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.

IL-6 secreted by astroglial cells regulates Na-K-Cl cotransport in brain microvessel endothelial cells

1997 ◽  
Vol 272 (6) ◽  
pp. C1829-C1835 ◽  
Author(s):  
D. Sun ◽  
C. Lytle ◽  
M. E. O'Donnell
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Astroglial Cell ◽  
Astroglial Cells ◽  
Microvessel Endothelial Cells ◽  
Brain Microvessel ◽  
Dose Dependent ◽  
Cell Conditioned Medium ◽  
Cerebral Microvessel

We showed previously that cerebral microvessel endothelial cells (CMEC) exhibit a prominent Na-K-Cl cotransporter that functions to regulate intracellular volume and may also mediate vectorial ion transport across the blood-brain barrier (BBB). Astrocytes and their conditioned media induce BBB properties of the endothelium. Our previous studies demonstrated that exposure of CMEC to astroglial cells markedly increases cotransport activity, upregulates cotransporter protein expression, and also increases cotransporter protein phosphorylation. In the present study, we evaluated the possibility that astroglial effects on the Na-K-Cl cotransporter are mediated by astroglial cell-secreted interleukin-6 (IL-6). Cotransporter activity was assessed as bumetanide-sensitive K influx, and protein expression was evaluated by Western blot analysis. Exposing CMEC to IL-6 for 48 h caused a dose-dependent stimulation of Na-K-Cl cotransport activity. Both C6 glial cell-conditioned medium (C6CM)-induced and IL-6-induced increases in cotransport activity were neutralized by anti-IL-6 antibodies. A 48-h exposure of cells to IL-6 or C6CM also resulted in increased cotransporter protein expression. Furthermore, using an enzyme-linked immunosorbent assay for IL-6, we found significant amounts of IL-6 in C6CM. These data suggest that astroglia-secreted IL-6 may mediate the observed astroglial cell effects on CMEC Na-K-Cl cotransport and further support the hypothesis that astrocytes participate in maintenance of cerebral ionic homeostasis by regulating Na-K-Cl cotransporter function at the BBB.

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.

Induction of ICAM-1 by TNF-alpha, IL-1 beta, and LPS in human endothelial cells after downregulation of PKC

1992 ◽  
Vol 263 (4) ◽  
pp. C767-C772 ◽  
Author(s):  
C. L. Myers ◽  
S. J. Wertheimer ◽  
J. Schembri-King ◽  
T. Parks ◽  
R. W. Wallace
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Protein Kinase Inhibitors ◽  
Intercellular Adhesion Molecule ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Western Blots ◽  
Mrna Induction

The intercellular adhesion molecule 1 (ICAM-1) is induced on endothelial cells by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and lipopolysaccharide (LPS). We have reported the sensitivity of cytokine-induced ICAM-1 expression to protein kinase inhibitors, including inhibitors of protein kinase C (PKC) [C. L. Myers, S. N. Desai, J. Schembri-King, G. L. Letts, and R. W. Wallace. Am. J. Physiol. 262 (Cell Physiol. 31): C365-C373, 1992]. To directly investigate the role of PKC in ICAM-1 induction, we downregulated PKC by pretreatment of human umbilical vein endothelial cells with phorbol 12-myristate 13-acetate (PMA) and assessed ICAM-1 protein and mRNA induction elicited by subsequent exposure to inflammatory stimuli. PMA treatment results in ICAM-1 protein induction that declines to basal levels by 3 days. Western blots of endothelial cell lysates reveal a nearly complete loss of immunologically reactive PKC. Subsequent activation with cytokine or LPS leads to reinduction of ICAM-1 protein and mRNA; however, the cells no longer produced substantial amounts of ICAM-1 protein or mRNA in response to PMA stimulation. Cross desensitization is observed with phorbol dibutyrate, while 4 alpha-phorbol has no desensitizing effect. The data indicate that PKC activation, while capable of inducing ICAM-1 expression, is not essential for ICAM-1 induction by the inflammatory mediators TNF-alpha, IL-1 beta, or LPS.

Regulation of calcium mobilization and entry in human platelets by endothelium-derived factors

1994 ◽  
Vol 267 (1) ◽  
pp. C236-C244 ◽  
Author(s):  
J. Geiger ◽  
C. Nolte ◽  
U. Walter
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Rapid Phase ◽  
No Donor ◽  
Cyclic Monophosphate ◽  
Dependent Protein ◽  
Camp And Cgmp ◽  
Stimulation Of

Stimulation of Ca2+ mobilization and entry by agonists such as ADP, thrombin, and thromboxane is an early step of platelet activation. Here, we compared the effects of adenosine 3',5'-cyclic monophosphate (cAMP)-elevating prostaglandins, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating nitrovasodilators, membrane-permeant selective activators of cAMP- or cGMP-dependent protein kinases, and physiological endothelium-derived factors on the agonist-evoked Ca2+ mobilization and entry in human platelets. Prostaglandin E1, the prostacyclin analogue Iloprost, the nitric oxide (NO) donor 3-morpholinosydnonimine hydrochloride, and selective activators of cGMP- or cAMP-dependent protein kinase strongly inhibited the agonist-evoked Ca2+ mobilization from intracellular stores and associated late Ca2+ entry but had little effects on the rapid (1st) phase of ADP-evoked Ca2+ entry. During coincubation of platelets with endothelial cells, endothelium-derived factors that were released strongly inhibited platelet agonist-evoked Ca2+ mobilization and only moderately affected the rapid phase of ADP-evoked Ca2+ entry. These effects were partially prevented when endothelial cells were preincubated with cyclooxygenase and/or NO synthase inhibitors. Endothelial cells therefore produce sufficient quantities of labile platelet inhibitors whose effects on the platelet Ca2+ response resemble those observed with selective cAMP- and cGMP-dependent protein kinase activators.

scholarly journals Thrombin Stimulation of the Vascular Cell Adhesion Molecule-1 Promoter in Endothelial Cells Is Mediated by Tandem Nuclear Factor-κB and GATA Motifs

2001 ◽  
Vol 276 (50) ◽  
pp. 47632-47641 ◽  
Author(s):  
Takashi Minami ◽  
William C. Aird
Keyword(s):  
Endothelial Cells ◽  
Adhesion Molecule ◽  
Umbilical Vein ◽  
Fold Increase ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Response Element ◽  
Vascular Adhesion ◽  
Stimulation Of

The goal of this study was to delineate the transcriptional mechanisms underlying thrombin-mediated induction of vascular adhesion molecule-1 (VCAM-1). Treatment of human umbilical vein endothelial cells with thrombin resulted in a 3.3-fold increase in VCAM-1 promoter activity. The upstream promoter region of VCAM-1 contains a thrombin response element, two nuclear factor κB (NF-κB) motifs, and a tandem GATA motif. In transient transfection assays, mutation of the thrombin response element had no effect on thrombin induction. In contrast, mutation of either NF-κB site resulted in a complete loss of induction, whereas a mutation of the two GATA motifs resulted in a significant reduction in thrombin stimulation. In electrophoretic mobility shift assays, nuclear extracts from thrombin-treated endothelial cells displayed markedly increased binding to the tandem NF-κB and GATA motifs. The NF-κB complex was supershifted with anti-p65 antibodies, but not with antibodies to RelB, c-Rel, p50, or p52. The GATA complex was supershifted with antibodies to GATA-2, but not GATA-3 or GATA-6. A construct containing tandem copies of the VCAM-1 GATA motifs linked to a minimal thymidine kinase promoter was induced 2.4-fold by thrombin. Taken together, these results suggest that thrombin stimulation of VCAM-1 in endothelial cells is mediated by the coordinate action of NF-κB and GATA transcription factors.

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.

scholarly journals Stimulation of tissue plasminogen activator production by retinoic acid: synergistic effect on protein kinase C-mediated activation

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 981-987 ◽  
Author(s):  
RD Medh ◽  
L Santell ◽  
EG Levin
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Retinoic Acid ◽  
Protein Kinase ◽  
Hela Cells ◽  
Additive Effect ◽  
Kinase C ◽  
Tissue Plasminogen ◽  
Camp Levels ◽  
Stimulation Of

Abstract Trans retinoic acid (t-RA) stimulated the production of tissue plasminogen activator (tPA) in HeLa-S3 and human umbilical vein endothelial cells (huvecs) in a dose-dependent manner with maximal release (four to five times control) at 40 nmol/L and 40 mumol/L, respectively. In endothelial cells, the stimulation of tPA production by phorbol 12-myristate 13-acetate (PMA) was potentiated 1.9-fold by 10 mumol/L t-RA, or 1.8 times the additive effect. In HeLa cells, total tPA secretion with 10 nmol/L PMA was increased from 43 ng/mL to 96 ng/mL by 40 nmol/L t-RA, which was two times the additive effect. Higher concentrations of t-RA (400 nmol/L) depressed tPA secretion by itself and also suppressed PMA-induced tPA production by 50%. Histamine and thrombin also synergized with t-RA. t-RA (40 nmol/L) and 10 micrograms/mL histamine or 10 U/mL thrombin combined to induce tPA production 3.4 and 1.3 times the additive effect in HeLa cells. Cyclic adenosine monophosphate (cAMP) levels were not significantly affected by 10 nmol/L to 10 mumol/L t-RA. Nor did 10 nmol/L PMA and 40 nmol/L t- RA together affect cAMP levels, suggesting that t-RA-mediated potentiation of PMA-induced tPA production occurred via a mechanism that was independent of cAMP levels. Downregulation of protein kinase C (PKC) by pretreatment of huvecs with 100 nmol/L PMA completely blocked a secondary response to PMA, but did not have a significant effect on t- RA induction. Pretreatment with 10 mumol/L t-RA, on the other hand, did not significantly affect a secondary stimulus by 100 nmol/L PMA, but completely suppressed a secondary stimulation by 10 mumol/L t-RA alone. These studies suggest that the mechanism mediating t-RA stimulation of tPA production interacts with the PKC pathway, resulting in synergism.

Regulation of arginine transport and metabolism by Protein Kinase Cα in endothelial cells: stimulation of CAT2 transporters and arginase activity

2010 ◽  
Vol 49 (2) ◽  
pp. 260-270 ◽  
Author(s):  
Rossana Visigalli ◽  
Amelia Barilli ◽  
Alessandro Parolari ◽  
Roberto Sala ◽  
Bianca Maria Rotoli ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Arginase Activity ◽  
Arginine Transport ◽  
Protein Kinase Cα ◽  
Stimulation Of

scholarly journals Stimulation of pancreatic β-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways

2006 ◽  
Vol 188 (3) ◽  
pp. 481-492 ◽  
Author(s):  
Birgitte N Friedrichsen ◽  
Nicole Neubauer ◽  
Ying C Lee ◽  
Vivian K Gram ◽  
Niels Blume ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclin D1 ◽  
Fold Increase ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
Cell Replication ◽  
Β Cell ◽  
Mitogen Activated Protein ◽  
Transcriptional Induction ◽  
Stimulation Of

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), have been suggested to act as β-cell growth factors and may therefore be of critical importance for the maintenance of a proper β-cell mass. We have investigated the molecular mechanism of incretin-induced β-cell replication in primary monolayer cultures of newborn rat islet cells. GLP-1, GIP and the long-acting GLP-1 derivative, lira-glutide, increased β-cell replication 50–80% at 10–100 nM upon a 24 h stimulus, whereas glucagon at a similar concentration had no significant effect. The stimulatory effect of GLP-1 and GIP was efficiently mimicked by the adenylate cyclase activator, forskolin, at 10 nM (~90% increase) and was additive (~170–250% increase) with the growth response to human growth hormone (hGH), indicating the use of distinct intracellular signalling pathways leading to mitosis by incretins and cytokines, respectively. The response to both GLP-1 and GIP was completely blocked by the protein kinase A (PKA) inhibitor, H89. In addition, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin and the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, both inhibited GLP-1- and GIP-stimulated proliferation. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, had no inhibitory effect on either GLP-1 or GIP stimulated proliferation. Cyclin Ds act as molecular switches for the G0/G1-S phase transition in many cell types and we have previously demonstrated hGH-induced cyclin D2 expression in the insulinoma cell line, INS-1. GLP-1 time-dependently induced the cyclin D1 mRNA and protein levels in INS-1E, whereas the cyclin D2 levels were unaffected. However, minor effect of GLP-1 stimulation was observed on the cyclin D3 mRNA levels. Transient transfection of a cyclin D1 promoter-luciferase reporter construct into islet monolayer cells or INS-1 cells revealed approximately a 2–3 fold increase of transcriptional activity in response to GLP-1 and GIP, and a 4–7 fold increase in response to forskolin. However, treatment of either cell type with hGH had no effect on cyclin D1 promoter activity. The stimulation of the cyclin D1 promoter by GLP-1 was inhibited by H89, wortmannin, and PD98059. We conclude that incretin-induced β-cell replication is dependent on cAMP/PKA, p42 MAPK and PI3K activities, which may involve transcriptional induction of cyclin D1. GLP-1, GIP and liraglutide may have the potential to increase β-cell replication in humans which would have significant impact on long-term diabetes treatment.

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