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)

We have identified a novel cellular action of thrombin on cultured rat adrenal medullary endothelial cells (RAMEC). Five-minute incubation of RAMEC with physiological concentrations of thrombin (<1 U/ml) caused within 3 h an increase in the basolateral deposition of the extracellular matrix (ECM) proteins fibronectin, laminin, and collagens IV and I, concomitant with a corresponding decrease in the apical release of these proteins into the medium. This shift in vectorial secretion of ECM proteins, quantitated with enzyme-linked immunoassays, was time dependent. Maximal stimulation of ECM protein deposition was observed after incubation of cells with thrombin for 5-15 min. Prolonged exposure (>1 h) to thrombin resulted in loss of proteins from the ECM. Thrombin-stimulated ECM protein deposition exhibited a bell-shaped dose dependence, peaking for all proteins at 0.25 U/ml of thrombin, and was independent of de novo mRNA or protein synthesis. Maximal amounts of deposited proteins increased between 2.5-fold (fibronectin) and 4-fold (collagen I) over baseline values. Similar results were obtained with thrombin receptor agonist peptide (TRAP), proteolytically active gamma-thrombin, and, to a lesser extent, other serine proteases such as trypsin and plasmin. A scrambled TRAP, proteolytically inactive PPACK-thrombin, DIP-thrombin, and type IV collagenase were ineffective. Together, these results suggest that the thrombin effects are mediated by proteolytic activation of the thrombin receptor. Possible involvement of the phospholipase C-signaling pathway in thrombin-mediated ECM protein deposition was also investigated. Inhibition or downregulation of protein kinase C (PKC) and chelation of intracellular or extracellular Ca2+ did not suppress, but rather enhanced, basal and thrombin-stimulated ECM protein deposition. Quantitative differences in augmentation of basolateral deposition by these treatments suggest differential regulatory pathways for individual ECM proteins. Our data indicate that, in cultured RAMEC, short-term activation of the thrombin receptor causes an increase in amounts of deposited ECM protein by a cellular signaling pathway that is independent of PKC activation and/or elevation of intracellular Ca2+.
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PMID:Thrombin modulates vectorial secretion of extracellular matrix proteins in cultured endothelial cells. 914 35

The serine proteases thrombin and trypsin are both powerful platelet agonists that act by cleaving the terminal portion of the thrombin receptor and allowing the new C-terminal to auto-stimulate the receptor. Synthetic peptides, termed thrombin receptor-activating peptides (TRAPs), have been shown to mimic many of the effects of thrombin. Here we have compared the effects of inhibitors on platelet aggregation and [14C]-arachidonic acid release in response to thrombin, trypsin and TRAP. Pretreatment of human platelets with BW755C (80 microM), which inhibits both cyclooxygenase and lipoxygenase, blocked trypsin (15-20 nM)- or TRAP (4-6 microM)-induced aggregation, but not thrombin (0.06-0.1 U/ml)-induced aggregation. The protease inhibitor leupeptin (10 micrograms/ml) abolished trypsin-induced aggregation and returned [14C]-arachidonic acid release from [14C]-arachidonic acid-prelabeled platelets to control levels. In contrast, leupeptin did not affect either aggregation or [14C]-arachidonic acid release in platelets stimulated by TRAP. Thrombin-induced aggregation and [14C]-arachidonic acid release were only partially inhibited by leupeptin. These data are consistent with the activation of platelets by both trypsin and TRAP occurring via the proteolytic receptor, whereas thrombin-induced platelet activation appears to occur by a dual mechanism of action. One component of thrombin-induced platelet activation is by a proteolytic action on the moderate-affinity receptor. This effect is sensitive to inhibition by leupeptin and is mimicked by trypsin and TRAP. The other component of thrombin is nonproteolytic and may occur by an action at a high-affinity receptor such as glycoprotein lb.
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PMID:Thrombin receptor-activating peptide releases arachidonic acid from human platelets: a comparison with thrombin and trypsin. 915 95

The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001-0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, gamma-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-alpha-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of G(i)-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca2+ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G(i)-protein.
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PMID:The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein. 919 75

Proteinase-activated receptor 2 (PAR-2) is a G protein-coupled receptor related to the thrombin receptor. PAR-2 can be activated by trypsin and by synthetic peptides corresponding to the new amino terminus generated by activating proteolytic cleavage. We show in this report that intravenous injection of PAR-2 agonist peptides has dramatic effects on arterial blood pressure in anesthetized rats. The peptide SLIGRLETQPPI, at 150 nmol/kg, transiently decreased the mean arterial pressure from 104 to 60 mm Hg. The hypotensive response was dose-dependent, and was not secondary to effects on central vasoregulatory systems, heart rate, or the kidneys. A nitric oxide synthase inhibitor attenuated the hypotensive response induced by the PAR-2 agonist peptide. Further experiments in vitro, on preparations of rat femoral artery and vein, showed that PAR-2 agonist peptide elicited a dose-dependent relaxation of both types of vessel. Removal of the endothelium abolished the agonist peptide-induced relaxation. Our results demonstrate that activation of PAR-2 can modulate vascular tone, and that this response was an effect mediated at least partly by nitric oxide. The effect on blood vessels further suggests that the physiological activator of this proteolytically activated receptor is an enzyme present and active in the blood, possibly after a vascular injury.
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PMID:Vascular effects of proteinase-activated receptor 2 agonist peptide. 925 86

Protease-activated receptor-2 (PAR-2) is a seven-transmembrane G protein-coupled receptor that possesses a structure and activation mechanism similar to those of the thrombin receptor. It is activated by low concentrations of trypsin (300 pM) and a synthetic hexapeptide [sequence of serine, leucine, isoleucine, glycine, arginine, leucine (SLIGRL), the rodent PAR-2 "tethered ligand"] representing the first six amino acids following the putative PAR-2 cleavage site. Previous studies have indicated that alpha-thrombin and SFLLRN (synthetic hexapeptide sequence of serine, phenylalanine, leucine, leucine, arginine, asparagine; the human thrombin receptor "tethered ligand") induce neurite retraction and neurotoxicity. Because of the strong similarities between thrombin receptor and PAR-2, we have proposed that PAR-2 may also participate in neurodegeneration. In the present study, we used reverse transcriptase polymerase chain reaction and immunocytochemistry to provide the first evidence that PAR-2 is present in the rat hippocampus. Moreover, we found SLIGRL to be toxic to hippocampal neurons in a concentration-dependent manner (> or = 100 microM). Calcium signaling studies were performed to aid in determining the mechanism by which PAR-2 activation is neurotoxic.
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PMID:Protease-activated receptor-2 (PAR-2) is present in the rat hippocampus and is associated with neurodegeneration. 934 32

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36 --> Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of approximately 80 micromol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares approximately 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr approximately 130, confirming the presence of secretable and functionally active homotetrameric alpha- and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at approximately 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36 --> Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.
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PMID:Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases. 935 58

Serine proteinases are involved in several physiological processes and elicit profound cellular effects in a variety of tissues. Besides the thrombin receptor a second receptor, activated by trypsin, the proteinase-activated receptor 2 (PAR-2), was cloned and characterized. Both enzymes generate a new extracellular N-terminus by limited proteolytic cleavage which functions as tethered ligand to activate the receptor. Synthetic peptides corresponding to the sequences of the newly generated N-terminus are able to mimic the effects of the enzymes. In porcine pulmonary arteries trypsin and the receptor-derived peptide SLIGRL elicited an endothelium-dependent transient relaxation of PGF2alpha-precontracted vessels. The EC50 values for trypsin and SLIGRL amounted to 1.1 +/- 0.2 nM and 5.4 +/- 0.6 microM, respectively. Trypsin and SLIGRL caused a homologous desensitization but thrombin and the thrombin receptor-activating peptide SFLLRN were still able to elicit pronounced relaxant effects. The trypsin- and SLIGRL-induced relaxant responses were markedly diminished after blockade of the nitric oxide synthesis by N(G)-nitro-L-arginine methyl ester (200 microM) and were absent in endothelium-denuded vessels. Indomethacin and hirudin did not influence the relaxant effects. The effect of trypsin but not that of SLIGRL was blocked by the proteinase inhibitor aprotinin suggesting that only proteolytically active trypsin activates the receptor. Benzamidine derivatives of the 3-amidinophenylalanine type with different affinity for trypsin and thrombin inhibited the vascular effects of trypsin (IC50 0.007-0.7 microM) correlating with its antitrypsin activity. The data suggest that the vascular effects of trypsin and SLIGRL are mediated through activation of PAR-2 which differs from the thrombin receptor.
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PMID:Trypsin- and SLIGRL-induced vascular relaxation and the inhibition by benzamidine derivatives. 940 26

The proteinase-activated receptor (PAR-2) which belongs to the family of proteolytically cleaved receptors is activated by trypsin and by a synthetic peptide (SLIGKV) derived from the new amino terminus. Here, we have studied the mitogenic effect of trypsin and of SLIGKV on human aortic smooth muscle cells (SMC). Like trypsin, SLIGKV was a potent mitogen for SMC and exhibited the same activity as that of SFLLRN, a peptide mimicking the new amino terminus created by cleavage of the thrombin receptor. SLIGKV stimulated the proliferation of growth-arrested SMCs with a half-maximum mitogenic response at 80 nM. Under the same experimental conditions, the retro analogue or SLIGKV (VKGILS) did not show any mitogenic activity. Two specific inhibitors of the enzymatic activity of trypsin, alpha 1-antitrypsin and aprotinin, specifically inhibited trypsin-induced SMC growth (IC50 = 0.87 +/- 0.09 and 0.74 +/- 0.11 microM, respectively) but remain without effect on the mitogenic effect of the agonist peptide. The mitogenic effect of trypsin and SLIGKV was due to the release of platelet derived growth factor as demonstrated by the inhibitory activity of a neutralizing monoclonal anti-PGDF-BB antibody. These results demonstrate that the mitogenic effect of trypsin for SMCs is intimately linked to its esterolytic activity and mediated by the activation of PAR-2.
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PMID:Induction of vascular smooth muscle cell growth by selective activation of the proteinase activated receptor-2 (PAR-2). 943 82

Activation of the G-protein-linked thrombin receptor in endothelial cells normally leads to an increase in free intracellular calcium, [Ca2+]i, which is the proximate stimulus for many important cell functions. We present evidence showing that signals from CD36, the thrombospondin (TSP) receptor, can inhibit this thrombin-mediated calcium response. Human endothelial cells preloaded with Indo-1 exhibited rapid calcium mobilization in response to thrombin. The presence of TSP inhibited the thrombin-stimulated calcium response in CD36-positive microvascular endothelial cells but not in CD36-negative umbilical vein endothelial cells. This TSP effect was mimicked by anti-CD36 antibodies and a TSP peptide (CSVTCG), but not by an alternative CD36 ligand (collagen IV) or an antibody to an alternative TSP receptor (alphavbeta3). TSP also inhibited the calcium response to the thrombin receptor-tethered ligand peptide, SFLLRN. In addition, TSP and anti-CD36 antibodies inhibited the calcium response of a closely related receptor, the trypsin/SLIGKVD-activated receptor PAR-2. TSP did not indiscriminately inhibit all calcium release pathways, since histamine- or VEGF-stimulated calcium responses were not inhibited by TSP. We conclude that cross-talk from the CD36 receptor influences the responsive state of the endothelial thrombin receptor family and/or its signaling pathway.
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PMID:Thrombin-stimulated calcium mobilization is inhibited by thrombospondin via CD36. 947 55

1. The vascular actions of the proteinase-activated receptor-2-activating peptides (PAR2APs), SLIGRL-NH2 (SL-NH2) and SLIGKV-NH2 (KV-NH2) as well as the reverse-sequence peptide, LSIGRL-NH2 (LS-NH2) and an N-acylated PAR2AP derivative, trans-cinnamoyl-LIGRLO-NH2 (tcLI-NH2), were studied in rat intact and endothelium-denuded artery ring preparations, primarily from the pulmonary artery (RPA). 2. In RPA rings with but not without a functional endothelium, SL-NH2 (but not LS-NH2) caused either an endothelium-dependent relaxation (at concentrations: < 10 microM) or (at higher concentrations: > 10 microM), an endothelium-dependent contraction. No contractile response was observed in endothelium-denuded preparations, that otherwise contracted in response to the PAR1AP, TFLLR-NH2. 3. The endothelium-dependent contractile response to SL-NH2 was not blocked by the alpha-adrenoceptor antagonist prazosin, the endothelin antagonist BQ123, the angiotensin II antagonist DuP753, by tetrodotoxin; nor by the enzyme inhibitors, N(omega)-nitro-L-arginine-methylester (NO-synthase), indomethacin (cyclo-oxygenase), SKF-525A (epoxygenase) and MK886 (leukotriene synthesis inhibitor). 4. In the relaxation assay, KV-NH2 was 5 fold less potent than SL-NH2, whereas in the contractile assay KV-NH2 was about equipotent with SL-NH2. However, the maximal contractile response to KV-NH2 was lower than that of SL-NH2. 5. The PAR2AP analogue, tcLI-NH2, was as active as SL-NH2 in the relaxation assay but was inactive as a contractile agonist in the endothelium-intact RPA. 6. The relaxant responses caused by SL-NH2 and trypsin, as well as the contractile response caused by SL-NH2, did not desensitize in the course of repeated exposures of the tissue to agonist; whereas the contractile response to trypsin, only observed at concentrations greater than 30 u ml(-1), was desensitized by previous exposure of the tissue to either thrombin or trypsin. 7. In a contractile assay, where the tissue was desensitized to a concentration of trypsin that would otherwise cause a relaxant response, the preparation still contracted in response to SL-NH2. However, the trypsin-desensitized preparations were no longer contracted by thrombin. 8. From the cross-desensitization by thrombin of the contractile response to trypsin (and vice versa), we concluded that the contractile effect of trypsin was due to activation of the thrombin receptor and not PAR2. 9. We concluded that the endothelium-dependent contraction caused by high concentrations of SL-NH2 is due to an as yet unidentified contracting factor; whereas the endothelium-dependent relaxation response observed at low concentrations of SL-NH2 (< or = 10 microM) is mediated by nitric oxide. 10. The distinct structure activity profiles for the contractile response (potency of KV-NH2 < or = SL-NH2) compared with the relaxant response (potency of KV-NH2 << SL-NH2); the contractile responsiveness to SL-NH2 of an endothelium-intact RPA preparation, that did not contract in response to trypsin; and the lack of contractile activity of the PAR2AP analogue tcLI-NH2, that was as active as SL-NH2 in the relaxation assay all argue in favour of receptor heterogeneity in the vasculature for the PAR2APs. It remains to be determined if the distinct endothelial receptor responsible for the contractile action of SL-NH2 can be proteolytically activated, like PAR1 and PAR2.
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PMID:Dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides: evidence for receptor heterogeneity. 957 40


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