James E. Bodmer

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces.

cAMP protects endothelial barrier function independent of inhibiting MLC20-dependent tension development

Exposure of cultured human umbilical vein endothelial cells to the cAMP agonists theophylline and forskolin decreased constitutive isometric tension of a confluent monolayer inoculated on a collagen membrane, but it did not prevent increased tension in cells exposed to thrombin. The inability of cAMP agonists to prevent tension development correlated with an inability of cAMP stimulation to prevent increased 20-kDa myosin light chain (MLC20) phosphorylation in response to thrombin. Although cAMP did not prevent tension development or increased MLC20phosphorylation, cAMP attenuated the effect of thrombin on transendothelial electrical resistance across a confluent monolayer inoculated on a gold microelectrode. Activation of cAMP-dependent signal transduction did not prevent a decline in resistance in thrombin-treated cells, but it more promptly restored transendothelial resistance to initial basal levels (10 min) compared with thrombin only (60 min). ML-7, an MLC kinase antagonist, at doses that attenuate increased MLC20 phosphorylation and tension development, did not prevent a decline in resistance in thrombin-treated cells. Yet, ML-7 also restored transendothelial resistance more rapidly than thrombin alone (20 min) but at a slower rate than cAMP. These data demonstrate that activation of cAMP-dependent signal transduction protects barrier function independent of inhibition of MLC20-dependent tension development.

Endothelial Cell Electrical Impedance Parameter Artifacts Produced by a Gold Electrode and Phase Sensitive Detection

Frequency dependent cellular micro-impedance estimates obtained from a gold two-electrode configuration using phase sensitive detection have become increasingly used to evaluate cellular barrier model parameters. The results of this study show that cellular barrier function parameter estimates optimized using measurements obtained from this biosensor are highly susceptible to both time dependent and systematic instrumental artifacts. Based on a power spectral analysis of experimentally measured microelectrode voltages, synchronous, 60 Hz, and white Gaussian noise were identified as the most significant time dependent instrumental artifacts. The reduction of these artifacts using digital filtering produced a corresponding reduction in the optimized model parameter fluctuations. Using a series of instrumental circuit models, this study also shows that electrode impedance voltage divider effects and circuit capacitances can produce systematic deviations in cellular barrier function parameter estimates. Although the implementation of an active current source reduced the voltage divider effects, artifacts produced by coaxial cable and other circuit capacitive elements at frequencies exceeding 1 kHz still remained. Reducing time dependent instrumental fluctuations and systematic errors produced a significant reduction in cellular model barrier parameter errors and improved the model fit to experimental data