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J Biol Chem, Vol. 274, Issue 50, 35693-35702, December 10, 1999
From the Endocrinology and Reproduction Research Branch, NICHD,
National Institutes of Health, Bethesda, Maryland 20892-4510
In excitable cells, oscillations in intracellular
free calcium concentrations ([Ca2+]i) can
arise from action-potential-driven Ca2+ influx, and such
signals can have either a localized or global form, depending on the
coupling of voltage-gated Ca2+ influx to intracellular
Ca2+ release pathway. Here we show that rat pituitary
somatotrophs generate spontaneous [Ca2+]i
oscillations, which rise from fluctuations in the influx of external
Ca2+ and propagate within the cytoplasm and nucleus. The
addition of caffeine and ryanodine, modulators of ryanodine-receptor
channels, and the depletion of intracellular Ca2+ stores by
thapsigargin and ionomycin did not affect the global nature of
spontaneous [Ca2+]i signals. Bay K 8644, an
L-type Ca2+ channel agonist, initiated
[Ca2+]i signaling in quiescent cells, increased
the amplitude of [Ca2+]i spikes in spontaneously
active cells, and stimulated growth hormone secretion in perifused
pituitary cells. Nifedipine, a blocker of L-type Ca2+
channels, decreased the amplitude of spikes and basal growth hormone
secretion, whereas Ni2+, a blocker of T-type
Ca2+ channels, abolished spontaneous
[Ca2+]i oscillations. Spiking was also abolished
by the removal of extracellular Na+ and by the addition of
10 mM Ca2+, Mg2+, or
Sr2+, the blockers of cyclic nucleotide-gated channels.
Reverse transcriptase-polymerase chain reaction and Southern blot
analyses indicated the expression of mRNAs for these channels in
mixed pituitary cells and purified somatotrophs. Growth
hormone-releasing hormone, an agonist that stimulated cAMP and cGMP
productions in a dose-dependent manner, initiated spiking
in quiescent cells and increased the frequency of spiking in
spontaneously active cells. These results indicate that in somatotrophs
a cyclic nucleotide-controlled plasma membrane Ca2+
oscillator is capable of generating global Ca2+ signals
spontaneously and in response to agonist stimulation. The
Ca2+-signaling activity of this oscillator is
dependent on voltage-gated Ca2+ influx but not on
Ca2+ release from intracellular stores.
The immortalized GH,1 GS, AtT-20, and At the present time, however, it is not clear to what extent the native
anterior pituitary cells differ from the immortalized cells. For
example, Kwiecien et al. (19) reported that cultured somatotrophs behave only as "conditional pacemakers." In other words, these cells are quiescent, and the addition of growth
hormone-releasing hormone (GHRH) is required to initiate firing. This
is in contrast with the results by Sims et al. (20) and Holl
et al. (21), who observed spontaneous firing of APs and
extracellular Ca2+-dependent fluctuation in
[Ca2+]i in a fraction of somatotrophs. Several
laboratories have also reported that gonadotrophs, lactotrophs, and
corticotrophs exhibit periods of spontaneous firing of APs (22-25). In
gonadotrophs, voltage-gated Ca2+ influx forms
membrane-localized [Ca2+]i signals, which are not
sufficient to trigger gonadotropin secretion (13). Basal ACTH, TSH, and
FSH secretion are also low and are unaffected by the removal of
extracellular Ca2+. In contrast, basal GH and prolactin
secretions are high and controlled by spontaneous Ca2+
influx through L-type Ca2+ channels (26). Since all these
cells exhibit spontaneous firing of APs (11), these observations raised
the possibility that AP-driven Ca2+ signals in somatotrophs
and lactotrophs, but not in other pituitary cells, are coupled to CICR,
leading to the generation of global [Ca2+]i
signals sufficient to trigger exocytosis. Here we studied the nature of
Ca2+ influx-dependent
[Ca2+]i transients in somatotrophs and their
relevance in growth hormone secretion.
Cell Cultures and Hormone Secretion--
Anterior pituitary
glands from adult female Harlan Sprague-Dawley rats were obtained from
Charles River Inc. (Wilmington, MA) and were dispersed into single
cells by a trypsin/DNase (Sigma) treatment procedure as described
previously (23). All experiments were performed in either identified
cells from mixed pituitary cell populations or purified somatotrophs.
The [Ca2+]i responses to GnRH, TRH, and GHRH,
applied at the end of experiments, were used for identification of
cells from a mixed population. Cell purification was done by a Percoll
discontinuous density gradient centrifugation (27).
For cell column perifusion, 12 × 106 mixed cells were
incubated with preswollen cytodex-1 beads (Amersham Pharmacia Biotech) in 60-mm culture dishes for 2 days. The cells were then loaded into
temperature-controlled chambers and perifused at 37 °C with Hanks'
M199 containing 20 mM HEPES and 0.1% bovine serum albumin for 60 min at a flow rate of 0.8 ml/min for 2 h, after which a stable basal secretion rate was established. During the test period, 1-min fractions were collected, and the perifusate was subsequently stored at
For cAMP and cGMP measurements, 1.0 × 106 cells per
well were cultured in 24-well plates for 24 h. Cultures were
washed and stimulated with GHRH in the phosphodiesterase-free Hanks'
M199 for 30 min. GH, cAMP, and cGMP contents were determined by
radioimmunoassay using the reagents and standards provided by Dr.
Parlow and the National Hormone and Pituitary Program (Torrance, CA),
Antibodies Inc. (Davis, CA), and Albert Baukal (NIH, Bethesda).
Calcium Imaging Measurements--
Cells were plated on
coverslips coated with poly-L-lysine and cultured in medium
199 containing Earle's salts, sodium bicarbonate, horse serum, and
antibiotics for 16-48 h. Both mixed populations of anterior pituitary
cells and highly purified somatotrophs were employed for
[Ca2+]i measurements. Cells were incubated for 60 min at 37 °C with 2 µM fura-2 AM (Molecular Probes,
Eugene, OR) in phenol red-free medium 199 containing Hanks' salts, 20 mM sodium bicarbonate, and 20 mM HEPES, pH 7.4. Coverslips with cells were washed with phenol red-free Krebs-Ringer
buffer and mounted on the stage of an Axiovert 135 microscope (Carl
Zeiss, Oberkochen, Germany) attached to an Attofluor Digital
Fluorescence Microscopy System (Atto Instruments, Rockville, MD). In
experiments without external Na+, this cation was replaced
with 120 mM N-methyl-D-glucamine
(NMDG). 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 505 nm was measured.
[Ca2+]i is shown as the ratio of intensities
measured at 340 and 380 nm
[F340/F380].
The intracellular distribution of Ca2+ signals was examined
by laser scanning confocal microscopy. Purified somatotrophs were kept
for 30 min at 37 °C and for additional 30 min at room temperature in
medium M199 with Hanks' salts and 4 µM fluo-3 AM
(Molecular Probes). The solution was then replaced with Krebs-Ringer
buffer with 2 mM CaCl2. Coverslips with cells
were mounted on a stage of an inverted microscope (Nikon Diaphot 300)
attached to a Bio-Rad MRC 1024 system (Bio-Rad). Laser line of 514 nm
was used for the excitation, and the emitted light was collected at 540 nm. Laser power was reduced to 1% in order to enable repetitive
scanning without damaging the cell. Images were collected under × 40 oil immersion objective and further zoom (× 2 and 2.5) was also
applied. Data acquisition was controlled by LaserSharp, version 3.2 time course software (Bio-Rad). The time period between two points in
planar scans (XY scans) was about 1.5 s, and 6-7 ms for line scans (XT scans). Each series of line scans lasted for 6.4 s. Data
are presented as increase in basal fluorescence
(F/F0).
RT-PCR Analysis of Cyclic Nucleotide-gated Channels
(CNGs)--
Total RNA was isolated from mixed and purified populations
of pituitary primary cell cultures using TRIZOLTM reagent
(Life Technologies, Inc.) and was treated with DNase I at 37 °C for
30 min. After heat inactivation of DNase I by incubating at 70 °C
for 15 min, first strand cDNA was synthesized from 5 µg of total
RNA, using Superscript II reverse transcriptase and oligo(dT)12-18 primers (Life Technologies, Inc.) in a
reaction volume of 20 µl. Aliquot (1 µl) of the resulting
single-strand cDNA was used for the PCRs, which were performed in
25-µl volume, containing 200 µM each of four
deoxynucleotide triphosphates, 50 mM KCl, 10 mM
Tris-HCl, pH 8.3, 2 mM MgCl2, and 0.5 units of Ex Taq polymerase (Panvera Corp., Madison, WI).
Nucleotide sequences for the PCR primers correspond to cytoplasmic
carboxyl regions of rat rod CNG1 (28), cone CNGgust (29), and olfactory
CNG2 (30) and have the following sequences: CNG1U (sense),
5'-TGCGAATTTGGGCAGTGACC-3', CNG1L (antisense),
5'-TCTCCTCCGGGTTCCTCAAG-3'; CNGgustU (sense), 5'-AGGGCAGATGCCAGGAACAT-3', CNGgustL (antisense),
5'-CGAGGCTGTAAAGTGTCTCA-3'; CNG2U (sense), 5'-GGAGGTAGATGTTCAGGAGA-3',
and CNG2L (antisense), 5'-CTTTGGGGAGAGTTCAGAGG-3'.
PCRs were run for 30 cycles at 94 °C, 1 min (denaturation);
55 °C, 35 s (annealing); 72 °C, 1 min (extension), followed
by a final extension for 10 min at 72 °C. Amplified DNA fragments were electrophoresed on 1% agarose gel and visualized with ethidium bromide. The same volumes of samples used for CNG mRNA analysis were also subjected to PCR using glyceraldehyde-3-phosphate
dehydrogenase-specific primers (31). PCR products separated on a gel
were then subjected to Southern blot analysis. Oligonucleotide probe
specific to rod CNG1 has the following sequence,
5'-CGAATCTTGGCTGAGTATGAATCGATGCAGCAG-3'. The probe was
labeled at the 5'-end with [ Extracellular Calcium Dependence of Spontaneous
[Ca2+]i Transients--
About 50% of
somatotrophs, examined 16-48 h after dispersion, exhibited no changes
in basal [Ca2+]i, and these cells are further
referred to as quiescent (Fig.
1A). The residual cells showed
fluctuations in [Ca2+]i and are referred to as
spontaneously active cells. As shown in Fig. 1, B-F, the
pattern of spontaneous [Ca2+]i oscillations was
variable among the cells. Some somatotrophs exhibited prolonged bursts
of elevated [Ca2+]i (Fig. 1, B-D),
and others exhibited shorter base-line-like [Ca2+]i transients (Fig. 1, E and
F). In spontaneously active cells, a transition from low
frequency (0.5-2 spikes per min) to higher frequency (3-10
spikes/min) of [Ca2+]i transients, and vice
versa, was frequently observed (Fig. 2,
A and B). Transitions from quiescent to active
status (Fig. 2C) and from active to quiescent status (Fig.
2D) were also observed.
Spontaneous [Ca2+]i transients were abolished by
the addition of 2 mM EGTA-containing medium, indicating a
role for Ca2+ influx in their generation (Fig.
3A, top tracing).
Removal of extracellular Ca2+ did not affect base-line
[Ca2+]i in quiescent cells (Fig. 3A, bottom
tracing). Depolarization of cells with 50 mM
K+ also abolished [Ca2+]i
oscillations (Fig. 3B, top tracing). In quiescent and
spontaneously active cells, 50 mM K+ induced a
peak in the [Ca2+]i response of comparable
amplitude (Fig. 3B). The response to K+ was
about twice as large as the peak of spontaneous
[Ca2+]i transients (Table
I). The average peak
[Ca2+]i of spontaneous transients in somatotrophs
was about 75% that observed in [Ca2+]i response
to the calcium-mobilizing agonist for these cells, endothelin-1 (Table
I). Thus, a plasma membrane Ca2+ oscillator is operative in
cultured somatotrophs and drives relatively high amplitude
[Ca2+]i transients.
Spontaneous [Ca2+]i transients were not unique to
somatotrophs, as they were also observed in other pituitary cells, including lactotrophs, and gonadotrophs. Spontaneous
[Ca2+]i transients were observed in about 40 and
20% of lactotrophs and gonadotrophs, respectively. In the same
preparation, the amplitudes of spontaneous
[Ca2+]i transients in somatotrophs were
significantly higher than those observed in the other two pituitary
cell types analyzed (Table I). In contrast, the amplitudes of
K+-induced [Ca2+]i responses were
comparable among these cells (Table I), indicating that somatotrophs,
lactotrophs, and gonadotrophs do not differ with respect to their
voltage-gated Ca2+ influx capacity.
Role of VGCCs in Spontaneous [Ca2+]i
Transients and GH Release--
To examine the role of VGCCs in
spontaneous [Ca2+]i transients in somatotrophs,
dihydropyridines, Cd2+, and Ni2+ were employed.
Bay K 8644, an L-type calcium channel agonist, was effective in
relatively high concentrations (Fig. 4).
In silent somatotrophs, 1 µM Bay K 8644 induced
[Ca2+]i transients, whose pattern was highly
comparable to that observed in spontaneously active cells (Fig.
4B, two top tracings). In a majority of oscillatory cells, 1 µM Bay K 8644 increased the amplitude of
[Ca2+]i transients (Fig. 4B, two bottom
tracings). Some somatotrophs also responded to 100 nM
Bay K 8644 by modulating the frequency of spontaneous
[Ca2+]i transients (Fig. 4A, two bottom
tracings), whereas this concentration was ineffective in
initiating [Ca2+]i transients in a majority of
quiescent cells (Fig. 4A, two top tracings). In all examined
cells, 10 nM Bay K 8644 was ineffective (not shown).
Like Bay K 8644, nifedipine, an L-type calcium channel blocker,
affected spontaneous [Ca2+]i transients only in
high concentrations. In about 50% of cells, 1 µM
nifedipine abolished Ca2+ spiking, whereas in the residual
cells it either had no effect or it changed the frequency and/or the
amplitude of [Ca2+]i transients (Fig.
5A). In a majority of cells,
100 nM nifedipine was ineffective or reduced the frequency
of [Ca2+]i transients (Fig. 5A).
Addition of Cd2+, a nonselective blocker of VGCCs,
abolished [Ca2+]i transients, but only in a small
fraction of the cells, and changed the pattern of spiking in a majority
of somatotrophs (Fig. 5B). In contrast, 100 µM
Ni2+, a relatively specific inhibitor of T-type
Ca2+ channels, inhibited spontaneous and Bay K 8644-induced
[Ca2+]i transients in a majority of the cells
(Fig. 5C).
The effects of dihydropyridines and Cd2+ on GH secretion
were evaluated in perifused pituitary cells. The addition of 1 µM nifedipine reduced basal GH secretion to about 50%
that observed in controls (Fig.
6A). In contrast, 1 µM Bay K 8644 increased GH secretion in a pulsatile-like
manner (Fig. 6A). At 100 nM concentrations, nifedipine and Bay K 8644 were practically ineffective (Fig.
6B), and 50 µM Cd2+ had only a
minor inhibitory effect (Fig. 6C).
Spatio-temporal Aspect of Spontaneous [Ca2+]i
Transients in Somatotrophs--
To characterize the spatio-temporal
aspects of Ca2+ signals in somatotrophs,
[Ca2+]i imaging was done by laser scanning
confocal microscopy. For these measurements, highly purified
somatotrophs were loaded with a Ca2+ indicator, fluo-3.
Fig. 7 shows the temporal and spatial
characteristics of Ca2+ signals measured simultaneously in
two spontaneously active somatotrophs, stimulated with Bay K 8644 and
subsequently by ionomycin. Measurements at the middle plane of these
somatotrophs revealed that [Ca2+]i rose in all
cellular regions analyzed during spontaneous transients (Fig. 7,
upper panel). In the somatotroph schematically shown on
left top panel (cell-I), there was no obvious difference in
the amplitude of [Ca2+]i transients in three
different regions of the cytoplasm (boxes and tracings
1, 2, and 4). A significant increase in
[Ca2+]i was also observed in the nucleoplasm,
with the amplitude of response higher than that observed in cytoplasm
(box and tracing 3).
Addition of Bay K 8644 increased the amplitude of
[Ca2+]i responses in all analyzed cell regions.
The [Ca2+]i profiles were highly comparable among
the tracings and specific for each cell (tracings 1-4 of
cell-I versus tracing 5 of cell-II).
Ionomycin-induced Ca2+ release was also detected in all
cellular regions analyzed, with the amplitude of
[Ca2+]i response higher than that observed during
Bay K 8644 stimulation. During continuous stimulation with ionomycin, a
recovery of the oscillatory response was observed in both cells but at different time points. The bottom panel in Fig. 7 shows the
central plane images for cell-I at four different time points,
indicated by arrows above trace 3 in the
upper panel. Images demonstrate the global nature of changes
in cytoplasmic and nuclear [Ca2+]i in a cell from
base line (A) to one of the peaks of the spontaneous
transients (B), one of the peaks of Bay K 8644-induced transients (C), and after ionomycin-induced discharge from
the intracellular Ca2+ pool (D).
To characterize further the generation of spontaneous
[Ca2+]i transients, a line scan mode of confocal
imaging was performed simultaneously in two cells during the transition
from quiescent to active stage. In a schematic representation of these two cells shown in Fig. 8, the
right-hand side of the middle panel, the
vertical line indicates where the recordings were done. The XT images, labeled as A-C, show the spatial nature of
[Ca2+]i signals during initiation of a
[Ca2+]i transient (A), during the peak
[Ca2+]i response (B), and between the
two [Ca2+]i peaks (C). These images
again demonstrate the global nature of spontaneous
[Ca2+]i transients in somatotrophs, with the rise
and the fall in [Ca2+]i being obvious in both
cytoplasmic and nucleoplasmic compartments.
Fluorescence intensities were also recorded at three line segments, one
in the sub-plasma membrane region (labeled as 3), another in
the central region of cytoplasm (labeled as 1) and a third
in the central region of nucleoplasm (labeled as 2). These data are presented as ratios between fluorescence intensity and the
average base-line intensity for the corresponding line segments (F/F0) in the upper panel
of Fig. 8. As in XY scan shown in Fig. 7, the
[Ca2+]i profiles resembled each other in all
monitored cell regions, suggesting that they were driven by the same
mechanism. Also, the values of F/F0
were still higher in the nucleoplasm (tracing 2 versus tracings 1 and 3). Finally, the
extended time scale analysis of F/F0
profiles shown in Fig. 8, upper panel, revealed that the
rise in [Ca2+]i occurred almost simultaneously in
cytoplasmic and nucleoplasmic regions.
Independence of Spontaneous [Ca2+]i
Transients on Ca2+ Release from Intracellular
Stores--
The ability of somatotrophs to resume
[Ca2+]i oscillations after the depletion of
intracellular Ca2+ pool by ionomycin (Fig. 7) argues
against the coupling of electrical activity to Ca2+ release
from intracellular stores. This conclusion was further supported in
three types of experiments. First, the addition of 1 µM
thapsigargin, a concentration sufficient to block completely Ca2+ uptake by endoplasmic reticulum
Ca2+-ATPase (32), did not abolish
[Ca2+]i oscillations in spontaneously active
cells. As shown in Fig. 9A, upper
panel, addition of thapsigargin increased the spiking frequency.
In quiescent cells, thapsigargin increased [Ca2+]i in a non-oscillatory manner, whereas Bay
K 8644 was able to initiate [Ca2+]i transients in
the presence of thapsigargin (Fig. 9A, bottom tracing).
Spontaneous (Fig. 9B, upper tracing) and Bay K 8644-induced
[Ca2+]i transients (Fig. 9B, bottom
tracing) were also observed in cells exposed to 10 µM thapsigargin for 20 min prior to
[Ca2+]i recording, further indicating that the
re-uptake of Ca2+ by endoplasmic reticulum is not required
to generate [Ca2+]i transients.
Second, the coupling of electrical activity to Ca2+ release
by activating a voltage-sensor was ruled out in experiments with depolarization of somatotrophs bathed in Ca2+-depleted
medium. As shown in Fig. 9C, depolarization of cells did not
affect base-line [Ca2+]i after the extracellular
Ca2+ was adjusted to about 100 nM. When cells
were bathed in 0.25 and 0.5 mM Ca2+-containing
medium, a non-oscillatory rise in [Ca2+]i was
observed, with the amplitude of responses lower than those observed in
physiological extracellular Ca2+ concentrations (not shown).
Third, the participation of RyRs in spontaneous and Bay K 8644-induced
[Ca2+]i transients was excluded in experiments
with caffeine and ryanodine, the modulators of these channels (33). As
shown in Fig. 10A, caffeine
was unable to initiate [Ca2+]i transients in
quiescent cells (upper tracing) or to change the rhythm of
Ca2+ spiking in spontaneously active cells (two
bottom tracings). Ryanodine, at its low stimulatory (5 µM) concentration, was also unable to initiate
[Ca2+]i transients (Fig. 10B, upper
tracing) or to change the pattern of spiking in spontaneously
active cells (two bottom tracings). This compound was also
ineffective at its high inhibitory (100 µM)
concentrations (Fig. 10C).
Cationic Channels and Spontaneous [Ca2+]i
Transients--
Addition of TTX, a specific blocker of voltage-gated
Na+ channels, did not affect the rhythm of the spontaneous
[Ca2+]i transients (Fig.
11A, top three tracings).
Also, in the presence of TTX, Bay K 8644 still modulated spontaneous
[Ca2+]i transients (Fig. 11A, the
2nd and 3rd tracings) and initiated oscillations
in quiescent cells (Fig. 11A, two bottom tracings). However,
in spontaneously active cells, [Ca2+]i transients
were immediately abolished by the removal of Na+,
suggesting that the influx of this cation through TTX-insensitive channels is involved in initiating Ca2+ spiking. In a
majority of cells, inhibition was transient and was followed by
recovery of Ca2+ spiking after 5-15 min of exposure to
Na+-deficient medium (Fig. 11B).
Elevation of extracellular [Ca2+] from 2 to 10 mM also abolished spontaneous [Ca2+]i
transients in a majority of the cells. Some cells responded to high
extracellular Ca2+ with a reduction in the amplitude and
frequency of [Ca2+]i transients (Fig.
12A, top tracing), whereas
in quiescent cells no changes in basal [Ca2+]i
were observed (not shown). Similarly, 10 mM
Mg2+ (Fig. 12B) and Sr2+ (Fig.
12C) abolished spiking or reduced the frequency and/or
amplitude of spontaneous [Ca2+]i transients,
suggesting the sensitivity of these Na+-conducting channels
to high concentrations of divalent cations. Such a cationic profile is
consistent with the presence of CNGs and their coupling to
Ca2+ signaling pathway in somatotrophs (34).
To address this hypothesis, RT-PCR analysis was performed, using
mRNAs from mixed population of anterior pituitary cells and purified somatotrophs and specific primers for three rat channels: rod,
cone, and olfactory. As in [Ca2+]i measurements,
dispersed cells were cultured for 24 h prior to RNA isolation. In
mixed pituitary cells, the expected size of PCR products for all three
CNGs was detected (Fig. 13). Moreover,
in purified somatotrophs, only the specific signal for rod type channel
was observed (Fig. 13, upper panels). Southern blot analysis
of the same gels further confirmed these results (Fig. 13, bottom
panels). No PCR products were detected from controls containing
all the components except for reverse transcriptase (RT), ruling out
the possibility of genomic DNA contamination.
GHRH-controlled [Ca2+]i Signals--
In
further experiments, cells were stimulated with GHRH, a well
established agonist for somatotrophs (27, 35, 36). Addition of 1 nM GHRH initiated Ca2+ spiking in quiescent
cells (Fig. 14A, bottom
tracing). In spontaneously active cells, 1 nM GHRH
increased frequency of [Ca2+]i transients (Fig.
14A, two middle tracings) or induced a sustained
non-oscillatory elevation in [Ca2+]i (Fig.
14A, two upper tracings). The majority of cells stimulated
with 100 nM GHRH responded with a non-oscillatory increase in [Ca2+]i that lasted as long as the agonist was
present in the medium (up to 20 min). GHRH action on Ca2+
signaling was accompanied by a dose-dependent increase in
cAMP production measured in the absence of phosphodiesterases, leading to a 50-fold increase in cAMP levels when cells were stimulated with
100 nM GHRH. In addition to cAMP, GHRH also stimulated cGMP production in a dose-dependent manner but with a shift in
the EC50 of about 1 log unit (Fig. 14B).
Oscillations in intracellular calcium concentrations
([Ca2+]i transients) can result from inositol
trisphosphate-induced release of intracellularly stored
Ca2+ and/or from voltage-gated and voltage-insensitive
Ca2+ influxes. The first type of
[Ca2+]i transients is well characterized in a
number of non-excitable and excitable cells, including hepatocytes,
acinar cells, oocytes, and pituitary gonadotrophs (23, 37-40).
Calcium-mobilizing agonist-induced [Ca2+]i
oscillations do not occur synchronously within the cell but originate
from a specific sub-plasma membrane locus and propagate throughout the
cell (41), including the nucleoplasm. The equilibrium between
cytoplasmic and nuclear [Ca2+] is probably achieved by
the release of Ca2+ from nuclear envelope in synchrony with
its release from endoplasmic reticulum (42-44).
In cells exhibiting spontaneous- or receptor-controlled firing of APs,
changes in plasma membrane potential can trigger Ca2+
influx through VGCCs. Since the activation of these channels is limited
by the duration of depolarization, AP-driven Ca2+ signals
are usually localized events that are relevant in the control of plasma
membrane-associated cellular functions, such as excitability and
neurotransmission (11). However, in cells expressing RyRs these
localized signals may also trigger CICR from intracellular stores,
leading to the formation of global Ca2+ signals (17, 18).
Thus, both calcium-mobilizing receptors and APs can generate local and
global Ca2+ signals and propagation of Ca2+
signals throughout the cell is coupled to release of Ca2+
from intracellular stores.
Here we show that somatotrophs are able to generate spontaneous
[Ca2+]i transients, with single spikes lasting
for several seconds. The elevations in [Ca2+]i
were detected in all regions of the cytoplasm, as well as in the
nucleoplasm, indicating the global nature of
[Ca2+]i transients. In the non-normalized images,
Ca2+ signal appeared to initiate in nucleoplasm and to
terminate in cytoplasm. However, the extended time scale analysis of
F/F0 profiles revealed that the rise
in [Ca2+]i occurred almost simultaneously in
cytoplasmic and nucleoplasmic regions. Also,
[Ca2+]i profiles in all monitored cell regions
were highly comparable, suggesting that they were driven by the same
mechanism. The apparent amplitudes of the Ca2+ signals,
expressed as F/F0 were similar
throughout the cytoplasm, including the sub-plasma membrane region, and
were higher in the nucleoplasm. These differences should be taken with
reservation, because of the controversies in comparing signals
originating from different cellular compartments (43-45). In addition,
the lack of difference in the amplitude of
[Ca2+]i responses close to the plasma membrane
and in the central cytoplasmic regions does not rule out the existence
of highly localized [Ca2+]i elevations in
sub-plasma membrane regions, since the resolution of our measurements
may not be sufficient to detect such changes.
High amplitude spontaneous [Ca2+]i transients are
not unique for somatotrophs, as immortalized pituitary cells also exhibit such changes (1-10). The role of voltage-gated
Ca2+ influx in spontaneous electrical activity and
[Ca2+]i transients in these cells is well
established (11). Several reports have also indicated the expression of
RyRs and their coupling to spontaneous electrical activity (7, 14, 15,
46, 47). The presence of specific mRNA for RyRs in a mixed
population of pituitary cells (46) is consistent with the coupling of
voltage-gated Ca2+ influx to CICR in native pituitary cells
as well. In further parallelism with immortalized cells, spontaneous
[Ca2+]i transients in somatotrophs were affected
by high concentration of dihydropyridines, Cd2+, and
Ni2+. Others have reported about the expression of VGCCs
and participation of voltage-gated Ca2+ influx in
spontaneous and GHRH-controlled electrical activity in somatotrophs
(19) and GC somatotroph lines (3).
However, the present data indicate that spontaneous electrical activity
in somatotrophs is not coupled to Ca2+ release from
intracellular stores. The lack of CICR in somatotrophs was documented
by the inability of two modulators of RyRs, caffeine and ryanodine
(33), to initiate [Ca2+]i transients in quiescent
cells and/or to alter the pattern of signaling in spontaneously active
cells. Also, spontaneous [Ca2+]i transients were
observed in cells treated with thapsigargin and ionomycin, showing that
the plasma membrane oscillator is operative even upon the depletion of
intracellular Ca2+ pools. Finally, in contrast to
hippocampal neurons (48), depolarization of somatotrophs bathed in
Ca2+-deficient medium did not generate Ca2+
signaling, arguing against the operation of a voltage sensor in these
cells. Thus, voltage-gated Ca2+ influx is able to generate
high amplitude [Ca2+]i oscillations without the
integration of a Ca2+ release mechanism.
The dependence of spontaneous [Ca2+]i transients
on voltage-gated Ca2+ influx and their independence on
Ca2+ release from intracellular stores implies a prolonged
opening of VGCCs, presumably by a sustained activation of a
depolarizing current needed to oppose the K+
channel-mediated repolarization of the cells. The expression of
depolarizing T-type Ca2+ and TTX-sensitive Na+
channels in somatotrophs (49, 50) could provide a transient but not a
prolonged depolarization of cells, because of their rapid and
voltage-dependent inactivation (51). Earlier studies have
indicated the TTX-insensitive Na+ dependence of spontaneous
[Ca2+]i oscillations in GH tumor somatotrophs
(3). Also, GHRH-induced electrical activity and Ca2+
signaling are dependent on extracellular Na+ (20, 35, 52).
In parallel to this, here we show that a substitution of
Na+ with NMDG leads to a temporal suppression of
spontaneous [Ca2+]i oscillations in cultured
somatotrophs. The transient nature of inhibition of spontaneous
[Ca2+]i oscillations by NMDG further suggests
that this compound or other ion(s) can substitute for Na+.
That is consistent with the role of a non-selective ion channel in
generating spontaneous [Ca2+]i oscillations.
A number of channel subtypes are non-selective and can generate the
pacemaking depolarizing currents, including CNGs that conduct
Na+, Ca2+, and K+ (34). Three lines
of evidence support the hypothesis that the rod type of CNGs function
as non-selective cationic channels in spontaneously active
somatotrophs. First, [Ca2+]i transients in
somatotrophs are inhibited by divalent cations in concentrations that
inhibit CNGs as well. Second, the specific PCR product of rod type CNGs
was obtained from highly purified somatotrophs. Third, GHRH initiated
spiking in quiescent cells and increased the frequency of oscillations
in spontaneously active somatotrophs, as well as increased the
production of two messengers needed for the activation of CNG channels,
cAMP and cGMP. However, to confirm the role of CNGs in the formation of [Ca2+]i transients, additional experiments are
required, including electrophysiological characterization of CNG
current, the use of specific intracellular blockers, such as
L-Cys-diltiazem, and Western blot analysis.
In conclusion, the presented results demonstrate that a plasma membrane
Ca2+ oscillator is operative in somatotrophs and is capable
of generating spontaneous high amplitude Ca2+ signals.
Although there is no coupling of electrical activity to intracellular
Ca2+ release, this oscillator induces global
Ca2+ signals. The results further suggest the physiological
relevance of the spontaneous activity in the control of basal GH
secretion. The operation of plasma membrane Ca2+ oscillator
in somatotrophs is dependent on TTX-insensitive Na+
conductance and voltage-gated Ca2+ influx. The oscillatory
activity was inhibited by a high concentration of divalent cations,
indicating a role of CNGs in pacemaker activity. Furthermore, the
specific messages for these channels were identified in pituitary
cells. In agreement with the hypothesis that cyclic nucleotides are
intracellular messengers controlling the plasma membrane
Ca2+ oscillator activity, GHRH, an agonist that signals
through adenylyl and guanlylyl cyclase pathways, initiated
[Ca2+]i spiking in quiescent cells and increased
the frequency of spiking in spontaneously active cells.
We thank Albert F. Parlow for help in
establishing radioimmunoassay for GH and Fredrick Van Goor and Ann
Katzur for their helpful discussions.
*
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.
The abbreviations used are:
GH, growth hormone;
APs, action potentials;
VGCCs, voltage-gated calcium channels;
CNGs, cyclic nucleotide-gated channels;
RyR, ryanodine receptor-channels;
TTX, tetrodotoxin;
[Ca2+]i, intracellular calcium
concentration;
GHRH, growth hormone-releasing hormone;
CICR, calcium-induced calcium release;
NMDG, N-methyl-D-glucamine;
RT-PCR, reverse
transcriptase-polymerase chain reaction.
Characterization of a Plasma Membrane Calcium Oscillator in Rat
Pituitary Somatotrophs*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
T3 pituitary
cells have been frequently used as cell
models for the characterization of electrical activity and the
associated Ca2+ signaling in lactotrophs, somatotrophs,
corticotrophs, and gonadotrophs, respectively (1-10). Simultaneous
measurements of electrical activity and intracellular free
Ca2+ concentrations ([Ca2+]i) in
GH3B6 pituitary cells confirmed the dependence of [Ca2+]i on action potential (AP) firing (1).
These cells express T-type and L-type voltage-gated Ca2+
channels (VGCCs), tetrodotoxin (TTX)-sensitive Na+
channels, and several voltage-gated and Ca2+-controlled
K+ channels, which are all involved in the spontaneous
firing of APs (11). Briefly, TTX-sensitive Na+ channels,
T-type Ca2+ channels, and dihydropyridine-sensitive L-type
Ca2+ channels control the slow pacemaker depolarization and
drive the rapid depolarizing phase of APs. On the other hand, several K+ channels control the repolarization phase of APs,
including delayed rectifier, BK, and SK channels (11-13). The
transient opening of VGCCs may provide a window for the coupling of
electrical activity to calcium-induced calcium release (CICR) through
ryanodine receptor channels (RyR) (7). In line with this, a
caffeine-sensitive intracellular Ca2+ pool (14) and the
cADP ribose-dependent signaling pathway were identified in
these cells (15). Thus, the plasma membrane oscillator in immortalized
pituitary cells resembles the one that is operative in cardiac and some
neuronal cells (16-18).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C.
-32P]ATP (5000 Ci/mmol)
using T4 polynucleotide kinase (PanVera Corp.). Pre-hybridization
was performed at 65 °C for 2 h in 6× SSPE, 0.01 M
sodium phosphate, pH 6.8, 1 mM EDTA, 0.5% SDS, 100 mg/ml
salmon sperm DNA, and 0.1% dried milk. Hybridization was performed
under the same conditions containing 0.5 pmol/ml radiolabeled probe for
3 h. Membranes were then washed at 42 °C for 10 min in 2× SSPE
and at 65 °C for 10 min and exposed to x-ray film (Eastman Kodak
Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Basal [Ca2+]i in
cultured somatotrophs. A, about 50% of somatotrophs
were silent. B-F, pattern of Ca2+ signals in
spontaneously active cells. The tracings shown are from
identified somatotrophs from mixed populations of anterior pituitary
cells. In this and the following figures, [Ca2+]i
tracings shown are representative of at least 20 recordings in three to
ten independent experiments. Fura 2 was employed as a dye for
[Ca2+]i recordings, if not otherwise
specified.
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Fig. 2.
The rhythm of Ca2+ signaling in
cultured somatotrophs. A and B, spontaneous
modulation of frequency of [Ca2+]i transients.
C and D, transitions from quiescent to active
(C) and from active to quiescent state (D).
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Fig. 3.
Characterization of spontaneous
[Ca2+]i transients. A,
extracellular Ca2+ dependence of spontaneous
[Ca2+]i transients. Cells were initially bathed
in 2 mM Ca2+-containing medium and subsequently
in Ca2+-deficient medium (free [Ca2+] about
100 nM), adjusted by EGTA. Bottom tracing
illustrates the lack of effects of extracellular Ca2+
depletion on [Ca2+]i in quiescent cells.
B, depolarization-induced [Ca2+]i
response in spontaneously active and quiescent somatotrophs. In this
and the following figures, arrows indicate the moment of
drug applications. The drugs were added in 1-ml volumes and were
present throughout the recording. The concentrations indicated
above the arrows are final.
Comparison of the amplitudes of spontaneous, agonist-induced, and
potassium-induced [Ca2+]i responses in pituitary
cells
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Fig. 4.
Effects of Bay K 8644, an L-type
Ca2+ channel agonist, on Ca2+ signaling in
somatotrophs. Initiation of [Ca2+]i
transients by 1 µM Bay K 8644 (B, two top
tracings) but not by 100 nM Bay K 8644 (A, two
top tracings) in quiescent cells. Modulation of the amplitude of
[Ca2+]i transients in spontaneously active cells
(A and B, two bottom tracings).
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Fig. 5.
Effects of voltage-gated calcium channel
blockers on spontaneous [Ca2+]i transients.
A, concentration-dependent effects of
nifedipine, an L-type Ca2+ channel antagonist, on the
pattern of [Ca2+]i transients. B,
effects of 50 µM Cd2+, a non-selective
blocker of voltage-gated Ca2+ channels, on the pattern of
[Ca2+]i transients. C, effects of 100 µM Ni2+, a relatively specific blocker of
T-type Ca2+ channels, on spontaneous (three top
tracings) and Bay K 8644 (1 µM)-induced
[Ca2+]i transients (two bottom
tracings). The arrows at the bottom indicate
the moments of drug application in all tracings shown above.
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Fig. 6.
Modulation of basal GH secretion by calcium
channel agonists and antagonists in perifused pituitary cells.
A and B, effects of dihydropyridines on basal GH
secretion. Open circles indicate the experiment with Bay K
8644 and closed circles experiment with nifedipine.
C, effects of Cd2+ on basal GH secretion. The
rectangle indicates duration of perifusion with Bay K 8644, nifedipine, or Cd2+. Fractions were collected every
minute.
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Fig. 7.
Spatio-temporal distribution of spontaneous
and Bay K 8644-induced [Ca2+]i transients in
purified somatotrophs. Confocal images of fluo-3-loaded cells were
recorded and fluorescence intensities measured simultaneously in five
different regions of two somatotrophs. As shown in the schematic
representation above the plots, cell-I regions denoted by 1, 2, and 4 are in the cytoplasm and region 3 is in the nucleoplasm, whereas the cell-II region is denoted by
5 and covers a part of cytoplasm and a part of nucleoplasm.
[Ca2+]i profiles from these regions are presented
as ratios between fluorescence intensities and basal fluorescence at
the corresponding region. The numbers on the
right correspond to the regions of interest shown above.
Bottom panel shows images for cell-I at four different time
points indicated by arrows above trace 3.
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Fig. 8.
Line scans of a somatotroph in transition
from quiescent to active state. As shown in the schematic
representation on the right, fluorescence was recorded along
a line that passed through a diameter of one cell and partially through
another cell. The line was scanned every 6-7 ms during 6.4 s; the
XT image was saved and a new series of scans started. Fluorescence
intensities were recorded at two line segments positioned in the
cytoplasm (labeled as 1 and 3) and one positioned
in the nucleoplasm (labeled as 2). Data are presented as
ratios between fluorescence intensity at given time and the average
base-line intensity for the corresponding line segments. The chosen
images show the initiation of a [Ca2+]i transient
(A), a period before and after the peak (B), and
a fall and subsequent rise in cytosolic and nuclear
[Ca2+] (C). The horizontal lines
above the [Ca2+]i profiles indicate
the time intervals at which presented XT images were recorded.
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Fig. 9.
Effects of thapsigargin and high potassium
concentrations on [Ca2+]i in cultured
somatotrophs. A, effects of thapsigargin, a blocker of
endoplasmic reticulum Ca2+-ATPase, on
[Ca2+]i in spontaneously active (upper
tracing) and quiescent cells (bottom tracing). Note the
ability of Bay K 8644 to initiate spiking in the presence of
thapsigargin. B, pattern of spontaneous
[Ca2+]i transients in somatotrophs treated with
10 µM thapsigargin for 20 min (upper tracings)
and Bay K 8644-induced [Ca2+]i transients
(bottom tracing). C, high potassium
concentration-induced depolarization. Somatotrophs bathed in
Ca2+-deficient medium (free [Ca2+]i
about 100 nM) were depolarized by 50 mM
potassium.
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Fig. 10.
Calcium influx is not coupled to
calcium-induced calcium release in somatotrophs. A, the
lack of effects of caffeine, an activator of ryanodine
receptor-channels, on the pattern of spontaneous
[Ca2+]i transients. B and
C, the lack of effects of a stimulatory (B) and
inhibitory (C) concentrations of ryanodine on
[Ca2+]i in somatotrophs. Bay K 8644 (1 µM)-induced [Ca2+]i transients are
shown in C, two upper tracings. The
arrows at the bottom indicate the moments of drug
application in all tracings shown above.
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Fig. 11.
Sodium dependence of Ca2+
transients in cultured somatotrophs. A, the lack of
effects of tetrodotoxin (TTX), a blocker of voltage-gated
Na+ channels, on spontaneous and Bay K 8644-induced
[Ca2+]i oscillations. B, transient
abolition of Ca2+ spiking in cells perfused with
Na+-deficient medium (NMDG was used to substitute for
Na+). The rectangle indicates the time when
cells were perfused with NMDG-containing medium.
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Fig. 12.
Inhibition of [Ca2+]i
transients by divalent cations. A-C, effects of 10 mM extracellular Ca2+ (A),
Mg2+ (B), and Sr2+ (C) on
Ca2+ spiking. Addition of 10 mM NaCl to bath
medium did not alter the pattern of [Ca2+]i
transients (not shown). The arrows at the bottom
indicate the moments of drug application in all tracings shown
above.
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Fig. 13.
Detection of CNG transcripts in mixed
anterior pituitary and purified somatotrophs. A, RT-PCR
analysis for rod (210 base pairs), cone (281 base pairs), and olfactory
(318 base pairs) CNGs in mixed pituitary cells and purified
somatotrophs. B, Southern blot analysis of the same gels
shown in upper panels. Blots were probed with 5'-end-labeled
oligonucleotide specific to the rod type CNGs.
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Fig. 14.
Effects of growth hormone-releasing hormone
(GHRH) on [Ca2+]i signaling and cyclic nucleotide
production in pituitary somatotrophs. A, stimulatory
effects of GHRH on [Ca2+]i in active (upper
tracings) and quiescent cells (bottom tracing).
B, dose-dependent effects of GHRH on cAMP and
cGMP production in pituitary cells. The results shown are mean ± S.E. from sextuplicate determination. The dotted lines
indicate the EC50 values derived from fitted logistic
curves. Cells were stimulated for 30 min with GHRH in the absence of
phosphodiesterase inhibitors.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence and reprint requests should be addressed:
Section on Cellular Signaling/ERRB/NICHD, Bldg. 49, Room 6A-36, 49 Convent Dr., Bethesda, MD 20892-4510. Tel.: 301-496-1638; Fax:
301-594-7031; E-mail: stankos@helix.nih.gov.
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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 A. E. Gonzalez-Iglesias, Y. Jiang, M. Tomic, K. Kretschmannova, S. A. Andric, H. Zemkova, and S. S. Stojilkovic Dependence of Electrical Activity and Calcium Influx-Controlled Prolactin Release on Adenylyl Cyclase Signaling Pathway in Pituitary Lactotrophs Mol. Endocrinol., September 1, 2006; 20(9): 2231 - 2246. [Abstract] [Full Text] [PDF]
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 S. A. Andric, T. S. Kostic, and S. S. Stojilkovic Contribution of Multidrug Resistance Protein MRP5 in Control of Cyclic Guanosine 5'-Monophosphate Intracellular Signaling in Anterior Pituitary Cells Endocrinology, July 1, 2006; 147(7): 3435 - 3445. [Abstract] [Full Text] [PDF]
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 A. Secondo, A. Pannaccione, M. Cataldi, R. Sirabella, L. Formisano, G. Di Renzo, and L. Annunziato Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents Am J Physiol Cell Physiol, January 1, 2006; 290(1): C233 - C243. [Abstract] [Full Text] [PDF]
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S. A. Andric, A. E. Gonzalez-Iglesias, F. Van Goor, M. Tomic, and S. S. Stojilkovic Nitric Oxide Inhibits Prolactin Secretion in Pituitary Cells Downstream of Voltage-Gated Calcium Influx Endocrinology, July 1, 2003; 144(7): 2912 - 2921. [Abstract] [Full Text] [PDF]
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 E. Perez-Reyes Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels Physiol Rev, January 1, 2003; 83(1): 117 - 161. [Abstract] [Full Text] [PDF]
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U. B. Kaupp and R. Seifert Cyclic Nucleotide-Gated Ion Channels Physiol Rev, July 1, 2002; 82(3): 769 - 824. [Abstract] [Full Text] [PDF]
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D. Zivadinovic, M. Tomic, D. Yuan, and S. S. Stojilkovic Cell-Type Specific Messenger Functions of Extracellular Calcium in the Anterior Pituitary Endocrinology, February 1, 2002; 143(2): 445 - 455. [Abstract] [Full Text] [PDF]
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 F. Van Goor, Y.-X. Li, and S. S. Stojilkovic Paradoxical Role of Large-Conductance Calcium-Activated K+ (BK) Channels in Controlling Action Potential-Driven Ca2+ Entry in Anterior Pituitary Cells J. Neurosci., August 15, 2001; 21(16): 5902 - 5915. [Abstract] [Full Text] [PDF]
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F. Van Goor, D. Zivadinovic, and S. S. Stojilkovic Differential Expression of Ionic Channels in Rat Anterior Pituitary Cells Mol. Endocrinol., July 1, 2001; 15(7): 1222 - 1236. [Abstract] [Full Text] [PDF]
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T. S. Kostic, S. A. Andric, and S. S. Stojilkovic Spontaneous and Receptor-Controlled Soluble Guanylyl Cyclase Activity in Anterior Pituitary Cells Mol. Endocrinol., June 1, 2001; 15(6): 1010 - 1022. [Abstract] [Full Text] [PDF]
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 C. J. H. Wong, J. D. Johnson, W. K. Yunker, and J. P. Chang Caffeine stores and dopamine differentially require Ca2+ channels in goldfish somatotropes Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2001; 280(2): R494 - R503. [Abstract] [Full Text] [PDF]
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 S. A. Andric, T. S. Kostic, M. Tomic', T.-a. Koshimizu, and S. S. Stojilkovic Dependence of Soluble Guanylyl Cyclase Activity on Calcium Signaling in Pituitary Cells J. Biol. Chem., January 5, 2001; 276(1): 844 - 849. [Abstract] [Full Text] [PDF]
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