Effect of chronic D-Ala², D-Leu⁵-enkephalin or pertussis toxin treatment on the high-affinity state of delta opioid receptor in neuroblastoma x glioma NG108-15 hybrid cells

Chronic treatment of neuroblastoma x glioma NG108-15 hybrid cells with the opioid agonist D-Ala², D-Leu⁵-enkephalin (DADLE) induces a homologous desensitization of the delta opioid receptors present in these cells. Since the K(d) value of the delta opioid receptor's high-affinity state reflects the potency of the agonist, we examined the effect of receptor desensitization in NG108-15 cells on the percentage of receptor in the high-affinity state. When NG108-15 hybrid cells were treated with 10 or 100 nM DADLE for 4 hr at 24°C, loss of DADLE's ability to inhibit adenylate cyclase was observed. However, when competition binding experiments were carried out with P₂P₃ membranes isolated from the delta opioid-desensitized hybrid cells, it was determined that 41.7 ± 3.4% of the total binding sites remained in the high-affinity state, with no apparent alteration in the K(d) value of either high- or low-affinity states. Similarly, when NG108-15 cells were treated with 100 ng/ml of pertussis toxin for 3 hr at 37°C, 39.9 ± 3.6% of the binding sites remained in the high-affinity state. This reduction in the percentage of receptor in high-affinity state was agonist specific, for chronic treatment of hybrid cells with levorphanol, a partial agonist, or the antagonist naloxone did not alter the percentage of opioid receptors in the high-affinity state. Furthermore, the delta opioid receptors remaining in the high-affinity state after chronic DADLE treatment were still sensitive to both Na⁺ and guanyldylimidodiphosphate, indicating that opioid ligand binding remained coupled to the G-proteins. On the other hand, complete elimination of the high-affinity states of the delta opioid receptor was accomplished by treating the hybrid cells with 100 nM DADLE for 4 hr at 24°C, followed by 100 ng/ml of pertussis toxin for 3 hr at 37°C, or vice versa. Cholera toxin treatment, which ADP-ribosylates the α-subunit (α(s)) of the stimulatory G-protein G(s), did not alter the percentage of receptor in high-affinity state. These binding data suggest that interaction between the delta opioid receptor and G-proteins remains after loss of measurable receptor activity, i.e., inhibition of adenylate cyclase. Only when alteration occurs in both ligand binding sites and in G(iα) is this interaction prevented.