Activation of bovine endothelial thromboxane receptors triggers release of prostacyclin but not EDRF.
OBJECTIVE
The aim was to examine the capacity of U46619 (a stable thromboxane A2 mimetic) to mediate release of endothelium derived relaxing factor (EDRF) from bovine aortic endothelial cells, and compare the response to the U46619 dependent release of prostacyclin (PGI2).


METHODS
Bovine aortic endothelial cells (AG4762) were cultured in vitro on microcarrier beads, which were then loaded onto a column and perfused. The cells were challenged with U46619, bradykinin, or the Ca2+ ionophore ionomycin in the perfusate, and measurements made of the release of 6-oxo-PGF1 alpha (the stable hydrolysis product of PGI2, measured by radioimmunoassay) and EDRF (bioassay). Cells were also cultured on glass cover slips, loaded with Fura 2-AM, and measurements made of the rise in intracellular Ca2+ after challenge with U46619, bradykinin or ionomycin.


RESULTS
U46619 triggered release of 6-oxo-PGF1 alpha but not EDRF from AG4762 cells, contrasting with bradykinin which released both 6-oxo-PGF1 alpha and EDRF. Ionomycin had little or no capacity to mimic and trigger release of 6-oxo-PGF1 alpha, although ionomycin mediated large increases in intracellular Ca2+. In contrast, staurosporine (a putative inhibitor of protein kinase C) substantially inhibited the U46619 and bradykinin dependent release of 6-oxo-PGF1 alpha.


CONCLUSIONS
In contrast to bradykinin linked receptors on AG4762 endothelial cells, which are coupled to the release of both prostacyclin and EDRF, activation of thromboxane A2 receptors on these cells selectively triggers release of PGI2 but not EDRF. Further, based on the distinct effects of ionomycin and staurosporine, it appears that agonist stimulated PGI2 release from these cells is mediated predominantly by protein kinase C, rather than by rises in intracellular Ca2+. This observation contrasts with previously described mechanisms of PGI2 release from endothelium obtained from other sources.
Clesham
G
G
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, United Kingdom.
Parsaee
H
H
Joseph
S
S
McEwan
J R
JR
MacDermot
J
J
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000818:Animals; D001011:Aorta; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism; D011464:Epoprostenol / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism*; D017482:Receptors, Thromboxane / Q000378:metabolism*
D011450:Prostaglandin Endoperoxides, Synthetic; D017482:Receptors, Thromboxane; D009569:Nitric Oxide; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D011464:Epoprostenol