L-nitroarginine Methyl Ester Research Articles

Assessment of heme oxygenase-1 (HO-1) activity in the cavernous tissues of sildenafil citrate-treated rats

To assess heme oxygenase-1 (HO-1) activity in the cavernous tissue of sildenafil citrate-treated rats. One hundred and ninety-two Sprague-Dawley male rats, divided into four equal groups, were investigated. Group 1, the control group, received regular animal chow; group 2 received sildenafil citrate by intragastric tube; group 3 received sildenafil and HO inhibitor (zinc protoporphyrin, ZnPP); and group 4 received sildenafil and nitric oxide synthase (NOS) inhibitor L-nitroarginine methyl ester (L-NAME). Twelve rats from each group were killed after 0.5 h, 1 h, 2 h and 3 h of drug administration. Then HO-1 activity, cGMP levels and NOS enzymatic activity in the cavernous tissues were estimated. In cavernous tissue, HO-1 activity, NOS enzymatic activity and cGMP concentration increased significantly in sildenafil-treated rats compared to other groups throughout the experiment. Rats receiving either HO or NOS inhibitors showed a significant decrease in these parameters. HO-1 cavernous tissue activity and NOS enzymatic activity demonstrated a positive significant correlation with cGMP levels (r = 0.646, r = 0.612 respectively; P < 0.001). The actions of PDE5 inhibitor sildenafil citrate in the cavernous tissue are partly mediated through the interdependent relationship between both HO-1 and NOS activities.

Investigation of the relaxant effects of propofol on ovalbumin-induced asthma in guinea pigs

Because the incidence of asthma appears to be increasing, the importance of proper perioperative management of individuals with asthma will also continue to increase. Although its mechanism of smooth muscle relaxation is unknown, propofol has been associated with less bronchoconstriction during anaesthetic induction. The aim of this study was to investigate the possible mechanism of these effects and the effects of propofol on the isolated trachea preparations from control and ovalbumin-sensitized guinea pigs. Adult male guinea pigs, weighing 280-330 g, were randomly allocated to two experimental groups, each consisting of 10 animals. Ten guinea pigs were sensitized by intramuscular injections of 0.30 mL of a 5% (w/v) ovalbumin/saline solution into each thigh (0.6 mL total) on days 1 and 4, whereas the remaining 10 served as controls receiving a total of 0.6 mL distilled water on days 1 and 4 as placebo. The isolated trachea preparations were mounted in tissue baths with modified Krebs-Henseleit solution and aerated with 95% oxygen and 5% carbon dioxide. We tested the effects of propofol (10(-7)-10(-3) M) on resting tension and after precontraction with carbachol and histamine on isolated trachea preparations from control and ovalbumin-sensitized guinea pigs. We also tested the effect of propofol on isolated trachea preparations precontracted with carbachol and histamine in the absence and presence of different inhibitors or antagonists. We investigated propofol responses in tracheal smooth muscle precontracted with CaCl2. Propofol (10(-7)-10(-3) M) produced a concentration-dependent relaxation of isolated tracheal preparations precontracted by carbachol (10(-6) M) and histamine (10(-6) M) in both groups. Preincubation with N(w)-nitro L-arginine methyl ester (3x10(-5) M), indomethacin (10(-5) M) or propranolol (10(-4) M) did not produce a significant alteration on propofol-induced relaxation responses (P>0.05), while preincubation with tetraethylammonium (3x10(-4) M) significantly decreased the propofol-induced relaxation responses in both groups (P<0.05). Propofol (10(-7)-10(-3) M) induced concentration-dependently relaxations in isolated trachea rings precontracted with CaCl2 in both the control and ovalbumin-sensitized groups. Propofol induced concentration-dependent relaxations in precontracted, isolated trachea smooth muscle of guinea pigs in both the control and ovalbumin-sensitized groups. These relaxations were independent of epithelial function and stimulation of beta adrenergic receptors. Opened Ca2+-sensitive K+ channels and inhibited L-type Ca2+ channels can contribute to these relaxations.

Mechanism of relaxation induced by nicotine in normal and ovalbumin-sensitized guinea-pig trachea

Nicotine is an irritant molecule in the cigarette that contributes airway hyper-reactivity. The aim of this study was to investigate the mechanism of these effects and effects of nicotine on the isolated trachea preparations from control and ovalbumin-sensitized guinea-pigs. Nicotine (3 × 10 − 5 to 3 × 10 − 4 M) produced concentration-dependent relaxation on isolated trachea preparations precontracted by carbachol (10 − 6 M) in both groups. We found that the relaxant effect of nicotine decreased in the presence of N(w)-nitro L-arginine methyl ester (L-NAME) (10 − 6 M), and hexamethonium (10 − 2 M) but not in the presence of α-bungarotoxin (10 − 3 M), and tetrodotoxin (3.1 × 10 − 6 M) in isolated trachea preparations in both groups. The relaxant effect of nicotine was less significant in isolated trachea preparations from ovalbumin-sensitized guinea-pigs than from control guinea-pigs ( P < 0.05). The contractions elicited by carbachol (10 − 6 M) were not significantly different in the ovalbumin-sensitized group than in the control group. Nicotine (10 − 4 M) significantly increased the cGMP levels in trachea preparations compared with the control preparations.( P < 0.05). These results suggest that nicotine-induced relaxation response in normal and ovalbumin sensitized guinea-pigs trachea is at least in part mediated by nitric oxide (NO) since it was significantly reduced in the presence of L-NAME. The decreased relaxation response to nicotine in ovalbumin sensitized guinea-pigs trachea may be due to impaired production and/or liberation of NO.

Endothelial Nitric Oxide Synthase Overexpression Provides a Functionally Relevant Angiogenic Switch in Hibernating Pig Myocardium

We investigated whether retroinfusion of liposomal endothelial nitric oxide synthase (eNOS) S1177D complementary deoxyribonucleic acid (cDNA) would affect neovascularization and function of the ischemic myocardium. Recently, we demonstrated the feasibility of liposomal eNOS cDNA transfection via retroinfusion in a model of acute myocardial ischemia/reperfusion. In the present study, we used this approach to target a phosphomimetic eNOS construct (eNOS S1177D) into chronic ischemic myocardium in a pig model of hibernation. Pigs (n = 6/group) were subjected to percutaneous implantation of a reduction stent graft into the left anterior descending artery (LAD), inducing total occlusion within 28 days. At day 28, retroinfusion of saline solution containing liposomal green fluorescent protein or eNOS S1177D cDNA (1.5 mg/animal, 2 x 10 min) was performed. Furthermore, L-nitroarginine-methylester (L-NAME) was applied orally from day 28, where indicated. At day 28 and day 49, fluorescent microspheres were injected into the left atrium for perfusion analysis. Regional functional reserve (at atrial pacing 140/min) was assessed at day 49 by subendocardial segment shortening (SES) (sonomicrometry, percent of ramus circumflexus region). The eNOS S1177D overexpression increased endothelial cell proliferation as well as capillary and collateral growth at day 49. Concomitantly, eNOS S1177D overexpression enhanced regional myocardial perfusion from 62 +/- 4% (control) to 77 +/- 3% of circumflex coronary artery-perfused myocardium, unless L-NAME was co-applied (69 +/- 5%). Similarly, eNOS S1177D cDNA improved functional reserve of the LAD (33 +/- 5% vs. 7 +/- 3% of circumflex coronary artery-perfused myocardium), except for L-NAME coapplication (13 +/- 6%). Retroinfusion of eNOS S1177D cDNA induces neovascularization via endothelial cell proliferation and collateral growth. The resulting gain of perfusion enables an improved functional reserve of the hibernating myocardium.

Short-term ethanol administration does not change the total pyramidal neuron number of the rat hippocampus: A stereologic study

In the hippocampus, short-term exposure to ethanol (EtOH) has been shown to inhibit some functions, and nitric oxide (NO) is an important modulator of physiologic processes. In this study, investigators explored the effects of EtOH on the total number of neurons in rat CA regions and the possible neuroprotective role of NO. The role of NO in rats given EtOH was examined with the use of a nonselective inhibitor of NOS (N[G]-nitro-L-arginine methyl ester [L-NAME], D[G]-nitro-Larginine methyl ester [D-NAME]), a central selective inhibitor of NOS (7-NI), and a donor of NO (L-arginine). Toward this end, rats were randomly divided into 6 groups: control (saline 3 g/kg intraperitoneal [ip]), ethanol (ethanol 3 g/kg ip), L-NAME (ethanol 3 g/kg ip + L- NAME 60 mg/kg ip), L-arginine (ethanol 3 g/kg ip + L-arginine 1 g/kg ip), D-NAME (ethanol 3 g/kg ip + D-NAME 60 mg/kg ip), and 7-NI (ethanol 3 g/kg ip + 7-NI 40 mg/kg ip). Blood ethanol concentrations were measured 90 min after EtOH administration. Means (value+/-standard deviation) of total pyramidal neuronal numbers in the right hippocampus were estimated using the optical fractionator counting method. Values were as follows: 446,558+/-6207, 483,517+/-20,311, 464,588+/-30,637, 479,688+/-10,780, 458,294+/-17,770, and 477,281+/-7641 in the control, ethanol, L-arginine, 7-NI, L-NAME, and D-NAME groups, respectively. No significant differences were observed between groups (P>.05). The results of the present study imply that short-term administration of EtOH does not affect total pyramidal neuronal number in the right hippocampus of the rat. Furthermore, NO does not change the effects of short-term EtOH on total pyramidal neuronal number in the right hippocampal CA regions of the rat. Additional studies are needed to clarify the role of NO and NOS inhibition in the effects associated with EtOH given on a short-term basis.

Matrix metalloproteinase 2–induced venous dilation via hyperpolarization and activation of K + channels: Relevance to varicose vein formation

Varicose veins are a common disorder of extensive venous dilation and remodeling with an as-yet unclear mechanism. Studies have shown increased plasma and tissue levels of matrix metalloproteinases (MMPs) in human varicose veins and animal models of venous hypertension. Although the effects of MMPs are generally attributed to extracellular matrix degradation, their effects on the mechanisms of venous contraction/relaxation are unclear. Our preliminary experiments have demonstrated that MMP-2 causes inhibition of phenylephrine-induced venous contraction. The purpose of this study was to determine whether MMP-induced inhibition of venous contraction involves an endothelium-dependent and/or -independent pathway. Circular segments of the inferior vena cava (IVC) were isolated from male Sprague-Dawley rats and suspended between two wire hooks in a tissue bath, and the effects of MMP-2 on phenylephrine- and KCl-induced contraction were measured. To study the role of endothelium-derived vasodilators, experiments were performed in the presence and absence of endothelium; N(G)-l-nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis; indomethacin, an inhibitor of prostacyclin synthesis; cromakalim, an activator of adenosine triphosphate-sensitive K+ channels (K(ATP)); and iberiotoxin, a blocker of large-conductance Ca2+-dependent K+ channels (BK(Ca)) and smooth muscle hyperpolarization. In endothelium-intact IVC segments, phenylephrine (10(-5) mol/L) caused significant contraction that slowly declined to 82.0% in 30 minutes. The addition of MMP-2 (1 microg/mL) caused a gradual decrease of phenylephrine contraction to 39.5% at 30 minutes. In endothelium-denuded IVC, MMP-2 induced a greater reduction of phenylephrine contraction, to 7.6%. In the presence of L-NAME (10(-4) mol/L), MMP-2 caused a marked decrease in phenylephrine contraction, to 4.4%. Large MMP-2-induced inhibition of phenylephrine contraction was also observed in IVC treated with L-NAME plus indomethacin. MMP-2 caused relaxation of phenylephrine contraction in IVC pretreated with cromakalim (10(-7) mol/L), an activator of K(ATP) channels. MMP-2-induced inhibition of phenylephrine contraction was abrogated in the presence of iberiotoxin (10(-8) mol/L), a blocker of BK(Ca). MMP-2 did not inhibit venous contraction during membrane depolarization by 96 mmol/L KCl, a condition that prevents outward K+ conductance and cell hyperpolarization. MMP-2 causes significant IVC relaxation that is potentiated in the absence of endothelium or during blockade of endothelium-mediated nitric oxide and prostacyclin synthesis. The lack of effects of MMP-2 on KCl contraction and in iberiotoxin-treated veins suggests MMP-2-induced smooth muscle hyperpolarization and activation of BK(Ca) channels--a novel effect of MMP that may play a role in the early stages of venous dilation and varicose vein formation.

Effect of vascular endothelial growth factor on pre-eclampsia in pregnant rats

To investigate relationship between the vascular endothelial growth factor (VEGF) and the pathogenesis of pre-eclampsia in pregnant rats. Pregnant rats were divided into two groups randomly. Saline solution or L-nitro arginine methyl ester (L-NAME) 125 mg/d was given subcutaneously from day 7 of gestation till establishing pre-eclampsia. Systolic blood pressure, urine protein, platelet count, and weight of pups and placenta were determined. The levels of VEGF in pregnant rats venous serum, placenta and decidual tissue from normal pregnancy and pre-eclampsia rats were detected by ELISA and immunohistochemistry, respectively. Pregnant rats which were given L-NAME produced physical signs similar to those of pre-eclampsia, such as increase in systolic blood pressure [(145.3 +/-4.6)mmHg] and urine protein [(814.3 +/-57.5)mg/L], and decrease in platelet count [(467.1 +/-76.3) x 10(9)/L] and weight of pups and placenta. Compared with controls, the intensity of VEGF immunostaining in trophoblast or decidual cells were significantly reduced. The serum levels of VEGF were significantly lower in pre-eclampsia group than in normal pregnancy. Decreased serum levels of VEGF and reduced expression of VEGF in placental tissues might in part explain the pathogenesis of pre-eclampsia in pregnant rats.

Role of carbon monoxide in electrically induced non-adrenergic, non-cholinergic relaxations in the guinea-pig isolated whole trachea.

Nitric oxide (NO) and vasoactive intestinal peptide (VIP) are considered transmitters of non-adrenergic, non-cholinergic (NANC) relaxations in guinea-pig trachea, whereas the role of carbon monoxide (CO) is unknown. This study was designed to assess the participation of CO, and to investigate the localization of haem oxygenase-2 (HO-2), the CO-producing enzyme, in tracheal neurons. NANC responses to electrical field stimulation (EFS) at 3 and 10 Hz were evaluated in epithelium-free whole tracheal segments as intraluminal pressure changes. Drugs used were: L-nitroarginine methyl ester (L-NAME, 100 microM) to inhibit NO synthase (NOS), alpha-chymotrypsin (2 U ml(-1)) to inactivate VIP, zinc protoporphyrin-IX (ZnPP-IX, 10 microM) to inhibit HO-2, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), a soluble guanylyl cyclase inhibitor. For immunohistochemistry, tissues were exposed to antibodies to PGP 9.5, a general neuronal marker, HO-2 and NOS, and processed with an indirect immunofluorescence method. alpha-Chymotrypsin did not affect NANC relaxations. ODQ inhibited NANC responses by about 60%, a value similar to that obtained by combining L-NAME and ZnPP-IX. The combination of ODQ, L-NAME and ZnPP-IX reduced the responses by 90%. Subpopulations of HO-2 positive neurons containing NOS were detected in tracheal sections. In the guinea-pig trachea, NANC inhibitory responses at 3 and 10 Hz use NO and CO as main transmitters. Their participation is revealed following inhibition of NOS, HO-2 and soluble guanylyl cyclase. The involvement of CO as a relaxing transmitter paves the way for novel therapeutic approaches in the treatment of airway obstruction.

Inhibitory Effects of Asiatic Acid on 7,12-Dimethylbenz[a]anthracene and 12-O-Tetradecanoylphorbol 13-Acetate-Induced Tumor Promotion in Mice

Asiatic acid, a pentacyclic triterpene, has been reported to induce apoptosis of various human cancer cells. In the present study, we assessed the anti-tumor promoting effect of asiatic acid against 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated skin tumorigenesis in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated ICR mice. Topical application of asiatic acid prior to each application of TPA resulted in a significant reduction in skin tumor formation. We also found that pre-application of asiatic acid alleviated TPA-induced [3H]thymidine incorporation, which is a conventional marker for skin tumor promotion. In addition, asiatic acid inhibited the TPA-induced generation of nitric oxide (NO) and expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), which are known to play important roles in tumor growth, especially in the promotion stage. In addition, topical application of aminoguanidine (AG), a selective iNOS inhibitor, and N(G)-nitro-L-arginine-methyl ester (NAME), another iNOS inhibitor, 30 min prior to TPA treatment significantly inhibited the TPA-induced COX-2 expression. These results suggest that asiatic acid may exert anti-tumorigenesis through inhibitory actions in NO and COX-2 signals.

Nicotine potentiates the nitrergic relaxation responses of rabbit corpus cavernosum tissue via nicotinic acetylcholine receptors

The presence of neuronal nicotinic acetylcholine receptors in rabbit corpus cavernosum tissue and possible mechanisms underlying the potentiation of electrical field stimulation induced relaxation by nicotine were analyzed. In corpus cavernosum tissue strips nicotine (3 × 10 − 5 M) and acetylcholine (10 − 3 M) produced potentiation on electrical field stimulation (amplitude 50 V; frequency 4 Hz; width 0.8 ms) induced relaxation responses. This nicotine-induced potentiation was not altered by atropine (10 − 6 M), guanethidine (5 × 10 − 6 M) and indomethacin (10 − 5 M), but abolished by hexamethonium chloride (10 − 5 M) and l-nitro arginine methyl ester (10 − 5 M). Nicotine did not cause any alteration on a single dose of carbachol (3 × 10 − 5 M) and sodium nitroprusside (10 − 5 M) induced relaxation responses. The results suggest that, nicotine-induced potentiation is NO and nicotinic acetylcholine receptor dependent but independent from prostaglandin synthesis, activation of muscarinic receptors and does not require intact adrenergic neurons. Nicotine did not affect smooth muscle and endothelium directly. In conclusion, in this study we showed for the first time that, nicotine acts on the nicotinic acetylcholine receptors located on the nitrergic nerves, thereby evoking the release of NO from these nerve terminals inducing relaxation response in rabbit corpus cavernosum tissue.

Interactions between apelin and angiotensin II on rat portal vein

Apelin (AP), an endogenous ligand of the APJ receptor, is involved in the regulation of cardiovascular homeostasis. Regardless the multiple similarities between AP and angiotensin II (Ang II), their roles seem to be different. We studied both the interactions between Apelin 13 (AP13) and Ang II and to what extent, if any, nitric oxide (NO) is involved. The experiments were performed in endothelium-denuded or endothelium-intact rat portal vein in the presence of 10 microM N(G)-nitro L-arginine methyl ester or 10 microM aminoguanidine. AP13 did not modify the isolated rat portal vein tone by itself, but inhibited the Ang II-induced contractions acting mainly by a NO-dependent mechanism. Our results sustain the hypothesis that AP13 could increase the activity of both constitutive and inducible NO synthase on either endothelium intact or endothelium denuded rat portal vein rings.

Endothelial Nitric Oxide Synthase–Dependent Tyrosine Nitration of Prostacyclin Synthase in Diabetes In Vivo

There is evidence that reactive nitrogen species are implicated in diabetic vascular complications, but their sources and targets remain largely unidentified. In the present study, we aimed to study the roles of endothelial nitric oxide synthase (eNOS) in diabetes. Exposure of isolated bovine coronary arteries to high glucose (30 mmol/l d-glucose) but not to osmotic control mannitol (30 mmol/l) switched angiotensin II-stimulated prostacyclin (PGI(2))-dependent relaxation into a persistent vasoconstriction that was sensitive to either indomethacin, a cyclooxygenase inhibitor, or SQ29548, a selective thromboxane receptor antagonist. In parallel, high glucose, but not mannitol, significantly increased superoxide and 3-nitrotyrosine in PGI(2) synthase (PGIS). Concurrent administration of polyethylene-glycolated superoxide dismutase (SOD), l-nitroarginine methyl ester, or sepiapterin not only reversed the effects of high glucose on both angiotensin II-induced relaxation and PGI(2) release but also abolished high-glucose-enhanced PGIS nitration, as well as its association with eNOS. Furthermore, diabetes significantly suppressed PGIS activity in parallel with increased superoxide and PGIS nitration in the aortas of diabetic C57BL6 mice but had less effect in diabetic mice either lacking eNOS or overexpressing human SOD (hSOD(+/+)), suggesting an eNOS-dependent PGIS nitration in vivo. We conclude that diabetes increases PGIS nitration in vivo, likely via dysfunctional eNOS.

Intravascular Neutrophil Activation Due to Carbon Monoxide Poisoning

We hypothesized that platelet-neutrophil interactions occur as a result of acute carbon monoxide (CO) poisoning, and subsequent neutrophil activation triggers events that cause neurologic sequelae. To identify platelet-neutrophil interactions and neutrophil activation in patients and in animal models, and to establish the association between these intravascular events and changes linked to CO-mediated neurologic sequelae in an animal model. Blood was obtained from 50 consecutive patients. Abnormalities were variable depending on the carboxyhemoglobin level at study admission and duration of CO exposure. Platelet-neutrophil aggregates were detected and plasma myeloperoxidase (MPO) concentration was significantly elevated in those with confirmed CO poisoning. Among patients exposed to CO for over 3 h, flow cytometry scans of neutrophils revealed increased surface expression of CD18 and, in some groups, MPO on the cell surface. Animal models revealed consistent evidence of platelet-neutrophil aggregates, neutrophil activation and surface MPO, and plasma MPO elevation. MPO was deposited along the brain vascular lining and colocalized with nitrotyrosine. CO poisoning caused abnormalities in the charge pattern of myelin basic protein (MBP), changes linked to adaptive immunologic responses responsible for neurologic sequelae in this model. Changes did not occur in thrombocytopenic rats, those receiving tirofiban to inhibit platelet-neutrophil interactions, or those receiving L-nitroarginine methyl ester to inhibit nitric oxide synthesis. Alterations in MBP did not occur in CO-poisoned knockout mice lacking MPO. Acute CO poisoning causes intravascular neutrophil activation due to interactions with platelets. MPO liberated by neutrophils mediates perivascular oxidative stress, which is linked to immune-mediated neurologic sequelae.

Muscle microvascular oxygenation in chronic heart failure: role of nitric oxide availability

To test the hypothesis that diminished vascular nitric oxide availability might explain the inability of individuals with chronic heart failure (CHF) to maintain the microvascular PO(2)'s (PO(2mv) proportional, variant O(2) delivery-to-uptake ratio) seen in healthy animals. We superfused sodium nitroprusside (SNP; 300 microm), Krebs-Henseleit (control, CON) and L-nitro arginine methyl ester (L-NAME; 1.5 mM) onto the spinotrapezius muscle and measured PO(2mv) by phosphorescence quenching in female Sprague-Dawley rats (n = 26) at rest and during twitch contractions (1 Hz). Seven rats served as controls (Sham) while CHF was induced by myocardial infarction. CHF rats were grouped as moderate (MOD; n = 15) and severe CHF (SEV; n = 4) according to morphological data and baseline PO(2mv). In contrast to Sham and MOD, L-NAME did not affect the PO(2mv) response (dynamics and steady-state) of SEV when compared with CON. SNP restored the PO(2mv) profile of SEV to that seen in Sham animals during CON. Specifically, the effect of L-NAME expressed as Delta(L-NAME - CON) were: Baseline PO(2mv) [in mmHg, DeltaSham = -7.0 +/- 1.6 (P < 0.05); DeltaSEV =-1.2 +/- 2.1], end-contractions PO(2mv) [in mmHg, DeltaSham = -5.0 +/- 1.0 (P < 0.05); DeltaSEV = -2.5 +/- 0.5] and time constant of PO(2mv) decrease [in s, DeltaSham = -6.5 +/- 3.0 (P < 0.05); DeltaSEV = -3.2 +/- 1.8]. These data provide the first direct evidence that the pathological profiles of PO(2mv) associated with severe CHF can be explained, in part, by a diminished vascular NO availability.

Crataegus Special Extract WS® 1442 Induces an Endothelium-Dependent, NO-mediated Vasorelaxation via eNOS-Phosphorylation at Serine 1177

This study investigates the influence of WS(R) 1442, a special extract of Crataegus leaves with flowers, on the relaxation of rat aorta and human mammarian artery (coronary bypass patients). Experiments were performed in the presence and absence (mechanical disruption) of endothelium. In addition, we investigated three fractions of WS(R) 1442 (fraction A: lipophilic, containing flavonoids and oligomeric procyanidins (OPC), fraction B: hydrophilic, containing flavonoids and low molecular weight OPC, fraction C: hydrophilic, essentially flavonoid-free and rich in high molecular weight OPC). WS 1442 induced a concentration-dependent vasodilation in isolated vessel rings that had been precontracted by 10 microM phenylephrine (concentration for halfmaximal relaxation (IC(50)): rat: 15.1 +/- 0.6 microg/ml (n = 7), human: 19.3 +/- 3.4 microg/ml (n = 6)). The maximal vasorelaxation induced after application of 100 microg of WS 1442 was 75.0 +/- 5.7% (rat) and 79.2 +/- 5.8% (human) of the papaverine (0.1 mM)-induced vasodilation. If the experiments were performed in the presence of L-nitroarginine methylester (10 microM, eNOS-inhibition) or after mechanical disruption of the endothelium, no vasorelaxation was observed in the presence of WS 1442. The vasorelaxant properties of WS 1442 were mediated by fraction C. WS 1442 induced an NO-liberation from human coronary artery endothelial cells as measured by diaminofluorescein. WS 1442 induced eNOS-activation was due to a phosphorylation at serine 1177. No eNOS-translocation or phosphorylation at serine 114 or threonine 495 was observed after application of WS 1442. It is concluded that WS 1442, induces an endothelium-dependent, NO-mediated vasorelaxation via eNOS phosphorylation at serine 1177.

Effect of inhibition of nitric oxide synthase on renal cyclooxygenase in the diabetic rat

Renal cyclooxygenase (COX)-2 expression and arachidonic acid-stimulated prostaglandin release are increased in streptozotocin-diabetic rats and are reduced by tempol treatment, indicating a role for superoxide. Generation of nitric oxide (NO) and its product with superoxide, peroxynitrite, is also increased in diabetes and can induce COX-2. To investigate a role of NO, rats were treated with l-nitroarginine methyl ester ( l-NAME; 100 mg/kg/day) to inhibit NO synthase (NOS) for 14–18 days. In isolated perfused kidneys from diabetic rats, prostaglandin release and vasoconstrictor responses to arachidonic acid were increased and renal cortical expression of COX-2 was 2-fold that of control rats. Treatment of diabetic rats with l-NAME reduced arachidonic acid-stimulated release of prostaglandins and the expression of COX-2. l-NAME increased vasoconstrictor responses to AA in diabetic and non-diabetic rats but abolished the differences between the two. These results, coupled with those using tempol, suggest that NO or its product with superoxide may contribute to the induction of renal COX-2 in the diabetic rat.

Effects of Altered Nitric Oxide Availability on Rat Muscle Microvascular Oxygenation During Contractions

Aging, chronic heart failure (CHF) and diabetes are all associated with vascular endothelial dysfunction. Compared with muscles from young healthy individuals, those from old and CHF/diabetic individuals exhibit a reduced muscle microvascular O pressure (POmv) at the onset of contractions that is expected to impair blood-myocyte O2 exchange. PURPOSE: To explore the putative role of nitric oxide (NO) in controlling PO2mv at rest and during contractions, we tested the hypotheses that at the onset of contractions (1 Hz) sodium nitroprusside (SNP, ↑ NO) would raise PO2mv and slow the kinetics of PO mv change whereas L-nitro arginine methyl ester (L-NAME, ↓ NO) would decrease PO2mv and speed its kinetics. This would assess the potential for derangements in NO production to induce the PO2mv and O2 kinetics impairments characteristic of aged and CHF/diabetic individuals. METHODS: Separately, we superfused the spinotrapezius muscle of female Sprague-Dawley rats (n = 7, body mass = 298 ± 10 g) with a NO donor SNP (300 uM) and the NO synthase inhibitor L-NAME (1.5 μM) during muscle contractions. Microvascular PO2 was measured by phosphorescence quenching. RESULTS: SNP decreased mean arterial pressure (92 ± 5 mmHg) below that of control (CON, 124 ± 4 mmHg) and L-NAME (120 ± 4 mmHg) conditions. SNP did not raise PO mv at rest but it did slow the kinetics of PO2mv by lengthening the time delay (TD, 14 ± 5 s), time constant (τ, 24 ± 10 s) and mean response time (MRT, 38 ± 4 s) of the response compared with CON (TD, 8.4 ± 3.3 s; τ, 16 ±4.5 s and MRT, 25 ± 1.5 s, P <: 0.05). In contrast, L-NAME decreased PO2mv at rest and speeded τ (10.1 ±3.8 s, P= 0.05), while TD (8.1 ± 1 s) was not significantly different. However, the MRT (18 ± 1 s) was faster for L-NAME compared to CON. Moreover, L-NAME caused PO2mv to transiently fall below steady-state values. CONCLUSION: These results indicate that NO availability can significantly affect microvascular O2 pressures, and therefore O2 transport, at rest and during contractions and suggests that such derangements in aging and chronic disease conditions may potentially result from impairments in NO availability.

Fluorescence Monitoring of ATP-Stimulated, Endothelium-Derived Nitric Oxide Production in Channels of a Poly(dimethylsiloxane)-Based Microfluidic Device

Intracellular nitric oxide (NO) production in a microfluidic endothelium is detected using fluorescence microscopy. Bovine pulmonary artery endothelial cells (bPAECs) were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent fluorescent DAF-FM DA/NO adduct was measured. Solutions of bradykinin, a well-known stimulus of endothelium-derived NO, activated nitric oxide synthase (NOS) in the immobilized bPAECs. This activation was inhibited using l-nitro arginine methyl ester (L-NAME), a competitive inhibitor of NOS. Importantly, the NO production was also stimulated with adenosine triphosphate (ATP) using concentrations as low as 1 microM. Previous reports on stimulating NO production using an immobilized endothelium in microfluidic channels were limited by the requirement of ATP concentrations of at least 100 microM, a value that is not physiologically relevant. The ability to monitor NO production with ATP concentrations that are similar to in vivo levels of ATP in the microcirculation represents a major advance in the use of microfluidic technology as an in vitro model of the microcirculation.

Involvement of endogenous nitric oxide in myeloperoxidase mediated benzo(a)pyrene induced polymorphonuclear leukocytes injury

The present study was undertaken to investigate the involvement of nitric oxide in the augmentation of benzo(a)pyrene induced cellular injury in polymorphonuclear leukocytes (PMNs). Polymorphs were isolated from the blood collected from Wistar rats treated with and without benzo(a)pyrene (50mg/kg, i.p.) through cardiac puncture. Catalase, superoxide dismutase (SOD), glutathione-s-transferase (GST), myeloperoxidase (MPO) and nitrite content were estimated in PMNs using standard procedures. Inducible nitric oxide synthase (iNOS) and cytochrome P-4501A1 (CYP1A1) expression in PMNs were also analyzed in presence or absence of nitric oxide synthase (NOS) inhibitors, aminoguanidine (AG, 5mM) and L-NG nitro L-arginine methyl ester (L-NAME, 1mM). A significant augmentation was observed in the nitrite content, activities of superoxide dismutase, MPO and GST and the expressions of iNOS and CYP1A1, however, catalase activity was attenuated in PMNs of benzo(a)pyrene treated rats as compared with their respective controls. AG and L-NAME resulted in a significant attenuation in nitrite content, MPO activity and iNOS expression; however, no significant alteration was observed in CYP1A1 expression. CYP1A1 inhibitor alpha-naphthoflavone inhibited the expression of iNOS in PMNs of benzo(a)pyrene treated animals significantly. The results obtained thus suggest that CYP1A1 induces iNOS expression leading to the generation of endogenous nitric oxide (NO) that could be responsible for the augmentation of myeloperoxidase-mediated benzo(a)pyrene-induced injury in PMNs.

Nitric Oxide Mediates the Blood-Pressure Lowering Effect of Garlic in the Rat Two-Kidney, One-Clip Model of Hypertension

Garlic reduces blood pressure (BP) in two-kidney, one-clip (2K-1C) rats, and enhances nitric oxide (NO) synthesis in in vivo and in vitro experiments. NO is an important modulator of BP in the 2K-1C model. This study investigated the role of NO in the BP-lowering effect of garlic in the 2K-1C model. BP readings (mm Hg) were obtained from 2K-1C rats in 4 groups treated intraperitoneally for 2 wk with either normal saline (NS), garlic, L-nitroarginine-methylester (L-NAME), or L-NAME+garlic (n=4x5). BP was determined using the tail-cuff method and compared with data of 4 similarly treated groups of normal (unclipped) rats (NRs). The BP readings of NR groups were 120+/-3 mm Hg for the NS-treated group, 120+/-2 mm Hg for the garlic-treated group, 167+/-3 mm Hg for the L-NAME treated group (higher than NS or garlic, P<0.001) and 128+/-5 mm Hg for the garlic+L-NAME-treated group (lower than L-NAME, P<0.001). The BP readings of 2K-1C rat groups were: for the NS group, 169+/-6 mm Hg (higher than NRs, P<0.001); for the garlic group, 116+/-7 mm Hg (lower than NS, P<0.001); for the L-NAME group: 184+/-8 mm Hg (higher than garlic, P<0.001), and for the L-NAME+garlic group: 130+/-6 mm Hg (lower than garlic or NS, P<0.001). The data show that L-NAME increases the BP of both NRs and 2K-1C rats, with the rise more evident in the NRs (39 vs. 9%, respectively). Garlic counteracts the hypertensive effect of L-NAME in NRs as well as 2K-1C rats. We conclude that the BP-lowering effect of garlic in the rat 2K-1C model may be partly mediated through the NO pathway.