Agonist-, antagonist-, and inverse agonist-regulated trafficking of the delta-opioid receptor correlates with, but does not require, G protein activation.

In this study, we explored the relationship between ligand-induced regulation of surface delta opioid receptors and G protein activation. G protein activation was assessed with [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) binding assays conducted at both 37 and 0 degrees C. Ligand-independent (constitutive) activity of the delta-receptor was readily observed when the [(35)S]GTP gamma S binding assay was performed at 37 degrees C. We identified a new class of alkaloid inverse agonists (RTI-5989-1, RTI-5989-23, RTI-5989-25), which are more potent than the previously described peptide inverse agonist ICI-174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu). Treatment with these inverse agonists for 18 h caused up-regulation of surface receptors. Eighteen-hour treatment with etorphine resulted in approximately 90% loss of surface receptor, whereas fentanyl, diprenorphine, and morphine caused between 20 and 50% loss. The abilities of ligands to modulate [(35)S]GTP gamma S binding at 37 degrees C showed a strong correlation with their abilities to regulate surface receptor number (r(2) = 0.86). Interestingly, the ability of fentanyl to activate G proteins was markedly temperature sensitive. Fentanyl showed no stimulation of [(35)S]GTP gamma S binding at 0 degrees C but was as efficacious as etorphine, morphine, and diprenorphine at 37 degrees C. Neither the ligand-induced receptor increases nor decreases were perturbed by pertussis toxin pretreatment, suggesting that functional G proteins are not required for ligand-regulated delta-opioid receptor trafficking.

Coactivation of beta-adrenergic and cholinergic receptors enhances the induction of long-term potentiation and synergistically activates mitogen-activated protein kinase in the hippocampal CA1 region.

Interactions between noradrenergic and cholinergic receptor signaling may be important in some forms of learning. To investigate whether noradrenergic and cholinergic receptor interactions regulate forms of synaptic plasticity thought to be involved in memory formation, we examined the effects of concurrent beta-adrenergic and cholinergic receptor activation on the induction of long-term potentiation (LTP) in the hippocampal CA1 region. Low concentrations of the beta-adrenergic receptor agonist isoproterenol (ISO) and the cholinergic receptor agonist carbachol had no effect on the induction of LTP by a brief train of 5 Hz stimulation when applied individually but dramatically facilitated LTP induction when coapplied. Although carbachol did not enhance ISO-induced increases in cAMP, coapplication of ISO and carbachol synergistically activated p42 mitogen-activated protein kinase (p42 MAPK). This suggests that concurrent beta-adrenergic and cholinergic receptor activation enhances LTP induction by activating MAPK and not by additive or synergistic effects on adenylyl cyclase. Consistent with this, blocking MAPK activation with MEK inhibitors suppressed the facilitation of LTP induction produced by concurrent beta-adrenergic and cholinergic receptor activation. Although MEK inhibitors also suppressed the induction of LTP by a stronger 5 Hz stimulation protocol that induced LTP in the absence of ISO and carbachol, they had no effect on LTP induced by high-frequency synaptic stimulation or low-frequency synaptic stimulation paired with postsynaptic depolarization. Our results indicate that MAPK activation has an important, modulatory role in the induction of LTP and suggest that coactivation of noradrenergic and cholinergic receptors regulates LTP induction via convergent effects on MAPK.

Ligand-induced changes in surface mu-opioid receptor number: relationship to G protein activation?

In this study, we explored the relationship between regulation of surface mu-opioid receptor number, ligand-induced G protein activation (measured by [(35)]S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding) and second messenger signaling (measured by the inhibition of cAMP accumulation). Etorphine and two isomers of cis-beta-hydroxy-3-methylfentanyl (RTI-1a and RTI-1b), which were full agonists for G protein activation and signaling, caused approximately a 50% loss of surface receptors after 1 h of treatment. Fentanyl and morphine were full agonists for inhibiting cAMP accumulation and partial agonists for stimulating [(35)S]GTPgammaS binding and internalization. Although both agonists were approximately 80% as efficacious as etorphine in stimulating [(35)S]GTPgammaS binding, fentanyl induced a 35% loss of surface receptors, whereas morphine only caused a 10% loss. Additionally, both long- and short-term treatment with the opioid antagonist naloxone caused increases in surface receptors. Unexpectedly, the weak partial agonists buprenorphine and one isomer of cis-beta-hydroxy-3-methylfentanyl (RTI-1d) also were found to cause an increase in surface receptors. Treatment with pertussis toxin (PTX) diminished agonist-induced loss of surface receptors. Furthermore, the abilities of morphine and fentanyl to cause internalization were more impaired after PTX treatment than that of etorphine. PTX treatment also significantly enhanced the increase in surface receptor number caused by 18-h treatment with naloxone and buprenorphine. The results of this study suggest that disruption of G protein coupling by PTX treatment affects ligand-regulated mu-receptor trafficking and that partial agonists for signaling can vary greatly in the ability to regulate the number of surface mu-opioid receptors.

mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain.

mu-Opioid receptors are the pharmacological targets of endogenous opioid peptides and morphine-like alkaloid drugs. Previous studies of transfected cells and peripheral neurons indicate that opioid receptors are rapidly internalized after activation by the alkaloid agonist etorphine but not after activation by morphine. To determine whether opioid receptors in the central nervous system are regulated by a similar process of agonist-selective internalization, mu-opioid receptors were examined in rat brain neurons after treatment of animals with opioid drugs. Internalized mu receptors were observed within 30 min after intraperitoneal injection of the alkaloid agonist etorphine, and this process was blocked by the antagonist naloxone. Colocalization of internalized opioid receptors with transferrin receptors in confocal optical sections indicated that receptor internalization observed in vivo is mediated by a membrane trafficking pathway similar to that observed previously in vitro using transfected human embryonic kidney 293 cells. Morphine failed to induce detectable rapid internalization of receptors, even when administered to animals at doses far in excess of those required to induce analgesia. To quantify these agonist-selective differences and to analyze an array of opioid ligands for their ability to trigger internalization, we used flow cytometry on stably transfected 293 cells. These studies indicated that the different effects of individual agonists are not correlated with their potencies for receptor activation and that a variety of clinically important agonists differ significantly in their relative abilities to stimulate the rapid internalization of opioid receptors.

Morphine activates opioid receptors without causing their rapid internalization.

We have examined the endocytic trafficking of epitope-tagged delta and mu opioid receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by peptide agonists (enkephalins) as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to internalize rapidly (t1/2 approximately 6 min) by a mechanism similar to that utilized by a number of other classes of receptor, as indicated by localization of internalized opioid receptors in transferrin-containing endosomes and inhibition of opioid receptor internalization by hypertonic media. Remarkably, morphine does not stimulate the rapid internalization of either delta or mu opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonist ligands, which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G protein-coupled receptor.

Opioid receptor types and subtypes: the delta receptor as a model.

Since the discovery of opioid receptors over two decades ago, an increasing body of work has emerged supporting the concept of multiple opioid receptors. Molecular cloning has identified three opioid receptor types--mu, delta, and kappa--confirming pharmacological studies that previously postulated the existence of these three receptors. The cloned opioid receptors are highly homologous and belong to the family of seven-transmembrane, G protein-coupled receptors. With the development of novel opioid ligands, subtypes of the mu, delta, and kappa receptors have been proposed, although the molecular basis of these subtypes has not been elucidated. In this review, we present the pharmacological data supporting the concept of multiple delta opioid receptor subtypes and offer hypothetical mechanisms which might generate these "subtypes."