L-canavanine, an inhibitor of inducible nitric oxide synthase, improves venous return in endotoxemic rats.

OBJECTIVE: To investigate the hemodynamic effects of L-canavanine (an inhibitor of inducible, but not of constitutive, nitric oxide synthase) in endotoxic shock. DESIGN: Controlled, randomized, experimental study. SETTING: Animal laboratory. SUBJECTS: Wistar rats. INTERVENTIONS: Rats were anesthetized with pentobarbital, and hemodynamically monitored. One hour after an intravenous challenge with 5 mg/kg of Escherichia coli endotoxin, the rats were randomized to receive a continuous infusion of either L-canavanine (20 mg/kg/hr; n = 8) or vehicle only (isotonic saline, n = 11). In all animals, the infusion was given over 5 hrs at a rate of 2 mL/kg/hr. These experiments were repeated in additional rats challenged with isotonic saline instead of endotoxin (sham experiments). MEASUREMENTS AND MAIN RESULTS: Arterial blood pressure, heart rate, thermodilution cardiac output, central venous pressure, mean systemic filling pressure, urine output, arterial blood gases, blood lactate concentration, and hematocrit were measured. In sham experiments, hemodynamic stability was maintained throughout and L-canavanine had no detectable effect. Animals challenged with endotoxin and not treated with L-canavanine developed progressive hypotension and low cardiac output. After 6 hrs of endotoxemia, both central venous pressure and mean systemic filling pressure were significantly below their baseline values, indicating relative hypovolemia as the main determinant of reduced cardiac output. In endotoxemic animals treated with L-canavanine, hypotension was less marked, while cardiac output, central venous pressure, and mean systemic filling pressure were maintained throughout the experiment. L-canavanine had no effect on the time-course of hematocrit. L-canavanine significantly increased urine output and reduced the severity of lactic acidosis. CONCLUSIONS: Six hours after an endotoxin challenge in rats, low cardiac output develops, which appears to be primarily related to relative hypovolemia. L-canavanine, a selective inhibitor of the inducible nitric oxide synthase, increases the mean systemic filling pressure, thereby improving venous return, under these conditions.

Long-term swimming exercise does not modulate the Akt-dependent endothelial nitric oxide synthase phosphorylation in healthy mice.

Molecular mechanisms by which exercise exerts cardiovascular benefits are poorly understood. Exercise-induced increase of endothelial NO synthase (eNOS) phosphorylation through the protein kinase Akt has been shown to be a key mechanism underlying the beneficial effect of exercise in coronary artery disease patients. We examined whether this protective pathway might also be activated in long-term-exercised healthy mice. C57BL/6 wild-type mice swam for 24 weeks. A group of sedentary animals were used as controls. Aortic levels of total protein kinase Akt (protein kinase B), phosphorylated Akt at ser473 (p-Akt), total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), and PECAM-1 (platelet endothelial cell adhesion molecule-1) were assessed by Western blotting. Protein expressions of Akt, p-Akt, eNOS, p-eNOS, and PECAM-1 were not modulated by 24 weeks of exercise. The Akt-dependent eNOS phosphorylation did not seem to be a primary molecular adaptation in response to long-term exercise in healthy mice.

Nitric oxide reduces Cl- absorption in the mouse cortical collecting duct through an ENaC-dependent mechanism.

Since nitric oxide (NO) participates in the renal regulation of blood pressure, in part, by modulating transport of Na(+) and Cl(-) in the kidney, we asked whether NO regulates net Cl(-) flux (JCl) in the cortical collecting duct (CCD) and determined the transporter(s) that mediate NO-sensitive Cl(-) absorption. Cl(-) absorption was measured in CCDs perfused in vitro that were taken from aldosterone-treated mice. Administration of an NO donor (10 μM MAHMA NONOate) reduced JCl and transepithelial voltage (VT) both in the presence or absence of angiotensin II. However, reducing endogenous NO production by inhibiting NO synthase (100 μM N(G)-nitro-l-arginine methyl ester) increased JCl only in the presence of angiotensin II, suggesting that angiotensin II stimulates NO synthase activity. To determine the transport process that mediates NO-sensitive changes in JCl, we examined the effect of NO on JCl following either genetic ablation or chemical inhibition of transporters in the CCD. Since the application of hydrochlorothiazide (100 μM) or bafilomycin (5 nM) to the perfusate or ablation of the gene encoding pendrin did not alter NO-sensitive JCl, NO modulates JCl independent of the Na(+)-dependent Cl(-)/HCO3(-) exchanger (NDCBE, Slc4a8), the A cell apical plasma membrane H(+)-ATPase and pendrin. In contrast, both total and NO-sensitive JCl and VT were abolished with application of an epithelial Na(+) channel (ENaC) inhibitor (3 μM benzamil) to the perfusate. We conclude that NO reduces Cl(-) absorption in the CCD through a mechanism that is ENaC-dependent.

Decreased intraocular pressure induced by nitric oxide donors is correlated to nitrite production in the rabbit eye.

PURPOSE: To evaluate the effect of intraocular administration of nitric oxide (NO) donors in the rabbit eye on intraocular pressure (IOP), inflammation, and toxicity. METHODS: Intravitreal and intracameral injections of two NO donors, SIN-1 and SNAP, and SIN-1C and BSS were performed. Clinical examination, IOP measurements, protein evaluation in aqueous humor, and histologic analysis of the ocular globes were realized. Nitric oxide release was demonstrated by nitrite production in the aqueous humor and in the vitreous using the Griess reaction. RESULTS: The drastic decrease of IOP, observed after a single NO donor injection, was correlated directly with nitrite production and, thus, to NO release. Injection of inactive metabolite of SIN-1, SIN-1C, which is not able to release NO, did not modulate IOP. When administered in the aqueous humor or in the vitreous, NO did not diffuse from one segment of the eye to another. No inflammation or histologic damage was observed as a result of a single NO donor administration. CONCLUSIONS: Nitric oxide is implicated directly in the regulation of IOP and its acute, and massive release into the rabbit eye did not induce inflammation or other growth toxic effects on the ocular tissues.

Nitric oxide activity through guanylate cyclase and phosphodiesterase modulation is impaired in fetal lambs with congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension and death. Administration of nitric oxide (NO) alone remains ineffective in CDH cases. We investigated in near full-term lambs with and without CDH the role of guanylate cyclase (GC), the enzyme activated by NO in increasing cyclic 3'-5'-guanylosine monophosphate, and the role of phosphodiesterase (PDE) 5, the enzyme-degrading cyclic 3'-5'-guanylosine monophosphate. METHODS: Congenital diaphragmatic hernia was surgically created in fetal lambs at 85 days of gestation. Pulmonary hemodynamics were assessed by means of pressure and blood flow catheters (135 days). In vitro, we tested drugs on rings of isolated pulmonary vessels. RESULTS: In vivo, sodium nitroprusside, a direct NO donor, and methyl-2(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5 trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032) and Zaprinast, both PDE 5 blockers, reduced pulmonary vascular resistance in CDH and non-CDH animals. The activation of GC by sodium nitroprusside and the inhibition of PDE 5 by T-1032 were less effective in CDH animals. In vitro, the stimulation of GC by 3(5'hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) (a benzyl indazole derivative) and the inhibition of PDE 5 by T-1032 were less effective in pulmonary vascular rings from CDH animals. The YC-1-induced vasodilation in rings from CDH animals was higher when associated with the PDE 5 inhibitor T-1032. CONCLUSIONS: Guanylate cyclase and PDE 5 play a role in controlling pulmonary vascular tone in fetal lambs with or without CDH. Both enzymes seem to be impaired in fetal lambs with CDH.

Ventricular but not atrial electro-mechanical delay of the embryonic heart is altered by anoxia-reoxygenation and improved by nitric oxide.

BACKGROUND/AIM: Excitation-contraction coupling is modulated by nitric oxide (NO) which otherwise has either beneficial or detrimental effects on myocardial function during hypoxia-reoxygenation. This work aimed at characterizing the variations of electromechanical delay (EMD) induced by anoxia-reoxygenation within the developing heart and determining whether atrial and ventricular EMD are modulated by NO to the same extent. METHODS: Hearts of 4 or 4.5-day-old chick embryos were excised and submitted in vitro to normoxia (45 min), anoxia (30 min) and reoxygenation (60 min). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout experiment. Anoxia-reoxygenation-induced chrono-, dromo-and inotropic disturbances and changes in EMD in atrium (EMDa) and ventricle (EMDv) were investigated in control hearts and in hearts exposed to 0.1, 1, 10, 50 and 100 microM of DETA-NONOate (a NO donating agent) or to 50 microM of L-NAME (a NOS inhibitor). RESULTS: Under normoxia, heart rate, PR interval, ventricular shortening velocity, EMDa and EMDv were similar in control, L-NAME-treated and DETA-NONOate-treated hearts. Under anoxia, cardiac activity became markedly erratic within less than 10 min in all groups. At the onset of reoxygenation, EMDv was increased by about 300% with respect to the preanoxic value while EMDa did not vary significatively. Compared to control conditions, L-NAME or DETA-NONOate had no influence on the negative chrono-, dromo- and inotropic effects induced by anoxia-reoxygenation. However, L-NAME prolonged EMDv during anoxia and delayed EMDv recovery during reoxygenation while 100 microM DETA-NONOate had the opposite effects. EMDa was neither affected by NOS inhibitor nor NO donor. At the end of reoxygenation, all the investigated parameters returned to their basal values. CONCLUSION: This work provides evidence that a NO-dependent pathway is involved in regulation of the ventricular excitation-contraction coupling in the anoxic-reoxygenated developing heart.

Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis.

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.

Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.

Local transient myocardial liposomal gene transfer of inducible nitric oxide synthase does not aggravate myocardial function and fibrosis and leads to moderate neovascularization in chronic myocardial ischemia in pigs.

Microcirculation (2010) 17, 69-78. doi: 10.1111/j.1549-8719.2010.00002.x Abstract Background: This study was designed to explore the effect of transient inducible nitric oxide synthase (iNOS) overexpression via cationic liposome-mediated gene transfer on cardiac function, fibrosis, and microvascular perfusion in a porcine model of chronic ischemia. Methods and Results: Chronic myocardial ischemia was induced using a minimally invasive model in 23 landrace pigs. Upon demonstration of heart failure, 10 animals were treated with liposome-mediated iNOS-gene-transfer by local intramyocardial injection and 13 animals received a sham procedure to serve as control. The efficacy of this iNOS-gene-transfer was demonstrated for up to 7 days by reverse transcriptase-polymerase chain reaction in preliminary studies. Four weeks after iNOS transfer, magnetic resonance imaging showed no effect of iNOS overexpression on cardiac contractility at rest and during dobutamine stress (resting ejection fraction: control 27%, iNOS 26%; P = ns). Late enhancement, infarct size, and the amount of fibrosis were similar between groups. Although perfusion and perfusion reserve in response to adenosine and dobutamine were not significantly modified by iNOS-transfer, both vessel number and diameter were significantly increased in the ischemic area in the iNOS-treated group versus control (point score: control 15.3, iNOS 34.7; P < 0.05). Conclusions: Our findings demonstrate that transient iNOS overexpression does not aggravate cardiac dysfunction or postischemic fibrosis, while potentially contributing to neovascularization in the chronically ischemic heart.

Intrauterine growth restriction is associated with structural alterations in human umbilical cord and decreased nitric oxide-induced relaxation of umbilical vein.

INTRODUCTION: Intrauterine growth restriction (IUGR) affects ∼8% of all pregnancies and is associated with major perinatal mortality and morbidity, and with an increased risk to develop cardiovascular diseases in adulthood. Despite identification of several risk factors, the mechanisms implicated in the development of IUGR remain poorly understood. In case of placental insufficiency, reduced delivery of oxygen and/or nutrients to the fetus could be associated with alterations in the umbilical circulation, contributing further to the impairment of maternal-fetal exchanges. We compared the structural and functional properties of umbilical cords from growth-restricted and appropriate for gestational age (AGA) term newborns, with particular attention to the umbilical vein (UV). METHODS: Human umbilical cords were collected at delivery. Morphological changes were investigated by histomorphometry, and UV's reactivity by pharmacological studies. RESULTS: Growth-restricted newborns displayed significantly lower growth parameters, placental weight and umbilical cord diameter than AGA controls. Total cross-section and smooth muscle areas were significantly smaller in UV of growth-restricted neonates than in controls. Maximal vasoconstriction achieved in isolated UV was lower in growth-restricted boys than in controls, whereas nitric oxide-induced relaxation was significantly reduced in UV of growth-restricted girls compared to controls. CONCLUSION: IUGR is associated with structural alterations of the UV in both genders, and with a decreased nitric oxide-induced relaxation in UV of newborn girls, whereas boys display impaired vasoconstriction. Further investigations will allow to better understand the regulation of umbilical circulation in growth-restricted neonates, which could contribute to devise potential novel therapeutic strategies to prevent or limit the development of IUGR.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro.

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Neuronal nitric oxide synthase does not contribute to the modulation of pulmonary vascular tone in fetal lambs with congenital diaphragmatic hernia (nNOS in CDH lambs).

AIM: The aim of this study was to determine the presence of the neuronal nitric oxide synthase (nNOS) in near full-term lambs with congenital diaphragmatic hernia (CDH) and its role in the modulation of pulmonary vascular basal tone. METHODS: We surgically created diaphragmatic hernia on the 85th day of gestation. On the 135th, catheters were used to measure pulmonary pressure and blood flow. We tested the effects of 7-nitroindazole (7-NINA), a specific nNOS antagonist and of N-nitro-L-arginine (L-NNA), a nonspecific nitric oxide synthase antagonist. In vitro, we tested the effects of the same drugs on isolated pulmonary vessels. The presence of nNOS protein in the lungs was detected by Western blot analysis. RESULTS: Neither 7-NINA nor L-NNA modified pulmonary vascular basal tone in vivo. After L-NNA injection, acetylcholine (ACh) did not decrease significantly pulmonary vascular resistance (PVR). In vitro, L-NNA increased the cholinergic contractile-response elicited by electric field stimulation (EFS) of vascular rings from lambs with diaphragmatic hernia. CONCLUSION: We conclude that nNOS protein is present in the lungs and pulmonary artery of near full-term lamb fetuses with diaphragmatic hernia, but that it does not contribute to the reduction of pulmonary vascular tone at birth

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide.

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1-5 mM), or NO donor (DETA NONOate 10-100 microM). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 microM had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 microM decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.

Increased Expression of Renal Cyclooxygenase-2 and Neuronal Nitric Oxide Synthase in Hypertensive Cx40-Deficient Mice.

Cx40-deficient mice (Cx40-/-) are hypertensive due to increased renin secretion. We evaluated the renal expression of neuronal nitric oxide synthase (nNOS) and cyclooxygenases COX-1 and COX-2, three macula densa enzymes. The levels of nNOS were increased in kidneys of Cx40-/- mice, as well as in those of wild-type (WT) mice subjected to the two-kidney one-clip model of hypertension. In contrast, the levels of COX-2 expression were only increased in the hypoperfused kidney of Cx40-/- mice. Treatment with indomethacin lowered blood pressure and renin mRNA in Cx40-/- mice without affecting renin levels, indicating that changes in COX-2 do not cause the altered secretion of renin. Suppression of NOS activity by N(G)-nitro-L-arginine methyl ester (L-NAME) decreased renin levels in Cx40-/- animals, indicating that NO regulates renin expression in the absence of Cx40. Treatment with candesartan normalized blood pressure in Cx40-/- mice, and decreased the levels of both COX-2 and nNOS. After a treatment combining candesartan and L-NAME, the blood pressure of Cx40-/- mice was higher than that of WT mice, showing that NO may counterbalance the vasoconstrictor effects of angiotensin II in Cx40-/- mice. These data document that renal COX-2 and nNOS are differentially regulated due to the elevation of renin-dependent blood pressure in mice lacking Cx40.