Figure 1. Simplified pathway of endogenous NO.

Presumably, NO has another unique event between the origin and the targets in its reaction scheme ( Fig.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 Fig.2  There are some important metabolites derived from NO that may have bioactivities similar to or different from those of NO. Fig.2shows 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.

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).

2-1. 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).

3-1. 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
Fig.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.

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. Fig.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.Fig.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  Fig.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.

Y. Kita et al. Fujisawa Pharmaceutical Co., Ltd.

NOR can also inhibit antiplatelet aggregation and thrombus formation (121). Aggregation of human platelet raising by ADP was inhibited effectively with NOR 3 ( IC50 = 0.75 mM ), while ISDN inhibited only 32 % of the total aggregation, even when 100 mM was used. In addition, NOR 3 provides cardioprotection in the ischemia/reperfusion system(126). A 32 mg kg-1 dose of the reagent prevented myocardial infarction following occlusion and reperfusion in a rat coronary artery. NOR 3 was also reported to have an antianginal effect. In a rat methacholin-induced coronary vasospasm model, 0.1mg Kg-1 dose of NOR 3 suppressed the elevation of the ST segment. On the other hand, ISDN suppressed it significantly at 3.2 mg kg-1(123). NOR 1, which has the shortest half-life time, is also a promising reagent for making NO standard solution for the NO calibrations in aqueous media by adding exact diluted NOR 1/DMSO solution to the buffer solutions. A good linear relationship between pM to mM levels was obtained by useing chemiluminescence (127).
NOR is usually used in DMSO or ethanol solution to dilute into the sample buffer solution because NOR is relatively stable in organic solutions if they are moisture free. NOR can also be used in oral doses using 0.5 % methylcellulose suspension. NORs are expected to be a useful tool for the continuous, rate-controlled addition of NO in biological samples without any additional side effects.

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).