Search PubMed⌕ Search

Biomedical subjects

Mitsuhiro Morita

Publications and source records attributed to Mitsuhiro Morita.

18 recordsLinked to original sources
PubMed2007

Dual regulation of astrocyte gap junction hemichannels by growth factors and a pro-inflammatory cytokine via the mitogen-activated protein kinase cascade.

Evidence that glutamate and ATP release from astrocytes can occur via gap junction hemichannels (GJHCs) is accumulating. However, the GJHC is still only one possible release mechanism and has not been detected in some studies, although this may be because the levels were below those detectable by the system used. Because of these conflicting results, we hypothesized that release from astrocyte GJHCs might depend on different astrocyte states, and screened for factors affecting astrocyte GJHC activity by measuring fluorescent dye leakage via GJHCs using a conventional method for GJHC acivation, i.e. removal of extracellular divalent cations. Astrocytes cultured in Dulbecco's minimal essential medium containing 10% fetal calf serum, a medium widely used for astrocyte studies, did not show dye leakage, whereas those cultured in a defined medium showed substantial dye leakage, which was confirmed pharmacologically to be due to GJHCs and not to P2x7 receptors. EGF and bFGF inhibited the GJHC activity via the mitogen-activated protein kinase cascade, and the effect of the growth factors was reversed by interleukin-1beta. These factors altered GJHC activity within 10 min, but did not affect connexin 43 expression. GJHC activity in hippocampal slice culture preparations was measured using the same methods and found to be regulated in a similar manner. These results indicate that astrocyte GJHC activity is regulated by brain environmental factors.

Animals↗
PubMed2006

Effects of bifemelane on the calcium level and ATP release of the human origin astrocyte clonal cell.

The effect of bifemelane hydrochloride (bifemelane) was examined on human origin astrocyte clonal cells (Kings-1). Bifemelane (125 - 1,000 microM) induced a dose-dependent increase in the intracellular calcium concentration ([Ca(2+)](i)). In the highest concentration (1,000 microM), the drug caused the second large increase in [Ca(2+)](i) during the washing. The increase that occurred during the administration partially remained in the Ca(2+)-free medium and was blocked by 2-aminoethoxydiphenyl borate (2-APB), an IP(3)-receptor blocker, indicating that the source of Ca(2+) for the increase could be ascribed to the intracellular store. The increase in [Ca(2+)](i) was not observed during washing with Ca(2+)-free medium, but was observed when the washing was performed with Ca(2+)-containing medium. Bifemelane caused a dose-dependent ATP release, but histamine and carbachol, which induced a large increase in [Ca(2+)](i), had no effects on the ATP release. The effects on the [Ca(2+)](i) were blocked by pretreatment with pyridoxal phosphate-6-azophenyl-2',4' disulfonic acid, a P2-receptor antagonist. Although the mechanisms of ATP release induced by the drug have not been elucidated yet, the present results demonstrate that the increase in [Ca(2+)](i) induced by bifemelane is not due to its direct effect on the cells, but is dependent upon the ATP released from the cells.

Adenosine Triphosphate↗
PubMed2006

A novel effect of bifemelane, a nootropic drug, on intracellular Ca2+ levels in rat cerebral astrocytes.

We investigated the effects of bifemelane, a nootropic drug, on the intracellular calcium concentration ([Ca2+]i) in rat cerebral astrocytes using a Ca2+ imaging device. At concentrations of 10 - 30 microM, bifemelane induced a slow onset and small increase in the [Ca2+]i, while at higher concentrations (100 - 300 microM), it induced a rapid transient increase in the [Ca2+]i during administration and a second large increase was seen during drug washout. The first peak was observed in Ca2+-free medium, but its onset was significantly delayed, and no second peak was seen. Neither of these effects was seen in cells treated with thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, in Ca2+-free medium. When thapsigargin-treated astrocytes were returned to normal medium containing Ca2+ (1.8 mM), the [Ca2+]i increased significantly, and this effect was reversely inhibited by bifemelane. We conclude that bifemelane causes the first peak by stimulating release from intracellular Ca2+ stores and the second by capacitive entry through store-operated Ca2+ channels. Although the detail mechanisms of action of the drug are still unknown, bifemelane will be provided as a pharmacological tool for basic studies on astrocytes.

Animals↗
PubMed2006

No effect of recombinase-mediated DNA transfer on production efficiency of transgenic rats.

It was reported that recombinase-A protein (RecA)-coated exogenous DNA was more likely to be integrated into mouse, goat and pig genomes. The objective of this study was to investigate whether integration of exogenous DNA into the rat genome is improved by the recombinase-mediated DNA transfer. Pronuclear microinjection of RecA-coated EGFP or OAMB DNA resulted in a production efficiency of transgenic rats of 1.4-2.9%, comparable with 0.9-2.6% when non-coated control DNA was used. Intracytoplasmic injection of the sperm heads exposed to RecA-coated EGFP DNA did not produce any transgenic rats (0 vs. 0-2.8% in control groups). Thus, the recombinase-mediated DNA transfer contributed very little to the production of transgenic rats by means of pronuclear microinjection and intracytoplasmic sperm injection.

Animals↗
PubMed2005

Lipopolysaccharide and proinflammatory cytokines require different astrocyte states to induce nitric oxide production.

Nitric oxide (NO) production by astrocytes is a significant factor affecting brain physiology and pathology, but the mechanism by which it is regulated is not known. Previous studies using different specimens and stimuli might have described different aspects of a complex system. We investigated the effect of culture and stimulus conditions on NO production by cultured astrocytes and identified two combinations of these allowing NO production. Lipopolysaccharide (LPS)-induced NO production required a high seeding cell density and was independent of the serum concentration, whereas that induced by proinflammatory cytokines required simultaneous treatment with interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma and low-serum conditions but was less affected by the seeding density. These two pathways showed differential sensitivity to protein kinase inhibitors. Both LPS and cytokines induced expression of inducible nitric oxide synthase (iNOS). Although LPS-induced iNOS expression required a high seeding cell density, cytokine-induced iNOS expression, in contrast to NO production, was not affected by the serum concentration. These results suggest that astrocytes interact with the environment and alter their responsiveness to NO production-inducing stimuli by regulating iNOS expression and activity. This is the first evidence for the selective use of two different regulatory pathways in any cell type.

Animals↗
PubMed2005

Autocrine activation of EGF receptor promotes oscillation of glutamate-induced calcium increase in astrocytes cultured in rat cerebral cortex.

We previously reported that astrocytes cultured for more than 2 days in a defined medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed calcium oscillation in response to glutamate, whereas the response pattern was transient in the absence of the exogenous growth factors. In the present study, we found that astrocytes showed glutamate-induced calcium oscillation, even in growth factor-free medium, if the cells had been cultured for more than 5 days. The calcium oscillation promoted by the prolonged culture period was suppressed by an inhibitor of EGF receptor tyrosine kinase, but not by a neutralizing antibody to bFGF, indicating that the accumulation of an autocrine factor that activates the EGF receptor leads to calcium oscillation. Astrocytes in our culture system expressed EGF, transforming growth factor alpha (TGFalpha), bFGF and acidic fibroblast growth factor (aFGF). Exogenous aFGF, which induced astrocyte immediate early gene expression to the same extent as EGF or bFGF, did not affect calcium oscillation. Exogenous EGF and bFGF promoted astrocyte hypertrophic morphology and proliferation, as well as calcium oscillation. In contrast, these properties did not accompany calcium oscillation induced by the prolonged culture period. These results suggest that astrocytes possess the ability to promote their own calcium oscillation, which is independent of hypertrophic changes to reactive astrocytes.

Animals↗
PubMed2005

A novel phospholipase C, PLC(eta)2, is a neuron-specific isozyme.

Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, beta-, gamma-, delta-, epsilon-, zeta-, and eta-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC(eta)2. PLC(eta)2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC(eta)2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC(eta)2 was more sensitive to calcium concentration than the PLC activity of the PLCdelta-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC(eta)2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC(eta)2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC(eta)2 in pyramidal cells of the hippocampus. Finally, we found that PLC(eta)2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC(eta)2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.

Amino Acid Sequence↗
PubMed2005

Ca2+-dependent induction of intracellular Ca2+ oscillation in hippocampal astrocytes during metabotropic glutamate receptor activation.

We have investigated whether the intracellular calcium concentration ([Ca(2+)](i)) oscillations induced in astrocytes using the metabotropic glutamate-receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) are Ca(2+)-dependent, using three different Ca(2+) indicators with different affinities for Ca(2+). When rat hippocampal cells in culture were loaded with fura-2 (K(d): 145 nM), two-thirds of the cells showed obvious oscillatory increase in [Ca (2+)](i) during t-ACPD-administration. Those cells were identified as astrocytes by immuno-histochemistry in our previous paper. In cells loaded with fura-2FF (K(d): 25,000 nM), a similar percentage of t-ACPD-responsive cells showed oscillatory [Ca(2+)](i) changes. However, in cells loaded with quin-2 (K(d): 60 nM), t-ACPD induced no oscillatory responses, but some cells showed a small transient increase in the [Ca(2+)](i). The same small transient [Ca(2+)](i) increase was seen in cells loaded with both fura-2FF and BAPTA, a Ca(2+) chelator (K(d): 135 nM). These findings indicate the involvement of [Ca(2+)](i)-dependent regulatory mechanisms in the induction of the t-ACPD-induced oscillatory change in the [Ca(2+)](i) in astrocytes.

Animals↗
PubMed2005

Joint biomarkers in idiopathic femoral head osteonecrosis: comparison with hip osteoarthritis.

OBJECTIVE: To compare concentrations of joint biomarkers in synovial fluid (SF) between idiopathic osteonecrosis of the femoral head (ION) and osteoarthritis (OA) of the hip joint. METHODS: Levels of the joint biomarkers cartilage oligomeric matrix protein (COMP), antigenic keratan sulfate (AgKS), and hyaluronan (HA) in SF samples from 21 cases of ION and their relationship to disease stage and history of steroid use were assessed and compared to the result of 29 cases of hip OA. RESULTS: In both the ION and hip OA groups, levels of COMP and AgKS in SF showed a significant positive correlation. The ION group had significantly higher levels of AgKS in SF than the hip OA group. In the ION group, stage II patients had significantly higher SF levels of both COMP and AgKS than those in stage III patients. No difference in level of HA in hip joint SF was found between steroid and non-steroid treated ION patients or between the stage II and III subgroups. CONCLUSION: SF levels of COMP and AgKS may serve as useful joint biomarkers that reflect cartilage metabolism not only in hip OA but also in ION.

Biomarkers↗
PubMed2004

Rotational acetabular osteotomy for acetabular dysplasia of the hip with a giant acetabular bone cyst: a case report.

In acetabular dysplasia of the hip joint accompanied by a giant acetabular bone cyst, rotational acetabular osteotomy may cause serious complications, such as bone necrosis after surgery or fracture of the fragile acetabulum during the operation. In a patient with this condition, we performed a two-stage operation: first, autogenous bone grafting supplemented with hydroxyapatite filling, then rotational acetabular osteotomy (after new bone formation had been assured). Radiographs and CT scans showed favorable fusion of the grafted bone. Some 18 months after the second operation, arthrograms showed no inflow of contrast medium from the articular cavity into the bone cyst region, although this had been observed before treatment. Thus, an effective remodeling of bony congruency was indicated in the mobile acetabulum 5 years after the second operation. This two-stage operation appears to be useful for correcting acetabular dysplasia accompanied by a giant bone cyst and to carry a reduced risk of serious complications, such as deterioration of the articular surface of the acetabulum or necrosis of the translocated acetabulum.

Acetabulum↗
PubMed2004

Telomere length, telomerase activity, and expression of human telomerase reverse transcriptase mRNA in growth plate of epiphyseal articular cartilage in femoral head during normal human development and in thanatophoric dysplasia.

Telomeres are important in chromosome structure and function, protecting against their degradation. However, few studies have examined telomeres in growth plates within articular cartilage during normal development. We investigated frozen sections that were obtained from 57 reference autopsy cases (aged from 16 weeks of gestation to 91 years) and from 2 patients with thanatophoric dysplasia. In the reference cases, telomere length was significantly longer in growth plates obtained from the 10 cases that were aged from 16 weeks of gestation to 10 years than in those from 47 of the adult cases (aged 20 to 91 years). In fetal, neonatal, and child cases, telomerase activity was significantly higher in the hypertrophied zone (HZ) in growth plates than in the other 3 zones. The hTERT mRNA staining intensity (staining area) was stronger (larger) in HZ and the proliferating zone than in the calcified zone and resting zone. In thanatophoric dysplasia, telomere length and telomerase activity were short and low, respectively, compared with those of normal growth plates at an equivalent age, and expression of hTERT mRNA was negative or weakly positive in all 4 zones within growth plates. These results suggest that telomere length and telomerase activity have significant effects in the growth plates of articular cartilage, particularly at developmental ages from fetus to child. We speculate that short telomere length and low telomerase activity may be important for chondrocyte differentiation in rhizomeric shortening of the limbs in thanatophoric dysplasia.

Adolescent↗
PubMed2004

A novel method to quantify calcium response pattern and oscillation using fura2 and acridine orange.

To study calcium imaging data of cell populations that have various response patterns in peak amplitude and frequency of calcium oscillation in response to stimulation, comprehensive characterization based on statistical analysis of each response is important. In cultures of cells that are flat and in contact with each other, it is difficult to distinguish individual cells in calcium imaging data. We have developed a novel method to determine areas corresponding to individual cells in calcium imaging data. Rat neonatal cerebral astrocytes were filled with the calcium indicator Fura2, stained with acridine orange, and illuminated with UV light. The cell nuclei were clearly visualized. In addition, the images of these nuclei were useful for analyzing concentration-dependent alteration of calcium oscillation of cultured astrocytes in response to glutamate. This novel method may be useful for studying factors affecting calcium response patterns of cultured cell populations, including culture conditions, stimulus paradigms, and synthetic compounds.

Acridine Orange↗
PubMed2004

A novel variant of ionotropic glutamate receptor regulates somatostatin secretion from delta-cells of islets of Langerhans.

Many metabolic factors affect the secretion of insulin from beta-cells and glucagon from alpha-cells of the islets of Langerhans to regulate blood glucose. Somatostatin from delta-cells, considered a local inhibitor of islet function, reduces insulin and glucagon secretion by activating somatostatin receptors in islet cells. Somatostatin secretion from delta-cells is increased by high glucose via glucose metabolism in a similar way to insulin secretion from beta-cells. However, it is unknown how low glucose triggers somatostatin secretion. Because L-glutamate is cosecreted with glucagon from alpha-cells under low-glucose conditions and acts as a primary intercellular messenger, we hypothesized that glutamate signaling triggers the secretion of somatostatin. In this study, we showed that delta-cells express GluR4c-flip, a newly identified splicing variant of GluR4, an (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptor of rat. After treatment with L-glutamate, AMPA, or kainate, secretion of somatostatin from isolated islets was significantly stimulated under low-glucose conditions. The glutamate-dependent somatostatin secretion was Ca(2+) dependent and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Somatostatin in turn inhibited the secretion of L-glutamate and glucagon from alpha-cells. These results indicate that L-glutamate triggers somatostatin secretion from delta-cells by way of the GluR4c-flip receptor under low-glucose conditions. The released somatostatin may complete the feedback inhibition of alpha-cells. Thus, alpha- and delta-cells may communicate with each other through L-glutamate and somatostatin signaling.

Animals↗
PubMed2003

Dual regulation of calcium oscillation in astrocytes by growth factors and pro-inflammatory cytokines via the mitogen-activated protein kinase cascade.

In response to neurotransmitters, astrocytes show various types of calcium increase (transient, oscillatory, and complex), the physiological significance of which is still controversial. To explore this variability, we examined factors affecting the calcium increase pattern in cultured astrocytes and investigated the consequences of the astrocytic calcium response in slice preparations. We found that growth factors (GFs) (EGF plus basic FGF) promoted calcium oscillation in response to glutamate, ATP, or thimerosal (which directly activates the inositol-1,4,5 triphosphate receptor) and that this effect was suppressed by pro-inflammatory cytokines (interleukin-1beta or tumor necrosis factor-alpha), lipopolysaccharide, or a MEK (mitogen-activated protein kinase kinase) inhibitor, suggesting dual regulation of calcium oscillation in astrocytes by factors affecting brain function and pathology via the mitogen-activated protein kinase (MAPK) cascade. The calcium oscillation was accompanied by enlargement of the calcium store, cell proliferation, and the development of a hypertrophic morphology. The cytokines suppressed GF-induced MAPK-dependent immediate early gene promoter activation, but not phosphorylation of extracellular signal-regulated kinase (ERK), showing that they affected gene regulation by acting on the MAPK cascade downstream of ERK. In slice preparations, a metabotropic glutamate receptor agonist converted the spontaneous neuronal calcium increase, attributable to synaptic transmission, to an oscillatory response similar to that seen in astrocytes in culture, indicating that the calcium response in astrocytes acted as a feedback mechanism on the activity of neighboring neurons. This is the first evidence for a dual regulation of calcium oscillation by physiological factors and for the control of calcium dynamics actually being used in physiological processes.

Adenosine Triphosphate↗
PubMed2003

Simultaneous imaging of phosphatidyl inositol metabolism and Ca2+ levels in PC12h cells.

To elucidate the spatial and temporal relationships between phosphatidyl inositol metabolism and changes in intracellular calcium levels, we developed a simultaneous imaging system using green fluorescent protein fused with the pleckstrin homology domain, and the fluorescent calcium indicator, FuraRed. The redistribution of the fusion protein, which represents the phosphatidyl inositol metabolism process, was quantified by calculating the coefficient of variance of the fluorescence over the entire cytosolic region, excluding the nucleus. This calculation increased the reproducibility, compared to the normalized fluorescence changes in arbitrarily selected cytosolic regions used in conventional analysis. The application of this method to analyzing the response of PC12h cells to a number of pharmacological stimuli showed that the extent of the phosphatidyl inositol metabolism was related to the calcium level, but not induced by calcium alone.

Animals↗
PubMed2003

Expression of group I metabotropic glutamate receptors in rat hippocampal cells in culture and their characterization by intracellular calcium ion dynamics.

The distribution of group I metabotropic glutamate receptors in rat hippocampal cells in culture was examined by calcium imaging and immunocytochemistry. To distinguish different cell types in the culture, the effects of t-ACPD ((1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid) and of NMDA (N-methyl-D-aspartate) were examined. About 40% of the cultured cells showed either a transient increase or a sustained or oscillatory increase in the intracellular calcium concentration ([Ca(2+)](i)) during t-ACPD administration, while about 60% of the cells showed a sustained [Ca(2+)](i) increase in response to NMDA. Cells that showed an oscillatory [Ca(2+)](i) change during t-ACPD administration did not respond to NMDA administration, while cells that showed a sustained [Ca(2+)](i) increase during NMDA administration did not show any oscillatory response to t-ACPD. After pharmacological examination using those two agonists, the cultured cells were subjected to immunocytochemistry using anti-GFAP and ant-MAP-2 antibodies to distinguish, respectively, astrocytes and neurons. All cells responding to NMDA with a sustained [Ca(2+)](i) increase were MAP-2-positive, whereas all cells showing either oscillatory or sustained [Ca(2+)](i) increase in response to t-ACPD were GFAP-positive. The present results show that, in these cultures, group I metabotropic glutamate receptors are mainly expressed on glial cells and contribute to dynamic [Ca(2+)](i) changes in astrocytes.

Animals↗
PubMed2002

Cell type-selective expression of green fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures.

A cell type-specific green fluorescent protein (GFP) expression system in rat cortical primary cultures has been developed for the fluorescence labeling of brain cells. Lipid-mediated transfection (lipofection) was employed, allowing the establishment of a convenient efficient system for the analysis of individual cells. To achieve cell type-specific labeling, GFP expression vectors containing the rat neuron-specific enolase (NSE) gene promoter, human glial fibril acidic protein (GFAP) gene promoter, human elongation factor (EF-1alpha) gene promoter, or human cytomegalovirus (CMV) immediate early promoter were constructed, and their specificities examined. Vectors containing the CMV or GFAP promoter resulted primarily in GFP expression in astrocytes, while those containing the EF1-alpha or NSE promoter resulted primarily in GFP expression in neurons. This labeling system was applied to the morphological analysis of living neurons and to cell type-selective calcium imaging. Confocal microscopy revealed that individual GFP-expressing neurons had processes, which were longer than 500 microm and bore spine-like protrusions. A calcium-indicating GFP variant, yellow cameleon (YC2.1), was expressed in the same system, and cell type-selective calcium imaging performed. On pharmacological stimulation, YC2.1-expressing neurons responded to depolarizing stimuli, but not to the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), while astrocytes responded only to tACPD.

Animals↗