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Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D, Ph.D, Division of Endocrinology, Metabolism & Molecular Medicine, Northwestern University Medical School, Tarry 15–703, 303 East Chicago Avenue, Chicago, Illinois 60611.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-LUC reporter
plasmid. RyR messenger RNA (mRNA) expression varied during the rat
estrous cycle with maximal levels following increases of progesterone.
The effects of gonadal steroids on pituitary RyR mRNA levels were
examined directly in ovariectomized rats that were treated with
estrogen (E), or estrogen and progesterone (P). In this paradigm, E
decreased, whereas E + P increased RyR3 mRNA levels. These results
indicate that RyR is expressed and hormonally regulated in the rat
pituitary and suggest that it might play a role in mediating
GnRH-induced gonadotropin synthesis and secretion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GnRH-mediated LH release from the pituitary is calcium-dependent (reviewed in Ref.4). LH secretion is biphasic and parallels changes in cytosolic calcium levels. The initial spike phase of LH release is dependent on intracellular calcium stores, whereas the later sustained phase appears to be primarily dependent on influx of extracellular calcium through the voltage-sensitive and -insensitive calcium channels (5, 6). In addition, increased intracellular calcium levels may also play a role in GnRH-stimulated gene transcription (7), which may involve activation of calcium-dependent isoforms of PKC or other calcium-dependent kinases.
The ryanodine and the IP3 receptors are Ca2+-permeable channels that are associated with intracellular organelles. Both types of receptors mediate the release of Ca2+ from intracellular stores and increase cytosolic Ca2+ in response to many different extracellular stimuli, including hormones and neurotransmitters (8, 9, 10). The RyR was initially described in the sarcoplasmic reticulum of skeletal muscle (RyR1), and subsequently in cardiac muscle (RyR2), and brain (RyR3) (11, 12, 13, 14, 15). Although several signaling pathways can activate the RyR, calcium-induced calcium release (CICR) is the primary mediator in most cell types (16). Recent evidence indicates that in addition to their ability to rapidly adapt to successive increases in the intracellular concentration of calcium, RyRs are also regulated by phosphorylation (17). Although there are no reports of RyR expression in the pituitary, the use of high doses of ryanodine to antagonize RyR function decreased GH releasing hexapeptide (GHRP)-stimulated calcium release (18). In addition, ruthenium red, a relatively selective RyR antagonist, has been shown to inhibit GnRH-mediated LH secretion (19). We hypothesized that the initial burst of GnRH-induced calcium release (3) might trigger CICR events involving the RyR in gonadotrope cells. In this report, we demonstrate that the RyR is expressed in the pituitary and performed functional studies to determine whether it might be involved in GnRH signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hormone replacements in an estrogen-induced gonadotropin surge model have been described previously (21). Briefly, animals were OVX bilaterally 10–12 days before the experiments. Three days before experiments, animals received either a blank or 17ß-estradiol-filled sc SILASTIC brand capsule (Dow Corning, Midland, MI; id, 0.062 in.; od, 0.125 in.; 5 mm in length). At 1130h on the morning of the experiments, rats received an injection of progesterone (P) (5 mg, sc) or an equivalent volume of sesame oil. At 1330 h, an ip injection of pentobarbital (40 mg/kg BW) or saline was administered. Anterior pituitary glands were removed at 1800 h on the day of experiments and frozen in liquid nitrogen. RyR3 messenger RNA (mRNA) levels were then examined in the following groups of animals (n = 3/group): 1) ovariectomized (OVX) animals; 2) OVX E-primed animals; 3) OVX E-primed animals that were given an injection of pentobarbital to block GnRH neurosecretion; and 4) OVX E-primed animals that were given an injection of P on the morning of experiments.
RT-PCR analyses
RNA was isolated from anterior pituitary glands by
centrifugation through cesium chloride or using Tri Reagent (Molecular
Research Center, Inc., Cincinnati, OH). In the cesium chloride
protocol, individual pituitaries were homogenized in 2 ml of 4
M guanidine thiocyanate (GTC) that was overlaid on a 5.7
M cesium chloride cushion (22). After centrifugation at
36,000 rpm for 4 h at 25 C, the RNA pellet was solubilized in
0.1% SDS, and purified by ethanol precipitation. For extraction of RNA
using Tri Reagent, individual pituitaries were homogenized in 1 ml of
Tri Reagent and total cellular RNA was isolated from pituitary
homogenates using the protocol of the supplier. RNA pellets were
resuspended and treated with RQ1 deoxyribonuclease (1 U/mg RNA;
Promega, Madison, WI) for 20 min at 37 C. Deoxyribonuclease was removed
by phenol-chloroform-isoamylalcohol extraction (pH 5.2). Final RNA
concentrations were determined by optical density at 260 nm, and
integrity was verified by ethidium bromide staining of ribosomal 28S
and 18S bands on an agarose gel.
RNA (1 µg) from each individual pituitary gland was converted into complementary DNA (cDNA) using 11.25 U of AMV reverse transcriptase (Promega) and random hexamers to prime the reaction. Control reactions were identical except for the omission of reverse transcriptase in one set of reactions to control for amplification of genomic DNA.
Oligonucleotide primers were used to amplify 480 bp of RyR1 and 430 bp of RyR2 and RyR3 cDNAs from RT products. The primers used for amplification of RyR1, RyR2, and RyR3 were based on the sequences for the corresponding receptors in the mouse (23): RyR1, 5'-GTTCCGG-TGCTGGGGACATG-3' and 5'-TGTGGGCTGTGATCTCCAGA-3'; RyR2, 5'-ACTGCTAAAGTGACCAACAG-3 and 5'-TTGCATCGCTGAAATCTAGT-3'; RyR3, 5'-GTTGCAAACCTGTGGAACTC-3' and 5'-CTACTGGGCTAAAGTCAAGG-3'. Primers for a similarly sized fragment (500 bp) of the rat ribosomal protein L19 (RPL19) cDNA were included to control for reaction efficiency and variations in mRNA concentration in the original RT reaction.
Before PCR amplification, 2, 4, 8, or 16 µl of each RT product were
brought to a volume of 20 µl with RT diluent that contained
concentrations of buffer, MgCl2 and dNTPs identical to
those of the original RT reaction. Preliminary experiments indicated
that these dilutions produced PCR products that were linear with
respect to starting concentrations of RyR3 and RPL19 cDNAs. Preliminary
assays were also conducted to determine the optimal number of cycles
necessary for producing PCR products within the exponential phase of
amplification. Based upon the results of these experiments, a two-step
PCR was chosen for amplification of RyR3. For the first round of
amplification, primers for RyR3 (20 pmol of each primer) were added to
the diluted RT product along with 0.1 µl
[-32P]deoxy-CTP (New England Nuclear Corp., Boston,
MA), 1 x Taq polymerase buffer (Promega), 1.25 U
Taq DNA polymerase (Promega), and the reaction was brought
to a volume of 50 µl in distilled water. The reaction mix was
overlaid with 75 µl mineral oil and amplified for six cycles in a
programmable thermocycler (M. J. Research, Cambridge, MA). The
amplification cycle consisted of denaturation at 94 C for 1 min,
annealing at 55 C for 1 min, and extension at 72 C for 1 min. In a
second round of amplification, primers for RPL19 (20 pmol of each
primer) and 1.25 U Taq polymerase, in 50 µl of 1 x
Taq polymerase buffer, were added to the product of the
first round of amplification and amplified for another 26 cycles under
the same conditions. An aliquot (25 µl) of each PCR reaction was
subjected to electrophoresis through a 6% polyacrylamide gel, and
bands were quantitated using a BAS1000 PhosphorImager (Fuji Photo Film
Co. Ltd., Japan). Data at each timepoint or treatment group was
calculated as the mean ratio of RyR3 to RPL19 mRNA for at least three
successive dilutions. Group means were generated, and one-way ANOVA was
used for estrous cycle measurements followed by post-hoc analyses using
Fisher’s protected least significant difference test (Statview
software, Berkeley, CA). P < 0.05 was considered
statistically significant.
Studies of LH secretion
Perifusion experiments were conducted as described previously
(24). Perifused primary pituitary cells were stimulated with 10 min
pulses of 10 nM GnRH every 20 min, either in the presence
or absence of a ryanodine receptor antagonist, ruthenium red (10
µM). Column effluent was collected in 1 min fractions and
assayed for rat LH (25). Secretory baselines were calculated as the
mean hormone released in the four fractions preceding a pulse of GnRH.
Secretory peaks and plateaus were calculated as hormone released in the
first 6 and 8 fractions following a GnRH pulse, respectively, minus the
preceding baseline. Perifusion experiments were repeated three times
with similar results. Peak and plateau values from control,
antagonist-treated, and washout groups were compared using Student’s
t test. P < 0.05 was considered
statistically significant.
Western blots
Anterior pituitary glands and heart tissue were isolated from
male or female Sprague Dawley rats and homogenized in a solution (0.9
M NaCl, 50 mM Na-phosphate, 2.5 mM
EDTA, 20 mM NaF, and 50 mM HEPES-Tris at pH
7.4; all from Sigma Chemical Co., St. Louis, MO) (NEHDPF solution)
containing protease inhibitors (1 mM
phenylmethylsulfonylfluoride, 1 mM leupeptin and 1
mM antipain; all from Sigma). The homogenate was spun at
13,000 x g for 15 min at 4 C and the supernatant was
centrifuged at 150,000 x g for 60 min at 4 C to
isolate the microsomal fraction. The pellet was sonicated in 1%
3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS) in
NEHDPF solution containing protease inhibitors. Cardiac and pituitary
microsomal proteins (100 µg) were subjected to electrophoresis
through a 4% denaturing polyacrylamide gel, and proteins were then
transferred to a nitrocellulose membrane by electrophoretic transfer at
1.3 A for 60 min. The membrane was incubated with G43 polyclonal
antibody (1:300 dilution; a gift from Dr. Kevin Campbell, University of
Iowa, Iowa City, IA) that recognizes the cardiac and brain isoforms of
the ryanodine receptor. Following subsequent incubations with a
secondary antibody (rabbit antigoat IgG at a dilution of 1:2000; Pierce
Immunochemicals, Rockford, IL) and antirabbit horse radish
peroxide-conjugated tertiary antibody (1:5000; Amersham Life Sciences,
Arlington Heights, IL), detection was performed with the enhanced
chemiluminescence system (Amersham).
Transient expression studies
Primary pituitary cells were cultured and transfected as
described previously (26). Cultures of primary pituitary cells were
transfected with a reporter gene containing 846 bp of 5'-flanking
sequence and 44 bp of exon 1 of the human glycoprotein -gene linked
to the luciferase gene in the plasmid pA3LUC (20 µg/well), by the
calcium phosphate precipitation method (27, 28). Cells were exposed to
the DNA precipitate in culture medium for 6 h at 37 C. After
24 h treatment without or with 10-8 M
GnRH analog (Des-Gly10,[D-Ala6]GnRH
ethylamide; Sigma; referred to as GnRH), in the presence or absence of
ruthenium red (10 µM), cells were harvested and
luciferase activity assays were performed as described before (26). The
values are expressed as relative light units.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). RyR2 and RyR3 were also expressed in T3–1 cells
(data not shown), a pituitary cell line that is derived from the
gonadotrope lineage (29).
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). Cardiac microsomal protein was used as a positive control
because the G43 antibody has been shown to recognize the cardiac
isoform in addition to the brain isoform (11). A band corresponding to
the ryanodine receptor (mol mass, 
560 kDa) was detected by the
antibody in both the cardiac and pituitary microsomal fractions. A
lower band (200 kDa) was seen in both lanes and may represent a
proteolytic fragment of the receptor that is still recognized by the
antibody.
Effect of ruthenium red on GnRH-induced LH secretion
The role of the RyR in GnRH-induced LH release was analyzed using
a perifusion system. Primary pituitary cells were treated with 10 min
pulses of GnRH every 20 min, either in the presence or absence of
ruthenium red (10 µM), an antagonist of the ryanodine
receptor (9) (Fig. 2A). Media fractions were collected
every minute throughout the experiment, to enable high resolution
monitoring of LH secretion. In the initial phase of the experiment,
cells received GnRH pulses alone. In the second phase, cells were
treated with ruthenium red in addition to receiving GnRH pulses. In the
final phase of the experiment, the antagonist was withdrawn to assess
the ability of the cells to recover from antagonist treatment. GnRH
treatment resulted in a biphasic pattern of LH release, consisting of a
rapid spike phase followed by a sustained plateau phase. Treatment with
ruthenium red resulted in a 40% decrease in the spike phase of
GnRH-induced LH release. This effect of ruthenium red was reversed upon
removal of the antagonist (the washout phase) (Fig. 2B). The plateau
phase of LH release was decreased 35% in presence of ruthenium red,
but this effect was not reversed after removal of the antagonist (Fig. 2B). The basal level of LH release was unchanged throughout the course
of the experiment.
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-LUC activity-promoter was next used to determine the
role of the RyR in GnRH-induced transcriptional activation (26).
Primary pituitary cells were transfected with the -846-LUC reporter
gene and were treated with GnRH (10 nM) either in the
presence or absence of ruthenium red (10 µM). Ruthenium
red caused an 85% decrease in GnRH responsiveness (Fig. 3A). The activity of a control viral promoter, RSV-LUC,
was not decreased by treatment with ruthenium red, indicating that the
antagonist did not cause a nonspecific reduction in reporter gene
expression (Fig. 3B).
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, A and B). Females
expressed 30% lower levels of RyR3 in the pituitary than males (Fig. 4C).
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). Also, the levels on Est 0900 h were 60%
higher than those on the morning of proestrus (Pro 0900 h). A
similar pattern of pituitary RyR2 mRNA expression was observed across
the estrous cycle (data not shown).
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). Also,
RyR3 mRNA expression was 1.8-fold higher in OVX E-primed animals that
received pentobarbital relative to those that were given a control
saline injection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We hypothesized that calcium released through the IP3
receptor might trigger the activation of the RyR, which has been shown
to be responsive to calcium-induced calcium release mechanisms (16).
Ruthenium red, a well-known inhibitor of the RyR, was used in the
current experiments to block calcium release that is mediated by the
RyR. Ruthenium red has been shown to bind to the RyR at sites that
overlap domains that are also involved in calcium and calmodulin
binding (46). Although it has been suggested that ruthenium red may
have poor membrane permeability (47), it has been shown to displace
more than half of 3H-ryanodine binding in competition
experiments in intact cells, indicating that there is at least partial
uptake of ruthenium red into the cells (48). Treatment with ruthenium
red caused a reversible decrease in the spike phase and an irreversible
decrease in the plateau phase of GnRH-induced LH secretion (Fig. 2),
without measurably altering basal secretion levels. Inhibition of
calcium release via the RyR likely accounts for the reduction in
secretion during the spike phase. However, these observations need to
be qualified by the possibility that ruthenium red might have other
cellular targets in addition to the RyR. For this reason, it will be of
interest to further explore the effects of other agents that act upon
the ryanodine receptor. For example, caffeine stimulates the ryanodine
receptor, and we have found that caffeine enhances GnRH-mediated LH
secretion (data not shown). And, ryanodine itself functions initially
as an agonist but acts as an antagonist at high doses and with
prolonged exposure (49).
It is interesting to note that the reduction in the plateau height of LH secretion was not reversed upon removal of ruthenium red. This result indicates that calcium release during the spike and plateau phases are differentially regulated, or that there may be more than one site of action for ruthenium red. Recently, a functional coupling between the ryanodine receptor and L-type calcium channels has been demonstrated in neurons (50). Thus, it is possible that ruthenium red may exert additional effects on calcium signaling indirectly by inhibiting the ryanodine receptor. It is unlikely that ruthenium red exerted a toxic effect at the dose administered, since LH secretion was reversible upon removal of the antagonist.
An independent endpoint of GnRH action is the activation of
gonadotropin gene expression. It has been proposed that in addition to
the PKC (37, 41) and mitogen-activated protein kinase pathways (51, 52), certain calcium-dependent kinases such as calcium-calmodulin
kinase (CaM kinase) may also be activated by GnRH (52). The
transcriptional effects of GnRH likely reflect the integration of
signals transmitted through these different pathways (2, 40). We used
the glycoprotein hormone -subunit promoter as a reporter gene for
GnRH-induced transcriptional responses (26, 28, 53). Data from our
transfection experiments indicate that treatment with ruthenium red
leads to a marked reduction in the stimulation of the -subunit gene
promoter by GnRH. This effect could result from the inhibition of a
calcium-dependent isoform of PKC or a calcium-calmodulin kinase, that
may be essential for transcriptional induction (40, 42, 51, 52). As
noted above, it remains possible that ruthenium red has effects on
proteins other than the ryanodine receptor. The loss of -LUC
activity was not due to cell toxicity as the activity of a control
viral promoter, RSV-LUC, was not decreased after antagonist treatment.
Thus, the ryanodine receptor may play a role in GnRH-mediated
transcription as well as LH secretion.
The RT-PCR studies show that the RyR2 and RyR3 mRNAs are expressed at
levels that are comparable with those seen in control tissues (heart,
muscle) (Fig. 1). The pituitary cell types that express the RyR remain
to be established. The fact that both types of RyRs are expressed in
T3–1 cells is consistent with expression in gonadotrope cells (data
not shown), but double-labeled immunohistological analyses will be
required to definitively establish cell type expression.
We considered the possibility that RyR expression might vary during
estrous cycle (Fig. 5). RyR3 mRNA levels declined between diestrus
0900 h and proestrus 0900 h, then increased gradually during
the day of proestrus before reaching a peak at 0900 h on estrus.
These changes in expression appeared to follow the changes in
progesterone levels that occur during the estrous cycle, suggesting a
possible role for progesterone in the regulation of RyR gene
expression. Whether these cyclic changes in RyR mRNA are reflected at
the protein level remains to be determined.
To further examine the roles of ovarian steroids in regulating RyR3 mRNA expression, a model of steroid replacement in ovariectomized rats was used (21). In this model, it is possible to study the effects of individual ovarian steroids in the absence of endogenous hormone production from the ovaries. The potential effects of GnRH on RyR3 mRNA expression were examined by using a pentobarbital block of hypothalamic neurosecretion. Estrogen priming alone suppressed RyR3 mRNA levels when compared with OVX rats, whereas additional treatment with progesterone caused a 2-fold increase over levels seen in OVX rats that were not given either estrogen or progesterone. These results are consistent with the idea that the preovulatory increase in progesterone mediates the rise in RyR3 mRNA expression seen on proestrus evening. A regulatory effect of progesterone on the pattern of cytosolic calcium signaling has been reported previously (54). A short-term progesterone treatment (3 h) increased cytosolic calcium signals in estrogen-treated gonadotropes from OVX animals, whereas prolonged exposure (48 h) decreased [Ca2+]i responses in these cells. These changes in cytosolic calcium levels paralleled the effects of estrogen and progesterone on GnRH-induced secretory responses (36, 55). The regulation of RyR mRNA levels by estrogen and progesterone observed in the current study provides one mechanism by which ovarian steroids may modulate the calcium signaling pathway in gonadotropes. Interestingly, pentobarbital treatment of OVX estrogen-primed rats caused an increase in RyR mRNA levels relative to those seen in OVX or OVX estrogen-primed rats. This effect suggests that, in addition to the suppression by estrogen, a surge in GnRH levels may also down-regulate RyR3 mRNA expression. The significance of the alterations in RyR3 mRNA levels across the estrous cycle requires further investigation. In addition, these studies of mRNA expression may not necessarily be paralleled by changes in RyR protein. Unfortunately, the large size of the RyR protein impairs transfer in Western blots, making it difficult to quantitate changes in protein levels. It will be of interest to determine whether blocking calcium release via the RyR in vivo will alter the nature of the gonadotropin surges.
In summary, our results demonstrate that the ryanodine receptor is expressed and hormonally regulated in the pituitary. In addition, the experiments employing ruthenium red suggest that it may play a functional role in mediating GnRH action. The mechanism by which RyR activity is regulated in gonadotropes remains to be addressed. For instance, the channel adaptation properties of the RyR have been shown to be regulated by PKA-phosphorylation in cardiac myocytes (17). In this system, phosphorylation by PKA is thought to increase the sensitivity of the RyR to a stimulus and to enhance RyR responsiveness to subsequent triggers. Whether such a phosphorylation mechanism may play a role in the activation-inactivation kinetics of the ryanodine and the IP3 receptors in gonadotropes is an intriguing possibility. These results also raise the possibility of the involvement of the RyR in secretory events stimulated by other agonists, which transduce their actions via G protein-coupled receptor systems.
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Acknowledgments |
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Received November 15, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit secretion mirrors luteinizing hormone and
follicle-stimulating hormone in perifused rat pituitary cells.
Endocrinology 127:2364–2371[Abstract]
-subunit
promoter by gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
-subunit promoter. Mol Endocrinol 6:1767–1773[Abstract]
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, P < 0.05 vs.
levels at Pro 0900 h. Serum progesterone and estradiol levels are
shown for these animals, as previously reported (20).