New insights into nitric oxide and coronary circulation

Nitric oxide: physiology and pharmacology.

Increased nitric oxide (NO) release has been implicated in the pathogenesis of the hyperdynamic circulation in portal hypertension. NOS 3 (eNOS) causes NO release from the endothelium in response to physical stimuli, such as increased blood flow and shear stress. We evaluated the functional activity of the endothelium in the superior mesenteric arterial bed of portal hypertensive rats through direct measurement of NO metabolites (NOx) during changes in flow and shear stress. The in vitro perfusion system (McGregor) was used in sham and portal vein-ligated (PVL) rats. Shear stress was applied gradually to superior mesenteric arterial beds by increasing the perfusion rate. Flow studies were performed serially before and after incubation with either Krebs solution alone or with the NO-inhibitor, NG-monomethyl-L-arginine (L-NMMA). NOx concentrations in the perfusate were measured using chemiluminescence. The slope of NOx release versus flow-induced shear stress was calculated. Before L-NMMA administration, NOx concentrations and release of NO in PVL rats were significantly elevated in comparison with sham rats at flow rates of 32, 40, and 48 mL/min. The slope of NOx production versus shear stress index was significantly higher in PVL than in sham rats. After L-NMMA incubation, the decrease in slope was significantly larger in PVL rats. This study provides direct evidences of an increased NO synthesis by the superior mesenteric arterial vascular endothelium of PVL animals in response to shear stress. The increased NO output in response to shear stress suggests an adaptative mechanism developed by the vascular endothelial cells in response to a chronic increase in flow-mediated shear stress.

EDF-1 contributes to the regulation of nitric oxide release in VEGF-treated human endothelial cells

Estrogen (E2) is reported to induce the rapid activation of endothelial nitric oxide synthase (eNOS) and NO release, resulting in vasodilation, decreased platelet adhesion and angiogenesis in the vessel wall. In this study, we have used a cell line (EA.hy926) derived from human umbilical vein endothelial cells (HUVECs) to investigate the cellular mechanism underlying E2-induced NO synthesis. Experimentally, NO production and cytosolic free [Ca2+] were monitored using the NO-sensitive dye DAF-FM and Fluo-3, respectively, and agonist-induced changes in membrane potential were recorded using patch clamp techniques. E2 dose-dependently induced rapid increases in NO synthesis, and only modest changes in cytosolic [Ca2+]. The PI3 kinase inhibitor LY294002, the E2 receptor antagonist ICI 182,780, and the eNOS inhibitor L-NAME all blocked E2-mediated NO synthesis. Interestingly, removal of extracellular Ca2+, buffering of cytosolic Ca2+ by BAPTA or inhibition of SK and IK Ca-activated K+ channels by apamin and charybdotoxin did not affect NO production in response to E2, but attenuated ATP-mediated NO synthesis. Unlike ATP or histamine, E2 did not induce hyperpolarization of endothelial cell membrane potential. These observations indicate that in human vascular endothelial cells, E2 induces rapid NO production via a cellular pathway distinct from that utilized by Ca-mobilizing stimuli. (Funded by CIHR)

Ethanol relaxes pulmonary artery by release of prostaglandin and nitric oxide

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin- and cadherin-mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO-dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex-comprised of PECAM-1, VE-cadherin and VEGFR2- and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

Mixed tocopherols inhibit platelet aggregation in humans: potential mechanisms

Recently, an isomeric mixture of herbal anti-inflammatory naphthoquinones shikonin and alkannin, and their derivatives, have been found to impair cellular responses involving nitric oxide (NO) and NO synthesis, like the acetylcholine-induced relaxation response of rat thoracic aorta and NO release from murine RAW 264.7 macrophages. However, the mechanisms of such effects, including whether NO synthase (NOS) activity is affected, remained unclear. We herein investigate possible targets of shikonin in these NOS-related events. Shikonin by itself dose-dependently inhibited the rat thoracic aorta relaxation in response to acetylcholine (pD′2 value: 6.29). Its optical enantiomer, alkannin, was equally inhibitory in the aorta relaxation–response assay. In RAW 264.7 cells, shikonin inhibited the lipopolysaccharide-induced NO production by 82% at 1 μM. A cell-free assay to verify direct effects on NOS activity showed that shikonin inhibits all isoforms of NOS (IC50 s, 4–7 μM), suggesting NOS as an inhibition target in both the events. Further possible targets of shikonin that might be involved in the inhibitions of the acetylcholine-induced aorta relaxation response and the NO generation by RAW 264.7 cells are also discussed. It is shown for the first time that shikonin inhibits NOS activity.

BackgroundNox-2 (also known as gp91phox), a subunit component of NADPH oxidases, generates reactive oxygen species (ROS). Nox-dependent ROS generation and nitric oxide (NO) release by microglia have been implicated in a variety of diseases in the central nervous system. Dexamethasone (Dex) has been shown to suppress the ROS production, NO release and inflammatory reaction of activated microglial cells. However, the underlying mechanisms remain unclear.ResultsThe present study showed that the increased ROS production and NO release in activated BV-2 microglial cells by LPS were associated with increased expression of Nox-2 and iNOS. Dex suppressed the upregulation of Nox-2 and iNOS, as well as the subsequent ROS production and NO synthesis in activated BV-2 cells. This inhibition caused by Dex appeared to be mediated by upregulation of MAPK phosphatase-1 (MKP-1), which antagonizes the activity of mitogen-activated protein kinases (MAPKs). Dex induced-suppression of Nox-2 and -upregulation of MKP-1 was also evident in the activated microglia from corpus callosum of postnatal rat brains. The overexpression of MKP-1 or inhibition of MAPKs (by specific inhibitors of JNK and p38 MAPKs), were found to downregulate the expression of Nox-2 and iNOS and thereby inhibit the synthesis of ROS and NO in activated BV-2 cells. Moreover, Dex was unable to suppress the LPS-induced synthesis of ROS and NO in BV-2 cells transfected with MKP-1 siRNA. On the other hand, knockdown of Nox-2 in BV-2 cells suppressed the LPS-induced ROS production and NO release.ConclusionIn conclusion, it is suggested that downregulation of Nox-2 and overexpression of MKP-1 that regulate ROS and NO may form the potential therapeutic strategy for the treatment of neuroinflammation in neurodegenerative diseases.

N-Nitrosamines are well established motifs to release nitric oxide (NO) under photoirradiation. Herein, a series of amphiphilic N-nitrosamine-based block copolymers (BCPx-NO) are developed to attain controlled NO release under photoirradiation (365 nm, 3.71 mW/cm2). The water-soluble BCPx-NO forms micellar architecture in aqueous medium and exhibits a sustained NO release of 92-160 μM within 11.5 h, which is 36.8-64.0% of the calculated value. To understand the NO release mechanism, a small molecular NO donor (NOD) resembling the NO releasing functional motif of BCPx-NO is synthesized, which displays a burst NO release in DMSO within 2.5 h. The radical nature of the released NO is confirmed by electron paramagnetic resonance (EPR) spectroscopy. The gradual NO release from micellar BCPx-NO enhances antibacterial activity over NOD and exhibits a superior bactericidal effect on Gram-positive Staphylococcus aureus. In relation to biomedical applications, this work offers a comprehensive insight into tuning light-triggered NO release to improve antibacterial activity.

Because the role of intracellular Ca2+ in the two-signal process for the induction of nitric oxide (NO) synthesis is controversial, this study was undertaken to examine the role of Ca2+ in the transcriptional regulation of inducible NO synthase (iNOS) in murine peritoneal macrophages. Treatment of the cells with thapsigargin (TG) or 2,5-di-(t-butyl)-1,4-benzodihydroquinone (tBuBHQ), which are the specific and potent Ca(2+)-ATPase inhibitors of endoplasmic reticulum (ER), showed modest effects on tumoricidal function, whereas TG or tBuBHQ in combination with interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) showed marked effects on tumoricidal function of the cells. The tumoricidal effects of the activated macrophages were correlated with the amount of NO synthesis, and totally abrogated by the use of NOS inhibitor, NG-monomethyl-L-arginine (NGMMA). The increases in NO synthesis was reflected as increased amounts of iNOS mRNA by Northern blotting. To confirm that iNOS induction was due to the changes in the intracellular Ca2+ level, the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca2+ chelator, was used. Blocking the increase of cytosolic free Ca2+ significantly decreased the induction of NO synthesis. To demonstrate that intracellular Ca2+ acts as a 'priming' signal rather than a 'triggering' signal on the induction of NO synthesis by murine peritoneal macrophages, we designed several experiments. When the cells were treated with TG 6 hr after the treatment with IFN-gamma, there was no increase in NO synthesis. In addition, when the cells were treated with TG or LPS 6 hr after treatment with tBuBHQ, a synergistic increase on NO synthesis was shown only in the case of LPS. When phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, was added to the cells 6 hr after the treatment with TG, there was a marked co-operative induction of NO synthesis, even though PMA alone has no effect. Based on the results obtained in this study, we suggest that cytosolic Ca2+ might be enough for the expression of iNOS gene as a priming signal and PKC might be involved in the induction of NO synthesis as a triggering signal by post-transcriptional modification of iNOS mRNA or iNOS itself in the activated murine peritoneal macrophages.

Advanced glycation end-products (AGEs) have been recognized as an important pathophysiological mechanism in endothelial dysfunction during diabetic atherogenesis. Homeobox (Hox) genes have been identified as playing a regulatory role in the adult cardiovascular system. Regulation of HoxA9EC is involved in diabetic endothelial dysfunction, but the mechanism of HoxA9EC regulation has remained undefined. Here, we sought to investigate how HoxA9EC is regulated in AGE-induced endothelial dysfunction and to explore the mechanism involved. We used human umbilical venous endothelial cells (HUVECs) cocultured with AGEs, and examined endothelial nitric oxide synthase (eNOS) activation, nitric oxide (NO) release, cell migration, and the expression of HoxA9EC and nuclear factor kappa B (NF-κB). AGEs suppressed eNOS activation, NO release, and the migration of HUVECs. Knockout of HoxA9EC also reduced eNOS activation, NO release, and the migration of HUVECs, and the enhancement of HoxA9EC improved the function of HUVECs. Furthermore, AGEs downregulated HoxA9EC expression and activated NF-κB, and the depression of HoxA9EC was significantly attenuated by the NF-κB inhibitor. On the other hand, knockout of HoxA9EC activated NF-κB and the enhancement of HoxA9EC suppressed NF-κB activation. In conclusion, AGEs could induce endothelial dysfunction through NF-κB-dependent HoxA9EC downregulation by reciprocal interaction, and the enhancement of HoxA9EC expression could attenuate the impairment.

The effects of high- and low-intensity percutaneous stimulation on nitric oxide levels and spike activity in the superficial laminae of the spinal cord

Blood coagulation response and bacterial adhesion to biomimetic polyurethane biomaterials prepared with surface texturing and nitric oxide release