Lysophosphatidylcholine stimulates phospholipase D in human coronary endothelial cells: role of PKC

1996 ◽  
Vol 271 (4) ◽  
pp. H1706-H1710 ◽  
Author(s):  
D. A. Cox ◽  
M. L. Cohen
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Human Coronary Artery ◽  
Gf 109203X ◽  
Messenger System ◽  
Stimulation Of

Lysophosphatidylcholine (lyso PC) mediates multiple potentially atherogenic effects on endothelial cells, although the cellular mechanism of these effects remains unclear. Phospholipase D (PLD) has been recognized as a novel second-messenger system that may regulate cellular function. The purpose of this study was to determine the effect of lyso PC on PLD activity in human coronary artery endothelial cells (HCAEC) by measuring [3H]phosphatidylethanol production in cells labeled with [3H]myristic acid. After incubation with lyso PC (20 microM) for 40 min, PLD activity was markedly stimulated from five- to sixfold. Stimulation of PLD activity by lyso PC was concentration dependent (half-maximum effective concentration of 7.6 microM) and was not mimicked by phosphatidylcholine (20 microM). Because PLD can be regulated by protein kinases, the effect of several protein kinase inhibitors on lyso PC-stimulated PLD activity was tested. The protein kinase A inhibitor H-89 (300 nM) and the tyrosine kinase inhibitors genistein (30 microM) and tyrphostin A25 (100 microM) had no effect on the stimulation of PLD by lyso PC (20 microM). The protein kinase C (PKC) inhibitor calphostin C (10-300 nM) affected neither lyso PC (20 microM)-nor 4 beta-phorbol 12,13-dibutyrate (PDBu, 300 nM)-stimulated PLD activity, suggesting that this agent may not inhibit PKC in these cells. In contrast, the selective PKC inhibitors GF-109203X (0.3-10 microM) and chelerythrine (1-30 microM) concentration dependently inhibited lyso PC (20 microM)-stimulated PLD activity and blocked PDBu (300 nM)-stimulated PLD activity. Together, these data document that lyso PC stimulated PLD in human endothelial cells, possibly by a PKC-dependent mechanism, and provide evidence that PLD activation in human endothelium is a novel and important mechanism by which lyso PC mediates its cellular and possibly atherogenic effects.

Astroglial-mediated phosphorylation of the Na-K-Cl cotransporter in brain microvessel endothelial cells

1996 ◽  
Vol 271 (2) ◽  
pp. C620-C627 ◽  
Author(s):  
D. Sun ◽  
M. E. O'Donnell
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Fold Increase ◽  
Protein Kinase Inhibitors ◽  
Astroglial Cell ◽  
Astroglial Cells ◽  
Hypertonic Medium ◽  
Phosphorylation Level ◽  
Microvessel Endothelial Cells ◽  
Stimulation Of

Our previous studies have shown that cerebral microvessel endothelial cells (CMEC) express a Na-K-Cl cotransporter and that exposure of CMEC to astroglial cells causes a nearly 2-fold increase in activity of the cotransporter but only 1.5-fold increase in expression of cotransport protein [D. Sun, C. Lytle, and M. E. O'Donnell. Am. J. Physiol. 269 (Cell Physiol. 38): C1506-C1512, 1995]. This finding suggests that the astroglial cell effects may be mediated by mechanisms involving cotransporter activation in addition to increased protein expression. In the present study, we evaluated the role of protein phosphorylation in elevation of CMEC cotransport activity by astroglial cells and extracellular hypertonicity. We also examined the effects of protein phosphatase and protein kinase inhibitors on both cotransporter activity and phosphorylation in CMEC. The phosphorylation level of Na-K-Cl cotransport protein was quantitatively evaluated by immunoprecipitation analysis with the use of a monoclonal antibody to the cotransporter after 32P labeling of cultured CMEC. Activity of the cotransporter was assessed as bumetanide-sensitive K influx. We found that the phosphatase inhibitors calyculin A and okadaic acid significantly increased both cotransport activity and phosphorylation of cotransport protein. Activity and phosphorylation level of the cotransporter were also markedly increased by exposing the cells to astroglial cell-conditioned or hypertonic medium. Moreover, the astroglial-induced stimulation of the CMEC cotransporter was inhibited by the protein kinase inhibitor K-252a. These findings suggest that phosphorylation of cotransport protein plays an important role in regulation of Na-K-Cl cotransport activity and that astroglial-induced elevation of cotransport activity involves both phosphorylation-associated stimulation of cotransport activity and increased expression of the cotransporter protein.

scholarly journals Genistein reduces tumor necrosis factor alpha-induced plasminogen activator inhibitor-1 transcription but not urokinase expression in human endothelial cells

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2984-2991 ◽  
Author(s):  
VW van Hinsbergh ◽  
M Vermeer ◽  
P Koolwijk ◽  
J Grimbergen ◽  
T Kooistra
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Plasminogen Activator ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Tyrosine Protein Kinase ◽  
Pai 1

Abstract The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF- alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55- kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u- PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also inhibited PAI-1 induction by thrombin and IL-4, it is likely that genistein does not act on a TNF alpha-receptor- coupled protein kinase but on the signal transduction pathway enhancing PAI-1 transcription. Our results suggest that the TNF alpha-induced signal transduction pathway of PAI-1 transcription involves a genistein- sensitive step that is not involved in the induction of u-PA by TNF alpha. Given the limited sensitivity to several other tyrosine protein kinase inhibitors, this genistein-sensitive step may be a potential target for pharmacologic intervention to reduce elevated plasma PAI-1 levels.

1,25-Dihydroxyvitamin D3 and TPA activate phospholipase D in Caco-2 cells: role of PKC-α

1999 ◽  
Vol 276 (4) ◽  
pp. G993-G1004 ◽  
Author(s):  
Sharad Khare ◽  
Marc Bissonnette ◽  
Beth Scaglione-Sewell ◽  
Ramesh K. Wali ◽  
Michael D. Sitrin ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Dihydroxyvitamin D3 ◽  
Dihydroxyvitamin D ◽  
1,25 Dihydroxyvitamin D ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Gf 109203X ◽  
Pkc Β ◽  
Stimulation Of

1,25-Dihydroxyvitamin D3[1,25(OH)2D3] and 12- O-tetradecanoylphorbol 13-acetate (TPA) both activated phospholipase D (PLD) in Caco-2 cells. GF-109203x, an inhibitor of protein kinase C (PKC) isoforms, inhibited this activation by both of these agonists. 1,25(OH)2D3activated PKC-α, but not PKC-β1, -βII, -δ, or -ζ, whereas TPA activated PKC-α, -β1, and -δ. Chronic treatment with TPA (1 μM, 24 h) significantly reduced the expression of PKC-α, -βI, and -δ and markedly reduced the ability of 1,25(OH)2D3or TPA to acutely stimulate PLD. Removal of Ca2+ from the medium, as well as preincubation of cells with Gö-6976, an inhibitor of Ca2+-dependent PKC isoforms, significantly reduced the stimulation of PLD by 1,25(OH)2D3or TPA. Treatment with 12-deoxyphorbol-13-phenylacetate-20-acetate, which specifically activates PKC-βI and -βII, however, failed to stimulate PLD. In addition, the activation of PLD by 1,25(OH)2D3or TPA was markedly reduced or accentuated in stably transfected cells with inhibited or amplified PKC-α expression, respectively. Taken together, these observations indicate that PKC-α is intimately involved in the stimulation of PLD in Caco-2 cells by 1,25(OH)2D3or TPA.

scholarly journals Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used.

1994 ◽  
Vol 5 (3) ◽  
pp. 313-322 ◽  
Author(s):  
J R Apgar
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Adenosine Receptor ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Filamentous Actin ◽  
Ige Receptor ◽  
Ige Mediated ◽  
Rbl Cells ◽  
Stimulation Of

Crosslinking of the IgE receptor on rat basophilic leukemia (RBL) cells using the multivalent antigen DNP-BSA leads to a rapid and sustained increase in the filamentous actin content of the cells. Stimulation of RBL cells through the adenosine receptor also induces a very rapid polymerization of actin, which peaks in 45-60 s and is equivalent in magnitude to the F-actin response elicited through stimulation of the IgE receptor. However, in contrast to the IgE mediated response, which remains elevated for over 30 min, the F-actin increase induced by the adenosine analogue 5'-(N-ethylcarboxamido)-adenosine (NECA) is relatively transient and returns to baseline values within 5-10 min. While previous work has shown that the polymerization of actin in RBL cells stimulated through the IgE receptor is mediated by protein kinase C (PKC), protein kinase inhibitors have no effect on the F-actin response activated through the adenosine receptor. In contrast, pretreatment of the cells with pertussis toxin completely inhibits the F-actin response to NECA but has relatively little effect on the response induced through the IgE receptor. Stimulation of RBL cells through either receptor causes increased production of phosphatidylinositol mono-phosphate (PIP) and phosphatidylinositol bis-phosphate (PIP2), which correlates with the F-actin response. Production of PIP and PIP2 may be important downstream signals since these polyphosphoinositides are able to regulate the interaction of gelsolin and profilin with actin. Thus the polymerization of actin can be triggered through either the adenosine receptor or the IgE receptor, but different upstream signaling pathways are being used. The IgE mediated response requires the activation of PKC while stimulation through the adenosine receptor is PKC independent but involves a G protein.

scholarly journals Protein kinase inhibitors--potential chemotherapeutic agents.

1995 ◽  
Vol 42 (4) ◽  
pp. 405-418 ◽  
Author(s):  
D Shugar
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Chemotherapeutic Agents ◽  
Protein Kinase Inhibitors ◽  
Donor And Acceptor ◽  
Catalytic Sites ◽  
Competitive Inhibitors ◽  
Preclinical Trials ◽  
Antiviral Chemotherapy

Protein kinase inhibitors, widely exploited for elucidation of the biological functions of kinases, have more recently come under active consideration as potential chemotherapeutic agents for tumour and other diseases. A brief overview is presented of diverse approaches to the design and development of selective protein kinase inhibitors, and related problems such as donor and acceptor specificities, stereochemical aspects, emerging relationships between protein, sugar and nucleoside kinases. In particular, and contrary to popular belief that ATP-competitive inhibitors cannot be selective because of the close homology of the ATP catalytic sites, numerous examples are presented of such inhibitors which are both potent and selective for a given kinase or class of kinases. Some of these are undergoing preclinical trials. Attention is also directed to the role of cellular and viral protein kinases in the life cycle of viruses, and the potential of these enzymes, especially those encoded by, and essential for replication of, a given virus as targets for antiviral chemotherapy.

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