Basic and Translational Science TRPV2 Activation Induces Apoptotic Cell Death in Human T24 Bladder Cancer Cells: A Potential Therapeutic Target for Bladder Cancer Takahiro Yamada, Takashi Ueda, Yasuhiro Shibata, Yosuke Ikegami, Masaki Saito, Yusuke Ishida, Shinya Ugawa, Kenjiro Kohri, and Shoichi Shimada OBJECTIVES To investigate the functional expression of the transient receptor potential vanilloid 2 (TRPV2) channel protein in human urothelial carcinoma (UC) cells and to determine whether calcium influx into UC cells through TRPV2 is involved in apoptotic cell death. MATERIAL AND METHODS The expression of TRPV2 mRNA in bladder cancer cell lines (T24, a poorly differentiated UC cell line and RT4, a well-differentiated UC cell line) was analyzed using reverse transcriptase- polymerase chain reaction. The calcium permeability of TRPV2 channels in T24 cells was investigated using a calcium imaging assay that used cannabidiol (CBD), a relatively selective TRPV2 agonist, and ruthenium red (RuR), a nonselective TRPV channel antagonist. The death of T24 or RT4 cells in the presence of CBD was evaluated using a cellular viability assay. Apoptosis of T24 cells caused by CBD was confirmed using an annexin-V assay and small interfering RNA (siRNA) silencing of TRPV2. RESULTS TRPV2 mRNA was abundantly expressed in T24 cells. The expression level in UC cells was correlated with high-grade disease. The administration of CBD increased intracellular calcium concentrations in T24 cells. In addition, the viability of T24 cells progressively decreased with increasing concentrations of CBD, whereas RT4 cells were mostly unaffected. Cell death occurred via apoptosis caused by continuous influx of calcium through TRPV2. CONCLUSIONS TRPV2 channels in UC cells are calcium-permeable and the regulation of calcium influx through these channels leads directly to the death of UC cells. TRPV2 channels in UC cells may be a potential new therapeutic target, especially in higher-grade UC cells. UROLOGY 76: 509.e1–509.e7, 2010. © 2010 Elsevier Inc.

ladder carcinoma is the second most common Transient receptor potential (TRP) channels are malignancy of the urinary tract and nearly 90% of Ca2ϩ-permeable channels that contribute to intracellular all primary tumors of the bladder are urothelial Ca2ϩ homeostasis. Recently, there has been increasing B 1 carcinomas (UCs). Currently, intravesical instillation of evidence for the association of TRP channels with can- bacillus Calmette-Guérin (BCG) is the most effective cer. The expression levels of members of the TRP Ca- and widely used agent for the treatment of superficial nonical (TRPC), Melastatine (TRPM), and vanilloid UC.2 In addition, other agents, including mitomycin C, (TRPV) families are correlated with the emergence have been used to prevent recurrence.3 However, recur- and/or progression of certain epithelial cancers.4,5 In UC, rence after intravesical instillation remains frequent. As a TRPV1 expression has been shown to progressively de- result, the development of new drugs that target only UC crease as tumor stage increases and cell differentiation cells is desirable. declines.6 In contrast, the expression of TRPV2 mRNA and proteins was enhanced in higher-grade UC speci- 7 The first two authors contributed equally to this work. mens and UC cell lines. Therefore, modulators of the From the Department of Neurobiology and Anatomy, Graduate School of Medical TRP channels expressed in high-grade UC cells may be Sciences, Nagoya City University, Nagoya, Japan; Department of Nephro-urology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan; and attractive targets for the medical treatment of malignant Department of Neuroscience and Cell Biology, Osaka University, Graduate School of UC. Medicine, Osaka, Japan Here, we examined whether TRPV2 channels are Reprint requests: T. Ueda, Department of Neurobiology and Anatomy, Graduate School of Medical Sciences, Nagoya City University, Kawasumi, Mizuho-cho, Mizuho- functional in 2 UC cell lines: T24, a poorly differentiated ku, Nagoya, Aichi 467–8601, Japan. E-mail: [email protected] line of UC cells, and RT4, which is well-differentiated. © 2010 Elsevier Inc. 0090-4295/10/$34.00 509.e1 All Rights Reserved doi:10.1016/j.urology.2010.03.029 We used both physiological and pharmacologic ap- Measurement of Intracellular 2؉ proaches. Although there is a lack of selective pharma- Calcium Concentrations ([Ca ]i) cologic tools specific to TRPV2, it has been recently Experiments were performed 48-72 hours after subculture. T24 2ϩ reported that cannabidiol (CBD) may be a relatively cells were incubated with a fluorescent Ca indicator (fura-2 acetoxymethyl ester, 10 ␮M, Life Technologies) in assay buffer selective TRPV2 agonist.8 We therefore used CBD as a for 45 minutes at room temperature. The loading solution was selective TRPV2 agonist in the present study. In addi- washed thoroughly for 10 minutes and the cells were stimulated tion, we investigated the association between TRPV2 with CBD (3 and 30 ␮M), 4␣-phorbol 12,13-didecanoate (4␣- and UC cell death. The most interesting finding was that PDD, 10 ␮M), capsaicin (1 ␮M), menthol (300 ␮M), carvacrol continuous exposure to CBD-mediated apoptotic cell (500 ␮M), or ruthenium red (RuR, 25-50 ␮M) using a bath death via TRPV2 in T24 cells. This investigation of the perfusion system at a flow rate of 2-3 mL/min. We recorded 2ϩ functional properties of UC cells is an important step in (Ca )i changes using an Olympus IX-70 microscope equipped the development of novel strategies for antitumor ther- with the Argus/HiSCA system (Hamamatsu Photonics, Hama- apeutics. matsu, Japan). Acquisition and analysis of the fluorescence images were performed with Argus/HiSCA, version 1.65 soft- ware.9 The assay buffer solution was prepared according to a previously described method.10 For further analysis, we selected MATERIAL AND METHODS cells showing similar internal calcium mobilization after 4␣- The Center for Experimental Animal Sciences at Nagoya City PDD was applied (10-15 cells). University approved the following experiments. Transfection of Small Interfering RNA (siRNA) T24 cells were seeded onto 60-mm dishes and incubated for 24 Cell Lines and Culture Conditions hours at 37 °C. The cells were then washed with McCoy’s 5 A modified medium and transfected with a siRNA for TRPV2 Bladder cancer cell lines (T24 and RT4) were obtained from (Silencer predesigned siRNA, ID s28081 or s28082) (Life Tech- the American Type Culture Collection (ATCC, Rockville, nologies) or scrambled siRNA as a negative control (Silencer MD). The T24 line is a poorly differentiated bladder UC cell negative control # 1 siRNA; AM4611) (Life Technologies) line, whereas the RT4 line is a well-differentiated bladder UC using lipofectamine 2000 (Life Technologies). At 48 hours after cell line. Both cell lines were cultured in McCoy’s 5 A modified transfection, transfected cells were replated, further incubated medium (Life Technologies, Carlsbad, CA), containing 5% for 48 hours, and used in RT-PCR, calcium imaging analysis, fetal calf serum, at 37 °C in a 5% CO atmosphere saturated 2 and an annexin V assay. with water vapor. Cellular Viability Assay Reverse Transcription Polymerase Chain Reaction (RT- The viabilities of T24 and RT4 cells were evaluated using the PCR). First, 3 ␮g of total RNA isolated from T24 or RT4 cells was Cell Counting Kit-8 (Dojin, Kumamoto, Japan), based on the subjected to random-primed reverse transcription using Super- 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro- Script II (Life Technologies). Next, 2.5% of the samples were mide (MTT) assay, according to the protocol described by amplified through 35 cycles of PCR with the following primers. For Yamamura et al. (2008).11 These cells were subcultured in human TRPV1 (product length ϭ 372 bp; GenBank accession 96-well plates and incubated at 37°C in a 5% CO2 atmosphere No. NM080706): 5=-CTGCGGACCCACTCCAAAAGGA-3= saturated with water vapor for 24-48 hour (approximately 5000 (sense) and 5=-AGAGCAGCAGGCTCTCCAGATC-3= (anti- cells/well); they were then incubated for 24 hour in medium sense); human TRPV2 (327 bp; NM016113): 5=-CTGCA- containing various concentrations of CBD with or without RuR CATCGCCATTGAGAAGA-3= (sense) and 5=-TTGGAG- (50 ␮M). Cellular viabilities in the absence and presence of GAGCCCATCATACATG-3= (antisense); human TRPV3 (288 CBD were quantitated colorimetrically by examining absor- bp; NM145068): 5=-GCTGAAGAAGCGCATCTTTGCA-3= bance at 450 nm (A450). Control cells were treated in exactly (sense) and 5=-TCATAGGCCTCCTCTGTGTACT-3= (anti- the same way, but with a solution lacking CBD (0.1%-2.0% sense); human TRPV4 (379 bp; NM021625): 5=-TACCTGT- methanol). We confirmed that methanol concentrations up to GTGCCATGGTCATCT-3= (sense) and 5=-TGCTATAG- 2.0% had no effect on cellular responses. The relative viabilities GTCCCCGTCAGCTT-3= (antisense); human TRPM8 (621 bp; were determined using the following equation: Relative viabil- ϭ ϫ NM024080): 5=-CCTGTTCCTCTTTGCGGTGTGGAT-3= ity (%) (A450 of CBD-treated cells/A450 of control cells) (sense) and 5=-TCCTCTGAGGTGTCGTTGGCTTT-3= (anti- 100. sense); human TRPA1 (541 bp; NM007332): 5=-GACCA- CAATGGCTGGACAGCT-3= (sense) and 5=-GTACCATT- Annexin-V Assay ␤ GCGTTGAGGGCTGT-3= (antisense); human -actin (298 bp; Apoptosis in T24 cells was evaluated using an annexin-V assay NM001101): 5=-GATCCTCACCGAGCGCGGCTACA-3= (FITC annexin V apoptosis Detection Kit I, 556 547) (BD (sense) and 5=-GCGGATGTCCACGTCACACTTCA-3= (an- Biosciences, San Jose, CA), which was performed according to tisense). Beta-actin was used for quantification of the samples. The the manufacturer’s protocol. Briefly, after incubation in a me- PCR products obtained were separated by electrophoresis in a 1% dium containing 30 ␮M CBD for 15 hour, a single-cell suspen- agarose gel. We checked the molecular identity and homogeneity sion (100 000 cells in 100 ␮L) was stained with annexin-V of the resulting PCR products using DNA sequencing and con- FITC and Propidium Iodide (PI) for 15 minutes at room tem- firmed through the BLAST database that the primers were specific perature in the dark. After this incubation, two-color (FL1 and for each TRP channel. FL2) flow cytometry analysis was conducted using a FacsCalibur

509.e2 UROLOGY 76 (2), 2010 Figure 1. RT-PCR analysis of the expression of mRNAs for transient receptor potential vanilloid 2 (TRPV2) and other TRP channels in T24 and RT4 cells. (A) TRP mRNA expression in T24 cells. The sizes of the DNA standards are indicated in the left margin. (B) TRPV2 and TRPV4 mRNA expression in T24 cells and RT4 cells. The controls, ␤-actin fragments, are shown in the lower panel. RT (Ϫ) indicates templates without reverse transcriptase. machine (BD Biosciences). Ten thousand cells per sample were presence of functional TRPV2 channels in the T24 cells. analyzed. We also confirmed that T24 cells failed to respond to capsaicin (a TRPV1-selective agonist), menthol (a TRPM8 Statistical Analysis agonist), or carvacrol (an agonist of TRPV3 or TRPA1) Pooled data are shown as mean Ϯ SE. Statistical significance (data not shown). These findings, together with the RT- was determined using analysis of variance with Bonferroni’s PCR data, imply that TRPV2 is abundantly expressed in comparison test. Significant differences are indicated in the T24 cells (high-grade UC cells), but not in RT4 cells P Ͻ P Ͻ figures as .01 or .001. (low-grade UC cells).

RESULTS Silencing of TRPV2 in T24 Cells Expression of TRP Channel mRNA in T24 Cells To determine the involvement of TRPV2 in CBD-in- To investigate the expression of TRPV2 and other TRP duced activation of T24 cells, we performed siRNA si- channels in poorly differentiated bladder UC cells (T24), lencing and evaluated the effect of TRPV2 knockdown we isolated total RNA from T24 cells and performed using RT-PCR and a calcium imaging assay. T24 cells RT-PCR with primer sets for various TRP channels. As were transfected with 2 different siRNA sequences. As shown in Fig. 1A, products with the predicted sizes and shown in Fig. 3(A), TRPV2 mRNA expression was sup- nucleotide sequences for TRPV2 and TRPV4 mRNA pressed by both siRNA1 and siRNA2, whereas neither were amplified from the cells (lanes 2 and 4, respec- the control nor the negative control siRNA had any tively). In contrast, PCR products corresponding to effect on TRPV2 mRNA expression. The invariable ex- TRPV1, TRPV3, TRPM8, or TRPA1 were not detected pression of TRPV4 mRNA indicates that these siRNA in the T24 cells (lanes 1, 3, 5, and 6, respectively). sequences selectively suppressed TRPV2 gene expression. Moreover, in a calcium imaging assay, siRNA1 and Previous reports have shown that the expression levels of ϩ 7 2 TRPV2 mRNA in UC cells increase with cancer grade. siRNA2 markedly suppressed CBD-induced [Ca ]i in- To confirm this finding, we compared the expression creases (Fig. 3B, 3C), whereas neither siRNA had any ␣ 2ϩ levels of TRPV2 mRNA in T24 cells to those in RT4 effect on 4 -PDD-induced [Ca ]i mobilization (Fig. 3B– cells. As shown in Fig. 1B, TRPV2 transcripts were more 3D). These results indicate that CBD induces an increase 2ϩ highly expressed in T24 cells than in RT4 cells. in [Ca ]i via TRPV2 channels in T24 cells. It should be noted that the siRNA fully inhibited the increase in 2ϩ ␮ ␮ Functional Analysis of TRPV2 in T24 Cells [Ca ]i that was induced by 30 M CBD as well as 3 M To confirm the presence of functional TRPV2 channels CBD (Fig. 3C), whereas RuR was less effective in sup- in T24 cells, we physiologically investigated the effects of pressing it (Fig. 2D). Thus, RuR treatment might be an agonist and an antagonist of TRPV2 on RT4 and T24 insufficient to suppress robust TRPV2 activation. cells. In RT4 cells, although 4␣-PDD (a TRPV4-selec- 2ϩ tive agonist) caused [Ca ]i to increase significantly Viability of T24 Cells in the ⌬ 2ϩ ϭ Ϯ ( [Ca ]i 0.62 0.06%, 36/42, 85.7% of the cells), Presence of a TRPV2 Agonist CBD displayed no apparent effect on any of the cells It has been shown that continuous Ca2ϩ influx through ⌬ 2ϩ ϭ Ϯ 2ϩ 2ϩ examined ( [Ca ]i 0.03 0.005, 0/42) (Fig. 2A). In Ca -permeable channels and high [Ca ]i kill cancer contrast, T24 cells responded to 4␣-PDD (60/69, 87.0%) cells via apoptosis and necrosis.4 We examined the effects (Fig. 2B). Moreover, almost all the cells exhibited a of Ca2ϩ influx through TRPV2 channels on the survival 2ϩ dose-dependent increase in [Ca ]i in response to CBD of T24 cells using a colorimetric quantitative kit based on ␮ ⌬ 2ϩ ϭ Ϯ (3 M, [Ca ]i 0.20 0.02%, 157/165, 95.2% and the MTT assay. T24 and RT4 cells were incubated for 24 ␮ ⌬ 2ϩ ϭ Ϯ 30 M, [Ca ]i 0.40 0.06%, 47/49, 95.9%) (Fig. hours in media containing various concentrations of 2C, 2D). These responses were fully or partially sup- CBD, as shown in Fig. 4A. The viability of T24 cells was pressed by 50 ␮M RuR (Fig. 2C, 2D), suggesting the found to be dependent on the concentration of CBD

UROLOGY 76 (2), 2010 509.e3 2ϩ 2ϩ Figure 2. Representative [Ca ]i response in RT4 (A) and T24 cells (B-D). (A) RT4 cells exhibited an increase in [Ca ]i in response to 4␣-PDD (10 ␮M), but not to cannabidiol (CBD, 3 ␮M). The 4␣-PDD–induced response was completely ␮ ␣ ␮ 2ϩ inhibited by extracellular ruthenium red (RuR, 25 M). (B) 4 -PDD (10 M) also induced an RuR-sensitive increase in [Ca ]i in T24 cells. (C) Moreover, in most T24 cells (approximately 95% of the cells examined), 3 ␮M CBD evoked a marked 2ϩ ␮ increase in [Ca ]I, which was fully suppressed by extracellular RuR (50 M). (D) In contrast, the responses induced by 30 ␮M CBD was reduced by approximately 50% with the addition of extracellular RuR (50 ␮M) in T24 cells.

Figure 3. siRNA-mediated knockdown of TRPV2 in T24 cells. The expression of TRPV2 and TRPV4 was analyzed at the mRNA level by RT-PCR (A) and at the functional level by a calcium imaging assay (B-D). (B) The effect of 3 ␮M CBD and 4␣-PDD on siRNA1-transfected T24 cells. (C) The effect of 30 ␮M CBD and 4␣-PDD on siRNA1-transfected T24 cells. Ionomycin (3 ␮M), a calcium ionophore, was used to prove that cells were viable. (B, C, and D) The siRNAs did not have any effects on ␣ 2ϩ Ͻ 4 -PDD–induced [Ca ]i responses. Statistically significant differences are expressed as **P .001 vs. scrambled siRNA. Experimental data were obtained from 10 cells.

509.e4 UROLOGY 76 (2), 2010 Figure 4. (A) Viability of T24 cells in the presence of a TRPV2 agonist. Cell death from exposure to different concentrations of CBD (1.5-60 ␮M) was observed in T24 cells after 24 hours of culture. Experimental data were obtained from 8-12 wells. (B) Exposure to 30 ␮M CBD for 24 hours did not affect the survival of RT4 cells, in contrast to T24 cells. Moreover, 50 ␮M RuR partially, but not fully, suppressed the effect of CBD on T24 cells. Statistically significant differences are expressed as *P Ͻ.01, **P Ͻ.001 vs. RT4 cells or T24 cells treated with RuR. Experimental data were obtained from 8-12 wells. (C and D) CBD-induced apoptosis of T24 cells as determined by the Annexin-V assay and effect of TRPV2 siRNA after exposure to 30 ␮M CBD for 15 hours. Cells in the lower left quadrant (Annexin-V-FITCϪ/PIϪ) are viable, those in the lower right quadrant (Annexin-V-FITCϩ/PIϪ) are early apoptotic, and those in the upper right quadrants (Annexin-V-FITCϩ/PIϩ) are late apoptotic or necrotic. Transfection of T24 cells with scrambled siRNA apparently caused apoptosis and necrosis in response to CBD (C), whereas transfection with TRPV2 siRNA1 did not (D). Data were obtained from 3 separate experiments and representative results are shown.

(ranging from 1.5-60 ␮M), with higher concentrations treatment with 30 ␮M CBD for 15-hour induced apo- resulting in more cell death. When comparing the sur- ptosis in T24 cells transfected with scrambled siRNA vival of T24 cells to the survival of RT4 cells, however, (annexin-V-FITCϩ/PIϪϭ11.4%, n ϭ 3), similar to 30 ␮M CBD was most effective in inducing T24-specific what occurred in control cells (data not shown). This cell death. CBD did not affect RT4 cells at a concentra- CBD-induced apoptosis decreased significantly in the tion of 30 ␮M, whereas it significantly reduced T24 cell cells transfected with siRNA1 (annexin-V-FITCϩ/PIϪ, viability, by 40%; 50 ␮M RuR significantly suppressed 1.31%, n ϭ 3) (Fig. 4D). Similar results were also ob- the effect of CBD (Fig. 4B). tained in the cells transfected with siRNA2 (data not shown). Moreover, the silencing was effective against Induction of Apoptosis Via TRPV2 in T24 Cells necrotic cell death (upper right quadrant; annexin-V- To investigate whether CBD induces apoptosis in T24 FITCϩ/PIϩ), indicating that CBD-induced apoptosis cells and whether TRPV2 is involved in this process, we and necrosis in T24 cells are the result of continuous 2ϩ combined an annexin-V assay and the siRNA strategy to Ca influx through TRPV2 channels. detect apoptosis. In the early stages of apoptosis, phos- phatidylserine (PS) is translocated from the inner side of the plasma membrane to the outer layer, causing PS to COMMENT become exposed on the external surface of the cells. In the present study, we focused on a Ca2ϩ-permeable Annexin-V is a Ca2ϩ-dependent phospholipid-binding TRP channels expressed abundantly in human bladder protein with a high affinity for PS.12 As shown in Fig. 4C, cancer cells, and we examined, using Ca2ϩ imaging anal-

UROLOGY 76 (2), 2010 509.e5 ysis and biochemical approaches, whether the regulation We further confirmed, using an annexin-V assay in of channel activity could lead to an inhibitory effect on combination with siRNA technology, that continuous the viability of UC cells. Our RT-PCR gene expression CBD treatment induces apoptotic cell death, and that analysis clearly showed that, among the TRP channels this effect is mediated by TRPV2 activation (Fig. 4C, examined, TRPV2 was abundantly expressed in the T24 4D). A previous study reported that, in human breast car- line, a line of poorly differentiated bladder UC cells; cinoma, CBD induces apoptosis via the direct and indirect however, it was not expressed in the RT4 line, which is activation of CB2 and TRPV1 receptors, cannabinoid/va- 2ϩ a differentiated UC cell line. Because TRPM8 and nilloid receptor-independent elevation of [Ca ]i, and re- TRPV1 transcripts, which are undetectable in T24 cells, active oxygen species.18 In T24 cells, however, silencing of have been reported to be upregulated in prostate can- TRPV2 mostly blocked CBD-induced apoptosis by 89% cers,13,14 TRP channels may be differentially expressed in (1.31% vs 11.4% of control), suggesting that TRPV2 chan- a variety of cancers. nels mainly contribute to CBD-induced apoptotic cell We also demonstrated that CBD induces the influx of death in high-grade UC cells. CBD has been shown to Ca2ϩ into human T24 UC cells, which express TRPV2 exert both central and peripheral actions with a broad endogenously, but not in RT4 cells, which lack TRPV2 spectrum of therapeutic effects on pain, neuroprotection, channel activity. Among the newly identified TRPV2 ago- anxiety, nausea, cerebral ischemia, type 1 diabetes, rheu- 21 nists, CBD is particularly interesting because it was the matoid arthritis, multiple sclerosis, and cancer. TRPV2 most potent and selective and its in vivo mechanism of has a wider distribution pattern: it is found in the brain, action and molecular targets remains unknown.8 CBD skin, spleen, lung, stomach, intestines, prostate, and pe- 22,23 was previously shown to activate human TRPV1 recep- ripheral blood. TRPV2 may not only constitute a tors.15 CBD acts as an allosteric modulator of ␮- and viable new drug target for high-grade bladder carcinoma, ␦-opioid receptors.16 It is an antagonist of CB1 and CB2 but may also comprise a mechanism by which CBD exerts cannabinoid receptors.17 In addition, the effect of CBD its clinically beneficial effects in vivo. was also observed in human MDA-MB-231 breast carci- noma, in which it acts through the direct and indirect CONCLUSIONS activation of the cannabinoid receptors CB2 and TRPV1 Our results clarified the differential TRPV2 expression in and the cannabinoid/vanilloid receptor-independent el- ϩ UC cells by demonstrating that they contribute to 2 18 ϩ evation of [Ca ]i. Because T24 cells lack capsaicin- 2 changes in the levels of [Ca ]i in high-grade UC cells, sensitive TRPV1 channel responses (our unpublished but not in low-grade UC cells. Moreover, continuous data) and silencing with TRPV2 siRNA almost abolished activation of TRPV2 triggered apoptosis in high-grade 2ϩ the stimulatory effect of CBD on [Ca ]i in T24 cells UC cells. This study provides new insight into the de- (Fig. 3B, 3C), we concluded that CBD predominantly velopment of novel strategies for antitumor therapeutics. 2ϩ stimulates TRPV2 channels and increases [Ca ]i in UC cells. References A growing number of studies have demonstrated that increases in [Ca2ϩ] regulate various signaling mecha- 1. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA i Cancer J Clin. 2005;55:10-30. nisms that control a variety of cellular processes such as 2. Alexandroff AB, Jackson AM, Odonnell MA, et al. BCG immu- proliferation, metabolism, and gene transcription; yet, notherapy of bladder cancer: 20 years on. Lancet. 1999;353:1689- 2ϩ under certain conditions, increases in [Ca ]i are cyto- 1694. toxic.4,19 Indeed, the activation of Ca2ϩ-permeable TRP 3. 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