Search

Objectives The theory of a red blood cell derived nitric oxide (NO) reserve conserving NO bioactivity and delivering NO as a function of oxygen demand has been the subject of much interest. We identified the human coronary circulation as an ideal model system in which to analyse NO metabolites because of its large physiological oxygen gradient. Our objective was to identify whether oxygen drove apportion between various NO metabolite species across a single vascular bed. Methods Plasma and red blood cell NO metabolites were assessed from the left main coronary artery, coronary sinus and pulmonary artery (providing cross heart and cross pulmonary analysis) of healthy subjects under resting conditions and following administration of an inhibitor of NO biosynthesis. Physiological parameters and angiographic data were monitored throughout the study. Results Under baseline conditions we observed significant metabolite flux upon the transit of blood across the coronary and pulmonary vascular beds. Whilst there was no net loss of NO through the coronary circulation ( p =0.0759), plasma nitrite/protein NO (excluding nitrate) ( p =0.0279) and red blood cell sulphanilamide labile signal ( p =0.0143) decreased whereas haemoglobin-bound NO increased three-fold ( p =0.005). These changes across the coronary circulation were reversed through the pulmonary circuit with red blood cell sulphanilamide labile signal ( p =0.0143) and plasma nitrite/protein NO ( p =0.0279) increasing and haemoglobin-bound NO decreasing. Blockade of NO synthesis increased mean arterial blood pressure ( p

We examined the effects of aspirin on basal and beta-adrenoceptor (beta-AR)-mediated nitric oxide synthase (NOS) activity in normal platelets.NOS activity was determined from the conversion of L-[3H]arginine to L-[3H]citrulline, both basally and following beta-AR stimulation, in platelets from healthy human subjects following both short- and long-term aspirin administration.Basal L-[3H]citrulline increased following aspirin 800 mg administered intravenously in vivo, from 0.31+/-0.12 to 0.76+/-0.14 pmol/10(8) platelets (P0.01). Isoproterenol at 1 micromol/l increased platelet NOS activity before but not following intravenous aspirin. After short-term in vitro treatment with aspirin 10 micromol/l, 400 micromol/l or 4 mmol/l, basal platelet L-[3H]citrulline increased similarly, an effect not seen with indomethacin 100 micromol/l or ibuprofen 10 micromol/l. Platelet NOS activity was not increased by albuterol 1 micromol/l, in the presence of indomethacin, ibuprofen or aspirin in vitro. By contrast, oral aspirin 75 mg daily for 14 days did not affect basal platelet NOS activity, but abolished beta-adrenergic NOS activation.Aspirin activates basal platelet NOS acutely, but not chronically, through a mechanism independent of cyclooxygenase (COX) inhibition. By contrast, both short- and long-term aspirin treatment inhibit platelet beta-adrenergic NOS activation by a COX-dependent mechanism. This indicates that aspirin exerts divergent effects on basal and beta-AR-stimulated platelet NOS activity, which are likely to be of clinical relevance.

Objective: We have analyzed the extent of endothelial dysfunction in cardiac resistance vessels of hyperlipidaemic apoE−/− mice and explored whether NO and/or prostacyclin dependent pathways are involved. Methods: Coronary resistance was measured in isolated perfused hearts from WT and apoE−/− mice. To discriminate between NO and PGI2-dependent flow responses, we made use of the finding that acetylcholine (ACh) predominantly activates the prostaglandin pathway whereas bradykinin (Bk) mainly acts via NO in murine cardiac resistance vessels. Results: Basal coronary flow as well as the ACh induced vasodilation (0.1–1 μM) were not different between WT and apoE−/− hearts (flow increase+100%). Similarly, vasodilation in response to the prostacyclin mimetic iloprost reached the same levels. In contrast, the Bk-stimulated [3.3 μM Bk] coronary flow was reduced from 31.6±4.2 in WT to 19.2±2.7 ml min−1 g−1 in apoE−/− hearts. NOS inhibition by ethylisothiourea (ETU, 10 μM) reduced basal as well as Bk-stimulated coronary flow in WT and apoE−/− hearts to the same extent. RT–PCR and Western analysis demonstrated that neither eNOS expression nor protein levels were reduced. Similarly, the flow response to the NO donor SNAP (0.3–33 μM) was not altered suggesting that soluble guanylyl cyclase was not affected. Intracoronary application of superoxide dismutase augmented the Bk-induced vasodilation of apoE−/− hearts almost back to WT levels (26.6±3.3 ml min−1 g−1). In line with this finding the NADPH induced O2− formation was enhanced in cardiac extracts from apoE−/− hearts. Conclusion: apoE−/− hearts develop a hemodynamically relevant endothelial dysfunction at the level of coronary resistance vessels most likely via inactivation of bioavailable NO by superoxide anions. The function of the prostacyclin system is not altered.

Objective: Apoptosis of cardiomyocytes may contribute to ischemia–reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. Methods and results: Isolated perfused rat hearts ( n =6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-l-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia–reperfusion. Conclusion: The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged.

The F2-isoprostanes are a family of novel prostaglandin isomers and a stable product of in vivo oxidative stress. 8-epi-prostaglandinF2 alpha, a member of this isoprostane family, is a vasoconstrictor and its local release may contribute to the abnormal vasomotor tone associated with hypercholesterolemia. We therefore aimed to outline the role of 8-epi-prostaglandinF2 alpha as a coronary vasoconstrictor in experimental hypercholesterolemia.Pigs were randomized to two experimental groups (each n = 9): normal (N) and high cholesterol (HC) diet. To determine the vasoconstrictive effects of 8-epi-prostaglandinF2 alpha in vitro, doses from 10(-9) to 10(-5) M were used to constrict coronary epicardial rings. Plasma total and LDL cholesterol levels were significantly higher in the HC group compared with the N group (P0.005) as were plasma 8-epi-prostaglandinF2 alpha levels (P0.001). 8-epi-prostaglandinF2 alpha immunoreactivity was present in the vessel wall in both groups. Normal vessels with intact endothelium (n = 8 rings) contracted to 8-epi-prostaglandinF2 alpha (maximal contraction 15.5 +/- 8.74%). In the HC group, rings with intact endothelium had a greater contractile response to 8-epi-prostaglandinF2 alpha compared to normals (72.3 +/- 7.9%; n = 8; P0.0001). This was reversed by preincubation with NOR-3, a NO donor (maximal contraction 6.7 +/- 1.56%; n = 5; P0.0001). Enhanced contraction in normal vessels occurred with endothelial denudation (98.4 +/- 3.56%; n = 6; P0.0001) and with preincubation of the endothelium-intact rings with L-NMMA (N-monomethyl-L-arginine), an NO synthase inhibitor (85.5 +/- 10.3%, n = 6, P0.001). The enhanced contraction seen with hypercholesterolemia did not occur with other prostanoid vasoconstrictors.Experimental hypercholesterolemia leads to a significant increase in 8-epi-prostaglandinF2 alpha levels in addition to enhanced 8-epi-prostaglandinF2 alpha-induced coronary vasoconstriction, in vitro. These findings support a role for the F2-isoprostanes in the regulation of coronary vasomotor tone in pathophysiologic states.

The guanidino-methylated arginine analogue N G monomethyl-l-arginine (l-NMMA) has been the standard nitric oxide synthase inhibitor used to evaluate the role of the l-arginine:nitric oxide pathway. However, l-NMMA and other methylated arginine residues are also synthesised in vivo by the action of a family of enzymes known as protein arginine methyltransferases. Proteolysis of proteins containing methylated arginine residues releases free methylarginine residues into the cytosol from where they may pass out of the cell into plasma. Of the three known methylarginine residues produced in mammals only asymmetrically methylated forms (l-NMMA and asymmetric dimethylarginine (ADMA)) but not symmetrically methylated arginine (symmetric dimethylarginine (SDMA)) inhibit nitric oxide synthase (NOS). We and others have proposed that endogenously produced asymmetrically methylated arginines may modulate NO production and that the accumulation of these residues in disease states may contribute to pathology. The activity of the enzyme dimethylarginine dimethylaminohydrolase that metabolises asymmetric methylarginines may be of critical importance in affecting NO pathways in health or disease.

OBJECTIVE: Venodilatation is a feature of endotoxaemia and sepsis. We have tested directly the hypothesis that three cytokines (IL-1 beta, TNF alpha and IL-6) generated during endotoxaemia affect venous tone in humans in vivo by increasing NO generation and explored whether the NO comes from the iNOS or eNOS isoform. DESIGN AND INTERVENTION: Cytokines were given into a superficial vein in very low doses sufficient only to produce changes in the study vessel. The effects of cytokines on the response to noradrenaline were examined. RESULTS: IL-1 beta increased basal NO-induced dilatation in the study vein, and this was sufficient to attenuate the constrictor response to exogenous noradrenaline or sympathetic stimulation. The effects were maximal at 6 h and both NG-monomethyl-L-arginine and aminoguanidine caused significant reversal of the IL-1 beta effects. However, no induction of iNOS mRNA was detected in the tissue samples. Instead, mRNA encoding eNOS and GTP cyclohydrolase-1 was detected in all vessels. CONCLUSION: The simplest explanation of these results is that IL-1 beta induces expression of GTP cyclohydrolase-1 which leads to increased generation of BH4 and activation of eNOS. This study identifies IL-1 beta as a key cytokine causing physiologically significant venodilatation in humans by increasing NO generation and suggests that this can occur even in the absence of iNOS expression.

Objective: Nitric oxide is a vasodilating and blood pressure lowering substance. To investigate whether calcium antagonists or angiotensin-converting enzyme (ACE) inhibitors increase vascular nitric oxide activity, we assessed systemic and renal vascular sensitivity to nitric oxide synthase inhibition in hypertensives on and off medication. Methods: Ten essential hypertensive patients, aged 22–51 years, were studied 3 times: ≥4 weeks off medication, after 3 weeks treatment with enalapril 20 mg twice a day and after 3 weeks nifedipine 60 mg/day. Each time, 24-h blood pressure registration was performed, followed by a clearance study to obtain a 3-h dose-response curve for intravenously infused NG-monomethyl-l-arginine (l-NMMA, respectively 0.75, 1.5 and 3.0 mg/kg/h). Results: l-NMMA dose-dependently increased mean arterial pressure with 5±2 mmHg and systemic vascular resistance with 24±5% at maximum dose, whereas cardiac output decreased (all P

Objective: Inducible nitric oxide synthase (iNOS) has been implicated to contribute to myocardial dysfunction in various settings, but considerable species differences have been noted in the levels of iNOS expression and its function in several tissues. The aim of this study was to elucidate evolutional changes in myocardial iNOS expression and function. Methods: An iNOS cDNA clone was isolated by RT-PCR from the 10-day old cultured chick embryonic ventricular myocytes stimulated with 10 μg/ml of lipopolysaccharide. Expression of the iNOS mRNA was analyzed with Northern blot analysis and RNase protection assay. The iNOS activity was estimated from conversion rates of l-arginine to l-citrulline and intracellular cGMP contents were measured with radioimmunoassay. Furthermore, both [Ca2+]i (fluorescent dye indo-1) and cell contraction (video motion detector) were simultaneously recorded. Results: Aside from the primer sequences, the insert (1026 bp) of the cDNA clone showed 66.4% identity at the deduced amino acid level to the human iNOS cDNAs. Northern blot analysis revealed that chicken iNOS mRNA of approximately 4.5 kb was induced by lipopolysaccharide within 6 h in the cultured myocytes. RNase protection assay also showed that lipopolysaccharide provoked 14.6±5.1-fold increases ( n =6, p

Objective : Effective endogenous fibrinolysis requires rapid release of endothelial tissue plasminogen activator (t-PA). Using the nitric oxide synthase inhibitor, l- N G-monomethylarginine (l-NMMA), we examined the contribution of endogenous nitric oxide to substance P-induced t-PA release in vivo in man. Methods : Blood flow and plasma fibrinolytic and haemostatic factors were measured in both forearms of 8 healthy male volunteers who received unilateral brachial artery infusions of substance P (2–8 pmol/min) and l-NMMA (1–4 μg/min). Results : Substance P caused dose-dependent increases in blood flow ( P

Objective: The strong angiogenic and mitogenic agents acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) share signalling pathways with known vasodilatory agonists. Therefore, we hypothesized that FGF's produce vasoactive responses. We also proposed that heparin would exert a similar action to FGF, because this proteoglycan not only binds to the FGF receptor, but also facilitates the release of FGF from the cardiac extracellular matrix and promotes its binding to a high-affinity receptor. To test these hypotheses, we examined the vasodilatory reactions of coronary arterioles to aFGF, bFGF, and heparin, and the effects of antagonists to nitric oxide synthase (l-NMMA), prostaglandins (indomethacin), ATP-sensitive potassium (K[ATP]) channels (glibenclamide), FGF and FGF receptors on the vasoactive responses. Methods: Arterioles (70–110 μm, internal diameter) were dissected from pig hearts and cannulated with micropipettes. Diameter was determined with videomicroscopy in response to bFGF and aFGF in concentrations of 1–100 ng/ml and to heparin (5–200 U/ml). Results: Basic FGF, but not aFGF, caused dose-dependent vasodilation with a maximum of 61±4%. Relaxation to bFGF was antagonized by pretreatment with l-NMMA, but was not affected by pretreatment with indomethacin or glibenclamide. Heparin caused dose-dependent vasodilation with a maximum of 100±3% which was partially blocked by either l-NMMA or glibenclamide, but not by indomethacin. Furthermore, the effect of bFGF could be significantly blocked by pretreatment with an FGF receptor antibody as well as with a monoclonal antibody against FGF. Pretreatment with both antibodies significantly inhibited also the effect of heparin. Conclusions: These results indicate that bFGF and heparin cause vasodilation of coronary arterioles via an increase in NO production and heparin additionally by other mechanisms such as by activating K[ATP] channels. Furthermore, the effect of heparin is partially mediated via FGF and FGF receptors. We therefore speculate that both substances may be involved in the regulation of coronary microvascular tone acting partially through the same signalling mechanisms.

Objective: To determine the involvement of prostaglandins, nitric oxide, and beta-adrenoceptor activation in insulin-induced vasodilation in the human forearm. Methods: Fifteen normal subjects were studied. Insulin was administered into the brachial artery in the presence of saline, the cyclo-oxygenase inhibitor indomethacin (0.65 μg/kg/min), the inhibitor of nitric oxide synthase N G-monomethyl-l-arginine (l-NMMA, 30 μg/kg/min), or the non-selective beta-adrenoceptor blocker propranolol (0.2 μg/kg/min). Forearm vascular resistance (FVR) was derived from forearm blood flow and concomitantly measured intra-arterial blood pressure. Results: Insulin decreased FVR by 32±5% ( P

Objective: The acute release of radicals upon reperfusion following myocardial ischemia may include both nitric oxide (NO) and superoxide anion (O2−⋅). The generation of peroxynitrite (ONOO−) from these radicals may contribute to ischemia-reperfusion injury. Our objective was to measure the generation of ONOO− during reperfusion of isolated hearts subjected to ischemia and to determine the effects of inhibition of NO synthase with N G-monomethyl-l-arginine (l-NMMA), or supplementation of NO with S -nitroso- N -acetyl-d,l-penicillamine (SNAP), on ONOO− generation and on the recovery of mechanical function. Methods and Results: Isolated rat hearts were perfused at constant pressure with Krebs' buffer containing l-tyrosine, which reacts with ONOO− to form dityrosine, a fluorescent product. Dityrosine was detected in the coronary effluent of hearts infused with synthetic ONOO−. In hearts subjected to 20 min of global, no-flow ischemia there was a marked rise in endogenous ONOO− formation which peaked at 30 s of reperfusion. Formation of ONOO− was dependent upon synthesis of both NO and O2−⋅, as dityrosine release was abolished by l-NMMA or superoxide dismutase, respectively. l-NMMA caused a concentration-dependent improvement in the recovery of mechanical function during reperfusion. Infusion of SNAP also abolished dityrosine release at reperfusion and improved the recovery of post-ischemic function. Conclusions: Our results show for the first time that reperfusion of the ischemic heart causes the acute production of ONOO−. Inhibiting the biosynthesis of ONOO− with l-NMMA or antagonizing its oxidant actions with SNAP are possible strategies to protect the heart from ischemia-reperfusion injury.

Objectives: The detrimental haemodynamic changes observed in septicaemia are generalised vasodilation, arterial hypotension, and hyporesponsiveness to vasopressor compounds, all of which could be explained by the release of an endogenous vasodilator. Experimental and clinical evidence suggests that tumour necrosis factor-α (TNF) induces the expression of vascular nitric oxide (NO) synthase within hours and that NO released from smooth muscle cells could be involved in the pathogenesis of septic shock. The aim of this study was to investigate the role of NO in the vascular effects of TNF. Methods: Using the dorsal hand vein compliance technique, the effect of the NO synthase inhibitor L-NG-monomethyl-arginine (L-NMMA) on α 1-adrenergic responsiveness (phenylephrine 1.25–8000 ng/min) was studied after prolonged local venous infusion of TNF (8.7 μg in 5 h) in 9 volunteers and in 6 volunteers without previous cytokine exposure. Results: Mean (±s.e.) maximum phenylephrine constriction (Emax) was 73 ± 6% and log dose-rates exerting 50% of Emax (log ED50) were 3.2 ± 0.09 (geometric mean: 1535 ng/min). Local co-administration of L-NMMA at a dose sufficiently high to block NO formation (3.4 μmol/min) increased venous sensitivity to phenylephrine threefold (log ED50 2.8 ± 0.1, P < 0.015; geometric mean: 574 ng/min) whereas Emax was similar (73 ± 5%). In the controls the phenylephrine dose-response relationship remained unaffected by simultaneous administration of L-NMMA. Conclusions: As no basal release of NO occurs in hand veins without previous exposure to TNF these results provide direct evidence for induction of NO formation in the human vasculature and consecutive resistance to α-adrenergic venoconstriction. NO might, therefore, be a key mediator of haemodynamic impairment in humans under conditions with known elevations of circulating TNF, such as a septic shock.

Objectives: Nitric oxide (NO) exerts both protective and detrimental actions in a variety of biological systems. During acute reperfusion following myocardial ischaemia, a rapid overproduction of free radicals, including NO, may occur. We investigated the effects of the NO synthase inhibitors N G-nitro-l-arginine methyl ester (l-NAME) and N G-monomethyl-l-arginine (l-NMMA), and the substrate for NO synthesis, l-arginine, on heart function during ischaemia and reperfusion injury. Methods: Spontaneously beating, isolated working rabbit hearts, perfused with modified Krebs-Henseleit buffer containing 1.2 mM palmitate bound to 3% bovine serum albumin, were subjected to 15 min of aerobic perfusion followed by 35 min of global, no-flow ischaemia and 30 min of aerobic reperfusion. Results: Throughout the reperfusion period there was a marked impairment in the recovery of mechanical function, measured as the product of heart rate × peak systolic pressure (rate-pressure product). Addition of l-NAME (3 μM) prior to the onset of ischaemia, but not at reperfusion, caused an immediate and significant increase in the recovery of mechanical function throughout the reperfusion period. The protective action of l-NAME was abolished by l- (but not d-) arginine (100 μM). l-NAME did not cause ischaemia as it did not alter glycogen or lactate content of aerobically perfused hearts. Furthermore, it did not prevent glycogen loss or lactate accumulation during 35 min of ischaemia, suggesting that the effects of l-NAME were not due to metabolic alterations during ischaemia itself. l-NMMA (30 μM) added prior to ischaemia, but not at reperfusion, also had a protective effect which was seen later in the reperfusion period. Addition of l- (but not d-) arginine (100 μM) prior to the onset of ischaemia resulted in an improved recovery of mechanical function only at 15 min of reperfusion. Conclusions: These results suggest that: (1) the recovery of mechanical function of hearts subjected to ischaemia-reperfusion injury can be improved by modulation of myocardial NO synthesis, (2) inhibition of NO synthesis (with l-NAME or l-NMMA) may offer prolonged protection whereas its stimulation (with l-arginine) provides only brief protection, and (3) the reasons for the pharmacological effectiveness of these divergent strategies may be due to the formation of peroxynitrite from NO and superoxide anion during reperfusion.

Objective: The aim was to examine the role of nitric oxide (NO) in the myocardial microcirculation. Methods: Open chest anaesthetised dogs received intracoronary infusion of NG-monomethyl-L-arginine (L-NMMA; 0.5 mg-kg−1 over 30 min). Myocardial microvascular extraction fraction of a small hydrophilic solute (technetium 99m labelled diethylenetriaminepenta-acetate [99mTc-DTPA]), and the regional myocardial plasma flow rate, were determined by the single injection, residue detection method, and the capillary permeability-surface area product was calculated. The tone in intramyocardial resistance vessels was assessed by the local 133Xe washout method during baseline conditions, in response to intracoronary acetylcholine, and during peak reactive hyperaemia after 10 s or 30 s of myocardial ischaemia, respectively. Results: In eight open chest dogs, L-NMMA attenuated the increase in myocardial plasma flow rate in response to intracoronary acetylcholine by 32(SEM 7)%, but failed to alter baseline myocardial plasma flow rate significantly, as determined by the local 133Xe washout method. L-NMMA did not influence myocardial microvascular permeability to 99mTc-DTPA. However, intracoronary L-NMMA decreased the peak reactive hyperaemic myocardial plasma flow rate after 10 s, but not 30 s, of coronary occlusion. Conclusions: In open chest dogs, microvascular NO synthesis is not a major determinant of baseline myocardial plasma flow rate, and does not appear to influence myocardial microvascular permeability significantly. In this model, NO intervenes in the regulation of the peak reactive hyperaemic plasma flow rate following brief, but not more prolonged, periods of coronary occlusion.

Aims Previously, we showed an enhanced excitatory ( N -methyl d-aspartate receptor-NR1) and decreased inhibitory neuronal nitric oxide (NO) synthase (nNOS) influence within the paraventricular nucleus (PVN) of rats with chronic heart failure (CHF). Although NR1 and nNOS are normally linked, they can be disconnected by nNOS sequestering with nNOS-associated protein (CAPON). The aim of this study was to elucidate the underlying mechanism for the disconnection between increased expression of NR1 and decreased nNOS in the PVN of rats with CHF which leads to enhanced sympathoexcitation. Methods and results CAPON expression was augmented while nNOS expression was decreased in the PVN of rats with CHF (6–8 weeks after left coronary artery ligation). Angiotensin II (Ang II) type I receptor (AT1) antagonist losartan (Los) treatment in rats with CHF reduced renal sympathetic nerve activity with concomitant normalization of protein expression of CAPON and nNOS in the PVN. Los treatment also reversed the blunting of endogenous NO-mediated sympatho-inhibition in rats with CHF. Moreover, Ang II-induced increase in CAPON expression in NG108 neuronal cells was also ameliorated by Los. Conclusion Blocking AT1 receptors prevents the overexpression of CAPON and concomitant decrease in nNOS in the PVN, resulting in attenuation of sympathoexcitation commonly observed in CHF. Taken together, our data highlight the importance of altered expression and subsequent interaction of nNOS and CAPON within the PVN, leading to increased sympathoexcitation in CHF. Identifying this crucial nNOS/CAPON interaction regulated by AT1 receptors may provide an important potential therapeutic target in CHF.

Objective: The aim was to investigate the release of nitric oxide (NO) from porcine mitral valves. Methods: The valves were sandwiched between gauze in a chamber constantly perfused by Holman solution. The presence of NO in the perfusate of the valves was detected by two methods: a bioassay cascade system using preconstricted rings of pig coronary artery as the detector tissue, and a chemiluminescent method, specific for NO. Results: Porcine mitral valves release NO under basal conditions. Release can be increased by the agonists ADP, substance P, bradykinin, thrombin, and the calcium ionophore A23187. ADP was by far the most effective. NO release under basal conditions was inhibited by each of the three NO synthase inhibitors studied, but only NG-monomethyl L-arginine (L-NMMA) reduced ADP stimulated release. A reversible inhibition of NO release occurred when valves were perfused with calcium-free Holman solution and NO release was inhibited by removal of the endocardial monolayer. Conclusions: The endocardial endothelium covering the mitral valves releases NO under basal conditions and following stimulation with several agonists. The NO synthase enzyme is calcium dependent. Different mechanisms may exist for the synthesis of basal and agonist dependent NO release in these cells. Cardiovascular Research 1993; 27 :1657-1661

The aim was to study the effects on coronary vascular tone of three inhibitors of nitric oxide (NO) synthesis.Studies were performed on isolated perfused hearts of 74 male New Zealand White rabbits fed normal laboratory diet. Resting coronary perfusion pressure was increased to 40-60 mm Hg with the thromboxane mimetic 9,11-dideoxy-9 alpha,11 alpha-methanoepoxy prostaglandin F2 alpha (U46619). The effects of NG-monomethyl-L-arginine (L-NMMA), N-iminoethyl-L-ornithine (L-NIO), and NG-nitro-L-arginine methyl ester (L-NAME) (0.3-300 microM) on resting coronary perfusion pressure were determined. The effects of these compounds, at concentrations that increased the resting perfusion pressure to a similar extent, on the fall in perfusion pressure induced by acetylcholine (0.1 microM) and glyceryl trinitrate (1 microM) were also investigated. In these studies the resting perfusion pressure was maintained at 40-60 mm Hg by reducing the concentration of U46619.L-NMMA, L-NIO, and L-NAME induced concentration dependent increases in resting coronary perfusion pressure (n = 3-9, p0.05). L-NAME had the greatest potency and efficacy, increasing the resting pressure by 48.0(SEM 9.6) mm Hg at 30 microM. L-NIO and L-NMMA increased perfusion pressure by 27.3(3.0) and 19.5(5.8) mm Hg respectively at the maximum concentration studied (300 microM). However, at concentrations that were equieffective on resting perfusion pressure (15 mm Hg increase), L-NMMA (100 microM), but not L-NIO (25 microM) or L-NAME (4 microM), significantly inhibited the fall in pressure induced by acetylcholine by 57.2(5.0)%, n = 6, p0.05. This effect of L-NMMA++ was attributed to a shorter duration of fall and was reversed by L-arginine (300 microM). L-NMMA (100 microM) and L-NIO (25 microM) potentiated the effect of glyceryl trinitrate by increasing the peak fall in perfusion pressure by 75.6(11.0)% and 68.8(24.1)% respectively (n = 6 for each, p0.05).The differential effects of the three inhibitors on resting coronary perfusion pressure and the acetylcholine induced fall in coronary perfusion pressure suggest that basal and stimulated NO synthesis may be differentially regulated. Reduction in the synthesis of endogenous NO by these compounds potentiates the glyceryl trinitrate induced fall in perfusion pressure, which may have important clinical implications.

Objective: The aim was to investigate the effects of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of both the constitutive (Ca2+ dependent) and inducible (Ca2+ independent) nitric oxide (NO) synthases, or of pretreatment with the glucocorticoid dexamethasone, an inhibitor of the induction of the Ca2+ independent NO synthase, on lipopolysaccharide induced shock in the anaesthetised rabbit. Methods: Mean arterial blood pressure, and blood flow in the portal vein, hepatic artery, and hindquarter vascular beds were measured in 49 halothane anaesthetised New Zealand White rabbits given lipopolysaccharide ( Salmonella minnesota, 500 μg·kg−1 intravenously). The effects of pre- or post-lipopolysaccharide treatment with L-NMMA (300 mg·kg−1 intravenously) and of pretreatment with dexamethasone (3 mg·kg−1 intravenously) were determined. The effect of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 300 μg·kg−1·h−1 intravenously) in animals treated with lipopolysaccharide and L-NMMA was also studied. Results: Lipopolysaccharide elicited an initial transient fall in mean arterial pressure and decreases in blood flow in the vascular beds, followed by a progressive fall in mean arterial pressure. L-NMMA when given either before or after lipopolysaccharide markedly exacerbated its effects and resulted in severe hypotension, intense vasoconstriction, and increased mortality. Pretreatment with dexamethasone had no effect on the initial haemodynamic changes following lipopolysaccharide, but prevented the subsequent fall in mean arterial pressure observed in animals treated with lipopolysaccharide alone. Dexamethasone failed, however, to protect animals also treated with L-NMMA before lipopolysaccharide. Animals pretreated with L-NMMA and SNAP showed reduced haemodynamic changes when compared with controls (lipopolysaccharide only) or lipopolysaccharide and L-NMMA treated animals. Conclusions: Inhibition of both constitutive and inducible NO synthases during endotoxaemia is deleterious. This can be overcome by replacing NO intravenously with a donor of NO. Selective inhibition of the inducible NO synthase may, however, be beneficial in shock.

Study objective – The aim was to investigate further the endothelium dependent vasodilator action of 5-hydroxytryptamine (5-HT) on the rat coronary circulation. Design – Using saponin to damage the endothelium and L-NMMA (NG -monomethyl-L-arginine), the selective inhibitor of nitric oxide formation, we examined the role of endothelium and nitric oxide in causing 5-HT induced vasodilatation in the isolated rat heart. Experimental material – 56 rat hearts were excised and perfused on a Langendorff preparation. Measurements and main results – 5-HT at (10−9 to 10−5 M) and glyceryl trinitrate at (10−5 to 10−3 M) (n = 24) induced a dose dependent increase in coronary flow. Chemical removal of the endothelium by exposure to saponin (30 μg-ml−1) (n = 8) abolished the 5-HT induced vasodilatation but had no effect on the response to glyceryl trinitrate. Pretreatment of rats (n = 8) with L-NMMA (100 mg·kg"1) unmasked a strong vasoconstrictor effect of 5-HT but did not affect the glyceryl trinitrate induced vasodilatation. Perfusion of L-NMMA pretreated hearts with L-arginine at 10−4 M (n = 8) restored the vasodilatation induced by 5-HT but L-arginine perfusion had no effect on the extent of 5-HT or glyceryl trinitrate induced vasodilatation in normal hearts (n = 8). Conclusions – In the coronary circulation of the isolated rat heart, 5-HT mediates its vasodilator effect via endothelium dependent release of nitric oxide.

Objective Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the underlying mechanisms are still unknown. In this study, we investigated in isolated rat cardiomyocytes subjected to hypoxia and reoxygenation whether administration of insulin during reoxygenation reduces reoxygenation-induced hypercontracture, a hallmark of acute reperfusion injury. The effects of insulin on potential pro-survival kinases, i.e., PI 3-kinase, NO synthase (eNOS), and cGMP-dependent protein kinase (PKG), and on cytosolic Ca2+ control in reoxygenated cardiomyocytes were investigated. Results Administration of insulin (10mU/L) during reoxygenation protected cardiomyocytes against hypercontracture development (cell length as % of end-hypoxic length: control 61.6±3.2; insulin 76.3±2.9*; n =26; * p

Objective: The mechanisms underlying hypercapnic coronary dilation remain unsettled. This study tests the hypothesis that flow dependent NO production is obligatory for the hypercapnic flow response. Methods/results: In isolated, constant pressure (CP) perfused guinea pig hearts a step change of arterial p CO2 from 38.6 to 61.4 mm Hg induced a bi-phasic flow response with an early transient (maximum 60 s) and a consecutive persisting flow rise (121.6 ± 6.6 (S.D.) % after 10 min). In contrast, when perfused with constant flow (CF), perfusion pressure only transiently (2 min) fell by 7.4 ± 4.8 % following the step change of arterial p CO2. In CP perfused hearts L-NAME (100 μmol/l) specifically abolished the delayed flow rise during hypercapnic acidosis (102.37 ± 2.9% after 10 min), whereas the inhibitor had no effect on perfusion pressure response in CF perfused hearts. Under CP perfusion arterial hypercapnia resulted in a transient rise of coronary cGMP release (from 0.69 ± 0.35 to 1.12 ± 0.68 pmol/ml), which was abolished after L-NAME. Surprisingly, the K+ATP channel blocker glibenclamide did not have any significant effect on the hypercapnic flow response but largely blunted reactive hyperemia after a 20 s flow stop. Conclusions: The delayed steady state hypercapnic flow response in guinea pig heart requires intact NO production. The absence of a persisting decrease in coronary resistance under CF perfusion points to an important role of shear stress dependent NO production.

Objectives : Our aim was to investigate mechanisms of inflammation-induced endothelial dysfunction in humans. Methods : Endothelial function in twenty-one healthy human volunteers was measured using forearm venous plethysmography before and 8 h after administration of typhoid vaccination to generate an inflammatory response. Basal and stimulated endothelial nitric oxide (NO) bioavailability was assessed by measurement of the responses to intra-arterial N G-monomethyl-l-arginine (l-NMMA) and bradykinin, respectively. The effects of supplementation with l-arginine or ascorbic acid were assessed to probe the effects of substrate deficiency and oxidative stress, respectively. Systemic effects were determined by measuring cytokine response, total anti-oxidant status (TAOS) and urinary protein excretion. Results : Vaccination induced a cytokine response, a fall in total anti-oxidant status and increased urinary albumin excretion (UAE). There was a reduction in the response to bradykinin (BK, P

The effects of in vivo gene transfer of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) genes on severe atherosclerosis were investigated in rabbits. The recombinant adenoviruses, Ad.eNOS and Ad.iNOS, which respectively express eNOS and iNOS, were constructed. Atherosclerosis was induced by a balloon injury followed by a high cholesterol diet for 12 weeks. The rabbits were divided into six groups: Gp cont (no treatment); Gp null (adenovirus sham-infected); Gp eNOS (Ad.eNOS); Gp iNOS (Ad.iNOS); Gp e+i (Ad.eNOS plus Ad.iNOS); and Gp heNOS (a high dose of Ad.eNOS). Examinations were carried out 7 days after gene transfer. Plasma lipid levels were not significantly changed, but transfection with Ad.eNOS (Gp eNOS and Gp heNOS) decreased the tissue cholesterol concentration and regressed atherosclerotic lesions. Vessels treated with Ad.iNOS (Gp iNOS and Gp e+i) showed iNOS staining in the atheroma, and slight staining at other parts of the vessels; those treated with Ad.eNOS showed eNOS staining in the endothelium and subintima, and slight staining at other parts. Ad.eNOS transfection, but not Ad.iNOS or Ad.eNOS+Ad.iNOS transfection, improved the impaired aortic endothelium-dependent relaxation (EDR) and basal NO-dependent response, increased tissue cyclic GMP (cGMP), and decreased the release of O2- from vessels. eNOS treatment showed a decreasing tendency in regions with peroxynitrite staining, MMP1 staining, and suspected apoptosis. In conclusion, in vivo gene transfer of eNOS, but not iNOS or eNOS plus iNOS, regressed atherosclerosis. The relations among NO, O2-, and peroxynitrite may be critical, and lipid resorption from the lesions may be responsible for the regression.

Objective: Postmenopausal estrogen replacement therapy lowers the incidence of cardiovascular disease, suggesting that estrogens support cardiovascular function. Estrogens dilate coronary arteries; however, little is known about the molecular basis of how estrogen affects the human coronary circulation. The cellular/molecular effects of estrogen action on human coronary smooth muscle were investigated in the present study. Methods: Patch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium. Results: Estrogen increased whole-cell currents over a range of membrane potentials, and further studies indicated that the large-conductance (186.5±3 pS), calcium- and voltage-activated potassium (BKCa) channel was the target of estrogen action. Channel activity was stimulated ∼15-fold by nanomolar concentrations of 17β-estradiol, and this stimulation was reversed >90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17β-Estradiol increased the level of cGMP and nitric oxide in human myocytes, and the stimulatory effect of estrogen on channel activity and NO production was reversed by inhibiting NO synthase with 10 μM N G-monomethyl-l-arginine. Conclusions: Our cellular and molecular studies identify the BKCa channel as a target of estrogen action in human coronary artery smooth muscle. This response to estrogen involves cGMP-dependent phosphorylation of the BKCa channel or a closely associated regulatory molecule, and further evidence suggests involvement of the NO/cGMP signaling system in coronary smooth muscle. These findings are the first to provide direct evidence for a molecular mechanism that can account for endothelium-independent effects of estrogen on human arteries, and may also help explain why estrogens reduce myocardial ischemia and stimulate coronary blood flow in patients with diseased coronary arteries.

Objective: Human urotensin II (hUII) is an endocrine hormone that acts as a potent arterial vasoconstrictor in both in vitro and in vivo studies in animals. We examined, for the first time, the local and systemic hemodynamic response to hUII in man in vivo. Methods: Four healthy male volunteers took part in pilot studies and 11 in definitive studies. Forearm blood flow (FBF) was measured in response to intra-arterial infusion of authentic, biologically active hUII (incremental rates of 0.001–300 pmol min−1) and saline placebo using venous occlusion plethysmography. Blood pressure, heart rate, cardiac output and hUII plasma concentrations were also measured. Forearm studies were repeated in five subjects with inhibition of endothelial mediators using aspirin and a “nitric oxide clamp”. Dorsal hand vein diameter was determined by a standard displacement technique in response to local administration of hUII (3–300 pmol min−1) with and without nitric oxide synthase inhibition. Results: There was no significant change in FBF during brachial infusion of saline or hUII (dose range, 0.001 to 300 pmol min−1). A nitric oxide clamp did not unmask vasoactive effects of hUII. Human UII infusions (100 and 300 pmol min−1) significantly increased plasma hUII concentrations from baseline (12±3 pmol l−1) to 106±15 and 307±98 pmol l−1, respectively. Despite high circulating hUII concentrations, no change was seen in systemic hemodynamics and ECGs were unchanged. Human UII had no effect on hand vein diameter ( n =6). Conclusions: In contrast to our hypothesised role of hUII, we found no vasoactive responses to hUII in vivo, consistent with recent in vitro studies in human blood vessels, but in contrast to non-human primate studies in vivo. Our data do not support a key role for hUII in the regulation of vascular tone and resting blood pressure in man. However, studies with hUII receptor antagonists are also needed before firm conclusions can be drawn.

Purpose: The hypolipidaemic agent, fenofibrate, has been shown to exert pleiotropic (lipid-independent) effects, of which the stimulation of the cardioprotective vasodilator, nitric oxide (NO), is particularly relevant. In this study, the NO generating properties of fenofibrate were investigated in cardiac microvascular endothelial cells (CMECs), with particular focus on the underlying role of NO synthase (NOS) isoforms. Methods: CMECs were treated with 50μM fenofibrate for 1 and 24h. Intracellular NO levels were measured by mean diaminofluorescein diacetate (DAF-2/DA) fluorescence intensity (FACS analysis) and nitrite levels by the Griess-method (platereader analysis). The putative involvement of NOS isoforms was investigated by administering the pharmacological inhibitors, L-NMMA (100μM; selective for eNOS/nNOS) or 1400W (80μM; selective for iNOS). Relevant signalling proteins were investigated by means of Western blotting. Results: DAF-2/DA measurements showed that fenofibrate increased NO-production significantly after 1 hour (Control 100%: Feno: 174.7%±11.03;p

Aims: It is well established that remote ischemic perconditioning (RIPerc) effectively protects from myocardial ischemia-reperfusion (IR) injury. The signaling events underlying the cardioprotective effect seem to involve nitric oxide (NO) but the exact mechanism remains unclear. We have recently demonstrated that IR injury is critically dependent on increased arginase activity resulting in reduced bioavailability of NO. The objective of this study was to investigate whether (1) oxidative/nitrosative stress-mediated RhoA/Rho kinase (ROCK) signaling pathway contributes to arginase upregulation following IR and (2) the inhibition of this pathway is involved as a cardioprotective mechanism of RIPerc. Methods: Anesthetized Sprague-Dawley rats subjected to 30 min left coronary artery ligation and 2 h reperfusion were randomized to: 1) no intervention (controls, n=10), 2) RIPerc induced by bilateral femoral artery occlusion during the last 15 min of myocardial ischemia (n=10), 3) RIPerc and administration of the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA; n=6), 4) administration of the ROCK inhibitor hydroxyfasudil (20 min prior to ischemia iv; n=7), or 5) administration of the peroxynitrite decomposition catalyst FeTPPS (10 min prior to reperfusion iv; n=6). Results: Infarct size (as a percentage of the area at risk) was significantly smaller in animals subjected to RIPerc (42±4%) compared to the controls (69±2%; P

Aging is a cardiovascular risk factor associated to an increase of reactive oxygen species (ROS) and Renin-Angiotensin System (RAS) activation, which in turn contributes to vascular dysfunction and remodelling. Both systems are known to be influenced by sex. In this regard, our study aims to determine whether sex-associated differences in ROS and RAS contribute to a differential regulation of vascular aging and associated dysfunction. Male and female outbreed CD-1 mouse were studied at 3, 7 and 12 months of age (mo). To determine the contribution of RAS, mice were treated from 3 to 12 months with Losartan (0.6 mg/L in the drinking water). Contraction to KCl and Angiotensin II (AngII) were determined in aortic rings by wire-myography. Morphometric analysis was performed by Haematoxylin-eosin staining. Oxidative stress was determined in aortic cross sections by dihydroethidiem (DHE). Sections were treated with AngII (10-6M), apocynin (10-4M), L-NMMA (3x10-4M) to determine signalling pathways involved in ROS production. At 12 mo, contractions to 60mM KCl were higher in males (15.4±0.4 mN) than in females (12.6±0.5 mN); P

OBJECTIVE: : In sinoatrial (SA) node cells, nitric oxide (NO) exerts a dual effect on the hyperpolarization-activated current, I(f), i.e. in basal conditions NO enhances I(f) whereas in the presence of beta-adrenergic stimulation it decreases it. Recent studies have shown that I(f) is present in ventricular myocytes from hypertrophied or failing hearts where it may promote abnormal automaticity. Since these pathological conditions are associated with increased sympathetic tone and upregulation of myocardial NO production, we set out to investigate whether I(f) is similarly modulated by NO in hypertrophied ventricular myocytes. METHODS: Left ventricular myocytes were isolated from 18-20-month-old spontaneously hypertensive rats (SHRs). Membrane current was measured under whole-cell or amphotericin-perforated patch-clamp conditions, at 35 degrees C. RESULTS: Application of diethylamine-NO (DEA-NO, 1-100 microM) did not alter the amplitude or voltage dependence of activation of I(f) under basal conditions (half-activation voltage, V(h): control -82.9+/-2.6, DEA-NO -84.0+/-2.6 mV). Similarly, I(f) was not affected by the inhibition of endogenous NO production (L-NMMA, 500 microM) or guanylate cyclase (ODQ, 10 microM). Forskolin (10 microM) or isoprenaline (100 nM) elicited a positive shift in V(h) but subsequent application of DEA-NO did not further affect the properties of I(f). CONCLUSIONS: Our results show that, unlike in SA node cells, in SHR ventricular myocytes basal and adrenergically stimulated I(f) is not modulated by exogenous NO or by constitutive NO or cGMP production.

Objective: We have recently shown that tumor necrosis factor-a (TNFa) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CMs). The aim of the present research was to investigate the pathways involved in this mitogenic response. Methods: CMs were isolated from 10-day-old chick embryos and grown to confluence. After 20 h of serum starvation the cells were treated with TNFa and LPS, and / or specific agonists and antagonists to manipulate the levels of polyamines, NO, cGMP and their biosynthetic enzymes ornithine decarboxylase (ODC), nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC). ODC, NOS, sGC activities and cGMP contents were determined by radiochemical procedures. DNA synthesis was determined by incorporation of 3 3 ( H)-thymidine. Results: Treatment of CMs with TNFa and LPS increased cell number and ( H)-thymidine incorporation. Addition of TNFa and LPS provoked an induction of ODC, with consequent polyamine accumulation, and a more delayed enhancement of NOS activity, which appeared to be independent of the activation of the ODC-polyamine system. TNFa and LPS treatment also enhanced cGMP level in CMs and both polyamine and NO biosyntheses appeared to be required. Experiments with specific inhibitors of ODC and NOS, as well as with inhibitors of sGC and cGMP-dependent protein kinase (PKG), showed that polyamine-, NO- and cGMP-dependent pathways are required for the mitogenic action of TNFa and LPS. Moreover, addition of exogenous polyamines to untreated cells raised 3 the cGMP level in a NO-dependent fashion, and enhanced ( H)-thymidine incorporation. The latter effect was inhibited by sGC or PKG inhibitors. Treatment of quiescent cells with NO donors, 8-bromo-cGMP or YC-1, an sGC activator, also promoted DNA synthesis. Furthermore, putrescine and NO donor can additively activate sGC in cell-free extracts. Conclusion: TNFa and LPS stimulate DNA synthesis in chick embryo CMs and this effect is mediated by polyamines, NO and intracellular cGMP. © 2001 Elsevier Science B.V. All rights reserved.

ATP-sensitive potassium channels (K+ATP) prominently contribute to basal coronary tone; however, flow reserve during exercise remains unchanged despite channel blockade with glibenclamide (GLI). We hypothesized that increasing perfusion pulsatility, as accompanies exercise, offsets vasoconstriction from K+ATP-channel blockade, and that this effect is blunted by nitric oxide synthase (NOS) inhibition.In 31 anaesthetized dogs the left anterior descending artery was blood-perfused by computer-controlled servo-pump, with real-time arterial perfusion pulse pressure (PP) varied from 40 and 100 mm Hg at a constant mean pressure and cardiac workload.At control PP (40 mm Hg), GLI (50 micrograms/min/kg, i.c.) lowered mean regional coronary flow from 37 +/- 5 to 25 +/- 4 ml/min (P0.001). However, this was not observed at 100 mm Hg PP (41 +/- 2 vs. 45 +/- 4). NOS inhibition by NG-monomethyl-L-arginine (L-NMMA) did not alter basal flow at 40 mm Hg PP, but modestly lowered flow (-5%, P0.001) at higher PP (100 mm Hg), reducing PP-flow augmentation by -36%, and acetylcholine (ACh) induced flow elevation by -39%. Co-infusion of L-NMMA with GLI resulted in net vasoconstriction at both PP levels (-60% and -40% at 40 and 100 mm Hg PP, respectively). Unlike GLI, vasoconstriction by vasopressin (-43 +/- 3% flow reduction at 40 mm Hg PP) or quinacrine (-23 +/- 7%) was not offset at higher pulsatility (-44 +/- 4 and -23 +/- 6%, respectively). Neither of the latter agents inhibited ACh- or PP-induced flow responses, nor did they modify the effect of L-NMMA on these responses.Increased coronary flow pulsatility offsets vasoconstriction from K+ATP blockade by likely enhancing NO release. This mechanism may assist exercise-mediated dilation in settings where K+ATP opening is partially compromised.

We have recently shown that exogenous nitric oxide (NO) elicits a positive chronotropic response by stimulating the hyperpolarization activated current, I(f).To examine whether L-arginine (L-Arg) can mimic the chronotropic effect of NO by enhancing its endogenous production.In spontaneously beating guinea pig atria we evaluated the heart rate (HR) response to increasing concentrations of L-Arg (1 mumol/l to 10 mmol/l), and compared it with that for D-Arg or L-lysine (L-Lys) (all in free base (FB) or hydrochloride (HCl) formulation).L-ArgFB100 mumol/l caused a reversible dose-dependent increase in HR (peak effect +64 +/- 7 bpm at 10 mmol/l, P0.05, n = 8). However, a similar HR response occurred with D-ArgFB (n = 7) or L-LysFB (n = 6). All FB formulations increased the perfusate pH (peak [pH]o = 8.61 +/- 0.03). Although alkalinization can stimulate NO release from the endothelium, this is unlikely to have contributed to HR changes in our preparation, since neither NG-methyl-L-arginine, (100-500 mumol/l, which per se reduced HR by 8 +/- 1%, P0.05, n = 9) nor NO scavenging (fresh 5% red blood cells, n = 9) caused a rightward shift of the concentration-response curve to L-ArgFB. Furthermore, as opposed to FB formulations, L-ArgHCl, D-ArgHCl or L-LysHCl1 mmol/l significantly decreased HR and [pH]o (n = 17). The chronotropic effects of L-ArFB or L-ArgHCl were reproduced by changing [pH]o with NaOH (n = 8) or HCl (n = 7), whereas the HR increase with L-ArgFB was prevented by clamping [pH]o at 7.42 +/- 0.07 (n = 10).In vitro, L-Arg can markedly affect HR through a pH-mediated, NO-independent mechanism. Our data show that the opposing changes in [pH]o induced by different formulations of L-Arg can importantly confound the assessment of the biological effects of this amino acid.

Objective: The aim of the present study was the investigation of the expression of NOS during cardiomyogenesis and its functional role. Design: The qualitative and quantitative expression of NOS isoforms during different stages of cardiac development was evaluated using immunocytochemistry and dot blots, respectively. The functional relevance of NOS expression during cardiomyogenesis was investigated using the in vitro ES cell-differentiation model and selective pharmacological agents. Results: On day 7.5 of embryonic development (E7.5) none of the NOS isoforms were expressed in the embryo, whereas the inducible (iNOS), as well as the endothelial (eNOS) isoforms were detected in the extraembryonic parts. In contrast, starting from E9.5 rat and murine embryos displayed prominent iNOS and eNOS expression. This was correlated with high expression of soluble guanylylcyclase (sGC) as well as high cyclic GMP (cGMP) content. During further development after E14.5 both, iNOS as well as eNOS, started to be downregulated and shortly prior to birth reduced staining for eNOS was found, whereas iNOS was hardly detectable. We further investigated whether NO plays a role for cardiomyogenesis, using in vitro ES cell-derived cardiomyocytes differentiating within embryoid bodies (EBs). The NOS expression pattern in these cells paralleled the one detected in vivo. We demonstrate that continuous incubation of EBs with the NOS inhibitors l-NMMA (2–10 mM) or l-NA (2–10 mM) for 4 to 9 days after plating resulted in a pronounced differentiation arrest of cardiomyocytes, whereas this effect could be reversed by coapplication of the NO-donor spermine-NONOate (10 μM). Conclusions: Both, iNOS and eNOS isoforms are prominently expressed during early stages of cardiomyogenesis. Around E14.5 NOS expression starts to decline. Moreover, the NO-generation is required for cardiomyogenesis since NOS inhibitors prevent the maturation of terminally differentiated cardiomyocytes using the ES cell system.

Hypercholesterolemia has been shown to impair endothelium-mediated, nitric oxide (NO)-dependent responses to acetylcholine (ACh), serotonin, substance P and flow-mediated dilation. We have recently shown that NO contributes to metabolic vasodilation in the human forearm. We sought to determine whether metabolic vasodilation is impaired in healthy subjects with hypercholesterolemia.We compared the forearm blood flow (FBF) responses to isotonic exercise, ACh and the endothelium-independent vasodilator sodium nitroprusside in young, otherwise healthy volunteers with hypercholesterolemia and controls before and after the NO inhibitor NG-monomethyl-L-arginine (L-NMMA). FBF was measured using venous occlusion plethysmography. Hypercholesterolemic (n = 20) and control (n = 20) subjects were age- and gender-matched.Total cholesterol (6.9 +/- 0.3 vs. 4.6 +/- 0.1 mmol/l, P0.0001), low density lipoprotein (4.9 +/- 0.4 vs. 2.7 +/- 0.1 mmol/l, P0.001) and triglyceride (1.3 +/- 0.2 vs. 0.8 +/- 0.1 mmol/l, P = 0.005) levels were higher in the hypercholesterolemic group. Basal FBF and resistance were similar in the two groups. Hypercholesterolemia impaired the peak FBF response to ACh (11.1 +/- 1.9 vs. 17.6 +/- 2.2 ml/100 ml/min, P = 0.03), and reduced the peak response to sodium nitroprusside (6.0 +/- 0.4 vs. 8.1 +/- 0.6 ml/100 ml/min, P0.01). However, hypercholesterolemia did not affect peak hyperemic FBF (13.1 +/- 1.0 vs. 13.2 +/- 1.0 ml/100 ml/min, P = 1.0) or the FBF volume repayment during the 1 or 5 min after exercise. Resting FBF was reduced by L-NMMA to a similar degree (by 33% vs. 40%, P = 0.17) in both groups. Although L-NMMA reduced peak hyperemic FBF (by 16% vs. 17%, P = 0.93) and the volume repaid after exercise in both groups, there were no differences between the two groups.Exercise-induced metabolic vasodilation is in part dependent on NO release. Hypercholesterolemia impairs NO-mediated vasodilation, but is not associated with a reduction in exercise-induced hyperemia. This may indicate that multiple compensatory mechanisms are operative in skeletal muscle metabolic vasodilation.

Objective: A reduced bioactivity of endothelial nitric oxide (NO) has been implicated in the pathogenesis of atherosclerosis. In humans, the endothelial l-arginine–NO pathway has been indirectly assessed via the flow response to endothelium-dependent vasodilators locally administered into the coronary, pulmonary or forearm circulation. However, biochemical quantification of endothelial NO formation in these organ circulations has been hampered so far because of the rapid metabolism of NO. Therefore, we aimed to work out a reliable biochemical index to assess endothelial NO formation in human circulation. Methods: In 33 healthy volunteers, forearm blood flow (FBF) was measured by standard techniques of venous occlusion plethysmography at rest, after local application of the endothelium-dependent vasodilator acetylcholine (ACH), the endothelium-independent vasodilator papaverine (PAP), the stereospecific inhibitor of endothelial NO synthase (eNOS) L-NMMA, and l-arginine (ARG), the natural substrate of eNOS. In parallel, nitrite and nitrate concentrations in blood samples taken from the antecubital vein were measured by HPLC using anion-exchange chromatography in combination with electrochemical and ultraviolet detection following a specific sample preparation method. Results: ACH dose-dependently increased resting FBF (from 3.0±0.3 to 10.4±0.9 ml/min per 100 ml tissue) and serum nitrite concentration (from 402±59 to 977±82 nmol/l, both p

Objective: Nitric oxide (NO) has been implicated in the depression of cardiac function in human heart failure. Some reports have identified iNOS (inducible nitric oxide synthase) within the myocyte component of the failing human heart, and NO is known to decrease the contraction amplitude of isolated ventricular myocytes. We have treated myocytes from failing human ventricle with a NOS inhibitor, N G-monomethyl-l-arginine (L-NMMA), in an attempt to restore contractile function. Methods and Results : Myocytes were isolated from failing and non-failing human ventricles and their contraction amplitude was measured during superfusion (32°C, 1–2 mmol/l Ca2+) and electrical stimulation (0.1–2 Hz). The contraction amplitude of myocytes from failing hearts was depressed in a frequency-dependent manner. At 1 Hz, the contraction amplitude of cells from non-failing heart was 4.70±0.53% cell shortening (mean±SEM, n =13 subjects), compared with 2.18±0.27% ( P

OBJECTIVE We have attempted to demonstrate the induction of inducible nitric oxide synthase in human vascular tissue and define the capacity of different cytokines to induce this enzyme. METHODS Segments of human arteries were stimulated with lipopolysaccharide (10 micrograms/ml), interleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (10 U/ml), and interferon-gamma (200 U/ml). Cytokines were either used alone or in certain combinations, as well as in the presence of L-NG-monomethyl-arginine (100 mumol/l) or cycloheximide (1 mumol/l). Induction was assessed by measurement of mRNA expression, immunocytochemical localisation of the expressed protein, nitric oxide synthase activity and levels of nitrite, a product of nitric oxide formation. RESULTS PCR analysis showed the presence of mRNA for iNOS in stimulated samples which could be inhibited by cycloheximide. There was positive staining with an antibody against human iNOS in the media of stimulated vessel segments. Stimulated segments were also shown to contain Ca(2+)-independent nitric oxide synthase activity. The cytokines and lipopolysaccharide together gave a significant rise in levels of nitrite in the medium after 36 and 48 h, which was inhibited by L-NG-monomethyl-arginine and cycloheximide. Only interferon-gamma incubated alone was capable of increasing nitrite levels. This effect was enhanced by co-incubation with either interleukin-1 beta, tumor necrosis factor-alpha or lipopolysaccharide. CONCLUSION We have shown that increased production of nitrite by human vascular tissue in response to cytokines is associated with induction of iNOS as shown at the molecular and protein levels, and further supported by the presence of increased Ca(2+)-independent nitric oxide synthase activity following cytokine stimulation.

Objective : The purpose of this study was to investigate the effects of vesnarinone on nitric oxide (NO) synthesis in cardiac myocytes. Methods : We measured the accumulation of nitrite, a stable oxidation product of NO, and the expression of inducible NO synthase (iNOS) protein in cultured neonatal rat cardiac myocytes. Results : Incubation of the cultures with interleukin-1β (IL-1β; 10 ng/ml) and tumor necrosis factor α (TNFα; 10 ng/ml) caused a marked increase in nitrite production. Although vesnarinone by itself showed no effect on nitrite accumulation, it enhanced cytokine-induced nitrite production by cardiac myocytes in a dose-dependent manner. The effect of vesnarinone was completely abolished in the presence of NG-monomethyl-l-arginine or actinomycin D. The vesnarinone-induced nitrite production was accompanied by increased iNOS protein expression. In the presence of dibutyryl-cAMP, cytokine-induced nitrite accumulation was further increased, but the stimulatory effect of vesnarinone on nitrite accumulation was diminished. The effect of vesnarinone was also inhibited by Rp-8-Br-cAMPS, a competitive inhibitor of protein kinase A, in a dose-dependent manner. Conclusions : These findings indicate that vesnarinone increases NO synthesis in cytokine-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.

Objective: The aims of the study were to establish the roles of angiotensin II and of the cardiopulmonary baroreceptor reflex in the regulation of peripheral vascular tone in patients with cirrhosis. Methods: Forearm blood flow responses to subsystemic, locally active intrabrachial infusions were measured in patients with Child's Grade C cirrhosis and matched controls using bilateral venous occlusion plethysmography. Responses were determined to the angiotensin II type I receptor antagonist, losartan, noradrenaline, angiotensin II and the nitric oxide synthase inhibitor, l- NG -monomethyl arginine. Results: Losartan at 30 and 90 μg/min caused no significant change in blood flow in controls, but caused 23±6% and 27±5% increases in patients respectively ( p

Objective : The concept of therapeutic angiogenesis with vascular endothelial growth factor (VEGF) has been validated in peripheral arterial disease. Its use in myocardial ischemia may be delayed as the result of the description in a porcine model of peripheral vasodilation after intraluminal injections of VEGF resulting in a 50% fatality rate by hypotension. We carried out this study to test whether VEGF-induced hypotension (1) is species specific, (2) is mediated by the receptor mediating angiogenesis, (3) is prevented by inhibition of nitric oxide synthase. Methods : In the rabbit corneal pocket assay we tested whether a previously published anti-idiotypic antibody (AIA) agonist of the VEGF receptor Flk-1/KDR could elicit angiogenesis. Various doses of recombinant VEGF or AIA were injected into anesthetized normotensive Wistar–Kyoto rats and the mean arterial blood pressure (MABP) was recorded. To test the implication of nitric oxide in VEGF-induced hypotension we treated the animals with a competitive inhibitor of nitric oxide synthase prior to the injection of VEGF. Results : Both VEGF and AIA induce angiogenesis but only intravenous injections of VEGF induced a rapid, transient and dose-dependent decrease in MABP. The ED50 was 0.5 μg. The interval between two VEGF injections required to lead to a decrease of MABP was 40 minutes. Nitric oxide synthesis inhibitor prevented, in a reversible fashion, the effect of VEGF. Conclusion : VEGF-induced hypotension is not species specific. It is prevented by nitric oxide inhibition. VEGF-induced angiogenesis and hypotension are not mediated in vivo by the same VEGF receptor

Objectives: Direct gene transfer of exogenous nitric oxide synthase, with the subsequent increase in nitric oxide production, could represent a potential therapeutic strategy in the treatment of vascular proliferative disorders. The goal of the present study was to determine if porcine coronary arteries could be transduced with an adenoviral vector encoding endothelial nitric oxide synthase (Ad.CMVeNOS) resulting in functional expression. Methods and Results: Segments of porcine right coronary artery were exposed for 1 h at 37°C to either replication-deficient adenovirus encoding bovine endothelial nitric oxide synthase (Ad.CMVeNOS, 5×109 pfu/ml) or control adenovirus encoding Escherichia coli β-galactosidase (Ad.CMVLacZ, 5×109 pfu/ml). Immunohistochemistry with a monoclonal antibody specific for nitric oxide synthase (NOS) demonstrated recombinant gene expression in both the endothelial and adventitial layers of Ad.CMVeNOS transduced coronaries with only endogenous NOS confirmed in the endothelium of Ad.CMVLacZ arteries. Coronary arteries transduced with Ad.CMVeNOS yielded 517±110 (mean±S.E.M.) nM/ng nitrite while vessels transduced with Ad.CMVLacZ yielded 126±84 nM/ng ( P

The detrimental haemodynamic changes observed in septicaemia are generalised vasodilation, arterial hypotension, and hyporesponsiveness to vasopressor compound, all of which could be explained by the release of an endogenous vasodilator. Experimental and clinical evidence suggests that tumour necrosis factor-alpha (TNF) induces the expression of vascular nitric oxide (NO) synthase within hours and that NO released from smooth muscle cells could be involved in the pathogenesis of septic shock. The aim of this study was to investigate the role of NO in the vascular effects of TNF.Using the dorsal hand vein compliance technique, the effect of the NO synthase inhibitor L-NG-monomethyl-arginine (L-NMMA) on alpha 1-adrenergic responsiveness (phenylephrine 1.25-8000 ng/min) was studied after prolonged local venous infusion of TNF (8.7 micrograms in 5 h) in 9 volunteers and in 6 volunteers without previous cytokine exposure.Mean (+/- s.e.) maximum phenylephrine constriction (Emax) was 73 +/- 6% and log dose-rates exerting 50% of Emax (log ED50) were 3.2 +/- 0.09 (geometric mean: 1535 ng/min). Local co-administration of L-NMMA at a dose sufficiently high to block NO formation (3.4 mumol/min) increased venous sensitivity to phenylephrine threefold (log ED50 2.8 +/- 0.1, P0.015; geometric mean: 574 ng/min) whereas Emax was similar (73 +/- 5%). In the controls the phenylephrine dose-response relationship remained unaffected by simultaneous administration of L-NMMA.As no basal release of NO occurs in hand veins without previous exposure to TNF these results provide direct evidence for induction of NO formation in the human vasculature and consecutive resistance to alpha-adrenergic venoconstriction. NO might, therefore, be a key mediator of haemodynamic impairment in humans under conditions with known elevations of circulating TNF, such as a septic shock.

Nitric oxide (NO) exerts both protective and detrimental actions in a variety of biological systems. During acute reperfusion following myocardial ischaemia, a rapid overproduction of free radicals, including NO, may occur. We investigated the effects of the NO synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), and the substrate for NO synthesis, L-arginine, on heart function during ischaemia and reperfusion injury.Spontaneously beating, isolated working rabbit hearts, perfused with modified Krebs-Henseleit buffer containing 1.2 mM palmitate bound to 3% bovine serum albumin, were subjected to 15 min of aerobic perfusion followed by 35 min of global, no-flow ischaemia and 30 min of aerobic reperfusion.Throughout the reperfusion period there was a marked impairment in the recovery of mechanical function, measured as the product of heart rate x peak systolic pressure (rate-pressure product). Addition of L-NAME (3 microM) prior to the onset of ischaemia, but not at reperfusion, caused an immediate and significant increase in the recovery of mechanical function throughout the reperfusion period. The protective action of L-NAME was abolished by L- (but not D-) arginine (100 microM). L-NAME did not cause ischaemia as it did not alter glycogen or lactate content of aerobically perfused hearts. Furthermore, it did not prevent glycogen loss or lactate accumulation during 35 min of ischaemia, suggesting that the effects of L-NAME were not due to metabolic alterations during ischaemia itself. L-NMMA (30 microM) added prior to ischaemia, but not at reperfusion, also had a protective effect which was seen later in the reperfusion period. Addition of L- (but not D-) arginine (100 microM) prior to the onset of ischaemia resulted in an improved recovery of mechanical function only at 15 min of reperfusion.These results suggest that: (1) the recovery of mechanical function of hearts subjected to ischaemia-reperfusion injury can be improved by modulation of myocardial NO synthesis, (2) inhibition of NO synthesis (with L-NAME or L-NMMA) may offer prolonged protection whereas its stimulation (with L-arginine) provides only brief protection, and (3) the reasons for the pharmacological effectiveness of these divergent strategies may be due to the formation of peroxynitrite from NO and superoxide anion during reperfusion.

To evaluate the effect of L-arginine, the physiological substrate of nitric oxide (NO), upon coronary flow (CF) and mechanical function during reperfusion following cardioplegic arrest.Two groups of isolated rat hearts were subjected to cardioplegic arrest for 4 h at 4 degrees C. In group 1 (n = 10) cardioplegic arrest was followed by 4 consecutive periods of reperfusion with Krebs buffer (control), Krebs plus L-lysine (1 mmol/1), Krebs plus L-arginine (1 mmol/1) and Krebs plus L-NGmonomethylarginine (L-NMMA), a specific inhibitor of NO synthesis, (0.5 mmol/1). In group 2, hearts (n = 8) were perfused by Krebs, then L-NMMA, during both pre- and postischaemic periods. In group 3, hearts (n = 8) were perfused by Krebs, then L-arginine (1, 2 and 4 mmol/1). In group 4 (n = 5), NO released into the perfusate was measured before ischaemia and during reperfusion.In group 1, L-arginine enhanced the postischaemic CF (ml/min +/- s.e.m.) from 15.0 +/- 0.4 to 17.2 +/-0.4. This was reduced by L-NMMA to 11.3 +/- 0.3. Postischaemic cardiac output (% of preischaemic value +/- s.e.m.) was increased from 55.8 +/- 2.4 to 80.1 +/- 2.5 by L-arginine and dropped to 54.3 +/- 2.3 with L-NMMA. In group 2, the pre- and postischaemic loss of coronary flow (CF) by L-NMMA was 51% and 31% respectively. In group 3, L-arginine did not modify CF. In group 4 the preischaemic level of NO (in nmol/ml/min) in the coronary effluent, measured by chemiluminescence, was 14.84 +/- 0.83 and dropped significantly (P0.05) to levels ranging from 3.80 +/- 0.56 to 4.75 +/- 0.51 during the postischaemic period.Exogenous administration of L-arginine improves low coronary reflow and postischaemic mechanical function.

The aim was to examine the role of nitric oxide (NO) in the myocardial microcirculation.Open chest anaesthetised dogs received intracoronary infusion of NG-monomethyl-L-arginine (L-NMMA; 0.5 mg.kg-1 over 30 min). Myocardial microvascular extraction fraction of a small hydrophilic solute (technetium 99m labelled diethylenetriaminepenta-acetate [99mTc-DTPA]), and the regional myocardial plasma flow rate, were determined by the single injection, residue detection method, and the capillary permeability-surface area product was calculated. The tone in intramyocardial resistance vessels was assessed by the local 133Xe washout method during baseline conditions, in response to intracoronary acetylcholine, and during peak reactive hyperaemia after 10 s or 30 s of myocardial ischaemia, respectively.In eight open chest dogs, L-NMMA attenuated the increase in myocardial plasma flow rate in response to intracoronary acetylcholine by 32(SEM 7)%, but failed to alter baseline myocardial plasma flow rate significantly, as determined by the local 133Xe washout method. L-NMMA did not influence myocardial microvascular permeability to 99mTc-DTPA. However, intracoronary L-NMMA decreased the peak reactive hyperaemic myocardial plasma flow rate after 10 s, but not 30 s, of coronary occlusion.In open chest dogs, microvascular NO synthesis is not a major determinant of baseline myocardial plasma flow rate, and does not appear to influence myocardial microvascular permeability significantly. In this model, NO intervenes in the regulation of the peak reactive hyperaemic plasma flow rate following brief, but not more prolonged, periods of coronary occlusion.

Objective: Studies in animals have indicated that increased production of nitric oxide from an inducible isoform of nitric oxide synthase contributes to the pathophysiology of endotoxic and cytokine induced shock. The aim of this study was to determine the role of nitric oxide in septic shock in humans. Methods: The study was a randomised, double blind, placebo controlled investigation of the effects of the nitric oxide synthase inhibitor NG monomefhyl-L-arginine (L-NMMA) in 12 patients with severe sepsis associated with hypotension. Measurements of haemodynamic, haematologieal, and biochemical variables were made. Results: L-NMMA (0.3 and 1 mg·kg−1) produced a dose dependent increase in mean arterial pressure, systemic vascular resistance, pulmonary vascular resistance, central venous pressure, and pulmonary artery occlusion pressure, and a decrease in cardiac output and heart rate. In response to the highest dose of L-NMMA systemic vascular resistance increased from 547(SEM 92) to 889(143) dyne·s·cm−5, mean arterial blood pressure increased from 80.9(2.9) to 100.5(6.1) mm Hg, and cardiac output decreased from 11.2(2.1) to 8.9(1.9) litres·min−1. Continuous infusion of L-NMMA (1 mg·kg−1·h−1) produced sustained haemodynamic changes. Platelet numbers decreased during the course of the study in both the L-NMMA and the placebo group and did not differ significantly between the groups. Conclusions: Low doses of L-NMMA cause a widespread increase in vascular tone and raise blood pressure in patients with septic shock. Overproduction of nitric oxide appears to be an important mechanism in septic shock in patients, and inhibition of nitric oxide synthesis might provide a novel therapeutic approach to this condition. However, L-NMMA produced a fall in cardiac output and it is possible that this would worsen tissue perfusion. Larger studies examining the effects of L-NMMA on mortality and morbidity are now required. Cardiovascular Research 1994; 28 :34-39