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Modulation of tyrosine hydroxylase gene expression in the rat adrenal gland by exercise: effects of age
Molecular Bram Research, 14 (1992) 51-56 (~) 1992 Elsevier Science Pubhshers B V All rights reserved 0169-328X/92/$0500
51
BRESM 70423
Modulation of tyrosine hydroxylase gene expression in the rat adrenal gland by exercise" effects of age N. Ttimer a'b, C. Hale d'e, J. Lawler c and R. Strong d'e "Gertatrtc Research, Educanon and Chntcal Center, Department of Veterans Affairs Medical Center, Gamesvdle, FL 32608-1197 (USA), bDepartment of Pharmacology and 'Department of Exercise Scwnces, College of Medicine, Umverstty of Florida, Gamesvtlle, FL 32610 (USA), dGertatrtc Research, Education and Chmcal Center, Department of Veterans Affairs Medical Center, St Louts, MO 63125 (USA) and eDepartments of Internal Medwme, Pharmacology and Physiological Science, St Louts University Medwal School, St Louts, MO 63104 (USA) (Accepted 31 December 1991)
Key words Tyroslne hydroxylase; Tyroslne hydroxylase mRNA, Chohne acetyltransferase; Glutamlc acid decarboxylase, Exercise, Training; Aging
Both aging and exercise are associated with alterations in circulating levels of catecholamlnes. To determine the interactions of age and exercise on tyroslne hydroxylase (TH) activity and TH mRNA, Fischer-344 female rats aged 5 months (young) and 25 months (old) were trained by treadmill runnmg for 10 weeks The elevation in maximum oxygen consumption in both groups was equivalent following exercise, indicating that training had occurred In control rats, both TH activity and TH mRNA were greater in the older groups when compared with the younger animals. In young rats, exercise decreased TH activity by 25% and TH mRNA by 27%. In older rats, exercise was not associated with a decrease m TH activity and TH mRNA Chohne acetyltransferase activity (CHAT) was decreased and glutamlc acid decarboxylase activity (GAD) was increased by exercise in young rats The decrease in ChAT activity and increase in GAD activity suggest that transsynaptlc mechanisms play a role in the exercise-induced alteration of TH gene expression. Neither ChAT nor GAD was altered by exercise m older groups Our data suggest that the previously reported diminution in catecholamxnes associated with exercise may be due to a decrease in TH mRNA and a resulting decrease in TH activity. There was no effect of exercise in the old rats, supporting previous observations that the plasticity of the sympathoadrenal system diminishes with age INTRODUCTION
role both in age-related diseases such as hypertension and in the beneficial effects of exercise on the cardio-
The peripheral sympathoadrenal system exerts a major integratwe function in homeostasis. Increased sympathoadrenal activity, as evidenced by increased circulating levels and turnover of catecholamines, occurs in response to stressful extremes in the external and internal e n v i r o n m e n t of the organism 2'3. There is also a considerable body of evidence indicating that plasma catecholamines are elevated with advancing age. Ageassociated increases in circulating catecholamines are well d o c u m e n t e d in h u m a n s and laboratory animals, both at rest and in response to stress 2'8'12'13'19'22. In addition,
vascular system. Hypertension is almost certainly a multifactorial process, involving hormonal and vascular alterations. However, age-related elevation of peripheral catecholamines has been implicated in the development and possible m a i n t e n a n c e of hypertension in humans and in several experimental models 14'18'25"43. O n the other
there is a decreased ability of the catecholaminergic system of older subjects to respond and adapt to environmental challenges, suggesting that control of sympathoadrenal function is altered during aging22'34. O n e consequence of the altered regulation of sympathoadrenal function may be the increased prevalence of hypertension during aging39. Alterations in catecholaminergic function may play a
hand, exercise may be beneficial in controlling hypertension 23. Exercise increases cardiovascular functional capacity and decreases myocardial oxygen d e m a n d in healthy people as well as most patients with cardiovascular disease 23. The effects of exercise training on the cardiovascular system may be, at least partly, a result of alterations in the peripheral catecholaminergic system 11. Turnover and circulating levels of peripheral catecholamines, measured both at rest and in response to exercise, is markedly reduced by chronic exercise in both humans and rodents 2°'21'27"28. Therefore, it is possible that exercise may prevent or reverse age-related increases in catecholamine synthesis and/or release. However,
Correspondence N Tumer. Geriatric Research, Education and Chnlcal Center (182), Department Veterans Affairs Medical Center, Gainesvllle, FL 32608-1197, USA
52 t h e r e are f e w r e p o r t s o n t h e e f f e c t s o f c h r o n i c e x e r c i s e o n t h e m o d i f i c a t i o n o f p e r i p h e r a l c a t e c h o l a m i n e s in a g e d populations We and others have r e p o r t e d that the expression of tyrosine hydroxylase (TH), the rate-limiting enzymatic s t e p in c a t e c h o l a m i n e b i o s y n t h e s i s 24, is i n c r e a s e d in t h e a d r e n a l g l a n d d u r i n g aging 17 36,40. F u r t h e r m o r e , t h e adr e n a l c o n t e n t o f d o p a m m e , t h e first c a t e c h o l a m i n e p r o d uct o f T H , was also i n c r e a s e d , i n d i c a t i n g t h a t a g e - r e l a t e d i n c r e a s e s In T H g e n e e x p r e s s i o n are f u n c t i o n a l l y significant ~6. T h e e x p e r i m e n t s d e s c r i b e d h e r e e x t e n d t h o s e o b s e r v a t i o n s to an i n v e s t i g a t i o n o f t h e i n t e r a c t i o n o f age a n d e x e r c i s e o n T H g e n e e x p r e s s i o n in t h e a d r e n a l g l a n d . W e m e a s u r e d T H activity a n d T H m R N A
levels in the
a d r e n a l g l a n d s o f y o u n g a n d old rats t h a t w e r e t r a i n e d o n a t r e a d m i l l for 10 w e e k s . F u r t h e r m o r e , since t h e adr e n a l G A B A e r g l c a n d c h o l i n e r g i c s y s t e m s play a role in m o d u l a t i n g T H g e n e e x p r e s s i o n 33'34, w e m e a s u r e d glut a m i c acid d e c a r b o x y l a s e ( G A D )
a n d c h o l i n e acetyl-
t r a n s f e r a s e (CHAT) a c t i v i t y , w h i c h are s y n t h e t i c e n z y m e s for ? ' - a m i n o b u t y r i c acid ( G A B A )
a n d a c e t y l c h o l i n e , re-
spectively. T h e r e s u l t s o f this s t u d y s u g g e s t t h e previously r e p o r t e d c h a n g e s m c a t e c h o l a m m e s f o l l o w i n g exercise
involve
alterations
at
the
level
of
TH
gene
expression.
MATERIALS AND METHODS
Antmals Fischer-344 (F-344) female rats 5 and 25 months old were obtained from a colony maintained for the National Institute on Aging at Harlan-Sprague-Dawley Labs, Inc (Indianapolis, IN) On arnval, rats were housed under bamer condmons in a temperatureregulated environment (21 + 1 °C) on a 12 h hght/dark cycle The animals were fed Purina Rat Chow diet and water ad hbltum The median hfe-span of F-344 rats is 27 months9 Trammg procedure Both young and old training groups were exercised by treadmill running 5 days/week for 10 weeks, each training session began w~th a 5-mm 'warm-up' at 15 meters/ram (0% grade) On day 1 (week 1) of training, the animals began exercising at 18 meters/min (10 min duration/0% grade) wxth the duration of exercise being mcreased by 2-3 min/day until the ammals reached 60 rain of exercise (including the warm-up) The exercise time period remained at 60 mm for the duration of the study During weeks 4-6 the treadmill speed was increased by 1 meter/ram per week until a speed of 20 meters/mm was reached; the speed remained constant through weeks 6-10 Begmmng on week 7, the treadmill grade was increased by 1 5%/week until reaching a zemth of a 6% grade during week 10 of training Non-exercised ammals were placed into the treadmill for equal lengths of time but the apparatus kept off Ammals were monitored continuously during exercise Measurement of Vomax At the completion of the training period, VOmax was measured on 4-6 animals in each experimental group using a flow-through open-circuit system Briefly. animals performed an incremental e\ercise protocol that began with a 4-ram warm-up and progressed with a work rate increase every four rain untd the ammals were unable to maintain the required power output VO, max was de-
fined as the h~ghest VO, obtained during the exercise test The protocol was designed such that VOmax was generally reached within 12-20 mm followmg the warm-up Room mr (P~O 2 = 142 mmHg) was pumped through the sealed treadmill chamber (Ommtech Electronics, Columbus, Ohio) at a flow rate of 4 5 hters/mm Gas was sampled (500 ml/min) from a small mmng chamber located at the back of the treadmill and analyzed for CO 2 and 0 2 concentrations using electronic gas analyzers (Beckman LB-2 and OM-I1, respectively, Sensor Medics, Anahmm, CA) The gas analyzers were cahbrated ~mmedmtely before and after each test using gravlmetrlcally standardized gases
Ttsaue pteparalton After ammals were anesthetized wtth pentobarbital (90 mg/kg), adrenal glands were removed qmckly, lmmedmtely frozen on dry me and stored at -80°C Each pmr of adrenal glands was decapsulated, weighed and homogemzed m 100/~1 of buffer (2 mM sodium phosphate, pH 6 0, fi 2c/c Triton) Aliquots of homogenate were removed for each assay as follows lfi ttl for TH assay, 10 ~1 for GAD and ChAT assays, 5 fd for protein assay, and the remainder (75 ~1) for determination of TH mRNA studies Tvrosme hvdrorylme assay Adrenal TH acnvity was measured by a coupled decarboxytase assay we previously described3~ The assay is based on trapping radmlabelled CO, evolved from the conversion of L-[~C]tyrosme to dopamlne by chemical decarboxylatlon26 Briefly, each 10-~1 sample of adrenal homogenate was diluted to 100 gl with homogenizing buffer The diluted sample was passed over Sephadex G50 columns to remove endogenous catecholamlnes Pomons of the eluate (6 ul) were assayed in duphcate m a final assay volume of 10 !d The concentration of the cofactor 6-MPH 4 was 3 mM, and the concentratmn of [14C]tyrosIne was 40 ~M, and the final pH of the reactmn mixture was 6 5 GAD assa~ GAD activity was determined m homogenates of adrenal glands using methods prewously described by us3~ GAD was measured by a decarboxylatlon assay using L-[l-t4C]glutamate as a substrate with subsequent trapping of ~4CO~ Ttssue homogenate (2 Fd) was added to the reaction m~x (5/d) containing 10 mM glutamate as a substrate ('hollne acetyltransferase assal ChAT actlwty was measured m 2 Fd of adrenal homogenate using [L-14C]acetyl CoA (0 2 mM) as a substrate, in a final volume of 7/d. as described previously by us ~v3s Tyrosme hvdroD'lase c D N A - m R N A hybrldtzatton The concentranon of total cellular RNA extracted from adrenal glands was determined from ethidmm bromide-stained gels using methods for small tissue samples described previously by Mallet and coworkers4 Total cellular RNA was subjected to agarose gel electrophoresis concurrently with RNA of known concentration, as determined by optmal density measured at wavelength 260 nm on a Beckman (Irvlne, CA) Spectrophotometer The ethldmm bromide-stained gels were exposed to ultraviolet light and photographed using Polaroid (Cambridge, MA) Type 55 film. The resultmg negative was scanned on a Blo-Rad (Richmond, CA) Model 620 scanning densltometer to obtain optical density measurements To quantify the concentration of RNA in the samples, the peak areas corresponding to the 18S and 28S ribosomal bands in the unknown were compared to those obtained from a group of sermlly diluted standards of known concentration The concentration of each sample was derived by hnear regression analysis Several concentrations of serially diluted RNA samples were immobilized on nylon membranes (Gene Screen. New England Nuclear, Boston, MA) using a Blo-Rad slot blot apparatus The baked filters were prehybndlzed using 25 mM potassmm phosphate, 5 × SSC, 5 .< Denhardt's solution, 100 mg/ml denatured salmon testes
53 TABLE I
5000
-
Effects of exercise traming and agmg on oxygen consumption 4000-
•
control
[]
tramed
Values represent m e a n s + S.E.M. of 9-12 rats. ="
Treatment
Maximum oxygen consumption (ml/kg/mm) 5 month
25 month
79.4 + 1 9 94 4 + 0 7*
59 4 + 3 0"* 72.8 + 1 2* **
~= ~
3000 -
2000 -
1000
Control Trained
-
0-
D N A , and 50% formamlde. After incubatmn for 14-16 h at 42°C, the filter was hybridized with a 32p nick-translated full-length rat T H 36 c D N A probe 5, kindly supplied to us by K a r e n O'Malley, Washington University School of Medicine. After incubation for 14-16 h at 42°C, the filter was washed and exposed to X-ray film (Kodak X - A R , Rochester, NY) for 72 h. T h e developed autoradiogram was scanned using a Blo-Rad Model 620 Video Densltometer The optical density was linear over the range of total cellular R N A used to calculate T H m R N A levels Values for T H m R N A content were derived from the slopes of hnes derived from plotting optical density versus total cellular R N A concentratmn
Data analysis Means and standard errors of the m e a n (S E . M ) were calculated from values obtained from replicate experiments in individual animals from each age and treatment group For T H , G A D and C h A T assays, each sample was assayed in duplicate. For T H m R N A m e a s u r e m e n t s , values were derived from m e a s u r e m e n t s m a d e on 3 - 4 different concentrations of total cellular R N A Comparisons of m e a n s a m o n g different age and treatment groups were m a d e by analys~s of variance D~fferences between individual group m e a n s were determined by Flscher's Protected Least Slgmficant Difference test i° Differences were considered significant when P < 0.05 (two-tailed test)
RESULTS
Table I shows that following 10 weeks of training, maximum oxygen consumption was significantly (P < 0.05) higher in trained animals of both age groups as compared to their respective control groups. Although the levels of oxygen consumption differed significantly
25
5
* Significantly different from age-matched control, P < 0.05. ** Slgmficantly different from 5 m o n t h control, P < 0.05.
AGE (too)
Fig. 1. Effects of age and exercise on adrenal T H activity Values represent m e a n s _+ S.E.M. of 9-11 rats assayed in duplicate. *Significantly different from age-matched controls, P < 0.01. tSignlficantly different from 5-month-old controls, P < 0.01
(P < 0.05) between the two age groups, both groups showed a similar magnitude of increase in oxygen consumption associated with training. These data indicate that training occurred equally in both groups. To determine the effect of training and age on the capacity for catecholamine biosynthesis, TH activity was assessed. Among control groups, T H activity was significantly greater in the 25-month-old compared with 5-month-old animals (Fig. 1). Among 5-month-old animals, T H activity was significantly lower (P < 0.01) in the trained group as compared to the young control group. However, in the older rats there was no significant difference in T H activity associated with training. To determine if alterations in TH activity were a consequence of changes in gene expression, we measured TH mRNA levels using a full-length TH cDNA probe. The results of T H mRNA quantification by slot blot analysis are shown in Fig. 2. Among control animals, a comparison between age groups revealed that T H mRNA was significantly (P < 0.01) elevated by 48% in adrenals from old rats compared with young rats. In the 5-monthold animals, TH mRNA in adrenals of exercised rats was significantly lower (P < 0.01) as compared to the young
T A B L E III T A B L E II
The effect of exercise and agmg on adrenal GAD activity
The effect of exercise and aging on adrenal ChAT activity Values represent m e a n s + S E M treatment group
Treatment
Control Trained
of 5-11 rats per age and
ChAt activity (nmol/mg protein~h)
Values represent the m e a n + S E.M. of samples assayed in duphcate. The n u m b e r of samples per group is shown in parentheses.
Treatment
5 month
25 month
9 06 + 0.58 6.57 + 0 41"
9.67 + 0 78 8.59 + 0 57
* Significantly different from 5 m o n t h control, P < 0 05
Control Trained
GAD activity (nmol/mg protem/h) 5 month
25 month
39 0 _+ 3 5 (8) 48 9 + 2.1" (8)
31 4 _+ 4.1 (7) 37 9 + 1.8 (7)
* Significantly different from age-matched control, P < 0 05.
54
15 [] []
control trained
10
o Z I= 'lr I-
05
O0 5
25
AGE (mo)
Fig 2 Effects of age and exercise on adrenal TH mRNA Values represent means + S E M of 9-11 samples, assessed at 3-4 concentratlons of total cellular RNA per sample. *Slgmficantly different from age-matched controls, P < 0 01 *Slgmficantly different from 5-month-old controls, P < 0 01
control group. On the other hand, there was no significant effect of training on T H m R N A in 25-month-old animals. Previous studies have shown that trans-synaptic induction of T H is mediated, at least partly, by acetyicholine 33. Therefore, we examined the functional status of the cholinerglc system by measuring C h A T actiwty. Table II shows that, in contrast to the findings for T H activity and T H m R N A , there was no significant effect of age on C h A T activity in control animals. On the other hand, c o m p a n s o n s between the 5-month-old control and trained groups revealed that C h A T activity was significantly (P < 0.05) lower by 30% in exercised rats as compared to the control group There was no significant effect associated with exercise training in the 25-month-old animals. The G A B A system has been shown to modulate catecholamine release and T H gene expression ls'1634 Therefore, we investigated the effect of age and exercise on G A D activity. As shown in Table III, there was no significant effect of aging on G A D activity in the control groups T h e r e was, however, an effect of training in the 5-month-old animals. G A D activity was significantly (P < 0.05) greater by 25% m the 5-month-old trained group as c o m p a r e d to the 5-month-old control group. O n the other hand, there was no significant effect of training on G A D activity in 25-month-old animals. DISCUSSION
The primary finding of the present investigation is that chrome exercise training is associated with a decrease m T H gene expression in the adrenal gland of young rats, but not old rats. M o r e o v e r , the results provide evidence that the exercise-associated changes in T H gene expression in young animals may involve alterations in neuro-
transmitter systems that participate in the trans-synaptlc regulation of T H gene expression. Previous studies have shown that chronic exercise training is associated with decreased turnover and circulating levels of catecholamines m both humans and rodents20 27 28 The present results extend those findings to show, for the first time, that exercise acts at the level ol the T H gene to alter the capacity for adrenal catecholamine biosynthesis. The reduction in the capacity for catecholamine synthesis may be linked to the beneficial effects of exercise in hypertenswe individuals u. A e r o b i c exercise for 16 weeks has been shown to reduce blood pressure in patients with hypertension ~ A m o n g those patients who had high levels of plasma catecholamines, the reduction in blood pressure was associated with changes m catecholamlne levels lI. Extrapolating the present findings to humans, a decline m T H gene expression may contribute to the benefits of exercise m s y m p a t h o a d r e n a l hypertension Since exercise may reduce catecholamme levels m con&tions of sympathetic hyperactwity, we were interested in knowing the extent to which exercise may reverse or attenuate age-associated increases in T H gene expression. Therefore, we c o m p a r e d measures of T H activity and T H m R N A in trained and untrained rats of different ages There was an age-associated increase m T H gene expression in control animals. The age-related increase in T H gene expression is in agreement with our prewous observations and those of o t h e r s 17'35'36'4° and is consistent with the higher catecholamine levels in adrenal glands of aged rats 2' 36 On the other hand, chronic exercise training was not associated with a change in T H gene expression in the older ammals. It ~s possible that older animals require a longer training period than younger animals to produce these alterations. Alternatwely, there may be a loss of plasticity of T H gene expression during aging In that regard, we have previously r e p o r t e d an age-associated decrease in T H induction by reserpine treatment 36 However, m that study, the agerelated alteration a p p e a r e d to be primarily at the posttranscriptional level In the present study, neither T H m R N A nor T H actw~ty was affected by exercise in the older groups. The mechamsms underlymg the age-related differences in regulation of T H gene expression are not enurely known H o w e v e r , results of measures oI C h A T and G A D m the present study p r o w d e evidence that alterations in trans-synaptIc regulation of T H gene expression may be revolved The cholinergic system plays a role in regulating T H gene expression in the adrenal medulla 33 Therefore, we determined the effect of exercise and aging on C h A T activity. C h A T acuvlty was reduced in the young exercised animals, whereas there was no effect of exercise in
55 the oldest group. O u r finding of a reduction in C h A T activity in young rats is consistent with a recent finding that exercise decreases C h A T activity in the brainstem, an area i m p o r t a n t in the control of autonomic function 31. R e d u c e d C h A T activity may reflect a reduction in cholinergic nerve activity. I n d e e d , changes in C h A T activity have b e e n shown to be associated with depolarizing conditions and altered acetylcholine release in brain and sympathetic ganglia ~'6. H o w e v e r , it is unlikely that a decrease in cholinergic activity alone is responsible for the decrease in T H gene expression. Previous studies have shown that severing the splanchnic nerve that innervates the adrenal medulla, a p r o c e d u r e that depletes the adrenal gland of CHAT, does not affect basal T H gene expression, although it blocks cold stress-induced increases in T H m R N A 3z. M o r e o v e r , although cholinergic antagonists prevent increases in T H m R N A p r o d u c e d by cold stress, they do not affect basal T H m R N A levels 33. H e n c e , an alteration in cholinergic function by itself may not be sufficient to explain the alterations in T H m R N A and T H activity associated with exercise. Therefore, we examined a n o t h e r n e u r o t r a n s m i t t e r system that regulates adrenal catecholaminergic function. Several reports indicate that G A B A receptors m a y play a role in regulation of catecholaminergic activity in the adrenal gland 7'15"16. G A B A receptors have b e e n shown to exist on adrenal chromaffin cellsTM. Glutamic acid decarboxylase ( G A D ) i m m u n o p o s i t w e axons have been d e t e c t e d in the splanchnic nerve innervating the adrenal medulla, and G A D has been detected in chromaffin cells 15'16. G A B A antagonists enhance and G A B A agonists inhibit the release of catecholamines in vivo p r o d u c e d by splanchnic nerve stimulation is. M o r e o v e r , we previously r e p o r t e d that the G A B A system plays a role in regulating adrenal T H gene expression 34. Therefore, by measuring G A D activity we examined the possibility that the G A B A e r g i c system may play a role in the exercise-induced alterations in T H gene expression. In contrast to the effects of exercise on C h A T activity, G A D activity was increased in the adrenal glands of young trained rats as c o m p a r e d to young controls. O n the other hand, there was no effect of exercise on G A D
REFERENCES 1 Ando, M , Iwata, M., Takahama, K and Nagata, Y., Effects of extracellular choline concentration and K + depolarization on chohne kinase and choline acetyltransferase actwmes m superior cervical gangha excised from rats. J Neurochem, 48 (1987) 1448-1453. 2 Avakxan, E V, Horvath, S.M. and Colburn, R W , Influence of age and cold stress on plasma catecholamme levels in rats, J Auton Nerv Syst , 10 (1984) 127-133 3 Axelrod, J and Reisine, T, Stress hormones: their interaction and regulaUon, Science, 224 (1984) 452-459
activity in old rats. This is the first r e p o r t that G A D activity is modified in the adrenal gland following exercise. The increase in the capacity for G A B A synthesis associated with chronic training suggests a further mechanism for the decrease in T H gene expression in young chronically exercised rats. M o r e o v e r , the increase in G A D activity indicates that the training-induced decreases in CHAT, T H , and T H m R N A that we observed are not simply a consequence of non-specific factors. These results, when considered with results of previous studies, provide evidence that the coordinate effects of decreased cholinergic activity and increased function of the adrenal G A B A system m a y combine to reduce T H gene expression consequent to chronic exercise training. The lack of effect of exercise training on T H gene expression in old rats may be explained in the same way, by the failure of exercise to modify the functional activity of adrenal acetylcholine and G A B A systems. However, we cannot rule out a role for other factors that m o d u l a t e adrenal catecholaminergic function. F o r example, vasoactive intestinal p o l y p e p t i d e (VIP) has been shown to play a role in regulating catecholamine release and T H activation in the adrenal medulla and in an adrenal chromaffin cell line 3°'41. M o r e o v e r , we recently rep o r t e d that V I P is capable of inducing T H m R N A and T H protein in PC12 cells, an adrenal chromaffin cell line 42. Therefore, additional investigations will be necessary to m o r e fully define the extracellular mechanisms involved in the trans-synaptic regulation of T H gene expression and how they are altered by exercise and aging.
Acknowledgements This work was supported by the Geriatric Research, Education and Chmcal Centers of the Gainesvdle, FL (N.T) and St. Louis, MO Department of Veterans Affairs Medical Centers (R.S.), by grants from the Merit Review Research program of the Department of Veterans Affmrs Medical Research Service (N.T, R S.), by a grant from the American Heart Assoclauon (N.T) and by Natmnal Institutes of Health Grant AG09557 (R S ). The authors gratefully acknowledge the assistance of Dr. Scott Power m training the rats and Dr. Karen O'Malley of Washington University School of Me&tree, St. Lores, MO for providing the TH 36 cDNA probe. We also thank Anne Crawford and Lmda Pntchett for typing and Janet Wootten for editing the manuscript
4 Blguet, N.E, Buda, M., Lamouroux, A , Samolyk, D and Mallet, J., Time course of the changes m TH mRNA levels in rat brain and adrenal medulla after a single rejection with reserpine, EMBO J., 5 (1986) 287-291. 5 Brown, E R , Coker, G T. and O'Malley, K L , OrganizaUon and evolution of the rat tyrosine hydroxylase gene, B,oehemtstry, 26 (1987) 5208-5212 6 Carroll, P T, Veratndme-induced activation of chohne-O-acetyltransferase activity m rat h~ppocampal ussue: relationship to veratndme-lnduced release of acetylcholine, Brain R e s , 414 (1987) 401-404 7 Castro, E , Oset-Gasque, M J , Canadas, S , Gimenez, G and
56 Gonzalez, M P , G A B A A and G A B A B sites in bovine adrenal medulla m e m b r a n e s , J Neurosct Res , 20 (1988) 241-245 8 Chiueh, C C , Nespor, S.M. and Rapoport, S.L., Cardiovascular, sympathetic and adrenal cortical responsiveness of aged Fischer-344 rats to stress, Neuroblol Aging, 1 (1980) 157-163 9 Coleman, G L , Barthold, S W , Osbal&ston, G W , Foster, S J and Jonas, W M , Pathological changes during aging m barh e r reared Fischer 344 male rats, J Geromol , 32 (1977) 258278 10 Dixon, W.J and Massey, F J , Introduction to Statistical Analysis. McGraw-Hill, New York, 1969 11 D u n c a n , J J., Farr, J E , Upton, S J , Hagan, R D , Ogelsby, M E and Blair, S N , The effects of aerobic exercise on plasma catecholammes and blood pressure in patients with mild essential hypertension, J A M A . 254 (1985) 2609-2613 12 Fleg, J L , Tzankoff, S P and Lakatta, E G , Age-related augmentation of plasma catecholamlnes during dynamic exercise in healthy males, J Appl Phystol, 59 (1985) 1033-1039 13 Ito, K , Sato, A , Sato, Y and Suzuki, H , Increases in adrenal catecholamlne secretion and adrenal sympathetic nerve unitary activities with aging in rats, Neuroscz L e t t , 69 (1986) 263-268 14 Julius, S , Schork, N and Schork, A , Sympathetic hyperactivity m early stages of hypertension the A n n Arbor data set, J Cardtovasc Pharrnacol, 12 (Suppl 3) (1988) S121-$129 15 Kataoka, Y , Fupmoto, M , Alho, H , Guldottl, A , Geffard, M., Kelly, G . D and H a n b a u e r , I , Intrinsic g a m m a aminobutyrIc a o d receptors modulate the release of catecholamine from canine adrenal gland in SltU, J Pharmacol Exp T h e r , 239 (1986) 584-590 16 Kataoka, Y., G u t m a n , Y , Guidottl, A , Panula, P , Wroblewskl, J , Cosenza-Murphy, D , Wu, J Y and Costa, E , Intrinsic G A B A e r g l c system of adrenal chromaffin cells, Proc Nail A c i d Sct USA, 81 (1984) 3218-3222 17 Kedzierski, W and Porter, J C , Quantitative study of tyrosme hydroxylase m R N A in catecholamlnergic neurons and adrenals during development and aging, Mol B r i m Re~ , 7 (1990) 4 5 51 18 Kubo, T and Hashlmoto, M., Effects of lntraventricular and intraspinal 6-hydroxydopamlne on blood pressure of spontaneously hypertensive rats, Arch Int Pharmacodyn , 232 (1978) 166-176 19 Kvetnansky, R , Johnova, E , Torda. T , Strbek, V , Balaz, V and Maoho, L , Changes of adrenal catecholamines and their synthesizing enzymes during ontogenesis and aging in rats, Mech Aging Dev , 7 (1978) 209-216 20 L e h m a n , M and Kuhl, J , Free plasma catecholamines, heart rates, lactate levels and oxygen uptake in competition weight lifters, cyclists and untrained control subjects, lnt J Sports Med , 7 (1986) 18-21 21 Mazzeo, R S . Colburn, R W and Horvath, S M , Effect of aging and endurance training on tissue catecholamine response to strenuous exercise in Fischer 344 rats. Metabohsm, 35 (1986) 602-607 22 McCarty, R , Age-related alterations in sympathetic-adrenal medullary responses to stress, Gerontology, 32 (1986) 172-183 23 McHenry, P L , Ellestad, M . H , Fletcher, G F., Froehcher, V , Hartley, H , Mitchell. J . H and Froellcher, E S S , A position for health professionals by the committee on exercise and cardiac rehabilitation of the council on chnlcal cardiology, A m e r ican Heart Association, Special Report, Circulation, 81(1) (1989) 396-398 24 Nagatsu, T , Levitt, M and Undenfrlend, S , Tyrosine hydroxylase. The initial step in norepinephrlne synthesis, J Btol Chem , 239 (1964) 2910-2917 25 O k a m o t o , K and Aokl, K , Development of a strain of spontaneously hypertensive rats, Jpn C~rc J , 27 (1963) 282-293 26 O k u n o , S and Fujisawa, H , Assay of tyrosine 3-monooxygenase using the coupled nonenzymatlc decarboxylation of D O P A ,
Anal Btochem , 129 (1983) 405-411 27 O s t m a n , I. and Nyback, H , Adaptive changes in central and peripheral noradrenergic neurons in rats following chronic exercise, Neuro~cience, 1 (1976) 41-47 28 O s t m a n , I , Sjostrand, N O and Swedln, G , Cardiac noradrenaline turnover and urinary catecholamme excretion in trained and untrained rats during rest and exercise, Acta Phystol Scand, 86 (1972) 299-308 29 Roberts, J and Turner, N , Age-related changes in autonomic function of catecholammes. In M. Rothstein ( E d ) , Review o] Biological Research m Aging. Vol 3, Alan R Liss, New York, 1987, pp 257-298 30 Roskoski, R , White, L , Knowlton, R and Roskoski, L M , Regulation of tyroslne hydroxylase activity m rat PC12 cells by neuropeptides of the secretion family, Mol Pharmacol, 36 (1989) 925-931 31 Somani, S.M., Babu, S R , Arneric, S.P and D u b e , S N , Effect o~ chohnesterase inhibitor and exercise on chohne acetyltransferase and acetylchohnesterase activities in rat brain regions, Pharmacol Btochem Behav, 39 (1991) 337-343 32 Stachowlak, M , Fluhary, S J , Strocker, E M , Zigmond, M J and Kaplan, B B,, Molecular adaptations in catecholamine biosynthesis induced by cold stress and sympathectomy, J Neuroscl Res , 16 (1986) 13-24 33 Stachowlak, M , Stricker, E M., Zigmond, M J and Kaplan, B B , A chohnergic antagonist blocks cold stress-induced alteratlons in rat adrenal tyroslne hydroxylase m R N A , Mol B r i m Re~, 3 (1988) 193-196 34 Strong, R , Hale, C , Moore, M A , Wessels-Reiker, M , A r m brecht, H J and Richardson, A , G A B A receptor modulation of tyrosine hydroxylase gene expression in rat adrenal gland, Neuro~ct L e t t , 117 (1990) 175-180 35 Strong, R., Moore, M A , Hale, C , Burke, W J., Armbrecht, J A and Richardson, A , Effects of age and dietary restriction on adrenal catecholamlne m e t a b o h s m and tyrosine hydroxylase gene expression In J A Armbrecht, R Coe, and N Wongsurawat ( E d s ) , Endocrmology of Agmg, Springer, New York, 1989, pp 218-227 36 Strong, R , Moore, M A , Hale, C , Wessels-Reiker, M , A r m brecht, H J and Richardson, A , Modulation of tyrosine hydroxylase gene expression m the rat adrenal gland by age and reserpine, B r i m R e s , 525 (1990) 126-132 37 Strong, R , Rehwaldt, C and Wood, W.G , Intra-regional ~ariatlons in the effect of aging on high affimty chohne uptake, chohne acetyltransferase and m u s c a n m c chohnerglc receptors In rat neostriatum, Exp Gerontol, 21 (1986) 177-186 38 Strong, R , Samorajski, T and Gottesfeld, Z , Regional mapping of neostriatal neurotransmltter systems as a function of aging, J Neurochem , 39 (1982) 831-836 39 Tuck, M L , Treatment of hypertension in the elderly In J A Armbrecht, R Coe and N Wongsurawat (Eds.), Endocrinology of Aging, Springer, New York, 1989, pp 147-160 40 Voogt, J.L . Arbogast, L A , Q u a d n , S K and Andrews, G , Tyrosine bydroxylase messenger R N A m the hypothalamus substantia mgra and adrenal medulla of old female rats, Mol B r i m Res , 8 (1990) 55-62 41 Wakade, A R , Nonchohnergic transmitter(s) maintains secretion of catecholamines from rat adrenal medulla for several hours of continuous stimulation of splanchnic neurons, J Neurochem , 50 (1988) 1302-1308 42 Wessels-Reiker, M , Haycock, J , Howlett, A C and Strong, R , Vasoactive intestinal peptide induces tyroslne hydroxylase gene expression in the PC12 chromaffin cell line, J Blol Chern , 266 (1991) 9347-9350 43 Westfall, T C and Meldrum, M J , Alterations in the release o1 norepinephrine at the vascular neuroeffector junction in hypertension, Annu ReI' Pharmacol , 25 (1985) 621-641
51
BRESM 70423
Modulation of tyrosine hydroxylase gene expression in the rat adrenal gland by exercise" effects of age N. Ttimer a'b, C. Hale d'e, J. Lawler c and R. Strong d'e "Gertatrtc Research, Educanon and Chntcal Center, Department of Veterans Affairs Medical Center, Gamesvdle, FL 32608-1197 (USA), bDepartment of Pharmacology and 'Department of Exercise Scwnces, College of Medicine, Umverstty of Florida, Gamesvtlle, FL 32610 (USA), dGertatrtc Research, Education and Chmcal Center, Department of Veterans Affairs Medical Center, St Louts, MO 63125 (USA) and eDepartments of Internal Medwme, Pharmacology and Physiological Science, St Louts University Medwal School, St Louts, MO 63104 (USA) (Accepted 31 December 1991)
Key words Tyroslne hydroxylase; Tyroslne hydroxylase mRNA, Chohne acetyltransferase; Glutamlc acid decarboxylase, Exercise, Training; Aging
Both aging and exercise are associated with alterations in circulating levels of catecholamlnes. To determine the interactions of age and exercise on tyroslne hydroxylase (TH) activity and TH mRNA, Fischer-344 female rats aged 5 months (young) and 25 months (old) were trained by treadmill runnmg for 10 weeks The elevation in maximum oxygen consumption in both groups was equivalent following exercise, indicating that training had occurred In control rats, both TH activity and TH mRNA were greater in the older groups when compared with the younger animals. In young rats, exercise decreased TH activity by 25% and TH mRNA by 27%. In older rats, exercise was not associated with a decrease m TH activity and TH mRNA Chohne acetyltransferase activity (CHAT) was decreased and glutamlc acid decarboxylase activity (GAD) was increased by exercise in young rats The decrease in ChAT activity and increase in GAD activity suggest that transsynaptlc mechanisms play a role in the exercise-induced alteration of TH gene expression. Neither ChAT nor GAD was altered by exercise m older groups Our data suggest that the previously reported diminution in catecholamxnes associated with exercise may be due to a decrease in TH mRNA and a resulting decrease in TH activity. There was no effect of exercise in the old rats, supporting previous observations that the plasticity of the sympathoadrenal system diminishes with age INTRODUCTION
role both in age-related diseases such as hypertension and in the beneficial effects of exercise on the cardio-
The peripheral sympathoadrenal system exerts a major integratwe function in homeostasis. Increased sympathoadrenal activity, as evidenced by increased circulating levels and turnover of catecholamines, occurs in response to stressful extremes in the external and internal e n v i r o n m e n t of the organism 2'3. There is also a considerable body of evidence indicating that plasma catecholamines are elevated with advancing age. Ageassociated increases in circulating catecholamines are well d o c u m e n t e d in h u m a n s and laboratory animals, both at rest and in response to stress 2'8'12'13'19'22. In addition,
vascular system. Hypertension is almost certainly a multifactorial process, involving hormonal and vascular alterations. However, age-related elevation of peripheral catecholamines has been implicated in the development and possible m a i n t e n a n c e of hypertension in humans and in several experimental models 14'18'25"43. O n the other
there is a decreased ability of the catecholaminergic system of older subjects to respond and adapt to environmental challenges, suggesting that control of sympathoadrenal function is altered during aging22'34. O n e consequence of the altered regulation of sympathoadrenal function may be the increased prevalence of hypertension during aging39. Alterations in catecholaminergic function may play a
hand, exercise may be beneficial in controlling hypertension 23. Exercise increases cardiovascular functional capacity and decreases myocardial oxygen d e m a n d in healthy people as well as most patients with cardiovascular disease 23. The effects of exercise training on the cardiovascular system may be, at least partly, a result of alterations in the peripheral catecholaminergic system 11. Turnover and circulating levels of peripheral catecholamines, measured both at rest and in response to exercise, is markedly reduced by chronic exercise in both humans and rodents 2°'21'27"28. Therefore, it is possible that exercise may prevent or reverse age-related increases in catecholamine synthesis and/or release. However,
Correspondence N Tumer. Geriatric Research, Education and Chnlcal Center (182), Department Veterans Affairs Medical Center, Gainesvllle, FL 32608-1197, USA
52 t h e r e are f e w r e p o r t s o n t h e e f f e c t s o f c h r o n i c e x e r c i s e o n t h e m o d i f i c a t i o n o f p e r i p h e r a l c a t e c h o l a m i n e s in a g e d populations We and others have r e p o r t e d that the expression of tyrosine hydroxylase (TH), the rate-limiting enzymatic s t e p in c a t e c h o l a m i n e b i o s y n t h e s i s 24, is i n c r e a s e d in t h e a d r e n a l g l a n d d u r i n g aging 17 36,40. F u r t h e r m o r e , t h e adr e n a l c o n t e n t o f d o p a m m e , t h e first c a t e c h o l a m i n e p r o d uct o f T H , was also i n c r e a s e d , i n d i c a t i n g t h a t a g e - r e l a t e d i n c r e a s e s In T H g e n e e x p r e s s i o n are f u n c t i o n a l l y significant ~6. T h e e x p e r i m e n t s d e s c r i b e d h e r e e x t e n d t h o s e o b s e r v a t i o n s to an i n v e s t i g a t i o n o f t h e i n t e r a c t i o n o f age a n d e x e r c i s e o n T H g e n e e x p r e s s i o n in t h e a d r e n a l g l a n d . W e m e a s u r e d T H activity a n d T H m R N A
levels in the
a d r e n a l g l a n d s o f y o u n g a n d old rats t h a t w e r e t r a i n e d o n a t r e a d m i l l for 10 w e e k s . F u r t h e r m o r e , since t h e adr e n a l G A B A e r g l c a n d c h o l i n e r g i c s y s t e m s play a role in m o d u l a t i n g T H g e n e e x p r e s s i o n 33'34, w e m e a s u r e d glut a m i c acid d e c a r b o x y l a s e ( G A D )
a n d c h o l i n e acetyl-
t r a n s f e r a s e (CHAT) a c t i v i t y , w h i c h are s y n t h e t i c e n z y m e s for ? ' - a m i n o b u t y r i c acid ( G A B A )
a n d a c e t y l c h o l i n e , re-
spectively. T h e r e s u l t s o f this s t u d y s u g g e s t t h e previously r e p o r t e d c h a n g e s m c a t e c h o l a m m e s f o l l o w i n g exercise
involve
alterations
at
the
level
of
TH
gene
expression.
MATERIALS AND METHODS
Antmals Fischer-344 (F-344) female rats 5 and 25 months old were obtained from a colony maintained for the National Institute on Aging at Harlan-Sprague-Dawley Labs, Inc (Indianapolis, IN) On arnval, rats were housed under bamer condmons in a temperatureregulated environment (21 + 1 °C) on a 12 h hght/dark cycle The animals were fed Purina Rat Chow diet and water ad hbltum The median hfe-span of F-344 rats is 27 months9 Trammg procedure Both young and old training groups were exercised by treadmill running 5 days/week for 10 weeks, each training session began w~th a 5-mm 'warm-up' at 15 meters/ram (0% grade) On day 1 (week 1) of training, the animals began exercising at 18 meters/min (10 min duration/0% grade) wxth the duration of exercise being mcreased by 2-3 min/day until the ammals reached 60 rain of exercise (including the warm-up) The exercise time period remained at 60 mm for the duration of the study During weeks 4-6 the treadmill speed was increased by 1 meter/ram per week until a speed of 20 meters/mm was reached; the speed remained constant through weeks 6-10 Begmmng on week 7, the treadmill grade was increased by 1 5%/week until reaching a zemth of a 6% grade during week 10 of training Non-exercised ammals were placed into the treadmill for equal lengths of time but the apparatus kept off Ammals were monitored continuously during exercise Measurement of Vomax At the completion of the training period, VOmax was measured on 4-6 animals in each experimental group using a flow-through open-circuit system Briefly. animals performed an incremental e\ercise protocol that began with a 4-ram warm-up and progressed with a work rate increase every four rain untd the ammals were unable to maintain the required power output VO, max was de-
fined as the h~ghest VO, obtained during the exercise test The protocol was designed such that VOmax was generally reached within 12-20 mm followmg the warm-up Room mr (P~O 2 = 142 mmHg) was pumped through the sealed treadmill chamber (Ommtech Electronics, Columbus, Ohio) at a flow rate of 4 5 hters/mm Gas was sampled (500 ml/min) from a small mmng chamber located at the back of the treadmill and analyzed for CO 2 and 0 2 concentrations using electronic gas analyzers (Beckman LB-2 and OM-I1, respectively, Sensor Medics, Anahmm, CA) The gas analyzers were cahbrated ~mmedmtely before and after each test using gravlmetrlcally standardized gases
Ttsaue pteparalton After ammals were anesthetized wtth pentobarbital (90 mg/kg), adrenal glands were removed qmckly, lmmedmtely frozen on dry me and stored at -80°C Each pmr of adrenal glands was decapsulated, weighed and homogemzed m 100/~1 of buffer (2 mM sodium phosphate, pH 6 0, fi 2c/c Triton) Aliquots of homogenate were removed for each assay as follows lfi ttl for TH assay, 10 ~1 for GAD and ChAT assays, 5 fd for protein assay, and the remainder (75 ~1) for determination of TH mRNA studies Tvrosme hvdrorylme assay Adrenal TH acnvity was measured by a coupled decarboxytase assay we previously described3~ The assay is based on trapping radmlabelled CO, evolved from the conversion of L-[~C]tyrosme to dopamlne by chemical decarboxylatlon26 Briefly, each 10-~1 sample of adrenal homogenate was diluted to 100 gl with homogenizing buffer The diluted sample was passed over Sephadex G50 columns to remove endogenous catecholamlnes Pomons of the eluate (6 ul) were assayed in duphcate m a final assay volume of 10 !d The concentration of the cofactor 6-MPH 4 was 3 mM, and the concentratmn of [14C]tyrosIne was 40 ~M, and the final pH of the reactmn mixture was 6 5 GAD assa~ GAD activity was determined m homogenates of adrenal glands using methods prewously described by us3~ GAD was measured by a decarboxylatlon assay using L-[l-t4C]glutamate as a substrate with subsequent trapping of ~4CO~ Ttssue homogenate (2 Fd) was added to the reaction m~x (5/d) containing 10 mM glutamate as a substrate ('hollne acetyltransferase assal ChAT actlwty was measured m 2 Fd of adrenal homogenate using [L-14C]acetyl CoA (0 2 mM) as a substrate, in a final volume of 7/d. as described previously by us ~v3s Tyrosme hvdroD'lase c D N A - m R N A hybrldtzatton The concentranon of total cellular RNA extracted from adrenal glands was determined from ethidmm bromide-stained gels using methods for small tissue samples described previously by Mallet and coworkers4 Total cellular RNA was subjected to agarose gel electrophoresis concurrently with RNA of known concentration, as determined by optmal density measured at wavelength 260 nm on a Beckman (Irvlne, CA) Spectrophotometer The ethldmm bromide-stained gels were exposed to ultraviolet light and photographed using Polaroid (Cambridge, MA) Type 55 film. The resultmg negative was scanned on a Blo-Rad (Richmond, CA) Model 620 scanning densltometer to obtain optical density measurements To quantify the concentration of RNA in the samples, the peak areas corresponding to the 18S and 28S ribosomal bands in the unknown were compared to those obtained from a group of sermlly diluted standards of known concentration The concentration of each sample was derived by hnear regression analysis Several concentrations of serially diluted RNA samples were immobilized on nylon membranes (Gene Screen. New England Nuclear, Boston, MA) using a Blo-Rad slot blot apparatus The baked filters were prehybndlzed using 25 mM potassmm phosphate, 5 × SSC, 5 .< Denhardt's solution, 100 mg/ml denatured salmon testes
53 TABLE I
5000
-
Effects of exercise traming and agmg on oxygen consumption 4000-
•
control
[]
tramed
Values represent m e a n s + S.E.M. of 9-12 rats. ="
Treatment
Maximum oxygen consumption (ml/kg/mm) 5 month
25 month
79.4 + 1 9 94 4 + 0 7*
59 4 + 3 0"* 72.8 + 1 2* **
~= ~
3000 -
2000 -
1000
Control Trained
-
0-
D N A , and 50% formamlde. After incubatmn for 14-16 h at 42°C, the filter was hybridized with a 32p nick-translated full-length rat T H 36 c D N A probe 5, kindly supplied to us by K a r e n O'Malley, Washington University School of Medicine. After incubation for 14-16 h at 42°C, the filter was washed and exposed to X-ray film (Kodak X - A R , Rochester, NY) for 72 h. T h e developed autoradiogram was scanned using a Blo-Rad Model 620 Video Densltometer The optical density was linear over the range of total cellular R N A used to calculate T H m R N A levels Values for T H m R N A content were derived from the slopes of hnes derived from plotting optical density versus total cellular R N A concentratmn
Data analysis Means and standard errors of the m e a n (S E . M ) were calculated from values obtained from replicate experiments in individual animals from each age and treatment group For T H , G A D and C h A T assays, each sample was assayed in duplicate. For T H m R N A m e a s u r e m e n t s , values were derived from m e a s u r e m e n t s m a d e on 3 - 4 different concentrations of total cellular R N A Comparisons of m e a n s a m o n g different age and treatment groups were m a d e by analys~s of variance D~fferences between individual group m e a n s were determined by Flscher's Protected Least Slgmficant Difference test i° Differences were considered significant when P < 0.05 (two-tailed test)
RESULTS
Table I shows that following 10 weeks of training, maximum oxygen consumption was significantly (P < 0.05) higher in trained animals of both age groups as compared to their respective control groups. Although the levels of oxygen consumption differed significantly
25
5
* Significantly different from age-matched control, P < 0.05. ** Slgmficantly different from 5 m o n t h control, P < 0.05.
AGE (too)
Fig. 1. Effects of age and exercise on adrenal T H activity Values represent m e a n s _+ S.E.M. of 9-11 rats assayed in duplicate. *Significantly different from age-matched controls, P < 0.01. tSignlficantly different from 5-month-old controls, P < 0.01
(P < 0.05) between the two age groups, both groups showed a similar magnitude of increase in oxygen consumption associated with training. These data indicate that training occurred equally in both groups. To determine the effect of training and age on the capacity for catecholamine biosynthesis, TH activity was assessed. Among control groups, T H activity was significantly greater in the 25-month-old compared with 5-month-old animals (Fig. 1). Among 5-month-old animals, T H activity was significantly lower (P < 0.01) in the trained group as compared to the young control group. However, in the older rats there was no significant difference in T H activity associated with training. To determine if alterations in TH activity were a consequence of changes in gene expression, we measured TH mRNA levels using a full-length TH cDNA probe. The results of T H mRNA quantification by slot blot analysis are shown in Fig. 2. Among control animals, a comparison between age groups revealed that T H mRNA was significantly (P < 0.01) elevated by 48% in adrenals from old rats compared with young rats. In the 5-monthold animals, TH mRNA in adrenals of exercised rats was significantly lower (P < 0.01) as compared to the young
T A B L E III T A B L E II
The effect of exercise and agmg on adrenal GAD activity
The effect of exercise and aging on adrenal ChAT activity Values represent m e a n s + S E M treatment group
Treatment
Control Trained
of 5-11 rats per age and
ChAt activity (nmol/mg protein~h)
Values represent the m e a n + S E.M. of samples assayed in duphcate. The n u m b e r of samples per group is shown in parentheses.
Treatment
5 month
25 month
9 06 + 0.58 6.57 + 0 41"
9.67 + 0 78 8.59 + 0 57
* Significantly different from 5 m o n t h control, P < 0 05
Control Trained
GAD activity (nmol/mg protem/h) 5 month
25 month
39 0 _+ 3 5 (8) 48 9 + 2.1" (8)
31 4 _+ 4.1 (7) 37 9 + 1.8 (7)
* Significantly different from age-matched control, P < 0 05.
54
15 [] []
control trained
10
o Z I= 'lr I-
05
O0 5
25
AGE (mo)
Fig 2 Effects of age and exercise on adrenal TH mRNA Values represent means + S E M of 9-11 samples, assessed at 3-4 concentratlons of total cellular RNA per sample. *Slgmficantly different from age-matched controls, P < 0 01 *Slgmficantly different from 5-month-old controls, P < 0 01
control group. On the other hand, there was no significant effect of training on T H m R N A in 25-month-old animals. Previous studies have shown that trans-synaptic induction of T H is mediated, at least partly, by acetyicholine 33. Therefore, we examined the functional status of the cholinerglc system by measuring C h A T actiwty. Table II shows that, in contrast to the findings for T H activity and T H m R N A , there was no significant effect of age on C h A T activity in control animals. On the other hand, c o m p a n s o n s between the 5-month-old control and trained groups revealed that C h A T activity was significantly (P < 0.05) lower by 30% in exercised rats as compared to the control group There was no significant effect associated with exercise training in the 25-month-old animals. The G A B A system has been shown to modulate catecholamine release and T H gene expression ls'1634 Therefore, we investigated the effect of age and exercise on G A D activity. As shown in Table III, there was no significant effect of aging on G A D activity in the control groups T h e r e was, however, an effect of training in the 5-month-old animals. G A D activity was significantly (P < 0.05) greater by 25% m the 5-month-old trained group as c o m p a r e d to the 5-month-old control group. O n the other hand, there was no significant effect of training on G A D activity in 25-month-old animals. DISCUSSION
The primary finding of the present investigation is that chrome exercise training is associated with a decrease m T H gene expression in the adrenal gland of young rats, but not old rats. M o r e o v e r , the results provide evidence that the exercise-associated changes in T H gene expression in young animals may involve alterations in neuro-
transmitter systems that participate in the trans-synaptlc regulation of T H gene expression. Previous studies have shown that chronic exercise training is associated with decreased turnover and circulating levels of catecholamines m both humans and rodents20 27 28 The present results extend those findings to show, for the first time, that exercise acts at the level ol the T H gene to alter the capacity for adrenal catecholamine biosynthesis. The reduction in the capacity for catecholamine synthesis may be linked to the beneficial effects of exercise in hypertenswe individuals u. A e r o b i c exercise for 16 weeks has been shown to reduce blood pressure in patients with hypertension ~ A m o n g those patients who had high levels of plasma catecholamines, the reduction in blood pressure was associated with changes m catecholamlne levels lI. Extrapolating the present findings to humans, a decline m T H gene expression may contribute to the benefits of exercise m s y m p a t h o a d r e n a l hypertension Since exercise may reduce catecholamme levels m con&tions of sympathetic hyperactwity, we were interested in knowing the extent to which exercise may reverse or attenuate age-associated increases in T H gene expression. Therefore, we c o m p a r e d measures of T H activity and T H m R N A in trained and untrained rats of different ages There was an age-associated increase m T H gene expression in control animals. The age-related increase in T H gene expression is in agreement with our prewous observations and those of o t h e r s 17'35'36'4° and is consistent with the higher catecholamine levels in adrenal glands of aged rats 2' 36 On the other hand, chronic exercise training was not associated with a change in T H gene expression in the older ammals. It ~s possible that older animals require a longer training period than younger animals to produce these alterations. Alternatwely, there may be a loss of plasticity of T H gene expression during aging In that regard, we have previously r e p o r t e d an age-associated decrease in T H induction by reserpine treatment 36 However, m that study, the agerelated alteration a p p e a r e d to be primarily at the posttranscriptional level In the present study, neither T H m R N A nor T H actw~ty was affected by exercise in the older groups. The mechamsms underlymg the age-related differences in regulation of T H gene expression are not enurely known H o w e v e r , results of measures oI C h A T and G A D m the present study p r o w d e evidence that alterations in trans-synaptIc regulation of T H gene expression may be revolved The cholinergic system plays a role in regulating T H gene expression in the adrenal medulla 33 Therefore, we determined the effect of exercise and aging on C h A T activity. C h A T acuvlty was reduced in the young exercised animals, whereas there was no effect of exercise in
55 the oldest group. O u r finding of a reduction in C h A T activity in young rats is consistent with a recent finding that exercise decreases C h A T activity in the brainstem, an area i m p o r t a n t in the control of autonomic function 31. R e d u c e d C h A T activity may reflect a reduction in cholinergic nerve activity. I n d e e d , changes in C h A T activity have b e e n shown to be associated with depolarizing conditions and altered acetylcholine release in brain and sympathetic ganglia ~'6. H o w e v e r , it is unlikely that a decrease in cholinergic activity alone is responsible for the decrease in T H gene expression. Previous studies have shown that severing the splanchnic nerve that innervates the adrenal medulla, a p r o c e d u r e that depletes the adrenal gland of CHAT, does not affect basal T H gene expression, although it blocks cold stress-induced increases in T H m R N A 3z. M o r e o v e r , although cholinergic antagonists prevent increases in T H m R N A p r o d u c e d by cold stress, they do not affect basal T H m R N A levels 33. H e n c e , an alteration in cholinergic function by itself may not be sufficient to explain the alterations in T H m R N A and T H activity associated with exercise. Therefore, we examined a n o t h e r n e u r o t r a n s m i t t e r system that regulates adrenal catecholaminergic function. Several reports indicate that G A B A receptors m a y play a role in regulation of catecholaminergic activity in the adrenal gland 7'15"16. G A B A receptors have b e e n shown to exist on adrenal chromaffin cellsTM. Glutamic acid decarboxylase ( G A D ) i m m u n o p o s i t w e axons have been d e t e c t e d in the splanchnic nerve innervating the adrenal medulla, and G A D has been detected in chromaffin cells 15'16. G A B A antagonists enhance and G A B A agonists inhibit the release of catecholamines in vivo p r o d u c e d by splanchnic nerve stimulation is. M o r e o v e r , we previously r e p o r t e d that the G A B A system plays a role in regulating adrenal T H gene expression 34. Therefore, by measuring G A D activity we examined the possibility that the G A B A e r g i c system may play a role in the exercise-induced alterations in T H gene expression. In contrast to the effects of exercise on C h A T activity, G A D activity was increased in the adrenal glands of young trained rats as c o m p a r e d to young controls. O n the other hand, there was no effect of exercise on G A D
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Acknowledgements This work was supported by the Geriatric Research, Education and Chmcal Centers of the Gainesvdle, FL (N.T) and St. Louis, MO Department of Veterans Affairs Medical Centers (R.S.), by grants from the Merit Review Research program of the Department of Veterans Affmrs Medical Research Service (N.T, R S.), by a grant from the American Heart Assoclauon (N.T) and by Natmnal Institutes of Health Grant AG09557 (R S ). The authors gratefully acknowledge the assistance of Dr. Scott Power m training the rats and Dr. Karen O'Malley of Washington University School of Me&tree, St. Lores, MO for providing the TH 36 cDNA probe. We also thank Anne Crawford and Lmda Pntchett for typing and Janet Wootten for editing the manuscript
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