We have explored the use of ECIS to monitor the signal transduction pathways activated by G protein coupled receptors (GPCR). This assay is based upon the widely accepted conjecture that GPCR activation, regardless of the second messenger, results in alterations of the cell's cytoskeletal elements. This culminates in morphological changes, and this is precisely the type of event detected in real time and with great sensitivity by the ECIS biosensor.
In preliminary studies using this approach, several receptors have been successfully studied, including the muscarinic receptor monitored in a dose response experiment reported in the figure above. To prepare for this experiment, CHO cells, engineered to over express the muscarinic receptor were first grown to confluence over a period of 24 hours in the ECIS wells. At time zero in the figure, the cell-covered electrodes exhibited resistance values from 4.1 to 5.4 k ohms normal variation in the measurement. At the arrow, the control received a small volume of balanced salt solution, whereas the other wells received the same vehicle but containing different concentrations of the muscarinic agonist, carbachol (final concentrations are listed in the figure). Following agonist addition, there is a steep rise in the impedance lasting for 15 to 20 minutes. The impedance then plateaus and begins a gradual return to the baseline value. The control shows only a slight response to the vehicle addition. The impedance changes are very significant- doubling in some cases.
The use of whole cell sensors for these signal transduction measurements is especially significant in drug discovery efforts. Drugs must function at the cellular level to ultimately have an effect upon tissue and the whole organism. The complex molecular events that result in a cellular response to a particular compound involve a series of signaling and feedback circuits. Simply detecting binding of a compound to a receptor does not assure the efficacy of the compound in other aspects of the transduction mechanism such as receptor activation, second messenger production, etc. Using whole cell sensors such as ECIS in drug assays eliminates these potential stumbling points. In addition, with the whole cell system one can also evaluate the toxicity of compounds and the effect of drugs upon different cell types.
Signal Transduction Assays
Related ECIS Publications
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Antipermeability Function of PEDF Involves Blockade of the MAP Kinase/GSK/β-Catenin Signaling Pathway and uPAR Expression. Jinling Yang, Elia J. Duh, Ruth B. Caldwell, and M. Ali Behzadian. Invest. Ophthalmol. Vis. Sci. 2010; 51:3273-3280.
Jingyan Han, Guoquan Liu, Jasmina Profirovic, Jiaxin Niu, and Tatyana Voyno-Yasenetskaya. Zyxin is involved in thrombin signaling via interaction with PAR-1 receptor. FASEB J. 2009; 23:4193-4206.
Dennis J. Grab, Jose C. Garcia-Garcia, Olga V. Nikolskaia, Yuri V. Kim, Amanda Brown, Carlos A. Pardo, Yongqing Zhang, Kevin G. Becker, Brenda A. Wilson, Ana Paula C. de A. Lima, Julio Scharfstein, J. Stephen Dumler, “Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells.” PLOS, July 2009, Volume 3, Issue 7, e479.
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Becker, P.M., Waltenberger, J., Yachechko, R., Mirzapoiazova, T., Sham, J.S.K., Lee, C.G., Elias, J.A., Verin, A.D., "Neuropilin-1 regulates vascular endothelial growth factor-mediated endothelial permeability." Circulation Research, 96:1257-1265, (2005).
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Weidenfeller, C., Schrot, S., Zozulya, A., Galla, H.J.,"Murine brain capillary endothelial cells exhibit improved barrier properties under the influence of hydrocortisone." [PDF] Brain Res. 1053(1-2):162-74 (2005).
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