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Effect of short-term constant light or constant darkness on the nyctohemeral rhythm of type-II iodothyronine 5′-deiodinase activity in rat anterior pituitary and pineal
Life Sciences, Vol. 42, pp. 1875-1879 Printed in the U.S.A.
Pergamon Press
EFFECT OF SHORT-TERM CONSTANT LIGHT OR CONSTANT DARKNESS ON THE NYCTOHEMKRAL RHYTHM OF TYPE-II IODOTHYRONINE 5'-DEIODINASE ACTIVITY IN RAT ANTERIOR PITUITARY AND PINEAL Masami Murakami, Monte A. Greer, Susan E. Greer, Staci Hjulstad, and Kiyoshi Tanaka Section of Endocrinology, Department of Medicine Oregon Health Sciences University, Portland, Oregon 97201 (Received in final form March 11, 1988) Summary Type-II iodothyronine 5'-deiodinase activity (5'-D) in both anterior pituitary and pineal was significantly elevated at 2400 h, approximately 0.5- and 20-fold higher than the noon value, respectively. The nocturnal rise in both organs was abolished by 6 h additional light. Short-term constant darkness did not alter 5'-D rhythmicity in either organ. These data suggest that environmental lighting plays an important role in the control of the 5'-D nyctohemeral rhythm in both anterior pituitary and pineal. Iodothyronine 5'-deiodinase (5 I-D) converts thyroxine (Td) to 3,5,3'-triiodothyronine (Ts) and has 2 main subgroups. Type-I is present in liver and kidney and many other organs. Type-II is found in the central nervous system, pituitary, brown adipose tissue (I), and pineal (2). Among the most distinctive features of Type-II 5'-D are the low Km for T* or reverse Ts (rTa), the relative insensitivity to inhibition by propylthiouracil (PTU), and the marked increase in its activity in hypothyroidism. Type-II primarily plays a role to provide local intracellular Ts, whereas Type-I contributes to plasma Ts. In the rat, there is a nyctohemeral rhythm with a zenith at midnight in Type-II 5'-D activity in both pituitary (3) and pineal (4). There are also nyctohemeral rhythms in plasma and pineal melatonin (5) and plasma TSH (6) with zeniths at midnight for melatonin and noon for TSH. In the pineal, N-acetyltransferase (NAT) catalyzes the acetylation of serotonin to N-acetylserotonin, an immediate precursor of melatonin (7). The nocturnal rise of NAT is considered responsible for the large nocturnal increase of melatonin synthesis in the rat pineal gland (8). The nocturnal rise of pineal melatonin content and NAT activity is abolished by continuous light but a circadian rhythmicity is maintained in constant darkness (9,10), and within one week of constant light the circadian plasma TSH rhythm is no longer correlated with clock time (11). Since there may be a relation between the quantity of intracellular T3 and the secretory activity of both the pineal (12) and the pituitary thyrotroph (13), we examined the effect of constant light and constant darkness on the nyctohemeral rhythm of anterior pituitary and pineal Type-II 5'-D activity. This material was presented in part at the 1987 Annual Meeting of the European Thyroid Association, Lausanne (14).
0024-3205188 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
1876
Light Effects on Thyroxine Deiodinase
~01.
42, NO. 19, 1988
Materials and Methods Animals: Adult male Sprague-Dawley-derived rats (200-250g) were obtained from Gn Laboratories. The rats were maintained 3 per cage on a controlled lighting schedule (lights on: 0600-1800 h) with free access to Purina Lab Chow and water for at least 2 weeks before the experiments. Rats were killed and tissues processed in a room immediately adjoining that in which the animals were caged. When the experiments were performed during hours of darkness, the adjoining room was lit only with a dim red incandescent light. Chemicals: T4, Ts, and DL-dithiothreitol (DTT) were purchased from Sigma (St. Louis, MO); rTs from Calbiochem (La Jolla, CA); 3,3'-diiodothyronine (3,3'-T2) from Henning (Berlin, West Germany)*6-n-propyl-2-thiouracil (PTU) from ICN Biochemicals (Cleveland, OH). Na121 I was purchased from Amersham. [3',5'-'251]T4 and [3',5'-12'I]rTs were labeled in our own laboratory from Ts and 3,3'-Tx respectively, by the chloramine-T method (15); contamination with free iodide was less than 2%. Processing of tissues: The rats were killed by decapitation within 10 seconds after removal from their home cage. The anterior pituitary and pineal glands were removed rapidly, frozen and stored at -70 C until assayed. Assay of 5'-D activity: Type-II 5'-D activity was measured as previously described (2,4). Briefly, tissues were homogenized in 100 mM pH 7.0 potassium phosphate buffer containing 1 mM EDTA and 10 mM DTT. After centrifugation at 3,000 rpm for 15 minutes the supernatants were incubated with a final concentration of 2 nM [ 1d51]rTs or [1251]T4 in a total volume of 100 l~l for 1 h at 37 C in the presence of 1 mM EDTA, 10 mM DTT and 1 mM PTU. Reaction was terminated by the addition of 100 ~1 of 2% BSA and 800 cl1of ice cold 10% TCA. The released 1251- was separated by Dowex-50 ion exchange resin and quantitated. Non-enzymatic deiodination was corrected by subtracting iodide released in tissue-free buffer. Protein concentration was determined by Bradford's method (16). The deiodinating activity was calculated as femtomoles of I- releasedfmg protein/h. T4 was used as the substrate for the pineal assay because of the low pineal 5'-D activity in the daytime. The relative nyctohemeral changes in enzyme activity were identical whether T 4 or rTs were used as the substrate. Statistics: Data are presented as mean 2 standard error. were made with Student's t-test.
Statistical analyses
Results Effect of 6-hours additional light on pineal and anterior pituitary Type-II 5'-D activity (Fig 1 and 2). Two groups of 6 rats each were killed at 1200 h and 2400 h under normal lighting conditions. A third group of 6 was killed at 2400 h after constant light for 18 h. In both pineal (PO.l).
vol. 42, No. 19, 1988
Light Effects on Thyroxine Deiodinase
1877
PINEAL S-D
ACTIVITY
mm
LIGHTING FIG. 1
Effect of constant light on pineal 5'-D activity. Pineal Type-II T4 5'-D activity is expressed as fmoles I- released/mg protein/h. The first and second groups were killed at 1200 and 2400 h under normal lighting conditions. The third group was killed at 2400 h after constant light for 18 hours. Each bar and vertical line indicate the mean and SE of the enzyme activity determined in 6 rats. Lighting conditions for the 24-h period are shown at the bottom of each vertical bar; an open horizontal bar indicates light and a solid bar, darkness.
2400
PITUITARY S-D
ACTIVITY
150
100
LIGHTING
FIG. 2 Effect of constant light on anterior pituitary 5'-D activity. Anterior pituitary Type-II rTs 5'-D activity is expressed as fmoles I-released/mg protein/h. The experimental procedure was the same as in Fig. 1.
1878
Light Effects on Thyroxine Deiodinase
PINEAL Y-D ACTIVITY
Vol. 42, No. 19, 1988
2000
LIGHTING
FIG. 3 Effect of constant darkness on pineal 5'-D activity. The enzyme activity is expressed as in Fig. 1. Two groups were killed at 1200 h or 2400 h under normal lighting conditions. The third group was killed at 1200 h after 6 hours additional darkness. The fourth.group was killed at 2400 h after 18 hours additional darkness. There were 5 rats in each group.
250 012OOH 200
PITUITARY
m2UJOH
1%
5'-D ACTMW 100
so
FIG. 4 Effect of constant darkness on anterior pituitary 5'-D activity. The enzyme activity is expressed as in Fig. 2. The experimental procedure was the same as in Fig. 3.
Vol. 42, No. 19, 1988
Light Effects on Thyroxine Deiodinase
1879
the thyrotroph (13). The nocturnal elevation of anterior pituitary 5'-D thus might account for the lower nocturnal than diurnal plasma TSH concentration (6). Within a week of constant light exposure, the circadian TSH rhythm is dissociated from its normal 24-h cycle (11). In our present study, the nocturnal rise of anterior pituitary Type-II 5'-D was abolished by 6 h constant light and was much less affected by a similar period of darkness. Although the mechanism by which the light information influences anterior pituitary 5'-D activity is not known, these data suggest that lighting conditions may play an important role in controlling the nyctohemeral rhythm of anterior pituitary 5'-D activity. Acknowledgements This work was supported by Research Grant DK-01447 from the NIDDK, National Institutes of Health and by Grant l-2-588-525 from the Medical Research Foundation of Oregon. References 1.
2. 3. 4. 5. 6. 7.
8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
M.M. KAPLAN, Neuroendocrinology 38 254-260 (1984). K. TANAKA, M. MURAKAMI and M.A. ?%ER, Biochem. Biophys. Res. Comm. 137 863-868 (1986). M. MURAKAMI, K. TANAKA and M.A. GREER, Abstracts 61st Ann. Mtg. Am. Thyroid Assoc. T-38 (1987) (Abstract). K. TANAKA, M. MURAKAMI and M.A. GREER, Endocrinology 121 74-77 (1987). D.C. KLEIN, In: Endocrine Rhythms, D.T. Krieger Ed., pp. 203-223, Raven Press, New York, 1979. H. FUKUDA, M.A. GREER, L. ROBERTS, C.F. ALLEN, V. CRITCHLOW and M. WILSON, Endocrinology 97 1424-1431 (1975). H. WEISSBACH, B-G. REDFIELD and J. AXELROD, Biochim. Biophys. Acta 43 352-353 (1960). D.C. KLEIN, In: The Hypothalamus, S. Reichlin, R.J. Baldessarini and J.B. Martin Eds., pp. 303-327, Raven Press, New York, 1978. C.L. RALPH, D. MULL, H.J. LYNCH and L. HEDLUND, Endocrinology -89 1361-1366 (1971). D.C. KLEIN and J.L. WELLER, Science 169 1093-1095 (1970). H. FUKUDA, M.A. GRF.ER, L. ROBERTS, S.E. GREER and P. PANTON, Endocrinology 101 1304-1309 (1977). CNIR and N. HIRSCHMANN, J. Neural Transmission 42 117-126 (1978). P.R. LARSEN, J.E. SILVA and M.M. KAPLAN, Endocr. Rev. 2 87-102 (1981). M. MURAKAMI, S.E. GREER, S. HJULSTAD, K. TANAKA and M.A. GREER, Ann. Endocrinol. $8 131-131 (1987). Y. NAKAMURA, I.J. CHOPRA and D.H. SOLOMON, J. Nucl. Med. -18 1112-1115 (1978). M.M. BRADFORD, Anal. Biochem. 72 248-254 (1976). T. DEGUCHI, J. Neurochem. 25 91-93 (1975). D.C. KLEIN and J.L. WELLER, Science 177 532-533 (1972).
Pergamon Press
EFFECT OF SHORT-TERM CONSTANT LIGHT OR CONSTANT DARKNESS ON THE NYCTOHEMKRAL RHYTHM OF TYPE-II IODOTHYRONINE 5'-DEIODINASE ACTIVITY IN RAT ANTERIOR PITUITARY AND PINEAL Masami Murakami, Monte A. Greer, Susan E. Greer, Staci Hjulstad, and Kiyoshi Tanaka Section of Endocrinology, Department of Medicine Oregon Health Sciences University, Portland, Oregon 97201 (Received in final form March 11, 1988) Summary Type-II iodothyronine 5'-deiodinase activity (5'-D) in both anterior pituitary and pineal was significantly elevated at 2400 h, approximately 0.5- and 20-fold higher than the noon value, respectively. The nocturnal rise in both organs was abolished by 6 h additional light. Short-term constant darkness did not alter 5'-D rhythmicity in either organ. These data suggest that environmental lighting plays an important role in the control of the 5'-D nyctohemeral rhythm in both anterior pituitary and pineal. Iodothyronine 5'-deiodinase (5 I-D) converts thyroxine (Td) to 3,5,3'-triiodothyronine (Ts) and has 2 main subgroups. Type-I is present in liver and kidney and many other organs. Type-II is found in the central nervous system, pituitary, brown adipose tissue (I), and pineal (2). Among the most distinctive features of Type-II 5'-D are the low Km for T* or reverse Ts (rTa), the relative insensitivity to inhibition by propylthiouracil (PTU), and the marked increase in its activity in hypothyroidism. Type-II primarily plays a role to provide local intracellular Ts, whereas Type-I contributes to plasma Ts. In the rat, there is a nyctohemeral rhythm with a zenith at midnight in Type-II 5'-D activity in both pituitary (3) and pineal (4). There are also nyctohemeral rhythms in plasma and pineal melatonin (5) and plasma TSH (6) with zeniths at midnight for melatonin and noon for TSH. In the pineal, N-acetyltransferase (NAT) catalyzes the acetylation of serotonin to N-acetylserotonin, an immediate precursor of melatonin (7). The nocturnal rise of NAT is considered responsible for the large nocturnal increase of melatonin synthesis in the rat pineal gland (8). The nocturnal rise of pineal melatonin content and NAT activity is abolished by continuous light but a circadian rhythmicity is maintained in constant darkness (9,10), and within one week of constant light the circadian plasma TSH rhythm is no longer correlated with clock time (11). Since there may be a relation between the quantity of intracellular T3 and the secretory activity of both the pineal (12) and the pituitary thyrotroph (13), we examined the effect of constant light and constant darkness on the nyctohemeral rhythm of anterior pituitary and pineal Type-II 5'-D activity. This material was presented in part at the 1987 Annual Meeting of the European Thyroid Association, Lausanne (14).
0024-3205188 $3.00 + .OO Copyright (c) 1988 Pergamon Press plc
1876
Light Effects on Thyroxine Deiodinase
~01.
42, NO. 19, 1988
Materials and Methods Animals: Adult male Sprague-Dawley-derived rats (200-250g) were obtained from Gn Laboratories. The rats were maintained 3 per cage on a controlled lighting schedule (lights on: 0600-1800 h) with free access to Purina Lab Chow and water for at least 2 weeks before the experiments. Rats were killed and tissues processed in a room immediately adjoining that in which the animals were caged. When the experiments were performed during hours of darkness, the adjoining room was lit only with a dim red incandescent light. Chemicals: T4, Ts, and DL-dithiothreitol (DTT) were purchased from Sigma (St. Louis, MO); rTs from Calbiochem (La Jolla, CA); 3,3'-diiodothyronine (3,3'-T2) from Henning (Berlin, West Germany)*6-n-propyl-2-thiouracil (PTU) from ICN Biochemicals (Cleveland, OH). Na121 I was purchased from Amersham. [3',5'-'251]T4 and [3',5'-12'I]rTs were labeled in our own laboratory from Ts and 3,3'-Tx respectively, by the chloramine-T method (15); contamination with free iodide was less than 2%. Processing of tissues: The rats were killed by decapitation within 10 seconds after removal from their home cage. The anterior pituitary and pineal glands were removed rapidly, frozen and stored at -70 C until assayed. Assay of 5'-D activity: Type-II 5'-D activity was measured as previously described (2,4). Briefly, tissues were homogenized in 100 mM pH 7.0 potassium phosphate buffer containing 1 mM EDTA and 10 mM DTT. After centrifugation at 3,000 rpm for 15 minutes the supernatants were incubated with a final concentration of 2 nM [ 1d51]rTs or [1251]T4 in a total volume of 100 l~l for 1 h at 37 C in the presence of 1 mM EDTA, 10 mM DTT and 1 mM PTU. Reaction was terminated by the addition of 100 ~1 of 2% BSA and 800 cl1of ice cold 10% TCA. The released 1251- was separated by Dowex-50 ion exchange resin and quantitated. Non-enzymatic deiodination was corrected by subtracting iodide released in tissue-free buffer. Protein concentration was determined by Bradford's method (16). The deiodinating activity was calculated as femtomoles of I- releasedfmg protein/h. T4 was used as the substrate for the pineal assay because of the low pineal 5'-D activity in the daytime. The relative nyctohemeral changes in enzyme activity were identical whether T 4 or rTs were used as the substrate. Statistics: Data are presented as mean 2 standard error. were made with Student's t-test.
Statistical analyses
Results Effect of 6-hours additional light on pineal and anterior pituitary Type-II 5'-D activity (Fig 1 and 2). Two groups of 6 rats each were killed at 1200 h and 2400 h under normal lighting conditions. A third group of 6 was killed at 2400 h after constant light for 18 h. In both pineal (P
vol. 42, No. 19, 1988
Light Effects on Thyroxine Deiodinase
1877
PINEAL S-D
ACTIVITY
mm
LIGHTING FIG. 1
Effect of constant light on pineal 5'-D activity. Pineal Type-II T4 5'-D activity is expressed as fmoles I- released/mg protein/h. The first and second groups were killed at 1200 and 2400 h under normal lighting conditions. The third group was killed at 2400 h after constant light for 18 hours. Each bar and vertical line indicate the mean and SE of the enzyme activity determined in 6 rats. Lighting conditions for the 24-h period are shown at the bottom of each vertical bar; an open horizontal bar indicates light and a solid bar, darkness.
2400
PITUITARY S-D
ACTIVITY
150
100
LIGHTING
FIG. 2 Effect of constant light on anterior pituitary 5'-D activity. Anterior pituitary Type-II rTs 5'-D activity is expressed as fmoles I-released/mg protein/h. The experimental procedure was the same as in Fig. 1.
1878
Light Effects on Thyroxine Deiodinase
PINEAL Y-D ACTIVITY
Vol. 42, No. 19, 1988
2000
LIGHTING
FIG. 3 Effect of constant darkness on pineal 5'-D activity. The enzyme activity is expressed as in Fig. 1. Two groups were killed at 1200 h or 2400 h under normal lighting conditions. The third group was killed at 1200 h after 6 hours additional darkness. The fourth.group was killed at 2400 h after 18 hours additional darkness. There were 5 rats in each group.
250 012OOH 200
PITUITARY
m2UJOH
1%
5'-D ACTMW 100
so
FIG. 4 Effect of constant darkness on anterior pituitary 5'-D activity. The enzyme activity is expressed as in Fig. 2. The experimental procedure was the same as in Fig. 3.
Vol. 42, No. 19, 1988
Light Effects on Thyroxine Deiodinase
1879
the thyrotroph (13). The nocturnal elevation of anterior pituitary 5'-D thus might account for the lower nocturnal than diurnal plasma TSH concentration (6). Within a week of constant light exposure, the circadian TSH rhythm is dissociated from its normal 24-h cycle (11). In our present study, the nocturnal rise of anterior pituitary Type-II 5'-D was abolished by 6 h constant light and was much less affected by a similar period of darkness. Although the mechanism by which the light information influences anterior pituitary 5'-D activity is not known, these data suggest that lighting conditions may play an important role in controlling the nyctohemeral rhythm of anterior pituitary 5'-D activity. Acknowledgements This work was supported by Research Grant DK-01447 from the NIDDK, National Institutes of Health and by Grant l-2-588-525 from the Medical Research Foundation of Oregon. References 1.
2. 3. 4. 5. 6. 7.
8. 9.
10. 11. 12. 13. 14. 15. 16. 17. 18.
M.M. KAPLAN, Neuroendocrinology 38 254-260 (1984). K. TANAKA, M. MURAKAMI and M.A. ?%ER, Biochem. Biophys. Res. Comm. 137 863-868 (1986). M. MURAKAMI, K. TANAKA and M.A. GREER, Abstracts 61st Ann. Mtg. Am. Thyroid Assoc. T-38 (1987) (Abstract). K. TANAKA, M. MURAKAMI and M.A. GREER, Endocrinology 121 74-77 (1987). D.C. KLEIN, In: Endocrine Rhythms, D.T. Krieger Ed., pp. 203-223, Raven Press, New York, 1979. H. FUKUDA, M.A. GREER, L. ROBERTS, C.F. ALLEN, V. CRITCHLOW and M. WILSON, Endocrinology 97 1424-1431 (1975). H. WEISSBACH, B-G. REDFIELD and J. AXELROD, Biochim. Biophys. Acta 43 352-353 (1960). D.C. KLEIN, In: The Hypothalamus, S. Reichlin, R.J. Baldessarini and J.B. Martin Eds., pp. 303-327, Raven Press, New York, 1978. C.L. RALPH, D. MULL, H.J. LYNCH and L. HEDLUND, Endocrinology -89 1361-1366 (1971). D.C. KLEIN and J.L. WELLER, Science 169 1093-1095 (1970). H. FUKUDA, M.A. GRF.ER, L. ROBERTS, S.E. GREER and P. PANTON, Endocrinology 101 1304-1309 (1977). CNIR and N. HIRSCHMANN, J. Neural Transmission 42 117-126 (1978). P.R. LARSEN, J.E. SILVA and M.M. KAPLAN, Endocr. Rev. 2 87-102 (1981). M. MURAKAMI, S.E. GREER, S. HJULSTAD, K. TANAKA and M.A. GREER, Ann. Endocrinol. $8 131-131 (1987). Y. NAKAMURA, I.J. CHOPRA and D.H. SOLOMON, J. Nucl. Med. -18 1112-1115 (1978). M.M. BRADFORD, Anal. Biochem. 72 248-254 (1976). T. DEGUCHI, J. Neurochem. 25 91-93 (1975). D.C. KLEIN and J.L. WELLER, Science 177 532-533 (1972).