Characteristics Na+-K+-ATPase
of thyroid-stimulated of rat heart
KENNETH D. PHILIPSON AND ISIDORE S. EDELMAN Cardiovascular Research Institute and Departments of Medicine and Biochemistry Biophysics, University of California School of Medicine, San Francisco, California
PHILIPSON, KENNETH D., AND ISID~RE S. EDELMAN. Characteristics of thyroid-stimulated Nu+-K+-ATPase of rat heart. Am. J. Physiol. 232(3): C202-C206, 1977 or Am. J. Physiol.: Cell Physiol. l(3): C202-C206, 1977. - The possibility that augmentation of cardiac Na+-K+-dependent adenosine triphosphatase (Na-K-ATPase) by L-3,5,3’-triiodothyronine (T3) was mediated by early changes in intracellular ion concentrations ([Na+] i, [K+] i> was explored by time-course analysis after a single injection of T3 in thyroid-ablated (Y> rats. At 6 and 16 h after injection, T, had no significant effect on cardiac [Na+J, [K+] iy or microsomal Na-K-ATPase activity. At 24 and 48 h, however, T, elicited proportionate increases in [K+]i and NaK-ATPase activity. Thus, no evidence was adduced that the T,-dependent increase in ventricular Na-K-ATPase activity is an adaptive response to prior changes in intracellular ion concentrations. The increase in [K+li is attributable to an increase in Na+ pump activity. Administration of T, to hypothyroid rats had no effect on the transition temperature or the activation energy of ventricular microsomal Na-K-ATPase, as analyzed by an Arrhenius plot. Thus, the lipid microenvironment and the properties of the enzyme may be independent of thyroid status. The latter inference was supported by kinetic analysis, in that T3 had no effect on the K, for ATP or the K& for Na+ and K+. Injection of T:, of the hypothyroid rat, however, signifcantly increased the Vmax’s for ATP, Na+, and K+ of ventricular microsomal Na-K-ATPase. These results are in accord with the inference of thyroidal induction of Na-KATPase indistinguishable from those present in the athyroid state.
triiodothyronine; sodium trophenyl phosphatase
pump; potassium
dependent;
parani-
and 94143
was the possibility that activation of cardiac Na-KATPase was secondary to a prior effect of thyroid hormone on intracellular ion concentrations, specifically, an increase in intracellular Na+ concentration ([Na]+i) or a decrease in intracellular K+ concentration ([K]+i). To explore this issue, we studied the time course of the changes in intracellular cation concentrations and in Na-K-ATPase activity in rat ventricle after a single injection of T,. The second issue we considered was the possible role of local changes in membrane phospholipids in thyroidal activation of cardiac Na-K-ATPase. This issue was raised by two findings: 1) stimulation of phospholipid synthesis by thyroid hormone (24), and 2) the dependence of Na-K-ATPase activity on membrane lipids (13). To explore this possibility we assessed temperatureactivity relationships of microsomal Na-K-ATPase analyzed in an Arrhenius plot which, for Na-K-ATPase, is biphasic with a unique transition temperature (5, 7). Previous workers have shown by fluorescence and electron spin resonance techniques that the transition temperature for Na-K-ATPase is related to a lipid-phase change and is sensitive to the lipid composition of the plasma membrane (5, 7). The third issue that was examined concerned the possibility that the T,-independent and T,-dependent cardiac Na-K-ATPases differed in their kinetic properties (K,‘s and V,,, ‘s for Na+, K+, and ATP). METHODS
indicate that increased energy utilization by the Na+ pump mediates much of the thyroid hormone-induced increase in the resting oxygen consumption (Qo,) (2, 7-11). In liver, kidney, and skeletal muscle, thyroid hormone increased both the fraction of respiration which is dependent on active Na+ transport [Qo2( t)] and the activity of the transport enzyme, Na+-K+-activated adenosine triphosphatase (Na-KATPase) (2, 9). A VARIETY
OF
FINDINGS
Heart muscle responds to thyroid hormone with increases in resting Qo, and in contractile activity (6). We reported previously that T, elevates Na-K-ATPase activity in rat cardiac tissue and was obtained even with
physiological dosesof T3, namely, 1 pg/lOO g body wt per day 1; the present
studies,
the first issue
we addressed
Hypothyroid male, Sprague-Dawley rats were used throughout these experiments. The techniques for inducing hypothyroidism with [1311]Na,preparing ventricular microsomes, and assaying for Na-K-ATPase activity were as described in the preceding paper (20). Intracellular ion concentrations. Thyroid-ablated rats were anesthetized with ether, and both kidneys were removed surgically after administering T, at times depending on the desired time of termination of the experiment,. Immediately following nephrectomy, 0.5 ml of saline containg 5 &i of [14C]inulin was injected into the tail vein. Five hours after injection of the [14C]inulin, the rats were killed by cervical dislocation. Blood was collected in a heparinized syringe by cardiac puncture and plasma prepared by centrifugation. Pieces of ventricular tissue were transferred to preweighed polyethylene tubes, weighed, dried for 48 h at 40°C in a vacuum
c202
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THYROID
HORMONE-INDUCED
C203
Na-K-ATPase
oven, and then reweighed. Tissue electrolytes were extracted in 1.4 ml of 0.1 N HNO, for 24 h at room temperature. The tissue extracts and plasma were analyzed for radioactivity in a Nuclear-Chicago Mark II liquid scintillation spectrometer and for Na+ and K+ content with a Perkin-Elmer model 303 atomic absorption spectrometer. In the calculation of extracellular Na+ and K+ concentrations, corrections were made for Donnan distribution ratios of ha+ = RK+ = 0.94 (18). Intracellular Na+ and K+ concentrations were calculated from the total electrolytes, the inulin space, and the extracellular ion concentrations (10). Temperature dependence of Na-K-ATPase activity. Ventricular microsomal fractions were prepared from T,-treated and diluent-treated, thryoid-ablated (1311) rats as described previously (20). Na-K-ATPase activity of these fractions was assayed in the standard incubation medium (see below) in constant-temperature water baths for 15 min, 18, 21, 25, 30, and 36°C. To obtain a sufficient number of aliquots for these determinations, five ventricles from T,-treated rats and five ventricles from control rats were pooled. Two such experiments were completed, and the mean values at each temperature computed for the two groups. Determination of V,,, and K,. To estimate the ATPdependent K, and V,,, of the Na-K-ATPase reaction, the standard incubation medium (NaCl, 100; KCl, 20; MgQ, 5; EDTA, 0.1; NaN,, 5; Tris, 50 (all in mM); pH 7.4) contained an ATP-regenerating system consisting of 2.5 mM phosphoenolypruvate, 40 pg pyruvate kinase, and variable concentrations of Tris-ATP (0.3-1-Z mM). The K1,2 and V,,, of the Na-K-ATPase system for Na+ were determined using the standard medium with varying quantities of Na+ (16-56 mM) and 3 mM Tris-ATP. The identical format was used to determine the&,, and V max for K+, except that NaCl and Tris-ATP concentrations were kept constant at 100 and 3 mM, respectively. The K+ concentration was varied from 0.5 to 4.0 mM. Ventricular microsomal fractions were prepared from T,-treated and control hypothyroid rats and incubated as described previously (20). Statistical calculations. The data are presented as means -+ SE. Since control and T,-treated animals were paired by age and weight and used for experiments at the same time, the results were analyzed for significance by the paired Student t test (23). MateriaZs. All conventional reagents were analytical grade. Tris-ATP, ouabain, phosphoenolpyruvate, and pyruvate kinase were purchased from Sigma Chemical Co., Na-L,3,5,3’-triiodothyronine (Sigma) was dissolved in saline containing 1% 0.1 N NaOH. [CarboxylTABLE
I. Effect of T3 on intracellular
14C]inulin, (2 &i/mg) land Nuclear Corp.
was purchased
from New
Eng-
RESULTS
Time course of effects of T, on ion composition and Na-K-ATPase activity. Pairs of hypothyroid rats were injected subcutaneously with 50 pg of TJlOO g body wt or the diluent. Bilateral nephrectomies were performed and [inulin]14C injected either 1, II, 19, or 43 h after administration of T,. Thus, the ventricles were removed at 6, 16, 24, or 48 h after T, was given, Aliquots of the ventricles were analyzed both for electrolyte content and Na-K-ATPase activity (microsomal fractions). Thyroid status had no significant effect on [Na+]i at any time point (Table 1). Significant increases, in both [K+]i and ventricular Na-K-ATPase activity were evident at 24 and 48 h, but not at the earlier times. The mean increases in [K+]i were 6.9 and 10.6 meq/liter of cell water at 24 and 48 h, respectively. The corresponding increases in Na-K-ATPase activity were 1.2 and 2.0 PM Pi per mg protein per h. The mean absolute changes in [K+J and Na-K-ATPase activity are displayed in Fig. 1. The correlation in the time courses of the response of these parameters to T3 is evident. Although the data are not given, we found no effect of thyroid status on plasma Na+ or K+ concentrations, in accord with earlier studies (10). Effect of T3 on temperature dependence of cardiac NaK-ATPase activity. Thyroid-ablated rats were injected subcutaneously with T3 (50 pg/lOO g body wt) or the diluent on alternate days for a total of three injections. The ventricles were removed 48 h after the last injection. Ventricular microsomal Na-K-ATPase activity was measured at temperatures of from 15 to 36°C. In an Arrhenius plot (l), the linear regressions were discontinuous at 24°C for Na-K-ATPase activity from both control and T,-treated ventricles (Fig. 2), The slopes of the linear segments give the activation energies for the reaction within those temperature limits, The activation energies were identical for both the hypothyroid and T,-treated Na-K-ATPases; 28,6 kcallmol below 24°C and 18+4kcallmol above the transition temperature. Effects of T, on K, and V,,, for ATP, Na+, and K+. To characterize the effect of thyroid hormone on the kinetic parameters of cardiac Na-K-ATPase, a study was made of the dependence of the reaction on the concentration of each of the reactants. With ATP as the variable, the preparations from both control and T3treated rats conform to Michaelis-Menten kinetics in a Lineweaver-Burk plot (14) (Fig. 3). Analogous linear
ion concentrations and Na-K-ATPase
activity
in hypothyroid
Downloaded from www.physiology.org/journal/ajpcell at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
rat heart
c204
K.
10.0
D. PHILIPSON
AND
I. S. EDELMAN
0 AIK+li - vd
A
I
ATPase
I
I
I
I
‘/[ATPI
6 16 24 48 HOURS AFTER T, TREATMENT FIG. 1. Time course of changes in ventricular [K+li and microsomal Na-K-ATPase activity in response to T, given to hypothyroid rats. These data are from Table 1. See legend of Table 1 for details,
- tI 3.2
I
1
I
3.3
3.4
3.5
l/T x 103("K-') 2. Effect of T3 on Arrhenius plot of Na-K-ATPase activity in ventricular microsomes. Units of Na-K-ATPase activity are pmol Pi per mg protein per h. Results are average of two experiments. Microsomal fraction of five hearts was pooled for each experiment (i.e., 10 rats per group). Pairs of rats were injected either with the diluent or with 50 pg of Ts per 100 g body wt on alternate days for a total of three doses. Hearts were collected 48 h after last injection. FIG.
regressions
on Lineweaver-Burk plots were obtained with Na+ or K+ as the variable. T, had no significant effect on the& for ATP or the Kli, for Na+ or K+ (Table 2). In contrast, T, augmented the V,,, for Na+ 45%, for K+ 82%, and for ATP 55%. DISCUSSION
Changes in intracellular ion concentrations may modulate a variety of intracellular processes, including the
FIG. 3* Effect of T3 on dependence of cardiac Na-K-ATPase activity on ATP concentration. Hypothyroid rats were injected with three doses of diluent or Ts (SO pg/lOO g body wt) at 48-h intervals. Assays performed on microsomal fractions in an ATP regenerating system. l/V is reciprocal of enzyme activity in PmoI Pi per mg protein per h and ATP concentration is in mM. N = 5 pairs of rats. Points are means + SE.
abundance of Na-K-ATPase, and RNA and protein synthesis (3, 17, 21, 22, 25). For example, Katz and Lindheimer (12) noted a correlation between the filtered Na+ load and renal Na-K-ATPase activity in response to thyroid hormone. These findings prompted us to explore the possibility that changes in intracellular cation concentrations may mediate the T,-dependent increase in cardiac Na-KATPase. This possibility is also raised by the relatively long latent period for induction of Na-K-ATPase as compared to other cellular events, such as the early increase (maximal at 6 h) in sensitivity to catecholamines evoked by T, (3). Thus, if T3 increases the permeability to Na +, thereby increasing ENa+],-early in the time course, this might induce an increase in the number of Na+ pumps at 24 h. This possibility was tested by analyzing the time course of the ionic and enzymic responses to a single injection of T, in the thyroid-ablated (Y) rat, No effect of T, on [Na+]i or intracellular water content was observed at 6-48 h after injection. In an earlier study, Ismail-Beigi and Edelman (10) found that administration of T3 in repeated doses over a I-wk period significantly lowered cardiac [Na+]i and raised [K+]i* In the present study, a single injection of T, did not elicit the change in [Na+]i but increased [K+]i significantly at 24 and 48 h. Moreover, the increases in [K+]i correlated well with the increases in ventricular Na-K-ATPase activity (Table 1 and Fig. 1). Thus, no evidence was adduced for a mediating role of changes in ion composition on the response of Na-KATPase to T,. The results are compatible with the inference that the changes in ion composition are a consequence of a primary increase in the activity of the Na+ Pump* If thyroid hormone induces the synthesis of Na-KATPase isozymes or of lipid constituents that are part of the microenvironment of the enzyme, the temperature
Downloaded from www.physiology.org/journal/ajpcell at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
THYROID TABLE
from
HORMONE-INDUCED
c205
Na-K-ATPase
2. Effect of T, on kinetic parameters for Na+, K+, and ATP uf Na-K-ATPase ventricular microsomal fructions Thyroid
Hypothyroid Hypothyroid P
K li2, mM
Status
+ T,
K 111,mM
Nat
K’
35.6 5 4.1 37.0 k 5.4
2.4 ir 0.2 3.1 2 0.3
NS
NS
Values are means * SE. Hypothyroid doses and the ventricles were analyzed
v mrlb, Fmol
ATP
0.42 0.38
f 0.09 2 0.05 NS
per h
K+
ATP
11.3 -t- 2.4 16.4 + 3.5
13.6 k 2.1 24.7 k 4.7
11.8 2 1.3 18.3 -t- 2.8
co.05
co.05
co.05
rats were injected with diluent or 50 pg of T:, per 100 g body 48 h after the final injection. N = 5 pairs of rats.
dependence of the reaction might be significantly altered. This possibility deserved consideration since NaK-ATPase activity is sensitive to the phospholipid microenvironment (5, 7) and thyroid hormone induces the synthesis of both proteins and membrane-associated phospholipids (24). Lipid fluidity influences enzyme activity and phase changes in membrane lipids have been detected as discontinuities in the Arrhenius plot of NaK-ATPase activity (5, 7). The results (Fig. 2) show no effect of T, on either the transition temperature or of the activation energy of the reaction in the Arrhenius plot. The fact that T, did not alter the transition temperature implies that the local lipid environment of Na-K-ATPase was not drastically modified. The lack of an effect of T3 on the activation energy (above or below the transition temperature) implies that the characteristics of the Na-K-ATPase system are independent of thyroid status. Thyroidal enhancement of Na-K-ATPase may result from synthesis of additional Na+ pump units or from increased reactivity of a fixed number of pump sites. In the former case, induction of an isozyme might be revealed by changes in the affinities of the enzyme for ATP, Na+, and K+. In the latter case, the affinities for these substrates might also be modified. LineweaverBurk plots, however, revealed no significant T,-dependent changes in the affinities; K, for ATP and K,,, for Na+ and K+ (Table 2, Fig. 3). The maximum velocities
Pi per m g Protein
Na’
wt on alternate
days for a total
of three
of the ATPase reaction, however, were significantly increased with all three substrates. Similar effects of T, on renal Na-K-ATPase have been reported by Lo et al. (15) for Na +, K+, and ATP, and by Asano et al. (2) on skeletal muscle for ATP. These results are consistent with TZ induction of the synthesis of Na+-transport enzymes with the same properties as those present in the athyroid state. That thyroid hormone induces the de novo synthesis of the catalytic subunit of renal cortical Na-K-ATPase has been inferred from experiments on the effects of T, on: 1) the number of [“Hlouabain binding sites, 2) the incorporation of .72Pfrom ATa2P(y) into the phosphorylated intermediate, and 3) the incorporation of radiolabeled amino acids into the large subunit of Na-K-ATPase (15, 16). The results obtained on the rat heart are consistent with these findings and with the presence of high-affinity nuclear receptors in the myocardium (19). The authors are grateful for the excellent technical assistance provided by Ms. Laurie Lippitt. This investigation was supported by Program Project Grant HL06285 from the National Heart and Lung Institute and was performed during the tenure of a Postdoctoral Traineeship provided by the National Heart and Lung Institute Grant No. HI,-05725 to K. D. Philipson. Present address of K. D. Philipson: Dept. of Physiology, University of California, Los Angeles, Calif. 90024. Received
for publication
19 August
1976.
REFERENCES 1. ARRHENIUS, S. 2. Physik. Chem. 4: 226, 1889. 2. ASANO, Y,, U. A. LIBERMAN, AND I, S. EDELMAN. Thyroid thermogenesis: relationships between Na+-dependent respiration and Na+ + K+-adenosine triphosphatase activity in rat skeletal muscle. J. CZirz. Invest. 57: 368-379, 1976. 3. BOARDMAN, L. J., J. F. LAMB, AND D. MCCALL. Uptake of [“HI ouabain and Na pump turnover rates in cells cultures in ouabain. J. Physiol., London 225: 619-635, 1972. 4. BRAY, G. A., AND H. M. GOLDMAN. Studies on the early effects of thyroid hormones. Endocrinology 76: 323-328, 1965. 5. CHARNOCK, J. S., AND C. L. BASHFORD. A fluorescent probe study of the lipid mobility of membranes containing sodiumand potassium-dependent adenosine triphosphatase. Mol. Pharmacol. 11: 766-774, 1975. 6. FREEDBERG, A. S., AND M. W. HAMOLSKY. Effects of thyroid hormones on certain nonendocrine organ systems. In: Handbook of Physiology. Endocrinology. Washington, D.C.: Am. Physiol. Sot. 1974, sect. 7, vol. III, chapt. 25, p. 435-468. 7. GRISHAM, C. M., AND R. E. BARNETT. The role of lipid-phase transitions in the regulation of the (sodium + potassium) adenosine triphosphatase. Biochemistry 12: 2635-2637, 1973. 8. ISMAIL-BEIGI, F., AND I. S. EDELMAN. The mechanism of thyroid calorigenesis: role of active sodium transport. Proc. N&l. Acad. Sci. U.S. 67: 10714078, 1970. 9. ISMAIL-BEIGI, F., AND I. 8. EDELMAN. The mechanism of the
10.
11.
12.
13.
14. 15.
16.
17.
calorigenic action of thyroid hormone: Stimulation of Na+ + K+activated adenosine triphosphatase activity. J. Gen. Physiol. 57: 710-722, 1971. ISMAIL-BEIGI, F., AND I. S. EDELMAN. Effects of thyroid status on electrolyte distribution in rat tissues. Am. J. Physiol. 225: 11721177, 1973. ISMAIL-BEIGX, F., AND I, S. EDELMAN. Time-course of the effects of thyroid hormone on respiration and Na+ + K+-ATPase activity in rat liver. Proc. Sot. Exptl. Biol. Med. 146: 983-988,1974. KATZ, A. I., AND M. D. LINDHEIMER. Renal NaK-ATPase and Na+ reabsorption in the hypothyroid rat. J. C&z. Invest. 52: 796804, 1973. KIMELBERG, H. K., AND D. PAPAHADJOPOULOS. Effects of phospholipid acyl chain fluidity, phase transitions, and cholesterol on (Na+ + K+t)-stimulated adenosine triphosphatase. J. BioZ. Chem. 249: 1071-1080, 1974. LINEWEAVER, H., AND D. BURK. The determination of enzyme dissociation constants. J. Am. Chem. Sot. 56: 658-666, 1934. Lo, C. S., T. R. AUGUST, U. A. LIBERMAN, AND I. S. EDELMAN. Dependence of renal (Na+ + K+)-adenosine triphosphatase activity on thyroid status. J. BioZ. Chem. 251: 7826-7833, 1976. Lo, C. S., AND I. S, EDELMAN. Effect of triiodothyronine on the synthesis and degradation of renal cortical (Na+ + K+)-adenosine triphosphatase. J. Biol. Chem. 251: 7834-7840, 1976. LUBIN, M. Intracellular potassium and macromolecular synthe-
Downloaded from www.physiology.org/journal/ajpcell at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
C206
K. D.
sis in mammalian cells. Nature 213: 451-453, 1967. 18. MANERY, J. F. Water and electrolyte metabolism. Physiol. Rev. 34: 334-417, 1954. 19. OPPENHEIMER, J. H., H. L. SCHWARTZ, AND M. I. SWRKS. Tissue differences in the concentration of triiodothyronine nuclear binding sites with rat liver, kidney, pituitary, heart, brain, spleen and testis. Endocrinology 95: W-903, 1974. 20. PHILIPSON, K. D., AND I. S. EDELMAN. Thyroid hormone control of Na+-K+-ATPase and K+-dependent phosphatase in rat heart. Am. J. Physiol. 232: C196-C201, 1977, 21. SARGENT, J. IL, AND A. J. THOMSON. The nature and properties of the inducible sodium-plus-potassium ion-dependent adenosine triphosphatase in the gills of eels (AnguiZZa anguiZZa) adapted to
PHILIPSON
AND
I. S. EDELMAN
fresh water and sea water. Biochem, J. 144: 69-75, 1974. 22. SILVA, P., J. P. HAYSLETT, AND F. H. EPSTEIN. The role of NaKactivated adenosine triphosphatase in potassium adaptation. J. CZin. Invest. 52: 2665-2671, 1973. 23. SNEDECOR, G. W., AND W. G. COCHRAN. Statistical Methods (6th ed.). Ames: Iowa State Univ. Press, 1967. 24. TATA, J, R. The formation and distribution of ribosomes during hormone-induced growth and development. Biochem. J. 104: l16, 1967. 25. VAUGHN, G. IL., AN-D J. S. COOK. Regeneration of cation-transport capacity in HeLa cell membranes after specific blockage by ouabain. Proc. AWL. Acad. Sci. U.S. 69: 2627-2631, 1972.
Downloaded from www.physiology.org/journal/ajpcell at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
of thyroid-stimulated of rat heart
KENNETH D. PHILIPSON AND ISIDORE S. EDELMAN Cardiovascular Research Institute and Departments of Medicine and Biochemistry Biophysics, University of California School of Medicine, San Francisco, California
PHILIPSON, KENNETH D., AND ISID~RE S. EDELMAN. Characteristics of thyroid-stimulated Nu+-K+-ATPase of rat heart. Am. J. Physiol. 232(3): C202-C206, 1977 or Am. J. Physiol.: Cell Physiol. l(3): C202-C206, 1977. - The possibility that augmentation of cardiac Na+-K+-dependent adenosine triphosphatase (Na-K-ATPase) by L-3,5,3’-triiodothyronine (T3) was mediated by early changes in intracellular ion concentrations ([Na+] i, [K+] i> was explored by time-course analysis after a single injection of T3 in thyroid-ablated (Y> rats. At 6 and 16 h after injection, T, had no significant effect on cardiac [Na+J, [K+] iy or microsomal Na-K-ATPase activity. At 24 and 48 h, however, T, elicited proportionate increases in [K+]i and NaK-ATPase activity. Thus, no evidence was adduced that the T,-dependent increase in ventricular Na-K-ATPase activity is an adaptive response to prior changes in intracellular ion concentrations. The increase in [K+li is attributable to an increase in Na+ pump activity. Administration of T, to hypothyroid rats had no effect on the transition temperature or the activation energy of ventricular microsomal Na-K-ATPase, as analyzed by an Arrhenius plot. Thus, the lipid microenvironment and the properties of the enzyme may be independent of thyroid status. The latter inference was supported by kinetic analysis, in that T3 had no effect on the K, for ATP or the K& for Na+ and K+. Injection of T:, of the hypothyroid rat, however, signifcantly increased the Vmax’s for ATP, Na+, and K+ of ventricular microsomal Na-K-ATPase. These results are in accord with the inference of thyroidal induction of Na-KATPase indistinguishable from those present in the athyroid state.
triiodothyronine; sodium trophenyl phosphatase
pump; potassium
dependent;
parani-
and 94143
was the possibility that activation of cardiac Na-KATPase was secondary to a prior effect of thyroid hormone on intracellular ion concentrations, specifically, an increase in intracellular Na+ concentration ([Na]+i) or a decrease in intracellular K+ concentration ([K]+i). To explore this issue, we studied the time course of the changes in intracellular cation concentrations and in Na-K-ATPase activity in rat ventricle after a single injection of T,. The second issue we considered was the possible role of local changes in membrane phospholipids in thyroidal activation of cardiac Na-K-ATPase. This issue was raised by two findings: 1) stimulation of phospholipid synthesis by thyroid hormone (24), and 2) the dependence of Na-K-ATPase activity on membrane lipids (13). To explore this possibility we assessed temperatureactivity relationships of microsomal Na-K-ATPase analyzed in an Arrhenius plot which, for Na-K-ATPase, is biphasic with a unique transition temperature (5, 7). Previous workers have shown by fluorescence and electron spin resonance techniques that the transition temperature for Na-K-ATPase is related to a lipid-phase change and is sensitive to the lipid composition of the plasma membrane (5, 7). The third issue that was examined concerned the possibility that the T,-independent and T,-dependent cardiac Na-K-ATPases differed in their kinetic properties (K,‘s and V,,, ‘s for Na+, K+, and ATP). METHODS
indicate that increased energy utilization by the Na+ pump mediates much of the thyroid hormone-induced increase in the resting oxygen consumption (Qo,) (2, 7-11). In liver, kidney, and skeletal muscle, thyroid hormone increased both the fraction of respiration which is dependent on active Na+ transport [Qo2( t)] and the activity of the transport enzyme, Na+-K+-activated adenosine triphosphatase (Na-KATPase) (2, 9). A VARIETY
OF
FINDINGS
Heart muscle responds to thyroid hormone with increases in resting Qo, and in contractile activity (6). We reported previously that T, elevates Na-K-ATPase activity in rat cardiac tissue and was obtained even with
physiological dosesof T3, namely, 1 pg/lOO g body wt per day 1; the present
studies,
the first issue
we addressed
Hypothyroid male, Sprague-Dawley rats were used throughout these experiments. The techniques for inducing hypothyroidism with [1311]Na,preparing ventricular microsomes, and assaying for Na-K-ATPase activity were as described in the preceding paper (20). Intracellular ion concentrations. Thyroid-ablated rats were anesthetized with ether, and both kidneys were removed surgically after administering T, at times depending on the desired time of termination of the experiment,. Immediately following nephrectomy, 0.5 ml of saline containg 5 &i of [14C]inulin was injected into the tail vein. Five hours after injection of the [14C]inulin, the rats were killed by cervical dislocation. Blood was collected in a heparinized syringe by cardiac puncture and plasma prepared by centrifugation. Pieces of ventricular tissue were transferred to preweighed polyethylene tubes, weighed, dried for 48 h at 40°C in a vacuum
c202
Downloaded from www.physiology.org/journal/ajpcell at Midwestern Univ Lib (132.174.254.157) on February 14, 2019.
THYROID
HORMONE-INDUCED
C203
Na-K-ATPase
oven, and then reweighed. Tissue electrolytes were extracted in 1.4 ml of 0.1 N HNO, for 24 h at room temperature. The tissue extracts and plasma were analyzed for radioactivity in a Nuclear-Chicago Mark II liquid scintillation spectrometer and for Na+ and K+ content with a Perkin-Elmer model 303 atomic absorption spectrometer. In the calculation of extracellular Na+ and K+ concentrations, corrections were made for Donnan distribution ratios of ha+ = RK+ = 0.94 (18). Intracellular Na+ and K+ concentrations were calculated from the total electrolytes, the inulin space, and the extracellular ion concentrations (10). Temperature dependence of Na-K-ATPase activity. Ventricular microsomal fractions were prepared from T,-treated and diluent-treated, thryoid-ablated (1311) rats as described previously (20). Na-K-ATPase activity of these fractions was assayed in the standard incubation medium (see below) in constant-temperature water baths for 15 min, 18, 21, 25, 30, and 36°C. To obtain a sufficient number of aliquots for these determinations, five ventricles from T,-treated rats and five ventricles from control rats were pooled. Two such experiments were completed, and the mean values at each temperature computed for the two groups. Determination of V,,, and K,. To estimate the ATPdependent K, and V,,, of the Na-K-ATPase reaction, the standard incubation medium (NaCl, 100; KCl, 20; MgQ, 5; EDTA, 0.1; NaN,, 5; Tris, 50 (all in mM); pH 7.4) contained an ATP-regenerating system consisting of 2.5 mM phosphoenolypruvate, 40 pg pyruvate kinase, and variable concentrations of Tris-ATP (0.3-1-Z mM). The K1,2 and V,,, of the Na-K-ATPase system for Na+ were determined using the standard medium with varying quantities of Na+ (16-56 mM) and 3 mM Tris-ATP. The identical format was used to determine the&,, and V max for K+, except that NaCl and Tris-ATP concentrations were kept constant at 100 and 3 mM, respectively. The K+ concentration was varied from 0.5 to 4.0 mM. Ventricular microsomal fractions were prepared from T,-treated and control hypothyroid rats and incubated as described previously (20). Statistical calculations. The data are presented as means -+ SE. Since control and T,-treated animals were paired by age and weight and used for experiments at the same time, the results were analyzed for significance by the paired Student t test (23). MateriaZs. All conventional reagents were analytical grade. Tris-ATP, ouabain, phosphoenolpyruvate, and pyruvate kinase were purchased from Sigma Chemical Co., Na-L,3,5,3’-triiodothyronine (Sigma) was dissolved in saline containing 1% 0.1 N NaOH. [CarboxylTABLE
I. Effect of T3 on intracellular
14C]inulin, (2 &i/mg) land Nuclear Corp.
was purchased
from New
Eng-
RESULTS
Time course of effects of T, on ion composition and Na-K-ATPase activity. Pairs of hypothyroid rats were injected subcutaneously with 50 pg of TJlOO g body wt or the diluent. Bilateral nephrectomies were performed and [inulin]14C injected either 1, II, 19, or 43 h after administration of T,. Thus, the ventricles were removed at 6, 16, 24, or 48 h after T, was given, Aliquots of the ventricles were analyzed both for electrolyte content and Na-K-ATPase activity (microsomal fractions). Thyroid status had no significant effect on [Na+]i at any time point (Table 1). Significant increases, in both [K+]i and ventricular Na-K-ATPase activity were evident at 24 and 48 h, but not at the earlier times. The mean increases in [K+]i were 6.9 and 10.6 meq/liter of cell water at 24 and 48 h, respectively. The corresponding increases in Na-K-ATPase activity were 1.2 and 2.0 PM Pi per mg protein per h. The mean absolute changes in [K+J and Na-K-ATPase activity are displayed in Fig. 1. The correlation in the time courses of the response of these parameters to T3 is evident. Although the data are not given, we found no effect of thyroid status on plasma Na+ or K+ concentrations, in accord with earlier studies (10). Effect of T3 on temperature dependence of cardiac NaK-ATPase activity. Thyroid-ablated rats were injected subcutaneously with T3 (50 pg/lOO g body wt) or the diluent on alternate days for a total of three injections. The ventricles were removed 48 h after the last injection. Ventricular microsomal Na-K-ATPase activity was measured at temperatures of from 15 to 36°C. In an Arrhenius plot (l), the linear regressions were discontinuous at 24°C for Na-K-ATPase activity from both control and T,-treated ventricles (Fig. 2), The slopes of the linear segments give the activation energies for the reaction within those temperature limits, The activation energies were identical for both the hypothyroid and T,-treated Na-K-ATPases; 28,6 kcallmol below 24°C and 18+4kcallmol above the transition temperature. Effects of T, on K, and V,,, for ATP, Na+, and K+. To characterize the effect of thyroid hormone on the kinetic parameters of cardiac Na-K-ATPase, a study was made of the dependence of the reaction on the concentration of each of the reactants. With ATP as the variable, the preparations from both control and T3treated rats conform to Michaelis-Menten kinetics in a Lineweaver-Burk plot (14) (Fig. 3). Analogous linear
ion concentrations and Na-K-ATPase
activity
in hypothyroid
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rat heart
c204
K.
10.0
D. PHILIPSON
AND
I. S. EDELMAN
0 AIK+li - vd
A
I
ATPase
I
I
I
I
‘/[ATPI
6 16 24 48 HOURS AFTER T, TREATMENT FIG. 1. Time course of changes in ventricular [K+li and microsomal Na-K-ATPase activity in response to T, given to hypothyroid rats. These data are from Table 1. See legend of Table 1 for details,
- tI 3.2
I
1
I
3.3
3.4
3.5
l/T x 103("K-') 2. Effect of T3 on Arrhenius plot of Na-K-ATPase activity in ventricular microsomes. Units of Na-K-ATPase activity are pmol Pi per mg protein per h. Results are average of two experiments. Microsomal fraction of five hearts was pooled for each experiment (i.e., 10 rats per group). Pairs of rats were injected either with the diluent or with 50 pg of Ts per 100 g body wt on alternate days for a total of three doses. Hearts were collected 48 h after last injection. FIG.
regressions
on Lineweaver-Burk plots were obtained with Na+ or K+ as the variable. T, had no significant effect on the& for ATP or the Kli, for Na+ or K+ (Table 2). In contrast, T, augmented the V,,, for Na+ 45%, for K+ 82%, and for ATP 55%. DISCUSSION
Changes in intracellular ion concentrations may modulate a variety of intracellular processes, including the
FIG. 3* Effect of T3 on dependence of cardiac Na-K-ATPase activity on ATP concentration. Hypothyroid rats were injected with three doses of diluent or Ts (SO pg/lOO g body wt) at 48-h intervals. Assays performed on microsomal fractions in an ATP regenerating system. l/V is reciprocal of enzyme activity in PmoI Pi per mg protein per h and ATP concentration is in mM. N = 5 pairs of rats. Points are means + SE.
abundance of Na-K-ATPase, and RNA and protein synthesis (3, 17, 21, 22, 25). For example, Katz and Lindheimer (12) noted a correlation between the filtered Na+ load and renal Na-K-ATPase activity in response to thyroid hormone. These findings prompted us to explore the possibility that changes in intracellular cation concentrations may mediate the T,-dependent increase in cardiac Na-KATPase. This possibility is also raised by the relatively long latent period for induction of Na-K-ATPase as compared to other cellular events, such as the early increase (maximal at 6 h) in sensitivity to catecholamines evoked by T, (3). Thus, if T3 increases the permeability to Na +, thereby increasing ENa+],-early in the time course, this might induce an increase in the number of Na+ pumps at 24 h. This possibility was tested by analyzing the time course of the ionic and enzymic responses to a single injection of T, in the thyroid-ablated (Y) rat, No effect of T, on [Na+]i or intracellular water content was observed at 6-48 h after injection. In an earlier study, Ismail-Beigi and Edelman (10) found that administration of T3 in repeated doses over a I-wk period significantly lowered cardiac [Na+]i and raised [K+]i* In the present study, a single injection of T, did not elicit the change in [Na+]i but increased [K+]i significantly at 24 and 48 h. Moreover, the increases in [K+]i correlated well with the increases in ventricular Na-K-ATPase activity (Table 1 and Fig. 1). Thus, no evidence was adduced for a mediating role of changes in ion composition on the response of Na-KATPase to T,. The results are compatible with the inference that the changes in ion composition are a consequence of a primary increase in the activity of the Na+ Pump* If thyroid hormone induces the synthesis of Na-KATPase isozymes or of lipid constituents that are part of the microenvironment of the enzyme, the temperature
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THYROID TABLE
from
HORMONE-INDUCED
c205
Na-K-ATPase
2. Effect of T, on kinetic parameters for Na+, K+, and ATP uf Na-K-ATPase ventricular microsomal fructions Thyroid
Hypothyroid Hypothyroid P
K li2, mM
Status
+ T,
K 111,mM
Nat
K’
35.6 5 4.1 37.0 k 5.4
2.4 ir 0.2 3.1 2 0.3
NS
NS
Values are means * SE. Hypothyroid doses and the ventricles were analyzed
v mrlb, Fmol
ATP
0.42 0.38
f 0.09 2 0.05 NS
per h
K+
ATP
11.3 -t- 2.4 16.4 + 3.5
13.6 k 2.1 24.7 k 4.7
11.8 2 1.3 18.3 -t- 2.8
co.05
co.05
co.05
rats were injected with diluent or 50 pg of T:, per 100 g body 48 h after the final injection. N = 5 pairs of rats.
dependence of the reaction might be significantly altered. This possibility deserved consideration since NaK-ATPase activity is sensitive to the phospholipid microenvironment (5, 7) and thyroid hormone induces the synthesis of both proteins and membrane-associated phospholipids (24). Lipid fluidity influences enzyme activity and phase changes in membrane lipids have been detected as discontinuities in the Arrhenius plot of NaK-ATPase activity (5, 7). The results (Fig. 2) show no effect of T, on either the transition temperature or of the activation energy of the reaction in the Arrhenius plot. The fact that T, did not alter the transition temperature implies that the local lipid environment of Na-K-ATPase was not drastically modified. The lack of an effect of T3 on the activation energy (above or below the transition temperature) implies that the characteristics of the Na-K-ATPase system are independent of thyroid status. Thyroidal enhancement of Na-K-ATPase may result from synthesis of additional Na+ pump units or from increased reactivity of a fixed number of pump sites. In the former case, induction of an isozyme might be revealed by changes in the affinities of the enzyme for ATP, Na+, and K+. In the latter case, the affinities for these substrates might also be modified. LineweaverBurk plots, however, revealed no significant T,-dependent changes in the affinities; K, for ATP and K,,, for Na+ and K+ (Table 2, Fig. 3). The maximum velocities
Pi per m g Protein
Na’
wt on alternate
days for a total
of three
of the ATPase reaction, however, were significantly increased with all three substrates. Similar effects of T, on renal Na-K-ATPase have been reported by Lo et al. (15) for Na +, K+, and ATP, and by Asano et al. (2) on skeletal muscle for ATP. These results are consistent with TZ induction of the synthesis of Na+-transport enzymes with the same properties as those present in the athyroid state. That thyroid hormone induces the de novo synthesis of the catalytic subunit of renal cortical Na-K-ATPase has been inferred from experiments on the effects of T, on: 1) the number of [“Hlouabain binding sites, 2) the incorporation of .72Pfrom ATa2P(y) into the phosphorylated intermediate, and 3) the incorporation of radiolabeled amino acids into the large subunit of Na-K-ATPase (15, 16). The results obtained on the rat heart are consistent with these findings and with the presence of high-affinity nuclear receptors in the myocardium (19). The authors are grateful for the excellent technical assistance provided by Ms. Laurie Lippitt. This investigation was supported by Program Project Grant HL06285 from the National Heart and Lung Institute and was performed during the tenure of a Postdoctoral Traineeship provided by the National Heart and Lung Institute Grant No. HI,-05725 to K. D. Philipson. Present address of K. D. Philipson: Dept. of Physiology, University of California, Los Angeles, Calif. 90024. Received
for publication
19 August
1976.
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