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NEUROPHARMACOLOGY
Medicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (E.D.M., S.D.B., L.H.L.); College of Pharmacy, Division of Medicinal and Natural Products Chemistry, S328, University of Iowa, Ames, Iowa (Y.J.); Department of Chemistry, Jilin University, Changchun, Jilin, People's Republic of China (T.L.); and Department of Medicinal Chemistry and High Technology Research Center, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe, Japan (Y.T., Y.O.)
Received May 15, 2007; accepted July 11, 2007.
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Abstract |
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; Suzuki and Misawa, 1997; Bardo, 1998; Cowen and Lawrence, 1999; Contet et al., 2004; Kas et al., 2004). MOP knockout animals provided evidence that this receptor system is intimately involved with alcohol consumption (Hall et al., 2001; Kieffer and Gaveriaux-Ruff, 2002), and genetic analyses revealed that the propensity for drug and alcohol abuse involves a single allele of MOP (Ide et al., 2004; Zhang et al., 2005). MOP belongs to the family of G-protein -coupled receptors and, as such, displays basal signaling, which is believed to play a role in opioid tolerance and physical dependence (Wang et al., 1994, 2001, 2004; Bilsky et al., 1996; Burford et al., 2000; Freye and Levy, 2005; Sadèe et al., 2005; Shoblock and Maidment, 2006). Ligands interacting with these type of receptors display an extensive range of activities from full agonism, partial agonism, and neutral antagonism to inverse agonism (Milligan et al., 1997; Wang et al., 2001; Zaki et al., 2001; Sadèe et al., 2005; Tryoen-Toth et al., 2005; Walker and Sterious, 2005). Inhibition (antagonism) of MOP has been applied to the suppression of the neural reward system for the clinical treatment of drug and ethanol addiction behaviors, although success was limited and complicated by the appearance of withdrawal symptoms (Cowen et al., 1999; Hyytiaä et al., 1999; Srisurapanont and Jarusuraisin, 2005). Compounds that exhibit neutral antagonist activities may offer distinct advantages over clinically approved naltrexone and naloxone, which behave as inverse agonists and decrease basal signaling resulting in severe withdrawal syndromes (Bilsky et al., 1996; Cruz et al., 1996; Wang et al., 2001; Raehal et al., 2005; Sadèe et al., 2005; Walker and Sterious, 2005).
[N-Allyl-Dmt1]-endomorphin-1 (N-allyl-Dmt-Pro-Trp-Phe-NH2) and [N-allyl-Dmt1]-endomorphin-2 (N-allyl-Dmt-Pro-Phe-Phe-NH2) are new MOP antagonists obtained by N-alkylation with a single allyl group (CH2=CH-CH2-) on the amino terminus of Dmt1 (2',6'-dimethyl-L-tyrosine), a residue that enhances peptide stability, alters ligand interaction with opioid receptors, and affects bioactivity to exhibit high MOP affinity (Kiµ = 0.26–0.45 nM) and MOP antagonism (pA2 = 8.18–8.59) (Li et al., 2007). Intracerebroventricular and subcutaneous injection of [N-allyl-Dmt1]-EM-1 or -2 inhibited morphine-induced analgesia in mice, whereas [N-allyl-Dmt1]-EM-2 also inhibited ethanol-induced frequency of spontaneous inhibitory postsynaptic currents (a component of GABAA receptor-mediated neuronal function) in hippocampal slices from rat brain, suggesting its potential application in treatment of alcohol dependence (Li et al., 2007). To further characterize the properties of [N-allyl-Dmt1]-EM-1 and -2, their intrinsic activities were assessed by functional [35S]GTP
S binding and forskolin-stimulated cyclic AMP accumulation assays in cell membranes isolated from vehicle, morphine, and ethanol-treated SK-N-SH cells (as model systems of naive and opiate or ethanol-dependent conditions), and in brain membranes isolated from opioid-naive and morphine-dependent mice and compared with the effects produced by naloxone and naltrexone. The properties of all compounds were also tested in mice in a model of withdrawal from an acute morphine-dependent state.
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Materials and Methods |
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S, dithiothreitol, loperamide hydrochloride, naltrexone hydrochloride, and morphine sulfate pentahydrate were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum was from HyClone (Logan, UT), and protease inhibitor cocktail was from Roche Diagnostics (Indianapolis, IN). Naloxone hydrochloride was obtained from Tocris Bioscience (Ellisville, MO). SK-N-SH cells (HTB-11) were from American Type Culture Collection (ATCC) (Manassas, VA). [35S]GTPS and Biotrak cAMP enzyme immunoassay kit were from GE Healthcare (Little Chalfont, Buckinghamshire, UK). BCA protein assay kit was from Pierce (Rockford, IL). Cell Culture. SK-N-SH cells were cultured as monolayers in minimal essential medium with Earle's salt, nonessential amino acids, 2 mM L-glutamine, and 1 mM sodium pyruvate supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Chronic opioid treatment was conducted by the addition of morphine solution (100 mM) in phosphate-buffered saline into the incubation medium (10 µM final concentration) to the 70 to 80% confluent cells and incubated for an additional 24 h. Treatment with ethanol was conducted by incubation of cells with 200 mM ethanol incorporated into the medium for 24 h.
Cell Membrane Preparation. Cells were washed twice with ice-cold phosphate-buffered saline, harvested in ice-cold 50 mM Tris buffer, pH 7.4, containing 0.2 mM EGTA, 1 mM MgCl2 and protease inhibitor cocktail (assay buffer), and centrifuged at 1500 rpm for 5 min, 4°C. Cell pellet was resuspended in 10 ml of ice-cold Tris buffer, homogenized for 10 s (Ultra turrax T8 homogenizer, setting 7) and centrifuged at low spin (1500 rpm, 5 min) followed by high-spin centrifugation (48,800g, 20 min). Membranes were washed once with 10 ml of the ice-cold Tris buffer and resuspended in the same buffer. Protein concentration was determined with the Bradford method using bovine serum albumin as the standard. Membrane suspension was aliquoted and stored until use at –80°C.
Preparation of Mouse Brain Membranes. Six-week-old male Swiss-Webster mice were obtained from Taconic Farms (German-town, NY) and were housed in a 12-h light/dark and temperature-controlled room with free access to food and water. All animal procedures were carried out according to protocols approved by the Animal Care and Use Committee (ACUC) at the National Institute of Environmental Health Sciences. Mice were divided into two groups and treated for 3 days. One group was administered three times per day by subcutaneous injection of vehicle (saline), and the second group was given morphine (100 mg/kg). Four hours after the final dosing, mice were euthanized with CO2, and brains were quickly removed, immediately frozen in liquid nitrogen, and kept at –80°C. To prepare membranes, brains were homogenized in 1 ml/brain of ice-cold buffer (0.32 M sucrose, 10 mM HEPES, pH 7.4, 2 mM EDTA, and a protease inhibitor cocktail) in a glass homogenizer with 15 strokes of a motor-driven Teflon pestle. Homogenate was centrifuged at 1500g for 10 min at 4°C, and the pellet was discarded and supernatant was centrifuged at 50,000g for 20 min at 4°C. This high-speed pellet was rehomogenized for 10 s (Ultra turrax T8 homogenizer, setting 7) in buffer (50 mM Tris buffer, pH 7.4, 1 mM EDTA, 1 mM MgCl2, 1 mM DTT, and 100 mM NaCl) and centrifuged at 50,000g for 20 min at 4°C and resuspended in the same solution. Protein concentration was determined with the BCA kit (Pierce). The membrane suspension was aliquoted and stored until use at –80°C.
[35S]GTPS Binding Assay. Membranes (50-µg protein) were incubated with varying concentrations of opioid compounds (in duplicate) in the Tris assay buffer containing 100 mM NaCl, 30 µM GDP, and 95 pM [35S]GTPS in a total volume of 0.5 ml for 60 min at room temperature (22 ± 1°C). Nonspecific binding was determined in the presence of unlabeled GTPS (10 µM). Reaction was stopped by vacuum filtration on GF/B filters presoaked overnight in 1% bovine serum albumin solution. Filters were washed twice with ice-cold 50 mM Tris buffer, pH 7.4, and bound [35S]GTPS was quantified by liquid scintillation counting (Packard Instrument Company Inc., Meriden, CT).
cAMP Measurement. SK-N-SH cells were plated in 96-well plates at 104 cells/well in 100 µl of medium. After overnight incubation, the cell cultures were treated with fresh medium (control) or medium containing either 10 µM morphine or 200 mM ethanol. After a 24-h incubation at 37°C, the medium was aspirated, cells were washed twice with PBS, and 100 µl of serum-free medium containing 100 µM 3-isobutyl-1-methylxanthine with or without 1 µM of test compound was added. After incubation for 20 min at 37°C, forskolin was added (5 µM final concentration), and cells were incubated at 37°C for additional 15 min. Reaction was stopped by removing incubation medium and adding cell lysis buffer from the Biotrak cAMP enzyme immunoassay kit (GE Healthcare). cAMP was determined following manufacturer's protocol, and protein concentration in cell lysates was determined using the BCA kit (Pierce).
In the case of mice brain membranes, 50 µg of membrane protein was incubated with 100 µM 3-isobutyl-1-methylxanthine in assay buffer (provided with the kit) with or without 1 µM of test compound for 20 min at 37°C and followed by stimulation with 5 µM forskolin for 15 min. cAMP was determined as given above.
Receptor Binding Assay. Each compound was analyzed in duplicate using five to eight different concentrations of peptide and repeated with different membrane preparations using 400 µg of protein/assay. Unlabeled compound (2 µM) determined nonspecific binding in the presence of 3.5 nM MOP-selective ligand [3H]DAMGO (50.0 Ci/mmol, KD = 1.5 nM; GE Healthcare). After an incubation for 2.5 h at room temperature (22°C), the membrane-radioligand complex was rapidly filtered through glass fiber filters (Whatman GFC) presoaked in 0.1% polyethylenimine to enhance the signal to noise ratio of the bound radiolabeled ligand-membrane complex and washed three times with ice-cold 50 mM Tris-HCl buffered 0.1% bovine serum albumin. The affinity constants (Ki) were calculated according to Cheng and Prusoff (1973).
Dependence Studies. Four hours after administration of morphine to mice (100 mg/kg s.c. in saline), test compounds were administered (i.p.) at different doses (five mice per dose) to assess their ability to precipitate acute opioid withdrawal. Naloxone (10 mg/kg i.p. in saline) was served as the antagonist control group. Doses of 10 and 30 mg/kg endomorphin analogs were chosen as equipotent with 10 mg/kg naloxone used for the blocking of morphine's spinal and supraspinal analgesia, respectively. Immediately after administration (i.p.) of test compounds or naloxone, mice were placed in a glass beaker fitted with a filter paper on the bottom, and the following parameters were recorded for 15 min: a) vertical jumps, b) fecal boli, c) "wet dog" shakes, and d) paw tremors (Yano and Takemori, 1977; Raehal et al., 2005). Observations were performed by two independent observers who were not aware of the nature of the treatment.
To assess effect of [N-allyl-Dmt1]-endomorphins on withdrawal elicited by naloxone or naltrexone, saline or compounds (10 mg/kg) were administered by s.c. injection 20 min before administration of naloxone (30 mg/kg i.p.) or naltrexone (10 mg/kg i.p.). Mice were observed both between injection of endomorphin analogs and naloxone or naltrexone and for 15 min following naloxone and naltrexone administration.
Statistical Analysis. Results were expressed as the means ± S.E.M. Statistical comparisons between groups were performed using analysis of variance followed by Student's t test. p values less than 0.05 were considered significant.
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Results |
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). The results reported herein demonstrated that they behave as neutral antagonists (Fig. 1) and bind with high affinity to the membranes isolated from SK-N-SH cells (Ki = 0.213 ± 0.023 nM and 0.218 ± 0.033 nM for [N-allyl-Dmt1]-endomorphin-1 and -2, respectively) (Table 1). Schild regression analysis (Arunlakshana and Schild, 1959) resulted in the following pA2 values: 8.083 ± 0.105 for [N-allyl-Dmt1]-endomorphin-1; 8.239 ± 0.086 for [N-allyl-Dmt1]-endomorphin-2; 8.639 ± 0.17 for naloxone; and 8.836 ± 0.152 for naltrexone (Table 1). Both compounds produced a concentration-dependent shift to the right in the dose-response curve of MOP agonist (loperamide)-stimulated [35S]GTPS binding in SK-N-SH cell membranes, similar to the effect of naloxone and naltrexone (Fig. 2, A–D).
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[35S]GTPS Binding in Membranes from Vehicle, Morphine, and Ethanol-Treated SK-N-SH Cells. In membranes obtained from vehicle-treated SK-N-SH cells, [N-allyl-Dmt1]-endomorphin-1 and -2 behaved similarly to naloxone and naltrexone (Figs. 1 and 3); neither compound significantly changed the basal binding of [35S]GTPS when analyzed at a concentration of 1 µM([N-allyl-Dmt1]-EM-1, 7.2 ± 3.1%; [N-allyl-Dmt1]-EM-2, 3.6 ± 3.7%; naloxone 1.4 ± 4.9%; naltrexone 5.9 ± 3.1%) (Fig. 3A).
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In membranes prepared from cells treated with morphine (10 µM, 24 h), both [N-allyl-Dmt1]-endomorphin-1 and -2 did not significantly change the basal [35S]GTPS binding (3.3 ± 2.7 and 0.6 ± 2.1%, respectively), whereas naloxone and naltrexone significantly decreased [35S]GTPS binding (–12.5 ± 2.2 and –7.9 ± 2.3%, respectively), which is characteristic of inverse agonism (Fig. 3B). However, the inhibition of agonist stimulated [35S]GTPS binding in membranes from cells treated with morphine (10 µM, 24 h), the endomorphin analogs behaved similarly to naloxone and naltrexone (Fig. 4).
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S binding level (6.4 ± 2.5 and –0.2 ± 1.9%, respectively), whereas once again naloxone and naltrexone markedly inhibited basal [35S]GTPS binding (–17.9 ± 4.4 and –4.5 ± 2.7%, respectively) (Fig. 3C). Treatment of SK-N-SH cells with morphine (10 µM, 24 h), or ethanol (200 mM, 24 h) did not affect cell growth, viability, or morphology in comparison with control (data not shown).
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S binding in comparison with control values (–0.69 ± 1.5% for [N-allyl-Dmt1]-EM-1 and 0.54 ± 2.5% for [N-allyl-Dmt1]-EM-2), whereas naltrexone had a tendency to decrease (–4.5 ± 3.1%) and naloxone significantly reduced [35S]GTPS binding (–8.91 ± 2.43%) (Fig. 3D). In brain membranes from morphine-dependent mice (morphine 100 mg/kg s.c., 3 times/day for 3 days), all compounds behaved similarly to membranes from morphine-treated SK-N-SH cells; i.e., naloxone and naltrexone significantly reduced [35S]GTPS binding (–20.33 ± 3.3 and –24.65 ± 6.7% respectively) (Fig. 3E), whereas the [N-allyl-Dmt1]-endomorphins had no significant influence on [35S]GTPS binding (–2.46 ± 1.24% for [N-allyl-Dmt1]-EM-1 and 0.1 ± 1.73% for [N-allyl-Dmt1]-EM-2) (Fig. 3E). In the same membranes, both [N-allyl-Dmt1]-endomorphins reversed the inhibitory effect of naloxone and naltrexone on [35S]GTPS binding (Fig. 5).
Changes in Forskolin-Stimulated cAMP Levels. In vehicle-treated cells and brain membranes from saline-treated mice, the endomorphin analogs lacked any significant effect on forskolin-stimulated cAMP accumulation (Fig. 6, A and D). In contrast, naloxone increased forskolin-stimulated cAMP formation in mice brain membranes (122.5 ± 0.7%), which is consistent with the effect of naloxone on [35S]GTPS binding in these preparations. In morphine-treated cells and brain membranes from morphine-dependent mice, the [N-allyl-Dmt1]-endomorphins did not have a significant influence on forskolin-stimulated cAMP accumulation, further confirming a behavior consonant with action as neutral antagonists. Naloxone and naltrexone increased forskolin-stimulated cAMP accumulation in both preparations (130.7 ± 0.9% for naloxone and 126 ± 2% for naltrexone in brain membranes from morphine-dependent mice) (Fig. 6, B and E). In ethanol-treated cells (Fig. 6C), naloxone and naltrexone increased forskolin-stimulated cAMP accumulation similar to the studies with morphine-treated cells, although this effect was consistently lower (106 ± 1 for naloxone and 111 ± 1.1% for naltrexone). As seen with the morphine-treated cells, the [N-allyl-Dmt1]-endomorphins did not have any effect on forskolin-stimulated cAMP accumulation in ethanol-treated cells (Fig. 6C).
Dependence Studies in Vivo. Severe withdrawal symptoms from acute morphine administration were observed in naloxone or naltrexone-treated mice (10 mg/kg i.p.). The withdrawal was manifested as vertical jumps (19 ± 5 for naloxone and 32 ± 5 for naltrexone), increased number of fecal boli, intensive paw tremors, and "wet dog" shakes (Table 2). Animals administered [N-allyl-Dmt1]-endomorphin-1 or -2 at doses of 10 and 30 mg/kg i.p. revealed none of the effects observed with naloxone or naltrexone. Furthermore, intracerebroventricular administration of [N-allyl-Dmt1]-endomorphin-1 or -2 also completely failed to evoke any withdrawal syndrome characteristic for naloxone or naltrexone (data not shown). Moreover, subcutaneous administration of [N-allyl-Dmt1]-endomorphin-1 or -2 (10 mg/kg) before naloxone or naltrexone treatment significantly reduced the number of jumps evoked by these compounds in the withdrawal from acute morphine dependence (Fig. 7) without showing any withdrawal symptoms by themselves.
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Discussion |
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). It is noteworthy that the Ki values obtained from SK-N-SH cell membranes were essentially identical to those obtained from rat brain synaptosomes (Li et al., 2007). Whereas [N-allyl-Dmt1]-endomorphin-1 and -2 displayed properties of MOP antagonists, although with only slightly lower potency than naloxone and naltrexone, the pA2 values determined in SK-N-SH cell membranes were comparable to those obtained in guinea pig ileum functional bioassay (Li et al., 2007), verifying that SK-N-SH cell membranes can be utilized as a reliable in vitro model system for evaluation of opioid mimetic properties.
Our results extend the utility of SK-N-SH cell membranes treated with morphine (10 µM, 24 h), ethanol (200 mM, 24 h), or vehicle, as a model of opiate- and ethanol-dependent states and naive condition, respectively (Zadina et al., 1993). Ethanol acts on multiple cellular sites without interacting with a predominant or specific receptor (Thibault et al., 2005). It is known that treatment of neuronal cells with ethanol causes distinct changes in cellular biochemical mechanisms, such as cAMP accumulation, function of the cAMP-response element-binding protein to regulate genes activated by the cAMP pathway, G protein expression, and increased expression of opioid receptors (Gordon et al., 1986, 1987; Charness et al., 1988, 1993; Thibault et al., 2005). Treatment of cells with morphine causes receptor down-regulation and an increase of receptor number in a constitutively active state (Zadina et al., 1993; Wang et al., 1994). Mechanism of these adaptations involves modification of and altered associations among signaling molecules. These changes shift MOP-coupled signaling from predominantly Gi
inhibitory to G
(Gi-derived) stimulatory adenylyl cyclase signaling (Chakrabarti et al., 1998). Chronic morphine also has been shown to enhance the interaction between MOP and Gs in connection with an increased G-protein expression (Gintzler and Chakrabarti, 2006).
Antagonists interacting with constitutively active receptors may behave either as neutral antagonists or antagonists with negative intrinsic activity (inverse agonists) (Milligan et al., 1997; Wang et al., 2001; Zaki et al., 2001; Sadèe et al., 2005; Tryoen-Toth et al., 2005; Walker and Sterious, 2005). In vehicle-treated cells, representing naive conditions, it is very difficult to observe negative intrinsic activity by antagonists because there is very low pool of constitutively active receptors that may explain why all tested compounds, including naloxone, were ineffective on basal signaling in membranes from vehicle-treated cells. In these membranes, [N-allyl-Dmt1]-EM-1 and -2, naloxone, and naltrexone each at 1 µM exhibited nonsignificant stimulation of [35S]GTPS binding (Fig. 3A); however, we observed a tendency by naloxone to inhibit agonist-stimulated [35S]GTPS binding below basal level (data not shown). Under those conditions, both [N-allyl-Dmt1]-EM-1, -2, and naltrexone blocked the effect of an agonist only to the initial basal signaling level. In membranes of morphine- or ethanol-treated cells, neither [N-allyl-Dmt1]-EM-1 nor [N-allyl-Dmt1]-EM-2 exerted a significant effect on basal [35S]GTPS binding. Under these conditions, naloxone and naltrexone inhibited basal [35S]GTPS binding, which is a characteristic feature of inverse agonists.
[N-Allyl-Dmt1]-endomorphin-1 and -2 behaved strictly as neutral MOP antagonists in membranes isolated from vehicle, morphine, or ethanol-treated cells. These results are identical to those obtained in isolated mouse brain membranes in which [N-allyl-Dmt1]-endomorphin-1 and -2 behaved as neutral MOP antagonists, regardless of opioid-naive and morphine-dependent state of the mice. In contrast, both naloxone and naltrexone inhibited basal [35S]GTPS binding not only in brain membranes from morphine-dependent mice but also from naive mice. In the latter conditions, the inhibition of [35S]GTPS binding by naloxone and naltrexone was much more pronounced than in membranes from the morphine-treated cells. Despite these differences, the results obtained in both systems are highly consistent and were further confirmed by the effect on cAMP levels and, more importantly, by the in vivo withdrawal from acute morphine dependence in mice.
[N-Allyl-Dmt1]-endomorphin-1 and -2 did not produce observable, measurable, or quantifiable withdrawal symptoms at doses of 10 and 30 mg/kg i.p., 100 nmol/mice i.c.v., or 10 mg/kg s.c. in contrast to those for naloxone and naltrexone (10 mg/kg i.p.), which caused severe withdrawal symptoms manifested in vertical jumps, paw tremors, wet dog shakes, and increased number of fecal boli. Moreover, [N-allyl-Dmt1]-endomorphin-1 and -2 inhibited withdrawal symptoms elicited by naloxone and naltrexone in vivo, consistent with their reversal of inhibitory effects of naloxone and naltrexone on [35S]GTPS binding in mice brain membranes from morphine-dependent animals.
[N-Allyl-Dmt1]-endomorphin-1 inhibited ethanol-induced frequency of spontaneous inhibitory postsynaptic currents in rat hippocampal neurons (a component in the promotion of GABAA receptor-mediated neuronal function) without affecting basal signaling level (Li et al., 2007); similar data were obtained with [N-allyl-Dmt1]-endomorphin-2 (Li and Swartzwelder, unpublished data). In those neurophysiological studies, [N-allyl-Dmt1]-endomorphin-1 and -2 were effective at nanomolar concentrations, whereas the comparable inhibitory effect of naltrexone could only be achieved at micromolar concentrations (data not shown).
These results suggest that the neutral antagonists [N-allyl-Dmt1]-endomorphins may have a potential therapeutic application for the treatment and amelioration of drug addiction to opiates and the alleviation of alcohol abuse without adverse side-effects observed with compounds exhibiting inverse agonist properties, such as naloxone and naltrexone.
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Footnotes |
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Part of this work was presented at the International Narcotics Research Conference; 2006 July 9–14; St. Paul, MN.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MOP, µ-opioid peptide receptor; Dmt, 2',6'-dimethyl-L-tyrosine; EM-1, endomorphin-1; EM-2, endomorphin-2; [35S]GTPS, guanosine 5'-O-(3-[35S]thiotriphosphate); DAMGO, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin.
Address correspondence to: Dr. Ewa D. Marczak, Medicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, P.O. Box 12233, MD C304, Research Triangle Park, NC 27709. E-mail address: marczake{at}niehs.nih.gov
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