Figure 1.
DMR characteristics of opioid ligands in HEK-MOR cells.
(a) Real time kinetic responses of DAMGO at different doses; each graph represents the mean ± s.d. of 2 independent measurements (n = 6); (b) Real time kinetic responses of β-funaltreamine at different doses; each graph represents the mean ± s.d. of 2 independent measurements (n = 4); (c) The maximal DMR amplitudes as a function of ligand doses for endomorphin-2, DAMGO, morphine and β-funaltreamine; (d) The maximal DMR amplitudes as a function of ligand doses for fentanyl and endomorphin-1. All dose responses represent the mean ± s.d. of 2 independent measurements (n = 4), except for DAMGO (n = 6).
Figure 2.
The blockage of MOR agonist-induced DMR by naloxone.
The maximal amplitudes of the MOR agonist-induced DMR as a function of naloxone doses. All three agonists, endomorphin-2, DAMGO and morphine, were assayed at 64 nM. The data represents the mean ± s.d. of 2 independent measurements, each in duplicate (n = 4).
Table 1.
Assay protocols and DMR signals used for similarity analysis.
Figure 3.
The numerical descriptor of opioid ligand pharmacology.
(a) A false colored heat map of opioid ligand-induced DMR in the native HEK-MOR cells. The responses at distinct time points were subject to similarity analysis. Three major time domains were seen. (b) The DAMGO DMR signal in the native HEK-MOR cells and its three representative time point descriptors (3 min, 9 min and 30 min post-stimulation, as indicated by 1, 2, and 3, respectively). The black arrow indicates the time (t = 0) when DAMGO was added. The graph represents the mean ± s.d. of 2 independent measurements, each in sixteen replicates (n = 32).
Figure 4.
A false colored heat map of all 42 opioid ligands.
The heat map was generated using similarity analysis of the DMR of all ligands under 13 assay conditions. 0.1% DMSO in the assay buffer was included as a control. In order to visualize the characteristics of a DMR for each assay, the three responses (3 min, 9 min, and 30 min) for each assay were grouped together. For each assay there are three adjacent columns to form a column group (A to M).
Figure 5.
The DMR characteristics of BNTX and the negative control (0.1% DMSO).
(a) The BNTX DMR in HEK-293 cells; (b) the BNTX DMR in the buffer treated (BNTX) and CTOP pretreated (CTOP – BNTX) HEK-MOR cells; and (c) the DAMGO DMR in the BNTX pretreated cells (BNTX – DAMGO) in comparison with the BNTX DMR in the DAMGO pretreated cells (DAMGO – BNTX); (d) the DMR induced by DMSO in HEK293; (e) the DMSO DMR in HEK-MOR or the CTOP pretreated HEK-MOR cells; (f) the DAMGO DMR after pretreatment with DMSO (DMSO – DAMGO) and the DMSO DMR after pretreatment with DAMGO (DAMGO –DMSO) in HEK-MOR cells. Each curve represents the average of duplicates.
Figure 6.
The DMR signals induced by levallorphan, β-funaltrexamine, and naltrindole in the DAMGO-activated HEK-MOR cells.
Each curve represents the average of duplicates.
Figure 7.
The ICI 199441-induced DMR in distinct molecule-treated cells.
The cells were HEK-293 cells (HEK293), HEK-MOR (HEK-MOR), or CTOP- (CTOP), or DAMGO- (DAMGO) pretreated HEK-MOR cells. Each curve represents the average of duplicates.
Figure 8.
The comparison between the DMR responses and the whole cAMP responses induced by opioid ligands.
Both were obtained in HEK-MOR cells. All data represent the mean ± s.d. of 2 independent measurements (n = 4).
Figure 9.
The sensitivity of opioid ligand-induced DMR to the pertussis toxin (PTx) pretreatment.
(a) the comparison between the DMR induced by each ligand in the untreated and PTx-pretreated HEK-MOR cells, in which the maximal amplitudes were plotted.; (b) The DMR arising from ICI 199441 and DAMGO in the PTx-pretreated HEK-MOR cells. All data represent the mean ± s.d. of 2 independent measurements (n = 4).
Figure 10.
The sensitivity of opioid ligand induced DMR to the pretreatment with forskolin (FSK) and cholera toxin (CTx).
The maximal DMR amplitudes of all opioid ligands were compared in HEK-MOR after pretreatment with: (a) buffer versus CTx; and (b) buffer versus forskolin.
Figure 11.
The comparison of the maximum responses of opioid ligands between the buffer- and inhibitor-pretreated HEK-MOR cells.
(a) U0126; (b) SB202190; (c) SP100625 and (d) LY294002. The broken circle referred to ICI199441, Leu5-enkephalin, DSLET, DAMME, and dynorphin A 1-13 from left to right, respectively.
Figure 12.
The comparison of the real responses (30 min post-stimulation) of opioid ligands between the buffer- and inhibitor-pretreated HEK-MOR cells.
(a) U0126; (b) SB202190; (c) SP100625 and (d) LY294002.