Release of endothelium-derived relaxing factor from freshly harvested porcine endothelial cells.

Vascular relaxation in rabbit aortic preparations induced by acetylcholine is endothelium-dependent. The nature of the endothelium-derived relaxing factor (EDRF) has not been ascertained because it is very labile (reported half-life 6-50 seconds). To obtain a stable source of EDRF, a system was developed in which the relaxing factor was continuously produced by freshly harvested porcine endothelial cells. Endothelial cells were collected from aortas by exposing the endothelial lining to collagenase 0.1%. Cells were washed and concentrated by repeated centrifugation to obtain a high cell count (7.2 X 10(6) cells/ml). Endothelium-deprived aortic strips from rabbits were incubated in these cells suspended in tissue culture medium and fetal calf serum. The strips were precontracted with histamine. Acetylcholine was added to induce EDRF release. Significant relaxation of endothelium-deprived aortic strips was observed. Superoxide dismutase, an enzyme known to protect EDRF against inactivation, caused further relaxation, which was inhibited by the addition of hemoglobin, an agent known to inhibit the relaxing action of EDRF. Even without the addition of acetylcholine, hemoglobin caused contraction of the denuded aortic strips in suspension of porcine endothelial cells, demonstrating spontaneous EDRF release. Hemoglobin had no effect in cell-free medium. Endothelial-cell-dependent relaxation occurred without attachment of endothelial cells to the endothelium-deprived aortic strips: when the cell suspension was replaced by cell-free medium, relaxation did not occur after acetylcholine. Scanning electron microscopy showed no attachment of endothelial cells to the subendothelial layer. It can be concluded that freshly harvested endothelial cells produce endothelium-derived relaxing factor with an without stimulation by acetylcholine.