Dojindo Newsletter No.1(1995)

Nitric Oxide: Mysterious Messenger

-Its chemistry, biology and new reagents for research-

Yoshiki Katayama

Molecular Systems Group
Department of Materials Physics and Chemistry
Graduate School of Engineering
Kyushu University

1. NO chemistry
2. Bioactivity of NO and NO metabolites.
3. Reagents for research on the NO pathway
4. SNAP (S-nitrosothiol)
5. SIN-1 (peroxynitrite donor)
6. Carboxy-PTIO (NO scavenger)
7. MGD-Fe2+ complex (water-soluble NO spin trapper)
8. References

Nitric oxide is an extremely important and versatile messenger in the biological system. At first, it was recognized as an endothelium-derived relaxing factor in the vascular system(1). It has also been identified as a neurotransmitter or neuromodurator in the neuronal system (2) and a cytotoxic factor in the immune system (3,4). Also, it is believed to be related to some tissue damage such as ischemia/reperfusion tissue(5-7) damage and excitatory neuronal death(8-10).
Figure 1 shows simplified NO reactions in a biological system. NO is synthesized by a unique enzyme, NO synthase, which is a self-sufficient P450 -type enzymatic complex(11). Then, it can diffuse into all direction passing through every cell membrane. Although the basic scheme in Figure 1A is very similar to any other messengers, including cyclic AMP, cyclic GMP, IP3, or hormones, the exact function of NO is unknown. For example, the function of NO in ischemia/reperfusional tissue damage has not been clarified, although NO should play an important role in the system.

Figure 1. Simplified pathway of endogenous NO.

Presumably, NO has another unique event between the origin and the targets in its reaction scheme (Figure 1B). Understanding the unique factor is important for clarifying the mysterious functions of NO. However, first we have to understand the chemistry of NO.
NO is a simple hydrophobic gaseous molecule that is highly diffusible.Even more important is its high reactivity. NO is a free radical that has a one-pair electron in a 2p-pai antibonding orbital. Thus, NO is extremely unstable; it cannot maintain its original form for very long in a biological environment. As a result, NO undergoes complex cahanges immediately after being released. The possible reactions of NO in physiological conditions are shown in Figure 2. There are some important metabolites derived from NO that may have bioactivities similar to or different from those of NO. Figure 2 shows that the reactions many be caused not by the NO molecule itself but by other NO-related metabolites. We need to understand the NO chemistry and each reaction. Thus, in this review I describe the NO chemistry, explain the bioactions of each NO metabolite, and introduce some new reagents for elucidating NO functions in a more elegant way.

Figure 2. Reaction of NO related compounds in biological environment.

1. NO chemistry
Because nitric oxide is a free radical, so it has dramatic reactivity. It is quite stable in absolute pure water and can be dissolved to 2 mM at room temperature. However, if molecular oxygen is dissolved in water, NO reacts with the oxygen very rapidly, as shown in Equations 1-5. Reaction 4 is much faster than reaction 2, so simple aqueous NO (12) solution gives nitrite anion as the decomposed product.

In fact, nitrite anion was obtained as the main product from the decomposition of NOC reagents that penetrate the NO molecule in pure form in aqueous media.
More important categories of NO chemistry relative to biological action are the following three forms, .NO, NO+, and NO-, as proposed by Stamler et al(12). NO has one electron in the 2p-pai antibonding orbital. If this electron is lost through oxidation, NO changes to the nitrosonium cation (NO+), and if another electron is added in the orbital through reduction, the nitronyl anion (NO-) is made.

1-1. .NO
The main reactivity of .NO is the donation to some transition metal cations. For example, NO binds to the ferrous ion (Fe2+) in deoxyhemoglobin to make a stable complex. The binding constant is 5E7 M-1s-1, while the dissociation constant is E-5s-1 (13). NO also reacts with other metalloproteins. In fact, it extracts the iron from the iron-sulfur center of some enzymes in the mitochondrial electron transfer system to generate stable iron nitrosyl, which inhibits respiration (13). Various iron-nitrosyl compounds are also formed in activated macrophages (14). One of the characteristic binding properties of NO is the ability to bind with Fe(III)-heme (12), which is different from O2 or CO. The complex produced, however, is so unstable that it tends to release NO+ equivalent with an attack of neucleophiles (thiol, amine etc.).

1-2. NO+
Although NO+ can be present in aqueous media, a major form of NO+ equivalent is nitroso compounds in biological environments. Nitroso compounds can be made chemically through the reaction of sodium nitrite and nucleophile in the presence of acid. However, biological production of nitroso compounds may occur in the following reactions: .NO2 and thiols or amines (Equation 6)(15)and disulfide (Equation 7) (16). .NO2 is generated by NO through a reaction with oxygen or from N2O3 or N2O4.

N2O3 could react with the nucleophile to give the nitrosocompound directly (Equation 8).

Another possibility is the nucleophilic attack of thiols or amines to the Fe(II)-NO+ complex, which occurs through the reaction of the NO and Fe(III)complex (17).
Nitrosocompounds act as NO+ equivalents and can be regarded as NO+ carriers in physiological conditions. The most important form of nitrosocompounds is S-nitrosothiols in a biological system. Amines can also be N-nitrosocompounds. However, ordinary amines are usually protonated in physiological pH to prohibit the nitrosation. S-Nitrosothiol can release NO spontaneously through the following homolytic cleavage (9). This NO release is accelerated in the presence of thiols.

S-nitrosothiol cannot be made by NO and thiol. In this case, the thiol is simply oxidized by NO to form disulfide (Eqation 10)(16).

1-3. NO-
NO can be reduced to a very stable form, NO-. NO- is formed biologically with 4-electron oxidation of guanidino nitrogen in arginine by NO synthase(18). It may also be generated from NO with SOD(Cu(I)). In a simple aqueous solution, NO- changes rapidly to N2O with the rate constant of 2E9 M-1s-1. NO- can also make an iron complex through a reaction with Fe(III)-heme. The physiological meaning of NO- has not been clarified, but Murphy et al. suggested NO- might act as a stabilized form of NO to prolong its lifetime with Superoxide dismutase(SOD)(19).

1-4. Peroxynitrite anion (ONOO-)
NO also reacts with the superoxide anion at the rate constant of 3.7E7 M-1s-1 to make peroxynitrite (Equation 11)(20). The anion starts to decompose with the protonation to make pernitrous acid.

The pKa value of the acid is 6.8, so about 20 % of the peroxynitrite anion is protonated at physiological pH(21). For example, the lifetime at pH 7.5 is reported to be about 1.9 s(22). The decomposition of ONOOH occurs either by intramolecular rearrangement, which forms nitric acid, or by homolytic cleavage, which generates .NO2 and .OH. In this case, a very radical oxidant, hydroxyl radical, is formed. The predominant cleavage is determined by the conformation of the ONOOH molecule(21). If the molecule takes cis conformation, intramolecular rearrangements occur, producing nitrate and proton. On the other hand, trans conformation leads to homolysis. Higher pH is said to result in nitrate production.

Although it has been reported that thermodynamic study tent to prohibit the homolysis of peroxynitrite. Others have reported capturing the hydroxyl radical, the product of the homolysis, indirectly(22,23).

2. Bioactivity of NO and NO metabolites
Figure 2 shows NO yields various metabolites in biological environment once it is formed. Although the bioaction of each metabolite has not been clarified yet, it may be similar to or different from the bioactivity of NO. In this chapter, the possible bioactions of each NO- derived metabolite are introduced. Among the NO-derived metabolites, S-nitrosothiol and peroxynitrite will be some of the most important compounds. NO2 and iron-nitrosyl should not be overlooked.

2-1. NO
(a) NO in the vascular system
Endothelium derived reluxing factor (EDRF) was discovered by Furchgott(24), and Moncada et al.(1) suggested that NO is one of the strongest candidates of EDRF. Through the work of both groups, the vasodilating activity of NO has been well characterized. The major source of NO that causes vasodilatation is endothelial NO synthase. Endothelial NOS is activated with intracellular calcium concentration increase by agonists like acetylcholine (25). NO is released to the neighboring smooth muscle, causing relaxation of the blood vessels(1). NO also diffuses to into the bloodstream to produce NO-hemoglobin(26) or met-hemoglobin and to inhibit platelet aggregation(27). Another important source of NO in the vasodilating system is NOergic neurons. Toda et al. reported that there are many noncholinergic, nonadrenergic neurons surrounding many blood vessels excepting coronary arteries. Those nerve cells are stained with neuronal NOS antibody(28-33). They have claimed that such toxic NO is an important factor in regulating blood flow, and EDRF is associated with the toxic NO.
The NO from endothelium and NOergic neurons may be related to certain pathological conditions. For example, some blood vessels, especially in the heart, lung and brain, seriously damage the surrounding tissue when ischemia reperfusion occurs in conjunction with accelerated NO production(34). Although it is believed that NO is related to the tissue damage, it has not been determined why excess NO is produced. Some researchers have proposed that NO is generated to protect the tissue from damage (34-37), but others claim that NO caused the tissue damage(7,38-41). In ischemic conditions, NO will be generated to protect the tissue and improve blood flow. However, the function of NO produced in a reperfusional system has not been clarified. It is also reported that the peroxynitrite anion, which is made by co-producing superoxide and NO, may be related to tissue damage(42). Kitakaze et al. reported that NOS is activated by acidosis in ischemic conditions. The acidosis activates Na+/H+ and then Na+/Ca+ pumps in endothelium to cause intracellular calcium ionic increase(43). NO from inducible NOS(iNOS) in macrophages, nucleophiles and smooth muscle cells in blood vessels, may be another source of this pathological NO production.

(b) Neuronal NO
NO is also generated in the neuron and the grial cell in central or peripheral nerve systems (44). NO made in the central nerve system has drawn the attention of many researchers because it may be closely related to neuronal plasticity(45). Such NO is released by neuronal NOS, another constitutive type that is soluble in cytosol(46,47). The NOS is also activated with an intracellular calcium increase through agonist-receptor interactions. In the hippocampus, induction of long term potenticution or depression(LTP or LTD) has been reported(45, 48-50). However, carbon monoxide may be the key mediator of the events(51,52), and the NO- generating system seems to have some interaction with CO generation(53). Thus, NO may play role. In the cerebellum, NO clearly is related to the formation of LTD (54,55). Cerebellum LTD can be induced by co-stimulation of the climbing fiber and the parallel fiber connected to the prukinje cell. If the bergman gria surrounding the neuron is intact, LT formation is regulated with NO. Shibuki et al.(56) hypothesized that the grial cell prohibited LT formation and NO unlocked the prohibition, while the direction of LT, so that LTP or LTD, was determined by the calcium concentration change in the prukinje cell. The participation of the grial cell must be considered to better understand the NO function in the nerve system.
NO also directly regulated the formation of ion channels or receptors. NO modulates the potassium channel(57-59) to regulate the neuronal transmission. Stamler et al. suggested that NO took back-regulation to the NMDA receptor through nitrosation in the redox regulatory site(10). Rao et al. indicated that intestinal ion transport got the tonic regulation by NO released from the non adrenergic noc cholinergic neuron(NANC neuron)(60). Okamoto et al. indicated that the response of the ionotropic glutamate receptor to AMPA is modulated by NO that is released with the stimulation of the metabotropic glutamate receptor in the cerebellum in chick embryo(61). However, the modulation of ion channels and receptors may be related not to the NO molecule but to the nitrosothiol structure.
In the peripheral nerve system, the mainfunction of NO is vasoregulation(62). The renal blood flow is suggested to be regulated indirectly with NO through the attenuation of sympathetic neuronal activity in addition to the direct action (63,64). Whether NO originates in the NANC neuron or in the central nervous system is unknown.
Another major area of study is the relationship between neuronal NO and excitatory neurotoxicity (8). Nerve cells tend to die from overstimulation of excitatory neurotransmitters or agonists. It is unclear whether NO is protective (10,65) or destructive (40,66). Although NO or the NO donor lead to marked neurotoxicity(67), the real cause of the toxicity may be not NO, but ONOO-. The nerve cell containing NOS activity is a quite large interneuron that has many neurofibers but few dendrites (68). It is believed that such neurons have some resistance to excitatory neurotoxicity. It may be caused by the attenuation of calcium sensitivity of enzymes relating to signal transduction, as reported by Murota et al. (69). Yamashita et al. did not recognize the glutamate tolerance of the NOS-containing neuron in cerebral ischemia with occlusion of the middle cerebral artery(70). It may be that the origin of the NO is iNOS in microgrias or macrophages in addition to neuronal NOS in the nerve cell.
Recently, the presence of neuronal NOS was reported in the skeletal muscle cell(71).

(c) NO in the immune system
Immune cells, including activated macrophage, nuclephile, monocyte, and kuppfer cells can release a greater amount of NO than endothelium or nerve cells. The origin of NO is in the different type of NOS from the constitutive NOS mentioned above. This type of NOS is induced with the stimulation of cytokines or lipopolysaccharide(LPS) and is called inducible NOS (iNOS)(72,73). The major function of the NO from iNOS is the cytostatic and cytotoxic effects on invading microorganisms or tumor cells (74,75). The mechanism of the cytotoxicity is in the following two categories. First is the inhibition of the respiration of mitochondria (76). NO strongly interacts with and inhibits some enzymes in the electron transfer system, because such enzymes contain an Iron-sulfur center in the catalytic site. Second is the direct modulation to the DNA synthesis to inhibit some enzymes(77,78). NO-releasing agents usually tend to inhibit cell proliferation and cell mitosis(79). The expected effect is thymidine uptake. NO is supposed to mediate the apoptosis of many cells (80). For example, macrophages activated with INF-gamma and LPS induce apoptosis with self-generating NO. NOS inhibits the phenomenon (81).
Although NO production of iNOS is essential for the defense system of an organism, this NO is sometimes related to pathological conditions, including sepsis, ischemia/reperfusion, acute pulmonary injury, multiple organ failure syndrome, and atherosclerosis. Septic shock is one of the most serious pathological conditions. LPS, which is a constituent of bacterial outer membrane, causes some serious damage to the biological system, including a decrease in blood pressure, cardiovascular and kidney damage, and bleeding in some organs. In such conditions, NO is formed in abnormally large amounts, causing a serious drop in blood pressure(82,83). It is still uncertain whether a complete scavenging of the NO is desirable for the improvement of the pathological condition. However, some researchers have found that certain NO scavenging agents increased the survival rate of the animals they tested(84).

2-2. S-Nitrosothiol
EDRF has two types of components: short-lived and long-acting. One of the strong candidates of the long-acting component is S-nitrosothiol(85). The chemical species that is formed with .NO2 or iron-nitrosyl and thiol is a spontaneous NO releaser. Nitrosothiol, mainly a S-nitrosoalbumin, a circulates in the bloodstream at uM levels(86). It is obvious that this type of compound has a potent vasorelaxing property. However, it is doubtful that the spontaneously released NO is related to the EDRF function(87). Because, there are only 6 times of the difference in vasorelaxability of various S-nitrosothiols in spite of the tremendous difference(50-100 times) of the amount of spontaneously released NO. In addition, there is reverse correlation between the vasodilating activity and NO releasing ability. The effects of some agents that influence the NO generation and lifetime are also reversed between vasorelaxation and NO-generating activity in S-nitrosothiols(17). N-acetylpenicillamine augmented the NO production from S-nitroso-N-acetylpenicillamine(SNAP), and SOD attenuated it. However, the vasodilating activity declined with N-acetylpenicillamine and increased in the presence of SOD(87). Kowaluk et al. reported that the metabolism of S-nitrosothiol in the cell membrane is important in the EDRF activity of S-nitrosothiols(87).
Another important S-nitrosothiol in intracellular space is S-nitrosoglutathione, because glutathione is present at mM levels in the inner cell. Depletion of intracellular glutathione and formation of S-nitrosoglutathione were shown in activated macrophages (88). This phenomenon may be important since S-nitrosoglutathione acts as a cytoprotective agent in the cytotoxic condition of NO and activated oxygen co-producing systems (see the section on ONOO- ) (89). Thiols may act as protecting agents in such pathological conditions to form S-nitrosothiols. Wu et al. reported that glutathione gives a vasodilating property to the peroxynitrite-generating system and demonstrated that the S-nitrosocompound was formed from glutathione and peroxynitrite (90). We should consider the physiological and pathological actions of NO-releasing systems in relation to NO, peroxynitrite, and S-nitrosothiol. S-Nitrosothiol may be an endogenous quencher of NO-related toxicity.
The bioaction of S-nitrosothiols was reported to be similar to that of NO in many cases (85). However, there are some differences in their physiological activities. Both S-nitroso-N-acetylpenicillamine (91) and pure NO-releasing agents, NOC (92) (see later section for NOC reagents), had similar potent vasorelaxation in the aorta ring sample of rats. Carboxy-PTIO, the NO scavenger (see section on Carboxy-PTIO) cannot completely attenuate the vasodilatation with SNAP in spite of total quenching of the relaxation with NO(93). Murayama et al. demonstrated the difference of the action of SNAP and NOC 18 in neurotransmitter release(94). Although SNAP and NOC 18 augmented the cyclic GMP level in the hippocampal cell equally, only SNAP could accelerate the noradrenaline release in the presence of thiol, DTT. Murayama supposed that the transmitter release might be accelerated not through cyclic GMP elevation but with a different mechanism in direct interaction with receptors or components in the cell through nitrosation of redox-sensitive sites. Thus, S-nitrosothiol may be another messenger in addition to the NO carrier to intracellular space.

2-3 Peroxynitrite
Peroxynitrite is an important chemical species relative to the cytotoxic effect of NO. This compound, formed with NO and superoxide anion(95), is a strong oxidant that damages cell membranes and proteins(22, 38,96,97). There is a report to suppose an another pathway that the peroxynitrite can be formed from hydrogen peroxide and nitrite, too(98). Thus, peroxynitrite may be able to be formed wherever both NO and activated oxygen are generated, which is often the case in many biological conditions. An immune cell that could release a great amount of NO and superoxide anion would be a major source of peroxynitrite (99). It has been reported that endothelium also releases peroxynitrite through the stimulation of agonists(100).
The most serious bioactivity of the anion may be related to endothelial damage (101). Loss of the barrier of endothelium is often seen in the first stage of various oxidative damages in the biological system. The authentic ONOO- or ONOO- donor, SIN-1, clearly damages the endothelial cell (22). The nerve cell is also damaged by the anion (10,102). Stamler et al. reported that neuronal death was caused not by the NO molecule but by peroxynitrite (10). On the other hand, Oguma et al. claimed that peroxynitrite accelerated the release of GABA or acetylcholine from the nerve cell without any cell damage(103). There was no leakage of Lactate dehydrogenase(LDH) from the sample cell. It means that action of peroxynitrite is not caused by a dameage of cell wall. It was also reported that ONOO- had vasodilating activity in coronary(104) and pulmonary arteries(90). This may be caused by an increase in the intracellular calcium concentration of smooth muscle cell in blood vessels because of the cell wall oxidative damage from peroxynitrite. However, Wu et al. (90) demonstrated that an S-nitrosothiol-like compound was formed by glutathione and peroxynitrite, and the NO released from the nitrosothiol was the real couse of vasorelaxation with ONOO-. High-performance liquid chromatography analysis showed that the nitrosothiol was not S-nitrosoglutathione. Moro et al.(89) reported that peroxynitrite inhibited platelet aggregation in the presence of glutathione. They showed that the cause was S-nitrosoglutathione formed by glutathione and peroxynitrite. Pure peroxynitrite accelerates the platelet aggregation.
Thus, it can be seen that NO and thiol reduce the cytotoxicity of activated oxygens in their synergistic action. The bioactivity of NO or ONOO-should be considered, along with environmental conditions.

2-4. Iron-nitrosyl complex
Many types of iron-nitrosyl complexes should be formed in a biological NO-generating system. The iron-nitrosyl complex of guanylyl cyclase is an activated form of the enzyme dissociating the axial imidazolyl ligand with the trans effect of NO(105). NO-hemoglobin is also a common iron-nitrosyl complex at work in the bloodstream and in brsathing(106), but its biological meaning is uncertain. Since some types of iron-nitrosyl complexes have vasodilating activity, it has been suggested that EDRF resembles the Fe-NO complex(107). Now, it may be difficult to recognize that the major part of EDRF is this type of compound. Akaike et al. showed that the MGD-Fe complex, the NO spin-trapping agent, had potent vasorelaxtion forming the nitrosyl complex with NO (93).

3. Reagents for research on the NO pathway
The chemical structure of NO changes immediately after it is released from NOS, as shown in Figure 1. One way to investigate the bioaction of NO in detail is to use the pharmacological tecnique of adding or scavenging NO and its related metabolites in pure form independently of other materials. Dojindo Labs offers some NO and NO-related compound donors and NO scavengers for this process. This chapter introduces some reagents for NO research, including classical and new types.

3-1. NO donors
The basic pharmacological technique is to add NO to the sample. The simplest way may be the addition of authentic NO solution. However, it is extremely difficult to get pure NO solution because NO is very unstable in aqueous media if it is dissolved with oxygen. In addition, if pure NO solution is added to sample, the concentration of NO will decrease rapidly. Thus, it will be impossible to add NO continuously and to mimic NO release from NOS. Chemical reagents that can release NO continuously in physiological conditions are increasingly important. Among the most widely used NO donors are glycerin trinitrate (GTN), sodium nitroprusside (SNP)(108), and SNAP(108).
(a) Classical NO donors
Figure 3 shows some classical NO donors that release NO in biological environments. These donors can be classified into the following categories: organic nitrate, organic nitrite, floxane derivatives, iron-nitrosyl, S-nitrosothiol, and sydnonimine.

Figure 3. Some examples of classical NO donors.

Organic nitrate
The reagents in this category have following general form.

This is ester of alcohol ROH and nitric acid. The chemical reactions of NO release are as follows(17).

The above reactions require special thiols such as cystein and N-acetylcystein. Other thiols result in nitrite anion, as in the following reactions. This reaction is 14 times faster than NO-releasing reaction when cystein is used as the cofactor, and 3 times faster when N-acetylcystein is used as the cofactor(17).

Organic nitrites
The general structure is RONO, which is ester of alcohol, ROH, and HNO2. This type of reagent releases NO in following reaction. The real NO releaser is S-nitrosothiol for this type of NO donor.

Floxanes
Floxane has following structure(109). This compound reacts with thiols to release NO.

The above three types of NO donors all require thiols as the cofactor for generating NO. In this case, NO moiety is transferred to thiol and then NO is released from the S-nitrosothiol or S-nitrososulfoxide. The real NO-releasing agent is a thiol derivative. Thus, these types of reagents consume endogenous thiols in biological samples to release NO. If thiol is depleted, further dosages of such reagents have no more NO activity, a condition called nitrate tolerance(110). For example, co-administration of N-acetylcystein suppresses the tolerance in gricerol trinitrate (GTN) (108). The thiol requirement means that the NO-releasing profile and the amount of NO generated may differ, depending on the site or type of samples. The real NO-releasing intermediates that are NO metabolites themselves have significant bioactivity similar to that of NO.

Iron-nitrosyl
SNP is the most popular reagent(111). It reagent reveals strong vasorelaxing activity and is believed to release NO, depending on the pH in the system (17). However, the time course of NO release is not linear and also has a property of NO+ or a bioactivity other than NO. In our investigations we have seen no evidence of spontaneous NO release . It is also sensitive to light and heat, and there is a danger of CN- release.

S-Nitrosothiol and Sydnonimine
These types of reagents can release NO spontaneously. However, S-nitrosothiols are other important messengers derived from NO in physiological environments(85). They are usually, but not always, unstable as chemical reagents. It is also reported that S-nitrosothiol releases NO through metabolism in the cell membrane (87), or NO release is accelerated by the presence of another thiol. Thus, S-nitrosothiol is no longer considered a pure spontaneous NO donor in biological samples. Sydnonimines can also release NO spontaneously, but superoxide is generated at the same time to make peroxynitrite anion (112). They should be peroxynitrite donors rather than NO donors. These two types of reagents are discussed in a later chapter on S-nitrosothiol and SIN-1.

Any classical NO donors cannot be considered ideal NO releasers that can generate pure NO spontaneously and independently of other materials in biological samples. As already mentioned, NO is a highly reactive molecule and can undergo various reactions. In this sense, the rate, amount, and period or profile of NO release are very important factors affecting NO bioactivity. Thus, the ideal NO donor should have the following capabilities:
* ability to control the amount of added NO
* ability to control the rate of NO release
* ability to estimate the concentration or rate of released NO
* ability to completely, spontaneously, and independently release NO on any environmental materials.
* ability to ensure that the by-products have no serious sideeffects.

We recently offered two new types of NO donors, NOCs and NORs, that meet the above conditions.

(b) NOC
Keefer et al. developed the following series of stabilized NO-amine complexes. NOC is our trademark of those reagents(113).

Amines usually form the following type of complex, which contains two NO molecules(114).

The complex is usually unstable and decomposes immediately releasing NO. The decomposition is triggered by protonation of the negative charge on oxygen of NO moiety. Thus, this type of complex can be stabilized with neutralization of the negative charge. Keefer et al. developed some new NO donors, called NONOates,which release pure NO spontaneously(115). They have been used successfully as good NO donors in various experiments(78, 115,117).

However, we are still concerned about the toxicity or bioactivity of their intermediate and by-product starting amine, i.e. N-nitrosodiethylamine or spermine. Keefer et al. later developed another type of NONOate (NOC)(113). These Drago-type complexes are intramolecular zwitter ions that are stabilized with an intramolecular hydrogen bond through dispersion of the negative charge, which prevents protonation. The rate of NO release depends on the weakness of the hydrogen bond. Thus, if NOC is used, the rate of adding NO to the sample can be controlled by selecting the compounds. Figure 4 shows the time course of NO release from our commercialized NOC 5, 7, 12,18, estimated by electron paramagnetic resonance spestroscopy (EPR) using Carboxy-PTIO(see the section on NO scavenger) at 37 ^oC and pH 7.4 in PBS buffer. The release of NO follows first-order kinetics, and their half-life times range from 5 min. to 21 hrs. The half-life times of each NOC reagent are as follows: NOC 5, 25 min; NOC 7, 5 min; NOC 12, 100 min; NOC 18, 21 hrs.

Figure 4. Time course of NO release from NOCs (0.1 mM) in PBS (0.1 M, pH 7.4) at 37 ^oC. NO was monitored with an EPR spectral change of Carboxy-PTIO (0.1 mM).

Sakurai et al. demonstrated that the NO-releasing rate was important in the secretion of insulin from the beta cell(118). Short-lived NOC 7 could not accelerate the secretion, but NOC 12, which was a slower NO releaser, could. Elevation of the cyclic GMP level is also dependent on the rate of the NO molecule. Obara et al. reported that a continuous dosage of NOC 7 reduced blood pressure without any decrease of blood flow to each organ(92).
Since the decomposition of NOC is triggered by protonation, the NO-releasing rate becomes faster as the pH level decreases. In the NOC experiment, it can be used easily to dilute NOC into alkaline solution, e.g. 0.1 M NaOH, then added into the sample buffer solution. The alkaline stock solution can be used for a day, but it decomposes 5 - 6 % after overnight storages, even in a freezer. NOC is stable for more than one year as a solid state at - 20c. The bottle should be opened after returning to room temperature, because it tends to decompose with moisture.

(C) NOR

The NOR series is a completely new and idealtype of NO donor developed by Fujisawa Pharmaceutical Industry(119). NORs are organic molecules, and although they have no ONO2 or ONO moiety, they can release NO spontaneously in a rate-controlling manner. It is also clear that the by-products have no significant bioactivity. Figure 5 shows the NO-releasing time coursefrom NORs. The profiles of NO release are very similar to that of NOCs. The release is followed by first-order kinetics. Half-life time and initial NO releasing rates from NORs at 37 ^oC, pH 7.4 are as follows: NOR 1, 1.8 min; NOR 2 , 2.8 min; NOR 3, 30 min; NOR 4, 60 min. Thus, the amount and rate of adding NO to the sample can be controlled by selecting the reagents from NOR 1-4 as well as NOCs.

Figure 5. Time course of NO release from NORs (0.1 mM) in PBS (0.1 M, pH 7.4) at 37 ^oC. NO was monitored with an EPR spectral change of Carboxy-PTIO (0.2 mM).

The vasodilating activity of NOR 3 (FK 409) was reported in rat aorta(119),rabbit aorta(120), and dog coronary arteries (121). The relaxing activity of NOR 3 (ED50 = 1 nM) is 300 times stronger than that of isosorbide dinitrate(ISDN)(ED50 = 310 nM) in a rat aorta ring sample(122) and 80 times stronger (ED50 = 16.7 nM) than ISDN (ED50 = 1340 nM) in a canine coronary artery (123). In this case, NOR 3 increased the plasma cyclic GMP level, wherseas ISDN had no increase in the level(124). The different NO-releasing rate was reflected even as an in vivo hypotensive effect (Figure 6)(125).

Figure 6. Time Course of the Effects of Intravenously Administered (A) NOR 4 and (B) NOR 3 ( : Vehicle, : 1.0 mg/kg, : 3.2 mg/kg) on Mean Blood Pressure of Rat. Changes in Mean Blood Pressure Were Expressed as Percentages of the Preadministration Value. Each Value Represents the Meam + SEM for Five Experiments. *P<0.05, **P<0.01 Compared with the Vehicle-Treated Group.

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4. SNAP (S-nitrosothiol)

As described above, S-nitrosothiols are important materials to consider releavant tothe bioactivity of NO. However, S-nitrosothiols are usually too unstable for use as chemical reagents. SNAP and S-nitrosogluitathione are the exceptions. Of course, SNAP is at least in part a spontaneous NO releaser with antiviral activity like that of NO (128).

Figure 7 shows the time course of spontaneous NO release from SNAP under certain conditions. The amount of spontaneous released NO was about 1/10 mol of used SNAP, and thiol (NAP) augmented the NO release.

Figure 7. Time course of spontanious NO release from SNAP in 0.1 M PBS (pH 7.4) at 37 ^oC. Generated NO was monitored with an EPR spectral change ofCarboxy-PTIO (0.1 M).

SNAP is potent vasodilator with little nitrate tolerance(129.130). For example, little attenuation of vasodilating activity of SNAP was seen after 24h use of GTN in rabbits(108). The order of vasodilating activity of various S-nitrosothiols was SNAP > GSNO = SNAC(S-nitroso-N-acetylcystein) > CoASNO(S- nitroso-coenzymeA) > CySNO(S-nitrosocystein) in a rat aorta ring sample. However, it was reported that the activity of SNAC was stronger than that of SNAP in a coronary ring sample(87). Figure 8 shows the vasodilating activity of SNAP in a rat aorta ring sample (131) in an organ bath and in the renal perfusion system of a rat.

Figure 8. Vasodilative Effect of SNAP.
(A)Rat Aorta Ring Sample Was Mounted Vertically in Organ Bath Filled With Krebs Solution, and Isometric Tension Development Was Recorded. The Tissue Was Precontracted with Phenylephrine, Then SNAP Was Added in Various Concentrations.
(B)Rat Kidney Was Prefused, and Perfusional Pressure Was Monitored. Blood Vessels Were Precontracted with Noradrenalin, Then SNAP Was Added in Various Concentrations.

T. Akaike, Kumamoto University, School of Medicine.

(Unpublished Date)

Although SNAP has been used as a spontaneous NO releaser, there are some examples demonstrating the difference in bioactivity between SNAP and NO. Cyclic GMP elevation by SNAP was not inhibited in rat hippocampus slices, while that by NO or NOC 18 was well attenuated with some NO scavengers (Table 1) (94). Murayama reported that SNAP accelerated the noradrenaline release in the hippocampus neuron in the presence of DTT (Figure 9)(94). On the other hand, the pure NO releaser, NOC 18, did not have any acceleration. It is also doubtful that spontaneously released NO is directly related to the bioactivity of SNAP (as described in the section on S-nitrosothiol).

Table 1. Inhibition of Cyclic GMP Production,That is Accerelate by NO Donors, with NO Quenchers in Rat Hippocampus Slice.

Figure 9. Enhancement of Noradrenalin Release from Rat Hippocampus with SNAP.
Both SNAP and NOC 18 (Releasing Pure NO) Did Not Accelarate the Transmitter Release, but SNAP potentiated the Release with the Presence of DTT.

T. Murayama, Faculty of Pharmaceutical Sciences, Hokkaido University.

Dose dependency of SNAP to augmentation of the intracellular cyclic GMP level of SNAP was examined in cerebellum slices(132). The efficiency was maximized at 1 mM. However, the inhibitory effect of cell proliferation was reported in 1-3 uM of SNAP. In this case, the effect did not relate to the inhibition of RNA or protein synthesis, because uridine and leusine uptake did not occur at a higher concentration of SNAP (more than 100 uM). SNAP exhibits some cytotoxicity at 100 uM, but it does not affect the mitochondrial function(133).

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5. SIN-1 (peroxynitrite donor)

Peroxynitrite is considered an endogenously formed cytotoxic factor derived from NO and the superoxide anion(22,95). It will be useful to add this compound continuously to the sample. SIN-1 is the metabolite form of molsidomine, the vasodilator. It decomposes spontaneously in neutral aqueous media-consuming oxygen to release NO and the superoxide anion simultaneously(122,134). Thus, the reagent can be used as a possible peroxynitrite donor. The mechanism of decomposition of SIN-1 is as follows(134). Hydrolysis of the mesoionic ring to SIN-1A is required for spontaneous decomposition. Thus, peroxynitrite release depends on the pH of the solution. The time course of the decomposition of SIN-1 was investigated(112). It was nonlinear, and some delay occurred because of the hydrolysis.

Releasing rates of NO and the superoxide anion have been reported as 3.68 uM/min and 7.02 uM/min, respectively, at pH 7.2, 37 ^oC(23). Another report claimed the NO releasing rate was 2.39 uM/min(134). SIN-1 generates nearly the same amount of nitrate and nitrite as the final products. Thiols tend to increase the ratio of nitrite/nitrate. SOD stabilizes the SIN-1A to decrease the production of these anions and increase the ratio(23). The activation of guanylyl cyclase of SIN-1 was weaker than SNAP in a cerebellum slice(132), but stronger in the isolated enzyme (134). This activity of SIN-1 was augmented with SOD, attenuated the superoxide producing system and independent on the presence of thiol(134).
SIN-1 may produce a hydroxyl radical with the homolytic cleavage of peroxynitrite. Although it is uncertain whether the hydroxy radical is formed in biological environments (96), the production of malondialdehyde from deoxyribose or phenolic fluorophore from benzoic acid was reported to demonstrate the formation of a hyroxyl radical-like oxidant indirectly (23). Production of those oxidative products was attenuated by hydroxyl radical scavengers (mannitol, ethanol, citric acid) or SOD. However, catalase did not inhibit the production of malondialdehyde or phenolic fluorophore. It means that the hydrogen peroxide does not account for the oxidatine phenomena.
Recently researchers used SIN-1 to show the relationship between peroxynitrite and neuronal cell death (10). In this case, NO helped to protect the cells from dying. Thus, SIN-1 will become more important for understanding the contributions of peroxynitrite in NO-related phenomena.

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6. Carboxy-PTIO (NO scavenger)

Carboxy-PTIO is a stable organic radical that was developed by Akaike et al. in Kumamoto University. This reagent reacts directly with the NO molecule to change it to the .NO2 radical, which is the closest metabolite of NO, as seen in the following reaction.

For scavenging endogenous NO, NOS inhibitors that are arginine derivatives have been used and hemoglobin has been used as an NO trapper. However, as shown in Figure 2, NO continuously undergoes complex changes. In addition, each metabolite derived from NO may have unique bioactivity. If ordinary NO scavengers such as arginine derivatives or hemoglobin are used, all other NO-derived metabolites are scavenged at the same time. The results obtained from NO scavenger experiments cannot determine which compuounds are realfactors, but they do indicate whether the NO-producing system is relevant to this phenomenon. On the other hand, Carboxy-PTIO makes it possible to determine the effect of pure NO molecule on the sample, and thus we can determine the effect of NO versus those of other NO metabolites, because the .NO2 radical is the closest metabolite of NO and can continue the intact reactions of NO-derived metabolites.
Akaike et al. demonstrated the inhibitory effect of Carboxy-PTIO on the vasodilatation of a rabbit aorta ring induced by acetylcholine(135). This effect was twice as strong as that of NG-nitroarginine. Yoshida et al. reported that Carboxy-PTIO augmented the antiviral activity of NO(136). They concluded that the antiviral activity was caused not by NO or peroxynitrite but possibly by .NO2 or .NO2-derived metabolites. Their result agree with those of Oury et al., who demonstrated that the neuronal toxicity of NO was potentiated by superoxide quenching in transgenic mouse with excess amounts f extracellular SOD (137). They concluded that the toxicity was in the NO molecule itself, but there were not any consideration of the contributions of other NO derivatives. Thus, Carboxy-PTIO offers useful methodology for understanding the effect of NO. Carboxy-PTIO attenuated the bioactivity of NO but was sometimes inefficient for that of SNAP(93).
Figure 10 and 11 show the inhibitory effect of Carboxy-PTIO on the vasodilatation by endogenous NO and exogenous NO donors. The toxicity of Carboxy-PTIO is lower than that of NOS inhibitors. In vivo excess administration of NOS inhibitors causes hypertension in animals, while Carboxy-PTIO raises their blood pressure to normal levels(84). In a cell culture system, Carboxy-PTIO shows a cytotoxicity concentration of more than 0.3-0.5 mM. It was reported that Carboxy-PTIO had a positive therapeutic effect on rats suffering from septic shock(84). Carboxy-PTIO prohibited the serious hypotension of sepsis and improved the renal function so greatly that there was a 100 % survival rate.

Figure 10. Antagonistic Effect of Carboxy-PTIO on the Relaxation of Rat Renal Blood Vessels with Endogenous NO. Rat Kidney was Perfused, and Perfusional Pressure Was Monitrored. Blood Vessels Were Precontracted with Noradrenalin, then Acetylcholine was Added in Various Concentrations.

T. Akaike, Kumamoto University, School of Medicine.

(Unpublished Date)

Figure 11. Antagonistic effect of Carboxy-PTIO on the relaxation of rat renal blood vessels with exogenous NO. Rat kidney was perfused, and perfusional pressure was monitored. Blood vessels were precontracted with noradrenalin, then SNAP was added in various concentrations.

T. Akaike, Kumamoto University, School of Medicine.

Carboxy-PTIO is a promising reagent for NO research and clinical applications, because of its unique pharmacological proparty. However, its chemical property should be understood prior to use. The compound is sensitive to reducing agents such as thiol or Fe2+ ion or ascorbate. The compound reduces reversibly with an endogenous reducing agent to produce a non-radical hydroxylamine-type compound. Thus, the effective concentration of Carboxy-PTIO is lower than that of initial doses, especially in vivo. Superoxide also reduces Carboxy-PTIO reversibly. The reducing rate varies. Ascorbate reduces it very rapidly, DTT reduction attenuates the radical completely for 30 min, and N-acetylpenicillamine has a hard time reducing Carboxy-PTIO in aqueous media. Because Carboxy-PTIO is decomposed irreversibly in acidic pH, the pH must be kept above 6.5 when dissolving Carboxy-PTIO. Ideally, more than 300 uM of Carboxy-PTIO should be used for the experiment. Carboxy-PTIO is highly water soluble, so more than 20 mM of aqueous solution can be made.
Carboxy-PTIO can be used with EPR to determine the amount of NO, because the EPR signal changes to a different pattern after reacting with NO. Figure 12 shows the EPR spectral change of Carboxy-PTIO in the reaction with NO from NOC 12. A weakening of signal A or an increase in signal B is usually used for the NO determination, because both signals do not overlap each other.

Figure 12. EPR spectral change ofCarboxy-PTIO in the reaction with NO. The reaction was monitored in 100mM PBS (pH 7.4) at 37 ^oC.

Figures 4,5 and 7 are estimated by using this technique. This methodology is very convenient in simple solutions because the NO concentration can be calculated by the relationship between the concentration of Carboxy-PTIO and the EPR signal height without any calibration, if there is no reducing agent. However, the lack of contact with the reducing agent must be considered if the technique is used for the determination of NO in biological samples, because there are various reducing activities in biological systems. Azu-ma et al. reported the estimation of NO release from endothelial cells that are stimulated with bradykinine using the cell-loaded column in the Carboxy-PTIO method(138). We have recently investigated the use of cationic-type Carboxy-PTIO packing in specialized liposome for endogenous NO trapping and have obtained good results.

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7. MGD-Fe2+ complex (water-soluble NO spin trapper)
The Spin-trapping technique is one of the most promising methods for determining NO, because NO is an unstable radical. At first, deoxy-hemoglobin was used as the NO spin trapper. Hemoglobin reacted with NO very rapidly to make NO-hemoglobin, which was able to detect NO in a characteristic three-line EPR signal (139), but NO2- also made a nitrosyl complex that interfered with NO detection. Kosaka et al. used CO-hemoglobin that did not react with NO2- and obtained better results(140). However, it was difficult to determine the origin of iron-nitrosyl and to prepare CO-hemoglobin. Vanin et al. developed the DETC(diethyldithiocarbamate)-Fe2+ complex technique for the NO spin trapping(141-145). The iron complex traps NO very well to form iron-nitrosyl, which can be detected as a three-line EPR signal (141). Although this technique is quite useful, poor solubility of the DETC-Fe2+ complex in aqueous media makes this method limited.

It have to be used after loading into killed yeast membrane, or used in a separate doseage of DETC and Fe2+ salt(146). Komarov et al. improved the technique using a water-soluble dithiocarbamate-Fe2+ complex, MGD2-Fe2+ (147).
This complex was able to detect NO from SNP or iNOS induced by LPS stimulation in vivo(148) using the S-band EPR technique.

Figure 13 shows the EPR signal increase of iron-nitrosyl of an MGD2-Fe2+ complex accompanied by NO release from NOC 5 solution.

Characteristic three-line signals are developed with NO generation. However, the complex trapped S-nitrosothiol and NO, resulting in a stabler complex than in the case of NO trapping (Figure 14). This result indicates that hemoglobin or guanylate cyclase could form a nitrosyl complex with S-nitrosothiol directly. Some reducing agents, DTT or ascorbate, tend to augment the EPR signal of the NO complex (Figures 13, 14). Komarov et al. reported that some EPR silent complexes (NO-MGD2-Fe2+-X, X = NO2 or Cl- etc.) were contained in the system and that the reducing agent changed them to the MGD2-Fe2+-NO type(147).

Figure 13. Development of Iron nitrosyl EPR signal in Fe2+(MGD2) (1 mM) aqueous solution in the presence of NOC 5 (500 uL). The reaction was monitored in 100 mM PBS (pH 7.4) at 37 ^oC.

Figure 14. Development of Iron nitrosyl EPR signal in Fe2+(MGD2) (1 mM) aqueous solution in the presence of SNAP (100 uL). The reaction was monitored in 100 mM PBS (pH 7.4) at 37 ^oC.

The MGD2-Fe2+complex is quite unstable, especially in the presence of dissolved oxygen. Thus, the complex should be used immediately after it is made. A 5:1 mixture of MGD and Fe2+ is used for making the complex with FeSO4 to get a more stable complex solution. Acidic conditions should be avoided because dithiocarbamate tents to decompose, forming toxic carbon disulfide. MGD and Fe(MGD)2 were reported as nontoxic up to levels of 8 mmol/kg and 0.3 mmol/kg, respectively(149).

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