Nitric Oxide Synthase-dependent Pathway Research Articles

Impact of prenatal and postnatal exposure to the pesticide chlorpyrifos on the contraction of rat ileal muscle strips: involvement of an inducible nitric oxide synthase-dependent pathway.

Prenatal/postnatal exposure to insecticides has been linked to developmental disorders in adulthood. Chlorpyrifos (CPF) is a widely used organophosphorus acetylcholinesterase (AChE)-inhibiting insecticide. The present study established whether prenatal and postnatal exposure to CPF is associated with intestinal motor dysfunction in adult rats. Three groups of pregnant rats were exposed to either CPF (1 or 5mg/kg/day; the CPF1 and CPF5 groups) or vehicle (the control group) by gavage from gestational day 1 until weaning. At weaning, the pups were separated from their dams and individually gavaged (with the same dose) until postnatal day 60. We then measured in vivo intestinal transit and the in vitro contractile responses of ileal smooth muscle strips to electrical field stimulation. Expression of inducible nitric oxide synthase (iNOS) in the ileum was determined using qRT-PCR and immunoblots. Morphometry and AChE assays were also performed. At adulthood, the mean body mass was lower in the CPF1 and CPF5 groups than in controls. CPF5 exposure was associated with weaker in vitro contraction of ileal muscle strips, which was reversed by adding the NOS inhibitor (L-NAME). There was no significant intergroup difference in the mean in vivo transit time. Exposure to CPF was associated with greater iNOS expression, lower AChE activity and reduced circular and longitudinal smooth muscle thickness. Prenatal and postnatal exposure to CPF in the rat is associated with weaker contraction of ileal longitudinal smooth muscle via a nitrergic mechanism with increased iNOS expression.

Enhancement of insulin-mediated rat muscle glucose uptake and microvascular perfusion by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside.

BackgroundInsulin-induced microvascular recruitment is important for optimal muscle glucose uptake. 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR, an activator of AMP-activated protein kinase), can also induce microvascular recruitment, at doses that do not acutely activate glucose transport in rat muscle. Whether low doses of AICAR can augment physiologic insulin action is unknown. In the present study we used the euglycemic hyperinsulinemic clamp to assess whether insulin action is augmented by low dose AICAR.MethodsAnesthetized rats were studied during saline infusion or euglycemic insulin (3 mU/kg/min) clamp for 2 h in the absence or presence of AICAR for the last hour (5 mg bolus followed by 3.75 mg/kg/min). Muscle glucose uptake (R’g) was determined radioisotopically with 14C-2-deoxyglucose and muscle microvascular perfusion by contrast-enhanced ultrasound with microbubbles.ResultsAICAR did not affect blood glucose, or lower leg R’g, although it significantly (p < 0.05) increased blood lactate levels and augmented muscle microvascular blood volume via a nitric oxide synthase dependent pathway. Insulin increased femoral blood flow, whole body glucose infusion rate (GIR), R’g, hindleg glucose uptake, and microvascular blood volume. Addition of AICAR during insulin infusion increased lactate production, further increased R’g in Type IIA (fast twitch oxidative) and IIB (fast twitch glycolytic) fiber containing muscles, and hindleg glucose uptake, but decreased R’g in the Type I (slow twitch oxidative) fiber muscle. AICAR also decreased GIR due to inhibition of insulin-mediated suppression of hepatic glucose output. AICAR augmented insulin-mediated microvascular perfusion.ConclusionsAICAR, at levels that have no direct effect on muscle glucose uptake, augments insulin-mediated microvascular blood flow and glucose uptake in white fiber type muscles. Agents targeted to endothelial AMPK activation are promising insulin sensitizers, however, the decrease in GIR and the propensity to increase blood lactate cautions against AICAR as an acute insulin sensitizer.

Apelin inhibits the activation of the nucleotide-binding domain and the leucine-rich, repeat-containing family, pyrin-containing 3 (NLRP3) inflammasome and ameliorates insulin resistance in severely burned rats

Hyperglycemia with insulin resistance remains a challenging problem in severely burned patients. Recent studies indicated the involvement of the nucleotide-binding domain and the leucine-rich, repeat-containing family, pyrin-containing 3 (NLRP3) inflammasome in insulin resistance and a beneficial role of apelin in insulin resistance. Our aim was to investigate whether apelin inhibits the activation of the NLRP3 inflammasome and ameliorates insulin resistance in severely burned rats. Male Wistar rats were subjected to a full-thickness burn injury comprising 40% of the total body surface area and were randomized to receive apelin, N(G)-methyl-L-arginine acetate salt (L-NMMA), and apelin plus treatments with L-NMMA. The following outcome measurements were assessed: apelin/APJ mRNA expression in white adipose tissue (WAT) and muscles, plasma apelin level, and activation of the NLRP3 inflammasome in WAT, Interleukin-1 β, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 levels in plasma, insulin resistance, survival rates, and endothelial nitric oxide synthase phosphorylation in soleus muscles. Severe burn induced a decreased expression of apelin/APJ mRNA in soleus muscles and a decrease in plasma apelin levels. Burn injury with apelin treatment restored plasma apelin level, inhibited NLRP3 inflammasome activity in WAT, and decreased inflammatory cytokine levels in plasma. Rats treated with apelin also showed improved insulin sensitivity and decreased mortality, accompanied by a remarkable induction of endothelial nitric oxide synthase phosphorylation in soleus muscle. Furthermore, the aforementioned effects of apelin were inhibited in part by treatment with L-NMMA. Apelin inhibits the activation of NLRP3 inflammasome, attenuates systemic inflammatory response, ameliorates insulin resistance, and promotes survival after severe burn, in part through an endothelial nitric oxide synthase-dependent pathway.

Oxidative stress induces claudin-2 nitration in experimental type 1 diabetic nephropathy

Renal complications in diabetes are severe and may lead to renal insufficiency. Early alterations in tight junction (TJ) proteins in diabetic nephropathy (DN) have not been explored and the role of oxidative stress in their disassembly has been poorly characterized. We investigated the expression and distribution of TJ proteins: claudin-5 in glomeruli (GL), occludin and claudin-2 in proximal tubules (PTs), and ZO-1 and claudin-1, -4, and -8 in distal tubules (DTs) of rats 21 days after streptozotocin injection. Redox status along the nephron segments was evaluated. Diabetes increased kidney injury molecule-1 expression. Expression of sodium glucose cotransporters (SGLT1 and SGLT2) and facilitative glucose transporter (GLUT2) was induced. Increased oxidative stress was present in GL and PTs and to a lesser extent in DTs (measured by superoxide production and PKCβ2 expression), owing to NADPH oxidase activation and uncoupling of the endothelial nitric oxide synthase-dependent pathway. Claudin-5, occludin, and claudin-2 expression was decreased, whereas claudin-4 and -8 expression increased. ZO-1 was redistributed from membrane to cytosol. Increased nitration of tyrosine residues in claudin-2 was found, which might contribute to decrement of this protein in proximal tubule. In contrast, occludin was not nitrated. We suggest that loss of claudin-2 is associated with increased natriuresis and that loss of glomerular claudin-5 might explain early presence of proteinuria. These findings suggest that oxidative stress is related to alterations in TJ proteins in the kidney that are relevant to the pathogenesis and progression of DN and for altered sodium regulation in diabetes.

Relaxin requires the angiotensin II type 2 receptor to abrogate renal interstitial fibrosis

Fibrosis is a hallmark of chronic kidney disease, for which there is currently no effective cure. The hormone relaxin is emerging as an effective antifibrotic therapy; however, its mechanism of action is poorly understood. Recent studies have shown that relaxin disrupts the profibrotic actions of transforming growth factor-β1 (TGF-β1) by its cognate receptor, relaxin family peptide receptor 1 (RXFP1), extracellular signal-regulated kinase phosphorylation, and a neuronal nitric oxide synthase-dependent pathway to abrogate Smad2 phosphorylation. Since angiotensin II also inhibits TGF-β1 activity through its AT2 receptor (AT2R), we investigated the extent to which relaxin interacts with the AT2R. The effects of the AT2R antagonist, PD123319, on relaxin activity were examined in primary rat kidney myofibroblasts, and in kidney tissue from relaxin-treated male wild-type and AT2R-knockout mice subjected to unilateral ureteric obstruction. Relaxin's antifibrotic actions were significantly blocked by PD123319 in vitro and in vivo, or when relaxin was administered to AT2R-knockout mice. While heterodimer complexes were formed between RXFP1 and AT2Rs independent of ligand binding, relaxin did not directly bind to AT2Rs but signaled through RXFP1-AT2R heterodimers to induce its antifibrotic actions. These findings highlight a hitherto unrecognized interaction that may be targeted to control fibrosis progression.

Neuropeptide FF activates ERK and NF kappa B signal pathways in differentiated SH-SY5Y cells

Neuropeptide FF (NPFF) has been reported to play important roles in regulating diverse biological processes. However, little attention has been focused on the downstream signal transduction pathway of NPFF. Here, we used the differentiated neuroblastoma cell line, dSH-SY5Y, which endogenously expresses hNPFF2 receptor, to investigate the signal transduction downstream of NPFF. In particular we investigated the regulation of the extracellular signal-regulated protein kinase (ERK) and the nuclear factor kappa B (NF-κB) pathways by NPFF in these cells. NPFF rapidly and transiently stimulated ERK. H89, a selective inhibitor of cyclic AMP-dependent protein kinase A (PKA), inhibited the NPFF-activated ERK pathway, indicating the involvement of PKA in the NPFF-induced ERK activation. Down-regulation of nitric oxide synthases also attenuated NPFF-induced ERK activation, suggesting that a nitric oxide synthase-dependent pathway is involved. Moreover, the core upstream components of the NF-κB pathway were also significantly activated in response to NPFF, suggesting that the NF-κB pathway is involved in the signal transduction pathway of NPFF. Collectively, these data demonstrate that nitric oxide synthases are involved in the signal transduction pathway of NPFF, and provide the first evidence for the interaction between NPFF and the NF-κB pathway. These advances in our interpretation of the NPFF pathway mechanism will aid the comprehensive understanding of its function and provide novel molecular insight for further study of the NPFF system.

Renoprotective effect of erythropoietin in ischemia/reperfusion injury: Possible roles of the Akt/endothelial nitric oxide synthase‐dependent pathway

It has been reported that erythropoietin protects the kidneys from ischemia/reperfusion injury. In the present study, we examined the role of Akt and endothelial nitric oxide synthase in the protective effect of erythropoietin on ischemia/reperfusion injury of the kidney. Erythropoietin was injected in the peritoneal space of ICR mice after ischemia/reperfusion injury and its effect was assessed by measuring blood urea nitrogen and creatinine, and by histological analysis. Phosphorylation of Akt and endothelial nitric oxide synthase was examined by western blot analysis. Endothelial nitric oxide synthase gene null mice were also used to examine the role of endothelial nitric oxide synthase in the renoprotective effect of erythropoietin. Erythropoietin administration significantly inhibited the increase in blood urea nitrogen and creatinine after ischemia/reperfusion injury compared with control mice. Accordingly, erythropoietin administration significantly ameliorated the histological damages, including apoptotic cell death. Erythropoietin significantly stimulated phosphorylation of Akt and endothelial nitric oxide synthase in the kidneys. When endothelial nitric oxide synthase gene null mice were subjected to ischemia/reperfusion injury, erythropoietin did not significantly suppress the increase in blood urea nitrogen or creatinine. Erythropoietin seems to activate the Akt/endothelial nitric oxide synthase-dependent pathway in the kidneys. This pathway might be implicated in the renoprotective effect of erythropoietin in the ischemia/reperfusion injury model.

Genistein enhances relaxation of the spontaneously hypertensive rat aorta by transactivation of epidermal growth factor receptor following binding to membrane estrogen receptors-α and activation of a G protein-coupled, endothelial nitric oxide synthase-dependent pathway

Genistein, a phytoestrogen present in soybeans, has well established vasodilator properties. The present study examined the mechanisms involved in the rapid vascular effects of genistein. Endothelium-dependent relaxations and contractions, induced by acetylcholine and the calcium ionophore A23187, were obtained in isolated aortic rings from male spontaneously hypertensive rats (SHR). Acute exposure to genistein potentiated relaxations and reduced contractions induced by the two agonists. Both effects of genistein were not affected by transcription- and translation-inhibitors or by tyrosine kinase inhibition. The potentiation of acetylcholine and A23187-induced relaxation by genistein was inhibited by NF023 and GP antagonist-2A, selective G i and G q α-subunit antagonists, respectively, but not by NF449, a selective G s α-subunit antagonist. These G protein antagonists did not alter the inhibitory effect of genistein on acetylcholine and A23187-induced contractions. The potentiation of A23187-induced relaxations by genistein was not inhibited by the conventional estrogen receptor (ER) antagonist, ICI 182,780, but inhibited by the specific ER-α antagonist, MPP, and by the epidermal growth factor receptor (EGFR) inhibitor, AG1478. It was mimicked by heparin-binding epidermal growth factor (HB-EGF). Activation of EGFR and endothelial nitric oxide synthase (eNOS) was detected in genistein-treated rings using Western blotting. These data suggest that the rapid vascular actions of genistein are mediated by non-genomic pathways and are unrelated to its tyrosine kinase inhibitory properties. Furthermore, genistein transactivates EGFR through membrane ERα via G protein-coupled pathways. This in turn enhances eNOS phosphorylation and hence endothelial function in the aorta of the SHR.

Mechanism of reactive oxygen species generation after opening of mitochondrial KATP channels

opening of the ATP-sensitive K+ (KATP) channel mediates the cardioprotective effect induced by pathophysiological stressors such as ischemic preconditioning (IPC) ([15][1], [29][2]), heat shock ([19][3]), and pharmacological agents, including adenosine ([5][4]), ACh ([26][5]), opioids ([12][6]),

Vascular Endothelial Growth Factor (VEGF)-D and VEGF-A Differentially Regulate KDR-mediated Signaling and Biological Function in Vascular Endothelial Cells

Vascular endothelial growth factor (VEGF)-D binds to VEGF receptors (VEGFR) VEGFR2/KDR and VEGFR3/Flt4, but the signaling mechanisms mediating its biological activities in endothelial cells are poorly understood. Here we investigated the mechanism of action of VEGF-D, and we compared the signaling pathways and biological responses induced by VEGF-D and VEGF-A in endothelial cells. VEGF-D induced KDR and phospholipase C-gamma tyrosine phosphorylation more slowly and less effectively than VEGF-A at early times but had a more sustained effect and was as effective as VEGF-A after 60 min. VEGF-D activated extracellular signal-regulated protein kinases 1 and 2 with similar efficacy but slower kinetics compared with VEGF-A, and this effect was blocked by inhibitors of protein kinase C and mitogen-activated protein kinase kinase. In contrast to VEGF-A, VEGF-D weakly stimulated prostacyclin production and gene expression, had little effect on cell proliferation, and stimulated a smaller and more transient increase in intracellular [Ca(2+)]. VEGF-D induced strong but more transient phosphatidylinositol 3-kinase (PI3K)-mediated Akt activation and increased PI3K-dependent endothelial nitric-oxide synthase phosphorylation and cell survival more weakly. VEGF-D stimulated chemotaxis via a PI3K/Akt- and endothelial nitric-oxide synthase-dependent pathway, enhanced protein kinase C- and PI3K-dependent endothelial tubulogenesis, and stimulated angiogenesis in a mouse sponge implant model less effectively than VEGF-A. VEGF-D-induced signaling and biological effects were blocked by the KDR inhibitor SU5614. The finding that differential KDR activation by VEGF-A and VEGF-D has distinct consequences for endothelial signaling and function has important implications for understanding how multiple ligands for the same VEGF receptors can generate ligand-specific biological responses.

Production of Vascular Endothelial Growth Factor by Murine Macrophages: Regulation by Hypoxia, Lactate, and the Inducible Nitric Oxide Synthase Pathway

Murine thioglycolate-induced peritoneal macrophages (MPMs) and the murine RAW264.7 macrophage-like cell line (RAW cells) constitutively produce vascular endothelial growth factor (VEGF). VEGF production is increased under hypoxic conditions or after cell activation with interferon-gamma (IFNgamma) and endotoxin (lipopolysaccharide, LPS). In contrast, tumor necrosis factor-alpha is produced only by IFNgamma/LPS-activated cells. Lactate (25 mmol/L) does not increase VEGF production by these cells. However, hypoxia, lactate, and IFNgamma/LPS-activated MPMs express angiogenic activity, whereas normoxic, nonactivated MPMs do not. Lack of angiogenic activity is not due to an antiangiogenic factor(s) in the medium of these cells. Angiogenic activity produced by hypoxia and lactate-treated MPMs is neutralized by anti-VEGF antibody, which also neutralizes most of the angiogenic activity produced by IFNgamma/LPS-activated MPMs. The inducible nitric oxide synthase inhibitors Ng-nitro-L-arginine-methyl ester (1.5 mmol/L) and aminoguanidine (1 mmol/L) block production of angiogenic activity by MPMs and RAW cells. In RAW cells, Ng-nitro-L-arginine-methyl ester and AG block IFNgamma/LPS-activated, but not constitutive, VEGF production, whereas in MPMs, neither constitutive nor IFNgamma/LPS-activated VEGF synthesis is affected. Synthesis of tumor necrosis factor-alpha is also unaffected. In contrast to normoxic, nonactivated MPMs, inducible nitric oxide synthase-inhibited, IFNgamma/LPS-activated MPMs produce an antiangiogenic factor(s). We conclude that VEGF is a major contributor to macrophage-derived angiogenic activity, and that activation by hypoxia, lactate, or IFNgamma/LPS switches macrophage-derived VEGF from a nonangiogenic to an angiogenic state. This switch may involve a posttranslational modification of VEGF, possibly by the process of ADP-ribosylation. ADP-ribosylation by MPM cytosolic extracts or by cholera toxin switches rVEGF165 from an angiogenic to a nonangiogenic state. In IFNgamma/LPS-activated MPMs, the inducible nitric oxide synthase-dependent pathway also regulates the expression of an antiangiogenic factor(s) that antagonizes the bioactivity of VEGF and provides an additional regulatory pathway controlling the angiogenic phenotype of macrophages.

Insulin acutely inhibits cultured vascular smooth muscle cell contraction by a nitric oxide synthase-dependent pathway.

Insulin acutely decreases contractile agonist-induced Ca2+ influx and contraction in endothelium-free cultured vascular smooth muscle (VSM) cells, but the mechanism is not known. Since it has been reported that insulin-induced vasodilation in humans is linked to nitric oxide synthase activity, we wished to determine whether insulin inhibits Ca2+ influx and contraction of cultured vascular smooth muscle cells by a nitric oxide synthase-dependent pathway. Primary cultures of endothelial cell-free VSM cells from canine femoral artery were preincubated with and without 1 nmol/L insulin for 30 minutes, and the 5-minute production of cGMP was measured. Insulin alone did not affect cGMP production, but in the presence of 10(-5) mol/L serotonin insulin stimulated cGMP production by 60%. N(G)-monomethyl-L-arginine (0.1 mmol/L), an inhibitor of nitric oxide synthase, inhibited the conversion of arginine to citrulline by these cells, blocked insulin-stimulated cGMP production, and blocked the inhibition by insulin of 5-hydroxytryptamine (5-HT)-stimulated Mn+2 (a Ca2+ surrogate) influx and contraction. Insulin did not affect contraction of VSM cells grown under conditions designed to deplete the cells of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase. These studies demonstrate that insulin acutely inhibits 5-HT-stimulated Ca2+ influx and contraction of endothelium-free cultured VSM cells by a nitric oxide synthase-dependent mechanism.