- Email: [email protected]
Effect of taurine and taurine + triiodothyronine combination on the development of anuran larvae
03WYb2Y
79 O-WI-WY5SU2.lKlO
EFFECT OF TAURINE AND TAURINE + TRIIODOTHYRONINE COMBINATION ON THE DEVELOPMENT OF ANURAN LARVAE L. FEUER, L. J. Tij~ij~ and G. CSABA Chinoin Pharmaceutical and Chemical Works Ltd. 1325 Budapest T6 u. 1, Hungary, and Department of Biology, Semmelweis University of Medicine, 1094 Budapest, Tiizolt(, u. 58. Hungary (Received 6 June 1978) Abstract-l. Shortening of the tail fin of frog larvae is stimulated in the early stages of development and is retarded in the late stages by taurine. 2. The effect of triiodothyronine on metamorphosis is antagonized by taurine. This antagonistic effect is dose-dependent and statistically significant. Taurine exerts a T, antagonistic effect in a concentration lo3 times higher than litoralon. 3. Taurine influences the development of frog larvae in the same manner as litoralon (gamma-L-glutamyl-taurine). Taurine is supposed to be the precursor of litoralon.
INTRODUCTION Litoralon (gamma-L-glutamyl-taurine), a newly detected hormone of the parathyroid (Feuer, 1975) stimulates the early development of frog larvae, accelerating tail resorption, enhancing the shortening of body length and the emergence of the forelimbs (Feuer et al., 197%~). At the same time, this hormone delays metamorphic transformation (Feuer et al., 1978a). This latter effect is probably due to the triiodothyronine (T3) antagonistic action of litoralon (Feuer et al., 1978b). Secretion of T3 is characteristic of the advanced stage of metamorphosis: .consequently, while prior to metamorphosis, vitamin A-like activity of litoralon (Feuer et al., 1977) and its enhancing effect on lysosomal activity (Feuer, 1977) dominates, resulting in the acceleration of the early phase of metamorphosis; an antagonism of T, delays metamorphic transformation, i.e. the later phase of this process. Based on the supposition that taurine was a structural element and a biochemical precursor of litoralon we have examined its effect on the metamorphosis of the frog in order to obtain proof of its possible triiodothyronine antagonistic effect.
(9) With the exception of the controls. the larvae were bathed for 2 hr daily in water containing 0.01 pg/ml T, (triiodothyronine, Sanabo) and were placed immediately after this, for an additional 2 hr period. into water containing 50. 200 or 5OOpg/ml taurine.
RESULTS (A) The applied concentrations of taurine have enhanced the shortening of the tail up to the 34th-36th experimental day. Following this, up to the 60th day, length of the taurine-treated larvae exceeded that of the controls, i.e. at this period taurine retarded further transformation. No significant difference could be found between the effects of the three taurine concentrations used. Values of the taurine curves on the llth, 15th, 21st. 30th and 36th day were significantly lower than the corresponding control values (P < 0.01, P < 0.01. P < 0.01, P < 0.01 and P < 0.05; Fig. 1).
_ _-__ -.-._
......
MATERIAL AND
Control Taurine 50 p/ml Taurine 2 c$ &ml Tourine 500&ml
METHODS
of Rana aroalis Wolterstorffi (100 in each group) tap water at a temperature of 20-25-C (room temperature). Experiments were started with larvae in stage 26 (Tyerentyev, 1950) and had a mean body length of 25-30 mm. Their water was exchanged daily and they were fed vegetable food (nettle powder, grated apple and lettuce) ad libitum and twice weekly ovalbumin. Ova
were kept in chlorine-free
Development of the larvae was controlled daily, their body length was measured by a phototechnical method. Growth of the control and experimental groups was represented in diagrams based on the mean values of body length (Feuer ef al., 1978~). Mortality was recorded with care and statistical significance of the differences was calculated by Student’s r-test. Two main experimental groups were formed : (A) With the exception of the controls, the animals were bathed for 2 hr daily in water containing 50, 200 or 500 pg/ml taurine (Merck), respectively.
L
IO
70
io”
s:
I%
50 II0
60 X120 xx
Days Fig. 1. Effect of taurine on the metamorphosis of frog larvae, expr&sed in body length (mm). Days: x . experimental days, x x, age of larvae. 995
L. FE~,cK. L. J.
Ti)~i)h
......... r
treated with T,. the difference reached the level of statistical significance only on the 15th and Zlst days (P < 0.05). Application of 50O~lg,ml resulted in no
Control -- - - Taurlne 50
-
100 90 80 70-
*g/W - Tawne 200 us/ml Tawne 500ig/ml
-
/
60-
z
50-
%
4030-
i
change on the 6th day. but a significant difference was observed between the group treated with T, only and that treated with taurine + T, (P c 0.01: Fig. 3) on the 11th. 15th and 2lst days.
/
I
g
&
DISCL’SSIO’L
&&. 1%
20IO-
,,.y’ ,,_... X..” p.” Y
; IO
2u I 60
I 70
1
“’ h’ ,
,x~,;Trs,y 30
I 40
I 50
I 60
I 90
I too
I I IO
’ Jxx 120
’
Fig. 2. ElTect of taurine on mortality of frog larvae t”,,). Days: X. experimental days: x x . age of larvae.
From day 40. death rate of the frog larvae treated with any concentration of taurine was significantly lower than that of the untreated controls (P < 0.01). No significant concentration-dependent difference could be detected among the effects of the three taurine concentrations, regardless of whether they were compared to each other or to the control (Fig. 2). (B) T, produced an extremely intensive shortening of the body length (shortening of the tail fin). It reached the level of statistical significance as early as the 6th day (P < 0.01) and remained significant also on the llth, 15th and 31st days (P < 0.01. P < 0.01 and P < 0.05. respectively): at later periods, due to the considerable reduction of the number of larvae in the groups, no statistics were calculated. Under the effect of SOpg/mI taurine, a significant difference was found, from the 6th day, compared to the untreated controls, but the difference was not significant with regard to the group treated with T3. In the case of treatment with 2OOpg/ml taurine. although between the 6th and 1 Ith days no significant difference was observed in comparison to the animals -----
Control Tourine 50 us/ml
and G. CSARA
+ T3
Results of the experiments clearly show that taurine enhanced the early stages of amphibian metamorphosis while it retarded later ones. The experiments also revealed that taurine antagonized the effects of triiodothyronine on the development of frog larvae. Even a IO-fold increase of taurine concentration did not produce a more marked development of normal frog larvae than the initial dose. The T,-antagonistic effect was. on the other hand. dose-dependent. The effects of taurine are in complete agreement with those described for litoralon (Feuer er al., 1978a-c). i.e. the parameters studied havr been influenced by both compounds in the same direction and to the same measure. Based on the supposition that taurine was the precursor of litoralon. we have applied taurine in concentrations two to three orders of magnitude higher (lO&lOOO times) than the effective litoralon concentrations. A strongly significant T,-antagonistic effect was observed with 5OO~g,‘ml taurine and 0.5 pg/ml htoralon from the 11th day (Feuer er ul., 1978b). Our experiments with “S taurine still in progress seem to confirm the hypothesis that litoralon (gamma-L-glutamyl taurine) is formed from taurine and gamma glutaminic dipeptides. in the presence of gamma-glutamyl-transpeptidase (GGTP) enzyme (Orlowski & Meister, 1965: Szisz. 1969: Griindig & Rummler. 1974). Gwynne and his coworkers (1976) observed the increase of taurine concentration in the tail tissue of Rana rtrtrsheiana tadpoles before the start of lysis. At the end of this process the taurine level returned to the pre-metamorphic value. In the triiodothyronine-treated tadpoles. taurine concentration remained unchanged. From this Gwynne et a/. came to the conclusion that metamorphosis was not regulated exclusively by the thyroid hormones. Baskin CT a/. (1973) observed the increase of pigmentation of Rana cutrsheiuna tadpoles under the effect of taurine and attributed it to an inhibition of melatonine production or release. We suppose that in this hormonal interaction. too, taurine may act as a precursor.
REFERENCES
*’ 1
IO I 70
I 65
I 15 I 75
Fig. 3. EtTect of T, (trtiodothyronine ine + T, expressed
I 20
I 25
l 30
I 80
I 65
1 lxx 90
0.01 pg/ml) and tauron the metamorphosis of frog larvae. length (mm). Days: x. experimental r. says: x x, age 01 rarvae.
treatment in body
x
BASKIN S. I. & DAGIRMANJIAN R. (1973) The effect of taurine on the pigmentation of the bullfrog tadpole. Camp. Biochcm. Physiol. 44A. 297-302. FEWER L. (1975) Br. Pat. 1404225: (1977) U.S. Pat. 4001396. FEUER L. (1977) Theoretical background of the recognition of a new bioactive substance. litoralon, isolated from the parathyroid. Further theoretical considerations. Biologia. Budapest 25, 3-33. FEUER L.. B~NYAI B. & HERCSEL J. (1977) Influence of protein free aqueous extract of parathyroid powder on berum vitamin
A level in rats. Experirnria
33. 1005~1006.
The development of anuran larvae FEUERL.. T6~ij~ L. J. & CSABAG. (1978a) Effect of litoraIon on the metamorphosis of Rana arcalis. Acta ho/. in press. FtuEa L., T~R~K L. & CSABA G. (1978b) The triiodothyronine antagonistic effect of gamma-L-glutamyl-taurine (Litoralon). Endokrinologie in press. Fr;ur:a L.. Tii~ii~ L. J.. KAPA E. & CSABAG. (1978~) The effect of gamma-L-glutamyl-taurine (Litoralon) on the amphibian metamorphosis. Cor~p. Biochrrll. Physiol. 6lC.
67-71.
GR~NIIIG C. A. & RUMMLISW. (1974) Gamma-glutamyltranspeptidase (GGTP). Dr. Grs. w\‘ys~~~. 29. l108ml 114.
997
GWYNNE H. & CASTROC. E. (1976) Taurine
levels in anuran tadpole tail during spontaneous and triiodothyronine induced metamorphosis. Cor~p. Biochrm. Ph.niol. S4A. 245-247..
ORLOWSKIM. & MEISTERA. (1965) Isolation of gammaglutamyl-transpeptidase from hog kidney. J. bio[. Chem. 240. 338-347. Sz.isz G. (1969) A kinetic photometric method for serum gammaglutamyl-transpeptidase. Clin. Chew 15, 124-136. TYERENTYEV P. B. (1950) In: Ljaguska (The Frog), pp 59-67. Goz. Izd.. Moscow.
79 O-WI-WY5SU2.lKlO
EFFECT OF TAURINE AND TAURINE + TRIIODOTHYRONINE COMBINATION ON THE DEVELOPMENT OF ANURAN LARVAE L. FEUER, L. J. Tij~ij~ and G. CSABA Chinoin Pharmaceutical and Chemical Works Ltd. 1325 Budapest T6 u. 1, Hungary, and Department of Biology, Semmelweis University of Medicine, 1094 Budapest, Tiizolt(, u. 58. Hungary (Received 6 June 1978) Abstract-l. Shortening of the tail fin of frog larvae is stimulated in the early stages of development and is retarded in the late stages by taurine. 2. The effect of triiodothyronine on metamorphosis is antagonized by taurine. This antagonistic effect is dose-dependent and statistically significant. Taurine exerts a T, antagonistic effect in a concentration lo3 times higher than litoralon. 3. Taurine influences the development of frog larvae in the same manner as litoralon (gamma-L-glutamyl-taurine). Taurine is supposed to be the precursor of litoralon.
INTRODUCTION Litoralon (gamma-L-glutamyl-taurine), a newly detected hormone of the parathyroid (Feuer, 1975) stimulates the early development of frog larvae, accelerating tail resorption, enhancing the shortening of body length and the emergence of the forelimbs (Feuer et al., 197%~). At the same time, this hormone delays metamorphic transformation (Feuer et al., 1978a). This latter effect is probably due to the triiodothyronine (T3) antagonistic action of litoralon (Feuer et al., 1978b). Secretion of T3 is characteristic of the advanced stage of metamorphosis: .consequently, while prior to metamorphosis, vitamin A-like activity of litoralon (Feuer et al., 1977) and its enhancing effect on lysosomal activity (Feuer, 1977) dominates, resulting in the acceleration of the early phase of metamorphosis; an antagonism of T, delays metamorphic transformation, i.e. the later phase of this process. Based on the supposition that taurine was a structural element and a biochemical precursor of litoralon we have examined its effect on the metamorphosis of the frog in order to obtain proof of its possible triiodothyronine antagonistic effect.
(9) With the exception of the controls. the larvae were bathed for 2 hr daily in water containing 0.01 pg/ml T, (triiodothyronine, Sanabo) and were placed immediately after this, for an additional 2 hr period. into water containing 50. 200 or 5OOpg/ml taurine.
RESULTS (A) The applied concentrations of taurine have enhanced the shortening of the tail up to the 34th-36th experimental day. Following this, up to the 60th day, length of the taurine-treated larvae exceeded that of the controls, i.e. at this period taurine retarded further transformation. No significant difference could be found between the effects of the three taurine concentrations used. Values of the taurine curves on the llth, 15th, 21st. 30th and 36th day were significantly lower than the corresponding control values (P < 0.01, P < 0.01. P < 0.01, P < 0.01 and P < 0.05; Fig. 1).
_ _-__ -.-._
......
MATERIAL AND
Control Taurine 50 p/ml Taurine 2 c$ &ml Tourine 500&ml
METHODS
of Rana aroalis Wolterstorffi (100 in each group) tap water at a temperature of 20-25-C (room temperature). Experiments were started with larvae in stage 26 (Tyerentyev, 1950) and had a mean body length of 25-30 mm. Their water was exchanged daily and they were fed vegetable food (nettle powder, grated apple and lettuce) ad libitum and twice weekly ovalbumin. Ova
were kept in chlorine-free
Development of the larvae was controlled daily, their body length was measured by a phototechnical method. Growth of the control and experimental groups was represented in diagrams based on the mean values of body length (Feuer ef al., 1978~). Mortality was recorded with care and statistical significance of the differences was calculated by Student’s r-test. Two main experimental groups were formed : (A) With the exception of the controls, the animals were bathed for 2 hr daily in water containing 50, 200 or 500 pg/ml taurine (Merck), respectively.
L
IO
70
io”
s:
I%
50 II0
60 X120 xx
Days Fig. 1. Effect of taurine on the metamorphosis of frog larvae, expr&sed in body length (mm). Days: x . experimental days, x x, age of larvae. 995
L. FE~,cK. L. J.
Ti)~i)h
......... r
treated with T,. the difference reached the level of statistical significance only on the 15th and Zlst days (P < 0.05). Application of 50O~lg,ml resulted in no
Control -- - - Taurlne 50
-
100 90 80 70-
*g/W - Tawne 200 us/ml Tawne 500ig/ml
-
/
60-
z
50-
%
4030-
i
change on the 6th day. but a significant difference was observed between the group treated with T, only and that treated with taurine + T, (P c 0.01: Fig. 3) on the 11th. 15th and 2lst days.
/
I
g
&
DISCL’SSIO’L
&&. 1%
20IO-
,,.y’ ,,_... X..” p.” Y
; IO
2u I 60
I 70
1
“’ h’ ,
,x~,;Trs,y 30
I 40
I 50
I 60
I 90
I too
I I IO
’ Jxx 120
’
Fig. 2. ElTect of taurine on mortality of frog larvae t”,,). Days: X. experimental days: x x . age of larvae.
From day 40. death rate of the frog larvae treated with any concentration of taurine was significantly lower than that of the untreated controls (P < 0.01). No significant concentration-dependent difference could be detected among the effects of the three taurine concentrations, regardless of whether they were compared to each other or to the control (Fig. 2). (B) T, produced an extremely intensive shortening of the body length (shortening of the tail fin). It reached the level of statistical significance as early as the 6th day (P < 0.01) and remained significant also on the llth, 15th and 31st days (P < 0.01. P < 0.01 and P < 0.05. respectively): at later periods, due to the considerable reduction of the number of larvae in the groups, no statistics were calculated. Under the effect of SOpg/mI taurine, a significant difference was found, from the 6th day, compared to the untreated controls, but the difference was not significant with regard to the group treated with T3. In the case of treatment with 2OOpg/ml taurine. although between the 6th and 1 Ith days no significant difference was observed in comparison to the animals -----
Control Tourine 50 us/ml
and G. CSARA
+ T3
Results of the experiments clearly show that taurine enhanced the early stages of amphibian metamorphosis while it retarded later ones. The experiments also revealed that taurine antagonized the effects of triiodothyronine on the development of frog larvae. Even a IO-fold increase of taurine concentration did not produce a more marked development of normal frog larvae than the initial dose. The T,-antagonistic effect was. on the other hand. dose-dependent. The effects of taurine are in complete agreement with those described for litoralon (Feuer er al., 1978a-c). i.e. the parameters studied havr been influenced by both compounds in the same direction and to the same measure. Based on the supposition that taurine was the precursor of litoralon. we have applied taurine in concentrations two to three orders of magnitude higher (lO&lOOO times) than the effective litoralon concentrations. A strongly significant T,-antagonistic effect was observed with 5OO~g,‘ml taurine and 0.5 pg/ml htoralon from the 11th day (Feuer er ul., 1978b). Our experiments with “S taurine still in progress seem to confirm the hypothesis that litoralon (gamma-L-glutamyl taurine) is formed from taurine and gamma glutaminic dipeptides. in the presence of gamma-glutamyl-transpeptidase (GGTP) enzyme (Orlowski & Meister, 1965: Szisz. 1969: Griindig & Rummler. 1974). Gwynne and his coworkers (1976) observed the increase of taurine concentration in the tail tissue of Rana rtrtrsheiana tadpoles before the start of lysis. At the end of this process the taurine level returned to the pre-metamorphic value. In the triiodothyronine-treated tadpoles. taurine concentration remained unchanged. From this Gwynne et a/. came to the conclusion that metamorphosis was not regulated exclusively by the thyroid hormones. Baskin CT a/. (1973) observed the increase of pigmentation of Rana cutrsheiuna tadpoles under the effect of taurine and attributed it to an inhibition of melatonine production or release. We suppose that in this hormonal interaction. too, taurine may act as a precursor.
REFERENCES
*’ 1
IO I 70
I 65
I 15 I 75
Fig. 3. EtTect of T, (trtiodothyronine ine + T, expressed
I 20
I 25
l 30
I 80
I 65
1 lxx 90
0.01 pg/ml) and tauron the metamorphosis of frog larvae. length (mm). Days: x. experimental r. says: x x, age 01 rarvae.
treatment in body
x
BASKIN S. I. & DAGIRMANJIAN R. (1973) The effect of taurine on the pigmentation of the bullfrog tadpole. Camp. Biochcm. Physiol. 44A. 297-302. FEWER L. (1975) Br. Pat. 1404225: (1977) U.S. Pat. 4001396. FEUER L. (1977) Theoretical background of the recognition of a new bioactive substance. litoralon, isolated from the parathyroid. Further theoretical considerations. Biologia. Budapest 25, 3-33. FEUER L.. B~NYAI B. & HERCSEL J. (1977) Influence of protein free aqueous extract of parathyroid powder on berum vitamin
A level in rats. Experirnria
33. 1005~1006.
The development of anuran larvae FEUERL.. T6~ij~ L. J. & CSABAG. (1978a) Effect of litoraIon on the metamorphosis of Rana arcalis. Acta ho/. in press. FtuEa L., T~R~K L. & CSABA G. (1978b) The triiodothyronine antagonistic effect of gamma-L-glutamyl-taurine (Litoralon). Endokrinologie in press. Fr;ur:a L.. Tii~ii~ L. J.. KAPA E. & CSABAG. (1978~) The effect of gamma-L-glutamyl-taurine (Litoralon) on the amphibian metamorphosis. Cor~p. Biochrrll. Physiol. 6lC.
67-71.
GR~NIIIG C. A. & RUMMLISW. (1974) Gamma-glutamyltranspeptidase (GGTP). Dr. Grs. w\‘ys~~~. 29. l108ml 114.
997
GWYNNE H. & CASTROC. E. (1976) Taurine
levels in anuran tadpole tail during spontaneous and triiodothyronine induced metamorphosis. Cor~p. Biochrm. Ph.niol. S4A. 245-247..
ORLOWSKIM. & MEISTERA. (1965) Isolation of gammaglutamyl-transpeptidase from hog kidney. J. bio[. Chem. 240. 338-347. Sz.isz G. (1969) A kinetic photometric method for serum gammaglutamyl-transpeptidase. Clin. Chew 15, 124-136. TYERENTYEV P. B. (1950) In: Ljaguska (The Frog), pp 59-67. Goz. Izd.. Moscow.