Cocaethylene affects human microvascular endothelial cell p38 mitogen-activated protein kinase activation and nuclear factor-kappaB DNA-binding activity.
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.
Tacker
Danyel Hermes
DH
Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Herzog
Norbert K
NK
Okorodudu
Anthony O
AO
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*
D016328:NF-kappa B; D048051:p38 Mitogen-Activated Protein Kinases; C066444:cocaethylene; D003042:Cocaine