Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1986 Apr;87(4):685–694. doi: 10.1111/j.1476-5381.1986.tb14586.x

Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells.

R J Gryglewski, S Moncada, R M Palmer
PMCID: PMC1916807  PMID: 3085757

Abstract

A cascade superfusion technique has been developed for the differential bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) released from porcine aortic endothelial cells cultured on microcarriers, packed into a column and perfused. Bradykinin (Bk; 20-100 nM) released prostacyclin (9.6 +/- 1.5 nM per 10(6) cells; mean +/- s.e.mean, n = 9) and prostaglandin E2 (PGE2; 2.1 +/- 0.6 nM per 10(6) cells) from the column measured by relaxation of strips of bovine coronary artery (BCA) and rabbit mesenteric or coeliac artery, respectively. The presence of these prostanoids in the effluent was confirmed by specific radioimmunoassays. A23187 (500-2000 nM) also released both prostacyclin and PGE2 from the cells. This release was long-lasting and not reproducible. Bk (20-100 nM) and A23187 (30-300 nM) released EDRF from the column. This was detected in a cascade of four rabbit aortic strips (RbA), denuded of endothelium and contracted with U46619 or phenylephrine. The relaxation of the RbA strips caused by EDRF was progressively attenuated down the cascade (half-life less than 7s) and was not affected by indomethacin. EDRF and prostacyclin could be differentially bioassayed in a cascade of alternating RbAs and BCAs as prostacyclin did not relax RbAs and the time delay to the BCAs destroys EDRF. EDRF could be bioassayed on its own when the endothelial cells were treated with indomethacin. 5-Hydroxytryptamine 0.2, noradrenaline 1.0, platelet-activating factor (Paf-acether) 1.0, formylmethionyl-leucyl-phenylalanine 1.0, acetylcholine 0.5, bethanecol 0.5, adenosine diphosphate 0.25 and angiotensin II 0.1 microM did not release either prostanoids or EDRF from the column.

Full text

PDF
685

Images in this article

686Figure 1
on p.686

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ager A., Gordon J. L., Moncada S., Pearson J. D., Salmon J. A., Trevethick M. A. Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells. J Cell Physiol. 1982 Jan;110(1):9–16. doi: 10.1002/jcp.1041100103. [DOI] [PubMed] [Google Scholar]
  2. Bakhle Y. S., Moncada S., de Nucci G., Salmon J. A. Differential release of eicosanoids by bradykinin, arachidonic acid and calcium ionophore A23187 in guinea-pig isolated perfused lung. Br J Pharmacol. 1985 Sep;86(1):55–62. doi: 10.1111/j.1476-5381.1985.tb09434.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beetens J. R., Van Hove C., Rampart M., Herman A. G. Acetylcholine stimulates the release of prostacyclin by rabbit aorta endothelium. J Pharm Pharmacol. 1983 Apr;35(4):251–252. doi: 10.1111/j.2042-7158.1983.tb02924.x. [DOI] [PubMed] [Google Scholar]
  4. Borgeat P., Samuelsson B. Arachidonic acid metabolism in polymorphonuclear leukocytes: effects of ionophore A23187. Proc Natl Acad Sci U S A. 1979 May;76(5):2148–2152. doi: 10.1073/pnas.76.5.2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bunting S., Moncada S., Vane J. R. The effects of prostagladin endoperoxides and thromboxane A2 on strips of rabbit coeliac artery and certain other smooth muscle preparations [proceedings]. Br J Pharmacol. 1976 Jul;57(3):462P–463P. [PMC free article] [PubMed] [Google Scholar]
  6. Cocks T. M., Angus J. A., Campbell J. H., Campbell G. R. Release and properties of endothelium-derived relaxing factor (EDRF) from endothelial cells in culture. J Cell Physiol. 1985 Jun;123(3):310–320. doi: 10.1002/jcp.1041230304. [DOI] [PubMed] [Google Scholar]
  7. Cocks T. M., Angus J. A. Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Nature. 1983 Oct 13;305(5935):627–630. doi: 10.1038/305627a0. [DOI] [PubMed] [Google Scholar]
  8. Davies P. F. Microcarrier culture of vascular endothelial cells on solid plastic beads. Exp Cell Res. 1981 Aug;134(2):367–376. doi: 10.1016/0014-4827(81)90436-5. [DOI] [PubMed] [Google Scholar]
  9. Dusting G. J., Moncada S., Vane J. R. Prostacyclin (PGX) is the endogenous metabolite responsible for relaxation of coronary arteries induced by arachindonic acid. Prostaglandins. 1977 Jan;13(1):3–15. doi: 10.1016/0090-6980(77)90037-5. [DOI] [PubMed] [Google Scholar]
  10. FURCHGOTT R. F. The pharmacology of vascular smooth muscle. Pharmacol Rev. 1955 Jun;7(2):183–265. [PubMed] [Google Scholar]
  11. Furchgott R. F., Cherry P. D., Zawadzki J. V., Jothianandan D. Endothelial cells as mediators of vasodilation of arteries. J Cardiovasc Pharmacol. 1984;6 (Suppl 2):S336–S343. doi: 10.1097/00005344-198406002-00008. [DOI] [PubMed] [Google Scholar]
  12. Furchgott R. F. The role of endothelium in the responses of vascular smooth muscle to drugs. Annu Rev Pharmacol Toxicol. 1984;24:175–197. doi: 10.1146/annurev.pa.24.040184.001135. [DOI] [PubMed] [Google Scholar]
  13. Furchgott R. F., Zawadzki J. V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980 Nov 27;288(5789):373–376. doi: 10.1038/288373a0. [DOI] [PubMed] [Google Scholar]
  14. Förstermann U., Neufang B. The endothelium-dependent vasodilator effect of acetylcholine: characterization of the endothelial relaxing factor with inhibitors of arachidonic acid metabolism. Eur J Pharmacol. 1984 Aug 3;103(1-2):65–70. doi: 10.1016/0014-2999(84)90190-0. [DOI] [PubMed] [Google Scholar]
  15. Gimbrone M. A., Jr, Alexander R. W. Angiotensin II stimulation of prostaglandin production in cultured human vascular endothelium. Science. 1975 Jul 18;189(4198):219–220. doi: 10.1126/science.1138377. [DOI] [PubMed] [Google Scholar]
  16. Gordon J. L., Martin W. Stimulation of endothelial prostacyclin production plays no role in endothelium-dependent relaxation of the pig aorta. Br J Pharmacol. 1983 Sep;80(1):179–186. doi: 10.1111/j.1476-5381.1983.tb11064.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Griffith T. M., Edwards D. H., Lewis M. J., Newby A. C., Henderson A. H. The nature of endothelium-derived vascular relaxant factor. Nature. 1984 Apr 12;308(5960):645–647. doi: 10.1038/308645a0. [DOI] [PubMed] [Google Scholar]
  18. Gryglewski R. J., Bunting S., Moncada S., Flower R. J., Vane J. R. Arterial walls are protected against deposition of platelet thrombi by a substance (prostaglandin X) which they make from prostaglandin endoperoxides. Prostaglandins. 1976 Nov;12(5):685–713. doi: 10.1016/0090-6980(76)90047-2. [DOI] [PubMed] [Google Scholar]
  19. Hong S. L. Effect of bradykinin and thrombin on prostacyclin synthesis in endothelial cells from calf and pig aorta and human umbilical cord vein. Thromb Res. 1980 Jun 15;18(6):787–795. doi: 10.1016/0049-3848(80)90201-7. [DOI] [PubMed] [Google Scholar]
  20. Johnson A. R. Human pulmonary endothelial cells in culture. Activities of cells from arteries and cells from veins. J Clin Invest. 1980 Apr;65(4):841–850. doi: 10.1172/JCI109736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Malick L. E., Wilson R. B. Modified thiocarbohydrazide procedure for scanning electron microscopy: routine use for normal, pathological, or experimental tissues. Stain Technol. 1975 Jul;50(4):265–269. doi: 10.3109/10520297509117069. [DOI] [PubMed] [Google Scholar]
  22. Moncada S., Ferreira S. H., Vane J. R. Bioassay of prostaglandins and biologically active substances derived from arachidonic acid. Adv Prostaglandin Thromboxane Res. 1978;5:211–236. [PubMed] [Google Scholar]
  23. Moncada S., Gryglewski R., Bunting S., Vane J. R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature. 1976 Oct 21;263(5579):663–665. doi: 10.1038/263663a0. [DOI] [PubMed] [Google Scholar]
  24. Palmer M. A., Piper P. J., Vane J. R. Release of rabbit aorta contracting substance (RCS) and prostaglandins induced by chemical or mechanical stimulation of guinea-pig lungs. Br J Pharmacol. 1973 Oct;49(2):226–242. doi: 10.1111/j.1476-5381.1973.tb08368.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Peach M. J., Singer H. A., Loeb A. L. Mechanisms of endothelium-dependent vascular smooth muscle relaxation. Biochem Pharmacol. 1985 Jun 1;34(11):1867–1874. doi: 10.1016/0006-2952(85)90300-4. [DOI] [PubMed] [Google Scholar]
  26. Pearson J. D., Carleton J. S., Hutchings A. Prostacyclin release stimulated by thrombin or bradykinin in porcine endothelial cells cultured from aorta and umbilical vein. Thromb Res. 1983 Jan 15;29(2):115–124. doi: 10.1016/0049-3848(83)90133-0. [DOI] [PubMed] [Google Scholar]
  27. Pearson J. D., Slakey L. L., Gordon J. L. Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells. Biochem J. 1983 Jul 15;214(1):273–276. doi: 10.1042/bj2140273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ryan U. S., Mortara M., Whitaker C. Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes. Tissue Cell. 1980;12(4):619–635. doi: 10.1016/0040-8166(80)90017-8. [DOI] [PubMed] [Google Scholar]
  29. SANFORD K. K., EARLE W. R., EVANS V. J., WALTZ H. K., SHANNON J. E. The measurement of proliferation in tissue cultures by enumeration of cell nuclei. J Natl Cancer Inst. 1951 Feb;11(4):773–795. [PubMed] [Google Scholar]
  30. Salmon J. A. A radioimmunoassay for 6-keto-prostaglandin F1alpha. Prostaglandins. 1978 Mar;15(3):383–397. doi: 10.1016/0090-6980(78)90122-3. [DOI] [PubMed] [Google Scholar]
  31. VANE J. R. A sensitive method for the assay of 5-hydroxytryptamine. Br J Pharmacol Chemother. 1957 Sep;12(3):344–349. doi: 10.1111/j.1476-5381.1957.tb00146.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. VANE J. R. THE USE OF ISOLATED ORGANS FOR DETECTING ACTIVE SUBSTANCES IN THE CIRCULATING BLOOD. Br J Pharmacol Chemother. 1964 Oct;23:360–373. doi: 10.1111/j.1476-5381.1964.tb01592.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vane J. R. The release and fate of vaso-active hormones in the circulation. Br J Pharmacol. 1969 Feb;35(2):209–242. doi: 10.1111/j.1476-5381.1969.tb07982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Weksler B. B., Ley C. W., Jaffe E. A. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A 23187. J Clin Invest. 1978 Nov;62(5):923–930. doi: 10.1172/JCI109220. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES