Modulation of the L-arginine/nitric oxide signalling pathway in vascular endothelial cells

Nitric oxide (NO) is synthesized from L-arginine, and in endothelial cells influx of L-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes L-arginine to NO and L-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of L-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of L-arginine despite having sufficient intracellular L-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17beta-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on L-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the L-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.     

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A₂ mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44^mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH₄) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O₂ ^•–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O₂ ^•– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH₄ level. Insulin and tempol blocked the high D-glucose–increased p42/44^mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O₂ ^•–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.     

Cytoskeletal regulation of the L-arginine/NO pathway in pulmonary artery endothelial cells

We investigated possible involvement of the actin cytoskeleton in the regulation of the L-arginine/nitric oxide (NO) pathway in pulmonary artery endothelial cells (PAEC). We exposed cultured PAEC to swinholide A (Swinh), which severs actin microfilaments, or jasplakinolide (Jasp), which stabilizes actin filaments and promotes actin polymerization, or both. After treatment, the state of the actin cytoskeleton, L-arginine uptake mediated by the cationic amino acid transporter-1 (CAT-1), Ca(2+)/calmodulin-dependent (endothelial) NO synthase (eNOS) activity and content, and NO production were examined. Jasp (50-100 nM, 2 h treatment) induced a reversible activation of L-[(3)H]arginine uptake by PAEC, whereas Swinh (10-50 nM) decreased L-[(3)H]arginine uptake. The two drugs could abrogate the effect of each other on L-[(3)H]arginine uptake. The effects of both drugs on L-[(3)H]arginine transport were not related to changes in expression of CAT-1 transporters. Swinh (50 nM, 2 h) and Jasp (100 nM, 2 h) did not change eNOS activities and contents in PAEC. Detection of NO in PAEC by the fluorescent probe 4,5-diaminofluorescein diacetate showed that Swinh (50 nM) decreased and Jasp (100 nM) increased NO production by PAEC. The stimulatory effect of Jasp on NO production was dependent on the availability of extracellular L-arginine. Our results indicate that the state of actin microfilaments in PAEC regulates L-arginine transport and that this regulation can affect NO production by PAEC.     

Ethanol inhibits L-arginine uptake and enhances NO formation in human placenta.

The acute effects of ethanol (20-60 mM) on L-arginine uptake and nitric oxide (NO) formation was investigated in human placental cotyledons perfused at constant flow. Ethanol (40 mM) decreased L-[3H]arginine uptake from 27.6 +/- 2.3 to 15.8 +/- 1.3 per cent (P < 0.05) of the injected dose and significantly enhanced NO levels in the perfusate from 0.88 +/- 0.11 to 2.80 +/- 0.39 microM. Ethanol also elicited the constriction of placental vessels. The effects of ethanol (20-60 mM) on L-arginine uptake and endothelial NO synthase (eNOS) activity were also investigated in cultured human umbilical vein endothelial cells (HUVEC). After 60 min of ethanol (40 mM) exposure, basal L-[3H]arginine uptake (4.7 +/- 0.3 pmol/microg protein/min) was inhibited by 60 per cent (P < 0.05). Basal eNOS activity in HUVEC determined under "no flow" (static) conditions was significantly increased (approximately 1.8 fold) by 60 mM ethanol. These data are consistent with a stimulatory effect of ethanol on eNOS activity in both basal and flow-stimulated conditions, which may serve a protective role against its vasoconstrictive acute effect. While acute ethanol administration inhibits L-arginine uptake, the present results do not allow us to speculate on the effects of chronic ethanol exposure on NO formation in the fetoplacental unity.     

Insulin restores glucose inhibition of adenosine transport by increasing the expression and activity of the equilibrative nucleoside transporter 2 in human umbilical vein endothelium

L-Arginine transport and nitric oxide (NO) synthesis (L-arginine/NO pathway) are stimulated by insulin, adenosine or elevated extracellular D-glucose in human umbilical vein endothelial cells (HUVEC). Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) has been proposed as a mechanism regulating adenosine plasma concentration, and therefore its vascular effects in human umbilical veins. Thus, altered expression and/or activity of hENT1 or hENT2 could lead to abnormal physiological plasma adenosine level. We have characterized insulin effect on adenosine transport in HUVEC cultured in normal (5 mM) or high (25 mM) D-glucose. Insulin (1 nM) increased overall adenosine transport associated with higher hENT2-, but lower hENT1-mediated transport in normal D-glucose. Insulin increased hENT2 protein abundance in normal or high D-glucose, but reduced hENT1 protein abundance in normal D-glucose. Insulin did not alter the reduced hENT1 protein abundance, but blocked the reduced hENT1 and hENT2 mRNA expression induced by high D-glucose. Insulin effect on hENT1 mRNA expression in normal D-glucose was blocked by N(G)-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and mimicked by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor). L-NAME did not block insulin effect on hENT2 expression. In conclusion, insulin stimulation of overall adenosine transport results from increased hENT2 expression and activity via a NO-independent mechanism. These findings could be important in hyperglycemia-associated pathological pregnancies, such as gestational diabetes, where plasma adenosine removal by the endothelium is reduced, a condition that could alter the blood flow from the placenta to the fetus affecting fetus growth and development.     

Epidermal growth factor up-regulates transforming growth factor-beta receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway

TGF-beta receptors (TbetaRs) are serine/threonine kinase receptors that bind to TGF-beta and propagate intracellular signaling through Smad proteins. TbetaRs are repressed in some human cancers and expressed at high levels in several fibrotic diseases. We demonstrated that epidermal growth factor (EGF) up-regulates type II TGF-beta receptor (TbetaRII) expression in human dermal fibroblasts. EGF-mediated induction of TbetaRII expression was inhibited by the treatment of fibroblasts with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, whereas MEK inhibitor PD98059 did not block the up-regulation of TbetaRII by EGF. EGF induced the TbetaRII promoter activity, and this induction was significantly blocked by SB203580, but not by PD98059. The overexpression of the dominant negative form of p38alpha or p38beta significantly reduced the induction of TbetaRII promoter activity by EGF. These results indicate that the EGF-mediated induction of TbetaRII expression involves the p38 MAPK signaling pathway. The EGF-mediated induction of TbetaRII expression may participate in a synergistic interplay between EGF and TGF-beta signaling pathway.     

Albumin endocytosis in endothelial cells induces TGF-beta receptor II signaling

Vascular endothelial cells undergo albumin endocytosis using a set of albumin binding proteins. This process is important for maintaining cellular homeostasis. We showed by several criteria that the previously described 73-kDa endothelial cell surface albumin binding protein is the 75-kDa transforming growth factor (TGF)-beta receptor type II (TbetaRII). Albumin coimmunoprecipitated with TbetaRII from a membrane fraction from rat lung microvascular endothelial cells. Albumin endocytosis-negative COS-7 cells became albumin endocytosis competent when transfected with wild-type TbetaRII but not when transfected with a domain-negative kinase mutant of TbetaRII. An antibody specific for TbetaRII inhibited albumin endocytosis. A mink lung epithelial cell line, which expresses both the TGF-beta receptor type I (TbetaRI) and the TbetaRII receptor, exhibited albumin binding to the cell surface and endocytosis. In contrast, mutant L-17 and DR-26 cells lacking TbetaRI or TbetaRII, respectively, each showed a dramatic reduction in binding and endocytosis. Albumin endocytosis induced Smad2 phosphorylation and Smad4 translocation as well as increased protein expression of the inhibitory Smad, Smad7. We identified regions of significant homology between amino acid sequences of albumin and TGF-beta, suggesting a structural basis for the interaction of albumin with the TGF-beta receptors and subsequent activation of TbetaRII signaling. The observed albumin-induced internalization of TbetaRII signaling may be an important mechanism in the vessel wall for controlling TGF-beta responses in endothelial cells.     

Leptin-induced endothelial dysfunction in obesity

Hyperleptinemia accompanying obesity affects endothelial nitric oxide (NO) and is a serious factor for vascular disorders. NO, superoxide (O(2)(-)), and peroxynitrite (ONOO(-)) nanosensors were placed near the surface (5+/-2 microm) of a single human umbilical vein endothelial cell (HUVEC) exposed to leptin or aortic endothelium of obese C57BL/6J mice, and concentrations of calcium ionophore (CaI)-stimulated NO, O(2)(-), ONOO(-) were recorded. Endothelial NO synthase (eNOS) expression and L-arginine concentrations in HUVEC and aortic endothelium were measured. Leptin did not directly stimulate NO, O(2)(-), or ONOO(-) release from HUVEC. However, a 12-h exposure of HUVEC to leptin increased eNOS expression and CaI-stimulated NO (625+/-30 vs. 500+/-24 nmol/l control) and dramatically increased cytotoxic O(2)(-) and ONOO(-) levels. The [NO]-to-[ONOO(-)] ratio ([NO]/[ONOO(-)]) decreased from 2.0+/-0.1 in normal to 1.30+/-0.1 in leptin-induced dysfunctional endothelium. In obese mice, a 2.5-fold increase in leptin concentration coincided with 100% increase in eNOS and about 30% decrease in intracellular L-arginine. The increased eNOS expression and a reduced l-arginine content led to eNOS uncoupling, a reduction in bioavailable NO (250+/-10 vs. 420+/-12 nmol/l control), and an elevated concentration of O(2)(-) (240%) and ONOO(-) (70%). L-Arginine and sepiapterin supplementation reversed eNOS uncoupling and partially restored [NO]/[ONOO(-)] balance in obese mice. In obesity, leptin increases eNOS expression and decreases intracellular l-arginine, resulting in eNOS an uncoupling and depletion of endothelial NO and an increase of cytotoxic ONOO(-). Hyperleptinemia triggers an endothelial NO/ONOO(-) imbalance characteristic of dysfunctional endothelium observed in other vascular disorders, i.e., atherosclerosis and diabetes.     

Antagonistic effects of TNF-alpha on TGF-beta signaling through down-regulation of TGF-beta receptor type II in human dermal fibroblasts

Transforming growth factor-beta stimulates the production of the extracellular matrix, whereas TNF-alpha has antifibrotic activity. Understanding the molecular mechanism underlying the antagonistic activities of TNF-alpha against TGF-beta is critical in the context of tissue repair and maintenance of tissue homeostasis. In the present study, we demonstrated a novel mechanism by which TNF-alpha blocks TGF-beta-induced gene and signaling pathways in human dermal fibroblasts. We showed that TNF-alpha prevents TGF-beta-induced gene trans activation, such as alpha2(I) collagen or tissue inhibitor of metalloproteinases 1, and TGF-beta signaling pathways, such as Smad3, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, without inducing levels of inhibitory Smad7 in human dermal fibroblasts. TNF-alpha down-regulates the expression of type II TGF-beta receptor (TbetaRII) proteins, but not type I TGF-beta receptor (TbetaRI), in human dermal fibroblasts. However, neither TbetaRII mRNA nor TbetaRII promoter activity was decreased by TNF-alpha. TNF-alpha-mediated decrease of TbetaRII protein expression was not inhibited by the treatment of fibroblasts with either a selective inhibitor of I-kappaB-alpha phosphorylation, BAY 11-7082, or a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Calpain inhibitor I (ALLN), a protease inhibitor, inhibits TNF-alpha-mediated down-regulation of TbetaRII. We found that TNF-alpha triggered down-regulation of TbetaRII, leading to desensitization of human dermal fibroblasts toward TGF-beta. Furthermore, these events seemed to cause a dramatic down-regulation of alpha2(I) collagen and tissue inhibitor of metalloproteinases 1 in systemic sclerosis fibroblasts. These results indicated that TNF-alpha impaired the response of the cells to TGF-beta by regulating the turnover of TbetaRII.     

Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts

Mitogen-activated protein kinases (p42mapk and p44mapk) are serine/threonine kinases that are activated rapidly in cells stimulated with various extracellular signals. This activation is mediated via MAP kinase kinase (p45mapkk), a dual specificity kinase which phosphorylates two key regulatory threonine and tyrosine residues of MAP kinases. We reported previously that the persistent phase of MAP kinase activation is essential for mitogenically stimulated cells to pass the "restriction point" of the cell cycle. Here, using specific polyclonal antibodies and transfection of epitope-tagged recombinant MAP kinases we demonstrate that these signaling protein kinases undergo distinct spatio-temporal localization in growth factor-stimulated cells. In G0-arrested hamster fibroblasts the activator p45mapkk and MAP kinases (p42mapk, p44mapk) are mainly cytoplasmic. Subsequent to mitogenic stimulation by serum or alpha-thrombin both MAP kinase isoforms translocate into the nucleus. This translocation is rapid (seen in 15 min), persistent (at least during the entire G1 period up to 6 h), reversible (by removal of the mitogenic stimulus) and apparently 'coupled' to the mitogenic potential; it does not occur in response to nonmitogenic agents such as alpha-thrombin-receptor synthetic peptides and phorbol esters that fail to activate MAP kinases persistently. When p42mapk and p44mapk are expressed stably at high levels, they are found in the nucleus of resting cells; this nuclear localization is also apparent with kinase-deficient mutants (p44mapk T192A or Y194F). In marked contrast the p45mapkk activator remains cytoplasmic even during prolonged growth factor stimulation and even after high expression levels achieved by transfection. We propose that the rapid and persistent nuclear transfer of p42mapk and p44mapk during the entire G0-G1 period is crucial for the function of these kinases in mediating the growth response.     

Gestational diabetes reduces adenosine transport in human placental microvascular endothelium, an effect reversed by insulin

Gestational diabetes mellitus (GDM) courses with increased fetal plasma adenosine concentration and reduced adenosine transport in placental macrovascular endothelium. Since insulin modulates human equilibrative nucleoside transporters (hENTs) expression/activity, we hypothesize that GDM will alter hENT2-mediated transport in human placental microvascular endothelium (hPMEC), and that insulin will restore GDM to a normal phenotype involving insulin receptors A (IR-A) and B (IR-B). GDM effect on hENTs expression and transport activity, and IR-A/IR-B expression and associated cell signalling cascades (p42/44 mitogen-activated protein kinases (p42/44(mapk)) and Akt) role in hPMEC primary cultures was assayed. GDM associates with elevated umbilical whole and vein, but not arteries blood adenosine, and reduced hENTs adenosine transport and expression. IR-A/IR-B mRNA expression and p42/44(mapk)/Akt ratios ('metabolic phenotype') were lower in GDM. Insulin reversed GDM-reduced hENT2 expression/activity, IR-A/IR-B mRNA expression and p42/44(mapk)/Akt ratios to normal pregnancies ('mitogenic phenotype'). It is suggested that insulin effects required IR-A and IR-B expression leading to differential modulation of signalling pathways restoring GDM-metabolic to a normal-mitogenic like phenotype. Insulin could be acting as protecting factor for placental microvascular endothelial dysfunction in GDM.     

Insulin rapidly stimulates L-arginine transport in human aortic endothelial cells via Akt

Insulin stimulates endothelial NO synthesis, at least in part mediated by phosphorylation and activation of endothelial NO synthase at Ser1177 and Ser615 by Akt. We have previously demonstrated that insulin-stimulated NO synthesis is inhibited under high culture glucose conditions, without altering Ca(2+)-stimulated NO synthesis or insulin-stimulated phosphorylation of eNOS. This indicates that stimulation of endothelial NO synthase phosphorylation may be required, yet not sufficient, for insulin-stimulated nitric oxide synthesis. In the current study we investigated the role of supply of the eNOS substrate, L-arginine as a candidate parallel mechanism underlying insulin-stimulated NO synthesis in cultured human aortic endothelial cells. Insulin rapidly stimulated L-arginine transport, an effect abrogated by incubation with inhibitors of phosphatidylinositol-3'-kinase or infection with adenoviruses expressing a dominant negative mutant Akt. Furthermore, supplementation of endothelial cells with extracellular L-arginine enhanced insulin-stimulated NO synthesis, an effect reversed by co-incubation with the L-arginine transport inhibitor, L-lysine. Basal L-arginine transport was significantly increased under high glucose culture conditions, yet insulin-stimulated L-arginine transport remained unaltered. The increase in L-arginine transport elicited by high glucose was independent of the expression of the cationic amino acid transporters, hCAT1 and hCAT2 and not associated with any changes in the activity of ERK1/2, Akt or protein kinase C (PKC). We propose that rapid stimulation of L-arginine transport contributes to insulin-stimulated NO synthesis in human endothelial cells, yet attenuation of this is unlikely to underlie the inhibition of insulin-stimulated NO synthesis under high glucose conditions.     

Human umbilical vein endothelium-derived exosomes play a role in foetoplacental endothelial dysfunction in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) characterizes by foetoplacental endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) from women with GDM show increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1). Moreover, expression of endothelial nitric oxide synthase (eNOS) and nitric oxide synthesis are increased. Exosomes are increased in maternal plasma from GDM. We evaluated the role of foetoplacental endothelial exosomes on endothelial dysfunction in GDM. Exosomes were isolated from HUVECs from normal (Ex_N) and GDM (Ex_GDM) pregnancies. HUVECs were exposed (8 h) to Ex_N or Ex_GDM and used for wound recovery assay (up to 8 h), L-arginine transport, hCAT-1 and eNOS expression and activity, reactive oxygen species (ROS) generation, and 44 and 42 kDa mitogen activated protein kinases (p44/42^mapk) and protein kinase B/Akt (Akt) activation. Wound recovery was slower in GDM compared with normal pregnancies and was recovered by Ex_N. However, Ex_GDM delayed wound recovery in cells from normal pregnancies. GDM-increased L-arginine transport, hCAT-1 and eNOS expression and activity, and p44/42^mapk activation were blocked by Ex_N, but Ex_GDM increased these parameters and ROS generation, and reduced eNOS phosphorylation at threonine⁴⁹⁵ in cells from normal pregnancies. Inhibition of p44/42^mapk, but not Akt reversed GDM-increased L-arginine uptake. In conclusion foetoplacental endothelial-released exosomes play a role in the maintenance of a GDM phenotype in HUVECs. It is suggested that Ex_N and Ex_GDM cargo are different with differential effects in cells from normal or GDM pregnancies. This phenomenon could contribute to the understanding of mechanisms behind foetoplacental endothelial dysfunction in GDM pregnancies.     

Transforming growth factor-beta induces formation of a dithiothreitol-resistant type I/Type II receptor complex in live cells

Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, the type I (TbetaRI) and type II (TbetaRII) receptors. We have used different and complementary techniques to study the physical nature and ligand dependence of the complex formed by TbetaRI and TbetaRII. Velocity centrifugation of endogenous receptors suggests that ligand-bound TbetaRI and TbetaRII form a heteromeric complex that is most likely a heterotetramer. Antibody-mediated immunofluorescence co-patching of epitope-tagged receptors provides the first evidence in live cells that TbetaRI. TbetaRII complex formation occurs at a low but measurable degree in the absence of ligand, increasing significantly after TGF-beta binding. In addition, we demonstrate that pretreatment of cells with dithiothreitol, which inhibits the binding of TGF-beta to TbetaRI, does not prevent formation of the TbetaRI.TbetaRII complex, but increases its sensitivity to detergent and prevents TGF-beta-activated TbetaRI from phosphorylating Smad3 in vitro. This indicates that either a specific conformation of the TbetaRI. TbetaRII complex, disrupted by dithiothreitol, or direct binding of TGF-beta to TbetaRI is required for signaling.     

Stable isotope dilution assay for liquid chromatography-tandem mass spectrometric determination of L-homoarginine in human plasma

Nitric oxide (NO), the endogenous modulator of vascular tone and structure, originates from oxidation of L-arginine catalysed by NO synthase (NOS). The L-arginine derivative L-homoarginine serves as an alternative NOS substrate releasing NO, competing with L-arginine for NOS, arginase, and arginine transport. In the present article we report a liquid chromatography-tandem mass spectrometric (LC-tandem MS) method for the determination of L-homoarginine in human plasma by stable-isotope dilution. L-[(13)C(6)]-Homoarginine was used as internal standard. This method provides high sample throughput of 25-μl aliquots of plasma with an analysis time of 4 min using LC-tandem MS electrospray ionisation in the positive mode (ESI+). Specific transitions for L-homoarginine and L-[(13)C(6)]-homoarginine were m/z 245 → m/z 211 and m/z 251 → m/z 217, respectively. The mean intra- and interassay CVs were 7.4 ± 4.5% (±SD) for 0.1-50 μmol/L and 7.5 ± 2.0% for 2 and 5 μmol/L, respectively. Applying this method, a mean plasma concentration of L-homoarginine of 2.5 ± 1.0 μmol/L was determined in 136 healthy humans.     

Cyclic AMP-independent involvement of Rap1/B-Raf in the angiotensin II AT2 receptor signaling pathway in NG108-15 cells

The angiotensin II (Ang II) type 2 (AT(2)) receptor is an atypical seven-transmembrane domain receptor. Controversy surrounding this receptor concerns both the nature of the second messengers produced as well as its associated signaling mechanisms. Using the neuronal cell line NG108-15, we have reported previously that activation of the AT(2) receptor induced morphological differentiation in a p21(ras)-independent, but p42/p44(mapk)-dependent mechanism. The activation of p42/p44(mapk) was delayed, sustained, and had been shown to be essential for neurite elongation. In the present report, we demonstrate that activation of the AT(2) receptor rapidly, but transiently, activated the Rap1/B-Raf complex of signaling proteins. In RapN17- and Rap1GAP-transfected cells, the effects induced by Ang II were abolished, demonstrating that activation of these proteins was responsible for the observed p42/p44(mapk) phosphorylation and for morphological differentiation. To assess whether cAMP was involved in the activation of Rap1/B-Raf and neuronal differentiation induced by Ang II, NG108-15 cells were treated with stimulators or inhibitors of the cAMP pathway. We found that dibutyryl cAMP and forskolin did not stimulate Rap1 or p42/p44(mapk) activity. Furthermore, adding H-89, an inhibitor of protein kinase A, or Rp-8-Br-cAMP-S, an inactive cAMP analog, failed to impair p42/p44(mapk) activity and neurite outgrowth induced by Ang II. The present observations clearly indicate that cAMP, a well known stimulus of neuronal differentiation, did not participate in the AT(2) receptor signaling pathways in the NG108-15 cells. Therefore, the AT(2) receptor of Ang II activates the signaling modules of Rap1/B-Raf and p42/p44(mapk) via a cAMP-independent pathway to induce morphological differentiation of NG108-15 cells.