Brain Research, 172 (1979) 131-138 © Elsevier/North-Holland Biomedical Press
131
T A U R I N E EFFECTS ON 45Ca2~ T R A N S P O R T IN RETINAL S U B C E L L U L A R FRACTIONS
H. PASANTES-MORALES, R. M. ADEME and A. M. LOPEZ-COLOMI~ Departamento de Biologia Experimental, lnstituto de Biologia, Universidad Nacional Aut6noma de Mdxico. Apartado Postal 70-600, Mdxico 20 D.F. (Mdxico)
(Accepted November 30th, 1978)
SUMMARY The effect of taurine on 45Ca2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45Ca2+ uptake was observed in retinal fractions incubated for 1-5 rain in a Krebs-bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50~o in 45Ca2+ uptake; in the crude synaptosomal fraction, taurine reduced 45Ca2+ accumulation by /, 70 oJo, the maximum inhibitory effect of taurine on 45Cae+ uptake (80 ~ ) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role for taurine in the regulation of calcium fluxes in the retina.
INTRODUCTION Taurine is the major constituent of the free amino acid pool in retinaa,19,22,26; yet its role in retinal function remains unknown. It has been suggested that taurine may act as a retinal inhibitory neurotransmitter20, but it is present in greater concentrations than such a putative function may require; moreover, most retinal taurine is localized in photoreceptorsla,14,21 in which the neurotransmitter released appears to be excitatory 7. Therefore, a role other than that of neurotransmitter should be considered for taurine. Taurine is released from the whole retina in response to light stimulation'~a,25. This light-induced release of taurine is also demonstrable in frog rod outer segments isolated from photoreceptors24; in consequence, an involvement of taurine in the mechanisms of light excitation might be envisaged. The process of transduction of photoexcitation into a nerve impulse in photoreceptors appears to implicate modifications in ionic gradients, in particular those of sodium, potassium and calcium9,15, '~7, and since some evidence exists suggesting an involvement of taurine in calcium
132 transport 6,1z,16, in the present study we examined the effects of taurine on 4~'Ca'~ transport into subcellular fractions of the chick retina. METHODS Retinas of 5-6-week-old chicks were removed and homogenized in 0.32 M sucrose containing 10 -4 M MgSOa (1:20 w/v). The crude nuclear fraction (P1) and the crude synaptosomal fraction (P2) were prepared according to the method of DeRobertis et al. 4, as previously described 17. Briefly, the sucrose homogenate was centrifuged at 900 × g for 10 min, and the washed pellet constituted the P1 fraction; the combined supernatants were centrifuged at 18,000 × g for 20 min to obtain the crude synaptosomal fraction (Pz). A fraction enriched in outer segments of photoreceptors (OS) was obtained by gently brushing the retinas, following the procedure of Cavaggioni et al.L for frog retinas. The outer segments were sedimented by centrifugation (900 × g, 10 min, 4 °C). The characterization of the isolated fraction was carried out by electron microscopy observation as previously described 17. The crude nuclear fraction (P1) contains nuclei, aggregates of mitochondria proceeding from the inner segments of the photoreceptors and photoreceptor terminals; the crude synaptosomal fraction (P2) contains mainly pinched off nerve terminals and some mitochondria. The OS fraction contains a large amount of cells from the pigmentary epithelium. The isolated particulate fractions were resuspended in 0.3 M sucrose containing 10 -4 M MgSO4. For measurements of 4SCa2+ uptake the resuspended fractions were incubated with 1 ml (final volume) of Krebs-bicarbonate medium (1 t8 mM NaC1; 4.7 m M KCI ; 1.2 m M KH2PO4; 2.5 m M CaCI2; 1.17 m M MgSO4 and 25 mM NaHCOz), p H 7.4. The incubation mixture contained 1 m M ATP, 2 /~Ci of 4~CaC12 (New England Nuclear), 0.2-0,4 mg of the particulate protein and taurine at the concentrations indicated in each experiment. The suspension was incubated at 37'=C for 5 min except as otherwise indicated. The reaction was stopped by centrifugation in a Beckman Microfuge; the pellet was washed superficially with cold distilled water, TABLE I Effects of l o w temperature, ATP, ruthenium red and 70 m M KCl on 45Ca2+ uptake by retbzal subcellular fractions
P1 and P~ fractions, isolated from chick retinas, were incubated in Krebs-bicarbonate medium without glucose for 5 min at 37 °C, except as otherwise indicated, in the presence of?/~Ci of 4sCaCh. Ruthenium red and 70 mM KCI were present from the beginning of the incubation. KCI was added to the incubation medium without substitutions to correct osmolarity. The results are means ± S.E.M. of the number of experiments indicated in parenthesis. P1
P2
(cpm/mg protein ~ 10 4)
Control, (37 °C, 1 mM ATP) ( 4 °C, 1 mM ATP) Without ATP ATP (2 mM) Ruthenium red (100/~M) KC1 (70 mM)
6.40 i 5.9 (12) 2.80 ~= 0.16 (4) 5.92 :L 0.31 (5) 6.39 ± 0.79 (3) 4.46 ~ 0.50 (3) 6.18 i 0.64 (4)
11.2 ± 1.16 (14) 2.7 ± 0.09 (4) 11.0 -[ 1.17 (5) 10.6 + 1.41 (3) 8.7 ± 1.01 (4) 10.6 -- 1.17 (4)
133 solubilized with 0.2 ml of NCS (Amersham, Searle) and transferred to scintillation vials. Radioactivity in the solubilized tissue was measured after addition of 5 ml of Tritosol s, in a Packard liquid scintillation counter. All determinations were carried out in duplicate. Protein was determined by the procedure of Lowry et al. TM. A correction was made for absorbed extracellular 45Ca2 ~- washing the pellet with medium containing 2.5 mM unlabeled calcium. Accumulation of 4~CaZ+ under these conditions decreased 8-12 ~. RESULTS 45Ca2~- uptake by retinal subcellular fractions was rapidly saturable, reaching equilibrium after 2 min of incubation. It was temperature dependent. Addition of 1-2 mM ATP did not increase the 45CaZ+ uptake appreciably. 4,5Ca2~ accumulation was slightly inhibited by ruthenium red. It was unaffected by increasing KC1 concentration to 70 mM or by 10 mM glutamate (Table I). Taurine concentration in the different chick retinal layers varies from 100 to 400 #moles/g dry weight21; therefore, taurine effects on 45Ca2+ uptake were studied at concentrations within this physiological range, 2.5 to 25 mM. At a concentration 0f25 mM taurine markedly inhibited 45Cae+ uptake in all the subcellular fractions examined. The maximum effect was observed in the OS fraction in which taurine produced a 45Ca2+ uptake decrease of 80 % of the control values' in the P~ fraction taurine reduced 45Ca2+ accumulation by more than 70 %; in the P1 fraction taurine inhibition was of 50 % of the control values (Fig. 1). Taurine effects on 45Ca2+ uptake
28- I
Control
24I '0
25 rnM Taurine
20-
x
._ "~ 16o.
E
12-
R o Q.
+ {::}
4-
i
OS
PI
Pz
Fig. 1. The effect of taurine (25 mM) on accumulation of 45Ca2+ by retinal subcellular fractions. Taurine was present in the incubation mixture containing Krebs-bicarbonate medium, pH 7.4, 1 mM neutralized ATP, 2/~Ci of 45CAC12and 0.7 1mg of protein in a final volume of 1 ml. Incubation time was 5 min at 37 C with shaking. The results are the means ± S.E.M. of 4 separate experiments for OS fraction, 12 experiments for PI and 14 experiments for Pz.
134 were not observed when 26 mM Tris.HCl substituted NaHCOa in the Krebs medium. The time course, specificity and dose dependence of taurine on 4'~Cae ' uptake were studied in the P1 and P2 fractions. Taurine effects on 45Ca2+ uptake were observed at the shortest time examined, 1 rain. At this time taurine inhibition was similar to that observed after 5 min of incubation (Fig. 2). The taurine-induced decrease of 4'~Ca2 ~ accumulation was observed to be dose-dependent. A decrease of 10'~';i of the control values was observed at 2.5 mM taurine concentration; maximum decrease was obtained at 25 mM taurine (Fig. 3). The specificity of taurine effects was investigated by the examination of the effects of GABA, glycine, fl-alanine and glutamate on 45CaZ+ accumulation. All the amino acids were tested at a concentration of 10 mM. GABA and glutamate did not alter 45Ca2~ uptake, whereas glycine and fl-alanine reduced it 15% and 25%, respectively. At the same concentration (10 mM) taurine decreased 45CaZ+ accumulation by 44 % and 74 ~/~ from the control values in P1 and Pz fractions respectively (Table II). In order to determine if taurine effects on 45CaZ~ accumulation were due to a decrease in calcium binding to cell membranes prior to the transport, 45Ca2+ incorporation was studied after submitting particles to osmotic lysis by homogenization in 20 vols of distilled water. Under these conditions ~'~Ca'~ binding to retinal membranes was observed but it was unaffected by taurine, suggesting that the amino acid produces its effects on 45CaZ~ transport associated to an osmotically sensitive particle. Taurine inhibition of ~SCa"~ accumulation might be due to an inhibitory effect on 45Ca2 ~ entry or to a decreased retention of labeled calcium accumulated by the retinal particles. Results in Fig. 4 show that addition of taurine after 2 min of incubation in the presence of 45Ca2 ~ produces a decrease in the radio-
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Time
{min)
Time ( m i n )
Fig. 2. T h e effect o f taurine (25 m M ) o n a c c u m u l a t i o n o f 4'5Ca~+ by P1 a n d P~ fractions, at different i n c u b a t i o n times. T h e i n c u b a t i o n m i x t u r e was as described i n Fig. 1. A l i q u o t s o f 0.3 ml of t h e s u s p e n s i o n were w i t h d r a w n at t h e i n c u b a t i o n t i m e s indicated. T h e results are the m e a n s ± S.E.M. or 4 - 6 separate experiments. Control, • • ; taurine, © - - - 0 -
135 80
70
==
6O
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~ 40 g .~.1o
3(?
z
~
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I
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Taurine
I
/
20
25
(raM)
Fig. 3. The effect of different concentrations of taurine on 45Ca2+ accumulation by P1 (C - - C) and Pz (O - - O ) fractions of retina. 45Ca2+ uptake was measured as described in Fig. 1 in incubation flasks containing 2.5, 5, 10 and 25 mM of taurine. The results are the means ± S.E.M. of 4 separate experiments.
TABLE I1
Effects o f various amino acids (10 mM) on 45Ca 2+ uptake by the crude nuclear (P1) and the crude synaptosomal (P~) fractions o f the chick retina P1 and P2 fractions isolated from the chick retina were incubated at 37 °C for 5 min in Krebs-bicarbonate medium, pH 7.4 in the presence of 10 mM amino acids. Control medium did not contain amino acids. 45Ca2+ uptake was measured as described in the text. Results are the means ± S.E.M. of the number of experiments indicated in parentheses.
Amino acid (10 raM)
P1 P2 (cpm/mgprotein x 10-4j
Control GABA Glycine Glutamate fi-Alanine Taurine
5.92 6.03 4.95 5.75 4.41 3.79
± 0.31 (12) ± 0.52 (4) ± 0.36 (4) zk 0.68 (4) ± 0.37 (6) ± 0.39 (4)
1 1 . 2 3± 10.71 ± 9.31 ± 10.68 ± 9.72 ± 5.66 ±
1.17 (14) 0.96 (4) 0.63 (4) 0.61 (4) 0.50 (6) 0.47 (7)
136 TAURINE
IO o
x
8
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~o I
I
~
I
b
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2
3
4
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Time
( rain )
Fig. 4. The effect of taurine (25 mM) on the concentration of 45Ca~+taken up by the Pz fraction of retina. The fraction was incubated as described in Fig. 1, and aliquots were withdrawn every min. After 2 min, taurine was added (final concentration 25 mM) and incubation continued for 3 more minutes. The results are the means ± S.E.M. of 3 separate experiments. activity accumulated by the fractions, suggesting that taurine may be affecting the retention of labeled calcium; however, the early effect of taurine on 4 ~ a " + accumulation suggests that the uptake of calcium is primarily affected. DISCUSSION
The present results show a direct effect of taurine on calcium transport in retinal subcellular fractions. This observation provides further support to the suggestion of a role for taurine as a modulator of calcium gradients. A variety of taurine effects on excitable tissues have been tentatively explained by modifications of calcium fluxes. The inotropic effect of taurine on heart has been related to taurine-induced modifications in the kinetics of calcium efflux from the different calcium intracellular poolsL The generalized anticonvulsant effect of taurine has also been thought to be in part associated with an alteration in calcium concentration in the central nervous system: disodium edetate a calcium chelator considerably reduces the protective effect of taurine against pentylenetetrazol-induced convulsions in mice t0,11. Taurine anticonvulsant effects may be subsequent to increased intracellular calcium levels in cerebral cortex; it has been described that taurine interferes with calcium binding to microsomes isolated from the rat cerebral cortex 12. Recently, a supressive effect of taurine on the K+-stimulated release of acetylcholine and norepinephrine has been explained by a taurine-increased retention of calcium within mitochondria 1~. In retina, a redistribution of the intracetlular calcium in photoreceptors seems to occur during photoexcitation 9, which in turn reduces the membrane sodium permeability and leads to the cell's hyperpolarization. The present results, together with the
137 p r e v i o u s o b s e r v a t i o n o f a l i g h t - i n d u c e d release o f t a u r i n e f r o m r e t i n a a n d f r o m r o d o u t e r s e g m e n t s , raises t h e p o s s i b i l i t y o f a role f o r t a u r i n e
in t h e r e g u l a t i o n
of
i n t r a c e l l u l a r c a l c i u m levels in retina. T h e d e c r e a s e d C a 2+ u p t a k e p r o d u c e d by t a u r i n e was o b s e r v e d in all retinal subc e l l u l a r f r a c t i o n s , i n c l u d i n g s y n a p t o s o m e s . T h i s m a y suggest a m o r e e x t e n d e d i n v o l v e m e n t o f t a u r i n e in t h e m o d u l a t i o n o f c a l c i u m fluxes in n e r v o u s tissue. ACKNOWLEDGEMENT T h i s r e s e a r c h was s u p p o r t e d in p a r t by G r a n t
1 RO1 EY02540-01 f r o m t h e
N a t i o n a l Eye I n s t i t u t e . REFERENCES I Bonting, S. L. and Daemen, F. J., Calcium as a transmitter in photoreceptor cells. In S. L. Bonting (Ed.), Transmitters in the Visual Proeess, Pergamon Press, Oxford, 1976, pp. 60 89. 2 Cavaggioni, A., Sorbi, R. T. and Turini, S., Efflux of potassium from isolated rod outer segments: a photic effect, J. Physiol. (Lond.), 232 (1973) 609-620. 3 Cohen, A. 1., McDaniel, M. and Orr, H. T., Absolute levels of some free amino acids in normal and biologically fractionated retinas, Invest. Ophthal., 12 (1973) 686 693. 4 DeRobertis, E., Pelegrino de Iraldi, A., Rodriguez de Lores Arnaiz, G. and Salganicoff, L., Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetyl choline and acetylcholinesterase, J. Neurochem., 9 (1962) 23 35. 5 Dolara, P., Agresti, A., Giotti, A. and Pasquini, G., Effect of taurine on calcium kinetics of guinea-pig heart, Europ. J. Pharrnaeol., 24 (1973) 352-358. 6 D~lara, P., Ledda, F., Mugelli, A., Mantelli, L., Zilletti, F., Franconi, F. and G iotti, A., Effect of taurine on calcium, inotropism and electrical activity of the heart. In A. Barbeau and R. J. Huxtable (Eds.), Taurine and Neurological Disorders, Raven Press, New York, 1978, pp. 151 160. 7 Dowling, J. E. and Ripps, H., Effect of magnesium on horizontal cell activity in the skate retina, Nature (Lond.), 242 (1973) 101-103. 8 Fricke, U., Tritosol: a new scintillation cocktail based on Triton X-100, AnJyt. Biochem., 63 (1975) 555 558. 9 Hagins, W. A. and Yoshikami, S., Role for calcium in excitation of retinal reds and cones, Exp. Eye Res., 18 (1974) 299-305. 10 Izumi, K., Igisu, H. and Fukuda, T., Supression of seizures by taurine -- specific or non-specific? Brain Research, 76 (1974) 171 173. I 1 lzumi, K., lgisu, H. and Fukuda, T., Effects ofedetate on seizure supressing actions of taurine and GABA, Brain Research, 88 (1975) 576 579. 12 Izumi, K., Butterworth, R. F. and Barbeau, A., Effect of taurine on calcium binding to microsomes isolated from rat cerebral cortex, Life Sci., 20 (1977) 943-950. 13 Keen, P. and Yates, R. A., Distribution of amino acids in subdivided rat retinae, Brit. J. Pharmacol., 52 (1974) 118p. 14 Kennedy, A. J. and Voaden, M. J., Free amino acids in the photoreceptor cells of the frog retina, J. Neurochem, 23 (1974) 1093 1095. 15 Korenbrot, J. 1. and Cone, R. A., Dark ionic flux and the effect of light in isolated rod outer segments, J. gen. Physiol., 60 (1972) 20~-5. 16 Kuriyama, K., Muramatso, M., Nakagawa, K. and Kakita, K., Modulating role of taurine on release of neurotransmitters and calcium transport in excitable tissues. In A. Barbeau and R. J. Huxtable (Eds.), Taurine and Neurological Disorders, Raven Press, New York, 1978, pp. 201 216. 17 Lopez-Colome, A. M., Salceda, R. and Pasantes-Morales, H., Potassium stimulated release of GABA, glycine and taurine from the chick retina, Neurochem. Res., 3 (1978) 431M41. 18 Lowry, O., Rosebrough, N., Farr, A. and Randall, R. J., Protein measurement with Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 19 Macaione, G., Ruggieri, P., De Luca, F. and Tucci, G., Free amino acids in developing rat retina, J. Neurochem., 22 (1974) 887 891.
138 20 Mandel, P. and Pasantes-Morales, H., Taurine, a putative neurotransmitter in retina. In S. L, Bonting (Ed.), Transmitters in the Visual Process. Pergamon Press, Oxford, 1976, pp. 89-105. 21 Orr, H. T., Cohen, A. L and Lowry, O. H., The distribution of taurine in the vertebrate retina, J. Neurochem., 26 (1976) 609-612. 22 Pasantes-Morales, H., Klethi, J., Ledig, M. and Mandel, P., Free amino acids in chicken and rat retina, Brain Reserach, 41 (1972) 494-497. 23 Pasantes-Morales, H., Urban, P. F., Kelthi, J. and Mandel, P., Light stimulated release of [3'~S]taurine from chicken retina, Brain Research, 51 (1973) 375-378. 24 Salceda, R., Lopez-Colome, A. M. and Pasantes-Morales, H., Light-stimulated release of p S ] taurine from frog retinal rod outer segments, Brain Reserach, 135 (1977) 186-191. 25 Schmidt, S. Y. and Berson, E. L., Taurine fluxes in isolated cat and rat retinas: effects of illumination, Exp. Eye Res., 26 (1978) 529-538. 26 Starr, M. S., Effect of dark adaptation of the GABA system in retina, Brain Research, 59 (1973) 331-338. 27 Yoshikami, S. and Hagins, W. A., Control of the dark current in vertebrate rods and cones. In. H. Langer (Ed.), Biochemistry and Physiology of Visual Pigments, Springer Verlag, New York, 1973, pp. 245-255.
131
T A U R I N E EFFECTS ON 45Ca2~ T R A N S P O R T IN RETINAL S U B C E L L U L A R FRACTIONS
H. PASANTES-MORALES, R. M. ADEME and A. M. LOPEZ-COLOMI~ Departamento de Biologia Experimental, lnstituto de Biologia, Universidad Nacional Aut6noma de Mdxico. Apartado Postal 70-600, Mdxico 20 D.F. (Mdxico)
(Accepted November 30th, 1978)
SUMMARY The effect of taurine on 45Ca2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45Ca2+ uptake was observed in retinal fractions incubated for 1-5 rain in a Krebs-bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50~o in 45Ca2+ uptake; in the crude synaptosomal fraction, taurine reduced 45Ca2+ accumulation by /, 70 oJo, the maximum inhibitory effect of taurine on 45Cae+ uptake (80 ~ ) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role for taurine in the regulation of calcium fluxes in the retina.
INTRODUCTION Taurine is the major constituent of the free amino acid pool in retinaa,19,22,26; yet its role in retinal function remains unknown. It has been suggested that taurine may act as a retinal inhibitory neurotransmitter20, but it is present in greater concentrations than such a putative function may require; moreover, most retinal taurine is localized in photoreceptorsla,14,21 in which the neurotransmitter released appears to be excitatory 7. Therefore, a role other than that of neurotransmitter should be considered for taurine. Taurine is released from the whole retina in response to light stimulation'~a,25. This light-induced release of taurine is also demonstrable in frog rod outer segments isolated from photoreceptors24; in consequence, an involvement of taurine in the mechanisms of light excitation might be envisaged. The process of transduction of photoexcitation into a nerve impulse in photoreceptors appears to implicate modifications in ionic gradients, in particular those of sodium, potassium and calcium9,15, '~7, and since some evidence exists suggesting an involvement of taurine in calcium
132 transport 6,1z,16, in the present study we examined the effects of taurine on 4~'Ca'~ transport into subcellular fractions of the chick retina. METHODS Retinas of 5-6-week-old chicks were removed and homogenized in 0.32 M sucrose containing 10 -4 M MgSOa (1:20 w/v). The crude nuclear fraction (P1) and the crude synaptosomal fraction (P2) were prepared according to the method of DeRobertis et al. 4, as previously described 17. Briefly, the sucrose homogenate was centrifuged at 900 × g for 10 min, and the washed pellet constituted the P1 fraction; the combined supernatants were centrifuged at 18,000 × g for 20 min to obtain the crude synaptosomal fraction (Pz). A fraction enriched in outer segments of photoreceptors (OS) was obtained by gently brushing the retinas, following the procedure of Cavaggioni et al.L for frog retinas. The outer segments were sedimented by centrifugation (900 × g, 10 min, 4 °C). The characterization of the isolated fraction was carried out by electron microscopy observation as previously described 17. The crude nuclear fraction (P1) contains nuclei, aggregates of mitochondria proceeding from the inner segments of the photoreceptors and photoreceptor terminals; the crude synaptosomal fraction (P2) contains mainly pinched off nerve terminals and some mitochondria. The OS fraction contains a large amount of cells from the pigmentary epithelium. The isolated particulate fractions were resuspended in 0.3 M sucrose containing 10 -4 M MgSO4. For measurements of 4SCa2+ uptake the resuspended fractions were incubated with 1 ml (final volume) of Krebs-bicarbonate medium (1 t8 mM NaC1; 4.7 m M KCI ; 1.2 m M KH2PO4; 2.5 m M CaCI2; 1.17 m M MgSO4 and 25 mM NaHCOz), p H 7.4. The incubation mixture contained 1 m M ATP, 2 /~Ci of 4~CaC12 (New England Nuclear), 0.2-0,4 mg of the particulate protein and taurine at the concentrations indicated in each experiment. The suspension was incubated at 37'=C for 5 min except as otherwise indicated. The reaction was stopped by centrifugation in a Beckman Microfuge; the pellet was washed superficially with cold distilled water, TABLE I Effects of l o w temperature, ATP, ruthenium red and 70 m M KCl on 45Ca2+ uptake by retbzal subcellular fractions
P1 and P~ fractions, isolated from chick retinas, were incubated in Krebs-bicarbonate medium without glucose for 5 min at 37 °C, except as otherwise indicated, in the presence of?/~Ci of 4sCaCh. Ruthenium red and 70 mM KCI were present from the beginning of the incubation. KCI was added to the incubation medium without substitutions to correct osmolarity. The results are means ± S.E.M. of the number of experiments indicated in parenthesis. P1
P2
(cpm/mg protein ~ 10 4)
Control, (37 °C, 1 mM ATP) ( 4 °C, 1 mM ATP) Without ATP ATP (2 mM) Ruthenium red (100/~M) KC1 (70 mM)
6.40 i 5.9 (12) 2.80 ~= 0.16 (4) 5.92 :L 0.31 (5) 6.39 ± 0.79 (3) 4.46 ~ 0.50 (3) 6.18 i 0.64 (4)
11.2 ± 1.16 (14) 2.7 ± 0.09 (4) 11.0 -[ 1.17 (5) 10.6 + 1.41 (3) 8.7 ± 1.01 (4) 10.6 -- 1.17 (4)
133 solubilized with 0.2 ml of NCS (Amersham, Searle) and transferred to scintillation vials. Radioactivity in the solubilized tissue was measured after addition of 5 ml of Tritosol s, in a Packard liquid scintillation counter. All determinations were carried out in duplicate. Protein was determined by the procedure of Lowry et al. TM. A correction was made for absorbed extracellular 45Ca2 ~- washing the pellet with medium containing 2.5 mM unlabeled calcium. Accumulation of 4~CaZ+ under these conditions decreased 8-12 ~. RESULTS 45Ca2~- uptake by retinal subcellular fractions was rapidly saturable, reaching equilibrium after 2 min of incubation. It was temperature dependent. Addition of 1-2 mM ATP did not increase the 45CaZ+ uptake appreciably. 4,5Ca2~ accumulation was slightly inhibited by ruthenium red. It was unaffected by increasing KC1 concentration to 70 mM or by 10 mM glutamate (Table I). Taurine concentration in the different chick retinal layers varies from 100 to 400 #moles/g dry weight21; therefore, taurine effects on 45Ca2+ uptake were studied at concentrations within this physiological range, 2.5 to 25 mM. At a concentration 0f25 mM taurine markedly inhibited 45Cae+ uptake in all the subcellular fractions examined. The maximum effect was observed in the OS fraction in which taurine produced a 45Ca2+ uptake decrease of 80 % of the control values' in the P~ fraction taurine reduced 45Ca2+ accumulation by more than 70 %; in the P1 fraction taurine inhibition was of 50 % of the control values (Fig. 1). Taurine effects on 45Ca2+ uptake
28- I
Control
24I '0
25 rnM Taurine
20-
x
._ "~ 16o.
E
12-
R o Q.
+ {::}
4-
i
OS
PI
Pz
Fig. 1. The effect of taurine (25 mM) on accumulation of 45Ca2+ by retinal subcellular fractions. Taurine was present in the incubation mixture containing Krebs-bicarbonate medium, pH 7.4, 1 mM neutralized ATP, 2/~Ci of 45CAC12and 0.7 1mg of protein in a final volume of 1 ml. Incubation time was 5 min at 37 C with shaking. The results are the means ± S.E.M. of 4 separate experiments for OS fraction, 12 experiments for PI and 14 experiments for Pz.
134 were not observed when 26 mM Tris.HCl substituted NaHCOa in the Krebs medium. The time course, specificity and dose dependence of taurine on 4'~Cae ' uptake were studied in the P1 and P2 fractions. Taurine effects on 45Ca2+ uptake were observed at the shortest time examined, 1 rain. At this time taurine inhibition was similar to that observed after 5 min of incubation (Fig. 2). The taurine-induced decrease of 4'~Ca2 ~ accumulation was observed to be dose-dependent. A decrease of 10'~';i of the control values was observed at 2.5 mM taurine concentration; maximum decrease was obtained at 25 mM taurine (Fig. 3). The specificity of taurine effects was investigated by the examination of the effects of GABA, glycine, fl-alanine and glutamate on 45CaZ+ accumulation. All the amino acids were tested at a concentration of 10 mM. GABA and glutamate did not alter 45Ca2~ uptake, whereas glycine and fl-alanine reduced it 15% and 25%, respectively. At the same concentration (10 mM) taurine decreased 45CaZ+ accumulation by 44 % and 74 ~/~ from the control values in P1 and Pz fractions respectively (Table II). In order to determine if taurine effects on 45CaZ~ accumulation were due to a decrease in calcium binding to cell membranes prior to the transport, 45Ca2+ incorporation was studied after submitting particles to osmotic lysis by homogenization in 20 vols of distilled water. Under these conditions ~'~Ca'~ binding to retinal membranes was observed but it was unaffected by taurine, suggesting that the amino acid produces its effects on 45CaZ~ transport associated to an osmotically sensitive particle. Taurine inhibition of ~SCa"~ accumulation might be due to an inhibitory effect on 45Ca2 ~ entry or to a decreased retention of labeled calcium accumulated by the retinal particles. Results in Fig. 4 show that addition of taurine after 2 min of incubation in the presence of 45Ca2 ~ produces a decrease in the radio-
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{min)
Time ( m i n )
Fig. 2. T h e effect o f taurine (25 m M ) o n a c c u m u l a t i o n o f 4'5Ca~+ by P1 a n d P~ fractions, at different i n c u b a t i o n times. T h e i n c u b a t i o n m i x t u r e was as described i n Fig. 1. A l i q u o t s o f 0.3 ml of t h e s u s p e n s i o n were w i t h d r a w n at t h e i n c u b a t i o n t i m e s indicated. T h e results are the m e a n s ± S.E.M. or 4 - 6 separate experiments. Control, • • ; taurine, © - - - 0 -
135 80
70
==
6O
50 E o
~ 40 g .~.1o
3(?
z
~
2o
I
I
5
I0
I
15
Taurine
I
/
20
25
(raM)
Fig. 3. The effect of different concentrations of taurine on 45Ca2+ accumulation by P1 (C - - C) and Pz (O - - O ) fractions of retina. 45Ca2+ uptake was measured as described in Fig. 1 in incubation flasks containing 2.5, 5, 10 and 25 mM of taurine. The results are the means ± S.E.M. of 4 separate experiments.
TABLE I1
Effects o f various amino acids (10 mM) on 45Ca 2+ uptake by the crude nuclear (P1) and the crude synaptosomal (P~) fractions o f the chick retina P1 and P2 fractions isolated from the chick retina were incubated at 37 °C for 5 min in Krebs-bicarbonate medium, pH 7.4 in the presence of 10 mM amino acids. Control medium did not contain amino acids. 45Ca2+ uptake was measured as described in the text. Results are the means ± S.E.M. of the number of experiments indicated in parentheses.
Amino acid (10 raM)
P1 P2 (cpm/mgprotein x 10-4j
Control GABA Glycine Glutamate fi-Alanine Taurine
5.92 6.03 4.95 5.75 4.41 3.79
± 0.31 (12) ± 0.52 (4) ± 0.36 (4) zk 0.68 (4) ± 0.37 (6) ± 0.39 (4)
1 1 . 2 3± 10.71 ± 9.31 ± 10.68 ± 9.72 ± 5.66 ±
1.17 (14) 0.96 (4) 0.63 (4) 0.61 (4) 0.50 (6) 0.47 (7)
136 TAURINE
IO o
x
8
{z
}6 c~ 4
~o I
I
~
I
b
I
2
3
4
5
Time
( rain )
Fig. 4. The effect of taurine (25 mM) on the concentration of 45Ca~+taken up by the Pz fraction of retina. The fraction was incubated as described in Fig. 1, and aliquots were withdrawn every min. After 2 min, taurine was added (final concentration 25 mM) and incubation continued for 3 more minutes. The results are the means ± S.E.M. of 3 separate experiments. activity accumulated by the fractions, suggesting that taurine may be affecting the retention of labeled calcium; however, the early effect of taurine on 4 ~ a " + accumulation suggests that the uptake of calcium is primarily affected. DISCUSSION
The present results show a direct effect of taurine on calcium transport in retinal subcellular fractions. This observation provides further support to the suggestion of a role for taurine as a modulator of calcium gradients. A variety of taurine effects on excitable tissues have been tentatively explained by modifications of calcium fluxes. The inotropic effect of taurine on heart has been related to taurine-induced modifications in the kinetics of calcium efflux from the different calcium intracellular poolsL The generalized anticonvulsant effect of taurine has also been thought to be in part associated with an alteration in calcium concentration in the central nervous system: disodium edetate a calcium chelator considerably reduces the protective effect of taurine against pentylenetetrazol-induced convulsions in mice t0,11. Taurine anticonvulsant effects may be subsequent to increased intracellular calcium levels in cerebral cortex; it has been described that taurine interferes with calcium binding to microsomes isolated from the rat cerebral cortex 12. Recently, a supressive effect of taurine on the K+-stimulated release of acetylcholine and norepinephrine has been explained by a taurine-increased retention of calcium within mitochondria 1~. In retina, a redistribution of the intracetlular calcium in photoreceptors seems to occur during photoexcitation 9, which in turn reduces the membrane sodium permeability and leads to the cell's hyperpolarization. The present results, together with the
137 p r e v i o u s o b s e r v a t i o n o f a l i g h t - i n d u c e d release o f t a u r i n e f r o m r e t i n a a n d f r o m r o d o u t e r s e g m e n t s , raises t h e p o s s i b i l i t y o f a role f o r t a u r i n e
in t h e r e g u l a t i o n
of
i n t r a c e l l u l a r c a l c i u m levels in retina. T h e d e c r e a s e d C a 2+ u p t a k e p r o d u c e d by t a u r i n e was o b s e r v e d in all retinal subc e l l u l a r f r a c t i o n s , i n c l u d i n g s y n a p t o s o m e s . T h i s m a y suggest a m o r e e x t e n d e d i n v o l v e m e n t o f t a u r i n e in t h e m o d u l a t i o n o f c a l c i u m fluxes in n e r v o u s tissue. ACKNOWLEDGEMENT T h i s r e s e a r c h was s u p p o r t e d in p a r t by G r a n t
1 RO1 EY02540-01 f r o m t h e
N a t i o n a l Eye I n s t i t u t e . REFERENCES I Bonting, S. L. and Daemen, F. J., Calcium as a transmitter in photoreceptor cells. In S. L. Bonting (Ed.), Transmitters in the Visual Proeess, Pergamon Press, Oxford, 1976, pp. 60 89. 2 Cavaggioni, A., Sorbi, R. T. and Turini, S., Efflux of potassium from isolated rod outer segments: a photic effect, J. Physiol. (Lond.), 232 (1973) 609-620. 3 Cohen, A. 1., McDaniel, M. and Orr, H. T., Absolute levels of some free amino acids in normal and biologically fractionated retinas, Invest. Ophthal., 12 (1973) 686 693. 4 DeRobertis, E., Pelegrino de Iraldi, A., Rodriguez de Lores Arnaiz, G. and Salganicoff, L., Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetyl choline and acetylcholinesterase, J. Neurochem., 9 (1962) 23 35. 5 Dolara, P., Agresti, A., Giotti, A. and Pasquini, G., Effect of taurine on calcium kinetics of guinea-pig heart, Europ. J. Pharrnaeol., 24 (1973) 352-358. 6 D~lara, P., Ledda, F., Mugelli, A., Mantelli, L., Zilletti, F., Franconi, F. and G iotti, A., Effect of taurine on calcium, inotropism and electrical activity of the heart. In A. Barbeau and R. J. Huxtable (Eds.), Taurine and Neurological Disorders, Raven Press, New York, 1978, pp. 151 160. 7 Dowling, J. E. and Ripps, H., Effect of magnesium on horizontal cell activity in the skate retina, Nature (Lond.), 242 (1973) 101-103. 8 Fricke, U., Tritosol: a new scintillation cocktail based on Triton X-100, AnJyt. Biochem., 63 (1975) 555 558. 9 Hagins, W. A. and Yoshikami, S., Role for calcium in excitation of retinal reds and cones, Exp. Eye Res., 18 (1974) 299-305. 10 Izumi, K., Igisu, H. and Fukuda, T., Supression of seizures by taurine -- specific or non-specific? Brain Research, 76 (1974) 171 173. I 1 lzumi, K., lgisu, H. and Fukuda, T., Effects ofedetate on seizure supressing actions of taurine and GABA, Brain Research, 88 (1975) 576 579. 12 Izumi, K., Butterworth, R. F. and Barbeau, A., Effect of taurine on calcium binding to microsomes isolated from rat cerebral cortex, Life Sci., 20 (1977) 943-950. 13 Keen, P. and Yates, R. A., Distribution of amino acids in subdivided rat retinae, Brit. J. Pharmacol., 52 (1974) 118p. 14 Kennedy, A. J. and Voaden, M. J., Free amino acids in the photoreceptor cells of the frog retina, J. Neurochem, 23 (1974) 1093 1095. 15 Korenbrot, J. 1. and Cone, R. A., Dark ionic flux and the effect of light in isolated rod outer segments, J. gen. Physiol., 60 (1972) 20~-5. 16 Kuriyama, K., Muramatso, M., Nakagawa, K. and Kakita, K., Modulating role of taurine on release of neurotransmitters and calcium transport in excitable tissues. In A. Barbeau and R. J. Huxtable (Eds.), Taurine and Neurological Disorders, Raven Press, New York, 1978, pp. 201 216. 17 Lopez-Colome, A. M., Salceda, R. and Pasantes-Morales, H., Potassium stimulated release of GABA, glycine and taurine from the chick retina, Neurochem. Res., 3 (1978) 431M41. 18 Lowry, O., Rosebrough, N., Farr, A. and Randall, R. J., Protein measurement with Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 19 Macaione, G., Ruggieri, P., De Luca, F. and Tucci, G., Free amino acids in developing rat retina, J. Neurochem., 22 (1974) 887 891.
138 20 Mandel, P. and Pasantes-Morales, H., Taurine, a putative neurotransmitter in retina. In S. L, Bonting (Ed.), Transmitters in the Visual Process. Pergamon Press, Oxford, 1976, pp. 89-105. 21 Orr, H. T., Cohen, A. L and Lowry, O. H., The distribution of taurine in the vertebrate retina, J. Neurochem., 26 (1976) 609-612. 22 Pasantes-Morales, H., Klethi, J., Ledig, M. and Mandel, P., Free amino acids in chicken and rat retina, Brain Reserach, 41 (1972) 494-497. 23 Pasantes-Morales, H., Urban, P. F., Kelthi, J. and Mandel, P., Light stimulated release of [3'~S]taurine from chicken retina, Brain Research, 51 (1973) 375-378. 24 Salceda, R., Lopez-Colome, A. M. and Pasantes-Morales, H., Light-stimulated release of p S ] taurine from frog retinal rod outer segments, Brain Reserach, 135 (1977) 186-191. 25 Schmidt, S. Y. and Berson, E. L., Taurine fluxes in isolated cat and rat retinas: effects of illumination, Exp. Eye Res., 26 (1978) 529-538. 26 Starr, M. S., Effect of dark adaptation of the GABA system in retina, Brain Research, 59 (1973) 331-338. 27 Yoshikami, S. and Hagins, W. A., Control of the dark current in vertebrate rods and cones. In. H. Langer (Ed.), Biochemistry and Physiology of Visual Pigments, Springer Verlag, New York, 1973, pp. 245-255.
