MK-886, a Leukotriene Biosynthesis Inhibitor, as an Activator of Ca Mobilization in Madin-Darby Canine Kidney (MDCK) Cells

The effect of 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886), a leukotriene biosynthesis inhibitor, on Ca mobilization in MadinDarby canine kidney cells has been examined by fluorimetry using fura-2 as a Ca indicator. MK-886 at 0.5 to 25 mM concentration dependently increased [Ca]i. The [Ca ]i increase comprised an immediate initial rise and a slowly decaying phase. Ca removal inhibited the Ca signals by reducing both the initial rise and the decay phase, suggesting that MK886 activated Ca influx and Ca release. In Ca-free medium, 10 mM MK-886 still increased [Ca]i after pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 mM), a mitochondrial uncoupler, and thapsigargin (1 mM), an endoplasmic reticulum Ca pump inhibitor. Conversely, pretreatment with MK-886 abolished CCCPand thapsigargin-induced Ca release. This suggests that 10 mM MK-886 released Ca from the endoplasmic reticulum, mitochondria, and other stores. The addition of 3 mM Ca increased [Ca]i after pretreatment with 10 mM MK-886 for 700 s in Ca-free medium, indicating that MK-886 induced capacitative Ca entry. This capacitative Ca entry was partly inhibited by SKF96365 (50 mM), by econazole (25 mM), and by inhibiting phospholipase A2 with aristolochic acid (40 mM) but not by inhibiting phospholipase D with 0.1 mM propranolol. MK-886 (10 mM)-induced Ca release was not altered by inhibiting phospholipase C with U73122 (2 mM) but was inhibited by 50% by suppressing phospholipase D and phospholipase A2 with propranolol (0.1 mM) and aristolochic acid (40 mM), respectively. Leukotrienes are potent biologically active compounds known to play a pivotal role in inflammation and other cell functions. Because leukotrienes are synthesized by 5-lipoxygenase in many cells, pharmacological inhibitors of 5-lipoxegenase prove to be useful in the study of leukotriene metabolism. 3-[1-(pChlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886) is one of the several compounds that have been used to block leukotriene biosynthesis both in vivo and in vitro. MK-886 is thought to act by inhibiting activation of 5-lipoxygenase enzyme by a protein termed 5-lipoxygenase activating protein (Rouzer et al., 1990). In vivo, it was shown that oral application of MK-886 in humans significantly inhibits leukotriene B4 biosynthesis (Depre et al., 1993) and blocks allergen-induced airway responses (Friedman et al., 1993). MK-886 inhibits leukotriene biosynthesis and antigeninduced bronchoconstriction in animal models and in asthmatic men (Young, 1991). In vitro, MK-886 exerts many effects, including preventing the translocation and activation of 5-lipoxygenase in human keratinocytes (Hegemann et al., 1995) and leukocytes (Rouzer et al., 1990), inhibiting voltage-gated K currents and activating Ca-activated K currents in rat arterial myocytes (Smirnov et al., 1998), and inhibiting DNA synthesis in leukemia cells (Khan et al., 1993). Moreover, MK886 was found to be a potent and specific inhibitor of both leukotriene B4 and leukotriene C4 synthesis in human phagocytes (Gillard et al., 1989; Menard et al., 1990) and to induce apoptosis and exhibit antiproliferative effects in HL-60 cells (Dittmann et al., 1998). The effect of MK-886 on Ca homeostasis is unexplored. A transient increase in [Ca]i is a crucial signal for numerous Received for publication August 2, 1999. 1 This work was supported by grants from the National Science Council (NSC88-2314-B-075B-003), Veterans General Hospital-Kaohsiung (VGHKS8913), and VTY Joint Research Program, Tsou’s Foundation (VTY88-P3-24) to C.-R.J. ABBREVIATIONS: MK-886, 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; fura-2/AM, 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(29-amino-59-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid pentaacetoxymethyl ester; IP3, inositol 1,4,5-trisphosphate; MDCK, Madin-Darby canine kidney; U73122, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; U73343, 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione; CCCP, carbonylcyanide m-chlorophenylhydrazone; SKF96365, 1-[b-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride. 0022-3565/00/2941-0096$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 294, No. 1 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 294:96–102, 2000 /1974/830096 96 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from cell events (Clapman, 1995; Berridge, 1997). A [Ca]i increase may occur on external stimulation as a result of Ca entry and/or Ca release. In nonexcitable cells that lack voltage-gated Ca channels, one of the principle Ca stores for the [Ca]i increase is the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca 21 store (Berridge, 1993). Binding of IP3 to its receptors on the internal stores causes active release of internal Ca. This discharge of the internal Ca store often triggers Ca influx, leading to a prolonged [Ca]i increase and refilling these stores. This Ca influx is termed “capacitative Ca entry” (Putney and Bird, 1993). Here we have investigated the effect of MK-886 on Ca signaling in Madin-Darby canine kidney (MDCK) cells. We have previously shown that in this epithelial cell, IP3-dependent agonists such as ATP (Jan et al., 1998a) and bradykinin (Jan et al., 1998b) increase [Ca]i by releasing Ca 21 from the endoplasmic reticulum (ER) Ca store, followed by capacitative Ca entry. Additionally, thapsigargin (Jan et al., 1999a) and 2,5-di-tert-butylhydroquinone (Jan et al., 1999b) increase [Ca]i by inhibiting the ER Ca 21 pump without increasing IP3 levels, leading to Ca 21 release and capacitative Ca entry. Thus, MDCK cells were chosen as a model to examine drug effects on Ca homeostasis in nonexcitable cells. Using fura-2 as a Ca probe, we have found that MK-886 concentration dependently increased [Ca]i in MDCK cells. We have established the concentration-response relationships both in the presence and absence of external Ca and have explored the possible mechanisms underlying MK-886induced [Ca]i signals. Materials and Methods Cell Culture. MDCK cells obtained from American Type Culture Collection (CRL-6253; Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heatinactivated fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in 5% CO2-containing humidified air. Solutions. Ca medium (pH 7.4) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 5 mM glucose. Ca-free medium contained no Ca plus 1 mM EGTA (calculated [Ca] , 0.1 nM). The experimental solution contained ,1% of solvent (ethanol), which did not affect [Ca]i (n 5 3). Optical Measurements of [Ca]i. Trypsinized cells (10 /ml) were loaded with 2 mM 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(29-amino-59-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid pentaacetoxymethyl ester (fura-2/AM) for 30 min at 25°C in DMEM. Cells were washed and resuspended in Ca medium before use. 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 (Tokyo, Japan) by continuously recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1-s intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 and 20 mM EGTA sequentially at the end of an experiment. [Ca]i was calculated as previously described (Grynkiewicz et al., 1985). Our studies showed that trypsinized cells prepared by this protocol respond to stimulation with ATP (Jan et al., 1998a), bradykinin (Jan et al., 1998b) or thapsigargin (Jan et al., 1999a) similarly to cells attached to coverslips. Chemical Reagents. The reagents for cell culture were from Life Technologies (Grand Island, NY). Fura-2/AM was from Molecular Probes (Eugene, OR). MK-886, 1-(6-((17b-3-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), 1-(6-((17b3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidinedione (U73343), and aristolochic acid were from BIOMOL (Plymouth Meeting, PA). The other reagents were from Sigma (St. Louis, MO). Statistical Analysis. All values are reported as means 6 S.E. of five or six experiments. Statistical comparisons were determined by using Student’s paired t test, and significance was accepted when