Detection Of Nitric Oxide Production Research Articles

268 - Quantitation of peroxynitrite production by macrophages

Reaction of peroxynitrite with coumarin boronic acid [1] was used for quantitation of peroxynitrite produced by activated murine macrophages (RAW 264.7) in cell culture. Highly fluorescent 7-hydroxycoumarin produced in reaction leads to fluorescence increase, affording real-time detection. Quiescent cells exhibit low rate of fluorescence increase which is further decreased in the presence of catalase, while catalase does not significantly affect the rate for activated cells. Specificity of the assay is supported by the dependence of the rate on the presence of arginine in the medium, inhibition by N6-(1-iminoethyl) L-lysine (inhibitor of iNOS) and by ebselen (peroxynitrite scavenger). The rate of peroxynitrite generation by stimulated RAW 264.7 was estimated as ~0.5 amol/(cell·s) that can be compared with NO generation rate of ~ 1.7 amol/(cell·s) determined earlier for the same cells [2]. Under the activation scheme used in this work (gamma-interferon + LPS), human THP-1 derived macrophages do not produce any peroxynitrite detectable by coumarin boronic acid. The level of iNOS mRNA in activated THP-1 derived macrophages does not exceed the level in control cells. 1 Zielonka, J.; Sikora, A.; Hardy, et al. Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides. Chemical Research in Toxicology. (2012), 25 (9), 1793–1799. 2 Woldman, Y. Y., Eubank, T. D., Mock, A. J., et al. Detection of nitric oxide production in cell cultures by luciferine-luciferase chemiluminescence. Biochemical and Biophysical Research Communications (2015), 465 232-238

Arginase expression modulates nitric oxide production in Leishmania (Leishmania) amazonensis.

BackgroundArginase is an enzyme that converts L-arginine to urea and L-ornithine, an essential substrate for the polyamine pathway supporting Leishmania (Leishmania) amazonensis replication and its survival in the mammalian host. L-arginine is also the substrate of macrophage nitric oxide synthase 2 (NOS2) to produce nitric oxide (NO) that kills the parasite. This competition can define the fate of Leishmania infection.Methodology/Principal findingsThe transcriptomic profiling identified a family of oxidoreductases in L. (L.) amazonensis wild-type (La-WT) and L. (L.) amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. We highlighted the identification of an oxidoreductase that could act as nitric oxide synthase-like (NOS-like), due to the following evidences: conserved domain composition, the participation of NO production during the time course of promastigotes growth and during the axenic amastigotes differentiation, regulation dependence on arginase activity, as well as reduction of NO amount through the NOS activity inhibition. NO quantification was measured by DAF-FM labeling analysis in a flow cytometry.Conclusions/SignificanceWe described an arginase-dependent NOS-like activity in L. (L.) amazonensis and its role in the parasite growth. The increased detection of NO production in the mid-stationary and late-stationary growth phases of La-WT promastigotes could suggest that this production is an important factor to metacyclogenesis triggering. On the other hand, La-arg- showed an earlier increase in NO production compared to La-WT, suggesting that NO production can be arginase-dependent. Interestingly, La-WT and La-arg- axenic amastigotes produced higher levels of NO than those observed in promastigotes. As a conclusion, our work suggested that NOS-like is expressed in Leishmania in the stationary growth phase promastigotes and amastigotes, and could be correlated to metacyclogenesis and amastigotes growth in a dependent way to the internal pool of L-arginine and arginase activity.

Peptide-Functionalized Fluorescent Particles for In Situ Detection of Nitric Oxide via Peroxynitrite-Mediated Nitration.

Nitric oxide (NO) is a free radical signaling molecule that plays a crucial role in modulating physiological homeostasis across multiple biological systems. NO dysregulation is linked to the pathogenesis of multiple diseases; therefore, its quantification is important for understanding pathophysiological processes. The detection of NO is challenging, typically limited by its reactive nature and short half‐life. Additionally, the presence of interfering analytes and accessibility to biological fluids in the native tissues make the measurement technically challenging and often unreliable. Here, a bio‐inspired peptide‐based NO sensor is developed, which detects NO‐derived oxidants, predominately peroxynitrite‐mediated nitration of tyrosine residues. It is demonstrated that these peptide‐based NO sensors can detect peroxynitrite‐mediated nitration in response to physiological shear stress by endothelial cells in vitro. Using the peptide‐conjugated fluorescent particle immunoassay, peroxynitrite‐mediated nitration activity with a detection limit of ≈100 × 10−9 m is detected. This study envisions that the NO detection platform can be applied to a multitude of applications including monitoring of NO activity in healthy and diseased tissues, localized detection of NO production of specific cells, and cell‐based/therapeutic screening of peroxynitrite levels to monitor pronitroxidative stress in biological samples.

Detection of nitric oxide production in cell cultures by luciferin–luciferase chemiluminescence

A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin–luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca2+ ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 102–104 cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM).

A bioassay for brassinosteroid activity based on the in vitro fluorimetric detection of nitric oxide production

Recent studies have shown that low concentrations of brassinolide induce a rapid generation of nitric oxide in mesophyll cells of maize leaves, which can be easily detected by fluorimetric methods. In this work we describe a series of natural and synthetic brassinosteroids that are able to trigger in vitro NO production in tomato cells that exhibits dose–response behavior. We propose that this effect can be used to develop a new rapid and very sensitive bioassay for brassinosteroid activity that offers several advantages when compared to the current methodologies.

Direct Nitric Oxide Detection in Aqueous Solution by Copper(II) Fluorescein Complexes

A series of FL(n) (n = 1-5) ligands, where FL(n) is a fluorescein modified with a functionalized 8-aminoquinoline group as a copper-binding moiety, were synthesized, and the chemical and photophysical properties of the free ligands and their copper complexes were investigated. UV-visible spectroscopy revealed a 1:1 binding stoichiometry for the Cu(II) complexes of FL(1), FL(3), and FL(5) in pH 7.0 buffered aqueous solutions. The reactions of FL(2) or FL(4) with CuCl(2), however, appear to produce a mixture of 1:1 and 1:2 complexes, as suggested by Job's plots. These binding modes were modeled by the synthesis and X-ray crystal structure determination of Cu(II) complexes of 2-[(quinolin-8-ylamino)methyl]phenol (modL), employed as a surrogate of the FL(n) ligand family. Two kinds of crystals, [Cu(modL)(2)](BF(4))(2) and [Cu(2)(modL')(2)(CH(3)OH)](BF(4))(2) (modL' = 2-[(quinolin-8-ylamino)methyl]phenolate), were obtained. The structures suggest that one oxygen and two nitrogen atoms of the FL(n) ligands most likely bind to Cu(II). Introduction of nitric oxide (NO) to pH 7.0 buffered aqueous solutions of Cu(FL(n)) (1 microM CuCl(2) and 1 microM FL(n)) at 37 degrees C induces an increase in fluorescence. The fluorescence response of Cu(FL(n)) to NO is direct and specific, which is a significant improvement over commercially available small molecule-based probes that are capable of detecting NO only indirectly. The NO-triggered fluorescence increase of Cu(FL(5)) occurs by reduction of Cu(II) to Cu(I) with concomitant dissociation of the N-nitrosated fluorophore ligand from copper. Spectroscopic and product analyses of the reaction of the FL(5) copper complex with NO indicated that the N-nitrosated fluorescein ligand (FL(5)-NO) is the species responsible for fluorescence turn-on. Density functional theory (DFT) calculations of FL(5) versus FL(5)-NO reveal how N-nitrosation of the fluorophore ligand brings about the fluorescence increase. The copper-based probes described in the present work form the basis for real-time detection of nitric oxide production in living cells.

Radiochemical HPLC detection of arginine metabolism: Measurement of nitric oxide synthesis and arginase activity in vascular tissue

Nitric oxide (NO) plays a key role in vascular homeostasis. Accurate measurement of NO production by endothelial nitric oxide synthase (eNOS) is critical for the investigation of vascular disease mechanisms using genetically modified animal models. Previous assays of NO production measuring the conversion of arginine to citrulline have required homogenisation of tissue and reconstitution with cofactors including NADPH and tetrahydrobiopterin. However, the activity and regulation of NOS in vivo is critically dependant on tissue levels of these cofactors. Therefore, understanding eNOS regulation requires assays of NO production in intact vascular tissue that do not depend on the addition of exogenous cofactors and have sufficient sensitivity and specificity. We describe a novel technique, using radiochemical detection of arginine to citrulline conversion, to measure NO production within intact mouse aortas, without exogenous cofactors. We demonstrate the presence of arginase activity in mouse aortas which has the potential to confound this assay. Furthermore, we describe the use of N-hydroxy-nor- l-arginine (nor-NOHA) to inhibit arginase and permit specific detection of NO production in intact mouse tissue. Using this technique we demonstrate a 2.4-fold increase in NO production in aortas of transgenic mice overexpressing eNOS in the endothelium, and show that this technique has high specificity and high sensitivity for detection of in situ NO synthesis by eNOS in mouse vascular tissue. These results have important implications for the investigation of NOS regulation in cells and tissues.

Effect of scutellarin on nitric oxide production in early stages of neuron damage induced by hydrogen peroxide

The aims of the present study were to investigate the regulatory function of scutellarin on production of nitric oxide (NO) as well as activities of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) in early stages of neuron damage induced by hydrogen peroxide. Direct detection of NO production was performed on primary cultures of living rat neuronal cells with an electrochemical sensor. Hydrogen peroxide significantly increased culture supernatant levels of NO, the total integral value of the defined areas (500–6500 s × pA) reached 3.68 × 10 6. Pre-treatment with scutellarin, caused the total integral value to decrease in a dose-dependent fashion (3.24 × 10 6, 2.15 × 10 6, 1.84 × 10 6 for groups 10, 50, and 100 uM scutellarin, respectively). After exposure to 2.0 mM hydrogen peroxide for 2 h, malondialdehyde (MDA) level, a marker of lipid peroxidation, was remarkably increased. The elevation can be suppressed by scutellarin. Hydrogen peroxide also caused significant loss of neuron viability. In comparison with the control group, scutellarin significant attenuated the loss. Results also showed that hydrogen peroxide increased activity of cNOS, which was markedly inhibited by scutellarin. However, exposure of neuronal cells to hydrogen peroxide did not lead to an increase in iNOS activity. In conclusion, our results suggest that NO production, which increased in early stages of neuron damage induced by hydrogen peroxide can be effectively inhibited by scutellarin. Moreover, our results indicate that increase in NO production is mediated by cNOS.

Nitric Oxide Production Within Rat Urothelial Cells

Recent studies have suggested that nitric oxide (NO) synthase (NOS) may be localized in the urothelium of the proximal part of the mammalian ureter. We investigated endogenous NO production in the proximal half of the rat ureter, localized its cellular source, characterized the NOS isoforms involved and assessed the impact of NO on ureteral motility. Direct detection of NO production was performed on primary cultures of living rat ureteral cells with the fluorescent indicator diaminofluorescein. Cultures were incubated with the NO precursor L-arginine or the NOS inhibitors L-NAME (N-nitro-L-arginine-methyl ester) and 1400W. NOS expression was determined by immunofluorescence and Western blot analysis. The functional effects of NO donors were assessed on isolated ureters. Significant basal NO production was demonstrated by the high fluorescence level detected in diaminofluorescein treated cell cultures. NO production was strictly limited to urothelial cells since no fluorescence was seen in smooth muscle cells. Pretreatment with L-NAME or 1400W resulted in a significant decrease in fluorescence. Constitutive and inducible NOS isoforms were detected in urothelial cultured cells and in lysates of the urothelial layer. NO donors inhibited in a concentration dependent manner the agonist induced contractile activity of isolated ureters. These results suggest that NO production stems from the urothelium and the NO pathway inhibits contractile activity in the proximal half of the rat ureter. Hence, the nitrergic pathway may be an important target for drugs producing relaxation of the mammalian ureter.

THE DELAYED HISTOLOGICAL CHANGES OF SEGMENTAL ISCHEMIA AND ISCHEMIA - REPERFUSION ON THE JEJUNAL VILLI 01

Acute intestinal ischemia represents a broad spectrum of diseases with variousclinical and pathological manifestations. The primary causes of insufficient bloodflow to the intestine arc diverse and include thromboembolism, neoplasms, vasculitis,abdominal inflammatory conditions, trauma, chemotherapy, radiation and corrosiveinjury (Rha et aI., 2000). Despite of the advances in the pathophysiological, laboratorydiagnosis and imaging techniques acute intestinal ischemiais still one ofthe major etiological causes of multiple organ failure (Schlichting et aI., 1995). Ischemiainduces continuous distrubance of hepatic microcirculation leading to liverdysfunction (Nakamura et al., 2001). Furthermore, it triggers the development oflung dysfunction (Ito et aI., 2003). Ischemia is often associated with mortality ratesmore than 60% and thus it represents a major challenging clinical problem (Lock,2001).Reviewing the literature re ,e1led that the intestinal mucosa in particular is oneof the most sensitive tissues to ischemia and ischemia reperfusion injury in the humanbody (Ikeda et aI., 1998; Kalia et aI., 2001). Although the histopathological alterationsin the intestinal mucosa after ischemic injury have been extensively described(Puglisi et al., 1995; Takeyoshi et al., 1996; Ikeda et al., 1998) yet most ofthe previous models of ischemia were achieved by complete occlusion of the superiormesenteric artery which often resulted in shock and early death of the experimentalanimalshence rendering it rather difficult to examine the delayed morphologicalchanges in the ischemic intestinal mucosa. Moreover. the intestinal segments distalto ischemia though clinically showed decreased contraction frequency. decreased relaxationresponses and altered responses to norepinephrine (Malone and Kanaan 2001) yet the morphological changes in locations distal to ischemia were not properlyinvestigated.Recently, the role ofmodulators particularly nitric oxide has received specialattention. It is hypothesized that nitric oxide might be involved in tissue protectionand provide partial preservation of intestinal mucosa as the first line of defense followingischemia reperfusion (Kalia et al., 20ot). Detection of nitric oxide could beachieved via the estimation of endothelial nitric oxide synthase (NOS) which is amember of a family of enzymes responsible for synthesis of nitric oxide from L arginineand molecular oxygen (Chan et al., 2002).Accordingly, it was the aim of the current work to illustrate the delayed morphologicaland morphometric changes following segmental ischemia and ischemiareperfusion produced by selective occlusion of terminal branches of superior mesentericartery. Moreover an attempt of detection of nitric oxide production wasachieved via the estimation of nitric oxide synthase. The changes in both the ischemicsegment as well as the segment distal to it were investigated. Such a study mightbe beneficial in post-ischemic resection anastomosis procedures, or in surgical revascularization.

Measurement of nitric oxide production by the lesioned rat retina with a sensitive nitric oxide electrode.

Nitric oxide (NO) plays multiple roles in the nervous system. It is produced as a result of damage or injury of the retina as a part of the central nervous system. Detailed knowledge of the extent and the time course of NO production is of great importance for the understanding of pathological processes and their appropriate medical treatment. Sections of rat retina were stained with antibodies against the three isoforms of NO synthase (NOS) at several time points after a lesion of the optic nerve. No significant changes of NOS expression could be seen at any of the checked time points. For the electrochemical detection of NO production, we modified small platinum electrodes with a NO-sensitive nickel porphyrin by electrochemical polymerisation. Compared to other substances, electrochemically polymerised eugenol was found to be most suitable for protection against interferences. For the measurements, differential pulse amperometry was used. The response to nitric oxide was linear.NO production of adult rat retinas was measured post axotomy after different time points with electrochemical electrodes ex vivo. With non-treated retinas, an NO concentration of approximately 15 microM was measured. NO concentration is elevated after an axotomy reaching its highest value of up to 30 microM 5 days after the lesion. The NO concentration is decreased below the initial value after 9-14 days post axotomy.

25] Fluorescent imaging of mitochondrial nitric oxide in living cells

Publisher Summary The chapter presents a study related to fluorescent imaging of mitochondrial nitric oxide in living cells. Nitric oxide (NO) is an important modulator of mitochondrial respiration and membrane potential. The presence of a mitochondrial NO synthase (mtNOS) has been described in the inner membrane of isolated rat liver mitochondria. However, because NO is a highly diffusible, short-lived (0.5–5 sec), highly reactive radical, its determination poses considerable technical problems. In fact, until a while ago, the production of NO by mtNOS has only been indirectly characterized in broken mitochondria using techniques, such as spectroscopy or the citrulline assay. Two novel fluorometric detection systems have been developed allowing the demonstration of the presence of NO within mitochondria via microscopy. 4,5-Diaminofluorescein diacetate (DAF-2/DA) permits the direct detection of NO production, and the MitoTracker allows the detection of mitochondrial potential changes. Thus, whereas DAF-2/DA can be used as a semiquantitative method of evaluation of NO production in living cells, the combination of these two systems provides an ideal tool for the spatial and direct visualization of NO within mitochondria. A variety of products from Molecular Probes allow the detection of mitochondrial activity.

Detection of nitric oxide production by fluorescent indicators

Detection of nitric oxide production by fluorescent indicators

Temporal and anatomical distribution of nitric oxide synthase mRNA expression and nitric oxide production during central nervous system inflammation

Nitric oxide (NO) has important roles in inflammatory processes. It was the aim of this study to ascertain whether changes in nitric oxide synthase (NOS) mRNA expression lead to similar temporal and anatomical changes in NO production in an experimental model of CNS inflammation. NOS-II (inducible NOS) mRNA expression was analyzed 2, 4, 6 and 24 h after intracerebroventricular (i.c.v.) injection of interleukin-1β (IL-1β) or vehicle. Increased expression of NOS-II mRNA was observed surrounding the microinjection site and meninges. The changes were significantly higher than controls at 4 and 6 h, returning to baseline at 24 h. Using the novel fluorometric NO detection system, 4,5-Diaminofluorescein diacetate (DAF-2/DA), for the direct detection of NO production, we observed a significant increase in NO production after 4 and 6 h. NO production was observed in areas surrounding the injection site, meninges surrounding the brain and perivascular cells and neuron-like cells throughout the cortex. However, increases in NO production in these areas remained significantly higher than controls at 24 h. These findings demonstrate for the first time that, in fresh frozen tissue, that the anatomical distribution of NOS-II mRNA is consistent with the distribution of NO production. We conclude that increases in NOS-II mRNA following i.c.v. administration of IL-1β lead to increases in NO production. While the mRNA is degraded by 24 h post treatment, the enzyme remains active. We propose that the DAF-2/DA method can be used as a potential marker in the diagnosis of CNS inflammation.

Cheletropic Traps for the Fluorescence Spectroscopic Detection of Nitric Oxide (Nitrogen Monoxide) in Biological Systems

Membrane-permeable, phenanthrene-derivedo-quinodimethanes of type 1 react with nitric oxide in a cheletropic fashion to produce fluorescent nitroxide radicals 2 and hydroxylamines 3. This method allows the sensitive and quantitative detection of nitric oxide production in biological samples by means of fluorescence microscopy, as is demonstrated by the monitoring of NO production from alveolar macrophages.

Electrochemical detection of nitric oxide production in human polymorphonuclear neutrophil leukocytes

The detection of nitric oxide (NO) release by human polymorphonuclear neutrophil leukocytes (PMNs) presents several difficulties, mainly due to concomitant production of O2- and H2O2, which could interfere with the measurements. A Nafion and nickel porphyrin-coated microelectrode was used to measure NO production in PMNs in vitro. It allowed detection of 6.3 +/- 1.9 nM NO in a PMN-containing system and was unaffected by added chemicals. Addition of the chemotactic oligopeptide f-met-leu-phe (fMLP; 100 nM) induced a NO release which reached a value of 71 +/- 30 pmol NO/10(6) PMN x ml(-1) 5 min after stimulation in the presence of SOD (150 U/ml). If SOD was omitted, the corresponding value was 36 +/- 20 pmol NO/10(6) PMN x ml(-1). Presence or absence of catalase did not alter the amount of NO measured. Addition of the NO-synthase inhibitor N(G)-monomethyl-L-arginine (LNMMA; 1 mM) reduced the current by 82 +/- 20%. These results agree with the rate of NO production in human PMNs when measured spectrophotometrically using the NO-dependent oxidation of oxyhaemoglobin to methaemoglobin. The NO production in human PMN was dependent on fMLP concentrations, but independent of cell-concentrations of 0.5-3.5 x 10(6)/ml. This paper shows that a electrochemical method, e.g. Nafion and porphyrin-coated microelectrode, is suitable for studies of NO release from stimulated human PMNs.

Electrochemical detection of nitric oxide production in perfused pig coronary artery: comparison of the performances of two electrochemical sensors

In situ direct measurement of nitric oxide (NO) in biological media is now possible by means of electrochemical detection. In the literature, there are principally two amperometric approaches based on the direct oxidation of NO either on a sensor made from platinum/iridium (Pt/Ir) alloy coated with a three-layered membrane or on a nickel porphyrin and Nafion-coated carbon fiber electrode. Nonetheless, the exact nature of the experimental amperometric signal obtained with the Pt/Ir system was never authenticated as being related to NO. This study compared responses of two sensors to the inhibition effect of N ω-nitro- l-arginine (L-NA) as the amperometric signals produced by 5-hydroxytryptamine (5-HT) on isolated pig coronary preparations. These amperometric signals could be attributed to NO only for the nickel porphyrin and Nafion-coated carbon fiber electrode. Indeed, voltammetric characterization of the electrochemical response demonstrated only variations of the baseline current upon additions of either SNAP or NO on the Pt/Ir electrode instead of anodic peak current displayed at 0.63–0.75 V for the other system. Nitrites induced baseline current variations with the Pt/Ir electrode, similar to those obtained with S-nitroso- N-acetyl- dl-penicillamine (SNAP) or NO. This study highlights the potential hazards and pitfalls that may be associated with the use of a Pt/Ir sensor calibrated with SNAP solutions for the detection of NO production in various biological systems.

Chemiluminescence detection of nitric oxide production from rat cerebral cortical endothelial cells in culture

Cultured brain microvascular endothelial cells have been used to study blood–brain barrier properties in vitro. Among several endothelial derived factors the gaseous free radical nitric oxide (NO) has been shown to regulate both vascular tone and blood–brain barrier permeability. Due to its short half-life and high reactivity with many other molecules, effects of NO have preferentially been investigated using NO synthase inhibitors, NO donors or, more recently, in NO synthase knock-out animals. In the present study we describe a rapid, sensitive and reliable protocol for chemiluminescence detection of NO 2 −, the stable oxydation product of NO, from the supernatant of rat cortical endothelial cells in culture. After suppression of inducible NOS activity brain endothelial cells showed low basal NO levels. After stimulation with calcium ionophore NO levels increased and were attenuated again by NOS blockade. NO release was also elicited by bradykinin or acetylcholine, albeit to a lesser extent. This protocol might prove useful to investigate calcium-dependent NO formation in cerebral endothelial cells and blood–brain barrier models in vitro. In principle, the same protocol can be applied to virtually any cell type in culture. Themes: Neurotransmitters, modulators, transporters, and receptors Topics: Other neurotransmitters

Mechanism of 5-hydroxytryptamine-induced coronary vasodilation assessed by direct detection of nitric oxide production in guinea-pig isolated heart.

1. We assessed whether a submaximal concentration (1 microM) of 5-hydroxytryptamine (5-HT) releases nitric oxide (NO) from the coronary endothelium in guinea-pig perfused heart (n = 5 or 6/group) by direct detection of NO in coronary effluent, and determined whether this accounts for the associated coronary dilation. We also tested whether saponin is a selective and specific tool for examining the role of this mechanism in mediating agonist-induced coronary dilatation. 2. Continuous 5 min perfusion with 5-HT, or acetylcholine (ACh; 1 microM), substance P (1 nM) or sodium nitroprusside (SNP; 1 microM) increased coronary flow from baseline by 3.6 +/- 0.2, 3.4 +/- 0.2, 1.8 +/- 0.1 and 4.1 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Coronary effluent NO content, detected by chemiluminescence, was correspondingly increased from baseline by 715 +/- 85, 920 +/- 136, 1019 +/- 58 and 2333 +/- 114 pmol min-1 g-1, respectively (all P < 0.05). 3. Continuous perfusion for 30 min with NG-nitro-L-arginine methyl ester (L-NAME) 100 microM reduced basal coronary effluent NO content by 370 +/- 32 pmol min-1 g-1 and coronary flow by 7.5 +/- 0.5 ml min-1 g-1 (both P < 0.05). Saponin (three cycles of 2 min of 30 micrograms ml-1 saponin perfusion interrupted by 2 min control perfusion) reduced basal coronary NO content by a similar amount (307 +/- 22 pmol min-1 g-1) but reduced basal coronary flow by only 0.6 +/- 0.2 ml min-1 g-1 (P < 0.05 versus the effect of L-NAME). 4. The increases in coronary flow in response to (5-HT), ACh and substance P were reduced (all P < 0.05) by 100 microM L-NAME to 1.2 +/- 0.3, 1.2 +/- 0.4 and 0.3 +/- 0.3 ml min-1 g-1, respectively. However, the flow increase in response to SNP was not reduced; it was in fact increased slightly to 4.8 +/- 0.4 ml min-1 g-1 (P < 0.05). 5. Similarly, after treatment with saponin, the increases in coronary flow in response to 5-HT, ACh and substance P were reduced to 2.1 +/- 0.3, 1.3 +/- 0.3 and 0.4 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Again, the response to SNP was increased slightly to 4.6 +/- 0.5 ml min-1 g-1 (P < 0.05). 6. L-NAME and saponin also inhibited 5-HT, ACh and substance P-induced NO release (P < 0.05), without affecting equivalent responses to SNP. 7. For substance P, the change in coronary flow (delta CF) correlated with log10 delta NO in the presence and absence of saponin and L-NAME; delta CF = 1.2(log delta NO) 1.9; r = 0.92; P < 0.05. For 5-HT the relationship was delta CF = 2.2(log delta NO-2.7; r = 0.79; P < 0.05, indicating that 5-HT causes a disproportionately greater increase in coronary flow per release of NO. This was taken to indicate that 5-HT relaxes coronary vasculature in part by releasing NO, but in part by additional mechanisms. ACh resembled 5-HT in this respect. 8. Saponin had no effect on cardiac systolic or diastolic contractile function assessed by the construction of Starling curves with an isochoric intraventricular balloon. 9. In conclusion, despite its minimal effect on basal coronary flow, saponin is an effective tool for revealing endothelium-dependent actions of coronary vasodilator substances and has selectivity in that it does not impair endothelium-independent vasodilatation or cardiac contractile function. 5-HT dilates guinea-pig coronary arteries largely by the release of NO from the coronary endothelium.

Detection of nitric oxide production and its role in pancreatic ischemia-reperfusion in rats.

Partial ischemia of rat pancreas body and tail was obtained by occlusion of the celiac axis for 1 h after gastrectomy. The plasma level of nitrite plus nitrate in both systemic and portal venous blood after reperfusion was significantly higher than that after sham operation and ischemia alone. The elevation after reperfusion was significantly decreased by NG-nitro-L-arginine methyl ester (L-NAME). Simultaneous administration of L-arginine counteracted the L-NAME-induced decrease in the level of nitric oxide (NO) end products. Generation of NO was further demonstrated by nitrosylhemoglobin detection by electron spin resonance in the blood after reperfusion. On the other hand, the plasma level of lipase, a marker of damage to pancreatic exocrine tissue, was significantly increased after ischemia-reperfusion and further increased by administration of L-NAME. This increase in lipase correlated with a decrease in tissue blood flow in the pancreas. These results suggest that NO is generated during and may have a protective role in ischemia-reperfusion of the rat pancreas.