Inflammation-induced changes in rostral ventromedial medulla mu and kappa opioid receptor mediated antinociception

Acute microinjection of mu-, delta-, or kappa-opioid receptor (MOPr, DOPr, KOPr) agonists into the rostral ventromedial medulla (RVM) produces antinociception. Thermal antinociception produced by MOPr and DOPr agonists is potentiated during inflammation [Hurley RW, Hammond DL. The analgesic effects of supraspinal mu and delta opioid receptor agonists are potentiated during persistent inflammation. J Neurosci 2000;20:1249-59]. Whether this potentiation extends to other stimulus modalities or to KOPr agonists is unknown. To examine these issues, rats received a unilateral intraplantar injection of complete Freund's adjuvant (CFA). Antinociception produced by RVM infusion of the KOPr agonist, U69593, and the MOPr agonist, DAMGO, was tested 4h-2 weeks thereafter. Thermal paw withdrawal latencies (PWLs) were assessed using the Hargreaves method. Mechanical thresholds were determined with the Von Frey and Randall-Selitto method. PWLs of the inflamed paw were reduced 4h-2 weeks after CFA injection. Infusion of either U69593 or DAMGO increased PWLs in CFA treated rats. A bilateral enhancement of the response to both agonists was observed 2 weeks relative to 4h post-CFA injection. Mechanical thresholds of the inflamed paw were decreased for >2 weeks post-CFA injection. Infusion of either agonist elevated thresholds of the inflamed and non-inflamed paws of CFA-treated rats. The magnitude of these effects was greater 2 weeks post-CFA injection for DAMGO and increased progressively for U69593. These data demonstrate that RVM infusion of MOPr or KOPr agonists attenuates CFA-evoked thermal and tactile allodynia and that these effects increase during prolonged inflammation. The augmented response of the non-inflamed paw to agonists suggests that inflammation induces centrally-mediated neuroplastic changes which enhance MOPr- and KOPr-mediated antinociception.     

Enhancement mu opioid antinociception by oral delta9-tetrahydrocannabinol: dose-response analysis and receptor identification

The antinociceptive effects of various mu opioids given p.o. alone and in combination with Delta-9-tetrahydrocannabinol (Delta9-THC) were evaluated using the tail-flick test. Morphine preceded by Delta9-THC treatment (20 mg/kg) was significantly more potent than morphine alone, with an ED50 shift from 28.8 to 13.1 mg/kg. Codeine showed the greatest shift in ED50 value when administered after Delta9-THC (139.9 to 5.9 mg/kg). The dose-response curves for oxymorphone and hydromorphone were shifted 5- and 12.6-fold, respectively. Methadone was enhanced 4-fold, whereas its derivative, l-alpha-acetylmethadol, was enhanced 3-fold. The potency ratios after pretreatment with Delta9-THC for heroin and meperidine indicated significant enhancement (4.1 and 8.9, respectively). Pentazocine did not show a parallel shift in its dose-response curve with Delta9-THC. Naloxone administration (1 mg/kg s.c.) completely blocked the antinociceptive effects of morphine p.o. and codeine p.o. The Delta9-THC-induced enhancement of morphine and codeine was also significantly decreased by naloxone administration. Naltrindole (2 mg/kg s.c.) did not affect morphine or codeine antinociception but did block the enhancement of these two opioids by Delta9-THC. No effect was seen when nor-binaltorphimine was administered 2 mg/kg s.c. before morphine or codeine. Furthermore, the enhancements of morphine and codeine were not blocked by nor-binaltorphimine. We find that many mu opioids are enhanced by an inactive dose of Delta9-THC p.o. The exact nature of this enhancement is unknown. We show evidence of involvement of mu and possibly delta opioid receptors as a portion of this signaling pathway that leads to a decrease in pain perception.     

Continuous intrathecal opioid treatment abolishes the regulatory effects of magnesium and guanine nucleotides on mu opioid receptor binding in rat spinal membranes.

The regulatory effects of cations and guanine nucleotides on mu receptor binding after opioid drug and pertussis toxin treatment were studied in the rat spinal cord model. Continuous intrathecal (i.t.) infusion with PL017 for 5 days induced tolerance in a dose-dependent manner. Maximal tolerance was observed at day 2. A single i.t. dose (1 microgram) of pertussis toxin also induced tolerance to opioid. When mu receptor binding of the high-affinity sites was determined by 125I-FK33824, spinal membrane preparations from morphine- and pertussis toxin-induced tolerant animals demonstrated approximately 30% less binding than control membranes. Analysis of equilibrium competition binding of FK33824 against [3H]naloxone under a variety of experimental conditions (i.e., cations and guanine nucleotides) revealed differences among control and treated membranes. With Na+ (100 mM) + GDP (100 microM) pretreated membranes and binding assays conducted in the presence of Mg++, all mu receptors were observed to be in a high-affinity state in control membranes, whereas about 30% of receptors were in the low-affinity state in membranes from opioid- and pertussis toxin-treated animals. The increase in the proportion of low-affinity sites was dependent upon the infusion dose of PL017, and the increase correlated well with the degree of opioid tolerance developed. The regulatory effect of 5'-guanylylimidodiphosphate on opioid agonist binding was reduced in membranes from pertussis toxin- or opioid-treated animals. In binding assays conducted in the presence of Na+ (100 mM) + Mg++ (5 mM) + 5'-guanylylimidodiphosphate (30 microM) or Na+ (100 mM) + GDP (100 microM), all mu receptors in control membranes were in a low affinity-state, while those from opioid- or pertussis toxin-treated animals existed in both the high- and the low-affinity states. Continuous i.t. infusion with PL017 at the high dose of 1 microgram/hr for 5 days also decreased significantly (about 40%) the total number of receptors. These studies indicate that continuous opioid infusion and pertussis toxin treatment results in impairment in the receptor-G-protein coupling. This is reflected by the decreased regulatory effects of Mg++ and guanine nucleotides. Thus, in addition to receptor down-regulation, which is induced by PL017 at high doses, receptor-G-protein uncoupling may play a role in opioid tolerance induced by continuous infusion with morphine and PL017.     

Antinociceptive and motor effects of delta/mu and kappa/mu combinations of intrathecal opioid agonists.

Interactions between selective opioid agonists acting at spinal mu-, delta-, and kappa-opioid receptors were evaluated by co-administering a low-antinociceptive dose of the selective delta-agonist, DPDPE, or the selective kappa-agonist, U50,488H, with sequentially increasing doses of the selective mu-agonist, DAMGO, intrathecally. Antinociceptive synergy (i.e., a more than additive antinociceptive effect) was observed with both combinations of opioid agonists tested. The demonstration of antinociceptive synergy suggests that the subtypes of spinal opioid receptors can act, at least in part, through a common neural circuit. Since our measure of antinociception, the Randall-Selitto paw-withdrawal test, is dependent on a normally functioning motor system, we also evaluated the effects of these same combinations of opioid peptides on motor coordination using a rotarod treadmill. A low-antinociceptive dose of DPDPE or U50,488H co-administered intrathecally, with sequentially increasing doses of DAMGO, did not worsen the decrement in rotarod performance observed with the same doses of DAMGO administered as a single agent. In fact, the low-antinociceptive dose of DPDPE significantly attenuated the decrease in rotarod performance produced when the same dose of DAMGO was administered as a single agent. The results of this study suggest that intrathecal combinations of selective mu- with both delta- or kappa-selective opioid agonists can produce antinociceptive synergy without producing an increase in motor side effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous intrathecal opioid analgesia: tolerance and cross-tolerance of mu and delta spinal opioid receptors

The tolerance effects of continuous intrathecal infusions of opioids at mu and delta receptors were studied in rats. These effects were compared to those of chronic systemic morphine. A chronic intrathecal infusion of the relatively selective delta agonist, [D-Ala2, D-Leu5]enkephalin (DADLE), produced a larger degree of tolerance to DADLE than to the highly specific mu-activating morphiceptin analog [N-methyl-Phe3, D-Pro4]morphiceptin (PL017). The slope of the analgesic dose-response curve for the highly specific delta agonist, cyclic [D-Penicillamine2, D-Penicillamine5]enkephalin (DPDPE), was significantly different (flatter) from those of mu agonists or DADLE. High-dose infusion of PL017 induced a 61-fold parallel shift of the dose-response curve for PL017. This same treatment also induced a corresponding flattened, nonparallel change of the dose-response curve for DADLE. This altered curve for DADLE was very similar in slope to that of DPDPE. Pretreatment with the irreversible mu antagonist, beta-funaltrexamine, caused a parallel rightward shift of the dose-response curve for PL017 but did not affect DPDPE activity. beta-Funaltrexamine treatment induced a nonparallel rightward shift of the dose-response curve for DADLE with a change of slope similar to that of DPDPE. These findings demonstrate a mixed mu-delta analgesic activity for the compound DADLE, which is often referred to as a prototypic delta agonist. These interactions differ from prior reports of none or minimal mu-ligand interactions with DADLE. Despite the cross-reactivity of DADLE to mu receptors, DADLE remains a more effective analgesic than do mu agonists in states of mu receptor tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of morphine with intrathecally administered calcium and calcium antagonists: evidence for supraspinal endogenous opioid mediation of intrathecal calcium-induced antinociception in mice

The interaction between morphine [i.p. and intrathecal (i.t.)] and calcium and its antagonists (i.t. and i.c.v.) was studied in the mouse tail-flick test for antinociception. Calcium (0.66 mumol i.t.) produced antinociception comparable to that of morphine (0.5 microgram i.t.) but had a significantly longer duration. A lower dose of calcium (0.16 mumol i.t.) significantly potentiated morphine (0.2 and 0.5 micrograms i.t.). The antinociceptive effect of i.p. morphine was also potentiated by i.t. calcium, but was antagonized by the i.t. administration of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (3.7-7.5 nmol), verapamil (15 micrograms), magnesium (9.4 nmol) and barium (1-2 nmol). In contrast, i.t. calcium and i.p. morphine were significantly potentiated by the i.c.v. administration of verapamil (15 micrograms) and antagonized by i.c.v. calcium (0.33 mumol). The antinociceptive effect of i.t. calcium was antagonized by naloxone administered s.c. (1 mg/kg) or i.c.v. (0.5 microgram), but not i.t. (0.5 and 10 microgram). It is concluded that the antinociceptive effect of i.t. calcium is mediated, at least partly, by a reflex supraspinal release of endogenous opioids, and that the administration of calcium and its antagonists modify the antinociceptive effect of morphine in opposite directions, depending upon whether they are administered by the i.t. or i.c.v. routes. Calcium may serve as a useful adjunct for opioid-induced analgesia via the i.t. route.     

Co-localization of mu opioid receptor and N-methyl-D-aspartate receptor in the trigeminal dorsal horn.

Antagonists acting at the N-methyl-D-aspartate (NMDA) receptor can block the development of tolerance to the analgesic effects of [mu ] opioid receptor (MOR) ligands, such as morphine, and can also enhance the analgesic efficacy of opioids. These findings have led to the hypothesis that interactions between NMDA receptor and MOR ligands may be due to the co-localization of these receptors on neurons in the dorsal horn. We used dual immunogold and immunoperoxidase immunocytochemistry for MOR1 and NMDAR1 to determine the degree of co-localization of these receptors in neurons of the trigeminal dorsal horn. By use of electron microscopy, we found that both receptors were primarily located in dendrites and to a lesser extent in perikarya, axons, axon terminals, and glia. With regard to the degree of co-localization in dendrites, 63% of MOR1-labeled dendrites also contained NMDAR1, whereas 61% of NMDAR1-labeled dendrites also contained MOR1. Most of the dual-labeled profiles (94%) were classified as dendrites, with the remainder being axons, axon terminals, or perikarya. These results suggest that direct interactions between MOR and NMDA receptor ligands are likely mediated through shared dendritic targets in the dorsal horn. Less frequently, we found evidence for modulation of afferents to MOR-containing neurons through presynaptic NMDA receptors.     

The role of opioid receptor density in morphine tolerance.

The effect of chronic opioid antagonist-induced functional supersensitivity and receptor upregulation on morphine tolerance was examined in Swiss Webster mice obtained from Taconic Farms and Charles River Laboratories. Mice were implanted s.c. with either naltrexone (NTX) or placebo pellets for 8 days. After 8 days, the pellets were removed, and 24 h later mice were either injected with morphine (50 or 100 mg/kg) or saline (acute tolerance protocol) or implanted with a 75-mg morphine or placebo pellet for 72 h (chronic tolerance protocol). Mice were tested for morphine analgesia 24 h after injections or 72 h after pellet implantations. Mice from Taconic Farms were more sensitive to morphine analgesia than Charles River mice, although the degree of tolerance in both strains was similar. Acute morphine produced a 1.7- and 1.9-fold increase in the ED50 for morphine analgesia in NTX and placebo pretreated mice, respectively. the chronic tolerance protocol produced the same shift (2.4-fold) in the ED50 in both NTX and placebo pretreated mice. In both tolerance protocols, NTX-pretreated mice were significantly more sensitive to the analgesic effects of morphine than placebo pretreated controls. Binding studies ([3H][D-Ala,2NMePhe4,Gly-ol5]enkephalin) indicated an approximately 40% increase in opioid receptor density with no significant alteration in affinity after chronic NTX treatment. These results indicate that acute and chronic tolerance to morphine develops comparably in control and upregulated, supersensitive mice. These findings suggest that new binding sites in upregulated mice mediate tolerance similarly to existing binding sites and that the degree of tolerance is unrelated to the density of opioid receptors.     

Peripheral opioid receptors mediating antinociception in inflammation. Evidence for involvement of mu, delta and kappa receptors

This study examined a possible peripheral site of action of opioids in the modulation of the response to noxious pressure on inflamed tissue. Rats developed a unilateral localized inflammation upon injection of Freund's complete adjuvant into one hindpaw. 4-6 days after inoculation, intraplantar administration of mu, delta and kappa selective agonists [D-Ala2,N-methyl-Phe4,Gly-ol5]-en-kephalin (1 micrograms), [D-Pen2,5]-enkephalin (40 micrograms) and U-50, 488H (50 micrograms) produced marked antinociceptive effects in inflamed but not noninflamed paws. Equivalent doses applied systemically (s.c. and i.v.) were without effect. Dose dependency and stereospecificity of these effects were demonstrated using (-)- and (+)-morphine and (-)- and (+)-tifluadom. Furthermore, by use of (-)- and (+)-naloxone, dose-dependent and stereospecific antagonism was shown. Lastly, reversal of effects of [D-Ala2,N-methyl-Phe4,Gly-Ol5]-enkephalin, [D-Pen2,5]-enkephalin and U-50,488H by mu, delta and kappa selective antagonists D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, ICI 174,864 and nor-BNI, respectively, indicated that these agents interact with discriminable populations of receptors. These observations suggest that several selective opioid agonists can modulate responses to noxious pressure through a peripheral opioid receptor-specific site of action in inflammation and that these receptors possess distinguishable pharmacological characteristics resembling those of mu, delta and kappa receptors.     

Gonadal hormone modulation of mu, kappa, and delta opioid antinociception in male and female rats.

Previous studies suggest that sex differences in morphine antinociception in rodents might be attributed to the activational effects of gonadal hormones. The present study determined whether hormonal modulation of opioid antinociception in adult rats extends to opioids other than the prototypic mu agonist morphine. Male and female rats were sham-gonadectomized (sham-GDX) or gonadectomized (GDX) and replaced with no hormone, estradiol (E2, females), progesterone (P4, females), E2+P4 (females), or testosterone (males). Approximately 28 days later, nociception was evaluated on the 50 degrees C hot plate and warm water tail withdrawal tests before and after subcutaneous administration of hydromorphone, buprenorphine, U50,488, or SNC 80. In sham-GDX (gonadally intact) rats, the mu agonists and U50,488 were less effective in females than in males in at least one nociceptive test, and the delta agonist SNC 80 was less effective in males than in females. In males, gonadectomy tended to decrease, and testosterone tended to increase antinociception produced by 3 of the 4 agonists. In females, gonadectomy and hormone treatment had more variable effects, although E2 tended to decrease mu opioid antinociception. The present results suggest that activational effects of gonadal hormones are relatively modest and somewhat inconsistent on antinociception produced by various opioid agonists in the adult rat. PERSPECTIVE: This study demonstrates that reproductive hormones such as testosterone in males and estradiol in females do not consistently modulate sensitivity to the analgesic effects of opioids in the adult organism.     

Peripheral opioid receptor blockade increases postoperative morphine demands—A randomized,double-blind,placebo-controlled trial

Experimental studies suggest that a large proportion of opioid analgesia can be mediated by peripheral opioid receptors. This trial examined the contribution of such receptors to clinical analgesia induced by intravenous morphine. We hypothesized that the selective blockade of peripheral opioid receptors by methylnaltrexone (MNX) would increase the patients’ demand for morphine to achieve satisfactory postoperative pain relief. In a double-blind, placebo-controlled, sequential 2-center trial, 50 patients undergoing knee replacement surgery were randomized (1:1) to receive either subcutaneous MNX (0.9 mg/kg) (hospital I: n = 14; hospital II: n = 11) or saline (hospital I: n = 13; hospital II: n = 12) at the end of surgery. The primary endpoint was the cumulative amount of intravenous morphine administered during the first 8 hours. Secondary endpoints were pain scores at rest and during movement (by numerical rating scale and McGill Questionnaire), vital signs, adverse side effects, and withdrawal symptoms. After MNX, demands for morphine were strongly (by about 40%) increased (hospital I: 35.31 ± 12.99 mg vs 25.51 ± 7.92 mg, P = 0.03; hospital II: 35.42 ± 11.73 mg vs 24.80 ± 7.84 mg, P = 0.02; pooled data: P < .001; means ± SD). Secondary endpoints were similar in all groups (P > .05). Thus, a significant proportion of analgesia produced by systemically administered morphine is mediated by peripheral opioid receptors. Drugs that selectively activate such receptors should have the potential to produce powerful clinical pain relief.     

Mediation of swim-stress antinociception by the opioid delta 2 receptor in the mouse.

The present study has characterized the antinociceptive response to cold water swim-stress (CWSS) in mice using opioid-selective antagonists as well as tolerance and cross-tolerance approaches. Mice subjected to CWSS using water at 5 degrees C for 3 min showed a marked antinociceptive response in the tail-flick test, which reached approximately 90% after +10 min, and which persisted for 15 to 20 min. This antinociceptive response (at +10 min) was antagonized by naloxone or by the delta antagonist ICI 174,864. Additionally, the CWSS response was antagonized by the opioid delta 2 antagonist, naltrindole-5'-isothiocyanate, but not by the delta 1 antagonist, [D-Ala2,Leu5,Cys6]enkephalin, or by the mu antagonist, beta-funaltrexamine or by the kappa antagonist, norbinaltorphimine. Although the CWSS-induced antinociceptive effect was blocked by some delta antagonists and tolerance resulted from the CWSS-induced response, the decrease in body temperature after each CWSS exposure was not affected by the opioid antagonists and reliably occurred in CWSS-tolerant mice, suggesting that the observed antinociception was independent of changes in body temperature. In mice rendered tolerant to the antinociceptive actions of the mu agonist, [D-Ala2,NMPhe4,Gly-ol] enkephalin, or to [D-Pen2,D-Pen5]enkephalin (predominantly a delta 1 agonist), the CWSS-induced antinociceptive response was unaltered. In contrast, in mice tolerant to the delta 2 agonist, [D-Ala2,Glu4]deltorphin, the CWSS-induced antinociceptive response was markedly and significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a bipartite recombinant adeno-associated viral vector for site-specific integration

Adeno-associated virus type 2 (AAV2) is the only virus known to integrate into a specific locus in the human genome. The locus, AAVS1, is on the q arm of chromosome 19 at position 13.4. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain two important elements needed for site-specific integration, that is, the rep gene or the P5 promoter, although they do integrate with low frequency at random locations in the human genome. We have designed a bipartite vector that does insert the transgene into AAVS1. One component, rAAVSVAV2, contains the rep gene, driven by the simian virus 40 early promoter rather than the P5 promoter. Thus, the integration enhancer element (IEE) within P5, which greatly enhances site-specific integration, has been deleted. The other component, rAAVP5UF11, contains the P5 IEE plus the transgene with associated regulatory elements. We have created clones of transduced HeLa cells, most of which appear to have the transgene inserted in AAVS1. We have not detected any clones that have rep inserted anywhere. With the optimal multiplicity of infection and ratio of rAAVSVAV2 and rAAVP5UF11, the transgene integrated specifically at AAVS1 with high efficiency (>60%). Most importantly, the cloned cell lines with the AAVS1 site-specific integrated green fluorescent protein (GFP) were healthy and stably expressed GFP for 35 passages. An AAV vector that would integrate at a specific site with high frequency could offer significant advantage in the transduction of progenitor cells and stem cells ex vivo and engineered cells could be used for human gene therapy. AAV site-specific integration gene therapy could provide a novel approach for diseases that need long-term gene expression.     

Renal mu opioid receptor mechanisms in regulation of renal function in rats.

Studies were performed in pentobarbital anesthetized Sprague-Dawley rats to determine whether mu opioid receptor agonists produce changes in renal function via intrarenal mechanisms. Left renal artery infusion of isotonic saline vehicle or the selective mu opioid receptor agonist, dermorphin (0.5 nmol/kg/min), did not alter mean arterial pressure or heart rate. In contrast, left renal artery dermorphin administration produced a significant decrease in left kidney urinary flow rate and sodium excretion without altering glomerular filtration rate or effective renal plasma flow; function of the right kidney was unaffected. Pretreatment of the left kidney with the opioid receptor antagonist naloxone, 50 micrograms/kg into left renal artery, prevented changes in urinary flow rate and sodium excretion induced by subsequent left renal artery dermorphin administration. Prior bilateral renal denervation abolished the antidiuretic and antinatriuretic responses to left renal artery dermorphin administration. These results suggest that mu opioid receptor agonists participate in the process of renal tubular sodium and water reabsorption via an intrarenal action that is dependent on an interaction with renal sympathetic nerves. This may occur via an action of mu opioid receptor agonists to facilitate the nerve terminal release and/or the direct tubular action of norepinephrine to affect renal tubular sodium and water reabsorption.     

Evidence for mu opioid receptor mediation of enkephalin-induced electroencephalographic seizures

The opioid receptor types involved in the mediation of enkephalin-induced electroencephalographic (EEG) seizures were studied in unanesthetized, freely moving rats. Four receptor-selective peptide ligands were evaluated for effectiveness in producing nonconvulsive EEG seizures after i.c.v. administration; these included the mu agonist, [D-Ala2-N-methyl-Phe4-Gly5-ol]enkephalin (DAGO), the mixed mu-delta agonist, [D-Ala2-D-Leu5]enkephalin (DADLE), and the selective delta agonists, [D-Pen2-D-Pen5]enkephalin and [D-Pen2-L-Pen5]enkephalin. Only DAGO and DADLE were found to produce EEG seizures, with DAGO being 9 times more potent than DADLE. DAGO produced a greater number of seizure episodes with a greater overall incidence compared with DADLE, reflecting its potent effect to elicit EEG seizure activity in these rats. Injections of [D-Pen2-D-Pen5]enkephalin or [D-Pen2-L-Pen5]enkephalin, even at the highest doses tested, failed to produce seizure activity. Behaviorally, the DAGO and DADLE EEG seizures were nonconvulsive but were temporally associated with episodic bursts of wet-dog shakes. The enkephalin-induced responses were extremely sensitive to antagonism by naloxone and completely blocked by pretreatment with the irreversible mu antagonist beta-funaltrexamine. The selective delta opioid receptor antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH) was ineffective. The use of the most selective agonists and antagonists for mu and delta opioid receptors suggests that, in rats, enkephalin-induced EEG seizures are mediated exclusively by mu opioid receptors and not by delta opioid systems.     

Dynamic temporal and spatial regulation of mu opioid receptor expression in primary afferent neurons following spinal nerve injury

Despite using prescribed pain medications, patients with neuropathic pain continue to experience moderate to severe pain. There is a growing recognition of a potent peripheral opioid analgesia in models of inflammatory and neuropathic pain. The goal of this study was to characterize the temporal and spatial expression of mu opioid receptor (mOR) mRNA and protein in primary afferent neurons in a rat L5 spinal nerve ligation model of persistent neuropathic pain. Bilateral L4 and L5 dorsal root ganglia (DRGs), L4 and L5 spinal cord segments, and hind paw plantar skins were collected on days 0 (naïve), 3, 7, 14, and 35 post‐spinal nerve ligation or post‐sham surgery. We found that expression of mOR mRNA and protein in primary afferent neurons changed dynamically and site‐specifically following L5 spinal nerve ligation. Real‐time RT—PCR, immunohistochemistry, and Western blot analysis demonstrated a down‐regulation of mOR mRNA and protein in the injured L5 DRG. In contrast, in the uninjured L4 DRG, mOR mRNA transiently decreased on day 7 and then increased significantly on day 14. Western blot analysis revealed a persistent increase in mOR protein expression, although immunohistochemistry showed no change in number of mOR‐positive neurons in the uninjured L4 DRG. Interestingly, mOR protein expression was reduced in the skin on days 14 and 35 post‐nerve injury and in the L4 and L5 spinal cord on day 35 post‐nerve injury. These temporal and anatomically specific changes in mOR expression following nerve injury are likely to have functional consequences on pain‐associated behaviors and opioid analgesia.     

Heroin acts on different opioid receptors than morphine in Swiss Webster and ICR mice to produce antinociception.

The opioid receptor types involved in supraspinal and spinal heroin-induced analgesia in Swiss Webster and ICR mice were determined by intracerebroventricular (i.c.v.) and intrathecal (i.t.) administration of opioid agonists and antagonists. Also, comparisons were made with morphine. Antinociception was measured by changes in tail-flick latency. In Swiss Webster mice, i.c.v. heroin like [D-Pen2-D-Pen5]enkephalin, a delta receptor opioid agonist, activated supraspinal delta opioid receptors as evidenced by inhibition of analgesia by coadministration of naltrindole, a delta receptor antagonist. Lack of effect of i.t. yohimbine and methysergide vs. i.c.v. heroin indicated that spinal descending noradrenergic and serotonergic systems were not involved. Heroin and [D-Pen2-D-Pen5]enkephalin were also matched in producing additive interactions with i.t. opioids. Also, i.c.v. heroin like [D-Pen2-D-Pen5]enkephalin did not activate a dynorphin-mediated antianalgesic system. In ICR mice, i.c.v. heroin receptor selectivity matched that of i.c.v. Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5, a selective mu receptor opioid agonist. Analgesia was inhibited by pretreatment with i.c.v. beta-funaltrexamine, a nonequilibrium mu receptor antagonist. Intrathecal administration of methysergide inhibited i.c.v. heroin-induced analgesia whereas i.t. yohimbine had no effect, which indicated that a descending serotonergic system but not a noradrenergic system was involved. Low doses of i.t. naloxone and nor-binaltorphimine increased the analgesic effect. This effect was consistent with activation of an antianalgesic system by i.c.v. heroin, which was mediated by dynorphin A in the spinal cord. Desensitization of the antianalgesic system also resulted in increased analgesia. In both Swiss Webster and ICR mice, i.t. heroin-induced analgesia involved spinal mu receptors like those stimulated by Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5. Analgesia was inhibited by i.t. naloxone. In both strains, i.t. heroin, like i.t. Tyr-D-Ala2-Gly-NMePhe4-Gly-ol5, produced an additive interaction with i.t. clonidine. In conclusion, the supraspinal receptors activated by heroin are different between Swiss Webster and ICR mice. In both strains, the receptor selectivities assigned to heroin did not match those for morphine. Heroin did not act by being biotransformed to morphine.     

In vivo receptor binding of opioid drugs at the mu site

The in vivo receptor binding of a series of opioid drugs was investigated in intact rats after s.c. administration of [3H]etorphine tracer, which selectively binds to mu sites in vivo. Receptor binding was determined by a membrane filtration assay immediately after sacrifice of the animals and brain homogenization. Coadministration of unlabeled opioid drugs together with tracer led to a dose-dependent decrease of in vivo tracer binding. Estimates of the doses required to occupy 50% of the mu sites in vivo established the following potency rank order: diprenorphine, naloxone, buprenorphine, etorphine, levallorphan, cyclazocine, sufentanil, nalorphine, ethylketocyclazocine, ketocyclazocine, pentazocine, morphine. In vivo-in vitro differences among the relative receptor binding potencies were only partially accounted for by differences in their access to the brain and the regulatory effects of Na+ and GTP, which are expected to reduce agonist affinities in vivo. The relationship among mu receptor occupancy in vivo and pharmacological effects of the opioid drugs is described.     

Contribution of mu and delta opioid receptors to the pharmacological profile of kappa opioid receptor subtypes

Molecular cloning has identified three opioid receptors: mu (MOR), delta (DOR) and kappa (KOR). Yet, cloning of these receptor types has offered little clarification to the diverse pharmacological profiles seen within the growing number of novel opioid ligands, which has led to the proposal of multiple subtypes. In the present study, utilizing in vitro and in vivo methods including the use of opioid receptor knockout mice, we find that certain antinociceptive effects of the KOR‐1 and KOR‐2 subtype‐selective ligands (+)‐(5α,7α,8β)‐N‐Methyl‐N‐[7‐(1‐pyrrolidinyl)‐1‐oxaspiro[4.5]dec‐8‐yl]‐benzene‐acetamide (U69, 593) and 4‐[(3,4‐Dichlorophenyl)acetyl]‐3‐(1‐pyrrolidinylmethyl)‐1‐piperazine‐carboxylic acid methyl ester fumarate (GR89, 696), respectively, are potentiated by antagonism of MOR and DOR receptors. We believe that our findings can be best explained by the existence of KOR‐DOR and KOR‐MOR heteromers. We only find evidence for the existence of these heteromers in neurons mediating mechanical nociception, but not thermal nociception. These findings have important clinical ramifications as they reveal new drug targets that may provide avenues for more effective pain therapies.