Direct Detection Of Nitric Oxide Research Articles

Fusing the Nagano’s and the Anslyn’s chemistry for lyso-specific NO detection

The biorelevance of nitric oxide (NO) has attracted intensive efforts in their in situ detection. Fluorescence-based methods have been established as the benchmark due to their sensitivity. The high stability and radical nature of NO contributed to the difficulties of their sensitive detection. Direct detection of NO has either involved the use of a heavy transition metal complex, or a sluggish cheletropic reaction with a diene. Therefore, detection of nitrosonium species, generated upon a formal one-electron oxidation of NO, has become the routine practice as a surrogate of biological NO. Amongst all, the Nagano chemistry using ortho-phenylenediamine and the Anslyn chemistry using 2-amino-3′-dialkylaminobiphenyl are the two predominant approaches. They differ from each in that a nitrogen-based or a carbon-based diazo-coupling occurs upon nitrosation of the primary amino group. Presumably, their cyclization kinetics are expected to differ and further impact the time required to turn-on the fluorescence. To address this mechanistic curiosity, we designed model molecular scaffolds (NOP1−3), via hybriding the Nagano and the Anslyn NO sensing chemistry, and showcased that the Anslyn chemistry exhibits a relatively higher cyclization kinetics. Based on that the detection product becomes fluorescent in lyso-relevent pH, we found that NOP3 have potentials for lyso-specific NO detection.

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Photophysical insights and guidelines for blue “turn‐on” fluorescent probes for the direct detection of nitric oxide (NO•) in biological systems

AbstractSynthesis and photophysical properties of a family of five blue fluorescent “turn‐on” probes for the direct detection of nitric oxide (NO•) is reported. The probes CB1‐5 feature a substituted 7‐hydroxy coumarin chromophore coupled to 2‐methyl‐8‐aminoquinoline, which act as tridentate ligand for Cu(II) and active site for monitoring NO• using a replacement strategy. The UV/vis absorption and fluorescence emission characteristics of the probes are significantly influenced by the substitution pattern on the coumarin ring, as well as by solvent polarity and pH. Time‐dependent density functional theory (TD‐DFT) calculations for CB4 and CB5 showed that the absorptions are due to π‐π* transitions localised on coumarin, with a small charge transfer contribution from the quinoline system at higher pH where the 7‐hydroxy coumarin moiety is deprotonated, which leads to a bathochromic shift of the absorption. Complexation of the probes with Cu(II) quenches the fluorescence, which is turned back on upon reaction with NO•, allowing selective detection of this important signalling molecule in μM concentrations. Preliminary experiments revealed that CB4 and CB5 enable to monitor endogenously produced NO• in living bacterial cells in multi‐dye imaging experiments.

Mannoside and 1,2-mannobioside β-cyclodextrin-scaffolded NO-photodonors for targeting antibiotic resistant bacteria

Two β-cyclodextrin derivatives randomly appended on the primary face with both the nitric oxide (NO) photodonor 4-nitro-3-(trifluoromethyl)aniline and a mannose or α(1→2)mannobioside residue are reported to construct targeted NO photoreleasing nanocarriers. 2D ROESY and PGSE NMR suggested supramolecular homodimerization in water by inclusion of the nitroaniline group into the facing macrocycle cavities. Isothermal titration calorimetry on their concanavalin A lectin binding showed an exothermic binding event to the lectin and an endothermic process during the dilution of the conjugates. Both α(1→2)mannobioside and the nitroaniline moieties significantly enhanced the binding to the lectin. These effects might arise from a better fit within the carbohydrate-recognition site in the former case and a multivalent effect caused by homodimerization in the latter. Direct detection of NO by amperometric technique shows that both β-cyclodextrin derivatives release this radical upon excitation with visible light with higher efficiency than the unfunctionalized NO photodonor.

Benign synthesis of N-(8-quinolyl)pyridine-2-carboxamide) ligand (Hbpq), and its Ni (II) and Cu (II) complexes. A fluorescent probe for direct detection of nitric oxide in acetonitrile solution based on Hbpq copper(II) acetate interaction

Abstract The ligand Hbpq = N-(8-quinolyl)pyridine-2-carboxamide) has been prepared using tetrabutylammonium bromide (TBAB) as an environmentally friendly reaction medium. Four new complexes of this ligand, [M(bpq)X] (M = Cu(II), X = SCN (1), N3 (2); M = Ni(II), X = SCN (3), N3 (4)), have also been synthesized and fully characterized. The crystal and molecular structures of [Cu(bpq)(NCS)]n (1) have been determined by X-ray crystallography. Copper(II) ion adopts a distorted square pyramidal (4 + 1) coordination in this complex. Hbpq ligand shows a strong emission at 500 nm in acetonitrile solution. The emission is quenched in the presence of copper(II) acetate, apparently because of the formation of [Cu(L)(OAc)(H2O)] complex. Introduction of nitric oxide (NO) into the acetonitrile solution at room temperature induces an increase in the fluorescence intensity, presumably due to the reduction of Cu(II) to Cu(I). This process is reversible and can form a basis for direct detection of NO.

Microchip electrophoresis with amperometric detection method for profiling cellular nitrosative stress markers.

The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2(-)) in cell lysates. There was a 2.5- to 4-fold increase in NO2(-) production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2(-) inside a single unstimulated macrophage cell was estimated to be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions.

Comparison of three different configurations of dual ultramicroelectrodes for the decomposition of S-Nitroso-L-glutathione and the direct detection of nitric oxide

We report on the characterization and use of three configurations of dual gold ultramicroelectrodes (UMEs), namely a ring-disc electrode, a disc-disc electrode, and band-band electrode. These can be used for the direct determination of nitric oxide that is released during the copper-catalyzed decomposition of S-nitroso-L-glutathione (GSNO). The dual UMEs were electrochemically characterized by using ferrocenemethanol as a soluble redox mediator during chronoamperometric measurements. The data are similar to those reported in the literature for various configurations and can be used to characterize the ability of copper ions to reach the vicinity of the NO sensor. One UME was electrochemically modified with layers of a poly(eugenol)/poly(phenol) composite to act as an NO sensor. The second UME was electrochemically coated with a copper layer that serves as a source for Cu(II) that is needed for the in-situ decomposition of GSNO. The mediated decomposition of GSNO is accomplished in presence of ascorbate that acts as reducing agent for Cu(II). The NO released from GSNO is detected at the same potential as applied to form the Cu(II)-based catalyst (+0.8 V vs Ag/AgCl).

Micro-ring disc ultramicroelectrodes array for direct detection of NO-release from S-nitrosoglutathione

In this work, we report on the design, microfabrication and use of a ring disk ultramicroelectrodes array (RD-UMEs) for direct detection of nitric oxide (NO)-release from decomposition of S-Nitrosoglutathione (GSNO) catalyzed by copper species. The electrochemical device includes seven individually addressable sets of gold RD-UMEs pairs and four gold counter electrodes. For each pair of RD-UMEs, the gold ring is electrochemically modified by poly(eugenol)/poly(phenol) layers to act as NO-sensor while the gold disc electrode is electrochemically coated by a copper layer serving as Cu2+ source for the catalysis of the in situ decomposition of GSNO. The mediated decomposition of GSNO by Cu2+ is achieved in presence of ascorbate. NO released from GSNO is detected on NO ring electrode at the same potential applied to form the Cu2+-based catalyst (+0.8V vs. Ag/AgCl) in phosphate buffer solution with or without serum.

Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase

Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases subnanomolar, physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescence approach to direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphosphate to guanosine 3′,5′-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases by about 200 times when NO binds with sGC and, in so doing, provides a sensor for nitric oxide. Luminescence detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from the ATP-dependent luciferin–luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurement of NO generated by a nitric oxide donor (PAPA-NONOate), nitric oxide synthase, and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.

Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase

Genetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2(-)), or both; these radicals may then react to form peroxynitrite. Alternatively, O2(-) radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE(-/-)) and apoE/iNOS double knockout (apoE(-/-)/ iNOS(-/-)) mice to assess vascular NO, O2(-), and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2(-), leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression.

Nitric oxide detection by DAF (diaminofluorescein) fluorescence in human myometrial tissue

Nitric oxide (NO) is considered to be involved in the modulation of uterine contractility. In the present pilot study, the direct detection of intracellular NO in pregnant human myometrial tissues has been investigated by using the fluorescent indicator 4,5-diaminofluorescein-2 diacetate (DAF-2DA). Pregnant myometrial tissue samples were obtained during Cesarean sections between week 34 and 40 of gestation before the onset of labor. Living explants were loaded with 10 μM DAF-2DA, stimulated with 1 mM arginine, subsequently fixed with glutaraldehyde and examined by fluorescence microscopy. The presence of NO synthases (NOS) was studied by immunohistochemistry. After application of DAF-2DA, DAF fluorescence was located primarily in blood vessels and to a minor extent in myometrial cells. By immunohistochemistry, strong endothelial NOS (eNOS) staining was found in vessel walls. In myometrial cells weak staining of eNOS and inducible NOS was observed. We conclude that the direct NO detection by DAF-2DA provides a new and independent method to identify sites of NO production in myometrium and other heterogeneous tissues.

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.

Direct detection of nitric oxide in human blood serum by use of 1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl)difluoroboradiaza-s-indacene with HPLC

To study the relationship between amounts of nitric oxide (NO) in blood and the development of ischemic cardio-cerebrovascular diseases, trace NO in human blood serum has, for the first time, been determined by use of a 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)difluoroboradiaza-s-indacene (TMDABODIPY)-based HPLC method. The proposed method is simple, rapid, and efficient, owing to its high sensitivity and good selectivity for NO. TMDABODIPY and its NO derivative are separated to baseline in 4 min, with simple separation conditions, on a C(18) column eluted with 50 mmol L(-1) ethanolamine in methanol. The derivative is detected by fluorescence at an emission wavelength of 507 nm with excitation at 498 nm. The response is a linear function of concentration in the range 0.8-800 nmol L(-1) NO. The detection limit can reach 2 x 10(-11) mol L(-1) (signal-to-noise ratio=3). The method has been used to detect NO in the serum of patients with five kinds of ischemic cardio-cerebrovascular disease and two diseases closely connected with ischemic cardio-cerebrovascular diseases. Recoveries of NO from spiked serum samples were between 96.58 and 105.71% and concentrations of NO observed in real samples were at 10(-7) mol L(-1) levels. Our studies indicate that the proposed TMDABODIPY-based HPLC technique can be developed into a sensitive and new method for clinical assay and pathology research.

Design, synthesis and characterization of a novel fluorescent probe for nitric oxide based on difluoroboradiaza- s-indacene fluorophore

Nitric oxide (NO) is a gaseous, free radical, which plays a role as an intracellular second messenger and a diffusible intercellular messenger. To obtain evidence for NO function in vivo, 1,3,5,7-tetramethyl-8-(4′-aminophenyl- N-(2′′-amino)-phenzyl)-difluoroboradiaza- s-indacene (TMAPABODIPY) was designed and synthesized as a fluorescent probe for nitric oxide, which features high photostability and no pH dependency over a wide pH range. The fluorescence of TMAPABODIPY itself is very strong. When TMAPABODIPY traps NO in the presence of dioxygen, the weak fluorescent triazole form is obtained, which offers the advantages of specificity, and sensitivity for direct detection of NO. The relationship was obtained between the quenching fluorescence intensity and NO concentration in the range 0.02–4.0 μmol l −1. The detection limit is 5 nmol l −1 (S/N = 3). The proposed method has been used to monitor the release of NO from S-nitrosocysteine, a NO-releasing agent.

Vasomodulating potential of mediterranean wild plant extracts.

The incidence of cardiovascular disease and endothelial dysfunction is low in the Mediterranean area, where the major proportion of daily calories comes from plant food, high in antioxidant polyphenols. It has been shown that a reduced production or enhanced inactivation of endothelium-derived nitric oxide (NO) is involved in the onset of endothelial dysfunction. We investigated the effects of Mediterranean wild plant, that is, wild artichoke and thyme, phenolic-rich extracts on NO release by porcine aortic endothelial cells (PAECs; by using indirect methods) and by cerebral cell membrane homogenates (by using direct NO detection). NO release by PAECs was significantly potentiated by 234% and 135% by wild artichoke and thyme extracts (10(-6) mol/L), respectively. Direct detection of NO release by brain membranes also showed significantly increased NO production after wild artichoke addition (+35.4%). Further, the release of another vasorelaxant factor by PAECs, that is, prostacyclin, was significantly increased by wild artichoke and thyme (10(-6) mol/L) (+269% and +190%, respectively). Investigation of the mechanism(s) of action of wild artichoke and thyme suggests maintenance of an intracellular reduced environment, as previously shown for ascorbate. Even though these data require in vivo confirmation, they suggest that regular intake of bioactive compounds from Mediterranean wild plants contributes to maintenance of proper vasomotion and to the low incidence of atherosclerosis and endothelial dysfunction recorded in the Mediterranean area.

Nitric oxide imaging in living neuronal tissues using fluorescent probes

Nitric oxide (NO) is a major modulator of neural functions. Since NO is a gaseous molecule with very short half-life, the spatial distribution of NO and its relationship to neuronal activity are difficult to resolve. Non-invasive and direct visualization of NO in neuronal tissues had been hampered by the lack of a suitable method to identify NO directly. A fluorescent indicator, which directly detects NO under physiological conditions, would be advantageous. Several indicators for direct detection of NO have been developed, which react with NO by forming a fluorescent complex. However, some of these dyes have cytotoxic properties or have been found to be rather unspecific under certain conditions. Fortunately, some of the indicators, which change their fluorescent pattern in the presence of NO, appear to be promising for the visualization of NO. Since little is known about the spatial spread and the temporal aspects of NO release after a specific stimulus, the use of the specific and non-toxic fluorescent NO indicators could provide a potentially powerful tool to study these aspects of NO release in neuronal tissues in vitro and in vivo. Such measurements, especially in combination with electrophysiological recordings, would greatly further NO research. In addition, based on their fluorescent pattern, these NO-sensitive dyes can be distinguished from the calcium-sensitive dye Fura-2, which allows NO-imaging together with calcium-imaging. This article summarizes recent advances and current trends in the visualization of NO in living neuronal tissues.

Direct Detection of Nitric Oxide and its Roles in Maintaining Gastric Mucosal Integrity Following Ethanol-induced Injury in Rats

In gastric mucosal injury, nitric oxide (NO) plays both cytoprotective and cytotoxic roles, and the NO level is one determinant of these dual roles. We employed electron paramagnetic resonance (EPR)-spectrometry combined with an NO-trapping technique to directly evaluate NO production in ethanol-induced gastric injury in rats. The rat stomach, mounted on an ex vivo chamber, was perfused with ethanol (12.5 and 43%), and NO levels in mucosal tissues were measured during perfusion. Luminal nitrite/nitrate (NOx) content, mucosal blood flow, area of mucosal injury, transmucosal potential difference (PD), and luminal pH were simultaneously monitored with/without preadministration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). NO levels in the gastric tissue increased during ethanol perfusion, and luminal NOx levels increased after the perfusion, accompanying an increase in the area of mucosal injury and changes in physiological parameters. Preadministration of L-NAME aggravated the gastric mucosal damage and suppressed increases in mucosal blood flow in a dose-dependent manner. These results demonstrate that endogenous NO produced in ethanol-induced gastric injury contributes to maintenance of mucosal integrity via regulation of mucosal blood flow.

In situ detection of reactive oxygen species and nitric oxide production in normal and pathological tissues: improvement by differential interference contrast

Reactive oxygen species (ROS), among which nitric oxide (NO) is currently included, play a plethora of (patho)physiological roles. Harman's free radical theory of aging put forth over 40 years ago received full support since then. A nitric oxide hypothesis of aging recently proposed by McCann, is very likely to be the object of widespread investigation in the near future. Therefore, the possibility of localizing at the (sub)cellular level under the light microscope the sites of ROS and NO production with simple and reliable methods appears as a powerful tool for analytic cytology and pathology. Various histochemical methods were developed for visualizing ROS production; a recently improved version to localize superoxide (and possibly also singlet oxygen), based on a DAB–Mn2+–Co2+ reaction, appears very promising. Since the direct detection of NO is still very difficult, the action sites of NO are currently localized by the identification of NO synthase (NOS). The most widespread method to reveal the catalytic activity of NOS is that of demonstrating the fixation-resistant NADPH diaphorase activity with the tetrazolium salt method. We have improved this method by using a tetrazolium salt whose formazan particles are very thin and lipid insoluble (tetranitroblue tetrazolium, TNBT) and by including a tissue protectant, polyvinyl alcohol, in the incubation medium. Here significant examples of application of the DAB–Mn2+–Co2+ technique for ROS and the TNBT–PVA method for NOS to normal liver and brain and to solid tumors are presented. We further document the usefulness of Nomarkis's differential interference contrast (DIC) to analyze wide tissue areas where ROS production or NOS activity is low or even nil. The improved version for NOS allowed for the first time to demonstrate NOS activity in liver fat-storing cells and in astrocyte-like cells in the brain.

Spectrofluorimetric determination of nitric oxide at trace levels with 5,6-diamino-1,3-naphthalene disulfonic acid

Based on the selective reaction that 5,6-diamino-1,3-naphthalene disulfonic acid (DANDS) traps nitric oxide (NO) in the presence of dioxygen to yield the highly fluorescent form, 1-[H]-naphthotriazole-6,8-disulfonic acid in moderately alkaline medium, a new spectrofluorimetric method for the determination of NO has been reported. The method offered the advantage of specificity, sensitivity and a simple protocol for the direct detection of NO in aqueous solution. The linear calibration range for NO was 0.04–1.44 μmol l −1 with a 3 σ detection limit of 0.6 nmol l −1. The proposed method has been used to monitor the release of NO from S-nitrosocysteine, a NO-releasing agent.

BLADDER DYSFUNCTION AFTER ACUTE URINARY RETENTION IN RATS

We investigated bladder function in acute urinary retention and subsequent catheterization in rat bladders. The penile urethra in rats was clamped with a small clip and cystostomy was performed to infuse 3 ml. of saline for inducing acute urinary retention. At 30 minutes after the induction of urinary retention the cystostomy was opened to empty the bladder. In functional studies contractile responses to carbachol were measured in group 1-before, group 2-at 3 ml. of urinary retention, group 3-at 3 ml. of urinary retention exposed for 30 minutes and group 4-30 minutes after catheterization. Moreover, in vivo real-time monitoring of blood flow and vesical pressure were measured in the bladders with a laser Doppler flowmeter and cystometrography, respectively. Malonaldehyde and 4-hydroxyalkenals were measured by colorimetric assay in these groups. In functional studies the mean maximum contractile response value plus or minus standard deviation of carbachol-to-bladder in groups 1 to 4 was 11.8 +/- 1.3, 11.9 +/- 1.7, 9.8 +/- 0.8 and 6.9 +/- 0.7 gm./mm.2, respectively. In real-time monitoring of blood flow and vesical pressure acute urinary retention significantly decreased blood flow and increased vesical pressure, and subsequent catheterization increased blood flow and decreased vesical pressure in the bladders. The concentrations of malonaldehyde and 4-hydroxyalkenals in the bladders in group 4 were significantly higher than in the other groups. Our data indicate that bladder dysfunction after catheterization is partially caused by free radicals, which have an important role in bladder dysfunction during acute urinary retention.