Differential Regulation of Histamine- and Bradykinin-Stimulated Phospholipase C in Adrenal Chromaffin Cells: Evidence for Involvement of Different Protein Kinase C Isoforms

Abstract: In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-α, ε, and ζ were identified. To characterize down-regulation of these enzymes, cells were incubated for 6–48 h with 1 µM phorbol myristate acetate (PMA). PKC-ε down-regulated more rapidly than PKC-α. At 12 h, PMA pretreatment, for example, PKC-ε was maximally down-regulated (23 ± 4% of controls), whereas PKC-α was unchanged. PKC-α showed partial down-regulation by 24 h of PMA pretreatment. PKC-ζ did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-ε than for PKC-α. The accumulation of total ³H-inositol (poly)phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 µM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-ε, whereas the restoration of the histamine response corresponds to the loss of PKC-α. This picture was confirmed with further studies on cytosolic Ca²⁺. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin cells are differentially regulated by PKC isoforms.     

Role of Protein Kinase C (PKC) in Agonist-Induced μ-Opioid Receptor Down-Regulation

Abstract : Agonist-induced down-regulation of opioid receptors appears to require the phosphorylation of the receptor protein. However, the identities of the specific protein kinases that perform this task remain uncertain. Protein kinase C (PKC) has been shown to catalyze the phosphorylation of several G protein-coupled receptors and potentiate their desensitization toward agonists. However, it is unknown whether opioid receptor agonists induce PKC activation under physiological conditions. Using cultured SH-SY5Y neuroblastoma cells, which naturally express μ- and δ-opioid receptors, we investigated whether μ-opioid receptor agonists can activate PKC by measuring enzyme translocation to the membrane fraction. PKC translocation and opioid receptor densities were simultaneously measured by ³H-phorbol ester and [³H]diprenorphine binding, respectively, to correlate alterations in PKC localization with changes in receptor binding sites. We observed that μ-opioid agonists have a dual effect on membrane PKC density depending on the period of drug exposure. Exposure for 2-6 h to [ d -Ala², N -Me-Phe⁴, Gly-ol]enkephalin or morphine promotes the translocation of PKC from the cytosol to the plasma membrane. Longer periods of opioid exposure (>12 h) produce a decrease in membrane-bound PKC density to a level well below basal. A significant decrease in [³H]diprenorphine binding sites is first observed at 2 h and continues to decline through the last time point measured (48 h). The opioid receptor antagonist naloxone attenuated both opioid-mediated PKC translocation and receptor down-regulation. These results demonstrate that opioids are capable of activating PKC, as evidenced by enhanced translocation of the enzyme to the cell membrane, and this finding suggests that PKC may have a physiological role in opioid receptor plasticity.     

Nociceptin (ORL-1) and μ-Opioid Receptors Mediate Mitogen-Activated Protein Kinase Activation in CHO Cells Through a Gi-Coupled Signaling Pathway: Evidence for Distinct Mechanisms of Agonist-Mediated Desensitization

Abstract: The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 receptor (OFQR) may be to modulate opioid-induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify opioid-stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction, indicating these receptors are capable of coupling to G_i/G_o proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen-activated protein (MAP) kinase in CHO cells expressing ORL-1 receptor (OFQR). Nociceptin (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of β-adrenergic receptor kinase (βARKct), which specifically blocks Gβγ-mediated signaling. Expression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21^ras, also attenuated nociceptin (OFQ)-stimulated MAP kinase activation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing μ-opioid receptor, [d -Ala²,N-Me-Phe⁴,Gly-ol]-enkephalin (DAMGO; a μ-opioid receptor-selective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, βARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL-1 (OFQR) and μ-opioid receptors mediate MAP kinase activation via a signaling pathway using the βγ-subunit of G_i, a PI-3K, and SOS, independent of PKC activity. In cells expressing both ORL-1 (OFQR) and μ-opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)- or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL-1 (OFQR) can modulate μ-opioid receptor signaling in a cellular system.     

Agonist-dependent μ-opioid receptor signaling can lead to heterologous desensitization

Desensitization of the µ-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Monitoring the release of intracellular Ca²⁺ ([Ca²⁺]_i), we reported that [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO)-induced receptor desensitization requires receptor phosphorylation and recruitment of β-arrestins (βArrs), while morphine-induced receptor desensitization does not. In current studies, we established that morphine-induced MOR desensitization is protein kinase C (PKC)-dependent. By using RNA interference techniques and subtype specific inhibitors, PKCε was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. In contrast, DAMGO did not increase PKCε activity and DAMGO-induced MOR desensitization was not affected by modulating PKCε activity. Among the various proteins within the receptor signaling complex, Gαi2 was phosphorylated by morphine-activated PKCε. Moreover, mutating three putative PKC phosphorylation sites, Ser⁴⁴, Ser¹⁴⁴ and Ser³⁰² on Gαi2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca²⁺]_i release, and this desensitization could be reversed by pretreating the cells with PKCε inhibitor or overexpressing Gαi2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Gαi-coupled receptors, such as the CB1.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Opioid-induced down-regulation of RGS4: role of ubiquitination and implications for receptor cross-talk

Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate μ- and δ- opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ～60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both Gα(i/o) and Gα(q) proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the Gα(i/o)-coupled DOR and the Gα(q)-coupled M(3) muscarinic receptor (M(3)R) was increased but not signaling of the α(2) adrenergic receptor or bradykinin BK(2) receptor, suggesting the development of cross-talk between the DOR and M(3)R involving RGS4.     

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.     

Agonist-induced, G protein-dependent and -independent down-regulation of the mu opioid receptor. The receptor is a direct substrate for protein-tyrosine kinase.

The mu opioid receptor (MOR) has been shown to desensitize after 1 h of exposure to the opioid peptide, [D-Ala(2), N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO), largely by the loss of receptors from the cell surface and receptor down-regulation. We have previously shown that the Thr(394) in the carboxyl tail is essential for agonist-induced early desensitization, presumably by serving as a primary phosphorylation site for G protein-coupled receptor kinase. Using a T394A mutant receptor, we determined that Thr(394) was also responsible for mu opioid receptor down-regulation. The T394A mutant receptor displayed 50% reduction of receptor down-regulation (14.8%) compared with wild type receptor (34%) upon 1 h of exposure to DAMGO. Agonist-induced T394A receptor down-regulation was unaffected by pertussis toxin treatment, indicating involvement of a mechanism independent of G protein function. Interestingly, pertussis toxin-insensitive T394A receptor down-regulation was completely inhibited by a tyrosine kinase inhibitor, genistein. Tyrosine kinase inhibition blocked wild type MOR down-regulation by 50%, and the genistein-resistant wild type MOR down-regulation was completely pertussis toxin-sensitive. Following DAMGO stimulation, MOR was shown to be phosphorylated at tyrosine residue(s), indicating that the receptor was a direct substrate for tyrosine kinase action. Mutagenesis of the four intracellular tyrosine residues resulted in complete inhibition of the G protein-insensitive MOR internalization. Therefore, agonist-induced MOR down-regulation appears to be mediated by two distinct cellular signal transduction pathways. One is G protein-dependent and GRK-dependent, which can be abolished by pertussis toxin treatment of wild type MOR or by mutagenesis of Thr(394). The other novel pathway is G protein-independent but tyrosine kinase-dependent, blocked by genistein treatment, and one in which Thr(394) has no regulatory role but phosphorylation of tyrosine residues appears essential.     

Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes

In hepatocytes, as in other cell types, Ca²⁺ signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP₃R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca²⁺ signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP₃ receptors (InsP₃R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP₃R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP₃-induced Ca²⁺ release in hepatocytes. This can be explained by the rather low expression level expression of InsP₃R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca²⁺ signaling via an inhibitory effect on InsP₃ synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP₃ synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.     

Activation of Type II Adenylyl Cyclase by the Cloned μ-Opioid Receptor: Coupling to Multiple G Proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ-opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ-opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ-opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ-selective agonist, [d -Ala², N-Me-Phe⁴,Gly⁵-ol]enkephalin, to stimulate cyclic AMP accumulation in a dose-dependent manner. The opioid-induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin-sensitive G_i proteins, because it was abolished completely by the toxin. Possible coupling between the μ-opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid-induced response became pertussis toxin-insensitive and was enhanced significantly upon co-expression with the α subunit of G_z, whereas those of G_q, G₁₂, or G₁₃ inhibited the opioid response. When pertussis toxin-sensitive G protein α subunits were tested under similar conditions, all three forms of α_i and both forms of α_o were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co-expression of α subunits reflects a functional coupling between α subunits and the μ-opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ-opioid receptor can couple to G_i1–3, G_o1–2, and G_z, but not to G_s, G_q, G₁₂, G₁₃, or G_t.     

Selective decrease of membrane-associated PKC-α and PKC-ε in response to elevated intracellular O-GlcNAc levels in transformed human glial cells

Increased flux through the hexosamine biosynthetic pathway (HBP) has been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. It has been suggested that this effect is due to increases in the β-O-linked N-acetylglucosamine (O-GlcNAc) modification. Herein, we demonstrate the effect of increasing the O-GlcNAc modification on the translocation of select PKC isozymes in a human astroglial cell line. Treating cells with either 8 mM d-glucosamine (GlcN), 5 mM streptozotocin (STZ), or 80 μM O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) produced a significant increase in the O-GlcNAc modification on both cytosolic and membrane proteins; however, both the level and rate of O-GlcNAc increase varied with the compound. GlcN treatment resulted in a rapid, transient translocation of PKC-βII that was maximal after 3 h (73±8%) and also produced a 48±15% decrease in membrane-associated PKC-ε after 9 h of treatment. Similar to GlcN treatment, STZ and PUGNAc treatment also resulted in decreased levels of PKC-ε in the membrane fraction. Significant decreases were seen as early as 5 h and, by 9 h of treatment, had decreased by 87±6% with STZ and 73±7% with PUGNAc. Unlike GlcN, both STZ and PUGNAc produced a decrease in PKC-α membrane levels by 9 h posttreatment (78±10% with STZ and 66±8% with PUGNAc) while neither compound produced any changes in PKC-βII translocation. In addition, none of the three compounds affected membrane levels of PKC-ι. Altogether, these results demonstrate a novel link between increased levels of the O-GlcNAc modification and the regulation of specific PKC isoforms.     

Site Mutation in the Rat μ-Opioid Receptor Demonstrates the Involvement of Calcium/Calmodulin-Dependent Protein Kinase II in Agonist-Mediated Desensitization

Abstract: The rat μ-opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, shows a desensitization to the inhibitory effect of the μ agonist DAMGO on adenylate cyclase activity within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin-dependent protein kinase II (CaM kinase II) on μ-opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase II in HEK293 cells. This coexpression led to a faster time course of agonist-induced desensitization of the μ-opioid receptor. The increase of desensitization could not be observed with a μ-opioid receptor mutant (S261A/S266A) that lacks two putative CaM kinase II phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase II in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261A/S266A receptor mutant. These results suggest that phosphorylation of Ser²⁶¹ and Ser²⁶⁶ by CaM kinase II is involved in the desensitization of the μ-opioid receptor.     

Discovery of N-substituted-endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenol and -phenyl carboxamide series of μ-opioid receptor antagonists

Gastrointestinal dysfunction as a consequence of the use of opioid analgesics is of significant clinical concern. First generation drugs to treat these opioid-induced side-effects were limited by their negative impact on opioid receptor agonist-induced analgesia. Second generation therapies target a localized, peripherally-restricted, non-CNS penetrant drug distribution of opioid receptor antagonists. Herein we describe the discovery of the N-substituted-endo-3-(8-aza-bicyclo[3.2.1]oct-3-yl)-phenol and -phenyl carboxamide series of μ-opioid receptor antagonists. This report highlights the discovery of the key μ-opioid receptor antagonist pharmacophore and the optimization of in vitro metabolic stability through the application of a phenol bioisostere. The compounds 27a and 31a with the most attractive in vitro profile, formed the basis for the application of Theravance Biopharma’s multivalent approach to drug discovery to afford the clinical compound axelopran (TD-1211), targeted for the treatment of opioid-induced constipation.     

Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.     

Agonist-Specific Regulation of δ-Opioid Receptor Trafficking by G Protein-Coupled Receptor Kinase and β-Arrestin

Abstract

Opioid receptors mediate multiple biological functions through their interaction with endogenous opioid peptides as well as opioid alkaloids including morphine and etorphine. Previously we have reported that the ability of distinct opioid agonists to differentially regulate μ-opioid receptor (μOR) responsiveness is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the receptor (1). In the present study, we further examined the role of GRK and β-arrestin in agonist-specific regulation of the δ-opioid receptor (δOR). While both etorphine and morphine effectively activate the δOR, only etorphine triggers robust δOR phosphorylation followed by plasma membrane translocation of β-arrestin and receptor internalization. In contrast, morphine is unable to either elicit δOR phosphorylation or stimulate β-arrestin translocation, correlating with its inability to cause δOR internalization. Unlike for the μOR, overexpression of GRK2 results in neither the enhancement of δOR sequestration nor the rescue of δOR-mediated β-arrestin translocation. Therefore, our findings not only point to the existence of marked differences in the ability of different opioid agonists to promote δOR phosphorylation by GRK and binding to β-arrestin, but also demonstrate differences in the regulation of two opioid receptor subtypes. These observations may have important implications for our understanding of the distinct ability of various opioids in inducing opioid tolerance and addiction.     

Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.     

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala² residue with d-Arg² makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg² have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg² may provide a new target molecule for developing novel analgesics that have fewer side effects.     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

Essential activation of PKC-delta in opioid-initiated cardioprotection

Stimulation of the delta(1)-opioid receptor confers cardioprotection to the ischemic myocardium. We examined the role of protein kinase C (PKC) after delta-opioid receptor stimulation with TAN-67 or D-Ala(2)-D-Leu(5)-enkephalin (DADLE) in a rat model of myocardial infarction induced by a 30-min coronary artery occlusion and 2-h reperfusion. Infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of the area at risk (IS/AAR). Control animals, subjected to ischemia and reperfusion, had an IS/AAR of 59.9 +/- 1.8. DADLE and TAN-67 administered before ischemia significantly reduced IS/AAR (36.9 +/- 3.9 and 36.7 +/- 4.7, respectively). The delta(1)-selective opioid antagonist 7-benzylidenenaltrexone (BNTX) abolished TAN-67-induced cardioprotection (54.4 +/- 1.3). Treatment with the PKC antagonist chelerythrine completely abolished DADLE- (61.8 +/- 3.2) and TAN-67-induced cardioprotection (55.4 +/- 4.0). Similarly, the PKC antagonist GF 109203X completely abolished TAN-67-induced cardioprotection (54.6 +/- 6.6). Immunofluorescent staining with antibodies directed against specific PKC isoforms was performed in myocardial biopsies obtained after 15 min of treatment with saline, chelerythrine, BNTX, or TAN-67 and chelerythrine or BNTX in the presence of TAN-67. TAN-67 induced the translocation of PKC-alpha to the sarcolemma, PKC-beta(1) to the nucleus, PKC-delta to the mitochondria, and PKC-epsilon to the intercalated disk and mitochondria. PKC translocation was abolished by chelerythrine and BNTX in TAN-67-treated rats. To more closely examine the role of these isoforms in cardioprotection, we utilized the PKC-delta selective antagonist rottlerin. Rottlerin abolished opioid-induced cardioprotection (48.9 +/- 4.8) and PKC-delta translocation without affecting the translocation of PKC-alpha, -beta(1), or -epsilon. These results suggest that PKC-delta is a key second messenger in the cardioprotective effects of delta(1)-opioid receptor stimulation in rats.     

Techniques for assessing knee joint pain in arthritis

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.