Chronic opioid antagonist treatment dose-dependently regulates μ-opioid receptors and trafficking proteins in vivo

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Chronic opioid antagonist treatment dose-dependently regulates μ-opioid receptors and trafficking proteins in vivo
  Chronic opioid antagonist treatment dose-dependently regulates m -opioid receptors and trafficking proteins in vivo Vikram Rajashekara, Chintan N. Patel, Kaushal Patel, Vishal Purohit, Byron C. Yoburn*  Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, 8000 Utopia Parkway,Queens, NY 11439, USA Received 25 March 2003; received in revised form 11 June 2003; accepted 13 June 2003 Abstract Chronic opioid antagonist treatment increases the density of   m -opioid receptors ( m OR) in many model systems. In previous studies,naltrexone treatment produced an increase in  m OR density accompanied by decreases in GRK-2 and DYN-2 protein abundance. To examinethe relationship between changes in receptor density and proteins involved in receptor trafficking, the dose-dependent effect of chronicnaloxone infusion was determined. Dose-dependent antagonism of morphine analgesia was also examined. Mice were infused with naloxone(0.1, 1.0, 5.0 mg/kg/day sc) for 7 days via osmotic pump. Controls were treated with placebo pellets. On the 7th day, morphine dose– response studies were determined using the tail flick. Other mice were sacrificed at the end of the treatment and spinal cords were collectedfor determination of   m OR density and GRK-2 and DYN-2 protein abundance. Naloxone infusion dose-dependently increased spinal  m OR density with no change in affinity. The increases in  m -receptor density were proportional to dose-dependent decreases in GRK-2 and DYN-2 protein levels. Furthermore, naloxone dose-dependently antagonized morphine. These data suggest that opioid antagonist-induced  m OR up-regulation in mouse spinal cord is associated with regulation of proteins involved in receptor trafficking and support suggestions that opioidantagonist-induced receptor up-regulation is due to reduced constitutive internalization of opioid receptors. D  2003 Elsevier Inc. All rights reserved.  Keywords:  m -Opioid receptor; Up-regulation; Trafficking proteins; Analgesia; Antagonist; Spinal cord 1. Introduction Chronic treatment with opioid receptor antagonists (e.g.,naloxone or naltrexone) increases the density of opioidreceptors in cell culture and in vivo models (Zukin andTempel, 1986; Zadina et al., 1994; Bhargava et al., 1993;Castelli et al., 1997; Yoburn et al., 1986; Duttaroy et al.,1999; Unterwald et al., 1995). Studies have demonstratedincreased  m ,  d  and  k  receptor density following chronicopioid antagonist treatment in both cell culture and in vivosystems, although up-regulation of   m -opioid receptorsappears to be of the largest magnitude (e.g., Yoburn et al.,1995; Zadina et al., 1994). Although opioid antagonist-induced up-regulation is a robust and highly reliable phe-nomenon, the mechanisms that account for the increase inopioid receptor density remain to be determined. Previousstudies have implicated proteins involved in receptor traf-ficking such as GRK, DYN and  b -arr in regulation of opioidreceptors (Patel et al., 2002b; Zhang et al., 1998; Tsao andvon Zastrow, 2001). GRKs and DYNs have been shown to play an important role in opioid agonist-induced receptor internalization in cell culture (Zhang et al., 1998; Whistler and von Zastrow, 1998). Recently, it has been proposed that these proteins also are important in opioid antagonist-induced receptor up-regulation in mouse spinal cord (Patelet al., 2002a).To date, opioid antagonist-induced regulation of traffick-ing proteins in the mouse spinal cord has been studied usingone antagonist (naltrexone) and a single chronic dosing protocol (Patel et al., 2002a). Therefore, the current study examined if regulation of trafficking proteins (GRK-2,DYN-2) occurs with a different opioid antagonist (nalox- 0091-3057/03/$ – see front matter   D  2003 Elsevier Inc. All rights reserved.doi:10.1016/S0091-3057(03)00166-7  Abbreviations:  GRK, G-protein receptor kinase; DYN, dynamin;  b -arr, b  arrestin; DAMGO, [D-Ala 2 , N-MePhe 4 , Gly 5 -ol]-enkephalin; GPCR, G- protein coupled receptor; PVDF, polyvinylidene difluoride;  m OR,  m -opioidreceptor.* Corresponding author. Tel.: +1-718-990-1623; fax: +1-718-990-6036.  E-mail address: (B.C. Yoburn), Biochemistry and Behavior 75 (2003) 909–913  one). We also examined the dose-dependent relationship between antagonism of morphine analgesia and regulationof   m OR density and trafficking protein abundance. This invivo study suggests that opioid antagonist-induced up-regulation of   m ORs depends upon regulation of intracellular  proteins implicated in receptor trafficking. 2. Materials and methods 2.1. Subjects Male, Swiss–Webster mice (22–24 g) (Taconic Farms,Germantown, NY) were used in all experiments. Animalswere housed 10 per cage for at least 24 h after arrival withfree access to food and water prior to experimentation.Each mouse was used only once. All procedures wereapproved by the St. John’s University Animal Care andUse Committee. 2.2. General procedure Osmotic minipumps (Alzet model 2001; Alza, Palo Alto,CA) that infused naloxone (0.1, 1.0, 5.0 mg/kg/day, 1  m l/h)were implanted subcutaneously in mice for 7 days. Toreduce costs, controls were implanted with an inert placebo pellet, which has no significant effect on any outcomemeasure (Duttaroy and Yoburn, 1995). The pellets and  pumps were implanted at the nape of the neck while micewere lightly anesthetized with halothane/oxygen (4:96). At the end of the 7 days naloxone or placebo treatment, micewere tested in morphine-induced analgesia assays or sacri-ficed and spinal cords were collected for receptor bindingand western blotting assays. 2.3.  m OR binding  Binding was performed as described previously (Yoburnet al., 1995). Briefly, mice ( n =12/group) were sacrificedand spinal cord removed, pooled and homogenized in 80volumes of ice-cold 50 mM Tris buffer (pH 7.4). Homoge-nates were centrifuged at 15,000 rpm (2–8   C) for 15 min,supernatants were discarded and pellets were resuspendedand incubated for 30 min at 25   C in 50 mM Tris buffer (pH7.4). Homogenates were centrifuged again and the pelletswere finally resuspended in 80 volumes of 50 mM phos- phate buffer (pH 7.2). An aliquot (200  m l) of homogenatewas assayed in triplicate in tubes containing 0.03–5 nM[ 3 H] [D-Ala 2 ,  N  -MePhe 4 , Gly 5 -ol] enkephalin (DAMGO) ( m ligand, New England Nuclear, Boston, MA). Nonspecific binding was determined in the presence of 1000 nM levor- phanol. Tubes were incubated for 90 min at 25   C and theincubation terminated by filtration of samples over GF/Bglass fiber filters (Brandel, Gaithersburg, MD). Filters werewashed three times with cold phosphate buffer and trans-ferred to vials containing scintillation cocktail and counted.Counts per minute (CPMs) were converted to disintegration per minute (DPMs) using the external standard method.Protein was assayed by the Bradford method (Bradford,1976) using reagent purchased from Bio-Rad (Richmond,CA). Independent saturation binding studies were repeated2–4 times for each dose of naloxone and controls. 2.4. Analgesia assay Analgesia testing was conducted with placebo pellets and pumps still implanted on the 7th day following the start of treatment. Analgesia was determined using the tail flick assay in which a beam of light was focused on the dorsal tailsurface approximately 2 cm from the tip of the tail. Theintensity of the light was adjusted so that baseline tail flick latencies were 2–4 s. Mice ( n = 7–9/treatment) wereinjected with 0.5 mg/kg sc morphine and tested for anti-nociception 30 min later. If a mouse failed to flick by 10 s, it was defined as analgesic. Mice that were not analgesic, wereinjected within 5 min of testing with a second dose of morphine (0.5 mg/kg) and tested again 30 min later. Testingwas continued in this manner until all mice were analgesic(cumulative dose range=0.5–5.5 mg/kg). All testing wasconducted in a blind manner. 2.5. Western-blotting assay Mice ( n =4/treatment) were sacrificed, individual spinalcords were rapidly removed on ice and homogenized(Brinkman Polytron Homogenizer, 20,000 rpm 30 s) in500  m l lysis buffer [2% SDS, 1 mM sodium orthovanadate,12.5 mM Tris (pH 7.4)], boiled for 5 min and centrifuged at 10,000 rpm (15   C) for 10 min. The supernatant wasremoved for analysis and protein concentration was deter-mined (Bradford, 1976). Samples were diluted using a mixture of equal volume of lysis and sample buffer (4%SDS, 1%  b -mercaptoethanol, 20% Glycerol, 125 mM Tris base, loading dye). An aliquot of the diluted sample (8  m l,0.25–2  m g of protein) was loaded on polyacrylamide gels(Pager Gels 10% Tris-Glycine, Biowhittaker Molecular Applications, Rockland, ME) and samples were separated by electrophoresis (150 V for 60 min). A sample from anindividual spinal cord was loaded on each lane. Proteinswere transferred to Immobilon-P PVDF membranes (Milli- pore, Bedford, MA) using the miniprotean II (Bio-Rad) at 100 V for 85 min. Nonspecific binding sites on themembrane were blocked by incubation (2 h at 24   C or overnight at 4   C) in blocking buffer (0.2% Aurora Block-ing Reagent; 1X Phosphate Buffered Saline: 0.058 M Na 2 HPO 4 , 0.017 M NaH 2 PO 4,  0.068 M NaCl; 0.05%Tween-20 from ICN Biomedicals, Costa Mesa, CA) fol-lowed by incubation (1 h, 24   C) with primary antibody in blocking buffer (Rabbit polyclonal IgG for GRK-2 (1:200);Goat polyclonal IgG for DYN-2 (1:300), Santa Cruz Bio-technology, Santa Cruz, CA). Membranes were washedtwice with blocking buffer and then incubated (1 h, 24 V. Rajashekara et al. / Pharmacology, Biochemistry and Behavior 75 (2003) 909–913 910   C) with secondary antibody in blocking buffer (Anti-rabbit IgG-AP for GRK-2 (1:5000), ICN Biomedicals; Anti-goat IgG-AP for DYN-2 (1:5000), Santa Cruz Biotechnology).Membranes were then washed thrice with blocking buffer,followed by two quick rinses with Assay buffer(20 mM Tris-HCl, pH 9.8, 1 mM MgCl 2 ). Bands were visualized using aChemiluminescence assay (CDP Star Substrate, Novagen,Madison, WI). A standard curve using increasing amounts of spinal cord protein from controls (0.25-2.0  m g/lane) wasincluded on each gel. This allowed conversion of opticaldensity into valid estimates of percent change in protein. Alldata are expressed as percent of control. Each experiment was replicated three times with new groups of mice. 2.6. Drugs  Naloxone HCl and corresponding placebo pellets werefrom Dupont Pharmaceuticals (Wilmington, DE) and Re-search Triangle Institute (Research Triangle Park, NC). [ 3 H]DAMGO was obtained from NEN Life Sciences (Boston,MA). Naloxone was dissolved in 0.9% saline and doses areexpressed as the free base. 2.7. Data analysis Gel images were captured using a FluorChem ver 2.0Imaging System (Alpha Innotech, San Leandro, CA). Theimages were digitized and analyzed for optical density usingGelPro image analysis software (ver 3.1, Media Cybernet-ics, Silver Spring, MD). Optical densities from Western blot data were converted to protein equivalents using the stan-dard curves and evaluated using ANOVA (  P  <.05). B max and K  D  values were estimated from saturation studies usingnonlinear regression (Prism ver 3.02, Graphpad Software,San Diego, CA). Binding data were best fit by a one-sitemodel. Significant differences (  P  <.05) among the groupswere analyzed using ANOVA with appropriate post hoccomparisons. Dose–response data were analyzed by Probit analysis (Finney, 1971) using a computerized program(BLISS 21, Department of Statistics, University of Edin- burgh, Edinburgh, Scotland) that estimates ED 50 s (  P  <.05)and relative potencies. Probit analysis was used to deter-mine significant differences among relative potencies of morphine. 3. Results In saturation binding studies, chronic naloxone treatment dose-dependently increased [  F  (3,11)=14.7,  P  <.0]  m OR density in mouse spinal cord (Fig. 1) without altering affinity[  F  (3,11) = 1.7,  P  >.05; mean K  D  ± S.E.M. = 0.71 ± 0.0,0.74±0.04, 0.71±0.02 and 0.71±0.01 nM; Placebo, Nalox-one 0.1, 1.0 and 5.0 mg/kg/day, respectively]. Chronicnaloxone treatment dose-dependently decreased GRK-2and DYN-2 protein abundance in mouse spinal cord[  F  (3,17)>10.5,  P  <.01]. The standard curves for GRK-2and DYN-2 proteins were linear and included the range of optical densities employed for unknowns. A representative Fig. 1. Effects of chronic naloxone on  m OR density, GRK-2 and DYN-2 protein abundance in mouse spinal cord. Mice ( n =4/treatment in threeindependent experiments) were chronically treated with naloxone (0.1, 1.0,5.0 mg/kg/day) or placebo (P) for 7 days. At the end of treatment, micewere sacrificed and spinal cords were collected. Binding data (ControlB max ±S.E.M.=189±17 fmol/mg protein) represents the mean percent of control from two to four experiments. Protein data are mean (±S.E.M. of all experiments) percent of placebo control from three independent experiments comprising four individual spinal cords per treatment (total n =12/treatment; mean±S.E.M. protein equivalents ( m g/lane) for each placebo control group=0.61±0.06 and 0.85±0.07 for GRK-2 and DYN-2,respectively). *Significantly different from placebo and 0.1 mg/kg/daynaloxone (  P  <.05). The inset presents representative blots from standardcurves (0.25, 0.5, 1.0, 2.0 ug total protein) and blots demonstrating thedose-dependent decrease in GRK-2 and DYN-2 protein abundance.Fig. 2. Effect of chronic naloxone on morphine analgesia. Mice wereimplanted with placebo pellets or infused with naloxone (0.1, 1.0, 5.0 mg/ kg/day sc) for 7 days. On the 7th day, cumulative dose–response studieswere conducted for morphine analgesia using the tail flick. Mice weresubcutaneously administered morphine and tested for analgesia 30 minlater. Testing was continued until all mice were analgesic. The relative potency data are summarized from four experiments. The inset depictsrepresentative cumulative dose–response functions for each condition.*Significantly different from placebo by Probit analysis (  P  <.05). V. Rajashekara et al. / Pharmacology, Biochemistry and Behavior 75 (2003) 909–913  911   blot for standard curves for GRK-2 and DYN-2 is shownin the insets in Fig. 1 (mean±S.E.M.,  r  2 =.96±.01 and.96±.02, three assays GRK-2 and DYN-2, respectively).Chronic naloxone treatment dose-dependently shifted themorphine analgesia dose response curve to the right (Inset, Fig. 2) and decreased morphine relative potency(Fig. 2). 4. Discussion Opioid antagonists reliably produce  m OR up-regulationin whole animal and in cell culture (Unterwald et al., 1995,1998; Zadina et al., 1994; Patel et al., 2002a; Narita et al.,2001). However, the mechanisms that mediate opioidantagonist-induced up-regulation of   m OR have not beendetermined. Recent studies have shown that increases in m OR density and potency of   m -agonists are associated withregulation of several proteins involved in GPCR trafficking.Specifically, GRK-2 and DYN-2 protein have shown to bedecreased following chronic naltrexone treatment  (Patel et al., 2002a). To date, only one dosing protocol and oneopioid antagonist have been utilized to regulate these proteins and  m OR density in the mouse spinal cord.Therefore, in this study, the effect of various infusion dosesof naloxone on GRK-2, DYN-2 and  m OR density weredetermined.The  m -opioid antagonist naloxone produced dose-depen-dent up-regulation of   m OR in mouse spinal cord and dose-dependent decreases in GRK-2 and DYN-2 protein abun-dance. At the lowest naloxone dose (0.1 mg/kg/day), therewere no significant changes in  m OR, GRK-2 or DYN-2. Inaddition, the lowest dose of chronic naloxone was ineffec-tive in antagonizing subcutaneous morphine analgesia. Withincreasing naloxone dose,  m OR density was up-regulated in parallel with decreases in GRK-2 and DYN-2 and signifi-cant antagonism of morphine potency.Previous reports suggest that opioid antagonist-inducedup-regulation in vivo may be due to inhibition of consti-tutive internalization (Patel et al., 2002a). Similarly, studies indicate that antagonist-induced up-regulation of some  m OR splice variants and mutants depend on proteins involved inGPCR trafficking and interference with constitutive inter-nalization (Koch et al., 2001; Li et al., 2001). The current  results are consistent with these data. The reduction inGRK-2 and DYN-2 protein abundance observed in the present study may be required for   m OR up-regulation;much like opioid agonist induced  m OR down-regulationdepends upon these trafficking proteins (Whistler and vonZastrow, 1998; Zhang et al., 1998; Patel et al., 2002b).Finally, there was no significant regulation of GRK-2 or DYN-2 or up-regulation of   m OR density at the lowest naloxone infusion dose. Since the low naloxone dose didnot antagonize opioid agonist effects, it may be the casethat regulation of proteins implicated in receptor traffickingand  m OR up-regulation requires a minimal level of receptor occupancy by opioid antagonists. It should be noted that Diaz et al. (2002) reported increases in GRKs followingantagonist treatment in rat brain. The apparent difference between those results and the present data may be due tothe 24-h interval between treatment and protein analysis.Studies have shown that opioid antagonist-induced up-regulation declines rapidly following treatment  (Tempel et al., 1982; Yoburn and Inturrisi, 1988). It is possible that GRK levels rebound above control soon after the end of treatment.In summary, the current data indicate that there is a closeassociation between opioid antagonist-induced up-regula-tion of   m OR and decreases GRK-2 and DYN-2 proteinabundance. These results support suggestions that chronicopioid antagonist induced up-regulation may depend uponinhibition of constitutive cycling of   m OR in mouse spinalcord. Acknowledgements The authors wish to thank Minesh Patel for patience andguidance throughout the course of these experiments. Wethank Dr. Tom Turnock, again.This work was supported by National Institutes of HealthGrant DA 12868. These data represent a portion of a thesis presented by Vikram Rajashekara to the faculty of theCollege of Pharmacy and Allied Health Professions, St.John’s University, in partial fulfillment of the requirementsfor the M.S. degree in Pharmaceutical Sciences. References Bhargava HN, Matwyshyn GA, Reddy PL, Veeranna. Effects of naltrexoneon the binding of [3H] D-Ala2, MePhe4, Gly-ol5-enkephalin to brainregions and spinal cord and pharmacological responses to morphine inthe rat. Gen Pharmacol 1993;24:1351–7.Bradford MM. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem 1976;72:248–54.Castelli MP, Melis M, Mameli M, Fadda P, Diaz G, Gessa GL. Chronicmorphine and naltrexone fail to modify mu-opioid receptor mRNAlevels in the rat brain. Brain Res Mol Brain Res 1997;45:149–53.Diaz A, Pazos A, Florez J, Ayesta FJ, Santana V, Hurle MA. 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