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PubMed1990

Mast cell chymase potentiates histamine-induced wheal formation in the skin of ragweed-allergic dogs.

Skin mast cells release the neutral protease chymase along with histamine during degranulation. To test the hypothesis that chymase modulates histamine-induced plasma extravasation, we measured wheal formation following intradermal injection of purified mast cell chymase and histamine into the skin of ragweed-allergic dogs. We found that chymase greatly augments histamine-induced wheal formation. The magnitude of the potentiating effect increases with increasing doses of chymase and becomes maximal approximately 30 min after administration. Injection of chymase without histamine does not evoke wheal formation. The chymase potentiation of histamine-induced skin responses is prevented completely by pretreatment with the H1-receptor antagonist pyrilamine, and is prevented by inactivation of chymase with soybean trypsin inhibitor, suggesting that both histamine and preserved catalytic activity are required for the effects of chymase. To examine the effects of histamine and chymase released in situ in further experiments, we measured wheal size following local degranulation of mast cells by intradermal injection of ragweed antigen or compound 48/80. We found that pretreatment with either soybean trypsin inhibitor or pyrilamine markedly reduces ragweed antigen- or 48/80-induced wheal formation, supporting the results obtained by injection of exogenous chymase and histamine. These findings suggest a novel and important proinflammatory role for chymase in modulating the effects of histamine on vascular permeability during mast cell activation.

Allergens↗
PubMed2006

Chymase inhibition prevents cardiac fibrosis and dysfunction after myocardial infarction in rats.

Human chymase activates not only angiotensin II but also transforming growth factor-beta, a major stimulator of myocardial fibrosis, while rat chymase activates transforming growth factor-beta, but not angiotensin II. To clarify the role of chymase-dependent transforming growth factor-beta activation, we evaluated whether chymase inhibition prevents cardiac fibrosis and cardiac dysfunction after myocardial infarction in rats. Myocardial infarction was induced by ligation of the left anterior descending coronary artery. One day after the ligation, rats were randomized into 2 groups: 1) a chymase-treated group that received 10 mg/kg per day of the chymase inhibitor NK3201 orally for 4 weeks; and 2) a vehicle group of non-treated rats with myocardial infarction. We also included a control group who underwent sham-operation and no treatment. Four weeks after ligation, echocardiography revealed that chymase inhibitor treatment reduced the akinetic area and increased fractional area change but did not significantly change left ventricular end-diastolic area. Chymase inhibition significantly reduced left ventricular end-diastolic pressure, increased the maximal end-systolic pressure-volume relationship and decreased the time constant of left ventricular relaxation. Chymase activity in the non-infarcted myocardium was significantly increased in the vehicle group, but it was significantly reduced by chymase inhibitor treatment. The fibrotic area in the cardiac tissues and the mRNA levels of collagen I and collagen III were also significantly lower in the chymase inhibitor-treated group than in the vehicle group. Therefore, the pathway forming chymase-dependent transforming growth factor-beta may play an important role in myocardial fibrosis and cardiac dysfunction rather than left ventricular dilatation after myocardial infarction.

Acetamides↗
PubMed1998

Chymase: its pathophysiological roles and inhibitors.

Chymase is a chymotrypsin-type serine protease mainly localized in mast cells (MCs). Human, primate, and dog chymase generate angiotensin II (Ang II) from Ang I, while mouse and rat chymases degrade Ang II. It is suggested that chymase generating Ang II might be an alternative Ang II-forming enzyme to angiotensin-converting enzyme (ACE) in the renin-angiotensin system in tissues, but not in blood, and cause hypertrophy and remodeling of cardiovascular tissues. Chymase also degrades extracellular matrix, and processes procollagenase, inflammatory cytokines and other bioactive peptides. As a result, chymase plays important roles in inflammatory tissues through its proteolytic activities to cause tissue remodeling, that is, a chymase inhibitor may have the ability to prevent diseases caused by the above inflammatory reactions. The investigation of chymase inhibitors by pharmaceutical companies has yielded peptide and peptide mimetic inhibitors. We also found potent non-peptide low molecular inhibitors. However, the in vivo functions of chymase have not been verified so far by applying a chymase inhibitor to in vivo pathological models. In this article, we overview the pathophysiological roles of chymase and chymase inhibitors proposed to date, and discuss the structure-activity relationships of substituted 3-phenylsulfonyl-1-phenylimidazolidine-2,4-dione derivatives.

Angiotensin I↗
PubMed1992

Mast cell proteoglycans modulate the secretagogue, proteoglycanase, and amidolytic activities of dog mast cell chymase.

Chymase, a potent secretagogue for airway gland serous cells, is stored in secretory granules and released from mast cells together with proteoglycans. To investigate the hypothesis tha tproteoglycans modulate chymase-induced effects, we studied the influence of proteoglycans purified from dog mastocytoma cells on chymase-induced secretion from cultured bovine airway gland serous cells. Heparin proteoglycans reduced the chymase-induced secretory response, whereas glycosaminoglycans and chondroitin sulfate proteoglycans had less of an effect. Chymase released together with proteoglycans from activated mast cells caused secretion comparable to that caused by purified chymase reconstituted with purified proteoglycans. Despite partial inhibition by exocytosed proteoglycans, the secretagogue activity of chymase remains substantial compared to that of histamine. However, proteoglycans virtually abolished chymase-induced degradation of the products of serous cell secretion. Although the secretagogue and proteoglycanase activities of chymase are inhibited by most classes of mast cell granule-associated glycans, the amidolytic activity of chymase toward tripeptide 4-nitroanilide substrates is augmented. These findings suggest that mast cell proteoglycans modulate the secretagogue, proteoglycanase, and peptidase activity of chymase, and the results predict that the extent of this modulation in vivo depends on the nature of the proteoglycans with which chymase is released from mast cells.

Animals↗
PubMed1990

Cleavage of a rat serosal mast cell membrane component during degranulation mediated by chymase, a secretory granule protease.

Exogenous addition of purified chymase, a rat serosal mast cell (RSMC) chymotryptic enzyme, results in RSMC degranulation at 37 degrees, but not at 1 degree. Chymase can cause an active site-dependent inducing event at 1 degree such that RSMC degranulation occurs if the cells are later incubated at 37 degrees. RSMC exposed to chymase or other stimuli were surface radiolabelled using 125I and Iodo-Gen, solubilized with 1% Nonidet-40, and the resulting 25,000 g supernatants analysed by SDS-PAGE and autoradiography. A 125I-labelled RSMC membrane protein of approximate 90,000 MW decreased upon exposure to either chymase or alpha-chymotrypsin (alpha-CT) for 5 min at 37 degrees or to chymase for 60 min at 1 degree. Exposure of RSMC to the secretagogues ionophore A23187, compound 48/80, and anti-IgE for 5 min at 37 degrees resulted in beta-hexosaminidase (a secretory granule enzyme) release, but did not cause a detectable change in the 90,000 MW surface-labelled protein. Lima bean trypsin inhibitor, which inhibits both the esterase and RSMC degranulation activities of chymase and alpha-CT, prevented the disappearance of the 125I-labelled 90,000 MW band when added with chymase or alpha-CT. Exposure of RSMC to chymase at 1 degree for 0-10 min, prior to addition of LBTI, led to a progressive disappearance of the 90,000 MW band, which corresponded to the kinetics of priming for subsequent RSMC degranulation at 37 degrees. When RSMC were exposed to trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree, a progressive disappearance of the 90,000 MW band occurred, in association with a loss of sensitivity to subsequent activation by chymase at 37 degrees. The disappearance of the 90,000 MW determinant in association with chymase-mediated priming for degranulation and the inability of chymase to mediate degranulation of trypsin-treated RSMC, which lack this membrane protein, suggests that it is involved in chymase-mediated RSMC degranulation.

Animals↗
PubMed1989

Modulation of chymase-mediated rat serosal mast cell degranulation by trypsin or diisopropyl fluorophosphate.

Exposure of rat serosal mast cells (RSMC) to chymase, an endogenous secretory granule serine protease, at 37 degrees results in exocytosis, as determined by beta-hexosaminidase release. As the number of RSMC is increased with a set amount of chymase, the net percentage beta-hexosaminidase release decreases linearly, implying a finite set of cellular interactions per chymase unit. Pretreatment of RSMC with trypsin at 37 degrees renders them refractory to subsequent exocytosis mediated by chymase in a dose- and time-dependent fashion, with complete refractiveness occurring by 15 min at 37 degrees with 2.5 micrograms trypsin/ml. Anti-IgE-mediated coupled activation-secretion of RSMC is not affected by the same trypsin pretreatment. When RSMC are pretreated with trypsin (2.5 micrograms/ml) for 0-120 min at 1 degree a progressive loss of sensitivity to activation by chymase at 37 degrees occurs. RSMC susceptibility to chymase-mediated degranulation after trypsin pretreatment can be partially regenerated by culturing the RSMC for about 24 hr in medium at 37 degrees. These findings suggest that a trypsin-sensitive constituent, possibly a receptor or substrate, is necessary for the functional interaction of chymase with RSMC. When added with diisopropyl fluorophosphate (DFP), chymase does not induce RSMC degranulation at 37 degrees. However, if the DFP is removed before addition of chymase at 37 degrees or is added after the chymase-priming event occurs at 1 degree, subsequent degranulation at 37 degrees is not inhibited. Thus, the induction and not the secretion phase is DFP-inhibitable in chymase-induced activation-secretion. In addition, the priming but not the exocytosis phase of chymase-initiated RSMC activation-secretion, which is not dependent on temperature and calcium ion concentration, involves a cellular trypsin-sensitive protein.

Animals↗
PubMed1997

Cloning and expression of the dog mast cell alpha-chymase gene.

Chymases are chymotrypsin-like serine proteinases secreted by mast cells. Alpha- and beta-chymases differ in structure, function, and mast cell subset- and species-specific expression. Seeking genetic regulatory elements shared by alpha-chymases, we sequenced the dog alpha-gene. Extensive homology was found in intronic and flanking sequences of the dog, human, and mouse alpha-chymase genes, but little in corresponding beta-chymase sequences. Repetitive elements probably derived from retroposons are unique features of the dog flank. DNA blots suggest that the dog alpha-gene, like its human counterpart, may be the genome's sole chymase, unlike in rodents, in which beta-chymases predominate. Nuclear runoff studies predict that transcriptional mechanisms explain differences in steady state chymase and tryptase mRNA levels between mastocytoma and non-mast cells. In dog BR mastocytoma cells incubated with phorbol ester, high steady state levels of alpha-chymase mRNA drop dramatically with little change in tryptase mRNA, whereas dexamethasone decreases expression of both mRNAs. Portions of the dog or human gene 5' flank transfected into BR cells drive expression of a reporter gene and define regions with active promoters. Thus, BR cells express high levels of alpha-chymase mRNA regulated independently of tryptase and support transcription using dog or human promoters. These studies reinforce the alphabeta-chymase dichotomy and suggest the utility of BR cells in probing regulation of alpha-chymase expression.

Animals↗
PubMed2006

Rapid lineage-specific diversification of the mast cell chymase locus during mammalian evolution.

Serine proteases constitute the major protein granule content of cells of several hematopoietic cell lineages. A subgroup of these proteases, including the mast cell chymases, neutrophil cathepsin G, and T cell granzymes B to F and N, are in all investigated mammals encoded in one locus, the chymase locus. It is interesting to note that this locus has diversified greatly during the last 95 Myr of mammalian evolution. This divergence is exemplified by the presence of Mcpt8-related genes and multiple beta-chymases in the mouse and rat, which lack direct counterparts in primates and in seven functional granzyme genes in the mouse where the human locus has only two. To study the expansion of the locus during rodent evolution and to better understand the evolutionary origin of beta-chymases and the Mcpt8-family, we have performed a detailed analysis of the chymase locus of four mammalian species, i.e., human, dog, mouse, and rat. As a result, we report here a second chymase-like gene in dog, Cma2, which clusters with beta-chymases in phylogenetic analyses. This finding supports a duplication of the common ancestor for alpha- and beta-chymases before the major radiation of placental mammals, and a loss of the ancestral beta-chymase gene sometime during primate evolution. Moreover, we show that in the rat, the Mcpt8-family diversified relatively recently together with sequences related to the beta-chymase Mcpt2. Eight novel genes were identified in the duplication region, four of which are predicted to be functional. Duplications of rat granzyme B- and C-like sequences occurred seemingly independently within a similar time frame, but did not give rise to functional genes. Due to the duplications in rat and deletions in the carnivore/primate lineage, the rat chymase locus is approximately 15 and 9 times larger than its counterparts in dog and human, respectively. These findings illustrate the importance of gene duplications in conferring rapid changes in mammalian genomes.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of mouse mast cell chymases.

Mouse mast cell chymases are granule-associated serine proteinases with chymotrypsin-like substrate specificities. cDNAs for two new chymases were isolated from a cDNA library constructed using mRNA from ABFTL-6 mouse mast cells by screening with a rat mast cell proteinase cDNA. The deduced amino acid sequence of mouse chymase 1 consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. Chymase 1 is unusual in that an Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine proteinase. Also, chymase 1 is expected to have a large positive charge (+13) at physiological pH. A partial cDNA for chymase 2 encodes 177 residues of the carboxy terminal portion of a second proteinase distinct from chymase 1. Chymase 2 cDNA contains a highly conserved intron/exon junction, a high positive charge (+17) and a novel, second potential N-glycosylation site. Transcripts for both chymases are found in ABFTL-6 mast cells, but only chymase 2 mRNA is in mouse connective tissue mast cells. These data suggest that these chymases have distinct enzymatic properties and tissue-specific patterns of gene expression.

Amino Acid Sequence↗
PubMed2005

Angiotensin converting enzyme-independent angiotensin ii production by chymase is up-regulated in the ischemic kidney in renovascular hypertension.

BACKGROUND: Tissue angiotensin II (ANG II) levels are elevated in both kidneys in renovascular hypertension (RVH). It has been demonstrated previously that intrarenal ANG II is augmented by an angiotensin converting enzyme (ACE) dependent mechanism in the non-ischemic kidney, but the role of ACE-independent production of ANG II in the kidney by the enzyme chymase is unknown. This study tested the hypothesis that intrarenal chymase activity is up-regulated in RVH. METHODS: A two-kidney, one-clip (2K1C) rat model was used to induce RVH (n = 6 rats/group). Regulation of intrarenal chymase activity by plasma ANG II was investigated using an ANG II-infusion model. At sacrifice 14 days post-operatively, steady-state ANG II levels in plasma and kidney were quantified by radioimmunoassay. ANG II production was quantified in kidney homogenates by incubating at 37 degrees C for 60 min with enzyme substrate (200 microm ANG I) alone or substrate containing the chymase inhibitor chymostatin. ANG II was separated and quantitated by HPLC. Chymase activity was defined as the fraction of ANG II production inhibited by Chymostatin. RESULTS: 2K1C and ANG II-infused rats developed significant hypertension, compared to control rats (P = 0.0001 and P = 0.001, respectively). Chymase-dependent ANG II production was increased in the ischemic kidney, but not the non-ischemic kidney, of 2K1C rats compared to control animals (*P < 0.05). Intrarenal chymase activity was unchanged by ANG II infusion (P = NS). CONCLUSIONS: Chymase activity is up-regulated in the ischemic kidney of 2K1C rats. Plasma ANG II does not appear to regulate intrarenal chymase activity, suggesting that ischemia per se up-regulates chymase activity in the kidney. ACE-independent ANG II production by chymase may provide a mechanism for augmenting intrarenal ANG II in the ischemic kidney in RVH.

Angiotensin II↗
PubMed2002

Chymase inhibitors may prevent postoperative adhesion formation.

OBJECTIVE: To clarify the role of chymase produced from mast cells in adhesion formation, we measured chymase activity level and investigated the preventive effect of a chymase inhibitor, Suc-Val-Pro-Phe(p)(OPh)(2), on the postoperative adhesion formation. DESIGN: Prospective randomized study using a surgical model for adhesion formation. SETTING: Clean hamsters in an academic research environment. ANIMAL(S): Sixty-seven female Syrian hamsters. INTERVENTION(S): Hamsters were given a lesion, produced by uterus scraping, and the chymase inhibitor (10 microM) or placebo was injected into the abdomen. Chymase activities in uteri were measured 3 days after the operation, and the scores of adhesion formations were assessed at 2 weeks. MAIN OUTCOME MEASURE(S): Measurement of chymase activity and scoring of adhesion formation were performed. RESULT(S): A significant increase of chymase activity in the injured uterus reduced by treatment with the chymase inhibitor. The scores of adhesion formations in the chymase inhibitor-treated group were significantly decreased in comparison with those in the placebo-treated group. CONCLUSION(S): Chymase contained in mast cells plays an important role in adhesion formation, and a chymase inhibitor may be a useful drug for prevention of adhesion formation.

Animals↗
PubMed2001

Mast cell tissue inhibitor of metalloproteinase-1 is cleaved and inactivated extracellularly by alpha-chymase.

We previously reported that mast cell alpha-chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. Alpha-chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1's alpha-chymase-mediated cleavage products reveals hydrolysis at Phe(12)-Cys(13) and Phe(23)-Val(24) in loop 1 and Phe(101)-Val(102) and Trp(105)-Asn(106) in loop 3 of the NH(2)-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to alpha-chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, alpha-chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with alpha-chymase increases gel B activity 2- to 5-fold, suggesting that alpha-chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of alpha-chymase blocks degranulation-induced TIMP-1 processing (absent in alpha-chymase-deficient HMC-1 cells). Purified alpha-chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell alpha-chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.

Acute-Phase Proteins↗
PubMed1986

Rat serosal mast cell degranulation mediated by chymase, an endogenous secretory granule protease: active site-dependent initiation at 1 degree C.

Exposure at 37 degrees C of rat serosal mast cells (RSMC) to chymase, an endogenous secretory granule serine protease, results in exocytosis as determined by the release of another secretory granule enzyme, beta-hexosaminidase. Chymase-mediated RSMC degranulation does not occur at 1 degree C; however, exposure of RSMC to chymase at 1 degree C followed by the removal of buffer and the resuspension of the cells in buffer alone at 37 degrees C results in exocytosis equivalent to that obtained by direct exposure of RSMC to chymase at 37 degrees C. Maximal chymase-mediated RSMC degranulation at 37 degrees C is Ca2+-dependent and Mg2+-independent. The dose-dependent degranulation-inducing interaction of chymase and alpha-chymotrypsin with RSMC at 1 degree C is Ca2+-independent, whereas subsequent exocytosis at 37 degrees C in new buffer without added enzyme still requires Ca2+. Specific binding of 125I-labeled alpha-chymotrypsin to RSMC does not occur at 1 degree C, implying that the inducing action of chymase is not a simple ligand-receptor binding. The enzyme inhibitors diisopropyl fluorophosphate and lima bean trypsin inhibitor inhibit subsequent exocytosis at 37 degrees C only if they are added within the first 10 min of the interaction of RSMC and chymase at 1 degree C, implying that an active site-dependent inducing event occurs between RSMC and chymase at 1 degree C. Thus, chymase-induced coupled activation-secretion can be divided into a cation- and temperature-independent initiation phase, which is dependent on the active site of exogenously added chymase and a subsequent temperature-dependent and calcium-augmented cellular secretion phase.

Animals↗
PubMed1985

Pharmacological modulation of activation-secretion of rat serosal mast cells by chymase, an endogenous secretory granule protease.

The action of pharmacologic agents on chymase-induced exocytosis of beta-hexosaminidase and arachidonic acid (AA) metabolism by rat serosal mast cells (RSMC) was determined and compared with their effects on anti-IgE induced activation. Indomethacin (INDO) (less than or equal to 10 microM), a cyclooxygenase inhibitor, did not affect chymase- or anti-IgE-mediated exocytosis, while completely inhibiting prostaglandin D2 (PGD2) release at 1.25 microM. Theophylline (THEO), mepacrine, 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW755C), and diethylcarbamazine (DEC), inhibitors of adenosine binding and phosphodiesterases, phospholipases, AA metabolism, and vesicular transport as well as leukotriene A4 formation, respectively, inhibited exocytosis with ID50 values of 3.4, 0.22, 3.4 and 1.9 mM for chymase and 2.4, 0.17, 2.8 and 5.2 mM for anti-IgE. These agents inhibited net PGD2 release with ID50 values of 2.1, 0.04, less than 0.05, and 1.5 mM for chymase and of 0.5, 0.1, less than 0.05, and 4 mM for anti-IgE. 5,6-Dehydroarachidonic acid (DHA) and arachidonyl hydroxylamine (AH), 5-lipoxygenase inhibitors, did not affect chymase-mediated exocytosis; anti-IgE-mediated exocytosis was not altered by AH but was suppressed by DHA (ID50 = 20 microM). Nordihydroguaiaretic acid (NDGA), an antioxidant, inhibited chymase-mediated exocytosis dose-dependently (ID50 less than or equal to 13.3 microM) while decreasing anti-IgE-mediated exocytosis by only 30% at 2.5-20 microM; net PGD2 release induced by both stimuli was inhibited dose-dependently. 2',5'-Dideoxyadenosine (DDA) and 1,6-di(0-(carbamoyl)cyclohexanone oxime)hexane (RHC 80267) and inhibitors of adenylate cyclase and of di-triglyceride lipases, respectively, had little effect on exocytosis induced by chymase but inhibited that induced by anti-IgE with ID50 values of 0.4 mM and 37 microM, respectively. With DDA the inhibition of net PGD2 release occurred with anti-IgE but not chymase, whereas RHC 80267 inhibited both chymase and anti-IgE-mediated PGD2 release. Differential inhibition of activation-secretion suggests either that chymase provides a step inhibited in IgE-mediated exocytosis by DDA, RHC 80267 and DHA, or that the activating pathway initiated by chymase is distinct.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1995

The chymase-angiotensin system in humans: biochemistry, molecular biology and potential role in cardiovascular diseases.

Angiotensin I-converting enzyme (ACE) inhibitors are highly effective in the treatment of cardiovascular diseases. However, the relationship among the antihypertensive effects of ACE inhibitors, ACE inhibition and plasma angiotensin II levels is complex. During chronic therapy with ACE inhibition, plasma angiotensin II levels return to normal despite a continued antihypertensive effect. Recent studies show that a conversion of angiotensin I to angiotensin II in tissues can proceed despite complete ACE inhibition. In the search for a potential ACE inhibitor-resistant angiotensin II-forming enzyme activity in human heart tissue, chymase was identified as a major angiotensin II-forming enzyme. In primates, chymase-like angiotensin II-forming activity is localized in a number of tissues including the heart, blood vessels and lungs. Within the human heart, mast cells and endothelial cells are the sites of synthesis and storage of chymase, but a high level of the secreted chymase is also found in the cardiac interstitium, associated with the extracellular matrix. Mammalian chymases may be divided into two distinct structural groups, alpha and beta. alpha-chymases, such as human chymase, are highly specific and efficient angiotensin II-forming enzymes. beta-chymases, including several rat and mouse chymases, have a broad substrate specificity like chymotrypsin and do not form angiotensin II. In humans and baboons only a single chm gene of the alpha-subtype can be identified. By using an angiotensin I analogue that is selectively converted to angiotensin II by chymase and not ACE, a functional chymase-dependent angiotensin II formation has recently been demonstrated in conscious baboons.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin I↗
PubMed2000

Secretory production of recombinant human chymase as an active form in Pichia pastoris.

We succeeded in expressing in a Pichia pastoris (P. pastoris) host a cDNA encoding a mature human chymase (h-chymase) which was secreted directly into the culture medium. Recombinant human heart chymase (rh-chymase) was purified from the culture medium via a single one-step heparin-agarose column chromatography tracing, using succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Suc-AAPF-pNA) hydrolysing activity. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the rh-chymase showed a diffused protein band with molecular weight of 32-37 kDa. After deglycosylation, however, rh-chymase changed to a sharp protein band with molecular weight 28 kDa, which is equal in size to deglycosylated h-chymase. The rh-chymase had an activity to convert one of the natural substrates, angiotensin I, to angiotensin II. Double reciprocal plot analysis revealed that the K(m) value ofrh-chymase against Suc-AAPF-pNA was approximately 5.1 mM, which is close to that of purified h-chymase.

Angiotensin I↗
PubMed2005

Mast cell chymase is increased in chronic atopic dermatitis but not in psoriasis.

Mast cell chymase is a chymotrypsin-like serine proteinase primarily stored in secretory mast cell granules. Mast cell chymase has various effects on angiotensin, metalloproteases, lipoproteins, procollagen, neuropeptides and cytokines. Recent studies have demonstrated that chymase inhibitors inhibit skin inflammation. In this study we sought to determine the role of mast cell chymase in atopic dermatitis (AD) in comparison with its role in psoriasis and normal skin. Skin biopsy specimens were obtained from non-lesional and lesional skin of patients with chronic AD and psoriasis and from normal skin of non-atopic and non-psoriatic controls. The number of mast cells containing chymase was determined by immunohistochemistry using a chymase-specific monoclonal antibody. A significantly (P < 0.05) enhanced number of chymase-positive cells was found in lesional AD skin as compared to normal skin as well as to lesional and non-lesional skin of patients with psoriasis. A significant (P < 0.05) increase in the number of chymase-positive cells was also found in non-lesional AD skin in comparison to psoriasis. An enhanced, albeit not statistically significant difference was noted in non-lesional AD skin as compared to normal skin. In conclusion, these results suggest that mast cell chymase may play an integral part in eliciting and maintaining cutaneous inflammation in AD but not in psoriasis. The increased proteinase activity of mast cell chymase may also be involved in promoting a skin barrier defect in AD, which subsequently enhances the skin's permeability to allergens and microbes and thereby aggravates the eczema.

Adult↗
PubMed2001

Chymase bound to heparin is resistant to its natural inhibitors and capable of proteolyzing high density lipoproteins in aortic intimal fluid.

Degranulated mast cells are present in the human arterial intima. After degranulation of rat serosal mast cells, the secreted neutral serine protease chymase remains bound to the heparin proteoglycan matrix of the exocytosed granules, forming granule remnants. Addition of granule remnants to human aortic intimal fluid results in proteolysis of the apoAI present in the intimal fluid, which contains physiological inhibitors of chymase. To study the physiological mechanism of this protection of granule remnant-bound chymase against its inhibitors, we performed experiments using HDL3 as substrate. Chymase, when bound to the heparin proteoglycans of granule remnants, but not when released from them, resisted inhibition by the mammalian protease inhibitors alpha1-antitrypsin, alpha2-antichymotrypsin, alpha2-macroglobulin, and eglin C. Importantly, the heparin proteoglycan-bound chymase, but not unbound chymase, degraded its inhibitor (alpha1-antitrypsin) in the presence of its substrate (HDL3). Finally, binding to heparin proteoglycans of a physiological inhibitor of chymase (mucus protease inhibitor (MPI)) or of another substrate of chymase (LDL) did not inhibit the degradation of HDL3 by granule remnant-bound chymase. This study demonstrates that binding of chymase to the heparin proteoglycan chains of the exocytosed mast cell granules allows the protease to remain active and degrade HDL3 in the presence of its physiological inhibitors and in the presence of high concentrations of LDL, such as are found in the interstitial fluid of the arterial intima.

Animals↗