Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells

1 Using fura-2 fluorometry of [Ca(2+)](i) in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca(2+)](i) elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was significantly (P<0.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaffected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP specifically attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher affinity PAR1-AP (haPAR1-AP) (Ala-pF-Arg-Cha-HArg-Tyr-NH(2)) specifically attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was significantly (P<0.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.     

PAR1, but not PAR4, activates human platelets through a Gi/o/phosphoinositide-3 kinase signaling axis

Thrombin-mediated activation of platelets is critical for hemostasis, but the signaling pathways responsible for this process are not completely understood. In addition, signaling within this cascade can also lead to thrombosis. In this study, we have defined a new signaling pathway for the thrombin receptor protease activated receptor-1 (PAR1) in human platelets. We show that PAR1 couples to G(i/o) in human platelets and activates phosphoinositide-3 kinase (PI3K). PI3K activation regulates platelet integrin alphaIIbbeta3 activation and platelet aggregation and potentiates the PAR1-mediated increase in intraplatelet calcium concentration. PI3K inhibitors eliminated these effects downstream of PAR1, but they had no effect on PAR4 signaling. This study has identified an important role for the direct activation of G(i/o) by PAR1 in human platelets. Given the efficacy of clopidogrel, which blocks the G(i/o)-coupled P2Y purinoceptor 12, as an antiplatelet/antithrombotic drug, our data suggest that specifically blocking only PAR1-mediated G(i/o) signaling could also be an effective therapeutic approach with the possibility of less unwanted bleeding.     

普萘洛尔通过磷脂酶D的活化诱导磷脂酸水平的增高

目的：考察普萘洛尔诱导细胞内磷脂酶D的活化,分析该酶的活化与普萘洛尔刺激引起胞内磷脂酸水平升高的关系,研究蛋白激酶C在此过程中的作用。方法：[^3H]-豆蔻脂酸放射性标记细胞,通过转磷脂酰反应,抽提细胞脂类分子用薄层层析法分析磷脂酶D的产物。用蛋白激酶C的抑制剂来抑制蛋白激酶C的活性或通过佛波酯长时间处理细胞来下调蛋白激酶C的水平。以免疫印记法分析蛋白激酶C的含量。结果：在正丁醇的存在下,普萘洛尔处理A－549细胞引起磷脂酶D的活性快速增高,普萘洛尔诱导磷脂酰丁醇的生成导致磷脂酸生成水平的下降。用蛋白激酶C的抑制剂星形孢菌素（staurosporine),Ro-31-8220及粗糖柴毒素（rottlerin)预处理细胞或通过佛波酯长时间处理下调胞内蛋白激酶C的水平对普萘洛尔诱导的磷脂酶D的活性没有抑制作用,而有增强效应。结论：普萘洛尔引起细胞内磷脂酸水平的提高是由于普萘洛尔对磷脂酶D活性的强烈诱导作用而导致的,蛋白激酶C对该过程可起到负调节作用。     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.     

Comparison of the effects of PAR1 antagonists, PAR4 antagonists, and their combinations on thrombin-induced human platelet activation

Thrombin activates human platelets through proteolytic activation of two protease-activated receptors (PARs), PAR1 and PAR4. In the present study, we show that, RWJ-56110, a potent synthetic PAR1 antagonist, inhibited platelet aggregation caused by a low concentration (0.05 U/ml) of thrombin, but lost its effectiveness when higher concentrations of thrombin were used as stimulators. YD-3, a non-peptide PAR4 antagonist, alone had little or no effect on thrombin-induced platelet aggregation, significantly enhanced the anti-aggregatory activity of PAR1 antagonist. In addition, we demonstrate for the first time that P-selectin expression in thrombin-stimulated platelets can be synergistically prevented by combined treatment of PAR1 antagonist and PAR4 antagonist. These results indicate that thrombin-induced platelet activation cannot be effectively inhibited by just blocking either single thrombin receptor pathway, and suggest a rationale for potential combination therapy in arterial thrombosis.     

The roles and mechanisms of PAR4 and P2Y12/phosphatidylinositol 3-kinase pathway in maintaining thrombin-induced platelet aggregation

BACKGROUND AND PURPOSE

Activation of human platelets by thrombin is mediated predominately through two proteinase-activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3-kinase (PI3K) inhibition leads to reversible PAR1-mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated.

EXPERIMENTAL APPROACH

The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC-1. Western blotting and fluo-3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca²⁺ mobilization respectively.

KEY RESULTS

When PAR4 function was inhibited either by the PAR4 antagonist YD-3 [1-benzyl-3-(ethoxycarbonylphenyl)-indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin-elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD-3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin-stimulated platelets. The aggregation-reversing activity mainly resulted from inhibition of both PI3K-dependent PKC activation and PAR4-mediated sustained intracellular Ca²⁺ rises. Blockade of ADP P2Y₁₂ receptor with 2-methylthioadenosine 5′-monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K-dependent PKC activation and its ability to reverse PAR1-activating peptide-induced platelet aggregation. Co-administration of 2-methylthioadenosine 5′-monophosphate triethylammonium salt with YD-3 also decreased the stability of thrombin-induced platelet aggregation.

CONCLUSIONS AND IMPLICATIONS

These results suggest that PAR4 acts in parallel with the P2Y₁₂/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.     

Synthesis of novel peptide inhibitors of thrombin-induced platelet activation

Inhibitors of the activation of platelet aggregation have promise as important therapeutic agents for the management of acute coronary syndrome (ACS). Platelet activation by thrombin, a serine protease, occurs by binding to and cleavage of the extracellular N-terminal domains of protease-activated receptors 1 and 4 (PAR1 and PAR4). The proteolysis of the PARs exposes new tethered ligands that then signal through transmembrane domains to initiate platelet activation as a downstream effect. A pentapeptide cleavage product of bradykinin with the sequence Arg-Pro-Pro-Gly-Phe serves as a thrombin inhibitor by blocking alpha- and gamma-thrombin-induced platelet aggregation. Analogs of RPPGF have been prepared that result in improved inhibition of thrombin activation of platelets. Specific amino acid residues required for activity against platelet aggregation have been identified, and a lead compound, rOicPaPhe(p-Me)-NH(2) (FM19), has been developed. FM19, which completely inhibits threshold gamma-thrombin-induced platelet aggregation at a concentration of 16 +/- 4 microm, represents an important lead compound in the development of inhibitors of thrombin-mediated platelet aggregation for treatment of ACS.     

Pulmonary fibrosis inducer, bleomycin, causes redox-sensitive activation of phospholipase D and cytotoxicity through formation of bioactive lipid signal mediator, phosphatidic acid, in lung microvascular endothelial cells

The mechanisms of lung microvascular complications and pulmonary hypertension known to be associated with idiopathic pulmonary fibrosis (IPF), a debilitating lung disease, are not known. Therefore, we investigated whether bleomycin, the widely used experimental IPF inducer, would be capable of activating phospholipase D (PLD) and generating the bioactive lipid signal-mediator phosphatidic acid (PA) in our established bovine lung microvascular endothelial cell (BLMVEC) model. Our results revealed that bleomycin induced the activation of PLD and generation of PA in a dose-dependent (5, 10, and 100 μg) and time-dependent (2-12 hours) fashion that were significantly attenuated by the PLD-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). PLD activation and PA generation induced by bleomycin (5 μg) were significantly attenuated by the thiol protectant (N-acetyl-L-cysteine), antioxidants, and iron chelators suggesting the role of reactive oxygen species (ROS), lipid peroxidation, and iron therein. Furthermore, our study demonstrated the formation of ROS and loss of glutathione (GSH) in cells following bleomycin treatment, confirming oxidative stress as a key player in the bleomycin-induced PLD activation and PA generation in ECs. More noticeably, PLD activation and PA generation were observed to happen upstream of bleomycin-induced cytotoxicity in BLMVECs, which was protected by FIPI. This was also supported by our current findings that exposure of cells to exogenous PA led to internalization of PA and cytotoxicity in BLMVECs. For the first time, this study revealed novel mechanism of the bleomycin-induced redox-sensitive activation of PLD that led to the generation of PA, which was capable of inducing lung EC cytotoxicity, thus suggesting possible bioactive lipid-signaling mechanism/mechanisms of microvascular disorders encountered in IPF.     

The signaling of protease-activated receptor-2 activating peptide-induced contraction in cat esophageal smooth muscle cells

Protease-activated receptors (PARs) are a family of G protein-coupled receptors with a unique activation mechanism involving proteolytic cleavage of the extracellular N-terminal domain of the receptor. PAR2 has a contractile effect on esophageal smooth muscle. We investigate the signaling pathways of the PAR2-activating peptide (PAR2-AP) induced contraction in cat esophageal smooth muscle cells. The length of freshly isolated smooth muscle cells and permeabilized cells from feline esophagus were measured by scanning micrometry, and by confirming molecular basis via western blot analysis. The responses to PAR2-AP were initial and sustained contractions, depending on time. The maximum contraction of the initial phase occurred at 60 s. The PAR2-AP-induced contraction was mediated by Gαi1, Gαi3, and Gαq protein activation, leading to phospholipase-c (PLC) and myosin light chain kinase (MLCK) activation. 20 kDa myosin light chain (MLC20) was phosphorylated by PAR2-AP. Rho kinase-2 (ROCK-2), an activator of 17 kDa C-kinase potentiated Protein phosphatase-1 Inhibitor (CPI-17), was increased by PAR2 receptor activation. In conclusion, PAR2-AP produced an initial contraction mediated by Gαi1, Gαi3, and Gαq protein activation, resulting in PLC and MLCK activation. The sustained contraction by PAR2-AP was mediated by the Rho/Rho kinase-dependent pathway.     

Statins and thrombin

L-Mevalonic acid is the distant precursor of cholesterol, in contrast to cholesterol, L-mevalonic acid, its distant precursor gives rise to farnesyl and geranylgeranyl pyrophosphates in relatively few metabolic steps. These isoprenyl pyrophophates covalently conjugate with specific G-proteins and serve as membrane anchors enabling them to carry out their function. Although farnesyl-proteins may participate in signal transduction, geranylgeranyl-proteins (e.g., Rho GTP binding proteins) are well known to downregulate signaling pathways by inhibiting L-mevalonic acid synthesis. Such inhibitors include 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, drugs (statins) and isoprenoids of dietary origins, where Rho protein activation appears to be necessary for cellular-mediated thrombin generation. Thrombin and other proteases (e.g., coagulation factor Xa, tryptase) upregulate protease-activated receptor (PAR) synthesis and PAR activation promotes synthesis and expression of other proteins [e.g., tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1)]. With the PAR-1 activating peptide SSFLRNP, we found that either cerivastatin or atorvastatin mitigated platelet stimulation in a time- and dose-dependent manner, as predicted if a statin-mediated Rho pathway is required. We also found that simvastatin decreased prothrombin fragments F1+2 in plasma from type 2 diabetics, demonstrating that statins downregulate thrombin generation. Thus, independent of cholesterol, statins and dietary isoprenoids behave as inhibitors of TF-dependent thrombin generation. Because thrombin has multiple physiological functions, the 20 pleiotropic effects reported for statins may reflect a common mechanism for downregulation of thrombin-mediated events, in particular at the cellular level.     

Mechanisms of action of proteinase-activated receptor agonists on human platelets

1. We studied the activation of human platelets by thrombin and proteinase activated receptor (PAR)-activating peptides (PAR-APs) [SFLLRNPNDKYEPF-amide (TRAP), TFLLR-amide (PAR1AP) and AYPGKF-amide (PAR4AP)]. 2. PAR agonist-induced platelet aggregation, glycoprotein (GP) Ib and GPIIb/IIIa surface expression and ADP release were measured by light aggregometry, flow cytometry and chemiluminescence. 3. Aggregation inhibitors, including prostacyclin (PGI(2)), nitric oxide-releasing agent (S-nitroso-glutathione, GSNO), aspirin, apyrase, and phenanthroline were used to study the susceptibility of PAR agonist-induced aggregation to pharmacological inhibition. 4. Thrombin was the most potent platelet agonist, followed by PAR1AP, TRAP and PAR4AP. 5. The aggregatory potencies of PAR-APs were not modified by the aminopeptidase inhibitor, amastatin. 6. Subthreshold concentrations of PAR1AP potentiated the effects of PAR4AP to stimulate maximal aggregation. 7. Both PGI(2) and GSNO reduced PAR agonist-induced aggregation and diminished GPIIb/IIIa up-regulation. 8. PAR agonist-induced aggregation was aspirin-insensitive indicating a minor role for TXA(2). 9. In contrast, phenanthroline and apyrase significantly enhanced the anti-aggregatory effects of aspirin against thrombin-, PAR1AP- and TRAP-induced aggregation suggesting the involvement of ADP- and MMP-2-dependent pathways. 10. PAR4AP-induced aggregation (but not PAR1AP-induced aggregation) was entirely ADP-dependent (abolished by apyrase) and resistant to phenanthroline (MMP-2-independent). 11. Thus, the mechanisms of PAR1 and 4-induced platelet aggregation are distinct and depend differentially on their ability to interact with pathways of aggregation, along with the subsequent activation of GPIIb/IIIa receptors.     

Activation of phospholipase D involved in both injury and survival in A549 alveolar epithelial cells exposed to H2O2

To determine the role of the phospholipase D (PLD) pathway in injury and survival of alveolar epithelial cells, A549 cells were exposed to H₂O₂ (500 μM) which resulted in time-dependent injury and bi-phasic increase of PLD activity at 5 min and at 3 h, respectively. n-Butanol (0.5%) inhibited PLD activation, attenuated cell injury at 5 min of H₂O₂ exposure, but enhanced injury at 3 h of exposure. This activation was inhibited by treatment with catalase (500 units/ml). Exogenous phosphatidic acid mimicked the effects of PLD activation, and diphenyliodonium (NADPH oxidase inhibitor) reversed the decline in cell viability induced by H₂O₂ exposure. Propranolol (phosphatidic acid phospholydrolase inhibitor) and quinacrine (phospholipase A2 inhibitor) had weak effects on H₂O₂-induced PLD activation but reversed H₂O₂-induced injury. We speculate that PLD activation at the initiation of H₂O₂ exposure predominantly results in NAPDH oxidase activation, which mediates A549 cell injury, but turns to mediating cell survival as the H₂O₂ attack continues, which might be mainly due to the accumulation of intracellular phosphatidic acid.     

Differential effects of quercetin,apigenin and genistein on signalling pathways of protease-activated receptors PAR1 and PAR4 in platelets

Background and purpose:

The modulation by flavonoids of platelet responses induced by thrombin has been little investigated, and the antiplatelet activity, as well as possible inhibitory mechanisms of these compounds on thrombin signalling, has not yet been elucidated. We explored whether flavonoids affect platelet signalling pathways triggered by thrombin and by the selective activation of its protease-activated receptors (PARs) 1 and 4, and analysed the antagonism of these polyphenols at thrombin receptors.

Experimental approach:

We investigated the effect of a range of polyphenolic compounds on platelet aggregation, 5-HT secretion, intracellular calcium mobilization, protein kinase activity and tyrosine phosphorylation, triggered by thrombin and PAR agonist peptides (PAR-APs). The ability of these flavonoids to bind to thrombin receptors was investigated by competitive radioligand binding assays using ¹²⁵I-thrombin.

Key results:

Quercetin, apigenin and genistein impaired platelet aggregation, as well as 5-HT release and calcium mobilization, induced by thrombin and PAR-APs. Quercetin and apigenin were inhibitors of protein kinases, but genistein exhibited a minimal ability to suppress platelet phosphorylation. Binding assays did not establish any kind of interaction between thrombin receptors and any of the flavonoids tested.

Conclusions and implications:

Quercetin, apigenin and genistein did not inhibit thrombin responses by interacting with thrombin receptors, but by interfering with intracellular signalling. While inhibition by genistein may be a consequence of affecting calcium mobilization, subsequent platelet secretion and aggregation, for quercetin and apigenin, inhibition of kinase activation may also be involved in the impairment of platelet responses.     

Thrombin-induced conversion of fibrinogen to fibrin results in rapid platelet trapping which is not dependent on platelet activation or GPIb

1. Activation of human platelets by thrombin is mediated by the proteolytic cleavage of two G-protein coupled protease-activated receptors, PAR-1 and PAR-4. However, thrombin also binds specifically to the platelet surface glycoprotein GPIb. It has been claimed that thrombin can induce aggregation of platelets via a novel GPIb-mediated pathway, which is independent of PAR activation and fibrinogen binding to alpha(IIb)beta(3) integrin, but dependent upon polymerizing fibrin and the generation of intracellular signals. 2. In the presence of both fibrinogen and the alpha(IIb)beta(3) receptor antagonist lotrafiban, thrombin induced a biphasic platelet aggregation response. The initial primary response was small but consistent and associated with the release of platelet granules. The delayed secondary response was more substantial and was abolished by the fibrin polymerization blocking peptide GPRP. 3. Cleavage of the extracellular portion of GPIb by mocarhagin partially inhibited thrombin-induced alpha(IIb)beta(3)-dependent aggregation and release, but had no effect on the secondary fibrin-dependent response. 4. Fixing of the platelets abolished alpha(IIb)beta(3)-dependent aggregation and release of adenine nucleotides, whereas the fibrin-dependent response remained, indicating that platelet activation and intracellular signalling are not necessary for this secondary 'aggregation'. 5. In conclusion, the secondary fibrin-dependent 'aggregation' response observed in the presence of fibrinogen and lotrafiban is a platelet trapping phenomenon dependent primarily on the conversion of soluble fibrinogen to polymerizing fibrin by thrombin.     

Protease-activated receptor-1 mediates thrombin-induced persistent sodium current in human cardiomyocytes

After the thrombus formation in cardiac cavities or coronaries, the serine protease thrombin is produced and can therefore reach the myocardial tissue by the active process of extravasation and binds to the G protein-coupled protease-activated receptor-1 (PAR1) expressed in human myocardium. The role of PAR1 was investigated in the thrombin effect on sodium current (I(Na)). I(Na) was recorded in freshly isolated human atrial myocytes by the whole-cell patch-clamp method. Action potentials (AP) were recorded in guinea pig ventricular tissue by the conventional glass microelectrode technique. Thrombin-activated PAR1 induced a tetrodotoxin-blocked persistent sodium current, I(NaP), in a concentration-dependent manner with an apparent EC(50) of 28 U/ml. The PAR1 agonist peptide SFLLR-NH(2) (50 microM) was able to mimic PAR1-thrombin action, whereas PAR1 antagonists N(3)-cyclopropyl-7-((4-(1-methylethyl)-phenyl)methyl)-7H-pyrrolo(3,2-f)quinazoline-1,3-diamine (SCH 203099; 10 microM) and 1-(3,5-di-tert-butyl-4-hydroxy-phenyl)-2-[3-(3-ethyl-3-hydroxy-pentyl)-2-imino-2,3-dihydro-imidazol-1-yl]-ethanone (ER 112787) (1 microM), completely inhibited it. The activated PAR1 involves the calcium-independent phospholipase-A(2) signaling pathway because two inhibitors of this cascade, bromoenol lactone (50 microM) and haloenol lactone suicide substrate (50 microM), block PAR1-thrombin-induced I(NaP).Asa consequence of I(NaP) activation, in guinea pig right ventricle papillary muscle, action potential duration (APD) were significantly increased by 20% and 15% under the respective action of 32 U/ml thrombin and 50 microM SFLLR-NH(2), and these increases in APD were prevented by 1 microM tetrodotoxin or markedly reduced by application of 1 microM SCH 203099 or ER 112787. Thrombin, through PAR1 activation, increases persistent component of the Na(+) current resulting in an uncontrolled sodium influx into the cardiomyocyte, which can contribute to cellular injuries observed during cardiac ischemia.     

Activation of mast cells induced by agonists of proteinase-activated receptors under normal conditions and during acute inflammation in rats

Functions of thrombin as a modulator of inflammation and tissue repair are mediated by the proteinase-activated receptor (PAR) family. Some of these effects may be induced by activation of mast cells. To characterize the degranulation of rat peritoneal mast cells in response to PAR agonists, the effects of thrombin, trypsin and peptide agonists of PARs (PAR-AP, proteinase-activated receptor-activating peptides) on secretion were investigated. The release of beta-hexosaminidase by thrombin (0.01-1 microM) was concentration-dependent and mediated via PAR(1), as evidenced by cathepsin G (100 microM)-induced inactivation of PAR(1) and thrombin-stimulated PAR(1) desensitization. Trypsin (1 microM) accelerated histamine secretion. The PAR(1)-AP, TRAP (SFFLRN, 1-100 microM) and the PAR(2)-AP SLIGRL (5-100 microM) caused the release of histamine, and beta-hexosaminidase from inflammatory mast cells were obtained from a model of acute peritonitis in rats. Relative to the response to compound 48/80, the thrombin- and TRAP-induced release of beta-hexosaminidase was higher in inflammatory mast cells than in the control. This suggests that additional exposure of PAR(1) on mast cells to PAR agonists or an increase in PARs sensitivity to PAR agonists probably occurred during acute inflammation.     

U-937细胞的磷脂酶D激活不联系前列腺素E_2的生物合成

目的研究磷脂酰胆碱特异性磷脂酶Ｄ（ＰＣ ＰＬＤ）在佛波酯诱导Ｕ ９３７细胞的前列腺素Ｅ２（ＰＧＥ２）生物合成调节中的作用。方法二甲亚砜（ＤＭＳＯ）分化的Ｕ ９３７细胞与［３Ｈ］ 烷基 溶血ＰＣ一起孵育，对细胞内ＰＣ池进行同位素标记。［３Ｈ］ 烷基磷脂酸（ＰＡ）、［３Ｈ］ 烷基 二脂酰甘油（ＤＡＧ）、［３Ｈ］ 烷基 磷脂酰乙醇（ＰＥｔ）采用薄层层析法分离，并以液体闪烁法测定含量。ＰＥｔ的形成被认为ＰＬＤ活性的可信的特异指标。培养液中ＰＧＥ２含量应用酶免疫法测量。结果１２ 十四烷酸 １３ 乙酸佛波酯（ＰＭＡ，１μｍｏｌ·Ｌ－１）兴奋Ｕ ９３７细胞产生［３Ｈ］ 烷基 ＰＡ与［３Ｈ］ 烷基 ＤＡＧ。孵育合剂中包含乙醇（２５～１０ｍｌ·Ｌ－１）引起浓度依赖性地减少ＰＭＡ诱导的ＰＡ与ＤＡＧ积聚，以及增加ＰＥｔ积聚。时相研究显示ＰＭＡ诱导的ＰＡ积聚先于ＤＡＧ积聚。ＰＡ磷酸水解酶抑制剂普萘洛尔（２００μｍｏｌ·Ｌ－１）增加ＰＡ积聚的同时减少ＰＭＡ诱导的ＤＡＧ的生成。乙醇与普萘洛尔均不影响ＰＭＡ增加Ｕ ９３７细胞的ＰＧＥ２生成。结论ＰＭＡ激活ＤＭＳＯ分化Ｕ ９３７细胞的ＰＬＤ，但它不联系该细胞的ＰＧＥ２生物合成。说明ＰＭＡ?