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 O. Okorodudu

Research output: Contribution to journalArticle

8 Scopus citations

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 1 2006

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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