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The effects of kynurenic acid, quinolinic acid and other metabolites of tryptophan on the development of the high pressure neurological syndrome in the rat
Neuropharmacology Vol. 28, No. I, pp. 4341, 1989 Printed in Great Britain. All rights reserved
0028-3908/89%3.00+ 0.00 Copyright Q 1989Pergamon Press plc
THE EFFECTS OF KYNURENIC ACID, QUINOLINIC ACID AND OTHER METABOLITES OF TRYPTOPHAN ON THE DEVELOPMENT OF THE HIGH PRESSURE NEUROLOGICAL SYNDROME IN THE RAT* BRIDGET WARDLEY-SMITH,’ M. J. HALSEY,’ DIANE HAWLEY’ and M. Ii. JOSEPH’ ‘HPNS Group, Clinical Research Centre, Watford Road, Harrow, Middlesex, HAI 3UJ, U.K. and 2Departments of Psychology and Biochemistry, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF. U.K. (Accepted
8 September
1988)
Summary-The effects of some biolo~cally active metabolites of tryptophan on the high pressure neurological syndrome (HPNS) were studied. Kynurenic acid, quinolinic acid, 5-hydroxytryptophan, kynurenine and 3-hydroxyanthranilic acid, at doses within the physiological range, were administered exogenously to rats prior to exposure to increased pressure and any effects on the tremor, myoclonus and convulsion end points of the high pressure neurological syndrome were observed. Quinolinic acid (25 and 50 mgjkg) and kynurenine (50mg/kg) reduced the onset pressure for tremor, but not myoclonus or convulsions. Kynurenic acid (100 mg/kg) increased tremor onset pressure; 5-hydroxytryptophan (20 mg/kg) slightly increased onset pressure for tremor but decreased that for myoclonus. 3-Hydroxyanthranilic acid (20 mg/kg) had no significant effect on any of the motor signs of the syndrome. These data provide further support for the idea that the motor events seen in the high pressure neurological syndrome are not produced by a single mechanism. Differences between the responses to related metabolites suggest that the precise balance between compounds such as kynurenic acid and quinohnic acid may be important in the appearance of the high pressure neurological syndrome. Key words-high pressure neurological syndrome, quinolinic acid, kynurenic acid, kynurenine pathway, tryptophan metabolites.
The high pressure neurological
syndrome (HPNS) occurs when man or animals are exposed to increased environmental pressure [above 20 atmospheres absolute, (ATA)]; behavioural signs include tremor, myoclonus and, at very high pressures, seizures. The underlying neurochemical mechanisms are unknown although there has been a considerable amount of work on the neurophysiology and neuropharmacology of the effects of high pressure on the central nervous system (Halsey, 1982; Angel, Halsey, Little, Meldrum, Ross, Rostain and Wardley-Smith, 1984). This syndrome continues to be one of the major limitations to deep diving (20&600m of sea water) and, in spite of an increase in the number of studies concerning the mechanisms of the high pressure neurological syndrome, there are no definitive data on the underlying neurochemical changes occurring. Pharmacological manipulations of most major neurotransmitter systems have been employed in studies of the mechanisms of the high pressure neurological syndrome. In the investigation of inhibitory amino acid neurotransmitters, it has been suggested that drugs that facilitate y-aminobutyric acid *Some of these results were presented at the Vth ISTRY meeting and are briefiy reported in the conference proceedings (Wardley-Smith, Halsey and Joseph, 1987). 43
(GABA)-mediated transmission provide some protection against the effects of high pressure in mice (Bichard and Little, 1982). However, another study showed that drugs that blocked the reuptake of GABA had no effect on the behavioural responses to pressure in the rat (Halsey, Rostain and WardleySmith, 1984). It has also been suggested that glycinergic mechanisms may be involved in the appearance of the high pressure neurological syndrome (Bowser-Riley, Daniels and Smith, 1988). It was demonstrated that excitatory amino acid neurotransmission may be particularly important in the tremor component of the syndrome. 2-Amino-7-phosphonoheptanoic acid, a potent antagonist of excitation produced by N-methyln-aspartate (NMDA), more than doubled the pressure at which tremor appeared in the rat (Meldrum, Wardley-Smith, Halsey and Rostain, 1983). This provided evidence that excitatory neurotransmitters, such as glutamate and aspartate, might be important in the high pressure neurological syndrome, and especially in the tremor component. However, there is still little information concerning the mechanism of hyperbaric seizures. Quinolinic acid and kynurenine are metabolites of tryptophan which have biological activity and have been proposed as endogenous convulsants (Lapin, 1978). In
BRIDGET WARDLEY-SMITHer al.
44
contrast, another metabolite of tryptophan, kynurenit acid, has been shown to have an anticonvulsant action (Meldrum, Chapman and Meilo, 1988). The interrelations between the various metabolites on the kynurenine pathway and excitatory amino acid receptors have been recently reviewed (Stone and Connick, 1985). These authors believe that the kynurenine group of compounds may well be involved in convulsive and degenerative disorders of the central nervous system. The possible contribution of these compounds to the convulsions and related events seen in the high pressure neurological syndrome were investigated. Preliminary experiments in which rats were pretreated with tryptophan gave results which varied greatly with dose and pretreatment time, suggesting more than one mechanism of action (Wardley-Smith, unpublished observations). Tryptophan is metabolized in the liver through the kynurenine pathway (Fig. 1) with nicotinamide adenine dinucleotide (NAD) as one final product. It is also metabolized to 5-hydroxytryptamine (S-HT) through the intermediate S-hydroxyt~ptophan (5-HTP). The latter pathway is also well established in the brain. Some of the enzymes and intermediates on the kynurenine pathway have been demonstrated in brain: kynurenine itself (Joseph, 1978; Gal and Sherman, 3978), its synthetic enzyme, indoleamine dioxygenase (Hayaishi, 1976), quinolinic acid (Wolfsenberger, Amsler, Cuenod, Foster, Whetsell and Schwartz, 1983; Moroni, Lombardi, Carla and Moneti, 1984) and its synthetic and degradative enzymes (Foster,
Tryptopha\
I
5Hydroxytryptophan \
N-Fbrmylkynurenine
\
\ 5Hydroxytryptamine
Kyn~ine -.----l Kynurenic
acid
3-Hydroxykynurenine
3-Hydroxyanthranilic
acid
I t
Z-Acroleyt-3-aminofumarate-
Other products
I
I
Quinolinic
acid
I
NAD t
Fig. 1. Major metabolites on the kynurenine and S-HT pathways of metabolism of tryptophan. Compounds used in this study are underlined.
White and Schwartz, 1986), and kynurenic acid (Moroni, Dussi, Lombardi, Beni and Carla, 1988). This paper reports results from experiments in which several of the biologically active metabolites of tryptophan have been administered to rats prior to exposure to high pressure. Any change in the appearance of the high pressure neurological syndrome could suggest which, if any, of these compounds might be important in this syndrome. METHODS (a) Lfrugs and doses
Male Sprague-Dawley rats (weight range 20&3OOg) were used in all experiments. They were pretreated with the appropriate drug or control solution, injected intraperitoneaily 30 min before the start of compression. Each rat was exposed to pressure individually, and was placed in a small cage mounted over a strain gauge, which was used to facilitate assessment of the motor events seen in the high pressure neurological syndrome (Baker, Halsey, Wardley-Smith and Wloch, 1981). The following drugs and doses were used: 5-hydroxytryptophan (5-HTP) 20 mg/kg (n = 8); L-kynurenine sulphate 50 mg/kg (n = 5); kynurenic acid SO and 100 mg/kg (n = 5) for each dose); 3-hydroxyanthranil~c acid 20 mgjkg (n = 6); quinolinic acid 25 (n = 3) and SO mg/kg (n = 5); saline controls (n = 14). Control rats were interspersed throughout the treatment with drugs in 3 groups; these were subsequently combined for analysis. 5-Hydroxytryptophan and kynurenine were dissolved in saline; kynurenic and quinolinic acids were dissolved in sodium hydroxide in saline (0.5-5 N N&H) and titrated back to pH 7-8 with hydrochloric acid. 3-HydroxyanthraniIic acid was dissolved in 0.1 N NaOH which had been deoxygenated with helium prior to use. The pH was adjusted to within the range 7.5-8.5 and the solution was subsequently protected from light and stored in an atmosphere of helium. All drugs were purchased from Sigma. (b) Pressurization After pretreatment, the rat was placed in a 251 pressure chamber rated to a maximum pressure of 400 atmospheres absolute (ATA). Helium was added to the pressure chamber at a rate of 3 atmospheres/min. The oxygen partial pressure was maintained at 0.6 atmospheres while the carbon dioxide partial pressure was reduced to less than 0.001 atmospheres. The environmental temperature was varied as required to maintain the rectai temperature between 36 and 38°C measured with a thermistor probe (YSI Ltd). Three endpoints were selected for assessment of the high pressure neurological syndrome: tremor, myoclonus and the first convulsion. The onset pressures for these endpoints were recorded for each rat; all observations were made by an observer who was unaware of the
45
Some tryptophan metabolites and HPNS pretreatment. The mean onset pressures (+ 1 standard error of the mean) were determined for each group of animals. The statistical comparisons were based on the Mann-Whitney U-test, except for kynurenic and quinolinic acids, where two different doses were tested and analysis of variance was also used to test for linear trends. The Minitab statistical computing system (Version 82.1) was used for all analyses. At the end of the experiment, each rat was humanely killed with an overdose of anaesthetic. In addition to the hyperbaric experiments, some rats were treated with S-HTP (20 mg/kg i.p.) and observed for a comparable period of time (l-1; hr) in the chamber at 1 atmosphere absolute for any behavioural changes. RESULTS
The results for the different drugs and doses used are shown in Table 1. Pretreatment with 5-HTP produced a small (12%) but significant increase in the threshold for tremor (P < 0.05) but in contrast produced a 20% decrease in the threshold for hyperbaric myoclonus (P < 0.02). The rats which received S-HTP at 1 atmosphere showed no behavioural changes; in particular there was no evidence of myoclonus or the 5-HT behavioural syndrome (Jacobs, 1976), thus the increase in myoclonus seen at pressure was not due to the effect of this dose of S-HTP alone. Kynurenine reduced the onset pressure for tremor by 27% (P < 0.005) but had no other effects. Kynurenic acid, at these doses, showed little effect on any of the endpoints and analysis of variance failed to show any statistical significance. With lOOmg/kg kynurenic acid, the tremor threshold pressure was increased by 17% (P < 0.02, Mann-Whitney U-test). However, testing for a linear trend for tremor in the 3 groups (50 and 100 mg/kg and controls) gave a P value of 0.02. 3-Hydroxyanthranilic acid had no significant effect on the motor signs of the high pressure neurological syndrome although the mean threshold pressure for convulsions was lower by 8% relative to controls.
Table I. Mean threshold pressures in atmospheres absolute (SEM) for tremor, myoclonus and convulsions after the different treatment with drugs. cornoared with saline controls Tremor
Myoclonus
Convulsion
Kynurenic acid 50 mglkg 100 mgikg
46.1 (4.1) 52.1 (2.5)’
78.9(1.7) 82.9 (3.7)
97.4(1.6) 95.9 (4.9)
Quinolinic acid 25 mgikg 50 mnikr! Kynure&e3-Hydroxyanthranilic 5-Hydroxytryptophan Saline controls
37.4 (0.8)’ 30.6 (2.2)* 32.1 (0.7)’ 44.3 (3.1) 46. I (0.9). 43.9(1.5)
87. I (2.4) 80.6(1.8) 75.3 (2.8) 83.0 (2.4) 68.9 (2.7)* 86.0 (I .6)
104.1 (1.7) 97.6 (0.9) 91.6(1.6) 87.9 (2.5) 105.9 (4.9) 95.2 (IS)
acid
*Indicates a statistically significant difference from the control data. The different P values are given in the text.
Quinolinic acid reduced the threshold for tremor in a dose-related manner. At 25 mg/kg quinolinic acid reduced the threshold for tremor by 15%; at 50 mg/kg by 30% (analysis of variance P < 0.005). Neither myoclonus nor convulsions were affected by quinolinic acid. DISCUSSION
These experiments were designed to investigate the effects of metabolites of tryptophan on the high pressure neurological syndrome. The compounds studied occur on the two main metabolic pathways of tryptophan, the 5-hydroxytryptamine pathway and the kynurenine pathway. The metabolites of the kynurenine pathway which were used are produced endogenously outside the C.N.S., and kynurenine, quinolinic acid and kynurenic acid have been shown to occur in the brain (Joseph, 1978; Gal and Sherman, 1978; Wolfsenberger et al., 1983; Moroni et al., 1988). Administration of 5-HTP (20 mg/kg) has been shown to raise the turnover of 5-HT and, to a lesser extent, 5-HT itself in the brain (Moir and Eccleston, 1968). This dose was chosen since it is likely to be similar in effect to lOOmg/kg tryptophan. In addition, larger doses of 5-HTP are more likely to enhance the release of catecholamines (Fuxe, Butcher and Engel, 1971), which would complicate the interpretation of any subsequent changes. This treatment had two opposing effects on the high pressure neurological syndrome, an increase in the threshold for tremor but a decrease in that for myoclonus. The increase in the threshold for tremor, through significant, was very small and of little practical value. However, the decrease in pressure for myoclonus was more marked (20%). This may simply represent the involvement of 5-HT in myoclonus (Growdon, 1979) although, not surprisingly, rats treated with this relatively small dose of 5-HTP at 1 atmosphere absolute showed no evidence of myoclonic jerking. These results could also be mediated by “kynurenine-like” metabolites of 5-HT or 5-HTP produced by indoleamine dioxygenase as well as by 5-HT itself. Pretreatment with 5-HTP produced no change in the threshold for convulsions which supports the view of others (Koblin, Little, Green, Daniels, Smith and Paton, 1980) that changes in 5-HT do not affect hyperbaric convulsions. However, in experiments where mice were pretreated with reserpine (5 mg/kg), which lowered thresholds for convulsions, subsequent treatment with tryptophan at fairly large doses (200-600 mg/kg) reversed about 20% of the latter, suggesting a small inhibitory role for 5-HT in high pressure convulsions (Brauer, Beaver and Sheehan, 1978). Results obtained with compounds occurring on the kynurenine pathway produced changes of greater magnitude. Pretreatment with kynurenine tended to reduce the pressure at which all 3 endpoints occurred, although the greatest effect was a reduction in the
46
BRIDGET WARDLEY-SMITH cl al.
threshold for tremor. Kynurenine has been proposed as an endogenous convulsant (Lapin, 1978) and has also been shown to facilitate the convulsions produced by strychnine (Lapin, 1980). In these studies, only the threshold for tremor was significantly reduced, suggesting either a possible potentiation or a direct action at the NMDA receptor. While kynurenine is taken up into the CNS after systemic administration, its effects on the high pressure neurological syndrome could be mediated by subsequent metabolites on the kynurenine pathway, either synthesized intracerebrally or taken up into the CNS after extracerebral synthesis. It is likely that a larger dose of kynurenine would have reduced the pressure for myoclonus and convulsions still further, but such doses would be completely non-physiological (Joseph, 1988). The two doses of kynurenic acid used in this study (50 or 100 mg/kg) had little effect. Kynurenic acid (100 mg/kg) increased the threshold for tremor by 17% but it was much less effective than the larger dose reported previously (500 mg/kg; Halsey, Hawley, Meldrum and Wardley-Smith, 1985). However, this dose-related effect, supported by the positive linear trend with dose found in the present study, provides further support for the involvement of the NMDA receptor in the genesis of tremor, since kynurenic acid has been shown to be a non-specific antagonist at the NMDA receptor and to antagonize excitation produced by quinolinic acid (Perkins and Stone, 1982). 3-Hydroxyanthranilic acid had no significant effect on any of the endpoints, although there was a tendency for convulsions to occur at a lower pressure. It proved impossible to give a larger dose due to problems of solubility and stability. Although 3-hydroxyanthranilic acid has been shown to be synthesised in brain (Gal, Young and Sherman, 1978), it is not known whether it can penetrate the blood-brain barrier. Quinolinic acid reduced the threshold for tremor in a significant dose-related manner (aov P < 0.005). Quinolinic acid has been proposed as a candidate for an endogenous transmitter at the NMDA receptor (Stone and Perkins, 1981) or alternatively at the “NMDA,” receptor (Perkins and Stone, 1983); however, the lack of a proven reuptake system remains a serious drawback to the establishment of quinolinic acid as an endogenous neurotransmitter (Stone and Connick, 1985). Quinolinic acid did not affect either myoclonus or convulsions, supporting the view that activity at the NMDA receptor influences mainly the tremor component of the high pressure neurological syndrome. All the studies involving competitive NMDA receptor antagonists have shown a preferential effect on tremor, with much less activity, possibly of a non-selective type, on myoclonus and convulsions (Meldrum et al., 1983; Wardley-Smith and Meldrum, 1984; Halsey et al., 1985).
The actions of small doses of quinolinic acid in lowering the threshold for tremor is surprising, in view of the reports that quinolinic acid is unable to penetrate the blood-brain barrier in mature rats (Foster, Miller, Oldendorf and Schwartz, 1984) and that substantial doses (80&2400 mg/kg i.p.) are required to produce seizures in mice (Czuczwar and Meldrum, 1982). The present results raise the possibility that the permeability of the blood-brain barrier may be increased at high pressure. This could be either a pre-convulsant phenomenon or possibly directly related to membrane changes resulting from high pressure itself, although such hypotheses have not been tested directly in the present study. Quinolinic acid may also affect tremor by a peripheral action. Fagni, Weiss, Pellet and Hugon (1982) have shown that spinal cats (transected at T99TlO) still exhibit a low frequency tremor at pressures above 50 atmospheres and these authors suggest that hyperbaric tremor is spinal or neuromuscular in origin. While it is unlikely that tremor is entirely non-central, it is possible that a component of it may be affected by peripherally active quinolinic acid. These results, taken as a whole, provide further support for the idea that the motor events seen in the high pressure neurological syndrome do not lie on a single continuum. It was proposed originally that the syndrome was a single entity with a common underlying mechanism (Miller, 1974) but the present results, together with others (Rowland-James, Wilson and Miller, 1981) make this hypothesis less likely. It is interesting that compounds occurring on the kynurenine pathway can influence the high pressure neurological syndrome in either direction, i.e. make the animal more or less sensitive to pressure. It may be that the precise balance between kynurenic acid and quinolinic acid is important in the appearance of the high pressure neurological syndrome. In conclusion, these data suggest that further investigation of the kynurenine pathway of metabolism of tryptophan in the high pressure neurological syndrome would be profitable. The effects seen were not great compared with some other compounds, such as 2-amino-7-phosphonoheptanoic acid (Meldrum et ul., 1983) but they significantly modified the syndrome at fairly small doses. It might be assumed that any compound with either excitatory or depressant properties would modify the syndrome; however, specific examples of compounds which are anaesthetic, anticonvulsant, sedative or muscle relaxant have all been shown to have no effect on the high pressure neurological syndrome (Green, Halsey and Wardley-Smith, 1977; Halsey and Wardley-Smith, 1981; Bowser-Riley er al., 1988). It would therefore seem that sensitive methods for investigating the changes in these metabolites. especially levels of kynurenic and quinolinic acid in the brain might provide useful information as to the role of these endogenously occurring compounds in the high pressure neurological syndrome.
Some tryptophan metabolites and HPNS REFERENCES
Aneel A.. Halsev M. J.. Little H.. Meldrum B. S., Ross J. AI. S., Rostain- J-C and Wardley-Smith B. (1984) Specific effects of drugs at pressure: animal investigations. Phil. Trans. R. Sot. 304B. 85-94. Baker J. A., Halsey M. J., Wardley-Smith B. and Wioch R. T. (1981) Assessment of the high pressure neurological syndrome (HPNS): a new method of measuring tremor in an animal model. In: Underwater Physiology VII. Proceedings of the Sevenrh Symposium on Underwater Physiology (Bachrach A. J. and Matzen M. M., Eds), pp.
415420. Undersea Medical Society, Bethesda. Bichard A. R. and Little H. J. (1982) Drugs that increase GABA transmission protect against the HPNS. Br. J. Pharmac. 76: 447452.
Bowser-Riley F., Daniels S. and Smith E. B. (1988) Investigations into the origin of the high pressure neurological syndrome: the interaction between pressure, strychnine and 1,2-propandiols in the mouse. Br. J. Pharmac. 94: 1069-1076.
Brauer R. W., Beaver R. W. and Sheehan M. E. (1978) Role of monoamine neurotransmitters in the compression-rate dependence of HPNS convulsions. In: Underwater Physiology VI. Proceedings of the Sixth Symposium on Underwater Physiology (Shilling, C. W. and Beckett, M. W.,
Eds), pp. 49-59. FASEB, Bethesda. Czuczwar S. J. and Meldrum B. S. (1982) Protection against chemically induced seizures by 2-amino-7-phosphonoheptanoic acid. Eur. J. Pharmac. 83: 335-338. Fagni L., Weiss M., Pellet J. and Hugon M. (1982) The possible mechanisms of the high pressure induced motor disturbances in the cat. Electroenceph. clin. Neurophysiol. 53: 59&601.
Foster A. C., Miller L. P., Oldendorf W. H. and Schwartz R. (1984) Studies on the dispositon of quinolinic acid after intracerebral or systemic administration in the rat. Expl Neural. 84: 42840.
Foster A. C., White R. J. and Schwartz R. (1986) Synthesis of quinolinic acid by 3-hydroxyanthranilic acid oxygenase in rat brian tissue in vitro. J. Neurochem. 47: 23-30. Fuxe K., Butcher L. L. and Engel J. (1971) DL-5-hydyroxytryptophan-induced in central monoamine neurons after peripheral decarboxylase inhibition. J. Pharm. Pharmac. 23: 42&424.
Gal E. M. and Sherman A. D. (1978) Synthesis and metabolism of L-kynurenine in rat brain. J. Neurochem. 30: 607413.
Gal E. M., Young R. B. and Sherman A. D. (1978) Tryptophan loading: consequent effects on the synthesis of kynurenine and 5-hydroxyindoles in rat brain. J. Neurochem. 31: 237-244.
Green C. J., Halsey M. J. and Wardley-Smith B. (1977) Possible protection against some of the physiological effects of hiah oressure. J. Phvsiol.. Land. 267: 502-503P. Growdon J. H. 11979) Serotonergic mechanisms in myoclonus. J. Neural. Trans., Suppl. 15: 209-216. Halsev M. J. (1982) Effects of high oressure on the central ner;ous system. Physiol. Rev. 62:’ 1342-1377. Halsey M. J., Hawley D., Meldrum B. S. and WardleySmith B. (1985) The effect of kynurenic acid on the HPNS in the rat. J. Physiol. 371: 64P. Halsey M. J., Rostain J. C. and Wardley-Smith B. (1984) Effect of GABA enhancine drues on the behavioural responses to high pressure yn the-rat. J. Physiol., Lond. 350: 25P.
47
Halsey M. J. and Wardley-Smith B. (1981) The high pressure neurological syndrome: do anticonvulsants~prdvent it? Br. J. Pharmac. 72: 502P-503P. Hayaishi 0. (1976) Properties and function of indoleamine2,3,-dioxygenase. J. Eiochem. 79: 13P-21P. Jacobs B. L. (1976) An animal behaviour model for studying central serotonergic synapses. Life Sci. 19: 777-786. Joseph M. H. (1978) Determination of kynurenine by a simple GLC method applicable to urine, plasma, brain and CSF. J. Chromatog. 146: 3341. Joseph M. H. (1988) The analysis of kynurenines and the biochemical pharmacology of kynurenine in the CNS. In: Quinolinic Acid and the Kynurenines (Stone T. W., Ed.). CRC Press, Boca Raton, FL. In press. Koblin D. D., Little H. J., Green A. R., Daniels S., Smith E. B. and Paton W. D. M. (1980) Brain monoamines and HPNS. Neuropharmacology 19: 1031-1038. Lapin I. P. (1978) Stimulant and convulsive effects of kynurenines injected into brain ventricles in mice. J. Neural Trans. 42: 3743.
Lapin I. P. (1980) Effect of kynurenine and quinolinic acid on the action of convulsants in mice. Pharmac. Biochem. Behau. 13: 17-20.
Meldrum B. S., Chapman A. G. and Mello L. (1988) Anticonvulsant action of kynurenic acid in reflex epilepsy in DBA/2 mice and in photosensitive baboons. Progress in Tryptophan and Seroionin Research II. In press. Meldrum B., Wardley-Smith B., Halsey M. and Rostain J-C. (1983) 2-Aminophosphonoheptanoic acid protects against the high pressure neurological syndrome. Eur. J. Pharmac. 87: 501-502.
Miller K. W. (1974) Inert gas narcosis, the high pressure neurological syndrome and the critical volume hypothesis. Science 185: 867-869. Moir A. T. B. and Eccleston D. (1968) The effects of precursor loading in the cerebral metabolism of 5-hydroxyindoles. J. Neurochem. 15: 1093-l 108. Moroni F., Lombardi G., Carla V. and Moneti G. (1984) The excitotoxin quinolinic acid is present and unevenly distributed in the brain. Brain Res. 295: 352-355. Moroni F., Russi P., Lombardi G., Beni M. and Carla V. (1988) Presence of kynurenic acid in the mammalian brain. J. Neurochem. 51: 177-180. Perkins M. N. and Stone T. W. (1982) An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid. Bruin Res. 247: 184-187. Perkins M. N. and Stone T. W. (1983) Ouinolinic acidregional variations in neuronal sensitivity. Brain Res. 259: 172-176.
Rowland-James P., Wilson M. W. and Miller K. W. (1981) Pharmacological evidence for multiple sites of action of pressure in mice. Undersea Biomed. Res. 8: l-l 1. Stone T. W. and Connick J. H. (1985) Quinolinic acid and other kynurenines in the central nervous system. Neuroscience 15: 597-617.
Stone T. W. and Perkins M. N. (1981) Quinolinic acid-a potent endogenous excitant at amino acid receptors in the CNS. Eur. j. Pharmac. 72: 411412. Wardley-Smith B. and Meldrum B. S. (1984) Effect of excitatory amino acid antagonists on the HPNS in rats. Eur. J. Pharmac. 105: 351-354.
Wolfsenberger M., Amsler U., Cuenod M., Foster A. C., Whetsell W. 0. and Schwartz R. (1983) Identification of quinolinic acid in rat and human brain tissue. Neurosci. Letr. 41: 247-252.
0028-3908/89%3.00+ 0.00 Copyright Q 1989Pergamon Press plc
THE EFFECTS OF KYNURENIC ACID, QUINOLINIC ACID AND OTHER METABOLITES OF TRYPTOPHAN ON THE DEVELOPMENT OF THE HIGH PRESSURE NEUROLOGICAL SYNDROME IN THE RAT* BRIDGET WARDLEY-SMITH,’ M. J. HALSEY,’ DIANE HAWLEY’ and M. Ii. JOSEPH’ ‘HPNS Group, Clinical Research Centre, Watford Road, Harrow, Middlesex, HAI 3UJ, U.K. and 2Departments of Psychology and Biochemistry, Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF. U.K. (Accepted
8 September
1988)
Summary-The effects of some biolo~cally active metabolites of tryptophan on the high pressure neurological syndrome (HPNS) were studied. Kynurenic acid, quinolinic acid, 5-hydroxytryptophan, kynurenine and 3-hydroxyanthranilic acid, at doses within the physiological range, were administered exogenously to rats prior to exposure to increased pressure and any effects on the tremor, myoclonus and convulsion end points of the high pressure neurological syndrome were observed. Quinolinic acid (25 and 50 mgjkg) and kynurenine (50mg/kg) reduced the onset pressure for tremor, but not myoclonus or convulsions. Kynurenic acid (100 mg/kg) increased tremor onset pressure; 5-hydroxytryptophan (20 mg/kg) slightly increased onset pressure for tremor but decreased that for myoclonus. 3-Hydroxyanthranilic acid (20 mg/kg) had no significant effect on any of the motor signs of the syndrome. These data provide further support for the idea that the motor events seen in the high pressure neurological syndrome are not produced by a single mechanism. Differences between the responses to related metabolites suggest that the precise balance between compounds such as kynurenic acid and quinohnic acid may be important in the appearance of the high pressure neurological syndrome. Key words-high pressure neurological syndrome, quinolinic acid, kynurenic acid, kynurenine pathway, tryptophan metabolites.
The high pressure neurological
syndrome (HPNS) occurs when man or animals are exposed to increased environmental pressure [above 20 atmospheres absolute, (ATA)]; behavioural signs include tremor, myoclonus and, at very high pressures, seizures. The underlying neurochemical mechanisms are unknown although there has been a considerable amount of work on the neurophysiology and neuropharmacology of the effects of high pressure on the central nervous system (Halsey, 1982; Angel, Halsey, Little, Meldrum, Ross, Rostain and Wardley-Smith, 1984). This syndrome continues to be one of the major limitations to deep diving (20&600m of sea water) and, in spite of an increase in the number of studies concerning the mechanisms of the high pressure neurological syndrome, there are no definitive data on the underlying neurochemical changes occurring. Pharmacological manipulations of most major neurotransmitter systems have been employed in studies of the mechanisms of the high pressure neurological syndrome. In the investigation of inhibitory amino acid neurotransmitters, it has been suggested that drugs that facilitate y-aminobutyric acid *Some of these results were presented at the Vth ISTRY meeting and are briefiy reported in the conference proceedings (Wardley-Smith, Halsey and Joseph, 1987). 43
(GABA)-mediated transmission provide some protection against the effects of high pressure in mice (Bichard and Little, 1982). However, another study showed that drugs that blocked the reuptake of GABA had no effect on the behavioural responses to pressure in the rat (Halsey, Rostain and WardleySmith, 1984). It has also been suggested that glycinergic mechanisms may be involved in the appearance of the high pressure neurological syndrome (Bowser-Riley, Daniels and Smith, 1988). It was demonstrated that excitatory amino acid neurotransmission may be particularly important in the tremor component of the syndrome. 2-Amino-7-phosphonoheptanoic acid, a potent antagonist of excitation produced by N-methyln-aspartate (NMDA), more than doubled the pressure at which tremor appeared in the rat (Meldrum, Wardley-Smith, Halsey and Rostain, 1983). This provided evidence that excitatory neurotransmitters, such as glutamate and aspartate, might be important in the high pressure neurological syndrome, and especially in the tremor component. However, there is still little information concerning the mechanism of hyperbaric seizures. Quinolinic acid and kynurenine are metabolites of tryptophan which have biological activity and have been proposed as endogenous convulsants (Lapin, 1978). In
BRIDGET WARDLEY-SMITHer al.
44
contrast, another metabolite of tryptophan, kynurenit acid, has been shown to have an anticonvulsant action (Meldrum, Chapman and Meilo, 1988). The interrelations between the various metabolites on the kynurenine pathway and excitatory amino acid receptors have been recently reviewed (Stone and Connick, 1985). These authors believe that the kynurenine group of compounds may well be involved in convulsive and degenerative disorders of the central nervous system. The possible contribution of these compounds to the convulsions and related events seen in the high pressure neurological syndrome were investigated. Preliminary experiments in which rats were pretreated with tryptophan gave results which varied greatly with dose and pretreatment time, suggesting more than one mechanism of action (Wardley-Smith, unpublished observations). Tryptophan is metabolized in the liver through the kynurenine pathway (Fig. 1) with nicotinamide adenine dinucleotide (NAD) as one final product. It is also metabolized to 5-hydroxytryptamine (S-HT) through the intermediate S-hydroxyt~ptophan (5-HTP). The latter pathway is also well established in the brain. Some of the enzymes and intermediates on the kynurenine pathway have been demonstrated in brain: kynurenine itself (Joseph, 1978; Gal and Sherman, 3978), its synthetic enzyme, indoleamine dioxygenase (Hayaishi, 1976), quinolinic acid (Wolfsenberger, Amsler, Cuenod, Foster, Whetsell and Schwartz, 1983; Moroni, Lombardi, Carla and Moneti, 1984) and its synthetic and degradative enzymes (Foster,
Tryptopha\
I
5Hydroxytryptophan \
N-Fbrmylkynurenine
\
\ 5Hydroxytryptamine
Kyn~ine -.----l Kynurenic
acid
3-Hydroxykynurenine
3-Hydroxyanthranilic
acid
I t
Z-Acroleyt-3-aminofumarate-
Other products
I
I
Quinolinic
acid
I
NAD t
Fig. 1. Major metabolites on the kynurenine and S-HT pathways of metabolism of tryptophan. Compounds used in this study are underlined.
White and Schwartz, 1986), and kynurenic acid (Moroni, Dussi, Lombardi, Beni and Carla, 1988). This paper reports results from experiments in which several of the biologically active metabolites of tryptophan have been administered to rats prior to exposure to high pressure. Any change in the appearance of the high pressure neurological syndrome could suggest which, if any, of these compounds might be important in this syndrome. METHODS (a) Lfrugs and doses
Male Sprague-Dawley rats (weight range 20&3OOg) were used in all experiments. They were pretreated with the appropriate drug or control solution, injected intraperitoneaily 30 min before the start of compression. Each rat was exposed to pressure individually, and was placed in a small cage mounted over a strain gauge, which was used to facilitate assessment of the motor events seen in the high pressure neurological syndrome (Baker, Halsey, Wardley-Smith and Wloch, 1981). The following drugs and doses were used: 5-hydroxytryptophan (5-HTP) 20 mg/kg (n = 8); L-kynurenine sulphate 50 mg/kg (n = 5); kynurenic acid SO and 100 mg/kg (n = 5) for each dose); 3-hydroxyanthranil~c acid 20 mgjkg (n = 6); quinolinic acid 25 (n = 3) and SO mg/kg (n = 5); saline controls (n = 14). Control rats were interspersed throughout the treatment with drugs in 3 groups; these were subsequently combined for analysis. 5-Hydroxytryptophan and kynurenine were dissolved in saline; kynurenic and quinolinic acids were dissolved in sodium hydroxide in saline (0.5-5 N N&H) and titrated back to pH 7-8 with hydrochloric acid. 3-HydroxyanthraniIic acid was dissolved in 0.1 N NaOH which had been deoxygenated with helium prior to use. The pH was adjusted to within the range 7.5-8.5 and the solution was subsequently protected from light and stored in an atmosphere of helium. All drugs were purchased from Sigma. (b) Pressurization After pretreatment, the rat was placed in a 251 pressure chamber rated to a maximum pressure of 400 atmospheres absolute (ATA). Helium was added to the pressure chamber at a rate of 3 atmospheres/min. The oxygen partial pressure was maintained at 0.6 atmospheres while the carbon dioxide partial pressure was reduced to less than 0.001 atmospheres. The environmental temperature was varied as required to maintain the rectai temperature between 36 and 38°C measured with a thermistor probe (YSI Ltd). Three endpoints were selected for assessment of the high pressure neurological syndrome: tremor, myoclonus and the first convulsion. The onset pressures for these endpoints were recorded for each rat; all observations were made by an observer who was unaware of the
45
Some tryptophan metabolites and HPNS pretreatment. The mean onset pressures (+ 1 standard error of the mean) were determined for each group of animals. The statistical comparisons were based on the Mann-Whitney U-test, except for kynurenic and quinolinic acids, where two different doses were tested and analysis of variance was also used to test for linear trends. The Minitab statistical computing system (Version 82.1) was used for all analyses. At the end of the experiment, each rat was humanely killed with an overdose of anaesthetic. In addition to the hyperbaric experiments, some rats were treated with S-HTP (20 mg/kg i.p.) and observed for a comparable period of time (l-1; hr) in the chamber at 1 atmosphere absolute for any behavioural changes. RESULTS
The results for the different drugs and doses used are shown in Table 1. Pretreatment with 5-HTP produced a small (12%) but significant increase in the threshold for tremor (P < 0.05) but in contrast produced a 20% decrease in the threshold for hyperbaric myoclonus (P < 0.02). The rats which received S-HTP at 1 atmosphere showed no behavioural changes; in particular there was no evidence of myoclonus or the 5-HT behavioural syndrome (Jacobs, 1976), thus the increase in myoclonus seen at pressure was not due to the effect of this dose of S-HTP alone. Kynurenine reduced the onset pressure for tremor by 27% (P < 0.005) but had no other effects. Kynurenic acid, at these doses, showed little effect on any of the endpoints and analysis of variance failed to show any statistical significance. With lOOmg/kg kynurenic acid, the tremor threshold pressure was increased by 17% (P < 0.02, Mann-Whitney U-test). However, testing for a linear trend for tremor in the 3 groups (50 and 100 mg/kg and controls) gave a P value of 0.02. 3-Hydroxyanthranilic acid had no significant effect on the motor signs of the high pressure neurological syndrome although the mean threshold pressure for convulsions was lower by 8% relative to controls.
Table I. Mean threshold pressures in atmospheres absolute (SEM) for tremor, myoclonus and convulsions after the different treatment with drugs. cornoared with saline controls Tremor
Myoclonus
Convulsion
Kynurenic acid 50 mglkg 100 mgikg
46.1 (4.1) 52.1 (2.5)’
78.9(1.7) 82.9 (3.7)
97.4(1.6) 95.9 (4.9)
Quinolinic acid 25 mgikg 50 mnikr! Kynure&e3-Hydroxyanthranilic 5-Hydroxytryptophan Saline controls
37.4 (0.8)’ 30.6 (2.2)* 32.1 (0.7)’ 44.3 (3.1) 46. I (0.9). 43.9(1.5)
87. I (2.4) 80.6(1.8) 75.3 (2.8) 83.0 (2.4) 68.9 (2.7)* 86.0 (I .6)
104.1 (1.7) 97.6 (0.9) 91.6(1.6) 87.9 (2.5) 105.9 (4.9) 95.2 (IS)
acid
*Indicates a statistically significant difference from the control data. The different P values are given in the text.
Quinolinic acid reduced the threshold for tremor in a dose-related manner. At 25 mg/kg quinolinic acid reduced the threshold for tremor by 15%; at 50 mg/kg by 30% (analysis of variance P < 0.005). Neither myoclonus nor convulsions were affected by quinolinic acid. DISCUSSION
These experiments were designed to investigate the effects of metabolites of tryptophan on the high pressure neurological syndrome. The compounds studied occur on the two main metabolic pathways of tryptophan, the 5-hydroxytryptamine pathway and the kynurenine pathway. The metabolites of the kynurenine pathway which were used are produced endogenously outside the C.N.S., and kynurenine, quinolinic acid and kynurenic acid have been shown to occur in the brain (Joseph, 1978; Gal and Sherman, 1978; Wolfsenberger et al., 1983; Moroni et al., 1988). Administration of 5-HTP (20 mg/kg) has been shown to raise the turnover of 5-HT and, to a lesser extent, 5-HT itself in the brain (Moir and Eccleston, 1968). This dose was chosen since it is likely to be similar in effect to lOOmg/kg tryptophan. In addition, larger doses of 5-HTP are more likely to enhance the release of catecholamines (Fuxe, Butcher and Engel, 1971), which would complicate the interpretation of any subsequent changes. This treatment had two opposing effects on the high pressure neurological syndrome, an increase in the threshold for tremor but a decrease in that for myoclonus. The increase in the threshold for tremor, through significant, was very small and of little practical value. However, the decrease in pressure for myoclonus was more marked (20%). This may simply represent the involvement of 5-HT in myoclonus (Growdon, 1979) although, not surprisingly, rats treated with this relatively small dose of 5-HTP at 1 atmosphere absolute showed no evidence of myoclonic jerking. These results could also be mediated by “kynurenine-like” metabolites of 5-HT or 5-HTP produced by indoleamine dioxygenase as well as by 5-HT itself. Pretreatment with 5-HTP produced no change in the threshold for convulsions which supports the view of others (Koblin, Little, Green, Daniels, Smith and Paton, 1980) that changes in 5-HT do not affect hyperbaric convulsions. However, in experiments where mice were pretreated with reserpine (5 mg/kg), which lowered thresholds for convulsions, subsequent treatment with tryptophan at fairly large doses (200-600 mg/kg) reversed about 20% of the latter, suggesting a small inhibitory role for 5-HT in high pressure convulsions (Brauer, Beaver and Sheehan, 1978). Results obtained with compounds occurring on the kynurenine pathway produced changes of greater magnitude. Pretreatment with kynurenine tended to reduce the pressure at which all 3 endpoints occurred, although the greatest effect was a reduction in the
46
BRIDGET WARDLEY-SMITH cl al.
threshold for tremor. Kynurenine has been proposed as an endogenous convulsant (Lapin, 1978) and has also been shown to facilitate the convulsions produced by strychnine (Lapin, 1980). In these studies, only the threshold for tremor was significantly reduced, suggesting either a possible potentiation or a direct action at the NMDA receptor. While kynurenine is taken up into the CNS after systemic administration, its effects on the high pressure neurological syndrome could be mediated by subsequent metabolites on the kynurenine pathway, either synthesized intracerebrally or taken up into the CNS after extracerebral synthesis. It is likely that a larger dose of kynurenine would have reduced the pressure for myoclonus and convulsions still further, but such doses would be completely non-physiological (Joseph, 1988). The two doses of kynurenic acid used in this study (50 or 100 mg/kg) had little effect. Kynurenic acid (100 mg/kg) increased the threshold for tremor by 17% but it was much less effective than the larger dose reported previously (500 mg/kg; Halsey, Hawley, Meldrum and Wardley-Smith, 1985). However, this dose-related effect, supported by the positive linear trend with dose found in the present study, provides further support for the involvement of the NMDA receptor in the genesis of tremor, since kynurenic acid has been shown to be a non-specific antagonist at the NMDA receptor and to antagonize excitation produced by quinolinic acid (Perkins and Stone, 1982). 3-Hydroxyanthranilic acid had no significant effect on any of the endpoints, although there was a tendency for convulsions to occur at a lower pressure. It proved impossible to give a larger dose due to problems of solubility and stability. Although 3-hydroxyanthranilic acid has been shown to be synthesised in brain (Gal, Young and Sherman, 1978), it is not known whether it can penetrate the blood-brain barrier. Quinolinic acid reduced the threshold for tremor in a significant dose-related manner (aov P < 0.005). Quinolinic acid has been proposed as a candidate for an endogenous transmitter at the NMDA receptor (Stone and Perkins, 1981) or alternatively at the “NMDA,” receptor (Perkins and Stone, 1983); however, the lack of a proven reuptake system remains a serious drawback to the establishment of quinolinic acid as an endogenous neurotransmitter (Stone and Connick, 1985). Quinolinic acid did not affect either myoclonus or convulsions, supporting the view that activity at the NMDA receptor influences mainly the tremor component of the high pressure neurological syndrome. All the studies involving competitive NMDA receptor antagonists have shown a preferential effect on tremor, with much less activity, possibly of a non-selective type, on myoclonus and convulsions (Meldrum et al., 1983; Wardley-Smith and Meldrum, 1984; Halsey et al., 1985).
The actions of small doses of quinolinic acid in lowering the threshold for tremor is surprising, in view of the reports that quinolinic acid is unable to penetrate the blood-brain barrier in mature rats (Foster, Miller, Oldendorf and Schwartz, 1984) and that substantial doses (80&2400 mg/kg i.p.) are required to produce seizures in mice (Czuczwar and Meldrum, 1982). The present results raise the possibility that the permeability of the blood-brain barrier may be increased at high pressure. This could be either a pre-convulsant phenomenon or possibly directly related to membrane changes resulting from high pressure itself, although such hypotheses have not been tested directly in the present study. Quinolinic acid may also affect tremor by a peripheral action. Fagni, Weiss, Pellet and Hugon (1982) have shown that spinal cats (transected at T99TlO) still exhibit a low frequency tremor at pressures above 50 atmospheres and these authors suggest that hyperbaric tremor is spinal or neuromuscular in origin. While it is unlikely that tremor is entirely non-central, it is possible that a component of it may be affected by peripherally active quinolinic acid. These results, taken as a whole, provide further support for the idea that the motor events seen in the high pressure neurological syndrome do not lie on a single continuum. It was proposed originally that the syndrome was a single entity with a common underlying mechanism (Miller, 1974) but the present results, together with others (Rowland-James, Wilson and Miller, 1981) make this hypothesis less likely. It is interesting that compounds occurring on the kynurenine pathway can influence the high pressure neurological syndrome in either direction, i.e. make the animal more or less sensitive to pressure. It may be that the precise balance between kynurenic acid and quinolinic acid is important in the appearance of the high pressure neurological syndrome. In conclusion, these data suggest that further investigation of the kynurenine pathway of metabolism of tryptophan in the high pressure neurological syndrome would be profitable. The effects seen were not great compared with some other compounds, such as 2-amino-7-phosphonoheptanoic acid (Meldrum et ul., 1983) but they significantly modified the syndrome at fairly small doses. It might be assumed that any compound with either excitatory or depressant properties would modify the syndrome; however, specific examples of compounds which are anaesthetic, anticonvulsant, sedative or muscle relaxant have all been shown to have no effect on the high pressure neurological syndrome (Green, Halsey and Wardley-Smith, 1977; Halsey and Wardley-Smith, 1981; Bowser-Riley er al., 1988). It would therefore seem that sensitive methods for investigating the changes in these metabolites. especially levels of kynurenic and quinolinic acid in the brain might provide useful information as to the role of these endogenously occurring compounds in the high pressure neurological syndrome.
Some tryptophan metabolites and HPNS REFERENCES
Aneel A.. Halsev M. J.. Little H.. Meldrum B. S., Ross J. AI. S., Rostain- J-C and Wardley-Smith B. (1984) Specific effects of drugs at pressure: animal investigations. Phil. Trans. R. Sot. 304B. 85-94. Baker J. A., Halsey M. J., Wardley-Smith B. and Wioch R. T. (1981) Assessment of the high pressure neurological syndrome (HPNS): a new method of measuring tremor in an animal model. In: Underwater Physiology VII. Proceedings of the Sevenrh Symposium on Underwater Physiology (Bachrach A. J. and Matzen M. M., Eds), pp.
415420. Undersea Medical Society, Bethesda. Bichard A. R. and Little H. J. (1982) Drugs that increase GABA transmission protect against the HPNS. Br. J. Pharmac. 76: 447452.
Bowser-Riley F., Daniels S. and Smith E. B. (1988) Investigations into the origin of the high pressure neurological syndrome: the interaction between pressure, strychnine and 1,2-propandiols in the mouse. Br. J. Pharmac. 94: 1069-1076.
Brauer R. W., Beaver R. W. and Sheehan M. E. (1978) Role of monoamine neurotransmitters in the compression-rate dependence of HPNS convulsions. In: Underwater Physiology VI. Proceedings of the Sixth Symposium on Underwater Physiology (Shilling, C. W. and Beckett, M. W.,
Eds), pp. 49-59. FASEB, Bethesda. Czuczwar S. J. and Meldrum B. S. (1982) Protection against chemically induced seizures by 2-amino-7-phosphonoheptanoic acid. Eur. J. Pharmac. 83: 335-338. Fagni L., Weiss M., Pellet J. and Hugon M. (1982) The possible mechanisms of the high pressure induced motor disturbances in the cat. Electroenceph. clin. Neurophysiol. 53: 59&601.
Foster A. C., Miller L. P., Oldendorf W. H. and Schwartz R. (1984) Studies on the dispositon of quinolinic acid after intracerebral or systemic administration in the rat. Expl Neural. 84: 42840.
Foster A. C., White R. J. and Schwartz R. (1986) Synthesis of quinolinic acid by 3-hydroxyanthranilic acid oxygenase in rat brian tissue in vitro. J. Neurochem. 47: 23-30. Fuxe K., Butcher L. L. and Engel J. (1971) DL-5-hydyroxytryptophan-induced in central monoamine neurons after peripheral decarboxylase inhibition. J. Pharm. Pharmac. 23: 42&424.
Gal E. M. and Sherman A. D. (1978) Synthesis and metabolism of L-kynurenine in rat brain. J. Neurochem. 30: 607413.
Gal E. M., Young R. B. and Sherman A. D. (1978) Tryptophan loading: consequent effects on the synthesis of kynurenine and 5-hydroxyindoles in rat brain. J. Neurochem. 31: 237-244.
Green C. J., Halsey M. J. and Wardley-Smith B. (1977) Possible protection against some of the physiological effects of hiah oressure. J. Phvsiol.. Land. 267: 502-503P. Growdon J. H. 11979) Serotonergic mechanisms in myoclonus. J. Neural. Trans., Suppl. 15: 209-216. Halsev M. J. (1982) Effects of high oressure on the central ner;ous system. Physiol. Rev. 62:’ 1342-1377. Halsey M. J., Hawley D., Meldrum B. S. and WardleySmith B. (1985) The effect of kynurenic acid on the HPNS in the rat. J. Physiol. 371: 64P. Halsey M. J., Rostain J. C. and Wardley-Smith B. (1984) Effect of GABA enhancine drues on the behavioural responses to high pressure yn the-rat. J. Physiol., Lond. 350: 25P.
47
Halsey M. J. and Wardley-Smith B. (1981) The high pressure neurological syndrome: do anticonvulsants~prdvent it? Br. J. Pharmac. 72: 502P-503P. Hayaishi 0. (1976) Properties and function of indoleamine2,3,-dioxygenase. J. Eiochem. 79: 13P-21P. Jacobs B. L. (1976) An animal behaviour model for studying central serotonergic synapses. Life Sci. 19: 777-786. Joseph M. H. (1978) Determination of kynurenine by a simple GLC method applicable to urine, plasma, brain and CSF. J. Chromatog. 146: 3341. Joseph M. H. (1988) The analysis of kynurenines and the biochemical pharmacology of kynurenine in the CNS. In: Quinolinic Acid and the Kynurenines (Stone T. W., Ed.). CRC Press, Boca Raton, FL. In press. Koblin D. D., Little H. J., Green A. R., Daniels S., Smith E. B. and Paton W. D. M. (1980) Brain monoamines and HPNS. Neuropharmacology 19: 1031-1038. Lapin I. P. (1978) Stimulant and convulsive effects of kynurenines injected into brain ventricles in mice. J. Neural Trans. 42: 3743.
Lapin I. P. (1980) Effect of kynurenine and quinolinic acid on the action of convulsants in mice. Pharmac. Biochem. Behau. 13: 17-20.
Meldrum B. S., Chapman A. G. and Mello L. (1988) Anticonvulsant action of kynurenic acid in reflex epilepsy in DBA/2 mice and in photosensitive baboons. Progress in Tryptophan and Seroionin Research II. In press. Meldrum B., Wardley-Smith B., Halsey M. and Rostain J-C. (1983) 2-Aminophosphonoheptanoic acid protects against the high pressure neurological syndrome. Eur. J. Pharmac. 87: 501-502.
Miller K. W. (1974) Inert gas narcosis, the high pressure neurological syndrome and the critical volume hypothesis. Science 185: 867-869. Moir A. T. B. and Eccleston D. (1968) The effects of precursor loading in the cerebral metabolism of 5-hydroxyindoles. J. Neurochem. 15: 1093-l 108. Moroni F., Lombardi G., Carla V. and Moneti G. (1984) The excitotoxin quinolinic acid is present and unevenly distributed in the brain. Brain Res. 295: 352-355. Moroni F., Russi P., Lombardi G., Beni M. and Carla V. (1988) Presence of kynurenic acid in the mammalian brain. J. Neurochem. 51: 177-180. Perkins M. N. and Stone T. W. (1982) An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid. Bruin Res. 247: 184-187. Perkins M. N. and Stone T. W. (1983) Ouinolinic acidregional variations in neuronal sensitivity. Brain Res. 259: 172-176.
Rowland-James P., Wilson M. W. and Miller K. W. (1981) Pharmacological evidence for multiple sites of action of pressure in mice. Undersea Biomed. Res. 8: l-l 1. Stone T. W. and Connick J. H. (1985) Quinolinic acid and other kynurenines in the central nervous system. Neuroscience 15: 597-617.
Stone T. W. and Perkins M. N. (1981) Quinolinic acid-a potent endogenous excitant at amino acid receptors in the CNS. Eur. j. Pharmac. 72: 411412. Wardley-Smith B. and Meldrum B. S. (1984) Effect of excitatory amino acid antagonists on the HPNS in rats. Eur. J. Pharmac. 105: 351-354.
Wolfsenberger M., Amsler U., Cuenod M., Foster A. C., Whetsell W. 0. and Schwartz R. (1983) Identification of quinolinic acid in rat and human brain tissue. Neurosci. Letr. 41: 247-252.