Dependence of Soluble Guanylyl Cyclase Activity on Calcium
Signaling in Pituitary Cells*
Silvana A.
Andric,
Tatjana S.
Kostic,
Melanija
Tomi
,
Taka-aki
Koshimizu, and
Stanko S.
Stojilkovic
From the Endocrinology and Reproduction Research Branch, NICHD,
National Institutes of Health, Bethesda, Maryland 20892-4510
Received for publication, May 22, 2000, and in revised form, October 11, 2000
 |
ABSTRACT |
The role of nitric oxide (NO) in the stimulation
of soluble guanylyl cyclase (sGC) is well established, but the
mechanism by which the enzyme is inactivated during the prolonged NO
stimulation has not been characterized. In this paper we studied the
interactions between NO and intracellular Ca2+ in the
control of sGC in rat anterior pituitary cells. Experiments were done
in cultured cells, which expressed neuronal and endothelial NO
synthases, and in cells with elevated NO levels induced by the
expression of inducible NO synthase and by the addition of several NO
donors. Basal sGC-dependent cGMP production was stimulated by the increase in NO levels in a time-dependent manner. In
contrast, depolarization of cells by high K+ and Bay K
8644, an L-type Ca2+ channel agonist, inhibited sGC
activity. Depolarization-induced down-regulation of sGC activity was
also observed in cells with inhibited cGMP-dependent
phosphodiesterases but not in cells bathed in
Ca2+-deficient medium. This inhibition was independent from
the pattern of Ca2+ signaling (oscillatory
versus nonoscillatory) and NO levels, and was determined by
averaged concentration of intracellular Ca2+. These results
indicate that inactivation of sGC by intracellular Ca2+
serves as a negative feedback to break the stimulatory action of NO on
enzyme activity in intact pituitary cells.
 |
INTRODUCTION |
Soluble guanylyl cyclases
(sGC)1 are heterodimeric
cytoplasmic proteins composed of
and
subunits that function
biologically as intracellular nitric oxide (NO) receptors and
effectors. Coexpression of both subunits is required to obtain enzyme
activity and NO regulation. The binding of NO to the sGC heme activates
the enzyme, resulting in the conversion of GTP to cGMP (1). Together
with particulate guanylyl cyclase and adenylyl cyclase (AC), these enzymes compose a family of proteins that is involved in a broad array
of cellular functions in cardiovascular, neuronal, neuroendocrine, and
other cell types (2). In this respect, sGC-generated cGMP production
allows this enzyme to transmit the NO signal to the downstream elements
of the signaling cascade, such as cGMP-dependent protein
kinase (3), cyclic nucleotide-gated channels (CNGs) (4), and
cGMP-regulated phosphodiesterases (5).
In contrast to the well established role of NO in the activation of
sGC, the mechanisms of inactivation for this enzyme have been
incompletely characterized. This is especially important for cells in
which the highly diffusible NO is intimately involved in the control of
Ca2+ signaling by facilitating Ca2+ release
and/or Ca2+ influx pathways, as in hepatocytes (6, 7),
glial cells (8), and neuroendocrine cells (9). Because the elevated intracellular calcium concentration
([Ca2+]i) stimulates the activity of two
NO-producing enzymes, neuronal NO synthase (nNOS) and endothelial NOS
(eNOS), such up-regulation would lead to overloading the cells with
Ca2+. Thus, it is reasonable to propose that the negative
feedback effect of Ca2+ on sGC would provide the necessary
mechanism to coordinate the regulation of intracellular cGMP and
Ca2+ concentrations.
To test this hypothesis, we chose rat anterior pituitary cells, the
majority of which exhibit spontaneous [Ca2+]i
transients of high amplitude that are sufficient to trigger hormone
secretion (10). These cells also express the messages for rod, cone,
and olfactory CNGs, which may participate in the generation of
spontaneous [Ca2+]i transients (11). Although not
fully characterized, the NO-signaling pathway is also expressed in
these cells (12-14) and is activated by several G protein-coupled
receptors (15-17). In our study, we initially characterized the NOS
subtypes expressed in pituitary cells and their participation in the
delivery of NO and sGC-controlled cGMP. We further characterized the
role of calcium in the control of sGC activity in intact 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 Farms (Germantown, NY). Pituitary cells
were dispersed as described previously (18) and cultured in medium 199 containing Earle's salts, sodium bicarbonate, 10% horse serum, and
antibiotics. Cell purification was done as described previously (18),
and further identification of gonadotrophs and lactotrophs was done by
the addition of specific Ca2+-mobilizing agonists
for these cells, gonadotropin-releasing hormone and
thyrotropin-releasing hormone (Peninsula, San Carlos, CA), respectively.
To express iNOS, cells (106/well) were treated for
16 h with 30 µg/ml lipopolysaccharide + 1000 IU/ml
interferon-
(LPS+IFN-
), both from Sigma. To elevate NO levels,
cells were treated with three NO donors: sodium nitroprusside (SNP)
from Research Biochemicals (Natick, MA) and
N-ethylethanamine:1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA) and 3,3'-(hydroxynitrosohydrazino)bis-1-propanamine
(DPTA), both from Alexis Biochemicals (San Diego, CA). Basal and
stimulated NOS activity was inhibited by aminoguanidine (RBI), an NOS
inhibitor. sGC activity was inhibited by
4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) and
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ), and cGMP-specific phosphodiesterases were inhibited by
vinpocetine and dipyridamole (all from Calbiochem).
Measurements of Intracellular Calcium Ion Concentration--
For
[Ca2+]i measurements, cells were incubated in
Krebs-Ringer buffer, supplemented with 2 µM fura-2/AM
(Molecular Probes, Eugene OR) at 37 °C for 60 min. Coverslips with
cells were washed with this buffer and mounted on the stage of an
Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to
the Attofluor digital fluorescence microscopy system (Atto
Instruments, Rockville, MD). Cells were examined under a ×40 oil
immersion objective during exposure to alternating 340- and 380-nm
light beams, and the intensity of light emission at 520 nm was
measured. The ratio of light intensities,
F340/F380, which reflects
changes in Ca2+ concentration, was followed in several
single cells simultaneously. To calculate the area under the
[Ca2+]i(t) curve, SigmaPlot
2000 software by SPSS was used. Average
[Ca2+]i(t) curves, shown in Fig. 8,
were calculated from traces obtained from 73 to 256 cells, base-line
values were subtracted, and the area was calculated using the
trapezoidal rule.
cGMP, cAMP, and Nitrite Measurements--
Cells (1 million/well)
were plated in 24-well plates and incubated overnight at 37 °C under
5% CO2-air and saturated humidity. Prior to the
experiments, the medium was removed and cells were washed with
Ca2+-containing medium 199 and stimulated at
37 °C under 5% CO2-air and saturated humidity. cAMP and
cGMP were measured in the medium and in dialyzed cells as
described previously (18), using specific antisera provided by Albert
Baukal (NICHD, Bethesda, MD). For measurement of total NO production
(NO2
+ NO3
), 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); the
mixture was incubated at room temperature for 10 min, and the
absorbance was measured at 546 nm (19). In experiments with
NO2
measurements, samples were not
treated with nitrate reductase. In both measurements, nitrite
concentrations were determined relative to a standard curve derived
from increasing concentrations of sodium nitrite. Concentrations of
cAMP, cGMP, and NO2 are expressed as combined values in
cell content and in medium.
Western Blot Analysis--
Postmitochondrial fractions of
anterior pituitary tissue and dispersed pituitary cells, cerebellum,
and aortic rings were obtained from adult female Sprague-Dawley rats.
Concentration of proteins was estimated by the Bradford method using
bovine serum albumin as a standard (20). Equal amounts of protein (22 µg) from each postmitochondrial fraction were run on one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a discontinuous buffer system (NOVEX, San Diego, CA). The
immunodetections on nNOS and iNOS were done with primary antibodies
from Affinity BioReagents, Inc. (Golden, CO); eNOS antibody was from
Transduction Laboratories (Lexington, KY); and
-actin antibody was
from Oncogene Research Products (Boston, MA). The secondary antibody
for all assays was an anti-mouse IgG (rabbit) from Life Technologies, Inc. (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.
 |
RESULTS |
The Characterization of NO-derived Enzymes in Pituitary
Cells--
Western blot analysis confirmed the presence of detectable
levels of nNOS in pituitary tissue (Fig.
1A, line 2),
cultured cells (line 3), and control tissue (cerebellum;
line 1). eNOS was also detectable in pituitary tissue
(line 5), dispersed pituitary cells (line 6), and
control tissue (aorta; line 4). iNOS was not detectable in
pituitary tissue (line 8) and dispersed cells (line 9), but was expressed in pituitary cells stimulated with
LPS+IFN-
for 16 h (line 7). In these experiments,
-actin expression was used as an internal standard (lines 10, 11, and 12). These results indicate that nNOS and eNOS
are constitutively expressed in mixed populations of anterior
pituitary cells and that the expression of iNOS can be induced in
dispersed pituitary cells.

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Fig. 1.
Characterization of the NO signaling pathway
in anterior pituitary cells. A, Western blot analysis
of NOS expressed in pituitary tissue (2, 5, and
8), cultured pituitary cells (3, 6,
and 9), pituitary cells stimulated with LPS+IFN-
(7), cerebellum (1), and aorta (4),
and -actin expression in aorta (10), pituitary tissue
(11), and cultured pituitary cells (12).
B, time dependence of cGMP production in
controls (basal) and in LPS+IFN- -, SNP-, DEA-, and DPTA-stimulated
cells. C, correlation between cGMP and nitrite levels in
samples collected 60 min after stimulation. In this and the following
figures, cells were cultured in medium not containing phosphodiesterase
inhibitors, if not otherwise specified. To express iNOS, pituitary
cells were stimulated with LPS+IFN- for 16 h prior to
experiment. SNP (1 mM), DEA, and DPTA (both 0.1 mM) were added immediately after replacing medium. The
results shown are mean ± S.E. from sextuplicates in one of at
least three similar experiments. All experiments were done in cultures
16 h after dispersion of cells.
|
|
The Characterization of NO-controlled cGMP Production--
Basal
cGMP production was analyzed in mixed populations of anterior pituitary
cells cultured in the absence of phosphodiesterase inhibitors. Under
these conditions, cGMP was produced in a time-dependent manner, reaching the steady-state plateau response 60-90 min after replacing medium in cultured cells (Fig. 1B, open
circles). Consistent with the role of the NO signaling pathway in
the control of cGMP production, the addition of NO donors, SNP, DEA,
and DPTA (21), was accompanied by a significant increase in cGMP
accumulation. In cells stimulated with LPS+IFN-
for 16 h, cGMP
production was also significantly elevated compared with controls and
was comparable with levels observed in cells stimulated with SNP.
Nitrite levels, which are commonly used as indicators of NO production
(22), were significantly elevated in treated cells. As expected, the profiles of nitrite levels expressed as the sum of both
NO2
and
NO3
versus
NO2
only (without nitrate reductase
treatment) were highly comparable. The levels of total nitrite measured
1 h after stimulation were similar in SNP- and
LPS+IFN-
-stimulated cells and significantly higher in cells treated
with DPTA and DEA. Finally, the levels of cGMP progressively increased
with the elevation in nitrite levels (Fig. 1C).
Both basal and LPS+IFN-
-induced cGMP productions were completely
inhibited by aminoguanidine, an NOS inhibitor (23), in the micromolar
to millimolar concentration range (Fig.
2A). In parallel to cGMP
production, nitrite accumulation in cultured medium was inhibited by
aminoguanidine (Fig. 2B). The blockers of sGC, NS 2028 (24),
and ODQ (25) also inhibited cGMP productions in a
dose-dependent manner. Fig.
3A illustrates the effect of NS 2028 on cGMP accumulation in pituitary cells during the first 60 min
of incubation. In the same samples, NS 2028 treatment did not
significantly affect cAMP production, indicating that AC is not
sensitive to this compound. NS 2028 also inhibited the SNP-induced cGMP
production (Fig. 3B) without affecting cAMP production.
Likewise, basal and SNP-induced cGMP production, but not cAMP
production, was significantly inhibited by ODQ (Fig.
4, A and B).

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Fig. 2.
Dose-dependent effects of
aminoguanidine, an NOS inhibitor, on cGMP (A) and
nitrite (B) production in pituitary cells.
Aminoguanidine was ineffective in inhibiting cGMP production induced by
1 mM SNP (not shown). Dotted lines, calculated
IC50 values. For other details see the legend of Fig.
1.
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Fig. 3.
Dependence of cAMP and cGMP production on sGC
activity (I). A, dose-dependent effects of
NS 2028, a specific inhibitor of sGC, on cGMP (open circles)
and cAMP (closed circles) production. B,
comparison of the effects of NS 2028 on basal and SNP-stimulated cAMP
and cGMP production. In this and the following figures, measurements
were done in cultures 60 min after stimulation, if not otherwise
specified. *, p < 0.01 versus
controls.
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Fig. 4.
Dependence of cAMP and cGMP production on sGC
activity (II). A, effects of ODQ, a hemoprotein
inhibitor, on cGMP production in controls and SNP-stimulated cells.
B, lack of effects of ODQ on cAMP production in controls and
SNP-stimulated cells. *, p < 0.01 versus controls.
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The Dependence of sGC Activity on
[Ca2+]i--
To study the effects of
cytosolic Ca2+ on sGC activity, the
[Ca2+]i was elevated by depolarizing cells with
high K+. Fig. 5A
illustrates that depolarization of cells with 50 mM K+ led to similar patterns of [Ca2+]i
response in somatotrophs, lactotrophs, and gonadotrophs (shown on
left panels), as well as all other unidentified cells (not
shown). A high K+-induced increase in
[Ca2+]i was accompanied by a significant and
dose-dependent inhibition of cGMP production to about 20%
of that observed in controls (Fig. 5B, left
panel). Both depolarization-induced elevation in
[Ca2+]i and inhibition of cGMP production were
almost abolished in cells bathed in Ca2+-deficient medium
(Fig. 5, A and B, right panels),
indicating that depolarization per se is not responsible for
the observed effects. Consistent with the role of
[Ca2+]i in the control of nNOS and eNOS,
depletion of extracellular Ca2+ was accompanied with a
significant decrease in cGMP production (illustrated by the upper
dashed line in Fig. 5B). Manipulation of
extracellular Ca2+ concentration did not obviously affect
cAMP production (Fig. 5B).

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Fig. 5.
Effects of high potassium-induced
depolarization of cells on Ca2+ signaling and cGMP
production in pituitary cells. A, extracellular
Ca2+-dependence of [Ca2+]i signaling.
S, somatotrophs, L, lactotrophs, G,
gonadotrophs. B, effects of high potassium on cAMP
(squares) and cGMP (circles) production in cells
bathed in Ca2+-containing and Ca2+-deficient
medium. Open circles and squares, controls bathed
in 5 mM K+-containing medium.
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The addition of two inhibitors of cGMP-specific phosphodiesterases,
vinpocetine and dipyridamole (26, 27), elevated basal cGMP levels (Fig.
6A). In cells with inhibited
phosphodiesterases, however, depolarization of cells inhibited cGMP
production with a comparable efficiency. High K+-induced
inhibition of cGMP production, but not cAMP production, was also
observed in cells expressing iNOS (Fig. 6B). Furthermore, high K+ inhibited cGMP production in control cells
treated with SNP, DEA, and DPTA (Fig. 6C). These results
indicate that elevated [Ca2+]i inhibits sGC
activity and that this inhibition occurs in the presence of elevated NO
levels.

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Fig. 6.
Characterization of depolarization-induced
inhibition of cGMP production. A, effects of high
K+ on cGMP production in controls and cells with inhibited
cGMP-dependent phosphodiesterases with 10 µM
vinpocetine and 1 µM dipyridamole. B,
dose-dependent effects of high K+ on cGMP and
cAMP production in cells expressing iNOS. C, effects of high
K+ on cGMP production in cultures stimulated with NO
donors. *, p < 0.01 versus controls.
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High K+-induced elevation in [Ca2+]i
(50, 75, and 100 mM) occurred in a nonoscillatory manner.
In contrast, depolarization of cells with 10-20 mM
K+ frequently led to the generation of an oscillatory
[Ca2+]i response after an initial spike response.
Fig. 7A illustrates the
effects of 12 mM K+ on the pattern of
[Ca2+]i signaling in identified somatotrophs,
lactotrophs, and gonadotrophs. At that particular concentration of
K+, a significant inhibition of cGMP production was
observed in control cells (Fig. 5B), as well as in cells
expressing iNOS (Fig. 6B). In further studies, the effects
of low K+ were compared with those induced by Bay K 8644, an L-type Ca2+ channel agonist. Addition of Bay K 8644 initiated [Ca2+]i oscillations in quiescent
somatotrophs, lactotrophs, gonadotrophs (Fig. 7B), and other
pituitary cells and increased the amplitude of transients in
oscillating cells (not shown). As in K+-stimulated cells,
the rise in [Ca2+]i induced by Bay K 8644 was
accompanied with the inhibition of cGMP production in controls and
vinpocetine-treated cells (Fig. 7C, left panels).
SNP-induced cGMP production was also inhibited by Bay K 8644 (Fig.
7C, right panel). Finally, Bay K 8644 inhibited cGMP production by LPS+IFN-
-treated cells in a
dose-dependent manner (Fig. 7D).

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Fig. 7.
Effects of Bay K 8644, an L-type
Ca2+ channel agonist, on Ca2+ signaling and
cGMP production in pituitary cells. A and B,
initiation of Ca2+ spiking in quiescent somatotrophs,
lactotrophs, and gonadotrophs by 12 mM
K+ and 1 µM Bay K 8644. C, inhibitory effects of Bay K 8644 on cGMP production in
control and in vinpocetine- and SNP-treated cells. D,
dose-dependent effects of Bay K 8644 on cGMP production in
pituitary cells expressing iNOS. *, p < 0.01 versus controls.
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To quantify the dependence of sGC activity on
[Ca2+]i, pituitary cells were stimulated with
increasing concentrations of K+ (10, 18, 30, 50, 75, and
100 mM) and Bay K 8644 (0.1 and 1 µM), and
the averaged area [Ca2+]i(t) curves
during 15 min of stimulation were calculated and compared with cGMP
production. Fig. 8, A and
B, illustrates the dependence of cGMP production and the
elevation in [Ca2+]i on the level of cell
depolarization. The relationship between [Ca2+]i
and cGMP in lower potassium (closed squares), high potassium
(open circles), and Bay K 8644-treated cells (closed circles) is shown in Fig. 8B. A linear relationship
between the [Ca2+]i and cGMP production indicates
that Ca2+-mediated inhibition of sGC was not dependent on
the pattern of signaling (nonoscillatory versus oscillatory)
but was determined by the averaged [Ca2+]i.

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Fig. 8.
Dependence of cGMP production on
[Ca2+]i. A,
dose-dependent effects of potassium on cGMP production
(upper panel) and [Ca2+]i response
(bottom panel). B, relationship between
[Ca2+]i and cGMP production in pituitary cells.
In A and B, [Ca2+]i was
expressed as a mean area per second under the curve subtracted for
basal [Ca2+]i during 15 min of stimulation with
high K+ and Bay K 8644. cGMP was measured in cells cultured
for 60 min. Cells were stimulated with 5 (controls), 10, 18, 30, 50, 75, and 100 mM KCl and with 0.1 and 1 µM Bay
K 8644.
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 |
DISCUSSION |
The NO-cGMP signaling pathway exhibits a complex action on
Ca2+ signaling in a number of cells. A cross-talk between
NO and Ca2+ is critical in cells with intra- and
intercellular Ca2+ oscillations, which are dependent on
Ca2+ release mechanisms, such as those reported recently in
hepatocytes (6). NO-cGMP also facilitates the Ca2+ influx
pathway by stimulating CNGs, channels that depolarize cells to the
level needed for activation of voltage-gated Ca2+ channels
(28). The same pathway may also inhibit Ca2+ influx by
activating Ca2+-controlled K+ channels in a
cGMP-dependent protein kinase-dependent manner (8, 29). Because the rise in [Ca2+]i is required
to activate nNOS and eNOS, the latter pathway would lead to the
coordinated regulation of [Ca2+]i and cGMP. That
is not the case with cells expressing CNGs, where the positive feedback
effects of Ca2+ on NOS would elevate
[Ca2+]i in an uncontrolled manner.
Two recent reports have indicated the expression of CNGs in pituitary
cells (11) and hypothalamic immortalized neurons (30); pituitary
somatotrophs express the message for rod CNGs, whereas gonadotropin-releasing hormone-secreting neurons express the olfactory subtype of these channels. In general, activation of these channels by
cAMP and cGMP leads to depolarization of cells and the subsequent facilitation of voltage-gated Ca2+ influx (28). These
channels may provide a rationale for the dual control of
Ca2+ signaling and secretion in neuroendocrine cells by the
NO signaling pathway and by AC-coupled receptors. The NO-cGMP pathway
is operative in pituitary and hypothalamic cells (12-17) and provides
a rationale for the control of spontaneous
[Ca2+]i fluctuations, which were observed in both
normal and immortalized pituitary cell types (31-36). Mixed
populations of pituitary cells express two enzymes, nNOS and eNOS (Fig.
1) (17, 37). These cells also express iNOS in response to LPS+IFN-
stimulation (Fig. 1) (38). AC-coupled receptors are also operative in
these cells and stimulate Ca2+ influx (39-42).
It was obvious that in cells where the NO-cGMP pathway is positively
coupled to Ca2+ influx, the inactivation of sGC is needed
to break the stimulatory action of NO on Ca2+ signaling and
Ca2+-controlled cellular functions. However, it was not
clear how sGC activity is controlled. Here we show that sCG activity in anterior pituitary cells is down-regulated by elevation in
intracellular Ca2+ and that the inhibition of the enzyme
activity is dependent on the [Ca2+]i. Our results
further indicate that Ca2+-dependent inhibition
of sGC activity occurs in the presence of elevated NO, i.e.
independently of the status of NOS, as well as when cGMP-specific
phosphodiesterases were inhibited.
Ca2+-dependent inhibition of sGC was observed
in cells exhibiting oscillatory and nonoscillatory signaling.
The effects of the nonoscillatory and high amplitude
[Ca2+]i signals, induced in our experiments with
depolarization of cells with high potassium, are unlikely to occur
under physiological situations and are here used to pharmacologically
establish the relationship between [Ca2+]i and
sGC activity. On the other hand, the effects of oscillatory
[Ca2+]i signals observed in low potassium and Bay
K 8644-stimulated cells are of relevance for the control of sGC
activity. Unstimulated pituitary cells frequently exhibit periods of
spontaneous and extracellular Ca2+-dependent
fluctuations in [Ca2+]i (11), which control basal
hormone secretion (10). The activation of several AC-coupled receptors
also leads to the initiation of extracellular
Ca2+-dependent [Ca2+]i
fluctuations in quiescent cells and to an increase in the frequency of
fluctuations in spontaneously active cells (39-42). Finally, when
activated, Ca2+-mobilizing receptors generate extracellular
Ca2+-independent oscillatory Ca2+ signals in
pituitary cells (10).
In accord with our observations concerning intact cells, it has also
been shown recently that Ca2+ inhibits sGC in crude cell
extract and immunopurified preparations (43). Such a role of
Ca2+ signaling is not unique for sGC; intracellular
Ca2+ also inhibits two other members of this family of
enzymes, particulate guanylyl cyclase (44, 45) and adenylyl cyclase,
particularly types V and VI (46). The common point in
[Ca2+]i-dependent inhibition of these
enzymes is in the reciprocal regulation of intracellular concentrations
of cyclic nucleotides and Ca2+. The unique characteristic
of the action of elevated [Ca2+]i on sGC
signaling pathway is its dual action; it stimulates cGMP production by
activating eNOS and nNOS but inhibits sGC even in the presence of
elevated NO. This inhibition is not only required to balance the
nNOS- and eNOS-generated NO production but also to protect the cells
from overloading with Ca2+ when NO production occurs in a
[Ca2+]i-independent manner, i.e. by
iNOS (22) and phosphorylated eNOS (47).
In summary, here we show that sGC and AC are operative in unstimulated
pituitary cells and differently regulated by
[Ca2+]i. The basal levels of cGMP are 2-4-fold
higher than cAMP when estimated in the absence of phosphodiesterase
inhibitors. Facilitation of voltage-gated Ca2+ influx and
removal of Ca2+ from medium were practically ineffective in
modulating cAMP production. This suggests that the participation of
[Ca2+]i-sensitive AC in unstimulated pituitary
cells is minor. In contrast, both treatments affected sGC activity.
Consistent with the role of [Ca2+]i in
facilitating nNOS and eNOS, cGMP production was inhibited but not
abolished by culturing cells in Ca2+-deficient medium.
Inhibition was not observed in cells with elevated NO. An increase in
[Ca2+]i also inhibited cGMP production but
independently of NO levels and the phosphodiesterase activity. The low
cAMP production and its independence of [Ca2+]i
and the high level of cGMP and its dependence on
[Ca2+]i are consistent with the coupling of the
NO signaling pathway, but not the AC signaling pathway, to spontaneous
Ca2+ signaling in cultured pituitary cells.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed: Section on Cellular
Signaling, ERRB/NICHD, Bldg. 49, Room 6A-36, 49 Convent Drive, Bethesda, MD 20892-4510. Tel: 301-496-1638; Fax: 301-594-7031; E-mail:
stankos@helix.nih.gov.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M004406200
 |
ABBREVIATIONS |
The abbreviations used are:
sGC, soluble
guanylyl cyclase(s);
NO, nitric oxide;
NOS, nitric acid synthase;
iNOS, inducible nitric oxide synthase;
nNOS, neuronal nitric oxide synthase;
eNOS, endothelial nitric oxide synthase;
AC, adenylyl cyclase;
LPS+IFN-
, 30 µg/ml lipopolysaccharide + 1000 IU/ml interferon-
;
SNP, sodium nitroprusside;
DEA, N-ethylethanamine:1,1-diethyl-2-hydroxy-2-nitrosohydrazine;
DPTA, 3,3'-(hydroxynitrosohydrazino)bis-1-propanamine;
NS 2028, 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one;
ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one;
Bay K 8644, 1,4,-dihydro-2,6-dimethyl-5-nitro-4;
and CNG, cyclic
nucleotide-gated channel.
 |
REFERENCES |
1.
|
Garbers, D. L.
(1992)
Cell
71,
1-4[Medline]
[Order article via Infotrieve]
|
2.
|
Denninger, J. W.,
and Marletta, M. A.
(1999)
Biochim. Biophys. Acta
1411,
334-350[Medline]
[Order article via Infotrieve]
|
3.
|
Smolenski, A.,
Burkhardt, A. M.,
Eigenthaler, M.,
Butt, E.,
Gambaryan, S.,
Lohmann, S. M.,
and Walter, U.
(1988)
Naunyn-Schmiedeberg's Arch. Pharmacol.
358,
134-139
|
4.
|
Zagotta, W. N.,
and Siegelbaum, S. A.
(1996)
Annu. Rev. Neurosci.
19,
235-263[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Beavo, J. A.
(1995)
Physiol. Rev.
75,
725-748[Abstract/Free Full Text]
|
6.
|
Patel, S.,
Robb-Gaspers, L. D.,
Stellato, K. A.,
Shon, M.,
and Thomas, A. P.
(1999)
Nat. Cell Biol.
1,
467-471[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Charles, A.
(1999)
Nat. Cell Biol.
1,
E193-195[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Willmott, N. J.,
Wong, K.,
and Strong, A. J.
(2000)
J. Neurosci.
20,
1767-1779[Abstract/Free Full Text]
|
9.
|
Yermolaieva, O.,
Brot, N.,
Weissbach, H.,
Heinemann, S. H.,
and Hoshi, T.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
448-453[Abstract/Free Full Text]
|
10.
|
Stojilkovic, S. S.,
and Catt, K. J.
(1992)
Endocr. Rev.
13,
256-280[Medline]
[Order article via Infotrieve]
|
11.
|
Tomic, M.,
Koshimizu, T.,
Yuan, D.,
Andric, S. A.,
Zivadinovic, D.,
and Stojilkovic, S. S.
(1999)
J. Biol. Chem.
274,
35693-35702[Abstract/Free Full Text]
|
12.
|
Hartt, D. J.,
Ogiwara, T.,
Ho, A. K.,
and Chik, C. L.
(1995)
Biochem. Biophys. Res. Commun.
214,
918-926[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Duvilanski, B. H.,
Zambruno, C.,
Seilicovich, A.,
Pisera, D.,
Lasaga, M.,
Diaz, M. C.,
Belova, N.,
Rettori, V.,
and McCann, S. M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
170-174[Abstract]
|
14.
|
Pinilla, L.,
Gonzalez, D.,
Tena-Sempre, M.,
and Aguilar, E.
(1998)
Neuroendocrinology
68,
180-186[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Kato, M.
(1992)
Endocrinology
131,
2133-2138[Abstract]
|
16.
|
Tsumori, M.,
Murakami, Y.,
Koshimura, K.,
and Kato, Y.
(1999)
J. Neuroendocrinol.
11,
451-456[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Lozach, A.,
Garrel, G.,
Lerrant, Y.,
Berault, A.,
and Counis, R.
(1998)
Mol. Cell. Endocrinol.
143,
43-51[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Tomic, M.,
Zivadinovic, D.,
Van Goor, F.,
Yuan, D.,
Koshimizu, T.,
and Stojilkovic, S. S.
(1999)
J. Neurosci.
19,
7721-7731[Abstract/Free Full Text]
|
19.
|
Green, L. C.,
Wagner, A. A.,
Glogowski, J.,
Skipper, P. L.,
Wishnok, J. S.,
and Tannenbaum, S. R.
(1982)
Anal. Biochem.
126,
131-138[Medline]
[Order article via Infotrieve]
|
20.
|
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Feelisch, M.
(1998)
Naunyn-Schmiedeberg's Arch. Pharmacol.
358,
113-122[Medline]
[Order article via Infotrieve]
|
22.
|
Knowles, R. G.,
and Moncada, S.
(1994)
Biochem. J.
298,
249-258[Medline]
[Order article via Infotrieve]
|
23.
|
Mayer, B.,
and Andrew, P.
(1998)
Naunyn-Schmiedeberg's Arch. Pharmacol.
358,
127-133[Medline]
[Order article via Infotrieve]
|
24.
|
Olesen, S.-P.,
Drejer, J.,
Axelsson, O.,
Moldt, P.,
Bang, L.,
Nielsen-Kudsk, J. E.,
Busse, R.,
and Mulsch, A.
(1998)
Br. J. Pharmacol.
123,
299-309[Abstract]
|
25.
|
Garthwaite, J.,
Southam, E.,
Boulton, C. L.,
Nielsen, E. B.,
Schmidt, K.,
and Mayer, B.
(1995)
Mol. Pharmacol.
48,
184-188[Abstract]
|
26.
|
Yamamoto, T.,
Yamamoto, S.,
Osborne, J. C.,
Manganiello, V. C.,
Vaughan, M.,
and Hidaka, H.
(1983)
J. Biol. Chem.
258,
14173-14177[Abstract/Free Full Text]
|
27.
|
Cataldi, M.,
Secondo, A.,
D'Alessio, A. D.,
Sarnacchiaro, F.,
Colao, A. M.,
Amoroso, S.,
Di Renzo, G. F.,
and Annunziato, L.
(1999)
Biochim. Biophys. Acta
1449,
186-193[Medline]
[Order article via Infotrieve]
|
28.
|
Wei, J.-Y.,
Samantra, R.,
Leconte, L.,
and Barnstable, C. J.
(1998)
Prog. Neurobiol. (Oxf.)
56,
37-64[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Griffith, O. W.,
and Stuehr, D. J.
(1995)
Annu. Rev. Physiol.
57,
707-736[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Vitalis, E. A.,
Costantin, J. L.,
Tsai, P.-S.,
Sakakibara, H.,
Paruthiyil, S.,
Liri, T.,
Martini, J.-P.,
Taga, M.,
Choi, A. L. H.,
Charles, A. C.,
and Weiner, R. I.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1861-1866[Abstract/Free Full Text]
|
31.
|
Kwiecien, R.,
and Hammond, C.
(1998)
Neuroendocrinology
68,
135-151[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Schlegel, W.,
Winiger, B. P.,
Mollard, P.,
Vacher, P.,
Wuarin, F.,
Zahnd, G. R.,
Wollheim, C. B.,
and Dufy, B.
(1987)
Nature
329,
719-721[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Wagner, K. A.,
Yacono, P. W.,
Golan, D. E.,
and Tashjian, A. H., Jr.
(1993)
Biochem. J.
292,
175-182[Medline]
[Order article via Infotrieve]
|
34.
|
Charles, A. C.,
Piros, E. T.,
Evans, C. J.,
and Hales, T. G.
(1999)
J. Biol. Chem.
274,
7508-7515[Abstract/Free Full Text]
|
35.
|
Holl, R. W.,
Thorner, M. O.,
Mandell, G. L.,
Sullivan, J. A.,
Sinha, Y. N.,
and Leong, D. A.
(1988)
J. Biol. Chem.
263,
9682-9685[Abstract/Free Full Text]
|
36.
|
Fiekers, J. F.,
and Konpoka, L. M.
(1996)
Cell Calcium
19,
327-336[Medline]
[Order article via Infotrieve]
|
37.
|
Wolff, D.,
and Datto, G. A.
(1992)
Biochem. J.
128,
201-206
|
38.
|
Vancelecom, H.,
Matthys, P.,
and Denef, C.
(1997)
J. Histochem. Cytochem.
45,
847-857[Abstract/Free Full Text]
|
39.
|
Kwiecien, R.,
Tseeb, V.,
Kurchikov, A.,
Kordon, C.,
and Hammond, C.
(1997)
J. Physiol. (Lond.)
499,
613-623[Abstract]
|
40.
|
Herrington, J.,
and Hille, B.
(1994)
Endocrinology
135,
1100-1108[Abstract]
|
41.
|
Lussier, B. T.,
French, M. B.,
Moor, B. C.,
and Kraicer, J.
(1991)
Endocrinology
128,
592-603[Abstract]
|
42.
|
Kuryshev, Y. A.,
Childs, G. V.,
and Ritchie, A. K.
(1995)
Endocrinology
136,
3925-3935[Abstract]
|
43.
|
Parkinson, S. J.,
Jovanovic, A.,
Jovanovic, S.,
Wagner, F.,
Terzic, A.,
and Waldman, S. A.
(1999)
Biochemistry
38,
6441-6448[CrossRef][Medline]
[Order article via Infotrieve]
|
44.
|
Wolbring, G.,
and Schnetkamp, P. P. M.
(1996)
Biochemistry
35,
11013-11018[CrossRef][Medline]
[Order article via Infotrieve]
|
45.
|
Dizhoor, A. M.,
Lowe, D. G.,
Olshevskaya, E. V.,
Laura, R. P.,
and Hurley, J. B.
(1994)
Neuron
12,
1345-1352[Medline]
[Order article via Infotrieve]
|
46.
|
Cooper, D. M. F.,
Mons, N.,
and Karpen, J. W.
(1995)
Nature
374,
421-424[CrossRef][Medline]
[Order article via Infotrieve]
|
47.
|
Butt, E.,
Bernhardt, M.,
Smolenski, A.,
Kotsonis, P.,
Frohlich, L. G.,
Sickmann, A.,
Meyer, H. E.,
Lohmann, S. M.,
and Schmidt, H. H. H. W.
(2000)
J. Biol. Chem.
275,
5179-5187[Abstract/Free Full Text]
|
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