Figure 1.
ACh-induced relaxation of mouse aortic rings is attenuated by LPC.
The rings were preincubated without (no LPC) or with 10 µM LPC 16:0 (A), 18:1 (B), 18:2 (C) or 20:4 (D) for 30 minutes, followed by precontraction with NE and cumulative addition of ACh. Relaxation values were expressed as a percentage of the NE-induced contraction. Results of each experimental condition are mean ± SEM of 24 rings for each case from 6 mice. *P<0.05, **P<0.01***P<0.001.
Figure 2.
Impact of COX-inhibition and prostacyclin-action on LPC-induced attenuation of relaxation.
The rings were preincubated without (no LPC) or with 10 µM of indicated LPC in the absence or presence of (A,C,D) indomethacin, a non-selective COX inhibitor (20 µM) or (B) CAY10441, a IP receptor antagonist (1 µM) for 30 minutes, followed by precontraction with NE and cumulative addition of ACh. Relaxation values were expressed as a percentage of the NE-induced contraction. Indomethacin improved relaxation attenuated by LPC 18:2 (C) and 20:4 (D). Relaxation attenuated by LPC 16:0 was exaggerated by indomethacin and CAY10441. Results are mean ± SEM of 20 rings for each case from 10 mice (A), 8 rings for each case from 3 mice (B) and 12 rings for each case from 6 mice (C,D).
Figure 3.
Blocking of TP receptor improves LPC 18:2- and 20:4-induced attenuation of relaxation.
The rings were preincubated without (no LPC) or with 10 µM LPC 18:2 (A) or 20:4 (B) in the absence or presence of 10 µM SQ29548, a TP receptor antagonist for 30 minutes, followed by precontraction with NE and cumulative addition of ACh. Results are mean ± SEM of 12 rings from 6 mice. *P<0.05, **P<0.01***P<0.001.
Figure 4.
Inhibition of TXA2- and PGI2- synthase improves relaxation attenuated by LPC 20:4.
The rings were preincubated without (no LPC) or with LPC 20:4 in the absence or presence of 10 µM furegrelate, a TXA2 synthase inhibitor (A) or 10 µM tranylcypromine, a PGI2 synthase inhibitor (B) or a combination of both (C) for 30 minutes, followed by precontraction with NE and cumulative addition of ACh. Results are mean ± SEM of 12 rings from 6 mice. *P<0.05, **P<0.01.
Figure 5.
Prostanoid release from LPC-treated aortic rings.
The rings were incubated in 200 µl aerated PSS under cell culture conditions at 37°C for 1 h. Thereafter, buffer was replaced with fresh PSS supplemented with PSS (control) or 10 µM LPC followed by further incubation under cell culture conditions at 37°C for 1 h. (A) 6-keto PGF1α, a stable degradation product of PGI2 (B), TXB2, a stable degradation product of TXA2, (C) PGE2 and (D) PGF2α were quantified by EIA assays and rings were solubilized for protein quantification. Results shown in A and B are means ± SD of four experiments and those in C and D of three experiments, done in triplicates. *P<0.05, **P<0.01***P<0.001.
Figure 6.
ROS are induced by LPC and contribute to LPC-induced impairment of relaxation.
A) The rings were equilibrated in 100 µl PSS buffer containing 10 µM DETCA and 10 µM lucigenin at 37°C for 30 minutes, followed by addition of PSS (control) or LPC (10 µM). Emitted light (RLU) was recorded every 10 seconds for 30 seconds. The RLU were normalised to protein content of respective aortic rings and expressed as percentage of control set to 100%. Results are means ± SEM of three separate experiments, each performed with three rings for each LPC. The rings were preincubated without (no LPC) or with 10 µM LPC 18:1 (B), 18:2 (C), 20:4 (D) or 16:0 (E) in the absence or presence of 200 µM Tempol for 30 minutes, followed by precontraction with NE and cumulative addition of ACh. Results for each condition are mean ± SEM of 12 rings from 6 mice. *P<0.05, **P<0.01.