Rapid Down-regulation of the Type I Inositol 1,4,5-Trisphosphate
Receptor and Desensitization of Gonadotropin-releasing
Hormone-mediated Ca2+ Responses in
T3-1
Gonadotropes*
Gary B.
Willars
§,
Jean E.
Royall
,
Stefan R.
Nahorski
,
Faraj
El-Gehani¶,
Helen
Everest¶, and
Craig A.
McArdle¶
From the
Department of Cell Physiology and
Pharmacology, University of Leicester, Medical Sciences Building,
P. O. Box 138, University Road, Leicester LE1 9HN,
United Kingdom and the ¶ University Neuroendocrine Unit,
Department of Medicine, University of Bristol, Marlborough Street,
Bristol BS2 8HW, United Kingdom
Received for publication, September 29, 2000, and in revised form, November 6, 2000
 |
ABSTRACT |
Despite no evidence for desensitization of
phospholipase C-coupled gonadotropin-releasing hormone (GnRH)
receptors, we previously reported marked suppression of GnRH-mediated
Ca2+ responses in
T3-1 cells by pre-exposure to
GnRH. This suppression could not be accounted for solely by reduced
inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) responses,
thereby implicating uncoupling of Ins(1,4,5)P3 production
and Ca2+ mobilization (McArdle, C. A., Willars,
G. B., Fowkes, R. C., Nahorski, S. R., Davidson, J. S., and Forrest-Owen, W. (1996) J. Biol. Chem. 271, 23711-23717). In the current study we demonstrate that GnRH causes a
homologous and heterologous desensitization of Ca2+
signaling in
T3-1 cells that is coincident with a rapid
(t1/2 < 20 min), marked, and
functionally relevant loss of type I Ins(1,4,5)P3 receptor
immunoreactivity and binding. Furthermore, using an
T3-1 cell line
expressing recombinant muscarinic M3 receptors we show that
the unique resistance of the GnRH receptor to rapid desensitization contributes to a fast, profound, and sustained loss of
Ins(1,4,5)P3 receptor immunoreactivity. These data
highlight a potential role for rapid Ins(1,4,5)P3 receptor
down-regulation in homologous and heterologous desensitization and in
particular suggest that this mechanism may contribute to the
suppression of the reproductive system that is exploited in the major
clinical applications of GnRH analogues.
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INTRODUCTION |
The decapeptide gonadotropin-releasing hormone
(GnRH)1 is released from the
hypothalamus of mammals in a pulsatile manner to regulate the
exocytotic release of luteinizing hormone and follicle-stimulating hormone from pituitary gonadotropes. These hormones are central to the
regulation of gonadal steroidogenesis and gamete maturation, and GnRH
therefore plays a vital role in the control of vertebrate reproduction.
GnRH acts on pituitary gonadotropes through a G-protein-coupled receptor that regulates phospholipase C (PLC) via G-proteins of the
G
q/11 family (1). GnRH-mediated activation of PLC
results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)
and diacylglycerol. These second messengers are able to mobilize
Ca2+ from intracellular stores and activate protein kinase
C, respectively (2), thereby propagating a signaling cascade that
accounts for the biological effects of GnRH.
In contrast to nearly all other known PLC-coupled G-protein-coupled
receptors (GPCRs), the GnRH receptor does not undergo rapid (seconds to
minutes) desensitization following exposure to agonist (3-8). Recent
evidence has suggested that this lack of acute regulation is related to
the lack of a C-terminal tail and the absence, therefore, of
appropriate regulatory phospho-acceptor sites (8, 9). From a functional
perspective, the resistance of the GnRH receptor to rapid
desensitization may serve to maintain cellular sensitivity and
responsiveness during events such as the pre-ovulatory gonadotropin
hormone surge and allow the frequency-encoded pattern of the
hypothalamic pulsatile GnRH release (10, 11) to be faithfully
maintained at the level of the pituitary gonadotropes.
Despite the lack of acute regulation of the GnRH receptor, sustained
exposure to GnRH is able to reduce GnRH-stimulated gonadotropin secretion, and this form of desensitization underlies the suppression of the reproductive system that is exploited in the major clinical applications of GnRH analogues (12). Given the importance of cytosolic
Ca2+ elevation in the mediation of GnRH-stimulated
gonadotropin secretion (1, 13-15), we have previously explored the
potential desensitization of this component of the GnRH
receptor-mediated signaling pathway in an immortalized mouse pituitary
cell line (
T3-1). Despite no evidence for rapid desensitization of
the GnRH receptor in these cells, pre-exposure to GnRH can cause a
marked suppression of subsequent GnRH-mediated elevations of
[Ca2+]i. Both the spike phase of the
response (which reflects Ins(1,4,5)P3-dependent
mobilization of intracellular Ca2+) and the sustained phase
of the response (which is dependent upon Ca2+ entry across
the plasma membrane through voltage-operated Ca2+ channels)
were attenuated by GnRH pretreatment (4, 5).
Desensitization of voltage-operated Ca2+ channels may
account for the desensitization of the plateau phase of the
GnRH-mediated response in
T3-1 cells (4), but the mechanism
underlying attenuated mobilization of Ca2+ from
intracellular stores is unclear. Although pre-exposure to GnRH reduces
both the number of plasma membrane GnRH receptors and the ability of
GnRH to generate Ins(1,4,5)P3, these effects are
insufficient to account for the reduced release of intracellular Ca2+ (5). Indeed, this desensitization is heterologous and
therefore most probably reflects post-receptor modification(s). Because desensitization of GnRH-stimulated Ca2+ mobilization from
intracellular stores cannot be attributed to attenuation of
Ins(1,4,5)P3 generation or depletion of hormone-mobilizable intracellular Ca2+ pools, it appears to reflect a reduction
in the efficiency with which Ins(1,4,5)P3 mobilizes
Ca2+ from intracellular stores (5). There is now
accumulating evidence that GPCR-mediated activation of PLC causes
down-regulation of Ins(1,4,5)P3 receptors (16-23), most
probably through proteolysis that is initiated as a consequence of
activation by Ins(1,4,5)P3 (20, 22, 23). However, this
down-regulation often requires several hours of agonist stimulation
(17, 23), whereas GnRH desensitizes Ca2+ responses in
T3-1 cells with pre-stimulation periods of 10-30 min (5). Thus, if
Ins(1,4,5)P3 receptor down-regulation underlies desensitization of GnRH-stimulated Ca2+ mobilization, it
would have to occur unusually rapidly in
T3-1 cells. The current
study was therefore undertaken to establish whether GnRH is able to
cause Ins(1,4,5)P3 receptor down-regulation and whether any
such effect underlies desensitization of Ca2+ mobilization
in these cells.
T3-1 cells stably transfected with recombinant
human M3 muscarinic receptors (7) were used in these
experiments to enable comparison of responses to PLC-activating GPCRs
that do (M3) and do not (GnRH) show rapid homologous
desensitization (7).
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EXPERIMENTAL PROCEDURES |
Materials and Cell Culture--
Reagents of analytical grade
were obtained from suppliers listed previously (5, 24-26), unless
stated, or alternatively from Sigma. Antibodies against PLC
isoforms and G
q/11 were from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA), with the exception of PLC
1, which was
from Upstate Biotechnology (Lake Placid, NY). The
T3-1
gonadotrope cell line was originally a gift from Dr. P. Mellon,
University of California, San Diego, CA, and in the current study we
used a cell line (
T3-1/M3) derived from this, which
also expresses the recombinant human muscarinic M3
receptor. Like the endogenously expressed GnRH receptor, this GPCR also couples to the activation of PLC in this cell line (7), and we have
demonstrated that muscarinic M3 receptors are subject to
rapid but partial desensitization, whereas the endogenously expressed
GnRH receptors show no evidence of such regulation (7). Cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin, 2 mM glutamine, and 10% (v/v) fetal calf serum. Cultures were maintained at 37 °C
in 5% CO2, humidified air and passaged weekly. For
experiments, cells were harvested with 10 mM HEPES, 154 mM NaCl, 0.54 mM EDTA (pH 7.4) and re-seeded
for use 1-2 days later. Cells were always maintained, and the
experimental manipulations were always performed, at 37 °C unless
stated otherwise.
Dynamic Video Imaging of Cytosolic Ca2+--
Video
imaging of fura-2-loaded cells was performed as described previously
(5). Briefly, cells grown on glass coverslips were loaded with the
acetoxymethyl ester of fura-2 (2 µM) for 30 min at
37 °C in 1 ml of buffer (pH 7.4, composition (mM): NaCl 127, CaCl2 1.8, KCl 5, MgCl2 2, NaH2PO4 0.5, NaHCO3 5, glucose 10, HEPES 10 with 0.1% bovine serum albumin). Cells were then washed
several times and placed within a heated (37 °C) perfusion stage of
a Nikon Diaphot inverted microscope. Image capture was performed using
MagiCal hardware following alternate excitation at 340 and 380 nm with
emission recorded at 510 nm. Values were averaged from 16 or 32 video
frames, and background fluorescence was subtracted prior to ratioing.
The ratio of fluorescence at 340 and 380 nm was calculated on a
pixel-by-pixel basis using maximum and minimum values defined by
treatment with 5 µM ionomycin in medium with either 10 mM CaCl2 or 10 mM EGTA and assuming
a dissociation constant of 225 nM for fura-2 and
Ca2+ at 37 °C, as described (14).
Western Blotting--
Cells were grown to confluence in 6-well
multiwell dishes. Medium was removed, and the cells were washed
(2 × 1 ml) with medium containing 0.1% bovine serum
albumin and incubated in a further 1 ml. GnRH was then added at the
appropriate concentration, and the cells were incubated at 37 °C in
5% CO2, humidified air. For immuno-detection of
Ins(1,4,5)P3 receptors, medium was aspirated after
the required time, the cell monolayer was washed once with ice-cold
Krebs/HEPES (7), and 1 ml of ice-cold TE buffer (10 mM
Tris, 10 mM EDTA, pH 7.4) was added. Cells were left for 5 min on ice and then scraped from the surface of the plate. Following trituration through a fine gauge needle the resulting suspension was
centrifuged (13,000 × g, 4 °C, 15 min). The
supernatant was then aspirated, and the pellet was resuspended in 50 µl of TE buffer. An equal volume of sample buffer (100 mM
Tris-HCl, 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 200 mM dithiothreitol) was then added, and the samples were
boiled for 3 min. Proteins were resolved by 5% SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and probed using
isotype-specific Ins(1,4,5)P3 receptor antibodies.
Immunoreactive bands were detected with ECL reagents and
exposure to Hyperfilm-ECL (Amersham Pharmacia Biotech). Where
required, densitometric analysis of the resulting bands was performed
using a Bio-Rad GS-670 imaging densitometer with Molecular Analyst
version 1.2 software. For immuno-detection of other proteins
(G
q/11 and PLC isoforms), Western blotting was carried
out as above with the exception that the cell monolayers were
solubilized in 200 µl of solubilization buffer (10 mM
Tris, 10 mM EDTA, 500 mM NaCl, 1% Nonidet
P-40, 0.1% SDS, 0.5% deoxycholate, 1 mM
phenylmethylsulfonyl fluoride, 100 µg/ml iodoacetamide, 100 µg/ml
benzamidine) for 30 min on ice before being processed as described
above. Details and use of the polyclonal antibody against the type I
Ins(1,4,5)P3 receptor and of the monoclonal antibodies against the type II and type III Ins(1,4,5)P3 receptors
have been described previously (26). Other antibodies were at dilutions according to the instructions of the suppliers.
Determination of Ins(1,4,5)P3 Receptor Density by
Binding of
[3H]Ins(1,4,5)P3--
Ins(1,4,5)P3
receptor binding was determined in membranes of
T3-1 cells as
described (16). Incubations were for 45 min at 4 °C in 200 µl of
incubation buffer with 2-10 µg of membrane protein, 10 nCi of
[3H]Ins(1,4,5)P3, and 0 or
10
9-10
5
M unlabeled Ins(1,4,5)P3. The incubations were
terminated by centrifugation and removal of supernatants by aspiration.
Pellets were then solubilized in NaOH and transferred to scintillant
for
-counting.
Ins(1,4,5)P3-mediated Release of
45Ca2+ from Intracellular Stores of
Permeabilized Cells--
45Ca2+ release assays
were performed in cytosol-like buffer (composition (mM): KCl 120, KH2PO4 2, (CH2COONa)2
5, MgCl2 2.4, HEPES 20, ATP 2, pH 7.2) using a previously
described method (27). The [Ca2+] of the cytosol-like
buffer was determined using fura-2 (28) and buffered to 120-190
nM with EGTA. Cells (2 ml containing 4-6 mg of protein)
were permeabilized by the addition of
-escin (25 µg/ml). The
suspension was then centrifuged (500 × g, 2 min) and resuspended in 6 ml of cytosol-like buffer, and
45Ca2+ was added (equivalent to 0.4 µl/ml
45Ca2+ at 1.98 mCi/ml). After gentle vortexing,
the cells were left for 15-20 min at room temperature. To initiate
release of loaded 45Ca2+, 50 µl of cells were
added to 50 µl of Ins(1,4,5)P3. After 60 s, 500 µl
of silicon oil was added, and the cells were centrifuged at 16,000 × g for 2 min. The aqueous phase and most of the silicon oil phase were aspirated, the tubes were inverted, and the remaining oil was allowed to drain. The pellets were solubilized in scintillant, and the unreleased 45Ca2+ was determined.
Release was calculated as a percentage of the total
45Ca2+ loaded. The size of the rapidly
releasable pool was also determined using 10 µM ionomycin
to indicate the amount of 45Ca2+ loaded into
the intracellular stores. This was ~80% of the total 45Ca2+.
 |
RESULTS |
Pretreatment of
T3-1/M3 cells for 1 h with
maximal concentrations (7) of either GnRH (1 µM) or
methacholine (1 mM) resulted in both homologous and
heterologous desensitization of agonist-mediated Ca2+
signaling (Fig. 1, a and
b). The homologous and heterologous loss of Ca2+
signaling as a result of GnRH or methacholine pretreatment were maximal
following 30-60 min of pretreatment and were sustained at this level
for at least 24 h of pretreatment (Fig. 1c).

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Fig. 1.
Homologous and heterologous desensitization
of Ca2+ signaling in
T3-1/M3 cells. Cells were
cultured, loaded with fura-2, and prepared for imaging as described
under "Experimental Procedures." Before imaging, they were
pretreated for 60 min (a and b) or for the times
indicated (c) in buffer containing 100 nM GnRH,
1 mM methacholine, or no addition (control). The cells were
then washed extensively and mounted on the microscope stage. During
imaging the cells were stimulated as indicated with either 100 nM GnRH (a and c) or 1 mM
methacholine (b). In panel c, showing the time
course of homologous and heterologous desensitization of GnRH-mediated
Ca2+ signaling, the GnRH-stimulated increase in
[Ca2+]i (calculated by subtraction of
pre-stimulation values from maximal post-stimulation values) is shown
as a function of pretreatment time. Control responses to GnRH were
determined at each time point but were not time-dependent
and have, therefore, been pooled and plotted at the 0 time point for
clarity. Each trace shows the mean ± S.E. derived from 3 separate
experiments (a and b), 3-7 separate experiments
(c), or >16 experiments (c, 0 h) with
20-50 cells imaged in each experiment.
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Expression of type I, II, and III Ins(1,4,5)P3 receptors
was examined by Western blotting of solubilized
T3-1/M3
cells using isoform-specific antibodies (26). Expression of all three
isoforms was detected, although bands representing type II and III
Ins(1,4,5)P3 receptors were faint and then only apparent
after exposure of the Western blot to film for longer periods of time
(data not shown). Although we are unable to quantitate precisely the
relative levels of the different types of Ins(1,4,5)P3
receptors, the predominant expression of type I is consistent with that
in the pituitary, which expresses type I, II, and III
Ins(1,4,5)P3 receptors at 73, 24, and 3% of the total
receptor population, respectively (19).
Challenge of
T3-1/M3 cells with 1 µM GnRH
resulted in a rapid and marked loss of type 1 Ins(1,4,5)P3
receptor immunoreactivity (Fig. 2,
a and c), which had a half-time of ~20 min, was
maximal by 60 min (<20% of immunoreactivity remaining), and sustained for at least 24 h in the continued presence of agonist (Fig. 2, a and c). Comparable data were obtained using 100 nM GnRH (Fig. 2c). Challenge of
T3-1/M3 cells with 1 mM methacholine also
resulted in a marked loss of type I Ins(1,4,5)P3 receptor
immunoreactivity, albeit with a rate and magnitude that was less than
that observed with GnRH (Fig. 2, b and c). In
contrast to treatment with 1 µM GnRH, there was some
recovery of type I Ins(1,4,5)P3 receptor immunoreactivity
during 24-h treatment with methacholine (Fig. 2, b and
c). The GnRH-mediated loss of type I
Ins(1,4,5)P3 receptor immunoreactivity was
concentration-dependent, with an EC50 of
9.91 ± 0.61 (log10, M;
n = 4; 0.12 nM) (all data with errors are
mean ± S.E.) at 60 min of treatment (data not shown).

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Fig. 2.
a-c, agonist-mediated down-regulation
of type I Ins(1,4,5)P3 receptor immunoreactivity in
T3-1/M3 cells. Cells were challenged with 1 µM GnRH, 100 nM GnRH, or 1 mM
methacholine for the times indicated, and Western blots for the type I
Ins(1,4,5)P3 receptor were performed. Western blots,
representative of three experiments, show the type I
Ins(1,4,5)P3 receptor in the absence of agonist treatment
or following treatment with either 1 µM GnRH
(a) or 1 mM methacholine (b) for the
indicated times. The density of the band representing the type I
Ins(1,4,5)P3 receptor was quantified and expressed as a
percentage of that in the absence of agonist (c). Data are
mean ± S.E.; n = 3. , 1 µM GnRH;
, 100 nM GnRH; , 1 mM methacholine.
d and e, time course of the recovery of type I
Ins(1,4,5)P3 receptor immunoreactivity following
down-regulation with GnRH. Following treatment with GnRH (1 µM) for 1 h, cells were washed, and the incubation
was continued in the presence of an antagonist of the GnRH receptor
(antide, 1 µM). Cells were then either solubilized
immediately or allowed the indicated recovery time before
solubilization. Western blotting for the type I
Ins(1,4,5)P3 receptor was then performed. A representative
Western blot is shown (d); below the blot are the mean
densitometric data (e). The density of the band
representing the type I Ins(1,4,5)P3 receptor was
quantified and expressed as a percentage of that under basal (no
agonist treatment) conditions. The data are the mean ± S.E.;
n = 3.
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In experiments designed to examine the rate of recovery of type I
Ins(1,4,5)P3 receptor immunoreactivity, cells were first treated with 1 µM GnRH for 1 h to induce
down-regulation. Agonist activation of GnRH receptors was then stopped
by washing the cell monolayer and continuing the incubation in the
presence of the GnRH receptor antagonist, antide (1 µM).
Type I Ins(1,4,5)P3 receptor immunoreactivity was then
examined over the subsequent 24 h. The 1-h pretreatment with GnRH
resulted in a marked loss of type I Ins(1,4,5)P3 receptor
immunoreactivity that was further reduced after 1 h of
"recovery" time (Fig. 2, d and e). Levels of
receptor immunoreactivity then increased back to basal levels by
24 h (Fig. 2, d and e). Similar results were
obtained when cells were treated with 1 µM GnRH and
washed, but antagonist was not added (data not shown).
In binding experiments, pretreatment of intact cells for 60 min with 1 µM GnRH reduced the binding of
[3H]Ins(1,4,5)P3 to
T3-1 membranes to
51 ± 7% (n = 4) of control without measurably
altering the Kd (Fig.
3).

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Fig. 3.
Loss of
[3H]Ins(1,4,5)P3 binding to membranes
prepared from T3-1/M3 cells
pretreated with GnRH. Cells were pretreated for 60 min in medium
with 0 ( ) or 1 µM GnRH ( ) as indicated, and
competition binding was then used to determine the number and affinity
of Ins(1,4,5)P3 receptors for
[3H]Ins(1,4,5)P3 as described under
"Experimental Procedures." The data shown are the mean ± S.E.
(n = 3) from a single representative experiment.
Pooling data from four such experiments revealed that GnRH pretreatment
reduced the Bmax to 51 ± 7% of control
without measurably altering the Kd (4.9 ± 2.2 nM).
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Permeabilization of
T3-1/M3 cells with
-escin
allowed intracellular Ca2+ stores to be loaded with tracer
45Ca2+, which could then be released by the
addition of Ins(1,4,5)P3. This exogenous
Ins(1,4,5)P3 was able to release a maximum of ~60% of
the 45Ca2+ that had been loaded over a 15-min
period, with an EC50 of
6.8 ± 0.2 (log10, M; n = 4; 0.16 µM) (Fig. 4). Treatment of
cells for 1 h with 1 µM GnRH significantly reduced
the magnitude of Ins(1,4,5)P3-mediated release of
45Ca2+ from the intracellular stores
(p < 0.001, two way analysis of variance) but had no
significant effect on the EC50 for
Ins(1,4,5)P3, which was
6.5 ± 0.3 (log10, M; n = 4; 0.32 µM)
(Fig. 4).

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Fig. 4.
Reduced ability of exogenous
Ins(1,4,5)P3 to release 45Ca2+ from
the intracellular stores of
T3-1/M3 cells following pretreatment
with GnRH. Cells were incubated in the presence or absence of 1 µM GnRH for 1 h and permeabilized with -escin,
and intracellular Ca2+ stores were loaded with
45Ca2+. Exogenous Ins(1,4,5)P3 was
then added, and the amount of 45Ca2+ release
was determined by measuring the radioactivity remaining associated with
the cell pellet. Results are expressed as the percentage of
45Ca2+ released by Ins(1,4,5)P3
compared with the total amount loaded. The data are the mean ± S.E.; n = 3.
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Thimerosal has been reported to increase Ins(1,4,5)P3
receptor sensitivity (29). In naive
T3-1/M3 cells, 100 µM thimerosal increased the spike
[Ca2+]i response to a sub-maximal (5 nM) (Fig. 5a) but
not maximal (1 µM) (Fig. 5c) concentration of
GnRH when cells were challenged in the absence of extracellular
Ca2+ (to assess the effects on Ca2+ release
only). This suggests that the Ins(1,4,5)P3 receptor does not limit the magnitude of the Ins(1,4,5)P3-mediated
Ca2+ release in naive cells stimulated with a maximal
concentration of GnRH but can limit the response to submaximal agonist
concentrations. When cells were pretreated for 1 h with 100 nM GnRH (in the presence of extracellular
Ca2+), thimerosal had little effect on the subsequent
response (again in the absence of extracellular Ca2+) to a
submaximal concentration of GnRH but markedly potentiated the response
to a maximal concentration (Fig. 5, b and d),
suggesting that, in GnRH-pretreated cells, Ins(1,4,5)P3
receptor activation is rate-limiting for GnRH-stimulated
Ca2+ mobilization.

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Fig. 5.
The effect of thimerosal on mobilization of
Ca2+ by GnRH in control and GnRH-pretreated
T3-1/M3 cells. Cells were
pretreated with 0 (a and c; Control) or 1 µM GnRH (b and d; GnRH pretreated)
for 60 min and then prepared for Ca2+ imaging as described
in the legend to Fig. 1. During imaging, the cells were transferred to
Ca2+-free medium (first vertical arrow) and then
to Ca2+-free medium containing 0 or 100 µM
thimerosal (second vertical arrow) as indicated. Finally,
the cells were stimulated with 5 nM (a and
b) or 1 µM (c and d)
GnRH (still in Ca2+-free medium with 0 or 100 µM thimerosal) as shown by the horizontal
arrows. Each trace shows the mean ± S.E. derived from three
separate experiments, with 20-50 cells imaged in each experiment.
After control pretreatments, thimerosal increased the response to 5 nM GnRH but not that to 1 µM GnRH, whereas in
GnRH-pretreated (desensitized) cells, thimerosal increased the response
to 1 µM GnRH but not that to 5 nM GnRH.
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Although challenge of cells with 1 µM GnRH resulted in a
dramatic reduction in type I Ins(1,4,5)P3 receptor
immunoreactivity (see above), exclusion of Ca2+ from the
extracellular buffer prevented the GnRH-mediated loss of
Ins(1,4,5)P3 receptors but had no significant effect on the basal (nonagonist-stimulated) levels over a 1-h period (Fig.
6a). Furthermore, the absence
of extracellular Ca2+ partially prevented the homologous
desensitization of GnRH-mediated spike and plateau Ca2+
signaling (Fig. 6, b and c). Incubation of cells
for 4 h with the cysteine protease (and proteasome) inhibitor
N-acetyl-Leu-Leu-norleucinal (ALLN, 100 µg/ml) (20, 22,
23) prior to and during a 1-h incubation with 1 µM GnRH
also markedly attenuated the agonist-induced loss of type I
Ins(1,4,5)P3 receptor immunoreactivity (Fig.
7a). The effects of ALLN on
the desensitization of the agonist-mediated Ca2+ response
were, however, difficult to interpret. Thus, even in the absence of
GnRH pretreatment, ALLN markedly inhibited the spike and plateau
[Ca2+]i responses to GnRH (data not shown), and
we therefore used the more specific proteasome inhibitor lactacystin
(10 µM, 4 h) (22). Lactacystin also markedly
protected type I Ins(1,4,5)P3 receptor immunoreactivity
against GnRH-mediated down-regulation (control (untreated), 100%;
1 h, 1 µM GnRH, 44.6 ± 4.5%; lactacystin, 80.6 ± 23.0%; 1 h, 1 µM GnRH + lactacystin,
102.5 ± 34.1%). Furthermore, lactacystin attenuated, but did not
completely prevent, GnRH-mediated desensitization of
[Ca2+]i mobilization (Fig. 7, b and
c). There was also some inhibition of GnRH-mediated spike
[Ca2+]i signaling in the presence of lactacystin
(Fig. 7b), although the plateau was unaffected (data not
shown).

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Fig. 6.
The influence of extracellular
Ca2+ on GnRH-mediated down-regulation of type I
Ins(1,4,5)P3 receptor immunoreactivity and GnRH-mediated
[Ca2+]i signaling in
T3-1/M3 cells. For determination
of Ins(1,4,5)P3 receptor immunoreactivity (a),
cells were challenged with GnRH (1 µM, 1 h) in
Krebs/HEPES buffer with either 1.3 mM Ca2+ or
no added Ca2+. Western blotting for the type I
Ins(1,4,5)P3 receptor was then performed. The density of
the bands representing the type I Ins(1,4,5)P3 receptor was
quantified and expressed as a percentage of that in the presence of
extracellular Ca2+ but in the absence of GnRH. The
data are the mean ± S.E.; n = 4. For
determination of [Ca2+]i signaling (b
and c), cells were pretreated with 0 (b) or 1 µM GnRH (c) for 60 min in buffer (filled
symbols) or Ca2+-free buffer (open
symbols), then washed in normal buffer, and prepared for
Ca2+ imaging as described in the legend to Fig. 1. During
imaging, the cells were transferred to Ca2+-free buffer
(vertical arrow) and then stimulated with 100 nM
GnRH (still in Ca2+-free buffer) as indicated by the
horizontal arrows. Each trace shows the mean ± S.E.
derived from three separate experiments, with 20-50 cells imaged in
each experiment.
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Fig. 7.
The influence of protease inhibition on
GnRH-mediated down-regulation of type I Ins(1,4,5)P3
immunoreactivity and homologous desensitization of
[Ca2+]i signaling in
T3-1/M3 cells. For determination
of Ins(1,4,5)P3 receptor immunoreactivity (a),
cells were incubated with (+) or without ( ) the
cysteine protease inhibitor ALLN (100 µg/ml) for 4 h. Agonist (1 µM GnRH or 1 mM methacholine) was then added,
and the incubation was continued for an additional 1 h. Western
blotting for the type I Ins(1,4,5)P3 receptor was then
performed. The densities of the bands representing the type I
Ins(1,4,5)P3 receptor were quantified and expressed as a
percentage of that under basal conditions (no ALLN or agonist). The
data shown are the mean ± S.E.; n = 4. For
determination of [Ca2+]i signaling (b
and c), cells were pretreated for 3 h in buffer with 0 (control, filled symbols) or 10 µM (open
symbols) lactacystin with 0 (b) or 100 nM
GnRH (c) added for the final 60 min of the pre-incubation
and fura-2-acetoxymethyl ester present for the final 30 min. The cells
were then washed in normal buffer and prepared for Ca2+
imaging as described in the legend to Fig. 1. During imaging, the
cells were transferred to Ca2+-free buffer and then
stimulated with 100 nM GnRH. Each trace shows the mean ± S.E. derived from three separate experiments, with 20-50 cells
imaged in each experiment.
|
|
Using commercially available antibodies against the PLC isoforms
1-4,
1-2, and
1-2, the
expression of PLC
1,
3,
1,
and
2 was demonstrated in
T3-1/M3 cells
(Fig. 8). The immunoreactivity of those
antibodies that did not detect proteins in
T3-1/M3
cells was confirmed using extracts from either SH-SY5Y neuroblastoma
cells or rat brain (data not shown). Given that GnRH receptor-mediated
responses are via G
q/11 and most likely, therefore, via
PLC
isoforms, we examined the influence of GnRH or methacholine
treatment on the expression of G
q/11, PLC
1, and
3. Exposure of
T3-1/M3 cells for up to 1 h with maximal concentrations of either GnRH (1 µM) or methacholine (1 mM) had no consistent effects on the levels of
G
q/11 or the PLC isoforms
1 and
3 (Fig. 8).

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|
Fig. 8.
Lack of effect of acute agonist treatment
( 1 h) on the expression of
G q/11,
PLC 1, and PLC 3
in T3-1/M3 cells. Western
blotting for the PLC isoforms 1-4, 1-2,
and 1-2 demonstrated the expression of
PLC 1, 3, 1, and
2 in T3-1/M3 cells. Agonist treatment
for up to 1 h had no effect on the expression levels of
G q/11, PLC 1, and PLC 3. This was also reflected in
the densitometric scan data from three separate experiments (data not
shown).
|
|
 |
DISCUSSION |
It has been known for over two decades that sustained stimulation
of gonadotropes with GnRH causes desensitization of GnRH-stimulated gonadotropin secretion (30), an effect that can be either exploited or
avoided in clinical applications of GnRH analogues (12). The recent
discovery that mammalian GnRH receptors do not show rapid homologous
desensitization (3-8) reveals that desensitization of GnRH-stimulated
gonadotropin secretion must reflect changes distal to the receptor, as
implied by earlier work showing that GnRH receptor regulation does not
explain desensitization of gonadotropin secretion (5). Our work has
revealed that pretreatment of
T3-1 cells with GnRH causes a
pronounced desensitization of GnRH-stimulated mobilization of
Ca2+ from intracellular stores (4, 5), which is of
particular interest in the light of the established importance of
Ca2+ mobilization in mediation of GnRH-stimulated
gonadotropin secretion. Because this desensitization is heterologous
(cross-desensitization is seen with responses to other PLC-activating
stimuli), it most likely reflects changes in the amount or activity of
effector proteins distal to the GnRH receptor.
Activation-dependent down-regulation of effector proteins
is emerging as an important mechanism for post-receptor adaptive responses, and such responses have already been observed in response to
GnRH. In
T3-1 cells, GnRH causes a loss of G
q (31)
and of regulatory and catalytic subunits of protein kinase A (32). It
also causes the apparent proteolysis of protein kinase C
and
(33). Here we have focused on the possible effects of GnRH on effector
proteins involved in Ca2+ metabolism and found that a
60-min pretreatment with GnRH causes pronounced desensitization of
GnRH-stimulated Ca2+ mobilization without measurably
altering cellular levels of PLC
1, PLC
3, or G
q.
These data are consistent with earlier studies demonstrating that GnRH
reduces G
q levels extremely slowly (half-time > 6 h) (31) and that reduced Ins(1,4,5)P3 generation
alone does not account for desensitization of Ca2+
mobilization in these cells (5). In contrast, we show that GnRH
pretreatment causes a pronounced down-regulation of
Ins(1,4,5)P3 receptors (as demonstrated by radioligand
binding and immunological quantification of type I
Ins(1,4,5)P3 receptors), that this effect is functionally
significant (as demonstrated by a reduction in Ins(1,4,5)P3-stimulated 45Ca2+
mobilization in permeabilized cells), and that the onset of this effect, and recovery from the effect, have similar kinetics to the
onset of, and recovery from, desensitization of Ca2+
mobilization (4, 5).
In several systems, activation of PLC-coupled GPCRs has been shown to
down-regulate Ins(1,4,5)P3 receptors, an effect attributed to Ca2+-dependent proteolysis of active
Ins(1,4,5)P3 receptors (17, 20, 22, 23). Activation of
these receptors has been shown to cause their ubiquitination
while still in the endoplasmic reticulum membrane (22). This is thought
to target Ins(1,4,5)P3 receptors for proteasomal
degradation, as demonstrated by the fact that proteasome inhibitors can
prevent GPCR-mediated down-regulation (20, 22, 23). Our data are in
accord with this model, because we have found that GnRH-mediated
down-regulation of type I Ins(1,4,5)P3 receptor
immunoreactivity is prevented in Ca2+-free medium and by
the two protease inhibitors ALLN and lactacystin. Interestingly, we
have found that the down-regulation of type I Ins(1,4,5)P3
receptors caused by GnRH is more rapid, more pronounced, and more
slowly reversed than that caused by methacholine (muscarinic M3 receptor activation). This is despite the fact that both
stimuli cause comparable increases in [Ca2+]i in
these cells and occur even when the concentrations of GnRH and
methacholine are matched to give comparable maximal increases in
Ins(1,4,5)P3 levels in these cells (100 nM
GnRH, 1 mM methacholine) (7). However, the muscarinic
M3 receptor undergoes a partial rapid homologous
desensitization and therefore causes a transient increase in
Ins(1,4,5)P3 mass, reducing to a sustained plateau after a
peak at 10 s, whereas the GnRH receptor does not rapidly
desensitize and therefore causes a sustained increase in
Ins(1,4,5)P3 mass, which reaches maximal levels within 20-30 s (7). This clearly implies that the Ins(1,4,5)P3
receptor down-regulation is sensitive not just to the magnitude of the Ins(1,4,5)P3 response but also to its duration, precisely
as expected if it is the Ins(1,4,5)P3 occupied (active)
receptor conformation that is sensitive to proteolysis (22). Thus, the
lack of GnRH receptor desensitization may contribute to the unusual
rapidity of Ins(1,4,5)P3 receptor down-regulation in these
cells. Typically, Ins(1,4,5)P3 receptor down-regulation
occurs with a half-time of 4-24 h (17, 23), as compared with <20 min
in GnRH-stimulated
T3-1/M3 cells (Fig. 2). Presumably
with other PLC-activating GPCRs, receptor desensitization attenuates
Ins(1,4,5)P3 responses and thereby reduces the rapidity
and/or magnitude of Ins(1,4,5)P3 receptor down-regulation.
It should be noted, however, that bombesin and cholecystokinin reduce
Ins(1,4,5)P3 receptor levels with a half-time of <30 min
in AR4-2J cells (19) and that methacholine caused
Ins(1,4,5)P3 receptor down-regulation with a half-time of
<60 min in
T3-1/M3 cells, demonstrating that
relatively rapid down-regulation can occur, even with receptors that do desensitize.
The major question raised by our data is whether down-regulation of
Ins(1,4,5)P3 receptors contributes to or underlies
desensitization of Ca2+ mobilization. Our investigations of
response kinetics are entirely compatible with this possibility because
we have found a) that the time-course of Ins(1,4,5)P3
receptor down-regulation in response to GnRH is comparable with that
for the onset of desensitization (Figs. 2 and 1c,
respectively) and b) that both effects are maintained as GnRH
pretreatment is extended to 24 h. Further support for the possible
causal relationship is provided by the demonstrations a) that the
GnRH-mediated Ins(1,4,5)P3 receptor loss and
desensitization of Ca2+ signaling are associated with
reduced Ins(1,4,5)P3-stimulated mobilization of
45Ca2+ from permeabilized cells (directly
establishing the functional significance of Ins(1,4,5)P3
receptor regulation in this system), b) that Ca2+-free
medium prevents and attenuates GnRH-mediated Ins(1,4,5)P3 receptor down-regulation and desensitization of Ca2+
mobilization, respectively, c) that lactacystin prevents and attenuates
GnRH-mediated Ins(1,4,5)P3 receptor down-regulation and
desensitization of Ca2+ mobilization, respectively, and d)
that thimerosal partially reverses desensitization of Ca2+
mobilization. Because thimerosal increases the affinity of
Ins(1,4,5)P3 receptors for Ins(1,4,5)P3 (29),
it would only be expected to influence responses to
Ins(1,4,5)P3 under conditions where
Ins(1,4,5)P3 receptor activation is limiting. Thus, the
ability of thimerosal to amplify Ca2+ mobilization by 5 nM GnRH, but not by 1 µM GnRH, implies that Ins(1,4,5)P3 receptor activation is limiting for the
response to the low concentration of GnRH but not to the high
concentration. In desensitized cells, however, thimerosal increased the
response to the high concentration of GnRH, demonstrating that in these cells Ins(1,4,5)P3 receptor activation has become limiting.
This is precisely what would be expected if Ins(1,4,5)P3
receptor loss leaves the desensitized cells with insufficient
Ins(1,4,5)P3 receptors for efficient mobilization of
Ca2+ even in the face of sufficient GnRH-stimulated
Ins(1,4,5)P3 levels.
Although our data are largely consistent with the possibility that
GnRH-mediated Ins(1,4,5)P3 receptor down-regulation
underlies desensitization of Ca2+ mobilization, several
lines of evidence might argue against this interpretation. Thus,
stimulation of muscarinic receptors results in a time course of
heterologous desensitization of GnRH-mediated [Ca2+]i elevation similar to the homologous
desensitization caused by GnRH pretreatment. This is despite the
finding that GnRH causes a more rapid and greater loss of
Ins(1,4,5)P3 receptor immunoreactivity than muscarinic
receptor stimulation. Furthermore, the retention of ~50% of
Ins(1,4,5)P3 receptors (Fig. 3) and the fact that maximal
Ins(1,4,5)P3-stimulated 45Ca2+
mobilization is only reduced by ~42% (Fig. 4) stand in contrast to
the almost complete loss of the spike phase
[Ca2+]i response to GnRH in desensitized cells
(Fig. 1). Similarly, complete inhibition of type I
Ins(1,4,5)P3 receptor down-regulation by pretreatment in
Ca2+-free medium, or in the presence of ALLN or
lactacystin, contrasts to only partial inhibition, or no measurable
inhibition, of desensitization. These apparent inconsistencies could
reflect contributions from other as yet unidentified mechanisms of
desensitization or may reflect differences in the relative
contributions of Ins(1,4,5)P3 receptor subtypes, or of
receptors in different cellular locations, to the end points
quantified. Thus, type II Ins(1,4,5)P3 receptors, which are
relatively resistant to down-regulation in other systems (19), may
contribute disproportionally to the 45Ca2+
mobilization response, and local down-regulation of
Ins(1,4,5)P3 receptors in the immediate vicinity of GnRH
receptors may be more extreme than that revealed by global measurements
of all Ins(1,4,5)P3 receptors. Alternatively, it is
possible that the 50% loss of Ins(1,4,5)P3 receptors and
the consequent increase in mean distance between functional
Ins(1,4,5)P3 receptors are sufficient to prevent propagation of Ca2+ mobilization by calcium-induced calcium
release (34) and therefore have a disproportionately large effect on
Ca2+ responses in intact cells (as compared with
permeabilized cells or membrane preparations). It is equally possible,
however, that other modifications of Ins(1,4,5)P3 receptors
(e.g. phosphorylation, ATP binding, ubiquitination)
inhibit Ins(1,4,5)P3 receptor signaling in the desensitized
cells without altering immunoreactivity or radioligand binding in
membrane preparations. Some of these modifications, particularly
ubiquitination, appear to be involved in the targeting of
Ins(1,4,5)P3 receptors for degradation (22). That such
targeting occurs is demonstrated by our finding that
Ins(1,4,5)P3 receptor immunoreactivity continues to decline
following removal of GPCR activation (Fig. 2).
Whereas a number of studies have demonstrated the principle of
agonist-induced Ins(1,4,5)P3 receptor down-regulation
(16-23), the current study provides evidence of a setting in which
such regulation may be functionally relevant. Thus, loss of
Ins(1,4,5)P3 receptors following either pre-ovulatory
surges in GnRH or, in particular, the clinical use of GnRH agonists may
play a part in the suppression of gonadotrope function. It should be
noted that such a mechanism would also result in a compromised function of other Ins(1,4,5)P3-dependent,
Ca2+-mobilizing receptors expressed on pituitary cells
(e.g. pituitary adenylyl cyclase-activating polypeptide
receptors). Such heterologous loss of function by this mechanism may be
less apparent in other systems in which GPCR desensitization may serve
to limit the down-regulation of signaling components shared with other receptors.
 |
FOOTNOTES |
*
This work was supported by Grants 16895/1.5 and 054949 from
the Wellcome Trust (to S. R. N. and C. A. M., respectively).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.
§
To whom correspondence should be addressed. Tel.: 0116-2523094;
Fax: 0116-2525045; E-mail: gbw2@leicester.ac.uk.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008916200
 |
ABBREVIATIONS |
The abbreviations used are:
GnRH, gonadotropin-releasing hormone;
PLC, phospholipase C;
Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate;
GPCR, G-protein-coupled receptor;
ALLN, N-acetyl-Leu-Leu-norleucinal.
 |
REFERENCES |
1.
|
Stojilkovic, S. S.,
Reinhart, J.,
and Catt, K. J.
(1994)
Endocr. Rev.
15,
462-499[Medline]
[Order article via Infotrieve]
|
2.
|
Berridge, M. J.
(1993)
Nature
361,
315-325[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Davidson, J. S.,
Wakefield, I. K.,
and Millar, R. P.
(1994)
Biochem. J.
300,
299-302[Medline]
[Order article via Infotrieve]
|
4.
|
McArdle, C. A.,
Forrest-Owen, W.,
Willars, G.,
Davidson, J. S.,
Poch, A.,
and Kratzmeier, M.
(1995)
Endocrinology
136,
4864-4871[Abstract]
|
5.
|
McArdle, C. A.,
Willars, G. B.,
Fowkes, R. C.,
Nahorski, S. R.,
Davidson, J. S.,
and Forrest-Owen, W.
(1996)
J. Biol. Chem.
271,
23711-23717[Abstract/Free Full Text]
|
6.
|
McArdle, C. A.,
Davidson, J. S.,
and Willars, G. B.
(1999)
Mol. Cell. Endocrinol.
151,
129-136[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Willars, G. B.,
McArdle, C. A.,
and Nahorski, S. R.
(1998)
Biochem. J.
333,
301-308[Medline]
[Order article via Infotrieve]
|
8.
|
Heding, A.,
Vrecl, M.,
Bogerd, J.,
McGregor, A.,
Sellar, R.,
Taylor, P. L.,
and Eidne, K. A.
(1998)
J. Biol. Chem.
273,
11472-11477[Abstract/Free Full Text]
|
9.
|
Willars, G. B.,
Heding, A.,
Vrecl, M.,
Sellar, R.,
Blomenröhr, M.,
Nahorski, S. R.,
and Eidne, K. A.
(1999)
J. Biol. Chem.
274,
30146-30153[Abstract/Free Full Text]
|
10.
|
Moenter, S. M.,
Caraty, A.,
Locatelli, A.,
and Karsch, F. J.
(1991)
Endocrinology
129,
1175-1182[Abstract]
|
11.
|
Moenter, S. M.,
Brand, R. M.,
Midgley, A. R.,
and Karsch, F. J.
(1992)
Endocrinology
130,
503-510[Abstract]
|
12.
|
Barbieri, R. L.
(1992)
Trends Endocrinol. Metab.
3,
30-34
|
13.
|
Hansen, J. R.,
McArdle, C. A.,
and Conn, P. M.
(1987)
Mol. Endocrinol.
1,
808-815[Abstract]
|
14.
|
McArdle, C. A.,
and Poch, A.
(1992)
Endocrinology
130,
3567-3574[Abstract]
|
15.
|
Tse, F. W.,
Tse, A.,
Hille, B.,
Horstmann, H.,
and Almers, W.
(1997)
Neuron
18,
121-132[Medline]
[Order article via Infotrieve]
|
16.
|
Wojcikiewicz, R. J. H.,
and Nahorski, S. R.
(1991)
J. Biol. Chem.
266,
22234-22241[Abstract/Free Full Text]
|
17.
|
Wojcikiewicz, R. J. H.,
Furuichi, T.,
Nakade, S.,
Mikoshiba, K.,
and Nahorski, S. R.
(1994)
J. Biol. Chem.
269,
7963-7969[Abstract/Free Full Text]
|
18.
|
Simpson, P. B.,
Challiss, R. A. J.,
and Nahorski, S. R.
(1994)
J. Neurochem.
63,
2369-2372[Medline]
[Order article via Infotrieve]
|
19.
|
Wojcikiewicz, R. J. H.
(1995)
J. Biol. Chem.
270,
11678-11683[Abstract/Free Full Text]
|
20.
|
Wojcikiewicz, R. J. H.,
and Oberdorf, J. A.
(1996)
J. Biol. Chem.
271,
16652-16655[Abstract/Free Full Text]
|
21.
|
Oberdorf, J.,
Vallano, M. L.,
and Wojcikiewicz, R. J. H.
(1997)
J. Neurochem.
69,
1897-1903[Medline]
[Order article via Infotrieve]
|
22.
|
Oberdorf, J.,
Webster, J. M.,
Zhu, C. C.,
Luo, S. G.,
and Wojcikiewicz, R. J. H.
(1999)
Biochem. J.
339,
453-461[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Sipma, H.,
Deelman, L.,
De Smedt, H.,
Missiaen, L.,
Parys, J. B.,
Vanlingen, S.,
Henning, R. H.,
and Casteels, R.
(1998)
Cell Calcium
23,
11-21[Medline]
[Order article via Infotrieve]
|
24.
|
Jenkinson, S.,
Nahorski, S. R.,
and Challiss, R. A. J.
(1994)
Mol. Pharmacol.
46,
1138-1148[Abstract]
|
25.
|
Willars, G. B.,
Nahorski, S. R.,
and Challiss, R. A. J.
(1998)
J. Biol. Chem.
273,
5037-5046[Abstract/Free Full Text]
|
26.
|
Mackrill, J. J.,
Wilcox, R. A.,
Miyawaki, A.,
Mikoshiba, K.,
Nahorski, S. R.,
and Challiss, R. A. J.
(1996)
Biochem. J.
318,
871-878[Medline]
[Order article via Infotrieve]
|
27.
|
Wilcox, R. A.,
Fauq, A.,
Kozikowski, A. P.,
and Nahorski, S. R.
(1997)
FEBS Lett.
402,
241-245[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Grynkiewicz, G.,
Poenie, M.,
and Tsien, R. Y.
(1985)
J. Biol. Chem.
260,
3440-3450[Abstract]
|
29.
|
Thrower, E. C.,
Duclohier, H.,
Lea, E. J.,
Molle, G.,
and Dawson, A. P.
(1996)
Biochem. J.
318,
61-66[Medline]
[Order article via Infotrieve]
|
30.
|
Belchetz, P. E.,
Plant, T. M.,
Nkai, Y.,
Keogh, E. J.,
and Knobil, E.
(1978)
Science
202,
631-633[Medline]
[Order article via Infotrieve]
|
31.
|
Shah, B. H.,
and Milligan, G.
(1994)
Mol. Pharmacol.
46,
1-7[Abstract]
|
32.
|
Garrel, G.,
McArdle, C. A.,
Hemmings, B. A.,
and Counis, R.
(1997)
Endocrinology
138,
2269-2266
|
33.
|
Harris, D.,
Reiss, N.,
and Naor, Z.
(1997)
J. Biol. Chem.
272,
13534-13540[Abstract/Free Full Text]
|
34.
|
Bootman, M. D.,
Berridge, M. J.,
and Lipp, P.
(1997)
Cell
91,
367-373[Medline]
[Order article via Infotrieve]
|
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