Practical nitric oxide measurement employing a nitric oxide‐selective electrode

Nitric oxide is an important bioregulatory molecule, being responsible, for example, for activity of endothelium-derived relaxing factor (EDRF). Acute hypertension, diabetes, ischaemia and atherosclerosis are associated with abnormalities of EDRF. Nitric oxide is thought to be a retrograde messenger in the central nervous system. The technology is not yet available for rapid detection of NO released by a single cell in the presence of oxygen and/or nitrite, so the release, distribution and reactivity of endogenous NO in biological systems cannot be analysed. Here we describe a porphyrinic microsensor that we have developed and applied to monitoring NO release in a microsystem. We selectively measured in situ the NO released from a single cell with a response time of less than 10 ms. The microsensor consists of p-type semiconducting polymeric porphyrin and a cationic exchanger (Nafion) deposited on a thermally sharpened carbon fibre with a tip diameter of approximately 0.5 microns. The microsensor, which can be operated in either the amperometric or voltammetric mode, is characterized by a linear response up to 300 microM and a detection limit of 10 nM. Nitric oxide at the level of 10(-20) mols can be detected in a single cell.

We measured, in vivo, the local concentration of nitric oxide (NO) in cerebral tissue, during and after transient middle cerebral artery occlusion in the rat (n = 8). Baseline concentration of NO was < 10(-8) M; upon initiation of ischemia, NO concentration increased to approximately 10(-6) M and then declined. Reperfusion likewise stimulated an increase in NO concentration to above baseline level. Administration of N-nitro-L-arginine methyl ester (n = 4), an inhibitor of nitric oxide synthase, before onset of ischemia, maintained NO at basal levels. Our data indicate that large increases in NO occur at onset of ischemia, which may affect tissue response to an ischemic insult.

To detect the release of nitric oxide (NO) in brain tissue, an electrochemical microprobe was developed. The output current of the probe correlated linearly with the NO concentration at the tip, and the sensitivity of the probe was between 3.5 and 106 pA per 1 microM change in NO concentration. This probe showed no sensitivity to oxygen or to oxidized derivatives of NO. The NO release from sodium nitroprusside solution was successfully detected by the probe. An NO probe inserted into the molecular layer of a rat cerebellar slice detected a response corresponding to 8-58 nM of NO concentration following electrical stimulation of the white matter. This response was blocked reversibly by tetrodotoxin (1 microM) and was attenuated in the medium containing hemoglobin (1 or 10 microM). The dependence of the response amplitude on the voltage at the cathode in the probe was the same as that of the NO-induced probe current. These results ensure that the NO probes developed in this study effectively detect the endogenous NO release in brain tissue.