* The University of New Mexico College of Pharmacy Toxicology Program, Albuquerque, New Mexico 871310001; University of California at Davis, Dept. of VM:Molecular Biosciences, and Center for Children's Environmental Health, Davis, California 95616; and
The University of Pennsylvania School of Medicine, Department of Pharmacology, Philadelphia, Pennsylvania 191046084
1 To whom correspondence should be addressed at 1 University of New MexicoMSC09 5360, Albuquerque, NM 871310001. Fax: (505) 272-6749. E-mail: sburchiel{at}salud.unm.edu.
Received June 16, 2005; accepted July 14, 2005
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ABSTRACT |
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Key Words: ryanodine receptor; calcium; 7,8-benzo(a)pyrene quinone.
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INTRODUCTION |
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Calcium is an important second messenger that regulates various biological functions in cells (Berridge, 2001). Calcium signaling in lymphocytes and other cells is influenced by such properties as the duration, intensity, or frequency of oscillations (Lewis, 2003
). We have found that several BaP metabolites alter Ca2+ signaling in Daudi human B cells, albeit by different mechanisms. Cytochrome P450 activation of BaP appears to be critical for immunotoxicity to human peripheral blood cells (Davila et al., 1996
). Our previous studies also found that BaP-7,8-diol alters intracellular calcium levels after several hours of treatment of normal human peripheral blood mononuclear cells (HPBMC) (Mounho and Burchiel, 1998
). Using Daudi human B cells as an in vitro model, we found increases in Ca2+ levels through activation of Src-like protein tyrosine kinases (Mounho and Burchiel, 1998
). Recently, we found that quinones of BaP (BPQs) also signal Ca2+, but through several different pathways. BP-quinones, including 1,6-BPQ, 3,6-BPQ, and 6,12-BPQ, are formed through cytochrome P450, peroxidase, and photochemical (UV) reactions (Reed et al., 2003
). BPQs are known to redox-cycle, leading to oxidative stress and energy depletion in mitochondria followed by loss of Ca2+ buffering capacity in cells (Zhu et al., 1995
). More recently we discovered another potential mechanism of Ca2+ elevation in cells due to a unique interaction of an ortho-BPQ (7,8-BPQ) with ryanodine receptors (RyR) (Pessah et al., 2001
). In these studies, we found that 7,8-BPQ causes Ca2+ release in rabbit skeletal muscle sarcoplasmic reticulum (SR) vesicles through a selective interaction with RyR.
RyR are homotetrameric calcium release channels of intracellular stores, which were first described on the sarcoplasmic reticulum (SR) in mammalian skeletal muscle cells and cardiac muscle cells (Fill and Copello, 2002; McPherson and Campbell, 1993
). More recently, RyR have been found on the endoplasmic reticulum of nonexcitable cells (Sei et al., 1999
). Mammalian RyRs are expressed as three widely distributed isoforms: RyR1, RyR2, and RyR3. Recent studies have shown that RyR1 and RyR2 are expressed in nonexcitable cells, such as lymphocytes (Sei et al., 1999
). RyR3 is widely expressed in many types of cells (Ogawa et al., 2000
). To investigate the role of RyR, ryanodine binding, which occurs only with the open channel, is frequently used as a probe of RyR function (Pessah et al., 1986
). Low concentrations of ryanodine (
1 µM) activate RyRs, whereas high concentrations (
100 µM) irreversibly inhibit RyR calcium release.
Based on previous studies of calcium responses produced by BPQs and other BaP metabolites in lymphoid and nonlymphoid cells, as well as rabbit muscle SR vesicles, we hypothesized that 7,8-BPQ would selectively (as compared to other BPQs) elevate intracellular Ca2+ in human and murine B and T cells via a RyR-dependent mechanism. In this report, we utilized Daudi human B cells, HPBMC, and murine spleen cells to analyze the immediate and delayed Ca2+ responses following exposure to 7,8-BPQ and various other BPQ and BaP metabolites. Our data show that only 7,8-BPQ produces a significant rapid increase in intracellular Ca2+. We found that this increase in Ca2+ was blocked by high concentrations of ryanodine. Therefore, we conclude that Ca2+ elevation by 7,8-BPQ in human and murine lymphoid cells is due to activation of RyR.
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MATERIALS AND METHODS |
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Cell line.
The Daudi human B cell line was obtained from American Type Culture Collection (Rockville, MD) and was grown in a humidified, 37°C, 5% CO2 incubator in complete RPMI 1640 medium containing 20% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin (Cambrex), and 2 mM L-glutamine.
Preparation of human peripheral blood mononuclear cells (HPBMC).
Human mononuclear cells were isolated from healthy blood donors under an approved IRB protocol using Fico/Lite LymphoH density gradient centrifugation method. Briefly, collected peripheral blood was placed in 6 x 10 ml Vacutainer® plasma tubes with heparin (VWR Cat. No.VT6480). Heparinized blood was diluted with equal volume of sterile phosphate-buffered saline (PBS) (without calcium and magnesium salts) (Sigma Chemical Co., St. Louis, MO), and then 30 ml of this mixture was overlayed on 15 ml Fico/Lite LymphoH at room temperature (RT). After density gradient centrifugation for 30 min at 200 x g without brake at RT, peripheral blood mononuclear cells were collected from the white layer between the plasma fraction and the Fico/Lite LymphoH fraction. The HPBMC were washed twice with PBS, and the cell pellet was then resuspended in 3 ml complete RPMI 1640 medium containing 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin (Cambrex), and 2 mM L-glutamine. Cells were counted and adjusted to 20 x 106 cells/ml.
Preparation of mouse spleen cells.
Spleens from female wild-type C57BL/6N mice (1314 weeks old) (Harlan laboratories, Indianapolis, IN) were aseptically removed and were dissociated in RPMI 1640 complete medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, 100 unit/ml penicillin, and 2 mM L-glutamine. Cells were centrifuged at 280 x g for 10 min, and pellets were resuspended and maintained in 2 ml RPMI 1640 complete medium on ice. The nucleated spleen cells were counted using the trypan blue (Sigma Chemical Co., St. Louis, MO) exclusion method.
Intracellular calcium measurement using flow cytometry.
The analysis of intracellular Ca2+ levels in Daudi human B cells, HPBMC, and mouse spleen cells was performed by flow cytometric methods (Burchiel et al., 1999, 2000
; Rethi et al., 2002
). For all experiments, cells were loaded with in 200 µl of 2 µM Fluo-3/AM (Molecular Probes) in FACS tubes and incubated at 37°C, 5% CO2 for 1 h, with cells being resuspended approximately every 20 min to ensure even dye loading. Then 800 µl of Dulbecco's phosphate buffered saline (DPBS) (with calcium and magnesium) were added to yield a 1 ml solution. Ca2+ responses were observed in two ways: (1) For rapid real-time analysis, Fluo-3 loaded cells were treated with various PAHs and continuously monitored for up to 30 min. (2) For longer incubations periods (1 h or more), after treatment with PAHs, cells were loaded with Fluo-3, and the fluorescence intensity was measured by flow cytometry. For Daudi human B cell experiments, cells were resuspended in complete RPMI medium at 2 x 105 cells/ml, and 1-ml cell aliquots were placed into three 12 x 75 mm tubes. After the centrifugation and aspiration, the cells were loaded with Fluo3. For HPBMC studies, 10 µl of 20 x 106 cells/ml HPBMC were loaded with Fluo-3 and stained with PE-conjugated mouse anti-human CD3 or mouse anti-human CD19 to label T or B cells, respectively. For murine spleen cell studies, 10 µl of 20 x 106 cells/ml murine spleen cells were put in 12 x 75 mm tubes and loaded with the addition of 200 µl of Fluo-3AM. After 1 h, 800 µl of DPBS solution was added to yield 2 x 105 cells/ml. Tubes were capped and covered with aluminum foil to transport to Flow Cytometry Facility for analysis. Fluorescence intensities were measured in the resuspended cells by FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA) to obtain baseline readings. Mean channel fluorescence intensities were calculated using CellQuest software.
Measurement of 3H-ryanodine binding.
High affinity binding of 3H-ryanodine (57 Ci/mmol; New England Nuclear, Boston, MA) to crude microsomal membrane extracts (0360 µg/ml) from cultured Daudi cells was performed in the presence of 500 mM KCl, 20 mM HEPES, 100µM CaCl2, 1 mM DTT, 100 µM PMSF, 100 µg/ml BSA, 1 µg/ml leupeptin, pH 7.4, and 10 nM 3H-ryanodine (Pessah et al., 1987). The binding reaction was initiated by the addition of microsomal membranes to the medium, and the mixture was permitted to equilibrate at 37°C for 2 h. Nonspecific binding was assessed in the presence of 10 µM unlabeled ryanodine. Separation of bound and free ligand was performed by rapid filtration through Whatman GF/B glass fiber filters using a Brandel cell harvester (Gaithersburg, MD). Filters were washed with two volumes of 0.5 ml ice-cold wash buffer containing 20 mM TrisHCl, 250 mM KCl, 15 mM NaCl, 50 µM CaCl2, pH 7.1, and placed into vials with 5 ml scintillation cocktail (Ready Safe; Beckman Instruments, Inc., Fullerton, CA). The 3H-ryanodine remaining on the filters was quantified by liquid scintillation spectrometry.
Statistical design and analysis.
All of the data reported in this paper were analyzed by SigmaStat software (SPSS, Point Richmond, CA). The statistical differences were determined by one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test, Dunnett's test and Student's t-test. A p-value of 0.05 between DMSO control and PAH-treated samples was considered significant.
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RESULTS |
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DISCUSSION |
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Our previous studies demonstrated that two P450-derived metabolites of BPDE and BaP-7,8-Diol increased tyrosine phosphorylation of Lyn and Syk (Mounho and Burchiel, 1998). This phosphorylation is known to result in the activation of PLC
1 and PLC
2 leading to cleavage of PIP2 and release of inositol-1,4,5-trisphosphate (IP3) (Winslow et al., 2003
). IP3-sensitive Ca2+ release receptors are located on the ER in lymphocytes. The current studies also demonstrated the presence of ryanodine receptors on microsomes (ER fraction) obtained from Daudi human B cells. Thus, there are apparently two closely related Ca2+ channels that control Ca2+ release from the ER of lymphoid cells, the IP3R and RyR (Grafton and Thwaite, 2001
). It has been proposed that IP3 induces a small amount of Ca2+ release from ER, which then triggers a Ca2+-induced Ca2+ release (CICR) mechanism (Fill and Copello, 2002
). RyR are also activated by increased cytosolic Ca2+ (Pessah et al., 2001
). Therefore, RyR may play an important role in lymphocytes to induce or maintain Ca2+ signaling responses.
The present study demonstrated that 7,8-BPQ produces a rapid increase in intracellular Ca2+ in Daudi human B cells, human peripheral blood B and T cells, and murine spleen cells. By contrast, the Ca2+ response of other BaP metabolites, including 1,6-BPQ, 3,6-BPQ, and BPDE, occurred at later time points (218 h). Therefore, our data suggest that 7,8-BPQ utilizes different signaling pathways to elicit early Ca2+ effects. Because we detected the presence of RyRs in Daudi microsomes, and we found that high concentrations of ryanodine prevented the 7,8-BPQ Ca2+ response in Daudi human B cells, human peripheral blood B and T cells, and murine spleen cells, we believe that RyR are responsible for the Ca2+ response of lymphoid cells to 7,8-BPQ. In our previous work discussed above with BaP-7,8-Diol, we presumed that BaP-7,8-diol was converted to BPDE to produce its effects on src kinases. However, we now know that Daudi and other human lymphocytes express the AKR1C1, and thus they have the ability to make the 7,8-BPQ. Other BPQs also increase intracellular Ca2+, but we believe that these much later effects are caused by redox-cycling and loss of Ca2+ buffering capacity by mitochondria due to ATP depletion, although this hypothesis was not tested in our current studies (Zhu et al., 1995). Because we found that 7,8-BPQ induces rapid increases in intracellular Ca2+ levels in human and murine B and T cells, we are now examining pathways for endogenous 7,8-BPQ formation, as well as the regulation of the human AKR1C1 gene in human cells and its homologue in mice.
In summary, we found that 7,8-BPQ is a potent and specific activator of RyR in the present studies leading to a rapid rise in intracellular Ca2+ in human and murine B and T cells. We believe that the early and rapid Ca2+ response produced by 7,8-BPQ may be immunomodulatory and may play a role in the immunotoxicity induced by BaP. BPQs have previously been found to activate murine splenic T cells following in vitro treatment at concentrations of 0.1 and 1 µM (Burchiel et al., 2004), although human leukocytes are inhibited following in vitro treatment with BPQs. Ca2+ elevation by 7,8-BPQ may also play a role in immune activation, in that many Ca2+-sensitive pathways regulate cell activation, such as adhesion molecular activation, enzymatic activation, exocytosis, migration and response to other ion channels activation (Panyi et al., 2004
). Changes in intracellular Ca2+ are also likely important in specific gene regulation, perhaps through PKCs (Lewis, 2003
). Pomorski et al. (2004)
found that intracellular calcium changes regulate cytokine expression, such as IL-1ß, and IL8, and effect the monocytes migration. However, this calcium-regulated specific gene expression occurs at relatively late time points. Thus, the early and rapid increase in Ca2+ produced by 7,8-BPQ suggests that RyR may play an important role in lymphocyte activation and may be a target of environmental agents.
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NOTES |
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ACKNOWLEDGMENTS |
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