Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 120 mg. kg-1. day-1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6-5.5 microM) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 microM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.     

Nitric oxide synthase activity in blood vessels of spontaneously hypertensive rats: antioxidant protection by gamma-tocotrienol.

Involvement of free radicals and nitric oxide (NO) has long been implicated to the pathogenesis of essential hypertension. Several studies using antioxidants as the radical scavenger have shown to confer protection against free radical mediated diseases. This study is designed to investigate the role of antioxidant gamma-tocotrienol on endothelial nitric oxide synthase (NOS) activity in spontaneously hypertensive rats (SHR). SHR's were divided into four groups namely untreated SHR (HC), treatment with 15 mg gamma-tocotrienol/kg diet (gammal), 30 mg gamma-tocotrienol/kg diet (gamma2) and 150 mg gamma-tocotrienol/kg diet (gamma3) and studied for three months. Wister Kyoto (WKY) rats were used as the control (C). Blood pressure was recorded every fortnightly by tail plethysmography. Animals were sacrificed and NOS activity in blood vessels was measured by [3H]arginine radioactive assay. Nitrite concentration in plasma was determined by Greis assay and lipid peroxides in the blood vessels by spectrofluorometry. This study showed that gamma-tocotrienol significantly reduced systolic blood pressure (SBP) in SHRs with a maximum reduction in group treated with gamma-tocotrienol 15 mg/kg diet (HC: 210 +/- 9 mmHg, gammal:123 +/- 19 mmHg). Blood vessels from untreated SHR showed a reduced NOS activity compare to that of WKY rats (C: 1.54 +/- 0.26 pmol/mg protein, HC: 0.87 +/- 0.23 pmol/mg protein; p<0.001). Gamma-tocotrienol improves NOS activity in all the groups with more significance in group gamma2 (p<0.001) and gamma3 (p<0.05). Plasma level of nitrite was reduced in SHR from 55 +/- 3 microM/ml in WKY to 26+/-2 muM/ml (p<0.001). Plasma nitrite level was reversed by treatment with gamma-tocotrienol. (gammal: p<0.001, gamma2: p<0.005, gamma3: p<0.001, respectively). In all the treatment groups, NOS activity showed significant negative correlation with blood pressure (gammal: r=-0.716, p<0.05; gamma2: r=-0.709, p<0.05; gamma3: r=-0.789, p<0.05). For plasma nitrite, although it shows a negative correlation with blood pressure it was significant only in gammal (r=-0.676, p<0.05) and gamma2 (r=-0.721, p<0.05). From this study we found that compared to WKY rats, SHR has lower NOS activity in blood vessels, which upon treatment with antioxidant gamma-tocotrienol increased the NO activity and concomitantly reduced the blood pressure. These findings further strengthen the hypothesis that free radicals and NO play critical role in pathogenesis of essential hypertension.     

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)alpha-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)alpha and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARalpha ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARalpha activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARalpha ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARalpha knockout (KO) mice compared with its wild type (WT) litter mates (130+/-10 mmHg versus 107+/-4 mmHg). L-NAME (100mg/L p.o.), the inhibitor of NO production abolished the difference between PPARalpha KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8+/-1.4 pM/mg versus 8.3+/-0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46+/-6%, p<0.05) and a approximately 3 fold greater Ca2+-dependent NOS activity in kidney homogenates of untreated PPARalpha WT compared with the KO mice. Clofibrate, a PPARalpha ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19+/-4%, p<0.05), increased urinary NO excretion (4.06+/-0.53-7.07+/-1.59 microM/24 h; p<0.05) and reduced plasma 8-isoprostane level (45.8+/-15 microM versus 31.4+/-8 microM), and NADP(H) oxidase activity (16+/-5%). Implantation of DOCA pellet (20mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193+/-13 mmHg versus 130+/-12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARalpha activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.     

Endothelial dysfunction and arterial pressure regulation during early diabetes in mice: roles for nitric oxide and endothelium-derived hyperpolarizing factor

We determined whether nitric oxide (NO) counters the development of hypertension at the onset of diabetes in mice, whether this is dependent on endothelial NO synthase (eNOS), and whether non-NO endothelium-dependent vasodilator mechanisms are altered in diabetes in mice. Male mice were instrumented for chronic measurement of mean arterial pressure (MAP). In wild-type mice, MAP was greater after 5 wk of N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 mg x kg(-1) x day(-1) in drinking water; 97 +/- 3 mmHg) than after vehicle treatment (88 +/- 3 mmHg). MAP was also elevated in eNOS null mice (113 +/- 4 mmHg). Seven days after streptozotocin treatment (200 mg/kg iv) MAP was further increased in L-NAME-treated mice (108 +/- 5 mmHg) but not in vehicle-treated mice (88 +/- 3 mmHg) nor eNOS null mice (104 +/- 3 mmHg). In wild-type mice, maximal vasorelaxation of mesenteric arteries to acetylcholine was not altered by chronic L-NAME or induction of diabetes but was reduced by 42 +/- 6% in L-NAME-treated diabetic mice. Furthermore, the relative roles of NO and endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced vasorelaxation were altered; the EDHF component was enhanced by L-NAME and blunted by diabetes. These data suggest that NO protects against the development of hypertension during early-stage diabetes in mice, even in the absence of eNOS. Furthermore, in mesenteric arteries, diabetes is associated with reduced EDHF function, with an apparent compensatory increase in NO function. Thus, prior inhibition of NOS results in endothelial dysfunction in early diabetes, since the diabetes-induced reduction in EDHF function cannot be compensated by increases in NO production.     

Systemic arterial pressure response to two weeks of Tempol therapy in SHR: involvement of NO, the RAS, and oxidative stress

The roles of nitric oxide (NO) and plasma renin activity (PRA) in the depressor response to chronic administration of Tempol in spontaneously hypertensive rats (SHR) are not clear. The present study was done to determine the effect of 2 wk of Tempol treatment on blood pressure [mean arterial pressure (MAP)], oxidative stress, and PRA in the presence or absence of chronic NO synthase inhibition. SHR were divided into four groups: control, Tempol (1 mmol/l) alone, nitro-L-arginine methyl ester (L-NAME, 4.5 mg x g(-1).day(-1)) alone, and Tempol + L-NAME or 2 wk. With Tempol, MAP decreased by 22%: 191 +/- 3 and 162 +/- 21 mmHg for control and Tempol, respectively (P < 0.05). L-NAME increased MAP by 16% (222 +/- 2 mmHg, P < 0.01), and L-NAME + Tempol abolished the depressor response to Tempol (215 +/- 3 mmHg, P < 0.01). PRA was not affected by Tempol but was increased slightly with L-NAME alone and 4.4-fold with L-NAME + Tempol. Urinary nitrate/nitrite increased with Tempol and decreased with L-NAME and L-NAME + Tempol. Tempol significantly reduced oxidative stress in the presence and absence of L-NAME. In conclusion, in SHR, Tempol administration for 2 wk reduces oxidative stress in the presence or absence of NO, but in the absence of NO, Tempol is unable to reduce MAP. Therefore, NO, but not changes in PRA, plays a major role in the blood pressure-lowering effects of Tempol. These data suggest that, in hypertensive individuals with endothelial damage and chronic NO deficiency, antioxidants may be able to reduce oxidative stress but not blood pressure.     

The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney

The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of N(omega)-propyl-L-arginine (L-NPA; 1 mg. kg(-1). h(-1)) and N(5)-(1-imino-3-butenyl)-L-ornithine (v-NIO; 1 mg. kg(-1). h(-1)), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (-11 +/- 1 mmHg with L-NPA and -7 +/- 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36-42%) and medulla (by 32-40%) following administration of L-NPA (n = 8) or v-NIO (n = 8). Subsequent infusion of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg. kg(-1). h(-1)) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38-45 mmHg and significantly decreased cortical (25-29%) and medullary (37-43%) blood flow. In addition, L-NAME further decreased NO in the renal cortex (73-77%) and medulla (62-71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose of L-NAME (5 mg. kg(-1). h(-1); n = 8) was administered to a separate group of rats. The low dose of L-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23-24%, medulla 31-33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.     

Treatment with tetrahydrobiopterin reduces blood pressure in male SHR by reducing testosterone synthesis

Treatment with tetrahydrobiopterin (BH(4)) reduces blood pressure in spontaneously hypertensive rats (SHR). In the present study, we tested the hypothesis that chronic BH(4) reduces blood pressure in male SHR by reducing testosterone biosynthesis mediated by increasing nitric oxide (NO). Male SHR, aged 17-18 wk, intact or castrated, were treated for 1 wk with BH(4) (20 mg.kg(-1).day(-1) ip). After 1 wk, mean arterial pressure (MAP), serum testosterone, and nitrate/nitrite excretion (NO(x)) were measured. MAP was significantly higher in intact males than castrated males (179 +/- 2 vs. 155 +/- 4 mmHg, P < 0.001). In intact males, BH(4) caused a 17% reduction in MAP (148 +/- 2 mmHg), had no effect on NO(x), and reduced serum testosterone by 85% (24.09 +/- 2.37 vs. 3.72 +/- 0.73 ng/dl; P < 0.001). In castrated males, BH(4) had no effect on MAP (152 +/- 5 mmHg) but increased NO(x) by 38%. When castrated males were supplemented with testosterone, MAP increased to the same level as in intact males (180 +/- 7 mmHg), and BH(4) had no effect on MAP (182 +/- 7 mmHg) or NO(x). NO has been shown to decrease testosterone biosynthesis. Chronic sodium nitrite (70 mg.kg(-1).day(-1) x 1 wk) decreased MAP in intact males (150 +/- 4 mmHg) but had no effect on serum testosterone (21.46 +/- 3.08 ng/dl). The data suggest that BH(4) reduces testosterone synthesis and thereby reduces MAP in male SHR, an androgen-dependent model of hypertension. The mechanism(s) by which BH(4) reduces serum testosterone levels are not clear, but the data do not support a role for NO as a mediator.     

Blood oxygen transport in rats under hypothermia combined with modification of the L-Arginine-NO pathway.

Nitric oxide (NO) has high affinity to heme and by interaction with oxyhemoglobin (HbO2) is converted into nitrate to form methemoglobin (MetHb) as a side product. In combining with deoxy-Hb NO yields a stable molecule of nitrosyl-hemoglobin (HbFe(II)NO) that can further be converted into nitrate and hemoglobin (Hb). In addition, Hb was shown to transport NO in a form of S-nitrosohemoglobin (SNO-Hb). These features of the Hb and NO interaction are important for blood oxygen transport including hemoglobin-oxygen affinity (HOA). The present investigation was aimed to study the blood oxygen transport indices (pO2, pCO2, pH, HOA, etc.) in rats under hypothermia combined with a modification of L-arginine-NO pathway. To modify the L-arginine-NO pathway, rats were administered with N(G)-nitro-L-arginine methyl ester (L-NAME), L-arginine, or sodium nitroprusside (SNP) intravenously before cooling. A substantial impairment of oxygen delivery and development of hypoxia, with an important contribution of HOA into the latter accompanied the deep hypothermia in rats. All the experimental groups developed metabolic acidosis, less pronounced in rats treated with L-arginine only. In the experiments with a modification of the L-arginine-NO pathway, an enhanced cold resistance, attenuated oxygen deficiency, and a weaker oxyhemoglobin dissociation curve (ODC) shift leftwards were observed only after the administration of L-arginine. Neither SNP nor L-NAME had not any protective effects. L-Arginine lowered the value of standard P50 (pO2, corresponding to 50% Hb saturation with oxygen at 37 degrees C, pH 7.4, and pCO2 = 40 mmHg). The actual P50 (at actual pH, pCO2 and temperature) decreased by approximately 15 mmHg and was significantly higher than that under hypothermia without the drug treatment (21.03 +/- 0.35 vs 17.45 +/- 0.60 mmHg). NO also can contribute to this system through different mechanisms (HOA modification, vascular tone regulation, peroxynitrite formation, and effects).     

Effects of losartan on the blood-brain barrier permeability in long-term nitric oxide blockade-induced hypertensive rats

Hypertension is closely associated with vascular endothelial dysfunction. The aim of this study was to investigate the effects of Angiotensin II (ANG II) receptor antagonist losartan on the blood-brain barrier (BBB) permeability in L-NAME-induced hypertension and/or in ANG II-induced acute hypertension in normotensive and hypertensive rats. Systolic blood pressure was measured by tail cuff method before, during and following L-NAME treatment (1 g/L). Losartan (3 mg/kg) was given to the animal for five days. Acute hypertension was induced by ANG II (60 microg/kg). Arterial blood pressure was directly measured on the day of the experiment. BBB disruption was quantified according to the extravasation of the albumin-bound Evans blue dye. Losartan significantly reduced the mean arterial blood pressure from 169 +/- 3.9 mmHg to 82 +/- 2.9 mmHg in L-NAME and from 171 +/- 2.9 mmHg to 84 +/- 2.9 in L-NAME plus losartan plus ANG II groups (p < 0.05). The content of Evans blue dye in the cerebral cortex significantly increased in L-NAME (p < 0.01). Moreover, the content of Evans blue dye markedly increased in the cerebellum (p < 0.001) and slightly increased in diencephalon region (p < 0.05) in L-NAME plus ANG II. Losartan reduced the increased BBB permeability to Evans blue dye in L-NAME (p < 0.01) and L-NAME plus ANG II (p < 0.001). These results indicate that L-NAME and L-NAME plus ANG II both lead to an increase in microvascular Evans blue dye efflux to brain, and losartan treatment attenuates this protein-bound dye transport into brain tissue presumably due to its protective effect on endothelial cells of brain vessels.     

Trapping of nitric oxide produced during denitrification by extracellular hemoglobin

A spectrophotometric method has been developed that uses extracellular hemoglobin (Hb) to trap nitric oxide (NO) released during denitrification as nitrosyl hemoglobin (HbNO). The rate of complexation of NO with Hb is about at the diffusion controlled limit for protein molecules and the product, HbNO, is essentially stable. Hb was added to an anaerobic bacterial suspension and denitrification was initiated with either KNO2 or KNO3. HbNO formation was observed for six species of denitrifying bacteria and showed isosbestic points at 544, 568, and 586 nm. Cellular NO production, presumably by nitrite reductase, was kinetically distinct from the much slower chemical reaction of Hb with KNO2 to form methemoglobin and HbNO. The rate of HbNO formation was proportional to cell density, essentially independent of pH from 6.8 to 7.4, nearly zero order in [Hb] and, at least with Paracoccus denitrificans, strongly inhibited by rotenone and antimycin A. The Cu chelator, diethyldithiocarbamate, had no effect on HbNO formation by Pa. denitrificans, but abolished that by Achromobacter cycloclastes which uses a Cu-containing nitrite reductase known to be inactivated by the chelator. HbNO formation did not occur with non-denitrifying bacteria. The stoichiometry at high [Hb] for conversion of Hb to HbNO was 1.3-1.8 KNO2 per Hb for Pa. denitrificans, Pseudomonas aeruginosa, and A. cycloclastes and about 3.4 for Pseudomonas stutzeri. The former range of values corresponds to a partition of about 2 N atoms in 3 toward trapping and 1 in 3 toward reduction on the pathway to N2. Nitrogen not trapped appeared largely as N2O in presence of acetylene. The results are consistent with a model in which NO is a freely diffusible intermediate between nitrite and N2O, providing that nitric oxide reductase is or nearly is a diffusion controlled enzyme.     

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N^ω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.     

Reversal of hemoglobin-induced vasoconstriction with sustained release of nitric oxide

Erythrocyte free hemoglobin (Hb) induces vasoconstriction due to nitric oxide (NO) scavenging, limiting the NO available for vascular smooth muscle. The central objective of this study was to restore NO bioavailability using long-lived circulating NO-releasing nanoparticles (NO-np) to reverse the vasoconstriction and hypertension induced by polymerized bovine Hb (PBH) NO scavenging. PBH (13 g/dl) was infused in a volume equal to 10% of the animal blood volume. Intravascular NO supplementation was provided with an infusion of NO-np (10 and 20 mg/kg body wt). This study was performed using the hamster window chamber model to concurrently access systemic and microvascular hemodynamics. Infusion of PBH increased blood pressure and induced vasoconstriction. Treatment with 10 and 20 mg/kg NO-np reduced the blood pressure and vasoconstriction induced by PBH. Moreover, the higher dose of NO-np decreased blood pressure and induced vasodilation compared with baseline, respectively. Treatment with NO-np to decrease PBH-induced vasoconstriction increased methemoglobin levels and plasma nitrite and nitrate. In conclusion, NO-np counteracted both systemic hypertension and decreased the vasoconstrictor effects of PBH infusion, improving systemic and microvascular function. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent to replenish NO in situations where NO production is impaired, insufficient, or consumed, thereby preventing vascular complications.     

Acute haemodynamic effects of IL-6 treatment in vivo: involvement of vagus nerve in NO-mediated negative inotropism

Interleukin-6 (IL-6) reduces myocardial haemodynamics. However, the intrinsic mechanisms of IL-6 effects are not known. We hypothesized that nitric oxide (NO) synthesised by neuronal synthase (nNOS) can be the molecular mediator of IL-6-mediated cardiac effects. Thus, we investigated in vivo after IL-6 acute administration: (1) the role of NO pathway; (2) the importance of NO derived from nNOS located in intracardiac vagal ganglion in the anterior surface of the left ventricle. Sprague-Dawley (SD) rats (225-250 g) were anaesthetized (sodium pentobarbital 30 mg/kg intraperitoneally administered) and ventilated. The effects of a single IL-6 bolus (100 microg/kg intravenously administered) were studied in four experimental groups: (a) IL-6 (n=6), (b) IL-6 plus 30 mg/kg of L-NAME (an eNOS and nNOS inhibitor; n=6), (c) IL-6 plus 25mg/kg of 7-NI (a specific nNOS inhibitor; n=6), (d) IL-6 plus vagal resection (n=6). We evaluated the following parameters: mean aortic pressure (MAP), left ventricular end systolic pressure (LVESP), left ventricular positive peak dP/dt (PP dP/dt). Data are expressed as mean+/-sem. IL-6 caused a transient but significant reduction of MAP (-21.8% of basal: p<0.05), LVESP (from 130+/-4.2 to 1056.5 mmHg: p<0.05) and PP dP/dt (from 5390+/-158 to 4400+/-223 mmHg/s, p<0.02). Concomitant treatment with L-NAME or 7-NI totally abolished IL-6 effects. Vagal resection significantly reduced the haemodynamic effects (MAP: -10% of basal: p=ns; LVEDS: from 125+/-7.3 to 117+/-6.8 mmHg, p<0.05; PP dP/dt from 5500+/-150 to 5000+/-143 mmHg/s, p<0.05). We conclude that acute administration of IL-6 caused transient but significant cardiac negative inotropism. IL-6 haemodynamic effects are partly due to NO synthesised by nNOS located in vagal left ventricular ganglia.     

Nitric oxide does not contribute to the hypotension of heatstroke.

The purpose of this study was to determine whether nitric oxide (NO) contributes to the hypotensive state induced by prolonged environmental heat (EH) stress. Ketamine-anesthetized rats were instrumented for the measurement of arterial blood pressure, electrocardiogram, and temperature at four sites. Rats were exposed to EH (ambient temperature, 40 +/- 1 degrees C) until mean arterial blood pressure (MAP) decreased to 75 mmHg, which was arbitrarily defined as the induction of heatstroke. In addition to cardiovascular and temperature measurements, the time required to reach this MAP end point and the subsequent survival time were measured. In three separate experimental series, the competitive NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) was administered (0, 10, or 100 mg/kg) either before, during (30 min after initiation of EH), or immediately after EH. L-NAME administered at any of these times transiently increased MAP. L-NAME infusion either before or during EH did not alter the EH time required to decrease MAP to 75 mmHg, but L-NAME pretreatment did decrease the colonic temperature at which this MAP end point was reached. L-NAME infusion before or after EH did not affect subsequent survival time, but L-NAME administered during EH significantly decreased survival time. The administration of L-NAME at any time point, therefore, did not prove beneficial in either preventing or reversing heatstroke. Taken together, these data suggest that NO does not mediate the hypotension associated with heatstroke.     

Angiotensin-(1-7) prevents development of severe hypertension and end-organ damage in spontaneously hypertensive rats treated with L-NAME

We examined the influence of chronic treatment with ANG-(1-7) on development of hypertension and end-organ damage in spontaneously hypertensive rats (SHR) chronically treated with the nitric oxide synthesis inhibitor L-NAME (SHR-L-NAME). L-NAME administered orally (80 mg/l) for 4 wk significantly elevated mean arterial pressure (MAP) compared with SHR controls drinking regular water (269 +/- 10 vs. 196 +/- 6 mmHg). ANG-(1-7) (24 microg x kg(-1) x h(-1)) or captopril (300 mg/l) significantly attenuated the elevation in MAP due to L-NAME (213 +/- 7 and 228 +/- 8 mmHg, respectively), and ANG-(1-7) + captopril completely reversed the L-NAME-dependent increase in MAP (193 +/- 5 mmHg). L-NAME-induced increases in urinary protein were significantly lower in ANG-(1-7)-treated animals (226 +/- 6 vs. 145 +/- 12 mg/day). Captopril was more effective (96 +/- 12 mg/day), and there was no additional effect of captopril + ANG-(1-7) (87 +/- 5 mg/day). The abnormal vascular responsiveness to endothelin-1, carbachol, and sodium nitroprusside in perfused mesenteric vascular bed of SHR-L-NAME was improved by ANG-(1-7) or captopril, with no additive effect of ANG-(1-7) + captopril. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1-7)- or captopril-treated SHR-L-NAME, with additive effects of combined treatment. The beneficial effects of ANG-(1-7) on MAP and cardiac function were inhibited when indomethacin was administered with ANG-(1-7), but indomethacin did not reverse the protective effects on proteinuria or vascular reactivity. The protective effects of the ANG-(1-7) analog AVE-0991 were qualitatively comparable to those of ANG-(1-7) but were not improved over those of captopril alone. Thus, during reduced nitric oxide availability, ANG-(1-7) attenuates development of severe hypertension and end-organ damage; prostaglandins participate in the MAP-lowering and cardioprotective effects of ANG-(1-7); and additive effects of captopril + ANG-(1-7) on MAP, but not proteinuria or endothelial function, suggest common, as well as different, mechanisms of action for the two treatments. Together, the results provide further evidence of a role for ANG-(1-7) in protective effects of angiotensin-converting enzyme inhibition and suggest dissociation of factors influencing MAP and those influencing end-organ damage.     

Structural alterations in the heart after long-term L-NAME and D-NAME treatment

N(G)-nitro-D-arginine methyl ester (D-NAME), considered as an inactive enantiomer of NAME, is generally used as a negative control for NO synthase inhibition with L-NAME. The aim of this work was to compare the effect of L-NAME (20 and 40 mg/kg/day), and D-NAME (40 mg/kg/day) on hemodynamic and structural parameters in the rat cardiovascular system. After 4 weeks of treatment, blood pressure and left ventricle weight/body weight ratio increased significantly in all studied groups versus control. Myocardial fibrosis (in %) represented 0.94 +/- 0.04 in control, 4.70 +/- 0.39 in L-NAME (20 mg/kg/day), 10.54 +/- 0.91 in L-NAME (40 mg/kg/day) and 5.25 +/- 0.46 in D-NAME (40 mg/kg/day) group. We conclude that in a long-term experiment D-NAME provokes similar changes in cardiovascular system like L-NAME.     

L-NAME-induced protein remodeling and fibrosis in the rat heart

The aim of the present study was to determine whether NO deficiency itself or rather the elevation of systolic blood pressure is responsible for the protein and structural remodeling of the heart during hypertension induced by long-term treatment by nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Three groups of rats were investigated. The first group served as control. In the second group L-NAME was given in the dose of 20 mg/kg/day in the drinking water and in the third group L-NAME was given in the dose of 40 mg/kg/day during 4 weeks. While L-NAME treatment in both doses caused essentially the same increase in systolic blood pressure (SBP), NO synthase activity and cGMP concentration in the left ventricle decreased by 17% and 13%, respectively in the 20 mg/kg/day L-NAME group and by 69% and 27%, respectively in the 40 mg/kg/day L-NAME group. The protein profile of the left ventricle in both L-NAME groups was characterized by an increased concentration of metabolic proteins. Nevertheless, a significant increase in the concentration of pepsin-soluble collagenous proteins and the concentration of hydroxyproline in pepsin-insoluble collagenous proteins was found only in the group receiving 40 mg/kg/day L-NAME. The morphometric evaluation revealed a significant increase in myocardial fibrosis in both L-NAME groups. However, this was more pronounced in the 40 mg/kg/day L-NAME group. It is concluded that NO deficiency resulted in significant enhancement of fibrotic tissue growth in proportion to the administered L-NAME dose, while SBP was increased similarly in both L-NAME groups. Thus, NO deficiency rather than hemodynamic changes appears to be crucially involved in collagenous protein and fibrotic tissue changes of the left ventricle in hypertension induced by L-NAME.     

Evidence of nitric oxide and angiotensin II regulation of circulation and cutaneous drinking in Bufo marinus

The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (l-NAME) increased vascular resistance (VR) 10% above baseline of 3.08+/-0.08 (n=11) mmHg/mL/min at 10 mg/kg and 20% above 3.05+/-0.08 (n=9) at 50 mg/kg in anesthetized toads (Bufo marinus). Blood pressure was unaffected by either dose of L-NAME. Blood flow decreased at the higher dose of L-NAME. L-arginine (300 mg/kg) reversed the effects of L-NAME on VR and blood flow in toads treated with 10 mg/kg but not with 50 mg/kg. Injection of 50 mg/kg L-NAME into empty-bladder toads produced a 10% decrease in water uptake, J(v), resulting in a J(v) of 1,267+/-11 cm(3)/cm(2)/s x 10(-7) (n=9) compared to 1,385+/-12 (n=8) for controls. Injection of 10 microg/kg angiotensin II (ANG II) increased J(v) 15% across the pelvic patch (J(v), cm(3)/cm(2)/s x 10(-7)), resulting in a J(v) of 1,723+/-12 cm(3)/cm(2)/s x 10(-7) (n=8) compared to 1,471+/-12 (n=8) for controls. It is hypothesized that during cutaneous drinking blood flow into the capillary bed of the pelvic patch is regulated by nitric oxide and ANG II.     

Hypotensive effect of agmatine, arginine metabolite, is affected by NO synthase

The metabolites of arginine were recently shown to be involved in cardiovascular control. The study addresses the general cardiovascular response of anaesthetized rats to agmatine, a decarboxylated arginine. The relation between two arginine metabolic pathways governed by arginine decarboxylase and nitric oxide synthase was investigated. Intravenous administration of agmatine 30 and 60 microM/0.1 ml saline elicited remarkable hypotension of 42.6+/-4.6 and 70.9+/-6.5 mm Hg, respectively. The hypotension was characterized by long duration with half-time of return 171.6+/-2.9 and 229.2+/-3.8 s, respectively. The time of total blood pressure BP recovery was about 10 min. Dose-dependent relaxation to agmatine was also found in aorta rings in vitro. Both doses of agmatine administered 60-180 min after NO synthase inhibition L-NAME 40 mg/kg i.v. caused greater hypotension 59.0+/-7.6 and 95.8 8.8 mm Hg P<0.01 both compared to animals with intact NO synthase, but this was accompanied by a significant shortening of the half-time of BP return. If agmatine was administered to hypertensive NO-deficient rats treated with 40 mg/kg/day L-NAME for 4 weeks, similar significant enhancement of hypotension was observed at both agmatine doses, again with a significant shortening of half-time of BP return. It can be summarized that the long-lasting hypotension elicited by agmatine was amplified after acute or chronic NO synthase inhibition, indicating a feedback relation between the two metabolic pathways of arginine.     

Oxidation of iron-nitrosyl-hemoglobin by dehydroascorbic acid releases nitric oxide to form nitrite in human erythrocytes

The reaction of deoxyhemoglobin with nitric oxide (NO) or nitrite ions (NO 2 (-)) produces iron-nitrosyl-hemoglobin (HbNO) in contrast to the reaction with oxyhemoglobin, which produces methemoglobin and nitrate (NO 3 (-)). HbNO has not been associated with the known bioactivities of NO. We hypothesized that HbNO in erythrocytes could be an important source of bioactive NO/nitrite if its oxidation was coupled to the ascorbic acid (ASC) cycle. Studied by absorption and electron paramagnetic resonance (EPR) spectroscopy, DHA oxidized HbNO to methemoglobin and liberated NO from HbNO as determined by chemiluminescence. Both DHA and ascorbate free radical (AFR), the intermediate between ASC and DHA, enhanced NO oxidation to nitrite, but not nitrate; nor did either oxidize nitrite to nitrate. DHA increased the basal levels of nitrite in erythrocytes, while the reactions of nitrite with hemoglobin are slow. In erythrocytes loaded with HbNO, HbNO disappeared after DHA addition, and the AFR signal was detected by EPR. We suggest that the ASC-AFR-DHA cycle may be coupled to that of HbNO-nitrite and provide a mechanism for the endocrine transport of NO via hemoglobin within erythrocytes, resulting in the production of intracellular nitrite. Additionally, intracellular nitrite and nitrate seem to be largely generated by independent pathways within the erythrocyte. These data provide a physiologically robust mechanism for erythrocytic transport of NO bioactivity allowing for hormone-like properties.