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.

Potentiation of morphine-induced antinociception and locomotion by citalopram is accompanied by anxiolytic-like effects

“…At this respect, we after sustained treatments (Berger andWhistler, 2010, Whistler, 2012). Moreover, we also described in these same cells that co-activation of 5-HT 2A receptors facilitated the endocytosis of MOP receptors upon morphine treatment similarly as we later A C C E P T E D M A N U S C R I P T 20 described in an analogous experimental model expressing 5-HT 2C receptors instead (Campa et al, 2015, Lopez-Gimenez et al, 2008. Although rather speculative in terms of translating in vitro results obtained from heterologous systems to native or physiological models, these results may suggest future approaches to further explore the biological mechanisms implicated in the improvement of morphine analgesia by enhancing serotonergic neurotransmission.…”

M3 muscarinic acetylcholine receptor facilitates the endocytosis of mu opioid receptor mediated by morphine independently of the formation of heteromeric complexes

“…In relation to the facilitation of the endocytosis, that result was equivalently obtained with the high homologous 5-HT2C receptor [14]. During the course of our studies with 5-HT2A receptors, we demonstrated that the inhibition of its coupling to Gαq/11 proteins by using the YM254890 compound aborted the facilitation of MOP receptor endocytosis by morphine [13].…”

“…Actually, the inability of morphine to endocytose MOP receptors upon activation may be overcome since under particular circumstances the internalisation of MOP receptors by morphine is possible, essentially by overexpression of accessory proteins involved in G-protein coupled receptor endocytosis mediated by agonist binding, principally GRK2 and -arrestin2 among others [5,11,12]. We recently described that the co-activation of either 5-HT2A or 5-HT2C serotonin receptor subtypes facilitates the endocytosis of MOP receptors promoted by morphine when co-expressed in Flp-In TM T-REx TM 293 cells [13,14]. In the case of 5-HT2A receptors, we demonstrated that this facilitation is dependent on receptor coupling to Gq/11 subunits and subsequent PKC activation.…”

“…This result also emulates the effects observed earlier in equivalent experiments conducted with cells co-expressing 5-HT2A and MOP receptors [13].In relation to the facilitation of MOP-YFP receptor internalisation by morphine upon the concurrent activation of FLAG-M3-Cerulean receptors with carbachol, both epifluorescence microscopy experiments conducted with living cells and biotinylation protection assays evidenced the endocytosis of MOP receptors apparently at the same extent than the observed when using DAMGO as activating agonist. However, a detailed examination of both endocytosis processes performed by using an image analysis method recently described in our laboratory[14] revealed a significant difference between these two experimental situations in terms of the kinetics of the process. Morphine plus carbachol treatment mediated internalisation of MOP-YFP receptors is notably slower that the equivalent observed upon DAMGO treatment, suggesting a different molecular machinery behind these cellular events.With respect the quantification of endocytosis as a pharmacological response, the number of endosomes per cell obtained when using DAMGO as agonist was about twice the value resulted from equivalent treatments using morphine plus carbachol to activate M3 muscarinic receptors endogenously expressed in Flp-In T-REx HEK293.…”

Nigritoxin is a bacterial toxin for crustaceans and insects

“…This suggests an intracellular mode of action of the nigritoxin. Consequently nigritoxin may not act as a host receptor agonist or antagonist, triggering signal transduction pathways 22 , although accumulating evidences revealed that the endocytosis of receptor–ligand contributes to generate cellular signals 23 , 24 . Finally although nigritoxin causes chromatin damage, it is not translocated into the nuclear compartment, such as cytolethal distending toxins 25 .…”