Lack of antinociceptive efficacy of intracerebroventricular [D-Ala2,Glu4]deltorphin, but not [D-Pen2,D-Pen5]enkephalin, in the mu-opioid receptor deficient CXBK mouse.

The antinociceptive efficacy of [D-Pen2,D-Pen5]enkephalin (DPDPE) (delta 1 agonist) and [D-Ala2,Glu4]deltorphin (delta 2 agonist) was evaluated following intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration in CD-1 and CXBK strains of mice using the radiant heat tail-flick test. Following i.c.v. administration, [D-Ala2,Glu4]deltorphin was effective in CD-1, but not CXBK, mice; DPDPE was approximately equiactive in both strains. While i.c.v. [D-Ala2,Glu4]deltorphin did not produce antinociception in the CXBK mouse, it effectively antagonized the antinociceptive actions of i.c.v. DPDPE. [D-Ala2,Glu4]deltorphin was effective following i.t. administration in both strains. These data suggest possible differences in the supraspinal populations of opioid delta receptor subtypes in the CXBK strain. On the basis of previously established selectivity of these agonists, the CXBK mouse may have a predominate population of supraspinal opioid delta 1, rather than delta 2, receptors.     

Induction of delta opioid receptor function by up-regulation of membrane receptors in mouse primary afferent neurons

It is not clear whether primary afferent neurons express functional cell-surface opioid receptors. We examined delta receptor coupling to Ca2+ channels in mouse dorsal root ganglion neurons under basal conditions and after receptor up-regulation. [D-Ala2,Phe4,Gly5-ol]-enkephalin (DAMGO), [D-Ala2,D-Leu5]-enkephalin (DADLE), trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzene-acetamide methanesulfonate (U-50,488H; 1 microM), and baclofen (50 microM) inhibited Ca2+ currents, whereas the -selective ligands [D-Pen2,Pen5]-enkephalin (DPDPE) and deltorphin II (1 microM) did not. The effect of DADLE (1 microM) was blocked by the mu-antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 300 nM) but not by the -antagonist Tyr-1,2,3,4-tetrahydroisoquinoline-Phe-Phe-OH (300 nM), implicating mu receptors. Despite a lack of functional delta receptors, flow cytometry revealed cell-surface receptors. We used this approach to identify conditions that up-regulate receptors, including mu receptor gene deletion in dorsal root ganglion neurons of mu-/- mice and 18-h incubation of mu+/+ neurons with CTAP followed by brief (10-min) DPDPE exposure. Under these conditions, the expression of cell-surface delta receptors was up-regulated to 149 +/- 9 and 139 +/- 5%, respectively; furthermore, DPDPE and deltorphin II (1 microM) inhibited Ca2+ currents in both cases. Viral replacement of mu receptors in mu-/- neurons reduced delta receptor expression to mu+/+ levels, restored the inhibition of Ca2+ currents by DAMGO, and abolished receptor coupling. Our observations suggest that receptor-Ca2+ channel coupling in primary afferent fibers may have little functional significance under basal conditions in which mu receptors predominate. However, up-regulation of cell-surface delta receptors induces their coupling to Ca2+ channels. Pharmacological approaches that increase functional delta receptor expression may reveal a novel target for analgesic therapy.     

The R7 subfamily of RGS proteins assists tachyphylaxis and acute tolerance at mu-opioid receptors.

Members of the R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11) are found in the mouse CNS. The expression of these proteins was effectively reduced in different neural structures by blocking their mRNA with antisense oligodeoxynucleotides (ODNs). This was achieved without noticeable changes in the binding characteristics of labeled beta-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO)-both agonists at mu-opioid receptors. The duration of morphine analgesia was greatly increased in RGS9-2 and in RGS11 knockdown mice. The impairment of R7 proteins brought about different changes in the analgesic activity of selective delta agonists. Knockdown of RGS11 reduced [D-Ala(2)]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala(2)]deltorphin II to produce a smoothened time-course curve-the peak effect blunted and analgesia extended during the declining phase. RGS9-2 impairment also promoted a similar pattern of change for [D-Pen(2,5)]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala(2)]deltorphin II and DPDPE analgesic effects. A single intracerebroventricular (i.c.v.) ED(80) analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at mu-opioid receptors, and show differences in their modulation of delta-opioid receptors.     

Contribution of supraspinal mu- and delta-opioid receptors to antinociception in the rat.

This study evaluated the contribution of supraspinal opioid receptors to the production of antinociception, in the rat. I.c.v. administration of a selective mu- (DAMGO) and a selective delta- (DPDPE), but not a selective kappa- (U50,488H) opioid receptor agonist, produced significant dose-dependent increase in mechanical nociceptive thresholds. ICI 174,864, a delta-opioid receptor antagonist, completely blocked the antinociceptive effects produced by DPDPE ([D-Pen2,D-Pen5]enkephalin) at a dose that had no effect on the increases in nociceptive thresholds produced by DAMGO ([D-Ala2,N-MePhe4,Gly5-ol]enkephalin). The simultaneous i.c.v. administration of a low-antinociceptive dose of DAMGO or DPDPE given in combination with sequentially increasing doses of the other opioid agonist, produced synergy (i.e., a more than additive antinociceptive effect), at the lower doses tested. The results of these experiments provide evidence to support the suggestion that both supraspinal mu- and delta-opioid receptors contribute to the production of antinociception, in the rat.     

Dexamethasone-induced selective inhibition of the central mu opioid receptor: functional in vivo and in vitro evidence in rodents.

1. Endogenous corticosteroids and opioids are involved in many functions of the organism, including analgesia, cerebral excitability, stress and others. Therefore, we considered it important to gain information on the functional interaction between corticosteroids and specific opioid receptor subpopulations. 2. We have found that systemic administration (i.p.) of the potent synthetic corticosteroid, dexamethasone, reduced the antinociception induced by the highly selective mu agonist, DAMGO or by less selective mu agonists morphine and beta-endorphin administered i.c.v.. On the contrary dexamethasone exerted little or no influence on the antinociception induced by a delta 1 agonist, DPDPE and a delta 2 agonist deltorphin II. Dexamethasone potentiated the antinociception induced by the kappa agonist, U50,488. 3. In experiments performed in an in vitro model of cerebral excitability in the rat hippocampal slice, dexamethasone strongly prevented both the increase of the duration of the field potential recorded in CA1, and the appearance and number of additional population spikes induced by mu receptor agonists. 4. In both models pretreatment with cycloheximide, a protein synthesis inhibitor, prevented the antagonism by dexamethasone of responses to the mu opioid agonists. 5. Our data indicate that in the rodent brain there is an important functional interaction between the corticosteroid and the opioid systems at least at the mu receptor level, while delta and kappa receptors are modulated in different ways.     

Analgesia produced by centrally administered DAGO, DPDPE and U50488H in the formalin test

Three opioid agonists ([D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) and U50488H) were tested independently for their ability to produce analgesia in the formalin test. These agonists were chosen based upon their ability to act selectively at mu, delta and kappa opioid receptor types respectively. Rats received one intracerebroventricular (i.c.v.) injection of an agonist 20 min after subcutaneous injection of 15% formalin into a rear paw. Formalin injection produces continuous pain that results in two stereotypic behaviors, paw licking and paw lifting. Ten minutes after i.c.v. injection rats were observed for an 8 min period and scored for formalin-induced behavior. All agonists produced analgesia as indicated by a dose-dependent attenuation of formalin-induced behavior. At the doses tested, the rank order of analgesic efficacy was DAGO greater than DPDPE greater than U50488H. We suggest that centrally located mu, delta and kappa opioid receptors can each modulate the perception of this clinically relevant form of continuous pain. Additionally, the highest dose of DPDPE tested significantly increased rearing whereas DAGO and U50488H failed to affect rearing.     

Selective opioid antagonist effects on opioid-induced inhibition of release of norepinephrine in guinea pig cortex

Opioid agonists with selectivity for mu, delta and kappa-receptors have each been shown to inhibit the K+-stimulated release of [3H]norepinephrine (NE) from slices of guinea pig cortex maintained in vitro. In order to provide further evidence that each of these types of opioid receptor can regulate the release of NE in this tissue, experiments with receptor-type selective opioid antagonists have been conducted. In initial experiments, the selectivity of the antagonists for specific types of opioid receptors in the cortex of the guinea pig in an incubation medium of the same composition as that used for release studies was confirmed. The delta-receptor selective antagonist, ICI 174,864, prevented the inhibitory actions of the delta-selective agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), but had little effect on the inhibitory actions of the mu-selective agonist, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO), or the kappa-selective agonist, U-50,488H. In contrast, the kappa-selective antagonist, nor-binaltorphimine (nor-BNI) prevented the inhibitory actions of U-50,488H, but had little effect on the inhibitory actions of DPDPE or DAMGO. The greater potency of the partially mu-selective antagonist, naloxone, in reversing the effects of DAMGO relative to those of DPDPE or U-50,488H was confirmed. These results support the conclusion that mu- delta- and kappa-opioid receptors each exert a negative regulatory effect on the stimulated release of NE in the cortex of the guinea pig.     

Maintenance of acute morphine tolerance in mice by selective blockage of kappa opioid receptors with norbinaltorphimine.

In this study we investigated the effect of the highly selective kappa opioid antagonist, norbinaltorphimine (norBNI) on the development of tolerance to a single dose of morphine. Mice were pretreated with 100 mg/kg of morphine sulfate (morphine), s.c. and 2 h later, norBNI (20 mg/kg s.c.) was administered and various times after this pretreatment, antinociceptive ED50 value of morphine was determined in the tail-flick assay. Twenty-four and 72 h after morphine injection, ED50 values of morphine were significantly increased by about 2.5-fold from those of their control mice that received saline instead of the tolerance-inducing dose of morphine. In a second set of experiments, animals were pretreated similarly with morphine and norBNI and 72 h after morphine injection, various opioid agonists were applied by the i.c.v. or i.t. route to see whether or not any cross-tolerance had developed to these agonists. The ED50 of i.c.v.-administered morphine was significantly greater than that of the non-pretreated controls. A small degree of cross-tolerance was observed with U-50,488H but not with DPDPE [D-Pen2,D-Pen5]enkephalin (DPDPE) at the supraspinal site. At the spinal site, tolerance to morphine was not observed. These results suggest that antagonism at kappa opioid sites after morphine administration, modulates positively the development of opioid tolerance.     

RGSZ1 and GAIP regulate mu- but not delta-opioid receptors in mouse CNS: role in tachyphylaxis and acute tolerance.

In the CNS, the regulators of G-protein signaling (RGS) proteins belonging to the Rz subfamily, RGS19 (G(alpha) interacting protein (GAIP)) and RGS20 (Z1), control the activity of opioid agonists at mu but not at delta receptors. Rz proteins show high selectivity in deactivating G(alpha)z-GTP subunits. After reducing the expression of RGSZ1 with antisense oligodeoxynucleotides (ODN), the supraspinal antinociception produced by morphine, heroin, DAMGO ([D-Ala2, N-MePhe4,Gly-ol5]-enkephalin), and endomorphin-1 was notably increased. No change was observed in the effect of endomorphin-2. This agrees with the proposed existence of different mu receptors for the endomorphins. The activities of DPDPE ([D-Pen2,5]-enkephalin) and [D-Ala2] deltorphin II, agonists at delta receptors, were also unchanged. Knockdown of GAIP and of the GAIP interacting protein C-terminus (GIPC) led to changes in agonist effects at mu but not at delta receptors. The impairment of RGSZ1 extended the duration of morphine analgesia by at least 1 h beyond that observed in control animals. CTOP (Cys2, Tyr3, Orn5, Pen7-amide) antagonized morphine analgesia when given during the period in which the effect of morphine was enhanced by RGSZ1 knockdown. Thus, in naive mice, morphine tachyphylaxis originated in the presence of the opioid agonist and during the analgesia time course. The knockdown of RGSZ1 facilitated the development of tolerance to a single dose of morphine and accelerated tolerance to continuous delivery of the opioid. These results indicate that mu but not delta receptors are linked to Rz regulation. The mu receptor-mediated activation of Gz proteins is effective at recruiting the adaptive mechanisms leading to the development of opioid desensitization.     

Effects of competitive and noncompetitive antagonists of the N-methyl-D-aspartate receptor on the analgesic action of delta 1- and delta 2-opioid receptor agonists in mice.

1. The effects of MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and LY 235959, a competitive antagonists of the NMDA receptor on the analgesic actions of [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2, Glu4] deltorphin II (deltorphin II), the putative delta 1- and delta 2-opioid receptor agonists, respectively, were determined in the male Swiss-Webster mice. 2. Intracerebroventricular administration of DPDPE or deltorphin II produced analgesia. MK-801 administered intraperitoneally 10 min before the injection of DPDPE or deltorphin II, dose-dependently antagonized the analgesic actions of both drugs. 3. LY 235959 also dose-dependently antagonized the analgesic actions of DPDPE and deltorphin II. 4. The effects of MK-801 and LY 235959 on the binding of [3H]-DPDPE to mouse brain membranes were also determined. Neither of the NMDA receptor antagonists had any effect on the binding of [3H]-DPDPE. 5. It is concluded that competitive and noncompetitive antagonists of the NMDA receptor antagonize the analgesic action of delta 1- and delta 2-opioid receptor agonists and that such effects are not mediated via a direct interaction with brain delta-opioid receptors.     

Modulation of mu-mediated antitussive activity in rats by a delta agonist.

Effects of selective mu and delta receptor agonists on capsaicin-induced cough reflex in rats were studied. Intracisternal injection (i.cist.) of a selective mu receptor agonist [D-Ala2,Mephe4,Gly-ol5]enkephalin (DAMGO) produced dose-related depression of coughs over the 0.003-0.03 nmol dose range. The antitussive potency of DAMGO was 100-fold more potent than morphine. The antitussive effects of DAMGO and morphine were significantly reduced by naloxone (1 nmol i.cist.). The selective delta receptor agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), at a dose of 10 nmol (i.cist.), had no significant effect on the number of coughs. When co-administered i.cist., DPDPE (10 nmol) consistently and significantly decreased the antitussive potencies of DAMGO and morphine. The decrease in the antitussive effects of DAMGO and morphine caused by DPDPE were prevented by selective delta receptor antagonist, naltrindole (3 nmol). These results suggest that the antitussive effects of opioids are mediated predominantly by mu receptors, and delta receptors may play an inhibitory role in antitussive processes that are mediated by the mu receptors.     

The role of delta-opioid receptor subtypes in neuropathic pain

A large body of evidence suggests an important role of delta-opioid receptor agonists in antinociception at the level of the spinal cord. Our study was undertaken to analyse the spinal antinociceptive and antiallodynic effects of delta(1)- and delta(2)-opioid receptor agonists and antagonist after their acute and chronic intrathecal administration in a neuropathic pain model in the rat. In rats with a crushed sciatic nerve, the delta(1)-opioid receptor agonist [D-Pen(2), D-Pen(5)]enkephalin (DPDPE, 5-25 microg i.t.) and the delta(2)-opioid receptor agonist deltorphin II (1.5-25 microg i.t.) dose dependently antagonized the cold-water allodynia which developed after sciatic nerve injury. These effects of DPDPE were antagonized by 7-benzylidenenaltrexon (BNTX, 1 microg i.t.) while the effects of deltorphin II were antagonized by 5'naltrindole izotiocyanate (5'NTII, 25 microg i.t.). Both agonists had a dose-dependent, statistically significant effect on the tail-flick latency in two tests, with focused light and cold water. Chronic administration of DPDPE (25 microg i.t.) and deltorphin II (15 microg i.t.) resulted in significant prolongation of the reaction time determined on days 2, 4 and 6 post-injury. In conclusion, our results show an antiallodynic and antinociceptive action of DPDPE and deltorphin II at the spinal cord level, which suggests that both delta-opioid receptor subtypes play a similar role in neuropathic pain. This indicates that not only delta(1)- but also delta(2)-opioid receptor agonists can be regarded as potential drugs for the therapy of neuropathic pain.     

Species differences in mu- and delta-opioid receptors.

The mu opioid receptor ligand [D-Ala2, NMePhe4, Gly-ol5]enkephalin (DAGO) and delta opioid receptor ligand [D-Pen2,D-Pen5]enkephalin (DPDPE) show similar specificity in competition binding studies in whole brain homogenate in rat and mouse. However, in saturation studies, the density and affinity of DPDPE binding sites were substantially greater in the mouse. There was no difference between the mouse and rat in the density and affinity of DAGO sites. Results from dose-response studies for analgesia using the same ligands administered i.c.v. in both species paralleled the binding studies. DAGO was approximately 2 times more potent in the mouse compared to the rat; while DPDPE was more than 15 times more potent in the mouse. Thus, binding capacity and affinity differences appear to be related to the functional potency of the mu and delta ligands in the two species. These results suggest that the difference in potency of DPDPE between rat and mouse is related to the differences in brain delta opioid receptors.     

Spinally mediated opioid antidiarrheal effects

To assess the role of opioid receptors in the spinal cord in regulation of functions of the intestinal mucosa in a secretory model, we evaluated the ability of i.t. administered mu (PL017), delta (DPDPE) and kappa (U50,488H) selective opioid agonists to inhibit diarrhea produced in mice by an injection of prostaglandin E2 (PGE2) (200 micrograms/mouse, i.p.). I.t. PL017 and DPDPE inhibited diarrhea in a dose-related fashion. U50,488H had only minimal antidiarrheal effects. The i.t. doses of PL017 and DPDPE required to inhibit diarrhea were higher than the doses required to produce antinociception and inhibit gastrointestinal transit. Spinally administered PL017 and DPDPE were considerably less potent in the diarrhea model than after i.c.v. administration but far more effective than after peripheral (s.c.) dosing. The antidiarrheal effects of spinally administered opioids were antagonized by concurrently administered naloxone. These data indicate that opioid chemosensitive sites in the spinal cord can modulate diarrhea produced by PGE2, and that the receptor specific opioids, PL017 and DPDPE, and to a lesser extent U50,488H, all possess antidiarrheal activity when administered i.t.     

Administration of mu-, kappa- or delta2-receptor agonists via osmotic minipumps suppresses murine splenic antibody responses

Previously, our laboratory has shown that morphine given by implantation of a 75-mg slow-release pellet for 48 h suppresses murine splenic antibody responses to sheep red blood cells (SRBCs) in a plaque-forming cell (PFC) assay. However, the use of slow-release pellets for such studies is limited, as these pellets are only available in fixed doses and similar pellets for kappa and delta agonists have not been developed. In the present study, we investigated the feasibility of administering opioids via Alzet osmotic minipumps to assess their immunomodulatory effects. Groups of mice received minipumps dispensing morphine sulfate, which has primary activity at the mu opioid receptor; U50,488H, which is a kappa-selective agonist; deltorphin II, which is a delta2-selective agonist; or DPDPE, which has greater selectivity for delta1 than delta, receptors. Morphine, U50,488H and deltorphin II were all immunosuppressive, with biphasic dose-response curves exhibiting maximal (approximately 50%) suppression of the PFC response at doses of 0.5 to 2 mg/kg/day 48 h after pump implantation. Further, immunosuppression by morphine sulfate, U50,488H or deltorphin II was blocked by simultaneous implantation of a minipump administering the opioid receptor-selective antagonists CTAP (1 mg/kg/day), nor-binaltorphimine (5 mg/kg/day), or naltriben (3 mg/kg/day), respectively. DPDPE was inactive at doses lower than 10 mg/kg/day. We conclude that osmotic minipumps are a practical and useful way of administering opioids to study their effects on the immune system, and give further evidence that immunosuppression induced in vivo by opioid agonists is mediated not only via mu, but also via kappa and delta2 opioid receptors.     

U50,488H differentially antagonizes the bladder effects of mu agonists at spinal sites

The mu antagonist property of the kappa agonist U50,488H was studied at the spinal level, using motility of the rat urinary bladder as an endpoint in vivo. Intrathecal (i.th.) administration of the mu agonists [D-Ala2,NMePhe4,Gly-ol]enkephalin (DAGO), [N-MePhe3,D-Pro4]enkephalin (PL017), morphine and normorphine, as well as the delta agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), resulted in an equieffective inhibition of volume-initiated contractions of the urinary bladder. In contrast, i.th. administration of U50,488H, a highly selective kappa agonist, had no effect on bladder motility. Pretreatment of rats with i.th. U50,488H prior to agonist administration, blocked the suppression of spontaneous bladder activity induced by equieffective i.th. does of morphine and normorphine, but failed to alter the inhibitory effect of the mu agonists DAGO and PL017, or that of the delta agonist DPDPE. The finding that U50,488H differentially antagonized the identical bladder effects of several mu agonists suggests the presence of mu receptor subtypes (mu isoreceptors) in the rat spinal cord, which may be involved in the regulation of bladder function.     

Effects of mu, kappa or delta opioids administered by pellet or pump on oral Salmonella infection and gastrointestinal transit

Our laboratory has shown previously that subcutaneously implanted, slow-release morphine pellets markedly enhanced susceptibility to oral infection with Salmonella typhimurium. Further, morphine, kappa and delta opioid receptor agonists infused via osmotic minipumps were immunosuppressive. The present study compared morphine pellets to morphine pumps and also examined the differential effects of morphine versus U50,488H (kappa agonist), deltorphin II (delta2 agonist), and (D-Pen2, D-Pen5)-enkephalin (DPDPE, delta1 agonist), administered via Alzet minipumps, on oral Salmonella infection and on gastrointestinal transit. The results show that all morphine-pelleted mice (26/26) had a marked increase in Salmonella burden in the Peyer's Patches, mesenteric lymph nodes and spleen. In comparison, only 8/20 mice receiving morphine by minipump at doses ranging from 1 to 25 mg/kg/day had any culturable Salmonella in their organs and the number of bacteria was very low. The level of Salmonella colonization correlated with blood morphine levels and gut transit measured using an intragastric charcoal meal. Morphine pellets inhibited gut transit by 38%, while mice receiving morphine by minipump at doses of 1 to 25 mg/kg/day showed only a dose-dependent 7% to 17% inhibition. Mice receiving various doses of U50,488H or DPDPE had no culturable Salmonella in the three sites. Deltorphin II given by minipump resulted in a moderate level of Salmonella in the spleen. Deltorphin II and U50,488H (0.1 to 10 mg/kg/day) did not suppress gut transit. The present studies indicate that a predominantly mu opioid receptor agonist, morphine, given by slow-release pellet, potentiated Salmonella infection and inhibited gastrointestinal transit. In contrast, morphine in pumps slightly inhibited intestinal transit, but did not sensitize to Salmonella infection. A delta1 opioid receptor agonist did not sensitize to infection, and a delta2 and a kappa opioid receptor agonist had minimal effects on either parameter.     

Receptor-selective antagonism of opioid antinociception in female versus male rats

This study was conducted to determine whether sex differences in opioid antinociception may be explained by sex differences in opioid receptor activation. The time course, dose-effect and selectivity of antagonists that have been previously shown to be relatively mu (beta-funaltrexamine, beta-FNA), kappa (norbinaltorphimine, norBNI), or delta (naltrindole, NTI) receptor selective in male animals were compared in female and male Sprague-Dawley rats using a 52 degrees C hotplate test. In both sexes, beta-FNA (10 or 20 microg intracerebroventricularly [i.c.v.]) dose-dependently blocked the antinociceptive effects of fentanyl (0.056 mg/kg subcutaneously); antagonism was observed 24 h after beta-FNA, and diminished within 7-14 days. In both sexes, norBNI (1 or 10 microg i.c.v.) dose-dependently blocked the antinociceptive effects of U69,593 (1.0 mg/kg subcutaneously); antagonism was maximal by 1-3 days post-norBNI and lasted longer than 56 days. NTI (1 or 10 microg i.c.v.) dose-dependently blocked the antinociceptive effects of [D-Pen2, D-Pen5]enkephalin (DPDPE, 100 nmol i.c.v.) in both sexes; however, the duration of action of NTI was shorter in females than in males. The antinociceptive effects of the mu receptor-preferring agonists fentanyl, morphine and buprenorphine were significantly and dose-dependently antagonized by beta-FNA, but not by norBNI or NTI, in both sexes. Beta-FNA antagonism was significantly greater in females compared with males given morphine, but not fentanyl or buprenorphine. The antinociceptive effects of the kappa receptor-preferring agonists U69,593 and U50,488 were significantly and dose-dependently antagonized by norBNI; U50,488 but not U69,593 was also antagonized to a lesser extent by NTI and beta-FNA, in both sexes. The antinociceptive effect of the delta receptor-preferring agonist SNC 80 was significantly antagonized by NTI, but not by norBNI or beta-FNA, in both sexes. The sex difference in beta-FNA antagonism of morphine suggests that there may be sex differences in functional mu opioid receptor reserve or signal transduction; however, the lack of consistency across all mu agonists weakens this hypothesis. Overall, the opioids tested had very similar receptor selectivity in male and female subjects.     

All three types of opioid receptors in the spinal cord are important for 2/15 Hz electroacupuncture analgesia.

The analgesic effect induced by 2/15 Hz electroacupuncture as shown by the increase in tail flick latency decreased steadily as electroacupuncture stimulation was given continuously for 6 h, showing the development of tolerance to electroacupuncture analgesia. These rats were then given an intrathecal (i.t.) injection of one of the following opioid agonists: the mu agonist, ohmefentanyl 7.5, 15 and 30 pmol, 10 min apart; the delta agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE) 5, 10 and 20 nmol, 10 min apart and the kappa agonist, dynorphin-(1-13) 2.5, 5 and 10 nmol, 10 min apart, respectively. The analgesic effect induced by ohmefentanyl, DPDPE or dynorphin was dramatically reduced in rats rendered tolerant to 2/15 Hz electroacupuncture analgesia. Rats were injected i.t. with one of the three specific opioid antagonists: the mu antagonist, beta-funaltrexamine (beta-FNA) (5, 10 and 20 nmol), the delta antagonist, ICI174,864 (1, 2 and 4 nmol) and the kappa antagonist, nor-binaltorphimine (nor-BNI) (3.125, 6.25 and 12.5 nmol). It was found that analgesia induced by 2/15 Hz electroacupuncture stimulation was significantly and almost totally blocked by any one of the three opioid antagonists. These results suggest that all three types of opioid receptors, the mu, delta and kappa receptors in the spinal cord of the rat play important roles in mediating analgesia induced by electroacupuncture of 2/15 Hz.     

Evidence for lack of modulation of mu-opioid agonist action by delta-opioid agonists in the mouse vas deferens and guinea-pig ileum.

1. There is evidence from in vivo studies for an interaction of mu- and delta-opioid ligands. In the present work this concept has been investigated using the mouse vas deferens and guinea-pig ileum myenteric plexus-longitudinal preparations. 2. In field stimulated vasa deferentia of the mouse, co-administration of sub-effective concentrations of the delta-opioid agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) and [Met5]- or [Leu5]enkephalin had no effect on the dose-response curves of the mu-agonists [D-Ala2,MePhe4, Gly-ol5]enkephalin (DAMGO) and morphine. Similarly, the delta-opioid agonists did not alter the potency of morphine and DAMGO when added at different times prior to the mu-opioid agonists, or when EC50 concentrations of delta-opioid ligands were co-administered. Compounds with preferred activity for the putative delta 1-(DPDPE) or delta 2-([D-Ala2,Glu4]deltorphin II (Delt II)) opioid receptors were ineffective in this respect. 3. The guinea-pig ileum contains delta-opioid receptors. No function of these receptors in mediating blockage of field-stimulated contractions was observed with ligands having affinity for the putative delta 1 or delta 2 subtypes nor were the agonists able to modulate responses to mu-opioid ligands in this tissue. 4. The results demonstrate the modulation of mu-opioid agonists by delta-opioid agonists does not occur in the isolated peripheral tissues examined. Thus the findings do not support the concept of a functional coupling of opioid receptors, though the results may be explained by differences between opioid systems in the brain and peripheral tissues examined.