Effect of phenethyl isothiocyanate on Ca 2+ movement and viability in MDCK canine renal tubular cells

Abstract

The effect of the natural compound phenethyl isothiocyanate (PEITC) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MDCK renal cells is unknown. This study explored whether PEITC changed [Ca²⁺]_i in MDCK cells using the Ca²⁺-sensitive fluorescent dye fura-2. PEITC at 200–700 μM increased [Ca²⁺]_i in a concentration-dependent manner. The signal was reduced by removing extracellular Ca²⁺. PEITC-induced Ca²⁺ influx was inhibited by nifedipine, econazole, SK&F 96365 and protein kinase C modulators. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited PEITC-induced rise in [Ca²⁺]_i. Incubation with PEITC also inhibited TG or BHQ-induced rise in [Ca²⁺]_i. Inhibition of phospholipase C with U73122 abolished PEITC-induced rise in [Ca²⁺]_i. At 15–75 μM, PEITC decreased viability. The cytotoxic effect of PEITC was enhanced by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester. Annexin V-FITC data suggest that 20 and 50 μM PEITC induced apoptosis. At 10 and 15 μM, PEITC did not increase reactive oxygen species (ROS) production. Together, in renal tubular cells, PEITC-induced rise in [Ca²⁺]_i by inducing phospholipase C-dependent Ca²⁺ release from endoplasmic reticulum and Ca²⁺ entry via store-operated Ca²⁺ channels. PEITC induced apoptosis in a concentration-dependent, ROS/Ca²⁺-independent manner.

Introduction

Dietary bioactive food components that interact with the immune response have considerable potential to reduce the risk of cancer. The upstream events by which isothiocyanates (ITCs) induce apoptosis have not been fully investigated. Numerous studies have reported that the apoptosis induced by ITCs was due to the generation of reactive oxygen species (ROS) as a result of conjugating with and, consequently, depleting cellular glutathione.^1,2 Phenethyl isothiocyanate (PEITC) induces cell cycle arrest and reduction of tubulin isotypes in human prostate cancer cells. ³ The role of reactive oxygen intermediates in cellular responses to PEITC is reported. ⁴ Induction of apoptosis by PEITC was shown in cells overexpressing Bcl-XL. ⁵ PEITC is found to cause G1 cell cycle arrest on HT-29 cells through the activation of p38 mitogen-activated protein kinase (MAPK) signaling pathway. ⁶ Induction of apoptosis by PEITC in human non–small-cell lung cancer cells involves protein binding. ⁷ PEITC triggers apoptosis in Jurkat cells made resistant by the overexpression of Bcl-2. ⁸ PEITC-induced apoptosis in PC3 human prostate cancer cells is mediated by ROS-dependent disruption of the mitochondrial membrane potential. ⁹ PEITC inhibits growth of ovarian cancer cells by inducing apoptosis via caspase and MAPK activation. ¹⁰ Mitochondria are the primary target in PEITC-induced apoptosis in human bladder cancer cells. ¹¹ Role of MAPKs in PEITC-induced apoptosis in human prostate cancer cells was noted. ⁹ Caspase-dependent apoptosis induced by PEITC was thought to be mediated by Bak and Bax. ¹² There is no research performed on the effect of PEITC in renal tubular cells.

Ca²⁺ ions play a pivotal role in various biological events. A rise in the intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) is a crucial trigger for numerous pathophysiological responses in cells. ¹³ However, an abnormal rise in [Ca²⁺]_i often causes interference of ion flux, dysfunction of proteins, apoptosis, proliferation, and so on. ¹⁴ In this regard, the effect of PEITC on [Ca²⁺]_i is unclear in any cell type.

Thus, we investigated the effect of PEITC on Ca²⁺ handling in MDCK canine renal cells. The MDCK cell line is a useful model for renal research. It has been shown that in this cell type, [Ca²⁺]_i can increase in response to the stimulation of exogenous ligands such as melittin, ¹⁵ anandamide ¹⁶ and celecoxib ¹⁷ and endogenous ligands like adenosine triphosphate (ATP) ¹⁸ and bradykinin. ¹⁹ In this study, fura-2 was used as a fluorescent Ca²⁺-sensitive dye to measure [Ca²⁺]_i. We show that PEITC induced both Ca²⁺ entry and Ca²⁺ release in MDCK cells. The rise in [Ca²⁺]_i is characterized, the concentration–response plots are established and the pathways underlying PEITC-evoked Ca²⁺ entry and Ca²⁺ release are explored. The effect of PEITC on cell viability was explored using the WST-1 (4-[3-(4-lodophenyl)-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate]) dye.

Materials and methods

Chemicals

The reagents for cell culture were from Gibco (Gaithersburg, Maryland, USA). PEITC and all other reagents were from Sigma-Aldrich (St Louis, Missouri, USA).

Cell culture

MDCK cells obtained from American Type Culture Collection were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin.

Solutions used in [Ca²⁺]_i measurements

Ca²⁺-containing medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 5 mM glucose. Ca²⁺-free medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 0.3 mM ethylenebis-oxyethylenenitrilotetraäcetic acid (EGTA), 10 mM HEPES and 5 mM glucose. PEITC was dissolved in dimethyl sulfoxide as a 1-M stock solution. The other agents were dissolved in water, ethanol or dimethyl sulfoxide. The concentration of organic solvents in the solution used in the experiments did not exceed 0.1% and did not alter viability or basal [Ca²⁺]_i.

[Ca²⁺]_i measurements

Confluent cells grown on 6 cm dishes were trypsinized and made into a suspension in culture medium at a density of 10⁶ per ml. Cell viability was determined by trypan blue exclusion (adding 0.2% trypan blue to 0.1 ml cell suspension. Dead cells would stain blue.). The viability was routinely greater than 95% after the treatment. Cells were subsequently loaded with 2 μM fura-2/AM for 30 min at 25°C in the same medium. After loading, cells were washed with Ca²⁺-containing medium twice and were made into a suspension in Ca²⁺-containing medium at a density of 10⁷ per ml. 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 immediately after 0.1 ml cell suspension was added to 0.9 ml Ca²⁺-containing or Ca²⁺-free medium, by recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1-s intervals. During the recording, reagents were added to the cuvette by pausing the recording for 2 s to open and close the cuvette-containing chamber. For the calibration of [Ca²⁺]_i, after completion of the experiments, the detergent Triton X-100 and 5 mM CaCl₂ were added to the cuvette to obtain the maximal fura-2 fluorescence. Then the Ca²⁺ chelator EGTA (10 mM) was added to chelate Ca²⁺ in the cuvette to obtain the minimal fura-2 fluorescence. [Ca²⁺]_i was calculated as described previously. ¹⁹ Mn²⁺ quench of fura-2 fluorescence was performed in Ca²⁺-containing medium containing 50 μM MnCl₂. MnCl₂ was added to the cell suspension in the cuvette 1 min before starting the fluorescence recoding. Data were recorded at excitation signal at 360 nm (Ca²⁺-insensitive) and emission signal at 510 nm at 1-s intervals as described previously. ²⁰

Cell viability assays

The measurement of cell viability was based on the ability of cells to cleave tetrazolium salts by dehydrogenases. Augmentation in the amount of developed color directly correlated with the number of live cells. Assays were performed according to manufacturer’s instructions specifically designed for this assay (Roche Molecular Biochemical, Indianapolis, Indiana, USA). Cells were seeded in 96-well plates at a density of 10,000 cells/well in the culture medium for 24 h in the presence of PEITC. The cell viability detecting reagent WST-1 (10 μl pure solution) was added to the samples after the PEITC treatment, and the cells were incubated for 30 min in a humidified atmosphere. In experiments using 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) to chelate cytosolic Ca²⁺, cells were treated with 10 μM BAPTA/AM for 1 h prior to incubation with PEITC. The cells were washed once with Ca²⁺-containing medium and incubated with/without PEITC for 24 h. The absorbance of the samples (A ₄₅₀) was determined using an enzyme-linked immunosorbent assay reader. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value.

Alex^® Flour 488 annexin V/PI staining for detection of apoptosis

Annexin V/propidium iodide (PI) staining assay was employed to further detect cells in early apoptosis stages. Cells were exposed to PEITC at the concentrations of 20 and 50 μM for 24 h. Cells were harvested after incubation and washed in cold phosphate-buffered saline (PBS). Cells were resuspended in 400 μl reaction solution with 10 mM of HEPES, 140 mM of NaC1 and 2.5 mM of CaC1₂ (pH 7.4). Alexa Fluor 488 annexin V/PI staining solution (Probes Invitrogen, Eugene, Oregon, USA) was added in the dark. After incubation for 15 min, the cells were collected and analyzed in a FACScan flow cytometry analyzer. Excitation wave was at 488 nm and the emitted green fluorescence of annexin V (FL1) and red fluorescence of PI (FL2) were collected using 530 and 575 nm band pass filters, respectively. A total of at least 20,000 cells were analyzed per sample. Light scatter was measured on a linear scale of 1024 channels and fluorescence intensity was on a logarithmic scale. The amount of early apoptosis and late apoptosis/necrosis were determined, respectively, as the percentage of annexin V⁺/PI⁻ or annexin V⁺/PI⁺ cells. Data were later analyzed using the flow cytometry analysis software WinMDI 2.8 (by Joe Trotter, freely distributed software). X and y coordinates refer to the intensity of fluorescence of annexin and PI, respectively.

Detection of intracellular ROS by flow cytometry

Cells were plated at a density of 2 × 10⁵ cells/well in 6-well plates (Falcon, BD Biosciences, Franklin Lakes, New Jersey, USA). After overnight incubation, cells were treated with sertraline for 24 h. Cells were harvested, washed twice with cold PBS and then 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and dihydroethidine (DHE) were added at a final concentration of 50 μg/ml in Ca²⁺-containing medium. Cells were incubated for 30 min at 37°C. After the cells were washed twice with cold PBS, 1 ml cold PBS was added. These two fluorescent probes were commonly used for the detection of intracellular oxidants. During an intracellular oxidative burst, ROS species are usually generated, leading to the conversion of the nonfluorescent probes into fluorescent molecules. The oxidation product of DCFH is dichlorofluorescein, with the green emission at 529 nm, while that of DHE is ethidium, emitting red fluorescence at 590 nm. The fluorescence was measured with a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA). Data were later analyzed using the flow cytometry analysis software WinMDI 2.8 (by Joe Trotter, freely distributed software) by gating 10²–10⁴ areas of the x and y coordinates.

Statistics

Data are reported as representative or mean SEM of the three experiments. Data were analyzed by two-way analysis of variance using the Statistical Analysis System (SAS^®, SAS Institute Inc., Cary, North Carolina, USA). Multiple comparisons between group means were performed by post hoc analysis using the Tukey’s honestly significant difference procedure. A p < 0.05 was considered significant.

Results

The effect of PEITC on basal [Ca²⁺]_i was explored. Figure 1(a) shows that the basal [Ca²⁺]_i level was 50 ± 2 nM. At the concentrations between 200 and 500 μM, PEITC evoked rise in [Ca²⁺]_i in a concentration-dependent manner in Ca²⁺-containing medium. At 300 μM, PEITC induced a gradual rise in [Ca²⁺]_i that attained to a net increase in 149 ± 2 nM (n = 3) at the time point of 350 s without showing a decay. The Ca²⁺ response is saturated at 500 μM PEITC because at a concentration of 700 μM, PEITC induced a similar response as that induced by 500 μM. Figure 1(b) shows that in the absence of extracellular Ca²⁺, 200–500 μM PEITC induced a rise in [Ca²⁺]_i. Figure 1(c) shows the concentration–response plot of PEITC-induced response. The half maximal effective concentration (EC₅₀) value was 250 ± 21 μM for both the responses.

Figure 1.

(a) Effect of PEITC on [Ca²⁺]_i in fura-2-loaded MDCK cells. PEITC was added at 30 s. The concentration of PEITC was indicated. The experiments were performed in Ca²⁺-containing medium. (b) Effect of PEITC on [Ca²⁺]_i in the absence of extracellular Ca²⁺. PEITC was added in Ca²⁺-free medium. (c) Concentration–response plots of PEITC-induced rise in [Ca²⁺]_i in the presence (filled circles) or absence (open circles) of extracellular Ca²⁺. Y-axis is the percentage of the net (baseline subtracted) area under the curve (30–350 s) of the rise in [Ca²⁺]_i induced by 500 μM PEITC in Ca²⁺-containing medium. Data are mean ± SEM of three experiments. *p < 0.05 when compared with open circles. [Ca²⁺]_i: Ca²⁺ concentrations; PEITC: phenethyl isothiocyanate.

Experiments were performed to confirm that PEITC-induced rise in [Ca²⁺]_i involved Ca²⁺ influx. Mn²⁺ enters cells through similar pathways as Ca²⁺ but quenches fura-2 fluorescence at all excitation wavelengths. ²⁰ Thus, quench of fura-2 fluorescence excited at the Ca²⁺-insensitive excitation wavelength of 360 nm by Mn²⁺ suggests Ca²⁺ entry. Figure 2 shows that 300 μM PEITC evoked an immediate decrease in the 360-nm excitation signal (compared with trace (a)). This implies that PEITC-induced rise in [Ca²⁺]_i involved Ca²⁺ entry. The decrease attained to a maximum of 110 ± 2 units (n = 3) at the time point of 360 s.

Figure 2.

Effect of PEITC on Ca²⁺ influx by measuring Mn²⁺ quench of fura-2 fluorescence. Experiments were performed in Ca²⁺-containing medium. MnCl₂ (50 μM) was added to cells 1 min before fluorescence measurements. The y-axis is fluorescence intensity (in arbitrary units) measured at the Ca²⁺-insensitive excitation wavelength of 360 nm and the emission wavelength of 510 nm. PEITC (200 μM) was added as indicated. Data are mean ± SEM of three experiments. PEITC: phenethyl isothiocyanate.

Experiments were conducted to explore the Ca²⁺ entry pathway of the PEITC-induced response. The store-operated Ca²⁺ influx inhibitors: nifedipine (1 μM), econazole (0.5 μM) and SK&F 96365 (5 μM); phorbol 12-myristate 13-acetate (a protein kinase C activator) as well as GF109203X (a protein kinase C inhibitor) were applied 1 min before PEITC. All these agents inhibited PEITC-induced rise in [Ca²⁺]_i by different degrees (Figure 3).

Figure 3.

Effect of Ca²⁺ channel blockers and PKC modulators on PEITC-induced rise in [Ca²⁺]_i. In blocker- or modulator-treated groups, the reagent was added 1 min before PEITC (200 μM). The concentration was 10 nM for PMA, 2 μM for GF109203X, 1 μM for nifedipine, 0.5 μM for econazole, and 5 μM for SK&F 96365. Data are expressed as the percentage of control (first column), which is the maximum value of 200 μM PEITC-induced rise in [Ca²⁺]_i, and are mean ± SEM of three experiments. *p < 0.05 when compared with first column. [Ca²⁺]_i: Ca²⁺ concentrations; PEITC: phenethyl isothiocyanate; PKC: protein kinase C; PMA: phorbol 12-myristate 13-acetate.

Efforts were made to explore the intracellular Ca²⁺ stores involved in PEITC-induced rise in [Ca²⁺]_i. Previous studies have shown that the endoplasmic reticulum is the major Ca²⁺ store in MDCK cells. Figure 4 shows that in Ca²⁺-free medium, addition of 50 μM of the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) ²¹ induced a rise in [Ca²⁺]_i to 75 ± 3 nM. PEITC (300 μM) added afterward induced a rise in [Ca²⁺]_i to 21 ± 2 nM (n = 3). Figure 4(b) shows that after pretreatment with PEITC for 470 s, BHQ induced a tiny rise in [Ca²⁺]_i of 8 ± 2 nM, which was smaller than the control BHQ response by 90%. Figure 4(c) shows that thapsigargin (TG; 1 μM), another inhibitor of endoplasmic reticulum Ca²⁺ pumps ²² evoked a rise in [Ca²⁺]_i to 31 ± 2 nM (n = 3). PEITC added afterward induced a rise in [Ca²⁺]_i to 20 ± 2 nM, which was smaller than control PEITC response (Figure 4(b)) by 50%. In contrast, Figure 4(d) shows that the addition of TG after PEITC incubation did not induce a rise in [Ca²⁺]_i.

Figure 4.

Intracellular Ca²⁺ stores of PEITC-induced Ca²⁺ release. Experiments were performed in Ca²⁺-free medium. PEITC (300 μM), thapsigargin (1 μM) and BHQ (50 μM) were added at time points indicated. Data are mean ± SEM of three experiments. PEITC: phenethyl isothiocyanate; BHQ: 2,5-di-tert-butylhydroquinone.

Phospholipase C-dependent production of inositol 1,4,5-trisphosphate is a key process for releasing Ca²⁺ from the endoplasmic reticulum. ¹³ Because PEITC released Ca²⁺ from the endoplasmic reticulum, the role of phospholipase C in this event was examined. U73122, a phospholipase C inhibitor, ²³ was used to see whether the activation of this enzyme was required for PEITC-induced Ca²⁺ release. Figure 5 shows that ATP (10 μM) induced a rise in [Ca²⁺]_i to 99 ± 2 nM. ATP is a well-known phospholipase C-dependent agonist that rises [Ca²⁺]_i in most cell types. ²⁴ Figure 5(b) shows that incubation with 2 μM U73122 did not change basal [Ca²⁺]_i but abolished ATP-induced rise in [Ca²⁺]_i. This suggests that under this condition U73122 effectively suppressed phospholipase C activity. Figure 5(b) also shows that the incubation with U73122 and ATP abolished PEITC-induced rise in [Ca²⁺]_i. U73343 (2 μM), a U73122 analogue, failed to have an inhibition (not shown).

Figure 5.

U73122-induced inhibition of PEITC-induced Ca²⁺ release. Experiments were performed in Ca²⁺-free medium. (a) ATP (10 μM) was added at 25 s. (b) First column is 300 μM PEITC-induced rise in [Ca²⁺]_i. Second column shows that 2 μM U73122 did not alter basal [Ca²⁺]_i. Third column shows the ATP-induced rise in [Ca²⁺]_i when compared with PEITC control. Fourth column shows that U73122 pretreatment for 200 s completely abolished ATP-induced rise in [Ca²⁺]_i (#p < 0.05 when compared with third column). Fifth column shows that U73122 (incubation for 200 s) and ATP (incubation for 50 s) pretreatment totally inhibited 300 μM PEITC-induced rise in [Ca²⁺]_i. Data are mean ± SEM of three experiments. [Ca²⁺]_i: Ca²⁺ concentrations; PEITC: phenethyl isothiocyanate; ATP: adenosine triphosphate.

Given that acute incubation with PEITC induces a substantial and lasting rise in [Ca²⁺]_i and that unregulated rise in [Ca²⁺]_i often alters cell viability, ¹⁴ experiments were performed to explore the effect of PEITC on viability of MDCK cells. Cells were treated with 0–75 μM PEITC for 24 h, and the tetrazolium assay was performed. In the presence of 15–75 μM PEITC, cell viability decreased in the concentration-dependent manner (Figure 6). The next question was whether the PEITC-evoked decrease in viability was induced by a preceding rise in [Ca²⁺]_i. The intracellular Ca²⁺ chelator BAPTA/AM ²⁵ was used to prevent a rise in [Ca²⁺]_i during PEITC pretreatment. BAPTA/AM loading did not alter control cell viability. In the presence of 10–50 μM PEITC, BAPTA/AM loading enhanced PEITC-induced decrease in viability (n = 3; p < 0.05).

Figure 6.

Cytotoxic effect of PEITC on the viability of MDCK cells. Cells were treated with 0–75 μM PEITC for 24 h, and the cell viability assay was performed. Data are mean ± SEM of three experiments. Each treatment had six replicates (wells). Data are expressed as percentage of control that is the increase in cell numbers in PEITC-free groups. Control had 10,898 ± 778 cells/well before experiments and had 13,569 ± 715 cells/well after incubation for 24 h. Cell numbers were counted by trypan blue exclusion as described in Materials and Methods section. *p < 0.05 when compared with control. In each group, the Ca²⁺ chelator BAPTA/AM (10 μM) was added to cells followed by treatment with PEITC in Ca²⁺-containing medium. Cell viability assay was subsequently performed using WST-1 assays. PEITC: phenethyl isothiocyanate; BAPTA/AM: 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester; WST-1: 4-[3-(4-lodophenyl)-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate].

Annexin V/PI staining was applied to detect apoptotic cells after PEITC treatment. Figure 7 shows that 20 and 50 μM PEITC induced apoptosis. At 50 μM, PEITC also induced necrosis.

Figure 7.

Apoptosis induced by PEITC measured by annexin V/PI staining. (a) Cells were treated with 0, 20 or 50 μM PEITC, respectively, for 24 h. Cells were then processed for annexin V/PI staining and analyzed by flow cytometry. (b) The percentage of apoptotic cells. *p < 0.05 when compared with control. PI: propidium iodide; PEITC: phenethyl isothiocyanate.

ROS are associated with multiple cellular functions such as cell proliferation, differentiation and apoptosis. To investigate whether PEITC induced oxidative stress in MDCK cells, the levels of intracellular ROS including superoxide anion (O₂ ⁻) and hydrogen peroxide (H₂O₂) in PEITC-treated cells was measured by flow cytometry using DHE and DCFH-DA fluorescent dyes, respectively. It was found that 10 and 15 μM PEITC treatment decreased intracellular levels of ROS (Figure 8).

Figure 8.

(a) Effect of PEITC on the hydrogen peroxide level in MDCK cells. DCFH-DA fluorescence was measured after the treatment with 0, 10 or 15 μM PEITC in serum-free culture media for 24 h. The fluorescence was quantified using the BD Cell Quest software. Data are mean ± SEM of four experiments. *p < 0.05 when compared with control. (b) Effect of PEITC on the superoxide anion level. DHE fluorescence in cells was measured after the treatment with 0, 10 or 15 μM PEITC in serum-free culture media for 24 h. The fluorescence was quantified using the BD Cell Quest software. Data are mean ± SEM of four experiments. *p < 0.05 when compared with control. The data are represented as DCFH-DA (or DHE) fluorescence percentage that refers to cells positive to DCFH-DA (or DHE). Controls are shown in the first column. PEITC: phenethyl isothiocyanate; DCFH-DA: 2′,7′-Dichlorofluorescein-diacetate; DHE: dihydroethidine.

Discussion

Our study shows that PEITC induced a rise in [Ca²⁺]_i in MDCK renal cells and examined the underlying mechanisms. This is the first report showing that the natural product PEITC can induce rise in [Ca²⁺]_i in renal cells. The data suggest that PEITC induced a concentration-dependent rise in [Ca²⁺]_i. PEITC increased [Ca²⁺]_i by depleting intracellular Ca²⁺ stores and causing Ca²⁺ influx from extracellular milieu because removing extracellular Ca²⁺ reduced a part of PEITC-induced rise in [Ca²⁺]_i. In MDCK cells, about 50% of 300 μM PEITC-induced rise in [Ca²⁺]_i was due to Ca²⁺ influx.

Removal of extracellular Ca²⁺ reduced the PEITC-induced response throughout the measurement period, suggesting that Ca²⁺ influx occurred during the whole stimulation period. The mechanism of PEITC-induced Ca²⁺ influx was examined. The results suggest that PEITC might cause Ca²⁺ influx via stimulating store-operated Ca²⁺ entry, which is induced by the depletion of intracellular Ca²⁺ stores ²⁶ based on the inhibition of PEITC-induced rise in [Ca²⁺]_i by nifedipine, econazole and SK&F 96365. These three compounds have been widely applied as the blockers of store-operated Ca²⁺ entry in different cell types.^{27 –29} Because activation of phospholipase C produces inositol triphosphate and diacylglycerol, which stimulates protein kinase C, the effect of regulation of protein kinase C activity on PEITC-induced rise in [Ca²⁺]_i was examined. Both the activation and inhibition of protein kinase C inhibited PEITC-induced rise in [Ca²⁺]_i. Activation or inhibition of protein kinase C has been shown to regulate Ca²⁺ channels in different preparations such as vascular smooth muscle, ³⁰ vascular myocytes, ³¹ vein myocytes, ³² HL60 cells ³³ and glomerular mesangial cells. ³⁴ Another possible mechanism that might cause PEITC-induced rise in [Ca²⁺]_i was that PEITC inhibited plasma membrane Ca²⁺ ATP pump so that cytosolic Ca²⁺ could not be pumped out of the cells and [Ca²⁺]_i would rise via leaks in the plasma membrane.

Regarding the Ca²⁺ stores involved in PEITC-induced Ca²⁺ release, the TG/BHQ-sensitive endoplasmic reticulum store might be the dominant store because TG/BHQ pretreatment greatly inhibited PEITC-induced rise in [Ca²⁺]_i; and conversely, PEITC pretreatment also inhibited TG/BHQ-induced Ca²⁺ release. Furthermore, it seems that phospholipase C-dependent pathways played a significant role in PEITC-induced Ca²⁺ release, because the response was abolished when phospholipase C activity was inhibited by U73122.

We show that PEITC inhibits viability of MDCK cells in a concentration-dependent manner. This is consistent with the cytotoxic effect of PEITC in other cell types. Ca²⁺ overloading is known to initiate processes leading to alteration in cell viability. ¹⁴ Because PEITC induced rise in [Ca²⁺]_i and inhibition of cell viability, it would be interesting to know whether the inhibition occurred in a Ca²⁺-dependent fashion. Our data show that PEITC-induced viability decrease was even enhanced when cytosolic Ca²⁺ was chelated by BAPTA. This implies that in this case, Ca²⁺ did not trigger PEITC-induced cell death. Emptying of intracellular Ca²⁺ stores and/or influx of extracellular Ca²⁺ can modulate cell viability in many cell types. ¹³ However, Ca²⁺-independent cell death could be found in some cell types such as thymic lymphoma cells, ³⁵ neutrophils, ³⁶ and so on. Furthermore, PEITC-induced cell death was found to involve apoptosis based on annexin V/PI staining, which is consistent with the apoptotic effect of PEITC observed in other cell lines. Our data also show that ROS-mediated mitochondrial pathway did not play a role in PEITC-induced apoptosis.

Together, the data show that the natural product PEITC induced Ca²⁺ release from endoplasmic reticulum in a phospholipase C-dependent manner and also caused Ca²⁺ influx via protein kinase C-modulated store-operated Ca²⁺ channels in renal cells. Because a rise in [Ca²⁺]_i can alter many cellular responses, caution should be applied in using PEITC in other in vitro studies.