Localization of opioid receptor antagonist [H]-LY255582 binding sites in mouse brain: Comparison with the distribution of mu, delta and kappa binding sites

Agonist stimulation of opioid receptors increases feeding in rodents, while opioid antagonists inhibit food intake. The pan-opioid antagonist, LY255582, produces a sustained reduction in food intake and body weight in rodent models of obesity. However, the specific receptor subtype(s) responsible for this activity is unknown. To better characterize the pharmacology of LY255582, we examined the binding of a radiolabeled version of the molecule, [(3)H]-LY255582, in mouse brain using autoradiography. In mouse brain homogenates, the K(d) and B(max) for [(3)H]-LY255582 were 0.156 +/- 0.07 nM and 249 +/- 14 fmol/mg protein, respectively. [(3)H]-LY255582 bound to slide mounted sections of mouse brain with high affinity and low non-specific binding. High levels of binding were seen in areas consistent with the known localization of opioid receptors. These areas included the caudate putamen, nucleus accumbens, claustrum, medial habenula, dorsal endopiriform nucleus, basolateral nucleus of the amygdala, hypothalamus, thalamus and ventral tegmental area. We compared the binding distribution of [(3)H]-LY255582 to the opioid receptor antagonist radioligands [(3)H]-naloxone (mu), [(3)H]-naltrindole (delta) and [(3)H]-norBNI (kappa). The overall distribution of [(3)H]-LY255582 binding sites was similar to that of the other ligands. No specific [(3)H]-LY255582 binding was noted in sections of mu-, delta- and kappa-receptor combinatorial knockout mice. Therefore, it is likely that LY255582 produces its effects on feeding and body weight gain through a combination of mu-, delta- and kappa-receptor activity.     

Localization of opioid receptor antagonist [3H]-LY255582 binding sites in mouse brain: comparison with the distribution of mu, delta and kappa binding sites

Agonist stimulation of opioid receptors increases feeding in rodents, while opioid antagonists inhibit food intake. The pan-opioid antagonist, LY255582, produces a sustained reduction in food intake and body weight in rodent models of obesity. However, the specific receptor subtype(s) responsible for this activity is unknown. To better characterize the pharmacology of LY255582, we examined the binding of a radiolabeled version of the molecule, [(3)H]-LY255582, in mouse brain using autoradiography. In mouse brain homogenates, the K(d) and B(max) for [(3)H]-LY255582 were 0.156 +/- 0.07 nM and 249 +/- 14 fmol/mg protein, respectively. [(3)H]-LY255582 bound to slide mounted sections of mouse brain with high affinity and low non-specific binding. High levels of binding were seen in areas consistent with the known localization of opioid receptors. These areas included the caudate putamen, nucleus accumbens, claustrum, medial habenula, dorsal endopiriform nucleus, basolateral nucleus of the amygdala, hypothalamus, thalamus and ventral tegmental area. We compared the binding distribution of [(3)H]-LY255582 to the opioid receptor antagonist radioligands [(3)H]-naloxone (mu), [(3)H]-naltrindole (delta) and [(3)H]-norBNI (kappa). The overall distribution of [(3)H]-LY255582 binding sites was similar to that of the other ligands. No specific [(3)H]-LY255582 binding was noted in sections of mu-, delta- and kappa-receptor combinatorial knockout mice. Therefore, it is likely that LY255582 produces its effects on feeding and body weight gain through a combination of mu-, delta- and kappa-receptor activity.     

Mercaptoacetate induces feeding through central opioid-mediated mechanisms in rats

The endogenous opioid system has been implicated in the mediation of food intake elicited by such regulatory challenges as glucoprivation induced by 2-deoxy-D-glucose (2DG) or food deprivation in rodents. Administration of the free fatty acid oxidation inhibitor, mercaptoacetate (MA), produces a potent short-term increase in feeding in rats, the mechanisms of which have been dissociated from that elicited by 2DG. The present study evaluated whether MA-induced feeding in rats was mediated by the endogenous opioid system through systemic administration of the general opioid antagonist, naltrexone, through central administration of either general, mu, mu(1), kappa(1) or delta opioid antagonists, and through central administration of antisense oligodeoxynucleotide (AS ODN) probes directed against specific exons of either the mu (MOR-1), kappa (KOR-1), kappa(3) (KOR-3/ORL-1) or delta (DOR-1) opioid receptor clones. MA-induced feeding was significantly and dose-dependently reduced by systemic naltrexone (0.005-5 mg/kg); these ingestive effects were quite selective since neither total, ambulatory nor stereotypic activity was affected by either MA itself or MA paired with naltrexone. MA-induced feeding was significantly reduced by central pretreatment with either naltrexone (0.1-20 microgram) or mu-selective (beta-funaltrexamine, 0.1-20 microgram), mu(1)-selective (naloxonazine, 1-20 microgram), kappa(1)-selective (nor-binaltorphamine, 0.1-20 microgram), or delta-selective (naltrindole, 1-20 microgram) opioid receptor antagonists. MA-induced feeding was significantly reduced by AS ODN probes directed against either exons 1, 2 or 3, but not exon 4 of the MOR-1 clone, exon 3, but not exons 1 or 2 of the KOR-1 clone, exons 1 or 2, but not exon 3 of the KOR-3/ORL-1 clone, and exon 1, but not exons 2 or 3 of the DOR-1 clone. These data are discussed in terms of opioid mediation of ingestive responses related to fat, and in terms of potential central sites of action at which lipoprivic ingestive responses might act.     

Reduction by central beta-funaltrexamine of food intake in rats under freely-feeding, deprivation and glucoprivic conditions

The present study evaluated the central effects of beta-funaltrexamine (B-FNA), a non-equilibrium antagonist of mu-opioid receptors and a reversible agonist of kappa-opioid receptors upon food intake in rats under freely-feeding, deprivation and glucoprivic conditions. B-FNA elicited distinct short-term and long-term actions, consistent with binding studies demonstrating its reversible kappa agonist actions and its irreversible mu receptor blockade. Whereas B-FNA (1-20 micrograms, i.c.v.) significantly stimulated free feeding for up to 6 h, B-FNA (10-20 micrograms) significantly inhibited (35-41%) free feeding at 24, 48 and 72 h after injection, a pattern temporally similar to its biochemical opioid effects. Pretreatment (24 h) with B-FNA (10-20 micrograms) significantly inhibited (33-49%) the increased intake following 24 h of food deprivation. Pretreatment (24 h) with B-FNA (10-20 micrograms) also significantly inhibited (75-100%) the increased glucoprivic intake induced by 2-deoxy-D-glucose. The short-term stimulation of food intake by central B-FNA was antagonized by the selective kappa antagonist, nor-binaltorphamine, but was unaffected by pretreatment 24 h earlier with the mu antagonist, B-FNA. Significant reductions in striatal (89%) and hypothalamic (46%) mu-opioid binding occurred in rats pretreated (24 h) with B-FNA; the low levels of delta binding in these structures precluded interpretation of B-FNA effects. These data indicate the importance of the mu-opioid receptor in the modulation of different forms of feeding behavior, and underscores the ability of selective opioid antagonists to delineate precise functional roles for different opioid receptor subtypes.     

Opioid receptor subtype antagonists differentially alter GABA agonist-induced feeding elicited from either the nucleus accumbens shell or ventral tegmental area regions in rats

Food intake is significantly increased by administration of either GABAA (e.g., muscimol) or GABAB (e.g., baclofen) agonists into either the shell region of the nucleus accumbens (NAC) or the ventral tegmental area (VTA); these responses are selectively blocked by pretreatment with corresponding GABAA and GABAB antagonists. Previous studies found that a single dose (5 microg) of the general opioid antagonist, naltrexone reduced feeding elicited by muscimol, but not baclofen in the NAC shell, and reduced feeding elicited by baclofen, but not muscimol in the VTA. The present study compared feeding responses elicited by either muscimol or baclofen in either the VTA and NAC shell following pretreatment with equimolar doses of selective mu (0.4, 4 microg), delta (0.4, 4 microg), or kappa (0.6, 6 microg) opioid receptor subtype antagonists. Muscimol (25 ng) and baclofen (200 microg) each significantly and equi-effectively increased food intake over 4 h following VTA or NAC shell microinjections. Muscimol-induced feeding elicited from the VTA was significantly enhanced by mu or delta antagonists, and was significantly reduced by kappa antagonists. Baclofen-induced feeding elicited from the VTA was significantly reduced by mu or kappa, but not delta antagonists. Muscimol-induced feeding elicited from the NAC was significantly reduced by either mu, kappa or delta antagonists. Baclofen-induced feeding elicited from the NAC was significantly reduced by kappa or delta, but not mu antagonists. These data indicate differential opioid receptor subtype antagonist-induced mediation of GABA receptor subtype agonist-induced feeding elicited from the VTA and NAC shell. This is consistent with previously demonstrated differential GABA receptor subtype antagonist-induced mediation of opioid-induced feeding elicited from these same sites. Thus, functional relationships exist for the elaborate anatomical and physiological interactions between these two neurochemical systems in the VTA and NAC shell.     

Cholecystokinin antagonists proglumide, lorglumide and benzotript, but not L-364,718, interact with brain opioid binding sites

It has been reported that proglumide and L-364,718 potentiate opioid-induced antinociception. However, their mode of action in pain modulation is still not understood. To evaluate a possible interaction with opioid receptors, we determined the affinities of the CCK antagonists proglumide, lorglumide, benzotript and L-364,718 on mu, delta and kappa binding sites, using guinea pig brain crude synaptosome preparations. These affinities were compared to that of the central CCK binding site, using rat brain slide-mounted sections. At 100 microM, proglumide competed for 13% and 17% of mu and kappa binding sites, but did not interact with delta and CCK sites. At this concentration, lorglumide reduced mu, delta, kappa and CCK specific binding by 44%, 69%, 35% and 88%, whereas benzotript diminished it by 16%, 13%, 38% and 48%, respectively. L-364,718 did not interact with opioid receptors (assay limit of solubility, 10 microM) but had a high affinity for CCK binding sites (IC50, 126nM). The lack of selectivity of proglumide, lorglumide and benzotript for CCK receptors suggests that their reported ability to potentiate morphine analgesia may be related to an interaction with opioid receptors.     

gamma-Aminobutyric acid receptor subtype antagonists differentially alter opioid-induced feeding in the shell region of the nucleus accumbens in rats.

Food intake is significantly increased by administration of mu-selective opioid agonists into the nucleus accumbens, particularly its shell region. Pretreatment with either opioid (mu, delta(1), delta(2) or kappa(1)) or dopaminergic (D(1)) receptor antagonists in the nucleus accumbens shell reduce mu opioid agonist-induced feeding. Selective GABA(A) (muscimol) and GABA(B) (baclofen) agonists administered into the nucleus accumbens shell each stimulate feeding which is respectively and selectively blocked by GABA(A) (bicuculline) and GABA(B) (saclofen) antagonists. The present study investigated whether feeding elicited by the mu-selective opioid agonist, [D-Ala(2),NMe(4),Gly-ol(5)]-enkephalin in the nucleus accumbens shell was decreased by intra-accumbens pretreatment with an equimolar dose range of either GABA(A) or GABA(B) antagonists, and further, whether general opioid or selective GABA antagonists decreased feeding elicited by GABA(A) or GABA(B) agonists in the nucleus accumbens shell. Feeding elicited by the mu-selective opioid agonist was dose-dependently increased following intra-accumbens pretreatment with GABA(A) (bicuculline) antagonism; this enhancement was significantly blocked by pretreatment with general or mu-selective opioid antagonists. In contrast, mu opioid agonist-induced feeding elicited from the nucleus accumbens shell was dose-dependently decreased by GABA(B) (saclofen) antagonism. Neither bicuculline nor saclofen in the nucleus accumbens shell altered baseline food intake. Whereas muscimol-induced feeding elicited from the nucleus accumbens shell was reduced by bicuculline and naltrexone, but not saclofen pretreatment, baclofen-induced feeding elicited from the nucleus accumbens shell was reduced by saclofen, but not by bicuculline or naltrexone. These data indicate that GABA(A) and GABA(B) receptor subtype antagonists differentially affect feeding elicited by mu opioid receptor agonists within the nucleus accumbens shell in rats.     

Naloxone benzoylhydrazone, a kappa 3 opioid agonist, stimulates food intake in rats.

Naloxone benzoylhydrazone (NalBzoH) is a selective, short-acting agonist at the kappa 3 opioid receptor and a slowly dissociating potent antagonist at the mu opioid receptor. Given the important role of kappa receptors in the opioid control of food intake, the present study examined the central and peripheral effects of NalBzoH upon food intake. Central administration of NalBzoH (1-20 micrograms, i.c.v.) significantly increased food intake for up to 12 h, but failed to alter intake or body weight after 24 or 48 h. The 12 h duration of NalBzoH-mediated effects may be due to either persistent kappa 3 receptor occupancy, and/or activation of an ingestive system which maintains its activity. Peripheral administration of NalBzoH (20 mg/kg, s.c.) significantly increased food intake for up to 1 h. To distinguish kappa 1 (U50,488H) and kappa 3 (NalBzoH) hyperphagic effects, these agonist effects were compared following pretreatment with either naltrexone or the kappa 1 antagonist, nor-binaltorphamine (Nor-BNI). Whereas naltrexone significantly reduced both U50,488H and NalBzoH hyperphagia, Nor-BNI blocked U50,448H, but not NalBzoH hyperphagia. These data indicate a distinct role for the kappa 3 receptor in ingestive behavior separable from that of kappa 1 effects.     

Reductions in body weight following chronic central opioid receptor subtype antagonists during development of dietary obesity in rats

Acute administration of long-acting general opioid antagonists reduces body weight and food intake in rats. In contrast, chronic administration of short-acting general opioid antagonists produces transient effects. The present study evaluated whether chronic central administration of selective long-acting antagonists of mu (beta-funaltrexamine, BFNA, 20 μg), mu₁ (naloxonazine, 50 μg), delta₁([d-Ala²,Leu⁵,Cys⁶]-enkephalin, DALCE, 50 μg), delta₂ (naltrindole isothiocyanate, NTII, 20 μg) or kappa (nor-binaltorphamine, NBNI, 20 μg) opioid receptor subtypes altered weight and intake of rats exposed to a palatable diet of pellets, fat, milk and water, relative to pellet-fed and diet-fed controls. Diet-fed rats receiving chronic vehicle injections significantly increased weight (7–10%) and intake over the 11-day time course. Weight was significantly reduced over the time course in rats administered either BFNA (9%), naloxonazine (12%), DALCE (7%) or NTII (6%). Initial weight reductions failed to persist following chronic NBNI. All antagonists chronically reduced fat intake, but did not systematically alter total intake, pellet intake or milk intake relative to the pattern of weight loss. These data indicate that central mu, mu₁, delta₁, delta₂, and, to a lesser degree, kappa receptors mediate long-term opioid modulation of weight even in animals maintained on diets that ultimately result in dietary obesity.     

Analysis of central opioid receptor subtype antagonism of hypotonic and hypertonic saline intake in water-deprived rats

Intake of either hypotonic or hypertonic saline solutions is modulated in part by the endogenous opioid system. Morphine and selective mu and delta opioid agonists increase saline intake, while general opioid antagonists reduce saline intake in rats. The present study evaluated whether intracerebroventricular administration of general (naltrexone) and selective mu (beta-funaltrexamins, 5–20 μg), mu₁ (naloxonazine, 50 μg), kappa (nor-binaltorphamine, 5–20 μg), delta (naltrindole, 20 μg), or delta, (DALCE, 40 μg) opioid receptor subtype antagonists altered water intake and either hypotonic (0.6%) or hypertonic (1.7%) saline intake in water-deprived (24 h) rats over a 3-h time course in a two-bottle choice test. Whereas peripheral naltrexone (0.5–2.5 mg/kg) significantly reduced water intake and hypertonic saline intake, central naltrexone (1–50 μg) significantly reduced water intake and hypotonic saline intake. Water intake was significantly reduced following mu and kappa receptor antagonism, but not following mu₁, delta, or delta₁ receptor antagonism. In contrast, neither hypotonic nor hypertonic saline intake was significantly altered by any selective antagonist. These data are discussed in terms of opioid receptor subtype control over saline intake relative to the animal's hydrational state and the roles of palatability and/or salt appetite.     

Methionine-enkephalin modulation of hydrogen peroxide (H2O2) release by rat peritoneal macrophages involves different types of opioid receptors

We investigated the involvement of specific types of opioid receptors in methionine-enkephalin (MET)-induced modulation of hydrogen peroxide (H2O2) release by rat macrophages primed with sub-optimal concentrations of phorbol myristate acetate (PMA). Peritoneal macrophages in vitro treated with different concentrations of MET were tested for H2O2 release in phenol red assay. In the antagonistic study macrophages were treated with MET and one opioid receptor antagonist, or combination of MET and two or three opioid receptor antagonists. MET decreased H2O2 release in eight individual macrophage samples, and increased it in 10 samples. The increase of H2O2 release induced by MET in macrophages was blocked with combination of opioid receptor antagonists specific delta1,2 and mu receptors, as well as with combination of antagonists specific for delta1,2 and kappa opioid receptors. MET-induced decrease of the H2O2 release in macrophages was prevented by opioid receptor antagonists specific for delta1,2 or mu receptors, and also with combination of two or three opioid receptor antagonists. MET-induced enhancement of H2O2 release was mediated via delta1 or delta2 opioid receptor subtypes, or by mu-kappa opioid receptor functional interactions, while MET-induced suppression involved functional interactions between delta1 and mu, delta2 and mu, or delta1 and kappa opioid receptors. It is possible that individual differences in basal or induced macrophage capacity to produce H2O2 might shape the repertoire of opioid receptors expression and in that way pre-determine the direction of MET-induced changes after the in vitro treatment.     

Central opioid receptor subtype antagonists differentially alter sucrose and deprivation-induced water intake in rats.

The present study compared the effectiveness of centrally-administered opioid receptor subtype antagonists to inhibit intake of either a 10% sucrose solution under ad libitum conditions, or water following 24 h of water deprivation. Full dose-response functions were evaluated over a 1 h period for the following antagonists: naltrexone (general: 1-50 micrograms), nor-binaltorphamine (Nor-BNI, kappa: 1-20 micrograms), beta-funaltrexamine (beta-FNA, mu: 1-20 micrograms), naltrindole (delta 2: 1-20 micrograms), [D-Ala2, Leu5, Cys6]-enkephalin (DALCE, delta 1: 10-40 micrograms) and naloxonazine (mu 1: 10-50 micrograms). Naltrexone significantly and dose-dependently inhibited both sucrose intake (64-67%) and deprivation-induced water intake (53-67%). Nor-BNI significantly and dose-dependently inhibited sucrose intake (53-55%), but failed to significantly affect (28%) deprivation-induced water intake. beta-FNA significantly and dose-dependently inhibited both sucrose intake (31-34%) and deprivation-induced water intake (36-50%). Naltrindole failed to significantly alter either sucrose intake (24%) or deprivation-induced water intake (16%). Whereas DALCE significantly, but transiently (15-20 min) inhibited sucrose intake (28%), it failed to significantly alter deprivation-induced water intake (14%). Naloxonazine significantly, but transiently (5-10 min) stimulated sucrose intake at low doses (26%), but non-significantly reduced sucrose intake at higher doses (20%). Naloxonazine failed to significantly alter deprivation-induced water intake (16% reduction). These data indicate that whereas the kappa and mu 2 binding sites participate in the opioid modulation of sucrose intake, the mu 2 binding site participates in the opioid modulation of deprivation-induced water intake.     

Selective opioid agonist and antagonist competition for [3H]-naloxone binding in amphibian spinal cord

Opioids elicit antinociception in mammals through three distinct types of receptors designated as mu, kappa and delta. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to characterize the site(s) of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid antagonist that has not been characterized in Rana pipiens. Using the non-selective opioid antagonist, [3H]-naloxone, opioid binding sites were characterized in amphibian spinal cord. Competitive binding assays were done using selective opioid agonists and highly-selective opioid antagonists. Naloxone bound to a single-site with an affinity of 11.3 nM and 18.7 nM for kinetic and saturation studies, respectively. A B(max) value of 2725 fmol/mg protein in spinal cord was observed. The competition constants (K(i)) of unlabeled mu, kappa and delta ranged from 2.58 nM to 84 microM. The highly-selective opioid antagonists yielded similar K(i) values ranging from 5.37 to 31.1 nM. These studies are the first to examine opioid binding in amphibian spinal cord. In conjunction with previous behavioral data, these results suggest that non-mammalian vertebrates express a unique opioid receptor which mediates the action of selective mu, kappa and delta opioid agonists.     

Intra-amygdalar injection of DAMGO: effects on c-Fos levels in brain sites associated with feeding behavior

It is well known that the mu opioid agonist, Tyr-D-Ala-Gly-(me) Phe-Gly-ol (DAMGO), increases food intake in rats when injected into a variety of brain sites including the central nucleus of the amygdala (CeA). Immunohistochemical studies measuring c-Fos immunoreactivity (IR) suggest that the CeA contributes to opioid-related feeding. In the current study, we injected 2 nmol of DAMGO and measured food intake, c-Fos IR levels in various brain sites involved in feeding behavior, and mu opioid receptor internalization. We also studied the effect of CeA-injected DAMGO on LiCl-induced increases in c-Fos IR in the amygdala. As was expected, intra-CeA injection of DAMGO increased food intake of rats over a 4-h period. DAMGO injection into the CeA also resulted in mu opioid receptor internalization in the CeA, indicating activation of mu opioid receptor expressing neurons in this site. Administration of DAMGO into the CeA increased c-Fos IR levels in the shell of the nucleus accumbens (NAcc), but not in 17 other brain sites that were studied. We also found that intra-CeA injection of DAMGO, prior to LiCl injection, decreased c-Fos IR levels in the CeA compared to vehicle-injected rats. Thus, intra-CeA administration of DAMGO may increase feeding, in part, by activating neurons in the shell of the nucleus accumbens and by inhibiting activity of selected neurons in the CeA.     

Opioid research in amphibians: an alternative pain model yielding insights on the evolution of opioid receptors

This review summarizes the work from our laboratory investigating mechanisms of opioid analgesia using the Northern grass frog, Rana pipiens. Over the last dozen years, we have accumulated data on the characterization of behavioral effects after opioid administration on radioligand binding by using opioid agonist and antagonist ligands in amphibian brain and spinal cord homogenates, and by cloning and sequencing opioid-like receptor cDNA from amphibian central nervous system (CNS) tissues. The relative analgesic potency of mu, delta, and kappa opioids is highly correlated between frogs and other mammals, including humans. Radioligand binding studies using selective opioid agonists show a similar selectivity profile in amphibians and mammals. In contrast, opioid antagonists that are highly selective for mammalian mu, delta, and kappa opioid receptors were not selective in behavioral and binding studies in amphibians. Three opioid-like receptor cDNAs were cloned and sequenced from amphibian brain tissues and are orthologs to mammalian mu, delta, and kappa opioid receptors. Bioinformatics analysis of the three types of opioid receptor cDNAs from all vertebrate species with full datasets gave a pattern of the molecular evolution of opioid receptors marked by the divergence of mu, delta, and kappa opioid receptor sequences during vertebrate evolution. This divergence in receptor amino acid sequence in later-evolved vertebrates underlies the hypothesis that opioid receptors are more type-selective in mammals than in nonmammalian vertebrates. The apparent order of receptor type evolution is kappa, then delta, and, most recently, the mu opioid receptor. Finally, novel bioinformatics analyses suggest that conserved extracellular receptor domains determine the type selectivity of vertebrate opioid receptors.     

Pre- and postnatal development of opiate receptor subtypes in rat spinal cord

We have studied the developmental expression of opiate binding sites in the rat spinal cord at various prenatal and postnatal stages. For each developmental stage, we have compared the expression pattern of kappa receptors with that of mu and delta receptor subtypes. Both mu and kappa receptors appear relatively early during spinal cord ontogeny (from the 15th prenatal day), while delta sites are expressed later at the postnatal period (starting at the 1st postnatal day). The number of kappa sites predominates throughout the development (55-80% of total opiate sites) with two peaks of binding activity: one at the 20th gestational day, and the other around the 7th postnatal day. mu sites represent 20-38% of the total opiate receptor population with one peak of binding activity appearing at the 1st postnatal day. The densities of mu and kappa receptors at the adult stage are lower by 40-50% than the peak values observed at the early postnatal periods. The relative amounts of delta sites remain low throughout the ontogeny (4-8% of the total opiate sites). The binding properties of neonatal (1 day after birth) kappa sites (ligand binding affinities, regulation of agonist binding by guanosine triphosphate and various cations) are similar to those displayed by kappa receptors in adult spinal cord.     

Analysis of opioid receptor subtype antagonist effects upon mu opioid agonist-induced feeding elicited from the ventral tegmental area of rats

The present study examined opioid receptor(s) mediation of feeding elicited by mu opioid agonists in the ventral tegmental area using general or selective opioid antagonist pretreatment. Naltrexone as well as equimolar doses of selective mu and kappa, but not delta opioid antagonists in the ventral tegmental area significantly reduced mu agonist-induced feeding, indicating a pivotal role for these receptor subtypes in the full expression of this response.     

Paradoxical and subtype-specific effects of opiate antagonists on the expression of opioid receptors in rat brain cultures

Cultures of aggregating fetal rat brain cells express mu, delta, and kappa opioid receptors. The potent and long-lasting opioid antagonist naltrexone was used to investigate whether different regulatory mechanisms are involved in the expression of the three receptor subtypes. In cultures treated for seven days, naltrexone increased dose-dependently the binding of 3H-diprenorphine to the three receptor subtypes, with the mu sites being affected at a lower concentration than the other two; A Scatchard analysis indicated that this receptor up-regulation was obtained by an increase in the BMax, with no significant change in the affinity of the ligand to the receptors. In contrast to these effects in cultures treated for 7 days, it was surprising to find that a 48 hr treatment with naltrexone had an apparent converse and subtype-specific influence; the antagonist decreased significantly the binding of 3H-diprenorphine to both mu and delta receptors but had no effect on kappa sites. Two other opioid antagonists, naloxone and levallorphan, had a similar effect. Further analysis of naltrexone's mode of action was obtained by studying its effect on the adenylate cyclase activity. Of several inducers of this enzyme, the beta-adrenergic inducer isoproterenol gave the highest increase in cyclic AMP. Naltrexone had no significant effect on the basal adenylate cyclase activity but it altered the pattern of cyclic AMP formation in isoproterenol-stimulated cultures. Overall, the results indicate that in addition to its classic antagonistic activity, naltrexone exhibits in fetal brain aggregates some properties associated with opiate agonists.     

Methionine-enkephalin stimulates hydrogen peroxide and nitric oxide production in rat peritoneal macrophages: interaction of mu, delta and kappa opioid receptors

OBJECTIVE: Methionine-enkephalin (MET) modulates various functions of macrophages related to both immune and inflammatory reactions in a naloxone reversible manner, suggesting that opioid receptors are involved in the regulation of macrophage activity. Since an endogenous opioid ligand might interact with more than one type of opioid receptor, the receptor interaction determines its effect on a particular function. METHODS: In the present study we have investigated the involvement of different opioid receptor types/subtypes in MET-induced modulation of H(2)O(2) and NO production in macrophages. Thioglycollate-elicited or resident rat peritoneal macrophages were treated in vitro with MET and/or specific antagonists of delta(1,2), delta(1), delta(2), mu and kappa opioid receptors. RESULTS: MET increased H(2)O(2)production in phorbol myristate acetate-stimulated rat peritoneal macrophages mainly through delta(1) opioid receptor. MET also enhanced NO production in rat peritoneal macrophages stimulated with lipopolysaccharide through delta(1) and mu opioid receptors. The blockade of mu and kappa receptor facilitated a potentiating effect of MET on H(2)O(2) release, and blockade of kappa receptor further raised the MET-induced increase of NO production in macrophages. CONCLUSION: It is concluded that both negative and positive functional interaction between delta, mu and kappa opioid receptors regulate the influence of MET on H(2)O(2) and NO production in rat peritoneal macrophages.     

Effect of opioid receptor ligands injected into the rostral lateral hypothalamus on c-fos and feeding behavior

The lateral hypothalamic area (LHa) is an important brain site for the regulation of food intake. Central injection of opioids increases food intake, and the LHa contains mu and kappa opioid receptors, both of which are involved in feeding behavior. It is unclear whether opioids impact feeding when injected directly into the rostral portion of the LHa (rLHa) in rats. We performed a series of studies in which free-feeding rLHa-cannulated rats were injected with opioid agonists (DAMGO, morphine, dynorphin, U-50488H) followed by the measurement of food intake at 1, 2, and 4 h postinjection. To determine whether opioid receptor ligands administered into the rLHa affect neuronal activation in this brain site, we studied cFos immunoreactivity (cFos IR) in response to rLHa stimulation with naltrexone. We found that the only compound that stimulated feeding behavior was morphine. The other agonists had no effect on food consumption. Naltrexone injection into the rLHa increased neural activation in the LHa, indicating the presence of functional opioid receptors in this region. These data suggest that although neuronal activity is affected by opioid agents acting in the rLHa, administration of selective mu and kappa opioid ligands in this subdivision of the LHa does not have a reliable effect on feeding behavior.