Endothelium-derived relaxing factor in cultured cells

Area of Science:

• Vascular biology
• Endothelial cell signaling
• Smooth muscle physiology

Background:

• Vasoactive agents stimulate endothelium-derived relaxing factor (EDRF) release.
• EDRF induces cyclic guanosine 3',5'-monophosphate (cGMP) accumulation and vascular smooth muscle relaxation.
• This mechanism mimics the effects of sodium nitroprusside.

Purpose of the Study:

• To investigate the role of endothelial cells in EDRF production and its downstream effects.
• To characterize the response of vascular smooth muscle cells to EDRF and sodium nitroprusside.
• To determine the necessity of cell-cell contact for EDRF-mediated responses.

Main Methods:

• Isolation of endothelium and vascular smooth muscle from porcine, bovine, and rat thoracic aorta.
• Culture of endothelial and smooth muscle cells, including mixed cultures and short-term cocultures on microcarriers.
• Measurement of cGMP accumulation in response to sodium nitroprusside and endothelium-dependent vasodilators.
• Assay of EDRF release from endothelial cells using a superfusion column system.

Main Results:

• Vascular smooth muscle's response to sodium nitroprusside increased with time in culture but decreased after passage 5 in bovine and porcine cells.
• Cultured endothelial cells did not respond to vasodilators or sodium nitroprusside with increased cGMP.
• Vascular smooth muscle cells responded solely to sodium nitroprusside.
• Mixed cultures and short-term cocultures demonstrated responsiveness to endothelium-dependent vasodilators.
• Bradykinin, A23187, melittin, and arachidonate released EDRF from endothelial cells, causing cGMP changes and relaxation.

Conclusions:

• Endothelial cells are essential for releasing EDRF, which modulates vascular smooth muscle tone via cGMP.
• Vascular smooth muscle cells require endothelial cell contact or factors to respond to endothelium-dependent vasodilators.
• EDRF release and its vasodilatory effects are confirmed and characterized.