Selective inhibition of [D-Ala2, Glu4]deltorphin antinociception by supraspinal, but not spinal, administration of an antisense oligodeoxynucleotide to an opioid delta receptor

Evidence in vivo has suggested the existence of subtypes of the delta opioid receptor (DOR), which have been termed delta 1 and delta 2. These proposed DOR subtypes are thought to be activated by [D-Pen2, D-Pen5]enkephalin (DPDPE, delta 1) and [D-Ala2, Glu4]deltorphin (delta 2). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin, but not of [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO, mu agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonists selective for delta, mu and kappa receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin but not of i.th. DAMGO or U69,593 (kappa agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, selectively inhibited the antinociceptive response to i.c.v. [D-Ala2, Glu4]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as delta 2 and further, suggest that this delta receptor subtype may play a major role in eliciting spinal delta-mediated antinociception.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by delta 1- but not delta 2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of delta-opioid receptor agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective delta 1-opioid receptor antagonist, but not with naltriben (NTB), a selective delta 2-opioid receptor antagonist. There were no significant differences in the antinociceptive effect of [D-Ala2]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala2]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal delta 1-opioid receptor-mediated antinociception, but are normally responsive to activation of delta 2-opioid receptors.     

Treatment with antisense oligodeoxynucleotide to the opioid delta receptor selectively inhibits delta 2-agonist antinociception

Using approaches emphasizing differential antagonism of receptor selective agonists and cross-tolerance paradigms, evidence in vivo has suggested the existence of subtypes of opioid delta receptors, which have been termed delta 1 and delta 2. Recent work has elucidated the structure of an opioid delta receptor. The present investigation attempted to continue to test the hypothesis of subtypes of delta receptors and to correlate the cloned delta receptor with the existing pharmacological classification. Synthetic oligodeoxynucleotides (oligos) complementary to the 5' end of the cloned delta receptor coding region (antisense) or its corresponding sequence (sense) were given by intracerebroventricular (i.c.v.) administration to mice, twice-daily for 3 days and antinociceptive responses to selective agonists at putative delta 1 and delta 2 receptors were subsequently determined. Treatment with antisense, but not sense, oligo significantly inhibited the response to [D-Ala2,Glu4]deltorphin (delta 2 agonist), but not to [D-Pen2,D-Pen5]enkephalin (DPDPE, delta 1 agonist). Further, subsequent administration of DPDPE elicited a full antinociceptive response in the same antisense oligo treated mice which did not show a significant response to [D-Ala2,Glu4]deltorphin while antisense oligo treated mice which responded to DPDPE did not show antinociception when tested subsequently with [D-Ala2,Glu4]deltorphin. The data suggest that the cloned delta receptor corresponds to that pharmacologically classified as delta 2 and continue to support the concept of subtypes of opioid delta receptors.     

The delta2-opioid receptor subtype stimulates phosphoinositide metabolism in mouse periaqueductal gray matter

The delta(delta)-opioid agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II increased the formation of inositol phosphates (IPs) in mice periaqueductal gray matter (PAG) slices pre-labeled with myo-[3H]inositol. Both delta-agonists caused an increase in IP accumulation in a dose-dependent manner (1-100 microM) and which was pertussis toxin (0.5 microg/mouse, i.c.v.) sensitive. This effect was blocked by the delta-antagonist ICI-174.864 (10 microM). The presence of subtypes of the delta-opioid receptor (delta1 and delta2) in PAG has been suggested by pharmacological studies. In this brain structure, naltrindrole 5'-isothiocyanate (5'-NTII), but not 7-benzylidenenaltrexone (BNTX), antagonized the effects of DPDPE and [D-Ala2]deltorphin II, suggesting the involvement of a population of delta receptors sensitive to the delta2-antagonist NT II on this effect. To further investigate the participation of delta-receptor subtypes in the stimulation of IPs formation, mice were injected with antisense oligodeoxynucleotides (ODNs) directed to nucleotides 7-26 or 2946 of the cloned delta-receptor mRNA, and PAG slices from these animals were used in in vitro assays. The results demonstrate that the reported increase of phosphoinositide (PI) hydrolysis depends on the agonist activation of the delta2-opioid receptor subtype in the PAG.     

delta Opioid receptor subtypes activate inositol-signaling pathways in the production of antinociception

To analyze the selectivity of delta receptor subtypes to regulate different classes of G proteins, the expression of the alpha-subunits of Gi2, Gi3, Go1, Go2, Gq and G11 transducer proteins was reduced by administration of oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. Mice receiving antisense ODNs to Gi2 alpha, Gi3 alpha, Go2 alpha and G11 alpha subunits showed an impaired antinociceptive response to all the delta agonists evaluated. An ODN to Go1 alpha specifically blocked the antinociceptive effect of the agonist of delta-1 receptors, [D-Pen2,5]enkephalin (DPDPE), without altering the activity of [D-Ala2]deltorphin II or [D-Ser2]-Leu-enkephalin-Thr (DSLET). In mice treated with an ODN to Gq alpha, the effects of the agonists of delta-2-opioid receptors were reduced, but not those of DPDPE. Thus, Go1 proteins are selectively linked to delta-1-mediated analgesia, and Gq proteins are related to delta-2-evoked antinociception. After impairing the synthesis of Go1 alpha subunits, DPDPE exhibited an antagonistic activity on the antinociception produced by [D-Ala2]deltorphin II. After treatment with ODNs complementary to sequences in Gq alpha or PLC-beta 1 mRNAs, the analgesic capacity of [D-Ala2]deltorphin II was diminished. However, the delta-2-agonist did not alter the antinociceptive activity of DPDPE. An ODN complementary to nucleotides 7 to 26 of the murine delta receptor reduced the analgesic potency of [D-Ala2]deltorphin II, but not that observed for DPDPE. In these mice, [D-Ala2]deltorphin II did not antagonize the effect of DPDPE. These results suggest the existence of different molecular forms of the delta opioid receptor, and the involvement of inositol-signaling pathways in the supraspinal antinociceptive effects of delta agonists.     

Binding of [3H][D-Ala2, MePhe4, Gly-ol5] enkephalin, [3H][D-Pen2, D-Pen5]enkephalin, and [3H]U-69,593 to airway and pulmonary tissues of normal and sensitized rats

The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [3H]labeled ligands of mu-([D-Ala2, MePhe4, Gly-ol5]enkephalin; DAMGO), delta ([D-Pen2, D-Pen5]enkephalin; DPDPE), and kappa-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (unsensitized) and actively IgE-sensitized rats acutely challenged with the specific antigen. [3H]DAMGO, [3H]DPDPE and [3H]U-69,593 bound to membranes of normal and sensitized tissues at a saturable, single high-affinity site. The rank order of receptor densities in normal tissues was delta- > or = kappa- > or = mu-, with lung parenchyma exhibiting the greatest binding capacity for delta- and mu- receptors compared to the other regions examined. The Kd values showed small differences between ligands and regions tested. The mu- and delta-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. By contrast, kappa-opioid receptor density was augmented in sensitized lung parenchyma but an increase in Kd values was also observed. These differential changes in the density and affinity of opioid receptor types may be related to alterations in endogenous opioid peptides during the process of sensitization.     

Interaction between the mu-agonist dermorphin and the delta-agonist [D-Ala2, Glu4]deltorphin in supraspinal antinociception and delta-opioid receptor binding

1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist.     

Postnatal development of delta-opioid receptor subtypes in mice

1. The density and affinity of binding sites for the delta-selective opioid ligands [3H]-[D-Ala2, Asp4]deltorphin (DELT-I), [3H]-[D-Ala2Glu4]-deltorphin (DELT-II), [3H]-[D-Pen2,D-Pen5]enkephalin (DPDPE), and [3H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice. 2. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (Bmax) labelled by [3H]-NTI was about one half that labelled by [3H]-DELT-I, [3H]-DELT-II and [3H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE Bmax was 25% less than the deltorphin-II Bmax. 3. In saturation experiments, specific binding of [3H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34%, high affinity site, Kd = 1.08 nM and 66% low affinity sites, Kd = 39.9 nM). 4. DPDPE produced a biphasic inhibition of specific [3H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice. 5. In adult mouse brain labelled with [3H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites DELT-I and DPDPE produced monophasic inhibition of specific [3H]-DELT-II binding to brain homogenates of adult mice. 6. These data suggest that a sub-population of delta-sites (probably the delta 2-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward. 7. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three delta-agonists was: DELT-I > DELT-II > DPDPE in 10 day-old mice: and DELT-I- DELT-II > DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the delta 2-subtype.     

Unchanged [3H]MK-801 binding and increased [3H]flunitrazepam binding in turtle forebrain during anoxia

In order to determine if functional changes in N-methyl-D-aspartate receptors and GABAA receptors play a role in the remarkable anoxia tolerance of freshwater turtle brain, we used autoradiographic techniques to assay [3H]MK-801 and [3H]flunitrazepam binding in turtle forebrain after turtles had been subjected to anoxia for 2 or 6 h. The effects of glutamate, glycine, competitive N-methyl-D-aspartate antagonists, glycine antagonists, polyamines, magnesium, and zinc on [3H]MK-801 binding were the same in anoxic and control turtle forebrains. These results indicate that NMDA receptor regulation plays no role in the adaptive responses to anoxia in turtle brain. In contrast, [3H]flunitrazepam binding was significantly increased in the anoxic dorsal cortex and striatum. The most parsimonious explanation for elevated benzodiazepine receptor binding is that the rise in extracellular GABA levels known to accompany anoxia enhances benzodiazepine receptor affinity. It is possible, however, that GABAA receptor upregulation during anoxia increases the effectiveness of the inhibitory action of released GABA and contributes to the anoxia tolerance of turtles.     

Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective mu or delta opioid agonists in mice

N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to block the development of antinociceptive tolerance to morphine. Assessment of the effects of NMDA antagonists on development of antinociceptive tolerance to selective opioid mu (mu) and delta (delta) agonists, however, has not been reported. In these experiments, selective mu and delta receptor agonists, and morphine, were repeatedly administered to mice either supraspinally (i.c.v.) or systemically (s.c.), alone or after pretreatment with systemic NMDA antagonists. Antinociception was evaluated using a warm-water tail-flick test. Repeated i.c.v. injections of mu agonists including morphine, fentanyl, [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO) and Tyr-Pro-NMePhe-D-Pro-NH2 (PL017) or [D-Ala2, Glu4]deltorphin, a delta agonist, or s.c. injections of morphine or fentanyl, produced antinociceptive tolerance as shown by a significant rightward displacement of the agonist dose-response curves compared to controls. Single injections or repeated administration of MK801 (a non-competitive NMDA antagonist) or LY235959 (a competitive NMDA antagonist) at the doses employed in this study did not produce behavioral toxicity, antinociception or alter the acute antinociceptive effects of the tested opioid agonists. Consistent with previous reports, pretreatment with MK801 or LY235959 (30 min prior to agonist administration throughout the tolerance regimen) prevented the development of antinociceptive tolerance to i.c.v. or s.c. morphine. Neither NMDA antagonist, however, affected the development of antinociceptive tolerance to i.c.v. fentanyl, DAMGO, or [D-Ala2, Glu4]deltorphin. Additionally, MK801 pretreatment did not affect the development of antinociceptive tolerance to i.c.v. PL017 or to s.c. fentanyl. Further, MK801 pretreatment also did not affect the development of tolerance to the antinociception resulting from a cold-water swim-stress episode, previously shown to be a delta-opioid mediated effect. These data lead to the suggestion that the mechanisms of tolerance to receptor selective mu and delta opioids may be regulated differently from those associated with morphine. Additionally, these findings emphasize that conclusions reached with studies employing morphine cannot always be extended to 'opiates' in general.     

Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction

Effects of activation of protein kinase C (PKC) on N-methyl-D-aspartate) NMDA receptor function were analyzed by quantitative autoradiography using [3H]MK-801 in rat brain slices. The density of [3H]MK-801 binding was highest in hippocampus and high levels were found in cortex, striatum and thalamus. Levels in brainstem and molecular layer of cerebellum were low. The receptor binding was markedly decreased in almost all areas by addition of 2. 5 mM Mg2+. After activation of PKC by 100 nM phorbol-12, 13-dibutyrate (PDBu), [3H]MK-801 binding was increased in most areas, but binding levels were not changed in brainstem and cerebellum. The elevated [3H]MK-801 binding produced by PDBu was significantly inhibited by addition of Mg2+ except in inferior colliculus and cerebellum. These results suggest that activation of PKC potentiates NMDA receptor function in a region-specific manner in the rat brain.     

Differences in NMDA receptor antagonist-induced locomotor activity and [3H]MK-801 binding sites in short-sleep and long-sleep mice

Short-Sleep (SS) and Long-Sleep (LS) mice differ in initial sensitivity to ethanol. Ethanol acts as an antagonist at N-methyl D-aspartate receptors (NMDARs). Therefore, we tested whether SS and LS mice also differ in initial sensitivity to NMDAR antagonists. Systemic injection (intraperitoneal) of either the noncompetitive NMDAR antagonist MK-801 (dizocilpine) or the competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) produced similar results. At lower drug doses, SS mice showed greater locomotor activation than LS mice; and at higher doses, SS mice continued to be activated whereas LS mice became sedated. Brain levels of [3H]MK-801 were 40% higher in SS, compared with LS, mice. However, blood levels of [3H]MK-801 and [3H]CPP and brain levels of [3H]CPP were similar in the two lines. NMDARs were measured using quantitative autoradiographic analysis of in vitro [3H]MK-801 binding to SS and LS mouse brains. Significantly higher (20 to 30%) receptor densities were observed in the hippocampus and cerebral cortex of SS mice. Our results support the hypothesis that SS and LS mice differ in initial sensitivity to NMDAR antagonists and suggest that the line differences in the dose-response relationships for MK-801- and CPP-induced locomotor activity are qualitatively similar to those reported for ethanol. Differences in pharmacokinetics and number of NMDARs may contribute to, but are unlikely to entirely account for, the differential behavioral responsiveness of SS and LS mice to MK-801 and CPP.     

Modulation by magnesium of N-methyl-D-aspartate receptors in developing human brain

AIM: To investigate age related alterations in glutamate N-methyl-D-aspartate (NMDA) receptor binding produced by the modulatory compounds glutamate, glycine, and magnesium (Mg2+) sulphate. METHODS: The effects produced by glutamate plus glycine, and Mg2+ on the binding of [3H]MK-801, a ligand for the N-methyl-D-aspartate ion channel phencyclidine site, were measured in membrane preparations made from prefrontal cortex from human neonate (n = 5), infant (n = 6), and adult (n = 6) necropsy brains. RESULTS: Neonatal brains had the least [3H]MK-801 binding, suggesting either a low density of NMDA receptors or a more restricted access of [3H]MK-801 to cation channel sites. Infant brains had the most [3H]MK-801 binding which was stimulated to a greater extent by L-glutamate (100 microM) and glycine (10 microM) than in neonatal and adult brains. MG2+ invariably inhibited [3H]MK-801 binding. However, the Mg2+ IC50 value was higher in neonatal brain (3.6 mM) than infant (1.4 mM) and adult (0.87 mM) brains. CONCLUSION: Infant brain may have excess NMDA receptors which are hyper responsive to glutamate and glycine. The lower potency of Mg2+ to inhibit [3H]MK-801 binding in neonatal cortex may be because newborn babies have NMDA receptors without the normal complement of Mg2+ sites. The findings suggest that therapeutic NMDA receptor block in neonates requires higher concentrations of magnesium sulphate in brain tissue.     

Analysis of the role of delta opioid receptors in gastroprotection in the rat

The effect of delta opioid agonists - [D-Ala2, D-Leu5]-enkephalin (DADLE), [D-Pen2, D-Pen5]-enkephalin (DPDPE) and deltorphin II - on acidified ethanol induced gastric mucosal lesions was studied in the rat compared with that of morphine. It was found that DADLE, DPDPE, deltorphin II and morphine exerted a dose-dependent inhibition on the mucosal lesions injected subcutaneously, their ID50 values were 0.037, 1.8, 3.5 and 0.35 micromoles/kg, respectively. Naltrindole (10 mg/kg sc.), the selective delta opioid receptor antagonist, inhibited the gastroprotective effect of DADLE, DPDPE and deltorphin II, but it failed to antagonise the effect of morphine. Our results suggest that 1. delta receptors are involved in opioid-mediated gastroprotection, 2. ethanol-induced gastric mucosal damage in the rat may be a quick, simple in vivo model for screening opioid delta receptor agonists and antagonists in the periphery.     

In vivo injection of antisense oligodeoxynucleotides to G alpha subunits and supraspinal analgesia evoked by mu and delta opioid agonists

For 5 consecutive days repeated intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotides (ODNs) to G alpha subunit mRNAs was used to impair the function of mouse Gi1, Gi2, Gi3 and Gx/z regulatory proteins. Decreases of 20 to 60% on the G alpha-like immunoreactivity could be observed in neural structures of mouse brain, an effect that was not produced by a random-sequence ODN used as a control. The ODN to Gi1 alpha subunits lacked effect on opioid-evoked analgesia. In mice injected with the ODN to Gi2 alpha subunits the antinociceptive activity of all the opioids studied appeared greatly impaired. The ODN to Gi3 alpha subunits reduced the effects of the selective agonists of delta opioid receptors, [D-Pen2,5]-enkephalin and [D-Ala2]deltorphin II. Conversely, the analgesia evoked by opioids binding mu opioid receptors, [D-Ala2, N-MePhe4,Gly-ol5]enkephalin and morphine, appeared consistently and significantly attenuated in mice injected with the ODN to Gx/z alpha. The effect of the neuropeptide beta-endorphine-(1-31) agonist at mu and delta receptors was also reduced by ODNs to Gi3 alpha or Gx/z alpha subunits. l.c.v. injection of antibodies directed to these G alpha subunits antagonized opioid-induced analgesia with a pattern similar to that observed for the ODNs. Thus, the mu and delta opiod receptors regulate different classes of G transducer proteins to mediate the analgesic effect of agonists. The in vivo antisense strategy and the use of specific antibodies to G alpha subunits gave comparable results, indicating that in the neural tissue the mRNAs and the G alpha subunits can be accessed by the corresponding ODNs and IgGs.     

Opioid peptide receptor studies. 9. Identification of a novel non-mu- non-delta-like opioid peptide binding site in rat brain

Quantitative binding studies resolved two high-affinity [3H][D-Ala2,D-Leu5]enkephalin binding sites in rat brain membranes depleted of mu binding sites by pretreatment with the irreversible agent BIT. The two binding sites had lower (delta ncx-2, Ki = 96.6 nM) and higher (delta ncx-1, Ki = 1.55 nM) affinity for DPDPE. The ligand-selectivity profile of the delta ncx-1 site was that of a classic delta binding site. The ligand-selectivity profile of the delta ncx-2 site was neither mu- or delta-like. The Ki values of selected agents for the delta ncx-2 site were: [pCl]DPDPE (3.9 nM), DPLPE (140 nM), and DAMGO (2.6 nM). Under these assay conditions, [3H][D-Ala2,D-Leu5]enkephalin binding to the cells expressing the cloned mu receptor is very low and pretreatment of cell membranes with BIT almost completely inhibits [3H]DAMGO and [3H][D-Ala2,D-Leu5]enkephalin binding. Intracerebroventricular administration of antisense DNA to the cloned delta receptor selectively decreased [3H][D-Ala2,D-Leu5]enkephalin binding to the delta ncx-1 site. Administration of buprenorphine to rats 24 h prior to preparation of membranes differentially affected mu, delta ncx-1, and delta ncx-2 binding sites. Viewed collectively, these studies have identified a novel non-mu- non-delta-like binding site in rat brain.     

Selective in vivo binding of [3H]naltriben to delta-opioid receptors in mouse brain

Naltriben (NTB) is a selective antagonist for the putative delta2-opioid receptor. We have determined the regional kinetics and pharmacological profile of [3H]naltriben in vivo in mouse brain. After i.v. administration to CD1 mice, [3H]naltriben uptake and retention were high in striatum, cortical regions and olfactory tubercles, and low in superior colliculi and cerebellum. Robust rank order correlation was found between [3H]naltriben uptake in discrete brain regions and prior delta-opioid receptor binding determinations in vitro and in vivo. [3H]Naltriben binding in vivo was saturable, and was blocked by the delta-opioid receptor antagonist naltrindole, but not by the mu-opioid receptor antagonist cyprodime or the K-opioid receptor agonist (trans)-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]ben zeneacetamide mesylate (U50,488H). (E)-7-Benzylidenenaltrexone (BNTX), a selective antagonist for the putative delta1-opioid receptor, was 9.6- to 12.9-fold less potent than naltriben as an inhibitor of [3H]naltriben binding. Thus, the sites labeled by [3H]naltriben in vivo may correspond to the delta2-opioid receptor subtype. Such assignment is not definitive, particularly considering the 4-fold higher brain uptake of naltriben as compared to (E)-7-benzylidenenaltrexone. Moreover, the regional distribution of [3H]naltriben in brains from CXB-7/BY (CXBK) mice, a strain that shows supraspinal delta1- but not delta2-opioid receptor agonist effects, was quite similar to that found for CD1 mice.     

Effect of 7-nitroindazole on tolerance to morphine, U-50,488H and [D-Pen2, D-Pen5] enkephalin in mice

The effects of 7-nitroindazole (7-NI), an inhibitor of the neuronal nitric oxide synthase (nNOS) which does not increase blood pressure, on tolerance to the antinociceptive activity of mu-(morphine), kappa-(U-50,488H) and delta-([D-Pen2, D-Pen5]enkephalin, DPDPE) opioid receptor agonists were determined in mice. Male Swiss-Webster mice were made tolerant by twice daily injections of morphine (20 mg/kg, s.c.), U-50,488H (25 mg/kg, i.p.) or DPDPE (20 micrograms/mouse, i.c.v.) for 4 days. When tested on day 5, tolerance to their antinociceptive activity was evidenced by decreased response in chronic drug treated mice in comparison to vehicle-injected mice. Concurrent administration of 7-NI (20, 40 or 80 mg/kg, i.p.) with DPDPE did not modify the development of tolerance to the antinociceptive action of DPDPE. However, 7-NI (40 or 80 mg/kg, i.p.) inhibited the development of tolerance to the antinociceptive activity of morphine and U-50,488H but the lower dose of 7-NI (20 mg/kg, i.p.) was not effective. Chronic administration of 7-NI by itself did not modify the acute response to morphine, U-50,488H or DPDPE. It is concluded that a specific inhibitor of nNOS can inhibit tolerance to the antinociceptive activity of mu- and kappa- but not of delta-opioid receptor agonists in mice.     

Research on factors affecting neonatal tetanus and its prevention through immunization

This study examined [3H]MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from cerebral cortex, hippocampus and corpus striatum of 3 week old rats exposed to 10 weeks of intermittent hypobaric hypoxia (4300 m; 450 Torr) and compared results with those of normoxic controls. The cortex, hippocampus and striatum of hypoxic animals had a 36, 35 and 31% reduction in binding sites (Bmax) and a 29, 32 and 17% decrease (reflecting increased affinity) in the dissociation constant (Kd) when compared to controls. In the cerebral cortex, both glutamate (100 microM) and glycine (10 microM) enhanced 3[H]MK-801 binding by two to 3-fold. Coagonist glutamate, however, had a higher EC50 (0.44 microM) in the hypoxic cortical membranes when compared to controls (0.28 microM). No significant differences were found in the EC50 of glycine. The results show that the NMDA receptor is altered in several brain regions of rats developing in a hypoxic environment.     

Global cerebral ischemia and reperfusion alters NMDA receptor binding in canine brain

We employed a canine model to test whether binding to the N-methyl-D-aspartate (NMDA) class of glutamate receptor channels is altered by global cerebral ischemia and/or reperfusion. Ischemia was induced by 10-min cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. In vitro autoradiography was performed on frozen brain sections with three radioligands: [3H]glutamate (under conditions to label the NMDA site), [3H]glycine, and [3H]MK-801. Modest decreases in [3H]glutamate and [3H]MK-801 binding were seen in several regions of hippocampus, and parietal and temporal cortex at early times after reperfusion, with values returning toward control by 24 h. In the striatum, a different pattern was seen: [3H]glutamate and [3H]MK-801 binding increased 50-200% at 0.5-4 h after the start of reperfusion, returning toward control levels by 24 h. These increases correlate with findings of increased sensitivity to NMDA-stimulated release of dopamine from striatal tissue in the same model (Werling et al., 1993), and suggest that changes in tissue receptors may contribute to the selective vulnerability to ischemic damage during the first hours following reperfusion.