Direct electrochemical measurement of nitric oxide in vascular endothelium.

The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium-derived nitric oxide (NO) is a vasodilator rapidly inactivated by superoxide and by Fe(II) and Fe(III), all found in significant quantities in biological systems. Thus due to the short life of NO in tissue (t1/2 = 3-6 s), in situ quantification of NO is a challenging problem. We designed the present study to perform direct measurements of nitric oxide using the electrochemical porphyrinic sensor. The most significant advantages of this sensor is small size (0.5-8 microm), rapid response time (0.1-1 ms), and low detection limit (10(-9) mol l(-1)). The porphyrinic sensor was used for in vitro and in vivo measurements of NO in an isolated single cell or tissue. Effects of hypertension, endotoxemia, and ischemia/reperfusion on the release of NO and/or its interaction with superoxide (O2-) were delineated. In the single endothelial cell (rabbit endocardium), NO concentration was highest at the cell membrane (950 +/- 50 nmol l(-1)), decreasing exponentially with distance from cell, and becoming undetectable at distances beyond 50 microm. The endothelium of spontaneously hypertensive rats (SHR) released 35% less NO (580 +/- 30 nmol l(-1)) than that of normotensive rats (920 +/- 50 nmol l(-1)), due to the higher production of O2- in SHR rats. Endothelial NO synthase (eNOS) generated NO (140 +/- 20 nmol l(-1)) in lung during the acute phase (first 10-15 min) of endotoxemia, followed by production of NO by inducible NOS. High production of O2- was observed during the entire period of endotoxemia. Ischemia (lower limb of rabbit) caused a significant increase of NO peaking at 15 min and decreasing thereafter, also due to O2- production.