Effect of fluoxetine on intracellular Ca2+levels in bladder female transitional carcinoma (BFTC) cells

Pharmacological Research, Vol. 43, No. 5, 2001 doi:10.1006/phrs.2001.0810, available online at http://www.idealibrary.com on

EFFECT OF FLUOXETINE ON INTRACELLULAR CA2+ LEVELS IN BLADDER FEMALE TRANSITIONAL CARCINOMA (BFTC) CELLS KWONG-YUI TANGa , TIM LUa , CHENG-HO CHANGa , YUK-KEUNG LOb , JIN-SHIUNG CHENGb , JUE-LONG WANGc , HONG-TAI CHANGd,e and CHUNG-REN JANf,g,∗ a Department

of Psychiatry, b Department of Internal Medicine, c Department of Rehabilitation, of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813, e School of Medicine, National Yang Ming University, Taipei, Taiwan 813, f Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan 813, g Department of Biology and Institute of Life Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan d Department

Accepted 27 February 2001

The effect of the antidepressant fluoxetine on Ca2+ signaling in cultured cells was largely unknown. The effect of various concentrations of fluoxetine on [Ca2+ ]i in populations of bladder female transitional cancer (BFTC) cells was evaluated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca2+ ]i concentration dependently (20–100 µM) with an EC50 value of 30 µM. The response was inhibited by 50–60% on extracellular Ca2+ removal. In Ca2+ -free medium, pretreatment with 1 µM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) abolished 50 µM fluoxetine-induced Ca2+ release; whereas pretreatment with fluoxetine did not alter the thapsigargin-induced Ca2+ response. Addition of 3 mM Ca2+ increased [Ca2+ ]i after pretreatment with 50 µM fluoxetine in Ca2+ -free medium, suggestive of fluoxetine-induced capacitative Ca2+ entry. Suppression of inositol 1,4,5-trisphosphate formation by 2 µM U73122 (a phospholipase C inhibitor) did not affect 50 µM fluoxetine-induced Ca2+ release. Collectively, this study shows that fluoxetine increased [Ca2+ ]i in bladder cancer cells in a concentration-dependent fashion, by releasing Ca2+ from thapsigargin-sensitive Ca2+ stores in an IP3 -independent manner, c 2001 Academic Press and by inducing Ca2+ influx from extracellular medium.

K EY WORDS : antidepressant, bladder cancer cells, Ca2+ , fluoxetine, fura-2, serotonin.

INTRODUCTION Fluoxetine (Prozac, Lilly 110140) was the first selective serotonin reuptake inhibitor to be synthesized and has been used as an effective antidepressant [1, 2]. Evidence shows that alterations in central serotonin 5-HT2 receptor function may explain the pathophysiology of depression and, thus, depression may be corrected by treatment with a selective serotonin reuptake inhibitor. However, recent observations cast doubt on the concept that the antidepressant effect of fluoxetine is solely attributed to serotonin reuptake inhibition in the central nervous system. For example, fluoxetine was found to increase cyctoplasmic free Ca2+ levels ([Ca2+ ]i ) in human frontal cortex [3]. Helmeste et al. [4] demonstrated that fluoxetine did not increase [Ca2+ ]i in platelets while another antidepressant sertraline did. Fluoxetine was ∗ Corresponding author. Department of Medical Education and Re-

search, Veterans General Hospital-Kaohsiung, 386 Ta Chung 1st Rd, Kaohsiung, Taiwan. E-mail: [email protected] 1043–6618/01/050503–06/$35.00/0

thought to dilate vascular smooth muscle by inhibiting Ca2+ channels or by interfering with Ca2+ signaling [5], and to inhibit depolarization-induced Ca2+ uptake in rat hippocampus synaptosomes [6]. Fluoxetine can block volume-regulated anion channels [7]. Fluoxetine has a dual effect on T cell proliferation by modulating the protein kinase C and protein kinase A pathways [8]. Together, fluoxetine appears to have multiple effects on functions of cells from neuronal and non-neuronal origins, which are dissociated from selective inhibition of serotonin uptake. In this study, fura-2 was used as a fluorescent dye for detecting [Ca2+ ]i in human bladder cancer cells. An increase in [Ca2+ ]i is a key signal in initiating and regulating various physiological and pathological cellular processes in all cell types; however, prolonged elevations in [Ca2+ ]i may injure the cells [9–11]. The [Ca2+ ]i signal could be triggered by intracellular Ca2+ release from Ca2+ stores and/or extracellular Ca2+ influx via selective pathways. Inositol 1,4,5-trisphosphate is an important c 2001 Academic Press

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messenger for intracellular Ca2+ release [9, 10]. Release of Ca2+ from intracellular stores often leads to the refilling of the stores and a sustained [Ca2+ ]i increase by causing extracellular Ca2+ influx via a process termed capacitative Ca2+ entry [12]. The exact mechanism of capacitative Ca2+ entry is controversial. Bladder female transitional cancer (BFTC) cells have been widely used as a model for biochemical research on human bladder cancer cells [13]. It was recently shown that [Ca2+ ]i in this cell could be increased by NPC15199, a novel anti-inflammatory agent [14]; however, the effect of other agents on [Ca2+ ]i in this cell has not been explored. The results in the present study show that fluoxetine induced significant increases in [Ca2+ ]i in BFTC cells. The concentration–response relationship was established, and the sources of the [Ca2+ ]i response and the underlying mechanisms were evaluated.

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was from RBI, and U73122 and U73343 were from Biomol. The other reagents were obtained from Sigma.

Statistical analysis The traces are typical of four or five similar responses. All values are reported as means ± SEM of four or five experiments. Because the data from each experiment were the average of responses from 0.5 million cells, the variation among experiments was typically small. This means that the mean ± SEM of four or five experiments can reveal significant results. Statistical comparisons were determined by using Student’s t-test, and significance was accepted when P< 0.05.

RESULTS

Effects of fluoxetine on [Ca2+ ]i in BFTC cells MATERIALS AND METHODS

Cell culture BFTC 905 human bladder female transitional cancer cells were cultured in RPMI-1640 medium supplemented with 5–10% heat-inactivated fetal bovine serum, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. Cells were kept at 37 ◦ C in 5% CO2 -containing humidified air.

Solutions Ca2+ medium (pH 7.4) contained (in mM): NaCl 140; KCl 5; MgCl2 1; CaCl2 2; Hepes 10; glucose 5. Ca2+ -free medium contained no Ca2+ plus 1 mM EGTA.

Optical measurements of [Ca2+ ]i

Trypsinized cells (106 ml−1 ) were allowed to recover in RPMI-1640 medium for 1 h before been loaded with 2 µM 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(20 -amino-50 -methylphenoxy)-ethaneN,N,N,N-tetraacetic acid pentaacetoxymethyl ester (fura-2/AM) for 30 min at 25 ◦ C in the same medium. The cells were washed and resuspended in Ca2+ medium. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 ◦ C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer by continuously recording excitation signals at 340 and 380 nm and emission signals at 510 nm at 1-s intervals. Maximum and minimum fluorescence values were obtained by adding Triton X-100 (0.1%) and EGTA (20 mM) sequentially at the end of an experiment. The ratio of excitation signals at 340 and 380 nm was used to calculate [Ca2+ ]i as described previously [15].

Chemical reagents The reagents for the cell culture were obtained from Gibco, fura-2/AM was from Molecular Probes, fluoxetine

At concentrations between 20–100 µM, fluoxetine increased [Ca2+ ]i in the presence of extracellular Ca2+ [Fig. 1(a)]. At a concentration of 10 µM, fluoxetine had no effect (trace c). The Ca2+ signal saturated at 50 µM of fluoxetine because 100 µM fluoxetine induced similar results. Over a time period of 250 s the [Ca2+ ]i increase consisted of an initial rise and an elevated phase. For example, at a concentration of 50 µM (trace a), fluoxetine induced an immediate, gradual increase in [Ca2+ ]i which peaked after a latency time of 31 ± 4 s (n = 4) at a net value of 150 ± 4 nM (baseline subtracted). This Ca2+ signal was followed by a gradual decay which reached a net plateau of 22 ± 3 nM at the time point of 140 s and the plateau remained for the rest of the measurement. The rise of the Ca2+ signal was slower in response to lower concentrations of fluoxetine. Extracellular Ca2+ removal significantly reduced 20–100 µM fluoxetine-induced Ca2+ signals. Figure 1(b) shows typical responses induced by 50 µM (trace a) and 30 µM (trace b) of fluoxetine. At a concentration of 10 µM, fluoxetine had no effect (trace c). Moreover, the plateau phase of the response was abolished by Ca2+ removal. The concentration–response plots both in the presence and absence of extracellular Ca2+ are illustrated in Fig. 1(c). Ca2+ removal reduced the [Ca2+ ]i increase induced by 20–100 µM fluoxetine by 50–60% in the net maximum [Ca2+ ]i (n = 4–6; P< 0.05). These data indicate an apparent EC50 value of about 30 µM.

Intracellular Ca2+ stores of fluoxetine-induced [Ca2+ ]i increases

The role of the endoplasmic reticulum Ca2+ store in fluoxetine-induced intracellular Ca2+ release was explored. Figure 2(a) shows that in Ca2+ -free medium 1 µM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump [16], induced a [Ca2+ ]i increase with a net maximum value of 31 ± 3 nM (n = 5). The response decayed to the baseline within 4 min. Subsequently, the addition of 50 µM fluoxetine failed

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Fig. 1. The concentration of fluoxetine was 50 µM in trace a, 30 µM in trace b, and 10 µM in trace c. The experiments were performed in Ca2+ medium. Fluoxetine was added at the time indicated by the arrows. (b) Similar to (a) except cells were bathed in Ca2+ -free medium. (c) Concentration–response plots of fluoxetine-induced Ca2+ signals in the presence (filled circles) or absence (open circles) of extracellular Ca2+ . The y-axis is the percentage of control which was the net maximum [Ca2+ ]i value (baseline subtracted) induced by 50 µM fluoxetine in Ca2+ medium. ∗ P< 0.05.

to increase [Ca2+ ]i . Figure 2(b) shows that in Ca2+ -free medium, after pretreatment with 50 µM fluoxetine for 5 min, addition of 1 µM thapsigargin induced a [Ca2+ ]i increase with a net peak value of 35 ± 5 nM which was indistinguishable from the control thapsigargin response shown in Fig. 2(a) (n = 4; P > 0.05).

Effect of fluoxetine on capacitative Ca2+ entry Experiments were performed to examine whether capacitative Ca2+ entry may play a role in fluoxetineinduced Ca2+ influx. Capacitative Ca2+ entry was measured by adding 3 mM Ca2+ to cells pretreated with fluoxetine in Ca2+ -free medium. Figure 3 shows that after depleting Ca2+ stores for 200 s with 50 µM fluoxetine, addition of 3 mM Ca2+ induced a [Ca2+ ]i increase with a net maximum [Ca2+ ]i value of 85 ± 4 nM

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(trace a; n = 5) which was three-fold higher than the control (adding Ca2+ alone without fluoxetine treatment; 28 ± 3 nM; trace b; P< 0.05).

Is inositol 1,4,5-trisphosphate involved in mediating fluoxetine-induced Ca2+ release? Figure 4(a) shows that in Ca2+ -free medium 10 µM ATP (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer) induced a [Ca2+ ]i increase, suggestive of the existence of an inositol 1,4,5-trisphosphatedependent Ca2+ -releasing machinery in BFTC cells. Figure 4(b) shows that incubation with 2 µM U73122 for 220 s to inhibit phospholipase C [17] prevented subsequently applied 10 µM ATP from increasing [Ca2+ ]i (n = 6). Conversely, 10 µM U73343 (an inactive U73122 analogue) had no effect on ATP-induced [Ca2+ ]i increases (not shown). This indicates that U73122 effectively inhibited inositol 1,4,5-trisphosphate formation. After U73122 pretreatment for 250 s, application of 50 µM fluoxetine induced a [Ca2+ ]i increase indistinguishable from the control shown in Fig. 2(b) (n = 4).

DISCUSSION This is the first report showing that fluoxetine increases [Ca2+ ]i in bladder cancer cells. Although a recent paper reported that fluoxetine increases [Ca2+ ]i in human frontal cortex [3], the underlying mechanism was not investigated. The present study examined the underlying mechanisms of the fluoxetine-induced [Ca2+ ]i increase in bladder cancer cells, and demonstrated that fluoxetine acts by releasing intracellular Ca2+ from the thapsigargin-sensitive endoplasmic reticulum Ca2+

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Fig. 4. Effect of U73122 on fluoxetine-induced intracellular Ca2+ release. In Ca2+ -free medium, (a) 10 µM ATP was added at 30 s. (b) Two µM U73122, 10 µM ATP, and 50 µM fluoxetine were added at 30 s, 220 s, and 290 s, respectively.

store without recruiting inositol 1,4,5-trisphosphate, and by triggering extracellular Ca2+ influx. Based on the present and previous data, it seems that fluoxetine alters Ca2+ signaling in different manners in different cell types. For example, although fluoxetine increases [Ca2+ ]i in some cells, it dilates vascular smooth muscle by inhibiting Ca2+ channels or by interfering with Ca2+ signaling [5], and inhibits depolarization-induced Ca2+ uptake in rat hippocampus synaptosomes [18]. Our data suggest that in bladder cancer cells, fluoxetine induced a concentration-dependent [Ca2+ ]i increase with an EC50 of 30 µM. Extracellular Ca2+ removal inhibited the maximum [Ca2+ ]i value by about half and abolished the plateau phase. This suggests that Ca2+ influx and Ca2+ release contributed about equally to the Ca2+ signal, and that the plateau phase was solely caused by Ca2+ influx.

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The intracellular Ca2+ store for fluoxetine-induced [Ca2+ ]i increases is most likely the thapsigarginsensitive endoplasmic reticulum store because fluoxetine failed to release more Ca2+ after the thapsigargininduced [Ca2+ ]i increase had subsided to baseline. The reason why thapsigargin-induced Ca2+ release was not altered after fluoxetine depleted the thapsigarginsensitive endoplasmic reticulum stores could be because fluoxetine-induced Ca2+ release was a transient event, and a substantial amount of the released Ca2+ ions had returned to the endoplasmic reticulum stores before addition of thapsigargin. It appears that inositol 1,4,5trisphosphate is not involved in fluoxetine-induced Ca2+ release because inhibiting inositol 1,4,5-trisphosphate formation did not affect the Ca2+ release. How exactly fluoxetine releases Ca2+ remains to be investigated. Our data suggest that fluoxetine may activate Ca2+ influx via capacitative Ca2+ entry. However, the data could also be interpreted as fluoxetine directly opening a plasmalemmal Ca2+ channel in a manner independent of Ca2+ store depletion. Collectively, the present study shows that fluoxetine increased [Ca2+ ]i in human bladder cancer cells. The concentrations used in this study were at the therapeutic range of fluoxetine. Koran et al. [19] reported that the mean plasma fluoxetine levels of 200 patients were 138 ± 59 µM after taking 60 mg per day of fluoxetine. Thus, the effect of fluoxetine on [Ca2+ ]i cannot be ignored when this drug is clinically used in treating depression. ACKNOWLEDGEMENTS This work was supported by grants from National Science Council (NSC89-2320-B-075B-009), Veterans General Hospital-Kaohsiung (VGHKS89-104) and VTY Joint Research Program, Tsou’s Foundation (VTY89-P3-21) to CRJ; VGHKS89-124 to KYT. REFERENCES 1. Wong DT, Bymaster FP, Engleman EA. Prozac (fluoxetine, Lilly 110140), the first selective serotonin uptake inhibitor and an antidepressant drug: twenty years since its first publication. Life Sci 1995; 57: 411–41.

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