Effects of guanyl nucleotides and ions on kappa opioid binding

Displacement curve analyses demonstrated that GTP and its nonhydrolyzable analog, GPP(NH)P, inhibited the binding of [3H]dihydromorphine (mu agonist), [3H]D-Ala2-D-Leu5-enkephalin (delta agonist), and [3H]ethylketocyclazocine (general agonist) but not [3H]diprenorphine (general antagonist). Using a paradigm to block mu and delta sites with specific cold ligands, [3H]ethylketocyclazocine labeled kappa sites which were less GTP sensitive than sites labeled by mu and delta agonists. Further, Na+ and Mg++, important in inhibitory adenylate cyclase systems, also decreased both unblocked and mu-/delta-blocked [3H]ethylketocyclazocine binding. Scatchard analyses revealed that the inhibitory effects of GTP result in a decrease in affinity without a significant change in binding capacity, and that the kappa component of [3H]ethylketocyclazocine binding was less sensitive to the effects of GTP than binding sites labeled by mu or delta agonists. In comparison to the effects of GTP, Na+ decreased binding affinity but increased the binding capacity of the kappa component. These data also suggest that the inhibitory effects of Na+ and GTP on binding affinity are not additive. Association and dissociation plots revealed that although both components of binding may be involved in these affinity changes, the dissociation rate represents the more significant factor. These data suggest that [3H]ethylketocyclazocine binding to kappa sites is GTP and Na+ sensitive. However, it should be noted that [3H]ethylketocyclazocine binding to kappa sites is less sensitive to GTP than its binding to other opiate sites, and that this kappa binding is differentially affected by Na+. The significance of these characteristics with regard to the effect of kappa opiates on adenylate cyclase activity remains to be determined.