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Division of Endocrinology, Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Dr. D. J. Haisenleder, Division of Endocrinology, Department of Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: djh2q{at}virginia.edu
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Abstract |
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, LHß, and FSHß messenger RNAs (mRNAs), and
only LHß mRNA was increased by continuous Ca2+. As
gonadotropin subunit and GnRH receptor (GnRH-R) mRNAs are
differentially regulated by alterations in GnRH pulse interval, we
aimed to determine whether changes in the frequency of Ca2+
signals play a role in this effect. Cultured adult female rat pituitary
cells in perifusion were given pulses of the Ca2+ channel
activator BayK 8644 (10 µM; with 10 mM KCl in
the injectate), at intervals of 16, 60, or 180 min for 24 h
(vehicle pulses or 100 pM GnRH to controls). Pulsatile
Ca2+ influx stimulated a rise in all mRNAs examined
(P < 0.05 vs. vehicle controls);
however, optimal pulse intervals differed. and LHß mRNAs were
maximally stimulated by 16- or 60-min pulses (57% and 74% increases,
respectively), with 180-min pulses being less effective. In contrast,
FSHß and GnRH-R mRNAs were selectively stimulated by 180-min pulses
(51% and 41% increases, respectively). Pulsatile GnRH produced
similar increases in GnRH-R and subunit mRNAs (53–78%
vs. controls). These results reveal that alterations in
the frequency of Ca2+ signals can regulate gonadotrope gene
expression in a differential manner, producing effects similar to
previous findings for GnRH. Thus, intermittent increases in
intracellular Ca2+ may be an important step in the
transmission of GnRH pulse signals from the plasma membrane to the
gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, LHß, and follistatin messenger RNAs
(mRNAs) are maximally stimulated by faster (15- to 30-min) pulse
intervals (1, 2), whereas FSHß and GnRH-R are increased by slower
(120- to 240-min) pulse intervals (1, 3). Other results have shown that
the effects of GnRH pulse frequency on gonadotropin subunit mRNA
expression involve increased gene transcription (4). However, the
site(s) in the intracellular signal transduction pathway that plays the
key role in frequency modulation remains to be determined. Calcium has been implicated as a major component in the mechanism of action of GnRH. Activation of the GnRH-R stimulates a transient increase in intracellular Ca2+ that results from inositol 1,4,5-trisphosphate-induced Ca2+ release from intracellular storage pools and Ca2+ influx from voltage sensitive (L-type) channels (5). Recently, we have shown that alterations in intracellular Ca2+ play a critical role in GnRH regulation of gonadotropin subunit genes (6). Specifically, administration of the Ca2+ channel blocker, verapamil, inhibits the GnRH-induced rise in gonadotropin subunit mRNAs. Also, pulsatile increases in intracellular Ca2+ via pulses of the L-type channel activator BayK 8644 are more effective in stimulating gonadotropin subunit mRNA expression than a continuous elevation in Ca2+ (via treatment with the Ca2+ ionophore A23187).
This study was conducted to investigate whether a critical site in GnRH frequency modulation is at the level of the plasma membrane receptor or at a downstream intracellular location(s). The strategy was to bypass the receptor and determine whether intermittent increases in intracellular Ca2+ (at various intervals) could reproduce the stimulatory effect of GnRH pulse patterns on gonadotropin subunit and GnRH-R mRNA expression. The measurement of GnRH-R mRNA was included because this receptor plays a critical role in gonadotrope physiology, and the expression of this gene has been shown to be to regulated by pulsatile GnRH in female rats in vivo in a frequency-dependent manner [i.e. maximal increases seen with 240-min pulses, whereas faster (8-min) pulses are ineffective (3)].
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental protocol
Chambers received pulses of the Ca2+ channel
activator, BayK 8644 plus potassium chloride [BK+KCl; peak chamber
concentrations, 10 µM (BK) and 10 mM (KCl)]
at intervals of 16, 60, or 180 min for 24 h. This treatment
paradigm was selected due to previous data showing that BK is more
effective in stimulating pituitary secretion or mRNA responses in the
presence of a threshold depolarization concentration of K in the medium
(6, 9). Control groups received either vehicle pulses (0.2%
ethanol/medium) or pulses of GnRH (peak chamber concentration, 100
pM) every 60 min. This GnRH treatment protocol was
selected, as previous experiments in our laboratory had shown that it
will stimulate both gonadotropin subunit and GnRH-R mRNA expression.
The complete study was conducted over 2 separate experiments (12
chambers/experiment). All treatment groups were represented in each
experiment. Data from both experiments are combined and shown in the
figures and table [total n per treatment group = 5, with the
exception of GnRH (n = 4); Exp 1: controls, n = 3; GnRH,
n = 2; BK+KCl (16 min), n = 3; BK+KCl (60 min), n = 2;
BK+KCl (180 min), n = 2; Exp 2: controls, n = 2; GnRH, n
= 2; BK+KCl (16 min), n = 2; BK+KCl (60 min), n = 3; BK+KCl
(180 min), n = 3].
Measurements
To assess secretory responses over the experimental duration,
perifusate samples (10-min fractions) were collected from each chamber
after 3 and 21 h of treatment. LH and FSH were measured by RIA
using reagents provided by the NIDDK. The RIA standards were NIDDK RP-3
(for LH) and RP-2 (for FSH).
After completion of each experiment, the cells were recovered, total
RNA was extracted with phenol, and , LHß, FSHß, and GnRH-R mRNAs
were determined by dot blot hybridization, as previously described (3, 4). mRNA concentrations were expressed as femtomoles of complementary
DNA bound per 100 µg pituitary DNA and are presented as the percent
increase vs. vehicle-pulsed controls.
Statistical analysis
The data were analyzed by one-way ANOVA, with differences
between treatment groups determined by Duncan’s multiple range
test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the effects of
vehicle, GnRH, and BK+KCl pulses on gonadotrope mRNA concentrations.
Pulsatile Ca2+ signals stimulated a significant increase in
gonadotropin subunit and GnRH-R mRNAs; however, differences were
observed in the optimal pulse interval. and LHß mRNAs were
stimulated by 16- and 60-min pulses, with maximal increases seen after
the more rapid (16-min) pulses (57% increase for and 74% increase
for LHß; P < 0.05 vs. vehicle controls).
In contrast, significant increases in FSHß and GnRH-R mRNAs were only
seen after slower (180-min) Ca2+ pulses (FSHß, 51%
increase vs. controls; GnRH-R, 41% increase;
P < 0.05). Significant differences between BK+KCl
pulse interval groups were not seen for gonadotropin subunit or GnRH-R
mRNAs. GnRH pulses also stimulated a rise in each mRNA examined
(53–78% vs. vehicle controls; P < 0.05).
With the exception of GnRH-R, the magnitude of responses to pulsatile
Ca2+ influx were similar to that seen for GnRH.
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shows the LH secretory
pattern to pulsatile BK+KCl. As we and other investigators have shown
previously (6, 10), LH secretory activity in perifused pituitary cells
decreased over the 24-h experimental duration; however, responsiveness
to BK+KCl or GnRH was maintained. Pulse intervals of 60 or 180 min
produced a pulsatile secretory pattern that was present throughout the
perifusion time course. Of interest, the pattern of the LH secretory
response in chambers that received 60-min pulses of BK+KCl was similar
to that seen for 60-min pulses of GnRH. A clear pulsatile LH release
pattern was not seen in the 16-min interval group, which may reflect
the 10-min duration over which perifusate fractions were collected. As
presented in Table 1, BK+KCl pulses
stimulated significant increases in LH and FSH secretion
(P < 0.05 vs. vehicle controls).
Although secretory responses were maintained over the experimental
duration, LH and FSH release vs. that in vehicle controls
decreased after 21 h of shorter (16- and 60-min) interval pulses
(P < 0.05 vs. 3 h point in the same
treatment group). As suggested in Fig. 2, the magnitude of LH
release responses to Ca2+ pulses after 3 h of
treatment were significantly greater than that seen for GnRH
(P < 0.05). Also, LH secretory responses to BK+KCl or
GnRH were greater (5- to 10-fold increases vs. controls)
than those of FSH (2- to 3-fold increases).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and LHß mRNAs
were stimulated by faster (16- to 60-min) pulse intervals, and FSHß
and GnRH-R were increased by the slower (180-min) interval. Although we
are not certain that the magnitude and duration of intracellular
Ca2+ responses to pulses of BK+KCl are the same as those
seen in response to GnRH, this treatment does result in a pulsatile LH
secretory pattern similar in profile to that observed after GnRH pulse
stimuli. As GnRH pulses produce similar mRNA responses, the results are
probably of physiological significance and suggest that transient
increases in intracellular Ca2+ may play a critical role in
transmitting frequency modulatory information from the plasma membrane
to the nucleus. In a previous report, we found that 60-min pulses of
BK+KCl stimulated a significant increase in FSHß mRNA levels (6). In
the present study, FSHß mRNA levels were also increased (28%) after
60-min pulses, but statistical significance was not obtained. Thus,
similar to previous in vivo findings for responses to
pulsatile GnRH (1), more rapid pulse intervals may be effective to some
degree; however, FSHß mRNA is optimally stimulated by the slower
BK+KCl pulse interval.
A recent study by Kaiser et al. (11) used GH3
cells cotransfected with plasmids containing gonadotropin subunit
promoter/reporter gene constructs and various concentrations of GnRH-R
expression vector. The results showed that maximal ratios of and
LHß to FSHß promoter responses to GnRH were seen in cells
expressing higher levels of GnRH-R. The hypothesis developed from these
findings was that and LHß are maximally stimulated by faster
interval GnRH pulses via the previously described frequency-dependent
rise in GnRH-R numbers (12). According to this hypothesis, the increase
in cell surface receptors would enhance the GnRH signal transduction
pathway for and LHß, but inhibit a pathway specific to FSHß.
The present data do not support the view that differential regulation
of gonadotropin subunit genes is mediated solely by the number of
GnRH-R. We were able to reproduce frequency-induced alterations in
gonadotrope gene expression using a model in which the stimulus
bypassed the GnRH receptor. Although our data clearly show that
frequency modulation can occur by actions downstream from the plasma
membrane, it does not exclude a role at the receptor level. Indeed,
recent information relating to GnRH-induced effects on G protein
expression, cross-talk between G protein-regulated systems, and
rapid/acute desensitization to GnRH stimulation support a role for
frequency modulation at the receptor level (13).
The mechanism(s) involved in the Ca2+ frequency effect on
gonadotrope gene expression remains to be determined. However,
alterations in intracellular Ca2+ can potentially influence
various signal transduction pathways. Ca2+ plays a critical
role in the activation of calmodulin kinase pathways and has been shown
to affect cAMP signaling in the pituitary (5, 14). Ca2+ is
also an important regulator of the protein kinase C pathway and is
required for the activation of specific protein kinase C isoforms
(i.e. and ß II) that have been identified within
gonadotrope cells (15). Data in gonadotrope-derived T-3 cells
reveal that an increase in intracellular Ca2+ stimulates a
rise in and ß II protein kinas C mRNAs (16). Recent unpublished
studies (in collaboration with Dr. Margaret Shupnik, University of
Virginia) have shown that BK stimulates an increase in
mitogen-activated protein kinase activity in T-3 cells, an
intracellular pathway that may mediate the -subunit transcriptional
response to GnRH (17). Thus, Ca2+ can influence several
signal transduction pathways that have been shown to regulate
gonadotrope gene expression. This provides the potential for
Ca2+ to play a pivotal role in gonadotrope responses to
alterations in GnRH pulse patterns, perhaps via regulatory actions on
cross-talk between signal transduction pathways.
Previous in vivo studies from our laboratory have shown that administering pulsatile GnRH to ovariectomized, GnRH-deficient female rats does not stimulate a rise in LHß mRNA levels unless the animals are treated with T (8). Other data reveal that the optimal serum concentration of T (0.5 ng/ml) was similar to the level observed on proestrus (7, 8), which may suggest that T plays an important role in the rapid rise in LHß mRNA levels seen during the GnRH-induced LH surge (1). The results of recent in vitro studies have also shown that pulsatile GnRH does not increase LHß mRNA expression in female rat pituitary cells when T is not added to the culture medium (6, 18, 19). However, the present findings reveal that pretreating the cells with a female physiological dose of T (0.5 ng/ml) allows for a LHß mRNA response to GnRH, demonstrating that T is acting at the pituitary level. Although the mechanism(s) for this effect of T remain to be determined, it is interesting to note that T is not required for pulsatile BK to stimulate an increase in LHß mRNA (6). This could suggest that a critical site of T action is at the plasma membrane. In support of this, a recent study found that T modulates intracellular Ca2+ responses to GnRH in male rat gonadotrope cells (20). If the critical site for T action is at the plasma membrane, further investigations will be needed to determine how this effect is LHß specific.
Based on the present findings, we conclude that intermittent increases in intracellular Ca2+ may be an important step in the transmission of GnRH pulse signals from the plasma membrane to the gene. Also, as Ca2+ pulses can generally reproduce the effect of pulsatile GnRH on gonadotropin subunit and GnRH-R mRNA expression, it is likely that frequency modulation occurs at a site(s) downstream from the plasma membrane receptor.
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Acknowledgments |
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Footnotes |
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Received June 4, 1997.
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