Endogenous opioids and their receptors in stress-induced analgesia

Stress can induce a naloxone reversible and a naloxone non reversible analgesia according to its parameters. We showed that naloxone non reversible analgesia can be reversed by antagonists of the kappa opiate receptor and that naloxone reversible analgesia can be related to mu receptors and beta-endorphin, while the kappa receptor mediated analgesia can be related to dynorphin. We have also shown that the characteristics of the receptors might change in consequence to stress and that the analgesic responses might be modulated by benzodiazepine agonists and antagonists.     

Increased food and water intake produced in rats by opiate receptor agonists

It has been suggested that endogenous opiate mechanisms may be involved in the physiological control of food and water intake. Support for this hypothesis has been obtained from studies of the effects of narcotic antagonists which reduce feeding and drinking, but it is also necessary to show that food and water intake can be facilitated by opiate agonists. In the present study the food intake of freely-feeding rats was increased by subcutaneous injections of morphine, a stabilised enkephalin analogue (RX 783030), and ethylketocyclazocine. Water intake was also increased but this effect was more variable than the increased eating. The increased food intake produced by the putative mu receptor agonists morphine and RX 783030 was blocked by a dose of naloxone which did not affect the facilitation of eating produced by ethylketocyclazocine, which may act at a separate population of receptors known as kappa receptors. These data are consistent with the possibility that opiate receptors are involved in the control of feeding and drinking.     

Withdrawal responses of guinea-pig isolated ileum following brief exposure to opiates and opioid peptides

Summary Withdrawal contractures following brief exposure of guinea-pig ileum to enkephalin analogues, dynorphin A-(1-13) and beta-endorphin and the opiate drugs morphine, ketocyclazocine, buprenorphine and MR2034 were investigated. Following 2 min contact with the ileum a withdrawal contracture was induced by washout of (Met⁵)- and (Leu⁵)-enkephalin and by several enkephalin analogues but not by washout of (D-Ala²,D-Leu⁵)-enkephalinamide or any of the other drugs tested. Addition of naloxone precipitated withdrawal to all opioids tested except the kappa receptor preferential drugs ketocyclazocine and MR2034 and the mu receptor partial agonist, buprenorphine.The heights of the withdrawal contractures to enkephalin analogues were found to be dependent on the concentration of agonist and of naloxone, and on the duration of the contact period of opioid with the ileum.The naloxone-precipitated withdrawal responses to morphine, dynorphin A-(1-13) and the washout withdrawal response to (Met⁵)-enkephalin were inhibited by substance P antagonists thus supporting the previous pro posal that substance P mediates the opiate withdrawal response.This study has shown that mu receptor agonists produced dependence in the guinea-pig ileum revealed by a withdrawal contracture on addition of naloxone, whereas dependence on kappa receptor agonists could not be revealed with naloxone. Since the guinea-pig ileum preparation has m and kappa receptors it was concluded that the endogenous opioid peptides, enkephalins, beta-endorphin and dynorphin A-(1-13), all induced dependence by acting on mu receptors in this preparation.     

Effects of tifluadom on food consumption compared with chlordiazepoxide and kappa agonists in the rat

Tifluadom (0.625-10.0 mg kg-1) was administered to non-deprived male rats which had been accustomed to eating a highly palatable diet in a 30 min test period. This compound, an opioid benzodiazepine, produced a significant increase in consumption of food when administered by the subcutaneous route, but not after intraperitoneal injection. Both chlordiazepoxide (1.25-20.0 mg kg-1) and the selective kappa opiate receptor agonist U-50,488 (0.3125-2.5 mg kg-1) also produced significant hyperphagic effects in the same feeding situation. In contrast, the two kappa opiate receptor agonists, ethylketocyclazocine (0.1-3.0 mg kg-1) and bremazocine (0.078-1.25 mg kg-1) brought about a dose-related suppression of food intake. Hence, the effects of kappa opiate receptor agonists in the feeding situation described here were not uniform. Furthermore, tifluadom could be likened either to a benzodiazepine or to a selective kappa receptor agonist. The hyperphagia induced by tifluadom was antagonized by naloxone, suggesting that the effect was mediated by an action at opiate receptors. It was not antagonized however by Ro15-1788 (10.0 and 20.0 mg kg-1), a selective benzodiazepine receptor antagonist, ruling out possible mediation by benzodiazepine receptors. The benzodiazepine receptor antagonist, CGS 8216, exhibited intrinsic activity when administered alone, and significantly reduced food consumption in tifluadom-treated and control animals.     

2',6'-Dimethyltyrosine]dynorphin A(1-11)-NH2 analogues lacking an N-terminal amino group: potent and selective kappa opioid antagonists.

Recent studies showed that dermorphin and enkephalin analogues containing two methyl groups at the 2',6'-positions of the Tyr(1) aromatic ring and lacking an N-terminal amino group were moderately potent delta and mu opioid antagonists. These results indicate that a positively charged N-terminal amino group may be essential for signal transduction but not for receptor binding and suggested that its deletion in agonist opioid peptides containing an N-terminal 2',6'-dimethyltyrosine (Dmt) residue may represent a general way to convert them into antagonists. In an attempt to develop dynorphin A (Dyn A)-derived kappa opioid antagonists, we prepared analogues of [Dmt(1)]Dyn A(1-11)-NH2 (1), in which the N-terminal amino group was either omitted or replaced with a methyl group. This was achieved by replacement of Tyr(1) with 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp]. Compounds were tested in the guinea pig ileum and mouse vas deferens bioassays and in rat and guinea pig brain membrane receptor binding assays. All analogues turned out to be potent kappa antagonists against Dyn A(1-13) and the non-peptide agonist U50,488 and showed only weak mu and delta antagonist activity. The most potent and most selective kappa antagonist of the series was [(2S)-Mdp(1)]Dyn A(1-11)-NH2 (5, dynantin), which showed subnanomolar kappa antagonist potency against Dyn A(1-13) and very high kappa selectivity both in terms of its K(e) values determined against kappa, mu, and delta agonists and in terms of its ratios of kappa, mu, and delta receptor binding affinity constants. Dynantin is the first potent and selective Dyn A-derived kappa antagonist known and may complement the non-peptide kappa antagonists norbinaltorphimine and GNTI as a pharmacological tool in opioid research.     

3H]dynorphin A binding and kappa selectivity of prodynorphin peptides in rat, guinea-pig and monkey brain

We have previously demonstrated that [3H]dynorphin A selectively labels kappa opioid receptors in guinea-pig whole brain. In these current studies, using protection from inactivation by beta-chloronaltrexamine (beta-CNA), we are able to demonstrate that although dynorphin A prefers kappa receptors, it will label mu receptors when kappa receptors are not available, or present in only a small number. Thus, differences in numbers of mu and kappa receptors present in brain preparations are critical in determining the receptor binding profile of [3H]dynorphin A across species. Additionally, although all the prodynorphin derived peptides show kappa preference, the ability of the other prodynorphin derived peptides to compete with [3H]dynorphin A for its receptor varies across species. Consequently, in a highly enriched kappa preparation such as monkey cerebral cortex, [3H]dynorphin A appears to label kappa receptors with substantial selectivity, and the other prodynorphin-derived peptides show less ability to compete with dynorphin A for its receptor. In contrast, in a kappa-poor tissue such as rat brain, all of the prodynorphin-derived peptides, including dynorphin A-(1-8), show very similar potency. Thus, differences in mu and kappa receptor numbers across brain regions and species lead to differences in the receptor binding profile of dynorphin A.     

Effects of receptor-selective opioids on operant behavior in morphine-treated and untreated rats.

Selective cross-tolerance is often used as evidence to differentiate opioid receptor subtypes. We used this strategy to study operant behavioral effects of the opioid peptides, [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAGO), [D-Pen2.5]-enkephalin (DPDPE) and dynorphin, agonists highly selective for mu, delta, and kappa receptors, respectively. Food-deprived rats were trained to lever-press on a fixed-interval 3-min schedule of food-reinforcement. Time-effect and dose-effect curves were generated for each of the peptides, as well as for morphine, administered ICV, 5 min prior to the 1-h operant session. Experiments were performed on untreated subjects and on subjects receiving chronic infusion of morphine (10 mg/kg/day) from osmotic pumps. In untreated animals, morphine and the mu-selective peptide, DAGO, induced relatively long-lasting dose-related decreases in responding, whereas the non-mu agonists, DPDPE and dynorphin, induced only transient effects: response rates increased at low doses and decreased at high doses. Animals receiving chronic morphine infusion were tolerant to the rate-decreasing effects of morphine and DAGO; ED50s increased by factors of 8 and 6, respectively. There was some evidence of cross-tolerance to DPDPE and of sensitization to dynorphin in the morphine-maintained animals.     

Spinal dynorphin involvement in the analgesia of pregnancy: effects of intrathecal dynorphin antisera

In both rats and humans there is an analgesia associated with pregnancy. This analgesia is spinally mediated and involves the kappa type of opiate receptor. The current study demonstrates that intrathecal administration of high affinity dynorphin antibodies produces a significant reduction in jump thresholds during pregnancy (day 20). The administration of pre-adsorbed antisera fails to produce this effect. These results support the hypothesis that a spinal dynorphin/kappa opiate receptor system is activated during gestation.     

Pharmacological characterization of human kappa/mu opioid receptor chimeras that retain high affinity for dynorphin A

Arylacetamide analgesics that stimulate kappa opioid receptors in the central nervous system mediate dysphoria and psychosis as well as analgesia. However, the naturally occurring peptide agonist, dynorphin A, is analgesic in the absence of dysphoria and psychosis, indicating that the therapeutic effects of kappa opioid agonists may be separated from their side effects. As part of our effort to discover kappa opioid receptor analgesics lacking side effects, we designed and constructed two mu/kappa chimeric receptors, composed primarily of amino acid residues derived from the mu opioid receptor, that were expected to bind dynorphin A with high affinity. In one, extracellular loop 2 and transmembrane domain 4 were derived from the kappa opioid receptor and in the other, only extracellular loop 2 was derived from the kappa opioid receptor. Most competitors of [(3)H]diprenorphine binding from a variety of structural classes bound to the chimeras with affinities similar to those with which they bound to the mu opioid receptor. In contrast, dynorphin A analogs bound to the chimeras with affinities similar to those with which they bound to the kappa opioid receptor. Pharmacological characterization of [(35)S]GTPgammaS binding mediated by the chimera with extracellular loop 2 derived from the kappa opioid receptor showed that it behaved as if it were mu opioid receptor with high affinity for dynorphin A analogs. These chimeras may be useful in identifying novel kappa receptor agonists that bind to the second extracellular loop of the receptor and share the desirable therapeutic profile of dynorphin A.     

Effect of centrally administered neurotensin on multiple feeding paradigms

Recent studies have suggested that the tridecapeptide, neurotensin, may be an endogenous satiety factor. The present study was undertaken to examine the effects of neurotensin on multiple paradigms known to stimulate feeding. Following a 30 hour starvation period, neurotensin suppressed feeding at the 20 microgram and 10 microgram dose, but not at the 1 microgram dose when compared to saline controls. Norepinephrine (20 micrograms ICV) induced feeding was suppressed at the 20 microgram neurotensin dose but not at the 10 microgram or 1 microgram dose. In contrast, neurotensin did not suppress muscimol induced feeding at any of the doses. Insulin induced feeding (10 units SC) also was not suppressed by neurotensin. Neurotensin suppressed dynorphin induced feeding at the 20 microgram and 10 microgram but not at the 1 microgram dose. Neurotensin suppressed spontaneous feeding (p less than 0.01) in vagotomized rats (2.5 +/- 0.3 g/2 hr) when compared with saline controls (4.2 +/- 0.5 g/2 hr) suggesting that an intact vagus is not necessary for neurotensin's anorectic effect. We conclude that neurotensin may play a role in short-term appetite regulation by a complex interaction with monoamines and neuropeptides, particularly norepinephrine and the kappa opiate agonist, dynorphin.     

The endogenous kappa agonist, dynorphin(1-13), does not alter basal or morphine-stimulated dopamine metabolism in the nigrostriatal pathway of the rat

Dopaminergic pathways in the brain of the rat have been shown to possess both mu and delta opioid regulatory inputs. In contrast, studies with synthetic kappa opiate agonists have demonstrated a lack of regulation of these dopaminergic systems by kappa opioids. The present authors have extended these observations, to study the effects of the putative endogenous kappa agonists, dynorphin (1-13), on the metabolism of nigrostriatal dopamine in the rat after intraventricular administration. The stability of the intraventricularly administered dynorphin was confirmed in vivo by measuring corticosterone in plasma in the same animals utilized for neurochemical analyses. This is a neuroendocrine parameter which has been demonstrated to possess central regulation by independent mu and kappa receptors. While morphine given parenterally elevated both the level of corticosterone in plasma and the central metabolism of dopamine, neither the parenteral administration of the kappa agonist, U50488H, or the intraventricular administration of dynorphin altered central metabolism of dopamine. However, in both cases, levels of corticosterone in plasma were dramatically elevated, clearly demonstrating the bioavailability of the kappa agonists. The actions of morphine on the metabolism of dopamine, which can be antagonized by pretreatment with synthetic kappa agonists, were not antagonized by dynorphin(1-13). To summarize, the present data indicated that the nigrostriatal dopaminergic pathway in the rat lacks kappa opioid regulation. In addition, while synthetic kappa agonists also possessed mu antagonist actions, the endogenous ligand, dynorphin, did not.     

Dynorphin-selective inhibition of adenylyl cyclase in guinea pig cerebellum membranes

Guinea pig cerebellum, which contains kappa opioid receptors uncontaminated by other opioid receptor types, was chosen to examine whether kappa receptors are coupled to adenylyl cyclase. Membranes were prepared from guinea pig cerebellum and pretreated at pH 4.5 to increase inhibitory activity, and adenylyl cyclase was assayed in the presence of dynorphin analogs as prototypical kappa agonists. Results showed that several dynorphin analogs inhibited adenylyl cyclase by 30-50%, whereas mu- and delta-preferring agonists had no effect. Dynorphin A and the kappa-selective compounds D-Pro10-dynorphin(1-11) and U-50,488H were the most potent agonists, with IC50 values of 0.03-0.05 microM, whereas other dynorphin gene products like dynorphin B and alpha-neo-endorphin were approximately 10-fold less potent. Like other Gi-coupled responses, dynorphin-inhibited adenylyl cyclase required GTP and sodium. Naloxone was a competitive antagonist for dynorphin-inhibited adenylyl cyclase, with 1 microM naloxone shifting the IC50 value of dynorphin A by 20-fold. The kappa-selective antagonist nor-binaltorphimine was even more potent, with 0.1 microM nor-binaltorphimine shifting the dynorphin IC50 value by 50-fold. These results suggest that dynorphin A and its analogs inhibit adenylyl cyclase by binding to a guanine nucleotide-binding protein-coupled opioid receptor whose pharmacological specificity matches those of kappa receptors.     

Dopamine receptors mediate alterations in striato-nigral dynorphin and substance P pathways

Peptides derived from prodynorphin, dynorphin A and B, (Leu)-enkephalin and (Leu)enkephalyl-Arg, as well as substance P, were measured in substantia nigra, striatum and globus pallidus, after subacute (5 doses at 6 hr intervals) treatment of rats with a number of dopamine receptor agonists and antagonists. Drugs selective for the dopamine D1 and D2 receptors, respectively, as well as unselective drugs were used. In the substantia nigra, levels of immunoreactive dynorphin A and dynorphin B were increased after treatment with a D2-antagonist (sulpiride) and a D1-agonist (SKF 38393), while a D1-antagonist (SCH 23390) reduced levels. The mixed D1 and D2 antagonist cis-flupenthixol reduced only the level of dynorphin A. A corresponding increase of the levels of (Leu)enkephalin in the nigra was found after treatment with sulpiride. In contrast to dynorphin peptides, the levels of (Leu)enkephalyl-Arg were markedly increased after both D1- and D2 (LY 171555)-stimulation. Substance P tended to be reduced after D1-stimulation and treatment with all the dopamine antagonists; the reduction was significant with sulpiride and cis-flupenthixol. Levels of peptides in striatum and globus pallidus were generally affected in the same direction as levels in the nigra. The results in this study present further evidence that dopamine receptor agents affect dynorphin peptides and substance P, differentially. Effects on (Leu)enkephalin and (Leu)enkephalyl-Arg only partly paralleled the effects on levels of dynorphin. Thus, the D1 and D2 receptors differentially affect levels of different products of prodynorphin, that is, seem to affect certain steps of the processing of prodynorphin selectively.     

Synthesis and biological activities of dynorphin A analogues with opioid antagonist properties

Dynorphin A, which displays a wide variety of physiological effects, binds to opioid receptors preferentially at the kappa receptor type. kappa-selective antagonists would be very useful as pharmacological and biochemical probes to study and better understand the action of dynorphin A at its preferred receptor. However, the development of such molecules has been elusive, and very few are known at this time. Taking these features into account, we have synthesized by the solid-phase procedure several analogues of dynorphin A containing various D-amino acid substitutions. The binding properties of the peptides have been examined at three main opioid binding sites (mu, delta, and kappa) and their kappa selectivity determined. Their biological activities have been tested in three specific pharmacological assays for agonist and/or antagonist properties. Introduction of D-Trp substitution leads to analogues, in particular [D- Trp2,8,D-Pro10]-, [D-Trp5,8,D-Pro10]-, and [D-Trp2,4,8,D-Pro10]dynorphin(1-11), showing antagonist properties in the isolated rabbit vas deferens preparation, a kappa specific bioassay. The antagonism against dynorphin A is weak, as indicated by the observed Ke values (433, 199, and 293 nM, respectively), and not very selective (kappa vs. mu). Such peptide analogues derived from the endogenous ligand and endowed with antagonist properties are the first ones reported to date and could open a promising way in designing more potent and selective kappa opioid antagonists.     

The mu opiate receptor is responsible for descending pain inhibition originating in the periaqueductal gray region of the rat brain

Opiate agonists exhibiting selectivity for mu, kappa, sigma, and delta opiate receptors were microinjected into the periaqueductal gray region (PAG) of the brain of rats to determine the receptor subtype(s) associated with the initiation of descending pain inhibition. The spinally organized, heat nociceptive tail-flick reflex was used to detect analgesia. Only morphine (mu) and [D-Ala2,D-Leu5]enkephalin (DADLE) (delta greater than mu) produced analgesia. However, both drugs appeared to be acting through the mu (morphine) receptor, since: (1) the action of DADLE was not inhibited by delta receptor antagonists, (2) a more highly selective delta agonist [D-Pen2,D-Pen5]enkephalin was ineffective and (3) agonists selective at other non-mu receptor sites (ethylketocyclazocine and U50,488H for kappa; n-allylnormetazocine for sigma) were also ineffective. It appeared that DADLE might be acting as a partial agonist at the morphine receptor in the PAG. The peptide was an agonist with low efficacy, and when a maximally effective dose of the peptide was administered simultaneously with morphine antagonism was observed. Ethylketocyclazocine and n-allylnormetazocine were also found to antagonize morphine, an observation that is consistent with the suggestion that they may act as mu receptor antagonists in addition to their agonistic action at kappa and sigma receptors, respectively. Thus, mu receptors appear to be responsible for the spinopetal analgesia from the PAG of the rat.     

Opiate receptor binding affected differentially by opiates and opioid peptides.

The potencies of various opiates in displacing several 3H-opiate ligands' binding to rat membranes vary depending on the nature of the ligand. Whereas opiate antagonists, as well as the opioid peptides and some agonists (etorphine, levorphanol and phenazocine) display similar affinities in displacing either 3H-opiate or 3H-methionine enkephalin binding, other agonists (such as morphine and oxymorphone) are considerably (20-50 times) weaker in displacing 3H-enkephalin than 3H-dihydromorphine binding. These agonists also compete for 3H-enkephalin binding with shallow displacement curves, and are greatly weakened in displacing 3H-naloxone binding in the presence of sodium. These agonists differ from the other opiate classes by possessing a relatively hydrophilic component in their C-ring moieties which may provide a basis for the differential interactions of drugs with the opiate receptor.     

Effects of opiate agonists and antagonists on aggressive encounters and subsequent opioid-induced analgesia, activity and feeding responses in male mice

The effects of peripheral administration of the mu, kappa and sigma opiate agonists, levorphanol (1.0 mg/kg), U-50,488 (1.0 and 10.0 mg/kg), (+/-) SKF-10,047 (10.0 and 30.0 mg/kg), respectively, as well as the delta opiate antagonists, ICI-154,129 (10.0 mg/kg), and the prototypic antagonist, naloxone (1.0 mg/kg), on the agonistic behaviors and subsequent analgesic, locomotory and ingestive responses of subordinate mice were examined in a "resident-intruder" paradigm. The latter behaviors were examined in both defeated and nondefeated mice that had received an equivalent level of aggression. The mu and delta opiate antagonists decreased, while the mu, kappa, and sigma opiate agonists selectively increased aggressive behavior (number of bouts of aggressive interactions, number of bites to defeat, time to defeat). Both naloxone and the delta antagonist suppressed defeat- and aggression-induced activity and feeding, while only naloxone blocked the analgesic response. Levorphanol enhanced, U-50,488 had variable dose related effects, and SKF-10,047 decreased the defeat and aggressive-induced responses. These results indicate that various opioid systems and opiate receptors are differentially involved in the mediation of various components of the agonistic encounters and in the expression of the consequences of social conflict and defeat-induced opioid activation.     

Effects of selective opioid receptor agonists and antagonists during myocardial ischaemia.

The antiarrhythmic activities of 16-methylcyprenorphine (M8008), nor-binaltorphimine (NBT) and naltrexone, which are relatively specific opioid receptor antagonists for delta, kappa and mu receptors, respectively, were examined during the 30 min following coronary artery occlusion in anaesthetised rats. The haemodynamic and electrocardiographic effects of the opioid receptor agonists [D-Ala2,D-Leu5]enkephalin (DADLE) (relatively selective for delta receptors), ICI-204448 (kappa) and glyol (mu) were also investigated over the 30-90 min post ligation period. When administered intravenously 5 min before ligation, M8008 (0.5 mg kg-1 and 2.5 mg kg-1) reduced the number of ventricular ectopic beats but had no effect on the incidence or duration of ventricular fibrillation. NBT and naltrexone were not antiarrhythmic at a dose of 0.5 mg kg-1 but at 2.5 mg kg-1 (a concentration at which both drugs block kappa receptors) the number of ventricular ectopic beats, the incidence of ventricular fibrillation and mortality were all reduced. All of the opioid receptor agonists caused a transient decrease in heart rate and in arterial blood pressure but none exhibited an arrhythmogenic effect. These studies suggest that the delta and kappa opioid receptor antagonists used may be antiarrhythmic as a result of blockade of the action of endogenously released peptides acting on these receptors or that they have a non-specific 'direct' antiarrhythmic action.     

Kappa Opioids,Salvinorin A and Major Depressive Disorder

Opioids are traditionally associated with pain, analgesia and drug abuse. It is now clear, however, that the opioids are central players in mood. The implications for mood disorders, particularly clinical depression, suggest a paradigm shift from the monoamine neurotransmitters to the opioids either alone or in interaction with monoamine neurons. We have a special interest in dynorphin, the last of the major endogenous opioids to be isolated and identified. Dynorphin is derived from the Greek word for power, dynamis, which hints at the expectation that the neuropeptide held for its discoverers. Yet, dynorphin and its opioid receptor subtype, kappa, has always taken a backseat to the endogenous b-endorphin and the exogenous morphine that both bind the mu opioid receptor subtype. That may be changing as the dynorphin/ kappa system has been shown to have different, often opposite, neurophysiological and behavioral influences. This includes major depressive disorder (MDD). Here, we have undertaken a review of dynorphin/ kappa neurobiology as related to behaviors, especially MDD. Highlights include the unique features of dynorphin and kappa receptors and the special relation of a plant-based agonist of the kappa receptor salvinorin A. In addition to acting as a kappa opioid agonist, we conclude that salvinorin A has a complex pharmacologic profile, with potential additional mechanisms of action. Its unique neurophysiological effects make Salvinorina A an ideal candidate for MDD treatment research.     

The role of opiate mechanisms in the development of tolerance to the anorectic effects of amphetamines

To study the role played by opiate mechanisms in the tolerance to the anorectic effects of amphetamines, the influence of chronic treatment with d,1-amphetamine (AMPH) on the effects of the selective kappa opiate agonist U50488H (U50), of morphine (MORPH) and of diazepam (DZP) on food and water intake was evaluated in rats. Since diuresis is selectively enhanced by kappa agonists, its sensitivity to chronic AMPH was also evaluated. On the first day of AMPH treatment the feeding response to U50 was depressed. On day 9, when tolerance to the anorectic effects of AMPH had developed, this response was enhanced and prolonged. U50-mediated diuresis was not increased in the AMPH group. AMPH however produced diuresis by itself and this effect may be responsible for the increased water intake that developed during chronic treatment. The administration of MORPH (on day 17), but not of DZP (on day 13), produced a similar pattern of response. Interruption of AMPH treatment brought about a slow normalization of response to U50, that appeared to be completed after 17 days. An increase in feeding response to U50 was also obtained after 14 days of cathinone administration, confirming the amphetamine-like properties of this drug. In order to evaluate the possibility that preventing sensitization of opiate mechanisms could also prevent tolerance to anorectic effects of AMPH, we chronically administered MORPH in combination with AMPH, obtaining a further reduction of feeding and an apparent slowing in tolerance development. However, such a reduction was also obtained acutely, although MORPH alone produced feeding stimulation. We suggest that opiates may both activate and inhibit feeding and that AMPH inhibits the activatory branch and works synergically with the inhibitory branch. The prolonged inhibition of the activatory branch causes its compensatory hypertrophy resulting in hypersensitivity to exogenous opiates.