Cocaethylene affects human microvascular endothelial cell p38 mitogen-activated protein kinase activation and nuclear factor-κB DNA-binding activity

Danyel Hermes Tacker, Norbert K. Herzog, Anthony Okorodudu

Research output: Contribution to journalArticle

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Abstract

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-κB (NF-κB) 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-κB 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-κB 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-κB 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-κ 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.

Original languageEnglish (US)
Pages (from-to)1926-1933
Number of pages8
JournalClinical Chemistry
Volume52
Issue number10
DOIs
StatePublished - Oct 2006

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Endothelial cells
p38 Mitogen-Activated Protein Kinases
Endothelial Cells
Chemical activation
DNA
Dimers
Lipopolysaccharides
Phosphorylation
Transcription Factors
Monolayers
Permeability
Therapeutics
Cell signaling
Electrophoretic mobility
Salmonella typhi
Salmonella
Calcium Signaling
cocaethylene
Poisons
Capillary Permeability

ASJC Scopus subject areas

  • Clinical Biochemistry

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Cocaethylene affects human microvascular endothelial cell p38 mitogen-activated protein kinase activation and nuclear factor-κB DNA-binding activity. / Tacker, Danyel Hermes; Herzog, Norbert K.; Okorodudu, Anthony.

In: Clinical Chemistry, Vol. 52, No. 10, 10.2006, p. 1926-1933.

Research output: Contribution to journalArticle

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abstract = "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-κB (NF-κB) 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-κB 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-κB 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-κB 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-κ 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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N2 - 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-κB (NF-κB) 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-κB 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-κB 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-κB 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-κ 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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