Neuroscience Vol. 4. pp. 1745 to 1749 Pcrpmon Ress Ltd 1979. Printed in Great
Britain
COMPONENTS OF TAURINE EFFLUX RAT BRAIN SYNAPTOSOMES
IN
P. KONTRO Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland Abt-The efBux of Cs5SJtaurine from isolated rat brain synaptosomes was studied in a superfusion system. The spontaneous cfBux of taurine was slow and could be described as comprising two first-order rata compownts, of which the slower (tl12 = 77.0 min) represents release from intrasynaptosomal spaces. Only a h&b tauriac concentration (10 mmol/l) in the medium enhanced the eillux of intrasynaptosomal taurine, which in&&es a rather stable intrasynaptosomal taurine compartment. Depolarizing concentrations of potassium ions stimulated taurine etilui and also induced the appearance of a new ‘intexmed&e’ eiBux coinponent. This component was absent when calcium ions were omitted from the medium. It is tl&eforc suggested that the component may originate from the emptying of synaptic vesicles.
TAURINE has been considered a neurotransmitter or neuromodulator in the CNS (see OJA, Komo & Lw 1976~; 1977; OJA & KONTRO, 1978). There is a high-affinity, strictly sodium-dependent uptake d taurine in brain synaptosomes (L&LXISA&& P&uLA & OJA, 1975; KONT~O & OJA, 1978u,bb
copy as described by KONTRO& @A (1978u) and their protein content was &term&l according to LOWRY, R~SEBR~GH, Fm & RANDALL(1951). The syaaptosomcs exhibited a constant oxygcol uptake for more than 2 h (Komno & OJA, 197&a). hubarions
Synaptosome suspensions (1.0-2.0 mg protein) were but the properties of taurine efl3ux have only been preincubated under shaking with urdabelled and [35S]partly cbwct&& The spontaneous et&ix of tautaurine (final concentrations: 5Oq1ol/l and 1 mCi/l) for rinc from brain slices is very slow (OJA, 1971), and 60 min under O1 at 310K in 2 ml of Krebs-Ringeralso in oiuo the average half-life of brain taurine is of HEPi% solution, pH 7.4, of the following composition: the order of days (CULLING, 1974; OJA, m & (mmol/l) NaCl 129, KCI 5.1, CaC& 2.7, MgS04 1.2, L&DE&K& 1933). High concentrations of potas- NaHIP04 1.2, KOH 8, HEPES 10 and ~glucose 10. sium ions and elect&al pulses stimulate the release of Synaptosome beds for e&x experiments were prepared by taurine from nervous tissue (JASPER & KOYAMA, 1969; a procedure modified from that of De BELLEROCHE & CLARK& C~LLN, 1976), as do light flashes from the BRADP~RD(1972). Synaptosomes were separated from preincubation solution by rapid tiltration through a Milliretina (Pm MowtBS, KLETHI,Uarw & MANpore filter (pore sixc 0.8 m) and rinsed with cold KrebsDEL, 1974). Such a stimulated release of taurine is possibly mod&d by calcium ions (COLLINS L TOPIWALA,Ringer-HEPES medium. The filter membrane with the attached synaptosome pellet was then rapidly transferred 1974; !GLCEDA & PASANTBS-MORALES, 1975; ti into a small plastic superfusion cwber (volume 0.8 ml) P~RD,DAVISON& W~e~ea, 1976; SIEOH~~RT & HECKL, connected through a rubber inlet tube with the incubation 1976), but it has not been shown unequivocally to be solution reservoir. An electro-mechanic pump (Desaga cakium&pendent, which propensity has generally 132100) was connected at silicone outlet tube and directed been cim&kcd essential for neurotransmitter release. the flow of medium from the bottom of the chamber I have now, t&&ore, studied taurine &ux from rat upwards. brain aynaptowmes, seeking to separate in a superThe flow of the preoxygenated Krebs-Ringer-HEPES fusion syrtom the possible eiIlux components in order medium was adjusted to 0.20 ml/min and superfusion conto see how exogenous taurine or potassium and cal- tinued for 9&12Omin. The effh~ents were fractionated directly into scintillation counting vials. In some expericium ions influence them individually.
EXPERIMENTALPROCEDURES Materials The purity of [“5S]taurine (sp. act. 21 Ci/mol, the RadiochemicalCkntrc,Amersham)was checked by ion exchange chromatography PI &swibed by OJA et al. (19766). The. br*down of [“Qnurine during the experiments was
purity of -meS
was checked by electron micros-
ments 1 or lOmmol/l unl&&d taurine were added to the super!hsion medium. In some further experiments the superfusate contained 25 or 55 mmol/l KCl to depolarize the synaptosomal membranes. Appropriate amounts of NaCl were omitted from the supplemented Krebs-RingerHEPES media to keep their osmolality from 293 to 302 mOsmol/l when checked with a halfmicro-osmometer. In some high-potassium (55 mmol/l) Krebs-RingerHEPES med&~CaClz was replaced by choline chloride. lkternumtion of radioactivity A&r supertusion the radioactivity of the eflluents and the SyMptosonle pellets on the 6Itet membranes was measured by an LKB-Wallac 81000 counter using 5 ml Instagel0 as the scintillation solution to sohrbilixe completely
1745
I746
P. KONTFW fitting and subtraction process was reputed and a ihm.! component extracted. The negative slopes of the strdighr lines are the rate constants k,, k, and k,i. The intercepts o: the extrapolated lines on the ordinate pave S,? S2 and S.?. which were proportionally adjusted to total 100::, (I.AAKSO. 1978). The half-times (tli2) equal O.G??,fi ft)r an) efflu?. component RESULTS
Spontaneous efjlux The release of E3%]taurine into unsupplemented Krebs-Ringer-HEPES medium was slow after an initial fast wash-out. The slower component of efflux became prominent only after 4Omin superfusion (Fig. 1). Analysis of the taurine efflux curVe yielded 20 40 60 80 loo 12Q two efflux components with rate constants of 9.0 x superfuhn thne min 10-3min-1 (k,) and 153B x 1W3 min-’ (k2) and FIG. 1. Spontaneous efaux of [%]taurine from rat whole half-times of 4.5 min and 77.0 min respectively brain synaptosomes. Synaptosomes were preincubated in (Table 1). The slow efflux component comprised only the presence of 50 pmol/i [“%]taurine for 30 min and then 14.6% of the total initial radioactivity accumulated in superfused for 120 min in small chambers at 310 K under the synaptosomes. O2 in Krebs-Ringer-HE= medium, pH 7.4. The total
o,lc
efflux curve was resolved into two first-order rate components: a fast component (+----@) with tl,z = 4.5min and a slow one (o----~) with tllZ = 77.0min. The graph shows the means of six experiments. Standard deviations for each point were l-15%. the membrane filters, synaptosome pellets and samples of incubation solution. The counting time was long enough to reduce the standard error of counting to Mow 2.0%. Any quenching was corrected by the external standard-channels ratio method. The average counting efficiency.for 3sS was 91%.
Calculations The initial radioactivity of the synaptosomes was calculated as the sum of the radioactivities in the efauent fractions and the residual radtiactivity in the synaptosome pellet after superfusion. The percentage of the radioactivity remaining in the synaptosomes at any time was calculated by subtracting the total radioactivity of the e6iuent medium from the initial symraptosomal radioactivity. In most experiments the e&x of [““S’Jtaurine could be
fitted by a differential equation with two exponential terms (OIA & VAHVELAINEN,1975): &(t) = Sle-kL’ + S2emkz’, in which S&t) is the radioactivity in the synaptosomes (per cent of the total initial radioactivity) at time t (min), S1 and S2 are the total initial &ctivities in the two components of eBux (per cent of the tot&l initial radioactivity in the synaptosomesj and kl and k2 the first-order ratcconstants (min-‘) of the two components of &lux. In higbpotassium media a third e&x component SJexp( -k,t) emerged. The efflux components were determined from the regression of the logarithm of the pekntage of radioactivity left in the synaptosomes on the incubation time (see gigs 1 and 3). The slowest component was first subtracted from the total efRux by fitting the least-squares straight like to the last experimental points. Ant&r Ie4M~ a@&@ line was then fitted to the point6 rq7reqiw~ttta rcs#ktsl e&x to obtain a faster component. when neccsaary, the above
Taurine homoexchange The addition of 1 mmoljl taurine to the superfusion medium did not have any significant effect on C3%]taurine release from synaptosomes (Table I), but a concentration of lOmmol/l slightly enhanced the [35S]taurine efflux (Fig. 2). The dAux stimulated by this homoexchange was also resolved into two components with half-times of 51.5 min and 4.6 min. the corresponding rate constants being 13.5 x 10-j min-’ (k,) and 150.0x 10W3min“ (k,) (Table 1). Only the slower efilux component was significantly enhanced by extrasynaptosomal taurine. The percentage contributions of the two efflux components remained unaltered. Potassium-stimulated e&x A depolarizing concentration of potassium ions caused a pronounced increase in the efHux of C3’S]taurine from synaptoaomes (Fig. 2). The pattern of release of [‘5S‘Jtaurine was also markedly modified at high potassium ion concentrations: a third ‘intermediate’ flux component emergbd’(Fig. 3). This third component was present in both 25 and 55 mmol/l K + concentrations; the rate constants WeTe 79.8 x 10W3min -’ and 136.4 x lo- 3 min- ’ respectively (Table 1). The component was calcium-dependent. since it disappeared in the absence of Ca*+ ions. The percentage contribution of the slow component to total etiux diminished when the intenndliate cornpoe nent emerged, while that of the fast component was not significantly altered. In the presence of 55 mmol;l K+ the rate constant of the slow d&a component but not at was increased (k = 17.4 x 10-3min-‘), 25mmol/l K+. Both high potassium ion concentrations markedly increased the re constant Qf the fast effhnt component (Table 1). The effect was more pronounced at 55 mmol/l K+ Tlk mts of both the slow and fast &ux composmnts by K’ ions
mia-’
153.0 f 10.6 4.y4.2-4.9) 85.4(76.8--96.2) 0.992
9.0 f 0.5 77.q73.3-81.1) 14.ql3.fi-15.6) 0.997
No addition (control)
141.6 f 7.9 4.9(4.6-5.2) 83.9(73.9-95.5) 0.996
9.3 f 0.3 74.q72.1-77.1) 16.1(15.3-16.9) 0.999
Taurine 1 mmo1p
150.0 f 8.2 4.6(4.4-4.9) 87.0(77.7-97.5) 0.997
13.5 f 1.2t 51.5(47.4--56.o)t 13.q11.9-14.2) 0.994
311.6 f 48.4t 2.1(1.9-2.6& 83.1(71.7-98.9) 0.996 136.4 f 15.8 5.1(4.6-5.7) 12.2(9.2-16.8) 0.988
79.8 f 10.3 8.7(7.7-10.0) 8.0(6.0-10.9) 0.992
17.4 f 0.8t 39.9(38.141.7)t 4.7(4.4-5.1H 0.998
K+ 55mmol,l
264.8 f 15.7t 2.q2.5-2.8fl 86.9(79.4-96.9) 0.996
7.8 f OS* 89.3(&W-94.8)t 5.1(4.9-5.3H 0.997
Suprrfusion media Taurine 10 mmol/l K+ 25 mmol/l
262.4 f 11.5t 2.q2.5-27)t 91.9(85.2-99.7) 0.998
17.8 f 0.4t 39.0(38.1-39.8n 8.1(7.W3,3)t 0.999
K+ 55mmol/Y :’ nocakium
Synaptosomes were preincubated and superfused as in Figs l-3 in Kreb~Ri~r-~EPES media modified as indicated. The e&x of taurine was resolved into first-order rate components as described in Experimental Procedures and shown in Figs 1 and 3. The rate constants (/cl, k2, k3), half-times (t& and the relative contributions as percentages (.S1, S,, Ss) of each efflux component are piven. The correlation coefficients indicate the fit of the least-squares straight regression lines with the experimental data. Mean value are given with their 95% confidence limits, which are unsymmetric for S,, S, and &. Statistically significant differences from the control experiments are indicated as follows: *P c 0.05; tP < 0.01.
corrdatioo coetkient Intermed& component ks x 10-3mjn-1 tt,z mm s3 % correlation coe&ient
s2 %
h/2 mill
ks x lo-
Fast compcnt
SI % corrtlation cwfkient
till mill
Slow cornpent kl x lo- min-1
Parametemofthe daux components
TAISLB 1.PARA~SOFTHEeFFLUXCOMPONENTSOFTAURINEiNRATBRAlNS~AP~~
1748
P.
’
’
20
’
’
I
’
40 60 superfusiontime min
’
’
80
KONTRO
’
FIG. 2. Efflux of [%jtaurine from rat whole brain synaptosomes in different superfusion media. Synaptosomes were preincubated as in Fig. 1 and then superfused in small chambers at 310 K under O2 in different media as follows: Spontaneous efflux of taurine in unsupplemented KrebsRinger-HEPES medium, pH 7.4 (O-_-O). Efllux of taurine in Krebs-Ringer-HEPES medium, pH 7.4, containing lOmmol/l taurine (H). Efflux of taurine in KrebsRinger-HEPES medium, pH 7.4, containing 55 mmol/l K’ (A---A). Efflux of taurine in Krebs-Ringer-HEPES medium, pH 7.4, containing 55 mmol/l K+ when calcium ions are omitted (A--A). Each curve is the mean of 3-6 experiments. Standard deviations for each point were l-16%. The calculated parameters of the efflux components are given in Table 1. were calcium-independent, for the effects persisted when CaZ+ ions were omitted from the medium.
concentration the observed enhancement IS not necessarily due to homoexchange, since hynaptosomes accumulate from superfusion medium unlabeiled taurine (OJA, 1971) which may then accelerate [,“S]taurine efflux from the inside uf ~ynap~osomal membranes (L&DESM&KI & OJA. i972). i’otassium-stimulated release of taurine has been I;,mn(i in brain slices. crude synaptosomal (P,) fractions and purified synaptosomes (DE BIXEROCHE & BKAIXIWW. 1073: Cot.I.INS & TOPIWALA. 1974; SlEGHARf & tiEt.KL. 1976: VI\RC;AS,DORM IX LOREWO S: Ow t;o. 19771. The earlier studies have not. howt*\~i tinequivocally established any calcium-dependeilcc‘ 01 taurine cfflus. SIEGHART& HIXXI. (1976) obser\cd GI attenuation in the potassium-stimulated efflux nf t;rurine from the PL fraction of a cerebral cortex homogenate in the presence of calcium ions. but the ~miv,l~ln c)f calcium ions has not affected the potassilinl.~~sllked release of taurine from cerebral cortex slices :,C’CII,~ INS & TOW wAL..4, 1974) or rat visual carter (Cd iNK &L Col.r.i>s_ 1976). The electrically stimulated K!GW of taurinc has been claimed to be calcium-dcl)~!.:lldent, howcvcr (COLLINS & TWIWALA, 1974; BRAIW~RR t’t trl.. 1976). My present results show that the ;wtassium-stimulated total efflux of taurine cannot k~ tirmonstrated lo be strictly calcium-dependent. I’oths~tum stimulation of both fast (possibly taurine from &maged synaptosomes and membrane binding sites! ,ind slow (taurine in the synaptoplasm) eR3.x occurs-ed also in the absence of Ca’ ’ ions. Only rhe intermediate efflux component *as calcium-dependent. Since this component emerged at the expense of the shkwest efflux com-
DISCUSSION The spontaneous efflux of C3%]taurine from rat brain synaptosomes was divided into two components with widely differing first-order rate constants. The fast component, with a half-time of 4.5 min, probably at least partially represents an initial wash-out of [3sS]taurine from an extrasynaptosomal, loosely bound taurine pool. Any [35S]taurine retained by damaged synaptosome fragments or attached to membranes is likewise rapidly released. The slower component, with a half-time of 77.Omin, then represents the actual release of taurine from the intracellular spaces of viable synaptosomes. Taurine appears to be rather effectively retained by intrasynaptosomal compartments. In the retina intracellular taurine may be still more firmly bound (STARR & VOADEN,1972;
0.1
’
20
8
40
’
60
80
’
’
100
120
superfusion time min
FIG. 3. E&IX components of f”‘S]taurine in a higb-potassium medium. Synaptosomes were prein&&ated and superof intrasynaptosomal fused as in Fig. 1, except that the superfusion medium taurine occurred with medium containing 1 mmol/l contained 25 mmol/l K+. The total efflux curve was taurine. This is in agreement with the results ob@ned divided into three first-order rate components: a fast efffux with crude synaptosomal fractions (!%E++T & component (A---A) with tliZ = 2.9mfn, a slow- efflux HECKL, 1976) and brain slices (CRNIC, HAFAMWSTADcomponent (O----O) with t,,* = 89.3 njn and .an inter& CUTLET, 1973). At the higher taurine co&entt%tlon mediate efflux component (t--e) with ti!2 = 8.7min: The graph is the mean of four experim@&. Standard devi(lOmmol/l) the first-order rate constant of the slower ations for each point were 2-167;. efflux component increased by 50%. At such a high
KENNEDY& VOADEN,1976). No apparent homoexchange
Taurine etilux from synaptosomes
ponent, it probably also originates in some intrasynaptosomal tauriae pool(s). The intrasynaptosomally accumulated [35S]tauriae could for instance be localized in a vesicular pool and a cytoplasmic pool. The involvement of calcium ions in the intermediate e&x component might be an indication that this very component is associated with the emptying of the contents
1149
of synaptic vesicles into the medium. I do not think, however, that this n ecessarily means that taurine is a neurotransmitter. Unassorted synaptic vesicles from the brain contain much taurine (DE BELLEROCHE & BRADFORD, 1973) which could be liberated together with some proper neurotransmitter(s) when synaptosome membranes are depolarized by potassium ions.
REFERENCES H. F., DA~I~~EIA. N. & WHEELERG. H. T. (1976) Taurine and synaptic transmission. In Tuurine (cds HUXT~LE R. t BARBEAU A.), pp. 303-310. Raven Press, New York. CLARKR. M. & Cc~~trns G. G. S. (1976) The release of endogenous amino acids from the rat visual cortex. J. Physiol., Lond. 262, 383400. C~LI_,IN~ G. G. S. (1974) The rates of synthesis, uptake and disappearance of [‘*CJtaurine in eight areas of the rat central nervous system. Brain Res. 76,447-459. (&LLIN~G. G. S. & TOPWALAS. H. (1974) The release of [‘*C]taurine from slices of rat cerebral cortex and spinal cord evoked by electrical stimulation and high potassium ion concentration. Br. J. Pharmac. SO, 45lP-452P. CYNIC D. M., HAMMERSTAD J. P. & Cum R. W. (1973) Accelerated efflux of [r4C] and C3H]amino acids fram superfused slices of rat brain. J. Neurochem. 20,203209. DE BELLEROCHE J. S. & BRADFORD H. F. (1972) Metabolism of beds of mammalian cortical synaptosomes: response to depolarizing influences. J. Neurochem.19, 585-602. DE BELLEROCHE J. S. & BRADIQRDH. F. (1973) Amino acids in synaptic vesicles from mammalian cerebral cortex: A reappraisal. J. Neurochem. 21,441-451. JASPER H. H. & KOYAMAI. (1969) Rate of release of amino acids from the cerebral cortex in the cat as affected by brainstem and thalamic stimulation. Can. J. Physiol. Pharmac. 47, 889-905. KENNEDYA. J. & VOADENM. J. (1976) Studies on the uptake and release of radioactive taurine by the frog retina. J. Neurochem. 27, 131-137. KONTROP. & OJA S. S. (1978~) Taurine uptake by rat brain synaptosomes. J. Neurochem. 30, 1297-1304. KONTROP. & OJA S. S. (1978b) Sodium dependence of taurine uptake in rat brain synaptosomes. Neuroscience 3,761-765. LAAKW M.-L. (1978) Efflux of phenylalanine and tryptophan from cerebral cortex slices of adult and 7-day-old rats. Acta physiol. stand. 102.74-83. LXHDESMXKI P. & KORHONENK. (1978) Comparative studies on the degradation of GABA and taurine in the brain. J. Neurochem. 30, 705-711. LXrtossMii~~ P. & OIA S. S. (1972) Effect of electrical stimulation on the influx and et&x of taurine in brain slices of newborn and adult rats. Expl Brain Res. 15, 4-38. L;~HD&KI P., Pasuw M. & OJA S. S. (1975) Effect of electrical stimulation and chlorpromazine on the uptake and relase of taurine, y-aminobutyric acid and glutamic acid in mouse brain synaptosomes. J. Neurochem. 25, 675680. LOWRY0. H., R~~~BROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193,265-275. OJA S. S. (1971) Exchange of taurine in brain slices of adult and 7-day-old rats. J. Neurochem. 18, 1847-1852. OJA S. S. & KONTROP. (1978) Neurotransmitter actions of taurine in the central nervous system. In Taurine and Neurological Disorders (eds BARBEAU A. 8r HUXTABLER.), pp. 181-200. Raven Press, New York. OJA S. S., KONTROP. & L&t~EsMji~t P. (1976a) Transport of taurine in the central nervous system. In Transport Phenomena in the Nervous System. Physiological and Pathological Aspects. Advances in Experimental Medicine (eds LEVI G., BATTISTIN L. Br LAITHAA.). Vol. 69, pp. 237-252. Plenum Press, New York. OJA S. S., KON~ROP. & Lji~~EsMji~l P. (1977) Amino acids as inhibitory neurotransmitters. Prq. Pharmucol. l(3), l-l 19. OJA S. S., L~~T~NENI. & LXHD~MXKIP. (1976b) Taurine transport rates between plasma and tissues in adult and 7-day-old mice. Q. J. exp. Physiol. 61, 133-143. OJA S. S. & VAHVELAINEN M.-L. (1975) Transport of amino acids in brain slices. In Research Methods of Neurochemistry (eds MARKSN. 8t RODNIGHT R.). Vol. 3, pp. 67-137. Plenum Press, New York. PASANTESMORALES H., KL.ETHIJ., URBANP. F. & MANDELP. (1974) The effect of electrical stimulation, light and amino acids on the eRlux of C3-%]taurine from the retina of domestic fowl. Expl Bruin Res. 19, 131-141. S.UXEDA R. & P.Q.wTES-MORALEYH. (1975) Calcium coupled release of [“Sltaurine from retina. Brain Res. W, 206211. SIEGHARTW. L HECKL K. (1976) Potassium-evoked release of taurine from synaptosomal fractions of cerebral cortex. Brain Res. 116, 538-543. STARRM. S. & VOADEN M. J. (1972)The uptake, metabolism and release of [‘“CJtaurine by rat retina in vitro. Vision Res. 12, 1261-1269. V.uo%s O., Dcnu~ oa Lc%tl~NzOM. C. & ORRFGOF. (1977) Effect of elevated extracellular potassium on the release of labelled noradrenaline, Edutamate,@y&w, B-ala&e and other amino acids from rat brain cortex slices. Neuroscience 2, 383390. WmrrA~~a V. P. & BAR= L. A. (1972) The ~ubcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. In Methods of Neurochemistry (ed. FRIEDR.), pp. 2-52. Marcel Dekker, New York. Bx~o~an
(Accepted 20 May 1979)
Britain
COMPONENTS OF TAURINE EFFLUX RAT BRAIN SYNAPTOSOMES
IN
P. KONTRO Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland Abt-The efBux of Cs5SJtaurine from isolated rat brain synaptosomes was studied in a superfusion system. The spontaneous cfBux of taurine was slow and could be described as comprising two first-order rata compownts, of which the slower (tl12 = 77.0 min) represents release from intrasynaptosomal spaces. Only a h&b tauriac concentration (10 mmol/l) in the medium enhanced the eillux of intrasynaptosomal taurine, which in&&es a rather stable intrasynaptosomal taurine compartment. Depolarizing concentrations of potassium ions stimulated taurine etilui and also induced the appearance of a new ‘intexmed&e’ eiBux coinponent. This component was absent when calcium ions were omitted from the medium. It is tl&eforc suggested that the component may originate from the emptying of synaptic vesicles.
TAURINE has been considered a neurotransmitter or neuromodulator in the CNS (see OJA, Komo & Lw 1976~; 1977; OJA & KONTRO, 1978). There is a high-affinity, strictly sodium-dependent uptake d taurine in brain synaptosomes (L&LXISA&& P&uLA & OJA, 1975; KONT~O & OJA, 1978u,bb
copy as described by KONTRO& @A (1978u) and their protein content was &term&l according to LOWRY, R~SEBR~GH, Fm & RANDALL(1951). The syaaptosomcs exhibited a constant oxygcol uptake for more than 2 h (Komno & OJA, 197&a). hubarions
Synaptosome suspensions (1.0-2.0 mg protein) were but the properties of taurine efl3ux have only been preincubated under shaking with urdabelled and [35S]partly cbwct&& The spontaneous et&ix of tautaurine (final concentrations: 5Oq1ol/l and 1 mCi/l) for rinc from brain slices is very slow (OJA, 1971), and 60 min under O1 at 310K in 2 ml of Krebs-Ringeralso in oiuo the average half-life of brain taurine is of HEPi% solution, pH 7.4, of the following composition: the order of days (CULLING, 1974; OJA, m & (mmol/l) NaCl 129, KCI 5.1, CaC& 2.7, MgS04 1.2, L&DE&K& 1933). High concentrations of potas- NaHIP04 1.2, KOH 8, HEPES 10 and ~glucose 10. sium ions and elect&al pulses stimulate the release of Synaptosome beds for e&x experiments were prepared by taurine from nervous tissue (JASPER & KOYAMA, 1969; a procedure modified from that of De BELLEROCHE & CLARK& C~LLN, 1976), as do light flashes from the BRADP~RD(1972). Synaptosomes were separated from preincubation solution by rapid tiltration through a Milliretina (Pm MowtBS, KLETHI,Uarw & MANpore filter (pore sixc 0.8 m) and rinsed with cold KrebsDEL, 1974). Such a stimulated release of taurine is possibly mod&d by calcium ions (COLLINS L TOPIWALA,Ringer-HEPES medium. The filter membrane with the attached synaptosome pellet was then rapidly transferred 1974; !GLCEDA & PASANTBS-MORALES, 1975; ti into a small plastic superfusion cwber (volume 0.8 ml) P~RD,DAVISON& W~e~ea, 1976; SIEOH~~RT & HECKL, connected through a rubber inlet tube with the incubation 1976), but it has not been shown unequivocally to be solution reservoir. An electro-mechanic pump (Desaga cakium&pendent, which propensity has generally 132100) was connected at silicone outlet tube and directed been cim&kcd essential for neurotransmitter release. the flow of medium from the bottom of the chamber I have now, t&&ore, studied taurine &ux from rat upwards. brain aynaptowmes, seeking to separate in a superThe flow of the preoxygenated Krebs-Ringer-HEPES fusion syrtom the possible eiIlux components in order medium was adjusted to 0.20 ml/min and superfusion conto see how exogenous taurine or potassium and cal- tinued for 9&12Omin. The effh~ents were fractionated directly into scintillation counting vials. In some expericium ions influence them individually.
EXPERIMENTALPROCEDURES Materials The purity of [“5S]taurine (sp. act. 21 Ci/mol, the RadiochemicalCkntrc,Amersham)was checked by ion exchange chromatography PI &swibed by OJA et al. (19766). The. br*down of [“Qnurine during the experiments was
purity of -meS
was checked by electron micros-
ments 1 or lOmmol/l unl&&d taurine were added to the super!hsion medium. In some further experiments the superfusate contained 25 or 55 mmol/l KCl to depolarize the synaptosomal membranes. Appropriate amounts of NaCl were omitted from the supplemented Krebs-RingerHEPES media to keep their osmolality from 293 to 302 mOsmol/l when checked with a halfmicro-osmometer. In some high-potassium (55 mmol/l) Krebs-RingerHEPES med&~CaClz was replaced by choline chloride. lkternumtion of radioactivity A&r supertusion the radioactivity of the eflluents and the SyMptosonle pellets on the 6Itet membranes was measured by an LKB-Wallac 81000 counter using 5 ml Instagel0 as the scintillation solution to sohrbilixe completely
1745
I746
P. KONTFW fitting and subtraction process was reputed and a ihm.! component extracted. The negative slopes of the strdighr lines are the rate constants k,, k, and k,i. The intercepts o: the extrapolated lines on the ordinate pave S,? S2 and S.?. which were proportionally adjusted to total 100::, (I.AAKSO. 1978). The half-times (tli2) equal O.G??,fi ft)r an) efflu?. component RESULTS
Spontaneous efjlux The release of E3%]taurine into unsupplemented Krebs-Ringer-HEPES medium was slow after an initial fast wash-out. The slower component of efflux became prominent only after 4Omin superfusion (Fig. 1). Analysis of the taurine efflux curVe yielded 20 40 60 80 loo 12Q two efflux components with rate constants of 9.0 x superfuhn thne min 10-3min-1 (k,) and 153B x 1W3 min-’ (k2) and FIG. 1. Spontaneous efaux of [%]taurine from rat whole half-times of 4.5 min and 77.0 min respectively brain synaptosomes. Synaptosomes were preincubated in (Table 1). The slow efflux component comprised only the presence of 50 pmol/i [“%]taurine for 30 min and then 14.6% of the total initial radioactivity accumulated in superfused for 120 min in small chambers at 310 K under the synaptosomes. O2 in Krebs-Ringer-HE= medium, pH 7.4. The total
o,lc
efflux curve was resolved into two first-order rate components: a fast component (+----@) with tl,z = 4.5min and a slow one (o----~) with tllZ = 77.0min. The graph shows the means of six experiments. Standard deviations for each point were l-15%. the membrane filters, synaptosome pellets and samples of incubation solution. The counting time was long enough to reduce the standard error of counting to Mow 2.0%. Any quenching was corrected by the external standard-channels ratio method. The average counting efficiency.for 3sS was 91%.
Calculations The initial radioactivity of the synaptosomes was calculated as the sum of the radioactivities in the efauent fractions and the residual radtiactivity in the synaptosome pellet after superfusion. The percentage of the radioactivity remaining in the synaptosomes at any time was calculated by subtracting the total radioactivity of the e6iuent medium from the initial symraptosomal radioactivity. In most experiments the e&x of [““S’Jtaurine could be
fitted by a differential equation with two exponential terms (OIA & VAHVELAINEN,1975): &(t) = Sle-kL’ + S2emkz’, in which S&t) is the radioactivity in the synaptosomes (per cent of the total initial radioactivity) at time t (min), S1 and S2 are the total initial &ctivities in the two components of eBux (per cent of the tot&l initial radioactivity in the synaptosomesj and kl and k2 the first-order ratcconstants (min-‘) of the two components of &lux. In higbpotassium media a third e&x component SJexp( -k,t) emerged. The efflux components were determined from the regression of the logarithm of the pekntage of radioactivity left in the synaptosomes on the incubation time (see gigs 1 and 3). The slowest component was first subtracted from the total efRux by fitting the least-squares straight like to the last experimental points. Ant&r Ie4M~ a@&@ line was then fitted to the point6 rq7reqiw~ttta rcs#ktsl e&x to obtain a faster component. when neccsaary, the above
Taurine homoexchange The addition of 1 mmoljl taurine to the superfusion medium did not have any significant effect on C3%]taurine release from synaptosomes (Table I), but a concentration of lOmmol/l slightly enhanced the [35S]taurine efflux (Fig. 2). The dAux stimulated by this homoexchange was also resolved into two components with half-times of 51.5 min and 4.6 min. the corresponding rate constants being 13.5 x 10-j min-’ (k,) and 150.0x 10W3min“ (k,) (Table 1). Only the slower efilux component was significantly enhanced by extrasynaptosomal taurine. The percentage contributions of the two efflux components remained unaltered. Potassium-stimulated e&x A depolarizing concentration of potassium ions caused a pronounced increase in the efHux of C3’S]taurine from synaptoaomes (Fig. 2). The pattern of release of [‘5S‘Jtaurine was also markedly modified at high potassium ion concentrations: a third ‘intermediate’ flux component emergbd’(Fig. 3). This third component was present in both 25 and 55 mmol/l K + concentrations; the rate constants WeTe 79.8 x 10W3min -’ and 136.4 x lo- 3 min- ’ respectively (Table 1). The component was calcium-dependent. since it disappeared in the absence of Ca*+ ions. The percentage contribution of the slow component to total etiux diminished when the intenndliate cornpoe nent emerged, while that of the fast component was not significantly altered. In the presence of 55 mmol;l K+ the rate constant of the slow d&a component but not at was increased (k = 17.4 x 10-3min-‘), 25mmol/l K+. Both high potassium ion concentrations markedly increased the re constant Qf the fast effhnt component (Table 1). The effect was more pronounced at 55 mmol/l K+ Tlk mts of both the slow and fast &ux composmnts by K’ ions
mia-’
153.0 f 10.6 4.y4.2-4.9) 85.4(76.8--96.2) 0.992
9.0 f 0.5 77.q73.3-81.1) 14.ql3.fi-15.6) 0.997
No addition (control)
141.6 f 7.9 4.9(4.6-5.2) 83.9(73.9-95.5) 0.996
9.3 f 0.3 74.q72.1-77.1) 16.1(15.3-16.9) 0.999
Taurine 1 mmo1p
150.0 f 8.2 4.6(4.4-4.9) 87.0(77.7-97.5) 0.997
13.5 f 1.2t 51.5(47.4--56.o)t 13.q11.9-14.2) 0.994
311.6 f 48.4t 2.1(1.9-2.6& 83.1(71.7-98.9) 0.996 136.4 f 15.8 5.1(4.6-5.7) 12.2(9.2-16.8) 0.988
79.8 f 10.3 8.7(7.7-10.0) 8.0(6.0-10.9) 0.992
17.4 f 0.8t 39.9(38.141.7)t 4.7(4.4-5.1H 0.998
K+ 55mmol,l
264.8 f 15.7t 2.q2.5-2.8fl 86.9(79.4-96.9) 0.996
7.8 f OS* 89.3(&W-94.8)t 5.1(4.9-5.3H 0.997
Suprrfusion media Taurine 10 mmol/l K+ 25 mmol/l
262.4 f 11.5t 2.q2.5-27)t 91.9(85.2-99.7) 0.998
17.8 f 0.4t 39.0(38.1-39.8n 8.1(7.W3,3)t 0.999
K+ 55mmol/Y :’ nocakium
Synaptosomes were preincubated and superfused as in Figs l-3 in Kreb~Ri~r-~EPES media modified as indicated. The e&x of taurine was resolved into first-order rate components as described in Experimental Procedures and shown in Figs 1 and 3. The rate constants (/cl, k2, k3), half-times (t& and the relative contributions as percentages (.S1, S,, Ss) of each efflux component are piven. The correlation coefficients indicate the fit of the least-squares straight regression lines with the experimental data. Mean value are given with their 95% confidence limits, which are unsymmetric for S,, S, and &. Statistically significant differences from the control experiments are indicated as follows: *P c 0.05; tP < 0.01.
corrdatioo coetkient Intermed& component ks x 10-3mjn-1 tt,z mm s3 % correlation coe&ient
s2 %
h/2 mill
ks x lo-
Fast compcnt
SI % corrtlation cwfkient
till mill
Slow cornpent kl x lo- min-1
Parametemofthe daux components
TAISLB 1.PARA~SOFTHEeFFLUXCOMPONENTSOFTAURINEiNRATBRAlNS~AP~~
1748
P.
’
’
20
’
’
I
’
40 60 superfusiontime min
’
’
80
KONTRO
’
FIG. 2. Efflux of [%jtaurine from rat whole brain synaptosomes in different superfusion media. Synaptosomes were preincubated as in Fig. 1 and then superfused in small chambers at 310 K under O2 in different media as follows: Spontaneous efflux of taurine in unsupplemented KrebsRinger-HEPES medium, pH 7.4 (O-_-O). Efllux of taurine in Krebs-Ringer-HEPES medium, pH 7.4, containing lOmmol/l taurine (H). Efflux of taurine in KrebsRinger-HEPES medium, pH 7.4, containing 55 mmol/l K’ (A---A). Efflux of taurine in Krebs-Ringer-HEPES medium, pH 7.4, containing 55 mmol/l K+ when calcium ions are omitted (A--A). Each curve is the mean of 3-6 experiments. Standard deviations for each point were l-16%. The calculated parameters of the efflux components are given in Table 1. were calcium-independent, for the effects persisted when CaZ+ ions were omitted from the medium.
concentration the observed enhancement IS not necessarily due to homoexchange, since hynaptosomes accumulate from superfusion medium unlabeiled taurine (OJA, 1971) which may then accelerate [,“S]taurine efflux from the inside uf ~ynap~osomal membranes (L&DESM&KI & OJA. i972). i’otassium-stimulated release of taurine has been I;,mn(i in brain slices. crude synaptosomal (P,) fractions and purified synaptosomes (DE BIXEROCHE & BKAIXIWW. 1073: Cot.I.INS & TOPIWALA. 1974; SlEGHARf & tiEt.KL. 1976: VI\RC;AS,DORM IX LOREWO S: Ow t;o. 19771. The earlier studies have not. howt*\~i tinequivocally established any calcium-dependeilcc‘ 01 taurine cfflus. SIEGHART& HIXXI. (1976) obser\cd GI attenuation in the potassium-stimulated efflux nf t;rurine from the PL fraction of a cerebral cortex homogenate in the presence of calcium ions. but the ~miv,l~ln c)f calcium ions has not affected the potassilinl.~~sllked release of taurine from cerebral cortex slices :,C’CII,~ INS & TOW wAL..4, 1974) or rat visual carter (Cd iNK &L Col.r.i>s_ 1976). The electrically stimulated K!GW of taurinc has been claimed to be calcium-dcl)~!.:lldent, howcvcr (COLLINS & TWIWALA, 1974; BRAIW~RR t’t trl.. 1976). My present results show that the ;wtassium-stimulated total efflux of taurine cannot k~ tirmonstrated lo be strictly calcium-dependent. I’oths~tum stimulation of both fast (possibly taurine from &maged synaptosomes and membrane binding sites! ,ind slow (taurine in the synaptoplasm) eR3.x occurs-ed also in the absence of Ca’ ’ ions. Only rhe intermediate efflux component *as calcium-dependent. Since this component emerged at the expense of the shkwest efflux com-
DISCUSSION The spontaneous efflux of C3%]taurine from rat brain synaptosomes was divided into two components with widely differing first-order rate constants. The fast component, with a half-time of 4.5 min, probably at least partially represents an initial wash-out of [3sS]taurine from an extrasynaptosomal, loosely bound taurine pool. Any [35S]taurine retained by damaged synaptosome fragments or attached to membranes is likewise rapidly released. The slower component, with a half-time of 77.Omin, then represents the actual release of taurine from the intracellular spaces of viable synaptosomes. Taurine appears to be rather effectively retained by intrasynaptosomal compartments. In the retina intracellular taurine may be still more firmly bound (STARR & VOADEN,1972;
0.1
’
20
8
40
’
60
80
’
’
100
120
superfusion time min
FIG. 3. E&IX components of f”‘S]taurine in a higb-potassium medium. Synaptosomes were prein&&ated and superof intrasynaptosomal fused as in Fig. 1, except that the superfusion medium taurine occurred with medium containing 1 mmol/l contained 25 mmol/l K+. The total efflux curve was taurine. This is in agreement with the results ob@ned divided into three first-order rate components: a fast efffux with crude synaptosomal fractions (!%E++T & component (A---A) with tliZ = 2.9mfn, a slow- efflux HECKL, 1976) and brain slices (CRNIC, HAFAMWSTADcomponent (O----O) with t,,* = 89.3 njn and .an inter& CUTLET, 1973). At the higher taurine co&entt%tlon mediate efflux component (t--e) with ti!2 = 8.7min: The graph is the mean of four experim@&. Standard devi(lOmmol/l) the first-order rate constant of the slower ations for each point were 2-167;. efflux component increased by 50%. At such a high
KENNEDY& VOADEN,1976). No apparent homoexchange
Taurine etilux from synaptosomes
ponent, it probably also originates in some intrasynaptosomal tauriae pool(s). The intrasynaptosomally accumulated [35S]tauriae could for instance be localized in a vesicular pool and a cytoplasmic pool. The involvement of calcium ions in the intermediate e&x component might be an indication that this very component is associated with the emptying of the contents
1149
of synaptic vesicles into the medium. I do not think, however, that this n ecessarily means that taurine is a neurotransmitter. Unassorted synaptic vesicles from the brain contain much taurine (DE BELLEROCHE & BRADFORD, 1973) which could be liberated together with some proper neurotransmitter(s) when synaptosome membranes are depolarized by potassium ions.
REFERENCES H. F., DA~I~~EIA. N. & WHEELERG. H. T. (1976) Taurine and synaptic transmission. In Tuurine (cds HUXT~LE R. t BARBEAU A.), pp. 303-310. Raven Press, New York. CLARKR. M. & Cc~~trns G. G. S. (1976) The release of endogenous amino acids from the rat visual cortex. J. Physiol., Lond. 262, 383400. C~LI_,IN~ G. G. S. (1974) The rates of synthesis, uptake and disappearance of [‘*CJtaurine in eight areas of the rat central nervous system. Brain Res. 76,447-459. (&LLIN~G. G. S. & TOPWALAS. H. (1974) The release of [‘*C]taurine from slices of rat cerebral cortex and spinal cord evoked by electrical stimulation and high potassium ion concentration. Br. J. Pharmac. SO, 45lP-452P. CYNIC D. M., HAMMERSTAD J. P. & Cum R. W. (1973) Accelerated efflux of [r4C] and C3H]amino acids fram superfused slices of rat brain. J. Neurochem. 20,203209. DE BELLEROCHE J. S. & BRADFORD H. F. (1972) Metabolism of beds of mammalian cortical synaptosomes: response to depolarizing influences. J. Neurochem.19, 585-602. DE BELLEROCHE J. S. & BRADIQRDH. F. (1973) Amino acids in synaptic vesicles from mammalian cerebral cortex: A reappraisal. J. Neurochem. 21,441-451. JASPER H. H. & KOYAMAI. (1969) Rate of release of amino acids from the cerebral cortex in the cat as affected by brainstem and thalamic stimulation. Can. J. Physiol. Pharmac. 47, 889-905. KENNEDYA. J. & VOADENM. J. (1976) Studies on the uptake and release of radioactive taurine by the frog retina. J. Neurochem. 27, 131-137. KONTROP. & OJA S. S. (1978~) Taurine uptake by rat brain synaptosomes. J. Neurochem. 30, 1297-1304. KONTROP. & OJA S. S. (1978b) Sodium dependence of taurine uptake in rat brain synaptosomes. Neuroscience 3,761-765. LAAKW M.-L. (1978) Efflux of phenylalanine and tryptophan from cerebral cortex slices of adult and 7-day-old rats. Acta physiol. stand. 102.74-83. LXHDESMXKI P. & KORHONENK. (1978) Comparative studies on the degradation of GABA and taurine in the brain. J. Neurochem. 30, 705-711. LXrtossMii~~ P. & OIA S. S. (1972) Effect of electrical stimulation on the influx and et&x of taurine in brain slices of newborn and adult rats. Expl Brain Res. 15, 4-38. L;~HD&KI P., Pasuw M. & OJA S. S. (1975) Effect of electrical stimulation and chlorpromazine on the uptake and relase of taurine, y-aminobutyric acid and glutamic acid in mouse brain synaptosomes. J. Neurochem. 25, 675680. LOWRY0. H., R~~~BROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193,265-275. OJA S. S. (1971) Exchange of taurine in brain slices of adult and 7-day-old rats. J. Neurochem. 18, 1847-1852. OJA S. S. & KONTROP. (1978) Neurotransmitter actions of taurine in the central nervous system. In Taurine and Neurological Disorders (eds BARBEAU A. 8r HUXTABLER.), pp. 181-200. Raven Press, New York. OJA S. S., KONTROP. & L&t~EsMji~t P. (1976a) Transport of taurine in the central nervous system. In Transport Phenomena in the Nervous System. Physiological and Pathological Aspects. Advances in Experimental Medicine (eds LEVI G., BATTISTIN L. Br LAITHAA.). Vol. 69, pp. 237-252. Plenum Press, New York. OJA S. S., KON~ROP. & Lji~~EsMji~l P. (1977) Amino acids as inhibitory neurotransmitters. Prq. Pharmucol. l(3), l-l 19. OJA S. S., L~~T~NENI. & LXHD~MXKIP. (1976b) Taurine transport rates between plasma and tissues in adult and 7-day-old mice. Q. J. exp. Physiol. 61, 133-143. OJA S. S. & VAHVELAINEN M.-L. (1975) Transport of amino acids in brain slices. In Research Methods of Neurochemistry (eds MARKSN. 8t RODNIGHT R.). Vol. 3, pp. 67-137. Plenum Press, New York. PASANTESMORALES H., KL.ETHIJ., URBANP. F. & MANDELP. (1974) The effect of electrical stimulation, light and amino acids on the eRlux of C3-%]taurine from the retina of domestic fowl. Expl Bruin Res. 19, 131-141. S.UXEDA R. & P.Q.wTES-MORALEYH. (1975) Calcium coupled release of [“Sltaurine from retina. Brain Res. W, 206211. SIEGHARTW. L HECKL K. (1976) Potassium-evoked release of taurine from synaptosomal fractions of cerebral cortex. Brain Res. 116, 538-543. STARRM. S. & VOADEN M. J. (1972)The uptake, metabolism and release of [‘“CJtaurine by rat retina in vitro. Vision Res. 12, 1261-1269. V.uo%s O., Dcnu~ oa Lc%tl~NzOM. C. & ORRFGOF. (1977) Effect of elevated extracellular potassium on the release of labelled noradrenaline, Edutamate,@y&w, B-ala&e and other amino acids from rat brain cortex slices. Neuroscience 2, 383390. WmrrA~~a V. P. & BAR= L. A. (1972) The ~ubcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles. In Methods of Neurochemistry (ed. FRIEDR.), pp. 2-52. Marcel Dekker, New York. Bx~o~an
(Accepted 20 May 1979)
