Am J Physiol Endocrinol Metab 282: E810–E819, 2002.
First published November 27, 2001; 10.1152/ajpendo.00038.2001.
Function-specific calcium stores selectively regulate
growth hormone secretion, storage, and mRNA level
JAMES D. JOHNSON,1 CHRISTIAN KLAUSEN,2 HAMID R. HABIBI,2 AND JOHN P. CHANG1
1
Department of Biological Sciences, University of Alberta, Edmonton, T6G 2E9;
and 2University of Calgary, Calgary, Alberta, Canada T2N 1N4
Received 26 January, 2001; accepted in final form 20 November, 2001
Address for reprint requests and other correspondence: J. P.
Chang, Dept. of Biological Sciences, Biological Sciences Bldg. CW
405, Univ. of Alberta, Edmonton, Alberta, T6G 2E9, Canada (E-mail:
[email protected]).
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.
pituitary; endoplasmic reticulum; exocytosis; thapsigargin;
ryanodine; goldfish
E810
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society
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CA2⫹ IS AN IMPORTANT REGULATOR of many components of
the protein-secretory pathway. It is well accepted that
localized domains of high cytosolic Ca2⫹ concentration
([Ca2⫹]c) are the trigger for the membrane fusion and
exocytosis that culminates this pathway (34). In addition, there is growing appreciation of the roles of both
[Ca2⫹]c and luminal Ca2⫹ ([Ca2⫹]L) homeostasis in the
regulation of upstream components of the extended
secretory pathway (i.e., from transcription to exocytosis; reviewed in Ref. 22). In neuroendocrine cells,
[Ca2⫹]c has been implicated as a regulator of secretory
vesicle/granule recruitment into the releasable pool
(44), whereas secretory granule [Ca2⫹]L has been
shown to modulate the condensation state of peptide
hormones and the probability of exocytosis (1, 18, 42).
Intracellular Ca2⫹ stores of the endoplasmic reticulum (ER) also have a dual function in the regulation of
hormone release. On the one hand, Ca2⫹ release from
the ER controls exocytosis in many nonexcitable, as
well as some excitable, cell types (35, 38, 49). On the
other hand, it has also been established that ER
[Ca2⫹]L must remain high for the maintenance of normal secretory pathway function. The Ca2⫹ milieu of
the ER lumen plays an important role in the posttranslational processing, sorting, and packaging of secreted
proteins (2, 6, 11). In many cases, proteins are misfolded in Ca2⫹-depleted ER. Cells in which ER [Ca2⫹]L
has been depleted with thapsigargin (Tg), a potent and
selective inhibitor of ER Ca2⫹-ATPase (29, 47, 52),
exhibit decreased levels of certain proteins within the
ER. This phenomenon is due to a stimulation of proteolysis and/or increased transport out of the ER (54).
Others have reported that Tg treatment inhibits the
export of proteins from the ER, an effect probably
related to errors in protein folding or other posttranslational modifications (11). Ca2⫹ has also been shown
to regulate mRNA translation through multiple mechanisms. Depletion of ER Ca2⫹, with Tg or Ca2⫹-mobilizing agonist, leads to a rapid (within minutes) inhibition of translational initiation (5, 40). Interestingly,
Preston and Berlin (39) noted that protein synthesis in
HeLa cells was sensitive only to specific Ca2⫹-mobilizing treatments, suggesting a functional heterogeneity
between multiple ER Ca2⫹ stores.
Finally, the ER communicates with the nucleus
through several routes to regulate gene transcription.
As is the case for translation, perturbed ER [Ca2⫹]L
homeostasis generates a signal, which acts independently of increased [Ca2⫹]c to control the expression of
several genes. These genes include calreticulin and
immunoglobulin heavy-chain-binding protein, which
encode ER resident Ca2⫹ buffering proteins and chaperones (29). The are many examples of cytosolic Ca2⫹
signal that stimulate the expression of specific genes
(e.g., Ref. 41). Both positive and negative transcription
Johnson, James D., Christian Klausen, Hamid R.
Habibi, and John P. Chang. Function-specific calcium
stores selectively regulate growth hormone secretion, storage, and mRNA level. Am J Physiol Endocrinol Metab 282:
E810–E819, 2002. First published November 27, 2001;
10.1152/ajpendo.00038.2001.—Ca⫹ stores may regulate multiple components of the secretory pathway. We examined the
roles of biochemically independent intracellular Ca2⫹ stores
on acute and long-term growth hormone (GH) release, storage, and mRNA levels in goldfish somatotropes. Thapsigargin-evoked intracellular Ca2⫹ concentration ([Ca2⫹]i) signal
amplitude was similar to the Ca2⫹-mobilizing agonist gonadotropin-releasing hormone, but thapsigargin (2 ␮M) did not
acutely increase GH release, suggesting uncoupling between
[Ca2⫹]i and exocytosis. However, 2 ␮M thapsigargin affected
long-term secretory function. Thapsigargin-treated cells displayed a steady secretion of GH (2, 12, and 24 h), which
decreased GH content (12 and 24 h), but not GH mRNA/
production (24 h). In contrast to the results with thapsigargin, activating the ryanodine (Ry) receptor (RyR) with 1 nM
Ry transiently increased GH release (2 h). Prolonged activation of RyR (24 h) reduced GH release, contents and apparent
production, without changing GH mRNA levels. Inhibiting
RyR with 10 ␮M Ry increased GH mRNA levels, production,
and storage (2 h). Increasing [Ca2⫹]i independently of Ca2⫹
stores with the use of 30 mM KCl decreased GH mRNA.
Collectively, these results suggest that parts of the secretory
pathway can be controlled independently by function-specific
Ca2⫹ stores.
FUNCTION-SPECIFIC CA2⫹ STORES
AJP-Endocrinol Metab • VOL
the extended secretory pathway and imparts insights
into the relative importance of cytosolic and luminal
Ca2⫹ signaling in these processes.
MATERIALS AND METHODS
Reagents. Stock solutions of sGnRH and cGnRH-II (Peninsula Laboratories, Belmont, CA) were made in distilled,
deionized water. Depolarizing medium (30 mM KCl) was
made by equimolar substitution of NaCl with KCl. Highpurity Ry (99.5%; Calbiochem, La Jolla, CA) was dissolved in
dimethyl sulfoxide (DMSO). Tg (Calbiochem) was dissolved
in ethanol. Cyclohexamide (ICN, Aurora, OH) was dissolved
directly into testing media. At the concentrations used in this
study (final concentrations ⬍0.1%), DMSO or ethanol did not
affect [Ca2⫹]c (data not shown), GH release, or GH contents.
Animals and cell preparation. Animal use protocols were
approved by the University of Alberta Animal Care Committee in accordance with national guidelines. Pituitaries from
goldfish (Aquatic Imports, Calgary, AB, Canada) were dispersed using a trypsin-DNase protocol that has been described previously (8). Dispersed cells were cultured overnight on poly-L-lysine-coated coverslips (0.25 million cells/
coverslip, 1-ml medium) for imaging experiments (24),
preswollen Cytodex beads (1.5 million/dish, 6-ml medium) for
cell column perifusion studies (8), or Primaria surface-modified tissue culture plates (Becton-Dickinson, Franklin
Lakes, NJ) for static incubation experiments designed to
measure long-term hormone secretion/contents (24-well
plates; 0.25 million cells/well; 1-ml medium) or GH mRNA
levels (6-well plates; 2 million cells/well; 4-ml medium) in
Medium 199 (M-199) with Earle’s salts (GIBCO, Grand Island, NY) supplemented with 2.2 g/l NaHCO3, 25 mM
HEPES, 100,000 U/l penicillin, 100 mg/l streptomycin, and
1% horse serum (pH adjusted to 7.2 with NaOH) at 28°C, 5%
CO2, and in saturated humidity.
Cell identification and Ca2⫹ imaging. Morphologically
identified somatotropes [⬎95% accuracy (50)] that had been
stably and evenly loaded with fura 2 by 35-min incubation in
10 ␮M of the acetoxymethyl ester form of the dye (Molecular
Probes, Eugene, OR), were imaged for our laboratory as
described previously (24). Cells were perifused with imaging
medium (testing medium without phenol red) at room temperature. The ratio of emission intensity (at 510 nm) from
alternate 340 nm and 380 nm excitation was converted to
intracellular Ca2⫹ concentration ([Ca2⫹]i) estimates off-line
using the formula of Grynkiewicz et al. (17) and empirically
derived constants as detailed previously (24).
Cellular GH content and hormone release experiments.
Prolonged cellular GH content and GH release were assayed
in testing medium (GIBCO M-199 with Hanks’ salts, supplemented with 2.2 g/l NaHCO3, 25 mM HEPES, 100,000 U/l
penicillin, 100 mg/l streptomycin, 20 mg/l phenol red, and
0.1% BSA, pH adjusted to 7.2 with NaOH) under static
incubation conditions [same as culture conditions described
above (8)]. After the incubation period, the medium was
aspirated, and cells were lysed and snap frozen. Prolonged
exposures to the treatments used in this study did not reduce
the cell number or the viability of the pituitary cell cultures
as assessed by trypan blue exclusion. Furthermore, microscopic examination of treated cells did not reveal morphological signs of apoptosis. For dynamic measurements of hormone release, cells were perifused at 18°C with testing
medium as described previously (8). Medium was collected in
5-min or 30-s fractions and stored at ⫺26°C. GH content of
samples from both static incubation and cell column perifu-
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factors have been identified (e.g., Refs. 7, 12, 43). However, the Ca2⫹-dependent repression of gene expression
seems to be less common. Collectively, the literature
indicates that the regulation of multiple components
of the extended secretory pathway by Ca2⫹ is complex. The ubiquity of Ca2⫹ as a signaling molecule
inevitably leads to the question: how can all of these
Ca2⫹-dependent cellular functions be regulated independently?
Multiple function-specific Ca2⫹ stores offer a solution to the problems associated with the independent
regulation of multiple cellular functions by Ca2⫹ (22).
However, whether certain ER Ca2⫹ stores regulate
exocytosis whereas others control gene expression or
protein processing is still poorly understood. Indeed,
multiple ER Ca2⫹ stores can be distinguished pharmacologically (22, 38). Most cell types contain agonistsensitive Ca2⫹ stores that are mobilized by inositol
1,4,5-triphosphate (IP3) and refilled by Tg-sensitive
sarco(endo)plasmic reticulum Ca2⫹-ATPase (SERCA)
Ca2⫹-ATPases (38). An additional Ca2⫹ store, which
can be modulated by ryanodine (Ry), coexists with the
IP3-sensitive Ca2⫹ pool(s) in a variety of cell types (e.g.,
Ref. 16). Some cell types possess at least three distinct
intracellular Ca2⫹ stores (reviewed in Ref. 22).
Whether other classes of ER Ca2⫹ stores, such as those
sensitive to Ry, modulate early components (gene expression, protein synthesis, or protein processing) of
the extended secretory pathway is poorly understood.
There are no reports of the effects of pharmacologically
distinct Ca2⫹ stores on multiple components of the
extended secretory pathway.
Goldfish somatotropes are an interesting subject for
such studies, because they appear to contain several
biochemical classes of intracellular Ca2⫹ stores that
may be attributable to the ER. The Ca2⫹-dependent
signal transduction pathways of two physiological
growth hormone (GH)-releasing neuropeptides, salmon gonadotropin-releasing hormone (sGnRH) or
chicken GnRH-II (cGnRH-II), have been characterized.
Acute GH release evoked by both GnRHs is initiated
entirely by rapidly exchanging intracellular Ca2⫹
stores, which are sensitive to TMB-8 and caffeine (25,
55). Interestingly, although cGnRH-II-stimulated GH
release can be abolished by 10 ␮M ryanodine (24), it is
insensitive to Tg and other inhibitors of SERCA (25),
suggesting the existence of additional, functionally independent Ca2⫹ stores. In the present study, we examined the role of Tg-sensitive Ca2⫹ stores in several
cellular functions by use of Ca2⫹ imaging, radioimmunoassay measurements of hormone secretion and cellular GH content, and Northern analysis. Although
Tg-sensitive Ca2⫹ stores did not regulate acute GH
release or GH mRNA levels, they were involved in
acute [Ca2⫹]c homeostasis as well as long-term GH
secretion and storage. In contrast, inhibition of RyR led
to a significant increase in GH mRNA. Increasing
[Ca2⫹]c without emptying intracellular Ca2⫹ stores led
to a robust decrease in GH mRNA levels. This study
provides novel evidence that certain classes of ER Ca2⫹
stores may be uncoupled from specific components of
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FUNCTION-SPECIFIC CA2⫹ STORES
RESULTS
Uncoupling of Tg-sensitive Ca2⫹ stores from acute
GH release. The principle physiological regulators of
GH release in goldfish somatotropes, sGnRH and
cGnRH-II, act by mobilizing intracellular Ca2⫹ stores
that are not sensitive to Tg (23). Previous studies have
shown that Tg can generate Ca2⫹ signals, under extracellular Ca2⫹-free conditions, in goldfish pituitary cells
(32). In the present study, we sought to confirm the
presence of Tg-sensitive Ca2⫹ stores in single identified
somatotropes and establish whether Ca2⫹ released
from these pools can evoke GH secretion. In cells
treated with 2 ␮M Tg, [Ca2⫹]i increased slowly and
steadily before gradually decaying to a higher baseline
value (Fig. 1); Tg-stimulated Ca2⫹ signals are presumAJP-Endocrinol Metab • VOL
Fig. 1. Comparison of Ca2⫹ signals evoked by thapsigargin (Tg; 2
␮M) and salmon gonadotropin-releasing hormone (sGnRH; 100 nM)
in morphologically identified, fura 2-loaded somatotropes. Treatment
durations are indicated by horizontal bars. Top: false-color images
from selected times throughout the experiment (gray dots). A schematic of the cell is included (inset) for reference. Results are representative of cells from 7 independent cultures. Amplitude of Ca2⫹
signals evoked by 100 nM chicken (c)GnRH-II are similar to those
induced by Tg and sGnRH (not shown; see Ref. 24).
ably initiated as a result of the uncompensated passive
leak from SERCA-containing intracellular Ca2⫹ stores
and sustained by store-operated Ca2⫹ entry. Compared
with Ca2⫹ signals evoked by Tg in the same cell, GnRH
generated Ca2⫹ signals with distinct kinetics. Ca2⫹
signals evoked by 100 nM sGnRH (Fig. 1) or 100 nM
cGnRH-II (data not shown) displayed a faster rate of
rise and a rapid decay during the application period,
consistent with a more “active” release of Ca2⫹ from
intracellular stores (23). At the resolution used in the
present study, the spatial pattern of Tg- and GnRHevoked Ca2⫹ signals were not substantially different
(Fig. 1).
Despite the fact that 2 ␮M Tg elicited Ca2⫹ signals
with similar maximal amplitudes compared with endogenous secretagogues such as sGnRH and cGnRH-II
(Figs. 1 and 2A, Ref. 23), Tg failed to stimulate the
release of GH in “rapid fraction” cell column perifusion
experiments (Fig. 2B). Thus Ca2⫹ signals generated by
2 ␮M Tg may be selectively uncoupled from the acute
regulation of GH secretion in goldfish somatotropes.
The inability of Tg treatment to cause acute GH release was also confirmed at 0.01–1 ␮M (data not
shown).
Characterization of basal GH release and storage. In
preparation for studies examining the effects of selective manipulations of Ca2⫹ stores on the long-term
regulation of basal GH secretion and storage, we first
characterized some features of these parameters in
unstimulated goldfish somatotropes. Cellular GH contents of unstimulated cells, maintained in testing medium, remained stable for ⱖ24 h (results from a representative experiment are shown in Fig. 3A).
Similarly, the rate of basal GH secretion did not
change significantly between the time points (2 h:
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sion experiments was determined by a validated radioimmunoassay (31).
Hormone release measurements from static incubation
studies were expressed as a percentage of controls. For studies of hormone secretion in perifusion conditions, values were
normalized to the mean of the first five measurements (%pretreatment). Where analyzed, net hormone release responses
of perifused cell columns to treatments were quantified by
integrating the area under the curve during the treatment
period. Each time point was subtracted from the prepulse
mean, defined as the average of the three time points before
the treatment period.
Quantification of GH mRNA levels. RNA extraction and
Northern blot analysis of dispersed goldfish pituitary cells
(cultured as in Cellular GH content and hormone release
experiments) were optimized from protocols designed for isolated pituitary fragments (20). Briefly, total RNA was isolated with TRIzol reagent (GIBCO), based on the acid-guanidinium thiocyanate phenol-chloroform method (10). RNA
was quantified and checked for purity with a spectrophotometer (A260/A280). Treatments did not affect the amount of
RNA isolated from individual wells. RNA (5 ␮g/lane) was
loaded and resolved on a 1.2% agarose-formaldehyde gel
before being transferred to a Hybond N⫹ membrane (Amersham, Arlington Heights, IL) by use of capillary action in the
presence of 20⫻ sodium chloride-sodium phosphate-EDTA
[1⫻ SSPE (in M) ⫽ 0.3 NaCl, 0.02 NaH2PO4 䡠 H2O, 0.002
EDTA, pH 7.4]. Membranes were prehybridized for 1 h and
hybridized for 2 h at 60°C with a specific cDNA probe for
goldfish GH (labeled with [␣-32P]deoxycytidine 5⬘-triphosphate by means of the random primer method, 3,000 Ci/mM,
Amersham) in rapid hybridization buffer (Amersham). Membranes were then subjected to a series of washes, with 0.1%
SDS, of increasing stringency [ⱕ0.1⫻ standard sodium citrate (SSC); 1⫻ SSC ⫽ 0.15 M NaCl, 15 mM sodium citrate].
Membranes were then exposed to film. Although ethidium
bromide-stained gels were imaged to ensure equal loading of
lanes, membranes were also stripped and rehybridized with
a probe for 18S ribosomal RNA, which served as an internal
standard. Treatments caused no observable change in 18S
levels. Autoradiograms were scanned and quantified using
the densitometry function of NIH Image 1.62 (National Institutes of Health, Bethesda, MD). GH mRNA values were
normalized to the internal control (18S levels) for each lane.
Statistical analysis. Statistical analyses were performed
using Student’s unpaired t-test or one-way ANOVA (followed
by Fisher’s protected least significant difference test), where
appropriate. Differences were considered to be significant
when P ⬍ 0.05. Results are presented as means ⫾ SE.
FUNCTION-SPECIFIC CA2⫹ STORES
370 ⫾ 97 ng/h; 12 h: 263 ⫾ 62 ng/h; 24 h: 271 ⫾ 52 ng/h;
pooled results from three separate experiments). Although the absolute amount of GH stored and released
varied among cell cultures, the relative proportion of
these two components was similar in different experiments. For this reason, GH values were normalized
with respect to controls in subsequent analysis.
Endocrine cells are known to release proteins, including hormones, through multiple routes, including
the regulated secretory pathway and the constitutive
secretory pathway (27). The latter pathway is characterized by small vesicles of newly produced proteins
that bud off from the trans-Golgi network, proceed
directly to the plasma membrane without being stored,
and immediately undergo exocytosis in the absence of a
measurable global Ca2⫹ signal (total time from protein
synthesis to exocytosis ⬍1 h; Refs. 27, 51). To determine the relative involvement of constitutive and regulated secretory pathways in somatotropes, we monitored the effects of cyclohexamide on basal GH
secretion and cellular contents. Treatment with the
protein synthesis inhibitor cyclohexamide (100 ␮M) did
not reduce GH release in 2-h static incubation studies
(Fig. 3B). Likewise, long exposures of cyclohexamide
did not affect basal GH release in perifusion experiments (Fig. 3C). These results suggest that the constitutive secretory pathway does not play a role in basal
GH secretion (ⱕ2 h) from goldfish somatotropes. The
AJP-Endocrinol Metab • VOL
observation that exocytosis evoked by ionomycin-mediated Ca2⫹ influx is also unaffected by cyclohexamide
indicates that somatotropes do not preferentially release newly synthesized GH through the regulated
secretory pathway.
Although prolonged treatment (12 and 24 h) with
cyclohexamide produced the expected decrease in cellular GH contents, a paradoxical increase in GH contents was observed after 2 h (Fig. 3B). At this time, we
can only speculate as to the possible underlying mechanism of this response. Perhaps cyclohexamide inhibited the formation of a certain pool of proteins, with
rapid turnover/synthesis rates, which are normally responsible for the degradation of some stored GH.
Differential involvement of Tg- and Ry-sensitive
Ca2⫹ stores in proximal components of the GH secretory
pathway. Having ruled out a direct coupling to acute
GH release, we asked whether Tg-sensitive Ca2⫹
stores regulate other aspects of the secretory pathway,
such as long-term GH release, cellular GH contents,
total GH (release ⫹ contents, an index of production),
and GH mRNA levels at three time points. In addition,
we compared the effects of Tg with those of Ry, a
treatment that is known to modulate the function of a
distinct subclass of ER Ca2⫹ stores in some cell types
(e.g., Ref. 16). Ry has the advantageous property of
locking the ryanodine receptor (RyR) Ca2⫹ release
channel in an open subconductance state, thereby
stimulating the release of Ca2⫹ at low (i.e., 1 nM) doses
(Ref. 13 and J. D. Johnson and J. P. Chang, unpublished data). Ry blocks Ca2⫹ release from RyR at
higher concentrations (i.e., 10 ␮M) and abolishes
cGnRH-II-stimulated GH release (24). Although both
Ry treatments selectively perturb Ca2⫹ flux from RyRcontaining Ca2⫹ stores, only the former would be expected to deplete these Ca2⫹ pools.
Overall, the long-term effect of Tg treatment (2 ␮M)
was the release of GH from the secretory pathway,
without a concomitant maintenance of cellular GH
contents (Fig. 4). In contrast, activation of RyR with 1
nM Ry produced a biphasic response. In the first 2 h,
Ca2⫹ release from Ry-sensitive store is positively coupled to GH release, contents, and net production,
whereas the net effect over 24 h was a decrease in all of
these parameters. On the other hand, blocking RyR
Ca2⫹ release channels with 10 ␮M Ry modulated GH
cellular contents (through a slight increase in production), in a manner that was only transient. Further
differences in the functional coupling of Tg-sensitive
and Ry-sensitive Ca2⫹ stores to the extended GH secretory pathway are revealed when the data are examined at each time point. After 2 h, Tg treatment had
initiated a sustained GH release response. Although 1
nM Ry also stimulated GH release over 2 h, this was
accompanied by an increase in cellular GH contents.
On the other hand, the inhibition of Ca2⫹ release from
these stores with 10 ␮M Ry increased GH contents
without affecting secretion. At the 12-h time point,
Tg-treated cells continued to release stored GH without an apparent compensatory increase in hormone
production. In the presence of Tg, cellular GH contents
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Fig. 2. Comparison of growth hormone (GH) release responses and
Ca2⫹ signals generated by 2 ␮M Tg. Treatment durations are indicated by horizontal bars. A: pooled results of Ca2⫹ signals from 10
single, 2 ␮M Tg-treated somatotropes from independent cultures. B:
Tg (2 ␮M), given under similar conditions, failed to stimulate GH
release from populations of somatotropes contained in mixed pituitary culture. Thirty-second samples from the cell column perifusion
were collected as indicated (n ⫽ 5).
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FUNCTION-SPECIFIC CA2⫹ STORES
were reduced to a level similar to that seen in experiments with cyclohexamide. In contrast, neither dose of
Ry affected GH secretion or GH contents over 12 h.
Over 24 h, the increase in GH release from Tg-treated
cultures, although still significant, was at its lowest
levels compared with controls. It is important to note
that, because our measurements are cumulative, these
latter determinations of GH release are still “contaminated” with the high responses seen after 2 h. Over
the course of the entire experiment, the stimulatory
dose of Ry had resulted in a reduction in basal GH
secretion, presumably due to the failure to compensate
with increases in GH contents and production. Nevertheless, the modest reduction in stored GH seen in 1
nM Ry-treated cultures was significantly different
from the more robust decrease seen in Tg-treated cells
during the same period (P ⬍ 0.05). On the other hand,
blocking of RyR had no significant effect on these
parameters. Taken together, these data suggest that
manipulations of Tg-sensitive and Ry-sensitive Ca2⫹
stores in goldfish somatotropes differentially control
the long-term secretion and replenishment of GH.
Next, in parallel experiments, we assessed the role of
these two intracellular Ca2⫹ stores in the control of GH
gene expression by Northern blot analysis of GH
AJP-Endocrinol Metab • VOL
mRNA levels. Neither 2 ␮M Tg nor 1 nM Ry significantly altered GH mRNA levels at any of the time
points tested (Fig. 5), despite their ability to evoke
robust GH secretion during the first 2 h. Surprisingly,
application of the blocking dose of Ry (10 ␮M) resulted
in a significant, but transient, elevation in GH gene
expression at 2 h, a time at which cellular GH contents
and GH production were also increased. These results
with 10 ␮M Ry suggest that perturbing Ry-sensitive
Ca2⫹ stores by blocking RyR can regulate GH biosynthesis. Although all of the treatments used in the
present study were shown to modulate the apparent
storage and production of GH, only the transient (2 h)
effects of 10 ␮M Ry are correlated with changes in GH
mRNA levels. It is unlikely that these effects of Tg and
Ry are attributable to changes in cell viability, because
the health and numbers of cells in these cultures were
not different, as determined by the trypan blue exclusion test. The fact that similar amounts of protein and
total mRNA (quantified spectrophotometrically) were
obtained from all cell cultures also attests to a lack of
cell loss.
Negative coupling of [Ca2⫹]c to GH gene expression.
To further evaluate the functional importance of specific Ca2⫹ signals, we examined the role of [Ca2⫹]c
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Fig. 3. Characterization of long-term
basal GH release and storage. A: mixed
populations of dispersed pituitary cells
release GH and maintain stable GH
contents over 24 h in testing media. A
representative example of 3 separate
experiments is shown. B: long-term
GH release and storage are not affected by 0.1% DMSO or 0.1% ETOH
(n ⫽ 8). Treatment with cyclohexamide
(Cyclo; 100 ␮M) significantly reduced
GH production and contents by 12 h
after an initial stimulation seen at 2 h
(n ⫽ 20). C: extended treatment with
100 ␮M cyclohexamide (horizontal bar)
did not reduce basal or 100 ␮M ionomycin (Iono)-stimulated GH release in
perifusion experiments (n ⫽ 6). 夹Significant difference compared with
time-matched controls.
FUNCTION-SPECIFIC CA2⫹ STORES
E815
Fig. 4. Differential coupling of endoplasmic reticulum Ca2⫹ stores
sensitive to Tg or ryanodine (Ry) to GH secretion, contents, and
production (secretion ⫹ contents) at 3 timepoints. GH values from
cultures treated with 2 ␮M Tg (n ⫽ 36), 1 nM Ry (n ⫽ 16), or 10 ␮M
Ry (n ⫽ 16) are normalized to time-matched controls (n ⫽ 48). See
Fig. 3A for typical absolute GH values (prenormalization). 夹Significant difference compared with time-matched controls.
signals generated independently of intracellular Ca2⫹
stores in the regulation of GH gene expression. To this
end, we treated cells with 30 mM KCl for 30 min to
activate voltage-gated Ca2⫹ channels (VGCCs) and
measured GH mRNA 12 h later. It has previously been
shown that KCl-stimulated Ca2⫹ signals in populations of goldfish pituitary cells are completely dependent on the influx of extracellular Ca2⫹ (21). A 30-min
treatment protocol was chosen because prolonged exposure to KCl (6–24 h) resulted in the detachment of
cells in our cultures (J. P. Chang, unpublished observations). The results of this experiment clearly show
that GH mRNA levels were reduced as a result of KCl
treatment (Fig. 6A), although the expected relationship between [Ca2⫹]c and hormone secretion is positive
in somatotropes (Fig. 6, B and C). This result suggests
that elevated [Ca2⫹]c initiates a signal that is inhibitory to GH gene expression in the goldfish.
DISCUSSION
Uncoupling of Tg-stimulated Ca2⫹ signals from
acute GH release. We have hypothesized that cells use
function-specific Ca2⫹ stores to generate a complex
array of Ca2⫹ signals capable of controlling different
components of the extended secretory pathway independently (23). A prediction of such a hypothesis is
that Ca2⫹ signals from certain intracellular stores
AJP-Endocrinol Metab • VOL
Fig. 5. Differential effects of Tg and Ry on GH mRNA levels at 3
timepoints. Results are presented as relative change from untreated
cultures at each time point (n ⫽ 4). GH mRNA was quantified using
densitometry and normalized to an internal standard (18S ribosomal
RNA). 夹Significant difference from time-matched control cultures.
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would fail to modulate certain Ca2⫹-dependent processes. The present study clearly indicates that such
functional specificity occurs. No GH release was seen
at a dose of Tg that evoked increases in [Ca2⫹]c of
similar maximal amplitude to those generated by
GnRH, suggesting that one class of ER Ca2⫹ stores is
functionally uncoupled from the acute control of exocytosis. Different degrees of uncoupling between Ca2⫹
signals and hormone release have been reported for
cell lines derived from the mammalian pituitary [e.g.,
␣T3–1 cells (48)]. In the present study, the comparison
between Tg and GnRH is valid, because acute GnRHstimulated hormone release relies exclusively on the
release intracellular Ca2⫹ stores, as is the case in
mammalian and fish gonadotropes (25, 47). Together
with the present data, we have shown that GnRH
signaling involves Ca2⫹ stores that are completely independent of those modulated by Tg (23), in contrast to
the situation in many other cell types (e.g., Ref. 49).
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FUNCTION-SPECIFIC CA2⫹ STORES
Instead, acute GnRH-stimulated GH release depends
on a rapidly depleting intracellular Ca2⫹ pool that is
sensitive to 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester and caffeine (23, 55). Recently, we
have identified the Ry-sensitive Ca2⫹ stores as an
essential mediator of cGnRH-II-stimulated GH release
(24). Thus somatotropes contain multiple agonist-sensitive intracellular Ca2⫹ stores, as we have shown to be
the case in goldfish gonadotropes (24, 25).
Tg-stimulated [Ca2⫹]L depletion induces the prolonged release of GH from the secretory pathway. What
is the role of Tg-sensitive Ca2⫹ stores in goldfish somatotropes if not to directly regulate acute GH release or
mediate agonist signaling? We suggest that prolonged
Tg treatment, which would deplete Tg-sensitive ER
Ca2⫹ stores, led to a long-term emptying of stored GH.
This response was not accompanied by a compensatory
increase in GH production (assessed by net hormone
present or GH mRNA levels). The net result was a
reduction in the cellular GH contents. Although the
decrease in cellular GH contents induced by Tg is
AJP-Endocrinol Metab • VOL
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Fig. 6. Cytosolic Ca2⫹ signals, induced by extracellular Ca2⫹ influx
through voltage-gated Ca2⫹ channels (VGCC) reduces GH mRNA
relative to untreated cells. A: cells were exposed to 30 mM KCl for 30
min. GH mRNA was measured after 12 h and quantified as in Fig. 5.
(n ⫽ 4). 夹Significant difference from control. B: activation of VGCCs
with 30 mM KCl evoked biphasic Ca2⫹ signals in identified somatotropes. Pooled results from 6 cells from different cultures are shown.
C: 30 mM KCl stimulated GH release (n ⫽ 6).
quantitatively similar to that seen in cyclohexamidetreated cells, the observation that Tg does not affect
GH production or mRNA levels suggests that Tg and
cyclohexamide have different mechanisms of action.
Therefore, Tg must reduce cellular contents at a site
that is distal to protein synthesis on the extended
secretory pathway. Although the exact mechanism of
Tg action remains to be determined, we hypothesize
that Tg accelerates GH traffic through the distal parts
of the secretory pathway. In light of the findings that
Tg does not stimulate acute exocytosis directly, we
propose that the prolonged secretion of GH in Tgtreated cultures results from an increased amount of
GH released through the “basal” mechanism. In some
endocrine cell types, basal secretion has been attributed to a portion of secretory granules, which undergo
random fusion in the steady state (51). We are not
aware of studies in other model systems that have
measured similar reductions in hormone contents in
Tg-treated cells.
Inhibition of Ry-sensitive Ca2⫹ stores transiently
stimulates GH production. It has been reported that
Tg-mobilizes only a fraction of stored Ca2⫹, equivalent
to between ⬃40 and 60%, depending on the cell type
(14, 37, 46). Thus we examined whether other components of the ER are functionally important in somatotropes. Previous studies have shown that Ry-sensitive
Ca2⫹ stores participate in acute agonist Ca2⫹ signaling
in mammalian somatotropes (19, 36). Despite the fact
that RyR is found in many tissues (45), the modulation
of gene expression and protein synthesis by Ry-sensitive Ca2⫹ stores is not well characterized in nonmuscle
cells. We report, for the first time, evidence of the
involvement of RyR in the long-term functioning of
somatotropes. Stimulation of ER Ca2⫹ release with 1
nM Ry evoked significant GH secretion after 2 h. This
response was transient and eventually resulted in the
inhibition of GH release over 24 h, unlike the prolonged secretory activity of Tg. On the other hand, the
inhibitory dose of Ry did not affect the release of GH.
Likewise, preliminary experiments have shown that
GnRH modulation of GH mRNA levels is also transient
(C. Klausen, J. P. Chang, and H. R. Habibi, unpublished observations). Collectively, these data suggest
that GH release is positively coupled to Ca2⫹ release
from Ry-sensitive stores, at least initially.
Interestingly, whereas both Ry treatments elevated
GH contents at 2 h, only 10 ␮M Ry increased GH
mRNA. This result suggests (indirectly) that part of
the increase in total GH seen in cells treated with 1 nM
Ry over 2 h results from some posttranscriptional enhancement of GH production. On the other hand, the
increased GH contents in 10 ␮M Ry-treated cells can be
accounted for by the significant increase in GH mRNA
expression. We propose that the inhibition of normal
Ca2⫹ homeostasis, and perhaps an elevated [Ca2⫹]L
rather than a cytosolic Ca2⫹ signal, mediates acute
changes in GH gene expression. Although others have
elegantly shown the importance of ER [Ca2⫹]L in the
control of ER regulatory genes (29), this is the first
time such a mechanism has been considered as a reg-
FUNCTION-SPECIFIC CA2⫹ STORES
AJP-Endocrinol Metab • VOL
(9). Recent preliminary experiments have shown that
sGnRH and cGnRH-II stimulated GH mRNA accumulation in vitro and in vivo over the same time period
when it was inhibited by KCl (28). Therefore, it seems
likely that VGCC-independent postreceptor pathways
are important in the long-term effects of GnRH on GH
gene expression.
Physiological significance and comparison with other
goldfish pituitary cell types. The fact that goldfish somatotropes are regulated by multiple agonists suggests that functional specificity of Ca2⫹ signaling must
be sufficiently complex to ensure the precise independent control of specific cellular functions by specific
extracellular signals. There has been a recent appreciation for the range of organelles participating in intracellular Ca2⫹ signaling, including the ER, nucleus,
Golgi, secretory granules, and mitochondria. This has
paralleled a growing understanding of the diversity of
intracellular Ca2⫹-release channels and associated second messengers, such as IP3, cADP-ribose, and nicotinic acid adenine dinucleotide phosphate, which make
up a multitude of pharmacological classes of Ca2⫹signaling systems (reviewed in Ref. 22). Future studies
should include the examination of novel classes of
intracellular Ca2⫹ stores and more mRNA species involved in different aspects of the secretory pathway.
Like somatotropes, goldfish gonadotropes are also
regulated by multiple agonists, including both GnRHs.
However, Ca2⫹-dependent signaling cascades differ
considerably between these cell types (9, 23–26). It is
likely that the actions of Tg may be cell type specific.
For example, studies of goldfish gonadotropes in which
Tg also evokes a reduction in cellular hormone contents have indicated that Tg significantly reduces
GTH-II mRNA levels at 12 and 24 h (J. D. Johnson, C.
Klausen, H. R. Habibi, and J. P. Chang, unpublished
results). By comparing the functional roles of intracellular Ca2⫹ stores between these two cell types, we may
learn important details about pituitary physiology.
It is important to note that pharmacological treatments used in the present study cannot replicate the
complexities of agonist stimulation. However, the observations that specific Ca2⫹-mobilizing treatments
can be uncoupled from specific Ca2⫹-dependent cellular functions is likely to have relevance to how pituitary function is controlled by multiple agonists. Similarly, the observation that specific components of the
extended secretory pathway can be modulated independently may eventually lead to strategies targeting
specific cellular functions in other cell types.
In conclusion, the present results provide new insights into the regulation of multiple aspects of the
extended secretory pathway by two intracellular Ca2⫹
stores and by cytosolic Ca2⫹ signals. Moreover, our
data support a model whereby both [Ca2⫹]L signals and
[Ca2⫹]c participate in the control of hormonal gene
expression. We propose that function-specific Ca2⫹
stores may represent a novel mechanism for the independent control of cellular functions by multiple agonists.
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ulator of hormonal mRNA in cultured pituitary cells. A
complete test of this hypothesis awaits the measurement of [Ca2⫹]L in the Ry-sensitive ER of intact somatotropes. Nevertheless, our results clearly demonstrate
that ER Ca2⫹ stores sensitive to Ry, but not Tg, can be
coupled to GH mRNA expression. This finding does not
rule out the possibility that Tg-sensitive Ca2⫹ pools
control the expression of other genes in somatotropes.
Indeed, the independent modulation of multiple gene
sets would offer additional levels of signaling specificity to the cell.
Role of [Ca2⫹]c in GH production. We directly examined the relationship between [Ca2⫹]c and GH mRNA
expression by use of depolarizing concentrations of KCl
to generate Ca2⫹ signals mediated by VGCC (but presumably not intracellular stores). Surprisingly, Ca2⫹
signals mediated by VGCC strongly inhibited GH
mRNA expression, although they were positively coupled to GH release. In other experiments, ionomycin,
which is also a potent GH secretagogue, dose-dependently decreased GH cellular contents and production
within 2 h (M. Volk and J. P. Chang, unpublished
results). Thus these effects of KCl on goldfish somatotropes are not likely due to the preferential signaling
by VGCC-mediated Ca2⫹ signals, as others have reported for some neurons and pituitary cells (3, 33), but
may be generalized to elevation of [Ca2⫹]c. A comparable dissociation between GH release and GH synthesis
has been noted in studies of KCl-treated mammalian
somatotropes. Treating rat somatotropes with high
KCl concentration for 1 h had no effect on GH mRNA
levels, despite a robust secretory response (4). Similarly, some treatments that would be expected to increase cellular Ca2⫹ dramatically, such as replacing
Ca2⫹-free medium with high-Ca2⫹ medium, fail to
stimulate increases in GH mRNA expression in GH3
cells and normal rat somatotropes [although prolactin
synthesis was stimulated in GH3 cells (15, 53)].
The finding that elevated [Ca2⫹]c is negatively coupled to mRNA expression is compatible with the observed differential effects of 1 nM Ry and 10 ␮M Ry on
GH mRNA levels. The major presumed difference between the two Ry treatments is that only the stimulatory dose activates the RyR to release Ca2⫹, whereas
both treatments would be expected to perturb the normal cycling of Ca2⫹ through the Ry-sensitive component of the ER. One might speculate that 1 nM Ry
generates two Ca2⫹ signals, one luminal and one cytosolic, which have opposing effects on GH mRNA levels,
thus resulting in no net change in GH mRNA accumulation. Additional work is needed to establish signaling
cascades coupling Ry-sensitive Ca2⫹ stores to GH gene
expression.
Future studies will also be required to determine
whether this unique relationship between [Ca2⫹]c and
GH mRNA plays a role in the long-term signaling of
the many endogenous agonists and antagonists that
regulate the function of goldfish somatotropes. sGnRH,
cGnRH-II, dopamine, and pituitary adenylate cyclaseactivating polypeptide are all known to require VGCC
activation to sustain long-term (2 h) evoked GH release
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E818
FUNCTION-SPECIFIC CA2⫹ STORES
This study was funded by Natural Science and Engineering Research Council of Canada operating grants to J. P. Chang (OGP121399) and H. R. Habibi (U-37946). J. D. Johnson received support
from a Province of Alberta Graduate Fellowship and the Andrew
Steward Memorial Prize. C. Klausen was supported by a studentship
from the Alberta Heritage Foundation for Medical Research.
Present address of J. D. Johnson: 815 Yalem Bldg., Barnes-Jewish
Hospital, Washington Univ. School of Medicine (Renal Division),
Mailstop: 9032648, 216 S. Kingshighway Blvd., St. Louis, MO 63110
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