Naunyn-Schmiedeberg's Naunyn-Schmiedeberg's Arch. Pharmacol. 301, 11
Archivesof
15 (1977)
Pharmacology 9 by Springer-Verlag1977
The Effect of CO2 on Monoamine Metabolism in Rat Brain J. GARCIA DE YEBENES PROUS*, A. CARLSSON, and M. A. MENA GOMEZ Department of Pharmacology, University of G6teborg, Fack, S-400 33 G6teborg 33, Sweden
Summary. White male albino rats were exposed to artificial atmosphere containing 20% of 02 and 5, 10 and 15 % of CO2, for periods of time ranging from 30 min to 2 h. Monoamine synthesis in different dissected brain areas was measured by estimating D o p a and 5-hydroxytryptophan (5-HTP) accumulation after inhibition of their decarboxylation with the L-aromatic aminoacid decarboxylase inhibitor N S D 1015 (3-hydroxybenzylhydrazine). Endogenous levels of noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), and N A and D A levels after inhibition of catecholamine synthesis with c~-methyltyrosine &-MT) were measured in dissected areas and whole brain. The synthesis of all three monoamines was found to be increased in all the cerebral regions. Endogenous dopamine levels with or without e-MT treatment were increased while N A levels decreased. 5-HT decreased in at least one area and 5 - H I A A increased in several brain areas. These observations indicate that the utilization of N A and 5-HT is increased and that of D A is diminished in acidosis, presumably as a consequence of a corresponding change in nerve impulse flow. The similarity between the monoamine changes induced by hypercarbia and by G A B A are pointed out. It is suggested that GABA-ergic activity is elevated in acidosis and that G A B A may be involved in the pH dependence of seizure thresholds, as well as in the increased tendency to respiratory depression induced by central nervous system depressant drugs in patients with chronic respiratory failure. Key words: Hypercarbia metabolism.
Acidosis -
Monoamine
Send offprint requests to Garcia de Yebenes' present address: Servicio de Neurologia, Hospital "Rfim6n y Cajal", Carretera de Colmenar Km 9,1, Madrid, Spain * On leave of absence from Hospital "Ramon y Cajal", Madrid, Spain
INTRODUCTION Previous work in rats has shown that hypoxia produces changes in monoamine synthesis and turnover and that these changes may play a role for the hypoxic disturbance of brain functions (Davis and Carlsson, 1973; Davis et al., 1973; Brown et al., 1975). Brown et al. (1974) showed that the effects of hypoxia on brain monoamine synthesis could be partly antagonized by adding 5 % CO2 to the inhaled gas mixture. Carlsson et al. (1977) observed that the hypercarbiainduced increase in the hydroxylation of tryptophan in 5 position was at least partly related to an increase in pO2, whereas the stimulation effect of hypercarbia on the hydroxylation of tyrosine in 3 position persisted even ifpO2 was kept constant. These observations prompted us to further investigate the effect of hypercarbia on the synthesis and utilization of brain monoamines.
MATERIAL AND METHODS Male Sprague Dawley rats (Anticimex, Stockholm) were used. The
animals were exposed to several gas mixtures in an hermetically closed box with a capacityof 401 and the gas mixture was let through the cage at a rate of 4 1/min. The cage was equipped with rubber gloves to allow handling of the animals. All the experiments were performed in a thermostat room at a temperature of 27~C and rectal temperatures were measured in a few animals, without observing any differences from the controls. The control animals were kept in another cage, open to room air in the same room. GasMixtures. The compositionof gas mixtureswas obtainedthrough different partial pressures of 02, CO2 and N2 in a flowmeter(Matheson and Co., East Rutherford, N. J.). The compositionwas checked by analysis of the gas mixtures collected in hermetically closed sacs. For monoamine studies we have used the following gas mixtures: for hypoxia." 8 % 02, 92 ~ N2 ; for asphyxia . 5 ~ CO2, 8 ~oo02 and 87~ Nz;for hypercarbia: 5% CO2, 20% Oz, 75% N2; 10% CO2, 20% Oz and 70% N2 and 15% CO2, 20% 02 and 65% N2. For studies of endogenous monoamine levels and utilization studies, we used hypercarbia with 5 and 15 % CO>
Schedule of Experiments. Synthesis of monoamines was measured after inhibiting L-aromaticamino acid decarboxylaseby NSD 1015 (3-hydroxybenzylhydrazineHC1) 100 mg/kg i.p. The animals were introduced in the cage immediately after NSD injection and killed
12
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977)
by decapitation 30 min later. Endogenous levels of catecholamines, 5-HT and 5-HIAA were measured after exposure of the animals to 15 ~ C Q for 1 h and 2 h. The animals were killed immediately after being taken out from the cage. Noradrenaline (NA) and doparnine (DA) utilization was measured after inhibition of tyrosine hydroxylase by c~-MT, 250 mg/kg i.p., immediately before exposure to the gas mixture for 1 or 2 h.
Biochemical Analysis. After death, the brains were quickly removed and dissected on an ice cold glass Petri dish according to Carlsson and Lindqvist (1973). In the first series of experiments, brains were divided in 4 portions: limbic, striatum, hemispheres and rest, containing the diencephalon and brain stem. In later experiments, these two parts were separately analyzed. In a few experiments, the spinal cord was also taken for analysis. After dissection, brain parts were frozen on dry ice, weighed and stored at - 80~C. The extraction of amines, precursors and metabolites was done after homogenization in 25 ml plastic tubes, containing ice could 10 ml of 0.4 N perchloric acid, 0.2 ml of 10 ~ EDTA and 0.1 ml of 5 ~ Na2S205. The extract was purified on strongly acidic cation exchange column (Dowex 50-X-4) according to Atack and Magnusson (1970). Fluorimetric analyses were made for: NA (Bertler et al., 1958), DA (Atack, 1973), 5-HT, 5-HIAA and 5-HTP (Atack and Lindqvist, 1973), Dopa (Kehr et al., 1972), tyrosine (Waalkes and Udenfriend, 1957) and tryptophan (B6dard et al., 1972). Statistics were done by Student's t-test or analysis of variance followed by Student's t-test and values were considered significant when P < 0.05.
RESULTS Single exposure for 30 rain to different gas mixtures produced significant changes in the activity in vivo of tyrosine and tryptophan hydroxylases, measured by the accumulation of Dopa and 5-HTP after inhibition by NSD 1015 (3-hydroxybenzylhydrazine) of their decarboxylation by L-aromatic amino acid decarboxylase. The results showed that 5 ~o CO2 produced a 3 0 - 60 ~o increase in the accumulation of Dopa and 5-HTP (Table 1). Hypoxia with or without simultaneous hypercarbia-tended to produce a decrease although significance was reached only for 5-HTP values in this limited material (Table 1). The
hypercarbia-induced increase in accumulation of Dopa tended to be somewhat higher in the dopaminerich regions, i.e. striatum and limbic forebrain than in the noradrenaline-predominated areas where statistical significance was reached only in the brain stem ("Rest" Table 1). With 5 ~o CO2, the hypercarbia-induced increase in 5-HTP accumulation peacked in the hemispheric and "rest" brain (Table 1). The effect of hypercarbia was more clearly seen when we increased the concentration of CO2 to 15 ~. In this case, the rate of Dopa synthesis was increased in most the cerebral regions to 200 ~ and in the diencephalic portion to 300 ~ (Table 2). In this series of experiments, we could observe that Dopa synthesis was increased in the spinal cord. Similar changes were observed in 5-HTP accumulation after 1 5 ~ COz, although the increase in 5-HTP in the diencephalon was less pronounced than in other brain parts (Table 2). (The levels of Dopa and 5-HTP are expressed in percentage of control values because the experiment with different CO2 concentration were performed on several days, and there are some differences between the controls.) Tyrosine and tryptophan levels were measured in the brain parts of animals exposed to various CO2 levels, but no significant differences were found except for the isolated elevation of tyrosine in limbic and striatum of 5 ~ CO2 exposed animals, what is of doubtful significance. We have measured catecholamine utilization, after inhibition of catecholamine synthesis by e-MT, and 1 h of exposure to 5 ~ CO2. No significant change in DA was found in any of the cerebral regions, but the NA content was decreased in the spinal cord from the control value of 212 _+ 12 ng/g (mean + S.E.M.; n = 12) to 169 _+ 8 ng/g (n = 12; P < 0.05) and in the rest portion from 578 _+ 31 ng/g (mean +_ S.E.M.; n = 3) to 392 + 2 ng/g (n = 2; P < 0.05). When we further dissected
Table 1. Dopa and 5-HTP accumulation in rat brain regions 30 rain after i.p. injection of NSD 1015 (3-hydroxy benzylhydrazine HC1) 100 mg/kg, and exposure to the following gas mixtures hypercarbia: 5 ~ CO2 + 20 ~ 02 + 75 ~ N2; asphyxia: 5 ~ CO2 + 8 ~ 02 + 87 ~ N2 ; hypoxia: 8 ~ 02 + 92 ~ N2; control: room air Dopa (ng/g) Control Limbic 439-+31 Striatum 591 -+ 31 Hemispheres 56_+ 5 Rest 137+ 7 No. of experiments 5
5-HTP (ng/g)
Hypercarbia
Asphyxia
Hypoxia
Control
Hypercarbia
641_+ 7 0 " • 920 _+ 109"*' • 78_+ 5 185_+ 11"*' • • • 5
467_+63 718 _+ 44 70_+ 9 172+ 8 •215 4
424_+63 619 _+ 70 59_+ 9 113-+10 3
136-+18 84 _+ 8 75-+ 4 154_+ 5 5
177 _+ 21 +'• 96_+ 13 82_+ 27 I31 _+ 12 +~ • • 52 +_ 14 26 • 13 111_+ 9 ++'•215215 73_+ 8 •215 28_+ 5** 205+_11 ++,•215215 125_+11 •21521574-+ 5*** 5 4 3
Results are means • S.E.M. Differs from control group. * : P < 0.05; **: P < 0.01; ***: P < 0.001 Differs from asphyxia group. +: P < 0.05; ++: P < 0.01; +++: P < 0.001 Differs from hypoxia group. • < 0.05; • • < 0.01; • • • < 0.001
Asphyxia
Hypoxia
J. Garcia de Yebenes Prous et al. : Monoamines and CO2 Table 2.
13
Percentage increases in cerebral Dopa and 5-HTP accumulation in rats exposed to 5 %, 10 % and 15 % CO2 5-HTP (% increase)
Dopa (% increase)
Limbic Striatum Hemispheres Diencephalon Brain stem Spinal cord No. of experiments
15% CO~
10% C Q
5% COa
15% CO~
10% C02
5 % C02
104 _+ 26 * 80 • 10" 150 4- 12" 200• 46 • 1 2 ' 91 • 10"** 3
30 • 19 61 • 22 * 90• 32• 21 -+ 16I 31 • 16 3
46 • 16 * 56 • 18"* 39• 9 3 9 + 8** -
94 • 10 *** 80 • 12 *** 8 2 • 3 *** 22• 2* 54 • 4 ** 60 -+ 16 * 3
63 • 5 *** 63 • ! *** 55 • 4 *** 18• I ;}Rest 29• 6' 29 • 10 3
3 0 • 15 56 • 14 48 • 12 ***
5
33_+ 7** 5
Shown are means • S.E.M. * Differs from control P < 0.05 ** Differs from control P < 0.01 *** Differs from control P < 0.001 Statistics done by analysis of variance followed by t-test For further details, see Table 1
Table 3.
Levels of DA and NA in rat brain regions 2 h after i.p. injection of e-MY (250 mg/kg) and 2 h exposure to 15 % CO2 NA (ng/g)
Limbic Striatum Hemispheres Diencephalon Brain stem No. of experiments
DA (ng/g)
Control
15 % CO2
Control
15 % CO2
312 • 16 151 • 16 112 • 4 622 _+ 13 318 • 8 4
226 113 77 400 232 4
474 • 16 1372 • 33
537 • 27 1719 • 53 **
• 4 ** • 15 • 4"* • 13 *** • 2 ***
4
4
Results are means + S.E.M. * Differs from control P < 0.05 ** Differs from control P < 0.01 *** Differs from control P < 0.001 Statistics were done by t-test
Table 4.
Endogenous levels of 5-HT and 5-HIAA in rat brain regions after 1 h exposure to 15 % CO2 5-HT (ng/g)
Limbic Striatum Hemispheres Diencephalon Brain stem Spinalcord No. of experiments
5-HIAA (ng/g)
Control
15 % CO2
Control
15 %CO2
258 • 8 211• 148 + 6 509 _+ 60 260 + 9 166 • 13 3
205 _+ 31 161• 126 • 10 289 • 1" 289 • 20 198 • 11 3
204 161 124 317 234 86 3
226 • 212• 162 • 379 • 308 • 86 • 3
• 18 • 4 • 3 • 6 • 9 • 3
18 1" 16" 11" 4
Results are means + S.E.M. Differs from control P < 0.05 Statistics done by t-test
t h e r e s t p o r t i o n , t h e d e c r e a s e w a s f o u n d i n t h e diencephalon where hypercarbia decreased the NA cont e n t f r o m t h e c o n t r o l v a l u e o f 6 5 J _+ 23 n g / g ( m e a n _+ S . E . M . ; n = 8) t o 559 + 10 (n = 8; P < 0.01). By increasing the length of exposure to 2 h and CO2
c o n c e n t r a t i o n t o 15 %, a n d a g a i n a f t e r e - M T i n h i b i tion of catecholamine synthesis we could observe a s i g n i f i c a n t d e c r e a s e o f N A c o n t e n t i n all t h e b r a i n areas, except in the striatum, and a significant increase i n D A i n t h e s t r i a t a l p o r t i o n ( T a b l e 3).
14
We also measured endogeneous levels of amines after 15~o CO2. An exposure of 2 h in 15~o CO2 decreased the whole brain concentration of NA from control value of 422 +_ 16 ng/g (mean _+ S.E.M. ; (n = 5) to 330 _+ 25 ng/g (n = 5 ; P < 0.05) and increased that of DA from a control value of 602_ 23 ng/g (n = 5) to 704 + 12 ng/g (n = 5 ; P < 0.01). 5-HT and 5-HIAA were measured in dissected parts, in order to get an insight into serotonin turnover (Table 4). 5-HIAA was in general increased in all the brain areas, though not significantly in the limbic. 5-HT was decreased in general though significantly only in the diencephalon. DISCUSSION Like pO2 variations, changes in pCO2 and in tissue pH may be expected to influence metabolic processes in the brain in two different ways, a) by direct effects on enzymatic and other mechanisms immediately dependent on pCO2 and pH, b) indirectly, though various homeostatic mechanisms activated via chemoreceptors. In the case of pO2 variations, an example of alternative a) is afforded by the dependence of tyrosine and tryptophan hydroxylases on the availability of 02 which serves as one of the substrates for the hydroxylation process. To what extent variations in pCO2 and pH induced in the present experiments exert similar direct actions contributing to the changes in monoamine synthesis and metabolism observed in the present experiments, cannot be decided from available data. That indirect mechanisms operate, is highly probable. For example, the increase in 5-HTP formation induced by hypercarbia is probably due at least partly to the increase in pO2 induced by adaptive changes, mainly in cerebral circulation (Carlsson et al., 1977). However, the increase in Dopa formation induced by hypercarbia could not be related to pO2, and thus it was concluded that pH may somehow exert a stimulatory influence on the hydroxylation of tyrosine. The present data further illustrate the differential action of hypercarbia on the monoaminergic systems. Whereas the endogenous levels of NA and 5-HT were decreased by hypercarbia, and in the case of NA this was demonstrated also after inhibition of its synthesis, the DA levels were elevated, this change being quite striking and statistically significant after inhibition of DA synthesis by e-MT. These observations indicate that the utilization of NA and 5-HT is enhanced and that of DA is retarted by hypercarbia. These differential actions of hypercarbia on the synthesis and utilization of the monoamines argue against a simple and direct action of the change in pH on the synthetic or catabolic enzymes involved as
Naunyn-Schmiedeberg's Arch. PharmacoI. 301 (1977)
being the sole or even the main mechanism by which hypercarbia influences these systems. It would appear more fruitful to look for actions on the physiological activity of the monoaminergic systems. Thus, the increased synthesis of NA and 5-HT in conjunction with an increased utilization of these amines might be at least partly secondary to an increased firing and/or firing-induced release of these transmitters, whereas the increased synthesis of DA coupled with a retarded utilization is typical for an inhibition of firing in this system (Carlsson, 1975). The pattern of changes induced by hypercarbia is rather similar to that caused by intraventricutar injection of GABA (Biswas and Carlsson, 1977). This invites the speculation that an enhanced release of GABA, or of another inhibitory putative transmitter such as taurine or/~-alanine, is involved in the changes observed. It is interesting in this context to recall the seizure threshold-elevating action of GABA as well as acidosis, which again invites the speculation that GABA is involved in the antiepileptic action of lowered pH. If this hypothetical involvement of GABA in acidosis could be proved, it can help to explain certain interesting phenomena that occur in patients with chronic respiratory insufficiency, such as their increased tendency to acute respiratory failure after small doses of central nervous system depressants (Bates et al., 1971). It is interesting to note that despite the contrast between hypercarbia and hypoxia as regards the action on monoamine synthesis, these two conditions show certain similarities in their actions on the utilization of monoamines: after hypoxia, too, the biochemical data suggest inhibition of dopaminergic and, if anything, stimulation of noradrenergic activity (Brown et al., 1975). The possibility may be considered that GABA is involved also in the hypoxia-induced changes in monoamine metabolism. In cerebral ischemia Mr~ulja et al. (1976) observed a fourfold increase in brain GABA levels. This increase might well contribute to an increased activation of GABA receptors in hypoxia and/or hypercarbia. Acknowledgements. This study was supported by grants from the Swedish Medical Research Council (no. 00155) and AB H~issle, M61ndal.
REFERENCES Atack, C. V. : The determination of dopamine by a modification of the dihydroxyindole fluorimetric assay. Br. J. Pharmacol. 48, 6 9 9 - 714 (1973) Atack, C. V., Lindqvist, M.: Conjoint native and orthophthaldialdehyde-condensate assays for the fluorimetric determination of 5-hydroxyindoles in brain. Naunyn-Schmiedeberg's Arch. Pharmacol. 279, 2 6 7 - 284 (1973)
J. Garcia de Yebenes Prous et al. : Monoamines and CO2 Atack, C. V., Magnusson, T. : Individual elution of noradrenaline (together with adrenaline), dopamine, 5-hydroxytryptamine and histamine from a single strong cation exchange column, by means of mineral acid-organic solvent mixtures. J. Pharm. Pharmacol. 22, 625 -- 627 (1970) Bates, D. V., MacKlem, P. T., Christie, R. V. : Respiratory failure. In: Respiratory function in disease, pp. 452. London: W. B. Saunder 1971 B~dard, R., Carlsson, A., Lindqvist, M.: Effect of a transverse cerebral hemisection on 5-hydroxytryptamine metaboIism in rat brain. Naunyn-Schmiedeberg's Arch. Pharmacol. 272, 1 - 15 (1972) Bertler, B., Carlsson, A., Rosengren, E.: A method for the fluorimetric determination of adrenaline and noradrenaline in tissues. Acta Physiol. Scand. 44, 273-292 (1958) Biswas, B., Carlsson, A.: The effect of intracerebroventricularly administered GABA on brain monoamine metabolism. NaunynSchmiedeberg's Arch. Pharmacol. 299, 4 7 - 5 1 (1977) Brown, R. M., Kehr, W., Carlsson, A. : Functional and biochemical aspects of catecholamine metabolism in brain under hypoxia. Brain Res. 85, 4 9 1 - 509 (1975) Brown, R. M., Snider, S. R., Carlsson, A.: Changes in biogenic amine synthesis and turnover induced by hypoxia and/or foot shock stress. II. The central nervous system. J. Neural Transm. 35, 293 - 305 (1974) Carlsson, A. : Receptor-mediated control of dopamine metabolism. In: Pre- and postsynaptic receptors. (E. Usdin and W. E. Bunney, Jr., eds.), pp. 4 9 - 6 5 . New York: Marcel Dekker, Inc. 1975
15 Carlsson, A., Lindqvist, M. : Effect of ethanol on the hydroxylation of tyrosine and tryptophan in rat brain in vivo. J. Pharm. Pharmacol. 25, 437-440 (1973) Carlsson, A., Holmin, T., Lindqvist, M., Siesj6, B. K.: Effect of hypercarbia and hypercapnia on tryptophan and tyrosine hydroxylation in rat brain. Acta Physiol. Scand. 99, 503-509 (1977) Davis, J. N., Carlsson, A. : The effect of hypoxia on monoamine synthesis levels and metabolism in rat brain. J. Neurochem. 21, 783-790 (1973) Davis, J.N., Carlsson, A., MacMillan, V., Siesj6, B.K.: Brain tryptophan hydroxylation Dependence on arterial oxygen tension. Science 182, 7 2 - 74 (1973) Kehr, W., Carlsson, A., Lindqvist, M. : A method for the determination of 3,4-dihydroxyphenylalanine (DOPA) in brain. NaunynSchmiedeberg's Arch. Pharmacol. 274, 273-280 (1972) Mrsulja, B.B., Lust, W.D., Mrsulja, B.J., Passonneau, J. U., Klatzo, I. : Post-ischemic changes in certain metabolites following prolonged ischemia in the gerbil cerebral cortex. J. Neurochem. 26, 1099-- 1103 (i976) Waalkes, T. P., Udenfriend, S. : A fluorimetric method for the estimation of tyrosine in plasma and tissue. J. Lab. Clin. Med. 50, 7 3 3 - 736 (1957)
Received June 23~Accepted September 23, 1977
Archivesof
15 (1977)
Pharmacology 9 by Springer-Verlag1977
The Effect of CO2 on Monoamine Metabolism in Rat Brain J. GARCIA DE YEBENES PROUS*, A. CARLSSON, and M. A. MENA GOMEZ Department of Pharmacology, University of G6teborg, Fack, S-400 33 G6teborg 33, Sweden
Summary. White male albino rats were exposed to artificial atmosphere containing 20% of 02 and 5, 10 and 15 % of CO2, for periods of time ranging from 30 min to 2 h. Monoamine synthesis in different dissected brain areas was measured by estimating D o p a and 5-hydroxytryptophan (5-HTP) accumulation after inhibition of their decarboxylation with the L-aromatic aminoacid decarboxylase inhibitor N S D 1015 (3-hydroxybenzylhydrazine). Endogenous levels of noradrenaline (NA), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), and N A and D A levels after inhibition of catecholamine synthesis with c~-methyltyrosine &-MT) were measured in dissected areas and whole brain. The synthesis of all three monoamines was found to be increased in all the cerebral regions. Endogenous dopamine levels with or without e-MT treatment were increased while N A levels decreased. 5-HT decreased in at least one area and 5 - H I A A increased in several brain areas. These observations indicate that the utilization of N A and 5-HT is increased and that of D A is diminished in acidosis, presumably as a consequence of a corresponding change in nerve impulse flow. The similarity between the monoamine changes induced by hypercarbia and by G A B A are pointed out. It is suggested that GABA-ergic activity is elevated in acidosis and that G A B A may be involved in the pH dependence of seizure thresholds, as well as in the increased tendency to respiratory depression induced by central nervous system depressant drugs in patients with chronic respiratory failure. Key words: Hypercarbia metabolism.
Acidosis -
Monoamine
Send offprint requests to Garcia de Yebenes' present address: Servicio de Neurologia, Hospital "Rfim6n y Cajal", Carretera de Colmenar Km 9,1, Madrid, Spain * On leave of absence from Hospital "Ramon y Cajal", Madrid, Spain
INTRODUCTION Previous work in rats has shown that hypoxia produces changes in monoamine synthesis and turnover and that these changes may play a role for the hypoxic disturbance of brain functions (Davis and Carlsson, 1973; Davis et al., 1973; Brown et al., 1975). Brown et al. (1974) showed that the effects of hypoxia on brain monoamine synthesis could be partly antagonized by adding 5 % CO2 to the inhaled gas mixture. Carlsson et al. (1977) observed that the hypercarbiainduced increase in the hydroxylation of tryptophan in 5 position was at least partly related to an increase in pO2, whereas the stimulation effect of hypercarbia on the hydroxylation of tyrosine in 3 position persisted even ifpO2 was kept constant. These observations prompted us to further investigate the effect of hypercarbia on the synthesis and utilization of brain monoamines.
MATERIAL AND METHODS Male Sprague Dawley rats (Anticimex, Stockholm) were used. The
animals were exposed to several gas mixtures in an hermetically closed box with a capacityof 401 and the gas mixture was let through the cage at a rate of 4 1/min. The cage was equipped with rubber gloves to allow handling of the animals. All the experiments were performed in a thermostat room at a temperature of 27~C and rectal temperatures were measured in a few animals, without observing any differences from the controls. The control animals were kept in another cage, open to room air in the same room. GasMixtures. The compositionof gas mixtureswas obtainedthrough different partial pressures of 02, CO2 and N2 in a flowmeter(Matheson and Co., East Rutherford, N. J.). The compositionwas checked by analysis of the gas mixtures collected in hermetically closed sacs. For monoamine studies we have used the following gas mixtures: for hypoxia." 8 % 02, 92 ~ N2 ; for asphyxia . 5 ~ CO2, 8 ~oo02 and 87~ Nz;for hypercarbia: 5% CO2, 20% Oz, 75% N2; 10% CO2, 20% Oz and 70% N2 and 15% CO2, 20% 02 and 65% N2. For studies of endogenous monoamine levels and utilization studies, we used hypercarbia with 5 and 15 % CO>
Schedule of Experiments. Synthesis of monoamines was measured after inhibiting L-aromaticamino acid decarboxylaseby NSD 1015 (3-hydroxybenzylhydrazineHC1) 100 mg/kg i.p. The animals were introduced in the cage immediately after NSD injection and killed
12
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977)
by decapitation 30 min later. Endogenous levels of catecholamines, 5-HT and 5-HIAA were measured after exposure of the animals to 15 ~ C Q for 1 h and 2 h. The animals were killed immediately after being taken out from the cage. Noradrenaline (NA) and doparnine (DA) utilization was measured after inhibition of tyrosine hydroxylase by c~-MT, 250 mg/kg i.p., immediately before exposure to the gas mixture for 1 or 2 h.
Biochemical Analysis. After death, the brains were quickly removed and dissected on an ice cold glass Petri dish according to Carlsson and Lindqvist (1973). In the first series of experiments, brains were divided in 4 portions: limbic, striatum, hemispheres and rest, containing the diencephalon and brain stem. In later experiments, these two parts were separately analyzed. In a few experiments, the spinal cord was also taken for analysis. After dissection, brain parts were frozen on dry ice, weighed and stored at - 80~C. The extraction of amines, precursors and metabolites was done after homogenization in 25 ml plastic tubes, containing ice could 10 ml of 0.4 N perchloric acid, 0.2 ml of 10 ~ EDTA and 0.1 ml of 5 ~ Na2S205. The extract was purified on strongly acidic cation exchange column (Dowex 50-X-4) according to Atack and Magnusson (1970). Fluorimetric analyses were made for: NA (Bertler et al., 1958), DA (Atack, 1973), 5-HT, 5-HIAA and 5-HTP (Atack and Lindqvist, 1973), Dopa (Kehr et al., 1972), tyrosine (Waalkes and Udenfriend, 1957) and tryptophan (B6dard et al., 1972). Statistics were done by Student's t-test or analysis of variance followed by Student's t-test and values were considered significant when P < 0.05.
RESULTS Single exposure for 30 rain to different gas mixtures produced significant changes in the activity in vivo of tyrosine and tryptophan hydroxylases, measured by the accumulation of Dopa and 5-HTP after inhibition by NSD 1015 (3-hydroxybenzylhydrazine) of their decarboxylation by L-aromatic amino acid decarboxylase. The results showed that 5 ~o CO2 produced a 3 0 - 60 ~o increase in the accumulation of Dopa and 5-HTP (Table 1). Hypoxia with or without simultaneous hypercarbia-tended to produce a decrease although significance was reached only for 5-HTP values in this limited material (Table 1). The
hypercarbia-induced increase in accumulation of Dopa tended to be somewhat higher in the dopaminerich regions, i.e. striatum and limbic forebrain than in the noradrenaline-predominated areas where statistical significance was reached only in the brain stem ("Rest" Table 1). With 5 ~o CO2, the hypercarbia-induced increase in 5-HTP accumulation peacked in the hemispheric and "rest" brain (Table 1). The effect of hypercarbia was more clearly seen when we increased the concentration of CO2 to 15 ~. In this case, the rate of Dopa synthesis was increased in most the cerebral regions to 200 ~ and in the diencephalic portion to 300 ~ (Table 2). In this series of experiments, we could observe that Dopa synthesis was increased in the spinal cord. Similar changes were observed in 5-HTP accumulation after 1 5 ~ COz, although the increase in 5-HTP in the diencephalon was less pronounced than in other brain parts (Table 2). (The levels of Dopa and 5-HTP are expressed in percentage of control values because the experiment with different CO2 concentration were performed on several days, and there are some differences between the controls.) Tyrosine and tryptophan levels were measured in the brain parts of animals exposed to various CO2 levels, but no significant differences were found except for the isolated elevation of tyrosine in limbic and striatum of 5 ~ CO2 exposed animals, what is of doubtful significance. We have measured catecholamine utilization, after inhibition of catecholamine synthesis by e-MT, and 1 h of exposure to 5 ~ CO2. No significant change in DA was found in any of the cerebral regions, but the NA content was decreased in the spinal cord from the control value of 212 _+ 12 ng/g (mean + S.E.M.; n = 12) to 169 _+ 8 ng/g (n = 12; P < 0.05) and in the rest portion from 578 _+ 31 ng/g (mean +_ S.E.M.; n = 3) to 392 + 2 ng/g (n = 2; P < 0.05). When we further dissected
Table 1. Dopa and 5-HTP accumulation in rat brain regions 30 rain after i.p. injection of NSD 1015 (3-hydroxy benzylhydrazine HC1) 100 mg/kg, and exposure to the following gas mixtures hypercarbia: 5 ~ CO2 + 20 ~ 02 + 75 ~ N2; asphyxia: 5 ~ CO2 + 8 ~ 02 + 87 ~ N2 ; hypoxia: 8 ~ 02 + 92 ~ N2; control: room air Dopa (ng/g) Control Limbic 439-+31 Striatum 591 -+ 31 Hemispheres 56_+ 5 Rest 137+ 7 No. of experiments 5
5-HTP (ng/g)
Hypercarbia
Asphyxia
Hypoxia
Control
Hypercarbia
641_+ 7 0 " • 920 _+ 109"*' • 78_+ 5 185_+ 11"*' • • • 5
467_+63 718 _+ 44 70_+ 9 172+ 8 •215 4
424_+63 619 _+ 70 59_+ 9 113-+10 3
136-+18 84 _+ 8 75-+ 4 154_+ 5 5
177 _+ 21 +'• 96_+ 13 82_+ 27 I31 _+ 12 +~ • • 52 +_ 14 26 • 13 111_+ 9 ++'•215215 73_+ 8 •215 28_+ 5** 205+_11 ++,•215215 125_+11 •21521574-+ 5*** 5 4 3
Results are means • S.E.M. Differs from control group. * : P < 0.05; **: P < 0.01; ***: P < 0.001 Differs from asphyxia group. +: P < 0.05; ++: P < 0.01; +++: P < 0.001 Differs from hypoxia group. • < 0.05; • • < 0.01; • • • < 0.001
Asphyxia
Hypoxia
J. Garcia de Yebenes Prous et al. : Monoamines and CO2 Table 2.
13
Percentage increases in cerebral Dopa and 5-HTP accumulation in rats exposed to 5 %, 10 % and 15 % CO2 5-HTP (% increase)
Dopa (% increase)
Limbic Striatum Hemispheres Diencephalon Brain stem Spinal cord No. of experiments
15% CO~
10% C Q
5% COa
15% CO~
10% C02
5 % C02
104 _+ 26 * 80 • 10" 150 4- 12" 200• 46 • 1 2 ' 91 • 10"** 3
30 • 19 61 • 22 * 90• 32• 21 -+ 16I 31 • 16 3
46 • 16 * 56 • 18"* 39• 9 3 9 + 8** -
94 • 10 *** 80 • 12 *** 8 2 • 3 *** 22• 2* 54 • 4 ** 60 -+ 16 * 3
63 • 5 *** 63 • ! *** 55 • 4 *** 18• I ;}Rest 29• 6' 29 • 10 3
3 0 • 15 56 • 14 48 • 12 ***
5
33_+ 7** 5
Shown are means • S.E.M. * Differs from control P < 0.05 ** Differs from control P < 0.01 *** Differs from control P < 0.001 Statistics done by analysis of variance followed by t-test For further details, see Table 1
Table 3.
Levels of DA and NA in rat brain regions 2 h after i.p. injection of e-MY (250 mg/kg) and 2 h exposure to 15 % CO2 NA (ng/g)
Limbic Striatum Hemispheres Diencephalon Brain stem No. of experiments
DA (ng/g)
Control
15 % CO2
Control
15 % CO2
312 • 16 151 • 16 112 • 4 622 _+ 13 318 • 8 4
226 113 77 400 232 4
474 • 16 1372 • 33
537 • 27 1719 • 53 **
• 4 ** • 15 • 4"* • 13 *** • 2 ***
4
4
Results are means + S.E.M. * Differs from control P < 0.05 ** Differs from control P < 0.01 *** Differs from control P < 0.001 Statistics were done by t-test
Table 4.
Endogenous levels of 5-HT and 5-HIAA in rat brain regions after 1 h exposure to 15 % CO2 5-HT (ng/g)
Limbic Striatum Hemispheres Diencephalon Brain stem Spinalcord No. of experiments
5-HIAA (ng/g)
Control
15 % CO2
Control
15 %CO2
258 • 8 211• 148 + 6 509 _+ 60 260 + 9 166 • 13 3
205 _+ 31 161• 126 • 10 289 • 1" 289 • 20 198 • 11 3
204 161 124 317 234 86 3
226 • 212• 162 • 379 • 308 • 86 • 3
• 18 • 4 • 3 • 6 • 9 • 3
18 1" 16" 11" 4
Results are means + S.E.M. Differs from control P < 0.05 Statistics done by t-test
t h e r e s t p o r t i o n , t h e d e c r e a s e w a s f o u n d i n t h e diencephalon where hypercarbia decreased the NA cont e n t f r o m t h e c o n t r o l v a l u e o f 6 5 J _+ 23 n g / g ( m e a n _+ S . E . M . ; n = 8) t o 559 + 10 (n = 8; P < 0.01). By increasing the length of exposure to 2 h and CO2
c o n c e n t r a t i o n t o 15 %, a n d a g a i n a f t e r e - M T i n h i b i tion of catecholamine synthesis we could observe a s i g n i f i c a n t d e c r e a s e o f N A c o n t e n t i n all t h e b r a i n areas, except in the striatum, and a significant increase i n D A i n t h e s t r i a t a l p o r t i o n ( T a b l e 3).
14
We also measured endogeneous levels of amines after 15~o CO2. An exposure of 2 h in 15~o CO2 decreased the whole brain concentration of NA from control value of 422 +_ 16 ng/g (mean _+ S.E.M. ; (n = 5) to 330 _+ 25 ng/g (n = 5 ; P < 0.05) and increased that of DA from a control value of 602_ 23 ng/g (n = 5) to 704 + 12 ng/g (n = 5 ; P < 0.01). 5-HT and 5-HIAA were measured in dissected parts, in order to get an insight into serotonin turnover (Table 4). 5-HIAA was in general increased in all the brain areas, though not significantly in the limbic. 5-HT was decreased in general though significantly only in the diencephalon. DISCUSSION Like pO2 variations, changes in pCO2 and in tissue pH may be expected to influence metabolic processes in the brain in two different ways, a) by direct effects on enzymatic and other mechanisms immediately dependent on pCO2 and pH, b) indirectly, though various homeostatic mechanisms activated via chemoreceptors. In the case of pO2 variations, an example of alternative a) is afforded by the dependence of tyrosine and tryptophan hydroxylases on the availability of 02 which serves as one of the substrates for the hydroxylation process. To what extent variations in pCO2 and pH induced in the present experiments exert similar direct actions contributing to the changes in monoamine synthesis and metabolism observed in the present experiments, cannot be decided from available data. That indirect mechanisms operate, is highly probable. For example, the increase in 5-HTP formation induced by hypercarbia is probably due at least partly to the increase in pO2 induced by adaptive changes, mainly in cerebral circulation (Carlsson et al., 1977). However, the increase in Dopa formation induced by hypercarbia could not be related to pO2, and thus it was concluded that pH may somehow exert a stimulatory influence on the hydroxylation of tyrosine. The present data further illustrate the differential action of hypercarbia on the monoaminergic systems. Whereas the endogenous levels of NA and 5-HT were decreased by hypercarbia, and in the case of NA this was demonstrated also after inhibition of its synthesis, the DA levels were elevated, this change being quite striking and statistically significant after inhibition of DA synthesis by e-MT. These observations indicate that the utilization of NA and 5-HT is enhanced and that of DA is retarted by hypercarbia. These differential actions of hypercarbia on the synthesis and utilization of the monoamines argue against a simple and direct action of the change in pH on the synthetic or catabolic enzymes involved as
Naunyn-Schmiedeberg's Arch. PharmacoI. 301 (1977)
being the sole or even the main mechanism by which hypercarbia influences these systems. It would appear more fruitful to look for actions on the physiological activity of the monoaminergic systems. Thus, the increased synthesis of NA and 5-HT in conjunction with an increased utilization of these amines might be at least partly secondary to an increased firing and/or firing-induced release of these transmitters, whereas the increased synthesis of DA coupled with a retarded utilization is typical for an inhibition of firing in this system (Carlsson, 1975). The pattern of changes induced by hypercarbia is rather similar to that caused by intraventricutar injection of GABA (Biswas and Carlsson, 1977). This invites the speculation that an enhanced release of GABA, or of another inhibitory putative transmitter such as taurine or/~-alanine, is involved in the changes observed. It is interesting in this context to recall the seizure threshold-elevating action of GABA as well as acidosis, which again invites the speculation that GABA is involved in the antiepileptic action of lowered pH. If this hypothetical involvement of GABA in acidosis could be proved, it can help to explain certain interesting phenomena that occur in patients with chronic respiratory insufficiency, such as their increased tendency to acute respiratory failure after small doses of central nervous system depressants (Bates et al., 1971). It is interesting to note that despite the contrast between hypercarbia and hypoxia as regards the action on monoamine synthesis, these two conditions show certain similarities in their actions on the utilization of monoamines: after hypoxia, too, the biochemical data suggest inhibition of dopaminergic and, if anything, stimulation of noradrenergic activity (Brown et al., 1975). The possibility may be considered that GABA is involved also in the hypoxia-induced changes in monoamine metabolism. In cerebral ischemia Mr~ulja et al. (1976) observed a fourfold increase in brain GABA levels. This increase might well contribute to an increased activation of GABA receptors in hypoxia and/or hypercarbia. Acknowledgements. This study was supported by grants from the Swedish Medical Research Council (no. 00155) and AB H~issle, M61ndal.
REFERENCES Atack, C. V. : The determination of dopamine by a modification of the dihydroxyindole fluorimetric assay. Br. J. Pharmacol. 48, 6 9 9 - 714 (1973) Atack, C. V., Lindqvist, M.: Conjoint native and orthophthaldialdehyde-condensate assays for the fluorimetric determination of 5-hydroxyindoles in brain. Naunyn-Schmiedeberg's Arch. Pharmacol. 279, 2 6 7 - 284 (1973)
J. Garcia de Yebenes Prous et al. : Monoamines and CO2 Atack, C. V., Magnusson, T. : Individual elution of noradrenaline (together with adrenaline), dopamine, 5-hydroxytryptamine and histamine from a single strong cation exchange column, by means of mineral acid-organic solvent mixtures. J. Pharm. Pharmacol. 22, 625 -- 627 (1970) Bates, D. V., MacKlem, P. T., Christie, R. V. : Respiratory failure. In: Respiratory function in disease, pp. 452. London: W. B. Saunder 1971 B~dard, R., Carlsson, A., Lindqvist, M.: Effect of a transverse cerebral hemisection on 5-hydroxytryptamine metaboIism in rat brain. Naunyn-Schmiedeberg's Arch. Pharmacol. 272, 1 - 15 (1972) Bertler, B., Carlsson, A., Rosengren, E.: A method for the fluorimetric determination of adrenaline and noradrenaline in tissues. Acta Physiol. Scand. 44, 273-292 (1958) Biswas, B., Carlsson, A.: The effect of intracerebroventricularly administered GABA on brain monoamine metabolism. NaunynSchmiedeberg's Arch. Pharmacol. 299, 4 7 - 5 1 (1977) Brown, R. M., Kehr, W., Carlsson, A. : Functional and biochemical aspects of catecholamine metabolism in brain under hypoxia. Brain Res. 85, 4 9 1 - 509 (1975) Brown, R. M., Snider, S. R., Carlsson, A.: Changes in biogenic amine synthesis and turnover induced by hypoxia and/or foot shock stress. II. The central nervous system. J. Neural Transm. 35, 293 - 305 (1974) Carlsson, A. : Receptor-mediated control of dopamine metabolism. In: Pre- and postsynaptic receptors. (E. Usdin and W. E. Bunney, Jr., eds.), pp. 4 9 - 6 5 . New York: Marcel Dekker, Inc. 1975
15 Carlsson, A., Lindqvist, M. : Effect of ethanol on the hydroxylation of tyrosine and tryptophan in rat brain in vivo. J. Pharm. Pharmacol. 25, 437-440 (1973) Carlsson, A., Holmin, T., Lindqvist, M., Siesj6, B. K.: Effect of hypercarbia and hypercapnia on tryptophan and tyrosine hydroxylation in rat brain. Acta Physiol. Scand. 99, 503-509 (1977) Davis, J. N., Carlsson, A. : The effect of hypoxia on monoamine synthesis levels and metabolism in rat brain. J. Neurochem. 21, 783-790 (1973) Davis, J.N., Carlsson, A., MacMillan, V., Siesj6, B.K.: Brain tryptophan hydroxylation Dependence on arterial oxygen tension. Science 182, 7 2 - 74 (1973) Kehr, W., Carlsson, A., Lindqvist, M. : A method for the determination of 3,4-dihydroxyphenylalanine (DOPA) in brain. NaunynSchmiedeberg's Arch. Pharmacol. 274, 273-280 (1972) Mrsulja, B.B., Lust, W.D., Mrsulja, B.J., Passonneau, J. U., Klatzo, I. : Post-ischemic changes in certain metabolites following prolonged ischemia in the gerbil cerebral cortex. J. Neurochem. 26, 1099-- 1103 (i976) Waalkes, T. P., Udenfriend, S. : A fluorimetric method for the estimation of tyrosine in plasma and tissue. J. Lab. Clin. Med. 50, 7 3 3 - 736 (1957)
Received June 23~Accepted September 23, 1977
