Endothelium modulates contractile response to simvastatin in rat aorta

Endothelium Modulates Contractile Response to Simvastatin in Rat Aorta Concepción Pérez-Guerrero*, Marí a Álvarez de Sotomayor, Maria Dolores Herrera and Elisa Marhuenda Department of Pharmacology, Faculty of Pharmacy, University of Seville, Profesor Garcia-Gonzalez s/n, 41012 Seville, Spain. Fax: 34-95-4233765. E-mail: [email protected] * Author for correspondence and reprints requests Z. Naturforsch. 55c, 121-124 (2000); received July 22/September 10/1999 HMG-CoA Reductase, Simvastatin, Endothelium, Rat Aorta Simvastatin is an inhibitor of HMG-CoA reductase used in the treatment of hypercholes terolemia. In the present study simvastatin-induced contraction was observed in rat aortic thoracic rings, this effect increased when the endothelium was removed and when NO syn thase was blocked by L-NOARG (3 x 10“5 m). The contractile effect of simvastatin on intact aortic rings diminished when cyclo-oxygenase was inhibited with indomethacin (10-5 m). Also in the presence of endothelium, pretreatment with mevalonate (1 mM), the product of HMGCoA reductase activity, significantly inhibited the contraction. In other experiments carried out on endothelium-removed preparations and in medium containing the calcium antagonist, diltiazem (10-5 and 10-6 m), the contraction dose-response curves were significantly reduced and the same happened in the presence of the inhibitor of sarcoplasmic reticulum Ca-2+- ATPase, cyclopiazonic acid (CPA) (3 x 10-6 m). The results suggest that simvastatin might increase intracellular calcium concentration. This effect could lead to an activation of NO synthase and cyclooxygenase pathways in endo thelial cells and to contraction in vascular smooth muscle cells. This rise in Ca2+ concentration could be due to an inhibition of isoprenoid synthesis prevented by mevalonate. Introduction Simvastatin is a drug widely used in the treat ment of hypercholesterolemia. This drug is an inhibitor of the rate-determining enzyme in the biosynthesis of cholesterol, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and has proved useful in the reduction of plasma low density lipoprotein (LDL). Clinical trials have demostrated that inhibitors of HMG-CoA reductase decrease cardiovascular-related morbidity and mortality (Vaughan et al., 1996). In previous studies of our laboratory, we found that another HMG-CoA reductase inhibitor, lovastatin, was able to decrease blood pressure and had vasodilatatory effects in precontracted iso lated rat thoracic aorta (Bravo et al., 1998). It has also been demostrated that chronic treatment with simvastatin was able to improve endothelial func tion in spontaneously hypertensive rats (Alvarez de Sotomayor et al., 1999). In order to elucidate the mechanism of action of simvastatin, the direct effect of this drug was investigated in rat thoracic aortic rings. Material and Methods Experimental procedure Wistar male rats (250-300 g) were killed by a blow on the head. The descending thoracic aorta was rapidly dissected and placed in a modified Krebs-Henseleit solution (PSS), containing ( m M ) : NaCl 118, KC1 4.75, N aH C03 25, MgS04 1.2, CaCl2 1.8, KH2P 0 4 1.2 and glucose 11. After ex cess fat and connective tissue were removed, the aortas were out cut into 2-3 mm rings. Aortic rings were mounted under the basal tension of 1 g in 20 ml organ baths containing PSS and at tached to a force-displacement transductor (Har vard UF-1) and the signal was recorded by a Powerlab® data acquisition system (ADInstruments) to measure the contractile force. The tissue bath was maintained at 37 °C and bubbled with a 95% 02-5% C 0 2 ( v /v ) gas mixture. In some experi ments, the endothelium of the aorta was mechani cally removed by gently rubbing the inner surface. The absence of endothelium was confirmed by the absence of relaxing effects of acetylcholine (ACh 1 0 ~ 6 m ) in aortic rings previously contracted by noradrenalin (NA 10' 5 m ). Each preparation was 0939-5075/2000/0100-0121 $ 06.00 © 2000 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • N Unauthenticated | Downloaded 05/14/20 03:18 PM UTC 122 C. Perez-Guerrero et al. • Effect of Simvastatin in Rat Aorta allowed to equilibrate for at least 90 min prior to start of experimental procedures, and during this period the incubation media was changed every 2 0 min. Dose-response curves of contraction were ob tained by addition of cumulative concentrations of simvastatin (107 to 10~ 4 m ) at 3-min intervals in the presence and absence of endothelium. The same experiments were carried out in the presence of N-w-nitro-L-arginine (L-NOARG, 3 x 10“ 5 m ), indomethacin (105 m ), mevalonate (1 m M ) , diltiazem (1 0 ~ 6 m or 1 0 - 5 m ) and cyclopiazonic acid (CPA, 3 x 106 m ) in aortic rings. All results are expressed as a percentage of the maximal con traction of noradrenalin-induced responses. Drugs Indomethacin (l-[/?-chlorobenzoyl]-5-methoxy2-methylindole-3-acetic acid), mevalonate ( d l - / 3 - hydroxy-/3-methyl-(5-valero-lactone), N-&»-nitro-Larginine (N5- [nitroamidino]-L-2,5-diaminopentanoic acid), diltiazem (ds-(+)-3-(acetyloxy)-5-[2- (dimethyl-amino)ethyl]-2,3-dihydro-2-(4-methoxy-phenyl)-l,l-benzothiazepin-4 (5H) one) hy drochloride, cyclopiazonic acid (C20H20N2O3) from Penicillium cyclopium, acetylcholine (C7H16N 0 2C1) and noradrenalin (-)-arterenol (CsHuNOs.C^öOö) were obtained from Sigma Chemical Co (St. Louis, MO, USA). Simvastatin (2,2-dimethylbutanoate 1,2,3,7,8,8ahexahydro-3,7-dimethyl-8-[2-tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-l-naphthalenyl), was generously provided by Merck laboratories (New Jersey, USA). All drugs were dissolved in dimethylsulfoxide (DMSO) except N-co-nitro-Larginine and diltiazem which were dissolved in dis tilled water. The final concentration of DMSO did not significantly affect the results. Statistical analysis Results are expressed as mean ± SEM of six preparations obtained from different animals. Analysis of variance (ANOVA) followed by the LSD (least significant difference) test were used for statistical analysis. P values <0.05 were con sidered to indicate a significant difference. Doseresponse slopes were analysed to give the concen tration of simvastatin producing 50% of the maxi mum contractile response (ED50) using a linear regression analysis. Results and Discussion Addition of cumulative concentrations of sim vastatin (1 0 ~ 7 to 1 0 ~ 4 m ) induced dose-dependent contractions in isolated rat thoracic aorta with and without endothelium, reaching maximal tension values of 1.22 ± 0.08 g and 1.94 ± 0.12 g and the ED5 0 values were 3.46 ± 0.56 x 105 m and 5.41 ± 0.25 x 106 m , respectively. In intact aortic rings, preincubation with L-NOARG (3 x 10~ 5 m ) signif icantly potentiated the contractions evoked by simvastatin (ED5 0 = 3.62 ± 0.27 x 106 m ). How ever, pretreatment of the aortic rings with indo methacin (1 0 ~ 5 m ) decreased the curve obtained with simvastatin alone underneath the control curve reaching only 30% of the maximal effect. In the presence of mevalonate (1 m M ), the pro duct of HMG-CoA reductase activity, simvastatininduced contraction was almost abolished. When the experiments were carried out on endotheliumremoved preparations and in a medium containing diltiazem (1 0 ~ 6 m or 1 0 ~ 5 m ), the curve obtained with simvastatin was significantly reduced, yield ing a higher effect at the highest dose of diltiazem assayed. Pretreatment with cyclopiazonic acid (3 x 1 0 - 6 m ) diminished the contractile effect of sim vastatin on endothelium-intact preparations. The percent data are summarized in Table I. The contractile effect induced by simvastatin in rat aortic thoracic rings increased when the endo thelium was removed, showing the involvement of endothelial factors in the mechanism of action of simvastatin. When NO synthase was blocked with L-NOARG, the contraction caused by simvastatin was enhanced, suggesting that NO is released in the presence of simvastatin, opposing its relaxant effect to the contraction. In this way, former expe riences have shown that NO is involved in some actions of statins (Laufs et al., 1998; HernändezPerera et al., 1998; Kaesemeyer et al., 1999). The contractile effect diminished when indomethacin inhibited cyclo-oxygenase, supporting the notion of a partial participation of contracting cyclo-oxygenase-derived factors (endoperoxides and throm boxanes) in this effect. Both results together might be explained by an action of simvastatin in a corvengent point of both, NO synthase and cyclo-oxyUnauthenticated | Downloaded 05/14/20 03:18 PM UTC C. Perez-Guerrero et al. ■ Effect of Simvastatin in Rat Aorta 123 Table I. Percentage effect reached in response to simvastatin (10-7 - 10-4 m) in aortic rings with (E(+)) and without endothelium (E (-)). In the absence of L-NOARG, indomethacin (INDO), cyclopiazonic acid (CPA), diltiazem (DIL) and mevalonate (MV). Values are mean ± SEM of n = 6 experiments. *P<0.05;**P<0.01; ***P<0.001 vs control with endothelium. #P<0.05; ##P <0.01; ###P 0.001 vs control without endothelium. Simvastatin (m) io-7 io-6 10 5 10"4 Control E(+) 0.7 ± 0.2 1.7 ± 1.0 28.7 ± 6.0 63.6 ± 7.7 Control E (- ) 1.2 ± 0.5 15.1 ± 4.0 64.8 ± 5.9 *** 89.8 ± 3.8 * L-NOARG (3 x 10~5 m) E(+) 0.7 ± 0.6 17.3 ± 7.4 76.9 ± 11.2 *** 107.9 ± 9.8 *** INDO (IO“5 m) E(+) 0.9 ± 0.2 0.9 ± 0.3 9.6 ± 4.4 30.0 ± 6.6 ** MV (IO“3 m) E(+) 1.8 ± 1.1 1.9 ± 1.1 2.4 ± 1.0 ** 13.9 ± 4.5 *** CPA (3 x 105 m) E (-) 1.7 ± 0.9 4.2 ± 1.1 #18.8 ± 2.5 ## 38.3 ± 4.3 ### CPA (3 x 106 m) E ( - ) 0.4 ± 0.2 0.6 ± 0.1 # 12.9 ± 2.2 # # # 44.7 ± 6.0 # # # DIL (10-5 m) E (-) 6.0 ± 3.0 3.8 ± 1.7 #9.2 ± 2.2 # # # 23.3 ± 6.1 # # # DIL (10-6 m) E (-) 3.1 ± 2.2 3.3 ± 1.4 # 15.2 ± 4.6 # # # 46.8 ± 6.8 # # # L-NOARG, N-co-nitro-L-arginine. INDO, indomethacin. MV, mevalonate. CPA, cyclopiazonic acid. DIL, diltiazem. E(+), with endothelium. E (-) , without endothelium. genase pathways, like Ca2+ increment in endothelial cell, necessary for activation of NO synthase (Moncada et al., 1991) and phospholipase A2 (Verbeij et al., 1981), and consequently cyclo-oxygenase. The presence of mevalonate, the metabolic product of HMG-CoA reductase (Goldstein and Brown 1990), almost abolished simvastatin-induced con traction, indicating the involvement of mevalonate pathway. In accordance with our hypothesis, some other reports showed that in vascular smooth mus cle cells, simvastatin has been able to increase tran siently cytosolic free Ca2+ concentration, being this effect prevented by the presence of mevalonate (Ng et al., 1994; Escobales et al., 1996). Diltiazem, a calcium entry antagonist, and cyclo piazonic acid, a Ca2+-ATPase inhibitor, were effec tive in inhibiting the contractile response to sim vastatin, leading us conclude that both: voltageoperated Ca2+ channels and calcium released from these stores are involved in the contractile mecha nism of simvastatin. In summary, simvastatin might increase intracellular calcium concentration in both endothelial and vascular smooth muscle cells. This effect lead to an activation of NO syn thase and cyclo-oxygenase pathway in endothelial cells and to contraction in vascular smooth mus cle cells. Unauthenticated | Downloaded 05/14/20 03:18 PM UTC 124 C. Perez-Guerrero et al. ■ Effect of Simvastatin in Rat Aorta Älvarez de Sotomayor M., Perez-Guerrero C., Herrera M. D. and Marhuenda E. (1999), Effects of chronic treatment with simvastatin on endothelial dysfunction in spontaneously hypertensive rats. J. Hypertens. 17, 769-776. Bravo L., Herrera M. D., Marhuenda E. and PerezGuerrero C. (1998), Cardiovascular effects of lovastatin in normotensive and spontaneously hypertensive rats. Gen. Pharmacol. 30, 331-336. Escobales N., Castro M., Altieri P. I. and Sanabria, P. (1996), Simvastatin releases Ca2+ from a thapsigarginsensitive pool and inhibits InsP3-depent Ca2+ mo bilization in vascular smooth muscle cells. J. Cardiovasc. Pharmacol. 27, 383-391. Goldstein J. L. and Brown M. S. (1990), Regulation of mevalonate pathway. Nature. 343, 425-430. Hernändez-Perera O., Perez-Sala D., Navarro-Antolin J., Sänchez-Pascuala R., Hernandez G., Diaz C. and La mas S. (1998), Effects of 3-hydroxy-3-methylglutarylCoA reductase inhibitors, atorvastatin and simvas tatin, on the expression of endothelin-1 and endothe lial nitric oxide synthase in vascular endothelial cells. J. Clin. Invest. 101, 2711-2719. Kaesemeyer W. H., Caidwell R. B., Huang J. and Cald well R. W. (1999), Pravastatin sodium activates endo thelial nitric oxide synthase independent of its cholesterol-lowering actions. J. Am. Coll. Cardiol. 33, 234241. Laufs U., La Fata V., Plutzky J. and Liao J. K. (1998), Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation. 97, 1129-1135. Moncada S., Palmer R. M. J. and Higg E. A. (1991), Nitric oxide: physiology, pathophysiology and phar macology. Pharmacol. Rev. 43, 109-142. Ng. L. L., Davies J. E. and Wojcikiewicz R. J. H. (1994), 3-Hydroxy-3-methylglutaryl Coenzyme A reductase inhibition modulates vasopressin-stimulated Ca2+ re sponses in rat A 10 vascular smooth muscle cells. Circ. Res. 74, 173-181. Vaughan C. J., Murphy M. B. and Buckley B. M. (1996), Statins do more than just lower cholesterol. Lancet 348, 1079-1082. Verbeij H. M., Slothoom A. J. and De Haas G. H. (1981), Structure and function of phospholipase A 2. Rev. Phy siol. Biochem. Pharmacol. 91, 91-203. Unauthenticated | Downloaded 05/14/20 03:18 PM UTC