Clinical Chemistry
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Clinical Chemistry 52: 1926-1933, 2006. First published August 17, 2006; 10.1373/clinchem.2005.065250
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(Clinical Chemistry. 2006;52:1926-1933.)
© 2006 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

Cocaethylene Affects Human Microvascular Endothelial Cell p38 Mitogen-Activated Protein Kinase Activation and Nuclear Factor-{kappa}B DNA-Binding Activity

Danyel Hermes Tacker, Norbert K. Herzog and Anthony O. Okorodudua

Department of Pathology, University of Texas Medical Branch, Galveston, TX.

aAddress correspondence to this author at: University of Texas Medical Branch, 301 University Blvd., Rte. 0551, Galveston, TX 77555-0551.

Background: Cocaethylene (CE) is known to increase the permeability of human microvascular endothelial cell monolayers. The molecular mechanism underlying this increase may involve calcium-modulated signaling pathways such as the p38 mitogen-activated protein kinase (p38 MAPK) and the nuclear factor-{kappa}B (NF-{kappa}B) family of transcription factors. The hypothesis of this study was that CE-mediated endothelial permeability change may be mediated by the p38 MAPK and consequently NF-{kappa}B dimers.

Methods: We used sandwich ELISA to detect phosphorylated p38 MAPK in the cell line human microvascular endothelial cell 1 (HMEC-1) after treatment with 1 mmol/L CE. We used electrophoretic mobility shift assay to detect changes in NF-{kappa}B dimers present in HMEC-1 and their DNA-binding activity after treatment with CE. Lipopolysaccharide (LPS) from Salmonella typhosa was used as a positive control for all experiments.

Results: Treatment with CE and LPS had similar effects on HMEC-1 p38 MAPK phosphorylation and NF-{kappa}B DNA-binding activity. Both treatments increased the phosphorylation of p38 MAPK, consistent with activation of proinflammatory cell signaling. Treatment of HMEC-1 with CE decreased DNA binding of both the RelA/p50 and p50/p50 dimers of the NF-{kappa}B transcription factor family, whereas treatment with LPS decreased and then increased the DNA binding of these dimers.

Conclusion: In addition to increasing HMEC-1 monolayer permeability, CE also alters transcription factor and kinase activity related to inflammation. Thus, CE causes endothelial activation that can elicit a prolonged and organized cellular response, rather than being directly toxic to endothelial cells.






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Copyright © 2006 by the American Association for Clinical Chemistry.