实电解质钙可诱发人肥大细胞组胺和类胰蛋白酶分泌

目的: 利用人大肠组织的肥大细胞和肥大细胞激活的体外研究系统,评价实电解质钙(calcium ionophore A23187, CI)诱导肥大细胞释放类胰蛋白酶和组胺的能力和机制.方法: 经酶悬浮的人大肠肥大细胞与CI共同培养后收集上清液,并用酶联免疫吸附试验(ELISA)的方法检测类胰蛋白酶分泌量,用以玻璃纤维为基础的荧光比色法检测组胺释放量.结果: 经过15 min的培养,CI可引起浓度相关性的组胺和类胰蛋白酶释放.其中组胺的最大分泌量比基础分泌量超出了5.3倍以上,而类胰蛋白酶的最大分泌量则比基础分泌量超出了2.8倍以上.CI在浓度高于1.0 μmol/L时引起的组胺释放量明显多于类胰蛋白酶释放量.时间关系曲线显示,CI的作用从加样后10 s开始,6 min后达高峰并至少持续15 min.百日咳毒素和代谢抑制剂均能抑制CI引起的组胺和类胰蛋白酶释放.结论: 人大肠肥大细胞在受到CI刺激时具有释放类胰蛋白酶和组胺的能力,这个过程与肥大细胞膜G蛋白偶联受体的激活有关,并消耗能量.     

蛋白酶激活受体2(PAR-2)激动剂对肥大细胞释放类胰蛋白酶的影响

研究反肉桂酰-亮-异亮-甘-精-亮-鸟-[酰胺]（tc-LIGRLO),一种PAR-2激动剂,对肥大细胞类胰蛋白酶释放的影响。结果显示,经过15min的培养,tc-LIGRLO可引起比基础分泌量增加1倍以上的类胰蛋白酶释放,作用强度超过抗IgE抗体和钙离子导入剂（calcium ionophore A23187,CI),而反PAR-2激动剂-反肉桂酰-鸟-亮-精-甘-异亮-亮-[酰胺]（tc-OLRGIL)无此作用,培养时间延长到30min时对tc-LIGRLO的作用无明显影响,其时间关系曲线表明,tc-LIGRLO的作用从1min开始,3min后达高峰,结果表明,PAR-2激动剂tc-LIGRLO是一种高效类胰蛋白酶释放刺激剂,在肥大细胞上可能有PAR-2存在。     

Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin.

Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.     

Protective role of mouse mast cell tryptase Mcpt6 in melanoma

Tryptase‐positive mast cells populate melanomas, but it is not known whether tryptase impacts on melanoma progression. Here we addressed this and show that melanoma growth is significantly higher in tryptase‐deficient (Mcpt6^?/?) versus wild‐type mice. Histochemical analysis showed that mast cells were frequent in the tumor stroma of both wild‐type and Mcpt6^?/? mice, and also revealed their presence within the tumor parenchyma. Confocal microscopy analysis revealed that tryptase was taken up by the tumor cells. Further, tryptase‐positive granules were released from mast cells and were widely distributed within the tumor tissue, suggesting that tryptase could impact on the tumor microenvironment. Indeed, gene expression analysis showed that the absence of Mcpt6 caused decreased expression of numerous genes, including Cxcl9, Tgtp2, and Gbp10, while the expression of 5p‐miR3098 was enhanced. The levels of CXCL9 were lower in serum from Mcpt6^?/? versus wild‐type mice. In further support of a functional impact of tryptase on melanoma, recombinant tryptase (Mcpt6) was taken up by cultured melanoma cells and caused reduced proliferation. Altogether, our results indicate a protective role of mast cell tryptase in melanoma growth.     

Dynamic changes and clinical significance of serum tryptase levels in STEMI patients treated with primary PCI

Abstract

Objective: To investigate the dynamic changes in serum tryptase levels and their association with clinical data in patients with acute ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI).

Methods: Serum tryptase levels were measured in 99 STEMI patients and 25 control subjects.

Results: Tryptase levels were significantly increased at admission, and descended after primary PCI. Tryptase levels at 0.5, 2 and 6?h after PCI were negatively correlated with the percentage of ST-segment resolution (STR) as well as left ventricular ejection fraction (LVEF).

Conclusions: High tryptase levels after PCI were associated with poor myocardial reperfusion and poor cardiac function.     

Histochemical method for demonstrating quail mast cell types simultaneously

Abstract

The development and application of selective staining methods for routine detection of mast cells are of considerable interest, because these cells play an important role in health and disease. The composition of cytoplasmic mast cell granules depends on the species and type of mast cell. The study reported here was conducted to investigate the combined use of aldehyde fuchsin (AF) and the Alcian blue-critical electrolyte concentration (AB-CEC) (pH 5.8, 0.3 M MgCl₂) techniques for differentiating avian mast cell subtypes. Tissue samples from skin, intestines, and lungs of six healthy adult quail and two control rats were fixed in Carnoy's solution and 10% formolin for routine histological processing. To determine the staining properties of sulfated glycosaminoglycans (GAGs), a three-step staining technique was applied using berberine sulfate, AF, and AB-CEC. In quail, AF positivity following application of the AB-CEC technique was found only in the lungs, mostly in cells that gave a berberine sulfate-positive reaction, and this positivity was determined to be localized particularly in the nucleus and perinuclear cytoplasm. In other regions, the pale AF staining of cells that did not emit fluorescence when stained with berberine sulfate was determined to be replaced by a blue color after application of AB-CEC. The AF/AB-CEC (pH 5.8, 0.3 M MgCl₂) technique demonstrated that rat and quail mast cells varied in both GAG types and their distribution within the cell. Especially in avian species, this technique can be applied to distinguish mast cells according to their GAG content. It can be used as an alternative to the AB/safranin O staining procedure for differentiating mast cells that contain and lack heparin.     

The fibrinogenolytic activity of purified tryptase from human lung mast cells

The capacity of purified tryptase from human lung mast cells to metabolize human fibrinogen, fibrin, and plasminogen was evaluated. Tryptase (5 micrograms/ml) inactivated the thrombin-induced clotting activity of fibrinogen (100 micrograms/ml) with essentially similar t 1/2 values of 4.6 min in the absence of heparin and 5.8 min in the presence of heparin (20 micrograms/ml) that were not appreciably different than with lysine-Sepharose-purified plasmin (5 micrograms/ml). Fibrinogen treated with tryptase together with heparin lost all detectable clotting activity by 4 hr at 37 degrees C, whereas fibrinogen treated with tryptase alone resulted in destruction of only 80% of fibrinogen clotting equivalents after 16 hr. Tryptase alone was observed to cleave only the alpha-chains of fibrinogen by electrophoresis of tryptase-treated, denatured, and reduced fibrinogen in polyacrylamide gradient gels. Tryptase together with heparin cleaved first the alpha-chain and then the beta-chain, the latter cleavage corresponding to complete loss of fibrinogen clotting activity by 4 hr. No fibrinogen fragments with anticoagulant activity were generated by tryptase. In contrast, plasmin left no residual clotting activity after 4 hr of incubation and generated fibrinogen fragments with anticoagulant activity. Plasmin sequentially cleaved the alpha, beta, and gamma subunits of fibrinogen. Tryptase alone (6 micrograms/ml) or together with heparin (20 micrograms/ml) failed to activate plasminogen (0.6 mg/ml) after a 60-min incubation at 37 degrees C. Addition of urokinase to tryptase-treated or untreated plasminogen resulted in essentially identical plasmin activities (0.32 and 0.34 U/ml, respectively), indicating that tryptase neither activates nor destroys plasminogen. Tryptase (700 ng) also failed to substantially solubilize cross-linked fibrin (2.6 micrograms) or the corresponding amount of fibrinogen bound to plastic microtiter plates with or without heparin. The failure to solubilize fibrinogen and, possibly, fibrin is consistent with the observation that the apparent m.w. by SDS polyacrylamide gel electrophoresis of unreduced fibrinogen is not appreciably altered by prior treatment with tryptase, even though cleavage of alpha-and beta-chains is revealed after reduction. Fibrinogenolysis by tryptase complements other mast cell mediators with anticoagulant properties such as heparin and suggests a significant prevention of coagulation by activated mast cells.     

A tick protein with a modified Kunitz fold inhibits human tryptase

TdPI, a tick salivary gland product related to Kunitz/BPTI proteins is a potent inhibitor of human beta-tryptase. Kinetic assays suggest that three of the four catalytic sites of tryptase are blocked by TdPI, and that the inhibition of one of these involves a peptide flanking the Kunitz head. In the course of the inhibition, tryptase cleaves TdPI at several positions. Crystal structures of the TdPI head, on its own and in complex with trypsin, reveal features that are not found in classical Kunitz/BPTI proteins and suggest the mode of interaction with tryptase. The loop of TdPI connecting the beta-sheet with the C-terminal alpha-helix is shortened, the disulphide-bridge pattern altered and N and C termini separated to produce a highly pointed molecule capable of penetrating the cramped active sites of tryptase. TdPI accumulates in the cytosolic granules of mast cells, presumably suppressing inflammation in the host animal's skin by tryptase inhibition.     

High density of tryptase‐positive mast cells in human colorectal cancer: a poor prognostic factor related to protease‐activated receptor 2 expression

Tryptase(+) mast cells (MCs), abundant in the invasive front of tumours, contribute to tissue remodelling. Indeed, protease‐activated receptor‐2 (PAR‐2) activation by MC‐tryptase is considered an oncogenic event in colorectal cancer (CRC). Recently, we have suggested NHERF1 as a potential new marker in CRC. In this study, we aimed to determine the distribution of tryptase(+) MCs and PAR‐2 and to examine the relationship between PAR‐2 and NHERF1, investigating their reputed usefulness as tumour markers. We studied a cohort of 115 CRC specimens including primary cancer (C) and adjacent normal mucosa (NM) by immunohistochemical double staining, analyzing the protein expression of MC‐tryptase, PAR‐2 and cytoplasmic NHERF1. MC density was higher in NM than in C. Tumours with high TNM stage and poor grade showed the highest MC density. A higher PAR‐2 immunoreactivity characterized tumours most infiltrated by MCs compared with samples with low MC density. Furthermore, PAR‐2 overexpression was associated with advanced TNM stage, poor grade and lymphovascular invasion (LVI). A positive correlation existed between tryptase(+) MC density and PAR‐2 expression. Cytoplasmic NHERF1 was higher in C than in NM and overexpressing tumours resulted associated with nodal and distant metastases, poor grade and LVI. PAR‐2 correlated with cytoplasmic NHERF1 and the PAR‐2(+)/cytoplasmic NHERF1(+) expression immunophenotype identified tumours associated with unfavourable prognosis and aggressive clinical parameters. Our data indicate that the high density of tryptase(+) MCs at invasive margins of tumours was associated with advanced stages of CRC and was strongly correlated with PAR‐2 expression.     

Pro-atherogenic lung and oral pathogens induce an inflammatory response in human and mouse mast cells

A broad variety of microbes are present in atherosclerotic plaques and chronic bacterial infection increases the risk of atherosclerosis by mechanisms that have remained vague. One possible mechanism is that bacteria or bacterial products activate plaque mast cells that are known to participate in the pathogenesis of atherosclerosis. Here, we show by real-time PCR analysis and ELISA that Chlamydia pneumoniae (Cpn) and a periodontal pathogen, Aggregatibacter actinomycetemcomitans (Aa), both induce a time and concentration-dependent expression and secretion of interleukin 8 (IL-8), tumour necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) by cultured human peripheral blood-derived mast cells, but not anti-inflammatory molecules, such as IL-10 or transforming growth factor β1 (TGF-β1). The IL-8 and MCP-1 responses were immediate, whereas the onset of TNF-α secretion was delayed. The Cpn-mediated pro-inflammatory effect was attenuated when the bacteria were inactivated by UV-treatment. Human monocyte-derived macrophages that were pre-infected with Cpn also induced a significant pro-inflammatory response in human mast cells, both in cocultures and when preconditioned media from Cpn-infected macrophages were used. Intranasal and intravenous administration of live Cpn and Aa, respectively induced an accumulation of activated mast cells in the aortic sinus of apolipoprotein E-deficient mice, however, with varying responses in the systemic levels of lipopolysaccharide (LPS) and TNF-α. Pro-atherogenic Cpn and Aa induce a pro-inflammatory response in cultured human connective tissue-type mast cells and activation of mouse aortic mast cells in vivo .     

VEGF concentration from plasma-activated platelets rich correlates with microvascular density and grading in canine mast cell tumour spontaneous model

Canine cutaneous mast cell tumour (CMCT) is a common cutaneous tumour in dog, with a higher incidence than in human. CMCT is classified in three subgroups, well and intermediately differentiated (G1 and G2), corresponding to a benign disease, and poorly differentiated (G3), corresponding to a malignant disease, which metastasize to lymph nodes, liver, spleen and bone marrow. In this study, we have evaluated serum (S), platelet-poor plasma (P-PP), plasma-activated platelet rich (P-APR) and cytosol vascular endothelial growth factor (VEGF) concentrations, microvascular density (MVD) and mast cell density (MCD) in a series of 86 CMCTs and we have correlated these parameters with each other, by means of ELISA detection of VEGF and immunohistochemistry. Results show that VEGF level from cytosol P-APR and MVD were significantly higher in G3 CMCTs as compared to G1 or G2 subgroups. Moreover, a significantly strong correlation among VEGF levels from P-PAR and cytosol, MVD and MCD was found in G3 subgroup. Because VEGF levels from P-APR well correlated with MVD and malignancy grade in CMCT, we suggest that VEGF might be secreted from MCs and it may be a suitable surrogate inter-species angiogenetic markers of tumour progression in CMCT. Finally, CMCT seems to be a useful model to study the role of MCs in tumour angiogenesis and inhibition of MCs degranulation or activation might be a new anti-angiogenic strategy worthy to further investigations.     

The immunohistochemical demonstration of chymase and tryptase in human intestinal mast cells

Summary An immunohistochemical double-labelling technique for the simultaneous identification of mast cells containing tryptase alone (MC_T) or chymase together with tryptase (MC_TC) was evaluated quantitatively using two monoclonal antibodies, mAb 1222A (antitryptase) and mAb 1254B (antichymase). Saturation conditions were established for the binding of the antibodies to the mast cell enzymes by counting labelled mast cells in consecutive sections of normal human intestine incubated with serial dilutions of the antibodies. When, under such conditions, the antitryptase was applied after saturation with mAb 1254B, the reproducibility of the double-labelling procedure was excellent. MC_T were located preferentially in the intestinal mucosa but, in contrast to what has previously been reported, they were not the predominant type of mast cell at this site. The percentage of MC_T of the total number of immunopositive mast cells varied considerably in the colonic mucosa (7–67%, average 30%), while this was not the case in the small intestinal mucosa (5–26%, average 10%). Mast cell chymase, unlike tryptase, was not recognized by the antichymase antibody after aldehyde fixation and a higher apparent fraction of MC_T therefore occurred after double labelling. These findings suggest that the proteinase composition of human mast cells, unlike that of murine mast cells, should not be taken as evidence of phenotypic heterogeneity. Taken together with previous observations, they suggest instead that the lack of chymase may be related to functional activity or stage of maturation of the mast cells.     

Effect of histamine and divalent cations on the activity and stability of tryptase from human mast cells

Tryptase from human mast cells is stabilized by negatively charged macromolecules such as heparin and is not affected by the protein inhibitors of serine proteinases normally present in human extracellular fluids. The current study demonstrated inhibition of tryptase-catalyzed cleavage of tosyl-Gly-Pro-Lys-p-nitroanilide by histamine and calcium, and destablization only by calcium. Calcium-mediated inhibition was competitive with a Ki of 30 mM. Cooperation of calcium with other extracellular cations or concentrations of calcium possible within cells or granules may permit calcium-mediated inhibition to occur in vivo. In contrast, only 5 mM calcium is needed to cause an irreversible 50% loss of tryptase activity after 60 min at room temperature. Histamine and N-methyl histamine concentrations of 2 mM to 10 mM inhibited tryptase activity by a different mechanism than calcium, resulting in sigmoid rather than hyperbolic kinetics. Whether this reflects cooperative binding of histamine to tryptase or conformational alterations of tryptase is not known. These concentrations of histamine are most relevant to those in mast cell secretory granules estimated at 100 mM, where tryptase is stored fully active and where histamine may play a role in attenuating tryptase activity.     

Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut

We tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.     

Establishment of the culture model system that reflects the process of terminal differentiation of connective tissue-type mast cells

To understand physiological roles of tissue mast cells, we established a culture system where bone marrow-derived immature mast cells differentiate into the connective tissue-type mast cell (CTMC)-like cells through modifying the previous co-culture system with Swiss 3T3 fibroblasts. Our system was found to reproducibly mimic the differentiation of CTMCs on the basis of several criteria, such as granule maturation and sensitivity to cationic secretagogues. The gene expression profile obtained by the microarray analyses was found to reflect many aspects of the differentiation. Our system is thus helpful to gain deeper insights into terminal differentiation of CTMCs.     

Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis

Background

Although mast cells are regarded as important regulators of inflammation and tissue remodelling, their role in cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) has remained less studied. This study investigates the densities and phenotypes of mast cell populations in multiple lung compartments from patients with CF, IPF and never smoking controls.

Methods

Small airways, pulmonary vessels, and lung parenchyma were subjected to detailed immunohistochemical analyses using lungs from patients with CF (20 lung regions; 5 patients), IPF (21 regions; 7 patients) and controls (16 regions; 8 subjects). In each compartment the densities and distribution of MC_T and MC_TC mast cell populations were studied as well as the mast cell expression of IL-6 and TGF-β.

Results

In the alveolar parenchyma in lungs from patients with CF, MC_TC numbers increased in areas showing cellular inflammation or fibrosis compared to controls. Apart from an altered balance between MC_TC and MC_T cells, mast cell in CF lungs showed elevated expression of IL-6. In CF, a decrease in total mast cell numbers was observed in small airways and pulmonary vessels. In patients with IPF, a significantly elevated MC_TC density was present in fibrotic areas of the alveolar parenchyma with increased mast cell expression of TGF-β. The total mast cell density was unchanged in small airways and decreased in pulmonary vessels in IPF. Both the density, as well as the percentage, of MC_TC correlated positively with the degree of fibrosis. The increased density of MC_TC, as well as MC_TC expression of TGF-β, correlated negatively with patient lung function.

Conclusions

The present study reveals that altered mast cell populations, with increased numbers of MC_TC in diseased alveolar parenchyma, represents a significant component of the histopathology in CF and IPF. The mast cell alterations correlated to the degree of tissue remodelling and to lung function parameters. Further investigations of mast cells in these diseases may open for new therapeutic strategies.     

Monoclonal antibodies against human mast cell tryptase demonstrate shared antigenic sites on subunits of tryptase and selective localization of the enzyme to mast cells

Two murine monoclonal antibodies were prepared against tryptase, the major neutral protease and protein component of human mast cells. The antibodies were termed G5 (IgG2B-kappa) and H4 (IgG1-kappa). They were specific for tryptase by an enzyme-linked immunosorbent assay and an immunotransblot technique. The latter procedure showed that H4 and G5 each bind to the 35,000 and 37,000 m.w. subunits of tryptase, indicating immunologic cross-reactivity between the subunits. The monoclonal antibodies reacted only with tryptase subunits in an extract of dispersed lung cells. By immunofluorescence microscopy, tryptase was further identified to be present only in cytoplasmic granules of Alcian Blue-stained mast cells in dispersed pulmonary cell preparations. No evidence for a mast cell subtype lacking tryptase was detected. In addition, a procedure for the purification of tryptase to homogeneity from dispersed pulmonary cells containing less than 10% mast cells was developed; this procedure involved high salt extraction, ammonium sulfate precipitation, and sequential chromatography with decyl-agarose, DEAE-agarose, and heparin-agarose. The procedure resulted in a higher yield even with less pure starting material than reported previously. Tryptase is a selective marker for mast cells in dispersed pulmonary cells, and can be detected with specific anti-tryptase antibodies.     

Mast cell tryptases: examination of unusual characteristics by multiple sequence alignment and molecular modeling.

Tryptases are trypsin-like serine proteinases found in the granules of mast cells. Although they show 40% sequence identity with trypsin and contain only 20 or 21 additional residues, tryptases display several unusual features. Unlike trypsin, the tryptases only make limited cleavages in a few proteins and are not inhibited by natural trypsin inhibitors, they form tetramers, bind heparin, and their activity on synthetic substrates is progressively inhibited as the concentration of salt increases above 0.2 M. Unique sequence features of seven tryptases were identified by comparison to other serine proteinases. The three-dimensional structures of the tryptases were then predicted by molecular modeling based on the crystal structure of bovine trypsin. The models show two large insertions to lie on either side of the active-site cleft, suggesting an explanation for the limited activity of tryptases on protein substrates and the lack of inhibition by natural inhibitors. A group of conserved Trp residues and a unique proline-rich region make two surface hydrophobic patches that may account for the formation of tetramers and/or inhibition with increasing salt. Although they contain no consensus heparin-binding sequence, the tryptases have 10-13 more His residues than trypsin, and these are positioned on the surface of the model. In addition, clustering of Arg and Lys residues may also contribute to heparin binding. Putative Asn-linked glycosylation sites are found on the opposite side of the model from the active site. The model provides structural explanations for some to the unusual characteristics of the tryptases and a rational basis for future experiments, such as site-directed mutagenesis.     

Immunolocalization of atrial natriuretic peptide in mast cells of human and rat pericardium

It is known that various heart disorders are accompanied by an elevated level of atrial natriuretic peptide (ANP), a regulator of cardiovascular homeostasis, in the pericardial fluid. Which cells produce ANP in the pericardial cavity is unclear. Using immunoelectron microscopy, we examined ANP localization in human and rat pericardium. ANP-immunobinding material was found in granules of mast cells (MC) localized in pericardial connective tissue. In rat pericardium, the average MC size is 6.5 × 12.5 μm and the MC density is about 50 cells per 1 mm² section area. For the human pericardium, these parameters are 9.1 × 13.6 μm and 10 cells per 1 mm², respectively. The results show that MCs are probably implicated in the pericardial endocrine function and in controlling the ANP level in the pericardial cavity.