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The functional importance of increased brain tryptophan in the serotonergic response to restraint stress
THE FUNCTIONAL IMPORTANCE OF INCREASED BRAIN TRYPTOPHAN IN THE SEROTONERGIC RESPONSE TO RESTRAINT STRESS G. A. Division
of Psychiatry.
KENNETT* and
M. H. JOSEPH
MRC Clinical Research Centre, Middlesex HA1 3UJ, England
Watford
Road,
Harrow,
(Accepretl19 September 1980) Summary-The increase in brain tryptophan induced by restraint stress in rats has been shown to be prevented by prior administration of valine, 200 mg/kg (i.p.). Brain 5hydroxytryptamine (5HT) was not depleted. but the stress-induced increase in S-hydroxyindoleacetic acid (5-HIAA) was prevented. A 5-HT-mediated functional response to stress, elevated plasma corticosterone, was however significantly attenuated by valine pretreatment, but was not affected by valine treatment alone. This suggests that the increase in 5-HIAA in brain is not merely secondary to increased brain tryptophan but indicates an increase in functional 5-HT activity, which is in turn at least partly dependent on the increase in brain tryptophan. Measurement of kynurenine levels in the same animals indicated an increase in synthesis in stress, but did not support the hypothesis that competition occurs between the kynurenine and 5-hydroxyindole patbways of tryptophan metabolism in brain.
It has been known for some time that experimental stresses, including restraint stress, in rats is accompanied by increases in brain levels of S-hydroxyindoleacetic acid (5-HIAA), the metabolic product of the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin), in the absence of a change in the concentration of the latter (Bliss, Ailion and Zwanziger, 1968: Curzon and Green, 1969; Curzon, Joseph and Knott. 1972). This has been interpreted as indicating that there is a stress-induced increase in 5-HT turnover, and has further been shown to be accompanied by an increase in brain tryptophan (the precursor of 5-HT) which is specific to this amino acid (Joseph, 1973; Knott, Joseph and Curzon, 1973). It has also been shown that exogenous tryptophan increases 5-HT synthesis in the rat brain (Eccleston, Ashcroft and Crawford, 1965; Fernstrom and Wurtman, 1971) presumably because tryptophan hydroxylase, the rate limiting enzyme for 5-HT synthesis, is not normally saturated by tryptophan (Freedman, Kappelman and Kaufman, 1972). Thus, the increase of tryptophan with stress would be consistent with the idea that increased synthesis of 5-HT is necessary to prevent its depletion due to increased release. However an alternative possibility is that the increase in 5-HIAA with stress reflects only increased intraneuronal metabolism of 5-HT due to the tryptophaninduced increase in synthesis, without any increase in 5-HT release and functional activity. This concept was advanced by Grahame-Smith (1971) to account
for the failure of tryptophan administration to induce behavioural changes in rats unless pretreated with a monoamine oxidase inhibitor to prevent this overspill metabolism. Increased brain tryptophan during stress has been shown to be accompanied by an increase in the proportion of free (i.e. not albumin bound) tryptophan in the plasma (Knott and Curzon, 1972) this free tryptophan being more available for uptake to the brain (Young and Sourkes, 1977; Green, 1978). Availability depends also on competition between tryptophan and other large neutral amino acids (including leucine, isoleucine, valine, phenylalanine and tyrosine) which share a common blood-brain transport system with tryptophan (Pardridge, 1977). Valine has previously been used to restrict tryptophan availability to the brain in rats (Messing, Fisher, Phebus and Lytle, 1976; Jacoby, Thomas, Poulakos and Siegal, 1979) and in human patients with coma due to hepatic encephalopathy (Riederer, Personal communication, and data presented at CINP, Goteborg 1980). Accordingly the effect of valine pretreatment on the metabolic and functional 5-HT response to stress has been studied. It was anticipated that valine pretreatment might lead to a stress-induced depletion of 5-HT, if indeed increased release of 5-HT occurred, and also perhaps to a loss of functional activity. As an index of functional 5-HT activity, in the hypothalamus at least, plasma corticosterone was measured as CRF release from the hypothalamus is, at least partially, dependent on 5-HT (Buckingham and Hodges, 1979; Jones, Hillhouse and Burden, 1976; Scapagnini, Moberg, Van Loon, De Groot and Ganong, 1971). Another pathway for tryptophan metabolism
* G.A.K. is an MRC Scholar. Key words: tryptophan, stress, serotonin, 5-hydroxytryptamine, kynurenine, corticosterone, valine, brain, response to stress. 39
40
G.
KENNETT
A.
and M. H. JOSEPH
known to be active in the rat brain is the kynurenine pathway (Gal, 1974; Hayaishi, 1976; Joseph, 1978a; Gal, Young and Sherman, 1978). It has been suggested that this pathway may have a regulatory role in relation to the 5-HT pathway, but its activity has not been studied during stress. Thus plasma and brain kynurenine were monitored during these experiments.
METHODS
Valine and trypophan metabolite standards were obtained from Sigma (Poole, U.K.) and reagents from BDH (Poole, U.K.) (AR grade when available). Corticosterone standard (4-Pregnen-11/?,21-diol-3,20 dione) was obtained from Koch Light (Colnbrook, U.K.). Male Sprague-Dawley rats (wt 200-280 g) were injected with isotonic saline, or valine in isotonic saline at a dose of 200 mg/kg as appropriate. Immobilization by tying to a grid was carried out as previously described (Curzon et al., 1972), using grids modified to facilitate subsequent sacrifice by decapitation. All experiments were conducted between 10.00 and 13.00 hr. Blood was collected in heparinized plastic
3OlT
(a) PT
tubes; plasma obtained by centrifugation was stored at -40°C. Brains were removed rapidly and dropped into a previously cooled vial standing in dry ice. All rats were housed before the experiment in a constant 12 hr day and 12 hr night cycle and fed on rat chow and water ad libitum. Plasma tryptophan and brain tryptophan, 5-HT and 5-HIAA were determined fluorometrically as previously described (Joseph, 1978b). Kynurenine was determined in the same samples of plasma and brain by gas liquid chromatography (GLC) as previously described (Joseph, 1978a, b) with the substitution of dibromo-p-xylene (100 ng/ml) (Aldrich Chemical Company) for dichloro-p-xylene (200 ng/ml), as internal standard. The longer retention of this compound allowed the GLC column to be operated at 140°C thus increasing the sensitivity of the method previously described (Joseph, 1978a). Plasma corticosterone was determined fluorometritally by the method of Mattingly (1962). RESULTS As expected from previous work, immobilization stress significantly (P < 0.001) elevated the levels of
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Hours of immobilization
stress
1
‘
Fig. 1. Effect of immobilization stress with (shaded columns) and without (open columns) vahne pretreatment (2OOmg/kg). (a) Plasma tryptophan; (b) plasma kynurenine: (c) plasma corticosterone; (d) brain tryptophan; (e) brain kynurenine; (f) brain 5-HT; (g) brain S-HIAA. n = 18 in col. 1; 8 in cot. 2 and 9 each in cols 3-6 except for (e) where n = 12 in col. 1; 8 in col. 2; 6 in cob 3, 5; 9 in cols 4, 6. Vertical bars indicate standard deviations, Significance of difference from column 1: *P < 0.01; **I’ < 0.005; ***P < 0.001: significance of difference from no valine pretreatment. ttP < 0.005; tttP < 0.001.
41
Brain tryptophan and S-HT function in stress brain ~yptophan (60.20/,at 1 hr, 39:7% at 2 fir, Fig. Id), and 5-HIAA (34.7% at 1 hr, 38.5% at 2 hr, Fig. lg) but 5-HT was not significantly altered (Fig. If). Brain kynurenine was also observed to be elevated (22.7% at 1 hr, P < 0.005; 34.4% at 2 hr P -Z 0.001, Fig. le). In plasma, stress resulted in the expected large rise in corticosterone (343% at 1 hr, 354% at 2 hr, P < 0.001 ineach case, Fig. lc) and marginal fall in total tryptophan (Fig. la). In addition, a rise in plasma kynurenine was observed (136% at 1 hr, P -c 0.001, 28.3% at 2 hr, P < 0.01, Fig. lb). It was also confirmed (Table 1) that valine, at doses of 100 mg/kg and 2OOmg/kg, reduced brain tryptophan (at 1 and 2 hr) and also 5-HT and S-HIAA at these times. In addition, valine at 2OOmgjkg had, no significant effect on plasma tryptophan, elevated corticosterone somewhat at 2 hr, and elevated plasma kynurenine but reduced brain kynurenine (Table 1). A dose of 2OOmg/kg at 1 hr was selected for further investigation. Administration of this dose immediately before immobilization completely prevented the stress-induced increase in brain tryptophan which had been seen 1 hr later (Fig. Id, compare columns 3 and 4). The increases in brain 5-HIAA and kynurenine were also prevented by valine pre-treatment (cols 3 and 4, Fig. lg and e respectively). Administration of valine 1 hr prior to sacrifice of animals immobilized for 2 hr, similarly prevented the rises in brain tryptophan (~01s 5 and 6 Fig. id). The increase in brain kynurenine was also prevented and that in 5-HIAA significantly attenuated (Fig. le and g). Plasma kynurenine was not however elevated more than by 1 hr stress alone, and plasma tryptophan was somewhat reduced by the combination of 2 hr stress and valine (27.1x, P < 0.01). The increase in plasma corticosterone induced by 1
or 2 hr stress was signi~~ntly attenuated by valine pretreatment (to 57% and 40% of the increase observed after stress alone at 1 and 2 hr, respectively, P < 0.001) although corticosterone was still significantly elevated above control levels. (197% at 1 hr, 137% at 2 hr, P < 0.001 for each, Fig. lc). DISCUSSION
The results show that administration of a competing amino acid can prevent the increase in brain tryp tophan which accompanies immobilization stress. They also show that blockade of this rise prevents, or at 2 hr markedly attenuates, the rise in S-HIAA. This was unexpected, since, as described in the introduction, it was anticipated that 5-HIAA would still be elevated, but at the expense of 5-HT depletion. At first sight this would appear to be consistent with the hypothesis that increased brain 5-HIAA on stress could simply be a reflection of increased brain tryptophan leading to increased 5-HT synthesis and catabolism, without any increase in 5-HT release or stimulation of receptors. This would be analogous to the situation for exogenous tryptophan described by GrahamtSmith (1971) and Green and Grahame-Smith (1976). However the plasma corti~osterone results show that the functional response to stress was attenuated by valine pretreatment. There was still a significant increase, and this may be attributable to the effects of other transmitters (Buckingham and Hodges, 1979) or to residual effects on 5-HT turnover, since one cannot extrapolate directly from metabolite levels to turnover rates; in addition, regional effects (e.g. in the hypothalmus) will not necessarily be reflected in the whole brain measurements. That 5-HT release in the hypothalamus may depend on increased tryptophan availability is also suggested by the increase in tryptophan
Table 1. The effect of valine (200 mg/kgf on tryptophan metabolism
n
9
8
9
8
Twptoph~ hdml)
18.1 & 4.7
15.7 & 2.6
Kynurenine (ng/ml)
941 j; 178
Corticosterone
8.49 * 3.35
16.1 + 3.1 (89%) 1268 i 204*** (134%) 9.94 * 5.54 (117%)
17.0 f 4.7 (108%) 1104 $- 200* (122%) 12.09 f 2.99** (147%)
Plasma
(fig/ml)
908 & 161 8.25 f 2.81
Brain
Twtophan Wg) Kynurenine 5-HT S-HIAA
2.31 & 0.42 137 & 23
rig/g
48.5 4 103 381 & 33
1.35 + 0.25**** (53.4%) 95 + 17**** (69.3%) 378 & SO** (77.9%) 321 + 24**** 04.3%)
2.30 + 0.19 115 f 19 449+63 407k65
1.38 f 0.70’*** (60.0%) 104 k 23 (90.4%) 372 &-56** (82.9%) 288 + 45**** (70.8%)
Results as mean 1: SD (% of saline control). Differences from saline injected: *P -c 0.05; **P <: 0.02; ***P <: 0.005; ****P < 0.001
42
G.
A.
KENNETT and
seen when 5-HT turnover was accelerated by electrical stimulation of the raphe nucleus (Marsden and Curzon, 1976). The conclusion that the functional 5-HT response to stress is at least partly dependent on an increase in brain tryptophan would be vitiated if valine could reduce the corticosterone response to stress by a nanserotonergic mechanism. One possibility would be a catecholaminergic mechanism, since valine would also be expected to compete with brain uptake of tyrosine and phenylalanine, the catecholamine precursors. However catecholamine synthesis in the brain is known to be less sensitive to precursor availability than is 5-HT synthesis, since marked end product regulation of tyrosine hydroxylase occurs (Shiman and Kaufman, 1970). In the present results the observation of a modest increuse of plasma corticosterone on treatment with valine alone (Table 1) tends also to argue against a 5-HT independent suppressive effect of valine on corticosterone secretion. A further point of interest in these studies concerns the effect of increases and decreases in brain tryptophan availability on the kynurenine pathway of tryptophan metabolism. It was found here that stress was accompanied by increases in kynurenine in the brain and in the plasma. These changes are not likely to be due to steroid induction of tryptophan pyrrolase, since this takes at least 3 hr (Curzon and Green, 1969; Joseph, 1973). The elevation in brain kynurenine could be due to increased tryptophan centrally; that in plasma to the increase in plasma-free tryptophan which occurs in stress, and is thought to at least partly underlie the increase in brain tryptophan (Knott and Curzon, 1972). However, the possibility that the increase in brain kynurenine represents uptake of peripherally produced kynurenine cannot be excluded, since brain kynurenine rises promptly in response to peripherally administered kynurenine (Joseph and Kadam, 1979). Valine on the other hand was found to reduce kynurenine centrally but to elevate it peripherally. While the former change might be expected from the reduction in brain tryptophan, this finding also does not contribute to the evidence for or against production of kynurenine from tryptophan centrally. Kynurenine is also presumably transported into the brain by the common large neutral amino acid carrier (Green and Curzon, 1970) and thus it would be expected that valine would reduce its uptake. It also follows from this that we cannot interpret the rise in plasma kynurenine on valine treatment in terms of increased tryptophan breakdown on the kynurenine pathway, perhaps consequent upon an alteration in the disposition of tryptophan. The failure of stress to further increase the valine-induced rise in plasma kynurenine may suggest that valine can reduce access of tryptophan to the sites at which it is converted to kynurenine, although the drop in plasma total tryptophan with valine plus 2 hr stress does suggest an altered disposition of tryptophan. If the activity of a central kynurenine pathway were
M. H. JOSEPH
regulated to divert less tryptophan from 5-HT synthesis when tryptophan availability decreased, and more when it increased, then it would be predicted that changes in kynurenine would be much more marked than those in 5-HIAA following a change in brain tryptophan accompanying stress or valine treatment. This was not found to be the case either in terms of magnitude, or in relation to time course at I and 2 hr. Thus, there is no evidence for such a regulatory role from these experiments, although of course a regulatory role over a shorter or longer time course cannot be excluded. In conclusion, these experiments have demonstrated that the increase in brain tryptophan in response to restraint stress can be blocked by prior administration of valine, a competing amino acid. The functional 5-HT response to stress is at least partly dependent on this rise in brain tryptophan. This supports the conclusion of Mueller, Twohy, Chen, Advis and Meites (1976) who demonstrated comparable endocrine responses in stressed, and in tryptophanand 5-HTP-treated rats. This argues against the hypothesis that increased brain 5-HIAA with stress is merely secondary to increased brain tryptophan. Interesting further questions include whether exogenous tryptophan can potentiate stress responses, and whether changes in 5-HT release in response to stress can be demonstrated in other parts of the brain. The present experiments have also demonstrated that stress increases kynurenine production in cico, but do not allow any conclusions to be drawn with regard to central production of kynurenine found in the brain. Finally they provide no evidence for any regulatory role of the kynurenine pathway in brain with respect to availability of tryptophan for 5-HT synthesis.
REFERENCES
Bliss, E. L., Ailion, J. and Zwanziger, J. (1968). Metabolism of norepinephrine serotonin and dopamine in rat brain with stress, J. Pharm. exp. Ther. 164: 122-134. Buckingham, J. C. and Hodges, J. R. (1979). Hypothalamic receptors influencing the secretion of corticotrophin releasing hormone in the rat. .I. Physic/. 290: 421431. Curzon, G. and Green, A. R. (1969). Effect of immobilization on rat liver tryptophan pyrrolase and brain 5hydroxytryptamine metabolism. Br. J. Pharmac. 37: 6899697. Curzon, G., Joseph, M. H. and Knott, P. J. (1972). Effects of immobilization and food deprivation on rat brain tryptophan metabolism. J. Nrur&hem. 19: 196771974. Eccleston. D.. Ashcroft. G. W. and Crawford. T. B. B. (1965). 5-FLydroxyindole metabolism in rat brain: a study of intermediate metabolism using the technique of tryptophan loading. J. Neurochem. 12: 493-503. Fernstrom, J. D. and Wurtman, R. J. (1971). Brain serotonin content: physiological dependence on plasma tryptophan levels. Science 173: 1499152. Freedman, P. A., Kappelman, A. H. and Kaufman, S. (1972). Partial purification and characterisation of tryptophan hydroxylase from rabbit hindbrain. J. hiol. Chem. 247: 41654173.
Brain tryptophan
and S-HT function
Gal. E. M. (1974). Ccrcbral tryptophan-2.3 dioxygenase and its induction in rat hrain. J. Ncrwo~~/~w. 22: 861 863. Gal. E. M.. Young. R. B. and Sherman. A. D. (1978). Tryptophan loading: consequent rlTects on the synthesis of kynurenine and 5hydroxyindoles in rat brain. J. Nrurothem. 31 : 237 244. Grahame-Smith. D. G. (1971). Studies in l?cw on the relationship hetwecn brain tryptophan, brain 5-HT synthesis and hyprractivity in rats treated with a monoamine oxidasr inhibitor and t_-tryptophan. J. Neurochenl. 18: 1053m 1066. Green. A. R. (1978). The effects of dietary tryptophan and its peripheral metabolism on brain 5hydroxytryptamine synthesis and function. In: Esstrrs in Neurochemisrrv rmd N~u~op/lurr,luco/~~~ (Youdim. M. B. H., Lovenberg. W.. Sharman. D. F. and Laanado. J. R.. Eds). Vol. 3. .op. . 103-128. Wiley. Chichester. Green. A. R. and Curzon. G. (1970). The effect of tryptophan metabolites on brain 5-hydroxytryptamine metabolism. Biochrrn. Phurmuc~. 19: 2061-2068. Green, A. R. and Grahame-Smith. D. G. (1976). Effect of drugs on processes regulating the functional activity of brain 5hydroxytryptamine. Narurv 260: 487491. Hayaishi. 0. (1976). Properties and function of indoleamine-2.3-dioxygenase. J. Biochern. 79: 1388218. Jacoby, J. H., Thomas, R. F.. Poulakos, J. J. and Siegel, A. (1979). Studies on trvptophan accumulation in brain during methiothepin induced enhancement of $hydroxyindole synthesis. Ntrun~,l-Schmiedebergs Arch. Pharmac. 307: 1433149. Jones, M. T.. Hillhouse. E. W. and Burden, J. (1976). Effect of various putative neurotransmitters on the secretion of corticotrophin releasing hormone from the rat hypothalamus in oitro. A model of the neurotransmitters involved. J. Endocrinol. 69: I-20. Joseph, M. H. (1973). Some relationships between intraand extra-cerebral tryptophan metabolism. Ph.D. Thesis, University of London. Joseph, M. H. (1978a). Determination of kvnurenine bv a simple gas-liquid chromatographic method applicable to urine, plasma, brain and cerebrospinal fluid. J. Chromaroyr. 146: 33341. Joseph, M. H. (1978b). Brain tryptophan metabolism on the 5-HT and kynurenine pathways in a strain of rats
with a deliciency 529
43
in stress in platelet
S-HT.
Br. J. Phmtrc~. 63:
533.
Joseph, M. H. and Kadam. B. V. (1979). Kynurenine: penetration to the brain. elfect on brain tryptophan and 5hydroxytryptamine metabolism. and binding to plasma albumin. Br. J. Phurmtrc. 66: 483P484P. Knott, P. J. and Curzon, G. (1972). Free tryptophan in plasma and brain tryptophan metabolism. Ntrtuw 239: 452-453. Knott, P. J., Joseph. M. H. and Curzon, G. (1973). Effects of food deprivation and immobilization on tryptophan and other amino acids in rat brain. J. Neurochcm. 20: 249-25 I. Marsden. C. A. and Curzon, G. (1976). ElTects of altered brain 5-hydroxytryptaminergic activity on brain tryptophan, 5-HT and 5-HIAA. Neurophrrrmucoloyy 15: 703-708.
Mattingly, D. (1962). A simple fluorimetric method for the estimation of free 11-hydroxycorticoids in human plasma. 1. c/in. Path. 15: 374379. Messing, R. B., Fisher, L. A., Phebus, L. and Lytle, L. D. (1976). Interaction of diet and drugs in the regulation of bra% 5-hydroxyindoles and the response to painful electric shock. Lfe Sci. 18: 707-714. Mueller, G. P., Twohy, C. P., Chen, H. T., Advis, J. P. and Meites, J. (1976). Effects of t,-tryptophan and restraint stress on hypothalamic and brain serotonergic turnover, and pituitary TSH and Prolactin release in rats. Life Sci. 18: 7155724. Pardridge, W. M. (1977). Regulation of amino acid availability to the brain. In: Nutrition and the Brain (Wurtman, R. J. and Wurtman, J. J., Eds), Vol. 1, pp. 141-204. Raven Press, New York. Scapagnini, U., Moberg, G. P., Van Loon, G. R., De Groat. J. and Ganong, W. F. (1971). Relation of brain 5-hydroxytryptamine content to the diurnal variation in plasma corticosterone in the rat. Neuroendocrinology 7: 90-96.
Shiman,
R. and Kaufman, S. (1970). Tyrosine hydroxylase. 17A: 6099615. Young, S. NY and Sourkes, T. L. (1977). Tryptophan in the central nervous system: regulation and significance. In: Adwnces in Neurochemistry (Agranoff, B. W. and Aprison, M. H., Eds), Vol. 2, pp. 133-191. Plenum Press, New York. Meth.
Enzvm.
of Psychiatry.
KENNETT* and
M. H. JOSEPH
MRC Clinical Research Centre, Middlesex HA1 3UJ, England
Watford
Road,
Harrow,
(Accepretl19 September 1980) Summary-The increase in brain tryptophan induced by restraint stress in rats has been shown to be prevented by prior administration of valine, 200 mg/kg (i.p.). Brain 5hydroxytryptamine (5HT) was not depleted. but the stress-induced increase in S-hydroxyindoleacetic acid (5-HIAA) was prevented. A 5-HT-mediated functional response to stress, elevated plasma corticosterone, was however significantly attenuated by valine pretreatment, but was not affected by valine treatment alone. This suggests that the increase in 5-HIAA in brain is not merely secondary to increased brain tryptophan but indicates an increase in functional 5-HT activity, which is in turn at least partly dependent on the increase in brain tryptophan. Measurement of kynurenine levels in the same animals indicated an increase in synthesis in stress, but did not support the hypothesis that competition occurs between the kynurenine and 5-hydroxyindole patbways of tryptophan metabolism in brain.
It has been known for some time that experimental stresses, including restraint stress, in rats is accompanied by increases in brain levels of S-hydroxyindoleacetic acid (5-HIAA), the metabolic product of the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin), in the absence of a change in the concentration of the latter (Bliss, Ailion and Zwanziger, 1968: Curzon and Green, 1969; Curzon, Joseph and Knott. 1972). This has been interpreted as indicating that there is a stress-induced increase in 5-HT turnover, and has further been shown to be accompanied by an increase in brain tryptophan (the precursor of 5-HT) which is specific to this amino acid (Joseph, 1973; Knott, Joseph and Curzon, 1973). It has also been shown that exogenous tryptophan increases 5-HT synthesis in the rat brain (Eccleston, Ashcroft and Crawford, 1965; Fernstrom and Wurtman, 1971) presumably because tryptophan hydroxylase, the rate limiting enzyme for 5-HT synthesis, is not normally saturated by tryptophan (Freedman, Kappelman and Kaufman, 1972). Thus, the increase of tryptophan with stress would be consistent with the idea that increased synthesis of 5-HT is necessary to prevent its depletion due to increased release. However an alternative possibility is that the increase in 5-HIAA with stress reflects only increased intraneuronal metabolism of 5-HT due to the tryptophaninduced increase in synthesis, without any increase in 5-HT release and functional activity. This concept was advanced by Grahame-Smith (1971) to account
for the failure of tryptophan administration to induce behavioural changes in rats unless pretreated with a monoamine oxidase inhibitor to prevent this overspill metabolism. Increased brain tryptophan during stress has been shown to be accompanied by an increase in the proportion of free (i.e. not albumin bound) tryptophan in the plasma (Knott and Curzon, 1972) this free tryptophan being more available for uptake to the brain (Young and Sourkes, 1977; Green, 1978). Availability depends also on competition between tryptophan and other large neutral amino acids (including leucine, isoleucine, valine, phenylalanine and tyrosine) which share a common blood-brain transport system with tryptophan (Pardridge, 1977). Valine has previously been used to restrict tryptophan availability to the brain in rats (Messing, Fisher, Phebus and Lytle, 1976; Jacoby, Thomas, Poulakos and Siegal, 1979) and in human patients with coma due to hepatic encephalopathy (Riederer, Personal communication, and data presented at CINP, Goteborg 1980). Accordingly the effect of valine pretreatment on the metabolic and functional 5-HT response to stress has been studied. It was anticipated that valine pretreatment might lead to a stress-induced depletion of 5-HT, if indeed increased release of 5-HT occurred, and also perhaps to a loss of functional activity. As an index of functional 5-HT activity, in the hypothalamus at least, plasma corticosterone was measured as CRF release from the hypothalamus is, at least partially, dependent on 5-HT (Buckingham and Hodges, 1979; Jones, Hillhouse and Burden, 1976; Scapagnini, Moberg, Van Loon, De Groot and Ganong, 1971). Another pathway for tryptophan metabolism
* G.A.K. is an MRC Scholar. Key words: tryptophan, stress, serotonin, 5-hydroxytryptamine, kynurenine, corticosterone, valine, brain, response to stress. 39
40
G.
KENNETT
A.
and M. H. JOSEPH
known to be active in the rat brain is the kynurenine pathway (Gal, 1974; Hayaishi, 1976; Joseph, 1978a; Gal, Young and Sherman, 1978). It has been suggested that this pathway may have a regulatory role in relation to the 5-HT pathway, but its activity has not been studied during stress. Thus plasma and brain kynurenine were monitored during these experiments.
METHODS
Valine and trypophan metabolite standards were obtained from Sigma (Poole, U.K.) and reagents from BDH (Poole, U.K.) (AR grade when available). Corticosterone standard (4-Pregnen-11/?,21-diol-3,20 dione) was obtained from Koch Light (Colnbrook, U.K.). Male Sprague-Dawley rats (wt 200-280 g) were injected with isotonic saline, or valine in isotonic saline at a dose of 200 mg/kg as appropriate. Immobilization by tying to a grid was carried out as previously described (Curzon et al., 1972), using grids modified to facilitate subsequent sacrifice by decapitation. All experiments were conducted between 10.00 and 13.00 hr. Blood was collected in heparinized plastic
3OlT
(a) PT
tubes; plasma obtained by centrifugation was stored at -40°C. Brains were removed rapidly and dropped into a previously cooled vial standing in dry ice. All rats were housed before the experiment in a constant 12 hr day and 12 hr night cycle and fed on rat chow and water ad libitum. Plasma tryptophan and brain tryptophan, 5-HT and 5-HIAA were determined fluorometrically as previously described (Joseph, 1978b). Kynurenine was determined in the same samples of plasma and brain by gas liquid chromatography (GLC) as previously described (Joseph, 1978a, b) with the substitution of dibromo-p-xylene (100 ng/ml) (Aldrich Chemical Company) for dichloro-p-xylene (200 ng/ml), as internal standard. The longer retention of this compound allowed the GLC column to be operated at 140°C thus increasing the sensitivity of the method previously described (Joseph, 1978a). Plasma corticosterone was determined fluorometritally by the method of Mattingly (1962). RESULTS As expected from previous work, immobilization stress significantly (P < 0.001) elevated the levels of
(d) BT
T
a**
:.: ::: ::: .::: ..
$;;i b!ibE
5 r
25001 zoo0 1500 loo0 500 01
454035 9
3O25 20 -
(bl PK
t-5 4
***
3 2 1
::i j :::
Q 5‘
0
(e) BK
*** iYllIl~ E..:. iii; ::: :.: *** ;;
50 Q loo 150 200 250 p
0
I
(f
***
I
r6CQ
5-HT
.:.: :::: :::: ..,.. .. 2:: ;j;:
::: ::: ::> .:.. :p ..... :>, ...
500 ::::il #: :::: 40a ,... .(0.. ... 300 5 .::: ;;i j!i cO 200
jji ttt ml]] (g 1 5-HIAA T
of41
3’: :::: .... .... i.. i.. .... .... ...
Li
::: :::
3
0
1
Hours of immobilization
stress
1
‘
Fig. 1. Effect of immobilization stress with (shaded columns) and without (open columns) vahne pretreatment (2OOmg/kg). (a) Plasma tryptophan; (b) plasma kynurenine: (c) plasma corticosterone; (d) brain tryptophan; (e) brain kynurenine; (f) brain 5-HT; (g) brain S-HIAA. n = 18 in col. 1; 8 in cot. 2 and 9 each in cols 3-6 except for (e) where n = 12 in col. 1; 8 in col. 2; 6 in cob 3, 5; 9 in cols 4, 6. Vertical bars indicate standard deviations, Significance of difference from column 1: *P < 0.01; **I’ < 0.005; ***P < 0.001: significance of difference from no valine pretreatment. ttP < 0.005; tttP < 0.001.
41
Brain tryptophan and S-HT function in stress brain ~yptophan (60.20/,at 1 hr, 39:7% at 2 fir, Fig. Id), and 5-HIAA (34.7% at 1 hr, 38.5% at 2 hr, Fig. lg) but 5-HT was not significantly altered (Fig. If). Brain kynurenine was also observed to be elevated (22.7% at 1 hr, P < 0.005; 34.4% at 2 hr P -Z 0.001, Fig. le). In plasma, stress resulted in the expected large rise in corticosterone (343% at 1 hr, 354% at 2 hr, P < 0.001 ineach case, Fig. lc) and marginal fall in total tryptophan (Fig. la). In addition, a rise in plasma kynurenine was observed (136% at 1 hr, P -c 0.001, 28.3% at 2 hr, P < 0.01, Fig. lb). It was also confirmed (Table 1) that valine, at doses of 100 mg/kg and 2OOmg/kg, reduced brain tryptophan (at 1 and 2 hr) and also 5-HT and S-HIAA at these times. In addition, valine at 2OOmgjkg had, no significant effect on plasma tryptophan, elevated corticosterone somewhat at 2 hr, and elevated plasma kynurenine but reduced brain kynurenine (Table 1). A dose of 2OOmg/kg at 1 hr was selected for further investigation. Administration of this dose immediately before immobilization completely prevented the stress-induced increase in brain tryptophan which had been seen 1 hr later (Fig. Id, compare columns 3 and 4). The increases in brain 5-HIAA and kynurenine were also prevented by valine pre-treatment (cols 3 and 4, Fig. lg and e respectively). Administration of valine 1 hr prior to sacrifice of animals immobilized for 2 hr, similarly prevented the rises in brain tryptophan (~01s 5 and 6 Fig. id). The increase in brain kynurenine was also prevented and that in 5-HIAA significantly attenuated (Fig. le and g). Plasma kynurenine was not however elevated more than by 1 hr stress alone, and plasma tryptophan was somewhat reduced by the combination of 2 hr stress and valine (27.1x, P < 0.01). The increase in plasma corticosterone induced by 1
or 2 hr stress was signi~~ntly attenuated by valine pretreatment (to 57% and 40% of the increase observed after stress alone at 1 and 2 hr, respectively, P < 0.001) although corticosterone was still significantly elevated above control levels. (197% at 1 hr, 137% at 2 hr, P < 0.001 for each, Fig. lc). DISCUSSION
The results show that administration of a competing amino acid can prevent the increase in brain tryp tophan which accompanies immobilization stress. They also show that blockade of this rise prevents, or at 2 hr markedly attenuates, the rise in S-HIAA. This was unexpected, since, as described in the introduction, it was anticipated that 5-HIAA would still be elevated, but at the expense of 5-HT depletion. At first sight this would appear to be consistent with the hypothesis that increased brain 5-HIAA on stress could simply be a reflection of increased brain tryptophan leading to increased 5-HT synthesis and catabolism, without any increase in 5-HT release or stimulation of receptors. This would be analogous to the situation for exogenous tryptophan described by GrahamtSmith (1971) and Green and Grahame-Smith (1976). However the plasma corti~osterone results show that the functional response to stress was attenuated by valine pretreatment. There was still a significant increase, and this may be attributable to the effects of other transmitters (Buckingham and Hodges, 1979) or to residual effects on 5-HT turnover, since one cannot extrapolate directly from metabolite levels to turnover rates; in addition, regional effects (e.g. in the hypothalmus) will not necessarily be reflected in the whole brain measurements. That 5-HT release in the hypothalamus may depend on increased tryptophan availability is also suggested by the increase in tryptophan
Table 1. The effect of valine (200 mg/kgf on tryptophan metabolism
n
9
8
9
8
Twptoph~ hdml)
18.1 & 4.7
15.7 & 2.6
Kynurenine (ng/ml)
941 j; 178
Corticosterone
8.49 * 3.35
16.1 + 3.1 (89%) 1268 i 204*** (134%) 9.94 * 5.54 (117%)
17.0 f 4.7 (108%) 1104 $- 200* (122%) 12.09 f 2.99** (147%)
Plasma
(fig/ml)
908 & 161 8.25 f 2.81
Brain
Twtophan Wg) Kynurenine 5-HT S-HIAA
2.31 & 0.42 137 & 23
rig/g
48.5 4 103 381 & 33
1.35 + 0.25**** (53.4%) 95 + 17**** (69.3%) 378 & SO** (77.9%) 321 + 24**** 04.3%)
2.30 + 0.19 115 f 19 449+63 407k65
1.38 f 0.70’*** (60.0%) 104 k 23 (90.4%) 372 &-56** (82.9%) 288 + 45**** (70.8%)
Results as mean 1: SD (% of saline control). Differences from saline injected: *P -c 0.05; **P <: 0.02; ***P <: 0.005; ****P < 0.001
42
G.
A.
KENNETT and
seen when 5-HT turnover was accelerated by electrical stimulation of the raphe nucleus (Marsden and Curzon, 1976). The conclusion that the functional 5-HT response to stress is at least partly dependent on an increase in brain tryptophan would be vitiated if valine could reduce the corticosterone response to stress by a nanserotonergic mechanism. One possibility would be a catecholaminergic mechanism, since valine would also be expected to compete with brain uptake of tyrosine and phenylalanine, the catecholamine precursors. However catecholamine synthesis in the brain is known to be less sensitive to precursor availability than is 5-HT synthesis, since marked end product regulation of tyrosine hydroxylase occurs (Shiman and Kaufman, 1970). In the present results the observation of a modest increuse of plasma corticosterone on treatment with valine alone (Table 1) tends also to argue against a 5-HT independent suppressive effect of valine on corticosterone secretion. A further point of interest in these studies concerns the effect of increases and decreases in brain tryptophan availability on the kynurenine pathway of tryptophan metabolism. It was found here that stress was accompanied by increases in kynurenine in the brain and in the plasma. These changes are not likely to be due to steroid induction of tryptophan pyrrolase, since this takes at least 3 hr (Curzon and Green, 1969; Joseph, 1973). The elevation in brain kynurenine could be due to increased tryptophan centrally; that in plasma to the increase in plasma-free tryptophan which occurs in stress, and is thought to at least partly underlie the increase in brain tryptophan (Knott and Curzon, 1972). However, the possibility that the increase in brain kynurenine represents uptake of peripherally produced kynurenine cannot be excluded, since brain kynurenine rises promptly in response to peripherally administered kynurenine (Joseph and Kadam, 1979). Valine on the other hand was found to reduce kynurenine centrally but to elevate it peripherally. While the former change might be expected from the reduction in brain tryptophan, this finding also does not contribute to the evidence for or against production of kynurenine from tryptophan centrally. Kynurenine is also presumably transported into the brain by the common large neutral amino acid carrier (Green and Curzon, 1970) and thus it would be expected that valine would reduce its uptake. It also follows from this that we cannot interpret the rise in plasma kynurenine on valine treatment in terms of increased tryptophan breakdown on the kynurenine pathway, perhaps consequent upon an alteration in the disposition of tryptophan. The failure of stress to further increase the valine-induced rise in plasma kynurenine may suggest that valine can reduce access of tryptophan to the sites at which it is converted to kynurenine, although the drop in plasma total tryptophan with valine plus 2 hr stress does suggest an altered disposition of tryptophan. If the activity of a central kynurenine pathway were
M. H. JOSEPH
regulated to divert less tryptophan from 5-HT synthesis when tryptophan availability decreased, and more when it increased, then it would be predicted that changes in kynurenine would be much more marked than those in 5-HIAA following a change in brain tryptophan accompanying stress or valine treatment. This was not found to be the case either in terms of magnitude, or in relation to time course at I and 2 hr. Thus, there is no evidence for such a regulatory role from these experiments, although of course a regulatory role over a shorter or longer time course cannot be excluded. In conclusion, these experiments have demonstrated that the increase in brain tryptophan in response to restraint stress can be blocked by prior administration of valine, a competing amino acid. The functional 5-HT response to stress is at least partly dependent on this rise in brain tryptophan. This supports the conclusion of Mueller, Twohy, Chen, Advis and Meites (1976) who demonstrated comparable endocrine responses in stressed, and in tryptophanand 5-HTP-treated rats. This argues against the hypothesis that increased brain 5-HIAA with stress is merely secondary to increased brain tryptophan. Interesting further questions include whether exogenous tryptophan can potentiate stress responses, and whether changes in 5-HT release in response to stress can be demonstrated in other parts of the brain. The present experiments have also demonstrated that stress increases kynurenine production in cico, but do not allow any conclusions to be drawn with regard to central production of kynurenine found in the brain. Finally they provide no evidence for any regulatory role of the kynurenine pathway in brain with respect to availability of tryptophan for 5-HT synthesis.
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