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-Amidating Monooxygenase Expression in Anterior Pituitary Gland
INSERM U297, Institut Federatif de Recherche Jean Roche, Faculté de Médecine Nord, 13916 Marseille, Cedex 20, France
Address all correspondence and requests for reprints to: Dr. L’Houcine Ouafik, INSERM U297, IFR Jean Roche, Faculté de Médecine Nord, Bd Pierre Dramard, 13916 Marseille Cedex 20, France.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-amidating
monooxygenase (PAM). This bifunctional protein contains peptidylglycine
-hydroxylating monooxygenase (PHM) and peptidyl--hydroxyglycine
-amidating lyase catalytic domains necessary for the two-step
formation of -amidated peptides from their COOH-terminal glycine
extended precursors. Expression of PAM was evaluated in the anterior
pituitary of intact cycling adult female rat and after experimental
manipulation of estrogen status. PAM messenger RNA (mRNA) levels showed
changes inversely related to the physiological variations of plasma
estrogen levels during the estrous cycle. Chronic treatment of
ovariectomized (OVX) rats with 17 ß-estradiol decreased PAM mRNA
levels to values comparable with those found in intact rats at
proestrus. In situ hybridization of anterior pituitary
sections using 35S-labeled full length RNA antisense
transcripts of rat PAM-1 complementary DNA showed that 17 ß-estradiol
treatment induced an overall decrease of the hybridization signal, as
compared with OVX rats. Progesterone treatment did not change PAM mRNA
levels both in OVX or OVX + E2 rats. Based on Northern blot
analysis and amplification of fragments derived from rat PAM-1 by
RT-PCR, it was found that estrogen status does not affect the
distribution of PAM mRNA among its various alternatively spliced forms.
In OVX 17 ß-estradiol treated rats, the specific activity of PAM in
the anterior pituitary decreased in both soluble and particulate
fractions compared with OVX animals. Western blot analysis demonstrated
a 105-kDa PAM protein in particulate fractions prepared from OVX and
OVX-17 ß-estradiol treated animals. The soluble fraction from OVX
animals contained major PAM proteins of 105, 95, 84, 75, and 45 kDa,
and 17 ß-estradiol treatment caused a decrease in the prevalence of
these proteins. These results indicate that estrogens are involved,
either directly or indirectly, in regulating the expression of PAM in
several cell types in the anterior pituitary gland. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amidated product (1, 2). Peptide -amidation is a
two-step reaction catalyzed by the bifunctional enzyme, peptidylglycine
-amidating monooxygenase (PAM; EC 1.14.17.3). The PAM precursor
protein encodes both of the enzymatic activities involved in peptide
amidation (3, 4). The first enzyme, peptidylglycine -hydroxylating
monooxygenase (PHM) produces an -hydroxylated intermediate in the
presence of copper, ascorbate, and molecular oxygen. At physiological
pH, the second enzyme, peptidyl--hydroxyglycine -amidating lyase
(PAL), catalyzes the conversion of peptidyl--hydroxyglycine
intermediates into -amidated peptide and glyoxylate (5, 6). The two
catalytic domains of the bifunctional PAM protein can be separated by
endoproteolysis and can act independently (7). Soluble and
membrane-associated PAM activities have been identified, and their
distribution is tissue specific (8). Complementary DNA (cDNA) clones
encoding 110-kDa bifunctional integral membrane protein form of PAM
have been isolated from many tissues in several species (2).
Tissue-specific alternative splicing of the single copy PAM gene can
generate at least seven forms of PAM messenger RNA (mRNA) in the rat
(2, 9).
Although the major anterior pituitary hormones such ACTH, TSH, FSH, LH,
PRL, and GH are not -amidated, PAM levels in the anterior pituitary
gland are among the highest in rat tissues (10, 11). Several
-amidated peptides, including substance P (SP), neuropeptide Y
(NPY), vasoactive intestinal peptide (VIP), galanin, neurotensin (NT),
joining peptide, GnRH, TRH, and pyroglutamyl-glutamyl-proline amide
have been identified in the rat anterior pituitary gland (12, 13, 14, 15, 16, 17, 18).
Evidence for PAM expression in the rat anterior pituitary gland was provided by immunocytochemistry and in situ hybridization (19). The data demonstrated that the anterior pituitary cells intensely immunostained for PAM represented a subpopulation of the gonadotrophs. Surgical ovariectomy has been shown to produce a significant rise in the pituitary content of SP immunoreactivity (IR), NPY-IR, and NT-IR, whereas VIP-IR showed a fall. These changes were reversed with estrogen replacement or high dose estrogen treatment (20). These data prompted us to investigate the effect of estrogens on PAM expression in the anterior pituitary gland.
Although the effects of estrogen status on many cellular metabolic processes have been well documented (21, 22), little is known about their effects on peptide-processing enzymes. In the present study, we have used several techniques to examine the effects of estrogen status on PAM expression in the anterior pituitary gland. Levels of PAM mRNA were evaluated using electrophoretic blot hybridization analysis, and changes in PAM mRNA forms were investigated using RT-PCR. Tissue levels of PAM activity were measured, and PAM protein forms were examined by Western blot analysis. In situ hybridization studies were conducted to determine whether changes in estrogen status altered PAM mRNA levels in all or only a fraction of the total cell population of the anterior pituitary gland. Our results strongly suggest that estrogen status is involved in the regulation of the PAM expression in this tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For studies on hormone replacement, rats were ovariectomized (OVX), and rested for 1 week. Thereafter, several groups of OVX rats received daily sc (sc) injections of 17 ß-estradiol (4, 20, 50, 100 or 1000 µg diluted in 100 µl of sesame oil; n = 3 for each group) (Merck, Germany) for 1 week. The experiment was repeated three times. To study the effect of progesterone on PAM expression, OVX animals received for 1 week daily sc injections of progesterone (P, 1 mg/rat) (Sigma Chemical Co., St. Louis, MO), alone or in combination with 17 ß-estradiol (4 µg/day) or sesame oil (controls).
At the end of each experiment, the animals were killed by decapitation and trunk blood was collected for serum LH and estradiol measurements. Anterior pituitaries were removed rapidly for determination of PAM activity, preparation of total RNA, or in situ hybridization studies.
LH RIA
Plasma levels of LH were measured in duplicate in a single assay
by a double antibody RIA and expressed as ng/ml of NIAMDD rat LH-RP 2.
Antirat LH serum CSU 120 was provided by Dr G. D. Niswender (Colorado
State University). The sensitivity of the assay was 0.25 ng/ml of
plasma. The intra and interassay coefficients of variation were below
10%.
Estradiol RIA
Plasma estradiol levels were measured using a RIA
(125I-Estradiol COATRIA, BioMerieux, Lyon, France). The
cross-reactivity of the antiestradiol antiserum with corticosterone was
less than 0.017%. The limit of sensitivity of the assay was 12 pg/ml
of plasma. The intra and interassay coefficients of variation were 6%
and 7%, respectively.
RNA isolation and Northern blot analysis
Total RNA was prepared from individual anterior pituitaries
using the acid guanidinium isothiocyanate-phenol-chloroform procedure
(23). RNA (10 µg) was denatured and then electrophoresed on a 1%
agarose formaldehyde denaturing gels. The denatured RNAs were
transferred to Hybond-N membrane (Amersham Corp, Les Ulis, Paris) by
capillary action in 20 x SSC (3.0 M NaCl, 0.3
M sodium citrate, pH 7.0), cross-linked by UV irradiation
and hybridized to [-32P]-labeled full length PAM cDNA
(3.8 kilobase pairs) (24). Filters were prehybridized, hybridized, and
washed as previously described (25). To correct for the actual amount
of RNA in each lane, blots were stripped and hybridized to cDNA probes
derived from frog ribosomal RNA (25). The autoradiograms were analyzed
by measurement of optical density by scanner-densitometer using NIH
Image 1.54 software (NIH, Bethesda, MD). The results are expressed as
optical density (OD) of PAM mRNA/OD 18S rRNA, with the panels
representing the mean ± SEM for each group of
rats.
Tissue preparation and amidation assays
Anterior pituitary tissue was homogenized in 20 mM
NaTES (N-Tris [hydroxy-methyl] methyl-2-amino-ethane sulfonic acid),
pH 7.4, 10 mM mannitol, containing 2 µg/ml leupeptine, 16
µg/ml benzamidine, and 300 µg/ml phenylmethylsulfonyl fluoride
using a ground glass homogenizer at 4 C. The homogenates were frozen
and thawed three times and separated into soluble and particulate
fractions, as previously described (25, 26). The crude particulate
fractions were solubilized by resuspension in the same buffer
containing 1% Triton X-100; after centrifugation for 60 min at
100,000 x g, the supernatants were used to measure
solubilized membrane associated PAM activity. All samples were stored
at -70 C until time of assay. Protein concentrations were determined
using the bicinchoninic acid protein assay reagent (Pierce Chemical
Co., Interchim, Paris) and BSA as standard.
Amidation assays were performed in duplicate essentially as previously described (11, 25). Unless indicated otherwise, assay tubes contained 25,000 - 35,000 cpm of mono-[125I]-D-Tyr-Val-Gly, 0.5 µM D-Tyr-Val-Gly, 500 µM ascorbate, 10 µM Cu2S04, catalase (100 µg/ml), and 2–6 µg protein in 150 mM NaTES buffer, pH 8.5. Reactions velocities are generally expressed as picomoles of product formed per microgram of protein per hour (specific activity). The variation between duplicate samples was less than 5%. The reaction velocities reported are initial velocities, using a concentration of substrate about 10-fold below the Km of the enzyme for peptide substrate. In general, no more than 10% of the substrate was converted into product in the assay.
Western blot analysis
Samples were prepared for electrophoresis by making them 2% in
SDS and 5% in 2-mercaptoethanol and boiling for 5 min. Samples of
soluble or particulate fractions were fractionated on slab gels
containing 10% acrylamide and 0.25%
N,N-methylene-bis-acrylamide using the buffer system of
Laemmli (27). Proteins were electrophoretically transferred to Hybond-C
membranes (Amersham Corp.) for 1 h at 210 mA and visualized with
Ponceau S (Sigma). Molecular weights were estimated by comparison with
standard protein (Rainbow markers, Amersham Corp.). Hybond-C strips
were blocked in PBS buffer containing 5% of nonfat dry milk and
immunostained using a 1:2000 dilution of rabbit antiserum raised
against purified bacterially expressed PHM [Ab 1764 to rPAM
(37–382)], PAL [Ab 471 to rPAM (463–864)] kindly provided by Dr.
B. A. Eipper (Johns Hopkins University, Baltimore, MD). Signals were
revealed using an enhanced chemiluminescence kit (ECL Kit, Amersham
Corp.).
Combined RT-PCR
Total anterior pituitary RNA (5 µg) from OVX or OVX rats
treated with 17 ß-estradiol, was reverse transcribed into cDNA using
1 µg oligo(deoxythymidine) 12–18 (Pharmacia PL,
Villejuif, Paris) as primer in a 20-µl reaction volume containing 50
mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl2, 0.5 mM each of four dNTPs
(Pharmacia), 20 U RNAsin (Promega, Lyon, France), and 400 U M-MLV
reverse transcriptase (GIBCO-BRL, Gaithersburg, MD) at 37 C for 90 min.
The synthetic oligonucleotide primers used in the PCR were all 17 mers.
Primers yielding sense cDNA were [all base pair numbers are for rPAM-1
(24)] no. 5 (366–382), no.4 (1359–1375), and no. 9 (3124–3140).
Primers yielding antisense cDNA were no. 19 (1455–1439), no. 10
(3188–3172) and no. 21 (3834–3816). PCRs were performed in a 50-µl
volume, with 20 mM Tris-HCl (pH 7.4, 25 C), 50
mM KCl, 1.5 mM MgCl2, 0.1% Triton
X-100, 200 µM each of four dNTPs, 1 µM each
primer, cDNA derived from the equivalent of 300 ng total RNA, and 2.5 U
Extrapol I DNA polymerase (Eurobio, Les Ulis, Paris), samples were
overlayed with one drop of light mineral oil and subjected to 35 cycles
in a Biometra Trio-Thermoblock cycler (Biometra). Cycling parameters
were as follows: the initial denaturation step was performed at 94 C
for 5 min; the repeat cycle consisted of annealing at 52 C for 50 sec,
followed by extension at 72 C for 2 min and 30 sec and then
denaturation at 94 C for 35 sec. The last extension time was lengthened
to 10 min. After thermal cycling, most of the oil was manually removed.
Samples were fractionated on agarose gels in 89 mM Tris, 89
mM boric acid, and 2.5 mM EDTA, pH 8.0. After
staining with ethidium bromide, the gels were photographed. It should
be emphasized that internal standards were not included during reverse
transcription of amplification, and the amplified products can be
compared only in a qualitative manner.
In situ hybridization
In situ hybridization using 35S-labeled
riboprobes was performed as previously described (28, 29). Cryostat
sections (10 µm) were mounted onto gelatin-coated slides. Labeled
probes were prepared using [-35S] UTP (New England
Nuclear, Dupont, Paris, France) and T3 or T7
RNA polymerase (Stratagene, Paris) to synthesize sense and antisense
RNA transcripts, respectively, from the rPAM-1 cDNA (ZAP 6) cloned into
Bluescripts (24). RNA sense and antisense transcripts were prepared by
linearizing the plasmid with the appropriate restriction enzyme
(XbaI for the antisense and Hind III for the
sense strand). Slides were fixed in 4% wt/vol paraformaldehyde and
acetylated with 0.5% vol/vol acetic anhydride in 0.9% wt/vol NaCl
containing 100 mM triethanolamine (pH 8.0).
35S-labeled riboprobe was added to the hybridization buffer
(50% formamide, 600 mM NaCl, 1 x Denhardt’s, 10
mM Tris-HCl (pH 7.4), 1 mM EDTA, 10
mM dithiothreitol, 0.2 µg/µl tRNA, and 10% wt/vol
dextran sulphate) to give a final concentration of 1 x
106 cpm/50 µl. Hybridization was carried out overnight at
56 C in moist sealed chambers. Coverslips were removed in 2 x
SSC. Slides were incubated in a solution of 2 x SSC (30 min, 37
C) containing 10 µg/ml RNase A (Eurogentec, Belgium). Slides were
subsequently rinsed in 2 x SSC at room temperature for 30 min,
dehydrated in graded ethanol solutions, air dried, and exposed to x-ray
film (Kodak X-OMAT AR) for 3 days. For higher resolution analysis,
slides were dipped in Ilford K5 photographic emulsion. The slides were
stored for 7 days at room temperature, developed, and lightly
counterstained with hematoxylin.
Statistical analysis
All results are expressed as the mean values ±
SEM. Statistical analysis was performed by a one-way ANOVA
followed by Fisher’s PLSD test (ANOVA, Statview 512, Brain Power Inc.,
Calabasas, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As
expected, mean concentrations of LH were greater (P <
0.0001) in rats at 1600 h during the proestrus period and were not
different among the other periods of estrous cycle. Similarly,
estradiol levels were highest at 1600 h during proestrus period.
Anterior pituitary PAM mRNA levels were assessed by Northern blot
analysis. Total RNA prepared from the pituitaries of individual rats
was subjected to Northern blot analysis, and PAM mRNA was visualized
using a radiolabeled rPAM-1 cDNA capable of detecting all of the known
forms of rat PAM mRNA (Fig. 1A). Pituitary PAM mRNA size
ranged from 3.6–3.8 kilobase pairs in size. The PAM cDNA probe was
removed from the blots, and the amount of ribosomal RNA present in each
sample was determined by hybridization to a cDNA probe for ribosomal
RNA (Fig. 1A). The amount of PAM mRNA in each sample was then
normalized to the amount of ribosomal RNA (Fig. 1B). There was a
significant change in anterior pituitary PAM mRNA levels during the
different periods in cycling female. Anterior pituitary PAM mRNA
levels at proestrus and diestrus were significantly lower than at
metestrus and estrus. For example PAM mRNA levels at proestrus (1000 h)
were 3.0 ± 0.5-fold lower than at metestrus (n = 4;
mean ± SEM).
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Effect of castration and 17 ß-Estradiol replacement
(dose-response curve) on PAM expression
To examine the above mentioned hypothesis, adult female rats were
OVX and the effect of 17 ß-estradiol replacement on PAM expression
was evaluated. Total RNA was prepared from the pituitaries of
individual animals and analyzed as described above (Fig. 2A). The amount of PAM mRNA in each sample was then
normalized to the amount of ribosomal RNA. The data demonstrate that
E2 treatment resulted in a 4.5 ± 0.5-fold (n =
3; mean ± SEM) decrease in the level of PAM mRNA in
the anterior pituitary gland compared with OVX rats (Fig. 2B).
Treatment with increasing doses of 17 ß-estradiol reduced PAM mRNA
levels to a proestrus value. However treatment with very high doses of
17 ß-estradiol failed to reduce PAM mRNA levels below the proestrus
value (Fig. 2B). As expected, when OVX animals were treated with
increasing doses of 17 ß-estradiol, plasma LH levels declined from
high levels to levels below those found in estrus animals (Fig. 2B).
Based on these results, the OVX animals were treated with 4 µg of 17
ß-estradiol for the subsequent experiments.
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and 2. No alteration in the
size distribution of PAM mRNA could be detected.
The effect of estrogen replacement on the anterior pituitary PAM
specific activity was also assessed (Fig. 3). In OVX
rats, total specific PAM activity increased by approximately 2 ±
0.08-fold and was reduced (0.21 ± 0.09 pmol/h) by treatment with
17 ß-estradiol (4 µg/day for 7 days) to levels observed at
proestrus period (0.16 ± 0.0015 pmol/h). The majority of the PAM
activity (70–80%) was recovered in the soluble fraction independently
of estrogen status. In both groups, the specific activity of the
soluble and particulate fractions mirrored the total specific activity
(Fig. 3).
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). Antibody specific to the PHM (Ab
1764) domain of the rat PAM-1 protein was used to identify PAM proteins
present in anterior pituitary extracts (Fig. 4). The major PAM protein
in particulate fractions from both groups of rats had a mass of
105 ± 3 kDa (Fig. 4A) and was also visualized by antiserum (Ab
471) to the PAL domain of the PAM precursor protein (data not shown).
The 105-kDa PAM protein found in anterior pituitary membranes is
thought to represent rPAM-2 and rPAM-3b. In preparations from OVX +
E2 rats, two proteins at 59 kDa and 56 kDa were also
present. They were not visualized with a PAL antiserum and their
relationship to PAM is unclear. Analysis of the soluble fraction
revealed the presence of multiple forms of PAM protein (Fig. 4B).
Consistent with the effect of estrogen status on PAM specific activity,
the soluble proteins from OVX animals yielded a more intense signal
than the same amount of protein from OVX + E2 animals. The
105 ± 3 kDa and 45 ± 3 kDa found in the soluble fraction,
must be generated by endoproteolytic cleavage between the PAL domain
and the putative trans-membrane domain; and between the PHM
and PAL domains respectively. Both proteins are products of rPAM-1,
whereas the 84 ± 2 kDa PAM protein is a product of PAM mRNA
lacking exon A (primarly rPAM-2) (30, 31). The 95 ± 3 kDa PAM
protein is thought to represent intact rPAM-3/3a. The 75 ± 2 kDa
PAM protein must arise from precursor forms of PAM (rPAM-1, -2, -3a,
-3b, or 3) by endoproteolytic cleavage. A 60-kDa protein is also
visualized by antibody to PHM, and its relationship to PAM is unclear.
The expression of the described PAM proteins was barely detectable in
OVX + E2 animals.
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).
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) (9, 30, 32). Forms of PAM mRNA lacking all or part of optional exon B [also
referred as exon 25 and 26 (9)] are more prevalent in the pituitary
tissue (30). The forms of PAM mRNA in the pituitary are poorly resolved
on denaturing agarose gels. To determine whether estrogen status
altered the forms of PAM mRNA present in the anterior pituitary, cDNA
was synthesized by reverse transcription of RNA prepared from OVX or
OVX + E2 animals. A qualitative measure of the forms of PAM
mRNA present was obtained by carrying out PCR amplification using pairs
of oligonucleotide primers that distinguish among the different
alternatively spliced forms of PAM mRNA (Fig. 6B). The primer pair
(5/19) spans the region common to all PAM mRNA forms from the 5' end of
rPAM-1 to immediately before optional exon A; the second primer pair
(4/10) spanning the regions referred to as exon A and B distinguishes
between mRNAs of the rPAM-1 or -2, -3a, -3b, and -3 types; the third
pair (9/21) extends from the 3' end of optional exon B to immediately
before the putative poly (A) addition signal. The results do not
provide a quantitative comparison of levels of PAM mRNA in different
samples. Amplified fragments were fractionated on agarose gels (Fig. 6C). Based on amplification using these three sets of primers, we found
no effect of estrogen status on the alternatively spliced forms of PAM
mRNA in the anterior pituitary; this observation is consistent with the
results of Northern blot analysis, which failed to demonstrate an
alteration in the molecular weight pattern of PAM mRNA after
manipulation of estrogen status. Adult anterior pituitary contains only
small amounts of rPAM-4 and -5 (30, 32). rPAM-4 and rPAM-5 mRNAs were
barely detectable by reverse transcription followed by PCR
amplification with form-specific primers and the results demonstrated
that estrogen status had no effect on these two forms (data not
shown).
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, in OVX rats PAM mRNA
was detected in majority of the cell bodies of the anterior pituitary
gland. E2 treatment of OVX rats induced an overall decrease
of the in situ hybridization signal, suggesting that
E2-induced PAM regulation is not restricted to a single
cell type.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As indicated in the introduction, several amidated peptides have been
identified in the anterior pituitary gland and may act locally in a
paracrine/autocrine way (34). Interestingly, the changes in the levels
of several -amidated peptides in the anterior pituitary gland have
been observed after chronic manipulation of estrogen status. Indeed,
ovariectomy produced a significant rise in the pituitary content of
SP-IR, NPY-IR, and NT-IR. This pattern is reversed with estrogen
replacement or high dose estrogen treatment. These changes in peptide
content are accompanied by parallel changes in the mRNA levels for each
peptide (20). The effect of ovariectomy on anterior pituitary VIP
levels are strikingly opposite to those described for SP, NT, and NPY
(20). The most dramatic effects were observed following treatment with
high dose estrogens, with a large increase in VIP and a significant
decrease in NT and SP concentrations. Like VIP, galanin levels increase
in the anterior pituitary gland of OVX female rats (15, 35). So far,
the simultaneous location of PAM and an amidated peptide in an
identified pituitary cell has not been possible. Immunochemical
staining of serial pituitary tissue sections for PAM and
well-established pituitary hormones has indicated that the anterior
pituitary cells intensively stained for PAM represent a subpopulation
of the gonadotrophs. PAM has also been identified at moderate levels in
corticotrophs and at lower levels in somatotrophs and lactotrophs (19).
The location of the amidated peptides in specific pituitary cell types
has not been fully established. SP has been detected in rat
gonadotrophs and lactotrophs (36); meanwhile, the attempts to
colocalize NT in any of the classical anterior
pituitary-hormone-producing cells have failed so far. NPY has been
localized in thyrotrophs (14) and VIP immunoreactivity has been
demonstrated only in lactotrophs (37). Galanin is possibly produced in
a number of different pituitary cells, but certainly in lactotrophs
(38). Receptors for SP (39), VIP (40), and NT (41) have been identified
in anterior pituitary cells. However, there is no clear pattern of
their physiological implications in the regulation of pituitary
functions. Because their expression has been shown to vary during
changes in estrogens levels, the influence of amidated peptides on the
pituitary control of reproduction has been investigated. There is now
some evidence for a physiological role of NPY in stimulating LH
secretion; the association between LH stimulation and increased
pituitary NPY expression after ovariectomy further supports a
functional role for this peptide in the control of gonadotrophins
secretion (42). In contrast, it has been suggested that SP inhibits LH
release and LH-induced GnRH release on the basis of passive
immunization anti-SP (43). VIP and galanin expression have been mainly
associated with PRL secretion. Estrogens stimulate VIP and PRL
expression and in vitro studies using dispersed rat
lactotrophs support an autocrine role for VIP in the control of PRL
secretion (44). Galanin may play a similar role because
immunoneutralization of this peptide decreases PRL release (45). The
demonstration of PAM regulation in the anterior pituitary gland as well
as the production of -amidated peptides by this tissue adds further
support for the intercellular communications within the anterior
pituitary. Indeed, so far, the physiological role of pituitary PAM
appears to be limited to the synthesis of amidated peptides which act
locally through autocrine and paracrine mechanisms of action (34).
Thus, the regulation of PAM activity may represent a third mechanism of
feedback of estrogens on pituitary function in addition to those
already established at the levels of the hypothalamus and of the
pituitary gonadotrophs. This concept may also be extrapolated to
thyroid hormones (29).
There is a coordinate regulation of PAM, NPY, SP, and NT expression as already described for PAM and amidated peptides like TRH in developing rat pancreas (46) and POMC (47) in the anterior pituitary under glucocorticoid changes. We have shown that PAM expression is regulated in rat anterior pituitary gland by thyroid hormone status and that pituitary levels of amidated peptides vary in the same direction (29). The opposite changes in PAM and VIP expression in the anterior pituitary gland do not exclude a coregulation of PAM and VIP expression at an individual cellular level. Indeed, a putative coregulation may be blunted by the low percentage of VIP-expressing cells in whole pituitary gland and the analysis of PAM expression in VIP-producing pituitary cells might be necessary for its demonstration.
In summary, the results reported in this study demonstrate that
estrogen status regulate PAM expression, a key processing enzyme in the
anterior pituitary gland. Our current efforts are directed toward the
determination of PAM colocalization with classical pituitary hormones
and -amidated peptides and the analysis of molecular mechanisms of
its regulation by estrogens. Both efforts should help in the
understanding of the physiological significance of these hormonal
alterations in PAM expression in the anterior pituitary gland.
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Acknowledgments |
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Received July 8, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-amidation. Annu Rev Neurosci 15:57–85[CrossRef][Medline]
-amidation are synthesized from a single mRNA. Biochem Biophys Res
Commun 172:197–203[CrossRef][Medline]
-amidating monooxygenase precursor contains two
separable enzymatic activities involved in peptide amidation. Biochem
Biophys Res Commun 171:926–932[CrossRef][Medline]
-hydroxyglycine derivatives, thereby functioning sequentially with
peptidylglycine -amidating monooxygenase in peptide amidation.
Biochemistry 26:6115–6120
-amidating reaction: evidence for a two-step
mechanism involving a stable intermediate at neutral pH. Biochem
Biophys Res Commun 169:524–530[CrossRef][Medline]
-amidating monooxygenase protein.
J Biol Chem 266:17004–17010-amidating
monooxygenase activity in rat pituitary. J Biol Chem 263:7550–7554-amidating monooxygenase gene: exon/intron
organization of catalytic, processing and routing domains. Mol
Endocrinol 6:1571–1584-amidating
activity in rat. Biochem Biophys Res Commun 140:230–236[CrossRef][Medline]
-amidating monooxygenase
activity and mRNA. Mol Endocrinol 3:1387–1398-amidating monooxygenase (PAM) in pituitary gland.
Endocrinology 127:358–364-amidating
monooxygenase in rat atrium. Proc Natl Acad Sci USA 86:735–739-amidating monooxygenase
(PAM) in rat heart atrium and ventricle. J Biol Chem 264:5839–5845-amidating
monooxygenase activity in rat pituitary. J Biol Chem 263:7550–7554
-amidating
monooxygenase expression in anterior pituitary gland. Mol Endocrinol 4:1497–1505-amidating monooxygenase. J Biol Chem 267:4008–4015-amidating monooxygenase (PAM) in primary
cultures of neonatal rat cardiocytes: a model for studying regulation
of PAM expression in the rat heart. Mol Endocrinol 6:1998–2008-amidation. J Biol Chem 266:1701–1707-amidating activity in the pancreas from neonatal rats. Proc Natl
Acad Sci USA 84:261–264-amidating monooxygenase expression.
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S. Fraboulet, F. Boudouresque, C. Delfino, and L'H. Ouafik Identification of a Novel cis-Element in the 3'-Untranslated Region of Mammalian Peptidylglycine {alpha}-Amidating Monooxygenase Messenger Ribonucleic Acid Endocrinology, March 1, 1998; 139(3): 894 - 904. [Abstract] [Full Text] [PDF]
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R. El Meskini, F. Boudouresque, and L'H. Ouafik Estrogen Regulation of Peptidylglycine {alpha}-Amidating Monooxygenase Messenger Ribonucleic Acid Levels by a Nuclear Posttranscriptional Event Endocrinology, December 1, 1997; 138(12): 5256 - 5265. [Abstract] [Full Text] [PDF]
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 B. Amir-Ahmady and L. M. Salati Regulation of the Processing of Glucose-6-phosphate Dehydrogenase mRNA by Nutritional Status J. Biol. Chem., March 23, 2001; 276(13): 10514 - 10523. [Abstract] [Full Text] [PDF]
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) and
particulate (squlo) fractions prepared from three pooled anterior
pituitary glands was measured as described in Materials and
Methods. Total PAM specific activity (squlf) was calculated by
taking into account the amount of protein in the two fractions. Data
from three experiments (n = 3 for each treatment group in three
experiments) were used to calculate the mean specific activity; each
sample was assayed in duplicate. Data are presented as mean ±
SEM. The asterisk indicates that the value
is significantly different between both groups (*,
P < 0.02).
