Endothelium-derived relaxing factor released from canine femoral artery by acetylcholine cannot be identified as free nitric oxide by electron paramagnetic resonance spectroscopy

Recent studies using chemiluminescence and spectrophotometry have shown that cultured and native endothelial cells release nitric oxide (NO). Pharmacological and biochemical evidence argue for and against the proposal that endothelium-derived relaxing factor (EDRF) is identical with free NO. In an attempt to identify EDRF as free NO, a bioassay technique was combined with an NO trap (hemoglobin bound to agarose; Ag-Hb), and electron paramagnetic resonance (EPR) spectroscopy was used to detect the resultant nitrosylhemoglobin (NO-Hb). Canine femoral arteries with or without endothelium were perfused with physiological saline solution containing superoxide dismutase and ibuprofen and were stimulated with acetylcholine. The relaxing activity of the effluent was monitored in canine coronary artery rings without endothelium (bioassay tissue) half-maximally contracted with U46619. Acetylcholine stimulated the release of EDRF from intact femoral arteries (but not from segments without endothelium), which relaxed the bioassay tissue by 63 +/- 5%. NO (approximately 1 and approximately 10 nM) infused directly over the bioassay tissue produced 34 +/- 8% and 96 +/- 3% relaxation, respectively (ED50, approximately 2 nM). Effluents were collected under vacuum in the absence of oxygen through a column containing Ag-Hb, and the samples were assayed for NO-Hb by EPR. Samples containing NO produced the triplet EPR signal characteristic of NO-Hb, but the effluent containing EDRF did not. Infusion of NO through the donor tissue in the presence of acetylcholine gave an EPR signal similar to that observed when NO had no contact with the tissue. Nitrite anion (up to 2.7 x 10(-2) M) produced no detectable NO-Hb in analogous experiments. Thus, EDRF released from the native endothelium of canine femoral artery cannot be identified as free NO. The present findings support a concept that EDRF may be a labile precursor of NO.