![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR AND MOLECULAR
-, and 
-Opioid Receptors with and without Agonist PretreatmentDepartment of Pharmacology, School of Medicine and Public Health, The Ohio State University, Columbus, Ohio
Received December 19, 2006; accepted January 29, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
-naltrexol, remain neutral antagonists at MOR under any condition. This study compares the regulation of basal signaling of MOR, 
-(DOR), and 
-(KOR) opioid receptors after pretreatment with morphine or receptor-selective agonists, in transfected human embryonic kidney 293 cell membranes. Moreover, naloxone, naltrexone, and related antagonists were compared for binding potency and effect on basal and agonist-stimulated receptor signaling, measuring guanosine 5'-O-(3-[35S]thio)triphosphate binding. The results demonstrate basal activity for each opioid receptor, which is modulated by pretreatment with agonists. Even closely related opioid antagonists display distinct patterns of neutral and inverse effects before and after agonist pretreatment, including distinct efficacies between naloxone and naltrexone at agonist-pretreated DOR and KOR. Pretreatment with different agonists has varying effects on inverse and neutral activities of some analogs tested. These results demonstrate that antagonist efficacy is context-dependent, possibly accounting for paradoxical pharmacological effects. Activity profiles at the three opioid receptors under different conditions could lead to antagonists with optimal clinical properties in treatment of addiction and adverse opioid effects.
). Antagonists with inverse agonist property potentially have distinct treatment outcomes compared with neutral antagonists, i.e., antagonists that block agonist activation but do not affect basal activity. For example, the classification of "typical" and "atypical" antipsychotics may in part be related to inverse agonism (atypical) and neutral antagonism (typical) at 5-hydroxytryptamine2C receptors (Herrick-Davis et al., 2000).
The opioid receptors belong to GPCR family and consist of three genes encoding µ-, -, and -opioid receptors (or MOR, DOR, and KOR, respectively). Although the basal signaling activity for DOR is readily detectable (Costa and Herz, 1989), being the first GPCR found to display basal signaling activity, this was more difficult to demonstrate for MOR, possibly due to the masking of basal MOR activity by interacting regulatory proteins, such as calmodulin (Wang et al., 1999). Our laboratory has demonstrated the presence of basal MOR signaling activity in various tissues in cell culture (Wang et al., 1994, 1999, 2000; Burford et al., 2000) and in mouse brain tissue (Wang et al., 2004), which was typically more prominent in opioid agonist-pretreated ("dependent") tissues. This was at first an unexpected finding, because MOR is thought to desensitize during agonist pretreatment; yet, we have shown that release of calmodulin from the receptor by agonist stimulation uncovered the innate basal activity of MOR in the dependent state (Wang et al., 1999, 2000). We have further identified several inverse agonists and neutral antagonists, the latter blocking both opioid agonist and inverse agonist effects, a strong indication that the observed effects are indeed elicited by binding to MOR (Bilsky et al., 1996; Wang et al., 2001, 2004). Constitutive MOR activity was independently confirmed (Liu et al., 2001), as was the unusual regulation of constitutive activity of MOR and DOR receptors by chronic agonists pretreatment (Liu and Prather, 2001, 2002). Moreover, basal opioid activity has been implicated in appetite (Emmerson et al., 2004), morphine tolerance (Heinzen et al., 2005), and methamphetamine-induced behavioral sensitization (Chiu et al., 2006).
An important finding came from the observation that some neutral antagonists, such as naloxone and naltrexone, turned into inverse agonists after agonists pretreatment, suggesting that the receptor has been modified in the dependent state. The conversion of naloxone and naltrexone from neutral antagonist to inverse agonist seems to contribute to precipitated withdrawal symptoms in opioid addicts (Wang et al., 2004; Raehal et al., 2005; Sadee et al., 2005). This was supported by our finding that neutral antagonist 6-naltrexol and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, which remain neutral in the dependent state, precipitate less withdrawal than naloxone in morphine-dependent mice, at equipotent pharmacological doses in blocking morphine-induced antinociception (Bilsky et al., 1996; Wang et al., 2004; Raehal et al., 2005). Others also reported that basal activity of MOR was related to conditioned aversive effect of naloxone in morphine-dependent mice (Shoblock and Maidment, 2006), opioid antagonists with different inverse agonist properties have different effects in precipitating withdrawal in acute morphine-dependent mice (Walker and Sterious, 2005), and constitutive opioid receptor activation is critically involved in acute opioid withdrawal (Freye and Levy, 2005).
The variable behavior of naloxone and naltrexone in naive and opioid-pretreated cells is consistent with the notion that ligand activity can vary with cellular context, i.e., the "protean" properties of the ligands (Gbahou et al., 2003). Thus, naltrexone and naloxone are protean antagonists at MOR, whereas 6-naltrexol is neutral under all conditions studied (Wang et al., 2001; Raehal et al., 2005). A thorough understanding of the malleable protean properties of opioid antagonists is important because of the dramatic precipitated withdrawal caused by naloxone and naltrexone, effects that may be avoided or ameliorated with a neutral antagonist such as 6-naltrexol. Moreover, these ligands also bind to DOR and KOR, but regulation of basal activity and protean ligand properties remain unknown at these subtypes.
In this study, we have investigated and compared the regulation of basal activity of MOR, DOR and KOR, and the effects of naloxone, naltrexone, and naltrexone derivatives 6-naltrexol (Raehal et al., 2005) and 6-naltrexamide (Fig. 1) on MOR, DOR, and KOR receptor with and without agonists pretreatments. We were particularly interested in 6-naltrexol and 6-naltrexamide, because these neutral antagonists represent potential therapeutic agents in the treatment of opioid side effect. We used BNTX (Wang et al., 2001) and ICI 174,864 (Costa and Herz, 1989) as full MOR and DOR inverse agonists, respectively. For KOR, we have identified nor-binaltorphimine (nor-BNI) and 5'-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5
-epoxy-3,14-dihydroxy-6,7-2'3'-indolomorphinan dihydrochloride (GNTI) as inverse agonist.
|
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-naltrexol, and 6-naltrexamide were obtained through the National Institute on Drug Abuse Drug Supply Program; U-69593, [D-Pen2,D-Pen5]-enkephalin (DPDPE), and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) were purchased from Sigma-Aldrich (St. Louis, MO); U50,488H, ICI 174,864, nor-BNI, and GNTI were purchased from Tocris Cookson Inc. (Ellisville, MO); [35S]GTP
S and [3H]diprenorphine were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Other reagents for cell culture were from Sigma-Aldrich or Fisher Scientific (Pittsburgh, PA). Cell Culture and Treatment. Human embryonic kidney (HEK) 293 cells stably transfected with human MOR (HEK-MOR), mouse DOR (HEK-DOR), and human KOR (HEK-KOR) were maintained in Dulbecco's modified Eagle's medium H16/F-12 supplemented with 10% fetal bovine serum, 100 µU/ml penicillin, 100 µg/ml streptomycin, and 200 µg/ml Geneticin (G-418; Invitrogen, Carlsbad, CA). The receptor expression levels were 1.2, 3.4, and 2.7pmol/mg protein for HEK-MOR, HEK-DOR, and HEK-KOR, respectively (measured by [3H]diprenorphine saturation binding assays in cell membranes). For agonist pretreatment, 80% confluent cells were cultured in the presence MOR-, DOR-, or KOR-specific agonists DAMGO (1 µM), DPDPE (1 or 10 µM), U50,488H (1 µM), or U-69593 (1 µM), or nonspecific agonist morphine (10 or 50 µM) for 24 h before harvest. Cells were then washed thoroughly with phosphate-buffered saline (PBS) to remove the treated drugs before membrane preparations.
[35S]GTPS Binding. Membrane preparation and [35S]GTPS binding assays were carried out as described previously (Wang et al., 2000). In brief, cells were harvested and washed with PBS, and then the cells were homogenized in buffer containing 10 mM Tris-HCl, pH 7.4, and 0.1 mM EDTA and centrifuged at 30,000g for 10 min. The pellets were resuspended in the same buffer and centrifuged again. The pellets from the second centrifugation were resuspended, aliquoted, and stored at –70°C. [35S]GTPS binding assays were performed using different conditions. For agonist effects, cell membranes (10 µg of protein) were incubated with drugs in 100 µl of assay buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 10 mM MgCl2, and 10 µM GDP at 30°C for 5 min. For inverse agonist effects, cell membranes (50 µg of protein) were incubated with drugs in 500 µl of different assay buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 10 µM GDP and 150 mM NaCl or KCl and different concentrations of MgCl2 (for MOR, 150 mM KCl, 1 mM MgCl2; for DOR, 150 mM KCl and 10 mM MgCl2; and for KOR, 150 NaCl and 10 mM MgCl2). The mixtures were incubated at 30°C for 30 min. After incubation, the reactions were stopped by adding 500 µl of ice-cold PBS followed by centrifugation at 13,000g for 10 min at 4°C. The pellets were washed once with 1 ml of PBS, and radioactivity was measured by liquid scintillation counter.
[3H]Diprenorphine Binding. For [3H]diprenorphine saturation binding assay, membranes (20 µg of protein) were incubated with different concentrations (0.5–5 nM) of [3H]diprenorphine in buffer containing 50 mM Tris-HCl, pH 7.4, and 5 mM EDTA at 23°C for 1 h. For competitive binding experiments, 0.5 nM [3H]diprenorphine was incubated with 20 µg of membranes in the absence or presence of different concentration of tested compounds at 23°C for 1 h. Incubations were terminated by rapid filtration onto glass fiber filters (Whatman Schleicher and Schuell, Keene, NH). The filters were washed with 10 ml of ice-cold PBS, and the radioactivity was quantified using a liquid scintillation counter.
Data Analysis. Results are expressed as means ± S.D. for at least three experiments, each performed in duplicate. Statistical analysis and curve fits of dose-response curves were performed using Prism (GraphPad Software Inc., San Diego, CA).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-naltrexol and 6-naltrexamide have highest affinity for MOR, followed by KOR, whereas they are 20 to 30 times less potent for DOR (Table 1). The binding affinity of 6-naltrexol for MOR and KOR is 2- to 5-fold higher than naloxone and 2-fold less potent than naltrexone, whereas it is 3- to 4-fold less potent than naloxone and naltrexone for DOR (Table 1). The binding affinity of 6-naltrexamide is 3-fold more potent than naloxone and 4-fold less potent than naltrexone at MOR, whereas it is 2- to 7-fold and 7- to 10-fold less potent than naloxone at KOR and DOR, respectively (Table 1).
|
|
S binding was determined in MOR, DOR, and KOR membranes. Naloxone, naltrexone, and analogs dose-dependently inhibited agonist-stimulated [35S]GTPS binding in MOR, DOR, and KOR membranes with similar potency order as observed for binding affinity (Table 1). This result indicates that naloxone, naltrexone, 6-naltrexol, and 6-naltrexamide all act as full antagonists at each of the three opioid receptors tested (Wang et al., 2001). On the other hand, the antagonistic potency (Ki') of BNTX, ICI 174,864, and nor-BNI is greater than the [3H]diprenorphine binding affinities (Ki) (Table 1). This result may be related to their status as full inverse agonists at the respective opioid receptors (see below).
Basal Signaling Activity of MOR and the Effects of Naltrexone Analogs. We have previously reported that pretreatment of MOR with morphine increases the inverse agonist effect of -chloronaltrexamine (Wang et al., 2000, 2001). To test the effects of DAMGO pretreatment on basal activity and inverse agonist effects, we pretreated MOR with DAMGO for 24 h, followed by removal of the agonist by washing the cells thoroughly. DAMGO pretreatment increased the EC50 value of DAMGO and decreased the Emax value (Fig. 2a; Table 2), indicating receptor desensitization and down-regulation has occurred. In contrast, both DAMGO and morphine pretreatment increased inverse agonist effects of BNTX and shifted the dose-response curve of BNTX to the left, with no difference observed between morphine and the receptor-selective agonist DAMGO (Fig. 2b; Table 2). These results demonstrate that morphine and DAMGO pretreatment increases the inverse agonist effects of BNTX, implying increased basal signal activity of MOR, consistent with previous results (Wang et al., 2001), and also sensitized MOR to the inverse agonist effects of BNTX, even though the receptor was partially desensitized to agonist activation. Similar to previous results (Wang et al., 2001), naloxone and naltrexone marginally increased basal [35S]GTPS binding in untreated MOR membranes, but decreased it after DAMGO or morphine pretreatment with similar EC50 values (Table 2). This is consistent with previous observations indicating that morphine or DAMGO pretreatment turns naloxone and naltrexone into inverse agonists. In contrast, 6-naltrexol and 6-naltrexamide did not decrease basal [35S]GTPS binding even in agonist-pretreated MOR membranes (Table 2), consistent with neutral antagonism without protean property as reported previously for 6-naltrexol (Wang et al., 2001).
|
|
|
-naltrexol and 6-naltrexamide are neutral antagonists regardless of agonist pretreatment. Although part of these results have been reported previously (except for 6-naltrexamide), they are essential in this report for direct comparison with subsequent results obtained under identical conditions with DOR and KOR.
Basal Signaling Activity of DOR and the Effects of Naltrexone Analogs. Pretreatment of DOR with DPDPE has been shown to decrease the inverse agonist effect of ICI 174,864 while turning naloxone into an inverse agonist (Liu and Prather, 2002). To test whether DPDPE and morphine exposure regulates DOR basal activity differently, we pretreated HEK-DOR cells with DPDPE or morphine for 24 h, followed by washout of the agonists. Agonist pretreatment increased the EC50 value and decreased the Emax value for DPDPE (Fig. 4a; Table 3), indicating receptor desensitization and down-regulation. Different from MOR, and consistent with previous results, pre-exposure of DOR to DPDPE decreased inverse agonist effects of ICI 174,864 without changing the EC50 value (Fig. 4b; Table 3). In contrast, pretreatment of DOR with morphine shifted the dose-response curve for ICI 174,864 to the left without affecting Emax (Fig. 4b; Table 3). These results show that DPDPE and morphine regulate basal DOR activity differently. Similar to MOR, in untreated DOR membranes, naltrexone and its analogs showed small increase (8–41%) in [35S]GTPS binding, indicating partial agonist activity in this membrane preparation (Table 3). In DPDPE-pretreated membranes, naloxone decreased basal [35S]GTPS binding, indicating conversion into an inverse agonist as reported previously (Liu and Prather, 2002). Likewise, morphine pretreatment also converted naloxone into inverse agonist (Table 3). However, in contrast to the findings with MOR, naltrexone did not turn into inverse agonist at DOR after either DPDPE or morphine pretreatment (Table 3). On the other hand, 6-naltrexol and 6-naltrexamide remained neutral after both agonists pretreatment (Table 3); therefore, they are neutral antagonists regardless of agonist pretreatment in DOR, as shown for MOR. The inverse agonist effect of ICI 174,864 in untreated DOR membranes was inhibited by all four antagonists, and the inverse agonist effect of naloxone in DPDPE-pretreated membranes was inhibited by naltrexone and analogs (Fig. 5). These results for the first time distinguish the effects of naloxone from those of naltrexone.
|
|
Basal Signaling Activity of KOR and the Effects of Naltrexone Analogs. Different from MOR and DOR, basal activity for KOR had not been fully demonstrated, although one KOR antagonist was found to have inverse agonist effect on KOR in a ligand screen (Becker et al., 1999). To test whether KOR displays basal activity, we tested the effects of KOR antagonists nor-BNI and GNTI on basal [35S]GTPS binding in KOR membranes. As shown in Fig. 6, nor-BNI and GNTI dose-dependently decreased basal [35S]GTPS binding with EC50 values in the femtomolar range and an Emax value of 
10% (Table 4). These results support the existence of basal activity of KOR and indicate that nor-BNI and GNTI are inverse agonists at KOR.
|
|
To test whether agonist pretreatment changes basal activity and/or inverse agonist effects in KOR, we pretreated HEK-KOR with the KOR-selective agonist U-69593, followed by washout. Pretreatment of KOR with 1 µM U-69593 shifted the dose-response curve of U-69593 to the right and decreased Emax (Fig. 7a; Table 4), indicating KOR desensitization and down-regulation. As observed for MOR, U-69593 pretreatment of KOR increased the inverse agonist effects of nor-BNI and GNTI, and additionally, decreased their EC50 value 3- to 4-fold (Fig. 7b; Table 4). Similar results were obtained by pretreatment with another KOR-selective agonist U50,488H (Table 4). Morphine also stimulated KOR with an EC50 value of 185 nM and an Emax value similar to that of U-69593, in this membrane preparation. Pretreatment of KOR with morphine (10 or 50 µM) also increased the EC50 value for U-69593, without affecting the Emax value (Table 4). Moreover, morphine pretreatment increased the inverse agonist effects of nor-BNI and GNTI, but in contrast to U-69593 or U50,488H pretreatment, it did not alter the EC50 values (Table 4). These results show that U-69593/U50,488H and morphine pretreatment regulate basal KOR activity differently.
|
To test whether the naltrexone analogs have inverse agonist effects at KOR, we measured their effects on [35S]GTPS binding in untreated KOR membranes. As with MOR, all four compounds showed no effect or small partial agonist effects, with Emax values ranging from 10 to 40% (Table 4). We then tested the effects of these compounds on agonist-pretreated KOR membranes. Naloxone decreased basal [35S]GTPS binding in U-69593, U50,488H, and morphine-pretreated KOR membranes (Table 4), as reported for MOR. In contrast to MOR, however, 6-naltrexol decreased basal [35S]GTPS binding in U-69593- and U50,488H-, but not morphine-pretreated KOR membranes (Table 4). Naltrexone and 6-naltremaxide remained neutral regardless of agonist pretreatment. These results demonstrate that different antagonists have distinct pharmacological properties at the three opioid receptors after different agonist pretreatments. Dose-response curves showed that the EC50 values were similar for 6-naltrexol, acting as partial agonist or inverse agonists in untreated compared with pretreated membranes, indicating that the same binding sites are involved (Table 4).
Naloxone, naltrexone, and its two analogs were able to inhibit the inverse agonist effects of nor-BNI in control KOR membranes (Fig. 8). Moreover, 6-naltrexol inhibited inverse agonist effect of naloxone in morphine-pretreated KOR membranes (Fig. 8). Likewise, 6-naltrexamide inhibited inverse agonist effect of 6-naltrexol in U-69593-pretreated membranes (Fig. 8).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
In contrast to agonist-induced receptor desensitization, our results indicate that agonist-pretreatment increases basal activity and/or sensitizes inverse agonist effects at MOR, DOR, and KOR, based on the following observations (Table 5). 1) For MOR, both potency and efficacy of BNTX increased, and naloxone and naltrexone turned into inverse agonist after agonist pretreatment. 2) For KOR, agonist pretreatment increased efficacy and potency of nor-BNI and GNTI (with the exception of morphine, which did not affect potency). In addition, naloxone and 6-naltrexol turned into inverse agonist after agonist pretreatment. 3) For DOR, although we observed a decrease in efficacy of ICI 174,864 after DPDPE pretreatment (possibly a result of partial receptor down-regulation), the potency of ICI 174,864 was increased after morphine pretreatment, indicating sensitization to inverse agonism after morphine pretreatment. Moreover, both morphine and DPDPE pretreatments turned naloxone into an inverse agonist.
It seems paradoxical that basal activity and inverse agonist properties of antagonists tend to increase after agonist pretreatment, whereas agonist-stimulation is desensitized. One possibility is the unmasking of basal receptor activity, for example by shedding calmodulin from binding sites in the i3 loop involved in G protein coupling (Wang et al., 1999). Increased constitutive state of receptor probably is associated with a change in receptor conformation after agonist pretreatment. This is supported by opioid receptor mutants that have enhanced constitutive activity (Brillet et al., 2003). The different pharmacological behavior of opioid antagonists may be caused by different affinities for various receptor conformations they act on. Previous studies have shown that pretreatment with the inverse DOR agonist (ICI 174,864) produces new receptor sites with 1000-fold higher affinity for naloxone at DOR (Pineyro et al., 2005). Alternatively, basal signaling could proceed via distinct signaling pathways that are activated while agonist-stimulated pathways are down-regulated. Switching of signaling pathways has been reported between different ligands and after pretreatment (Chakrabarti et al., 2001; Dupre et al., 2004). In either case, agonist pretreatment alters the opioid receptor system, modulating both agonist-stimulated and basal signaling.
Our current results are consistent with our previous reports (Wang et al., 2000, 2001, 2004) as well as a report by Liu and Prather (2001), showing that the effect of the inverse MOR agonist -chloronaltrexamine was increased after morphine pretreatment. Our results did not show differences in inverse agonist effects between morphine and DAMGO pretreatment, inconsistent with a previous report showing DAMGO pretreatment increased inverse agonist effects more strongly than morphine (Liu and Prather, 2001). The discrepancy may be caused by the different cell culture (GH3 cell in the previous study) or by different DAMGO concentrations used for pretreatment. In this study, we have chosen concentration of agonists based on their ability to produce similar response (1 µM for DAMGO and 10 µM for morphine), whereas the previous report used the same dose (10 µM for both). Because DAMGO is known to be more efficacious than morphine, we would expect it to have greater effects on receptor regulation at equal doses. However, whereas agonist-stimulated responses are more strongly affected by DAMGO than morphine, this was not the case for the regulation of basal activity in this case. We propose that different mechanisms play a role in these two processes, an interesting possibility, because it then might be feasible to develop ligands that have differential effects and improved in vivo pharmacology. For DOR, our results are consistent with those of others (Liu and Prather, 2002), showing the effect of ICI 174,864 decreased after DPDPE pretreatment, whereas naloxone turned into an inverse agonist. The decrease in the Emax value of ICI 174,864 might have been caused by substantial receptor internalization and down-regulation after DPDPE pretreatment, which does not occur after morphine pretreatment. It is also possible that internalized receptor may not be available to the hydrophilic peptide ligand ICI 174,864. This requires further study using a nonpeptide inverse agonist of DOR, such as RTI compounds (Zaki et al., 2001). The emergence of inverse agonist properties of naloxone after DPDPE indicates that the receptor is more sensitive to the antagonist, as observed with MOR. The increased potency of ICI 174,864 after morphine pretreatment also supports a process of sensitization of inverse agonism after morphine pretreatment of DOR, similar to that of MOR (calmodulin is likely to bind to DOR as well, having an identical i3 loop).
In interpreting the results of this report, one needs to consider that transfected cell lines and membrane preparations may not reflect the true pharmacological properties encountered in vivo. Nevertheless, we have taken the results from such in vitro studies, and similar data obtained with mouse brain membrane preparations, to predict pharmacological properties in vivo. Specifically, antagonists found to be neutral even after agonist pretreatment of MOR were subsequently shown to cause significantly less withdrawal in morphine-dependent mice (Bilsky et al., 1996; Wang et al., 2001, 2004; Raehal et al., 2005). No such in vitro-in vivo correlations exist for DOR and KOR, but the present in vitro data can form a foundation for testing different opioid antagonist properties in vivo. One further needs to consider that the cell lines are expressing high levels of opioid receptors, and the membrane incubations are done under conditions that facilitate measurements of basal activity. Under these conditions, even antagonists considered devoid of agonist activity did show some stimulation of G protein coupling, as has been observed previously (Wang et al., 2001, 2004). Last, the magnitude of the measured inverse effects is typically less than agonist-stimulated effects. For the latter, one chooses conditions that minimize basal GTP binding by adding high GDP concentrations, whereas one lowers GDP levels to observe basal coupling. Yet, this increases background noise and hence reduces the percentage of decrease that can be observed for inverse agonists. Our in vivo data indicate that the basal signaling levels and inverse effects observed for MOR are relevant to measured antagonist effects in morphine-dependent mice (Wang et al., 2004). Similar relationships may hold for DOR and KOR. It will be of interest to determine how these varying antagonist properties translate into differential pharmacological properties in vivo, in particular in eliciting withdrawal, both centrally and in the peripheral nervous system.
This study also demonstrates qualitative differences between naloxone and naltrexone, both thought to represent prototypical opioid antagonists. Conversion of these two antagonists into inverse agonists at MOR is thought to underlie at least in part their potent ability to precipitate withdrawal in an opioid-dependent state (Wang et al., 2004). However, only naloxone converted into an inverse agonist at DOR and KOR, whereas naltrexone did not. Upon titrating naloxone and naltrexone dose-response curves in measuring various withdrawal effects, clear differences emerge at higher dose levels (E. J. Bilsky, unpublished data). Our results point toward developing safer and more effective opioid antagonists targeting a variety of clinical needs, including long-term treatment of addiction, and opioid-induced gastrointestinal dysfunction.
The opioid antagonist naltrexone has been used to treat opioid overdose, opioid addiction (Gonzalez et al., 2004), and addictions to other drugs of abuse, such as alcohol (Davidson et al., 1999; Chick et al., 2000). Aversive effects of naltrexone, which is similar to opioid withdrawal and occurs even in patients without pre-exposure to opioids (Hollister et al., 1981), limit its widespread use. Neutral opioid antagonists, such as 6-naltrexol, hold promise for causing less aversive effects in opioid-dependent subjects. As a metabolite of naltrexone in humans, but not in rodents, 6-naltrexol has been suggested to contribute to the long-term duration of naltrexone action in human (Cone et al., 1974; Verebey et al., 1976). Moreover, serum 6-naltrexol levels are related to alcohol responses in heavy drinkers after naltrexone administration (McCaul et al., 2000), and 6-naltrexol also reduces alcohol consumption in rats (Rukstalis et al., 2000; Stromberg et al., 2002). Although MOR is the main target receptor in narcotic analgesia and dependence, DOR and KOR also contribute to these processes, either through heterodimerization with MOR or through presynaptic/postsynaptic regulation of MOR (Narita et al., 2001; Khotib et al., 2004; Wang et al., 2005). Whether antagonists with neutral or inverse agonist property at DOR and KOR are related to side effects of antagonists in vivo requires further investigation.
|
Acknowledgements |
|---|
-naltrexol, and 6-naltrexmide were obtained from the National Institute on Drug Abuse drug supply program. |
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GPCR, G protein-coupled receptor; MOR, µ-opioid receptor; DOR, -opioid receptor; KOR, -opioid receptor; BNTX, 7-benzylidenenaltrexone; ICI 174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; nor-BNI, nor-binaltorphimine; GNTI, 5'-guanidinyl-17-(cyclopropylmethyl)-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7–2'3'-indolomorphinan dihydrochloride; U-69593, (+)-(5,7,8)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide; DPDPE, [D-Pen2,D-Pen5]-enkephalin; DAMGO, [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin; U50,488H, trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate; GTPS, guanosine 5'-O-(3-thio)triphosphate; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Nal, naloxone; 6-nal, 6-naltrexol; 6-NXM, 6-naltrexamide; ANOVA, analysis of variance.
Address correspondence to: Dr. Wolfgang Sadée, Department of Pharmacology, Graves Hall 5072, College of Medicine, The Ohio State University, 333 West 10th Ave., Columbus, OH 43210. E-mail: wolfgang.sadee{at}osumc.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Becker JA, Wallace A, Garzon A, Ingallinella P, Bianchi E, Cortese R, Simonin F, Kieffer BL, and Pessi A (1999) Ligands for -opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library. J Biol Chem 274: 27513–27522.
Bilsky EJ, Bernstein RN, Wang Z, Sadee W, and Porreca F (1996) Effects of naloxone and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 and the protein kinase inhibitors H7 and H8 on acute morphine dependence and antinociceptive tolerance in mice. J Pharmacol Exp Ther 277: 484–490.
Brillet K, Kieffer BL, and Massotte D (2003) Enhanced spontaneous activity of the mu opioid receptor by cysteine mutations: characterization of a tool for inverse agonist screening. BMC Pharmacol 3: 14.[CrossRef][Medline]
Burford NT, Wang D, and Sadee W (2000) G-protein coupling of mu-opioid receptors (OP3): elevated basal signalling activity. Biochem J 348: 531–537.[CrossRef][Medline]
Chakrabarti S, Oppermann M, and Gintzler AR (2001) Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cell signaling. Proc Natl Acad Sci USA 98: 4209–4214.
Chick J, Anton R, Checinski K, Croop R, Drummond DC, Farmer R, Labriola D, Marshall J, Moncrieff J, Morgan MY, et al. (2000) A multicentre, randomized, double-blind, placebo-controlled trial of naltrexone in the treatment of alcohol dependence or abuse. Alcohol Alcohol 35: 587–593.
Chiu CT, Ma T, and Ho IK (2006) Methamphetamine-induced behavioral sensitization in mice: alterations in mu-opioid receptor. J Biomed Sci 13: 797–811.[CrossRef][Medline]
Cone EJ, Gorodetzky CW, and Yeh SY (1974) The urinary excretion profile of naltrexone and metabolites in man. Drug Metab Dispos 2: 506–512.[Abstract]
Costa T and Herz A (1989) Antagonists with negative intrinsic activity at delta opioid receptors coupled to GTP-binding proteins. Proc Natl Acad Sci USA 86: 7321–7325.
Davidson D, Palfai T, Bird C, and Swift R (1999) Effects of naltrexone on alcohol self-administration in heavy drinkers. Alcohol Clin Exp Res 23: 195–203.[CrossRef][Medline]
Dupre DJ, Rola-Pleszczynski M, and Stankova J (2004) Inverse agonism: more than reverting constitutively active receptor signaling. Biochem Cell Biol 82: 676–680.[CrossRef][Medline]
Emmerson PJ, McKinzie JH, Surface PL, Suter TM, Mitch CH, and Statnick MA (2004) Na+ modulation, inverse agonism, and anorectic potency of 4-phenylpiperidine opioid antagonists. Eur J Pharmacol 494: 121–130.[CrossRef][Medline]
Freye E and Levy J (2005) Constitutive opioid receptor activation: a prerequisite mechanism involved in acute opioid withdrawal. Addict Biol 10: 131–137.[CrossRef][Medline]
Gbahou F, Rouleau A, Morisset S, Parmentier R, Crochet S, Lin JS, Ligneau X, Tardivel-Lacombe J, Stark H, Schunack W, et al. (2003) Protean agonism at histamine H3 receptors in vitro and in vivo. Proc Natl Acad Sci USA 100: 11086–11091.
Gonzalez G, Oliveto A, and Kosten TR (2004) Combating opiate dependence: a comparison among the available pharmacological options. Expert Opin Pharmacother 5: 713–725.[CrossRef][Medline]
Heinzen EL, Booth RG, and Pollack GM (2005) Neuronal nitric oxide modulates morphine antinociceptive tolerance by enhancing constitutive activity of the muopioid receptor. Biochem Pharmacol 69: 679–688.[CrossRef][Medline]
Herrick-Davis K, Grinde E, and Teitler M (2000) Inverse agonist activity of atypical antipsychotic drugs at human 5-hydroxytryptamine2C receptors. J Pharmacol Exp Ther 295: 226–232.
Hollister LE, Johnson K, Boukhabza D, and Gillespie HK (1981) Aversive effects of naltrexone in subjects not dependent on opiates. Drug Alcohol Depend 8: 37–41.[CrossRef][Medline]
Kenakin T (2004) Efficacy as a vector: the relative prevalence and paucity of inverse agonism. Mol Pharmacol 65: 2–11.
Khotib J, Narita M, Suzuki M, Yajima Y, and Suzuki T (2004) Functional interaction among opioid receptor types: up-regulation of mu- and delta-opioid receptor functions after repeated stimulation of kappa-opioid receptors. Neuropharmacology 46: 531–540.[CrossRef][Medline]
Liu JG and Prather PL (2001) Chronic exposure to µ-opioid agonists produces constitutive activation of µ-opioid receptors in direct proportion to the efficacy of the agonist used for pretreatment. Mol Pharmacol 60: 53–62.
Liu JG and Prather PL (2002) Chronic agonist treatment converts antagonists into inverse agonists at -opioid receptors. J Pharmacol Exp Ther 302: 1070–1079.
Liu JG, Ruckle MB, and Prather PL (2001) Constitutively active µ-opioid receptors inhibit adenylyl cyclase activity in intact cells and activate G-proteins differently than the agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin. J Biol Chem 276: 37779–37786.
McCaul ME, Wand GS, Rohde C, and Lee SM (2000) Serum 6-beta-naltrexol levels are related to alcohol responses in heavy drinkers. Alcohol Clin Exp Res 24: 1385–1391.[CrossRef][Medline]
Narita M, Funada M, and Suzuki T (2001) Regulations of opioid dependence by opioid receptor types. Pharmacol Ther 89: 1–15.[CrossRef][Medline]
Pineyro G, Azzi M, deLean A, Schiller PW, and Bouvier M (2005) Reciprocal regulation of agonist and inverse agonist signaling efficacy upon short-term treatment of the human -opioid receptor with an inverse agonist. Mol Pharmacol 67: 336–348.
Raehal KM, Lowery JJ, Bhamidipati CM, Paolino RM, Blair JR, Wang D, Sadee W, and Bilsky EJ (2005) In vivo characterization of 6-naltrexol, an opioid ligand with less inverse agonist activity compared with naltrexone and naloxone in opioid-dependent mice. J Pharmacol Exp Ther 313: 1150–1162.
Rukstalis MR, Stromberg MF, O'Brien CP, and Volpicelli JR (2000) 6--Naltrexol reduces alcohol consumption in rats. Alcohol Clin Exp Res 24: 1593–1596.[Medline]
Sadee W, Wang D, and Bilsky EJ (2005) Basal opioid receptor activity, neutral antagonists, and therapeutic opportunities. Life Sci 76: 1427–1437.[CrossRef][Medline]
Shoblock JR and Maidment NT (2006) Constitutively active mu opioid receptors mediate the enhanced conditioned aversive effect of naloxone in morphine-dependent mice. Neuropsychopharmacology 31: 171–177.[Medline]
Stromberg MF, Rukstalis MR, Mackler SA, Volpicelli JR, and O'Brien CP (2002) A comparison of the effects of 6-beta naltrexol and naltrexone on the consumption of ethanol or sucrose using a limited-access procedure in rats. Pharmacol Biochem Behav 72: 483–490.[CrossRef][Medline]
Verebey K, Volavka J, Mule SJ, and Resnick RB (1976) Naltrexone: disposition, metabolism, and effects after acute and chronic dosing. Clin Pharmacol Ther 20: 315–328.[Medline]
Walker EA and Sterious SN (2005) Opioid antagonists differ according to negative intrinsic efficacy in a mouse model of acute dependence. Br J Pharmacol 145: 975–983.[CrossRef][Medline]
Wang D, Raehal KM, Bilsky EJ, and Sadee W (2001) Inverse agonists and neutral antagonists at mu opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence. J Neurochem 77: 1590–1600.[CrossRef][Medline]
Wang D, Raehal KM, Lin ET, Lowery JJ, Kieffer BL, Bilsky EJ, and Sadee W (2004) Basal signaling activity of mu opioid receptor in mouse brain: role in narcotic dependence. J Pharmacol Exp Ther 308: 512–520.
Wang D, Sadee W, and Quillan JM (1999) Calmodulin binding to G protein-coupling domain of opioid receptors. J Biol Chem 274: 22081–22088.
Wang D, Sun X, Bohn LM, and Sadee W (2005) Opioid receptor homo- and heterodimerization in living cells by quantitative bioluminescence resonance energy transfer. Mol Pharmacol 67: 2173–2184.
Wang D, Surratt CK, and Sadee W (2000) Calmodulin regulation of basal and agonist-stimulated G protein coupling by the mu-opioid receptor (OP(3)) in morphine-pretreated cell. J Neurochem 75: 763–771.[CrossRef][Medline]
Wang Z, Bilsky EJ, Porreca F, and Sadee W (1994) Constitutive mu opioid receptor activation as a regulatory mechanism underlying narcotic tolerance and dependence. Life Sci 54: PL339–PL350.[CrossRef][Medline]
Zaki PA, Keith DE Jr, Thomas JB, Carroll FI, and Evans CJ (2001) Agonist-, antagonist-, and inverse agonist-regulated trafficking of the -opioid receptor correlates with, but does not require, G protein activation. J Pharmacol Exp Ther 298: 1015–1020.
This article has been cited by other articles:
|
|
 |
|
  S. Sirohi, P. Kumar, and B. C. Yoburn {micro}-Opioid Receptor Up-Regulation and Functional Supersensitivity Are Independent of Antagonist Efficacy J. Pharmacol. Exp. Ther., November 1, 2007; 323(2): 701 - 707. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
