The effects of inhibiting K_Ca channels in n-3 PUFA-induced relaxation of rat mesenteric artery preconstricted with U46619.

<p>(A) DHA- and (B) EPA-induced relaxation of rat mesenteric artery in the presence of L-NAME (300 μM) followed by the subsequent addition of K_Ca inhibitors; apamin (50 μM), TRAM-34 (1 μM) and paxilline (Pax, 1 μM) (<i>n</i> = 5). Data are expressed as mean±SEM. *Indicates P<0.05, significant difference from control curve assessed by one-way ANOVA followed by Bonferroni post-test.</p

The effects of inhibiting K_Ca channels in n-3 PUFA-induced relaxation of rat aorta preconstricted with U46619.

<p>(A) DHA- and (B) EPA-induced relaxation of rat aorta in the presence of L-NAME (300 μM) followed by the subsequent addition of K_Ca inhibitors; apamin (50 μM), TRAM-34 (1 μM) and paxilline (Pax, 1 μM) (<i>n</i> = 5). Data are expressed as mean ± SEM. * Indicates P<0.05, significant difference from control curve was assessed by one-way ANOVA followed by Bonferroni post-test.</p

The effects of L-NAME and indomethacin in n-3 PUFA-induced relaxation of rat arteries preconstricted with U46619.

<p>(A) DHA- and (B) EPA-induced relaxation of rat mesenteric artery in the presence of L-NAME (300 μM) and subsequent addition of indomethacin (10 μM) (<i>n</i> = 5). (C) DHA- and (D) EPA-induced relaxation of rat aorta in the presence of L-NAME and subsequent addition of indomethacin (<i>n</i> = 5). Data are expressed as mean±SEM. * Indicates P<0.05, significant difference from control curve was assessed by one-way ANOVA followed by Bonferroni post-test.</p

The effect of endothelium removal in n-3 PUFA-induced relaxation of rat arteries preconstricted with U46619.

<p>(A) DHA (<i>n</i> = 5) and (B) EPA-induced relaxation of rat mesenteric artery following endothelium removal (<i>n</i> = 6). (C) DHA (<i>n</i> = 7) and (D) EPA-induced relaxation of rat aorta following endothelium removal (<i>n</i> = 6). Data are expressed as mean±SEM. * Indicates P<0.05, significant difference from control curve was assessed by one-way ANOVA followed by Bonferroni post-test.</p

The effect of clotrimazole in n-3 PUFA-induced relaxation of rat arteries preconstricted with U46619.

<p>(A) DHA- and (B) EPA-induced relaxation of rat mesenteric artery in the presence of clotrimazole (1 μM) (<i>n</i> = 5–6). (C) DHA- and (D) EPA-induced relaxation of rat aorta in the presence of clotrimazole (<i>n</i> = 5). Data are expressed as mean±SEM. * Indicates P<0.05, significant difference from control curve was assessed by one-way ANOVA followed by Bonferroni post-test.</p

A schematic showing potential mechanisms involved in n-3 PUFA-induced relaxation of rat mesenteric artery and aorta preconstricted with U46619.

<p>Solid arrows represent pathways that have been previously investigated whereas dotted arrows represent hypothetical pathways based on our findings. In mesenteric arteries n-3 PUFAs enters the cytosol of the endothelium via diffusion from the plasma or release from the phospholipid bilayer via the activity of phospholipase A2 (PLA2); (1) EPA can be converted into EpETEs by CYP450, activating BK_Ca. IK_Ca may be potentially activated by (2) EpETEs, (3) EPA and (4) DHA. (5) Similar to EpETEs, EPA might also be involved in the direct activation of BK_Ca. (6) DHA directly activates BK_Ca (62). (7) Both n-3 PUFAs may enter the cytosol of VSMCs via diffusion or release from the phospholipid bilayer due to the activity of phospholipase A2 (PLA2) and directly activate BK_Ca. (8) CYP450 derived metabolites of EPA such as EpETEs may induce vasodilation through K_Ca independent mechanisms which could involve other potassium channels such as K_ATP.</p

Effect of simvastatin on isolated EDH responses obtained in the presence of a NO synthase inhibitor.

<p>(A–D) Original traces showing the effect of 100 nM simvastatin (A) on isolated SLIGRL-induced EDH-mediated responses (hyperpolarization, upper panels; relaxation, lower panels) obtained in rat MCAs treated with the NOS inhibitor L-NAME (100 µM). Also shown is the effect of block of K_Ca3.1 (TRAM-34; B); combined block of K_Ca2.3 and 3.1 with apamin and TRAM-34 (C) and the further blockade of K_Ca1.1, 2.3 and 3.1 with iberiotoxin, apamin and TRAM-34 (D). Also shown (E–G) are histograms of the mean data for SLIGRL-induced EDH mediated responses (hyperpolarization, upper panels; relaxation, lower panels) in the presence of 100 nM simvastatin (E), 1 µM simvastatin (F). Normally in the presence of L-NAME inhibition of K_Ca3.1 alone is sufficient to block the EDH response. However, statins revealed a K_Ca2.3 component to the EDH response. *P<0.05 indicates a difference from control, ^φP<0.05 indicates a significant difference from simvastatin (100 nM or 1 µM) as determined by one-way ANOVA with Tukey’s post-test, n = 5–8.</p

Histograms showing EDH evoked by SLIGRL (20 µM) in rat MCAs that are able to synthesise NO but treated with the TP receptor agonist, U46619 (50–100 nM).

<p>Also shown is the effect of the HMG-CoA reductase inhibitor simvastatin at either 100 nM (A) or 1 µM (B) on EDH and the effect of blocking K_Ca3.1 alone (TRAM-34, 1 µM), in the subsequent presence of blockade of both K_Ca2.3 (apamin 100 nM) and K_Ca3.1 as well as the combined of blockade of K_Ca1.1 (iberiotoxin 100 nM), K­_Ca2.3 and K_Ca3.1. *P<0.05 indicates a significant difference from control using one way ANOVA with Tukey’s post-test, n = 4–7. ^φP<0.05 indicates a significant difference from simvastatin (100 nM or 1 µM) as determined by one-way ANOVA with Tukey’s post-test, n = 4–7.</p

Expression of the TP receptor in the endothelium of rat middle cerebral arteries.

<p>A) RT-PCR amplification of mRNA transcripts for TP receptor (439 bp) and K_Ca2.3 channels (514 bp) from rat MCA. bp = base pairs B) Localization of TP receptor and PECAM-1-immunoreactivity in whole mount preparations of rat MCA. TP receptor-immunoreactivity was present in the endothelial cell layer (PECAM-1-positive). Orientation of cell nuclei was determined using DAPI. The merged image demonstrates coexpression of TP receptors and PECAM-1, indicating TP receptor expression in the endothelial cells of rat MCAs. Scale bar, 20 µm.</p

Effect of restoring the isoprenoid signalling pathway on EDH responses obtained in the presence of simvastatin and a NO synthase inhibitor.

<p>(A–D) original traces showing isolated SLIGRL-induced, EDH-mediated hyperpolarizations (upper panels) and relaxations (lower panels) obtained from rat MCAs treated with the NOS inhibitor L-NAME (100 µM; A). Also shown is the additional effect of simvastatin (100 nM), addition of GGPP (1 µM; C) and the combination of simvastatin and GGPP with the K_Ca3.1 inhibitor TRAM-34 (1 µM; D). (E) Histogram showing the mean data for isolated EDH-mediated responses (hyperpolarization, upper panel; relaxation, lower panel). While GGPP did not alter the total EDH mediated response it reversed the ability of simvastatin to protect K_Ca2.3 function as inhibition of K_Ca3.1 with TRAM-34 alone was sufficient to significantly inhibit the EDH response. *P<0.05 indicates a significant difference from control using one-way ANOVA with Tukey’s post-test, n = 4.^φP<0.05 indicates a significant difference from simvastatin, as determined by one-way ANOVA with Tukey’s post-test, n = 4.</p