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Endocrinology, doi:10.1210/en.2004-0632
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Endocrinology Vol. 145, No. 12 5638-5645
Copyright © 2004 by The Endocrine Society

Expression and Regulation of Type 2 Iodothyronine Deiodinase in Rat Aorta Media

Saori Yasuzawa-Amano, Nagaoki Toyoda, Akimasa Maeda, Atsushi Kosaki, Yasukiyo Mori, Toshiji Iwasaka and Mitsushige Nishikawa

Department of Medicine II, Kansai Medical University, Moriguchi-City, Osaka 570, Japan

Address all correspondence and requests for reprints to: Nagaoki Toyoda, M.D., Department of Medicine II, Kansai Medical University, 10-15 Fumizono-Cho, Moriguchi-City, Osaka 570, Japan. E-mail: toyoda{at}takii.kmu.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Here, we have found that type 2 iodothyronine deiodinase (D2) is present in rat aorta media and that there is a circadian variation in the D2 expression. The D2 mRNA was approximately 4-fold higher at 0900 h than at 2100 h, and the activity was approximately 6-fold higher at noon than at 2100 h. The increase in aorta media D2 activity is preceded by the increase in its mRNA. The increase in D2 mRNA and activity in the circadian variation was reduced by the administration of prazosin, an {alpha}1-adrenergic antagonist, and propranolol, a ß- adrenergic antagonist. Furthermore, phenylephrine, an {alpha}1-adrenergic agonist, and isoproterenol, a ß-adrenergic agonist, caused a significant increase in D2 mRNA and activity. In the hypothyroid rats, aorta mediae D2 mRNA at both 0900 and 2100 h were not significantly different when compared with those in the euthyroid rats. On the other hand, aorta mediae D2 activity at both 1200 and 2100 h in the hypothyroid rats were approximately 2-fold higher. From these results, we suggest that D2 activity of rat aorta media is increased by both {alpha}1- and ß-adrenergic stimulation, at least partly, at the pretranslational level. We also suggest that both {alpha}1- and ß-adrenergic mechanisms may be involved, at least partly, in the circadian variation of the activity. In the hypothyroid state, the aorta media D2 activity is increased mainly by the posttranslational mechanism, and the similar circadian variation of the D2 expression is present as in the euthyroid state.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROXINE (T4), A MAJOR secretory product of the thyroid gland, needs to be converted to T3 by iodothyronine deiodinase to exert its biological activity. There are two isoenzymes that catalyze the conversion, type 1 and type 2 iodothyronine deiodinase (D1 and D2, respectively) (1, 2). Both enzymes are selenoproteins and contain the rare amino acid selenocysteine at their active sites. D1 is present in liver, kidney, thyroid gland, and many other tissues, whereas D2 is present in a limited number of tissues, including anterior pituitary, brain, brown adipose tissue (BAT), pineal, and harderian gland in the rat. In human, D2 is expressed not only in pituitary and brain, as it is in the rat, but also in thyroid, myocardium, and skeletal muscle, which is not the case in the rodent (3, 4, 5, 6, 7). The Michaelis-Menten constant (Km) of D2 is approximately 1–10 nM for T4, which is 100 times lower than that of D1. D2 is relatively insensitive to inhibition by 6-propyl-2-thiouracil (PTU), which inhibits D1. It has been thought that D1 is the major contributor to the circulating plasma T3 levels, whereas catalysis of T4 by D2 is primarily responsible for the production of T3 within specialized tissues where D2 exists. Recently, mice with targeted disruption of the D2 gene (D2 knockout mice) were established (8). From the investigations of the mouse, the physiological importance of D2 was confirmed in the regulation of TSH secretion by thyroid hormones and in adaptive thermogenesis in BAT (9).

D2 activity is expressed in cultured human vascular smooth muscle cells (10, 11). However, to our knowledge, it has not been demonstrated whether D2 is expressed in the vessel using the animal model. Vascular smooth muscle cells are a major component of the media of the artery. Therefore, in the present study, we have investigated whether D2 is expressed in the rat aorta media. We have also studied its circadian variation during the day. Furthermore, we have investigated the effects of adrenergic agonists and antagonists and the effect of the hypothyroid state on D2 expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[{alpha}-32P]UTP, [{alpha}-32P]dCTP, and [125I]T4 were purchased from NEN Life Science Products Corp. (Boston, MA), Sephadex LH20 from Pharmacia Biotech (Uppsala, Sweden), T7 RNA polymerase from Promega Corp. (Madison, WI), and AG 50W-X2 from Bio-Rad Laboratories, Inc. (Hercules, CA). All other chemicals of the highest quality were obtained from Sigma Chemical Co. (St. Louis, MO) or Nacalai Tesque (Kyoto, Japan) unless otherwise indicated.

Animals
All procedures were performed in accordance with institutional guidelines for animal research at Kansai Medical University. Male Sprague Dawley rats ranging in age from 8–10 wk were used. Rats were acclimated to a 12-h light, 12-h dark schedule (lights between 0800 and 2000 h) and controlled temperature (25 ± 1 C). Rat chow and tap water were provided ad libitum. For injection experiments, all of the drugs were dissolved in saline, and control groups received the same volume of saline vehicle. The doses and time lags for each adrenergic agonist and antagonist were chosen according to the previous reports (12, 13, 14, 15, 16). Hypothyroidism was induced by the addition of 0.03% methimazole to the drinking water for 3 wk. Hypothyroidism was assessed clinically by failure to gain weight at the expected rate and by measuring the serum T4 and T3 concentrations.

Preparation of aorta media
Rats were anesthetized with ether, and their thoracic aortas were dissected. Each was cleaned of fat and connective tissue. After the removal of tunica intima and adventitia, mediae were isolated and immediately frozen and stored at –70 C.

Measurement of iodothyronine deiodinase activity
Tissue was homogenized in a 10-fold volume of ice-cold buffer containing 100 mM potassium phosphate (pH 7.0), 20 mM dithiothreitol, and 1 mM EDTA (homogenization buffer). After centrifugation at 3000 rpm for 10 min, the supernatants were used for the deiodination assay. Iodothyronine deiodinase activity was measured as previously described (4, 7, 11). Tissue homogenates were incubated with 2 nM [125I]T4, which was purified using LH-20 column chromatography on the day of the experiment in the presence of 1 mM PTU in homogenization buffer in a total volume of 300 µl at 37 C for 2 h. The reaction was terminated by adding 200 µl horse serum and 100 µl of 50% trichloroacetic acid. The released 125I– was separated by column chromatography using AG 50W-X2 resin and counted with a {gamma}-counter. The protein concentration was measured according to the method of Bradford using BSA as a standard (17). The deiodinating activity was calculated as femtomoles of I– released per milligram of protein per hour. Kinetic analyses were performed with Lineweaver-Burk plots. Slopes and intercepts for the double-reciprocal plots were calculated by least-squares analysis. Maximum velocity (Vmax) and apparent Km values were determined from the intercepts with the ordinate and abscissa, respectively.

RNA preparation and Northern analysis
Total RNA was extracted from tissues by the acid guanidinium thiocyanate phenol-chloroform method according to the method of Chomczynski and Sacchi (18). Plasmid rDII 5-1/pBluescript SK, which contains rat D2 cDNA, was kindly provided by Dr. St. Germain (3). Briefly, rat D2 cRNA probe was synthesized by in vitro transcription of linearized rDII 5-1/pBluescript SK using T7 RNA polymerase and [{alpha}-32P]UTP. Total RNA (30 µg/lane) was electrophoresed on a 0.8% agarose gel and transferred to nylon membranes (Biodyne, Pall, NY). The membrane was prehybridized with the hybridization buffer [50% formamide, 5x SSC (1x SSC contains 150 mM NaCl and 15 mM sodium citrate), 200 µg/ml denatured salmon sperm DNA, 0.2% SDS, 5% dextran sulfate, and 1x Denhardt’s solution (1x contains 0.2% BSA, 0.2% Ficoll, and 0.2% polyvinyl pyrrolidone)] at 68 C for 3 h. Subsequently, the membrane was hybridized at 68 C overnight with the hybridization buffer containing a rat D2 cRNA probe. The membrane was washed twice in 2x SSC-0.1% SDS at 25 C for 15 min and twice in 0.1x SSC-0.1% SDS at 68 C for 1 h. D2 mRNA level was determined by Fujix Bioimage Analyzer (BAS 2000; Fuji Photo Film, Tokyo, Japan). After detection of D2 mRNA, the probe was stripped off, and blots were rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe, which was synthesized using [{alpha}-32P]dCTP, as a control as previously described (11). D2 mRNA level was corrected by the mRNA for GAPDH.

Determination of serum T4 and T3 concentrations
Serum T4 and T3 concentrations were assayed by commercial kits (Elecsys Electrochemiluminescence, Roche Diagnostics Corp., Indianapolis, IN) (19).

Statistical analysis
All results are presented as means ± SD. Differences between groups were analyzed by ANOVA with multiple comparison using Dunnett’s method. Statistical significance was accepted at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of iodothyronine deiodinase in aorta media
The deiodinating activity in the homogenates of rat aorta media was measured by the release of I– from 2 nM [125I]T4 in the presence of 20 mM dithiothreitol. T4 deiodination was dependent on an incubation period and a protein concentration of tissue homogenate. The increase of T4 deiodination was almost linear up to 2 h of incubation period and up to 4 mg/ml protein concentration. Incubation at 4 C or preheating the homogenate at 56 C for 30 min completely abolished the deiodination. These results suggest that the T4 deiodinating activity of rat aorta media is enzymic in nature. Next, kinetic studies using different concentrations of T4 were performed (Table 1Go). From the double-reciprocal plot of the data in Table 1Go, kinetic constants for T4 were calculated to be Km = 4.4 nM, and Vmax= 62.5 fmol of I– released/mg protein per hour. Furthermore, the T4 deiodinating activity was not influenced by 1 mM PTU but was completely inhibited by 1 mM iopanoic acid. These results indicate that the characteristics of the deiodinating activity in the aorta media are compatible with D2.


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  		TABLE 1. Deiodinating activity in rat aorta media

 
Variation of D2 mRNA and activity in aorta media during the day
Variation in aorta media D2 mRNA and activity during the day were investigated. Groups of four rats each were killed every 3 h during a whole 24-h period. As shown in Fig. 1AGo, Northern analysis of D2 mRNA in four pooled aorta mediae demonstrated the hybridization signal with approximately 7.5 kb. A distinct circadian variation of D2 mRNA was observed (Fig. 1Go, A and B). The D2 mRNA exhibited the low level between 1500 and 2400 h, increasing its level at 0300 h, and reaching the maximal level at 0900 h (approximately 4-fold above the level at 2100 h). Furthermore, a definite circadian variation in aorta media D2 activity was also observed (Fig. 1BGo). The D2 activity exhibited low values between 1800 and 0300 h, increasing its activity at 0600 h, and reaching the maximal value at 1200 h (approximately 6-fold above the value at 2100 h), as shown in Fig. 1BGo. These results suggest the increase in D2 activity is preceded approximately 3 h by the increase in D2 mRNA.



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  		FIG. 1. Circadian variation in D2 mRNA and activity in the rat aorta media. A, Northern analysis of D2 mRNA and GAPDH mRNA in the rat aorta media. Four aorta mediae were obtained at the indicated hours (lights on, 0800–2000 h). Each lane represents four pooled aorta mediae. B, D2 mRNA (D2 mRNA/GAPDH mRNA ratio; black bars) and D2 activity (white bars) in aorta mediae. The optical density of the D2 mRNA was corrected for GAPDH mRNA, and results were expressed as a percentage of the value obtained at 2100 h. The results of D2 mRNA shown represent the mean ± SD of three different experiments. The D2 activity shown represents the mean ± SD of four animals. *, P < 0.05 compared with the value obtained at 2100 h.

 
Effects of {alpha}- and ß-adrenergic antagonists on the circadian variation of D2 mRNA and activity in aorta media
To understand the mechanism causing the circadian variation in aorta media D2 mRNA and activity, we examined whether {alpha}- and ß-adrenergic pathways were involved. Rats were injected sc with prazosin [(4.0 mg/kg body weight (BW)], an {alpha}1-adrenergic antagonist; yohimbine (4.0 mg/kg BW), an {alpha}2-adrenergic antagonist; propranolol (10 mg/kg BW), a ß-adrenergic antagonist; both prazosin and propranolol; or vehicle at 2200 h. Because the peaks for D2 mRNA and activity were demonstrated at 0900 and 1200 h, respectively, the aorta mediae were obtained at 0900 h for Northern analysis and at 1200 h for the measurement of D2 activity. Prazosin and propranolol significantly reduced the D2 mRNA by approximately 70 and 50%, respectively (Fig. 2Go, A and B). Prazosin and propranolol also significantly reduced the D2 activity by approximately 70 and 50%, respectively (Fig. 2BGo). On the other hand, yohimbine did not change the D2 mRNA (Fig. 2Go, A and B) or the activity (Fig. 2BGo). Both prazosin and propranolol administration reduced the D2 mRNA and activity by 90%. These results suggest that both {alpha}1- and ß-adrenergic pathways may be involved, at least partly, in the circadian variation of rat aorta media D2 mRNA and its activity.



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  		FIG. 2. Effects of prazosin (Praz), yohimbine (Yoh), and propranolol (Pro) on the increase in aorta media D2 mRNA and activity. Rats were injected with Praz, an {alpha}1-adrenergic antagonist (4.0 mg/kg BW); Yoh, an {alpha}2-adrenergic antagonist (4.0 mg/kg BW); Pro, a ß-adrenergic antagonist (10 mg/kg BW); Praz plus Pro; or vehicle at 2200 h, and four aorta mediae were obtained at 0900 h for Northern analysis and at 1200 h for D2 activity. A, Northern analysis of D2 mRNA in aorta media. Each lane represents four pooled aorta mediae. B, D2 mRNA (D2 mRNA/GAPDH mRNA ratio; black bars) and D2 activity (white bars) in aorta media obtained as described above. The OD of the D2 mRNA was corrected for GAPDH mRNA, and the results were expressed as a percentage of the value obtained at 0900 h. The results of D2 mRNA shown represents the mean ± SD of three different experiments. The D2 activity was expressed as a percentage of the value obtained at 1200 h. The results of D2 activity shown represents the mean ± SD of four animals. *, P < 0.05 compared with control at 0900 h (D2 mRNA) or at 1200 h (D2 activity).

 
Effects of {alpha}1- and ß-adrenergic agonists on the D2 mRNA and activity in aorta media
To study the effects of {alpha}1- and ß-adrenergic pathways on aorta media D2 mRNA and activity, rats were injected sc with phenylephrine (1.0 mg/kg BW), an {alpha}1-adrenergic agonist; isoproterenol (0.3 mg/kg BW), a ß-adrenergic agonist; both phenylephrine and isoproterenol; or vehicle. Because the D2 mRNA and activity are low between 1500 and 2100 h, rats were injected at 1500 h and were killed at various time intervals (0, 1.5, 3, and 6 h) between 1500 and 2100 h. Phenylephrine caused an approximately 4-fold increase in the D2 mRNA and activity 6 h after injection (Fig. 3Go, A and B). On the other hand, isoproterenol caused an approximately 3-fold increase in the D2 mRNA and activity 3 h after injection (Fig. 4Go, A and B).



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  		FIG. 3. Effects of phenylephrine on the aorta media D2 mRNA and activity. A, Northern analysis of D2 mRNA in aorta media of rats injected with phenylephrine, an {alpha}1-adrenergic agonist (1.0 mg/kg BW at 1500 h). Four aorta mediae were obtained at the indicated hours after phenylephrine administration. Each lane represents four pooled aorta mediae. B, D2 mRNA (D2 mRNA/GAPDH mRNA ratio; black bars) and D2 activity (white bars) in aorta media obtained as described above. The OD of the D2 mRNA was corrected for GAPDH mRNA, and the results were expressed as a percentage of the value obtained for control rats at 1500 h. The results of D2 mRNA shown represent the mean ± SD of three different experiments. The D2 activity shown represents the mean ± SD of four animals. *, P < 0.05 compared with control rats.

 


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  		FIG. 4. Effects of isoproterenol on the aorta media D2 mRNA and activity. A, Northern analysis of D2 mRNA in aorta media of rats injected with isoproterenol, a ß-adrenergic agonist (0.3 mg/kg BW at 1500 h). Four aorta mediae were obtained at the indicated hours after isoproterenol administration. Each lane represents four pooled aorta mediae. B, D2 mRNA (D2 mRNA/GAPDH mRNA ratio; black bars) and D2 activity (white bars) in aorta media obtained as described above. The OD of the D2 mRNA was corrected for GAPDH mRNA, and the results were expressed as a percentage of the value obtained for control rats at 1500 h. The results of D2 mRNA shown represent the mean ± SD of three different experiments. The D2 activity shown represents the mean ± SD of four animals. *, P < 0.05 compared with control rats.

 
To examine the relationship between {alpha}1- and ß-adrenergic stimulation, rats were injected sc with both phenylephrine (1.0 mg/kg BW) and isoproterenol (0.3 mg /kg BW) at 1500 h and were killed 3 h after the injection. The D2 mRNA and activity of rats administrated with both phenylephrine and isoproterenol were higher than those of phenylephrine or isoproterenol alone (Fig. 5Go, A and B).



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  		FIG. 5. Effects of combination of phenylephrine (Phe) and isoproterenol (Iso) on the aorta media D2 mRNA and activity. A, Northern analysis of D2 mRNA in aorta media. Rats were injected with Phe, an {alpha}1-adrenergic agonist (1.0 mg/kg BW), alone; Iso, a ß-adrenergic agonist (0.3 mg/kg BW), alone; or Phe plus Iso at 1500 h. Four aorta mediae were obtained after 3 h. Each lane represents four pooled aorta mediae. B, D2 mRNA (D2 mRNA/GAPDH mRNA ratio; black bars) and D2 activity (white bars) in aorta media obtained as described above. The OD of the D2 mRNA was corrected for GAPDH mRNA, and the results were expressed as a percentage of the value obtained for control rats at 1800 h. The results of D2 mRNA shown represent the mean ± SD of three different experiments. The D2 activity shown represents the mean ± SD of three animals. *, P < 0.05 compared with control rats.

 
Furthermore, to investigate whether a weak ß-adrenergic pathway is involved in the effect of phenylephrine on D2 expression, rats were injected sc with phenylephrine (1.0 mg/kg BW) alone or in combination with propranolol (10 mg/kg BW) at 1500 h and were killed 3 h after the injection. The D2 mRNA and activity of the rats administered phenylephrine in combination with propranolol were not significantly different compared with phenylephrine alone (Fig. 6Go, A and B). Next, to investigate the {alpha}1-adrenergic pathway in the effect of isoproterenol on D2 expression, rats were injected sc with isoproterenol (1.0 mg/kg BW) alone or in combination with prazosin (4.0 mg/kg BW) at 1500 h and were killed 3 h after the injection. The D2 mRNA and activity of rats administered isoproterenol in combination with prazosin were not significantly different compared with isoproterenol alone (Fig. 6Go, A and B).



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  		FIG. 6. Effects of combination of phenylephrine (Phe) plus propranolol (Pro) or isoproterenol (Iso) plus prazosin (Praz) on the aorta media D2 mRNA and activity. A, Northern analysis of D2 mRNA in aorta media. Rats were injected with Phe, an {alpha}1-adrenergic agonist (1.0 mg /kg BW), alone; Phe plus Pro, a ß-adrenergic antagonist (10 mg/kg BW); Iso, a ß-adrenergic agonist (1.0 mg/kg BW), alone; Iso plus Praz, an {alpha}1-adrenergic antagonist (4.0 mg/kg BW), at 1500 h. Four aorta mediae were obtained after 3 h. Each lane represents four pooled aorta mediae. B, D2 mRNA (D2 mRNA/GAPDH mRNA ratio; black bars) and D2 activity (white bars) in aorta media obtained as described above. The OD of the D2 mRNA was corrected for GAPDH mRNA, and the results were expressed as a percentage of the value obtained for control rats at 1800 h. The results of D2 mRNA shown represent the mean ± SD of three different experiments. The D2 activity shown represents the mean ± SD of four animals. *, P < 0.05 compared with control rats.

 
Effect of hypothyroid state on the D2 mRNA and activity in aorta media
To study the effect of the hypothyroid state on the aorta media D2 expression, the D2 mRNA and activity of hypothyroid rats were measured and compared with those of euthyroid control. The serum T4 and T3 concentrations in the hypothyroid rats were 1.4 ± 0.2 µg/dl and 61 ± 5 ng/dl, respectively. Aorta mediae were obtained at 0900 and 2100 h for Northern analysis and at 1200 and 2100 h for the measurement of D2 activity. The aorta media D2 mRNA of hypothyroid rats was not significantly different when compared with those of euthyroid rats at both 0900 and 2100 h (Fig. 7AGo). On the other hand, the D2 activity of hypothyroid rats was approximately 2-fold higher compared with those of euthyroid rats at both 1200 and 2100 h (Fig. 7BGo).



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  		FIG. 7. Comparisons of the aorta media D2 mRNA and activity of the euthyroid and hypothyroid rats. A, Four aorta mediae were obtained from euthyroid (white bars) and hypothyroid (black bars) rats at 0900 and 2100 h. The OD of the D2 mRNA was corrected for GAPDH mRNA, and the results were expressed as a percentage of the value obtained for euthyroid rats at 2100 h. The results of D2 mRNA shown represent the mean ± SD of three different experiments. B, Four aorta mediae were obtained from euthyroid (white bars) and hypothyroid (black bars) rats at 1200 and 2100 h. The D2 activity shown represents the mean ± SD of four animals. *, P < 0.05 compared with euthyroid rats.

 
Daily variations of serum T4 and T3 concentrations
In rat, it has been reported that there are circadian variations in the serum T4 and T3 concentrations (20). These results suggest that the circadian rhythmicity of serum T4 and T3 concentrations may induce the changes of the D2 expression of aorta media. Therefore, we measured the serum T4 and T3 concentrations during the day to confirm the circadian rhythmicity of them. As shown in Table 2Go, there were circadian variations in the serum T4 and T3 concentrations. The peak serum T4 and T3 concentrations at 1600 h were approximately 138% (T4) and 137% (T3) of those values at 2400 h, respectively. When we investigated the effects of the adrenergic compounds on D2 expression in the present study, we investigated the effects of the adrenergic compounds on serum T4 and T3 concentrations. Serum T4 and T3 concentrations were not significantly changed by the administration of the adrenergic compounds in the present study (data not shown).


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  		TABLE 2. Daily variations of serum T4 and T3 concentrations

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results have clearly demonstrated that T4 deiodinating activity is present in rat aorta media. The activity was not inhibited by 1 mM PTU and had low Km for T4. Therefore, the T4 deiodinating activity has characteristics compatible with D2. Northern analysis using rat D2 cRNA probe demonstrated a hybridization signal approximately 7.5 kb in size in rat aorta media. The size of D2 mRNA is the same as that of rat pituitary, brain, and BAT (3). The present results are the first demonstration of the expression of D2 activity and mRNA in rat aorta media. The Km value of rat aorta media is similar to that of human aortic smooth muscle cells, and the Vmax value is approximately 30% of the cells (10, 11). The media is the muscular wall of the artery, which consists of spiraling layers of smooth muscle cells. Therefore, we consider that the source of the D2 of rat aorta media is mostly smooth muscle cells.

Interestingly, we have found that a circadian variation is present in the D2 mRNA and activity of rat aorta media. The aorta media D2 activity at 1200 h was approximately 6-fold higher than that at 2100 h. Northern analysis clearly demonstrated that the aorta media D2 mRNA was highly expressed at between 0300 and 0900 h. It is noteworthy that the increase and the peak in aorta media D2 activity are preceded approximately 3 h by those in the D2 mRNA. The significant rise and fall in the aorta media D2 mRNA followed by the change in the D2 activity suggests that the transcriptional regulation may be a primary determinant of the rhythmic D2 expression in the rat aorta media.

In the present study, both prazosin, an {alpha}1-adrenergic antagonist, and propranolol, a ß-adrenergic antagonist, depressed the rise of aorta media D2 mRNA and activity. Interestingly, a combination of prazosin and propranolol depressed D2 mRNA and activity more strongly. These results suggest that both {alpha}1- and ß-adrenergic mechanisms may be involved, at least partly, in the circadian variation of rat aorta media D2 mRNA and activity. Furthermore, rat aorta media D2 mRNA and activities were increased by phenylephrine, an {alpha}1-adrenergic agonist, and isoproterenol, a ß-adrenergic agonist. The effects of both phenylephrine and isoproterenol on the D2 mRNA and activity were more than those of each alone. These results suggest that {alpha}1- adrenergic agonist and ß-adrenergic agonist may cooperatively work to induce the D2 expression. Phenylephrine has a weak ß-adrenergic effect, and isoproterenol has a weak {alpha}1-adrenergic effect. Therefore, to understand whether {alpha}1-adrenergic effect alone or ß-adrenergic effect alone is able to cause an increase in D2 mRNA and activity, we investigated the effects of phenylephrine in combination with propranolol, and isoproterenol in combination with prazosin. Neither propranolol nor prazosin significantly changed the effect of phenylephrine or isoproterenol on the D2 expression. These results suggest that both {alpha}1-adrenergic stimulation and ß-adrenergic stimulation are able to cause the increase in D2 mRNA and activity. Additional investigations are necessary to understand the mechanism by which {alpha}1- and ß-adrenergic pathways regulate aorta media D2 expression. The involvement of both {alpha}1- and ß-adrenergic pathways in the regulation of D2 expression was reported in brown adipocytes (12, 21). Although the circadian variation in the aorta media D2 activity has an approximately 3-h delay with respect to D2 mRNA expression, this is not observed after treatment with phenylephrine or isoproterenol. We do not have a clear explanation for the delay of the D2 activity in the circadian variation. The translational and posttranslational mechanisms, which are not involved in the induction by adrenergic stimulation, may be involved in the induction of the circadian variation of the D2 activity.

Nyctohemeral variation of D2 activity is observed in rat pineal, harderian gland, and central nervous system (13, 14, 20). It is reported that the rhythmic changes in rat pineal and harderian gland D2 activities are regulated mainly at the pretranslational level by a ß-adrenergic mechanism transmitted through superior cervical ganglia (16, 22). It is also reported that rat pineal D2 mRNA reached a peak 3 h after the onset of darkness (16). In the present study, the aorta media D2 mRNA began to rise approximately 7 h after the beginning of darkness. Thus, it appears unlikely that the same controlling mechanism is involved in the circadian variation of the aorta media D2 mRNA as those of pineal and harderian gland.

It has been reported that urinary excretion of catecholamine in rats is higher in the dark period than in the light period, suggesting the existence of circadian rhythms of catecholamine excretion in the rat (23). The circadian catecholamine rhythm in rats could be partially explained by the enhanced activity of the sympatho-adrenomedullary system, which is associated with a longer period during which rats are awake and their higher locomotor activity in the dark phase (24). In the present study, the significant increment of aorta media D2 mRNA was observed at 0300 h (active period). Therefore, we suggest that the possible circadian variations of serum catecholamine levels may be, at least partly, involved in the circadian variation in the D2 expression, although we do not have real evidence in the present study.

D2 activity is regulated by thyroid hormone status both at the pre- and posttranslational levels (3, 25, 26). T4 suppresses D2 activity mainly at the posttranslational level through acceleration of the degradation rate of D2 protein (27). A ubiquitin-proteasomal-mediated mechanism has been demonstrated to be involved in the posttranslational regulation of D2 activity by substrates (28, 29, 30). In contrast, T3 suppresses D2 activity by decreasing D2 mRNA without affecting its half-life, indicating that this effect is because of suppression of transcription of the D2 gene through thyroid hormone receptors (26). In the present study, aorta media D2 activities of hypothyroid rats at both 1200 and 2100 h were approximately 2-fold higher when compared with those of euthyroid rats. On the other hand, aorta media D2 mRNA of hypothyroid rats at both 0900 and 2100 h were not altered significantly. From these results, we suggest that the decrease of the serum T4 level may increase the D2 activity by decreasing the posttranslational effect of T4. In the present study, we observed that there were circadian variations in the rat serum T4 and T3 concentrations as previously described (20). The serum T4 concentration is near the peak at noon in the present study, indicating that D2 activity is depressed to near the maximum degree by a posttranslational mechanism at noon. However, the aorta media D2 activity is highest at noon in the present study. Therefore, we suggest that the variation of serum T4 concentration may not be the factor to induce the variation of the aorta media D2 activity. Interestingly, the similar circadian variation of aorta media D2 mRNA was observed in the hypothyroid rats as in the euthyroid rats. These results suggest that the change of the thyroid hormone level may not be the factor to induce the circadian variation of aorta media D2 mRNA. It has been reported that D2 mRNA in pituitary, BAT, and cerebral cortex in the hypothyroid rat increased more than 2-fold when compared with the euthyroid rat (3, 25). Therefore, further examinations are necessary to clarify the reason for the absence of the increment of the aorta media D2 mRNA in the hypothyroid state.

T3 decreases systemic vascular resistance by dilating the resistance arterioles of the peripheral circulation (31). The vasodilation is caused by a direct effect of T3 on vascular smooth muscle cells that promotes relaxation (32). It has been reported that the 12-h mean values of systolic and diastolic blood pressure were significantly lower in the light phase than in the dark phase (33). It seems likely that the T3 concentration in the vascular smooth muscle may be higher in the light phase when compared with the dark phase, because the aorta media D2 activity is higher in the light phase. Therefore, we suggest that the lower blood pressure in the light phase may be, at least partly, caused by the higher T3 level in the vascular smooth muscle. Additional investigations using D2 knockout mice are necessary to identify the role of vascular D2 activity.

In conclusion, we have found the presence of D2 mRNA and activity in rat aorta media and have demonstrated a circadian variation of D2 mRNA and activity. The D2 activity of rat aorta media is increased by both {alpha}1- and ß-adrenergic agonists, at least partly, at the pretranslational level. Although the generating signal for the circadian variation of D2 mRNA is currently unknown, we suggest that both {alpha}1- and ß-adrenergic mechanisms may be involved, at least partly, in the circadian variation. In the hypothyroid state, the aorta media D2 activity is increased mainly by the posttranslational mechanism, and the similar circadian variation of the D2 expression is present as in the euthyroid state.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Science Research from the Ministry of Education, Science, and Culture of Japan and the Smoking Research Foundation.

This study was presented in part at the 75th Annual Meeting of the American Thyroid Association, September 16–21, 2003.

Abbreviations: BAT, Brown adipose tissue; BW, body weight; D1, type 1 iodothyronine deiodinase; D2, type 2 iodothyronine deiodinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PTU, 6-propyl-2-thiouracil.

Received May 17, 2004.

Accepted for publication August 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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