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Effect of cardiotoxic concentrations of catecholamines on Na+Ca2+ exchange in cardiac sarcolemmal vesicles
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
40, 206-2 13 (1984)
Effect of Cardiotoxic Concentrations of Catecholamines on Na+-Ca*+ Exchange in Cardiac Sarcolemmal Vesicles’ SAMUEL
MALLOV
Department of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York 13210 Received July 25, 1983, and in revised form October 3, 1983 The mechanism by which the administration of large doses of catecholamines produces myocardial necrosis in experimental animals and humans has not yet been ascertained. One of the consequences of such administration is the accumulation of high concentrations of Ca in the heart and it has therefore been suggested that this is the factor that leads to cell injury. The elevated intracellular Ca appears to be due in part to enhanced Ca influx resulting from catecholamine-induced opening of membrane Ca channels and in part to increased membrane permeability caused by membrane damage. It is not clear, however, why the myocardial cells are unable to extrude their extra load of Ca, at least initially, so as to maintain normal Ca concentrations. The effects of high concentrations of epinephrine and isoproterenol on Na+Ca*+ exchange transport in isolated sarcolemmal vesicles prepared from hearts of SpragueDawley rats were determined. It was found that catecholamine concentrations of 10m4to lo-* M inhibited the exchange in a dose-related manner while choline, in the same concentrations, had no effect. It is therefore possible that cardiotoxic concentrations of catecholamines also interfere with Na+-Ca2+ exchange transport across the myocardial sarcolemma in vivo and thereby inhibit the efflux of intracellular Ca.
INTRODUCTION The administration of large doses of sympathomimetic amines, including catecholamines, to experimental animals (Rona et al., 1959; Ferrans et al., 1964; Rosenblum et al., 1965a, b) or human patients (Szakacs and Cannon, 1958; Winsor and Berger, 1975), or the presence of high levels of endogenous catecholamines in humans (Kline, 196 1; Gupta, 1975) tends to produce cardiac injury leading to myocardial necrosis. The mechanism of the production of this type of injury is not yet clear, although a number of hypotheses have been offered (Stanton and Schwartz, 1967; Fleckenstein, 1971). Thus, it has been reported that administration of a cardiotoxic dose of catecholamine is followed by the increased influx of Ca into and deposition in myocardial cells (Fleckenstein, 197 1; Bloom and Davis, 1972), and it has been suggested that the cardiac damage results from Ca-induced reduction of intracellular ATP concentrations (Fleckenstein, 1971; Fleckenstein et al., 1974). The increased rate of accumulation of plasma Ca in myocardial cells begins shortly after catecholamine administration but continues long after the catecholamine has been cleared from the circulation and appears to be due to plasma membrane damage at the latter time (Mallov, 1983). It is not clear, however, why the initial increased Ca influx, presumably due to the normal physiological action of catecholamines, i.e., the opening of Ca channels in plasma membranes during the plateau phase of the action potential in beating heart, is not handled adequately by the cells so that normal intracellular Ca concentration can be maintained and Ca accumulation not occur. Ca efflux from ’ Supported by a grant from AHA-Upstate
New York Chapter, Inc. 206
0014-4800/84 $3.00 Copyright 63 1984 by Academic Press, Inc. Au rights of reproduction in any form reserved.
Na+-Ca*+
EXCHANGE
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myocardial cells takes place by passive leak, by active extrusion via an ATPase pump, and by a Na+-Ca2+ exchange mechanism (Bar&t, 198 1). We have examined the effect of high concentrations of catecholamines on the latter, using isolated, purified cardiac sarcolemmal vesicles as a test preparation, and have found that the former inhibit Na+-Ca2+ exchange in a dose-related fashion. METHODS Preparation of membrane vesicles. Cardiac sarcolemmal vesicles were prepared from the hearts of male Sprague-Dawley rats, 300-350 g in weight, by the method of Pitts (1979), but scaled down so that each preparation was begun with 2.5 g of ventricular tissue (obtained from three animals) instead of 60 g. After removing the hearts following the induction of anesthesia by ip administration of Na pentobarbital, 40 mg/kg, the ventricles were dissected out, washed in ice-cold homogenizing medium (0.6 M sucrose, 10 mM imidazole/HCl, pH 7.0), transferred to 8 ml of fresh cold homogenizing medium, minced with a fine scissors, and homogenized with a Polytron PT-20 tissue grinder (five 20-second bursts at setting 5). The homogenate was centrifuged at 12,000g for 30 min. The supernatant obtained was diluted with 12 ml of 160 mM KCl, 20 mM MOPS (morpholinopropanesulfonic acid), pH 7.4, (KCl/MOPS), and centrifuged at 96,000g for 60 min. The pellet was resuspended in 2 ml KCl/MOPS and layered over 29 ml of 30% sucrose solution containing 0.3 M KCl, 50 mM sodium pyrophosphate, and 0.1 M Tris-HCl, pH 8.3, and centrifuged at 95,000g for 120 min. The band at the sample-sucrose interface was removed, diluted with 3 vol of KCl/MOPS, and centrifuged at 100,OOOg for 90 min. The surface of the pellet was washed with NaCl/MOPS and the pellet then resuspended in NaCl/MOPS in a final volume of 0.5 ml. Cardiac vesicles prepared in this manner have been shown to be enriched in Na+, K+-ATPase, and Na+-Ca2+ exchange activities and to be only very slightly contaminated with mitochondria or sarcoplasmic reticulum (Pitts, 1979). Measurement of Na+-Ca2+ exchange. Vesicles were first loaded with NaCl by incubating them in 160 mM NaCl/MOPS at 37” for 20 min in a shaking incubator. Each loading mixture contained 40 ~1 of the vesicle preparation, 20 ~1 of NaCl/ MOPS, 5 mg% ascorbic acid, and no catecholamine or a catecholamine (or choline) in a concentration of 10F4 to 10U2 M. The ascorbic acid, added to inhibit the oxidation of catecholamine, did not, in any concentration tested (O-20 mg%), alter the results. After the loading period, 30 ~1 of the loading mixture was added to 470 ~1 of incubation medium at 37°C containing 40 pm 45CaC12, 160 M KCl/ MOPS, 0.6 mg% ascorbic acid, and a catecholamine (or choline) in the same concentration as in the loading mixture, to give a final volume of 500 ~1. The catecholamines were added as the bitartrate salts (Sigma Chemical Co.) and the choline as the chloride salt (MCB Chemicals). Aliquots (50 ~1) of the total incubation mixture were withdrawn after the desired intervals of time (10 set to 4 min), rapidly filtered through Millipore filters (0.45 pm) and washed 3 times with 5 ml of KCYMOPS. The filters were dissolved in 10 ml of Filtron-X (National Diagnostics) and counted in a liquid scintillation counter. Filter blanks were determined at the same time by adding 50 ~1 of the same incubation mixtures minus vesicles to the filters, washing the latter and counting them, in order to correct for any radioactivity on the filters not associated with vesicles. Control incubations were also carried out simultaneously in which 160 mM NaCLIMOPS was substituted for the KClIMOPS in the incubation medium so that there was
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no Na+ gradient from inside to outside the vesicles. A low level of 45Ca2+ uptake was normally observed (5- 10% of that in KCl/MOPS) which was subtracted from the values obtained in KCVMOPS in order to estimate the uptake of 45Ca2+ due to Na+-Ca2+ exchange. Protein determination. Protein contents of the various preparations of vesicles were determined by the method of Lowry et al. (195 1). RESULTS Figure 1 shows a typical curve obtained for uptake of 45Ca by vesicles in the absence of any agent, when the vesicles were incubated in KCl/MOPS and NaCl/ MOPS media. The uptake due to Na+-Ca2+ exchange was rapid, maximum vesicle 45Ca2+ concentrations being reached in 2 min. The initial rate of exchange was determined as the Ca2+ uptake during the first minute of incubation, per milligram vesicle protein. Figure 2 shows the effect of the presence of epinephrine in concentrations of 10V3 to 10T2 M on the uptake in KCl/MOPS. Little change occurred in the uptakes in NaCl/MOPS medium. Appreciable inhibition could be discerned at the concentration of 10e3 M and the degree of inhibition increased with the concentration, reaching about 90% at the concentration of 1Oe2M epinephrine. Figure 3 illustrates the inhibiting effect of different doses of epinephrine in two ways. Isoproterenol exerted similar inhibitory effects on the Na+-Ca2+ exchange (Table I), an initial inhibitory effect occurring at a lower concentration than with epinephrine ( 1O-4 M). When the nitrogenous base, choline, was substituted as a nonspecific control for the catecholamines, no effect on Na+-Ca2+ exchange occurred (Table I). DISCUSSION It has been suggested that excessive influx of Ca into cells is the final common pathway by which a variety of cytotoxic agents cause cell death (Schanne et al., 1979; Farber, 198 1). Overload of myocardial cells with Ca has been implicated as an etiological factor in the cardiac injury and necrosis produced by a number
FIG. 1. Na, Ca exchange measured by ‘%a influx into cardiac sarcolemmal vesicles. Vesicles loaded with NaCl were diluted, 30 to 500, into medium containing 40 p&f 45CaC12 and either 160 mM KC1 or 160 mM NaCI. Aliquots of the incubation mixture were filtered through 0.45 pm Millipore filters, washed, and counted at the indicated times.
Na+-Caz+
EXCHANGE
IN CATECHOLAMINE
2
CARDIOTOXICITY
3
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4
Minutes FIG. 2. Effect of increasing doses of epinephrine on Ca uptake in KC1 medium by cardiac sarcolemmal vesicles previously loaded with Na. Uptake in NaCl medium changed very little with the addition of epinephrine, and is not shown.
of different agents and situations (Alto and Dhalla, 1979; Barzu et al., 1979; Boink et al., 1976; Buja et al., 1979; Holland and Olson, 1975; Kovacs et al., 1972; Shen and Jennings, 1972; Wrzolkowa and Zydowo, 1980) including high concentrations of catecholamines (Bloom and Cincilla, 1969; Fleckenstein, 197 1). Bloom and Cincilla (1969) noticed a very rapid accumulation of deposits of a material which they believed to be Ca salts, in the mitochondria of myocardial cells, after injecting rats ip with a large dose of isoproterenol. After 8 hr, intracellular damage was clearly present. Bloom and Davis (1972, 1974) claimed to be able to distinguish three different rates of increase in cardiac Ca, following isoproterenol administration to rats, and suggested that the first was due to the release of endogenous myocardial norepinephrine, the second to isoproterenolstimulated increased Ca influx into myocardial cells, and the third to increased passive Ca influx resulting from cell necrosis and the breakdown of membrane permeability barriers. After injecting 45Ca2+ iv and infusing epinephrine iv into dogs for 6 hr, Kraikitpanitch et al. (1976) observed the occurrence of elevated cardiac 45Ca concentrations, elevated total cardiac Ca levels, and myocardial cell damage. They ascribed the increased Ca uptake and deposition to the myocardial damage produced. Fleckenstein (1971) and Fleckenstein et al. (1974,1975) reported the occurrence of cell necrosis, Ca deposition, and reduced ATP and creatine phosphate concentrations in the hearts of rats treated with large doses of isoproterenol. All of
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TABLE I Effect of Catecholamines on Na+-Ca *+ Exchange in Cardiac Sarcolemmal Vesicles Vesicle preparation
Agent
Na+-Ca2+ exchange (nmole Ca/mg protein/min)
Change induced by agent (%)
0 1o-3 1o-3 1o-3 10-j 1O-3 1o-2
6.56 6.22 5.24 3.83 2.98 1.63 0.43
-5.2 -20.1 -41.6 -54.6 -15.2 -93.5
0 10-r 1o-3 10-r lo-’ lo-’ 10-r
4.43 4.00 3.12 2.96 1.34 1.12 0.52
-9.1 -16.0 -33.2 -69.8 -74.1 -88.3
0 1o-4 10-3 4 x 10-r 8 x 1O-3 1o-2
5.31 4.90 4.57 3.97 1.66 0.98
-1.1 -13.9 -25.2 -68.7 -81.5
0 1o-3 4 x 1o-3 8 x IO-’ 1O-2
6.36 6.12 6.34 6.61 6.24
-3.8 -0.3 +3.9 -1.9
Concentration of)
Epinephrine 2 4 6 8
x x X x
Epinephrine 2 4 6 8
A
Isoproterenol
Choline
:,
x x x x
o Numbers in parentheses indicate numbers of rats used in each preparation.
these effects were reduced or prevented by the prior administration of Ca channelblocking agents. The authors concluded that increased Ca influx into myocardial cells, produced directly by isoproterenol, led to decreased ATP concentrations as a consequence of the activation of Ca-ATPases and the interference by Ca with mitochondrial ATP synthesis, and that inadequate ATP concentrations then resulted in cell breakdown. Increased Ca influx into beating myocardial cells is a normal consequence of physiological doses of epinephrine and other catecholamines, and is believed to be due to the opening of slow Ca channels in the plasma membrane. However, Ca does not accumulate to the extent that it does after large, cardiotoxic doses, and intracellular Ca concentrations are maintained within the normal range due to compensatory Ca efflux. The latter occurs as the result of the exchange of internal Ca2+ for external Na+ and other mechanisms (Barritt, 198 1). The exchange type of Ca transport is driven by the Na+ gradient from outside to inside the cell. We have tried to determine whether high cardiotoxic concentrations of catecholamines not only increase the rate of Ca influx into myocardial cells but also reduce the rate of Ca efflux from the cells so that unusually high and damaging
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FIG. 3. Dose-related inhibition by epinephrine of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.
intracellular Ca levels are reached. Our examination of the effects of the former on the Na+-Ca*+ exchange system indicates that they do indeed interfere with this type of Ca transport. We have employed the isolated sarcolemmal vesicle as the assay preparation. Such vesicles have been shown to possess an active Na+Ca*+ exchange transport system whose activity can be modified by a number of different parameters (Reeves and Sutko, 1978, 1980; Pitts, 1979; Miyamato and Racker, 1980; Philipson and Nishimoto, 1980). The direction of net Ca*+ flow by such exchange depends on the direction of the Na+ gradient across the membrane. If the inhibition that we have observed in vitro also occurs in vivo, it may promote intracellular Ca deposition and cell damage that eventually leads to myocardial necrosis. The concentration of catecholamine necessary to inhibit the Na+-Ca’+ exchange in vitro appears to be within the same range as that produced by a cardiotoxic dose in vivo. For example, the lowest concentration of isoproterenol found to produce significant inhibition in vitro was 10e4 M. We usually administer a SC dose of 3 mg/kg to a 300-350 g rat to produce myocardial necrosis. Others have employed higher doses. If all of the doses we administered were rapidly absorbed into the circulation and distributed in a plasma volume of 20 ml, the resulting concentration to which the heart would be initially exposed would be 2.3 X 10m4 M. While not all of the isoproterenol is immediately absorbed, the myocardial cells are exposed not only to the exogenous catecholamine but to catecholamines released from the adrenal and sympathetic nerve endings so that the in vivo concentrations may well be large enough to inhibit Na+-Ca*+ exchange. Fleckenstein et al. (1974) have suggested that Ca’+ efflux from Ca overloaded myocardial fibers may decline as a result of Ca*+-induced decreases in ATP concentrations. We believe that high concentrations of catecholamines may also have a more direct inhibiting effect on Ca*+ efflux.
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REFERENCES ALTO, L. E., and DHALLA, N. S. (1979). Defects in microsomal calcium transport due to intracellular calcium overload. J. Mol. Cell. Cardiol. 11 (Suppl. l), 3. BARRIT~, G. J. (1981). Calcium transport across cell membranes: Progress toward molecular mechanisms. Trends Biochem. Sci. 6, 322-325. BARZU, T., DAM, D. T., CUPARENCU, B., DANCEA, S., and SANDBOHB, B. (1979). Ultrastructural and biochemical investigations on the effect of calcium overload in the rat heart. Agressologie 20, 149-154. BLOOM, S., and CINCILLA, P. A. (1969). Myocytolysis and mitochondrial calcification in rat myocardium after low doses of isoproterenol. Amer. J. Pathol. 54, 373-391. BLOOM, S., and DAVIS, D. L. (1972). Calcium as a mediator of isoproterenol-induced myocardial necrosis. Amer. J. Pathol. 69, 459-470. BLOOM, S., and DAVIS, D. L. (1974). Isoproterenol myocytolysis and myocardial calcium. In “Recent Advances in Studies on Cardiac Structure and Metabolism” (N. S. Dhalla, ed.), Vol. 4, pp. 581590. Univ. Park Press, Baltimore, Md. BOINK, A. B. T. J., RUIGROK, T. J. C., MAAS, A. H. J., and ZIMMERMAN, A. N. E. (1976). Changes in high energy phosphate compounds of isolated rat hearts during calcium-free perfusion and reperfusion with calcium. J. Mol. Cell Cardiol. 8, 973-979. BUJA, L. M., HAGLER, H. K., BURTON, K. P., and WILLERSON, J. T. (1979). Analytical electron microscopic study of mitochondrial calcium accumulation in ischemic myocardial injury. J. Mol. Cell. Cardiol. 11 (Suppl. 1), 11. FARBER, J. L. (1981). The role of calcium in cell death. Life Sci. 29, 1289-1295. FERRANS, V. J., HIBBS, R. G., BLACK, W. C., and WEILBAECHER, D. G. (1964). Isoproterenol-induced myocardial necrosis. A histochemical and electron microscopic study. Amer. Heart J. 68, 71-76. FLECKENSTEIN, A. (1971). Specific inhibitors and promoters of calcium action in excitation-contraction coupling of heart muscle and their role in the prevention or production of myocardial lesions. In “Calcium and the Heart” (P. Harris and L. Opie, eds.), pp. 135-188. Academic Press, New York. FLECKENSTEIN, A., JANKE, J., DORING, H. J., and LEDER, 0. (1974). Myocardial fiber necrosis due to intracellular calcium overload-A new principle in cardiac pathophysiology. In “Recent Advances in Studies on Cardiac Structure and Metabolism” (N. S. Dhalla, ed.), Vol. 4, pp. 563-580. Univ. Park Press, Baltimore, Md. GUPTA, K. K. (1975). Pheochromocytoma and myocardial infarction. Lancet 1, 281-282. HOLLAND, C. E., JR., and OLSON, R. E. (1975). Prevention by hypothermia of paradoxical calcium necrosis in cardiac muscle. J. Mol. Cell. Cardiol. 7, 917-928. KLINE, I. K. (1961). Myocardial alterations associated with pheochromocytomas. Amer. J. Clin. Pathol. 38, 539-547.
KOVACS, K., GARDELL, C., and BLASCHEK, J. A. (1972). Ultrastructural studies of various forms of experimentally induced cardiac necrosis. In “Recent Advances in Studies on Cardiac Structure and Metabolism” (E. Bajusz and G. Rona, eds.), Vol. 1, pp. 551-562. Univ. Park Press, Baltimore, Md. KRAIKITPANITCH, S., HAYGOOD, C. C., BAXTER, D. J., YUNICE, A. A., and LINKEMAN, R. D. (1976). Effects of acetylsalicylic acid, dipyridamole and hydrocortisone on epinephrine-induced myocardial injury. Amer. Heart J. 92, 615-622. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (195 1). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MALLOV, S. (1983). Role of calcium and free fatty acids in epinephrine-induced myocardial necrosis. Toxicol. Appl. Pharmacol.. in press. MIYAMOTO, H., and RACKER, E. (1980). Solubilization and partial purification of the CaZ+/Na+ antiporter from the plasma membrane of bovine heart. J. Biol. Chem. 255, 2656-2658. PHILIPSON, K. D., and NISHIMOTO, A. Y. (1980). Na+-Ca*+ exchange is affected by membrane potential in cardiac sarcolemmal vesicles. J. Biof. Chem. 255, 6880-6882. Purrs, B. J. R. (1979). Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. J. Biol. Chem. 254,6232-6235. REEVES, J. P., and SUTKO, J. L. (1978). Sodium-calcium ion exchange in cardiac membrane vesicles. Proc. Nat. Acad. Sci, USA 76, 590-594. REEVES, J. P., and SUTKO, J. L. (1980). Sodium-calcium exchange activity generates a current in cardiac membrane vesicles. Science 208, 146 I- 1464. RONA, G., CHAPPEL, C. I., BALAZS, T., and GAUDRY, R. (1959). An infarct-like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. Arch. Pathol. 67, 443-455.
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ROSENBLUM, I., WOHL, A., and STEIN, A. A. (1965a). Studies in cardiac necrosis. I. Production of cardiac lesions with sympathomimetic amines. Toxicol. Appf. Pharmacol. 7, l-14. ROSENBLUM, I., WOHL, A., and STEIN, A. A. (1965b). Studies in cardiac necrosis. III. Metabolic effects of sympathomimetic amines producing cardiac lesions. Toxicol. Appl. Pharmacol. 7, 344351. SCHANNE, F. A. X., KANE, A. B., YOUNG, E. E., and FARBER, J. L. (1979). Calcium dependence of toxic cell death: A final common pathway. Science 206,700-702. SHEN, A. C., and JENNINGS, R. B. (1972). Myocardial calcium and magnesium in acute ischemic injury. Amer. J. Pathol. 67, 417440. STANTON, H. C., and SCHWARTZ, A. (1967). Effects of a hydrazine monoamine oxidase inhibitor (phenelzine) on isoproterenol-induced myocardiopathies in the rat. J. Phurmacol. Exp. Ther. 157, 649-658.
SZAKACS, J. E., and CANNON, A. (1958). L-Norepinephrine
myocarditis. Amer. J. Clin. Pathol. 30,
425-434.
WINSOR, T., and BERGER, H. J. (1975). Isoproterenol toxicity. Amer. Heart J. 89, 814-817. WRZOLKOWA, T., and ZYDOWO, M. (1980). Ultrastructural studies on the vitamin D induced heart lesions in the rat. J. Mol. Cell. Cardiol. 12, 1117-l 133.
AND
MOLECULAR
PATHOLOGY
40, 206-2 13 (1984)
Effect of Cardiotoxic Concentrations of Catecholamines on Na+-Ca*+ Exchange in Cardiac Sarcolemmal Vesicles’ SAMUEL
MALLOV
Department of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York 13210 Received July 25, 1983, and in revised form October 3, 1983 The mechanism by which the administration of large doses of catecholamines produces myocardial necrosis in experimental animals and humans has not yet been ascertained. One of the consequences of such administration is the accumulation of high concentrations of Ca in the heart and it has therefore been suggested that this is the factor that leads to cell injury. The elevated intracellular Ca appears to be due in part to enhanced Ca influx resulting from catecholamine-induced opening of membrane Ca channels and in part to increased membrane permeability caused by membrane damage. It is not clear, however, why the myocardial cells are unable to extrude their extra load of Ca, at least initially, so as to maintain normal Ca concentrations. The effects of high concentrations of epinephrine and isoproterenol on Na+Ca*+ exchange transport in isolated sarcolemmal vesicles prepared from hearts of SpragueDawley rats were determined. It was found that catecholamine concentrations of 10m4to lo-* M inhibited the exchange in a dose-related manner while choline, in the same concentrations, had no effect. It is therefore possible that cardiotoxic concentrations of catecholamines also interfere with Na+-Ca2+ exchange transport across the myocardial sarcolemma in vivo and thereby inhibit the efflux of intracellular Ca.
INTRODUCTION The administration of large doses of sympathomimetic amines, including catecholamines, to experimental animals (Rona et al., 1959; Ferrans et al., 1964; Rosenblum et al., 1965a, b) or human patients (Szakacs and Cannon, 1958; Winsor and Berger, 1975), or the presence of high levels of endogenous catecholamines in humans (Kline, 196 1; Gupta, 1975) tends to produce cardiac injury leading to myocardial necrosis. The mechanism of the production of this type of injury is not yet clear, although a number of hypotheses have been offered (Stanton and Schwartz, 1967; Fleckenstein, 1971). Thus, it has been reported that administration of a cardiotoxic dose of catecholamine is followed by the increased influx of Ca into and deposition in myocardial cells (Fleckenstein, 197 1; Bloom and Davis, 1972), and it has been suggested that the cardiac damage results from Ca-induced reduction of intracellular ATP concentrations (Fleckenstein, 1971; Fleckenstein et al., 1974). The increased rate of accumulation of plasma Ca in myocardial cells begins shortly after catecholamine administration but continues long after the catecholamine has been cleared from the circulation and appears to be due to plasma membrane damage at the latter time (Mallov, 1983). It is not clear, however, why the initial increased Ca influx, presumably due to the normal physiological action of catecholamines, i.e., the opening of Ca channels in plasma membranes during the plateau phase of the action potential in beating heart, is not handled adequately by the cells so that normal intracellular Ca concentration can be maintained and Ca accumulation not occur. Ca efflux from ’ Supported by a grant from AHA-Upstate
New York Chapter, Inc. 206
0014-4800/84 $3.00 Copyright 63 1984 by Academic Press, Inc. Au rights of reproduction in any form reserved.
Na+-Ca*+
EXCHANGE
IN CATECHOLAMINE
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myocardial cells takes place by passive leak, by active extrusion via an ATPase pump, and by a Na+-Ca2+ exchange mechanism (Bar&t, 198 1). We have examined the effect of high concentrations of catecholamines on the latter, using isolated, purified cardiac sarcolemmal vesicles as a test preparation, and have found that the former inhibit Na+-Ca2+ exchange in a dose-related fashion. METHODS Preparation of membrane vesicles. Cardiac sarcolemmal vesicles were prepared from the hearts of male Sprague-Dawley rats, 300-350 g in weight, by the method of Pitts (1979), but scaled down so that each preparation was begun with 2.5 g of ventricular tissue (obtained from three animals) instead of 60 g. After removing the hearts following the induction of anesthesia by ip administration of Na pentobarbital, 40 mg/kg, the ventricles were dissected out, washed in ice-cold homogenizing medium (0.6 M sucrose, 10 mM imidazole/HCl, pH 7.0), transferred to 8 ml of fresh cold homogenizing medium, minced with a fine scissors, and homogenized with a Polytron PT-20 tissue grinder (five 20-second bursts at setting 5). The homogenate was centrifuged at 12,000g for 30 min. The supernatant obtained was diluted with 12 ml of 160 mM KCl, 20 mM MOPS (morpholinopropanesulfonic acid), pH 7.4, (KCl/MOPS), and centrifuged at 96,000g for 60 min. The pellet was resuspended in 2 ml KCl/MOPS and layered over 29 ml of 30% sucrose solution containing 0.3 M KCl, 50 mM sodium pyrophosphate, and 0.1 M Tris-HCl, pH 8.3, and centrifuged at 95,000g for 120 min. The band at the sample-sucrose interface was removed, diluted with 3 vol of KCl/MOPS, and centrifuged at 100,OOOg for 90 min. The surface of the pellet was washed with NaCl/MOPS and the pellet then resuspended in NaCl/MOPS in a final volume of 0.5 ml. Cardiac vesicles prepared in this manner have been shown to be enriched in Na+, K+-ATPase, and Na+-Ca2+ exchange activities and to be only very slightly contaminated with mitochondria or sarcoplasmic reticulum (Pitts, 1979). Measurement of Na+-Ca2+ exchange. Vesicles were first loaded with NaCl by incubating them in 160 mM NaCl/MOPS at 37” for 20 min in a shaking incubator. Each loading mixture contained 40 ~1 of the vesicle preparation, 20 ~1 of NaCl/ MOPS, 5 mg% ascorbic acid, and no catecholamine or a catecholamine (or choline) in a concentration of 10F4 to 10U2 M. The ascorbic acid, added to inhibit the oxidation of catecholamine, did not, in any concentration tested (O-20 mg%), alter the results. After the loading period, 30 ~1 of the loading mixture was added to 470 ~1 of incubation medium at 37°C containing 40 pm 45CaC12, 160 M KCl/ MOPS, 0.6 mg% ascorbic acid, and a catecholamine (or choline) in the same concentration as in the loading mixture, to give a final volume of 500 ~1. The catecholamines were added as the bitartrate salts (Sigma Chemical Co.) and the choline as the chloride salt (MCB Chemicals). Aliquots (50 ~1) of the total incubation mixture were withdrawn after the desired intervals of time (10 set to 4 min), rapidly filtered through Millipore filters (0.45 pm) and washed 3 times with 5 ml of KCYMOPS. The filters were dissolved in 10 ml of Filtron-X (National Diagnostics) and counted in a liquid scintillation counter. Filter blanks were determined at the same time by adding 50 ~1 of the same incubation mixtures minus vesicles to the filters, washing the latter and counting them, in order to correct for any radioactivity on the filters not associated with vesicles. Control incubations were also carried out simultaneously in which 160 mM NaCLIMOPS was substituted for the KClIMOPS in the incubation medium so that there was
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no Na+ gradient from inside to outside the vesicles. A low level of 45Ca2+ uptake was normally observed (5- 10% of that in KCl/MOPS) which was subtracted from the values obtained in KCVMOPS in order to estimate the uptake of 45Ca2+ due to Na+-Ca2+ exchange. Protein determination. Protein contents of the various preparations of vesicles were determined by the method of Lowry et al. (195 1). RESULTS Figure 1 shows a typical curve obtained for uptake of 45Ca by vesicles in the absence of any agent, when the vesicles were incubated in KCl/MOPS and NaCl/ MOPS media. The uptake due to Na+-Ca2+ exchange was rapid, maximum vesicle 45Ca2+ concentrations being reached in 2 min. The initial rate of exchange was determined as the Ca2+ uptake during the first minute of incubation, per milligram vesicle protein. Figure 2 shows the effect of the presence of epinephrine in concentrations of 10V3 to 10T2 M on the uptake in KCl/MOPS. Little change occurred in the uptakes in NaCl/MOPS medium. Appreciable inhibition could be discerned at the concentration of 10e3 M and the degree of inhibition increased with the concentration, reaching about 90% at the concentration of 1Oe2M epinephrine. Figure 3 illustrates the inhibiting effect of different doses of epinephrine in two ways. Isoproterenol exerted similar inhibitory effects on the Na+-Ca2+ exchange (Table I), an initial inhibitory effect occurring at a lower concentration than with epinephrine ( 1O-4 M). When the nitrogenous base, choline, was substituted as a nonspecific control for the catecholamines, no effect on Na+-Ca2+ exchange occurred (Table I). DISCUSSION It has been suggested that excessive influx of Ca into cells is the final common pathway by which a variety of cytotoxic agents cause cell death (Schanne et al., 1979; Farber, 198 1). Overload of myocardial cells with Ca has been implicated as an etiological factor in the cardiac injury and necrosis produced by a number
FIG. 1. Na, Ca exchange measured by ‘%a influx into cardiac sarcolemmal vesicles. Vesicles loaded with NaCl were diluted, 30 to 500, into medium containing 40 p&f 45CaC12 and either 160 mM KC1 or 160 mM NaCI. Aliquots of the incubation mixture were filtered through 0.45 pm Millipore filters, washed, and counted at the indicated times.
Na+-Caz+
EXCHANGE
IN CATECHOLAMINE
2
CARDIOTOXICITY
3
209
4
Minutes FIG. 2. Effect of increasing doses of epinephrine on Ca uptake in KC1 medium by cardiac sarcolemmal vesicles previously loaded with Na. Uptake in NaCl medium changed very little with the addition of epinephrine, and is not shown.
of different agents and situations (Alto and Dhalla, 1979; Barzu et al., 1979; Boink et al., 1976; Buja et al., 1979; Holland and Olson, 1975; Kovacs et al., 1972; Shen and Jennings, 1972; Wrzolkowa and Zydowo, 1980) including high concentrations of catecholamines (Bloom and Cincilla, 1969; Fleckenstein, 197 1). Bloom and Cincilla (1969) noticed a very rapid accumulation of deposits of a material which they believed to be Ca salts, in the mitochondria of myocardial cells, after injecting rats ip with a large dose of isoproterenol. After 8 hr, intracellular damage was clearly present. Bloom and Davis (1972, 1974) claimed to be able to distinguish three different rates of increase in cardiac Ca, following isoproterenol administration to rats, and suggested that the first was due to the release of endogenous myocardial norepinephrine, the second to isoproterenolstimulated increased Ca influx into myocardial cells, and the third to increased passive Ca influx resulting from cell necrosis and the breakdown of membrane permeability barriers. After injecting 45Ca2+ iv and infusing epinephrine iv into dogs for 6 hr, Kraikitpanitch et al. (1976) observed the occurrence of elevated cardiac 45Ca concentrations, elevated total cardiac Ca levels, and myocardial cell damage. They ascribed the increased Ca uptake and deposition to the myocardial damage produced. Fleckenstein (1971) and Fleckenstein et al. (1974,1975) reported the occurrence of cell necrosis, Ca deposition, and reduced ATP and creatine phosphate concentrations in the hearts of rats treated with large doses of isoproterenol. All of
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TABLE I Effect of Catecholamines on Na+-Ca *+ Exchange in Cardiac Sarcolemmal Vesicles Vesicle preparation
Agent
Na+-Ca2+ exchange (nmole Ca/mg protein/min)
Change induced by agent (%)
0 1o-3 1o-3 1o-3 10-j 1O-3 1o-2
6.56 6.22 5.24 3.83 2.98 1.63 0.43
-5.2 -20.1 -41.6 -54.6 -15.2 -93.5
0 10-r 1o-3 10-r lo-’ lo-’ 10-r
4.43 4.00 3.12 2.96 1.34 1.12 0.52
-9.1 -16.0 -33.2 -69.8 -74.1 -88.3
0 1o-4 10-3 4 x 10-r 8 x 1O-3 1o-2
5.31 4.90 4.57 3.97 1.66 0.98
-1.1 -13.9 -25.2 -68.7 -81.5
0 1o-3 4 x 1o-3 8 x IO-’ 1O-2
6.36 6.12 6.34 6.61 6.24
-3.8 -0.3 +3.9 -1.9
Concentration of)
Epinephrine 2 4 6 8
x x X x
Epinephrine 2 4 6 8
A
Isoproterenol
Choline
:,
x x x x
o Numbers in parentheses indicate numbers of rats used in each preparation.
these effects were reduced or prevented by the prior administration of Ca channelblocking agents. The authors concluded that increased Ca influx into myocardial cells, produced directly by isoproterenol, led to decreased ATP concentrations as a consequence of the activation of Ca-ATPases and the interference by Ca with mitochondrial ATP synthesis, and that inadequate ATP concentrations then resulted in cell breakdown. Increased Ca influx into beating myocardial cells is a normal consequence of physiological doses of epinephrine and other catecholamines, and is believed to be due to the opening of slow Ca channels in the plasma membrane. However, Ca does not accumulate to the extent that it does after large, cardiotoxic doses, and intracellular Ca concentrations are maintained within the normal range due to compensatory Ca efflux. The latter occurs as the result of the exchange of internal Ca2+ for external Na+ and other mechanisms (Barritt, 198 1). The exchange type of Ca transport is driven by the Na+ gradient from outside to inside the cell. We have tried to determine whether high cardiotoxic concentrations of catecholamines not only increase the rate of Ca influx into myocardial cells but also reduce the rate of Ca efflux from the cells so that unusually high and damaging
Na*-Caz+
EXCHANGE
IN CATECHOLAMINE
CARDIOTOXICITY
211
FIG. 3. Dose-related inhibition by epinephrine of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.
intracellular Ca levels are reached. Our examination of the effects of the former on the Na+-Ca*+ exchange system indicates that they do indeed interfere with this type of Ca transport. We have employed the isolated sarcolemmal vesicle as the assay preparation. Such vesicles have been shown to possess an active Na+Ca*+ exchange transport system whose activity can be modified by a number of different parameters (Reeves and Sutko, 1978, 1980; Pitts, 1979; Miyamato and Racker, 1980; Philipson and Nishimoto, 1980). The direction of net Ca*+ flow by such exchange depends on the direction of the Na+ gradient across the membrane. If the inhibition that we have observed in vitro also occurs in vivo, it may promote intracellular Ca deposition and cell damage that eventually leads to myocardial necrosis. The concentration of catecholamine necessary to inhibit the Na+-Ca’+ exchange in vitro appears to be within the same range as that produced by a cardiotoxic dose in vivo. For example, the lowest concentration of isoproterenol found to produce significant inhibition in vitro was 10e4 M. We usually administer a SC dose of 3 mg/kg to a 300-350 g rat to produce myocardial necrosis. Others have employed higher doses. If all of the doses we administered were rapidly absorbed into the circulation and distributed in a plasma volume of 20 ml, the resulting concentration to which the heart would be initially exposed would be 2.3 X 10m4 M. While not all of the isoproterenol is immediately absorbed, the myocardial cells are exposed not only to the exogenous catecholamine but to catecholamines released from the adrenal and sympathetic nerve endings so that the in vivo concentrations may well be large enough to inhibit Na+-Ca*+ exchange. Fleckenstein et al. (1974) have suggested that Ca’+ efflux from Ca overloaded myocardial fibers may decline as a result of Ca*+-induced decreases in ATP concentrations. We believe that high concentrations of catecholamines may also have a more direct inhibiting effect on Ca*+ efflux.
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