Stimulation Of Nitric Oxide Production Research Articles

Luminal flow regulates NO and O2(-) along the nephron.

Urinary flow is not constant but in fact highly variable, altering the mechanical forces (shear stress, stretch, and pressure) exerted on the epithelial cells of the nephron as well as solute delivery. Nitric oxide (NO) and superoxide (O(2)(-)) play important roles in various processes within the kidney. Reductions in NO and increases in O(2)(-) lead to abnormal NaCl and water absorption and hypertension. In the last few years, luminal flow has been shown to be a regulator of NO and O(2)(-) production along the nephron. Increases in luminal flow enhance fluid, Na, and bicarbonate transport in the proximal tubule. However, we know of no reports directly addressing flow regulation of NO and O(2)(-) in this segment. In the thick ascending limb, flow-stimulated NO and O(2)(-) formation has been extensively studied. Luminal flow stimulates NO production by nitric oxide synthase type 3 and its translocation to the apical membrane in medullary thick ascending limbs. These effects are mediated by flow-induced shear stress. In contrast, flow-induced stretch and NaCl delivery stimulate O(2)(-) production by NADPH oxidase in this segment. The interaction between flow-induced NO and O(2)(-) is complex and involves more than one simply scavenging the other. Flow-induced NO prevents flow from increasing O(2)(-) production via cGMP-dependent protein kinase in thick ascending limbs. In macula densa cells, shear stress increases NO production and this requires that the primary cilia be intact. The role of luminal flow in NO and O(2)(-) production in the distal tubule is not known. In cultured inner medullary collecting duct cells, shear stress enhances nitrite accumulation, a measure of NO production. Although much progress has been made on this subject in the last few years, there are still many unanswered questions.

Effects of Curcumin on LPS-induced Nitric Oxide Production from Murine Macrophages and PBMC of Humans with Respiratory Allergic Disease

RATIONALE: Curcumin(Cur), found in curry, is a dietary pigment giving curry its unique color and has anti-inflammatory properties. Airway macrophages of asthmatics are a source of exhaled nitric oxide(NO), which is elevated in active asthma. We determined if Cur suppressed NO production from both a murine macrophage cell line (RAW264.7) and PBMC from allergic asthmatic adult humans(AAAH).METHODS: RAW264.7 cells were stimulated ± LPS (100 ng/ml) and ± Cur (1.4-5.4uM). Cur was also added to cells at 1 and 4 days before and after LPS activation to determine time course of suppression. PBMC from AAAH (n=4) ± LPS or allergen were treated ± Cur. NO2-, a measure of NO, was measured by Greiss reagent on days 3-6.RESULTS: Baseline LPS stimulated NO production from RAW264.7 cells(22.8uM) and was suppressed 73% with maximal Cur concentration(6.2uM); suppression was observed without LPS but not dose-dependently. Cur induced suppression was still present 1 and 4 days after LPS (60% and 9% respectively). NO levels were variable when Cur was added 1 day before LPS and not suppressed when Cur added 4 days before. (p<0.05) PBMC of AAAH produced variable amounts of NO (0.44-7uM) which were not increased with allergen/LPS(2.0-5.85uM) nor suppressed by Cur (2.0-7.42uM) (p=0.9).CONCLUSIONS: Cur suppresses LPS-induced NO production in RAW264.7 macrophages in a dose-dependent manner. Neither LPS or allergen stimulated NO production from PBMC of AAAH so evaluation of NO suppressive capacity of Cur was not optimal. The antioxidant activity of Cur on NO production by airway macrophages in asthma needs further investigation. RATIONALE: Curcumin(Cur), found in curry, is a dietary pigment giving curry its unique color and has anti-inflammatory properties. Airway macrophages of asthmatics are a source of exhaled nitric oxide(NO), which is elevated in active asthma. We determined if Cur suppressed NO production from both a murine macrophage cell line (RAW264.7) and PBMC from allergic asthmatic adult humans(AAAH). METHODS: RAW264.7 cells were stimulated ± LPS (100 ng/ml) and ± Cur (1.4-5.4uM). Cur was also added to cells at 1 and 4 days before and after LPS activation to determine time course of suppression. PBMC from AAAH (n=4) ± LPS or allergen were treated ± Cur. NO2-, a measure of NO, was measured by Greiss reagent on days 3-6. RESULTS: Baseline LPS stimulated NO production from RAW264.7 cells(22.8uM) and was suppressed 73% with maximal Cur concentration(6.2uM); suppression was observed without LPS but not dose-dependently. Cur induced suppression was still present 1 and 4 days after LPS (60% and 9% respectively). NO levels were variable when Cur was added 1 day before LPS and not suppressed when Cur added 4 days before. (p<0.05) PBMC of AAAH produced variable amounts of NO (0.44-7uM) which were not increased with allergen/LPS(2.0-5.85uM) nor suppressed by Cur (2.0-7.42uM) (p=0.9). CONCLUSIONS: Cur suppresses LPS-induced NO production in RAW264.7 macrophages in a dose-dependent manner. Neither LPS or allergen stimulated NO production from PBMC of AAAH so evaluation of NO suppressive capacity of Cur was not optimal. The antioxidant activity of Cur on NO production by airway macrophages in asthma needs further investigation.

Nitric oxide: a key factor behind the dysfunctionality of endothelial progenitor cells in diabetes mellitus type-2

Diabetes mellitus type-2 (DM-2) contributes to atherogenesis by inducing endothelial cell injury and dysfunction. Endothelial progenitor cells (EPCs) are essential to blood vessel formation, can differentiate into mature endothelial cells, and promote the repair of damaged endothelium. In DM-2, the circulating EPC count is low and their functionality is impaired. The mechanisms that underlie this reduced count and impaired functionality are poorly understood. Nitric oxide (NO) is a short-lived signalling molecule that is produced by vascular endothelial cells and participates in the maintenance of vascular tone. NO is also known to participate in other physiological processes, such as cell survival, proliferation, and migration. The bioavailability of NO is reduced in EPCs from DM-2 patients. Interestingly, an inverse relationship exists between the reduction in NO bioavailability in EPCs and the patient's plasma glucose and glycated haemoglobin levels. In addition, NO bioavailability in EPCs correlates with plasma oxidized low-density lipoprotein levels in DM-2. Although this reduction in NO bioavailability could be attributed to oxidative stress in DM-2 patients, it also may be due to impairment of one or more members of the protein signalling cascades that are responsible for NO production. The stimulation of NO production or its signalling cascades in EPCs may increase their numbers and improve their function, thus attenuating endothelium damage, independent of the vasodilatory effects of NO. This review summarizes the metabolic alterations that underlie the molecular mechanisms that may be responsible for EPC decrease and dysfunction in DM-2 with emphasis on the involvement of the NO system.

Regulation of Inducible Nitric Oxide Synthase Expression in Bovine Granulosa Cells and Its Involvement in Follicular Dominance.

Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Nitric oxide (NO), a free-radical gas, has been implicated in follicular growth in rodents, but the cow is an intriguing enigma: NO is produced by bovine granulosa cells and is regulated by FSH, but the enzymes responsible are reported to be absent. Three genes encode NO synthase proteins, one of which - iNOS - is widely considered to be regulated by extracellular ligands. We hypothesize that granulosa cells express the gene encoding iNOS, and that the expression of this gene is under hormonal regulation. The objectives of the present study were to determine the abundance of iNOS mRNA in growing and non-growing follicles in cattle, to determine the regulation of iNOS expression by FSH and growth factors, and to determine the action of NOS in bovine granulosa cells. The two largest follicles on days 1 to 5 of the first follicular wave of the cycle were collected from each pair of ovaries from six cows. Messenger RNA encoding iNOS was detected in granulosa and theca cells of bovine follicles by real-time PCR. The granulosa cells of dominant follicles contained higher levels of mRNA for iNOS compared with subordinate follicles (P<0.01). The regulation of iNOS expression was evaluated in a serum-free granulosa cell culture system. FSH (P<0.05) and IGF1 (P<0.01) stimulated E2 secretion iNOS mRNA abundance in a dose-dependent manner. Moreover, both FGF2 and EGF (P<0.01) decreased iNOS expression and E2 secretion in FSH and in IGF1-treated cells. Owing to the parallel changes in estradiol secretion and iNOS mRNA, we assessed the role of estradiol in the regulation of iNOS mRNA; replacement of estrogen precursor with a non-aromatizable androgen (DHT) or the addition of an estrogen receptor antagonist inhibited FSH-stimulated iNOS mRNA abundance, and addition of E2 alone stimulated NO production in vitro. To assess the role of endogenous iNOS, cells were treated with an iNOS-selective inhibitor, which resulted in increased FasL mRNA levels (P<0.01), induction of caspase-3 activation (P<0.01) and an increase in the incidence of cell death (P<0.05). In conclusion, bovine graulosa cells express iNOS, and increased iNOS mRNA levels are associated with growth of the dominant follicle. Gonadotropic factors stimulate iNOS mRNA levels through increased E2 secretion, and physiological levels of NOS activity may contribute to growth and survival of granulosa cells. Supported by NSERC Canada and CNPq Brazil. (poster)

Is oxidative stress a therapeutic target in cardiovascular disease?

An abnormal production of reactive oxygen species (ROS) and the subsequent decrease in vascular bioavailability of nitric oxide (NO) have long been proposed to be the common pathogenetic mechanism of the endothelial dysfunction, resulting from diverse cardiovascular risk factors such as hypercholesterolaemia, diabetes mellitus, chronic smoking, metabolic syndrome, and hypertension. Superoxide produced by the nicotinamide dinucleotide phosphate (NADPH) oxidase, mitochondrial sources, or the xanthine oxidase may react with NO, thereby resulting in excessive formation of peroxynitrite, a reactive nitrogen species that has been demonstrated to accelerate the atherosclerotic process by causing direct structural damage and by causing further ROS production. Despite this sound biological rationale and a number of pre-clinical and clinical lines of evidence, studies testing the effects of classical antioxidants such as vitamin C, vitamin E, or folic acid in combination with vitamin E have been disappointing. Rather, substances such as statins, angiotensin-converting enzyme inhibitors, or AT1-receptor blockers, which possess indirect antioxidant properties mediated by the stimulation of NO production and simultaneous inhibition of superoxide production (e.g. from the NADPH oxidase), have been shown to improve vascular function in pre-clinical and clinical studies and to reduce the incidence of cardiovascular events in patients with cardiovascular disease. Today, oxidative stress remains an attractive target for cardiovascular prevention and therapy. However, a deeper understanding of its source, and of its role in vascular pathology, is necessary before new trials are attempted.

Shear stress increases nitric oxide production in thick ascending limbs

We showed that luminal flow stimulates nitric oxide (NO) production in thick ascending limbs. Ion delivery, stretch, pressure, and shear stress all increase when flow is enhanced. We hypothesized that shear stress stimulates NO in thick ascending limbs, whereas stretch, pressure, and ion delivery do not. We measured NO in isolated, perfused rat thick ascending limbs using the NO-sensitive dye DAF FM-DA. NO production rose from 21 ± 7 to 58 ± 12 AU/min (P < 0.02; n = 7) when we increased luminal flow from 0 to 20 nl/min, but dropped to 16 ± 8 AU/min (P < 0.02; n = 7) 10 min after flow was stopped. Flow did not increase NO in tubules from mice lacking NO synthase 3 (NOS 3). Flow stimulated NO production by the same extent in tubules perfused with ion-free solution and physiological saline (20 ± 7 vs. 24 ± 6 AU/min; n = 7). Increasing stretch while reducing shear stress and pressure lowered NO generation from 42 ± 9 to 17 ± 6 AU/min (P < 0.03; n = 6). In the absence of shear stress, increasing pressure and stretch had no effect on NO production (2 ± 8 vs. 8 ± 8 AU/min; n = 6). Similar results were obtained in the presence of tempol (100 μmol/l), a O(2)(-) scavenger. Primary cultures of thick ascending limb cells subjected to shear stresses of 0.02 and 0.55 dyne/cm(2) produced NO at rates of 55 ± 10 and 315 ± 93 AU/s, respectively (P < 0.002; n = 7). Pretreatment with the NOS inhibitor l-NAME (5 mmol/l) blocked the shear stress-induced increase in NO production. We concluded that shear stress rather than pressure, stretch, or ion delivery mediates flow-induced stimulation of NO by NOS 3 in thick ascending limbs.

Hyaluronan inhibits p38 mitogen-activated protein kinase via the receptors in rheumatoid arthritis chondrocytes stimulated with fibronectin fragment

This study was aimed to examine the inhibitory mechanism of high molecular weight hyaluronan (HA) on nitric oxide (NO) production by NH2-terminal heparin-binding fibronectin fragment (FN-f) in rheumatoid arthritis (RA) chondrocytes. When the RA cartilage explants or the isolated RA chondrocytes in monolayer were incubated with FN-f, the fragment stimulated NO production with induction of inducible nitric oxide synthase (iNOS) and activation of p38 mitogen-activated protein kinase. Pretreatment with 2,700 kDa HA resulted in significant suppression of FN-f-stimulated NO production in RA cartilage as well as in chondrocyte monolayer cultures in association with iNOS down-regulation. Inhibition studies with p38 inhibitor indicated the requirement of p38 for FN-f-induced NO production. HA suppressed p38 activation by the FN-f, leading to a decrease in NO production. Immunofluorescence cytochemistry revealed HA association with intercellular adhesion molecule-1 (ICAM-1) and CD44. While the individual antibody to ICAM-1 or CD44 partially reversed HA effect on the FN-f action, both antibodies in combination completely blocked the HA effect. The present study clearly demonstrated that the high molecular weight of HA suppressed the FN-f-activated p38 via ICAM-1 and the CD44 in RA chondrocytes. HA could down-regulate the catabolic action of FN-f in RA joints through the mechanism demonstrated in this study.

NITRIC OXIDE PRODUCTION DISORDERS IN LEUKOCYTES OF PATIENTS WITH RECURRENT FURUNCULOSIS

The propensity for certain individuals to develop staphylococcal recurrent furunculosis (RF) is not fully understood. But among the reasons of its development the immune system dysfunctions are described. As in the staphylococcus elimination the main role is played by neutrophils, the objective of this study was to determine nitric oxide (NO) and reactive oxygen species (ROS) production by polymo rphonuclear leukocytes (PMNs) of patients with RF and to compare them with the response of normal cells to stimulation. The spontaneous and pyrogenal-stimulated nitric oxide production was evaluated in leukocyte cell cultures by Griess reaction, and ROS generation was determined in the stimulated and unstimulated NBT-test. In this study we have demonstrated that leukocytes of healthy subjects respond on stimulation by the increase of both NO and ROS production. In contrast, leukocytes of patients with RF react by depression of NO formation at stimulation, and are characterized by decrease of ROS production reserve with the increasing of spontaneous ROS generation. Described disorders are revealed in the remission period as well as at exacerbation of furunculosis. The present study suggests that leukocytes of patients with RF have stable defect of stimulated NO production increase, which can be the reason for recurrent and severe course of furunculosis.

Characterization of a putative AS regulatory phosphorylation site implicated in vascular endothelial nitric oxide production

Argininosuccinate synthase (AS) catalyzes the rate‐limiting step of the nitric oxide (NO) cycle in vascular endothelial cells (EC); however, little is known about its regulation. Recently, we reported that AS is regulated by phosphorylation. Using in silico, as well as proteomic analysis, we have identified a putative phosphorylation site at serine 328 which is located adjacent to a caveolin binding motif located in the central region of AS tertiary structure. In this report, we examined the role of the S328 site relative to supporting NO production in ECs by mutation analysis, mutating S328 to either an alanine (S328A) or to the phospho‐mimetic aspartate (S328D). These constructs were overexpressed in ECs, and NO production was measured after stimulation with either the calcium ionophore plus ortho‐vanadate, or vascular endothelial growth factor (VEGF). In both cases, the S328D AS mutant produced significantly higher NO than wild type AS (n=3, p&lt;0.05), while the S328A mutant had no effect over background. Notably, the S328A mutant also did not support cell viability when the BAECs were serum starved before VEGF incubation. These data suggest that the S328D mutant promotes stimulated NO production and maintains cell viability, and supports the proposal that S328 may be a biologically significant site for AS regulation.

ATP mediates flow‐induced nitric oxide production in thick ascending limbs

Mechanical stimulation caused by increasing flow induces the release of ATP and other nucleotides. Both luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in the thick ascending limb (TAL). We hypothesized that luminal flow‐induced NO production in TALs is mediated by ATP. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. The rate of increase in fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nL/min stimulated NO production from 17±15 to 130±34 arbitrary units (AU)/min (p &lt; 0.04; n=7). When an ATP scavenging system (hexokinase; 10 U/ml plus 5.5 mM glucose) was added to the luminal perfusate and basolateral bath, luminal flow did not increase NO production (control: 32±5; after ATP scavenging: 19±4 AU/min; n=6). When luminal flow was increased with the ATPase apyrase (5 U/ml) in the luminal perfusate and basolateral bath, NO levels did not change significantly (control: 41±12; after apyrase: 59±11; n=6). The P2 receptor antagonist suramin (300 umol/L) in the lumen and bath reduced flow‐induced NO production by 73% (control: 56±11; with suramin: 13±12; n=5). We concluded that ATP mediates flow‐induced NO production in thick ascending limbs via P2 receptors.Support: PO1 HL 090550, RO1 HL 070985

Involvement of ethylene and nitric oxide in cell death in mastoparan‐treated unicellular alga Chlamydomonas reinhardtii

This work demonstrates a contribution of ethylene and NO (nitric oxide) in MP (mastoparan)-induced cell death in the green algae Chlamydomonas reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of PCD (programmed cell death). A pharmacological approach involving combined treatments with MP and ethylene- and NO-interacting compounds indicated the requirement of trace amounts of both ethylene and NO in MP-induced cell death. By employing a carbon dioxide laser-based photoacoustic detector to measure ethylene and a QCL (quantum cascade laser)-based spectrometer for NO detection, simultaneous increases in the production of both ethylene and NO were observed following MP application. Our results show a tight regulation of the levels of both signalling molecules in which ethylene stimulates NO production and NO stimulates ethylene production. This suggests that, in conjunction with the elicitor, NO and ethylene cooperate and act synchronously in the mediation of MP-induced PCD in C. reinhardtii. To the best of our knowledge, this is the first report on the functional significance of ethylene and NO in MP-induced cell death.

Oestrogenic Compounds Modulate Cytokine-induced Nitric Oxide Production in Mouse Osteoblast-like Cells

Nitric oxide (NO) is a mediator of bone metabolism with effects on both bone resorption and formation. Its production by both the constitutive and inducible isoforms of nitric oxide synthase (NOS) is affected by oestrogen in several types of cell and in tissues other than bone cells. Recently, oestrogens were found to increase basal NO production by osteoblasts via enhanced activity or expression, or both, of NOS-3. Inflammatory cytokines, however, increase NO by increasing the expression of NOS-2. In this study we have examined whether cytokine-induced NO production by osteoblastic cells was affected by oestrogenic compounds by studying the effect of 17beta-oestradiol and the anti-oestrogens ICI164,384 and 4-hydroxytamoxifen on cytokine-induced NO production in oestrogen receptor positive MC3T3-E1 osteoblast-like cells. Combinations of the inflammatory cytokines interleukin-1beta, tumour necrosis factor-alpha, and interferon-gamma with lipopolysaccharide stimulated NO production up to 11-fold. This cytokine-induced NO production was further increased dose-dependently by the anti-oestrogens ICI164,384 and 4-hydroxytamoxifen (133.3 +/- 3.2% and 146.0 +/- 13.2%, respectively). 17Beta-oestradiol either had no effect on or slightly inhibited cytokine-induced NO production. It did, however, dose-dependently counteract the stimulatory effect of the anti-oestrogens. Concentrations of 17beta-oestradiol needed to prevent the stimulatory effect of 4-hydroxytamoxifen were ca tenfold that of ICI164,384. These findings show that, in addition to the stimulatory effect of oestrogen on basal NO production by NOS-3, cytokine-induced NO production is also affected by oestrogenic compounds in osteoblasts.

Extracellular Vesicles from Cryptococcus neoformans Modulate Macrophage Functions

Cryptococcus neoformans and distantly related fungal species release extracellular vesicles that traverse the cell wall and contain a varied assortment of components, some of which have been associated with virulence. Previous studies have suggested that these extracellular vesicles are produced in vitro and during animal infection, but the role of vesicular secretion during the interaction of fungi with host cells remains unknown. In this report, we demonstrate by fluorescence microscopy that mammalian macrophages can incorporate extracellular vesicles produced by C. neoformans. Incubation of cryptococcal vesicles with murine macrophages resulted in increased levels of extracellular tumor necrosis factor alpha (TNF-alpha), interleukin-10 (IL-10), and transforming growth factor beta (TGF-beta). Vesicle preparations also resulted in a dose-dependent stimulation of nitric oxide production by phagocytes, suggesting that vesicle components stimulate macrophages to produce antimicrobial compounds. Treated macrophages were more effective at killing C. neoformans yeast. Our results indicate that the extracellular vesicles of C. neoformans can stimulate macrophage function, apparently activating these phagocytic cells to enhance their antimicrobial activity. These results establish that cryptococcal vesicles are biologically active.

Discovery of a benzoxazine derivative promoting angiogenesis in vitro and in vivo

Angiogenesis is a multi-step process that refers to the growth of new vessels from pre-existing ones. Endothelial proliferation, migration, and tube formation constitute a critical step in angiogenesis. Recently, we demonstrated that a novel benzoxazine derivative, 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (ABO) could improve the proliferation of human umbilical vein endothelial cells (HUVECs) without basic fibroblast growth factor (bFGF) and serum. In this study, we further tested its effect on endothelial angiogenesis with Matrigel assay, migration assay, and in vivo chick chorioallantoic membrane (CAM) assay. Our results showed that ABO effectively facilitated cell migration and promoted capillary-like tube formation in vitro and in vivo. To elucidate the underlying mechanisms, we examined intracellular reactive oxygen species (ROS) level/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and superoxide dismutase (SOD) activities, nitric oxide (NO) level/endothelial nitric oxide synthase (eNOS) activity, and mitochondrial membrane potential (MMP). Our data indicated that ABO depressed ROS with inhibition of NADPH oxidase instead of SOD activity, stimulated NO production and eNOS activation, and restored MMP in HUVECs. Our findings suggest that ABO is a promising tool for exploring the mechanisms of angiogenesis and may have a therapeutic potential in ischemic pathologies.

Nitric Oxide Can Mediate Beta-Adrenergic- and CaMKII-Dependent Spontaneous Ca2+ Waves in Cardiac Myocytes, Independent of PKA Activation

Increased diastolic SR Ca leak can initiate spontaneous Ca waves (SCaWs). SCaWs activate inward Na/Ca exchanger current causing an arrhythmogenic delayed afterdepolarization. Here we examine SCaWs in ventricular myocytes isolated from rabbit hearts. Myocytes did not exhibit SCaWs at baseline conditions, but 43% did when exposed to isoproterenol (ISO). This ISO-induced increase in activity was reversed by inhibition of CaMKII by KN93, but not with PKA inhibition by H89. At similar [Ca]SRT (121 μM) myocytes treated with ISO plus KN93 had significantly fewer SCaWs versus those treated with ISO or ISO plus H89 (0.2±0.28 vs. 1.1±0.28 & 1.29±0.39 SCaWs cell−1, respectively). We attribute this increase in activity to the previously characterized CaMKII-dependent increase in RyR-dependent leak. We also find that SR Ca leak is increased by the nitric oxide (NO) donor, SNAP; and this NO-dependent effect is also completely reversed by KN-93. We also show the increase in leak to be dependent on nitric oxide synthase 1 (NOS1) activity. At comparable SR Ca load (132 μM) ISO treated myocytes have significantly higher leak vs. control (8.4 vs. 3.8 μM). The ISO-induced leak (at constant SR Ca load) was attenuated by the NOS1 inhibitor, SMLT, but not the NOS3 inhibitor, L-NIO (3.5 vs. 6.8 μM). Moreover, ISO causes an upward trend in myocyte [NO] (sensed by the NO-dependent dye, DAF-2 A), and the NOS inhibitor, L-NAME significantly attenuated the development of SCaWs. Together this data suggests a novel pathway in which β1-adrenergic receptor activation stimulates NO production via NOS1, which in turn activates CaMKII to increase RyR gating, SR Ca leak, SCaWs and delayed afterdepolarizations.

Netrin-1 prevents ischemia/reperfusion-induced myocardial infarction via a DCC/ERK1/2/eNOSs1177/NO/DCC feed-forward mechanism

We have recently shown that a novel endothelial mitogen netrin-1 potently stimulates nitric oxide (NO) production via a DCC-ERK1/2 dependent mechanism. In view of the well-established cardioprotective role of NO, the present study investigated whether netrin-1 is cardioprotective via NO signaling in the heart. Netrin-1 receptor DCC was abundantly expressed in the C57BL/6J mouse hearts. Perfusion of heart with netrin-1 (100 ng/mL) using a Langendorff system significantly increased NO production. Under ischemia/reperfusion (I/R), netrin-1 induced a substantial reduction in infarct size (21.8±4.9% from 42.5±3.6% in the controls), which was accompanied by an augmented production of NO. Pre-perfusion with DCC-antibody, U0126 (MEK1/2 inhibitor), L-NAME or PTIO (NO scavenger) attenuated protective effects of netrin-1 on infarct size and NO production, indicating upstream roles of DCC and ERK1/2 in NO production, as well as an essential role of NO in cardioprotection. Netrin-1 induced reduction in infarct size was significantly attenuated in DCC+/− mice, confirming an intermediate role of DCC. In additional experiments we found netrin-1 increased ERK1/2 and eNOSs1177 phosphorylation, and DCC protein expression, which was diminished by I/R. Furthermore, netrin-1-induced DCC upregulation was NO and ERK1/2-dependent, implicating a feed-forward mechanism. DAF-AM staining revealed enhanced NO production in both cardiac endothelial cells (ECs) and myocytes. In primarily isolated cardiomyocytes, netrin-1 also increased NO production, DCC abundance and ERK1/2 phosphorylation. Of note, cardiac apoptosis was significantly attenuated by netrin-1, which was reversed by DCC-antibody, U0126, L-NAME or PTIO. In summary, our data clearly demonstrate that netrin-1 potently protects the heart from I/R injury by stimulating NO production from cardiac ECs and myocytes. This potent effect is mediated by a DCC/ERK1/2/eNOSs1177/NO/DCC feed-forward mechanism in both cell types.

P2X purinoceptors mediate an endothelium‐dependent hyperpolarization in longitudinal smooth muscle of anterior mesenteric artery in young chickens

The chicken anterior mesenteric artery contains an outer longitudinal smooth muscle layer, whose neural regulation remains to be elucidated. ATP evokes a depolarization in the smooth muscle through P2Y purinoceptors. However, there may be an additional inhibitory regulation because blockade of P2Y purinoceptors converts the depolarization to hyperpolarization. The objective of the present study was to examine the mechanism underlying this hyperpolarization. Membrane potentials of longitudinal smooth muscle of the chicken mesenteric artery were recorded with a microelectrode technique. Perivascular nerves were stimulated by applying electrical field stimulation (EFS). EFS induced a hyperpolarization in preparations obtained from 5-week-old chickens, whereas it evoked a depolarization in those from 12-week-old chickens. The EFS-evoked hyperpolarization in 5-week-old chickens was blocked by a non-specific purinoceptor antagonist, suramin, and by a specific P2X purinoceptor antagonist, pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid. Desensitization of the P2X purinoceptor with its agonist alpha,beta-MeATP significantly suppressed EFS-evoked hyperpolarization. Blockade of the P2Y purinoceptor did not affect EFS-evoked hyperpolarization. The application of the NOS inhibitor Nomega-nitro-L-arginine methyl ester or the removal of the endothelium inhibited the hyperpolarization. The application of the nitric oxide (NO) donor sodium nitroprusside mimicked the hyperpolarization. Reverse transcriptase-PCR showed that P2X purinoceptors are expressed in the endothelium of the anterior mesenteric artery. Hyperpolarization in the longitudinal smooth muscle of the chicken anterior mesenteric artery was induced by ATP. ATP released from perivascular nerves may act on P2X purinoceptors in the endothelium and thereby stimulate NO production.

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO2-) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 microM). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Nitric oxide/cGMP signalling inducesEscherichia coliK1 receptor expression and modulates the permeability in human brain endothelial cell monolayers during invasion

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) mediated by outer membrane protein A (OmpA) results in the leakage of HBMEC monolayers. Despite the influence of nitric oxide (NO) in endothelial cell tight junction integrity, its role in E. coli-induced HBMEC monolayer permeability is poorly defined. Here, we demonstrate that E. coli invasion of HBMEC stimulates NO production by increasing the inducible nitric oxide synthase (iNOS) expression. Exposure to NO-producing agents enhanced the invasion of OmpA(+)E. coli and thereby increased the permeability of HBMEC. OmpA(+)E. coli-induced NO production lead to increased generation of cGMP and triggered the expression of OmpA receptor, Ec-gp96 in HBMEC. Pre-treatment of HBMEC with iNOS inhibitors or by introducing siRNA to iNOS, but not to eNOS or cGMP inhibitors abrogated the E. coli-induced expression of Ec-gp96. Overexpression of the C-terminal truncated Ec-gp96 in HBMEC prevented NO production and its downstream effector, cGMP generation and consequently, the invasion of OmpA(+)E. coli. NO/cGMP production also activates PKC-alpha, which is previously shown to be involved in HBMEC monolayer leakage. These results indicate that NO/cGMP signalling pathway plays a novel role in OmpA(+)E. coli invasion of HBMEC by enhancing the surface expression of Ec-gp96.

Effects of Tumor Necrosis Factor-α and Nitric Oxide on Prostaglandins Secretion by the Bovine Oviduct Differ in the Isthmus and Ampulla and Depend on the Phase of the Estrous Cycle

To determine the possible roles of tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO) in the bovine oviduct, ampulla and isthmus collected during the estrous cycle were exposed for 18 h to TNFalpha, NO donor (NONOate), NO synthase inhibitors (L-NOARG, L-NAME and AMT) and oxytocin (OT) as a positive control. Prostaglandins (PGs) and NO(2)/NO(3) in conditioned media were measured. TNFalpha stimulated PGF(2alpha) secretion on Day 0 (onset of estrus = Day 0) and Days 2-3, in both the ampulla and isthmus, but on Days 18-20 only in ampulla. TNFalpha increased PGE(2) secretion in both fragments in each phase. NONOate did not affect PGF(2alpha) secretion on Days 18-20, whereas this NO donor stimulated PGF(2alpha) secretion in both fragments on Day 0 and Days 2-3. TNFalpha increased NO(2)/NO(3) production in every examined phase in the ampulla and on Days 2-3 in the isthmus. L-NAME lowered NO(2)/NO(3) production regardless of phase or fragment. L-NOARG and AMT lowered NO(2)/NO(3) production in both fragments on Day 0 and Days 2-3. The possible role of TNFalpha, NO or PGs on the oviductal contractility during the early-luteal phase was also examined. Neither TNFalpha nor NONOate influenced contractility in either fragment. Although PGF(2alpha) stimulated the contraction in both fragments, PGE(2) decreased it. When taken together, TNFalpha seems to play some role as a modulator of PGF(2alpha) and PGE(2) production and for transferring the embryo from the oviduct to the uterus by stimulating NO production in the bovine oviduct.