Angela Monopoli

Persistent Behavioral Sensitization to Chronic l-DOPA Requires A2A Adenosine Receptors

To investigate the role of A2A adenosine receptors in adaptive responses to chronic intermittent dopamine receptor stimulation, we compared the behavioral sensitization elicited by repeated l-DOPA treatment in hemiparkinsonian wild-type (WT) and A2A adenosine receptor knock-out (A2AKO) mice. Although the unilateral nigrostriatal lesion produced by intrastriatal injection of 6-hydroxydopamine was indistinguishable between WT and A2A KO mice, they developed strikingly different patterns of behavioral sensitization after daily treatment with low doses of l-DOPA for 3 weeks. WT mice initially displayed modest contralateral rotational responses and then developed progressively greater responses that reached a maximum within 1 week and persisted for the duration of the treatment. In contrast, any rotational behavioral sensitization in A2A KO mice was transient and completely reversed within 2 weeks. Similarly, the time to reach the peak rotation was progressively shortened in WT mice but remained unchanged in A2A KO mice. Furthermore, dailyl-DOPA treatment produced gradually sensitized grooming in WT mice but failed to induce any sensitized grooming in A2AKO mice. Finally, repeated l-DOPA treatment reversed the 6-OHDA-induced reduction of striatal dynorphin mRNA in WT but not A2A KO mice, raising the possibility that the A2A receptor may contribute to l-DOPA-induced behavioral sensitization by facilitating adaptations within the dynorphin-expressing striatonigral pathway. Together these results demonstrate that the A2A receptor plays a critical role in the development and particularly the persistence of behavioral sensitization to repeated l-DOPA treatment. Furthermore, they raise the possibility that the maladaptive dyskinetic responses to chronic l-DOPA treatment in Parkinsons disease may be attenuated by A2A receptor inactivation.

The selective A2A receptor antagonist SCH 58261 reduces striatal transmitter outflow, turning behavior and ischemic brain damage induced by permanent focal ischemia in the rat.

Adenosine A(2A) receptor antagonists have been proved protective in different ischemia models. In this study we verified if the protective effect of the selective A(2A) antagonist, SCH 58261, could be attributed to the reduction of the excitatory amino acid outflow induced by cerebral focal ischemia. A vertical microdialysis probe was inserted into the striatum of male Wistar rats and, after 24 h, permanent right intraluminal middle cerebral artery occlusion (MCAo) was induced. Soon after waking, rats showed a definite contralateral turning behavior, which persisted up to 7 h after MCAo. During 4 h after MCAo, glutamate, aspartate, GABA, adenosine and taurine outflow increased. SCH 58261 (0.01 mg/kg, i.p.), administered 5 min after MCAo, suppressed turning behavior and significantly reduced the outflow of glutamate, aspartate, GABA and adenosine. At 24 h after MCAo, the rats showed severe sensorimotor deficit and damage in both the striatum and cortex. SCH 58261 significantly reduced cortical damage but did not protect against the sensorimotor deficit. The protective effect of SCH 58261 against turning behavior and increased outflow of excitatory amino acids in the first hours after MCAo suggests the potential utility of selective adenosine A(2A) antagonists when administered in the first hours after ischemia. Furthermore, this study, for the first time, proposes that turning behavior after permanent intraluminal MCAo, be used as a precocious index of neurological deficit and neuronal damage.

Production of leukotrienes in a model of focal cerebral ischaemia in the rat

The aim of this work was to evaluate the role of leukotrienes in brain damage in vivo in a model of focal cerebral ischaemia in the rat, obtained by permanent occlusion of middle cerebral artery. A significant (P<0.01) elevation of LTC4, LTD4 and LTE4 (cysteinyl‐leukotrienes) levels occurred 4 h after ischaemia induction in the ipsilateral cortices of ischaemic compared to sham‐operated animals (3998±475 and 897±170 fmol g−1 tissue, respectively, P<0.01). The NMDA receptor antagonist MK‐801 and the adenosine A2A receptor antagonist SCH 58261 were administered in vivo at doses known to reduce infarct size and compared with the leukotriene biosynthesis inhibitor MK‐886. MK‐886 (0.3 and 2 mg kg−1 i.v.) and MK‐801 (3 mg kg−1 i.p.) decreased cysteinyl‐leukotriene levels (−78%, P<0.05; −100%, P<0.01; −92%, P<0.01, respectively) 4 h after permanent occlusion of the middle cerebral artery, whereas SCH 58261 (0.01 mg kg−1 i.v.) had no significant effects. MK‐886 (2 mg kg−1 i.v.) was also able to significantly reduce the cortical infarct size by 30% (P<0.05). We conclude that cysteinyl‐leukotriene formation is associated with NMDA receptor activation, and that it represents a neurotoxic event, the inhibition of which is able to reduce brain infarct area in a focal ischaemic event.

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices.

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.

Effects of selective A1 and A2 adenosine receptor agonists on cardiovascular tissues

SummaryWe investigated the negative chronotropic and vasodilating properties of new selective A1 and A2 adenosine agonists such as 2-chloro-N6-cyclopentyladenosine (CCPA) and 2-hexynyl-5′-N-ethyl-carboxamidoadenosine (2-hexynyl-NECA) as compared with reference adenosine analogues. The potency of these compounds on heart rate was assessed in the rat atrial preparation and their activity on the vascular tone was determined in both rat aorta and bovine coronary artery. CCPA was found to be the most potent At agonist of those currently available in producing negative chronotropic effects (EC50 = 8.2 nM). The A1 antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) blocked CCPA activity in a dose-dependent manner. There was also a significant correlation between its biological effect and the affinity for A1 receptors as measured in the rat brain by [3H]-N6-cyclohexyladenosine (3[H]-CHA) binding. The A2 selective agonist 2-hexynyl-NECA showed vasodilating properties comparable with those observed with the reference compounds, CGS 21680 and NECA. EC50 values were 596 and 569 nM in rat aorta and bovine coronary artery, respectively. Moreover, the rank order of potency was similar in the two vascular districts examined, suggesting that the rat aorta is a useful model for studying the effects of adenosine derivatives on vascular tone. In addition, the potency of the compounds in inducing vasodilation was found to be correlated with their affinity for A2 receptors as measured in the rat striatum by 3[H]-CGS 21680 binding.These data further support that A1 receptors are involved in depressing cardiac activity and A2 receptors in inducing vasorelaxation.

The dopamine D1 receptor is involved in the regulation of REM sleep in the rat

The dopamine D1 receptor agonist, SKF 38393, and the D1 antagonist, SCH 23390, were studied for their effects on sleep in the rat. Over 6 h, SKF 38393 (0.1-10 mg/kg s.c.) dose dependently reduced the amount of rapid eye movement (REM) sleep and enhanced the duration of wakefulness. The drug affected REM at low doses (ED50 = 0.4 mg/kg) at which wakefulness was unchanged and the characteristic grooming behavior was not apparent. REM changes were characterized by a decrease in the number of episodes with no alteration of latency to the first episode. Over a very low dose range (0.003-0.3 mg/kg s.c.), SCH 23390 enhanced the amount of REM by increasing both number and average duration of episodes. There was also a moderate increase of non-REM sleep but the percent change was less marked than that occurring for REM. Given at 0.003 mg/kg, SCH 23390 prevented the REM changes induced by SKF 38393 (0.3-3 mg/kg). It is suggested that D1 receptors are involved in the regulation of the REM sleep process.

Adenosine receptors in neurological disorders

Adenosine is present in the extacellular space of organs and tissues in the mammalian organism where it modulates a variety of physiological processes through specific cell membrane receptors which have been identified as members of the G-protein coupled receptors (GPCRs). Currently, four receptors have been cloned and characterised, namely A1, A2A, A2B, A3 adenosine receptors. The different distribution of receptor populations in organs and tissues together with their importance in normal and altered conditions make each individual adenosine receptor an attractive target for the discovery of new pharmacological agents. Major efforts have involved the A1 and A2A receptors with the central nervous (and cardiovascular) systems being the medical areas of highest interest. Thus, a variety of selective receptor agonists and antagonists have been discovered and some are currently under development for the treatment of neurological disorders. Treatment of pain, neurodegenerative diseases, such as Parkinson’s (PD) and Huntington’s disease (HD), cognitive impairment and epilepsy are all potential therapy areas of future drug development. This review describes the scientific background information currently available together with the rationale underlying the innovative strategies associated with the modulation of each individual adenosine receptor.

Selective adenosine A2A receptor antagonists.

In the early 1990s it became clear that the A2A adenosine receptor had characteristics that made it distinct from the other A1, A2B and A3 adenosine receptors. Great progress has been made with the discovery of selective A2A receptor antagonists. A variety of synthetic substitutions on the xanthine moiety led the chemists of Kyowa-Hakko to discover that introduction of the styryl group in the 8 position of xanthines was critical in achieving compounds endowed with selective A2A receptor antagonistic properties. One compound, KW 6002, (E)1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine, is currently being developed for treatment of Parkinsons disease. A number of non-xanthine heterocycles have also been synthesized starting from the non-selective adenosine antagonist CGS 15943, a triazoloquinazoline. Thus, replacement of the phenyl ring of CGS 15943 with a heterocyclic ring such as pyrazole or imidazole, led to a series of interesting compounds whose prototype, SCH 58261, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, has become a reference A2A receptor antagonist. Modification of N7 substituents has progressed to optimize A2A receptor selectivity and pharmacokinetic characteristics. A related class of compounds having a bicyclic instead of the tricyclic ring structure is also of interest. The prototype of these triazintriazolo derivatives, ZM 241385, is a potent A2A receptor antagonist; however, it also shows interactions with A2B receptors. The relevance of the A2A receptors in specific disease states, especially in the central nervous system, makes this class of adenosine receptor blockers of interest for treatment of neurodegenerative disorders such as Parkinsons disease.

Rapamycin, but not FK506 and GPI-1046, increases neurite outgrowth in PC12 cells by inhibiting cell cycle progression.

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.

Nonsteroidal anti-inflammatory drugs increase tumor necrosis factor production in the periphery but not in the central nervous system in mice and rats

Abstract: Nonsteroidal anti‐inflammatory drugs (NSAIDs), which inhibit prostaglandin (PG) synthesis, augment production of tumor necrosis factor (TNF) in most experimental models. We investigated the effect of two NSAIDs, indomethacin and ibuprofen, on the production of TNF in the CNS induced by intracerebroventricular injection of lipopolysaccharide (LPS). Indomethacin and ibuprofen, administered intraperitoneally, augmented (three‐ to ninefold) the levels of TNF in serum and peripheral organs of mice injected intraperitoneally with LPS and in rats with adjuvant arthritis (up to a sevenfold increase). However, NSAIDs (intraperitoneally or intracerebroventricularly) did not increase brain TNF production induced by intravenous LPS. In fact, indomethacin decreased (1.4–1.8‐fold) TNF levels in the spinal cord of rats with experimental autoimmune encephalomyelitis and in the cortex of rats with focal cerebral ischemia. Systemic administration of iloprost inhibited serum TNF levels after intraperitoneal LPS, whereas intracerebroventricular injection of iloprost or PGE2 did not inhibit brain TNF induced by intracerebroventricular LPS. Both peripheral and central TNF productions were inhibited by cyclic AMP level‐elevating agents or dexamethasone. Thus, a PG‐driven negative feedback controls TNF production in the periphery but not in the CNS.