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Sedimentation and release properties of glial particles present in P2-fractions isolated from rat cerebral cortex
Brain Research, 170 (1979) 203-208 © Elsevier/North-Holland Biomedical Press
203
Sedimentation and release properties of gliai particles present in P2-fractions isolated from rat cerebral cortex
W. SIEGHART and E. SINGER Psychiatrische Universitiitsklinik, Department of Biochemical Psychiatry, A-1097 Vienna and Institute o f Pharmacology, University o f Vienna, A-1090 Vienna (Austria)
(Accepted March 8th, 1979)
Recently it has been demonstrated that glial cells may possess several properties previously thought to play a specific role in synaptic activity. Thus, glial cells possess Na+-dependent high affinity transport systems for GABA 6,9,17,19,2°, glutamate 7,12,1n and taurinel,n, 19 and release radiolabeled GABA by a K+-stimulated process2,13, 21. The stimulated release of radiolabeled amino acids from several glial systems seems to be independent of exogenous Ca 2+ (refs. 11, 21 and 26). However, the K+-stimulated release of [3H]GABA from dorsal root glial cells is enhanced in the presence of Ca 2÷ (ref. 13). On homogenization, glial cells form particles3,7,23, 25 which are able to accumulate radiolabeled glutamate 7 or taurine z3 similarly to intact cells. Since even highly purified synaptosomal fractions may be contaminated up to 40 ~o with glial material s, in a previous report it was investigated whether there are any differences in the sedimentation characteristics of synaptosomes and particles derived from bulk isolated glia, C-6 glioma cells or dorsal root glia cells 25. It was found that the buoyant density of glial particles depended on the source of the glial material and that particles derived from bulk isolated glial cells sedimented nearly identically to synaptosomes. In the present investigation a different approach was used to characterize the sedimentation properties of glial particles contaminating P2-fractions isolated from rat cerebral cortex. It has been demonstrated that [3H]fl-alanine is selectively accumulated into cortical glial cells when brain slices are incubated with this radiolabeled amino acid is. In contrast, radiolabeled GABA is accumulated selectively into nerve endings during a short incubation of slices prepared from rat cerebral cortex ~0. Thus, P2fractions were prepared from slices previously incubated with [3H]fl-alanine or [14C]GABA and the sedimentation properties of [ZH]fl-alanine-containing glial particles and [14C]GABA-containing nerve ending particles were compared. In addition, the K+-evoked release of radiolabeled fl-alanine or GABA from these particles and its modification by the presence of Ca z÷ was investigated. Sprague-Dawley rats (150-250 g) were killed by decapitation and cerebral cortex slices were prepared as previously described 22. The slices were suspended in ice-cold oxygenated Krebs-Ringer medium (KR-medium) (composition: 118.5 mM NaC1, 4.75 mM KCI, 0.88 mM CaCI2, 1.18 mM MgSO4, 5.0 mM D-glucose and 16.2 mM so-
204 dium phosphate buffer, pH 7.4) and centrifuged at 1000 × g for 5 min. Slices originating from 1 g of cortex tissue were then resuspended in 10 ml of oxygenated K R medium. After 20 min of preincubation at 37 °C, portions of the suspension equivalent to 100 mg of tissue wet weight were incubated for 30 min with [3H]fl-alanine (fl-[33H]alanine, 0.3 ~M, 32 Ci/mmol, Radiochemical Center, Amersham) or for 5 min with either [14C]GABA (4-amino-n[U-14C]butyric acid, 0.6/~M, 224 mCi/mmol, Radiochemical Center, Amersham) or [3H]GABA (4-amino-n-[2,3-3H]butyric acid, 0.1 #M, 66 Ci/mmol, Radiochemical Center, Amersham). The short incubation time of cortex slices with radiolabeled GABA was used to ensure a specific accumulation of GABA into nerve endings since with longer incubation times appreciable amounts of GABA may be accumulated by the slow glial uptake system 17. At the end of the incubation period 5 ml of ice-cold 0.32 M sucrose containing 2 mM Tris.HCl, pH 7.4, were added to the suspensions and the tubes were centrifuged for 5 min at 4000 × g. Slices were resuspended in 1 ml 0.32 M sucrose-2 mM Tris-HC1, pH 7.4, P2-fractions were prepared as described previously 22 and washed once by centrifugation of the resuspended pellets at 10,000 × g for 20 min. In some experiments crude microsomal (P3) fractions were isolated by centrifugation of the supernatant of the Pz-fractions at 100,000 × g for 60 min. Density gradient centrifugation, fractionation of the gradients, and estimation of MAO activity and radioactivity in the fractions were performed as previously describedZL Homogenization of slices previously incubated with [aH]fl-alanine or [14C]GABA and differential centrifugation resulted in P2-fractions which retained 25 i 1.3 ~ (mean ± S.E.M., n = 11) of the [aH]fl-alanine or 33 I- 1,4 ~ (mean ± S.E.M., n = 14) of the [14C]GABA radioactivity accumulated into the slices. This [aH]fl-alanine or [14C]GABA radioactivity seemed to be inside particles rather than adsorbed to membranes since sonication or resuspension of the P2-fractions in distilled water resulted in a nearly complete loss of radioactivity from sedimentable membrane fragments. Microsomes did not seem to constitute a significant portion of these [3H]fl-alanine or [14C]GABA-containing particles, since crude microsomal fractions contained only 3.2 ± 0.4 ~ (mean _-k S.E.M., n -- 8) of the [SH] or [14C]radioactivity accumulated into the slices. Furthermore, when [aH]fl-alanine or [14C]GABA was added to slices immediately prior to homogenization, the resulting P2-fractions contained only about 8 ~ of the radioactivity of Pz-fractions recovered from slices previously incubated with the radiolabeled amino acids. This indicated that redistribution of radioactivity during homogenization was negligible. Since autoradiographic evidence indicates a selective accumulation of [aH]-fl-alanine into glial cells is and of [14C]GABA into nerve endings 10, these results suggest, that on homogenization of cortical slices glial particles arise which retain a rather large part of the previously accumulated [aH]fl-alanine and which sediment similar to synaptosomes in P2-fractions. In order to compare the buoyant density of [aH]fl-alanine-containing particles with that of synaptosomes, slices of rat cerebral cortex, previously incubated with [14C]GABA, were co-homogenized with slices previously incubated with [aH]fl-alanine. P2-fractions were isolated and sedimented through a linear sucrose density gradient, ranging from 0.32-1.5 M sucrose, for 2 h at 25,000 r.p.m. Under these condi-
205 tions, [3H]fl-alanine-containing particles sedimented to a buoyant density similar to [14C]GABA-containing synaptosomes (Fig. 1). Mitochondria as localized by their M A O activity sedimented to a higher buoyant density than both [3H]fl-alanine and [14C]GABA-containing particles. Identical results were obtained, when slices previously incubated with [3H]fl-alanine or with [14C]GABA were homogenized separately, and the resulting P~-fractions were mixed just before sedimentation through a density gradient. P2-fractions isolated from slices previously incubated with [3H]GABA or [aH]/5alanine were used to investigate the release properties of [3H]GABA- or [3H]fl-alaninecontaining particles. Release was investigated using a superfusion apparatus as previously described 15,24. Thus, the washed and resuspended P2-fractions isolated from 70-100 mg tissue slices previously incubated with the radiolabeled amino acids were immobilized on W h a t m a n G F / B filters and continuously superfused at 0.5 ml/min with an oxygenated superfusion medium 24 containing either 0.5 m M E G T A (ethyleneglycolbis (fl-aminoethylether) N, Nl-tetraacetic acid) or 0.88 m M CaCl2, and kept at 37 °C with a thermostatted jacket. T w o m i n fractions of the superfusates were collected directly into liquid scintillation counting vials. After 11 min of superfusion the medium was replaced either by a medium of the same composition or by a high K+-medium containing 56 m M KC1 (replacing an equimolar concentration of NaCI). Liquid scin-
.3
["]OABA .2
//
,]/// 2/
i Frm:tkm
Fig. 1. Density gradient centrifugation of P~-fractions isolated from slices previously incubated with laH]fl-alanine or [14C]GABA. Slices of rat cerebral cortex, previously incubated with [aH]fl-alanine were co-homogenized with slices previously incubated with [14C]GABA. P2-fractions were isolated and centrifuged for 2 h at 25,000 r.p.m, in a Beckman SW 27-2 rotor, in a linear sucrose density gradient ranging for 0.32-1.5 M sucrose. The gradient was pierced at the bottom and radioactivity or MAO activity was determined in aliquots of the collected gradient fractions as previously described22. MAO activity was expressed as nmol [t4C]indoleacetic acid formed in 60 min.
206
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7 6
!, 56ram K+
I
o
!
8
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4
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3H]8-Alanine __ : 0,88mMCa2+ \ ~ , 5 mM EGTA
3
2
1
56mM K+ I
I
I
I
0
8
16
24
I
32
min
Fig. 2. Effect of depolarizing concentrations of KCI in the absence or presence of CaCle on [3H]GABA or [3H]fl-alanine release from P2-fractions isolated from slices previously incubated with the radiolabeled amino acids. Slices of rat cerebral cortex, previously incubated with [aH]GABA or [aH]fl-alanine were homogenized, P2-fractions were isolated and continuously superfused with a superfusion medium24 containing either 0.5 mM EGTA or 0.88 mM CaCI2. During the time period indicated by the bar the superfusion medium was rapidly replaced by a superfusion medium in which part of the NaC1 had been iso-osmotically replaced by KC1. Replacement of the superfusion medium with a medium of the same composition did not change the spontaneous efflux of [SH]GABA or [SH]fl-alanine. Two-min fractions of the superfusates were collected. Radioactivity released during a 2-min period was expressed as percentage of total radioactivity. Total radioactivity was calculated by adding the radioactivity released during the whole superfusion experiment to the radioactivity remaining on the filters. Each point represents the mean of at least 6 separate experiments.
tillation c o u n t i n g of superfusates a n d filters was performed as described previously 24. M e t a b o l i s m of radiolabeled fl-alanine a n d G A B A was measured by means of paperc h r o m a t o g r a p h y la,z2. More t b a n 80 ~ of [3H]GABA or 98 ~ of [aH]fl-alanine released during superfusion or present o n the filters at the e n d of the experiments were f o u n d to remain unmetabolized. O n c o n t i n u o u s superfusion of Pa-fractions there was a
207 spontaneous effux of [aH]GABA or [aH]fl-alanine which was reversibly increased by rapid replacement of the superfusion medium with a medium containing 56 m M K + (Fig. 2). In agreement with previous results4,14, the K+-evoked release of [aH]GABA was increased in the presence of 0.88 m M Ca 2+. In contrast, the K+-evoked release of [aH]fl-alanine was not changed in the presence of Ca 2+ (Fig. 2). Since [aH]fl-alaninecontaining particles presumably are derived from glial cells, this result is in agreement with several reports indicating that the stimulus-induced release of amino acids from glial cells seems to be independent of exogenous Ca 2+ (refs. 11, 21 and 26). Thus, the Ca 2+independency of the K+-evoked release might be a criterion to distinguish glial particles from synaptosomes. In conclusion, the present results confirm previous evidence, that on homogenization of brain tissue glial particles arise, which behave similarly to synaptosomes during differential sedimentation or density gradient centrifugationa, 7,23,2z. It has been shown previously that these particles are still able to accumulate putative transmitter substances 7,2a and the present study indicates that in addition these particles are able to release amino acids by a K+-dependent, Ca2+-independent mechanism. Thus, glial particles not only contaminate Pz-fractions to an appreciable extent, but also in many respects resemble nerve ending particles and therefore care must be exercised in interpreting results obtained with synaptosomal preparations. We gratefully acknowledge the skillful technical assistance of Martina Scherer and Eva Kotai and thank J. Reisenhofer for typing the manuscript. This work was supported by 'Fonds zur F6rderung der wissenschaftlichen Forschung in 0sterreich'. 1 Borg, J., Balcar, V. J., and Mandel, P., High affinity uptake of taurine in neuronal and glial cells, Brain Research, 118 (1976) 514-516. 2 Bowery, N. G. and Brown, D. A., ?-aminobutyric acid uptake by sympathetic ganglia, Nature New Biol., 238 (1972) 89-91. 3 Cotman, C. W., Herschman, H. and Taylor, D., Subcellular fractionation of cultured glial cells, J. Neurobiol., 2 (1971) 169-180. 4 Cotman, C. W., Haycock, J. W. and Frost White, W., Stimulus-secretion coupling processes in brain: analysis of noradrenaline and gamma-aminobutyric acid release, J. Physiol. (Lond.), 254 (1976) 475-505. 5 Ehinger, B., Glial uptake of taurine in the rabbit retina, Brain Reserach, 60 (1973) 512-516. 6 Henn, F. A. and Hamberger, A., Glial cell function: uptake of transmitter substances, Proc. nat. Acad. Sci. Wash., 68 (1971) 2686-2690. 7 Henn, F. A., Goldstein, M. N. and Hamberger, A., Uptake of the neurotransmitter candidate glutamate by glia, Nature (Lond.), 249 (1974) 663-664. 8 Henn, F. A., Anderson, D. J. and Rustad, D. G., Glial contamination of synaptosomal fractions, Brain Research, 101 (1976) 341-344. 9 Hutchison, H. T., Werrbach, K., Vance, C. and Haber, B., Uptakeof neurotransmitters by clonal lines of astrocytoma and neuroblastoma in culture. I. Transport of ?-arninobutyric acid, Brain Research, 66 (1974) 265-274. 10 Iversen, L. L. and Bloom, F. E., Studies of the uptake of [aH]GABA and [aH]glycinein slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography, Brain Research, 41 (1972) 131-143. 11 Johnston, G. A. R., Effects of calcium on the potassium-stimulated release of radioactive fl-alanine and ?-arninobutyric acid from slices of rat cerebral cortex and spinal cord, Brain Research, 121 (1977) 179-181.
208 12 McLennan, H., The autoradiographic localization of L-[3H]glutarnate in rat brain tissue, Brain Research, 115 (1976) 139-144. 13 Minchin, M. C. W. and Iversen, L. L., Release of [3H]gamma-aminobutyric acid from glial cells in rat dorsal root ganglia, J. Neurochem., 23 (1974) 533-540. 14 Placheta, P., Singer, E., Sieghart, W. and Karobath, M., submitted for publication. 15 Raiteri, M. Angelini, F. and Levi, G., A simple apparatus for studying the release of neurotransmitters from synaptosomes, Europ. J. PharmacoL, 25 (1974) 411-414. 16 Schon, F. and Kelly, J. S., Autoradiographic localization of [ZH]GABA and [all]glutamate over satellite glial cells, Brain Research, 66 (1974) 275-288. 17 Schon, F. and Kelly, J. S., The characterization of [3H]GABA uptake into the satellite glial cells of rat sensory ganglia, Brain Research, 66 (1974) 289-300. 18 Schon, F. and Kelly, J. S., Selective uptake of [3H]fl-alanine by glia: association with the glial uptake system for GABA, Brain Research, 86 (1975) 243-257. 19 Schrier, B. K. and Thompson E. J., On the role of glial cells in the mammalian nervous system. Uptake, excretion and metabolism of putative neurotransmitters by cultured glial tumor cells, J. biol. Chem., 249 (1974) 1769-1780. 20 Sellstrtim, A. and Hamberger, A., Neuronal and glial systems for 7'-aminobutyric acid transport, J. Neurochem., 24 (1975) 847-852. 21 Sellstr~m, A. and Hamberger, A., Potassium-stimulated 7'-aminobutyric acid release from neurons and glia, Brain Research, 119 (1977) 189-198. 22 Sieghart, W. and Karobath, M., Evidence for specific synaptosomal localization of exogenous accumulated taurine, J. Neurochem., 23 (1974) 911-915. 23 Sieghart, W. and Karobath, M., Uptake of taurine into subcetlular fractions of C-6 glioma cells, J. Neurochem., 26 (1976) 981-986. 24 Sieghart, W. and Heckl, K., Potassium-evoked release of taurine from synaptosomal fractions of rat cerebral cortex, Brain Research, 116 (1976) 538-543. 25 Sieghart, W., Sellstr6m, A. and Henn, F., Sedimentation characteristics of subcellular vesicles derived from three glial systems, J. Neurochem., 30 (1978) 1587-1589. 26 Weinreich, D. and Hammerschlag, R., Nerve impulse-enhanced release of amino acids from nonsynaptic regions of peripheral and central nerve trunks of bullfrog, Brain Research, 84 (1975) 137-142.
203
Sedimentation and release properties of gliai particles present in P2-fractions isolated from rat cerebral cortex
W. SIEGHART and E. SINGER Psychiatrische Universitiitsklinik, Department of Biochemical Psychiatry, A-1097 Vienna and Institute o f Pharmacology, University o f Vienna, A-1090 Vienna (Austria)
(Accepted March 8th, 1979)
Recently it has been demonstrated that glial cells may possess several properties previously thought to play a specific role in synaptic activity. Thus, glial cells possess Na+-dependent high affinity transport systems for GABA 6,9,17,19,2°, glutamate 7,12,1n and taurinel,n, 19 and release radiolabeled GABA by a K+-stimulated process2,13, 21. The stimulated release of radiolabeled amino acids from several glial systems seems to be independent of exogenous Ca 2+ (refs. 11, 21 and 26). However, the K+-stimulated release of [3H]GABA from dorsal root glial cells is enhanced in the presence of Ca 2÷ (ref. 13). On homogenization, glial cells form particles3,7,23, 25 which are able to accumulate radiolabeled glutamate 7 or taurine z3 similarly to intact cells. Since even highly purified synaptosomal fractions may be contaminated up to 40 ~o with glial material s, in a previous report it was investigated whether there are any differences in the sedimentation characteristics of synaptosomes and particles derived from bulk isolated glia, C-6 glioma cells or dorsal root glia cells 25. It was found that the buoyant density of glial particles depended on the source of the glial material and that particles derived from bulk isolated glial cells sedimented nearly identically to synaptosomes. In the present investigation a different approach was used to characterize the sedimentation properties of glial particles contaminating P2-fractions isolated from rat cerebral cortex. It has been demonstrated that [3H]fl-alanine is selectively accumulated into cortical glial cells when brain slices are incubated with this radiolabeled amino acid is. In contrast, radiolabeled GABA is accumulated selectively into nerve endings during a short incubation of slices prepared from rat cerebral cortex ~0. Thus, P2fractions were prepared from slices previously incubated with [3H]fl-alanine or [14C]GABA and the sedimentation properties of [ZH]fl-alanine-containing glial particles and [14C]GABA-containing nerve ending particles were compared. In addition, the K+-evoked release of radiolabeled fl-alanine or GABA from these particles and its modification by the presence of Ca z÷ was investigated. Sprague-Dawley rats (150-250 g) were killed by decapitation and cerebral cortex slices were prepared as previously described 22. The slices were suspended in ice-cold oxygenated Krebs-Ringer medium (KR-medium) (composition: 118.5 mM NaC1, 4.75 mM KCI, 0.88 mM CaCI2, 1.18 mM MgSO4, 5.0 mM D-glucose and 16.2 mM so-
204 dium phosphate buffer, pH 7.4) and centrifuged at 1000 × g for 5 min. Slices originating from 1 g of cortex tissue were then resuspended in 10 ml of oxygenated K R medium. After 20 min of preincubation at 37 °C, portions of the suspension equivalent to 100 mg of tissue wet weight were incubated for 30 min with [3H]fl-alanine (fl-[33H]alanine, 0.3 ~M, 32 Ci/mmol, Radiochemical Center, Amersham) or for 5 min with either [14C]GABA (4-amino-n[U-14C]butyric acid, 0.6/~M, 224 mCi/mmol, Radiochemical Center, Amersham) or [3H]GABA (4-amino-n-[2,3-3H]butyric acid, 0.1 #M, 66 Ci/mmol, Radiochemical Center, Amersham). The short incubation time of cortex slices with radiolabeled GABA was used to ensure a specific accumulation of GABA into nerve endings since with longer incubation times appreciable amounts of GABA may be accumulated by the slow glial uptake system 17. At the end of the incubation period 5 ml of ice-cold 0.32 M sucrose containing 2 mM Tris.HCl, pH 7.4, were added to the suspensions and the tubes were centrifuged for 5 min at 4000 × g. Slices were resuspended in 1 ml 0.32 M sucrose-2 mM Tris-HC1, pH 7.4, P2-fractions were prepared as described previously 22 and washed once by centrifugation of the resuspended pellets at 10,000 × g for 20 min. In some experiments crude microsomal (P3) fractions were isolated by centrifugation of the supernatant of the Pz-fractions at 100,000 × g for 60 min. Density gradient centrifugation, fractionation of the gradients, and estimation of MAO activity and radioactivity in the fractions were performed as previously describedZL Homogenization of slices previously incubated with [aH]fl-alanine or [14C]GABA and differential centrifugation resulted in P2-fractions which retained 25 i 1.3 ~ (mean ± S.E.M., n = 11) of the [aH]fl-alanine or 33 I- 1,4 ~ (mean ± S.E.M., n = 14) of the [14C]GABA radioactivity accumulated into the slices. This [aH]fl-alanine or [14C]GABA radioactivity seemed to be inside particles rather than adsorbed to membranes since sonication or resuspension of the P2-fractions in distilled water resulted in a nearly complete loss of radioactivity from sedimentable membrane fragments. Microsomes did not seem to constitute a significant portion of these [3H]fl-alanine or [14C]GABA-containing particles, since crude microsomal fractions contained only 3.2 ± 0.4 ~ (mean _-k S.E.M., n -- 8) of the [SH] or [14C]radioactivity accumulated into the slices. Furthermore, when [aH]fl-alanine or [14C]GABA was added to slices immediately prior to homogenization, the resulting P2-fractions contained only about 8 ~ of the radioactivity of Pz-fractions recovered from slices previously incubated with the radiolabeled amino acids. This indicated that redistribution of radioactivity during homogenization was negligible. Since autoradiographic evidence indicates a selective accumulation of [aH]-fl-alanine into glial cells is and of [14C]GABA into nerve endings 10, these results suggest, that on homogenization of cortical slices glial particles arise which retain a rather large part of the previously accumulated [aH]fl-alanine and which sediment similar to synaptosomes in P2-fractions. In order to compare the buoyant density of [aH]fl-alanine-containing particles with that of synaptosomes, slices of rat cerebral cortex, previously incubated with [14C]GABA, were co-homogenized with slices previously incubated with [aH]fl-alanine. P2-fractions were isolated and sedimented through a linear sucrose density gradient, ranging from 0.32-1.5 M sucrose, for 2 h at 25,000 r.p.m. Under these condi-
205 tions, [3H]fl-alanine-containing particles sedimented to a buoyant density similar to [14C]GABA-containing synaptosomes (Fig. 1). Mitochondria as localized by their M A O activity sedimented to a higher buoyant density than both [3H]fl-alanine and [14C]GABA-containing particles. Identical results were obtained, when slices previously incubated with [3H]fl-alanine or with [14C]GABA were homogenized separately, and the resulting P~-fractions were mixed just before sedimentation through a density gradient. P2-fractions isolated from slices previously incubated with [3H]GABA or [aH]/5alanine were used to investigate the release properties of [3H]GABA- or [3H]fl-alaninecontaining particles. Release was investigated using a superfusion apparatus as previously described 15,24. Thus, the washed and resuspended P2-fractions isolated from 70-100 mg tissue slices previously incubated with the radiolabeled amino acids were immobilized on W h a t m a n G F / B filters and continuously superfused at 0.5 ml/min with an oxygenated superfusion medium 24 containing either 0.5 m M E G T A (ethyleneglycolbis (fl-aminoethylether) N, Nl-tetraacetic acid) or 0.88 m M CaCl2, and kept at 37 °C with a thermostatted jacket. T w o m i n fractions of the superfusates were collected directly into liquid scintillation counting vials. After 11 min of superfusion the medium was replaced either by a medium of the same composition or by a high K+-medium containing 56 m M KC1 (replacing an equimolar concentration of NaCI). Liquid scin-
.3
["]OABA .2
//
,]/// 2/
i Frm:tkm
Fig. 1. Density gradient centrifugation of P~-fractions isolated from slices previously incubated with laH]fl-alanine or [14C]GABA. Slices of rat cerebral cortex, previously incubated with [aH]fl-alanine were co-homogenized with slices previously incubated with [14C]GABA. P2-fractions were isolated and centrifuged for 2 h at 25,000 r.p.m, in a Beckman SW 27-2 rotor, in a linear sucrose density gradient ranging for 0.32-1.5 M sucrose. The gradient was pierced at the bottom and radioactivity or MAO activity was determined in aliquots of the collected gradient fractions as previously described22. MAO activity was expressed as nmol [t4C]indoleacetic acid formed in 60 min.
206
[3.]Q,.
7 6
!, 56ram K+
I
o
!
8
2'4
4
3'2
3H]8-Alanine __ : 0,88mMCa2+ \ ~ , 5 mM EGTA
3
2
1
56mM K+ I
I
I
I
0
8
16
24
I
32
min
Fig. 2. Effect of depolarizing concentrations of KCI in the absence or presence of CaCle on [3H]GABA or [3H]fl-alanine release from P2-fractions isolated from slices previously incubated with the radiolabeled amino acids. Slices of rat cerebral cortex, previously incubated with [aH]GABA or [aH]fl-alanine were homogenized, P2-fractions were isolated and continuously superfused with a superfusion medium24 containing either 0.5 mM EGTA or 0.88 mM CaCI2. During the time period indicated by the bar the superfusion medium was rapidly replaced by a superfusion medium in which part of the NaC1 had been iso-osmotically replaced by KC1. Replacement of the superfusion medium with a medium of the same composition did not change the spontaneous efflux of [SH]GABA or [SH]fl-alanine. Two-min fractions of the superfusates were collected. Radioactivity released during a 2-min period was expressed as percentage of total radioactivity. Total radioactivity was calculated by adding the radioactivity released during the whole superfusion experiment to the radioactivity remaining on the filters. Each point represents the mean of at least 6 separate experiments.
tillation c o u n t i n g of superfusates a n d filters was performed as described previously 24. M e t a b o l i s m of radiolabeled fl-alanine a n d G A B A was measured by means of paperc h r o m a t o g r a p h y la,z2. More t b a n 80 ~ of [3H]GABA or 98 ~ of [aH]fl-alanine released during superfusion or present o n the filters at the e n d of the experiments were f o u n d to remain unmetabolized. O n c o n t i n u o u s superfusion of Pa-fractions there was a
207 spontaneous effux of [aH]GABA or [aH]fl-alanine which was reversibly increased by rapid replacement of the superfusion medium with a medium containing 56 m M K + (Fig. 2). In agreement with previous results4,14, the K+-evoked release of [aH]GABA was increased in the presence of 0.88 m M Ca 2+. In contrast, the K+-evoked release of [aH]fl-alanine was not changed in the presence of Ca 2+ (Fig. 2). Since [aH]fl-alaninecontaining particles presumably are derived from glial cells, this result is in agreement with several reports indicating that the stimulus-induced release of amino acids from glial cells seems to be independent of exogenous Ca 2+ (refs. 11, 21 and 26). Thus, the Ca 2+independency of the K+-evoked release might be a criterion to distinguish glial particles from synaptosomes. In conclusion, the present results confirm previous evidence, that on homogenization of brain tissue glial particles arise, which behave similarly to synaptosomes during differential sedimentation or density gradient centrifugationa, 7,23,2z. It has been shown previously that these particles are still able to accumulate putative transmitter substances 7,2a and the present study indicates that in addition these particles are able to release amino acids by a K+-dependent, Ca2+-independent mechanism. Thus, glial particles not only contaminate Pz-fractions to an appreciable extent, but also in many respects resemble nerve ending particles and therefore care must be exercised in interpreting results obtained with synaptosomal preparations. We gratefully acknowledge the skillful technical assistance of Martina Scherer and Eva Kotai and thank J. Reisenhofer for typing the manuscript. This work was supported by 'Fonds zur F6rderung der wissenschaftlichen Forschung in 0sterreich'. 1 Borg, J., Balcar, V. J., and Mandel, P., High affinity uptake of taurine in neuronal and glial cells, Brain Research, 118 (1976) 514-516. 2 Bowery, N. G. and Brown, D. A., ?-aminobutyric acid uptake by sympathetic ganglia, Nature New Biol., 238 (1972) 89-91. 3 Cotman, C. W., Herschman, H. and Taylor, D., Subcellular fractionation of cultured glial cells, J. Neurobiol., 2 (1971) 169-180. 4 Cotman, C. W., Haycock, J. W. and Frost White, W., Stimulus-secretion coupling processes in brain: analysis of noradrenaline and gamma-aminobutyric acid release, J. Physiol. (Lond.), 254 (1976) 475-505. 5 Ehinger, B., Glial uptake of taurine in the rabbit retina, Brain Reserach, 60 (1973) 512-516. 6 Henn, F. A. and Hamberger, A., Glial cell function: uptake of transmitter substances, Proc. nat. Acad. Sci. Wash., 68 (1971) 2686-2690. 7 Henn, F. A., Goldstein, M. N. and Hamberger, A., Uptake of the neurotransmitter candidate glutamate by glia, Nature (Lond.), 249 (1974) 663-664. 8 Henn, F. A., Anderson, D. J. and Rustad, D. G., Glial contamination of synaptosomal fractions, Brain Research, 101 (1976) 341-344. 9 Hutchison, H. T., Werrbach, K., Vance, C. and Haber, B., Uptakeof neurotransmitters by clonal lines of astrocytoma and neuroblastoma in culture. I. Transport of ?-arninobutyric acid, Brain Research, 66 (1974) 265-274. 10 Iversen, L. L. and Bloom, F. E., Studies of the uptake of [aH]GABA and [aH]glycinein slices and homogenates of rat brain and spinal cord by electron microscopic autoradiography, Brain Research, 41 (1972) 131-143. 11 Johnston, G. A. R., Effects of calcium on the potassium-stimulated release of radioactive fl-alanine and ?-arninobutyric acid from slices of rat cerebral cortex and spinal cord, Brain Research, 121 (1977) 179-181.
208 12 McLennan, H., The autoradiographic localization of L-[3H]glutarnate in rat brain tissue, Brain Research, 115 (1976) 139-144. 13 Minchin, M. C. W. and Iversen, L. L., Release of [3H]gamma-aminobutyric acid from glial cells in rat dorsal root ganglia, J. Neurochem., 23 (1974) 533-540. 14 Placheta, P., Singer, E., Sieghart, W. and Karobath, M., submitted for publication. 15 Raiteri, M. Angelini, F. and Levi, G., A simple apparatus for studying the release of neurotransmitters from synaptosomes, Europ. J. PharmacoL, 25 (1974) 411-414. 16 Schon, F. and Kelly, J. S., Autoradiographic localization of [ZH]GABA and [all]glutamate over satellite glial cells, Brain Research, 66 (1974) 275-288. 17 Schon, F. and Kelly, J. S., The characterization of [3H]GABA uptake into the satellite glial cells of rat sensory ganglia, Brain Research, 66 (1974) 289-300. 18 Schon, F. and Kelly, J. S., Selective uptake of [3H]fl-alanine by glia: association with the glial uptake system for GABA, Brain Research, 86 (1975) 243-257. 19 Schrier, B. K. and Thompson E. J., On the role of glial cells in the mammalian nervous system. Uptake, excretion and metabolism of putative neurotransmitters by cultured glial tumor cells, J. biol. Chem., 249 (1974) 1769-1780. 20 Sellstrtim, A. and Hamberger, A., Neuronal and glial systems for 7'-aminobutyric acid transport, J. Neurochem., 24 (1975) 847-852. 21 Sellstr~m, A. and Hamberger, A., Potassium-stimulated 7'-aminobutyric acid release from neurons and glia, Brain Research, 119 (1977) 189-198. 22 Sieghart, W. and Karobath, M., Evidence for specific synaptosomal localization of exogenous accumulated taurine, J. Neurochem., 23 (1974) 911-915. 23 Sieghart, W. and Karobath, M., Uptake of taurine into subcetlular fractions of C-6 glioma cells, J. Neurochem., 26 (1976) 981-986. 24 Sieghart, W. and Heckl, K., Potassium-evoked release of taurine from synaptosomal fractions of rat cerebral cortex, Brain Research, 116 (1976) 538-543. 25 Sieghart, W., Sellstr6m, A. and Henn, F., Sedimentation characteristics of subcellular vesicles derived from three glial systems, J. Neurochem., 30 (1978) 1587-1589. 26 Weinreich, D. and Hammerschlag, R., Nerve impulse-enhanced release of amino acids from nonsynaptic regions of peripheral and central nerve trunks of bullfrog, Brain Research, 84 (1975) 137-142.