G-receptor antagonists increased the activating effect of mastoparan on low Km GTPase of mouse PAG.

Mastoparan activated in a concentration-dependent manner the low Km GTPase activity in P2 fractions from mouse periaquedultal grey matter (PAG). This peptide at 1-10 mM produced increases of 30-70% over the basal value of 90-120 pmol Pi/mg/min. A series of substances displaying antagonist activity at cellular receptors and not modifying the GTPase function, when used at nanomolar and micromolar concentrations enhanced the effect of mastoparan upon this enzyme. These included antagonists of receptors coupling G proteins: naloxone (non selective opioid antagonist), CTOP (m opioid receptors), ICI 174,864 (d opioid receptors), nor-BNI (k opioid receptors), sulpiride (D2 dopaminergic antagonist), idazoxan (a2 adrenergic antagonist). Bicuculline, antagonist of a receptor not linked to G proteins, GABAA, did not alter the effect of mastoparan on the GTPase. The m opioid agonist, DAMGO, prevented naloxone from increasing the function of the mastoparan-activated enzyme. Thus, mastoparan appears to act on Gi/Go proteins at a site not directly related to the receptor binding domain.

In vivo modulation of G proteins and opioid receptor function by antisense oligodeoxynucleotides.

The work in our laboratory has been designed to characterize the transducer mechanisms coupled to neurotransmitter receptors in the plasma membrane. Particular attention has been paid to the physiological/pharmacological effects mediated by the opioid system. Antisense oligodeoxynucleotides have proved useful in correlating opioid receptor clones with those defined pharmacologically. The involvement of the cloned opioid receptors mu, delta, and kappa in analgesia has been determined by means of in vivo injection of ODNs directed to the receptor mRNAs. Using this strategy the classes of G-transducer proteins regulated by each type/subtype of opioid receptor in the promotion of antinociception have also been characterized. After displaying different patterns of binding to their receptors, opioids trigger a variety of intracellular signals. The physiological implications and therapeutic potential of these findings merit consideration.

Administration of myr(+)-G(i2)alpha subunits prevents acute tolerance (tachyphylaxis) to mu-opioid effects in mice.

The administration of efficacious doses of morphine or beta-endorphin causes acute tolerance (tachyphylaxis) to the effects of additional administrations of these opioids. Mice intracerebroventricularly (icv)-injected with biologically active myristoylated (myr(+))-G(i2)alpha subunits developed no tachyphylaxis to morphine antinociception in the tail-flick test. This treatment increased the potency of opioid-induced analgesia during the declining phase. Moreover, animals showing tachyphylaxis to opioid effects exhibited normal responses to the agonists after icv-administration of myr(+)-G(i2)alpha subunits. In morphine tolerant/dependent mice, an icv dose of 12 pmol/mouse myr(+)-G(i2)alpha subunits facilitated complete restoration of morphine antinociception in only 4 or 5 days instead of the 10 to 11 days required for post-dependent mice. This was observed when myr(+)-G alpha subunits were injected within the first 24 h of chronic morphine administration -- but not later when long-term tolerance takes place. These results suggest that during the course of an opioid effect a progressive reduction of receptor-regulated G-proteins occurs, and hence tachyphylaxis develops. Exogenous administration of myr(+)-G alpha subunits may be of therapeutic potential in improving agonist activity and accelerating the recovery of post-dependent receptors.

Myr+-Gi2 alpha and Go alpha subunits restore the efficacy of opioids, clonidine and neurotensin giving rise to antinociception in G-protein knock-down mice.

In mice whose Gi/o-protein function had been impaired by antisense 'knock-down' or pertussis toxin treatment, i.c.v. injection of myr+-Gi/o alpha subunits restored the effectiveness of beta-endorphin, morphine, DPDPE, clonidine and neurotensin to produce antinociception. Myr+-G alpha subunits of the class of G-proteins actually impaired were more effective than unlike but related myr+-G alpha subunits. Selectivity was noted in that only exogenous myr+-G alpha subunits affected (enhanced) the activity of agonists in G alpha-deficient signalling systems. This treatment had little effect on agonist potency when the impairment resided at the receptor level. The potential of the opioids, clonidine and R-PIA to increase G alpha-related in vitro hydrolysis of GTP was also re-established after injecting myr+-Gi2 alpha subunits into Gi2-knocked-down mice. Myr+-Gi2 alpha subunits pre-incubated with GTPgammaS or GDPbetaS before i.c.v. injection did not improve the activity of agonists in vivo (antinociception) or in vitro (regulation of low Km GTPase). After impairing the function of PKCbeta1 by antisense treatment or with the inhibitor H7, the effect of myr+-G alpha subunits on agonist potency was prevented. Electron microscope analysis showed the entry of gold-conjugated myr+-G alpha subunits into neural cells. These particles were found in the cytoplasm, associated with the plasma membranes of different neuronal processes and also in synaptic junctions. In cultured neurons and astrocytes myr+-Gi2 alpha-associated fluorescence was internalised in a dose-dependent manner and distributed in the plasma membrane and cytosol, as well as in nuclei of dividing astrocytes. Thus, G alpha subunits in CSF enter into neurons and functionally couple to the receptor-triggered signalling cascade. As G-proteins have been implicated in the pathophysiology of several neural disorders, this finding may be valuable in the therapy of such dysfunctions.

Glycosylated phosducin-like protein long regulates opioid receptor function in mouse brain.

Phosducin (Phd), a protein that in retina regulates rhodopsin desensitization by controlling the activity of Gt beta gamma-dependent G-protein-coupled receptor kinases (GRKs), is present in very low levels in the CNS of mammals. However, this tissue contains proteins of related sequence and function. This paper reports the presence of N-glycosylated phosducin-like protein long (PhLP(L)) in all structures of mouse CNS, mainly in synaptic plasma membranes and associated with G beta subunits and 14-3-3 proteins. To analyze the role PhLP(L) in opioid receptor desensitization, its expression was reduced by the use of antisense oligodeoxynucleotides (ODNs). The antinociception induced by morphine, [D-Ala(2), N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO), beta-endorphin, [D-Ala(2)]deltorphin II, [D-Pen(2,5)]-enkephalin (DPDPE) or clonidine in the tail-flick test was reduced in PhLP(L)-knock-down mice. A single intracerebroventricular (icv)-ED(80) analgesic dose of morphine gave rise to acute tolerance that lasted for 4 days, but which was prevented or reversed by icv-injection of myristoylated (myr(+)) G(i2)alpha subunits. PhLP(L) knock-down brought about a myr(+)-G(i2)alpha subunit-insensitive acute tolerance to morphine that was still present after 8 days. It also diminished the specific binding of (125)I-Tyr(27)-beta-endorphin-(1-31) (human) to mouse periaqueductal gray matter membranes. After being exposed to chronic morphine treatment, post-dependent mice required about 10 days for complete recovery of morphine antinociception. The impairment of PhLP(L) extended this period beyond 17 days. It is concluded that PhLP(L) knock-down facilitates desensitization and uncoupling of opioid receptors.

Activation of I(2)-imidazoline receptors enhances supraspinal morphine analgesia in mice: a model to detect agonist and antagonist activities at these receptors.

This work investigates the receptor acted upon by imidazoline compounds in the modulation of morphine analgesia. The effects of highly selective imidazoline ligands on the supraspinal antinociception induced by morphine in mice were determined. 2. Intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of ligands selective for the I(2)-imidazoline receptor, 2-BFI, LSL 60101, LSL 61122 and aganodine, and the non selective ligand agmatine, increased morphine antinociception in a dose-dependent manner. Neither moxonidine, a mixed I(1)-imidazoline and alpha(2)-adrenoceptor agonist, RX821002, a potent alpha(2)-adrenoceptor antagonist that displays low affinity at I(2)-imidazoline receptors, nor the selective non-imidazoline alpha(2)-adrenoceptor antagonist RS-15385-197, modified the analgesic responses to morphine. 3. Administration of pertussis toxin (0.25 microg per mouse, i.c.v.) 6 days before the analgesic test blocked the ability of the I(2)-imidazoline ligands to potentiate morphine antinociception. 4. The increased effect of morphine induced by I(2)-imidazoline ligands (agonists) was completely reversed by idazoxan and BU 224. Identical results were obtained with IBI, which alkylates I(2)-imidazoline binding sites. Thus, both agonist and antagonist properties of imidazoline ligands at the I(2)-imidazoline receptors were observed. 5. Pre-treatment (30 min) with deprenyl, an irreversible inhibitor of monoamine oxidase B (IMAO-B), produced an increase of morphine antinociception. Clorgyline, an irreversible IMAO-A, given 30 min before morphine did not alter the effect of the opioid. At longer intervals (24 h) a single dose of either clorgyline or deprenyl reduced the density of I(2)-imidazoline receptors and prevented the I(2)-mediated potentiation of morphine analgesia. 6. These results demonstrate functional interaction between I(2)-imidazoline and opioid receptors. The involvement of G(i)-G(o) transducer proteins in this modulatory effect is also suggested.

Antibodies and antisense oligodeoxynucleotides to mu-opioid receptors, selectively block the effects of mu-opioid agonists on intestinal transit and permeability in mice.

1. We have studied the effects of mu and delta opioids on intestinal function (permeability, PER; gastrointestinal transit, GIT), and their antagonism after the intracerebroventricular (i.c.v.) administration of specific antibodies (ABs) or antisense oligodeoxynucleotides (ODN) to mu-receptors (OR). Central versus peripheral site/s of action of subcutaneous (s.c.) mu-opioids, were also assessed. 2. Male Swiss CD-1 mice were used. GIT was measured with charcoal and PER by the passage of 51Cr-EDTA from blood to lumen. 3. Morphine and fentanyl (i.c.v. and s.c.) inhibited GIT and PER in a dose-related manner; they were more potent by i.c.v. route, both on GIT and PER (70 and 17 times for morphine and fentanyl). They also had a greater effect on GIT than PER (4.3 and 1.6 times). DPDPE had a lower potency than mu-agonists in all experiments, and no dose-response could be obtained after s.c. administration on GIT. 4. Pretreatment with i.c.v. ABs (24 h) or antisense ODN (5 days), decreased the effects (GIT and PER) of i.c.v. morphine and fentanyl, while those of DPDPE remained unchanged. The ABs did not alter the peripheral effects of mu-opioids. 5. The results show that (i.c.v. or s.c.) mu opioids produce dose-related inhibitions of PER and GIT, being more potent by the i.c.v. route. Delta-opioids had a greater effect on PER than GIT, while the opposite occurred for mu-agonists. Pretreatment with ABs or ODN to mu-OR, blocked the central effects of mu (but not delta) agonists on GIT and PER.

Dynorphin A: inhibitory effect on other opiate ligand bindings in the mouse brain.

The opioid peptide dynorphin A antagonizes morphine-induced analgesia in vivo and inhibits opiate binding in vitro, although most of it is rapidly degraded under both conditions. The inhibitory effect was present even in tissue treated in vivo with dynorphin A and assayed in vitro without it. Shorter fragments of this peptide lacked these effects, indicating that the apparent potency did not result from a metabolite. Na+ ion specifically reversed both agonist and antagonist binding from in vitro inhibition by dynorphin A. These results are discussed in terms of current opioid receptor theories.

Transport of CSF antibodies to Galpha subunits across neural membranes requires binding to the target protein and protein kinase C activity.

In the light of functional studies, it has been suggested that antibodies directed to alpha subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2alpha IgGs were labeled with 125I, fluorescein or with gold particles. The expression of Galpha subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2alpha IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Galpha IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2alpha 125I-IgGs was almost absent in Gi2alpha knocked-down mice, but not after decreasing the quantity of Gzalpha subunits. The immunosignals of CSF anti-Galpha 125I-IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cbeta1 (PKCbeta1). In mice treated with an ODN to PKCbeta1 mRNA, 125I-IgGs to Gi2alpha subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Galpha subunits that translocate to the external side of the neural membrane before being internalized by a PKCbeta1-dependent mechanism.

Impairment by apomorphine of one-trial passive avoidance learning in mice: the opposing roles of the dopamine and noradrenaline systems.

Pretraining administration of the dopaminergic stimulant apomorphine (0.25--16 mg/kg) impaired retention performance of mice on a one-trial passive avoidance task. Only with a very high dose (16 mg/kg) of this drug did the effect seem related to an interference with memory formation processes. Of the dopamine receptor-blocking agents used, haloperidol (0.125--1 mg/kg), but not chlorpromazine or clozapine (0.25--4 mg/kg), prevented the apomorphine effect. Phenoxybenzamine (8 mg/kg), a noradrenaline receptor-blocker, antagonized the haloperidol effect and, when combined with a subeffective dose of apomorphine, impaired passive avoidance learning. The results obtained are interpreted in terms of the proposed inhibitory actions exerted by central noradrenaline on dopamine systems of the brain.

Neuropeptide Y is an inhibitor of neural function in the myenteric plexus of the guinea-pig ileum.

Neuropeptide Y (NPY) reduced the resting tension of the myenteric plexus-longitudinal muscle preparation (MP-LM) of the guinea-pig ileum (GPI). NPY in a dose-dependent manner also reduced the neurally-mediated excitatory effect of cholecystokinin octapeptide (CCK8) sulfated form on this preparation. However, NPY, at the concentration used in the study, did not modify the effect of exogenous acetylcholine (ACh). All these features were also shared by other inhibitory peptides, like somatostatin (SOM) and the enkephalin derivative FK 33-824. The preparation developed a degree of tachyphylaxis to the inhibitory effect of NPY more rapidly than it did to SOM. Moreover, the inhibitory effect of neuropeptide Y was of longer duration than the one seen for somatostatin. A faster metabolic rate might account for the lower development of tachyphylaxis to somatostatin. The presence of the opioid antagonist naloxone did not alter the inhibitory features of NPY or SOM. Therefore, the involvement of any endogenous opioid in the action of these two inhibitory peptides can be disregarded.

Opiate activity of peptides derived from the three opioid peptide families on the rat vas deferens.

The inhibitory activity of opioid peptides derived from pro-opiomelanocortin (POMC), pro-enkephalin A and pro-enkephalin B (= pro-dynorphin) on the electrically evoked twitch of the rat vas deferens (RVD) was evaluated. The POMC-derived beta-endorphin exhibits the greatest potency on this preparation. In addition, all peptides derived from pro-enkephalin A show full agonistic activity with BAM-22P and peptide E as the most potent peptides. In contrast, the majority of peptides derived from pro-enkephalin B (= pro-dynorphin) were essentially inactive on this tissue. Moreover, no antagonistic properties of these peptides were demonstrable in this preparation; thus the opioid receptors present in the RVD (putative epsilon receptors) might not possess any particular affinity for the pro-enkephalin B derived peptides.

Dynorphin B-29: chemical synthesis and pharmacological properties in opioid systems in vitro.

Porcine dynorphin B-29 was synthesized by solid phase procedures and purified using gel filtration, ion exchange, reverse phase liquid chromatography and preparative high pressure liquid chromatography (HPLC). Binding experiments using brain tissue indicated this peptide displaced mu and k ligands equally well and had significant, though less, affinity for delta. In isolated tissue systems, its potency ranked as follows: guinea pig ileum, mouse vas, rabbit vas, and rat vas. Interestingly, all IC50s were reduced in the presence of peptidase inhibitors, particularly in the rabbit vas. These results indicate that dynorphin B-29 like dynorphin, interacts with multiple receptors in the brain, as well as in isolated tissue systems.

Dynorphin1-13: interaction with other opiate ligand bindings in vitro.

Dynorphin1-13 is a potent inhibitor of electrically-induced contractions in the guinea pig ileum, where it has the properties of kappa-(ethylketocyclazocine) type opioids. In the brain, however, it has no analgesic potency, yet inhibits that induced by morphine. To gain further insight into its mechanism of action in the latter system, we tested its ability to compete for the binding of several opiates to brain membranes in vitro. Dynorphin1-13 inhibited the binding of all ligands examined, including dihydromorphine, D-Ala2-D-Leu5-enkephalin, ethylketacyclazocine (EKC) and naloxone. In all cases, it reduced the number of high affinity sites and, in the case of EKC, it also increased the Kd. We conclude that the mechanism of dynorphin inhibition is not simple rapidly reversible competition and is certainly not identical with respect to all opiate ligands.

Pertussis toxin differentially reduces the efficacy of opioids to produce supraspinal analgesia in the mouse.

The i.c.v. administration of 0.5 microgram pertussis toxin to mice led to a non-competitive reduction (approximately 60 to 70%) of the supraspinal analgesia evoked by i.c.v. injection of ED90 doses of [D-Ala2,N-MePhe4,Gly-ol5]enkephalin, [D-Ala2,N-MePhe4,Met-(O)5-ol]enkephalin, [D-Ala2,Met5]enkephalinamide, [D-Ala2,D-Leu5]enkephalin or [D-Pen2,D-Pen5]enkephalin, whereas the analgesic effect of ED90 doses of morphine, etorphine, beta-casomorphin-(1-4) amide or human beta-endorphin was reduced to a lesser extent (about 20 to 30%). The co-administration of any of the opioids from the first group together with morphine resulted in antagonism of the effect elicited by the alkaloid. It is suggested that pertussis toxin treatment reduces differentially the efficacy displayed by various opioids when acting via mu receptors to produce supraspinal analgesia.

Evaluation of delta receptor mediation of supraspinal opioid analgesia by in vivo protection against the beta-funaltrexamine antagonist effect.

The involvement of delta opioid receptors in supraspinal analgesia was investigated. With this aim, opioids that produced analgesia in the tail immersion test were administered i.c.v. to mice a few minutes before the irreversible antagonist, beta-funaltrexamine (beta-FNA). Protection of the respective analgesic effects from beta-FNA blockade was obtained when evaluated 24 h later. Moreover, mu ligands protected the analgesia evoked by ED50s of morphine, [D-Ala2,N-Me-Phe4,Met-(o)5-ol]enkephalin (FK 33-824), [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAGO) and human beta-endorphin at doses (ED50s) lower than those required for delta ligands (approximately ED90s) to reach a similar protection. delta Preferential ligands effectively protected the analgesia induced by ED50s of [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Thr2,Leu5]enkephalin-Thr6 (DTLET) and [D-Pen2,D-Pen5]enkephalin (DPDPE) from the beta-FNA-deteriorating effect. FK 33-824 and DAGO also provided good protection of the analgesia elicited by these delta ligands whereas morphine protected much less. Binding studies after i.c.v. injection of beta-FNA showed that its alkylating effect on opioid receptors was restricted to periventricular areas. In PAG, where the mu/delta receptor ratio is about 10, [3H]DADLE specific binding was still present after ED50s of DPDPE, DAGO, morphine and DADLE as protecting agents. [3H]Dihydromorphine [( 3H]DHM) binding was well protected by ED90s of morphine and DAGO, and to a lesser extent by DPDPE and DADLE. These results suggest that delta ligands, after binding to delta receptors, also need to act upon mu receptors to produce high levels of supraspinal analgesia in the tail immersion test.

Mastoparan reduces the supraspinal analgesia mediated by mu/delta-opioid receptors in mice.

Intracerebroventricular (i.c.v.) administration of the venom peptide, mastoparan, to mice decreased to a limited extent opioid-induced supraspinal analgesia in a non-competitive fashion. The mu-opioid receptor agonists, [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) and morphine, the mu/delta-opioid receptor ligands, human beta-endorphin-(1-31) and [D-Ala2,D-Leu5]-enkephalin (DADLE), and the selective ligands of delta-opioid receptors, [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II, showed an impaired analgesic effect in mice given mastoparan. Mastoparan diminished the analgesic activity of DPDPE and [D-Ala2]deltorphin II to the same extent as observed after giving the delta-opioid receptor-selective antagonist, ICI 174864. The mu-opioid receptor-mediated analgesia that remained after mastoparan was abolished in the presence of the opioid antagonist, naloxone. Mastoparan after binding to Gi alpha/Go alpha subunits could block opioid antinociception. The existence of a class of G protein functionally coupled to mu-opioid receptors, but resistant to the effect of mastoparan is suggested.

Intracerebroventricular N-ethylmaleimide differentially reduces supraspinal opioid analgesia in mice.

I.c.v. injection of 1 nmol N-ethylmaleimide (NEM) into mice interfered with opioid-induced supraspinal analgesia, as assessed in the warm water tail-flick test. This effect of NEM was long-lasting (more than 3 days), non-competitive and differentially inhibited by the opioids studied. The analgesia induced by [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2,Met5]enkephalinamide (DAME) and [D-Pen2,D-Pen5]enkephalin (DPDPE) was greatly reduced in NEM-treated mice. The antinociception elicited by [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO) was also impaired although to a lesser extent. In contrast, the activity of morphine and etorphine remained unchanged. NEM-sensitive opioids coadministered with morphine antagonized the analgesia elicited by the alkaloid in NEM-treated mice. The administration of naltrexone or DADLE, DAGO, [D-Ala2,N-MePhe4,Met-(O)5-ol]enkephalin (FK-33824) and morphine in doses equivalent to the ED90 doses for inducing analgesia, a few minutes before NEM prevented it from interfering with DADLE-elicited supraspinal analgesia when evaluated 24 h later. In contrast, the selective delta antagonist, ICI 174864, did not protect the DADLE-induced analgesia against the effect of NEM. We suggest that NEM produced its effect by acting upon a site that appears to be distal to the receptor binding site, presumably located on the guanine nucleotide binding regulatory proteins, Gi/Go. Therefore, these transducer proteins might play a key role in the effects displayed by opioids when acting via the mu receptor-Gi/Go complexes.

[Leu5]enkephalin containing peptides derived from a common precursor: evaluation of opioid activity in in vitro bioassays.

Opioid peptides containing the sequence of [Leu5]enkephalin were studied in two isolated organ preparations sensitive to opiates, the guinea pig ileum (GPI) and the mouse was deferens (MVD). All peptides tested were able to inhibit the electrically stimulated contraction in both tissues by interacting with specific receptors sensitive to the antagonist naloxone. Some of these peptides, mainly the shorter sequences, showed considerable potency differences in the two systems, suggesting that at least two different types of receptors are involved. Dynorphin-(1-17) displayed the highest agonistic potency in both preparations. In its case, there were no differences in IC50s nor in the shapes of the dose response curves in the two systems, suggesting a common receptor type; however, the reversal of its inhibitory effect following washout of the peptide was much more complete in the MVD than in GPI. Dynorphin B exhibited a higher potency in the GPI. Extension to dynorphin B-29 peptide did not induce changes in the agonistic activity in either system. An increase in one amino acid residue, dynorphin-(1-9) to -(1-10) or beta-neo-endorphin to alpha-neo-endorphin, resulted in a large potency increase in GPI and an opposite effect in MVD. While it has been reported that dynorphin interacts with the kappa opiate receptor in both mouse vas deferens and guinea pig ileum, our results suggest that the observable differences in the kinetics of the interaction in these systems could be due to the presence of different receptor types in these tissues.

Use of selective antagonists and antisense oligonucleotides to evaluate the mechanisms of BUBU antinociception.

Evidence suggests that the antinociceptive effects of selective delta-opioid receptor agonists may involve an activation of the mu-receptor in some experimental conditions. The aim of this study was to clarify the receptors involved in the antinociceptive responses of the selective and systemically active delta-opioid receptor agonist Tyr-D-Ser-(O-tert-butyl)-Gly-Phe-Leu-Thr-(O-tert-butyl) (BUBU). The antinociception induced by systemic (i.v.) or central (i.c.v.) administration of BUBU was measured in the hot plate (jumping and paw lick latencies) and tail immersion tests in mice. In both tests, the responses were more intense when BUBU was administered by central route. The pre-treatment with the mu-opioid receptor antagonist cyprodime blocked the effects induced by central BUBU in the hot plate and tail immersion tests. The delta-opioid receptor antagonist naltrindole had no effect on BUBU-induced antinociception in the hot plate but decreased BUBU responses in the tail immersion test. Further evidence for this dual receptor action of BUBU was demonstrated by using antisense oligodeoxynucleotides. Thus, a reduction in central BUBU-induced antinociception was observed in the tail immersion test after the administration of antisense probes that selectively blocked the expression of mu- or delta-opioid receptors. These findings clearly indicate using a dual pharmacological and molecular approach that BUBU mediates its antinociceptive effects via activation of both mu- and delta-opioid receptors.