General Aspects and History

Nitric oxide (NO) was the first gaseous moiety to be identified as a messenger molecule with highly unorthodox properties. The major sources of NO are endothelial cells, macrophages and neurons. In contrast to neurotransmitters and classic neuromodulators, nitric oxide - because of its gaseous nature - cannot be stored in vesicles. Conventional neurotransmitters are released by exocytosis from the presynaptic site, whereas nitric oxide is synthesized by nitric oxide synthase (NOS) and simply diffuses from the nerve terminals into the surrounding tissue.

Conventional neurotransmitters and neuromodulators undergo reversible interactions with cell surface receptors; and their lifetime is terminated by presynaptic re-uptake or by enzymatic degradation. Nitric oxide does not need such mechanisms. It reaches its targets simply by diffusion; and a specific re-uptake system seems not to exist.

Nitric oxide is a paracrine messenger, which was first described in the vascular system by Furchgott and Zawadski in 1980. In blood vessels, NO plays a crucial role. The well known vasodilatatory properties of acetylcholine and bradyki-nin are coupled to the release of NO, which in smooth muscle cells triggers an increase in the intracellular concentration of cyclic guanosine monophosphate (cGMP). This increase in cGMP is essential for the mediation of muscle relaxation. In addition, NO mediates some of the bactericidal and tumoricidal effects of macrophages.

The idea that the gaseous substance NO plays a role as an intracellular second messenger in the central nervous system was developed in the late 1980s. Garthwaite and coworkers (1989) demonstrated that nitric oxide as well as NMDA increases intraneuronal concentrations of cGMP. In addition, they showed that, in the presence of an inhibitor of nitric oxide synthase (NOS), NMDA fails to increase cGMP levels. Nitric oxide is not exclusively an intracellular second messenger, but is also a diffusable retrograde messenger. When synthesized in neurons, NO appears to modulate several physiological and pathophysiological processes. In addition to its involvement in various neuronal mechanisms, including regulation of cerebrovascular perfusion, modulation of wakefulness, mediation of nociception, olfaction food intake and drinking, NO seems also to contribute to mechanisms attributed to learning and memory.

Since NO lacks a vesicular storage site and is exclusively produced on demand by the activity of NOS, attempts to gain information on the distribution of NO in living systems have failed. However, by purification and cloning neuronal NOS (nNOS), it has been possible to generate monospecific antibodies against nNOS; and these have been extensively utilized as markers for NO-synthesizing cells.

In living cells, nitric oxide is produced in very low concentrations and has an extremely short lifetime, making direct measurements difficult. Physiological attempts to monitor local concentrations of nitric oxide in vivo or in vitro have been hampered by the lack of a suitable means to identify it directly.

To date, many issues related to nitric oxide metabolism remain unanswered because most common techniques for nitric oxide determination depend on indirect methods: i.e. the so-called Griess reaction, which utilizes a NADPH-dia-phorase technique, and immunocytochemical approaches, which depend on NOS localization. Substances suitable for direct labeling of NO-producing cells, analogous for example to fura-2, which changes its fluorescent behavior with changes in Ca2+ concentrations, would be beneficial for further NO research. Two fluorochromes are regarded as promising candidates: 1,2-diaminoanthra-quinone (DAQ) and 4,5-diaminofluorescein diacetate (DAF-2 DA). DAQ reacts specifically with NO by forming a fluorescent triazole. DAF-2 DA does not directly interact with nitric oxide, because the ester bonds of the dye must be hy-drolyzed by intracellular esterase to generate DAF-2, which then reacts with NO to form the corresponding triazole ring (DAF-2 T). Suitable protocols for the application of these substances will ultimately further cell biological NO research.

Aside from NO, there is another small gaseous molecule that can act as a neuromodulator. This gaseous molecule is carbon monoxide (CO). Carbon monoxide is produced by heme oxygenase which cleaves the heme ring into CO and biliverdin, which is rapidly reduced to bilirubin. Since CO is a gaseous substance, the distribution of this molecule, like in case of NO, cannot be visualized directly. Since CO is produced by hemeoxygenase - the distribution of this enzyme can be examined.

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