eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1

Reports on the role of AMP-activated protein kinase (AMPK) in thrombin-mediated activation of endothelial nitric-oxide synthase (eNOS) in endothelial cells have been conflicting. Previously, we have shown that under culture conditions that allow reduction of ATP-levels after stimulation, activation of AMPK contributes to eNOS phosphorylation and activation in endothelial cells after treatment with thrombin. In this paper we examined the signaling pathways mediating phosphorylation and activation of eNOS after stimulation of cultured human umbilical vein endothelial cells (HUVEC) with histamine and the role of LKB1-AMPK in the signaling. In Morgan's medium 199 intracellular ATP was lowered by treatment with histamine or the ionophore A23187 while in medium RMPI 1640 ATP was unchanged after identical treatment. In medium 199 inhibition of Ca^+ 2/CaM kinase kinase (CaMKK) by STO-609 only partially inhibited AMPK phosphorylation but after gene silencing of LKB1 with siRNA there was a total inhibition of AMPK phosphorylation by STO-609 after treatment with either histamine or thrombin, demonstrating phosphorylation of AMPK by both upstream kinases, LKB1 and CaMKK. Downregulation of AMPK with siRNA partially inhibited eNOS phosphorylation caused by histamine in cells maintained in medium 199. Downregulation of LKB1 by siRNA inhibited both phosphorylation and activity of eNOS and addition of the AMPK inhibitor Compound C had no further effect on eNOS phosphorylation. When experiments were carried out in medium 1640, STO-609 totally prevented the phosphorylation of AMPK without affecting eNOS phosphorylation. AMPKα2 downregulation resulted in a loss of the integrity of the endothelial monolayer and increased expression of GRP78, indicative of endoplasmic reticular (ER) stress. Downregulation of AMPKα1 had no such effect. The results show that culture conditions affect endothelial signal transduction pathways after histamine stimulation. Under conditions where intracellular ATP is lowered by histamine, AMPK is activated by both LKB1 and CaMKK and, in turn, mediates eNOS phosphorylation in an LKB1 dependent manner. Both AMPKα1 and − α2 are involved in the signaling. Under conditions where intracellular ATP is unchanged after histamine treatment, CaMKK alone activates AMPK and eNOS is phosphorylated and activated independent of AMPK.     

eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca2+ in human atrial myocardium

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser¹¹⁷⁷ (Ser¹¹⁷⁷ eNOS). We have recently shown that agonist-induced Ser¹¹⁷⁷ eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca²⁺ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser¹¹⁷⁷ eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca²⁺ chelator BAPTA before and after application of BRL 37344 (BRL), a ₃-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser¹¹⁷⁷ eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca²⁺ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser¹¹⁷⁷ eNOS phosphorylation is dependent on intracellular Ca²⁺. 2) In atrial myocardium, eNOS-translocation and not Ser¹¹⁷⁷ eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca²⁺ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation. ₃-adrenoceptor; BRL 37344; cardiomyocyte; heart; Ca²⁺ regulation     

l-Theanine promotes nitric oxide production in endothelial cells through eNOS phosphorylation

Consumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the key regulator of vascular functions in endothelium. In this study, we establish that l-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO) production in endothelial cells. l-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). l-Theanine induced NO production was partially attenuated in presence of l-NAME or l-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca^2 + and Akt independent, as assessed by fluo-4AM and immunoblotting experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor, PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that l-theanine administration in vitro activated ERK/eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of l-theanine mediated vascular health benefits of tea.     

Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation

The presentstudy was performed to characterize thrombin-stimulated phospholipaseA₂(PLA₂) activity and theresultant release of lysophospholipids from endothelial cells. Themajority of PLA₂ activity inendothelial cells was membrane associated,Ca²⁺ independent, and arachidonateselective. Incubation with thrombin increased membrane-associatedPLA₂ activity using bothplasmenylcholine and alkylacyl glycerophosphocholine substrates in theabsence of Ca²⁺, with no increasein activity observed with phosphatidylcholine substrate. The increasedPLA₂ activity was accompanied byarachidonic acid and lysoplasmenylcholine (LPlasC) release fromendothelial cells into the surrounding medium. Thrombin-induced changeswere duplicated by stimulation with the thrombin-receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with theCa²⁺-independentPLA₂ inhibitor bromoenol lactoneblocked thrombin-stimulated increases inPLA₂ activity, arachidonic acid,and LPlasC release. Stimulation of protein kinase C (PKC) increasedbasal PLA₂ activity and LPlasCproduction. Thrombin-stimulatedPLA₂ activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cellsactivates a PKC-activated, membrane-associated,Ca²⁺-independentPLA₂ that selectively hydrolyzesarachidonylated, ether-linked phospholipid substrates, resulting inLPlasC and arachidonic acid release.

     

Effect of long-term piceatannol treatment on eNOS levels in cultured endothelial cells

Piceatannol (3, 3′, 4, 5′-tetrahydroxy-trans-stilbene) is a naturally occurring phytochemical found in passion fruit (Passiflora edulis) seeds. Previously, we demonstrated that piceatannol has acute vasorelaxant effects in rat thoracic aorta. It was suggested that endothelial NO synthase (eNOS) might be involved in piceatannol-induced acute vasorelaxation. Here, we investigated the expression of eNOS in EA.hy926 human umbilical vein cells after long-term treatment with piceatannol, and compared this effect with that of resveratrol, an analog of piceatannol. Long-term treatment with piceatannol up-regulated eNOS mRNA expression and increased eNOS protein expression in a dose-dependent manner. Moreover, piceatannol increased the levels of phosphorylated eNOS. Treatment with resveratrol also increased eNOS expression, but to a lesser degree than piceatannol. These findings indicate that piceatannol may improve vascular function by up-regulating eNOS expression.     

Omeprazole suppresses endothelial calcium response and eNOS Ser1177 phosphorylation in porcine aortic endothelial cells

Molecular Biology Reports - Although high doses of proton pump inhibitors can elicit an anticancer effect, this strategy may impair vascular biology. In particular, their effects on endothelial...     

Intercellular communication upon mechanical stimulation of CPAE- endothelial cells is mediated by nucleotides

Intercellular Ca(2+)-signaling, after mechanical stimulation of calf pulmonary artery endothelial cells (CPAE), was investigated with fluorescence video imaging. Mechanical stimulation evoked an intracellular Ca(2+)-response in the mechanically stimulated (MS) cell, proceeding to the neighboring (NB) cells as a Ca(2+)-wave. The intercellular propagation of the Ca(2+)-wave was unaffected by the gap junction blockers halothane or heptanol. Therefore the intercellular communication (IC) pathway of the Ca(2+)-wave in CPAE cells does not depend on gap junctional communication but is most likely mediated by release of an extracellular mediator. Continuous unilateral flow experiments confirmed the presence of a diffusible mediator: the Ca(2+)-rise in upstream NB cells is significantly lower than in control experiments. After desensitization of purinergic receptors by pretreatment of CPAE cells with ATP (100mM), UTP (100 microM), 2MeSATP (100microM) or ADPbS (100 microM), the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation was significantly inhibited. Also suramin (200 and 400 microM), a non-specific purinergic receptor blocker, reduced the IC. Application of the nucleotidase apyrase VI (10U/ml), which has a high ATPase/ADPase ratio, enhanced Ca(2+)-signaling and IC. In contrast, apyrase VII (10U/ml), which has a high ADPase/ATPase ratio, significantly depressed the propagation of the intercellular Ca(2+)-wave upon mechanical stimulation. Our experiments therefore demonstrate that the IC, evoked by a mechanical stimulus of CPAE cells, is mediated via release of nucleotides in the extracellular space. The data indicate that the diffusible messenger, responsible for the propagation of a Ca(2+)-wave, is mainly ADP or a combination of ADP/ATP.     

Desensitization of inositol phosphate production after agonist stimulation of endothelial cells is not mediated by protein kinase C

To investigate the possible role of protein kinase C activation in the desensitization of inositol phosphate production in endothelial cells we compared desensitization induced by agonists to that induced by the phorbol ester TPA. While histamine or thrombin induced desensitization of inositol phosphate production is homologous TPA induced desensitization is heterologous. The protein kinase C inhibitor H-7 reduced TPA desensitization but had no effect on the agonist induced desensitization. While downregulation of protein kinase C by long term (24 hr) treatment of the cells with TPA reduced the desensitization mediated by short term TPA-treatment it did not affect the agonist induced desensitization. These results suggest that desensitization of inositol phosphate production after agonist stimulation of endothelial cells is not mediated by protein kinase C.     

The role of caveolin-1 in PCB77-induced eNOS phosphorylation in human-derived endothelial cells

Polychlorinated biphenyls (PCBs) may contribute to the pathology of atherosclerosis by activating inflammatory responses in vascular endothelial cells. Endothelial nitric oxide synthase (eNOS) is colocalized with caveolae and is a critical regulator of vascular homeostasis. PCBs may be proatherogenic by causing dysfunctional eNOS signaling. The objective of this study was to investigate the role of caveolin-1 in PCB-induced endothelial dysfunction with a focus on mechanisms associated with eNOS signaling. Cells derived from an immortalized human vascular endothelial cell line were treated with PCB77 to study nitrotyrosine formation through eNOS signaling. Phosphorylation studies of eNOS, caveolin-1, and kinases, such as Src, phosphatidylinositol 3-kinase (PI3K), and Akt, were conducted in cells containing either functional or small-interfering RNA-silenced caveolin-1 protein. We also investigated caveolin-1-regulated mechanisms associated with PCB-induced markers of peroxynitrite formation and DNA binding of NF-kappaB. Cellular exposure to PCB77 increased eNOS phosphorylation and nitric oxide production, as well as peroxynitrite levels. A subsequent PCB-induced increase in NF-kappaB DNA binding may have implications in oxidative stress-mediated inflammatory mechanisms. The activation of eNOS by PCB77 treatment was blocked by inhibitors of the Src/PI3K/Akt pathway. PCB77 also increased phosphorylation of caveolin-1, indicating caveolae-dependent endocytosis. Caveolin-1 silencing abolished both the PCB-stimulated Akt and eNOS phosphorylation, suggesting a regulatory role of caveolae in PCB-induced eNOS signaling. These findings suggest that PCB77 induces eNOS phosphorylation in endothelial cells through a Src/PI3K/Akt-dependent mechanism, events regulated by functional caveolin-1. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs.     

Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions.

Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP --> ADP --> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.     

Vasostatin 1 activates eNOS in endothelial cells through a proteoglycan‐dependent mechanism

Accumulating evidences point to a significant role for the chromogranin A (CgA)‐derived peptide vasostatin 1 (VS‐1) in the protective modulation of the cardiovascular activity, because of its ability to counteract the adrenergic signal. We have recently shown that VS‐1 induces a PI3K‐dependent‐nitric oxide (NO) release by endothelial cells, contributing to explain the mechanism of its cardio‐suppressive and vasodilator properties. However, the cellular processes upstream the eNOS activation exerted by this peptide are still unknown, as typical high‐affinity receptors have not been identified. Here we hypothesize that in endothelial cells VS‐1 acts, on the basis of its cationic and amphipathic properties, as a cell penetrating peptide, binding to heparan sulfate proteoglycans (HSPGs) and activating eNOS phosphorylation (Ser1179) through a PI3K‐dependent, endocytosis‐coupled mechanism. In bovine aortic endothelial cells (BAE‐1 cells) endocytotic vesicles trafficking was quantified by confocal microscopy with a water‐soluble membrane dye; caveolin 1 (Cav1) shift from plasma membrane was studied by immunofluorescence staining; VS‐1‐dependent eNOS phosphorylation was assessed by immunofluorescence and immunoblot analysis. Our experiments demonstrate that VS‐1 induces a marked increase in the caveolae‐dependent endocytosis, (115 ± 23% endocytotic spots/cell/field in VS‐1‐treated cells with respect to control cells), that is significantly reduced by both heparinase III (HEP, 17 ± 15% above control) and Wortmannin (Wm, 7 ± 22% above control). Heparinase, Wortmannin, and methyl‐β‐cyclodextrin (MβCD) abolish the VS‐1‐dependent eNOS phosphorylation (P^Ser1179eNOS). These results suggest a novel signal transduction pathway for endogenous cationic and amphipathic peptides in endothelial cells: HSPGs interaction and caveolae endocytosis, coupled with a PI3K‐dependent eNOS phosphorylation. J. Cell. Biochem. 110: 70–79, 2010. © 2010 Wiley‐Liss, Inc.     

Pertussis toxin-sensitive and -insensitive thrombin stimulation of Shc phosphorylation and mitogenesis are mediated through distinct pathways

Activation of both receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) result in phosphorylation of the adaptor protein Shc, providing sites of interaction for proteins in downstream signal transduction cascades. The mechanism of Shc phosphorylation and its function in G protein signaling pathways is still unclear. By examining Shc phosphorylation in response to thrombin in two cell lines, we have defined distinct pertussis toxin (PTX)-sensitive and -insensitive mechanisms by which GPCRs can stimulate tyrosine phosphorylation of Shc. By mutating the tyrosines in Shc, we show that the three sites of tyrosine phosphorylation, Y239, Y240, and Y317, are necessary for thrombin signaling in both systems. The SH2 (src homology 2) domain of Shc is also critical for signaling, but not required for phosphorylation of Shc. In both cell types, inhibition of src family member kinases by chemical inhibitors or microinjection block Shc phosphorylation and bromodeoxyuridine (BrdU) incorporation in response to thrombin. However, in the PTX-sensitive thrombin pathway, both betagamma function and the epidermal growth factor receptor (EGFR) are necessary for Shc phosphorylation and BrdU incorporation. In contrast, signaling in the PTX-insensitive pathway is not mediated through betagamma or the EGFR. Thus, while phosphorylation and function of Shc appear to be the same in both thrombin pathways, the mechanism of tyrosine kinase activation proximal to Shc is different. The differences in signaling between the two thrombin pathways may be representative of mechanisms used by other PTX-sensitive and -insensitive GPCRs to mediate specific responses. In addition, transactivation of RTKs may be a manner by which GPCRs can amplify their signal.     

The phosphorylation state of MRLC is polyamine dependent in intestinal epithelial cells

Cell migration is important to the integrity of the gastrointestinal tract for the normal movement of cells from crypt to villi and the healing of wounds. Polyamines are essential to cell migration, mucosal restitution, and, hence, healing. Polyamine depletion by α-difluoromethyl ornithine (DFMO) inhibited migration by decreasing lamellipodia and stress fiber formation and preventing the activation of Rho-GTPases. Polyamine depletion increased the association of the thick F-actin cortex with phosphorylated myosin regulatory light chain (pMRLC). In this study, we determined why MRLC is constitutively phosphorylated as part of the actin cortex. Inhibition of myosin light chain kinase (MLCK) decreased RhoA and Rac1 activities and significantly inhibited migration. Polyamine depletion increased phosphorylation of MRLC (Thr18/Ser19) and stabilized the actin cortex and focal adhesions. The Rho-kinase inhibitor Y27632 increased spreading and migration by decreasing the phosphorylation of MRLC, remodeling focal adhesions, and by activating Rho-GTPases. Thus phosphorylation of MRLC appears to be the rate-limiting step during the migration of IEC-6 cells. In addition, increased localization of RhoA with the actin cortex in polyamine-depleted cells appears to activate Rho-kinase. In the absence of polyamines, activated Rho-kinase phosphorylates myosin phosphatase targeting subunit 1 (MYPT1) at serine-668 leading to its inactivation and preventing the recruitment of phosphatase (protein phosphastase, PP1cδ) to the actomyosin cortex. In this condition, MRLC is constitutively phosphorylated and cycling does not occur. Thus activated myosin binds F-actin stress fibers and prevents focal adhesion turnover, Rho-GTPase activation, and the remodeling of the cytoskeleton required for migration.     

NHERF2 is crucial in ERM phosphorylation in pulmonary endothelial cells

Background

EBP50 and NHERF2 adaptor proteins are incriminated in various signaling pathways of the cell. They can bind ERM proteins and mediate ERM-membrane protein interactions.

Results

Binding of ERM to EBP50 and NHERF2 was compared in pulmonary artery endothelial cells by immunoprecipitation. NHERF2 associates with all three ERM, but EBP50 appeared to be a weak binding partner if at all. Furthermore, we detected co-localization of NHERF2 and phospho-ERM at the cell membrane and in the filopodia of dividing cells. Silencing of NHERF2 prevented agonist or angiogenesis induced phosphorylation of ERM, while overexpression of the adaptor elevated the phosphorylation level of ERM, likely catalyzed by Rho kinase 2, which co-immunoprecipitated with NHERF2/ERM in control EC, but did not bind to ERM in NHERF2 depleted cells. Dependence of ERM phosphorylation on NHERF2 was also shown in Matrigel tube formation assay, and NHERF2 was proved to be important in angiogenesis as well. Furthermore, when NHERF2 was depleted or cells were overexpressing a mutant form of NHERF2 unable to bind ERM, we found attenuated cell attachment with ECIS measurements, while it was supported by overexpression of wild type NHERF2.

Conclusions

Pivotal role of NHERF2 in the phosphorylation process of ERM in pulmonary artery endothelial cells is shown. We propose that NHERF2 provides a common anchoring surface for ERM and Rho kinase 2. Our results demonstrate the essential role of NHERF2 in endothelial cell adhesion/migration and angiogenesis.

     

Developmental downregulation of Xenopus cyclin E is phosphorylation and nuclear import dependent and is mediated by ubiquitination

Cyclins are regulatory subunits that bind to and activate catalytic Cdks. Cyclin E associates with Cdk2 to mediate the G1/S transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how cyclin E is normally downregulated during development in order to determine if disruption of similar mechanisms could either contribute to its overexpression in cancer, or be exploited to decrease its expression. In Xenopus laevis embryos, cyclin E protein level is high and constant until its abrupt destabilization by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT) and remodeling of the embryonic to the adult cell cycle. Since degradation of mammalian cyclin E is regulated by the ubiquitin proteasome system and is phosphorylation dependent, we examined the role of phosphorylation in Xenopus cyclin E turnover. We show that similarly to human cyclin E, phosphorylation of serine 398 and threonine 394 plays a role in cyclin E turnover at the MBT. Immunofluorescence analysis shows that cyclin E relocalizes from the cytoplasm to the nucleus preceding its degradation. When nuclear import is inhibited, cyclin E stability is markedly increased after the MBT. To investigate whether degradation of Xenopus cyclin E is mediated by the proteasomal pathway, we used proteasome inhibitors and observed a progressive accumulation of cyclin E in the cytoplasm after the MBT. Ubiquitination of cyclin E precedes its proteasomal degradation at the MBT. These results show that cyclin E destruction at the MBT requires both phosphorylation and nuclear import, as well as proteasomal activity.     

Degradation of ornithine decarboxylase in mammalian cells is ATP dependent but ubiquitin independent

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines in mammalian cells is characterized by an extremely short half-life. In the present study, ODC degradation was investigated in 653-1 mouse myeloma cells that overproduce ODC and in ts85 cells that are thermosensitive for conjunction of ubiquitin to target proteins. Addition of 2-deoxyglucose and dinitrophenol (agents that efficiently deplete cellular ATP) to the growth medium of these cells inhibited ODC degradation. In contrast, chloroquine and leupeptin, inhibitors of intralysosomal proteolysis, did not affect ODC degradation. Shifting ts85 cells to 42 degrees C (a non-permissive temperature that inhibited conjugation of ubiquitin to target proteins) did not prevent ODC degradation. The ATP-dependent degradation of ODC in 653-1 cells was inhibited substantially by N alpha-tosyl-L-lysine chloromethane (TosPheMeCl), iodoacetamide and o-phenanthroline. These results suggest that ODC degradation occurs via a non-lysosomal. ATP-requiring and ubiquitin-independent cellular proteolytic mechanism, and that serine proteases and enzymes containing sulphydryl groups and metalloenzyme(s) may be involved in this process.