Masamichi Okada

Glutamate receptors: brain function and signal transduction

Glutamate receptors are important in neural plasticity, neural development and neurodegeneration. N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors act as glutamate-gated cation channels, whereas metabotropic receptors (mGluRs) modulate the production of second messengers via G proteins. Molecular studies from our and other laboratories indicated that NMDA receptors and mGluRs exist as multiple subunits (NMDAR1 and NMDAR2A-2D) and multiple subtypes (mGluR1-mGluR8). In light of the molecular diversity of glutamate receptors, we explored the function and intracellular signaling mechanisms of different members of glutamate receptors. In the visual system, retinal bipolar cells receive glutamate transmission from photoreceptors and contribute to segregating visual signals into ON and OFF pathways. The molecularly cloned mGluR6 is restrictedly expressed at the postsynaptic site of ON-bipolar cells in both rod and cone systems. Gene targeting of mGluR6 results in a loss of ON responses without changing OFF responses and severely impairs detecting visual contrasts. Since AMPA receptors mediate OFF responses in OFF-bipolar cells, two distinct types of glutamate receptors effectively operate for ON and OFF responses. mGluR1 and mGluR5 are both coupled to inositol triphosphate (IP3)/calcium signal transduction with an identical agonist selectivity. Single-cell intracellular calcium ([Ca2+]i) recordings indicated that glutamate evokes a non-oscillatory and oscillatory [Ca2+]i response in mGluR1-expressing and mGluR5-expressing cells, respectively. This difference results from a single amino acid substitution, aspartate of mGluR1 or threonine of mGluR5, at the G protein-interacting carboxy-terminal domains. Protein kinase C phosphorylation of the threonine of mGluR5 is responsible for inducing [Ca2+]i oscillations in mGluR5-expressing cells and cultured glial cells. Thus, the two closely related mGluR subtypes mediate diverging intracellular signaling in glutamate transmission.

Neuroprotective effects of the selective type 1 metabotropic glutamate receptor antagonist YM-202074 in rat stroke models

We describe in vitro properties and in vivo neuroprotective effects of a newly synthesized, high-affinity, selective allosteric metabotropic glutamate receptor type 1 (mGluR(1)) antagonist, N-cyclohexyl-6-{[(2-methoxyethyl)(methyl)amino]methyl}-N-methylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM-202074). YM-202074 bound an allosteric site of rat mGluR(1) with a K(i) value of 4.8+/-0.37 nM. YM-202074 also inhibited the mGluR(1)-mediated inositol phosphates production in rat cerebellar granule cells with an IC(50) value of 8.6+/-0.9 nM, while showing selectivity over mGluR(2-7). When YM-202074 was infused intravenously at an initial dose of 20 mg/kg/h for 0.5 h followed by a dose of 5 mg/kg/h for 7.5 h, the free concentration of YM-202074 in the brain rapidly (<12 min) reached approximately 0.3 microM, reaching a steady-state phase within 1.5 h. We first treated rats such that they developed transient middle cerebral artery (MCA) occlusion. Results clearly demonstrate a dose-dependent improvement of neurological deficit and reduction of the infarct volume in both the hemisphere and cortex when YM-202074 was infused intravenously immediately after occlusion at a dose of 10 or 20 mg/kg/h for 0.5 h followed by a dose of 2.5 or 5 mg/kg/h for 23.5 h, respectively. Significant neuroprotection was maintained even when the administration of drugs was delayed by up to 2 h following the onset of ischemia. Furthermore, the improvement of neurological deficit and the reduction of infarct volume were sustained for 1 week following the onset of ischemia. These results suggest that YM-202074 exhibits great potential as a novel neuroprotective agent for the treatment of stroke.

Vascular calcification and secondary hyperparathyroidism of severe chronic kidney disease and its relation to serum phosphate and calcium levels.

Background and purpose:u2002 Various complications consequent on disordered calcium and phosphate homeostasis occur frequently in chronic kidney disease (CKD) patients. Particularly, vascular calcification has high morbidity and mortality rates. There is a clear need for a better CKD model to examine various aspects of this disordered homeostasis.

Effects of a new thyrotropin releasing hormone analogue, YM-14673, on the in vivo release of acetylcholine as measured by intracerebral dialysis in rats

Abstract: The effects of a new thyrotropin releasing hormone (TRH) analogue, YM‐14673 (Nα‐[[(S)‐4‐oxo‐2‐azetidi‐nyl]carbonyl]‐l‐histidyl‐l‐prolinamide dihydrate), on the release of acetylcholine (ACh) in free‐moving rats were examined in vivo by intracerebral dialysis. In the frontal cortex, YM‐14673 (0.1–0.3 mg/kg) caused a significant dose‐dependent increase in the extracellular levels of ACh, suggesting that YM‐14673 stimulated the ACh release. These actions of YM‐14673 were about 50 times more potent than those of TRH. On the other hand, extracellular levels of ACh in caudate nucleus were not changed following injection of YM‐14673 even at 3 mg/kg. TRH and methamphetamine also increased the release of ACh in frontal cortex. Haloperidol prevented the increase in the methamphetamine‐induced release of ACh, whereas the increased release of ACh produced by YM‐14673 was partially antagonized by haloperidol. These results suggest that the dopaminergic system affects the fa‐cilitatory effects on the ACh release in the frontal cortex and that the stimulatory effect of YM‐14673 on the frontal cholinergic neurons is partially mediated by dopaminergic neurons.

Renoprotective properties of pirfenidone in subtotally nephrectomized rats.

Renal fibrosis is the final common pathway of chronic kidney disease, and its progression predicts the degree of renal dysfunction. We investigated the renoprotective properties of pirfenidone in a remnant kidney model of chronic renal failure to determine its pharmacological potency compared to enalapril. Five-sixths nephrectomized rats were fed diet containing pirfenidone (approximately 700mg/kg/day) for 8weeks. Pirfenidone steadily inhibited the progression of proteinuria, but not to a significant degree. Pirfenidone prevented the elevation of plasma creatinine and blood urea nitrogen. At the end of the experiment, pirfenidone had reduced systolic blood pressure by means of its renoprotective effect. In a histological study, pirfenidone improved interstitial fibrosis in the renal cortex. These effects were supported by the suppression of the expression of TGF-beta and fibronectin in the mRNA of the kidney. In contrast, pirfenidone had little effect on the expression of alpha-smooth muscle actin, which is one of the proteins responsible for epithelial-mesenchymal transition. This property was confirmed by the TGF-beta-induced transdifferentiation observed in cultured normal rat kidney tubular epithelial NRK52E cells. These results suggest that pirfenidone improves the progression of chronic renal failure via its antifibrotic action, although pirfenidone has less effective TGF-beta-induced epithelial to mesenchymal transdifferentiation.

Comparison of chronic renal failure rats and modification of the preparation protocol as a hyperphosphataemia model

Background:u2003 Several animal models with chronic renal failure have been established and used for demonstrating complications including hyperphosphataemia. Although long‐time feeding is required to cause hyperphosphataemia in animals, a few modifications have been reported to provide more useful models for research.

Minodronic acid, a third-generation bisphosphonate, antagonizes purinergic P2X2/3 receptor function and exerts an analgesic effect in pain models

The P2X(2/3) receptor has an important role in the nociceptive transmission. Minodronic acid is a third third-generation bisphosphonate and a potent inhibitor of bone resorption. We found that minodronic acid inhibited alpha,beta-methylene ATP-induced cation uptake with the potency higher than that of suramin in the P2X(2/3) receptor receptor-expressing cells. Other bisphosphonates did not show such activity. Subcutaneously administered (10-50 mg/kg) minodronic acid significantly inhibited the alpha,beta-methylene ATP-, acetic acid- and formalin-induced nociceptive behaviors in mice. These unique effects of minodronic acid would be beneficial for the treatment of accelerated bone turnover diseases accompanied by bone pain, including bone metastases.

ASP6537, a novel highly selective cyclooxygenase-1 inhibitor, exerts potent antithrombotic effect without "aspirin dilemma".

INTRODUCTIONnAspirin inhibits both the cyclooxygenase (COX)-1-dependent production of thromboxane A2 (TXA2) in platelets and COX-2-dependent production of anti-aggregatory prostaglandin I2 (PGI2) in vessel walls, resulting in aspirin dilemma. Our objective is to investigate whether ASP6537 can overcome aspirin dilemma and exert a potent antithrombotic effect without a concurrent ulcerogenic effect.nnnMETHODSnWe evaluated the inhibitory effects of ASP6537 on recombinant human COX-1 (rhCOX-1) and rhCOX-2 activities using a COX-1/2 selectivity test. To determine whether ASP6537 induces aspirin dilemma, we examined the effects of ASP6537 on in vitro TXA2 and PGI2 metabolite production from platelets and isolated aorta of guinea pigs, and on plasma concentrations of TXA2 and PGI2 metabolites in aged rats. Finally, we evaluated the antithrombotic effects and ulcerogenic activity of ASP6537 using an electrically induced carotid arterial thrombosis model and a gastric ulcer model in guinea pigs.nnnRESULTSnThe IC50 ratios of rhCOX-2 to rhCOX-1 for ASP6537 and aspirin were >142,000 and 1.63 fold, respectively. ASP6537 inhibited TXA2 production more selectively than aspirin in in vitro and in vivo TXA2/PGI2 production studies. ASP6537 exerted a significant antithrombotic effect at ≥3 mg/kg, while aspirin tended to inhibit thrombosis at 300 mg/kg but it was not statistically significant. Further, ASP6537 did not induce ulcer formation at 100 mg/kg, whereas aspirin exhibited an ulcerogenic effect at doses of ≥100 mg/kg.nnnCONCLUSIONSnASP6537 functions as a highly selective COX-1 inhibitor with a superior ability to aspirin for normalizing TXA2/PGI2 balance, and exerts antithrombotic effect without ulcerogenic effect.

Possible involvement of central cholinergic system in ameliorating effects of indeloxazine, a cerebral activator, on disturbance of learning behavior in rats

Effects of indeloxazine, a cerebral activator, on passive avoidance learning by disruption of cholinergic transmission were studied in rats. The shortened latency was prolonged when indeloxazine was administered before training in rats subjected to scopolamine-injection, ethylcholine aziridinium ion (AF64A)-treatment and nucleus basalis magnocellularis (NBM) lesion. Indeloxazine administered immediately after training also showed ameliorating effects on passive avoidance in NBM-lesioned rats. Indeloxazine increased extracellular acetylcholine concentration in the frontal cortex of rats. Indeloxazine possesses facilitatory effects on cerebral function in part due to activation of the central cholinergic system.