Signal Transduction


The ECIS Method

Many compounds of interest in biological and medical research are those molecules that specifically bind to cell surface receptors. When the receptor receives these ligands, subsequent changes in a signal transduction pathway result in the enhancement or attenuation of a cellular response. Assays capable of detecting and quantifying the receptor-ligand interaction are valuable tools both in fundamental studies of cell behavior and in the development of new drugs. Many modern assays are based upon detection of receptor-ligand binding event directly. Although binding of a compound is an essential component of signal transduction, this event alone does not assure that the biological response affected by the receptor is altered. Assays where one monitors cellular response directly, can avoid these potential false positives and provide more reliable information regarding the efficacy of compounds.

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.

To be certain the response monitored by ECIS is in fact the specific activation of the muscarinic receptor, we first estimated the EC50 value from the dose response data. Using the maximum value obtained following carbachol addition as indicative of the response, the data gave an EC50 of 1 micromolar, consistent with values reported in the literature for this receptor-ligand pair. Next, we carried out experiments with an antagonist for the receptor, perenzipine (PZP), to see if we could block the carbachol response. In the figure below these results are presented. Here, at the first arrow, we first administer different concentrations of PZP, followed in 15 minutes (the second arrow) with a fixed dose of 20 mM carbachol. Controls are included with only buffer addition at both times and with no PZP addition to see the normal carbachol response. The ability of PZP to block the carbachol activation is clearly demonstrated and indicates that we in fact are seeing morphological changes associated with activation of the muscarinic receptor and the ensuing signal transduction cascade.

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

ADAM15 regulates endothelial permeability and neutrophil migration via Src/ERK1/2 signaling. Chongxiu Sun, Mack H. Wu, Mingzhang Guo, Mark L. Day, Eugene S. Lee, and Sarah Y. Yuan. Cardiovasc Res. published 10 April 2010, 10.1093/cvr/cvq060.

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.

ten Klooster, J. P., Jeffer, Z.M., Chernoff, J., Hordijk, P.L., "Targeting and activation of Rac1 are mediated by the exchange factor B-Pix." J. Cell Biology. 172 (5): 759-769 (2006).

Qiao, J., Huang, F., Naikawadi, R.P., Kim, K.S., Said, T., Hazel Lum, H., "Lysophosphatidylcholine Impairs Endothelial Barrier Function through the G Protein-Coupled Receptor, GPR4." AJP Lung Cell Mol Physiol 0: 00508.2005v1 (2006).

Meyer, M.C., McHowat, J., "Calcium-Independent Phospholipase A2-Catalyzed Plasmalogen Hydrolysis in Hypoxic Human Coronary Artery Endothelial Cells,” [PDF] Am J Physiol Cell Physiol (2006).

ten Klooster, J. P., Jeffer, Z.M., Chernoff, J., Hordijk, P.L. "Targeting and activation of Rac1 are mediated by the exchange factor B-Pix." J. Cell Biology. 172 (5): 759-769 (2006).

Ren, J., Xiao, Y., Singh, L.S., Zhao, X., Zhao, Z., Feng, L., Rose, T.M., Prestwich, G.D., Xu, Y.,"Lysophosphatidic Acid Is Constitutively Produced by Human Peritoneal Mesothelial Cells and Enhances Adhesion, Migration, and Invasion of Ovarian Cancer Cells." [PDF] Cancer Res 2006; 66: (6) (2006).

Meyer, M.C., Kell, P.J., Creer, M.H., McHowat, J., "Calcium-independent phospholipase A2 is regulated by a novel protein kinase C in human coronary artery endothelial cells,” Am J Physiol Cell Physiol 288: C475-C482, (2005).

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).

Charrier, L., Yan, Y., Driss, A., Laboisse, C.L., Sitaraman, S.V. and Merlin, D.,"ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers." [PDF] AJP Gastrointest Liver Physiol. 288:346-353, (2005).

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).

McLaughlin, J.N., Shen, L., Holinstat, M., Brooks, J.D., DiBenedetto, E., Hamm, H.E., "Functional Selectivity of G Protein Signaling by Agonist Peptides and Thrombin for the Protease-activated Receptor-1." J. Biol. Chem. 280(26): 25048-25059 (2005).

McLaughlin, J.N., Mazzoni, M.R., Cleator, J.H., Earls, L., Perdigoto, A.L., Brooks, J.D., Muldowney, J.A.S., Vaughan, D.E., Hamm, H.E., "Thrombin Modulates the Expression of a Set of Genes Including Thrombospondin-1 in Human Microvascular Endothelial Cells." J. Biol. Chem. 280(23): 22172-22180 (2005).

Wu, M.H. "Endothelial focal adhesions and barrier function." J. Physiology. 569(2): 359 (2005).

Huang, F.,Subbaiah, P., Holian, O.,Zhang, J.,Johnson, A.,Gertzberg, N.,Lum, H., "Lysophosphatidylcholine Increases Endothelial Permeability: Role of PKC and RhoA Cross-talk" Am J Physiol Lung Cell Mol Physiol 289: L176-L185, (2005).

Kilani, M.M., Mohammed, K.A., Nasreen, N., Tepper, R.S., Antony, V.B., "RSV causes HIF-1alpha stabilization via NO release in primary bronchial epithelial cells." Inflammation. 28(5):245-5, (2004).

Zeng, W., Matter, W.F., Yan, S.B., Um, S.L., Vlahos, C.J., Liu, L., "Effect of drotrecogin alfa (activated) on human endothelial cell permeability and Rho kinase signaling." Critical Care Medicine. 32(5): S302-S308 (2004).

Regard, J.B., Scheek, S., Borbiev, T., Lanahan, A.A., Schneider, A., Demetriades, A.M., Hiemisch, H., Barnes, C.A., Verin, A.D., Worley, P.F., "Verge: A Novel Vascular Early Reponse Gene." The Journal of Neuroscience. 24(16):4092-4103 (2004)

Clements, R., Minnear, R., Singer, H., Keller, R., Vincent, P., "RhoA and Rho-kinase dependent and independent signals mediate TGF--induced pulmonary endothelial cytoskeletal reorganization and permeability." Am J Physiol Lung Cell Mol Physiol 288: L294-L306, (2004).

Ramirez-Icaza G, Mohammed KA, Nasreen N, Van Horn RD, Hardwick JA, Sanders KL, Tian J, Ramirez-Icaza C, Johnson MT, Antony VB. "Th2 cytokines IL-4 and IL-13 downregulate paxillin expression in bronchial airway epithelial cells." J Clin Immunol. 24(4):426-34 (2004).

Wang Q, Turlington A, Heo S, Blanco A, Tian J, Xie Z, Yan B, Wan Y., "Extracellular matrix activity and caveolae events contribute to cell surface receptor activation that leads to MAP kinase activation in response to UV irradiation in cultured human keratinocytes." Int J Mol Med. Apr;15(4):633-40 (2005)

De Blasio, B.F., Rottingen, J.A., Sand, K.L., Giaever, I., Iversen, J.G. "Global synchronous oscillations in cytosolic calcium and adherence in bradykinin- stimulated Madin-Darby canine kidney cells." Acta Physiol Scand 180: 335-346 (2004)

Kim, Yuri V., Di Cello, Francescopaolo, Hillaire, Coryse S., Kim, Kwang Sik. "Differential Ca+ signaling by thrombin and protease-activated receptor-1-activating peptide in human brain microvascular endothelial cells." [PDF] American Journal of Physiology-Cell Physiology, 286: C31-C42 (2004).

Hug, T.S. "Biophysical methods for monitoring cell-substrate interactions in drug discovery." Assay Drug Dev Technol. 1(3):479-88. Review (2003).

Schmidt, M.H., Chen, B., Randazzo, L.M., Bogler, O. "SETA/CIN85/Ruk and its binding partner AIP1 associate with diverse cytoskeletal elements, including FAKs, and modulate cell adhesion." J Cell Sci. 116(Pt 14):2845-55 (2003).

Sawhney RS, Sharma B, Humphrey LE, Brattain MG. "Integrin alpha2 and extracellular signal-regulated kinase are functionally linked in highly malignant autocrine transforming growth factor-alpha-driven colon cancer cells." J Biol Chem. 278(22):19861-9 (2003).

Reaas, V., Torbla, S., Andersen, M.H., Jensen, J., Rustan, A.C. "Electrical stimulation improves insulin responses in a human skeletal muscle cell model of hyperglycemia." Ann N Y Acad Sci. 967:506-15 (2002).

Hillebrandt, H. Abdelghani, A. Abdelghani-Jacquin, C. Aepfelbacher, M. Sackmann, "Electrical and optical characterization of thrombin-induced permeability of cultured endothelial cell monolayers on semiconductor electrode arrays,” Applied Physics A-Material Science & Processing, 73 (5): 539-546, 2001.

Tiruppathi, C., W. Yan, R. Sandoval., T. Naqvi., A. N. Pronin., J.L. Benovic, and A. B. Malik. "G protein-coupled receptor kinase-5 regulates Thrombin-Activated signaling in endothelial cells,” Proc. Natl. Acad. Sci. USA. 97:7440-7445, 2000.

Wegener, J., Zink, S., Rosen, P., GAlla, H., "Use of Electrochemical Impedance measurements to monitor B-adrenergic stimulation of bovine aortic endothelial cells." [PDF] Eur J Physiol 437: 925-934, 1999.

Reddy, L., Wang, H.S., Keese, C.R., Giaever, I., Smith, T.J., "Assessment of rapid morphological changes associated with elevated cAMP levels in human orbital fibroblasts,” Experimental Cell Research, 245 (2): 360-367 (1998).