Sadayo Nakajima-Iijima

DEVELOPMENTAL CHANGES IN DISTRIBUTION OF DEATH-ASSOCIATED PROTEIN KINASE MRNAS

Death‐associated protein kinase (DAP‐kinase) is Ca2+/calmodulin‐dependent serine/threonine kinase that contains ankyrin repeats and the death domain. It has been isolated as a positive mediator of interferon‐γ‐induced apoptotic cell death of HeLa cells. In order to reveal the physiological role of DAP‐kinase, the tissue distribution and developmental changes in mRNA expression of DAP‐kinase were investigated by Northern blot and in situ hybridization analyses. DAP‐kinase mRNA was predominantly expressed in brain and lung. In brain, DAP‐kinase mRNA had already appeared at embryonic day 13 (E13) and was, thereafter, detected throughout the entire embryonic period. High levels of expression were detected in proliferative and postmitotic regions within cerebral cortex, hippocampus, and cerebellar Purkinje cells. These findings suggest that DAP‐kinase may play an important role in neurogenesis where a physiological type of cell death takes place. The overall expression of DAP‐kinase mRNA in the brain gradually declined at postnatal stages, and the expression became restricted to hippocampus, in which different expression patterns were observed among rostral, central, and caudal coronal sections, suggesting that DAP‐kinase may be implicated in some neuronal functions. Furthermore, it was found that the expression of DAP‐kinase mRNA was increased prior to a certain cell death induced by transient forebrain ischemia, indicating a possible relationship between DAP‐kinase and neuronal cell death. J. Neurosci. Res. 58:674–683, 1999.

Inducible expression of N-methyl-d-aspartate (NMDA) receptor channels from cloned cDNAs in CHO cells

To develop a drug screening system, we introduced expression vectors carrying the mouse N-methyl-D-aspartate (NMDA) receptor channel epsilon1 and zeta1 subunit cDNAs under the promoter of the Drosophila heat shock protein hsp70 into Chinese hamster ovary (CHO) cells. We selected clonal cell lines by means of RNA blot hybridization and fura-2 fluorometry. One of these cell lines, ZE1-1, optimally expressed the epsilon1 and zeta1 subunit mRNAs when induced by an incubation at 43 degrees C for 2 h. Heated ZE1-1 cells exhibited the NMDA-induced intracellular Ca2+ elevation, whereas unheated they showed no such response. NMDA and L-glutamate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, induced an increase in the intracellular Ca2+ concentration. The response to the agonists was marginal in the absence of glycine, and diminished by Mg2+ and NMDA receptor antagonists. Furthermore, exposure to agonists of ZE1-1 cells expressing the epsilon1/zeta1 NMDA receptor channel resulted in the release of lactate dehydrogenase (LDH) activity in the culture medium indicating agonist-induced cell death. NMDA receptor antagonists inhibited the LDH activity release. These results suggest that ZE1-1 cells will provide a useful screening system for novel drugs acting on the epsilon1/zeta1 NMDA receptor channel.

Evaluation of agonist selectivity for the NMDA receptor ion channel in bilayer lipid membranes based on integrated single-channel currents

A new method for evaluating chemical selectivity of agonists to activate the N-methyl-D-aspartate (NMDA) receptor was presented by using typical agonists NMDA, L-glutamate and (2S, 3R, 4S)-2-(carboxycyclopropyl)glycine (L-CCG-IV) and the mouse epsilon1/zeta1 NMDA receptor incorporated in bilayer lipid membranes (BLMs) as an illustrative example. The method was based on the magnitude of an agonist-induced integrated single-channel current corresponding to the number of total ions passed through the open channel. The very magnitudes of the integrated single-channel currents were compared with the different BLMs as a new measure of agonist selectivity. The epsilon1/zeta1 NMDA receptor was partially purified from Chinese hamster ovary (CHO) cells expressing the epsilon1/zeta1 NMDA receptor and incorporated in BLMs formed by the tip-dip method. The agonist-induced integrated single-channel currents were obtained at 50 microM agonist concentration, where the integrated current for NMDA was shown to reach its saturated value. The obtained integrated currents were found to be (4.5 +/- 0.55) x 10(-13) C/s for NMDA, (5.8 +/- 0.72) x 10(-13) C/s for L-glutamate and (6.6 +/- 0.61) x 10(-13) C/s for L-CCG-IV, respectively. These results suggest that the agonist selectivity in terms of the total ion flux through the single epsilon1/zeta1 NMDA receptor is in the order of L-CCG-IV approximately = L-glutamate > NMDA.

Analysis of the glycine binding domain of the NMDA receptor channel ζ1 subunit

IN an attempt to examine glycine binding domain of the ζ1 subunit of the mouse N-methyl-D-aspartate (NMDA) receptor channel, we constructed N-terminal or C-terminal deletion mutants of the ζ1 subunit cDNA and expressed them in Spodoptera frugiperda cells using a baculovirus system. Analysis of binding of a glycine binding site antagonist, 5,7-[3-3H]dichlorokynurenate ([3H]DCKA) to the deleted ζ1 mutants provided the first direct experimental evidence that the regions of N-terminal 282 and C-terminal 102 amino acid residues do not significantly affect glycine binding, and that both the region of ∼260 amino acid residues preceding the putative transmembrane segment M1 and the region between the segments M3 and M4 are required to form the glycine binding domain.

Establishment of CHO cell lines expressing four N-methyl-D-aspartate receptor subtypes and characterization of a novel antagonist PPDC

To develop an assay system that allows the N‐methyl‐D‐aspartate (NMDA) receptor subtype‐selective antagonistic potency of drugs, we have established Chinese hamster ovary cell lines expressing the four NMDA receptor subtypes (GluRϵ1/ζ1–GluRϵ4/ζ1) heat‐indelibly. Using these clonal cells, we found that a novel antagonist, (1S,2R)‐1‐phenyl‐2[(S)‐1‐aminopropyl]‐N,N‐diethylcyclopropanecarboxamide, was less selective for the GluRϵ1/ζ1: the IC50 values for the GluRϵ1/ζ1–GluRϵ4/ζ1 were 41.7, 13.3, 12.6 and 11.5 μM, respectively, while two well‐known antagonists, DL‐2‐amino‐5‐phosphonovaleric acid and ifenprodil, showed the known potency and selectivity for each subtype. Thus, the established clonal cells are of use in characterizing the pharmacological properties of drugs that act on NMDA receptors.

Real‐time, two‐dimensional visualization of ischaemia‐induced glutamate release from hippocampal slices

The involvement of excitatory amino acid (EAA) toxicity in ischaemia‐induced neuronal cell death has long been suggested. However, in the hippocampus, the brain site most vulnerable to ischaemia, the detailed spatial and temporal patterns of EAA release are not yet known. To address this issue, we have developed a novel strategy for the continuous, real‐time, two‐dimensional monitoring of EAA release from brain slices. As EAA detector, we used a cell line transformed with the N‐methyl‐d‐aspartate (NMDA) receptor, which is exclusively activated by EAAs, leading to an increase in the intracellular Ca2+ level. Combined with a calcium imaging technique, the use of this cell line allowed the temporal and regional analysis of EAA release from a brain slice placed directly on top of the clonal cells in a culture dish. Using this strategy, we demonstrated ischaemia‐induced EAA release in rat hippocampal slices. Increased EAA release was seen initially in the CA1 region, about 3 min after the beginning of ischaemia, then in the CA3 region and dentate gyrus, and, finally, throughout the hippocampal slice. Regional differences in extracellular EAA levels were also seen, with more EAA being released from the CA1 region than from the middle dentate gyrus. The present results are especially interesting as neurons in the CA1 region are more vulnerable to ischaemia than those in the CA3 region and dentate gyrus.

Immortalization of human endothelial cells by origin-defective simian virus 40 DNA

Human endothelial cells isolated from an umbilical cord vein were transfected with origin-defective simian virus 40 (SV40) DNA. Among several of the SV40 transfected clones isolated, cell lines SV-2 and SV-3 showed a normal endothelial cell morphology and extended life span, and could survive almost 100 generations. Just before crisis, the morphology of SV-3 changed. SV-3T cell line was isolated from this SV-3 culture, which acquired an almost infinite life span, rapid growth rate and the ability to grow in soft agar. At the same time, the SV-3T cell line lost the factor VIII-related antigen and normal endothelial cell morphology, and showed an abnormal chromosome number. Further characterization showed the ability of SV-2 and SV-3T to produce increasing amounts of tissue plasminogen activator and a similar level of a plasminogen activator inhibitor compared with normal human endothelial cells. These results indicate that the SV-3T cell line was transformed and acquired an infinite life span while still r...

A single-channel method for evaluation of very magnitudes of Ca2+ ion fluxes through ε4/ζ1 N-methyl-d-aspartate receptor channels in bilayer lipid membranes

Abstract A single-channel method for evaluating agonist selectivity in terms of the very number of Ca 2+ ions passed through the e4/ζ1 N -methyl- d -aspartate (NMDA) receptor ion channel in bilayer lipid membranes (BLMs) is described. The number of Ca 2+ passed through the single-channel was obtained from single-channel recordings in a medium where the primary permeant ion is Ca 2+ . The recombinant e4/ζ1 NMDA channel was partially purified from Chinese hamster ovary cells expressing the channel and incorporated in BLMs formed by the tip-dip method. It was found that the e4/ζ1 channel in BLMs is permeable to Ca 2+ and Na + , but the number of Ca 2+ passed through the channel is much fewer than that of Na + . The integrated Ca 2+ currents induced by three typical agonists NMDA, l -glutamate and l -CCG-IV were obtained at concentration of 50 μM, where the integrated currents for all the agonists reached their saturated values. The integrated Ca 2+ currents obtained are (3.1±0.21)×10 −13 C/s for NMDA, (4.6±0.31)×10 −13 C/s for l -glutamate and (5.7±0.25)×10 −13 C/s for l -CCG-IV, respectively, suggesting that the three kinds of agonists have different efficacies to induce permeation of Ca 2+ . The range of the agonist selectivity thus obtained is much narrower than that of binding affinities for the NMDA receptors from rat brain. The present method is able to detect Ca 2+ permeation with a detection limit of ≈10 5 Ca 2+ ions/s.