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Molecular Neuroendocrinology Unit, Rowett Research Institute (J.V.F., P.B., P.J.M.), Bucksburn, Aberdeen, Scotland AB21 9SB, United Kingdom; and the Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health (S.L.C., D.C.K.), Bethesda, Maryland 20892-4480
Address all correspondence and requests for reprints to: Dr. Peter J. Morgan, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, Scotland AB21 9SB, United Kingdom. E-mail: p.morgan{at}rri.sari.ac.uk
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Abstract |
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Introduction |
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Although this function appears to be valid for the enzyme in the pineal gland and retina, the function of the enzyme is not known in those tissues containing low levels of AANAT messenger RNA (mRNA) (1), especially the pituitary gland (1, 2). The issue of AANAT in the pituitary gland is of special interest because this tissue mediates some of the photoperiodic effects of melatonin (6, 7, 8, 9); one region, the pars tuberalis (PT), has one of the highest concentrations of melatonin receptors (10). The pars tuberalis has been shown to secrete a PRL-releasing factor that has been called tuberalin, and it is believed that this factor may contribute to the seasonal changes in plasma PRL expressed in photoperodically sensitive species (7, 8, 9). Thus, melatonin receptors on the PT may indirectly mediate the seasonal effects of melatonin on the output of PRL from the ovine pars distalis (PD) (6, 7, 8, 11).
The paradoxical presence of a key melatonin-synthesizing enzyme in a melatonin target site stimulated us to extend previous observations that the AANAT gene is expressed in the pituitary. We confirmed this and have gone on to establish that AANAT mRNA and enzyme activity occurs in the PT at levels that are severalfold higher than those in the PD. However, we found no evidence to indicate that the pituitary can synthesize melatonin, suggesting a totally distinct function for AANAT within this tissue. We also found marked differences in the regulation of AANAT mRNA by cAMP between the pineal and pituitary glands. These findings lead us to suspect that AANAT plays a role in the pituitary gland that is not directly linked to melatonin synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR and cloning of PCR products
Total RNA was extracted from pineal, PT, and PD tissue using
Trizol solution (Sigma Chemical Co., Poole, UK) and
treated for 2 h with 10 U RQ1-deoxyribonuclease (Promega Corp., Southampton, UK). Total RNA (1 µg) was reverse
transcribed using 500 ng oligo(deoxythymidine) primer (Promega Corp.) and 200 U Superscript reverse transcriptase (Life
Sciences BRL, Paisley, UK). Negative control reactions were performed
without reverse transcriptase to demonstrate the absence of
contaminating genomic DNA. PCR was performed using Pfu DNA polymerase
(Stratagene, Cambridge, UK) over 40 cycles on a
thermocycler (30-sec denaturation at 92 C, 40-sec annealing at 60 C,
1-min 45-sec elongation at 74 C). PCR primers for ovine AANAT
complementary DNA (cDNA) amplification were designed against regions
7–26 and 410–430 of the published sequence (GenBank accession no.
U29663; sense, 5'-ACAGCTCCCGGAGTGGTGG-3'; antisense,
5'-CCGATGATGAAGGCCACGAG-3'). Primers for
hydroxyindole-O-methyltransferase (HIOMT) cDNA amplification
were designed against regions 369–392 and 853–873 of the bovine HIOMT
cDNA sequence (GenBank accession no. M81862; sense,
5'-CAGGGAAGGGAGGAACCAGTATCT-3'; antisense,
5'-GCCCCGCCCGTCCGTGTCCAG-3'). Glyceraldehyde-3-phosphate dehydrogenase
(G3PDH) cDNA amplification primers were designed against bp 10–34 and
614–638 of the human sequence (GenBank accession no. X01677; sense,
5'-TGAAGGTCGGAGTGAACGGATTTG-3'; antisense,
5'-GCGCCAGTAGAAGTAGGGATGATG-3'). Primers for ovine inducible cAMP early
repressor (ICER) cDNA amplification were designed against bp 16–36 and
132–149 of the mouse ICER sequence (GenBank accession no. S66024;
sense, 5'-ATGGCTGTAACTGGAGATGAA-3'; antisense,
5'-GTTGCTGGGGACTGTGC-3'). The HIOMT and G3PDH PCR products were
amplified from ovine pineal cDNA, and the AANAT and ICER PCR products
were amplified from ovine PT cDNA. PCR products were blunt end cloned
into the SrfI site of the pCRscript vector as advised by the
manufacturer (Stratagene). The sequences of the cloned
inserts were verified by sequencing in both directions using the
AmpliTaq sequencing kit (ABI, Warrington, UK). PCR primers were
designed, and sequence comparisons were performed using the Lasergene
computer package (DNAstar, Inc., Madison, WI).
Assay of melatonin production by cultured cells
Six-well plates were seeded with primary pineal cells
(106), primary PT cells (3 x 106), or
primary PD cells (3 x 106) cells in DMEM-12% lamb
serum and stimulated with 10 µM forskolin. Six hours
later the cell suspensions were collected by centrifugation at
1500 x g for 10 min at 4 C, and the supernatant was
recovered. Melatonin in the supernatant was assayed using a
modification of the RIA of a published method (13). Briefly, a rabbit
antimelatonin antibody (Stockgrand Ltd., Surrey, UK) was diluted
1:30,000 in assay buffer (100 mM Tricine, 154
mM NaCl, and 0.2% gelatin, pH 8.0) and incubated with 500
µl sample supernatant and 3.6 fmol [125I]melatonin
(2200 Ci/mmol; Amersham) overnight at 4 C. The next day 100 µl of
1:100 diluted antirabbit IgG (SAPU, Paisley, Scotland), 400 µl 0.2%
-globulin, and 1 ml 24% polyethylene glycol
(Mr, 8000) were added, and precipitated proteins
were pelleted at 3000 rpm for 30 min at 4 C. The pellets were washed
once with 2 ml 0.2% -globulin-24% polyethylene glycol (1:1) and
repelleted at 3000 rpm for 30 min at 4 C. Samples were counted using a
Packard -counter (Downers Grove, IL) and compared with a standard
curve.
Assay of N-acetyltransferase activity
AANAT activity was determined using a modification of the method
of Namboodiri et al. (14). Freshly frozen pineal, PT, and PD
tissues (collected during the day) were minced and homogenized (
100
mg/ml) in 10 mM ammonium acetate buffer, pH 6.5. To 25 µl
diluted homogenate were added 25 µl substrate mix (40 mM
tryptamine-HCl, 1 mM [1-14C]acetyl coenyzme A
(Amersham, Aylesbury, UK; 8.4 µCi/µmol), 0.1 M
Na2HPO4/NaH2PO4, pH
6.8). After 30-min incubation at 37 C, the reaction was stopped by the
addition of 1 ml water-saturated chloroform, which partitioned
14C-labeled N-acetyltryptamine into the organic
layer. The chloroform extract was washed (three times) with 0.1
M phosphate buffer, pH 6.8, and 500-µl samples were
transferred to a scintillation vial and evaporated at 40 C.
Scintillation fluid was added, and the samples were counted. All
samples were normalized to total protein content, and the protein
concentration was determined using the method of Bradford (15).
RNA analysis
Trizol (Sigma Chemical Co., Poole, UK) and RNeasy
kits (Qiagen, Dorking, UK) were used to extract total RNA from tissues
and primary cells, respectively. Total RNA was fractionated on 1%
agarose denaturing formaldehyde gels, and the RNA was blotted to Nylon
membranes (Boehringer Mannheim, Lewes, UK) using a pressure blotting
station (Stratagene). DNA probes for Northern blot
analysis were labeled with [
-32P]deoxy-CTP (3000
mCi/mmol; Amersham) using a random primer labeling kit (Megaprime,
Amersham). The templates used in this study corresponded to the cloned
PCR products described above (see RT-PCR and cloning of PCR products)
except in the case of G3PDH probe labeling, where a commercially
available human G3PDH probe was used (Clontech, Cambridge,
UK). Hybridizations were performed at 65 C using Quickhyb solution
(Stratagene) following the manufacturer’s instructions
and washed to high stringency (0.1 x SSC-0.1% SDS at 55 C).
After exposure to BioMax or X-Omat LS (Kodak, Hemel Hempstead, UK)
autoradiographic film, blots were stripped with 0.1% SDS at 95 C for 5
min before rehybridization with other probes.
Ribonuclease (RNase) protection assays were performed to detect ICER
mRNA using a RNase protection assay II kit as described by Barrett
et al. (16). A radiolabeled ovine ICER antisense riboprobe
was generated from the plasmid described above, digested with
BamHI (see RT-PCR and cloning of PCR products) using T3 RNA
polymerase (Promega Corp., Southampton, UK), and
[-32P] UTP (8000 Ci/mmol; Amersham). The
specific activity of the riboprobe was generally in the range of
2–3 x 105 cpm/ng. Typically, 8 µg total RNA were
mixed with 2 fmol ICER antisense riboprobe and incubated overnight at
47 C. On the next day the RNA/probe mixture was digested for 2 h
at 37 C with a 1:25 dilution of RNase T1. After ethanol precipitation
the samples were denatured, and protected fragments were visualized by
electrophoresis on 8% polyacrylamide gels and autoradiography. The
ICER riboprobe used in this study is specific for the sheep ICER
sequence (see RT-PCR and cloning of PCR products) and is therefore
expected to protect a 142-base region of sheep ICER mRNA.
In situ hybridization
In situ hybridization (10-µm sections) was
performed as described by Morgan et al. (7). Generation of
radiolabeled ([35S] UTP; 1000 Ci/mmol; DuPont-New
England Nuclear, Stevenage, UK) Mel1a receptor riboprobes were
generated as described by Drew et al. (17). Digoxigenin
(DIG)-labeled Mel1a receptor riboprobes were generated from the
same template, except DIG-1-UTP (Boehringer Mannheim, Indianapolis, IN)
was substituted for the radiolabeled nucleotide (7). The plasmid
templates used to generate radiolabeled AANAT riboprobes were
previously described by Coon et al. (2). Sense riboprobes
were generated using T3 RNA polymerase and XcmI-digested
plasmids, and antisense riboprobes were made using T7 RNA polymerase
and EcoRI-digested plasmids. Before hybridization sections
were fixed in 4% paraformaldehyde and acetylated with 26
mM acetic anhydride in 0.1 M
triethylamine. After washing in PBS and ethanol dehydration,
slides were mixed with the hybridization cocktail (50% formamide, 10%
dextran sulfate, 0.3 M NaCl, 1 x Denhardt’s
solution, 0.01 M Tris, 1 mM EDTA. 0.01
M dithiothreitol, 0.5 mg/ml transfer RNA, and 0.3
µg/ml riboprobe) and incubated overnight at 57 C. After
hybridization, the sections were treated with RNase A [0.02 mg/ml in
0.5 M NaCl, 0.01 M Tris (pH 8), and 1
mM EDTA] at 37 C for 30 min and washed to high stringency
(0.1 x SSC-1 mM dithiothreitol, 60 C). The sections
were dehydrated in ethanol and dried under vacuum before exposure to
Hyperfilm ß-max film (Amersham) for 2–6 weeks. The protocol for
analyzing sections hybridized with both radiolabeled and DIG-labeled
probes has been described previously (7).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); expression in the ovine PD was also
detected, but at substantially lower levels (1/20th of PT). The
abundance of PT AANAT mRNA was 1/15th lower than that in the pineal
gland (Fig. 1a). The transcripts detected in the pituitary and pineal
glands (1.4 kb) were the same size. In addition, the sequence of a
440-bp PCR product obtained using PT cDNA and AANAT primers (Fig. 1b)
was identical to the corresponding region of authentic ovine AANAT.
In situ hybridization confirmed that the PT contained a
higher abundance of AANAT mRNA than the PD (Fig. 2a). Together, these observations confirm
that AANAT is expressed in the pituitary gland and establish that
expression is higher in the PT.
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). Where single cells could be
identified, silver grains from the AANAT probe could seen overlying
cells that expressed Mel1a mRNA, visualized by DIG labeling (see Fig. 2b). These data indicate that the AANAT gene appears to be expressed in
the majority of Mel1a mRNA-expressing PT cells (Fig. 2b).
To determine whether the differential levels of AANAT mRNA were
translated into differential levels of AANAT activity, PT and PD were
assayed (Fig. 3). AANAT activity in the
pituitary was about 1/10th (PT) to 1/20th (PD) that in the pineal
gland. The differential levels of AANAT activity between the PT and
pineal are similar in magnitude (1/10th) to the comparative levels of
AANAT mRNA between the two glands (1/15th). By contrast, the relative
levels of AANAT activity between the PD and pineal (1/20th) show less
correspondence to the relative levels of AANAT mRNA expression between
the two glands (1/300th).
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First, secretion of melatonin by primary cultures of ovine PT and PD
cells was examined (Fig. 4). Melatonin
was undetectable in PT or PD culture medium from control cultures or
cultures treated with forskolin. In the same series of experiments,
melatonin was detected in medium from pinealocyte cultures (Fig. 4).
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), although HIOMT and TOH
transcripts were detected in extracts of the pineal gland. RT-PCR
detected very weak expression of ovine HIOMT in PT mRNA, but not in PD
(Fig. 5a); this method generated a single 600-bp product from pineal
mRNA (Fig. 5a) that was 95% identical to the corresponding region of
bovine HIOMT. These data indicate that the pituitary gland does not
appear to have the capacity of convert tryptophan to detectable amounts
of melatonin.
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). In contrast, in PT, forskolin (10
µM; 4, 8, and 12 h) treatment suppressed AANAT mRNA
levels (Figs. 7 and 8a).
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), consistent with the conclusion that these effects of forskolin are
mediated by cAMP.
AANAT mRNA levels in the rat exhibit a marked day/night rhythm, which
is thought to be regulated by cAMP acting via cAMP response element
(CRE)-binding proteins that interact with a CRE in the AANAT promoter.
In this system, the initial effects of cAMP are to stimulate the
transcription of the AANAT gene. As cAMP also induces the expression of
ICER (20), it is thought that a secondary effect is to modulate the
amplitude and duration of AANAT gene expression. The proposal that ICER
acts as an inducible repressor of cAMP-dependent gene expression is
based on several lines of evidence, including the demonstration that
ICER mRNA is elevated by treatments that elevate cAMP and that ICER
suppresses cAMP-dependent gene expression (21). Here we examined
whether elevation of cAMP with forskolin increases ICER mRNA in ovine
pituitary and pineal tissues. Forskolin treatment increased ICER mRNA
levels in the ovine PT in a time-dependent manner and remained elevated
up to at least 8 h (Fig. 8b). Melatonin (10 nM)
suppressed the increase in ICER at 8 h (Fig. 8b). These
observations are consistent with the hypothesis that cAMP suppresses
expression of PT AANAT mRNA by increasing ICER protein. Forskolin
treatment also increased ICER mRNA levels in the pineal gland (Fig. 6).
However, as indicated above, forskolin does not alter pinealocyte AANAT
mRNA levels in this tissue, providing evidence that the effects of cAMP
on AANAT mRNA levels in these tissues differs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The best-established function of AANAT is to participate in the synthesis of melatonin. However, this does not appear to be the role of the enzyme in the pituitary gland, because it was not possible to detect synthesis of melatonin or to detect significant levels of mRNA encoding the first and last enzymes in melatonin synthesis, TOH and HIOMT. Accordingly, it would appear that AANAT plays another role. One possibility is that it serves to acetylate serotonin or another potential arylalkylamine substrate, including tyramine, phenylethylamine, tryptamine, and methoxytryptamine.
The discovery that AANAT is coexpressed with Mel1a in PT cells is not the first indication that both the AANAT and Mel1a receptor genes are expressed in the same cell. AANAT, the Mel1a receptor, and a melatonin-related receptor are coexpressed in the ovine retina (17). The expression of both genes in the same tissue or cell leads to the speculation that AANAT might act locally. One hypothetical relationship might involve the melatonin-related receptor, an orphan receptor with close structural similarity to the melatonin receptor (17), which may bind a melatonin-related indole. A second hypothesis involves N-acetylserotonin, which has been shown to act as a partial agonist of the melatonin receptor (22) and therefore could modulate melatonin signal transduction within the pituitary.
This study revealed marked differences in the regulation of AANAT mRNA expression between the sheep pineal gland and PT; differences from the rat pineal gland are also evident. In contrast to the rat pineal gland (1), the levels of AANAT mRNA in the sheep pineal gland do not appear to be regulated through cAMP. In view of this, the robust and unequivocal repression of AANAT mRNA levels in ovine PT in response to forskolin represents a distinct tissue-specific difference in the role of cAMP in the regulation of AANAT expression between the ovine PT and pineal gland.
One possible explanation of the differential influences of cAMP on AANAT expression in the pituitary and pineal glands is that cAMP induces ICER in one but not the other. However, this was found not to be correct, because forskolin elevated ICER in both tissues. The dissociation of the ICER and AANAT mRNA responses in the sheep pineal gland is consistent with the conclusion that cAMP and ICER are relatively unimportant to the regulation of the ovine pineal AANAT mRNA expression. Forskolin increases ICER protein in the PT (Fleming, J. V., J. H. Stehle, and P. J. Morgan, unpublished observations), and so it is not unreasonable to suspect that ICER could play a role in the repression of AANAT in the PT. In contrast, we found that in the pineal gland forskolin can elevate ICER mRNA without associated changes in AANAT mRNA levels. This raises a question about the role of CREs in the regulation of the expression of the ICER and AANAT genes.
Whereas it is known that CREs play an important role in the regulation of expression of the rat AANAT gene (23), it is not known whether such sites are also present in the sheep AANAT promoter or whether other sites participate in the regulation of expression of this gene. Differences in regulation between the pineal and pituitary gland might reflect differences in the relative concentrations of transcription factors that control expression. It is important to note that the experiments reported here were conducted upon pineal and PT tissue collected during the daytime. Both the pineal and the PT are influenced by the light-dark cycle, and in the pineal gland AANAT activity is highest at night. Thus, similar experiments performed on pineal and pituitary glands collected during the night may reveal a different pattern of regulation.
The ability of melatonin to block forskolin-stimulated repression of AANAT mRNA expression suggests that the expression of AANAT within the PT is likely to be photoperiodically regulated because melatonin exhibits a marked daily rhythm that is altered by changes in day length. This photoperiodic transduction within the PT may be related to the seasonal endocrine role of this tissue in sheep (6, 7, 8, 9).
These studies may be of more general importance in understanding the role of AANAT expressed in extrapineal sites, including the brain. The colocalization of AANAT and the melatonin receptor in the retina and PT raises the possibility that these genes might be commonly coexpressed in others as part of a molecular regulatory module that functions in other areas.
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Acknowledgments |
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Footnotes |
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Received July 7, 1998.
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