Figure 1.
Expression of the TP receptor in the endothelium of rat middle cerebral arteries.
A) RT-PCR amplification of mRNA transcripts for TP receptor (439 bp) and KCa2.3 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.
Figure 2.
Effect of inhibition of Rho Kinase on TP induced constriction and EDH responses obtained in the presence of TP stimulation in the rat middle cerebral artery.
(A) Concentration response curve showing the vasoconstrictor response produced by the thromboxane A2 mimetic U46619 (1 nM-1 µM; n = 5) in rat middle cerebral arteries in the presence and absence of the selective Rho kinase inhibitor Y27632 (1 and 10 µM; n = 5). Inhibition of Rho kinase significantly and concentration dependently reduced the maximum constriction produced by U46619 and significantly shifted the concentration response curve to the right. (B) Histogram showing SLIGRL (20 µM) induced EDH evoked in the presence of U46619 (50–100 nM) in vessels able to synthesise NO. Also shown are EDH in the presence of KCa3.1 blockade (1 µM, TRAM-34), blockade of both KCa2.3 and 3.1 (100 nM apamin+TRAM-34) and blockade of KCa1.1, 2.3 and 3.1 (100 nM Iberiotoxin+apamin+TRAM-34). Block of KCa3.1 alone was sufficient to significantly reduce EDH; subsequent block of KCa2.3 had no further effect indicating this channel was not functional. Residual EDH was inhibited by further blockade of KCa1.1. (C) Histogram showing SLIGRL induced EDH in the presence of U46619 and the Rho Kinase inhibitor Y27632 in vessels able to synthesise NO. EDH was only significantly reduced following combined blockade of both KCa3.1 and 2.3, indicating that the KCa2.3 channel was now functional. *P<0.05 indicates a significant difference from control (U46619 alone) using one-way ANOVA with Tukey’s post-test, n = 5–6) φP<0.05 indicates a significant difference from Y27632 as determined by one-way ANOVA with Tukey’s post-test, n = 5–6.
Figure 3.
Original traces showing the isolated EDH response evoked by SLIGRL (20 µM) in rat MCAs treated with the NOS inhibitor L-NAME (100 µM).
Upper panels show Em, lower panels tension. (A) Control EDH response. (B) EDH response in the presence of the Rho kinase inhibitor Y27632 (10 µM). (C) EDH response in the presence of Y27632 and the KCa3.1 blocker, TRAM-34 (1 µM). (D) EDH response in the presence of Y27632, TRAM-34 and the KCa2.3 blocker apamin (100 nM). (E) Histogram showing SLIGRL-induced EDH in the presence of Y27632 (10 µM), Y27632 and TRAM-34 and the combination of Y27632, TRAM-34 and apamin. (F) Histogram showing SLIGRL-induced EDH mediated hyperpolarization in the presence of SR5037 (1 µM), SR5037 and TRAM-34 and the combination of SR5037, TRAM-34 and apamin. Both Y27632 and SR5037 fully relaxed L-NAME induced tone, hyperpolarization was unaffected. Normally in the presence of L-NAME blockade of KCa3.1 is sufficient to block the EDH response, however following inhibition of Rho kinase subsequent inhibition of KCa2.3 is required to fully block the EDH response. *P<0.05 indicates a significant difference from control, one-way ANOVA with Tukey’s post-test, n = 4–6. φP<0.05 indicates a significant difference from Rho kinase inhibitor alone (Y27632 or SR5037) as determined by one-way ANOVA with Tukey’s post-test, n = 4–6.
Figure 4.
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).
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 KCa3.1 alone (TRAM-34, 1 µM), in the subsequent presence of blockade of both KCa2.3 (apamin 100 nM) and KCa3.1 as well as the combined of blockade of KCa1.1 (iberiotoxin 100 nM), KCa2.3 and KCa3.1. *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.
Figure 5.
Effect of simvastatin on isolated EDH responses obtained in the presence of a NO synthase inhibitor.
(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 KCa3.1 (TRAM-34; B); combined block of KCa2.3 and 3.1 with apamin and TRAM-34 (C) and the further blockade of KCa1.1, 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 KCa3.1 alone is sufficient to block the EDH response. However, statins revealed a KCa2.3 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.
Figure 6.
Effect of restoring the isoprenoid signalling pathway on EDH responses obtained in the presence of simvastatin and a NO synthase inhibitor.
(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 KCa3.1 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 KCa2.3 function as inhibition of KCa3.1 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.
Figure 7.
Schematic diagram showing potential regulatory mechanisms of KCa2.3 channels by TP receptor/rho mediated signalling.
Increased stimulation of the TP receptor with the agonist U46619 or following inhibition of NOS (eNOS) with L-NAME (NO can supress the action of TP receptors or synthesis of metabolites that activate TP) (1) results in activation of rhoA and stimulation of Rho kinase (2). Rho kinase (or associated signalling) inhibits KCa2.3 function (3) and inhibitors of Rho kinase (Y27632 or SR5037) restore or protect the KCa2.3 component of EDH (4). Statins by inhibiting HMG-CoA prevent formation of the isoprenoid GGPP (5). This reduces Rho mediated signalling by preventing GGPP dependent translocation of rhoA to the plasma membrane (6). Therefore statins protect KCa2.3 function by inhibiting Rho-mediated signalling via the TP receptor (1–3.). Red arrows/text indicates an inhibitory mechanism. Green text/arrows represent a stimulatory mechanism. Blue arrows indicate synthetic pathways; dashed blue line indicates translocation to the plasma membrane.