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Effect of repeated stress on plasma catecholamines and taurine in young and old rats
Neurobiologyof Aging, Vol. 14, pp. 359--366, 1993
0197-4580/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
Effect of Repeated Stress on Plasma Catecholamines and Taurine in Young and Old Rats L O U I S M I L A K O F S K Y , *1 N I N A H A R R I S , " A N D W O L F G A N G
H. V O G E L t
*Department of Chemistry, Penn State University, Berks Campus, Reading, PA 19610 ~Department of Pharmacology and Psychiatry and Human Behavior, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 R e c e i v e d 21 O c t o b e r 1992; R e v i s e d 5 February 1993; A c c e p t e d 22 February 1993 MILAKOFSKY, L., N. HARRIS AND W. H. VOGEL. Effect of repeatedstresson plasma catecholaminesand taurine in youngand old rats. NEUROBIOL AGING 14(4) 359-366, 1993.--The effect of age and multiple stress responses on plasma norepinephrine (NE), epinephrine (EPI), and taurine (TAU) levels were determined in F344 rats. Blood samples obtained from catheterized young (3 months) and old (24 months) animals were used to examine plasma levels of NE, EPI, and TAU under baseline conditions and in the same animals after a 30-min immobilization stress. Rats were again immobilized and blood drawn (Day 3) following a l-day rest period and, after an additional 4-day rest period (Day 7). Age differences seen between young and old rats were not the same for the three neurochemicals measured but were relatively unique for the specific biochemicals. In old animals baseline values of NE but not EPI and TAU were higher then young animals and all three values did not change for the baseline during the two additional stress exposures. Initial stress responses were similar for all three biochemicals in both age groups. Although no signs of adaptation were evident in the old animals, adaptation to immobilization was seen for EPI and TAU but not NE on the third occasion in the young animals. Correlations seen between NE and EPI in young and old rats on the first day disappeared during the second stress period but were again seen during the third stress exposure suggesting subtle indicators of repeated stress subject to adaptation. TAU values for young but not old rats correlated positively with EPI concentrations during the first stress exposure and negatively after the immobilization was terminated indicating a regulatory interaction between EPI and TAU present in young but lost in old animals. Thus, changes in levels and interrelationships of specific biochemicals during repeated stress experiences in rats may provide a good model of the aging process. Aging
Aged rats
Stress
Plasma
Norepinephrine
THE response to stress of plasma catecholamines (CA), norepinephrine (NE), and epinephrine (EPI) has been examined as a function of age. Both basal levels and stress responses in old animals have been found to differ from those seen in young animals. However, published data are rather inconsistent and sometimes contradictory. While one study reported no change in basal NE but an increase in EPI levels during aging (1), another study found increased levels of NE (14), and a third study observed no increases in both CA (13). Similarly, stress responses of the plasma CA in old animals have been reported to be increased, decreased, or unchanged as compared to young animals (1,13,14). Unfortunately, such data do not lead to general conclusions about the basal levels of the plasma CA and their stress responses as a function of age. In addition to the CA, plasma taurine (TAU) has recently been shown to be significantly affected by age and stress (16,22,26) and to attenuate the immobilized stress-induced EPI release from the adrenal glands (11). Unfortunately, these results must be inter-
Epinephrine
Taurine
Adaptation
preted with caution because in most studies blood was obtained following decapitation; a method now known to produce artefactually altered levels of TAU (15). For these reasons, we felt it necessary to re-evaluate the effects of age on basal levels of plasma NE, E, and TAU and their stress responses. In addition, young (3 months) and old (24 months) rats were each stressed three times by immobilization (30 min) to observe initial adaptation responses in individual animals as well as to search for possible relationships among the three biochemicals during rest and stress at these two ages. METHOD
Animals and Catheterization Male 3- or 24-month-old Fischer 344 rats were obtained from the National Institute of Aging and group housed in animal quarters (about 24°C, light from 8:00--20:00 h and dark from 20:00-
To whom requests for reprints should be addressed. 359
360
MILAKOFSKY, HARRIS AND VOGEL
TABLE 1 EFFECT OF STRESS AND AGING ON PLASMANE OF F344 RATS Day (pg/mL ± SEM) Time (min)
1
3 Months N 0 15 30 60 120 180 240 24 Months N 0 15 30 60 120 180 240
3
7
274 1256 955 585 506 406 373
7 ± 24 ± 134t + 97t --- 66 +- 49 4- 46 -+ 46
6 264_+ 57 1476 -+ 287; 1330 -+ 55%¶ 780 -+ 91 492 ± 44 476 - 42 374 -+ 50
580 2269 1769 1032 818 705 632
8 -+ 70:[: --- 268t'§ +- 207f'§ ± 94§ ± 73§ +- 69§ ± 71:]:
660 2737 2511 1400 868 665 803
8 - 145:[: - 335~':~ +- 297t'§'¶ +-- 182:~ ± 90§ ± 91 +- 1265
5 3 3 9 - 70 1102 ± 138t'# 1147 : 1331 641 ± 102 402 ± 72 394 ± 87 421 -+ 132
696 2475 2123 1463 911 733 759
8 +- 152 --- 240t'§ -+ 213%§ + 166"§ -+ 96§ ± 91:~ - 115
* p < 0.05 with respect to time = 0 min for each day and age; fp < 0.001 with respect to time = 0 min for each day and age; l:p < 0.05 between 3 and 24 months at the same time point; §p < 0.01 between 3 and 24 months at the same time point; ¶p < 0.01 between Day 1 and 3 at the same time point; CAp< 0.01 between Day 3 and 7 at the same time point.
8:00 h, water and Purina chow ad lib) to acclimate lbr at least I week before the experiment. In preparation for the stress experiments, animals were anesthetized with ketamine and acepromacine (100 ÷ 1 mg/kg, IP) and the right external jugular vein implanted with a silastic catheter as described previously (20,23). Catheters were exteriorized through the back and the portion outside of the animal was protected by a steel spring. Catheter and steel spring were supported by a swivel pulley above the cage. After surgery rats were housed individually with free access to food and water. Catheter patency was assured by flushing the catheter at least twice daily with heparin (1000 units/ml). The animals were then allowed to recover for 48 h before further testing.
Immobilization and Preparation of Plasma Samples At the time of the experiment, blood (about 0.3 mL) was obtained in the home cage and the animal immediately immobilized by being taped with the four paws to the laboratory bench. Blood was obtained at 15 and 30 min during this stress experience. The animal was then released, returned to its home cage, and blood drawings performed after 30, 60, 90, and 210 additional min. After 1 day of rest, the animal was again immobilized and blood drawn as described above on the 3rd day and after a 4-day rest period, again on the 7th day. All immobilizations occurred between 10:00 a.m. and noon. As reported earlier, it has been established that repeated blood samplings over the experimental period do not affect plasma TAU or CA levels (15,19,20). All blood samples were centrifuged to obtained plasma, which was stored at - 80°C in vials containing 0.04 mL of an anticoagulant solution of EGTA and reduced glutathione (Amersham Co.).
1800
2:
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1500
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1400
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AGE 3 m o
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DAY 1
-
DAY 3 -
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500 400
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30
60
120
180
240
15
30
60
120
180
240
15
30
60
120
180
240
TIME (min.) FIG. 1. The percentage changes from baseline values of plasma norepinephrine (NE) during and after repeated immobilization stress. Values are a percentage of baseline --- SEM. *p < 0.05 with respect to time = 0 rain for each day and age; **p < 0.01 with respect to time = 0 min for each day and age; ***p < 0.001 with respect to time = 0 min for each day and age.
AGE-STRESS ON CATECHOLAMINES AND TAURINE
TABLE 2 THE EFFECT OF STRESS AND AGING ON PLASMA EPI OF F344 RATS
361
efficient and linear regression analyses were used to compare individual data. Values o f p < 0.05 were considered significant. RESULTS
Day (pg/mL +- SEM) Time (rain) 3Months N 0 15 30 60 120 180 240 24Months N 0 15 30 60 120 180 240
1
3
7
7 234± 2356 ± 1745 -+ 1010 -+ 563 ± 323 -+ 239 ±
36 142, 337* 204* 150 52 52
108 2286 2688 910 539 235 245
6 ± 26 -+ 527* -+ 458*'¶ -+ 177 -+ 110 -+ 28 ± 93
218 1583 1482 679 325 394 396
5 ± 72 ± 291*'#'* ± 271t'## -+ 81 ± 151 -+ 145 ± 240
8 ± ± ± -+ -+ ± ±
73 1112~ 686~ 139 119 123 90
269 3563 3963 886 607 426 583
8 -+ 93 -+ 70~ -+ 899~ -+ 118 -+ 126 -+ 85 -+ 164
561 3247 3147 1169 689 427 478
8 ± 207 -+ 505$'§ -+ 500,'§ -+ 263 -+ 136 -+ 109 -+ 57
258 3805 3450 866 568 497 335
* p < 0.05 with respect to time = 0 rain for each day and age; tp < 0.01 with respect to time = 0 min for each day and age; :~p < 0.001 with respect to time = 0 min for each day and age; §p < 0.05 between 3 mos and 24 mos at the same time point; ¶p < 0.01 between Day 1 and Day 3 at the same time point; # p < 0.01 between Day 3 and Day 7 at the same time point; # # p < 0.001 between Day 3 and Day 7 at the same time point; *p < 0.05 between Day 1 and Day 7 at the same time point.
Plasma TAU Analysis At the time of analysis, plasma TAU samples were thawed and an aliquot (0.05 mL) deproteinized immediately with 0.40 M perchloric acid as previously described (15). Concentrations of TAU in deproteinized samples were determined by ion-exchange high performance liquid chromatography (HPLC) with fluorometric detection as previously described (15). This procedure utilized a meter long microbore HPLC stainless steel column containing spherical cation-exchange resin and fluorescence detection following post-column reaction with o-phthaldialdehyde, Lithium citrate elution buffers were used to separate TAU from other amino acids and related compounds in rat plasma. This technique has been shown to be reliable, sensitive, and valid (15).
Plasma CA Analysis The plasma levels of NE and EPI were determined by radioenzymatic analysis (Upjohn Co. " C A T - A - K I T " ) as previously described (5).
Evaluation The T A U concentrations were quantitatively determined by relating chromatographic peak heights to peak heights from a known TAU standard and an internal standard, m-fluorophenylalanine (MFP). Total mean T A U values (nmol/mL) were determined (15). CA results were expressed as mean values (pg/mL). The trapezoidal rule was used to find the area under the stress curve (AUC) for TAU, EPI, and NE curves. Statistical analysis of the data (SAS software) was performed by a three-way analysis of variance (ANOVA) for two repeated measurements (time, day) with a Tukey post hoc test or by A_NOVA with log transformations (to minimize variations among animals). Pearson correlation co-
Plasma concentrations of NE before, during, and after immobilization are shown in Table 1. For young rats, baseline levels of NE remained unchanged over the three restraints. Levels of NE rose rapidly and markedly during the stress period but returned quickly to baseline values after cessation of the stressor. Similar patterns were observed during all subsequent stress experiences with some minor but significant differences. For old rats, base values also remained unchanged but were higher on the first two occasions as compared to the young rats. During stress, levels also rose quickly and markedly but were significantly higher than those seen in the young rats. Percentage changes from baseline values for NE during and after immobilization are shown in Fig. 1. Increases were seen during and sometimes after stress but no significant differences were found between the different days or the two age groups. Plasma concentrations of EPI before, during, and after immobilization are shown in Table 2. Baseline values remained unchanged for both age groups over the 3 experimental days and were not different between these two groups. Similarly, the rise and fall of EPI levels during and after stress were similar for both age groups on the first two occasions. However, on the third day, the intensity of the stress response was less in young rats while it remained higher in the old rats. Percentage changes from baseline values for EPI during and after immobilization are shown in Fig. 2 to be increased in both age groups during stress. In the young animals, the stress response increased on the second occasion but fell to first exposure stress levels on the third stress experience. In the old animals a slow, but statistically not significant, decline in the stress values can be seen over the last two stress experiences. TABLE 3 THE EFFECT OF STRESS AND AGING ON PLASMA TAU OF F344 RATS Day (nmol/mL -+ SEM) Time (min) 3 Months N 0 15 30 60 120 180 240 24 months N 0 15 30 60 120 180 240
1
7 36.1117 -+ 84.2 -+ 60.7 ± 40.5 --54.2 -+ 43.2 -+
36.9 109 101 85.9 43.7 40.6 39.1
2.9 13, 14.4" 5.6 3.9 13.8 5.8
8 -+ 5.7 -+ 23t -+ 16" -+ 17.3 -+ 5.8 -+ 4.8 -+ 4.2
3
7
6 44.0--- 4.9 96.1 ± 12.2t 88.4 -+ 14.7" 66.4 -+ 8.3 57.3 -+ 2.7 54.3 -+ 7.1 51.9 --+ 3.6
5 32.7+ - 2.1 54.7 -+ 3 . 6 t t t ' # 46.1 ± 4 . 5 t t ' # 44.4 -+ 5.6 40.0 --- 6.0 63.6 -+ 23.5 69.3 -+ 30.7
28.4 62.6 69.8 65.7 35.5 34.8 32.3
8 -+ -+ ± -+ -+ -+ ±
4.7§ 10.5¶ 13.0" 12.3 7.2§ 4.8§ 5.3§
48.4 104 100 107 81.0 60.8 51.5
8 -+ 11.1 -+ 26.8 -+ 25 --- 24 -+ 29.1# ± 11.1 -+ 8.2
* p < 0.05 with respect to time = 0 min for each day and age; tp < 0.01 with respect to time = 0 min for each day and age; ~:p < 0.001 with respect to time = 0 min for each day and age; §p < 0.05 between 3 mos and 24 months at the same time point; ¶p < 0.05 between Day 1 and Day 3 at the same time point; # p < 0.05 between Day 3 and Day 7 at the same time point; tp < 0.05 between Day 1 and Day 7 at the same time point; t t t p < 0.001 between Day 1 and Day 7 at the same time point.
362
MILAKOFSKY, HARRIS AND VOGEL
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TIME (rain.) FIG. 2. The percentage changes from baseline values of plasma epinephrine (EPI) during and after repeated immobilization stress. Values are a percentage of baseline --- SEM. *p < 0.05 with respect to time = 0 min for each day and age; **p < 0.01 with respect to time = 0 min for each day and age; ***p < 0.001 with respect to time = 0 min for each day and age; Sp < 0.05 between Day 1 and Day 3 at the same time point; SSp < 0.0l between Day 1 and Day 3 at the same time point; #p < 0.05 between Day 3 and Day 7 at the same time point; ##p < 0.01 between Day 3 and Day 7 at the same time point.
Plasma concentrations of TAU before, during, and after immobilization are shown in Table 3. Baseline levels remained the same on all three occasions within both age groups with the exception of a lower value on the second exposure to stress in the older rats. During and after stress, TAU levels rose and fell similarly in both age groups. On the third occasion, stress values were lower in the young animals but remained higher in old animals. Percentage changes from baseline values for TAU during and after immobilization are shown in Fig. 3. Stress induced increases were significant for the young animals on the first two occasions but not the third exposure. For the old animals, increases were significant on all three occasions. AUC for all three compounds during the 30-min stress period and a 60-min period (including 30 min of stress followed by a 30 min release period) are shown in Fig. 4. For NE, AUC is relatively constant for all three immobilizations in both age groups but significantly higher for the old rats. However, the AUC for the 60rain experimental period is higher on Day 3 indicating a slower return to baseline for both age groups. The AUC for EPI is also relatively constant for the first two occasions in both age groups but shows higher values on the third occasion for the old rats. Again, on the second stress exposure return to baseline is slower for young animals only. The AUC for TAU decreases over subsequent immobilizations and is markedly lower on the third occasion for the young animals; in contrast, the values for the old rats remain relatively stable.
Correlation analyses among the three biochemicals at the three stress experiences were performed uncovering the significant correlations summarized in Table 4. Significant correlations between EPI and NE were found at different times for the young as well as the old animals mostly on the first and last stress experience. Fewer correlations were found between TAU and EPI and even fewer between TAU and NE. Specifically, TAU was correlated for the most part with EPI on the first and third exposure to stress. No major correlations were apparent for a specific biochemical in individual young animals over the 3 days of stress. However, in the old animals some consistent correlations were found. The 15 min stress values for NE correlated between Day 1 and Day 3 (r = 0.85, p < 0.01) and between Day 3 and Day 7 (r = 0.78, p < 0.05). The 15-min stress values for EPI correlated for the first two exposures (r = 0.90, p < 0.01). For TAU, only some post-stress values (time = 60, 120, and 180 min) correlated between the first and last stress exposure (r = 0.95, p < 0.001; r = 0.74, p < 0.05 and r = 0.85, p < 0.001). DISCUSSION
It is difficult to uncover general conclusions about stressinduced biochemical differences between young and old rats since different strains of rats, variations in the nature or intensity of the stressor employed, or a particular biochemical selected can give rise to different interpretations (13,16). In addition, presentation of the results can lead to different conclusions. Absolute values
AGE-STRESS ON CATECHOLAMINES AND TAURINE
363
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AGE 2 4
DAY 1
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DAY 1
AGE 3 m o -
400
Z
AGE 24
DAY 3
mo
-
DAY 3
a5o AG~ ~ ma
.,~
-
nAY 7
soo
~5o O ~oo I 150
.< 100
50
15
30 6 0 120 180 2 4 0
15
3 0 6 0 120 180 2 4 0
15
30 60 120 160 2 4 0
TIME (min.) FIG. 3. The percentage changes from baselinevalues of plasma taurine (TAU)during and after repeated immobilizationstress. Values are a percentage of baseline --+ SEM. *p < 0.05 with respect to time = 0 min for each day and age; **p < 0.01 with respect to time = 0 rain for each day and age; ***p < 0.001 with respect to time = 0 min for each day and age; Sp < 0.05 between Day 1 and Day 3 at the same time point; Ap < 0.05 betweenDay 1 and Day 7 at the same time point; /X/X/Xp < 0.001 between Day 1 and Day 7 at the same time point; @@p < 0.0l between 3 and 24 months at the same time point. including baseline values represent the actual impact of a chemical on the body during stress whereas percentages or AUC represent only the stress-induced change from the original homeostasis. Both presentations may lead to different conclusions. In this article, we consider both options. A comparison of baseline values alone for Day 1 shows that only the baseline concentrations of NE (Table 1) are higher in old animals whereas those for EPI (Table 2) and TAU (Table 3) are generally similar between both age groups. These data do not confirm the reported age difference for TAU (26) perhaps due to the fact that we obtain blood through the catheter shown to be more accurate than blood collection after decapitations (15). Elevated NE levels in resting old animals have also been reported (14) although other studies have shown either no differences (6,10,13, 27) or elevated levels of EPI (2). Similar to our studies, it has been shown in humans that older individuals exhibit higher sympathetic activity with higher plasma NE levels (21,24); probably resulting from increased NE appearance and not decreased clearance (24). Although baseline levels of all three biochemicals did not change during the sequential repeated stresses, other studies using longer periods of stress have either reported increases (6) or no increase in these resting concentrations (10). These increased levels of NE are indicative of increased sympathetic activity under resting conditions in older animals; although it could be argued that older animals may be more stressed under apparently resting conditions, and thus, show the observed higher NE levels.
As expected, levels of all three biochemicals rise significantly during the first stress exposure in both age groups. The actual values of NE (Table l) reach much higher levels in old animals resulting in a greater impact of the CA and on their target organs. However, percentage increases above baseline (Fig. 1) are not statistically different in young and old animals. In contrast, a comparison of the general stress experience in terms of AUC values shows that old animals do indeed experience a higher stress response (Fig. 4). No major differences in the stress response were seen for EPI (Table 2 and Fig. 2) and for TAU (Table 3 and Fig. 3) between the age groups regardless of the method of comparison used. Data in the literature are in partial agreement with our findings. For example, varied stress responses in young and old rats have been reported in that immobilization produced larger increases in plasma NE in young rats (14), cold stress caused larger increases in old rats (2), brief footsbock induced lower CA responses in old rats (13), and cold water immersion or shaker stress brought about the same responses in young and old rats (13,27). In humans, NE levels during upright posture were higher in older subjects as compared to younger individuals (24). Thus, the impact of EPI and TAU on target organs and their actual stress responses were the same for both age groups on the first stress experience. However, the stress response for NE was slightly higher and the actual impact of NE on the target organs was much higher in old animals. Repeated stress experiences over longer periods of time can
364
MILAKOFSKY. HARRIS AND VOGEL r.1 Z
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I r.1 >-
[ ~i~!AGE - 3 MO
80000
50000 40000
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DAY 1 I
DAY 3
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160000
120000
80000
Z <
40000 30 r a i n 60 r a i n
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4000 3000 2000
Z tO00 .< "~
<
0
30 rain 60 mln
DAY 1
30 r a i n 60 m l n
DAY 3
30 m i n 60 m i n
DAY 7
FIG. 4. The areas under the curve (AUC) for NE, EPI, and TAU during a 30-min stress period and a 30-min stress period plus a 30-min release period. Values are AUC -+ SEM. @p < 0.05 between 3 and 24 months at the same time point; $p < 0.05 between Day 1 $$ and Day 3 at the same time point; p < 0.01 between Day 1 and Day 3 at the same time point; Ap < 0.05 between Day 1 and Day 7 at the same time point;/X/Xp < 0.01 between Day 1 and Day 7 at the same time point; #p < 0.05 between Day 3 and Day 7 at the same time point; #*p < 0.01 between Day 3 and Day 7 at the same time point.
involve mechanisms of adaptation (1,6,10). In our case, animals were stressed three times but rest periods varied. In the first case, there was 1 day of rest between the first and second stress session and in the second case, there were 3 days of rest between the second and third stress session. We noticed adaptation already at the third stress exposure in spite of the fact that the animals had 4 days of rest between stress experiences. In the case of NE, the actual peak values of the stress responses (Table 1) did not change markedly in either young or old animals and remained higher in the old rats. A comparison of percentage (Fig. 1) and AUC values (Fig. 4) over the three stress experiences also did not show difference within each age group; although the return to baseline was slowed on the second stress exposure. On the other hand, in the case of EPI actual stress values (Table 2) were lower on the third
stress occasion in the young rats but remained at high levels in the old animals. This difference was not apparent when evaluated in term of percentages (Fig. 2) but was evident with AUC values (Fig. 4) where young rats showed significantly lower values on the third exposure to stress and a retarded return to baseline onDay 3. Similarly, actual stress values for TAU (Table 3) in young rats were much lower on the third occasion as compared to the first exposure and did not differ from baseline values. In the old rats, no differences are seen among all three occasions. The same conclusions were reached when comparing percentages (Fig. 3) or AUC values (Fig. 4) among the three occasions and between the two age groups. Thus, both EPI and TAU show signs of adaptation in young rats already at the third stress exposure. This was not the case for old rats where stress responses remain essentially unchanged.
AGE-STRESS ON CATECHOLAMINES AND TAURINE
365
TABLE 4 CORRELATION ANALYSIS BETWEEN PLASMA NE, EPI, AND TAU IN YOUNG AND OLD F344 RATS
Day 1
TAU
Time (Min)
3 months N 0 15 30 60 120 180 240 24 months N 0 15 30 60 120 180 240
3
EPI
TAU
7 TAU
--
NE
ns
TAU
--
NE
ns
TAU
--
7
EPI
TAU
6 ns O. 8 3 " ns
0.76* 0.84* 0.75 * - 0.79*
EPI
5
--
ns
--
ns
ns
ns
ns
ns
--
ns
--
ns
ns -ns --
0.88*
ns
ns
ns
ns
0.92*
ns
ns
ns
ns
--
ns
ns
ns
ns
ns
--
ns
--
ns
ns
ns
--
ns
--
ns
ns
ns
ns
--
ns
ns
ns
-0.94"
0.94* 0.91"
NE
ns
TAU NE TAU NE TAU NE TAU NE
-ns -ns --
TAU
--
ns
--
8 0.86**
--
NE
ns
ns
ns
ns
ns
TAU
--
ns
--
ns
--
NE
ns
ns
ns
ns
ns
TAU NE TAU NE TAU NE
--
--
ns
--
ns
ns
ns ns ns 0.79*
ns
--
ns
0.905
ns
8
0.855. ns
ns
0.975. ns
8
ns
0.71 *
ns
ns
ns
--
0.885.
--
ns
--
0.865. 0.845. 0.72*
ns
ns
ns
ns
ns
ns
0.72* 0.905.
--
ns
--
ns
--
0.90t
ns
ns
ns
ns
ns
0.79*
TAU
--
ns
--
ns
--
ns
NE
ns
ns
ns
ns
ns
ns
TAU NE
--
ns
--
ns
--
ns
ns
ns
ns
ns
ns
ns
Values are correlation coefficients; ns = not significant; *p < 0.05; 5.p < 0.01. In addition to the CA, our findings establish TAU as a sensitive stress chemical which responds quickly to stress and shows rapid adaptation in young but not old animals. This adaptation processes clearly distinguished the young from the old animals. It has been shown that various hormones including the CA affect aging (8). For example, stress-induced increases of the related stress chemical, corticosterone (CORT) were found to be similar in young and old rats on the first stress exposure but the stress response of this hormone was more prolonged and attenuated, less in old animals during chronic stress (7,17). No correlations between CORT and NE or EPI levels have been found (25) and to the best of our knowledge, no correlation between CORT and TAU has been performed. Thus, it is conceivable that stress induced increases in these hormones accelerate the aging process. On the other hand, young animals seem to protect themselves from the deleterious effects of such high levels over prolonged periods of time by adaptation which possibly reduces dangerous levels of these biochemicals, a process evidently impaired in the old organ-
isms. Thus, older individuals would be at increased risk since they would not adapt as readily and must endure higher levels of these biochemicals over time which could accelerate aging and might be responsible for the faster progression of aging seen at old ages. In the old animals, higher baseline and stress levels of NE have been shown to occur. Although the exact cause is unknown at present, two explanations can be offered. It has been shown that the NE content of blood vessels is the same in young and old rats but that there is an age-related increase in NE release due to a decline in the negative feedback mediated by prejunctional alpha-2 receptors (3). Furthermore, the release of EPI and NE from the adrenal glands increases with a decrease in phenylethylamine-Nmethyl transferase (PNMT) favoring the amount of NE released during stress (9). Thus, higher levels of NE in the old animals most probably result from changes in the biology of blood vessels and adrenal glands. Because all the measured biochemicals were determined in the same animals on each occasion, correlations of these biochemicals within each age group (Table 4) were calculated. As shown previously for young rats, baseline and stress values between NE and EPI were highly correlated (12). Again, we find this correlation on Day 1 but lose most of these correlations for the second and third stress experiences. Old rats did not show this baseline correlation between EPI and NE on the first day but exhibited this relationship during stress. Correlations were again lost on the second exposure to stress but re-emerged on the third occasion. Correlations between NE or EPI and TAU are more sporadic. For young rats, TAU values correlated positively with EPI concentrations during the first stress exposure but negatively after immobilization was terminated. These results support the suggestion (11,16) that there may be a regulatory interaction between EPI and TAU in young animals. In contrast, old animals did not show this pattern. Further evaluation of correlations among different biochemicals and their changes during repeated stress experiences might provide additional information on biochemical changes as they occur during aging. No significant correlations were observed for each biochemical over the three occasions in individual young animals. In old animals, some correlations were found indicating that old animals responded more characteristically within the three immobilizations. Studies were carried out to see if differences in the biochemical stress responses of individual rats correlated with differences in stress-induced behavioral responses, recovery from stress and, in our old rats, with time of survival. No differences were observed by global assessment of struggling during immobilization and in the recovery of the young and old rats after release from immobilization. All old rats died between 3 to 6 months after the experiment but no correlation was found between the extent of the stress response of any of the biochemicals and the subsequent time of survival. Moreover, one young rat showed normal stress responses during the first and second stress experiences for all three biochemicals studied but a drastically increased stress-response in EPI, NE, and TAU on Day 7. Average stress values for the animals after 30 rain of stress for EPI, NE and TAU on the third occasion were 1482 pg/mL, 1147 pg/mL, and 46.1 nmol/mL, respectively whereas values for this rat on Day 7 were 16000 pg/mL, 9770 pg/mL, and 391 nmol/mL. This animal died shortly after the last immobilization. While it is rare for animals to show serious health consequences after short periods of immobilization (18), it has been reported that some animals die after 30 rain of immobilization (4). Thus, the possibility that a very few animals respond to a repeated stress experiences by releasing unusually high stress levels of NE, EPI, or TAU could constitute a useful marker to predict future harm to or even death of the animal.
366
MILAKOFSKY, HARRIS AND VOGEL
In conclusion, the age differences seen between young and old rats did not manifest themselves uniformly but were relatively unique for a specific biochemical. Baseline values for NE but not for EPI and TAU were higher in old animals but did not change during the three brief stress exposures. Initial stress responses were higher for NE in old animals but similar for TAU and EPI in both age groups. Adaptation to immobilization was seen for EPI and TAU but not NE already on the third occasion in the young animals while no signs of adaptation were evident for the old animals. Correlations between NE and EPI in young and old rats seen on the first day were not observed on the second stress period
but were seen again on the third stress exposure indicating subtle effects of stress which also show some degree of adaptation. ACKNOWLEDGEMENTS We gratefully acknowledge the Penn State University Biomedical Research Support Grant, the Penn State University Gerontology Center Faculty Development Projects, the Penn State University Berks Campus Faculty Development Fund, and the Public Health Grant (AA06017) for Financial Support. We also thank Heidi Weiss and Betty M. Nsubuga for their technical assistance.
REFERENCES 1. Algeri, S.; Calderini, G.; Lomuscio, G; Rocchetti, M.; Sacchetti, G.; Toffano, G.; Ponzio, F. Differential response to immobilization stress of striatal dopaminergic and hippocampal noradrenergic systems in aged rats. Neurobiol. Aging 92:213-216; 1988. 2. Avakian, E. V., Horvath, S. M.; Volbum, R. W. Influence of age and cold stress on plasma catecholamine levels in rats. J. Auton. Nerv. Syst. 10:127-133; 1984. 3. Buchholz, J.; Duckies, S. P. Effect of age on prejunctional modulation of norepinephrine release. J. Pharmacol. Exper. Ther. 252:159165; 1990. 4. Chiueh, C. C.; Nespor, S. M.; Rapoport, S. 1. Cardiovascular, sympathetic and adrenal cortical responsiveness of aged Fisher-344 rats to stress. Neurobiol. Aging 1:157-63; 1980. 5. Coyle, J. T.; Henry D. Catecholamines in fetal and newborn rat brain. J. Neurochem. 21:61-67; 1973. 6. Dobrakovova, M.; Kvetnansky, R.; Oprsalova, Z.; Macho, L. The effect of chronic stress on the activity of the symphatheticadrenomedullary system. Bratislavske Lekarske Listy 91:587-592; 1990. 7. Erisman, S.; Carness, M.; Takahashi, L. K.; Lent, S. J. The effects of stress on plasma ACTH and corticosterone in young and aging pregnant rats and their fetuses. Life Sci. 47:1527-1533; 1990. 8. Everitt, A.; Meites, J. Minireview: Aging and anti-aging effects of hormones. J. Gerontol. Biol. Sci. 44:B139-147; 1989. 9. Fernandez-Knz, J.; Bukhari, A. R.; Hernandes, M. L.; Alemany, J.; Ramos, J. A. Sex- and age-related changes in catecholamine metabolism and release of rat adrenal gland. Neurobiol. Aging 10:331-335; 1989. 10. Konarska, M.; Stewart, R. E.; McCarty. R. Habituation of plasma catecholamine responses to chronic intermittent restraint stress. Psychobiol. 18:30-34; 1990. 11. Kuriyama, K.; Nakagawa, K. Role of taurine in adrenal gland: A preventive effect on stress-induced release of catecholamines from chromaffin granules. In: Huxtable, R.; Barbeau, A., eds. Taurine. New York: Raven Press, 1976:335-345. 12. Livezey, G. T.; Balabkins, N.; Vogel, W. H. The effect of ethanol (alcohol) and stress on plasma catecholamine levels in individual female and male rats. Neuropsychobiology 17:193-198; 1987.
13. McCarty, R. Age-related alterations in sympathetic-adrenal medullary responses tostress. Gerontology 32:172-183; 1986. 14. Michalikova, S.; Balazova, H.; Jezova, D.; Kvetnansky, R. Changes in circulating catecholamine levels in old rats under basel conditions and during stress. Bratislavske Lekarske Listy 91:689-693; 1990. 15. Milakofsky, L.; Hare, T. A.; Miller, J. M.; Vogel, W. H. Comparison of amino acid levels in rat blood obtained by catheterization and decapitation. Life Sci. 34:1333-1340; 1984. 16. Milakofsky, L.; Hare, T. A.; Miller, J. M.; Vogel, W. H. Rat plasma levels of amino acids and related compounds during stress. Life Sci. 36:753--761; 1985. 17. Odio, M.; Brodish, A. Age-related adaption of pituitaryadrenocortical responses to stress. Neuroendocrinology. 49:382-388; 1989. 18. Pare, W. P. Immobilization stress not necessarily fatal for aged rats. Exp. Aging Res. 11:179--181; 1985. 19. Pashko, S. Studies with amphetamine using the chronically catheterized rat as a model. Thesis Thomas Jefferson University; 1979. 20. Pashko, S.; Vogel, W. H. Factors influencing the plasma levels of amphetamine and its metabolites in catheterized rats. Biochem. Pharmacol. 29:221-225; 1980. 21. Roubein, I. F.; Embree, L. J.; Jackson, D. W. Changes in catecholamine levels in discrete regions of rat brain during aging. Exp. Aging Res. 12:193-196; 1986. 22. Scholz, N. Significance of the taurine-glycine ratio as an indicator of stress. Bull. Environ. Contain. Toxicol. 38:15-21; 1987. 23. Upton, R. A. Simple and reliable method for serial sampling of blood from rats. J. Pharm. Sci. 64:112-114; 1975. 24. Veith, R. C.; Featherstone, J. A.; Linares, O. A.; Halter, J. B. Age differences in plasma norepinephrine kinetics in humans. J. Gerontology 41:319-324; 1986. 25. Vogel, W. H.; Jensh, R. Chronic stress and plasma catecholamine and corticosterone levels in male rats. Neurosci. Let. 87:183-188; 1988. 26. Wallace, D. R.; Dawson, Jr., R. Decreased plasma taurine in aged rats. Gerontology 36:19-27; 1990. 27. Yamamoto, J.; Nakai, M.; Natsume, T. Cardiovascular responses to acute stress in young-to-old spontaneously hypertensive rats. Hypertension 9:362-370; 1987.
0197-4580/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
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Effect of Repeated Stress on Plasma Catecholamines and Taurine in Young and Old Rats L O U I S M I L A K O F S K Y , *1 N I N A H A R R I S , " A N D W O L F G A N G
H. V O G E L t
*Department of Chemistry, Penn State University, Berks Campus, Reading, PA 19610 ~Department of Pharmacology and Psychiatry and Human Behavior, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 R e c e i v e d 21 O c t o b e r 1992; R e v i s e d 5 February 1993; A c c e p t e d 22 February 1993 MILAKOFSKY, L., N. HARRIS AND W. H. VOGEL. Effect of repeatedstresson plasma catecholaminesand taurine in youngand old rats. NEUROBIOL AGING 14(4) 359-366, 1993.--The effect of age and multiple stress responses on plasma norepinephrine (NE), epinephrine (EPI), and taurine (TAU) levels were determined in F344 rats. Blood samples obtained from catheterized young (3 months) and old (24 months) animals were used to examine plasma levels of NE, EPI, and TAU under baseline conditions and in the same animals after a 30-min immobilization stress. Rats were again immobilized and blood drawn (Day 3) following a l-day rest period and, after an additional 4-day rest period (Day 7). Age differences seen between young and old rats were not the same for the three neurochemicals measured but were relatively unique for the specific biochemicals. In old animals baseline values of NE but not EPI and TAU were higher then young animals and all three values did not change for the baseline during the two additional stress exposures. Initial stress responses were similar for all three biochemicals in both age groups. Although no signs of adaptation were evident in the old animals, adaptation to immobilization was seen for EPI and TAU but not NE on the third occasion in the young animals. Correlations seen between NE and EPI in young and old rats on the first day disappeared during the second stress period but were again seen during the third stress exposure suggesting subtle indicators of repeated stress subject to adaptation. TAU values for young but not old rats correlated positively with EPI concentrations during the first stress exposure and negatively after the immobilization was terminated indicating a regulatory interaction between EPI and TAU present in young but lost in old animals. Thus, changes in levels and interrelationships of specific biochemicals during repeated stress experiences in rats may provide a good model of the aging process. Aging
Aged rats
Stress
Plasma
Norepinephrine
THE response to stress of plasma catecholamines (CA), norepinephrine (NE), and epinephrine (EPI) has been examined as a function of age. Both basal levels and stress responses in old animals have been found to differ from those seen in young animals. However, published data are rather inconsistent and sometimes contradictory. While one study reported no change in basal NE but an increase in EPI levels during aging (1), another study found increased levels of NE (14), and a third study observed no increases in both CA (13). Similarly, stress responses of the plasma CA in old animals have been reported to be increased, decreased, or unchanged as compared to young animals (1,13,14). Unfortunately, such data do not lead to general conclusions about the basal levels of the plasma CA and their stress responses as a function of age. In addition to the CA, plasma taurine (TAU) has recently been shown to be significantly affected by age and stress (16,22,26) and to attenuate the immobilized stress-induced EPI release from the adrenal glands (11). Unfortunately, these results must be inter-
Epinephrine
Taurine
Adaptation
preted with caution because in most studies blood was obtained following decapitation; a method now known to produce artefactually altered levels of TAU (15). For these reasons, we felt it necessary to re-evaluate the effects of age on basal levels of plasma NE, E, and TAU and their stress responses. In addition, young (3 months) and old (24 months) rats were each stressed three times by immobilization (30 min) to observe initial adaptation responses in individual animals as well as to search for possible relationships among the three biochemicals during rest and stress at these two ages. METHOD
Animals and Catheterization Male 3- or 24-month-old Fischer 344 rats were obtained from the National Institute of Aging and group housed in animal quarters (about 24°C, light from 8:00--20:00 h and dark from 20:00-
To whom requests for reprints should be addressed. 359
360
MILAKOFSKY, HARRIS AND VOGEL
TABLE 1 EFFECT OF STRESS AND AGING ON PLASMANE OF F344 RATS Day (pg/mL ± SEM) Time (min)
1
3 Months N 0 15 30 60 120 180 240 24 Months N 0 15 30 60 120 180 240
3
7
274 1256 955 585 506 406 373
7 ± 24 ± 134t + 97t --- 66 +- 49 4- 46 -+ 46
6 264_+ 57 1476 -+ 287; 1330 -+ 55%¶ 780 -+ 91 492 ± 44 476 - 42 374 -+ 50
580 2269 1769 1032 818 705 632
8 -+ 70:[: --- 268t'§ +- 207f'§ ± 94§ ± 73§ +- 69§ ± 71:]:
660 2737 2511 1400 868 665 803
8 - 145:[: - 335~':~ +- 297t'§'¶ +-- 182:~ ± 90§ ± 91 +- 1265
5 3 3 9 - 70 1102 ± 138t'# 1147 : 1331 641 ± 102 402 ± 72 394 ± 87 421 -+ 132
696 2475 2123 1463 911 733 759
8 +- 152 --- 240t'§ -+ 213%§ + 166"§ -+ 96§ ± 91:~ - 115
* p < 0.05 with respect to time = 0 min for each day and age; fp < 0.001 with respect to time = 0 min for each day and age; l:p < 0.05 between 3 and 24 months at the same time point; §p < 0.01 between 3 and 24 months at the same time point; ¶p < 0.01 between Day 1 and 3 at the same time point; CAp< 0.01 between Day 3 and 7 at the same time point.
8:00 h, water and Purina chow ad lib) to acclimate lbr at least I week before the experiment. In preparation for the stress experiments, animals were anesthetized with ketamine and acepromacine (100 ÷ 1 mg/kg, IP) and the right external jugular vein implanted with a silastic catheter as described previously (20,23). Catheters were exteriorized through the back and the portion outside of the animal was protected by a steel spring. Catheter and steel spring were supported by a swivel pulley above the cage. After surgery rats were housed individually with free access to food and water. Catheter patency was assured by flushing the catheter at least twice daily with heparin (1000 units/ml). The animals were then allowed to recover for 48 h before further testing.
Immobilization and Preparation of Plasma Samples At the time of the experiment, blood (about 0.3 mL) was obtained in the home cage and the animal immediately immobilized by being taped with the four paws to the laboratory bench. Blood was obtained at 15 and 30 min during this stress experience. The animal was then released, returned to its home cage, and blood drawings performed after 30, 60, 90, and 210 additional min. After 1 day of rest, the animal was again immobilized and blood drawn as described above on the 3rd day and after a 4-day rest period, again on the 7th day. All immobilizations occurred between 10:00 a.m. and noon. As reported earlier, it has been established that repeated blood samplings over the experimental period do not affect plasma TAU or CA levels (15,19,20). All blood samples were centrifuged to obtained plasma, which was stored at - 80°C in vials containing 0.04 mL of an anticoagulant solution of EGTA and reduced glutathione (Amersham Co.).
1800
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TIME (min.) FIG. 1. The percentage changes from baseline values of plasma norepinephrine (NE) during and after repeated immobilization stress. Values are a percentage of baseline --- SEM. *p < 0.05 with respect to time = 0 rain for each day and age; **p < 0.01 with respect to time = 0 min for each day and age; ***p < 0.001 with respect to time = 0 min for each day and age.
AGE-STRESS ON CATECHOLAMINES AND TAURINE
TABLE 2 THE EFFECT OF STRESS AND AGING ON PLASMA EPI OF F344 RATS
361
efficient and linear regression analyses were used to compare individual data. Values o f p < 0.05 were considered significant. RESULTS
Day (pg/mL +- SEM) Time (rain) 3Months N 0 15 30 60 120 180 240 24Months N 0 15 30 60 120 180 240
1
3
7
7 234± 2356 ± 1745 -+ 1010 -+ 563 ± 323 -+ 239 ±
36 142, 337* 204* 150 52 52
108 2286 2688 910 539 235 245
6 ± 26 -+ 527* -+ 458*'¶ -+ 177 -+ 110 -+ 28 ± 93
218 1583 1482 679 325 394 396
5 ± 72 ± 291*'#'* ± 271t'## -+ 81 ± 151 -+ 145 ± 240
8 ± ± ± -+ -+ ± ±
73 1112~ 686~ 139 119 123 90
269 3563 3963 886 607 426 583
8 -+ 93 -+ 70~ -+ 899~ -+ 118 -+ 126 -+ 85 -+ 164
561 3247 3147 1169 689 427 478
8 ± 207 -+ 505$'§ -+ 500,'§ -+ 263 -+ 136 -+ 109 -+ 57
258 3805 3450 866 568 497 335
* p < 0.05 with respect to time = 0 rain for each day and age; tp < 0.01 with respect to time = 0 min for each day and age; :~p < 0.001 with respect to time = 0 min for each day and age; §p < 0.05 between 3 mos and 24 mos at the same time point; ¶p < 0.01 between Day 1 and Day 3 at the same time point; # p < 0.01 between Day 3 and Day 7 at the same time point; # # p < 0.001 between Day 3 and Day 7 at the same time point; *p < 0.05 between Day 1 and Day 7 at the same time point.
Plasma TAU Analysis At the time of analysis, plasma TAU samples were thawed and an aliquot (0.05 mL) deproteinized immediately with 0.40 M perchloric acid as previously described (15). Concentrations of TAU in deproteinized samples were determined by ion-exchange high performance liquid chromatography (HPLC) with fluorometric detection as previously described (15). This procedure utilized a meter long microbore HPLC stainless steel column containing spherical cation-exchange resin and fluorescence detection following post-column reaction with o-phthaldialdehyde, Lithium citrate elution buffers were used to separate TAU from other amino acids and related compounds in rat plasma. This technique has been shown to be reliable, sensitive, and valid (15).
Plasma CA Analysis The plasma levels of NE and EPI were determined by radioenzymatic analysis (Upjohn Co. " C A T - A - K I T " ) as previously described (5).
Evaluation The T A U concentrations were quantitatively determined by relating chromatographic peak heights to peak heights from a known TAU standard and an internal standard, m-fluorophenylalanine (MFP). Total mean T A U values (nmol/mL) were determined (15). CA results were expressed as mean values (pg/mL). The trapezoidal rule was used to find the area under the stress curve (AUC) for TAU, EPI, and NE curves. Statistical analysis of the data (SAS software) was performed by a three-way analysis of variance (ANOVA) for two repeated measurements (time, day) with a Tukey post hoc test or by A_NOVA with log transformations (to minimize variations among animals). Pearson correlation co-
Plasma concentrations of NE before, during, and after immobilization are shown in Table 1. For young rats, baseline levels of NE remained unchanged over the three restraints. Levels of NE rose rapidly and markedly during the stress period but returned quickly to baseline values after cessation of the stressor. Similar patterns were observed during all subsequent stress experiences with some minor but significant differences. For old rats, base values also remained unchanged but were higher on the first two occasions as compared to the young rats. During stress, levels also rose quickly and markedly but were significantly higher than those seen in the young rats. Percentage changes from baseline values for NE during and after immobilization are shown in Fig. 1. Increases were seen during and sometimes after stress but no significant differences were found between the different days or the two age groups. Plasma concentrations of EPI before, during, and after immobilization are shown in Table 2. Baseline values remained unchanged for both age groups over the 3 experimental days and were not different between these two groups. Similarly, the rise and fall of EPI levels during and after stress were similar for both age groups on the first two occasions. However, on the third day, the intensity of the stress response was less in young rats while it remained higher in the old rats. Percentage changes from baseline values for EPI during and after immobilization are shown in Fig. 2 to be increased in both age groups during stress. In the young animals, the stress response increased on the second occasion but fell to first exposure stress levels on the third stress experience. In the old animals a slow, but statistically not significant, decline in the stress values can be seen over the last two stress experiences. TABLE 3 THE EFFECT OF STRESS AND AGING ON PLASMA TAU OF F344 RATS Day (nmol/mL -+ SEM) Time (min) 3 Months N 0 15 30 60 120 180 240 24 months N 0 15 30 60 120 180 240
1
7 36.1117 -+ 84.2 -+ 60.7 ± 40.5 --54.2 -+ 43.2 -+
36.9 109 101 85.9 43.7 40.6 39.1
2.9 13, 14.4" 5.6 3.9 13.8 5.8
8 -+ 5.7 -+ 23t -+ 16" -+ 17.3 -+ 5.8 -+ 4.8 -+ 4.2
3
7
6 44.0--- 4.9 96.1 ± 12.2t 88.4 -+ 14.7" 66.4 -+ 8.3 57.3 -+ 2.7 54.3 -+ 7.1 51.9 --+ 3.6
5 32.7+ - 2.1 54.7 -+ 3 . 6 t t t ' # 46.1 ± 4 . 5 t t ' # 44.4 -+ 5.6 40.0 --- 6.0 63.6 -+ 23.5 69.3 -+ 30.7
28.4 62.6 69.8 65.7 35.5 34.8 32.3
8 -+ -+ ± -+ -+ -+ ±
4.7§ 10.5¶ 13.0" 12.3 7.2§ 4.8§ 5.3§
48.4 104 100 107 81.0 60.8 51.5
8 -+ 11.1 -+ 26.8 -+ 25 --- 24 -+ 29.1# ± 11.1 -+ 8.2
* p < 0.05 with respect to time = 0 min for each day and age; tp < 0.01 with respect to time = 0 min for each day and age; ~:p < 0.001 with respect to time = 0 min for each day and age; §p < 0.05 between 3 mos and 24 months at the same time point; ¶p < 0.05 between Day 1 and Day 3 at the same time point; # p < 0.05 between Day 3 and Day 7 at the same time point; tp < 0.05 between Day 1 and Day 7 at the same time point; t t t p < 0.001 between Day 1 and Day 7 at the same time point.
362
MILAKOFSKY, HARRIS AND VOGEL
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Plasma concentrations of TAU before, during, and after immobilization are shown in Table 3. Baseline levels remained the same on all three occasions within both age groups with the exception of a lower value on the second exposure to stress in the older rats. During and after stress, TAU levels rose and fell similarly in both age groups. On the third occasion, stress values were lower in the young animals but remained higher in old animals. Percentage changes from baseline values for TAU during and after immobilization are shown in Fig. 3. Stress induced increases were significant for the young animals on the first two occasions but not the third exposure. For the old animals, increases were significant on all three occasions. AUC for all three compounds during the 30-min stress period and a 60-min period (including 30 min of stress followed by a 30 min release period) are shown in Fig. 4. For NE, AUC is relatively constant for all three immobilizations in both age groups but significantly higher for the old rats. However, the AUC for the 60rain experimental period is higher on Day 3 indicating a slower return to baseline for both age groups. The AUC for EPI is also relatively constant for the first two occasions in both age groups but shows higher values on the third occasion for the old rats. Again, on the second stress exposure return to baseline is slower for young animals only. The AUC for TAU decreases over subsequent immobilizations and is markedly lower on the third occasion for the young animals; in contrast, the values for the old rats remain relatively stable.
Correlation analyses among the three biochemicals at the three stress experiences were performed uncovering the significant correlations summarized in Table 4. Significant correlations between EPI and NE were found at different times for the young as well as the old animals mostly on the first and last stress experience. Fewer correlations were found between TAU and EPI and even fewer between TAU and NE. Specifically, TAU was correlated for the most part with EPI on the first and third exposure to stress. No major correlations were apparent for a specific biochemical in individual young animals over the 3 days of stress. However, in the old animals some consistent correlations were found. The 15 min stress values for NE correlated between Day 1 and Day 3 (r = 0.85, p < 0.01) and between Day 3 and Day 7 (r = 0.78, p < 0.05). The 15-min stress values for EPI correlated for the first two exposures (r = 0.90, p < 0.01). For TAU, only some post-stress values (time = 60, 120, and 180 min) correlated between the first and last stress exposure (r = 0.95, p < 0.001; r = 0.74, p < 0.05 and r = 0.85, p < 0.001). DISCUSSION
It is difficult to uncover general conclusions about stressinduced biochemical differences between young and old rats since different strains of rats, variations in the nature or intensity of the stressor employed, or a particular biochemical selected can give rise to different interpretations (13,16). In addition, presentation of the results can lead to different conclusions. Absolute values
AGE-STRESS ON CATECHOLAMINES AND TAURINE
363
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15
30 60 120 160 2 4 0
TIME (min.) FIG. 3. The percentage changes from baselinevalues of plasma taurine (TAU)during and after repeated immobilizationstress. Values are a percentage of baseline --+ SEM. *p < 0.05 with respect to time = 0 min for each day and age; **p < 0.01 with respect to time = 0 rain for each day and age; ***p < 0.001 with respect to time = 0 min for each day and age; Sp < 0.05 between Day 1 and Day 3 at the same time point; Ap < 0.05 betweenDay 1 and Day 7 at the same time point; /X/X/Xp < 0.001 between Day 1 and Day 7 at the same time point; @@p < 0.0l between 3 and 24 months at the same time point. including baseline values represent the actual impact of a chemical on the body during stress whereas percentages or AUC represent only the stress-induced change from the original homeostasis. Both presentations may lead to different conclusions. In this article, we consider both options. A comparison of baseline values alone for Day 1 shows that only the baseline concentrations of NE (Table 1) are higher in old animals whereas those for EPI (Table 2) and TAU (Table 3) are generally similar between both age groups. These data do not confirm the reported age difference for TAU (26) perhaps due to the fact that we obtain blood through the catheter shown to be more accurate than blood collection after decapitations (15). Elevated NE levels in resting old animals have also been reported (14) although other studies have shown either no differences (6,10,13, 27) or elevated levels of EPI (2). Similar to our studies, it has been shown in humans that older individuals exhibit higher sympathetic activity with higher plasma NE levels (21,24); probably resulting from increased NE appearance and not decreased clearance (24). Although baseline levels of all three biochemicals did not change during the sequential repeated stresses, other studies using longer periods of stress have either reported increases (6) or no increase in these resting concentrations (10). These increased levels of NE are indicative of increased sympathetic activity under resting conditions in older animals; although it could be argued that older animals may be more stressed under apparently resting conditions, and thus, show the observed higher NE levels.
As expected, levels of all three biochemicals rise significantly during the first stress exposure in both age groups. The actual values of NE (Table l) reach much higher levels in old animals resulting in a greater impact of the CA and on their target organs. However, percentage increases above baseline (Fig. 1) are not statistically different in young and old animals. In contrast, a comparison of the general stress experience in terms of AUC values shows that old animals do indeed experience a higher stress response (Fig. 4). No major differences in the stress response were seen for EPI (Table 2 and Fig. 2) and for TAU (Table 3 and Fig. 3) between the age groups regardless of the method of comparison used. Data in the literature are in partial agreement with our findings. For example, varied stress responses in young and old rats have been reported in that immobilization produced larger increases in plasma NE in young rats (14), cold stress caused larger increases in old rats (2), brief footsbock induced lower CA responses in old rats (13), and cold water immersion or shaker stress brought about the same responses in young and old rats (13,27). In humans, NE levels during upright posture were higher in older subjects as compared to younger individuals (24). Thus, the impact of EPI and TAU on target organs and their actual stress responses were the same for both age groups on the first stress experience. However, the stress response for NE was slightly higher and the actual impact of NE on the target organs was much higher in old animals. Repeated stress experiences over longer periods of time can
364
MILAKOFSKY. HARRIS AND VOGEL r.1 Z
tO0000
I r.1 >-
[ ~i~!AGE - 3 MO
80000
50000 40000
Z
20000 <
30 rain 60 rain
DAY 1 I
DAY 3
DAY 7
160000
120000
80000
Z <
40000 30 r a i n 60 r a i n
DAY 1
30 r a i n 80 r a i n
30 r a i n 60 m i n
DAY 3
DAY 7
5000
I
AGE - 3 MO
4000 3000 2000
Z tO00 .< "~
<
0
30 rain 60 mln
DAY 1
30 r a i n 60 m l n
DAY 3
30 m i n 60 m i n
DAY 7
FIG. 4. The areas under the curve (AUC) for NE, EPI, and TAU during a 30-min stress period and a 30-min stress period plus a 30-min release period. Values are AUC -+ SEM. @p < 0.05 between 3 and 24 months at the same time point; $p < 0.05 between Day 1 $$ and Day 3 at the same time point; p < 0.01 between Day 1 and Day 3 at the same time point; Ap < 0.05 between Day 1 and Day 7 at the same time point;/X/Xp < 0.01 between Day 1 and Day 7 at the same time point; #p < 0.05 between Day 3 and Day 7 at the same time point; #*p < 0.01 between Day 3 and Day 7 at the same time point.
involve mechanisms of adaptation (1,6,10). In our case, animals were stressed three times but rest periods varied. In the first case, there was 1 day of rest between the first and second stress session and in the second case, there were 3 days of rest between the second and third stress session. We noticed adaptation already at the third stress exposure in spite of the fact that the animals had 4 days of rest between stress experiences. In the case of NE, the actual peak values of the stress responses (Table 1) did not change markedly in either young or old animals and remained higher in the old rats. A comparison of percentage (Fig. 1) and AUC values (Fig. 4) over the three stress experiences also did not show difference within each age group; although the return to baseline was slowed on the second stress exposure. On the other hand, in the case of EPI actual stress values (Table 2) were lower on the third
stress occasion in the young rats but remained at high levels in the old animals. This difference was not apparent when evaluated in term of percentages (Fig. 2) but was evident with AUC values (Fig. 4) where young rats showed significantly lower values on the third exposure to stress and a retarded return to baseline onDay 3. Similarly, actual stress values for TAU (Table 3) in young rats were much lower on the third occasion as compared to the first exposure and did not differ from baseline values. In the old rats, no differences are seen among all three occasions. The same conclusions were reached when comparing percentages (Fig. 3) or AUC values (Fig. 4) among the three occasions and between the two age groups. Thus, both EPI and TAU show signs of adaptation in young rats already at the third stress exposure. This was not the case for old rats where stress responses remain essentially unchanged.
AGE-STRESS ON CATECHOLAMINES AND TAURINE
365
TABLE 4 CORRELATION ANALYSIS BETWEEN PLASMA NE, EPI, AND TAU IN YOUNG AND OLD F344 RATS
Day 1
TAU
Time (Min)
3 months N 0 15 30 60 120 180 240 24 months N 0 15 30 60 120 180 240
3
EPI
TAU
7 TAU
--
NE
ns
TAU
--
NE
ns
TAU
--
7
EPI
TAU
6 ns O. 8 3 " ns
0.76* 0.84* 0.75 * - 0.79*
EPI
5
--
ns
--
ns
ns
ns
ns
ns
--
ns
--
ns
ns -ns --
0.88*
ns
ns
ns
ns
0.92*
ns
ns
ns
ns
--
ns
ns
ns
ns
ns
--
ns
--
ns
ns
ns
--
ns
--
ns
ns
ns
ns
--
ns
ns
ns
-0.94"
0.94* 0.91"
NE
ns
TAU NE TAU NE TAU NE TAU NE
-ns -ns --
TAU
--
ns
--
8 0.86**
--
NE
ns
ns
ns
ns
ns
TAU
--
ns
--
ns
--
NE
ns
ns
ns
ns
ns
TAU NE TAU NE TAU NE
--
--
ns
--
ns
ns
ns ns ns 0.79*
ns
--
ns
0.905
ns
8
0.855. ns
ns
0.975. ns
8
ns
0.71 *
ns
ns
ns
--
0.885.
--
ns
--
0.865. 0.845. 0.72*
ns
ns
ns
ns
ns
ns
0.72* 0.905.
--
ns
--
ns
--
0.90t
ns
ns
ns
ns
ns
0.79*
TAU
--
ns
--
ns
--
ns
NE
ns
ns
ns
ns
ns
ns
TAU NE
--
ns
--
ns
--
ns
ns
ns
ns
ns
ns
ns
Values are correlation coefficients; ns = not significant; *p < 0.05; 5.p < 0.01. In addition to the CA, our findings establish TAU as a sensitive stress chemical which responds quickly to stress and shows rapid adaptation in young but not old animals. This adaptation processes clearly distinguished the young from the old animals. It has been shown that various hormones including the CA affect aging (8). For example, stress-induced increases of the related stress chemical, corticosterone (CORT) were found to be similar in young and old rats on the first stress exposure but the stress response of this hormone was more prolonged and attenuated, less in old animals during chronic stress (7,17). No correlations between CORT and NE or EPI levels have been found (25) and to the best of our knowledge, no correlation between CORT and TAU has been performed. Thus, it is conceivable that stress induced increases in these hormones accelerate the aging process. On the other hand, young animals seem to protect themselves from the deleterious effects of such high levels over prolonged periods of time by adaptation which possibly reduces dangerous levels of these biochemicals, a process evidently impaired in the old organ-
isms. Thus, older individuals would be at increased risk since they would not adapt as readily and must endure higher levels of these biochemicals over time which could accelerate aging and might be responsible for the faster progression of aging seen at old ages. In the old animals, higher baseline and stress levels of NE have been shown to occur. Although the exact cause is unknown at present, two explanations can be offered. It has been shown that the NE content of blood vessels is the same in young and old rats but that there is an age-related increase in NE release due to a decline in the negative feedback mediated by prejunctional alpha-2 receptors (3). Furthermore, the release of EPI and NE from the adrenal glands increases with a decrease in phenylethylamine-Nmethyl transferase (PNMT) favoring the amount of NE released during stress (9). Thus, higher levels of NE in the old animals most probably result from changes in the biology of blood vessels and adrenal glands. Because all the measured biochemicals were determined in the same animals on each occasion, correlations of these biochemicals within each age group (Table 4) were calculated. As shown previously for young rats, baseline and stress values between NE and EPI were highly correlated (12). Again, we find this correlation on Day 1 but lose most of these correlations for the second and third stress experiences. Old rats did not show this baseline correlation between EPI and NE on the first day but exhibited this relationship during stress. Correlations were again lost on the second exposure to stress but re-emerged on the third occasion. Correlations between NE or EPI and TAU are more sporadic. For young rats, TAU values correlated positively with EPI concentrations during the first stress exposure but negatively after immobilization was terminated. These results support the suggestion (11,16) that there may be a regulatory interaction between EPI and TAU in young animals. In contrast, old animals did not show this pattern. Further evaluation of correlations among different biochemicals and their changes during repeated stress experiences might provide additional information on biochemical changes as they occur during aging. No significant correlations were observed for each biochemical over the three occasions in individual young animals. In old animals, some correlations were found indicating that old animals responded more characteristically within the three immobilizations. Studies were carried out to see if differences in the biochemical stress responses of individual rats correlated with differences in stress-induced behavioral responses, recovery from stress and, in our old rats, with time of survival. No differences were observed by global assessment of struggling during immobilization and in the recovery of the young and old rats after release from immobilization. All old rats died between 3 to 6 months after the experiment but no correlation was found between the extent of the stress response of any of the biochemicals and the subsequent time of survival. Moreover, one young rat showed normal stress responses during the first and second stress experiences for all three biochemicals studied but a drastically increased stress-response in EPI, NE, and TAU on Day 7. Average stress values for the animals after 30 rain of stress for EPI, NE and TAU on the third occasion were 1482 pg/mL, 1147 pg/mL, and 46.1 nmol/mL, respectively whereas values for this rat on Day 7 were 16000 pg/mL, 9770 pg/mL, and 391 nmol/mL. This animal died shortly after the last immobilization. While it is rare for animals to show serious health consequences after short periods of immobilization (18), it has been reported that some animals die after 30 rain of immobilization (4). Thus, the possibility that a very few animals respond to a repeated stress experiences by releasing unusually high stress levels of NE, EPI, or TAU could constitute a useful marker to predict future harm to or even death of the animal.
366
MILAKOFSKY, HARRIS AND VOGEL
In conclusion, the age differences seen between young and old rats did not manifest themselves uniformly but were relatively unique for a specific biochemical. Baseline values for NE but not for EPI and TAU were higher in old animals but did not change during the three brief stress exposures. Initial stress responses were higher for NE in old animals but similar for TAU and EPI in both age groups. Adaptation to immobilization was seen for EPI and TAU but not NE already on the third occasion in the young animals while no signs of adaptation were evident for the old animals. Correlations between NE and EPI in young and old rats seen on the first day were not observed on the second stress period
but were seen again on the third stress exposure indicating subtle effects of stress which also show some degree of adaptation. ACKNOWLEDGEMENTS We gratefully acknowledge the Penn State University Biomedical Research Support Grant, the Penn State University Gerontology Center Faculty Development Projects, the Penn State University Berks Campus Faculty Development Fund, and the Public Health Grant (AA06017) for Financial Support. We also thank Heidi Weiss and Betty M. Nsubuga for their technical assistance.
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