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Taurine levels in the anuran tadpole tail during spontaneous and triiodothyronine-induced metamorphosis
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T A U R I N E LEVELS IN T H E A N U R A N T A D P O L E TAIL D U R I N G S P O N T A N E O U S A N D TR I I O D O T H Y R O N I N E - I N D U C E D M E T A M O R P H O S I S G~.VYNNE n . LI'I-rLE AND CARMEN E, CAffnlo Department of Biochemistry, Texas Technical University School of Medicine, P.O. Box 4569, Lubbock, TX 79409, U.S.A. (Receired 15 At,lUSt 1975)
Ahstraet---l. Taurine levels were determined in tail tissue of R u n s c , tesbei, n , tadpoles during spontaneous and triiodolhyroninc-induced metamorphosis. 2. Taurine levels increased prior to the beginning of tail regression in spontaneous metamorphosis and then declined to premetamorphic levels. 3. No changes in taurine levels occurred in triiodothyIonine-treated animals. 4. Implications of these findings are discussed. on a diet of chopped mixed greens. All chemicals were the best grade commercially available and were used without further purification. Animals at various stages of metamorphosis (Taylor & KoUros, 1946) were anesthetized by immersion in ice water and as much tail tissue as possible was excised. The tails were weighed and homogenized in 1'0 N potassium hydroxide and the homogenales were allowed to stand overnight to solubZtize the tissue. An excess of perchloric acid was then added to precipitate potassium ions and macromolecules. The precipitate was removed by centrifugation and washed once with 0-5 M perchl0rie acid which was combined with the supernatant. Duplicate 0-25 ml aliquots of the supernatant were analyzed for taurine by means of a Beckman Model 121 automatic amino acid analyzer. In the triiodothyronine (T3) experiments, premetamorphic animals (stages X-Xll) were given a single injection of 0-3 n-mole of T3 in 0"I~£ NaOH/g body wt and sacrificed at 2-day intervals for 14 days. Controls were injected with the vehicle and sacrificed at the same lime intervals as the treated animals. Both control and experimental animals were fasted during the course of the experiment. Protein was determined by the biuret method.
INTRODUCTION
ANU|',AN amphibians undergo extensive morphologi~tl and physiological modifications during the metam o r p h i c transition from aquatic larva to terrestrial adult. These moditications include growth in some tissues, of which limbs and lungs are typical examples and regression in other tissues, represented by the gills and tail. The biochemical changes which occur in various tissues during m e t a m o r p h o s i s have been reviewed by Frieden & Just (1970). T h e tail, which consists mainly of skeletal muscle, has been the most thoroughly investigated of the several regressing tissues. T h e most striking biochemical events occurring in the tadpole tail during m e t a m o r p h o s i s are a marked increase in the total activity and specific activity of proteolytic enzymes (Weber, 1969) and an increase in the rate of incorporation of labeled amino acids into protein (Little et al., 1973). Many, but not all, metamorphic, biochemical changes can be elicited in premet~unorphie animals by injection of thyroid hormones (Tonoue & Frieden, 1970; W a n g & Frieden, 1973). Increased incorporation o f a m i n o acids into protein (Weinstock et al., 1969) and increased activities of pro:.eolytic enzymes (Iodice et aL, 1972; K a r & Pearson, 1973) have also been reported in patients and experimental animals with muscular dystrophy. These biochemical similarities between the pathological regression of skeletal muscle which occurs in muscular dystrophy and the physiological regression of anuran m e t a m o r p h o s i s suggest that the regressing tadpole tail might provide a useful model system for at least some aspects o f neuromuscular disease. Several reports in the literature suggesting a role for taurine in the etiology of muscular dystrophy (Bank et al., 1971; Wilson et al., 1965: Baskin & Dagirmanjiar, 1973) led us to u n d e r t a k e this study of taurine levels in metamorphosis.
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MATERIALS A N D M E T H O D S
Rana catesbeiana tadpoles were obtained from a commercial supplier and were maintained in the laboratc~ry
x
xvll
of
xmll
xx
xxll
metamorphosis
Fig. I. Taurine levels (mea n ± S.E.) in tail tissue of Rana caresbeiana tadpoles during spontaneous metamorphosis. 245
246
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E. C'ASTB.(~)
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~ O,E'O-
~
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l I
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metamorphosis
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RESULTS AND DISCUSSION
Figure l illustrates the changes in taurine concentration in tail tissue at various selected stages of spontaneous metamorphosis. In Fig. 2 the changes in the ratio of tail weight to body w e i g h t a r e shown as an i n d e x of the degree of tail regression. Taurine levels are quite low in animals up to stage X; By stages XVII-XVIII ~taurine levels were doubled and then declined rapidly to below premetamorphic levels by stage XXII. A significant decrease in tail wt/body wt was not observed until stage XX. In Fig. 3 the effects of Ta on taurine levels are shown, and Fig. 4 shows the changes in tail wt/body wt at various times after T3 injection. Significant regression had occurred by day 12 however n o differences in taurine concentrations between T 3 treated animals and controls were observed at any time. The a n u r a n tadpole during metamorphosis, provides an excellent model system for the investigation of basic mechanisms of tissue regression, particularly hormone-induced regression. Reports of pos~;ible involvement of taurine in the regression of skeletal muscle in muscular dystrophies of man ,and some ant-
2Z-
t.o-
J
4
o~ter
"T= i n j e e t l o n
Fig. 4. Changes in the tail weight relative to total body weight (mean 4- S.E.) of Ram7 cate.sheiana tadpoles after a single injection of 0.3 n-mole of triiodolhyronine/g body wl I.~i controls. [3 T 3 treated). reals suggested its determination in the regressing tadpole tail. O u r data indicate a rise in taurine levels in the tall of Ram~ cttleshehm~t tadpoles during spontaneous metamorphosis. This increase in taurine concentration occurred prior to the beginning of tail regression however. Taurine levels were also determined in tadpoles injected with triiodothyronine. While tail regression occurred in these animals, as indicated by a significant decrease in the tail wt/body wt ratio, n o change in taurine levels occurred. This observation indicates that a concomitant increase in taurine concentration is not required for tail regression to occur. If the regression of skeletal muscle in anuran metamorphosis shares a c o m m o n mechanism with the regression which occurs in muscular dystrophy, then a cause-effect relationship between tautine levels and muscular dystrophy as proposed by Baskin & Dagirmanjian (1973) is questionable. While many of the biochemical events of a n u r a n metamorphosis can be elicited by thyroid hormones, a number of differences between spontaneous a n d induced metamorphosis have been reported (Wang & Frieden, 1973; Just & Atkinson. 1972; Atkinson & Little, 1972). O u r observation that taurine levels increase in the tadpole tail during spontaneous but not during Ta-induced metamorphosis is another addition to the growing body of evidence that factors other than thyroid hormones may be involved in directing the progress of anuran metamorphosis.
Its-
a,
~
0
Doys
Fig. 2. Changes in fail weight relative to total body weight (mean + S,E.) in tail tissue of Rcma c'atesheiana tadpoles during spontaneous metamorphosis.
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t !I
REFERENCES
0~-
1"- 0 6 -
A'rKtYSONB. G. & LITTLEG. H. (1972) Growth and regression in tadpole pancreas during spontaneous and thyroid hormone ind,ced metamorphosis. Mech. Age. Devl 32, 299-312. BANK W. J., ROWLAND L. P. & If'SEN J. (1971) Amino acids of plasma and urine in diseases of muscle. Arch. NeuroL 24, 176--186. BASKIN S. 1. d~ ~a;31RMANJIAN R. (1973) Possible involvement of taurm~ in the genesis of muscular dystrophy. Nature. Load. 245, 464-465. FRIEDEN E. & JUST J. J. (1970) Hormonal responses in amphibian metamorphosis. In Biochemical Actions o f Hormones (Edited by LIIWACKG.), VoL I, pp. 1-52. Academic Press. New York.
mMmmm 2
4
Doys
S
aft6r
a
I0
~
~
Ta i n j e c t i o n
Fig. 3. Taurine levels (mean _+ S.E.) in tail tissue of Rana catesheiana tadpoles after a single injection of 0-3 n-mole of triiodothyronine/g body wt ( I controls, El T 3 treated).
Taurine levels during metamorphosis loDtc~ A. A., CHINJ., PImKEI~S. & Wr.qNSTOCKI. M. 11972) Cathepsins A,B,C,D and autolysis during development of breast muscle of normal and dystrophic chickens. Archs Biochem. Biophys. 152, 166-174. Jt~s'r J. J. & ATKINSONB. G. (1972) Hemoglobin transitions in the bullfrog Rana catesbeiana during spontaneous and induced metamorphosis. J. exp. Zool. 182, 271-281. Kaa N. (7.. & PI~ARSONC. M. (1973) Acid, neutral and alkaline cathepsins in normal and diseased human muscle. Enzymes 13, 188-196. Lt'rrt.E G. H., ATKINSON B. G. & FRIEDEN E. (1973) Changes in the rates of protein synthesis and degradation in the tail of Rana catesbeiana during metamorphosis. Devl. Biol. 30, 366-373. r,,,','Loa A. C. & KOLLROSJ. J. (1946) Stages in the normal development of Rtma pipiens larvae. Anat. Rec. 94, 7-23. TONOUt~T. & FRIEDENE. (1970) The effect of T3 on leucine incorporation into tail and other tadpole tissues, d. biol. Chem. 245, 2359-2362.
247
WANG V. B. & FRIEDI~NE. (1973) Changes in cathepsit~ C activity during spontaneous and induced metamorphosis of the bullfrog. Gen. comp. Endocr. 21, 381-389. WIttIER R. (1969) Tissue involution and lysosmnal enzymes during anuran metamorphosis. In L.rsomes #~ Bioh~yy aml Pathoh~D" (Edited by DINGLL:J. T. t~ FEI.I.H.B.), Vol. II, pp. 437-461. North-Holland Publ., Amsterdam. WE'INSTOCK l, M., Soil T. S., FREEDMANH. A. & CLITI.EI~. M. E. (1969) Amino acid incorporation into proteins of developing breast muscle of the normal and dystrophic chicken. Biochem. Meal. 2, 345--356. WILSON B. W., PETL"RSOND. W. & LILYIILADF.A. L. (1965} Free amino acids of developing skeletal musculature of normal and genetic~dly dystrophic chickens. Proc. Soc. exp. Biol. Med. 119, t04-108.
Ih'i~ai*l
T A U R I N E LEVELS IN T H E A N U R A N T A D P O L E TAIL D U R I N G S P O N T A N E O U S A N D TR I I O D O T H Y R O N I N E - I N D U C E D M E T A M O R P H O S I S G~.VYNNE n . LI'I-rLE AND CARMEN E, CAffnlo Department of Biochemistry, Texas Technical University School of Medicine, P.O. Box 4569, Lubbock, TX 79409, U.S.A. (Receired 15 At,lUSt 1975)
Ahstraet---l. Taurine levels were determined in tail tissue of R u n s c , tesbei, n , tadpoles during spontaneous and triiodolhyroninc-induced metamorphosis. 2. Taurine levels increased prior to the beginning of tail regression in spontaneous metamorphosis and then declined to premetamorphic levels. 3. No changes in taurine levels occurred in triiodothyIonine-treated animals. 4. Implications of these findings are discussed. on a diet of chopped mixed greens. All chemicals were the best grade commercially available and were used without further purification. Animals at various stages of metamorphosis (Taylor & KoUros, 1946) were anesthetized by immersion in ice water and as much tail tissue as possible was excised. The tails were weighed and homogenized in 1'0 N potassium hydroxide and the homogenales were allowed to stand overnight to solubZtize the tissue. An excess of perchloric acid was then added to precipitate potassium ions and macromolecules. The precipitate was removed by centrifugation and washed once with 0-5 M perchl0rie acid which was combined with the supernatant. Duplicate 0-25 ml aliquots of the supernatant were analyzed for taurine by means of a Beckman Model 121 automatic amino acid analyzer. In the triiodothyronine (T3) experiments, premetamorphic animals (stages X-Xll) were given a single injection of 0-3 n-mole of T3 in 0"I~£ NaOH/g body wt and sacrificed at 2-day intervals for 14 days. Controls were injected with the vehicle and sacrificed at the same lime intervals as the treated animals. Both control and experimental animals were fasted during the course of the experiment. Protein was determined by the biuret method.
INTRODUCTION
ANU|',AN amphibians undergo extensive morphologi~tl and physiological modifications during the metam o r p h i c transition from aquatic larva to terrestrial adult. These moditications include growth in some tissues, of which limbs and lungs are typical examples and regression in other tissues, represented by the gills and tail. The biochemical changes which occur in various tissues during m e t a m o r p h o s i s have been reviewed by Frieden & Just (1970). T h e tail, which consists mainly of skeletal muscle, has been the most thoroughly investigated of the several regressing tissues. T h e most striking biochemical events occurring in the tadpole tail during m e t a m o r p h o s i s are a marked increase in the total activity and specific activity of proteolytic enzymes (Weber, 1969) and an increase in the rate of incorporation of labeled amino acids into protein (Little et al., 1973). Many, but not all, metamorphic, biochemical changes can be elicited in premet~unorphie animals by injection of thyroid hormones (Tonoue & Frieden, 1970; W a n g & Frieden, 1973). Increased incorporation o f a m i n o acids into protein (Weinstock et al., 1969) and increased activities of pro:.eolytic enzymes (Iodice et aL, 1972; K a r & Pearson, 1973) have also been reported in patients and experimental animals with muscular dystrophy. These biochemical similarities between the pathological regression of skeletal muscle which occurs in muscular dystrophy and the physiological regression of anuran m e t a m o r p h o s i s suggest that the regressing tadpole tail might provide a useful model system for at least some aspects o f neuromuscular disease. Several reports in the literature suggesting a role for taurine in the etiology of muscular dystrophy (Bank et al., 1971; Wilson et al., 1965: Baskin & Dagirmanjiar, 1973) led us to u n d e r t a k e this study of taurine levels in metamorphosis.
2220-
.EE J z -
o402o.©
~
Sto~es
MATERIALS A N D M E T H O D S
Rana catesbeiana tadpoles were obtained from a commercial supplier and were maintained in the laboratc~ry
x
xvll
of
xmll
xx
xxll
metamorphosis
Fig. I. Taurine levels (mea n ± S.E.) in tail tissue of Rana caresbeiana tadpoles during spontaneous metamorphosis. 245
246
G w v n . , , ; r ; t l . LII"trLI': A N t ) C A R M E N
E. C'ASTB.(~)
o 1o.
"17 --
+
~ O,E'O-
~
"[
i
oJ8-
l I
005-
0.0~
togas
metamorphosis
of
RESULTS AND DISCUSSION
Figure l illustrates the changes in taurine concentration in tail tissue at various selected stages of spontaneous metamorphosis. In Fig. 2 the changes in the ratio of tail weight to body w e i g h t a r e shown as an i n d e x of the degree of tail regression. Taurine levels are quite low in animals up to stage X; By stages XVII-XVIII ~taurine levels were doubled and then declined rapidly to below premetamorphic levels by stage XXII. A significant decrease in tail wt/body wt was not observed until stage XX. In Fig. 3 the effects of Ta on taurine levels are shown, and Fig. 4 shows the changes in tail wt/body wt at various times after T3 injection. Significant regression had occurred by day 12 however n o differences in taurine concentrations between T 3 treated animals and controls were observed at any time. The a n u r a n tadpole during metamorphosis, provides an excellent model system for the investigation of basic mechanisms of tissue regression, particularly hormone-induced regression. Reports of pos~;ible involvement of taurine in the regression of skeletal muscle in muscular dystrophies of man ,and some ant-
2Z-
t.o-
J
4
o~ter
"T= i n j e e t l o n
Fig. 4. Changes in the tail weight relative to total body weight (mean 4- S.E.) of Ram7 cate.sheiana tadpoles after a single injection of 0.3 n-mole of triiodolhyronine/g body wl I.~i controls. [3 T 3 treated). reals suggested its determination in the regressing tadpole tail. O u r data indicate a rise in taurine levels in the tall of Ram~ cttleshehm~t tadpoles during spontaneous metamorphosis. This increase in taurine concentration occurred prior to the beginning of tail regression however. Taurine levels were also determined in tadpoles injected with triiodothyronine. While tail regression occurred in these animals, as indicated by a significant decrease in the tail wt/body wt ratio, n o change in taurine levels occurred. This observation indicates that a concomitant increase in taurine concentration is not required for tail regression to occur. If the regression of skeletal muscle in anuran metamorphosis shares a c o m m o n mechanism with the regression which occurs in muscular dystrophy, then a cause-effect relationship between tautine levels and muscular dystrophy as proposed by Baskin & Dagirmanjian (1973) is questionable. While many of the biochemical events of a n u r a n metamorphosis can be elicited by thyroid hormones, a number of differences between spontaneous a n d induced metamorphosis have been reported (Wang & Frieden, 1973; Just & Atkinson. 1972; Atkinson & Little, 1972). O u r observation that taurine levels increase in the tadpole tail during spontaneous but not during Ta-induced metamorphosis is another addition to the growing body of evidence that factors other than thyroid hormones may be involved in directing the progress of anuran metamorphosis.
Its-
a,
~
0
Doys
Fig. 2. Changes in fail weight relative to total body weight (mean + S,E.) in tail tissue of Rcma c'atesheiana tadpoles during spontaneous metamorphosis.
u ~
t !I
REFERENCES
0~-
1"- 0 6 -
A'rKtYSONB. G. & LITTLEG. H. (1972) Growth and regression in tadpole pancreas during spontaneous and thyroid hormone ind,ced metamorphosis. Mech. Age. Devl 32, 299-312. BANK W. J., ROWLAND L. P. & If'SEN J. (1971) Amino acids of plasma and urine in diseases of muscle. Arch. NeuroL 24, 176--186. BASKIN S. 1. d~ ~a;31RMANJIAN R. (1973) Possible involvement of taurm~ in the genesis of muscular dystrophy. Nature. Load. 245, 464-465. FRIEDEN E. & JUST J. J. (1970) Hormonal responses in amphibian metamorphosis. In Biochemical Actions o f Hormones (Edited by LIIWACKG.), VoL I, pp. 1-52. Academic Press. New York.
mMmmm 2
4
Doys
S
aft6r
a
I0
~
~
Ta i n j e c t i o n
Fig. 3. Taurine levels (mean _+ S.E.) in tail tissue of Rana catesheiana tadpoles after a single injection of 0-3 n-mole of triiodothyronine/g body wt ( I controls, El T 3 treated).
Taurine levels during metamorphosis loDtc~ A. A., CHINJ., PImKEI~S. & Wr.qNSTOCKI. M. 11972) Cathepsins A,B,C,D and autolysis during development of breast muscle of normal and dystrophic chickens. Archs Biochem. Biophys. 152, 166-174. Jt~s'r J. J. & ATKINSONB. G. (1972) Hemoglobin transitions in the bullfrog Rana catesbeiana during spontaneous and induced metamorphosis. J. exp. Zool. 182, 271-281. Kaa N. (7.. & PI~ARSONC. M. (1973) Acid, neutral and alkaline cathepsins in normal and diseased human muscle. Enzymes 13, 188-196. Lt'rrt.E G. H., ATKINSON B. G. & FRIEDEN E. (1973) Changes in the rates of protein synthesis and degradation in the tail of Rana catesbeiana during metamorphosis. Devl. Biol. 30, 366-373. r,,,','Loa A. C. & KOLLROSJ. J. (1946) Stages in the normal development of Rtma pipiens larvae. Anat. Rec. 94, 7-23. TONOUt~T. & FRIEDENE. (1970) The effect of T3 on leucine incorporation into tail and other tadpole tissues, d. biol. Chem. 245, 2359-2362.
247
WANG V. B. & FRIEDI~NE. (1973) Changes in cathepsit~ C activity during spontaneous and induced metamorphosis of the bullfrog. Gen. comp. Endocr. 21, 381-389. WIttIER R. (1969) Tissue involution and lysosmnal enzymes during anuran metamorphosis. In L.rsomes #~ Bioh~yy aml Pathoh~D" (Edited by DINGLL:J. T. t~ FEI.I.H.B.), Vol. II, pp. 437-461. North-Holland Publ., Amsterdam. WE'INSTOCK l, M., Soil T. S., FREEDMANH. A. & CLITI.EI~. M. E. (1969) Amino acid incorporation into proteins of developing breast muscle of the normal and dystrophic chicken. Biochem. Meal. 2, 345--356. WILSON B. W., PETL"RSOND. W. & LILYIILADF.A. L. (1965} Free amino acids of developing skeletal musculature of normal and genetic~dly dystrophic chickens. Proc. Soc. exp. Biol. Med. 119, t04-108.