INTRODUCTION

Nitric oxide (^·NO)¹ is an ubiquitous endogenously produced free radical. The physicochemical properties allow ^·NO to serve as a biological messenger. ^·NO can exert cytoprotective and cytotoxic effects depending on its concentration, site of generation, and the reactions it undergoes (1-3). Released from endothelial cells, it acts as an endothelium-derived relaxing factor (EDRF) with anticoagulant and antithrombotic properties (4). Disturbances in ^·NO release and its decreased stability and bioactivity are proposed to be a major part of vascular diseases such as atherosclerosis, ischemia, or hypertension (5-7).

Like ^·NO, superoxide anion (O₂) is a free radical with a relatively low overall reactivity (e.g. compared with the hydroxyl radical, ^·OH). It is formed as an intermediate in a variety of enzymatic reactions and is kept within a physiological concentration range by superoxide dismutase (SOD, EC 1.15.1.1) (8, 9). It has been shown that SOD increases the half-life of EDRF released from isolated arteries (10). Elevated O₂ production (e.g. via the xanthine oxidase, arachidonic acid, or NADH oxidoreductase pathway) and a concomitant increase in cytotoxicity are described for different pathological conditions (11). The reaction of O₂ with ^·NO at a diffusion controlled rate not only depletes both radicals, it also leads to the formation of more toxic species, such as peroxynitrite (12, 13). Thus, superoxide dismutase-mimetic compounds could be of therapeutic interest in the conventional context (e.g. as antiinflammatory agents; Ref. 14) but also by increasing the lifetime and biological activity of ^·NO. 3-Morpholinosydnonimine (SIN-1), a well known nitrovasodilator simultaneously releasing ^·NO and O₂ (15), provides a model system for evaluation of the interaction of both species and the influence of SOD-mimetic compounds.

Among SOD-mimetic compounds, low molecular weight copper or iron complexes were found to be very effective (16, 17). Cytoprotection, at least partly due to SOD-like activity, was also reported for nitroxides, a class of free radicals widely applied as tools in electron paramagnetic resonance (EPR) spectroscopy (18). A subgroup of nitroxides, nitronyl nitroxides, can be used for ^·NO detection (19); however, reduction by O₂ and other reducing agents limits its application (20). As shown in Scheme 1, nitroxides can oxidize O₂ to molecular oxygen (I). The resulting hydroxylamine is EPR-silent and is oxidized back to the nitroxide by reducing another O₂ to hydrogen peroxide (H₂O₂) (II) (21).

Scheme 1.

Cell culture studies revealed an inhibition of superoxide-mediated cytotoxicity and mutagenicity by nitroxides similar to SOD (22, 23). Although SOD activity of nitroxides could not be found by stopped-flow kinetic analysis (24), nitroxides have been identified as genuine SOD mimetics rather than O₂ scavengers by direct and indirect physicochemical methods (25). Additionally, protection against oxidative damage independent of O₂ and H₂O₂ was found and proposed to result from nitroxide-mediated oxidation of redox-active trace metal ions (26). Because of the predominantly intracellular generation of O₂, membrane permeation by SOD mimetics or superoxide scavengers applied to biological systems is an important aspect. Moreover, stability and toxicity have to be considered. Nitroxides fulfill these requirements (27); they are relatively stable low molecular weight compounds with non-immunogenic properties, their toxicity is low, and, most important, their synthesis allows large variations of physicochemical properties, such as lipophilicity.

However, the effect of nitroxides on ^·NO generated by model systems (e.g. SIN-1) or endothelial cells has not been investigated so far. This study demonstrates that, similar to SOD (28, 29), nitroxides augment the detectable amount of ^·NO released from SIN-1 and cultured endothelial cells by selectively removing O₂.

EXPERIMENTAL PROCEDURES

Materials

SOD was purchased from Boehringer Mannheim and 5,5-dimethylpyrroline-N-oxide (DMPO, used as described previously; Ref. 30) from Aldrich. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and 3-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl (3-carboxy-proxyl, CP) were obtained from Sigma; 3-ethoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (3-ethoxycarbonyl-proxyl, ECP) was synthesized starting from CP (31). SIN-1 was supplied by Alexis (San Diego, CA). Gases were provided by Middleton Bay Airgas (San Leandro, CA) and Messer Griessheim (Berlin, Germany). All commercial chemicals were of the highest quality available and purchased from Sigma if not stated otherwise.

Endothelial Cell Culture

Bovine aortic endothelial cells (BAEC, passages 14-18) (32) were cultivated on 25-cm² flasks (Corning Costar, Cambridge, MA) in minimal essential medium (Eagle's salt) supplemented with 10% fetal calf serum, 0.2 M glutamine, without antibiotics at 37 °C in 5% CO₂ and 95% air. Subcultivation was made twice a week with trypsin/EDTA (0.25% v/v each).

Bovine atrial endothelial cells (BAtEC) were prepared as described by Ryan et al. (33). The cells were subcultivated to confluence on Corning flasks (162 cm²), seeded at passage 14 in a ratio of 1:3 on six-well plates (Corning), and used at confluence after 3 days of culture. The culture medium was Medium 199 (Earle's salts) containing 2.2 g/liter sodium bicarbonate and 25 mM HEPES buffer (Life Technologies, Inc.) supplemented with 10% fetal calf serum (HyClone, Logan, UT) and 10% newborn calf serum (Life Technologies, Inc.), 1.0 µM thymidine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 0.25 µg/ml amphotericin B (Life Technologies, Inc.).

Quality of the endothelial cell cultures was verified by phase contrast microscope-detected cobblestone appearance at confluence, presence of factor VIII antigen, contents of alkaline phosphatase, and angiotensin-converting enzyme.

All cell experiments were performed in physiological salt solution (PSS) supplemented with 1 mM L-arginine. For BAEC phosphate-buffered PSS (composition in mM: 136.9 NaCl, 2.7 KCl, 9.6 Na₂HPO₄, 1.5 KH₂PO₄, 0.5 MgCl₂, 0.9 CaCl₂, pH 7.4) (Biochrom, Berlin, Germany) was used, whereas experiments with BAtEC were performed in 10 mM HEPES-buffered PSS (composition in mM: 140.0 NaCl, 5.0 KCl, 4.5 NaHEPES, 5.5 HEPES, 1.0 MgCl₂, 0.0034 EDTA, 1.5 CaCl₂, pH 7.4). Prior to the experiments, cells were washed two times with PSS.

^·NO Chemiluminescence

The determination of ^·NO was performed using a ^·NO analyzer NOA270B (BAEC experiments) or NOA280 (BAtEC experiments) (Sievers, Boulder, CO) without use of a reducing agent. The output signal (mV) is proportional to the amount of ^·NO. The signal was recorded using a chart recorder (Gould, Cleveland, OH) and a modified high performance liquid chromatography data acquisition and calculation system (ACCESS CHROM, Perkin Elmer Nelson Systems, Cupertino, CA) and either expressed as mV (headspace) or as nanomolar concentration of ^·NO (supernatant). The ^·NO concentration (nM) calculation was performed only with samples from the supernatant solution, where ^·NO standards were used as a basis of calculation.

Detection of ^·NO Released from Cultured Endothelial Cells

The ^·NO release from endothelial cells was measured (a) in samples of the supernatant solution (for BAtEC) and (b) continuously in the headspace (for BAEC). All cell experiments were performed at 37 °C.

Cells grown in six-well plates were washed twice with PSS and covered with 1 ml of PSS containing the required agents. After incubation for 30 min, 0.8 ml of the supernatant was injected into a special purge vessel with a gas-tight syringe (Hamilton, Reno, NV). ^·NO was expelled and transported to the reaction chamber by a stream of helium. The area under the resulting peak was calculated and calibrated with peak areas from ^·NO standards.

Cells grown in flasks (25 cm²) were washed as described for (a) and covered with 2 ml of PSS. Through a tube placed above the supernatant, headspace gas was drawn by vacuum continuously into the reaction chamber. Under these conditions, determination of ^·NO is based on the fixed distribution of gases between liquid and gas phase depending on their solubility. During the experiment the flask was shaken gently in a shaking incubator (Boekel Industries, Feasterville-Trevose, PA).

Detection of ^·NO released from SIN-1

For experiments with SIN-1, 3 ml of freshly prepared aqueous solution was placed into a sealed glass vessel and stirred with a magnetic stirrer. The rubber seal contained two tubes: one for headspace gas that was drawn into the reaction chamber and one inlet for gas exchange. The cell-free experiments were performed at room temperature (22 °C).

^·NO Standard

^·NO released into the supernatant was quantified by comparison with ^·NO standards. Diluted ^·NO gases (65 and 6.8 ppm in N₂) were used as standards. For preparation of ^·NO solutions, deionized water was deaerated, saturated with argon, and gassed with a continuous stream of diluted ^·NO (65 ppm) for at least 20 min, resulting in a concentration of 130 nM at 22 °C. There was no significant difference in the area under the curve using gaseous (6.8 ppm) or liquid standards containing the same amount of ^·NO. Standard curves were recorded each day. The detection limit was less than 1 pmol (1 nM ^·NO concentration for an injected sample volume of 1 ml) at a signal-to-noise ratio of 3 for single injections of ^·NO. The response was linear at least up to 1 µM ^·NO concentration.

EPR Spectroscopy

EPR experiments were carried out at room temperature on a Bruker ECS 106 X-band spectrometer (equipped with a high sensitivity rectangular-mode cavity ER 4102 ST). The samples were placed into a flat quartz cell, and standard experimental conditions were as follows: modulation frequency, 100 kHz; modulation amplitude, 0.1 mT; field set, 347.5 mT; scan range, 10.0 mT; microwave power, 10 milliwatts.

Protein Determination

The protein content of BAtEC was measured for selected experiments with a kit for protein determination (per procedure no. TPRO-562, Sigma). Cells were lysed by incubation with 1% Triton X-100 (v/v) for 30 min. Bovine serum albumin was used as standard.

Cytotoxicity Detection

Cell death was evaluated by quantification of lactate dehydrogenase (LDH) release into the cell supernatant as an index for plasma membrane damage (34). The concentration of LDH was measured with a commercially available cytotoxicity detection kit (Boehringer Mannheim).

Calculations and Statistical Analysis

The dose response of SOD and nitroxides on ^·NO release was done in sets of n = 4-6 (n represents the number of experiments performed on different cell cultures or model systems). ^·NO concentration was expressed as mean ± S.E. Significant difference between means of control and treatment groups was calculated by Student's t test; a value of p < 0.01 was accepted for statistically significant difference.

RESULTS

SIN-1 was chosen to study the interaction of ^·NO with O₂ in a cell-free system. The ^·NO release from freshly prepared aqueous solutions of SIN-1 increased during the first 30-40 min and reached afterwards a stable plateau (Fig. 1, inset) as detected by ozone-mediated chemiluminescence. During the plateau phase, SOD was added to remove O₂ produced by SIN-1. The detectable amount of ^·NO was rapidly elevated by a factor of 16.7 (Fig. 1, trace a; Table I). A similar but less prominent effect was observed when the nitroxides were added under the same conditions (Fig. 1, traces b-d; Table I).

Table I. Increase of detectable ·NO released from SIN-1 and from BAtEC by SOD and nitroxides Compound SIN-1a BAtECb Superoxide dismutase 16.7 2.7 TEMPOL 6.2 1.6 3-Ethoxycarbonyl-proxyl 3.7 1.4 3-Carboxy-proxyl 4.5 1.5 a The values for SIN-1 represent the -fold increase of detectable ·NO in the headspace 15 min after addition of SOD (100 units/ml) or nitroxides (10 µM). b The values for BAtEC are the -fold increase of detectable ·NO (measured in the supernatant) by SOD or nitroxides (100 µM) after 30 min of coincubation with 10 µM A23187.

Addition of 1 mM SIN-1 to a 0.1 M DMPO solution resulted in the appearance of a spin adduct spectrum with a_N = a_H = 1.49 mT, indicating the formation of the DMPO-hydroxyl radical adduct (DMPO/^·OH, Fig. 2a). In the presence of either 100 units/ml SOD or 10 µM TEMPOL (giving a strong triplet signal superimposition), a significant increase of the spin adduct signal intensity was found (Fig. 2, b and c). When catalase (0.5 mg/ml) was added to the reaction mixtures, formation of DMPO spin adducts was prevented both in the presence of SOD (Fig. 2d) and nitroxides (spectra not shown).

Endothelial cell monolayers grown in a culture flask are not exposed to flow or shear stress; unstimulated ("basal") ^·NO release from BAEC was not detected in the headspace of this system. A sustained and reproducible ^·NO signal was measured after incubation with 1-10 µM Ca²⁺ ionophore A23187 (5 µM, Fig. 3). ^·NO release was abolished by 0.1 mM N-nitro-L-arginine methyl ester (L-NAME) in the absence of exogenous L-arginine.

Similar to BAEC, exposure of BAtEC to 10 µM A23187 in a six-well plate resulted in an increase of ^·NO concentration in the supernatant reaching steady state after approximately 15 min and lasting for at least 30 min (12.4 ± 0.7 nM, corresponding to a production rate of approximately 2.3 pmol of ^·NO/min/mg of protein).

Addition of SOD resulted in an increase of the amount of ^·NO detected using both BAEC and BAtEC after stimulation with A23187. Fig. 3 displays a representative experiment with BAEC, whereas Fig. 4 illustrates SOD-induced concentration-dependent augmentation of detectable ^·NO released from BAtEC stimulated with different concentrations of A23187. Higher concentrations of SOD (up to 300 units/ml) did not cause a further increase of detectable ^·NO (data not shown).

Addition of TEMPOL to the supernatant of endothelial cells resulted in an effect similar to SOD (see Fig. 3). Fig. 5 shows the dose dependence of the effect of nitroxides on ^·NO released from BAtEC. Starting at concentrations of 10 µM for TEMPOL and 1 µM for ECP and CP, the nitroxides significantly (p < 0.01) increased the detectable ^·NO concentration in the supernatant. The maximum effects of SOD and nitroxides on ^·NO release from both SIN-1 and endothelial cells are summarized in Table I.

DISCUSSION

The study of the interaction of O₂ and ^·NO is complicated by several factors; both species are unstable, the rate constant of their reaction is exceptionally high, and the concomitant formation of other reactive species has to be considered. These restrictions were taken into account by application of appropriate methods for detection of these radicals: ozone-mediated chemiluminescence (for authentic ^·NO) and EPR spectroscopy (for other radical species). This approach allowed us to study an effect of SOD-mimetic nitroxides that has not been investigated so far: the influence of these SOD-mimetic compounds on ^·NO released from SIN-1 and cultured endothelial cells.

Two different modes of ^·NO analysis were used in this study: (a) headspace measurements for qualitative time-course assessment, and (b) discrete sample collection from the supernatant of cells at single time points. The assay of the ^·NO detection in solution represents absolute values for dissolved ^·NO, whereas headspace measurements allow resolution of the time course of the ^·NO generation within a period of 5 s.

Aqueous solutions of 1 mM SIN-1 resulted in a stable flux of ^·NO into the headspace after an initial lag phase of approximately 30-40 min. Similarly, spontaneous ^·NO release measured by the conversion of oxyhemoglobin to methemoglobin has been described by Feelisch et al. (15). In contrast, using a chemiluminescence technique with a helium-flushed, gas-permeable tubing inserted into the SIN-1 solution for sample collection, Beckman et al. did not observe ^·NO release in vitro in the absence of SOD (35). An effective removal of ^·NO from the headspace, as it occurs by reaction with hemoglobin, reduces its depletion by O₂. Since O₂ and ^·OH enhance sydnonimine decomposition (conversion of SIN-1 to SIN-1A as the rate-limiting step; Ref. 15), an increase in O₂ lifetime would potentially favor a higher ^·NO liberation until a new equilibrium between ^·NO release into the headspace and ^·NO reaction with O₂ is reached. Furthermore, the oxygen-dependent breakdown of SIN-1A might decline under conditions where no oxygen supply is guaranteed, as it would happen in an airtight sealed reaction vessel.

Addition of 100 units/ml SOD to SIN-1 solution at the plateau phase of ^·NO release caused a significant 16.7-fold increase in the headspace concentration of ^·NO (Table I). This effect was also observed after addition of the nitroxides (10 µM). The order of efficacy was as follows: SOD > TEMPOL > CP > ECP (Table I).

EPR spin trapping experiments using DMPO were performed to characterize the radical species formed during the decomposition of SIN-1 and to verify the SOD-mimetic activity of nitroxides (26, 27). Only DMPO/^·OH adducts were detected. Since the reaction rate of ^·NO with O₂ (6.7 × 10⁹ M¹ s¹) and the rate for the spontaneous dismutation of O₂ to H₂O₂ (in the range of 10⁵ M¹ s¹ at pH 7.4; Ref. 36) is much higher than that of O₂ with DMPO (in the range of 10¹ M¹ s¹; Ref. 37), virtually no O₂ can be trapped by DMPO under these conditions. In the presence of trace amounts of metal ions, in particular ferrous ions, reaction of O₂ with H₂O₂ leads to the formation of ^·OH (Fenton reaction), which in turn reacts with DMPO (in control experiments, addition of iron chelators resulted in a significant reduction of spin adduct formation). In the presence of SOD, the dismutation of O₂ (leading to formation of H₂O₂) is accelerated, increasing the formation of ^·OH via the Fenton reaction. In the same way, nitroxides elevated the signal intensity of the DMPO/^·OH adducts. Addition of catalase effectively removes H₂O₂, and the formation of DMPO/^·OH is prevented both in the presence and absence of SOD or nitroxides. These results are supported by a recent study showing that SIN-1-mediated cytotoxicity of HepG2 cells is increased by SOD but completely abolished in the presence of catalase (38). Our data clearly demonstrate the SOD-mimetic activity of nitroxides in the SIN-1 model system. Furthermore, there is no indication of peroxynitrite-dependent ^·OH formation, which would be independent from catalase activity (35, 39).

To study the effect of nitroxides on ^·NO produced by cultured endothelial cells, a stable and maintained ^·NO release was induced by stimulation of the cells with the Ca²⁺ ionophore A23187 for up to 30 min. Within this time frame, there was no sign of decreased cell viability as measured by release of LDH into the extracellular space (data not shown). Increase of intracellular free calcium concentration is a stimulus not only for ^·NO production (i.e. activation of endothelial ^·NO synthase, ecNOS), but for other processes as well, including production of O₂ (40, 41), which could decrease the detectable amount of ^·NO (28). Additionally, tetrahydrobiopterin, a cofactor of NOS, was found to rapidly oxidize ^·NO through the generation of O₂ (42). The present study shows that SOD as well as three different nitroxides significantly enhanced the detectable amount of ^·NO released from cultured endothelial cells by removing O₂. Continuous monitoring showed an instantaneous elevation of the steady-state ^·NO concentration in the headspace gas. This observation has been confirmed by experiments measuring ^·NO accumulated in the supernatant of cells. The maximum increase in ^·NO concentration was observed at 30 units/ml SOD (200 nM) and 30-100 µM nitroxides. The higher concentration required for nitroxides versus SOD (factor > 10²), may be explained by the difference in the rate constants for the reaction of O₂ with SOD or TEMPOL (i.e. 2 × 10⁹ M¹ s¹ (43) versus 4.0 × 10⁵ M¹ s¹ (44), respectively). The rank order of efficacy for the four compounds tested was the same in the cell culture and SIN-1 experiments: SOD > TEMPOL > CP > ECP (Table I).

The increased detectable concentration of ^·NO in the presence of the nitroxides could be explained either by enhanced ^·NO release, decreased ^·NO removal, or a combination of both. Considering the chemistry of SIN-1 degradation (45), there is no reaction known by which SOD or the nitroxides would selectively increase ^·NO liberation without a concomitant increase in O₂ release. Similarly, an activation of ecNOS (direct or indirect via increase of intracellular free calcium concentration or cofactors) leading to an elevated ^·NO biosynthesis is rather unlikely. Therefore, the most probable explanation is that the nitroxides increase the detectable amount of NO via reducing ^·NO degradation by removal of O₂ from the system. The EPR data clearly demonstrated an SOD-like action of the investigated nitroxides in the SIN-1 model. Thus, the most probable explanation for nitroxide-induced increase in detectable ^·NO release from endothelial cells is the selective removal of O₂ by these compounds.

Genetically elevated amounts of endogenous SOD (46), SOD derivatives (47), or SOD-mimetic nitroxides (18) have been reported to reduce the cytotoxic effect of oxygen free radicals in vitro and in vivo. On the other hand, it must be mentioned that decreasing the O₂ concentration alone by catalyzing its dismutation does not necessarily reduce an elevated cytotoxic potential (38). The spin trapping experiments point out that catalase activity is required for the further destruction of H₂O₂ to nontoxic species.

In summary, this study demonstrates that the SOD-mimetic activity of nitroxides increase the amount of bioavailable and detectable ^·NO in vitro. Since O₂ is a main cause of ^·NO degradation, its removal would be beneficial especially in situations when O₂ is increased leading to the inactivation of ^·NO and formation of toxic species such as peroxynitrite (i.e. in arteriosclerosis or inflammation). Since the discovery of SOD-mediated increase in stability of EDRF or ^·NO in in vitro systems (28, 29), the body of evidence is growing supporting a role of O₂ and its effective removal by superoxide dismutation as determinants of ^·NO bioavailability under physiological and pathological conditions. Nitroxides exert the properties of potential pharmacological agents since they are (compared with SOD) relatively stable low molecular weight compounds without immunogenic properties and with low toxicity (26, 48). Their structure allows synthetic modifications necessary for adaptation to the intended use. Nevertheless, it has to be determined in further in vitro and in vivo studies whether the SOD-mimetic properties of nitroxides could be of therapeutic importance against O₂-mediated lowering of bioavailable ^·NO.