Mutsumi Maruyama

Effects of acromelic acid A on the binding of [3H]glutamic acid and [3H]kainic acid to synaptic membranes and on the depolarization at the frog spinal cord

Triton treatment of synaptic membranes from the frog spinal cord enhanced the specific binding of [3H]glutamic acid compared with non-treated fresh and frozen ones, but not that of [3H]kainic acid. Acromelic acid A specifically inhibited the binding of [3H]kainic acid, and was approximately 100 times more potent than kainic acid. Acromelic acid A and excitatory amino acids caused a depolarization in the ventral root of the frog spinal cord in a dose-dependent manner, and the effect of acromelic acid A was much superior to that of kainic acid or domoic acid. Acromelic acid A is one of the most potent kainic acid agonist at the frog spinal cord.

Selective solubilization of physalaemin-type substance P binding sites from rat brain membranes by glycodeoxycholate and NaCl

Active substance P binding sites were solubilized from rat brain membranes by treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. About 50% of the binding activity in membrane-bound binding sites was recovered in the solubilized fraction after centrifugation at 105,000 g for 1 h. [3H]Substance P absorbed extensively to glass tubes and glass filters, but the absorption was greatly reduced by siliconizing glass tubes and preincubating glass filters in a solution containing poly-D-lysine and bovine serum albumin. [3H]Substance P was found to bind the solubilized receptors in a saturable fashion with a Bmax of 145 fmol/mg protein and a Kd of 4.6 nM, and these bindings were completely replaced by low concentrations of unlabeled substance P and physalaemin.

Characteristics of brain injury-derived neurotrophic peptide-binding sites on rat brain synaptosomes and neurons in culture.

Brain injury-derived neurotrophic peptide is the fragmental 13-mer peptide of the novel neurotrophic factor which was extracted and purified from Sponge Gelform made of gelatin implanted at the mechanically-induced injury site in neonatal rat brains. Brain injury-derived neurotrophic peptide supports survival of septal cholinergic and mesencephalic dopaminergic neurons in culture, and rescues hippocampal neurons in culture from glutamate neurotoxicity. Here we studied the binding characteristics of brain injury-derived neurotrophic peptide to synaptosomes from normal adult rat brains and neurons in culture from neonatal rat brains. [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide binding to rat brain synaptosomes was specific and saturable. Equilibrium binding studies revealed that [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide bound to 1.1 pmol/mg protein with a Kd (dissociation constant) of 0.17 microM in hippocampal synaptosomes and to 2.0 pmol/mg protein with a Kd of 0.38 microM in septal synaptosomes. [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide could bind to a subpopulation of hippocampal neurons in culture from embryonic rat brains. Affinity cross-linking with the carboxyl-reactive cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-HCl and [125I]Asp-[Tyr11]-brain injury-derived neurotrophic peptide produced radiolabeled bands corresponding to 100,000, 50,000 and 40,000 mol. wt molecules on hippocampal neurons in culture. These results suggest that the 13-mer sequence of brain injury-derived neurotrophic peptide plays a crucial role in expressing the neurotrophic properties of the factor.

Binding characteristics of [3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [3H]peptidergic opioid ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [3H]-[D-Ala2-, Met5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl2 and 100 mM NaCl at 0 degree C (on ice) for 3 h. With this assay condition, the significant binding of following [3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin (mu-type), [3H]-[D-Ala2, D-Leu5]enkephalin (delta-type) and [3H]dynorphin1-9 (kappa-type). The number of binding sites in solubilized preparations for each [3H]ligand corresponded to 40-50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [3H]ligand. Apparent dissociation constants between solubilized receptors and [3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycodeoxycholate and NaCl and the dilution process.

Inhibition of transmitter release by TI233, a calmodulin antagonist, from clonal neural cells and a presumed site of action.

Effects of TI233, a calmodulin antagonist, on transmitter release were studied using a clonal pheochromocytoma cell line (PC12h). TI233, at a concentration of 30 microM, completely suppressed the release of preloaded [3H]NE and [3H]DA. The 50% suppression dose was around 3 microM. TI233 did not inhibit the [3H]NE release evoked by the calcium ionophore A23187. Electrophysiological examinations using a clonal neuroblastoma x glioma hybrid cell line (NG108-15) revealed that TI233 blocked the voltage-sensitive calcium channel of the membrane in the same concentration range. Thus it was suggested that TI233 inhibited transmitter release from neuronal cells by blocking the entry of calcium to the cytoplasm.

Evidence that [3H]glutamate binding sites are masked by biologically relevant endogenous factor on cell membranes of frog spinal cord

We identified the possible endogenous factor effective to modulate the binding of [3H]-labeled excitatory amino acid agonists and antagonists in the 100,000 x g supernatant of Triton X-100 (0.01%)-treated cell membranes from frog spinal cords. The factor inhibited the binding of [3H]glutamate to Triton X-100-treated cell membranes, to which the binding capacity of [3H]glutamate increased much more than that to intact cell membranes. The binding capacities of [3H]AMPA (an AMPA type agonist) and [3H]CPP (an NMDA type antagonist) to cell membranes remained low by Triton treatment, but they were enhanced significantly by the addition of the factor. The effect of the factor on the [3H]kainate binding was hardly observable. The factor may provide key information on receptor structures and the classification of receptor types concerning excitatory amino acids in the mammalian central nervous system.

Preparation and properties of trimethylammonium group-containing analogues of [D-Ala2, Leu5]enkephalin and gramicidin S

Quaternization of primary amino groups of derivatives of [D-Ala2, Leu5]enkephalin and gramicidin S with Mel–KHCO3 in MeOH afforded new biologically active analogues possessing trimethylammonium group(s).

Development of an antibody against a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein and identification of a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein in hippocampal neurons.

Brain injury-derived neurotrophic peptide is a 13-amino acid peptide derived from a 15,000 mol. wt neurotrophic factor released from sites of mechanical injury in neonatal rat brain. This peptide promotes survival of septal cholinergic neurons and mesencephalic dopaminergic neurons, and protects hippocampal neurons from glutamate-induced neurotoxicity. In this study, we have developed a monoclonal antibody against a brain injury-derived neurotrophic peptide-binding protein by immunizing mice with septal synaptosomes from five-week-old rat brain. Monoclonal antibodies were screened for inhibition of the binding of a 125I-labeled analogue of brain injury-derived neurotrophic peptide to rat brain synaptosomes. The monoclonal antibody 6A22 suppressed the biological activity of brain injury-derived neurotrophic peptide and abolished the protective effect of the neurotrophic peptide against glutamate-induced neurotoxicity. This monoclonal antibody recognized a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein, which was also identified by cross-linking experiments. Immunohistochemical studies showed that the 6A22 antibody bound to the cell surfaces of a subpopulation (about 60%) of hippocampal neurons in culture. These results are consistent with the possibility that the 40,000 mol. wt protein belongs to brain injury-derived neurotrophic peptide receptors.

A new system for studying neuronal cell death using NG-108 cell and BINP

SHIGEKI FURUYA’, TOSHIHIDE TABATA 2.3,4, JUNYA MITOMA’, ASAMI MAKINO’, MASANOBU KANO**‘, 4. YOSHIO HIRABAYASHI’ ‘Lab. for Cellular Glycobiol., Frontier Res. Prog.. RIKEN, Wako, Saitama 35 I-0198, 2CREST. Japan Sci. & Tech. Corp. 3Lab. for Cellular Neurophysiol., Brain Sci. Inst., RIKEN, Wake, Saitama 35 I-019X. 4Dept. of Physiol., Kanazawa Univ. Schl. of Med.. Kanazawa, lshikawa 920-X640 Purkinje cell survival and dendritic development appear to be regulated by granule cell afferents. It has been demonstrated that purified Purkinje cells survive poorly and fail to extend dendrites in the absence of granule cells under a monolayer culture condition. Despite these observations, the molecular mechanism underlying the survival and dendritic development of Purkinje cells remains largely unknown. We observed poor Purkinje cell survival with undeveloped dendrites under a certain serum-free culture condition, even in the presence of granule cells. This impaired growth was overcome by the addition of a conditioned medium prepared from cerebellar astrocyte cultures (CACM), but not neurotrophins and other known neurotrophic factors. The addition of CACM produced a 3.0 to 3.5-fold increase in the number of Purkinje cells at 14 days in vitro. A conditioned medium prepared from hippocampal astrocytes also improved Purkinje cell survival and dendritic development to a similar extent. The growth-promoting activity in CACM is heat stable and recovered in the low molecular weight fraction (Mr < -3000). These results strongly suggest that Purkinje cells require an additional factor that is supplied by astrocytes for their survival and dendritic development. The molecular nature of the astrocyte-derived Purkinje cell growth-promoting factor is currently being investigated.

1316 Characterization of BINP (brain injury-derived neurotrophic peptide) binding protein in synaptosomes and cultures from rat brain

Sheng-Tian Li’ , Kunio Kato3, Katsuhiko Mikoshiba’ Long-term depression (LTD) in the CA1 area of rat hippocampal slices was studied. We employed a special perfusion solution composed of variable concentrations of Ca 2+ Mg2+ and K+ to substitute for low frequency , stimulation in order to induce LTD without electrical stimulation. LTD was successfully induced with this conditioning solution (Ca 2f: 4 mM; Mg2+: 0.1 mM; Kf: 5mM) applied for 20min (-28.5h8.8’30; N=12). The advantage of this protocol is that the whole slice is exposed to conditioning solution, and therefore LTD should be induced in all of the synapses, possibly yielding clearer results in biochemical studies than electrical stimulation-induced LTD. We tested the effect of protein phosphatase inhibitors, such as FK-506 and cyclosporine A on the LTD induced by the conditioning solution. We also investigated phosphatase activity during LTD induction by biochemical methods, and compared protein phosphatase activity before and after the induction of LTD.