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Natural Science Research Univ. TokushimaVol. 23, No.1 (2009) p.1-6 Effect of econazole on membrane calcium transport in rat thymocytes Jun Akiyama, Hiroshi Ogasa, Yoshiko Hirata, Kohei Yamashita, Haruka Shibagaki, Fumika Tanaka, Yoko Sakanashi, Yasuo Oyama* Laboratory of Cellular Signaling, Faculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan * Corresponding author: Yasuo Oyama, Ph.D. E-mail: [email protected] __________________________________________________________________________________________ ABSTRACT
Econazole, one of azole antifungals, is proven to exhibit an inhibitory action on Mycobateriumtuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions. However, econazole hasbeen used as a pharmacological tool for inhibiting capacitative Ca2+ influx and exerts multiple effects on cellularCa2+ circumstance. Therefore, to suggest the toxic effect of econazole at therapeutic concentrations, we havetested on the effect on membrane Ca2+ transport in rat thymocytes by using a flow cytometer with Fluo-3, anindicator of intracellular Ca2+. Econazole at concentrations of 1-3 µM increased membrane Ca2+ permeabilityand inhibited capacitative Ca2+ influx without affecting passive Ca2+ influx, Ca2+ release from intracellular Ca2+store sites, and membrane Ca2+ pump. Econazole at 0.3 µM, a therapeutic concentration against tuberculosiscaused by multidrug-resistant and latent M. tuberculosis, did not affect membrane Ca2+ transport. It may besuggested that econazole at therapeutic concentrations exerts no side effect related to Ca2+.
Keywords: econazole; calcium; cytotoxicity; lymphocyte __________________________________________________________________________________________ 1. INTRODUCTION
inhibiting Ca2+-ATPase (Mason et al., 1993) andactivates extracellular Ca2+ influx (Jan et al., 1999).
Thus, econazole seems to exert multiple effects on Mycobaterium tuberculosis is similar in sequence to cellular calcium signaling. It is known that Ca2+ plays physiological and pathological roles in various types of cells. Therefore, econazole at therapeutic (Guardiola-Diaz et al., 2001). Several azole antifungals bind CYP51 with high affinity, suggesting intracellular Ca2+ homeostasis, leading to harmful (or that CYP51 of M. tuberculosis is targeted for drug cytotoxic) effect. To test the possibility, the effect of action (Guardiola-Diaz et al., 2001). Econazole, one econazole at concentrations of 3 µM or less on of azole antifungals, is proven to exhibit an inhibitory intracellular Ca2+ level under normal Ca2+ and Ca2+- action on M. tuberculosis and its multidrug-resistant strains under in vitro and ex vivo conditions (Ahmad In present study, the effects of econazole have been et al., 2005, 2006, 2007, 2008). Thus, there is a examined on rat thymocytes by means of flow- chemotherapeutic potential of econazole against cytometry with appropriate fluorescent probes. Rat tuberculosis caused by multidrug-resistant and latent thymocytes were used for the test because of following reasons. First, the cell membranes of In pharmacological cellular studies, econazole has thymocytes remain intact because single cells are been used as an inhibitor of capacitative Ca2+ influx in prepared without an enzymatic treatment. Second, many cells (Sargeant et al., 1992; Mason et al., 1993; the Ca2+-dependent process of cell death is Vostal et al., 1993; Koch et al., 1994). This type of Ca2+ influx is very important for refilling intracellular (McConkey et al., 1994; Winoto, 1997). Third, Ca2+ stores (Putney and Bird, 1993). Furthermore, many chemicals affect intracellular Ca2+ level via econazole increases intracellular Ca2+ level by various mechanisms in thymocytes (Chikahisa and releasing Ca2+ from endoplasmic reticulum via Oyama, 1992; Oyama et al., 1994, 1995; Okazaki et Effect of econazole on membrane calcium transport in rat thymocytes al., 1996; Hirama et al., 2004; Oyama et al., 2006).
The methods for measurements of cellular andmembrane parameters using a flow cytometer 2. MATERIALS AND METHODS
equipped with an argon laser (CytoACE-150, JASCO,Tokyo, Japan) and fluorescent probes were similar to those previously described (Chikahisa et al., 1996).
The fluorescence was analyzed by JASCO software Econazole and dimethyl sulfoxide (DMSO) were (Ver.3XX, JASCO). As to chemicals used in this obtained from Sigma Chemical Co. (St. Louis, USA).
study, there was no fluorescence detected under our Econazole was initially dissolved in DMSO and then added to cell suspension. The final concentration of To assess membrane integrity, propidium iodide was added to the cell suspension to achieve a final incubation with DMSO at 0.3 % or less did not affect concentration of 5 µM. Since propidium stains the Fluorescent probes, propidium iodide and Fluo-3, measurement of propidium fluorescence from the were obtained from Molecular Probes (Eugene, USA).
cells provides a clue to select the cells with intact Other chemicals (NaCl, CaCl , MgCl , KCl, glucose, membranes. Propidium fluorescence was measured HEPES, NaOH, and ZnCl ) were purchased from at 2 min after the application by a flow cytometer.
Excitation wavelength for propidium was 488 nm andemission was detected at 600 ± 20 nm.
To estimate the change in intracellular Ca2+concentration of rat thymocytes, Fluo-3-AM (Kao et This study was approved by the Committee for al., 1989) was used with propidium iodide. The Animal Experiments in the University of Tokushima dyes were added to the cell suspension to achieve a (Registered No. 05279 for Y. Oyama).
final concentration of 500 nM for Fluo-3-AM and 5 µM for propidium iodide. The cells were incubated Laboratories, Yokohama, Japan) were provided with with Fluo-3-AM for 60 min and propidium iodide for water automatically and a commercial diet (MF, 2 min before any fluorescence measurements, Oriental Yeast, Tokyo, Japan) ad libitum. The respectively. Fluo-3 fluorescence was measured animal room was maintained at a temperature of 23 ± only from the cells that were not stained with 2 °C and a relative humidity of 55 ± 5 %, and it was propidium. Excitation wavelength for Fluo-3 was artificially illuminated with fluorescent light on a 12- 488 nm and emission was detected at 530 ± 15 nm.
h light/dark cycle (08:00-20:00 h).
The procedure to prepare cell suspension was similar to that previously reported (Chikahisa et al.,1996). In brief, thymus glands dissected from ether- Statistical analysis was performed by using Tukey anesthetized rats were sliced at a thickness of 400- multivariate analysis. A P value of < 0.05 was 500 µm with razor under ice-cold condition (1-4 °C).
considered significant. Values are mean ± standard The slices were gently triturated by shaking in chilled Tyrode's solution (in mM: NaCl 150, KCl 5, CaCl 2, MgCl 1, glucose 5, HEPES 5, with an appropriate amount of NaOH to adjust pH to 7.3-7.4) to 3. RESULTS
dissociate thymocytes. Thereafter, the Tyrode'ssolution containing the cells was passed through a 3.1. Effect of econazole on Fluo-3 fluorescence of rat mesh (a diameter of 10 µm) to prepare the cell thymocytes in absence and presence of external Ca2+ suspension (about 106 cells/ml). The beakercontaining the cell suspension was water-bathed at Under external Ca2+-free condition where Ca2+ was 36 °C for 1 h before the experiment.
replaced with equimolar Mg2+, the incubation of cells Some thymocytes spontaneously undergo apoptosis with econazole at concentrations ranging from 0.3 during a prolonged incubation for 6 h or more.
µM to 3 µM for 1 hr did not increased the mean Therefore, we used thymocytes freshly isolated from intensity of Fluo-3 fluorescence (Fig. 1). Results thymus glands for this study. The total number of 8- suggest that econazole at therapeutic concentrations 12 week old rats sacrificed under ether inhalation does not induce the release of Ca2+ from intracellular anesthesia was 10. Each experiment was completed Ca2+ store sites. In the presence of 2 mM Ca2+, within 6 h after the isolation of cells from thymus econazole at 0.3 µM did not affect the intensity of Fluo-3 fluorescence. However, the incubation with1 µM econazole for 1 hr increased the intensity of 2.3. Fluorescence measurements of cellular and Fluo-3 fluorescence. Further augmentation of Fluo- 3 fluorescence was observed in the case of 3 µM Akiyama, Ogasa, Hirata, Yamashita, Shibagaki, Tanaka, Sakanashi, Oyama econazole (Fig. 1). It is suggested that econazole at similarly occurred. Thus, it is suggested that passive Ca2+ influx is not affected by econazole at therapeutic intracellular Ca2+ concentration. Thus, since the increase by econazole is dependent on external Ca2+, Under external Ca2+-free condition, the incubation econazole at micromolar concentrations may increase of cells with 100 nM thapsigargin depletes Ca2+ from endoplasmic reticulum, leading to an activation ofcapacitative Ca2+ influx (Oyama et al., 1994).
3.2. Effects of econazole on Fluo-3 fluorescence Therefore, the augmentation of Fluo-3 fluorescence augmented by reintroduction of Ca2+ in absence and by the reintroduction of external Ca2+ is due to the combination of passive and capacitative Ca2+ influx inthe presence of thapsigargin. As shown in Fig. 2, Under Ca2+-free condition, the reintroduction of while the incubation with 0.3 µM econazole did not external Ca2+ increased the mean intensity of Fluo-3 affect the augmentation of Fluo-3 fluorescence by the fluorescence (Fig. 2). The augmentation of Fluo-3 reintroduction, the augmentation was attenuated by 1 fluorescence by the reintroduction of external Ca2+ µM and 3 µM econazole. Since econazole did not may be due to passive Ca2+ influx. Even in the affect the passive Ca2+ influx, it is likely that the presence of econazole at concentrations ranging from attenuation of Fluo-3 fluorescence by econazole is 0.3 µM to 3 µM, the augmentation of Fluo-3 due to the decrease in capacitative Ca2+ influx.
fluorescence by the reintroduction of external Ca2+ Fig.1. Effect of econazole on mean intensity of Fluo-3 fluorescence in the absence and presence of external Ca2+.
Column and bar indicate average and SD of 4 experiments. Asterisks (* and **) show significant increase (P <
0.05 and P < 0.01, respectively) in the intensity, comparing with control.

Effect of econazole on membrane calcium transport in rat thymocytes Fig. 2. Effect of econazole on mean intensity of Fluo-3 fluorescence augmented by reintroduction of external Ca2+
in absence and presence of thapsigargin. Asterisk (*) shows significant attenuation (P < 0.05 and P < 0.01,
respectively) of the intensity, comparing with control augmentation. The bar indicates the control level after
the reintroduction.

Fig. 3. Effect of econazole on mean intensity of Fluo-3 fluorescence at 30 min after the start of application of
A23187. The increase at 1 min after the start of A23187 application was significant (P < 0.01), comparing with
control. The bar indicates the A23187-induced level at 1 min in the presence of econazole.

3.3. Effect of econazole on Fluo-3 fluorescence suggested that econazole exerts no inhibitory action augmented by A23187 under external Ca2+-free Under external Ca2+-free condition, A23187 at 100 4. DISCUSSION
nM increased mean intensity of Fluo-3 fluorescenceand the intensity gradually returned to control level Econazole, an imidazole antifungal, is proposed to within 30 min in continued presence of A23187 (Fig.
possess a therapeutic potential against tuberculosis 3, Oyama et al., 1994). This change in the (Ahmad et al., 2005, 2006. 2007. 2008). However, fluorescence intensity is due to the release of Ca2+ this agent inhibits capacitative Ca2+ influx (Mason et from intracellular Ca2+ stores by A23187 and the al., 1993; Hornestein et al., 1996; Christian et al., extrusion of Ca2+ by membrane Ca2+ pump (Oyama et 1996). Furthermore, econazole exerts multiple al., 1994). The intensity of Fluo-3 fluorescence at effects on cellular Ca2+ signal (Jan et al., 1999).
30 min after the start of application of A23187 in the Therefore, one may argue the possibility that presence of 0.3-3 µM econazole was similar to that in the absence of econazole (Fig. 3). Thus, it is cytotoxic effect that is related to Ca2+ because Akiyama, Ogasa, Hirata, Yamashita, Shibagaki, Tanaka, Sakanashi, Oyama intracellular Ca2+ is known to play physiological, homeostasis, resulting in some cytotoxic effects.
pathological, and toxicological roles.
Possible therapeutic concentration of econazole In present study, econazole at micromolar against tuberculosis is 0.1-0.3 µM (Ahmad et al., concentrations (1-3 µM) increased membrane Ca2+ 2005). As shown in Figs. 1-3, 0.3 µM econazole did permeability, resulting in the increase in intracellular not change Fluo-3 fluorescence in rat thymocytes.
Ca2+ concentration under normal Ca2+ condition (Fig.
Thus, econazole at submicromolar concentrations, 1), and attenuated the thapsigargin-related increase in intracellular Ca2+ concentration via reintroducing intracellular Ca2+ homeostasis. Therefore, it is external Ca2+ by inhibiting capacitative Ca2+ influx (Fig. 2). Of two reciprocal effects described above, concentrations exerts no cytotoxic effect that is related to Ca2+. However, our recent study by using permeability is predominant in affecting intracellular FluoZin-3 reveals that submicromolar econazole Ca2+ concentration because of larger augmentation of greatly increases mean intensity of FluoZin-3 Fluo-3 fluorescence by econazole under normal Ca2+ fluorescence in the presence of physiological condition (Fig. 1). On the contrary, econazole at 1-3 concentrations of Zn2+ (Kinazaki et al., In submitted).
µM did not induce Ca2+ release from intracellular Therefore, it may be suggested that the side effect of Ca2+ site (Fig. 1) and did not affect membrane Ca2+ submicromolar econazole is related to Zn2+, but not pump (Fig. 3). Thus, econazole at micromolar Ca2+. In this aspect, further study will be necessary.
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cell hybridomas. Seminars in Immunology 9, 51- Clotrimazole, an antifungal drug possessing Article history:Received MS - 20 January 2009Received revised MS - 28 January 2009Accepted MS - 28 January 2009

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