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

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)710-716
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume256
Issue number2
StatePublished - 1991

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