Selective Protection of Benzomorphan Binding Sites Against Inactivation by N-Ethylmaleimide. Evidence for k-Opioid Receptors in Frog Brain

Selective binding of [3H]bremazocine and [3H]-ethylketocyclazocine to kappa-opioid receptor sites in frog (Rana esculenta) brain membranes is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide (NEM). Pretreatment of the membranes with kappa-selective compounds [ethylketocyclazocine (EKC), dynorphin (1-13), or U-50,488H] but not with [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO; mu specific ligand) or [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DADLE; delta specific ligand) strongly protects the binding of the radioligands against NEM inactivation. These results provide more evidence for the existence of kappa-opioid receptors in frog brain. The relatively high concentrations of NEM that are needed to decrease the specific binding of [3H]bremazocine together with the observation of an almost complete protection of its binding sites by NaCl suggest that bremazocine may act as an opioid antagonist in frog brain.     

Modification by Cholecystokinin Octapeptide of the Binding ofμ-, δ-, and K-Opioid Receptors

Previous study has shown that cholecystokinin (CCK) octapeptide (CCK-8) suppressed the binding of opioid receptors to the universal opioid agonist [3H]etorphine. In the present study, highly selective tritium-labeled agonists for the mu-[(tryrosyl-3,5-3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAGO], delta- ([tyrosyl-3,5-3H][D-Pen2,5]enkephalin ([3H]DPDPE], and kappa- ([3H]U69,593) opioid receptors were used to clarify which type(s) of opioid receptor in rat brain homogenates is suppressed by CCK-8. In the competition experiments, CCK-8 suppressed the binding of [3H]DAGO and [3H]U69,593 but not that of [3H]DPDPE to the respective opioid receptor. This effect was blocked by the CCK antagonist proglumide at 1 mumol/L. In the saturation experiments, CCK-8 at concentrations of 0.1 nmol/L to 1 mumol/L decreased the Bmax of [3H]DAGO binding sites without affecting the KD; on the other hand, CCK-8 increased the KD of [3H]U69,593 binding without changing the Bmax. The results suggest that CCK-8 inhibits the binding of mu- and kappa-opioid receptors via the activation of CCK receptors.     

Separation of kappa-opioid receptor subtype from frog brain

Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) from tritiated Tyr-D-Ala2-Me-Phe4-Gly-ol5 enkephalin (DAGO) and Tyr-D-Ala2-L-Leu5-enkephalin (DALA) binding (mu-and delta-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (s20.w) for the kappa receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas mu- and delta-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular entity from the mu and delta receptor sites.     

Interaction of Opiates with Opioid Binding Sites in the Bovine Adrenal Medulla: I. Interaction with δ and μ Sites

In the present study we examined the interaction of opiates with the delta and mu opioid binding sites in the bovine adrenal medulla. [3H][D-Ala2, D-Leu5]-enkephalin ( [3H]DADLE) in the presence of saturating concentrations of morphiceptin was used to analyze delta site interactions, whereas either [3H]DADLE in the presence of saturation concentrations of [D-Ser2, Leu5]-enkephalin-Thr6 (DSLET) or [3H][D-Ala2, Me-Phe4, Gly5-ol]-enkephalin ( [3H]DAGO) was used for the determination of mu sites. Both binding sites were found to interact stereoselectively with opiates. The binding was affected differentially by proteolytic enzymes (trypsin, alpha-chymotrypsin, pepsin), N-ethylmaleimide, and A2-phospholipase. Kinetic and equilibrium binding studies revealed that in each case radiolabeled opiates interact with one class of binding sites, following simple second-order bimolecular kinetics. Competition for binding by opiates and opioid peptides confirmed the delta and mu selectivity of these sites. Monovalent (Na+, Li+, K+) and divalent (Mg2+, Mn2+, Ca2+) ions interacted differentially with these two binding sites: In general, monovalent cations affected preferentially the apparent number of binding sites, whereas divalent ions modified the equilibrium dissociation constant. Furthermore, positive or negative cooperativity and an apparent heterogeneity of binding sites were detected under some ionic conditions.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD = 8.8 +/- 1.7 nM; Bmax = 2.3 +/- 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a mu-selective ligand: KD = 150 +/- 34 nM; Bmax = 3.0 +/- 0.6 pmol/mg of protein. Trypsin or heat treatment (100 degrees C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by greater than 80%. The rank order of potency of drugs at this site was levorphanol greater than DHM greater than normorphine greater than naloxone much greater than dextrorphan; the mu-specific peptide [D-Ala2,Gly-ol5]-enkephalin and delta-, kappa-, and sigma-ligands were inactive at this site. The other site was labeled by (-)-[3H]ethylketocyclazocine ((-)-[3H]EKC), a kappa-opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD = 212 +/- 25 nM; Bmax = 1.9 +/- 0.2 pmol/mg of protein). (-)-[3H]EKC binding could be displaced by kappa-opioids but not by mu-, delta-, or sigma-opioids or by the kappa-peptide dynorphin. Specific binding constituted approximately 70% of total binding at 1 nM and approximately 50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the delta-ligands [3H][D-Ala2,D-Leu5]-enkephalin and [3H][D-Pen2,D-Pen5]-enkephalin was undetectable in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coupling of adrenal medullary opioid receptors to islet-activating protein-sensitive GTP-binding proteins

Possible coupling of bovine adrenal medullary opioid receptors to islet-activating protein (IAP, pertussis toxin)-sensitive GTP-binding proteins was investigated by studying effects of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and IAP treatment of membranes on opioid binding. Gpp(NH)p inhibited [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) binding by increasing the dissociation constant of [3H]DADLE and membranes, and enhanced slightly [3H]diprenorphine binding. IAP treatment of membranes reduced [3H]DADLE binding and abolished almost completely the Gpp(NH)p inhibition of [3H]DADLE binding. Treatment of membranes with IAP and [32P]NAD resulted in radio-labeling of membrane proteins of approximately 39,000 dalton. DADLE inhibited adenylate cyclase activity in rat brain caudate nucleus. However, DADLE, beta-endorphin, levorphanol and dynorphin A(1-13) did not show any significant inhibitory action on bovine adrenal medullary adenylate cyclase activity. These results suggest that bovine adrenal medullary opioid (DADLE) receptors are linked to IAP-sensitive GTP-binding proteins which are not directly coupled to adenylate cyclase.     

The occurrence of different kappa opioid receptors (K1 and K2) in frog (Rana esculenta) brain membranes

Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively high ethylketocyclazocine (EKC) binding properties and by their low affinity to mu and delta ligands when D-Ala2-(Me)Phe4-Gly5-ol enkephalin (DAGO) and D-Ala2-Leu5-enkephalin (DALE) is used. In competition experiments it has been established that EKC and N-cyclopropylmethyl-norazidomorphine (CAM), which are non-selective kappa-ligands, have relatively high affinity to frog brain as well as the kappa 2 (which is DALE sensitive subpopulation of the kappa receptor) ligands etorphine and Metenkephalin-Arg6-Phe7 (1.). The kappa 2 subtype in frog brain resembles more to the mu subtype than to the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and DAGO.     

14-beta-Methyl-8-oxacyclorphan (BC-3016), a morphinan derivative with high affinity for kappa opioid receptor: comparison with dynorphin-A(1-13)

14-beta-Methyl-8-oxacyclorphan (BC-3016) was tested for its ability to depress the electrically evoked contractions of the guinea pig ileum (GPI) and of the mouse vas deferens (MVD) and to compete with the binding of prototype ligands selective for kappa-, mu-, or delta-opioid receptors in membrane preparations of rat brain and guinea pig cerebellum. BC-3016 was a very potent agonist in the GPI and MVD preparations, with ID50 of 0.7 and 31 nM, respectively. The activity of levorphanol, a standard alkaloid related to BC-3016, was much lower in both assays with ID50 values of 44 and 86 nM, respectively. Conversely, the activity of BC-3016 was quite comparable to that of dynorphin-A(1-13) in both preparations. In the GPI assay, a putative kappa-receptor antagonist, MR-2266, was 6.6 and 5.5 times more potent than naloxone in blocking the activity of BC-3016 and dynorphin-A(1-13), respectively. BC-3016 was also very potent in displacing bound [3H]ethylketocyclazocine ([3H]EKC) to membrane preparations of the guinea pig cerebellum, a brain component containing predominantly kappa-opioid receptors (Ki of 0.58 nM). Its potency in the displacement of the bound mu-ligand, 3H-labelled (D-Ala2,MePhe4,Gly-OH5)-enkephalin ([3H]DAGO), to rat brain homogenates was somewhat lower (Ki of 0.8 nM) but still high when compared with its ability to displace the delta-ligand, 3H-labelled (D-Ser2, Thr6)-Leu-enkephalin ([3H]DSLET) to rat brain homogenates (Ki of 4.45 nM). The affinity of BC-3016 for the opioid receptor was 2.1-fold higher than that of U-50488H, a selective kappa-opioid ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. II. Binding characteristics in frog brain membranes

3H-D-Ala2-Leu5-enkephalin chloromethyl ketone (3H-DALECK) was used to label opioid receptors of frog brain membranes. We have previously shown (15) that 70% of the opioid receptors are of kappa type in this preparation. The binding of 3H-DALECK was of high affinity, half maximal binding being achieved by 0.9 nM of the radioligand. The number of sites labeled was calculated to be 108 fmol/mg protein. Opioid ligands, incubated with the membranes prior to the label, inhibited 3H-DALECK binding with the following rank order:etorphine greater than EKC greater than DAGO greater than DALECK greater than DADLE. Dissociation experiments showed that 70% of the binding is irreversible. Fluorography performed after SDS-PAGE revealed specific covalent labeling of protein subunits of 90, 58 and 20 kD molecular weights. Results will be compared to those obtained in rat brain (13). Our two studies demonstrate that 3H-DALECK is a useful probe for investigation the subunit structure of opioid receptors.     

Effect of sodium on [3H]ethylketocyclazocine binding to opioid receptors in frog brain membranes

The specific binding of (³H)ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 °C and at room temperature. The optimal NaCl concentration was 25 mM at 0 °C and 50 mM at 24 °C. MgCl₂ inhibited the [³H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [³H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the B_max of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 °C than at 24 °C. TheK _D values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [³H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.Abbreviations used DAGO D-Ala²-(Me)Phe⁴-Gly-ol⁵-enkephalin - DALE d-Ala²-l-Leu⁵-enkephalin - DADLE d-Ala²-d-Leu⁵-enkephalin - EKC Ethylketocyclazocine - DHM Dihydromorphine - BIT 2-(p-ethoxybenzyl)1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate - FIT Fentanyl isothiocyanate     

Synthesis and biological activity of dynorphin-(1-13) and analogs substituted in positions 8 and 10

Dynorphin-(1-13) (Dyn-(1-13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [3H]-ethylketocyclazocine (EKC, kappa ligand), [3H]-[D-Ala2, MePhe4-Gly-ol5]-enkephalin (DAGO, mu ligand) and [3H]-[D-Ser2, Thr6]-Leu-enkephalin (DSLET, delta ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the kappa receptor ([3H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1-13)). On the other hand, the ability of [Ala8] Dyn-(1-13) to displace the binding of [3H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the mu receptor was not greatly affected ([3H]-DAGO displacement; Ki of 0.44 nM as compared with 0.50 nM for Dyn-(1-13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the kappa site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro10]-Dyn-(1-13) was equipotent to [Ala8]-Dyn-(1-13) in the GPI but its affinity for the mu binding site was decreased by a factor of 2.7 as compared with Dyn-(1-13).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Effect of Intrastriatal Kainic Acid on the Modulation of Dopamine Release by μ- and δ-Opioid Peptides: A Microdialysis Study

The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by mu- and delta-opioid peptides. The effects of [D-Pen2,D-Pen5]-enkephalin (DPDPE) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO), two highly selective delta- and mu-opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the delta agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that delta-opioid receptors modulating the striatal DA release, in contrast to mu receptors, are not located on neurons that may be lesioned by kainic acid.     

Differential Effects of Mg2+ and Other Divalent Cations on the Binding of Tritiated Opioid Ligands

The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.     

A photoactivatable naltrexone derivative labels glycoproteins of different molecular weight corresponding to the mu- and kappa-opioid receptors.

The synthesis and characterization of a novel opioid receptor photoaffinity probe [3H]naltrexyl urea phenylazido derivative ([3H]NUPA) is described. In the absence of light, [3H]NUPA binds with high affinity in a reversible and saturable manner to rat brain and guinea pig cerebellum membranes. Dissociation constants and binding capacities (Scatchard plots) are 0.11 nM and 250 fmol/mg of protein for rat brain and 0.24 nM and 135 fmol/mg of protein for guinea pig cerebellum. Competition experiments indicate that this ligand interacts with high affinity at both mu- and kappa-opioid binding sites while exhibiting low affinity at delta sites (Ki = 21 nM). On irradiation, [3H]NUPA incorporates irreversibly into rat brain and guinea pig cerebellum membranes. SDS gel electrophoresis of rat brain membranes reveals specific photolabeling of a 67-kDa molecular mass band. Conversely, a major component of 58 kDa and a minor component of 36 kDa are obtained from [3H]NUPA-labeled guinea pig cerebellum membranes. Different photolabeling patterns are obtained in rat brain (mu/delta/kappa, 4/5/1) and guinea pig cerebellum (mu+delta/kappa, 1,5/8,5) membranes in the presence of selective opioid ligands indicating labeling of mu and kappa sites, respectively. Thus, [3H]NUPA behaves as an efficient photoaffinity probe of mu- and kappa-opioid receptors, which are probably represented by distinct glycoproteins of 67 and 58 kDa, respectively.     

Distinct Subtypes of the Opioid Receptor with Allosteric Interactions in Brain Membranes

The binding isotherms of opioid receptors in rat brain membranes with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE), [3H]dihydromorphine ([3H]DHM), and [3H]etorphine were analysed to show the effects of Mg2+, Na+, and guanine nucleotides. Four opioid receptor subtypes of delta, kappa, mu 1, and mu 2 specificities were differentiated, where necessary with the aid of specific displacing ligands. Both a guanine nucleotide [guanosine-5'-(beta, gamma-imido)triphosphate] and the cations (Na+, Mg2+) affect the affinity state of all four subtypes of the receptor. The opioid binding behaviour is found on detailed inspection to be complex, with cases of "half-of-the-sites" reactivity and of cooperativity. By their behaviour under the various ionic conditions noted, it was concluded that these subtypes are distinct, without the need to assume interconvertibility by such agents. The evidence suggests that the formation of heterologous kappa-delta or mu 1-mu 2 receptor complexes is required for stabilization of the high-affinity conformational state of the receptor. Important effects of cations in increasing the binding and regulating the equilibria of receptor association-dissociation were observed when these studies were conducted, not in the Tris-HCl buffer commonly used in opioid binding assays, but in N-tris[hydroxymethyl]-methyl-2-aminoethanesulphonate (K+) buffer (TES-KOH; 10 mM, pH 7.5): it was found that ionic species of Tris can substitute for divalent cations. Dithiothreitol effects on agonist binding in the presence and absence of the cations suggested that those cation effects involve the exchange of -SH/-SS- bonds between receptor subunits. All of the behaviour is interpreted in terms of a model involving association-dissociation equilibria of homologous and/or heterologous receptor subunits of an oligomeric opioid receptor structure.     

14 beta-(Bromoacetamido)morphine irreversibly labels mu opioid receptors in rat brain membranes

The binding properties of 14 beta-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H] [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the mu binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The mu receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the delta-selective peptide [3H] [D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of mu and delta blockers, selective for kappa binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled beta h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the mu site did not afford protection.2+hese studies have demonstrated that when a disulfide bond     

Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.     

Activation of central opioid receptors may induce gastric mucosal defence in the rat.

The effect of different opioid peptides on acidified ethanol- and indomethacin-induced gastric mucosal lesions was studied following intracerebroventricular (i.c.v.) administration. It was found that both the selective delta opioid receptor agonists--deltorphin II, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE)-, mu-opioid receptor agonist--[D-Ala(2), Phe(4), GlyT-ol]-enkephalin (DAGO)--as well as beta-endorphin inhibited the mucosal damage induced by both ethanol and indomethacin in pmolar dose range. In contrast, the gastric acid secretion was not influenced by DADLE in the dose of 16 nmol/rat and only a slight reduction (40%) was induced by DAGO in the dose of 1.9 nmol/rat. The protective effect was abolished in both ulcer models by bilateral cervical vagotomy. N(G)-nitro-L-arginine, an inhibitor of NO synthase, reduced the protective action in ethanol-induced, but not in indomethacin-induced gastric damage. The results suggest that activation of supraspinal delta and mu-opioid receptors resulted in inhibition of gastric mucosal lesions elicited by ethanol or indomethacin. The gastroprotective action is independent from the effect of opioids on acid secretion. Vagal nerve is involved in conveying the central action to the periphery. The mechanism of the gastroprotective effect of opioids is different in ethanol- and indomethacin-ulcer models: prostaglandins and nitric oxide are likely to be involved in the protective action of opioid peptides in ethanol-, but not in the indomethacin-ulcer model.     

Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.