Thienopyridines: Effects on cultured endothelial cells

In cultured endothelial cells harvested from human umbilical vein (HUVEC) o r bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 mu M) or ticlopidine (100 mu M) caused an increase in nitrite generation in HUVEC from basal 227+/-37 to 372+/-60 or to 325+/-33 pmoles per 10(6) c ells, respectively. md in BAEC from basal 182+/-17 to 378+/-18 or to 423+/- 66,pmoles per 10(6) cells (n = 6), respectively. Calcium ionophore A 23187 (1 mu M):or ticlopidine (100 mu M) next to 30 min incubation with BAEC incr eased release of 6-keto-PGF(1 alpha) from basal level of 9.4+/-1.8 to 96.2/-5.1 or to 99.5+/-10.2 pmoles per 10(6) cells: respectively. The pretreatm ent with aspirin (300 mu M) cut down this rise to 4.2+/-0.1 pmoles per 10(6 ) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7+/-3.3 nM (n:= 53), 68.3 +/-5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cul tured endothelial cells calcium ionophore A 23187 (0.1 mu M) produced net m aximum rise in [Ca2+]i by 157+/-27 nM (n = 16)[ ECV304], by 107+/-58 nM (n= 4) [HUVEC], and by 231.0+/-41.3 nM (n = 8) [BAEC], respectively, while ticl opidine (30 mu M) produced net maximum rise in [Ca2+]i by 30.0+/-3.2 nM (n = 9)[ECV304], 48.8+/-15.6 nM (n = 4)[HUVEC] and 28.4+/-5.4 nM (n = 8)[BAEC] , respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than t hat of calcium A 23187 but also its maximum appeared after a lag period tha t was 2-3 times longer than that for A23187. In ECV304 clopidogrel at conce ntrations of 10, 30 and 100 mu M produced maximum increment of [Ca2+]i by 1 6.5+/-3.8 nM (n = 7), 47.0+/-6.9 nM (n = 8) and 67.2+/-8.3 nM (n= 8), respe ctively. Incubation of BAEC with A23187 (1 mu M), ticlopidine Or clopidogre l (100 mu M) for 2 h did not influence viability of cultured endothelial ce lls. We claim that thienopyridines, independently of their delayed anti-pla telet properties ex vivo do release NO and PGI(2) from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be m ediated by a rise in [Ca2+]i, however, this possibility has not been proved .