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 language | English (US) |
|---|---|
| Pages (from-to) | 1926-1933 |
| Number of pages | 8 |
| Journal | Clinical Chemistry |
| Volume | 52 |
| Issue number | 10 |
| DOIs | |
| State | Published - Oct 2006 |
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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 journal › Article
}
TY - JOUR
T1 - Cocaethylene affects human microvascular endothelial cell p38 mitogen-activated protein kinase activation and nuclear factor-κB DNA-binding activity
AU - Tacker, Danyel Hermes
AU - Herzog, Norbert K.
AU - Okorodudu, Anthony
PY - 2006/10
Y1 - 2006/10
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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33749146967&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33749146967&partnerID=8YFLogxK
U2 - 10.1373/clinchem.2005.065250
DO - 10.1373/clinchem.2005.065250
M3 - Article
C2 - 16916993
AN - SCOPUS:33749146967
VL - 52
SP - 1926
EP - 1933
JO - Clinical Chemistry
JF - Clinical Chemistry
SN - 0009-9147
IS - 10
ER -