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Sarcolemmal ATPase activities of the rat heart ventricle—dependence on age and sodium ion
Comp. Biochem. Physiol. Vol. 8611,No. 4, pp. 815-820, 1987 Printed in Great Britain
0305-0491/87 $3.00+0.00 PergamonJournals Ltd
SARCOLEMMAL ATPase ACTIVITIES OF THE RAT HEART V E N T R I C L E - - D E P E N D E N C E ON AGE A N D SODIUM ION P.~IVI K A R ~ N and MATTI VORNANENt *Laboratory of Animal Physiology, Department of Biology, University of Turku, SF-20500 Turku 50, and ~'Department of Biology, University of Joensuu, P.O. Box 11l, SF-80101 Joensuu 10, Finland (Recewed 16 June 1986)
Abstract--1. Na-K-ATPase activity of the rat heart was similar throughout the postnatal growth when measured from crude unpurified fraction. 2. Instead in the cardiac sarcolemmal fraction, isolated by hypotonic shock LiBr-treatment method, the activity was over two times higher in 10-day old neonates than in adult rats. 3. The conflicting results are partly explained by different effects of the isolation procedure on neonatal and adult tissues. 4. Na concentration for half-maximal activity of the Na-K-ATPase was similar in neonates (7.0 mM) and adults (6.4 raM). 5. Ca-ATPase activity was not affected by Na concentration (2-100 mM) in the two age-groups studied.
INTRODUCTION Contraction of the cardiac cell is triggered by a rapid rise of intracellular Ca concentration i.e. Ca-transient (Allen and Kurihara, 1980) and the intensity of the contractile state is proportional to its magnitude. Sarcolemma plays a crucial role in the regulation of myocardial contractility by controlling the exchange of ions between extracellular fluid and cytosol of the cardiac cell (Chapman, 1979; Langer et al., 1982). Along with voltage gated ionic channels sarcolemmal ATPases are intimately involved in the rapid changes of myoplasmic ion concentrations. Ca-activated Mgdependent ATPase (Ca-Mg-ATPase) is directly involved in lowering myoplasmic Ca concentration by extruding Ca through the sarcolemma (Caroni and Carafoli, 1980). Na-K-ATPase participates in the active transport of Na and K maintaining relatively low Na-level within the cell (Skou, 1965). Furthermore Na-K-ATPase is indirectly associated with the regulation of myoplasmic Ca, since intracellular Na and Ca concentrations are coupled by the Na~Caexchange mechanism (Reuter and Seitz, 1968). The presence of Ca- or Mg-activated ATPase (Ca/MgATPase) in the heart sarcolemma has been also well established by biochemical (Sulakhe and Dhalla, 1971, DhaUa et al., 1981) as well as histochemical methods (Malouf and Meissner, 1980). This ATPase is suggested to be involved in the regulation of Ca influx perhaps as a part of the gating mechanism of the slow inward calcium channel (Dhalla et aL, 1982, Vrbjar et aL, 1985). Although sarcolemmal ATPases of the adult mammals have been the object of intense investigation there are only a few studies concerning the activities and kinetic properties of cardiac sarcolemmal ATPases from maturing animals (Nagatomo and Sasaki 1981; Khatter, 1985). The aim of the present study was to compare the activities of sarcolemmal
ATPases from neonatal and adult rat heart. Especially, we tried to measure Na-K-ATPase activity and to determine its dependence on Na ion concentration. Rat is particularly interesting in this respect, because in this species the contractile properties of the cardiac tissue change markedly during the first weeks of postnatal life (Hopkins et al., 1973; Vornanen, 1984, 1985). MATERIALS AND METHODS
Isolation of sarcolemma Male and female Wistar rats between 1 day and 6 months of age were used. Animals were bred on a 12 hr dark, 12 hr light cycle and fed ad libitum with normal animal chow. Young rats were decapitated with scissors and adult animals were killed by cervical dislocation after slight ether anesthesia. Ventricular muscle was excised and homogenized in ice-cold buffer (10mM Tris-HC1, 1 mMEDTA, pH7.4). Several hearts were pooled from younger animals but only one heart was needed in the case of adults. Sarcolemmal fractions were isolated from 10-day old neonates and adult rats by the hypotonic shock-LiBr treatment method as described by Panagia et al. (1982). This method was chosen for its high yield, enabling extraction of purified sarcolemmal fraction from the small tissues of neonatal animals (Dhalla and Pierce 1984), Na-K-ATPase activity was also determined at the beginning of the purification procedure, i.e. in the pellet of first centrifugation (1000g for 10 min), which was suspended in 1 mM Tris-HCl (pH 7.0) for ATPase determinations.
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Treatment of sarcolemma with deoxycholate In order to expose latent Na-K-ATPase activity sarcolemmal membranes were treated with deoxycholate. Five to eight ml of sarcolemmal suspension was diluted with the same volume of 0.2% Na-deoxycholate in 1 mM Tris-HC1 (pH 7.0) and incubated for 20 min in an ice-water bath with gentle mixing. Deoxycholate to protein ratio varied between 0.1-0.2. The sarcolemmal fraction was recollected by centrifugation at 5000g for 5 min. The pellet was suspended in l mM Tris-HC1 (pH 7.0) for enzyme assays.
P.~IVI KARTrUNEN and MATTIVORNANEN
816 A TPase determinations
Three different ATP hydrolysing activities were measured. Na-K-ATPase activity was determined as the difference of total ATPase obtained in the presence of 50 mM Tris-HCl (pH 7.5), 4 mM Tris-ATP, 4 mM MgCI2, 20 mM KCI and 100 mM NaCI and Mg-ATPase measured in the presence of 50mM Tris-HC1 (pH7.5), 4 m M Tris-ATP, and 4 mM MgC12. Alternatively Na-K-ATPase was also determined as the portion of total ATPase which was inhibited by I mM ouabain. Mg/Ca-ATPase activity was estimated in a reaction mixture containing 50 mM Tris-HC1 (pH 7.5), 4 mM Tris-ATP and 4 mM MgC12 or CaCI 2. ATPase hydrolysis occurring in the absence of activating cations was subtracted from these ATPase activities to obtain the final values. Myofibrillar myosin ATPase was measured in 450 mM KC1, 45 mM imidazole (pH 7.5), 5 mM Na2-ATP, 10 mM NaN 3 and 10 mM EDTA (K-EDTA-ATPase; Martin et al., 1982). The assays were performed at 37°C in a total volume of 1.5 ml and in the presence of 20-300#g protein. The reaction was started after 5 min preincubation by ATP addition. After 10 min incubation the inorganic phosphate liberated by the different ATPases was measured in the clear supernatant by the method of Atkinson et al. (1973). The protein content was assayed by the procedure of Lowry et al. (1951) using fatty acid free bovine serum albumin as the standard protein. Electron microscopy
Pellets of membranes were fixed in 1.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.0) overnight at 4°C. They were washed with 0.1 M phosphate buffer 8-10 times and postfixed in 1% OsO4 for 2.5 hr at 4°C. After dehydration in a graded ethanol series (30-100%) the sample was embedded in Epon. Thin sections were cut and stained with uranylacetate and lead citrate (LKM-ultrastainer) and examined with JEM 100 CX electron microscope. Statistics
Mean values among several groups were compared by one-way analysis of variance (ANOVA) and significant differences between individual groups were tested after ANOVA by Scheffe's multiple test. Student's t-test for independent and paired samples was used to compare mean values of two groups or two treatments, respectively. RESULTS The high yield by the h y p o t o n i c s h o c k - L i B r treatm e n t m e t h o d , requiring relatively small a m o u n t s of original tissue, allowed purification of sarcolemmal m e m b r a n e s from cardiac tissues o f b o t h n e o n a t a l (10-day old) a n d adult rats. Electron microscopy showed t h a t in b o t h age groups the isolated fraction consisted of m e m b r a n e vesicles or sacs of variable size. T h e vesicles were coated in several places by fuzzy glycocalyx, thus showing their sarcolemmal
Table 1. Effect of Na-azide and Na-orthovanadate on sarcolemmal Ca-ATPase and Mg-ATPase activities Ca-ATPase Mg-ATPase Additions A N A N Na-azide 5mM -26.8 -21.5 -55.8 -55.0 Vanadate 2#M +1.4 +1.6 -3.0 -5.0 200/~M -16.7 -11.0 -9.7 -6.8 The values are % change of the ATPase activities (+ = stimulation, - = inhibition) of adult (A) and neonatal (N = 10-days old)
sarcolemmal preparation. The results are means of 2-5 preparations.
origin (Fig. 1). M i t o c h o n d r i a l c o n t a m i n a t i o n was n o t observed b u t some r e m n a n t s of myofibrils were occasionally found, suggesting t h a t 0.6 M LiBr is unable to extract myofibrillar protein totally. Sodium azide (5 m M ) inhibited 2 1 ~ 7 % of the C a - A T P a s e a n d a b o u t 55% o f the M g - A T P a s e activity o f the present sarcolemmal fraction (Table 1). Azide is generally regarded as an inhibitor of mitoc h o n d r i a l ATPase, thereby suggesting a significant c o n t a m i n a t i o n o f o u r p r e p a r a t i o n by mitochondria. However, even in highly purified sarcolemmal memb r a n e preparations, M g - A T P a s e activity is f o u n d to be 50% sensitive to azide (Jones et al., 1980). It seems t h a t azide sensitive A T P a s e exists in addition to m i t o c h o n d r i a also in the sarcolemma of muscle cells ( K w a n a n d R a m l a l 1982; D h a l l a a n d Pierce, 1984, Turi a n d T b r f k , 1985). In any case the azide sensitive p o r t i o n o f the C a / M g - A T P a s e activity was similar in b o t h age groups studied. N a - o r t h o v a n a d a t e , a p o t e n t inhibitor of the CaM g - A T P a s e of the sarcoplasmic reticulum, exerted slight a n d equal effect o n the basic C a / M g - A T P a s e activity o f the two p r e p a r a t i o n s (Table 1). In accordance with the electron microscopic findings some myofibrillar c o n t a m i n a t i o n as myosin A T P a s e activity was f o u n d in the sarcolemmal fraction. However K - E D T A - A T P a s e activities were similar in b o t h preparations, showing equal myofibrillar c o n t a m i n a t i o n in the two age groups. W h e n N a - K - A T P a s e activity was m e a s u r e d at the beginning o f the purification procedure, i.e. in the pellet of the first centrifugation, n o significant differences existed between the four age groups studied. T h e activities were similar from n e w b o r n s (3-4 day old) to y o u n g adults (5-6 m o n t h old). Instead in the final sarcolemmal fraction N a - K - A T P a s e activity of the 10-day old rats was over twice t h a t of the adult animals (Table 2). T h u s the difference a p p e a r e d in the course of the isolation procedure. Because the purity of the fractions seemed to be similar in the two age groups, o t h e r causes for the conflicting results were sought. A possibility existed t h a t all of the
Table 2. Age-dependence of sarcolemmal Na-K-ATPase activity in initial crude fraction and final sarcolemmal preparation Na-K-ATPase activity Age of animal Crude fraction N Sarcolemmalfraction N Enrichment 3 Days 4.8 ~ 0.32 6 --10 Days 3.8 -T-0.49 7 13.4 -T-2.50 9 3.6 15 Days 4.4 -T-0.30 6 --Adult 3.5 -T-0.42 7 6.2 -T-0.43* 18 1.7 Na-K-ATPase activity was measured as ouabain (1 raM) inhibitable portion of the total ATPase activity. Crude fraction is the pellet from first eentrifugation of the homogenate, suspended in l mM Tris-HCl (pH7.0). Results are means+S.E.M, of 6--18 experiments as indicated. *Significantly (p < 0.05) different from the value of the 10-day old group.
Heart sarcolemmal ATPase
Fig. 1. Electron micrographs of sarcolemmal fractions from adult (a) and lO-day old (b) rats. Scale bar 1 #m.
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P.~IVI KARTTUNEN and MATTI VORNANEN
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adult rat heart ventricles. Km-valuesare 7.0 and 6.4 mM for neonatal and adult animals, respectively. The results are means of 9-27 separate experiments and the lines were fitted by linear regression.
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Fig. 2. Sarcolemmal ATPase activities of adult (I-9) and 10-day old (r~) rat heart ventricles before (control) and after deoxycholate (doc) treatment. Na-K-ATPase activity was measured as ouabain inhibitable portion of total ATPase (O) and as the difference between total and Mg-ATPases (Mg). One S.E.M and number of preparations are shown above each bar. Asterisks (*p < 0.05, **p < 0.01) indicate significant differences between two means ( N S = n o t significant).
Na-K-ATPase activity was not expressed and therefore it was attempted to reveal latent Na-K-ATPase activity by deoxycholate treatment. Deoxycholate increased the Na-K-ATPase activity of the adult rat heart but slightly depressed it in the neonates. So the difference between the age-groups was no more statistically significant if expressed as an oubain inhibitable portion of the total ATPase but still reached a statistically significant level when measured against Mg-ATPase activity (Fig. 2). The sarcolemmal preparations of the two age groups showed differing behaviour in relation to ouabain. In the adult the Na-K-ATPase activity was higher when measured as a ouabain sensitive component of the total ATPase than if determined as the difference between total ATPase and Mg-ATPase. In the neonates the situation was reversed. The dependence of the Na-K-ATPase activity on Na concentration (2-200 mM) is shown in Fig. 3. In this regard the enzymes of the two age-groups were similar. Half maximal activity of the Na-K-ATPase was reached at 6.4 and 7.0 mM NaCI for neonatal and adult rats, respectively. V~x was, as expected on the basis of their specific activities, far higher in the
neonates (16.6pmol Pi/mg prot./hr) than in the adults (5.6/~mol Pi/mg prot./hr). Sarcolemmal Mg-ATPase and Ca-ATPase activities were much higher in neonates than in adults (Fig. 2). However, activity ratios Mg-ATPase/Na-KATPase and Ca-ATPase/Na-K-ATPase were similar, being 4.2 and 5.3 for neonatal and 4.1. and 4.0 for adult rats, respectively. This suggests that Mg/Ca-ATPase is not a contaminant from other cellular organelles but copurifies with Na-K-ATPase. Deoxycholate decreased Mg- and Ca-ATPase activities significantly in the neonates but did not markedly affect on those of the adults (Fig. 2). Sarcolemmal Ca-ATPase activity was not dependent on sodium concentration in the range 2-100mM (Fig. 4). DISCUSSION
Among the cardiac tissues of common laboratory mammals the adult rat heart is exceptional in several respects. In normal physiological solution the rat
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No Fig. 4. Effect of N a concentration on the sarcolemmal C a - A T P a s e activity of neonatal (N = 5) and adult (N = 7) rat heart ventricles. S.E.M. were smaller than the size of symbols.
Heart sarcolemmal ATPas¢ heart ventricle functions as if its cellular Ca stores were maximally loaded (Forester and Mainwood, 1974). Instead the neonatal cardiac tissue, up to the age of 2 weeks, behaves like that of other adult mammals, showing no signs of Ca overload (Vornanen, 1984). The cellular basis for this difference between adult and maturing tissues is not clear. Sarcolemmal Na-K-ATPase is one possible candidate, for it regulates the level of cytosolic Ca in addition to intracellular Na and K concentrations. This is because concentration gradients of Na and Ca are coupled through the Na-Ca exchange mechanism (Reuter and Seitz, 1968). The relation between intracellular Na and twitch tension is steep with 1 mM increase in Na concentration producing about a 100% increase in the twitch magnitude (Wasserstrom et aL, 1983). Therefore activity and kinetics of the sarcolemmal Na-K-ATPase are important determinants in the regulation of cardiac contractility. Sarcolemmal Na-K-ATPase activity of the adult rat heart was less than half from that of the neonates. The difference was not, however, seen at the beginning of the purification procedure. The total protein content of 10-day old and adult rat hearts were 71 and 76 pg/mg tissue wet wt respectively, the activities of crude unpurified fraction thus being comparable on the basis of total protein. Therefore a question arises, whether the final difference in Na-K-ATPase activity is real or is it an artefact created by the isolation procedure. First of all, the difference between the age-groups could be due to variable degree of contamination of the sarcolemmal fraction by other cellular structures. This is not evident on the basis of K-EDTA-ATPase activity or by azide and vanadate sensitivity of the basic ATPases nor by morphological criteria of the membrane vesicles. The preparations may also differ in regard to the proportion of sealed vesicles and their sidedness (inside-out/outside-out). Latent Na-K-ATPase activity was revealed by deoxycholate treatment in the sarcolemmal fraction of the adult but not in the preparation of the neonatal rats, suggesting that a greater number of the vesicles were sealed in the former group. Higher susceptility of the Ca/Mg-ATPase of the neonatal rat heart to deoxycholate treatment further supports the notion that in the neonates the membrane faces are more directly exposed to surrounding medium. The sensitivity of the Mg-ATPase of the adult rat heart to I mM ouabain also suggests that some portion of the Na-K-ATPase activity is latent in the membrane vesicles of this age-group. Thus in some degree the difference in sarcolemmal Na-K-ATPase activity may be explained on the basis of variable effect of the hypotonic shock LiBr-treatment method on adult and neonatal tissues. On the other hand the low Na-K-ATPase activity of the adult rat heart can be partly explained also on morphological grounds. Approximately one-third of the sarcolemma in the adult rat heart is located at the level of T-tubules (Page et al., 1974), which may be almost totally devoid of Na-K-ATPase activity (Almers and Stirling, 1984). In 10-day-old neonates T-tubules are just beginning to develop (Schiebler and Wolff, 1966; Hirakow and Gotoh, 1975). Thus
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although the density of Na-K-ATPase molecules were similar in the peripheral sarcolemma of the myocyte the total Na-K-ATPase activity per sarcolemmal protein would be lower in the adults due to the contribution of T-tubular protein. Because surface-to-volume ratio of the cardiac cells decreases during the postnatal life of the rat (Page et al., 1974; Olivetti et al., 1980), an equal pump density in the peripheral sarcolemma would mean a lower Na-K-ATPase activity per unit cell volume in the adult rat heart. Thus our results, that sarcolemmal Na-K-ATPase activity is higher in the neonates than in the adults, is in agreement with the finding of Awad and Clay (1982), who found higher Na-K-ATPase activity in young (1-1.5 months) than in senescent (10-12 months) rats. Similar results have also been reported for other mammals including dog and guinea-pig (Miller and Gilliland, 1972; Marsh et al., 1981; Khatter, 1985) suggesting that this is a general trend among mammals. However, in the rabbit Na-K-ATPase activity is higher in adults than in newborn (0-3 days) animals (Nagatomo and Sasaki 1981). Care must, however, be taken when interpreting the results because isolation procedure can have profoundly different effects on neonatal and adult tissues. The dependence of the Na-K-ATPase activity on Na was similar in the two age-groups studied. The half-maximal activity was reached at about 7 mM Na, which is close to the values of 8.95 and 8.47 measured for adult (3 month old) and senescent (26 month old) rat heart, respectively (Katano et al., 1985). Because intracellular Na activity is in the range of 6-9 mM in the adult heart (Sheu and Fozzard, 1982; Grupp et al., 1985), the enzyme seems to be half-maximally activated in vivo, and thus possesses ample reserve capacity. Ca-ATPase activity was not dependent on Na concentration. This is in accordance with the findings of Tuana and Dhalla (1982) with the sarcolemmal preparation of the adult rat heart but contrary to those of McNamara et al. (1974) on dog heart. Thus, our findings do not support the proposal that this enzyme is the site of Na-Ca antagonism that regulates cardiac contractility at sarcolemmal level (Tuana and Dhalla, 1982). In conclusion, our results suggest that the Na-K-ATPase activity of the neonatal rat heart ventricles may be somewhat higher than that of the adults, whereas Na-kinetics of the enzyme is independent of age. Therefore in the myocytes of young rat heart the reserve capacity of the sodium pump may be higher than in the adults, which could result in different type of contractile behaviour in situations where cellular Na load or Na-pump activity are altered. REFERENCES
Allen D. G. and Kurihara S. (1980) Calcium transients in mammalian ventricular muscle. Eur. Heart J. 1 (Supplement A), 5-15. Almers W. and Stirling C. (1984) Distribution of transport proteins over animal cell membranes. J. Memb. Biol. 77, 169-186.
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Atkinson A. Gatenby A. D. and Lowe A. G. (1973) The determination of inorganic orthophosphate in biological systems. Biochem. Biophys. Acta 320, 195--204. Awad B. A. and Clay S. W. (1982) Age-dependent alterations in lipids and function of rat heart sarcolemma. Mech. Ageing Devl. 19, 333-342. Caroni P. and Carafoli E. (1980) An ATP-dependent Capumping system in dog heart sarcolemma. Nature 283, 765-767. Chapman R. A. (1979) Excitation-contraction coupling in cardiac muscle. Prog. Biophys. molec. Biol. 35, 1-52. Dhalla N. S. and Pierce G. N. (1984) Isolation and characterization of the sarcolemmal membrane from the heart. In Methods in Studying Cardiac Membranes, Volume I. (Edited by Dhalla N. S.) pp. 3-18. CRC Press, Florida. Dhalla N. S., Anand-Srivastava M. B., Tuana B. S. and Khandelwal R. L. (1981) Solubilization of a calcium dependent adenosine triphosphatase from rat heart sarcolemma. J. molec. Cell. Cardiol. 13, 418-423. Dhalla N. S., Pierce G. N., Panagia V., Singal P. K. and Beamish R. E. (1982) Calcium movements in relation to heart function. Bask Res. Cardiol. 77, 117-139. Forester G. V. and Mainwood G. W. (1974) Interval dependent inotropic effects in the rat myocardium and the effect of calcium. Pflfiegers Arch. ges. PhysioL 352, 189-196. Grupp J., Wook-Bin J., Lee C. O., Lee S-W. Pecker M. S. and Schwarz A. (1985) Relation of sodium pump inhibition to positive inotropy at low concentrations of ouabain in rat heart muscle. J. Physiol. 360, 149-160. Hirakow R. and Gotoh T. (1975) A quantitative ultrastructural study on the developing rat heart. In Developmental and Physiological Correlates of Cardiac Muscle (Edited by Lieberman M. and Sano T.), pp. 37-48. Raven Press, New York. Hopkins S. F., McCutcheon E. P. and Wekstein D. R. (1973) Postnatal changes in rat ventricular function. Circ. Res. 32, 685-691. Jones L. R., Maddock S. W. and Besch H. R. Jr. (1980) Unmasking effect of alamethicin on the (Na+,K+)ATPase, fl-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles. J. bioL Chem. 255, 9971-9980. Katano Y., Kennedy R. H., Stemmer P. M., Temma K. and Akera T. (1985) Ageing and digitalis sensitivity of cardiac muscle in rats. Eur. J. Pharmacol 113, 167-178. Khatter J. C. (1985) Mechanism of age-related differences in the cardiotoxic action of digitalis. J. Cardiovasc. Pharmacol. 7, 258-261. Kwan C. Y. and Ramlal T. (1982) On the inhibition of smooth muscle membrane ATPase by sodium azide. Biochem. Int. 4, 439-449. Langer G. A., Frank J. S. and Philipson K. D. (1982) Ultrastructure and calcium exchange of the sarcolemma, sarcoplasmic reticulum and mitochondria of the myocardium. Pharmac. Ther. 16, 331-376. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-275. Malouf N. N. and Meissner G. (1980) Cytochemical localization of a "basic" ATPase to canine myocardial surface membrane. J. Histochem. Cytochem. 12, 1286-1294. Marsh A. J., Lloyd B. L. and Taylor R. R. (1981) Age dependence of myocardial Na+-K+-ATPase activity and
digitalis intoxication in the dog and guinea pig. Circ. Res. 48, 329-333. Martin A. F., Pagani E. D. and Solaro R. J. (1982) Thyroxine-induced redistribution of isoenzymes of rabbit ventricular myosin. Circ. Res. 50, 117-124. McNamara D. B., Singh J. N. and Dhalla N. S. (1974) Properties of some heart sarcolemmal bound enzymes. J. Biochem. 76, 603-609. Miller W. W. and Gilliland K. (1972) Developmental differences in canine myocardial adenosine triphosphatase activity (Abstr.). Circulation 45 (Suppl. II), 36. Nagatomo T. and Sasaki M. (1981) (Na+-K+)-ATPase activity of newborn rabbit cardiac muscle. Biochem. Med. 26, 286-293. Olivetti G., Anversa P. and Loud A. V. (1980) Morphometric study of early postnatal development in the left and right ventricular myocardium of the rat. Circ. Res. 46, 503-512. Page E., Earley J. and Power B. (1974) Normal growth of ultrastructures in rat left ventricular myocardial cells. Circ. Res. 34/35 (Supplement II), 12-16. Panagia V., Lamers J. M. J., Singal P. K. and Dhalla N. S. (1982) Ca 2+- and Mg2+-dependent ATPase activities in the deoxycholate-treated rat heart sarcolemma. Int. J. Bioehem. 14, 387-397. Reuter H. and Seitz N. (1968) The dependence of calcium etttux from cardiac muscle on temperature and external ion composition. J. Physiol. (Lond.) 195, 451-470. Schiebler T. H. and Wolff H. H. (1966) Elektronenmikroskopische Untersuchungen am Herzmuskel der Ratte wahrend der Entwicklung. Z. Zellforsch. mikrosk. Anat. 69, 22-44. Sheu S.-S. and Fozzard H. A. (1982) Transmembrane Na + and Ca 2+ electrochemical gradients in cardiac muscle and their relationship to force development. J. gen. Physiol. 80, 325-351. Skou J. C. (1965) Enzymatic basis for active transport of Na + and K + across cell membrane. Physiol. Rev. 45, 596-617. Sulakhe P. V. and Dhalla N. S. (1971) Excitationcontraction coupling in heart. VI, Demonstration of calcium activated ATPase in the dog heart sarcolemma. Life Sci. 10, 185-191. Tuana B. S. and Dhalla N. S. (1982) Purification and characterization of a Ca2+-dependent ATPase from rat heart sarcolemma. J. biol. Chem. 257, 14440-14445. Turi A. and T6r6k K. (1985) Myometrial (Na+-K+)-ac tivated ATPase and its Ca 2+ sensitivity. Biochim. Biophys. Acta 818, 123-131. Vornanen M. (1984) Activation of contractility and sarcolemmal Ca2+-ATPase by Ca 2+ during postnatal development of the rat heart. Comp. Biochem. Physiol. 78A, 691-695. Vornanen (1985) Effect of strontium on the contractile properties of postnatally developing rat heart ventricles. Can. J. Physiol. Pharmacol. 63, 9-17. Vrbjar N., Ziegelh6ffer A., Breier A., Soos J., Dzuba A. and Monosikova R. (1985) Quantitative relationship between the protein secondary structure in cardiac sarcolemma and the activity of the membrane-bound Ca2+-ATPase. Gen. Physiol. Biophys. 4, 411-416. Wasserstrom J. A., Schwartz D. J. and Fozzard H. J. (1983) Relation between intracellular sodium and twitch tension in sheep cardiac Purkinje strands exposed to cardiac glycosides. Circ. Res. 52, 697-705.
0305-0491/87 $3.00+0.00 PergamonJournals Ltd
SARCOLEMMAL ATPase ACTIVITIES OF THE RAT HEART V E N T R I C L E - - D E P E N D E N C E ON AGE A N D SODIUM ION P.~IVI K A R ~ N and MATTI VORNANENt *Laboratory of Animal Physiology, Department of Biology, University of Turku, SF-20500 Turku 50, and ~'Department of Biology, University of Joensuu, P.O. Box 11l, SF-80101 Joensuu 10, Finland (Recewed 16 June 1986)
Abstract--1. Na-K-ATPase activity of the rat heart was similar throughout the postnatal growth when measured from crude unpurified fraction. 2. Instead in the cardiac sarcolemmal fraction, isolated by hypotonic shock LiBr-treatment method, the activity was over two times higher in 10-day old neonates than in adult rats. 3. The conflicting results are partly explained by different effects of the isolation procedure on neonatal and adult tissues. 4. Na concentration for half-maximal activity of the Na-K-ATPase was similar in neonates (7.0 mM) and adults (6.4 raM). 5. Ca-ATPase activity was not affected by Na concentration (2-100 mM) in the two age-groups studied.
INTRODUCTION Contraction of the cardiac cell is triggered by a rapid rise of intracellular Ca concentration i.e. Ca-transient (Allen and Kurihara, 1980) and the intensity of the contractile state is proportional to its magnitude. Sarcolemma plays a crucial role in the regulation of myocardial contractility by controlling the exchange of ions between extracellular fluid and cytosol of the cardiac cell (Chapman, 1979; Langer et al., 1982). Along with voltage gated ionic channels sarcolemmal ATPases are intimately involved in the rapid changes of myoplasmic ion concentrations. Ca-activated Mgdependent ATPase (Ca-Mg-ATPase) is directly involved in lowering myoplasmic Ca concentration by extruding Ca through the sarcolemma (Caroni and Carafoli, 1980). Na-K-ATPase participates in the active transport of Na and K maintaining relatively low Na-level within the cell (Skou, 1965). Furthermore Na-K-ATPase is indirectly associated with the regulation of myoplasmic Ca, since intracellular Na and Ca concentrations are coupled by the Na~Caexchange mechanism (Reuter and Seitz, 1968). The presence of Ca- or Mg-activated ATPase (Ca/MgATPase) in the heart sarcolemma has been also well established by biochemical (Sulakhe and Dhalla, 1971, DhaUa et al., 1981) as well as histochemical methods (Malouf and Meissner, 1980). This ATPase is suggested to be involved in the regulation of Ca influx perhaps as a part of the gating mechanism of the slow inward calcium channel (Dhalla et aL, 1982, Vrbjar et aL, 1985). Although sarcolemmal ATPases of the adult mammals have been the object of intense investigation there are only a few studies concerning the activities and kinetic properties of cardiac sarcolemmal ATPases from maturing animals (Nagatomo and Sasaki 1981; Khatter, 1985). The aim of the present study was to compare the activities of sarcolemmal
ATPases from neonatal and adult rat heart. Especially, we tried to measure Na-K-ATPase activity and to determine its dependence on Na ion concentration. Rat is particularly interesting in this respect, because in this species the contractile properties of the cardiac tissue change markedly during the first weeks of postnatal life (Hopkins et al., 1973; Vornanen, 1984, 1985). MATERIALS AND METHODS
Isolation of sarcolemma Male and female Wistar rats between 1 day and 6 months of age were used. Animals were bred on a 12 hr dark, 12 hr light cycle and fed ad libitum with normal animal chow. Young rats were decapitated with scissors and adult animals were killed by cervical dislocation after slight ether anesthesia. Ventricular muscle was excised and homogenized in ice-cold buffer (10mM Tris-HC1, 1 mMEDTA, pH7.4). Several hearts were pooled from younger animals but only one heart was needed in the case of adults. Sarcolemmal fractions were isolated from 10-day old neonates and adult rats by the hypotonic shock-LiBr treatment method as described by Panagia et al. (1982). This method was chosen for its high yield, enabling extraction of purified sarcolemmal fraction from the small tissues of neonatal animals (Dhalla and Pierce 1984), Na-K-ATPase activity was also determined at the beginning of the purification procedure, i.e. in the pellet of first centrifugation (1000g for 10 min), which was suspended in 1 mM Tris-HCl (pH 7.0) for ATPase determinations.
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Treatment of sarcolemma with deoxycholate In order to expose latent Na-K-ATPase activity sarcolemmal membranes were treated with deoxycholate. Five to eight ml of sarcolemmal suspension was diluted with the same volume of 0.2% Na-deoxycholate in 1 mM Tris-HC1 (pH 7.0) and incubated for 20 min in an ice-water bath with gentle mixing. Deoxycholate to protein ratio varied between 0.1-0.2. The sarcolemmal fraction was recollected by centrifugation at 5000g for 5 min. The pellet was suspended in l mM Tris-HC1 (pH 7.0) for enzyme assays.
P.~IVI KARTrUNEN and MATTIVORNANEN
816 A TPase determinations
Three different ATP hydrolysing activities were measured. Na-K-ATPase activity was determined as the difference of total ATPase obtained in the presence of 50 mM Tris-HCl (pH 7.5), 4 mM Tris-ATP, 4 mM MgCI2, 20 mM KCI and 100 mM NaCI and Mg-ATPase measured in the presence of 50mM Tris-HC1 (pH7.5), 4 m M Tris-ATP, and 4 mM MgC12. Alternatively Na-K-ATPase was also determined as the portion of total ATPase which was inhibited by I mM ouabain. Mg/Ca-ATPase activity was estimated in a reaction mixture containing 50 mM Tris-HC1 (pH 7.5), 4 mM Tris-ATP and 4 mM MgC12 or CaCI 2. ATPase hydrolysis occurring in the absence of activating cations was subtracted from these ATPase activities to obtain the final values. Myofibrillar myosin ATPase was measured in 450 mM KC1, 45 mM imidazole (pH 7.5), 5 mM Na2-ATP, 10 mM NaN 3 and 10 mM EDTA (K-EDTA-ATPase; Martin et al., 1982). The assays were performed at 37°C in a total volume of 1.5 ml and in the presence of 20-300#g protein. The reaction was started after 5 min preincubation by ATP addition. After 10 min incubation the inorganic phosphate liberated by the different ATPases was measured in the clear supernatant by the method of Atkinson et al. (1973). The protein content was assayed by the procedure of Lowry et al. (1951) using fatty acid free bovine serum albumin as the standard protein. Electron microscopy
Pellets of membranes were fixed in 1.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.0) overnight at 4°C. They were washed with 0.1 M phosphate buffer 8-10 times and postfixed in 1% OsO4 for 2.5 hr at 4°C. After dehydration in a graded ethanol series (30-100%) the sample was embedded in Epon. Thin sections were cut and stained with uranylacetate and lead citrate (LKM-ultrastainer) and examined with JEM 100 CX electron microscope. Statistics
Mean values among several groups were compared by one-way analysis of variance (ANOVA) and significant differences between individual groups were tested after ANOVA by Scheffe's multiple test. Student's t-test for independent and paired samples was used to compare mean values of two groups or two treatments, respectively. RESULTS The high yield by the h y p o t o n i c s h o c k - L i B r treatm e n t m e t h o d , requiring relatively small a m o u n t s of original tissue, allowed purification of sarcolemmal m e m b r a n e s from cardiac tissues o f b o t h n e o n a t a l (10-day old) a n d adult rats. Electron microscopy showed t h a t in b o t h age groups the isolated fraction consisted of m e m b r a n e vesicles or sacs of variable size. T h e vesicles were coated in several places by fuzzy glycocalyx, thus showing their sarcolemmal
Table 1. Effect of Na-azide and Na-orthovanadate on sarcolemmal Ca-ATPase and Mg-ATPase activities Ca-ATPase Mg-ATPase Additions A N A N Na-azide 5mM -26.8 -21.5 -55.8 -55.0 Vanadate 2#M +1.4 +1.6 -3.0 -5.0 200/~M -16.7 -11.0 -9.7 -6.8 The values are % change of the ATPase activities (+ = stimulation, - = inhibition) of adult (A) and neonatal (N = 10-days old)
sarcolemmal preparation. The results are means of 2-5 preparations.
origin (Fig. 1). M i t o c h o n d r i a l c o n t a m i n a t i o n was n o t observed b u t some r e m n a n t s of myofibrils were occasionally found, suggesting t h a t 0.6 M LiBr is unable to extract myofibrillar protein totally. Sodium azide (5 m M ) inhibited 2 1 ~ 7 % of the C a - A T P a s e a n d a b o u t 55% o f the M g - A T P a s e activity o f the present sarcolemmal fraction (Table 1). Azide is generally regarded as an inhibitor of mitoc h o n d r i a l ATPase, thereby suggesting a significant c o n t a m i n a t i o n o f o u r p r e p a r a t i o n by mitochondria. However, even in highly purified sarcolemmal memb r a n e preparations, M g - A T P a s e activity is f o u n d to be 50% sensitive to azide (Jones et al., 1980). It seems t h a t azide sensitive A T P a s e exists in addition to m i t o c h o n d r i a also in the sarcolemma of muscle cells ( K w a n a n d R a m l a l 1982; D h a l l a a n d Pierce, 1984, Turi a n d T b r f k , 1985). In any case the azide sensitive p o r t i o n o f the C a / M g - A T P a s e activity was similar in b o t h age groups studied. N a - o r t h o v a n a d a t e , a p o t e n t inhibitor of the CaM g - A T P a s e of the sarcoplasmic reticulum, exerted slight a n d equal effect o n the basic C a / M g - A T P a s e activity o f the two p r e p a r a t i o n s (Table 1). In accordance with the electron microscopic findings some myofibrillar c o n t a m i n a t i o n as myosin A T P a s e activity was f o u n d in the sarcolemmal fraction. However K - E D T A - A T P a s e activities were similar in b o t h preparations, showing equal myofibrillar c o n t a m i n a t i o n in the two age groups. W h e n N a - K - A T P a s e activity was m e a s u r e d at the beginning o f the purification procedure, i.e. in the pellet of the first centrifugation, n o significant differences existed between the four age groups studied. T h e activities were similar from n e w b o r n s (3-4 day old) to y o u n g adults (5-6 m o n t h old). Instead in the final sarcolemmal fraction N a - K - A T P a s e activity of the 10-day old rats was over twice t h a t of the adult animals (Table 2). T h u s the difference a p p e a r e d in the course of the isolation procedure. Because the purity of the fractions seemed to be similar in the two age groups, o t h e r causes for the conflicting results were sought. A possibility existed t h a t all of the
Table 2. Age-dependence of sarcolemmal Na-K-ATPase activity in initial crude fraction and final sarcolemmal preparation Na-K-ATPase activity Age of animal Crude fraction N Sarcolemmalfraction N Enrichment 3 Days 4.8 ~ 0.32 6 --10 Days 3.8 -T-0.49 7 13.4 -T-2.50 9 3.6 15 Days 4.4 -T-0.30 6 --Adult 3.5 -T-0.42 7 6.2 -T-0.43* 18 1.7 Na-K-ATPase activity was measured as ouabain (1 raM) inhibitable portion of the total ATPase activity. Crude fraction is the pellet from first eentrifugation of the homogenate, suspended in l mM Tris-HCl (pH7.0). Results are means+S.E.M, of 6--18 experiments as indicated. *Significantly (p < 0.05) different from the value of the 10-day old group.
Heart sarcolemmal ATPase
Fig. 1. Electron micrographs of sarcolemmal fractions from adult (a) and lO-day old (b) rats. Scale bar 1 #m.
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P.~IVI KARTTUNEN and MATTI VORNANEN
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adult rat heart ventricles. Km-valuesare 7.0 and 6.4 mM for neonatal and adult animals, respectively. The results are means of 9-27 separate experiments and the lines were fitted by linear regression.
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Fig. 2. Sarcolemmal ATPase activities of adult (I-9) and 10-day old (r~) rat heart ventricles before (control) and after deoxycholate (doc) treatment. Na-K-ATPase activity was measured as ouabain inhibitable portion of total ATPase (O) and as the difference between total and Mg-ATPases (Mg). One S.E.M and number of preparations are shown above each bar. Asterisks (*p < 0.05, **p < 0.01) indicate significant differences between two means ( N S = n o t significant).
Na-K-ATPase activity was not expressed and therefore it was attempted to reveal latent Na-K-ATPase activity by deoxycholate treatment. Deoxycholate increased the Na-K-ATPase activity of the adult rat heart but slightly depressed it in the neonates. So the difference between the age-groups was no more statistically significant if expressed as an oubain inhibitable portion of the total ATPase but still reached a statistically significant level when measured against Mg-ATPase activity (Fig. 2). The sarcolemmal preparations of the two age groups showed differing behaviour in relation to ouabain. In the adult the Na-K-ATPase activity was higher when measured as a ouabain sensitive component of the total ATPase than if determined as the difference between total ATPase and Mg-ATPase. In the neonates the situation was reversed. The dependence of the Na-K-ATPase activity on Na concentration (2-200 mM) is shown in Fig. 3. In this regard the enzymes of the two age-groups were similar. Half maximal activity of the Na-K-ATPase was reached at 6.4 and 7.0 mM NaCI for neonatal and adult rats, respectively. V~x was, as expected on the basis of their specific activities, far higher in the
neonates (16.6pmol Pi/mg prot./hr) than in the adults (5.6/~mol Pi/mg prot./hr). Sarcolemmal Mg-ATPase and Ca-ATPase activities were much higher in neonates than in adults (Fig. 2). However, activity ratios Mg-ATPase/Na-KATPase and Ca-ATPase/Na-K-ATPase were similar, being 4.2 and 5.3 for neonatal and 4.1. and 4.0 for adult rats, respectively. This suggests that Mg/Ca-ATPase is not a contaminant from other cellular organelles but copurifies with Na-K-ATPase. Deoxycholate decreased Mg- and Ca-ATPase activities significantly in the neonates but did not markedly affect on those of the adults (Fig. 2). Sarcolemmal Ca-ATPase activity was not dependent on sodium concentration in the range 2-100mM (Fig. 4). DISCUSSION
Among the cardiac tissues of common laboratory mammals the adult rat heart is exceptional in several respects. In normal physiological solution the rat
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Heart sarcolemmal ATPas¢ heart ventricle functions as if its cellular Ca stores were maximally loaded (Forester and Mainwood, 1974). Instead the neonatal cardiac tissue, up to the age of 2 weeks, behaves like that of other adult mammals, showing no signs of Ca overload (Vornanen, 1984). The cellular basis for this difference between adult and maturing tissues is not clear. Sarcolemmal Na-K-ATPase is one possible candidate, for it regulates the level of cytosolic Ca in addition to intracellular Na and K concentrations. This is because concentration gradients of Na and Ca are coupled through the Na-Ca exchange mechanism (Reuter and Seitz, 1968). The relation between intracellular Na and twitch tension is steep with 1 mM increase in Na concentration producing about a 100% increase in the twitch magnitude (Wasserstrom et aL, 1983). Therefore activity and kinetics of the sarcolemmal Na-K-ATPase are important determinants in the regulation of cardiac contractility. Sarcolemmal Na-K-ATPase activity of the adult rat heart was less than half from that of the neonates. The difference was not, however, seen at the beginning of the purification procedure. The total protein content of 10-day old and adult rat hearts were 71 and 76 pg/mg tissue wet wt respectively, the activities of crude unpurified fraction thus being comparable on the basis of total protein. Therefore a question arises, whether the final difference in Na-K-ATPase activity is real or is it an artefact created by the isolation procedure. First of all, the difference between the age-groups could be due to variable degree of contamination of the sarcolemmal fraction by other cellular structures. This is not evident on the basis of K-EDTA-ATPase activity or by azide and vanadate sensitivity of the basic ATPases nor by morphological criteria of the membrane vesicles. The preparations may also differ in regard to the proportion of sealed vesicles and their sidedness (inside-out/outside-out). Latent Na-K-ATPase activity was revealed by deoxycholate treatment in the sarcolemmal fraction of the adult but not in the preparation of the neonatal rats, suggesting that a greater number of the vesicles were sealed in the former group. Higher susceptility of the Ca/Mg-ATPase of the neonatal rat heart to deoxycholate treatment further supports the notion that in the neonates the membrane faces are more directly exposed to surrounding medium. The sensitivity of the Mg-ATPase of the adult rat heart to I mM ouabain also suggests that some portion of the Na-K-ATPase activity is latent in the membrane vesicles of this age-group. Thus in some degree the difference in sarcolemmal Na-K-ATPase activity may be explained on the basis of variable effect of the hypotonic shock LiBr-treatment method on adult and neonatal tissues. On the other hand the low Na-K-ATPase activity of the adult rat heart can be partly explained also on morphological grounds. Approximately one-third of the sarcolemma in the adult rat heart is located at the level of T-tubules (Page et al., 1974), which may be almost totally devoid of Na-K-ATPase activity (Almers and Stirling, 1984). In 10-day-old neonates T-tubules are just beginning to develop (Schiebler and Wolff, 1966; Hirakow and Gotoh, 1975). Thus
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although the density of Na-K-ATPase molecules were similar in the peripheral sarcolemma of the myocyte the total Na-K-ATPase activity per sarcolemmal protein would be lower in the adults due to the contribution of T-tubular protein. Because surface-to-volume ratio of the cardiac cells decreases during the postnatal life of the rat (Page et al., 1974; Olivetti et al., 1980), an equal pump density in the peripheral sarcolemma would mean a lower Na-K-ATPase activity per unit cell volume in the adult rat heart. Thus our results, that sarcolemmal Na-K-ATPase activity is higher in the neonates than in the adults, is in agreement with the finding of Awad and Clay (1982), who found higher Na-K-ATPase activity in young (1-1.5 months) than in senescent (10-12 months) rats. Similar results have also been reported for other mammals including dog and guinea-pig (Miller and Gilliland, 1972; Marsh et al., 1981; Khatter, 1985) suggesting that this is a general trend among mammals. However, in the rabbit Na-K-ATPase activity is higher in adults than in newborn (0-3 days) animals (Nagatomo and Sasaki 1981). Care must, however, be taken when interpreting the results because isolation procedure can have profoundly different effects on neonatal and adult tissues. The dependence of the Na-K-ATPase activity on Na was similar in the two age-groups studied. The half-maximal activity was reached at about 7 mM Na, which is close to the values of 8.95 and 8.47 measured for adult (3 month old) and senescent (26 month old) rat heart, respectively (Katano et al., 1985). Because intracellular Na activity is in the range of 6-9 mM in the adult heart (Sheu and Fozzard, 1982; Grupp et al., 1985), the enzyme seems to be half-maximally activated in vivo, and thus possesses ample reserve capacity. Ca-ATPase activity was not dependent on Na concentration. This is in accordance with the findings of Tuana and Dhalla (1982) with the sarcolemmal preparation of the adult rat heart but contrary to those of McNamara et al. (1974) on dog heart. Thus, our findings do not support the proposal that this enzyme is the site of Na-Ca antagonism that regulates cardiac contractility at sarcolemmal level (Tuana and Dhalla, 1982). In conclusion, our results suggest that the Na-K-ATPase activity of the neonatal rat heart ventricles may be somewhat higher than that of the adults, whereas Na-kinetics of the enzyme is independent of age. Therefore in the myocytes of young rat heart the reserve capacity of the sodium pump may be higher than in the adults, which could result in different type of contractile behaviour in situations where cellular Na load or Na-pump activity are altered. REFERENCES
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