Effects of statins on vascular function of endothelin-1

Abstract

1. Although statins have been reported to inhibit the prepro-endothelin-1 (ET-1) gene transcription in endothelial cells, their effects on the vascular function of ET-1 have not been explored. We, therefore, examined the effects of statins on contraction and DNA synthesis mediated by ET-1 in vascular smooth muscle. The effects of statins on contraction induced by ET-1 were compared to those mediated by noradrenaline (NA) and KCl. 2. Simvastatin (SV) induced a concentration-dependent relaxation of tonic contraction mediated by ET-1 (10 nM) (IC50 value of 1.3 microM). The relaxation was also observed in rings precontracted with NA (0.1 microM) and KCl (60 mM). In contrast, pravastatin did not have any effect on the contractions. 3. Endothelial denudation or pretreatment with L-NAME did not prevent the relaxation, but did reduce the relaxant activity of SV. 4. SV prevented Rho activation caused by ET-1 and KCl in aortic homogenates, as assessed by a Rho pulldown assay. 5. The Rho kinase inhibitor HA-1077 mimicked the effects of SV on tonic contractions induced by ET-1, NA and KCl. 6. Pretreatment with the Kv channels inhibitor, 4-aminopyridine, attenuated the ability of SV to relax contractions mediated by ET-1 and NA. 7. In quiescent VSM cells, SV significantly inhibited DNA synthesis and Rho translocation stimulated by ET-1, as assessed by [3H]thymidine incorporation and Western blot, respectively. 8. Inhibition of Rho geranylgeranylation by GGTI-297, or treatment with HA-1077, mimicked the effects of SV on DNA synthesis stimulated by ET-1. 9. The results show that the statin potently inhibits both ET-1-mediated contraction and DNA synthesis via multiple mechanisms. Clinical benefits of statins may result, in part, from their effects on vascular function of ET-1.

Figure 1

Effects of SV on tonic contractions induced by ET-1, NA and KCl. Aortic rings were precontracted with either ET-1 (10 nM), NA (0.1 μM), or KCl (60 mM) prior to obtaining cumulative concentration–response curves to SV. Results are expressed as a percentage of maximum tonic contraction produced by the vasoconstrictors, and are expressed as means±s.e.m. for experiments with 10 or more rings.

Figure 2

Effects of endothelial denudation and L-NAME on relaxation of rat aortic rings caused by SV. Aortic rings were pretreated for 30 min with L-NAME (200 μM) in the presence (E+) or absence (E−) of endothelium, and then precontracted with either ET-1 (10 nM) (panel a) or NA (0.1 μM) (panel b), prior to obtaining cumulative concentration–effect curves for SV. Exposure to L-NAME resulted in an increase in tension to ET-1 and NA by about 20%. Results in panels a and b are expressed as a percentage of maximum contraction induced by either ET-1 or NA, and are presented as means±s.e.m. for experiments with 10 rings. Panel c shows representative traces of the effects of bradykinin (0.001–1 μM) on rings of rat aorta with preserved and denuded endothelium. The rings were precontracted with NA (0.1 μM). Bradykinin produced concentration-dependent relaxations in preparations, which had been precontracted with NA, in the presence (upper trace) but not in the absence (lower trace) of endothelium. Each trace illustrates the response of an individual ring preparation and is representative of eight or more independent experiments.

Figure 3

Effects of mevalonate on relaxation mediated by SV in rat aortic rings precontracted with ET-1. Aortic rings were pretreated with either mevalonate (200 μM) or vehicle for 2 h, and then precontracted with ET-1 (10 nM) prior to obtaining cumulative concentration–effect curves for SV. Results are expressed as a percentage of the maximum tonic contraction to ET-1, and are presented as means±s.e.m. for experiments with six rings.

Figure 4

Effects of HA-1077 on tonic contractions developed in response to ET-1, NA and KCl. HA-1077 was administered in increasing cumulative concentrations (0.1–1 μM) to preparations, in which tonic contractions to the vasoconstrictors had developed. Results are expressed as a percentage of maximum tonic contraction produced by either ET-1 (10 nM), NA (0.1 μM) or KCl (60 mM), and are presented as means±s.e.m. for experiments with six rings.

Figure 5

Effects of SV and nimodipine on Rho activation in rat aorta stimulated with either ET-1 or KCl. The endothelium-denuded rat aorta preparations were stimulated with either ET-1 (10 nM) or KCl (60 mM), and the muscle was snap frozen after 5 and 30 min of stimulation. SV (2 μM) or nimodipine (0.1 μM) were administered 10 min before the administration of ET or KCl. Rho activation was determined by using an affinity precipitation assay with GST-fusion protein of the RBD of the Rho effector rhotekin, as described in Methods. Panel a shows the effects of SV (2 μM) and nimodipine (0.1 μM) on Rho activation in rat aorta stimulated for 5 min (left) and 30 min (right) with ET-1 (10 nM). Panel b shows the effects of SV and nimodipine on Rho activation in rat aorta stimulated with KCl (60 mM) for 5 min (left) and 30 min (right). The position of GTP-Rho is indicated on the right of the representative immunoblots. Quantification of intensities of GTP-Rho bands was performed by scanning densitometry. Results are expressed as a percentage of respective controls, and are means±s.e.m. of duplicate determinations from five experiments.

Figure 6

Effects of nimodipine on tonic contraction developed in response to ET-1, NA and KCl. Aortic rings were precontracted with either ET-1 (10 nM), NA (0.1 μM) or KCl (60 mM) and nimodipine was administered in increasing cumulative concentrations (0.1 nM–1 μM) to preparations in which tonic contractions to the vasoconstrictors had developed. Results are expressed as a percentage of maximum tonic contraction produced by ET-1, NA or KCl, and are presented as means±s.e.m. for experiments with six rings.

Figure 7

Effects of the inhibitor of Kv channels, 4-AP, on SV-induced relaxation of rat aortic rings. Preparations were pretreated for 30 min with 4-AP (1 mM) in the presence (E+) or absence (E−) of endothelium, and then precontracted with either ET-1(10 nM) (panel a) or NA (0.1 μM) (panel b), prior to obtaining cumulative concentration–response curves for SV. Results are expressed as a percentage of maximum tonic contraction induced by either ET-1 or NA and are presented as means±s.e.m. for experiments with six rings.

Figure 8

Effects of SV, GGTI-297 and HA-1077 on [³H]thymidine incorporation stimulated by ET-1 in VSM cells. [³H]thymidine incorporation was used to assess DNA synthesis. Subconfluent, quiescent cells were incubated with either vehicle (0.3% DMSO) or ET-1 (10 nM) for 48 h. SV (0.1–10 μM) (panel a), was added to quiescent cells 24 h before administration of ET-1.GGTI-297 (200 nM) (panel b) and HA-1077 (1 μM) (panel c) were administered 30 min prior to addition of ET-1 (10 nM) for 48 h. Data represent the means±s.e.m. of six experiments performed in triplicate. ^***P<0.001 versus control; ^##P<0.01 and ^###P<0.001 versus ET-1.

Figure 9

Effects of SV on Rho translocation mediated by ET in VSM cells. Subconfluent, quiescent cells were stimulated with ET-1 (10 nM) for 10 min. SV was administered for 24 h before treatment with ET-1. Protein-matched samples from cytosolic (C) and membrane (M) fractions were resolved by SDS–PAGE and immunoblotted with a monoclonal Rho antibody as described in Methods. Upper panel shows a representative Western blot illustrating Rho translocation induced with ET-1 in the absence or in the presence of SV. Quantification of intensities of Rho bands was performed by densitometry (arbitrary units). Bars (lower panel) represent Rho distribution in the cytosol and membrane fractions, expressed as a percentage of respective controls. Results are expressed as means±s.e.m. from three experiments performed in duplicate. ^**P<0.01 versus control; ^##P<0.01 versus ET-1.