Doxycycline ameliorates 2K-1C hypertension-induced vascular dysfunction in rats by attenuating oxidative stress and improving nitric oxide bioavailability

Vascular dysfunction associated with two-kidney, one-clip (2K-1C) hypertension may result from both altered matrix metalloproteinase (MMP) activity and higher concentrations of reactive oxygen species (ROS). Doxycycline is considering the most potent MMP inhibitor of tetracyclines and attenuates 2K-1C hypertension-induced high blood pressure and chronic vascular remodeling. Doxycycline might also act as a ROS scavenger and this may contribute to the amelioration of some cardiovascular diseases associated with increased concentrations of ROS. We hypothesized that in addition to its MMP inhibitory effect, doxycycline attenuates oxidative stress and improves nitric oxide (NO) bioavailability in 2K-1C hypertension, thus improving hypertension-induced arterial endothelial dysfunction. Sham operated or 2K-1C hypertensive rats were treated with doxycycline 30 mg/kg/day (or vehicle). After 8 weeks of treatment, aortic rings were isolated to assess endothelium dependent vasorelaxation to A23187. Arterial and systemic levels of ROS were respectively measured using dihydroethidine (DHE) and thiobarbituric acid reactive substances (TBARS). Neutrophils-derived ROS were tested in vitro using the fluoroprobe Carboxy-H(2)DCFDA and human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). NO levels were assessed in rat aortic endothelial cells by confocal microscopy. Aortic MMP activity was determined by in situ zymography. Doxycycline attenuated 2K-1C hypertension (169 +/- 17.3 versus 209 +/- 10.9 mm Hg in hypertensive controls, p < 0.05) and protected against hypertension-induced reduction in endothelium-dependent vasorelaxation to A23187 (p < 0.05). Doxycycline also decreased hypertension-induced oxidative stress (p <= 0.05), higher MMP activity (p < 0.01) and improved NO levels in aortic endothelial cells (p < 0.01). Therefore, doxycycline ameliorates 2K-1C hypertension-induced endothelial dysfunction in aortas by inhibiting oxidative stress generation and improving NO bioavailability, in addition to its inhibitory effects on MMP activity. (C) 2012 Elsevier Inc. All rights reserved.

alpha 1-Acid Glycoprotein Decreases Neutrophil Migration and Increases Susceptibility to Sepsis in Diabetic Mice

The mechanisms underlying immune deficiency in diabetes are largely unknown. In the present study, we demonstrate that diabetic mice are highly susceptible to polymicrobial sepsis due to reduction in rolling, adhesion, and migration of leukocytes to the focus of infection. In addition, after sepsis induction, CXCR2 was strongly downregulated in neutrophils from diabetic mice compared with nondiabetic mice. Furthermore, CXCR2 downregulation was associated with increased G-protein coupled receptor kinase 2 (GRK2) expression in these cells. Different from nondiabetic mice, diabetic animals submitted to mild sepsis displayed a significant augment in alpha 1-acid glycoprotein (AGP) hepatic mRNA expression and serum protein levels. Administration of AGP in nondiabetic mice subjected to mild sepsis inhibited the neutrophil migration to the focus of infection, as well as induced t-selectin shedding and rise in CD11b of blood neutrophils. Insulin treatment of diabetic mice reduced mortality rate, prevented the failure of neutrophil migration, impaired GRK2-mediated CXCR2 downregulation, and decreased the generation of AGP. Finally, administration of AGP abolished the effect of insulin treatment in diabetic mice. Together, these data suggest that AGP may be involved in reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice. Diabetes 61:1584-1591, 2012

Activation of Survival and Apoptotic Signaling Pathways in Lymphocytes Exposed to Palmitic Acid

The toxicity of palmitic acid (PA) towards a human T-lymphocyte cell line (Jurkat) has been previously investigated but the mechanism(s) of PA action were unknown. In the current study, Jurkat cells were treated with sub-lethal concentrations of PA (50-150 mu M) and the activity of various signaling proteins was investigated. PA-induced apoptosis and mitochondrial dysfunction in a dose-dependent manner as evaluated by DNA fragmentation assay and depolarization of the mitochondrial membrane, respectively. PA treatment provoked release of cytochrome c from the inner mitochondrial membrane to the cytosol, activated members of the MAPK protein family JNK, p38, ERK, activated caspases 3/9, and increased oxidative/nitrosative stress. Exposure of cells to PA for 12 h increased insulin receptor (IR) and GLUT-4 levels in the plasma membrane. Insulin treatment (10 mU/ml/30 min) increased the phosphorylation of the IR beta-subunit and Akt. A correlation was found between DNA fragmentation and expression levels of both IR and GLUT-4. Similar results were obtained for PA-treated lymphocytes from healthy human donors and from mesenteric lymph nodes of 48-h starved rats. PA stimulated glucose uptake by Jurkat cells (in the absence of insulin), stimulated accumulation of neutral lipids (triglyceride), and other lipid classes (phospholipids and cholesterol ester) but reduced glucose oxidation. Our results suggest that parameters of insulin signaling and non-oxidative glucose metabolism are stimulated as part of a coordinated response to prompt survival in lymphocytes exposed to PA but at higher concentrations, apoptosis prevails. These findings may explain aspects of lymphocyte dysfunction associated with diabetes. J. Cell. Physiol. 227: 339-350, 2012. (C) 2011 Wiley Periodicals, Inc.

Differential role of CB1 and TRPV1 receptors on anandamide modulation of defensive responses induced by nitric oxide in the dorsolateral periaqueductal gray

CB1, TRPV1 and NO can regulate glutamate release and modify defensive behaviors in regions related to defensive behavior such as the dorsolateral periaqueductal gray (dIPAG). A possible interaction between the endocannabinoid and nitrergic systems in this area, however, has not been investigated yet. The objective of the present work was to verify if activation of CB1 or TRPV1 receptors could interfere in the flight responses induced in rats by the injection of SIN-1, an NO donor, into the dIPAG. The results showed that local administration of a low dose (5 pmol) of anandamide (AEA) attenuated the flight responses, measured by the total distance moved and maximum speed in an open arena, induced by intra-dIPAG microinjection of SIN-1 (150 nmol). URB597 (0.1 nmol), an inhibitor of anandamide metabolism, produced similar effects. When animals were locally treated with the CB1 receptor antagonist AM251 the effective AEA dose (5 pmol) increased, rather than decreased, the flight reactions induced by SIN1-1. Higher (50-200 nmol) doses of AEA were ineffective and even tended to potentiate the SIN-1 effect. The TRPV1 antagonist capsazepine (CPZ, 30 nmol) prevented SIN-1 effects and attenuated the potentiation of its effect by the higher (200 nmol) AEA dose. The results indicate that AEA can modulate in a dual way the pro-aversive effects of NO in the dIPAG by activating CB1 or TRPV1 receptors. (C) 2012 Elsevier Ltd. All rights reserved.

The relationship between training status, blood pressure and uric acid in adults and elderly

Abstract Background Hypertension can be generated by a great number of mechanisms including elevated uric acid (UA) that contribute to the anion superoxide production. However, physical exercise is recommended to prevent and/or control high blood pressure (BP). The purpose of this study was to investigate the relationship between BP and UA and whether this relationship may be mediated by the functional fitness index. Methods All participants (n = 123) performed the following tests: indirect maximal oxygen uptake (VO2max), AAHPERD Functional Fitness Battery Test to determine the general fitness functional index (GFFI), systolic and diastolic blood pressure (SBP and DBP), body mass index (BMI) and blood sample collection to evaluate the total-cholesterol (CHOL), LDL-cholesterol (LDL-c), HDL-cholesterol (HDL-c), triglycerides (TG), uric acid (UA), nitrite (NO2) and thiobarbituric acid reactive substances (T-BARS). After the physical, hemodynamic and metabolic evaluations, all participants were allocated into three groups according to their GFFI: G1 (regular), G2 (good) and G3 (very good). Results Baseline blood pressure was higher in G1 when compared to G3 (+12% and +11%, for SBP and DBP, respectively, p<0.05) and the subjects who had higher values of BP also presented higher values of UA. Although UA was not different among GFFI groups, it presented a significant correlation with GFFI and VO2max. Also, nitrite concentration was elevated in G3 compared to G1 (140±29 μM vs 111± 29 μM, for G3 and G1, respectively, p<0.0001). As far as the lipid profile, participants in G3 presented better values of CHOL and TG when compared to those in G1. Conclusions Taking together the findings that subjects with higher BP had elevated values of UA and lower values of nitrite, it can be suggested that the relationship between blood pressure and the oxidative stress produced by acid uric may be mediated by training status.

NCX-1000, a nitric oxide-releasing derivative of UDCA, does not decrease portal pressure in patients with cirrhosis: results of a randomized, double-blind, dose-escalating study

NCX-1000 (2(acetyloxy) benzoic acid-3(nitrooxymethyl)phenyl ester) is an nitric oxide (NO)-releasing derivative of ursodeoxycholic acid (UDCA), which showed selective vasodilatory effect on intrahepatic circulation in animal models of cirrhosis. This study was aimed at testing the efficacy and tolerability of this compound in patients with cirrhosis and portal hypertension.

Neurotoxicity of glia activated by gram-positive bacterial products depends on nitric oxide production

The present study examined the mechanism by which bacterial cell walls from two gram-positive meningeal pathogens, Streptococcus pneumoniae and the group B streptococcus, induced neuronal injury in primary cultures of rat brain cells. Cell walls from both organisms produced cellular injury to similar degrees in pure astrocyte cultures but not in pure neuronal cultures. Cell walls also induced nitric oxide production in cultures of astrocytes or microglia. When neurons were cultured together with astrocytes or microglia, the cell walls of both organisms became toxic to neurons. L-NAME, a nitric oxide synthase inhibitor, protected neurons from cell wall-induced toxicity in mixed cultures with glia, as did dexamethasone. In contrast, an excitatory amino acid antagonist (MK801) had no effect. Low concentrations of cell walls from either gram-positive pathogen added together with the excitatory amino acid glutamate resulted in synergistic neurotoxicity that was inhibited by L-NAME. The induction of nitric oxide production and neurotoxicity by cell walls was independent of the presence of serum, whereas endotoxin exhibited these effects only in the presence of serum. We conclude that gram-positive cell walls can cause toxicity in neurons by inducing the production of nitric oxide in astrocytes and microglia.

The SLC3 and SLC7 families of amino acid transporters

Amino acids are necessary for all living cells and organisms. Specialized transporters mediate the transfer of amino acids across plasma membranes. Malfunction of these proteins can affect whole-body homoeostasis giving raise to diverse human diseases. Here, we review the main features of the SLC3 and SLC7 families of amino acid transporters. The SLC7 family is divided into two subfamilies, the cationic amino acid transporters (CATs), and the L-type amino acid transporters (LATs). The latter are the light or catalytic subunits of the heteromeric amino acid transporters (HATs), which are associated by a disulfide bridge with the heavy subunits 4F2hc or rBAT. These two subunits are glycoproteins and form the SLC3 family. Most CAT subfamily members were functionally characterized and shown to function as facilitated diffusers mediating the entry and efflux of cationic amino acids. In certain cells, CATs play an important role in the delivery of L-arginine for the synthesis of nitric oxide. HATs are mostly exchangers with a broad spectrum of substrates and are crucial in renal and intestinal re-absorption and cell redox balance. Furthermore, the role of the HAT 4F2hc/LAT1 in tumor growth and the application of LAT1 inhibitors and PET tracers for reduction of tumor progression and imaging of tumors are discussed. Finally, we describe the link between specific mutations in HATs and the primary inherited aminoacidurias, cystinuria and lysinuric protein intolerance.

T-786C polymorphism of the endothelial nitric oxide synthase gene and neuralgia-inducing cavitational osteonecrosis of the jaws.

OBJECTIVE: We hypothesized that, similar to idiopathic hip osteonecrosis, the T-786C mutation of the endothelial nitric oxide synthase (eNOS) gene affecting nitric oxide (NO) production was associated with neuralgia-inducing cavitational osteonecrosis of the jaws (NICO). DESIGN: In 22 NICO patients, not having taken bisphosphonates, mutations affecting NO production (eNOS T-786C, stromelysin 5A6A) were measured by polymerase chain reaction. Two healthy normal control subjects were matched per case by race and gender. RESULTS: Homozygosity for the mutant eNOS allele (TT) was present in 6 out of 22 patients (27%) with NICO compared with 0 out of 44 (0%) race and gender-matched control subjects; heterozygosity (TC) was present in 8 patients (36%) versus 15 control subjects (34%); and the wild-type normal genotype (CC) was present in 9 patients (36%) versus 29 controls (66%) (P = .0008). The mutant eNOS T-786C allele was more common in cases (20 out of 44 [45%]) than in control subjects (15 out of 88 [17%]) (P = .0005). The distribution of the stromelysin 5A6A genotype in cases did not differ from control subjects (P = .13). CONCLUSIONS: The eNOS T-786C polymorphism affecting NO production is associated with NICO, may contribute to the pathogenesis of NICO, and may open therapeutic medical approaches to treatment of NICO through provision of L-arginine, the amino-acid precursor of NO.

Glutamate and nitric oxide as regulatory transmitters in developing rabbit retina

Glutamate is the major excitatory neurotransmitter in the retina and serves as the synaptic messenger for the three classes of neurons which constitute the vertical pathway--the photoreceptors, bipolar cells and ganglion cells. In addition, the glutamate system has been localized morphologically, pharmacologically as well as molecularly during the first postnatal week of development before synaptogenesis occurs. The role which glutamate plays in the maturing visual system is complex but ranges from mediating developmental neurotoxicity to inducing neurite outgrowth.^ Nitric oxide/cGMP is a novel intercellular messenger which is thought to act in concert with the glutamate system in regulating a variety of cellular processes in the brain as well as retina, most notably neurotoxicity. Several developmental activities including programmed cell death, synapse elimination and synaptic reorganization are possible functions of cellular regulation modulated by nitric oxide as well as glutamate.^ The purpose of this thesis is to (1) biochemically characterize the endogenous pools of glutamate and determine what fraction exists extracellularly; (2) examine the morphological expression of NO producing cells in developing retina; (3) test the functional coupling of the NMDA subtype of glutamate receptor to the NO system by examining neurotoxicity which has roles in both the maturing and adult retina.^ Biochemical sampling of perfusates collected from the photoreceptor surface of ex vivo retina demonstrated that although the total pool of glutamate present at birth is relatively modest, a high percentage resides in extracellular pools. As a result, immature neurons without significant synaptic connections survive and develop in a highly glutamatergic environment which has been shown to be toxic in the adult retina.^ The interaction of the glutamate system with the NO system has been postulated to regulate neuronal survival. We therefore examined the developmental expression of the enzyme responsible for producing NO, nitric oxide synthase (NOS), using an antibody to the constitutive form of NOS found in the brain. The neurons thought to produce the majority of NO in the adult retina, a subpopulation of widefield amacrine cells, were not immunoreactive until the end of the second postnatal week. However, a unique developmental expression was observed in the ganglion cell layer and developing outer nuclear layer of the retina during the first postnatal week. We postulate NO producing neurons may not be present in a mature configuration therefore permitting neuronal survival in a highly glutamatergic microenvironment and allowing NO to play a development-specific role at this time.^ The next set of experiments constituted a functional test of the hypothesis that the absence of the prototypic NO producing cells in developing retina protects immature neurons against glutamate toxicity. An explant culture system developed in order to examine cellular responses of immature and adult neurons to glutamate toxicity showed that immature neurons were affected by NMDA but were less responsive to NMDA and NO mediated toxicity. In contrast, adult explants exhibited significant NMDA toxicity which was attenuated by NMDA antagonists, 2-amino-5-phosphonovaleric acid (APV), dextromethorphan (Dex) and N$\rm\sp{G}$-D-methyl arginine (metARG). These results indicated that pan-retinal neurotoxicity via the NMDA receptor and/or NO activation occurred in the adult retina but was not significant in the neonate. (Abstract shortened by UMI.) ^

Calcium-independent activation of endothelial nitric oxide synthase by ceramide

The endothelial isoform of NO synthase (eNOS) is targeted to sphingolipid-enriched signal-transducing microdomains in the plasma membrane termed caveolae. Among the caveolae-targeted sphingolipids are the ceramides, a class of acylated sphingosine compounds that have been implicated in diverse cellular responses. We have explored the role of ceramide analogues in eNOS signaling in cultured bovine aortic endothelial cells (BAEC). Addition of the ceramide analogue N-acetylsphingosine (C2-ceramide; 5 μM) to intact BAEC leads to a significant increase in NO synthase activity (assayed by using the fluorescent indicator 4,5-diaminofluorescein) and translocation of eNOS from the endothelial cell membrane to intracellular sites (measured by using quantitative immunofluorescence techniques); the biologically inactive ceramide N-acetyldihydrosphingosine is entirely without effect. C2-ceramide-induced eNOS activation and translocation are unaffected by the intracellular calcium chelator 1,2-bis-o-aminophenoxyethane-N,N,N′,N′-tetraacetic acid (BAPTA). Using the calcium-specific fluorescent indicator fluo-3, we also found that C2-ceramide activation of eNOS is unaccompanied by a drug-induced increase in intracellular calcium. These findings stand in sharp contrast to the mechanism by which bradykinin, estradiol, and other mediators acutely activate eNOS, in which a rapid, agonist-promoted increase in intracellular calcium is required. Finally, we show that treatment of BAEC with bradykinin causes a significant increase in cellular ceramide content; the response to bradykinin has an EC50 of 3 nM and is blocked by the bradykinin B2-receptor antagonist HOE140. Bradykinin-induced ceramide generation could represent a mechanism for longer-term regulation of eNOS activity. Our results suggest that ceramide functions independently of Ca2+-regulated pathways to promote activation and translocation of eNOS, and that this lipid mediator may represent a physiological regulator of eNOS in vascular endothelial cells.

Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis

Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes’ peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or by in situ generation of peroxynitrite from NO coupling to superoxide anion. Cu,Zn-superoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generated in situ from superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid)2, and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inflammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.

Microtubule dysfunction by posttranslational nitrotyrosination of α-tubulin: A nitric oxide-dependent mechanism of cellular injury

NO2Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO2Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO2Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of α-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin–tyrosine ligase. In contrast to the enzymatically regulated carboxyl-terminal tyrosination/detyrosination cycle of α-tubulin, incorporation of NO2Tyr shows apparent irreversibility. Nitrotyrosination of α-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelial-barrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of α-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of α-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motor- and microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO2Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the α-tubulin tyrosination/detyrosination cycle plays in microtubule function.

Nerve growth factor rapidly suppresses basal, NMDA-evoked, and AMPA-evoked nitric oxide synthase activity in rat hippocampus in vivo

In adult forebrain, nerve growth factor (NGF) influences neuronal maintenance and axon sprouting and is neuroprotective in several injury models through mechanisms that are incompletely understood. Most NGF signaling is thought to occur after internalization and retrograde transport of trkA receptor and be mediated through the nucleus. However, NGF expression in hippocampus is rapidly and sensitively regulated by synaptic activity, suggesting that NGF exerts local effects more dynamically than possible through signaling requiring retrograde transport to distant afferent neurons. Interactions have been reported between NGF and nitric oxide (NO). Because NO affects both neural plasticity and degeneration, and trk receptors can mediate signaling within minutes, we hypothesized that NGF might rapidly modulate NO production. Using in vivo microdialysis we measured conversion of l-[14C]arginine to l-[14C]citrulline as an accurate reflection of NO synthase (NOS) activity in adult rat hippocampus. NGF significantly reduced NOS activity to 61% of basal levels within 20 min of onset of delivery and maintained NOS activity at less than 50% of baseline throughout 3 hr of delivery. This effect did not occur with control protein (cytochrome c) and was not mediated by an effect of NGF on glutamate levels. In addition, simultaneous delivery of NGF prevented significant increases in NOS activity triggered by the glutamate receptor agonists N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Rapid suppression by NGF of basal and glutamate-stimulated NOS activity may regulate neuromodulatory functions of NO or protect neurons from NO toxicity and suggests a novel mechanism for rapidly mediating functions of NGF and other neurotrophins.

An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity

Caspases are key mediators in liver inflammation and apoptosis. In the present study we provide evidence that a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA), NCX-1000 ([2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester]), protects against liver damage in murine models of autoimmune hepatitis induced by i.v. injection of Con A or a Fas agonistic antibody, Jo2. Con A administration causes CD4+ T lymphocytes to accumulate in the liver and up-regulates FasL expression, resulting in FasL-mediated cytotoxicity. Cotreating mice with NCX-1000, but not with UDCA, protected against liver damage induced by Con A and Jo2, inhibited IL-1β, IL-18, and IFN-γ release and caspase 3, 8, and 9 activation. Studies on HepG2 cells demonstrated that NCX-1000, but not UDCA, directly prevented multiple caspase activation induced by Jo2. Incubating HepG2 cells with NCX-1000 resulted in intracellular NO formation and a DTT-reversible inhibition of proapoptotic caspases, suggesting that cysteine S-nitrosylation was the main mechanism responsible for caspase inhibition. Collectively, these data suggest that NCX-1000 protects against T helper 1-mediated liver injury by inhibiting both the proapoptotic and the proinflammatory branches of the caspase superfamily.