Characterization of Calcium Oscillations in Normal and Benzo[a]pyrene-Treated Clone 9 Cells

Rola Barhoumi*,{ddagger}, Youssef Mouneimne{dagger}, Igbal Awooda*, Stephen H. Safe{dagger},{ddagger}, Kirby C. Donnelly*,{ddagger} and Robert C. Burghardt*,{ddagger},1

* Department of Veterinary Anatomy and Public Health, {dagger} Department of Physiology and Pharmacology, and {ddagger} Faculty of Toxicology, Texas A&M University, College Station, Texas 77843

Received February 8, 2002; accepted April 15, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular Ca2+ oscillations induced by oxytocin and vasopressin were analyzed in a rat liver cell line (Clone 9) in order to identify mechanisms by which benzo[a]pyrene (BaP) alters Ca2+signaling patterns in these cells. Clone 9 cells exhibit an initial Ca2+ spike, followed by Ca2+ oscillations upon oxytocin or vasopressin treatment. The range of frequencies (maximum 110 mHz) was dependent on agonist concentration with a constant amplitude less than or equal to the amount of Ca2+ generated from the inositol trisphosphate (InsP3)-sensitive pool. This study examined contributions of extracellular and intracellular pools to the frequency of Ca2+ oscillations and the role of membrane channels, second messengers, and different pharmacological reagents on the regulation of oscillation frequency in both control and BaP-treated cells. Results indicated that the Ca2+ oscillations are mainly due to inositol 1,4,5-triphosphate (InsP3)-sensitive stores and that extracellular Ca2+ contributes to refilling of this intracellular Ca2+ pool. The frequency of Ca2+ oscillations is also sharply affected by protein kinase C activated by phospholipase C. In BaP-treated Clone 9 cells, basal Ca2+ levels were elevated and the frequency of Ca2+ oscillations was suppressed in a dose-dependent fashion. Suppression of Ca2+ oscillations is due, at least in part, to an effect of BaP on enhanced opening of K+ channels. This was confirmed by showing that inhibition of the K+ channel opening by tetraethylammonium chloride can reverse the effect of BaP on oxytocin-induced Ca2+ oscillations, and potentially decrease the toxicity of BaP.

Key Words: Ca2+ oscillations; oxytocin; vasopressin; fluorescence microscopy; laser cytometry; BaP.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium signaling in many excitable and nonexcitable cell types frequently occurs as repetitive increases in cytoplasmic-free Ca2+ concentration ([Ca2+]i) referred to as Ca2+ oscillations. The periodic [Ca2+]i spikes, which increase with increasing agonist concentration, are thought to constitute a frequency-encoded signal with a high signal-to-noise ratio, which limits prolonged exposure of cells to high [Ca2+]i (Sneyd et al., 1995Go). Analysis of mechanisms that underlie the spatiotemporal patterns of [Ca2+]i has led to an understanding of the role of plasma membrane Ca2+ channels in triggering external Ca2+ influx and inositol 1,4,5-trisphosphate (InsP3) receptors and/or ryanodine receptors in the release of Ca2+ from intracellular stores (Berridge, 1997Go; Berridge et al., 2000Go; Putney and Bird, 1993Go).

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., 1993Go; Stojilkovic et al., 1994Go; Tse et al., 1993Go). 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, 1998Go; Oancea and Meyer, 1998Go). Similarly, the activities of Ca2+-sensitive mitochondrial dehydrogenases in hepatocytes that drive ATP production are controlled by Ca2+ oscillations (Hajnóczky et al., 1995Go). 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., 1997Go; Hu et al., 1999Go; Li et al., 1998Go).

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., 1996Go, 2000Go). The cell line exhibits uniform properties and is widely used for in vitro toxicity studies ranging from oxidative injury (Grune et al., 1995Go) to chemical carcinogenesis (Na et al., 1995Go). Like normal liver cells (Ariño et al., 1989Go; Thomas et al., 1995Go) 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., 2000Go). 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, 1996Go). 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., 2000Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Culture media, Dulbecco’s phosphate-buffered saline (PBS), serum, nifedipine, thapsigargin, oxytocin, vasopressin, ethylene glycol-O,O‘-bis-(ß-aminoethyl ether) N,N,N'N'-tetraacetic acid (EGTA), tetraethylammonium chloride (TEA), 8-bromo-cyclic adenosine-3':5'-monophosphoric acid (8-Br-cAMP), 8-bromo-cyclic guanosine-3':5'-monophosphoric acid (8-Br-cGMP) and all general chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Chelerythrine chloride was purchased from A.G. Scientific, Inc. (San Diego, CA). Tissue culture flasks were obtained from Corning (Oneonta, NY) and 2-well Lab-Tek Chambered Coverglass slides were purchased from Nunc, Inc. (Naperville, IL). Fluo-4, AM and the Calcium Calibration Buffer kit #2 were purchased from Molecular Probes, Inc. (Eugene, OR).

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 Ham’s 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 (0–20 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., 2000Go). 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 10–15 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., 1991Go). It is also used as an indicator for capacitative Ca2+ entry (e.g., Broad et al., 2001Go). Chelerithrine chloride is a potent and selective inhibitor of group A and B PKC isoforms (Herbert et al., 1990Go). 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., 2000Go). TEA is an inhibitor of all known K+ channels (Hille, 1992Go).

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 10–15 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., 1999Go). Control experiments to monitor any effects of solvent for each test substance were performed similarly. For each experiment, data were recorded from 10–15 cells per well of the culture dish and 2–4 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 Dunnett’s or Tukey’s test for multiple comparisons at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of oxytocin- and vasopressin-induced Ca2+ oscillations in normal and BaP-treated Clone 9 cells.
Oxytocin and vasopressin were used to induce Ca2+ oscillations in Clone 9 cells, examples of which are shown in Figure 1Go. Uniform oscillations were established within 15 min of addition of hormone and remained uniform for at least 30 min. The frequency of these oscillations was concentration-dependent, although Clone 9 cells were more sensitive to vasopressin than to oxytocin (Fig. 2Go). For example, analysis of data obtained from individual cells revealed that the average frequency of oscillation in control Clone 9 cells treated with 100 nM oxytocin became stable with a value of 47.25 ± 3.55 mHz (i.e., one oscillation every 21 s) when Ca2+ was present in the culture medium. The maximum increase in the oscillating Ca2+ signal was approximately 136 nM above basal [Ca2+]i represented by a normalized value of about twice the basal [Ca2+]i level detected prior to initiation of oscillations (Fig. 1Go). The steady state [Ca2+]i, about which cells oscillate, did not change from the basal [Ca2+]i detected prior to addition of oxytocin. However, stable oscillations with an average frequency of 94.56 ± 8.27 mHz (i.e., one oscillation every 11 s) developed when cells were treated with 1.0 nM vasopressin. The increase in the oscillating Ca2+ signal was about 67 nM above basal [Ca2+]i, represented by a normalized value of approximately 1.5 times the basal [Ca2+]i detected prior to initiation of oscillations (Fig. 1Go). Clone 9 cells exhibited no spontaneous Ca2+ oscillations (data not shown).



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 1. An example of the highly uniform Ca2+ oscillations in Clone 9 cells that became stable after about 15 min after treatment with 100 nM oxytocin (left) and 0.5 nM vasopressin (right). The normalized value represents fluo-4 fluorescence intensity at each time point, divided by the fluorescence intensity at time zero. Normalized values of 2 and 1.5 indicate a 136 nM and 67 nM increase over basal [Ca2+]i levels, respectively.

 


View larger version (10K):
[in this window]
[in a new window]
 
FIG. 2. The [Ca2+]i frequency response of Clone 9 cells to graded concentrations of oxytocin (0–10 µM, left) and vasopressin (0–10 nM, right). The maximum oscillation frequency was 72.4 ± 4.0 mHz for oxytocin and 109.5 ± 7.1 mHz for vasopressin. Values shown represent average frequency of oscillations ± SEM of at least 26–43 cells at each concentration, collected from 2–4 different samples.

 
Addition of EGTA (1.0 mM) to the culture medium completely eliminated the oxytocin-induced oscillations and decreased the vasopressin-induced oscillation frequency to 13.25 ± 0.96 mHz. Further, the steady state [Ca2+]i within cells decreased to approximately 73% of the basal [Ca2+]i prior to addition of the hormone.

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. 3Go).



View larger version (12K):
[in this window]
[in a new window]
 
FIG. 3. Effect of 24-h BaP exposure (0–20 µM) on oxytocin (100 nM)- and vasopressin (1.0 nM)-induced Ca2+ oscillations in Clone 9 cells. Values represent mean frequencies ± SEM (mHz) of at least 20–40 cells per BaP concentration. *Significantly different from control at p < 0.05.

 
The contribution of the InsP3-sensitive pool to Ca2+ oscillations in normal and BaP-treated Clone 9 cells was determined following the establishment of stable, high frequency oscillations with 100 nM oxytocin or 1 nM vasopressin and subsequent treatment of cells with thapsigargin to cause leakage of Ca2+ from intracellular InsP3-sensitive stores while oscillations were ongoing. Data analysis indicated that 50 nM thapsigargin decreased the frequency of oscillation from 47.25 ± 3.55 mHz to 6.71 ± 3.54 mHz in oxytocin-stimulated cells within 6.4 min while 100 nM thapsigargin was needed to decrease the oscillation frequency from 94.56 ± 8.27 to 24.22 ± 7.96 mHz within the same time interval. One µM thapsigargin completely eliminated all agonist-induced Ca2+ oscillations. The magnitude of the Ca2+ peak during thapsigargin treatment was equal to or greater than the peak of Ca2+ oscillation induced by hormone alone (data not shown). Cells exposed to 10 µM BaP exhibited increased sensitivity to thapsigargin as addition of 50 nM of this microsomal Ca2+-ATPase pump inhibitor to cultures with ongoing 100 nM oxytocin- or 0.5 nM vasopressin-induced Ca2+ oscillations completely eliminated all oscillations.

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. 4Go). 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).



View larger version (12K):
[in this window]
[in a new window]
 
FIG. 4. Analysis of actions of PKC inhibitor and cyclic nucleotide second-messenger pathways on the frequency of Ca2+ oscillations in Clone 9 cells. Values represent mean ± SEM (mHz) of at least 20–50 cells for each concentration. Effects of the PKC inhibitor chelerythrine chloride (0–50 µM), of 8-Br-cGMP (0–1.0 mM), and of 8-Br-cAMP (0–1.0 mM) on oxytocin-induced Ca2+ oscillation frequencies are shown. Treatments marked with different letters are significantly different from each other at p < 0.05.

 
To further investigate the basis for the earlier observation of suppression of plasma membrane potential by BaP (Barhoumi et al., 2000Go), the action of the K+ channel blocker, TEA, was examined. Addition of TEA did not significantly alter the frequency of agonist-induced Ca2+ oscillations in untreated Clone 9 cells. In contrast, addition of the K+ channel blocker, TEA (10 mM) to the ongoing oxytocin- or vasopressin-induced Ca2+ oscillations restored the amplitude as well as the frequency of Ca2+ oscillations in BaP-treated cells to that of control levels. To examine the responses of individual cells in BaP-treated cultures to TEA, a subset of cells that exhibited loss of oscillatory behavior were examined. An example of the restoration of Ca2+ oscillations caused by addition of TEA is shown in Figure 5Go, and quantitative assessment of these cells is shown in Figure 6Go. These data suggest that the open state of K+ channels in Clone 9 cells exposed to BaP is enhanced resulting in suppression of Ca2+ oscillations. Further, restoration of Ca2+ oscillations can be achieved by reversing the open state of these plasma membrane channels.



View larger version (23K):
[in this window]
[in a new window]
 
FIG. 5. An example of the effect of the K+ channel blocker, TEA, on Ca2+ oscillations in a single Clone 9 cell treated for 24 h with10 µM BaP, followed by 100 nM oxytocin stimulation. In this example, there was complete suppression of oscillations. The vertical line indicates the addition of TEA. Note the decrease in [Ca2+]i and the restoration of the oscillation frequency and amplitude of individual [Ca2+]i peaks.

 


View larger version (25K):
[in this window]
[in a new window]
 
FIG. 6. Reversal of effect of BaP on oxytocin- and vasopressin-induced Ca2+ oscillations by TEA in Clone 9 cells in which Ca2+ oscillations were suppressed. Values represent mean frequencies ± SEM (mHz) of at least 20–40 cells per treatment group. Treatments with different letters are significantly different from each other at p < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Free [Ca2+]i is a versatile and universal signaling agent that plays a pivotal role in cellular homeostasis in virtually every cell type, e.g., as a second messenger involved in signal transduction, and as point of convergence for other second messenger pathways linking events initiated at the cell membrane to biological responses. Intracellular Ca2+ is consequently in a position to accumulate and integrate information from multiple signaling systems and to convert information into a form or code that regulates downstream events ranging from secretion to gene expression. The existence of diverse Ca2+ signaling patterns that vary in frequency and/or amplitude has led to the concept that many Ca2+-regulated processes are controlled by these codes (Berridge, 1997Go). A number of cellular and mitochondrial enzymes and transcription factors have been identified as decoders of Ca2+ signals. For example, CaM kinase II can discriminate between low and high frequency Ca2+ signals by decoding Ca2+ oscillations into graded levels of kinase activity (De Koninck and Schulman, 1998Go). Other decoders include certain isoforms of PKC, PLC, mitochondrial dehydrogenases and transcription factors including CREB, CREM, NF-AT, and NF-{kappa}B (Dale et al., 2001Go; Dolmetsch et al., 1997Go; Hajnóczky et al., 1995Go; Hardingham et al., 2001Go; Hu et al., 1999Go; Oancea and Meyer, 1998Go). In the case of Ca2+-sensitive transcription factors, numerous target genes are affected.

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., 1995Go; Zingg, 1996Go).

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., 2001Go). 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, 2001Go).

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., 2000Go). Ca2+-activated K+ channels can be stimulated by both cAMP and activation of soluble guanylate cyclase to oppose depolarization stimuli (Berridge et al., 2000Go; Lee and Kang, 2001Go) 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., 2000Go).

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., 2000Go). 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., 2000Go). 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, 2000Go). These nondioxin-like PCBs accumulate in the brain and can interfere with Ca2+ homeostasis via multiple targets, including ryanodine receptors (Wong et al., 1997Go), L-type and/or voltage-operated Ca2+ channels (Bae et al., 1999Go), and Ca2+ buffering systems (Yang and Kodavanti, 2001Go) 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., 1993Go; Romero et al., 1997Go; Tannheimer et al., 1999Go). BaP metabolites were more effective in elevating Ca2+ than the parent compound (Mounho and Burchiel, 1998Go; Romero et al., 1997Go; Tannheimer et al., 1999Go). 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., 1996Go; Mouneimne et al., 1999Go). 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., 1998Go).

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.


    ACKNOWLEDGMENTS
 
This work was supported in part by NIH grants P42-ES04917 and P30-ES09106 ES and funding from the Texas Agricultural Experiment Station.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (979) 847-8981. E-mail: rburghardt{at}cvm.tamu.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ariño, J., Bosch F., Gomez-Foix, A. M., and Guinovart, J. J. (1989). Oxytocin inactivates and phosphorylates hepatocyte glycogen synthase. Biochem. J. 261, 827–830.[ISI][Medline]

Bae, J., Stuenkel, E. L., and Loch-Caruso, R. (1999). Stimulation of oscillatory uterine contraction by the PCB mixture Aroclor 1242 may involve increased [Ca2+]i through voltage-operated calcium channels. Toxicol. Appl. Pharmacol. 155, 261–272.[ISI][Medline]

Barhoumi, R., Mouneimne, T., Phillips, T. D., Safe, S. H., and Burghardt, R. C. (1996). Alteration of oxytocin-induced calcium oscillations in clone 9 cells by toxin exposure. Fundam. Appl. Toxicol. 33, 220–228.[ISI][Medline]

Barhoumi, R., Mouneimne, Y., Ramos, K. S., Safe, S. H., Phillips, T. D., Centonze, V. E., Ainley, C., Gupta, M. S., and Burghardt R. C. (2000). Analysis of benzo[a]pyrene partitioning and cellular homeostasis in a rat liver cell line. Toxicol. Sci. 53, 264–270.[Abstract/Free Full Text]

Berridge, M. J. (1997). The AM and FM of calcium signalling. Nature 386, 759–760.[ISI][Medline]

Berridge M. J., Lipp, P., and Bootman, M. D. (2000). The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell. Biol. 1,11–21.[ISI][Medline]

Broad, L. M., Braun, F. J., Lievremont, J. P., Bird, G. S., Kurosaki, T., and Putney, J. W., Jr. (2001). Role of the phospholipase C-inositol 1,4,5-trisphosphate pathway in calcium release-activated calcium current and capacitative calcium entry. J. Biol. Chem. 276, 15945–15952.[Abstract/Free Full Text]

Burghardt, R. C., Barhoumi, R., Sanborn, B. M., and Andersen, J. (1999). Oxytocin-induced Ca2+ responses in human myometrial cells. Biol. Reprod. 60,777–782.[Abstract/Free Full Text]

Codazzi, F., Teruel, M. N., and Meyer, T. (2001). Control of astrocyte Ca(2+) oscillations and waves by oscillating translocation and activation of protein kinase C. Curr. Biol. 11, 1089–1097.[ISI][Medline]

Dale, L. B., Babwah, A. V., Bhattacharya, M., Kelvin, D. J., and Ferguson, S. S. G. (2001). Spatial-temporal patterning of metabotropic glutamate receptor-mediated inositol 1,4,5-triphosphate, calcium, and protein kinase C oscillations. Protein kinase C-dependent receptor phosphorylation is not required. J. Biol. Chem. 276, 35900–35908.[Abstract/Free Full Text]

De Koninck, P., and Schulman, H. (1998). Sensitivity of CaM Kinase II to the frequency of Ca2+ oscillations. Science 279, 227–230.[Abstract/Free Full Text]

Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C., and Healy, J. I. (1997). Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386, 855–858.[ISI][Medline]

Gee, K. R., Brown, K. A., Chen, W. N., Bishop-Stewart, J., Gray, D., and Johnson, I. (2000). Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium 27, 97–106.[ISI][Medline]

Grune, T., Reinheckel, T., Joshi, M., and Davies, K. J. (1995). Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome. J. Biol. Chem. 270, 2344–2351.[Abstract/Free Full Text]

Hajnóczky, G., Robb-Gaspers, L. D., Seitz, M. B., and Thomas, A. P. (1995). Decoding of cytosolic calcium oscillations in the mitochondria. Cell 82, 415–424.[ISI][Medline]

Hanneman, W., Legare, M., Barhoumi, R., Burghardt, R., Tiffany-Castiglioni, E., and Safe, S. (1993).2,3,7,8-tetrachlorodibenzo-p-dioxin–induced elevation of intracellular calcium ions in cultured hippocampal neurons and astroglia. Organohal. Comp. 13, 353–356.

Hardingham, G. E., Arnold, F. J. L., and Bading, H. (2001). Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity. Nat. Neurosci. 4, 261–267.[ISI][Medline]

Herbert, J. M., Augereau, J. M., Gleye, J., and Maffrand, J. P. (1990). Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 172, 993–999.[ISI][Medline]

Hille, B. (1992). Ionic Channels of Excitable Membranes. Sinaur Associates, Sunderland, MA.

Hu, Q., Deshpande, S., Irani, K., and Ziegelstein, R. C. (1999). [Ca2+]i oscillation frequency regulates agonist-stimulated NF-{kappa}B transcriptional activity. J. Biol. Chem. 274, 33995–33998.[Abstract/Free Full Text]

Kodavanti, P. R., and Tilson, H. A. (2000). Neurochemical effects of environmental chemicals: In vitro and in vivo correlations on second messenger pathways. Ann. N.Y. Acad. Sci. 919, 97–105.[Abstract/Free Full Text]

Lee, S. W., and Kang, T. M. (2001). Effects of nitric oxide on the Ca2+-activated potassium channels in smooth muscle cells of the human corpus cavernosum. Urol. Res. 29, 359–365.[ISI][Medline]

Li, W., Llopis, J., Whitney, M., Zlokarnik, G., and Tsien, R. (1998). Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature 392, 936–941.[ISI][Medline]

Lytton, J., Westlin, M., and Hanley, M. R. (1991). Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J. Biol. Chem. 266, 17067–17071.[Abstract/Free Full Text]

Mouneimne, Y., Barhoumi, R., Phillips, T. D., Safe, S. H., and Burghardt, R. C. (1999). Alteration of oxytocin- and vasopressin-induced calcium oscillations in a rat liver cell line by TCDD. Toxicologist 48, 64 (Abstract).

Mounho, B. J., and Burchiel, S. W. (1998). Alterations in human B cell calcium homeostasis by polycyclic aromatic hydrocarbons: Possible associations with cytochrome P450 metabolism and increased protein tyrosine phosphorylation. Toxicol. Appl. Pharmacol. 149, 80–89.[ISI][Medline]

Na, M. R., Koo, S. K., Kim, D. Y., Park, S. D., Rhee, S. K., Kang, K. W., and Joe, C. O. (1995). In vitro inhibition of gap junctional intercellular communication by chemical carcinogens. Toxicology 98, 199–206.[ISI][Medline]

Oancea E., and Meyer, T. (1998). Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95, 307–318.[ISI][Medline]

Parrish, A. R., Fisher, R. L., Bral, C. M., Burghardt, R. C., Gandolfi, A. J., Brendel, K., and Ramos K. S. (1998). Benzo(a)pyrene-induced alterations in growth-related gene expression and signaling in precision-cut adult rat liver and kidney slices. Toxicol. Appl. Pharmacol. 152, 302–308.[ISI][Medline]

Putney, J. W., Jr., and Bird, G. St. J. (1993). The inositol phosphate-calcium signaling system in nonexcitable cells. Endocr. Rev. 14, 610–631.[ISI][Medline]

Romero, D. L., Mounho, B. J., Lauer, F. T., Born, J. L, and Burchiel, S. W. (1997). Depletion of glutathione by benzo(a)pyrene metabolites, ionomycin, thapsigargin, and phorbol myristate in human peripheral blood mononuclear cells. Toxicol. Appl. Pharmacol. 144, 62–69.[ISI][Medline]

Sneyd, J., Keizer, J., and Sanderson, M. J. (1995). Mechanisms of calcium oscillations and waves: A quantitative analysis. FASEB J. 9, 1463–1472.[Abstract/Free Full Text]

Stauffer, P. L., Zhao, H., Luby-Phelps, K., Moss, R. L., Star, R. A., and Muallem, S. (1993). Gap junction communication modulates [Ca2+]i oscillations and enzyme secretion in pancreatic acini. J. Biol. Chem. 268, 19769–19775.[Abstract/Free Full Text]

Stojilkovic, S. S., Tomic, M., Kukuljan, M., and Catt, K. J. (1994). Control of calcium spiking frequency in pituitary gonadotrophs by a single-pool cytoplasmic oscillator. Molec. Pharmacol. 45, 1013–1021.[Abstract]

Tannheimer, S. L., Lauer, F. T., Lane, J., and Burchiel, S. W. (1999). Factors influencing elevation of intracellular Ca2+ in the MCF-10A human mammary epithelial cell line by carcinogenic polycyclic aromatic hydrocarbons. Mol. Carcinog. 25, 48–54.[ISI][Medline]

Taylor, C. W., and Thorn, P. (2001). Calcium signalling: IP3 rises again ...and again. Curr. Biol. 11, R352–355.[ISI][Medline]

Thomas, A. P., Renard-Rooney, D. C., Hajnóczky, G., Robb-Gaspers, D., Lin, C., and Rooney, T. A. (1995). Subcellular organization of calcium signalling in hepatocytes and the intact liver. In Calcium Waves, Gradients and Oscillation (G.R. Bock and K. Ackrill, Eds.), pp. 18–49. Ciba Foundation Symposium 188. John Wiley & Sons, West Sussex, England.

Tse, A., Tse, F. W., Almers, W., and Hille B. (1993). Rhythmic exocytosis stimulated by GnRH-induced calcium oscillations in rat gonadotropes. Science 260, 82–84.[ISI][Medline]

Tsien, R. (1989) Measurement of cytosolic free Ca2+ with quin 2. Methods Enzymol 172, 230–262.[ISI][Medline]

Wong, P. W., Brackney, W. R., and Pessah, I. N. (1997). Ortho-substituted polychlorinated biphenyls alter microsomal calcium transport by direct interaction with ryanodine receptors of mammalian brain. J. Biol. Chem. 272, 15145–15153.[Abstract/Free Full Text]

Yang, J.-H., and Kodavanti, P. R. S. (2001). Possible molecular targets of halogenated aromatic hydrocarbons in neuronal cells. Biochem. Biophys. Res. Com. 280, 1372–1377.[ISI][Medline]

Yue, C., Ku, C.-Y., Liu, M., Simon, M. I., and Sanborn, B. M. (2000). Molecular mechanism of the inhibition of phospholipase C ß3 by protein kinase C. J. Biol. Chem. 275, 30220–30225.[Abstract/Free Full Text]

Zingg, H. H. (1996). Vasopressin and oxytocin receptors. Bailliere’s Clin. Endocrinol. Metab. 10, 75–96.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Barhoumi, R.
Articles by Burghardt, R. C.
PubMed
PubMed Citation
Articles by Barhoumi, R.
Articles by Burghardt, R. C.