Hiroshi Kase

K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases.

K-252 compounds (K-252a and b isolated from Nocardiopsis sp. (1) and their synthetic derivatives) were found to inhibit cyclic nucleotide-dependent protein kinases and protein kinase C to various extents. The inhibitions were of the competitive type with respect to ATP. K-252a was a non-selective inhibitor for these three protein kinases with Ki values 18-25 nM. K-252b showed a comparable potency for protein kinase C (Ki, 20nM), whereas inhibitory potencies for cyclic nucleotide-dependent protein kinases were reduced. KT5720 and KT5822 selectively inhibited cAMP-dependent (Ki, 60nM) and cGMP-dependent (Ki, 2.4nM) protein kinases, respectively.

Adenosine A2A receptor antagonists as new agents for the treatment of Parkinson's disease

There is now good reason to believe that blockade of the adenosine A2A receptor could be of value in the treatment of Parkinsons disease. Peter J. Richardson, Hiroshi Kase and Peter G. Jenner review the actions of this receptor in the striatum, emphasizing its ability to modulate the neuronal activity of striatal GABA-releasing output neurones, and showing that recently developed A2A receptor antagonists are capable of reducing the disabling effects of nigral cell degeneration in primates. They conclude that such antagonists may be useful as novel therapeutic agents for the treatment of Parkinsons disease.

Combined Use of the Adenosine A2A Antagonist KW-6002 with l-DOPA or with Selective D1 or D2 Dopamine Agonists Increases Antiparkinsonian Activity but Not Dyskinesia in MPTP-Treated Monkeys

The novel selective adenosine A(2A) receptor antagonist KW-6002 improves motor disability in MPTP-treated parkinsonian marmosets without provoking dyskinesia. In this study we have investigated whether KW-6002 in combination with l-DOPA or selective D1 or D2 dopamine receptor agonists enhances antiparkinsonian activity in MPTP-treated common marmosets. Combination of KW-6002 with the selective dopamine D2 receptor agonist quinpirole or the D1 receptor agonist SKF80723 produced an additive improvement in motor disability. Coadministration of KW-6002 with a low dose of L-DOPA also produced an additive improvement in motor disability, and increased locomotor activity. The ability of KW-6002 to enhance antiparkinsonian activity was more marked with L-DOPA and quinpirole than with the D1 agonist. However, despite producing an enhanced antiparkinsonian response KW-6002 did not exacerbate L-DOPA-induced dyskinesia in MPTP-treated common marmosets previously primed to exhibit dyskinesia by prior exposure to L-DOPA. Selective adenosine A(2A) receptor antagonists, such as KW-6002, may be one means of reducing the dosage of L-DOPA used in treating Parkinsons disease and are potentially a novel approach to treating the illness both as monotherapy and in combination with dopaminergic drugs.

Microbial polysaccharide, HS-142-1, competitively and selectively inhibits ANP binding to its guanylyl cyclase-containing receptor

During the search for ANP receptor ligands of microbial origin, we isolated a novel polysaccharide, HS-142-1, from culture broth of Aureobasidium sp. HS-142-1 inhibited [125I]-rANP binding to ANP receptor in rabbit kidney cortex membranes with an IC50 of 0.3 mu g/ml, but gave no effects on specific binding of [125I]-Endothelin nor [125I]-Angiotensin II to their respective receptors in bovine lung membranes. HS-142-1 competitively and selectively inhibited ANP binding to its guanylyl cyclase-containing receptor purified from solubilized bovine adrenocortical membranes and blocked cGMP production elicited by ANP. HS-142-1 is the first non-peptide antagonist selective for ANP functional receptor and will be a powerful tool to elucidate the physiological functions of ANP.

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.

Adenosine A2A antagonists with potent anti-cataleptic activity

Abstract Structure-activity relationship of 8-styrylxanthines for in vivo adenosine A2A antagonism were explored. Diethyl substitution both at the 1- and 3-position was found to dramatically potentiate the anti-cataleptic activity.

Adenosine A2A receptor enhances GABAA‐mediated IPSCs in the rat globus pallidus

1 The actions of adenosine A2A 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 A2A receptor agonist 2‐[p‐(2‐carboxyethyl)phenethylamino]‐5′‐N‐ethylcarboxamido‐ adenosine (CGS21680; 0.3‐3 μm) enhanced IPSCs evoked by stimulation within the GP. The actions of CGS21680 were blocked by the A2A 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 μm) 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, A2A receptors. 4 The results suggest that A2A receptors in the GP serve to inhibit GP neuronal activity, thereby disinhibiting subthalamic nucleus neurone activity. Thus, the A2A receptor‐mediated presynaptic regulation in the GP, together with the A2A receptor‐mediated intrastriatal presynaptic control of GABAergic neurotransmission described previously, may play a crucial role in controlling the neuronal functions of basal ganglia. This A2A receptor‐mediated presynaptic dual control in the striatopallidal pathway could also afford the mode of action of A2A antagonists for ameliorating the symptoms of Parkinsons disease in an animal model.

Presynaptic adenosine A2A receptors enhance GABAergic synaptic transmission via a cyclic AMP dependent mechanism in the rat globus pallidus

We previously reported a presynaptic facilitatory action of A2A receptors on GABAergic synaptic transmission in the rat globus pallidus (GP). In the present study we identify the intracellular signalling mechanisms responsible for this facilitatory action of A2A receptors, using biochemical and patch‐clamp methods in rat GP slices. The adenosine A2A receptor selective agonist CGS21680 (1, 10 μM) and the adenylyl cyclase activator forskolin (1, 10 μM) both significantly increased cyclic AMP accumulation in GP slices. The CGS21680 (1 μM)‐mediated increase in cyclic AMP was inhibited by the A2A receptor selective antagonist KF17837 (10 μM). In an analysis of miniature inhibitory postsynaptic currents (mIPSCs), forskolin (10 μM) increased the mIPSC frequency without affecting their amplitude distribution, a result similar to that previously reported with CGS21680. The adenylyl cyclase inhibitor 9‐(tetrahydro‐2‐furanyl)‐9H‐purin‐6‐amine (SQ22,536, 300 μM) abolished the CGS21680‐induced enhancement in the frequency of mIPSCs. H‐89 (10 μM), a selective inhibitor for cyclic AMP‐dependent protein kinase (PKA), blocked the CGS21680‐induced enhancement of the mIPSC frequency. The calcium channel blocker CdCl2 (100 μM) did not prevent CGS21680 from increasing the frequency of mIPSCs. These results indicate that A2A receptor‐mediated potentiation of mIPSCs in the GP involves the sequential activation of the A2A receptor, adenylyl cyclase, and then PKA, and that this facilitatory modulation could occur independently of presynaptic Ca2+ influx.

K252a, a new inhibitor of protein kinase C, concomitantly inhibits 40K protein phosphorylation and seroton in secretion in a phorbol ester-stimulated platelets

K252a isolated from microbial origin was found to potently inhibit protein kinase C in vitro (1). This agent inhibits phosphorylation of 40,000 dalton protein (40K protein) induced by 12-0-tetradecanoylphorbol-13-acetate(TPA) in intact rabbit platelets. This indicates that K252a exhibits the inhibition of protein kinase C in intact cells. The serotonin secretion induced by TPA was inhibited by K252a at nearly equal concentrations required to inhibit the phosphorylation of 40K protein. This provides the evidence to support the cause-effect relationship between the protein phosphorylation and the secretion in TPA-stimulated platelets.

KW‐3902, a selective high affinity antagonist for adenosine A1 receptors

1 We demonstrate that 8‐(noradamantan‐3‐yl)‐1,3‐dipropylxanthine (KW‐3902) is a very potent and selective adenosine A1 receptor antagonist, assessed by radioligand binding and cyclic AMP response in cells. 2 In rat forebrain adenosine A1 receptors labelled with [3H]‐cyclohexyladenosine (CHA), KW‐3902 had a Ki value of 0.19 nM, whereas it showed a Ki value of 170 nM in rat striatal A2A receptors labelled with [3H]‐2‐[p‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine (CGS21680), indicating 890 fold A1 receptor selectivity versus the A2A receptor. KW‐3902 at 10 μm showed no effect on recombinant rat A3 receptors expressed on CHO cells. 3 Saturation studies with [3H]‐KW‐3902 revealed that it bound with high affinity (Kd = 77 pM) and limited capacity (Bmax = 470 fmol mg−1 of protein) to a single class of recognition sites. A high positive correlation was observed between the pharmacological profile of adenosine ligands inhibiting the binding of [3H]‐KW‐3902 and that of [3H]‐CHA. 4 KW‐3902 showed potent A1 antagonism against the inhibition of forskolin‐induced cyclic AMP accumulation in DDT1 MF‐2 cells by the Arselective agonist, cyclopentyladenosine with a dissociation constant (KB value) of 0.34 nM. KW‐3902 antagonized 5′‐N‐ethylcarboxamidoadenosine‐elicited cyclic AMP accumulation via A2B receptors with a KB value of 52 nM. 5 KW‐3902 exhibited marked species‐dependent differences in the binding affinities. The highest affinity was for the rat A1 receptor (Ki = 0.19 nM) and these values for guinea‐pig and dog A1 receptors were 1.3 and 10 nM, respectively.