Mu-opioid receptor desensitization in mature rat neurons: lack of interaction between DAMGO and morphine

Abstract

Mu-opioid receptors (MORs) exhibit rapid desensitization and internalization during exposure to various opioid agonists. In some studies, however, morphine has been observed to produce little MOR desensitization or internalization. We examined desensitization in mature rat locus ceruleus (LC) neurons and confirmed that morphine is a very poor desensitizing agent, whereas [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), a high-efficacy agonist, and methadone, an agonist we observed to be of equivalent efficacy to morphine, produced profound rapid desensitization. Similarly, by measuring plasma membrane receptor levels in HEK293 cells stably expressing T7-epitope-tagged rat MOR1 at near physiological levels (HEK293-MOR1 cells), DAMGO and methadone but not morphine caused rapid MOR internalization. It has been reported that a low concentration of DAMGO, coapplied with morphine, caused morphine to induce MOR internalization. We examined whether this interaction occurred in mature mammalian neurons at the level of receptor desensitization. Coapplication of low concentrations of DAMGO did not increase morphine-induced desensitization in LC neurons but caused a lesser degree of desensitization than DAMGO alone. We also failed to observe an enhancement by DAMGO of morphine-induced desensitization in the electrically stimulated guinea pig ileum myenteric plexus-longitudinal muscle preparation. In HEK293-MOR1 cells, low concentrations of DAMGO did not convert morphine into a receptor-internalizing agent. The data presented here fail to support the theory that low concentrations of DAMGO can increase morphine-induced MOR desensitization or internalization.

Figure 1.

DAMGO and methadone produced greater rapid desensitization than morphine in LC neurons. A, Whole-cell patch-clamp recordings from three individual LC neurons. Calibration: 50 pA, 10 min. DAMGO (10 μm) and methadone (30 μm), but not morphine (30 μm), induced rapid desensitization. B, Rate of decay of responses. Pooled data of percentage maximum response plotted against time after maximum response was attained (n = 4 for DAMGO and methadone; n = 4-13 for morphine). Data were fitted to a single exponential (r² > 0.999 for each). C, Table summarizing kinetics of desensitization (n values as in B; ^*p < 0.05 vs DAMGO; ^†p < 0.05 vs DAMGO or methadone). D, E, Percentage desensitization at 7 and 30 min after peak. Morphine induced less desensitization than DAMGO or methadone (^*p < 0.05 morphine vs methadone or DAMGO; n = 4-13 for morphine; n = 4 for DAMGO and methadone).

Figure 2.

DAMGO coapplied with morphine did not induce MOR desensitization. A-C, Data from LC neurons. A, Representative current traces from two LC neurons. Calibration: 100 pA, 10 min). B, MOR desensitization induced by DAMGO (100 nm) was inhibited by morphine (5 μm). Pooled data (n = 4-7) taken 30 min after peak response. DAMGO alone induced more desensitization than morphine alone or morphine plus DAMGO (^*p < 0.05 for both). C, Desensitization induced by morphine (30 μm) was not increased by coadministration with DAMGO (500 nm) (^*p < 0.05 DAMGO vs morphine or morphine plus DAMGO; n = 4). D, DAMGO did not reveal morphine as a desensitizing agent in GPILM-MP preparations. Pooled data (n = 3) showing percentage desensitization after 15 min of drug exposure.

Figure 3.

DAMGO did not convert morphine into a MOR internalizing agent in HEK293 cells. A, Percentage loss of surface MOR expression after various periods of opioid exposure (^*p < 0.05 morphine vs DAMGO or methadone; n = 5-13). B, Coapplication of DAMGO (10 nm) did not enhance 5 μm morphine-induced MOR loss (n = 7). Coapplication of 100 nm DAMGO and 5 μm morphine (C) or 500 nm DAMGO and 30 μm morphine (D) did not significantly increase receptor loss compared with morphine alone. Although the mean percentage receptor loss in each case with DAMGO and morphine was greater compared with morphine alone, this effect was not statistically significant and was lower than the mean receptor loss with DAMGO alone (n = 5-6). E, In HEK293 cells with high MOR expression levels, 100 nm DAMGO and 5 μm morphine either alone or combined caused negligible receptor loss (n = 4).