Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The serine protease thrombin is formed at sites of coagulation and inflammation and has been shown to have important proinflammatory cellular effects relevant to the pathogenesis of periodontal disease. Thrombin acts via specific cell surface receptors termed protease-activated receptor-1 (PAR-1) and PAR-3, which have a distinctive method of activation. Proteolytic cleavage of the extracellular domain by thrombin reveals a hidden amino terminus which then acts as a "tethered ligand". A short synthetic peptide (SFLLRN) can also mimic the tethered ligand and activate PAR-1 but not PAR-3. Also, a trypsin-sensitive receptor termed PAR-2 has been described which is activated by the PAR-1 activating peptide SFLLRN. Here we show conclusively by flow cytometric and Northern blot analysis that human gingival fibroblasts (HGF) express PAR-1 but not PAR-2. In functional studies we also show that thrombin and SFLLRN stimulated increased expression of mRNA encoding nuclear transcription factor NF-IL-6 and IL-6 in vitro. At optimal concentrations, thrombin (10(-7) M) induced 7.6 +/- 0.01 ng/ml immunoactive IL-6 and PAR-1 activating peptide (5 x 10(-5) M) induced 2.2 +/- 0.2 ng/ml (mean +/- standard error of mean). A proteolytically inactive recombinant thrombin (serine 195 to alanine) was without activity. These data show that HGF express PAR-1 and suggest that PAR-1 activation stimulates increased NF-IL-6 and IL-6 gene expression and IL-6 secretion by HGF in vitro. Whether HGF express PAR-3 is unknown, but the fact that SFLLRN was not a complete replacement for thrombin raises the possibility that HGF may express additional thrombin receptors. These findings add weight to the importance of the cytokine-like role played by thrombin and raise the possibility that protease-activated receptors may play a role in the pathogenesis of inflammatory periodontal disease.
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PMID:Protease-activated receptors and their role in IL-6 and NF-IL-6 expression in human gingival fibroblasts. 968 16

Although serine proteases are usually considered to act principally as degradative enzymes, certain proteases are signaling molecules that specifically regulate cells by cleaving and triggering members of a new family of proteinase-activated receptors (PARs). There are three members of this family, PAR-1 and PAR-3, which are receptors for thrombin, and PAR-2, a receptor for trypsin and mast cell tryptase. Proteases cleave within the extracellular NH2-terminus of their receptors to expose a new NH2-terminus. Specific residues within this tethered ligand domain interact with extracellular domains of the cleaved receptor, resulting in activation. In common with many G protein-coupled receptors, PARs couple to multiple G proteins and thereby activate many parallel mechanisms of signal transduction. PARs are expressed in multiple tissues by a wide variety of cells, where they are involved in several pathophysiological processes, including growth and development, mitogenesis, and inflammation. Because the cleaved receptor is physically coupled to its agonist, efficient mechanisms exist to terminate signaling and prevent uncontrolled stimulation. These include cleavage of the tethered ligand, receptor phosphorylation and uncoupling from G proteins, and endocytosis and lysosomal degradation of activated receptors.
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PMID:Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. 969 85

The present study examined the effect of intraplantar (i.pl.) administration of a selective agonist of protease-activated receptor (PAR)-2, SLIGRL-NH2(PP6-NH2), on vascular permeability in rat hindpaw. PP6-NH2, administered i.pl. at 10-100 nmol per paw, enhanced vascular permeability and caused oedema formation in rat hindpaw. SLIGRL (PP6-OH) and trypsin, by i.pl. administration, also elicited an increase in vascular permeability, although i.pl. administration of the mixture of constituent amino acids of PP6-OH at an equivalent dose did not. The PP6-NH2-induced increase in vascular permeability was abolished by repeated pretreatment with compound 48/80 to deplete bioactive amines in mast cells. These findings suggest that the activation of PAR-2 induces acute inflammation, at least partially, via mast cell degranulation in rat hindpaw.
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PMID:Increased vascular permeability by a specific agonist of protease-activated receptor-2 in rat hindpaw. 980 21

Protease-activated receptors (PARs) are receptors which require proteolytic cleavage to be self-activated by newly exposed N-terminal 'tethered ligands', and hence serve as sensors for protelytic enzymes. While both the thrombin receptor (PAR-1) and PAR-2 (activated by tryptic enzymes) have been shown to mediate endothelium-dependent vasorelaxation, only PAR-1 has been shown to cause direct vascular smooth muscle contraction. In this study, we report that trypsin and the PAR-2 selective peptide ligand SLIGRL-NH2 not only caused endothelium-dependent relaxation of mouse renal arteries but also direct smooth muscle contraction if endothelial nitric oxide synthase was inhibited or if the endothelium was removed.
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PMID:Endothelium-dependent and -independent responses to protease-activated receptor-2 (PAR-2) activation in mouse isolated renal arteries. 983 89

A second protease-activated receptor (PAR-2) that could be activated by trypsin or more physiologically by mast cell tryptase has been recently cloned. Both the structure and activation mechanism of PAR-2 was similar to the functional thrombin receptor (PAR-1). Although many effects of the coagulation protease thrombin on the vascular endothelium could be attributed to PAR-1 activation, very little is known about the physiological and pathophysiological role of PAR-2. We investigated whether stimulation of PAR-2 on endothelial cells induced two cellular responses that play a central role in primary and secondary haemostasis: the release of high molecular weight von Willebrand factor (hmw-VWF) from Weibel-Palade bodies and the de novo synthesis of tissue factor (TF) mRNA and protein. Human umbilical vein endothelial cells (HUVEC) were incubated with agonists for PAR-2 at 37 degrees C. Both trypsin and SLIGKV increased TF mRNA and activity and induced the release of hmw-VWF due to elevated levels of cytosolic Ca2+. Trypsin (10 nm) induced a 6-fold increase of TF mRNA and reduced time until fibrin clot formation to 37%, indicating trebling of the cell surface located TF activity. Stimulation of HUVEC with the PAR-2 agonist peptide SLIGKV induced a dose-dependent increase of TF mRNA up to 6 times and TF activity up to 3 times. Release of hmw-VWF was achieved both after incubation of HUVEC with trypsin and SLIGKV and was directly depending on intracellular Ca2+ mobilization. To make results comparable to the functional thrombin receptor, homologous experiments were carried out using the PAR-1 agonists thrombin and SFLLRN.
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PMID:Endothelial protease-activated receptor-2 induces tissue factor expression and von Willebrand factor release. 1023 35

1. Proteases regulate cells by cleaving proteinase-activated receptors (PARs). Thrombin and trypsin cleave PAR-1 and PAR-2 on neurons and astrocytes of the brain to regulate morphology, growth and survival. We hypothesized that thrombin and mast cell tryptase, which are generated and released during trauma and inflammation, regulate enteric neurons by cleaving PAR-1 and PAR-2. 2. We detected immunoreactive PAR-1 and PAR-2 in > 60 % of neurons from the myenteric plexus of guinea-pig small intestine in primary culture. A large proportion of neurons that expressed substance P, vasoactive intestinal peptide or nitric oxide synthase also expressed PAR-1 and PAR-2. We confirmed expression of PAR-1 and PAR-2 in the myenteric plexus by RT-PCR using primers based on sequences of cloned guinea-pig receptors. 3. Thrombin, trypsin, tryptase, a filtrate from degranulated mast cells, and peptides corresponding to the tethered ligand domains of PAR-1 and PAR-2 increased [Ca2+]i in > 50 % of cultured myenteric neurons. Approximately 60 % of neurons that responded to PAR-1 agonists responded to PAR-2 agonists, and > 90 % of PAR-1 and PAR-2 responsive neurons responded to ATP. 4. These results indicate that a large proportion of myenteric neurons that express excitatory and inhibitory neurotransmitters and purinoceptors also express PAR-1 and PAR-2. Thrombin and tryptase may excite myenteric neurons during trauma and inflammation when prothrombin is activated and mast cells degranulate. This novel action of serine proteases probably contributes to abnormal neurotransmission and motility in the inflamed intestine.
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PMID:Thrombin and mast cell tryptase regulate guinea-pig myenteric neurons through proteinase-activated receptors-1 and -2. 1035 15

1. Protease-activated receptors (PARs) are activated by an irreversible proteolytic mechanism which renders cleaved receptors unresponsive to subsequent challenges with activating enzymes. Non-specific proteolysis of PARs downstream of the activation site also prevents subsequent enzymic activation. Therefore, we investigated the effects of non-activating amino-terminal proteolysis with the bacterial protease thermolysin on PAR-mediated relaxation of porcine coronary artery ring preparations contracted with the thromboxane A2 mimetic U46619 (1-10 nM). 2. Treatment of contracted artery ring segments with thermolysin (0.01-1 u ml-1, 20 min) caused no response, but abolished endothelium-dependent relaxations induced by the enzymic activators of PAR-1, and PAR-2, thrombin (0.01-0.3 u ml-1) and trypsin (0.003-0.1 u ml-1) respectively. The same treatment, however, did not affect similar responses to the proteolysis-independent PAR-1 and PAR-2 activating peptides, SFLLRN-NH2 and SLIGRL-NH2 respectively (0.1-10 microM). 3. The inhibition of responsiveness to trypsin after thermolysin treatment recovered in a time-dependent manner, with maximal recovery (77.3 +/- 8.0% of time controls) occurring 150 min after thermolysin treatment. No recovery of responsiveness to thrombin after thermolysin treatment was observed within this time, however, the thrombin response returned to control levels after 20 h. 4. The recovery of responsiveness to trypsin was inhibited by the translation inhibitor cycloheximide (100 microM; 17.3 +/- 4.7%) and the protein trafficking inhibitor brefeldin A (10 microM; 12.1 +/- 4.8%) but was unaffected by the transcription inhibitor actinomycin D (2 microM; 65.1 +/- 3.6%), which did, however, abolish upregulation of B1-kinin receptors in this preparation. 5. In conclusion, our findings indicate that activation-independent amino-terminal proteolysis of PARs stimulates selective recovery of endothelial cell PAR-2 responsiveness, which appears to be regulated by translation. Such a novel mechanism for the maintenance of responsiveness to enzymic PAR-2 activators may imply that these receptors play important roles in vascular homeostasis.
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PMID:Protease-activated receptor-2 turnover stimulated independently of receptor activation in porcine coronary endothelial cells. 1040 51

Proteinase-activated receptors (PARs), ubiquitous surface molecules participating on many biological processes have been recently discovered. Specific receptors for thrombin (PAR-1 and PAR-3) and trypsin (PAR-2) are described in this review. They belong to a family of G protein-coupled receptors activated by amino acid sequence of N-terminal part of bound ligand revealed by site-specific proteolysis. PARs participate in tissue growth and differentiation, regeneration and reparation, inflammatory response regulation, malignant transformation, but even in vascular tonus and blood pressure regulation. (Fig. 5, Ref. 35.)
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PMID:Thrombin and trypsin receptors: the same mechanism of signalling on cellular surfaces. 1049 1

1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.
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PMID:Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells. 1056 30

Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary permeability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury-related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endothelial (RBCE) cells and astrocytes derived from rat brain express two different thrombin receptors. The first is proteolytically activated receptor (PAR)-1, the receptor responsible for the vast majority of the thrombin's cellular activation functions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identified. Functional significance of thrombin receptors was indicated by changes in [Ca2+]i in response to thrombin, as measured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmol/L induced an abrupt increase in [Ca2+]i whereas, upon addition of active site-blocked thrombin or plasmin, [Ca2+]i remained unchanged. The [Ca2+]i signal attributable to thrombin was smaller in a low Ca2+-containing medium, indicating that an influx of Ca2+ from the extracellular medium makes a contribution to the overall [Ca2+]i rise. The amplitude of the transient [Ca2+]i signal was dependent on the concentration of thrombin, and repeated application of the enzyme caused an essentially complete and long-term desensitization of the receptor. The PAR-1 agonist peptide SFLLRN also elicited a transient increase in [Ca2+]i. After activation by SFLLRN, cells showed a diminished response to thrombin, but the response was not absent, indicating that PAR-3 might contribute to the generation of the [Ca2+]i signal. Pretreatment of RBCE cells with 100 nmol/L plasmin completely prevented [Ca2+]i rise attributable to thrombin. These data show that RBCE cells and astrocytes express at least two receptors for thrombin, PAR-1 and PAR-3, and probably both receptors are involved in thrombin-induced [Ca2+]i signals. Plasmin itself does not elevate [Ca2+]i but prevents the activation of receptors by thrombin.
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PMID:Identification of thrombin receptors in rat brain capillary endothelial cells. 1061 6


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