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J. Biol. Chem., Vol. 277, Issue 48, 46391-46401, November 29, 2002
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From the Signalisation cellulaire, Régulation de gènes
et Physiologie de l'Axe gonadotrope, UMR CNRS 7079, Physiologie et
Physiopathologie, Université Pierre et Marie Curie,
75252 Paris Cedex 05, France
Received for publication, April 18, 2002, and in revised form, August 9, 2002
Nitric-oxide synthase type I (NOS I) is expressed
primarily in gonadotrophs and in folliculo-stellate cells of the
anterior pituitary. In gonadotrophs, the expression and the activity of NOS I are stimulated by gonadotropin-releasing hormone (GnRH) under
both experimental and physiological conditions. In the present study,
we show that pituitary adenylate cyclase-activating polypeptide (PACAP)
is twice as potent as GnRH at increasing NOS I levels in cultured rat
anterior pituitary cells. The action of PACAP is detectable after 4-6
h and maximal at 24 h, this effect is mimicked by 8-bromo-cAMP and
cholera toxin and suppressed by H89 suggesting a mediation through the
cAMP pathway. Surprisingly, NADPH diaphorase staining revealed that
these changes occurred in gonadotrophs exclusively although PACAP and
cAMP, in contrast to GnRH, have the potential to target several types
of pituitary cells including folliculo-stellate cells. There was no
measurable alteration in NOS I mRNA levels after cAMP or PACAP
induction. PACAP also stimulated cGMP synthesis, which was maximal
within 15 min and independent of cAMP, however, only part resulted from NOS I/soluble guanylate cyclase activation implying that in contrast to
GnRH, PACAP has a dual mechanism in cGMP production. Interestingly, induction of NOS I by PACAP markedly enhanced the capacity of gonadotrophs to produce cGMP in response to GnRH. The fact that PACAP
may act on gonadotrophs to alter NOS I levels, generate cGMP, and
potentiate the cGMP response to GnRH, suggests that cGMP could play
important cellular functions.
Anterior pituitary gonadotrophs are endocrine cells that produce
and secrete luteinizing hormone
(LH)1 and
follicle-stimulating hormone (FSH), two gonadotropins that are crucial
for the control of the reproductive function. The secretory action of
pituitary gonadotrophs is under a complex neuroendocrine control
network that includes hypothalamic, gonadal, and locally produced
hormones and other factors (1) of which gonadotropin-releasing hormone
(GnRH), a decapeptide, is the most prominent among the hypothalamic
regulatory peptides. In fact it is well established that GnRH acting
through a specific G protein-coupled receptor present on the surface of
gonadotrophs is crucial for gene expression, synthesis, and release of
biologically active gonadotropins (2, 3). GnRH also regulates a number
of genes involved in GnRH signaling including its own receptor gene
(4).
Compared with GnRH, the function and mechanisms of action of pituitary
adenylate cyclase-activating polypeptide (PACAP), another potential
regulator of gonadotrophs is much less documented. PACAP is a 27- or
38-amino acid hypophysiotropic peptide first isolated from sheep
hypothalamus by its ability to stimulate cAMP production in rat
pituitary cells (5). Intra-atrial injection of PACAP induces LH
secretion in male rats. In vitro, PACAP stimulates only a
weak secretion of LH and FSH by primary cultures of pituitary cells
over 3-5 h (6, 7), however, longer stimulation periods appear more
effective (8). PACAP also regulates the mRNA expression of the
gonadotropin Similar to PACAP and suggesting possible interconnections between
transduction mechanisms of both neurohormones, GnRH initiates several
intracellular signaling pathways among which are the activation of
phospholipase C resulting in the production of diacylglycerol and
inositol phosphate that are responsible for protein kinase C (PKC)
activation and intracellular Ca2+ mobilization. GnRH also
induces the activation of the mitogen-activated protein kinase (MAPK)
cascade and the production of cGMP. Recent studies have demonstrated
that the latter two effects are indirect, the first resulting from the
activation of PKC (16), and the second from the acute elevation of
Ca2+ resulting in the activation of nitric-oxide synthase
(NOS) type I (17, 18).
NOS I is one of the three NOS isoforms that have been described to
date. Each is encoded by a distinct gene: type I (neuronal) and type
III (endothelial) NOS are thought to be constitutive Ca2+-calmodulin-dependent enzymes, whereas type
II, primarily found in macrophages, is inducible and
Ca2+-independent. These enzymes catalyze the formation of
nitric oxide (NO), a highly reactive and diffusible gas that plays an
important role as an inter- or intracellular messenger and exerts, at
least in part, its effects via the activation of soluble guanylate
cyclase (sGC) (19, 20). NOS I has been shown to mediate the
N-methyl-D-aspartate action on GnRH secretion in
the hypothalamus (21, 22), whereas an endothelial NOS III is involved
in the estradiol-induced secretion of GnRH (23). The identification of
NOS I in pituitary gonadotrophs and folliculo-stellate cells (24) has
raised the possibility that NO may act as a regulator of pituitary
activity. In mammals, the exact role of NO and cGMP on gonadotropin
secretion is still controversial (18, 24-28), whereas in amphibians
both basal and GnRH-induced pituitary gonadotropin secretion seem to be
up-regulated by NO (29). An effect of NO and cGMP on growth
hormone and prolactin secretion has been described in mammals
(28, 30-33).
Recent studies (27) from our laboratory have provided evidence that
GnRH stimulated NOS I gene expression and the consequent increase in
NOS I protein in rat pituitary gonadotrophs. Moreover, this
GnRH-dependent up-regulation of NOS I was distinct during an important physiologic event such as proestrus in female rats, leading to an amplified production of pituitary cGMP (18).
Complementary to this, we have isolated a pituitary-specific promoter
of NOS I that conferred responsiveness to GnRH as well as cAMP (34). The effect of PACAP on the GnRH signaling pathway (12, 14, 35), its
role during proestrus in gonadotrope/GnRH responsiveness (36, 37), and
its ability to act through cAMP and Ca2+ prompted us to
analyze the effect of PACAP on NOS I in the pituitary. We show that,
like GnRH, PACAP augments pituitary levels of NOS I in
vitro, via the cAMP/PKA-dependent transduction
pathway, and that this NOS I is restricted to the gonadotrope cells. In
addition, besides its stimulating effect on NOS I protein that occurs
after a protracted period, PACAP also induces a rapid, cAMP-independent production of cGMP. The PACAP-induced NOS I appears fully functional because pretreatment with PACAP enhances the capacity of pituitary cells to produce cGMP and potentiates the cGMP response of gonadotrophs to GnRH. Collectively, data suggest that PACAP and GnRH operate together to regulate the NOS/cGMP signaling pathway in these cells.
Materials--
PACAP27, PACAP38, 8-Br-cAMP, H89, and
NG-monomethyl-L-arginine
(L-NMMA) were obtained from Alexis Corporation (Coger,
Paris, France). TPA (12-O-tetradecanoylphorbol-13-acetate),
PD98059 (2'-amino-3'-methoxyflavone), GF109203X (bisindolylmaleimide
I), ODQ
(1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one), and A23187 were purchased from Calbiochem (San Diego, CA). Cholera
toxin, IBMX (3-isobutyl-1-methylxanthine), and triptorelin ([D-Trp6]GnRH) were provided by Sigma.
Anterior Pituitary Cells Culture--
Anterior pituitary glands
were removed from male Wistar rats (200-220 g; Janvier, Le
Genest-Saint-Isle, France). The cells were enzymatically dispersed
using the trypsin dissociation procedure described previously (38).
Cells in 300-350 µl of Ham's F-10 medium (Biomedia, Boussens,
France) supplemented with 10% fetal calf serum (Biomedia) and
gentamycin (20 µg/ml, Sigma) were plated in 20-mm diameter Nunc
multiwell culture dishes (Poly-labo, France) at a density 3 × 106 cells for Western blot analysis. After the 30-45 min
required for cell attachment, the culture medium volume was adjusted to 2 ml and the cells were further incubated for 2 days at 37 °C in a
humidified atmosphere with 5% CO2. Drugs were added in
serum-free Ham's F-10 medium as described in the text and when
required, inhibitors were added 1 h before incubation with
secretagogues. At the end of the incubation period, the medium
was removed and stored at Preparation of Cell Extracts and Western Blot Analysis of NOS I,
NOS II, and NOS III--
Protein extraction and Western analysis were
performed as previously described (27). The pituitary cells (3 × 106 cells) were homogenized in 10 mM Tris-HCl,
pH 7.4, containing 2 mM EDTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 20 µg/ml leupeptin.
Homogenates were centrifuged for 45 min at 20,000 × g
at 4 °C and protein concentration in the supernatant was determined
according to Bradford (39). Proteins were separated in slab gel
electrophoresis using 7% polyacrylamide separating gel in a
Mini-Protean-3 apparatus (Bio-Rad). Protein molecular weight markers
(Kaleidoscope standards, Bio-Rad) were co-electrophoresed.
After electrotransfer onto nitrocellulose membrane (Sartorius,
Göttingen, Germany), NOS I, NOS II, or NOS III were
immunodetected using specific affinity purified antibodies
(Transduction Laboratories, Lexington, KY) at dilutions of 1/200,
1/1000, and 1/500, respectively, and the enhanced chemiluminescence
system (ECL, Amersham). Blots were exposed to Kodak XAR-5 films
(Eastman Kodak Co., Rochester, NY).
Analysis of NOS I Promoter Activity: Cell Culture, Transfection,
and Luciferase Assay--
The pituitary-specific promoter used in this
study was previously isolated and characterized (34). The construct
consisted of the full-length promoter ( Extraction of mRNA and Dot Blot Hybridization--
Total RNA
was prepared from cultured rat pituitary cells (3 × 106) using Tri-Insta-Pure (Eurogentec, Seraing, Belgium).
Dots and hybridization were performed as previously described (27, 43) using a 1.2-kb rat NOS I complementary DNA (Alexis Corp.) as probe and
cyclophilin mRNA for standardization.
NADPH Diaphorase Cytochemistry and
Immunocytochemistry--
Pituitary cells (6 × 105)
were plated in poly-L-lysine-coated (Sigma) chambers of
Lab-Tek slides (Nalge Nunc Int., Poly-labo) in Ham's F-10
medium containing 10% fetal calf serum. After 3 days, the medium was
replaced and 20 nM PACAP38 or 1 mM 8-Br-cAMP was added to cells previously treated (1 h) or not with 30 µM H89. After 24 h, cells were washed with cold PBS,
then fixed for 20 min with 4% paraformaldehyde, and rinsed three times
for 10 min with PBS before permeabilization by incubation with 0.1%
saponin for 15 min. After a further three washes with PBS, the NADPH
diaphorase cytochemical procedure was performed as described previously
(27). Cells were incubated in the dark at 37 °C for 30 min in 50 mM Tris-HCl, pH 8, containing 1 mg/ml
Following cytochemistry, the cells were further processed for
immunochemistry. Gonadotrophs were identified with a mouse monoclonal immunoaffinity purified anti-bovine LH Measurement of LH and FSH--
RIA, using kits provided by Dr.
A. F. Parlow, National Hormone and Peptide Program
(Harbor-UCLA Medical Center, Torrance, CA) and NIDDK (NIH, Baltimore,
MD), determined LH and FSH in the culture. Highly purified rat LH
(NIDDK LH I-9) and rat FSH (NIDDK FSH I-8) were used for iodination
(46), and rLH-RP3 and rFSH-RP2 were used as the references.
Anti-rLH-S11 and anti-rFSH-S11 antisera were used at the appropriate
dilution (1/750,000 and 1/125,000, respectively). Bound and free
hormone were separated using immobilized protein A (47).
In Vitro Incubation of Anterior Pituitary or Dispersed Cells and
cGMP Assay--
Anterior hemipituitaries dissected from male Wistar
rats were incubated 1 h at 37 °C in culture medium 199 (Biomedia, Boussens, France). The tissues were then incubated for
1 h in the presence of 0.3 mM IBMX alone or in
combination with appropriate concentrations of EGTA and inhibitors of
NOS, sGC, or PKA as indicated in the text. Then, 50 nM
PACAP38, 3 nM cholera toxin, or 10 nM
triptorelin was added and the incubation was continued for the
indicated periods of time (from 15 to a maximum 60 min). Tissue and
medium were separated and stored, at Data Analysis--
Western blots were scanned and numeric images
were analyzed with a computer image processing system (NIH Image
software for densitometric analysis of gels). All given values are the
mean ± S.E. of at least three separate experiments typically with
three replicates for each experimental group. Differences between means were assessed by analysis of variance followed by Dunnett's
t test. *, p PACAP38/27 in Vitro Stimulates Expression of Pituitary NOS I in a
Time- and Concentration-dependent Manner--
The
potential influence of PACAP on NOS I expression was examined over a
96-h period using primary cultures of rat anterior pituitary cells.
According to the autoradiogram shown in Fig. 1A, pituitary cells contained
detectable levels of NOS I under basal (nonstimulated) conditions,
whereas neither NOS II nor NOS III were detected. As illustrated in the
time course (Fig. 1B) and concentration dependence (Fig.
2) curves, PACAP38 and PACAP27 were both
equally potent in increasing NOS I protein. The increase was detectable
as early as 4-6 h after the addition of an optimal concentration of
PACAP (
Because GnRH was previously shown to up-regulate NOS I in
vivo (18, 27), we also examined as a control the effects of the
GnRH agonist triptorelin. Fig. 1B shows that triptorelin
also increased in vitro the level of NOS I. The time course
was similar to that of PACAP, however, at the maximal effective
concentration, GnRH was about two times less potent than PACAP
(Vmax = 2.48 ± 0.15 versus
3.59 ± 0.08%).
The release of LH and FSH under PACAP and GnRH treatments was
systematically assayed as a comparative functional index. Figs. 1C and 2 (inset) show that PACAP38 and PACAP27
had a similar effect on both LH and FSH secretion, however, this was
much lower than that observed with GnRH. This was the opposite to the
observed PACAP and GnRH effects on the NOS I. The secretion in response to PACAP appeared somewhat delayed compared with that under GnRH. The
EC50 for LH and FSH release was, respectively, 1.58 ± 0.64 and 2.28 ± 0.2 nM, thus not significantly
different from that measured for NOS I up-regulation.
Implication of the cAMP/PKA Transduction Pathway in PACAP
Up-regulation of NOS I--
To identify the intracellular signaling
mediating the PACAP induced up-regulation of NOS I, activators and/or
inhibitors of the PKA, PKC, MAPK, or Ca2+ pathways were
used. As shown in Fig. 3, both 8-Br-cAMP
(a permeant analog of cAMP) and cholera toxin (an endogenous cAMP
generator) caused at optimal concentrations (1 mM and 3 nM, respectively) an increase in the concentration of NOS I
similar to that observed with 50 nM PACAP. In contrast,
neither the phorbol ester TPA (5 nM) nor the
Ca2+ ionophore A23187 (5 µM) affected NOS I
levels (Fig. 3A), whereas both of these substances were
clearly active taking into account their well established stimulatory
action on LH release (Fig. 3A, inset).
Pretreatment of pituitary cells with the PKC inhibitor GF109203X (2 µM) had no influence on both the basal as well as
PACAP-induced levels of NOS I (Fig. 4),
which was coherent with and reinforced the absence of an effect with
TPA on NOS I induction. A similar lack of influence of the MAPK cascade
was observed with the MAPK inhibitor PD98059 (20 µM). In
contrast, the PACAP action on NOS I was quasi-totally abolished by H89,
a potent inhibitor of PKA that acted via a competitive interaction at
the ATP-binding site (50). The concentration dependence of H89 effects
is further depicted in Fig. 5. As
mentioned above (Fig. 3A), A23187 had no effect on NOS I
synthesis, however, the addition of EGTA resulted in a partial (50%)
reduction in the stimulation of NOS I levels by PACAP (Fig. 4),
suggesting an involvement of extracellular Ca2+ in the
mechanism of PACAP action. This was at variance with the LH release
that was induced by A23187 in the absence of PACAP (Fig. 3A,
inset), and strongly inhibited by EGTA in the presence of
PACAP (Fig. 4, inset). The PACAP-induced LH release was also inhibited to various degrees by GF109203X and H89 (Figs. 4 and 5,
insets) illustrative of the complex contribution of the PKC and PKA pathways in mediating the releasing action of PACAP within gonadotrophs. These data suggested a major involvement of the cAMP/PKA
pathway in the PACAP induction of NOS I with a possible, partial
contribution of extracellular Ca2+ but not of
Ca2+ entry. Finally, direct activation of the PKA pathway
with 8-Br-cAMP induced NOS I with a time course profile similar to that
of PACAP as illustrated in Fig. 3B.
NADPH Diaphorase Reveals an Exclusive Increase in Staining of
Gonadotrophs under PACAP Stimulation or Direct Activation of the
cAMP/PKA Pathway--
Our results argue in favor of a major
implication of PACAP acting via cAMP to up-regulate pituitary NOS I
levels. Because PACAP has the potential to target several cell types in
the anterior pituitary, in particular gonadotrope and
folliculo-stellate cells that contain NOS I, the question arose whether
these (or/and other) cell types were concerned by PACAP and cAMP
actions. To address this question, NADPH diaphorase activity together
with immunoidentification of the stained cells were done. The data in
Fig. 6 show the immunodetection of the
S100 protein identifying folliculo-stellate cells and LH PACAP Stimulates Pituitary Production of cGMP, a Rapid Effect
Mediated in Part by NOS I and Independent of cAMP--
Because,
similar to GnRH, PACAP was able to regulate NOS I levels, we examined
whether it was capable of inducing the rapid generation of cGMP and
whether this action occurred through the NOS/NO/sGC cascade as shown
for GnRH (18). For this, anterior hemipituitaries were incubated with
or without PACAP for varying periods of time from 0 to 60 min. As shown
in Fig. 7, 50 nM PACAP38 readily induced a very rapid and marked increase in cGMP with a maximum
attained at 15 min. When compared with the effects of 10 nM
GnRH used as a reference, both profiles were quite similar.
The relationship between cGMP production, PACAP, and NOS I was further
examined using the NOS inhibitor, L-NMMA, and the sGC inhibitor, ODQ. Fig. 8 shows that the
maximal effective concentrations of L-NMMA (1 mM) or ODQ (6 µM) caused only a partial but
similar (50%) reduction in cGMP produced under stimulation by PACAP.
In contrast, under GnRH stimulation, either drugs completely abolished cGMP production. These data suggest that, unlike GnRH, PACAP stimulates the production of cGMP in the pituitary through
NO/NOS/sGC-dependent and -independent mechanisms. Under the
experimental conditions used, these two systems could contribute
equally to the PACAP-induced production of cGMP taking into
consideration the similar degree (50%) of inhibition observed using
either L-NMMA or ODQ, which both block the NO/NOS/sGC
system.
The possible influence of cAMP on the short term stimulation (15 min)
of the pituitary guanylate cyclase activity was tested. As shown on the
Fig. 8, 3 nM cholera toxin was unable to induce cGMP
production, whereas 30 µM H89 did not alter the
PACAP-induced production of cGMP, demonstrating an absence of influence
of any cAMP-dependent mediatory mechanisms.
Pretreatment with PACAP Enhances the Capacity of Pituitary Cells to
Produce cGMP and Potentiates the cGMP Response of Gonadotrophs to
GnRH--
Because PACAP elevates NOS I protein levels in the anterior
pituitary and particularly in gonadotrophs as attested by the data in
Fig. 6, whether the NOS-dependent production of cGMP could similarly be affected was examined: 1) after hormone removal (cessation of PACAP stimulation) and 2) under stimulation with the gonadotrope cell-specific stimulator GnRH. For this, 2-day pituitary cell cultures
were treated as described under "Experimental Procedures," with or
without 20 nM PACAP38 for 24 h to induce an optimal
NOS I synthesis. PACAP was then removed and the capacity of treated and
untreated cells to produce cGMP was measured over 1 h in the absence of any hormone or in response to 1 nM triptorelin.
Because of its abundant release in pituitary cell cultures, cGMP was
assayed in both cell extracts and medium and the total
production was reconstituted accordingly (Fig.
9A). From this study it was
apparent that: 1) in both compartments the cGMP production was higher
when cells were pretreated with PACAP, whether GnRH was absent or
present during the 1-h incubation and 2) in the presence of the NOS
inhibitor L-NMMA (hatched bars), the cGMP
production was notably reduced under all conditions, in particular,
with GnRH. A significant difference in L-NMMA modulation
was observed between PACAP pretreated and untreated cells, which was
essentially visible in the medium. This was consistent with the
PACAP-induced NOS-independent cGMP production described in Fig. 8. This
value was relatively modest in comparison to the cGMP produced in the
absence or presence of GnRH probably because of the preferential
induction of NOS I over elements of the NOS-independent system.
Fig. 9B shows the total (cells plus medium) cGMP after
subtraction of the respective values in the presence of
L-NMMA and thus implies a NOS-dependent cGMP
production. Compared with untreated (vehicle treated) cells, PACAP
pretreatment caused a 3.5 ± 0.13-fold increase in cGMP
production. The response to GnRH also was more intense after PACAP
pretreatment, representing 4.6 ± 0.4-fold of that in untreated
cells. These data thus indicate that the NOS I protein accumulated
under PACAP induction is fully active and its activity in terms of cGMP
production augments in a comparable degree as does NOS I protein
(~3.6-fold, cf. Fig. 1). In addition the large increase in
NOS-dependent cGMP production under GnRH suggests that the
PACAP induction primarily concerns gonadotrophs.
PACAP and NOS I Gene Expression--
The capacity of PACAP and
cAMP to induce NOS I protein was abolished by actinomycin D (data not
shown). Two complementary approaches were used to determine whether or
not PACAP regulates NOS I gene expression. Having recently
characterized a pituitary-specific NOS I promoter that was capable of
responding to the stimulation by GnRH or cAMP/PKA activators in the
gonadotrope L
A topic that has been very recently debated (52) is whether L
To complement the previous study, the effects of PACAP38, 8-Br-cAMP,
and cholera toxin on steady-state levels of NOS I mRNA in cultured
rat pituitary cells were examined by blot analysis of total RNA with a
32P-labeled cDNA probe. Fig. 10C shows that
no change was apparent in the labeling of the spots. This was confirmed
after correction with reference to the cyclophilin mRNA standard.
In the present study we show for the first time that PACAP, like
GnRH, readily up-regulated NOS I protein in the anterior pituitary and
induces cGMP production. The effect of PACAP on NOS I expression is
selective with respect to other NOS isoforms. It requires a few hours
to develop under the continual presence of the neurohormone, occurs
through mediation of the cAMP/PKA transduction pathway, and concerns
only the gonadotrophs. In contrast, the action of PACAP on cGMP
production is rapid, i.e. develops within minutes, is
independent of cAMP and unexpectedly, involves the activation of not
only the NOS/NO/sGC system but also another, nonidentified process.
Consistent with the induction of a potentially active NOS I, PACAP
enhanced the capacity of pituitary cells to produce cGMP and
potentiated the cGMP response of gonadotrophs to GnRH. Thus it can be
postulated that PACAP and GnRH operate in concert to regulate in
gonadotrophs the NOS I level as well as NOS-dependent
production of cGMP.
Previous studies based on immunohistochemical, in situ
hybridization, and/or NADPH diaphorase histochemical techniques have established that detectable levels of NOS I were expressed in gonadotrophs and folliculo-stellate cells in rat and human anterior pituitaries (24, 54). Moreover, a distinct augmentation in NOS I
protein and activity by GnRH, which targets gonadotrophs exclusively,
was demonstrated in vivo in rats under a variety of
experimental and physiological conditions (18, 24, 27). Specifically,
castrated rats (a model in which the pituitary is hyperstimulated by
endogenous GnRH because of central depression of its secretion), or
intact rats injected with a GnRH agonist, expressed high levels of NOS
I (mRNA and protein). In contrast, blocking of GnRH receptors (and,
therefore, prevention of the endogenous GnRH action) by a GnRH
antagonist readily caused depression of pituitary NOS I (both
parameters) in intact as well as in castrated rats. From these data it
could be assumed that ~75% NOS I that was detected in intact rat
pituitary was expressed in gonadotrophs and the residual 25% could be
present in folliculo-stellate cells, in addition to possibly other
endocrine cells such as the somatotrophs and lactotrophs although at
much lower levels (54, 55). In castrated rats the latter percentage was
further reduced to 5-6% as a result of the GnRH-induced up-regulation
of NOS I only in gonadotrophs. The present data showing that in
vitro GnRH induced a substantial, time-dependent
elevation of NOS I levels in anterior pituitary cells are in complete
agreement with, and further complement, our previous in vivo
observations (18, 27, 34).
In contrast to GnRH, which targets gonadotrophs exclusively, PACAP
receptors are present on most cell types present in the anterior
pituitary (7). These include the somatotrophs and lactotrophs, which in
addition to the gonadotrophs and the folliculo-stellate cells, could
express NOS I. The coexpression of NOS I and PACAP receptors in various
pituitary cell types raise questions about the nature of the cells
capable of responding to PACAP. In particular, because at optimal
concentrations PACAP induced NOS I to levels about twice that observed
with GnRH, could PACAP be more potent than GnRH in the stimulation of
NOS I production in gonadotrophs only, in cells other than
gonadotrophs, or cumulatively in several cell types including the
gonadotrophs. The observation that NADPH diaphorase staining, a method
that quantitatively reflects in situ the presence of NOS
(56, 57), increased only in gonadotrophs strongly argues in favor of
the first hypothesis. The PACAP-induced NOS I appears fully functional
because the capacity of cells to produce cGMP in a
NOS-dependent manner is increased to a similar degree as
the NOS I protein (~3.6-fold). Furthermore, the fact that
(NOS-dependent) cGMP formation in response to GnRH was
similarly amplified following PACAP treatment reinforces the notion
that gonadotrophs are the major targets of NOS I induction and
demonstrates that NOS I induced in these cells: 1) is active and 2)
potentiates the GnRH action. Interestingly, whereas PACAP appears more
efficient than GnRH to induce NOS I, the inverse situation is noted
concerning gonadotropin release. This could reflect differences in the
preferential transduction pathways used by each hormone and their
relative importance in regulating either process.
In this respect, the data demonstrating the involvement of the cAMP/PKA
pathway in the action of PACAP on NOS I synthesis provides important
information. It is well established that cAMP is an important
intracellular mediator of PACAP action (14, 15) and in gonadotrophs,
PACAP is a more potent stimulator than GnRH of the cAMP/PKA pathway
(13). The anterior pituitary expresses different forms of PACAP
receptors (7, 58). The fact that, in this study, PACAP38 and PACAP27
are equally potent in stimulating NOS I is indicative of the
implication of PAC1 receptors, known to be the form expressed in
gonadotrophs (7, 15, 59). PAC1 receptors are capable of signaling via
cAMP and to a lesser extent via inositol phosphate and
Ca2+, however, an involvement of the cAMP/PKA pathway is
supported by the following: 1) both 8-Br-cAMP and cholera toxin mimic
in a similar manner the effects of PACAP on NOS I; and 2) in a
dose-dependent way, H89 prevented these PACAP actions. In
contrast, neither PKC nor the MAPK cascade (the activation of which may
occur via PKC in gonadotrophs) are involved in this process because
TPA, a potent activator of PKC, is unable to generate NOS I. In
addition, both the PKC inhibitor GF109203X and the MAPK inhibitor
PD98059 are unable to inhibit the production of NOS I by PACAP.
Interestingly, there could be a possible contribution of extracellular
Ca2+ in this process taking into consideration the partial
inhibition of PACAP induction of NOS I by EGTA and the lack of
induction of NOS I in response to the Ca2+ ionophore A23187
when added alone. The latter is a potent stimulator of LH and FSH release.
Because PACAP increases the NOS I level via cAMP and a strong increase
in NADPH diaphorase staining is apparent only in gonadotrophs that, in
addition, can be accompanied by an amplified
L-NMMA-sensitive cGMP formation under GnRH, this effect of
PACAP seems highly specific to gonadotrophs. In particular because
folliculo-stellate cells show no change in NADPH diaphorase staining
even though they contain detectable levels of NOS I and are known to
respond to PACAP through cAMP (51), suggesting that at least one
element required for PACAP/cAMP induction of NOS I in gonadotrophs is
lacking in folliculo-stellate cells. Indeed, in contrast to
gonadotrophs, folliculo-stellate cells did not stain more intensely for
NADPH diaphorase when 8-Br-cAMP or cholera toxin was added instead of
PACAP, supporting this hypothesis and further indicating that the
eventual missing factor could be localized downstream of PKA.
The mechanism by which PACAP may induce NOS I in gonadotrophs appears
complex. Whereas cAMP, as well as GnRH (34), was shown to increase the
transcriptional activity of a pituitary-specific NOS I promoter in the
murine gonadotrope cell line L The NADPH diaphorase detected in this study clearly relies on NOS I
because of the absence in nonstimulated cells and the lack of induction
with PACAP of either NOS II or NOS III (see Fig. 1). In agreement with
our results, NOS II was not found in normal rat and human pituitary
tissue or long term rat pituitary cell cultures (27, 32, 62, 63),
however, some authors (62, 63) have detected a weak expression of NOS
III probably originating from blood vessels.
Based on previous studies (17, 18) and using hemipituitaries from
normal rats we found that PACAP is as potent as GnRH in generating a
quasi-immediate production of cGMP. Nevertheless, when investigating
the implication of the NOS/NO/sGC cascade in PACAP action using
classical inhibitors such as L-NMMA for NOS I and ODQ for
sGC, an unexpected major divergence was observed. GnRH generates cGMP
exclusively through the activation of the NOS/NO/sGC system, however,
PACAP induces, in addition to the latter system, another mechanism for
cGMP production. Surprisingly considering the implication of either a
single or a double mechanism, both GnRH and PACAP produce at maximum
concentrations the same elevation in cGMP. Whether this is fortuitous
or not is impossible to determine presently. Nevertheless, as a
consequence, it can be concluded based on the effects of NOS or sGC
inhibitors on cGMP that PACAP is in fact two times less efficient than
GnRH at activating the NOS/NO/sGC system. These differences in efficacy could be explained by the respective potencies of each hormone to
elevate intracellular Ca2+ in the gonadotrophs (13).
Nevertheless, whether the gonadotrophs are the only site of cGMP
production under PACAP stimulation is an additional subject of
interrogation, as well as the two mechanisms of production,
i.e. NOS/sGC-dependent and -independent involved in the process.
Recent studies have provided some evidence for expression of sGC in the
whole population or enriched pituitary cell types including enriched
somatotrophs and lactotrophs (55). In the latter cell preparations,
however, thyrotropin-releasing hormone (that can act on
thyrotrophs and lactotrophs) and most surprisingly, GnRH, are
unable to induce a marked increase in cGMP production. The reduced
effect of GnRH on cGMP in this study could be the result of
experimental conditions because the cells were used 16 h after
plating compared with the 48 h usually preferred by us and others
for a complete recovery of the cell response to GnRH. Indeed, it has
long been established that GnRH, GnRH analogs, or NO donors can induce
a rapid, concentration-dependent formation of cGMP in
cultured pituitary cells as well as in pituitary tissue (17, 64, 65).
This is further documented in our present study that, in addition,
shows clearly the dependence of the GnRH response upon the degree of
NOS I expression (cf. Fig. 9B). Using a direct
in situ immunodetection technique to reveal cGMP-producing cells in response to NO donors in rat pituitary slices, Yamada et
al. (66) identified essentially gonadotrophs, suggesting that
these cells primarily contained a functionally reactive sGC, or enough
sGC to produce detectable levels of cGMP under these conditions.
Surprisingly, in such studies no cGMP response to NO donors could be
detected in folliculo-stellate cells (66). The same was recently
observed by the McArdle's group (67) using a folliculo-stellate cell
line (TtT-GF) and classical cGMP assay. The observation that GH3 cells,
which readily express sGC (55), did not elicit a cGMP response to NO
donors (67) reinforces the idea that activation of sGC could be
complex. Collectively, the data strongly argue in favor of a major
influence of PACAP acting as a NO/cGMP elicitor in gonadotrophs. This
idea is further reinforced by the fact that gonadotrophs also express
high levels and can regulate NOS I in rat pituitary.
The question remains as to the mechanisms and cell type(s) involved in
the NOS/NO-independent production of cGMP. Whether sGC might be
activated via cAMP by phosphorylation through PKA has been evoked (68).
We evaluated this eventuality using 8-Br-cAMP or cholera toxin. Both
substances, however, revealed to be ineffective in generating cGMP.
Complementary to this, H89 had no effect on the cGMP produced in
response to PACAP. Thus in our conditions, a role for cAMP in the rapid
production of cGMP could be totally excluded, whether
NOS/NO-dependent or not. These data further reciprocally
(and implicitly) demonstrate that NOS I protein up-regulation is not
implicated in, or required for, the PACAP-induced elevation of cGMP
over the very short period of time concerned.
The only other potential mechanism for the production of cGMP involves
the membrane-bound guanylate cyclases that can be activated by
natriuretic peptides (69). Consistently, the anterior pituitary and in
particular gonadotrophs, contain high concentrations of C-type
natriuretic peptide (CNP), whereas a body of experimental data
indicates that gonadotrophs, lactotrophs, corticotrophs, and
folliculo-stellate cells but neither somatotrophs nor thyrotrophs express transmembrane type B natriuretic peptide receptor and respond
to CNP by increasing cGMP production (67, 70). How could PACAP
stimulate the membrane-linked GC and induce the NOS/NO/sGC-independent production of cGMP remains to be determined. One possibility could rely
on the direct alteration by PACAP of natriuretic peptide receptor type
B activity as reported by Murthy et al. (71) for the
natriuretic peptide receptor type C in smooth muscle. Another could
come from an initial, direct or indirect PACAP-induced release of CNP
resulting in the auto- and/or paracrine activation of its specific
GC-bound receptor. As gonadotrophs contain the vast majority of
pituitary CNP, such an eventuality implies that GnRH, in contrast to
PACAP, is unable to induce CNP release.
In conclusion, the fact that cGMP can be generated under hormonal
stimulation in gonadotrophs and other cells, moreover through single or
double cascade mechanisms, suggests that this cyclic nucleotide may
have an important role in the anterior pituitary. In gonadotrophs this
idea is further reinforced by the possibility that NOS I protein can be
up-regulated by at least two neurohormones, GnRH and PACAP, that can
occur during an important physiological event such as the midcycle
surge in the female rat (18). To date, there has been no clear
demonstration of a biological function of the cGMP in these cells
especially with regards to gonadotropin release, whether the cyclic
nucleotide is produced through NOS/NO/sGC or CNP/natriuretic peptide
receptor type B (25, 27, 64, 72). Indeed the redundancy of the hormonal
transduction pathways in gonadotrophs together with the pituitary cell
heterogeneity might be responsible for such a situation because it
makes its study complicated. Based on other systems, cGMP may
play a role in the regulation of cellular Ca2+ metabolism
and gene transcription through a small group of effectors, i.e. cGMP-dependent kinases and
phosphodiesterases and cGMP-gated ion channels (67), the implication
and roles of which remains to be explored in gonadotrophs.
We thank Dr. Albert F. Parlow and the
NIDDK's National Hormone and Peptide Program for kind provisions with
highly potent antisera against FSH *
This work was supported in part by grants from the CNRS and
the Université Pierre et Marie Curie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of funds from the Ministère de l'Education
Nationale, de la Recherche et de la Technologie, and the Association pour la Recherche sur le Cancer.
¶
To whom correspondence should be addressed: UMR-CNRS, 7079 Physiologie et Physiopathologie, Université P. & M. Curie, Case 256, 75252 Paris Cedex 05, France. Tel.: 33-1-44-27-26-48; Fax: 33-1-44-27-26-50; E-mail: Raymond.Counis@snv.jussieu.fr.
Published, JBC Papers in Press, September 18, 2002,DOI 10.1074/jbc.M203763200
The abbreviations used are:
LH, luteinizing
hormone;
CNP, C-type natriuretic peptide;
GnRH, gonadotropin-releasing
hormone;
FSH, follicle-stimulating hormone;
MAPK, mitogen-activated
protein kinase;
NO, nitric oxide;
NOS, nitric-oxide synthase;
NOS I, NOS type I or neuronal NOS;
NOS II, NOS type II or inducible NOS;
NOS
III, NOS type III or endothelial NOS;
L-NMMA, NOS
inhibitor;
ODQ, soluble guanylate cyclase inhibitor;
PACAP, pituitary
adenylate cyclase-activating polypeptide;
PACAP38, the 38-amino acid
form of PACAP;
PACAP27, the 27-amino acid form of PACAP;
PKA, protein
kinase A;
PKC, protein kinase C;
sGC, soluble guanylate cyclase;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
8-Br-cAMP, 8-bromo-cAMP;
IBMX, 3-isobutyl-1-methylxanthine;
PBS, phosphate-buffered saline;
RIA, radioimmunoassay.
Pituitary Adenylate Cyclase-activating Polypeptide Stimulates
Nitric-oxide Synthase Type I Expression and Potentiates the cGMP
Response to Gonadotropin-releasing Hormone of Rat Pituitary
Gonadotrophs*
,
ABSTRACT
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INTRODUCTION
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, LH
, and FSH subunits (9, 10). There is
significant evidence that PACAP may act in synergy with GnRH to induce
gonadotropin release with at least several of these effects mediated
through cAMP (10-13). In addition, recent studies in our laboratory
have demonstrated that PACAP stimulates the promoter activity of rat
GnRH receptor via the cAMP/protein kinase A (PKA) pathway (14).
Finally, most of the pituitary endocrine cell types as well as
folliculo-stellate cells have been shown to possess receptors to PACAP
(7). Gonadotrophs would express essentially, if not exclusively,
PACAP-specific type 1 (PAC1) receptors that respond to PACAP via
activation of phospholipase C in addition to adenylate cyclase, and
Ca2+ mobilization (7). In these cells PACAP would be a more
potent stimulator of cAMP (EC50 
3 nM) than
of inositol phosphate production (EC50 20 nM) (15).
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20 °C for quantitative analysis of
gonadotropin release and the cells were washed with cold
phosphate-buffered saline (PBS; 10 mM sodium phosphate, 150 mM NaCl, pH 7.4) before protein extraction.
1523 to +387) placed upstream
of the firefly luciferase (Luc) reporter gene. Transfection assays were
performed using the pituitary gonadotrope cell lines LT2 and T3-1
generated by P. Mellon (40, 41). The cells were cultured in Dulbecco's
modified Eagle's medium (Sigma) with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate. Cells, grown
at 37 °C in a humidified atmosphere with 5% CO2, were
transfected using the LipofectAMINE Plus assay according to the
manufacturer's recommendations (Invitrogen). Briefly, 1 × 105 cells were plated in 24-well plates in triplicate wells
24 h before transfection. 200 ng of promoter construct and 100 ng
of pTK-Renilla (Promega Corp., Lyon, France) were combined with 0.6 µl of LipofectAMINE and 0.42 µl of Plus reagent in 250 µl of
Opti-MEM (Invitrogen). The mixture was incubated for 15 min at room
temperature before being added to the cells. After 6 h, the medium
was replaced by Dulbecco's modified Eagle's medium, 2% fetal calf
serum, and penicillin/streptomycin, in the presence or absence of
either substances to be tested. After 18 h, cells were harvested,
lysed, and luciferase (firefly and renilla) activities were measured using the dual-luciferase reporter assay system (Promega) (42). The
ratio of firefly luciferase to renilla luciferase activity served as a
measure of normalized luciferase activity.
-NADPH, 0.1 mg/ml
nitro blue tetrazolium salt, and 0.3% Triton X-100.
antibody (number 518B7) used
at a 1/300 dilution (44). All the other cell types were identified
using rabbit polyclonal antibodies: folliculo-stellate cells with an
anti-S100 protein (Immunotech, Marseille, France; dilution 1/600),
lactotrophs with an anti-rat prolactin (number 27B14, dilution 1/200)
(45), somatotrophs with an anti-synthetic human growth hormone (NIDDK,
National Institutes of Health (NIH), number IC-4, AFP-1613102481;
dilution 1/100), corticotrophs with an anti-human ACTH (NIDDK (NIH),
number IC-2, AFP-39013082; dilution 1/200), and thyrotrophs with
an anti-rat TSH (NIDDK (NIH), number IC-1, AFP-1274789; dilution
1/200). Occasionally, FSH-containing gonadotrophs were immunoidentified
with a polyclonal anti-rat FSH (NIDDK (NIH), number IC-2, AFP-HFSH6;
dilution 1/100). After a 1-h incubation, cells were washed with PBS and
then incubated with biotinylated donkey anti-rabbit Ig
F(ab')2 (dilution 1/500) or rhodamine goat anti-mouse Ig
antibodies (dilution 1/200) for 45 min each, followed by 30 min with
streptavidin-fluorescein complex (dilution 1/100). The cells were then
washed in PBS and mounted with Vectashield (Biosys, Compiègne,
France). Controls omitting primary antibodies were also performed.
80 and 20 °C, respectively,
until use. Cells were used to test the effects of prolonged treatments
with PACAP on cGMP production. Two-day cultures (1 × 106 cells/well) were incubated for 24 h in the absence
or presence of 20 nM PACAP38 in serum-free Ham's F-10
culture medium. After medium removal, cells were incubated for 1 h
without PACAP but in the presence of 0.3 mM IBMX alone or
in combination with 1 mM L-NMMA. The medium was
renewed and the capacity of cells to produce cGMP was measured after an
incubation of 1 h in the absence or presence of 1 nM
triptorelin. Cells and medium were collected and immediately
used for cGMP assay. The cGMP was determined according to Steiner
et al. (48) using a commercial RIA kit (PerkinElmer Life
Sciences, Le Blanc-Mesnil, France) that required acetylation of
the samples (49).
0.05 was considered significant.
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10 nM). The maximum level was attained at 24 h representing a 3.59 ± 0.08-fold augmentation as compared with
nonstimulated cells and remained elevated for the rest of the period
examined (96 h). The EC50 deduced from the concentration dependence curves determined during a 24-h period was 1.36 ± 0.32 nM (Fig. 2). Neither NOS II nor NOS III were induced
by 50 nM PACAP38 or PACAP27 over 24 h (Fig.
1A) indicative of a selective action of the neuropeptide on
isoform I.
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Fig. 1.
PACAP specifically stimulates expression of
NOS I in a time-dependent manner in primary cultures of rat
anterior pituitary cells. Comparison with GnRH. A,
Western analysis of PACAP effects on NOS isoforms. Proteins (40 µg)
were extracted from rat anterior pituitary cells cultured for 24 h
in the absence ( ) or presence (+) of 50 nM PACAP38 and
resolved in SDS-PAGE. After blotting, NOS isoforms were submitted to
immunodetection with specific, immunoaffinity purified commercial
antibodies (Transduction Laboratories). Complexes on filters were
revealed using the enhanced chemiluminescence system as described under
"Experimental Procedures" and autoradiography. Reference extracts
(Ref.) for NOS I (post-hypophysis), NOS II (mouse macrophage
cells), and NOS III (human endothelial cells), respectively.
B and C, time course of PACAP38, PACAP27, and
GnRH effects on NOS I. Cultured rat anterior pituitary cells were
incubated in the absence (
) and presence of 50 nM
PACAP38 (
) or PACAP27 (
), and 1 nM GnRH agonist
triptorelin (
) for various periods of time (0-96 h). B,
proteins were extracted in parallel from stimulated and nonstimulated
cells (3 × 106) and 40-µg samples were resolved by
Western blotting as in A. Autoradiographs were analyzed by
laser densitometry. Results were expressed relative to untreated
control cells (zero time) and represent the mean ± S.E. of
triplicate samples in three independent determinations. C,
LH and FSH release into the media were determined using a RIA kit.
Values were expressed in nanograms/ml of medium. *, p 0.05; **, p 0.01; ***, p 0.001 (compared with untreated cells).
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Fig. 2.
Concentration dependence of the stimulatory
effects of PACAP on NOS I protein in rat pituitary cells. Rat
anterior pituitary cells (3 × 106) were cultured for
24 h with increasing concentrations of PACAP38 or PACAP27 (range
0-100 nM). The NOS I level was determined by Western
analysis and densitometric analysis of autoradiographs as indicated in
Fig. 1. Values were expressed relative to nonstimulated cells.
Inset shows LH and FSH released in medium (expressed
as nanograms/ml). All the results are the mean ± S.E. of
triplicate samples in three independent experiments. *,
p 0.05; **, p 0.01 (compared with
untreated cells).
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Fig. 3.
Effects of PACAP and various transduction
pathway activators on NOS I pituitary cell content. A, rat
anterior pituitary cells (3 × 106) were incubated for
24 h in the absence and presence of 50 nM PACAP38, 1 mM 8-Br-cAMP, 3 nM cholera toxin, 5 µM A23187, or 5 nM TPA. Cell protein extracts
were analyzed by Western blotting and the NOS I level was determined as
indicated in the legend to Fig. 1. Inset shows LH release
into medium as determined by RIA, to serve as comparative
functional index. B, comparison of the time course effects
of 8-Br-cAMP and PACAP on NOS I protein expression. Cells were
incubated for various times (0-96 h) in the absence ( ) and presence
of 1 mM 8-Br-cAMP () or 50 nM PACAP38 ().
All values are expressed relative to untreated cells in A,
and to untreated cells at zero time in B. Data represent the
mean ± S.E. of three different experiments. *, p 0.05; **, p 0.01 (compared with the corresponding
time point value in untreated cells).
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Fig. 4.
Influence of EGTA and various transduction
pathway inhibitors on PACAP-induced up-regulation of NOS I in cultured
rat pituitary cells. Rat anterior pituitary cells (3 × 106) were incubated for 24 h in the absence and
presence of 50 nM PACAP38 without or with 30 µM H89, 2.5 mM EGTA, 20 µM
PD98059, or 2 µM GF109203X. Cell extracts were analyzed
by Western blotting and the NOS I levels were determined as described
in the legend to Fig. 1. Inset shows LH release into
medium as determined by RIA, to serve as comparative functional
index. All values are expressed relative to untreated cells and
represent the mean ± S.E. of three different experiments. **,
p 0.01 (compared with untreated cells).
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Fig. 5.
Concentration-dependent
inhibition by H89 of PACAP effects on NOS I expression. Rat
anterior pituitary cells (3 × 106) were exposed to
H89 at the indicated concentrations (0-30 µM) without
( ) or with () 50 nM PACAP38 for 24 h, then
protein extracts were further processed for quantification of NOS I
levels as indicated in the legend to Fig. 1. Parallel changes in LH
release (measured in medium by RIA) are shown in the inset.
In each case data were pooled from three separate experiments
(mean ± S.E.) and are expressed as a percentage taking as
reference the values measured in untreated cells (absence of PACAP and
H89). *, p 0.05; **, p 0.01, as
compared with controls without H89; a, p 0.05, as compared with the corresponding basal value.
present in
the very large majority (about 90%) of gonadotrophs (the rest of
gonadotrophs express only FSH and are thus identifiable with an
anti-FSH, not shown). Under basal conditions a very faint cytoplasmic diaphorase activity was visible (Aa), however,
after a 24-h treatment with 20 nM PACAP38 or 1 mM Br-cAMP, a marked elevation in the staining was observed
(Ba and Ca, respectively) that localized
exclusively to gonadotrope cells (Bb and Cb).
Similar data were obtained after cholera toxin treatment (data not
shown). Moreover, H89 (30 µM) completely abolished the
PACAP-induced elevation of diaphorase activity seen in gonadotrophs
(Da and Db) thus confirming the immunoblot
results (Fig. 5) and further demonstrating a PKA-mediated induction of
a potentially active NOS I by PACAP in these cells. No change could be
detected in other endocrine cells identified with anti-prolactin,
anti-somatotropin, anti-thyrotropin, anti-adrenocorticotropin (not
shown), or in folliculo-stellate cells (Bc and
Cc) despite the presence of PACAP receptors on the latter
cells (7, 51).
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Fig. 6.
Change in NADPH diaphorase staining in rat
anterior pituitary cells in response to PACAP, 8-Br-cAMP, and H89:
evidence of co-localization with gonadotrope cells.
Pituitary cells (6 × 105) were plated in Lab-Tek
slides and incubated in the absence (A) and presence of 20 nM PACAP38 (B), 1 mM 8-Br-cAMP
(C), or 30 µM H89 + 20 nM
PACAP38 (D). After fixation with paraformaldehyde,
cells were processed for NADPH diaphorase (a) and
subsequently stained with a series of specific antibodies to identify
the different pituitary cell types (see "Experimental Procedures").
For clarity only the double detection of LH (b) and S100
protein (c) immunoreactive cells (i.e. the very
large majority of gonadotrophs that express LH or LH plus FSH,
and folliculo-stellate cells, respectively), is shown. LH is
visualized with rhodamine and S100 protein with fluorescein. Note the
intense NADPH diaphorase staining of glandular cells
(arrowheads) with 20 nM PACAP38 (Ba)
or 1 mM 8-Br-cAMP (Da), contrasting with the
absence of (or weak) staining in cells co-treated with both PACAP38 and
H89 (Da). As denoted by arrowheads the only
glandular cells responsive to PACAP or the cAMP analog were those
positive for LH. In contrast NADPH diaphorase staining in
folliculo-stellate cells (complete arrows) was never
significantly altered (Ac, Bc, Cc, and
Dc). The scale bar (on Aa) represents
50 µm.
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Fig. 7.
Effects of PACAP38 on pituitary cGMP
production. Rat anterior hemipituitaries were preincubated 1 h in the presence of 0.3 mM IBMX, then the medium
was renewed and hemipituitaries were further incubated for various
periods of time (15-60 min) with IBMX in the absence and presence of
50 nM PACAP38 or 10 nM triptorelin as
reference. Pituitary cGMP content was determined using an appropriate
RIA as indicated under "Experimental Procedures." Values were
corrected for variability in sampling using an external standard and
expressed in total picomoles. Data represent the mean ± S.E. of
three individual experiments performed with triplicates. **,
p 0.01, as compared with control.
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Fig. 8.
Evaluation of the cAMP/PKA and NOS/NO
pathways in the mediation of PACAP effects on pituitary production of
cGMP. Comparison with GnRH. Rat anterior hemipituitaries were
pretreated 1 h with 0.3 mM IBMX in the absence and
presence of 1 mM L-NMMA, 6 µM
ODQ, or 30 µM H89 and then incubated for a further 15 min
in the absence or presence of the same compounds or in different
combinations with 50 nM PACAP38 or 10 nM
triptorelin. Cholera toxin (3 nM) was added to
IBMX-pretreated cells only. The cGMP content was then determined as
described in the legend to Fig. 7. Data expressed as total picomoles
are the mean ± S.E. of three individual experiments performed
with triplicates. **, p 0.01, as compared with
control untreated cells; a, p 0.01 as
compared with cells treated with PACAP38 alone.
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Fig. 9.
Basal and GnRH stimulated cGMP production
after NOS I induction by PACAP. Two-day cultures (1 × 106 cells) were incubated in the absence (vehicle) or
presence of 20 nM PACAP38 for 24 h. The medium
was changed and cells were incubated a further 1 h with or without
1 nM triptorelin, in the presence of 0.3 mM
IBMX and in the absence or presence of 1 mM
L-NNMA as described under "Experimental Procedures."
The cGMP was determined in cells and media using RIA as described in
the legend to Fig. 7. A, columns represent the cGMP measured
in cell extracts or medium. The portion corresponding to the level in
the presence of L-NMMA is represented in hatched
bars. B, total (cells plus medium) cGMP after deducting the
L-NMMA values (represent NOS-dependent cGMP).
Data expressed as picomoles/106 cells are the mean ± S.E. of three individual experiments performed in triplicate.
a, p 0.01, as compared with the
unpretreated cells incubated without GnRH; b,
p 0.001 as compared with PACAP-pretreated cells
incubated without GnRH. c, p 0.05 (comparing the difference between L-NMMA values from
PACAP-treated and untreated cells).
T2 cell line we examined the effects of PACAP in the
same system. A construct consisting of the full-length NOS I promoter
placed upstream of the luciferase reporter gene was used for the
transfection studies. As shown on Fig.
10A, there was no increase
in luciferase activity in LT2 cells in response to stimulation with
50 nM PACAP38, whereas a ~2.5- and 3-fold increase was
noted in the presence of 2 mM 8-Br-cAMP and 3 nM cholera toxin, respectively. TPA (50 nM) was without effect.
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Fig. 10.
Transcriptional activity of NOS I promoter
in a gonadotrope cell line and pituitary cell content of NOS I mRNA
in response to PACAP and cAMP/PKA activation. A, L T2
cells were transiently transfected with full-length rat NOS I promoter
construct (1523 to +387) and treated with maximally effective
concentrations of PACAP38 (50 nM), 8-Br-cAMP (2 mM), cholera toxin (3 nM), or TPA (50 nM). B, LT2 and T3-1 cells were
transfected with the 
MMTV-Luc(wtCRE) vector and treated with PACAP,
cholera toxin, or TPA. Controls were treated with the vehicle alone.
Luciferase activity was normalized to the activity of TK-renilla
luciferase expression vector and expressed as fold-stimulation over
control. Each bar represents the mean ± S.D. for at
least five separate experiments, each performed in duplicate. In each
experiment, treated and control cells were compared, and different
letters indicate significant differences between treatments.
p 0.001. C, dot blot analysis of
mRNAs extracted from rat anterior pituitary cells cultured for
24 h in the absence and presence of cholera toxin (3 nM) and PACAP38 (50 nM). A representative
autoradiogram shows the NOS I and cyclophilin mRNAs after
hybridization to their respective 32P-labeled cDNA
probes.
T2
cells are able to respond to PACAP and activate the PKA pathway. This
was evaluated using a cAMP-responsive promoter containing several
copies of the canonical cAMP-responsive enhancer (TGACGTCA) placed
upstream of the MMTV-Luc(wtCRE) promoter (53). LT2 cells and, for
comparison, the better characterized T3-1 cells (15) were
transfected with this artificial promoter. Fig. 10B shows
that PACAP was capable of inducing a ~2-fold increase in the activity
of this promoter in the LT2 cells, compared with a 5-fold increase
in T3-1 cells. Similarly the response to cholera toxin was higher in
the T3-1 (8-fold) than in the LT2 cells (4.5-fold). Surprisingly
in the latter cells, TPA was as potent as PACAP in stimulating
luciferase activity. No such an effect of TPA was observed in T3-1
cells. These data thus indicate that the LT2 cell line, like the
T3-1 cell line, can respond to PACAP. However, the ability of PACAP
to activate the MMTV-Luc(wtCRE) reference construct is much lower
for the LT2 cells, and can be mimicked by PKC activation, suggesting
major differences in the intracellular signaling properties of cell lines.
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T2, surprisingly, no such an effect of
PACAP was observed. PACAP was similarly ineffective in stimulating NOS
I promoter activity in T3-1 cells (data not shown), but in this cell
line, the NOS I promoter was also unresponsive to GnRH. In addition, no
measurable changes in NOS I mRNA levels after PACAP or cAMP
induction were detected by classical blot analysis of total RNA
extracted from the cultured normal rat pituitary cells, an obvious
conclusion is that PACAP may stimulate translational efficiency rather
than transcription of NOS I mRNA. Nevertheless, in complementary
experiments we observed that both PACAP and cAMP induction of NOS I
protein in cultured pituitary cells were totally abolished by
actinomycin D. This implicates a transcriptional process in PACAP
induction. The latter process may, of course, concern a gene(s)
distinct from NOS I. However, it remains unclear why PACAP did not
increase NOS I promoter activity in LT2 cells, whereas a response
was observed after stimulation with the cAMP analog 8-Br-cAMP, or
cholera toxin, a potent inductor of the PACAP second messenger, cAMP.
An explanation for the discordant results may be the relatively low
efficacy of PACAP in activating the cAMP/PKA pathway in these cells, as
revealed by the use of the MMTV-Luc(wtCRE) reference construct.
Perhaps even more likely is an action of PACAP through the sole PLC/PKC
pathway, which does not induce the NOS I promoter. In support of this
hypothesis, it has been recently reported that PACAP was unable to
stimulate cAMP production in LT2 cells (52). Therefore, taking into
consideration all our data and the extremely complex, multifaceted
regulation of the NOS I, as has been previously described (60), the
occurrence of multiple mechanisms of NOS I induction cannot be
excluded. One possibility is the simultaneous increase of transcription and degradation of NOS I mRNA, resulting in the steady-state
accumulation of newly synthesized, more efficiently translated mRNA
molecules (60, 61).
ACKNOWLEDGEMENTS
, TSH, growth hormone, and
ACTH, and RIA kits for LH and FSH. We also express our warmest thanks
to Dr. Janet Roser, Department of Animal Science, University of
California, Davis, CA, and Dr. Dominique Grouselle, CNRS, Paris,
France, for the generous gift of purified anti-LH monoclonal
antibody and anti-rat PRL antiserum, respectively. We are grateful to
Dr. Pamela Mellon, University of California, San Diego, for kindly
providing the LT2 and T3-1 cell lines, and Dr. Dietmar Spengler,
Max-Planck Institute of Psychiatry, Munich, Germany, for the gift of
the MMTV-Luc(wtCRE) plasmid. We thank Marie-Claude Chenut, Danielle Duchêne, and Pierrette Thouvenot for their contribution in the preparation of the manuscript, cell cultures, and rat care,
respectively. We are grateful to Dr. Lisa Oliver (U-419 INSERM, Nantes,
France) for correction of the English text and editorial assistance.
FOOTNOTES
Recipient of funds from the Chancellerie des Universités de
Paris, the Association pour la Recherche sur le Cancer, and the Fondation pour la Recherche Médicale.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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