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Biomedical subjects

T Masaki

Publications and source records attributed to T Masaki.

At least 163 records · Page 9Linked to original sources
PubMed1999

Transforming growth factor-beta1 may be involved in shunt obstruction in patients on chronic hemodialysis.

Obstructed shunt vessels were studied immunohistochemically to clarify the mechanism of shunt obstruction in hemodialysis patients. The subjects were 12 hemodialysis patients with shunt obstruction, and 8 patients newly started on hemodialysis were used as the controls. Cryosections of shunt tissue were prepared and stained for thrombomodulin as well as transforming growth factor-beta1 using the enzyme antibody method. In the obstructed shunt group, the intima was significantly thicker than in the control group. In addition, staining of the intima for thrombomodulin was decreased in the obstructed shunt group when compared with the controls. Staining for transforming growth factor-beta1 was related to intimal thickening and cell proliferation. These results indicate that release of thrombomodulin occurs with vascular endothelial cell damage and that transforming growth factor-beta1 may be involved in intimal hypertrophic change and shunt obstruction.

Adult↗
PubMed1999

[Analysis of Ca2+ entry channels in endothelin-1-induced contraction of rat aorta using new blockers].

1) We have recently shown that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). These channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. Here we characterized Ca2+ entry channels involved in ET-1-induced contractions of rat thoracic aortic rings and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of single smooth muscle cells using these blockers. 2) LOE 908 or a blocker of voltage-operated Ca2+ channel nifedipine had no effect on the contractions and increases in [Ca2+]i induced by thapsigargin, whereas SK&F 96365 abolished them. 3) The contractions and increases in [Ca2+]i induced by ET-1 depended on extracellular Ca2+ but were resistant to nifedipine. The responses to lower concentrations (< or = 0.1 nM) of ET-1 were abolished by either SK&F 96365 or LOE 908. The responses to higher concentrations (> or = 1 nM) were abolished by SK&F 96365, but were partially resistant to LOE 908. 4) These results show that the contractions and increases in [Ca2+]i of rat aortic smooth muscles at lower concentrations of ET-1 involve only one Ca2+ entry channel which is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those at higher concentrations of ET-1 involve another Ca2+ entry channel which is sensitive to SK&F 96365 but resistant to LOE 908 (SOCC) in addition to the former channel.

Acetamides↗
PubMed1999

[Characterization of voltage-independent Ca2+ channels activated by endothelin-1].

To clarify Ca2+ entry channels involved in the endothelin-1 (ET-1)-induced increase in the intracellular concentration ([Ca2+]i), we performed whole-cell recordings of patch-clamp techniques and monitoring of [Ca2+]i with Ca2+ indicators fura-2 and fluo-3 in A7r5 cells (a cell line derived from rat thoracic aortic smooth muscle cells). With whole-cell recordings, lower concentrations (< or = 1 nM) of ET-1 activated a Ca(2+)-permeable nonselective cation channel (designated NSCC-1). In contrast, higher concentrations (> or = 1 nM) of ET-1 activated two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). Importantly, we found that these Ca2+ channels can be pharmacologically discriminated using blockers of the so-called receptor operated Ca2+ influx such as SK&F 96365 and LOE 908. That is, NSCC-1 is resistant to SK&F 96365 but sensitive to LOE 908; NSCC-2 is sensitive to both SK&F 96365 and LOE 908; SOCC is sensitive to SK&F 96365 but resistant to LOE 908. Using these blockers, we analyzed the ET-1-induced increase in [Ca2+]i. The increase in [Ca2+]i induced by lower concentrations of ET-1 was resistant to SK&F 96365 but sensitive to LOE 908. In contrast, the increase in [Ca2+]i induced by higher concentrations of ET-1 was partially suppressed to approximately 30% of controls by either SK&F 96365 or LOE 908 alone, and it was abolished by their combination. These results show that the increase in [Ca2+]i induced by lower concentrations (< or = 1 nM) of ET-1 results from Ca2+ influx through NSCC-1, whereas the increase in [Ca2+]i induced by higher concentrations (> or = 10 nM) of ET-1 results from Ca2+ influx through NSCC-1, NSCC-2 and SOCC.

Acetamides↗
PubMed1999

Ca2+ entry channels in rat thoracic aortic smooth muscle cells activated by endothelin-1.

The contraction of the rat aorta induced by endothelin-1 (ET-1) requires entry of extracellular Ca2+, but involvement of voltage-operated Ca2+ channel is minor. Using whole-cell recordings of patch-clamp and monitoring of the intracellular free Ca2+ concentration ([Ca2+]i), we characterized Ca2+ entry channels in A7r5 cells activated by ET-1. ET-1 activates three types of voltage-independent Ca2+ entry channels: two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC). Furthermore, it was found that these channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is resistant to SK&F 96365, but sensitive to LOE 908, whereas NSCC-2 is sensitive to both SK&F 96365 and LOE 908. SOCC is sensitive to SK&F 96365, but resistant to LOE 908. Using these channel blockers, we analyzed Ca2+ entry channels involved in the ET-1-induced contractions of rat thoracic aorta and increases in [Ca2+]i of single smooth muscle cells. The responses to lower concentrations of ET-1 (< or = 0.1 nM) were abolished by either SK&F 96365 or LOE 908 alone. In contrast, the responses to higher concentrations of ET-1 (> or = 1 nM) were suppressed by SK&F 96365 or LOE 908 to about 10% and 35% of controls, respectively, and abolished by combined treatment with SK&F 96365 and LOE 908. These results show that the responses of rat aorta to lower concentrations of ET-1 involve only one Ca2+ channel that is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those to higher concentrations of ET-1 involve NSCC-1, NSCC-2 and SOCC, contributing 10%, 55% and 35%, respectively, to total Ca2+ entry.

Acetamides↗
PubMed1999

Factors influencing arteriovenous fistula dysfunction in Japanese patients on chronic hemodialysis.

Arteriovenous fistula dysfunction is a constant problem in chronic hemodialysis patients. We investigated the factors influencing fistula dysfunction in 184 patients on chronic hemodialysis. Stepwise regression analysis and Cox proportional hazards model were used to assess the relationship between fistula dysfunction and age, sex, duration of hemodialysis, diabetes mellitus, hematocrit, serum creatinine, blood urea nitrogen, Kt/V, prothrombin time, blood pressure, anticoagulant therapy, dose of erythropoietin, calcium channel blocker therapy, and angiotensin-converting enzyme inhibitor therapy. Fistula dysfunction showed a significant relationship with a low systolic blood pressure, a low hematocrit, the presence of diabetes mellitus, and angiotensin-converting enzyme inhibitor therapy. These results suggested that treatment with angiotensin-converting enzyme inhibitors may help to prevent fistula dysfunction.

Adult↗
PubMed1999

Structural basis of the function of endothelin receptor.

Endothelin receptor is a good model for analysis of the function of heptahelical G-protein coupled receptor. In ligand binding to the heptahelical receptor, the receptor has two functions, i.e. 'message' and 'address' functions. Each function has been assigned to different domain of the receptor. A different part of the ligand structure also corresponds to each domain of the receptor. Classically, classification of receptor has been done according to the difference of address domain, i.e. affinity difference of the receptor. However, present results predict that the classification of receptor is also possible according to the message domain. After stimulation of ET receptor by a ligand, the receptor transmits a signal to G-protein. Several kinds of G-proteins can possibly be activated. Different structural domains of the receptor are assigned to the coupling of the different Galpha-protein. Activated G-protein transmits the message to effector. Each Galpha-protein acts on different target molecules, resulting in different responses. However, the activation of each Galpha-protein presumably depends on its intracellular level. Even if the same receptor is activated with the same ligand, resulting final response is different from cell to cell. Therefore, classification of receptor according to the function of the receptor is difficult.

Animals↗
PubMed1999

[A case of myasthenia gravis accompanied by steroid-resistant nephritic syndrome and CD57+ lymphocytes expansion in peripheral blood].

A 38-year-old woman showed symptoms of myasthenia gravis (MG) three months after receiving thymectomy for malignant thymoma. She was treated with anti-acetylcholine esterase drugs and azathioprine over 10 years with two exacerbations, which were controlled by plasmapheresis and large amounts of steroid. Nephrotic syndrome developed suddenly at the age of 48, was progressive even after azathioprine withdrawal, and resistant to several immunosuppressive therapies such as steroids and cyclosporine A, and plasmapheresis. She died of systemic infection one-and-a-half years after the onset of nephrotic syndrome. Immunological studies revealed several abnormalities of cellular immunity. The expansion of gamma-delta T cells and CD57+ lymphocytes in peripheral blood was characteristic findings. These cells are thought to originate from the extrathymic process. Nephrotic syndrome has been thought to be sometimes complicated with thymoma. Although some pathogenetic possibilities about combination of nephrotic syndrome and thymoma were supposed, none has yet been clarified. As we noticed the remarkable increase in the number of CD57+ cells, we examined its proportion in the peripheral blood of patients with MG and/or thymoma, as well as in individuals without sickness. The study revealed the expansion of CD57+ cells in MG thymoma patients (32.3 +/- 15.9%), compared with healthy controls (15.2 +/- 5.4%), MG non-thymoma patients (20.3 +/- 11.5%), and thymoma non-MG patients (15.2 +/- 12.0%) statistically (Mann-Whitney's U test). Therefore, we supposed that the peripheral CD57+ cell expansion was associated with parathymic immunological abnormalities, such as MG, thymoma, and nephrotic syndrome.

Adult↗
PubMed1998

Genomic structure and chromosomal mapping of the human and mouse hippocalcin genes.

In an attempt to elucidate the possible relationship of hippocalcin to neurological disorders, we isolated and analyzed the human and mouse hippocalcin genes. The human and mouse hippocalcin genes contain three exons and two introns, and span approximately 7 and 8kb, respectively. The exon/intron splice junctions of the human and mouse genes are all situated in exactly the same position and are not consistently placed with respect to the coding regions of the tandemly repeated EF-hand motifs. The amino acid sequences of human and mouse hippocalcins deduced from the genes are 100% identical. Within the 2-kb 3'-flanking sequences of the human and mouse genes, one conserved polyadenylation signal was identified at positions 762 and 823bp downstream from TAG, respectively. Within the 2.6-kb 5'-flanking sequences of the human and mouse genes, neither a canonical 'TATA' box nor a 'CAAT' box was found. Southern blot analysis of the human and mouse genomic DNAs demonstrated that the positive bands coincide exactly with those expected from the sequence of the cloned genes, indicating that the human and mouse hippocalcin genes are present as a single-copy gene. Fluorescence in-situ hybridization revealed that the human hippocalcin gene is located at chromosome 1 p34.2-35 and the mouse hippocalcin gene at chromosome 4 D2-D3.

Animals↗
PubMed1998

High-affinity arginine transport of bovine aortic endothelial cells is impaired by lysophosphatidylcholine.

The mechanisms of endothelial dysfunction characterized by the impaired nitric oxide (NO) release have not yet been clarified. Because the phenomenon is mimicked in vitro by the application of oxidized LDL and its major lipid constituent, lysophosphatidylcholine (LPC), we analyzed their effects on the arginine-NO system, especially on the arginine transport system. LPC inhibited NO release induced by ADP in cultured bovine aortic endothelial cells. The inhibition was attenuated by the excess amount of extracellular arginine. LPC was found to inhibit the arginine transport in bovine aortic endothelial cells, which is mediated by high- and low-affinity components. LPC predominantly impaired the high-affinity component. In the presence of a high concentration of arginine, LPC showed apparently no inhibition of arginine transport, because the low-affinity transporter compensated for the activity. Taken together, the impairment of the high-affinity transport system might account for the inhibition of NO release by LPC. LPC also inhibited arginine transport in the intima of intact bovine aorta. Furthermore, LPC inhibited the activity of the high-affinity arginine transporter in endothelial cells, in the cationic amino acid transporter-1 expressed in COS-7 cells. The activity of cationic amino acid transporter-1 might be important for the prevention of endothelial dysfunction.

Adenosine Diphosphate↗
PubMed1998

Expression of lectin-like oxidized low density lipoprotein receptor-1 in human and murine macrophages: upregulated expression by TNF-alpha.

Uptake of oxidized low density lipoprotein (Ox-LDL) and subsequent foam cell transformation have been implicated in early atherogenesis. Although multiple molecules, including class A and B scavenger receptors, have been identified as Ox-LDL receptors, additional receptors may also be involved in this process. Here, we provide evidence that lectin-like Ox-LDL receptor-1 (LOX-1), a novel Ox-LDL receptor initially identified in vascular endothelial cells, is also expressed in macrophages in humans and mice. Expression of LOX-1 can be induced after macrophage-like differentiation in vitro in human peripheral blood monocytes and the related cell line THP-1 cells. Furthermore, LOX-1 expression can also be detected in resident peritoneal macrophages, and can be upregulated by an inflammatory cytokine TNF-alpha. These results suggest that LOX-1 in macrophages may play an important role in Ox-LDL uptake and subsequent foam cell formation in this cell type.

Animals↗
PubMed1998

Inducible expression of lectin-like oxidized LDL receptor-1 in vascular endothelial cells.

Endothelial dysfunction, or activation, elicited by oxidized LDL (Ox-LDL) or its lipid constituent, has been implicated in the initiation and progression of atherosclerosis. We have recently identified a C-type lectin-like molecule, designated lectin-like Ox-LDL receptor-1 (LOX-1), which acts as a cell-surface receptor for Ox-LDL in cultured vascular endothelial cells. In this study, we provide evidence that LOX-1 expression can be upregulated by tumor necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA) in cultured bovine aortic endothelial cells. TNF-alpha and PMA upregulated LOX-1 protein and mRNA in a time- and dose-dependent manner. Nuclear runoff assay revealed that TNF-alpha stimulates transcription of the LOX-1 gene. Chinese hamster ovary K1 cells stably expressing LOX-1 internalized 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled Ox-LDL but did not significantly internalize acetylated LDL (Ac-LDL), which was effectively suppressed by excess amounts of unlabeled Ox-LDL but not by Ac-LDL. Upregulated expression of LOX-1 by TNF-alpha and PMA was associated with increased uptake of DiI-Ox-LDL that cannot be blocked by excess amounts of unlabeled Ac-LDL. Taken together, LOX-1 is a receptor specific for Ox-LDL, and enhanced uptake of Ox-LDL via this novel receptor on vascular endothelial cells may play an important role in endothelial activation in atherogenesis.

Animals↗
PubMed1998

Fluid shear stress transcriptionally induces lectin-like oxidized LDL receptor-1 in vascular endothelial cells.

Fluid shear stress has been shown to modulate various endothelial functions, including gene expression. In this study, we examined the effect of fluid shear stress on the expression of lectin-like oxidized LDL receptor-1 (LOX-1), a novel receptor for atherogenic oxidized LDL in cultured bovine aortic endothelial cells (BAECs). Exposure of BAECs to the physiological range of shear stress (1 to 15 dyne/cm2) upregulated LOX-1 protein and mRNA in a time-dependent fashion. LOX-1 mRNA levels peaked at 4 hours, and LOX-1 protein levels peaked at 8 hours. Inhibition of de novo RNA synthesis by actinomycin D totally abolished shear stress-induced LOX-1 mRNA expression. Furthermore, nuclear runoff assay showed that shear stress directly stimulates transcription of the LOX-1 gene. Chelation of intracellular Ca2+ with quin 2-AM completely reduced shear stress-induced LOX-1 mRNA expression; furthermore, the treatment of BAECs with ionomycin upregulated LOX-1 mRNA levels in a dose-dependent manner. Taken together, physiological levels of fluid shear stress can regulate LOX-1 expression by a mechanism dependent on intracellular Ca2+ mobilization. Inducible expression of LOX-1 by fluid mechanics may play a role in localized expression of LOX-1 and atherosclerotic lesion formation in vivo.

Animals↗
PubMed1998

Lectin-like oxidized low-density lipoprotein receptor 1 mediates phagocytosis of aged/apoptotic cells in endothelial cells.

Recognition of the exposure of phosphatidylserine (PS) on the outer surface of plasma membrane has been implicated in the phagocytosis of aged/apoptotic cells. Because oxidized low-density lipoprotein (OxLDL) has been reported to block the phagocytosis, here we examined whether lectin-like OxLDL receptor 1 (LOX-1), the OxLDL receptor in endothelial cells, mediates phagocytosis of aged/apoptotic cells by endothelial cells. Cultured bovine aortic endothelial cells (BAE) and Chinese hamster ovary (CHO) cells expressing bovine LOX-1 (BLOX-1-CHO), but not wild-type CHO-K1 cells, bound aged red blood cells (RBC) and apoptotic cells, which were further phagocytosed. The binding of aged RBC and the phagocytosis of apoptotic cells were inhibited by OxLDL, acetyl LDL, and other LOX-1 ligands in both BAE and BLOX-1-CHO. mAb against LOX-1 blocked the binding of aged RBC to BAE, suggesting a role for LOX-1 in the recognition of aged cells. The recombinant soluble LOX-1 inhibited the interactions of aged/apoptotic cells with both BLOX-1-CHO and BAE and distinguished aged RBC from native RBC and apoptotic cells from native cells. PS liposome inhibited these LOX-1-mediated interactions with aged/apoptotic cells, suggesting LOX-1 recognizes PS of the apoptotic cells. PS exposed on the surface of apoptotic cells is known to be procoagulant. Accordingly, increased OxLDL may be one of the reasons for enhanced coagulation in atherosclerosis, inhibiting the removal of apoptotic cells.

Animals↗
PubMed1998

Role of nonselective cation channels as Ca2+ entry pathway in endothelin-1-induced contraction and their suppression by nitric oxide.

The present study was carried out to clarify the role of nonselective cation channels as a Ca2+ entry pathway in the contraction and the increase in [Ca2+]i induced by endothelin- in endothelium-denuded rat thoracic aorta rings, and their suppression by nitric oxide (NO). In Ca2+-free medium, the endothelin-1-induced contraction was suppressed to about 20% of control values, although the increase in [Ca2+]i became negligible. The contraction and the increase in [Ca2+]i monitored by fura 2 fluorescence were unaffected by a blocker of L-type voltage-operated Ca2+ channels nifedipine. A blocker of nonselective cation channels 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imida zole . HCl(SK&F 96365) suppressed the endothelin-1-induced contraction and increase in [Ca2+]i to the level similar to that after removal of extracellular Ca2+. SK&F 96365 had no further effect on the endothelin-1-induced contraction in the absence of extracellular Ca2+. The endothelin-1-induced contraction and increase in [Ca2+]i were abolished by a donor of NO sodium nitroprusside. The effects of another NO donor 3-morpholinosydnonimine (SIN-1) were also tested and yielded essentially similar results to those for sodium nitroprusside on the endothelin-1-induced contraction. Furthermore, the inhibitory effects of sodium nitroprusside could be blocked with a guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) at 30 microM. These findings suggest that Ca2+ entry through nonselective cation channels but not voltage-operated Ca2+ channels plays a critical role in the endothelin-1-induced increase in [Ca2+]i and the resulting contraction and that inhibition by NO of the endothelin-1-induced contraction is mainly the result of blockade of Ca2+ entry through these channels.

Animals↗
PubMed1998

Increased Cat3-mediated cationic amino acid transport functionally compensates in Cat1 knockout cell lines.

Arginine transport is important for a number of biological processes in vertebrates, and its transport may be rate-limiting for the production of nitric oxide. The majority of L-Arg transport is mediated by System y+, although several other carriers have been kinetically defined. System y+ cationic amino acid transport is mediated by proteins encoded by a family of genes, Cat1, Cat2, and Cat3. High affinity L-arginine transport was investigated in embryonic fibroblast cells derived from Cat1 knockout mice that lack functional Cat1. Both wild type and knockout cells transport arginine with comparable Km and Vmax. However, the apparent affinity for lysine transport was 2.4 times lower in Cat1(-/-) cells when compared with wild type cells, a property characteristic of Cat3-mediated transport. Northern analysis-documented Cat2 mRNA increased 2-fold, whereas Cat3 mRNA levels increased 11-fold in Cat1(-/-) relative to Cat1(+/+) cells. The low affinity Cat2a mRNA was not detectably expressed in these cells. Even though Cat3 expression is normally limited to adult brain, there was a large increase in the amount of Cat3 protein present at the plasma membrane of Cat1(-/-) embryonic fibroblast cells. These results suggest that Cat3 compensates for the loss of functional Cat1 in cells derived from Cat1 knockout mice and mediates the majority of high affinity arginine transport.

Amino Acid Transport Systems, Basic↗
PubMed1998

Purification of a novel endothelin-converting enzyme specific for big endothelin-3.

Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big ET-1, with a Km value of 0.14 microM for big ET-3. The conversion to ET-3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO-K1 cells expressing recombinant human ET(B) receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3.

Animals↗
PubMed1998

Endothelin-induced apoptosis of A375 human melanoma cells.

Endothelin-1 (ET-1) inhibited serum-dependent growth of asynchronized A375 human melanoma cells, and the growth inhibitory effect was markedly enhanced when ET-1 was applied to the cells synchronized at G1/S boundary by double thymidine blocks. Flow cytometric analysis revealed that ET-1 did not inhibit the cell cycle progression after the release of the block but caused a significant increase of the hypodiploid cell population that is characteristic of apoptotic cell death. ET-1-induced apoptosis was confirmed by the appearance of chromatin condensation on nuclear staining and DNA fragmentation on gel electrophoresis. The increase in the hypodiploid cell peak was manifest within 16 h of exposure to 5 nM ET-1. Within the same time range, ET-1 caused actin reorganization and drastic morphological changes of the surviving cells from epithelioid to an elongated bipolar shape. These phenotypical changes were preceded by ET-1-induced increase and nuclear accumulation of the tumor suppressor protein p53. All of these effects of ET-1 were mediated by ETB via a pertussis toxin-sensitive G protein. Flow cytometric analysis with fluorescent dye-labeled ET-1 revealed up-regulation of ETB expressed by the cells in G1/early S phases, and overexpression of the receptor protein by cDNA microinjection conferred the responsiveness (both apoptosis and morphological changes) to ET-1 irrespective of the position of the cell in the cell cycle. These results indicated the presence of ETB-mediated signaling pathways to apoptotic cell machinery and cytoskeletal organization. Furthermore, the densities of ETB expressed by individual A375 melanoma cells appeared to be regulated by a cell cycle-dependent mechanism, and the receptor density can be a limiting factor to control the apoptotic and cytoskeletal responses of the cells to ET-1. Although the molecular mechanisms remain to be elucidated, these findings added a new dimension to the diverse biological activities of ETs and also indicated a novel mechanism to control the responsiveness of the cell to the peptides.

Cell Division↗