title: Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy.
abstract: The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.
authors: Institute of Biomedicine and Biotechnology of Cantabria, (IBBTEC), CSIC, UC, Santander, Spain.
authors: Víctor M
authors: VM
authors: Campa
authors: Department of Biological and Health Psychology, Autonoma University of Madrid (UAM), Madrid, Spain.
authors: Almudena
authors: A
authors: Capilla
authors: Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela (USC), Santiago de Compostela, Spain.
authors: María J
authors: MJ
authors: Varela
authors: Institute of Biomedicine and Biotechnology of Cantabria, (IBBTEC), CSIC, UC, Santander, Spain.
authors: Arlet M Acanda
authors: AM
authors: de la Rocha
authors: Institute of Biomedicine and Biotechnology of Cantabria, (IBBTEC), CSIC, UC, Santander, Spain.
authors: Juan C
authors: JC
authors: Fernandez-Troyano
authors: Institute of Physics of Cantabria, (IFCA), CSIC, UC, Santander, Spain.
authors: R Belén
authors: RB
authors: Barreiro
authors: Institute of Biomedicine and Biotechnology of Cantabria, (IBBTEC), CSIC, UC, Santander, Spain.
authors: Juan F
authors: JF
authors: Lopez-Gimenez
mesh_terms: D004705:Endocytosis / Q000187:drug effects*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D057809:HEK293 Cells; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D044405:Receptor, Serotonin, 5-HT2C / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology; D058826:Serotonin 5-HT2 Receptor Agonists / Q000494:pharmacology; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: D044405:Receptor, Serotonin, 5-HT2C; D058826:Serotonin 5-HT2 Receptor Agonists; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D012701:Serotonin