Inositol 1,4,5-Triphosphate Receptor-Sensitive Ca2+Release, Store-Operated Ca2+ Entry, and cAMP Responsive Element Binding Protein Phosphorylation in Developing Cortical Cells following Exposure to Polychlorinated Biphenyls
- Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Abstract
The present study assessed intracellular Ca2+signaling pathways sensitive to polychlorinated biphenyls (PCBs), xenobiotics that perturb neural development and plasticity. Mobilization of intracellular Ca2+ stores after acute exposure to a PCB mixture, Aroclor 1254 (A1254), as well as selected PCB congeners, was studied in P0 rat cortical neuronal culture using fluorescence microscopy. Ca2+ responses to A1254 progressed from a transient intracellular Ca2+ increase (lasting 3–5 min) at 1 to 2 μM (0.3–0.6 ppm) to a Ca2+ transient with store-operated Ca2+ influx and later disturbances of basal Ca2+ concentration; this latter pattern occurred more often with 10 to 20 μM (3–6 ppm) A1254. Thapsigargin, xestospongin C, and carbachol/Ca2+-free buffer blocked significantly the PCB-induced Ca2+ transient, whereas both ryanodine (to deplete ryanodine-sensitive stores) and the L-type Ca2+channel blocker nifedipine were without effect on the A1254 initial Ca2+ transient. Both thapsigargin and xestospongin also blocked latent elevations (at 0.5 h) in Ca2+, disturbances that depend upon extracellular Ca2+ entry via ion channels. Two possible consequences were explored. Phosphorylation of cAMP responsive element binding protein, a Ca2+-activated nuclear transcription factor (CREB), occurred in an A1254 concentration-dependent manner and persisted at least 1 h. Cell viability following a 24-h exposure to A1254 (2–20 μM) was decreased at 20 μM, but only in cells cultured >6 days. This cell death did not occur via an apoptotic mechanism. These results indicate that Ca2+ disturbances following PCB exposure are associated with 1) discrete alterations in IP3receptor-mediated signals and 2) activation of downstream events that impact developing cortical cells.
Footnotes
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Send reprint requests to: Dr. Timothy J. Shafer, Neurotoxicology Division, MD-74B, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. E-mail:Shafer.Tim{at}epa.gov
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The research described in this article has been funded, reviewed, and approved by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency. Approval does not signify that the contents necessarily reflect the views and policies of the agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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Portions of this work were presented at the combined annual meeting of the American Society of Biochemistry and Molecular Biology and the American Society of Pharmacology and Experimental Therapeutics, and have been published in abstract form (Inglefield et al., 2000).
- Abbreviations:
- PCB
- polychlorinated biphenyl
- [Ca2+]i
- intracellular calcium concentration
- IP3
- inositol 1,4,5-triphosphate
- A1254
- Aroclor 1254
- fura-2-AM
- fura-2-acetoxymethyl ester
- DCB
- 2,2′-dichlorobiphenyl (PCB 4)
- PCB 15
- 4,4′-dichlorobiphenyl
- PCB 126
- 3,3′,4,4′,5-pentachlorobiphenyl
- PCB 138
- 2,2′,3,4,4′,5′-hexachlorobiphenyl
- DMSO
- dimethyl sulfoxide
- DIV
- days in vitro
- TUNEL
- terminal-deoxynucleotidyl-transferase-mediated dATP biotin nick-end labeling
- CREB
- cAMP responsive element binding protein
- pCREB
- phospho-cAMP responsive element binding protein
- SOC
- store-operated channel
- VGCC
- voltage-gated Ca2+ channel
- MAP
- mitogen-activated protein
- ERK
- extracellular signal-regulated kinase
- GABAA
- γ-aminobutyric acid receptor, type A
- AFC
- 7-amino-4-trifluoromethylcoumarin
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- Received October 11, 2000.
- Accepted December 19, 2000.
- U.S. Government
