Characterization of the Synergistic Effect between Ligands of Opioid and Free Fatty Acid Receptors in the Mouse Model of Colitis.

BACKGROUND: Recent studies suggest that lipids, including free fatty acids (FFAs), are necessary for proper µ opioid receptor (MOR) binding and that activation of opioid receptors (ORs) improves intestinal inflammation. The objective of the study was to investigate a possible interaction between the ORs and FFA receptors (FFARs) ligands in the colitis. METHODS: The potential synergistic effect of ORs and FFARs ligands was evaluated using mouse model of acute colitis induced by dextran sulfate sodium (DSS, 4%). Compounds were injected intraperitoneally (i.p.) once or twice daily at the doses of 0.01 or 0.02 mg/kg body weight (BW) (DAMGO-an MOR agonist), 0.3 mg/kg BW (DPDPE-a δ OR (DOR) agonist) and 1 mg/kg BW (naloxone-a non-selective OR antagonist, GLPG 0974-a FFAR2 antagonist, GSK 137647-a FFAR4 agonist and AH 7614-a FFAR4 antagonist) for 4 days. RESULTS: Myeloperoxidase (MPO) activity was significantly decreased after DAMGO (0.02 mg/kg BW) and GSK 137647 (1 mg/kg BW) administration and co-administration as compared to DSS group. CONCLUSIONS: Treatment with ligands of ORs and FFARs may affect the immune cells in the inflammation; however, no significant influence on the severity of colitis and no synergistic effect were observed.

Suppression of Human Natural Killer Cells by Different Classes of Opioids.

BACKGROUND: The use of regional and other opioid-sparing forms of anesthesia has been associated with a decrease in the recurrence of certain malignancies. Direct suppression of human natural killer cells by opioids has been postulated to explain this observation. However, the effect of different classes of opioids on suppression of natural killer cell cytotoxicity has not been systematically characterized. METHODS: After confirming that freshly isolated natural killer cells from peripheral human blood express opioid receptors, cells were incubated with increasing concentrations of clinically used or receptor-specific opioid agonists. We also evaluated the effect of pretreatment with receptor-specific antagonists or naloxone. Treated natural killer cells were then coincubated with a carboxyfluorescein succinimidyl ester-labeled target tumor cell line, K562. Annexin V staining was used to compare the percent of tumor cell apoptosis in the presence of opioid-pretreated and untreated natural killer cells. Treated samples were compared to untreated samples using Kruskal-Wallis tests with a post hoc Dunn correction. RESULTS: Morphine, methadone, buprenorphine, loperamide, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin, and U-50488 significantly decreased natural killer cell cytotoxicity. When natural killer cells were pretreated with naloxone, cyprodime, and nor-binaltorphimine before exposure to morphine, there was no difference in natural killer cytotoxicity, compared to the amount observed by untreated natural killer cells. Fentanyl, O-desmethyltramadol, and [D-Pen2,D-Pen5] enkephalin did not change natural killer cell cytotoxicity compare to untreated natural killer cells. CONCLUSIONS: Incubation of isolated natural killer cells with certain opioids causes a decrease in activity that is not observed after naloxone pretreatment. Suppression of natural killer cell cytotoxicity was observed with µ- and κ-receptor agonists but not δ-receptor agonists. These data suggest that the effect is mediated by µ- and κ-receptor agonism and that suppression is similar with many clinically used opioids.

Opposite effects of mu and delta opioid receptor agonists on excitatory propagation induced in rat somatosensory and insular cortices by dental pulp stimulation.

The insular cortex (IC) contributes to nociceptive information processing. IC neurons express opioid receptors, including the mu (MOR), kappa (KOR), and delta (DOR) subtypes. Opioidergic agonists suppress excitatory synaptic transmission in the cerebral cortex. In addition, morphine injection into the IC reduces responses to noxious thermal stimuli. However, the mechanisms of the opioid-dependent modulation of cortical excitation at the macroscopic level, which bridge the cellular and behavioral findings, have remained unknown. The present in vivo optical imaging study aimed to examine the effects of the agonists of each subtype on cortical excitatory propagation in the IC and the neighboring cortices, the primary (S1) and secondary somatosensory (S2) areas. To assess the opioidergic effects on the cortical circuits, we applied electrical stimulation to the maxillary 1st molar pulp, which induced excitation in the ventral part of S1 and the S2/insular oral region (IOR). The initial excitatory response was observed 10-14ms after stimulation, and then excitation propagated concentrically. DAMGO (10-100µM), an MOR agonist, suppressed the amplitude of cortical excitation and shrank the maximum excitation areas in S1 and S2/IOR. In contrast, 10-100µM DPDPE, a DOR agonist, increased the amplitude of excitation and expanded the area of maximum excitation. U50488 (10-100µM), a KOR agonist, had little effect on cortical excitation. These results suggest that MOR-induced suppression of excitatory propagation in the IC is an underlying mechanism of the powerful analgesic effects of MOR agonists. In contrast, DOR may play a minor role in suppressing acute pain.

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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The inhibition of the nitric oxide-cGMP-PKG-JNK signaling pathway avoids the development of tolerance to the local antiallodynic effects produced by morphine during neuropathic pain.

Tolerance to the local antiallodynic effects of morphine, DPDPE ([D-Pen(2),D-Pen(5)]-Enkephalin) or JWH-015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone) after their repeated administration during neuropathic pain was evaluated. The role of the nitric oxide-cGMP-protein kinase G (PKG)-c-Jun N-terminal kinase (JNK) signaling pathway on the peripheral morphine-induced tolerance after the chronic constriction of sciatic nerve in mice was also assessed. The mechanical and thermal antiallodynic effects produced by a high dose of morphine, DPDPE or JWH-015 subplantarly administered daily from days 10 to 20 after nerve injury were estimated with the von Frey filaments and cold plate tests. The antiallodynic effects of the repeated administration of morphine combined with a sub-analgesic dose of a selective inducible nitric oxide synthase (NOS2) (L-N(6)-(1-iminoethyl)-lysine; L-NIL), L-guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ), PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) or JNK (anthra[1,9-cd]pyrazol-6(2H)-one; SP600125) inhibitor from days 10 to 20 after injury were also evaluated. The repeated administration of morphine, but not DPDPE or JWH-015, produced a rapid development of tolerance to its mechanical and thermal antiallodynic effects in sciatic nerve-injured mice. The co-administration of morphine with L-NIL, ODQ, Rp-8-pCPT-cGMPs or SP600125 avoided the development of morphine antiallodynic tolerance after nerve injury. These findings reveal that the repeated local administration of DPDPE or JWH-015 did not induce antinociceptive tolerance after sciatic nerve injury-induced neuropathic pain. Our data also indicate that the peripheral nitric oxide-cGMP-PKG-JNK signaling pathway participates in the development of morphine tolerance after nerve injury and propose the inactivation of this pathway as a promising strategy to avoid morphine tolerance during neuropathic pain.

Involvement of peripheral mu opioid receptors in scratching behavior in mice.

Pruritus is a common adverse effect of opioid treatment. However, the mechanism by which pruritus is induced by opioid administration is unclear. In this study, we examined the effects of the intradermal injection of loperamide, a peripherally restricted opioid receptor agonist, on the itch sensation. When injected intradermally into the rostral part of the back in mice, loperamide elicited scratching behavior. We also examined the effects of the selective mu opioid receptor agonist [d-Ala², N-Me-Phe4, Gly5-ol]-enkephalin acetate (DAMGO), the selective delta opioid receptor agonist [d-Pen(2,5)]-enkephalin (DPDPE), and the selective kappa opioid receptor agonist U-50488H on scratching behavior in mice in order to determine which subtype is involved in opioid-induced pruritus. Following intradermal injection into the rostral part of the back in mice, DAMGO elicited scratching behavior, while DPDPE and U-50488H did not. This suggests that peripheral mu opioid activation elicits the itch sensation. Next, we focused on the treatment of opioid-induced itch sensation without central adverse effects. Naloxone methiodide is a peripherally restricted opioid receptor antagonist. In the present study, naloxone methiodide significantly suppressed scratching behavior induced by loperamide and DAMGO. These findings suggest that mu opioid receptors play a primary role in peripheral pruritus and that naloxone methiodide may represent a possible remedy for opioid-induced itching.

A role for delta opioid receptors in the central nucleus of the amygdala in anxiety-like behaviors.

RATIONALE: Compounds acting on delta opioid receptors (DOR) modulate anxiety-like behaviors, yet the site of action underlying this effect is unknown. DOR mRNA and protein are expressed in the central nucleus of the amygdala, a region that plays an important role in processing fear, stress, and anxiety. We hypothesized that this brain region may contribute to the modulation of anxiety by DOR drugs. OBJECTIVE: The present study investigated the role of DOR in the central amygdala in anxiety-like behaviors. METHODS: The selective DOR agonist [D-Pen 2,5]-enkephalin (DPDPE) or antagonist naltrindole was bilaterally microinjected into the central nucleus of the amygdala of adult male Sprague Dawley rats and anxiety-like behaviors were assessed using the elevated plus maze. The effects of DOR agonists on heightened anxiety produced by stress were also investigated. RESULTS: Rats injected with DPDPE into the central nucleus of the amygdala demonstrated less anxiety-like behavior, as evidenced by significantly greater number of open-arm entries and time spent in the open arms than controls. Naltrindole administered alone did not affect the duration or number of entries onto the open arms; however, naltrindole pre-treatment blocked the anxiolytic effects produced by DPDPE. Systemic administration of the selective DOR agonist, SNC80, or microinjection of DPDPE into the central amygdala prior to a swim stress blocked the anxiogenic effect produced by the swim stress. CONCLUSIONS: These findings provide direct evidence that activation of DOR in the central amygdala reduces anxiety-like behavior and suggest that DOR in this area are important for regulating anxious states.

Functional characterization of mouse organic anion transporting peptide 1a4 in the uptake and efflux of drugs across the blood-brain barrier.

This study investigated the role of a multispecific organic anion transporter, Oatp1a4/Slco1a4, in drug transport across the blood-brain barrier. In vitro transport studies using human embryonic kidney 293 cells expressing mouse Oatp1a4 identified the following compounds as Oatp1a4 substrates: pitavastatin (K(m) = 8.3 microM), rosuvastatin (K(m) = 12 microM), pravastatin, taurocholate (K(m) = 40 microM), digoxin, ochratoxin A, and [d-penicillamine(2,5)]-enkephalin. Double immunohistochemical staining of Oatp1a4 with P-glycoprotein (P-gp) or glial fibrillary acidic protein demonstrated that Oatp1a4 signals colocalized with P-gp signals partly but not with glial fibrillary acidic protein, suggesting that Oatp1a4 is expressed in both the luminal and the abluminal membranes of mouse brain capillary endothelial cells. The brain-to-blood transport of pitavastatin, rosuvastatin, pravastatin, and taurocholate after microinjection into the cerebral cortex was significantly decreased in Oatp1a4(-/-) mice compared with that in wild-type mice. The blood-to-brain transport of pitavastatin, rosuvastatin, taurocholate, and ochratoxin A, determined by in situ brain perfusion, was significantly lower in Oatp1a4(-/-) mice than in wild-type mice, whereas transport of pravastatin and [D-penicillamine(2,5)]-enkephalin was unchanged. The blood-to-brain transport of digoxin was significantly lower in Oatp1a4(-/-) mice than in wild-type mice only when P-gp was inhibited by 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 (GF120918). Taken together, these results show that Oatp1a4 can mediate the brain-to-blood and blood-to-brain transport of its substrate drugs across the blood-brain barrier. The brain-to-plasma ratio of taurocholate, pitavastatin, and rosuvastatin was close to the capillary volume in wild-type mice, and it was not affected by Oatp1a4 dysfunction. Whether Oatp1a4 can deliver drugs from the blood to the brain remains controversial.

The peripheral administration of a nitric oxide donor potentiates the local antinociceptive effects of a DOR agonist during chronic inflammatory pain in mice.

Several works reveal that nitric oxide could enhance the peripheral antinociception induced by opioids during acute inflammation. Nonetheless, the role of nitric oxide in the local antinociceptive effects of delta-opioid receptor (DOR) agonists during chronic peripheral inflammation is not known. The aim of this study is to evaluate whether nitric oxide would enhance the local antinociceptive effects of a DOR agonist during chronic inflammatory pain in mice. Chronic inflammatory pain was induced by the subplantar administration of complete Freund's adjuvant (CFA; 30 microl) and thermal hyperalgesia assessed by plantar test. In C57BL/6J mice, we evaluated the local antinociceptive effects of a DOR agonist, [D-Pen2,5]-enkephalin (DPDPE) and a nitric oxide donor, DETA NONOate DETA/NO 2,2'-(hydroxynitrosohydrazino) Bis-Ethanamine (NOC-18) alone or combined (DPDPE plus NOC-18) at 1, 4, 7, and 10 days after CFA injection. The reversibility of the peripheral antinociceptive effects of DPDPE, alone or combined with NOC-18, was assessed with the local administration of selective (naltrindole) and non-selective (naloxone methiodide) DOR antagonists. The local administration of DPDPE or NOC-18 alone dose-dependently inhibited the thermal hyperalgesia induced by peripheral inflammation. Moreover, the co-administration of NOC-18 with DPDPE significantly increased the antinociceptive effects produced by DPDPE from 1 to 10 days of CFA-induced inflammatory pain (P < 0.05). These effects were completely blocked by naltrindole and naloxone methiodide. Our results demonstrate that nitric oxide might enhance the local antinociceptive effects of a DOR agonist during chronic inflammatory pain by interaction with peripheral DOR, representing a useful strategy for an efficient antinociceptive treatment of peripheral inflammatory pain.

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 24h 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 2h, then hourly up to 6h post-injection. Naltrexone dose-dependently suppressed fluid consumption 24h 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 4h 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.

Neuroprotection and functional recovery conferred by administration of kappa- and delta 1-opioid agonists in a rat model of global ischemia.

Studies that have evaluated the beneficial effect of pre-ischemic treatment of kappa-opioid receptor agonists have used short-term reperfusion intervals. We examined the long-term impact of the pre-ischemic peripheral injection of U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide), a selective kappa-opioid receptor agonist, on neuronal damage and behavioral deficits following global ischemia in rats. Four groups of ischemic rats were pretreated with various doses of U50,488H (i.p. 0, 5, 15, 30 mg/kg) 15 min prior to vessel occlusion. Two groups of sham-operated animals that received either saline or U50,488H (30 mg/kg) acted as controls. The injection of 30 mg/kg U50,488H led to a 65% increase in CA1 neuron survival 35 days post-ischemia. CA1 neuronal protection translated into significant improvement of ischemia-induced spatial memory deficits assessed in the 8-arm radial maze. However, there was no difference in activity in the open field. We also found that the pre-ischemic intracerebroventricular injection of 5 mug of the delta1-opioid receptor agonist DPDPE ([d-Pen(2,5)]-enkephalin) produced a 59% increase in CA1 neuron survival 7 days post-ischemia. Similar to U50,488H, DPDPE had no significant impact on locomotor activity. These findings support a role for kappa- and delta-opioid receptors in attenuation of ischemia-induced hippocampal damage and cognitive impairments.

Selectivity of delta- and kappa-opioid ligands depends on the route of central administration in mice.

The existence of heterodimeric opioid receptors has introduced greater complexity to the in vivo characterization of pharmacological selectivity of agonists by antagonists. Because of the possibility of cooperativity between receptors organized as heterodimers, it is conceivable that selective antagonists may antagonize an agonist bound to a neighboring, allosterically coupled receptor. As a consequence, the in vivo selectivity of an opioid antagonist may depend on the organizational state of receptors that mediate analgesia. In this regard, phenotypic delta- and kappa-opioid receptors have been proposed to arise from different organizational states that include oligomeric delta-kappa heterodimers and homomeric delta and kappa receptors. In view of the evidence for analgesia mediated by delta-kappa heterodimers in the spinal cord, but not the brain, we have investigated the selectivity of pharmacologically selective delta and kappa antagonists in mice by both i.t. and i.c.v. routes of administration to evaluate changes in selectivity. Using pharmacologically selective delta (benzylidenenaltrexone, naltrindole, and naltriben) and kappa (norbinaltorphimine) antagonists versus delta ([D-Pen(2),D-Pen(5)]-enkephalin and deltorphin II) and kappa [3,4-dichloro-N-methyl-N-[(1R,2R)-2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and bremazocine] agonists, the delta-1/delta-2 selectivity ratios were found to be dependent on the route of administration (i.t. versus i.c.v.). The data from different routes of administration suggest that differences in molecular recognition between spinal delta-kappa heterodimers and supraspinal homomeric delta and kappa receptors may contribute to the divergent selectivity ratios of selective antagonists. In view of the observed tissue-dependent selectivity, we suggest that multiple opioid antagonists be employed routinely in establishing agonist selectivity in vivo.

Effects of opioid subtypes on detrusor overactivity in rats with cerebral infarction.

AIM: In order to determine the influence of different opioid receptor subtypes on detrusor overactivity after left middle cerebral artery (MCA) occlusion, cystometric recordings were obtained in conscious rats. METHODS: Female Sprague-Dawley rats were used in this study. Control cystometrography was followed by left MCA occlusion. The sham-operated (SO) rats underwent the same procedures except for MCA occlusion. [D-Ala(2), Phe(4), Gly(5)]-enkephalin (DAGO; mu-opioid agonist), [D-Pen(2,5)]-enkephalin (DPDPE; delta1-opioid agonist), deltorpin II (delta2-opioid agonist), and U-50488 (kappa-opioid agonist) were administered intracerebroventricularly at graded doses. The bladder capacity, residual volume, micturition threshold pressure, and bladder contraction pressure were determined. Finally, the volume of the infarction was measured. RESULTS: The intracerebroventricular administration of DAGO and DPDPE significantly increased the bladder capacity in the cerebrally infarcted (CI) and SO rats, but differences in the changes in bladder capacity between the CI and SO rats were not significant. Deltorpin II did not produce any changes in the bladder capacity in the CI or SO rats at any dose examined. However, the intracerebroventricular administration of U-50488 significantly increased the bladder capacity in the CI rats but not in the SO rats. None of the drugs affected the residual volume, micturition threshold pressure or bladder contraction pressure at any dosage examined. The mean infarcted volumes were not significantly different from those in the vehicle-treated rats. CONCLUSION: These results suggest that the opioid receptor subtypes, mu and delta1 in the brain, are related to the micturition reflex. Furthermore, the kappa opioid agonist might be useful for the suppression of detrusor overactivity caused by cerebral infarction.

Mediating delta-opioid-initiated heart protection via the beta2-adrenergic receptor: role of the intrinsic cardiac adrenergic cell.

Stimulation of cardiac beta(2)-adrenergic receptor (beta(2)-AR) or delta-opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that delta-opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity that was colocalized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective delta-opioid agonist D-[Pen(2,5)]enkephalin (DPDPE) to ICA cells increased [Ca(2+)](i) transients in a concentration-dependent manner. Such an effect was abolished by the Ca(2+) channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of the ICA cell and its epinephrine release in delta-opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-ventricular myocyte coculture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54 +/- 15% or myocyte death by 26 +/- 4%, respectively. beta(2)-AR blockade markedly attenuated delta-opioid-initiated infarct size-limiting effect and abolished delta-opioid-initiated myocyte survival protection in rat ICA cell-myocyte coculture. Furthermore, delta-opioid agonist exerted no myocyte survival protection in the absence of cocultured ICA cells during ischemia-reperfusion. We conclude that delta-opioid-initiated myocardial infarct size reduction is primarily mediated via endogenous epinephrine/beta(2)-AR signaling pathway as a result of ICA cell activation.

The role of progestin receptors and the mitogen-activated protein kinase pathway in delta 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 delta 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 delta 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 delta opioid receptors.

Peptidic delta opioid receptor agonists produce antidepressant-like effects in the forced swim test and regulate BDNF mRNA expression in rats.

Systemically active, nonpeptidic delta opioid receptor agonists have been shown to produce antidepressant and anxiolytic effects in animal models in rodents. In addition, delta agonists have been shown to increase expression of brain-derived neurotrophic factor (BDNF) mRNA, an effect of some antidepressants, which may be important for the clinical efficacy of antidepressant drugs. The present study examined whether a variety of peptidic delta agonists, DPDPE, JOM-13, a systemically active derivative of DPDPE, deltorphin II, and H-Dmt-Tic-NH-CH2-Bid could produce convulsions and antidepressant-like effects in the forced swim test. In addition, some of these compounds were examined for their influence on BDNF mRNA expression. All four agonists dose-dependently decreased immobility in the forced swim test, indicating an antidepressant-like effect. Only JOM-13 produced convulsions at doses required for antidepressant-like effects. In addition, DPDPE increased BDNF mRNA expression, as measured by in situ hybridization, in the frontal cortex. The antidepressant-like effect of the agonists in the forced swim test and the increase in BDNF mRNA expression produced by DPDPE were blocked by the delta antagonist naltrindole. Therefore, activation of the delta receptor by centrally administered peptidic agonists and intravenously administered JOM-13 produces behavioral antidepressant-like effects without producing convulsions, and some peptidic agonists can increase BDNF mRNA expression, however, not as consistently as the systemically active nonpeptidic agonists.

Intracerebroventricular D-Pen2, D-Pen5-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-Pen2, D-Pen5-enkephalin (DPDPE) (0 microg, 0.005 microg, 1.0 microg or 2.5 microg) 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 microg 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 microg 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 microg 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.

Effects of chronic intrathecal infusion of a partial differential opioid agonist in dogs.

To define the effects of chronic spinal exposure to a highly selective partial differential opioid agonist c[DPen(2),DPen(5)]enkephalin (DPDPE), adult beagles were prepared with chronic lumbar intrathecal catheters. Groups of dogs received intrathecal infusions (100 micro l/h) of saline (vehicle), DPDPE 3 mg/ml or 6 mg/ml for 28 days. Over the 28-day period, saline or 3 mg/ml showed minimal changes in neurological function, whereas in the 6 mg/ml animals, prominent hind limb dysfunction evolved over the 28-day interval. Histopathology in control animals displayed a modest pericatheter reaction considered normal for this model. Dogs receiving DPDPE (three of four at 6 mg/ml and one of four at 3 mg/ml) but not saline (zero of four) developed large inflammatory masses (granulomas) in the intrathecal space located proximal to the catheter tip. In these masses, severe chronic inflammatory changes in combination with necrosis and fibrosis was detected. Occasional focal destruction of neuropil was detected also in the adjacent spinal cord parenchyma. These masses contained extensive accumulation of mouse antihuman macrophages (MAC)-positive inflammatory cells expressing tumor necrosis factor-alpha (TNF-alpha), revealing infiltration of macrophages, granulocytes, and monocytes. In separate animals, prepared with dual intrathecal catheters, lumbar CSF was sampled at specified time points following intrathecal bolus (3 mg/ml) and 24 h DPDPE infusion (3 mg/ml and 6 mg/ml). Steady-state cerebrospinal fluid (CSF) DPDPE levels were 18.6 +/- 1.0 and 22.6 +/- 4.0 micro g/ml for 3 mg/ml and 6 mg/ml infusions respectively. These results indicate that this partial differential opioid agonist DPDPE produces a concentration and time-dependent formation of an intrathecal inflammatory mass.

Spinal nitric oxide contributes to the analgesic effect of intrathecal [d-pen2,d-pen5]-enkephalin in normal and diabetic rats.

BACKGROUND: Spinal nitric oxide (NO) is important for the analgesic actions of morphine and cholinergic agents. Its role in the analgesic effect of delta-opioid receptor agonists is not known. In the present study, the authors determined the role of spinal endogenous NO in the antinociceptive effect of intrathecal [D-Pen2, D-Pen5 ]-enkephalin (DPDPE), a delta-opioid receptor agonist, in normal rats and a rat model of diabetic neuropathic pain. METHODS: Rats were rendered diabetic with streptozotocin (50 mg/kg, intraperitoneal). Intrathecal catheters were implanted in age-matched normal and diabetic rats. Nociceptive thresholds were determined by application of a noxious pressure stimulus to the hind paw. The dose-dependent effect of intrathecal DPDPE was first determined. The role of spinal NO in the analgesic effect of intrathecal DPDPE was then examined through intrathecal treatments with NO synthase inhibitors (NMMA and TRIM) and a specific NO scavenger (carboxy-PTIO). RESULTS: The diabetic rats developed a sustained mechanical hyperalgesia within 3 weeks after streptozotocin injection. Intrathecal DPDPE, 2-20 micro g, dose-dependently increased the withdrawal threshold in response to the noxious pressure in normal and diabetic rats. However, the ED(50) of DPDPE in diabetic rats was about twofold higher than that in normal rats. Intrathecal pretreatment with NMMA, TRIM, or carboxy-PTIO diminished the analgesic effect of DPDPE in both normal and diabetic rats. Furthermore, the inhibitory effect of NMMA on the action of intrathecal DPDPE was reversed by intrathecal l-arginine but not d-arginine. CONCLUSIONS: Intrathecal DPDPE produces an antinociceptive effect in normal rats and a rat model of diabetic neuropathic pain. Spinal endogenous NO contributes importantly to the analgesic action of intrathecal DPDPE in both normal and diabetic neuropathic pain conditions.

Role of spinal nitric oxide in the inhibitory effect of [D-Pen2, D-Pen5]-enkephalin on ascending dorsal horn neurons in normal and diabetic rats.

Intrathecal [D-Pen2,D-Pen5]-enkephalin (DPDPE; a delta-opioid agonist) has a profound antinociceptive effect in neuropathic pain. Spinal nitric oxide (NO) has been implicated in the analgesic effect of several G protein-coupled receptor agonists. Little, however, is known about the role of spinal NO in the inhibitory effect of DPDPE on spinal dorsal horn neurons. In the present study, we determined the role of NO in the inhibitory effect of DPDPE on ascending dorsal horn neurons in normal rats and in a rat model of diabetic neuropathic pain. Single-unit activity of ascending dorsal horn neurons was recorded in anesthetized rats. The responses of dorsal horn neurons to graded mechanical stimuli and von Frey filaments were determined before and after local spinal application of 0.1 to 5 microM DPDPE. The influence of an NO synthase inhibitor, 1-(2-trifluoromethylphenyl) imidazole (TRIM; 30 microM), on the effect of DPDPE was then studied in separate groups of dorsal horn neurons in normal and diabetic rats. DPDPE inhibited the response of dorsal horn neurons in both normal and diabetic rats in a concentration-dependent fashion. The inhibitory effect of 1 microM DPDPE was abolished by 1 microM naltrindole, a delta-opioid antagonist. Furthermore, the inhibitory effect of DPDPE on the evoked response of dorsal horn neurons was largely eliminated by TRIM in normal and diabetic rats. These data suggest that DPDPE has a profound inhibitory effect on dorsal horn neurons in normal and diabetic rats. Spinal endogenous NO is essential for the inhibitory effect of DPDPE on ascending dorsal horn neurons in both normal and diabetic rats.