Vascular connexins have differing responses to tumor necrosis factor
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KeywordsTumor Necrosis Factor HeLa Cell Label Cell Vascular Tone Cell Transfer
Cell-to-cell communication via gap junctions has been implicated in the control of vascular tone and may be altered in sepsis. Endothelial cells express connexin (Cx) 37, Cx40 and Cx43 and cytokines may modulate their function in sepsis resulting in altered gap junctional intercellular communication. Our hypothesis is that tumor necrosis factor (TNF) will decrease gap-junction-dependent cell-to-cell communication of vascular connexin.
Transformed HeLa cells expressing vascular Cx37, Cx40 or Cx43 were used in these experiments. HeLa cells were treated with TNF (20 ng/ml) for up to 2 hours. In dye-transfer experiments, carboxyfluroscein (HeLaCx40 and HeLaCx43) or Alexa Fluor-480 (HeLaCx37) was injected into one cell for 10 seconds and cell transfer was allowed to proceed for 10 minutes and the number of labeled cells counted. Cell lysates were prepared in Triton X-100 lysate buffer and detergent-soluble fractions collected. Cx37, Cx40 and Cx43 were detected by western blot.
After 1 hour, TNF treatment resulted in near total loss of dye-coupling in HeLaCx37 and HeLaCx43 (P < 0.02, n = 14 to 16) and remained constant up to 2 hours. Dye coupling in HeLaCx40 cells remained unchanged after 1 hour and decreased after 2 hours (P < 0.05, n = 10). Western blots indicated that TNF treatment did not affect detergent solubility of Cx40 and Cx43. However, TNF caused a significant increase in detergent solubility of Cx37.
These results suggest that inflammatory mediators affect connexins differently. The loss of Cx37 function may be due to the loss of detergent resistance, suggesting internalization of Cx37 in response to TNF.
Transformed HeLa cells were a gift from Dr K. Willecke.
This article is published under license to BioMed Central Ltd.