Progress in pursuit of therapeutic A2A antagonists: the adenosine A2A receptor selective antagonist KW6002: research and development toward a novel nondopaminergic therapy for Parkinson's disease.

Research and development of the adenosine A2A receptor selective antagonist KW6002 have focused on developing a novel nondopaminergic therapy for Parkinson's disease (PD). Salient pharmacologic features of KW6002 were investigated in several animal models of PD. In rodent and primate models, KW6002 provides symptomatic relief from parkinsonian motor deficits without provoking dyskinesia or exacerbating existing dyskinesias. The major target neurons of the A2A receptor antagonist were identified as GABAergic striatopallidal medium spiny neurons. A possible mechanism of A2A receptor antagonist action in PD has been proposed based on the involvement of striatal and pallidal presynaptic A2A receptors in the "dual" modulation of GABAergic synaptic transmission. Experiments with dopamine D2 receptor knockout mice showed that A2A receptors can function and anti-PD activities of A2A antagonists can occur independent of the dopaminergic system. Clinical studies of KW6002 in patients with advanced PD with L-dopa-related motor complications yielded promising results with regard to motor symptom relief without motor side effects. The development of KW6002 represents the first time that a concept gleaned from A2A biologic research has been applied successfully to "proof of concept" clinical studies. The selective A2A antagonist should provide a novel nondopaminergic approach to PD therapy.

Adenosine A(2A) receptor enhances GABA(A)-mediated IPSCs in the rat globus pallidus.

1. The actions of adenosine A(2A) receptor agonists were examined on GABAergic synaptic transmission in the globus pallidus (GP) in rat brain slices using whole-cell patch-clamp recording. GP neurones were characterized into two major groups, type I and type II, according to the degree of time-dependent hyperpolarization-activated inward rectification and the size of input resistance. 2. The A(2A) receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido- adenosine (CGS21680; 0.3-3 microM) enhanced IPSCs evoked by stimulation within the GP. The actions of CGS21680 were blocked by the A(2A) antagonists (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). 3. The CGS21680-induced increase in IPSCs was associated with a reduction in paired-pulse facilitation. CGS21680 (0.3 microM) increased the frequency of miniature IPSCs (mIPSCs) without affecting mIPSC amplitude. These observations demonstrated that the enhancement of IPSCs in the GP was attributable to presynaptic, but not postsynaptic, A(2A) receptors. 4. The results suggest that A(2A) receptors in the GP serve to inhibit GP neuronal activity, thereby disinhibiting subthalamic nucleus neurone activity. Thus, the A(2A) receptor-mediated presynaptic regulation in the GP, together with the A(2A) receptor-mediated intrastriatal presynaptic control of GABAergic neurotransmission described previously, may play a crucial role in controlling the neuronal functions of basal ganglia. This A(2A) receptor-mediated presynaptic dual control in the striatopallidal pathway could also afford the mode of action of A(2A) antagonists for ameliorating the symptoms of Parkinson's disease in an animal model.

Noradrenergic excitation and inhibition of GABAergic cell types in rat frontal cortex.

Noradrenaline (NA) from the locus coeruleus and GABA from intracortical nonpyramidal cells exert strong influences on cortical activity. To assess possible interaction between the two, the effects of noradrenergic agonists on spontaneous GABAergic IPSCs as well as on the activity of identified GABAergic cell types were investigated by in vitro whole-cell recordings from the frontal cortex of 18- to 22-d-old rats. NA (3-50 microM) and an alpha-adrenergic agonist, 6-fluoronorepinephrine (FNE; 30-50 microM), induced an increase of IPSC frequency in pyramidal cells, but a beta-adrenergic agonist did not. This increase was reduced by tetrodotoxin, bicuculline, and alpha-adrenergic antagonists, suggesting that GABAergic cells are excited via alpha-adrenoceptors. Fast-spiking or late-spiking cells were depolarized by application of NA or FNE, but none demonstrated spike firings. The former morphologically included common multipolar cells with extended axonal arborizations as well as chandelier cells, and the latter neurogliaform cells. Most somatostatin-immunoreactive regular or burst-spiking cells, including Martinotti cells and wide arbor cells, were depolarized and accompanied by spike firing. In a few cases this was preceded by hyperpolarization. Cholecystokinin-immunoreactive regular or burst-spiking nonpyramidal cells, including large basket cells, were affected heterogeneously: depolarization, hyperpolarization followed by depolarization, or hyperpolarization resulted. The findings suggest that, similar to the effects of acetylcholine, the excitability of cortical GABAergic cell types is differentially regulated by NA and that NA actions are similar to cholinergic ones in some GABAergic cell types but not in others.

The role of adenosine A2a receptors in regulating GABAergic synaptic transmission in striatal medium spiny neurons.

We demonstrated an adenosine A2a receptor-mediated disinhibition of medium spiny projection neurons using intracellular recording and the whole-cell patch-clamp recording applied to these cells, visually identified in thin rat striatal slices. The A2a receptor agonist 2-[p-(2-carboxyethyl) phenylethylamino]-5'-N- ethylcarboxamido adenosine (CGS-21680; 0.3-10 microM) suppressed GABAergic synaptic transmission onto these cells in a manner inhibited by the A2a receptor-selective antagonist (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (0.1-1.0 microM). The A1 receptor antagonists had no effect on the CGS-21680-induced suppression. Analysis of spontaneous miniature inhibitory synaptic currents indicated that suppression of intrastriatal GABAergic synaptic transmission was attributable to presynaptic, but not postsynaptic, A2a receptors. Therefore, the A2a receptor may regulate striatal output activity by relieving GABA-mediated inhibition of the medium spiny projection neurons, which explains the ability of purinergic agents to affect motor control.

Calcium channel blockers improve hypoxia/hypoglycemia-induced impairment of rat hippocampal 2-deoxyglucose uptake in vitro after ethanol withdrawal.

The aim of the present study was to determine whether calcium channel antagonists attenuated hypoxia/hypoglycemia- or glutamate-induced reduction in 2-deoxyglucose (2-DG) uptake of hippocampal slices obtained from ethanol withdrawal rats. Ethanol withdrawal significantly potentiated the hypoxia/hypoglycemia- and glutamate-induced reductions in 2-DG uptake of hippocampal slices. Both nifedipine and flunarizine exhibited attenuating effects on ethanol withdrawal-induced potentiation of impairment of 2-DG uptake caused by hypoxia/hypoglycemia or glutamate. Hypoxia/hypoglycemia-induced deficit of 2-DG uptake was prevented by ethanol, but chronic consumption of ethanol resulted in the development of tolerance to neuroprotective effect. These findings suggest that the increased sensitivity of neurons to ischemic damage by ischemia may involve in the increased activity of calcium channels in the hippocampus.

Binding of [3H]KF17837S, a selective adenosine A2 receptor antagonist, to rat brain membranes.

The potential of 8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-[3H] methylxanthine ([3H]Kf17837S) as a highly selective antagonist radioligand for the adenosine A2A receptor was examined and compared with the properties of the adenosine A2A receptor agonist radioligand 2-[p-(2-[3H]carboxyethyl)phenethylamino]-5'-N-ethyl- carboxamidoadenosine ([3H]CGS21680). [3H]KF17837S specific binding to rat striatal membranes was saturable and reversible. Saturation studies showed that the binding of [3H]KF17837S occurred at a single site, with high affinity (Kd, 7.1 +/- 0.91 nM) and limited capacity (Bmax, 1.3 +/- 0.23 pmol/mg of protein). Adenosine receptor antagonist ligands competed with the binding of 1 nM [3H]KF17837S with the following order of activity: CGS15943 > KF17837S > N-[2-(dimethylamino)ethyl]-N-methyl- 4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)benzenesul fonamide > or = xanthine amine congener > 8-cyclopentyl-1,3-dipropylxanthine > 8-(noradamantan-3-yl)-1,3-dipropylxanthine > caffeine. Adenosine receptor agonists inhibited [3H] KF17837S binding in the following order: 5'-N-ethylcarboxamidoadenosine > or = CGS21680 > 2-phenylaminoadenosine > or = (R)- N6-phenylisopropyladenosine > N6-cyclopentyladenosine > (S)- N6-phenylisopropyladenosine. The Ki values of the antagonists for [3H]KF17837S binding and the rank order of potency were similar to those for [3H]CGS21680 binding. The affinities of the agonists were lower with [3H]KF17837S binding than with [3H] CGS21680 binding. However, a strong positive correlation (r = 0.98) was observed between the pharmacological profiles for these two radioligand assays. The inhibition curve for CGS21680 was best fitted to a two-component binding model and addition of GTP shifted the inhibition curve to the right, suggesting that [3H]KF17837S labeled two agonist coupling states. Other pharmacological agents had negligible affinities for the [3H]KF17837S binding site. Autoradiographic study of [3H]KF17837S binding using rat brain sections revealed that the binding site was highly enriched in the striatal region. These data indicate that [3H] KF17837S labels the adenosine A2A receptor in rat brain.

Calcium-dependent hyperexcitability of hippocampal CA1 pyramidal cells in an in vitro slice after ethanol withdrawal of the rat.

The physiological profiles contributing to hyperexcitability of hippocampal CA1 neurons following ethanol withdrawal (EW) were examined in an in vitro slice preparation obtained from EW rats. Sixty-two percent of CA1 neurons in slices from EW rats exhibited intrinsic burst property which was rarely observed in those from control animals. The mean duration of plateau component of calcium (Ca) spikes was significantly increased after EW. The burst response evoked by either synaptic or direct stimulation in hippocampal CA1 neurons from EW rats was markedly depressed by high Mg solution but not by flunarizine. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA receptor antagonist, markedly depressed the synaptically evoked burst response, while [(+/-)-2-carboxypiperazine-4-yl-]-propyl-1-phosphonic acid, a selective N-methyl-D-aspartate (NMDA) receptor antagonist, slightly delayed the onset of the response. The results indicate that an increase in the number of bursting hippocampal CA1 neurons associated with an augmentation of the plateau component of Ca spike contributes to the genesis of hyperexcitability in EW rats. Furthermore, non-NMDA receptor-mediated EPSP is mainly responsible for a synaptic induction of the burst response. These results are consistent with the involvement of high-threshold Ca channels in EW hyperexcitability.

NMDA receptor-dependent formation of long-term potentiation in the rat medial amygdala neuron in an in vitro slice preparation.

Population spike was recorded from the medial and central nucleus of the amygdala (AMG) in an in vitro slice preparation following stimulation of the stria terminalis (ST). The population spike was completely suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), a non-NMDA receptor antagonist, but not affected by 3-[(+)-2-carboxypiperazine-4-yl-]-propyl-1-phosphonic acid (CPP) (20 microM), a competitive NMDA receptor antagonist. Tetanic stimulation to the ST induced long-term potentiation (LTP) in both the medial and central AMG. Tetanic stimulation of 20 s in duration and 50 Hz in frequency most effectively potentiated the population spike. All the NMDA antagonists tested, such as CPP (20 microM), (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5,10- imine-hydrogen maleate (30 microM) and 7-chlorokynurenic acid (30 microM), significantly depressed the formation of LTP in the medial AMG. 2-Amino-3-phosphonopropionate (1 mM), a metabotropic glutamate receptor antagonist, and intracerebral ventricular injection of ilet activating protein had no significant effects on the formation of LTP. Furthermore, the LTP formation of EPSP was completely blocked in BAPTA [bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid]-loaded medial AMG neurons. These results indicate that Ca2+ influx mainly through NMDA receptors is crucial for the induction of LTP in the ST-medial AMG neuron synapses.

Inhibitory effect of NMDA on dopaminergic transmission in a slice preparation of rat globus pallidus.

The effect of N-methyl-D-aspartate (NMDA) on KCl-evoked endogenous dopamine (DA) release from slices of rat globus pallidus (GP) was examined. NMDA inhibited the KCl-evoked DA release in a dose-dependent manner. This inhibition was blocked by CPP, an NMDA antagonist. Tetrodotoxin partially antagonized the effect of NMDA. The NMDA-induced inhibition was also partially antagonized by bicuculline methiodide and was mimicked by muscimol. These results strongly suggest that 1) an activation of NMDA receptors exerts an inhibitory effect on dopaminergic transmission in GP and 2) GABAergic transmission is involved in the effect of NMDA.

Identification of an alpha 2-adrenoceptor in human coronary arteries by radioligand binding assay.

A single high affinity binding site for an alpha 2-adrenoceptor in human coronary arteries was identified by radioligand binding assay. Human coronary arteries were obtained at autopsy within 6 hours of death. A crude membrane solution was incubated with (3H)-rauwolscine at 25 degrees C for 30 min. The binding of (3H)-rauwolscine was rapidly saturable and reversible. Kd was 1.2 +/- 0.2 (SE) nM and Bmax 22 +/- 3 fmol/mg protein. This is the first study which has shown the presence of an alpha 2-adrenoceptor in human coronary arteries using a radioligand binding assay method.

Identification of an atrial natriuretic peptide specific receptor in human kidney.

A specific receptor for human atrial natriuretic peptide (h-ANP) was identified in the human kidney using the radioligand binding assay. Samples were prepared from non-malignant renal tissues obtained at nephrectomy of patients with renal carcinoma. Binding studies using [125I]hANP were performed at 0 degree C for 20 minutes and terminated by a rapid filtration technique. Scatchard plot analysis revealed [125I]hANP bound to a single class of binding site (Kd = 0.4 +/- 0.2 nM) with a density of 16 +/- 4 fmol/mg protein in the renal cortex (n = 7). The binding was rapid and maximal binding was obtained within 20 minutes after the start of incubation. Radioligand displacement was observed in a dose dependent fashion when cold hANP was entered into the reaction mixture. However, unrelated agents, such as angiotensin II or 1-epinephrine, did not affect the binding. This is the first time characterization of the hANP receptor in the human kidney has been conducted using a Scatchard plot analysis.