* Department of Veterinary Anatomy and Public Health,
Department of Physiology and Pharmacology, and
Faculty of Toxicology, Texas A&M University, College Station, Texas 77843
Received February 8, 2002; accepted April 15, 2002
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ABSTRACT |
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Key Words: Ca2+ oscillations; oxytocin; vasopressin; fluorescence microscopy; laser cytometry; BaP.
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INTRODUCTION |
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Ca2+ oscillations appear to be a fundamental mechanism of cell signaling. In systems such as pancreatic acinar cells and pituitary gonadotrophs, where agonists induce oscillatory changes in [Ca2+]i and also stimulate maximal secretion, it appears that frequency-encoded Ca2+ signals exert a physiological role (Stauffer et al., 1993; Stojilkovic et al., 1994
; Tse et al., 1993
). A number of cellular targets have been identified that are involved in the decoding of these Ca2+ signals. The activity of calcium-calmodulin kinase II (CAM Kinase II) and protein kinase C (PKC) are tuned to the frequency of Ca2+ oscillations (De Koninck and Schulman, 1998
; Oancea and Meyer, 1998
). Similarly, the activities of Ca2+-sensitive mitochondrial dehydrogenases in hepatocytes that drive ATP production are controlled by Ca2+ oscillations (Hajnóczky et al., 1995
). The selective activation of different transcription factors that can fine tune gene expression appear to be regulated by both amplitude and frequency encoded Ca2+ signals (Dolmetsch et al., 1997
; Hu et al., 1999
; Li et al., 1998
).
In earlier studies, a communication-competent rat liver cell line (Clone 9), which exhibited a complex pattern of oxytocin-induced Ca2+ oscillations, was examined as a potential model to explore effects of selected toxicants on cellular Ca2+ homeostasis (Barhoumi et al., 1996, 2000
). The cell line exhibits uniform properties and is widely used for in vitro toxicity studies ranging from oxidative injury (Grune et al., 1995
) to chemical carcinogenesis (Na et al., 1995
). Like normal liver cells (Ariño et al., 1989
; Thomas et al., 1995
) Clone 9 cells respond to hormones such as oxytocin and vasopressin, which are important glycogenolytic effectors in liver cells and exhibit induction of microsomal ethoxyresorufin-O-deethylase (EROD) activity by BaP (Barhoumi et al., 2000
). Oxytocin and vasopressin act primarily by the G protein-coupled formation of InsP3 via the phosphoinositide-specific phospholipase C (PLC) and the subsequent release of [Ca2+]i from intracellular stores (Zingg, 1996
). Exposure of these cells to the carcinogenic polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BaP) was found to suppress gap junction-mediated intercellular communication (GJIC) and plasma membrane potential, while elevating basal [Ca2+]i (Barhoumi et al., 2000
). Given the diversity of cellular functions that are regulated by [Ca2+]i and the contributions of plasma membrane potential and GJIC to the regulation and propagation of Ca2+ signals, the present study was designed to investigate the actions of BaP on oxytocin- and vasopressin-induced Ca2+ oscillations in Clone 9 cells. The objectives of this study were to: (1) characterize the stable, long-term, agonist-induced Ca2+ oscillations in Clone 9 cells; (2) investigate the mechanisms by which BaP may affect agonist-induced Ca2+ oscillations; and (3) identify experimental treatments that may partially reverse the effects of BaP.
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MATERIALS AND METHODS |
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Stock solution of 1.0 mM fluo-4, AM was prepared in DMSO and diluted with medium to 3.0 µM (0.3% final DMSO concentration) for loading in cultured cells. Ten mM nifedipine stock was prepared in DMSO and a 10 µM concentration was used for experiments. Thapsigargin stock (1.0 mM) was prepared in DMSO and used at concentrations of 50 nM to 1.0 µM ( 0.1% DMSO). Chelerythrine chloride was prepared as 10 mM stock in DMSO. Ten mM stock solutions of 8-Br-cAMP and 8-Br-cGMP were prepared in PBS and used at concentrations of 10 µM to 1.0 mM. TEA was prepared as a 1.0 M stock in PBS and used at concentrations ranging from 1.0 to 100 mM for cell treatments. Clone 9 (ATCC, CRL 1439) rat liver cells were obtained at passage 17 and used between passages 25 to 35. Cells were grown in Hams Nutrient Mixture F-12 with 10% fetal bovine serum on 2-well Lab-Tek Chambered Coverglass slides following plating at a density of 105 cells per well for 48 h prior to analysis. To study the effects of BaP on Ca2+ oscillations, actively growing Clone 9 cells were treated with graded concentrations of BaP (020 µM) in the same culture medium for 24 h prior to analysis of [Ca2+]i when cultures were approximately 80% confluent.
Methods.
Fluo-4, AM, used to monitor [Ca2+]i, is a nonratiometric visible wavelength probe that exhibits about a 40-fold enhancement of fluorescence intensity with Ca2+ binding (Gee et al., 2000). To minimize differences in fluo-4 loading from experiment to experiment, cells were seeded at the same density (105 cells per well); all experiments were performed with the same fluo-4, AM stock; and each treatment was compared to a separate control. Cells were loaded for 1 h with 3.0 µM fluo-4, AM at 37°C and then washed with serum- and phenol red-free medium. Cells were then placed on the stage of a Meridian Ultima confocal microscope (Meridian Instruments, Okemos, MI) and an area of the well containing groups of 1015 cells was selected. For image collection, scan parameters were adjusted for maximum detection of fluorescence with minimal cellular photobleaching. Fluorescence was generated in the cells by excitation at 488 nm, and fluorescence emission from scanned individual cells was collected (530 nm) by means of a photomultiplier tube with a 3-s sampling interval. The basal [Ca2+]i was determined prior to addition of the hormones and/or pharmacologic agents and fluorescence intensity changes were normalized to this basal [Ca2+]i level. Ca2+ oscillations were analyzed between 15 and 30 min of addition of hormones when oscillations were highly uniform. Calibration of intracellular [Ca2+]i was performed as described by Tsien (1989) using the Calcium Calibration Buffer kit #2 (Molecular Probes).
To determine the source of Ca2+ pools involved in oscillations and their corresponding input to the frequency, a variety of pharmacologic agents were employed. Thapsigargin is an inhibitor of the microsomal Ca2+-ATPase pump, which causes leakage of Ca2+ from intracellular InsP3-sensitive stores and prevents their refilling (Lytton et al., 1991). It is also used as an indicator for capacitative Ca2+ entry (e.g., Broad et al., 2001
). Chelerithrine chloride is a potent and selective inhibitor of group A and B PKC isoforms (Herbert et al., 1990
). Both 8-Br-cAMP and 8-Br-cGMP are second messengers that can influence [Ca2+]i by acting on both Ca2+ and K+ channels and pumps (Berridge et al., 2000
). TEA is an inhibitor of all known K+ channels (Hille, 1992
).
To test each of these agents, cells loaded with fluo-4 were placed on the Ultima confocal microscope stage and basal fluorescence intensity was obtained from 5 image scans recorded from about 1015 cells. Following the fifth scan, cells were exposed to vasopressin or oxytocin and scanning continued at the same sampling interval until the cells established a uniform pattern of oscillation (approximately 15 min). Oscillation frequency was then determined over the next 7 min of the Ca2+ response, a pharmacologic agent was added, and the frequency was then determined for a similar time interval (Burghardt et al., 1999). Control experiments to monitor any effects of solvent for each test substance were performed similarly. For each experiment, data were recorded from 1015 cells per well of the culture dish and 24 wells were analyzed. Two replicate experiments on consecutive days were performed.
Ca2+ oscillation frequency data are expressed as means ± SEM mHz. Differences between treatments performed on the same day were evaluated by ANOVA, followed by Dunnetts or Tukeys test for multiple comparisons at p < 0.05.
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RESULTS |
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When Clone 9 cells were treated with 10 µM BaP for 24 h, the basal [Ca2+]i level in cells increased, while the transient Ca2+ peak induced by oxytocin or vasopressin was unchanged. Similar results were obtained in the presence or absence of the voltage-operated Ca2+ channel blocker, nifedipine. In addition, when BaP-treated cells were exposed to thapsigargin, the InsP3-sensitive Ca2+ release, as well as capacitative Ca2+ entry, were identical to control cells (data not shown). Further, BaP caused nonuniformity of Ca2+ oscillations that resulted in a dose-dependent reduction in the frequency of 100 nM oxytocin- and 1 nM vasopressin-induced Ca2+ oscillations (Fig. 3).
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Regulation of Ca2+oscillations in normal and BaP-treated clone 9 cells by protein kinase C, second messengers, and K+ channels.
To study the contribution of PKC, and the cGMP- and cAMP-second messenger pathways to the frequency of Ca2+ oscillations in control and BaP-treated Clone 9 cells, the PKC inhibitor chelerythrine chloride and second messenger analogs (8-Br-cGMP and 8-Br-cAMP) were used. Rapid, dose-dependent suppression of oxytocin-induced Ca2+ oscillations was recorded with each reagent, although the relative sensitivity of the oscillations to each reagent differed (Fig. 4). Addition of chelerythrine chloride (PKC inhibitor), 8-Br-cGMP or 8-Br-cAMP to BaP-treated cells exhibited a similar response to control Clone 9 cells (data not shown).
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DISCUSSION |
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In the present study, analysis of the frequency of Ca2+ oscillations was used to characterize the dose-dependence and cytoplasmic mechanisms controlling oxytocin- and vasopressin-induced frequency encoded Ca2+ signals in an immortalized rat liver cell line and to determine the effects of BaP on these processes. Both oxytocin and vasopressin induce uniform, stable oscillations and the frequencies of these oscillations are concentration-dependent, with vasopressin having a steeper concentration-frequency relationship, greater potency, and maximum effect. The highest frequencies obtained for oxytocin and vasopressin were 72.4 ± 4.0 mHz and 109.5 ± 7.1 mHz, respectively. Differences between hormone-induced frequencies may be due to the number of receptors and/or the relative affinity of the receptors for these vasoactive hormones with actions on glycogenolysis and mitochondrial energy metabolism in liver (Hajnóczky et al., 1995; Zingg, 1996
).
The main intracellular source of Ca2+ involved in these oscillations is the InsP3-sensitive pool activated through stimulation of PLC-ß via G-protein-linked receptors. The presence of extracellular Ca2+ is required to maintain the oscillations, indicating the importance of plasma membrane Ca2+ fluxes in refilling the intracellular pools. Activation of PKC as a result of PLC-ß activation constitutes a potential inhibitory feedback loop on this pathway that may contribute to Ca2+ oscillations. Using the PKC inhibitor, chelerythrine chloride, the frequency of oxytocin-induced Ca2+ oscillations dropped at a high rate; oscillations were rapidly abolished at 20 µM. These data suggest the importance of the PKC feedback mechanism in controlling Ca2+ oscillations in Clone 9 cells where PKC is repetitively activated by Ca2+ and diacylglycerol as the result of PLC-ß activation to terminate individual Ca2+ spikes (Codazzi et al., 2001). This is accomplished by PKC phosphorylation of oxytocin or vasopressin receptors to uncouple them from activation of Gq and/or phosphorylation of PLC-ß and prevent its activation by Gq (Taylor and Thorn, 2001
).
The frequency of Ca2+ oscillations in Clone 9 cells is also influenced by cytoplasmic levels of cAMP and cGMP, since both 8-Br-cAMP and 8-Br-cGMP cause a dose-dependent suppression of oscillations. The specific targets of these second messengers in Clone 9 cells, which modulate oscillation frequency, have not been defined; however, there are numerous ways in which their effects can be mediated. Both 8-Br-cAMP and 8-Br-cGMP can influence [Ca2+]i by acting on both Ca2+ channels and pumps (Berridge et al., 2000). Ca2+-activated K+ channels can be stimulated by both cAMP and activation of soluble guanylate cyclase to oppose depolarization stimuli (Berridge et al., 2000
; Lee and Kang, 2001
) and cAMP can inhibit oxytocin-induced phosphatidyl inositol turnover and increases in [Ca2+]i through protein kinase A-mediated phosphorylation of certain isoforms of PLC (Yue et al., 2000
).
In this investigation, use of Ca2+ oscillation frequency analysis was useful to better define the adverse effects of BaP on intracellular Ca2+ homeostasis and signaling. We previously reported the rapid uptake and partitioning of BaP into cellular membranes that resulted in uncoupling of gap junctions, increases in [Ca2+]i, and hyperpolarization of cells (Barhoumi et al., 2000). These effects suggested a direct action of BaP on multiple membrane targets which could alter signaling mechanisms involving [Ca2+]i as a second messenger. The present studies confirm this adverse effect of BaP resulting from suppression of oxytocin- and vasopressin-induced frequency-encoded Ca2+ signals. Treatment of Clone 9 cells with 10 µM BaP for 24 h altered the frequency response for both hormones with a significantly greater impact on vasopressin-induced Ca2+ oscillations. This agonist-specific impact of BaP may be due to the different dose frequency and amplitude patterns of Ca2+ oscillations exhibited by both hormones in control Clone 9 cells.
The contribution of PKC to the frequency of Ca2+ oscillation did not change in BaP-treated cells as the PKC inhibitor reduced the frequency at the same rate of control cells. Both 8-Br-cGMP and 8-Br-cAMP had the same effect on frequency in BaP-treated cells when compared to control cells, which indicates that PKC, cGMP, and cAMP pathways were not detectably altered by BaP treatments.
The effects of BaP were noticeable on the pools involved in determining the initial Ca2+ transient and the frequency of Ca2+ oscillations. These effects may be due in part to the increase in basal [Ca2+]i caused by a direct action of BaP on Ca2+ and/or K+ channels. Analysis of the initial Ca2+ transients induced by oxytocin identified no significant effects of BaP on voltage-operated Ca2+ channels (nifedipine-sensitive), InsP3-sensitive stores, or stores operated Ca2+ channels (thapsigargin-sensitive). However, a direct action of BaP on plasma membrane K+ channels may account for previous observations of hyperpolarization of the plasma membrane within 30 min of addition of BaP to culture medium (Barhoumi et al., 2000). Direct evidence in support of this suggestion was provided in the present study by the action of the K+ channel blocker, TEA, which reestablished a uniform pattern of Ca2+ oscillations with a frequency comparable to that in control cells, by partial cellular depolarization and possibly by mediating the sequestration of Ca2+ into intracellular stores. Collectively, these studies suggest that membrane channels, including gap junctions and K+ channels, are important targets of BaP toxicity. If BaP exerts similar effects in vivo, the consequence may be impairment of tissue homeostasis and function.
It is noteworthy that alterations of Ca2+ homeostasis in cells exposed to PAHs have been most extensively investigated in other persistent environmental pollutants such as polychlorinated biphenyls (PCBs). Among PCBs, perturbation of Ca2+ signaling appears to be greatest among the noncoplanar, non-Ah receptor-activating, ortho-substituted PCB congeners (Kodavanti and Tilson, 2000). These nondioxin-like PCBs accumulate in the brain and can interfere with Ca2+ homeostasis via multiple targets, including ryanodine receptors (Wong et al., 1997
), L-type and/or voltage-operated Ca2+ channels (Bae et al., 1999
), and Ca2+ buffering systems (Yang and Kodavanti, 2001
) leading to translocation of PKC.
In contrast, the actions of dioxin-like PAHs on Ca2+ homeostasis appear to be subtler. BaP and 2,3,7,8-tetrachlorodibenzo-p-dioxin can induce sustained elevation of Ca2+, which is thought to involve cytochrome P450-mediated metabolism (Hanneman et al., 1993; Romero et al., 1997
; Tannheimer et al., 1999
). BaP metabolites were more effective in elevating Ca2+ than the parent compound (Mounho and Burchiel, 1998
; Romero et al., 1997
; Tannheimer et al., 1999
). Oxidative metabolism of BaP can result in redox stress, which could alter voltage-sensitive pathways in membranes. Similar suppressive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on Ca2+ oscillations in Clone 9 cells have been observed, whereas the weak aryl hydrocarbon agonist 1,2,3,4-TCDD had no effect on oscillations (Barhoumi et al., 1996
; Mouneimne et al., 1999
). Further studies are needed to clarify how BaP modulation of redox pathways in cells may affect K+ channel activity in Clone 9 cells. It is also possible that alterations in Ca2+ homeostasis in Clone 9 cells observed in the present study may involve cytochrome P450-independent mechanisms. It has been reported that BaP-induced alterations in signal transduction pathways may contribute to tumor promotion and progression through nongenotoxic mechanisms (Parrish et al., 1998
).
Given the importance of [Ca2+]i in signal transduction, cellular proliferation, mitochondrial energy production, and gene expression, a better understanding of (1) how the conversion of signal transduction events initiated at the cell surface are converted into code that regulates downstream events, and (2) what the consequences are of toxicant-altered, encoded Ca2+ signals. The present studies provide a powerful experimental approach to begin to address these issues. Studies are currently underway to define the functional consequences of BaP-altered amplitude and frequency-encoded Ca2+ signals.
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ACKNOWLEDGMENTS |
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NOTES |
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