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The chimeric peptide EN-9 was reported as a κ-opioid/neuropeptide FF receptors bifunctional agonist that modulated chronic pain with no tolerance. Many lines of evidence have shown that the effect of the κ-opioid receptor is mediated by not only its specific activation but also downstream events participation, especially interaction with the μ-opioid receptor pathway in antinociception and tolerance on most occasions. The present study investigated the acute and chronic cross-tolerance of EN-9 with μ-opioid receptor agonist EM-2, DAMGO, and morphine after intracerebroventricularly (i.c.v) injection in the mouse tail-flick test. In the acute tolerance test, EN-9 showed symmetrical acute cross-tolerance to DAMGO but no cross-tolerance to EM2. In the chronic tolerance test, EN-9 had no tolerance after eight days of repeated administration. However, EN-9 illustrated complete cross-tolerance to morphine and symmetrical cross-tolerance to EM2. In addition, inhibition of NPFF receptor could induce the tolerance development of EN-9. These findings indicated that supraspinal EN-9-induced antinociception contains additional components, which are mediated by the downstream μ-opioid receptor pathway both in acute and chronic treatment, whereas the subtypes of μ-opioid receptor or NPFF system pathway involved in antinociceptive effects induced by EN-9 are complex. Identifying the receptor mechanism could help design preferable bifunctional opioid compounds., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Environmental factors contribute to autism spectrum disorder. Fentanyl, one of the most widely used opioid analgesics in anaesthesia, can induce neurotoxicity, but its role in autism remains unknown. We determined whether fentanyl induced autism-like behaviours in young mice and the underlying mechanisms., Methods: Young male and female mice received fentanyl at postnatal days 6, 8, and 10, and performed behavioural tests, including three-chamber social preference, elevated plus maze, grooming behaviour, and open-field test, from postnatal days 30-32. Expression of Grin2b, the gene encoding the GluN2B subunit of the N-methyl-d-aspartate receptor, was assessed in the anterior cingulate cortex of male mice using fluorescence in situ hybridisation histochemistry. We used bisulfite target sequencing to determine Grin2b hypermethylation sites after fentanyl treatment. In the specific activation and rescue experiments, we injected the mu opioid receptor agonist [D-Ala, 2 N-MePhe, 4 Gly-ol]-enkephalin (DAMGO) or Grin2b overexpression lentivirus into the anterior cingulate cortex of male mice., Results: Fentanyl induced autism-like behaviours in both young male and female mice, and downregulated Grin2b expression (0.49-fold [0.08] vs 1.00-fold [0.09]; P<0.01) and GluN2B protein amounts (0.38-fold [0.07] vs 1.00-fold [0.12]; P<0.01) in the anterior cingulate cortex through hypermethylation of Grin2b. The mu-opioid receptor antagonist naloxone and overexpression of Grin2b in anterior cingulate cortex attenuated the fentanyl-induced effects, whereas DAMGO injection into the anterior cingulate cortex induced autism-like behaviours., Conclusions: These data suggest that fentanyl induces autism-like behaviours in young mice via an epigenetic mechanism. Further research is required to determine possible clinical relevance to autism risk., (Copyright © 2022 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Radioactive ligand binding assays are the most commonly applied method for the determination of binding affinities of compounds at a particular receptor. While they are highly sensitive and high-throughput capable they come with major disadvantages due to the radioactive ligands utilized. Here we present the development of a mass-spectrometry-based binding assay for the determination of binding affinities at the human μ-opioid receptor using non-labelled DAMGO ([D-Ala 2 , N-MePhe 4 , Gly 5 -ol]-enkephalin). The runtime of the LC-MS/MS method was 5.5 min per data point and allowed for the highly sensitive detection of 38.5 fg DAMGO on column. The assay shows low non-specific binding and the equilibrium dissociation constant of DAMGO was 0.57 nM. The assay was applied to determine the K i values of 17 opiates/opioids and the results were in good agreement with the data from radioactive receptor binding assays published in the literature. Additionally, the K i value of six 2-benzylbenzimidazoles, including the widely abused designer opioid isotonitazene, were determined ranging from 0.654 to 72.9 nM. Consequently, the developed assay provides a suitable alternative to radioactive binding assays as it allows for a reliable and rapid determination of receptor binding affinities of e.g. newly emerging designer opioids., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A number of G protein-coupled receptors have been shown to stimulate tuberin phosphorylation, which is critical for the regulation of translation and is apparently involved in neurotrophin-promoted survival of serum-deprived cells. Here, in HEK 293 cells transiently expressing the delta-, kappa-, or mu-opioid receptors, Western blotting analysis using a phosphospecific anti-tuberin antibody revealed a dose- and time-dependent increase in tuberin phosphorylation upon stimulation by specific opioid agonists. In NG108-15, PC12, and SH-SY5Y cells that endogenously express delta-, kappa-, and mu-opioid receptors, respectively, specific opioid agonists also stimulated tuberin phosphorylation in a dose- and time-dependent manner. Pretreatment of cells with pertussis toxin or PI3K inhibitor wortmannin blocked the opioid-stimulated tuberin phosphorylation, implicating the possible involvement of the G(i/o) proteins and the phosphatidylinositol-3 kinase/Akt pathway in opioid-induced tuberin phosphorylation. This is the first study that demonstrates the regulatory role of opioid receptors on tuberin.

Opioid binding properties of Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalin-framed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [(3)H][Ile(5,6)]deltorphin II (K(i) = 8 nM), whereas its binding to the mu ([(3)H]DAMGO) and kappa ([(3)H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [(3)H][Ile(5,6)]deltorphin II binding sites (96% blocking at 10 microM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile(5, 6)]deltorphin II-stimulated [(35)S]GTPgammaS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [(35)S]GTPgammaS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors., (Copyright 1999 Academic Press.)

Enkephalins are opioid peptides that bridge the neuroendocrine and immune systems. Using flow cytometry and a fluorescein conjugate of the endogenous pentapeptide methionine-enkephalin (ME), we have identified enkephalin receptors on cultured human lymphocytes. Cell surface recognition sites that bound ME with high affinity and specificity were detected for NALM 6 (pre-B acute lymphoblastic leukemia) and Jurkat (T lymphoma) cells. Brain-like enkephalin receptors were measured for these lymphocytes using conventional radioligand-receptor assays and the highly delta opioid receptor-selective enkephalin analog [3H]DPDPE. Upon activation, the lymphocyte enkephalin receptors transmitted signals that enhanced the accumulation of intracellular cAMP. These studies provide evidence that cultured human lymphocytes of the B (NALM 6 cells) and T (Jurkat cells) lineages express functional enkephalin receptors and suggest that such receptors may be instrumental in the lymphocyte response to opioid peptides and alkaloids., (Copyright 1999 Academic Press.)

These studies were undertaken to determine the effects of mu-opioid receptor depletion through irreversible alkylation on the dose-effect curve for heroin self-administration. Heroin maintained responding in rats with an inverted U-shaped dose-effect curve and administration of 10 nmol of beta-funaltrexamine i.c.v. (beta-FNA) significantly increased the ED50 on the ascending limb from 1.9 to 5.3 micrograms/infusion, and from 24.3 to 211.8 micrograms/infusion on the descending limb. Administration of saline i.c.v. produced no effect on heroin self-administration. Administration of 40 nmol of beta-FNA increased the ED50S from 5.1 to 33.9 and from 14.4 to 502.8 micrograms/infusion on the ascending and descending portions of heroin's dose-effect curve, respectively. beta-FNA (40 nmol, i.c.v.) had no effect on cocaine self-administration. [3H]DAMGO binding density was decreased in the caudate and nucleus accumbens by 29 or 54% 24 h after administration of 10 or 40 nmol of beta-FNA i.c.v., respectively. The effects of beta-FNA on heroin self-administration were completely overcome by increasing the dose of heroin however, as the shape and slope of the self-administration dose-effect curve was not different when higher doses of heroin were made available for self-administration compared to control data or saline administration. Therefore, there appear to be spare mu-opioid receptors for heroin for the production of its reinforcing effects in rats. Furthermore, the self-administration dose-effect curves returned to control values prior to the return of [3H]DAMGO binding, further suggesting that the full complement of mu-opioid receptors is not necessary for heroin to produce its reinforcing effects. These findings support the existence of spare mu-opioid receptors for heroin in maintaining self-administration in rats.

A new series of rigid analogues (1a-g, 2a-g) of the previously reported analgesic 3-cinnamyl-8-propionyl-3,8-diazabicyclo[3.2.1]octane (I) and its reverted isomer 3-propionyl-8-cinnamyl (II) were synthesized, in which the cinnamyl substituent is incorporated in benzocondensed bicyclic systems. Binding assays for the affinity towards mu receptors indicated that, while in the reverted series 2 the beta-naphthylmethyl (2d) and the benzocycloheptenylmethyl derivative (2g) favorably compared with II, all compounds 1 displayed a mu-affinity lower than that of the parent I. Modeling studies suggest that the flexibility of the cinnamyl side chain plays an important role for activity.

Endogenous opioids exert a variety of functions outwith the central nervous system, including modulation of some murine lymphocyte functions. The results of this study indicate that mu-, delta- and kappa-receptor selective agonists are potent in vitro stimulators of mitogen-induced proliferation of murine T-lymphocytes. Moreover, the observed enhancement of mitogen-induced proliferation was reversed by mu-, delta- and kappa-receptor class selective antagonists, beta-funaltrexamine, ICI 174,864 and nor-binaltorphimine, respectively. An additional study has revealed that repeated administration (four injections) of the opioid receptor selective agonists DAGO, DPDPE and U-50488 also enhanced the concanavalin A-induced proliferation of lymphocytes. These results suggest that there are three classes of opioid receptors on T-lymphocytes and that all these receptor classes are involved in the stimulation of concanavalin A-induced proliferation.

The effects of methionine-enkephalin and the selective agonists of mu-, delta- and kappa-opioid receptor subtypes [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, [D-Pen(2,5)]enkephalin U-50488 on the production of nitrite by activated peritoneal murine macrophages were studied. Macrophages were activated with interferon-gamma plus lipopolysaccharide in the presence or absence of graded concentrations of opioids. Methionine-enkephalin and mu-; delta- and kappa-agonists combined with interferon-gamma plus lipopolysaccharide caused an increase in nitrite release from cultured macrophages. Only 10 mM U-50488 led to a decrease in nitrite release from interferon-gamma and LPS-stimulated macrophages. This effect was not produced in a naloxone-sensitive manner. The opioids added to the fresh culture 8 h after the stimulation of macrophages by interferon-gamma plus lipopolysaccharide--when an inducible form of nitric oxide synthase activity is presumably expressed--did not alter the rate of nitrite production. This suggests that the effect of opioids on nitric oxide synthase is produced at the transcriptional level. The opioid receptor antagonist naloxone reduced the stimulatory effect of opioids on nitrite production by stimulated macrophages. Opioids added to the culture of resting macrophages did not change nitrite release from macrophages which were later induced with interferon-gamma plus lipopolysaccharide. The results of this study suggest that methionine-enkephalin can modulate the immune response by controlling, via opioid receptors, the production of nitric oxide.

Opioid agonists either potentiate or suppress basal cAMP production in SK-N-SH cells. The inhibitory effect is mediated by PTX-sensitive GTP-binding proteins, while the stimulatory effect involves Ca++ entry and calmodulin activation. Both pathways can be activated simultaneously by opioid agonists. Low (nM) concentrations of either mu (DAMGO) or delta (DPDPE) selective opioids potentiate cAMP formation. At higher (100 nM) concentrations, however, a net suppression takes over; this suppression can be eliminated by PTX, and the underlying stimulatory effect is disclosed. Micromolar concentrations of either mu or delta selective agonists cross-activate the other (delta or mu) receptors, and augment the stimulatory pathway. The overall outcome (either stimulation or inhibition of cAMP production) is dependent on the balance between the two overlapping pathways, and can be modified by blocking either of the two opposing mechanisms.

Animals from two substrains of AB mice, i.e., ABH/Md and ABG/Md, differ in the occurrence of aggressive behavior. After maturation, male ABH mice regularly exhibited abnormal aggressive behavior making group-housing impossible. In contrast, ABG animals never showed such behavioral patterns. To elucidate the role of opioid mechanisms, we tested the reaction of these animals to morphine in the hot plate test. Moreover, specific DAMGO binding was measured. It was shown that mice from control groups differed significantly in reaction to the thermal stimulus. ABH mice had significantly longer reaction times. With increasing doses of morphine this difference disappeared, suggesting different levels of basal activity in endogenous opioid systems. This is underlined by significantly lower DAMGO binding in aggressive ABH mice. The results suggest that differences in endogenous opioid systems may account for differences in aggressiveness.

The highly potent and efficacious mu-opioid agonist fentanyl was SC infused into rats with submaximal analgesic doses (0-1.14 mumol/kg/day) continuously for 8 days, checked by the constant daily urinary recovery of intact drug (0.43 +/- 0.031% of the daily dose). Tail-flick latencies measured at 24 (day 1) and 48 h (day 2) after starting the infusion were increased in a dose-dependent fashion compared with those before the infusion (day 0). However, at day 8, the latencies were increased only weakly, not significantly, revealing tolerance to the antinociceptive activity of fentanyl. Fentanyl at all doses showed no significant effect on the capacity (Bmax) and affinity (Kd) of the mu-opioid receptor binding of DAMGO to whole brain (Bmax 126.2 +/- 3.00 fmol/mg protein, Kd 1.00 +/- 0.04 nM) and spinal cord (Bmax 48.24 +/- 2.71 fmol/mg protein, Kd 1.93 +/- 0.13 nM) membranes gained from the rats after killing them at day 8. Gpp(NH)p increased the Kd for brain and spinal cord sites by 3.09 and 2.65, respectively, independent of the fentanyl dose. The infusion with fentanyl did not after the basal and forskolin-stimulated adenylate cyclase activity in the whole brain membranes, nor did it change the inhibition of the forskolin-stimulated activity by DAMGO. It is concluded that, in rats, constant long-term body levels of highly potent mu-agonists result in a tolerant state that, however, does not produce overall changes in the parameters of their specific receptor sites in the CNS, i.e., receptor capacity and affinity, and in the events closely related to them, i.e., their regulation by GTP and of adenylate cyclase. This does not exclude such possible changes to be restricted to specific regions in the CNS.

The ability of neuropeptide FF (NPFF) to modulate mu- and delta-opioid-induced analgesia by intracerebroventricular administration was compared in adults and 14-day-old mice. In adults, opioid-induced analgesia was predominantly mediated by mu-receptors whereas mu- and delta-receptors were equally involved in pups. An NPFF analog, 1 DMe, reduced the analgesic effect of DAGO and [D.Ala2]deltorphin-I, mu and delta selective agonists respectively. However, a high dose of 1DMe (22 nmol) increased both morphine and [D.Ala2]deltorphin-I-induced analgesia. Dose-response curves for 1DMe in the presence of naltrindole or naltrexone, delta- and mu-opioid selective antagonists respectively, indicate that 1DMe preferentially reversed mu-receptor-mediated but increased delta-receptor-mediated analgesia. These findings demonstrate differences in control of mu- and delta-induced analgesia by NPFF receptors.

The present study examines the stimulatory effect of opioids on adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in the human neuroblastoma cell line SK-N-SH, and its dependence on calcium. We show that, in this culture, the mu-opioid selective agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin stimulates cyclic AMP production by 30% in a naloxone-reversible manner. This stimulation is completely dependent on calcium and involves the activation of calcium/calmodulin since it is abolished in the presence of EGTA, calcium channel blockers or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). The results suggest that the activation of calcium/calmodulin dependent adenylyl cyclases by opioids in SK-N-SH cells is secondary to the induction of calcium influx and the consequent elevation of intracellular calcium level.

The hypothesis that mu-opioid agonists having low antinociceptive efficacy might be more susceptible to interference with G-protein coupling than mu-opioid agonists having higher antinociceptive efficacy was tested. Supraspinal antinociceptive efficacy for the three mu-opioid agonists morphine, [D-Ala2, NMePhe4, Gly5-ol]-enkephalin (DAMGO) and sufentanil in the mouse 55 degrees C warm-water tail-flick test was evaluated 18-24 h after intracerebroventricular (i.c.v.) administration of beta-funaltrexamine (beta-FNA). The beta-FNA pretreatment (0.2-2.0 nmol) attenuated antinociception in the order morphine > DAMGO > sufentanil, consistent with previous reports of their relative antinociceptive efficacy. The association of efficacy with G-protein coupling was then assessed by determining sensitivity to i.c.v. (0.1-3.0 micrograms) pertussis toxin (PTX) or cholera toxin (CTX). The effect of PTX on equiantinociceptive doses was in the inverse order of agonist efficacy. CTX augmented sufentanil-induced antinociception. Morphine- and DAMGO-induced antinociception were unaffected by CTX. These data suggest that: (i) highly efficacious mu agonists (viz., sufentanil) couple more efficiently to PTX-sensitive inhibitory Gi-proteins than do agonists of lower efficacy (viz., morphine, DAMGO) and (ii) highly efficacious mu agonists have greater capacity to utilize CTX-sensitive stimulatory Gs-proteins than do mu-agonists with lower efficacy.

C57BL/6J (B6) mice self-administer substantial quantities of morphine compared to DBA/2J (D2) mice, and most of the genetic component of this strain difference has been attributed to a locus on chromosome 10 in the vicinity of the mu opioid receptor gene. To compare binding characteristics of mu opioid receptor populations between the two strains, mice were given single daily injections of a long-acting preparation of morphine sulfate (80 mg/kg, s.c.) or saline for a period of seven days, and euthanatized six hours after the last injection. Brains were removed and dissected into specific regions. Receptor binding studies were performed on frontal cortex and striatum. Data were analyzed using non-linear regression, and Kd and Bmax comparisons made between strains and treatments. Specific [3H]DAMGO binding in striatum indicates that the density of mu opioid receptors in saline-treated B6 mice and saline-treated D2 mice does not differ significantly. After repeated morphine injection, B6 mice exhibited a decrease in striatal [3H]DAMGO binding, indicating a downregulation of receptor density by approximately 45% (p=.0003 vs saline-treated B6), a phenomenon not observed in D2 mice. In frontal cortex, no differences in [3H]DAMGO binding were observed between strains or treatment groups. These results demonstrate a significant difference between mu opioid receptor regulation in B6 and D2 mice, and may underlie well documented strain differences in specific opioid-related behaviors.

Studies of human and animal central nervous system receptor degradation and measurement postmortem are important as human tissue can rarely be collected under ideal experimental conditions. Correlating the change in binding of opioid receptor subtypes over time will help define the conditions under which human studies may be valid. The present study was designed to investigate the rate at which opioid receptor subtypes degrade postmortem. Brains from rats or cerebelli from guinea pigs were kept at 22 degrees C or 4 degrees C at times from zero to 24 hours to simulate two common human collection techniques; room temperature and morgue refrigeration. Tissue homogenates from these brains were analysed for mu1, mu2, delta, kappa1 and kappa3 opioid receptor binding using standard radioligand binding techniques. At room temperature mu1, mu2 and delta opioid receptor binding was reduced between 6 and 12 hours, whereas kappa1 and kappa3 binding was reduced after 24 hours. At 4 degrees C mu1, mu2, delta, kappa1 and kappa3 binding remained constant over the 24 hour period.

Guinea pig ileum longitudinal muscle/myenteric plexus preparations contain functional mu- and kappa-opioid receptors. A preparation with blocked kappa receptors was obtained by pretreating guinea pigs with 0.1-10.0 mg/kg intraperitoneal doses of a new kappa-selective affinity label, DIPPA (2-(3,4-dichlorophenyl)-N-methyl-N-[1S-1-(-3-isothiocyanatophenyl)-2-(-1 -pyrrolidinyl) ethyl] acetamide). Determination of IC50 values for the mu-selective agonist DAMGO ([D-Ala2,MePhe4,Gly(ol)5]-enkephalin) and the kappa-selective agonist U 69,593 ([5alpha,7alpha,8beta]-(+)-N-methyl-N-[7-(1-pyrrolidin yl)-1-oxaspiro-(4,5)-dec-8-yl]) showed that 0.5 mg/kg DIPPA at 48 h produced reliable, near-complete blockade of kappa-opioid activity but a minimal shift for kappa-opioid agonism. This new assay should be useful for studying mixed agonists/antagonists that produce strong kappa-opioid receptor agonism, which prevents determination of mu-opioid receptor antagonism.

The tail-flick response is a spinal reflex that can be modulated by administration of antinociceptive agents supraspinally through activation of descending systems and involvement of the action of neurotransmitters in the spinal cord. Descending noradrenergic and serotonergic systems are involved in morphine (and other mu opioid receptor agonists)-induced antinociception. These descending systems, however, are not involved in supraspinal delta opioid receptor agonist-induced antinociception. Recently, a descending system mediated by spinal gamma-aminobutyric acid (GABA) A and B receptors has been demonstrated to be involved in the antinociceptive action of delta 1 opioid receptor agonists ([D-Pen2,5]enkephalin in ICR mice and [D-Pen2,5]enkephalin and heroin in Swiss-Webster mice). In the present study, the involvement of spinal GABAA receptors in the antinociceptive action of supraspinal delta 2 opioid receptor agonists, [D-Ser2]-Leu-enkephalin-Thr and 6-monoacetylmorphine, action was demonstrated. The intrathecal administration of GABAA receptor antagonists, bicuculline and picrotoxin, inhibited the antinociceptive action of both [D-Ser2]-Leu-enkephalin-Thr and 6-monoacetylmorphine given intracerebroventricularly. The intrathecal administration of 2-hydroxysaclofen, a GABAB receptor antagonist, had no effect. These studies suggest that supraspinal delta 2, like delta 1, opioid receptor action involves spinal GABAA receptors, but delta 2, unlike delta 1, action does not involve GABAB receptors. Thus, the supraspinal delta 1 agonist action (heroin, DPDPE) and the delta 2 agonist action (6MAM, DSLET) can be further differentiated by the selectivity of the spinal GABA receptors involved in Swiss-Webster mice.

Two experiments, using centrally administered [D-Ala2-MePhe4-Gly(ol)5]enkephalin (DAMGO), a selective mu-opioid agonist, assessed the thermoregulatory consequences of cold acclimation. Experiment 1 assessed whether cold acclimation influenced DAMGO hyperthermia at room temperature. Sialo-adenectomized rats were implanted with ICV cannulae and IP Mini-Mitters. After 3 weeks of exposure to 5 degrees C (cold acclimation) or 22 degrees C (non-cold acclimation) rats were pretreated with IP naltrexone HCl (2 mg/kg b.wt.) or vehicle (0.15 M saline) and later administered a 5-microliters ICV injection of 0.15 M saline, 0.1, or 1.0 microgram DAMGO. Cold acclimation exerted little effect on core temperature but potentiated DAMGO hyperthermia in a dose-dependent, naltrexone-reversible, activity-independent manner. Experiment 2 assessed the effect these same manipulations exerted on operant escape from a convective source of mild heat (37 degrees C). Duration of heat escape increased with cold acclimation in a naltrexone-resistant manner, yet was not influenced by DAMGO in either non-cold-acclimated or cold-acclimated rats. These findings suggest that two central adaptations occur with cold acclimation: A non-mu-opioid process that increases heat sensitivity and a mu-opioid process that potentiates hyperthermia but fails to alter heat escape due to mu-opioid-mediated analgesia.

To elucidate further the role of mu-opioid receptors in mediating analgesia and cardiovascular function at rest and during stress, rats were pretreated ICV with either saline (5 microliters) or beta-funaltrexamine (beta-FNA, 5 nmol/5 microliters), a noncompetitive opioid receptor antagonist that inactivates irreversibly mu receptors, 2 days prior to [D-Ala2, N MePhe4, Gly5-ol]enkephalin (DAMPGO, 1 nmol, ICV) administration. Pretreatment with beta-FNA blocked DAMPGO-induced analgesia as measured by the tail-flick test. DAMPGO also produced an increase in blood pressure (BP), sympathoadrenal outflow, and a bradycardia. Pretreatment with beta-FNA converted the DAMPGO-induced bradycardia to a tachycardia, significantly reduced the DAMPGO-induced increase in epinephrine by 60%, and the norepinephrine response by 45%, and attenuated mildly the increase in BP due to DAMPGO. In saline-treated rats, restraint stress evoked an increase in HR, BP, and plasma catecholamines. Pretreatment with beta-FNA partially attenuated the increase in HR in response to stress. In the presence of DAMPGO, restraint stress resulted in a further bradycardia, which was significantly blocked by pretreatment with beta-FNA. Stress also produced increases in BP and plasma catecholamines, which were not prevented by pretreating rats with beta-FNA. These results indicate that beta-FNA may not have inactivated all the receptors accessible to DAMPGO which control BP, or alternatively, beta-FNA may selectively inactivate a subtype of mu receptors. In addition, brain mu opioid receptors appear to be significantly involved in mediating supraspinal analgesia and regulating parasympathetic outflow to the heart and sympathoadrenal release of catecholamines.

The effect of intact enkephalin (MENK) molecule or its metabolite Tyr-Gly-Gly (TGG) as well as the effect of synthetic agonist for opioid receptor subtypes (DADLE and DAGO) on superoxide anion release from human neutrophils has been investigated. In lower MENK concentrations, where MENK alone had no effect on O2- release, inhibition of enkephalinase by thiorphan significantly increased O2- production, while in higher concentrations, where MENK alone was effective, inhibition of enkephalinase had no effect. Aminopeptidase inhibition by bestatin did not influence O2- release from MENK treated PMNs. While MENK predominantly stimulated, TGG suppressed O2- release. Opioid antagonist naloxone (10(-5) M) abrogated the effect of MENK on O2- release. DADLE (delta receptor agonist) increased O2- release in 10(-11) M concentration, while DAGO (mu receptor agonist) had no effect in any concentration examined. Enkephalinase inhibition increased O2- production from DADLE but not from DAGO treated PMNs. It seems, therefore, that free radical production is mainly associated with the delta subtype of the opioid receptor. Also, our observations support the hypothesis that enkephalinase might be the enzyme selectively responsible for regulating effects of enkephalins.

The site of interaction of opioids and inhalation anaesthetic agents is unknown, but may be at the level of the opioid receptor. In this study we have used SH-SY5Y human neuroblastoma cells, which express both mu and delta receptors, to examine the effects of halothane on the receptor binding profiles of [3H]diprenorphine (DPN), an opioid receptor antagonist, and [3H] [D-Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO), a mu receptor selective agonist. Binding of [3H]DPN and [3H]DAMGO was performed at 37 degrees C for 60 min in the presence of air, nitrous oxide (75%) or air containing halothane (0.5-5.0% v/v). Compared with air controls, neither 75% nitrous oxide nor 0.5, 1.0, 2.0 and 5.0% halothane influenced DPN binding variables. Binding of [3H]DAMGO was unaffected by 1.0% halothane, but 5.0% halothane reduced the affinity, with a modest increase in Kd (1.15 (0.16) to 1.7 (0.2) nmol litre-1) without effect on Bmax. Our data suggest that the site of opioid and volatile anaesthetic interaction is not at the opioid receptor.

The present investigation was aimed at elucidating if mu-, delta- and kappa-opioid receptors are involved in the effects of DAMGO (selective mu-agonist), DPDPE (selective delta-agonist) and U-50,488H (selective kappa-agonist) in isolated electrically driven human right atria strips. The negative inotropic effects induced by the opioid agonists used in this study were not antagonized in presence of naloxone (preferentially mu-antagonist), naltrindole (selective delta-antagonist) and norbinaltorphimine (selective kappa-antagonist). These data suggest that the opioid receptors are not involved in the cardiac depressant effects induced by mu-, delta- and kappa-opioid agonists.

The effect of opioids on NO production by LPS-stimulated murine macrophages J774 has been investigated. Morphine (mu and k opioid receptor agonist), DAGO (selective mu receptor agonist) and U50-488H (selective k receptor agonist), added (10(-10)-10(-6) M) to the cells 0.5 h before activation with LPS, significantly inhibited NO production. This effect was reverted by naloxone (10(-12)-10(-8) M), an opioid specific antagonist. In contrast, DPDPE and deltorphin II respectively delta 1 and delta 2 receptor agonists (10(-10)-10(-6) M) did not affect NO generation. Morphine was not able to inhibit NO production when added after LPS challenge. The results of the present study indicate that opioids are able to inhibit NO formation in LPS-activated macrophages through the involvement of specific opioid receptors. Moreover, the ability of morphine to inhibit NO production only when given before LPS challenge suggests that the opiate inhibits the induction but not the activity of the inducible NO synthase.

We examined the effects of an exogenous mu opioid agonist and antagonist on systemic physiology and neurological outcome following TBI in the rat. Experiment I: [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) (0.1 nMol or 0.3 nMol in 5 microliters) (n = 10) or artificial CSF (n = 10) was administered 5 min prior to fluid-percussion brain injury (2.1 atmospheres). Motor performance was assessed on days 1-5 after TBI. The mu receptor agonist, DAMGO significantly reduced both beam-walking latency and body weight loss after injury (p < 0.05). DAMGO-treated rats (n = 5) did not differ from CSF-treated rats (n = 5) on either systemic arterial blood pressure or heart rate responses to injury. Experiment II: Beta-funaltrexamine (beta-FNA) (20.0 nMol in 5.0 microliters) (n = 10) or artificial CSF (n = 10) was administered (icv) to rats 5 min prior to fluid-percussion brain injury (1.8 atmospheres). Motor performance was assessed on days 1-5 after TBI. The mu receptor antagonist, beta-FNA, significantly increased beam-walking latency after injury (p < 0.05). beta-FNA-treated rats (n = 5) did not differ from CSF-treated rats (n = 5) on either systemic arterial blood pressure or heart rate responses to injury. Experiment III: Neither beta-FNA nor DAMGO affected motor performance in uninjured rats. These results suggest that activation of mu opioid receptors by exogenous agonists may provide protection against deficits in motor performance produced by fluid percussion brain injury.

The effect of chronic opioid antagonist treatment on the analgesic potency of six opioid agonists was compared to changes in opioid receptor density and the selectivity of each agonist for mu (DAMGO), delta (DPDPE) and kappa (U69,593) opioid receptors. Mice were implanted SC with a 15-mg naltrexone or placebo pellet for 8 days. The pellets were removed and 24 h later, mice were sacrificed and binding studies were conducted, or mice were tested in analgesia (tail-flick) dose-response studies. All six analgesics acted as full agonists for both placebo and naltrexone-treated mice. Naltrexone increased the analgesic potency of methadone, etorphine, fentanyl, meperidine, and oxycodone by 1.9-3.2-fold. The analgesic potency of propoxyphene was not increased significantly (1.3-fold). In saturation binding studies in brain homogenate, naltrexone increased the Bmax of mu, delta, and kappa opioid receptors by 86, 43, and 33%, respectively, without altering Kd. Competition binding studies for each receptor type were conducted in brains from untreated mice, and KIs were determined for each agonist. All agonists had greatest selectivity toward mu compared with delta and kappa receptors. There did not appear to be an obvious relationship between receptor selectivity and the magnitude of supersensitivity. These studies indicate that supersensitivity occurs for a broad range of opioid analgesics following chronic opioid antagonist treatment in the mouse. However, the selectivity of these agonists for mu, delta, and kappa receptors does not appear to correlate with differences in supersensitivity.

Evaluation of the effects of tolerance to morphine in vivo on free intracellular calcium levels [(Ca++)i] in whole brain synaptosomes from rats and synaptosomes from brain regions and spinal cords of both rats and mice indicated that in whole brain and spinal cord synaptosomes, [Ca++]i levels are significantly higher in morphine-tolerant mice. Although levels do not differ between nontolerant and tolerant preparations from the rat spinal cord, [Ca++]i brain levels are significantly higher in morphine-tolerant rats. Evaluation of three brain regions from both nontolerant and tolerant rats and two brain regions from mice indicated no significant differences in basal [Ca++]i, with the exception that lower basal levels of calcium were observed in the midbrain of the tolerant rat. In vivo, mice tolerant to morphine (SC) were cross tolerant to DAMGO (IT and ICV). Cross tolerance between morphine and DAMGO was observed in vitro. No cross-tolerance was observed between morphine (SC) and the delta-agonist, DPDPE (IT or ICV), or the kappa-agonist, U50,488H (U50, IT or ICV) in vivo. Similarly, no cross tolerance was observed between morphine (SC) and DPDPE or U50 in vitro. These data indicate that tolerance in vivo induced a change in synaptosomal calcium modulation such that tolerance could be observed in vitro. Thus, a membrane component may be altered by the development of tolerance to morphine leading to an alteration in [Ca++]i in both the brain and spinal cord.

We investigated the potential influence of opioid and melanotropic peptides on endogenous norepinephrine (NE) release from the A2 noradrenergic cell group of rats using a static, fixed-volume incubation procedure. Norepinephrine release from slices of caudal dorsomedial medulla (CDMM) was evoked by high potassium concentrations (20 and 60 mM) in a Ca(2+)-dependent and dose-related manner. Treatment with the potent melanotropin agonist [Nle4,D-Phe7] alpha-MSH(NDP-MSH) had no effect on K(+)-induced NE release. In contrast, human beta-endorphin1-31 significantly reduced K(+)-stimulated NE release, but not in the presence of naloxone. The highly-selective mu-opioid agonist [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) also significantly reduced evoked NE release. The inhibitory effect of DAMGO was completely blocked by naloxone. Naloxone alone did not alter evoked NE release. The inhibitory effect of DAMGO was not enhanced by reducing the stimulatory concentration of K+. None of the peptides tested influenced basal NE release. These data indicate that melanotropin receptors do not regulate NE release in CDMM. In contrast, the opioid peptides DAMGO and beta-endorphin inhibit K(+)-stimulated release of endogenous NE. These data suggest a role for mu-opioid receptors in controlling NE release from A2 noradrenergic neurons.

Two opioid peptides were generated by in vitro pepsin treatment of bovine hemoglobin. These peptides were identified using a GPI test and purified using HPLC chromatographic techniques. They correspond to fragments 31-40 (LVV-hemorphin-7) and 32-40 (VV-hemorphin-7) of the beta-chain of bovine hemoglobin. Binding experiments strongly confirm that VV-hemorphin-7 and LVV-hemorphin-7 are opioid peptides since they inhibited [3H]naloxone binding to rat brain membranes. Our results indicate that VV-hemorphin-7 and LVV-hemorphin-7 exhibit a lesser potency both in GPI and binding tests. Selectivity and affinity of these purified peptides and synthetic hemorphin-7 for opioid receptors is discussed.

Patients receiving opioids report feeling sleepy, but opioids actually inhibit the rapid eye movement phase of sleep (REM). Inhibition of REM sleep is followed by a rebound increase in REM sleep associated with cardiopulmonary complications. The medial pontine reticular formation (mPRF) is a brain region from which morphine can inhibit REM sleep. The present study tested the hypothesis that specific subtypes of opioid receptors within the mPRF mediate inhibition of REM sleep. Synthetic opioid agonists selective for mu, delta and kappa subtypes were microinjected into the mPRF of four awake cats and polygraphic recordings of sleep and breathing were obtained. An enkephalinase inhibitor was microinjected into the mPRF to assess the contribution of endogenous opioids to the control of sleep and breathing. Only the mu agonist significantly inhibited REM sleep, and no opioid depressed breathing. These results demonstrate that opioid-induced REM sleep inhibition is mediated by mu receptor subtypes in the mPRF.

Enhanced sensitivity to some of the behavioral effects of the opioid antagonist naltrexone (NTX) develops following once-weekly injections of cumulative doses of the drug. Rats treated with this regimen of NTX injections show enhanced sensitivity to the operant response rate decreasing effects of NTX and NTX-induced salivation. The enhanced sensitivity is long-lasting and appears to be produced through conditioning processes. We have conducted saturation binding assays to assess possible changes in the number and affinity of mu and delta opioid receptors in cortical, midbrain and hindbrain membrane preparations from Long-Evans rats treated once weekly for 8 weeks with cumulative doses of the drug (1, 3, 10, 30 and 100 mg/kg). 3H-DAMGO (0.5-21 nM) and 3H-pCl-DPDPE (0.04-4 nM) were used to characterize mu and delta receptors, respectively. NTX treatment had no effect on 3H-DAMGO binding in cortex, but decreased binding in midbrain and increased binding in hindbrain relative to saline-treated controls. Saturation analyses revealed that these differences reflected changes in the number, but not the affinity of mu receptors. NTX treatment also increased the amount of 3H-pCl-DPDPE bound to delta receptors in midbrain and hindbrain, but not in cortex. Again, these changes were due to changes in the number of receptors. Thus, chronic NTX differentially affects the number of mu and delta opioid receptors in various brain regions.

The intracerebroventricular (i.c.v.) injection to mice of a polyclonal antibody raised against the peptide sequence 208-216 (TKYRQGSID) of cloned rat mu opioid receptor, reduced the analgesic potency of DAMGO, morphine and beta-endorphin-(1-31) when studied 48 h later in the tail-flick test. Antinociception elicited by delta agonists, DPDPE and [D-Ala2]-Deltorphin II, and by the kappa agonist U-50488H, was fully expressed in mice undergoing this treatment. The specific binding displayed by 0.6 nM [3H]-DAMGO was reduced in membranes preincubated with the antiserum, whereas no change could be detected for 3 nM [3H]-DPDPE or 2 nM [3H]-U-69593 labelling delta and kappa opioid receptors respectively. Naloxonazine, irreversible antagonist of the pharmacologically defined mu 1 opioid receptor, and beta-funaltrexamine (beta-FNA), that also displays irreversible antagonism at mu 1/2 receptors, when injected i.c.v. 24 h before the opioids significantly reduced the activity of DAMGO and morphine. In mice treated with naloxonazine, but not with beta-FNA, the antibody further reduced the remaining analgesic effect of DAMGO and morphine. Thus, both the antibody and beta-FNA blocked a wider population of mu opioid receptors than that tagged by naloxonazine.

The affinity of morphine, codeine, dihydrocodeine and their glucuronides for mu-, delta-, and kappa-opioid receptors was investigated. Binding was studied on guinea-pig brain homogenates with [3H]DAMGO, [3H]DPDPE, and [3H]U69593. The substitution of the free phenolic group of morphine caused a decrease in binding at opioid receptors without affecting the mu/delta-ratio nor that of mu/kappa. Glucuronidation of the 6-hydroxyl group of morphine, codeine or dihydrocodeine did not affect the affinity to mu-receptors, slightly increased the affinity for delta-receptors and reduced the affinity for kappa-receptors. The 6-glucuronides possess a decreased selectivity for mu-receptors over delta-receptors whereas that for mu- over kappa-receptors was increased. It is concluded that chemical variations at 3- and 6-position of morphine independently affect the affinity to opioid receptor subtypes.

Spinally administered mu opioid agonists produce potent antinociception and inhibition of gastrointestinal transit. Blockade of 5-hydroxytryptamine (5-HT) or norepinephrine (NE) uptake potentiates intrathecal (i.t.) DAMGO antinociception. To determine whether 5-HT and NE uptake blockade will also potentiate the gastrointestinal inhibition, mice were treated with zimelidine, desipramine or saline, followed by i.t. DAMGO and tested for tailflick antinociception or inhibition of gastrointestinal transit. DAMGO produced antinociception dose-dependently (ED50 = 4.6 ng). Zimelidine (10 mg/kg, s.c., 1 hr before DAMGO) produced a 6.2-fold leftward shift in the antinociceptive dose-response curve (ED50 = 0.73 ng). Desipramine produced a 5.3-fold shift (ED50 = 1.4 ng). DAMGO also produced a dose-dependent inhibition of gastrointestinal transit (ED50 = 117 ng). However, zimelidine or desipramine treatment did not affect DAMGO inhibition of gastrointestinal transit (ED50 = 80 ng.).

The goal of this study was to compare the characteristics of mu- and delta-opioid receptors in the cortex of DBA/2 and C57BL/6 mice, which differ in sensitivity to the long- and short-term effects of morphine. The characteristics of mu-opiate receptors were not different in the cortex of both strains. Both high- and low-affinity binding sites of DSLET, a specific ligand of delta-opiate receptors, were present in the cortex of C57BL/6 mice, whereas the high-affinity binding sites were not found in the cortex of DBA/2 mice. The absence of high-affinity DSLET binding sites, which are similar to the delta 2 type of opioid receptors, may explain the less intensive naloxone-precipitated withdrawal reaction of DBA/2 as compared with C57BL/6 mice.

Stress produced by pinching the tail is known to increase feeding behavior in rats, and endogenous opioids have been implicated in the mediation of this effect. We have reported previously that a nonspecific opioid antagonist and a mu-selective antagonist decrease this stress-induced eating (SIE) when they are microinjected into the substantia nigra (SN). The present study investigated the possibility that activation of opioid receptors in the SN might also alter SIE. Because oral stereotypy and nociception are affected by opioid mechanisms in the SN, measurements of gnawing and of tail flick and hot plate response latencies were also made. Bilateral injection of morphine (0.1-20 nmol) and the mu-selective agonist D-Ala2,N-Me-Phe4,Gly5-ol-enkephalin (DAGO; 0.03-1 nmol) increased response latency on the hot plate test and decreased gnawing produced by tail pinch. Tail flick latency and SIE were not affected. It is concluded that activation of opioid receptors in the SN does not produce an alteration in SIE as has been seen with opioid antagonists.

Chlornaltrexamine (beta-CNA, 0.5 micrograms) alone or beta-CNA plus either mu-agonist, D-Ala2-NMePhe4-Gly-ol-enkephalin (DAMGO, 500 ng) or delta-agonist, D-Pen2-D-Pen5-enkephalin (DPDPE, 10 micrograms) was injected intrathecally (i.t.) to protect mu- or delta-opioid receptors, respectively, for 24 h in male ICR mice. The antinociception was assessed by the tail-flick and hot-plate test. DPDPE or DAMGO injected i.t. increased inhibition of the tail-flick and hot-plate response in a dose-dependent manner. The dose-response curve for tail-flick and hot-plate response induced by DPDPE or DAMGO in i.t. saline-treated group significantly shifted to the right in i.t. beta-CNA alone treated group but returned to the control level in the group treated with i.t. beta-CNA coadministered with DPDPE or DAMGO, respectively. The effects of protection of mu- and delta-opioid receptor in the spinal cord on inhibition of the tail-flick and hot-plate response induced by beta-endorphin and morphine administered intracerebroventricularly (i.c.v.) were then studied. Intrathecal pretreatment with beta-CNA or beta-CNA coadministered with DAMGO attenuated inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. However, i.t. treatment with beta-CNA coadministered with DPDPE did not affect inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. Intrathecal pretreatment with beta-CNA or beta-CNA coadministered with either DPDPE or DAMGO did not alter inhibition of the hot-plate response induced by beta-endorphin administered i.c.v.(ABSTRACT TRUNCATED AT 250 WORDS)

The modulation by morphine of the spinal release of met-enkephalin-like material (MELM) was investigated in anaesthetized rats whose intrathecal space was perfused with an artificial CSF (ACSF). Morphine (10 microM in the ACSF), as well as a mu- (DAGO, 10 microM) or delta opioid receptor agonist (DTLET, 10 microM), significantly decreased the outflow of MELM. The effects of morphine and DTLET were prevented by the delta antagonist, naltrindole (10 microM), but not by naloxone (10 microM). Conversely, naloxone, but not naltrindole, prevented the inhibitory effect of DAGO. Although neither the kappa 1 agonist, U 50488H (10 microM), nor the kappa 1 antagonist, norbinaltorphimine (10 microM), exerted on their own any significant effect, norbinaltorphimine enhanced the inhibitory action of morphine. In contrast to the inhibition induced by morphine (with or without naloxone) which was preventable by 10 microM naltrindole, the inhibition of MELM release by morphine plus norbinaltorphimine was only partly reduced by naltrindole. Thus, concomitant stimulation of mu, delta and kappa 1 receptors might account for the apparent delta opioid receptor-dependent inhibition of MELM release by morphine. Indeed, its potential inhibitory effect through the stimulation of mu receptors (normally prevented by the concomitant stimulation of kappa 1 receptors) becomes efficient only when kappa 1 receptors are blocked.

The present study examined the effects of morphine, DAMGO, DPDPE and U-50, 488H on auricular rate on isolated right atria of the rat. All the opioid agonists tested induced a decrease of auricular rate. The maximal effect obtained with U-50,488H (75 +/- 8.3%) was significantly (p < 0.001) higher than that obtained with morphine (12 +/- 2.7%), DAMGO (8 +/- 0.6%) or DPDPE (11 +/- 1.8%). The inhibitory effects of U-50,488H were not antagonized by the presence of naloxone (10(-7) or 5 x 10(-7) M) or MR-2266 (10(-7) or 5 x 10 (-7) M). Moreover, U-50,488H did not change the auricular chronotropism in the presence of atropine (5 x 10(-7) M). In this case the maximal inhibitory effect was 79 +/- 6.7%, similar to that obtained with the kappa-agonist alone (75 +/- 8.3%). Propranolol (10(-8) or 5 x 10(-8) M) modified the inhibitory effect of U-50,488H. The maximal effect obtained by the kappa-agonist in presence of propranolol was 100 +/- 0 significantly (p < 0.01) higher than that obtained with U-50,488H alone. These results demonstrated that the depressant action of U-50,488H was not blocked in the presence of opioid receptor antagonists and probably does not involve opioid receptors. Furthermore, propranolol caused a dose-dependent potentiation of the effects of the kappa-agonist supporting the conclusion that it is not mediated by opioid receptors.

Opioid binding sites specific for [3H]dynorphin1-8 were characterized in the particulate membrane fraction of frog (Rana esculenta) brain. The degradation of the radioligand during the assay was prevented by the use of a broad spectrum of peptidase inhibitors. The binding of [3H]dynorphin1-8 to frog brain membranes was stereoselective, reversible, saturable, and displaceable by a series of opioid ligands including dynorphin1-13, bremazocine, levorphanol and naloxone. The specific binding of [3H]dynorphin1-8 can be significantly inhibited by Na+ ions and/or guanine nucleotides confirming the agonist property of the ligand in vitro. A single set of high affinity opioid binding sites with a Kd approximately 7.5 nM is present in the membranes. The maximum density of binding sites (Bmax approximately 1.1 pmol [3H]dynorphin1-8 per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated opioid peptides selective for the mu- and delta-types of opioid receptor showed that in the frog brain most of the sites labelled by [3H]dynorphin1-8 are kappa-sites and that this is a rich source of such sites.

A 58 kDa protein which was phosphorylated on tyrosine residues with morphine was found in human neuroblastoma cells (SK-N-SH cells) by immunoblot with monoclonal anti-phosphotyrosine antibody. The tyrosine phosphorylation was induced by morphine in 5 min in a dose-dependent manner and the increment was completely inhibited by naloxone. A Delta (d) agonist, [D-Pen2,Pen5]-enkephalin (DPDPE), but not a m agonist, [D-Ala2,N-Me-Phe,Gly5-ol]-enkephalin (DAGO), stimulated the phosphorylation and treatment of the cells with pertussis toxin inhibited the phosphorylation by morphine. These data suggest that d receptor-stimulation increases tyrosine phosphorylation of the 58 kDa protein through Gi protein in SK-N-SH cells.

Previous work in our laboratory has shown that DAMGO (ICV) will cause an elevation in plasma corticosterone (CS). The effect was blocked by pretreatment with beta-FNA but not by naloxonazine, suggesting indirectly that DAMGO's effect was via a mu 2-opioid receptor. TRIMU-5, a mu 2 agonist/mu 1 antagonist, was tested in a similar series of experiments to show more directly that the effect of DAMGO to increase plasma CS was via the mu 2 receptor. Experiments were conducted on conscious, unrestrained, male Sprague-Dawley rats with chronic IV catheters and ICV cannula guides allowing for serial blood sampling and drug injection into the right lateral ventricle. During this process, animals remained isolated in sound-attenuated one-way vision boxes. TRIMU-5, 50 micrograms, produced a sustained increase in plasma CS for a 3-h period. The response peaked at 30 min, showing a plasma CS level of 19.7 +/- 1.4 micrograms/dl. A lower dose, 10 micrograms, did not produce a significant response. A higher dose, 100 micrograms, produced an elevated hormone response in a pilot study but was lethal in half the animals. The plasma CS increase was blocked by pretreatment with beta-FNA, 20 micrograms ICV, given 18 h before TRIMU-5, but was unaffected by naloxonazine pretreatment, 20 mg/kg i.v., also administered 18 h before TRIMU-5. These data confirm our earlier conclusion that the effect of DAMGO to elevate plasma CS was through a mu 2-opioid receptor.

The effects of bilateral microinjections of mu-opioid receptor agonist DAMGO (0.00, 0.01, 0.1, or 1.0 microgram/side) were tested in rats for 120 min in activity monitors. The horizontal movement, rearing, and stereotypy times in seconds were measured during 12 consecutive 10-min time blocks. DAMGO (0.01, 0.1, and 1.0 microgram) resulted in biphasic effects, inhibition followed by activation for each of the three measures. These data replicate the behavioral effects of ICV DAMGO except that the duration of the behavioral effects were longer with Acb injections.

Initial experiments were conducted to determine whether or not the aging process alters the ability of young, mature, or aged male Fischer 344 rats (5- to 6-, 15- to 16-, and 25- to 26-months-old, respectively) to respond to thermal nociceptive stimuli. Using the tail-flick analgesiometric assay, 25- to 26-month-old rats responded significantly faster to the heat source than 15- to 16-month-old animals, but no significant differences were noted between the 5- to 6-month-old and aged rats. Another series of investigations compared the effects of aging on the spinal antinociceptive properties of the mu opioid agonist [D-Ala2,N-methyl-Phe4,Gly5-ol] enkephalin (DAMPGO) and the delta agonist [D-Pen2,D-Pen5] enkephalin (DPDPE). In these studies, young, mature, and aged rats were injected intrathecally (IT) with different doses of DAMPGO or DPDPE, and opioid-induced antinociception was tested on the tail-flick test. All three age groups responded to IT DAMPGO in a dose-dependent manner but, for the most part, higher spinal doses were required to produce significant elevations in tail-flick latency in the aged cohort of rats. The spinal analgesic effects of DPDPE also declined with advanced age. The aging process apparently alters the pain-inhibitory function of mu and delta opioid receptors in the rat spinal cord.

This study compared the effects of receptor-selective peptide and nonpeptide opioid agonists administered intramuscularly to squirrel monkeys responding under a fixed-interval 3-min schedule of stimulus termination. The mu opioid receptor agonist morphine (0.1-3.0 mg/kg) increased response rate at low doses and decreased it and quarter-life at higher doses. [D-Ala2,N-Me-Phe4,Gly-ol]Enkephalin (DAMGO; 0.3-3.0 mg/kg) reduced quarter-life at the highest dose. The kappa dose. The kappa opioid receptor agonist U50,488H (0.1-1.0 mg/kg) elevated response rate transiently and dose-dependently decreased quarter-life. Dynorphin A(1-13) (0.3-10 mg/kg), a purported endogenous ligand of the kappa opioid receptor, decreased response rate slightly but significantly at 3.0 mg/kg and had no effect on quarter-life. Thus, the behavior of squirrel monkeys was affected by systemically administered peptide as well as by nonpeptide opioid drugs. The two alkaloids were much more effective than the two peptides, presumably because of greater ability to penetrate the blood-brain barrier. Quarter-life was often a more sensitive measure of drug effects than was response rate.

The surprising change of selectivity induced by the change of chirality in peptides containing the tetrahydro-3-isoquinoline carboxylic acid (Tic) in second position, interpreted as a conformational preference induced on the Tyr-Xaa-Phe domain, can instead be attributed to the Tyr-Tic message domain. The relative spatial disposition of the aromatic ring of delta-selective non peptidic opiates is compatible with a message domain, in opioid peptides, of only two residues. This hypothesis was tested through the synthesis of Tyr-L-Tic-NH2, Tyr-D-Tic-NH2, Tyr-L-Tic-Ala-NH2, Tyr-L-Tic-Ala-OH and Tyr-D-Tic-Ala-NH2. Peptides containing Tyr-L-Tic- behave as very selective delta antagonists and those containing Tyr-DTic- as non selective agonists. This is the first case of opioid peptides containing a two-residue message domain and of opioid dipeptides with substantial opioid activity.

We have examined the effect of fentanyl on [3H]noradrenaline release in a human neuroblastoma cell preparation, SH-SY5Y. Fentanyl produced a significant, concentration-dependent inhibition of [3H]noradrenaline release with IC50 values of 5.5 x 10(-6) mol litre-1 and 15.5 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively. The small difference in IC50 between the two evoking stimuli may be explained by the weak binding affinity of fentanyl to muscarinic receptors (Ki = 570 nmol litre-1). The minimum concentrations at which a significant effect was observed were 0.3 x 0.10(-6) mol litre-1 and 10.0 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively; these values are considerably in excess of the serum concentration of fentanyl required to produce analgesia. Naloxone failed to antagonize the fentanyl inhibition and, furthermore, morphine and an enkephalin had no effect on evoked release, implying a non-opioid receptor mediated effect.

Characteristics of opioid receptors binding in the Mongolian gerbil cerebellum were determined by in vitro radioligand binding technique. Homogenate of cerebellar membranes possessed a binding capacity for 3H-DAMGO, a mu-agonist, and for 3H-diprenorphine, an antagonist of mu, delta and kappa-receptors. These bindings were saturable, and characterized by high affinity (Kd values: 1.55 +/- 0.43 nM for 3H-DAMGO and 0.56 +/- 0.20 nM for 3H-diprenorphine) and high density (Bmax: 127.8 +/- 23.8 fmoles/mg protein for 3H-DAMGO and 135.8 +/- 9.03 fmoles/mg protein for 3H-diprenorphine). Autoradiographically, the binding sites were predominantly located in the molecular layer of the cerebellum. 3H-DPDPE, a delta-agonist, showed only very small binding capacity to the cerebellar membranes. The kappa-agonist, 3H-U-69593, also showed very small binding capacity to the cerebellar membranes except in the early postnatal period. Thus, the gerbil cerebellum can be considered as a tissue containing a homogeneous population of mu-opioid receptors.