Impaired shear stress-induced nitric oxide production through decreased NOS phosphorylation contributes to age-related vascular stiffness.

Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and sphingosine 1-phosphate, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- 13.9%, while the old aorta response was significantly attenuated (23.6 +/- 11.3%, P < 0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P < 0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P < 0.001). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.191 m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.118 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P < 0.001). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P < 0.001). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.     

Regional and whole-body markers of nitric oxide production following hyperemic stimuli

The measurement of nitric oxide (NO) bioavailability is of great clinical interest in the assessment of vascular health. However, NO is rapidly oxidized to form nitrite and nitrate and thus its direct detection in biological systems is difficult. Venous plasma nitrite (nM concentrations) has been shown to be a marker of forearm NO production following pharmacological stimulation of the endothelium utilizing acetylcholine (Ach). In the present study, we demonstrate, within 15 apparently healthy subjects (34.1 +/- 7.3 years), that reactive hyperemia of the forearm, a physiological endothelial stimulus, results in a 52.5% increase in mean plasma nitrite concentrations (415 +/- 64.0 to 634 +/- 57.1 nM, P = 0.015). However, plasma nitrite is readily oxidized to nitrate within plasma, and thus its utility as a marker of NO production within the clinical setting may be limited. Alternatively, NOx (predominantly nitrate) is relatively stable in plasma (microM concentrations), but is produced by sources other than the vasculature and has been shown to be unsuitable as a measure of localized NO production. We reasoned that the principle source of NOx generation during exercise is NO production and thus have examined the change in NOx following treadmill exercise stress. In this study, 12 apparently healthy subjects showed an increase (from baseline) in venous NOx at peak effort and during recovery (12 +/- 9.1 and 17 +/- 15.3 microM respectively, P < 0.05). In contrast, 10 subjects with cardiovascular disease showed no significant increases. Additionally, a correlation between VO(2peak) and the change in circulating NOx (r(2) = 0.4585, P < or = 0.01) indicated the subjects who could exercise hardest also produced the most NO.     

Reduced perivascular PO2 increases nitric oxide release from endothelial cells

Many studies have suggested that endothelial cells can act as "oxygen sensors" to large reductions in oxygen availability by increasing nitric oxide (NO) production. This study determined whether small reductions in oxygen availability enhanced NO production from in vivo intestinal arterioles, venules, and parenchymal cells. In vivo measurements of perivascular NO concentration ([NO]) were made with NO-sensitive microelectrodes during normoxic and reduced oxygen availability. During normoxia, intestinal first-order arteriolar [NO] was 397 +/- 26 nM (n = 5), paired venular [NO] was 298 +/- 34 nM (n = 5), and parenchymal cell [NO] was 138 +/- 36 nM (n = 3). During reduced oxygen availability, arteriolar and venular [NO] significantly increased to 695 +/- 79 nM (n = 5) and 534 +/- 66 nM (n = 5), respectively, whereas parenchymal [NO] remained unchanged at 144 +/- 34 nM (n = 4). During reduced oxygenation, arteriolar and venular diameters increased by 15 +/- 3% and 14 +/- 5%, respectively: NG-nitro-L-arginine methyl ester strongly suppressed the dilation to lower periarteriolar Po2. Micropipette injection of a CO2 embolus into arterioles significantly attenuated arteriolar dilation and suppressed NO release in response to reduced oxygen availability. These results indicated that in rat intestine, reduced oxygen availability increased both arteriolar and venular NO and that the main site of NO release under these conditions was from endothelial cells.     

Endothelium-dependent vasorelaxation in inhibited by in vivo depletion of vascular thiol levels: role of endothelial nitric oxide synthase.

Thiols like glutathione may serve as reducing cofactors in the production of nitric oxide (NO) and protect NO from inactivation by radical oxygen species. Depletion of thiol compounds reduces NO-mediated vascular effects in vitro and in vivo. The mechanisms underlying these actions are not clear, but may involve decreased synthesis of NO and/or increased degradation of NO. This study investigates the effect of glutathione depletion on the response to NO-mediated vasodilation induced by acetylcholine (Ach, 10 micrograms/kg), endothelial NO synthase (eNOS) activity and potential markers of vascular superoxide anion (O2.-) production in conscious chronically catheterized rats. Thiol depletion induced by buthionine sulfoximine (BSO, 1 g i.p. within 24 h) decreased the hypotensive effect of Ach by 30% (MAP reduction before BSO 27 +/- 3 mmHg, 19 +/- 3 mmHg after BSO, (mean +/- SEM), p < .05, n = 8). The impaired effect of Ach was associated with a significant reduction in eNOS activity (control: 7.7 +/- 0.8, BSO: 3.9 +/- 0.4 pmol/min/mg protein (p < .05), n = 6). In contrast, neither NADH/NADPH driven membrane-associated oxidases nor lucigenin reductase activity were significantly (p < .05) affected by BSO (BSO: 4415 +/- 123, control: 4105 +/- 455 counts/mg; n = 6) in rat aorta. It is concluded that in vivo thiol depletion results in endothelial dysfunction and a reduced receptor-mediated vascular relaxation. This effect is caused by reduced endothelial NO formation.     

Glucose-induced release of nitric oxide from mouse pancreatic islets as detected with nitric oxide-selective glass microelectrodes

Nitric oxide (NO) is believed to play an important role in pancreatic islet physiology and pathophysiology. Research in this area has been hampered, however, by the use of indirect methods to measure islet NO. To investigate the role of NO in islet function, we positioned NO-sensitive, recessed-tip microelectrodes in close proximity to individual islets and monitored oxidation current to detect subnanomolar NO in the bath. NO release from islets consisted of a series of rapid bursts lasting several seconds and/or slow oscillations with a period of approximately 100-300 s. Average baseline NO near the islets in 2.8 mM glucose was 524+/-59 nM (n=12). Raising glucose from 2.8 to 11.1 mM augmented NO release by 429+/-133 nM (n=12, P<0.05), an effect blocked by the NO synthase inhibitor L-NAME (n=3). We also observed that glucose-stimulated increases in NO release were contemporaneous with changes in NAD(P)H and O2 but occurred well before increases in calcium associated with glucose-stimulated insulin secretion. In summary, we demonstrate that NO release from islets is oscillatory and rapidly augmented by glucose, suggesting that NO release occurs early following an increase in glucose metabolism and may contribute to the stimulated insulin secretion triggered by suprathreshold glucose.     

Influence of nitric oxide donors and peroxynitrite on the contractile effect and concentration of norepinephrine

Nitric oxide (NO) and peroxynitrite (ONOO) are said to destroy norepinephrine (NE). We studied the role of NE decomposition by NO donors and ONOO as they affect the contractile activity of NE in rat denuded thoracic aorta. First, we determined the relaxing effect of NO donors (SNAP, PROLI/NO, Sodium nitrite, SIN-1) and ONOO after precontraction by NE (1 microM). SNAP and SIN-1 (EC(50) 50-110 nM) were more active than PROLI/NO, Sodium nitrite or ONOO (EC(50) 19-30 microM). The relaxing effect of NO donors and ONOO were decreased by ODQ (10 microM), a guanylate cyclase inhibitor. Second, we compared the contractile activity of NE before and after preincubation with NO donors or ONOO in presence of ODQ. NE (1 microM) was incubated with NO donors or ONOO at the concentrations of 0.1 mM in both Krebs solution or phosphate buffer (pH 7.4; 0.1 M) for 10 minutes at 37 degrees C. NE evoked the aorta contraction in the same concentrations before and after preincubation with NO donors. In contrast, ONOO decreased effect of NE, EC(50) was measured at 4.3+/-0.3 nM and 13.4+/-1.6 nM, before and after preincubation of NE with ONOO respectively. Third, we measured the NE concentration using the HPLC method. We revealed that the concentration of NE after preincubation with NO donors was unaltered. However HPLC measurement revealed that NE concentration after preincubation with ONOO was reduced 2-3-fold. Therefore, under these experimental conditions ONOO, but not NO donors, was capable of destroying NE.     

Real-time monitoring of NO release from single cells using carbon fiber microdisk electrodes modified with single-walled carbon nanotubes

In this paper, a novel NO electrochemical microsensor, which is fabricated by modifying the surface of a carbon fiber microdisk electrode (CFMDE, diameter: 5-7mum) with single-walled carbon nanotubes (SWNTs) and Nafion membrane, is reported for the first time. The modification of SWNTs dramatically improves the sensitivity of CFMDEs, and the detection limit for NO is 4.3nM that is nearly 10 times lower than that from the bare one and lower than most NO electrochemical sensors reported before. The Nafion membrane offers a good barrier to some interferents such as nitrite and ascorbic acid without losing response speed to NO. The sensor has been successfully applied to the measurement of NO release from single isolated human umbilical vein endothelial cells (HUVECs). Real-time amperometric data show that the addition of l-arginine (l-arg) or acetylcholine (ACh) can cause a quick increase in NO production with a maximum concentration of 232+/-44nM (n=5) and 159+/-29nM (n=5), respectively.     

Myeloperoxidase enhances nitric oxide catabolism during myocardial ischemia and reperfusion

Impaired microvascular function during myocardial ischemia and reperfusion is associated with recruitment of polymorphonuclear neutrophils (PMN) and has been attributed to decreased bioavailability of nitric oxide (NO). Whereas myeloperoxidase (MPO), a highly abundant, PMN-derived heme protein facilitates oxidative NO consumption and impairs vascular function in animal models of acute inflammation, its capacity to function in this regard during human myocardial ischemia and reperfusion remains unknown. Plasma samples from 30 consecutive patients (61 +/- 14 years, 80% male) presenting with acute myocardial infarction were collected 9 +/- 4 h after vessel recanalization and compared to plasma from healthy control subjects (n = 12). Plasma levels of MPO were higher in patients than in control subjects (1.4 +/- 0.9 vs 0.3 +/- 0.2 ng/mg protein, respectively, p < 0.0001). The addition of hydrogen peroxide to patient plasma resulted in accelerated rates of NO consumption compared to control subjects (0.53 +/- 0.25 vs 0.068 +/- 0.039 nM/s/mg protein, respectively, p < 0.0001). Myocardial tissue from patients with the same pathology revealed intense recruitment of MPO-positive PMN localized along infarct-related vessels as well as diffuse endothelial distribution of non-PMN-associated MPO immunoreactivity. Endothelium-dependent microvascular function, as assessed by an acetylcholine-dependent increase in forearm blood flow in 75 patients with symptomatic coronary artery disease, inversely correlated with MPO plasma levels (r = -0.75, p < 0.005). Plasma from patients undergoing myocardial reperfusion contained increased levels of MPO, which catalytically consumed NO in the presence of H(2)O(2). Given the correlation between intravascular MPO levels and forearm vasomotor function in patients with coronary artery disease, MPO appears to be an important modulator of vasomotor function in inflammatory vascular disease and a potential therapeutic target for treatment.     

Increased expression of endothelial nitric oxide synthase and caveolin-1 in the aorta of rats with isoproterenol-induced cardiac hypertrophy

Isoproterenol-induced cardiac hypertrophy is associated with increased expression of endothelial nitric oxide synthase in the aorta but without signs of improved endothelial function. The aim was to examine the hypothesis that increased expression of eNOS allosteric inhibitor caveolin-1 could be associated with unimproved endothelium-dependent relaxations. Rats received isoproterenol (5 mg/kg body mass, i.p., n = 13) or its vehicle (n = 14) during 1 week. Systolic blood pressure (SBP) and heart rate (HR) were measured by the tail-cuff method. Expression of eNOS and caveolin-1 was measured using immunoblotting analysis. Relaxations of isolated aorta to acetylcholine and sodium nitroprusside were evaluated ex vivo. After 1 week of isoproterenol administration, basal SBP and HR were decreased (SBP 110 +/- 3 vs. 126 +/- 3 mmHg, p < 0.05; HR 342 +/- 8 vs. 366 +/- 6 beats/min, p < 0.05). Isoproterenol increased the mass of the left ventricle (+33% +/- 4% vs. control; p < 0.05) and right ventricle (+40% +/- 9%; p < 0.05). Isoproterenol administration increased the expression of eNOS (+53% +/- 12%; p < 0.05) and caveolin-1 (+54% +/- 20%, p < 0.05) in the aorta. Relaxation of isolated aorta to acetylcholine and sodium nitroprusside showed a trend towards a worsened endothelial function and a lower sensitivity to exogenous NO. Thus, 1 week of isoproterenol administration led to increased eNOS expression in the aorta without amelioration of endothelial vasorelaxation function. Concomitant increase in caveolin-1 expression may be responsible for this paradox.     

Elevated plasma viscosity in extreme hemodilution increases perivascular nitric oxide concentration and microvascular perfusion

We tested the hypothesis that high-viscosity (HV) plasma in extreme hemodilution causes wall shear stress to be greater than low-viscosity (LV) plasma, leading to enhanced production of nitric oxide (NO). The perivascular concentration of NO was measured in arterioles and venules and the tissue of the hamster chamber window model, subjected to acute extreme hemodilution, with a hematocrit (Hct) of 11% using Dextran 500 (n = 6) or Dextran 70 (n = 5) with final plasma viscosities of 1.99 +/- 0.11 and 1.33 +/- 0.04 cp, respectively. HV plasma significantly increased the periarteriolar, perivenular, and tissue NO concentration by 2.0, 1.9, and 1.4 times the control (n = 7). The NO concentration with LV plasma was not statistically different from control. Arteriolar shear stress was significantly increased in HV plasma relative to LV plasma in arterioles but not in venules. Aortic endothelial NO synthase (eNOS) protein expression was increased with HV plasma but not with LV plasma. There was a weak correlation between perivascular NO concentration and the locally calculated shear stress induced by the procedures, when blood viscosity was corrected according to Hct values previously determined in studies of microvascular Hct distribution. The finding that the periarteriolar and venular NO concentration in HV plasma was the same although arteriolar shear stress was significantly greater than venular shear stress maybe be due to differences in vessel wall metabolism between arterioles and venules and the presence of NO transport through the blood stream in the microcirculation. Results support the concept that in extreme hemodilution HV plasma maintains functional capillary density through a NO-mediated vasodilatation.     

Simvastatin upregulates coronary vascular endothelial nitric oxide production in conscious dogs

Statin drugs can upregulate endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial cells independent of lipid-lowering effects. We investigated the effect of short-term simvastatin administration on coronary vascular eNOS and NO production in conscious dogs and canine tissues. Mongrel dogs were instrumented under general anesthesia to measure coronary blood flow (CBF). Simvastatin (20 mg. kg(-1). day(-1)) was administered orally for 2 wk; afterward, resting CBF was found to be higher compared with control (P < 0.05) and veratrine- (activator of reflex cholinergic NO-dependent coronary vasodilation) and ACh-mediated coronary vasodilation were enhanced (P < 0.05). Response to endothelium-independent vasodilators, adenosine and nitroglycerin, was not potentiated. After simvastatin administration, plasma nitrate and nitrite (NO(x)) levels increased from 5.22 +/- 1.2 to 7. 79 +/- 1.3 microM (P < 0.05); baseline and agonist-stimulated NO production in isolated coronary microvessels were augmented (P < 0.05); resting in vivo myocardial oxygen consumption (MVO(2)) decreased from 6.8 +/- 0.6 to 5.9 +/- 0.4 ml/min (P < 0.05); NO-dependent regulation of MVO(2) in response to NO agonists was augmented in isolated myocardial segments (P < 0.05); and eNOS protein increased 29% and eNOS mRNA decreased 50% in aortas and coronary vascular endothelium. Short-term administration of simvastatin in dogs increases coronary endothelial NO production to enhance NO-dependent coronary vasodilation and NO-mediated regulation of MVO(2).     

In vivo assessment of microvascular nitric oxide production and its relation with blood flow

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100-120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 +/- 7% membrane bound. Basal NO release was 60.1 +/- 5.1 pM/min (n = 35), and plasma flow was 2.95 +/- 0.27 microl/min (n = 29). Local NO synthase inhibition with 30 microM N(omega)-nitro-L-arginine reduced NO production to 8.6 +/- 2.6 pmol/min (-83 +/- 5%, n = 9) and plasma flow to 1.95 +/- 0.15 microl/min (-28 +/- 12%, n = 17) within 30-45 min, in parallel with constriction of arterioles (9-14%) and venules (19-25%). The effects of N(omega)-nitro-L-arginine (10-30 microM) were proportional to basal microvascular conductance (r = 0.7, P < 0.05) and fully prevented by 1 mM L-arginine. We conclude that in this tissue, NO production contributes to 35-50% of resting microvascular conductance and plasma-tissue exchange.     

Thiopental inhibits nitric oxide production in rat aorta

We studied whether thiopental affects endothelial nitric oxide dependent vasodilator responses and nitrite production (an indicator of nitric oxide production) elicited by acetylcholine, histamine, and A23187 in rat aorta (artery in which nitric oxide is the main endothelial relaxant factor). In addition, we evaluated the barbiturate effect on nitric oxide synthase (NOS) activity in both rat aorta and kidney homogenates. Thiopental (10-100 microg/mL) reversibly inhibited the endothelium-dependent relaxation elicited by acetylcholine, histamine, and A23187. On the contrary, this anesthetic did not modify the endothelium-independent but cGMP-dependent relaxation elicited by sodium nitroprusside (1 nM - 1 microM) and nitroglycerin (1 nM - 1 microM), thus excluding an effect of thiopental on guanylate cyclase of vascular smooth muscle. Thiopental (100 microg/mL) inhibited both basal (87.8+/-14.3%) and acetylcholine- or A23187-stimulated (78.6+/-3.9 and 39.7+/-5.6%, respectively) production of nitrites in aortic rings. In addition the barbiturate inhibited (100 microg/mL) the NOS (45+/-4 and 42.8+/-9%) in aortic and kidney homogenates, respectively (measured as 14C-labeled citrulline production). In conclusion, thiopental inhibition of endothelium-dependent relaxation and nitrite production in aortic rings strongly suggests an inhibitory effect on NOS. Thiopental inhibition of the NOS provides further support to this contention.     

Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release

Endothelium-derived nitric oxide (NO) is synthesized in response to chemical and physical stimuli. Here, we investigated a possible role of the endothelial cell glycocalyx as a biomechanical sensor that triggers endothelial NO production by transmitting flow-related shear forces to the endothelial membrane. Isolated canine femoral arteries were perfused with a Krebs-Henseleit solution at a wide range of perfusion rates with and without pretreatment with hyaluronidase to degrade hyaluronic acid glycosaminoglycans within the glycocalyx layer. NO production rate was evaluated as the product of nitrite concentration in the perfusate and steady-state perfusion rate. The slope that correlates the linear relation between perfusion rate and NO production rate was taken as a measure for flow-induced NO production. Hyaluronidase treatment significantly decreased flow-induced NO production to 19 +/- 9% of control (mean +/- SD; P < 0.0001 vs. control; n = 11), whereas it did not affect acetylcholine-induced NO production (88 +/- 17% of pretreatment level, P = not significant; n = 10). We conclude that hyaluronic acid glycosaminoglycans within the glycocalyx play a pivotal role in detecting and amplifying the shear force of flowing blood that triggers endothelium-derived NO production in isolated canine femoral arteries.     

Neuropeptide Y-Y1 receptor modulates nitric oxide level during stroke in the rat

In a rat endovascular middle cerebral artery occlusion (MCAO) stroke model, we previously showed that intracerebroventricular (ICV) injection of neuropeptide Y (NPY) or an Y1 receptor agonist, [Leu(31),Pro(34)]-NPY, increased the infarct volume, that an Y1 receptor antagonist, BIBP3226, reduced the infarct volume, and that an Y2 receptor agonist, NPY3-36, had no effect. In this study, we used electron paramagnetic resonance (EPR) spectroscopy to measure nitric oxide (NO) and examined how ICV administration of NPY or its receptor analogs would modulate the brain NO level between the bregma levels +2 and -4 mm during MCAO, since excessive NO mediates ischemic damage. The relative brain NO concentration was increased to 131.94 +/- 7.99% (mean +/- SEM; n = 8) at 15 min of MCAO. NPY treatment further increased the relative brain NO concentration to 250.94 +/- 50.48% (n = 8), whereas BIBP3226 significantly reduced the brain NO concentration to 69.63 +/- 8.84% (n = 8). [Leu(31),Pro(34)]-NPY (137.61 +/- 14.54%; n = 7) or NPY3-36 (129.23 +/- 21.77%; n = 8) did not affect the brain NO concentration at 15 min of MCAO. Our results suggest that the NPY-Y1 receptor activation mediates ischemic injury via NO overproduction and that inhibition of the Y1 receptor may confer protection via suppression of excessive NO production during ischemia.     

Biphasic modulation of vascular nitric oxide catabolism by oxygen

Endothelium-derived nitric oxide (NO) plays an important role in the regulation of vascular tone. Lack of NO bioavailability can result in cardiovascular disease. NO bioavailability is determined by its rates of generation and catabolism; however, it is not known how the NO catabolism rate is regulated in the vascular wall under normoxic, hypoxic, and anaerobic conditions. To investigate NO catabolism under different oxygen concentrations, studies of NO and O2 consumption by the isolated rat aorta were performed using electrochemical sensors. Under normoxic conditions, the rate of NO consumption in solution was enhanced in the presence of the rat aorta. Under hypoxic conditions, NO consumption decreased in parallel with the O2 concentration. Like the inhibition of mitochondrial respiration by NO, the inhibitory effects of NO on aortic O2 consumption increased as O2 concentration decreased. Under anaerobic conditions, however, a paradoxical reacceleration of NO consumption occurred. This increased anaerobic NO consumption was inhibited by the cytochrome c oxidase inhibitor NaCN but not by the free iron chelator deferoxamine, the flavoprotein inhibitor diphenylene iodonium (10 microM), or superoxide dismutase (200 U/ml). The effect of O2 on the NO consumption could be reproduced by purified cytochrome c oxidase (CcO), implying that CcO is involved in aortic NO catabolism. This reduced NO catabolism at low O2 tensions supports the maintenance of effective NO levels in the vascular wall, reducing the resistance of blood vessels. The increased anaerobic NO catabolism may be important for removing excess NO accumulation in ischemic tissues.     

Inhibition of nitric oxide synthase enhances superoxide activity in canine kidney

To evaluate the role of a potential interaction between superoxide anion (O(2)(-)) and nitric oxide (NO) in regulating kidney function, we examined the renal responses to intra-arterial infusion of a superoxide dismutase mimetic, tempol (0.5 mg.kg(-1).min(-1)), in anesthetized dogs treated with or without NO synthase inhibitor, N(omega)-nitro-l-arginine (NLA; 50 microg.kg(-1).min(-1)). In one group of dogs (n = 10), tempol infusion alone for 30 min before NLA infusion did not cause any significant changes in renal blood flow (RBF; 5.2 +/- 0.4 to 5.0 +/- 0.4 ml.min(-1).g(-1)), glomerular filtration rate (GFR; 0.79 +/- 0.04 to 0.77 +/- 0.04 ml.min(-1).g(-1)), urine flow (V; 13.6 +/- 2.1 to 13.9 +/- 2.5 microl.min(-1).g(-1)), or sodium excretion (U(Na)V; 2.4 +/- 0.3 to 2.2 +/- 0.3 micromol.min(-1).g(-1)). Interestingly, when tempol was infused in another group of dogs (n = 12) pretreated with NLA, it caused increases in V (4.4 +/- 0.4 to 9.7 +/- 1.4 microl.min(-1).g(-1)) and in U(Na)V (0.7 +/- 0.1 to 1.3 +/- 0.2 micromol.min(-1).g(-1)) without affecting RBF or GFR. Although NO inhibition caused usual qualitative responses in both groups of dogs, the antidiuretic (47 +/- 5 vs. 26 +/- 4%) and antinatriuretic (67 +/- 4 vs. 45 +/- 11%) responses to NLA were seen much less in dogs pretreated with tempol. NLA infusion alone increased urinary excretion of 8-isoprostane (13.9 +/- 2.7 to 22.8 +/- 3.6 pg.min(-1).g(-1); n = 7), which returned to the control levels (11.6 +/- 3.4 pg.min(-1).g(-1)) during coadministration of tempol. These data suggest that NO synthase inhibition causes enhancement of endogenous O(2)(-) levels and support the hypothesis that NO plays a protective role against the actions of O(2)(-) in the kidney.     

Real-time measurement of nitric oxide by luminol-hydrogen peroxide reaction in crystalloid perfused rat heart

The objective of this study was to develop an assay system that allows continuous monitoring of nitric oxide (NO) released from crystalloid perfused hearts. We utilized chemiluminescence reaction between NO and luminol-H(2)O(2) to quantify the NO level in coronary effluent. Isolated rat hearts were subjected to ordinary Langendorff's perfusion, and the right ventricle was cannulated to sample coronary effluent. After equilibration, the coronary flow rate was set constant and the hearts were paced at 300 bpm. Coronary effluent was continuously sampled and mixed with the chemiluminescent probe containing 0.018 mmol/l luminol plus 10 mmol/l H(2)O(2). Chemiluminescence from the mixture of coronary effluent and the probe was continuously measured. NO concentration was calibrated by various concentrations (0.5-400 pmol/l) of standard NO solution. The lower detection limit of NO was 1 pmol/l. Basal NO release from isolated perfused rat heart was 0.41 +/- 0.17 pmol/min/g of heart weight, and that was significantly suppressed by 0.1 mmol/l of L-NAME to 0.18 +/- 0.10 pmol/min/g of heart weight (n = 7). Application of 0.1 and 0.3 micromol/l acetylcholine increased NO level in the coronary effluent, in a concentration-dependent manner, from 6.6 +/- 1.7 in a baseline condition to 16.3 +/- 7.4 and 30.3 +/- 16.1 pmol/l at each peak, respectively. Thrombin at 1 and 10 U/ml also increased NO level from 17.6 +/- 4.3 in control to 35.5 +/- 10.4 and 48.7 +/- 8.7 pmol/l at each peak, respectively (n = 7). Thus, this assay system is applicable to the continuous real-time measurement of NO released from crystalloid perfused hearts, and it may be useful for the study of physiological or pathophysiological role of NO in coronary circulation.     

Dynamics of nitric oxide release in the cardiovascular system

The endothelium plays a critical role in maintaining vascular tone by releasing nitric oxide (NO). Endothelium derived NO diffuses to smooth muscles, triggering their relaxation. The dynamic of NO production is a determining factor in signal transduction. The present studies were designed to elucidate dynamics of NO release from normal and dysfunctional endothelium. The nanosensors (diameter 100-300 nm) exhibiting a response time better than 100 micros and detection limit of 1.0 x 10(-9) mol L(-1) were used for in vitro monitoring of NO release from single endothelial cells from the iliac artery of normotensive (WKY) rats, hypertensive (SHR) rats, and normal and cholesterolemic rabbits. Also, the dynamics and distribution of NO in left ventricular wall of rabbit heart were measured. The rate of NO release was much higher (1200 +/- 50 nmol L(-1) s(-1)) for WKY than for SHR (460 +/- 10 nmol L(-1) s(-1)). Also, the peak NO concentration was about three times higher for WKY than SHR. Similar decrease in the dynamics of NO release was observed for cholesterolemic rabbits. The dynamics of NO release changed dramatically along the wall of rabbit aorta, being highest (0.86 +/- 0.12 micromol L(-1)) for the ascending aorta, and lowest for the iliac aorta (0.48 +/- 0.15 micromol L(-1)). The distribution of NO in the left ventricular wall of rabbit heart was not uniform and varied from 1.23 +/- 0.20 micromol L(-1) (center) to 0.90 +/- 0.15 micromol L(-1) (apex). Both, the maximal concentration and the dynamics of NO release can be useful diagnostic tools in estimating the level of endothelial dysfunction and cardiovascular system efficiency.     

Effect of 5 alpha-pregnan-3 alpha-ol-20-one on nitric oxide biosynthesis and plasma volume in rats

Plasma 5 alpha-pregnan-3 alpha-ol-20-one (pregnan) levels and nitric oxide (NO) biosynthesis increase during pregnancy. These factors have independently been implicated in the control of blood pressure and volume. We wished to determine whether pregnan might be responsible both for the increase in NO biosynthesis and for the increase in plasma volume observed during pregnancy. Virgin female Long-Evans rats were implanted with indwelling cannulas and maintained on a low nitrate/nitrite diet. After the rats recovered from surgery, 500 microg of pregnan or vehicle were given daily for 2 days. NO biosynthesis and plasma volume were measured in conscious animals before and after treatment. Pregnan caused a significant increase in NO biosynthesis (1.9 +/- 0.8 micromol/24 h, n = 10) compared with the vehicle-treated control group (0.3 +/- 0.4 micromol/24 h, n = 10, P < 0.05). Similarly, there was a significant increase in plasma volume in the pregnan-treated group (0.7 +/- 0.2 ml/100 g, n = 11) compared with the vehicle-treated control group (0.2 +/- 0.1 ml/100 g, n = 11, P < 0.05). These results confirm that pregnan can mimic pregnancy by its ability to increase both NO biosynthesis and plasma volume.