Effect of Nortriptyline on Ca2+ Handling in SIRC Rabbit Corneal Epithelial Cells
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Chinese Journal of Physiology 53(3): 178-184, 2010 DOI: 10.4077/CJP.2010.AMK044 Effect of Nortriptyline on Ca2+ Handling in SIRC Rabbit Corneal Epithelial Cells Kai-Hsien Chang1, Hung-Pin Tan1, Chun-Chi Kuo2, Daih-Huang Kuo3, Pochuen Shieh3, Fu-An Chen3, and Chung-Ren Jan4 1Department of Psychiatry, Yung-Kang Veterans Hospital, Tainan 71051, Taiwan, ROC 2Department of Pharmacy, Tajen University, Pingtung 90741 3Department of Nursing, Tzu Hui Institute of Technology, Pingtung 92641 and 4Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan, Repnblic of China Abstract To explore the effect of nortriptyline, a tricyclic antidepressant, on cytosolic free Ca2+ concentrations 2+ 2+ ([Ca ]i) in corneal epithelial cells, [Ca ]i levels in suspended SIRC rabbit corneal epithelial cells were measured by using fura-2 as a Ca2+-sensitive fluorescent dye. Nortriptyline at concentrations between 2+ 2+ 20-200 µM increased [Ca ]i in a concentration-dependent manner. The Ca signal was reduced partly by removing extracellular Ca2+. Nortriptyline-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and alteration of activity of protein kinase C. In Ca2+-free medium, 200 µM nortriptyline pretreatment 2+ 2+ greatly inhibited the rise of [Ca ]i induced by the endoplasmic reticulum Ca pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ; 2+ 2+ another endoplasmic reticulum Ca pump inhibitor) nearly abolished nortriptyline-induced [Ca ]i 2+ rise. Inhibition of phospholipase C with U73122 decreased nortriptyline-induced [Ca ]i rise by 75%. 2+ Taken together, nortriptyline induced [Ca ]i rises in SIRC cells by causing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels. Key Words: Ca2+, corneal epithelial cells, nortriptyline, SIRC cells Introduction A regulated rise in cytosolic free Ca2+ levels 2+ ([Ca ]i) is a pivotal messenger in all cell types and Nortriptyline is a classic tricyclic antidepressant can trigger many physio-pathological processes (5). 2+ that is used in treatment of various psychological However, an abnormal elevation in [Ca ]i is often disorders such as migrane (26), smoking cessation cytotoxic (8). Thus, it is important to examine the (15), Parkinson’s disease (32) and depression (30). In effect of an agent on cellular Ca2+ signaling in order in vitro studies, most of the effects exerted by nortrip- to understand its in vitro effect. Nortriptyline has 2+ tyline are inhibitory, including inhibition of opiod been shown to increase [Ca ]i and decrease via- receptors (42), Ca2+-activated K+ channel (39), prim- bility in MG63 human osteosarcoma cells (13) and ing of human neutrophils (36), human cytochrome renal tubular cells (6). The effect of nortriptyline on 2+ P450 enzymes (33), human neutrophil phagocytosis [Ca ]i in corneal cells is unknown. SIRC rabbit and oxidative burst (25), and astroglial inwardly corneal epithelial cells were applied in this study. rectifying Kir4.1 channels (37). SIRC cells have characteristics similar to human Corresponding author: Dr. Chung-Ren Jan, Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan, R.O.C. Fax: +886-7-3468056, E-mail: [email protected] Received: July 15, 2009; Revised: September 18, 2009; Accepted: September 30, 2009. 2010 by The Chinese Physiological Society. ISSN : 0304-4920. http://www.cps.org.tw 178 Nortriptyline and Corneal Cells 179 300 A 300 B a, 200 µM 250 250 200 200 b, 100 µM a, 200 µM (nM) (nM) i i ] 150 ] 150 2+ 2+ b, 100 µM [Ca µ [Ca 100 c, 50 M 100 d, 20 µM 50 50 c, 50 µM nortriptyline e, 0 or 10 µM nortriptyline 0 0 0 50 100 150 200 250 0 50 100 150 200 250 Time (sec) Time (sec) C * * 100 + Ca2+ − Ca2+ 80 * 60 40 [area under the curve] 20 0 % of 200 mM Nortriptyline-Induced Response 0 100 200 300 400 500 Nortriptyline (µM) 2+ Fig. 1. A. Effect of nortriptyline on [Ca ]i in fura-2-loaded SIRC cells. Nortriptyline was added at 25 sec. The concentration of nortriptyline was indicated. The experiments were performed in a Ca2+-containing medium. B. Effect of removal of Ca2+ on 2+ 2+ 2+ nortriptyline-induced [Ca ]i rise. Experiments were performed in a Ca -free medium (Ca was replaced with 0.3 mM EGTA). In A & B, data shown are representatives of three experiments. C. Concentration-response plots of nortriptyline-induced 2+ 2+ [Ca ]i rise in the presence (filled circles) or absence (open circles) of extracellular Ca . Y axis is the percentage of control 2+ which is the net (baseline subtracted) area under the curve (25-200 sec) of the [Ca ]i rise induced by 200 µM nortriptyline in Ca2+-containing medium. Data are means ± SEM of three experiments. *P < 0.05 compared with open circles. corneal epithelial cells (2, 29), and, due to active this study shows that nortriptyline induced a signifi- 2+ responsiveness under experimental conditions, have cant [Ca ]i increase in SIRC cells. The time course been used as a model for corneal epithelial cell and the concentration-response relationship, the Ca2+ research. Several agents, such as tamoxifen (14), sources of the Ca2+ signal and the role of phospholipase ketoconazole (21), fendiline (22) and econazole (7), C in the signal have been explored. have been found to stimulate SIRC cells by causing 2+ a [Ca ]i increase. The inositol 1,4,5-trisphosphate Materials and Methods (IP3)-sensitive Ca2+ store is the major Ca2+ store that releases Ca2+ into the cytosol when cells are stimulated Chemicals by endogenous agents such as ATP (14). But exoge- nous agents can release Ca2+ from IP3-insensitive The reagents for cell culture were purchased stores (7, 21, 22). The Ca2+ release may induce Ca2+ from Gibco (Gaithersburg, MD, USA). Fura-2/AM influx across the plasma membrane via the process of was obtained from Molecular Probes (Eugene, OR, store-operated Ca2+ entry (27). USA). Nortriptyline and other reagents were from Using fura-2 as a fluorescent Ca2+ indicator, Sigma-Aldrich (St. Louis, MO, USA). 180 Chang, Tan, Kuo, Kuo, Shieh, Chen and Jan Cell Culture 100 SIRC rabbit corneal epithelia cells obtained * 80 from American Type Culture Collection were cultured in Dulbecco’s modified Eagle medium supplemented * with 10% heat-inactivated fetal bovine serum, 100 60 * U/ml penicillin and 100 µg/ml streptomycin at 37°C * * 40 in a humidified 5% CO2 atmosphere. 2+ % of Control Response 20 Solutions Used in [Ca ]i Measurements Ca2+-containing medium (pH 7.4) contained 140 0 mM NaCl, 5 mM KCl, 1 mM MgCl , 2 mM CaCl , 10 2 2 PMA mM Hepes, 5 mM glucose. The experimental reagents EconazoleNifedipineVerapamil were dissolved in water, ethanol or dimethyl sulfoxide. Nortriptyline GF109203XSK&F96365 The concentration of organic solvents in the solution Aristochic Acid used in experiments did not exceed 0.1%, and did not Fig. 2. Effect of Ca2+ channel blockers and phospholipase 2+ 2+ alter basal [Ca ]i. A2 inhibitor on nortriptyline-induced [Ca ]i rise. The 2+ [Ca ]i rise induced by 200 µM nortriptyline was taken as 2+ the control. In blocker- or modulator-treated groups, the [Ca ]i Measurements reagent was added 1 min before nortriptyline. The µ Confluent cells grown on 6-cm dishes were concentration was 1 M for nifedipine and verapamil; 0.5 µM for econazole; 5 µM for SK&F96365; 20 µM for trypsinized and made into a suspension in culture 6 aristolochic acid, 10 nM for phorbol 12-myristate 13- medium at a density of 10 cells/ml. Cells were sub- acetate (PMA) and 2 µM for GF109203X. Data are µ sequently loaded with 2 M fura-2/AM for 30 min at expressed as the percentage of control (1st column from 25°C in the same medium. After loading, cells were the left) that is the maximum value of 200 µM nortrip- 2+ 2+ washed with Ca -containing medium twice and were tyline-induced [Ca ]i rise, and are means ± SEM of three made into a suspension in Ca2+-containing medium experiments. *P < 0.05 compared to the control. at a density of 107 cells/ml. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring; the cuvette group means were performed by post-hoc analysis contained 1 ml of medium and 0.5 million cells. using the Tukey’s HSD (honestly significant dif- Fluorescence was monitored with a Shimadzu RF- ference) procedure. A P-value less than 0.05 was 5301PC spectrofluorophotometer immediately after considered significant. 0.1 ml cell suspension was added to 0.9 ml Ca2+- containing or Ca2+-free medium by recording excita- Results tion signals at 340 nm and 380 nm and emission signal 2+ at 510 nm at 1-sec intervals. During the recording, Fig. 1A shows that the basal [Ca ]i level was reagents were added to the cuvette by pausing the approximately 50 nM. At concentrations between 20 2+ recording for 2 sec to open and close the cuvette- and 200 µM, nortriptyline evoked [Ca ]i rises in a 2+ 2+ containing chamber. For calibration of [Ca ]i, after concentration-dependent manner in a Ca -containing completion of the experiments, the detergent Triton medium. At 10 µM, nortriptyline did not cause a 2+ 2+ X-100 and 5 mM CaCl2 were added to the cuvette to [Ca ]i rise (data not shown). The [Ca ]i rise induced obtain the maximal fura-2 fluorescence. The Ca2+ by 200 µM nortriptyline attained to 225 ± 2 nM (n = chelator EGTA (10 mM) was subsequently added to 3) followed by a slow decay.