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Synergistic effect of nicorandil and amlodipine on lysosomal hydrolases during experimental myocardial infarction in rats
Biomedicine & Pharmacotherapy 57 (2003) 309–313 www.elsevier.com/locate/biopha
Original article
Synergistic effect of nicorandil and amlodipine on lysosomal hydrolases during experimental myocardial infarction in rats Venkatachalem Sathish, Kesavarao Kumar Ebenezar, Thiruvengadam Devaki * Department of Biochemistry and Molecular Biology, University of Madras, Guindy Campus, Chennai 600 025, India Received 15 January 2003; accepted 27 March 2003
Abstract The synergistic effect of nicorandil (KATP channel opener) and amlodipine (calcium channel blocker) on lysosomal hydrolases in serum and heart was examined by determining the activity of b-glucuronidase, b-N-acetyl glucosaminidase, b-galactosidase, cathepsin-D and acid phosphatase on isoproterenol-induced myocardial infarction in rats. The rats given isoproterenol (150 mg kg–1 daily, i.p.) for 2 d showed significant increase in serum and heart lysosomal hydrolases activity. Isoproterenol administration to rats resulted in decreased stability of the membranes, which was reflected by the lowered activity of cathepsin-D and b-glucuronidase in mitochondrial, nuclear, lysosomal and microsomal fractions. Pretreatment with nicorandil (2.5 mg kg–1 daily, p.o.) and amlodipine (5.0 mg kg–1 daily, p.o.) for 3 d significantly prevented these alterations and restored the enzyme activity to near normal. These findings demonstrate that the pretreatment with nicorandil and amlodipine could preserve lysosomal integrity and hence establish the cardioprotective effect of the combination. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Myocardial infarction; Nicorandil; Amlodipine; Lysosomal hydrolases
1. Introduction Nicorandil is a unique drug with a dual mechanism of action that acts not only as an ATP-sensitive potassium channel opener, but also as a nitrate by activating guanylate cyclase [1]. It is currently used for the treatment of angina pectoris. Nicorandil increases membrane K+ conductance, leading to cellular hyperpolarization and consequently to closure of voltage-operated Ca2+ channels [2]. It has been demonstrated that the nicorandil scavenges hydroxyl radicals and inhibits superoxide anion production in neutrophils in vitro [3]. Amlodipine, a calcium channel antagonist, has been used as an effective antihypertensive agent. The inhibition of lipoprotein oxidation is considered as an important aspect of their mechanism of action, whereby the formation of potentially atherogenic modified lipoproteins is prevented [4]. Lysosomes are membrane bound structures that contain hydrolytic enzymes capable of degrading most of the cellular constituents. In addition, lysosomes play a major role in secretion and transport processes. It has been postulated that the intracellular release of lysosomal enzymes and their sub* Corresponding author. E-mail address: [email protected] (T. Devaki). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0753-3322(03)00036-2
sequent extralysosomal activity may exercise a pivotal role in the progressive modifications that lead from reversible myocardial ischaemia to irreversible infarction [5]. Isoproterenol, a synthetic catecholamine and b-adrenergic agonist, causes severe stress in the myocardium, resulting in infarct-like necrosis of the heart muscle [6]. Isoproterenol-induced myocardial infarction results in increased lysosomal hydrolases activity that may be responsible for tissue damage and infarcted heart [7]. Intracellular release of lysosomal enzymes following myocardial ischaemia may directly [8] or through activation of the complement pathway result in cell injury and death. Isoproterenol-induced myocardial necrosis involves membrane permeability alterations that bring about loss of function and integrity of myocardial membranes [9]. In myocardial ischaemia, the damage caused by the enzymes of lysosomal and mitochondrial origin and the modification of tissue constituents by these enzymes play an important role. Recently we have reported the synergistic effect of nicorandil and amlodipine on myocardial marker enzymes, mitochondrial enzymes and antioxidant status during isoproterenol-induced myocardial infarction in rats [10,11]. Therefore, the present investigation was undertaken to study the alteration in the activity of lysosomal hydrolases on cardiac function during isoproterenol-induced myocardial
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Table 1 Activities of lysosomal hydrolases in serum of control and experimental groups of rats Group (treatment) 1 (Control) 2 (Isoproterenol) 3 (Nicorandil only) 4 (Amlodipine only) 5 (Nicorandil + amlodipine) 6 (Nicorandil + isoproterenol) 7 (Amlodipine + isoproterenol) 8 (Nicorandil + amlodipine + isoproterenol)
b-Glucuronidase 8.49 ± 0.60 12.10 ± 0.52a 8.52 ± 0.45 8.59 ± 0.47 8.46 ± 0.38 9.86 ± 0.66a,b 9.93 ± 0.74a,b 8.81 ± 0.65a,b,c,d
b-N-acetyl glucosaminidase 18.07 ± 0.89 24.63 ± 0.94a 17.89 ± 0.76 18.03 ± 0.83 17.93 ± 0.91 21.37 ± 0.93a,b 21.45 ± 0.87a,b 18.87 ± 0.79b,c,d
b-Galactosidases 12.33 ± 1.02 22.61 ± 1.53a 12.28 ± 0.98 12.51 ± 1.08 12.22 ± 0.86 16.06 ± 1.23a,b 15.79 ± 1.31a,b 12.09 ± 1.07b,c,d
Cathepsin-D 12.73 ± 1.27 18.35 ± 1.61a 12.43 ± 1.12 12.59 ± 0.98 12.92 ± 1.20 15.82 ± 1.43a,b 15.52 ± 1.37a,b 13.10 ± 1.04b,c,d
Acid phosphatase 77.34 ± 5.56 115.10 ± 7.78a 78.56 ± 4.93 78.09 ± 4.66 77.63 ± 5.02 90.12 ± 6.22a,b 91.27 ± 6.58a,b 80.15 ± 5.23b,c,d
Results are means ± S.D. (n = 6). P < 0.05 compared with a group l (control), b group 2 (isoproterenol), c group 6 (nicorandil + isoproterenol), or d group 7 (amlodipine + isoproterenol). Activity is expressed as: µmol P-nitrophenol per hour per 100 mg protein for b-glucuronidase, b-N-acetylglucosaminidase and b-galactosidases; µmol of tyrosine liberated per hour per 100 mg protein for cathepsin-D; and µmol of phenol released per hour per 100 mg proteins for acid phosphatases.
infarction and the synergistic effect of nicorandil and amlodipine in reducing the extent of lysosomal damage in the myocardium. Distribution of the major cardiac lysosomal proteinase, cathepsin-D, which is localized predominantly in myocytes rather than in interstitial cells [12] also was studied.
dose of isoproterenol), the animals were sacrificed. Blood was collected and serum separated was used for enzyme assays. 2.4. Separation of subcellular fractions
Nicorandil and amlodipine were procured from Sun Pharmaceutical Ltd., India. Isoproterenol hydrochloride, P-nitrophenyl-N-acetyl-b-D-glucosaminide and P-nitrophenyl b-D-glucuronide were purchased from Sigma Chemical Co., St. Louis, MO. All other chemicals used were of analytical grade.
The heart tissue sample was cut open and placed in isotonic saline to remove the blood. Then the heart tissue was rinsed in ice cold 0.25 M sucrose, blotted, weighed and minced. The enzyme extracts were prepared by homogenizing the tissue samples in 0.25 M sucrose at 4 °C. A portion of this preparation was used to determine the total activity. Another portion of the homogenate was subjected to differential centrifugation, and the different fractions were separated as follows: structural proteins, nucleus, and cell debris at 600 g for 10 min; mitochondria at 5000 g for 10 min; lysosomes at 15,000 g for 10 min; microsomes at 120,000 g for 30 min and supernatant cytosol.
2.2. Animals
2.5. Measurement of enzyme activity
Adult male albino rats of Wistar strain, weighing approximately 120–140 g, were obtained from King Institute of Preventive Medicine, Chennai, India. They were acclimatized to animal-house conditions, were fed commercial pelleted rat chow (Hindustan Lever Ltd, Bangalore, India), and had free access to water. All the procedures were as per the guidelines of human/animal ethical committee.
Myocardial subfractions were treated with Triton X-100 (final concentration 0.2% v/v) in ice for 15 min prior to the determination of enzymatic activity. The activities of b-glucuronidase [14], b-N-acetyl glucosaminidase [15], b-galactosidase [16], cathepsin-D [17] and acid phosphatase [18] were assayed.
2. Materials and methods 2.1. Chemicals
2.6. Statistical analysis 2.3. Experimental protocols The rats were divided into eight groups (n = 6 in each group): group 1, control; group 2, isoproterenol-induced; group 3, nicorandil only; group 4, amlodipine only; group 5, nicorandil + amlodipine; group 6, nicorandil + isoproterenol; group 7, amlodipine + isoproterenol; group 8, nicorandil + amlodipine + isoproterenol. Drug administration was as follows: isoproterenol given intraperitoneally for 2 d (150 mg kg–1 daily) [13]; nicorandil given orally (2.5 mg kg–1 daily) for 3 d; amlodipine given orally (5.0 mg kg–1 daily) for 3 d [10]. After the experimental period (24 h after the second
The data were analysed using one-way analysis of variance followed by least significant difference (LSD) test at P < 0.05. The values are expressed as means ± S.D.
3. Results Table 1 shows the activities of serum lysosomal hydrolases in control and experimental groups of rats. The activities of serum lysosomal hydrolases, b-glucuronidase, b-Nacetyl glucosaminidase, b-galactosidase, cathepsin-D and
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Table 2 Activities of lysosomal hydrolases in heart of control and experimental groups of rats Group (treatment) 1 (Control) 2 (Isoproterenol) 3 (Nicorandil only) 4 (Amlodipine only) 5 (Nicorandil + amlodipine) 6 (Nicorandil + isoproterenol) 7 (Amlodipine + isoproterenol) 8 (Nicorandil + amlodipine + isoproterenol)
b-Glucuronidase 20.79 ± 1.39 32.06 ± 2.07a 20.87 ± 1.28 21.02 ± 1.81 20.93 ± 1.66 25.69 ± 2.34a,b 25.92 ± 2.57a,b 21.43 ± 1.97b,c,d
b-N-acetyl glucosaminidase 42.93 ± 3.23 54.38 ± 4.12a 43.11 ± 3.51 43.39 ± 3.67 42.73 ± 3.77 48.35 ± 3.97a,b 48.69 ± 4.10a,b 43.21 ± 3.49b,c,d
b-Galactosidases 34.28 ± 2.63 43.19 ± 3.47a 34.79 ± 2.91 34.61 ± 2.78 34.66 ± 2.53 38.76 ± 3.08a,b 38.72 ± 3.22a,b 34.57 ± 2.88b,c,d
Cathepsin-D 23.51 ± 1.91 30.93 ± 2.41a 23.74 ± 1.63 23.87 ± 1.52 23.98 ± 1.83 27.02 ± 2.26a,b 26.89 ± 2.18a,b 24.09 ± 1.89b,c,d
Acid phosphatase 121.2 ± 6.71 153.1 ± 8.81a 122.6 ± 5.93 121.9 ± 6.07 122.3 ± 6.27 135.2 ± 7.28a,b 136.2 ± 7.51a,b 124.5 ± 6.83b,c,d
Results are means ± S.D. (n = 6). P < 0.05 compared with a group l (control), b group 2 (isoproterenol), c group 6 (nicorandil + isoproterenol), or d group 7 (amlodipine + isoproterenol). Activity is expressed as: µmol P-nitrophenol per hour per 100 mg protein for b-glucuronidase, b-N-acetylglucosaminidase and b-galactosidases; µmol of tyrosine liberated per hour per 100 mg protein for cathepsin-D; and µmol of phenol released per hour per 100 mg proteins for acid phosphatases.
acid phosphatase were found to be elevated significantly (P < 0.05) following isoproterenol-induced myocardial infarction (group 2) when compared to control group. When the rats were treated with nicorandil or amlodipine alone any per se effect (groups 3 and 4) on the activities of the lysosomal hydrolases was not produced as compared to control animals. Pretreatment with nicorandil plus amlodipine (group 8) resulted in a significant reduction (P < 0.05) in the activities of these enzymes towards near normal as compared with the rats given nicorandil and amlodipine (groups 6 and 7) monotherapy as well as isoproterenol intoxicated rats (group 2). Table 2 shows the activities of lysosomal hydrolases in the heart of control and experimental groups of rats. A significant increase (P < 0.05) in the activities of b-glucuronidase, b-N-acetyl glucosaminidase, b-galactosidase, cathepsin-D and acid phosphatase in the heart was noticed in rats that received isoproterenol when compared with those in control (group 1). The rats treated with nicorandil and amlodipine (group 8) in our study showed significant reduction in the activities of b-glucuronidase, b-N-acetyl glucosaminidase, b-galactosidase, cathepsin-D and acid phosphatase (P < 0.05) when compared with rats given the drugs individually (groups 6 and 7). Tables 3 and 4 show the distribution and changes in the activities of b-glucuronidase and cathepsin-D in the subcellular fractions of the heart, respectively. There was a signifi-
cant decrease (P < 0.05) in the activities of b-glucuronidase and cathepsin-D in the nuclear, mitochondrial, lysosomal and microsomal fractions along with a significant increase (P < 0.05) in the cytosolic fraction of the rats administered with isoproterenol when compared with control group. The combined administration of nicorandil and amlodipine (group 8) in our study significantly (P < 0.05) alleviated the altered activities of these enzymes in the mitochondrial, lysosomal, microsomal and cytosolic fractions of heart when compared with the rats given nicorandil and amlodipine alone (groups 6 and 7). Increased ratio of cytosol to bound lysosomal activities and cytosol to total activities for b-glucuronidase and cathepsin-D observed with isoproterenol administration was found to be decreased in combined treatment with nicorandil and amlodipine.
4. Discussion Hypoxia or ischaemia induced alterations in the membrane integrity of individual lysosomes might result in the hydrolysis of multiple mitochondria. Thus, these alterations may be primary structural lesions responsible for the genesis of the process of ischaemic myocardial injury [19]. It was reported that the localization of acid hydrolase in cardiac myocytes is in the lysosome and that the release of these
Table 3 Activities of b-glucuronidase in the subcellular distribution of heart of control and experimental groups of rats Group (treatment)
Nuclear fraction
1 (Control) 2 (Isoproterenol) 3 (Nicorandil only) 4 (Amlodipine only) 5 (Nicorandil + amlodipine) 6 (Nicorandil + isoproterenol) 7 (Amlodipine + isoproterenol) 8 (Nicorandil + amlodipine + isoproterenol)
9.18 ± 0.28 7.92 ± 0.21a 9.19 ± 0.25 9.17 ± 0.25 9.18 ± 0.19 9.02 ± 0.32b 8.99 ± 0.24b 9.08 ± 0.31b
Mitochondrial fraction 6.44 ± 0.32 4.89 ± 0.28a 6.48 ± 0.36 6.43 ± 0.39 6.45 ± 0.30 5.63 ± 0.28a,b 5.59 ± 0.25a,b 6.37 ± 0.33b,c,d
Lysosomal fraction 10.20 ± 0.36 7.21 ± 0.29a 10.24 ± 0.54 10.22 ± 0.49 10.18 ± 0.47 8.84 ± 0.43a,b 8.79 ± 0.37a,b 10.04 ± 0.40b,c,d
Microsomal fraction 4.78 ± 0.22 1.81 ± 0.12a 4.83 ± 0.28 4.86 ± 0.30 4.81 ± 0.29 3.46 ± 0.25a,b 3.40 ± 0.21a,b 4.53 ± 0.24a,b,c,d
Cytosolic fraction A•
B••
3.42 ± 0.17 6.48 ± 0.34a 3.38 ± 0.14 3.40 ± 0.18 3.36 ± 0.14 4.31 ± 0.16a,b 4.24 ± 0.12a,b 3.52 ± 0.19b,c,d
0.165 0.202 0.162 0.162 0.161 0.168 0.164 0.164
0.335 0.899 0.330 0.333 0.330 0.488 0.482 0.350
Results are means ± S.D. (n = 6). P < 0.05 compared with a group l (control), b group 2 (isoproterenol), c group 6 (nicorandil + isoproterenol), or d group 7 (amlodipine + isoproterenol). Activity is expressed as: µmol P-nitrophenol liberated per hour per 100 mg of protein. A•: ratio of cytosol (free) to lysosomal (bound) activity. B••: ratio of cytosol (free) to total activity.
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Table 4 Activity of cathepsin-D in the subcellular distribution of heart of control and experimental groups of rats Group (treatment)
Nuclear fraction
1 (Control) 2 (Isoproterenol) 3 (Nicorandil only) 4 (Amlodipine only) 5 (Nicorandil + amlodipine) 6 (Nicorandil + isoproterenol) 7 (Amlodipine + isoproterenol) 8 (Nicorandil + amlodipine + isoproterenol)
12.11 ± 1.17 9.57 ± 0.82a 12.01 ± 1.06 12.21 ± 0.95 12.13 ± 0.91 11.04 ± 1.03a,b 10.92 ± 0.94a,b 11.83 ± 1.16 b,c,d
Mitochondrial fraction 13.19 ± 1.19 8.28 ± 0.67a 13.18 ± 0.86 13.05 ± 0.94 13.32 ± 0.79 11.26 ± 0.87a,b 10.98 ± 1.09a,b 12.39 ± 1.13b,c,d
Lysosomal fraction 26.29 ± 1.93 16.28 ± 1.42a 25.95 ± 2.03 26.13 ± 2.39 25.99 ± 2.27 21.09 ± 1.69a,b 20.93 ± 1.73a,b 26.95 ± 2.42b,c,d
Microsomal fraction 7.38 ± 0.34 3.22 ± 0.22a 7.21 ± 0.34 7.13 ± 0.41 7.27 ± 0.39 4.82 ± 0.19a,b 4.67 ± 0.41a,b 6.18 ± 0.57a,b,c,d
Cytosolic fraction A•
B••
16.10 ± 1.43 26.20 ± 2.18a 15.98 ± 1.16 16.07 ± 1.21 15.90 ± 1.33 21.26 ± 1.81a,b 20.96 ± 1.73a,b 17.27 ± 1.38a,b,c,d
0.684 0.847 0.673 0.673 0.663 0.787 0.779 0.716
0.612 1.610 0.696 0.615 0.612 1.010 1.001 0.641
Results are means ± S.D. (n = 6). P < 0.05 compared with a group l (control), b group 2 (isoproterenol), c group 6 (nicorandil + isoproterenol), or d group 7 (amlodipine + isoproterenol). Activity is expressed as: µmol tyrosine liberated per hour per 100 mg of protein. A•: ratio of cytosol (free) to lysosomal (bound) activity. B••: ratio of cytosol (free) to total activity.
enzymes from the lysosome to the cytosol leads to myocardial cellular injury and death in the ischaemic state of the heart [5,19,20]. This is in agreement with our findings showing that in isoproterenol administered rats, the activity of serum and heart lysosomal acid hydrolases were increased. Lysosomal enzymes are important mediators of acute myocardial infarction and their release into the cytoplasm stimulates the formation of inflammatory mediators such as oxygen radicals and prostaglandins [21]. It has been suggested that oxygen free radicals generated during ischaemia in addition to the direct myocardial damaging effect may also be responsible for the cardiac damage through the release of lysosomal enzymes [22]. The membrane deterioration of lysosomes by isoproterenol-induced lipid peroxidation could have resulted in the leakage of enzymes from the enclosed sacs. Lysosomal membranes are reported to contain large amounts of glycoproteins which play an important role in maintaining lysosomal structure and functions [23]. The activities of serum and heart lysosomal hydrolases significantly increased in myocardial infarcted rats. These changes in our study are compatible with other studies [24,25]. Kennett and Weglicki [26] reported that the cytosolic acid hydrolases released from lysosomes and from the sarcoplasmic reticulum induce the dysfunction and distribution of mitochondria, sarcolemma and other organelle. Nicorandil plus amlodipine treatment reversed the increased activities of lysosomal enzymes by its inhibitory effect on lipid peroxidation [11] thereby reducing the extent of lysosomal damage induced by isoproterenol in the myocardium. Lipophilic calcium antagonists can trap free radicals by electron donating and radical resonating mechanisms, thereby blocking the lipid peroxidation chain reaction [27–30]. Isoproterenol administration to rats resulted in decreased stability of the membranes, which was reflected by the lowered activities of b-glucuronidase and cathepsin-D in nuclear, mitochondrial, lysosomal and microsomal fractions. Nicorandil plus amlodipine treatment in isoproterenol administered rats could inhibit the release of these enzymes from the lysosomal and microsomal fractions, which could be due to the stabilizing effect of nicorandil and amlodipine on the lysosomal and microsomal membranes. The distribu-
tion of enzyme activities between the cytosol to lysosomal and cytosol to total activities reported in our studies indicates that there is a decreased lysosomal stability in isoproterenol treated rats. In addition, the results of this study demonstrate that nicorandil plus amlodipine might inhibit the release of lysosomal enzymes as well as decrease the activity of the total lysosomal acid hydrolases, thereby enhancing the stability of lysosomes. Nicorandil produces the antioxidative action through forcing structural interactions with lipid membranes and the nitro group of nicorandil may play a role in the interaction [31]. Both in vitro and in vivo studies have shown that amlodipine inhibits oxidative damage to lipids associated with cellular membranes and lipoprotein particles [32–34]. The result of this study implies that the lysosomal hydrolases play an important role in isoproterenol-induced myocardial infarction. The combined treatment with nicorandil and amlodipine proved to be more protective in reducing the extent of lysosomal membrane damage and preserve the lysosomal integrity in experimentally induced myocardial infarction in rats.
Acknowledgements The first author wishes to thank the Council of Scientific and Industrial Research (CSIR), India for their financial assistance provided in the form of SRF.
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Original article
Synergistic effect of nicorandil and amlodipine on lysosomal hydrolases during experimental myocardial infarction in rats Venkatachalem Sathish, Kesavarao Kumar Ebenezar, Thiruvengadam Devaki * Department of Biochemistry and Molecular Biology, University of Madras, Guindy Campus, Chennai 600 025, India Received 15 January 2003; accepted 27 March 2003
Abstract The synergistic effect of nicorandil (KATP channel opener) and amlodipine (calcium channel blocker) on lysosomal hydrolases in serum and heart was examined by determining the activity of b-glucuronidase, b-N-acetyl glucosaminidase, b-galactosidase, cathepsin-D and acid phosphatase on isoproterenol-induced myocardial infarction in rats. The rats given isoproterenol (150 mg kg–1 daily, i.p.) for 2 d showed significant increase in serum and heart lysosomal hydrolases activity. Isoproterenol administration to rats resulted in decreased stability of the membranes, which was reflected by the lowered activity of cathepsin-D and b-glucuronidase in mitochondrial, nuclear, lysosomal and microsomal fractions. Pretreatment with nicorandil (2.5 mg kg–1 daily, p.o.) and amlodipine (5.0 mg kg–1 daily, p.o.) for 3 d significantly prevented these alterations and restored the enzyme activity to near normal. These findings demonstrate that the pretreatment with nicorandil and amlodipine could preserve lysosomal integrity and hence establish the cardioprotective effect of the combination. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Myocardial infarction; Nicorandil; Amlodipine; Lysosomal hydrolases
1. Introduction Nicorandil is a unique drug with a dual mechanism of action that acts not only as an ATP-sensitive potassium channel opener, but also as a nitrate by activating guanylate cyclase [1]. It is currently used for the treatment of angina pectoris. Nicorandil increases membrane K+ conductance, leading to cellular hyperpolarization and consequently to closure of voltage-operated Ca2+ channels [2]. It has been demonstrated that the nicorandil scavenges hydroxyl radicals and inhibits superoxide anion production in neutrophils in vitro [3]. Amlodipine, a calcium channel antagonist, has been used as an effective antihypertensive agent. The inhibition of lipoprotein oxidation is considered as an important aspect of their mechanism of action, whereby the formation of potentially atherogenic modified lipoproteins is prevented [4]. Lysosomes are membrane bound structures that contain hydrolytic enzymes capable of degrading most of the cellular constituents. In addition, lysosomes play a major role in secretion and transport processes. It has been postulated that the intracellular release of lysosomal enzymes and their sub* Corresponding author. E-mail address: [email protected] (T. Devaki). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0753-3322(03)00036-2
sequent extralysosomal activity may exercise a pivotal role in the progressive modifications that lead from reversible myocardial ischaemia to irreversible infarction [5]. Isoproterenol, a synthetic catecholamine and b-adrenergic agonist, causes severe stress in the myocardium, resulting in infarct-like necrosis of the heart muscle [6]. Isoproterenol-induced myocardial infarction results in increased lysosomal hydrolases activity that may be responsible for tissue damage and infarcted heart [7]. Intracellular release of lysosomal enzymes following myocardial ischaemia may directly [8] or through activation of the complement pathway result in cell injury and death. Isoproterenol-induced myocardial necrosis involves membrane permeability alterations that bring about loss of function and integrity of myocardial membranes [9]. In myocardial ischaemia, the damage caused by the enzymes of lysosomal and mitochondrial origin and the modification of tissue constituents by these enzymes play an important role. Recently we have reported the synergistic effect of nicorandil and amlodipine on myocardial marker enzymes, mitochondrial enzymes and antioxidant status during isoproterenol-induced myocardial infarction in rats [10,11]. Therefore, the present investigation was undertaken to study the alteration in the activity of lysosomal hydrolases on cardiac function during isoproterenol-induced myocardial
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Table 1 Activities of lysosomal hydrolases in serum of control and experimental groups of rats Group (treatment) 1 (Control) 2 (Isoproterenol) 3 (Nicorandil only) 4 (Amlodipine only) 5 (Nicorandil + amlodipine) 6 (Nicorandil + isoproterenol) 7 (Amlodipine + isoproterenol) 8 (Nicorandil + amlodipine + isoproterenol)
b-Glucuronidase 8.49 ± 0.60 12.10 ± 0.52a 8.52 ± 0.45 8.59 ± 0.47 8.46 ± 0.38 9.86 ± 0.66a,b 9.93 ± 0.74a,b 8.81 ± 0.65a,b,c,d
b-N-acetyl glucosaminidase 18.07 ± 0.89 24.63 ± 0.94a 17.89 ± 0.76 18.03 ± 0.83 17.93 ± 0.91 21.37 ± 0.93a,b 21.45 ± 0.87a,b 18.87 ± 0.79b,c,d
b-Galactosidases 12.33 ± 1.02 22.61 ± 1.53a 12.28 ± 0.98 12.51 ± 1.08 12.22 ± 0.86 16.06 ± 1.23a,b 15.79 ± 1.31a,b 12.09 ± 1.07b,c,d
Cathepsin-D 12.73 ± 1.27 18.35 ± 1.61a 12.43 ± 1.12 12.59 ± 0.98 12.92 ± 1.20 15.82 ± 1.43a,b 15.52 ± 1.37a,b 13.10 ± 1.04b,c,d
Acid phosphatase 77.34 ± 5.56 115.10 ± 7.78a 78.56 ± 4.93 78.09 ± 4.66 77.63 ± 5.02 90.12 ± 6.22a,b 91.27 ± 6.58a,b 80.15 ± 5.23b,c,d
Results are means ± S.D. (n = 6). P < 0.05 compared with a group l (control), b group 2 (isoproterenol), c group 6 (nicorandil + isoproterenol), or d group 7 (amlodipine + isoproterenol). Activity is expressed as: µmol P-nitrophenol per hour per 100 mg protein for b-glucuronidase, b-N-acetylglucosaminidase and b-galactosidases; µmol of tyrosine liberated per hour per 100 mg protein for cathepsin-D; and µmol of phenol released per hour per 100 mg proteins for acid phosphatases.
infarction and the synergistic effect of nicorandil and amlodipine in reducing the extent of lysosomal damage in the myocardium. Distribution of the major cardiac lysosomal proteinase, cathepsin-D, which is localized predominantly in myocytes rather than in interstitial cells [12] also was studied.
dose of isoproterenol), the animals were sacrificed. Blood was collected and serum separated was used for enzyme assays. 2.4. Separation of subcellular fractions
Nicorandil and amlodipine were procured from Sun Pharmaceutical Ltd., India. Isoproterenol hydrochloride, P-nitrophenyl-N-acetyl-b-D-glucosaminide and P-nitrophenyl b-D-glucuronide were purchased from Sigma Chemical Co., St. Louis, MO. All other chemicals used were of analytical grade.
The heart tissue sample was cut open and placed in isotonic saline to remove the blood. Then the heart tissue was rinsed in ice cold 0.25 M sucrose, blotted, weighed and minced. The enzyme extracts were prepared by homogenizing the tissue samples in 0.25 M sucrose at 4 °C. A portion of this preparation was used to determine the total activity. Another portion of the homogenate was subjected to differential centrifugation, and the different fractions were separated as follows: structural proteins, nucleus, and cell debris at 600 g for 10 min; mitochondria at 5000 g for 10 min; lysosomes at 15,000 g for 10 min; microsomes at 120,000 g for 30 min and supernatant cytosol.
2.2. Animals
2.5. Measurement of enzyme activity
Adult male albino rats of Wistar strain, weighing approximately 120–140 g, were obtained from King Institute of Preventive Medicine, Chennai, India. They were acclimatized to animal-house conditions, were fed commercial pelleted rat chow (Hindustan Lever Ltd, Bangalore, India), and had free access to water. All the procedures were as per the guidelines of human/animal ethical committee.
Myocardial subfractions were treated with Triton X-100 (final concentration 0.2% v/v) in ice for 15 min prior to the determination of enzymatic activity. The activities of b-glucuronidase [14], b-N-acetyl glucosaminidase [15], b-galactosidase [16], cathepsin-D [17] and acid phosphatase [18] were assayed.
2. Materials and methods 2.1. Chemicals
2.6. Statistical analysis 2.3. Experimental protocols The rats were divided into eight groups (n = 6 in each group): group 1, control; group 2, isoproterenol-induced; group 3, nicorandil only; group 4, amlodipine only; group 5, nicorandil + amlodipine; group 6, nicorandil + isoproterenol; group 7, amlodipine + isoproterenol; group 8, nicorandil + amlodipine + isoproterenol. Drug administration was as follows: isoproterenol given intraperitoneally for 2 d (150 mg kg–1 daily) [13]; nicorandil given orally (2.5 mg kg–1 daily) for 3 d; amlodipine given orally (5.0 mg kg–1 daily) for 3 d [10]. After the experimental period (24 h after the second
The data were analysed using one-way analysis of variance followed by least significant difference (LSD) test at P < 0.05. The values are expressed as means ± S.D.
3. Results Table 1 shows the activities of serum lysosomal hydrolases in control and experimental groups of rats. The activities of serum lysosomal hydrolases, b-glucuronidase, b-Nacetyl glucosaminidase, b-galactosidase, cathepsin-D and
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Table 2 Activities of lysosomal hydrolases in heart of control and experimental groups of rats Group (treatment) 1 (Control) 2 (Isoproterenol) 3 (Nicorandil only) 4 (Amlodipine only) 5 (Nicorandil + amlodipine) 6 (Nicorandil + isoproterenol) 7 (Amlodipine + isoproterenol) 8 (Nicorandil + amlodipine + isoproterenol)
b-Glucuronidase 20.79 ± 1.39 32.06 ± 2.07a 20.87 ± 1.28 21.02 ± 1.81 20.93 ± 1.66 25.69 ± 2.34a,b 25.92 ± 2.57a,b 21.43 ± 1.97b,c,d
b-N-acetyl glucosaminidase 42.93 ± 3.23 54.38 ± 4.12a 43.11 ± 3.51 43.39 ± 3.67 42.73 ± 3.77 48.35 ± 3.97a,b 48.69 ± 4.10a,b 43.21 ± 3.49b,c,d
b-Galactosidases 34.28 ± 2.63 43.19 ± 3.47a 34.79 ± 2.91 34.61 ± 2.78 34.66 ± 2.53 38.76 ± 3.08a,b 38.72 ± 3.22a,b 34.57 ± 2.88b,c,d
Cathepsin-D 23.51 ± 1.91 30.93 ± 2.41a 23.74 ± 1.63 23.87 ± 1.52 23.98 ± 1.83 27.02 ± 2.26a,b 26.89 ± 2.18a,b 24.09 ± 1.89b,c,d
Acid phosphatase 121.2 ± 6.71 153.1 ± 8.81a 122.6 ± 5.93 121.9 ± 6.07 122.3 ± 6.27 135.2 ± 7.28a,b 136.2 ± 7.51a,b 124.5 ± 6.83b,c,d
Results are means ± S.D. (n = 6). P < 0.05 compared with a group l (control), b group 2 (isoproterenol), c group 6 (nicorandil + isoproterenol), or d group 7 (amlodipine + isoproterenol). Activity is expressed as: µmol P-nitrophenol per hour per 100 mg protein for b-glucuronidase, b-N-acetylglucosaminidase and b-galactosidases; µmol of tyrosine liberated per hour per 100 mg protein for cathepsin-D; and µmol of phenol released per hour per 100 mg proteins for acid phosphatases.
acid phosphatase were found to be elevated significantly (P < 0.05) following isoproterenol-induced myocardial infarction (group 2) when compared to control group. When the rats were treated with nicorandil or amlodipine alone any per se effect (groups 3 and 4) on the activities of the lysosomal hydrolases was not produced as compared to control animals. Pretreatment with nicorandil plus amlodipine (group 8) resulted in a significant reduction (P < 0.05) in the activities of these enzymes towards near normal as compared with the rats given nicorandil and amlodipine (groups 6 and 7) monotherapy as well as isoproterenol intoxicated rats (group 2). Table 2 shows the activities of lysosomal hydrolases in the heart of control and experimental groups of rats. A significant increase (P < 0.05) in the activities of b-glucuronidase, b-N-acetyl glucosaminidase, b-galactosidase, cathepsin-D and acid phosphatase in the heart was noticed in rats that received isoproterenol when compared with those in control (group 1). The rats treated with nicorandil and amlodipine (group 8) in our study showed significant reduction in the activities of b-glucuronidase, b-N-acetyl glucosaminidase, b-galactosidase, cathepsin-D and acid phosphatase (P < 0.05) when compared with rats given the drugs individually (groups 6 and 7). Tables 3 and 4 show the distribution and changes in the activities of b-glucuronidase and cathepsin-D in the subcellular fractions of the heart, respectively. There was a signifi-
cant decrease (P < 0.05) in the activities of b-glucuronidase and cathepsin-D in the nuclear, mitochondrial, lysosomal and microsomal fractions along with a significant increase (P < 0.05) in the cytosolic fraction of the rats administered with isoproterenol when compared with control group. The combined administration of nicorandil and amlodipine (group 8) in our study significantly (P < 0.05) alleviated the altered activities of these enzymes in the mitochondrial, lysosomal, microsomal and cytosolic fractions of heart when compared with the rats given nicorandil and amlodipine alone (groups 6 and 7). Increased ratio of cytosol to bound lysosomal activities and cytosol to total activities for b-glucuronidase and cathepsin-D observed with isoproterenol administration was found to be decreased in combined treatment with nicorandil and amlodipine.
4. Discussion Hypoxia or ischaemia induced alterations in the membrane integrity of individual lysosomes might result in the hydrolysis of multiple mitochondria. Thus, these alterations may be primary structural lesions responsible for the genesis of the process of ischaemic myocardial injury [19]. It was reported that the localization of acid hydrolase in cardiac myocytes is in the lysosome and that the release of these
Table 3 Activities of b-glucuronidase in the subcellular distribution of heart of control and experimental groups of rats Group (treatment)
Nuclear fraction
1 (Control) 2 (Isoproterenol) 3 (Nicorandil only) 4 (Amlodipine only) 5 (Nicorandil + amlodipine) 6 (Nicorandil + isoproterenol) 7 (Amlodipine + isoproterenol) 8 (Nicorandil + amlodipine + isoproterenol)
9.18 ± 0.28 7.92 ± 0.21a 9.19 ± 0.25 9.17 ± 0.25 9.18 ± 0.19 9.02 ± 0.32b 8.99 ± 0.24b 9.08 ± 0.31b
Mitochondrial fraction 6.44 ± 0.32 4.89 ± 0.28a 6.48 ± 0.36 6.43 ± 0.39 6.45 ± 0.30 5.63 ± 0.28a,b 5.59 ± 0.25a,b 6.37 ± 0.33b,c,d
Lysosomal fraction 10.20 ± 0.36 7.21 ± 0.29a 10.24 ± 0.54 10.22 ± 0.49 10.18 ± 0.47 8.84 ± 0.43a,b 8.79 ± 0.37a,b 10.04 ± 0.40b,c,d
Microsomal fraction 4.78 ± 0.22 1.81 ± 0.12a 4.83 ± 0.28 4.86 ± 0.30 4.81 ± 0.29 3.46 ± 0.25a,b 3.40 ± 0.21a,b 4.53 ± 0.24a,b,c,d
Cytosolic fraction A•
B••
3.42 ± 0.17 6.48 ± 0.34a 3.38 ± 0.14 3.40 ± 0.18 3.36 ± 0.14 4.31 ± 0.16a,b 4.24 ± 0.12a,b 3.52 ± 0.19b,c,d
0.165 0.202 0.162 0.162 0.161 0.168 0.164 0.164
0.335 0.899 0.330 0.333 0.330 0.488 0.482 0.350
Results are means ± S.D. (n = 6). P < 0.05 compared with a group l (control), b group 2 (isoproterenol), c group 6 (nicorandil + isoproterenol), or d group 7 (amlodipine + isoproterenol). Activity is expressed as: µmol P-nitrophenol liberated per hour per 100 mg of protein. A•: ratio of cytosol (free) to lysosomal (bound) activity. B••: ratio of cytosol (free) to total activity.
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Table 4 Activity of cathepsin-D in the subcellular distribution of heart of control and experimental groups of rats Group (treatment)
Nuclear fraction
1 (Control) 2 (Isoproterenol) 3 (Nicorandil only) 4 (Amlodipine only) 5 (Nicorandil + amlodipine) 6 (Nicorandil + isoproterenol) 7 (Amlodipine + isoproterenol) 8 (Nicorandil + amlodipine + isoproterenol)
12.11 ± 1.17 9.57 ± 0.82a 12.01 ± 1.06 12.21 ± 0.95 12.13 ± 0.91 11.04 ± 1.03a,b 10.92 ± 0.94a,b 11.83 ± 1.16 b,c,d
Mitochondrial fraction 13.19 ± 1.19 8.28 ± 0.67a 13.18 ± 0.86 13.05 ± 0.94 13.32 ± 0.79 11.26 ± 0.87a,b 10.98 ± 1.09a,b 12.39 ± 1.13b,c,d
Lysosomal fraction 26.29 ± 1.93 16.28 ± 1.42a 25.95 ± 2.03 26.13 ± 2.39 25.99 ± 2.27 21.09 ± 1.69a,b 20.93 ± 1.73a,b 26.95 ± 2.42b,c,d
Microsomal fraction 7.38 ± 0.34 3.22 ± 0.22a 7.21 ± 0.34 7.13 ± 0.41 7.27 ± 0.39 4.82 ± 0.19a,b 4.67 ± 0.41a,b 6.18 ± 0.57a,b,c,d
Cytosolic fraction A•
B••
16.10 ± 1.43 26.20 ± 2.18a 15.98 ± 1.16 16.07 ± 1.21 15.90 ± 1.33 21.26 ± 1.81a,b 20.96 ± 1.73a,b 17.27 ± 1.38a,b,c,d
0.684 0.847 0.673 0.673 0.663 0.787 0.779 0.716
0.612 1.610 0.696 0.615 0.612 1.010 1.001 0.641
Results are means ± S.D. (n = 6). P < 0.05 compared with a group l (control), b group 2 (isoproterenol), c group 6 (nicorandil + isoproterenol), or d group 7 (amlodipine + isoproterenol). Activity is expressed as: µmol tyrosine liberated per hour per 100 mg of protein. A•: ratio of cytosol (free) to lysosomal (bound) activity. B••: ratio of cytosol (free) to total activity.
enzymes from the lysosome to the cytosol leads to myocardial cellular injury and death in the ischaemic state of the heart [5,19,20]. This is in agreement with our findings showing that in isoproterenol administered rats, the activity of serum and heart lysosomal acid hydrolases were increased. Lysosomal enzymes are important mediators of acute myocardial infarction and their release into the cytoplasm stimulates the formation of inflammatory mediators such as oxygen radicals and prostaglandins [21]. It has been suggested that oxygen free radicals generated during ischaemia in addition to the direct myocardial damaging effect may also be responsible for the cardiac damage through the release of lysosomal enzymes [22]. The membrane deterioration of lysosomes by isoproterenol-induced lipid peroxidation could have resulted in the leakage of enzymes from the enclosed sacs. Lysosomal membranes are reported to contain large amounts of glycoproteins which play an important role in maintaining lysosomal structure and functions [23]. The activities of serum and heart lysosomal hydrolases significantly increased in myocardial infarcted rats. These changes in our study are compatible with other studies [24,25]. Kennett and Weglicki [26] reported that the cytosolic acid hydrolases released from lysosomes and from the sarcoplasmic reticulum induce the dysfunction and distribution of mitochondria, sarcolemma and other organelle. Nicorandil plus amlodipine treatment reversed the increased activities of lysosomal enzymes by its inhibitory effect on lipid peroxidation [11] thereby reducing the extent of lysosomal damage induced by isoproterenol in the myocardium. Lipophilic calcium antagonists can trap free radicals by electron donating and radical resonating mechanisms, thereby blocking the lipid peroxidation chain reaction [27–30]. Isoproterenol administration to rats resulted in decreased stability of the membranes, which was reflected by the lowered activities of b-glucuronidase and cathepsin-D in nuclear, mitochondrial, lysosomal and microsomal fractions. Nicorandil plus amlodipine treatment in isoproterenol administered rats could inhibit the release of these enzymes from the lysosomal and microsomal fractions, which could be due to the stabilizing effect of nicorandil and amlodipine on the lysosomal and microsomal membranes. The distribu-
tion of enzyme activities between the cytosol to lysosomal and cytosol to total activities reported in our studies indicates that there is a decreased lysosomal stability in isoproterenol treated rats. In addition, the results of this study demonstrate that nicorandil plus amlodipine might inhibit the release of lysosomal enzymes as well as decrease the activity of the total lysosomal acid hydrolases, thereby enhancing the stability of lysosomes. Nicorandil produces the antioxidative action through forcing structural interactions with lipid membranes and the nitro group of nicorandil may play a role in the interaction [31]. Both in vitro and in vivo studies have shown that amlodipine inhibits oxidative damage to lipids associated with cellular membranes and lipoprotein particles [32–34]. The result of this study implies that the lysosomal hydrolases play an important role in isoproterenol-induced myocardial infarction. The combined treatment with nicorandil and amlodipine proved to be more protective in reducing the extent of lysosomal membrane damage and preserve the lysosomal integrity in experimentally induced myocardial infarction in rats.
Acknowledgements The first author wishes to thank the Council of Scientific and Industrial Research (CSIR), India for their financial assistance provided in the form of SRF.
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