Adaptation capacity of biogenic amines turnover to hypoxia in different brain areas of old rats

Neurochem. Int. Vol. 17, No. 2, pp. 281 289, 1990 Printed in Great Britain. All rights reserved

0197-0186/9053.00+0.00 Copyright © 1990Pergamon Press plc

ADAPTATION CAPACITY OF BIOGENIC AMINES T U R N O V E R TO HYPOXIA IN D I F F E R E N T BRAIN AREAS OF OLD RATS* G. SCHULZE, H. COPER a n d CH. FAHNDRICH Institute of Neuropsychopharmacology, Free University of Berlin, Ulmenallee 30, 1000 Berlin 19, F.R.G. (Received 15 January 1990 ; accepted 9 March 1990) Ahstract--Catecholamines and indoleamines serve in the CNS as neurotransmitters in a great number of functional pathways. In order to contribute findings which might help to understand differences in functioning and behavioral performances with aging, the concentrations of dopa, dopamine, noradrenaline, 5-hydroxytryptophane (5-HTP), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) in four brain regions (hypothalamus, c. striatum, hippocampus, cortex) of young and old rats were determined 30 min after i.p. injection of NSD 1015 (3-hydroxybenzylhydrazine hydrochloride). The influence of 12 h hypoxia of 10 and 8% 02 as well as 36 h hypoxia of 10% was investigated. Accumulation of dopa and 5HTP under normoxic conditions is reduced in old rats compared to young rats; the concentration of dopamine, noradrenaline and 5-HT is not significantly different between the age groups. After 12 h of 10% 02 in young rats a drop of dopa accumulation occurs, only at 8% 02 in both groups can a significant reduction be observed; dopamine and noradrenaline do not show a uniform tendency. Under the same conditions 5-HTP accumulation is reduced in both groups, 5-HT and 5-HIAA decrease at 10% 02 but are in the range of controls at 8% 02. After 36 h 10% O2 hypoxia dopa accumulation in young rats returns to normal whereas in the striatum of old rats the decrease continues, but in the hypothalamus an increase above normal occurs. Dopamine and noradrenaline return to normal. Besides, in the hypothalamus of young rats 5-HTP accumulation is compensated. 5-HT and 5-HIAA rise even above control values.

F o r several years n o w a high level o f a g r e e m e n t has existed o n the view that, in functional terms, the m o s t significant impact o f the aging process o f a n organism is its progressively diminishing adaptability to e n d o g e n o u s a n d exogenous stimuli. The individual cell, o r g a n a n d system functions in a regulated state, i.e. a state of reciprocal control, react to these stimuli t h r o u g h various mechanisms. This state is generally referred to as homeostasis a n d defined as a steady state equilibrium t h a t c a n occur as a response to a given situation or r e q u i r e m e n t within a more or less b r o a d n o r m a l range. It ensures continuity, b u t also provides the possibility for change. H o m e o s t a s i s o n the cellular level as well as o n the level of the entire o r g a n i s m can be m a i n t a i n e d only if the d y n a m i c range o f the various functions possesses sufficient stability a n d capacity. All processes involved in m a i n t a i n i n g homeostasis can be s u b s u m e d u n d e r the term adap-

tivity. W i t h increasing age the control of homeostasis becomes m o r e a n d m o r e sluggish, its range narrows, its stability a n d capacity diminish. One expression of the diminished stability o f regulated systems is t h a t their susceptibility to disorders increases, This can be detected a n d nearly quantified by m e a n s of the deviation from standard, w i t h o u t the exact cause (always) being known. T h e same is true o f the capacity for c o m p e n s a t i o n . It is determined by the scope a n d speed o f the c o m p e n s a t o r y or controlling m e c h a n i s m s a n d can be tested by determining the critical value required to overstrain the system in question (Coper et al., 1986). This concept has been verified repeatedly in studies in the field o f behavioral biology (Schulze et aL, 1988 ; J/inicke et al., 1988; F r e e m a n n a n d G i b s o n , 1988) a n d is, in general terms, a p p a r e n t l y even valid for cognitive functions o f m a n (Baltes, 1987). Schulze a n d J/inicke (1986) f o u n d in aged rats a p r o t r a c t e d a d a p t a t i o n period for s p o n t a n e o u s m o t o r b e h a v i o r a n d food intake u n d e r the influence o f a mild n o r m o * Dedicated to Professor Hermann Blaschko on the occasion of his 90th birthday. baric hypoxia (10 v o l % 02) o f 72 h duration. The 281

282

G. SCHULZF,et al.

aged animals with chronic 0 2 deficiency were also unable to cope adequately with the requirements of an o p e r a n t b e h a v i o r test (J/inicke a n d Schulze, 1987). On the molecular level, the concept of diminishing adaptivity in old age has only begun to be pursued. But ample findings do exist which point in the direction of an increased susceptibility of n e u r o n a l transmission in old age. This expresses itself in an in part decreased q u a n t i t y of transmitters, in a loss of receptors or diminished plasticity, i.e. capacity for up or down regulation or in modified enzymatic control activity (Carfagna et al., 1985; Rogers and Bloom, 1985: Sparks et al., 1985; Algeri a n d Ponzio, 1986; Greenberg, 1986; Ponzio a n d Algeri, 1986; Bloom, 1988; Scarpace, 1988). In recent years the focus of interest has been the cholinergic system (choline deficiency hypothesis o f S D A T ) (Crook et al., 1986; Pedigo, 1986: H a g a n a n d Morris, 1988; Pilch a n d Mfiller, 1988). On the special stress conditions o f mild hypoxia transmitters have not been investigated as t h o r o u g h l y and systematically for purposes of c o m p a r i s o n under consideration of the a b o v e - m e n t i o n e d concept (Davis and Carlsson, 1973; F r e e m a n a n d Gibson, 1988). Brown et al. (1975) a n d J/iggi a n d Loew (1976) found that hypoxia-related behavioral deficits can in part be countered by applying substances with a d o p a m i n e agonistic effect. F o u r hypotheses on the adaptivity of the central neuronal d o p a m i n e / n o r a d r e n e r g i c a n d serotonergic system in old age were tested in the present study. (1) The synthesis of D O P A a n d 5-HTP is slowed down in the brains o f aged animals. (2) The c o n c e n t r a t i o n s of dopamine, N A or 5H T a n d 5-HIA show a lower level in aged animals in a steady state. (3) The age effect on D O P A and 5 - H T P synthesis, with c o r r e s p o n d i n g consequences for amine concentration, is intensified by a 12 h hypoxia as a function of the degree of 0 2 deficiency. (4) The tendency toward normalization of synthesis activity (adaptivity) is delayed in aged animals during a hypoxia lasting 36 h.

EXPERIMENTAL PROCEDURES

Animals

Female Wistar rats were used. The young adult animals were 4 6 months old and weighed between 200 220 g. The aged animals were 29-month-old exbreeders weighing 305 _+35 g. The animals of this age group are the 50% survivors of an original population. The age-related spon-

taneous mortality of the animals amounted to 68 per 1000 a month. Up to the onset of the experiment the animals were kept in groups of five in a 12/12 light/dark cycle with food and water ad libitum. Hypoxia exposure

The experimental animals were placed in pairs into chambers in which it was possible to produce a normobaric reduction of pOe by means of a controlled introduction of nitrogen (cE Schulze and J~nicke, 1986 for details). The exposure was performed at 10% O2 for 12 and 36 h and at 8% 02 for 12 h. Normoxic controls were kept in chambers of the same type at 20.9 vol% O_,. Thirty minutes before the end of the exposure, the animals were injected with 100 mg/kg of NSD l015 (3-hydroxybenzylhydrazine hydrochloride) i.p. Ti,ssue preparation, extraction, separation and detection o/ catechols and indoh,s

The rats were decapitated at the end of the exposure period, their brains removed and the regions (1) hypothalamus, (2) corpus striatum, (3) hippocampus and (4) cerebral cortex prepared on an ice-cooled glass plate. For ( 1), (2) and (3), the preparation followed given morphological structures; cerebral cortex was obtained as a c. 0.5 mm thick slice with the aid of a tissue slicer. The tissue of each region was divided for the separate extraction of catechols and indoles and stored on dry ice until it was further processed. The weights of the semi-regions were: hypothalamus 16.8_+3.6 rag; c. striatum 28.5_+6.1 mg; hippocampus 39.7_+ 7.3 mg : cortex cerebri 47.7 +_ 10 mg. Extraction

The procedure is basically in accordance with the method of Wagner et al. (1979) with minor modifications : (a/ Catechols. Up to 100 mg of tissue was ultrasonically homogenized (Sonifire, microtip) in 0.5 rnl of a 0.4 mol/1 HCIO4 solution containing 5.4 mmol/l of EDTA and 2.5 mmol/l of Na-metabisulphite. Having been centrifuged for 5 rain at 12,000 g, the supernatant was adjusted to pH 8.6 with 400 id of Tris buffer, I tool/l, and an adsorption to A1203 performed. After having been washed twice with distilled water, the elution from the AI,O3 was carried out with 300 #1 of 0.1 mol/l acetic acid : the acetic acid was removed by lyophilization and the sample dissolved in 200 #1 of mobile phase (elution buffer) of the subsequent reversed phase HPLC. fh) lndoles. Up to 50 rng of tissue was ultrasonically homogenized in 100 #1 of a 0.62 mol/1 ZnSO4 solution containing 1. I mmol/l ofascorbic acid. Separation and delection

tIPLC device : HP 1084B, electrochemical detector : BAS LC 4A. Catechol column : 200 x 4 mm; stationary phase : Shahdon MOS 5 u ; mobile phase : phosphate/citrate buffer, 0.02 tool/l, pH 3.3, 20% methanol, 1.8 mmol/1 Na-octysulphate, 0.02% EDTA; column temperature 33':C, flow 1 ml/min; oxidation potential of the working electrode 720 mV against an Ag/AgCI reference electrode. Indole column : 200 x 4 mm : stationary phase : Shandon ODS 5 u ; mobile phase: phosphate/citrate buffer 0.1 mol/I, pH 3.0, 10% methanol, 0.2% EDTA, column temperature 33 C, flow 1 ml/min: oxidation potential of the working electrode 720 mV against Ag/AgCI reference electrode.

Adaptation capacity of biogenic amines turnover to hypoxia

283

Hypothakamus

IF

130. 120. %

110.

A

100.

M I N

90.

E

7060.

C 0

N C

+.

I

,

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50. 40302010. 0 .315 .257

.267 .269

1.52 1.39

.225 .173

1.66 1.81

.263 .316

.018 .017

,021 .021

.080 .065

.019 .013

.069 .080

.014.024

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YNG OLD

YNG OLD

YNG OLD

YNG OLD

YNG OLD

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90. 80. 70. 60, 50. 40. 30. 20. 10O.932 .860

11.4 10.0

.096 .078

.112 .069

.697 .639

.289 .301

.058 .045

.548 .456

.010 .009

.009 .005

.039 .042

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YNG OLD

YNG OLD

YNG OLD

YNG OLD

YNG OLD

DOPA

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5--HT

5HIAA

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Fig. 1. Concentration of DOPA, dopamine (DA), noradrenaline (NA), 5-hydroxytryptophane (5-HTP), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-acetic acid (5-HIAA) in two regions (hypothalamus and c. striatum) of brains of young and old rats. Each column represents the mean_+_SEM of 10 rats expressed as percent values. The numbers below the columns represent mean and SEM expressed as #g/g wet weight. 4tp < 0.05 (Scheff6).

284

G. SCHULZEel al.

The quantity of an amine in the injection volume was calculated by referring the surface units beneath the chromatogram peak to the surface values obtained from standard solutions carried through the entire extraction procedure. In determining the tissue concentration, the fact that the extraction volume was increased by 75% of the tissue weight was taken into account.

Statistics The tissue concentrations of the amines examined were subjected to a two-factor analysis of variance per region and amine with the factors of age (2 levels) and 02 concentrations in the air inspired (3 levels). Single comparisons in pairs were performed as per Scheff6 (H0: M i = M2). An overall significance level of 5% was defined. RESULTS

(1) Accumulation of DOPA and 5-HTP The D O P A synthesis rate, as detected with the aid of the method employed, differs considerably in the regions examined. Low rates are found in cortex and hippocampus ( < 0.1/~g/g wet wt/30 min ; not demonstrated), mean rates in the hypothalamus (~0.3/~g/g wet wt/30 min) and highest rates in the striatum (0.9 #g/g wet wt/30 rain). The values for aged animals are lower throughout in the corresponding regions (Fig. 1) For 5-HTP accumulation, a factor of c. 3 is found between the lowest value (cortex : 0.077 ~g/g wet wt/30 rain; hippocampus: 0.091 /~g/g wet wt/30 min) and the highest value (hypothalamus : 0.23/~g/g wet wt/30 rain). In old age, the synthesis rate in the hypothalamus is diminished by 27%, by c. 40% in the three other regions. Hypothesis (1) is thus verified.

(2) Amine concentration in younq and aged animals under normoxia The concentration of the transmitters dopamine, noradrenaline and 5-HT, which are metabolically dependent on D O P A or 5-HTP, show no significant age-related changes (Fig. 1 ; cortex and hippocampus

not demonstrated). Hypothesis (2) must therefore be rejected.

(3) Influence oJ 12 h of hypoxia on (a) the synthesis of DOPA and 5-HTP and (b) on the catechol and indole concentration in different brain regions in youn 9 and aged animals The matter at hand now was to test whether and to what extent the dependent variable "O2 content of the air" has an influence on the synthesis and concentration of catechols and indoles in different brain regions of young and aged animals. Since the individual brain regions in principle react the same way to the hypoxia, the following remarks and data are limited to changes in the striatum and the hypothalamus. (a) For 10% O: in the air inspired by young animals, the accumulation of D O P A is found to be distinctly lower- by 25% in the hypothalamus and by 32% in the striatum, whereas it remains largely constant in the aged animals (Fig. 2). When the 02 content of the air is further reduced, the D O P A accumulation likewise sinks by up to 50% in both young and aged animals. Ten percent 02 in the inspiration air leads to a similar result with regard to formation of 5-HTP. A reduction of the 5-HTP accumulation (e. 30%) is found in all brain regions of the young animals. In the aged animals the H T P synthesis is significantly more impaired. A pronounced 02 deficit (8% in the inspiration air) leads, in analogy to the D O P A synthesis (except striatum of young rats), to a further reduction of 5-HTP formation in young and aged animals. (b) The concentrations of transmitters are not a mirror image of the synthesis activity of tyrosine hydroxylase or tryptophan hydroxylase. N A does decrease in the hypothalamus of young and aged animals at 10% 02 but D O P A supply is diminished only in the young. DA remains constant in the striatum

Fig. 2 (opposite). Concentration of DOPA, dopamine, noradrenaline, 5-hydroxytryptophane, 5-hydroxytryptamine, 5-hydroxyindole-acetic acid in two regions of brains of young and old rats after 12 h exposure to 21, 10 and 8% 02 in the inhalation air. Each point represents the mean_+SEM of 10 rats expressed as percent values of young controls.

Hypothalamus C. striatum

Young

Old

~ []

0 A

~ P < 0.05 vs young controls (Scheff6). * P < 0.05 vs old controls (Scheff6).

285

Adaptation capacity of biogenic amines turnover to hypoxia DOPA

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286

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of young and aged animals in spite of reduced or unchanged D O P A synthesis. 5-HT and 5-HIAA show only a change in hypothalamus in old rats. A more intensive hypoxia (8%) hardly leads to any changes of D A concentration in young and aged animals, nor does it follow the sharp drop of D O P A accumulation. The N A content, on the other hand, sinks systematically only in the hypothalamus. The indoles remain stable in the young animals; in the aged animals the decrease at 10% O , level, is compensated more or less at 8% O2. Hypothesis 3 must thus be rejected and is in need of a much more differentiated examination. (4) Effects ~[ 36 h o f 10% O : on the turnover and concentration 0[" catechols and indoles

The D O P A accumulation, which had dropped in the young animals following 12 h of 10% normobaric hypoxia, shows a tendency toward normalization after 36 h have elapsed. No adaptation can be observed in the aged animals, in which synthesis activity in the striatum decreases further. An increase to 140% of the initial value occurs in the hypothalamus. The case tends more to the opposite for the indoles. As opposed to the reaction in the striatum, the synthesis of 5-HTP in the hypothalamus of the young animals, which is diminished following 12 h of hypoxia, does not return to normal. In the aged animals, the initially decreased turnover in both regions moves towards the starting level (Fig. 3). As was mentioned above, no pronounced agerelated adaptive insufficiency was observed in the concentration ofcatechols. The negative indole deflection in the aged animals after 12 h is overcompensated after 36 h. Hypothesis (4) can thus be seen as having been verified in part. It, too, will have to be reform ulated. DISCUSSION

amines which he has strongly influenced for the past 50 years. Nevertheless, many findings obtained in investigations on the concentration of catecholamine or indolamine neurotransmitters and their precursors and catabolic products in the brains of young and aged rats fail to tally. N o r are the findings of investigations of the in t~itro activity of a number of brain enzymes involved in catecholamine metabolism without contradictions (Rogers and Bloom, 1985). The deviations, however, seldom amount to more than 25%. But the dimension of changes in enzyme activities likewise always remains under 25% (Reis et al., 1977). Since even the findings within a species depend on a number of variables like breed, sex, age, preparation of brain regions etc., the changes observed are evidently not a universal phenomenon of aging. The data obtained in investigations should thus be seen as having the character of an approximate value for the changes induced by hypoxia ("testing the limits"). As Davis and Carlsson (1973) already pointed out, a relative hypoxia of 10% retards D O P A synthesis, which, in the sense of an adaptation, again returns to normal following a more or less protracted period of time (36 h) (Davis. 1975. 1976). But, contrary to expectations, a reduction of D O P A accumulation evidently occurs only in the young animals. For both intermediary metabolites ( D O P A and 5-HTP) however it is valid that their synthesis drops in all regions except the striatum in animals of both age groups as the availability of 02 further decreases. Tyrosinehydroxylase ( " T H " ; EC 1.14.16.2) is the key enzyme and at the same time the rate-limiting step in catecholamine synthesis, as is tryptophanhydroxylase ( " T r H " : EC 1.t4.16.4) for the metabolism of serotonin. The turnover of tyrosine into dihydroxyphenylalanine (DOPA) or of tryptophan into 5-HTP is regulated by a number of factors (Mandell, 1978). The most familiar of these l:actors are :

Recently Blaschko (1987) reviewed the history of the pharmacology and biochemistry of catechol-

(l) PO, = normally 76 mm Hg or pAO2 = 55 mm Hg ;

Fig. 3 r opposite ). Concentration of DOPA, dopamine, noradrenalinc, 5-hydroxytryptophanc, 5-hydroxytryptamine, 5-hydroxyindole-acetic acid in two regions of brains of young and old rats after 0, 12.36 11 exposure to 10% 02 in the inhalation air. Each point represents the mean+SEM of 10 rats expressed as percent values of young controls.

Hypothalamus C. striatum

Young

Old

~ [~

C' i~

~ P < 0.05 vs young controls (Scheff6). ~'P < 0.05 vs old control (Scheff6).

Adaptation capacity of biogenic amines turnover to hypoxia

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With a physiological substrate concentration and tetrahydrobiopterin (BH4) as a co-factor (Katz, 1980, 1981), the Km of the T H for 02 is 2 3 mm Hg, and that of the TrH amounts to 3~4 mm Hg. If, as was the case here, the partial 02 pressure is lowered, this should have repercussions for the activity of the enzymes, as it did in the young animals. Why the effect does not occur in the aged animals is, for the present, difficult to understand. N o r can the data be explained by modified end product inhibition (Carlsson et al., 1976), since a formal relation of this type can be observed only in the hypothalamus of aged animals. The findings cannot even be explained by regulatory influences like the BH4 co-factor being systematically altered in old age or by 02 deficiency, as these have not yet been investigated. They are, however, by no means surprising within the concept of declining adaptivity in old age. This means for the present findings : (a) The metabolism of young animals is retarded under mild hypoxia, but this retardation has little effect on the availability of transmitters. It is an appropriate adaptation to a given situation. Aged animals respond less flexibly and less economically. As a response to the stimulus of hypoxia, they keep up the turnover of catechols, or even increase it, without there being a need to compensate for a transmitter deficit. What in part occurs is production, which is not required in functional terms and remains without consequences. (b) When the 02 supply is further diminished, an adapted or surplus counterregulation is more restricted. Young and aged animals respond more or less uniformly by lowering their synthesis activity, which becomes insufficient to sustain D A and N A (except NA in the striatum). The 5-HT level is evidently kept up even more energetically. (c) The somewhat reduced capacity for compensation in old age also becomes evident in adaptivity in the face of more protracted stress. It is, however, less distinct in the relatively low deviation from the standard in the catechol concentrations than in the 5-HT system. The differences in behavior or performance mentioned in the introduction for young and aged animals under the stress of hypoxia appear to have no direct bearing on the turnover and concentration of catecholamines and indolamines. The greater attenuation of motility and the sharper reduction of food

and fluid intake as well as the reduced learning capacity in an operant behavior paradigm observed for the aged animals is accompanied by almost normal D O P A synthesis and slightly reduced 5-HTP synthesis as compared with normoxic conditions. Similar discrepancies were also demonstrated in the cholinergic system (Schulze and Siebel, 1990). This provides confirmation of the fact that the complex regulatory and control functions cannot be assigned to one specific biochemically determined function. At the same time it is also the basis of the adaptation processes. The stability and capacity of the ability to compensate evidently depend on age. As age increases, the processing of information in the central nervous system is subject to progressive control losses which are not reflected in moderate or "significant" increases or decreases of transmitters, receptors etc, but which find expression in the susceptibility or the stability of their response capacity. The losses are more pronounced in cases in which an attempt was made to test the limits of regulation capacity.

REFERENCES

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