Department of Molecular Biosciences, and Center for Childrens Environmental Health and Disease Prevention, University of California, Davis, California 95616
Received August 1, 2003; accepted September 23, 2003
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ABSTRACT |
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Key Words: polychlorinated biphenyls; cerebellar granule neurons; calcium signaling; glutamate excitotoxicity; ryanodine receptors.
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INTRODUCTION |
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A major physiological ligand for activation of RyR is intracellular Ca2+ itself. Enhancement of RyR activity by intracellular Ca2+ amplifies signals through a process termed calcium-induced calcium release (CICR). Chemical probes that enhance or inhibit RyR channel activity, such as caffeine, ryanodine, and ruthenium red, have been demonstrated to influence Ca2+ signaling events in a number of neuronal cell types (Kano et al., 1995; Llano et al., 1994
; Usachev and Thayer, 1997
). In this regard, non-coplanar polychlorinated biphenyls (PCBs) are anthropogenic chemicals of concern to human health that have been shown to disrupt Ca2+ homeostasis in cultured primary neurons and a neuronal cell line (Kodavanti et al., 1993
; Wong et al., 2001
). One mechanism by which non-coplanar PCB congeners alter Ca2+ regulation is a result of their interaction with RyR complexes that alter the fidelity of Ca2+ release from ER/SR stores (Wong et al., 1996
, 1997
). One of the most active modulators of RyR identified to date, 2,2',3,5',6-pentachlorobiphenyl (PCB 95), has been shown to promote RyR-mediated Ca2+ release by dramatically enhancing channel sensitivity to CICR and diminishing the inhibitory potency of physiological negative modulators Ca2+ and Mg2+. These actions of PCB 95 depend on the integrity of the complex formed between FKBP12 and RyR (Wong and Pessah 1997b
). Immunosuppressants such as FK506 and rapamycin, which specifically bind to FKBP12 and promote its dissociation from RyR, selectively eliminate responses to non-coplanar PCBs and related compounds in subcellular preparations and in intact PC12 cells (Pessah and Wong, 2001
).
Although the molecular mechanisms by which non-coplanar PCBs alter RyR function have been studied in subcellular preparations and PC12 cells, there are no studies to date that have investigated the consequence of these mechanisms on excitatory signaling events in primary neuronal cells. The objectives of the present study are to better understand how PCB 95 sensitizes Ca2+ signaling in primary cerebellar granule neuronal cultures, and to define their relationship to altered RyR function. PCB 95 at 110 µM is found to significantly enhance sensitivity of RyR towards caffeine, and these actions are similar to those of the immunosuppressant rapamycin, suggesting a common mechanism of sensitization. Furthermore, NMDA- and AMPA-elicited Ca2+ signals were significantly enhanced by PCB 95 or rapamycin, revealing a functional link between amplification of RyR signaling and sensitivity to excitatory amino acids. These results may explain, at least in part, the neurotoxicity of non-coplanar PCBs to neurons, including recently identified excitotoxicity that promotes apoptosis (Howard et al., 2003).
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MATERIALS AND METHODS |
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For experiments aimed at testing if the sensitizing actions of PCB 95 were dependent on the composition of the neuronal cultures (Fig. 3AC), the method of Schilling and coworkers was utilized (Schilling et al., 1991
). Cerebella from 16-day-old embryos (C57BL/6) were incubated in 0.1% trypsin in PBS for 15 min at room temperature. Two volumes of 15% heat-inactivated fetal bovine serum was added to halt digestion, tissue was triturated with a plastic pipette, and the cell suspension was then filtered through a 210-µm nylon mesh. The cells were collected by centrifugation (500 x g, 15 min) and resuspended in basal Eagles Medium supplemented with 2 mM sodium pyruvate and hormones (Fischers growth medium). Neurons were plated at a density of approximately 2 x 105 cells per cm2 on poly-D-lysine-coated MatTek culture dishes in Fischers growth medium supplemented with 5% heat-inactivated horse serum. Two-thirds of the volume in each dish was replaced with serum-free Fischers growth medium 24 h later, and cells were kept in a 37°C humidified chamber with 5% CO2 up to day four in culture. These cultures were comprised of granule and Purkinje neurons and glia (Schilling et al., 1991
).
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Unless otherwise noted, preincubations of cells with PCB 95, PCB 66, rapamycin, thapsigargin, ryanodine, or control solvent (DMSO) were performed by replacing 250 µl of drug containing BSS into the culture dishes 20 min prior to commencing acquisition of images. In experiments designed to study the acute effect of PCBs and rapamycin, chemicals were introduced singly or in combination by manual perfusion using a glass pipette. Caffeine, NMDA, or AMPA was introduced by a gravity-driven, valve-controlled perfusion system (ALA Scientific, New York, NY) with the inlet placed a fixed distance from the cells imaged to reduce between-dish variability.
Analysis.
Post acquisition analyses of temporal changes in fluorescence ratio (R340/380 nm) from individual cell somas were calculated using Imagemaster software (PTI). R340/380 were calculated using circular regions of interest placed within the somatic region of individual cells, based on the methods previously described (Grynkiewicz et al., 1985; also see below). In order to acquire data from granule neurons, imaging analysis was only performed on small-bodied (approximately 10-µm diameter soma) neurons. This eliminated acquisition of data from other cell types, since all other cells in the preparation (glia, Purkinje neurons, and inhibitory interneurons) contain larger somas (approximately 20-µm diameter). For experiments with mixed cerebellar cultures (Figs. 3A
3C
), all cells in the field were imaged, regardless of cell diameter. Cells responding with a base-to-peak change
5 SD over the baseline recording were scored as responders and included for statistical analysis. Unless otherwise indicated, data obtained from individual neurons within each microscopic field were pooled from several dishes between days 2 and 4 in culture. In some experiments (e.g., responses to NMDA), the amplitude of each cells response was expressed as a fraction of the mean control response obtained from all the cells analyzed within a microscopic field. For each treatment, data was collected from three or more dishes from at least two different culture sets. Differences in mean responses were tested for significance using Mann-Whitney U or Kruskal-Wallis statistical tests followed by a post hoc Dunns test to determine significant differences between treatments. For the Dunns test, the p-value for significant difference was determined by dividing 0.05 by the number of groups x the number of groups - 1. For experiments scoring percentage cells responding, data were fitted to a logit model, and differences between treatments and days in culture tested for significance by a post hoc
2 test of contrasts. Standard errors are not indicated for data analyzed using this statistical test since they are not symmetrical. The p-value for significance was calculated by dividing 0.05 by the number of comparisons and is indicated in each figure legend.
Materials.
Cell culture reagents (media, serum, L-glutamine, penicillin-streptomycin, and trypsin) were purchased from Life Technologies (Grand Island, NY). DNase I was purchased from Worthington Biochemical Corporation (Lakewood, NJ), and poly-D-lysine was from Sigma (St Louis, MO). 5-Fluoro-2-deoxyuridine was purchased from Calbiochem (La Jolla, CA), Fura2-AM and pluronic F-127 were from Molecular Probes (Eugene, OR), PCB congeners were from Ultra Scientific (North Kensington, RI), ryanodine and NMDA were from Calbiochem (La Jolla, CA), rapamycin, AMPA, and caffeine were from Sigma (St Louis, MO), and thapsigargin was from Research Biochemicals (Natick, MA).
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RESULTS |
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Relationship Between Rapamycin- and PCB 95-induced RyR Sensitization
Immunophilin FKBP12, a cis/trans peptidylprolyl isomerase, plays an important signaling function in neurons (Snyder et al., 1998), and has been shown to be a required component of PCB 95-mediated sensitization of RyR (Pessah and Wong, 2001
; Wong and Pessah, 1997
; Wong et al., 2001
). We investigated the influence of the immunosuppressant rapamycin, a high-affinity ligand for FKBP12, and its possible relationship to sensitization of RyR by PCB 95 in intact granule neurons in culture. Pretreatment of cerebellar granule neurons with rapamycin (20 µM, 20 min) sensitized responses to 5 mM caffeine in a manner similar to that seen with PCB 95 (increased the fraction of cells responding over three-fold (p < 0.008; Fig. 4
)). PCB 95 (10 µM) and rapamycin (50 µM) introduced in combination slightly elevated the number of neurons responding to caffeine compared to either treatment alone, suggesting a common mechanism of action (Fig. 4
). The percentage of neurons responding to caffeine in the presence of PCB 95 and rapamycin in combination was not significantly different compared with responses obtained when the compounds were tested singly (p > 0.008; Fig. 4
).
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Our attempts to measure Ca2+ responses mediated by the metabotropic glutamate receptor agonist, t-ACPD (400 µM) in granule cells in culture were unsuccessful (data not shown). Mixed success in eliciting t-ACPD-induced Ca2+ fluxes has been reported previously (del Río et al., 1999). Since t-ACPD-elicited Ca2+ responses have been measured at 25°C following cellular depolarization (Irving et al., 1992b
), attempts were made to elicit t-ACPD-induced Ca2+ signals directly following a 10-s depolarization with 40 mM KCl with no added success. In addition, preincubation with PCB 95 (10 µM, 20 min) did not produce a measurable t-ACPD-induced Ca2+ response. In comparison, the responses to perfusion of NMDA or AMPA were robust. Cells pretreated with PCB 95 exhibited enhanced responses to a 10-s exposure of 5 µM NMDA or AMPA, with the peak Ca2+ response increasing by 40 and 87%, respectively, relative to control measurements (Figs. 6A
6D
).
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DISCUSSION |
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In contrast to the temporal changes in caffeine sensitivity seen with cerebellar granule neurons in culture, PCB 95 enhanced the sensitivity to caffeine with short incubation times (20 min), suggesting a direct interaction with existing RyR complexes resulting in signal amplification. Interestingly the resting [Ca2+]i and the Ca2+-store levels were not measurably altered by PCB 95, indicating an allosteric mechanism altering the sensitivity to direct RyR activators. Also evident from the present studies is the similar nature by which rapamycin and PCB 95 enhanced the sensitivity of granule neurons to caffeine, indicating a common mechanism. Undifferentiated PC12 cells, perfused with nM to low mM PCB 95, elicited a transient rise of [Ca2+]i that was not completely dependent on extracellular Ca2+ (Wong et al., 2001). In addition, preincubation with FK506 completely eliminated PCB 95-induced rises in cytoplasmic Ca2+ in PC12 cells. The differences in eliciting direct responses to PCB 95 and the ability of immunosuppressant to eliminate the effects of PCB 95 in PC12 cells and granule neurons may reflect differential expression of RyR isoforms in the two cell types and/or how FKBP12 modulates RyR channel function. In support for this interpretation, mammalian type 1 (RyR1) and type 3 (RyR3) ryanodine receptors exhibit fundamental differences in pharmacology and gating behavior that may stem from differences in key protein-protein interactions (Fessenden et al., 2000
; Rousseau and Proteau, 2001). Although these results suggest a common primary mechanism mediated by the FKBP12/RyR-sensitive store, there appears to be a fundamental difference in how FKBP12 regulates RyR function in the two cell types, especially when probed with PCB 95 and immunosuppressant.
Previous studies with skeletal fibers and isolated SR have shown that preincubation with FK506 or rapamycin increased the sensitivity of RyR1 to caffeine (Ahern et al., 1997; Brillantes et al., 1994
; Lamb and Stephenson, 1996
; Mayrleitner et al., 1994
; Timerman et al., 1993; Wong and Pessah, 1997
). These actions have been attributed to the dissociation of FKBP12 from the RyR complex by the immunosuppressant. However, the actions of PCB 95 have been shown to require the integrity of the FKBP12/RyR1 complex, since both FK506 and rapamycin eliminated responses to non-coplanar PCB (Wong et al., 1997b
). Work with non-coplanar brominated tyrosine macrocyclic bastadins, isolated from the marine sponge Ianthella basta, have also been shown to possess activity that requires an intact FKBP12/RyR complex, but bastadin 5 itself failed to dissociate FKBP12 from skeletal SR membranes (Mack et al., 1994
). Moreover, bastadin 10 lacked RyR activity until the complex was reconstituted with recombinant FKBP12 (Chen et al., 1999
). These results indicate that PCB 95 and bastadins interact with the FKBP12/RyR complex to enhance sensitivity to caffeine and RyR-mediated Ca2+ signaling. Whether PCB 95 sensitizes the RyR by weakening the FKBP12/RyR complex in culture granule neurons remains to be determined.
PCB 95 and Rapamycin Amplify Signaling through Ionotropic Glutamate Receptors
In addition to sensitizing RyR-mediated Ca2+ release, a new finding was that PCB 95 and rapamycin significantly enhanced responses to agonists (NMDA and AMPA) of ionotropic glutamate receptors. In addition to typical Ca2+ influx mediated through NMDA receptor activation, AMPA receptors expressed in cerebellar granule neurons have been reported to also conduct Ca2+ (Jones et al., 2000). Our results indicated that PCB 95 sensitizes both receptor subtypes to their respective agonists. Unexpectedly, these actions of PCB 95 were not significantly altered by store depletion (thapsigargin) and RyR block (Ry). In fact, thapsigargin or Ry alone enhanced NMDA responses and were seen in addition to the enhancement produced by PCB 95. Since PCB 95, rapamycin, thapsigargin, and ryanodine all either directly or indirectly affect CICR, it is possible that all four reagents enhance NMDA-elicited Ca2+ signals through convergent mechanisms that modify RyR conformation. Ca2+ influx through store- and voltage-operated Ca2+ influx channels (SOCCs and VOCCs) have been shown to amplify Ca2+ signals mediated by NMDA (Netzeband et al., 1999
; Qiu et al., 1995
Qiu et al., 1998; Simpson et al., 1993
). Furthermore, SOCCs and VOCCs have been shown to physically interact with RyRs in a manner that enhances Ca2+ entry in response to RyR activation (Chavis et al., 1996
; Kiselyov et al., 2000
; Nakai et al., 1996
). These results indicate that the amplification mechanism may be mediated through conformational states of RyR resulting from direct binding of SOCCs or VOCCs to the FKBP12/RyR complex (caffeine, ryanodine, rapamycin, or PCB 95) or indirectly as a consequence of depletion of Ca2+ stores (thapsigargin). Considering that PCB 95 amplifies NMDA-mediated signaling, even when the RyR channels are blocked by ryanodine, the possibility is raised that conformational coupling between RyR and SOCCs or VOCCs could amplify excitatory signals in cerebellar granule neurons. Therefore, ligands that alter the interaction between RyR and FKBP12 may in turn modulate functional interactions with plasma membrane Ca2+ entry channels responsible for signal amplification and refilling of stores. In support of this interpretation, rapamycin has been shown to alter functional communication between the RyR and L-type VOCC (Lamb and Stephenson, 1996
).
While there is evidence in the literature that non-coplanar PCBs may affect Ca2+ regulation through RyR-independent mechanisms (Kodavanti et al., 1993; Mundy et al., 1999
), it is unlikely these interactions are responsible for the PCB 95 enhancement of the NMDA and AMPA receptor-mediated Ca2+ signal observed in the present study. Kodavanti et al. (1993)
originally reported that non-coplanar 2,2'-dichlorobiphenyl (PCB 4; 3050 µM) caused an increase in [Ca2+]i when perfused onto cerebellar granule neurons, and Mundy et al.(1999)
determined this intracellular Ca2+ rise to be due to an increase in Ca2+ influx, likely due to diminished microsomal and mitochondrial Ca2+ buffering. While this is a plausible explanation for their data, it should be noted that in our studies, we did not observe an increase in [Ca2+]i upon PCB 95 addition (Fig. 5A
). Several experimental differences should be noted in the present study, including the age of the neurons in culture on experimental challenge (days 24 vs. days 68), and the substantially higher levels (3050 µM) of a different non-coplanar PCB congener (2,2'-dichlorobiphenyl) needed to alter resting [Ca2+]i in the previous studies. More recently, Inglefield and coworkers tested the actions of A1254 and non-coplanar ortho-substituted PCBs (10 µM) on cortical neurons in culture (Inglefield et al., 2001
, 2002
). Aroclor 1254 and the non-coplanar PCBs tested (but not those having coplanar structures) elicited intracellular Ca2+ transients that were not altered either by ryanodine or the L-type Ca2+ channel blocker, nifedipine. Rather, these effects seemed to be mediated through activation of IP3Rs. It is interesting to note that Howard and coworkers recently found that Aroclor 1254 and non-coplanar PCB 47 (110 µM) significantly enhanced caspase-mediated apoptosis in hippocampal, but not cortical, neurons in culture (Howard et al., 2003
). Although inhibitors of Ca2+ entry and IP3R-mediated Ca2+ release failed to ameliorate PCB 47-induced apoptosis, benzeneethanamine 2,6-dichloro-4-(dimethylamino)-
-methyl-9-chloride, FLA 365, a selective cell-permeable blocker of RyRs (Calviello and Chiesi, 1989; Mack et al., 1992
), completely prevented excitotoxicity mediated by this non-coplanar PCB (Howard et al., 2003
). Collectively, these data indicate that the initial molecular target(s) of non-coplanar PCBs are likely to reside within the endoplasmic reticulum (structurally and functionally related RyR and IP3R channel complexes). How these compounds alter Ca2+ signaling depends not only on the pattern of expression of these channels within cells but the context in which they are expressed. Considering that both RyR and IP3R channels are highly regulated by both small ligands and accessory proteins, it is not surprising that the net actions of non-coplanar PCBs on Ca2+ signaling are highly dependent on cellular context.
Implications for Non-coplanar Ortho-substituted PCB Neurotoxicity
Multiple lines of evidence support the preferential neurotoxicity of non-coplanar, ortho-substituted PCBs. Decreased catecholamine levels in certain brain regions in mammals (Seegal et al., 1990, 1991a
,Seegal et al., b
) and reduced dopamine levels in rat pheochromocytoma cells (Shain et al., 1991
) have been attributed to certain ortho-substituted PCB congeners. Perinatal exposure of monkeys and rodents to PCBs induced behavioral abnormalities consisting of delayed reflex development, altered activity patterns, learning deficits, and impaired memory, problems currently of concern to human exposure (Schantz, et al., 1995
, 1997
, 2003
). Kodavanti et al. (1993)
first linked non-coplanar PCB toxicity to its ability to disrupt Ca2+ regulation in cerebellar granule cell studies. Evidence for a direct interaction of ortho-substituted PCBs with the RyR (Wong et al., 1996
, 1997
), suggests this may be a selective mechanism for the disruption of Ca2+ homeostasis and toxicity in neurons.
The finding that low levels of PCB 95 significantly enhanced Ca2+ signals elicited by NMDA and AMPA glutamate receptor activation, without altering basal Ca2+ levels, lends support for a selective, receptor-mediated mechanism of neurotoxicity. An alteration in the tightly regulated Ca2+ signaling pathways may induce profound changes in cellular functioning that eventually leads to the neurotoxicity described in numerous experimental and epidemiological studies. In conclusion, ortho-substituted PCBs are shown for the first time to directly interact with the RyR to sensitize RyR-mediated Ca2+ release in cerebellar granule neurons. Sensitization of responses to caffeine by rapamycin was similar to that of PCB 95, supporting a common mechanism of sensitization that could involve weakening of the RyR/FKBP12 complex. In addition, PCB 95 was shown to enhance NMDA- and AMPA-elicited Ca2+ signals, suggesting that the PCB 95/RyR/FKBP12 complex may be responsible for directly or indirectly altering Ca2+ influx through VOCCs or SOCCs. The PCB 95-induced alterations in Ca2+ signaling presented here provide insight into the mechanism of non-coplanar PCB toxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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