The molecular mechanisms of cellular tolerance to delta-opioid agonists. A minireview

Chronic treatment with deltaopioid agonists, similar to other agonist drugs, causes tolerance. Tolerance is a complex adaptation process that consists of multiple, cellular and neural-system adaptations. Cellular tolerance to delta-opioid agonists involves feedback-regulation of the function, concentration, and localization of the delta-opioid receptors (receptor desensitization) as well as of intracellular effectors (functional desensitization). We are using a recombinant Chinese hamster ovary cell line expressing the human delta-opioid receptors (hDOR/CHO) to investigate the molecular mechanisms of cellular tolerance. We found that the structurally distinct delta-opioid agonists mediate receptor down-regulation by different mechanisms. Thus, truncation of the last 35 C-terminal amino acids of the hDOR completely abolished DPDPE, but not SNC 80-mediated receptor down-regulation. In addition, down-regulation of the wild type-, and the truncated hDORs exhibited different inhibitor sensitivity-profile. Chronic delta-opioid agonist treatment also causes functional desensitization of forskolin-stimulated cAMP formation and cAMP overshoot in the hDOR/CHO cells. We have demonstrated that chronic SNC 80 treatment also causes concurrent phosphorylation of the adenylyl cyclase (AC) VI isoenzyme hDOR/CHO cells. Both AC superactivation and AC VI phosphorylation were SNC 80 dose-dependent, naltrindole-sensitive, and exhibited similar time course-, and protein kinase inhibitor-sensitivity profile. We hypothesize that phosphorylation of AC VI plays an important role in delta-opioid agonist-mediated AC superactivation in hDOR/CHO cells.     

Regulation of adenylyl cyclase, ERK1/2, and CREB by Gz following acute and chronic activation of the delta-opioid receptor

Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive Gz (a member of the Gi subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing delta-opioid receptors with or without Gz to examine the role of Gz in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta-selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element-binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signal-regulated protein kinases (ERKs) 1 and 2. In cells coexpressing Gz, DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but Gz could not replace Gi to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both Gi and Gz mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although Gz mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, Gz alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.     

Differential sorting of human delta-opioid receptors after internalization by peptide and alkaloid agonists

Desensitization and internalization of G protein-coupled receptors observed after agonist activation are considered two important regulatory processes of receptor transduction. Endogenous human delta-opioid receptors (hDOR) are differentially regulated in terms of desensitization by peptide ([d-Pen2,5]enkephalin (DPDPE) and Deltorphin I) and alkaloid (etorphine) agonists in the neuroblastoma cell line SK-N-BE (Allouche, S., Roussel, M., Marie, N., and Jauzac, P. (1999) Eur. J. Pharmacol. 371, 235-240). In the present study, we examined the role of hDOR internalization and down-regulation in this differential desensitization. Sustained activation by peptides for 30 min caused a marked decrease of both [3H]diprenorphine binding sites and hDOR immunoreactivity, observed in a Western blot, whereas a moderate reduction by 30% was observed after a 30- and 60-min etorphine exposure in binding experiments without opioid receptor degradation. Using fluorescence microscopy, we visualized hDOR internalization promoted by different agonists in SK-N-BE cells expressing FLAG-tagged hDOR. Agonist withdrawal results in a greater recycling process correlated with a stronger hDOR resensitization after etorphine treatment compared with DPDPE or Deltorphin I, as shown in binding, immunocytochemical, and functional experiments. This suggests a distinct sorting of opioid receptors after their internalization. We demonstrated a lysosomal hDOR targeting upon peptides by using chloroquine in binding, Western blot, and immunocytochemical experiments and by colocalization of this receptor with a late endosome marker. In contrast, when the recycling endosome blocker monensin was used, acceleration of desensitization associated with a strong intracellular immunostaining was observed upon etorphine treatment. The possibility of separate endocytic pathways responsible for the differential sorting of hDOR upon peptide and alkaloid ligand exposure was ruled out by binding and immunocytochemical experiments using sucrose hypertonic solution. First, these results showed complex relationships between hDOR internalization/down-regulation and desensitization. Second, we demonstrated for the first time that the same receptor could undergo a distinct sorting after internalization by peptide and alkaloid agonists.     

Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation

Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.     

Opioid receptor-coupled second messenger systems

Although pharmacological data provide strong evidence for different types of opioid receptors (e.g., mu, delta, and kappa), they share many common properties in their ability to couple to second messenger systems. All opioid receptor types are coupled to G-proteins, since agonist binding is diminished by guanine nucleotides and agonist-stimulated GTPase activity has been identified in several preparations. Moreover, all three types inhibit adenylyl cyclase. This second messenger system has been identified for opioid receptors in both isolated brain membranes and in transformed cell culture. Studies with chronic treatment with opioid agonists suggest that the coupling of receptors with G-proteins and second messenger effectors may play important roles in development of opioid tolerance.     

The role of opioid receptor phosphorylation and trafficking in adaptations to persistent opioid treatment

Mu-opioid receptor activation underpins clinical analgesia and is the central event in the abuse of narcotics. Continued opioid use produces tolerance to the acute effects of the drug and adaptations that lead to physical and psychological dependence. Continued mu-receptor signaling provides the engine for these adaptations, with most evidence suggesting that chronic agonist treatment produces only limited alterations in primary mu-opioid receptor signaling. Here we examine agonist regulation of mu-opioid receptor function, and whether this is altered by chronic treatment. Receptor phosphorylation is thought to be the key initial event in agonist regulation of the mu-opioid receptor, providing a signal for acute receptor desensitization and also subsequent receptor resensitization. Morphine appears to produce qualitatively and quantitatively different mu-receptor phosphorylation than other agonists, but the consequences of this remain obscure, at least in neurons. There is no evidence that agonist-induced mu-opioid receptor phosphorylation changes in chronically morphine-treated animals, although receptor regulation appears to be altered. Thus, as receptor phosphorylation and resensitization appear to maintain continued signaling through the mu-opioid receptor, these two events are crucial in facilitating adaptations to chronic opioid treatment, and the possibility that agonist-specific phosphorylation can contribute to the development of different adaptations remains open.     

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory G_i proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-x_L, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.     

Modulatory effects of Gs-coupled excitatory opioid receptor functions on opioid analgesia,tolerance, and dependence

Electrophysiologic studies of opioid effects on nociceptive types of dorsal root ganglion (DRG) neurons in organotypic cultures have shown that morphine and mostμ, δ, and κ opioid agonists can elicit bimodal excitatory as well as inhibitory modulation of the action potential duration (APD) of these cells. Excitatory opioid effects have been shown to be mediated by opioid receptors that are coupled via Gs to cyclic AMP-dependent ionic conductances that prolong the APD, whereas inhibitory opioid effects are mediated by opioid receptors coupled via Gi/Go to ionic conductuances that shorten the APD. Selective blockade of excitatory opioid receptor functions by low (ca. pM) concentrations of naloxone, naltrexone, etorphine and other specific agents markedly increases the inhibitory potency of morphine or other bimodally acting agonists and attenuates development of tolerance/dependence. These in vitro studies have been confirmed by tail-flick assays showin that acute co-treatment of mice with morphine plus ultra-low-dose naltrexone or etorphine remarkably enhances the antinociceptive potency of morphine whereas chronic co-treatment attenuates development of tolerance and naloxone-precipitated withdrawal-jumping symptoms. Special issue dedicated to Dr. Eric J. Simon.     

Differential effects of opioid receptor agonists on nociception and cAMP level in the spinal cord of monoarthritic rats.

Changes in functional responsiveness of spinal opioid receptors in monoarthritic rats were investigated at the behavioral and the molecular level. After intrathecal administration of morphine, D-Ala2-D-Leu5-enkephalin (DADLE), D-Pen2-D-Pen5-enkephalin (DPDPE) and dynorphin monoarthritic rats showed an enhanced antinociceptive response as measured by a tail-flick latency. No such changes were observed following administration of the selective kappa agonists U50,488H and U69,593. The opioid mu and delta receptor agonists (0.1-1.0 microM) inhibited the basal, as well as the forskolin-stimulated cAMP formation in spinal cord slices obtained from monoarthritic rats, whereas no significant changes were found in control animals. Higher concentrations of the mu and delta opioid receptor agonists were required to attenuate the cAMP level in spinal cord of control animals. The selective kappa agonists U50,488H and U69,593 did not influence the cAMP formation in monoarthritic or control animals. Additionally, we found that the GppNHp-stimulated level of cAMP was higher in the spinal cord slices of monoarthritic rats, which points to an enhanced responsiveness of the adenylate cyclase effector system to the action of this GTP analog. Our data suggest that the enhanced antinociceptive response to intrathecally administered opioids in monoarthritic rats may be connected with the increased sensitivity of adenylate cyclase to the inhibitory effects of mu and delta agonists.     

The role of mu opioid receptor desensitization and endocytosis in morphine tolerance and dependence

Following activation, most G protein coupled receptors undergo regulation by a cascade of events that promote receptor desensitization and endocytosis. Following endocytosis, receptors can then be recycled to the plasma membrane, retained in an intracellular compartment, or targeted for degradation. For receptors that are recycled, like the mu opioid receptor (MOR), endocytosis serves as the first step toward resensitizing receptors. For receptors that are degraded, endocytosis serves as the first step toward receptor downregulation. Thus, for receptors like the MOR, the desensitization-endocytosis-resensitization cycle serves as a rapid and dynamic means to titrate signaling through the receptor. However, not all agonist ligands at the MOR promote the same degree of receptor desensitization and endocytosis. For example, the endogenous peptide ligands at the MOR induce rapid desensitization, endocytosis, and recycling. By contrast, morphine induces only weak or partial desensitization and little to no endocytosis. As a consequence, signal transduction promoted by morphine is less dynamic than that induced by endogenous ligands as well as other opioid agonists that promote endocytosis. The resulting imbalance of desensitization-endocytosis-resensitization has at least two consequences: (1) in cell types where morphine induces desensitization but not endocytosis and/or resensitization, desensitization is protracted; (2) in cell types where morphine induces neither desensitization nor endocytosis, prolonged signaling through the receptor leads to multiple cellular adaptations downstream of receptor-G protein coupling. Both protracted desensitization and adaptive cellular changes probably contribute to the pronounced in vivo tolerance and dependence that occur with chronic morphine treatment. As a consequence, facilitating receptor endocytosis, using either genetic or pharmacological approaches, can restore the balance of signaling through the receptor and affect the development of tolerance and dependence.     

Benzylideneoxymorphone: A new lead for development of bifunctional mu/delta opioid receptor ligands

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.     

Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.     

Cloning and functional analysis of a gene encoding a novel muscarinic acetylcholine receptor expressed in chick heart and brain

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChR) expressed in chick heart are pharmacologically, immunologically, and biochemically distinct from mAChR expressed in mammalian heart. A chicken genomic clone encoding a mAChR whose deduced amino acid sequence is most homologous to the mammalian m4 receptor has been isolated. Northern blot analysis demonstrated that this gene is expressed in both chick heart and brain. The receptor encoded by this gene was expressed in stably transfected Chinese hamster ovary (CHO) and Y1 adrenal carcinoma cells in order to examine its ligand binding and functional properties. The receptor expressed in CHO and Y1 cells exhibits high affinity binding for the muscarinic antagonists quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the cloned chick m4 receptor exhibits pharmacological properties similar to those previously reported for the chick cardiac receptor. This m4 receptor was able to mediate both agonist-dependent inhibition of forskolin-stimulated cAMP accumulation and agonist-dependent stimulation of phosphoinositide metabolism when expressed in CHO cells. In contrast, when expressed in Y1 cells, the chick m4 receptor mediated agonist-dependent inhibition of forskolin-stimulated cAMP accumulation, but not stimulation of phosphoinositide metabolism. Thus, as with the mammalian cardiac (m2) receptor, the functional specificity of the chick cardiac receptor appears to be dependent on the cell type in which it is expressed.     

Opioid Modulation of Extracellular Signal-Regulated Protein Kinase Activity Is Ras-Dependent and Involves Gβγ Subunits

Abstract: Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with μ-, δ-, or κ-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ- ([ d -Ala²,Me-Phe⁴,Gly-ol⁵]enkephalin)-, δ- ([ d -Pen², d -Pen⁵]enkephalin)-, or κ- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a βγ scavenger, CD8-β-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-β-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of G_i/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.     

MOR Is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.     

Internalization and recycling of human mu opioid receptors expressed in Sf9 insect cells

Internalization and recycling of G protein-coupled receptors (GPCRs), such as the mu-opioid receptor, largely depend on agonist stimulation. Agonist-promoted internalization of some GPCRs has been shown to mediate receptor desensitization, resensitization, and down-regulation. In this study, we investigated whether different mu opioid agonists displayed different effects in receptor internalization and recycling, the potential mechanisms involved in ohmefentanyl-induced internalization process. In transfected Sf9 insect cells expressing 6His-tagged wild type mu opioid receptor, exposure to 100 nM ohmefentanyl caused a maximum internalization of the receptor at 30 min and receptors seemed to reappear at the cell membrane after 60 min as determined by radioligand binding assay. Ohmefentanyl-induced human mu opioid receptor internalization was concentration-dependent, with about 40% of the receptors internalized following a 30-min exposure to 1 microM ohmefentanyl. 10 microM morphine and 1 microM DAMGO could also induce about 40% internalization. The antagonist naloxone and pretreatment with pertussis toxin both blocked ohmefentanyl-induced internalization without affecting internalization themselves. Incubation with sucrose 0.45 M significantly inhibited ohmefentanyl-induced internalization of the mu receptor. The removal of agonists ohmefentanyl and morphine resulted in the receptors gradually returning to the cell surface over a 60 min period, while the removal of agonist DAMGO only partly resulted in the receptor recycling. The results of this study suggest that ohmefentanyl-induced internalization of human mu opioid receptor in Sf9 insect cells occurs via Gi/o protein-dependent process that likely involves clathrin-coated pits. In addition, the recycling process displays the differential modes of action of different agonists.     

Developmental Changes in the Modulation of Cyclic AMP Formation by the Metabotropic Glutamate Receptor Agonist 1S,3R-Aminocyclopentane-1,3-Dicarboxylic Acid in Brain Slices

Metabotropic glutamate receptors (mGluRs) have been recently described as a family of guanine nucleotide-binding regulatory protein-coupled receptors with multiple signal transduction pathways. At least one of these receptors appears to be negatively coupled to adenylyl cyclase when stably expressed in transfected cells. We have studied how activation of native mGluRs modulates cyclic AMP (cAMP) formation in brain slices prepared from rats at different ages. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (1S,1R-ACPD), a selective agonist of mGluRs, slightly increased basal cAMP formation but reduced forskolin-stimulated cAMP formation in adult hippocampal slices, in agreement with previous results. The action of 1S,3R-ACPD on basal cAMP formation was not reproduced by the ionotropic receptor agonists N-methyl-D-aspartate, kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and was antagonised by L-2-amino-3-phosphonopropionate (L-AP-3). L-AP-3, however, did not prevent but rather mimicked the inhibitory action of 1S,3R-ACPD on forskolin-stimulated cAMP formation. In hippocampal slices from 1-, 8-, or 15-day-old rats, 1S,3R-ACPD increased basal cAMP formation but failed to reduce the action of forskolin. A similar development pattern of modulation was observed in hypothalamic slices with the difference that 1S,3R-ACPD did not stimulate basal cAMP formation in the hypothalamus of adult animals. These results suggest that inhibition of forskolin-stimulated cAMP formation by 1S,3R-ACPD is mediated by a specific mGluR subtype that is preferentially expressed in the adult.