Life Sciences Vol . 18, pp . Printed in the U .S .A.
Pergamon Press
75-80
CY~TEINE SULFINATE CARBOXYLYASE IN THE VISUAL PATHI+IAY OF ADULT CHICKEN R .L . Mathur, J . Rlethi, Vii . Ledig* and P . Mandel Centre de Neurochimie du CNRS, 11 Rue Humaaa, 67085 Strasbourg Cedes, France (~teceived in f1na1 form December 2, 1975) SU[~L~IARY Cysteine sulfinate (CSA) carbosylyase, the enzyme which synthesizes taurine through hypotaurine, shown a higher activity in the inner plexiform and nuclear layer of adult chick retina compared to the outer plexiform and nuclear layers whereas the outer segments of photoreceptors do not show any activity of this enzyme . Thése observations suggest an endogenous synthesis of taurine preferentially in certain layers of retina . Therefore, taurine fulfills one more criteria which is required by a substance to be accepted as a neurotransmitter in an organ . Studies on the distribution of CSAcarboxylyase in the visual pathway and other brain areas show a very high activity of this enzyme in optic tectum followed by cerebral cortex, cerebellum, retina, lateral geniculate body and optic nerve, taken with chiasma and tract in decreasing order . On the other hand, analysis of the free amino acid pool reveals a very high content of taurine in retina as compared to optic tectum . Cysteine sulfinate carboxylyase activity and the content of taurine therefore do not seem to bear a good correlation and other mechanisms of release, uptake and degradation might be involved in regulating the taurine content in these tissues . Taurine has been suggested to be a transmitter in retina (1) . The evidence for its new role in retinal function include the depression of the amplitude of b-wave of ERG (2,3), reversal of its inhibitory effect by the antagonist strych nine (4), release by photic, electrical and chemical stimulation (5,6) and the presence of a high affinity uptake system (2) . Although retina is known to contain very high amounts of taurine in various animal species (7,8), the evidence of its site of synthesis in different cells remained obscure . Taurine is known to be synthesized through the decarboxylatioa of L-cysteiae sulfinate is various animal tissues, and the reaction is catalyzed by the enzyme cyateina sulfinate carboaylyase (EC 4 .1 .1 .29) which is a pyridoxal phosphate dependent enzyme containing essential -SH groups (9-11) . The present investigation deals with the distribution of the enzyme CSA-carbosylyase in different layers of retina and in the higher visual centres of domestic fowl . Free amino acids of the optic tectum have also been analysed to study whether taurine could also play a role in the higher visual centres . NfATERLALS AND METHODS Adult chicken were sacrificed by decapitation and the eyes and tissues of the visual pathway removed . The whole procedure was completed within 15 min in cold environment . Chargé de Recherche au CNRS .
75
76
Taurine Synthesis in Visual Pathway
Vol . 18, No . 1
Separation of different Zayera of retina The enucleated eyes were frozen in dry ice for at least 4 h and frozen tangential sections of 8 u thickness were cut in a IEC microtome cryostat at -28°C . The sections were lyophilized at -60°C and different layers of the retina dis sected by free hand microdissection under a binocular dissecting microscope as described by Lowry et aZ . (12) . The microdissected material was pooled until we obtained 200-500 ug of protein for each layer of retina . The lyophilized material was stored at -60 ° C until utilized . Estimation of the enzyme activity The microdissected material was homogenized in glass homogenizes with a teflon pestle in 0 .8 ml of ice cold 0 .67 mlt Sorensen's phosphate buffer pH 6.8 containing 2 .5 mg of pyridoxal phosphate and 0 .2 ml triton X-100 per 100 ml . Fresh tissues were homogenized in 5-10 ml of the buffer and 0 .8 ml aliquots were used for enzyme assay essentially as described by Smith et aZ . (13) with the modification that TCA was used to stop the reaction instead of sulphuric acid . The homogenates for retina were incubated for 30 min at 37 °C in 5 mM of cold cyateine sulfinate and 1 uCi of 14C-cyateine sulfinate labelled at the carboxyl group (specific activity 11 mCi/mM) . In the initial experiments conducted to study the effect of substrate concentration on enzyme activity, we observed maximal activity at 5 mM concentration of substrate . It was not worthwhile to use a higher concentration of substrate as that would dilute further the radioactivity which is not desirable . All the other tissues taken were incubated in a 10 m~I concentration of cold cyateine sulfinate and 0 .2 uCi of radioactive substrate, as the amount of tissue was relatively larger . Cyateine sulfinate, cold and radioactive, was injected in incubation flask in 0 .2 ml solution pH 6 .5-7 .0 so that the final Volume was 1 .0 ml . After the incubation period the reaction was stopped by injecting 0 .2 ml of 40 % TCA . The labelled C02 evolved was absorbed in 0 .15 ml of hyamine hydroxide (the flasks remained covered for another 90 min to allow maximal absorption of COp in hyamine hydroxide) and used for counting in 2 ml of absolute ethanol and 10 ml of 0 .4 % omnifluor in toluene . The vials were counted in the Intertechaique liquid scintillation spectrometer model SL 30 . Estimation of proteins The precipitated proteins in the incubation flasks were centrifuged and the pellets dissolved in 1 N NaOH . The proteins were estimated according to the method of Lowry et aZ . (14) . AnaZyais of free amino acids Four fresh optic lobes were used in each estimation . Free amino acids were extracted in 0 .6 N perchloric acid . The extract was brought to pH 2 with 1 N RAH and centrifuged to remove perchlorate precipitate . The free amino acids we re analysed in a Technicon analyzer model NCI according to the method of Efron (15) . Light 8t1.mLlZation The animals were kept in a cage illuminated by fluorescent lights for 4 h. The amount of light that fell in the cage was 600 lux from any direction . After the period of light exposure, the animals were sacrificed and the tissues dis sected out in the same environment and then used for homogenization and enzyme assay in the normal day light . Animals in the control group were kept in normal day light conditions and were exposed to the ordinary day-night cycle .
Vol . 18, No . 1
Taurine Synthesis in Visual Pathway
77
Reagents Potassium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethanol, Prolabo, France . Triton R-100, Roth, West Germany . Hyamine hydroxide, Koch Light, England . Omnifluor, NEN Chemicals, West Germany . Toluene, Carlo Erba . Cysteine aulfinate cold, Sigma, USA . Cysteine sulfinate 14C was prepared by Mr . L . Pichat from Commissariat à l'Energie Atomique, France . RESULTS AND DISCUSSION The outer segments of photoreceptors do not show any activity of Cysteine sulfinate carboxylyase whereas the outer nuclear layer and the outer plexiform layer taken together show the presence of the enzyme in this region (Table 1) . TABLE 1 Distribution of Cysteine Sulfinate Carboxylyase in Different Layers of Adult Chicken Retina Layers of Retina
mumoles C02/h/mg protein
Outer segments of photoreceptors
Nil
Outer nuclear and plexiform layer
10 .2
Inner nuclear and plexiform layer
18 .5
Ganglion celle
N .D .
N .D . Nôt determiäéd (preparatiôn was spoiled during enzyme assay) . Material obtained from 75 eyes was used for the experiment . The inner nuclear layer and the inner plexiform layer taken together show a comparatively higher activity of the enzyme . It is interesting to note that Yaminobutyric acid, which also depresses the brwave of ERG (16-18) accumulates preferentially in the inner part of the inner pleaiform layers of avian retina (19) while some selective accumulation of this amino acid could also be seen in the horizontal cells . It becomes evident from the present data that the ability of chick retina to synthesize taurine lies largely in the area rich in amacrine cells (the inner plexiform layer taken together with inner nuclear layer) whereas the outer layers which include the horizontal cell layer show relatively less activity of this enzyme . Therefore, taurine fulfills another criteria which is required for a substance to be accepted as a possible neurotransmitter i .e . the presence of synthesizing enzyme in the regions of its suggested site of action . Studies on the distribution of CSA-carboxylyase in different regions of the visual pathway and different areas of brain show a very high activity of this enzyme in the optic tectum . Cerebellum and cerebral cortex show about half of the activity compared to optic tectum while retina and lateral geniculate body show relatively leas activity (Table 2) . The distribution of CSA-carboxylyase in different parts of brain and visual pathway of chicken exposed to continuous light for 4 h remains unchanged in the optic tectum, lateral geniculate body, cerebral cortex and cerebellum . On the other hand no measurable activity of this enzyme could be observed in the optic nerve (Table 2) . Electrical stimulation of rat brain slices has been shown to enhance the activity of CSA-carboxylyase (11) . However, exposure to light for 4 h is a relatively mild stimulus compared to electrical stimulation . Therefore, studies with longer exposure of repeated light stimulation are definitely required before one can make a conclusion regarding the sensitivity of this enzyme
78
Vol . 18, No . 1
Taurine Syntheaie is Visual Pathway
to physiolôgical stimulation . TABLE 2 Distribution of Cyateine Sulfinate Carboxylyase Activity in Different Regions of the Visual Pathway of Adult Chicken Tissue
umoles C02/h/g tissue Control
Continuous light
Retina
3 .51 10 .34 (7)
Optic nerve with chiasma and tract
1 .78 ±1 .01
(5)
2 .63
Lateral geniculate body
Nil
(3)
(2)
2 .68 10 .37 (3)
Optic tectum left
22 .44 13 .69 (4)
26 .19 t3 .5ß (3)
Right
23 .95 13 .14 (4)
26 .79 ±4 .68 (3)
Cerebral cortex
12 .51 ±0 .62 (4)
12 .16 10 .87 (3)
Cerebellum
10 .25 ±0 .88 (4)
9 .55 ±2 .03 (3)
Figures in parenthesis indicate the number of experiments in each case . The distribution of taurine has not before been measured in different rerions of the visual pathway, however, earlier work from our laboratory has shown that taurine is present in very high amounts in retina compared to brain in adult chicken (ß) . We observed that the content of taurine in retina is 9 .6 umoles/g fresh weight and constitutes 41 .5 S of the total free amino acid pool . On the other hand, the content of taurine in optic tectum is 0 .37 umoles/g fresh weight and constitutes only 1 .7 X of the total free amino acid pool . The activity of CSA-carboxylyase and the content of taurine therefore do not show a good correlation in the optic tectum and retina of adult chick . A similar obaervation has been reported for developing rabbit brain (20) . It is probable that CSA-carboxylyase activity may not be the only factor regulating the content of taurine in different tissues ; other processes like release, reuptake and degradation mechanisms might also play an important role . It is known that degradation processes are important in the regulation of cyclic GMP (21) . Reup- " take mechanisms for taurine have been demonstrated for retina and brain dices (2,22) . ACKNOWLEDGEMENTS The skillful technical assistance of Mra . A . Poilpré is gratefully acknowledged . This work was supported by a grant from the French Commissariat à l'Energie Atomique, Département de Biologie . REFERENCES H . PASANTES-MORALES, P .F . URBAN, J . KLETHI, and P . MANDEL, J . PhyBioZ . (Paris) 87 211A (1973) . H . PASANTSS-MORALES, J . RLETHI, P .F . URBAN, and P . MANDEL, PhysioZ. Chem . Phus . _4 339-348 (1972) . H . PASANTES-?TORALES, H . BONAVENTURE, N . WIOLAND, and P . MANDEL, Intern . J . Neurosci . _5 235-241 (1973) . N . BONAVENTURE, N . WIOLAND, and P . MANDEL, Brain Res . 80 281-289 (1974) .
Vol . 18, No . 1
Taurine Synthesis in Visual Pathway
79
5 . H . PASANTES-MORALES, J . KLETHI, P .F . URBAN, and P . MANDEL, E~ptZ . Brain Res . _19 131-141 (1974) . 6 . H . PASANTES-MORALES, P .F . URBAN, J . KLETHI, and P . MANDEL, Brain Res . _51 375-378 (1973) . 7 . R. KUBIERR, and A . DOLENR, J. Chromatogr . _1 266-268 (1958) . 8 . H. PASANTES-MORALES, J . KLETHI, P .F . URBAN, and P . MANDEL, Brain Res . 41 494-497 (1972) . 9 . J .G . JACOBSON, and L .H . SMITH Jr ., Nature (Lund.) _200 575-578 (1963) . 10 . J .G . JACOBSON, L .L . GHOMAS, and L .H . SMITH Jr ., Bioehim. Biophya . Acta _85 103-115 (1964) . 11 . S .S . OJA, M .L . KARVONEN, and P . LÄHUESMÄKI, Brain Res. _55 173-178 (1973) . 12 . O .H . LOWRY, N .R . ROBERTS, and C.H . LEWEIS, J. BioZ . Chem . 220 879-892 (1956) . 13 . L .H . SMITH Jr ., M. SULLIVAN, and C .M . HUGULEY Jr ., J. CZin. Invest . _40 656-665 (1961) . 14 . O .H . LOWRY, N.J . ROSEBROUGH, A .L . FARR, and R.J . RANDALL, J. BioZ . Chem . 193 265-275 (1951) . 15 . M .L . EFRON, in Technicon Symposium on Automztion in AnaZyticaZ Chemistry, ed . L .T . SREGGS (pp . 637-642), Mediad Inc ., New York (1966) . 16 . S .Z . KRA1tER, P .A . SHERafAN, and J . SEIFTE':, Intern . J. Neuro_pharmacol . _6 463-472 (1967) . 17 . E . ROBERTS, and K. RURIYAMA, Brain Res . 8 1-35 (1968) . 18 . N .W . SCHOLES, and E . ROBERTS, Biochem. PharmacoZ . 13 1319-1329 (1964) . 19 . J . MARSCHALL, and M. VOADEN, Invest . Ophthal. _13 60F607 (1974) . 20 . H .C . AGRAWAL, A .N . DAVISON, and L .R . KACZMAREK, Biochem . J. _122 759-763 (1971) . 21 . C . GORIDIS, and N. VIRMAUX, Nature (Loud.) 248 57-59 (1973) . 22 . S .S . OJA, J . Neurochem . 18 1847-1852 (1971)
Pergamon Press
75-80
CY~TEINE SULFINATE CARBOXYLYASE IN THE VISUAL PATHI+IAY OF ADULT CHICKEN R .L . Mathur, J . Rlethi, Vii . Ledig* and P . Mandel Centre de Neurochimie du CNRS, 11 Rue Humaaa, 67085 Strasbourg Cedes, France (~teceived in f1na1 form December 2, 1975) SU[~L~IARY Cysteine sulfinate (CSA) carbosylyase, the enzyme which synthesizes taurine through hypotaurine, shown a higher activity in the inner plexiform and nuclear layer of adult chick retina compared to the outer plexiform and nuclear layers whereas the outer segments of photoreceptors do not show any activity of this enzyme . Thése observations suggest an endogenous synthesis of taurine preferentially in certain layers of retina . Therefore, taurine fulfills one more criteria which is required by a substance to be accepted as a neurotransmitter in an organ . Studies on the distribution of CSAcarboxylyase in the visual pathway and other brain areas show a very high activity of this enzyme in optic tectum followed by cerebral cortex, cerebellum, retina, lateral geniculate body and optic nerve, taken with chiasma and tract in decreasing order . On the other hand, analysis of the free amino acid pool reveals a very high content of taurine in retina as compared to optic tectum . Cysteine sulfinate carboxylyase activity and the content of taurine therefore do not seem to bear a good correlation and other mechanisms of release, uptake and degradation might be involved in regulating the taurine content in these tissues . Taurine has been suggested to be a transmitter in retina (1) . The evidence for its new role in retinal function include the depression of the amplitude of b-wave of ERG (2,3), reversal of its inhibitory effect by the antagonist strych nine (4), release by photic, electrical and chemical stimulation (5,6) and the presence of a high affinity uptake system (2) . Although retina is known to contain very high amounts of taurine in various animal species (7,8), the evidence of its site of synthesis in different cells remained obscure . Taurine is known to be synthesized through the decarboxylatioa of L-cysteiae sulfinate is various animal tissues, and the reaction is catalyzed by the enzyme cyateina sulfinate carboaylyase (EC 4 .1 .1 .29) which is a pyridoxal phosphate dependent enzyme containing essential -SH groups (9-11) . The present investigation deals with the distribution of the enzyme CSA-carbosylyase in different layers of retina and in the higher visual centres of domestic fowl . Free amino acids of the optic tectum have also been analysed to study whether taurine could also play a role in the higher visual centres . NfATERLALS AND METHODS Adult chicken were sacrificed by decapitation and the eyes and tissues of the visual pathway removed . The whole procedure was completed within 15 min in cold environment . Chargé de Recherche au CNRS .
75
76
Taurine Synthesis in Visual Pathway
Vol . 18, No . 1
Separation of different Zayera of retina The enucleated eyes were frozen in dry ice for at least 4 h and frozen tangential sections of 8 u thickness were cut in a IEC microtome cryostat at -28°C . The sections were lyophilized at -60°C and different layers of the retina dis sected by free hand microdissection under a binocular dissecting microscope as described by Lowry et aZ . (12) . The microdissected material was pooled until we obtained 200-500 ug of protein for each layer of retina . The lyophilized material was stored at -60 ° C until utilized . Estimation of the enzyme activity The microdissected material was homogenized in glass homogenizes with a teflon pestle in 0 .8 ml of ice cold 0 .67 mlt Sorensen's phosphate buffer pH 6.8 containing 2 .5 mg of pyridoxal phosphate and 0 .2 ml triton X-100 per 100 ml . Fresh tissues were homogenized in 5-10 ml of the buffer and 0 .8 ml aliquots were used for enzyme assay essentially as described by Smith et aZ . (13) with the modification that TCA was used to stop the reaction instead of sulphuric acid . The homogenates for retina were incubated for 30 min at 37 °C in 5 mM of cold cyateine sulfinate and 1 uCi of 14C-cyateine sulfinate labelled at the carboxyl group (specific activity 11 mCi/mM) . In the initial experiments conducted to study the effect of substrate concentration on enzyme activity, we observed maximal activity at 5 mM concentration of substrate . It was not worthwhile to use a higher concentration of substrate as that would dilute further the radioactivity which is not desirable . All the other tissues taken were incubated in a 10 m~I concentration of cold cyateine sulfinate and 0 .2 uCi of radioactive substrate, as the amount of tissue was relatively larger . Cyateine sulfinate, cold and radioactive, was injected in incubation flask in 0 .2 ml solution pH 6 .5-7 .0 so that the final Volume was 1 .0 ml . After the incubation period the reaction was stopped by injecting 0 .2 ml of 40 % TCA . The labelled C02 evolved was absorbed in 0 .15 ml of hyamine hydroxide (the flasks remained covered for another 90 min to allow maximal absorption of COp in hyamine hydroxide) and used for counting in 2 ml of absolute ethanol and 10 ml of 0 .4 % omnifluor in toluene . The vials were counted in the Intertechaique liquid scintillation spectrometer model SL 30 . Estimation of proteins The precipitated proteins in the incubation flasks were centrifuged and the pellets dissolved in 1 N NaOH . The proteins were estimated according to the method of Lowry et aZ . (14) . AnaZyais of free amino acids Four fresh optic lobes were used in each estimation . Free amino acids were extracted in 0 .6 N perchloric acid . The extract was brought to pH 2 with 1 N RAH and centrifuged to remove perchlorate precipitate . The free amino acids we re analysed in a Technicon analyzer model NCI according to the method of Efron (15) . Light 8t1.mLlZation The animals were kept in a cage illuminated by fluorescent lights for 4 h. The amount of light that fell in the cage was 600 lux from any direction . After the period of light exposure, the animals were sacrificed and the tissues dis sected out in the same environment and then used for homogenization and enzyme assay in the normal day light . Animals in the control group were kept in normal day light conditions and were exposed to the ordinary day-night cycle .
Vol . 18, No . 1
Taurine Synthesis in Visual Pathway
77
Reagents Potassium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethanol, Prolabo, France . Triton R-100, Roth, West Germany . Hyamine hydroxide, Koch Light, England . Omnifluor, NEN Chemicals, West Germany . Toluene, Carlo Erba . Cysteine aulfinate cold, Sigma, USA . Cysteine sulfinate 14C was prepared by Mr . L . Pichat from Commissariat à l'Energie Atomique, France . RESULTS AND DISCUSSION The outer segments of photoreceptors do not show any activity of Cysteine sulfinate carboxylyase whereas the outer nuclear layer and the outer plexiform layer taken together show the presence of the enzyme in this region (Table 1) . TABLE 1 Distribution of Cysteine Sulfinate Carboxylyase in Different Layers of Adult Chicken Retina Layers of Retina
mumoles C02/h/mg protein
Outer segments of photoreceptors
Nil
Outer nuclear and plexiform layer
10 .2
Inner nuclear and plexiform layer
18 .5
Ganglion celle
N .D .
N .D . Nôt determiäéd (preparatiôn was spoiled during enzyme assay) . Material obtained from 75 eyes was used for the experiment . The inner nuclear layer and the inner plexiform layer taken together show a comparatively higher activity of the enzyme . It is interesting to note that Yaminobutyric acid, which also depresses the brwave of ERG (16-18) accumulates preferentially in the inner part of the inner pleaiform layers of avian retina (19) while some selective accumulation of this amino acid could also be seen in the horizontal cells . It becomes evident from the present data that the ability of chick retina to synthesize taurine lies largely in the area rich in amacrine cells (the inner plexiform layer taken together with inner nuclear layer) whereas the outer layers which include the horizontal cell layer show relatively less activity of this enzyme . Therefore, taurine fulfills another criteria which is required for a substance to be accepted as a possible neurotransmitter i .e . the presence of synthesizing enzyme in the regions of its suggested site of action . Studies on the distribution of CSA-carboxylyase in different regions of the visual pathway and different areas of brain show a very high activity of this enzyme in the optic tectum . Cerebellum and cerebral cortex show about half of the activity compared to optic tectum while retina and lateral geniculate body show relatively leas activity (Table 2) . The distribution of CSA-carboxylyase in different parts of brain and visual pathway of chicken exposed to continuous light for 4 h remains unchanged in the optic tectum, lateral geniculate body, cerebral cortex and cerebellum . On the other hand no measurable activity of this enzyme could be observed in the optic nerve (Table 2) . Electrical stimulation of rat brain slices has been shown to enhance the activity of CSA-carboxylyase (11) . However, exposure to light for 4 h is a relatively mild stimulus compared to electrical stimulation . Therefore, studies with longer exposure of repeated light stimulation are definitely required before one can make a conclusion regarding the sensitivity of this enzyme
78
Vol . 18, No . 1
Taurine Syntheaie is Visual Pathway
to physiolôgical stimulation . TABLE 2 Distribution of Cyateine Sulfinate Carboxylyase Activity in Different Regions of the Visual Pathway of Adult Chicken Tissue
umoles C02/h/g tissue Control
Continuous light
Retina
3 .51 10 .34 (7)
Optic nerve with chiasma and tract
1 .78 ±1 .01
(5)
2 .63
Lateral geniculate body
Nil
(3)
(2)
2 .68 10 .37 (3)
Optic tectum left
22 .44 13 .69 (4)
26 .19 t3 .5ß (3)
Right
23 .95 13 .14 (4)
26 .79 ±4 .68 (3)
Cerebral cortex
12 .51 ±0 .62 (4)
12 .16 10 .87 (3)
Cerebellum
10 .25 ±0 .88 (4)
9 .55 ±2 .03 (3)
Figures in parenthesis indicate the number of experiments in each case . The distribution of taurine has not before been measured in different rerions of the visual pathway, however, earlier work from our laboratory has shown that taurine is present in very high amounts in retina compared to brain in adult chicken (ß) . We observed that the content of taurine in retina is 9 .6 umoles/g fresh weight and constitutes 41 .5 S of the total free amino acid pool . On the other hand, the content of taurine in optic tectum is 0 .37 umoles/g fresh weight and constitutes only 1 .7 X of the total free amino acid pool . The activity of CSA-carboxylyase and the content of taurine therefore do not show a good correlation in the optic tectum and retina of adult chick . A similar obaervation has been reported for developing rabbit brain (20) . It is probable that CSA-carboxylyase activity may not be the only factor regulating the content of taurine in different tissues ; other processes like release, reuptake and degradation mechanisms might also play an important role . It is known that degradation processes are important in the regulation of cyclic GMP (21) . Reup- " take mechanisms for taurine have been demonstrated for retina and brain dices (2,22) . ACKNOWLEDGEMENTS The skillful technical assistance of Mra . A . Poilpré is gratefully acknowledged . This work was supported by a grant from the French Commissariat à l'Energie Atomique, Département de Biologie . REFERENCES H . PASANTES-MORALES, P .F . URBAN, J . KLETHI, and P . MANDEL, J . PhyBioZ . (Paris) 87 211A (1973) . H . PASANTSS-MORALES, J . RLETHI, P .F . URBAN, and P . MANDEL, PhysioZ. Chem . Phus . _4 339-348 (1972) . H . PASANTES-?TORALES, H . BONAVENTURE, N . WIOLAND, and P . MANDEL, Intern . J . Neurosci . _5 235-241 (1973) . N . BONAVENTURE, N . WIOLAND, and P . MANDEL, Brain Res . 80 281-289 (1974) .
Vol . 18, No . 1
Taurine Synthesis in Visual Pathway
79
5 . H . PASANTES-MORALES, J . KLETHI, P .F . URBAN, and P . MANDEL, E~ptZ . Brain Res . _19 131-141 (1974) . 6 . H . PASANTES-MORALES, P .F . URBAN, J . KLETHI, and P . MANDEL, Brain Res . _51 375-378 (1973) . 7 . R. KUBIERR, and A . DOLENR, J. Chromatogr . _1 266-268 (1958) . 8 . H. PASANTES-MORALES, J . KLETHI, P .F . URBAN, and P . MANDEL, Brain Res . 41 494-497 (1972) . 9 . J .G . JACOBSON, and L .H . SMITH Jr ., Nature (Lund.) _200 575-578 (1963) . 10 . J .G . JACOBSON, L .L . GHOMAS, and L .H . SMITH Jr ., Bioehim. Biophya . Acta _85 103-115 (1964) . 11 . S .S . OJA, M .L . KARVONEN, and P . LÄHUESMÄKI, Brain Res. _55 173-178 (1973) . 12 . O .H . LOWRY, N .R . ROBERTS, and C.H . LEWEIS, J. BioZ . Chem . 220 879-892 (1956) . 13 . L .H . SMITH Jr ., M. SULLIVAN, and C .M . HUGULEY Jr ., J. CZin. Invest . _40 656-665 (1961) . 14 . O .H . LOWRY, N.J . ROSEBROUGH, A .L . FARR, and R.J . RANDALL, J. BioZ . Chem . 193 265-275 (1951) . 15 . M .L . EFRON, in Technicon Symposium on Automztion in AnaZyticaZ Chemistry, ed . L .T . SREGGS (pp . 637-642), Mediad Inc ., New York (1966) . 16 . S .Z . KRA1tER, P .A . SHERafAN, and J . SEIFTE':, Intern . J. Neuro_pharmacol . _6 463-472 (1967) . 17 . E . ROBERTS, and K. RURIYAMA, Brain Res . 8 1-35 (1968) . 18 . N .W . SCHOLES, and E . ROBERTS, Biochem. PharmacoZ . 13 1319-1329 (1964) . 19 . J . MARSCHALL, and M. VOADEN, Invest . Ophthal. _13 60F607 (1974) . 20 . H .C . AGRAWAL, A .N . DAVISON, and L .R . KACZMAREK, Biochem . J. _122 759-763 (1971) . 21 . C . GORIDIS, and N. VIRMAUX, Nature (Loud.) 248 57-59 (1973) . 22 . S .S . OJA, J . Neurochem . 18 1847-1852 (1971)
