Biomedical subjects

M Takaoka

Publications and source records attributed to M Takaoka.

At least 127 records · Page 7Linked to original sources

PubMed1991

Conversion of big endothelin-1 to endothelin-1 by two-types of metalloproteinases of cultured porcine vascular smooth muscle cells.

Incubation of big endothelin-1 (big ET-1, 1-39) with the membrane fraction obtained from cultured vascular smooth muscle cells (VSMCs) resulted in an increase in immunoreactive-ET (IR-ET), which was inhibited by EDTA but not by phosphoramidon, a metalloproteinase inhibitor. When the incubation was performed in the presence of N-ethylmaleimide (NEM), the generation of IR-ET was markedly augmented and this augmentation was abolished by phosphoramidon. The pH profile for IR-ET generation in the presence of NEM was apparently distinct from that observed in the absence of NEM. Reverse-phase HPLC of the incubation mixture with or without NEM revealed one major IR-ET component corresponding to the elution position of synthetic ET-1 (1-21). When the cultured VSMCs were incubated with big ET-1, a conversion to the mature ET-1 was observed. This ET-1 generation from exogenously applied big ET-1 was markedly inhibited by the addition of phosphoramidon, although the inhibitor did not influence the basal secretion of ET-1-like materials. These results suggest the presence of two types of metalloproteinases, which can generate ET-1, in VSMCs. The possibility that ET-1 functions in an autocrine manner to control the cardiovascular system warrants further attention.

Animals↗

PubMed1991

N-ethylmaleimide differentiates endothelin converting activity by two types of metalloproteinases derived from vascular endothelial cells.

We have recently found that cultured vascular endothelial cells (ECs) contain two types of metalloproteinases which convert big endothelin-1 (big ET-1) to endothelin-1 (ET-1) via a single cleavage between Trp21 and Val22. In the present study, two enzymes were clearly differentiated by using sulfhydryl blocking reagents and anion-exchange HPLC. As reported, the converting activity of the membrane fraction of ECs was specifically inhibited by phosphoramidon. N-ethylmaleimide (NEM) markedly enhanced the apparent converting activity of the membrane fraction. This enhancement was not due to the direct action on the converting enzyme, but rather to inhibition of the degradation of big ET-1 and/or ET-1. In contrast, the converting activity of the cytosolic fraction was abolished by NEM treatment. Effects of phosphoramidon and NEM on converting activities of both fractions were confirmed after anion-exchange HPLC of each fraction, using a COSMOGEL QA column. Our results provide new information on two types of metalloproteinases which convert big ET-1 to ET-1, in vascular ECs.

Animals↗

PubMed1991

Phosphoramidon inhibits the conversion of intracisternally administered big endothelin-1 to endothelin-1.

It is suggested that endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). We examined the effects of intracisternal administration of big ET-1 on the cerebral arteries in the absence or presence of pretreatment with phosphoramidon, an inhibitor of ET converting enzyme, in anesthetized dogs. After intracisternal administration of big ET-1 (10 micrograms/dog), the caliber of the basilar artery on the angiogram was decreased to about 59% of the control. This was accompanied by a marked increase in immunoreactive ET in the cerebrospinal fluid. Systemic arterial pressure was markedly elevated following big ET-1 injection. All changes induced by big ET-1 were effectively prevented with phosphoramidon. These data suggest that intracisternally administered big ET-1 is converted to ET-1 and that the generated ET-1 produces cerebral vasospasm and hypertension. A phosphoramidon-sensitive metalloproteinase appears to contribute to this conversion.

Animals↗

PubMed1991

Evidence for phosphoramidon-sensitive conversion of big endothelin-1 to endothelin-1 in isolated rat mesenteric artery.

The effect of phosphoramidon, a metalloproteinase inhibitor, on the pressor response to big endothelin-1 (big ET-1) in the isolated perfused rat mesenteric artery was examined. Big ET-1 (10(-9)-10(-7) M) caused a concentration-dependent increase in the perfusion pressure. The pressor response was markedly suppressed by phosphoramidon (10(-5) M). The inhibitor did not influence the ET-1 (5 X 10(-11)-10(-8) M)-induced pressor action. Big ET-1 (5 X 10(-8) M)-induced pressor action was accompanied by an increase in immunoreactive (IR)-ET in the perfusate, and this increase was suppressed by the addition of phosphoramidon. IR-ET in the perfusate was confirmed to be ET-1, as determined by reverse-phase HPLC. These findings strongly suggest that phosphoramidon-sensitive metalloproteinase contributes to the conversion of big ET-1 to ET-1, in vivo.

Animals↗

PubMed1991

Phosphoramidon prevents cerebral vasospasm following subarachnoid hemorrhage in dogs: the relationship to endothelin-1 levels in the cerebrospinal fluid.

There is increasing evidence that the conversion of big endothelin-1 (big ET-1) to endothelin-1 (ET-1) is specifically inhibited by the metalloproteinase inhibitor phosphoramidon. We investigated the effect of phosphoramidon on delayed cerebral vasospasm from subarachnoid hemorrhage (SAH) using a two-hemorrhage canine model. The magnitude of the vasospasm and the drug effect were determined angiographically. On SAH Day 7, diameter of the basilar artery decreased to about 55% of the control value obtained before SAH (on Day 0). Immunoreactive ET (IR-ET) in the cerebrospinal fluid (CSF) significantly increased after SAH (on Day 7). The intracisternal pretreatment of phosphoramidon potently suppressed the decrease in diameter of the basilar artery after SAH, i.e., observed decrease was only about 20%, compared with the value before SAH. In the phosphoramidon group, IR-ET in CSF markedly increased (on SAH Day 2), but the increased levels of IR-ET significantly declined on SAH Day 7. These results clearly indicate that phosphoramidon effectively prevents delayed cerebral vasospasm. Whether the prevention is due to the inhibition of conversion of big ET-1 to ET-1 is now under study.

Animals↗

PubMed1991

Isolation and structural studies of human milk oligosaccharides that are reactive with a monoclonal antibody MSW 113.

We have determined the structures of six oligosaccharides isolated from human milk using a monoclonal antibody, MSW 113. The isolation involved affinity chromatography on a column of the immobilized monoclonal antibody and high-performance liquid chromatography. From the results of 500 and 600 mHz 1H NMR spectroscopy and fast atom bombardment-mass spectrometry their structures were deduced to be: [formula; see text] Two of these oligosaccharides, numbers 4 and 5, have not previously been described. All of them bound to MSW 113, but their reactivities are weaker than those of sialyl-Le(a) oligosaccharides. The results indicate that MSW 113 reacts with oligosaccharides with the mono- and disialyl-Le(a), and other sialyl type 1 structures.

Antibodies, Monoclonal↗

PubMed1991

Conversion of big endothelin-1 to endothelin-1 by phosphoramidon-sensitive metalloproteinase derived from aortic endothelial cells.

When big endothelin-1 (big ET-1, 1-39) was incubated with the membrane fraction obtained from cultured endothelial cells (ECs) at pH 7.0 for 6 h, the immunoreactive (ir) ET in the reaction mixture was markedly increased. Phosphoramidon, a metalloproteinase inhibitor, as well as metal chelators specifically suppressed the above increase. Using reverse-phase high-performance liquid chromatography, ir-ET was confirmed to be ET-1[1-21]. In addition, we noted that the alterations in ET-1 correlated with those in the C-terminal fragment (CTF, 22-39) of big ET-1. When cultured ECs were incubated with phosphoramidon, time-dependent secretion of ET-1 and CTF from the cells was markedly suppressed. In contrast, the secretion of big ET-1 was increased by phosphoramidon. Thiorphan, a specific inhibitor of neutral endopeptidase 24.11, was without effect on the secretion of ET-related peptides. Moreover, phosphoramidon potently inhibited the hypertensive effect of big ET-1 without affecting the ET-1-induced hypertension in anesthetized rats. From these findings, it seems reasonable to consider that phosphoramidon-sensitive and membrane-bound metalloproteinase, which is not a neutral endopeptidase 24.11, is the most plausible candidate for big ET-1-converting enzyme in vivo.

Animals↗

PubMed1991

Mode of cleavage of porcine big endothelin-1 by aspartic proteinases.

Cleavage sites of porcine big endothelin-1 (big ET-1, 1-39) by cathepsin D were examined and compared with those by other aspartic proteinases including pepsin. Cathepsin D cleaved not only the Trp21-Val22 bond, but also the Asp18-Ile19 bond of big ET-1[1-39]. The mature ET-1[1-21], generated by the cleavage between Trp21 and Val22, was subsequently degraded by removal of the C-terminal tripeptide (Ile19-Ile20-Trp21). On the other hand, pepsin cleaved the Trp21-Val22 bond of big ET-1[1-39] to produce ET-1[1-21], but did not degrade the generated ET-1[1-21]. These results indicate that aspartic proteinases such as cathepsin D and pepsin are capable of converting big ET-1[1-39] to ET-1[1-21], whereas the former proteinase is by no means specific for the Trp21-Val22 bond of big ET-1[1-39].

Amino Acid Sequence↗

PubMed1991

Phosphoramidon-sensitive endothelin converting enzyme in rat lung.

A neutral proteinase with endothelin (ET)-1 converting activity was identified in cytosol and membrane fractions prepared from rat lung, in a ratio of 1:4, respectively. The membrane-bound proteinase was solubilized by 0.5% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) with an increase in specific activity, and then was characterized. The solubilized proteinase was capable of converting big ET-1 to ET-1 with an optimum pH of 6.5, and the conversion was dose-dependently suppressed by phosphoramidon (IC50 = 0.5 microM). The molecular mass of the proteinase was estimated to be about 500 kDa by gel filtration in the presence of 0.5% CHAPS. These results indicate that rat lung contains a phosphoramidon-sensitive neutral proteinase catalyzing conversion of big ET-1 to ET-1. The proteinase may be involved in the biosynthetic pathway of ET-1 in the lung and/or the conversion of circulating big ET-1.

Animals↗

PubMed1990

Proteolytic processing of porcine big endothelin-1 catalyzed by cathepsin D.

Cathepsin D, a candidate for endothelin-converting enzyme (ECE), was allowed to act on porcine big endothelin-1 (big ET-1, 1-39). The proteinase primarily cleaved the Asp18-Ile19 and Trp21-Val22 bonds of big ET-1(1-39), with a optimum pH of 3.5. The mature ET-1(1-21), generated by the cleavage between Trp21 and Val22, was subsequently degraded by removal of most of the C-terminal tripeptide (Ile19-Ile20-Trp21). Therefore, cathepsin D is by no means a specific ECE, although the proteinase does cleave the Trp21-Val22 bond of big ET-1(1-39) to produce mature ET-1(1-21). The possibility that cathepsin D can act as an endothelin-degrading enzyme in vivo warrants consideration.

Amino Acid Sequence↗

PubMed1990

Conversion of big endothelin-1 to endothelin-1 by two types of metalloproteinases derived from porcine aortic endothelial cells.

Incubation of big endothelin-1 (big ET-1(1-39] with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive-endothelin (IR-ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction-induced increase in IR-ET, whereas the increase in IR-ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse-phase (RP)-HPLC of the incubation mixture of big ET-1 with the cytosolic or membrane fraction revealed one major IR-ET component corresponding to the elution position of synthetic ET-1(1-21). Simultaneously, immunoreactivities like the C-terminal fragment (CTF22-39) of big ET-1 were present, as deduced from the RP-HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET-1 to ET-1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.

Animals↗

PubMed1990

Phosphoramidon, a metalloproteinase inhibitor, suppresses the secretion of endothelin-1 from cultured endothelial cells by inhibiting a big endothelin-1 converting enzyme.

Time-dependent secretion of immunoreactive-endothelin (IR-ET) from cultured porcine aortic endothelial cells was markedly suppressed by phosphoramidon is due to proteinase inhibitor. Analysis of the culture supernatant with or without phosphoramidon by reverse-phase high performance liquid chromatography confirmed that the suppression of IR-ET secretion by phosphoramidon is due to a decreae in secretion of endothelin-1-like materials. The secretion of the C-terminal fragment (CTF, 22-39)-like materials of big ET-1 was also decreased by phosphoramidon, whereas there was an increased secretion of big ET-1-like materials. These data strongly suggest that phosphoramidon suppresses the secretion of ET-1 from cultured endothelial cells by inhibiting the conversion of big ET-1 to ET-1. It is most likely that phosphoramidon-sensitive metalloproteinase is responsible for the processing of big ET-1 in vascular endothelial cells.

Animals↗

PubMed1990

Mode of cleavage of pig big endothelin-1 by chymotrypsin. Production and degradation of mature endothelin-1.

Pig endothelin-1 [ET-1-(1-21)] seems to be produced via proteolytic processing between Trp-21 and Val-22 of an intermediate form consisting of 39 amino acid residues, termed big ET-1-(1-39), by a chymotrypsin-like proteinase. We examined the chymotryptic-cleavage sites of big ET-1-(1-39) by reverse-phase h.p.l.c. and sequence analysis, and found that chymotrypsin cleaved initially the Tyr-31-Gly-32 bond of big ET-1-(1-39), followed by cleavage between Trp-21 and Val-22. Furthermore, chymotrypsin hydrolysed the generated ET-1-(1-21), producing a single major product that had the same amino acid sequence as ET-1-(1-21) with a cleavage between Tyr-13 and Phe-14. The disulphide bridge between Cys-1 and Cys-15 remained intact. These results indicate that the conversion of big ET-1-(1-39) into ET-1-(1-21) catalysed by chymotrypsin requires hydrolysis of the Tyr-31-Gly-32 bond before that of the Trp-21-Val-22 bond, an event followed by cleavage between Tyr-13 and Phe-14 within the loop of ET-1-(1-21). Thus a chymotrypsin-like proteinase might be involved not only in the production but also in the degradation of ET-1-(1-21) in vivo.

Amino Acid Sequence↗

PubMed1990

Phosphoramidon, a metalloproteinase inhibitor, suppresses the hypertensive effect of big endothelin-1.

Intravenous (i.v.) injection of big endothelin-1 (1-39, 0.05-1.0 nmol/kg) to anaesthetized rats produced a dose-dependent and long-lasting hypertensive effect, the magnitude of which was similar to that evoked by ET-1 (1-21). In animals given phosphoramidon (0.25 mg/kg per min i.v.), a metalloproteinase inhibitor, the hypertensive effect of big endothelin-1 was markedly attenuated. The same dose of phosphoramidon did not influence the endothelin-1-induced hypertensive effect. The possibility that big endothelin-1 is converted to endothelin-1 by a phosphoramidon-sensitive metalloproteinase in vivo warrants further attention.

Anesthesia↗

PubMed1990

Evidence for pepstatin-sensitive conversion of porcine big endothelin-1 to endothelin-1 by the endothelial cell extract.

We designed a radioimmunoassay (RIA) specific for the C-terminal fragment (CTF, 22-39) of porcine big endothelin-1 (big ET-1, 1-39), and investigated whether the generation of ET-1 (1-21) is concomitant with that of the CTF during incubation of big ET-1 with the endothelial cell (EC)-extract. When the incubation mixture was applied to reverse-phase high performance liquid chromatography coupled with the RIAs for CTF and ET, immunoreactive (IR)-CTF eluted as one major and one minor peak, and IR-ET as one major peak. The retention times of each peak corresponded to those of synthetic CTF, big ET-1 and ET-1, respectively. An aspartic protease inhibitor pepstatin-A completely inhibited the generation of CTF- and ET-1-like materials. Both materials were also detected in culture medium of porcine aortic ECs. These findings strongly suggest that ET-1 is generated from big ET-1 via a single cleavage between Trp21 and Val22 in vascular ECs and that a pepstatin-sensitive aspartic protease is a possible candidate for big ET-1 converting enzyme.

Animals↗

PubMed1990

Conversion of porcine big endothelin to endothelin by an extract from the porcine aortic endothelial cells.

Conversion of porcine big endothelin (big ET) to endothelin (ET) by an extract from cultured porcine aortic endothelial cells was investigated using a radioimmunoassay (RIA) specific for ET and reverse-phase high performance liquid chromatography (RP-HPLC). When big ET was incubated with the extract at an acid pH in the presence of E-64, a cysteine protease inhibitor, the amount of immunoreactive-ET (IR-ET) in the incubation mixture was greatly increased and the optimum pH for this increased reaction was 4.0. The extract-induced increase in IR-ET was completely inhibited by pepstatin-A. These immunoreactive alterations correlated with those in the vasoconstrictor activity. When the incubation mixture of big ET with the cell extract was applied to the RP-HPLC, the IR-ET eluted at the same retention time as seen with synthetic porcine ET. We suggest that a pepstatin-sensitive aspartic protease is involved in the conversion of big ET to ET in vascular endothelial cells.

Animals↗

PubMed1990

Pepsin, an aspartic protease, converts porcine big endothelin to 21-residue endothelin.

Porcine big endothelin (big ET-39) at 1 nM, a concentration with no influence on contractile activity in isolated rat aorta, induced a slow-onset and sustained contraction by the pre-incubation with pepsin. When the incubation mixture of big ET-39 with pepsin was analyzed by high-performance liquid chromatography on an octadecyl silica column, two major products of pepsin hydrolysis were obtained; their amino acid sequences were identical with those of 21-residue endothelin (ET-21) and a C-terminal peptide of big ET-39, big ET (22-39), respectively. On the other hand, no degradation of ET-21 was observed by pepsin treatment. These results indicate that pepsin specifically cleaves a Trp21-Val22 bond in the big ET-39 molecule, producing ET-21 and big ET (22-39). Thus, the possibility that pepsin-like aspartic protease may participate in the conversion of big ET-39 to ET-21 in vivo warrants further attention.

Animals↗

PubMed1990

Guanine levels in house dust as a means of estimating the mite population.

House dust has recently been incriminated as an important causative allergen in bronchial asthma and allergic rhinitis. In particular attention is being focused on the mites contained in house dust. Previously, the number of mites contained in house dust was determined by counting under a microscope. This method is not appropriate, however, for epidemiological investigations which require large numbers of specimens, as it is time-consuming and labor intensive. Guanine makes up about 0.3% of the body weight of mites and is present in large amounts in mite excrement, and by using an improved method for quantifying guanine in house dust we obtained a correlation coefficient of 0.776 between the number of mites and amount of guanine. In addition, because virtually no guanine is contained in human nails and hair, thus eliminating possible sources of error, it became clear that the amount of guanine in house dust can be used to estimate the total amount of mite allergen resulting from the bodies and excrement of the acarids.

Animals↗