Stimulation Of Nitric Oxide Production Research Articles

Crosstalk between hydrogen sulfide and nitric oxide in endothelial cells

Hydrogen sulfide (H2S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2S, stimulated NO production whereas blockage of the activity of H2S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2S on ECs. Our results suggest that H2S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2S-induced angiogenesis.

AB0107 AKT activation by fibronectin fragment leads to NF-KB up-regulation in rheumatoid arthritic chondrocytes

BackgroundIncreased fibronectin fragments are thought to contribute to joint destruction in rheumatoid arthritis (RA). However, the mechanism whereby fibronectin fragments cause catabolic activities is not totally understood. While COOH-terminal heparin-binding...

Abstract 503: Ixmyelocel-T, a Unique Mixed Cellular Therapy for the Treatment of Vascular Diseases Associated with Endothelial Dysfunction

Ixmyelocel-T, an expanded autologous multicellular therapy cultured from bone marrow mononuclear cells (BMMNCs), is under investigation for the treatment of vascular diseases associated with endothelial dysfunction. This study examines the mechanisms by which ixmyelocel-T treatment may exert beneficial effects on the endothelium in comparison to BMMNCs. To determine the effects on endothelial cells, ixmyelocel-T and BMMNCs were co-cultured with human umbilical vein endothelial cells (HUVEC) in non-contacting Transwell inserts. Co-culture with ixmyelocel-T resulted in increased eNOS (1730±141, vs. 1371±135 pg/mL, p < 0.05) measured by ELISA, and nitric oxide (NO) production (1.97±0.2, vs. 1±0.1 relative fluorescence, p < 0.001) measured by DAF-2DA. Co-culture with BMMNCs did not have an effect on eNOS or NO in HUVECs. In TNFα stimulated HUVECs, ixmyelocel-T decreased the generation of reactive oxygen species (46±4 vs. 100±3 % of HUVEC, p < 0.01) measured with DCFH-DA and increased SOD activity (1.3±0.1, vs. 1±0.1 % of HUVEC, p < 0.05), whereas co-culture with BMMNCs didn’t have an effect on ROS or SOD in HUVECs. Apoptosis analyzed by a caspase 3/7 assay demonstrated that ixmyelocel-T decreased apoptosis in TNFα treated HUVECs (0.78±0.02, vs. 1±0.05 relative to HUVEC, p < 0.001). Co-culture with BMMNCs had no effect on HUVEC apoptosis. Real time PCR analysis revealed that ixmyelocel-T decreased the expression of inflammatory markers (ICAM1, VCAM1, and MCP-1) in TNFα treated HUVECs, whereas treatment with BMMNCs had no effect. ELISA analysis indicated that ixmyelocel-T decreased MCP-1 (11983±5357 vs. 23312±11044 pg/mL, p < 0.05) and increased IL-10 secretion (61.3±11.2 vs. 1.2±0.5 pg/mL, p < 0.001), whereas treatment with BMMNCs had no effect. Ixmyelocel-T stimulated NO production, reduced oxidative stress and inflammation, and prevented apoptosis in endothelial cells. BMMNCs did not exhibit similar results. This is most likely due to the anti-inflammatory cell phenotypes associated with ixmyelocel-T’s expansion process. This study indicates that ixmyelocel-T may be superior to BMMNCs in the treatment of diseases associated with endothelial dysfunction and vascular inflammation.

Abstract 208: Altered Cerebrovascular Reactivity Following One Month of Obstructive Sleep Apnea

Obstructive sleep apnea (OSA) has been identified as a significant risk factor for stroke. However, little is known regarding the effects of OSA on the cerebrovascular wall. Using a novel rodent model of OSA we assessed the hypothesis that (1) OSA augments endothelin (ET-1) induced constrictions of cerebral arteries and (2) OSA attenuates dilations of cerebral arteries by agonist-induced nitric oxide (NO) release from the cerebrovascular endothelium. The repetitive airway closures associated with OSA lead to intermittent hypoxia/hypercapnia and reoxygenation, increased negative intrathoracic pressures, and arousals. In order to model the physiological consequences of OSA, we have chronically instrumented rats with inflatable endotracheal obstruction devices. Unanesthetized freely-ranging rats underwent 30 apneas/ hour for 8 hours/ day (sleep phase) for 1 month. During apnea pO 2 decreased from 122±3 to 67±3 mm Hg; pCO 2 increased from 43±1 to 51±1 mm Hg; pH decreased from 7.46±0.00 to 7.38±0.01; and hemoglobin O 2 saturation decreased from 94±1 to 82±1 % (n=5 and p<0.05 for each). Following 1 month of OSA blood pressure, plasma ET-1 and NO levels were similar in sham and OSA rats. Using the pressurized cerebral artery preparation, we observed a 17.5-fold increase in sensitivity to ET-1 (n=5-6, p<0.05) after 1 month of OSA. The increased sensitivity of OSA cerebral arteries to ET-1 was abolished by the ET-B receptor antagonist BQ-788 (n=6, NS). Additionally, constrictions to the ET-B specific agonist IRL-1620 were significantly greater in OSA, versus sham, cerebral arteries (n=6, p<0.05). Dilations to ATP (a P2Y 2 agonist which stimulates NO production in the endothelium) were attenuated in cerebral arteries from OSA rats by 40% (n=5-8, p<0.05). However dilations to the NO-donor MAHMA-NOnoate were similar between groups. In conclusion, 1 month of OSA results in (1) increased sensitivity of cerebral arteries to ET-1, likely through upregulation of ET-B receptors on the vascular smooth muscle and (2) decreased endothelial-derived NO production. These data suggest that OSA results in significant alterations to the cerebrovascular wall in the absence of hypertension.

Intramuscular and interstitial nitric oxide concentrations following doxorubicin administration in nude mice.

The purpose of the present study was to examine intramuscular (IM) nitric oxide (NO) concentrations in the gastrocnemius (G) and quadriceps (Q) muscles as well as interstitial NO levels in the G of immunodeficient nude mice (n=6) following the administration of doxorubicin (10mg/kg). Muscles were collected at day 3 and 7 post injection and interstitial concentrations were determined using the microdialysis technique. Gastrocnemius IM concentrations of NO were increased (P<0.05) from control (626±18 μmol/kg dw) to 823 ±48 and 831±22 μmol/kg dw at day 3 and 7, respectively. Similarly, IM quadriceps concentrations of NO increased (P<0.05) from control (599±13 μmol/kg dw) to 738 ±18 and 756±46 μmol/kg dw at day 3 and 7, respectively. In contrast, G interstitial NO concentrations did not change (P>;0.05) from control values (14.0±2.5 μmol/l) for either collection period (day 3 = 16.4±1.7. day 7 = 15.7 ±1.6 μmol/l). These data clearly demonstrate that intramuscular NO concentrations are increased as a result of doxorubicin administration, and remain elevated for up to 7 days following initial dosing. Interestingly, doxorubicin administration did not alter interstitial NO concentrations suggesting that the effects of anthracyclines on stimulating NO production was isolated to intramuscular mechanisms. Overall, this study suggests that doxorubicin administration stimulates intramuscular NO production and may be a result of increased oxidative stress.Supported by NSERC

Contribution of α-Adrenoceptor Stimulation by Phenylephrine to Basal Nitric Oxide Production in the Isolated Mouse Aorta

In the mouse aorta, contractions evoked by the α(1)-adrenoceptor agonist phenylephrine are strongly suppressed by the continuous production of nitric oxide (NO). We investigated whether phenylephrine itself stimulated NO production by activating endothelial α(2)-adrenoceptors. On a prostaglandin F(2α) contraction, the α(2)-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) induced 29.3 ± 7.4% relaxation, which was inhibited by 0.1 μM 2-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL44408) with a pKB' corresponding to α(2)-antagonism. In the presence of NO synthase blockers, UK14304 elicited small contractions above 1 μM that were inhibited by 0.1 μM prazosin, but not influenced by 0.1 μM rauwolscine. At 3 μM or higher concentrations, phenylephrine caused only modest relaxation (up to 7.4 ± 2.3%) of segments constricted with prostaglandin F(2α) in the presence of prazosin, which was abolished with 0.1 μM BRL44408. Furthermore, BRL44408 did not increase contractions induced with 1 μM phenylephrine. These results confirm that α(1)- but not α(2)-adrenoceptors are expressed on aortic smooth muscle cells, whereas endothelial cells only express α(2)-adrenoceptors. Moreover, phenylephrine exerted a very modest relaxing effect through nonspecific stimulation of α(2)-adrenoceptors, but only at concentrations higher than 1 μM. It is concluded that the high basal output of NO in the isolated mouse aorta is not due to stimulation of α-adrenoceptors.

Stimulated Nitric Oxide Production and Arginine Deficiency in Children with Cystic Fibrosis with Nutritional Failure

To determine whether upregulated whole body de novo arginine synthesis and protein breakdown are present as a compensatory mechanism to meet the increased demand for arginine and nitric oxide (NO) production in pediatric patients with cystic fibrosis (CF) and nutritional failure. In 16 children with CF, studied at the end of antibiotic treatment for a pulmonary exacerbation, and 17 healthy controls, whole body arginine, citrulline (Cit), and protein turnover were assessed by stable isotope methodology and de novo arginine synthesis, arginine clearance, NO synthesis, protein synthesis and breakdown, and net protein balance were calculated. The plasma isotopic enrichments and amino acid concentrations were measured by liquid chromatography-tandem mass spectrometry. Increased arginine clearance was found in patients with CF (P < .001), whereas whole body NO production rate and plasma arginine levels were not different. Whole body arginine production (P < .001), de novo arginine synthesis, and protein breakdown and synthesis (P < .05) were increased in patients with CF, but net protein balance was comparable. Patients with CF with nutritional failure (n = 7) had significantly higher NO production (P < .05), de novo arginine synthesis, Cit production (P < .001), and plasma Cit concentration (P < .05) and lower plasma arginine concentration (P < .05) than those without nutritional failure (n = 9). Nutritional failure in CF is associated with increased NO production. However, up-regulation of de novo arginine synthesis and Cit production was not sufficient to meet the increased arginine needs leading to arginine deficiency.

C‐peptide‐stimulated nitric oxide production in a cultured pulmonary artery endothelium is erythrocyte mediated and requires Zn2+

C-peptide has been shown to stimulate the production of nitric oxide (NO) in aortic endothelial cells via activation of endothelial nitric oxide synthase (eNOS) through an increased calcium influx. Here, results obtained using cultured bovine pulmonary artery endothelial cells (bPAECs) suggest that C-peptide does not induce eNOS activation directly in cultured pulmonary artery endothelium. However, C-peptide has been shown to stimulate the release of ATP from erythrocytes, a well-documented stimulus of eNOS activity in the pulmonary endothelium. Therefore, studies were performed to examine if C-peptide can indirectly stimulate NO production in a cultured pulmonary endothelium that is erythrocyte mediated. NO production and free intracellular calcium changes were monitored in immobilized bPAECs using specific intracellular fluorescent probes after stimulation with adenosine triphosphate (ATP), calcium ionophore A23187, or C-peptide. A microfluidic device enabled immobilized bPAECs to interact with flowing erythrocytes in the presence and absence of C-peptide to determine the role of the erythrocyte in C-peptide-stimulated NO production in cultured bPAECs. ATP and the calcium ionophore stimulate significant increases in both intracellular NO production and influx of free calcium in cultured bPAECs. In contrast, C-peptide, ranging from physiological to above physiological concentrations, was unable to stimulate NO production or calcium influx in the bPAECs. However, when erythrocytes were pre-incubated with a mixture containing physiological concentrations of C-peptide with Zn(2+) and haemodynamically pumped beneath bPAECs cultured on a microfluidic device, an 88.6 ± 7.5% increase in endothelial NO production was observed. C-peptide does not affect NO production in bPAECs directly but can impact NO production through an erythrocyte-mediated mechanism. Furthermore, in the absence of Zn(2+), C-peptide does not stimulate this NO production directly or indirectly. These results suggest that C-peptide, in the presence of Zn(2+), may be a determinant in purinergic receptor signalling via its ability to stimulate the release of ATP from erythrocytes.

Inhibition of Nitric Oxide and Tumour Necrosis Factor-α Production in Peritoneal Macrophages by &lt;i&gt;Aspergillus nidulans&lt;/i&gt; Melanin

The naturally occurring pigment, melanin is found in organisms of all phylogenetic kingdoms, including fungi, and exhibits a wide range of biological activities. Our objective was to investigate the effects of melanin extracted from the fungus Aspergillus nidulans on the production of the pro-inflammatory mediators nitric oxide (NO) and tumour necrosis factor-α (TNF-α) in peritoneal macrophages and on the viability of McCoy mouse fibroblasts. The results showed that A. nidulans melanin did not stimulate NO production in macrophages, but it inhibited the NO production in lipopolysaccharide (LPS)-stimulated macrophages by approximately 82%. Similarly, A. nidulans melanin inhibited LPS-stimulated TNF-α production by 52% and showed a slight stimulatory effect on TNF-α production in macrophages. In addition, the toxicity of A. nidulans melanin to McCoy cells was much lesser (IC50=373.5±2.4 µg/mL) than that of known agents such as cisplatin (IC50=41.2 µg/mL). The viability of peritoneal macrophages was greater than 90% at the highest melanin concentration tested (100 µg/mL). Thus, the combination of low cytotoxicity and marked inhibition of TNF-α and NO production suggests that A. nidulans melanin has potential as an anti-inflammatory agent and may be used in the future for development of new drugs with therapeutic utility.

Generation of avirulent Leishmania parasites and induction of nitric oxide production in macrophages by using polyacrylic acid

Polyacrylic acid (PAA) is one of the anionic synthetic polyelectrolytes and is used in various immunological and pharmaceutical applications. PAA has been used as adjuvant in veterinary vaccines, in particular. However, to our knowledge, there are no studies that document immunostimulant properties of PAA in Leishmania infection. The main aim of this study was to investigate the interaction of Leishmania parasites with PAA: the possible effects on the infectivity of Leishmania promastigotes; and, induction of nitric oxide (NO) production in macrophages in vitro. The cytotoxicity of PAA on both macrophages and Leishmania infantum promastigotes were determined by MTT assay. NO production in the macrophage culture supernatant was measured by the Griess method. A significant, dose-dependent and time-dependent decrease in the infection index was observed after PAA exposure. The value of this decrease was found to be between 93% and 100% for all concentration and time points. PAA (molecular weight (MW) 30, 100 kDa at 1mg/1h)-exposed parasites stimulate NO production significantly at 48 h post-infection (PI), when compared to the control. This study demonstrates that Leishmania parasites lost their virulence upon interaction with PAA, and this interaction induced NO production in infected macrophages. These results may have important implications in the development of anti-leishmanial vaccines and amelioration of immune response.

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin [IL]-1 beta, IL-6, IL-12, IL-10 and macrophage inflammatory protein-2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up-regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.

In vitro effects of nitric oxide donors on apoptosis and oxidative/nitrative protein modifications in ADP-activated platelets

Nitric oxide (NO) is an important physiological signaling molecule. However, when produced in excessive amounts, NO can also have toxic effects. The aim of this study is to investigate the effects of exogenous- and endogenous-derived NO on oxidative modifications of proteins and apoptosis in activated platelets. Washed platelets were incubated with L-arginine or nitroso-glutathione (GSNO) in the presence of adenosine diphosphate (ADP). After incubation, caspase-3 activity, phosphatidylserine (PS) externalization and the potential of mitochondrial membrane as markers of apoptosis were measured. In addition, the alterations in protein carbonylation (PCO) and nitrotyrosine (NT) formation as markers of protein oxidation were examined. Platelet activation with ADP (20 µM) significantly increased PCO and NT levels and apoptotic events. After incubation with L-arginine, platelet NO production increased significantly. This L-arginine-induced increase caused decreases in formerly increased PCO and NT levels associated with ADP-induced platelet activation. Stimulation of NO production with L-arginine protected platelets from apoptosis. GSNO caused an increase in protein NT levels. Despite this change, GSNO was effective in inhibition of P-selectin expression, platelet aggregation, protein carbonylation and apoptosis. The results suggest that L-arginine and GSNO-mediated NO leads to the inhibition of key apoptotic processes including caspase-3 activation, PS exposure and low mitochondrial membrane potential in washed platelets. The inhibitory effect of platelet clearance of L-arginine and GSNO may be a novel useful therapeutic property in clinical application.

The effect of probiotic‐fermented soy milk on enhancing the NO‐mediated vascular relaxation factors

Soy milk is one of the common soy-based foods in Asia. In this study the effects of soy milk fermented with selected probiotics on nitric oxide (NO)-mediated vascular relaxation factors in cell model systems were investigated. Soy milk fermented with Lactobacillus plantarum TWK10 or Streptococcus thermophilus BCRC 14085 for 48 h showed a greater transformation of glucoside isoflavones to aglycone isoflavones (P < 0.05). An increase in aglycone isoflavones in ethanol extracts from fermented soy milk stimulated NO production and endothelial NO synthase (eNOS) activity in human umbilical vein endothelial cells. It also had a stimulating effect on superoxide anion scavenging and prostaglandin E₂ production. In addition, it enhanced mRNA expression of the E-prostanoid 4 receptor in rat thoracic aorta smooth muscle cells. Moreover, a small amount of O₂⁻ induced by water extracts from fermented soy milk at low concentration (1 mg mL⁻¹) increased the content of calcium ions and activated eNOS, thereby promoting NO production and the coupling state of eNOS. Soy milk fermented with selected probiotics promotes the relaxation factors of vascular endothelial cells and can be applied in the development of functional foods.

Calcium-permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors Trigger Neuronal Nitric-oxide Synthase Activation to Promote Nerve Cell Death in an Src Kinase-dependent Fashion

In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.

The Control of Stress Induced Type I Diabetes Mellitus in Humans through the Hepatic Synthesis of Insulin by the Stimulation of Nitric Oxide Production

The role of stress induced development of Type-1 diabetes mellitus (T1DM) as opposed to autoimmunity remains obscure. It has been reported that a stress induced protein, identified to be dermcidin isoform 2 (dermcidin) inhibited insulin synthesis in both the pancreatic β cells and the hepatic cells. As dermcidin effect could be neutralized by the increased production of systemic nitric oxide (NO), investigations were carried out to determine the feasibility of controlling stress induced T1DM through the neutralization of dermcidin by systemic increase of NO. To determine the role of plasma dermcidin level in T1DM subjects (n=45), if any, when the plasma dermcidin level were determined, it was found that the protein level was increased in 65% of the participating volunteers. Efforts were made to normalize the plasma glucose level (median=175 mg/dL) in these T1DM subjects by systemic increase of NO by applying a cotton pad containing 0.28mmol sodium nitroprusside on the abdominal skin. It was found that the systemic increase of NO level decreased the blood glucose level of 275 mg/dL (median) to 115 mg/dL (median) in these volunteers within 24 h with concomitant increase of plasma insulin level from 7.5 μunits/dL to 101 μunits/dL at the same time. The increase of plasma insulin level was accompanied by the decrease of dermcidin level of 124.5 nM to 18 nM with increase of NO from 0.43 ± 0.19 nM to 4.1 ± 1.56 nM. The results suggested that the stress induced T1DM by dermcidin could be controlled by the systemic increase of NO which in consequence led to increased synthesis of insulin.

Abstract 632: Flow-induced TRPV4 Activation Mediates Calcium Influx In The Thick Ascending Limb.

Background: The thick ascending limb is critical in the regulation of sodium balance and therefore blood pressure. We previously showed that nitric oxide (NO), which inhibits sodium chloride transport and therefore blood pressure is regulated by luminal flow in this segment. Transient receptor potential vanilloid 4 (TRPV4), a member of the TRPV family of cation channels, is expressed in the thick ascending limb. This calcium permeable channel can be activated by luminal flow in other cell types. Therefore, we hypothesized that in the thick ascending limb luminal flow induces TRPV4 activation thereby increasing calcium influx and NO production. METHODS: We used the TRPV4 antagonists Ruthenium red and RN 1734 and the TRPV4 agonist GSK1016790A and measured intracellular calcium and NO production in the absence and presence of luminal flow in isolated and perfused thick ascending limbs from Sprague Dawley rats. NO production was measured using the NO-sensitive dye DAF FM da. Intracellular calcium was measured using the ratiometric calcium sensitive dye Fura-2 AM. Results: Increasing luminal flow from 0 to 20 nL/min stimulated NO production from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min ( p &lt;0.05, n =5). Increasing luminal flow in the presence of the TRPV4 antagonist Ruthenium red (15 μM) eliminated NO production (from 18 ± 5 to 16 ± 9 AU/min; n = 4). Flow-induced NO production was also prevented by the selective TRPV4 antagonist RN 1734 (10 μM) (from 11 ± 7 to 9 ± 2 AU/min; n = 4). Increasing luminal flow increased Fura 2 ratio units by 113 ± 20 %. However, in the presence of the TRPV4 antagonist Ruthenium red (15 μM) Fura 2 ratio units increased only by 55 ± 12 % in response to luminal flow ( p &lt;0.04, n =6). In the absence of luminal flow, activating TRPV4 using the TRPV4 selective agonist GSK1016790A (1 ηM) increased Fura 2 ratio units by 204 ± 43 %. Conclusion: From these data we conclude that flow-induced TRPV4 activation mediates calcium influx and NO production in the thick ascending limb.

Endothelial Insulin and IGF-1 Receptors: When Yes Means NO

Cardiovascular (CV) disease is the leading cause of morbidity and mortality and a major driver of health care costs in patients with type 2 diabetes. Observational studies suggest that insulin resistance and hyperglycemia independently predict atherosclerosis (1,2). However, recent clinical trials have been disappointing in that intensive glycemic control does not reduce the risk of CV events in individuals with diabetes (3). Consequently, it has been suggested that therapies targeting hyperinsulinemia and/or insulin resistance (e.g., metformin) may lead to CV risk reduction (2). In addition to its metabolic actions, insulin has important vascular actions that stimulate endothelial production of nitric oxide (NO), an anti-inflammatory and antiatheroslcerotic molecule (4). In turn, endothelial insulin resistance leads to diminished glucose disposal, endothelial dysfunction, and atherosclerosis. Strategies that ameliorate endothelial insulin resistance may simultaneously augment metabolic and vascular actions of insulin, thereby reducing CV risk. However, molecular mechanisms regulating endothelial insulin action are still unclear. Elegant studies from various laboratories have elucidated insulin signaling pathways that regulate NO production in the endothelium (4). Insulin binding to its receptor increases receptor tyrosine kinase activity and results in phosphorylation of insulin receptor (IR) substrate (IRS)-1 and sequential activation of phosphatidylinositol 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase (PDK)-1. PDK-1, in turn, activates Akt, which then directly phosphorylates endothelial NO synthase (eNOS) at Ser1177, resulting in increased eNOS activity and NO production (Fig. 1). Although less potent, IGF-1, like insulin, activates the PI3K-Akt-eNOS pathway and stimulates NO production in endothelial cells (5,6). FIG. 1. Relative distribution of insulin (INS) and IGF-1R …

The role of sex steroids on cellular events involved in vascular disease

In this work we checked the hypothesis whether estrone, progesterone, and testosterone are able to modulate the interactions between platelets, monocytes, and endothelial cells either under basal or inflammatory conditions. Using adhesion assays we demonstrated that pretreatment of endothelial cells with estrone, progesterone, or testosterone prevented monocytes and platelets adhesion induced by the proinflammatory agent bacterial lipopolysaccharide. The hormones reduced the expression of mRNA of ICAM-1, VCAM-1, and P-selectin, endothelial surface proteins that mediate monocytes and platelets adhesion respectively. Integrins are the main leukocyte proteins that allow firm adhesion. Using flow cytometry we showed that estrone treatment of monocytes reduced CD11b and CD11c expression, either under basal or injury (lipopolysaccharide) conditions. The three steroids inhibited platelet aggregation in a nitric oxide dependent manner. Platelet function was not affected by the steroid treatment. The molecular mechanisms of action exerted by the steroids included the participation of the intracellular signaling pathways PKC, MAPK, and PI3K, which selectively and differentially mediate the stimulation of nitric oxide release. We evidence that estrone, progesterone, and testosterone modulate monocyte and platelet adhesion to endothelial cells, events that play a major role in the initiation and progression of vascular lesions. The steroid action was evidenced under basal or inflammatory conditions. The mechanisms of action exerted by the steroids included stimulation of nitric oxide production and the participation of PKC, MAPK, and PI3K systems.

Albumin upregulates eNOS mRNA through ETRA/B in human proximal tubular epithelial cells

Proximal tubular cells respond to proteinuria by expressing several cytokines and inflammatory molecules that induce interstitial fibrosis. Increased attention has been drawn toward the systems of endothelin (ET) and nitric oxide (NO). This work contributes to the elucidation of the interplay between these two systems in proximal tubular epithelial cells (PTECs) after exposure in proteinuric conditions. HK-2 cells, a human PTEC line, were incubated with albumin, simulating proteinuric conditions. Cells were then lysed and either total RNA was isolated or whole cell extracts were prepared. PreproET-1, ET receptors (ETRA and ETRB) and NO synthases (eNOS, iNOS) mRNA accumulation was estimated by RT-PCR, and proteins by Western blot analysis. NO production was assessed using Griess reaction. Furthermore, we treated HK-2 cells with NO donor sodium nitroprusside, NO inhibitor L-NAME, ETRA inhibitor BQ123, ETRB inhibitor BQ788 and purified ET-1, and investigated the potential interplay between albumin-induced stimulation of NO or ET-1 systems. We found that albumin upregulates preproET-1, ETRA, ETRB, eNOS and iNOS mRNA as well as protein and stimulates NO production. Additionally, we recorded an ETRA/B dependent regulation of albumin-induced eNOS expression. For the first time an in vitro albumin-induced ET-1 and NO interplay was revealed.

Adrenomedullin and cardiovascular diseases

The cardiovascular system is regulated by the autonomic nervous system, the renin–angiotensin–aldosterone system, nitric oxide (NO) and other factors including neuropeptides. Research in neurohumoral factors has led to the development of many cardiovascular drugs. Adrenomedullin (ADM), initially isolated from the adrenal gland, has diverse physiological and pathophysiological functions in the cardiovascular system. It is produced in many organs and tissues including the vasculature. ADM has numerous actions, including vasodilation, natriuresis, antiapoptosis and stimulation of NO production. It might play a protective role in various cardiovascular pathologies, and its plasma level is elevated in patients with hypertension and heart failure. Administration of ADM is a possible therapeutic approach for treating cardiovascular diseases. A number of studies have investigated the infusion of ADM in humans, which seems to be benficial in heart failure and myocardial infarction. Instead of ADM infusion, augmentation of its endogenous level is another possible strategy. Gene therapy is feasible in animal models, but its application in humans is limited. At present, the most promising clinical application of ADM is the use of the plasma level of mid-regional proadrenomedullin as a biomarker in cardiovascular diseases. It is a good marker of prognosis and survival in patients with coronary aretery disease or heart failure.