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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.
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PMID:Differential regulation of phosphoinositide and phosphatidylcholine hydrolysis by protein kinase C-beta 1 overexpression. Effects on stimulation by alpha-thrombin, guanosine 5'-O-(thiotriphosphate), and calcium. 131 71

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.
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PMID:Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1. 180 96

Intracellular killing of Staphylococcus aureus by human monocytes after cross-linking Fc gamma R is known to be a phospholipase C (PLC)-dependent process. Activation of PLC leads to the formation of second messengers that synergistically activate protein kinase C (PKC). The aim of this study was to obtain more insight into the role of PKC in Fc gamma R-mediated killing process. PKC inhibitors H-7 and staurosporine markedly suppressed the killing of S. aureus by monocytes stimulated by cross-linking Fc gamma RI or -II. Cross-linking Fc gamma R caused a transient increase in PKC activity in the membranes of monocytes, as measured by Ca2+/phospholipid-dependent phosphorylation of histone. Western blot analysis revealed that cross-linking Fc gamma R stimulated a transient increase in PKC-beta in the membranes of monocytes with kinetics that correlated closely with the translocation of PKC activity. Cross-linking Fc gamma R on monocytes also stimulated the translocation of PKC-epsilon but not PKC-alpha. PMA and 1-oleoyl-2-acetylglycerol (OAG), which caused translocation of PKC-alpha, -beta, and -epsilon, did not stimulate the killing process. Incubation with these PKC activators for 10 min rendered monocytes unresponsive to stimulation of killing of S. aureus via Fc gamma R. It could be that activation of certain PKC isozymes, probably PKC-alpha and -epsilon, by these activators causes feedback inhibition of PLC and, consequently, the killing in monocytes, because PMA blocks the Fc gamma R-mediated intracellular inositol(1,4,5)P3 formation and PKC translocation. Together, our results indicate that PKC isozymes play an important role in both stimulation and inhibition of the Fc gamma R-mediated intracellular killing of bacteria by monocytes.
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PMID:Role of protein kinase C isozymes in Fc gamma receptor-mediated intracellular killing of Staphylococcus aureus by human monocytes. 760 54

Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.
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PMID:1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i. 794 45

Prompted by the reversal of skin hyperproliferation to normal by 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid, we investigated a possible mechanism for this antiproliferative action. To address this we first demonstrated that 13-HODE is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns4,5-P2) and released as 13-HODE-containing diacylglycerol by epidermal phospholipase C. Secondly, we tested the possibility whether this putative 13-HODE-containing DAG (13HODE-DAG) could exert a modulatory effect on epidermal protein kinase C (PKC) activity which previously has been associated with skin hyperproliferation. Unlabeled 13HODE-DAG was generated from 13-HODE-containing phosphatidylcholine after phospholipase C hydrolytic cleavage. The effects of the 13HODE-DAG were determined on: i) total epidermal PKC activity; ii) diolein-activated PKC activity; and iii) the two identified epidermal PKC-isozymes (PKC-beta and PKC-alpha). Our data revealed over a twofold activation of total basal PKC activity by diolein. In contrast, replacement of diolein (1,2-dioleoylglycerol) with 13HODE-DAG (1-palmitoyl,2-13HODE-glycerol) in the incubation mixture exerted no effect on total basal PKC activity. In an another experiment, 13HODE-DAG inhibited diolein-activated PKC activity in a dose-dependent manner. To determine whether the effects of 13HODE-DAG are selective, we tested its effects on DEAE-Sephacel-purified and Western blot-confirmed PKC isozymes. Our data revealed that 13HODE-DAG selectively inhibited the activity of PKC-beta isozyme, while exerting negligible effect on the PKC-alpha isozyme. This selective inhibitory effect of 13HODE-DAG on a major epidermal PKC isozyme activity suggests that 13HODE-containing DAG seemingly can modulate epidermal PKC activity, which purportedly is associated with epidermal hyperproliferation.
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PMID:Expression of protein kinase C isozymes in guinea pig epidermis: selective inhibition of PKC-beta activity by 13-hydroxyoctadecadienoic acid-containing diacylglycerol. 807 13

In glomerular endothelial cells, extracellular ATP stimulates a phospholipase C with subsequent hydrolysis of polyphosphoinositides and an increase in cytosolic free Ca2+ concentration ([Ca2+]i). Short-term (30 min) pretreatment of endothelial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C (PKC), decreases the ATP-stimulated phosphoinositide degradation and Ca2+ mobilization. However, this inhibition was lost after incubating the cells for four hours with TPA. Longer-term pretreatment (10 to 48 hr) even potentiated ATP-induced phosphoinositide breakdown and Ca2+ mobilization. In addition, pretreating the cells for 30 minutes with the specific PKC inhibitor Ro 31-8220 dose-dependently increased ATP-stimulated phosphoinositide hydrolysis, thus clearly indicating a regulatory role for PKC in the inositol lipid signaling pathway in glomerular endothelial cells. By using specific antibodies recognizing the different PKC isoenzymes, it is observed that glomerular endothelial cells express five isoenzymes: PKC-alpha, -delta, -epsilon, -zeta and -theta. No PKC-beta, -gamma, -eta and -mu isoenzymes were detected. On exposure to TPA, a complete depletion of PKC-alpha is observed within four hours. In contrast, PKC-epsilon was more resistant to phorbol ester, and even after 48 hours of TPA treatment, only 60% of PKC-epsilon was down-regulated. PKC-theta decreased very slowly from the cytosol (47% left after 24 hr of phorbol ester treatment) and translocated to the Triton X100-insoluble fraction. Moreover, PKC-delta and PKC-zeta were not significantly affected by 48 hours of phorbol ester incubation. Thus, only PKC-alpha is depleted with a kinetic that corresponds to the loss of feedback inhibition of ATP-stimulated phosphoinositide turnover. In the next step, [Ca2+]i changes were measured in single cells loaded with Fura-2 after microinjection of neutralizing PKC isoenzyme-specific antibodies. Injection of antibodies specific for PKC-alpha potently increased Ca2+ mobilization in response to ATP stimulation when compared to cells injected with buffer only or antibodies specific for PKC-epsilon. These results provide evidence that PKC-alpha mediates feedback inhibition of ATP-stimulated phosphoinositide hydrolysis in glomerular endothelial cells.
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PMID:Feedback regulation of extracellular ATP-stimulated phosphoinositide hydrolysis by protein kinase C-alpha in bovine glomerular endothelial cells. 926 87

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O2*- generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.
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PMID:Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity. 1021 46

We have investigated the inhibitory effect of 2-hydroxymethyl-1-naphthol diacetate (TAC) on the respiratory burst of rat neutrophils and the underlying mechanism of action was also assessed in this study. TAC caused concentration-related inhibition of the formylmethionyl-leucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2*-) generation (IC50 10.2+/-2.3 and 14.1+/-2.4 microM, respectively) and O2 consumption (IC50 9.6+/-2.9 and 13.3+/-2.7 microM, respectively) of neutrophils. TAC did not scavenge the generated O2*- during dihydroxyfumaric acid autoxidation. TAC inhibited both the transient elevation of [Ca2+]i in the presence or absence of [Ca2+]o (IC50 75.9+/-8.9 and 84.7+/-7.9 microM, respectively) and the generation of inositol trisphosphate (IP3) (IC50 72.0+/-9.7 microM) in response to fMLP. Cytosolic phospholipase C (PLC) activity was also reduced by TAC at a same range of concentrations. The PMA-induced PKC-beta associated to membrane was attenuated by TAC (about 80% inhibition at 30 microM). Upon exposure to fMLP, the cellular cyclic AMP level was decreased in neutrophils pretreated with TAC. TAC attenuated fMLP-induced phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 (IC50 17.4+/-1.7 microM), but not p38. The cellular formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) induced by fMLP was inhibited by TAC in a concentration-dependent manner (IC50 25.4+/-2.4 and 25.9+/-1.4 microM, respectively). TAC had no effect on the O2*- generation of PMA-stimulated and arachidonic acid (AA)-stimulated NADPH oxidase preparations. However, TAC caused concentration-related decrease of the membrane associated p47phoX in PMA-stimulated neutrophils (about 80% inhibition at 30 microM). We conclude that inhibition by TAC of the neutrophil respiratory burst is probably attributable to the blockade of the p42/44 MAPK and phospholipase D (PLD) pathways, the membrane translocation of PKC, and to the failure in assembly of a functional NADPH oxidase complex. Blockade of the PLC pathway by TAC probably plays a minor role.
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PMID:2-Hydroxymethyl-1-naphthol diacetate (TAC) suppresses the superoxide anion generation in rat neutrophils. 1023 46

Activation of the protein kinase C (PKC) family is a potential signaling mechanism by which high ambient glucose concentration modulates the phenotype and physiological function of cells. Recently, the cardiac renin angiotensin system (RAS) has been reported to promote PKC translocation in the diabetic heart via the angiotensin (ANG) II type 1 receptor (AT-1R). To evaluate the molecular events coupled with high glucose-induced PKC translocation and to examine the role of endogenously released ANG II in myocyte PKC signaling, primary cultures of adult rat ventricular myocytes were exposed to normal (5 mmol/l) or high (25 mmol/l) glucose for 12-24 h. Western blot analysis indicated that adult rat ventricular myocytes coexpress six PKC isozymes (alpha, beta(1,) beta(2,) delta, epsilon, and zeta). Translocation of five PKC isozymes (beta(1), beta(2), delta, epsilon, and zeta) was detected in response to 25 mmol/l glucose. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate blocked glucose-induced translocation of PKC-beta(2), -delta, and -zeta. Inhibition of tyrosine kinase with genistein blocked glucose-induced translocation of PKC-beta(1) and -delta, whereas chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane N,N,N,'N'-tetraacetic acid blocked translocation of PKC-beta(1) and -beta(2). Enzyme-linked immunosorbent assay performed on culture media from myocytes maintained in 25 mmol/l glucose detected a twofold increase in ANG II. Addition of an AT-1R antagonist (losartan; 100 nmol/l) to myocyte cultures blocked translocation of PKC-beta(1), -beta(2), -delta, and -epsilon. Phosphorylation of troponin (Tn) I was increased in myocytes exposed to 25 mmol/l glucose. Losartan selectively inhibited Tn I serine phosphorylation but did not affect phosphorylation at threonine residues. We concluded that 1) 25 mmol/l glucose triggers the release of ANG II by myocytes, resulting in activation of the ANG II autocrine pathway; 2) differential translocation of myocyte PKC isozymes occurs in response to 25 mmol/l glucose and ANG II; and 3) AT-1R-dependent PKC isozymes (beta(1), beta(2), delta, and epsilon) target Tn I serine residues.
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PMID:Angiotensin II promotes glucose-induced activation of cardiac protein kinase C isozymes and phosphorylation of troponin I. 1147 56

Previous results have shown that the human promyelocytic leukemia HL-60 cell line responds to either proliferating or differentiating stimuli. When these cells are induced to proliferate, protein kinase C (PKC)-beta II migrates toward the nucleus, whereas when they are exposed to differentiating agents, there is a nuclear translocation of the alpha isoform of PKC. As a step toward the elucidation of the early intranuclear events that regulate the proliferation or the differentiation process, we show that in the HL-60 cells, a proliferating stimulus (i.e., insulin-like growth factor-I [IGF-I]) increased nuclear diacylglycerol (DAG) production derived from phosphatidylinositol (4,5) bisphosphate, as indicated by the inhibition exerted by 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine and U-73122 (1-[6((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione), which are pharmacological inhibitors of phosphoinositide-specific phospholipase C. In contrast, when HL-60 cells were induced to differentiate along the granulocytic lineage by dimethyl sulfoxide, we observed a rise in the nuclear DAG mass, which was sensitive to either neomycin or propranolol, two compounds with inhibitory effect on phospholipase D (PLD)-mediated DAG generation. In nuclei of dimethyl sulfoxide-treated HL-60 cells, we observed a rise in the amount of a 90-kDa PLD, distinct from PLD1 or PLD2. When a phosphatidylinositol (4,5) bisphosphate-derived DAG pool was generated in the nucleus, a selective translocation of PKC-beta II occurred. On the other hand, nuclear DAG derived through PLD, recruited PKC-alpha to the nucleus. Both of these PKC isoforms were phosphorylated on serine residues. These results provide support for the proposal that in the HL-60 cell nucleus there are two independently regulated sources of DAG, both of which are capable of acting as the driving force that attracts to this organelle distinct, DAG-dependent PKC isozymes. Our results assume a particular significance in light of the proposed use of pharmacological inhibitors of PKC-dependent biochemical pathways for the therapy of cancer disease.
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PMID:Proliferating or differentiating stimuli act on different lipid-dependent signaling pathways in nuclei of human leukemia cells. 1190 74


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