Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy

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

Umbilical cord blood coagulability, acidosis and intracranial hemorrhage

Peer Reviewe

Generation of an inducible double-stable cell line expressing MOP-YFP and c-myc-5-HT_2C-Cerulean receptors.

<p>Images obtained by confocal microscopy from living Flp-In HEK293 cells expressing permanently MOP-YFP receptors and c-myc-5-HT_2C-Cerulean receptors in an inducible manner. The right column corresponds to the images resulting after exciting the cells with the YFP light settings whereas in the left column are the same microscopy field illuminated for CFP visualization. When indicated, +DOX corresponds to treatment with doxycycline (0.01 μg/ml) for 24 hours prior to microscope observation.</p

Effect of different doses of serotonin (5-HT) on the facilitation of the endocytosis of MOP-YFP receptors by morphine.

<p><b>A.</b> Data graph display representative results from time-course MOP-YFP receptor endocytosis experiments conducted by co-treatment with morphine at 10 μM plus 5-HT at different concentrations (see symbol legends). <b>B.</b> Dose-response curve (continuous line) obtained after non-linear analysis of the maximal effect (number of vesicles per cell) generated by morphine (10 μM) plus different concentrations of 5-HT. Each point represents the mean ± SEM of three independent experiments. Dotted line display the characteristic bell shaped profile observed when connecting these points in an increasing dose-dependent manner.</p

Kinetic parameters obtained from endocytosis assays performed with HeLa cells transiently expressing MOP-YFP receptors.

<p>Cells were treated with DAMGO (10 μM), sufentanyl (1 μM) or morphine (10 μM) for 15 minutes. Data indicate mean ± SEM of (n) independent experiments. t_1/2: time to reach the 50% of the maximal response (minutes). Emax: maximal response (number of vesicles per cell). n.d.: not detected.</p><p>Kinetic parameters obtained from endocytosis assays performed with HeLa cells transiently expressing MOP-YFP receptors.</p

Effect of different doses of DAMGO on the time-course generation of endocytic vesicles containing MOP-YFP receptors.

<p><b>A.</b> Data graph show representative results from time-course MOP-YFP receptor endocytosis experiments performed with DAMGO at different concentrations (see symbol legends). <b>B.</b> Dose-response curve obtained after non-linear analysis of data corresponding to the maximal effect in terms of number of vesicles per cells generated by different concentrations of DAMGO. Each point represents the mean ± SEM of three independent experiments.</p

Kinetic parameters obtained from endocytosis assays performed with cells heterologously co-expressing MOP-YFP and c-myc-5-HT2C-Cerulean receptors.

<p>Cells were treated with DAMGO (10 μM), sufentanyl (1 μM), methadone (10 μM), morphine (10 μM), morphine (10 μM) plus 5-HT (10 μM), morphine (10 μM) plus DOI (10 μM), morphine (10 μM) plus WAY161503 (1 μM), oxycodone (10 μM) plus 5-HT (10 μM) or buprenorphine (10 μM) plus 5-HT (10 μM). Data indicate mean ± SEM of (n) independent experiments. t_1/2: time to reach the 50% of the maximal response (minutes). Emax: maximal response (number of vesicles per cell). n.d.: not detected.</p><p>Kinetic parameters obtained from endocytosis assays performed with cells heterologously co-expressing MOP-YFP and c-myc-5-HT2C-Cerulean receptors.</p