• Abstract and Figures
• Public Full-text

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1740 (2005) 481 – 488 http://www.elsevier.com/locate/bba Calcium ionophore A23187 action on cardiac myocytes is accompanied by enhanced production of reactive oxygen species Tomasz Przygodzkia,*, Adam Sokalb, Maria Bryszewskaa aDepartment of General Biophysics, University of Lodz, Poland bFirst Department and Clinic of Cardiology of Silesian Medical Academy, Zabrze, Poland Received 14 July 2004; received in revised form 18 March 2005; accepted 22 March 2005 Available online 9 April 2005 Abstract We show that rat neonatal cardiac myocytes exposed to 1 Amol/l of the calcium ionophore A23187 respond with an enhanced production of reactive oxygen species (ROS). This dose is not cytotoxic to the myocytes. A higher concentration (10 Amol/l) evokes less ROS production and is significantly cytotoxic 24 h after exposure, but not immediately after removal of the A23187, when ROS are measured. Both cell death and the decrease in mitochondrial potential are only partially sensitive to MPT inhibitor cyclosporin A. Experiments performed to elucidate the sources of ROS included use of the nitric oxide synthase (NOS) inhibitor L-NAME; NOS involvement was excluded. Experiments with the oxidative phosphorylation uncoupler CCCP revealed that mitochondria are at least partially responsible for the observed effect. Further studies with cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors (indomethacin and MK886, respectively) showed that these enzymes could also be sources of ROS when the calcium level is elevated. Their effect appeared to be independent of phospholipase A2 inhibition, suggesting that COX and LOX stimulation is not due to elevated substrate (arachidonic acid) concentration but rather to a direct effect of calcium. D 2005 Elsevier B.V. All rights reserved. Keywords: Calcium; Reactive oxygen species; Cardiac myocyte; Ischemia and reperfusion 1. Introduction sarcoplasmic/endoplasmic calcium ATPase (SERCA) and the ryanodine receptors (RyR) are oxidized [13,14]. The Calcium overload and oxidative stress accompany, question arises as to whether the opposite interaction takes among other processes, cardiac tissue hypoxia and reoxy- place; the paper of Sharikabad et al. [16] supports this genation [1–4]. The mechanisms leading to calcium over- possibility. These authors showed that the hypoxia/reoxyge- load under these conditions are well understood, but those nation-induced elevation of ROS production in isolated adult responsible for oxidative stress remain unclear. Most rat cardiac myocytes correlates with the external Ca2+ authors consider mitochondria to be the main source of concentration. The disadvantage of this model is that under reactive oxygen species (ROS) during both ischemia and hypoxic conditions, it is impossible to distinguish between reperfusion [1,5,6]; other possible sources are NO synthases the effect of elevated calcium and the possible effects of [7], NADPH oxidase [15] and xanthine oxidase [8]. lowered O2 and (as a consequence) the ATP supply. Studies of General knowledge of biological phenomena suggests that the Ca2+ effect on isolated mitochondria show that the ions the processes mentioned above do not develop independently, may act directly on the organelle membrane, changing its existing studies support this presumption. It has been shown properties and leading to enhanced ROS production [21,22]. that oxidative stress contributes to the impairment of calcium Considerations based on these facts prompted us to inves- turnover in cardiac cells because proteins such as the tigate whether increased intracellular calcium affects ROS production in normoxic cardiac myocytes. * Corresponding author. Our approach was to use the calcium ionophore A23187 2+ E-mail address: [email protected] (T. Przygodzki). to increase the [Ca ]i level directly. Neonatal rat cardiac 0925-4439/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2005.03.009 482 T. Przygodzki et al. / Biochimica et Biophysica Acta 1740 (2005) 481–488 myocytes were used as model cells. Using the fluorescent replaced to allow complete de-estrification of the loaded probe dichlorofluorescin diacetate (H2DCFDA), we meas- probe. The fluorescence was measured using a fluorescence ured ROS production in the cells exposed to A23187 for plate reader (Fluoroskan, Ascent) at excitation wavelength various periods. The probe fluoresces after enzyme-depend- 485 nm and emission wavelength 538 nm. Results are ent oxidation by hydrogen peroxide or peroxynitrite [17].It presented as percentages of the fluorescence intensities of is located mainly in and around the mitochondria in neonatal the control (stained and A23187-untreated cells) T S.D. rat cardiomyocytes [18]. To follow the effect of the iono- phore on the metabolic status of the cells, the mitochondrial 2.4. Intracellular ROS production assays potential was assayed by means of JC-1 fluorescence. We also investigated the effect of reduced glutathione, a well Dichlorofluorescin diacetate (H2DCFDA) (20 mM stock known antioxidant, on A23187-induced cytotoxicity. solution in DMSO) in incubation buffer was added to cells plated on black plates (15,000 per well), pretreated for 3 h with the substances under test, to reach a final concentration 2. Methods of 10 AM. The fluorescence was measured with fluores- cence plate reader at the excitation wavelength 485 nm and 2.1. Cell culture emission wavelength 538 nm; each measurement was performed in 15–20 min. The fluorescence values were The hearts from 20 to 30 neonatal (1–3 days old) plotted against time and the linear parts of the curves were Wistar rats were minced in Krebs–Ringer buffer. The used to estimate the initial velocity of fluorescin oxidation tissue was digested (5 cycles) with a collagenase type II/ defined as ( Ft ÀF0)/t, where Ft is the fluorescence value at pancreatin mixture (80 units/ml and 0.6 mg/ml, respec- time t (min) and F0 is the fluorescence value immediately tively). The cell suspension was incubated with DNAse after the addition of H2DCFDA (Fig. 1). (0.01 mg/ml) for 10 min, filtered through nylon mesh and centrifuged (350 Â g, 40 min) in a discontinuous Percoll 2.5. Mitochondrial potential measurements gradient to obtain a cardiomyocyte-enriched fraction [21]. This fraction was washed twice in Dulbecco’s Modified The changes in mitochondrial membrane potential (wmit) Eagle Medium (DMEM) by centrifugation (200 Âg,10 were assayed by 1,1V,3,3V-tetraethylbenzimidazolcarbocya- min) and the cells were plated on Petri dish for 1 h to allow nine iodide (JC-1) fluorescence intensity measurements. the remaining fibroblasts to attach. Non-attached cardio- This carbocyanine dye accumulates in mitochondria in a myocytes were then plated on 96-well plates (15,000 per potential-dependent manner. The negative potential of the well) covered with gelatin in the culture medium (DMEM/ inner mitochondrial membrane facilitates the formation of M 199 4:1, 10% horse serum, 5% foetal calf serum, 1% dye aggregates (J-aggregates), with both the excitation and penicillin/streptomycin and 10 Ag/ml cytosine-h-d-arabi- emission peaks red-shifted (530 nm/590 nm) compared to nofuranoside). After 48 h the medium was replaced with monomers (485 nm/538 nm). This excludes the possibility fresh culture medium without cytosine-h-d-arabinofurano- that a given change in mitochondrial fluorescence results side. Experiments were performed after a further 24 h. from impaired dye accumulation by the whole cell [25]. The cells were incubated for 30 min with 5 AM JC-1 in the 2.2. Viability assays incubation buffer. Prior to measurements the cells were washed twice with the incubation buffer. The fluorescence The cells were incubated with MTT (50 Al, 0.25 mg/ml, was measured on the Fluoroskan Ascent plate reader with the in culture medium) for 2 h. The medium was then removed filter pairs 485 nm/538 nm and 530 nm/590 nm. The results and the wells were filled with 50 Al DMSO to dissolve the formazan crystals. The absorbance was measured at 580 nm, with a correction at 720 nm using an ELISA plate reader (Awareness Technology Inc.), Stat Fax Type. 2.3. Intracellular calcium assays The intracellular calcium level was measured using Ca2+- sensitive fluorescence probe Fluo-3 AM (5 mM stock solution in DMSO). For fluorescence measurements, the cardiomyocytes (15,000 per well) were cultured on 96-well black plates. The cells were stained with Fluo-3 AM (5 AM) in incubation buffer (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 0.9 mM NaH2PO4, 20 mM HEPES, Fig. 1. Typical plot showing the time dependence of DCF fluorescence glucose 3.8 mg/ml, pH 7.4) for 1 h, then the buffer was intensity in cells treated with various concentrations of A23187. T. Przygodzki et al. / Biochimica et Biophysica Acta 1740 (2005) 481–488 483 Fig. 2. Changes in wmit in cells treated with 10 Amol/l CCCP. n =3,j— P <0.05 (compared to control). Fig. 4. ROS production in cells incubated for 3 h with the given concentrations of A23187. Measurements were performed immediately after the incubation. n =5,j—P <0.05 (compared to control). are shown as dimer-to-monomer fluorescence intensity ratios. The dye loading conditions were shown to respond showed increased ROS production, and the effect did not to mitochondrial potential depolarization with CCCP (Fig. 2). correlate linearly with concentration (Fig. 4); concentra- tions of 5 Amol/l and 10 Amol/l produced less effect than 2.6. Statistics lower doses. The assays of cell viability and wmit, which are reported in next subsections, revealed that higher Results are presented as meanTstandard deviation. ionophore doses decrease the values of both parameters. Statistical analysis were performed by one-way ANOVA However, this decrease is delayed in comparison with that followed by comparison of means by the Tukey test at a in ROS production.

Load more

  8

Copy Link