Mechanisms of opioid signal transduction in SH-SY5Y neural cells.

Abstract

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 $\mu$ 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$\sb{\rm D}$ similar to the high-affinity K$\sb{\rm D}$ in cells and the addition of GTP reduced the B$\sb{\rm max}$ by 87%. In intact cells and isolated membranes, the kinetic constants for opioid stimulation of low-K$\sb{\rm m}$ GTPase and inhibition of AC were similar to the high affinity K$\sb{\rm D}$, 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$\alpha$o, while antibodies to G$\alpha$i$\sb{1,2}$ or G$\alpha$i$\sb3$ had marginal effects. In contrast, inhibition by the $\delta$ agonist DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen-OH) was most sensitive to G$\alpha$i$\sb{1,2}$ 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$\alpha$o levels by 26% without altering, G$\alpha$i$\sb{1,2}$, G$\alpha$i$\sb3$ or G$\alpha$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 $\mu$ and $\delta$ 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$\sb{\rm max}$ of antagonist binding by 80-95%, increased the K$\sb{\rm s}$ for GTPase stimulation 10- to 14-fold, and the K$\sb{\rm i}$ for AC inhibition 2- to 3-fold. These parameters were only marginally affected by agonists of lower acute potency, regardless of their I$\sb{\rm max}$. 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 $\beta$-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$\sb{\rm max}$ of $\mu$, but not $\delta$ opioid binding, while not affecting receptor coupling to AC. These results indicate potential regulation of opioid signal transduction by specific differentiating agents.