Administration Of L-NMMA Research Articles

Chronic stress-induced lymphocyte reduction in a nitric oxide dependent manner

Abstract Psychological and physical stress can suppress the immune system in both humans and animals. The mechanism by which stress affects immune responses, however, remains poorly defined. Recent evidence demonstrated that nitric oxide (NO) modulates cell-mediated immune responses. Inducible nitric oxide synthase (iNOS) plays a major role in immune regulation. To investigate the role of NO in the chronic stress-induced lymphocyte depletion, we utilized a selective inhibitor of iNOS activity, NG-monomethyl-L-arginine (L-NMMA), which has been widely used, including from our laboratory and others, to evaluate the role of NO in immune responses both in vitro and in vivo. Here we demonstrate that administration of L-NMMA prevents chronic stress-induced lymphocyte reduction. Moreover, inhibition of iNOS prevents the alterations of cleaved-caspase-1 and p-JNK following chronic stress. These results reveal that chronic stress-induced lymphocyte reduction in a NO dependent manner.

Choline attenuates inflammatory hyperalgesia activating nitric oxide/cGMP/ATP-sensitive potassium channels pathway

New findings on neural regulation of immunity are allowing the design of novel pharmacological strategies to control inflammation and nociception. Herein, we report that choline, a 7-nicotinic acetylcholine receptor (α7nAChRs) agonist, prevents carrageenan-induced hyperalgesia without affecting inflammatory parameters (neutrophil migration or cytokine/chemokines production) or inducing sedation or even motor impairment. Choline also attenuates prostaglandin-E2 (PGE2)-induced hyperalgesia via α7nAChR activation and this antinociceptive effect was abrogated by administration of LNMMA (a nitric oxide synthase inhibitor), ODQ (an inhibitor of soluble guanylate cyclase; cGMP), andglibenclamide(an inhibitor of ATP-sensitive potassium channels). Furthermore, choline attenuates long-lasting Complete Freund’s Adjuvant and incision-induced hyperalgesia suggesting its therapeutic potential to treat pain in rheumatoid arthritis or post-operative recovery, respectively. Our results suggest that choline modulates inflammatory hyperalgesia by activating the nitric oxide/cGMP/ATP-sensitive potassium channels without interfering in inflammatory events, and could be used in persistent pain conditions.

Nitric Oxide Contributes to Vasomotor Tone in Hypertensive African Americans Treated With Nebivolol and Metoprolol.

Endothelial dysfunction is more prevalent in African Americans (AAs) compared with whites. The authors hypothesized that nebivolol, a selective β1 -antagonist that stimulates nitric oxide (NO), will improve endothelial function in AAs with hypertension when compared with metoprolol. In a double-blind, randomized, crossover study, 19 AA hypertensive patients were randomized to a 12-week treatment period with either nebivolol 10 mg or metoprolol succinate 100 mg daily. Forearm blood flow (FBF) was measured using plethysmography at rest and after intra-arterial infusion of acetylcholine and sodium nitroprusside to estimate endothelium-dependent and independent vasodilation, respectively. Physiologic vasodilation was assessed during hand-grip exercise. Measurements were repeated after NO blockade with L-N(G) -monomethylarginine (L-NMMA) and after inhibition of endothelium-derived hyperpolarizing factor (EDHF) with tetraethylammonium chloride (TEA). NO blockade with L-NMMA produced a trend toward greater vasoconstriction during nebivolol compared with metoprolol treatment (21% vs 12% reduction in FBF, P=.06, respectively). This difference was more significant after combined administration of L-NMMA and TEA (P<.001). Similarly, there was a contribution of NO to exercise-induced vasodilation during nebivolol but not during metoprolol treatment. There were significantly greater contributions of NO and EDHF to resting vasodilator tone and of NO to exercise-induced vasodilation with nebivolol compared with metoprolol in AAs with hypertension.

Acute glucagon induces postprandial peripheral insulin resistance.

Glucagon levels are often moderately elevated in diabetes. It is known that glucagon leads to a decrease in hepatic glutathione (GSH) synthesis that in turn is associated with decreased postprandial insulin sensitivity. Given that cAMP pathway controls GSH levels we tested whether insulin sensitivity decreases after intraportal (ipv) administration of a cAMP analog (DBcAMP), and investigated whether glucagon promotes insulin resistance through decreasing hepatic GSH levels.Insulin sensitivity was determined in fed male Sprague-Dawley rats using a modified euglycemic hyperinsulinemic clamp in the postprandial state upon ipv administration of DBcAMP as well as glucagon infusion. Glucagon effects on insulin sensitivity was assessed in the presence or absence of postprandial insulin sensitivity inhibition by administration of L-NMMA. Hepatic GSH and NO content and plasma levels of NO were measured after acute ipv glucagon infusion. Insulin sensitivity was assessed in the fed state and after ipv glucagon infusion in the presence of GSH-E. We founf that DBcAMP and glucagon produce a decrease of insulin sensitivity, in a dose-dependent manner. Glucagon-induced decrease of postprandial insulin sensitivity correlated with decreased hepatic GSH content and was restored by administration of GSH-E. Furthermore, inhibition of postprandial decrease of insulin sensitivity L-NMMA was not overcome by glucagon, but glucagon did not affect hepatic and plasma levels of NO. These results show that glucagon decreases postprandial insulin sensitivity through reducing hepatic GSH levels, an effect that is mimicked by increasing cAMP hepatic levels and requires physiological NO levels. These observations support the hypothesis that glucagon acts via adenylate cyclase to decrease hepatic GSH levels and induce insulin resistance. We suggest that the glucagon-cAMP-GSH axis is a potential therapeutic target to address insulin resistance in pathological conditions.

Role of nitric oxide in optic nerve head blood flow regulation during an experimental increase in intraocular pressure in healthy humans

The present study set out to investigate whether nitric oxide, a potent vasodilator, is involved in the regulatory processes in optic nerve head blood flow during an experimental increase in intraocular pressure (IOP). The study was conducted in a randomized, double-masked, placebo-controlled, three way cross-over design. 12 healthy subjects were scheduled to receive either L-NMMA (an unspecific nitric oxide synthase inhibitor), phenylephrine (an α-adrenoceptor agonist) or placebo on three different study days. Optic nerve head blood flow was measured using laser Doppler flowmetry and IOP was increased stepwise with a suction cup. Mean arterial pressure (MAP) and IOP were measured non-invasively and ocular perfusion pressure (OPP) was calculated as OPP = 2/3 MAP-IOP. Administration of L-NMMA and phenylephrine significantly increased MAP and therefore OPP at rest (p < 0.01). L-NMMA significantly reduced baseline blood flow in the optic nerve head (p < 0.01). Application of the suction cup induced a significant increase in IOP and a decrease in OPP (p < 0.01). During the stepwise increase in IOP, some autoregulatory potential was observed until OPP decreased approximately −30% below baseline. None of the administered substances had an effect on this autoregulatory behavior (p = 0.49). The results of the present study confirm that the human optic nerve head shows some regulatory capacity during a decrease in OPP. Nitric oxide is involved in the regulation of basal vascular tone in the optic nerve head but does not seem to be involved in the regulatory mechanisms during an acute increase in IOP in young healthy subjects.

Myocardial protection by co-administration of l-arginine and tetrahydrobiopterin during ischemia and reperfusion

BackgroundReduced bioavailability of nitric oxide (NO) is a key factor contributing to myocardial ischemia and reperfusion injury. The mechanism behind the reduction of NO is related to deficiency of the NO synthase (NOS) substrate l-arginine and cofactor tetrahydrobiopterin (BH4) resulting in NOS uncoupling. The aim of the study was to investigate if the combination of l-arginine and BH4 given iv or intracoronary before reperfusion protects from reperfusion injury. MethodsSprague–Dawley rats and pigs were subjected to myocardial ischemia and reperfusion. Rats received vehicle, l-arginine, BH4, l-arginine+BH4 with or without the NOS-inhibitor L-NMMA iv 5min before reperfusion. Pigs received infusion of vehicle, l-arginine, BH4 or l-arginine+BH4 into the left main coronary artery for 30min starting 10min before reperfusion. ResultsInfarct size was significantly smaller in the rats (50±2%) and pigs (54±5%) given l-arginine+BH4 in comparison with the vehicle groups (rats 65±3% and pigs 86±5%, P<0.05). Neither l-arginine nor BH4 alone significantly reduced infarct size. Administration of L-NMMA abrogated the cardioprotective effect of l-arginine+BH4. Myocardial BH4 levels were 3.5- to 5-fold higher in pigs given l-arginine+BH4 and BH4 alone. The generation of superoxide in the ischemic-reperfused myocardium was reduced in pigs treated with intracoronary l-arginine+BH4 versus the vehicle group (P<0.05). ConclusionAdministration of l-arginine+BH4 before reperfusion protects the heart from ischemia–reperfusion injury. The cardioprotective effect is mediated via NOS-dependent pathway resulting in diminished superoxide generation.

Myocardial protection by co-administration of L-arginine and tetrahydrobiopterin during ischemia and reperfusion

Abstract Background Reduced bioavailability of nitric oxide (NO) is a key factor contributing to myocardial ischemia and reperfusion injury. The mechanism behind the reduction of NO is related to deficiency of the NO synthase (NOS) substrate l-arginine and cofactor tetrahydrobiopterin (BH 4 ) resulting in NOS uncoupling. The aim of the study was to investigate if the combination of l-arginine and BH 4 given iv or intracoronary before reperfusion protects from reperfusion injury. Methods Sprague–Dawley rats and pigs were subjected to myocardial ischemia and reperfusion. Rats received vehicle, l-arginine, BH 4 , l-arginine+BH 4 with or without the NOS-inhibitor L-NMMA iv 5min before reperfusion. Pigs received infusion of vehicle, l-arginine, BH 4 or l-arginine+BH 4 into the left main coronary artery for 30min starting 10min before reperfusion. Results Infarct size was significantly smaller in the rats (50±2%) and pigs (54±5%) given l-arginine+BH 4 in comparison with the vehicle groups (rats 65±3% and pigs 86±5%, P 4 alone significantly reduced infarct size. Administration of L-NMMA abrogated the cardioprotective effect of l-arginine+BH 4 . Myocardial BH 4 levels were 3.5- to 5-fold higher in pigs given l-arginine+BH 4 and BH 4 alone. The generation of superoxide in the ischemic-reperfused myocardium was reduced in pigs treated with intracoronary l-arginine+BH 4 versus the vehicle group (P Conclusion Administration of l-arginine+BH 4 before reperfusion protects the heart from ischemia–reperfusion injury. The cardioprotective effect is mediated via NOS-dependent pathway resulting in diminished superoxide generation.

Role of Nitric Oxide in Optic Nerve Head Blood Flow Regulation during Isometric Exercise in Healthy Humans

We determined whether administration of a nitric oxide synthase (NOS) inhibitor alters optic nerve head blood flow (ONHBF) regulation during isometric exercise in healthy subjects. Our study was done in a randomized, placebo-controlled, double-masked, three-way crossover design. A total of 18 healthy subjects was randomized to receive either placebo, phenylephrine, or an inhibitor of NOS (L-NMMA) on three different study days. ONHBF was measured with laser Doppler flowmetry while the study participants performed isometric exercise (squatting). This was done before drug administration and during infusion of the study drugs. Mean arterial pressure (MAP) and IOP were measured noninvasively, and ocular perfusion pressure (OPP) was calculated as 2/3 MAP - IOP. The response in ONHBF to isometric exercise was less pronounced than the response in OPP, indicating for some autoregulatory capacity in the ONH. Administration of L-NMMA significantly decreased ONHBF at rest (P < 0.01). In contrast, inhibition of NOS did not alter the pressure-flow relationship in the ONH during an experimental increase in OPP compared to phenylephrine and placebo (P = 0.37 between groups). The data of our study support previous findings that ONHBF is autoregulated during an experimental increase in OPP. Nitric oxide has an important role in basal ONHBF regulation, but seems not to be involved in the autoregulatory response during an increase in OPP induced by isometric exercise. (ClinicalTrials.gov number, NCT00806741.).

Effect of NO synthase inhibition on retinal vessel reaction to isometric exercise in healthy humans

It has been shown that retinal blood flow is autoregulated, meaning that flow is independent of perfusion pressure within a certain range. We tested the hypothesis that nitric oxide (NO) synthase inhibition alters the response of retinal arterial and venous vessels during isometric exercise. In this study, nine healthy subjects were included. Each subject received the NO synthase inhibitor Ng-monomethyl-l-Arginine (l-NMMA, the α-receptor agonist phenylephrine or placebo intravenously on three study days. Retinal vessel diameter was assessed with the retinal vessel analyser (RVA), at baseline and during a squatting period of 6-7 min in absence or presence of l-NMMA, phenylephrine or placebo. Mean arterial pressure (MAP) and pulse rate (PR) increased significantly during all pretreatment squatting periods (p < 0.001) Retinal venous and arterial diameters showed a continuous decrease during squatting (p < 0.001). Phenylephrine increased MAP and PR but did not alter the retinal vessel diameter response to squatting. Administration of l-NMMA lead to a significant decrease in venous diameter before isometric exercise (p = 0.004). In addition, the retinal venous diameter response during administration of the NO synthase inhibitor was less pronounced than during phenylephrine or placebo (p < 0.001). Our study confirms that NO plays an important role in the control of retinal vascular tone at rest. In addition, the present data indicate a role of NO in retinal autoregulation, because the response of retinal venous diameters was altered after NO synthase inhibition. The nature of involvement, however, appears to be complex and requires further studies.

Ageing uncompensated: exercise, nitric oxide and hypoxia

Ageing uncompensated: exercise, nitric oxide and hypoxia

Endothelium in polycythemia secondary to obstructive lung disease

The object of this editorial is the report of Boyer et al in this issue of the Journal of Applied Physiology .It has two merits. One, endothelial function was studied in individuals with polycythemia secondary to obstructive lung disease, a condition not commonly addressed by cardiovascular investigators, although its presence is associated with a clearcut increase in cardiovascular risk .Two, the authors made use of virtually all techniques available in man to determine endothelial-dependent modulation of vascular tone, an unusual effort that differentiates this study from most other studies so far published. The results provide evidence that in polycythemic patients with obstructive lung disease the forearm vasoconstriction induced by L-NMMA administration was not reduced as compared to controls, while the brachial artery dilatation in response to post-ischemic hyperemia was increased.

Effects of central administration of insulin or l-NMMA on rat skeletal muscle microvascular perfusion

Intracerebroventricular (ICV) administration of a nitric oxide synthase (NOS) inhibitor to rats has been reported to raise blood pressure (BP) and cause insulin resistance, suggestive of a central effect of insulin that is NO dependent. Herein we test whether ICV insulin has peripheral haemodynamic and metabolic effects and whether peripheral effects of systemic insulin are affected by the ICV administration of the NOS inhibitor N(G) -methyl-l-arginine (l-NMMA). Anaesthetized rats were fitted with an ICV cannula for insulin, artificial cerebrospinal fluid (aCSF) or l-NMMA infusion. Rats receiving ICV l-NMMA (500 µg) underwent systemic insulin clamp (10 mU/min/kg) or saline treatment for 70 min and were compared with animals receiving an equal amount of l-NMMA infused systemically. ICV aCSF or insulin (135 mU/min/kg brain) for 70 min or systemic l-NMMA (500 µg) had no effect on BP, heart rate (HR), femoral blood flow (FBF), glucose infusion rate, muscle 2-deoxyglucose uptake, microvascular perfusion or plasma insulin. However, ICV l-NMMA reduced systemic insulin-mediated increases in FBF (2.05 ± 0.08 to 1.55 ± 0.15 ml/min), 2-deoxyglucose uptake (17.7 ± 0.15 to 10.0 ± 0.03 µg/g/min) and microvascular perfusion (10.5 ± 0.5 to 6.6 ± 1.1 mol/min) (each mean ± SE, p < 0.05); plasma insulin, HR and BP were unaffected. Central insulin administration had no effect on skeletal muscle haemodynamics or glucose metabolism. However, systemic insulin-mediated increases in limb blood flow, muscle microvascular perfusion and glucose uptake may be regulated by a central pathway that is NO dependent.

Hocus pocus hypoxia – NO and augmented vasodilatation in the systemic vasculature during hypoxic exercise

Evidence clearly points to an enhanced vasodilatation in systemic vessels during hypoxic exercise that serves to defend oxygen delivery to active musculature in the face of a reduced inspired fraction of O2 (Wilkins et al. 2008). Exercise-induced hyperaemia is a complex process with redundant mechanisms that can be called upon when required. During hypoxic exercise there is an additional dilator response in human skeletal muscle attributable to the reduction in arterial oxygen content rather than arterial O2 tension per se. Importantly, prevailing vascular tone results from both neural and metabolic factors acting on the vascular smooth muscle and endothelium. At the vascular endothelium adrenergic and non-adrenergic vasoactive pathways play a regulatory role in normal vascular function. The overall effect on haemodynamics and arterial pressure will be determined by how these various pathways and mechanisms integrate at the level of the vascular smooth muscle cell. Over recent years various research groups, including ours (Bailey et al. 2009), have combined a number of invasive experimental protocols across isolated vascular beds in conjunction with pharmacological blockade of vasoactive metabolite receptors or the actual metabolite itself to elucidate any potential contribution to exercise and/or hypoxic vasodilatation. One of the most intensely studied candidates for both exercise hyperaemia and hypoxic vasodilatation is nitric oxide (NO•). Increases in blood flow, cyclic wall stress due to pulsatile blood flow and catecholamines produce an up-regulation and release of NO• from the vascular endothelium (Busse & Fleming, 2006) via the enzyme endothelial nitric oxide synthase (eNOS). Hypoxia has been associated with additional sources of NO• release from deoxyhaemoglobin, β-adrenergic and adensosine receptor stimulation (Stamler et al. 1997; Bryan & Marshall, 1999; Wilkins et al. 2008). The NO• released toward the vascular lumen is a powerful vasodilator responsible for mediating basal vascular tone (Stamler et al. 1997). However, not all vascular beds respond in a similar manner with the pulmonary vasculature demonstrating a strong hypoxia-induced vasoconstriction whereas the cerebral vasculature responds in a similar fashion to the systemic vessels with a vasodilatation (Bailey et al. 2009). Metabolism of NO• within the vasculature to the more biochemically stable moiety nitrite serves as a means to determine circulating bioavailability of NO•. It appears that whilst this metabolic pathway of NO• was initially considered unidirectional, exogenous nitrite can induce sustained vasodilatation especially when the local vascular environment is hypoxic or ischaemic (Maher et al. 2008). It is within this environment that deoxygenated haemoglobin appears to convert nitrite to NO• (Stamler et al. 1997). Our laboratory, in collaboration with others, recently reported a reduced pulmonary vasoconstriction with systemic infusion of sodium nitrite (Ingram et al. 2010) while others have also reported augmented systemic arterial hypoxic vasodilatation with the same agent (Maher et al. 2008). Thus, with this background it is evident that during hypoxic exercise there is a compensatory vasodilatation that is sustained during increased exercise intensity and a clear contender for mediating the response is NO• either from enhanced endothelial release and/or circulating deoxyhaemoglobin. Casey et al. (2010), in a recent article in the The Journal of Physiology, sought to address this issue by examining the effects of hypoxic forearm exercise whilst simultaneously infusing the NOS inhibitor NG-monomethyl-l-arginine (l-NMMA) to investigate the influence of endothelial-derived NO•. In a parallel branch of the investigation, the authors also attempted to glean further information regarding hypoxic-induced NO• release via adenosine receptor stimulation by exogenous administration of combined l-NMMA and aminophylline, an adenosine receptor antagonist (Casey et al. 2010). Efficacy of eNOS blockade was established via intra-arterial acetylcholine infusion. Casey and colleagues (2010) utilised the isolated forearm exercise model with 22 healthy young adults. Subjects performed rhythmic forearm exercise in the non-dominant arm at 10% and 20% of individual maximal voluntary contraction. Twelve subjects completed protocol 1 (saline or l-NMMA infusion) and ten subjects completed protocol 2 (saline or l-NMMA–aminophylline infusion). Due to the long half-life of l-NMMA, study drugs were administered in the same order. Exercise was performed in normoxia and normocapnic hypoxia. Hypoxic inspiration rendered systemic arterial O2 saturations at ∼80%. Arterial pressure responses were monitored with an indwelling pressure transducer in the brachial artery whilst forearm blood flow was determined in the brachial artery via ultrasound. Forearm vascular conductance was calculated by the quotient of forearm blood flow and arterial pressure (Casey et al. 2010). The paper highlights three key findings of importance regarding the role of NO• in hypoxic vasodilatation.

Inhibition of nitric oxide synthases prevents and reverses α,β-meATP-induced neck muscle nociception in mice

Tension-type headache (TTH) is associated with noxious input from neck muscles. Intravenous administration of the unspecific nitric oxide synthase inhibitor L-NMMA in chronic TTH patients caused analgesia and reduction of neck muscle tenderness. The unspecific nitric oxide synthase inhibitor L-NMMA was applied in an experimental model for neck muscle nociception in anesthetized mice (N = 25). Local injection of α,β-meATP into semispinal neck muscles induced sustained facilitation of brainstem nociception as monitored by the jaw-opening reflex. Preceding intraperitoneal administration of L-NMMA (0.05, 0.1, 1 mg/kg) prevented reflex facilitation evoked by α,β-meATP in a dose-dependent manner. Intraperitoneal injection of L-NMMA subsequent to intramuscular α,β-meATP application reversed established brainstem reflex facilitation back to baseline values. Both experiments with preceding and subsequent L-NMMA indicate the involvement of nitric oxide synthases in the induction and maintenance of facilitation. However, future experiments will have to address the involvement of various isoenzymes in order to provide for new therapeutic concepts in TTH.

Nitric oxide contributes to the augmented vasodilatation during hypoxic exercise

We tested the hypotheses that (1) nitric oxide (NO) contributes to augmented skeletal muscle vasodilatation during hypoxic exercise and (2) the combined inhibition of NO production and adenosine receptor activation would attenuate the augmented vasodilatation during hypoxic exercise more than NO inhibition alone. In separate protocols subjects performed forearm exercise (10% and 20% of maximum) during normoxia and normocapnic hypoxia (80% arterial O(2) saturation). In protocol 1 (n = 12), subjects received intra-arterial administration of saline (control) and the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). In protocol 2 (n = 10), subjects received intra-arterial saline (control) and combined L-NMMA-aminophylline (adenosine receptor antagonist) administration. Forearm vascular conductance (FVC; ml min(-1) (100 mmHg)(-1)) was calculated from forearm blood flow (ml min(-1)) and blood pressure (mmHg). In protocol 1, the change in FVC (Delta from normoxic baseline) due to hypoxia under resting conditions and during hypoxic exercise was substantially lower with L-NMMA administration compared to saline (control; P < 0.01). In protocol 2, administration of combined L-NMMA-aminophylline reduced the DeltaFVC due to hypoxic exercise compared to saline (control; P < 0.01). However, the relative reduction in DeltaFVC compared to the respective control (saline) conditions was similar between L-NMMA only (protocol 1) and combined L-NMMA-aminophylline (protocol 2) at 10% (-17.5 +/- 3.7 vs. -21.4 +/- 5.2%; P = 0.28) and 20% (-13.4 +/- 3.5 vs. -18.8 +/- 4.5%; P = 0.18) hypoxic exercise. These findings suggest that NO contributes to the augmented vasodilatation observed during hypoxic exercise independent of adenosine.

NOS inhibition blunts and delays the compensatory dilation in hypoperfused contracting human muscles

We previously demonstrated that skeletal muscle blood flow is restored in the exercising forearm during experimental hypoperfusion via local dilator and/or myogenic mechanisms. This study examined the role of nitric oxide (NO) in the restoration of blood flow to the active muscles during hypoperfusion. Eleven healthy subjects (10 men/1 woman; 25 +/- 1 yr of age) performed rhythmic forearm exercise (10% and 20% of maximum) while hypoperfusion was evoked by balloon inflation in the brachial artery above the elbow. Each trial included baseline, exercise, exercise with inflation, and exercise after deflation (3 min each). Forearm blood flow (FBF; ultrasound) and local (brachial artery catheter pressure, BAP) and systemic arterial pressure [mean arterial pressure (MAP); Finometer] were measured. The exercise bouts were repeated during N(G)-monomethyl-L-arginine (L-NMMA) infusion (NO synthase inhibition). Forearm vascular conductance (FVC; ml x min(-1) x 100 mmHg(-1)) was calculated from BF (ml/min) and BAP (mmHg). FBF and FVC fell acutely with balloon inflation during all trials (P < 0.01). Recovery of FBF and FVC [(inflation - nadir)/(steady-state exercise - nadir)] with L-NMMA administration was reduced during 20% exercise (FBF = 77 +/- 7% vs. 88 +/- 8%; FVC = 71 +/- 8% vs. 90 +/- 9%; P < 0.01) but not 10% exercise (FBF = 83 +/- 4% vs. 81 +/- 5%, P = 0.37; FVC = 75 +/- 10% vs. 76 +/- 7%; P = 0.44) compared with the respective control trial. The time to steady-state vasodilator response was substantially longer during the l-NMMA trials (10% = 74 +/- 4 s vs. 61 +/- 6 s; 20% = 53 +/- 4 s vs. 41 +/- 4 s; P < 0.05). Thus the magnitude and timing of the NO contribution to compensatory dilation during forearm exercise with hypoperfusion was dependent on exercise intensity. These observations suggest that NO is released by contracting muscles or that a portion of the dilation caused by ischemic metabolites is NO dependent.

Arginase inhibition mediates cardioprotection during ischaemia-reperfusion

Nitric oxide (NO) is vital for the integrity of the cardiovascular system and protection against ischaemic heart disease. Arginase is up-regulated during ischaemia-reperfusion (IR) and this enzyme might compete with NO synthase (NOS) for arginine. The present study investigated whether arginase blockade protects from myocardial IR injury and whether such an effect is coupled to increased NO bioavailability. Sprague-Dawley rats were subjected to 30 min of coronary artery ligation, followed by 2 h of reperfusion. The animals were given either saline, or the arginase inhibitor N-omega-hydroxy-nor-l-arginine (nor-NOHA) with or without the NO scavenger carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) or the NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) iv 15 min before ischaemia. The infarct size was 79 +/- 4% of the area at risk in the control group. Nor-NOHA treatment reduced the infarct size to 39 +/- 7% (P < 0.001). Administration of cPTIO or l-NMMA completely abolished the protective effect of nor-NOHA. Expression of arginase I was significantly (P < 0.05) increased in ischaemic myocardium. Nor-NOHA treatment resulted in higher plasma levels of nitrite (P < 0.05) and a 10-fold increase in the citrulline/ornithine ratio (P < 0.001), indicating a shift in arginine utilization towards NOS. Inhibition of arginase protects from myocardial infarction by a mechanism that is dependent on NOS activity and bioavailability of NO by shifting arginine utilization from arginase towards NOS. These findings suggest that targeting of arginase is a promising future therapeutic strategy for protection against myocardial IR injury.

In vivo human lower extremity saphenous vein bypass grafts manifest flow mediated vasodilation

As in arteries, venous endothelium modulates vessel homeostasis and tone. The effect of an arterialized environment on venous endothelial function remains poorly understood. In particular, regulation of saphenous vein graft (SVG) blood flow and lumen caliber remains undefined. We hypothesized that mature SVGs would exhibit endothelium-dependent, flow-mediated vasodilation (FMD). We further hypothesized that endothelium-derived nitric oxide (NO) was an important mediator. Patients with femoral to popliteal artery SVGs that had maintained primary patency and were at least 1 year from surgery were enrolled. High-resolution, B-mode ultrasound scans were used to measure vein graft diameter before and 1 minute after reactive hyperemia (RH) to determine FMD. RH was created through application of 220 mm Hg to the calf for 5 minutes with a sphygmomanometric cuff. After a 10-minute recovery period, nitroglycerin-mediated, endothelium-independent vasodilation was measured 3 minutes after administration of nitroglycerin 0.4 mg sublingually. Brachial artery FMD was determined by validated techniques. L-N(G)monomethyl arginine (L-NMMA; 1 mg/kg infusion over 10 minutes) was used in a subset of patients (n = 6) to competitively inhibit endothelial NO synthase. Nineteen subjects were enrolled. The median age of the SVGs was 34.6 (21.0-49.7) months. SVG flow-mediated, endothelium-dependent vasodilation was measured at 5.28% +/- 3.1% mean change in lumen diameter (range, 1.99%-9.36%; P < .0001 for diameter change). Nitroglycerin-mediated vasodilation was 3.7% +/- 1.0%, (range, 16%-10.04%; P < .005). Intravenous administration of L-NMMA abolished SVG FMD (5.7 +/- 1.4% before L-NMMA vs 0.01 +/- 0.01% during L-NMMA infusion; P = .0088) and attenuated brachial artery FMD (7.54% +/- 1.0% vs 5.7 +/- 1.4; P = .05). SVGs manifest flow-mediated, endothelium-dependent, and nitroglycerin-mediated endothelium-independent vasodilation. Vein graft endothelium-dependent FMD is likely mediated by NO. Further investigation will be required to determine the role of endothelial function in vein graft patency.

Spinal and peripheral mechanisms involved in the enhancement of morphine analgesia in acutely inflamed mice.

The analgesic effect induced by opiates is often potentiated during experimental inflammatory processes. We describe here that lower doses of systemic morphine are necessary to increase thermal withdrawal latencies measured in both hind paws of mice acutely inflamed with carrageenan than in healthy ones. This bilateral potentiation seems mediated through spinal opioid receptors since it is inhibited by the intrathecal (i.t.), but not intraplantar (i.pl.) administration of the opioid receptor antagonist naloxone-methiodide, and also appears when morphine is i.t. administered. Furthermore, the i.pl. administration of the nitric oxide (NO) synthase inhibitor, L-NMMA, or the K (ATP) (+) -channel blocker, glibenclamide, to carrageenan-inflamed mice inhibits the enhanced effect of systemic morphine in the paw that receives the injection of the drug, without affecting the potentiation observed in the contralateral one. The i.pl. administration of L-NMMA also partially antagonised the analgesic effect induced by i.t. morphine in inflamed mice. Finally, the increased analgesic effect evoked by the i.pl. administration of the NO donor SIN-1 either in the inflamed or in the contralateral paw of carrageenan-inflamed mice suggests that enhanced responsiveness to the peripheral analgesic effect of NO may be also underlying the bilateral potentiation of morphine-induced analgesia in acutely inflamed mice.

Prominent role of NF-κB in the induction of endothelial activation by endogenous nitric oxide inhibition

Decreased endothelial nitric oxide (NO) production and increased expression of vascular cell adhesion molecule-1 (VCAM-1) are early features of atherosclerosis. We investigated the effects of suppressing endogenous NO production by the NO synthase inhibitor l-mono-methyl-arginine (L-NMMA), given alone or in combination with interleukin(IL)-1alpha, on VCAM-1 expression by human umbilical vein endothelial cells (HUVEC). VCAM-1 expression (by enzyme immunoassay), barely detectable at baseline, was significantly increased by L-NMMA (by no more than 20% over control compared with IL-1alpha induction). This was paralleled by an increase in U937 monocytoid cell adhesion. When HUVEC incubated with L-NMMA were stimulated with low concentrations of IL-1alpha (0.05-0.5ng/mL), these determined a higher VCAM-1 expression than in the presence of L-NMMA or IL-1alpha alone. Northern analysis indicated that VCAM-1 mRNA was induced by L-NMMA alone, and that the effects of L-NMMA and IL-1alpha were, again, at least additive. Nuclear factor-kappaB (NF-kappaB), GATA, activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF-1), transcription factors all involved in VCAM-1 gene expression, were all activated at electrophoretic mobility shift assay and at chromatin immunoprecipitation assay by L-NMMA, but additive effects with the combined administration of L-NMMA and IL-1alpha only occurred for NF-kappaB. These results support the view that endogenous NO mantains a normal endothelial non-reactivity towards circulating monocytes, and that suppression of this endogenous brake for endothelial activation results in the activation of multiple transcription factors even in the absence of other endothelial activators, with a prominent role of NF-kappaB in the presence or absence of other inflammatory mediators.