Ischemia-induced alteration of myocardial Na+-K+-ATPase activity and ouabain binding sites in hypercholesterolemic rabbits

ATHEROSCLEROSIS Atherosclerosis127(1996)59-64

ELSEVIER

Ische-mia.-induced alteration of myocardial Na + -K + -ATPase activity and ouabain binding sites in hypercholesterolemic rabbits Wen-Jone Chen”, Shoei-Yn Lin-Shiaub,

Huei-Chen

Huangb, Yuan-Teh

Lee’,“.*

“Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taill.an bDepartment of Pharmacology. College of Medicine, National Taiwan Unirersity. Taipei, Taiwan

Received3 August 1995;revised20 May 1996;accepted26 June 1996

Abstract

The purpose of this study was to explore the effect of &hernia on the Na + -K + -ATPase activity and ouabain receptor of the myocardial sarcdlemma in hypercholesterolemic rabbits. Male New Zealand white rabbits were fed with either standard chow or standard chow supplemented with 0.5% (w/w) cholesterol and 10% (w/w) coconut oil. After an 8 week feeding period, the rabbits underwent ;i thoracotomy and myocardial ischemia was induced by occlusion of the coronary artery. Myocardial samples from the ischemic and non-ischemic regions of the left ventricle of control and cholesterol-fed rabbits were taken for study. The cholesterol-fed group showed a decreasein both Na + -K + -ATPase activity and [3H]ouabain binding sites as compared to the control group. Ischemia caused a reduction in both Na + -K + -ATPase activity and [3H]ouabain binding sites in both control and cholesterol-fed rarbbits. The combination of ischemia and hypercholesterolemia produced an additive effect, with a further decreasein both Na + -K + -ATPase activity and [3H]ouabain binding sites. Neither the activity of Mg + + -ATPase nor the binding affinity for C3H]ouabainwas affected by either hypercholesterolemia or ischemia. These findings indicate that hypercholesterolemia may exaggerate certain aspects of functional deterioration arising during myocardial ischemia. Keywords: Ischemia; Hypercholesterolemia; Na + -K + -ATPase; Ouabain receptor

1. Introduction The importance of dietary cholesterol in affecting the physiology and pathology of the cardiovascular system has been emphasized in a number of studies both on animals [l-3] and humans [4-61. A high cholesterol diet results in increased cholesterol levels, not only in the plasma, but also in the myocardium [3,7]. Cholesterol plays an important role in the regulation of myocardial sarcolemmal function. A high membrane cholesterol content alters the physical properties of the membrane, producing changes in phase transition,

* Correspondingauthor. ’ Presentaddress:7, Chung-ShanS. Road, Taipei, Taiwan. Tel./ ‘ax: + 886 2 39599 I I.

fluidity, homogeneity and permeability [8- lo], which are thought to be related to changes in certain membrane-bound enzymes and receptors [l-3]. Thus, the mechanism of action of dietary cholesterol on cardiac function could be attributed to the development of atherosclerosis, leading to the occlusion of essential coronary arteries feeding the heart muscle, and to alterations in structural lipid composition, resulting in changes in enzymatic activities and receptor functions of the myocardial sarcolemma [l-6]. In tact, these two mechanisms could operate simultaneously in cases of long-standing hypercholesterolemia, which is a relatively common pathological condition in industrialized countries today. For this reason, we decided to study their combined effect on certain functions of the myocardial sarcolemma by occluding the coronary artery in cholesterol-fed rabbits.

0021-9l50~9C~/$15.0~3Cl 1996 Elsebier Science Ireland Ltd. All rights reserved PII SO021 -9150(96)05964-7

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W.-J. Chen et al. I Atherosclerosis

Na + -K+ -ATPase has been localized in the cardiac sarcolemma and is considered to be involved in the maintenance of intracellular Na+ and K+ concentrations in the myocardium [l 1,121.It is believed that this enzyme is a receptor for cardiac glycosides and that the glycoside-mediated positive inotropic effect on the myocardium could be a consequent effect of the inhibition of Na + -K + -ATPase activity [ 11,121. Earlier studies have shown that Na + -K+ -ATPase activity is significantly decreased in the ischemic heart [13- 161. However, all of these studies were performed under conditions of physiological normo-cholesterolemic state. In order to study ischemic effects on certain myocardial sarcolemmal functions in the pathophysiological hypercholesterolemic state, we measured the changes in Na + -K + -ATPase activity and ouabain binding sites by occlusion of the coronary artery in hypercholesterolemic rabbits. 2. Materials and methods 2.1. Preparation of animals

Male New Zealand white rabbits with an initial weight of 1.2- 1.5 kg were randomly assigned to two groups. The control group received standard rabbit chow (Purina 5321, St Louis, MO, USA), while the study group received standard rabbit chow supplemented with 0.5% cholesterol (w/w) and 10% coconut oil (w/w). All rabbits were housed in individual cages with raised screen bases in a room maintained at a constant temperature of 25°C and on a 12:12 hour light-dark cycle, Food and water were given ad libiturn. At the end of the 8 week feeding period, the animals were sacrificed. After a 12 h fasting, the rabbits were anesthetized with pentobarbital (25 mg/kg i.v.) and anesthesia maintained during the experiment by intravenous injection of small amounts of pentobarbital (5 mg/kg), sufficient to abolish the cornea1 reflex. The rabbits were intubated through a tracheostomy and ventilated with room air using a constant-volume respirator (Harvard Apparatus, Cambridge, MA, USA). A polyethylene catheter was introduced into the aorta via the right femoral artery in order to monitor arterial pressure and another catheter placed into the right femoral vein for blood sampling. Thoracotomy was performed through the fifth left intercostal space and the heart was suspended in a pericardial cradle. A 5-O silk surgical ligature was placed around the circumflex branch of the left coronary artery. Acute coronary occlusion by tightening the ligature was maintained for 30 min. The lead II of electrocardiogram and systemic arterial pressure (Statham P23 DB pressure transducer) were recorded continuously during the experiment (Gould Instruments). Myocardial ischemia was confi-

127 (1996) 59-64

rmed by ST segment elevation of the ECG, as well as by observation of regional cyanosis over the myocardial surface. Rabbits which did not complete the experiment were excluded from analysis. At the end of the procedure, the rabbits were immediately sacrificed by rapid intravenous injection of potassium chloride. Myocardial samples were taken from the ischemic and non-ischemic myocardium of the left ventricle and stored in liquid nitrogen until analysis. The investigation conformed with the Guide for the care and use of laboratory animals published by the US National Institutes of Health. 2.2. Membrane preparation

Crude membrane preparations were obtained from the myocardial samples according to the procedure of Alam et al. [17]. In brief, myocardial samples (about 0.6 g) were minced with scissors and homogenized using a Polytron with 9-10 volumes of 0.05 M Tris-HCl, pH 7.4, containing 0.32 M sucrose and 1 mM MgCl,. All procedures were performed at 0-4°C. The homogenate was filtered through four layers of cheese cloth and a crude sarcolemmal membrane fraction was prepared by differential centrifugation. The purity of the membrane preparation was determined by monitoring 5’-nucleotidase activity using an assay kit purchased from Sigma (St. Louis, MO, USA) [18]. 2.3. Assay of Na+-K+-ATPase

activity

The assay was performed as described previously [19,20], with minor modifications. The sarcolemmal fraction (1 ,ug of protein) was incubated for 20 min at 37°C in modified Krebs solution (118 mM NaCl, 4.7 mM KCl, 1.1 mM MgCl,, 12.5 mM NaHCO,, 0.5 mM EGTA and 11.1 mM glucose), containing 5 mM sodium azide and 3 mM ATP. All experiments were performed in triplicate. The Na + -K + -Mg + + -ATPase activity was estimated by colormetric determination of the amount of inorganic phosphate released from ATP. The Mg+ +- ATPase activity, assayed in the presence of 1 mM ouabain in modified Krebs solution, was subtracted from the Na + -K + -Mg + + -ATPase activity to obtain the Na + -K + -ATPase activity. 2.4. [3H]ouabain binding studies

[3H]ouabain binding studies were performed as described previously [19,20], with minor modifications. [3H]ouabain (specific activity 15.0 Ci/mmol) was obtained from Amersham (Amersham, Buckinghamshire, UK). The total binding of [3H]ouabain to the myocardial sarcolemma was estimated at [3H]ouabain concentrations of 8-512 nM. Sarcolemmal fractions (120 pg protein/100 ~1) plus 200 ~1 of reaction mixture contain-

W.-J. Chen et al. I Atherosclerosis 127 (1996) 59-64

ing [3H]ouabain, with or without unlabelled ouabain, were incubated at 37°C for 30 min. After incubation, 750 ~1 of polyethylene glycol (17% w/v in Tris-HCl buffer) was added to precipitate protein-bound [3H]ouabain, as described by Plourde et al. [21]. The mixture was centrifuged at 3000 x g for 30 min at 0-4°C to separate bound and free ouabain. The pellet (bound ouabain) was washed and mixed with scintillation counting cocktail (Ready GelTM,Beckman, Fullerton, CA, USA) and the radioactivity measured in a Beckman liquid scintillation spectrometer (Beckman LS-5801, Beckman, Fullerton, CA, USA). Non-specific binding was determined as the bound radioactivity measured in the presence of an excess (1 mM) of non-labelled ouabain. Specific binding was estimated from the difference between total and non-specific binding. The maximum binding site concentration (B,,,) and the affinity for ouabain (KJ were determined by Scatchard analysis [22,23]. 2.5. Protein determination and statistical analysis Protein was determined by the method of Lowry [24], using bovine serum albumin as the standard. Values in the text, as well as in the Figures and Tables, are expressed as the mean &-S.E.M. Difference between groups was determined using multigroup analysis of variance according to Bonferroni correction [25]. When F was significant, means were compared by Student’s t test: paired t test for intragroup comparison of ischemic effects and unpaired t test for intergroup comparison. Statistical significance was defined as a value of P < 0.05.

3. Results The rabbits fe:d for 8 weeks with the high cholesterol diet had a significantly higher plasma level of cholesterol than the c’ontrol group (57.8 + 3.8 mmol/l versus I .4 + 0.1 mmoljl, 12= 10, I’ < 0.001). Other variables, such as body weight, heart rate and blood pressure, showed no significant difference between the two groups (Table 1). As shown in Table 1, coronary artery

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Table I Changes in heart rate and blood pressure following coronary artery ligation in rabbits Control (n = 10)

Chol-fed (n = IO)

HR (bpm)

MBP (mm&9

HR (bpm)

MBP (mmJ%)

92 +2 70 + 3
285 Ifr 5 281 +6 NS

93 i: 1 68 i 3
Baseline 294 + I Post-ligation 299 + 6 P value NS

Chol, cholesterol; HR, heart rate; MBP, mean blood pressure; bpm, beats/min; NS, not significant

ligation significantly decreasedthe mean blood pressure as compared to baseline in both groups, but did not significantly affect heart rate. In order to evaluate whether or not the non-ischemic region of the ventricle could be used as the control for the ischemic region, the yield of protein, specific activity of the Na+ -K+ -ATPase and the 13H]ouabain binding properties were measured in the non-ischemic region and a normal ventricle. As shown in Table 2, there was no significant difference in these criteria between the non-ischemic region of the heart and a normal ventricle. No significant difference in yield of membrane protein was seen between control and cholesterol-fed animals. The 5’-nucleotidase activity in the isolated myocardial sarcolemma increased by 5-6 fold in both groups when compared with whole homogenate. The relative purity of the membranes in the two groups was also not significantly different. The effects of myocardial ischemia on sarcolemmal Na + -K + -ATPase and Mg + + -ATPase activities in control and cholesterol-fed rabbits are shown in Fig. 1. Ischemia caused a significant decreasein Na + -K + -ATPase activity in both groups (control, 4.07 ) 0.23 versus 3.23 + 0.12 pmol Pi/mg protein per h, P < 0.005; cholesterol-fed, 3.31 f- 0.12 versus 2.66 + 0.18 pmol Pi/ mg protein per h, P < 0.001). The ratio of Na+ -K+ ATPase activity in the ischemic region relative to that in the non-ischemic region was not significantly different between the control and cholesterol-fed groups

Table 2 Na+-K+-ATPase activity and [‘Hjouabain binding properties in non-ischemic region of the heart and a normal ventricle

Na+ -K + -ATPase activity (pmol Pi/mg protein per h) Bmaxof [3H]ouabairl (pmol/mg protein) & of [3H]ouabain (nM)

Control

Chol-fed

Non-IR (n = 10) Normal (n = 8) P value

Non-IR (n = 10) Normal (n = 10) P value

4.07 f 0.23

4.16 f 0.25

NS

3.31 *0.12

3.22 +0.13

NS

10.4+ 0.3 117*3

10.8+ 0.5 120* 3

NS NS

8.9 + 0.3 118&2

9.1 +0.5 121 k2

NS NS

Non-IR, non-ischemic region; B,,,,.,, maximal binding sites; Kd, dissociation constant. Other abbreviations used are the same as in Table I

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(A) LI

lC+,ed

Fig. 1. (A). Na + -K + -ATPase activity in control and cholesterol-fed rabbits. Ischemia causesa significantly decreasein Na + -K + -ATPase activity in both two groups. The Na+-K+-ATPase activity, in both the ischemic and non-ischemic region, is significantly less in the cholesterol-fed group as compared with the control. (B). Mg + +-ATPase activity in control and cholesterol-fed rabbits. Mg+ +-ATPase activity was the same in all groups studied. Non-IR, non-ischemic region; IR, ischemic region; Chol, cholesterol; NS, not significant.

(control, 81 It 4%; cholesterol-fed, 80 _+3%; P = NS). The Na+ -K+ -ATPase activity, in either the non-ischemic or ischemic region, was significantly less in the cholesterol-fed group as compared with the control group. Ischemia did not cause a significant change in Mg + + -ATPase activities in the control or cholesterolfed groups and there was no significant difference between the two groups. The effects of ischemia on [3H]ouabain binding sites in control and cholesterol-fed rabbits are shown in Fig. 2. Ischemia caused a significant decrease in the maximum number of ouabain binding sites (B,,,) in both groups (control, 10.4 f 0.3 versus 8.5 f 0.3 pmol/mg protein, P < 0.001; cholesterol-fed, 8.9 + 0.3 versus 7.4 + 0.2 pmol/mg protein, P < 0.001). The ratio of the B max value for [3H]ouabain binding in the ischemic

P=NS

Fig. 2. [3H]-ouabain binding study in control and cholesterol-fed rabbits. (A). Ischemia causes a significant decrease in the maximum number of binding sites (B,,,,,) for [3H]ouabain in both groups. The B lTX3Xlin both the ischemic and non-ischemic region, is significantly lower in the cholesterol-fed group as compared with the control. (B). The dissociation constant (KJ for [3H]ouabain was the same in all groups studied. The abbreviations used are the same as in Fig. 1.

127 (1996) 59-64

region relative to that in the non-ischemic region showed no significant difference in the control and cholesterol-fed groups (control, 82 + 3%; cholesterolfed, 84 + 2%; P = NS); however, the actual value for B maxin either the non-ischemic or ischemic region, was significantly less in the cholesterol-fed group as compared with the control group. In terms of the dissociation constant (&) for [3H]ouabain binding, ischemia caused no significant change in the control or cholesterol-fed groups and there was no significant difference between the two groups. 4. Discussion Dietary cholesterol exerts a profound influence on the physiology and pathology of the cardiovascular system [l-6]. The mechanisms of action of dietary cholesterol on cardiac function is not only through the process of atherosclerosis of coronary artery [4-61, but also through the perturbation of the myocardial membrane lipid composition and membrane related functions [l-3]. Cholesterol is a major component of the myocardial sarcolemma membrane lipids [26] and plays an important role in the regulation of sarcolemmal function [27-291. An increase in cholesterol content of the cell membrane results in a decrease in membrane fluidity and decreases membrane permeability [9,30], thus altering the function of membrane-bound enzymes, ion channels and receptors [l-3,27-29]. In our study, we have demonstrated that rabbits fed a high cholesterol diet show a significant hypercholesterolemia and a decreasedactivity of the myocardial sarcolemmal Na + K + -ATPase, which is accompanied by a decreasein the maximum number of [3H]ouabain binding sites in both the non-ischemic and ischemic regions, as compared with the control group. These results indicate that cholesterol exerts an inhibitory role on myocardial sarcolemmal Na + -K + -ATPase activity. A high cholesterol content of the cell membrane has been shown to inhibit Na + -K + -ATPase activity in other tissues, such as the erythrocyte, liver and kidney [31-331. Several mechanisms have been proposed to account for the inhibitory effect of membrane cholesterol on the Na + K+ -ATPase activity. For example, the rigid sterol structure of cholesterol might reduce the capability of Na + -K + -ATPase to undergo a conformational change [lo]. A second possibility would involve a direct interaction between cholesterol and the Na + -K + -ATPase, leading to modulation of its activity [lo]. A third possible mechanism is lipid peroxidation occurring after cholesterol enrichment of the cell membrane [31]. In the present study, 30 min of ischemia induced by coronary artery occlusion caused a significant reduction in myocardial sarcolemmal Na + -K + -ATPase activity and specific [3H]ouabain binding, in both the control

W.-J. Chen et ul. 1Atheroscltwnis

and cholesterol-fed groups. These observations are in agreement with previous studies [13- 161performed in the normo-cholesterolemic state. In the hypercholesterolemic state, a commonly encountered pathological condition, the effects of ischemia on the sarcolemmal Na + -K + -ATPase and [“Hlouabain binding have never been reported. We have demonstrated that, in hypercholesterolemia, ischemia can cause a further significant reduction in m.yocardial sarcolemmal Na + -K + -ATPase activity and specific [3H]ouabain binding. This indicates that the mechanisms responsible for the changes in sarcolemmal Na ’ -K + -ATPase activity seen during ischemia still operate in certain pathological conditions, such1as hypercholesterolemia, and that two pathological factors (ischemia and hypercholesterolemia) occurring simultaneously can have a greater effect on the sarcolemmal Na + -K + -ATPase activity. During myocardial ischemia, no significant difference in the reduction of sarcolemmal Na+ -K + -ATPase activity was seen between the control and cholesterol-fed groups implying that the mechanisms responsible for the reduction might be independent of cholesterol level. Several mechanisms have been proposed to explain the decrease in Na + -K+ -ATPase activity seen during ischemia. Depletion of the cellular ATP pool, which occurs rapidly during myocardial ischemia, has been suggested as one such factor in the reduction of Na+K + -ATPase activity and glycoside binding [34]. A second possible mechanism is the accumulation of toxic metabolites resulting from ischemia, which could play an important role in injury to the sarcolemmal Na+K+-ATPase [35]. A third mechanism could be the elevation of the intracellular Ca2+ concentration during myocardial ischemia, which might result in activation of the Ca’+-dependent protease and inhibit the Na+ -K+ -ATPase activity 1341.Acidosis, due to lactate accumulation, rnight also cause inactivation of the Na + -K + -ATPase [36]. Na+ -K+ -ATPase is responsible for pumping Na+ out of the cell in exchange for extracellular K+ against their respective concentration gradients and is essential for the regulation of ionic content and membrane excitability of the myocardial cell. A decrease in Na + K+ -ATPase activity would lead to accumulation of intracellular Na + , thereby favoring an increase in levels of intracellular Ca’ + due to increased Na + /Ca” + exchange, and possibly resulting in intracellular Ca”+ overload. Since a decreasein Na + -K + -ATPase activity and [3H]ouabam binding sites are strongly correlated with myocardial dysfunction [37,38]; the long-term hypercholesterolemic state, because of coronary atherosclerosis and/or perturbation of myocardial membrane-related functions, leading to a decrease in Na + -K + -ATPase activity and [3H]ouabain specific binding, may be of importance in the regulation of myocardial function. The myocardial Na +-K + -AT-

127 (1996) 59-64

63

Pase is suggested to be a cellular receptor for digitalis glycosides. In acute myocardial ischemia, the sensitivity of the myocardium to the arrhythmogenic action of digitalis dramatically increases and this is known to be associated with an inhibition of the myocardial Na+ K + -ATPase [39,40]. Since hypercholesterolemia itself induces a reduction in Na +-K+ -ATPase activity, should ischemia develop in the hypercholesterolemic state, the sensitivity of the myocardium to the arrhythmogenic action of digitalis might be even higher. The implications of our findings in this study are that: (i) High dietary cholesterol may directly influence the physiology and pharmacology of the heart by altering myocdrdial sarcolemmal function. Cholesterol enrichment of the membrane may induce alternation of functions of membrane-bound enzymes, ion channels and receptors. [l-3,27-29] (ii) Since cholesterol plays an important role in the process of coronary atherosclerosis, it may cause occlusion of the coronary artery and myocardial ischemia in long-term hypercholesterolemia [4-61. The combined effect of hypercholesterolemia and acute myocardial ischemia on the pathophysiology of the heart deserves further investigations. (iii) Either ischemia or hypercholesterolemia, alone can cause a decrease in myocardial Na + -K + -ATPase activity and specific [3H]ouabain binding site, and together, they have an additive effect on Na +-K+ -ATPase activity and [3H]ouabain binding. Hypercholesterolemia may exaggerate the changes of the Na+-K+-ATPase activity seen during myocardial ischemia. However, there was some limitation in this study. High cholesterol diet given to New Zealand white rabbits for 8 weeks resulted in a very marked increase in cholesterol (to 57.8 mmol/l). It would be important to acknowledge this in any attempts to extrapolate to human hypercholesterolemia. In summary, we have demonstrated that high dietary cholesterol may result in hypercholesterolemia and lead to a decrease in myocardial Na + -K + -ATPase activity accompanied by a reduction in [‘Hlouabain binding sites. Acute myocardial ischemia induced by coronary artery occlusion results in a further reduction in myocardial Na + -K + -ATPase activity and [‘Hlouabain binding sites in hypercholesterolemic rabbits. These results imply that hypercholesterolemia may exaggerate certain functional deterioration arising during myocardial ischemia. The clinical implication of this study is that the link between hypercholesterolemia and cardiovascular disease is not only in terms of the process of atherosclerosis but also in terms of lipid perturbation of membrane-related functions. These two pathological factors might operate simultaneously and produce synergistic and/or additional effects, which may have an important impact on cardiovascular pathophysiology seen in hypercholesterolemic patients.

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Acknowledgements We thank Ms. Hsiao-Ju Cheng and Ms. Yu-Hong Wu for technical assistance and Ms. Tsai-Hsia Lee for excellent secretarial assistance. This work was supported in part by a grant from the National Science Council of Taiwan (NSC 82-0115-B-002-479). References Ul McMurchie EJ, Patten GS, Chamock JS, McLannan PL. The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart. Biochim Biophys Acta 1987;898:137-153. 121Tsuji K, Tsutsumi S, Ogawa K, Miyazaki Y, Satake T. Cardiac alpha, and beta adrenoreceptors in rabbits: effects of dietary sodium and cholesterol. Cardiovasc Res 1987;21:39-44. [31 McMurchie EJ, Patten GS. Dietary cholesterol influences cardiac B-adrenergic receptor adenylate cyclase activity in the marmoset monkey by changes in membrane cholesterol status. Biochim Biophys Acta 1988;942:324-332. [41 Simons LA. Interactions of lipids and lipoproteins with coronary artery disease mortality in 19 countries. Am J Cardiol 1986;57:5G-106. PI Gotto Jr AM, LaRosa JC, Hunninghake D, et al. The cholesterol facts. A summary of the evidence relating dietary fats, serum cholesterol, and coronary heart disease. Circulation 1990;81:1721-1733. Fl Stason WB. Cost and benefits of risk factor reduction for coronary heart disease: insights from screening and treatment of serum cholesterol. Am Heart J 1990;119:718-724. [71 Ho JL, Pang LC, Taylor CB. Modes of cholesterol accumulation in various tissue of rabbits with prolonged exposure to various serum cholesterol levels. Atherosclerosis 1974;19:561566.

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