title: Cocaethylene affects human microvascular endothelial cell p38 mitogen-activated protein kinase activation and nuclear factor-kappaB DNA-binding activity.
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-kappaB (NF-kappaB) 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-kappaB 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-kappaB 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-kappaB 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-kappaB transcription factor family, whereas treatment with LPS decreased and then increased the DNA binding of these dimers.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Danyel Hermes
authors.initials: DH
authors.lastname: Tacker
authors.forename: Norbert K
authors.initials: NK
authors.lastname: Herzog
authors.forename: Anthony O
authors.initials: AO
authors.lastname: Okorodudu
mesh_terms: D002199:Capillary Permeability; D002460:Cell Line; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008833:Microcirculation / Q000166:cytology; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D016328:NF-kappa B; D048051:p38 Mitogen-Activated Protein Kinases; C066444:cocaethylene; D003042:Cocaine