Lymphocyte Na,K-ATPase is reduced in aged people

Lymphocyte Na,K-ATPase is reduced in aged people

Lymphocyte Na,K-ATPase Chiarella BOZZO, Maria Goria, Claudio Marengo, Is Reduced in Aged People Saverio Marena, Fabrizio Veglia, and Gianfranco Pa...

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Lymphocyte Na,K-ATPase Chiarella BOZZO, Maria Goria, Claudio Marengo,

Is Reduced in Aged People

Saverio Marena,

Fabrizio Veglia, and Gianfranco

Pagan0

Na,K-ATPase-dependent %b uptake, maximum velocity (V,,,), Michaelis constant (K,) of the uptake, and [3H]-ouabain binding were investigated in the lymphocytes of 10 elderly subjects (age > 60 years). and in 10 middle-aged (41 to 60 years) and 10 young controls (age 5 40 years). %b uptake was reduced in elderly versus both middle-aged and young subjects (20.14 ? 3.30 ~35.50 * 2.67, P = 602, and w 36.53 * 4.49 nmol, P = .012), as was the number of [3H]-ouabain binding sites per cell (32,662 f 2,215 Y 40,420 f 1 ,184, P = .Ol 1, and v 46,596 + 1,349, P = .014). V,,, was reduced in elderly v young subjects (1.20 + 0.10 Y 1.64 & 0.13, P = .034), but not versus the middle-age group (1.20 + 0.10 v 1.54 ? 0.12 nmol - min-’ , NS). K, was no different among the three groups. No differences were found between middle-aged and young subjects. Significant correlations were observed between age and Na,K-ATPase-dependent %b uptake (r = -.620, (r = -439, P = .024), and [‘HI-ouabain binding sites (r = -646, P = .002). Moreover, the site P = 60009). v,,, number was positively correlated with both uptake (r = .635, P = .002) and V,, (r = .554, P = .Ol 1 I. These differences were observed both in women and men. We conclude that there is an age-dependent reduction in lymphocyte Na,K-ATPase activity, which is fully manifested over 60 years, and that this alteration is probably due to the reduced number of functional units of Na,K-ATPase in advancing age. 0 7990 by W. B. Saunders Company.

P

HYSIOLOGICAL AGING is accompanied by metabolic defects, including reduced insulin sensitivity with impaired glucose tolerance, ‘,2likely reduced resting oxygen consumption,3.4 and a slight decrease in T3 and T4 production rates.576 Membrane Na,K-ATPase is stimulated by insu1in7z8and makes a significant contribution to basal’ and, to a lesser extent, thyroid hormone-induced” cell thermogenesis. Thus, it may be supposed to be altered in advancing age. Moreover, Na,K-ATPase is very sensitive to changes in membrane lipid fluidity, which is itself reduced in aging as a result of the increased cholesterol/phospholipid ratio.“.” Our previous studies have shown that both in vitro insulin binding’ and glucose transport2 are membrane events that are impaired in normal aging, together with reduced in vivo insulin sensitivity.‘.2 It has been reported that Na,K-ATPase levels decrease in tissue homogenates from old anima1s,‘3”5 but this phenomenon does not seem uniform in all organ systems. Studies of Na,K-ATPase in elderly subjects provide contrasting views on human aging. Reduced Na,K-ATPase activity and/or higher intracellular sodium values with increasing age have been reported for both sexes’6-‘8or for women only,‘9’2’ whereas other investigators failed to find any differences between young and elderly subjects.22-24More recent investigations 25.26 demonstrated an altered response in the induction of Na,K-ATPase by thyroid hormone in human senescence. This report describes a further study of this topic, in which Na,K-ATPase levels (as reflected by rubidium 86 uptake and

From the Institute of Internal Medicine, University of Turin, Italy. Supported by a grant from the Minister0 Pubblica Istruzione (MPI60% 1987). Address reprint requests to Gianfranco Pagano, MD, Istituto di Medicina Interna. Universitb di Torino, Corso Polonia 14. 10126 Torino. Italy. a 1990 by W.B. Saunders Company. 00260495/90/3908-0005$03.00/0

808

‘H-ouabain binding) were measured in human peripheral lymphocytes from young, middle-aged, and elderly subjects. Fresh peripheral lymphocytes were chosen as the cell model, because they show higher ionic flux than erythrocytes.27~28 MATERIALS AND METHODS

The subjects were 30 normotensive white volunteers free from endocrine dysfunctions, divided into three age groups of 10 subjects: elderly (~60 years), middle-age (41 to 60 years), and young (17 to 40 years). None of them were on drug therapy. All young subjects displayed a normal response to the oral glucose tolerance test, as did seven of 10 middle-aged subjects and three of 10 elderly subjects: the other subjects showed an impaired glucose tolerance according to the National Diabetes Data Group?9 Normal plasma T3 and T4 levels were checked in the three groups. All subjects were on a balanced diet (30 kcal/kg: 60% carbohydrates, 30% lipids, 10% proteins, -30 g fibers) and engaged in moderate physical activity. All subjects gave informed consent. The clinical features of the three groups are shown in Table 1. After an overnight fast, 150 mL of venous blood was withdrawn, collected in tubes containing preservative-free heparin (20 IE/mL), and centrifuged on a Ficoll-Sodium Metrizoate gradient (d = 1.077 g/mL, Nycomed, Oslo, Norway) in accordance with Boyurn’s method.” The mononuclear cell layer was deprived of monocytes by adherence to plastic flasks at 37OC, 5% CO, for 1 hour. Contamination with monocytes in the final lymphocyte suspension was less than 2%. Parameters evaluated as an index of lymphocyte Na,K-ATPase activity were the uptake of 86Rb (an analogue of potassium) and the kinetic parameters maximum velocity (V,,,) and Michaelis’ constant (K,). The number of Na,K-ATPase per cell was established by [‘HI-ouabain binding. Specimens for evaluating of plasma T3 and T4 concentration (RIA Method, Nuclear Medical Laboratories, Irving, TX), insulin (RIA Method, Corning, Medfield, MA), and glucose (Glucoseanalyzer 2, Beckman Instruments, Fullerton, CA) levels were also collected. 86Rb Uptake

s6Rb uptake was measured as previously described,7,“,32 and used as in our previous study.13 All assays were performed in duplicate at 37“C in Hepes buffer

Metebotism, Vol39, No 8 (August). 1990: pp 808-814

LYMPHOCME

Na,K-ATPase AND AGING

809

Table 1. Clinical Data of Elderly, Middle-Aged,

and Young Subjects

Plasma Glucose Fasting

Age

Subject

Sex

(v)

8MI

RI

Z-HOW

Fasting

(mg/dL)

Z-Hour

(plJ/mL)

T3

(ng/mL)

T4 WmL)

Elderly C.I.

F

91

19

86

189

B

74

107

D.A.

F

89

21

83

149

6

75

170

9.0

G.G.

M

82

21

98

151

6

53

163

10.6

L.G.

M

88

23

100

193

11

65

122

8.3

L.M.T.

F

68

27

85

110

13

55

184

11.1

M.M.

F

65

22

75

86

11

79

102

5.2

M.O.

F

78

22

93

199

7

193

117

15.2.

R.G.

M

75

21

114

197

15

96

143

6.5

RX.

F

76

17

76

90

5

55

120

7.8

S.V.

M

81

21

89

195

5

123

177

11.0

77.1

21.4

89.9

155.9

8.7

86.8

140.5

9.6

2.6

0.7

3.5

13.7

1.1

12.9

9.2

0.9

Mean SEM

11.5

Middle-aged N.N.

F

55

19

80

120

12

68

142

9.3

D.lG.

M

54

18

75

133

20

107

115

8.7

C.E.

F

44

25

90

92

9

99

138

6.7

M.M.

M

52

25

68

99

14

89

185

11.8

F.G.

M

48

20

72

125

15

43

120

9.4

F.C.

F

60

21

91

112

7

30

103

7.7

R.I.

M

56

20

70

160

18

122

93

8.3

L.J.

F

58

18

93

162

8

72

85

10.8

C.‘V.

M

41

19

76

100

6

25

150

9.5

G.B.

M

59

25

101

155

10

64

156

8.4

52.7

21.0

81.6

125.8

11.9

71.9

128.7

9.1

2.0

0.9

3.4

7.8

1.4

9.8

9.3

0.4

Mean SEM Young B.M.

M

32

20

75

92

6

15

137

8.2

B.M.L.

F

27

20

72

97

8

41

140

8.0

C.G.

M

23

24

92

115

9

36

115

9.0

C.P.

F

39

19

81

121

13

38

139

8.3

D.A.

M

37

20

74

148

11

60

128

7.6

G.J.

M

38

24

66

139

12

60

117

9.3

L.S.

M

17

19

95

133

18

85

120

6.2

R.W.

F

37

19

94

99

11

48

98

5.0

R.A.

F

40

26

71

124

16

102

133

8.0

V.F.

M

31

26

89

125

18

65

152

8.3

32.1

21.7

80.9

119.3

12.2

55.0

127.9

7.8

2.3

0.8

3.2

5.6

1.2

7.5

4.7

0.4

Mean SEM

NOTE. Statistical differences among groups ere not significant for all parameters, except for age (P = .00009),

in all comparison) and 2-hour plasma

glucose values between elderly and young (P = .024). Abbreviations: BMI, bodY mass index (weight in kg/height in m*); IRI, immunoreactive insulin. *Normal values of free T3 end free T4 were recorded in patient M.O. (FT3 = 17.5 pg/mL: FT4 = 1.3 pg/mL).

(Hepes 100 mmol/L, glucose 10 mmol/L, MgSO, 1.2 mmol/L, NaCll20 mmol/L, CH,COONa 15 mmol/L, EDTA-Na, 1 mmol/ L, KC1 S mmol/L, human serum albumin l%, pH 7.4) on 4 x lo6 fresh lymphocytes, unless otherwise stated. Transport was begun with the addition of a tracer dose (30 rmol/L) of 86RbC1(specific radioactivity, 0.037 to 1.29 TBq/g, New England Nuclear, Boston, MA). Fifteen minutes later, 86Rbuptake was stopped by transferring the assay tubes into an ice bath. Cells were separated from the radioactive medium by the di-n-butyl phthalate centrifugation technique and then counted in a Packard scintillation counter.‘.” The Na,K-ATPase-dependent uptake was therefore obtained by subtracting the amount taken up in the presence of a high ouabain concentration (lo-’ mol/L) from the total uptake. The highly

purified ouabain was provided by Sigma Chemical (St Louis, MO). For the purpose of determining the V,,, and K, of the *6Rb transport, the lymphocytes were washed with a K+-free buffer and then resuspended in a Hepes buffer containing from 0 to 10 mmol/L RbCl, instead of KCl.‘,” Choline chloride was used to keep constant ionic strength and osmolarity. 86RbC1 was then added and the experiment continued as described. The reciprocal of the rate of 86Rb uptake in relation to the reciprocal of the unlabeled RbCl concentration was then plotted in the linear diagram of Lineweaver-Burk to calculate the kinetic parameters V,,, and K,. Since 86Rb was used as a potassium tracer, in keeping with the demonstration of a direct relation between its uptake and that of 42K,‘4 and since the potassium concentration in the incubation

80220

810

medium proved to be constant in our experiments, the two parameters may be considered as equivalent.

rP.

r-P..Oval

90

rNs1pT .

f3H]Ouabain Binding

ET AL

.0(2-j

-P=JJo21

Ft=

.

[‘HI-ouabain binding was measured by a saturation binding technique. r5~r6The lymphocytes were washed with K+-free Hepes buffer, then suspended at the concentration of 4 x lo6 and incubated in the same buffer with increasing amounts (from 2.25 to 135 nmol/L) of [3H(G)]-ouabain at 37OCfor 3 hours in a water shaking bath. The cells were then processed as described above. Nonspecific binding was determined in parallel by measuring residual radioactivity in the presence of 10’ higher concentrations of unlabeled ouabain (IO’ x 2.25 to 10’ x 135 nmol/L). The nonspecific binding was below 5%. The same [‘H(G)]-ouabain batch (specific radioactivity, 0.540 TBq/mmol, New England Nuclear) was used for all assays. All measurements were performed in duplicate. The number of specific ouabain binding sites per cell and the dissociation constant (K,J were calculated by Scatchard analysis.37

h * f ”

60

% ; x 5 I "

Statistical Methods The data were expressed both as individual values and means 2 SEM. Statistical analysis was performed with one-way ANOVA, Student’s r test for independent samples, and Pearson’s analysis of linear regression.” Two-tailed Pvalues c.05 were regarded as significant.

Fig 1. Lymphocyte %b uptake in 10 elderly aged (A), and 10 young subjects (0).

(0).

10 middle-

RESULTS

Mean results in elderly, middle-aged, and young subjects are reported in Table 2; total and Na,K-ATPase-dependent 86Rb uptake were reduced in elderly versus middle-aged and young groups as were the number of [3H]-ouabain binding sites. No differences were recorded between middle-aged and young groups for these parameters. The passive (non-Na,KATPasedependent) uptake measured in the presence of 10m3 mol/L ouabain was similar in all groups. Individual data of 86Rb uptake are shown in Fig 1. The Scatchard analysis of [3H]-ouabain binding curves are represented in Fig 2.

V,,, was reduced in elderly subjects versus young controls, but not versus middle-aged subjects, and no differences were recorded between middle-aged and young groups (Table 2). Lineweaver-Burk plot is shown in Fig 3. The constants K, and K, were similar in the three groups (Table 2). Variances among the three groups, as assessed with ANOVA test, were significant for total (P = .013) and Na,K-ATPase-dependent 86Rb uptake (P = .006) and for [3H]-ouabain binding sites (P = .006). Significant correlations were observed between age and

Table 2. Mean Results in Elderly, Middle-Aged,

and Young Subjects

%b Uptake* Total lnmoll

Non-Na,K-ATPaseDependent (nmol)

Na,K-ATPaseDependent (nmoll

%0uabain Binding Vma): (nmol . min-‘)

K, (mmol/L)

3H-Ouabain BindingSites hI./Cdl~

K, 110-9 mol/L)

Elderly subjects (age > 60 yr) 4.27

20.14

1.20

3.96

32,662

7.49

SEM 3.79 0.96 Middle-aged subjects (age 4 l-60 yr)

Mean

3.30

0.10

0.78

2,215

1.35

40,420

9.09

Meall

24.42

39.12

3.50

35.60

1.54

2.62

0.16

2.67

0.12

0.28

40.79

4.26

36.53

1.64

3.56

5.10

0.49

4.49

0.13

0.35

SEM 2.69 Young subjects (age 5 40 yr) Mean SEM P valuest

1,184 40,596 1,349

1.40 9.94 1.95

a b

,005 NS

NS NS

,002 NS

NS

NS

,011

NS

NS

NS

NS

NS

c

,019

NS

,012

.034

NS

,014

NS

+The values are referred to 4 x lo6 lymphocytes. tThe differences were evaluated by t test for unpaired samples. a, Elderly “middle-aged subjects; b, middle-aged “young subjects; c, elderly vyoung subjects.

LYMPHOCYTE

Na,K-ATPase

AND AGING

811

The number of Na,K-ATPase per cell was measured by the saturation binding curve (not shown) with increasing

200

100 Bound

(fmol

300

- 4x1oeC~IS)

Fig 2. Scatchard plot of [3H]-ouabain binding data in 10 elderly (0). 10 middle-aged (A), and 10 young subjects (0): the intersections on the X-axis represent the values of maximal specific ouabain binding.

Na,K-.4TPase-dependent 86Rb uptake (Fig 4), V,,, (r = -.439, P = .024), and [3H]-ouabain binding sites (r = -.648, P = .002). Moreover, the site number was positively correlated with both uptake (r = .635, P = .002) and V,,, (r = .554, P = ,011). Data of Na,K-ATPase-dependent 86Rb uptake were analyzed separate for males and females: differences among the three age groups were not sex-dependent (Table 3) and correlations between age and uptake were significant both in women (r = -.634, P = .014) and men (r = -.646, P = ,006).

concentration of [3H]-ouabain (2.25 to 135 nmol/L), a specific inhibitor of this enzyme. The Scatchard piot of the binding data gives a rectilinear diagram, from which the binding sites per cell are extrapolated (Fig 2). This method for calculating the number of Na,K-ATPase per cell is generally accepted, even though variations in the composition of the membrane lipid bilayer may have different effects on the transmembrane ion flux and the ouabain bound on the outer surface of the membrane.42 The concomitant decrease of 86Rb uptake and number of [3H]-ouabain binding sites and the significant correlations between binding sites and both uptake and V,,, observed in this study indicate that the two phenomena are concordant. Since a reduction in the affinity or the molecular activity of Na,K-ATPase, or the presence of functionally different sites, should have altered the K, for Rb/K uptake, and the K, is unchanged in this study, the decreased number of Na,KATPase units in our aged subjects seems to be the primary event in depressing Na,K-ATPase activity. Therefore, the reduced Na,K-ATPase number in elderly subjects led to the impairment of 86Rb uptake through the reduced V,,,, which is mainly dependent on the number of Na,K-ATPase. It has been shown that sex, as well as race:’ can influence Na,K-ATPase activity. However, no difference was observed between males and females in our series as a whole, nor within each group (Table 3). A negative correlation between age and Na,K-ATPasedependent 86Rb uptake was found in all subjects (r = ,620 P = .00009), as in both women (r = -.634 P = .014) and men (r = -.646P = .006). It should be emphasized that our results were obtained with an isolated, purified lymphocyte preparation free from other polymorphonuclear and mononuclear leukocytes. This ensures a greater uniformity in the results compared with the

DISCUSSION I

These findings are in line with the view that Na,K-ATPase is less active in subjects over 60 years of age compared with those under 60 years. It is important to emphasize that the two other age groups (range, 41 to 60 years and 17 to 40 years) presented similar data and no alteration of Na,KATPase activity occurred before the sixth decade in our subjects. Some reports described a similar result on insulin sensitivity, which is decreased over 60 years,39v40whereas a most marked decline between ages 20 and 45 years was reported by DeFronzo.4’ The reduction in 86Rb uptake observed in the elderly group corresponds to the active transport rate (Na,K-ATPasedependent 86Rb uptake) (Fig 1). In particular, this reduction depends on a lower V,,, (Table 2), measured in presence of increasing concentration of unlabeled RbCl (0 to 10 mmol/ L), as shown in Fig 3 (ie, mean V,,, was decreased in elderly towards both young, P = .034, and middle-aged subjects, P = .067, even if the latter difference was not statistically significant). Affinity for Rb+, which is regarded as a K+ analogue,34 is not altered by aging, since there is no significant change in K, (Fig 3, Table 2).

/

/o

Fig 3. Linear diagrams (Lineweaver-Burk plot) of the rate of “Rb uptake as a function of RbCl concentration on lymphocytes from 10 elderly (0). 10 middle-aged (A), and 10 young subjects (0): the intersections on the X-axis represent K,-’ and those on the Y-axis define V,,,-’ .

BOUO

812

r = -0.a20

.

Pco.ocol

30

do yr.

a0

40 Fig 4. Relationship between Na.K-ATPase-dependent “Rb uptake and age in all subjects (0 elderly; A middle-aged; 0 young subjects).

Table 3. Na.K-ATPase-Dependent

%b

ElderlySubjects

MiddleAged (4 l-60

(60 yr)

Subject

Uptake*

use of either all the mononuclear cells,25 or red blood cells, which are anuclear, lack mechanisms for the repair of cell damage, and display reduced metabolism and ion flu~.~‘,~* It should also be emphasized that our subjects were neither hospitalized nor overweight. They were negative for family history of diabetes, and their glucose load (75 g) curves were nondiabetic: no subjects reached the limit value of 200 mg/dL at the second hour after load,29 as shown in Table 1. Our observation of reduced Na,K-ATPase in the elderly is in line with the findings obtained on red blood ceils by Beilin et alI9 and Naylor et al”‘.” m . aged females. A similar result was also obtained by Gambert et alI6 in males, but these data must be carefully analyzed, since they were obtained on red blood cells from nonfasted subjects. Witkowski et al” reported that both active (Na,K-ATPase-dependent) and passive lymphocyte transport of 86Rb were already lower in subjects of both sexes over 30 years, but they did not go into detail, since neither V,,, and K, nor [3H]-ouabain binding sites were investigated. Recently, Gambert25 noted a reduced Na,K-ATPase response to T3 on mononuclear cells from both men and women, but no difference in basal activity. However, Gambert’s study must be considered with some caution, since T3 concentration (10-j mol/L) was greatly over the physiological range, and yet these findings were achieved on mem-

Uptake in 10 Elderly, 10 Middle-Aged,

Data Examined Separately

and 10 Young Subjects:

for Males and Females Subjects

Young Subjects

yr)

Subject

(540 Uptake*

VT)

Subject

Uptake*

26.31

b.1.

20.43

N.N.

40.34

B.M.L.

D.A.

10.54

C.E.

39.14

C.P.

39.50

L.M.T.

30.55

F.C.

28.96

R.W.

35.62

M.M.

35.00 20.15

L.J.

43.61

R.A.

29.43

32.72

M.O. A.C.

17.10

Mean

22.30

SEM

3.66

Mean

38.0 1

Mean

SEM

3.16

SEM

2.98

Males G.G.

5.45

D.G.

40.93

B.M.

31.73

L.G.

34.76

M.M.

24.14

C.G.

25.40

KG.

22.42

F.G.

21.14

D.A.

30.34

S.V.

5.01

R.I.

46.12

G.J.

26.58

C.V.

39.00

L.S.

75.15

G.B.

32.65

V.F.

45.25

Mean

34.00

Mean

39.08

Mean SEM P valuest

16.91 7.20

SEM

4.02

NS All Subjects

7.77 NS

Uptake 14)

Mean SEM

Males (n =

SEM

NS

Females (n =

16)

Mean SEM

P veluest *The uptake was expressed es nmol of Rb for 4 x 1Or lymphocytes. TThe difference was evaluated by t test for unpaired samples.

ET AL

29.76 2.64 31.63 4.16 NS

LYMPHOCZYTE Na,K-ATPase AND AGING

813

branes from heterogeneous mononuclear cell population (86% lymphocytes). Guernsey et a126reported extreme alteration in thyroid hormone regulation of Na,K-ATPase during human senescence in cultured fibroblasts using three experimental models: genetic premature aging syndrome, aging in vitro, and early passage cells from aged patients. Although relevant, these data are not conclusive, because of the paucity of the case series. Finally, other investigators found no age-related difference in Na,K-ATPase.22-24 It is possible that these discordances, like those in recent studies on obesity,32*33*42 may be partially ascribed to differences in method and technique. In fact, some investigators measured Na,K-ATPase changes from the hydrolysis of 32P-ATP or the release of inorganic phosphate, whereas others measured the transport of ions mediated by Na,KATPase and/or the [3H]-ouabain binding (as in our study), or confined themselves to measure the amount of intracellular Na’ and K’. As far as the relationship between age and *6Rb uptake by peripheral lymphocytes is concerned (Fig 4), it is evident that although a good correlation is present (r = -.620, P = .00009), the uptake in the two groups under 60 years is quite the same (r = - .222, P = .347), while in subjects over 60 years the correlation is stronger (r = -.747, P = .013). This corresponds to a impairment of Na,K-ATPase activity, which is strictly related to the advanced age, rather than a progressive age-dependent decline. Briefly, there is a trend toward decreasing Na,K-ATPase activity over 40 years, which becomes more evident and fully manifested over 60 years. The data disagree with the report of Witkowski et al,” who studied a large group of blood donors and showed a significant decrease of both active and passive 86Rb uptake in both middle-aged and older subjects in comparison to young subjects. The lack of information about the clinical characteristics of the subjects (weight, diet, blood pressure, drug consumption, etc) leaves uncertainty about the quality of standardization of the case series. We can underline that diet, physical training, and no drug consumption were standardized in our subjects. Our previous reports have shown a reduction in insulin receptor,s’,* and in glucose carriers in physiological aging,* as reported also by other investigatorsSM This phenomenon

could be secondary to a generalized, though not clearly demonstrated, reduction of protein synthesis. A more plausible explanation is an alteration of the chemical composition of the cell membrane (increased cholesterol:phospholipid ratio),‘* and hence the decreased plasma membrane microviscosity” observed in old age. This would lead to limitation of the lateral diffusion of proteins in the membrane, and thus to masking some Na,K-ATPase units, as well as receptors and carriers. Furthermore, increased free radical reactions, occurring in old age,45S46 may lead to membrane

alteration. We would also underline the importance of endogenous Na,K-ATPase regulation factors, such as insulin, T3 and T4, and catecholamines4’ The insulin resistance at the receptor and postreceptor level displayed by the elderly subjects is a well-known phenomenon.‘.*.” Reduced insulin binding could lead to reduced stimulation of Na,K-ATPase. The stimulation effects of catecholamines and thyroid hormones on this enzyme have been reported, and the importance of these steps on the aging process is still unknown. The consequences of depressed Na,K-ATPase activity on different cells could be of great importance, and will depend on the organ involved, and to a large extent the interaction of the pump with other cell functions: interference with the activity of enzymes (eg, pyruvic dehydrogenase) requiring K’ as cofactor; in brain and peripheral nerves support of the ion gradient required for nerve impulse; in endocrine glands secretion; in cardiac muscle - interaction with Ca++ATPase and muscle contractility; in kidney - involvement in the active reabsorption of sodium chloride; in gut involvement in the sodium-driven uptake of nutrients. At the lymphocyte level, reduced Na,K-ATPase activity could result in reduced activation in immune responses. It has already been shown that lymphocytes display a reduced proliferative response in the aged, owing to increased membrane microviscosity, which is primarily evident from the fifth decade.” In conclusion, our findings support the hypothesis that there is an age-dependent decline in lymphocyte Na,KATPase, which is fully manifested over age 60 years, and that this alteration is probably due to the reduced number of functional units of Na,K-ATPase in advancing age.

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