Simultaneous measurement of endothelium-derived relaxing factor by bioassay and guanylate cyclase stimulation.
1. Endothelium-derived relaxing factor (EDRF) released by cultured endothelial cells (EC) from bovine aortae was measured by bioassay using pre-contracted strips of rabbit aorta and by radioimmunoassay of guanosine 3':5'-cyclic monophosphate (cyclic GMP) produced by stimulation of bovine lung soluble guanylate cyclase. 2. Bradykinin (Bk, 3 and 30 pmol) injected through a column of EC caused release of EDRF as detected by bioassay and increased cyclic GMP concentrations. Superoxide dismutase (SOD, 15 u ml-1) increased the amount of EDRF detected by the activation of soluble guanylate cyclase. 3. In the absence of endothelial cells, nitric oxide (NO, 1-2 microM), arachidonic acid (AA, 3-30 microM) or sodium nitroprusside (SNP, 1-100 microM) stimulated guanylate cyclase. Superoxide dismutase strongly increased the stimulation of guanylate cyclase induced by NO, but had little effect on the stimulation induced by SNP and no effect on the stimulation induced by AA. 4. Oxyhaemoglobin (10-300 microM) abolished the stimulation of guanylate cyclase by EDRF, NO or SNP but was much less effective as an inhibitor of AA-induced stimulation of guanylate cyclase. 5. These results demonstrate that measurement of guanylate cyclase stimulation by radioimmunoassay is a viable method for detecting EDRF release, especially useful when the drugs used interfere with bioassay tissues.
Kondo
K
K
William Harvey Research Institute, St Bartholomew's Hospital Medical College, London.
Mitchell
J A
JA
de Nucci
G
G
Vane
J R
JR
D000818:Animals; D002417:Cattle; D006152:Cyclic GMP / Q000032:analysis; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D066298:In Vitro Techniques; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000032:analysis* / Q000494:pharmacology; D010108:Oxyhemoglobins / Q000032:analysis / Q000302:isolation & purification; D011863:Radioimmunoassay; D013268:Stimulation, Chemical
D010108:Oxyhemoglobins; D009569:Nitric Oxide; D006162:Guanylate Cyclase; D006152:Cyclic GMP