EPAC1/RAP1 ACTIVATION AMELIORATES ALCOHOL-INDUCED BARRIER DYSFUNCTION.
Document Type
Presentation
Start Date
22-10-2010 3:00 PM
End Date
22-10-2010 4:30 PM
Description
Abnormal microvascular leakage can lead to edema and tissue dysfunction. Alcohol worsens outcome in trauma victims, however the microvascular response to alcohol is not known. We tested the hypothesis that acute exposure to alcohol disrupts endothelial barrier function. We also investigated whether alcohol mediates its effects through: 1) PLO-dependent mechanisms, 2) local generation of superoxide, and 3) inhibition of cAMP-dependent pathways. We evaluated the barrier function of human umbilical vein endothelial cells (HUVEC) by determining the transendothelial electrical resistance (TER) using Electric Cell-substrate Impedance Sensing (ECIS). We tested if alcohol was acting through a PLO-dependent pathway using 1-butanol vs. 2butanol. We tested if the barrier dysfunction was dependent upon superoxide formation using two superoxide dismutase mimetics, MnTBAP (2 & 10 uM) and MnTBPyP (4 & 40 uM). We then tested the effect of two cAMP analogs, 8-Br-cAMP (100 uM) and the Epac1/Rap1 selective activator 8-CPT-2'-O-Me-cAMP (8-CPT) (100 uM), on barrier function. Alcohol induced an acute decrease in TER in a dose dependent manner. 1-butanol, like ethanol, alters PLD signaling, while 2-butanol does not. Both 1-butanol and 2-butanol produced a similar drop in TER. MnTBAP and MnTBPyP both failed to inhibit the ethanol (EtOH)-induced decrease in TER. Treatment of cells with either of the cAMP analogs caused an increase in TER. However, the magnitude of the decrease in TER following addition of EtOH was not different between the cells treated with cAMP analogs or vehicle. The time course of the alcohol-induced increase in barrier dysfunction was significantly shortened by addition of 8-CPT 5 or 10 minutes after EtOH treatment (58 ± 8min for control vs. 16 ± 0.4 min for 8-CPT 5 min-post EtOH and 19 ± 0.6 min for 8-CPT 10 min-post EtOH, p<0.01 ). The results suggest that the alcohol-induced decrease in barrier integrity is not dependent upon PLO signaling, local production of superoxide, or inhibition of cAMP-dependent pathways. However, pharmacological elevation of intracellular cAMP levels in endothelial cells can resolve alcohol induced barrier dysfunction via an Epac1/Rap1-dependent pathway. Supported by NIH P20 RR018766 and a grant from the American Heart Association.
Recommended Citation
Doggett, Travis and Breslin, J. W., "EPAC1/RAP1 ACTIVATION AMELIORATES ALCOHOL-INDUCED BARRIER DYSFUNCTION." (2010). Dr. Joseph M. Moerschbaecher, III Graduate Research Day. 7.
https://digitalscholar.lsuhsc.edu/grad_rs/2010/poster2/7
EPAC1/RAP1 ACTIVATION AMELIORATES ALCOHOL-INDUCED BARRIER DYSFUNCTION.
Abnormal microvascular leakage can lead to edema and tissue dysfunction. Alcohol worsens outcome in trauma victims, however the microvascular response to alcohol is not known. We tested the hypothesis that acute exposure to alcohol disrupts endothelial barrier function. We also investigated whether alcohol mediates its effects through: 1) PLO-dependent mechanisms, 2) local generation of superoxide, and 3) inhibition of cAMP-dependent pathways. We evaluated the barrier function of human umbilical vein endothelial cells (HUVEC) by determining the transendothelial electrical resistance (TER) using Electric Cell-substrate Impedance Sensing (ECIS). We tested if alcohol was acting through a PLO-dependent pathway using 1-butanol vs. 2butanol. We tested if the barrier dysfunction was dependent upon superoxide formation using two superoxide dismutase mimetics, MnTBAP (2 & 10 uM) and MnTBPyP (4 & 40 uM). We then tested the effect of two cAMP analogs, 8-Br-cAMP (100 uM) and the Epac1/Rap1 selective activator 8-CPT-2'-O-Me-cAMP (8-CPT) (100 uM), on barrier function. Alcohol induced an acute decrease in TER in a dose dependent manner. 1-butanol, like ethanol, alters PLD signaling, while 2-butanol does not. Both 1-butanol and 2-butanol produced a similar drop in TER. MnTBAP and MnTBPyP both failed to inhibit the ethanol (EtOH)-induced decrease in TER. Treatment of cells with either of the cAMP analogs caused an increase in TER. However, the magnitude of the decrease in TER following addition of EtOH was not different between the cells treated with cAMP analogs or vehicle. The time course of the alcohol-induced increase in barrier dysfunction was significantly shortened by addition of 8-CPT 5 or 10 minutes after EtOH treatment (58 ± 8min for control vs. 16 ± 0.4 min for 8-CPT 5 min-post EtOH and 19 ± 0.6 min for 8-CPT 10 min-post EtOH, p<0.01 ). The results suggest that the alcohol-induced decrease in barrier integrity is not dependent upon PLO signaling, local production of superoxide, or inhibition of cAMP-dependent pathways. However, pharmacological elevation of intracellular cAMP levels in endothelial cells can resolve alcohol induced barrier dysfunction via an Epac1/Rap1-dependent pathway. Supported by NIH P20 RR018766 and a grant from the American Heart Association.
Comments
See abstract book page 51