D-Pen2,D-Pen5]enkephalin Research Articles

Age-related changes in peripheral nociceptor function

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4–5 months) and aged (26–27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E2 (PGE2). Intraplantar injection of TRP channel activators, capsaicin (TRPV1), mustard oil (TRPA1) and menthol (TRPM8) each resulted in greater mechanical allodynia in aged versus young rats and capsaicin-induced heat allodynia was also greater in aged rats. A treatment-induced allodynia that was greater in young rats was never observed. The anti-allodynic effects of intraplantar injection of kappa and delta opioid receptor agonists, salvinorin-A and D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, were greater in aged than young rats, whereas mu opioid receptor agonists, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine, were not effective in aged rats. Consistent with these observations, in primary cultures of peripheral sensory neurons, inhibition of cAMP signaling in response to delta and kappa receptor agonists was greater in cultures derived from aged rats. By contrast, mu receptor agonists did not inhibit cAMP signaling in aged rats. Thus, age-related changes in nociceptors generally favor increased pain signaling in aged versus young rats, suggesting that changes in nociceptor sensitivity may play a role in the increased incidence of pain in the elderly population. These results also suggest that development of peripherally-restricted kappa or delta opioid receptor agonists may provide safer and effective pain relief for the elderly.

Impact of T161, Y318 and S363 alanine mutations on regulation of the human delta-opioid receptor (hDOPr) induced by peptidic and alkaloid agonists

Previously, we showed a differential regulation of the human delta-opioid receptor (hDOPr) by etorphine and [D-Pen2, D-Pen5] enkephalin (DPDPE). To understand the molecular basis of such differences, we introduced 3 alanine mutations at the residues T161. Y318 and S363.Both wild type (WT) and hDOPr mutants were expressed in HEK cells containing endogenous arrestins or CFP-tagged arrestin 3, then desensitization, internalization, recycling and phosphorylation were studied.In a context of endogenous arrestin expression, a major difference in DOPr desensitization was observed between agonists that was modified with the T161A mutation upon etorphine and with the S363A substitution upon DPDPE exposure. While both agonists induced a major receptor internalization, T161A and S363A impaired DOPr sequestration only for etorphine. However, similar level of S363 phosphorylation was measured between agonists. When CFP-tagged arrestin 3 was over-expressed, a similar profile of desensitization was measured for both agonists. In this context, all the 3 alanine mutations decreased etorphine-induced receptor desensitization. Using FRET, we showed similar interactions between WT hDOPr and arrestin 3 under DPDPE and etorphine stimulation which were delayed by both the Y318A and the S363A substitutions for etorphine. Finally, hDOPr recycling was qualitatively evaluated by microscopy and showed neither arrestin 3/hDOPr colocalization nor major impact of alanine mutations except for the S363A which impaired internalization and recycling for etorphine.The T161, Y318 and S363 residues of hDOPr could underlie the differential regulation promoted by DPDPE and etorphine.

Opioid Receptor Activity and Analgesic Potency of DPDPE Peptide Analogues Containing a Xylene Bridge.

d-Pen2,d-Pen5 enkephalin (DPDPE) is one of the most selective synthetic peptide agonists targeting the δ-opioid receptor. Three cyclic analogues of DPDPE containing a xylene bridge in place of disulfide bond have been synthesized and fully characterized as opioid receptors agonists. The in vitro activity was investigated showing a good affinity of 7a-c for μ- and δ-receptors. In vivo biological assays revealed that 7b is the most potent analogue with the ability to maintain high level of analgesia from 15 to 60 min following intracerebroventricular (i.c.v.) administration, whereas DPDPE was slightly active until 45 min. Compound 7b induced long lasting analgesia also after subcutaneous administration, whereas DPDPE was inactive.

Influence of the hypothalamic arcuate nucleus on intraocular pressure and the role of opioid peptides.

BackgroundAn opioid peptide neuron/humoral feedback regulation might be involved in changes of intraocular pressure (IOP). The aims of this study are to investigate the effects of arcuate nucleus (ARC) and opioid peptides on intraocular pressure (IOP).MethodsFifty-four healthy purebred New Zealand white rabbits (108eyes) were randomly divided into 4 groups, including control group, electrical stimulation group, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) group, and [D-Pen 2, D-Pen5]- enkephalin (DPDPE) group. Bilateral IOP was measured after unilateral electrical stimulation of the ARC or unilateral microinjection into the ARC of the selective μ-opioid receptor agonist DAMGO or the selective δ opioid receptor agonist DPDPE, both alone and after pre-administration of either the non-selective opioid receptor antagonist naloxone or saline.ResultsBoth electrical stimulation in ARC and micro-injection either <mu> or <delta> opioid receptor agonists, DAMGO or DPDPE, respectively, caused a significant bilateral reduction in IOP (P<0.05) which was more pronounced in the ipsilateral than in the contralateral eye. Pretreatment with naloxone prevented some, but not all IOP reductions.ConclusionThe ARC takes part in the negative regulation of IOP, an action that may involve opioid neurons.

Intra-VTA Deltorphin, But Not DPDPE, Induces Place Preference in Ethanol-Drinking Rats: Distinct DOR-1 and DOR-2 Mechanisms Control Ethanol Consumption and Reward

While there is a growing body of evidence that the delta opioid receptor (DOR) modulates ethanol (EtOH) consumption, development of DOR-based medications is limited in part because there are 2 pharmacologically distinct DOR subtypes (DOR-1 and DOR-2) that can have opposing actions on behavior. We studied the behavioral influence of the DOR-1-selective agonist [D-Pen(2) ,D-Pen(5) ]-Enkephalin (DPDPE) and the DOR-2-selective agonist deltorphin microinjected into the ventral tegmental area (VTA) on EtOH consumption and conditioned place preference (CPP) and the physiological effects of these 2 DOR agonists on GABAergic synaptic transmission in VTA-containing brain slices from Lewis rats. Neither deltorphin nor DPDPE induced a significant place preference in EtOH-naïve Lewis rats. However, deltorphin (but not DPDPE) induced a significant CPP in EtOH-drinking rats. In contrast to the previous finding that intra-VTA DOR-1 activity inhibits EtOH consumption and that this inhibition correlates with a DPDPE-induced inhibition of GABA release, here we found no effect of DOR-2 activity on EtOH consumption nor was there a correlation between level of drinking and deltorphin-induced change in GABAergic synaptic transmission. These data indicate that the therapeutic potential of DOR agonists for alcohol abuse is through a selective action at the DOR-1 form of the receptor.

Factors that Restrict the Cell Permeation of Cyclic Prodrugs of an Opioid Peptide, Part 3: Synthesis of Analogs Designed to have Improved Stability to Oxidative Metabolism

Previously, our laboratory reported that cyclic peptide prodrugs of the opioid peptide H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE) are metabolized by cytochrome P450 (CYP450) enzymes, which limits their systemic exposure after oral dosing to animals. In an attempt to design more metabolically stable cyclic prodrugs of DADLE, we synthesized analogs of DADLE cyclized with a coumarinic acid linker (CA; CA-DADLE), which contained modifications in the amino acid residues known to be susceptible to CYP450 oxidation. Metabolic stability and metabolite identification studies of CA-DADLE and its analogs were then compared using rat liver microsomes (RLM), guinea pig liver microsomes (GPLM), and human liver microsomes (HLM), as well as recombinant human recombinant cytochrome P450 3A4 (hCYP3A4). Similar to the results observed for CA-DADLE, incubation of its analogs with RLM, GPLM, and HLM resulted in monohydroxylation of an amino acid side chain on these cyclic prodrugs. When CA-DADLE was incubated with hCYP3A4, similar oxidative metabolism of the peptide was observed. In contrast, incubation of the CA-DADLE analogs with hCYP3A4 showed that these amino-acid-modified analogs are not substrates for this CYP450 isozyme. These results suggest that the amino-acid-modified analogs of CA-DADLE prepared in this study could be stable to metabolic oxidation by CYP3A4 expressed in human intestinal mucosal cells.

Influence of corticostriatal δ-opioid receptors on abnormal involuntary movements induced by L-DOPA in hemiparkinsonian rats

Chronic l-3,4-dihydroxyphenylalanine (l-DOPA) treatment of Parkinson's disease induces in time numerous side effects, such as abnormal involuntary movements called l-DOPA-induced dyskinesias (LIDs). An involvement of glutamate transmission, dopamine transmission and opioid transmission in striatal output pathways has been hypothesized for the induction of LIDs. Interestingly, our previous experiments indicated that some striatal δ-opioid receptors are located on terminals of glutamatergic corticostriatal neurons and that stimulation of these receptors modulates the release of glutamate and dopamine. The present study was performed to test the involvement of δ-opioid receptors, and more precisely of those located on corticostriatal neurons, in abnormal involuntary movements induced by l-DOPA in hemiparkinsonian rats. The effects of a selective agonist, [d-Pen2, d-Pen5]-enkephalin (DPDPE) and a selective antagonist (naltrindole) of δ-opioid receptors on LIDs were investigated in animals submitted or not to a corticostriatal deafferentation. Our results indicate that DPDPE and naltrindole respectively enhanced and reduced LIDs in animals in which the ipsilateral cortex was preserved intact. However, the lesion of the ipsilateral cortex prevented the stimulant effect of DPDPE on LIDs. The [3H]-DPDPE binding to striatal membranes prepared from the whole striatum was also studied. A significant increase in density of δ-opioid receptors was found in the striatum of dyskinetic animals as compared to non-dyskinetic animals but this difference was abolished by the corticostriatal deafferentation. These results indicate that δ-opioid transmission modulates the expression of LIDs in rodents and suggest that the δ-opioid receptors involved in this effect are located on terminals of corticostriatal neurons.

Nucleus Accumbens μ-Opioid Receptors Mediate Social Reward

Positive social interactions are essential for emotional well-being and proper behavioral development of young individuals. Here, we studied the neural underpinnings of social reward by investigating the involvement of opioid neurotransmission in the nucleus accumbens (NAc) in social play behavior, a highly rewarding social interaction in adolescent rats. Intra-NAc infusion of morphine (0.05-0.1 μg) increased pinning and pouncing, characteristic elements of social play behavior in rats, and blockade of NAc opioid receptors with naloxone (0.5 μg) prevented the play-enhancing effects of systemic morphine (1 mg/kg, s.c.) administration. Thus, stimulation of opioid receptors in the NAc was necessary and sufficient for morphine to increase social play. Intra-NAc treatment with the selective μ-opioid receptor agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) (0.1-10 ng) and the μ-opioid receptor antagonist Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) (0.3-3 μg) increased and decreased social play, respectively. The δ-opioid receptor agonist DPDPE ([D-Pen(2),D-Pen(5)]-enkephalin) (0.3-3 μg) had no effects, whereas the κ-opioid receptor agonist U69593 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]acetamide) (0.01-1 μg) decreased social play. Intra-NAc treatment with β-endorphin (0.01-1 μg) increased social play, but met-enkephalin (0.1-5 μg) and the enkephalinase inhibitor thiorphan (0.1-1 μg) were ineffective. DAMGO (0.1-10 ng) increased social play after infusion into both the shell and core subregions of the NAc. Last, intra-NAc infusion of CTAP (3 μg) prevented the development of social play-induced conditioned place preference. These findings identify NAc μ-opioid receptor stimulation as an important neural mechanism for the attribution of positive value to social interactions in adolescent rats. Altered NAc μ-opioid receptor function may underlie social impairments in psychiatric disorders such as autism, schizophrenia, or personality disorders.

Shell/core differences in mu- and delta-opioid receptor modulation of dopamine efflux in nucleus accumbens

The mu- and delta-opioid receptors located at the terminal level in nucleus accumbens are involved in the opiate modulation of dopamine release in this brain area. However, recent studies suggest that the effects of opioid drugs on the core subregion of nucleus accumbens may completely differ from those observed in the shell. We used in vivo microdialysis to simultaneously apply selective mu- and delta-opioid receptor agonists and to measure extracellular levels of dopamine in three subregions of the accumbens, namely shell, core, and the transition zone between them. The regional analysis of these subregions of the accumbens demonstrated that basal levels of dopamine and its metabolites were higher in the core, and decreased from this subregion to the shell. Retrodialysis application to the core of both the selective mu-receptor agonist ([ d-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin (DAMGO) (1 μmol/L)) and of the selective delta-opioid receptor agonist ([ d-Pen 2, d-Pen 5]-enkephalin (DPDPE) (50 nmol/L)) increased the dialysate levels of dopamine. However, the application of these drugs to the shell significantly reduced the dopamine levels in this subregion. Local application of the same doses of these drugs in the transition zone between the shell and the core did not significantly affect the dopamine levels in dialysates. These results suggest that the opioid circuits modulating dopaminergic activity in the shell could differ from those in the core of the nucleus accumbens.

Acute delta- and kappa-opioid agonist pretreatment potentiates opioid antagonist-induced suppression of water consumption

The primary objective of this study was to determine whether pretreatment with kappa- and delta-opioid agonists potentiates naltrexone-induced suppression of water consumption following 24 h of deprivation. This study also examined the temporal effects of agonist-induced antinociception using the tail-flick and hot-plate tests. Adult male Sprague-Dawley rats were water deprived 20 h and then given an injection (s.c. or i.c.) of an opioid agonist or saline. Drugs included the mu-opioid agonists morphine and DAMGO ([ d-Ala2, NMePhe4,Gly-ol5]-enkephalin), the kappa-opioid agonists spiradoline, bremazocine, and U69,593, and the delta-opioid agonists BW 373U86 and DPDPE ([D-Pen2, D-Pen5]-enkephalin). Three hours and forty-five minutes later, animals received a single dose of naltrexone (0.1–30 mg/kg, s.c.) or saline. Fifteen minutes later, animals were allowed free access to water for 30 min. For the tail-flick and hot-plate tests, animals were given a single injection of agonist and tested in both procedures every 30 min for up to 2 h, then hourly up to 6 h post-injection. Naltrexone dose-dependently suppressed fluid consumption 24 h after deprivation. The effects of naltrexone on drinking were potentiated following pretreatment with at least one dose of the agonists tested except BW 373U86. With the exception of BW 373U86, DAMGO, and DPDPE, all of the opioid agonists produced significant antinociception in the hot-plate test. Only BW 373U86 failed to have an antinociceptive effect in the tail-flick test. By 4 h after treatment, drug-induced antinociception had largely waned, suggesting the potentiation of naltrexone-induced drinking suppression was not a result of a direct interaction with the agonists. In conclusion, kappa-opioid and delta-opioid receptors appear to contribute to the manifestation of acute opioid dependence, albeit to a lesser degree than mu-opioid receptors.

Distinct inhibition of voltage-activated Ca2+ channels by δ-opioid agonists in dorsal root ganglion neurons devoid of functional T-type Ca2+ currents

Both μ- and δ-opioid agonists selectively inhibit nociception but have little effect on other sensory modalities. Voltage-activated Ca2+ channels in the primary sensory neurons are important for the regulation of nociceptive transmission. In this study, we determined the effect of δ-opioid agonists on voltage-activated Ca2+ channel currents (ICa) in small-diameter rat dorsal root ganglion (DRG) neurons that do and do not bind isolectin B4 (IB4). The δ-opioid agonists [d-Pen2,d-Pen5]-enkephalin (DPDPE) and deltorphin II produced a greater inhibition of high voltage–activated ICa in IB4-negative than IB4-positive neurons. Furthermore, DPDPE produced a greater inhibition of N-, P/Q-, and L-type ICa in IB4-negative than IB4-positive neurons. However, DPDPE had no significant effect on the R-type ICa in either type of cells. We were surprised to find that DPDPE failed to inhibit either the T-type or high voltage–activated ICa in all the DRG neurons with T-type ICa. Double immunofluorescence labeling showed that the majority of the δ-opioid receptor-immunoreactive DRG neurons had IB4 labeling, while all DRG neurons immunoreactive to δ-opioid receptors exhibited Cav3.2 immunoreactivity. Additionally, DPDPE significantly inhibited high voltage–activated ICa in Tyrode's or N-methyl-d-glucamine solution but not in tetraethylammonium solution. This study provides new information that δ-opioid agonists have a distinct effect on voltage-activated Ca2+ channels in different phenotypes of primary sensory neurons. High voltage–activated Ca2+ channels are more sensitive to inhibition by δ-opioid agonists in IB4-negative than IB4-positive neurons, and this opioid effect is restricted to DRG neurons devoid of functional T-type Ca2+ currents.

Influence of glial cells in the dopamine releasing effect resulting from the stimulation of striatal δ-opioid receptors

Recent data indicate that striatal dopamine release induced by stimulation of δ-opioid receptors is a consequence of glutamate release. However, glial cells, which mainly support glutamate uptake and are involved in glutamate signaling and potentially express δ-opioid receptors, could participate to this effect. The present study investigates the contribution of glial cells in the releasing effects of [ d-Pen2, d-Pen5]-enkephalin (DPDPE) by using the gliotoxin l-α-aminoadipate ( l-αAA). Initially, we evaluated the early influence of l-αAA local infusion (10 μg/μL) on dialysate levels of glutamate and dopamine under basal or DPDPE treatment conditions. l-αAA produced a significant increase of glutamate and dopamine in dialysates (+76% and +50% respectively) and the concomitant infusion of DPDPE (10 μM) significantly enhanced this effect in an additive manner (+110% and +44% respectively). Secondly, we assessed the DPDPE effects on striatal glutamate and dopamine dialysate levels, 2 days after an intra-striatal injection of l-αAA which produced destruction of glial cells. This lesion, decreasing the basal glutamate dialysate level as well as its tissue content (by 55% and 36% respectively), prevented the increase in glutamate and dopamine extracellular levels induced by DPDPE. This result confirmed that the DPDPE-induced dopamine release requires an initial glutamate release. However, this effect could reflect a major disruption of glutamatergic transmission caused by the toxin, as suggested by the local infusion of glutamine (2.5 mM) which, in lesioned rats, prevented the decrease in the basal extracellular content of glutamate and restored the DPDPE-induced increase in glutamate and dopamine dialysate levels. Therefore, these results indicate that, although glial cells are essential to maintain functional glutamatergic neurotransmission, they are not directly involved in the process by which stimulation of striatal δ-opioid receptors induces extracellular glutamate release and, consecutively, dopamine release.

Presynaptic modulation of 5-HT release in the rat septal region

5-HT released from serotonergic axon terminals in the septal nuclei modulates the activity of septal output neurons (e.g. septohippocampal cholinergic neurons) bearing somatodendritic 5-HT receptors. Therefore, we studied the mechanisms involved in the presynaptic modulation of 5-HT release in the lateral (LS) and medial septum (MS), and the diagonal band of Broca (DB). HPLC analysis showed that tissue concentrations of noradrenaline, dopamine and 5-HT were highest in DB (DB>MS>LS). Slices prepared from LS, MS and DB regions were preincubated with [3H]5-HT, superfused in the presence of 6-nitro-2-(1-piperazinyl)-quinoline (6-nitroquipazine) and electrically stimulated up to three times (first electrical stimulation period (S1), S2, S3; 360 pulses, 3 Hz, 2 ms, 26–28 mA). In all septal regions the Ca2+-dependent and tetrodotoxin-sensitive electrically-evoked overflow of [3H] was inhibited by the 5-HT1B agonist CP-93,129 and the α2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline tartrate (UK-14,304). Also the μ- and κ-opioid receptor agonists (d-Ala2, N-Me-Phe4, glycinol5)-enkephalin (DAMGO) and [trans-(1S,2S(-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzenacetamide hydro-chloride] (U-50,488H), respectively, acted inhibitory (although less potently), whereas the δ-opioid receptor agonist (d-Pen2, d-Pen5)-enkephalin (DPDPE), the dopamine D2 receptor agonist quinpirole and the adenosine A1 receptor agonist N6-cyclopentyladenosine were all ineffective; the GABAB receptor agonist baclofen had weak effects. All inhibitory effects of the agonists were antagonized by the corresponding antagonists (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR-55,562), idazoxan, naloxone, nor-binaltorphimine), which also significantly enhanced the evoked release of 5-HT at S1. It is concluded that 5-HT release in septal nuclei of the rat is modulated by presynaptic 5-HT1B autoreceptors, as well as by α2-, μ- and κ-opioid heteroreceptors. All of these receptors seem to be under a tonic inhibitory influence of the corresponding endogenous agonists and show qualitatively comparable modulatory properties along the dorso-ventral distribution of the 5-HT terminals.

Beta‐arrestin 1 and beta‐arrestin 2 differentially direct the phosphorylation‐dependent and ‐independent internalization and desensitization of delta‐opioid receptor

δ-opioid receptor (DOR) and its phosphorylation-deficient mutant S363A (DORS363A) underwent internalization and desensitization during the long-term activation by [D-Pen2,D-Pen5]enkephalin (DPDPE). Co-expression of dominant negative mutant of dynamin (DynK44E) blocked the internalization of both DOR and DORS363A without significant influence on their desensitization. Confocal microscopy studies using β-arrestinGFP indicated that both DOR and DORS363A activation could induce transient translocation of the β-arrestins to the plasma membrane. Internalization and desensitization of both DOR and its phosphorylation-deficient mutant DTS were blocked in mouse embryonic fibroblasts (MEF) cells lacking β-arrestin1 and β-arrestin 2 (β arr1−/−/β arr2−/−) upon long-term treatment of DPDPE, suggesting that β-arrestins is a key determinant in triggering receptor desensitization. Internalization of DOR was impaired similarly in MEF cells lacking β-arr1 (β arr1−/−) or cells lacking β-arr2 (β arr2−/−). However, desensitization of DOR in β arr1−/− cells was slightly reduced, while the desensitization of DOR was significantly impaired in β arr2−/− cells. On the other hand, both internalization and desensitization of DTS is not significantly impaired in both β arr1−/− and β arr2−/− cells. These results indicate that β-arrestin 1 and β-arrestin 2 are differentially required in the phosphorylation-dependent and -independent internalization and desensitization of DOR. (These studies are supported by NIDA grants DA007339, DA016674, DA000564, DA011806)

The role of progestin receptors and the mitogen-activated protein kinase pathway in δ opioid receptor facilitation of female reproductive behaviors

The present study investigated the role of the progestin receptor (PR) and the mitogen-activated protein kinase (MAPK) pathway in the facilitation of lordosis behavior by the δ opioid receptor agonist [ d-Pen 2, d-Pen 5]-enkephalin (DPDPE). Ovariectomized, estrogen-primed rats were treated with the PR antagonist RU486 or the MAPK inhibitor PD98059 prior to intraventricular (icv) infusion of DPDPE. Both RU486 and PD98059 blocked receptive and proceptive behaviors induced by DPDPE at 60 min, and RU486 continued to inhibit estrous behavior at 90 min. Because δ opioid receptors can activate the p42/44 MAPKs, extracellular signal regulated kinases (ERK), we determined the effects of DPDPE on ERK phosphorylation. Icv infusion of DPDPE increased the levels of phosphorylated ERK in the hypothalamus and preoptic area of female rats, assessed by immunoblotting. These results support the participation of the PR and the MAPK pathway in the facilitation of lordosis behavior by δ opioid receptors.

Intracerebroventricular d-Pen 2, d-Pen 5-enkephalin administration soon after stressor imposition influences behavioral responsivity to a subsequent stressor encounter in CD-1 mice

Endogenous opioid peptide systems diminish stress-induced autonomic nervous system, neuroendocrine (hypothalamic-pituitary-adrenal axis) and behavioral responses, attenuating a collection of physiological symptoms basic to emotional and affective states. Neurogenic stressors may incite specific central changes in opioid peptide availability as well as changes in mu and delta-opioid receptor function. The present investigation evaluated the proactive influence of an intracerebroventricular injection of the opioid receptor agonist d-Pen 2, d-Pen 5-enkephalin (DPDPE) (0 μg, 0.005 μg, 1.0 μg or 2.5 μg) on locomotor behavior of mice following uncontrollable footshock (Shock) or novel shock chamber exposure (No Shock). It was expected that DPDPE administration following Shock on Day 1 would restore locomotor activity up to 1 week and prevent shock-associated behavior of mice encountering a brief session of footshock 18 days later. Exposure to Shock reduced horizontal locomotor and vertical locomotor (rearing) activity of mice while 2.5 μg DPDPE restored behavior. Eighteen days following Shock and DPDPE challenge, mice were exposed to either an abbreviated session of footshock (Mild Stress) or the shock chamber (Cues). Mice in the No Shock and Shock groups administered 2.5 μg DPDPE on Day 1 did not exhibit any locomotor deficits in response to Mild Stress on Day 18. Mice in the Shock group administered 0.005 μg DPDPE on Day 1, did not exhibit exaggerated rearing deficits following ensuing Mild Stressor encounter relative to mice reexposed to Cues on Day 18. Taken together, these data show that (a) footshock differentially affects rearing and locomotor activity, (b) DPDPE administration increases locomotor activity for up to 1 week following footshock and DPDPE administration, (c) reexposure to Mild Stress affects rearing and locomotor performance differently depending on previous stressor history and DPDPE dose, (d) DPDPE affords long-lasting protection to previously non-stressed mice against the deleterious effects of subsequent mild stress on locomotor activity, while a low dose of DPDE is sufficient to prevent shock-induced sensitization of rearing deficits, 18 days following original stressor and drug presentation. Finally, our investigation demonstrates that DPDPE administration alters the behavioral impact of future stressful encounters and emphasizes the importance of investigating opioid mechanisms in chronic stress disorders.

Involvement of corticostriatal glutamatergic terminals in striatal dopamine release elicited by stimulation of δ‐opioid receptors

We have previously shown that striatal dopamine release induced locally by a delta-opioid receptor agonist was totally inhibited by a glutamate N-methyl-D-aspartate receptor antagonist, indicating the involvement of glutamatergic receptors in this effect. The aim of the present study was to specify this mechanism. Firstly, we investigated the effect of [D-Pen2,D-Pen5]-enkephalin (DPDPE) on glutamate release in rats by intrastriatal microdialysis. The infusion of DPDPE (10 microm) enhanced the glutamate content in dialysate by approximately 34%, an effect which did not appear to result from inhibition of glutamate uptake. We then considered the consequences of a unilateral thermocoagulation of the frontal cortex on either glutamate or dopamine release induced by stimulation of delta-opioid receptors 2 days later. This lesion, which decreased the glutamate content in ipsilateral striatum by approximately 30%, totally prevented the increase in dialysate levels of glutamate induced by DPDPE. Moreover, whereas DPDPE (10 microm) was found to increase the striatal dopamine release in intact animals by approximately 59%, this effect was also completely suppressed by the cortical lesion. Finally, we studied the effect of the lesion on the [3H]-DPDPE binding to striatal membranes prepared from the whole striatum. In the ipsilateral striatum a significant decrease in this [3H]-DPDPE binding (by approximately 18%) was found 2 days after the lesion. Our results indicate that the increase in striatal dopamine release induced by DPDPE probably depends on glutamate release from corticostriatal glutamatergic afferents in response to the stimulation of delta-opioid receptors located on terminals of these neurons.

Opioid profiles of Cys 2-containing enkephalin analogues

To elucidate the structural features determining δ-opioid receptor properties of enkephalin analogues containing Cys(O 2NH 2) in position 2, a series of Cys 2-containing derivatives were synthesized and tested for their effectiveness in depressing electrically evoked contractions of the mouse vas deferens (predominantly enkephalin-selective δ-opioid receptors) and the guinea-pig ileum (μ- and κ-opioid receptors). The peptidase resistance of the compounds was also tested. The ratio IC 50 in the guinea-pig ileum/IC 50 in the mouse vas deferens, indicating selectivity for δ-opioid receptors, was high for Cys(O 2NH 2) 2-containing analogues and especially for [Cys(O 2NH 2) 2, Leu 5]enkephalin, which was about seven times more selective than δ-opioid receptor selective ligand cyclic [ d-Pen 2, d-Pen 5]enkephalin (DPDPE). The dissociation constant ( K A) and relative efficacy ( e rel) of the compounds in the mouse-isolated vas deferens were determined using explicit formulae derived by fitting of the data points with two-parametric hyperbolic function. The obtained values for K A and e rel suggest that: (i) incorporation of Cys(O 2NH 2) 2 in the molecule of [Leu 5]enkephalin highly increases the efficacy and does not change significantly the affinity of the respective analogues to δ-opioid receptors; [Cys(O 2NH 2) 2, Leu 5]enkephalin has higher affinity than DPDPE, but is less resistant to enzyme degradation; the effect of this modification on the efficacy is decreased when methionine is in position 5; (ii) d-configuration of Cys(O 2NH 2) 2-containing analogues increases their peptidase resistance, but reduces efficacy and affinity of the peptides towards δ-opioid receptors; (iii) the substitution of Cys(O 2NH 2) with Hcy(O 2NH 2) reduces the efficacy, affinity and potency of the respective analogues and maintains their sensitivity to endogenous peptidases; (iv) the substitution of the sulfonamide group with benzyl group in the molecule of Cys in position 2 decreases their efficacy and affinity toward δ-opioid receptors, but attaches resistance to enzyme degradation. The results obtained in this study allow: (i) to involve the receptor affinity and agonist efficacy as drug-design consideration for δ-opioid receptor properties of newly synthesized compounds and (ii) to characterize some of the structural features, which set the pattern for their opioid profiles.

Hepatobiliary disposition of the metabolically stable opioid peptide [D-Pen2, D-Pen5]-enkephalin (DPDPE): pharmacokinetic consequences of the interplay between multiple transport systems.

[D-Pen2,D-Pen5]-Enkephalin (DPDPE) is excreted extensively into the bile. Although DPDPE is transported by P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (Mrp2) has been identified as an important mechanism for DPDPE transport across the canalicular membrane of the hepatocyte. The present studies determined the relative impact of Mrp2 and P-gp on the hepatobiliary disposition of [3H]DPDPE in isolated perfused rat livers (IPLs). Perfusate clearance of [3H]DPDPE was not different between livers from control and Mrp2-deficient (TR-) rats. Biliary excretion of [3H]DPDPE in IPLs from Wistar control rats was rapid and extensive. However, when [3H]DPDPE was administered to livers from TR- rats, the rate and extent of excretion decreased significantly. Surprisingly, in the presence of the P-gp inhibitor GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], biliary excretion of [3H]DPDPE was not inhibited in control livers. In contrast, administration of GF120918 to TR- livers further reduced the maximal excretion rate and decreased net biliary excretion of [3H]DPDPE by 87%. GF120918 administration caused an unexpected increase in perfusate clearance in both control and TR- rat livers. At distribution equilibrium, [3H]DPDPE liver/perfusate partitioning was higher in GF120918-treated livers. Results of pharmacokinetic modeling were consistent with the hypothesis that GF120918 inhibited a [3H]DPDPE basolateral excretion mechanism. Mrp2 is the primary mechanism for [3H]DPDPE biliary excretion, and P-gp facilitates excretion of [3H]DPDPE only in the absence of functional Mrp2. [3H]DPDPE is a substrate for a basolateral efflux mechanism that is sensitive to inhibition by GF120918. These data emphasize the importance of using appropriate model systems and comprehensive pharmacokinetic modeling in elucidating the complex interplay between multiple transport systems.

Medial preoptic area δ-opioid receptors inhibit lordosis

Endogenous opioid peptides that activate the δ-opioid receptor (DOR) are thought to facilitate female receptive behavior. This facilitation of lordosis has been demonstrated by intracerebroventricular infusions and injection of DOR-active ligands into the ventromedial hypothalamic nucleus, an area with robust DOR binding. However, DOR binding is distributed throughout the hypothalamus, and the role of DOR in other areas of the hypothalamus has not been examined. In the current study, we demonstrated DOR immunoreactivity in the medial preoptic area (MPO), in particular medial preoptic nucleus (MPN) of the preoptic area. DOR immunoreactive processes were sparsely distributed in the medial and lateral parts of the MPN. Larger DOR immunoreactive fibers were localized in the ventrolateral aspect of the lateral MPN. The MPN is involved in the modulation of female sexual receptivity and the distribution of DOR in this area suggested to us that DOR may regulate lordosis. Ovariectomized rats with unilateral cannulae aimed at the MPN were given 5 μg 17β-estradiol benzoate (EB), once every 4 days and tested for lordosis. [D-Pen 2, D-Pen 5]-enkephalin (DPDPE), a DOR agonist, microinfused into the MPO, 52–54 h after EB-priming, inhibited lordosis when compared with the aCSF (vehicle) control ( P ≤ 0.05). The inhibitory effects of DPDPE were reversed by microinjection of naltrindole, a DOR antagonist ( P ≤ 0.05). Interestingly, the DOR inhibition of lordosis is similar to the μ-opioid receptor inhibition of lordosis in the MPN. These results indicate that DOR in the MPO, particularly in the MPNm, plays an important role in the regulation of lordosis.