Nitric oxide ( NO ) is a reactive radical gas, which plays a wide variety of roles in many biological processes. For example the regulation of blood pressure and digestion, cytoxicity of macrophages, penile erection and modulation of blood clotting. NO is generated during the conversion of L-arginine to citrulline by NO - synthase ( NOS ) in many cell types. NO leads to an activation of guanylate-cyclase in it,s target cells. The subsequent increase of cyclic GMP leads induces the biological reaction.
In the central nervous system (CNS) NO is identified as a second messenger. It also appears to regulate the blood flow in the brain and has been implicated as a mediator of long-term potentiation.
In previous investigations NO was mainly indirectly measured. The NOS was blocked with false substrates like L-nitro-arginine. From the resulting loss of function it is possible to speculate about the way of NO-action.
Our aim is to directly measure NO from CNS - cells by ozon-chemoluminescence. In this method the reaction of NO with O3 leads to a detectable generation of photons, which are converted into electrical signals by means of a photomultiplier. Changes of the electrical current are registered as a function of time and stored. The area under the resulting curve equals the NO-concentration, which can be quantitatively determined, if compared with Nitrite-standards. This method is also used as a standard method for detection of NO in environmental studies. It is very sensitive (5-20 pMol) and highly specific for NO. For detection of NO from biological effluates the containing NO is released by Helium and then transferred into the Sievers NO-analyzer. Thus it is possible to measure NO, produced by cultured cells or isolated organs. Because of the short biological half-life of NO ( which is quickly oxidized to NO2- ) it is sometimes necessary to use a reduction system (1,1,Dimethylferrocyene) reduces NO2- to NO.
NO-measurement in vivo: A closed cranial window in an anesthetized rat (see cranial window technique in vivo) is superfused with artificial cerebrospinal fluid (aCSF). At the same time we can register the blood flow or the reaction of leucocytes. The superfused medium is used for NO-measurement.
NO-measurement in vitro: These are the subsequent investigations in primary and secondary cell cultures to identify, which type of cells is responsible for the NO-content change in the superfused medium. Cells are incubated in culture wells with medium for different time intervals. Additives are given into the medium to induce different physiological and pathological conditions.
The results allow conclusions about the role of NO in the brain and about the cell types of the CNS which might be the source of the NO-Production.