Differential regulation of inducible and endothelial nitric oxide synthase by kinin B1 and B2 receptors.

Kinins are vasoactive peptides that play important roles in cardiovascular homeostasis, pain and inflammation. After release from their precursor kininogens, kinins or their C-terminal des-Arg metabolites activate two distinct G protein-coupled receptors (GPCR), called B2 (B2R) or B1 (B1R). The B2R is expressed constitutively with a wide tissue distribution. In contrast, the B1R is not expressed under normal conditions but is upregulated by tissue insult or inflammatory mediators. The B2R is considered to mediate many of the acute effects of kinins while the B1R is more responsible for chronic responses in inflammation. Both receptors can couple to Galphai and Galphaq families of G proteins to release mediators such as nitric oxide (NO), arachidonic acid, prostaglandins, leukotrienes and endothelium-derived hyperpolarizing factor and can induce the release of other inflammatory agents. The focus of this review is on the different transduction events that take place upon B2R and B1R activation in human endothelial cells that leads to generation of NO via activation of different NOS isoforms. Importantly, B2R-mediated eNOS activation leads to a transient ( approximately 5min) output of NO in control endothelial cells whereas in cytokine-treated endothelial cells, B1R activation leads to very high and prolonged ( approximately 90min) NO production that is mediated by a novel signal transduction pathway leading to post-translational activation of iNOS.

Nitric oxide measurements during endotoxemia.

BACKGROUND: Excessive continuous NO release from inducible NO synthase over prolonged periods under pathological conditions, such as endotoxemia, contributes significantly to circulatory failure, hypotension, and septic shock. This NO production during endotoxemia is accompanied by superoxide release, which contributes to the fast decay of NO. Therefore, the amount of NO that diffuses to target sites may be much lower than the total amount released under pathological conditions. METHODS: We performed in vivo and ex vivo measurements of NO (electrochemical) and ex vivo in situ measurements of superoxide, peroxynitrite (chemiluminescence), and nitrite and nitrate (ultraviolet-visible spectroscopy). We determined the effect of lipopolysaccharide administration (20 mg/kg) on diffusible NO, total NO (diffusible plus consumed in chemical reactions), and superoxide and peroxynitrite release in the pulmonary arteries of rats. RESULTS: An increase in diffusible NO generated by constitutive NO synthase was observed immediately after administration of lipopolysaccharide, reaching a plateau (145 +/- 18 nmol/L) after 540 +/- 25 s. The plateau was followed by a decrease in NO concentration and its subsequent gradual increase after 45 min because of NO production by inducible NO synthase. The concentration of superoxide increased from 16 +/- 2 nmol/L to 30 +/- 3 nmol/L after 1 h and reached a plateau of 41 +/- 4 nmol/L after 6 h. In contrast to the periodic changes in the concentration of diffusible NO, the total concentration of NO measured as a sum of nitrite and nitrate increased steadily during the entire period of endotoxemia, from 2.8 +/- 0.2 micromol/L to 10 +/- 1.8 micromol/L. CONCLUSIONS: The direct measurement of NO concentrations in the rat pulmonary artery demonstrates dynamic changes throughout endotoxemia, which are related to the production of superoxide and the subsequent increase in peroxynitrite. Monitoring endotoxemia with total nitrate plus nitrite is not sensitive to these fluctuations in NO concentration.

Prostaglandin E1 reduces ischemia/reperfusion injury by normalizing nitric oxide and superoxide release.

To test the effects of prostaglandin E1 on 2.5 h of ischemia followed by 2 h of reperfusion, continuous nitric oxide measurements (electrochemical) were correlated with intermittent assays of superoxide and peroxynitrite levels (chemiluminescence) and ischemia/reperfusion injury in rabbit adductor magnus muscle. Administering prostaglandin E1 (1 microg/kg) before or during ischemia/reperfusion caused normalization of the release of nitric oxide, superoxide, and peroxynitrite to slightly above preischemic levels. This pattern was dramatically different from that observed during ischemia/reperfusion alone, where nitric oxide concentration increased three times above its basal level. Normalization of constitutive nitric oxide synthase activity in the presence of prostaglandin E1 was associated with a significant reduction of superoxide and peroxynitrite production and subsequent reduction of ischemia/reperfusion injury. At 2 h of reperfusion, vasoconstriction associated with ischemia/reperfusion injury was eliminated, and edema was significantly mollified but still apparent. Prostaglandin E1 treatment does not directly inhibit constitutive nitric oxide synthase, like the inhibitor N(omega)-monomethyl-L-arginine. Some phenomenon associated with ischemia turns on endothelial constitutive nitric oxide synthase to start transforming L-arginine and oxygen into nitric oxide, but prostaglandin E1 seems to inhibit this phenomenon. Thus, essential local L-arginine pools are not depleted, and normal basal levels of essential nitric oxide are maintained, whereas cytotoxic superoxide and peroxynitrite production by L-arginine-deficient constitutive nitric oxide synthase is prevented.

Nitric oxide deficiency contributes to large cerebral infarct size.

The purpose of this study was to examine the role played by a deficit in nitric oxide (NO) in contributing to the large cerebral infarcts seen in hypertension. Cerebral infarction was produced in rats by occlusion of the middle cerebral artery (MCA). Studies were performed in Sprague-Dawley (SD) rats subjected to NO synthase blockade (N(G)-nitro-L-arginine [L-NNA], 20 mg x kg(-1) x d(-1) in drinking water) and in spontaneously hypertensive stroke-prone rats (SHRSP). NO released in the brain in response to MCA occlusion was monitored with a porphyrinic microsensor in Wistar-Kyoto rats. The increment in NO released with MCA occlusion was 1.31+/-0.05 micromol/L in L-NNA-treated rats, 1.25+/-0.04 micromol/L in SHRSP, 2. 24+/-0.07 micromol/L in control SD rats, and 2.25+/-0.06 micromol/L in Wistar-Kyoto rats (P<0.0001 for control versus the other groups). Infarct sizes in the L-NNA-treated and control SD rats were 8.50+/-0. 8% and 5.22+/-0.7% of the brain weights, respectively (P<0.05). The basilar arterial wall was significantly thicker in L-NNA-treated rats compared with their controls. We conclude that both the deficit in NO and the greater wall thickness contribute to the larger infarct size resulting from MCA occlusion in SHRSP and in L-NNA-treated rats compared with their respective controls.

cAMP pulse during preservation inhibits the late development of cardiac isograft and allograft vasculopathy.

The causes of transplant-associated coronary artery disease remain obscure, and there is no known treatment. Preservation injury of murine heterotopic vascularized cardiac isografts caused a small, albeit significant, increase in neointimal formation; preservation injury of allografts markedly increased both the incidence and severity of transplant-associated coronary artery disease. As cAMP is an important vascular homeostatic mediator the levels of which decline during organ preservation, buttressing cAMP levels solely during initial preservation both improved acute allograft function and reduced the severity of transplant-associated coronary artery disease in grafts examined 2 months later. Inhibiting the cAMP-dependent protein kinase abrogated these beneficial effects. cAMP treatment was associated with an early reduction in leukocyte infiltration and a reciprocal decrease in superoxide and increase in NO levels. These data indicate that alloantigen-independent injury to the graft, which occurs at the time of cardiac preservation, can set in motion pathological vascular events that are manifest months later. Furthermore, a cAMP pulse during cardiac preservation reduces the incidence and severity of transplant-associated coronary artery disease.

Effect of native and oxidized low-density lipoprotein on endothelial nitric oxide and superoxide production : key role of L-arginine availability.

BACKGROUND: Native and oxidized LDLs (n-LDL and ox-LDL) are involved in the atherogenic process and affect endothelium-dependent vascular tone through their interaction with nitric oxide (NO). METHODS AND RESULTS: In this study we evaluated directly, by using a porphyrinic microsensor, the effect of increasing lipoprotein concentrations on endothelial NO and superoxide (O(2)(-)) production. We investigated where lipoproteins may affect the L-arginine-NO pathway by pretreating cells with L-arginine, L-N-arginine methyl ester (L-NAME), and superoxide dismutase. Bovine aortic endothelial cells were exposed for 1 hour to increasing concentrations of n-LDL (from 0 to 240 mg cholesterol/dL) and ox-LDL (from 0 to 140 mg cholesterol/dL). A stimulated (calcium ionophore) NO concentration decreased to 29% of the control at n-LDL concentration of 80 mg cholesterol/dL and to 15% of the control at 20 mg cholesterol/dL of ox-LDL. L-Arginine partially neutralized the inhibitory effect of n-LDL and ox-LDL on the NO generation. Superoxide dismutase pretreatment did not modify NO production, whereas L-NAME blunted NO generation at all LDL concentrations. O(2)(-) production was increased at low n-LDL and very low ox-LDL concentrations; this was reversed by L-arginine. CONCLUSIONS: These findings confirm the inhibitory role of n-LDL and ox-LDL on NO generation and suggest that lipoproteins may induce a decreased uptake of L-arginine. The local depletion of the L-arginine substrate may derange the NO synthase, leading to overproduction of O(2)(-) from oxygen, the other substrate of NO synthase.

Direct electrochemical measurement of nitric oxide in vascular endothelium.

The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium-derived nitric oxide (NO) is a vasodilator rapidly inactivated by superoxide and by Fe(II) and Fe(III), all found in significant quantities in biological systems. Thus due to the short life of NO in tissue (t1/2 = 3-6 s), in situ quantification of NO is a challenging problem. We designed the present study to perform direct measurements of nitric oxide using the electrochemical porphyrinic sensor. The most significant advantages of this sensor is small size (0.5-8 microm), rapid response time (0.1-1 ms), and low detection limit (10(-9) mol l(-1)). The porphyrinic sensor was used for in vitro and in vivo measurements of NO in an isolated single cell or tissue. Effects of hypertension, endotoxemia, and ischemia/reperfusion on the release of NO and/or its interaction with superoxide (O2-) were delineated. In the single endothelial cell (rabbit endocardium), NO concentration was highest at the cell membrane (950 +/- 50 nmol l(-1)), decreasing exponentially with distance from cell, and becoming undetectable at distances beyond 50 microm. The endothelium of spontaneously hypertensive rats (SHR) released 35% less NO (580 +/- 30 nmol l(-1)) than that of normotensive rats (920 +/- 50 nmol l(-1)), due to the higher production of O2- in SHR rats. Endothelial NO synthase (eNOS) generated NO (140 +/- 20 nmol l(-1)) in lung during the acute phase (first 10-15 min) of endotoxemia, followed by production of NO by inducible NOS. High production of O2- was observed during the entire period of endotoxemia. Ischemia (lower limb of rabbit) caused a significant increase of NO peaking at 15 min and decreasing thereafter, also due to O2- production.

Nitric oxide release from normal and dysfunctional endothelium.

The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium - derived nitric oxide (NO) is a vasodilator rapidly inactivated by superoxide (O2-) found in significant quantities. The porphyrinic sensor (0.5-8 microm diameter) and chemiluminescence methods were used to measure NO and (O2-) respectively. Effects of hypertension, low density lipoprotein (LDL), and heart preservation on the release of NO and O2- were delineated. In the single endothelial cell (rat aorta) NO concentration was the highest in the cell membrane decreasing exponentially with distance from cell, and becoming undetectable beyond 50 microm and 25 microm for normotensive (WKY) and hypertensive (SHR) rats respectively. The endothelium of SHR released 40% less NO (300+/-25 nmol L(-1)) than that of normotensive rats (500+20 nmol L(-1)), due to the higher production of O2- in SHR rats. An exponentially decreasing NO production (from 1.20 +/- 0.15 to 0.16 +/- 0.05 micromol (L-1)) and concomitant increase of O2- generation (from 10 +/- 0.3 to 300 +/- 25 nmol L(-1) were observed in left ventricle of stored (eight hours) rabbit heart. Native and oxidized low density lipoproteins (nLDL and oxLDL) inhibited NO generation and increased O2- production. The local depletion of the L-arginine substrate may disarrange the nitric oxide synthase, leading to production of O2- from oxygen.

Bioflavonoid quercetin scavenges superoxide and increases nitric oxide concentration in ischaemia-reperfusion injury: an experimental study.

BACKGROUND: L-Arginine-depleted environments accompanying ischaemia-reperfusion enhance superoxide production, leading to the formation of reactive oxygen species and a concomitant reduction in basal nitric oxide levels. The bioflavonoid quercetin may prevent these undesirable effects by scavenging superoxide. METHODS: Untreated rabbits were compared with those infused with quercetin (5 mg/kg for 2 min) during hindlimb ischaemia (2.5 h) and reperfusion (2 h). In both groups, nitric oxide concentration was measured (porphyrinic microsensor) in the femoral artery wall. Microvasculature changes (morphometry) and superoxide concentration (chemiluminescence) were measured intermittently in biopsies. RESULTS: Approximately 6 min into the period of ischaemia a rapid increase in nitric oxide level from a mean(s.e.m.) basal level of 50(20) to 450(30) nmol/l was observed. In untreated animals, nitric oxide concentration dropped to an undetectable level (less than 1 nmol/l) during reperfusion. In quercetin-treated animals, the decrease in nitric oxide concentration was slower, such that substantial amounts (60(20) nmol/l) accumulated during reperfusion. In biopsies after ischaemia-reperfusion maximal calcium ionophore A23187-stimulated nitric oxide concentration increased (25-30 per cent) in the presence of quercetin, while the superoxide concentration decreased. CONCLUSION: Quercetin treatment mollified ischaemia-reperfusion injury to skeletal muscle by scavenging destructive superoxide and enhancing the cytoprotective nitric oxide concentration.

Protective role of pulmonary nitric oxide in the acute phase of endotoxemia in rats.

We present for the first time direct continuous assay of NO concentration (porphyrinic sensor) in the lung parenchyma of Sprague-Dawley rats in vivo during endotoxemia. Intravenous infusion of lipopolysaccharide (LPS, 2 mg x kg(-1) x min(-1) for 10 minutes) stimulated an acute burst of NO from constitutive NO synthase (NOS) that peaked 10 to 15 minutes after the start of LPS infusion, mirroring a coincident peak drop in arterial pressure. NO concentration declined over the next hour to twice above pre-LPS infusion NO levels, where it remained until the rats died, 5 to 6 hours after LPS infusion. The chronic drop in arterial pressure observed from 70 minutes to 6 hours after the start of LPS infusion was not convincingly mirrored by a chronic increase in NO concentration, even though indirect NO assay (Griess method, assaying NO decay products NO2-/NO3-) showed that NO production was increasing as a result of continuous NO release by inducible NOS. A NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA, 10 mg/kg i.v.) injected 45 minutes before LPS infusion, resulted in sudden death accompanied by macroscopically/microscopically diagnosed symptoms similar to acute respiratory distress syndrome <25 minutes after the start of LPS infusion. Pharmacological analysis of this L-NNA+LPS model by replacing L-NNA with 1-amino-2-hydroxy-guanidine (selective inhibitor of inducible NOS) or by pretreatment with S-nitroso-N-acetyl-penicillamine (NO donor), camonagrel (thromboxane synthase inhibitor), or WEB2170 (platelet-activating factor receptor antagonist) indicated that in the early acute phase of endotoxemia, LPS stimulated the production of cytoprotective NO, cytotoxic thromboxane A2, and platelet-activating factor.

Mechanical transduction of nitric oxide synthesis in the beating heart.

NO alters contractile and relaxant properties of the heart. However, it is not known whether changes in ventricular loading conditions affect cardiac NO synthesis. To understand this potential contractile-relaxant autoregulatory mechanism, production of cardiac NO in response to mechanical stimuli was measured in vivo using a porphyrinic sensor placed in the left ventricular myocardium. The beating rabbit heart exhibited cyclic changes in [NO], peaking at 2.7+/-0.1 micromol/L near the endocardium and 0.93+/-0.20 micromol/L in the midventricular myocardium (concentrations were 15+/-4% lower in the rat heart). In the present study, we demonstrate for the first time that increasing or decreasing ventricular preload in vivo is followed by parallel changes in [NO], which may represent a novel autoregulatory mechanism to adjust cardiac performance or perfusion on a beat-to-beat basis. To quantify the relationship between applied force and NO synthesis, intermittent compressive or distending forces applied to ex vivo nonbeating hearts were shown to cause bursts of NO synthesis, with peak [NO] linearly related to ventricular transmural pressure. Experiments in which denuding cardiac endothelial and endocardial cells abrogated the NO signal indicate that these cells transduce mechanical stimulation into NO production in the heart. Taken together, these studies may help explain load-dependent relaxation, cardiac memory for mechanical events of preceding beats, diseases associated with myocardial distension, autoregulation of myocardial perfusion, and protection from thrombosis in the turbulent flow environment within the beating heart.

In situ measurement of nitric oxide, superoxide and peroxynitrite during endotoxemia.

We report in vivo and ex vivo measurements of nitric oxide (electrochemical), ex vivo in situ measurements of superoxide (chemiluminescence) and peroxynitrite (chemiluminescence) and delineate the effect of endotoxemia on nitric oxide, superoxide and peroxynitrite release in aorta of rats. Nitric oxide release was measured in the aorta wall. An increase of nitric oxide concentration was observed immediately after administration of lipopolysaccharide (Escherichia coli serotype 0127: B8, 20 mg/kg), reaching a plateau ((50 nmol/L) after 180 +/- 50 seconds; the plateau was followed by decreasing nitric oxide concentration and its subsequent gradual small increase after 45 minutes. Superoxide and peroxynitrite production increased dramatically during endotoxemia. Superoxide concentration increased from 10 +/- 2 nmol/L to 28 +/- 3 nmol/L at one hour, and reached a 50 nmol/L plateau at 4 hours. The pattern of peroxynitrite release paralleled the pattern of superoxide release during the time course of endotoxemia. Diametrical alteration of nitric oxide and superoxide concentration with subsequent production of peroxynitrite may be a major cause of endothelial cell injury during endotoxemia.