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)

Relaxation of rat aorta segments with sodium nitroprusside and endothelium-dependent vasodilators, such as acetylcholine, histamine, A23187, ATP, thrombin, and trypsin, is associated with cyclic-GMP (cGMP) accumulation in a concentration- and time-dependent fashion. With rat aorta segments, these agents also increase cyclic GMP-dependent protein-kinase activity and alter the incorporation of 32P into numerous smooth-muscle proteins. Identical patterns of protein phosphorylation were observed with both classes of relaxants on two-dimensional gel electrophoresis and autoradiography. The effects of nitroprusside were observed with or without the endothelium present. In contrast, the effects of the endothelium-dependent agents on all of these parameters (cGMP, cGMP-dependent protein kinase and protein phosphorylation) required the integrity of the endothelium. Various inhibitors of phospholipase and lypoxygenase prevented the effects of the endothelium-dependent agents, suggesting that a metabolite of arachidonic acid is the endothelium-relaxant factor and responsible for guanylate-cyclase activation. A smooth-muscle protein with decreased 32P incorporation after treatment with either class of relaxants has been identified as myosin light chain. A model is presented suggesting that the effects of endothelium-dependent vasodilators and directly acting nitrovasodilators converge at the level of guanylate-cyclase activation and cGMP accumulation, which explains the common biochemical and physiological effects on smooth muscle of these two classes of vasodilators.
J Cardiovasc Pharmacol 1985
PMID:Role of cyclic-GMP in relaxations of vascular smooth muscle. 240 83

We have recently reported that exogenous thrombin produced a dose- and endothelium-dependent coronary vasodilation in both intact open-chested dogs and in isolated dog coronary artery preparations. To determine whether the observed vasodilatory effect may be related to thrombin proteolytic enzymatic activity, effects of other proteases, such as trypsin, chymotrypsin, and pepsin, on the mechanical responses of isolated dog coronary arteries were studied. Among the four proteases evaluated, only thrombin (0.01-0.1 U/ml) and trypsin (0.03-0.67 U/ml) consistently produced a potent dose- and endothelium-dependent relaxation, that was reproducible with repeated testings. Addition of chymotrypsin (0.01-1.0 U/ml) produced only a minimal effect and was not reproducible, while addition of pepsin, as much as 10 U/ml, did not produce any effect. The specific soybean trypsin inhibitor and aprotinin, but not heparin and hirudin, competitively shifted the trypsin dose-response to the right, whereas heparin, hirudin, and antithrombin III proved to be more effective than trypsin inhibitors in inhibiting the thrombin-induced vasodilation. In all cases, the thrombin- and trypsin-induced vasodilation were equally sensitive to inhibition by the specific synthetic thrombin inhibitor, PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone, 1-30 nM). PPACK, however, had no effect on the other endothelium-dependent coronary vasodilators, such as acetylcholine and adenosine triphosphate, in our isolated dog coronary artery preparations. Biochemical determinations of the amidolytic activity of thrombin, using Tosylglycyl-L-prolyl-L-arginine-p-nitroanilide as a chromogen, also indicated a similar PPACK and heparin-antithrombin III dose-dependent inhibition of the thrombin enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
J Cardiovasc Pharmacol
PMID:Mechanism of thrombin-induced endothelium-dependent coronary vasodilation in dogs: role of its proteolytic enzymatic activity. 241 89

Using site-directed mutagenesis we have created an altered calmodulin in which Gln-3 and Thr-146 have both been replaced by cysteines. We have reacted this protein with the bifunctional reagent, bismaleimidohexane, forming an intramolecular cross-link between the two cysteines. In the crystal structure of native calmodulin alpha-carbons at positions 3 and 146 are 37 A apart. In the bismaleimidohexane cross-linked protein these atoms can be no more than 19 A apart, and model building studies indicate that there is probably a bend in the central helix of calmodulin. A second modified calmodulin was generated by cleaving the central helix of the cross-linked protein at Lys-77 with trypsin. In this molecule, the two lobes of calmodulin are joined solely by the bismaleimidohexane cross-link, which bridges Cys-3 and Cys-146. Vm and Kact values for activation of myosin light chain kinase activity by the cross-linked and cross-linked/trypsinized proteins are not significantly different from those for the control protein. This result indicates that one role for the central helix may be to serve as a flexible tether between the calmodulin lobes. This is consistent with a model calmodulin-enzyme complex in which the central helix is bent, and the two lobes exert a concerted effect. A detailed model of this type has been proposed for the calmodulin-myosin light chain kinase complex (Persechini, A. and Kretsinger, R.H. (1988) J. Cardiovasc. Pharmacol., in press).
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PMID:The central helix of calmodulin functions as a flexible tether. 313 20

Ventricular cells from adult rats were isolated enzymatically and used as a model system for determining what factors affect the release of adenosine triphosphate (ATP) from myocardial cells. The enzyme systems used to isolate cells were trypsin:collagenase; hyaluronidase:collagenase and dispase:collagenase. Adenosine triphosphate was released in greater amounts in response to hypoxia from cells freed by each of the enzymatic procedures. This occurred while the intracellular concentration of ATP remained constant. Experiments were then performed to determine whether the conditions that occur during myocardial ischaemia or hypoxia altered the release of ATP. Cells suspended in either oxygenated or anoxic buffer at a pH of 6.8 released a significantly lower amount of ATP than cells suspended in either condition at pH 7.4. To test the possibility that ATP was released from nucleotide-protein-Ca2+ complexes located in the sarcolemma, artificial disruption of these structures was carried out. Incubation of oxygenated cells with the chelating agent, ethyleneglycol-bis (B-aminoethyl ether)-N, N-tetraacetic acid (EGTA), stimulated the release of ATP in a hyperbolic relationship while incubation of anoxic cells with ethylenediamine tetraacetate (EDTA) stimulated the release of ATP in such a way that the pattern of release followed a sigmoid response with maximal amounts of ATP, 995 +/- 55 pmol.mg-1 protein, occurring in the presence of 0.1 to 2.0 mmol.litre-1 EDTA. By incubating cells with radioactive EDTA, there was no indication that EDTA entered the cells. No release of ATP above control levels occurred when EDTA was chelated with Ca2+ before being applied to isolated cells. These data suggest that the source of ATP found extracellularly may have been nucleotide-protein-Ca2+ complexes located in the sarcolemma, and further support the role of ATP as a coronary vasodilator during hypoxic conditions.
Cardiovasc Res 1983 May
PMID:Possible source of adenosine triphosphate released from rat myocytes in response to hypoxia and acidosis. 641 42

We investigated the effects of the thrombin inhibitor, argatroban ((2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8- quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid) on the endothelium-derived relaxing factor-nitric oxide (EDRF-NO)-dependent relaxant, and the endothelial cell-independent constrictor actions of thrombin. Experiments were performed in isolated rings of canine coronary arteries. Argatroban inhibited thrombin-induced relaxation (range of thrombin activity 0.003-0.3 U/ml), with an ED50 of 0.3 microM. The ED50 value was not different from inhibition of thrombin amidolytic cleavage of the chromogenic substrate N-p-tosylgly-pro-arg-p-nitroanilide acetate (TOGSPAN 0.28 microM), but inhibition was highly selective. Argatroban did not block EDRF-NO-dependent relaxations to trypsin (0.003-0.3 U/ml; Emax -88.7 + 2.0% without vs. -88.1 +/- 2.7% with argatroban), acetylcholine (ACh 1 nM to 1 microM; Emax -90.5 +/- 4.7% and -88.6 +/- 3.1%, with and without argatroban, respectively), or the calcium ionophore A23187 (1 nM to 1 microM; Emax -98.5 +/- 1.2 vs. -99.4 +/- 0.6%). The inhibitory effects of argatroban on thrombin-induced constriction were then compared with those of the irreversible thrombin inhibitor D-phenylalanyl-L-prolyl L-arginine chloromethyl ketone (PPACK). The highest concentration of argatroban (10 microM) inhibited the vasoconstrictor effects of thrombin but did not completely block the effects (Emax 21.4 +/- 8.1% of KCl constriction without argatroban and Emax 14.0 +/- 5.2% of KCl-induced constriction with argatroban). In contrast, both a 10- and a 100-fold lower concentration of PPACK (0.1-1 microM) prevented the thrombin-induced increase in tension. Thrombin-induced constriction therefore appeared to disclose mechanistic differences between the two thrombin inhibitors. Thrombin vasomotor actions were inhibited by argatroban, however, and this may contribute significantly to the therapeutic effect of argatroban.
J Cardiovasc Pharmacol 1993 Nov
PMID:Argatroban and inhibition of the vasomotor actions of thrombin. 750 29

In human placenta, endothelin (ET) could derive from maternal, fetal, and/or endogenous sources. Therefore, localization of ET-1 was investigated by immunohistochemistry in human term placenta and in cultured trophoblasts. In sections of placenta, ET-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the vessels and in the syncytiotrophoblasts of the villi. ET-1 IR was also detected in the decidual cells and in the extravillous cytotrophoblasts of the basal plate. The extravillous cytotrophoblasts of the chorionic plate and of the placental septa also exhibited strong ET-1 IR. For trophoblast culture, cytotrophoblastic cells were obtained from placental villi by trypsin-DNAse dispersion, further purified on a Percoll gradient, and enriched by employing a monoclonal anti-HLA class I antibody. After different periods of culture of purified cytotrophoblastic cells (1-5 days), ET-1 IR was observed in 95% of cells: cytotrophoblastic cells, trophoblast aggregates, and syncytiotrophoblasts. The presence of ET-1,2 immunoreactivity (ET-1,2 IR) in the culture media was demonstrated by radioimmunoassay. A uniform daily production of the peptide was observed over at least 5 days (approximately 50 fmol/10(6) cells/24 h). Furthermore, trophoblastic cells that had been cultured for 5 days contained significant amounts of ET-1,2 IR (24 fmol/10(6) cells). These results suggest a trophoblastic origin for ET-1 and support the hypothesis of a paracrine and autocrine action of the peptide in the physiology of the trophoblast and placenta.
J Cardiovasc Pharmacol 1993
PMID:Endothelin-1 binding sites and immunoreactivity in the cultured human placental trophoblast: evidence for an autocrine and paracrine role for endothelin-1. 750 48

Parathyroid hypertensive factor (PHF) has been purified from two sources of material: plasma of spontaneously hypertensive rats (SHRs) and culture medium from organ culture of SHR parathyroid glands. Chromatographic characteristics of PHF from these two sources are identical. Biological activity of PHF (assayed as the characteristic delayed hypertensive response in normotensive rats) is sensitive to degradation by treatment in base, and the enzymes trypsin, chymotrypsin, phospholipase C, and phospholipase D. PHF activity may also be extracted from source material with chloroform: methanol (4:1). A hypothetical structure for the active component of PHF is suggested. This is comprised of a peptide liked to a lysophospholipid.
J Cardiovasc Pharmacol 1994
PMID:Purification and structural characterization of parathyroid hypertensive factor. 751 47

Activation and reactivation of the ATP-sensitive K+ channel (IK.ATP) were studied with the patch-clamp technique in guinea-pig ventricular myocytes. The K+ channel openers, nicorandil and pinacidil, activated IK.ATP in an internal ATP-dependent manner. Both drugs increased the open probability of IK.ATP without changing the channel conductance. They prolonged lifetimes of bursts and shortened interburst intervals without influencing the fast gating within bursts. These effects were the opposite of those of internal ATP. However, the interaction between ATP and either nicorandil or pinacidil appeared not to be simple competition. We found that three carbonyl compounds--3,4-dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, and 2,4-dihydroxyacetophenone--could activate IK.ATP through an intracellular mechanism that was dependent upon the presence of ADP and Mg2+. It has been suggested that these three carbonyl compounds bind covalently to proteins to form a Schiff base, which may be responsible for their effects upon IK.ATP. Internal application of the proteolytic enzyme trypsin prevented both the spontaneous and Ca(2+)-induced rundown of the KK.ATP channel. Tryptic digestion did not change either the channel's sensitivity to inhibition by ATP nor the fast gating kinetics of IK.ATP. Internal application of an exopeptidase, carboxypeptidase A, but not leu-aminopeptidase, prevented the spontaneous and Ca(2+)-induced rundown of the IK/ATP channel, effects similar to those of trypsin treatment. These results suggest that the target site of trypsin digestion may be located on the carboxy (C)-terminal of the channel proteins or associated regulatory units.
Cardiovasc Drugs Ther 1993 Aug
PMID:Activation and reactivation of the ATP-sensitive K+ channel of the heart can be modified by drugs. 825 28

Endothelial protease-activated receptors (PARs) may be important sensors of vascular inflammation and injury. Activation of endothelial PAR1 and PAR2 causes nitric oxide-mediated arterial smooth muscle relaxation in a number of species and PAR4 activation causes similar responses in isolated rat aorta. However, it is unclear whether these receptors mediate such responses in human arteries because the most potent activators of PAR1, PAR2, and PAR4, thrombin and trypsin, cause endothelium-dependent relaxation of human coronary arteries through a common PAR1-like receptor. This study aimed to determine whether this unique pharmacology of PARs in human coronary arteries extends to human pulmonary arteries. PAR1 and PAR2 mRNA and protein were detected in human pulmonary arteries via reverse transcription polymerase chain reaction and immunohistochemistry, respectively. PAR4 mRNA was also detected in human pulmonary arteries. Contracted human pulmonary artery ring segments suspended for isometric tension measurement relaxed in a concentration- and endothelium-dependent manner to thrombin (0.001-0.1 U/ml), trypsin (0.01-1 U/ml), and the PAR1-activating peptide, SFLLRN (0.1-10 microM). By contrast, the PAR2- and PAR4-activating peptides, SLIGKV and GYPGQV, respectively, caused neither contraction nor relaxation of precontracted human pulmonary arteries. Relaxations to thrombin and trypsin cross-desensitized, while tachyphylaxis to SFLLRN abolished subsequent relaxations to both thrombin and trypsin. We conclude that human pulmonary arteries express PAR1, PAR2, and PAR4, but that only PAR1, or a PAR1-like receptor, is coupled to endothelium-dependent relaxation.
J Cardiovasc Pharmacol 2001 Jul
PMID:Protease-activated receptor (PAR) 1 but not PAR2 or PAR4 mediates endothelium-dependent relaxation to thrombin and trypsin in human pulmonary arteries. 1144 93

DPC423, 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide, is a synthetic, orally bioavailable, competitive, and selective inhibitor of human coagulation factor Xa (K(i) [nM]: factor Xa, 0.15; trypsin, 60; thrombin, 6000; plasma kallikrein, 61; activated protein C, 1800; factor IXa, 2200; factor VIIa, >15,000; chymotrypsin, >17,000; urokinase, >19,000; plasmin, >35,000; tissue plasminogen activator, >45,000; complement factor I, 44,000 [IC(50)]). In vitro, DPC423 produced anticoagulant effects in human plasma in which it doubled prothrombin time, activated partial thromboplastin time, and Heptest clotting time at 3.1 +/- 0.4, 3.1 +/- 0.4, and 1.1 +/- 0.5 microM, respectively. In dogs, DPC423 had a good pharmacokinetic profile with an oral bioavailability of 57%, a plasma clearance of 0.24 L/kg/h, and a plasma half-life of 7.5 h. In rabbit and rat models of arteriovenous shunt thrombosis, DPC423 was an effective antithrombotic agent with an IC(50) of 150 and 470 nM, respectively. The antithrombotic effect of DPC423 is likely to be related to the inhibition of factor Xa but not to the inhibition of thrombin or due to direct inhibition of platelet aggregation. Therefore, based on potency, selectivity, efficacy, and oral bioavailability, DPC423 was selected for clinical development as an oral anticoagulant for the potential treatment of thrombotic disorders. Preliminary human data suggest that DPC423 is orally bioavailable in humans and has a long plasma half-life.
Cardiovasc Drug Rev 2002
PMID:Nonpeptide factor Xa inhibitors: DPC423, a highly potent and orally bioavailable pyrazole antithrombotic agent. 1217 91


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