GTPase and adenylate cyclase desensitize at different rates in NG108-15 cells.
The time course of opioid receptor binding disappearance and loss of responsiveness of the opioid-controlled GTPase and adenylate cyclase were compared in membranes derived from NG108-15 cells pretreated with the opioid peptide agonist [D-Ala2,D-Leu5]enkephalin (DADLE). Upon pretreatment with DADLE, a rapid desensitization of the opioid-stimulated GTPase occurred with a time course distinguishable as two exponential components having respective half-lives of 5-9 and 60-80 min. Opioid receptor binding activity, as assessed using [3H]diprenorphine, also decayed as two exponential components whose half-lives were similar to those for GTPase desensitization (7 and 120 min). However, when [3H]diprenorphine binding was measured in the presence of sodium and GTP, only the second, slow component was apparent. In contrast, desensitization of the opioid-controlled adenylate cyclase occurred as only one exponential decaying process, displaying a half-life of 57 min. Whereas the loss of responsiveness of GTPase to DADLE was entirely accounted for by a reduction in the maximal stimulation produced acutely by DADLE, desensitization of adenylate cyclase was characterized by both a decrease in maximal inhibition and a shift to the right of the EC50 of the agonist in inhibiting acutely the enzyme. In addition, after 1 hr of pretreatment with DADLE, the opioid-stimulated GTPase was desensitized by 65%, whereas 80% of maximal inhibition of adenylate cyclase could still be achieved. We suggest that: the rapid loss of responsiveness of the opioid-GTPase system results from an uncoupling between the receptor and the nucleotide-binding regulatory protein (N); the fast decaying GTPase activity appears to be not directly related to the opioid-mediated inhibition of adenylate cyclase; and the slow decaying GTPase activity, as well as the desensitization of the opioid-adenylate cyclase, is most likely accounted for by down-regulation of the opioid receptor. These findings may indicate that part of the opioid-stimulated GTPase in the membrane is not involved in inhibition of the cyclase and could reflect the activity of a regulatory protein which couples opioid receptors to another membrane effector. Alternatively, they might be interpreted on the basis of a model which involves a tight coupling between receptor activation and N protein and a large amplification mechanism between N protein and adenylate cyclase.
D000067956:Adenylyl Cyclase Inhibitors; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D004174:Diprenorphine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D016308:Enkephalin, Leucine-2-Alanine; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D011957:Receptors, Opioid / Q000187:drug effects* / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D011957:Receptors, Opioid; D004174:Diprenorphine; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins