0022-3042/7?10701-033 1so2 00'0
Journol o j Nrumchrmisrr,, Vol. 33. pp. 331 to 337
Pergamon Press Ltd 1979. Printed in Great Britain 0 International Society for Neurochemistry Ltd
UPTAKE A N D RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS G. H. T. WHELER,'H.F.
A. N. DAVISON3 and E. J. THOMPSON3
'Laboratory of Developmental Neurobiologv, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, Maryland 20014, U.S.A. 'Biochemistry Department, Imperial College of Science and Technology, London, SW7 2A2, U.K. and 3Department of Neurochemistry, Institute of Neurology, London, WC1 3BG, U.K. (Received 3 Novrniher 1978. Rrvised 26 January 1979. Accepted 2 Frhrimry 1979)
Abstract-Cortex slices, synaptosomes and C-6 glioma cells were used to study [35S]taurine uptake and its electrically-stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41 of the particle-bound taurine and 16% of the total in the tissue. The uptake of [14C]GABA by C-6 glioma cells was inhibited 3-fold more by p-alanine than by L-DABA, whilst synaptosome preparations showed the opposite pattern, L-DABA being 2 or 3 times more effective than 8-alanine. [35S]Taurine uptake inhibition by L-DABA was low for synaptosomes and C-6 glioma. whereas j?-alanine showed considerable effect on C-6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with p-alanine. When 30 min rather than 5 min incubations were employed, j?-alanine depressed [35S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium-dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.
TAURINE is present in neural tissue at high concentrations (e.g. about 7-8pmol/g cortex in rats, LOMBARDINI, 1976) but the functions it may perform remain obscure, though its potent neuronal inhibitory properties suggest that one of its functions may be to serve as a neurotransmitter (CURTIS & WATKINS, 1960, 1965; OKAMOTO & QUASTEL, 1973). The biochemical properties associated with amino acid transmitter function should include a tissue capacity for high affinity uptake of the proposed transmitter and calcium-dependent release of the substance (IVERSENet a!., 1973). Inactivation by re-uptake could be expected as a special feature for taurine in view of its slow metabolism (PECK& AWAPARA,1967). Further, the differential actions of L-DABA and p-alanine on neuronal and glial uptake of GABA suggest a means of locating the compartments involved in taurine uptake, as 8-alanine is also a competitiveuptake inhibitor for taurine (LAHDESMAKI & OJA, 1973; KACZMAREK & DAVISON, 1972). In this paper we present further evidence for the presence in cerebral cortex of a stimulus-coupled taurine release process which is calcium-sensitive. In addition we have attempted to define the tissue com-
partments which are involved in these activities. Calcium is also shown to influence the uptake of taurine. EXPERIMENTAL PROCEDURES Cortex slices
Top slices of cerebral cortex (40-80 mg weight, 0.3 mm thickness) from female Sprague-Dawley rats (200-250 g body wt) held in Quick Transfer holders (MCILWAIN, 1975) were preincubated for 15min at 37°C in 5ml of Krehs bicarbonate medium and gassed with 0 , / C 0 2 (95:5, v/v). Krebs-bicarbonate has the following composition (mM): NaCI, 124; KCI, 5 ; K H zP 0 4 . 1.2; CaCI,. 0.75; MgSO?. 1.3; NaHCO,, 26.0; glucose, 10. They were then drained and incubated in 20 or 540pM-isotopic taurine (final specific radioactivity 48 mCi/mmol) in 5 ml of the same medium. After 5 min incubation the slice was removed in its holder, drained and rapidly rinsed twice by immersion in 100 ml incubation medium containing non-isotopic taurine at the same concentration (i.e. 20 or 5 4 0 ~ ~It) .was then transferred to 1.5 ml of 10% TCA, homogenized, centrifuged at 1400 y for 5 min and 1 ml was taken for radioactivity counting.
Subcellular fractionurion of slices ufrrr [35S]taurine uptukv
Top slices of cerebral cortex were taken as above. Individual slices were incubated in medium conpaining ~ ~ concentration for [%]taurine at 2 O p ~or 5 4 0 final To whom all reprint requests should be sent. 30min. They were then drained and rapidly rinsed in Abbreviutwns used: L-DABA, ~-2,4-diaminobutyrate; 0.32 M-SUCTOSe at room temperature before homogenization TCA, trichloracetic acid; PCA, perchloric acid; PBD, and subcelluar fraction by the method of GRAY& WHIT2-Phenyl-5-(4-biphenylyl)-1,3,4-oxadiazole; RSA, relative TAKER (1962) as modified by BRADFORD rf ul. (1973). Six specific activity. pooled slices were homogenized in 9 ml of 0.32 M-sucrose 331
332
G . H. T. WHELEK, H. F. BRADFORD. A. N. DAVISON and E. J. THOMIWN
at 5 C, with four up-and-down strokes repeated 3 times using a plastic homogenizer with a radial clearance of 2SOpM. The homogenate was then centrifuged at l O O O g for 10 rnin in a SO rotor in a Beckman L2 6 5 8 ultracentrifuge to yield a supernatant and a pellet (P,). The supernatant was carcfully removed with a Pasteur pipette and recentrifuged in the 50 rotor at 20,000 y for 20 rnin to yield the crude mitochondrial pellet (P,) and combined second supernatant and microsomes (S + P3). P, was resuspended in 2 ml of 0.3 M-sucrose at S’C by gentle homogenization. 1.6ml of the resuspended P 2 was taken and layered on top of a discontinuous gradient of 1.6 ml 0.8 M-sucrose above 1.6 ml 1.2 M-sucrose. The gradients were then centrifugcd in an SW39 head for I h at 76,OOOy at 5°C. Subsequently three fractions were collected: P,A, or myelin fraction at the boundary of the 0.32 M and 0.8 M-sucrose, PZB, or synaptosome fraction at the boundary of the 0.8 M and 1.2 M-sucrose, and P2C. the mitochondrial pellet. resuspended in 0.32 M-SUCTOSe. 0.8 ml of each fraction was added to 0.2ml 2S”/,TCA, centrifuged at 14009 for lOmin, and 1 ml of the supernatant was taken for liquid scintillation counting. Aliquots of each fraction were also taken for protein estimation (LOWRYet uf., 1951). All procedures during the centrifugation of the slices were done at & S T .
insoluble pellet was dissolved in 4.5 N - N ~ O Hand assayed for protein (LOWKY c>t ul.. 1951). C-6 glioinu rissur,. This was suspended in incubation medium using ten culture plates per 15 ml of mcdium. giving 0.9mg proteinjml. This was incubated in the same manner as for thc synaptosome suspensions. counted in the same way. and aliquots taken for protein estimation. Ependyinul (white rnutter) .slicc,s. These were prepared by placing one cerebral hemisphere. cortex downwards, on a moistened filter paper on a surgical block. An incision was made in the white matter uppermost to thc level of the lateral ventricle. The white matter was then folded open and thin slices were taken from the lining of the ventriclc (ependyma) with the aid of a McIlwain slicing guide with 4 0 fresh a recess set at 300 jtm. The slices obtained ( 3 0 ~ ~ mg wt) were rapidly transferred to incubation medium at room temperature and placed between the jaws of quick transfer holders. They were then incubated and treated as for cortex slices. Reltwse c~rprriinenfs
From rortc’x slices. Top slices of cortex wcre cut as dcscribed above and incubated for 30min in incubation medium containing calcium and isotopic taurine at 20 f I M final concentration. at 3 7 ’ C and gassed with 02/C0,(9S:S, v/v). They were then drained, rinsed 3 times C‘pruke studirs in thr presence of P-ulunine und L-DABA in medium containing non-isotopic taurine at 20 pM and The various tissue preparations. i.e. cortex slices, C-6 transferred t o another beaker containing 5 ml of medium glioma cells. ependymal slices and synaptosomes were in- at 37°C for 15 min. They were then transferred to a second cubated for 5 or 30 rnin with either p-alanine or L-DABA beaker and electrically stimulated for I S min. Electrical at a final concentration of 1 mM and with [35S]taurine stimulation was applied to each slice using the McIlwain at 20 PM. The incubation medium ( 5 ml) was at 37‘C and Quick Transfer holder. Stimulation was by application of square wave pulses alternating in polarity, of 0.4ms was gassed with O,/CO, (95:5, v/v). Individual tissue preparations were treated as follows: duration, SO Hz and of mean current 3CSO mA. Slices were then drained and transferred consecutively to two beakers Cortex slices. These were prepared as described above. After incubation slices were removed and rapidly rinsed containing incubation medium as before, for 15 min twice by immersion in IOOml of incubation medium con- periods. Slices were then rinsed rapidly 3 times in incubataining non-isotopic taurine at 20 PM. They were homogen- tion medium, and cach slice was homogenized in 3 ml of i7ed in 3 ml of 0.32 M-sucrose, and 0.6 rnl was added to 0.S M-PCA.The homogenate was centrifuged at 1400y for IOmin and 1 ml of the supernatant was takcn for liquid O.2ml of 20%TCA, centrifuged as described above and scintillation counting. taken for liquid scintillation counting. Some slices were incubated and stimulated in iso-osnioSpnuptosonit,suspensions and heds. The method for preparing the synaptosomes described was modified as fol- tic incubation medium without calcium ions but with 1 mM-EGTA present (neutralized to pH 7.4 with NaOH). lows. Whole cerebral cortex was homogenized at ice temperature as a 10% (w/v) suspension in 0.32 M-sucrose. The Other slices were pre-incubated in Ca2+-containing isotopic taurine medium. Subsequently these slices were et procedure was then as described elsewhere (BRADFORD ul., 1973). Synaptosomes were collected for incubation in rapidly rinsed 3 times in incubation medium containing Krehs-bicarbonate medium either as suspensions in glass no calcium but 1 mM-EGTA. They were then incubated vials or as synaptosome beds in O2/CO,(9S:5, v/v) at as before in calcium free medium for 15 min periods and 37 C. Synaptosome beds consist of deposits of synapto- stimulated as before during the second period (B). Srrhrrllulur fructions o j cor/vx slices. Cortex slices were somes sandwiched between nylon gauzes and were pre& BRAD- preincubated under the same general conditions as above pared as described previously (Dt BELLEROCHF. FORD 1972). in Krebs-bicarbonate medium containing calcium ions. Synaptosome beds were incubated as descl-;bed for They were stimulated for 20 rnin in 2 groups, one at 48 mA 30min. drained, rinsed twice by immersion in l00ml of mean current, and the other at 36 mA mean current using non-isotopic medium, and extracted in 10% TCA. Synapto- alternating square wave pulses as 5Oc.p.s. and 0.4ms some suspensions (2-3 mg protein/ml) were incubated duration. A t the end of the incubation period the slices under the same conditions. Samples of 0.5 ml were taken were rapidly rinsed 4 times in 0.32 M-sucrose at room temat 5, 10, 20 and 30min and centrifuged in a bench-top peraturc. They were then homogenized and subfracultracentrifuge at 18,000 y for 2 min. The supernatant was tionated as described for cortex slices above and were subremoved, and the synaptosome pellet was extracted with sequently extracted in TCA, aliquots of each fraction being 102, TCA, agitated on a vortex mixer, centrifuged for 5 min taken for liquid scintillation counting and protein estimaas above and the supernatant taken for liquid scintillation tion as described above. counting. The incubation medium above the synaptosome Liquid scinrillution counting. TCA supernatants of tissue pellet was also taken for radioactivity counting. The TCA samples (501. to I ml) were added to 19ml of toluene
333
Taurine uptake and release from cerebral cortex
T A B L1.~ U P T A K r O r
[24S]TAURINI
TO SUUCLLLLLAK FRACTIONS
nmol Taurine/g wet wt. of slice taken up/30 rnin 20 /cM-Taurine 540 p - T a u r i n e
Fraction
Protein content ~E/E
8796 k 1061 914 f 49 2460 f 796 5783 k 825 515 f 127 1145 If: 145 371 f 67
108.0 f 1.6 31.3 k 1.2 61.3 f 1.5 18.7 0.8 3.9 k 0.3 24.6 f 0.3 21.5 f 1.2
Relative specific activity
Percentage of nonsupernatant taurine
10.7 f 0.9 29.5 f 1.2 59.9 f 0.9 6.6 k 1.6 16.5 f 0.9 4.5 f 0.7
0.4 f 0. I0 0.5 f 0.02 3.6 f 0.10 1.9 +_ 0.40 0.8 f 0.03 0.3 f 0.03
26 k 2 74 f 2
10.6 1.1 24.7 f 0.8 64.8 f 0.6 5.6 5 0 . 8 12.9 k 0.9 4.5 If: 0.7
0.4 f 0.03 0.5 k 0.01 3.9 & 0.03 1.6 k 0.30 0.6 f 0.03 0.2 f 0.03
30 f 3 70 k 3
495 f 9 54 f 4 156 f 30 314 f 50 37 f 14 83 k 19 23 f 3 Pcrcentagc recovery
17f4 41 f 2 11 1 2
16 i 3 37 f 2 13 f 2
Results are ~ s . L . M .Relative specific activity is percentage of taurine uptake/percentage of protein in the fraction. Results are for four separate estimations. Key to table: H. homogenate: P , , nuclear pellet: P,, crude mitochondrial pellet: P, + S, microsomes and soluble supernatant: PZA, myelin fraction: P2B, synaptosome fraction; P,C, mitochondria1 pellet.
PBD-2-methoxy ethanol used in the ratio of 5:4. vjv, and counted on a LS-200B Beckman Liquid Scintillation counter. Quench correction was achieved using an internal standard. Sources for materials used were: [35S]taurine. Amersham, U.K , L-DABA, Sigma: EGTA, Sigma.
RESULTS
Uprakc. of [35S]taurine into suhcrllular coriipartnients of intact cortex slices T a b l e I shows the relative recovery of [35S]taurine
in different subcellular fractions following the incubation of cortex slices in isotopic taurine at 20 and 540 PM. C3'S]Taurine content of the different fractions is given as a percentage of the total isotope present in the homogenate. The [35S]taurine content varied widely among the different fractions. At 20 pwtaurine most of the [35S]taurine was recovered in the microsomes and soluble supernatant (S, + P3), and the crude mitochondrial pellet (P,) took up the next highest percentage of taurine. Within the crude mitochondrial pellet the synaptosome fraction of P,B was most enriched. A similar pattern was obtained at 5 4 0 p ~ , although in this case a smaller proportion of [3'S]taurine was found in the synaptosomal fraction, and more was recovered in the soluble supernatant fraction.
When protein content was taken into account a similar distribution of [35S]taurine was observed (RSA). The only difference was that the myelin fraction (P,A) showed a higher [35S]taurine content than before, possibly reflecting leakage of from synaptosomes. When the [35S]taurine uptake \/as described as a percentage of the sedimentable o r particle bound [35S]taurine, 41% was found to occur in the , 37% at 540 FM. synaptosomal fraction at 20 p ~ and Recoveries of c.p.m. and protein were 103% and 93% respectively. Taurinci und GABA uptake in the presence of ~-aluiiinc~ and dianiinohutyrate
Figure 1 shows the effect of b-alanine (I mM) on ) by preparations of differing taurine (20 p ~ uptake neuronal and glial content, and by synaptosome preparations. Results are from incubation periods of 30 rnin and 5 min. After 30 rnin incubation it is seen that /3-alanine depressed by 50% the uptake of [35S]taurine by white matter slices (ependyma). This may be compared with a 30% and 28% inhibition of this uptake by cortex slices and synaptosome beds respectively. After 5 min incubation, taurine (20 PM) uptake is not depressed for synaptosome suspensions. but C-6 glioma suspensions showed a 41% inhibition under these conditions. L-DABA (1 mM) produced little inhibition of taurine uptake by synaptosome suspensions
G. H. T. W H L L I RH. , F. BRADFORD, A. N. DAVISON and E. J. THOMPSON
334
or by C-6 glioma suspensions over an incubation period of 5min. Figure 2 shows 5min uptake periods for [I4C]GABA (20 p ~ in) the presence of p-alanine (1 mM) or L-DABA (1 mM). For synaptosome suspensions L-DABA depressed GABA uptake by 84%. However, in C-6 glioma suspensions uptake is only depressed by 13%. 8-Alanine depressed [I4C]GABA uptake by 26% in synaptosome suspensions, and by 39% in C-6 glioma suspensions. Thus fl-alanine and L-DABA affect the two preparations differently. 13-alanine having its main effect on uptake by f - 6 glioma suspensions and L-DABA having its main effect on uptake by synaptosome suspensions.
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Culciuni dependence of [35S]tauriiw rrleasr from cortex slices
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FIG. I . The uptake of ["S]taurine t o different tissue samples in the presence of 1 mM-L-DABA (0)and 1 mM-fialanine (H). The time of incubation is described below. Four to ten measurements were made for each value. VertiAhhreriurions used: A, synaptosome cal bars indicate s.L..M. beds: B. C6-glioma (both 5 min in incubation); C, cortex slices: D. synaptosome beds; E. ependymal slices (all 30 min incubations). Control uptake was in the range 5@~80 nmol taurine/g wet wt.
Figure 3 shows the efRux of preloaded [35S]taurine from incubated cerebral cortex slices. Points A. B, C and D represent 15min periods of [35S]ta~irine efflux into consecutive beakers containing taurine-free incubation medium f Ca2+ and EGTA. The proportion of total [35S]taurine recovered in the medium after incubation for 1 h with or without applied electrical stimulation was high (approx 40%). This suggests that either a large proportion of the total tissue content of taurine is released or that both uptake and release occur largely from a special tissue compartment.
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FIG. 2. Comparison of the uptake of [14C]GABA to synaptosomal suspensions and C6-glioma suspensions over a 5 min pcriod of incubation, in the presence of 1 mM-LDABA (0) or I mM-/l-alanine (W). Each histogram is the mean of four to ten samples. Vertical bars indicate S.F.M. A h h r w i u t i o m u s c d : A, synaptosome suspensions; B, C6-glioma.
rnin
...--.-.--,
on electrically stimukdled [3sS]taurine release. Electrical stimulation was for I5 min (period B). Symbols used are: ---tc,electrically stimulated slices in medium containing Ca2+; control slices in same medium; ' . . . .. slices preloaded in Ca2'-free (EGTA containing) medium and subsequently electrically stimulated in Ca' '-free medium. Vertical lines are S.E.M. from five or more separate measurements. ["SITaurine efflux was calculated as nmol using the specific radioactivity of ["Sltaurine prescnt in the incubation medium used for preloading the slices.
335
Taurine uptake and release from cerebral cortex TABLI2.
[35S]TAURlNL
I N SUBFRACTIONS OF CORTEX SLlCtS AND INCUBATION MFIIIUM INE/g WET WT TISSUE)
Fraction
Release into incubation medium
(nm0l [3ss]TAUR-
Control
Stimulated
Difference
25.7 f 1.7 132.0 f 10.9 150.0 f 4.4 21.3 f 3.3 78.9 f 6.4 16.3 rt 1.7
25.8 f 1.9 99.7 f 10.6 143.2 f 3.3 22.1 f 1.0 55.6 f 7.0 15.8 f 1.6
- 32.3% - 6.8$ + 0.8t - 23.3t -0.5t
11.6
113.2 _+ 14.5
+ 25.3
88.1
+0.1$
* 93% recovery of homogenate. Slices were stimulated at 36 mA mean current for 20 min, in the presence of C a z + .Released [j*S]taurine is corrected for levels in control medium. Difference significant with t P < 0.05 or $ P < 0.01. Results are ~ s . E . M . Each result is the mean of 4 separate estimations. Change in [35S]taurine content of slice suh-conipartiwnrs during electrical stiniu/ation
Table 2 shows the comparison of the changes in content of the subcellular compartments of slices electrically stimulated at 36 mA mean current. The changes in the synaptosome fraction of 23.3 nmol/g of [35S]taurine was commensurate with the increase in [35S]taurine recovered in the incubation mediums, which was equivalent to 25.3 nmol and was significant ( P < 0.05). The other fraction showing most change was the soluble cytoplasmic fraction and microsomes (S P3), in which there was a much smaller decrease (6.8 nmol/g), but this was not significant ( P < 0.1). At higher currents (48 mA) the soluble fraction showed a 10-fold increase in its loss of [35S]taurine and exceeded the synaptosome fraction in magnitude by a factor of two. Thus at the lower current the synaptic terminal compartment of the tissue could be contributing the larger proportion of released [35S]taurine, though re-uptake during the period of the experiment would have diminished the absolute amounts in the medium.
+
DISCUSSION Taurine uptake to cortex slices
The studies of KACZMERAK & DAVISON(1972); & OJA(1973); LAHDESMAKI et al. (1975) LAHDESMAKI and LOMBARDINI(1977) have shown that taurine uptake to cortex slices is a complex process with saturable and unsaturable components. There appear to be both high (5&60 PM) and low (6 mM) affinity saturable systems at work. The nature of the tissue compartments taking up taurine remain uncertain, but glial cells in retina (EHINGER,1973) or in culture, C-6 glioma (SCHRIER& THOMPSON,1974) and neuroblastoma cells appear to do so readily. It seems likely therefore that both newones and glial cells of cortex slices will be actively accumulating taurine. The finding that among the sub-fractions of the crude mitochondria1 fraction, the synaptosomes carried the greatest content of C3%]taurine indicates that
the presynaptic region of the slice does accummulate taurine. This probably represents true uptake, as recent studies in which taurine has been added to homogenate have shown that there is no significant uptake of taurine by subcellular fractions during the subsequent subfractionation procedure (SIEGHART & KAROBATH, 1974: RASSINet a/., 1977). The enzyme producing taurine (cysteinesulphinate decarboxylase. EC 4.1.1.29) has a synaptic localization (AGRAWALet a/., 1971) and synaptic vesicles appear to be enriched in taurine (DEBELLEROCHE & BRADFORD, 1973; RASSIN or a/.. 1977). In addition, evidence has been published for the existence of a synaptosomal sub-population which accumulates [35S]taurine (SIEGHART & KAROBATH, 1974; SIEGHART & HECKL,1976). The large proportion of taurine recovered in the soluble cytoplasmic fraction presumably reflects the considerable amount of uptake by glial and neuronal cell bodies. In order to differentiate between taurine uptake to glial and neuronal elements in the slice we used p-alanine as a blocker for taurine uptake. Recent experiments have shown that p-alanine specifically enters glial cells in the brain cortex (SCHON& KELLY, 1975; IVERSEN & BLOOM,1972); moreover, it competes with the uptake of taurine, and also with the uptake of GABA. L-DABA, on the other hand, is specifically taken up into nerve-endings (DICK& KELLY,1975) and competes with GABA uptake (TVERSEN & JOHNSTON, 1971). Hence we compared the affects of p-alanine and L-DABA on taurine and GABA high affinity uptake. It was notable that p-alanine depressed taurine uptake over a 5min period far more in glial cells (C-6 glioma) than in synaptosome preparations, on which it had little effect. Uptake was depressed by 50% or more in ependymal slices over a longer period (30 min) when synaptosomes and cortex slices were also showing a substantial (28-30%) reduction in their taurine uptake. LAHDESMAKI & OJA (1973) have reported a similar potency of action of b-alanine on taurine uptake to cortex slices under approximately equivalent conditions. Ependymal and cortex slices contain both glial and neuronal elements (nerve fibres in ependymal slices)
336
G . H.T.
WHtLLR.
H. F. BRADFORD, A. N. DAVISON and E. J. THOMI’SON
though the latter are likely to be more enriched in neuronal components than the former. Thus, only relative differences can be expected between the two kinds of preparation. With these results in mind, and arguing by analogy with GABA transport systems (see below), the relatively greater potency of p-alanine in blocking taurine uptake to preparations richer in glial than in neuronal elements could well indicate that taurine is entering mainly synaptosomes themselves rather than any cytoplasmic particles of glial origin present in synaptosome preparations.
(IVERSEN& KELLY,1975). Also, the fraction studied by SCHMIDet a / . (1975) was not a pure synaptosomal preparation, but a crude fraction which contained other elements including mitochondria (P2). Release experirwnts
Release of transmitter compounds has been shown to occur from both neurons and glial cells (BOWI-RY & BROWN.1972) though glial cells do not show Ca” dependence in their release (SELLSTROM & HAMBERGER, 1977) or atypical Ca” dependence (MINCHIN & IVERSEN. 1974). Thus, C a 2 + dependence has become an important criterion in distinguishing G A BA uptakr between release from the two cell types. Non-calcium The uptake of GABA to synaptosomes and C-6 dependent release of putative amino acid transmitters glioma was compared (Figure 2) in the presence of including glutamate has been observed to occur from & I mM-p-alanine or 1 mM-L-DABA in an attempt to electrically stimulated nerve trunks (WEINREICH 1975), and K + stimulation caused gain a measure of the glial contamination of synapto- HAMMERSCHLAG, ~ some fractions. Since L-DABA depressed synaptoso- similar release of GABA from glia ( B o w r : ~& ma1 GABA uptake by 84% but had little effect on BROWN,1972). Previous studies have shown that elecreducing C-6 glioma GABA uptake, synaptosome trical stimulation can release taurine from brain slices preparations would appear to contain little glial con- and that it is not metabolised during short term incutamination. The inverse pattern was obtained using bation and electrical stimulation experiments (KACZ/I-alanine. Thus, this compound depressed C-6 glioma MAREK & DAVISON, 1972). uptake of GABA to a greater extent (40%) than to The release of [3’S]taurine into the medium by the synaptosomal suspension (27%) when 5 min incu- electrical stimulation reported here was Ca*+-depenbation periods were used. With longer periods of in- dent. Since stimulus-induced release of taurine from cubation (30min) the same pattern of depression of slices was higher in calcium-free medium than passive synaptosomal uptake by L-DABA and 8-alanine was release from control slices in the same medium, it maintained (Fig. 2). These results suggest that the is possible that some endogenous Ca2+ is still availsynaptosome preparation is mainly of neuronal origin able for the process. Alternatively this non Ca2 and is not heavily contaminated with cytoplasmic dependent efflux of [35S]taurine could be from glial bodies of glial origin. R t U B U R N (1978) obtained simi- cells. lar relative potencies of L-DABA and /I-alanine inhiSlices which were stimulated electrically and then bition of [14C]GABA uptake to synaptosomes. subfractionated to locate the pool of taurine respon& HAM- sible for stimulated release showed two main results. L-DABA has been reported (SELLSTROM H ~ R G E R 1975) , to inhibit GABA uptake to a cortical First, at the lower stimulating current of 36mA the glial preparation to the same extent (50%) that it electrically stimulated release was mainly from the blocks GABA uptake to synaptosome preparations, synaptosomal pool. Second, at higher currents which might be thought to conflict with the above (48mA) release of taurine occurred from other fracassessment of the purity of synaptosome preparations. tions, including the soluble supernatant. Thus, in However, autoradiographic studies of cerebral cortex order to stimulate release specifically from synaptic uptake and of sympathetic ganglia show DABA local- regions stimulation must not be too extensive. ized (retained) in neuronal elements, mainly nerve Change in the soluble supernatant fraction may rependings (DICK& KELLY, 1975). Also, the IC,, resent release from other tissue components such as reported for DABA in blocking GABA uptake to the glial cells or neuronal cell bodies. glial cells of sympathetic ganglia (0.7 mM) is much higher than its counterpart for cortex slices ( 5 0 ~ ~Acknowledynttents-This ) work was supported by an MRC (IVERSEN& KELLY,1975). For these reasons it seems project grant. likely that the glial preparation of SELLsmoM & HAMRERGER (1975) contained synaptosomes as contaminants. It has been reported that p-alanine blocks REFERENCES GABA and taurine uptake to synaptosomes by et a/., 1973; SCHMIDet al., 1975) 6G80% (SNODGRASS A. N. & KACZMAREK L. K. but this does not correlate with the autoradiographic ACRAWAL H. C., DAVISON (1971) Subcellular distribution of taurine and cysteinsulevidence which implies specific uptake of 8-alanine phinate decarboxylase in developing rat brain. Biochrm to glial cells. This was also indicated by the greater J. 122, 759-163. effectiveness of p-alanine in blocking GABA uptake BOWERYN. G . & BROWND. A. (1972) y-Aminobutyric to ganglia rather than cortex slices, the IC5,values acid uptake by sympathetic ganglia. Nature, N e w Biol. for this inhibition being 120 PM and 2 mM, respectively 238, 89-91. +
Taurine uptake and release from cerebral cortex G . W. & THOMAS A. J. (1973) BKAI)FORD H. F., BENNETT Dcpolarizing stimuli and thc rclease of physiologically active amino acids from suspensions of mammalian synwptosomes. J . Noirrocherii. 21. 495S505. J. C. (1960) The excitation and CURTIS D. R. & WATRINS dcpression of spinal neurones by structurally related amino acids. J . Neurochrrii. 6, 1 177141. J. C. (1965) The pharmacology CURTISD. R. & WATKINS of amino acids related to gamma-aminobutyric acid. Phurriiuc. K r r . 17. 3 4 7 ~391. J. S. & BRAIXWRI) H. F. (1972) MetaboDE BI-LLLROCHI. lism of beds of mammalian cortical synaptosomes: response to depolarizing influences. J . Neurochnri. 19. 585 602. J . S. & BRAI)FORD H. F. (1973) Amino DE BLLLIWWCHI: acids in synaptic vesicles from mammalian cerebral cortex: a reappraisal. J . Nrurochrrri. 21, 441-451. DICKF. & KELLYJ. S. (1975) L-2.4-diaminobutyric acid (i.-DABA) a s a selcctivc marker for inhibitory nerve terminals in rat brain. Br. J . Phurriiuc. 53. 439. EkiK1NGi.R B. (1973) Glial uptake of taurine in the rabbit rctina. Bruin Res. 60. 512-516. V. P. (1962) The isolation of G R A YE. G. & WHITTAKIR nerve endings from brain ; an clectron-microscopic study of cell fragments derived by homogenisation and centrifugation. J . Aiiur. %. 79-88. IVI.RS[:N L. L. & BLOOMF. E. (1972) Studies of the uptake of I13H]GABA and [3H]glycine in slices and homogcnates of rat brain and spinal cord by electron microscopic autoradiography. Brriiri R c r s . 41. 131 143. IVEKSEN L. L. & J(1HNSTON G. A. R . (1971) GABA uptake in rat central nervous system: comparison of uptake in sliccs and homogenates and the effects of some inhibitors. J . Ntwrochrrii. 18, 1939 1950. IVEKSEN L. L. & KELLYJ. S. (1975) Uptake and metabolism of p-aminobutyric acid by neurones and glial cells. Bioc-him. Phurriiuc. 24, 933- 938. IVEKSEN L. L., KI.LLY J. S., MINCHINM., SCHONF. & SNOIXRASS S. R. (1973) Role of amino acids and peptides in synaptic transmisstion. Bruin R e x 62. 567-576. A. N. (1972) Uptake and KA(ZMARI:K L. K. & DAVISON rclease of taurine from rat brain slices. J . Neurochern. 19. 2355-2362. LAHIXSMAKI P. & OJAS. S. (1973) O n the mechanism of taurine transport at brain cell membranes. J . Nrurochern. 20. 1411-1417. LAHIIESMAKI P., PASULAM. & OJAS. S. (1975) Effect of clectrical stimulation and chlorpromazine on the uptake and release of taurine. GABA and glutamic acid in mouse brain synaptosomes. J . Nrurochrm. 25, 675-680. o M i i A R i m i 3. B. (1976) Regional and subce~iukdrstudies R. on taurine in the rat CNS, in Tuurine (HUXTABLE & BARREArJ A.. eds), pp.311-326. Raven Press, New York. .OMHARI)INI J. B. (1977) High affinity uptake systems for taurine in tissue slices and synaptosome fractions pre-
337
pared from various regions of the rat CNS. Correction of transport data by different experimental procedures. 1. Nerrmchrn7. 29, 305-3 12. LOWRY0. H., ROSI'IIROUGH N. J., FARRA. L. & RANDALL R. J. (1951) Protein measurement with the Fohn phenol reagent. J . bid. Cherii. 193. 265-275. M C ~ L W A IH. N (1975) Metabolic experiments with neural tissues, in P rnctical Neurochemistry (MCILWAINH., ed.) p. 150. Churchill Livingstone. MINCHINM. C. W. & IVERSENL. L. (1974) Release ol [3H]gamma-aminobutyric acid from glial cell in rat dorsal root ganglia. J . Neurochrrii. 23. 533-541. OKAMOTO K. & QUASTEL J. H. (1973) Spontaneous action potentials in isolated guinea pig cerebellar sliccs; efectr of amino acids and conditions affecting sodium and water uptake. Proc. R . SOC. B. 184. 83-90. PECKE. J. & AWAPARA J. (1967) Formation of taurine and isethionic acid in rat brain. Biochirn. biophys. Actu 141. 499-506. RASSIND. K.. STURMAN J. A. & G A U L LG . E. (1977) Taurine in developing rat brain; subcellular distribution and in association with synaptic vesicles of ["S]taurine maternal. fetal and neonatal rat brain. J . Nc,urocheiii. 28. 41-50. R I IXJURN D. A. (1978) Relationship between synaptosomal uptake and release of ['4C]GABA; [14C]diaminobutyric acid and ['4C]j-alanine. J . Nc~urochcwi. 31. 939-946. SCHMIUR., SIEGHART W. & KAHOBATH M. (1975) Taurinc uptake in synaptosomal fractions of rat cerebral cortex. J . Neurocherli. 25. 5-9. SCHONF. & KELLYJ. S. (1975) Selective uptake of [-'HI/]ssociation with the glial uptake system for GABA. Bruin Res. 86. 243-257. E. J. (1974) O n the role of SCHRERN. K. & THOMPSON glial cells in the mammalian nervous system. J . hiol. Cherii. 249, 1769-1 780. A. & HAMBI:RGI:R A. (1975) Neuronal and glial SELLSTROM systems for 7-aminobutyric acid transport. J . Nrurocherii. 24, 847-852. SELLSTROM A. & HAMBtRGER A. (1977) Potassium-stimulated y-aminobutyric acid release from neurons and glia. Brain Res. 119. 189-198. SIECHART W. & HECKLK. (1976) Potassium-evoked release of taurine from synaptosomal fractions of rat cerebral cortex. Bruiri Res. 116, 538-543. M. (1974) Evidence for specific SIECHART W. & KARORATH synaptosomal localization of exogenous accumulated taurine. J . Nrurocherii. 23. 91 1-915. S. R.. HEVLEY-WHYTE T. E. & LORENZO A. V. SNOVGRASS (1973) GABA transport by nerve ending-fractions of rat brain. J . Nrurocherii. 20. 771-782. D. & HAMMERSCHLAC R. (1975) Nerve impulseWEINREICH enhanced release of amino acids from non-synaptic regions of peripheral and central nerve trunks of bullfrog. Bruin Res. 84, 137-142.
Journol o j Nrumchrmisrr,, Vol. 33. pp. 331 to 337
Pergamon Press Ltd 1979. Printed in Great Britain 0 International Society for Neurochemistry Ltd
UPTAKE A N D RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS G. H. T. WHELER,'H.F.
A. N. DAVISON3 and E. J. THOMPSON3
'Laboratory of Developmental Neurobiologv, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, Maryland 20014, U.S.A. 'Biochemistry Department, Imperial College of Science and Technology, London, SW7 2A2, U.K. and 3Department of Neurochemistry, Institute of Neurology, London, WC1 3BG, U.K. (Received 3 Novrniher 1978. Rrvised 26 January 1979. Accepted 2 Frhrimry 1979)
Abstract-Cortex slices, synaptosomes and C-6 glioma cells were used to study [35S]taurine uptake and its electrically-stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41 of the particle-bound taurine and 16% of the total in the tissue. The uptake of [14C]GABA by C-6 glioma cells was inhibited 3-fold more by p-alanine than by L-DABA, whilst synaptosome preparations showed the opposite pattern, L-DABA being 2 or 3 times more effective than 8-alanine. [35S]Taurine uptake inhibition by L-DABA was low for synaptosomes and C-6 glioma. whereas j?-alanine showed considerable effect on C-6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with p-alanine. When 30 min rather than 5 min incubations were employed, j?-alanine depressed [35S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium-dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.
TAURINE is present in neural tissue at high concentrations (e.g. about 7-8pmol/g cortex in rats, LOMBARDINI, 1976) but the functions it may perform remain obscure, though its potent neuronal inhibitory properties suggest that one of its functions may be to serve as a neurotransmitter (CURTIS & WATKINS, 1960, 1965; OKAMOTO & QUASTEL, 1973). The biochemical properties associated with amino acid transmitter function should include a tissue capacity for high affinity uptake of the proposed transmitter and calcium-dependent release of the substance (IVERSENet a!., 1973). Inactivation by re-uptake could be expected as a special feature for taurine in view of its slow metabolism (PECK& AWAPARA,1967). Further, the differential actions of L-DABA and p-alanine on neuronal and glial uptake of GABA suggest a means of locating the compartments involved in taurine uptake, as 8-alanine is also a competitiveuptake inhibitor for taurine (LAHDESMAKI & OJA, 1973; KACZMAREK & DAVISON, 1972). In this paper we present further evidence for the presence in cerebral cortex of a stimulus-coupled taurine release process which is calcium-sensitive. In addition we have attempted to define the tissue com-
partments which are involved in these activities. Calcium is also shown to influence the uptake of taurine. EXPERIMENTAL PROCEDURES Cortex slices
Top slices of cerebral cortex (40-80 mg weight, 0.3 mm thickness) from female Sprague-Dawley rats (200-250 g body wt) held in Quick Transfer holders (MCILWAIN, 1975) were preincubated for 15min at 37°C in 5ml of Krehs bicarbonate medium and gassed with 0 , / C 0 2 (95:5, v/v). Krebs-bicarbonate has the following composition (mM): NaCI, 124; KCI, 5 ; K H zP 0 4 . 1.2; CaCI,. 0.75; MgSO?. 1.3; NaHCO,, 26.0; glucose, 10. They were then drained and incubated in 20 or 540pM-isotopic taurine (final specific radioactivity 48 mCi/mmol) in 5 ml of the same medium. After 5 min incubation the slice was removed in its holder, drained and rapidly rinsed twice by immersion in 100 ml incubation medium containing non-isotopic taurine at the same concentration (i.e. 20 or 5 4 0 ~ ~It) .was then transferred to 1.5 ml of 10% TCA, homogenized, centrifuged at 1400 y for 5 min and 1 ml was taken for radioactivity counting.
Subcellular fractionurion of slices ufrrr [35S]taurine uptukv
Top slices of cerebral cortex were taken as above. Individual slices were incubated in medium conpaining ~ ~ concentration for [%]taurine at 2 O p ~or 5 4 0 final To whom all reprint requests should be sent. 30min. They were then drained and rapidly rinsed in Abbreviutwns used: L-DABA, ~-2,4-diaminobutyrate; 0.32 M-SUCTOSe at room temperature before homogenization TCA, trichloracetic acid; PCA, perchloric acid; PBD, and subcelluar fraction by the method of GRAY& WHIT2-Phenyl-5-(4-biphenylyl)-1,3,4-oxadiazole; RSA, relative TAKER (1962) as modified by BRADFORD rf ul. (1973). Six specific activity. pooled slices were homogenized in 9 ml of 0.32 M-sucrose 331
332
G . H. T. WHELEK, H. F. BRADFORD. A. N. DAVISON and E. J. THOMIWN
at 5 C, with four up-and-down strokes repeated 3 times using a plastic homogenizer with a radial clearance of 2SOpM. The homogenate was then centrifuged at l O O O g for 10 rnin in a SO rotor in a Beckman L2 6 5 8 ultracentrifuge to yield a supernatant and a pellet (P,). The supernatant was carcfully removed with a Pasteur pipette and recentrifuged in the 50 rotor at 20,000 y for 20 rnin to yield the crude mitochondrial pellet (P,) and combined second supernatant and microsomes (S + P3). P, was resuspended in 2 ml of 0.3 M-sucrose at S’C by gentle homogenization. 1.6ml of the resuspended P 2 was taken and layered on top of a discontinuous gradient of 1.6 ml 0.8 M-sucrose above 1.6 ml 1.2 M-sucrose. The gradients were then centrifugcd in an SW39 head for I h at 76,OOOy at 5°C. Subsequently three fractions were collected: P,A, or myelin fraction at the boundary of the 0.32 M and 0.8 M-sucrose, PZB, or synaptosome fraction at the boundary of the 0.8 M and 1.2 M-sucrose, and P2C. the mitochondrial pellet. resuspended in 0.32 M-SUCTOSe. 0.8 ml of each fraction was added to 0.2ml 2S”/,TCA, centrifuged at 14009 for lOmin, and 1 ml of the supernatant was taken for liquid scintillation counting. Aliquots of each fraction were also taken for protein estimation (LOWRYet uf., 1951). All procedures during the centrifugation of the slices were done at & S T .
insoluble pellet was dissolved in 4.5 N - N ~ O Hand assayed for protein (LOWKY c>t ul.. 1951). C-6 glioinu rissur,. This was suspended in incubation medium using ten culture plates per 15 ml of mcdium. giving 0.9mg proteinjml. This was incubated in the same manner as for thc synaptosome suspensions. counted in the same way. and aliquots taken for protein estimation. Ependyinul (white rnutter) .slicc,s. These were prepared by placing one cerebral hemisphere. cortex downwards, on a moistened filter paper on a surgical block. An incision was made in the white matter uppermost to thc level of the lateral ventricle. The white matter was then folded open and thin slices were taken from the lining of the ventriclc (ependyma) with the aid of a McIlwain slicing guide with 4 0 fresh a recess set at 300 jtm. The slices obtained ( 3 0 ~ ~ mg wt) were rapidly transferred to incubation medium at room temperature and placed between the jaws of quick transfer holders. They were then incubated and treated as for cortex slices. Reltwse c~rprriinenfs
From rortc’x slices. Top slices of cortex wcre cut as dcscribed above and incubated for 30min in incubation medium containing calcium and isotopic taurine at 20 f I M final concentration. at 3 7 ’ C and gassed with 02/C0,(9S:S, v/v). They were then drained, rinsed 3 times C‘pruke studirs in thr presence of P-ulunine und L-DABA in medium containing non-isotopic taurine at 20 pM and The various tissue preparations. i.e. cortex slices, C-6 transferred t o another beaker containing 5 ml of medium glioma cells. ependymal slices and synaptosomes were in- at 37°C for 15 min. They were then transferred to a second cubated for 5 or 30 rnin with either p-alanine or L-DABA beaker and electrically stimulated for I S min. Electrical at a final concentration of 1 mM and with [35S]taurine stimulation was applied to each slice using the McIlwain at 20 PM. The incubation medium ( 5 ml) was at 37‘C and Quick Transfer holder. Stimulation was by application of square wave pulses alternating in polarity, of 0.4ms was gassed with O,/CO, (95:5, v/v). Individual tissue preparations were treated as follows: duration, SO Hz and of mean current 3CSO mA. Slices were then drained and transferred consecutively to two beakers Cortex slices. These were prepared as described above. After incubation slices were removed and rapidly rinsed containing incubation medium as before, for 15 min twice by immersion in IOOml of incubation medium con- periods. Slices were then rinsed rapidly 3 times in incubataining non-isotopic taurine at 20 PM. They were homogen- tion medium, and cach slice was homogenized in 3 ml of i7ed in 3 ml of 0.32 M-sucrose, and 0.6 rnl was added to 0.S M-PCA.The homogenate was centrifuged at 1400y for IOmin and 1 ml of the supernatant was takcn for liquid O.2ml of 20%TCA, centrifuged as described above and scintillation counting. taken for liquid scintillation counting. Some slices were incubated and stimulated in iso-osnioSpnuptosonit,suspensions and heds. The method for preparing the synaptosomes described was modified as fol- tic incubation medium without calcium ions but with 1 mM-EGTA present (neutralized to pH 7.4 with NaOH). lows. Whole cerebral cortex was homogenized at ice temperature as a 10% (w/v) suspension in 0.32 M-sucrose. The Other slices were pre-incubated in Ca2+-containing isotopic taurine medium. Subsequently these slices were et procedure was then as described elsewhere (BRADFORD ul., 1973). Synaptosomes were collected for incubation in rapidly rinsed 3 times in incubation medium containing Krehs-bicarbonate medium either as suspensions in glass no calcium but 1 mM-EGTA. They were then incubated vials or as synaptosome beds in O2/CO,(9S:5, v/v) at as before in calcium free medium for 15 min periods and 37 C. Synaptosome beds consist of deposits of synapto- stimulated as before during the second period (B). Srrhrrllulur fructions o j cor/vx slices. Cortex slices were somes sandwiched between nylon gauzes and were pre& BRAD- preincubated under the same general conditions as above pared as described previously (Dt BELLEROCHF. FORD 1972). in Krebs-bicarbonate medium containing calcium ions. Synaptosome beds were incubated as descl-;bed for They were stimulated for 20 rnin in 2 groups, one at 48 mA 30min. drained, rinsed twice by immersion in l00ml of mean current, and the other at 36 mA mean current using non-isotopic medium, and extracted in 10% TCA. Synapto- alternating square wave pulses as 5Oc.p.s. and 0.4ms some suspensions (2-3 mg protein/ml) were incubated duration. A t the end of the incubation period the slices under the same conditions. Samples of 0.5 ml were taken were rapidly rinsed 4 times in 0.32 M-sucrose at room temat 5, 10, 20 and 30min and centrifuged in a bench-top peraturc. They were then homogenized and subfracultracentrifuge at 18,000 y for 2 min. The supernatant was tionated as described for cortex slices above and were subremoved, and the synaptosome pellet was extracted with sequently extracted in TCA, aliquots of each fraction being 102, TCA, agitated on a vortex mixer, centrifuged for 5 min taken for liquid scintillation counting and protein estimaas above and the supernatant taken for liquid scintillation tion as described above. counting. The incubation medium above the synaptosome Liquid scinrillution counting. TCA supernatants of tissue pellet was also taken for radioactivity counting. The TCA samples (501. to I ml) were added to 19ml of toluene
333
Taurine uptake and release from cerebral cortex
T A B L1.~ U P T A K r O r
[24S]TAURINI
TO SUUCLLLLLAK FRACTIONS
nmol Taurine/g wet wt. of slice taken up/30 rnin 20 /cM-Taurine 540 p - T a u r i n e
Fraction
Protein content ~E/E
8796 k 1061 914 f 49 2460 f 796 5783 k 825 515 f 127 1145 If: 145 371 f 67
108.0 f 1.6 31.3 k 1.2 61.3 f 1.5 18.7 0.8 3.9 k 0.3 24.6 f 0.3 21.5 f 1.2
Relative specific activity
Percentage of nonsupernatant taurine
10.7 f 0.9 29.5 f 1.2 59.9 f 0.9 6.6 k 1.6 16.5 f 0.9 4.5 f 0.7
0.4 f 0. I0 0.5 f 0.02 3.6 f 0.10 1.9 +_ 0.40 0.8 f 0.03 0.3 f 0.03
26 k 2 74 f 2
10.6 1.1 24.7 f 0.8 64.8 f 0.6 5.6 5 0 . 8 12.9 k 0.9 4.5 If: 0.7
0.4 f 0.03 0.5 k 0.01 3.9 & 0.03 1.6 k 0.30 0.6 f 0.03 0.2 f 0.03
30 f 3 70 k 3
495 f 9 54 f 4 156 f 30 314 f 50 37 f 14 83 k 19 23 f 3 Pcrcentagc recovery
17f4 41 f 2 11 1 2
16 i 3 37 f 2 13 f 2
Results are ~ s . L . M .Relative specific activity is percentage of taurine uptake/percentage of protein in the fraction. Results are for four separate estimations. Key to table: H. homogenate: P , , nuclear pellet: P,, crude mitochondrial pellet: P, + S, microsomes and soluble supernatant: PZA, myelin fraction: P2B, synaptosome fraction; P,C, mitochondria1 pellet.
PBD-2-methoxy ethanol used in the ratio of 5:4. vjv, and counted on a LS-200B Beckman Liquid Scintillation counter. Quench correction was achieved using an internal standard. Sources for materials used were: [35S]taurine. Amersham, U.K , L-DABA, Sigma: EGTA, Sigma.
RESULTS
Uprakc. of [35S]taurine into suhcrllular coriipartnients of intact cortex slices T a b l e I shows the relative recovery of [35S]taurine
in different subcellular fractions following the incubation of cortex slices in isotopic taurine at 20 and 540 PM. C3'S]Taurine content of the different fractions is given as a percentage of the total isotope present in the homogenate. The [35S]taurine content varied widely among the different fractions. At 20 pwtaurine most of the [35S]taurine was recovered in the microsomes and soluble supernatant (S, + P3), and the crude mitochondrial pellet (P,) took up the next highest percentage of taurine. Within the crude mitochondrial pellet the synaptosome fraction of P,B was most enriched. A similar pattern was obtained at 5 4 0 p ~ , although in this case a smaller proportion of [3'S]taurine was found in the synaptosomal fraction, and more was recovered in the soluble supernatant fraction.
When protein content was taken into account a similar distribution of [35S]taurine was observed (RSA). The only difference was that the myelin fraction (P,A) showed a higher [35S]taurine content than before, possibly reflecting leakage of from synaptosomes. When the [35S]taurine uptake \/as described as a percentage of the sedimentable o r particle bound [35S]taurine, 41% was found to occur in the , 37% at 540 FM. synaptosomal fraction at 20 p ~ and Recoveries of c.p.m. and protein were 103% and 93% respectively. Taurinci und GABA uptake in the presence of ~-aluiiinc~ and dianiinohutyrate
Figure 1 shows the effect of b-alanine (I mM) on ) by preparations of differing taurine (20 p ~ uptake neuronal and glial content, and by synaptosome preparations. Results are from incubation periods of 30 rnin and 5 min. After 30 rnin incubation it is seen that /3-alanine depressed by 50% the uptake of [35S]taurine by white matter slices (ependyma). This may be compared with a 30% and 28% inhibition of this uptake by cortex slices and synaptosome beds respectively. After 5 min incubation, taurine (20 PM) uptake is not depressed for synaptosome suspensions. but C-6 glioma suspensions showed a 41% inhibition under these conditions. L-DABA (1 mM) produced little inhibition of taurine uptake by synaptosome suspensions
G. H. T. W H L L I RH. , F. BRADFORD, A. N. DAVISON and E. J. THOMPSON
334
or by C-6 glioma suspensions over an incubation period of 5min. Figure 2 shows 5min uptake periods for [I4C]GABA (20 p ~ in) the presence of p-alanine (1 mM) or L-DABA (1 mM). For synaptosome suspensions L-DABA depressed GABA uptake by 84%. However, in C-6 glioma suspensions uptake is only depressed by 13%. 8-Alanine depressed [I4C]GABA uptake by 26% in synaptosome suspensions, and by 39% in C-6 glioma suspensions. Thus fl-alanine and L-DABA affect the two preparations differently. 13-alanine having its main effect on uptake by f - 6 glioma suspensions and L-DABA having its main effect on uptake by synaptosome suspensions.
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FIG. I . The uptake of ["S]taurine t o different tissue samples in the presence of 1 mM-L-DABA (0)and 1 mM-fialanine (H). The time of incubation is described below. Four to ten measurements were made for each value. VertiAhhreriurions used: A, synaptosome cal bars indicate s.L..M. beds: B. C6-glioma (both 5 min in incubation); C, cortex slices: D. synaptosome beds; E. ependymal slices (all 30 min incubations). Control uptake was in the range 5@~80 nmol taurine/g wet wt.
Figure 3 shows the efRux of preloaded [35S]taurine from incubated cerebral cortex slices. Points A. B, C and D represent 15min periods of [35S]ta~irine efflux into consecutive beakers containing taurine-free incubation medium f Ca2+ and EGTA. The proportion of total [35S]taurine recovered in the medium after incubation for 1 h with or without applied electrical stimulation was high (approx 40%). This suggests that either a large proportion of the total tissue content of taurine is released or that both uptake and release occur largely from a special tissue compartment.
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10
A
FIG. 2. Comparison of the uptake of [14C]GABA to synaptosomal suspensions and C6-glioma suspensions over a 5 min pcriod of incubation, in the presence of 1 mM-LDABA (0) or I mM-/l-alanine (W). Each histogram is the mean of four to ten samples. Vertical bars indicate S.F.M. A h h r w i u t i o m u s c d : A, synaptosome suspensions; B, C6-glioma.
rnin
...--.-.--,
on electrically stimukdled [3sS]taurine release. Electrical stimulation was for I5 min (period B). Symbols used are: ---tc,electrically stimulated slices in medium containing Ca2+; control slices in same medium; ' . . . .. slices preloaded in Ca2'-free (EGTA containing) medium and subsequently electrically stimulated in Ca' '-free medium. Vertical lines are S.E.M. from five or more separate measurements. ["SITaurine efflux was calculated as nmol using the specific radioactivity of ["Sltaurine prescnt in the incubation medium used for preloading the slices.
335
Taurine uptake and release from cerebral cortex TABLI2.
[35S]TAURlNL
I N SUBFRACTIONS OF CORTEX SLlCtS AND INCUBATION MFIIIUM INE/g WET WT TISSUE)
Fraction
Release into incubation medium
(nm0l [3ss]TAUR-
Control
Stimulated
Difference
25.7 f 1.7 132.0 f 10.9 150.0 f 4.4 21.3 f 3.3 78.9 f 6.4 16.3 rt 1.7
25.8 f 1.9 99.7 f 10.6 143.2 f 3.3 22.1 f 1.0 55.6 f 7.0 15.8 f 1.6
- 32.3% - 6.8$ + 0.8t - 23.3t -0.5t
11.6
113.2 _+ 14.5
+ 25.3
88.1
+0.1$
* 93% recovery of homogenate. Slices were stimulated at 36 mA mean current for 20 min, in the presence of C a z + .Released [j*S]taurine is corrected for levels in control medium. Difference significant with t P < 0.05 or $ P < 0.01. Results are ~ s . E . M . Each result is the mean of 4 separate estimations. Change in [35S]taurine content of slice suh-conipartiwnrs during electrical stiniu/ation
Table 2 shows the comparison of the changes in content of the subcellular compartments of slices electrically stimulated at 36 mA mean current. The changes in the synaptosome fraction of 23.3 nmol/g of [35S]taurine was commensurate with the increase in [35S]taurine recovered in the incubation mediums, which was equivalent to 25.3 nmol and was significant ( P < 0.05). The other fraction showing most change was the soluble cytoplasmic fraction and microsomes (S P3), in which there was a much smaller decrease (6.8 nmol/g), but this was not significant ( P < 0.1). At higher currents (48 mA) the soluble fraction showed a 10-fold increase in its loss of [35S]taurine and exceeded the synaptosome fraction in magnitude by a factor of two. Thus at the lower current the synaptic terminal compartment of the tissue could be contributing the larger proportion of released [35S]taurine, though re-uptake during the period of the experiment would have diminished the absolute amounts in the medium.
+
DISCUSSION Taurine uptake to cortex slices
The studies of KACZMERAK & DAVISON(1972); & OJA(1973); LAHDESMAKI et al. (1975) LAHDESMAKI and LOMBARDINI(1977) have shown that taurine uptake to cortex slices is a complex process with saturable and unsaturable components. There appear to be both high (5&60 PM) and low (6 mM) affinity saturable systems at work. The nature of the tissue compartments taking up taurine remain uncertain, but glial cells in retina (EHINGER,1973) or in culture, C-6 glioma (SCHRIER& THOMPSON,1974) and neuroblastoma cells appear to do so readily. It seems likely therefore that both newones and glial cells of cortex slices will be actively accumulating taurine. The finding that among the sub-fractions of the crude mitochondria1 fraction, the synaptosomes carried the greatest content of C3%]taurine indicates that
the presynaptic region of the slice does accummulate taurine. This probably represents true uptake, as recent studies in which taurine has been added to homogenate have shown that there is no significant uptake of taurine by subcellular fractions during the subsequent subfractionation procedure (SIEGHART & KAROBATH, 1974: RASSINet a/., 1977). The enzyme producing taurine (cysteinesulphinate decarboxylase. EC 4.1.1.29) has a synaptic localization (AGRAWALet a/., 1971) and synaptic vesicles appear to be enriched in taurine (DEBELLEROCHE & BRADFORD, 1973; RASSIN or a/.. 1977). In addition, evidence has been published for the existence of a synaptosomal sub-population which accumulates [35S]taurine (SIEGHART & KAROBATH, 1974; SIEGHART & HECKL,1976). The large proportion of taurine recovered in the soluble cytoplasmic fraction presumably reflects the considerable amount of uptake by glial and neuronal cell bodies. In order to differentiate between taurine uptake to glial and neuronal elements in the slice we used p-alanine as a blocker for taurine uptake. Recent experiments have shown that p-alanine specifically enters glial cells in the brain cortex (SCHON& KELLY, 1975; IVERSEN & BLOOM,1972); moreover, it competes with the uptake of taurine, and also with the uptake of GABA. L-DABA, on the other hand, is specifically taken up into nerve-endings (DICK& KELLY,1975) and competes with GABA uptake (TVERSEN & JOHNSTON, 1971). Hence we compared the affects of p-alanine and L-DABA on taurine and GABA high affinity uptake. It was notable that p-alanine depressed taurine uptake over a 5min period far more in glial cells (C-6 glioma) than in synaptosome preparations, on which it had little effect. Uptake was depressed by 50% or more in ependymal slices over a longer period (30 min) when synaptosomes and cortex slices were also showing a substantial (28-30%) reduction in their taurine uptake. LAHDESMAKI & OJA (1973) have reported a similar potency of action of b-alanine on taurine uptake to cortex slices under approximately equivalent conditions. Ependymal and cortex slices contain both glial and neuronal elements (nerve fibres in ependymal slices)
336
G . H.T.
WHtLLR.
H. F. BRADFORD, A. N. DAVISON and E. J. THOMI’SON
though the latter are likely to be more enriched in neuronal components than the former. Thus, only relative differences can be expected between the two kinds of preparation. With these results in mind, and arguing by analogy with GABA transport systems (see below), the relatively greater potency of p-alanine in blocking taurine uptake to preparations richer in glial than in neuronal elements could well indicate that taurine is entering mainly synaptosomes themselves rather than any cytoplasmic particles of glial origin present in synaptosome preparations.
(IVERSEN& KELLY,1975). Also, the fraction studied by SCHMIDet a / . (1975) was not a pure synaptosomal preparation, but a crude fraction which contained other elements including mitochondria (P2). Release experirwnts
Release of transmitter compounds has been shown to occur from both neurons and glial cells (BOWI-RY & BROWN.1972) though glial cells do not show Ca” dependence in their release (SELLSTROM & HAMBERGER, 1977) or atypical Ca” dependence (MINCHIN & IVERSEN. 1974). Thus, C a 2 + dependence has become an important criterion in distinguishing G A BA uptakr between release from the two cell types. Non-calcium The uptake of GABA to synaptosomes and C-6 dependent release of putative amino acid transmitters glioma was compared (Figure 2) in the presence of including glutamate has been observed to occur from & I mM-p-alanine or 1 mM-L-DABA in an attempt to electrically stimulated nerve trunks (WEINREICH 1975), and K + stimulation caused gain a measure of the glial contamination of synapto- HAMMERSCHLAG, ~ some fractions. Since L-DABA depressed synaptoso- similar release of GABA from glia ( B o w r : ~& ma1 GABA uptake by 84% but had little effect on BROWN,1972). Previous studies have shown that elecreducing C-6 glioma GABA uptake, synaptosome trical stimulation can release taurine from brain slices preparations would appear to contain little glial con- and that it is not metabolised during short term incutamination. The inverse pattern was obtained using bation and electrical stimulation experiments (KACZ/I-alanine. Thus, this compound depressed C-6 glioma MAREK & DAVISON, 1972). uptake of GABA to a greater extent (40%) than to The release of [3’S]taurine into the medium by the synaptosomal suspension (27%) when 5 min incu- electrical stimulation reported here was Ca*+-depenbation periods were used. With longer periods of in- dent. Since stimulus-induced release of taurine from cubation (30min) the same pattern of depression of slices was higher in calcium-free medium than passive synaptosomal uptake by L-DABA and 8-alanine was release from control slices in the same medium, it maintained (Fig. 2). These results suggest that the is possible that some endogenous Ca2+ is still availsynaptosome preparation is mainly of neuronal origin able for the process. Alternatively this non Ca2 and is not heavily contaminated with cytoplasmic dependent efflux of [35S]taurine could be from glial bodies of glial origin. R t U B U R N (1978) obtained simi- cells. lar relative potencies of L-DABA and /I-alanine inhiSlices which were stimulated electrically and then bition of [14C]GABA uptake to synaptosomes. subfractionated to locate the pool of taurine respon& HAM- sible for stimulated release showed two main results. L-DABA has been reported (SELLSTROM H ~ R G E R 1975) , to inhibit GABA uptake to a cortical First, at the lower stimulating current of 36mA the glial preparation to the same extent (50%) that it electrically stimulated release was mainly from the blocks GABA uptake to synaptosome preparations, synaptosomal pool. Second, at higher currents which might be thought to conflict with the above (48mA) release of taurine occurred from other fracassessment of the purity of synaptosome preparations. tions, including the soluble supernatant. Thus, in However, autoradiographic studies of cerebral cortex order to stimulate release specifically from synaptic uptake and of sympathetic ganglia show DABA local- regions stimulation must not be too extensive. ized (retained) in neuronal elements, mainly nerve Change in the soluble supernatant fraction may rependings (DICK& KELLY, 1975). Also, the IC,, resent release from other tissue components such as reported for DABA in blocking GABA uptake to the glial cells or neuronal cell bodies. glial cells of sympathetic ganglia (0.7 mM) is much higher than its counterpart for cortex slices ( 5 0 ~ ~Acknowledynttents-This ) work was supported by an MRC (IVERSEN& KELLY,1975). For these reasons it seems project grant. likely that the glial preparation of SELLsmoM & HAMRERGER (1975) contained synaptosomes as contaminants. It has been reported that p-alanine blocks REFERENCES GABA and taurine uptake to synaptosomes by et a/., 1973; SCHMIDet al., 1975) 6G80% (SNODGRASS A. N. & KACZMAREK L. K. but this does not correlate with the autoradiographic ACRAWAL H. C., DAVISON (1971) Subcellular distribution of taurine and cysteinsulevidence which implies specific uptake of 8-alanine phinate decarboxylase in developing rat brain. Biochrm to glial cells. This was also indicated by the greater J. 122, 759-163. effectiveness of p-alanine in blocking GABA uptake BOWERYN. G . & BROWND. A. (1972) y-Aminobutyric to ganglia rather than cortex slices, the IC5,values acid uptake by sympathetic ganglia. Nature, N e w Biol. for this inhibition being 120 PM and 2 mM, respectively 238, 89-91. +
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