Reduced antinociceptive responsiveness to hyperbaric oxygen in opioid-tolerant mice.

BACKGROUND: Hyperbaric oxygen (HBO2 ) therapy can produce analgesia in patients experiencing various conditions of chronic pain. Previously, we reported that naloxone antagonized the acute antinociceptive effect of both brief (11 min) and longer (60 min) HBO2 treatments. This implied a possible role for opioid receptors in the antinociceptive effects of HBO2 . OBJECTIVES: The aim of this study was to determine whether mice previously rendered as tolerant to opioid agonists would exhibit a reduced antinociceptive responsiveness to HBO2 . METHODS: Male NIH Swiss mice were given repeated injections of the opioid agonists morphine, fentanyl or (-)-U50488H over 4 days to induce tolerance at their respective opioid receptors. Mice receiving saline according to a similar injection schedule served as a vehicle control group. On day 5, 15 h after the last injection, mice received either an antinociceptive challenge dose of the opioid agonists or a 30-min HBO2 exposure at 3.5 atmosphere absolute. The antinociception was then assessed by the 0.6% acetic acid-induced abdominal constriction test. RESULTS: The results showed that mice rendered as tolerant to morphine, fentanyl or (-)-U50488H pretreatment all exhibited reduced antinociceptive responsiveness to themselves and HBO2 . CONCLUSIONS: These results demonstrated that both µ- and κ-opioid receptors are involved in mediation of the acute antinociceptive response to HBO2 .

Involvement of the protein kinase Cgamma isoform in development of tolerance to nitrous oxide-induced antinociception in mice.

Prolonged exposure to nitrous oxide (N2O) results in development of acute tolerance to its antinociceptive effect. Cross-tolerance to N2O-induced antinociception is also observed in morphine-tolerant animals. Despite increasing evidence of tolerance development to N2O-induced antinociception, the details of the mechanisms that underlie this tolerance remain unknown. The present study was conducted to investigate the involvement of brain protein kinase C (PKC) isoform in these two types of tolerance to N2O-induced antinociception in mice. Prolonged exposure (41 min in total, including 30 min pre-exposure and 11 min of antinociceptive testing) to 70% N2O produced a reduction in N2O-induced antinociception, indicating development of acute tolerance. The prolonged exposure to 70% N2O caused an activation of PKCgamma isoform in the brain, but not the PKCepsilon isoform. Pretreatment with a PKCgamma-antisense oligonucleotide but not the corresponding mismatch oligonucleotide (i.c.v.) prevented the development of acute tolerance to N2O-induced antinociception. Chronic morphine treatment (10 mg/kg, s.c., b.i.d. for 5 days) resulted in development of tolerance to morphine-induced antinociception and cross-tolerance to N2O-induced antinociception. The development of tolerance to morphine and cross-tolerance to N2O were both inhibited by pretreatment with PKC inhibitor, chelerythrine (1 nmol, i.c.v.). Morphine-tolerant mice showed an activation of PKC within the brain, which was suppressed by pretreatment with chelerythrine (1 nmol, i.c.v.). Thus, activation of brain PKC, in particular, the PKCgamma isoform, appears to play an important role in the development of both acute tolerance and cross-tolerance to N2O-induced antinociception in mice.

Involvement of brain protein kinase C in nitrous oxide-induced antinociception in mice.

Exposure of mice to the anesthetic gas nitrous oxide (N(2)O) produces a marked antinociceptive effect. Protein kinase C is a key regulatory enzyme that may be targeted by general anesthetics. However, a relationship between N(2)O-induced antinociception and protein kinase C has yet to be established. The present study was conducted to identify whether protein kinase C might influence N(2)O-induced antinociception in mice. Regular exposure (11 min) to N(2)O produced concentration-dependent antinociception in mice, as determined using the abdominal constriction test. N(2)O-induced antinociception was attenuated by i.c.v. pretreatment with phorbol 12,13-dibutyrate, a protein kinase C activator. This phorbol 12,13-dibutyrate antagonism of N(2)O-induced antinociception was reversed by i.c.v. pretreatment with calphostin C, a protein kinase C inhibitor. Long-term exposure (41 min in total, including 30 min prior to, and 11 min of analgesic testing) to 70% N(2)O produced reduced analgesic effects, compared with regular exposure to 70% N(2)O, thus indicating acute tolerance to N(2)O-induced antinociception. However, mice pretreated with calphostin C, chelerythrine, which is another protein kinase C inhibitor, and phorbol 12,13-dibutyrate, did not develop acute tolerance. Regarding activation of protein kinase C, regular exposure to 70% N(2)O did not increase protein kinase C within the membrane fraction of brain tissue, as determined by immunoblot analysis, but long-term exposure to 70% N(2)O did. The i.c.v. pretreatment with calphostin C and phorbol 12,13-dibutyrate prevented the increase in protein kinase C observed with long-term exposure to 70% N(2)O. These results suggest that brain protein kinase C negatively regulates the antinociceptive effect of N(2)O, and that activation of brain protein kinase C is related to the development of acute tolerance to N(2)O-induced antinociception in mice.

Detection and mapping of quantitative trait loci that determine responsiveness of mice to nitrous oxide antinociception.

Exposure to 70% N(2)O evokes a robust antinociceptive effect in C57BL/6 (B6) but not in DBA/2 (D2) inbred mice. This study was conducted to identify quantitative trait loci (QTL) in the mouse genome that might determine responsiveness to N(2)O. Offspring from the F(2) generation bred from B6 and D2 progenitors exhibited a broad range of responsiveness to N(2)O antinociception as determined by the acetic acid-induced abdominal constriction test. QTL analysis was then used to dissect this continuous trait distribution into component loci, and to map them to broad chromosomal regions. To this end, 24 spleens were collected from each of the following four groups: male and female F(2) mice responding to 70% N(2)O in oxygen with 100% response (high-responders); and male and female F(2) mice responding with 0% response (low-responders). Genomic DNA was extracted from the spleens and genotyped with simple sequence length polymorphism MapPairs markers. Findings were combined with findings from the earlier QTL analysis from BXD recombinant inbred mice [Brain Res 725 (1996) 23]. Combined results revealed two significant QTL that influence responsiveness to nitrous oxide on proximal chromosome 2 and distal chromosome 5, and one suggestive QTL on midchromosome 18. The chromosome 2 QTL was evident only in males. A significant interaction was found between a locus on chromosome 6 and another on chromosome 13 with a substantial effect on N(2)O antinociception.

Comparison of N2O- and chlordiazepoxide-induced behaviors in the light/dark exploration test.

Earlier research has demonstrated similarities in the behavioral effects of nitrous oxide (N2O) and benzodiazepine (BZ) drugs such as chlordiazepoxide (CP). The present research was conducted to compare the behavioral effects of N2O and CP in mice in the light/dark exploration test. When challenged with either N2O or CP, mice exhibited significant dose-dependent increases in the time spent in the light compartment and also in the number of transitions between the light and dark compartments. Pretreatment with BZ receptor antagonist flumazenil (FLU), the GABA(A) receptor antagonist SR-95531 or the selective neuronal nitric oxide (NO) synthase (nNOS) inhibitor 7-nitroindazole (7-NI) all antagonized anxiolytic effects of N20 and CP. Based on these findings, it was concluded that N20 and CP evoke similar behavioral effects in the light/dark exploration test that are similar in their interaction with BZ and GABA(A) receptor antagonists. There also appears to be a specific role for nNOS in generating the NO involved in mediation of these effects.

Antagonism of nitrous oxide antinociception in mice by intrathecally administered antisera to endogenous opioid peptides.

Previously it was demonstrated that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by kappa-opioid receptors. Since nitrous oxide is thought to cause the neuronal release of endogenous opioid peptide to stimulate opioid receptors, this study was designed to identify the opioid peptides involved, especially in the spinal cord, by determining whether nitrous oxide antinociception can be differentially inhibited by intrathecally (i. t.) administered antisera to different opioid peptides. Male NIH Swiss mice were pretreated i.t. with rabbit antisera to opioid peptides then exposed 24 h later to one of three different concentrations of nitrous oxide in oxygen. Dose-response curves constructed from the data indicated that the antinociceptive effect of nitrous oxide was significantly antagonized by antisera to various dynorphins (DYNs) and methionine-enkephalin (ME), but not by antiserum to beta-endorphin (beta-EP). The AD(50) values for nitrous oxide antinociception were significantly elevated by antisera to DYNs and ME but not beta-EP. These findings of this study support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test involves the neuronal release of DYN and ME in the spinal cord.

Role of brain dynorphin in nitrous oxide antinociception in mice.

Earlier studies indicate that nitrous oxide antinociception is mediated by opioid receptors, and we have hypothesized that nitrous oxide stimulates a neuronal release of an endogenous opioid peptide (EOP) that stimulates opioid receptors. To further test this hypothesis, male NIH Swiss mice were pretreated intracerebroventricularly with rabbit antisera to opioid peptides or with various inhibitors of peptidases involved in the degradation of EOPs. Mice were subsequently exposed to three different concentrations of nitrous oxide in oxygen, and their antinociceptive responsiveness was measured using the acetic acid abdominal constriction test. Nitrous oxide antinociception was significantly attenuated by 24-h pretreatment with antisera to various fragments of dynorphin (DYN) but not by antisera against methionine-enkephalin (ME) or beta-endorphin (beta-EP). In other experiments, nitrous oxide antinociception was significantly enhanced by 30-min pretreatment with phosphoramidon, an inhibitor of endopeptidase 24.11, which has been implicated in DYN degradation, but not bestatin or captopril, which inhibit aminopeptidase and angiotensin-converting enzyme, respectively. The latter enzymes have been implicated in degradation of certain EOPs albeit not DYN. These findings support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by endogenous DYN acting in the central nervous system.

Increased production of nitric oxide metabolites in the hippocampus under isoflurane anaesthesia in rats.

There is disagreement concerning the role of nitric oxide (NO) in general anaesthesia. The present study was conducted to determine whether the anaesthetic drug isoflurane alters levels of NO metabolites, NOx (NO2 and NO3), in the hippocampus of rats during and after anaesthesia. Results showed resting hippocampal NOx levels of about 20 pmol in freely moving control rats. Five minutes after the induction of isoflurane anaesthesia (4.72% = 4 minimum alveolar concentrations, detected by righting reflex), there was loss of the righting reflex coincident with a significant elevation in hippocampal NOx levels. During isoflurane anaesthesia, the maximum NOx concentration rose approximately 2.4-fold higher than control levels; the NO3 level increased about 5-fold higher than resting levels. NOx levels returned to control levels following discontinuation of the anaesthetic. When rats were pretreated with L-NG-nitro arginine methyl ester, an NO synthase-inhibitor, the isoflurane-induced increases in NOx were markedly suppressed. D-NG-nitro arginine methyl ester was ineffective in preventing these neurochemical changes, thus indicating the stereo-selective nature of the inhibition by L-NG-nitro arginine methyl ester Furthermore, L-NG-nitro arginine methyl ester, pre-treatment likewise prevented increases in both NO2 and NO3 levels. When rats were exposed to 80% nitrous oxide in oxygen, there was loss of the righting reflex but no change in hippocampal NOx levels. These findings indicate that isoflurane increases production of hippocampal NO and that this may be pertinent to general anaesthetic drug effects.

The efficacy of delta-opioid receptor-selective drugs.

Delta-opioid receptor-selective drugs may provide an alternative to mu-opioid-selective drugs currently used for the relief of pain. To develop improved delta-opioid receptor-selective drugs, better measures of drug activity are necessary. In this review we suggest that efficacy calculations provide a superior measure of drug activity as compared to dissociation constants and drug potencies in functional assays. Efficacy, as discussed in this review, is defined as a quantitative measurement of the ability of a drug to stimulate second messenger systems or measurable functional responses in cells or tissues under standard conditions. Efficacy values will allow medicinal chemists to understand the contributions of both the coupling efficiency and dissociation constant to drug potencies in the development of new delta-opioid receptor-selective drugs.

Relative efficacies of delta-opioid receptor agonists at the cloned human delta-opioid receptor.

The present study was conducted to determine the relative efficacies of the selective delta-opioid receptor agonists SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[D-Pen2,4'-ClPhe4,D-Pen5]enkephalin) and (-)-TAN67 ((-)-2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPgammaS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human delta-opioid receptor. Efficacy was determined according to the formula: efficacy = (E(max-A)/Emax)(A'/A + 1) X 0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6-7 times greater than that of (-)-TAN67.

delta 9-Tetrahydrocannabinol is a partial agonist of cannabinoid receptors in mouse brain.

We measured the ability of the cannabinoid agonists delta 9-tetrahydrocannabinol and R(+)-[2,3,-dihydro-5-methyl-3- [(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-napht halenyl) methanone mesylate (WIN 55,212-2) to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding in mouse brain membranes. delta 9-Tetrahydrocannabinol stimulated [35S]GTP gamma S binding by about 25% as compared to WIN 55,212-2. This is the first report demonstrating that delta 9-tetrahydrocannabinol acts as a partial agonist in stimulating [35S]GTP gamma S binding in the mouse brain.

AM630 antagonism of cannabinoid-stimulated [35S]GTP gamma S binding in the mouse brain.

This research was designed to determine the action of the novel aminoalkylindole AM630 (6-iodo-pravadoline) at the cannabinoid receptor by studying its interaction with the cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-y]-(1-naphthalenyl)methanone mesylate) on guanosine-5'-O-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding in mouse brain. WIN 55,212-2 stimulated [35S]GTP gamma S binding, while AM630 had no effect. AM630 antagonized WIN 55,212-2-2induced [35S]GTP gamma S binding and shifted the WIN 55,212-dose-response curve to the right. These results clearly demonstrate that AM630 exerts cannabinoid receptor antagonist properties in the brain.

AM630 is a competitive cannabinoid receptor antagonist in the guinea pig brain.

AM630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [35S]GTPgammaS binding suggesting that difference in AM630 activity in different tissues is not due to species variation.

Relative efficacies of cannabinoid CB1 receptor agonists in the mouse brain.

We measured (-)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohe xyl]-phenol (CP 55,940)-, (-)11-OH-delta8-tetrahydrocannabinol-dimethylheptyl (HU-210)-, anandamide- and delta9-tetrahydrocannabinol-stimulated G protein activation in mouse brain using the [35S]GTPgammaS functional assay. The Ki values for these drugs were determined by agonist competition binding with the cannabinoid CB1 receptor antagonist [3H]N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride ([3H]SR141716A). This information was used to calculate the efficacy for drug stimulation of G protein activity. The rank order of efficacy was CP 55,940 > HU-210 > anandamide > delta9-tetrahydrocannabinol with the latter two drugs being partial agonists. Since efficacy values relate receptor occupancy to functional responses, we believe efficacy values are a better measure of drug-mediated functional responses compared with measurements of drug potency.

Nitrous oxide antinociception in BXD recombinant inbred mouse strains and identification of quantitative trait loci.

Among inbred mouse strains, DBA/2 mice are unique because of their poor responsiveness to nitrous oxide (N2O) antinociception. As a first step towards identifying candidate genes involved in determining antinociceptive responsiveness to N2O, male mice from the DBA/2 strain, the more responsive C57BL/6 strain, their B6D2F1 offspring, and 22 BXD recombinant inbred (RI) strains derived from DBA/2 and C57BL/6 mice were exposed to N2O and evaluated using the acetic acid abdominal constriction test. When exposed to 70% N2O, C57BL/6, DBA/2 and B6D2F1 mice exhibited antinociceptive responses of 78, 22 and 55%, respectively. The BXD RI strains demonstrated varying degrees of responsiveness to N2O. Cluster analysis revealed one cluster of 16 strains approximating the C57BL/6 progenitor (61.9-100% antinociceptive response to 70% N2O) and another of six strains around the DBA/2 progenitor (9.1-40% antinociceptive response to 70% N2O). The robust strain differences permitted screening the strain means with 1492 marker loci previously mapped in BXD RI strains. Using a QTL analysis specifically tailored to existing mouse RI strains, we found associations at the 0.01 level on seven chromosomes with the most promising marker loci being Il2ra, Hbb, Hmg1rs7 and Gsl5 on chromosomes 2, 7, 16 and 19, respectively (P < 0.002).

Use of antisense oligodeoxynucleotide to determine delta-opioid receptor involvement in [D-Ala2]deltorphin II-induced locomotor hyperactivity.

Intracerebroventricularly (i.c.v.)-administered [D-Ala2]deltorphin II (20 micrograms) produced a marked locomotor hyperactivity in male ICR mice. The locomotor hyperactivity induced in response to i.c.v. [D-Ala2]deltorphin II (20 micrograms) was suppressed by pretreatment with naltriben (NTB, 10 micrograms) but not 7-benzylidene naltrexone (BNTX, 1 microgram) and D-Phe-Cys-Tyr-D-Try-Orn-Thr-Phe-Thr-NH2 (CTOP, 100 ng). The influence of antisense oligodeoxynucleotide to delta-opioid receptor mRNA (delta-AS oligo) or a mismatch oligodeoxynucleotide (MM oligo) on the locomotor hyperactivity induced by [D-Ala2]deltorphin II was determined. Groups of mice pretreated i.c.v. with delta-AS oligo (1 microgram), MM oligo (1 microgram) or saline (4 microliters) once a day for 3 days, were injected i.c.v. [D-Ala2]deltorphin II (10 or 20 micrograms) and the locomotor response to [D-Ala2]deltorphin II was measured. The locomotor hyperactivity of i.c.v. [D-Ala2]deltorphin II (10 or 20 micrograms) were significantly suppressed by i.c.v. pretreatment with delta-AS oligo but not MM oligo. The present results indicate that pretreatment with delta-AS oligo suppresses mouse locomotor hyperactivity produced by stimulation of delta 2-opioid receptors in the brain.

Involvement of nitric oxide in intracerebroventricular beta-endorphin-induced neuronal release of methionine-enkephalin.

Previous work has suggested that the antinociceptive effect of nitrous oxide (N2O) in rats is mediated, at least in part, by beta-endorphin (beta-EP) and that centrally administered beta-EP stimulates release of methionine-enkephalin (ME) in the rat spinal cord. Since inhibition of central nitric oxide (NO) production has been found to suppress N2O antinociception, we examined the possible involvement of NO in the release of spinal cord ME by i.c.v. beta-EP. Urethane-anesthetized, male Sprague-Dawley rats were intrathecally (i.t.) perfused with artificial cerebrospinal fluid (aCSF) and fractions of perfusate were assayed for immunoreactive (i.r.) ME. The beta-EP-induced increase in ME concentration in the i.t. perfusate was significantly suppressed by perfusing the animal with aCSF containing 100 microM L-NG-nitro arginine (L-NOARG), an inhibitor of NO synthase (NOS). The further addition of 50 microM L-arginine (L-ARG), but not D-arginine (D-ARG), to the aCSF reversed the suppression of the ME change by L-NOARG. However, the potency of L-ARG decreased with increasing concentrations of L-ARG. On the other hand, increasing the concentration of L-NOARG in the aCSF to 250 microM failed to produce a greater suppression of the beta-EP-induced increase in ME. These findings suggest that NO may mediate the beta-EP-induced release of ME in the spinal cord and that interference with this mechanism might be an explanation for the antagonism of N2O antinociception in rats by NOS inhibitors.

Involvement of nitric oxide in nitrous oxide anxiolysis in the elevated plus-maze.

We recently reported that inhibition of nitric oxide (NO) production by the NO synthase (NOS) inhibitor L-NG-nitro arginine (L-NOARG) antagonized the behavioral effects of a benzodiazepine (BZ) in a mouse paradigm for screening anxiolytic drug activity. Because other research has found that the anesthetic gas nitrous oxide (N2O) also produces BZ-like behavioral effects, the present research was conducted to ascertain whether NO might also be involved in N2O anxiolysis. Male Swiss-Webster mice were tested in an elevated plus-maze inside an inflatable glovebag. Exposure to N2O significantly increased exploratory activity on the open arms of the plus-maze, as measured by the number of entries into the open arms and the time spent on the open arms. Pretreatment with L-NOARG significantly reduced the N2O-induced elevation in open arm activity. This antagonism of the N2O effect was reversed by ICV treatment of L-NOARG-pretreated mice with L-arginine but not D-arginine. These findings indicate that NO possibly mediates behavioral effects of N2O in an animal model for anxiety.

Effect of opioid peptide antisera on nitrous oxide antinociception in rats.

This study was performed to examine the effects of ICV injection of antiserum against beta-endorphin (beta-EP) or methionine-enkephalin (ME) on nitrous oxide-induced antinociception in rats using the hot plate test. The injection of beta-EP antiserum reversed the antinociceptive effect of nitrous oxide in a dose-related manner up to 200 micrograms/rat. However, antagonism of nitrous oxide by 400 micrograms beta-EP antiserum was comparable to that produced by 200 micrograms. On the other hand, similar amounts of ME antiserum had little effect against nitrous oxide antinociception. These findings suggest that beta-EP may play an important role in the antinociceptive effect of nitrous oxide.