Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inhibition of NO synthesis promotes P-selectin expression on endothelial cells; however, the precise mechanism is unclear. Because No has been shown to inhibit protein kinase C (PKC) activity, we examined the hypothesis that the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) stimulates P-selectin expression on platelets via PKC activation. Ten-minute incubation with either phorbol 12-myristate 13-acetate (PMA), thrombin, or L-NAME significantly increased P-selectin expression on platelets (as assessed by flow-cytometric analysis) and PKC activity of platelet membranes. Increased P-selectin expression induced by either PMA, thrombin, or L-NAME was significantly attenuated by the selective PKC inhibitor UCN-01 (7-hydroxystaurosporine). Furthermore, L-NAME-induced P-selectin expression was significantly attenuated by either L-arginine, 8-bromo-cGMP, or sodium nitroprusside (SNP). Interestingly, L-NAME further potentiated P-selectin upregulation by thrombin. L-NAME, thrombin, and PMA also significantly increased polymorphonuclear leukocyte adherence to the coronary artery endothelium, an effect that was significantly attenuated by the anti-P-selectin monoclonal antibody PB1.3 or by UCN-01, L-arginine, 8-bromo-cGMP or SNP but not by D-arginine or he nonblocking anti-P-selectin monoclonal antibody NBP1.6. These results indicate that inhibition of NO synthesis induces rapid P-selectin expression, which appears to be at least partially mediated by PKC activation in platelets. Similar effects and mechanisms of L-NAME on P-selectin function were also observed in endothelial cells, another site of P-selectin expression.
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PMID:Inhibition of nitric oxide biosynthesis promotes P-selectin expression in platelets. Role of protein kinase C. 758 91

P-selectin is an adhesion receptor for leukocytes that is redistributed from secretory granule membranes to the surfaces of activated platelets and endothelial cells. The cytoplasmic domain of P-selectin contains two serines, two threonines, and one tyrosine that could potentially be phosphorylated. We found that P-selectin was phosphorylated in both platelets and endothelial cells and that phosphorylation rapidly increased after cell activation. Approximately 0.02, 0.05, and 0.08 mol of phosphate/mol of P-selectin were incorporated, respectively, into resting, thrombin-activated, and phorbol ester-activated platelets. Phosphorylation was completely inhibited by the protein kinase C inhibitors, staurosporine, H-7, and chelerythrine, and was enhanced by the phosphatase inhibitors, okadaic acid and calyculin-A. Phosphoamino acid analysis of 32P-labeled P-selectin showed that phosphorylation occurred predominantly on serine with lesser amounts on threonine. When expressed in transfected Chinese hamster ovary cells, P-selectin was also phosphorylated. Mutagenesis studies showed that Ser788 was the principal site of phosphorylation, with minor sites on the other serine and threonine residues of the cytoplasmic domain. Phosphorylation may regulate membrane trafficking or other functions of P-selectin.
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PMID:The cytoplasmic domain of P-selectin is phosphorylated on serine and threonine residues. 769 Dec 35

Polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia/reperfusion (MI/R) injury. We examined the cardioprotective effects of N,N,N-trimethylsphingosine (TMS) in a murine model of MI (20 min) and R (24 h) injury in vivo, focusing on leukocyte-endothelial interactions. TMS is a synthetic N-methylated sphingosine derivative that has protein kinase C inhibitory activity and has been shown to prevent leukocyte activation. TMS (18 microgram/kg), administered intravenously 1 min prior to reperfusion, significantly attenuated myocardial necrotic injury assessed by myocardial creatine kinase loss compared with MI/R rats receiving only vehicle (P<0.001). Cardiac myeloperoxidase activity, an index of PMN accumulation in the ischemic myocardium, was also significantly attenuated by TMS compared with rats receiving vehicle (P<0.001). We further examined whether TMS can attenuate leukocyte-endothelial interaction by intravital microscopy. TMS significantly attenuated NG-nitro-L-arginine-methyl ester (L-NAME)-stimulated PMN rolling and adherence to the rat microvascular endothelium. This action of TMS appears to be mediated by reduction of P-selectin expression because immunohistochemical analysis demonstrated that TMS significantly attenuated endothelial P-selectin expression in the L-NAME-superfused rat mesenteric microvasculature. Similarly, TMS markedly attenuated rapid P-selectin expression in rat platelets stimulated with either thrombin or L-NAME assessed by flow cytometry. In conclusion, TMS seems to be an effective cardioprotective agent by inhibiting early leukocyte-endothelial interaction, thus preventing leukocyte accumulation in the ischemic reperfused myocardium.
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PMID:Myocardial protection by N,N,N-trimethylsphingosine in ischemia reperfusion injury is mediated by inhibition of P-selectin. 860 8

Lysophosphatidylcholine (LysoPC), an atherogenic lysophospholipid contained in oxidized low-density lipoprotein (LDL), has been shown to stimulate protein kinase C (PKC). Since PKC activators are suggested to elicit rapid P-selectin expression in platelets and endothelial cells, we examined whether LysoPc promotes P-selectin expression in platelets and P-selectin-mediated leukocyte adherence to endothelial cells via a mechanism involving PKC activation. LysoPc, but not phosphatidylcholine (PC), which is a major phospholipid component in native LDL, significantly upregulated P-selectin on cat platelets by flow cytometric analysis. This P-selectin upregulation by LysoPC was significantly attenuated by two PKC inhibitors, 7-hydroxystaurosporine (UCN-01) and N,N,N-trimethylsphingosine, and by two NO donors, CAS1609 and sodium nitroprusside. Submicellar concentrations of LysoPc significantly activated PKC in platelets, and this was inhibited by either UCN-01 or CAS1609. LysoPC, but not PC, significantly increased adherence of autologous cat polymorphonuclear leukocytes to coronary vascular endothelium, which was also markedly attenuated by UCN-01 and by CAS1609. LysoPC induced P-selectin expression on the surface of cat coronary vascular endothelium as assessed by immunohistochemical analysis. These results suggest that LysoPC, an atherogenic lysophospholipid contained in oxidized LDL, rapidly induces P-selectin expression in both platelets and endothelial cells at least partially via PKC activation. Furthermore, NO-generating agents may inhibit P-selectin upregulation by LysoPC. Since P-selectin may play an important role in initiating atherosclerosis, our data provide further insight into the mechanism of early stages of atherogenesis and of NO-mediated inhibition of atherosclerosis.
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PMID:Lysophosphatidylcholine promotes P-selectin expression in platelets and endothelial cells. Possible involvement of protein kinase C activation and its inhibition by nitric oxide donors. 862 May 97

Small cell lung carcinoma (SCLC) frequently metastasizes early in the course of the disease. P-selectin (P-sel), a cell adhesion molecule expressed on activated platelets and endothelial cells (EC), has previously been demonstrated to mediate binding of platelets to SCLC. We hypothesized that P-sel facilitates attachment of SCLC to EC, acting as an important factor in SCLC metastasis. To test this hypothesis, attachment of H82 cells (SCLC cell line) to EC was quantified. Attachment of H82 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated EC was increased compared with control EC. Increased attachment of H82 cells to EC was apparent after 10 min of TPA activation, reached a peak after 30 min, and returned to baseline after 120 min of exposure. The TPA-induced increase in H82 cell attachment to EC was inhibited by addition of anti-P-sel antibodies but not by addition of anti-E-selectin antibodies. The TPA-induced increase in H82 cell attachment was likely mediated by activation of EC protein kinase C (PKC). Pretreatment of the EC with PKC inhibitors effectively blocked the TPA-mediated increase in H82 cell attachment. In addition, prolonged exposure of EC to TPA resulted in decreased expression of the PKC-alpha and PKC-epsilon isoforms. These data indicate for the first time that attachment of SCLC to activated EC appears to be mediated by increased expression of P-sel on the EC surface, which may result from activation of specific isoforms of PKC.
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PMID:P-selectin-mediated attachment of small cell lung carcinoma to endothelial cells. 899 61

Alpha toxin from Clostridium perfringens type A, a phospholipase C, has been implicated in many of the localized and systemic features of gas gangrene. We demonstrated that human endothelial cells synthesize two vasoactive lipids, platelet-activating factor (PAF) and prostacyclin, in response to alpha toxin treatment. The stimulated synthesis of PAF required the enzymatic activity of the toxin and subsequent protein kinase C activation. Alpha toxin-treated endothelial cells accumulated the products of the phospholipase C reaction, diacylglycerol and ceramide, and exhibited a decrease in the enzymatic precursors phosphatidylcholine and sphingomyelin. Furthermore, the temporal accumulation of PAF depended on the concentration of the toxin in the overlying medium and was blocked in the presence of a neutralizing antibody. The cultured endothelial cells also exhibited enhanced neutrophil adhesion in response to alpha toxin which was mediated through the PAF receptor and P-selectin. P-selectin expression by endothelial cells and extravascular neutrophil accumulation were also observed in tissue sections from alpha toxin-injected Sprague-Dawley rats. These endothelial cell-mediated processes are important in maintaining vascular homeostasis and, when activated in a dysregulated manner by C. perfringens alpha toxin, may contribute to localized and systemic manifestations of gas gangrene including enhanced vascular permeability, localized neutrophil accumulation, and myocardial dysfunction.
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PMID:Alpha toxin from Clostridium perfringens induces proinflammatory changes in endothelial cells. 923 3

Ischemic preconditioning (IPC) refers to a phenomenon in which a tissue is rendered resistant to the deleterious effects of prolonged ischemia by previous exposure to brief periods of vascular occlusion. While the beneficial effects of IPC were first demonstrated in the myocardium, it is now clear that preconditioning protects postischemic skeletal muscle, brain, and small intestine and may also occur in humans. Although first described over a decade ago, the mechanisms underlying the powerful protective effects of IPC remain uncertain. However, a growing body of evidence indicates that the beneficial actions of IPC involve the activation of adenosine A1 receptors during the period of preconditioning ischemia in most organs and species. Adenosine A1 receptor stimulation is thought to promote the translocation and activation of specific isoforms of protein kinase C1 which in turn phosphorylate as yet unidentified cellular effector molecules. In the heart, it has been suggested that ATP-sensitive potassium channels may represent important effectors of the preconditioning phenomenon. In contrast, ATP-sensitive potassium channel activation does not seem to contribute to the beneficial effects of IPC in the small bowel and seems to play only a limited role in skeletal muscle. In these peripheral tissues, the beneficial effects of IPC are related to inhibition of leukocyte adhesion and emigration. In the small intestine, IPC seems to prevent postischemic leukocyte adhesion by maintaining the bioavailability of nitric oxide (a potent endogenous anti-adhesive agent) and preventing, the expression of P-selectin (an adhesive molecule expressed by endothelial cells that is thought to modulate leukocyte rolling). In skeletal muscle, these actions are mediated by an effect of IPC to augment the production of adenosine (another potent endogenous anti-adhesive agent) during reperfusion. Thus, although adenosine-induced protein kinase C activation seems to play an important role in initiating the beneficial actions of IPC in most tissues, the effector of the preconditioning phenomenon seems to differ among tissues. Understanding the mechanisms of IPC has led to the recognition that tissues may also be preconditioned by administration of agents that act via the same signaling cascade (e.g., adenosine, bradykinin, alpha 1-adrenergic agonists). The purpose of this review is to summarize the evidence regarding the mechanisms of IPC in different organs.
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PMID:Mechanisms of ischemic preconditioning. 926 97

Thrombin cleaves its G-protein-linked seven-transmembrane domain receptor, thereby releasing a 41-aa peptide and generating a new amino terminus that acts as a tethered ligand for the receptor. Peptides corresponding to the new amino terminal end of the proteolyzed seven-transmembrane domain thrombin receptor [TR42-55, SFLLRNPNDKYEPF, also known as TRAP (thrombin receptor-activating peptide)], previously have been demonstrated to activate the receptor. In this study, we demonstrate that the 41-aa cleaved peptide, TR1-41 (MGPRRLLLVAACFSLCGPLLSARTRARRPESKATNATLDPR) is a strong platelet agonist. TR1-41 induces platelet aggregation. In whole-blood flow cytometric studies, TR1-41 was shown to be more potent than TR42-55 and almost as potent as thrombin, as determined by the degree of increase in: (i) platelet surface expression of P-selectin (reflecting alpha granule secretion); (ii) exposure of the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex; and (iii) fibrinogen binding to the activated GPIIb-IIIa complex. As determined by experiments with inhibitors [prostaglandin I2, staurosporine, wortmannin, the endothelium-derived relaxing factor congener S-nitroso-N-acetylcysteine (SNAC), EDTA, EGTA, and genestein], and with Bernard-Soulier or Glanzmann's platelets, we demonstrated that TR1-41-induced platelet activation is: (i) inhibited by cyclic AMP; (ii) mediated by protein kinase C, phosphatidyl inositol-3-kinase, myosin light chain kinase, and intracellular protein tyrosine kinases; (iii) dependent on extracellular calcium; and (iv) independent of the GPIb-IX and GPIIb-IIIa complexes. TR1-41-induced platelet activation was synergistic with TR42-55. In summary, the cleaved peptide of the seven-transmembrane domain TR (TR1-41) is a strong platelet agonist.
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PMID:The cleaved peptide of the thrombin receptor is a strong platelet agonist. 950 Dec 19

Lymphocyte adhesion to endothelial cells and the extravascular deposition of fibrin are 2 important processes during pathologic situations such as allograft rejection. Tissue factor (TF) expression was therefore measured on human umbilical vein endothelial cells (HUVECs) after coculture with allogeneic lymphocytes (PBLs) by a factor Xa generation assay. When cocultured with PBLs, HUVECs expressed strong procoagulant activity related to the TF/factor VII-dependent pathway, which was enhanced when endothelial cells were treated with interferon-gamma (IFN-gamma). The highest TF activity was measured when 10(5) lymphocytes were incubated with 10(4) HUVECs (ratio 10: 1) for 4 hours, a time-dependent course similar to that obtained with tumor necrosis factor-alpha (TNF-alpha), and direct contact between the 2 cell types was necessary. PBL-induced TF activity was inhibited by cycloheximide or actinomycin D, indicating active protein synthesis that was confirmed by the increase in TF mRNA detected by reverse transcription-polymerase chain reaction. It was then demonstrated that 1 of the primary signaling pathways leading to endothelial cell TF expression was a rapid initial interaction between membrane TNF expressed on PBLs and the 75-kd TNF receptor, with subsequent involvement of platelet-activating factor and P-selectin. Finally, we showed that the transduction of external signals involving the activation of protein kinase C and protein tyrosine kinases also contributed to the regulation of TF expression.
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PMID:Human allogeneic lymphocytes trigger endothelial cell tissue factor expression by a tumor necrosis factor-dependent pathway. 985 44

Activated platelets and endothelium surface express the cell adhesion molecule P-selectin (CD62P), which plays an important role in mediating interactions with leukocytes. Increased levels of a functional soluble form of P-selectin (sP-selectin) have been reported in several pathological states but it is not clear whether this circulating sP-selectin originates from platelets and/or endothelial cells. Here we describe the concurrent kinetics of intracellular storage, surface expression and release of platelet P-selectin induced by thrombin or the protein kinase C activator PMA. Platelet activation with submaximal concentrations of thrombin (0.1 U/ml) resulted in a rapid decrease of intracellular P-selectin. This decrease of intracellular P-selectin concurred with a gradual increase of surface expression and an initial increase of sP-selectin. Our results indicate that intracellular stores of P-selectin were only partly mobilized upon activation with submaximal concentrations of thrombin. A high concentration of thrombin (1.0 U/ml) induced a rapid and nearly total decrease of intracellular stores and a more pronounced, but transient, increase of surface expression. The release of P-selectin was fast and occurred during the initial activation phase. The NO donor SNAP inhibited both surface expression and release of platelet P-selectin in a similar manner. PMA (0.1-1.0 microM) mediated a more slow, gradual and sustained surface expression and release of P-selectin than thrombin. Thus, surface expression and release of platelet P-selectin show different kinetics depending on the mode of activation.
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PMID:Kinetics of platelet P-selectin mobilization: concurrent surface expression and release induced by thrombin or PMA, and inhibition by the NO donor SNAP. 986 63


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