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T3–1 Cell Line1
Department of Obstetrics and Gynaecology, Christchurch School of Medicine (J.J.E.), Christchurch, New Zealand; and Department of Medicine, University of Bristol (W.F.-O., C.A.McA.), Bristol, BS2 8HW, United Kingdom
Address all correspondence and requests for reprints to: J. J. Evans, University Department of Obstetrics and Gynaecology, Christchurch Women’s Hospital, Private Bag 4711, Christchurch, New Zealand. E-mail: jevans{at}chmeds.ac.nz
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Abstract |
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T3–1 cells, a gonadotrope-derived cell line.
Oxytocin, vasopressin, and vasotocin each stimulated accumulation of
[3H]inositol phosphates in cells prelabeled with
[3H]inositol, indicating activation of PIC. The rank
order of potency (oxytocin > vasotocin > vasopressin) and
sensitivity to inhibition by oxytocin and vasopressin receptor
antagonists, revealed the effect to be mediated by oxytocin-selective
receptors. Like other PIC activators, these nonapeptides caused
biphasic (spike-plateau) increases in the cytosolic Ca2+.
The spike response to oxytocin and GnRH were both retained in
Ca2+-free medium, reflecting mobilization of intracellular
Ca2+, and were comparably reduced by thapsigargin, implying
mobilization of Ca2+ from a shared thapsigargin-sensitive
intracellular pool. Brief stimulation with oxytocin, vasopressin, or
vasotocin prevented subsequent Ca2+ responses to oxytocin,
but not to GnRH, suggesting that the oxytocin receptor undergoes rapid
homologous desensitization and reinforcing the interpretation that the
nonapeptides act via the same receptor type. Oxytocin did not increase
Ca2+ in cells stimulated with GnRH, whereas GnRH caused a
spike Ca2+ increase even in the presence of oxytocin,
implying that different mechanisms of desensitization (Ca2+
pool depletion and receptor uncoupling) are operating for two distinct
PIC-coupled receptors in these cells. The demonstration that oxytocin
acts directly via PIC-linked, oxytocin-selective receptors to increase
cytosolic Ca2+ in a gonadotrope-derived cell line is
consistent with the possibility that oxytocin has a comparable effect
on nonimmortalized gonadotropes. |
Introduction |
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There is abundant evidence that a number of other local or hormonal regulators, in addition to GnRH, apparently participate in the control of LH during the ovulatory cycle (6, 7). These include oxytocin. Administration of an oxytocin antagonist to rats at proestrus inhibits the development of the preovulatory LH surge (8, 9), and oxytocin causes a dose-dependent stimulation of LH secretion from dispersed anterior pituitary cells of female rats in vitro (10). These observations are consistent with oxytocin playing a key role in the generation of the preovulatory LH surge, apparently by acting directly on pituitary cells.
Pharmacological studies have demonstrated that the receptors mediating oxytocin-stimulated LH secretion from rat pituitary cell cultures are oxytocin selective and distinct from GnRH receptors (10, 11), but the identity and location of these receptors remains controversial. Binding studies have revealed the presence of oxytocin receptors in the anterior pituitary (12), but these appeared to be expressed on lactotropes (13) (rather than on gonadotropes), consistent with reports of oxytocin-mediated stimulation of PRL secretion (14). The failure to detect oxytocin receptors on gonadotropes is somewhat surprising given the fact that oxytocin stimulates gonadotropin secretion from pituitary cell cultures and also increases the cytosolic Ca2+ concentration and activates Ca2+-sensitive K+ channels in individual gonadotropes (15).
In this study, we examined the effects of oxytocin on T3–1 cells, a
murine gonadotrope-derived cell line, reasoning that any
oxytocin-induced effect would both support the argument that
gonadotropes are direct targets for oxytocin action and provide a model
system for characterization of the receptors and effector systems
mediating the effects of neurohypophyseal hormones on gonadotropin
secretion.
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Materials and Methods |
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Cell culture
T3–1 cells were cultured in serum-supplemented DMEM as
described previously (16, 17). For the experiments, they were harvested
by trypsinization and then incubated for 1–3 days in 12- or 24-well
culture plates (2 or 1 ml medium/well), which for Ca2+
imaging experiments contained untreated round glass coverslips.
Dynamic video imaging of cytosolic Ca2+
Dynamic video imaging of fura 2-loaded T3–1 cells was
performed to measure cytosolic Ca2+ as described previously
(18, 19). Briefly, the cells were washed in a physiological salt
solution (PSS: 127 mM NaCl, 1.8 mM
CaCl2, 5 mM KCl, 2 mM
MgCl2, 0.5 nM NaH2PO4,
5 mM NaHCO3, 10 mM glucose, 0.1%
BSA, and 10 mM HEPES, pH 7.4) and then loaded by incubation
for 30 min in PSS containing 2 µM fura 2/AM. The cells
were then washed in PSS to remove excess fura 2/AM, and the cover slips
were loaded into a stainless steel holder that was fitted into a
heating chamber at 37 C. Image capture was performed within 10–25 min
of loading in approximately 500 µl PSS or in a modified PSS
containing 100 µM EGTA and no CaC12
(Ca2+-free medium) using MagiCal hardware, Tardis software
and a Nikon Diaphot microscope (Kingston-upon-Thames, Surrey, UK).
Details of the cell stimulations are given in the figures and figure
legends. The cells were excited alternately at 340 and 380 nm, and
emitted light was collected at 510 nm, averaging the data from 8 or 16
video frames and subtracting background values before ratioing. The
ratio of florescence at 340 and 380 nm was calculated on a
pixel-by-pixel basis and used to determine ionized Ca2+
concentration assuming a dissociation constant of 225 nM
for fura-2 and Ca2+ at 37 C (18).
Determination of total inositol phosphate (IP) accumulation
Total [3H]IP accumulation was quantified in
[3H]inositol-labeled T3–1 cells as described
previously (17). Briefly, the cells were cultured in 24-well culture
plates and then washed and incubated for 20–24 h in 0.5 ml medium 199
with 10 mM HEPES (pH 7.4), 0.3% BSA, 2.0 µCi/ml
myo-[2-3H]inositol, and antibiotics (penicillin and
streptomycin). The cells were then washed extensively in PSS (4 times,
over a period of 20 min) and stimulated for 15 min in PSS supplemented
with 10 mM LiCl, and the peptides indicated in the figures
and figure legends. Incubations were terminated by removal of the media
and by adding 1 ml water at 95 C. After freezing and thawing, the
[3H]IPs in the cell lysates were separated from free
[3H]inositol by anion exchange chromatography on Dowex-1
columns (Sigma Chemical Co., Poole, Dorset, UK) (formate form). The
[3H]IPs were eluted with 1 M ammonium formate
in 0.1 M formic acid, and the amount of 3H
eluted in this fraction was determined by liquid scintillation
spectroscopy.
Statistical analysis and data presentation
The figures show the mean ± SEM of data pooled from
the specific number of independent experiments (raw data or data
normalized as described in the figure legends). Statistical analysis
was performed by Student’s t test with P <
0.5 as the limit of statistical significance. For Ca2+
imaging experiments, software-based image analysis was used to quantify
the ionized Ca2+, the whole fields of view that typically
contained 10–50 cells, over the time course of each experiment. The
figures either show data for individual cells or show the mean (±
SEM) of data pooled from the indicated number of fields of
view such that a figure showing data from three independent experiments
(n = 3) actually shows the mean response of 30–150 cells. Where
spike and plateau Ca2+ values are reported, these were
defined arbitrarily as the maximum response within 10 sec of
stimulation and the response after 1 min, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells
labeled with [3H]inositol and then stimulated in the
presence of 10 mM LiCl (Fig. 1
). These
effects were, however, relatively modest compared with the 15-fold
increase caused by GnRH, with maximal concentrations approximately
doubling basal levels of [3H]IPtotal
accumulation. Although there was a tendency for vasopressin to have
greater efficacy than oxytocin (which might reflect simultaneous
activation by vasopressin of both oxytocin and vasopressin-selective
receptors), it was not statistically significant (P >
0.1). However oxytocin was the most potent of these peptides
(EC50 
0.1 nM), indicating that the effects
observed are mediated predominantly or solely by oxytocin-selective
receptors.
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, the stimulatory effects of oxytocin and
vasopressin (both at 10-7 M) were both
inhibited by 10-6 M
d(CH2)5[Tyr(Me)2,
Thr4,
Tyr-NH29]ornithine8 vasotocin (a
selective oxytocin receptor antagonist), but neither was measurably
altered by 10-6 M
Phae-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (a selective
V1 vasopressin receptor antagonist), again indicating mediation by
oxytocin receptors.
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, GnRH and oxytocin (10-7 M) both caused rapid
increases in [Ca2+]i in T3–1 cells. The
spike phases of the responses were of comparable amplitude, but whereas
the response to GnRH was clearly biphasic (spike-plateau), the response
to oxytocin reduced more rapidly with a much less pronounced plateau
phase. The spike phase of the response to GnRH reflects mobilization of
[Ca2+]i and was therefore seen in
Ca2+-free medium, whereas the plateau phase reflects
Ca2+ entry and is absolutely dependent on the presence of
extracellular Ca2+ (Fig. 4) (17). The same
is apparently true for oxytocin, which caused a clear spike increase in
[Ca2+]i in the T3–1 cells in
Ca2+-free medium, and there was an increase in
[Ca2+]i in a plateau-type response when the
cells returned to normal Ca2+-containing medium (Fig. 4).
Again, in Ca2+-free medium, the spike phases of the
responses to GnRH and oxytocin were of comparable magnitude, whereas
the plateau response to oxytocin was much lower than that to GnRH after
return to normal medium.
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T3–1
cells (20, 21, 22) and to inhibit GnRH-stimulated gonadotropin secretion
(2). To determine whether oxytocin mobilizes
[Ca2+]i from the same thapsigargin-sensitive
pool, T3–1 cells were treated with 0 or 2 µM
thapsigargin in Ca2+-free medium and then stimulated with
GnRH or oxytocin (both at 10-7 M). As
expected, GnRH, oxytocin, and thapsigargin all caused transient
increases in [Ca2+]i in Ca2+-free
medium, and pretreatment with thapsigargin clearly attenuated the spike
response to GnRH (Fig. 5A). The spike response to
oxytocin was similarly reduced by pretreatment with thapsigargin,
indicating that oxytocin, like GnRH, increases
[Ca2+]i in Ca2+-free medium by
mobilization of Ca2+ from a thapsigargin-sensitive
intracellular store.
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, each of the
nonapeptides increased [Ca2+]i in T3–1
cells, with the effects of oxytocin and vasotocin being more pronounced
than that of vasopressin. In each case, the response was rapid in onset
and consisted of a pronounced spike-type increase with little plateau.
After extensive washing, oxytocin failed to elicit a
[Ca2+]i increase in cells previously
stimulated with any of the nonapeptides. However the responsiveness to
GnRH was retained.
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). The
T3–1 cells had no response to oxytocin if there had been a
preceding exposure to GnRH. In contrast, the response to GnRH was
unaffected by whether cells had been previously stimulated by oxytocin
(Fig. 7B). These figures show mean responses obtained in three imaging
experiments (e.g. average data derived from 50 cells),
and we were therefore concerned that the spike response to GnRH might
only have occurred in a subset of cells not responding to the prior
stimulation with oxytocin. However analysis of data from individual
experiments revealed that this was not the case (Fig. 8). It was found that virtually all cells that responded
to oxytocin were also stimulated by GnRH.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells. The demonstration of such effects in this
gonadotrope-derived cell line clearly supports the notion that
gonadotropes are direct targets for neurohypophyseal hormones and
provides a model system for further characterization of the receptors
involved.
The fact that all three neurohypophyseal peptides act on T3–1
cells raises the question of whether they cross-react at a single
receptor or act on distinct receptor subtypes in these cells. Assuming
mediation by a single receptor type, the rank order of potency for
stimulation of [3H]IPtotal accumulation
(oxytocin > vasotocin >> vasopressin) and the inhibitory
effect of the oxytocin receptor antagonist,
d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]ornithine8
vasotocin (Fig. 2
) clearly suggest this to be an oxytocin-selective
receptor. Moreover, the vasopressin response was inhibited by an
oxytocin antagonist (Fig. 2) but not by a vasopressin antagonist,
implying that the vasopressin effect in these cells is also mediated by
oxytocin receptors. The effects of these antagonists, with previously
established activity, at an order of magnitude of excess, reinforce the
argument that T3–1 cells express a single major neurohypophyseal
receptor type. The existence of oxytocin receptor subtypes has been
postulated after studies of uterus and hippocampus (23, 24, 25, 26). However
only a single class of oxytocin receptor has been detected in the
anterior pituitary gland (12).
In Ca2+ imaging experiments, oxytocin, vasotocin, and
vasopressin (each at 10-7 M) each caused
comparable rapid and transient increases in
[Ca2+]i. After washing, subsequent
stimulation with oxytocin failed to increase
[Ca2+]i, suggesting the occurrence of both
homologous and heterologous desensitization (Fig. 6). The occurrence of
homologous desensitization (but not heterologous desensitization) of
[Ca2+]i responses to oxytocin and vasopressin
in renal medulla cells was taken as evidence for action via distinct
receptors (27). The same logic and the demonstration of heterologous
desensitization in this study implies that T3–1 cells express a
single class of Ca2+-mobilizing receptor that is stimulated
by oxytocin, vasotocin, and vasopressin. The alternative explanation,
that desensitization is not receptor-specific because it involves a
biochemical lesion occurring distal to the receptor
(e.g. Ins(1, 4, 5)P3 receptor loss)
is unlikely because cells desensitized to oxytocin remained responsive
to GnRH (Fig. 6).
The Ca2+ increase caused by 10-7 M
oxytocin was rapid in onset, with a spike-type increase achieved within
10 sec of stimulation followed by a gradual reduction to near basal
levels (Fig. 3). Stimulation with oxytocin in Ca2+-free and
then in Ca2+-containing medium confirmed two distinct
phases of the Ca2+ response (Fig. 4). The rapid and
transient increase following oxytocin stimulation is seen in
Ca2+-free medium and must therefore reflect mobilization of
[Ca2+]i pools. Replacement of medium with
calcium-containing solution induced a sustained increase of
[Ca2+]i. The dependence of the second phase
on extracellular Ca2+ presumably reflects entry of
Ca2+ across the plasma membrane. Because oxytocin receptors
are known to activate PIC in several systems and increase
[3H]IPtotal accumulation in T3–1 cells
loaded with [3H]inositol (Figs. 1 and 2), it is likely
that the spike phase of the Ca2+ response to
neurohypophyseal peptides in these cells reflects PIC activation,
Ins(1, 4, 5)P3 generation, and consequent mobilization of
Ca2+ from Ins(1, 4, 5)P3-sensitive
intracellular stores. This interpretation is supported by studies with
thapsigargin, a specific inhibitor of the endoplasmic reticulum
Ca2+-sequestering ATPase, which depletes the Ins(1, 4, 5)P3-sensitive Ca2+ pool in many systems (28).
The fact that thapsigargin caused comparable reductions of the spike
responses to oxytocin and GnRH (Fig. 5) implies that oxytocin, like
GnRH (29) mobilizes Ca2+ from a thapsigargin- and Ins(1, 4, 5)P3-sensitive [Ca2+]i pool.
Although 10-7 M GnRH and 10-7
M oxytocin caused comparable spike increases in
[Ca2+]i, the plateau response to oxytocin was
clearly less pronounced than that to GnRH (Figs. 3 and 4). Because the
plateau response to GnRH reflects activation of Ca2+ entry
via voltage-operated Ca2+ channels, it seems that oxytocin
must activate such entry inefficiently, if at all, in T3–1 cells.
The lower plateau could reflect differential coupling of oxytocin- and
GnRH-receptors to distinct effector systems. Alternatively there might
be different rates of receptor desensitization. Agonist occupancy of
many G protein-coupled receptors causes desensitization within several
seconds to minutes, involving activation of G-protein receptor kinases,
phosphorylation of the receptor, and consequent uncoupling from
adenylyl cyclase or PIC (30, 31). This G-protein receptor
kinase-mediated phosphorylation very often occurs on specific amino
acids of the C-terminal tail, but the GnRH receptor lacks any such
tail, and the recent demonstration of sustained
[3H]IPtotal accumulation for 10–15 min in
GnRH-stimulated cells (32) suggests that such mechanisms do not cause
rapid desensitization of GnRH action. In the absence of rapid receptor
desensitization, the transient spike Ca2+ response to GnRH
in these cells most likely reflects depletion of the GnRH-mobilizable
[Ca2+]i pool, as verified by the lack of
effect of Ca2+ ionophores on cytosolic Ca2+
when administered after GnRH in either Ca2+-free or
Ca2+-containing medium (2, 29). Such depletion presumably
underlies the lack of spike Ca2+ response to oxytocin in
cells stimulated with GnRH (Fig. 7), and is in direct contrast to the
retention of the spike response to GnRH in cells stimulated with
oxytocin (Fig. 7). One possible explanation for retention of the spike
response to GnRH in cells treated with oxytocin is that the spike
response to GnRH occurs in a subset of T3–1 cells that are
unresponsive to oxytocin. However analysis of single-cell data (Fig. 8)
argues against this interpretation; numerous individual cells were
identified showing spike-type responses to both oxytocin and then GnRH.
The clear implication is that the initial response to oxytocin is
transient, not because of Ca2+ pool depletion, but rather
because the oxytocin receptor undergoes rapid desensitization
(uncoupling from PIC) so that [Ca2+]i returns
to basal levels before depletion of the
[Ca2+]i pool. If so, then distinct mechanisms
are operating in this cell type for desensitization to distinct types
of PIC-coupled G protein-coupled receptor.
In spite of oxytocin and GnRH inducing similar spike increases in
[Ca2+]i, the maximum effect of GnRH on
[3H]IPtotal accumulation was 10- to 20-fold
greater than the maximal effect of the neurohypophyseal peptides (Fig. 1). This may reflect positive coupling within the system. The
concentration response curve for GnRH effects on
[Ca2+]i lies to the left of that for
elevation of IP3 in these cells (33) such that
concentrations of GnRH, which cause submaximal Ins(1, 4, 5)P3 increase, can maximally elevate
[Ca2+]i. The same apparently holds true for
oxytocin, which can elicit a maximal increase in
[Ca2+]i at a concentration causing a
relatively modest increase in [3H]IPtotal
accumulation. Oxytocin and GnRH have also been shown to cause
comparable increases in [Ca2+]i of
nonimmortalized rat pituitary cells (34), yet, compared with GnRH,
oxytocin is a relatively poor stimulus for gonadotropin secretion. This
may reflect rapid desensitization of oxytocin receptors, but an
alternative explanation is that oxytocin is an inefficient acute
secretagogue because it rapidly activates only one branch of the
bifurcating PIC-mediated signal transduction cascade. It has been
suggested that the elevation of [Ca2+]i
caused by GnRH in gonadotropes is alone insufficient to stimulate
exocytosis. In permeabilized gonadotropes, stimulation of exocytosis by
physiological concentrations of Ca2+ requires the
concomitant increase in sensitivity to Ca2+ caused by
activation of protein kinase C (35), and protein kinase C activators
can enhance sensitivity of intact gonadotropes to GnRH and to
Ca2+ ionophores (36). The relatively low efficacy of
oxytocin in stimulating [3H]IPtotal
accumulation suggests that oxytocin may activate protein kinase C less
effectively than GnRH such that even a maximal oxytocin-induced
increase in [Ca2+]i is insufficient to cause
maximal gonadotropin secretion.
In summary, we have shown that oxytocin, vasopressin, and vasotocin all
stimulate [3H]IPtotal accumulation and
elevate [Ca2+]i in a gonadotrope-derived cell
line, supporting the possibility that nonimmortalized gonadotropes are
direct targets for the neurohypophyseal peptides. In this regard it is
worthwhile noting that T3–1 cells are known to be directly
stimulated by GnRH, endothelin-1, pituitary adenylate
cyclase-activating polypeptide, and ATP (and now oxytocin), results
that parallel recent electrophysiological studies that have
unambiguously demonstrated direct effects of a number of stimuli on
isolated gonadotropes or membranes (15). These observations, in which
responsive consistency between nonimmortalized gonadotropes and
T3–1 cells is demonstrated, taken together with the present data
and the previously reported effects on gonadotropin secretion (10) and
Ca2+ signaling in nonimmortalized gonadotropes, suggest
that gonadotropes are likely to express oxytocin receptors. The spike
phase of the Ca2+ response to oxytocin is largely retained
in Ca2+-free medium and is reduced by thapsigargin. It
therefore most likely reflects activation of PIC and mobilization of
[Ca2+]i by Ins(1, 4, 5)P3.
Cross-desensitization between the neurohypophyseal peptides indicates
action via a single receptor type, and the rank order of potency
(oxytocin > vasotocin > vasopressin) along with sensitivity
to an oxytocin receptor antagonist suggest mediation by an
oxytocin-selective receptor. Cross-desensitization studies using GnRH
and oxytocin imply that distinct mechanisms underlie desensitization of
the spike [Ca2+]i responses to oxytocin and
GnRH in these cells.
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Acknowledgments |
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T3–1 cells. |
Footnotes |
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Received November 12, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells. Mol Cell
Endocrinol 87:95–103[CrossRef][Medline]
T3–1,
gonadotrope-derived cell line. Mol Cell Neurosci 3:124–132[CrossRef]
T3–1 pituitary gonadotroph cells. J Endocrinol 136:51–58T3–1 cells due to uncoupling of inositol 1,4,5
trisphosphate generation and Ca2+ mobilization. J Biol
Chem 271:23711–23717T3-1 cells: does store-dependent
Ca2+ influx mediate gonadotrophin-releasing hormone action?
J Endocrinol 149:155–169
T3–1 cells line.
Endocrinology 136:4864–4871
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