Analysis of delta-opioid receptor activities stably expressed in CHO cell lines: Function of receptor density?

The question of whether short- and long-term opioid agonist activities could be affected by receptor density is being addressed with Chinese hamster ovary (CHO) cell lines stably expressing delta-opioid receptors. CHO cells expressing different levels of delta-opioid binding sites were isolated and characterized. The opioid binding sites in these cell lines have stereoselective high affinity for ³H-diprenorphine (0.18 to 0.91 nM), with agonist binding sensitive to both Na⁺ and GTPγS, and are of the delta-2- opioid receptor subtype. This is concluded from observations that the delta- 2-opioid receptor-selective agonist naltriben (NTB) has higher affinity than the delta-1-opioid receptor-selective agonist 7-benzylidenenaltrexone (BNTX). It also could be demonstrated that delta-opioid agonists inhibited forskolin- stimulated intracellular ³H-cAMP production and that agonist inhibition could be blocked by pretreating cells with pertussis toxin. Again, NTB is more potent than BNTX or naloxone in reversing DPDPE inhibition of intracellular ³H-cAMP production. When the ability of [D-Ala²,D- Leu⁵]enkephalin (DADLE) to regulate adenylyl cyclase activity was examined in three separate CHO cell lines: CHODORX1-15, CHODORX1-8 and CHODORX1-4, which express 1.42 ± 0.08, 0.74 ± 0.07 and 0.27 ± 0.04 pmol/mg-protein of receptor, respectively, maximal inhibitory levels in clones X1-15 and X1-8 were similar, whereas maximal inhibitory level for clone X1-4 was 66% of the other two clones. However, when maximal inhibitory levels of other opioid receptor-selective agonists were examined, different levels of inhibition were observed among these three CHO clones. Mu- and kappa-opioid receptor- selective agonists exhibited partial agonistic properties in X1-4. Furthermore, the potencies of various agonists in inhibiting adenylyl cyclase activity in these three clones were dissimilar and were related to affinity for receptor only in the case of clone X1-4. Therefore, observed agonist effect is not simply a function of receptor density in these CHO clones expressing delta-opioid receptor. Long-term exposure of these CHO cell lines to DADLE resulted in a time-dependent down-regulation of opioid binding sites and loss in agonist ability to regulate intracellular ³H-cAMP level. Receptor down-regulation in these cell lines did not involve changes in opioid receptor mRNA levels as demonstrated by quantitative polymerase chain reaction analysis. The percentage of receptor being down-regulated in clone X1-4 was 38% compared with 60% to 67% in the other two clones. In contrast, receptor desensitization was maximal in clone X1-4, whereas receptor desensitization was minimal in clone X1-8. Interestingly, compensatory increase in adenylyl cyclase activity was maximal in clone X1-15 (360 ± 4.5% above the control level), which had minimal receptor desensitization, whereas in clone X1-4, which had complete receptor desensitization, only a 21 ± 5.0% increase from control adenylyl cyclase level was observed. These data suggest a receptor concentration dependency in long-term opioid responses and also suggest likely problems in interpretation of future receptor mutagenesis studies on short- and long-term action of delta-opioid agonists.