- Email: [email protected]
Northern analysis of type II iodothyronine deiodinase mRNA in rat harderian gland
Life Sciences, Vol. 63, No. 21, pp. 1843-1848, 1998
ELSEVIER
NORTHERN
PI1 SOO24-3205(98)00471-8
ANALYSIS OF TYPE II IODOTHYRONINE mRNA IN RAT HARDERIAN GLAND
Copyright o 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/98 $19.00 + .oO
DEIODINASE
Osamu Araki, Masami Murakami I, Y uji Kamiya, Y asuhiro Hosoi, Takayuki Ogiwara, Haruo Mizuma, Tokuji Iriuchijima and Masatomo Mori
First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 37 l-85 11, Japan (Received in final form September 7, 19%) Summary It has been known that type II iodothyronine deiodinase activity is present in rat Harderian gland and the activity is significantly increased by isoproterenol administration. We have performed Northern analyses to study whether the transcript for type II iodothyronine deiodinase is expressed in rat Harderian gland and whether the isoproterenol stimulation of type II iodothyronine deicdinase activity in rat Harderian gland is due to the change in its mRNA level. Northern analyses have demonstrated that type II iodothyronine deiodinase mRNA, approximately 7.5 kb in size, is expressed in rat Harderian gland, and the mRNA levels as well as the deiodinase activities are greater in hypothyroid rats than those in euthyroid rats. Type II iodothyronine deiodinase mRNA levels and the deiodinase activities in Harderian gland were increased by isoproterenol administration, and the increase in the mRNA levels preceded that in the deiodinase activities. These results indicate that 7.5 kb transcript for type II iodothyronine deiodinase is expressed in rat Harderian gland and fl-adrenergic stimulation of type II iodothyronine deiodinase activity is due at least in part to the increase in its mRNA level. Key Words: thyroid hormone, adrenergic receptor, hypothyroidism, isoproterenol
Thyroxine (Ta), which is a major secretory product of thyroid gland, needs to be converted to 3,5,3’-triiodothyronine (T3) by iodothyronine deiodinase to exert its biological activity. Type I iodothyronine deiodinase (DI) activity is present in thyroid gland, liver and kidney, while type II iodothyronine deiodinase (DII) activity is present in brain, anterior pituitary, brown fat, pineal gland and Harderian gland in the rat (1). DI activity is known to decrease in hypothyroid state and mainly contribute to circulating T3 level. In contrast, DII activity increases in hypothyroid state and plays a critical role to provide a local intracellular T3. Furthermore, DII activity in brown fat (2), pineal gland (3) and Harderian gland (4) has been demonstrated to be significantly increased by adrenergic stimulation. Recently, a cDNA encoding DII has been cloned from Rana catesbeiana tissues (5), and subsequently from rat brown fat (6). Expression of DII mRNA, approximately 7.5 kb in size, was observed in brain, anterior pituitary, brown fat and pineal gland in the rat by Northern analysis
‘Corresponding author: M. Murakami, First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 37 l-85 11, Japan Phone:+8 1 27 2208126; FAX:+8 127 2208 136; E-mail:[email protected]
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Harderian Gland Dciodinase mRNA
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(6,7). Although Northern analysis of DII mRNA in Harderian gland has not been reported, GarciaMacias et al. determined the size of the mRNA expressing the DII activity to be I.4 - 2.4 kb using Xenopus laevis oocytes which were injected with poly (A)+ RNA from rat Harderian gland (8). Thus, it has not been known whether the 7.5 kb transcript for DII is present in rat Harderian gland. In the present report, we have performed Northern analyses to study whether the 7.5 kb transcript for DII mRNA is also expressed in rat Harderian gland and whether the adrenergic stimulation of DII activities is due to the increase in its mRNA levels.
Methods Animals Two-month-old, male Wistar rats were maintained two per cage on a 12-hour light, 12-hour dark schedule (lights on at 0600 h) at 25kl”C and fed standard laboratory chow and tap water ad lib&urn. The rats were acclimated to this condition for at least one week before the experiment. Hypothyroid rats were produced by administering 0.03% methimazole in their drinking water for 4 weeks. Hypothyroidism was judged by a decreased weight gain of the animals. Some rats were injected subcutaneously with 0.3 mg/kg body weight of isoproterenol at 1200 h. The Harderian glands were rapidly removed, divided into two pieces, frozen and stored at -70°C until the RNA isolation or the quantitation of DII activity. RNA preparation and Northern analysis
Total RNA was isolated from each Harderian gland by modified acid guanidinium thiocyanatephenol-chloroform method according to Chomczynski and Sacchi (9). Northern analyses were performed as previously described (7,lO). Plasmid rDI1 5 I/pBluescript SK, which contains rat DII cDNA, was kindly provided by Dr. St. Germain (6). Briefly, rat DII cRNA probe was synthesized by in vitro transcription of linearized rDI1 5-l/pBluescript SK using T7 polymerase (Boehringer Mannheim GmbH, Mannheim, Germany) and [a-32P]UTP (New England Nuclear Corp., Boston, MA). Twenty mg of total RNA per each lane was electrophoresed on a 1.4% agarose gel containing 2M formaldehyde and transferred overnight in 20 x SSC (1 x SSC: 150 mM sodium chloride and 15 mM trisodium citrate) to a nylon membrane (Biodyne, Pall BioSupport Corp., East Hills, NY). RNA was cross-linked to the nylon membrane with a UV Stratalinker. The membrane was prehybridized with the hybridization buffer (50% formamide, 0.2% SDS, 5% dextran sulfate, 5OmM HEPES, 5 x SSC, 5 x Denharts’ solution and 250 mg/ml denatured salmon sperm DNA) at 68°C for 2 hours. Subsequently, the membrane was hybridized at 68°C overnight with the hybridization buffer containing a rat DII cRNA probe. The membrane was washed twice in 2 x SSC, 0.1% SDS at 25°C for 15 mitt, and twice in 0.1 x SSC, 0.1% SDS at 68°C for 1 hour. Autoradiography was established by exposing the filters for 6 to 24 hours to X-ray film (Kodak XAR-2, Eastman Kodak Co., Rochester, NY) at -70°C. After the detection of DII mRNA, the probe was stripped off and blots rehybridized with a control p-actin cRNA probe, which was synthesized in vitro using T7 RNA polymerase and [a-32Pj UTP. Hybridization and washing were performed as described above and the membrane was exposed for 2 to 6 hours. Messenger RNA levels were quantitated by densitometry using an NIH Image Version 1.61, and the optical density of the DII band with 7.5 kb in length was corrected for @actin. RNA samples for comparison were analyzed on the same blot. Quantitation oj’DI1 activity Harderian gland DII activity was measured as previously described (11) with minor modifications (12). Briefly, each Harderian gland was homogenized separately in a 20-fold volume of homogenizing buffer (100 mM potassium phosphate, pH 7.0, containing 1 mM EDTA and 20 mM dithiothreitol). Homogenates were centrifuged at 3,000 rpm for 15 min and resultant infranatants were incubated in a total volume of 100 ml, containing 2 nM [1251]T4(New England Nuclear Corp.), which was purified using LH-20 (Pharmacia, Uppsala, Sweden) column chromatography on the day of experiment, 1 mM EDTA, 20 mM dithiothreitol and 1 mM propylthiouracil, pH 7.0, for one hour at 37°C. The reaction was terminated by the addition of 100 ml 2% bovine serum albumin (BSA) and 800 ml 10% trichloroacetic acid. After centrifugation at 3,000 rpm for 10 mitt, the supematant was applied to a small column packed with Dowex-50 ion exchange resin (Bio-Rad Laboratories,
Vol. 63, No. 21, 1998
Harderian Gland Deiodinase mRNA
(a) Euthyroid
Hypothyroid
-7.5kb
28s 18S-
/3-actin (b) *
r^ 600 5
T
6 8
100
-
0
7
EuthyroidHypothyroid
Euthyroid Hypothyroid
Fig.1 Effect of hypothyroidism on DII mRNA and DII activity in rat Harderian gland. (a) Northern analyses of DII mRNA levels in Harderian glands in euthyroid and hypothyroid rats. Each lane represents individual rat. (b) DII mRNA (DII mRNA/& actin mRNA ratio) levels in Harderian glands in euthyroid and hypothyroid rats. The results are expressed as a percentage of the value obtained for euthyroid control rats. Data shown represent the mean f SE of four animals. (c) DII activities in Harderian glands in euthyroid and hypothyroid rats. Data shown represent the mean f SE of four animals. * p-zO.01, compared with euthyroid control rats by Student’s t test.
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Hercules, CA, bed volume 1 ml) and eluted with 2 ml 10% glacial acetic acid. Separated 1251was counted with a y -counter. Non-enzymatic deiodination was corrected by subtracting I‘ released in tissue-free tubes. The protein concentration was determined by Bradford’s method using BSA as a standard (13). Deiodinating activity was linear within the range of the protein concentration used (2 IO mg/ml), and expressed as femtomoles I- released per mg protein/hour after multiplication by a factor of 2 to correct random labeling at the equivalent 3’ and 5’ positions. Sfutistics
Statistical differences were evaluated by Student’s t test or Newman-Keuls STONE International, Tokyo, Japan).
test (WinSTAT,
LIGHT
Results Results of Northern analyses of DII mRNA and DII activities in Harderian gland in euthyroid and hypothyroid rats are shown in Fig. 1. Northern analyses of DII mRNA demonstrated the hybridization signal with approximately 7.5 kb in Harderian gland and the signal increased 3.5fold in hypothyroid rats compared with euthyroid rats as shown in Fig. I (a) and (b). DII activities also increased 7-fold in hypothyroid rats as shown in Fig. 1 (c). In contrast, p-actin mRNA levels in Harderian gland did not show a significant difference between euthyroid and hypothyroid rats. These results indicate that mRNA levels for DII as well as DII activities in Harderian gland increase in hypothyroid rats. which is in agreement with the characteristic of DII as described in other tissues (6914). In the next experiment, euthyroid rats were injected with 0.3 mgikg body weight of isoproterenol which have been known as a potent stimulus for Dll activities in rat Harderian gland (4). Harderian glands were removed at different hours after the administration of isoproterenol for the quantitation of DII mRNA levels and DII activities. Northern analyses of DII mRNA demonstrated the prominent hybridization signal with approximately 7.5 kb in Harderian gland, and the hybridization signal was increased 5-fold by isoproterenol injection as shown in Fig. 2 (a) and (b). DII mRNA levels in Harderian gland increased as early as one hour after the isoproterenol injection, and reached the peak level by 3 hours after the treatment. Although DII activities in Harderian gland increased Cfold by 3 to 4 hours after the isoproterenol injection, DII activities did not increased by one hour after the treatment as shown in Fig. 2 (c). DII mRNA levels and DII activities did not change significantly 3 hours after the administration of vehicle only, and p-actin mRNA levels did not change after isoproterenol injection in Harderian gland as shown in Fig. 2 (a), (b) and (c). These results indicate that mRNA levels for DII as well as DII activities are increased by isoproterenol administration and that the increase in DII mRNA levels precedes the increase in DII activities in rat Harderian gland.
Discussion Expression of DII mRNA, approximately 7.5 kb in size, has been demonstrated in brain, anterior pituitary, brown fat and pineal gland in the rat by Northern analyses (6,7), and DII mRNA levels have been reported to increase in cerebral cortex, anterior pituitary and brown fat in hypothyroid rats (6,14). In the present study, Northern analyses using reported rat DII cRNA probe (6) have clearly demonstrated the presence of the hybridization signal with approximately 7.5 kb in rat Harderian gland, and the mRNA levels as well as DII activities were significantly increased in hypothyroid rats compared with euthyroid rats. These results suggest that the 7.5 kb DII mRNA is also expressed in rat Harderian gland, and that the increase in DII activities in Harderian gland in hypothyroid rats is due at least in part to the increase in DII mRNA levels. We have also investigated whether Harderian gland DII mRNA levels are increased by isoproterenol administration, since it has been shown that DII activities are significantly stimulated by isoproterenol in rat Harderian gland (4). The present data have demonstrated that DII mRNA levels significantly increased within one hour after isoproterenol injection, and the increase in DII mRNA levels preceded the increase in DII activities in rat Harderian gland. The rapid induction of DII
Vol. 63, No. 21, 1998
Harderian Gland Dciodinase mRNA
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3
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28s 18S-
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_ 600-
** T **
j 500< : 400EEt: 300E 5 200-
0
1 2
3 4
6 -3 hours lsoproterenol Vehicle
-
-
0
1
2
3
4
6 -3 hours lsoproterenol Vehicle
Fig.2 Effect of isoproterenol administration on DII mRNA and DII activity in rat Harderian gland. (a) Northern analyses of DII mRNA in Harderian glands of isoproterenol (0.3 mg/kg BW) or vehicle injected rats. Harderian glands were obtained at indicated hours after isoproterenol or vehicle injection. The representative results are shown. (b) DII mRNA (DII mRNA/&actin mRNA ratio) levels in Harderian glands of isoproterenol or vehicle injected rats. The results are expressed as a percentage of the value obtained for control (0 hour) rats. Data shown represent the mean f SE of four animals. (c) DII activities in Harderian glands of isoproterenol or vehicle injected rats. Data shown represent the mean f SE of four animals. * p&05, ** p&01, compared with control (0 hour) rats by Newman-Keuls test.
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mRNA
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mRNA by isoproterenol administration, presumably through the activation of adenylate cyclase ( 15) is in agreement with the recent observation that DII mRNA in cultured rat astrocytes are significantly increased within one hour by forskolin or 8-bromo-CAMP stimulation (16). These results suggest that the expression of DII is regulated through p-adrenergic mechanism at the pretranslational level in rat Harderian gland. A significant p-adrenergic stimulation of DlI mRNA in rat Harderian gland is basically in agreement with the results by Garcia-Macias et al. (8) who determined the DII transcripts by using Xenopus luevis oocytes which were injected with poly (A)+ RNA from rat Harderian gland. However, they demonstrated the size of mRNA for DII in rat Harderian gland to be 1.4 - 2.4 kb that is different from our present results, possibly due to the methodological difference. Although our Northern analyses of Harderian gland DII mRNA have demonstrated the predominant and prominent band approximately 7.5 kb in length, less prominent bands corresponding to I .6 - 4.3 kb were also detected especially in isoproterenol treated rats as reported in other tissues (6). It is not known whether those mRNAs in smaller size have any contribution to DII activities in rat Harderian gland as well as in other tissues. In summary, the present results suggest that Harderian gland may provide a useful model to investigate the mechanisms involved in the adrenergic regulation of DII expression. Further studies are required to elucidate the physiological significance of the adrenergic stimulation of TA to T3 conversion in Harderian gland.
Acknowledgments We are indebted to Dr. Donald L. St. Germain for the kind gift of the cDNA probe for rat DII. The present work was supported in part by a grant-in-aid (#09671024 to M. Murakami) for scientific research from the Ministry of Education. Science and Culture, Japan.
References I.
2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16.
J.L. LEONARD and J. KOEHRLE, Werner and fngbar’s The Thyroid: A Fundamental und Clinical Text, L.E. Braverman and R.D. Utiger (Eds.). 125-161, Lippincott-Raven. Philadelphia, PA ( 1996). J.E. SILVA, S.A. MELLEN and P.R. LARSEN, Nature 305 712-713 (1983). K. TANAKA, M. MURAKAMI and M.A. GREER, Endocrinology 12 174-77 (1987). A. RUBIO, J.M. GUERRERO, M.A. GONZALEZ, M.A. LOPEZ-GONZALEZ and C. OSUNA. Life Sci. 49 1523-1530 (1991). J.C. DAVEY, K.B. BECKER, M.J. SCHNEIDER, D.L. ST. GERMAIN and V.A. GALTON, J. Biol. Chem. 270 26786-26789 (1995). W. CROTEAU, J.C. DAVEY. V.A. GALTON and D.L. ST. GERMAIN. J. Clin. Invest. 98 405-417 (1996) M. MURAKAMI, Y. HOSOI, T. NEGISHI, Y. KAMIYA,T. OGIWARA, H. MIZUMA, M. YAMADA, T. IRIUCHIJIMA and M. MORI. Neurosci. Lett. 227 65-67 (1997). J.F. GARCIA-MACIAS. P. MOLINERO. A. RUBIO. J.M. GUERRERO and C. OSUNA. Life Sci. 61 181.192(1997). P. CHOMCZYNSKI and N. SACCHI. Anal. Biochem., 162 156-159 (1987). K. MIYASHITA. M. MURAKAMI, T. IRIUCHIJIMA, T. TAKEUCHI and M. MORI, Mol. Cell. Endocrinol. 115 161-167 (1995). J.L. LEONARD and I.N. ROSENBERG, Endocrinology 107 l376- I383 (1980). M. MURAKAMI, K. TANAKA, M.A. GREER and M. MORI, Endocrinology 123 16761681 (1988). M.M. BRADFORD, Anal. Biochem. 7 2 248-254 ( 1976). L.A. BURMEISTER, J. PACHUCKI and D.L. ST. GERMAIN, Endocrinology 138 523 l5237 (1997). M. MURAKAMI, S.E. GREER, S. MCADAMS and M.A. GREER, Life Sci. 44 425-429 (1989). S. PALLUD. A. LENNON, M. RAMAUGE, J. GAVARET, W. CROTEAU, M. PIERRE, F. COURTIN and D.L. ST. GERMAIN. J. Biol. Chem. 272 18104181 IO (1997).
ELSEVIER
NORTHERN
PI1 SOO24-3205(98)00471-8
ANALYSIS OF TYPE II IODOTHYRONINE mRNA IN RAT HARDERIAN GLAND
Copyright o 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/98 $19.00 + .oO
DEIODINASE
Osamu Araki, Masami Murakami I, Y uji Kamiya, Y asuhiro Hosoi, Takayuki Ogiwara, Haruo Mizuma, Tokuji Iriuchijima and Masatomo Mori
First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 37 l-85 11, Japan (Received in final form September 7, 19%) Summary It has been known that type II iodothyronine deiodinase activity is present in rat Harderian gland and the activity is significantly increased by isoproterenol administration. We have performed Northern analyses to study whether the transcript for type II iodothyronine deiodinase is expressed in rat Harderian gland and whether the isoproterenol stimulation of type II iodothyronine deicdinase activity in rat Harderian gland is due to the change in its mRNA level. Northern analyses have demonstrated that type II iodothyronine deiodinase mRNA, approximately 7.5 kb in size, is expressed in rat Harderian gland, and the mRNA levels as well as the deiodinase activities are greater in hypothyroid rats than those in euthyroid rats. Type II iodothyronine deiodinase mRNA levels and the deiodinase activities in Harderian gland were increased by isoproterenol administration, and the increase in the mRNA levels preceded that in the deiodinase activities. These results indicate that 7.5 kb transcript for type II iodothyronine deiodinase is expressed in rat Harderian gland and fl-adrenergic stimulation of type II iodothyronine deiodinase activity is due at least in part to the increase in its mRNA level. Key Words: thyroid hormone, adrenergic receptor, hypothyroidism, isoproterenol
Thyroxine (Ta), which is a major secretory product of thyroid gland, needs to be converted to 3,5,3’-triiodothyronine (T3) by iodothyronine deiodinase to exert its biological activity. Type I iodothyronine deiodinase (DI) activity is present in thyroid gland, liver and kidney, while type II iodothyronine deiodinase (DII) activity is present in brain, anterior pituitary, brown fat, pineal gland and Harderian gland in the rat (1). DI activity is known to decrease in hypothyroid state and mainly contribute to circulating T3 level. In contrast, DII activity increases in hypothyroid state and plays a critical role to provide a local intracellular T3. Furthermore, DII activity in brown fat (2), pineal gland (3) and Harderian gland (4) has been demonstrated to be significantly increased by adrenergic stimulation. Recently, a cDNA encoding DII has been cloned from Rana catesbeiana tissues (5), and subsequently from rat brown fat (6). Expression of DII mRNA, approximately 7.5 kb in size, was observed in brain, anterior pituitary, brown fat and pineal gland in the rat by Northern analysis
‘Corresponding author: M. Murakami, First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 37 l-85 11, Japan Phone:+8 1 27 2208126; FAX:+8 127 2208 136; E-mail:[email protected]
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(6,7). Although Northern analysis of DII mRNA in Harderian gland has not been reported, GarciaMacias et al. determined the size of the mRNA expressing the DII activity to be I.4 - 2.4 kb using Xenopus laevis oocytes which were injected with poly (A)+ RNA from rat Harderian gland (8). Thus, it has not been known whether the 7.5 kb transcript for DII is present in rat Harderian gland. In the present report, we have performed Northern analyses to study whether the 7.5 kb transcript for DII mRNA is also expressed in rat Harderian gland and whether the adrenergic stimulation of DII activities is due to the increase in its mRNA levels.
Methods Animals Two-month-old, male Wistar rats were maintained two per cage on a 12-hour light, 12-hour dark schedule (lights on at 0600 h) at 25kl”C and fed standard laboratory chow and tap water ad lib&urn. The rats were acclimated to this condition for at least one week before the experiment. Hypothyroid rats were produced by administering 0.03% methimazole in their drinking water for 4 weeks. Hypothyroidism was judged by a decreased weight gain of the animals. Some rats were injected subcutaneously with 0.3 mg/kg body weight of isoproterenol at 1200 h. The Harderian glands were rapidly removed, divided into two pieces, frozen and stored at -70°C until the RNA isolation or the quantitation of DII activity. RNA preparation and Northern analysis
Total RNA was isolated from each Harderian gland by modified acid guanidinium thiocyanatephenol-chloroform method according to Chomczynski and Sacchi (9). Northern analyses were performed as previously described (7,lO). Plasmid rDI1 5 I/pBluescript SK, which contains rat DII cDNA, was kindly provided by Dr. St. Germain (6). Briefly, rat DII cRNA probe was synthesized by in vitro transcription of linearized rDI1 5-l/pBluescript SK using T7 polymerase (Boehringer Mannheim GmbH, Mannheim, Germany) and [a-32P]UTP (New England Nuclear Corp., Boston, MA). Twenty mg of total RNA per each lane was electrophoresed on a 1.4% agarose gel containing 2M formaldehyde and transferred overnight in 20 x SSC (1 x SSC: 150 mM sodium chloride and 15 mM trisodium citrate) to a nylon membrane (Biodyne, Pall BioSupport Corp., East Hills, NY). RNA was cross-linked to the nylon membrane with a UV Stratalinker. The membrane was prehybridized with the hybridization buffer (50% formamide, 0.2% SDS, 5% dextran sulfate, 5OmM HEPES, 5 x SSC, 5 x Denharts’ solution and 250 mg/ml denatured salmon sperm DNA) at 68°C for 2 hours. Subsequently, the membrane was hybridized at 68°C overnight with the hybridization buffer containing a rat DII cRNA probe. The membrane was washed twice in 2 x SSC, 0.1% SDS at 25°C for 15 mitt, and twice in 0.1 x SSC, 0.1% SDS at 68°C for 1 hour. Autoradiography was established by exposing the filters for 6 to 24 hours to X-ray film (Kodak XAR-2, Eastman Kodak Co., Rochester, NY) at -70°C. After the detection of DII mRNA, the probe was stripped off and blots rehybridized with a control p-actin cRNA probe, which was synthesized in vitro using T7 RNA polymerase and [a-32Pj UTP. Hybridization and washing were performed as described above and the membrane was exposed for 2 to 6 hours. Messenger RNA levels were quantitated by densitometry using an NIH Image Version 1.61, and the optical density of the DII band with 7.5 kb in length was corrected for @actin. RNA samples for comparison were analyzed on the same blot. Quantitation oj’DI1 activity Harderian gland DII activity was measured as previously described (11) with minor modifications (12). Briefly, each Harderian gland was homogenized separately in a 20-fold volume of homogenizing buffer (100 mM potassium phosphate, pH 7.0, containing 1 mM EDTA and 20 mM dithiothreitol). Homogenates were centrifuged at 3,000 rpm for 15 min and resultant infranatants were incubated in a total volume of 100 ml, containing 2 nM [1251]T4(New England Nuclear Corp.), which was purified using LH-20 (Pharmacia, Uppsala, Sweden) column chromatography on the day of experiment, 1 mM EDTA, 20 mM dithiothreitol and 1 mM propylthiouracil, pH 7.0, for one hour at 37°C. The reaction was terminated by the addition of 100 ml 2% bovine serum albumin (BSA) and 800 ml 10% trichloroacetic acid. After centrifugation at 3,000 rpm for 10 mitt, the supematant was applied to a small column packed with Dowex-50 ion exchange resin (Bio-Rad Laboratories,
Vol. 63, No. 21, 1998
Harderian Gland Deiodinase mRNA
(a) Euthyroid
Hypothyroid
-7.5kb
28s 18S-
/3-actin (b) *
r^ 600 5
T
6 8
100
-
0
7
EuthyroidHypothyroid
Euthyroid Hypothyroid
Fig.1 Effect of hypothyroidism on DII mRNA and DII activity in rat Harderian gland. (a) Northern analyses of DII mRNA levels in Harderian glands in euthyroid and hypothyroid rats. Each lane represents individual rat. (b) DII mRNA (DII mRNA/& actin mRNA ratio) levels in Harderian glands in euthyroid and hypothyroid rats. The results are expressed as a percentage of the value obtained for euthyroid control rats. Data shown represent the mean f SE of four animals. (c) DII activities in Harderian glands in euthyroid and hypothyroid rats. Data shown represent the mean f SE of four animals. * p-zO.01, compared with euthyroid control rats by Student’s t test.
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Hercules, CA, bed volume 1 ml) and eluted with 2 ml 10% glacial acetic acid. Separated 1251was counted with a y -counter. Non-enzymatic deiodination was corrected by subtracting I‘ released in tissue-free tubes. The protein concentration was determined by Bradford’s method using BSA as a standard (13). Deiodinating activity was linear within the range of the protein concentration used (2 IO mg/ml), and expressed as femtomoles I- released per mg protein/hour after multiplication by a factor of 2 to correct random labeling at the equivalent 3’ and 5’ positions. Sfutistics
Statistical differences were evaluated by Student’s t test or Newman-Keuls STONE International, Tokyo, Japan).
test (WinSTAT,
LIGHT
Results Results of Northern analyses of DII mRNA and DII activities in Harderian gland in euthyroid and hypothyroid rats are shown in Fig. 1. Northern analyses of DII mRNA demonstrated the hybridization signal with approximately 7.5 kb in Harderian gland and the signal increased 3.5fold in hypothyroid rats compared with euthyroid rats as shown in Fig. I (a) and (b). DII activities also increased 7-fold in hypothyroid rats as shown in Fig. 1 (c). In contrast, p-actin mRNA levels in Harderian gland did not show a significant difference between euthyroid and hypothyroid rats. These results indicate that mRNA levels for DII as well as DII activities in Harderian gland increase in hypothyroid rats. which is in agreement with the characteristic of DII as described in other tissues (6914). In the next experiment, euthyroid rats were injected with 0.3 mgikg body weight of isoproterenol which have been known as a potent stimulus for Dll activities in rat Harderian gland (4). Harderian glands were removed at different hours after the administration of isoproterenol for the quantitation of DII mRNA levels and DII activities. Northern analyses of DII mRNA demonstrated the prominent hybridization signal with approximately 7.5 kb in Harderian gland, and the hybridization signal was increased 5-fold by isoproterenol injection as shown in Fig. 2 (a) and (b). DII mRNA levels in Harderian gland increased as early as one hour after the isoproterenol injection, and reached the peak level by 3 hours after the treatment. Although DII activities in Harderian gland increased Cfold by 3 to 4 hours after the isoproterenol injection, DII activities did not increased by one hour after the treatment as shown in Fig. 2 (c). DII mRNA levels and DII activities did not change significantly 3 hours after the administration of vehicle only, and p-actin mRNA levels did not change after isoproterenol injection in Harderian gland as shown in Fig. 2 (a), (b) and (c). These results indicate that mRNA levels for DII as well as DII activities are increased by isoproterenol administration and that the increase in DII mRNA levels precedes the increase in DII activities in rat Harderian gland.
Discussion Expression of DII mRNA, approximately 7.5 kb in size, has been demonstrated in brain, anterior pituitary, brown fat and pineal gland in the rat by Northern analyses (6,7), and DII mRNA levels have been reported to increase in cerebral cortex, anterior pituitary and brown fat in hypothyroid rats (6,14). In the present study, Northern analyses using reported rat DII cRNA probe (6) have clearly demonstrated the presence of the hybridization signal with approximately 7.5 kb in rat Harderian gland, and the mRNA levels as well as DII activities were significantly increased in hypothyroid rats compared with euthyroid rats. These results suggest that the 7.5 kb DII mRNA is also expressed in rat Harderian gland, and that the increase in DII activities in Harderian gland in hypothyroid rats is due at least in part to the increase in DII mRNA levels. We have also investigated whether Harderian gland DII mRNA levels are increased by isoproterenol administration, since it has been shown that DII activities are significantly stimulated by isoproterenol in rat Harderian gland (4). The present data have demonstrated that DII mRNA levels significantly increased within one hour after isoproterenol injection, and the increase in DII mRNA levels preceded the increase in DII activities in rat Harderian gland. The rapid induction of DII
Vol. 63, No. 21, 1998
Harderian Gland Dciodinase mRNA
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28s 18S-
(b)
_ 600-
** T **
j 500< : 400EEt: 300E 5 200-
0
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3 4
6 -3 hours lsoproterenol Vehicle
-
-
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1
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3
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6 -3 hours lsoproterenol Vehicle
Fig.2 Effect of isoproterenol administration on DII mRNA and DII activity in rat Harderian gland. (a) Northern analyses of DII mRNA in Harderian glands of isoproterenol (0.3 mg/kg BW) or vehicle injected rats. Harderian glands were obtained at indicated hours after isoproterenol or vehicle injection. The representative results are shown. (b) DII mRNA (DII mRNA/&actin mRNA ratio) levels in Harderian glands of isoproterenol or vehicle injected rats. The results are expressed as a percentage of the value obtained for control (0 hour) rats. Data shown represent the mean f SE of four animals. (c) DII activities in Harderian glands of isoproterenol or vehicle injected rats. Data shown represent the mean f SE of four animals. * p&05, ** p&01, compared with control (0 hour) rats by Newman-Keuls test.
1848
Harderian Gland Deiodinase
mRNA
Vol. 63, No. 21, 1998
mRNA by isoproterenol administration, presumably through the activation of adenylate cyclase ( 15) is in agreement with the recent observation that DII mRNA in cultured rat astrocytes are significantly increased within one hour by forskolin or 8-bromo-CAMP stimulation (16). These results suggest that the expression of DII is regulated through p-adrenergic mechanism at the pretranslational level in rat Harderian gland. A significant p-adrenergic stimulation of DlI mRNA in rat Harderian gland is basically in agreement with the results by Garcia-Macias et al. (8) who determined the DII transcripts by using Xenopus luevis oocytes which were injected with poly (A)+ RNA from rat Harderian gland. However, they demonstrated the size of mRNA for DII in rat Harderian gland to be 1.4 - 2.4 kb that is different from our present results, possibly due to the methodological difference. Although our Northern analyses of Harderian gland DII mRNA have demonstrated the predominant and prominent band approximately 7.5 kb in length, less prominent bands corresponding to I .6 - 4.3 kb were also detected especially in isoproterenol treated rats as reported in other tissues (6). It is not known whether those mRNAs in smaller size have any contribution to DII activities in rat Harderian gland as well as in other tissues. In summary, the present results suggest that Harderian gland may provide a useful model to investigate the mechanisms involved in the adrenergic regulation of DII expression. Further studies are required to elucidate the physiological significance of the adrenergic stimulation of TA to T3 conversion in Harderian gland.
Acknowledgments We are indebted to Dr. Donald L. St. Germain for the kind gift of the cDNA probe for rat DII. The present work was supported in part by a grant-in-aid (#09671024 to M. Murakami) for scientific research from the Ministry of Education. Science and Culture, Japan.
References I.
2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16.
J.L. LEONARD and J. KOEHRLE, Werner and fngbar’s The Thyroid: A Fundamental und Clinical Text, L.E. Braverman and R.D. Utiger (Eds.). 125-161, Lippincott-Raven. Philadelphia, PA ( 1996). J.E. SILVA, S.A. MELLEN and P.R. LARSEN, Nature 305 712-713 (1983). K. TANAKA, M. MURAKAMI and M.A. GREER, Endocrinology 12 174-77 (1987). A. RUBIO, J.M. GUERRERO, M.A. GONZALEZ, M.A. LOPEZ-GONZALEZ and C. OSUNA. Life Sci. 49 1523-1530 (1991). J.C. DAVEY, K.B. BECKER, M.J. SCHNEIDER, D.L. ST. GERMAIN and V.A. GALTON, J. Biol. Chem. 270 26786-26789 (1995). W. CROTEAU, J.C. DAVEY. V.A. GALTON and D.L. ST. GERMAIN. J. Clin. Invest. 98 405-417 (1996) M. MURAKAMI, Y. HOSOI, T. NEGISHI, Y. KAMIYA,T. OGIWARA, H. MIZUMA, M. YAMADA, T. IRIUCHIJIMA and M. MORI. Neurosci. Lett. 227 65-67 (1997). J.F. GARCIA-MACIAS. P. MOLINERO. A. RUBIO. J.M. GUERRERO and C. OSUNA. Life Sci. 61 181.192(1997). P. CHOMCZYNSKI and N. SACCHI. Anal. Biochem., 162 156-159 (1987). K. MIYASHITA. M. MURAKAMI, T. IRIUCHIJIMA, T. TAKEUCHI and M. MORI, Mol. Cell. Endocrinol. 115 161-167 (1995). J.L. LEONARD and I.N. ROSENBERG, Endocrinology 107 l376- I383 (1980). M. MURAKAMI, K. TANAKA, M.A. GREER and M. MORI, Endocrinology 123 16761681 (1988). M.M. BRADFORD, Anal. Biochem. 7 2 248-254 ( 1976). L.A. BURMEISTER, J. PACHUCKI and D.L. ST. GERMAIN, Endocrinology 138 523 l5237 (1997). M. MURAKAMI, S.E. GREER, S. MCADAMS and M.A. GREER, Life Sci. 44 425-429 (1989). S. PALLUD. A. LENNON, M. RAMAUGE, J. GAVARET, W. CROTEAU, M. PIERRE, F. COURTIN and D.L. ST. GERMAIN. J. Biol. Chem. 272 18104181 IO (1997).