Optically pure (-)-4-[(N-allyl-3-methyl-4-piperidinyl)phenyl-amino]-N,N-diethylbenzami de displays selective binding and full agonist activity for the delta opioid receptor

The optical isomers of 4-[(N-allyl-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzamide+ ++ (3) have been prepared and tested in both binding and functional assays. The data show that (-)-3 is responsible for the delta opioid activity demonstrated by the racemic material. This compound displays a binding affinity of 5.5 nM for the delta opioid receptor as well as a 470-fold delta versus mu selectivity. Importantly, (-)-3 is a full agonist at the delta receptor in comparison with SNC-80 (2). Taken together, the data suggest that (-)-3 behaves more like the prototypical delta agonists, BW373U86 or SNC-80, and less like the peptidomimetic compound SL-3111 (5).     

Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.     

Immunofluorescence detection of delta opioid receptors (DOR) on human peripheral blood CD4+ T cells and DOR-dependent suppression of HIV-1 expression.

The delta opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover, in DOR-transfected Jurkat cells, delta opioids have been shown to suppress HIV-1 p24 Ag expression. These observations led us to characterize the expression of DORs by human peripheral blood T cells and to determine whether a specific DOR agonist, benzamide,4-([2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]-N,-,(2S[1(S*),2alpha,5beta])-(9Cl) (SNC-80), can suppress p24 Ag expression by HIV-1-infected CD4+ T cells obtained from normal donors. By immunofluorescence flow cytometry, PHA stimulated the expression of DOR from 1.94 +/- 0.70 (mean +/- SEM) to 20.70 +/- 1.88% of the PBMC population by 48 h (p < 0.0001). DOR expression was approximately 40% of both the PHA-stimulated CD4+ and CD8+ T cell subsets, and virtually all DORs were found on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA, and then CD4+ T cells were purified, pretreated with SNC-80, and infected with HIV-1. In a concentration-dependent manner, SNC-80 inhibited production of p24 Ag. SNC-80 10(-10) M maximally suppressed (approximately 50%) both lymphocytotropic (HIV-1 MN) and monocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished the inhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection with HIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD4+ T cells significantly suppressed the expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.     

Single residue modifications of the delta opioid receptor selective peptide, [D-Pen2,D-Pen5]-enkephalin (DPDPE). Correlation of pharmacological effects with structural and conformational features

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [D-Pen2,D-Pen5]enkephalin, Tyr-D-Pen-Gly-Phe-D-PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by 1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1-DPDPE ([DAT1]DPDPE) and phenylglycine4-DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced mu receptor binding affinity of the carboxamide terminal DPDPE-NH2 appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the alpha-aminoisobutyric acid substituted analog [Aib3]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the delta opioid receptor.     

Ligand binding profiles of delta-opioid receptor in human cerebral cortex membranes: evidence of delta-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.     

Synthesis of novel analogues of the delta opioid ligand SNC-80 using AlCl3-promoted aminolysis.

Two focused libraries of delta opioid ligands were synthesised using AlCl3 facilitated aminolysis. Several compounds were identified with DOR binding affinities higher or similar to SNC-80. A novel acyclic derivative of SNC-80 produced antinociception in the acetic acid abdominal constriction test, which is at least partially mediated via the delta-opioid receptor.     

Antinociceptive interactions of opioid delta receptor agonists with morphine in mice: supra- and sub-additivity.

In this study, the antinociceptive interactions of fixed ratio combinations of intracerebroventricularly (i.c.v.) given morphine and subantinociceptive doses of the delta agonists, [D-Pen2, D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin (DELT) or [Met5]enkephalin (MET) were examined using the mouse warm water tail flick test. When morphine was coadministered with DPDPE or DELT in a 4:1 and 9:1 mixture, respectively, a synergistic antinociceptive effect was observed. In contrast, when morphine was coadministered with MET in a 1:2 fixed ratio mixture, a subadditive interaction occurred. These results demonstrate both positive and negative modulatory interactions of delta agonists with morphine in an antinociceptive endpoint and that these interactions can be either supra- or subadditive. The data support the concept of a functional interaction between opioid mu and delta receptors and a potential regulatory role for the endogenous ligands of the opioid delta receptor.     

Steroid delta 4-5 beta-reductase in human mammary tumors.

Steroid delta 4-5 alpha- and delta 4-5 beta-reductase activity was determined in 16 human mammary tumors and 8 DMBA-induced rat mammary tumors using a spectrophotometric assay. Steroid delta 4-5 alpha-reductase was present in all tumors investigated while delta 4-5 beta-reductase was detected in only 6 estrogen receptor negative human breast tumors and absent in all estrogen receptor positive human breast tumors as well as in all rat mammary tumors. Further support for the presence of delta 4-5 beta-reductase was established by using a dual-labelling technique consisting of incubating tumor slices with [14C] testosterone and adding [3H] etiocholanolone, [3H] testosterone and [3H]-5 alpha-dihydrotestosterone at the end of the reaction. Following extraction and chromic acid oxidation, 4-androstenedione, 5 beta-androstanedione and 5 alpha-androstanedione were isolated and purified, and the constancy of the 14C/3H ratio was used as proof of 5 alpha-reductase and 5 beta-reductase. These results were shown to be consistent with the data obtained using the spectrophotometric assay.     

Differential knockdown of delta-opioid receptor subtypes in the rat brain by antisense oligodeoxynucleotides targeting mRNA.

Two antisense oligodeoxynucleotides (A-ODN), targeting delta-opioid receptor mRNA (DOR) and two mismatch ODN sequences (mODN) were continuously infused for 24 days into the lateral brain ventricles of Wistar rats. The density of delta-opioid receptors in rat brain homogenates was measured by saturation binding experiments using four selective ligands, two agonists ([D-Ala2, Glu4]-deltorphin and DPDPE) and two antagonists (Dmt-Tic-OH and naltrindole), and by immunoblotting SDS solubilized receptor protein. In brain membranes of mODN or saline-infused rats, the rank order of delta-opioid receptor density, calculated by Bmax values of the four delta-opioid receptor ligands, was: [D-Ala2, Glu4]deltorphin approximately Dmt-Tic-OH approximately naltrindole (86-118 fmo/mg protein) > DPDPE (73.6+/-6.3 fmol/mg protein). At the end of the 24 day infusion of A-ODN targeting DOR nucleotide sequence 280299 (A-ODN280-299), the Bmax of DPDPE (62.4+/-3.2 fmol/mg protein) was significantly higher than that of Dmt-Tic-OH (31.5+/-3.9 fmol/mg protein). Moreover, both the Kd value for DPDPE saturation binding and the Ki value for Dmt-Tic-OH displacement by DPDPE were halved. In contrast, an A-ODN treatment targeting exon 3 (A-ODN741-760) decreased the specific binding of [D-Ala2, Glu4]deltorphin and Dmt-Tic-OH significantly less (67%-81%) than the binding of DPDPE (53%), without changes in DPDPE Ki and KD values. No A-ODN treatment modified the specific binding of the micro-opioid agonist DAMGO and of the k-selective opioid receptor ligand U69593. On the Western blot of solubilized striatum proteins, A-ODN(280-299) and A-ODN(741-760) downregulated the levels of the DOR protein, whereas the corresponding mODN were inactive. The 24-day infusion of A-ODN(280-299) inhibited the rat locomotor response to [D-Ala2, Glu4]deltorphin but not to DPDPE. Intracerebroventricular (i.c.v.) infusion of A-ODN(741-760) reduced the locomotor responses to both delta-opioid receptor agonists, whereas mODN infusion never affected agonist potencies. In conclusion, these results demonstrate that 24-day continuous i.c.v. infusion of A-ODN targeting the nucleotide sequence 280-299 of DOR can differentially knockdown delta1 and delta2 binding sites in the rat brain.     

长时间给予高选择性δ阿片受体激动剂抑制δ受体mRNA表达

我们采用反转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法,观察了δ阿片受体肽类激动剂「Ｄ－Ｐｅｎ＾２．５」ｅｎｋｅｐｈａｌｉｎ（ＤＰＤＰＥ）及非肽类激动剂ＢＷ３７３Ｕ８６对δ受体ｍＲＮＡ表达的影响,并比较了两者作用的差异性。结果如下：（１）ＤＰＤＰＥ作用２４及４８ｈ可使δ受体ｍＲＮＡ表达水平显著降低,则ＢＷ３７３Ｕ８６只在２４ｈ有显著抑制作用;（２）ＤＰＤＰＥ在１０＾－６ｍｏｌ／Ｌ时即可使δ受体的ｍＲＮＡ     

Sigma and opioid receptors in human brain tumors

Human brain tumors (obtained as surgical specimens) and nude mouse-borne human neuroblastomas and gliomas were analyzed for sigma and opioid receptor content. Sigma binding was assessed using [3H]1,3-di-o-tolylguanidine (DTG), whereas opoid receptor subtypes were measured with tritiated forms of the following: mu, [D-ala2,mePhe4,gly-ol5]enkephalin (DAMGE); kappa, ethylketocyclazocine (EKC) or U69,593; delta, [D-pen2,D-pen5]enkephalin (DPDPE) or [D-ala2,D-leu5]enkephalin (DADLE) with mu suppressor present. Binding parameters were estimated by homologous displacement assays followed by analysis using the LIGAND program. Sigma binding was detected in 15 of 16 tumors examined with very high levels (pmol/mg protein) found in a brain metastasis from an adenocarcinoma of lung and a human neuroblastoma (SK-N-MC) passaged in nude mice. kappa opioid receptor binding was detected in 4 of 4 glioblastoma multiforme specimens and 2 of 2 human astrocytoma cell lines tested but not in the other brain tumors analyzed.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

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     

Opioid receptor selectivity reversal in deltorphin tetrapeptide analogues.

Deltorphin N-terminal tetrapeptides [DEL A: H-Tyr-D-Met-Phe-His-R, where R = -NH2, -NH-NH2, -OCH3, -OH, -NH-NH-CO-R' (R' = -CH3 or adamantane); DEL C: H-Tyr-D-Ala-Asp-R (R = -OH, -NHCH3)], were used in a receptor binding assay with [3H]DADLE and [3H]DPDPE for delta sites, and [3H]DAGO for mu sites; tetrapeptide Ki delta values were similar with either [3H]-delta ligand. DEL A tetrapeptides C-terminally substituted with -NH2, -NH-NH2, -OCH3, and -OH had 10 to greater than 1,000-fold decreased Ki delta values, while Ki mu increased 5 to 100-fold to yield mu selectivity. C-Terminal substitution with -NH-NH2 and -OCH3 conferred highest mu selectivities; adamantyl and acetyl hydrazide derivatives were non-selective. DEL-(1-4)-OH peptides had decreased delta and mu affinities: DEL A-[Asp4]-(1-4)-OH and DEL C-(1-4)-OH had low affinities (greater than 1 microM), however, the Ki delta of the former was 5-fold greater than the latter, and the Ki mu was less by 15-fold. The data suggest that the "message" domain of DEL exhibits receptor selectivity different from that of the heptapeptide.     

Activation of peripheral delta opioid receptors eliminates cardiac electrical instability in a rat model of post-infarction cardiosclerosis via mitochondrial ATP-dependent K+ channels

The effects of the selective delta-1 (delta(1)) opioid receptor agonist, DPDPE, and the selective delta(2) opioid receptor agonist, DSLET, have been studied on the ventricular fibrillation threshold (VFT) in rats with an experimental post-infarction cardiosclerosis (CS). It has been found that CS induced a significant decrease in VFT. This CS-induced decrease in VFT was significantly reversed by intravenous administration of DPDPE (0.1 mg/kg) 10 min before VFT measurement. On the contrary, intravenous injection of DSLET (0.5 mg/kg) exacerbated the CS-induced cardiac electrical instability. Pretreatment with the selective delta opioid receptor antagonist, ICI 174,864 (0.5 mg/kg), completely abolished the changes in VFT produced by both DPDPE and DSLET. Previous administration of a nonselective peripherally acting opioid receptor antagonist, naloxone methiodide (5 mg/kg) also completely reversed the antifibrillatory action of DPDPE. Naloxone methiodide and ICI 174,864 alone had no effect on VFT. Pretreatment with the nonselective K(ATP) channel blocker, glibenclamide (0.3 mg/kg), or with the mitochondrial selective K(ATP) channel blocker, 5-hydroxydecanoic acid (5-HD, 5 mg/kg), completely abolished the DPDPE-induced increase in cardiac electrical stability. Glibenclamide and 5-HD alone had no effect on VFT. These results demonstrate that the delta opioid receptor plays an important role in the regulation of electrical stability in rats with post-infarction cardiosclerosis. We propose that peripheral delta(1) opioid receptor stimulation reverses CS-induced electrical instability via mitochondrial K(ATP) channels. On the contrary, delta(2) opioid receptor stimulation may exacerbate the CS-induced decrease in VFT. Further studies are necessary to determine the delta opioid receptor subtype which mediates the antifibrillatory effect of DPDPE and pro-fibrillatory effect of DSLET.     

Pharmacological characterization of AR-M1000390 at human delta opioid receptors

We investigated the pharmacological properties of a newly synthesised delta agonist AR-M1000390, derived from SNC-80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide), in the neuroblastoma cell line SK-N-BE expressing only human delta-opioid receptors. Binding and functional experiments showed a weak affinity (K(i) = 106 +/- 34 nM) correlated with a weak potency (EC(50) = 111 +/- 31 nM) to inhibit the forskolin-stimulated cAMP accumulation. Sustained activation of opioid receptors in the presence of the maximal inhibitory concentration of AR-M1000390 produced a rapid and strong desensitization. In order to examine the contribution of internalization and down-regulation in the desensitization processes, binding and functional experiments were conducted in the presence or in the absence of hypertonic sucrose solution to block clathrin-dependent opioid receptor endocytosis. We observed both the inability of AR-M1000390 to down-regulate opioid receptors and the absence of any effect of sucrose on desensitization. The lack of delta-opioid receptor internalization by AR-M1000390 was further corroborated by confocal microscopy using antibodies directed either against the endogenous delta-opioid receptors or the FLAG-tagged delta-opioid receptors stably expressed in the SK-N-BE cells. These data suggest that uncoupling rather than internalization is responsible for delta-opioid receptors desensitization by AR-M1000390.     

4-Hydroxy-1-[3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl]piperidines: selective h5-HT1D agonists for the treatment of migraine

A series of 4-hydroxy-1-[3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl] piperidines was investigated as potential selective h5-HT1D agonists for the treatment of migraine. The 4-[(N-benzyl-N-methyl)amino]methyl analog 12a was found to be a full agonist at the h5-HT1D receptor with good binding selectivity over the h5-HT1B receptor.     

Discrete mapping of brain Mu and delta opioid receptors using selective peptides: quantitative autoradiography, species differences and comparison with kappa receptors

The opioid peptides, [3H]DAGO and [3H]DPDPE, bound to rat and guinea pig brain homogenates with a high, nanomolar affinity and to a high density of mu and delta receptors, respectively. [3H]DAGO binding to mu receptors was competitively inhibited by unlabelled opioids with the following rank order of potency: DAGO greater than morphine greater than DADLE greater than naloxone greater than etorphine much greater than U50488 much greater than DPDPE. In contrast, [3H]DPDPE binding to delta receptors was inhibited by compounds with the following rank order of potency: DPDPE greater than DADLE greater than etorphine greater than dynorphin(1-8) greater than naloxone much greater than U50488 much greater than DAGO. These profiles were consistent with specific labelling of the mu and delta opioid receptors, respectively. In vitro autoradiographic techniques coupled with computer-assisted image analyses revealed a discrete but differential anatomical localization of mu and delta receptors in the rat and guinea pig brain. In general, mu and delta receptor density in the rat exceeded that in the guinea pig brain and differed markedly from that of kappa receptors in these species. However, while mu receptors were distributed throughout the brain with "hotspots" in the fore-, mid- and hindbrain of the two rodents, the delta sites were relatively diffusely distributed, and were mainly concentrated in the forebrain with particularly high levels within the olfactory bulb (OB), n. accumbens and striatum. Notable regions of high density of mu receptors in the rat and guinea pig brain were the accessory olfactory bulb, striatal "patches" and "streaks," amygdaloid nuclei, ventral hippocampal subiculum and dentate gyrus, numerous thalamic nuclei, geniculate bodies, central grey, superior and inferior colliculi, solitary and pontine nuclei and s. nigra. Tissues of high delta receptor concentration included, OB (external plexiform layer), striatum, n. accumbens, amygdala and cortex (layers I-II and V-VI). Delta receptors in the guinea pig were, in general, similarly distributed to the rat, but in contrast to the latter, the hindbrain regions such as the thalamus, geniculate bodies, central grey and superior and inferior colliculi of the guinea pig were apparently more enriched than the rat. These patterns of mu and delta site distribution differed dramatically from that of the kappa opioid sites in these species studied with the peptide [125I]dynorphin(1-8).     

Prejunctional effects of opioids in the perfused mesentery of the rat and rabbit: interactions with alpha 2-adrenoceptors.

Prejunctional effects of opioids were examined in the perfused mesentery of two species: the rat and rabbit. Use of agonists selective for subtypes of mu, delta, and kappa opioid receptors produced no effect on contractile responses to adrenergic nerve stimulation in the rat perfused mesentery, except for small effects of the kappa agonist EKC, which may be non specific. In contrast, mu, delta and kappa receptors appear to be present in the rabbit. The mu selective agonist, DAMGO, kappa agonist, ethylketocyclazocine, and delta agonists, DPDPE and [Leu5]-enkephalin, all produced significant inhibition of contractile responses to transmural nerve stimulation. The inhibitory effect was greatest for ethylketocyclazocine. To test the possibility that prejunctional activation of alpha 2 adrenoceptors with endogenous norepinephrine might decrease the activity of prejunctional opioid receptors in the rabbit, inhibitory effects of delta and kappa selective agonists were tested in the presence of 10(-7) M yohimbine. Inhibitory responses of the kappa selective agonist ethylketocyclazocine were enhanced, while that of delta selective agonists [Leu5]-enkephalin and DPDPE remained unchanged when yohimbine was present. Thus, the effects of opioids vary and depend on the tissue and receptor subtypes they act upon. Furthermore, the enhanced inhibitory effect of opioid receptor activation in the presence of yohimbine is not found for all opioid receptors.