The rates of synthesis, uptake and disappearance of [14C]taurine in eight areas of the rat central nervous system

Brain Researck, 76 (1974) 44%459

447

iL~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

T H E RATES OF SYNTHESIS, U P T A K E A N D D I S A P P E A R A N C E OF [a4C]T A U R I N E IN E I G H T AREAS OF T H E R A T C E N T R A L N E R V O U S SYSTEM

G. G. S. COLLINS

Department o[' Pharmacology, The School of Pkarmacy, University of London, London WCIN 1 4X (Great Britain) (Accepted March 18th, 1974)

SUMMARY

A study has been made of the uptake, metabolism and half-life of [14C]taurine in 8 areas of the rat central nervous system. When tissue slices were incubated with radioactive taurine (1.31 # M ) the rate of accumulation was approximately linear with time for at least 20 min. The differences in uptake for the various areas when measured in this way were not related to the endogenous taurine concentrations. Radioactive cysteine and cystine were converted to [14C]taurine in vivo at equal rates although there were significant differences in the percentage conversion in various brain areas; there was a direct relationship between the extent of formation of [14C]taurine from these precursors and endogenous taurine levels. The in vivo efflux of [t4C]taurine from all areas of the rat central nervous system investigated was multiphasic, half-lives varying from about 9 to 240 h; there were significant differences in the rates of disappearance of the labelled taurine in the areas studied. It is concluded that the metabolism of taurine differs markedly in the various areas of the rat central nervous system and the possible physiological significance of this finding is discussed.

1NTRODUCTION

Taurine (2-aminoethanesulphonic acid) has been found in nearly all animal tissues investigated and is often present in high concentrations (for references, see Jacobsen and Smith2a). Recently, an increasing interest has been shown in the possibility that taurine may have a role in the central nervous system as either a 'modulator' or as an inhibitory transmitter in its own right. For example, when taurine is administered microiontophoretically onto single neurones of the cerebral cortex 9, spinal cord s,1°,1~, or medulla iv it causes depression of firing rates. The efflux of radioactively

448

G.G.S. COLtaNS

labelled taurine from slices of cerebral cortex is markedly increased by electrical stimulation7,~2,1s,26 and more recently, high potassium ion concentrations have been shown to be effective in releasing endogenous taurine from synaptosomal preparations of cerebral cortex 13. In a series of in vivo experiments, Jasper and Koyama24 demonstrated an increased release of taurine, glutamate, aspartate and gtycine from the cortex of cats on activation of the midbrain reticular formation and in addition, the light-evoked release of [~S]taurine from retinae has been demonstrated~ Moreover, several studies have been carried out to elucidate the characteristics of taurine uptake by neural tissues2,1z,la,26,zv,29,30,'3a,37. The heterogeneous distribution pattern exhibited by taurine in the brains of many mammalian species ~,~,27,~6,39 suggests that a detailed study of the metabolism of taurine in different brain areas would be rewarding, in the present paper, possible regional differences in the uptake, metabolism and rates of disappearance of [14C]taurine in the rat central nervous system have been investigated and related to the tissue concentrations of endogenous taurine. METHODS

Dissection of rat brains. Rats were killed by cervical dislocation, the brains and cervical spinal cord rapidly removed, washed with ice-cold normal saline solution and placed on glass petri dishes which were standing on ice. The brains were dissected into 7 areas (as described by Glowinski and Iversenl~); pons-medulla, cerebellum, striatum, hypothalamus, midbrain, cerebral cortex-hippocampus and olfactory bulbs. After weighing, the tissue samples were either extracted with perchloric acid or used to measure the uptake of [14C]taurine. Measurement of 1-14Cjtaurine uptake. Two distinct saturable uptake systems for taurine have been postulated23; the fl-system has a high affinity for taurine whereas the co-system has a very much lower affinity. In these experiments the concentrations of [14C]taurine in the incubation flasks has been kept so low (1.31 #M) that any uptake detected would be mainly due to influx mediated by the fl-system. The uptake of [14C]taurine into slices of rat brain has been estimated using the method devised by Iversen and Neal 2z. Briefly, the dissected tissue fragments were sliced into 0.05 mm cubes using a mechanical chopper 31 and 10 mg suspended in 10 ml of oxygenated Ringer solution at 37 °C in sealed flasks (composition of Ringer solution: sodium chloride, l18.5mM; potassium chloride, 4.75mM; calcium chloride; 1.77 raM; magnesium sulphate, 1.18raM; sodium phosphate buffer, pH 7.4, 16.2 mM: Dglucose, 1 g/l). After 10 min preincubation, 0.05 #Ci of [14C]taurine was added giving a final concentration of 1.31 #M and incubation allowed to continue for 5, 10 or 20 rain. The slices were recovered by rapid filtration, the radioactivity released by the addition of 0.3 ml of distilled water and estimated in a scintillation spectrometer. Suitable blank samples were also carried through the entire procedure. The amount of [~4C]taurine accumulated has been expressed as nmoles/g wet weight of tissue. It is known that the only amino acids to inhibit taurine uptake by the fi-system are fi-alanine and hypotaurine26. If these substances exhibit a heterogeneous distri-

TAURINE METABOLISM IN RAT C N S

449

bution, they might interfere with taurine influx to varying extents in the different brain areas under investigation, thereby making comparative studies difficult to interpret. In order to determine whether these amino acids or any other endogenous substances influence taurine transport, an experiment was carried out in which the uptake of [14C]taurine by 20 and 40 mg of slices in 10 ml Ringer solution was compared with that by 10 mg of tissue over a 20-rain time period. In all 8 areas of the rat central nervous system investigated the amount of [14C]taurine accumulated by the slices was directly proportional to the wet weight of tissue used, suggesting that any endogenous inhibitors of taurine transport were not interfering with taurine uptake. Measurement of endogenous taurine levels. In some experiments the free amino acid content of the various brain areas was measured. Tissues were extracted using 6 ~ v/v perchloric acid 3s and the amino acid contents estimated as previously described 6 using an LKB-Biocal amino acid analyser. The taurine content of the samples has been expressed as/~moles/g wet weight of tissue. Estimation of the half-life oftaurine. The half-life of taurine in 8 areas of the rat central nervous system was estimated by measuring the rate of disappearance of intracisternally administered [14C]taurine. Groups of rats were lightly anaesthetised with ether, injected with 1/~Ci of [14C]taurine in 20 #1 of Merlis solution 32 into the cisterna magna and were killed after 0.25, 0.5, 1, 2, 4 and 6 h and I, 2, 3 and 4 days. The brains were removed and where applicable rapidly dissected into their constituent areas. The free amino acids were extracted by homogenising the tissue samples in 5 ml of 6 ~ v/v perchloric acid and after removing the denatured protein by centrifugation (2000 × g for 15 rain), the perchlorate was precipitated by the dropwise addition of potassium hydroxide solution until the pH value rose to 4.0. The samples were then stored at 0 °C for 2 h and the precipitated potassium perchlorate removed by centrifugation (2000 x g for 5 rain). The supernatant was evaporated to dryness under vacuo and the residue dissolved in 2.0 ml of distilled water. The [l~C]taurine was separated from other amino acids and from possible radioactive metabolites using an ion exchange procedure based on that described by Huxtable and Bressler 20. All glass columns were packed with 10 mm ),: 70 mm of Dowex AG-1-X2 resin, 100-200 mesh, chloride form, onto which a 10 mm )< 30 mm layer of Amberlite CGI20, 100mesh, hydrogen form was carefully pipetted. The column was washed with 10 ml of distilled water prior to use. The pH value of each sample was adjusted to 7.0 using sodium hydroxide solution and a 1.0 ml aliquot placed on the column. Taurine was eluted by passing 15.0 ml of distilled water through the column ('taurine fraction'); 8 ml of this eluate was mixed with 10 ml of 'Instagel' (Packard Instrument Company) and the radioactivity estimated in a scintillation spectrometer. The purity of the 'taurine fraction' was ascertained using two methods. First, a series of the 'taurine fractions' were passed through an LKB-Biocal amino acid analyser. In each case, only one peak, corresponding with that of authentic taurine appeared on the tracing. Secondly, thin-layer chromatography of some samples was carried out using 3 solvent systems ( 9 6 ~ ethanol-34 ~,, ammonia, 7:3; ethyl acetatemethanol-acetic acid-water, 6:2:1:I; butanol-acetic acid-water, 4:1:1) on silica gel thin-layer plates. The amino acids were visualised using a 0.25/o°r w/v solution of"

450

G.G.S.

COLHNS

ninhydrin in acetone. In addition, 5 mm horizontal strips of the silica gel were scraped off the plate, the radioactivity eluted with 0.5 ml water and was counted in 10 ml o f a 0.5 ~,~ w/v solution of butyl PBD in toluene containing 6 ml of 2-ethoxyethanol. in a series of experiments in which 20 tissue extracts were submitted to chromatography using all 3 solvent systems, only one ninhydrin-positive spot corresponding to authentic taurine was detected; in addition, only one peak of radioactivity was present which again corresponded with that of authentic [14C]taurine. It was of particular importance to ensure that the taurine fraction was not contaminated with radioactive isethionic acid (2-hydroxyethanesulphonic acid), the major metabolite of taurine in the braina'L Solutions of pure sodium isethionate (5 mg in 1 ml of distilled water adjusted to pH 7.0) were passed through the ionexchange resins and the column washed with 15 ml of distilled water. The presence or absence of isethionic acid in the eluate was determined by measuring the release of free sulphate in the presence of acid. Aliquots (1.0 ml) of the eluate were boiled to dryness with 0.2 ml concentrated nitric acid, the residue dissolved in 1.0 ml o f distilled water and 0.1 ml of I N barium chloride solution added. The presence of sulphate was detected by the appearance of a cloudy white precipitate; using this method, 50 #g of isethionic acid was detectable. When 1.0 ml aliquots of the water washing were tested for the presence of isethionic acid, none was detectable. However, when 25 ml of 2 N HCI was passed through the resin beds and the eluate ('metabolite fraction') treated as described above, the presence of free sulphate was detected. The absence of isethionate in the 'taurine fraction' and its presence in the 'metabotite fraction' was also confirmed using thin-layer chromatography. A mixture of 0.05/zCi p~C]taurine, 50/zg non-radioactive taurine and 100/zg o f isethionic acid in 1.0 ml distilled water was placed on a resin column after which 15 ml of distilled water followed by 15 ml of 2 N HCI were passed through the resin beds. After evaporation to dryness, ascending thin-layer chromatography on Silica gel using the ethyl acetate-methanol acetic acid-water (6:2:1:1 by volume) solvent system was carried out on each of the two fractions; the presence of [14C]taurine was detected as described earlier, ,,o w/v solution of potassium whereas isethionic acid was detected using a spray of a 1 °/"""" permanganate in l N sulphuric acid 2°. No isethionic acid could be detected in the "taurine fraction' which contained a single peak of radioactivity associated with the ninhydrin-positive spot. No radioactivity or ninhydrin positive spot was detectable on chromatography o f the pooled acid eluate fractions. From these t w o series o f experiments it was concluded that the 'taurine fraction' contained neither isethionic acid or any other amino acid contaminant.

The rate of formation of /~4C/taurine from

24C/cysteine and :~l~C/cystitw~

Groups of rats were lightly anaesthetised with ether and injected intracisternally with either 1.43/~Ci of L-[U-14C]cysteine or 1.43 ~Ci of L-[U-14C]cystine in 20/~1 of Merlis 32 solution. Animals were killed at 0.25, 0.5, 1, 2 and 4 h after injection, the brains rapidly removed, dissected as previously described and the amino acids extracted using 5 ml 0.6 ~ v/v perchloric acid. The [14C]taurine formed was separated by ion-exchange chromatography as described earlier. In these experiments it was necessary to determine that the taurine fraction was not contaminated with the possible

TAUR1NE METABOLISM IN RAT C N S

451

intermediates produced in the conversion of p4C]cysteine and p4C]cystine to [14C]taurine. For this reason, thin-layer chromatography of the 'taurine fraction' was carried out using all 3 solvent systems. Standard solutions of taurine, hypotaurine, cysteine, cystine, cysteic acid and isethionic acid were also chromatographed alone and also with the 'taurine fraction'. The 'taurine fractions' from extracts of brains from animals injected either with p4C]cysteine or p4C]cystine were found to contain a single spot of radioactivity corresponding to authentic [14C]taurine and also only one ninhydrin-positive spot, again corresponding with that o f authentic taurine. Estimation of the half-life of [14Csurea. The half-life of urea was estimated in 3 brains areas (pons-medulla, midbrain and cerebral cortex-hippocampus) by measuring the rate of disappearance of [l:*C]urea. Groups of rats were lightly anaesthetised with ether and 5/~Ci of [14C]urea injected intracisternally. The animals were killed 1, 2, 4, 6, 10 and 12 h later, dissected as previously described and homogenised with perchloric acid to extract the labelled urea. Aliquots (5.0 ml) of the extract were mixed with 10 ml 'lnstagel' (Packard Instrument Company) and the radioactivity estimated by scintillation spectrometry. MATERIALS

[1,2-1aC]taurine (2.11 mCi/mmole) was obtained from the New England Nuclear Company, Dreieichenhain, G.F.R.; p4C]urea (61 mCi/mmole), L-[U-I'~C]cystine (39 mCi/mmole) and t-[U-14C]cysteine (38.6 mCi/mmole) were obtained from the Radiochemical Centre, Amersham, U.K. All chemicals used were of the highest obtainable purity. RESULTS

Endogenous taurine levels. The endogenous taurine content of 8 areas of the rat central nervous system is shown in Table I; concentrations vary from between 7.33 /,moles/g in the olfactory bulbs to only 1.20/zmoles/g in the cervical cord. These values are similar to those previously published'~,16,27,a
452

G.o.S.

COLLINS

TABLE I ENDOGENOUS TAURINE CONTENT AND UPTAKE OF [14C]TAUR1NE IN VARIOUS AREAS OF THE RAT CENTRAL NERVOUS SYSTEM

Tissue slices (10 mg wet weight) were incubated at 37 C for 5, l0 or 20 min in I0 ml of Ringer solution containing [14C]taurine (1.31 nmolesfml). Slices were recovered by rapid filtration and assayed for radioactivity. Results are mean values :[: S.E.M. for between 4 and 6 experiments.

Area

Taurine content (#moles~g) n= 6

Uptake of/14Cjtaurine (nmoles/g) .......................................... 5 rain 10 rain 20 mill

Pons-medulla Cerebellum Hypothalamus Striatum Midbrain Cerebralcortex-hippocampus Olfactory bulbs Cervicalcord Whole brain

1.83 .~_~0.071 4.47 ::i: 0.239* 1.93 ::: 0.111 4.35 - 0.167' 1.95 ± 0.120 4.24 ~ 0.136 7.33 :+: 0.367'~ 1.20 Z 0.140 3.67 0.182

0.312 0.261 0.571 0.416 0.771 0.891 0.402 0.308 . .

_h 0.019 [ 0.018 _~- 0.103 -: 0.071 ~!: 0,093 ~[ 0.063 ::!: 0.051 k 0.052 . . .

0.602 0.542 1.16 0.822 1.48 1.67 0.862 0.62l

~ 0.024 4.:-0.098 2_ 0.102 5:0.091 ~- 0.149 4 0.188 ± 0.112 5 0.069

1.08 1.21 2.08 1.61 2.92 2.94 1.64 0,993

L 0.087 ! 0.149 : 0.147'* 0.193 0.262*** : 0.312'** : 0.181 !~: 0.102

* Significantly different (P < 0.001) compared with pons-medulla, hypothalamus, midbrain and cervical cord. ** Significantly higher (P < 0.05) compared with pons-medulla, cerebellum and cervical cord. *** Significantly different (P < 0.005) compared with other regions. § Significantly different (P < 0.001) compared with all other regions.

94 ~,~ of the total tissue radioactivity was present as [14C]taurine; thus, either metabolism of the taurine is minimal or any metabolites formed leave the brain rapidly. The disappearance of [14C]taurine was also studied in 8 areas of the rat central nervous system over a time range of between 0.25 h and 4 days after injection of the radioactive amino acid. In all areas investigated, the rate of disappearance of [uC]taurine was exponential during the first 4 h after injection. There were no significant differences between any of the fractional rate constants (see Table II), the corresponding half-lives (t~ values) varying between 9.1 h (striatum)and 16.1 h (pons-medulla). I00

5O

E ~

lO

TIME AFTER INJECTION (DAYS)

Fig. 1. Changes in radioactivity in whole rat brain after the intracisternal injection of l t~Ci [l~C]taurine. Each point is the mean of 8 experiments 4+ S . E M . Total radioactivity ( 0 ) ; [l'~C]taurine (C).

TAURINE METABOLISMIN RAT CNS

453

TABLE II THE RATE OF DISAPPEARANCE OF [14CITAURINE AND [I'ICIUREA FROM VARIOUS AREAS OF THE RAT CENTRAL NERVOUS SYSTEM

Groups of between 4 and 6 rats were injected intracisternally with either 1.0/~Ci of [l,2-~C]taurine or 5/~Ci of [14CJurea and the rate of disappearance of the solutes measured (see Materials and Methods). Fractional rate constants (k values) were calculated from the regression coefficients and turnover rates from t½ values and size of endogenous taurine pool (see Table 1). The disappearance of [~C]taurine from the midbrain, hypothalamus, olfactory bulbs and whole brain between 6 h and 4 days after injection was non-linear so that k values could not be calculated. Taurine Area

0.25 4 h after I ttCi of 2 HCjtaurine

6 h ~ days after 1 ,uCi of :' ~4C Jtaurine

k (perh)

k (perh)

t½ (h) Turnover (nmo&~/h)

Cervicalcord Pons-medulla Cerebellum Striatum Cerebral cortexbippocampus Midbrain Hypothalamus Olfactory bulbs Whole brain

0.075 ~ 0.062 k 0.092 ~ O.l IO i.

Urea

1-12 h after 5/~Ci oJ; l~C]urea

Area

Pons-medulla Midbrain Cerebral cortexhippocampus

0.034 13.3 45.0 0.047 16.1 57.0 0.039 10.5 212 0.038 9.10 239

0.102:k 0.021 9.85 0.071 i 0.032 14.1 0.083:~ 0.037 12.0 0.106-- 0.021 9.43 0.103 -: 0.027 9.71

k {perh)

215 69.0 80.0 389 189

0.022 ~ 0.025 ± 0.0080 ± 0.0042 5:

t½ (h) Turnow,r (mno&/g/h)

0.0035 0.0026 0.0019" 0.0017"

0.0060 5 0.0033* --

45.7 13.1 40.0 22.9 125 17.9 238 9.1 167

12.7

--

t½ (h)

0.699 ~ 0.027 1.43 0.752 ? 0.050 1.33 0.602~ 0.058 1.66

* Significantly different (P < O.OI) compared with cord and pons-medulla.

O f the total dose o f [14C]taurine injected, 54.7 :+: 1.7 ~ (mean -L- S.E.M. for 6 animals) was recovered as u n c h a n g e d taurine 0.25 h after injection, whereas 6 h after injection, 27.9 :~ 2.8 (mean ~ S.E.M. for 6 animals) of the initial dose was present. Between 6 h a n d 4 days after injection o f the labelled taurine, areas o f the central nervous system could be classified into two types depending on the rate of disappearance of the [~4C]taurine. In the midbrain, h y p o t h a l a m u s a n d olfactory bulbs (Table II), the efftux over this time period was n o t exponential but progressively slowed in a m a n n e r similar to that shown in Fig. 1 for the whole brain. F o r this reason, fractional rate constants could be n o t calculated. However, the rate o f disappearance o f radioactive taurine from the other areas was exponential and fractional rate constants were calculated. The rate o f disappearance o f [14C]taurine from the cervical cord and p o n s medulla regions gave half-lives o f 45.7 and 40.0 h respectively, whereas the values in cerebellum, striatum and cerebral cortex were in excess of 120 h (see Table ll).

454

O. O. S. COLLINS

It was possible that one or both of the efflux components was artefactual and, indeed, that the significant differences between the fractional rate constants of the slow second phase shown in Table I1 were the result of the experimental procedure employed. For these reasons, the rate of efftux of [14C]urea was measured from the pons-medul la, an area exhibiting a rapid second exponential efflux, the cerebral cortexhippocampus which exhibited a slow second exponential phase (t~ 167 h) and the midbrain in which efftux was non-exponential. The etttux of [14C]urea was followed between 1 and 12 h after injection but not beyond this time as levels of radioactivity were too low to be accurately assayed at later time intervals. The efflux of [t~C]urea in all 3 brain areas showed a single exponential phase there being no significant differences between the fractional rate constants (see Table 11). The rate of disappearance of labelled urea was very much more rapid than even the fastest efftux of [~C]taurine. Therefore, the differences in the disappearance of taurine from the areas investigated and the multiphasic nature of this release are unlikely to be artefacts. Formation of/HC]taurine from 14C cvstine and ,; taC/cysteine. An attempt was made to measure the ability of rat brain in vivo to metabolise [~C]cysteine and [14C]cystine into labelled taurine. The total radioactivity in the different brain areas after the intracisternal injection of the radioactive precursors was assumed to reflect their intracellular localisation. It was also assumed that the exogenous precursors freely equilibrated with their endogenous stores. In order that the differential accumulation of the precursors in the various brain areas would not limit the observed rate of formation of [~4C]taurine, the results have been expressed not as the absolute amounts of radioactive taurine formed but as percentages of the total radioactivity present. The in vivo rate of formation of [14C]taurine from labelled cystine and cysteine by whole rat brain is shown in Fig. 2. Rat central nervous tissue is seen to be able to metabolise both precursors into taurine, there being no signiticant preference for either substrate. Maximum [14C]taurine levels occur between I and 2 h

10

bJ U~ bJ

~z Q

O

/

m

Y

I

0

4

T I M E AFTER INJECTION (Hr$)

Fig. 2. The increase in [~4C]taurine content o f whole rat brain after the intracisternal injection o f 1.43/~Ci o f [14C]cysteine ( x ) or [l~Clcystine ((~). Each point is the mean of 5 experiments ::i S.E.M,

TAURINE METABOLISM IN RAT C N S

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TABLE I11 T H E F O R M A T I O N OF [ 1 4 C ] T A U R I N E

FROM [14C]CYSTEINE

AND

[IIC]('YSTINE

BY R A T C E N T R A l _ N E R V O U S

SYSTEMin vivo Groups of rats were injected intracisternally with either [l'~C]cysteine or [HC]cystine and the amount or [HC]taurine formed measured 0.25, 0.5, l, 2 and 4 h after injection. The values are the maximum amounts of [~4C]taurine formed expressed as a percentage of the total disint./min/g of the tissue under investigation and are the means ~ S.E.M. of between 4 and 6 observations. Area

Precursor 14C ) cystehw

Pons-medulla Hypothalamus Striatum Midbrain Cerebral cortex-hippocampus Olfactory bulbs Whole brain

8.6 5 0.79 6.8 L 0.39 12.1 ~ 1.36" 6.9 ~ 0.81 13.2 a 0.74* 18.2 I: 2.18" 8.1 : 0.92

[ 14Cj (')'stilze

8.8 7.1 13.3 7.47 12.7 20.3 8.5

~: 0.59 ~_ 0.85 i 1.48" :~ 0.69 L 0.98* k 2.78* ± 1.02

* Significantly different (P < 0.05) compared with pons-medulla, hypothalamus, midbrain and whole brain.

a f t e r i n j e c t i o n o f e i t h e r p r e c u r s o r . N o a t t e m p t was m a d e to identify a n y possible interm e d i a t e c o m p o u n d s f o r m e d . T h e rate o f f o r m a t i o n o f [l~C]taurine f r o m t h e t w o prec u r s o r s was also m e a s u r e d in v a r i o u s b r a i n areas ( T a b l e l II). A g a i n , t h e r e is no significant difference in the c o n v e r s i o n o f either cysteine o r cystine to taurine. H o w e v e r , there are significant differences b e t w e e n the p e r c e n t a g e c o n v e r s i o n o f p r e c u r s o r s to t a u r i n e in the v a r i o u s b r a i n areas. W h e n a g r a p h o f the e n d o g e n o u s t a u r i n e c o n t e n t is p l o t t e d a g a i n s t t h e p e r c e n t a g e o f total d i s i n t , / m i n / g p r e s e n t as [l~C]taurine, a d i r e c t r e l a t i o n s h i p is seen (Fig. 3).

8

O8 ~o /

/ tu 5

WB

z

/

/

STR

E

3 M8

2 I oD z o 123

z

uJ

HYP

1

0

2 4 6 8 10 12 14 16 18 °/o OF TOTAL D P M / g PRESENT

AS

[14C]TAURINE

Fig. 3. The relationship between the endogenous taurine content and the maximum amount of [~4C]taurine formed from [14C]cysteine ( × ) or [laC]cystine (O) in various areas of the rat brain. CC, cerebral cortex hippocampus; HYP, hypothalamus; MB, midbrain; OB, olfactory bulbs; P-M, pons-medulla; STR, striatum; WB, whole brain,

456

(~. (i. ~. COLLINS

DISCUSSION

There are at least 3 factors that may be involved in maintaining the high concentrations of free taurine present in the mammalian central nervous system. First, tissues might possess a particularly avid system for transport of taurine from extracellular to intracellular spaces. It is well known that at least two saturable transport systems for taurine exist - - the so-called fi- and co-systems23. The fi-system has a relatively low KM (approximately 50-60#M)20, 30 compared with the ,,system (approximately 6-10 mM) 29 and shows a greater structural specificity. [ndeed, it has previously been noted 26 that the fi-transport system for taurine is similar to that of GABA and glycine in areas where these compounds are thought to have a transmitter role 2~,22,-~'~. For this reason, the concentration o f taurine in the incubation medium of the present experiments was purposely kept low (1.31 #M). The results show that regional differences occur in the accumulation of taurine (see Table I) but that uptake does not correlate with the endogenous levels of taurine (see also Kandera et al.27). This suggests that the high affinity uptake system may only play a minor role in maintenance o f taurine levels. However, the limitations of these experiments must be stressed and it is clear that a detailed regional study of taurine uptake by the rat brain would be rewarding. Second, neural tissue might possess an active biosynthetic capacity lot taurine. There appear to be 5 possible pathways by which taurine may be formed l~c)a. Unfortunately, most of the reported in vivo investigations of taurine synthesis have been confined to peripheral tissues and those that have been extended to include the central nervous system did not exclude the possibility that taurine was formed in the periphery and thence transported into the brain. However, in a series of in vitro experiments, Peck and Awapara :~5 clearly demonstrated the ability of slices of rat cerebral cortex to metabolise methionine to taurine via cysteine, cysteine sulphinate and hypotaurine. The present experiments demonstrate that, in vivo, taurine may be formed from cysteine or cystine in the rat brain. In addition, there is a direct correlation between the activity of this biosynthetic pathway and the levels of taurine in the various areas of the central nervous system (see Fig. 3). However, it must be remembered that the above interpretation rests on a series of assumptions that may or may not be valid. First, it assumes that the pathway of conversion of cysteine to taurine is physiologically irreversible and second, that the [14C]cysteine and [14C]cystine accumulated by the brain in vivo have the same subcellular and biochemical distribution patterns as do the endogenous amino acids. Finally, the rate of formation of [14C]taurine is assumed to be very much greater than its rate of" metabolism (see below). It is not known whether these assumptions are justified in the present in vivo situation. If they are reasonable assumptions, the results would suggest that the rate o f formation oftaurine in the rat central nervous system may be a major factor in regulating tissue taurine levels, it is interesting to note that there is no direct relationship between cysteine sulphinate decarboxylase activity (the enzyme that catalyses the decarboxylation of cysteine sulphinate to hypotaurine) and taurine levels t whereas GABA levels in the brain area are a function of glutamate decarboxylase activity 5 (the enzyme that cata-

TAUR1NE METABOLISM 1N RAT C N S

457

lyses the decarboxylation of glutamate to GABA). Clearly, the decarboxylation of cysteine sulphinate is not rate-limiting in the synthesis of taurine. The third factor that would tend to conserve taurine levels would be a slow breakdown to isethionate, the major metabolite of taurine in the central nervous system. Although in the present experiments no effort was made to isolate any radioactive isethionate that might have been formed, the fact that in all areas of the central nervous system investigated the 'metabolite fraction' after injection of [14C]taurine constituted less than 6 ~ of the total radioactivity indicates either that breakdown of taurine in the brain is slow or that once formed, isethionate rapidly leaves the brain; this latter suggestion may be discarded, for Huxtable and Bressler 20 showed that the half-life of isethionate in whole rat brain was approximately 24 h. Peck and Awapara 35 even stated that the rate of metabolism of taurine to isethionate by slices of rat brain 'is at such a low rate that it has no significant effect upon taurine concentration'. However, such a slow rate of metabolism may itself aid conservation of the taurine. In an attempt to investigate the dynamic aspects of taurine metabolism, the rate of disappearance ofintracisternalty administered [HC]taurine has been measured. During the initial faster efflux, that is, between 0.25 and 4 h after injection of [14C]taurine, the fractional rate constants for all areas investigated were similar (see Table I1). However, because of the heterogeneous distribution of taurine, the calculated turnover rates varied from 389 nmoles/g/h in the olfactory bulbs to only 45.0 nmoles/g/h in the cervical cord. The slower efflux phase, between 6 h and 4 days after treatment, showed significant differences between the areas investigated. First, there were those tissues in which the rate of disappearance of [14C]taurine was non-exponential but progressively slowed with time. The second group of areas were those in which the rate of disappearance of [14C]taurine was exponential; this group could be further subdivided into those areas in which the half-life was less than 50 h (cervical cord and pons-medulla) and those in which t~ values exceeded 120 h (cerebellum, striatum and cerebral cortex-hippocampus). It is tempting to speculate that the nonexponential efflux from the midbrain hypothalamus and olfactory bulbs is the result of the presence of both the slow and faster second efflux phase exhibited by the other brain areas. In any case, the rate of turnover of taurine in the central nervous system is more rapid than that occurring in peripheral tissues where half-lives of between 288 and 312 h have been reported 4. On the other hand the half-life of GABA, another putative neurotransmitter substance, varies from only 0.51 to 4.72 h (see ref. 5). The significance o f the multiphasic disappearance of [14CJtaurine from the various areas of the rat central nervous system is difficult to assess. That the efflux of [~4CJurea is monophasic and is similar in all areas investigated (see Table 11), strongly suggests that the differences in taurine half-lives are real and not simply a reflection of the experimental procedure employed. The function taurine plays in the brain is unknown. Unlike other amino acid neurotransmitter candidates such as GABA and glutamate a,4°, taurine does not play a role in intermediary metabolism in the central nervous system; indeed, so far as is known, the only catabolic pathway for taurine in the brain is its conversion to isethionate a'5. The regional heterogeneity in taurine

458

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m e t a b o l i s m d e s c r i b e d in this p a p e r suggests a signilicant role l o r ' h e -~-~a~l~ aci~i. a l t h o u g h n o t necessarily as t h a i o f a n e u r o t r a n s m i t t e r substance. It i:, p~,:,sibic that t a u r i n e in the c e n t r a l n e r v o u s system is i m p o r t a n t o n l y as a s u b s t r a t e for the I'ormat~o~'~ o f i s e t h i o n a t e . T h e r e are high c o n c e n t r a t i o n s o f i s e t h i o n a t e in the s q u i d giant ~tXOl/>" and r e c e n t l y a c o n c e n t r a t i o n o f 3

4 / , m o l e s / g in the rat c e r e b r a l corte~, has i~cen

r e p o r t e d 19, In a d d i t i o n , in the presence o f a d r e n a l i n e , the rate o f synthesis ~t" b o t h t a u r i n e a n d i s e t h i o n a t e f r o m cystine in d o g h e a r t were increased ~md a c c o m p a n i e d by r e t e n t i o n o f K ~ w h e r e a s a c e t y l c h o l i n e d e p r e s s e d the c o n v e r s i o n *~. t! v, o u l d be useful to relate the t u r n o v e r o f t a u r i n e a n d its rate o f m e t a b o l i s m to iseth~ouate with the c o n c e n t r a t i o n s o f a c e t y l c h o l i n e a n d m o n o a m i n e

transmitter substances ~ithm

v a r i o u s r e g i o n s o f the c e n t r a l n e r v o u s system. Clearly, a detailed stud 3 ,,,]" regionai difference in the m e t a b o l i s m o f t a u r i n e is o f i m p o r t a n c e .

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