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Effect of maprotiline on Ca2+ movement in human neuroblastoma cells
Life Sciences 75 (2004) 1105 – 1112 www.elsevier.com/locate/lifescie
Effect of maprotiline on Ca2+ movement in human neuroblastoma cells Shu-Shong Hsu a,b, Wei-Chuan Chen c, Yuk-Keung Lo d, Jin-Shiung Cheng d, Jeng-Hsien Yeh d, He-Hsing Cheng d, Jin-Shyr Chen a, Hong-Tai Chang a, Bang-Ping Jiann a, Jong-Khing Huang a, Chung-Ren Jan e,* a
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan b Department of Radiological Technology, I-Shou, University, Kaohsiung 840, Taiwan c Department of Surgery, Ping Tung Christian Hospital, Ping Tung 900, Taiwan d Department of Medicine, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan e Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan Received 14 November 2003; accepted 20 February 2004
Abstract In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2 + concentrations ([Ca2 +]i) was explored by using the Ca2 +-sensitive probe fura-2. Maprotiline at concentrations greater than 100 AM caused a rapid rise in [Ca2 +]i in a concentration-dependent manner (EC50 = 200 AM). Maprotiline-induced [Ca2 +]i rise was reduced by 50% by removal of extracellular Ca2 +. Maprotiline-induced [Ca2 +]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. In Ca2 +-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2 +-ATPase, caused a monophasic [Ca2 +]i rise, after which the increasing effect of maprotiline on [Ca2 +]i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2 +]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2 +]i by stimulating extracellular Ca2 + influx and also by causing intracellular Ca2 + release from the endoplasmic reticulum via a phospholiase C-independent manner. D 2004 Elsevier Inc. All rights reserved. Keywords: Ca2+; Ca2+ stores; Fura-2; IMR32; Maprotiline; Neuroblastoma cells
* Corresponding author. Tel.: +886-7-3422121-1509; fax: +11-886-7-3468056. E-mail address: [email protected] (C.-R. Jan). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.02.022
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Introduction Maprotiline is an antidepressant that is thought to act mainly by selectively inhibiting noradrenaline reuptake (Humble, 2000). Several other in vitro effects have been attributed to maprotiline. Maprotiline was shown to inhibit gamma-aminobutyric acid- and N-methyl-D-aspartate-induced intracellular Ca2 + concentration ([Ca2 +]i) increases in primary cultured rat cortical neurons (Takebayashi et al., 2000), but to amplify serotonin 2A receptor-induced [Ca2 +]i mobilization in C6 glioma cells through a calmodulin-dependent pathway (Muraoka et al., 1998), to inhibit 22Na+ influx in bovine chromaffin cells (Arita et al., 1987), and to block delayed rectifier potassium current in ventricular myocytes (Casis et al., 2002). The other effects of maprotiline are less documented. The present study was aimed to explore the effect of maprotiline on baseline [Ca2 +]i in human neuroblastoma cells. Despite inhibitory or amplifying effects were shown previously in different types of cells, a possible effect of maprotiline on baseline [Ca2 +]i has never been considered. A regulated rise in [Ca2 +]i is a key signal in all cell types, and can trigger many physio-pathological events (Berridge, 1997, 1993; Bootman et al., 1993); but an unregulated elevation in [Ca2 +]i is often cytotoxic (Annunziato et al., 2003). Thus it is important to examine the effect of an agent on baseline [Ca2 +]i and on agents-induced Ca2 + movement in order to understand its in vitro effect. In the present study, IMR32 human neuroblastoma cells were used to investigate the effect of maprotiline on baseline [Ca2 +]i. Using fura-2 as a fluorescent Ca2 + indicator, this study shows that maprotiline induced a significant rise in baseline [Ca2 +]i in a concentration-dependent manner. The time course and the concentration-response relationship, the Ca2 + sources of the Ca2 + signal, and the role of phospholipase C in the signal have been explored.
Materials and methods Cell culture IMR32 human neuroblastoma cells were obtained from American Type Culture Collection and were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 Ag/ml streptomycin. Cells were kept at 37 jC in 5% CO2-containing humidified air. Solutions Ca2 +-containing medium (pH 7.4) had (in mM): NaCl 140; KCl 5; MgCl2 1; CaCl2 2; Hepes 10; glucose 5. Ca2 +-free medium contained similar components as Ca2 +-containing medium except that CaCl2 was substituted with 0.1 mM EGTA. Agents were dissolved in water, ethanol or dimethyl superoxide as stock solutions. Maprotiline was dissolved in water as stock solutions. Final concentrations of organic solvents in the [Ca2 +]i measurements were less than 0.1% and did not alter basal [Ca2 +]i. [Ca2+]i measurements Trypsinized cells (106/ml) were allowed to recover in culture medium for 1 hour before being loaded with 2 AM fura-2/acetoxy methyl (fura-2/AM) for 30 min at 25 jC. The cells were washed and
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re-suspended in Ca2 +-containing medium. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 jC) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer (Kyoto, Japan) by recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1s intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 and 10 mM EGTA sequentially at the end of each experiment. [Ca2 +]i was calculated as described previously assuming a Kd of 155 nM (Grynkiewicz et al., 1985). Chemicals The reagents for cell culture were from Gibco (Gaithersburg, MD, USA). Fura-2/AM was from Molecular Probes (Eugene, OR, USA). U73122 (1-(6-((17h-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) was from Biomol (Plymouth Meeting, PA, USA). The other reagents were from Sigma (St. Louis, MO, USA). Statistics Statistical comparisons were determined by using Student’s t test, and significance was accepted when P < 0.05.
Results Effect of maprotiline on [Ca2+]i In Ca2 +-containing medium, the basal [Ca2 +]i was 50 F 3 nM (n = 5). Addition of maprotiline ( z 100 AM) caused an immediate rise in [Ca2 +]i, which lasted for, at least, 200 s after the addition of maprotiline (Fig. 1A); e.g. maprotiline (500 AM)-induced [Ca2 +]i rise attained to 185 F 3 nM (n = 5; trace a) over the baseline. The Ca2 + signal was followed by a gradual decay that reached a level of 47 F 2 nM over the baseline at the time point of 250 s. The increasing effect of maprotiline was concentration-dependent with an EC50 of about 200 AM by fitting to a Hill equation (Fig. 1C; filled circles). Sources of Ca2+ of maprotiline-induced [Ca2+]i rise To examine whether/how influx of extracellular Ca2 + and/or mobilization of Ca2 + from the intracellular store site(s) may contribute to maprotiline-induced [Ca2 +]i rise, the effect of maprotiline on [Ca2 +]i was measured in the absence of extracellular Ca2 +. Fig. 1B shows that the [Ca2 +]i rise caused by maprotiline was attenuated, with no change in baseline [Ca2 +]i (50 F 3 nM, n = 5). Maprotiline (500 AM) increased [Ca2 +]i by 96 F 2 nM at the time point of 62 s. The Ca2 + signal returned to baseline at the time point of 200 s. These data suggest that maprotiline induced both extracellular Ca2 + influx and intracellular Ca2 + release. The concentration-response curves of maprotiline-induced [Ca2 +]i rises in Ca2 +-containing medium and in Ca2 +-free medium are shown in Fig. 1C.
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Fig. 1. Maprotiline-induced concentration-dependent [Ca2 +]i rises in IMR32 cells. (A) In Ca2 +-containing medium, maprotiline was added at 25 s. The concentration of maprotiline was indicated. (B) Effect of removal of extracellular Ca2 + on maprotilineinduced response. The experiments were performed in Ca2 +-free medium (no added Ca2 + plus 0.1 mM EGTA). The concentration of maprotiline was indicated. (C) The concentration-response plots of maprotiline-induced Ca2 + signals. The y axis is the percentage of control. Control was the net (baseline subtracted) area under the curve between 25 – 250 s of 500 AM maprotiline-induced [Ca2 +]i rise. Data are means F S.E.M. of five experiments. *P < 0.05.
Effect of Ca2+ blockers on maprotiline-induced [Ca2+]i rise Further experiments were performed to explore the Ca2 + influx pathways of maprotiline-induced [Ca2 +]i rise. Fig. 2 shows that pretreatment with 10 AM nifedipine inhibited 502% of the maximum level of 500 AM maprotiline-induced [Ca2 +]i rise in Ca2 +-containing medium (P < 0.05; n = 5); whereas 10 AM of verapamil or diltiazem failed to have an effect (P > 0.05; n = 5). Intracellular Ca2+stores of maprotiline-induced Ca2+ release We examined whether maprotiline-induced [Ca2 +]i rise involves the mobilization of intracellular Ca2 + stored within the endoplasmic reticulum. Fig. 3A shows that in Ca2 +-free medium, addition of
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Fig. 2. Effect of Ca2 + blockers on maprotiline-induced [Ca2 +]i rises. Experiments were performed in Ca2 +-containing medium. Blockers (10 AM) were added to cells 1 min before fluorescence measurements. The y axis is percentage of control response that was the net area under the curve (25 – 250 s) of the [Ca2 +]i rise induced by 500 AM maprotiline. Data are mean F S.E.M. of five experiments. *P < 0.05.
thapsigargin, an inhibitor of endoplasmic reticulum ATPases (Thastrup et al., 1990), increased [Ca2 +]i by 49 F 2 nM (n = 5) in a rapid monophasic manner. Addition of maprotiline at the time point of 500 s did not affect the thapsigargin-induced [Ca2 +]i rises (n = 5; P > 0.05).
Fig. 3. Intracellular Ca2 + stores of maprotiline-induced [Ca2 +]i rise. The experiments were performed in Ca2 +-free medium. The agents were added at the time points indicated by arrows. The concentration of agents was 1 AM for thapsigargin and 500 AM for maprotiline. Data are means F S.E.M. of five experiments.
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Fig. 4. Lack of involvement of phospholipase C in maprotiline-induced [Ca2 +]i rise. The experiments were performed in Ca2 +free medium. U73122 (2 AM) and maprotiline (500 AM) were added at the time points indicated by arrows. Data are mean F S.E.M. of five experiments.
Lack of involvement of phospholipase C in maprotiline-induced Ca2+ release The possibility that phospholipase C-inositol 1,4,5-trisphosphate pathway is involved in maprotilineinduced Ca2 + release was examined. Fig. 4 shows that after the pretreatment with 2 AM U73122, an inhibitor of phospholipase C (Thompson et al., 1991), 500 AM maprotiline induced a [Ca2 +]i rise that was indistinguishable from the control shown in Fig. 1B (n = 5; P > 0.05).
Discussion The novel finding of the present study is that the antidepressant maprotiline can induce significant increases in baseline [Ca2 +]i in human neuroblastoma cells. It has been previously reported that maprotiline inhibits gamma-aminobutyric acid- and N-methyl-D-aspartate-induced intracellular Ca2 + concentration ([Ca2 +]i) increases in primary cultured rat cortical neurons (Takebayashi et al., 2000), but to amplify serotonin 2A receptor-induced [Ca2 +]i mobilization in C6 glioma cells through a calmodulindependent pathway (Muraoka et al., 1998); however, we are the first to show that maprotiline at concentrations greater than 100 AM can trigger a rise in baseline [Ca2 +]i. The maprotiline-induced [Ca2 +]i rise is equally contributed by extracellular Ca2 + influx and intracellular Ca2 + release, as the Ca2 + signal was reduced by half by removal of extracellular Ca2 +. Our data suggest that maprotilineinduced Ca2 + influx portion is mediated by nifedipine-sensitive Ca2 + channels, because the maprotilineinduced [Ca2 +]i rise in Ca2 +-containing medium was reduced by 50% by nifedipine. This Ca2 + influx channel appears to be selective for nifedipine as verapamil and diltiazem failed to have an effect. Consistently, the Ca2 + influx induced by riluzole in the same neuroblastoma cell line was also blocked by nifedipine, but was not altered by verapamil (Wang et al., 2001). Nifedipine-sensitive Ca2 + channels
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and N-type Ca2 + channels have been reported previously (Grassi et al., 1994; Carabelli et al., 1996) in this cell line. In Ca2 +-containing medium, maprotiline-induced [Ca2 +]i increase displayed a gradual rise and a stable, flat plateau. In contrast, in Ca2 +-free medium, maprotiline induced a smaller [Ca2 +]i increase followed by a clear decrease. This suggests that extracellular Ca2 + influx contributes not only to the initial increase, but also to the prolonged phase of maprotiline-induced [Ca2 +]i ruse in Ca2 +containing medium. The endoplasmic reticulum Ca2 + stores have been shown to play an important role in a [Ca2 +]i rise in IMR32 cells (Wang et al., 2001). Regarding the Ca2 + stores of the maprotiline-induced response, the thapsigargin-sensitive endoplasmic reticulum store appears to be dominant, because maprotiline could not release more intracellular Ca2 + after thapsigargin had depleted the endoplasmic reticulum Ca2 + store. The maprotiline -induced Ca2 + releasing process may be independent of inositol 1,4,5-trisphosphate since suppression of phospholipase C did not affect maprotiline-induced Ca2 + release. Taken together, these findings suggest that in human IMR32 neuroblastoma cells, maprotiline increases baseline [Ca2 +]i by stimulating extracellular Ca2 + influx and also by causing intracellular Ca2 + release from the endoplasmic reticulum via a phospholiase C-independent manner. Since a rise in baseline [Ca2 +]i or depletion of intracellular Ca2 + can interfere with many cellular responses, caution must be exercised in using maprotiline in other in vitro studies. Furthermore, previous studies of maprotiline commonly focused on the drug’s selective inhibition of norepinephrine reuptake, and ignored its possible other effects. Here we show evidence that maprotiline may alter signal transduction in neuronal cells, and this may supplement its antidepressant action or may contribute to its unwanted clinical side effects.
Acknowledgements This work was supported by grants from Veterans General Hospital-Kaohsiung (VGHKS92G-11) and NSC92-2320-B-075B-003 to CR Jan, and VGHKS93-92 to SS Hsu.
References Annunziato, L., Amoroso, S., Pannaccione, A., Cataldi, M., Pignataro, G., D’Alessio, A., Sirabella, R., Secondo, A., Sibaud, L., Di Renzo, G.F., 2003. Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions. Toxicology Letters 139, 125 – 133. Arita, M., Wada, A., Takara, H., Izumi, F., 1987. Inhibition of 22Na+ influx by tricyclic and tetracyclic antidepressants and binding of [3H]imipramine in bovine adrenal medullary cells. Journal of Pharmacology and Experimental Therapy 243 (1), 342 – 348. Berridge, M.J., 1997. Elementary and global aspects of calcium signalling. Journal of Physiology (London) 499, 291 – 306. Berridge, M.J., 1993. Inositol trisphosphate and calcium signaling. Nature 361, 315 – 325. Bootman, M.D., Berridge, M.J., Lipp, P., 1993. Cooking with calcium: the recipes for composing global signals from elementary events. Cell 91, 367 – 373. Carabelli, V., Lovallo, M., Magnelli, V., Zucker, H., Carbone, E., 1996. Voltage-dependent modulation of single N-Type Ca2 + channel kinetics by receptor agonists in IMR32 cells. Biophysics Journal 70 (5), 2144 – 2154. Casis, O., Gallego, M., Sanchez-Chapula, J.A., 2002. Imipramine, mianserine and maprotiline block delayed rectifier potassium current in ventricular myocytes. Pharmacological Research 45 (2), 141 – 146.
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Grassi, C., Magnelli, V., Carabelli, V., Sher, E., Carbone, E., 1994. Inhibition of low- and high-threshold Ca2 + channels of human neuroblastoma IMR32 cells by Lambert-Eaton myasthenic syndrome (LEMS) IgGs. Neuroscience Letters 181 (1 – 2), 50 – 56. Grynkiewicz, G., Poenie, M., Tsien, R.Y., 1985. A new generation of Ca2 + indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440 – 3450. Humble, M., 2000. Noradrenaline and serotonin reuptake inhibition as clinical principles: a review of antidepressant efficacy. Acta of Psychiatry Scandinavia Supplement 402, 28 – 36. Muraoka, S., Kamei, K., Muneoka, K., Takigawa, M., 1998. Chronic imipramine administration amplifies the serotonin2A receptor-induced intracellular Ca2 + mobilization in C6 glioma cells through a calmodulin-dependent pathway. Journal of Neurochemistry 71 (4), 1709 – 1718. Takebayashi, M., Kagaya, A., Inagaki, M., Kozuru, T., Jitsuiki, H., Kurata, K., Okamoto, Y., Yamawaki, S., 2000. Effects of antidepressants on gamma-aminobutyric acid- and N-methyl-D-aspartate-induced intracellular Ca2 + concentration increases in primary cultured rat cortical neurons. Neuropsychobiology 42 (3), 120 – 126. Thastrup, O., Cullen, P.T., Drobak, B.K., Hanley, M.R., Dawson, A.P., 1990. Thapsigargin, a tumor promoter, discharges intracellular Ca2 + stores by specific inhibition of the endoplasmic reticulum Ca2 +-ATPase. Proceedings of National Academy of Sciences of USA 87, 2466 – 2470. Thompson, A.K., Mostafapour, S.P., Denlinger, L.C., Bleasdale, J.E., Fisher, S.K., 1991. The aminosteroid U73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. Journal of Biological Chemistry 266, 23856 – 23862. Wang, J.L., Lee, K.C., Tang, K.Y., Lu, T., Chang, C.H., Chow, C.K., Chen, W.C., Su, W., Law, Y.P., Jan, C.R., 2001. Effect of the neuroprotective agent riluzole on intracellular Ca2 + levels in IMR32 neuroblastoma cells. Archives of Toxicology 75 (4), 214 – 220.
Effect of maprotiline on Ca2+ movement in human neuroblastoma cells Shu-Shong Hsu a,b, Wei-Chuan Chen c, Yuk-Keung Lo d, Jin-Shiung Cheng d, Jeng-Hsien Yeh d, He-Hsing Cheng d, Jin-Shyr Chen a, Hong-Tai Chang a, Bang-Ping Jiann a, Jong-Khing Huang a, Chung-Ren Jan e,* a
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan b Department of Radiological Technology, I-Shou, University, Kaohsiung 840, Taiwan c Department of Surgery, Ping Tung Christian Hospital, Ping Tung 900, Taiwan d Department of Medicine, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan e Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 813, Taiwan Received 14 November 2003; accepted 20 February 2004
Abstract In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2 + concentrations ([Ca2 +]i) was explored by using the Ca2 +-sensitive probe fura-2. Maprotiline at concentrations greater than 100 AM caused a rapid rise in [Ca2 +]i in a concentration-dependent manner (EC50 = 200 AM). Maprotiline-induced [Ca2 +]i rise was reduced by 50% by removal of extracellular Ca2 +. Maprotiline-induced [Ca2 +]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. In Ca2 +-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2 +-ATPase, caused a monophasic [Ca2 +]i rise, after which the increasing effect of maprotiline on [Ca2 +]i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2 +]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2 +]i by stimulating extracellular Ca2 + influx and also by causing intracellular Ca2 + release from the endoplasmic reticulum via a phospholiase C-independent manner. D 2004 Elsevier Inc. All rights reserved. Keywords: Ca2+; Ca2+ stores; Fura-2; IMR32; Maprotiline; Neuroblastoma cells
* Corresponding author. Tel.: +886-7-3422121-1509; fax: +11-886-7-3468056. E-mail address: [email protected] (C.-R. Jan). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.02.022
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Introduction Maprotiline is an antidepressant that is thought to act mainly by selectively inhibiting noradrenaline reuptake (Humble, 2000). Several other in vitro effects have been attributed to maprotiline. Maprotiline was shown to inhibit gamma-aminobutyric acid- and N-methyl-D-aspartate-induced intracellular Ca2 + concentration ([Ca2 +]i) increases in primary cultured rat cortical neurons (Takebayashi et al., 2000), but to amplify serotonin 2A receptor-induced [Ca2 +]i mobilization in C6 glioma cells through a calmodulin-dependent pathway (Muraoka et al., 1998), to inhibit 22Na+ influx in bovine chromaffin cells (Arita et al., 1987), and to block delayed rectifier potassium current in ventricular myocytes (Casis et al., 2002). The other effects of maprotiline are less documented. The present study was aimed to explore the effect of maprotiline on baseline [Ca2 +]i in human neuroblastoma cells. Despite inhibitory or amplifying effects were shown previously in different types of cells, a possible effect of maprotiline on baseline [Ca2 +]i has never been considered. A regulated rise in [Ca2 +]i is a key signal in all cell types, and can trigger many physio-pathological events (Berridge, 1997, 1993; Bootman et al., 1993); but an unregulated elevation in [Ca2 +]i is often cytotoxic (Annunziato et al., 2003). Thus it is important to examine the effect of an agent on baseline [Ca2 +]i and on agents-induced Ca2 + movement in order to understand its in vitro effect. In the present study, IMR32 human neuroblastoma cells were used to investigate the effect of maprotiline on baseline [Ca2 +]i. Using fura-2 as a fluorescent Ca2 + indicator, this study shows that maprotiline induced a significant rise in baseline [Ca2 +]i in a concentration-dependent manner. The time course and the concentration-response relationship, the Ca2 + sources of the Ca2 + signal, and the role of phospholipase C in the signal have been explored.
Materials and methods Cell culture IMR32 human neuroblastoma cells were obtained from American Type Culture Collection and were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 Ag/ml streptomycin. Cells were kept at 37 jC in 5% CO2-containing humidified air. Solutions Ca2 +-containing medium (pH 7.4) had (in mM): NaCl 140; KCl 5; MgCl2 1; CaCl2 2; Hepes 10; glucose 5. Ca2 +-free medium contained similar components as Ca2 +-containing medium except that CaCl2 was substituted with 0.1 mM EGTA. Agents were dissolved in water, ethanol or dimethyl superoxide as stock solutions. Maprotiline was dissolved in water as stock solutions. Final concentrations of organic solvents in the [Ca2 +]i measurements were less than 0.1% and did not alter basal [Ca2 +]i. [Ca2+]i measurements Trypsinized cells (106/ml) were allowed to recover in culture medium for 1 hour before being loaded with 2 AM fura-2/acetoxy methyl (fura-2/AM) for 30 min at 25 jC. The cells were washed and
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re-suspended in Ca2 +-containing medium. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 jC) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer (Kyoto, Japan) by recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1s intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 and 10 mM EGTA sequentially at the end of each experiment. [Ca2 +]i was calculated as described previously assuming a Kd of 155 nM (Grynkiewicz et al., 1985). Chemicals The reagents for cell culture were from Gibco (Gaithersburg, MD, USA). Fura-2/AM was from Molecular Probes (Eugene, OR, USA). U73122 (1-(6-((17h-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) was from Biomol (Plymouth Meeting, PA, USA). The other reagents were from Sigma (St. Louis, MO, USA). Statistics Statistical comparisons were determined by using Student’s t test, and significance was accepted when P < 0.05.
Results Effect of maprotiline on [Ca2+]i In Ca2 +-containing medium, the basal [Ca2 +]i was 50 F 3 nM (n = 5). Addition of maprotiline ( z 100 AM) caused an immediate rise in [Ca2 +]i, which lasted for, at least, 200 s after the addition of maprotiline (Fig. 1A); e.g. maprotiline (500 AM)-induced [Ca2 +]i rise attained to 185 F 3 nM (n = 5; trace a) over the baseline. The Ca2 + signal was followed by a gradual decay that reached a level of 47 F 2 nM over the baseline at the time point of 250 s. The increasing effect of maprotiline was concentration-dependent with an EC50 of about 200 AM by fitting to a Hill equation (Fig. 1C; filled circles). Sources of Ca2+ of maprotiline-induced [Ca2+]i rise To examine whether/how influx of extracellular Ca2 + and/or mobilization of Ca2 + from the intracellular store site(s) may contribute to maprotiline-induced [Ca2 +]i rise, the effect of maprotiline on [Ca2 +]i was measured in the absence of extracellular Ca2 +. Fig. 1B shows that the [Ca2 +]i rise caused by maprotiline was attenuated, with no change in baseline [Ca2 +]i (50 F 3 nM, n = 5). Maprotiline (500 AM) increased [Ca2 +]i by 96 F 2 nM at the time point of 62 s. The Ca2 + signal returned to baseline at the time point of 200 s. These data suggest that maprotiline induced both extracellular Ca2 + influx and intracellular Ca2 + release. The concentration-response curves of maprotiline-induced [Ca2 +]i rises in Ca2 +-containing medium and in Ca2 +-free medium are shown in Fig. 1C.
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Fig. 1. Maprotiline-induced concentration-dependent [Ca2 +]i rises in IMR32 cells. (A) In Ca2 +-containing medium, maprotiline was added at 25 s. The concentration of maprotiline was indicated. (B) Effect of removal of extracellular Ca2 + on maprotilineinduced response. The experiments were performed in Ca2 +-free medium (no added Ca2 + plus 0.1 mM EGTA). The concentration of maprotiline was indicated. (C) The concentration-response plots of maprotiline-induced Ca2 + signals. The y axis is the percentage of control. Control was the net (baseline subtracted) area under the curve between 25 – 250 s of 500 AM maprotiline-induced [Ca2 +]i rise. Data are means F S.E.M. of five experiments. *P < 0.05.
Effect of Ca2+ blockers on maprotiline-induced [Ca2+]i rise Further experiments were performed to explore the Ca2 + influx pathways of maprotiline-induced [Ca2 +]i rise. Fig. 2 shows that pretreatment with 10 AM nifedipine inhibited 502% of the maximum level of 500 AM maprotiline-induced [Ca2 +]i rise in Ca2 +-containing medium (P < 0.05; n = 5); whereas 10 AM of verapamil or diltiazem failed to have an effect (P > 0.05; n = 5). Intracellular Ca2+stores of maprotiline-induced Ca2+ release We examined whether maprotiline-induced [Ca2 +]i rise involves the mobilization of intracellular Ca2 + stored within the endoplasmic reticulum. Fig. 3A shows that in Ca2 +-free medium, addition of
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Fig. 2. Effect of Ca2 + blockers on maprotiline-induced [Ca2 +]i rises. Experiments were performed in Ca2 +-containing medium. Blockers (10 AM) were added to cells 1 min before fluorescence measurements. The y axis is percentage of control response that was the net area under the curve (25 – 250 s) of the [Ca2 +]i rise induced by 500 AM maprotiline. Data are mean F S.E.M. of five experiments. *P < 0.05.
thapsigargin, an inhibitor of endoplasmic reticulum ATPases (Thastrup et al., 1990), increased [Ca2 +]i by 49 F 2 nM (n = 5) in a rapid monophasic manner. Addition of maprotiline at the time point of 500 s did not affect the thapsigargin-induced [Ca2 +]i rises (n = 5; P > 0.05).
Fig. 3. Intracellular Ca2 + stores of maprotiline-induced [Ca2 +]i rise. The experiments were performed in Ca2 +-free medium. The agents were added at the time points indicated by arrows. The concentration of agents was 1 AM for thapsigargin and 500 AM for maprotiline. Data are means F S.E.M. of five experiments.
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Fig. 4. Lack of involvement of phospholipase C in maprotiline-induced [Ca2 +]i rise. The experiments were performed in Ca2 +free medium. U73122 (2 AM) and maprotiline (500 AM) were added at the time points indicated by arrows. Data are mean F S.E.M. of five experiments.
Lack of involvement of phospholipase C in maprotiline-induced Ca2+ release The possibility that phospholipase C-inositol 1,4,5-trisphosphate pathway is involved in maprotilineinduced Ca2 + release was examined. Fig. 4 shows that after the pretreatment with 2 AM U73122, an inhibitor of phospholipase C (Thompson et al., 1991), 500 AM maprotiline induced a [Ca2 +]i rise that was indistinguishable from the control shown in Fig. 1B (n = 5; P > 0.05).
Discussion The novel finding of the present study is that the antidepressant maprotiline can induce significant increases in baseline [Ca2 +]i in human neuroblastoma cells. It has been previously reported that maprotiline inhibits gamma-aminobutyric acid- and N-methyl-D-aspartate-induced intracellular Ca2 + concentration ([Ca2 +]i) increases in primary cultured rat cortical neurons (Takebayashi et al., 2000), but to amplify serotonin 2A receptor-induced [Ca2 +]i mobilization in C6 glioma cells through a calmodulindependent pathway (Muraoka et al., 1998); however, we are the first to show that maprotiline at concentrations greater than 100 AM can trigger a rise in baseline [Ca2 +]i. The maprotiline-induced [Ca2 +]i rise is equally contributed by extracellular Ca2 + influx and intracellular Ca2 + release, as the Ca2 + signal was reduced by half by removal of extracellular Ca2 +. Our data suggest that maprotilineinduced Ca2 + influx portion is mediated by nifedipine-sensitive Ca2 + channels, because the maprotilineinduced [Ca2 +]i rise in Ca2 +-containing medium was reduced by 50% by nifedipine. This Ca2 + influx channel appears to be selective for nifedipine as verapamil and diltiazem failed to have an effect. Consistently, the Ca2 + influx induced by riluzole in the same neuroblastoma cell line was also blocked by nifedipine, but was not altered by verapamil (Wang et al., 2001). Nifedipine-sensitive Ca2 + channels
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and N-type Ca2 + channels have been reported previously (Grassi et al., 1994; Carabelli et al., 1996) in this cell line. In Ca2 +-containing medium, maprotiline-induced [Ca2 +]i increase displayed a gradual rise and a stable, flat plateau. In contrast, in Ca2 +-free medium, maprotiline induced a smaller [Ca2 +]i increase followed by a clear decrease. This suggests that extracellular Ca2 + influx contributes not only to the initial increase, but also to the prolonged phase of maprotiline-induced [Ca2 +]i ruse in Ca2 +containing medium. The endoplasmic reticulum Ca2 + stores have been shown to play an important role in a [Ca2 +]i rise in IMR32 cells (Wang et al., 2001). Regarding the Ca2 + stores of the maprotiline-induced response, the thapsigargin-sensitive endoplasmic reticulum store appears to be dominant, because maprotiline could not release more intracellular Ca2 + after thapsigargin had depleted the endoplasmic reticulum Ca2 + store. The maprotiline -induced Ca2 + releasing process may be independent of inositol 1,4,5-trisphosphate since suppression of phospholipase C did not affect maprotiline-induced Ca2 + release. Taken together, these findings suggest that in human IMR32 neuroblastoma cells, maprotiline increases baseline [Ca2 +]i by stimulating extracellular Ca2 + influx and also by causing intracellular Ca2 + release from the endoplasmic reticulum via a phospholiase C-independent manner. Since a rise in baseline [Ca2 +]i or depletion of intracellular Ca2 + can interfere with many cellular responses, caution must be exercised in using maprotiline in other in vitro studies. Furthermore, previous studies of maprotiline commonly focused on the drug’s selective inhibition of norepinephrine reuptake, and ignored its possible other effects. Here we show evidence that maprotiline may alter signal transduction in neuronal cells, and this may supplement its antidepressant action or may contribute to its unwanted clinical side effects.
Acknowledgements This work was supported by grants from Veterans General Hospital-Kaohsiung (VGHKS92G-11) and NSC92-2320-B-075B-003 to CR Jan, and VGHKS93-92 to SS Hsu.
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