Endothelium-Derived Relaxing Factor in Cultured Cells

Many vasoactive agents stimulate release of an endothelium-derived relaxing factor (EDRF). EDRF stimulates cyclic guanosine 3′,5′-monophosphate (cGMP) accumulation and relaxation of vascular smooth muscle in a manner similar to that produced by sodium nitroprusside. Endothelium and vascular smooth muscle were isolated from porcine, bovine, and rat thoracic aorta. The capacity of sodium nitroprusside to stimulate cGMP accumulation in cultured bovine, porcine, and rat vascular smooth muscle was found to increase with time in culture to a maximum of 12 to 14 days after plating. In addition, bovine and porcine vascular smooth muscle, but not rat vascular smooth muscle, lost the sodium nitroprusside–stimulated cGMP response after the fifth passage. Cultured endothelial cells did not respond to endothelium-dependent vasodilators or sodium nitroprusside with increased cGMP levels. Vascular smooth muscle cells responded only to sodium nitroprusside. Mixed cultures of porcine and bovine endothelium and vascular smooth muscle and bovine endothelium and rat vascular smooth muscle responded to endothelium-dependent vasodilators with increased cGMP levels. Short-term (4 hours) coculture experiments using bovine endothelium grown on microcarriers to assess the need for long-term contact between the two cell types produced similar results. Release of EDRF from bovine endothelium was studied by loading endothelium-covered microcarrier beads into a column superfused with physiological buffer. Treatment of the column with bradykinin, the calcium ionophore A23187, melittin, and arachidonate released EDRF from the column as measured by cGMP changes in denuded aortic rings and vascular smooth muscle cells and by relaxation of rings when bathed in column effluent. The time course of cGMP changes and relaxation were similar and could be reversed by hydroquinone. These findings demonstrate that EDRF can be released from cultured cells and define a variety of cell culture techniques that can be used in the further study of EDRF.