| The mechanisms of opioid receptor coupling through G protein to adenylate cyclase (AC) during acute and long-term receptor occupancy by ligand were investigated in human neuroblastoma SH-SY5Y cells. In intact cells, the {dollar}mu{dollar} agonist DAMGO (Tyr-D-Ala-Gly-(Me)Phe-Gly-ol) bound to two sites of which less than 10% were of high-affinity and pertussis toxin (PTX) sensitive. In isolated membranes, DAMGO bound to a single population of sites with a K{dollar}sb{lcub}rm D{rcub}{dollar} similar to the high-affinity K{dollar}sb{lcub}rm D{rcub}{dollar} in cells and the addition of GTP reduced the B{dollar}sb{lcub}rm max{rcub}{dollar} by 87%. In intact cells and isolated membranes, the kinetic constants for opioid stimulation of low-K{dollar}sb{lcub}rm m{rcub}{dollar} GTPase and inhibition of AC were similar to the high affinity K{dollar}sb{lcub}rm D{rcub}{dollar}, indicating analogous efficiency of signal transduction in both preparations with a large reserve of uncoupled receptors whose population in cells is regulated by endogenous GTP.; Inhibition of AC in SH-SY5Y membranes by DAMGO was blocked 50-60% by either of two antibodies to G{dollar}alpha{dollar}o, while antibodies to G{dollar}alpha{dollar}i{dollar}sb{lcub}1,2{rcub}{dollar} or G{dollar}alpha{dollar}i{dollar}sb3{dollar} had marginal effects. In contrast, inhibition by the {dollar}delta{dollar} agonist DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen-OH) was most sensitive to G{dollar}alpha{dollar}i{dollar}sb{lcub}1,2{rcub}{dollar} antibodies. Similar results were observed with DAMGO in membranes from rat brain stratium. Treatment of SH-SY5Y cells for 24 hrs with DAMGO reduced G{dollar}alpha{dollar}o levels by 26% without altering, G{dollar}alpha{dollar}i{dollar}sb{lcub}1,2{rcub}{dollar}, G{dollar}alpha{dollar}i{dollar}sb3{dollar} or G{dollar}alpha{dollar}s content. Addition of purified Go to membranes from PTX-treated cells restored DAMGO inhibition of AC by 70%, while recovery of DPDPE effects required much higher concentrations. These results describe a novel role for Go, preferential G protein coupling of {dollar}mu{dollar} and {dollar}delta{dollar} receptors, and reduced levels of Go during opioid tolerance.; Long-term treatment of the cells with agonists which were potent in acutely inhibiting AC decreased the B{dollar}sb{lcub}rm max{rcub}{dollar} of antagonist binding by 80-95%, increased the K{dollar}sb{lcub}rm s{rcub}{dollar} for GTPase stimulation 10- to 14-fold, and the K{dollar}sb{lcub}rm i{rcub}{dollar} for AC inhibition 2- to 3-fold. These parameters were only marginally affected by agonists of lower acute potency, regardless of their I{dollar}sb{lcub}rm max{rcub}{dollar}. DAMGO, a potent agonist, retained its ability to induce tolerance even under sub-maximal signal, produced by lower concentrations or by PTX pretreatment. Alkylation of 50% of the receptors by {dollar}beta{dollar}-chlornaltrexamine did not significantly alter the rank order of opioids based on their maximum acute effect. The results show that receptor down-regulation and effector desensitization correlate with the acute potency, not the maximum effect of an opioid in interacting with the effector.; Among differentiating agents investigated, phorbol ester increased the B{dollar}sb{lcub}rm max{rcub}{dollar} of {dollar}mu{dollar}, but not {dollar}delta{dollar} opioid binding, while not affecting receptor coupling to AC. These results indicate potential regulation of opioid signal transduction by specific differentiating agents. |
