Changes in cerebrovascular disease-related deaths and their location during the COVID-19 pandemic in Japan.

OBJECTIVE: The COVID-19 pandemic placed an enormous strain on healthcare systems and raised concerns for delays in the management of patients with acute cerebrovascular events. In this study, we investigated cerebrovascular excess deaths in Japan. STUDY DESIGN: Vital mortality statistics from January 2012 to May 2022 were obtained from the Japanese Ministry of Health, Labour and Welfare. METHODS: Using quasi-Poisson regression models, we estimated the expected weekly number of cerebrovascular deaths in Japan from January 2020 through May 2022 by place of death. Estimates were calculated for deaths in all locations, as well as for deaths in hospitals, in geriatric health service facilities, and at home. The age subgroups of ≥75 and <75 years were also considered. Weeks with a statistically significant excess of cerebrovascular deaths were determined when the weekly number of observed deaths exceeded the upper bound of 97.5% prediction interval. RESULTS: Excess deaths were noted in June 2021 and became more pronounced from February 2022 onward. The trend was notable among those aged ≥75 years and for those who died in hospitals. With respect to the location of deaths, the excess was significant in geriatric health services facilities from April 2020 to June 2021, whereas no evidence of excess hospital deaths was observed during the same period. CONCLUSIONS: Beginning in the late 2021, excess cerebrovascular deaths coincided with the spread of the Omicron variant and may be associated with increased healthcare burden. In 2020, COVID-19 altered the geography of cerebrovascular deaths, with fewer people dying in hospitals and more dying in geriatric health service facilities and at home.

B-type natriuretic peptide levels are decreased by reducing dietary salt intake in patients with compensated heart failure with preserved ejection fraction.

BACKGROUND: Dietary salt restriction is believed to be a mainstay in the management of patients with heart failure. However, the effect of salt intake on heart failure has not been well evaluated in outpatient medical practice. AIMS: The aim of the present study was to assess the hypothesis that B-type natriuretic peptide (BNP) level, as an objective marker of heart failure, is associated with salt intake in patients with heart failure. METHODS: One hundred and thirteen consecutive patients with mild compensated heart failure (77 ± 10 years old, 51 female) were included. We estimated dietary salt intake by the concentration of sodium and creatinine in spot urine. We measured BNP at the time of urine sampling and assessed the relationship between the % changes in BNP levels (%ΔBNP) and the changes in the estimated daily salt excretion (ΔNaCl) during the follow-up period. RESULTS: The baseline median BNP level was 150 (interquartile range: 83-263) pg/mL and the estimated daily salt excretion was 162 ± 45 mmol/day. There was a positive correlation between %ΔBNP and ΔNaCl (r = 0.61, P < 0.01). Multiple regression analysis revealed that %ΔBNP was associated with ΔNaCl (P < 0.01), but not with changes in systolic blood pressure and bodyweight. CONCLUSIONS: Changes in BNP levels were associated with changes in the estimated daily salt excretion in outpatients with compensated heart failure. Salt restriction may be beneficial for the management of patients with heart failure.

Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration.

Prolonged or high-intensity exposure to visible light leads to photoreceptor cell death. In this study, we demonstrate a novel pathway of light-induced photoreceptor apoptosis involving the low-affinity neurotrophin receptor p75 (p75NTR). Retinal degeneration upregulated both p75NTR and the high-affinity neurotrophin receptor TrkC in different parts of Müller glial cells. Exogenous neurotrophin-3 (NT-3) increased, but nerve growth factor (NGF) decreased basic fibroblast growth factor (bFGF) production in Müller cells, which can directly rescue photoreceptor apoptosis. Blockade of p75NTR prevented bFGF reduction and resulted in both structural and functional photoreceptor survival in vivo. Furthermore, the absence of p75NTR significantly prevented light-induced photoreceptor apoptosis. These observations implicate glial cells in the determination of neural cell survival, and suggest functional glial-neuronal cell interactions as new therapeutic targets for neurodegeneration.

The origin and cell lineage of microglia: new concepts.

Despite intense study, the precise origin and cell lineage of microglia, the resident mononuclear phagocytes of the nervous system, are still a matter for debate. Unlike macroglia (astrocytes and oligodendrocytes) and neurons, which are derived from neuroectoderm, microglial progenitors arise from peripheral mesodermal (myeloid) tissue. The view still commonly held is that tissue-resident mononuclear phagocytes (including microglia) are derived from circulating blood monocytes and these take up residence late in gestation and postnatally. However, microglial progenitors colonise the nervous system primarily during embryonic and fetal periods of development. Recent evidence indicates differences between the lineage of mononuclear phagocytes during the embryonic and fetal period from that in the neonate and adult-mononuclear phagocytes that take up residence within tissues are derived from a lineage of myeloid cells that is independent of the monocyte lineage. Our own findings on the development and differentiation of microglial progenitors, taken together with findings by other investigators, and in the context of the heterogeneity between myeloid differentiation in the fetus and in the adult, support the view that microglia are derived prenatally from mesodermal progenitors that are distinct from monocytes. Furthermore, microglial progenitors colonise the nervous system via extravascular routes initially. These findings challenge the concept that resident microglia in the nervous system are derived from circulating blood monocytes. Work is still underway to establish the tissue of origin and lineage of microglial progenitors in vivo. This information is critical not only from a developmental perspective, but significantly from a therapeutic viewpoint, as (i) the unique property of microglial progenitors to colonise the nervous system from the periphery allows these cells to be exploited as a biological and non-invasive means for cell therapy by delivering genes to the nervous system (microglial engraftment), and (ii) there are indications that microglial progenitors are specifically able to home to the nervous system. Use of microglial progenitors for therapeutic purposes becomes feasible only if the origin and cell lineage of these microglial progenitors are known and these cells can be isolated and manipulated in vitro (i.e., to express specific trophic factors) prior to therapeutic transfer (e.g., intravenously) in vivo. In this paper, we shall briefly consider the existing concepts on the origin and lineage of microglial progenitors and discuss new hypotheses in the light of emerging data that suggest clear differences between fetal and adult ontogeny of myeloid cells.

The mutation in the ATP-binding region of JAK1, identified in human uterine leiomyosarcomas, results in defective interferon-gamma inducibility of TAP1 and LMP2.

The presentation of human leukocyte antigens (HLA) class I requires the coordinated expression of numerous components involved in antigen presentation. Tumor cells may alter the antigen presentation by HLA class I, allowing them to evade antitumor immunity. In many cases, the lack of antigen presentation can be attributed to the downregulation of genes needed for antigen processing, such as the transporters associated with antigen processing (TAP) 1, and the proteasomal component, low molecular weight proteins (LMP) 2. The TAP1 and LMP2 genes are transcribed from a shared bidirectional promoter containing an interferon (IFN)-gamma-response factor element; thus, the IFN-gamma-signal strongly induces both TAP1 and LMP2 expression. Low molecular weight proteins2-deficient mice exhibited the development of uterine leiomyosarcomas. Here, the differential responsiveness to IFN-gamma of the SKN human uterine leiomyosarcomas cell line was investigated. We now identify the G871E mutation in the ATP-binding region of Janus kinases 1, suggesting that the loss of TAP1 and LMP2 induction is a defect in the earliest steps of the IFN-gamma-signal pathway, resulting in the inability of SKN cells to upregulate the antigen-processing pathway. Understanding the mechanisms by which these tumors circumvent cytokine signalling, thereby evading antitumor-specific immunity, would greatly aid the efficacy of immunotherapy for treating uterine leiomyosarcomas.

A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions.

We have previously purified a novel GTPase-activating protein (GAP) for Ras which is immunologically distinct from the known Ras GAPs, p120GAP and neurofibromin (M. Maekawa, S. Nakamura, and S. Hattori, J. Biol. Chem. 268:22948-22952, 1993). On the basis of the partial amino acid sequence, we have obtained a cDNA which encodes the novel Ras GAP. The predicted protein consists of 847 amino acids whose calculated molecular mass, 96,369 Da, is close to the apparent molecular mass of the novel Ras GAP, 100 kDa. The amino acid sequence shows a high degree of similarity to the entire sequence of the Drosophila melanogaster Gap1 gene. When the catalytic domain of the novel GAP was compared with that of Drosophila Gap1, p120GAP, and neurofibromin, the highest degree of similarity was again observed with Gap1. Thus, we designated this gene Gap1m, a mammalian counterpart of the Drosophila Gap1 gene. Expression of Gap1m was relatively high in brain, placenta, and kidney tissues, and it was expressed at low levels in other tissues. A recombinant protein consisting of glutathione-S-transferase and the GAP-related domain of Gap1m stimulated GTPase of normal Ras but not that of Ras having valine at the 12th residue. Expression of the same region in Saccharomyces cerevisiae suppressed the ira2- phenotype. In addition to the GAP catalytic domain, Gap1m has two domains with sequence closely related to those of the phospholipid-binding domain of synaptotagmin and a region with similarity to the unique domain of Btk tyrosine kinase. These results clearly show that Gap1m is a novel Ras GAP molecule of mammalian cells.

Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors.

The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, suramin, or adenosine-3'-phosphate-5'-phosphosulfate did not inhibit ATP- or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G(i/o)-coupled P2Y receptors.

Microglia and the development of spongiform change in Creutzfeldt-Jakob disease.

Recent in vitro experiments suggest that neurotoxicity of the prion protein is dependent on the presence of microglia. We have studied 11 cases of Creutzfeldt-Jakob disease (CJD) using immunocytochemistry in combination with computerized image analysis to clarify the relationship between spongiform change and microglial activation. MHC class II-positive microglia were almost exclusively confined to cortical gray matter where the neuropil area occupied by these cells exceeded that of controls more than 350-fold. In cortical regions with a bimodal distribution of spongiform degeneration, the presence of class II-positive microglia correlated well with the presence of vacuolation in layer V, but significantly less with spongiform change in layers II and III. In areas where spongiform degeneration affected the entire depth of the cortex, activated microglia were predominantly located in the inner one-half of the cortex or were evenly distributed throughout all cortical laminae. Here, microglia exhibited atypical, tortuous cell processes and occasionally intracytoplasmic vacuoles, suggesting that microglia themselves may become a disease target. Taken together, our results provide indirect evidence against an early causative involvement of microglia in the development of spongiform change. At later stages, however, diseased microglia could produce harmful factors which mediate both astrogliosis and neuronal injury.

Expression of the alpha 2-macroglobulin-encoding gene in rat brain and cultured astrocytes.

The alpha 2-macroglobulin (alpha 2M), a protease inhibitor, is a major acute-phase protein in rats, and is produced in the liver during acute inflammation. Recently, it has been demonstrated that alpha 2M is also produced by cultured astrocytes from newborn rat brain and has neurite-promoting activity. Here, we found that the expression of the alpha 2M gene was significantly enhanced in the brain following intraperitoneal injection of the neurotoxicant, kainic acid (KA), suggesting that alpha 2M acts as an acute-phase protein in the brain, as in the case of the liver, and may be involved in neural repair processes. Expression of alpha 2M in cultured astrocytes was shown to be stimulated by interleukin-6 (IL-6) and/or leukemia inhibitory factor (LIF) in the presence of glucocorticoid. The amount of mRNAs for IL-6 and LIF increased in the brain of KA-injected rats prior to alpha 2M induction. These results strongly suggested that IL-6 and LIF are involved in alpha 2M induction in the brain, as in the case of the liver. Analysis of the cis-acting element(s) and the trans-acting factor(s) suggested that the regulatory mechanism for alpha 2M expression in astrocytes was similar to that in inflamed liver.

Retinoic acid enhances the secretion of plasminogen from cultured rat microglia.

To determine the amount of plasminogen in microglial conditioned medium, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for rat plasminogen was established. Weak cross-reactivity with human serum plasminogen was observed, while no reactivity was detected with frog and carp plasminogen. The specificity of the immunosorbent assay was confirmed by Western blotting. The secretion of plasminogen into the microglial culture medium was quantified by using the established ELISA and was found to be increased depending on the culture time and number of microglia. The secretion was increased about 5-fold by stimulation with retinoic acid, while interleukin-1, and basic fibroblast growth factor showed no significant effect.

Production and secretion of plasminogen in cultured rat brain microglia.

The production and secretion of plasminogen in cultured rat brain microglia was investigated. Urokinase-dependent caseinolytic activity was detected by zymography in microglial conditioned medium with a molecular weight of about 90 kDa. The 90-kDa protein was also detected by Western blotting with anti-rat plasminogen antiserum in the non-reducing condition. Immunoprecipitation with plasminogen antiserum following [35S]methionine labelling revealed that the plasminogen detected in microglial conditioned medium is synthesized in microglia. The amount of plasminogen in the conditioned medium was increased by stimulation with lipopolysaccharide. These results show that cultured microglia produce plasminogen and secrete it into the culture medium.

Plasminogen-binding protein associated with the plasma membrane of cultured embryonic rat neocortical neurons.

To investigate the receptor-like molecule(s) for plasminogen (PGn) on the neuronal surface, the properties of binding of PGn to the plasma membrane of cultured embryonic rat neocortical neurons were investigated. [125I]PGn was found to specifically bind to the plasma membrane depending on the incubation temperature and time. The binding was also affected strongly by ionic strength and slightly by Ca2+. Furthermore, ligand blotting analysis revealed that [125I]PGn binds to a major protein with an apparent molecular weight of 45 kDa among plasma membrane proteins. These results suggest that the 45-kDa protein is a PGn receptor-like molecule on the neuronal surface.

Induction of CHOP and apoptosis by nitric oxide in p53-deficient microglial cells.

Excessive nitric oxide (NO) has been implicated in neurotoxicity after stresses such as ischemia. NO toxicity is generally thought to be mediated by the DNA damage-p53 pathway or mitochondrial dysfunction. We investigated the mechanism of NO toxicity by using murine microglial MG5 cells established from p53-deficient mice. When MG5 cells were exposed to bacterial lipopolysaccharide plus interferon-gamma, mRNA and protein for inducible NO synthase (iNOS) were markedly induced, and apoptosis occurred. Under these conditions, we found that mRNA and protein for CHOP/GADD153, a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress-induced apoptosis, are induced. iNOS mRNA was induced 2 h after treatment, whereas CHOP mRNA began to increase at 6 h with a time lag. CHOP mRNA was also induced by NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP) or NOC18, or a peroxynitrite generator 3-(4-morpholinyl)-sydnonimine hydrochloride (SIN-1). Bip/GRP78, an ER chaperone which is known to be induced by ER stress, was also induced by SNAP or SIN-1, indicating that NO causes ER stress. These results suggest that NO-induced apoptosis in MG5 cells occurs through the ER stress pathway involving CHOP, but is independent of p53.

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 (GluRepsilon1/zeta1-GluRepsilon4/zeta1) 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 GluRepsilon1/zeta1: the IC(50) values for the GluRepsilon1/zeta1-GluRepsilon4/zeta1 were 41.7, 13.3, 12.6 and 11.5 microM, 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.

Microglia and the early phase of immune surveillance in the axotomized facial motor nucleus: impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage-colony stimulating factor-deficient mice.

Activation of microglia is among the first cellular changes in the injured CNS. However, little is known about their specific contribution to secondary damage or repair processes in neighboring neurons and nonneuronal cells or to the immune surveillance of the damaged tissue. Animal models with defective microglial response such as osteopetrosis provide an approach to explore these effects. Osteopetrosis (op) is an autosomal recessive mutation with a complete deficiency of the macrophage-colony stimulating factor (MCSF; CSF-1), an important mitogen for brain microglia. In the current study we examined the effects of this MCSF deficiency on the microglial reaction and the overall cellular response to nerve injury in the mouse axotomized facial motor nucleus. In the brain, MCSF receptor immunoreactivity was found only on microglia and was strongly up-regulated following injury. MCSF deficiency led to a failure of microglia to show a normal increase in early activation markers (thrombospondin, MCSF receptor, alpha M beta 2- and alpha 5 beta 1-integrins), to spread on the surface of axotomized motoneurons, and to proliferate after injury. Early recruitment of CD3(+) T-lymphocytes to the facial nucleus 24 hours after injury was reduced by 60%. In contrast, the neuronal and astrocyte response was not affected. There was a normal increase in the neuropeptides calcitonin gene-related peptide and galanin, neuronal c-JUN, and NADPH-diaphorase and a decrease in choline acetyltransferase and acetylcholinesterase. Astrocyte glial fibrillary acidic protein immunoreactivity also showed a normal increase. There was a normal influx of macrophages and granulocytes into the injured facial nerve. Synaptic stripping, neuronal survival, and speed of axonal regeneration were also not affected. The current results show a strong, selective effect of MCSF on the early activation of microglia and, indirectly, on lymphocyte recruitment. This early phase of microglial activation appears not to be involved in the process of repair following peripheral nerve injury. However, it is important in the initiation of inflammatory changes in the brain and in the interaction with the immune system.

Widespread activation of the brainstem preceding the recruiting rhythm in human epilepsies.

The excitability change of the brainstem was investigated before and during the conspicuous epileptic discharge in six patients with generalized convulsive seizures. The discharge consisted of a short duration of recruiting rhythm, which was considered equivalent to the seizure discharge on electroencephalogram. The excitability of the brainstem was measured with the parameters (amplitude and area) of component waves (wave-III and -V) of brainstem auditory evoked potentials. The theoretical background of the analysis is that brainstem auditory evoked potentials are 'far-field' potentials, by which they convey the information on the activity change of the brainstem even during the paroxysmal discharge within the cortex. The excitability of both the ventral (parameters of wave-III) and the dorsal brainstem (parameters of wave-V) exhibited a synchronized change (activation-inactivation). They were enhanced from -2.4+/-0.4 s, reaching the maxima before the onset of the seizure discharge, and decayed corresponding to the emergence of the recruiting rhythm. The results suggest the possibility that the widespread (ventral and dorsal) and synchronized activation of the brainstem triggers the seizure discharge in human generalized epilepsy. During the widespread activation of the brainstem, both the thalamus and the cortex probably undergo a suppressed inhibitory state through the cholinergic activation, precipitating the seizure discharge.

Distribution of the galectin-1 mRNA in the rat nervous system: its transient upregulation in rat facial motor neurons after facial nerve axotomy.

Galectin-1 is a member of the animal lectin family that displays conserved consensus sequences and similar carbohydrate binding specificities. Recent analyses revealed that galectin-1 plays an important role in the process of nerve regeneration. We analyzed the topological expression of galectin-1 mRNA in adult rat nervous system. Galectin-1 mRNA was predominantly observed in the cell bodies of neurons such as oculomotor nucleus (III), trochlear nucleus (IV), trigeminal motor nucleus (V), abducens nucleus (VI), facial nucleus (VII), hypoglossal nucleus (XII), red nucleus, and locus ceruleus. Neurons in pineal gland and dorsal root ganglia expressed galectin-1 mRNA. We next tested whether the axotomy of facial nerve altered the expression of galectin-1 mRNA in motor neurons. In the adult rats, the axotomy of facial nerve induced transient upregulation of galectin-1 mRNA around 6 h after axotomy. These results indicate that galectin-1 may play roles in the early event of the nerve injury and regeneration through the transient change of its expression level.

The effect of a secreted form of beta-amyloid-precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones.

1. The effects of secreted forms of beta-amyloid-precursor proteins (APP(S)s) on the intracellular Ca2+ concentration ([Ca2+]i) were investigated in rat cultured hippocampal neurones. APP695S, a secretory form of APP695, attenuated the increase in [Ca2+]i evoked by glutamate. In addition, APP695S itself evoked an increase in [Ca2+]i in 1 or 2 day-cultured hippocampal cells, but not in 7 to 13 day-cultured cells. 2. Eighty-one percent of neurones which were immunocytochemically positive for microtubule-associated protein 2 responded to APP695S with an increase in [Ca2+]i. 3. APP695S induced a transient rise in [Ca2+]i even in the absence of extracellular Ca2+ and produced an elevation in inositol-1,4,5-trisphosphate (IP3) in a concentration-dependent manner from 100 to 500 ng ml(-1). In the presence of extracellular Ca2+, APP695S caused a transient rise in [Ca2+]i followed by a sustained phase at high [Ca2+]i, suggesting Ca2+ entry from the extracellular space. 4. The [Ca2+]i elevation was mimicked by amino terminal peptides of APPs, but not by carboxy terminal peptides. 5. These results taken together suggest that APP695S induces an increase in [Ca2+]i in hippocampal neurones through an IP3-dependent mechanism that changes according to the stage of development.

Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures.

1. In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difluoro-phenoxyaceta mide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors. 2. PEPA dose-dependently potentiated AMPA-induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell-to-cell variation in rat hippocampal cultures. 3. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (> or = 2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested. 4. Reverse transcriptase-polymerase chain reaction analysis suggested that GluR2-flip, GluR1-flip, GluR2-flop, and GluR1-flop were abundantly expressed (in this rank order) in the cultures used. 5. In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice-variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high. 6. These results suggest that the potentiation by PEPA alone reveals cell-to-cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia.

1. ATP (10-100 microM), but not glutamate (100 microM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 microM) nor glutamate (100 microM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 microM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 microM). The Ca2+ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 microM to 10 microM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 microM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 microM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but alpha,beta-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 microM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 microM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 microM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 microM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.