The vascular and cardioprotective effects of liriodenine in ischemia–reperfusion injury via NO-dependent pathway

Nitric Oxide 11 (2004) 307–315 www.elsevier.com/locate/yniox

The vascular and cardioprotective eVects of liriodenine in ischemia–reperfusion injury via NO-dependent pathway Wei-Luen Changa, Ching-Hu Chunga, Yang-Chang Wub, Ming-Jai Sua,¤ a

Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan b Graduate Institute of Natural Products, Kaohsiung Medical College, Kaohsiung, Taiwan Received 6 September 2004 Available online 19 November 2004

Abstract Liriodenine is an aporphine derivative isolated from the plant Fissistigma glaucescens. Electrophysiological action, particularly the blockage of Na+ and K+ channels, contributes to the drug’s well-known anti-arrhythmic action. However, liriodenine’s cardioprotective eYcacy and the relation of the channel blockages to the eYcacy are poorly known, as is the drug’s eVect on coronary Xow and endothelial function. The present study evaluated the protection conveyed by liriodenine to myocardium and coronary endothelial cells under conditions of ischemia–reperfusion and to assess the involvement of a nitric oxide (NO)-dependent mechanism. In the LangendorV model utilizing Sprague–Dawley rat hearts, the left main coronary artery was occluded for 30 min and reperfusion for 120 min. Liriodenine (1
M) signiWcantly promoted the recovery of coronary Xow and decreased myocardial infarction compared with vehicle-treated hearts. The drug attenuated the reduction of endothelial reactivity and NO release. To simulate the condition that occurs in the ischemic stage, human umbilical vein endothelial cells (HUVEC) were cultured in serum free conditions. Liriodenine showed concentration-dependent eVects on cell viability associated with anti-apoptosis under serum-deprivation. Liriodenine prevented eNOS reduction in serum-deprived HUVEC and ischemia–reperfusion hearts. The vascular and cardioprotective eVects were reversed by NG-nitro-L-arginine methyl ester. Another Na+ and K+ channel blocker with similar activities as liriodenine (quinidine) failed to protect endothelial cells and myocytes. These results demonstrate that liriodenine reduces the extent of cardiovascular injuries under ischemia–reperfusion conditions mainly by preserving the eNOS and the NO production.  2004 Elsevier Inc. All rights reserved. Keywords: eNOS; HUVEC; Ischemia–reperfusion heart; Liriodenine; Nitric oxide; Quinidine

Prolonged myocardial ischemia without reperfusion inevitably results in myocardial cell death. Paradoxically, the reperfusion of an ischemic heart, although providing the cells with oxygen and trophic substances, may contribute to additional tissue injury. Myocardial ischemia–reperfusion represents a clinically relevant event associated with thrombolysis, angioplasty, and coronary bypass surgery. It induces a spectrum of events including arrhythmias, transient mechanical dysfunction or ‘myocardial stunning’, and cell death [1,2]. The eVective pro*

Corresponding author. Fax: +886 2 23971403. E-mail address: [email protected] (M.-J. Su).

1089-8603/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.10.004

tection of the heart against ischemia–reperfusion injury is one of the paramount goals of cardiology research. In the last two decades, considerable eVort has focused on limiting infarct size and other myocardial manifestations of ischemia–reperfusion injury. In addition to damaging myocytes, the consequences of myocardial ischemia–reperfusion extend to the coronary vascular wall and especially to the endothelial cells [3]. Marked structural injury to these cells is induced [4], which is accompanied by impairment of endotheliumdependent protective eVects [5,6]. The injury is associated with reduced bioavailability of endothelium nitric oxide (NO), which promotes vasodilation [7,8], inhibits

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platelet adhesion and aggregation [9,10], regulates leukocyte-endothelial cell interactions [11], and may modulate cardiac myocyte function. Based on these Wndings, it has been speculated that the loss of endothelial function and NO-mediated eVects may be important in the development of myocardial ischemia–reperfusion injuries [12]. Liriodenine is an aporphine derivative isolated from the plant Fissistigma glaucescens [13]. It induces vasodilation in rat thoracic aorta [14] and exerts positive inotropic and negative chronotropic eVects in isolated rat cardiac muscle [15,16]. Reduction of ischemia–reperfusion-induced arrhythmia occurs both in vitro [16] and in vivo [17]. Through the inhibition of Na+ and transient outward K+ channels, liriodenine is able to suppress ventricular arrhythmia induced by myocardial ischemia– reperfusion [16]. Although the foregoing electrophysiological actions of liriodenine have been well studied, the drug’s cardioprotective eYcacy has until recently been poorly understood, as has the relationship between the cardiac protection and the Na+ and K+ channel blockages. Indeed, since myocardial ischemia–reperfusion injuries involve multiple essential molecules and cell types, it is possible that liriodenine may modulate components other than these channels. The main goals of the present study were to assess whether liriodenine limited the infarct size in LangendorV-perfused rat hearts subjected to ischemia–reperfusion, and to evaluate whether the protective eVect of liriodenine also extended to coronary endothelial cells. The role of the channel blockages and the NO pathway in liriodenine protection from myocardial injury and endothelial dysfunction following myocardial ischemia–reperfusion was investigated via the comparison of liriodenine activity to that of another channel blocker, quinidine.

Materials and methods Endothelial cell culture Human umbilical vein endothelial cells (HUVEC)1 were prepared as previously described [18]. Umbilical cord veins were cannulated and Xushed with cold buVer to remove blood and then were Wlled with 0.1% collagenase (type I) for 10 min at 37 °C. Isolated endothelial cells, identiWed by their positive immunoXuorescent staining for von Willebrand factor antigen (Dako, Carpenteria, CA), were maintained in M199 medium containing 20% FBS, 30
g/ml ECGS, 4 mM L-glutamine, 100 U/ml penicillin, and 100
g/ml streptomycin, and 1 Abbreviations used: DMSO, dimethyl sulfoxide; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; HUVEC, human umbilical vein endothelial cells; L-NAME, NG-nitro-L-arginine methyl ester; MTT, 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide.

incubated at 37 °C in 5% CO2. The cells were used after 2–4 passages. Cell viability Cell viability was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) [19]. HUVEC were grown in serum free M199 medium in the absence or presence of various concentrations of liriodenine for 48 h prior to incubation with MTT (0.5 mg/ml) for 4 h. After incubation and aspiration of the medium, the cells were dissolved in dimethyl sulfoxide (DMSO) and the absorbance of the developed color was measured at 550 nm. HUVEC grown in M199 medium with 20% FBS for 48 h served as the control. Detection of HUVEC apoptosis For the morphological staining of apoptotic cells, cells were centrifuged (10 min, 700g), Wxed in 4% paraglutaraldehyde for 30 min, and stained with annexin VFITC (BioVision Research Products, Mountain View, CA) at 4 °C for 1 h with a continuous shaking. After incubation, cells were washed twice, resuspended in PBS, and analyzed immediately by FACalibur (Becton–Dickinson, Franklin Lakes, NJ) at excitation and emission wavelengths of 488 and 525 nm, respectively. Fluorescence signals from 5000 cells were collected to calculate the mean Xuorescence intensity of single cell and the percentage of positively staining cells. LangendorV heart model The investigation was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Adult male Sprague–Dawley rats (National Laboratory Animal Center, Taipei, Taiwan), weighing 250–300 g, were intraperitoneally anesthetized with sodium pentobarbital (50 mg kg¡1) and given heparin (300 U kg¡1) by the same route. The LangendorV-perfused heart model, with constant perfusion pressure instead of constant Xow, was used [20]. Hearts were rapidly excised and immersed in ice-cold perfusion medium. The aorta was cannulated and perfused at 70 mmHg with Tyrode’s solution containing 120 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.2 mM NaH2PO4, 2.5 mM CaCl2, 1.2 mM MgCl2, and 1.2, and 5.5 mM glucose. The perfusate was equilibrated with 95% O2, 5% CO2 at 37 °C, giving a pH of 7.4. Perfusion pressure was monitored using a MLT844/D pressure transducer (Capto, Horten, Norway) connected to a PowerLab (ADInstruments, Castle Hill, Australia). The coronary eZuent was collected for the measurement of coronary Xow. A 7/0 silk ligature was placed under the left main coronary artery. A small plastic snare formed

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from a Portex P-270 cannula was threaded through the ligature and placed in contact with the heart. Tightening the ligature could then occlude the artery and reperfusion was achieved by releasing the tension applying to the ligature. The establishment of ischemia and reperfusion was ascertained by the amount of coronary eZuent. A successful occlusion was conWrmed by 40–50% reduction in coronary Xow as compared with preischemia values. Experimental groups All hearts were allowed to stabilize for 20 min. Hearts subjected to 30 min of regional ischemia and 120 min of reperfusion were randomized into six groups. In the Wrst group (vehicle group), hearts were given 0.02% DMSO. In the Lir 0.1 and 1.0 groups, hearts received 0.1 and 1
M liriodenine, respectively. In the Q group, hearts were given 1
M Quinidine. In the Wrst four treatment groups, drug or vehicle infusion was started 20 min prior to the ischemia and was continued throughout the rest of the ischemia–reperfusion protocol. To investigate the NO-dependent pathway induced by liriodenine, an NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was used in the two remaining groups. In the L-NAME group, hearts were given a further 15 min perfusion with L-NAME alone followed by a combination with DMSO infusion. In the Wnal group (Lir 1 + L-NAME group), hearts were given a further 15 min perfusion with L-NAME followed by perfusion with L-NAME and 1
M liriodenine. The sham group underwent a further 170 min aerobic perfusion. Estimation of myocardial damage Hearts used for infarct size calculation (n D 8–10) were harvested immediately after termination of the experiment and the infarct area was determined by the triphenyltetrazolium chloride staining [21]. The ventricular tissue was sliced into 1 mm sections and incubated in 10 mg of 2,3,5-triphenyltetrazolium chloride ml¡1 0.9% NaCl, pH 7.4, at 37 °C for 40 min. Sections were then placed in 10% formaldehyde in saline for two days before infarct (white) tissue was excised. The weight of infarct tissue was expressed as the percentage of left ventricular weight.

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NO analysis Samples of coronary perfusate were collected from hearts in the vehicle, Lir 1, and Q groups to determine NO levels. The concentrations of NO were determined in 25
l volumes with a chemiluminescence method using a NO analyzer (Model NOA 280, Sievers Instruments, Boulder, CO) based on a gas-phase chemiluminescence reaction between NO and ozone [23]. Because NO is rapidly oxidized to nitrite then nitrate, the samples were reduced to NO with vanadium chloride in hydrochloric acid at 90 °C. The detection range was set between 0.2 and 10
mol/L. Western blotting Following LangendorV perfusion, the hearts were frozen in liquid nitrogen. Tissue was homogenized and lysed with lysis buVer consisting of tissue protein extraction reagent (Pierce Biotechnology, Rockford, IL) supplemented with 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 10
g/ml aprotinin, and 10
g/ml leupeptin (pH 7.4). HUVEC incubated with diVerent concentrations of liriodenine under serum-deprivation for 24 h were washed in PBS, harvested, and pelleted at 1000g for 10 s in 1.5 ml microfuge tubes. The supernatant was removed. Cells were lysed with lysis buVer (15 mM Tris–HCl, 50 mM NaCl, 5 mM EGTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 10
g/ml aprotinin, and 10 mg/ml leupeptin, pH 7.4) and sonicated on ice for 30 s using a sonicator (Sonics and Materials, Danbury, CT) at 30% power. Lysate samples from tissue or cell (30
g protein/lane) were separated by SDS–PAGE using 8% gels and transferred to polyvinylidene diXuoride membranes (Perkin–Elmer Life Sciences, Boston, MA). The membranes were blocked in 5% (w/v) 0-fat milk dissolved in Tris/phosphate/saline/ Tween (TPST) and incubated with primary antibodies (rabbit anti-mouse, rat, and human eNOS N-terminus polyclonal antibody; N-20, or mouse anti-mouse, rat, and human -tubulin monoclonal antibody; Ab-1) and secondary antibodies, which were diluted in Tris/phosphate/ saline/Tween (TPSP) containing 1% non-fat milk. After incubation, blots were washed three times in TPST and developed using an enhanced ECL detection system (Millipore, Bedfors, MA). Primary and horseradish peroxidase-conjugated secondary antibodies were used at a concentration of 1
g/ml.

Evaluation of coronary endothelium-dependent vascular function

Statistical analysis

Coronary Xow before and after histamine perfusion were measured. The increase of coronary Xow after histamine perfusion was normalized to the coronary Xow before histamine perfusion and was used as an index of endothelium-dependent vascular function [22].

All values are presented as means § SE. DiVerences between groups were assessed by one-way ANOVA and Newman–Keuls multiple comparison test where appropriate. p values equal to or less than 0.05 (p < 0.05) were considered as signiWcant diVerence.

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Chemicals Liriodenine was isolated from plant F. glaucescens as previous described [13]. It was dissolved in dimethyl sulfoxide (DMSO). The Wnal concentration of DMSO in the bathing solution did not exceed 0.02%. The following drugs were used: quinidine, L-NAME, sodium nitrite, histamine, 2,3,5-triphenyl-tetrazolium chloride (TTC), 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), collagen type I, and bovine serum albumin (BSA), which were purchased from Sigma (St. Louis, MO). Vanadium (III) was obtained from Merck Biosciences (Darmstadt, Germany). Anti-eNOS antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti -tubulin monoclonal antibody (Ab-1) was from Oncogene Research (Cambridge, MA); and Medium 199 (M199), fetal bovine serum (FBS), and all culture reagents were purchased from Gibco (Grand Island, NY). Endothelial cell growth supplement (ECGS) was purchased from Upstate Biotechnology (Lake Placid, NY).

Administration of either 1
M quinidine or L-NAME alone did not aVect infarct size compared to the vehicle group. EVect on coronary Xow in ischemia–reperfusion Before the coronary ligation, coronary Xow was slightly decreased after vehicle, liriodenine, and quinidine treatments, but no signiWcant diVerence existed among these groups. However, the administration of the NOS inhibitor L-NAME (100
M) markedly decreased coronary Xow (71.9 § 6.8%, p < 0.01 vs vehicle group) (Fig. 2A). After the coronary ligation, coronary Xow was decreased upon reperfusion in relation to the respective basal values (30.2 § 2.6%) (Fig. 2B). Hearts pretreated with 1
M liriodenine recovered a signiWcant amount of post-ischemia myocardial coronary Xow (52.9 § 3.0%, p < 0.01 vs vehicle group). No signiWcant improvement in

Results Infarct size in ischemia–reperfusion rat hearts Fig. 1 shows the infarct size expressed as a percentage of the left ventricle in all experimental groups. Infarct size was 48.6 § 2.0% of the left ventricle in the vehicle group, but was reduced to 21.2 § 2.1% in the group given 1
M liriodenine (p < 0.01). When liriodenine was administered together with L-NAME, the resulting infarct size (46.4 § 2.5%) was not signiWcantly diVerent from that of the vehicle group, but was signiWcantly larger than that of the group given 1
M liriodenine alone (p < 0.01).

Fig. 1. Infarct size expressed as the percentage of left ventricle in isolated rat hearts subjected to ischemia–reperfusion in the groups given vehicle, liriodenine, quinidine, L-NAME or L-NAME plus liriodenine. Results are expressed as means § SE. ** Denotes signiWcant diVerence (p < 0.01) from vehicle group (lane 1). 99 Denotes signiWcant diVerence (p < 0.01) from 1
M liriodenine treatment group (lane 3).

Fig. 2. Coronary Xow before (A) and after (B) regional ischemia–reperfusion of the rat hearts given vehicle, liriodenine, quinidine, LNAME or L-NAME plus liriodenine. Lanes 5 and 6 in (B) are expressed as the percentage of the preischemic coronary Xow in hearts given L-NAME. Results are expressed as means § SE. ** Denotes signiWcant diVerence (p < 0.01) from vehicle group (lane 1). 99 Denotes signiWcant diVerence (p < 0.01) from 1
M liriodenine treatment group (lane 3).

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post-ischemic coronary Xow was observed in the other treatment groups. The improvement in post-ischemia coronary Xow by liriodenine was eliminated by LNAME (36.7 § 4.6%, p < 0.01 vs L1 group).

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1 h and 1.38 § 0.1
M, p < 0.01 reXow 2 h vs vehicle group). Measurement of alterations in endothelial-dependent vascular reactivity

NO release in ischemia–reperfusion No signiWcant diVerences in NO concentration were evident between the vehicle, liriodenine, and quinidine groups before the ischemia period (Fig. 3). However, ischemia–reperfusion decreased the concentration of NO, especially in the reperfusion phase (0.85 § 0.08
M, p < 0.01, reXow 1 h and 0.79 § 0.04
M, p < 0.01, reXow 2 h vs 1.34 § 0.03
M, basal). Pretreatment with 1
M liriodenine but not quinidine attenuated the decrease of NO in reperfused hearts (1.28 § 0.08
M, p < 0.01, reXow

Infusion of the endothelium-dependent vasodilator histamine (1
M) in the coronary circulation of rat hearts in the sham group induced a rapid increase in coronary Xow (20.3 § 2.5%). After the hearts were exposed to ischemia–reperfusion, the histamine-induced enhancement of Xow was attenuated in the vehicle (2.8 § 2.0%, p < 0.01 vs sham group) and quinidine (7.1 § 3.3%, p < 0.05 vs sham group) groups. However, an improvement of the endothelial response was observed in the liriodenine group (17.6 § 3.5%, p < 0.01 vs vehicle group) (Fig. 4).

Fig. 3. Time courses of NO production during ischemia–reperfusion in coronary eZuent of the groups given vehicle, liriodenine or quinidine. Values are presented as means § SE per groups. ** Denotes signiWcant diVerence (p < 0.01) from vehicle group.

Fig. 4. EVect of the endothelial-dependent vasodilator histamine on coronary Xow at the end of ischemia–reperfusion in the groups given vehicle, liriodenine or quinidine. Values are presented as means § SE per groups. * Denotes signiWcant diVerence (p < 0.05) from sham group. ** Denotes signiWcant diVerence (p < 0.01) from sham group. 99 Denotes signiWcant diVerence (p < 0.01) from vehicle group.

Fig. 5. Liriodenine inXuence on the death of serum-deprived HUVEC. HUVEC were incubated with liriodenine, quinidine or L-NAME plus liriodenine under serum-deprived conditions for 48 h. (A) Cell viability was determined by a MTT assay. (B) Apoptosis was determined by annexin V analysis. ** Denotes signiWcant diVerence (p < 0.01) from control (lane 1) cells. 99 Denotes signiWcant diVerence (p < 0.01) from serum-deprived (lane 2) cells. § Denotes signiWcant diVerence (p < 0.01) from 1
M liriodenine treatment in serum-deprived (lane 3) cells.

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InXuence of liriodenine on HUVEC serum-deprived apoptosis Serum deprivation decreased MTT-assessed cell viability to 40.4 § 1.5% of the level displayed by control cells grown in the presence of serum (Fig. 5A). The presence of 0.1–1
M liriodenine reversed the serumdeprivation eVects in a dose-dependent manner. The maximal eVect was observed using 1
M liriodenine (63.2 § 4.1% of control; p < 0.01), therefore, this concentration was used in an annexin V analysis to detect apoptotic cells in serum-deprived HUVEC. As shown in Fig. 5B, liriodenine prevented endothelial cell apoptosis. This eVect was signiWcantly reversed by LNAME. eNOS expression in HUVEC To determine whether changes in eNOS protein levels correlated with the protective eVects of liriodenine, eNOS Western blotting in serum-deprived HUVEC with and without liriodenine was conducted. eNOS protein level decreased signiWcantly after exposure to serum-deprived conditions for 24 h (Fig. 6). Liriodenine prevented reduction of eNOS in serumdeprived HUVEC in a dose-dependent manner, which reached signiWcance after pretreatment with 1
M of the drug.

Fig. 7. The eVects of liriodenine or quinidine on eNOS protein expression in isolated rat hearts after ischemia–reperfusion. Original Western blots are reported in the upper panels and results of densitometry in the lower panels. Levels of -tubulin are shown for comparison. ** Denotes signiWcant diVerence (p < 0.01) from sham-operated (lane 1) hearts. 99 Denotes signiWcant diVerence (p < 0.01) from the occluded zone of vehicle-treated (lane 3) hearts.

eNOS expression in ischemia–reperfusion rat hearts The decrease in HUVEC eNOS expression and NO release in ischemia–reperfusion hearts (Fig. 3) was indicative of a decrease in eNOS protein. Such a decrease was observed in ischemia–reperfusion hearts, especially in the occluded zone (Fig. 7). Exposure to 1
M liriodenine prevented the decrease. Moreover, quinidine produced only a minimal prevention against this decrease.

Discussion

Fig. 6. The eVect of liriodenine on eNOS protein expression in serumdeprivated HUVEC. HUVEC were incubated with diVerent concentrations of liriodenine under serum-deprivation for 24 h. The typical traces shown are representative of three independent experiments. Levels of -tubulin are shown for comparison. ** Denotes signiWcant diVerence (p < 0.01) from control (lane 1) cells. 99 Denotes signiWcant diVerence (p < 0.01) from serum-deprived (lane 2) cells.

The present study, performed using a LangendorV model of myocardial ischemia–reperfusion, shows that liriodenine induces signiWcant protective eVects, as assessed by a marked limitation of infarct size, recovery of coronary Xow, and prevention of reperfusion-induced coronary endothelial dysfunction. Concurrent with these beneWcial eVects, liriodenine prevents eNOS reduction induced by ischemia–reperfusion. The action of liriodenine on eNOS catalyzing NO production appears to be causally linked to the beneWcial eVects reported in this study, since it is abrogated by the treatment with the NOS inhibitor L-NAME. Cardioprotective eVects of liriodenine via NO pathway Myocardial damage induced by ischemia–reperfusion has already been extensively characterized. Infarct size is

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a major determinant of complication. In this study, ischemia–reperfusion-induced infarct size was reduced by liriodenine at a dose consistent with that required to show an anti-arrhythmic eVect in the rat heart LangendorV perfusion model. Anti-arrhythmic agents like lidocaine and disopyramide produce a Na+ channel blocking activity, which results in lessened ionic imbalance, and which may mediate the infarct size reducing eVect on ischemia–reperfusion hearts [24]. Moreover, 30
M quinidine exerts a cardioprotective eVect [25]. This concentration, however, far exceeds that used clinically. Indeed, previously we found that quinidine at concentrations higher than 10
M had strong Ca2+ channel blocking activity, which may contribute to the reduction of Ca2+ overload and injury in ischemia–reperfusion myocardium [26]. By contrast, we observed that 1
M quinidine, which has an ion channel blocking activity and anti-arrhythmic eYcacy similar to liriodenine [16], fails to exert anti-infarct action and NO release in ischemia–reperfusion hearts. Therefore, the mechanism involved in the myocardial protective eVect of liriodenine appears to be independent of its Na+ or K+ channel blocking activity. An increased concentration of NO limits myocardial ischemia–reperfusion injury [27–29]. The present study demonstrates that the decrease of infarct size in the presence of liriodenine coincides with the increase of NO in coronary eZuent compared with the untreated group. Moreover, the infarct limiting eVect of liriodenine is attenuated by the NOS inhibitor L-NAME. These observations suggest that the NO-dependent pathway is related to the cardioprotective eVects of liriodenine. Mechanisms of the cardioprotective eVect of NO NO may exert cardioprotection in ischemia–reperfusion, both directly on cardiac myocytes and indirectly through the improvement of coronary perfusion [30,31]. In the present study, we observed liriodenine treatment under basal conditions did not aVect basal coronary Xow. However, in ischemia–reperfusion conditions with impaired endogenous NO formation, the treatment of liriodenine enhanced NO formation, which may dilate the vessels. Thus, the coronary Xow was higher in the liriodenine group, suggesting that the cardioprotection by liriodenine may be due in part to the maintenance of coronary blood Xow. However, whether the production of NO by liriodenine has direct protective eVect on the myocardium remains to be examined. Endothelium protective eVect of liriodenine and NO In this experiment, we observed that the protective eVects of liriodenine extend to endothelial cells in

313

ischemia–reperfusion. The vasodilator response to histamine after ischemia–reperfusion was signiWcantly larger in the liriodenine group than in the vehicle group. The poor response to histamine in the vehicle group was the result of endothelium injury under ischemia–reperfusion conditions and that endotheliumdependent relaxation, and thus the release of NO, was impaired. This suggestion supports the Wndings of previous studies [5,6,32]. Therefore, liriodenine preserving the endothelium-dependent vasodilation may be the result of coronary endothelial protective eVect of liriodenine. In addition, the present study shows that liriodenine inhibits endothelial cell apoptosis induced by serum deprivation. The beneWcial eVect of liriodenine parallels the eNOS preservation and is abrogated by L-NAME treatment. NO protects endothelial cell from apoptosis induced by many stimulation, such as tropic factor withdrawal, TNF-, or reactive oxygen species [33–36]. Thus, NO derived from eNOS must somehow limit endothelial cell apoptosis that is probably linked to the recovery of endothelial dysfunction by liriodenine in intact heart subjected ischemia–reperfusion. Mechanisms for the preservation of NO production by liriodenine Hearts subjected to ischemia–reperfusion display marked coronary functional alterations and a consistent decrease of NO in the coronary eZuent. Under these conditions, we observed that the cardiac level of eNOS was signiWcantly reduced with respect to the vehicle group. One possible reason for the lowering of eNOS content is the increased protein degradation that occurs under ischemic–reperfusion conditions, due to the activation of cellular proteases [22,37]. Presently, the eNOS reduction in ischemia–reperfusion hearts was preserved by liriodenine, which contributes to the maintenance of bioavailable NO. However, whether liriodenine preserves eNOS reduction in ischemia–reperfusion hearts via protease inhibition or via directly upregulation of eNOS in other signaling pathway, such as MAPK and PKC [38] or CaMKII [39] pathway, is not clear. Furthermore, the cellular location of the increased expression of eNOS cannot be determined. However, the preservation of eNOS reduction in serum deprived endothelial cells and the increase of endothelium-dependent vasodilator response suggest that endothelial cell eNOS is preserved or upregulated by liriodenine. In conclusion, our experiments show that liriodenine is able to exert potent protective eVects in ischemia–reperfusion conditions, which are evident in both myocardial and coronary endothelial cells, and which appear to be mediated mainly through the preservation of eNOS protein and maintenance of functional NO release.

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Acknowledgment This study was supported by grant from the National Science Council of Taiwan (NSC 90-2320-B-002-081).

References [1] D.J. Hearse, R. Bolli, Reperfusion induced injury: manifestations, mechanisms, and clinical relevance, Cardiovasc. Res. 26 (1992) 101–108. [2] E. Braunwald, R.A. Kloner, Myocardial reperfusion: a doubleedged sword?, J. Clin. Invest. 76 (1985) 713–719. [3] T.M. Scarabelli, R.A. Knight, N.B. Rayment, T.J. Cooper, A. Stephanou, B.K. Brar, K.M. Lawrence, G. Santilli, D.S. Latchman, G.F. Baxter, D.M. Yellon, Quantitative assessment of cardiac myocyte apoptosis in tissue sections using the Xuorescence-based tunel technique enhanced with counterstains, J. Immunol. Methods 228 (1999) 23–28. [4] N. KaeVer, V. Richard, A. Francois, F. Lallemand, J.P. Henry, C. Thuillez, Preconditioning prevents chronic reperfusion-induced coronary endothelial dysfunction in rats, Am. J. Physiol. 271 (1996) H842–H849. [5] D.D. ku, Coronary vascular reactively after acute myocardial ischemia, Science 218 (1982) 576–578. [6] K.M. Van Benthuysen, I.F. McMurtry, L.D. Horwitz, Reperfusion after acute coronary occlusion in dogs impairs endotheliumdependent relaxation to acetylcholine and augments contractile reactivity in vitro, J. Clin. Invest. 79 (1987) 265–274. [7] R.F. Furchgott, J.V. Zawadzki, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288 (1980) 373–376. [8] L.J. Ignarro, H. Lippton, J.C. Edwards, W.H. Baricos, A.L. Hyman, P.J. Kadowtiz, C.A. Gruetter, Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates, J. Pharmacol. Exp. Ther. 218 (1981) 739–749. [9] L.J. Ignarro, C. Napoli, J. Loscalzo, Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview, Circ. Res. 90 (2002) 21–28. [10] B.T. Mellion, L.J. Ignarro, E.H. Ohlstein, E.G. Pontecorovo, A.L. Hyman, P.J. Kadowitz, Evidence for the inhibitory role of guanosine 3⬘,5⬘-monophosphate in ADP-induced human platelet aggregation in the presence of nitric oxide and related vasodilators, Blood 57 (1981) 946–955. [11] P. Kubes, D.N Granger, Nitric oxide: an endogenous modulator of leukocyte adhesion, Proc. Natl. Acad. Sci. USA 88 (1991) 4651– 4655. [12] K. Laude, C. Thuillez, V. Richard, Coronary endothelial dysfunction after ischemia and reperfusion: a new therapeutic target?, Braz. J. Med. Biol. Res. 34 (2001) 1–7. [13] S.T. Lu, Y.C. Wu, S.P. Leou, Alkaloid of Fissistigma glaucescens and Goniothalamas species, Phytochemistry 24 (1985) 1829– 1834. [14] S. Chulia, M.A. Noguera, M.D. Ivorra, D. Cortes, M.P. D’Ocon, Vasodilator eVects of liriodenine and norushinsunine, two aporphine alkaloids isolated from Annona cherimolia, in rat aorta, Pharmacology 50 (1995) 380–387. [15] C.H. Lin, G.J. Chang, M.J. Su, Y.C. Wu, C.M. Teng, F.N. Ko, Pharmacological characteristics of liriodenine, isolated from Fissistigma glaucescens, a novel muscarinic receptor antagonist in guinea-pigs, Br. J. Pharmacol. 113 (1994) 275–281. [16] G..J. Chang, M.H. Wu, Y.C. Wu, M.J. Su, Electrophysiological mechanisms for antiarrhythmic eYcacy and positive inotropy of

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

liriodenine, a natural aporphine alkaloid from Fissistigma glaucescens, Br. J. Pharmacol. 118 (1996) 1571–1583. W.L. Chang, Y.C. Wu, M.J. Su, Ischemia and/or reperfusioninduced arrhythmia in anesthetized rats: prevention by liriodenine, in: Third International Congress on Coronary Artery Disease from Prevention to Intervention, 2000, Abstract 23. E.A. JaVe, R.L. Nachman, C.G. Becker, C.R. Minick, Culture of human endothelial cells derived from umbilical veins, J. Clin. Invest. 52 (1973) 2745–2756. O. Huet, J.M. Petit, M.H. Ratinaud, R. Julien, NADHdependent dehydrogenase activity estimation by Xow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, Cytometry 13 (1992) 532– 539. M.J. Curtis, D.J. Hearse, Ischaemia-induced and reperfusioninduced arrhythmias diVer in their sensitivity to potassium: implications for mechanisms of initiation and maintenance of ventricular Wbrillation, J. Mol. Cell. Cardiol. 21 (1989) 21–40. D.C. Warltier, M.G. Zyvoloski, G.J. Gross, H.F. Hardman, H.L. Brooks, Determination of experimental myocardial infarct size, J. Pharmacol. Methods 6 (1981) 199–210. R.R. Giraldez, A. Panda, Y. Xia, S.P. Sanders, J.L. Zweier, Decreased nitric-oxide synthase activity causes impaired endothelium-dependent relaxation in the postischemic heart, J. Biol. Chem. 272 (1997) 21420–21426. F. Yang, E. Troncy, M. Francoeur, B. Vinet, P. Vinay, G. Czaika, G. Blaise, EVects of reducing reagents and temperature on conversion of nitrite and nitrate to nitric oxide and detection by chemiluminescence, Clin. Chem. 43 (1997) 657–662. S. Takeo, K. Tanonaka, K. Shimizu, K. Hirai, K. Miyake, R. Minematsu, BeneWcial eVects of lidocaine and disopyramide on oxygen-deWciency-induced contractile failure and metabolic disturbance in isolated rabbit hearts, J. Pharmacol. Exp. Ther. 248 (1989) 306–314. J.X. Liu, K. Tanonaka, A. Sanbe, K. Yamamoto, S. Takeo, BeneWcial eVects of quinidine on post-ischemic contractile failure of isolated rat hearts, J. Mol. Cell. Cardiol. 25 (1993) 1249– 1263. M.J. Su, Y.C. Nieh, H.W. Huang, C.C. Chen, Dicentrine, an alphaadrenoceptor antagonist with sodium and potassium channel blocking activities, Naunyn. Schmiedebergs. Arch. Pharmacol. 349 (1994) 42–49. Y. Maejima, S. Adachi, H. Ito, K. Nobori, M. Tamamori-Adachi, M. Isobe, Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity, Cardiovasc. Res. 59 (2003) 308–320. C.L. Wainwright, A.M. Miller, L.M. Work, P. Del Soldato, NCX4016 (NO-aspirin) reduces infarct size and suppresses arrhythmias following myocardial ischaemia/reperfusion in pigs, Br. J. Pharmacol. 135 (2002) 1882–1888. J. Pernow, Y. Uriuda, Q.D. Wang, X.S. Li, R. Nordlander, L. Rydén, The protective eVect of L-arginine on myocardial injury and endothelial function following ischemia and reperfusion in the pig, Eur. Heart J. 15 (1994) 1712–1719. R. Pabla, A.J. Buda, D.M. Flynn, D.B. Salzberg, D.J. Lefer, Intracoronary nitric oxide improves postischemic coronary blood Xow and myocardial contractile function, Am. J. Physiol. 269 (1995) H1113–H1121. M. Kitakaze, K. Node, T. Minamino, H. Kosaka, Y. Shinozaki, H. Mori, M. Inoue, M. Hori, T. Kamada, Role of nitric oxide in regulation of coronary blood Xow during myocardial ischemia in dogs, J. Am. Coll. Cardiol. 27 (1996) 1804–1812. V. Richard, K. Laude, C. Artigues, N. KaeVer, J.P. Henry, C. Thuillez, Heat stress increases endothelium-dependent relaxations and prevents reperfusion-induced endothelial dysfunction, Clin. Exp. Pharmacol. Physiol. (2002) 956–962.

W.-L. Chang et al. / Nitric Oxide 11 (2004) 307–315 [33] J. Haendeler, U. Weiland, A.M. Zeiher, S. Dimmeler, EVects of redox-related congeners of NO on apoptosis and caspase-3 activity, Nitric Oxide 1 (1997) 282–293. [34] S. Dimmeler, J. Haendeler, M. Nehls, A.M. Zeiher, Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32like proteases, J. Exp. Med. 185 (1997) 601–608. [35] L. Rossig, J. Haendeler, C. Hermann, P. Malchow, C. Urbich, A.M. Zeiher, S. Dimmeler, Nitric oxide down-regulates MKP-3 mRNA levels. Involvement in endothelial cell protection from apoptosis, J. Biol. Chem. 275 (2000) 25502–25507. [36] C. Hermann, A.M. Zeiher, Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the gluta-

315

thione redox cycle and nitric oxide synthase, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 3588–3592. [37] J.M. McCord, Oxygen-derived free radicals in postischemic tissue injury, N. Engl. J. Med. 312 (1985) 159–163. [38] H. Wang, L. Lin, J. Jiang, Y. Wang, Z.Y. Lu, J.A. Bradbury, F.B. Lih, D.W. Wang, D.C. Zeldin, Upregulation of endothelial nitric oxide synthase by endothelium-derived hyperpolarizing factor involves MAPK and PKC signaling pathways, J. Pharmacol. Exp. Ther. 307 (2003) 753–764. [39] H. Li, C. Burkhardt, U.R. Heinrich, I. Brausch, N. Xia, U. Forstermann, Histamine upregulates gene expression of endothelial nitric oxide synthase in human vascular endothelial cells, Circulation 107 (2003) 2348–2354.