。若钙信号失调导致平衡被打破,则会造成神经元功能异常甚至死亡。近年来多项研究表明,钙稳态失衡与神经退行性疾病,例如阿尔茨海默病等的产生和发展密切相关,由此发展出关于阿尔茨海默病的钙假说。该假说认为,神经元钙稳态调节机制的持续性改变是神经元功能失常、大脑产生慢性疾病的重要因素。阿尔茨海默病发生发展过程中,神经元胞浆钙水平异常增高,致使多种钙依赖性酶的活性异常,进而影响基因转录。虽然内质网钙稳态的变化目前仍存在一定的争议,但较为确定的是线粒体中存在着钙超载的现象,导致氧化磷酸化反应下调,活性氧的产量增加,进而引发细胞凋亡。本文主要介绍了神经元钙信号系统及其功能,简要梳理了阿尔茨海默病钙假说的相关研究,并对后续研究进行了展望。     

Inhibitors of Na+/Ca2+ exchanger prevent oxidant-induced intracellular Ca2+ increase and apoptosis in a human hepatoma cell line

Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca²⁺ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca²⁺ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2',7'-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N'-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca²⁺ concentration, which was completely prevented by the antioxidants. An intracellular Ca²⁺ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca²⁺ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca²⁺ increase and induction of apoptosis were significantly inhibited by an extracellular Ca²⁺ chelator or Na⁺/Ca²⁺ exchanger blockers (bepridil and benzamil), whereas neither Ca²⁺ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca²⁺ through the activation of reverse mode of Na⁺/Ca²⁺ exchanger. However, tBOOH decreased intracellular Na⁺ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca²⁺ concentration by direct activation of the reverse mode of Na⁺/Ca>²⁺ exchanger, rather than indirect elevation of intracellular Na⁺ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca²⁺ may mediate this action of tBOOH. These results further suggest that Na⁺/Ca²⁺ exchanger may be a target for the management of oxidative hepatotoxicity.