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)

In experimental membranous nephropathy, C5b-9 induces noncytolytic glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by metabolites of arachidonic acid. In cultured GEC, sublytic C5b-9 increases cytosolic Ca2+ concentration ([Ca2+]i), activates phospholipase C (PLC), and releases arachidonic acid and eicosanoids. This study examined mechanisms of arachidonic acid production by C5b-9. In GEC labeled with [3H]arachidonate C5b-9 increased free [3H]arachidonic acid and 1,2-[3H]-arachidonoyl-diacylglycerol (DAG), an endogenous activator of protein kinase C (PKC). Elevated [Ca2+]i was not sufficient to account for increased free arachidonic acid. Moreover, in GEC that had been depleted of PKC by preincubation for 18 h with 2 microM phorbol myristate acetate, the C5b-9-induced arachidonate release was inhibited by greater than 75%. Reacylation of phospholipids was not decreased by C5b-9. Homogenates of GEC that had been stimulated with C5b-9 released more [14C]arachidonate from exogenously added 2-[14C]arachidonoyl-phosphatidyl-ethanolamine or 2-[14C]arachidonoyl-phosphatidylcholine than homogenates of unstimulated cells (assayed at a Ca2+ concentration of 2 mM). These experiments demonstrate directly that C5b-9 increased phospholipase A2 (PLA2) activity. PLA2 appeared to be stimulated as a result of PKC activation (probably secondary to increased DAG) in association with elevated [Ca2+]i. The C5b-9-induced activation of PLA2 may lead to release of eicosanoids, which may contribute toward impaired glomerular capillary wall permselectivity in experimental membranous nephropathy.
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PMID:Release of arachidonic acid by complement C5b-9 complex in glomerular epithelial cells. 190 97

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which, in some models, is partially mediated by eicosanoids. By analogy, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids in cultured rat GEC. In this study, we demonstrate that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which we identified as "cPLA2." This increase was abolished with inhibitors of protein kinase C. C5b-9 did not affect low-molecular-mass membrane-associated or secretory PLA2 activities. In GEC that stably overexpress cPLA2 activity and protein (produced by transfection of cPLA2 cDNA), immunoblot analysis showed that sublytic C5b-9 induced a decreased mobility of cPLA2, consistent with cPLA2 phosphorylation. Incubation of cPLA2-transfected GEC with sublytic C5b-9 significantly increased production of free AA and prostaglandin E2, whereas, in control GEC, the C5b-9-induced changes in free AA and prostaglandin E2 were small. Furthermore, both C5b-9-dependent sublytic cytotoxicity and cytolysis were enhanced in GEC overexpressing cPLA2, compared with control cells. Thus C5b-9 increased cPLA2 activity, probably via phosphorylation involving a protein kinase C-dependent pathway. Phospholipid hydrolysis by cPLA2 resulted in release of substrate for eicosanoid synthesis and in enhancement of C5b-9-dependent GEC injury. Both processes may facilitate glomerular damage in membranous nephropathy.
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PMID:Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells. 750 41

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria. In cultured rat GEC, C5b-9 stimulates a phosphoinositide-directed phospholipase (PL) C and products of PLC downregulate C5b-9-mediated GEC injury. We now report that C5b-9-induced hydrolysis of phosphatidylcholine (PC) provides an additional source of 1,2-diacylglycerol (DAG). PC was labeled in intact GEC by brief incubation with 1-O-[alkyl-3H]2-lyso-PC. Assembly of C5b-9 stimulated an increase in PC-derived [3H]DAG (173 +/- 18% control), which was reduced in GEC depleted of protein kinase C (PKC) by prolonged preincubation with phorbol 12-myristate 13-acetate (PMA). Similar to C5b-9, [3H]DAG was released from PC after brief incubation of GEC with Ca2+ ionophore A23187 plus PMA. The increases in [3H]DAG induced by C5b-9 and A23187 plus PMA were paralleled by increases in DAG mass. C5b-9 also increased [3H]phosphatidic acid (PA; 182 +/- 37% control), but there was no significant interconversion of DAG and PA. Thus DAG probably originated via PLC. PC-directed PLC activity was also studied in GEC homogenates by release of [14C]DAG from exogenous 1-palmitoyl-2-[arachidonoyl-14C]PC. PLC activity was present at physiological Ca2+ concentration (200-1,200 nM), and PMA stimulated PLC activity in cell homogenates (in presence of ATP). These results demonstrate directly that PMA stimulates release of DAG from PC and are in keeping with the effect of PMA in [3H]lyso-PC-labeled GEC. Thus GEC contain a PC-directed PLC, whose activity is physiologically regulated and is present at nanomolar Ca2+ concentration. C5b-9 stimulates PC-directed PLC, leading to production of DAG. This DAG might trigger a mechanism for limiting injury during complement attack.
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PMID:Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9. 823 84

Diabetic late complications are characterized by morphological and biochemical alterations of the extracellular matrix. In particular, longstanding diabetes causes quantitative and qualitative changes in basement membrane structure of retinal and renal capillaries. Immunohistochemical investigations of diabetic kidneys with diffuse glomerulosclerosis show increased collagen type IV deposition in the mesangial matrix and decreased heparan sulfate proteoglycan content in the mesangial matrix and glomerular basement membrane as well. In nodular glomerulosclerosis normal basement membrane components are decreased or absent while the occurrence of collagen type III in this stage has been interpreted as an irreversible alteration of the glomerular structure. These changes seem to be the underlying cause for the alterations in renal functions like persistent albuminuria and proteinuria. Increased intra- and extracellular levels of glucose and its derivatives are thought to be responsible for diabetic tissue dysfunction although there are reports on possible genetic defects causing increased susceptibility to develop diabetic nephropathy. Recent results, however, focus on the role of glucose-induced cytokine secretion as mediator for altered metabolism of glomerular matrix proteins. In vitro studies with cultured kidney cells have shown that the glucose-induced dysregulation of the basement membrane synthesis may be mediated by a glucose dependent activation of protein kinase C. Alternatively or synergistically, the formation of AGE products formed after prolonged exposure of matrix proteins to elevated glucose may also lead to cytokine secretion subsequently inducing synthesis of extracellular matrix proteins. Studies in experimental animals confirm the diabetes induced dysregulation of the synthesis of extracellular matrix components on the molecular level.
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PMID:Alterations of glomerular matrix proteins in the pathogenesis of diabetic nephropathy. 851 35

Platelet-activating factor (PAF) is a potent inflammatory mediator that participates in the pathogenesis of proteinuria and glomerular damage. However, the role of this lipid in glomerular sclerosis remains unknown. This study examines the effect of PAF on the regulation of extracellular matrix proteins by rat and human mesangial cells. PAF increased in a dose-dependent manner the gene expression of fibronectin and type IV collagen, but not type I collagen. Moreover, an increase in cell-associated and soluble fibronectin synthesis was also seen. These effects were abolished by BN52021 and WEB2086, two different PAF receptor antagonists. Because transforming growth factor (TGF)-beta has been considered a profibrogenic cytokine, this study also evaluated whether PAF effects might be mediated by the production of endogenous TGF-beta. PAF caused an increase in TGF-beta1 mRNA expression (by a protein kinase C-dependent pathway) and TGF-beta activity. Moreover, PAF-induced fibronectin synthesis was totally abolished when an anti-TGF-beta-neutralizing antibody was added to the culture medium, suggesting that PAF stimulates fibronectin synthesis, at least in part, through the induction of TGF-beta. Addition of cycloheximide, a protein synthesis inhibitor, upregulated PAF-induced fibronectin mRNA expression but downregulated PAF-induced TGF-beta1 gene expression, suggesting the existence of different regulatory transcriptional factors of the two proteins. These results suggest that PAF may be implicated in matrix accumulation during renal injury and therefore contribute to the pathogenesis of glomerulosclerosis.
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PMID:Platelet-activating factor stimulates gene expression and synthesis of matrix proteins in cultured rat and human mesangial cells: role of TGF-beta. 925 53

In rat membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. In cultured rat GEC, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids, due to activation of phospholipase A2 (PLA2). To address mechanisms of PLA2 activation, GEC were stably transfected with cDNAs of wild-type cytosolic PLA2 (cPLA2-wt), or group II secretory PLA2, producing overexpression of PLA2 activity. Sublytic C5b-9 markedly increased free [3H]AA in cPLA2-wt-transfected GEC, but only trivial increases were evident in secretory PLA2-transfected, or neo (control) GEC. In cPLA2-wt-transfected GEC, reduction of extracellular free Ca2+ or down-regulation of protein kinase C inhibited [3H]AA release. To further address the regulation of cPLA2, we stably expressed a mutant cPLA2 in which the Ca2+-dependent lipid binding domain was deleted (deltaCaLB). In GEC that express cPLA2-deltaCaLB, the C5b-9-induced increase in free [3H]AA was comparable with neo, despite expression of cPLA2-deltaCaLB at levels similar to cPLA2-wt. We then stably expressed another cPLA2 mutant (cPLA2-srcmyr) in which the CaLB domain was replaced by the N-terminal myristoylation domain of c-Src. cPLA2-srcmyr is permanently membrane associated. At low extracellular free Ca2+, C5b-9 increased free [3H]AA significantly in GEC that express cPLA2-srcmyr, while in neo GEC, the change was negligible. Thus, C5b-9 activates the cPLA2 isoform. Activation is dependent on the CaLB domain, and is mediated by phosphorylation, Ca2+ influx, and membrane association.
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PMID:Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells. 931 58

Angiotensin-converting enzyme (ACE) inhibitors are antihypertensive drugs that have been shown to reduce proteinuria and to slow down the progression of renal function deterioration in different models of chronic glomerular disease. Major pathogenetic features of progressive glomerular injury leading to glomerulosclerosis are mesangial cell proliferation and platelet-derived growth factor (PDGF) expression. The aim of the present study was to evaluate the effect of ramipril, an ACE inhibitor, on these two potential therapeutic targets. Thus, the effect of ramipril on DNA synthesis, cell proliferation and PDGF A and B chain gene expression in fetal calf serum (FCS)-activated cultured human glomerular mesangial cells was investigated. DNA synthesis was evaluated by tritiated thymidine incorporation, cell proliferation by direct cell counting and cell viability by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). PDGF A and B chain gene expressions were studied by Northern blot and RT-PCR, respectively. In a dose-dependent manner ramipril inhibited the FCS-induced DNA synthesis and cell proliferation. This effect was not dependent upon a toxic effect as demonstrated by MTT. The antiproliferative effect of ramipril was most likely independent of its ability to inhibit ACE present in the FCS and/or expressed by the cells, since a synthetic peptide that specifically inhibits ACE, at the same molar concentrations, did not inhibit FCS-stimulated DNA synthesis. Moreover, ramipril significantly reduced FCS-induced PDGF A and B chain gene expression. Finally, ramipril completely abolished the PDGF A and B chain gene expression induced by phorbol 12-myristate 13-acetate, a specific protein kinase C activator, suggesting a site of action downstream of this enzyme in the mitogenic signal transduction pathway. Our study would suggest that the modulatory action of ramipril on activated mesangial cell proliferation and PDGF expression is independent of its ability to inhibit ACE and could represent an additional mechanism in the renal protective effects of this drug.
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PMID:Ramipril inhibits in vitro human mesangial cell proliferation and platelet-derived growth factor expression. 1035 63

Nephrotic-range proteinuria is associated with a several-fold increase risk of cardiovascular infarction. This increased risk is accompanied by endothelial dysfunction, which is not related to increased blood pressure and is not correctable by acute administration of L-arginine. The latter is in direct contrast to what has been found in patients with primary hypercholesterolemia, suggesting that either hypoalbuminemia itself or other aspects of the dyslipidemia characteristic of the nephrotic syndrome impair endothelial function. Lysophosphatidylcholine (lyso-PC) is formed during oxidative modification of cholesterol, and lyso-PC in oxidized low-density lipoprotein (LDL) is responsible for reduced endothelial function in vitro. However, in the circulation, lyso-PC is tightly bound to albumin. Indeed, the addition of albumin can restore endothelial function, which was previously disturbed by lyso-PC. Hypoalbuminemia induces a shift in lyso-PC to lipoproteins, notably LDL, and to erythrocytes. The latter directly induces a reduction in deformability that can also be corrected by the addition of albumin. Hypoalbuminemia may disturb endothelial function, either by directly affecting Gi-protein-dependent signal transduction or indirectly by changing the configuration of the cell membrane. Such a change in cell membrane configuration will disturb binding of ligands to receptors and of endothelial nitric oxide (NO) synthase to caveolin. However, other pathways have been suggested, such as stimulation by lyso-PC of vasoconstriction mediated by protein kinase C. It remains to be shown whether lipid-lowering and antiproteinuric strategies have independent positive effects on endothelial function in nephrotic subjects.
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PMID:Endothelial function in proteinuric renal disease. 1041 39

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The increased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In this paper, we investigate the role of protein kinase C (PKC) on the PAN induced reactive oxygen generation in isolated rat hepatocytes. Isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA, 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat hepatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, at 10 and 100 microM significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dependent from 10 to 100 microM. H1004, a reagent used as control for H-7, did not affect (at 10 microM) or increased little (at 100 microM) the synthesis of MG and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited (at 10 microM) MG synthesis in the presence of PAN. PKC in the membrane fraction, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with PAN, while it changed little without PAN. These results indicate that PAN activates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.
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PMID:The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside. 1079 14

Diabetic nephropathy associated with hyperglycemia is characterized by glomerular hyperfiltration and endothelial dysfunction. Vascular endothelial growth factor (VEGF) is known to be primarily involved in neoangiogenesis and increased endothelial permeability. The purpose of this study was to investigate VEGF expression in response to high glucose in rat cultured mesangial cells and to identify its signal pathway via protein kinase C (PKC). Rat mesangial cells were cultured with different concentrations of glucose: normal (5 mM d-glucose), medium (15 mM d-glucose) and high (30 mm d-glucose). Calphostin-C as a PKC inhibitor and phorbol myristate acetate (PMA) as a PKC downregulator were instillated into culture media to evaluate the role of PKC in mediating the glucose-induced increase in VEGF expression. High glucose increased expression of VEGF at the mRNA and protein levels, identified by semi-quantitative RT-PCR and western blotting, within 3 h and in a time- and glucose concentration-dependent manner. Calphostin-C and PMA inhibited glucose-induced increases in VEGF expression at the mRNA and protein levels. In conclusion, high glucose can directly increase VEGF expression in rat mesangial cells via a PKC-dependent mechanism. These results suggest that VEGF could be a potential mediator of glomerular hyperfiltration and proteinuria in diabetic nephropathy.
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PMID:Expression of vascular endothelial growth factor in response to high glucose in rat mesangial cells. 1082 45


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