Rapid assessment of G protein signaling of four opioid receptors using a real-time fluorescence-based membrane potential assay.

Opioids are the most powerful analgesics used clinically; however, severe side effects limit their long-term use. Various concepts involving biased intracellular signaling, partial agonism or multi-receptor targeting have been proposed to identify novel opioids with increased analgesic efficacy but reduced side effects. The search for such 'better opioids' implies screening of huge compound libraries and requires highly reliable, easy to perform and high throughput screening (HTS) assays. Here, we utilize an established membrane potential assay to monitor activation of G protein-coupled inwardly rectifying potassium (GIRK) channels, one of the main effectors of opioid receptor signaling, as readout to determine pharmacological profiles of opioids in a non-invasive manner. Specifically, in this study, we optimize assay conditions and extend the application of this assay to screen all four members of the opioid receptor family, stably expressed in AtT-20 and HEK293 cells. This ultra-sensitive system yielded EC50 values in the nano-molar range. We further validate this system for screening cells stably co-expressing two opioid receptors, which could be a valuable tool for investigating bi-functional ligands and studying interactions between receptors. Additionally, we demonstrate the utility of this assay to study antagonists as well as ligands with varying efficacies. Our results suggest that this assay could easily be up-scaled to HTS assay in order to efficiently study receptor activation and screen for novel opioids.

GTPγS-Autoradiography for Studies of Opioid Receptor Functionality.

The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR ) that are Gi coupled which make them suitable for studying the receptor functionality. The [35S]GTP γS autoradiography assay is a good option that has the benefit of generating both anatomical and functional data in the area of interest. It is based on the first step of the signaling mechanism of GPCRs. When a ligand binds to the receptor GTP will replace GDP on the a-subunit of the G-protein, leading to a dissociation of the ßγ-subunit. These subunits will start a cascade of second messengers and subsequently a physiological response.

Pharmacological and molecular docking assessment of cryptotanshinone as natural-derived analgesic compound.

Medicinal plants from traditional chinese medicine are used increasingly worldwide for their benefits to health and quality of life for the relevant clinical symptoms related to pain. Among them, Salvia miltiorrhiza Bunge is traditionally used in asian countries as antioxidant, anticancer, anti-inflammatory and analgesic agent. In this context, several evidences support the hypothesis that some tanshinones, in particular cryptotanshinone (CRY), extracted from the roots (Danshen) of this plant exhibit analgesic actions. However, it is surprisingly noted that no pharmacological studies have been carried out to explore the possible analgesic action of this compound in terms of modulation of peripheral and/or central pain. Therefore, in the present study, by using peripheral and central pain models of nociception, such as tail flick and hot plate test, the analgesic effect of CRY in mice was evaluated. Successively, by the aim of a computational approach, we have evaluated the interaction mode of this diterpenoid on opioid and cannabinoid system. Finally, CRY was dosed in mice serum by an HPLC method validated according to European Medicines Agency guidelines validation rules. Here, we report that CRY displayed anti-nociceptive activity on both hot plate and tail flick test, with a prominent long-lasting peripheral analgesic effect. These evidences were indirectly confirmed after the daily administration of the tanshinone for 7 and 14 days. In addition, the analgesic effect of CRY was reverted by naloxone and cannabinoid antagonists and amplified by arginine administration. These findings were finally supported by HPLC and docking studies, that revealed a noteworthy presence of CRY on mice serum 1 h after its intraperitoneal administration and a possible interaction of tested compound on µ and k receptors. Taken together, these results provide a new line of evidences showing that CRY can produce analgesia against various phenotypes of nociception with a mechanism that seems to be related to an agonistic activity on opioid system.

Opioidergic and dopaminergic modulation of cost/benefit decision-making in Long Evans Rats.

Eating disorders are associated with impaired decision-making and dysfunctional reward-related neurochemistry. The present study examined the potential contributions of dopamine and opioid signaling to these processes using two different decision-making tasks. In one task, Long Evans Rats chose between working for a preferred food (high-carbohydrate banana-flavored sucrose pellets) by lever pressing on a progressive-ratio schedule of reinforcement vs. obtaining less preferred laboratory chow that was concurrently available. In a second (effort-free) task, rats chose between the same two reinforcers when they were both available freely. Rats were trained in these tasks before receiving haloperidol (0.00, 0.05, 0.10mg/kg, intraperitoneally (i.p.)) or naloxone (0.0, 1.5, 3.0mg/kg, i.p.). In the first task, haloperidol decreased breakpoint, lever presses, number of reinforcers earned, and increased chow intake, whereas naloxone decreased breakpoint and number of reinforcers earned but had no effect on chow consumption. In the effort-free task, haloperidol reduced intakes of both foods without affecting preference, whereas naloxone selectively reduced the consumption of banana-pellets. The present findings support converging evidence suggesting that DA signaling affects processes more closely related to appetitive motivation, leaving other components of motivation unchanged. By contrast, opioid signaling appears to mediate aspects of hedonic feeding by selectively altering intakes of highly palatable foods. For preferred foods, both appetitive and consummatory aspects of food intake were altered by opioid receptor antagonism. Our findings argue against a general suppression of appetite by either compound, as appetite manipulations have been shown to unselectively alter intakes of both types of food regardless of the task employed.

[(35)S]GTPγS autoradiography for studies of opioid receptor functionality.

The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR) that are Gi-coupled which make them suitable for studying the receptor functionality. The [(35)S]GTPγS autoradiography assay is a good option that has the benefit of generating both anatomical and functional data in the area of interest. It is based on the first step of the signaling mechanism of GPCRs. When a ligand binds to the receptor GTP will replace GDP on the α-subunit of the G protein, leading to a dissociation of the ßγ-subunit. These subunits will start a cascade of second messengers and subsequently a physiological response.

Assessment of the analgesic effect of centhaquin in mouse tail flick and hot-plate tests.

BACKGROUND: Centhaquin is a centrally acting hypotensive drug like clonidine. Clonidine also produces analgesia and hypothermia in mice and potentiates morphine analgesia. Clonidine analgesia is blocked by idazoxan and naloxone while it is potentiated by BQ123 and sulfisoxazole. This study was conducted to determine the analgesic and hypothermic properties of centhaquin, and to assess whether it potentiates morphine analgesia. Yohimbine (α(2)-adrenergic antagonist), idazoxan (imidazoline/α(2)-adrenergic antagonist), naloxone (opioid antagonist), and BQ123 and sulfisoxazole (endothelin ET(A) antagonists) were used to study the involvement of these receptors in centhaquin analgesia and hypothermia. METHODS: Analgesic (tail flick and hot-plate tests) latencies and body temperatures were measured in male Swiss Webster mice treated with vehicle plus centhaquin, antagonists plus centhaquin or centhaquin plus morphine. RESULTS: Centhaquin produced dose-dependent analgesia which was partially blocked by yohimbine, idazoxan and naloxone. BQ123 and sulfisoxazole did not affect centhaquin analgesia. Morphine analgesia was not potentiated by centhaquin. Centhaquin produced mild hypothermia which was not blocked by yohimbine, idazoxan, naloxone, BQ123 or sulfisoxazole. CONCLUSIONS: This is the first report demonstrating the analgesic activity of centhaquin. The α(2)-adrenergic, imidazoline and opioid receptors are involved in mediating centhaquin analgesia. Endothelin ET(A) receptors do not play a role in centhaquin analgesia; centhaquin does not augment morphine analgesia.

Preclinical assessment of drug combinations for the treatment of pain: isobolographic and dose-addition analysis of the opioidergic system.

Opioid analgesics are the most frequently prescribed medications for the treatment of moderate or severe pain; however, their use is constrained by unwanted side effects. One therapeutic approach used to improve the side effect profile of opioids is the administration of a second drug in an opioid-containing mixture. Preclinical studies designed to predict the therapeutic potential of novel opioid-containing drug combinations are currently underway, and must rely on quantitative methods to assess their interactive effects. In this manuscript, an overview of isobolographic analysis is presented along with recent advances in isobolographic theory pertaining to drugs that differ in efficacy and to the statistical analysis of dose-addition. Next, studies using these analyses to assess the interactive effects of opioids and novel adjunct drugs, including selective COX-2 inhibitors, α(2)-adrenergic receptor agonists, δ-opioids, glutamate receptor antagonists and cannabinoid receptor agonists, are reviewed. Finally, comments on the future assessment of drug combinations for the treatment of pain-related disorders are made.

Low sensitivity of the positron emission tomography ligand [11C]diprenorphine to agonist opiates.

Previously, we reported minimal opioid receptor occupancy following a clinical dose of the micro-opioid agonist, methadone, measured in vivo using positron emission tomography (PET) with [(11)C]diprenorphine and subsequently used rats to obtain experimental data in support of a high receptor reserve hypothesis (Melichar et al., 2005). Here, we report on further preclinical studies investigating opioid receptor occupancy with oxycodone (micro- and kappa-receptor agonist), morphine (micro-receptor agonist), and buprenorphine (partial agonist at the micro-receptor and antagonist at the delta- and kappa-receptors), each given at antinociceptive doses. In vivo binding of [(11)C]diprenorphine was not significantly reduced after treatment with the full agonists but was reduced by approximately 90% by buprenorphine. In addition, given that [(11)C]diprenorphine is a non-subtype-specific PET tracer, there was no regional variation that might feasibly be interpreted as due to differences in opioid subtype distribution. The data support minimal competition between the high-efficacy agonists and the non-subtype-selective antagonist radioligand and highlight the limitations of [(11)C]diprenorphine PET to monitor in vivo occupancy. Alternative means may be needed to address clinical issues regarding opioid receptor occupancy that are required to optimize treatment strategies.

Assessment of the activity of a novel nociceptin/orphanin FQ analogue at recombinant human nociceptin/orphanin FQ receptors expressed in Chinese hamster ovary cells.

The neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the nociceptin receptor (NOP). In an attempt to identify high potency NOP agonists for use in the brain we have compared the activity of a novel N/OFQ analogue [Phe(1)Psi(CH(2)-O)Gly(2)]N/OFQ(1-13)NH(2) ([F/G-O]) with the existing [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) ([F/G]). Both peptides are modified between the first two N-terminal amino acids and are further compared with the agonist template N/OFQ(1-13)NH(2) in [(3)H]N/OFQ binding, GTPgamma[(35)S] binding and cAMP inhibition studies using Chinese hamster ovary cells expressing the recombinant human NOP. All peptides displaced [(3)H]N/OFQ, stimulated GTPgamma[(35)S] binding and inhibited cAMP formation. In [(3)H]N/OFQ binding and GTPgamma[(35)S] binding the rank order affinity and potency was N/OFQ(1-13)NH(2)>[F/G-O]>[F/G]. In GTPgamma[(35)S] binding [F/G] was a clear partial agonist with intrinsic activity (E(max) stimulation factor, mean+/-SEM, n=4) of 7.75+/-1.02 compared with N/OFQ(1-13)NH(2) of 11.13+/-1.76. The efficacy of [F/G-O] (10.17+/-1.88) approached that of the full agonist N/OFQ(1-13)NH(2). Downstream, at the level of cAMP formation, all peptides were full agonists with the following rank order potency: N/OFQ(1-13)NH(2)>[F/G-O]=[F/G]. The enhanced potency and intrinsic activity of the novel [F/G-O] modification makes this an interesting peptide for further in vivo analysis.

Glycopeptide-membrane interactions: glycosyl enkephalin analogues adopt turn conformations by NMR and CD in amphipathic media.

Four enkephalin analogues (Tyr-D-Thr-Gly-Phe-Leu-Ser-CONH(2), 1, and the related O-linked glycopeptides bearing the monosaccharide beta-glucose, 2, the disaccharide beta-maltose, 3, and the trisaccharide beta-maltotriose, 4) were synthesized, purified by HPLC, and biophysical studies were conducted to examine their interactions with membrane model systems. Glycopeptide 2 has been previously reported to penetrate the blood-brain barrier (BBB), and produce potent analgesia superior to morphine in mice (J. Med. Chem.2000, 43, 2586-90 and J. Pharm. Exp. Ther. 2001, 299, 967-972). The parent peptide and its three glycopeptide derivatives were studied in aqueous solution and in the presence of micelles using 2-D NMR, CD, and molecular mechanics (Monte Carlo studies). Consistent with previous conformational studies on cyclic opioid agonist glycopeptides, it was seen that glycosylation did not significantly perturb the peptide backbone in aqueous solution, but all four compounds strongly associated with 5-30 mM SDS or DPC micelles, and underwent profound membrane-induced conformational changes. Interaction was also observed with POPC:POPE:cholesterol lipid vesicles (LUV) in equilibrium dialysis experiments. Although the peptide backbones of 1-4 possessed random coil structures in water, in the presence of the lipid phase they each formed a nearly identical pair of structures, all with a stable beta-turn motif at the C-terminus. Use of spin labels (Mn(2+) and 5-DOXYL-stearic acid) allowed for the determination of the position and orientation of the compounds relative to the surface of the micelle.

Modulation of morphine tolerance by the competitive N-methyl-D-aspartate receptor antagonist LY274614: assessment of opioid receptor changes.

Recent reports have demonstrated that the coadministration of morphine with an N-methyl-D-aspartate (NMDA) receptor antagonist can attenuate and/or reverse the development of morphine tolerance. In the present study we used an experimental tolerance paradigm using morphine pellets (75 mg) to produce an 1-fold shift in the morphine dose-response curve in rats. Coadministration of the competitive NMDA receptor antagonist LY274614 [(+-)-6-phosphonomethyl-decahydroisoquinolin-3-carboxylic acid] via continuous s.c. infusion (24 mg/kg/24 hr) significantly attenuated the development of morphine tolerance. In addition, animals made tolerant to morphine and then infused with LY274614 (24 mg/kg/24 hr) regained their analgesic sensitivity to morphine more rapidly than morphine-tolerant animals given a saline infusion. To determine whether LY274614 treatment modifies the subsequent development of tolerance, LY274614 was administered to nontolerant animals for 1 week. One week after LY274614 treatment was discontinued the animals were challenged with morphine and then implanted with morphine pellets. Neither the expression of morphine analgesia nor the development of morphine tolerance differed when LY274614- and saline-treated animals were compared. The infusion of LY274614 for 7 days did not increase the affinity or density of mu, delta, kappa-1 or kappa-3 opioid receptors in rat brain homogenates as measured by ligand binding assays. Additionally, the IC50 values for LY274614 in mu-1, mu-2, delta, kappa-1 or kappa-3 ligand binding assays were greater than 10 microM. Taken together these results demonstrate that the competitive NMDA receptor antagonist LY274614 can both attenuate and reverse the development of morphine tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin receptor distribution reflects social organization in monogamous and polygamous voles.

The neuropeptide oxytocin has been implicated in the mediation of several forms of affiliative behavior including parental care, grooming, and sex behavior. Here we demonstrate that species from the genus Microtus (voles) selected for differences in social affiliation show contrasting patterns of oxytocin receptor expression in brain. By in vitro receptor autoradiography with an iodinated oxytocin analogue, specific binding to brain oxytocin receptors was observed in both the monogamous prairie vole (Microtus ochrogaster) and the polygamous montane vole (Microtus montanus). In the prairie vole, oxytocin receptor density was highest in the prelimbic cortex, bed nucleus of the stria terminalis, nucleus accumbens, midline nuclei of the thalamus, and the lateral aspects of the amygdala. These brain areas showed little binding in the montane vole, in which oxytocin receptors were localized to the lateral septum, ventromedial nucleus of the hypothalamus, and cortical nucleus of the amygdala. Similar differences in brain oxytocin receptor distribution were observed in two additional species, the monogamous pine vole (Microtus pinetorum) and the polygamous meadow vole (Microtus pennsylvanicus). Receptor distributions for two other neurotransmitter systems implicated in the mediation of social behavior, benzodiazepines, and mu opioids did not show comparable species differences. Furthermore, in the montane vole, which shows little affiliative behavior except during the postpartum period, brain oxytocin receptor distribution changed within 24 hr of parturition, concurrent with the onset of maternal behavior. We suggest that variable expression of the oxytocin receptor in brain may be an important mechanism in evolution of species-typical differences in social bonding and affiliative behavior.

Assessment of the in vivo and in vitro opioid activity of bridged hexahydroaporphine and isoquinoline molecules.

Four novel racemic bridged hexahydroaporphine (1 and 2) and isoquinoline (3 and 4) analogues have been synthesized in an attempt to generate bicyclic derivatives of the morphinan ring system. The opioid activity of these analogues has been assessed through membrane-binding studies, in vitro studies in isolated guinea pig ileum and mouse vas deferens, and in vivo studies utilizing the mouse hot plate technique. The bridged isoquinoline precursor molecules were inactive as antinociceptives. Both the racemic phenolic hexahydroaporphine 1 and its 10-methoxy congener 2 demonstrated dose-dependent, albeit weak, antinociceptive activity when administered icv, but they induced lethal convulsions when given subcutaneously. The antinociception elicited by 1 appeared to show very weak opioid character while that caused by 2 was totally nonopioid.

Assessment of the kappa-opioid activity of a series of 6,7-benzomorphans in the rabbit vas deferens.

The purpose of this study was to re-assess the classification in vivo of a series of (-)-normetazocine derivatives as kappa-opioid agonists. From their ability to inhibit contractions of the electrically stimulated rabbit vas deferens, the (2"S)-N-(2"-methoxypropyl) and N-(2"-methoxyisobutyl) derivatives were identified as full agonists. Several partial agonists were discerned, including the (2"S)-N-tetrahydrofurfuryl derivative, Mr2034. (+)-tifluadom, (+/-)-bremazocine, (-)-ketazocine and (+/-)-U-50,488 were used as reference compounds. Structure-activity relationships are discussed.

An assessment of the role of opioid receptors in the response to cannabimimetic drugs.

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs delta 9-tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D-Ala2-Met5-enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108-15 neuroblastoma X glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.

Simultaneous assessment of the opiate-induced modifications in the vertical and horizontal components of locomotor activity in mice.

The effects of some opiates on the horizontal and vertical components of locomotor activity in mice were measured in actometers fitted with photoelectric cells counting horizontal displacements and the striding over a low partition wall. Morphine and fentanyl, used at low doses which did not modify the horizontal locomotor activity, decreased the striding over of the partition wall. Such a dissociation was not observed with either kappa or sigma agonists which affected in the same way the two components of the locomotor activity: both components were inhibited by kappa agonists and slightly stimulated by the sigma agonist SKF 10047. Although the opiate-induced inhibition of the striding over was induced by low doses of morphine, relatively high doses of naloxone were necessary for its antagonism. This apparently difficult antagonism could result in fact from the sedation which appeared when morphine was associated with naloxone.

Assessment of the opiate properties of two constituents of a toxic illicit drug mixture.

The intravenous use of an illicit synthetic drug preparation has caused permanent parkinsonism in a number of addicts. Chemical analysis has revealed the ingredients to be two related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-4-propionoxypiperidine (MPPP). The opiate properties of these two compounds have been assessed using in vitro receptor binding techniques as well as behavioral tests indicative of opiate action, including analgesia, catatonia, respiratory depression and the loss of righting and corneal reflexes. All opiate activity was found to reside with MPPP, which proved to be a potent mu-type agonist. It is concluded that the opiate properties of MPPP alone explain repeated abuse of MPTP/MPPP mixtures by heroin addicts.

Multiple opiate receptor affinities of kappa and agonist/antagonist analgesics: in vivo assessment.

The affinities of kappa and agonist/antagonist (Ag/Ant) analgesics for mu and delta opiate receptors were examined in vivo in the rat. In the case of kappa agonists, these agents appear to be mu and delta antagonists in vivo. The mu antagonist activity appears to involve a specific isoreceptor population, namely mu-2 receptors. With Ag/Ant analgesics, a more complex pharmacology is evident such that at mu and delta receptor populations these agents can exhibit pure Ag, pure Ant or a combination of Ag and Ant actions. These activities vary with the neuronal localization of the receptor population being examined. In addition, complex species differences are evident with Ag/Ant actions.

Differential regulation of the mu-, delta-, and kappa-opiate receptor subtypes by guanyl nucleotides and metal ions.

The effects of Na+, guanyl-5'-yl imidodiphosphate (Gpp(MH)p), and Mn2+ on the binding of dihydromorphine, ethylketocyclazocine, D-Ala2, D-Leu5-enkephalin, and diprenorphine to the opiate receptor were investigated. Three distinct binding sites, mu, delta, and kappa sites, were identified with the use of multiple tracer displacement curves. Moreover, this approach was used to determine the effects of Gpp (NH)p and metal ions on each individual binding site. At the mu and delta sites, Na+ and Gpp(NH)p each decreased and Mn2+ increased agonist binding affinities, with the exception of D-Ala2, D-Leu-enkephalin affinity which was not affected by Gpp(NH)p. None of these conditions markedly altered dihydromorphine and D-Ala2, Leu5-enkephalin binding to kappa sites, whereas the affinity of ethylke-tocyclazocine for kappa sites was decreased by Gpp(NH)p. Sodium ions lowered the capacity of mu sites and Gpp(NH)p reduced that of delta sites, while both agents increased the capacity of apparent kappa sites. These results demonstrate that each of the kinetically distinguishable binding sites is regulated differentially by metal ions and guanyl nucleotides. Simultaneous addition of Na+ and Gpp(NH)p greatly reduced the binding affinity of all three agonists at their respective high affinity sites (dihydromorphine at the mu site, D-Ala2, D-Leu5-enkephalin at the delta site, and ethylketocyclazocine at the kappa and mu sites). This result confirms previous observations that agonist binding is characterized by a large affinity reduction in the presence of both Na+ and guanyl nucleotides, and it extends this concept to each of the opiate receptor subtypes.

Identification of beta-endorphin-6(16-17) as the principal metabolite of des-tyrosin-gamma-endorphin (DTgammaE) in vitro and assessment of its activity in neurotransmitter receptor binding assays.

Des-tyrosine-gamma-endorphin (beta-endorphin-(2-17); DTgamma E) lacks direct in vitro activity at dopaminergic receptors, but does inhibit in vivo [3H]spiperone binding in various rat brain areas. The principal objective of these studies was to test the hypothesis that DTgammaE may exert its selective, neuroleptic-like activity through an active metabolite. Accordingly, DTgammaE was incubated at 37 degrees C in a whole rat brain homogenate of neutral pH after which samples were prepared for HPLC analysis. The major, heat-stable metabolite of DTgammaE was identified as the clinically active, beta-endorphin related fragment, beta-endorphine-(6-17). The beta-endorphin sequences 4-17, 5-17, l0-17, 12-17 and 2-16 were also present but in minor amounts. Identical results were obtained studying DTgammaE metabolism using rat striatal tissue slices. Neurotransmitter receptor binding experiments showed that beta-endorphin-(6-17) was inactive at central dopaminergic, serotonergic, muscarinic, benzodiazepine and opiate receptors measured in vitro. Thus, like DTgammaE, beta-endorphin-(6-17) differs from classical neuroleptics in that it does not inhibit in vitro [3H]spiperone binding in the corpus striatum, frontal cortex or mesolimbic areas of the rat brain. It may be that DTgammaE and beta-endorphine-(66-17) exert their selective neuroleptic-like activity through an indirect inhibition of central dopaminergic activity, possibly in combination with an in vivo antagonism of the postsynaptic dopamine receptor.