- Email: [email protected]
Effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on nitric oxide-induced hydroxyl radical generation in the rat heart
Biochimica et Biophysica Acta 1536 (2001) 55^63 www.bba-direct.com
E¡ect of £uvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on nitric oxide-induced hydroxyl radical generation in the rat heart Toshio Obata a
a;
*, Akio Ebihara b , Yasumitsu Yamanaka
a
Department of Pharmacology, Oita Medical University, Hasama-machi, Oita 879-5593, Japan b Kaminokawa Hospital, Kaminokawa-machi, Tochigi 329-0611, Japan Received 27 July 2000; received in revised form 2 October 2000; accepted 18 October 2000
Abstract We examined the effect of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the production of hydroxyl radical (c OH) generation via nitric oxide synthase (NOS) activation by an in vivo microdialysis technique. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and tissue was perfused with Ringer's solution through the microdialysis probe at a rate of 1 Wl/min. Sodium salicylate in Ringer's solution (0.5 nmol/Wl/min) was infused directly through a microdialysis probe to detect the generation of c OH. Induction of [K ]o (70 mM) or tyramine (1 mM), significantly increased the formation of c OH trapped as 2,3-dihydroxybenzoic acid (DHBA). The application of NG -nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, significantly decreased the K depolarization-induced c OH formation, but the effect of tyramine significantly increased the level of 2,3-DHBA. When fluvastatin (100 WM), an inhibitor of low-density lipoprotein (LDL) oxidation, was administered to L-NAME-pretreated animals, both KCl and tyramine failed to increase the level of 2,3-DHBA formation. The effect of fluvastatin may be unrelated to K depolarization-induced c OH generation. To examine the effect of fluvastatin on ischemic/reperfused rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed. However, in the presence of fluvastatin (100 WM), the elevation of 2,3-DHBA was not observed in ischemia/reperfused rat heart. Fluvastatin, orally at a dose of 3 mg/kg/day for 4 weeks, significantly blunted the rise of serum creatine phosphokinase and improved the electrocardiogram 2 h after coronary occlusion. These results suggest that fluvastatin is associated with a cardioprotective effect due to the suppression of noradrenaline-induced c OH generation by inhibiting LDL oxidation in the heart. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Fluvastatin; 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; Low-density lipoprotein; NG -Nitro-L-arginine methyl ester; Hydroxyl radical; Microdialysis
Abbreviations: LDL, low-density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; L-NAME, NG -nitro-L-arginine methyl ester; CPK, creatine phosphokinase; DHBA, dihydroxybenzoic acid; LAD, left anterior descending coronary artery; NOS, nitric oxide synthase; XO, xanthine oxidase * Corresponding author. Fax: +81-97-586-5729; E-mail: [email protected] 0925-4439 / 00 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 0 0 ) 0 0 0 9 0 - 9
BBADIS 62003 24-4-01
56
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
1. Introduction Oxidative modi¢cation of low-density lipoprotein (LDL) is a key event in early atherogenesis, which contributes to cholesterol accumulation in the arterial wall and the development of the atherosclerotic lesion [1,2]. Fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor [3,4], protects against the progression of atherosclerosis and it is known to be a potent inhibitor of LDL oxidation [5,6]. LDL is oxidized by transition metal ions, such as copper and iron [5,7]. Although free radical reactions are a part of normal metabolism, the overproduction of reactive oxygen species such as superoxide anion (O3 2 ), hydrogen peroxide (H2 O2 ) and hydroxyl radical (c OH) may contribute to cellular injury [8]. Oxygen-derived free radicals have been implicated in the mediation of myocardial ischemia/ reperfusion injury [9]. Although £uvastatin has been shown to have antioxidative properties [10^13], the e¡ect of £uvastatin on nitric oxide (NO) is not well known. NO is a free radical that regulates a variety of biological functions and also has a role of in the pathogenesis of cellular injury [14^16]. Cytotoxic free radicals such as peroxinitrite (ONOO3 ) and c OH may also be implicated in NO-mediated cell injury [17]. NO is synthesized from L-arginine by NO synthase (NOS) [18]. To con¢rm the cardioprotective e¡ect of £uvastatin, we investigated the e¡ect of NG -nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, on c OH generation by an in vivo microdialysis technique [19,20]. To achieve this goal, we measured c OH formation in in vivo hearts, with the use of a £exibly mounted microdialysis technique that we developed [19]. 2. Materials and methods 2.1. Animal preparation Adult male Wistar rats weighing 300^400 g were kept in an environmentally controlled room (20^ 23³C, 50^60% humidity, illuminated from 7.00 to 19.00 h) and fed with food and water ad libitum. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.; Sigma, St. Louis, MO, USA) and
the level of anesthesia was maintained by intraperitoneal injection of chloral hydrate (20 mg/kg). After intubation, the rat was mechanically ventilated with room air supplemented with oxygen. The chest was opened at the left ¢fth intercostal space, and the pericardium was removed to expose the left ventricle. The heart rate, arterial blood pressure, and electrocardiogram (ECG) were monitored and recorded continuously. At the end of the experiments the rats were killed by an overdose of anesthetic. All procedures in dealing with the experimental animals met the guideline principles stipulated by the Physiological Society of Japan and the Animal Ethics Committee of the Oita Medical University. 2.2. Microdialysis technique Details of the £exibly mounted microdialysis technique and its application to measure biological substances in the interstitial space have been described previously [19]. We created a suitable microdialysis probe. The tubes of the dialysis probe (approx. 15 cm long) were supported loosely at the midpoint on a rotatable stainless steel wire, so that their movement was totally synchronized with a rapid up-and-down movement of the tip caused by the heart beats. The probe was implanted from the epicardial surface into the left ventricular myocardium to a depth of 3 mm and perfused through an inlet tube. The synchronized movement of the tip of the microdialysis probe with the beating ventricle minimized the tissue injury that would otherwise be caused by friction between the probe and the muscle tissue. The tip of a microdialysis probe (3 mm length and 220 Wm o.d. with the distal end closed) was made of dialysis membrane (cellulose hollow ¢ber of 10 Wm thick with 50 000 molecular weight cuto¡). Two ¢ne silica tubes (150 Wm o.d.) were inserted from the open end into the tip of the microdialysis tube consisting of a cylinder-shaped dialysis membrane which served as the inlet for the perfusate and the outlet for the dialysate, respectively. The inlet tube was connected to a microinjection pump (Carnegie Medicine, CMA/100 Stockholm, Sweden), and the outlet tube was led to a high performance liquid chromatography (HPLC) pump.
BBADIS 62003 24-4-01
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
2.3. Experimental protocol To ¢nd out the most appropriate perfusion rate, we bathed the microdialysis probe in vitro in a 37³C Ringer's solution that contained 1.0 WM noradrenaline and the samples were collected at di¡erent perfusion speeds (0.5^5 Wl/min). Based on the data collected from such experiments, we chose the most pertinent perfusion rate of 1.0 Wl/min in the present measurements of noradrenaline and the relative recovery of noradrenaline under this perfusion rate was estimated to be 17.0 þ 0.7%. Oxygen free radicals are very reactive, and the non-enzymatic c OH adduct of salicylate, 2,3-dihydroxybenzoic acid (DHBA), provides an assay of c OH formation [20,21]. The c OH captured as the hydroxylated derivative of salicylic acid was measured by HPLC with an electrochemical (EC) procedure. For trapping c OH radicals [20,21] in the myocardium, sodium salicylate in Ringer's solution (0.5 nmol/Wl/min) was perfused by a microinjection pump and the basal level of 2,3DHBA during a de¢nite period of time was determined. Fluvastatin, KCl or tyramine was dissolved in Ringer's solution for perfusion through a microdialysis probe into the myocardium. After a 30 min washout with Ringer's solution, £uvastatin was introduced through the probe. Under a constant supply of £uvastatin, KCl or tyramine was perfused. Samples (1.0 Wl/min) were collected into small collecting tubes containing 0.1 N HClO4 and assayed immediately for 2,3-DHBA by HPLC-EC. In the case of the L-NAME-treated rats, L-NAME (5 mg/kg) was injected intravenously into the rats before the experiments. In order to ascertain the protective e¡ect of £uvastatin on myocardial infarction and reperfusion damage, the serum creatine phosphokinase (CPK) assay was measured. We divided the animals into two groups of ¢ve rats each: a non-treatment group (control group) and a £uvastatin-pretreated group. In the £uvastatin-pretreated group, £uvastatin was orally administered once a day in a dose of 3 mg/kg for 4 weeks. Blood was collected from catheterization of the carotid artery analyzed for CPK using Iatrontec CK rate (A) (Iatron Laboratories). 2.4. Preparation of ischemic rats After microdialysis probe implantation in the is-
57
chemic zone, the left anterior descending coronary artery branch (LAD) was clamped by a thread through a tube surrounding the coronary artery. The heart was subjected to regional ischemia for 15 min by the occlusion of LAD followed by reperfusion for 60 min. 2.5. Analytical procedures The dialysate samples were immediately injected for analysis into an HPLC-EC system equipped with a glassy carbon working electrode (Eicom, Kyoto, Japan) and an analytic reverse-phase column on an Eicompak MA-5ODS column (5 Wm 4.6U150 mm; Eicom). The working electrode was set at a detector potential of 0.75 V. Each liter of mobile phase contained 1.5 g 1-heptanesulfonic acid sodium salt (Sigma), 0.1 g Na2 EDTA, 3 ml triethylamine (Wako Pure Chemical Industries, Japan) and 125 ml acetonitrile (Wako) dissolved in H2 O. The pH of the solution was adjusted to 2.8 with 3 ml phosphoric acid (Wako).
Fig. 1. E¡ect of £uvastatin on KCl-induced c OH formation after treatment with L-NAME. Animals treated with KCl (hatched bar; 70 mM) after the application of L-NAME (50 mg/kg i.v.) plus £uvastatin (solid bar; 100 WM) (closed circles) were compared with animals treated with KCl only (open circles). Di¡erences between the time courses of 2,3-DHBA were studied with the Mann-Whitney U-test. Values are means þ S.E.M. for six animals; *P 6 0.05 versus pre-KCl value.
BBADIS 62003 24-4-01
58
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
2.6. Materials Fluvastatin sodium (XU-62-320) was donated by the Discovery Research Laboratory, Tanabe Seiyaku (Saitama, Japan). Tyramine-HCl and sodium salicylate and its metabolites were purchased from Sigma. These drugs were dissolved in Ringer's solution containing 147 mM NaCl, 2.3 mM CaCl2 and 4 mM KCl, pH 7.4. L-NAME was purchased from Dojindo Laboratories (Kumamoto, Japan). 2.7. Statistical analysis All values are presented as means þ S.E.M. Di¡erences between the time courses of the levels of 2,3DHBA were studied by means of the Mann-Whitney U-test. The signi¢cance of di¡erence was determined using ANOVA with Fisher's post hoc test. A P value of less than 0.05 was regarded as statistically signi¢cant. 3. Results 3.1. E¡ect of £uvastatin on high KCl-induced c OH formation after L-NAME treatment The c OH captured as the hydroxylated derivative of salicylic acid was measured by the HPLC-EC procedure. The basal level of 2,3-DHBA in the heart dialysate from control animals following infusion of sodium salicylate (0.5 nmol/Wl/min) was 0.033 þ 0.007 WM. Time-dependent changes in the formation of 2,3-DHBA were monitored in the dialysate from rat heart. Fluvastatin alone did not a¡ect the level of 2,3-DHBA in the absence of sodium salicylate (data not shown). The e¡ects of £uvastatin, an inhibitor of LDL oxidation, on extracellular potassium ion concentration, [K ]o (70 mM)-induced c OH formation were examined. Equivalent increases in the osmotic concentration of the Ringer's solution by adding sucrose (150 mM) did not a¡ect the level of 2,3-DHBA (data not shown). The introduction of high KCl (70 mM) was begun. KCl signi¢cantly increased c OH formation trapped as 2,3-DHBA at 15^ 30 min after the beginning of KCl application (n = 6, P 6 0.05). However, no increase in 2,3-DHBA level
Fig. 2. E¡ect of £uvastatin on tyramine-induced c OH formation after treatment with L-NAME. Animals treated with tyramine (hatched bar; 1 mM) after the application of L-NAME (50 mg/kg i.v.) plus £uvastatin (solid bar; 100 WM) (closed circles) were compared with animals treated with tyramine only (open circles). Di¡erences between the time courses of 2,3-DHBA were studied with the Mann-Whitney U-test. Values are means þ S.E.M. for six animals; *P 6 0.05 versus pre-tyramine value.
in the absence of KCl was observed. After removal of KCl from the perfusate, the level of 2,3-DHBA signi¢cantly decreased. In the presence of £uvastatin (100 WM), when corresponding experiments were performed on animals pretreated with L-NAME (5 mg/ kg i.v.), KCl (70 mM) failed to increase the level of 2,3-DHBA at 180 min application of £uvastatin (Fig. 1). However, in the absence of £uvastatin, L-NAME inhibited [K ]o -induced 2,3-DHBA formation and the results are summarized in Fig. 3A. The introduction of tyramine (1 mM) signi¢cantly increased the level of 2,3-DHBA at 15^30 min after the beginning of tyramine application (n = 6, P 6 0.05) (Fig. 2). After removal of tyramine from the perfusate, the level of 2,3-DHBA signi¢cantly decreased. In the presence of £uvastatin (100 WM), when corresponding experiments were performed on animals pretreated with L-NAME (5 mg/kg i.v.), the same results were obtained. However, tyramine (1 mM) failed to increase the level of 2,3-DHBA formation at 180 min after application of £uvastatin and the results are summarized in Fig. 3B.
BBADIS 62003 24-4-01
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
59
Fig. 3. E¡ect of L-NAME and £uvastatin on KCl- or tyramine-induced c OH formation. High KCl (70 mM; A) or tyramine (1 mM; B) was added to animals pretreated with L-NAME (5 mg/kg i.v.) or tyramine (1 mM). The levels of 2,3-DHBA are given as a percentage of the value measured just before the application of KCl or tyramine. Each column and vertical bar indicates the mean þ S.E.M. for six animals; *P 6 0.05 versus the level of 2,3-DHBA of the control (ANOVA and Fisher's test). ns, not signi¢cant.
3.2. E¡ect of £uvastatin on c OH formation in ischemia/reperfusion The presence of c OH was con¢rmed in ischemia/ reperfused rat heart. The heart was subjected to regional ischemia for 15 min (210 min after probe implantation) by LAD occlusion followed by reperfusion for 60 min. Sodium salicylate (0.5 nmol/Wl/min) was infused for 90 min to trap c OH, which was formed by ischemia/reperfusion of myocardium. After the dialysate probe was implanted in the left ventricular myocardium, the level of 2,3-DHBA remained unchanged until reperfusion. When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed in the heart dialysate. This elevation was not observed outside the ischemic area. However, in the presence of £uvastatin (100 WM), no elevation of 2,3-DHBA in ischemia/ reperfusion was observed at 180 min after application of £uvastatin (Fig. 4). When corresponding experiments were performed with rats orally treated with £uvastatin (at a dose of 3 mg/kg/day for 4 weeks), the same results were obtained (data not shown). To con¢rm myocardial ischemia and reper-
Fig. 4. E¡ect of £uvastatin in ischemia/reperfused rat heart on the formation of c OH products of salicylate. The time course of in vivo trapping of highly reactive c OH (2,3-DHBA) in extracellular £uid of myocardium is described in the text. The £uvastatin (closed circles ; 100 WM)-treated group of perfusion-ischemia (solid bar)-reperfusion was compared with the control group (open circles). Values are means þ S.E.M. for six animals. Differences between the time courses of the levels of 2,3-DHBA were studied by means of the Mann-Whitney U-test. *P 6 0.05 versus the level of 2,3-DHBA before ischemia.
BBADIS 62003 24-4-01
60
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
fusion damage, the changes in serum CPK after reperfusion were examined. Oral £uvastatin blunted the rise in CPK (228 þ 32 versus 142 þ 19 U/ml, n = 5, P 6 0.05). 4. Discussion The present study indicated that £uvastatin, a HMG-CoA reductase inhibitor, is associated with the cardioprotective e¡ect by suppressing noradrenaline-induced c OH generation in the heart, with the use of a £exibly mounted microdialysis technique [19]. With this technique it is feasible to make stable and long-term measurements of c OH. The control of 2,3-DHBA formation in the heart dialysate was about 10% higher than in the in vitro perfusion reagent background (data not shown). The drugs were administered through the microdialysis probe. Accordingly, the concentration pro¢le of the administered compounds in the surrounding interstitial space is unknown; in general, the extracellular concentration of a compound given through the probe would never reach the concentration present in the dialysis probe [22]. This is an unavoidable limitation of the microdialysis technique that should be kept in mind when interpreting the experimental data. Although the bene¢cial e¡ect of £uvastatin was antioxidative properties [11,12], the exact mechanism is not clearly demonstrated until now. In the present study, to test a possible link between LDL oxidation and c OH production, an HMG-CoA inhibitor (£uvastatin) was used. Antioxidant substances have been shown to attenuate myocardial dysfunction [23^25]. Oxidative modi¢cation of LDL is thought to contribute to the production of oxygen-derived free radicals [26]. It is known that reactive oxygen species such as c OH may be related to the oxidation of LDL [27]. Therefore, we examined whether inhibition of LDL oxidation can reduce c OH generation. High [K ]o (70 mM) signi¢cantly increased the level of 2,3DHBA (Fig. 1). To determine whether L-NAME has a radical scavenging or antioxidant e¡ect, we examined the e¡ect of L-NAME on K depolarization-induced c OH generation. The application of L-NAME (5 mg/kg i.v.) signi¢cantly decreased the level of 2,3-DHBA by the action of high [K ]o . Therefore, it is possible that [K ]o -induced depolari-
zation evokes c OH generation via NOS activation. However, when corresponding experiments were performed on animals pretreated with £uvastatin (100 WM), the same results were obtained (Figs. 1 and 3A). Moreover, we con¢rmed that £uvastatin reduced c OH formation by the action of K depolarization (data not shown). These results indicate that LDL oxidation may not be mediated with NOS activation. Although L-NAME did not a¡ect the tyramine-induced c OH formation, the e¡ect of L-NAME on [K ]o -induced c OH formation was abolished (Fig. 3A,B). The precise mechanism of depolarization-induced c OH generation is obscure. It is known that NOS inhibition may inhibit depolarization-induced NOS activation by reducing Ca2 in£ux through the blockade of Na and Ca2 channels [28]. The reactive oxygen species causes excessive Na entry through the fast Na channel, leading to intracellular Ca2 overload through the Na -Ca2 exchange system [29]. Intracellular Ca2 overload is then considered to lead to cell death under physiological conditions such as ischemia/reperfusion injury [30,31]. It is known that O3 2 and NO rapidly react to form the stable peroxinitrite (ONOO3 ) and then its decomposition generates c OH [17,32]. However, this idea is under discussion [33]. The interaction between depolarization and oxygen free radicals in myocardium is not clear. However, the release of noradrenaline was induced by nerve depolarization [34]. Noradrenaline may also have a deleterious e¡ect on the myocardium by serving as a source of free radicals [35]. It is known that noradrenaline is released from cardiac sympathetic nerve terminals [36,37]. Therefore, when tyramine (1 mM), a catecholamine releaser [38], was infused through a microdialysis probe, a marked elevation in the level of 2,3-DHBA was observed. When tyramine was administered to animals pretreated with L-NAME (5 mg/kg i.v.), L-NAME did not a¡ect the tyramine-induced c OH formation (Fig. 3B). However, in the presence of £uvastatin (100 M), tyramine failed to increase 2,3-DHBA formation. These results indicated that LDL oxidation may be related to noradrenaline-induced c OH generation, but LDL oxidation may be unrelated to c OH generation via NOS activation. The therapeutic e¡ect of £uvastatin is controversial, mainly due to a potent inhibitor of LDL oxida-
BBADIS 62003 24-4-01
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
61
Fig. 5. The reaction pathway in rat heart illustrates the formation of hydroxyl radicals by depolarization-induced NO. NO, nitric oxc ide; NOS, nitric oxide synthase; L-NAME, NG -nitro-L-arginine methyl ester; XO, xanthine oxidase; O3 2 , superoxide anion; OH, hydroxyl radical; MAO, monoamine oxidase; DOPGAL, 3,4-dihydroxyphenylglycolaldehyde.
tion with which to test this hypothesis experimentally. Several experimental studies have shown that oxygen radicals contribute to myocardial damage induced by ischemia/reperfusion [39,40]. It is well known that ischemia induces depolarization [41,42]. Therefore, we con¢rmed the e¡ect of £uvastatin on ischemia/reperfused rat heart. Fluvastatin (100 WM) attenuated c OH generation induced by ischemia/reperfusion (Fig. 4). When rats were orally treated with £uvastatin (at a dose of 3 mg/kg/day for 4 weeks), the same results were obtained (data not shown). Based on the present studies, it is possible that LDL oxidation mediates ischemia/reperfusioninduced c OH generation via depolarization in ventricular muscle. According to the reaction pathway in Fig. 5, c OH may be formed in vivo during enzyme oxidation. The enzyme xanthine oxidase (XO) catalyzes the conversion of hypoxanthine to xanthine and simultaneously generates O3 2 [43]. XO resulting from xanthine dehydrogenase during ischemia [8] is thought to be a potential source of O3 2 in rat myocardium. It has an extremely short half-life [20] and rapidly undergoes dismutation yielding H2 O2 . Furthermore, it undergoes a Fenton-type reaction in the presence of iron and yields highly cytotoxic c OH [44,45]. Radicals can cause tissue injury. Myocardial rat heart [46,47] injury was determined by measuring the level of CPK from the ventricular, a technique reported previously to be correlated with
histologic determination of infarct size rat hearts. The protective e¡ect of £uvastatin on myocardial ischemia and reperfusion damage was examined in rat hearts. In the group pretreated with £uvastatin (orally at a dose of 3 mg/kg/day for 4 weeks), serum CPK was signi¢cantly lower than that in the control group (n = 5, P 6 0.05). It is possible that inhibition of LDL oxidation may reduce c OH generation and hence ameliorate myocardial injury. The ECG manifestations of ischemia after ligation in £uvastatin appeared to improve. The abnormal Q waves of the £uvastatin-pretreated group were signi¢cantly less than in the control group (P 6 0.05, n = 5) (data not shown). Although the precise mechanism of c OH generation in the ischemic heart is obscure, we previously found a concomitant increase of noradrenaline and c OH generation on myocardial injury [19]. Further investigation is necessary to con¢rm the relationship between free radical generation and LDL oxidation. This ¢nding shows that LDL oxidation may be relevant to ischemia/reperfusion-induced c OH generation. These results suggest that inhibition of LDL oxidation is associated with a cardioprotective e¡ect due to the suppression of noradrenalineinduced c OH generation. The results of the present study may be useful in elucidating the actual mechanism of free radical formation in heart disorders. These experiments in cardiac microdialysis have versatile applications and of-
BBADIS 62003 24-4-01
62
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
fer new possibilities for the in vivo study of cardiac physiology. In the future, cardiac microdialysis heart perfusion experiments using the hydroxylation of salicylate to detect c OH generation may be useful in answering some of the fundamental questions concerning the relevance of oxidant damage in the pathogenesis of heart disorders, such as infarction. References [1] M. Aviram, Modi¢ed forms of low density lipoprotein and atherosclerosis, Atherosclerosis 98 (1993) 1^9. [2] R. Ross, The pathogenesis or atherosclerosis: a perspective for the 1990's, Nature 362 (1993) 801^809. [3] J. Kurokawa, K. Hayashi, Y. Toyota, T. Shingu, M. Shiomi, G. Kajiyama, High dose of £uvastatin sodium (XU62-320), a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, lowers plasma cholesterol levels in homozygous Watanabe-heritable hyperlipidemic rabbits, Biochim. Biophys. Acta 1259 (1995) 99^104. [4] K. Hayashi, J. Kurokawa, S. Nomura, Y. Kuga, Y. Ohkura, G. Kajiyama, E¡ect of £uvastatin sodium (XU62-320), a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the induction of low-density lipoprotein receptor in HepG2 cells, Biochim. Biophys. Acta 1167 (1993) 223^ 225. [5] O. Hussein, S. Schlezinger, M. Rosenblat, S. Keidar, M. Aviram, Reduced susceptibility of low density lipoprotein (LDL) to lipid peroxidation after £uvastatin therapy is associated with the hypocholesterolemic e¡ect of the drug and its binding to the LDL, Atherosclerosis 128 (1997) 11^18. [6] W. Leonhardt, T. Kurktschiev, D. Meissner, P. Lattke, C. Abletshauser, G. Weidinger, W. Jaross, M. Hanefeld, E¡ects of £uvastatin therapy on lipids, antioxidants, oxidation of low density lipoproteins and trace metals, Eur. J. Clin. Pharmacol. 53 (1997) 65^69. [7] P.A. Belinky, M. Aviram, S. Mahmood, J. Vaya, Structural aspects of the inhibitory e¡ect of glabridin on LDL oxidation, Free Radic. Biol. Med. 24 (1998) 1419^1429. [8] J.M. McCord, Oxygen-derived free radicals in postischemic tissue injury, New Engl. J. Med. 312 (1985) 159^163. [9] D.K. Das, R.M. Engelman, R. Clement, H. Otani, M.R. Prasad, P.S. Rao, Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage, Biochem. Biophys. Res. Commun. 148 (1987) 314^319. [10] A. Yamamoto, K. Hoshi, K. Ichihara, Fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes, Eur. J. Pharmacol. 361 (1998) 143^149. [11] K. Suzumura, M. Yasuhara, H. Narita, Superoxide anion scavenging properties of £uvastatin and its metabolites, Chem. Pharm. Bull. 47 (1999) 1477^1480.
[12] K. Suzumura, M. Yasuhara, K. Tanaka, A. Odawara, H. Narita, T. Suzuki, An in vitro study of the hydroxyl radical scavenging property of £uvastatin, an HMG-CoA reductase inhibitor, Chem. Pharm. Bull. 47 (1999) 1010^1012. [13] T. Nakamura, H. Nishi, Y. Kokusenya, K. Hirota, Y. Miura, Mechanism of antioxidative activity of £uvastatindetermination of the active position, Chem. Pharm. Bull. 48 (2000) 235^237. [14] K.Y. Xu, D.L. Huso, T.M. Dawson, D.S. Bredt, L.C. Becker, Nitric oxide synthase in cardiac sarcoplasmic reticulum, Proc. Natl. Acad. Sci. USA 86 (1999) 657^662. [15] J.L. Zweier, P. Wang, A. Samouilov, P. Kuppusamy, Enzyme-independent formation of nitric oxide in biological tissues, Nat. Med. 1 (1995) 804^809. [16] R. Varin, P. Mulder, V. Richard, F. Tamion, C. Devaux, J.P. Henry, F. Lallemand, G. Lerebours, C. Thuillez, Exercise improves £ow-mediated vasodilatation of skeletal muscle arteries in rats with chronic heart failure. Role of nitric oxide, prostanoids, and oxidant stress, Circulation 99 (1999) 2951^2957. [17] J.S. Beckman, T.W. Beckman, J. Chen, P.A. Marshall, B.A. Freeman, Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide, Proc. Natl. Acad. Sci. USA 87 (1990) 1620^ 1624. [18] L. Comini, T. Bachetti, G. Gaia, E. Pasini, L. Agnoletti, P. Pepi, C. Ceconi, S. Curello, R. Ferrari, Aorta and skeletal muscle NO synthase expression in experimental heart failure, J. Mol. Cell. Cardiol. 28 (1996) 2241^2248. [19] T. Obata, H. Hosokawa, Y. Yamanaka, In vivo monitoring of norepinephrine and c OH generation on myocardial ischemic injury by dialysis technique, Am. J. Physiol. 266 (1994) H903^H908. [20] B. Halliwell, H. Kaur, M. Ingleman-Sundberg, Hydroxylation of salicylate as an assay for hydroxyl radicals: a cautionary note, Free Radic. Biol. Med. 10 (1991) 439^441. [21] R.A. Floyd, J.J. Watson, P.K. Wong, Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography with electrochemical detection of phenol and salicylate hydroxylation products, J. Biochem. Biophys. Methods 10 (1984) 221^235. [22] H. Benveniste, Brain microdialysis, J. Neurochem. 52 (1989) 1667^1679. [23] S. Pietri, E. Maurelli, K. Drieu, M. Culcasi, Cardioprotective and anti-oxidant e¡ects of the terpenoid constituents of Ginkgo biloba extract (EGb 761), J. Mol. Cell. Cardiol. 29 (1997) 733^742. [24] J. Vaage, M. Antonelli, M. Bu¢, O. Irtun, R.A. DeBlasi, G.G. Corbucci, A. Gasparetto, A.G. Semb, Exogenous reactive oxygen species deplete the isolated rat heart of antioxidants, Free Radic. Biol. Med. 22 (1997) 85^92. [25] J. Slezak, N. Tribulova, J. Pristacova, B. Uhrik, T. Thomas, N. Khaper, N. Kaul, P.K. Singal, Hydrogen peroxide changes in ischemic and reperfused heart. Cytochemistry and biochemical and X-ray microanalysis, Am. J. Pathol. 147 (1995) 772^781.
BBADIS 62003 24-4-01
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63 [26] M. Sugawa, S. Ikeda, Y. Kushima, Y. Takashima, O. Cynshi, Oxidized low density lipoprotein caused CNS neuron cell death, Brain Res. 761 (1997) 165^172. [27] C. Smith, M.J. Mitchinson, O.I. Aruoma, B. Halliwell, Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions, Biochem. J. 286 (1992) 901^905. [28] S. Tatsumi, Y. Itoh, F.H. Ma, H. Higashira, Y. Ukai, Y. Yoshikuni, K. Kimura, Inhibition of depolarization-induced nitric oxide synthase activation by NS-7, a phenylpyrimidine derivative, in primary neuronal culture, J. Neurochem. 70 (1998) 59^65. [29] L. Verdonck, M. Borgers, Myocardial protection by R 56865: a new principle based on prevention of ion channel pathology, Am. J. Physiol. 266 (1991) H903^H908. [30] J.L. Farber, Biology of disease: membrane injury and calcium homeostasis in the pathogenesis of coagulative necrosis, Lab. Invest. 47 (1982) 114^123. [31] M. Tani, Mechanisms of Ca2 overload in reperfused ischemic myocardium, Annu. Rev. Physiol. 52 (1990) 543^559. [32] X. Shi, A. Lenhart, Y. Mao, ESR spin trapping investigation on peroxynitrite decomposition: no evidence for hydroxyl radical production, Biochem. Biophys. Res. Commun. 203 (1994) 1515^1521. [33] V. Darley-Usmar, H. Wiseman, B. Halliwell, Nitric oxide and oxygen radicals: a question of balance, FEBS Lett. 369 (1995) 131^135. [34] K.U. Malik, E. Sehic, Prostaglandins and the release of the adrenergic transmitter, Ann. NY Acad. Sci. 604 (1990) 222^ 236. [35] A.M. Wheatley, F.T. Thandroyen, L.H. Opie, Catecholamine-induced myocardial cell damage: catecholamines or adrenochrome, J. Mol. Cell. Cardiol. 17 (1985) 349^359. [36] T. Akiyama, T. Yamazaki, I. Ninomiya, In vivo monitoring of myocardial interstitial norepinephrine by dialysis technique, Am. J. Physiol. 261 (1991) H1643^H1647. [37] T. Yamazaki, T. Akiyama, H. Kitagawa, Y. Takauchi, T. Kawada, K. Sunagawa, A new, concise dialysis approach to assessment of cardiac sympathetic nerve terminal abnormalities, Am. J. Physiol. 272 (1997) H1182^H1187.
63
[38] S.E. Downing, V. Chen, Myocardial injury following endogenous catecholamine release in rabbits, J. Mol. Cell. Cardiol. 17 (1985) 377^387. [39] M. Karmazyn, Contribution of prostaglandins to reperfusion-induced ventricular failure in isolated rat heart, Am. J. Physiol. 251 (1986) H133^H140. [40] E. Okabe, R. Fujimaki, M. Murayama, H. Ito, Possible mechanism responsible for mechanical dysfunction of ischemic myocardium. A role of oxygen free radicals, Jpn. Circ. J. 53 (1989) 1132^1137. [41] A.K. Snabaitis, M.J. Shattock, D.J. Chambers, Comparison of polarized and depolarized arrest in the isolated rat heart for long-term preservation, Circulation 96 (1997) 3148^3156. [42] N. Seyedi, T. Win, H.M. Lander, R. Levi, Bradykinin B2receptor activation augments norepinephrine exocytosis from cardiac sympathetic nerve endings. Mediation by autocrine/ paracrine mechanisms, Circ. Res. 81 (1997) 774^784. [43] S. Marubayashi, K. Dohi, K. Yamada, T. Kawasaki, Role of conversion of xanthine dehydrogenase to oxidase in ischemic rat liver cell injury, Surgery 110 (1991) 537^543. [44] A.M. Seacat, P. Kuppusamy, J.L. Zweier, J.D. Yager, ESR identi¢cation of free radicals formed from the oxidation of catechol estrogens by Cu2 , Arch. Biochem. Biophys. 347 (1997) 45^52. [45] T. Obata, A. Ebihara, Y. Yamanaka, Fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, resists hydroxyl radical generation in the rat myocardium, J. Pharm. Pharmacol. 52 (2000) 425^430. [46] M. Chiariello, G. Brevetti, G. DeRosa, R. Acunzo, F. Petillo, F. Rengo, M. Condorelli, Protective e¡ects of simultaneous alpha and beta adrenergic receptor blockade on myocardial cell necrosis after coronary arterial occlusion in rats, Am. J. Cardiol. 46 (1980) 249^254. [47] D. Maclean, M.C. Fishbein, E. Braunwald, P.R. Maroko, Long-term preservation of ischemic myocardium after experimental coronary artery occlusion, J. Clin. Invest. 61 (1978) 541^553.
BBADIS 62003 24-4-01
E¡ect of £uvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on nitric oxide-induced hydroxyl radical generation in the rat heart Toshio Obata a
a;
*, Akio Ebihara b , Yasumitsu Yamanaka
a
Department of Pharmacology, Oita Medical University, Hasama-machi, Oita 879-5593, Japan b Kaminokawa Hospital, Kaminokawa-machi, Tochigi 329-0611, Japan Received 27 July 2000; received in revised form 2 October 2000; accepted 18 October 2000
Abstract We examined the effect of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the production of hydroxyl radical (c OH) generation via nitric oxide synthase (NOS) activation by an in vivo microdialysis technique. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and tissue was perfused with Ringer's solution through the microdialysis probe at a rate of 1 Wl/min. Sodium salicylate in Ringer's solution (0.5 nmol/Wl/min) was infused directly through a microdialysis probe to detect the generation of c OH. Induction of [K ]o (70 mM) or tyramine (1 mM), significantly increased the formation of c OH trapped as 2,3-dihydroxybenzoic acid (DHBA). The application of NG -nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, significantly decreased the K depolarization-induced c OH formation, but the effect of tyramine significantly increased the level of 2,3-DHBA. When fluvastatin (100 WM), an inhibitor of low-density lipoprotein (LDL) oxidation, was administered to L-NAME-pretreated animals, both KCl and tyramine failed to increase the level of 2,3-DHBA formation. The effect of fluvastatin may be unrelated to K depolarization-induced c OH generation. To examine the effect of fluvastatin on ischemic/reperfused rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed. However, in the presence of fluvastatin (100 WM), the elevation of 2,3-DHBA was not observed in ischemia/reperfused rat heart. Fluvastatin, orally at a dose of 3 mg/kg/day for 4 weeks, significantly blunted the rise of serum creatine phosphokinase and improved the electrocardiogram 2 h after coronary occlusion. These results suggest that fluvastatin is associated with a cardioprotective effect due to the suppression of noradrenaline-induced c OH generation by inhibiting LDL oxidation in the heart. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Fluvastatin; 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; Low-density lipoprotein; NG -Nitro-L-arginine methyl ester; Hydroxyl radical; Microdialysis
Abbreviations: LDL, low-density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; L-NAME, NG -nitro-L-arginine methyl ester; CPK, creatine phosphokinase; DHBA, dihydroxybenzoic acid; LAD, left anterior descending coronary artery; NOS, nitric oxide synthase; XO, xanthine oxidase * Corresponding author. Fax: +81-97-586-5729; E-mail: [email protected] 0925-4439 / 00 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 0 0 ) 0 0 0 9 0 - 9
BBADIS 62003 24-4-01
56
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
1. Introduction Oxidative modi¢cation of low-density lipoprotein (LDL) is a key event in early atherogenesis, which contributes to cholesterol accumulation in the arterial wall and the development of the atherosclerotic lesion [1,2]. Fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor [3,4], protects against the progression of atherosclerosis and it is known to be a potent inhibitor of LDL oxidation [5,6]. LDL is oxidized by transition metal ions, such as copper and iron [5,7]. Although free radical reactions are a part of normal metabolism, the overproduction of reactive oxygen species such as superoxide anion (O3 2 ), hydrogen peroxide (H2 O2 ) and hydroxyl radical (c OH) may contribute to cellular injury [8]. Oxygen-derived free radicals have been implicated in the mediation of myocardial ischemia/ reperfusion injury [9]. Although £uvastatin has been shown to have antioxidative properties [10^13], the e¡ect of £uvastatin on nitric oxide (NO) is not well known. NO is a free radical that regulates a variety of biological functions and also has a role of in the pathogenesis of cellular injury [14^16]. Cytotoxic free radicals such as peroxinitrite (ONOO3 ) and c OH may also be implicated in NO-mediated cell injury [17]. NO is synthesized from L-arginine by NO synthase (NOS) [18]. To con¢rm the cardioprotective e¡ect of £uvastatin, we investigated the e¡ect of NG -nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, on c OH generation by an in vivo microdialysis technique [19,20]. To achieve this goal, we measured c OH formation in in vivo hearts, with the use of a £exibly mounted microdialysis technique that we developed [19]. 2. Materials and methods 2.1. Animal preparation Adult male Wistar rats weighing 300^400 g were kept in an environmentally controlled room (20^ 23³C, 50^60% humidity, illuminated from 7.00 to 19.00 h) and fed with food and water ad libitum. The rats were anesthetized with chloral hydrate (400 mg/kg, i.p.; Sigma, St. Louis, MO, USA) and
the level of anesthesia was maintained by intraperitoneal injection of chloral hydrate (20 mg/kg). After intubation, the rat was mechanically ventilated with room air supplemented with oxygen. The chest was opened at the left ¢fth intercostal space, and the pericardium was removed to expose the left ventricle. The heart rate, arterial blood pressure, and electrocardiogram (ECG) were monitored and recorded continuously. At the end of the experiments the rats were killed by an overdose of anesthetic. All procedures in dealing with the experimental animals met the guideline principles stipulated by the Physiological Society of Japan and the Animal Ethics Committee of the Oita Medical University. 2.2. Microdialysis technique Details of the £exibly mounted microdialysis technique and its application to measure biological substances in the interstitial space have been described previously [19]. We created a suitable microdialysis probe. The tubes of the dialysis probe (approx. 15 cm long) were supported loosely at the midpoint on a rotatable stainless steel wire, so that their movement was totally synchronized with a rapid up-and-down movement of the tip caused by the heart beats. The probe was implanted from the epicardial surface into the left ventricular myocardium to a depth of 3 mm and perfused through an inlet tube. The synchronized movement of the tip of the microdialysis probe with the beating ventricle minimized the tissue injury that would otherwise be caused by friction between the probe and the muscle tissue. The tip of a microdialysis probe (3 mm length and 220 Wm o.d. with the distal end closed) was made of dialysis membrane (cellulose hollow ¢ber of 10 Wm thick with 50 000 molecular weight cuto¡). Two ¢ne silica tubes (150 Wm o.d.) were inserted from the open end into the tip of the microdialysis tube consisting of a cylinder-shaped dialysis membrane which served as the inlet for the perfusate and the outlet for the dialysate, respectively. The inlet tube was connected to a microinjection pump (Carnegie Medicine, CMA/100 Stockholm, Sweden), and the outlet tube was led to a high performance liquid chromatography (HPLC) pump.
BBADIS 62003 24-4-01
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
2.3. Experimental protocol To ¢nd out the most appropriate perfusion rate, we bathed the microdialysis probe in vitro in a 37³C Ringer's solution that contained 1.0 WM noradrenaline and the samples were collected at di¡erent perfusion speeds (0.5^5 Wl/min). Based on the data collected from such experiments, we chose the most pertinent perfusion rate of 1.0 Wl/min in the present measurements of noradrenaline and the relative recovery of noradrenaline under this perfusion rate was estimated to be 17.0 þ 0.7%. Oxygen free radicals are very reactive, and the non-enzymatic c OH adduct of salicylate, 2,3-dihydroxybenzoic acid (DHBA), provides an assay of c OH formation [20,21]. The c OH captured as the hydroxylated derivative of salicylic acid was measured by HPLC with an electrochemical (EC) procedure. For trapping c OH radicals [20,21] in the myocardium, sodium salicylate in Ringer's solution (0.5 nmol/Wl/min) was perfused by a microinjection pump and the basal level of 2,3DHBA during a de¢nite period of time was determined. Fluvastatin, KCl or tyramine was dissolved in Ringer's solution for perfusion through a microdialysis probe into the myocardium. After a 30 min washout with Ringer's solution, £uvastatin was introduced through the probe. Under a constant supply of £uvastatin, KCl or tyramine was perfused. Samples (1.0 Wl/min) were collected into small collecting tubes containing 0.1 N HClO4 and assayed immediately for 2,3-DHBA by HPLC-EC. In the case of the L-NAME-treated rats, L-NAME (5 mg/kg) was injected intravenously into the rats before the experiments. In order to ascertain the protective e¡ect of £uvastatin on myocardial infarction and reperfusion damage, the serum creatine phosphokinase (CPK) assay was measured. We divided the animals into two groups of ¢ve rats each: a non-treatment group (control group) and a £uvastatin-pretreated group. In the £uvastatin-pretreated group, £uvastatin was orally administered once a day in a dose of 3 mg/kg for 4 weeks. Blood was collected from catheterization of the carotid artery analyzed for CPK using Iatrontec CK rate (A) (Iatron Laboratories). 2.4. Preparation of ischemic rats After microdialysis probe implantation in the is-
57
chemic zone, the left anterior descending coronary artery branch (LAD) was clamped by a thread through a tube surrounding the coronary artery. The heart was subjected to regional ischemia for 15 min by the occlusion of LAD followed by reperfusion for 60 min. 2.5. Analytical procedures The dialysate samples were immediately injected for analysis into an HPLC-EC system equipped with a glassy carbon working electrode (Eicom, Kyoto, Japan) and an analytic reverse-phase column on an Eicompak MA-5ODS column (5 Wm 4.6U150 mm; Eicom). The working electrode was set at a detector potential of 0.75 V. Each liter of mobile phase contained 1.5 g 1-heptanesulfonic acid sodium salt (Sigma), 0.1 g Na2 EDTA, 3 ml triethylamine (Wako Pure Chemical Industries, Japan) and 125 ml acetonitrile (Wako) dissolved in H2 O. The pH of the solution was adjusted to 2.8 with 3 ml phosphoric acid (Wako).
Fig. 1. E¡ect of £uvastatin on KCl-induced c OH formation after treatment with L-NAME. Animals treated with KCl (hatched bar; 70 mM) after the application of L-NAME (50 mg/kg i.v.) plus £uvastatin (solid bar; 100 WM) (closed circles) were compared with animals treated with KCl only (open circles). Di¡erences between the time courses of 2,3-DHBA were studied with the Mann-Whitney U-test. Values are means þ S.E.M. for six animals; *P 6 0.05 versus pre-KCl value.
BBADIS 62003 24-4-01
58
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
2.6. Materials Fluvastatin sodium (XU-62-320) was donated by the Discovery Research Laboratory, Tanabe Seiyaku (Saitama, Japan). Tyramine-HCl and sodium salicylate and its metabolites were purchased from Sigma. These drugs were dissolved in Ringer's solution containing 147 mM NaCl, 2.3 mM CaCl2 and 4 mM KCl, pH 7.4. L-NAME was purchased from Dojindo Laboratories (Kumamoto, Japan). 2.7. Statistical analysis All values are presented as means þ S.E.M. Di¡erences between the time courses of the levels of 2,3DHBA were studied by means of the Mann-Whitney U-test. The signi¢cance of di¡erence was determined using ANOVA with Fisher's post hoc test. A P value of less than 0.05 was regarded as statistically signi¢cant. 3. Results 3.1. E¡ect of £uvastatin on high KCl-induced c OH formation after L-NAME treatment The c OH captured as the hydroxylated derivative of salicylic acid was measured by the HPLC-EC procedure. The basal level of 2,3-DHBA in the heart dialysate from control animals following infusion of sodium salicylate (0.5 nmol/Wl/min) was 0.033 þ 0.007 WM. Time-dependent changes in the formation of 2,3-DHBA were monitored in the dialysate from rat heart. Fluvastatin alone did not a¡ect the level of 2,3-DHBA in the absence of sodium salicylate (data not shown). The e¡ects of £uvastatin, an inhibitor of LDL oxidation, on extracellular potassium ion concentration, [K ]o (70 mM)-induced c OH formation were examined. Equivalent increases in the osmotic concentration of the Ringer's solution by adding sucrose (150 mM) did not a¡ect the level of 2,3-DHBA (data not shown). The introduction of high KCl (70 mM) was begun. KCl signi¢cantly increased c OH formation trapped as 2,3-DHBA at 15^ 30 min after the beginning of KCl application (n = 6, P 6 0.05). However, no increase in 2,3-DHBA level
Fig. 2. E¡ect of £uvastatin on tyramine-induced c OH formation after treatment with L-NAME. Animals treated with tyramine (hatched bar; 1 mM) after the application of L-NAME (50 mg/kg i.v.) plus £uvastatin (solid bar; 100 WM) (closed circles) were compared with animals treated with tyramine only (open circles). Di¡erences between the time courses of 2,3-DHBA were studied with the Mann-Whitney U-test. Values are means þ S.E.M. for six animals; *P 6 0.05 versus pre-tyramine value.
in the absence of KCl was observed. After removal of KCl from the perfusate, the level of 2,3-DHBA signi¢cantly decreased. In the presence of £uvastatin (100 WM), when corresponding experiments were performed on animals pretreated with L-NAME (5 mg/ kg i.v.), KCl (70 mM) failed to increase the level of 2,3-DHBA at 180 min application of £uvastatin (Fig. 1). However, in the absence of £uvastatin, L-NAME inhibited [K ]o -induced 2,3-DHBA formation and the results are summarized in Fig. 3A. The introduction of tyramine (1 mM) signi¢cantly increased the level of 2,3-DHBA at 15^30 min after the beginning of tyramine application (n = 6, P 6 0.05) (Fig. 2). After removal of tyramine from the perfusate, the level of 2,3-DHBA signi¢cantly decreased. In the presence of £uvastatin (100 WM), when corresponding experiments were performed on animals pretreated with L-NAME (5 mg/kg i.v.), the same results were obtained. However, tyramine (1 mM) failed to increase the level of 2,3-DHBA formation at 180 min after application of £uvastatin and the results are summarized in Fig. 3B.
BBADIS 62003 24-4-01
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
59
Fig. 3. E¡ect of L-NAME and £uvastatin on KCl- or tyramine-induced c OH formation. High KCl (70 mM; A) or tyramine (1 mM; B) was added to animals pretreated with L-NAME (5 mg/kg i.v.) or tyramine (1 mM). The levels of 2,3-DHBA are given as a percentage of the value measured just before the application of KCl or tyramine. Each column and vertical bar indicates the mean þ S.E.M. for six animals; *P 6 0.05 versus the level of 2,3-DHBA of the control (ANOVA and Fisher's test). ns, not signi¢cant.
3.2. E¡ect of £uvastatin on c OH formation in ischemia/reperfusion The presence of c OH was con¢rmed in ischemia/ reperfused rat heart. The heart was subjected to regional ischemia for 15 min (210 min after probe implantation) by LAD occlusion followed by reperfusion for 60 min. Sodium salicylate (0.5 nmol/Wl/min) was infused for 90 min to trap c OH, which was formed by ischemia/reperfusion of myocardium. After the dialysate probe was implanted in the left ventricular myocardium, the level of 2,3-DHBA remained unchanged until reperfusion. When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed in the heart dialysate. This elevation was not observed outside the ischemic area. However, in the presence of £uvastatin (100 WM), no elevation of 2,3-DHBA in ischemia/ reperfusion was observed at 180 min after application of £uvastatin (Fig. 4). When corresponding experiments were performed with rats orally treated with £uvastatin (at a dose of 3 mg/kg/day for 4 weeks), the same results were obtained (data not shown). To con¢rm myocardial ischemia and reper-
Fig. 4. E¡ect of £uvastatin in ischemia/reperfused rat heart on the formation of c OH products of salicylate. The time course of in vivo trapping of highly reactive c OH (2,3-DHBA) in extracellular £uid of myocardium is described in the text. The £uvastatin (closed circles ; 100 WM)-treated group of perfusion-ischemia (solid bar)-reperfusion was compared with the control group (open circles). Values are means þ S.E.M. for six animals. Differences between the time courses of the levels of 2,3-DHBA were studied by means of the Mann-Whitney U-test. *P 6 0.05 versus the level of 2,3-DHBA before ischemia.
BBADIS 62003 24-4-01
60
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
fusion damage, the changes in serum CPK after reperfusion were examined. Oral £uvastatin blunted the rise in CPK (228 þ 32 versus 142 þ 19 U/ml, n = 5, P 6 0.05). 4. Discussion The present study indicated that £uvastatin, a HMG-CoA reductase inhibitor, is associated with the cardioprotective e¡ect by suppressing noradrenaline-induced c OH generation in the heart, with the use of a £exibly mounted microdialysis technique [19]. With this technique it is feasible to make stable and long-term measurements of c OH. The control of 2,3-DHBA formation in the heart dialysate was about 10% higher than in the in vitro perfusion reagent background (data not shown). The drugs were administered through the microdialysis probe. Accordingly, the concentration pro¢le of the administered compounds in the surrounding interstitial space is unknown; in general, the extracellular concentration of a compound given through the probe would never reach the concentration present in the dialysis probe [22]. This is an unavoidable limitation of the microdialysis technique that should be kept in mind when interpreting the experimental data. Although the bene¢cial e¡ect of £uvastatin was antioxidative properties [11,12], the exact mechanism is not clearly demonstrated until now. In the present study, to test a possible link between LDL oxidation and c OH production, an HMG-CoA inhibitor (£uvastatin) was used. Antioxidant substances have been shown to attenuate myocardial dysfunction [23^25]. Oxidative modi¢cation of LDL is thought to contribute to the production of oxygen-derived free radicals [26]. It is known that reactive oxygen species such as c OH may be related to the oxidation of LDL [27]. Therefore, we examined whether inhibition of LDL oxidation can reduce c OH generation. High [K ]o (70 mM) signi¢cantly increased the level of 2,3DHBA (Fig. 1). To determine whether L-NAME has a radical scavenging or antioxidant e¡ect, we examined the e¡ect of L-NAME on K depolarization-induced c OH generation. The application of L-NAME (5 mg/kg i.v.) signi¢cantly decreased the level of 2,3-DHBA by the action of high [K ]o . Therefore, it is possible that [K ]o -induced depolari-
zation evokes c OH generation via NOS activation. However, when corresponding experiments were performed on animals pretreated with £uvastatin (100 WM), the same results were obtained (Figs. 1 and 3A). Moreover, we con¢rmed that £uvastatin reduced c OH formation by the action of K depolarization (data not shown). These results indicate that LDL oxidation may not be mediated with NOS activation. Although L-NAME did not a¡ect the tyramine-induced c OH formation, the e¡ect of L-NAME on [K ]o -induced c OH formation was abolished (Fig. 3A,B). The precise mechanism of depolarization-induced c OH generation is obscure. It is known that NOS inhibition may inhibit depolarization-induced NOS activation by reducing Ca2 in£ux through the blockade of Na and Ca2 channels [28]. The reactive oxygen species causes excessive Na entry through the fast Na channel, leading to intracellular Ca2 overload through the Na -Ca2 exchange system [29]. Intracellular Ca2 overload is then considered to lead to cell death under physiological conditions such as ischemia/reperfusion injury [30,31]. It is known that O3 2 and NO rapidly react to form the stable peroxinitrite (ONOO3 ) and then its decomposition generates c OH [17,32]. However, this idea is under discussion [33]. The interaction between depolarization and oxygen free radicals in myocardium is not clear. However, the release of noradrenaline was induced by nerve depolarization [34]. Noradrenaline may also have a deleterious e¡ect on the myocardium by serving as a source of free radicals [35]. It is known that noradrenaline is released from cardiac sympathetic nerve terminals [36,37]. Therefore, when tyramine (1 mM), a catecholamine releaser [38], was infused through a microdialysis probe, a marked elevation in the level of 2,3-DHBA was observed. When tyramine was administered to animals pretreated with L-NAME (5 mg/kg i.v.), L-NAME did not a¡ect the tyramine-induced c OH formation (Fig. 3B). However, in the presence of £uvastatin (100 M), tyramine failed to increase 2,3-DHBA formation. These results indicated that LDL oxidation may be related to noradrenaline-induced c OH generation, but LDL oxidation may be unrelated to c OH generation via NOS activation. The therapeutic e¡ect of £uvastatin is controversial, mainly due to a potent inhibitor of LDL oxida-
BBADIS 62003 24-4-01
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
61
Fig. 5. The reaction pathway in rat heart illustrates the formation of hydroxyl radicals by depolarization-induced NO. NO, nitric oxc ide; NOS, nitric oxide synthase; L-NAME, NG -nitro-L-arginine methyl ester; XO, xanthine oxidase; O3 2 , superoxide anion; OH, hydroxyl radical; MAO, monoamine oxidase; DOPGAL, 3,4-dihydroxyphenylglycolaldehyde.
tion with which to test this hypothesis experimentally. Several experimental studies have shown that oxygen radicals contribute to myocardial damage induced by ischemia/reperfusion [39,40]. It is well known that ischemia induces depolarization [41,42]. Therefore, we con¢rmed the e¡ect of £uvastatin on ischemia/reperfused rat heart. Fluvastatin (100 WM) attenuated c OH generation induced by ischemia/reperfusion (Fig. 4). When rats were orally treated with £uvastatin (at a dose of 3 mg/kg/day for 4 weeks), the same results were obtained (data not shown). Based on the present studies, it is possible that LDL oxidation mediates ischemia/reperfusioninduced c OH generation via depolarization in ventricular muscle. According to the reaction pathway in Fig. 5, c OH may be formed in vivo during enzyme oxidation. The enzyme xanthine oxidase (XO) catalyzes the conversion of hypoxanthine to xanthine and simultaneously generates O3 2 [43]. XO resulting from xanthine dehydrogenase during ischemia [8] is thought to be a potential source of O3 2 in rat myocardium. It has an extremely short half-life [20] and rapidly undergoes dismutation yielding H2 O2 . Furthermore, it undergoes a Fenton-type reaction in the presence of iron and yields highly cytotoxic c OH [44,45]. Radicals can cause tissue injury. Myocardial rat heart [46,47] injury was determined by measuring the level of CPK from the ventricular, a technique reported previously to be correlated with
histologic determination of infarct size rat hearts. The protective e¡ect of £uvastatin on myocardial ischemia and reperfusion damage was examined in rat hearts. In the group pretreated with £uvastatin (orally at a dose of 3 mg/kg/day for 4 weeks), serum CPK was signi¢cantly lower than that in the control group (n = 5, P 6 0.05). It is possible that inhibition of LDL oxidation may reduce c OH generation and hence ameliorate myocardial injury. The ECG manifestations of ischemia after ligation in £uvastatin appeared to improve. The abnormal Q waves of the £uvastatin-pretreated group were signi¢cantly less than in the control group (P 6 0.05, n = 5) (data not shown). Although the precise mechanism of c OH generation in the ischemic heart is obscure, we previously found a concomitant increase of noradrenaline and c OH generation on myocardial injury [19]. Further investigation is necessary to con¢rm the relationship between free radical generation and LDL oxidation. This ¢nding shows that LDL oxidation may be relevant to ischemia/reperfusion-induced c OH generation. These results suggest that inhibition of LDL oxidation is associated with a cardioprotective e¡ect due to the suppression of noradrenalineinduced c OH generation. The results of the present study may be useful in elucidating the actual mechanism of free radical formation in heart disorders. These experiments in cardiac microdialysis have versatile applications and of-
BBADIS 62003 24-4-01
62
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63
fer new possibilities for the in vivo study of cardiac physiology. In the future, cardiac microdialysis heart perfusion experiments using the hydroxylation of salicylate to detect c OH generation may be useful in answering some of the fundamental questions concerning the relevance of oxidant damage in the pathogenesis of heart disorders, such as infarction. References [1] M. Aviram, Modi¢ed forms of low density lipoprotein and atherosclerosis, Atherosclerosis 98 (1993) 1^9. [2] R. Ross, The pathogenesis or atherosclerosis: a perspective for the 1990's, Nature 362 (1993) 801^809. [3] J. Kurokawa, K. Hayashi, Y. Toyota, T. Shingu, M. Shiomi, G. Kajiyama, High dose of £uvastatin sodium (XU62-320), a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, lowers plasma cholesterol levels in homozygous Watanabe-heritable hyperlipidemic rabbits, Biochim. Biophys. Acta 1259 (1995) 99^104. [4] K. Hayashi, J. Kurokawa, S. Nomura, Y. Kuga, Y. Ohkura, G. Kajiyama, E¡ect of £uvastatin sodium (XU62-320), a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the induction of low-density lipoprotein receptor in HepG2 cells, Biochim. Biophys. Acta 1167 (1993) 223^ 225. [5] O. Hussein, S. Schlezinger, M. Rosenblat, S. Keidar, M. Aviram, Reduced susceptibility of low density lipoprotein (LDL) to lipid peroxidation after £uvastatin therapy is associated with the hypocholesterolemic e¡ect of the drug and its binding to the LDL, Atherosclerosis 128 (1997) 11^18. [6] W. Leonhardt, T. Kurktschiev, D. Meissner, P. Lattke, C. Abletshauser, G. Weidinger, W. Jaross, M. Hanefeld, E¡ects of £uvastatin therapy on lipids, antioxidants, oxidation of low density lipoproteins and trace metals, Eur. J. Clin. Pharmacol. 53 (1997) 65^69. [7] P.A. Belinky, M. Aviram, S. Mahmood, J. Vaya, Structural aspects of the inhibitory e¡ect of glabridin on LDL oxidation, Free Radic. Biol. Med. 24 (1998) 1419^1429. [8] J.M. McCord, Oxygen-derived free radicals in postischemic tissue injury, New Engl. J. Med. 312 (1985) 159^163. [9] D.K. Das, R.M. Engelman, R. Clement, H. Otani, M.R. Prasad, P.S. Rao, Role of xanthine oxidase inhibitor as free radical scavenger: a novel mechanism of action of allopurinol and oxypurinol in myocardial salvage, Biochem. Biophys. Res. Commun. 148 (1987) 314^319. [10] A. Yamamoto, K. Hoshi, K. Ichihara, Fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes, Eur. J. Pharmacol. 361 (1998) 143^149. [11] K. Suzumura, M. Yasuhara, H. Narita, Superoxide anion scavenging properties of £uvastatin and its metabolites, Chem. Pharm. Bull. 47 (1999) 1477^1480.
[12] K. Suzumura, M. Yasuhara, K. Tanaka, A. Odawara, H. Narita, T. Suzuki, An in vitro study of the hydroxyl radical scavenging property of £uvastatin, an HMG-CoA reductase inhibitor, Chem. Pharm. Bull. 47 (1999) 1010^1012. [13] T. Nakamura, H. Nishi, Y. Kokusenya, K. Hirota, Y. Miura, Mechanism of antioxidative activity of £uvastatindetermination of the active position, Chem. Pharm. Bull. 48 (2000) 235^237. [14] K.Y. Xu, D.L. Huso, T.M. Dawson, D.S. Bredt, L.C. Becker, Nitric oxide synthase in cardiac sarcoplasmic reticulum, Proc. Natl. Acad. Sci. USA 86 (1999) 657^662. [15] J.L. Zweier, P. Wang, A. Samouilov, P. Kuppusamy, Enzyme-independent formation of nitric oxide in biological tissues, Nat. Med. 1 (1995) 804^809. [16] R. Varin, P. Mulder, V. Richard, F. Tamion, C. Devaux, J.P. Henry, F. Lallemand, G. Lerebours, C. Thuillez, Exercise improves £ow-mediated vasodilatation of skeletal muscle arteries in rats with chronic heart failure. Role of nitric oxide, prostanoids, and oxidant stress, Circulation 99 (1999) 2951^2957. [17] J.S. Beckman, T.W. Beckman, J. Chen, P.A. Marshall, B.A. Freeman, Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide, Proc. Natl. Acad. Sci. USA 87 (1990) 1620^ 1624. [18] L. Comini, T. Bachetti, G. Gaia, E. Pasini, L. Agnoletti, P. Pepi, C. Ceconi, S. Curello, R. Ferrari, Aorta and skeletal muscle NO synthase expression in experimental heart failure, J. Mol. Cell. Cardiol. 28 (1996) 2241^2248. [19] T. Obata, H. Hosokawa, Y. Yamanaka, In vivo monitoring of norepinephrine and c OH generation on myocardial ischemic injury by dialysis technique, Am. J. Physiol. 266 (1994) H903^H908. [20] B. Halliwell, H. Kaur, M. Ingleman-Sundberg, Hydroxylation of salicylate as an assay for hydroxyl radicals: a cautionary note, Free Radic. Biol. Med. 10 (1991) 439^441. [21] R.A. Floyd, J.J. Watson, P.K. Wong, Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography with electrochemical detection of phenol and salicylate hydroxylation products, J. Biochem. Biophys. Methods 10 (1984) 221^235. [22] H. Benveniste, Brain microdialysis, J. Neurochem. 52 (1989) 1667^1679. [23] S. Pietri, E. Maurelli, K. Drieu, M. Culcasi, Cardioprotective and anti-oxidant e¡ects of the terpenoid constituents of Ginkgo biloba extract (EGb 761), J. Mol. Cell. Cardiol. 29 (1997) 733^742. [24] J. Vaage, M. Antonelli, M. Bu¢, O. Irtun, R.A. DeBlasi, G.G. Corbucci, A. Gasparetto, A.G. Semb, Exogenous reactive oxygen species deplete the isolated rat heart of antioxidants, Free Radic. Biol. Med. 22 (1997) 85^92. [25] J. Slezak, N. Tribulova, J. Pristacova, B. Uhrik, T. Thomas, N. Khaper, N. Kaul, P.K. Singal, Hydrogen peroxide changes in ischemic and reperfused heart. Cytochemistry and biochemical and X-ray microanalysis, Am. J. Pathol. 147 (1995) 772^781.
BBADIS 62003 24-4-01
T. Obata et al. / Biochimica et Biophysica Acta 1536 (2001) 55^63 [26] M. Sugawa, S. Ikeda, Y. Kushima, Y. Takashima, O. Cynshi, Oxidized low density lipoprotein caused CNS neuron cell death, Brain Res. 761 (1997) 165^172. [27] C. Smith, M.J. Mitchinson, O.I. Aruoma, B. Halliwell, Stimulation of lipid peroxidation and hydroxyl-radical generation by the contents of human atherosclerotic lesions, Biochem. J. 286 (1992) 901^905. [28] S. Tatsumi, Y. Itoh, F.H. Ma, H. Higashira, Y. Ukai, Y. Yoshikuni, K. Kimura, Inhibition of depolarization-induced nitric oxide synthase activation by NS-7, a phenylpyrimidine derivative, in primary neuronal culture, J. Neurochem. 70 (1998) 59^65. [29] L. Verdonck, M. Borgers, Myocardial protection by R 56865: a new principle based on prevention of ion channel pathology, Am. J. Physiol. 266 (1991) H903^H908. [30] J.L. Farber, Biology of disease: membrane injury and calcium homeostasis in the pathogenesis of coagulative necrosis, Lab. Invest. 47 (1982) 114^123. [31] M. Tani, Mechanisms of Ca2 overload in reperfused ischemic myocardium, Annu. Rev. Physiol. 52 (1990) 543^559. [32] X. Shi, A. Lenhart, Y. Mao, ESR spin trapping investigation on peroxynitrite decomposition: no evidence for hydroxyl radical production, Biochem. Biophys. Res. Commun. 203 (1994) 1515^1521. [33] V. Darley-Usmar, H. Wiseman, B. Halliwell, Nitric oxide and oxygen radicals: a question of balance, FEBS Lett. 369 (1995) 131^135. [34] K.U. Malik, E. Sehic, Prostaglandins and the release of the adrenergic transmitter, Ann. NY Acad. Sci. 604 (1990) 222^ 236. [35] A.M. Wheatley, F.T. Thandroyen, L.H. Opie, Catecholamine-induced myocardial cell damage: catecholamines or adrenochrome, J. Mol. Cell. Cardiol. 17 (1985) 349^359. [36] T. Akiyama, T. Yamazaki, I. Ninomiya, In vivo monitoring of myocardial interstitial norepinephrine by dialysis technique, Am. J. Physiol. 261 (1991) H1643^H1647. [37] T. Yamazaki, T. Akiyama, H. Kitagawa, Y. Takauchi, T. Kawada, K. Sunagawa, A new, concise dialysis approach to assessment of cardiac sympathetic nerve terminal abnormalities, Am. J. Physiol. 272 (1997) H1182^H1187.
63
[38] S.E. Downing, V. Chen, Myocardial injury following endogenous catecholamine release in rabbits, J. Mol. Cell. Cardiol. 17 (1985) 377^387. [39] M. Karmazyn, Contribution of prostaglandins to reperfusion-induced ventricular failure in isolated rat heart, Am. J. Physiol. 251 (1986) H133^H140. [40] E. Okabe, R. Fujimaki, M. Murayama, H. Ito, Possible mechanism responsible for mechanical dysfunction of ischemic myocardium. A role of oxygen free radicals, Jpn. Circ. J. 53 (1989) 1132^1137. [41] A.K. Snabaitis, M.J. Shattock, D.J. Chambers, Comparison of polarized and depolarized arrest in the isolated rat heart for long-term preservation, Circulation 96 (1997) 3148^3156. [42] N. Seyedi, T. Win, H.M. Lander, R. Levi, Bradykinin B2receptor activation augments norepinephrine exocytosis from cardiac sympathetic nerve endings. Mediation by autocrine/ paracrine mechanisms, Circ. Res. 81 (1997) 774^784. [43] S. Marubayashi, K. Dohi, K. Yamada, T. Kawasaki, Role of conversion of xanthine dehydrogenase to oxidase in ischemic rat liver cell injury, Surgery 110 (1991) 537^543. [44] A.M. Seacat, P. Kuppusamy, J.L. Zweier, J.D. Yager, ESR identi¢cation of free radicals formed from the oxidation of catechol estrogens by Cu2 , Arch. Biochem. Biophys. 347 (1997) 45^52. [45] T. Obata, A. Ebihara, Y. Yamanaka, Fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, resists hydroxyl radical generation in the rat myocardium, J. Pharm. Pharmacol. 52 (2000) 425^430. [46] M. Chiariello, G. Brevetti, G. DeRosa, R. Acunzo, F. Petillo, F. Rengo, M. Condorelli, Protective e¡ects of simultaneous alpha and beta adrenergic receptor blockade on myocardial cell necrosis after coronary arterial occlusion in rats, Am. J. Cardiol. 46 (1980) 249^254. [47] D. Maclean, M.C. Fishbein, E. Braunwald, P.R. Maroko, Long-term preservation of ischemic myocardium after experimental coronary artery occlusion, J. Clin. Invest. 61 (1978) 541^553.
BBADIS 62003 24-4-01