دانلود رایگان مقاله آنالیز مبتنی بر امپدانس سیگنالینگ گیرنده مواد مخدر مو

Abstract

The mu opioid receptor is a G-protein coupled receptor able to signal through the Gαi/o class of G-protein and β-arrestin pathways, stimulating down-stream effector pathways. Signaling bias occurs when different receptor agonists lead to different signaling outcomes. Traditionally these have been studied using end-point assays. Real-time cellular analysis platforms allow for the analysis of the holistic effects of receptor activation as an integrated output. While this allows for different ligands to be compared rapidly, the cellular mechanisms underlying the signal are not well described. Using an impedance based system, the impedance responses for two opioid ligands, morphine and DAMGO were examined. The impedance responses for these two agonists, while showing similar features, were distinct from each other. Some of the mechanisms underlying the mu opioid receptor coupled impedance changes were investigated. It was found that the response is a result of discrete cellular processes, including G-protein signaling and protein kinase phosphorylation.

3.2. Kinase phosphorylation

The activation of MOR is known to lead to the activation of MAPK pathways. While ERK1/2 is the most widely described, other pathways have also been implicated in the opioid response [22–24]. The activation of several MAPK, the NF-κB and AKT signaling pathways in response to the opioid treatments were measured using Alphascreen antibody-based assays. There was no evidence of the activation of p38, JNK or NF-κB (data not shown), whereas ERK1/2 and AKT1/2/3 displayed phosphorylation in response to MOR activation in a time dependent manner (Fig. 3). Following both DAMGO and morphine treatments, the phosphorylation of ERK1/2 was detected using an antibody binding to the Threonine 202/Tyrosine 204 phosphorylated epitope. Phosphorylation of ERK1/2 was detected after two minutes and peaked at five minutes (Fig. 3A and D). The activation of the AKT1/2/3 pathway was investigated using two assays to detect either the phosphorylated Threonine 308 (Thr308) or Serine 473 (Ser473) sites of AKT1, 2 and 3. The phosphorylation at both of these sites occurred rapidly in cells treated with DAMGO or morphine. For cells treated with DAMGO, AKT phosphorylation peaked at two minutes and rapidly decreased (Fig. 3B and C). For cells treated with morphine, the peak in AKT phosphorylation at both sites occurred at five minutes and was sustained for longer (Fig. 3E and F).