3H]U-69593 labels a subtype of kappa opiate receptor with characteristics different from that labeled by [3H]ethylketocyclazocine

[3H]U-69593 is an opiate agonist that has been reported to bind in vitro with high affinity and selectivity to the kappa receptor subtype. The studies reported here were designed to determine the optimal conditions for labeling kappa receptors with [3H]U-69593 and to further characterize the binding site. The effects of temperature and NaCl on [3H]U-69593 binding were of particular interest because previous studies reported that [3H]ethylketocyclazocine ([3H]EKC) and [3H]bremazocine binding to kappa receptors was optimal at 4 degrees C in the presence of NaCl. Those conditions were not found to be optimal for [3H]U-69593 binding. Although the pharmacological specificity and Bmax of [3H]U-69593 binding was similar at room temperature and at 4 degrees C, the binding affinity was approximately three times lower at 4 degrees C than at room temperature. In addition, NaCl had an effect on [3H]U-69593 binding that was opposite that on [3H]EKC binding at 4 degrees C (100 nM DAGO and 100 nM DADLE were included in all [3H]EKC assays to prevent binding to mu and delta receptors), i.e. NaCl decreased, rather than increased, [3H]U-69593 binding at 4 degrees C. These differences between [3H]U-69593 and [3H]EKC binding at 4 degrees C were accentuated by a vast difference in the density of the binding sites [Bmax approximately equal to 12 fmol/mg protein for [3H]U-69593 vs approximately equal to 375 fmol/mg protein for [3H]EKC at 4 degrees C in the presence of NaCl) and suggested that [3H]U-69593 might bind selectively to a kappa receptor subtype. This concept was supported by competition experiments. In particular, the site labeled by [3H]EKC at 4 degrees C was found to be relatively insensitive (compared to [3H]U-69593 and [3H]EKC binding at room temperature) to the kappa agonist U-50488H, a close analog to U-69593. Based on these findings, we propose that [3H]U-69593 (and U-50488H) labels a kappa receptor subtype which differs from that labeled by [3H]EKC at 4 degrees C.     

Opioids and hibernation. II. Effects of kappa opioid U69593 on induction of hibernation in summer-active ground squirrels by "hibernation induction trigger" (HIT)

A "Hibernation Induction Trigger" (HIT) isolated from plasma of winter-hibernating woodchucks induced hibernation in summer-active ground squirrels (Citellus tridecemlineatus). Effects of kappa opioid U69593 on the HIT-induced hibernation were examined. U69593 alone did not elicit marked behavioral alteration or hibernation in summer-active ground squirrels. U69593, however, antagonized hibernation induced by HIT in summer active ground squirrels. In the guinea pig ileum myenteric plexus-longitudinal muscle preparation, woodchuck HIT depressed the electrically-induced contraction. The depression was, however, neither reversed nor blocked by naloxone even when naloxone was used at high doses. This study demonstrates that kappa opioid, at least in the case of U69593, was unable to induce hibernation in the summer-active ground squirrels. The results also demonstrate that woodchuck HIT, like the bear HIT, did not act directly at opioid receptors. Together with our previous observation that naloxone blocked summer hibernation induced by HIT (Bruce et al., Life Sci.., this issue), it is tempting to suggest that HIT may not mediate its effects through kappa opioid receptors but may do so through other types of opioid receptors such as mu or delta. U69593 may antagonize HIT-induced hibernation as a mu or delta receptor antagonist.     

Effects of U-50,488H and U-50,488H withdrawal on catecholaminergic neurons of the rat hypothalamus

Previous report from our laboratory showed that morphine produces a stimulatory effect of hypothalamic noradrenaline (NA) turnover concurrently with enhanced pituitary-adrenal response after its acute injection and during withdrawal. In the present work we have studied the effects of acute and chronic administration of the kappa agonist U-50,488H as well as the influence of U-50,488H withdrawal on the activity of hypothalamic NA and dopamine (DA) neurons and on the activity of hypothalamic-pituitary-adrenal (HPA) axis. A single dose of U-50,488H (15 mg/kg i.p.) significantly increased hypothalamic NA and decreased DA turnover at the time of an enhanced corticosterone release. Rats rendered tolerant to the kappa agonist by administration of U-50,488H twice a day for 4 days showed no changes in corticosterone secretion. Additionally, a decrease in both hypothalamic MHPG (the cerebral NA metabolite) production and NA turnover was observed, whereas DOPAC concentration and DA turnover were enhanced, which indicate the development of tolerance towards the neuronal and endocrine actions of U-50,488H. After naloxone (3 mg/kg s.c.) administration to U-50,488H-tolerant rats, we found neither behavioural signs of physical dependence nor changes in hypothalamic catecholaminergic neurotransmission. In addition, corticosterone secretion was not altered in U-50,488H withdrawn rats. Present data clearly indicate that tolerance develops towards the NA turnover accelerating and DA turnover decreasing effect of U-50,488H. Importantly and by contrast to mu agonists, present results demonstrate that U-50,488H withdrawal produce no changes in hypothalamic catecholamines turnover or in corticosterone release (an index of the hypothalamus-pituitary-adrenal activity), which indicate the absence of neuroendocrine dependence on the kappa agonist. As has been proposed, this would suggest that the mu and the kappa receptor be regulated through different cellular mechanisms, as kappa agonists have a lower proclivity to induce dependence.     

Exploration of universal cysteines in the binding sites of three opioid receptor subtypes by disulfide-bonding affinity labeling with chemically activated thiol-containing dynorphin A analogs.

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.     

Selective activation of opioid receptors differentially affects lordosis behavior in female rats

The effects of opioid peptides that are highly selective ligands for mu receptors (morphiceptin). delta receptors (delta-receptor peptide), kappa receptors (dynorphin 1-9), and the mu/delta complex (beta-endorphin), were tested on lordosis behavior in ovariectomized rats primed with estrogen and progesterone. Intracerebroventricular infusions of beta-endorphin or morphiceptin both inhibited and facilitated lordosis in a dose-dependent fashion whereas all doses of delta-receptor peptide facilitated lordosis. Dynorphin 1-9 had no significant effect at any dose, although a trend toward increased lordosis quotients was observed 30 min after infusion. The effects of beta-endorphin, morphiceptin, and delta-receptor peptide were reversed with naloxone, although naloxone alone had no effect on lordosis behavior. These results indicate that the specific activation of opioid receptor subtypes differentially affects lordosis behavior. It appears that binding to high-affinity mu 1 receptors exerts an inhibitory influence on lordosis, whereas binding to low-affinity mu 2 receptors or delta receptors exerts a facilitatory influence. Binding to kappa receptors does not appear to affect lordosis behavior.     

Characterization of kappa1-opioid receptor binding in human insular cortex.

Mesolimbic dopaminergic neurotransmission is modulated by dynorphin peptides binding to kappa-opioid receptors. The interaction between dynorphin and dopamine systems makes the kappa-opioid receptor a potential drug discovery target for the development of therapeutic agents for schizophrenia and drug abuse. This study reports the specificity and parameters of [3H]U69593 binding in the insular cortex, a representative corticolimbic area of the human brain. The results demonstrate that the radioligand [3H]U69593 labels a single population of receptors in human insular cortex with an affinity in the low nanomolar range. The pharmacological profile for inhibition of [3H]U69593 binding was determined in this brain region using drugs known to bind to mu, kappa and delta opioid receptors. The results show that kappa-opioid selective agonists and antagonists inhibit binding of this ligand in human brain with comparable affinities and rank order as previously described for rat and guinea pig brain and the cloned kappa1-opioid receptor subtype.     

Opioid receptor selectivity of peptide models of beta-endorphin

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic alpha-helical domain (residues 13-29) in beta-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for mu- and delta-opioid receptors than for kappa opioid receptors. Relative to beta-endorphin, the peptide models were 2-3 times more potent in the mu and kappa receptor binding assays, and about equipotent in the delta receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to beta-endorphin and the other was twice as potent. Like beta-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by mu receptors and not kappa receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of beta-endorphin.     

Effect of the anticonvulsant U-54494A on cortical neuron excitability: comparison to the kappa agonist U-50488H.

U-54494A, a 1,2-diamine anticonvulsant, and U-50488H, a structurally related agonist for opiate kappa receptors, were tested for effects on spontaneous and glutamate-evoked firing rates in cerebral cortex of urethane-anesthetized male Sprague-Dawley rats. Iontophoretic application of 1,2-diamines, glutamate diethyl ether (GDEE), or procaine depressed spontaneous and amino acid-induced firing of cortical neurones. With continued ejection of 1,2-diamines or procaine, firing was silenced completely, but GDEE could maintain a partial suppression. A rapid rebound of excitation followed cessation of procaine ejections, but not of other agents. Procaine, but not U-54494A, blocked axonal conduction of rabbit sciatic nerve. Intravenous U-54494A and U-50488H significantly depressed spontaneous firing rates of cortical neurones, but only the U-50488H effects were antagonized by naloxone. It is concluded that U-54494A inhibits neuronal excitability by a mechanism independent of the analgesic kappa receptor. Biochemical and physiological studies have demonstrated that U-54494A and the kappa opioid agonist U-50488H (a structurally related diamine) (1) have anticonvulsant activity (2, 3). U-54494A lacks kappa analgesic and sedative properties, and it has been suggested that the mechanism of action of this compound may be mediated by a subtype of kappa opioid receptor (3). The effects of kappa analgesics on neuronal firing in nociceptive pathways have been described (4, 5). However, no previous electrophysiological studies on U-54494A have been done. Since U-54494A antagonizes amino acid-induced seizures (3), the interactions of this compound with glutamate are of interest. In the present study, the antagonist efficacies of U-54494A and U-50488H for inhibiting spontaneous and 1-glutamate stimulated neurons of the rat prefrontal cerebral cortex were assessed after i.v. and microiontophoretic administration of the compounds. Effects observed with these routes of administration allow the observation of neuronal changes occurring immediately after administration and take advantage of the high temporal resolution provided by the electrophysiological recording techniques of single cells. A preliminary account of portions of this work have been previously disclosed (6).     

Species differences in the stereoselectivity of kappa opioid binding sites for [3H]U-69593 and [3H]ethylketocyclazocine.

Stereoselectivity of the binding sites for the specific kappa-opioid agonist [3H]U-69593, a benzeneacetamido based ligand was investigated in membrane suspension prepared from frog and rat brain, as well as guinea pig cerebellum, using the pure chiral forms of different unlabelled opiates. The ligand binding sites showed stereospecificity with at least three orders of magnitude differences in the affinities (measured as Ki values) of the opioid stereoisomer pairs both in rat and guinea pig membrane fractions. However, in frog brain membranes there was no substantial difference in potencies of the (-) and (+) isomers competing for the [3H]U-69593 binding sites. Another type of the kappa-site preferring opioid ligand, [3H]ethylketocyclazocine, a benzomorphan derivative was able to discriminate between (-) and (+) forms of the same compounds even in frog brain membrane preparation. Our data concerning binding profile of [3H]U-69593 in frog brain membranes are consistent with the observation that kappa opioid binding sites in frog (Rana esculenta) brain differ from those kappa-sites found in mammalian brains.     

TENA, a selective kappa opioid receptor antagonist

A number of opioid antagonists (TENA, naloxone, Mr 2266, WIN 44441) were evaluated for their selectivity in antagonizing the effect of mu, kappa, and delta agonists in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Among these four antagonists, TENA was the most potent and the only ligand which was selective for kappa receptors. In this regard TENA was approximately 27-times more effective in antagonizing the kappa agonist, U-50488H, relative to the mu agonist, morphine, and it was about 5-times more effective against ethylketazocine (EK) relative to morphine. At the same concentration (20 nM) TENA did not significantly antagonize the delta agonist, [D-Ala2,D-Ala5]enkephalin (DADLE), in the MVD. Also, TENA was more effective than naloxone, EK, or U-50488H in protecting kappa receptors from irreversible blockage by beta-CNA. The results of this study indicate that TENA is the most selective kappa antagonist yet reported.     

Resolution in the receptor binding of putative mu and kappa opiates

Conditions for the stereospecific binding of 3H-ethylketocyclazocine and 3H-etorphine were established in membranes from rat and pigeon brain. In displacing the specific binding of the radiolabeled ligands, putative mu and kappa opiates displayed different sensitivity toward sodium. In membranes from both species, the ratio of the sodium responses exhibited by a given drug in displacing 3H-ethylketocyclazocine and 3H-etorphine, respectively, ("double sodium ratio" = DSR) distinguished between mu and kappa opiates. Compounds characterized on the basis of their pharmacological effects as kappa opiates had DSR values between 0.3 and 2.2, regardless of their nature as agonists or antagonists. In contrast, the DSR for mu opiates ranged from 3.4 to 11. In rat brain membranes, UM 1382 (U-50,488, a compound with pronounced kappa activity) exhibited a DSR of 0.3, while the corresponding value for morphine was 7.4. Dynorphin-(1-13) had a DSR of 1. Within each of the two groups, the simple sodium ratio continued to serve as an index for the agonist or antagonist property of the tested opiates.     

Effects of the opiate antagonists diprenorphine and naloxone and of selected opiate agonists on feeding behavior in guinea pigs

Opiate-sensitive feeding behavior has now been demonstrated in a number of species. We sought information on which opioid receptors might be involved in the observed feeding behaviors. Guinea pigs are known to have higher concentrations of the opioid kappa receptor than any other laboratory animal, so we compared the feeding suppressive potency of the general opiate antagonist, diprenorphine to that of the relatively more mu-specific antagonist, naloxone in that species. We found that diprenorphine was over twenty times more effective than naloxone in suppressing feeding in guinea pigs, suggesting the importance of receptors other than mu in feeding initiation in the guinea pig. Confirmatory evidence for the role of kappa receptors was sought, but not found, in comparisons of the effectiveness of different types of opiate agonists in promoting feeding in these animals. These agonists suppressed, rather than stimulated feeding. We conclude that no feeding stimulatory effects of opiates can be demonstrated in guinea pigs. This observation may indicate that opioids play little role in the natural regulation of feeding in this species or that opioids result in prolonged sedation during which the animals fail to eat. The greater feeding suppressive potency of diprenorphine, a general opiate antagonist, versus naloxone, a mu-preferential antagonist, indicates that to whatever extent opiates are involved in guinea pig feeding, the opiate effect is probably not a mu receptor effect.     

κ Receptor Activation Attenuates l-trans-Pyrrolidine-2,4-Dicarboxylic Acid-Evoked Glutamate Levels in the Striatum

Abstract: The effects of local κ receptor activation and blockade on extracellular striatal glutamate levels evoked by reverse microdialysis of l - trans -pyrrolidine-2,4-dicarboxylic acid ( l - trans -PDC) were investigated. l - trans -PDC elevates extracellular glutamate levels in vivo by acting as a competitive substrate for plasma membrane excitatory amino acid transporters. The selective κ-opioid receptor agonist U-69593 (1-100 n M ) significantly attenuated l - trans -PDC-stimulated glutamate levels in a concentration-dependent manner. The selective κ receptor antagonist nor -binaltorphimine (1-100 n M ) reversed the U-69593-induced decrease in l - trans -PDC-evoked glutamate levels also in a concentration-dependent manner, indicating that the U-69593-induced reduction was mediated by κ receptor activation. In addition, nor -binaltorphimine significantly elevated basal extracellular glutamate levels, implying that κ receptors tonically regulate glutamate efflux in the striatum. Previous data from this laboratory have shown that l - trans -PDC-evoked extracellular glutamate levels are partially calcium-sensitive. The present study demonstrated that the inhibition of l - trans -PDC-evoked glutamate levels by reduced calcium perfusion was not altered by U-69593. Therefore, κ receptors regulate the calcium-dependent component of l - trans -PDC-evoked extracellular glutamate levels in the striatum.     

Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to multiple sites in rat and guinea pig cerebellum

[3H]Met-enkephalin-Arg6-Phe7 (MERF) has been shown to label opioid (kappa2 and delta) and sigma2 sites in rat and frog brain membrane preparations, and no specific binding to kappa1 opioid receptors could be established (refs. 6 and 8). In this study the binding was examined in rat cerebellar membranes which are relatively rich in kappa2-sites, and in guinea pig cerebellar preparations where kappa1 opioid receptors are almost exclusively present. In accordance with our previous results, [3H]MERF binding could not be displaced in guinea pig cerebellar membranes neither with U-69,593 nor with naloxone or levorphanol suggesting no interaction with opioid sites, nevertheless a Kd of 2.8 nM was calculated in cold saturation experiments. In rat cerebellar membrane fractions about the half of the specific [3H]MERF binding sites was inhibited by opiate alkaloids such as naloxone, ethylketocyclazocine, or bremazocine. This portion of the heptapeptide binding sites was stereoselective as demonstrated by the difference in the affinities of the enantiomeric compounds levorphanol and dextrorphan, therefore it would represent an opioid site. In both tissues (-)N-allyl-normetazocine (SKF-10,047), which is also considered as sigma2 ligand, displayed the highest affinities. Among opioid peptides beta-endorphin and dynorphin(1-13) showed the highest potencies, displacing [3H]MERF also from its non-opioid sites. It was concluded therefore that [3H]MERF does not bind to kappa1 sites, and besides kappa2-opioid sites substantial binding to peptide preferring non-opioid sites, and/or sigma2 receptors also occurs.     

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.     

High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.     

Kyotorphin (tyrosine-arginine): Further evidence for indirect opiate receptor activation

Analgesia, opiate receptor binding, and neurochemical effects of kyotorphin (tyrosine-arginine) were studied in the rat. It was found that while kyotorphin, $\text{in vivo}$, causes naloxone reversible analgesia, and affects dopamine metabolism and acetylcholine turnover in the same manner as do morphine and other opiate agents, the dipeptide does not bind to mu, delta or kappa opiate receptors $\text{in vitro}$. Taken together, these data support the concept that there is an indirect action of kyotorphin on opiate receptors.     

Kappa receptor mediated opioid dependence in rhesus monkeys

The kappa receptor-selective agonist U-50, 488 was administered chronically to rhesus monkeys. Tolerance developed to the overt behavioral effects of U-50,488 without cross-tolerance to morphine. Withdrawal behaviors produced by deprivation, naloxone or quadazocine administration in U-50, 488-dependent monkeys consisted of hyperactivity, excessive grooming, and yawning. The syndrome was suppressed in a dose-related manner by a kappa agonist, ethylketazocine, but not by doses of morphine that suppressed its own withdrawal. The mu-selective antagonist, beta-funaltrexamine, at doses which are active in morphine-dependent monkeys, did not precipitate withdrawal in U50, 488-dependent monkeys. Dependence, which is the result of activity at the kappa receptor, was distinct from morphine dependence.     

Opioid control of oxytocin secretion: evidence of distinct regulatory actions of two opiate receptor types

Stress induced oxytocin (OT) secretion was measured in female rats following treatment with various opiate antagonists selective for different types of opiate receptor. Naloxone (mu selective) and MR2266 BS (kappa selective) potentiated the OT response to an emotional stress (1 min. immobilization) whereas the delta selective antagonist ICI 154129 was without effect. Similarly, naloxone and MR2266 BS, but not ICI 154129, potentiated the response to a physical stress (i.p. hypertonic saline). A dose response comparison of the actions of naloxone and MR2266 BS revealed that naloxone was most effective in potentiating the immobilization response whereas MR2266 BS elicited greater responses than naloxone when administered prior to hypertonic saline. The results indicate that the opioid regulation of stress induced OT secretion is primarily mediated via mu and kappa opiate receptor types, the two types differentially regulating the OT response to two different stressors.     

Opioid agonist/antagonist effect of naloxone in modulating rabbit jejunum contractility in vitro.

Opioid peptides are the most effective drugs in controlling pain; their action is elicited by binding to specific membrane receptors. The gastrointestinal tract represents, after the nervous system, the site in which the opioid receptors are expressed at high levels. The opioid agonist morphine has a significant inhibitory effect on intestinal motility, this action is blocked by naloxone an opioid antagonist mainly active at mu and kappa receptors. In this study the presence of mu opioid receptor on rabbit jejunum was investigated by western blot. The effects of beta-endorphin, the endogenous opioid peptide with the highest affinity to the mu opioid receptor and those of naloxone on spontaneous rabbit jejunum contractions were evaluated. Beta-endorphin (10(-6) M) showed a relaxant effect on jejunum contractility while naloxone showed a dual effect inducing an increase of spontaneous contractility at low concentrations (10(-6) M, 10(-7) M, 10(-8) M) and a decrease when high concentrations (10(-3) M, 10(-4) M, 10(-5) M) were utilized. The obtained results demonstrate that mu opioid receptor is expressed in rabbit jejunum and suggest that this receptor may be involved in mediating the effects of both opioid agonist and antagonist on jejunum contractions.