Statins and selective inhibition of Rho kinase protect small conductance calcium-activated potassium channel function (KCa2.3) in cerebral arteries

Background: In rat middle cerebral and mesenteric arteries the KCa2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, KCa2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain KCa2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA). Methods: MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence. Results: Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of KCa2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. KCa2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation. Conclusions: Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell KCa2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of KCa2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment

Statins and selective inhibition of Rho kinase protect small conductance calcium-activated potassium channel function (KCa2.3) in cerebral arteries

Background: In rat middle cerebral and mesenteric arteries the KCa2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, KCa2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain KCa2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA). Methods: MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence. Results: Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of KCa2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. KCa2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation. Conclusions: Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell KCa2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of KCa2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment

Evidence both L-type and non-L-type voltage-dependent calcium channels contribute to cerebral artery vasospasm following loss of NO in the rat

We recently found block of NO synthase in rat middle cerebral artery caused spasm, associated with depolarizing oscillations in membrane potential (Em) similar in form but faster in frequency (circa 1 Hz) to vasomotion. T-type voltage-gated Ca2+ channels contribute to cerebral myogenic tone and vasomotion, so we investigated the significance of T-type and other ion channels for membrane potential oscillations underlying arterial spasm. Smooth muscle cell membrane potential (Em) and tension were measured simultaneously in rat middle cerebral artery. NO synthase blockade caused temporally coupled depolarizing oscillations in cerebrovascular Em with associated vasoconstriction. Both events were accentuated by block of smooth muscle BKCa. Block of T-type channels or inhibition of Na+/K+-ATPase abolished the oscillations in Em and reduced vasoconstriction. Oscillations in Em were either attenuated or accentuated by reducing [Ca2+]o or block of KV, respectively. TRAM-34 attenuated oscillations in both Em and tone, apparently independent of effects against KCa3.1. Thus, rapid depolarizing oscillations in Em and tone observed after endothelial function has been disrupted reflect input from T-type calcium channels in addition to L-type channels, while other depolarizing currents appear to be unimportant. These data suggest that combined block of T and L-type channels may represent an effective approach to reverse cerebral vasospasm

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

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.

<p>(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 K_Ca3.1 blockade (1 µM, TRAM-34), blockade of both K_Ca2.3 and 3.1 (100 nM apamin+TRAM-34) and blockade of K_Ca1.1, 2.3 and 3.1 (100 nM Iberiotoxin+apamin+TRAM-34). Block of K_Ca3.1 alone was sufficient to significantly reduce EDH; subsequent block of K_Ca2.3 had no further effect indicating this channel was not functional. Residual EDH was inhibited by further blockade of K_Ca1.1. (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 K_Ca3.1 and 2.3, indicating that the K_Ca2.3 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.</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

Original traces showing the isolated EDH response evoked by SLIGRL (20 µM) in rat MCAs treated with the NOS inhibitor L-NAME (100 µM).

<p>Upper panels show E_m, 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 K_Ca3.1 blocker, TRAM-34 (1 µM). (D) EDH response in the presence of Y27632, TRAM-34 and the K_Ca2.3 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 K_Ca3.1 is sufficient to block the EDH response, however following inhibition of Rho kinase subsequent inhibition of K_Ca2.3 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.</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

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