A critical role of striatal A2A R-mGlu5 R interactions in modulating the psychomotor and drug-seeking effects of methamphetamine.

Addiction to psychostimulants is a major public health problem with no available treatment. Adenosine A2A receptors (A2A R) co-localize with metabotropic glutamate 5 receptors (mGlu5 R) in the striatum and functionally interact to modulate behaviours induced by addictive substances, such as alcohol. Using genetic and pharmacological antagonism of A2A R in mice, we investigated whether A2A R-mGlu5 R interaction can regulate the locomotor, stereotypic and drug-seeking effect of methamphetamine and cocaine, two drugs that exhibit distinct mechanism of action. Genetic deletion of A2A R, as well as combined administration of sub-threshold doses of the selective A2A R antagonist (SCH 58261, 0.01 mg/kg, i.p.) with the mGlu5 R antagonist, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (0.01 mg/kg, i.p.), prevented methamphetamine- but not cocaine-induced hyperactivity and stereotypic rearing behaviour. This drug combination also prevented methamphetamine-rewarding effects in a conditioned-place preference paradigm. Moreover, mGlu5 R binding was reduced in the nucleus accumbens core of A2A R knockout (KO) mice supporting an interaction between these receptors in a brain region crucial in mediating addiction processes. Chronic methamphetamine, but not cocaine administration, resulted in a significant increase in striatal mGlu5 R binding in wild-type mice, which was absent in the A2A R KO mice. These data are in support of a critical role of striatal A2A R-mGlu5 R functional interaction in mediating the ambulatory, stereotypic and reinforcing effects of methamphetamine but not cocaine-induced hyperlocomotion or stereotypy. The present study highlights a distinct and selective mechanistic role for this receptor interaction in regulating methamphetamine-induced behaviours and suggests that combined antagonism of A2A R and mGlu5 R may represent a novel therapy for methamphetamine addiction.

Smoking enhances the proinflammatory effects of nucleotides on cytokine release from human lung.

Nucleotides have effects on immune cells which are complex but generally proinflammatory, and have been suggested to play a role in smoking-related lung diseases. However, there have been no studies directly measuring functional responses to nucleotides in human lungs taken from smokers. We used fragments of post mortem human lung from smokers and non-smokers, incubated them with a range of nucleotides (4-1000 µM) in the presence of lipopolysaccharide (LPS; 10 µg/ml) for 24 hours and measured cytokines (IL-1ß, IFNγ, IL-17, TNFα, IL-6, IL-8, IL-2 and IL-10) in the supernatants using multiplex immunoassays. Although the basal cytokine levels in the smokers were generally higher in the smokers than the non-smokers, there were no significant differences in either the basal release or the LPS-stimulated release of any of the cytokines when lungs from smokers and non-smokers were compared. There were no significant effects of ATP, ADP, AMP, UTP, α,ß-methylene-ATP, P1, P4-diATP, 2-methylthio-ATP or Bz-ATP on the release of cytokines from the lungs. However, the stable ATP analogue ATPγS increased the release of IL-1ß and IFNγ, and the effect was greatly increased in lungs from smokers. In non-smokers but not in smokers ATPγS increased the release of IL-17. Overall these results clearly demonstrate for the first time that in normal human lung a stable ATP analogue can enhance LPS-induced pro-inflammatory cytokine release, and that these effects are greatly altered by a prior history of smoking. This provides strong support for the suggestion that nucleotides are involved in the pathogenesis of smoking-related diseases.

Chronic methamphetamine treatment induces oxytocin receptor up-regulation in the amygdala and hypothalamus via an adenosine A2A receptor-independent mechanism.

There is mounting evidence that the neuropeptide oxytocin is a possible candidate for the treatment of drug addiction. Oxytocin was shown to reduce methamphetamine self-administration, conditioned place-preference, hyperactivity and reinstatement in rodents, highlighting its potential for the management of methamphetamine addiction. Thus, we hypothesised that the central endogenous oxytocinergic system is dysregulated following chronic methamphetamine administration. We tested this hypothesis by examining the effect of chronic methamphetamine administration on oxytocin receptor density in mice brains with the use of quantitative receptor autoradiographic binding. Saline (4ml/kg/day, i.p.) or methamphetamine (1mg/kg/day, i.p.) was administered daily for 10 days to male, CD1 mice. Quantitative autoradiographic mapping of oxytocin receptors was carried out with the use of [(125)I]-vasotocin in brain sections of these animals. Chronic methamphetamine administration induced a region specific upregulation of oxytocin receptor density in the amygdala and hypothalamus, but not in the nucleus accumbens and caudate putamen. As there is evidence suggesting an involvement of central adenosine A2A receptors on central endogenous oxytocinergic function, we investigated whether these methamphetamine-induced oxytocinergic neuroadaptations are mediated via an A2A receptor-dependent mechanism. To test this hypothesis, autoradiographic oxytocin receptor binding was carried out in brain sections of male CD1 mice lacking A2A receptors which were chronically treated with methamphetamine (1mg/kg/day, i.p. for 10 days) or saline. Similar to wild-type animals, chronic methamphetamine administration induced a region-specific upregulation of oxytocin receptor binding in the amygdala and hypothalamus of A2A receptor knockout mice and no genotype effect was observed. These results indicate that chronic methamphetamine use can induce profound neuroadaptations of the oxytocinergic receptor system in brain regions associated with stress, emotionality and social bonding and that these neuroadaptations are independent on the presence of A2A receptors. These results may at least partly explain some of the behavioural consequences of chronic methamphetamine use.

The oxytocin analogue carbetocin prevents emotional impairment and stress-induced reinstatement of opioid-seeking in morphine-abstinent mice.

The main challenge in treating opioid addicts is to maintain abstinence due to the affective consequences associated with withdrawal which may trigger relapse. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin (OT) in the modulation of mood disorders as well as drug addiction. However, its involvement in the emotional consequences of drug abstinence remains unclear. We investigated the effect of 7-day opioid abstinence on the oxytocinergic system and assessed the effect of the OT analogue carbetocin (CBT) on the emotional consequences of opioid abstinence, as well as relapse. Male C57BL/6J mice were treated with a chronic escalating-dose morphine regimen (20-100 mg/kg/day, i.p.). Seven days withdrawal from this administration paradigm induced a decrease of hypothalamic OT levels and a concomitant increase of oxytocin receptor (OTR) binding in the lateral septum and amygdala. Although no physical withdrawal symptoms or alterations in the plasma corticosterone levels were observed after 7 days of abstinence, mice exhibited increased anxiety-like and depressive-like behaviors and impaired sociability. CBT (6.4 mg/kg, i.p.) attenuated the observed negative emotional consequences of opioid withdrawal. Furthermore, in the conditioned place preference paradigm with 10 mg/kg morphine conditioning, CBT (6.4 mg/kg, i.p.) was able to prevent the stress-induced reinstatement to morphine-seeking following extinction. Overall, our results suggest that alterations of the oxytocinergic system contribute to the mechanisms underlying anxiety, depression, and social deficits observed during opioid abstinence. This study also highlights the oxytocinergic system as a target for developing pharmacotherapy for the treatment of emotional impairment associated with abstinence and thereby prevention of relapse.

Paracetamol inhibits nitric oxide synthesis in murine spinal cord slices.

Paracetamol is an effective analgesic but its mechanism of action is unclear. We investigated the effect of paracetamol and the analgesic adjuvant caffeine on the activity of NO synthase in mouse spinal cord and cerebellar slices in vitro, by measuring the conversion of [(3)H]arginine to [(3)H]citrulline. Paracetamol (100 microM) had no effect on NO synthase activity in cerebellum, but in the spinal cord both paracetamol (100 microM) and caffeine (30 microM) attenuated glutamate (5 mM)-induced [(3)H]citrulline production and in combination they abolished it. In conclusion paracetamol inhibits spinal cord NO synthesis and this may be related to its analgesic effects.

Reduced response to the formalin test and lowered spinal NMDA glutamate receptor binding in adenosine A2A receptor knockout mice.

Adenosine is a neuromodulator with complex effects on pain pathways. Mice lacking the adenosine A2A receptor are hypoalgesic, and have altered analgesic responses to receptor-selective opioid agonists. These and other findings suggest a role for the adenosine A2A receptor in sensitizing afferent fibres projecting to the spinal cord. To test this hypothesis formalin (20 microl, 5%) was injected into the paw and nociceptive responses were measured in wildtype and adenosine A2A receptor knockout mice. There was a significant reduction in nociception associated with sensory nerve activation in the knockout mice as measured by time spent biting/licking the formalin-injected paw and number of flinches seen during the first phase, but only the number of flinches was reduced during the second inflammatory phase. In addition, the selective adenosine A2A antagonist SCH58261 (3 and 10 mg/kg) also antagonised both phases of the formalin test. We also labelled NMDA glutamate and NK1 receptors in spinal cord sections as an indirect measure of nociceptive transmission from peripheral sites to the spinal cord. [3H]-Substance P binding to NK1 receptors was unaltered but there was a substantial reduction in binding of [3H]-MK801 to NMDA glutamate receptors in all regions of the spinal cord from knockout mice. The decrease in NMDA glutamate receptor binding may reflect reduced peripheral sensory input to the spinal cord during development and could relate to the hypoalgesia in this genotype. These results support a key role for the adenosine A2A receptor in peripheral nociceptive pathways.

Modulation of paracetamol antinociception by caffeine and by selective adenosine A2 receptor antagonists in mice.

This study investigated the involvement of adenosine receptors in the interaction between paracetamol and caffeine in mice, using the adenosine A2A receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261) and the adenosine A2B receptor antagonist 1-propyl-8-p-sulfophenylxanthine (PSB1115), in the tail immersion and hot-plate tests. Paracetamol (10-200 mg/kg) was antinociceptive in both tests, but, in contrast to previous studies, caffeine (10 mg/kg) was pronociceptive in the tail immersion test, and reduced the effects of paracetamol in both tests. SCH58261 (3 mg/kg) was antinociceptive in both tests and in its presence paracetamol (50 mg/kg) had no further effect. PSB1115 (10 mg/kg) had little effect alone but potentiated the effect of paracetamol (50 mg/kg) in the hot-plate test and abolished it in the tail immersion test. These results suggest that adenosine A2B receptors may be involved in the action of paracetamol in a pathway-dependent manner, and also support the existence of pronociceptive adenosine A2A receptors.

Dietary copper supplements modulate aortic superoxide dismutase, nitric oxide and atherosclerosis.

The objective was to test the hypothesis that dietary copper inhibits atherosclerosis by inducing superoxide dismutase (SOD) and potentiating nitric oxide (NO). New Zealand White rabbits were fed either a cholesterol diet (n = 8) or a cholesterol diet containing 0.02% copper acetate (n = 8) for 13 weeks. We found that the intimal area was significantly smaller in the animals supplemented with copper (P < 0.005), although integrated plasma cholesterol levels were not significantly different. This was associated with a significant increase in aortic copper content (P < 0.05), SOD activity (P < 0.05) and Cu/Zn SOD mRNA (P < 0.05) and a significant decrease in nitrotyrosine content (P < 0.05). Furthermore, there was a positive correlation between aortic copper content and SOD activity (P < 0.005, R(2) = 0.83) and a negative correlation between aortic superoxide dimutase activity and nitrotyrosine content (P < 0.005, R(2) = 0.93). In organ bath experiments, the relaxation of precontracted carotid artery rings to calcium ionophore was greater in animals supplemented with copper. No difference in response to sodium nitroprusside was observed. These data suggest that in the cholesterol-fed rabbit, copper supplements inhibit the progression of atherosclerosis by increasing SOD expression, thereby reducing the interaction of NO with superoxide, and hence potentiating NO-mediated pathways that may protect against atherosclerosis.

An investigation of binding sites for paracetamol in the mouse brain and spinal cord.

Quantitative autoradiography has been used to assess whether [3H]paracetamol (3 microM) binds specifically to any area of the murine brain and spinal cord and to investigate whether paracetamol (1-100 microM) competes for binding to the nociceptin opioid peptide (NOP) receptor or to the nitrobenzylthioinosine (NBTI)-sensitive adenosine transporter in the brains of mice. [3H]paracetamol binding was homogenous and, although there was some indication of specific binding overall, this binding in most individual regions failed to reach statistical significance. However, thoracic segments of the spinal cord were found to have significantly higher specific binding than cervical and lumbar regions. Paracetamol did not significantly compete for binding to the NOP receptor or to the NBTI-sensitive adenosine transporter, showing that it does not mediate its effect via these sites. Although paracetamol did bind specifically to the murine brain and spinal cord, the binding was not region-specific, suggesting binding is not related to any particular neurotransmitter system.

Quantitative autoradiography of adenosine receptors and NBTI-sensitive adenosine transporters in the brains of mice deficient in the preproenkephalin gene.

There is a large body of evidence indicating important interactions between the adenosine and the opioid systems in regulating pain, opioid dependence and withdrawal. Mice lacking the proenkephalin gene and therefore lacking the endogenous enkephalin peptides have been successfully developed and exhibit decreased locomotor activity, are hyperalgesic and show enhanced anxiety and aggression. In addition, an upregulation of mu and delta receptors was also observed in the brains of knockout mice. To investigate if there are any compensatory alterations in adenosine systems in the brains of mutant mice, we have carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors and nitrobenzylthioinosine (NBTI)-sensitive adenosine transporters in the brains of wild-type and homozygous enkephalin knockout mice. Adjacent coronal brain sections were cut from brains of +/+ and -/- mice for the determination of binding of [(3)H]1,3-dipropyl-8-cyclopentylxanthine ([(3)H]DPCPX), [(3)H]2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([(3)H]CGS21680) or [(3)H]NBTI to A(1) and A(2A) adenosine receptors and NBTI-sensitive adenosine transporters, respectively. A small but significant increase in [(3)H]DPCPX and [(3)H]NBTI binding but no significant change in [(3)H]CGS21680 binding was detected in enkephalin knockout brains. The results provide further evidence of functional interactions in the brain between opioid receptors and A(1) adenosine receptors as well as NBTI-sensitive adenosine transporters but not A(2A) receptors.

Enhanced morphine withdrawal and micro -opioid receptor G-protein coupling in A2A adenosine receptor knockout mice.

Much evidence supports the hypothesis that A2A adenosine receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might also be involved. We have evaluated morphine withdrawal signs in wild-type and A2A receptor knockout mice and shown a significant enhancement in some withdrawal signs in the knockout mice. In addition, micro -opioid and dopamine D2 receptor autoradiography, as well as micro -opioid receptor-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTPgammaS) autoradiography was carried out in brain sections of withdrawn wild-type and knockout mice. No significant changes in D2 and micro -opioid receptor binding were observed in any of the brain regions analysed. However, a significant increase in the level of micro receptor-stimulated [35S]GTPgammaS binding was observed in the nucleus accumbens of withdrawn knockout mice. These data indicate that the A2A receptor plays a role in opioid withdrawal related to functional receptor activation.

Impairment of vascular function following BCG immunisation is associated with immune responses to HSP-60 in the cholesterol-fed rabbit.

An immune response to heat shock protein (HSP)-60/65 has recently been implicated in atherogenesis. The aim of this study was to determine whether this effect may be mediated by impairment of endothelial function. Rabbits were injected with bacillus Calmette-Guerin (BCG) vaccine (n=12) or saline (n=12). A further injection of BCG or saline was administered after 2 weeks. After a further 2 weeks, animals were fed either a 0.25-1% cholesterol diet or a chow diet for 16 weeks. Blood cholesterol levels were maintained at 10-12mmol/l by altering the dietary cholesterol content. Plasma levels of anti-mycobacterial antibodies rose following BCG immunisation, but anti-HSP antibodies developed only in the BCG-immunised, cholesterol-fed rabbits. Aortic endothelium from cholesterol-fed, but not chow-fed, rabbits stained positively for HSP-60, independently of the immunisation protocol. Endothelial function was impaired in the BCG immunised, cholesterol-fed rabbits as measured by acetylcholine-mediated relaxation of isolated non-atherosclerotic carotid artery rings (P<0.05). This impairment was positively associated with the level of plasma anti-HSP-60 antibodies (P<0.01). These results suggest that BCG immunisation impairs endothelial responses, at least in part, by immune responses against mycobacterial and vascular HSP.

Quantitative autoradiography of adenosine receptors in brains of chronic naltrexone-treated mice.

1. Manipulation of micro opioid receptor expression either by chronic morphine treatment or by deletion of the gene encoding micro opioid receptors leads to changes in adenosine receptor expression. Chronic administration of the opioid receptor antagonist naltrexone leads to upregulation of micro receptor binding in the brain. 2. To investigate if there are any compensatory alterations in adenosine systems in the brains of chronic naltrexone-treated mice, we carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors in the brains of mice treated for 1 week with naltrexone (8 mg(-1) kg(-1) day(-1)), administered subcutaneously via osmotic minipump. 3. Adjacent coronal brain sections were cut from chronic saline- and naltrexone-treated mice for the determination of binding of [(3)H] D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin ([(3)H] DAMGO), [(3)H]1,3-dipropyl-8-cyclopentylxanthine ([(3)H] DPCPX) or [(3)H] 2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([(3)H] CGS21680) to micro, A(1) and A(2A) receptors, respectively. 4. A significant increase in micro and A(1) receptor binding was detected in chronic naltrexone-treated brains. The changes in micro receptors were significant in several regions, but changes in A(1) were relatively smaller but showed significant upregulation collectively. No significant change in A(2A) receptor binding was detected in chronic naltrexone-treated brains. 5. The results show that blockade of opioid receptors causes upregulation of A(1) receptors, but not A(2A) receptors, by as yet undefined mechanisms.

Changes in spinal delta and kappa opioid systems in mice deficient in the A2A receptor gene.

A large body of evidence indicates important interactions between the adenosine and opioid systems in regulating pain at both the spinal and supraspinal level. Mice lacking the A(2A) receptor gene have been developed successfully, and these animals were shown to be hypoalgesic. To investigate whether there are any compensatory alterations in opioid systems in mutant animals, we have performed quantitative autoradiographic mapping of mu, delta, kappa, and opioid receptor-like (ORL1) opioid receptors in the brains and spinal cords of wild-type and homozygous A(2A) receptor knock-out mice. In addition, mu-, delta-, and kappa-mediated antinociception using the tail immersion test was tested in wild-type and homozygous A(2A) receptor knock-out mice. A significant reduction in [3H]deltorphin-I binding to delta receptors and a significant increase in [3H]CI-977 binding to kappa receptors was detected in the spinal cords but not in the brains of the knock-out mice. Mu and ORL1 receptor expression were not altered significantly. Moreover, a significant reduction in delta-mediated antinociception and a significant increase in kappa-mediated antinociception were detected in mutant mice, whereas mu-mediated antinociception was unaffected. Comparison of basal nociceptive latencies showed a significant hypoalgesia in knock-out mice when tested at 55 degrees C but not at 52 degrees C. The results suggest a functional interaction between the spinal delta and kappa opioid and the peripheral adenosine system in the control of pain pathways.

Quantitative autoradiography of adenosine receptors and NBTI-sensitive adenosine transporters in the brains and spinal cords of mice deficient in the mu-opioid receptor gene.

There is a large body of evidence indicating important interactions between the adenosine and opioid systems in regulating pain at both the spinal and supraspinal level. Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed and the animals show complete loss of analgesic responses to morphine as well as differences in pain sensitivity. To investigate if there are any compensatory alterations in adenosine systems in mutant animals, we have carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors and nitrobenzylthioinosine (NBTI) sensitive adenosine transporters in the brains and spinal cords of wild type, heterozygous and homozygous mu-opioid receptor knockout mice. Adjacent coronal sections were cut from the brains and spinal cords of +/+, +/- and -/- mice for the determination of binding of [3H]DPCPX, [3H]CGS21680 or [3H]NBTI to A(1) and A(2A) adenosine receptors and NBTI-sensitive adenosine transporters, respectively. A small but significant reduction in [3H]DPCPX and [3H]NBTI binding was detected in mutant mice brains but not in spinal cords. No significant change in A(2A) binding was detected in mu-opioid receptor knockout brains. The results suggest there may be functional interactions between mu-receptors and A(1) adenosine receptors as well as NBTI-sensitive adenosine transporters in the brain but not in the spinal cord.