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First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan
Address all correspondence and requests for reprints to: Masami Murakami, M.D., First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan. E-mail: mmurakam{at}sb.gunma-u.ac.jp
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Abstract |
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Introduction |
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It has been shown that pineal DII activity significantly increases during the hours of darkness, primarily through ß-adrenergic mechanism (3). The dramatic nocturnal increase in pineal DII activity is comparable to that in pineal arylalkylamine N-acetyltransferase (AANAT) that generates a circadian rhythm of melatonin synthesis derived from activation of the pineal sympathetic innervation (4). Although the mechanisms involved in the nocturnal increase in pineal DII activity remain to be elucidated, transcriptional regulation of the nocturnal increase in pineal AANAT has been described (5, 6, 7, 8).
Recently, a complementary DNA (cDNA) encoding DII has been cloned from Rana catesbeana tissues (9) and subsequently from rat brown fat (10), which has enabled us to study the mechanisms involved in the regulation of DII expression. Northern analysis and RT-PCR analysis suggested that pineal DII messenger RNA (mRNA) at 2400 h is greater than that at 1200 h (11). As the temporal profile of the activation of pineal DII mRNA during the hours of darkness or after ß-adrenergic stimulation has not been described, it is not known whether pineal DII mRNA correlates with DII activity under those conditions.
In the present study, we have evaluated the relationship between DII mRNA and DII activity in pineal gland in various experimental conditions to study the mechanisms involved in the regulation of pineal DII expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]UTP and
[125I]T4 were purchased from New England
Nuclear Corp. (Boston, MA). AG 50W-X2 resin and protein assay kit were
obtained from Bio-Rad Laboratories, Inc. (Hercules, CA).
Fitton-Jackson modification of BGJb medium was purchased from
Life Technologies (Grand Island, NY). T7 RNA polymerase
was obtained from Boehringer Mannheim GmbH (Mannheim, Germany). All
other chemicals at the highest quality were obtained from Sigma Chemical Co. (St. Louis, MO) or Wako Pure Chemical Industries Ltd. (Osaka, Japan) unless otherwise indicated.
Animals and experimental procedures
Two-month-old male Wistar rats were maintained two per cage on a
12-h light, 12-h dark schedule (lights on, 0600–1800 h) at 25 ±
1 C and fed standard laboratory chow and tap water ad
libitum. The rats were acclimated to this condition for at least 1
week before the experiment. For injection experiments, all of the drugs
were dissolved in saline, and control groups received the same volume
of saline vehicle. To determine the effect of superior cervical
ganglionectomy (SCGx), bilateral SCGx was performed as previously
described (12), and rats were killed 1 week postoperatively. Dim red
light was used when the experiments were performed during the hours of
darkness. Rats were killed by decapitation, and the pineal glands were
removed, immediately frozen and stored at -70 C until RNA isolation or
measurement of DII activity. All experiments were performed in
accordance with the guidelines of the Gunma University animal care and
use committee.
Organ culture of pineal gland
Rat pineal gland was cultured by the method of Klein and Weller
(13) with minor modifications (14). In brief, rats were killed by
decapitation between 1100–1300 h, and pineal glands were removed
quickly and chilled in culture medium on ice. Two or three pineal
glands were placed on a nylon mesh in a 24-well culture plate
containing 300 µl culture medium. The culture medium consisted of a
Fitton-Jackson modification of BGJb medium supplemented with 0.05 mg/ml
ascorbic acid, 100 µg/ml streptomycin, 100 U/ml penicillin, and 0.1%
BSA. After 1 h of preincubation, the pineals were transferred to
medium containing the compounds for the periods indicated.
Preincubation and incubation were performed at 37 C under 95%
O2 and 5% CO2. After the incubation period,
pineal glands were immediately frozen and stored at -70 C until RNA
isolation or measurement of DII activity.
RNA preparation and Northern analysis
Total RNA was isolated from five pooled pineal glands using the
modified acid guanidinium thiocyanate-phenol-chloroform method
according to Chomczynski and Sacchi (15). Northern analysis was
performed as previously described (11, 16). Plasmid rDII
5–1/pBluescript SK, which contains rat DII cDNA, was provided by Dr.
St. Germain (10). Briefly, rat DII complementary RNA (cRNA) probe was
synthesized by in vitro transcription of linearized rDII
5–1/pBluescript SK using T7 RNA polymerase and
[-32P]UTP. Twenty micrograms of total RNA/lane were
electrophoresed on a 1.4% agarose gel containing 2 M
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 (Stratagene, La Jolla, CA). The membrane was prehybridized
with the hybridization buffer (50% formamide, 0.2% SDS, 5% dextran
sulfate, 50 mM HEPES, 5 x SSC, 5 x Denhart’s
solution, and 250 mg/ml denatured salmon sperm DNA) at 68 C for 2
h. 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 min and twice in
0.1 x SSC-0.1% SDS at 68 C for 1 h. Autoradiography was
established by exposing the filters for 6–24 h 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 were
rehybridized with a control ß-actin cRNA probe, which was synthesized
in vitro using T7 RNA polymerase and
[-32P]UTP. Hybridization and washing were performed as
described above, and the membrane was exposed for 2–6 h. The mRNA
level was quantitated by densitometry using NIH Image Version 1.61, and
the optical density of the DII band was corrected for ß-actin.
Measurement of DII activity
Pineal DII activity was measured as previously described (17)
with minor modifications (12). Briefly, each pineal gland was
homogenized by sonication in 100 µl homogenizing buffer (100
mM potassium phosphate, pH 7.0, containing 1 mM
EDTA and 20 mM dithiothreitol). Homogenates were incubated
in a total volume of 50 µl containing 2 nM
[125I]T4, which was purified using LH-20
(Pharmacia Biotech, Uppsala, Sweden) column chromatography
on the day of experiment, 1 mM EDTA, 20 mM
dithiothreitol, and 1 mM 6-propyl-2-thiouracil, pH 7.0, for
1 h at 37 C. The reaction was terminated by the addition of 100
µl 2% BSA and 800 µl 10% trichloroacetic acid. After
centrifugation at 3000 rpm for 10 min, the supernatant was applied to a
small column packed with AG 50W-X2 resin (bed volume, 1 ml) and eluted
with 2 ml 10% glacial acetic acid. Separated 125I was
counted with a 
-counter. Nonenzymatic deiodination was corrected by
subtracting the I- released in tissue-free tubes. The
protein concentration was determined by Bradford’s method using BSA as
a standard (18). Deiodinating activity was linear within the range of
the protein concentration used and was expressed as femtomoles of
I- released per mg protein/h after multiplication by a
factor of 2 to correct random labeling at the equivalent 3'- and
5'-positions.
Statistics
Statistical differences were evaluated by Newman-Keuls test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. As shown in Fig. 1b, DII mRNA, which
was corrected for ß-actin (closed circles), increased
during the hours of darkness (1800–0600 h) and reached a peak at
2100 h. Although DII activity (open circles) also
increased during the hours of darkness, the activity reached the peak
level at 2400 h. These results suggest that the nocturnal increase
in DII activity is preceded by an increase in DII mRNA in the rat
pineal gland.
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. As shown in Fig. 2b, pineal DII mRNA
(closed circles) increased as early as 1 h after the
isoproterenol injection and reached a peak level by 2 h after the
treatment. Pineal DII activity (open circles) significantly
increased by 2 h after the isoproterenol injection and reached a
peak by 3 h after the treatment. These results suggest that pineal
DII mRNA is rapidly increased by isoproterenol administration and that
the isoproterenol-stimulated increase in DII activity is preceded by
the increase in DII mRNA in the rat pineal gland.
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, a and b. This treatment also significantly decreased the nocturnal rise
in pineal DII activity at 2400 h, as shown in Fig. 3c. These
results indicate that DII mRNA and DII activity are spontaneously
stimulated through a ß-adrenergic mechanism during the hours of
darkness in the rat pineal gland.
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, a and b,
bilateral SCGx abolished the nocturnal rise in pineal DII mRNA at
2100 h. Bilateral SCGx also abolished the nocturnal increase in
pineal DII activity at 2400 h, as shown in Fig. 4c. These results
indicate that the nocturnal rise in DII mRNA and that in DII activity
are regulated by the sympathetic innervation transmitted through SCG in
the rat pineal gland.
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, a and b, constant light
exposure abolished the nocturnal rise in DII mRNA at 2100 h.
Constant light exposure also abolished the nocturnal increase in DII
activity at 2400 h, as shown in Fig. 5c. These results suggest
that the nyctohemeral rhythm in DII mRNA and that in DII activity could
be entrained by environmental lighting conditions in the rat pineal
gland.
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, a and b, 1 µM
isoproterenol or 1 mM (Bu)2cAMP significantly
stimulated DII mRNA in cultured pineal glands. Isoproterenol or
(Bu)2cAMP also stimulated DII activity in cultured pineal
gland, as shown in Fig. 6c. Although treatment with 25 µM
cycloheximide markedly decreased isoproterenol-stimulated DII activity
in cultured pineal glands, as shown in Fig. 6c, treatment with
cycloheximide for 6 h markedly increased isoproterenol-stimulated
DII mRNA in cultured pineal glands, as shown in Fig. 6, a and b. These
results suggest that isoproterenol stimulation of DII mRNA does not
require protein synthesis, and inhibition of protein synthesis enhanced
the increase in DII mRNA after 6 h compared with treatment with
isoproterenol alone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, Northern analysis clearly demonstrated that pineal DII mRNA markedly increased during the hours of darkness. It is noteworthy that the increase and the peak in DII activity are preceded by the increase and the peak in DII mRNA during the hours of darkness in pineal gland. The significant rise and fall in pineal DII mRNA followed by the change in DII activity suggest that transcriptional regulation may be a primary determinant of rhythmic DII expression in the rat pineal gland, as suggested for the nocturnal increase in pineal AANAT (5, 6).
We have also investigated whether pineal DII mRNA is increased by isoproterenol administration, as it has been shown that DII activity is significantly stimulated by a ß-adrenergic agonist in the rat pineal gland (20). The present data have demonstrated that DII mRNA significantly increases within 1 h after isoproterenol injection, and the increase in DII activity is preceded by the increase in DII mRNA in the rat pineal gland. The rapid induction of DII mRNA by isoproterenol administration, presumably through the activation of adenylate cyclase (14), is in agreement with the recent observation that DII mRNA in cultured rat astrocytes is significantly increased within 1 h by forskolin or 8-bromo-cAMP stimulation (23). These results suggest that the expression of DII is stimulated by a ß-adrenergic mechanism at the pretranslational level in the rat pineal gland.
We have further evaluated the possible relationship between the nocturnal increase in pineal DII mRNA and that in DII activity under different experimental conditions. We have studied the effect of a ß-adrenergic antagonist, bilateral SCGx, or constant light exposure, all of which have been known to suppress the nocturnal rise in pineal DII activity (12, 20, 21). Propranolol treatment significantly decreased the nocturnal rise in DII mRNA as well as that in DII activity, indicating that pineal DII expression is spontaneously stimulated through a ß-adrenergic mechanism at the pretranslational level during the hours of darkness. Bilateral SCGx abolished the nocturnal rise in pineal DII mRNA as well as DII activity, suggesting that pineal DII expression is regulated at the pretranslational level by sympathetic innervation transmitted through SCG. Constant light exposure abolished the nocturnal rise in DII mRNA as well as that in DII activity, suggesting that the circadian rhythm of DII expression could be entrained by environmental lighting conditions (4) at the pretranslational level.
Isoproterenol or (Bu)2cAMP treatment significantly stimulated DII mRNA and DII activity in cultured pineal glands, further indicating that pineal DII expression is regulated at the pretranslational level through a ß-adrenergic mechanism coupled with a cAMP regulatory cascade. Although treatment with cycloheximide markedly decreased isoproterenol-stimulated DII activity in cultured pineal glands, treatment with cycloheximide for 6 h markedly increased isoproterenol-stimulated DII mRNA in cultured pineal glands. These results suggest that isoproterenol stimulation of pineal DII mRNA does not require protein synthesis, and inhibition of protein synthesis enhanced the increase in pineal DII mRNA after 6 h compared with the effect of treatment with isoproterenol alone, which is consistent with the proposal that ß-adrenergic stimulation induces the synthesis of one or more inhibitory transcription factors in the rat pineal gland (24). To clarify the mechanism involved in the cAMP stimulation of pineal DII mRNA, it is necessary to analyze a possible cAMP response element in the promoter region of the rat DII gene.
It has been reported that the nocturnal increase in pineal AANAT activity is preceded by an increase in AANAT mRNA (24), which is in agreement with the present findings for pineal DII expression. Although the size of the pineal NAT transcript has been reported to decrease during the hours of darkness or after adrenergic stimulation, possibly due to the decrease in polyadenylate tail length (24), the size of the pineal DII transcript did not significantly change during the hours of darkness or after adrenergic stimulation in the present study. Different mechanisms, therefore, might be involved in the regulation of DII expression and that of AANAT expression in the rat pineal gland.
Nucleotide sequence analysis of cloned DII cDNA revealed that DII contains unique in-frame TGA codons that code for selenocysteine (10), which was demonstrated in rat DI (25) and type III iodothyronine deiodinase (DIII) (26), which functions as an inner ring deiodinase to convert T4 and T3 to inactive metabolites (rT3 and 3,3'-diiodothyronine, respectively). A stem-loop selenocysteine insertion sequence (SECIS element) is required for the read-through and translation of the TGA codons into selenocysteine (27). However, the reported rat DII cDNA lacks the classical SECIS element and is able to express deiodinase activity only when fused to the SECIS-containing 3'-untranslated region of the rat DIII cDNA (10). Although the SECIS element has not been described for 3'-untranslated region of rat DII, significant correlations between the results of Northern analysis and DII activity in various experimental conditions in the present study strongly suggest that the 7.5-kb transcript hybridized with the reported rat DII cRNA probe is the major mRNA for DII in the rat pineal gland.
A significant nocturnal increase in pineal DII expression through a ß-adrenergic mechanism, which is abolished by bilateral SCGx or constant light exposure, may provide a useful model to investigate the regulatory mechanism of DII expression. Although thyroid hormones have been suggested to be involved in physiological regulation of pineal function (28, 29), further studies are required to elucidate the physiological significance of the nyctohemeral rhythm of pineal T3 production, which is generated by rhythmic DII expression.
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Acknowledgments |
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Footnotes |
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Received April 6, 1998.
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,
0 h control; •, 1 µM Iso; 
, 1 µM
Iso and 25 µM CHX; 
, 1 mM dbcAMP; 
,
medium only; 
, 25 µM CHX. *, P <
0.05; **, P < 0.01.