ADP-ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced mu-opioid receptor endocytosis

Agonist exposure of many G protein-coupled receptors induces a rapid receptor phosphorylation and uncoupling from G proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Using a yeast two-hybrid screen, the rat mu-opioid receptor (MOR1, also termed MOP) was found to be associated with phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane, which has been implicated in the formation of endocytotic vesicles. Coimmunoprecipitation experiments in HEK293 cells coexpressing MOR1 and PLD2 confirmed that MOR1 constitutively interacts with PLD2. Treatment with the mu receptor agonist DAMGO ([d-Ala(2), Me Phe(4), Glyol(5)]enkephalin) led to an increase in PLD2 activity, whereas morphine, which does not induce MOR1 receptor internalization, failed to induce PLD2 activation. The DAMGO-mediated PLD2 activation was inhibited by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) but not by the protein kinase C (PKC) inhibitor calphostin C indicating that opioid receptor-mediated activation of PLD2 is ARF- but not PKC-dependent. Furthermore, heterologous stimulation of PLD2 by phorbol ester led to an accelerated internalization of the mu-opioid receptor after both DAMGO and morphine exposure. Conversely the inhibition of PLD2-mediated phosphatidic acid formation by 1-butanol or overexpression of a negative mutant of PLD2 prevented agonist-mediated endocytosis of MOR1. Together, these data suggest that PLD2 play a key role in the regulation of agonist-induced endocytosis of the mu-opioid receptor.     

Role of phospholipase D2 in the agonist-induced and constitutive endocytosis of G-protein coupled receptors

We have recently shown that the mu-opioid receptor [MOR1, also termed mu-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the mu-opioid receptor (MOR1D, also termed MOP(1D)) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the delta-opioid receptor [DOR, also termed delta-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis.     

RGS14 prevents morphine from internalizing Mu-opioid receptors in periaqueductal gray neurons

Opioid agonists display different capacities to stimulate mu-opioid receptor (MOR) endocytosis, which is related to their ability to provoke the phosphorylation of specific cytosolic residues in the MORs. Generally, opioids that efficiently promote MOR endocytosis and recycling produce little tolerance, as is the case for [d-Ala², N-MePhe⁴,Gly-ol⁵] encephalin (DAMGO). However, morphine produces rapid and profound antinociceptive desensitization in the adult mouse brain associated with little MOR internalization. The regulator of G-protein signaling, the RGS14 protein, associates with MORs in periaqueductal gray matter (PAG) neurons, and when RGS14 is silenced morphine increased the serine 375 phosphorylation in the C terminus of the MOR, a GRK substrate. Subsequently, these receptors were internalized and recycled back to the membrane where they accumulated on cessation of antinociception. These mice now exhibited a resensitized response to morphine and little tolerance developed. Thus, in morphine-activated MORs the RGS14 prevents GRKs from phosphorylating those residues required for β-arresting-mediated endocytosis. Moreover morphine but not DAMGO triggered a process involving calcium/calmodulin-dependent kinase II (CaMKII) in naïve mice, which contributes to MOR desensitization in the plasma membrane. In RGS14 knockdown mice morphine failed to activate this kinase. It therefore appears that phosphorylation and internalization of MORs disrupts the CaMKII-mediated negative regulation of these opioid receptors.     

Endocytosis as a Biological Response in Receptor Pharmacology: Evaluation by Fluorescence Microscopy

The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method –the Q-Endosomes algorithm– that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT_2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.     

G protein-coupled receptor kinase 2 mediates mu-opioid receptor desensitization in GABAergic neurons of the nucleus raphe magnus

Nucleus raphe magnus (NRM) sends the projection to spinal dorsal horn and inhibits nociceptive transmission. Analgesic effect produced by mu-opioid receptor agonists including morphine partially results from activating the NRM-spinal cord pathway. It is generally believed that mu-opioid receptor agonists disinhibit spinally projecting neurons of the NRM and produce analgesia by hyperpolarizing GABAergic interneurons. In the present study, whole-cell patch-clamp recordings combined with single-cell RT-PCR analysis were used to test the hypothesis that DAMGO ([D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin), a specific mu-opioid receptor agonist, selectively hyperpolarizes NRM neurons expressing mRNA of glutamate decarboxylase (GAD(67)). Homologous desensitization of mu-opioid receptors in NRM neurons could result in the development of morphine-induced tolerance. G protein-coupled receptor kinase (GRK) is believed to mediate mu-opioid receptor desensitization in vivo. Therefore, we also investigated the involvement of GRK in mediating homologous desensitization of DAMAMGO-induced electrophysiological effects on NRM neurons by using two experimental strategies. First, single-cell RT-PCR assay was used to study the expression of GRK2 and GRK3 mRNAs in individual DAMGO-responsive NRM neurons. Whole-cell recording was also performed with an internal solution containing the synthetic peptide, which corresponds to G(betagamma)-binding domain of GRK and inhibits G(betagamma) activation of GRK. Our results suggest that DAMGO selectively hyperpolarizes NRM GABAergic neurons by opening inwardly rectifying K(+) channels and that GRK2 mediates short-term homologous desensitization of mu-opioid receptors in NRM GABAergic neurons.     

C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization

The main analgesic effects of the opioid alkaloid morphine are mediated by the mu-opioid receptor. In contrast to endogenous opioid peptides, morphine activates the mu-opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse mu-opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four mu-receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell- and tissue-specific C-terminal splicing of the mu-opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.     

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.     

An opiate cocktail that reduces morphine tolerance and dependence

Morphine is an exceptionally effective analgesic whose utility is compromised by the development of tolerance and dependence to the drug. Morphine analgesia and dependence are mediated by its activity at the mu opioid peptide (MOP) receptor [1]. The MOP receptor is activated not only by morphine, but also by other opiate drugs such as methadone and endogenous opioids such as endorphins. Morphine, however, is a unique opioid agonist ligand because it fails to induce endocytic trafficking of the MOP receptor [2], whereas the endogenous ligands and methadone do facilitate endocytosis [3]. Using the unique pharmacology of the MOP receptor and its proposed existence as an oligomeric structure [4], we designed a pharmacological cocktail that facilitates endocytosis of the MOP receptor in response to morphine. This cocktail consists of morphine and a small dose of methadone. Importantly, this cocktail, while retaining full analgesic potency, does not promote morphine dependence. We further demonstrate that dependence is reduced, at least in part, because endocytosis of the MOP receptor in response to morphine prevents the upregulation of N-methyl-D-aspartate (NMDA) receptors.     

Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells

Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat micro-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the mu-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced mu-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted mu-receptor phosphorylation with an IC50 of 20 microM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on mu-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because mu-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and intemalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the mu-opioid receptor.     

Phospholipase D2 modulates agonist-induced mu-opioid receptor desensitization and resensitization

Receptor phosphorylation, arrestin binding, uncoupling from G protein and subsequent endocytosis have been implicated in G protein-coupled receptor desensitization after chronic agonist exposure. In search of proteins regulating the mu-opioid receptor endocytosis, we have recently established that activation of phospholipase D (PLD)2 is required for agonist-induced mu-opioid receptor endocytosis. In this study, we determined the effect of PLD2 activity on the desensitization and resensitization rate of the mu-opioid receptor. We clearly demonstrated that inhibition of PLD2-mediated phosphatidic acid formation by alcohol (1-butanol or ethanol) or overexpression of a dominant negative mutant of PLD2 prevented agonist-mediated endocytosis and resulted in a faster desensitization rate of the mu-opioid receptor after chronic (D-Ala2, Me Phe4, Glyol5)enkephalin treatment in human embryonic kidney 293 cells. Moreover, inhibition of PLD2 activity led to an impairment of the resensitization rate of the mu-opioid receptor. In summary, our data strongly suggest that PLD2 is a modulator of agonist-induced endocytosis, desensitization and resensitization of the mu-opioid receptor.     

Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine

Background

Although the systemic administration of cannabinoids produces antinociception, their chronic use leads to analgesic tolerance as well as cross-tolerance to morphine. These effects are mediated by cannabinoids binding to peripheral, spinal and supraspinal CB1 and CB2 receptors, making it difficult to determine the relevance of each receptor type to these phenomena. However, in the brain, the CB1 receptors (CB1Rs) are expressed at high levels in neurons, whereas the expression of CB2Rs is marginal. Thus, CB1Rs mediate the effects of smoked cannabis and are also implicated in emotional behaviors. We have analyzed the production of supraspinal analgesia and the development of tolerance at CB1Rs by the direct injection of a series of cannabinoids into the brain. The influence of the activation of CB1Rs on supraspinal analgesia evoked by morphine was also evaluated.

Results

Intracerebroventricular (icv) administration of cannabinoid receptor agonists, WIN55,212-2, ACEA or methanandamide, generated a dose-dependent analgesia. Notably, a single administration of these compounds brought about profound analgesic tolerance that lasted for more than 14 days. This decrease in the effect of cannabinoid receptor agonists was not mediated by depletion of CB1Rs or the loss of regulated G proteins, but, nevertheless, it was accompanied by reduced morphine analgesia. On the other hand, acute morphine administration produced tolerance that lasted only 3 days and did not affect the CB1R. We found that both neural mu-opioid receptors (MORs) and CB1Rs interact with the HINT1-RGSZ module, thereby regulating pertussis toxin-insensitive Gz proteins. In mice with reduced levels of these Gz proteins, the CB1R agonists produced no such desensitization or morphine cross-tolerance. On the other hand, experimental enhancement of Gz signaling enabled an acute icv administration of morphine to produce a long-lasting tolerance at MORs that persisted for more than 2 weeks, and it also impaired the analgesic effects of cannabinoids.

Conclusion

In the brain, cannabinoids can produce analgesic tolerance that is not associated with the loss of surface CB1Rs or their uncoupling from regulated transduction. Neural specific Gz proteins are essential mediators of the analgesic effects of supraspinal CB1R agonists and morphine. These Gz proteins are also responsible for the long-term analgesic tolerance produced by single doses of these agonists, as well as for the cross-tolerance between CB1Rs and MORs.     

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Background

In general, opioids that induce the recycling of μ-opioid receptors (MORs) promote little desensitization, although morphine is one exception to this rule. While morphine fails to provoke significant internalization of MORs in cultured cells, it does stimulate profound desensitization. In contrast, morphine does promote some internalization of MORs in neurons although this does not prevent this opioid from inducing strong antinociceptive tolerance.

Results

In neurons, morphine stimulates the long-lasting transfer of MOR-activated Gα subunits to proteins of the RGS-R7 and RGS-Rz subfamilies. We investigated the influence of this regulatory process on the capacity of morphine to promote desensitization and its association with MOR recycling in the mature nervous system. In parallel, we also studied the effects of [D-Ala², N-MePhe⁴, Gly-ol⁵] encephalin (DAMGO), a potent inducer of MOR internalization that promotes little tolerance. We observed that the initial exposure to icv morphine caused no significant internalization of MORs but rather, a fraction of the Gα subunits was stably transferred to RGS proteins in a time-dependent manner. As a result, the antinociception produced by a second dose of morphine administered 6 h after the first was weaker. However, this opioid now stimulated the phosphorylation, internalization and recycling of MORs, and further exposure to morphine promoted little tolerance to this moderate antinociception. In contrast, the initial dose of DAMGO stimulated intense phosphorylation and internalization of the MORs associated with a transient transfer of Gα subunits to the RGS proteins, recovering MOR control shortly after the effects of the opioid had ceased. Accordingly, the recycled MORs re-established their association with G proteins and the neurons were rapidly resensitized to DAMGO.

Conclusion

In the nervous system, morphine induces a strong desensitization before promoting the phosphorylation and recycling of MORs. The long-term sequestering of morphine-activated Gα subunits by certain RGS proteins reduces the responses to this opioid in neurons. This phenomenon probably increases free Gβγ dimers in the receptor environment and leads to GRK phosphorylation and internalization of the MORs. Although, the internalization of the MORs permits the transfer of opioid-activated Gα subunits to the RGSZ2 proteins, it interferes with the stabilization of this regulatory process and recycled MORs recover the control on these Gα subunits and opioid tolerance develops slowly.     

Effect of the A118G polymorphism on binding affinity, potency and agonist-mediated endocytosis, desensitization, and resensitization of the human mu-opioid receptor

The most prevalent single-nucleotide polymorphism (SNP) A118G in the human mu-opioid receptor gene predicts an amino acid change from an asparagine residue to an aspartatic residue in amino acid position 40. This N40D mutation, which has been implicated in the development of opioid addiction, was previously reported to result in an increased beta-endorphin binding affinity and a decreased potency of morphine-6-glucuronide. Therefore, in the present study we have investigated whether this mutation might affect the binding affinity, potency, and/or the agonist-induced desensitization, internalization and resensitization of the human mu-opioid receptor stably expressed in human embryonic kidney 293 cells. With the exception of a reduced expression level of N40D compared to human mu-opioid receptor (hMOR) in HEK293 cells, our analyses revealed no marked functional differences between N40D and wild-type receptor. Morphine, morphine-6-glucuronide and beta-endorphin revealed similar binding affinities and potencies for both receptors. Both the N40D-variant receptor and hMOR exhibited robust receptor internalization in the presence of the opioid peptide [d-Ala(2),N-MePhe(4),Glyol(5)]enkephalin (DAMGO) and beta-endorphin but not in response to morphine or morphine-6-glucuronide. After prolonged treatment with morphine, morphine-6-glucuronide or beta-endorphin both receptors showed similiar desensitization time courses. In addition, the receptor resensitization rates were nearly identical for both receptor types.     

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.     

GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors

Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.     

μ-Opioid receptor-stimulated synthesis of reactive oxygen species is mediated via phospholipase D2

We have recently shown that the activation of the rat μ-opioid receptor (MOPr, also termed MOR1) by the μ-agonist [ d -Ala², Me Phe⁴, Glyol⁵]enkephalin (DAMGO) leads to an increase in phospholipase D2 (PLD2) activity and an induction of receptor endocytosis, whereas the agonist morphine which does not induce opioid receptor endocytosis fails to activate PLD2. We report here that MOPr-mediated activation of PLD2 stimulates production of reactive oxygen molecules via NADH/NADPH oxidase. Oxidative stress was measured with the fluorescent probe dichlorodihydrofluorescein diacetate and the role of PLD2 was assessed by the PLD inhibitor d -erythro-sphingosine (sphinganine) and by PLD2-small interfering RNA transfection. To determine whether NADH/NADPH oxidase contributes to opioid-induced production of reactive oxygen species, μ-agonist-stimulated cells were pre-treated with the flavoprotein inhibitor, diphenylene iodonium, or the specific NADPH oxidase inhibitor, apocynin. Our results demonstrate that receptor-internalizing agonists (like DAMGO, β-endorphin, methadone, piritramide, fentanyl, sufentanil, and etonitazene) strongly induce NADH/NADPH-mediated ROS synthesis via PLD-dependent signaling pathways, whereas agonists that do not induce MOPr endocytosis and PLD2 activation (like morphine, buprenorphine, hydromorphone, and oxycodone) failed to activate ROS synthesis in transfected human embryonic kidney 293 cells. These findings indicate that the agonist-selective PLD2 activation plays a key role in the regulation of NADH/NADPH-mediated ROS formation by opioids.     

Mediation of adenylyl cyclase sensitization by PTX-insensitive GalphaoA, Galphai1, Galphai2 or Galphai3

Chronic activation of mu-opioid receptors, which couple to pertussis toxin-sensitive Galphai/o proteins to inhibit adenylyl cyclase (AC), leads to a compensatory sensitization of AC. Pertussis toxin-insensitive mutations of Galphai/o subtypes, in which the pertussis toxin-sensitive cysteine is mutated to isoleucine (Galpha ), were used to determine whether each of the Galphai/o subtypes is able to mediate sensitization of AC. Galpha , G , G or G were individually transiently transfected into C6 glioma cells stably expressing the mu-opioid receptor, or transiently co-expressed with the mu-opioid receptor into human embryonic kidney (HEK)293T cells. Cells were treated with pertussis toxin to uncouple endogenous Galphai/o proteins, followed by acute or chronic treatment with the mu-opioid agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO). Each Galphai/o subtype mediated acute DAMGO inhibition of AC and DAMGO-induced sensitization of AC. The potency for DAMGO to stimulate sensitization was independent of the Galphai/o subtype, but the level of sensitization was increased in clones expressing higher levels of Galphai/o subunits. Sensitization of AC mediated by a component of fetal bovine serum, which was also dependent on the level of functional Galphai/o subunits in the cell, was observed. This serum-mediated sensitization partially masked mu-opioid-mediated sensitization when expressed as percentage overshoot due to an apparent increase in AC activity.     

Sympathetic denervation-induced changes in G protein expression in enteric neurons of the guinea pig colon

Chronic sympathetic denervation entails subsensitivity to alpha(2)-adrenoceptor agonists and supersensitivity to kappa- and mu-opioid receptor agonists modulating cholinergic neurons in the guinea pig colon. A possible role for signal transduction G proteins in contributing to development of these sensitivity changes was investigated. Pertussis toxin (PTX), a blocker of the G(i/o)-type family of G proteins significantly reduced the inhibitory effects of UK14,304 (alpha(2)-adrenoceptor agonist), U69593 (kappa-opioid receptor agonist) and DAMGO (mu-opioid receptor agonist) on acetylcholine (ACh) overflow in preparations obtained from normal animals, but not in those obtained from sympathetically denervated animals. In this experimental condition, immunoblot analysis revealed reduced levels of G(alphao), G(alphai2), G(alphai3) and G(beta) in myenteric plexus synaptosomes. On reverse, synaptosomal levels of G(alphai1) and G(alphaz), a PTX-insensitive G-protein, increased after chronic ablation of the sympathetic pathways. These data suggest that changes in the function and expression of inhibitory G proteins coupled to alpha(2)-adrenoceptors, kappa- and mu-opioid receptors occur in the myenteric plexus of the guinea pig colon after chronic sympathetic denervation. The possibility that regulation of G proteins represents one of the biochemical mechanisms at the basis of the changes in sensitivity of enteric cholinergic neurons to alpha(2)-adrenoceptor, kappa- and mu-opioid receptor agonists is discussed.     

Fast modulation of μ-opioid receptor (MOR) recycling is mediated by receptor agonists

The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.     

Morphine-induced mu-opioid receptor rapid desensitization is independent of receptor phosphorylation and beta-arrestins

Receptor desensitization involving receptor phosphorylation and subsequent betaArrestin (betaArr) recruitment has been implicated in the tolerance development mediated by mu-opioid receptor (OPRM1). However, the roles of receptor phosphorylation and betaArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca(2+) ([Ca(2+)](i))release to monitor receptor activation, as predicted, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a receptor phosphorylation- and betaArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from betaArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from betaArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca(2+)](i) release to a greater extent than that of DAMGO and could induce the phosphorylation of Ser(375) residue, morphine-induced desensitization was not influenced by mutating the phosphorylation sites or in MEF cells lacking betaArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of betaArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of betaArr.