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.
Brovkovych
V
V
Department of Chemistry, Oakland University, Rochester, MI 48309-4477, USA.
Stolarczyk
E
E
Oman
J
J
Tomboulian
P
P
Malinski
T
T
D000818:Animals; D001013:Aorta, Thoracic / Q000502:physiology; D015374:Biosensing Techniques; D004563:Electrochemistry; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005263:Femoral Artery / Q000502:physiology; D007700:Kinetics; D008168:Lung / Q000737:chemistry; D008297:Male; D008839:Microelectrodes; D009206:Myocardium / Q000737:chemistry / Q000166:cytology; D009569:Nitric Oxide / Q000032:analysis* / Q000096:biosynthesis / Q000097:blood; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011817:Rabbits; D051381:Rats; D011921:Rats, Inbred WKY; D013481:Superoxides / Q000737:chemistry; D019323:omega-N-Methylarginine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D013481:Superoxides; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat