Search PubMed⌕ Search

Biomedical subjects

T Masaki

Publications and source records attributed to T Masaki.

At least 271 records · Page 15Linked to original sources
PubMed1995

Endothelin-1-induced downregulation of ETB receptor mRNA: participation of cAMP.

In this study we examined the participation of cAMP formation in endothelin-1 (ET-1)-induced downregulation of ETB receptor mRNA in ROS 17/2 rat osteosarcoma cells. Dibutyryl cAMP induced downregulation of ETB receptor mRNA in a time-dependent manner. ET-1 induced production of inositol phosphates and an increase of cAMP level in ROS 17/2 cells. A stimulatory effect on cAMP level was also observed when A23187 plus PMA was added to the cells. The increase in cAMP level induced either by ET-1 or by A23187 plus PMA was inhibited by indomethacin. The downregulation of ETB receptor mRNA induced by ET-1 was significantly inhibited by indomethacin. These results suggest that the ET-1-induced downregulation of ETB receptor mRNA in ROS 17/2 cells may be partly mediated through the increase in cAMP level secondary to the activation of the phosphoinositide hydrolysis/Ca2+ transduction cascade.

Animals↗
PubMed1995

Inhibition of convertase-related processing of proendothelin-1.

The biologically inactive precursor proendothelin-1 (proET-1) is initially processed intracellularly to the intermediate big endothelin peptide (big ET-1) before its conversion to the endothelin-1 (ET-1) peptide by the endothelin-converting enzyme (ECE). We recently demonstrated that purified furin, a calcium-dependent serine endoprotease belonging to the family of mammalian convertases, cleaved proET-1 in vitro and hence produced the physiologically relevant big ET-1 peptide. Therefore, furin becomes a candidate proET-1-cleaving enzyme responsible for the initial biosynthetic processing steps of this precursor. In this study we examined the inhibitory properties of two convertase inhibitors, i.e., the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk) and the alpha 1-antitrypsin Portland (AT-PDX) on proET-1 processing. When purified furin or PACE4, another mammalian convertase, was incubated in the presence of dec-RVKR-cmk, proET-1 processing was completely abolished. In the presence of purified AT-PDX, furin-related cleavage of proET-1 was abolished but not PACE4 processing. In addition, incubation of endothelial cells with dec-RVKR-cmk inhibited production of ET-1 but did not significantly alter the levels of the prostanoid PGI2. These results indicate that convertase-related processing of proET-1 can be inhibited in vitro and in vivo, and that convertase-specific processing may be prevented with AT-PDX.

Amino Acid Sequence↗
PubMed1995

Possible sources of endothelin-1 in damaged rat brain.

We investigated the sources of endothelin-1 (ET-1) detected in the damaged brain tissue of rats after cold injury. ET-1 in the injured sites was increased 1-5 days after the injury. Physiologic observations revealed that breakdown of the blood-brain barrier occurred and that plasma-derived albumin exuded into the brain parenchyma after cold injury. Radiolabeled ET-1, which was injected into the left cardiac ventricle, was detected in the tissue at the injury site, and ET-1-like immunoreactivity was observed in the brain capillaries in the damaged tissues. TGF-beta 1, which is a tissue regeneration factor, potently induced the expression of ET-1 mRNA in cultured astrocytes. These results suggest that ET-1 is produced through a variety of ways in the damaged brain.

Animals↗
PubMed1994

Endothelin-1 activates mitogen-activated protein kinases through two independent signalling pathways in rat astrocytes.

Northern blot analysis and displacement study revealed that the endothelin (ET) receptor functionally expressed in rat primary cultured astrocytes is the ETB receptor. Mitogen-activated protein kinases (MAP kinases) in the cells were activated by 10 nM ET-1, a dose that maximally stimulated phosphoinositide hydrolysis. This activation was potently inhibited by pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) which leads to protein kinase C (PKC) down-regulation and was slightly inhibited by pretreatment with pertussis toxin (PTX). Pretreatment of the cells with PMA plus PTX completely inhibited the ET-1-augmented MAP kinase activity. Activation of MAP kinases was also induced by 0.1 nM ET-1, which hardly stimulated phosphoinositide hydrolysis. This activation was fully inhibited by pretreatment with PTX but insensitive to pretreatment with PMA. ET-1-stimulated production of inositol phosphates was not affected by pretreatment with PTX. These results suggest that activation of MAP kinases secondary to stimulation of the ETB receptor with ET-1 in rat primary cultured astrocytes was mediated through two independent signalling pathways. PKC-dependent pathway and PTX-sensitive G protein-mediated pathway.

Animals↗
PubMed1994

Genetic transfer of endothelin converting enzyme activity to CHO-K1 cells: detection of positive cells by reverse hemolytic plaque assay.

We have established a novel method of molecular cloning of endothelin converting enzyme, a key enzyme in the production of a potent vasoconstrictor endothelin-1, by modification of the reverse hemolytic plaque assay. Also, we demonstrated that a cell line, CHO-K1, showed no detectable activity of endothelin converting enzyme. This cell line was transfected with a cDNA library of bovine endothelial cells. The modified reverse hemolytic plaque assay was shown to detect even a single CHO-K1 cell that was changed to produce mature ET-1 by transfection. Thus, this novel method is suggested to be useful for the molecular cloning of other secreted antigens and their processing enzyme.

Animals↗
PubMed1994

Endothelin-A receptor mediates cardiac inhibition by regulating calcium and potassium currents.

Voltage-sensitive ion channels play fundamental roles in the regulation of cardiac function by various neurotransmitters. Endothelins have strong positive inotropic and chronotropic effects, for which recent studies have implicated various intracellular mechanisms. However, very little is known about the underlying ion-channel regulation by the peptide. We report here that endothelin-1 consistently hyperpolarizes the membrane and shortens the duration of the action potential in mammalian atrial myocytes, leading to suppression of electrical excitability of the heart. Endothelin-1, but not endothelin-3, inhibited the L-type calcium current by decreasing cyclic AMP accumulation and activated the muscarinic potassium current by stimulating a pertussis toxin-sensitive GTP-binding protein. Consistent with these results, endothelin-1 strongly reduced the heart rate when it was increased by beta-adrenoceptor stimulation. These effects were blocked by an ETA (endothelin-1-selective) receptor-selective antagonist, BQ123 (refs 8-11). The ETA receptor-mediated regulation of cardiac ion channels gives new insight into our understanding of the physiological and pathophysiological roles of endothelins in the control of cardiac function.

Action Potentials↗
PubMed1994

Molecular cloning of low-Ca(2+)-sensitive-type non-muscle alpha-actinin.

We previously reported the purification and characterization of a novel non-muscle alpha-actinin from chicken lung [Imamura, M. & Masaki, T, (1992) J. Biol. Chem. 267, 25927-25933]. The Ca2+ sensitivity of the lung alpha-actinin for the interaction with polymerized actin (F-actin) was much lower than those of the other reported non-muscle alpha-actinins. Here, we isolated a cDNA clone encoding the novel alpha-actinin by screening a chicken lung lambda g11 cDNA library with antibody specific for the low-Ca(2+)-sensitive alpha-actinin. The deduced amino acid sequence of the lung alpha-actinin showed 76%, 82% and 83% identity to those of chicken skeletal muscle, smooth-muscle and fibroblast-type alpha-actinin, respectively. Marked difference in the structure between the lung-type and the other alpha-actinins was found in the extreme NH2-terminal and in the COOH-terminal half; in the third and fourth regions of four spectrin-like repeats, and in two Ca(2+)-binding EF-hand consensus regions. The NH2-terminal-side EF-hand contained a notable defect in one of the five oxygen-containing amino acid side chains involved in chelating Ca2+, suggesting that the lower Ca2+ sensitivity of the lung alpha-actinin is ascribable to this defect. Northern blot analysis showed that the expression pattern of lung-type alpha-actinin mRNA in various non-muscle tissues differed from that of the other known non-muscle-type (fibroblast-type) alpha-actinin. The present results clearly demonstrate the existence of two structurally and functionally different types of non-muscle alpha-actinin; high-Ca(2+)-sensitive-type (NM1) and low-Ca(2+)-sensitive-type (NM2) alpha-actinin.

Actinin↗
PubMed1994

Determination of 9 alpha, 11 beta-prostaglandin F2 in human urine and plasma by gas chromatography-mass spectrometry.

Sensitive and specific assay methods for 9 alpha, 11 beta-prostaglandin F2 (9 alpha, 11 beta-PGF2) by gas chromatography-mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9 alpha, 11 beta-PGF2 was based on the use of the methyl ester-dimethylisopropylsilyl ether derivative, and pentadeuterated PGF2 alpha as a convenient internal standard. The calibration graph for 9 alpha, 11 beta-PGF2 was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise ratio = 2.3). The method was applied to the determination of 9 alpha, 11 beta-PGF2 in urine and plasma samples from patients with bronchial asthma.

Adolescent↗
PubMed1994

Pseudo-noncompetitive antagonism by BQ123 of intracellular calcium transients mediated by human ETA endothelin receptor.

The family of endothelins, endothelin-1 (ET-1), ET-2 and ET-3, act on two subtypes of receptors called ETA and ETB receptors. BQ123 is an ETA-selective competitive antagonist. In this study, however, BQ123 inhibited ET-1-induced transients of cytosolic Ca2+ concentrations ([Ca2+]i) in an apparently noncompetitive manner in mouse L cell stably expressing cloned human ETA receptor. BQ123 (3-30 nM) did not change the EC50 of ET-1 (6.6 nM), but reduced the maximum responses down to 15.0%. In an non-equilibrium binding assay which mimicks the conditions of [Ca2+]i transient assay (cells were incubated with [125I]ET-1 only for 30 s), BQ123 (30 nM) constantly decreased the specific [125I]ET-1 binding to approximately 13% of that without the antagonist. Thus, the apparent noncompetitive antagonism by BQ123 of ETA receptor-mediated [Ca2+]i transient is due to the assay condition where the interactions of the agonist, antagonist and receptor remained non-equilibrium state.

Animals↗
PubMed1994

Ischemia-induced changes in catecholamine release and their mechanisms: a study using cultured bovine adrenal chromaffin cells.

Ischemia-induced changes in neurotransmitter release and their mechanisms were examined using cultured bovine adrenal chromaffin cells. When the cells were incubated in glucose-free media equilibrated with 0% O2/100% N2 (ischemia), ATP content decreased and reached the minimum level within 40 min. Control incubation was done in media equilibrated with 21% O2 in N2. After 10-min incubation under ischemic conditions, basal catecholamine (CA) release was elevated and the elevation persisted up to 90 min. High K(+)-evoked CA release was transiently enhanced at 10 min, but after that, it decreased to reach the minimum level at 60 min. At 10 min, cytosolic free Ca2+ concentration ([Ca2+]i) and 45Ca2+ uptake of the resting cells (basal values) and high K(+)-evoked increases in these two parameters were unchanged, but CA release from permeabilized cells in response to Ca2+ in media was augmented. After 60-min incubation under ischemic conditions, basal [Ca2+]i was elevated: the elevation was observed even in the absence of extracellular Ca2+. In contrast, high K(+)-evoked increases in [Ca2+]i and in 45Ca2+ uptake were suppressed, but basal 45Ca2+ uptake into intact cells and CA release from permeabilized cells were unchanged. These results suggest that in an early phase (10 min) of ischemia, both basal and stimulation-evoked CA release are augmented because of increased sensitivity of exocytotic machinery to Ca2+. In the late phase (60 min), basal CA release is augmented because of an increase in basal [Ca2+]i, which is due to accumulation of Ca2+ derived from intracellular Ca2+ pools: stimulation-evoked CA release is suppressed because of inhibition of stimulation-evoked increase in [Ca2+]i, which is due to functional disturbance of voltage-dependent Ca2+ channels.

Adenosine Triphosphate↗
PubMed1994

Identification of a 115-kDa protein from muscle tissues: expression of a novel nonmuscle alpha-actinin in vascular endothelial cells.

A novel nonmuscle alpha-actinin from chicken lung tissue is distinct from reported nonmuscle and muscle alpha-actinins in its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 6 M urea (apparent molecular mass is 115 kDa) and its Ca(2+)-sensitivity in actin binding (M. Imamura and T. Masaki, J. Biol. Chem. 267, 25927-25933, 1992). SDS-PAGE analysis of extract from chicken gizzard smooth muscle tissue in the presence of urea showed the existence of a protein migrating at 115 kDa. Analyses of proteolytic digestion and partial amino acid sequence revealed that the latter 115-kDa protein is identical or very similar to the Ca(2+)-insensitive lung alpha-actinin. We prepared an antibody specific for the 115-kDa protein by affinity column coupled with the purified lung alpha-actinin, and the expression and distribution of the 115-kDa protein in muscle tissues were examined. Immunoblotting and immunofluorescence microscopy demonstrated that the 115-kDa protein was expressed in heart and pectoralis muscle tissues as well as in gizzard. Double-fluorescence staining with the anti-115-kDa protein antibody and anti-muscle alpha-actinin antibody indicated that the 115-kDa protein was localized in tubular structures along the axes of muscle fibers, whose diameter was 3.5-5 microns. The tubular structure did not respond to the muscle cells. Vascular-labeling analysis with carbon dye showed that the staining with anti-115-kDa protein antibody was located along the inside of the vascular wall. These results indicated that the Ca(2+)-insensitive alpha-actinin was expressed in vascular endothelial cells and suggested that its Ca(2+)-sensitivity was related to the function of the endothelial cells.

Actinin↗
PubMed1994

Non-polypoid adenoma of the large intestine.

In order to find non-polypoid (flat) adenomas in the large intestine, one hundred and twenty-six consecutively resected large bowels were stained with methylene blue or haematoxylin and examined using a stereoscopic microscope with special attention to pit pattern abnormalities. The primary diseases were classified into familial adenomatous polyposis (FAP) in 9 cases, multiple adenomas in 2 cases, colorectal cancers in 90 cases, Crohn's disease in 5 cases, ulcerative colitis in 2 cases, constipation in 9 cases, diverticular disease in 3 cases and others in 6 cases, respectively. Thirty-five flat adenomas were detected in 14 colons (14/126 = 11%). Twenty-one flat adenomas were found in 9 non-FAP cases (7 colons removed for cancer and 2 for multiple adenomas) and the remaining 14 flat adenomas were found in 5 FAP colons. No flat adenomas were found in any of the 25 colons removed for non-neoplastic disease. Nine flat adenomas (26%) had a depressed shape. The mean age of the non-FAP cases was 57 years and that of the FAP cases was 29 years (P < 0.005). The mean size of non-FAP flat adenomas was 6.1 mm and that of FAP flat adenomas was 3.1 mm (P < 0.005). Non-FAP flat adenomas were more frequently right-sided than those in FAP. Epithelial dysplasia was graded as mild in 31 (89%) and moderate in 4 (11%), respectively. Moderate dysplasia was found in non-FAP cases exclusively. These results suggest that flat adenomas may be heterogeneous. The significance of these lesions is discussed.

Adenoma↗
PubMed1994

Multicatalytic proteinase is associated with characteristic oval structures in cortical Lewy bodies: an immunocytochemical study with light and electron microscopy.

The ATP-ubiquitin-dependent proteolytic pathway (ubiquitin pathway) is believed to be involved in the formation of various neuronal inclusion bodies including Lewy bodies (LBs), a pathological hallmark of Parkinson disease and diffuse Lewy body disease (DLBD). Since multicatalytic proteinase (MCP) is involved in the ubiquitin pathway, an investigation of whether MCP is involved in neuronal inclusion bodies would provide a clue to the mechanism underlying the formation of neuronal inclusion bodies as well as to the pathogenesis of degenerative neurological disorders. In this study, we investigated detailed immunolocalization of MCP in LBs in DLBD brains using light and electron microscopy. We raised three different monoclonal antibodies against purified human MCP. Each of them recognized different sets of MCP subunits on Western blotting. Immunohistochemically, anti-MCP antibodies recognized all ubiquitin-positive cortical LBs in situ as well as those isolated from frozen DLBD cortices, suggesting that MCP is present in LBs as a whole molecule exhibiting protease activity. In electron microscopy, MCP immunoreactivity (MCP-IR) was exclusively localized on a characteristic oval structure with an approximate diameter of 100 nm. This structure was distributed throughout the LBs and was devoid of ubiquitin immunoreactivity. Treatment of isolated LBs with 2% SDS, but not with 0.5% Triton X-100, removed this structure from LBs in which fibrous materials predominated. Ubiquitin immunoreactivity was also decreased in isolated LBs treated with 2% SDS, suggesting that the fibrous structures in LBs were not ubiquitinated in situ. Thus, it is suggested that LBs are subjected to a proteolytic process in which MCP plays a role via processing of specific components of LBs.

Aged↗
PubMed1994

Lower incidence of K-ras codon 12 mutation in flat colorectal adenomas than in polypoid adenomas.

In order to clarify genetic changes in flat adenomas, K-ras codon 12 point mutations were examined in 56 flat adenomas, 81 polypoid adenomas and 42 cancers of colon and rectum. The mutation frequency in flat adenomas was 23% (13/56), significantly lower than that in polypoid adenomas (67%: 54/81) and cancers (76%: 32/42). Even mildly dysplastic adenomas or small (less than 5 mm) adenomas showed higher mutation incidence in polypoid type (62%, 57%) than in flat type (23%, 19%). Among flat adenomas, flat elevated lesions exhibited relatively higher mutation frequency than completely flat or depressed ones. As for cancers, 14 tumors (33%) contained mutations only in a minor tumor cell population, indicating that these mutations occur at a late stage of tumorigenesis. These results suggest that the adenoma-carcinoma sequence through flat adenomas may be different from that through polypoid adenomas, and genetic changes may be heterogeneous in colorectal carcinogenesis.

Adenoma↗
PubMed1994

Loss of contractile activity of endothelin-1 induced by electrical field stimulation-generated free radicals.

1. Electrical field stimulation (EFS; 10 V, 10 Hz, 2 ms) of porcine coronary artery strips precontracted with 10 nM endothelin-1 (ET-1) for 5 min caused a biphasic response, consisting of a slight contraction during EFS and a marked and irreversible relaxation just after EFS. This irreversible relaxation after EFS has never been investigated. In the present study, we have investigated the mechanism of the relaxation after EFS. 2. The EFS-induced response was not affected by the presence or absence of endothelium and was insensitive to 10 microM tetrodotoxin (TTX). 3. In the presence of free radical scavengers (40 u ml-1 superoxide dismutase (SOD), 1200 u ml-1 catalase or 80 mM D-mannitol), the relaxation after EFS was significantly inhibited. Moreover, relaxation after EFS was not observed in porcine coronary artery strips precontracted with 20 mM KCl. 4. In a cascade experiment, EFS of Krebs-Ringer solution containing 10 nM ET-1 induced marked suppression of the contractile activity of ET-1 in porcine coronary artery strips, which was in accord with the observed decrease in release of immunoreactive ET-1 (ir-ET-1). This effect of EFS was significantly inhibited by each of the free radical scavengers, 3 mM vitamin C, 40 u ml-1 SOD, 1200 u ml-1 catalase and 80 mM D-mannitol. 5. The exchange of 95% O2/5% CO2 gas for 95% N2/5% CO2 gas significantly inhibited the EFS-induced decrease in release of ir-ET-1. 6. Neither superoxide anions generated by xanthine (10 JM) plus xanthine oxidase (0.1 micro ml-1) nor hydrogen peroxide (10 microM) exogenously added to Krebs-Ringer solution containing 10 nM ET-1 affected the level of ir-ET-1.7. Generation of hydroxyl radicals was detected in the EFS-applied Krebs-Ringer solution. The EFS-induced generation of hydroxyl radicals was dependent on the period of stimulation and 02-bubbling, and significant generation of hydroxyl radicals was detectable with stimulation of over 5 min.Moreover, hydroxyl radicals generated in 50 mM NaCl solution containing 10 nM ET-1 by H202 plus Fe2 , i.e. the Fenton reaction, significantly decreased the level of ir-ET-l.8. These findings suggest that oxygen-derived hydroxyl radicals generated by EFS of porcine coronary artery strips inactivate ET-1, probably by structural modification. Thus, porcine coronary artery strips precontracted with ET-1 are potently relaxed by EFS.

Animals↗