Masako Kurokawa

Neuroprotection by adenosine A2A receptor blockade in experimental models of Parkinson's disease

Adenosine A2A receptors are abundant in the caudate‐putamen and involved in the motor control in several species. In MPTP‐treated monkeys, A2A receptor‐blockade with an antagonist alleviates parkinsonian symptoms without provoking dyskinesia, suggesting this receptor may offer a new target for the antisymptomatic therapy of Parkinsons disease. In the present study, a significant neuroprotective effect of A2A receptor antagonists is shown in experimental models of Parkinsons disease. Oral administration of A2A receptor antagonists protected against the loss of nigral dopaminergic neuronal cells induced by 6‐hydroxydopamine in rats. A2A antagonists also prevented the functional loss of dopaminergic nerve terminals in the striatum and the ensuing gliosis caused by MPTP in mice. The neuroprotective property of A2A receptor antagonists may be exerted by altering the packaging of these neurotoxins into vesicles, thus reducing their effective intracellular concentration. We therefore conclude that the adenosine A2A receptor may provide a novel target for the long‐term medication of Parkinsons disease, because blockade of this receptor exerts both acutely antisymptomatic and chronically neuroprotective activities.

Inhibition by KF17837 of adenosine A2A receptor-mediated modulation of striatal GABA and ACh release.

1 The effect of the A2A adenosine receptor agonist, 2‐p‐(‐2‐carboxyethyl)phenethyl‐amino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) on the potassium evoked release of [3H]‐γ‐aminobutyric acid ([3H]‐GABA) from nerve terminals derived from the caudate‐putamen and the globus pallidus of the rat was compared. In both preparations CGS 21680 (1 nm) inhibited the [3H]‐GABA release evoked by 15 mm KCl but had no effect on that evoked by 30 mm KCl. 2 The ability of CGS 21680 (1 nm) to inhibit the release of [3H]‐GABA from striatal nerve terminals was unaffected by the presence of the GABA receptor antagonists, bicuculline (10 μm), phaclofen (100 μm) and 2‐hydroxysaclofen (100 μm). Similarly the opioid receptor antagonist, naloxone (10 μm), the adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 40 nm), and the cholinoceptor antagonists, mecamylamine (10 μm) and atropine (100 nm) had no effect on this inhibition. 3 The ability of CGS 21680 (0.1 nm) to stimulate the release of [3H]‐acetylcholine ([3H]‐ACh) from striatal nerve terminals was unaffected by the presence of bicuculline (10 μm), 2‐hydroxysaclofen (100 μm), phaclofen (100 μm), naloxone (10 μm) and DPCPX (4 nM). 4 The novel A2A receptor antagonist, (E)‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropyl‐7‐methylxanthine (KF 17837), blocked the CGS 21680 (1 nm)‐induced inhibition of [3H]‐GABA efflux with an EC50 of approximately 30 nm and also antagonized the CGS 21680 (0.1 nm)‐induced stimulation of [3H]‐ACh release with an EC50 of approximately 0.3 nm. 5 It is concluded that the A2A adenosine receptor is present on both GABAergic and cholinergic nerve terminals of the rat striatum and that in both the caudate‐putamen and the globus pallidus this receptor inhibits [3H]‐GABA release. No evidence was seen for a difference in the ligand binding sites of this receptor in the two groups of nerve terminals.

Adenosine A2a Receptor‐Mediated Modulation of Striatal Acetylcholine Release In Vivo

Abstract: To determine the functions of striatal adenosine A2a receptors in vivo, the effects of a selective agonist, 2‐[4‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine hydrochloride (CGS 21680), and an antagonist, (E)‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropyl‐7‐methylxanthine (KF17837), on acetylcholine release were investigated in the striatum of awake freely moving rats using microdialysis. Intracerebroventricular injection of CGS 21680 (10 µg) increased acetylcholine release in striatum and KF17837 (30 mg/kg p.o.) antagonized the CGS 21680‐induced acetylcholine elevation. To investigate the contribution of dopaminergic and GABAergic neurons on A2a receptor‐mediated acetylcholine release, the effects of CGS 21680 were studied by using dopamine‐depleted rats in the presence or absence of GABA antagonists. In the dopamine‐depleted striatum, the intrastriatal application of CGS 21680 (0.3–30 µM) increased extracellular acetylcholine, which was significantly greater than that in normal striatum. The CGS 21680‐induced elevation of acetylcholine release was still observed in the presence of GABA antagonists bicuculline (30 µM) and 2‐hydroxysaclofen (100 µM) and was similar in both normal and dopamine‐depleted striatum. These results suggest that A2a agonist stimulates acetylcholine release in vivo, and this effect of A2a agonist is modulated by dopaminergic and GABAergic neurotransmission.

Dual signalling by the adenosine A2a receptor involves activation of both N- and P-type calcium channels by different G proteins and protein kinases in the same striatal nerve terminals

Abstract: Many Gs‐linked receptors have been reported to use multiple signalling pathways in transfected cells but few in their normal cell environment. We show that the adenosine A2a receptor uses two signalling pathways to increase the release of acetylcholine from striatal nerve terminals. One pathway involves activation of Gs, adenylyl cyclase, protein kinase A, and P‐type calcium channels; the other is mediated by a cholera toxin‐insensitive G protein, protein kinase C, and N‐type calcium channels. The effects of these two pathways are not additive, the second pathway being inhibited by the first; but they are equally sensitive to the A2a receptor antagonist KF17837. This demonstrates that the A2a receptor activates two signalling systems in striatal cholinergic neurons.

In vivo regulation of acetylcholine release via adenosine A1 receptor in rat cerebral cortex

The roles of the endogenous adenosine on acetylcholine release via adenosine A1 receptor were investigated in rat cerebral cortex using brain microdialysis. Oral administration of KF15372 (8-dicyclopropylmethyl-1,3-dipropylxanthine), a novel selective adenosine A1 receptor antagonist, at doses of 1.25, 5, and 20 mg/kg, significantly increased the extracellular levels of acetylcholine in rat cerebral cortex. Selective A1 agonist N6-((R)-phenylisopropyl) adenosine (R-PIA) did not affect the extracellular level of acetylcholine by both oral (1.25 mg/kg) and intracortical administrations (0.3 microM) via dialysis probe. These results suggest that the extracellular level of acetylcholine is under tonic inhibitory control of endogenous adenosine via the A1 receptor.

Regulation of Neurotransmitter Release in Basal Ganglia by Adenosine Receptor Agonists and Antagonists in Vitro and in Vivo

Publisher Summary This chapter discusses that the control of neurotransmitter release is a basic and fundamental property of neuromodulators such as adenosine and can be used to localize receptors and at the same time, increase the overall understanding of adenosine action. In combination with other techniques, which include electrophysiology, receptor autoradiography and hybridization, the fundamental mechanisms by which adenosine and its receptors control the motor behavior can be elucidated. Adenosine receptor ligands have profound and diversified effects on transmitter release in the central nervous system (CNS), which notifies additional roles for this nucleoside in the control of neuronal activity. This chapter also reiterates that adenosine A1 and A2A receptors have innumerable effects on neurotransmitter release in the striatum which are consistent with the control of the striatopallidal pathway in particular. Proper analysis of the relative importance of the A2A receptors—expressed in rectifying the imbalance of striatal activity observed in Parkinsons disease—will greatly enhance the understanding of this area of the brain.

249 Adenosine A2A receptor modulates GABA release in the globus pallidus of 6-OHDA-lesioned rats: A microdialysis study

Shun Hamada’, Kouji Senzaki’ , Kayoko Hamaguchi-Hamada ‘, Katsuhiko Tabuchi2, Hideko Yamamoto3, Toshifumi Yamamoto4, Hideyuki Okano2, Nobuo Okado’ Two polyclonal antibodies were raised against the N-terminus peptide (NH2-IPNSLMQLGDGPRLYHNDFN) of rat 5-HT2A receptor in chickens (5-HT2A-N) and a glutathione S-transferase fusion protein that contained the 65 amino acids of the carboxy-terminal of mouse 5-HT2A receptor protein in rabbits (5-HT2A-C). 5HT2A-N and -C antibodies reacted strongly with a single band of 77-78 kDa in postsynaptic density proteins. The highest levels of immunoreactivity were observed in the olfactory bulb, neocortex, claustrum, piriform cortex, pontine nuclei, red nucleus and cranial motor nuclei. In the olfactory bulb, mitral and tufted cells were intensely labeled. Although many immunoreactive neurons were found in layers II-VI in the neocortex, pyramidal neurons in layer V were most intensely labeled. In layer IV, strong neuropil labeling was observed. By immunoelectron microscopy, 5-HT2A receptor immunoreaction was localized just beneath the postsynaptic membrane thickening of asymmetric synapses.