Effects of chronic opioid exposure on guinea pig mu opioid receptor in Chinese hamster ovary cells: comparison with human and rat receptor

Abstract

Chronic opioid treatment leads to agonist-specific effects at the mu opioid receptor. The molecular mechanisms resulting from chronic opioid exposure include desensitization, internalization and down-regulation of membrane-bound mu opioid receptors (MOP). The purpose of this study was to compare the cellular regulation of guinea pig, human and rat MOP expressed in Chinese hamster ovary (CHO) cells, following exposure to two clinically important opioids, morphine and methadone. MOP expressing CHO cells were treated in culture with methadone or morphine for up to 48 h. Radioligand diprenorphine and [D-AIa(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO)-stimulated GTP gamma S binding assays were carried out using paired control and opioid-exposed CHO cells. Methadone induced downregulation of the mu opioid receptor, while morphine induced desensitization of the receptor for all three species. Furthermore, morphine predominantly decreased the potency of DAMGO to stimulate GTP gamma S binding, whereas methadone primarily reduced its efficacy. Changes in DAMGO potency and efficacy differed among species and depended on the opioid used to treat the cells. Our results showed similarities between guinea pig and human MOP for morphine-induced desensitization, but identified differences between the two for methadone-induced desensitization. In contrast, human and rat MOP differed in response to morphine treatment, but were not distinct in their response to methadone treatment. The guinea pig is an excellent and established animal model to study opioid effects, but its molecular opioid pharmacology has not been investigated thus far. These results can assist in understanding species differences in the effects of opioid ligands activating the mu opioid receptor.

Figure 1

Competition of specific [³H]-diprenorphine binding (0.3 nM) by unlabeled compounds was measured using homogenized membranes prepared from guinea pig mu opioid receptor CHO cells. Each curve was plotted using data from one representative experiment with each measurement determined in triplicates. Each competitor was evaluated in three independent experiments. A K_i value was calculated for each ligand and is represented in Table 2. (NSB, non-specific binding, TB, total binding)

Figure 2

A-C:Opioid-stimulated GTPγS binding was performed using DAMGO, methadone and morphine (Table 3) for guinea pig (A), human (B), and rat (C) mu opioid receptor in membranes isolated from CHO cells. Curves are plotted using data from one representative experiment. D: The efficacies of the different opioids were normalized to the efficacy of the DAMGO-stimulated assay of the same experiment. E_max of morphine was significantly lower than E_max of DAMGO and methadone for all species. E_max of morphine for rat mu opioid receptor was significantly lower than for guinea pig and human. Data are mean ± S.E.M of three independent experiments (D). * P < 0.05 vs. DAMGO and methadone ^# P < 0.05 vs. guinea pig and human

Figure 3

Receptor binding in opioid treated mu opioid receptor expressing CHO cells. Cells were exposed in culture to 20 μM morphine, 10 μM methadone, or 5 μM (-)- or (+)-methadone for 3 h to 48 h. Homogenized membrane preparations were assayed for their ability to bind [³H]-diprenorphine (0.6 nM). Receptor binding is plotted as percent of untreated control. Data is mean ± S.E.M of three independent experiments performed in triplicates. No significant changes in receptor binding were observed for chronic morphine or (+)-methadone treatment. Both rac- and (-)-methadone caused reduced receptor binding after 3 h of exposure that was sustained up to 48 h (P < 0.001 vs. control). No statistical differences were found after 12 h of treatment for each drug.

Figure 4

DAMGO-stimulated GTPγS binding in opioid treated CHO cells. Cells were treated in culture as in Figure 3 with either morphine or methadone for 3 or 12 h. Dose response curves for DAMGO-stimulated GTPγS binding with homogenized membranes were generated. EC₅₀ (panel A,C) and E_max (panel B,D) were calculated and are graphed as percent of paired control. Data is mean ± S.E.M of three independent experiments performed in duplicates for each concentration. Methadone-induced tolerance was different for the guinea pig mu opioid receptor compared to the other two species, as was the case for the rat receptor following morphine treatment. Significant time-dependent changes for both treatments are indicated. ^x P < 0.05 vs. untreated control ** P < 0.01 vs. guinea pig and human ^## P < 0.01 vs. guinea pig ^# P = 0.05 vs. rat *** P < 0.001 vs. human and rat

Figure 5

Effects of methadone isomers on GTPγS binding at the guinea pig mu opioid receptor. DAMGO-stimulated GTPγS binding curves were generated using membrane preparations from guinea pig mu opioid receptor expressing-CHO cells pretreated with 10 μM rac-methadone, or 5 μM (-)- or (+)-methadone for the times indicated. EC₅₀ (Figure 5A) and E_max (Figure 5B) are plotted as percent of control. Data is mean ± S.E.M of three or four independent experiments performed in duplicates for each concentration of DAMGO. Two-way ANOVA for factors of drug and time for both values revealed no significant differences between rac- and (-)-methadone. Both drugs significantly decreased potency and efficacy compared to control after 3 h (P < 0.001 for all comparisons). No significant change in either value with time was observed for (+)methadone treated cells. Differences between methadone isomers are denoted. *** P < 0.001 vs. untreated control ^### P ≤ 0.001 vs. rac- and (-)-methadone