Apomorphine: modification of its hyperthermic effect in rabbits by p-chlorophenylalanine.

The hyperthermic response of rabbits to apomorphine, a dopaminergic agonist, is abolished by prior treatment with p-chlorophenylalanine. If such 5-hydroxytryptamine (5-HT)-depleted animals are administered a peripherally acting decarboxylase inhibitor plus 5-hydroxytryptophan, central stores of 5-HT are regenerated and the hyperthermic response to apomorphine is restored in part. The effects of apomorphine in rabbits with elevated concentrations of 5-HT are not different from those in control animals. The behavioral effects of apomnorphine appear to be constant in all groups of animals tested. It is suggested that the hyperthermic effects of apomorphine in rabbits require the presence of 5-HT.

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.

Kinetic modeling of hormone release: application to time-averaged met-enkephalin release.

The time course of hormone concentrations in response to a stimulus can be characterized with kinetic methods to express rates of hormone release and elimination, total amount of hormone release and expected best times for blood sampling. Routine application of kinetic methods should increase the sensitivity and applicability of hormonal challenge tests. Applications to the TRH test and to prolactin release measurements are reviewed. A new kinetic model for continuously collected samples is applied to measurements of met-enkephalin release in rats. Calculations indicated an initial concentration of 1876 pg/ml and a ventricular volume of distribution of 0.25 ml. After exposure of rats to nitrous oxide, the rate of release of met-enkephalin-like activity into the ventricles rose from 3.4 to 8.3 pg/min.

Effects of benzodiazepine agonist, inverse agonist and antagonist drugs in the mouse staircase test.

This study examined the effects of the benzodiazepine agonist midazolam and inverse agonist noreleagnine independently and in conjunction with the antagonist flumazenil in the mouse staircase test. According to this paradigm, the numbers of steps ascended (NSA) and rears (NR) reflect locomotor activity and anxiety, respectively. Midazolam reduced NR at doses that did not affect NSA; this NR-lowering effect was blocked by flumazenil. Noreleagnine increased NR at doses that did not affect NSA; this NR-elevating effect was also blocked by flumazenil. Effective antagonist doses of flumazenil alone had no effect on NR or NSA. The exactly opposite effects of midazolam and noreleagnine on NR and their antagonism by flumazenil are consistent with the postulated activities of these drugs at benzodiazepine receptors.

Nitrous oxide induces an anxiolytic-like effect in the conditioned defensive burying paradigm, which can be reversed with a benzodiazepine receptor blocker.

To investigate the anxiolytic effects of nitrous oxide (N2O), male hooded rats were tested in the conditioned defensive burying (CDB) test, a paradigm that exploits a propensity of rats to bury objects associated with aversive stimulation. A single, brief electrical shock was delivered to rats upon contact with an electrified prod, before exposure to one of four mixtures of N2O and oxygen (O2) (10-40% N2O) or room air (RA). Compared to RA-exposed animals, rats exposed to N2O exhibited a concentration-related reduction in duration and height of prod-directed "defensive" burying with floor bedding material; these measures reached statistical significance at 30% N2O. Pretreatment with 20 mg/kg of the benzodiazepine receptor blocker flumazenil, which alone had no effect, effectively antagonized a 30% N2O-induced decrease in burying. Horizontal locomotion and rearing were not significantly affected at concentrations of N2O that attenuated prod-directed burying. Treatment with the benzodiazepine anxiolytic standard, chlordiazepoxide (2.5-10.0 mg/kg) also resulted in dose-related attenuation of burying behavior. These findings show that N2O can induce effects similar to those of known anxiolytics in this paradigm and suggest a benzodiazepine mechanism in its mediation.

Nitrous oxide anxiolytic effect in mice in the elevated plus maze: mediation by benzodiazepine receptors.

In earlier research, we have hypothesized that exposure to nitrous oxide (N2O) produces an anxiolytic effect that is mediated by benzodiazepine (BZ) receptors. The present research was conducted to characterize pharmacologically the behavioral effects of N2O in comparison with a BZ standard, chlordiazepoxide (CP), in the mouse elevated plus maze. Exposure to increasing levels of N2O produced a concentration-related increase in the percent of total entries into and the percent of total time spent on the open arms, a pattern of response similar to that induced by CP. These effects on N2O and CP were both antagonized by pretreatment with the BZ receptor blocker flumazenil (FLU). In another experiment, mice made tolerant to CP also exhibited a cross-tolerance to N2O. These results support the hypothesis that the anxiolytic effect of N2O is mediated by BZ receptors.

Comparison of nitrous oxide, morphine and diazepam effects in the mouse staircase test.

Mice were exposed for 10-20 min to room air, 100% oxygen (O2) or increasing concentrations of nitrous oxide (N2O) in O2, then tested for 3 min in a staircase inside a glovebag. N2O produced a concentration-dependent increase in the number of steps ascended (NSA) but no change in the number of rears (NR). Pretreatment with naloxone reversed the increase in NSA and also unmasked N2O reduction in NR. By comparison, increasing doses of the narcotic standard morphine reduced NSA and NR; these changes in NSA and NR were sensitive to antagonism by naloxone. The benzodiazepine standard diazepam produced a dose-related reduction in NR while reducing NSAd only at higher doses. These data indicate that N2O influences on NSA and NR resemble neither morphine nor diazepam. In addition, it appears that the opioid activity of N2O might mask its antianxiety activity in this particular paradigm.

Benzodiazepine receptor mediation of behavioral effects of nitrous oxide in mice.

Nitrous oxide produces behavioral effects, the underlying mechanism of which is not known. In the mouse staircase test, exposure to nitrous oxide caused a reduction in rearing activity, an effect similar to that produced by benzodiazepines in this paradigm, when its opioid action on locomotion is blocked by naloxone. In this study, we tested whether effects of nitrous oxide might be mediated by benzodiazepine receptors, using chlordiazepoxide as a control. The abilities of nitrous oxide and chlordiazepoxide to reduce rearing were significantly attenuated in mice pretreated with the benzodiazepine receptor blocker flumazenil or rendered tolerant to benzodiazepines. These findings suggest an involvement of benzodiazepine receptors in mediation of certain behavioral effects of nitrous oxide.

Microwave facilitation of methylatropine antagonism of central cholinomimetic drug effects.

Pilocarpine-induced hypothermia and oxotremorine-induced tremors in mice are central cholinomimetic drug effects that are readily blocked by the muscarinic antagonist atropine. However, the quaternary ammonium derivative of atropine, methylatropine, is unable to block these cholinomimetic drug effects by virture of its inability to penetrate the blood-brain barrier (BBB) and blood-cerebral spinal fluid barrier (B-CSFB). Dose-response curves for pilocarpine and oxotremorine effects are not appreciably affected either by pretreatment with methylatropine (1.0 mg/kg) or by exposure to moderate-level microwave irradiation (2.45 GHz, 23.7 W/kg, CW, 10-min exposure). However, in mice receiving both the methylatropine pretreatment and microwave irradiation, the dose-response curves for both pilocarpine and oxotremorine effects were significantly shifted to the right, signifying a central anticholinergic action by methylatropine. These data indicate that a single acute exposure to a thermogenic level of microwave irradiation facilitates methylatropine antagonism of centrally mediated cholinomimetic drug effects. One possible explanation for this observation is that microwave radiation may enhance passage of quaternary ammonium compounds like methylatropine across the BBB and B-CSFB.

Protection by U-50,488H against beta-chlornaltrexamine antagonism of nitrous oxide antinociception in mice.

Nitrous oxide antinociception in the abdominal constriction test was significantly reduced in mice pretreated intracerebroventricularly (i.c.v.) with beta-chlornaltrexamine (beta-CNA). However, this antagonism was reversed when the beta-CNA was co-administered i.c.v. with the kappa-opioid ligand U-50,488H but not the mu-opioid ligand CTOP. These findings further demonstrate that nitrous oxide antinociception in the mouse abdominal constriction paradigm is mediated by kappa- but not mu-opioid receptors.

Influence of narcotic antagonist drugs upon nitrous oxide analgesia in mice.

Nitrous oxide produced a concentration-related suppression of phenylquinone-induced abdominal constriction in mice. This analgesic effect was significantly reduced (but not abolished) by systemic pretreatment with (-)-naloxone or naltrexone but not (+)-naloxone. Systemic pretreatment with methylnaltrexone failed to appreciably influence nitrous oxide analgesia; however, methylnatrexone, administered centrally, significantly attenuated the drug effect. Furthermore, nitrous oxide analgesia was significantly reduced by MR-2266 (which is relatively selective for kappa-opioid receptors) but not by beta-funaltrexamine (which is selective for mu-opioid receptors) at the doses employed in this study. These findings suggest that nitrous oxide analgesia might involve an activation of kappa-opioid receptors in the central nervous system; however, a possible involvement of mu-opioid receptors is not absolutely precluded by this study.

Strain-dependent differences in responsiveness of mice to nitrous oxide (N2O) antinociception.

N2O antinociception was assessed in eight inbred and two outbred mouse strains. Results indicated the following order of responsiveness among the 10 strains: A/J (most sensitive), C57BL/6ByJ, C57BL/6J, BALB/cByJ, C3H/HeJ, Swiss-Webster, CXBK/ByJ, ICR, CBA/J and DBA/2J (least sensitive). These results demonstrate significant strain-dependent differences in antinociceptive responsiveness to N2O. The weak antinociceptive response to N2O in the DBA/2J strain, which is sensitive to morphine and U-50, 488H, indicates some underlying neurobiological difference in the DBA/2J mouse that imparts resistance to N2O. The responsiveness of CXBK/ByJ mice to N2O indicates that mu-opioid receptors may not play an important role in N2O antinociception in mice.

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).

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.

Potentiation by naloxone of the anti-oxotremorine effect of L-DOPA.

Pretreatment with the catecholamine precursor L-DOPA but not the narcotic antagonist naloxone suppressed the tremorigenic effect of oxotremorine in mice. However, when animals were pretreated with both L-DOPA and several different doses of naloxone, there was a dose-related potentiation of the antitremor effect of L-DOPA. Naloxone also produced dose-dependent potentiation of the antitremor activity of lower doses of L-DOPA in the presence of the peripheral decarboxylase inhibitor carbidopa. These findings suggest a possible therapeutic application for naloxone in treatment of dopamine dysfunction disorders.

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.

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.