0736-5748/84 $113.110+ O.0~1 Pergamon Press ktd. © 1984 ISDN
lnt. J. Devl. Neuroscience, Vo[. 2, No. 6, pp. 529-534, 1984. Printed in Great Britain.
M O D I F I C A T I O N OF T A U R I N E A N D H Y P O T A U R I N E U P T A K E S Y S T E M S IN C U L T U R E D P R I M A R Y A S T R O C Y T E S BY SERUM-FREE MEDIUM AND DIBUTYRYL CYCLIC AMP TREATMENT I. HOLOPAINEN
Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 1tl, Finland (Accepted 4 April 1984)
Abstract--The characteristics of taurine and hypotaurine uptake were studied during astrocyte maturation in cultures grown in normal, serum-containing medium or in serum-free medium in the presence of 0.1 mmol/l dibutyryl cyclic AMP (dBcAMP). The uptake of both amino acids consisted of one saturable high-affinity component in both control and treated cultures. The dBcAMP treatment produced no marked modification of the transport systems. In the treated cultures the kinetic parameters of taurine and hypotaurine uptake remained unaltered during maturation. In the control cells the transport constant and maximal velocity of taurine uptake were greater in 21-day-old than in 16-day-old cultures, while the changes in hypotaurine transport were the opposite. The uptakes were strictly sodium-dependent and also considerably decreased when potassium ions were omitted from incubation medium. The uptake of both amino acids was affected more by potassium omission in the dBcAMPtreated than in the control cultures. Also the results with metabolic poisons suggest that physiological ion gradients sustained by an active Na +, K+-pump are essential for the normal uptake of taurine and hypotaurine. Key words: Astrocyte culture, Taurine, Hypotaurine, Transport.
T a u r i n e ( 2 - a m i n o e t h a n e s u l p h o n i c acid) a n d its p r e c u r s o r h y p o t a u r i n e ( 2 - a m i n o e t h a n e s u l p h i n i c acid) inhibit firing of c e n t r a l n e u r o n s . 21'27 T h e y a r e r e l e a s e d f r o m b r a i n slices a n d s y n a p t o s o m e s by d e p o l a r i z i n g stimuli. 11'16 T h e p o s s i b l e s y n a p t i c actions of t a u r i n e a n d h y p o t a u r i n e c o u l d be t e r m i n a t e d by a s o d i u m - d e p e n d e n t high-affinity r e - u p t a k e into s u r r o u n d i n g glial a n d / o r n e u r o n a l cells. A l t h o u g h the u p t a k e s y s t e m s h a v e b e e n s t u d i e d in v a r i o u s n e r v o u s tissue p r e p a r a tions,4,s,9,~3 ~5,23,2s the role o f glial cells in h y p o t a u r i n e t r a n s p o r t has r e m a i n e d u n d e f i n e d . L i k e w i s e o n l y v e r y little is k n o w n of t h e u p t a k e s y s t e m s d u r i n g cell m a t u r a t i o n . 4 T a u r i n e a n d h y p o t a u r i n e u p t a k e in c u l t u r e d p r i m a r y a s t r o c y t e s was n o w s t u d i e d at d i f f e r e n t stages of cell m a t u r a t i o n . A n u m b e r of s p e c i m e n s w e r e also s u b j e c t e d to d i b u t y r y l cyclic A M P ( d B c A M P ) c o m b i n e d with s e r u m w i t h d r a w a l f r o m m e d i u m . This t r e a t m e n t has b e e n shown to i n d u c e m o r p h o l o g i c a l a n d b i o c h e m i c a l c h a n g e s in c u l t u r e d a s t r o c y t e s , io,~9,22,29
EXPERIMENTAL
PROCEDURES
Materials T h e r a d i o l a b e l l e d a m i n o acids a n d cell c u l t u r e m e d i a u s e d have b e e n d e s c r i b e d in detail e a r l i e r . 8~9 N 6 , O 2 ' - d i b u t y r y l - a d e n o s i n e 3 ' , 5 ' - c y c l i c m o n o p h o s p h a t e ( d B c A M P ) was p r o d u c e d b y S i g m a (St. L o u i s ) .
Cell culture A l l e x p e r i m e n t s w e r e c a r r i e d o u t using t h e glial cell c u l t u r e s y s t e m d e v e l o p e d by B o o h e r & S e n s e n b r e n n e r , 1 with c e r t a i n m o d i f i c a t i o n s . 3° C e r e b r a l h e m i s p h e r e s of n e w b o r n W i s t a r rats w e r e e x c i s e d , m e n i n g e s c a r e f u l l y r e m o v e d a n d t h e tissue b l o c k s r i n s e d in c u l t u r e m e d i u m . T w o h e m i s p h e r e s w e r e p r e s s e d with t e f l o n p e s t l e t h r o u g h a sterile 80 ~xm p o r e - s i z e n y l o n sieve into 10 ml o f c u l t u r e m e d i u m . T h e cell s u s p e n s i o n was c a r e f u l l y t r i t u r a t e d a n d a s a m p l e of 0.5 ml i n t r o d u c e d into tissue c u l t u r e dishes (35 x 10 m m , N u n c A / S , D e n m a r k ) c o n t a i n i n g 2.0 ml of c u l t u r e m e d i u m , E a g l e ' s m i n i m u m e s s e n t i a l m e d i u m s u p p l e m e n t e d with s o d i u m b i c a r b o n a t e (2.2 g/l), h e a t - i n a c t i v a t e d f o e t a l calf s e r u m ( 1 0 % ) a n d L - g l u t a m i n e (final c o n c e n t r a t i o n 2 m m o l / l ) . T h e 529
530
1. t t~lopainen
dishes were kept at 310 K in humidified atmospheric air containing 5'7o (v/v) CO2. Antibiotics. penicillin (200000 IU/1) and streptomycin (200 rag/l) were added to prevent bacterial contamination. The culture medium was first changed after 2 days and subsequently three times a week. In some cultures foetal calf serum was removed after 2 weeks and 0.1 retool/1 dBcAMP added to medium. These treated specimens were further cultured for 2 or 7 days prior to experiments. Uptake experiments The uptake of labelled taurine and hypotaurine was assayed as previously described, s'~ Briefly, the experiments were carried out at 310 K, pH 7.4, in freshly prepared, oxygenated KrebsRinger-Hepes glucose medium. After preincubation (10 min), the final incubation (10 rain) was started by adding [3H]taurine (6.2-18.5 MBq/1) or [35S]hypotaurine (4.4-18.5 MBq/I) together with unlabelled taurine (1.0 ixmol/l-1.0 mmol/1) or hypotaurine (4.7 p,mol/1-1.0 mmol/1). Changes in ionic composition of media and additions of metabolic poisons were made during both preincubation and final incubation. Total protein content per dish was measured by the method of Lowry et al. TM The kinetics of taurine and hypotaurine uptake were analyzed as in Kontro & Oja. 14
RESULTS Cellular morphology The morphology of the cultures was monitored daily during the entire culture period. After 2 weeks the cell layers were usually confluent and composed mainly of similar flat, polygonalshaped glial cells as described by Hansson et al. ~ Only a few processes were visible. After withdrawal of foetal calf serum and addition of dBcAMP most cells assumed within 2 days a star-like appearance with retraction of cell bodies and extension of processes. The processes tended to be elongated and more numerous when cells were cultured for 1 week in this medium. In untreated control cultures the cells remained polygonal-shaped during the entire culture period of 3 weeks. Protein content The protein contents in the 16- and 21-day-old control cultures were 0. i0 _q_ _ 0.01 and 0.15 _-_0.01 mg/dish, and in the 16- and 21-day-old treated cultures 0.08---0.01 and 0.09--+0.01 mg/dish, respectively. Kinetics of uptake Taurine (10 ixmol/l) and hypotaurine (5 p,mol/l) uptakes were linear during the entire incubation time studied (60 min) both in t6-day-old treated (control cultures were not studied) and 21day-old treated and control cultures (data not shown). A final incubation time of 10 min was chosen for kinetic studies, during which time the cells had already accumulated enough radioactivity for precise measurements. Transport of [3H]taurine and [35S]hypotaurine consisted of one saturable high-affinity component both at early (16 days) and late (21 days) stages of astrocyte maturation. The transport constant (Km) of taurine uptake was significantly lower in 16-dayold untreated (control) than in treated cultures, but later, at 21 days, the maximal velocity (1I) was significantly reduced in treated samples (Table 1). The longer cultivation period increased ( P < 0.001) both Km and V of taurine transport in the control cultures. In dBcAMP-treated cells no significant changes occurred in the kinetic parameters of taurine uptake during maturation. The Km of hypotaurine uptake was significantly higher in treated astrocytes than in controls at both maturation stages, whereas the maximal velocity was highest in 16-day-old untreated cultures (Table 1). In 16-day-old untreated cultures both Km and V were higher (P<0.001) than in 21-day-old specimens. Analogously to taurine, the dBcAMP treatment did not significantly affect the kinetic constants at either stage of astrocyte cultivation. Effects of cations There were no significant differences between control and dBcAMP-treated cultures in the responses of taurine and hypotaurine uptakes to total omission of sodium, calcium and magnesium ions from medium or to a reduction of the sodium concentration to 60 mmol/l in medium.
531
T a u r i n e a n d h y p o t a u r i n e u p t a k e in a s t r o c y t e s Table 1. Kinetic parameters of high-affinity taurine and hypotaurine uptake in cultured astrocytes Culture conditions Taurine Control Treated Control Treated
Culture time (days)
Km (Ixmol/1)
V (ixmol/s x kg)
Kd (10 3 x s ~)
16
12.2 ~-:(1.9 22.5 ± 2.7+ 18.3 -~:0.9 19.9 _~:2.3
12.(I + (/.4 12.9 + 0.9 17.3 _+(1.4 12.0 + (1.6,
2.4 6.0 4.3 3.8
30.5 ~_:3.0 43.8_~: 3.4 * 17.0-+ 1.3 55.7-+ 7.8~
15.0 -+ 0.7 11.1 _+0.5, 10.9-+0.3 10.l+0.9
4.7 2.7 3.5 3.5
21
Hypotaurine Control Treated Control Treated
16 21
The results (means +- S.E.M.) were calculated as described in Experimental Procedures. Control cultures were grown in normal, serum-containing m e d i u m , treated cultures in serum-free m e d i u m in the presence of 0.1 mmol/1 d B c A M P Significance of differences from the corresponding controls (Student's independent two-tailed t-test): * P < 0 . 0 5 . t P < ( / . 0 1 . $P<11.001.
The data obtained from both cultures were therefore pooled. On the other hand, the responses to the omission of potassium ions were different in the two groups (Table 2). Taurine and hypotaurine uptakes were strictly sodium-dependent in all cultures, being almost totally abolished upon removal of extracellular Na+-ions (Table 2). At 60 mmol/l Na + the uptakes were restored to approximately one half. The omission of calcium ions only weakly inhibited taurine and hypotaurine uptake, while the absence of magnesium ions had no measurable effect at all. The effects of omission of sodium, calcium and magnesium were strikingly similar in both 16- and 21-day-old cultures. The uptakes of both amino acids were also considerably reduced when Table 2. Effects of cation omission on taurine and hypotaurine uptake in cultured astrocytes Uptake (% of control) Experimental conditions
Taurine
Hypotaurine
Culture time: 16 days -Na + Na+ 60mmol/1 - K + (control) - K +(treated) - C a 2+ - M g ~+
1.1+(1.2(13)$ 41.7-+4.2(13).:-53.7-+2.2 (6)$ 34.2 + 1.6 (7)$ 70.6+-3.5(14)* 98.1 +5.3(121
1.2-+0.2(11)42.1 -+2.5(11)$ 61.(I-+5.5 (6)" 33.9+-5.3 (5)$ 78.4+6.8(11) 8(1.4-+5.5(12)
Culture time: 21 days -Na ~ Na+ 60 mmol/I - K + (control) - K + (treated) - C a 2+ -Mg ~
2.4+0.8(20)$ 44.5+4.1(1815 48.1 +- 3.4 (13)~ 23.3+-2.1(101, 80.1 +- 5.8 (19)+ 99.7-+4.0(24)
2.4+-0.6(1615 40.2-+1.6(21), 43.1 -*-3.8(101, 28.6-+2.2 (8), 79.2+-3.4(171+ 90.2+7.3(15)
The cells were incubated for 10 min at 310 K with labelled taurine (10 ixmol/I) or hypotaurine (8 ixmol/I) in K r e b s - R i n g e r - H e p e s glucose m e d i u m (pH 7.4). The cations omitted were replaced by equimolar a m o u n t s of choline. The results (mean +- S.E.M.) in either age group represent pooled data from the control and treated cultures, except with potassium-free media. Taurine uptake in standard m e d i u m was in ixmol/(s × kg) 4.7±(I.2 and 3.5 + 0.4 (16 days); 5.4 +- 0.6 and 13.3 -+ 0.2 (21 days) in the control and treated cultures, respectively. Hypotaurine uptake was similarly in ixmol/(s × kg) 4.1 + 0.2 and 2.5 -+ 0.2 ( 16 days) ; 3.1 + 0.1 and 2.8 4__0.3 (21 days) in the control and treated cultures. N u m b e r of determinations in parentheses. Statistical significance of differences from the corresponding controls: * P < 0 . 0 5 , +P
532
I. Hotopainen
potassium ions were omitted from the medium. In the absence of potassium the uptakcs wclc always significantly lower in the dBcAMP-treated cultures than in the controls (taurine uptake: P<0.001, hypotaurine uptake: P<0.01, in both age groups). The longer the culture period, the greater was the decrease in uptake in K ~-free medium.
Effects of metabolic poisons Taurine and hypotaurine uptakes were equally affected by metabolic poisons in the dBcAMPtreated and control cultures. The results in Table 3 are thus pooled data from the two culture conditions. Ouabain reduced taurine and hypotaurine uptake to approximately one half. 2,4-Dinitrophenol (DNP) was also a potent inhibitor, whereas NaCN had only a minimal effect. There were no significant differences in the inhibitory potency of the metabolic poisons with respect to taurine uptake at different maturation stages (data not shown). In 21-day-old cultures the inhibition was also of the same order of magnitude in both taurine and hypotaurine uptakes.
Fable 3. Effects of metabolic poisons on taurine and hypotaurine uptake in cultured astrocytes Inhibitor concentration (mmol/1) Culture time: 21 days None (control) DNP(1.0) NaCN (0.5) Ouabain (3.0)
Uptake (24, of control) Taurine
Hypotaurine
100.0 -+ 7.6 (4) 68.4+5.0(4)+ 94,1 ±4.3(4) 46,7+4.4(4)+
100.0 -+4.3 (3) 82.2-+3.7(3) * 100.8±7.6(3) 59.3± 1,2 (3)+
The cells were preincubated for 10 min at 31(I K in KrebsRinger-Hepes glucose medium with the effectors indicated, then [3H]taurine (10 ~mol/I) or [35S]hypotaurine (8 btmol/l) was added and incubations continued for 10 rain. The results (mean -+ S.E.M.) are pooled from the control and treated cultures. Number of quadruplicate experiments in parentheses. Statistical significance of differences from the corresponding controls: *P<0.05, tP<0.(101.
DISCUSSION In both control and treated cultures the high-affinity uptake of taurine and hypotaurine was strictly sodium-dependent. This observation of absolute sodium dependence agrees with earlier reports on taurine uptake in cell lines and primary astrocytes2'9'17'3° and on hypotaurine uptake in brain slices, synaptosomes and neuroblastoma cells. 9"~3'~5 In earlier studies taurine and hypotaurine uptakes have also been reduced in the absence of potassium ions from incubation medium. 2"1e'13 The activity of Na +, K+-ATPase diminishes in potassium-free medium24 and the altered ion gradients across cell membranes may be responsible for reduced amino acid transport. This assumption is also supported by the ouabain sensitivity of the uptake. Differentiation induced by the dBcAMP treatment increases the activity of Na ÷, K+-ATPase in cultures and the sensitivity of astrocytes to changes in the extracellular K ÷ concentration.24 Therefore taurine and hypotaurine uptake decreased here more in dBcAMP-treated than in control cells. Modification of the Km values of taurine and hypotaurine were the most striking effect of serum withdrawal combined with addition of dBcAMP. Km for hypotaurine was three times higher in the 21-day-old treated cultures than in the controls but in the 16-day-old cultures the changes in gm were small. Schousboe et al. 30 have reported similar V but higher Km for taurine in primary astrocytes under experimental conditions comparable to ours. In a glioma cell line cyclic nucleotides have not had marked effects on the kinetic parameters of taurine uptake, 3 whereas in primary astrocytes they have decreased V of taurine but only minimally affected Km.4 This is consistent with the present data on 21-day-old primary astrocytes, in spite of differences in the concentration of cyclic nucleotides and duration of treatment. In control cultures, maturation of
Taurine and hypotaurine uptake in astrocytes
533
a s t r o c y t e s s e e m s to i n c r e a s e the affinity a n d c a p a c i t y o f t a u r i n e u p t a k e . B o r g e t al 4 h a v e also rep o r t e d a m a r k e d i n c r e a s e in t a u r i n e t r a n s p o r t in p r i m a r y a s t r o c y t e s d u r i n g m a t u r a t i o n . F u r t h e r m o r e , in rat b r a i n slices t a u r i n e u p t a k e has b e e n f o u n d h i g h e r in a d u l t t h a n in n e w b o r n a n i m a l s , ts In c o n t r a s t to the p r e s e n t s t u d y , Km for h y p o t a u r i n e has b e e n h i g h e r in b r a i n p r e p a r a t i o n s f r o m a d u l t t h a n f r o m d e v e l o p i n g mice. ~3'26 In b r a i n slices f r o m a d u l t mice the t r a n s p o r t c a p a c i t y for h y p o t a u r i n e has b e e n h i g h e r 26 b u t l o w e r in s y n a p t o s o m a l p r e p a r a t i o n s ~3 t h a n in c o r r e s p o n d i n g p r e p a r a t i o n s f r o m d e v e l o p i n g b r a i n . M o r e o v e r , in t h e s e e a r l i e r studies V was c o n s i d e r a b l y l o w e r t h a n h e r e , w h e r e a s Km has b e e n of the s a m e o r d e r o f m a g n i t u d e . T h e d i f f e r e n t d e v e l o p m e n t a l p a t t e r n s are a p p a r e n t l y d u e to the c o n t r i b u t i o n of n e u r o n a l a n d glial c o m p o n e n t s to t r a n s p o r t in slices. A s s u g g e s t e d for G A B A a n d t a u r i n e u p t a k e , 4 t h e t r a n s p o r t s y s t e m s c o u l d m a t u r e dissimilarly in n e u r o n s a n d glial cells. T h e d B c A M P t r e a t m e n t of cells, in spite of c o n s p i c u o u s m o r p h o l o g i c a l c h a n g e s , did not p r o duce t h e e x p e c t e d m o d i f i c a t i o n s in t r a n s p o r t p r o c e s s e s u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s . In t r e a t e d cells the kinetic p a r a m e t e r s of t a u r i n e a n d h y p o t a u r i n e u p t a k e r e m a i n e d u n a l t e r e d at diff e r e n t m a t u r a t i o n stages, w h e r e a s o n l y in c o n t r o l c u l t u r e s w e r e statistically significant c h a n g e s d i s c e r n i b l e . Cells c u l t u r e d for 16 d a y s ( a f t e r a 2 - d a y t r e a t m e n t ) s e e m to p o s s e s s the s a m e transp o r t p r o p e r t i e s as t h o s e c u l t u r e d for 21 d a y s ( a f t e r a 1-week t r e a t m e n t ) . T h e fast a n d p r o m i n e n t m o r p h o l o g i c a l r e s p o n s e , t o g e t h e r with b i o c h e m i c a l changes,~°'31 of c u l t u r e d a s t r o c y t e s to s e r u m w i t h d r a w a l a n d d B c A M P t r e a t m e n t m a y also r a p i d l y l e a d to a l t e r e d f u n c t i o n a l propertie:;. T h e e x t e n s i v e i n c r e a s e in glial fibrillary acidic ( G F A ) p r o t e i n in the d B c A M P - t r e a t e d cells f u r t h e r suggests that the cells u n d e r g o c h a n g e s c o m p a r a b l e to the p a t h o l o g i c a l p h e n o m e n o n r e p r e s e n t i n g a r e a c t i v e a s t r o c y t e . 52s32 T a u r i n e a n d h y p o t a u r i n e u p t a k e in c u l t u r e d p r i m a r y a s t r o c y t e s c o n s i s t e d of only o n e s a t u r a b l e high-affinity s o d i u m - d e p e n d e n t t r a n s p o r t s y s t e m . T h e close r e s e m b l a n c e of t h e s e u p t a k e s y s t e m s to each o t h e r in r e s p e c t of t r a n s p o r t kinetics a n d c a t i o n r e q u i r e m e n t s c o r r o b o r a t e s the a s s u m p tion of o n e c o m m o n t r a n s p o r t s y s t e m for t h e s e a m i n o acids. 7 M o r e o v e r , t h e u p t a k e s y s t e m s res e m b l e d t h o s e o f n e u r o t r a n s m i t t e r s , f u r t h e r suggesting t h a t t a u r i n e a n d h y p o t a u r i n e might have s o m e role in the r e g u l a t i o n of n e u r o n a l activity. A c k n o w l e d g e m e n t s - - I would like to thank Miss P/iivi Kyr615.for skillful technical assistance and Miss Raija Mattila and
Eija Kyr61/ifor expert secretarial aid. Dr./~. Sellstr6m willingly advised me on the principles of cultures of primary astrocytes. The study was supported by a grant from the Emil Aaltonen Foundation and the Research and Science Foundation of Lfifike Oy.
REFERENCES 1. Booher J. & Sensenbrenner M. (1972) Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology 2, 97-105. 2. Borg J., Balcar V. J., Mark J. & Mandel P. (1979) Characterization of taurine uptake by neuronal and glial cells in culture. J. Neurochern. 32, 1801-1805. 3. Borg J., Balcar V. J. & Mandel P. (1979) Effect of cyclic nucleotides on high affinity uptake of L-glutamate and taurine in glial and neuroblastoma cells. Brain Res. 166, 113-120. 4. Borg J., Ramaharobandro N., Mark J. & Mandel P. (1980) Changes in the uptake of GABA and taurine during neuronal and glial maturation. J. Neurochern. 34, 1113-1122. 5. Fedoroff S., White R., Neal J., Subrahmanyan L. & Kalnins V. 1. (1983) Astrocyte cell lineage, lI, Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin- and GFP-containing intermediate filaments. Devl Brain Res. 7, 303-315. 6. Hansson E., Sellstr6m/~., Persson L. I. & R6nnbfick L. (1980) Brain primary culture--a characterization. Brain Res. 188, 233-246. 7. Holopainen I. & Kontro P. (1984) Taurine and hypotaurine transport by a single system in cultured neuroblastoma cells. Acta physiol, scand., in press. 8. Holopainen 1., Kontro P., Frey H. J. & Oja S. S. (1983) Taurine, hypotaurine, and GABA uptake by cultured neuroblastoma cells. J. Neurosci. Res. 10, 83-92. 9. Holopainen I., Kontro P. & Oja S. S. (1984) Taurine and hypotaurine transport in neuroblastoma cells: effects of cations. Neurochem. Int., 6, 217-222. 10, Kimelberg H. K., Narumi S. & Bourke R. S. (1978) Enzymatic and morphological properties of primary rat brain astrocyte cultures, and enzyme development in vivo. Brain Res. 153, 55-77. 11. Kontro P. (1979) Components of taurine effiux in rat brain synaptosomes. Neuroscience 4, 1745-1749. 12. Kontro P. (1982) Effects of cations on taurine, hypotaurine and GABA uptake in mouse brain slices. Neurochern. Res. 7, 1391-1401. 13. Kontro P. (1983) Hypotaurine transport in mouse brain synaptosomal preparations. Acta physiol, scand. ! 18, 57-60. 14. Kontro P. & Oja S. S. (1978) Taurine uptake by rat brain synaptosomes. J. Neurochem. 30, 1297-131/4.
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1. H o l o p a i n e n
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