Takashi Masuko

Polyamine transport, accumulation, and release in brain.

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential‐dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradient‐dependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.

Toll-like Receptor 3 Stimulation Causes Corticosteroid-Refractory Airway Neutrophilia and Hyperresponsiveness in Mice

BACKGROUND RNA virus infections, such as rhinovirus and respiratory syncytial virus, induce exacerbations in patients with COPD and asthma, and the inflammation is corticosteroid refractory. The main aim of this study is to establish a murine model induced by a Toll-like receptor 3 (TLR3) agonist, an RNA virus mimic, and investigate the response to corticosteroid. METHODS A/J mice were given polyinosinic-polycytidylic acid (poly[I:C]), a TLR3 agonist, intranasally, in the presence or absence of cigarette smoke exposure. Inflammatory cell accumulation and C-X-C motif chemokine (CXCL) 1, interferon (IFN), and CXCL10 production in BAL fluid (BALF) were determined by flow cytometry and enzyme-linked immunosorbent assay, respectively, and airway hyperresponsiveness (AHR) to histamine/methacholine was determined by a two-chambered, double-flow plethysmography system. BALB/c and C57BL/6J mice were also used for comparisons. RESULTS Intranasal treatment of poly(I:C) significantly induced airway neutrophilia; production of CXCL1, IFN-β, and CXCL10; and necrotic cell accumulation in BALF. It also increased airway responsiveness to histamine or methacholine inhalation. This poly(I:C)-dependent airway inflammation and AHR was not inhibited by the corticosteroid fluticasone propionate (FP) (up to 0.5 mg/mL intranasal), although FP strongly inhibited lipopolysaccharide (TLR4 agonist)-induced airway neutrophilia. Furthermore, cigarette smoke exposure significantly increased TLR3 expression in murine lung tissue and exacerbated poly(I:C)-induced neutrophilia and AHR. CONCLUSIONS These results suggest that TLR3 stimulation is involved in corticosteroid-refractory airway inflammation in lung, which is enhanced by cigarette smoking, and this may provide a model for understanding virus-induced exacerbations in COPD and their therapy.

Monoamines directly inhibit N-methyl-d-aspartate receptors expressed in Xenopus oocytes in a voltage-dependent manner

Dopamine has numerous functions in the brain and has been shown to modulate responses of N-methyl-D-aspartate (NMDA) receptors on thalamic and hippocampus neurons [N.G. Castro, M.C.F. de Mello, F.G. de Mello, Y. Aracava, Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393, Br. J. Pharmacol. 126 (1999) 1847-1855]. Thus, the effects of dopamine, serotonin, tyramine, epinephrine, norepinephrine, and octopamine on NMDA receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. Serotonin and tyramine, in addition to dopamine, were found to inhibit macroscopic currents at heteromeric NMDA receptors, but not AMPA (GluR1/GluR2) receptors. Epinephrine, norepinephrine and octopamine also weakly inhibited macroscopic currents at NR1/NR2A and NR1/NR2B receptors. The inhibitory effects of these monoamines became prominent at -100 mV comparing those at -20 mV. Mutations at NR1 N616, NR2B N615, and NR2B N616, but not at NR1 W563 and NR1 N650, reduced the inhibitory effects by monoamines. These results indicate that these monoamines directly act on the narrowest region of channel pore.

Cyclophane and acyclic cyclophane: Novel channel blockers of N-methyl-d-aspartate receptor

The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on various glutamate receptors were studied with these receptors expressed in Xenopus oocytes using voltage-clamp recording. CPCn, CPPy, TGDMAP and ATGDMAP were found to inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2), but not Ca(2+)-permeable AMPA receptors (GluR1), Ca(2+)-nonpermeable AMPA receptors (GluR1/GluR2) and metabotropic glutamate receptors (mGluR1alpha). The inhibition of NR1/NR2A receptors by these compounds was more potent than those of the other NMDA receptor subtypes. At a resting potential (-70 mV), the IC(50) values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5+/-0.1, 1.0+/-0.2, 8.0+/-0.8 and 4.9+/-0.5 microM, respectively. The inhibition by these compounds was voltage-dependent, that is, the degree of inhibition was in the order of negative holding potentials, -100 mV>-70 mV>-20 mV. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by CPCn. The inhibition by CPCn was not altered significantly in the mutants at the critical asparagines in the M2 loop, NR1 N616, NR2B N615 and NR2B N616, these residues are known to form the narrowest region of the channel and the binding site of Mg(2+). However, mutations at NR1 N650, located in the vestibule of channel pore, and NR1 D669, located in the extracellular region, reduced the inhibition by CPCn, suggesting that these amino acid residues interact with CPCn. These results suggest that CPCn interacts directly with the mouth or vestibule of the ion channel, like a lid.

Structural changes of regulatory domain heterodimer of N-methyl-D-aspartate receptor subunits GluN1 and GluN2B through the binding of spermine and ifenprodil.

Modeling the binding sites for spermine and ifenprodil on the regulatory (R) domains of the N-methyl-d-aspartate receptor GluN1 and GluN2B subunits was carried out after measuring spermine stimulation and ifenprodil inhibition at receptors containing GluN1 and GluN2B R domain mutants. Models were constructed based on the published crystal structure of the GluN1 and GluN2B R domains, which form a heterodimer (Nature 475:249–253, 2011). The experimental results and modeling suggest that a binding site for spermine was formed by the residues near the cleft between the R1 and R2 lobes of the GluN1 R domain (GluN1R) together with residues on the surface of the R2 (C-terminal side) lobe of the GluN2B R domain (GluN2BR). The ifenprodil binding site included residues on the surface of the R1 lobe (N-terminal side) of GluN1R together with residues near the cleft between the R1 and R2 lobes of GluN2BR. It was confirmed using a Western blot analysis that GluN1R and GluN2BR formed a heterodimer. Models of spermine and ifenprodil binding to the heterodimer were constructed. The modeling suggests that an open space between the two R1 lobes of GluN1R and GluN2BR is promoted through spermine binding and that the R1 lobes of GluN1R and GluN2BR approach each other through ifenprodil binding—an effect opposite to that seen with the binding of spermine.

Cleft-type cyclophanes confer neuroprotection against excitatory neurotoxicity in vitro and in vivo through inhibition of NMDA receptors.

The cleft-type cyclophanes (ACCn, DNCn and TsDCn) were found to strongly inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2) but not AMPA receptors expressed in Xenopus oocytes at voltage-clamp recording. The inhibition by cleft-type cyclophanes was voltage-dependent, because the inhibition was larger at -100 mV than at -20 mV. Mutations at NR1 N650, located in the vestibule of the channel pore, reduced the inhibition by DNCn and TsDCn, suggesting that the residue (N650) interacts with these cleft-type cyclophanes. Cell toxicity of TsDCn on SH-SY5Y cells was slightly weaker than that of memantine. The neuroprotective effects of cleft-type cyclophanes against cell damage caused by NMDA were investigated in cultured rat hippocampal neurons. Addition of 10 microM DNCn or TsDCn into the medium ablated the neurotoxicity induced by NMDA, and a similar effect was also observed with memantine. The neuroprotective effects of cleft-type cyclophanes were then assayed on NMDA-induced seizures in mice. Intracerebroventricular injection of TsDCn (5 mg/mouse) decreased the seizure induced by intraperitoneal injection of NMDA (115 mg/kg) in mice. The results demonstrate that these cleft-type cyclophanes interact directly with the extracellular mouth of the NMDA channel pore and exhibit neuroprotective effects on NMDA-induced excitatory toxicity in primary cultured neurons and mice.

Antagonism of NMDA receptors by butanesulfonyl-homospermine guanidine and neuroprotective effects in in vitro and in vivo

The polyamine derivative BsHSPMG (butanesulfonyl-homospermine with guanidine group) was found to inhibit macroscopic currents strongly at heteromeric N-methyl-D-aspartate (NMDA) receptors (NR1/NR2A and NR1/NR2B) and Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (homomeric glutamate receptor 1) receptors expressed in Xenopus laevis oocytes on voltage-clamp recording. The IC(50) values of BsHSPMG for NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors were 0.016, 0.021, 5.4, and 9.0 μM, respectively. BsHSPMG inhibited the activity of NR1/NR2A and NR1/NR2B receptors more strongly and did it for those of NR1/NR2C and NR1/NR2D receptors more weakly than a therapeutic drug of Alzheimers disease, memantine. The inhibition by BsHSPMG was voltage-dependent, since it was prominent at -100 mV compared to that at -20 mV. Mutations including NR1 N616Q, E621Q, N650A, L655A, T807C, NR2B W559L, M562S, W607L, N616Q, and V620E, among others, reduced the inhibition by BsHSPMG, suggesting that BsHSPMG penetrates the channel pore of NMDA receptors deeply. The toxicity of BsHSPMG in neuroblastoma SH-SY5Y cells was much weaker than that of memantine. The effect of BsHSPMG was measured on the focal cerebral ischemia induced by occlusion (1 h) of the middle cerebral artery in mice. BsHSPMG applied before or after occlusion greatly reduced the volume of infarct in mice. These findings demonstrate that BsHSPMG penetrates the NMDA channel pore and exhibits neuroprotective effects against excitatory toxicity in mice.

Inhibitory Action of Antidepressants on Mouse Betaine/GABA Transporter (BGT1) Heterologously Expressed in Cell Cultures

Betaine/γ-aminobutyric acid (GABA) transporter (BGT1, SLC6A12) is a member of the Na+- and Cl−-dependent neurotransmitter transporter gene family with a homology to the GABA transporters (GATs), GAT1 (SLC6A1), GAT2 (SLC6A13) and GAT3 (SLC6A11) (HUGO nomenclature). Since antidepressants have been reported to inhibit GABA uptake, we examined those effects on mouse BGT1 (mBGT1) in comparison with other mouse GAT (mGAT) subtypes in the heterologously expressed cell cultures. All antidepressants tested here inhibited the [3H]GABA uptake through mBGT1 and mGATs in a rank order of potency with mBGT1 > mGAT1-3. Kinetic analyses for maprotilline, mianserine and trimipramine revealed that they inhibited mBGT1 and mGAT1 noncompetitively, except that mianserine competitively inhibited mBGT1. These results provided a clue to investigate the structure-function relationship of mBGT1 using antidepressants as a tool, leading to the identification of potential candidates for selective and specific inhibitors of mBGT1.

Neuroprotection by Tosyl-Polyamine Derivatives through the Inhibition of Ionotropic Glutamate Receptors

Tosyl-polyamine derivatives such as N-{4-[4-(guanidinobutylamino)-butylamino]butyl}-4-methylbenzenesulfonamide trihydrochroride (TsHSPMG) have been found to strongly inhibit macroscopic currents through heteromeric N-methyl-d-aspartate (NMDA) receptors (NR1/NR2A, NR1/NR2B) and Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (homomeric glutamate receptor 1) receptors expressed in Xenopus laevis oocytes on voltage-clamp recording. In the present study, it was found that the inhibition of NMDA receptor activity induced by tosyl-polyamine derivatives was voltage-dependent. Some mutations located in the intracellular region of the channel pore, such as NR1 E621Q and NR2B W607L, reduced the inhibition by tosyl-polyamine derivatives, suggesting that tosyl-polyamine derivatives penetrate deeply into the channel pore of NMDA receptors. The neuroprotective effects of tosyl-polyamine derivatives against cell injury caused by NMDA were investigated in cultured rat hippocampal neurons. Addition of 1 μM TsHSPMG to medium ablated the neurotoxicity induced by NMDA, and a similar effect was observed with 30 μM memantine. The neuroprotective effects of tosyl-polyamine derivatives on NMDA-induced seizures in mice were also assayed. Intracerebroventricular or intravenous injection of TsHSPMG (0.1 or 0.5 mg/kg) decreased the seizures induced by intraperitoneal injection of NMDA in mice. These findings indicate that tosyl-polyamine derivatives exhibit neuroprotective effects not only in primary cultured neurons but also in mice.

Ribulose-1,5-bisphosphate carboxylase/oxygenase from a nitrite-oxidizing chemoautotroph, Nitrobacter agilis ATCC 14123: Purification and properties

Abstract Nitrite-oxidizing chemoautotrophic Nitrobacter agilis ATCC 14123 exhibited remarkedly strong ribulose-1,5-bisphosphate carboxylase (RuBisCO) activity. RuBisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 480 kDa by gel filtration, suggesting that the enzyme consisted of two different subunits [Large (L): 50 kDa, Small (S): 10 kDa], as demonstrated by SDS-PAGE. This confirmed that the enzyme has a L 8 S 8 structure. The K m values of the enzymef for RuBP, NaHCO 3 , and Mg 2+ were estimated to be 0.039 mM, 6.39 mM and 1.36 mM, respectively. The optimum pH and temperature for its activity were approximately 8.0 and 40°C, respectively. The enzyme was stable up to 45°C and in a pH range of 7.0–9.0 (4°C, 48 h). The enzyme activity was inhibited by Mo 2+ , Cu 2+ , Hg 2+ , N -ethylmaleimide, p -chloromercuribenzoate and SDS (1 mM). The N-terminal amino acid sequence of the large subunit is AVKSYQAGVTQYRQSYWQPDYMPL, and that of the small subunit is AVQAYRSLKKYETFSYLPQ.