Spontaneous and Receptor-Controlled Soluble Guanylyl Cyclase Activity in Anterior Pituitary Cells
Tatjana S. Kostic,
Silvana A. Andric and
Stanko S. Stojilkovic
Endocrinology and Reproduction Research Branch National
Institute of Child Health and Human Development National Institutes
of Health Bethesda, Maryland 20892-4510
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ABSTRACT
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Nitric oxide (NO)-dependent soluble guanylyl
cyclase (sGC) is operative in mammalian cells, but its presence and the
role in cGMP production in pituitary cells have been incompletely
characterized. Here we show that sGC is expressed in pituitary tissue
and dispersed cells, enriched lactotrophs and somatotrophs, and
GH3 immortalized cells, and that this enzyme is
exclusively responsible for cGMP production in unstimulated cells.
Basal sGC activity was partially dependent on voltage-gated calcium
influx, and both calcium-sensitive NO synthases (NOS), neuronal and
endothelial, were expressed in pituitary tissue and mixed cells,
enriched lactotrophs and somatotrophs, and GH3
cells. Calcium-independent inducible NOS was transiently expressed in
cultured lactotrophs and somatotrophs after the dispersion of cells,
but not in GH3 cells and pituitary tissue. This
enzyme participated in the control of basal sGC activity in cultured
pituitary cells. The overexpression of inducible NOS by
lipopolysaccharide + interferon-
further increased NO and cGMP
levels, and the majority of de novo produced cGMP was
rapidly released. Addition of an NO donor to perifused pituitary cells
also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and
GnRH slightly increased basal cGMP production, but only when added in
high concentrations. In contrast, adenylyl cyclase agonists GHRH and
CRF induced a robust increase in cGMP production, with
EC50s in the physiological concentration range.
As in cells overexpressing inducible NOS, the stimulatory action of
GHRH and CRF was preserved in cells bathed in calcium-deficient medium,
but was not associated with a measurable increase in NO production.
These results indicate that sGC is present in secretory anterior
pituitary cells and is regulated in an NO-dependent manner through
constitutively expressed neuronal and endothelial NOS and transiently
expressed inducible NOS, as well as independently of NO by adenylyl
cyclase coupled-receptors.
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INTRODUCTION
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Guanylyl cyclases (GCs) catalyze the formation of cGMP, an
intracellular messenger molecule that is involved in regulation of
various cellular functions by activating specific cGMP-receptor
proteins. These include cGMP-dependent protein kinases I and II (1),
cyclic nucleotide-gated (CNG) channels (2), and cyclic
nucleotide-regulated phosphodiesterases (PDEs) (3). Whereas in
mammalian cells, adenylyl cyclases (ACs) exist only in membrane-bound
forms, GCs occur in both particulate (pGC) and soluble (sGC) forms.
Receptor-linked pGCs are stimulated by peptide hormones (4), and sGC
functions biologically as intracellular nitric oxide (NO) receptor and
effector (5). sGC is a heterodimeric cytoplasmic protein composed of
- and ß-subunits, and coexpression of both subunits is required to
obtain enzyme activity. In the absence of NO, sGC-derived cGMP
production is negligible, and three members of NO synthases (NOS),
neuronal (nNOS), endothelial (eNOS), and inducible (iNOS), supply
cells with NO by catalyzing the oxidation of
L-arginine to L-citrulline and NO (6).
The first two enzymes are constitutively expressed and their activation
is dependent on a rise in intracellular Ca2+
concentration ([Ca2+]i),
whereas iNOS does not require elevation in
[Ca2+]i to generate NO.
The released NO stimulates sGC by binding to the heme part of the
enzyme, causing an elevation in cGMP production (6, 7, 8).
Earlier studies have suggested that pituitary cells express pGC, and
that this enzymes activity is regulated by natriuretic peptides (9, 10). A lot of indirect evidence has also suggested the operation of sGC
in these cells. Two NOS subtypes, nNOS and iNOS, were detected in
normal and immortalized pituitary cells (11, 12, 13, 14, 15, 16, 17) and were confirmed to
be active by measurements of NO, NO2, and
NO3 under different experimental paradigms (18, 19). Consistent with the Ca2+-calmodulin
sensitivity of nNOS (6), agonists that increase
[Ca2+]i, including the
Ca2+-mobilizing TRH and
Ca2+-influx-dependent GHRH (20), were found to
modulate NO levels and hormone secretion (19, 21). However, the effects
of NO donors and NOS inhibitors on basal and agonist-induced hormone
secretion (22, 23, 24, 25, 26, 27) are not necessarily good indicators of the status of
sGC in these cells. For example, in the absence of cGMP measurements,
it is difficult to separate the direct messenger roles of NO (28) from
those mediated by cGMP.
Here we studied the expression and regulation of sGC in rat anterior
pituitary cells. Specifically, we addressed the coupling of sGC
activity with spontaneous voltage-gated Ca2+
influx. We also studied the role of AC-coupled and
Ca2+-mobilizing receptors and inflammatory
factors on sGC activity. The results of these investigations indicate
that sGC is operative in unstimulated cells and is partially controlled
by NOS. Calcium-insensitive iNOS also participates in the control of
basal sGC activity. sGC activity is further stimulated by elevation of
iNOS expression, which leads to an increase in NO production, and by
activation of adenylyl cyclase-coupled receptors, which act in an
NO-independent manner.
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RESULTS
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Dependence of Basal cGMP Production on sGC
Total cyclic nucleotide levels (cell content + released in medium)
were estimated in anterior pituitary cells 16 h after dispersion.
Cells were washed and incubated in serum-free medium in the absence or
presence of 1 mM 3-isobutyl-1-methylxanthine (IBMX), a
nonselective PDE inhibitor. At the end of the incubation period, cells
were dialyzed in an IBMX-containing medium, and cyclic nucleotide
concentrations were determined by RIA. As shown in Fig. 1A
, in the absence of IBMX during
incubation in serum-free medium pituitary cells produce cGMP in a
time-dependent manner, with the calculated half-time of 45 min. cAMP
levels also increased, but more gradually, with the calculated
half-time of 80 min. In five experiments done under similar
experimental conditions, the steady-state levels of cGMP, estimated
2 h after incubation, were 3- to 8-fold higher than cAMP.

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Figure 1. Characterization of Basal Cyclic Nucleotide
Production in Pituitary Cells
A and B, Time course of total (cell content and released) cGMP and cAMP
production in the absence (A) and presence (B) of 1 mM
IBMX, a nonselective PDE inhibitor. In this and the following figures,
data points are means ± SEM from six replicates in
one of at least three similar experiments.
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When cultured in the presence of IBMX, accumulation of cyclic
nucleotides dramatically increased over time, with the calculated
half-times of 55 and 75 min for cGMP and cAMP, respectively (Fig. 1B
).
In further parallelism to experiments without IBMX, cGMP production was
48 times higher than cAMP when cells were bathed in the presence of 1
mM IBMX. These results indicate that both enzymes, GC and
AC, are operative in unstimulated cells, and de novo cGMP
production dominates over cAMP production. Thus, cGMP, rather than
cAMP, has a potential to act as a messenger in unstimulated pituitary
cells.
In general, cGMP production in unstimulated pituitary cells could
reflect basal pGC and/or basal sGC activity, the latter controlled by
basal NOS activity. To dissociate between these two possibilities,
cells were bathed in medium containing nonselective NOS inhibitors,
Nw-monomethyl-L-arginine
(L-NMMA) and
Nw-nitro-L-arginine
methyl ester (L-NAME) (7). As shown in Fig. 2
, A and B, both compounds inhibited
basal cGMP production in a concentration-dependent manner. Consistent
with the specificity of their action, only a minor decrease in basal
cAMP levels was observed in L-NMMA- and
L-NAME-stimulated cells. The residual cGMP
production observed in the presence of high NOS inhibitor
concentrations was less than 10%, indicating that the majority of
basal cGMP production is derived by NO-sensitive sGC. In line with
this, RT-PCR and Western blot analysis revealed the presence of sGC in
pituitary tissue and mixed cells, enriched lactotrophs and
somatotrophs, and GH3 immortalized cells (Fig. 2
, C and D). These results indicate that in a mixed population of
pituitary cells NOS are active and produce NO to levels that are
sufficient to trigger cGMP production by sGC.

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Figure 2. Dependence of Basal cGMP Production on sGC Activity
A and B, Concentration-dependent effects of two nonselective NOS
inhibitors, L-NAME (A) and L-NMMA (B), on cAMP
and cGMP accumulation in pituitary cells during a 30-min incubation in
the absence of PDE inhibitors. C, Expression of sGC transcripts in
pituitary cells. Reverse transcriptional PCR analysis was performed
using the sGC-ß1 (upper panel) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH, bottom
panel) specific primers. D, Western blot analysis of sGC
expressed in pituitary cells.
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Expression of NOS in Pituitary Cells
To examine the expression of NOS, RT-PCR analysis was done in
pituitary tissue, dispersed pituitary cells, enriched somatotrophs and
lactotrophs, GH3 immortalized cells, and control
tissues. The transcripts for nNOS were present in both cultured
pituitary cells (Fig. 3A
, lane 1) and
pituitary tissue (lane 3). Our study also revealed the presence of nNOS
transcripts in GH3 cells (Fig. 3B
, lane 1) and
enriched lactotrophs (lane 3) and somatotrophs (lane 5). The
transcripts for eNOS were also detected in pituitary tissue (Fig. 4A
, lane 3), mixed populations of
pituitary cells (lane 1), as well as in lactotrophs (Fig. 4B
, lane 3),
but not somatotrophs (lane 5). In further agreement with the expression
of eNOS in secretory cells, the transcripts for eNOS were also detected
in GH3 cells (Fig. 4B
, lane 1).

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Figure 3. Expression of nNOS Transcripts in Pituitary
A and B, Reverse transcriptional PCR analysis was performed using the
nNOS (upper panel) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH, bottom panel) specific primers in
pituitary tissue and cultured cells, as well as in control tissue
(cerebellum). Negative controls were performed in the absence of enzyme
in the RT reaction. The RT-PCR products were separated in 1% agarose
gel and visualized with ethidium bromide.
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Figure 4. Expression of eNOS Transcripts in Pituitary
A and B, RT-PCR analysis was performed using the eNOS (upper
panel) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH,
bottom panel) specific primers in pituitary tissue and
cultured cells, as well as in cerebellum and aorta (control tissues).
For details see Fig. 3 legend.
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mRNA for iNOS was also observed in mixed population of pituitary cells
(Fig. 5A
, lane 3) and enriched
lactotrophs (Fig. 5B
, lane 3) and somatotrophs (lane 5), all cultured
for 16 h. However, no transcripts for iNOS were detected in
pituitary tissue (Fig. 5A
, lane 1) or GH3 cells
(Fig. 5B
, lane 1). Time course study with mixed anterior pituitary
cells revealed that iNOS expression began during the initial 2-h
incubation at 37 C (Fig. 5C
, lane 8) and progressed during the next
4 h (lanes 6 and 4). In 24-h-old cultures, the message was hardly
detectable (line 2), indicating the transient nature of iNOS expression
in pituitary cells.

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Figure 5. Expression of iNOS Transcripts in Pituitary
A, Expression of iNOS transcripts in pituitary tissue, and mixed
pituitary cells stimulated with and without LPS + IFN for 16 h. B,
Expression of iNOS transcripts in GH3 cells and enriched
lactotrophs and somatotrophs. C, Time course of iNOS expression in
mixed anterior pituitary cells. Tissue collection and dispersion of
cells was done at 4 C, whereas trypsin digestion (15 min) and trypsin
inhibitor incubations (15 min) were done at 37 C. After cell dispersion
and their counting, a fraction of cells was immediately dialyzed (0
time point). The residual cells were plated and incubated at 37 C for
indicated times.
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Extracellular Calcium Dependence of Cyclic Nucleotide
Production
Earlier studies have shown that the majority of anterior pituitary
cells exhibit periods of spontaneous firing of action potentials, which
are sufficient to generate global calcium signals (reviewed in Ref.
29). Because the activity of several ACs and two NOS is known to depend
on [Ca2+]i (30), we
tested the dependence of pituitary AC and sGC activity on extracellular
Ca2+. Cells cultured for 16 h were bathed
for 60 min in IBMX-free (Fig. 6A
) and
IBMX-containing media (Fig. 6B
), both supplemented with variable
Ca2+ concentrations. In cells bathed in
Ca2+-deficient medium (free
Ca2+ about 200 nM), cGMP levels were
2- to 3-fold higher than cAMP, and an increase in
Ca2+ concentration in medium led to a progressive
increase in cGMP production. This pattern was observed in both
experimental conditions, with and without inhibited PDEs. In contrast,
cAMP levels were not obviously affected by changing
Ca2+ concentration in IBMX-free medium (Fig. 6A
),
but increased in cultures bathed in IBMX-containing medium (Fig. 6B
).

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Figure 6. Dependence of AC and GC Activity on Extracellular
Ca2+ Influx
A and B, Concentration-dependent effects of extracellular
Ca2+ on basal cAMP and cGMP production in cells bathed for
60 min in the absence (A) and presence (B) of 1 mM IBMX. C,
Comparison of the effects of nifedipine, a specific blocker of L-type
voltage-gated Ca2+ channels, with depletion of
extracellular Ca2+ on cAMP and cGMP accumulation during
60-min incubation. Asterisks indicate
P < 0.05 vs. control
(+Ca2+).
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In further experiments, cells were bathed in IBMX-free and
Ca2+-containing medium supplemented with 1
µM nifedipine, a specific blocker of L-type
voltage-gated Ca2+ channels. Figure 6C
shows that
the blockade of voltage-gated Ca2+ influx was
accompanied by a significant decrease in cGMP level, comparable to that
observed in cells bathed in Ca2+-deficient
medium. On the other hand, cAMP levels were not significantly affected
by addition of nifedipine. These results indicate that pituitary GC
activity is partially dependent on spontaneous voltage-gated
Ca2+ influx, and that a significant portion of
cGMP production occurs independently of the status of extracellular
Ca2+. Adenylyl cyclase is also positively coupled
to Ca2+ influx, but the basal PDE activity is
sufficient to keep cAMP levels low.
Consistent with the expression of functional nNOS and its participation
in control of basal sGC activity in unstimulated cells, addition of
N5-(1-imino-3-butenyl)-L-ornithine
(vinyl-L-NIO), a relatively specific nNOS blocker
(31), led to a dose-dependent decrease in basal cGMP production (Fig. 7A
). The level of inhibition of cGMP
production by high (10 µM) concentration of
this blocker was similar to that observed in cells after removal of
extracellular calcium, suggesting that nNOS mediates the coupling of
voltage-gated Ca2+ influx with sGC signaling
pathway. It is also likely that eNOS expressed in lactotrophs
participates in the control of basal sGC activity. Since we do not know
of a specific inhibitor of this enzyme, this hypothesis was not
tested.

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Figure 7. Effects of NOS-Specific Inhibitors on Basal cGMP
and NO Production
A, Concentration-dependent effects of vinyl-L-NIO, a
relatively specific nNOS inhibitor, on basal cGMP production in
pituitary cells incubated for 60 min in the absence of PDE inhibitors.
Asterisks indicate P < 0.05
vs. control. B, Concentration-dependent effects of
aminoguanidine, a relatively specific iNOS inhibitor, on basal cAMP and
cGMP production in pituitary cells incubated for 60 min in the absence
of PDE inhibitors. To exclude extracellular Ca2+-dependent
cGMP production, cells were bathed in medium containing about 200
nM Ca2+. Inset, Effects of
aminoguanidine (AG) on nitrite production in pituitary cells cultured
overnight. Dotted lines illustrate the calculated
EC50 values.
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As described above, depletion of extracellular
Ca2+ did not reduce cGMP production to the levels
observed in cells bathed in the presence of high concentrations of
nonselective NOS inhibitors. These results suggest that sGC in
unstimulated pituitary cells used in our studies also produce cGMP
independently of voltage-gated Ca2+ influx,
i.e. through transiently expressed iNOS. Furthermore,
extracellular Ca2+-independent cGMP production
was inhibited by aminoguanidine, a specific blocker of iNOS (32), in a
concentration-dependent manner, with an EC50 of
about 70 µM (Fig. 7A
), comparable to that
observed in other tissues expressing iNOS (33). Basal NO production was
also inhibited by aminoguanidine in a concentration-dependent manner
and with an EC50 comparable to that observed in
cGMP measurements (Fig. 7B
, inset).
Dependence of sGC Activity on NO Levels
To increase NO levels above the basal, pituitary cells were
treated with lipopolysaccharide + interferon-
(LPS + IFN) for
16 h. As expected, these cells exhibited an elevated expression of
mRNA for iNOS compared with untreated cells (Fig. 5A
, line 5
vs. line 3, respectively). Consistent with the functional
expression of this enzyme, nitrite accumulation in cells overexpressing
iNOS was significantly elevated compared with control cells in all time
points examined (Fig. 8A
). cGMP
production was also significantly elevated in LPS + IFN-treated cells
compared with untreated cells (Fig. 8B
). Under these experimental
conditions, there was a linear relationship between
NO2, a stable product of NO, and cGMP levels
(Fig. 8C
). Aminoguanidine inhibited NO production in cells
overexpressing iNOS in a concentration-dependent manner (Fig. 8D
).
Finally, removal of extracellular Ca2+ only
slightly (
10%) reduced NO and cGMP production in cells
overexpressing iNOS. Thus, the increase in NO production in pituitary
cells is accompanied with proportional elevation in cGMP production.
Furthermore, pituitary cells have a potential to respond to
inflammation by expressing iNOS, an action that leads to increase of
two messengers, NO and cGMP.

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Figure 8. Overexpression of iNOS in Pituitary Cells
A and B, Time course of nitrite (A) and cGMP (B) accumulation in
controls (open circles) and cells overexpressing iNOS
(closed circles). C, Relationship between nitrite levels
and cGMP accumulation. D, Concentration-dependent effects of
aminoguanidine (AG) on nitrite production in pituitary cells
overexpressing iNOS. Data points are derived from panels A and B;
r = 0.97. Basal cGMP levels present in cells at the
beginning of stimulation (0 time point) were subtracted. Experiments
were done in pituitary cells incubated in medium without PDE
inhibitors.
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We also examined the kinetics of cGMP release in controls and cells
overexpressing iNOS. The time course study indicated that the majority
of de novo produced cGMP in control cells was released in
medium (Fig. 9A
, left panel).
A similar ratio of released vs. intracellular cGMP levels
was observed in cells overexpressing iNOS (Fig. 9A
, right
panel). Nitrite accumulation in and outside of cells was also
analyzed. As shown in Fig. 9B
, cell content of nitrite in unstimulated
and LPS + IFN-treated cells was high and further increased during
incubation. On the other hand, no nitrites were detected in medium
immediately after washing the cells, and there was a progressive
increase during incubation. Because NO easily diffuses between
intracellular and extracellular medium, these results indicate that the
majority of NO was transformed to NO2 in medium.
Thus, in both unstimulated and stimulated cells medium nitrite
concentration reflects the activity of NOS better than its cellular
concentration.

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Figure 9. Relationship between Cell Content and Released cGMP
in Unstimulated Cells
A and B, Time course of cGMP (A) and NO (B) accumulation in cells and
medium in unstimulated cells (left panel) and cells
overexpressing iNOS (right panel), cultured in medium
without PDE inhibitors. Asterisks indicate
P < 0.05 vs. control (0 time
point).
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These experiments in static cultures raised the possibility that the
kinetics of liberation of cGMP could be examined in perifusion system
in a manner comparable to that commonly used for analysis of kinetics
of pituitary hormone secretion. To do this, pituitary cells attached to
beads (15 x 106/column) were perifused with
medium containing 0.1 mM
N-ethylethanamine:1,1-diethyl-2-hydroxy-2-nitrosohydrazine
(DEA), a rapidly releasable NO donor. Figure 10A
(open circles)
illustrates nitrite levels measured in medium after addition of DEA,
and Fig. 10B
(open circles) illustrates cGMP levels measured
in the same samples. The profiles of DEA-derived nitrite were similar
in columns with and without cells (not shown), confirming that
transformation of NO into NO2 does not require
cells and occurs rapidly in water solution. Thus, the measured
NO2 levels reflect the kinetics of its
accumulation in medium.

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Figure 10. Release of cGMP by Perifused Pituitary Cells
A, Time course of nitrite accumulation in medium from perifused cells
(open circles). To calculate the rate of nitrite
production, a sigmoidal curve (solid line) was employed
to fit experimental data. B, Comparison of the rate of nitrite
production and cGMP release. The first derivative of sigmoidal function
(solid line) reflects the calculated rate of
NO2 production after addition of DEA in medium. Open
circles indicate the measured cGMP in medium from perifused
pituitary cells. Samples were collected every minute during a 2-h
perifusion.
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To fit experimental data (open circles, Fig. 10A
) and
calculate the rate of nitrite production, a sigmoidal curve
(full line, Fig. 10A
) was employed. The first derivative of
this function (shown in Fig. 10B
as full line) reflects the
rate of NO2 production after addition of DEA in
medium. There was an 8-min delay between the initial increase in
NO2 accumulation and initial cGMP release, and a
22-min delay in peak NO2 and cGMP production
(Fig. 10B
). In the same samples, cAMP was not detectable. These results
indicate that the kinetics of cGMP production by sGC reflect the
kinetics of NO release and that rapid release of this nucleotide into
medium protects against the action of PDEs.
Receptor-Regulation of sGC Activity
To examine the potential role of sGC in receptor-mediated
signaling, cells were stimulated with several
Ca2+-mobilizing and AC-coupled receptors. The
Ca2+-mobilizing TRH receptors are expressed in
thyrotrophs and lactotrophs, whereas GnRH receptors are expressed only
in gonadotrophs. Their activation by TRH and GnRH also led to a small,
but significant elevation in cGMP production when added in a
submicromolar to micromolar concentration range (Fig. 11
, A and B, right panels).
Consistent with the cross-coupling of these receptors to AC, cAMP
production was also elevated (left panels).

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Figure 11. Concentration-Dependent Effect of TRH and GnRH on
Cyclic Nucleotide Accumulation
Cells were bathed in medium with IBMX for 60 min.
Asterisks indicate P < 0.05
vs. control.
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AC-coupled GHRH and CRF receptors are expressed in somatotrophs and
corticotrophs, respectively. As expected, their activation led to an
increase in cAMP production in a concentration-dependent manner (Fig. 12
, A and B, left panels).
GHRH and CRF also induced a concentration-dependent increase in cGMP
production (right panels), indicating the cross-coupling of
these receptors to sGC signaling pathway. In further accord with this,
addition of NS 2028, a specific blocker of sGC, abolished GHRH and
CRF-induced cGMP production, without affecting cAMP production (Fig. 12C
). In parallel to LPS + IFN-stimulated cells, GHRH-induced
stimulation of cGMP production was also observed in cells bathed in
Ca2+-deficient medium (not shown). In contrast to
LPS + IFN-stimulated cells, no increase in NO production was
detected in GHRH-stimulated cells (Table 1
). These results indicate that
AC-coupled receptors also stimulate sGC at basal NO production,
i.e. independently of their action on
Ca2+ signaling and NOS activation.

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Figure 12. Activation of AC and sGC by GHRH and CRF
A and B, Concentration-dependent effect of GHRH and CRF on cyclic
nucleotide accumulation. C, Effects of NS2028, a specific inhibitor of
sGC, on GHRH and CRF-induced cGMP production. Cells were bathed in
medium with IBMX for 60 min. Asterisks indicate
P < 0.05 vs. control.
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DISCUSSION
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The hypotheses addressed in this study emerged from investigations
on cyclic nucleotides and calcium signaling in pituitary and other
neuroendocrine cells. Two recent reports suggest the expression of
CNG-like channels in pituitary and hypothalamic cells and their
potential roles in the control of Ca2+ signaling
and secretion (34, 35). There are three types of CNG channels: rod,
cone, and olfactory; cGMP and cAMP are equipotent in the activation of
cone and olfactory CNG channels, whereas cGMP is more potent in
regulating rod CNG channels (2). Pituitary cells also express NOS (14, 15, 36), and the majority of dispersed and in situ pituitary
cells exhibit periods of spontaneous electrical activity associated
with voltage-gated Ca2+ influx (29, 37). Thus, it
is possible that the relationship between cGMP and
Ca2+ signaling pathways in pituitary cells is
bidirectional, i.e. the rise in
[Ca2+]i may represent an
effective signal for the activation of NOS, and the NO-controlled sCG
may supply the cells with sufficient cGMP to activate CNG channels.
Once elevated, [Ca2+]i
can also modulate the activity of several other proteins involved in
cyclic nucleotide signaling pathway. Two AC isozymes, ACI and ACVIII,
are stimulated by Ca2+, whereas ACV and ACVI are
inhibited by this cation (30), suggesting that cAMP has a potential to
act as a messenger in control of Ca2+ signaling
in unstimulated cells, as it does in GHRH- and CRF-stimulated cells
(29). Calcium also inhibits sGC directly (38, 39) and enhances the
activity of PDEs (3), leading to the termination of cyclic nucleotide
intracellular signaling functions. Furthermore,
Ca2+ inhibits CNG channels (2) and stimulates NO-
and cGMP kinase-regulated K+ channels (28, 40),
and both actions should inhibit electrical activity and voltage-gated
Ca2+ influx. These observations indicate that the
interactions between Ca2+ and cyclic nucleotide
signaling pathways are extremely complex and cell specific,
i.e. they depend on the subtypes of enzymes and channels
expressed in a particular cell type.
In general, Ca2+ sensitivity of ACI and ACVIII in
excitable cells provides a potential coupling mechanism between
voltage-gated Ca2+ influx and cAMP production in
the absence of receptor stimulation. Here we show that pituitary AC
activity is facilitated by spontaneous voltage-gated
Ca2+ influx, but the impact of
Ca2+ influx-dependent cAMP production is
diminished by PDEs. Only during receptor activation does AC-derived
cAMP production dominate over PDE activity, as documented in
experiments with GHRH. Basal cGMP production in pituitary cells is also
dependent on spontaneous voltage-gated Ca2+
influx, and steady-state cGMP levels are 3- to 8-fold higher than cAMP
levels. This finding suggests that cGMP, rather than cAMP, plays a
messenger role in unstimulated cells, including control of
voltage-gated Ca2+ influx.
Several lines of evidence demonstrate that basal cGMP production in
pituitary cells is mediated by sGC. First, basal cGMP production was
dramatically reduced by addition of several NOS inhibitors, indicating
its dependence on NO signaling. Second, the addition of NO donors led
to an increase in cGMP production, with a delay in cGMP release of
about 8 min. Third, inhibition of sGC by two specific inhibitors,
1H-(1, 2, 4)oxadiazolo(4, 3,1)quinoxalin-1-one and
4H-8-bromo-1,2,4-oxadiazolo(3, 4-d)benz(b)(1, 4)oxazin-1-one, decreased basal cGMP (38). In accord with these
findings, RT-PCR and Western blot analyses revealed the presence of sGC
in pituitary tissue, mixed pituitary cells, enriched lactotrophs and
somatotrophs, and GH3 cells.
Our results indicate that the coupling of voltage-gated
Ca2+ influx to sGC occurs indirectly, through
activation of Ca2+-sensitive NOS. The present
data further indicate that nNOS is expressed in a mixed population of
pituitary cells. The enzyme is also expressed in intact pituitary
tissue, suggesting the potential in vivo relevance of the
nNOS-sGC signaling pathway in normal physiological conditions. Several
previously published studies have shown the expression of nNOS in
folliculo-stellate and secretory pituitary cells, as well as in
immortalized pituitary cells (14, 15, 16, 17, 36). Here we show that enriched
somatotrophs and lactotrophs also express nNOS. This enzyme probably
mediates the coupling of voltage-gated Ca2+
influx with sGC, since extracellular
Ca2+-dependent cGMP production was inhibited by
vinyl-L-NIO, an nNOS-selective inhibitor (31). In
line with this idea, in situ measurements of electrical
activity and [Ca2+]i
indicate that these cells are spontaneously active and can generate
intra- and intercellular Ca2+ signals (29, 37).
Furthermore, Western blot analysis indicates that nNOS expression in
pituitary cells varies during the estrous cycle and that the proestrus
rise in nNOS expression is accompanied by elevation of basal cGMP
levels (12). Also, estrogens were found to down-regulate nNOS in
anterior pituitary cells and GH3 tumors (41).
The finding that eNOS is expressed in pituitary tissue and mixed
pituitary cells is novel. It was believed that eNOS expression is
restricted exclusively to vascular endothelium and that the presence of
this enzyme in most tissues is generally attributable to the vascular
endothelium contained in those tissues (6). However, here we show that
eNOS transcripts are also present in enriched lactotrophs, as well as
in GH3 cells, demonstrating that secretory cells
can also express this enzyme. In the absence of a specific blocker for
this subtype of the enzyme, we were unable to estimate its
participation in the control of basal sGC activity in secretory
cells.
In addition to eNOS and nNOS, cultured pituitary cells also express
transcripts for Ca2+-insensitive iNOS. The
expression of functional enzyme in cultured cells is indicated by the
incomplete inhibition of cGMP productions in cells bathed in
Ca2+-deficient medium and in cells with inhibited
nNOS. Extracellular Ca2+-independent NO and cGMP
production was inhibited by aminoguanidine, in concentrations that are
specific to iNOS (32). The expression of iNOS in pituitary cells, but
not in pituitary tissue, reflects a reaction of cells to dispersion
and/or culturing conditions and may affect spontaneous
Ca2+ signaling in these cells. Transcripts for
iNOS were first observed after incubation for 2 h after the cell
dispersion and reached peak in expression after incubation for 6
h. During longer incubation times, a gradual decrease and disappearance
of the transcripts for iNOS were observed. When cultured in
serum-containing medium, immortalized P11 cells also express iNOS and
produce a proinflammatory-like factor (11). The ability of mixed
anterior pituitary cells to overexpress functional iNOS in response to
LPS + IFN stimulation is also in accord with published data (11, 13, 18), suggesting that this enzyme may mediate the interactions between
immune and endocrine systems.
The ability of AC-coupled GHRH receptors to stimulate sGC is in line
with previously published findings that GHRH stimulates the synthesis
of NO. This synthesis occurs at least partially through cAMP (21),
suggesting that phosphorylation of NOS may account for elevation in NO
production. GHRH also stimulates electrical activity and voltage-gated
Ca2+ influx in pituitary somatotrophs (34, 42, 43, 44), suggesting that Ca2+ may mediate the
activation of NOS by this agonist. Purified somatotrophs express the
mRNA message for rod CNG channels (34), further indicating that both
cAMP and cGMP may participate in GHRH-stimulated electrical activity in
these cells. However, the present data clearly indicate that GHRH, as
well as CRF, the other agonist acting through an AC signaling pathway,
can induce a robust increase in cGMP production in extracellular
Ca2+ and NO-independent manner.
Ca2+-mobilizing TRH and GnRH receptors were less
potent in activating sGC, further indicating that signals other than
Ca2+/NO can elevate sGC activity. In accord with
this, protein kinase A and protein kinase C were found to stimulate sGC
activity in vitro (45, 46). Further studies are required to
demonstrate the role of cAMP-protein kinase A in activation of sGC.
In summary, we show here that sGC is expressed in secretory pituitary
cells and controls basal cGMP production. Our results indicate that the
basal enzyme activity is high and partially dependent on voltage-gated
Ca2+ influx and
Ca2+-dependent NOS, the rest being controlled by
Ca2+-independent iNOS. In contrast, basal cAMP
levels are very low, and thus it is unlikely that cAMP acts as an
intracellular messenger in unstimulated cells. nNOS was identified in
enriched somatotrophs and lactotrophs, as well as in
GH3 cells, whereas eNOS was found in lactotrophs
and GH3 cells, but not in somatotrophs. Cell
dispersion and/or culturing conditions lead to transient expression of
iNOS in mixed pituitary cells, as well as in lactotrophs and
somatotrophs. This enzyme participates in control of sGC activity
in vitro during the initial 16-h incubation. Basal sGC
activity is elevated by overexpression of iNOS, indicating the
relevance of this enzymatic pathway in pathophysiological conditions.
Activation of AC-coupled receptors also leads to a robust increase in
sGC activity, the majority of which occurs in an extracellular
Ca2+ and NO-independent manner. Finally, the
presence of NOS in somatotrophs and lactotrophs and the ability of
GHRH, TRH, GHRH, and CRF to elevate sGC activity indicate that the sGC
signaling pathway is common for all secretory anterior pituitary
cells.
 |
MATERIALS AND METHODS
|
---|
Cell Cultures and Treatments
Experiments were performed on anterior pituitary cells from
normal female Sprague Dawley rats obtained from Taconic Farm
(Germantown, NY). Pituitary cells were dispersed as described
previously (38), and cultured as mixed cells or enriched lactotrophs
and somatotrophs in medium 199 containing Earles salts, sodium
bicarbonate, 10% heat-inactivated horse serum, and antibiotics.
Purification of lactotrophs and somatotrophs was done by a two-stage
Percoll discontinuous density gradient centrifugation as described in
Refs. 34, 42 . The PRL-secreting GH3 cells
(obtained from ATCC, Manassas, VA) were cultured in Hams
F12K medium supplemented with 2 mM
L-glutamine, 1.5 g/liter sodium bicarbonate, 15%
heat-inactivated horse serum, and 2.5% FBS.
To express iNOS, cells (106/well) were treated
for 16 h with 30 µg/well lipopolysaccharide + 1,000 IU/well
interferon-
(LPS + IFN), both from Sigma (St. Luis,
MO). To elevate NO levels, cells were treated with NO donors DEA and
DPTA, both from Alexis Biochemicals (San Diego, CA). Basal NOS activity
was inhibited by L-NMMA), L-NAME, and
vinyl-L-NIO) (RBI, Natick, MA). Phosphodiesterases were
inhibited by IBMX (from Calbiochem, La Jolla, CA).
cGMP, cAMP, and Nitrite Measurements
Cells (1 million per well) were plated in 24-well plates in
serum-containing M199 and incubated overnight at 37 C under 5%
CO2-air and saturated humidity. Before
experiments, medium was removed and cells were washed with serum-free
M199 and stimulated at 37 C under 5% CO2-air and
saturated humidity. cAMP/cGMP was measured in medium and in dialyzed
cells as previously described (38), using specific antisera provided by
Albert Baukal (NICHD, Bethesda, MD). The antisera used in our RIAs are
highly specific for cAMP and cGMP, i.e. there was not any
cross-reactivity at 100 pM and lower
concentrations. All samples were diluted 2- to 5-fold and cyclic
nucleotide concentrations were estimated using standard curves ranging
from 5 fmol to 1 pmol.
For measurement of total NO production, samples were initially treated
with nitrate reductase (Alexis Biochemicals) to convert nitrate to
nitrite. Sample aliquots were then mixed with an equal volume of Greiss
reagent containing 0.5% sulfanilamide and 0.05%
naphthylethylenediamine in 2.5% phosphoric acid (all from
Sigma), and the mixture was incubated at room temperature
for 10 min and the absorbance measured at 546 nm (38). Nitrite
concentrations were determined relative to a standard curve derived
from increasing concentrations of sodium nitrite.
RNA Isolation and RT-PCR
Total RNA was extracted from a mixed population of anterior
pituitary cells before and after purification using TRIZOL reagent
(Life Technologies, Inc., Gaithersburg, MD), and its
purity was determined spectrophotometrically. RNA samples were
subjected to RT-PCR to determine whether these cells contain nNOS, iNOS
and/or eNOS mRNA, as well as sGC-ß1. To eliminate residual genomic
DNA, RNA samples were treated with DNase I. Two micrograms of total RNA
from each sample with DNase I treatment, were reverse transcribed into
cDNA in a 20 µl reaction mixture containing oligo (dT)18 primer and
Superscript II reverse transcriptase (Life Technologies, Inc.) according to the suppliers instructions. An aliquot of 5
µl of the RT reaction was amplified with PCR reagent system
(Life Technologies, Inc.) in a final volume of 50 µl
containing 1.5 mM MgCl2, 0.4
µM of each primer, 0.2 mM of each
deoxynucleoside triphosphate, and 1.25 U of TaqDNA polymerase.
Sequences for sense and antisense primers, respectively, were: iNOS,
5'-ATGGCTTGCCCCTGGAAGTTTCTC-3' and
5'-CCTCTGATGGTGCCATCGGGCATCTG-3';
eNOS, 5'-TACGGAGCAGCAAATCCAC-3' and 5'-CAGGCTGCAGTCCTTTGAT-3';
nNOS, 5'-CCGTCCTTTGAATACCAGCCTGATCCATG-3' and
5'-CAGTTCCTCCAGGAGGGTGTCCACCGCAT-3'; and sGC-ß1,
5''-GATCCGCAATTACGGCG-3' and 5'-TGGAGAGGGATGTCACTACTCAG-3'.
The PCR products were analyzed by agarose gel (1%) electrophoresis and
visualized with ethidium bromide. The same volumes of samples used for
NOS analysis were also subjected to PCR reaction using GAPDH-specific
primers. Sequences for sense and antisense primers were:
5'-GGCATCCTGGGCTACACTG-3' and 5'-TGAGGTCCACCACCCTGTT-3', respectively.
Samples of RNA isolated from rat cerebellum, aortic tissue, and a mixed
population of anterior pituitary cells stimulated with LPS + IFN were
used as positive controls for nNOS, eNOS, and iNOS, respectively.
Reaction without RNA sample or RT served as negative controls.
Western Blot Analysis
Postmitochondrial fractions of anterior pituitary tissue and
dispersed pituitary cells were obtained from adult female Sprague
Dawley rats. Concentration of proteins was estimated by Bradford method
using BSA as a standard (47). Equal amounts of protein (20 µg) from
each postmitochondrial fraction were run on one-dimensional SDS-PAGE,
using a discontinuous buffer system (Novex, San Diego,
CA). The immunodetections on sGC were done with an antibody specific
for ß1-subunit (Cayman Chemical, Ann Arbor, MI). The secondary
antibody for all assays was an antirabbit IgG (goat) from
Kirkegaard & Perry Laboratories (Gaithersburg, MD) linked
to horseradish peroxidase. The reactive bands were always determined
with a luminol-based kit, and the reaction was detected by an enhanced
chemiluminescence system, using x-ray film.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Stanko Stojilkovic, Section on Cellular Signaling, Endocrinology and Reproduction Research Branch/National Institute of Child Health and Human Development, Building 49, Room 6A-36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail: stankos{at}helix.nih.gov
Received for publication December 1, 2000.
Revision received February 21, 2001.
Accepted for publication February 27, 2001.
 |
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