Target-cell contact activates a highly selective capacitative calcium entry pathway in cytotoxic T lymphocytes

Calcium influx is critical for T cell activation. Evidence has been presented that T cell receptor-stimulated calcium influx in helper T lymphocytes occurs via channels activated as a consequence of depletion of intracellular calcium stores, a mechanism known as capacitative Ca(2+) entry (CCE). However, two key questions have not been addressed. First, the mechanism of calcium influx in cytotoxic T cells has not been examined. While the T cell receptor-mediated early signals in helper and cytotoxic T cells are similar, the physiology of the cells is strikingly different, raising the possibility that the mechanism of calcium influx is also different. Second, contact of T cells with antigen-presenting cells or targets involves a host of intercellular interactions in addition to those between antigen-MHC and the T cell receptor. The possibility that calcium influx pathways in addition to those activated via the T cell receptor may be activated by contact with relevant cells has not been addressed. We have used imaging techniques to show that target-cell-stimulated calcium influx in CTLs occurs primarily through CCE. We investigated the permeability of the CTL influx pathway for divalent cations, and compared it to the permeability of CCE in Jurkat human leukemic T cells. CCE in CTLs shows a similar ability to discriminate between calcium, barium, and strontium as CCE in Jurkat human leukemic T lymphocytes, where CCE is likely to mediated by Ca(2+) release-activated Ca(2+) current (CRAC) channels, suggesting that CRAC channels also underlie CCE in CTLs. These results are the first determination of the mechanism of calcium influx in cytotoxic T cells and the first demonstration that cell contact-mediated calcium signals in T cells occur via depletion-activated channels.     

Interactions of mechanotransduction pathways

Integrins may serve as mechanosensors in endothelial cells (ECs): shear stress causes integrin-Shc association, assembly of the signaling complex and then leads to JNK activation. Flow also mediates selective and cell-specific alterations in vascular cell G-protein expression that correlate with changes in cell-signalling, G-protein functionality and modulate Ca2+ concentration. In this study, we explored the cross-talks between EC membrane mechanosensors, such as integrins, ion channels, and G-proteins in shear stress-induced signal transduction by their specific inhibition. Confluent monolayer of bovine aortic endothelial cells (BAECs) were incubated with or without specific inhibitors prior to shearing experiments. Our results showed an attenuation of integrin-Shc association under shear stress with RGD, and with PTX, but not with BAPTA/AM. The inhibitions of shear-activated JNK are similar for RGD and PTX. However, unlike for integrin association, the chelation of calcium reduced JNK activation. These results provide several lines of evidence of the interactions between different mechanosensors in ECs. First, integrin-Shc association required cell attachment and G-protein activity, but not intracellular calcium. Second, shear-induced JNK activation is regulated by multiple mechano-sensing mechanisms such as integrin, G-protein and calcium concentration.     

Flow-activated ion channels in vascular endothelium

The ability of vascular endothelial, cells (ECs) to respond to fluid mechanical forces associated with blood flow is essential for flow-mediated vasoregulation and arterial wall remodeling. Abnormalities in endothelial responses to flow also play a role in the development of atherosclerosis. Although our understanding of the endothelial signaling pathways stimulated by flow has greatly increased over the past two decades, the mechanisms by which ECs sense flow remain largely unknown. Activation of flow-sensitive ion channels is among the fastest known endothelial responses to flow; therefore, these ion channels have been proposed as candidate flow sensors. This review focuses on: 1) describing the various types of flow-sensitive ion channels that have been reported in ECs, 2) discussing the implications of activation of these ion channels for endothelial function, and 3) proposing candidate mechanisms for activation of flow-sensitive ion channels.     

Ion channels and transporters in cancer. 6. Vascularizing the tumor: TRP channels as molecular targets

Tumor vascularization is a critical process that determines tumor growth and metastasis. In the last decade new experimental evidence obtained from in vitro and in vivo studies have challenged the classical angiogenesis model forcing us to consider new scenarios for tumor neovascularization. In particular, the genetic stability of tumor-derived endothelial cells (TECs) has been recently questioned in several studies, which show that TECs, as well as pericytes, differ significantly from their normal counterparts at genetic and functional levels. In addition to such an epigenetic action of tumor microenvironment on endothelial cells (ECs) commitment, the distinct characteristics of TECs could be due to differences in their origin compared with preexisting differentiated ECs. Intracellular Ca(2+) signals are involved at different critical phases in the regulation of the complex process of angiogenesis and tumor progression. These signals are generated by a wide variety of intrinsic and extrinsic factors. Several key components of Ca(2+) signaling including Ca(2+) channels in the plasma membrane, endoplasmic reticulum, calcium pumps, and mitochondria contribute to the generation, amplitude, and frequency of these Ca(2+) change. In particular, several members of the transient receptor potential (TRP) family of calcium-permeable channels have profound effects on the function of ECs. Because of its multifaceted role in the control of cell function, proliferation, and motility, TRP channels have been suggested as a potential molecular target for control of tumor neovascularization. Since plasma membrane Ca(2+) channels are easily and directly accessible via the bloodstream, they are potential targets for a number of pharmacological and antibody-targeted therapeutic strategies, with specificity being the main limitation. In this review we discuss recent advances in understanding the role of Ca(2+) channels, with specific reference to TRP channels, in tumor vascularization process.     

Receptor-regulated dynamic interaction between endothelial nitric oxide synthase and calmodulin revealed by fluorescence resonance energy transfer in living cells

The endothelial isoform of nitric oxide synthase (eNOS), a key regulator of vascular tone, is activated in endothelial cells by diverse Ca(2+)-mobilizing agonists, including vascular endothelial growth factor (VEGF). Although the activation state of eNOS and the subcellular localization of the enzyme are both highly regulated, the relationship between enzyme activity and subcellular targeting remains obscure. We aim here to elucidate this relationship by direct dynamic imaging analysis of Ca(2+)/CaM-dependent eNOS activation in living endothelial cells, using high-resolution confocal microscopy and donor dequenching fluorescence resonance energy transfer (FRET) techniques. Confocal images show a complex pattern of eNOS subcellular distribution; the enzyme is concentrated in both the plasma membrane and internal membranes, with robust expression in the perinuclear region. We construct a fusion protein between eNOS and the FRET-based calcium sensor cameleon, and analyze the temporal and spatial pattern of VEGF-mediated calcium mobilization using donor dequenching FRET methods. We find that VEGF promotes rapid mobilization of intracellular calcium throughout the regions of the cell in which eNOS is distributed. We further create a series of fusion proteins and use FRET imaging methods to study the interactions between eNOS and its obligate allosteric activator protein calmodulin. We clone the FRET acceptor EYFP (enhanced yellow fluorescent protein) at the C-terminus of calmodulin, and the FRET donor ECFP (enhanced cyan fluorescent protein) into eNOS at a site adjacent to its calmodulin-binding domain. FRET imaging analysis of individual endothelial cells cotransfected with eNOS-ECFP and calmodulin-EYFP shows that VEGF induces interactions between eNOS and calmodulin wherever both are present in the cell. Our studies provide evidence that the pool of rapidly responsive receptor-activated eNOS is distributed throughout endothelial cells in both plasma membrane and internal membrane structures, and that this distribution parallels the localization of agonist-induced intracellular Ca(2+) changes in the vicinity of eNOS.     

Differential effects of intravenous anesthetics on capacitative calcium entry in human pulmonary artery smooth muscle cells

We assessed the roles of the protein kinase C (PKC) and the tyrosine kinase (TK) signaling pathways in regulating capacitative calcium entry (CCE) in human pulmonary artery smooth muscle cells (PASMCs) and investigated the effects of intravenous anesthetics (midazolam, propofol, thiopental, ketamine, etomidate, morphine, and fentanyl) on CCE in human PASMCs. Fura-2-loaded human PASMCs were placed in a dish (37 degrees C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration ([Ca2+]i) was measured as the 340/380 fluorescence ratio in individual PASMCs. Thapsigargin, a sarcoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was then activated by restoring extracellular Ca2+ (2.2 mM). The effects of PKC activation and inhibition, TK inhibition, and the intravenous anesthetics on CCE were assessed. Thapsigargin caused a transient increase in [Ca2+]i. Restoring extracellular Ca2+ caused a rapid peak increase in [Ca2+]i, followed by a sustained increase in [Ca2+]i; i.e., CCE was stimulated in human PASMCs. PKC activation attenuated (P < 0.05), whereas PKC inhibition potentiated (P < 0.05), both peak and sustained CCE. TK inhibition attenuated (P < 0.05) both peak and sustained CCE. Midazolam, propofol, and thiopental each attenuated (P < 0.05) both peak and sustained CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. Our results suggest that CCE in human PASMCs is influenced by both the TK and PKC signaling pathways. Midazolam, propofol, and thiopental each attenuated CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE.     

Simultaneous imaging of [Ca2+]i and intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Pregnancy and the follicular phase of the ovarian cycle show elevation of uterine blood flow and associated increases in uterine artery endothelium (UAE) endothelial nitric oxide (NO) synthase (eNOS) expression. Nonetheless, a role for increased NO production during pregnancy and the follicular phase has only been inferred by indirect measures. The recent development of a uterine artery endothelial cell model further suggests that pregnancy is associated with reprogramming of cell signaling, such that eNOS may become more Ca(2+) sensitive and be subject to regulation by Ca(2+)-independent kinases. This study describes for the first time the direct and simultaneous monitoring of NO production and intracellular free Ca(2+) concentration ([Ca(2+)](i)) in freshly isolated UAE from pregnant, follicular, and luteal sheep. The pharmacological agonists ionomycin (calcium ionophore) and thapsigargin (TG; endoplasmic reticulum Ca(2+) pump inhibitor) were used to maximally elevate [Ca(2+)](i) and fully activate eNOS as a measure of eNOS expression. NO production stimulated by ionomycin (5 microM) and TG (10 microM) were 1.95- and 2.05-fold, respectively, in pregnant-UAE and 1.34- and 1.37-fold in follicular-UAE compared with luteal-UAE. In contrast, the physiological agonist ATP (100 microM) stimulated a 3.43-fold increase in NO in pregnant-UAE and a 1.90-fold increase in follicular-UAE compared with luteal-UAE, suggesting that pregnancy and follicular phase enhance eNOS activation beyond changes in expression in vivo. 2-aminoethoxydiphenyl borate (APB; an inositol 1,4,5-trisphosphate receptor blocker) totally prevented the ATP-induced [Ca(2+)](i) response but only partially inhibited NO production. Thus pregnancy-enhanced eNOS activation in UAE is mediated through [Ca(2+)](i)-insensitive pathways as well as through a greater eNOS sensitivity to [Ca(2+)](i).     

Role of LOX‐1 in monocyte adhesion‐triggered redox,Akt/eNOS and Ca2+ signaling pathways in endothelial cells

This study was conducted to examine the role of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) in monocyte adhesion‐induced redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in endothelial cells (ECs). LOX‐1 was blocked by an antibody‐neutralizing LOX‐1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47^phox, and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30 min and NF‐κB phosphorylation within 1 h, resulting in redox‐sensitive gene expression. Akt and eNOS phosphorylation was induced 15 min after adding monocytes and returned to control level after 30 min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX‐1 blunted the monocyte adhesion‐triggered redox‐sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca²⁺ mobilization and Ca²⁺ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX‐1 is involved in redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in monocyte adhesion to ECs independent of oxidized low‐density lipoprotein (ox‐LDL). Furthermore, blockade of Ca²⁺ inhibited monocyte adhesion‐triggered Rac1 and p47^phox activation and ROS generation in ECs, whereas Ca²⁺ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor‐α or ox‐LDL. We provide evidence that LOX‐1 plays a role in redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in monocyte adhesion to ECs independent of the ox‐LDL–LOX‐1 axis. J. Cell. Physiol. 220: 706–715, 2009. © 2009 Wiley‐Liss, Inc.     

Role of capacitative calcium entry on glutamate-induced calcium influx in type-I rat cortical astrocytes

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross-talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL-2H3), a model where calcium release-activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL-2H3 cells and (ii) glutamate-induced Ca2+ influx in astrocytes plays a primary role in glutamate-dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL-2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.     

Effect of superoxide and lipid peroxide on cytosolic free calcium concentration in cultured pig aortic endothelial cells

The effect of reactive oxygen on cytosolic free calcium concentration [( Ca++]i) in pig aortic endothelial cells (ECs) was studied. Linoleate hydroperoxide (LHO) and superoxide radicals generated from xanthine with xanthine oxidase (X-XO) were used as sources of reactive oxygen. [Ca++]i in ECs was measured with quin 2 and the value for quiescent ECs was 112 +/- 11 nM. Both LHO and X-XO increased [Ca++]i in a dose-dependent manner without accompanying the significant cellular damage. Nifedipine suppressed the increase in [Ca++]i provoked by LHO and X-XO. Thus, the biological effects of reactive oxygen might be mediated, at least in part, by the activation of voltage-dependent calcium channels in ECs.     

Biomechanical regulation of planar cell polarity in endothelial cells

Cell polarity refers to the uneven distribution of certain cytoplasmic components in a cell with a spatial order. The planar cell polarity (PCP), the cell aligns perpendicular to the polar plane, in endothelial cells (ECs) has become a research hot spot. The planar polarity of ECs has a positive significance on the regulation of cardiovascular dysfunction, pathological angiogenesis, and ischemic stroke. The endothelial polarity is stimulated and regulated by biomechanical force. Mechanical stimuli promote endothelial polarization and make ECs produce PCP to maintain the normal physiological and biochemical functions. Here, we overview recent advances in understanding the interplay and mechanism between PCP and ECs function involved in mechanical forces, with a focus on PCP signaling pathways and organelles in regulating the polarity of ECs. And then showed the related diseases caused by ECs polarity dysfunction. This study provides new ideas and therapeutic targets for the treatment of endothelial PCP-related diseases.     

Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 microM) and nifedipine (5 microM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 microM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 microM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.     

Identifying the Ca(++) signalling sources activating chloride currents in Xenopus oocytes using ionomycin and thapsigargin

The calcium ionophore, ionomycin (IM), and the sarcoplasmic/endoplasmic reticulum (SER) calcium pump inhibitor, thapsigargin (TG), were used to study the roles of Ca(++) from different sources in regulating Ca(++)-dependent Cl(-) currents in Xenopus oocytes. The Ca(++)-dependent Cl(-) currents, Ic, were measured in voltage-clamped oocytes (Vc = -60 mV). In the presence of extracellular Ca(++), both TG (0.1 to 10 microM) and IM (0.1 to 10 microM) induce release of Ca(++) from SER and activated capacitative Ca(++) entry (CCE) across the plasma membrane leading to activation of both "fast" and "slow" Cl(-) currents. The fast Ic was produced by Ca(++) release from SER while Ca(++) entry across the plasma membrane activated the slow Ic. Intracellular application of the calcium buffer, BAPTA, blocked activation of the slow Ic due to Ca(++) entry via CCE pathways, but not via IM-mediated movement across the plasma membrane. It is concluded that predominantly Ca(++) release from stores regulates a fast Ic while Ca(++) entry through CCE pathways regulates a slow Ic. Further, the CCE and slow Ic pathways must be located in spatially separated compartments since BAPTA can effectively abolish the effects of Ca(++) entry via the CCE pathway, but not by the IM-mediated entry pathway.     

Calcium signal transduction from caveolae

Caveolae are specialized membrane microdomains that are found on the plasma membrane of most cells. Recent studies indicate that a variety of signaling molecules are highly organized in caveolae, where their interactions initiate specific signaling cascades. Molecules enriched in this membrane include G protein-coupled receptors, heterotrimeric GTP binding proteins, IP3 receptor-like protein, Ca2+ ATPase, eNOS, and several PKC isoforms. Direct measurements of calcium changes in endothelial cells suggest that caveolae may be sites that regulate intracellular Ca2+ concentration and Ca2+ dependent signal transduction. This review will focus on the role of caveolae in controlling the spatial and temporal pattern of intracellular Ca2+ signaling.     

The molecular interaction of a copper chelate with human P-glycoprotein

We previously reported that the vasoactive peptide 1 (P1, "SSWRRKRKESS") modulates the tension of pulmonary artery vessels through caveolar endothelial nitric oxide synthase (eNOS) activation in intact lung endothelial cells (ECs). Since PKC-α is a caveolae resident protein and caveolae play a critical role in the peptide internalization process, we determined whether modulation of caveolae and/or caveolar PKC-α phosphorylation regulates internalization of P1 in lung ECs. Cell monolayers were incubated in culture medium containing Rhodamine red-labeled P1 (100 μM) for 0-120 min. Confocal examinations indicate that P1 internalization is time-dependent and reaches a plateau at 60 min. Caveolae disruption by methyl-β-cyclodextrin (CD) and filipin (FIL) inhibited the internalization of P1 in ECs suggesting that P1 internalizes via caveolae. P1-stimulation also enhances phosphorylation of caveolar PKC-α and increases intracellular calcium (Ca(2+)) release in intact cells suggesting that P1 internalization is regulated by PKC-α in ECs. To confirm the roles of increased phosphorylation of PKC-α and Ca(2+) release in internalization of P1, PKC-α modulation by phorbol ester (PMA), PKC-α knockdown, and Ca(2+) scavenger BAPTA-AM model systems were used. PMA-stimulated phosphorylation of caveolar PKC-α is associated with significant reduction in P1 internalization. In contrast, PKC-α deficiency and reduced phosphorylation of PKC-α enhanced P1 internalization. P1-mediated increased phosphorylation of PKC-α appears to be associated with increased intracellular calcium (Ca(2+)) release since the Ca(2+) scavenger BAPTA-AM enhanced P1 internalization. These data indicate that caveolar integrity and P1-mediated increased phosphorylation of caveolar PKC-α play crucial roles in the regulation of P1 internalization in lung ECs.     

Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S1P activation of eNOS is approximately 10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(1179) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.     

Update on vascular endothelial Ca(2+) signalling: A tale of ion channels, pumps and transporters

A monolayer of endothelial cells (ECs) lines the lumen of blood vessels and forms a multifunctional transducing organ that mediates a plethora of cardiovascular processes. The activation of ECs from as state of quiescence is, therefore, regarded among the early events leading to the onset and progression of potentially lethal diseases, such as hypertension, myocardial infarction, brain stroke, and tumor. Intracellular Ca2+ signals have long been know to play a central role in the complex network of signaling pathways regulating the endothelial functions. Notably, recent work has outlined how any change in the pattern of expression of endothelial channels, transporters and pumps involved in the modulation of intracellular Ca2+ levels may dramatically affect whole body homeostasis. Vascular ECs may react to both mechanical and chemical stimuli by generating a variety of intracellular Ca2+ signals, ranging from brief, localized Ca2+ pulses to prolonged Ca2+ oscillations engulfing the whole cytoplasm. The well-defined spatiotemporal profile of the subcellular Ca2+ signals elicited in ECs by specific extracellular inputs depends on the interaction between Ca2+ releasing channels, which arelocated both on the plasma membrane and in a number of intracellular organelles, and Ca2+ removing systems. The present article aims to summarize both the past and recent literature in the field to provide a clear-cut picture of our current knowledge on the molecular nature and the role played by the components of the Ca2+ machinery in vascular ECs under both physiological and pathological conditions.     

Decorin derived antiangiogenic peptide LRR5 inhibits endothelial cell migration by interfering with VEGF-stimulated NO release

Excessive angiogenesis plays critical roles in many human diseases including cancer. We have previously shown that human decorin derived 26 amino acids peptide Leucine Rich Repeat 5 (LRR5) inhibits multiple aspects of angiogenesis including vascular endothelial growth factor (VEGF) stimulated migration of endothelial cells (ECs). In this study, we have characterized the molecular mechanism of LRR5 which reveals that its anti-migratory effect on ECs is mediated by inhibiting VEGF-stimulated endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) release. LRR5 carried out this function through signaling pathways that involves PI3 kinase and Akt, but not ERK. This anti-NO release effect is mediated by the C-terminal 13 amino acids of LRR5, correlating with the anti-migratory function of this region.     

Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt

Endothelin-1 has dual vasoactive effects, mediating vasoconstriction via ETA receptor activation of vascular smooth muscle cells and vasorelaxation via ETB receptor activation of endothelial cells. Although it is commonly accepted that endothelin-1 binding to endothelial cell ETB receptors stimulates nitric oxide (NO) synthesis and subsequent smooth muscle relaxation, the signaling pathways downstream of ETB receptor activation are unknown. Here, using a model in which we have utilized isolated primary endothelial cells, we demonstrate that ET-1 binding to sinusoidal endothelial cell ETB receptors led to increased protein kinase B/Akt phosphorylation, endothelial cell nitric-oxide synthase (eNOS) phosphorylation, and NO synthesis. Furthermore, eNOS activation was not dependent on tyrosine phosphorylation, and pretreatment of endothelial cells with pertussis toxin as well as overexpression of a dominant negative G-protein-coupled receptor kinase construct that sequesters betagamma subunits inhibited Akt phosphorylation and NO synthesis. Taken together, the data elucidate a G-protein-coupled receptor signaling pathway for ETB receptor-mediated NO production and call attention to the absolute requirement for heterotrimeric G-protein betagamma subunits in this cascade.