Nitric Oxide-dependent Mechanism Research Articles

Pharmacological and biological screening of ascorbigen: protection against glucose‐induced endothelial cell toxicity

Cruciferous vegetables contain significant amounts of ascorbigen and related substances with known molecular structures. This study tested the hypothesis that ascorbigen demonstrates antioxidant properties and protects human umbilical cord endothelial cells against hyperglycemic toxicity in vitro. It was observed that ascorbigen, in micromolar concentrations, protected against endothelial cell death from glucose toxicity. Additionally, ascorbigen at 3.0 mm shifted the concentration response curve of l-phenylephrine to the right, with a reduction in the maximal contractile effects of the agonist. This action was not related to alpha-adrenoceptor blockade. Ascorbigen also relaxed the vascular tone induced by l-phenylephrine, which is not mediated by an endothelial cell nitric oxide-dependent mechanism. On the guinea-pig ileum, the spasmogenic effects of carbachol, histamine and serotonin were reduced in the presence of 3 mM ascorbigen. Spasm of the gut induced by the acetylcholinesterase inhibitor, physostigmine, was antagonized by ascorbigen with an IC50 of 286 microM. This natural product also has a weak antiparasitic activity. The cytoprotective effects of ascorbigen may be highly relevant in the optimum physiological regulation of the function and the therapeutic value of this substance in disease settings needs to be further investigated.

Selective estrogen receptor-α and estrogen receptor-β agonists rapidly decrease pulmonary artery vasoconstriction by a nitric oxide-dependent mechanism

Both endogenous and exogenous estrogen decrease pulmonary artery (PA) vasoconstriction. Whether these effects are mediated via estrogen receptor (ER)-alpha or ER-beta, and whether the contribution of ERs is stimulus-dependent, remains unknown. We hypothesized that administration of the selective ER-alpha agonist propylpyrazole triol (PPT) and/or the selective ER-beta agonist diarylpropiolnitrile (DPN) rapidly decreases PA vasoconstriction induced by pharmacologic and hypoxic stimuli via a nitric oxide (NO)-dependent mechanism. PA rings (n = 3-10/group) from adult male Sprague-Dawley rats were suspended in physiologic organ baths. Force displacement was measured. Vasoconstrictor responses to phenylephrine (10(-8)M - 10(-5)M) and hypoxia (Po(2) 35-45 mmHg) were determined. Endothelium-dependent and -independent vasorelaxation were measured by generating dose-response curves to acetylcholine (10(-8)M - 10(-4)M) and sodium nitroprusside (10(-9)M - 10(-5)M). PPT or DPN (10(-9)M - 5 x 10(-5)M) were added to the organ bath in the presence and absence of the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) (10(-4)M). Selective ER-alpha activation (PPT, 5 x 10(-5)M) rapidly (<20 min) decreased phenylephrine-induced vasoconstriction. This effect, as well as PPT's effects on endothelium-dependent vasorelaxation, were neutralized by l-NAME. In contrast, selective ER-beta activation (DPN, 5 x 10(-5)M) rapidly decreased phase II of hypoxic pulmonary vasoconstriction (HPV). l-NAME eliminated this phenomenon. Lower PPT or DPN concentrations were less effective. We conclude that both ER-alpha and ER-beta decrease PA vasoconstriction. The immediate onset of effect suggests a nongenomic mechanism. The contribution of specific ERs appears to be stimulus specific, with ER-alpha primarily modulating phenylephrine-induced vasoconstriction, and ER-beta inhibiting HPV. NO inhibition eliminates these effects, suggesting a central role for NO in mediating the pulmonary vascular effects of both ER-alpha and ER-beta.

Near infrared light protects cardiomyocytes from hypoxia and reoxygenation injury by a nitric oxide dependent mechanism

Photobiomodulation with near infrared light (NIR) provides cellular protection in various disease models. Previously, infrared light emitted by a low-energy laser has been shown to significantly improve recovery from ischemic injury of the canine heart. The goal of this investigation was to test the hypothesis that NIR (670 nm) from light emitting diodes produces cellular protection against hypoxia and reoxygenation-induced cardiomyocyte injury. Additionally, nitric oxide (NO) was investigated as a potential cellular mediator of NIR. Our results demonstrate that exposure to NIR at the time of reoxygenation protects neonatal rat cardiomyocytes and HL-1 cells from injury, as assessed by lactate dehydrogenase release and MTT assay. Similarly, indices of apoptosis, including caspase 3 activity, annexin binding and the release of cytochrome c from mitochondria into the cytosol, were decreased after NIR treatment. NIR increased NO in cardiomyocytes, and the protective effect of NIR was completely reversed by the NO scavengers carboxy-PTIO and oxyhemoglobin, but only partially blocked by the NO synthase (NOS) inhibitor L-NMMA. Mitochondrial metabolism, measured by ATP synthase activity, was increased by NIR, and NO-induced inhibition of oxygen consumption with substrates for complex I or complex IV was reversed by exposure to NIR. Taken together these data provide evidence for protection against hypoxia and reoxygenation injury in cardiomyocytes by NIR in a manner that is dependent upon NO derived from NOS and non-NOS sources.

Inhibition of rat aortic smooth muscle contraction by 2-methoxyestradiol

Recent studies suggest that 2-methoxyestradiol (2-ME), an estrogen metabolite, has a similar inhibitory effect as 17beta-estradiol (E2) on vascular tone. However, it is not known whether 2-ME mediates the effects of E2 or by what mechanism 2-ME regulates smooth muscle contraction. Therefore, we compared the effects of 2-ME and E2 on rat aortic smooth muscle contraction. A preincubation with 2-ME (10 microM) for 1 h inhibited phenylephrine (PE)-induced tension in endothelium-intact, but not -denuded, tissues, whereas E2 inhibited PE-induced contraction in both preparations. The effects of 2-ME and E2 on endothelium-intact preparations were prevented by L-NAME hydrochloride (a nitric oxide synthase inhibitor). The 2-ME treatment reduced PE-induced phosphorylation of the 20-kDa myosin regulatory light chain. The inhibitory effects of 2-ME and E2 were not affected by ICI-182780 (an estrogen receptor antagonist) or actinomycin D (a gene transcription inhibitor); however, the effect of 2-ME, but not E2, was prevented by cycloheximide (a protein synthesis inhibitor). Furthermore, the effect of E2 was not blocked by 1-aminobenzotriazole (a cytochrome P-450 inhibitor) or Ro 41-0960 (a catechol-O-methyltransferase inhibitor). The effect of 2-ME was not mimicked by microtubule-interfering agents (nocodazole or Taxol). We conclude that 2-ME inhibits smooth muscle contractility through an endothelium- and nitric oxide-dependent mechanism, which does not involve estrogen receptors or microtubule disruption. The effect of 2-ME, but not E2, involves de novo protein synthesis. 2-ME does not mediate the inhibitory effect of E2 on smooth muscle contraction. These results support a potentially important role of 2-ME in the regulation of smooth muscle tone in the vasculature.

The involvement of norepinephrine, neuropeptide Y, and nitric oxide in the cutaneous vasodilator response to local heating in humans

Presynaptic blockade of cutaneous vasoconstrictor nerves (VCN) abolishes the axon reflex (AR) during slow local heating (SLH) and reduces the vasodilator response. In a two-part study, forearm sites were instrumented with microdialysis fibers, local heaters, and laser-Doppler flow probes. Sites were locally heated from 33 to 40 degrees C over 70 min. In part 1, we tested whether this effect of VCN acted via nitric oxide synthase (NOS). In five subjects, treatments were as follows: 1) untreated; 2) bretylium, preventing neurotransmitter release; 3) N(G)-nitro-L-arginine methyl ester (L-NAME) to inhibit NOS; and 4) combined bretylium + L-NAME. At treated sites, the AR was absent, and there was an attenuation of the ultimate vasodilation (P < 0.05), which was not different among those sites (P > 0.05). In part 2, we tested whether norepinephrine and/or neuropeptide Y is involved in the cutaneous vasodilator response to SLH. In seven subjects, treatments were as follows: 1) untreated; 2) propranolol and yohimbine to antagonize alpha- and beta-receptors; 3) BIBP-3226 to antagonize Y(1) receptors; and 4) combined propranolol + yohimbine + BIBP-3226. Treatment with propranolol + yohimbine or BIBP-3226 significantly increased the temperature at which AR occurred (n = 4) or abolished it (n = 3). The combination treatment consistently eliminated it. Importantly, ultimate vasodilation with SLH at the treated sites was significantly (P < 0.05) less than at the control. These data suggest that norepinephrine and neuropeptide Y are important in the initiation of the AR and for achieving a complete vasodilator response. Since VCN and NOS blockade in combination do not have an inhibition greater than either alone, these data suggest that VCN promote heat-induced vasodilation via a nitric oxide-dependent mechanism.

M1732 Lipopolysaccharide (LPS) Sensitizes Bile Duct Epithelial Cells for Tight Junction (TJ) Disruption By Hydrogen Peroxide and TNFα By Nitric Oxide-Dependent Mechanism

M1732 Lipopolysaccharide (LPS) Sensitizes Bile Duct Epithelial Cells for Tight Junction (TJ) Disruption By Hydrogen Peroxide and TNFα By Nitric Oxide-Dependent Mechanism

Evidence for the participation of calcium in non‐genomic relaxations induced by androgenic steroids in rat vas deferens

Androgens cause non-genomic relaxation in several smooth muscle preparations. However, such an effect has not been investigated in rat vas deferens yet. Our purpose was to study the effect of testosterone and derivatives in this tissue. The influence of androgens was tested on contraction and translocation of intracellular Ca(2+) induced by KCl in rat vas deferens in vitro. The testosterone derivative 5alpha-dihydrotestosterone produced a rapid and reversible concentration-dependent relaxation of KCl-induced contractions. Other androgens were also effective, showing the following rank order of potency: androsterone >5beta-dihydrotestosterone >androstenedione >5alpha-dihydrotestosterone >testosterone. Calcium-induced contractions were also inhibited (about 45%) by 5alpha-dihydrotestosterone (30 microM). Moreover 5alpha-dihydrotestosterone blocked the increase of intracellular Ca(2+) induced by KCl, measured by the fluorescent dye fura-2. Relaxation to 5alpha-dihydrotestosterone was resistant to the K(+) channel antagonists glibenclamide, 4-aminopyridine and charybdotoxin. It was not affected by removal of epithelium or by L-NNA (300 microM), an inhibitor of nitric oxide biosynthesis, nor by selective inhibitors of soluble guanylate cyclase, ODQ or LY 83583, indicating that nitrergic or cGMP mediated mechanisms were not involved. The androgen-induced relaxation was also not blocked by the protein synthesis inhibitor cycloheximide (300 microM) or by the classical androgen receptor flutamide (up to 100 microM), corroborating that the effect is non-genomic. Testosterone derivatives caused relaxation of the rat vas deferens, that did not involve epithelial tissue, K(+) channels, or nitric oxide-dependent mechanisms, but was related to a partial blockade of Ca(2+) influx.

IFNγ Enhances Efferocytosis by Macrophages (MΦ) in Chronic Granulomatous Disease (CGD) by a Nitric Oxide (NO) Dependent Mechanism

IFNγ Enhances Efferocytosis by Macrophages (MΦ) in Chronic Granulomatous Disease (CGD) by a Nitric Oxide (NO) Dependent Mechanism

Nitric oxide and peroxynitrite signalling triggers homocysteine-mediated apoptosis in trigeminal sensory neurons in vitro

The neurotoxic actions of homocysteine on central nervous system neurons have been well established, yet its effects on the neurons of the peripheral nervous system remain largely unknown. We analysed the consequences of homocysteine exposure for the in vitro survival of embryonic and postnatal murine trigeminal sensory neurons from E14 to P1, and also quantified the effects of homocysteine exposure on neurite outgrowth. We discovered that homocysteine was toxic to these neurons when they were grown with NGF, or, in the case of P1 trigeminal neurons, with CNTF. Cell death induced by homocysteine was blocked using caspase inhibitors indicating that this cell loss was apoptotic. In addition, we demonstrated that homocysteine toxicity was mediated through the actions of the NMDA receptor, nitric oxide and peroxynitrite. We found that homocysteine had no effect on neurite outgrowth. Taken together our data show that homocysteine induces apoptosis in trigeminal sensory neurons via a nitric oxide-dependent mechanism. These data represent the first demonstration that homocysteine is toxic to a population of cranial sensory neurons and elucidate key components of the signalling pathway engaged to bring this about. These findings are of importance to our understanding of homocysteine's influence on neurodevelopment and on peripheral neuropathies.

Acute-phase protein α-1-acid glycoprotein mediates neutrophil migration failure in sepsis by a nitric oxide-dependent mechanism

The reduction of circulating neutrophil migration to infection sites is associated with a poor outcome of severe sepsis. alpha-1-Acid glycoprotein (AGP) was isolated from the sera of severely septic patients by HPLC and acrylamide gel electrophoresis and identified by mass spectrometry. Both the isolated protein and commercial AGP inhibited carrageenin-induced neutrophil migration into the rat peritoneal cavity when administered i.v. at a dose of 4.0 microg per rat (95 pmol per rat). Analysis by intravital microscopy demonstrated that both proteins inhibited the rolling and adhesion of leukocytes in the mesenteric microcirculation. The inhibitory activity was blocked by 50 mg/kg aminoguanidine, s.c., and was not demonstrable in inducible nitric oxide synthase (iNOS) knockout mice. Incubation of AGP with neutrophils from healthy subjects induced the production of NO and inhibited the neutrophil chemotaxis by an iNOS/NO/cyclic guanosine 3,5-monophosphate-dependent pathway. In addition, AGP induced the l-selectin shedding by neutrophils. The administration of AGP to rats with mild cecal ligation puncture sepsis inhibited neutrophil migration and reduced 7-day survival from approximately 80% to 20%. These data demonstrate that AGP, an acute-phase protein, inhibits neutrophil migration by an NO-dependent process and suggest that AGP also participates in human sepsis.

Cardiac growth and angiogenesis coordinated by intertissue interactions

Cardiac hypertrophy and angiogenesis are coordinately regulated during physiological or adaptive cardiac growth, and disruption of the balanced growth and angiogenesis leads to contractile dysfunction and heart failure. Coordination of growth and angiogenesis is in part mediated by the secretion of angiogenic growth factors from myocytes in response to hypertrophic stimuli, which enables the vasculature to "catch up" to the growth of the myocardium. In this issue of the JCI, two studies provide novel insights into the regulatory mechanisms of cardiac growth and coronary angiogenesis. Heineke et al. demonstrate that GATA4 acts as a stress-responsive transcription factor in murine cardiac myocytes that induces the expression of angiogenic growth factors (see the related article beginning on page 3198). Tirziu et al. show that enhanced coronary angiogenesis per se leads to hypertrophic growth of myocytes through a nitric oxide-dependent mechanism (see the related article beginning on page 3188). These studies, together with previous reports, suggest the existence of reciprocal signals between the myocardium and the vasculature that promote the growth of each other in a paracrine fashion.

Morphine-induced Neuroimmunomodulation in Murine Visceral Leishmaniasis: The Role(s) of Cytokines and Nitric Oxide

Opioid modulation of host resistance to infectious diseases is well documented; however, not much is known during visceral leishmaniasis (VL). Low doses of morphine, administered subcutaneously in Leishmania donovani-infected BALB/c mice, on days 0 and +15, significantly (p < 0.05) suppressed (1 mg/kg/day) or even sterile-cleared (2 mg/kg/day) the infection; paradoxically, high doses (10 and 30 mg/kg/day) exacerbated the infection. In vitro, low concentration (1 x 10(-9) and 1 x 10(-11) M) morphine treatment of L. donovani-infected mouse peritoneal macrophages (PM), endowed them with significant (p < 0.05) leishmanicidal activity, whereas a high-concentration (1 x 10(-5) M) treatment augmented intramacrophage parasite growth. Naloxone pre-treatment of infected-mice (4 mg/kg x 2) and of infected-PM (1 x 10(-5) M), blocked only the morphine low dose/concentration-induced protective effect. The splenocytes from protected mice and morphine low concentration-treated infected-PM, elaborated significantly (p < 0.05) enhanced levels of interleukin-12, interferon-gamma, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor and nitrite in the culture medium; a high dose/concentration suppressed their elaboration. Curiously, only morphine high dose/concentration-treated infected mice splenocytes and infected PM, produced significantly (p < 0.05) increased quantity of transforming growth factor-beta1. Aminoguanidine, significantly (p < 0.05) blocked the morphine low dose/concentration-induced protective effect, in vivo and in vitro. This first study demonstrates dose-dependent biphasic modulatory effects of morphine in L. donovani-infected mice and PM, in vitro, apparently via nitric oxide-dependent mechanisms. These results thus demonstrate the implications of opiate abuse on the efficacy assessment of antileishmanial drugs and vaccines, and on the reactivation of latent VL in areas where both drug abuse and VL are rampant.

Transendothelial flow inhibits neutrophil transmigration through a nitric oxide-dependent mechanism: potential role for cleft shear stress

Endothelial cells in vivo are well known to respond to parallel shear stress induced by luminal blood flow. In addition, fluid filtration across endothelium (transendothelial flow) may trigger nitric oxide (NO) production, presumably via shear stress within intercellular clefts. Since NO regulates neutrophil-endothelial interactions, we determined whether transendothelial flow regulates neutrophil transmigration. Interleukin-1beta-treated human umbilical vein endothelial cell (HUVEC) monolayers cultured on a polycarbonate filter were placed in a custom chamber with or without a modest hydrostatic pressure gradient (DeltaP, 10 cm H(2)O) to induce transendothelial flow. In other experiments, cells were studied in a parallel plate flow chamber at various transendothelial flows (DeltaP = 0, 5, and 10 cm H(2)O) and luminal flows (shear stress of 0, 1, and 2 dyn/cm(2)). In the absence of luminal flow, transendothelial flow reduced transmigration of freshly isolated human neutrophils from 57% to 14% (P < 0.05) and induced an increase in NO detected with a fluorescent assay (DAF-2DA). The NO synthase inhibitor L-NAME prevented the effects of transendothelial flow on neutrophil transmigration, while a NO donor (DETA/NO, 1 mM) inhibited neutrophil transmigration. Finally, in the presence of luminal flow (1 and 2 dyn/cm(2)), transendothelial flow also inhibited transmigration. On the basis of HUVEC morphometry and measured transendothelial volume flow, we estimated cleft shear stress to range from 49 to 198 dyn/cm(2). These shear stress estimates, while substantial, are of similar magnitude to those reported by others with similar analyses. These data are consistent with the hypothesis that endothelial cleft shear stress inhibits neutrophil transmigration via a NO-dependent mechanism.

Frontiers in glucagon-like peptide-2: multiple actions, multiple mediators

Glucagon-like peptide-2 (GLP-2) is a pleiotropic hormone that affects multiple facets of intestinal physiology, including growth, barrier function, digestion, absorption, motility, and blood flow. The mechanisms through which GLP-2 produces these actions are complex, involving unique signaling mechanisms and multiple indirect mediators. As clinical trials have begun for the use of GLP-2 in a variety of intestinal disorders, the elucidation of such mechanisms is vital. The GLP-2 receptor (GLP-2R) is a G protein-coupled receptor, signaling through multiple G proteins to affect the cAMP and mitogen-activated protein kinase pathways, leading to both proliferative and antiapoptotic cellular responses. The GLP-2R also demonstrates unique mechanisms for receptor trafficking. Expression of the GLP-2R in discrete sets of intestinal cells, including endocrine cells, subepithelial myofibroblasts, and enteric neurons, has led to the hypothesis that GLP-2 acts indirectly through multiple mediators to produce its biological effects. Indeed, several studies have now provided important mechanistic data illustrating several of the indirect pathways of GLP-2 action. Thus, insulin-like growth factor I has been demonstrated to be required for GLP-2-induced crypt cell proliferation, likely involving activation of beta-catenin signaling. Furthermore, vasoactive intestinal polypeptide modulates the actions of GLP-2 in models of intestinal inflammation, while keratinocyte growth factor is required for GLP-2-induced colonic mucosal growth and mucin expression. Finally, enteric neural GLP-2R signaling affects intestinal blood flow through a nitric oxide-dependent mechanism. Determining how GLP-2 produces its full range of biological effects, which mediators are involved, and how these mediators interact is a continuing area of active research.

Flavonoid-rich fraction of Maytenus ilicifolia Mart. ex. Reiss protects the gastric mucosa of rodents through inhibition of both H +,K +-ATPase activity and formation of nitric oxide

Maytenus ilicifolia Mart. ex. Reissek (Celastraceae), a medicinal plant known in Brazil as “espinheira-santa” is commonly used to treat gastric disorders. The effect of the flavonoid-rich fraction separated from the leaves was evaluated for its gastroprotective properties and the mechanism(s) involved in this activity. Intraperitoneal administration of the flavonoid-rich fraction potently protected rats from experimentally induced chronic (ED 50 = 79 mg/kg) and acute gastric lesions by ethanol (ED 50 = 25 mg/kg) and indomethacin (ED 50 = 4 mg/kg) without altering the decreased amount of cytoprotective glutathione and mucus amount in the injured gastric mucosa. A potent reduction of gastric acid hypersecretion (ED 50 = 7 mg/kg, i.p.) was accompanied by a reduction of nitric oxide release (ED 50 = 1.6 mg/kg, i.p.) in the gastric secretion of 2 h pylorus ligated rats which suggests an important role for nitric oxide-dependent mechanisms. Inhibition of gastric acid secretion in vivo was correlated with the in vitro inhibition of rabbit gastric H +,K +-ATPase activity (IC 50 = 41 μg/mL). Chemical investigation of the fraction showed galactitol (25%), epicatechin (3.1%) and catechin (2%) as the majoritary components. Collectively, the results show that the flavonoid-rich fraction of Maytenus ilicifolia potently protects animals from gastric lesions with high potency through inhibition of gastric acid secretion.

Direct stimulatory effect of ghrelin on pituitary release of LH through a nitric oxide-dependent mechanism that is modulated by estrogen

Ghrelin, a gut peptide with key actions on food intake and GH secretion, has been recently recognized as potential regulator of reproductive function. Thus, in adult female rats, ghrelin has been proven to modulate GnRH/LH secretion, with predominant inhibitory effects in vivo. We analyze herein potential direct pituitary effects of ghrelin on basal and GnRH-stimulated gonadotropin secretion in prepubertal female rats, and its interplay with ovarian inputs, nitric oxide (NO), and hypothalamic differentiation. In the experimental setting, pituitaries from intact and ovariectomized prepubertal female rats were challenged with ghrelin in vitro and LH secretion was monitored. Our results demonstrate that 1) ghrelin consistently stimulated in vitro pituitary LH secretion under different experimental conditions; 2) the sensitivity to ghrelin, expressed either as the minimal effective dose or the amplitude of the LH response, was modulated by ovarian inputs; 3) the blockade of estrogen action significantly augmented the stimulatory effect of ghrelin; 4) the stimulatory effect of ghrelin on LH secretion required proper NO synthesis; and 5) the ability of ghrelin to elicit LH secretion in vitro was preserved after alteration (masculinization) of brain sexual differentiation. Overall, our present data reinforce the concept that ghrelin participates in the control of LH secretion, with potential stimulatory actions at the pituitary level that require the presence of NO and are modulated by ovarian signals.

Preserved reflex cutaneous vasodilation in cystic fibrosis does not include an enhanced nitric oxide-dependent mechanism

In humans, vasoactive intestinal peptide (VIP) may play a role in reflex cutaneous vasodilation during body heating. We tested the hypothesis that the nitric oxide (NO)-dependent contribution to active vasodilation is enhanced in the skin of subjects with cystic fibrosis (CF), compensating for sparse levels of VIP. In 2 parallel protocols, microdialysis fibers were placed in the skin of 11 subjects with CF and 12 controls. Lactated Ringer was perfused at one microdialysis site and NG-nitro-L-arginine methyl ester (2.7 mg/ml) was perfused at a second microdialysis site. Skin blood flow was monitored over each site with laser-Doppler flowmetry. In protocol 1, local skin temperature was increased 0.5 degrees C every 5 s to 42 degrees C, and then it maintained at 42 degrees C for approximately 45 min. In protocol 2, subjects wore a tube-lined suit perfused with water at 50 degrees C, sufficient to increase oral temperature (Tor) 0.8 degrees C. Cutaneous vascular conductance (CVC) was calculated (flux/mean arterial pressure) and scaled as percent maximal CVC (sodium nitroprusside; 8.3 mg/ml). Vasodilation to local heating was similar between groups. The change (Delta%CVCmax) in CVC with NO synthase inhibition on the peak (9+/-3 vs. 12+/-5%CVCmax; P=0.6) and the plateau (45+/-3 vs. 35+/-5%CVCmax; P=0.1) phase of the skin blood flow response to local heating was similar in CF subjects and controls, respectively. Reflex cutaneous vasodilation increased CVC in CF subjects (58+/-4%CVCmax) and controls (53+/-4%CVCmax; P=0.37) and NO synthase inhibition attenuated CVC in subjects with CF (37+/-6%CVCmax) and controls (35+/-5%CVCmax; P=0.8) to a similar degree. Thus the preservation of cutaneous active vasodilation in subjects with CF is not associated with an enhanced NO-dependent vasodilation.

CARDIOPROTECTIVE EFFECTS OF A NON‐ALCOHOLIC EXTRACT OF RED WINE DURING ISCHAEMIA AND REPERFUSION IN SPONTANEOUSLY HYPERTENSIVE RATS

1. We reported recently the cardioprotection conferred by a non-alcoholic extract of Cabernet-Sauvignon red wine (RWE) against alterations derived from ischaemia and reperfusion in normotensive rats. The aim of the present study was to assess the effects of RWE on ischaemia/reperfusion injury in hearts isolated from spontaneously hypertensive rats (SHR). 2. After stabilization, rat isovolumic perfused hearts were exposed to a 20 min global ischaemic period followed by 30 min reperfusion in the absence (ischaemic control (IC) hearts) or presence of RWE infused prior to ischaemia and early in reperfusion. In other hearts, N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, was administered prior to RWE infusion (L-NAME + RWE). 3. Left ventricular developed pressure (LVDP), dP/dt(max) and left ventricular end-diastolic pressure (LVEDP) were used to assess myocardial function. 4. At the end of reperfusion, LVDP and dP/dt(max) decreased to 47 +/- 9 and 46 +/- 9% of baseline values, respectively, in IC. Treatment with the RWE significantly improved systolic postischaemic recovery (LVDP = 85 +/- 8%; dP/dt(max) = 83 +/- 5%) and attenuated the increase in LVEDP (23 +/- 6 and 53 +/- 8 mmHg in RWE and IC, respectively; P < 0.05). 5. In the L-NAME + RWE group, L-NAME completely abolished the systolic and diastolic protection induced by RWE (LVDP = 44 +/- 13%; dP/dt(max) = 43 +/- 13%; LVEDP = 60 +/- 10 mmHg). 6. These data are the first demonstration that a non-alcoholic extract of Cabernet-Sauvignon red wine protects SHR hearts from systolic and diastolic alterations induced by ischaemia and reperfusion through a nitric oxide-dependent mechanism.

Role of iNOS‐derived nitric oxide (NO) on hind limb ischemia/reperfusion (I/R)‐induced remote injury to the gut

Systemic inflammatory response syndrome (SIRS), as a consequence of ischemia/reperfusion (I/R) negatively influences the function of the affected organs. In this study we assessed the potential mechanisms (role of NO) of remote intestinal inflammatory response elicited by hind limb I/R. To this end C57BL/6 (WT) and iNOS−/− mice were subjected to 1h of bilateral hind limb ischemia followed by 6h of reperfusion. Some WT mice were injected (s.c.) with iNOS inhibitor, 1400W (5mg/kg) immediately before induction of ischemia. The obtained results indicate that hind limb I/R results in dysfunction of the gut as assessed by the increase in total protein levels (Bradford assay), TNF-a protein (ELISA) and NO (Griess reaction) in the lumen of small intestine. In addition, hind limb I/R results in an increase in leukocyte accumulation (intravital microscopy and MPO assay), TNF-a and iNOS protein expression (Western blots) in the mucosa of small intestine. Important to note, that the most profound changes with respect to the gut dysfunction were found in jejunum and ileum, whereas duodenum was affected the least. Interfering with iNOS activity (1400W and iNOS−/− mice) significantly attenuated hind limb I/R-induced leukocyte recruitment to the gut mucosa and dysfunction of the small intestine. Taken together these data indicate that hind limb I/R induces remote inflammatory response in the small intestine through iNOS-derived NO dependent mechanism (HSFO-NA5580 and MOP-68848)

THE EFFECT OF HYPERBARIC OXYGEN ON ENDOTHELIAL NITRIC OXIDE SYNTHASE EXPRESSION IN ISCHEMIA-REPERFUSION INJURY.

Introduction The beneficial effects of hyperbaric oxygen (HBO) in the amelioration of ischemia-reperfusion (IR) injury have been shown to occur through a nitric oxide-dependent mechanism. Previous work has shown an early increase in eNOS activity caused by HBO. There is also a late up-regulation of eNOS mRNA. The purpose of this experiment is to determine if there is an increase in eNOS protein following HBO treatment. α2-Antiplasmin (α2-antiplasmin) was administered to determine the source of eNOS. Methods The gracilis muscle flap was raised in 40 male Wistar rats (n = 10/group). Rats were divided into four groups: a nonischemic control, IR, IR + HBO (100% O2 at 2.5 ATA) and an IR + HBO + α2-antiplasmin group each with an n = 10. Ischemia consisted of 4 hours of global ischemia on the gracilis muscle flap. The α2-antiplasmin was administered by direct continuous infusion into the gracilis muscle prior to HBO. The HBO treatment consisted 100% O2 at 2.5 ATA during the last 90 minutes of ischemia. The gracilis muscle and pulmonary vasculature were harvested 30 minutes after reperfusion, weighed, and flash frozen in liquid nitrogen. A Bradford assay was performed to determine the total protein quantity. The quantity of eNOS protein was measured using Western immunoblotting. Results IR did not significantly increase eNOS protein compared with the nonischemic control (35.6 ± 1.3 vs 38.6 ± 1.7 ng). HBO did not change the level of eNOS protein in skeletal muscle (38.8 ± 2.0 ng). In addition, the blockade of the plasmin/eNOS cascade had no effect on the level of eNOS protein (35.8 ± 2.7 ng). Discussions These results offer evidence that the immediate increase in eNOS activity seen in HBO treated IR injury is not due to an increase in eNOS protein. Instead, it may be related to an increase in VEGF protein or mechanotransduction of the VEGFR2 caused by the sheer stress of reperfusion.