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PubMed2004

Stimulation of mucin secretion from human bronchial epithelial cells by mast cell chymase.

AIM: To investigate the effect of chymase on the mucin secretion from human bronchial epithelial cells. METHODS: Primarily-cultured human bronchial epithelial (PCHBE) cells and normal human bronchial epithelial (NHBE) cells were cultured with chymase or other stimulus in a mixture of bronchial epithelial growth medium (BEGM) and Dulbecco's modified Eagle's medium (DMEM), and the quantities of stimulatory mucin release were recorded. MUC5AC mucin was measured with an ELISA and dolichos biflorus agglutinin (DBA) mucin was determined with an enzyme linked DBA assay. RESULTS: A dose-dependent secretion of DBA mucin from PCHBE cells was observed with chymase with a maximum secretion of 98 % above baseline being achieved following 3 h incubation. The action of chymase started from 1 h, peaked at 3 h and dramatically decreased at 20 h following incubation. Chymase was able to also stimulate approximately 38 % increase in MUC5AC mucin release from PCHBE cells, and about 121 % increase in DBA mucin release from NHBE cells. A chymase inhibitor soybean trypsin inhibitor (SBTI) was able to inhibit up to 85 % chymase induced mucin release, indicating that the enzymatic activity was essential for the actions of chymase on bronchial epithelial cells. CONCLUSION: Chymase is a potent stimulus of mucin secretion from human bronchial epithelial cells. It can contribute to mucus hypersecretion process in the patients with chronic obstructive pulmonary disease or asthma.

Bronchi↗
PubMed1987

Quantitation of histamine, tryptase, and chymase in dispersed human T and TC mast cells.

Levels of histamine, chymase, and tryptase were assessed in preparations of dispersed human TC (tryptase+, chymase+) mast cells obtained from foreskin and of dispersed human T (tryptase+, chymase-) mast cells obtained from lung. Consistent with previous immunohistochemical results, extracts of T mast cells, the predominant mast cell type in lung (93% T and 7% TC mast cells), were deficient in human chymase (less than 0.3 microgram and 0.04 U/10(6) mast cells) but not tryptase (10.8 micrograms and 0.3 U/10(6) mast cells) by corresponding immunologic and enzymatic (suc-L-ala-ala-pro-phe-p-nitroanilide in the presence of aprotinin and tosyl-L-gly-pro-lys-p-nitroanilide in the presence of soybean trypsin inhibitor, respectively) assays. The minor presence of chymase activity in lung could be accounted for by the minor presence of lung TC mast cells. Extracts of TC mast cells, the predominant mast cell type (1% T and 99% TC mast cells) in foreskin, contained both proteases. However, TC mast cells from adult foreskin contained eightfold to 10-fold higher levels of chymase (4.5 micrograms and 1.01 U/10(6) mast cells) and twofold to threefold higher levels of tryptase (11.5 micrograms and 0.27 U/10(6) mast cells) than did TC mast cells from newborn foreskin (less than 0.6 microgram and 0.09 U of chymase and 35 micrograms and 0.62 U of tryptase/10(6) mast cells). In contrast, histamine levels were not significantly different in adult foreskin TC (1.9 microgram/10(6) mast cells), newborn foreskin TC (1.6 microgram/10(6) mast cells), and adult lung T (1.5 microgram/10(6) mast cells) mast cells. The relative ratio of each mediator in newborn foreskin mast cells to that in adult foreskin mast cells is highest for histamine, followed by tryptase and then chymase. Tryptase from TC and T mast cells had identical subunit compositions by Western blot analysis and similar apparent specific activities. This study extends the previously reported immunohistochemical distinction between human T and TC mast cells in tissue sections by direct quantitation of chymase and tryptase in dispersed preparations of T and TC mast cells.

Adult↗
PubMed1977

Preparative purification of the rat mast cell chymase: characterization and interaction with granule components.

The rat mast cell granule chymotrypsinlike enzyme was purified to homogeneity from 1 M NaCl solubilized membrane and granule-rich fractions of concentrated rat peritoneal mast cells by a preparative technique utilizing chromatography on Dowex 1, filtration on Sephadex G-75, and affinity chromatography with D-tryptophan methyl ester. Acid disk gel electrophoresis of the purified chymase disclosed a single stained band with activity being eluted from a replicate sliced gel in the same region. SDS-polyacrylamide gel electrophoresis of purified protein gave a single stained band that did not change in position with reduction and alkylation. Mast cell chymase is thus a cationic protein of 25,000 mol wt composed of a single polypeptide chain. The apparent K(m) of the chymase for BTEE was 1.5 x 10(-3) M and the V(max) was 67.8 mumol/min per mg. The enzyme was inhibited by TPCK and not by TLCK. The chymase complexed with native macromolecular rat mast cell heparin in molar ratios of 12:1 and 16:1, and complete heparin uptake occurred at a 40:1 ratio of chymase to heparin. Chymase activity was partially masked by combination with heparin in the isolated granule or experimental chymase-heparin complex, and soluble purified chymase was inhibited by concentrations of 5-HT comparable to those present in mast cells. It is therefore possible that the active site of chymase in the mast cell granule is largely masked by the combined effects of macromolecular heparin and 5-HT.

Animals↗
PubMed2002

Recent chymase inhibitors and their effects in in vivo models.

Recent efforts to discover novel chymase inhibitors have produced orally bioavailable compounds. Studies using such inhibitors have shed light on the pathophysiological roles of chymase, eg, a chymase inhibitor has prevented atherosclerosis, restenosis and myocardial infarction in respective animal models. In these cardiovascular diseases, angiotensin I is likely involved as a substrate for chymase. The studies using chymase inhibitors have also shown the potential role of chymase in other diseases, including atopic dermatitis, tissue fibrosis and rheumatoid arthritis; a chymase inhibitor also reduced ischemic reperfusion injury in the small intestine. These results suggest the existence of physiological substrates for chymase other than angiotensin I. Chymase inhibitors are promising for the treatment of cardiovascular as well as inflammatory diseases.

Journal Article↗
PubMed1999

The detection of mast cell subpopulations in formalin-fixed human tissues using a new monoclonal antibody specific for chymase.

Chymase is an important marker for human mast cells as well as a mediator of inflammation and matrix remodelling, but research into chymase-containing mast cell subpopulations has been hampered by the lack of reagents suitable for use with formalin-fixed tissue. A monoclonal antibody to chymase (designated CC1) was prepared by immunizing a mouse with chymase purified from human skin, fusing the splenocytes with NS-1 myeloma cells, and screening the hybridoma supernatants by ELISA with recombinant human prochymase isolated from a baculovirus expression system. This antibody bound to chymase in western blots and bound selectively to cells with the morphology and distribution of mast cells in paraffin wax sections of skin, synovium, lung, and heart. In sequential sections and with double-labelling experiments, chymase was localized to cells which contained mast cell tryptase; in contrast to previous reports, no evidence was found for its presence in endothelial cells or any other cell type. The antibody permitted chymase-containing mast cells to be detected in formalin-fixed tissues, and the numbers identified were similar to those in tissues fixed with Carnoy's or ethanol fixatives. Immunocytochemistry with antibody CC1 provides for the first time a sensitive and specific means for the detection of chymase in routinely fixed tissues and should prove valuable in studying mast cell subsets in disease.

Animals↗
PubMed1997

Soybean Bowman-Birk protease inhibitor is a highly effective inhibitor of human mast cell chymase.

Soybean Bowman-Birk protease inhibitor (BBI) is an inhibitor of serine proteases with two functional inhibitory domains of different specificities: one is specific for chymotrypsin-like proteases, the other for trypsin-like proteases. Chymase and tryptase are serine proteases which are stored in mast cell granules and released upon degranulation. This work investigated the inhibition of human chymase and tryptase by BBI. Active-site titration of human skin chymase by BBI demonstrated that BBI was a highly effective inhibitor of human chymase. Virtually stoichiometric inhibition of chymase by BBI was observed at 10 nM chymase. Kinetic studies of the inhibition reaction yielded an association rate constant of 4.0 x 10(5) M(-1) s(-1) and a dissociation rate constant of 1.7 x 10(-5) s(-1). From these two constants we estimate a K(i) of 50 pM. Chymase/BBI complexes did not dissociate in SDS-PAGE analyses under nonreducing conditions, consistent with the formation of a very tight complex with little tendency to dissociate. In contrast to chymase, human tryptase was not inhibited by BBI. These studies demonstrate that BBI is a good inhibitor of human chymase, exhibiting reaction properties better than physiological inhibitors described to date.

Binding Sites↗
PubMed1999

Highly efficient inhibition of human chymase by alpha(2)-macroglobulin.

The inhibition of human chymase by the protease inhibitor alpha(2)-macroglobulin (alpha2M) was investigated. Titration of chymase hydrolytic activity with purified alpha2M showed that approximately 1 mol of alpha2M tetramer inhibits 1 mol of chymase. Inhibition was associated with cleavage of the alpha2M bait region and formation of a 200-kDa covalent complex. NH(2)-terminal sequencing of chymase-treated alpha2M revealed cleavage at bonds Phe684-Tyr685 and Tyr685-Glu686 of the bait region. alpha2M pretreated with methylamine, an inactivator of alpha2M, did not inhibit chymase. The apparent second-order rate constant for inhibition (k(ass)) was 5 x 10(6) M(-1) s(-1), making alpha2M the most efficient natural protein protease inhibitor of chymase so far described. The k(ass) value for inhibition was decreased approximately 10-fold by addition of heparin, a glycosaminoglycan produced by mast cells that binds to chymase. Heparin did not change significantly the stoichiometry of inhibition or block covalent complex formation. These results indicate that alpha2M is an important inhibitor to consider in the regulation of human chymase.

Chymases↗
PubMed2001

Inhibitory mechanism of daphnodorins for human chymase.

We investigated the inhibitory mechanisms of daphnodorins for human chymase using three-dimensional molecular modeling. In daphnodorin A-human chymase complex, daphnodorin A was fixed to the active site via hydrogen bonds with Ala177, Phe29, and Gly199 in human chymase, and it formed hydrogen bonds with Ser182 and Gly180, and this complex was formed stably. In daphnodorin B-human chymase complex, daphnodorin B formed hydrogen bonds with Lys28 and Phe29 in human chymase, but it could not form hydrogen bonds with Gly199, Ala177, and Lys179. The phenyl group of daphnodorin B shifted from the P1 hole in human chymase in comparison with that of daphnodorin A. For the inhibition of human chymase by daphnodorins, we indicated that it was significant whether daphnodorins formed hydrogen bonds with Ala177 located in the P1 hole, Ser182 located in the active site, Gly180 located in the anion hole, and with Gly199, Phe29, and Lys28 in human chymase.

Benzopyrans↗
PubMed2002

Chymase inhibitor, BCEAB, suppressed peritoneal adhesion formation in hamster.

BACKGROUND: Mast cells are closely related to adhesion formation, while it has been unclear which factor in mast cells plays an important role in the development of adhesion formation. To clarify the role of chymase produced from mast cells in adhesion formation, we investigated the preventive effect of a specific chymase inhibitor, BCEAB, on adhesion formation in a hamster experimental model. MATERIALS AND METHODS: Hamsters were administered orally once daily with 100 mg/kg of BCEAB or placebo from the operated day to 1 week after the operation. The uterus was grasped and denuded by a swab. RESULTS: One week after the operation, the scores for adhesion formation in the chymase inhibitor-treated group were significantly decreased in comparison with those in the placebo-treated group (placebo-treated group, 2.80 +/- 0.20; chymase inhibitor-treated group 1.60 +/- 0.31: P < 0.01). The chymase activity in the injured uterus was also significantly suppressed in the chymase inhibitor-treated group (placebo-treated group, 17.3 +/- 2.69 mU/mg protein; chymase inhibitor-treated group 9.60 +/- 0.89: P < 0.05). After scraping the utelus, the level of transforming growth factor-beta in the peritoneal fluid was significantly increased in the placebo-treated group, while it was suppressed to 70% by the treatment with BCEAB. CONCLUSIONS: The specific chymase inhibitor BCEAB may be a useful drug for prevention of adhesion formation.

Animals↗
PubMed1990

Quantitative enzyme-histochemical analysis of tryptase- and chymase-containing mast cells in psoriatic skin.

Tryptase-containing mast cells have recently been found to be increased in the upper dermis of psoriatic lesions. In the present study, the distribution of chymase- and tryptase-containing mast cells was morphometrically analysed at different dermal levels of lesional and non-lesional psoriatic skin (12 patients) as well as normal human skin. Mast cell tryptase was identified enzyme-histochemically, using Z-Gly-Pro-Arg-MNA as the substrate. For demonstrating mast cell chymase, a simple and specific enzyme-histochemical staining method was developed, using Suc-Val-Pro-Phe-MNA as the substrate. All mast cells positive for chymase were also positive for tryptase and Giemsa stain. Although the number of tryptase-positive mast cells was slightly increased throughout the dermis of lesional psoriatic skin, this increase was most pronounced in the upper dermis immediately beneath, and in close contact with, the epidermis. In contrast, the number of chymase-positive mast cells was clearly decreased in the upper dermis of psoriatic lesions, but not in the deeper dermis, as compared with non-lesional psoriatic skin. In addition, all chymase-positive mast cells observed in the upper dermis were very weakly stained when compared with those in the deeper dermis. No differences were found between non-lesional psoriatic skin and normal skin in which the number of mast cells containing chymase was 72-73% of the number containing tryptase. The present results suggest that T mast cells particularly, containing tryptase but no chymase, proliferate in psoriatic lesions, and that the increase in tryptase activity and the decrease in chymase activity in the upper dermis may lead to an imbalance in the biochemical regulatory systems.

Adolescent↗
PubMed2005

Application of novel tissue microarrays to investigate expression of tryptase, chymase and KIT protein in placental mast cells.

INTRODUCTION: Tissue microarrays comprise numerous small representative tissue samples from hundreds of different cases assembled on a single histologic slide, and therefore allow high throughput analysis of multiple specimens at the same time. Mast cells are paracrine cells found ubiquitously in connective tissue. Expression of the serine proteases tryptase and chymase, as well as KIT protein, the receptor for stem cell factor (SCF), has been demonstrated in mast cells. Because little is known about the role of mast cells in the placenta, we investigated the number and expression of chymase, tryptase, and KIT protein in placental mast cells using newly developed tissue microarrays. MATERIALS AND METHODS: Tissue microarrays were prepared from archival paraffin tissue blocks of 90 placentae, including 15 normal ones as a control group. Gestational age of the placentae ranged from 7 to 42 weeks. Sections of formalin-fixed paraffin-embedded material were prepared on chemically activated cover-slides. The slides were cut in 4-mm(2) squares containing representative areas, and transferred to a tissue microarray. Hematoxylin and eosin (H&E), chloroacetate esterase (CAE), toluidine blue, periodic acid--Schiff (PAS), and immunohistochemical staining were performed. The number of mast cells and expression of chymase, tryptase, and KIT protein were evaluated in each case. RESULTS: Mast cell numbers in placentae with inflammation/abortion exceeded that of normal placentae. Although statistically not significant, we furthermore observed an increase in chymase-positive mast cells in the group of placentae associated with fetal malformations/chromosomal aberrations compared with normal placentae. DISCUSSION: Novel tissue microarray technique has been introduced into placental research, and allows multiple placental tissue samples to be effectively analyzed simultaneously. This study indicated an increased number of chymase-positive mast cells in placentae with fetal malformation/chromosomal aberration. Activation of angiotensin II by chymase may play a role in fetal malformation. Moreover, it has been speculated that mast cells may only express chymase (MC(C)). Our findings denote the presence of placental MC(C). However, further studies are needed to elucidate more precisely the role of mast cell chymase in the placenta.

Adult↗
PubMed2005

Eosinophil migration induced by mast cell chymase is mediated by extracellular signal-regulated kinase pathway.

Mast cell chymase is known to induce eosinophil migration in vivo and in vitro. In the present study, we investigated possible involvement of mitogen-activated protein (MAP) kinases; extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, in the chymase-induced eosinophil migration. Human chymase induced a rapid phosphorylation of ERK1/2 and p38 in human eosinophilic leukemia EoL-1 cells, while no phosphorylation was detected in JNK. The chymase-induced phosphorylation of ERK and p38 was inhibited by pertussis toxin. Similar results were obtained in the experiments using mouse chymase and eosinophils. U0126 (the inhibitor for MAP/ERK kinase) suppressed chymase-induced migration of EoL-1 cells and mouse eosinophils. However, SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) showed little effect on the migration. It is suggested therefore that chymase activates ERK and p38 probably through G-protein-coupled receptor, and that ERK but not p38 cascade may have a crucial role in chymase-induced migration of eosinophils.

Animals↗
PubMed2004

Therapeutic applications of chymase inhibitors in cardiovascular diseases and fibrosis.

Chymase activates not only angiotensin I to angiotensin II but also latent transforming growth factor-beta-binding protein to transforming growth factor-beta. In dog grafted veins, chymase activity and angiotensin II concentration along with vascular proliferation were significantly increased, while they were significantly suppressed by a chymase inhibitor. After balloon injury in dog arteries, chymase activity was significantly increased in the injured artery, and a chymase inhibitor and an angiotensin AT(1) receptor antagonist were effective in preventing the vascular proliferation, but an angiotensin-converting enzyme inhibitor was ineffective. In fibrotic models, the tissue fibrosis was reduced by chymase inhibitors. In adhesion models, the transforming growth factor-beta concentration and adhesion formation were suppressed by chymase inhibitors. Therefore, chymase inhibitors may be useful for preventing cardiovascular diseases and fibrosis via inhibition of angiotensin II formation and transforming growth factor-beta activation.

Animals↗
PubMed2005

Effect of chymase on intraocular pressure in rabbits.

Chymase is a chymotrypsin-like serine protease that is stored exclusively in the secretory granules of mast cells and converts big endothelins to endothelin-1 (1-31). The aim of this study was to evaluate the effect of chymase on intraocular pressure in rabbits. Chymase injection (3 and 10 mU) resulted in a trend toward increased intraocular pressure and a significant increase in intraocular pressure at a dose of 10 mU compared with the control. A specific chymase inhibitor, Suc-Val-Pro-Phe(P)(OPh)(2), attenuated the ocular hypertension induced by chymase. Endothelin-1 (1-31) also caused ocular hypertension, which was inhibited by a selective endothelin ET(A) receptor antagonist, cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ-123). Moreover, chymase-induced ocular hypertension was inhibited by BQ-123. These results suggest that chymase influences the regulation of intraocular pressure, and it is likely that the formation of endothelin-1 (1-31) and subsequent activation of endothelin ET(A) receptors are involved in the development of ocular hypertension induced by chymase.

Animals↗
PubMed1997

Induction of chymase that forms angiotensin II in the monkey atherosclerotic aorta.

Chymase shows a catalytic efficiency in the formation of angiotensin (Ang) II. In the present study, the characterization and primary structure of monkey chymase were determined, and the pathophysiological role of chymase was investigated on the atherosclerotic monkey aorta. Monkey chymase was purified from cheek pouch vascular tissue using heparin affinity and gel filtration columns. The enzyme rapidly converted Ang I to Ang II (Km = 98 microM, k(cat) = 6203/min) but did not degrade several peptide hormones such as Ang II, substance P, vasoactive intestinal peptide and bradykinin. The primary structure, which was deduced from monkey chymase cDNA, showed a high homology to that of human chymase (98%). The mRNA levels of the aorta chymase were significantly increased in the atherosclerotic aorta of monkeys fed a high-cholesterol diet. These results indicate that monkey chymase has a highly specific Ang II-forming activity and may be related to the pathogenesis of atherosclerosis.

Amino Acid Sequence↗
PubMed2001

Oral administration of a specific chymase inhibitor, NK3201, inhibits vascular proliferation in grafted vein.

Chymase may play an important role in vascular proliferation, as shown by in-vitro experiments, but the role of chymase in vivo has been unclear. In this study, we investigated the effect of a novel chymase inhibitor, NK3201, on this proliferation in dog grafted veins. NK3201 inhibited human and dog chymases, but not rabbit ACE. NK3201 suppressed the Ang I-induced vascular contraction in isolated dog arteries in the presence of an ACE inhibitor, and the IC50 value of chymostatin and NK3201 in dog artery was 320 nM. In dog, the concentration of NK3201 in blood was about 10 microM at 24 h after oral administration of the drug (5 mg/kg). In the group treated with NK3201, each dog was administered orally 5 mg/kg per day from 5 days before to the day before the removal of the grafted veins. Each dog underwent right common carotid artery bypass grafting with the ipsilaterial external jugular vein. By 28 days after grafting, a significant vascular proliferation was observed in the grafted veins and the chymase activity was also increased significantly. Treatment with chymase inhibitor significantly suppressed the proliferation of the grafted veins and the increased chymase activity. In this study, we demonstrate for the first time that oral administration of a specific chymase inhibitor, NK3201, appears useful for preventing vascular proliferation.

Acetamides↗
PubMed1990

Dog mast cell chymase: molecular cloning and characterization.

We cloned and characterized a cDNA coding for the complete amino acid sequence of dog mast cell chymase. The cDNA was identified by screening a dog mastocytoma cDNA library with an oligonucleotide probe based on the amino acid sequence of a fragment of dog mastocytoma chymase. The deduced amino acid sequence reveals a putative 21-residue prepropeptide followed by a catalytic domain of 228 residues. The primary structure of the preproenzyme shares features with rat mucosal mast cell chymase (RMCP II), several lymphocyte-associated proteases, and neutrophil cathepsin G. The common characteristics include an apparent activation peptide terminating in glutamic acid, strict conservation of an octapeptide (residues 9-16) in the N-terminal portion of the catalytic domain, and the presence of only six cysteines available for intramolecular disulfide bond formation. However, dog chymase differs in being modified by N-glycosylation. Although the dog chymase catalytic domain exhibits a similar level of sequence identity when compared with both RMCP II and the rat connective tissue mast cell chymase RMCP I (58% and 61%, respectively), the dog enzyme most closely resembles RMCP I in its high predicted net charge (+16) and in the presence of serine at the base of its putative primary substrate binding pocket. The dog chymase differs strikingly from dog mast cell tryptase in the preprosequence and in the structure of the catalytic domain. Therefore, chymase appears not to be closely related to tryptase and may not share a mechanism of activation, even though both enzymes are packaged and released together.

Amino Acid Sequence↗
PubMed2003

Structure of human pro-chymase: a model for the activating transition of granule-associated proteases.

Human chymase is a protease involved in physiological processes ranging from inflammation to hypertension. As are all proteases of the trypsin fold, chymase is synthesized as an inactive "zymogen" with an N-terminal pro region that prevents the transition of the zymogen to an activated conformation. The 1.8 A structure of pro-chymase, reported here, is the first zymogen with a dipeptide pro region (glycine-glutamate) to be characterized at atomic resolution. Three segments of the pro-chymase structure differ from that of the activated enzyme: the N-terminus (Gly14-Gly19), the autolysis loop (Gly142-Thr154), and the 180s loop (Pro185A-Asp194). The four N-terminal residues (Gly14-Glu15-Ile16-Ile17) are disordered. The autolysis loop occupies a position up to 10 A closer to the active site than is seen in the activated enzyme, thereby forming a hydrogen bond with the catalytic residue Ser195 and occluding the S1' binding pocket. Nevertheless, the catalytic triad (Asp102-His57-Ser195) is arrayed in a geometry close to that seen in activated chymase (all atom rmsd of 0.52 A). The 180s loop of pro-chymase is, on average, 4 A removed from its conformation in the activated enzyme. This conformation disconnects the oxyanion hole (the amides of Gly193 and Ser195) from the active site and positions only approximately 35% of the S1-S3 binding pockets in the active conformation. The backbone of residue Asp194 is rotated 180 degrees when compared to its conformation in the activated enzyme, allowing a hydrogen bond between the main-chain amide of residue Trp141 and the carboxylate of Asp194. The side chains of residues Phe191 and Lys192 of pro-chymase fill the Ile16 binding pocket and the base of the S1 binding pocket, respectively. The zymogen positioning of both the 180s and autolysis loops are synergistic structural elements that appear to prevent premature proteolysis by chymase and, quite possibly, by other dipeptide zymogens.

Autolysis↗