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T3-1 gonadotrophs via the cAMP/PKA signaling
system
Fondation pour Recherches Médicales, University of Geneva, CH-1211 Geneva 4, Switzerland
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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To investigate the regulation of free cytosolic
calcium concentration
([Ca2+]i)
by the adenosine 3',5'-cyclic monophosphate (cAMP)
signaling system in clonal gonadotrophs, microfluorimetric recordings
were made in single indo 1-loaded 
T3-1 cells. Forskolin,
8-bromoadenosine 3',5'-cyclic monophosphate, or a low
concentration (100 pM) of the hypothalamic factor pituitary adenylate
cyclase-activating polypeptide (PACAP) stimulated
Ca2+ step responses or repetitive
Ca2+ transients, which were
blocked by the removal of extracellular Ca2+ by the dihydropyridine (DHP)
(+)PN 200-110 or by preincubation with the protein kinase A (PKA)
antagonist H-89 (10 µM). Thus activation of the cAMP/PKA system in
T3-1 gonadotrophs stimulates Ca2+ influx through DHP-sensitive
(L-type) Ca2+ channels. In
contrast, high PACAP concentrations (100 nM) stimulated biphasic
Ca2+ spike-plateau responses. The
Ca2+ spike was independent of
extracellular Ca2+, and similar
responses were observed by microperfusion of individual cells with
D-myo-inositol
1,4,5-trisphosphate, suggesting the involvement of the phospholipase C
(PLC) signaling pathway. The Ca2+
plateau depended on Ca2+ influx,
was blocked by (+)PN 200-110, but was only partially blocked by
H-89 pretreatment. In conclusion, PACAP stimulates [Ca2+]i
increases in T3-1 gonadotrophs through both the PLC and
adenylate cyclase signaling pathways. Furthermore, this is the first
clear demonstration that the cAMP/PKA system can mediate changes in [Ca2+]i
in gonadotroph-like cells.
pituitary adenylate cyclase-activating polypeptide; adenosine 3',5'-cyclic monophosphate; protein kinase A; vasoactive intestinal polypeptide; H-89; anterior pituitary cells; L-type calcium channels
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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ANTERIOR PITUITARY GONADOTROPHS synthesize and secrete
luteinizing hormone (LH) and follicle-stimulating hormone (FSH) under the regulation of the hypothalamic factor LH-releasing hormone (LHRH or
GnRH) and gonadal steroids. However, studies on the activity of this
cell type have been hampered by the fact that the anterior pituitary
gland contains at least six different cell types, and the gonadotrophs
make up only 5-10% of the total population. Recently targeted
oncogenesis was used to produce the T3-1 cell line, which
possesses gonadotroph-like characteristics (30). These cells express
the LHRH receptor and the -subunit common to LH and FSH, although
they do not express the LH and FSH-specific 
-subunits (30). The
T3-1 cell line has proven very useful in biochemical studies of
the regulation of gonadotroph-like cells (1, 4, 9, 12, 27, 31), and in
particular on the regulation of -subunit and early gene expression
(27, 31). However, relatively little is known about the regulation of
intracellular signaling in T3-1 cells. In particular, although
the adenosine 3',5'-cyclic monophosphate (cAMP)/protein
kinase-A (PKA) intracellular signaling pathway has been implicated in
the regulation of -subunit expression in both normal and clonal
gonadotrophs (13, 31), the effects of this system on other
intracellular signaling pathways, such as changes in free cytosolic
Ca2+ concentration
([Ca2+]i)
in T3-1 cells, have not been widely studied.
The present article describes the effects of activation of the cAMP/PKA
system on changes in
[Ca2+]i
in T3-1 gonadotrophs. We tested the effects of forskolin, an
activator of adenylyl cyclase (AC), the membrane- permeable cAMP analog
8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), and
the hypothalamic factor pituitary adenylate cyclase-activating polypeptide (PACAP) on Ca2+
changes in these cells. PACAP, which has been proposed to act as a
hypophysiotropic factor regulating anterior pituitary cell activity
(24), exists in both 38-amino acid (PACAP-38) and
NH2-terminally shortened 27-amino
acid (PACAP-27) forms and shares significant sequence homology with
vasoactive intestinal polypeptide (VIP) (2). There are at least three
subtypes of PACAP/VIP receptors (PVRs). The type 1 receptor PVR1 (also
known as the PACAP-R) is selective for PACAP over VIP and couples to
the activation of both AC and phospholipase C (PLC). In contrast, the
PVR2 (VIP1R) and PVR3
(VIP2R) receptors do not
distinguish between PACAP and VIP and couple to the activation of AC
but not PLC (24). In the present study, PACAP was shown to be 100- to
1,000-fold more potent than VIP in stimulating
Ca2+ changes in T3-1
cells, suggesting the involvement of the PVR1. Low concentrations of
PACAP, as well as forskolin and 8-BrcAMP, stimulated
Ca2+ influx through
dihydropyridine (DHP)-sensitive
Ca2+ channels in T3-1
cells via the activation of the cAMP/PKA system. In contrast, higher
PACAP concentrations stimulated both
Ca2+ mobilization and
Ca2+ influx responses, which were
in part mediated by the action of the
D-myo-inositol
1,4,5-trisphosphate
[Ins(1,4,5)P3]/diacylglycerol (DG) signaling system. Such results have important implications for
understanding the ways in which the activation of the cAMP/PKA system
may modulate gonadotroph activity, both alone and in concert with the
Ins(1,4,5)P3/DG system (9, 24,
31).
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cell culture.
The technique used for the T3-1 cell culture has been
previously reported (25). Briefly, the T3-1 cells were grown as a monolayer culture in Dulbecco's modified Eagle's medium containing 4.5 mg/ml glucose, 5% fetal bovine serum, and 5% horse serum at 37°C in a humidified atmosphere of 5%
CO2-95% air. Medium was exchanged
every 2-3 days, and cells were passaged at 7-day intervals. For
the Ca2+ experiments, the cells
were harvested by treatment with trypsin and then cultured for 3-4
days on round coverslips in six-well culture plates.
Measurement of
[Ca2+]i.
The technique used for the measurement of
[Ca2+]i
changes in pituitary cells has been described in detail elsewhere (25). Briefly, cells were loaded with 4.4 µM of the membrane-permeable acetyloxymethyl ester form of the
Ca2+ fluorescent dye indo 1 (indo
1-AM) in standard medium [S medium: (in mM) 127 NaCl, 5 KCl, 2 MgCl2, 1.8 CaCl2, 5 NaHCO3, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 glucose, pH 7.4] containing 0.04% pluronic F-127 for 30 min at room temperature. After the cells were washed three
times with S medium, the coverslip containing cells was placed onto the
stage of an inverted epifluorescence microscope (Diaphot, Nikon, Tokyo,
Japan). The indo 1 within the cells was excited by ultraviolet light,
and the resultant Ca2+-dependent
fluorescence was collected by an oil immersion objective (Nikon Fluor)
(40), split into two components by a dichroic mirror (
crit 455 nm), and detected by
separate photomultipliers after passing through interference filters of
either 405 or 480 nm wavelength. The outputs of the two
photomultipliers were recorded at 33 Hz by use of an acquisition system
(Acqui, SICMU, University of Geneva, Switzerland) run on an
IBM-compatible computer. The fluorescence values obtained at the two
wavelengths were ratioed (R = F405/F480) and then converted to Ca2+ values
using the formula
[Ca2+]i = Kd × × (R 
Rmin)/(Rmax R), where
Kd is the
dissociation constant. The calibration constants were empirically
determined from 
50 individual cells with indo 1-loaded cells exposed
to the calcium ionophore ionomycin (2 µM) in S medium containing either 10 mM [Ca2+]
(Rmax) or essentially
Ca2+ free with 10 mM ethylene
glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) (Rmin). The value of
was calculated as the ratio of fluorescence at 480 nm for indo 1 in
minimal and maximal Ca2+
concentrations. The
Kd for indo
1/Ca2+ binding was taken as 250 nM.
Microperfusion of Ins(1,4,5)P3 into the
cells.
The technique used for the microperfusion of compounds into single
anterior pituitary cells has been previously described (23). Briefly,
the whole cell configuration of the patch-clamp technique was used to
microperfuse Ins(1,4,5)P3
(20-40 µM) into individual T3-1 cells. The standard
pipette solution contained (in mM) 120 potassium aspartate, 20 KCl, 2 MgCl2, 20 HEPES-NaOH, 0.1 GTP, 2 ATP, and 0.04 indo 1 (pH 7.4), and it had a free
[Ca2+] of 70-85
nM as measured with a
Ca2+-sensitive electrode (23).
Data analysis.
The data presented in this paper were gathered from a total of 42 experimental days. For any particular experimental manipulation, data
were pooled from 3 separate days of experiments. Baseline [Ca2+]i
values were calculated as an average over a 1-min period.
[Ca2+]i
values for the Ca2+-step,
Ca2+-transient, and
Ca2+-plateau responses were
calculated as an average over the 60-s period between 1 and 2 min after
the start of the Ca2+ response.
[Ca2+]i
values for the spike response were taken as the peak
[Ca2+]i
value in each case. The
[Ca2+]i
values are expressed in this paper as means ± SE. Student's t-test and Fisher's Exact Test were
used to test for statistical differences between
[Ca2+]i
values and numbers of responding cells, respectively. For all analyses
P 
0.05 was taken as statistically
significant.
Materials.
The T3-1 cells were kindly provided by Dr. P. L. Mellon (University of California, La Jolla, CA). PACAP-38,
PACAP-27, VIP, and
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89) were purchased from Calbiochem (Laufelfingen, Switzerland), and
indo 1, indo 1-AM, and pluronic F-127 were obtained from Molecular Probes (Leiden, the Netherlands). Isradipine [(+)PN
200-110] and its inactive isoform [()PN
200-110] were a generous gift from Sandoz. All other
reagents were obtained from Sigma Chemical (St. Louis, MO).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Concentration-dependent effects of PACAP and VIP on
[Ca2+]i.
We first tested the effects of the hypothalamic factor PACAP-38 on
[Ca2+]i
changes in T3-1 gonadotrophs by use of microfluorimetric
recordings in single indo 1-loaded cells. At low concentrations (100
pM) PACAP-38 stimulated "step" or "repetitive transient"
Ca2+ responses (Fig.
1, A and
B), which will be described in more detail below. However, as the PACAP-38 concentration was increased, not
only did more cells respond
(Fig.1E), but an increasing
proportion of cells exhibited biphasic "spike-plateau"
Ca2+ responses (Fig.
1C). At the highest concentration
tested (100 nM), PACAP increased
[Ca2+]i
in 23 of 28 cells (82%), and 21 of the 23 responding cells showed a
Ca2+ spike-plateau response. The
other two cells exhibited a Ca2+
step response typically observed at lower (100 pM)
PACAP-38 concentrations (Fig. 1A).
Biphasic spike-plateau responses to PACAP-38 (100 nM) started from a
mean
[Ca2+]i
value of 110 ± 10 nM and reached a
Ca2+ peak of 450 ± 70 nM before
decreasing to a Ca2+ plateau of
170 ± 20 nM measured between 1 and 2 min after the peak response
(n = 21).
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T3-1 cells express mRNA
for both the PACAP-preferring PACAP/VIP type 1 receptor (PVR1) and the
PVR3, which has similar affinity for PACAP and VIP (25). In an attempt
to identify the dominant receptor contributing to the PACAP-stimulated
Ca2+ responses in these cells, we
recorded changes in
[Ca2+]i
at the single cell level in response to a range of PACAP-38, PACAP-27,
and VIP concentrations (100 fM to 100 nM). To compare the sensitivity
of the cells to PACAP-38, PACAP-27, and VIP, concentration-response curves were based on the number of cells responding to a particular peptide concentration (Fig. 1). Analysis of the curves so produced revealed that PACAP-27 and PACAP-38 had similar potencies, with ~25%
of cells exhibiting Ca2+ responses
at 100 fM PACAP, a proportion of responsive cells that increased to
80-90% at the top concentrations (10-100 nM) of PACAP tested
(Fig. 1E). Both PACAP-27 and
PACAP-38, however, were 100- to 1,000-fold more potent than VIP in
stimulating changes in
[Ca2+]i.
The relative potency of PACAP-38, PACAP-27, and VIP suggests the action
on the PVR1 in T3-1 cells.
At all concentrations tested, PACAP-27 was equivalent to PACAP-38 in
stimulating
[Ca2+]i
responses in T3-1 cells (Fig.
1E). Furthermore, PACAP-27 caused similar changes in
[Ca2+]i,
i.e., predominantly spike-plateau response patterns at 100 nM and step
responses or Ca2+ transients at
concentrations 100 pM. In contrast, VIP even at the maximal dose of
100 nM stimulated either Ca2+ step
or repetitive Ca2+ transients
(Fig. 1D), and the biphasic
Ca2+ spike-plateau response
pattern was never observed in response to this peptide. Responses to
VIP at 100 nM were observed in 10 of 16 cells (63%), producing a mean
increase in
[Ca2+]i
over basal of 90 ± 10 nM (P 0.05). All VIP-stimulated Ca2+
responses were dependent on extracellular
Ca2+ (25).
In summary, PACAP stimulates Ca2+
changes in T3-1 cells through an action on the PVR1. High
concentrations of PACAP-38 (100 nM) stimulate biphasic
Ca2+ spike-plateau responses,
whereas low concentrations of PACAP-38 (100 pM) stimulate
Ca2+ step or
Ca2+ transient responses. Because
low concentrations of PACAP-38 have previously been shown to
preferentially stimulate cAMP production in T3-1 cells (25,
26), the following series of experiments were designed to test the
hypothesis that the Ca2+ responses
stimulated by low (100 pM) PACAP-38 concentrations could be mediated
through the cAMP signaling system.
Effect of 8-BrcAMP, forskolin, and low concentrations of PACAP on
[Ca2+]i
in T3-1 cells.
The effect of activation of the cAMP/PKA system on changes in
[Ca2+]i
was probed by raising intracellular cAMP concentration in T3-1 cells by three independent mechanisms:
1) addition of the
membrane-permeable cAMP analog 8-BrcAMP;
2) activation of endogenous AC by
the addition of forskolin (12, 25); or
3) addition of a low concentration of PACAP-38, which activates an AC-coupled cell surface receptor expressed in these cells (25, 26).
T3-1 cells was 110 ± 10 nM
(n = 25). All three treatments,
8-BrcAMP (1 mM), forskolin (2 µM), and PACAP-38 (100 pM), stimulated
increases in
[Ca2+]i
in single T3-1 gonadotrophs, which were observed either as Ca2+ step responses (see Figs.
1A and
3A) or repetitive
Ca2+ transients (Figs.
1B and 2,
A and
C). Eight of 16 cells (50%) responded to 1 mM 8-BrcAMP, producing a mean increase in
[Ca2+]i
over basal of 70 ± 10 nM (n = 8;
P 0.05, Student's
t-test). Eleven of 17 cells (65%)
responded to 2 µM forskolin, showing a mean increase in
[Ca2+]i
of 170 ± 50 nM (n = 11;
P 0.05). Eleven of 26 cells (42%) responded to 100 pM PACAP-38, producing a mean increase in
[Ca2+]i
of 100 ± 20 nM (n = 11;
P 0.05).
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Involvement of
Ca2+ influx
through cell membrane
Ca2+ channels.
The Ca2+ responses stimulated by
8-BrcAMP, forskolin, and 100 pM PACAP-38 were absolutely dependent on
extracellular Ca2+, because they
were never observed in Ca2+-free
medium (Fig. 2B; 8-BrcAMP:
n = 8; forskolin:
n = 9; 100 pM PACAP-38:
n = 12; in all cases
P 0.05, Fisher's Exact Test).
0.05). To more specifically
identify the Ca2+ channel type
involved, we tested the effect of (+)PN 200-110 (isradipine), a
specific inhibitor of DHP-sensitive (L-type) voltage-activated Ca2+ channels expressed in
T3-1 cells (4). As shown in Fig. 3, the inactive PN 200-110 isoform [()PN 200-110;
100 nM] was without effect on the
Ca2+ responses stimulated by
forskolin (2 µM; n = 5; Fig.
3A) or PACAP-38 (100 pM;
n = 5; Fig.
3B). In contrast, the
Ca2+ responses stimulated by
forskolin (2 µM) or PACAP-38 (100 pM) were completely blocked by
addition of the active (+) isoform of PN 200-110 (100 nM)
{forskolin: n = 5; PACAP-38:
n = 5;
P 0.05 compared with controls
[()PN 200-110; Fig. 3,
A and
B]}. Similar
effects were also observed with another L-type
Ca2+ channel blocker, nifedipine
(1-10 µM; data not shown).
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Involvement of PKA in the
Ca2+ responses.
Taken together, the results detailed above suggest that a rise in
intracellular cAMP concentration can lead to an increase in
[Ca2+]i
through the activation of Ca2+
influx via DHP-sensitive Ca2+
channels. To test whether this is a direct effect of cAMP, or an effect
mediated by a cAMP-dependent protein kinase such as PKA, we performed a
series of experiments with H-89, a competitive inhibitor of the
ATP-binding site of PKA (5). This antagonist has been previously used
in anterior pituitary cells to probe for the involvement of the
cAMP/PKA system in corticotropin-releasing hormone (CRH)-stimulated
ionic currents in adrenocorticotropic hormone (ACTH)-secreting
pituitary cells (28), and PACAP-stimulated interleukin-6 release from
rat folliculo-stellate cells (29). We also attempted to use the PKA
antagonist RpcAMPS, the Rp diastereoma of adenosine
3',5'-cyclic monophosphothioate [1 mM; (22)]
but found that preincubation with this compound had an appreciable effect to increase basal
[Ca2+]i
levels in T3-1 cells (data not shown), possibly as a result of
partial activation of the RII subunit of PKA (10) expressed in this
cell type (9). For this reason we did not employ RpcAMPS in the present
studies.
T3-1 cells are mediated through the cAMP/PKA
signaling system.
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What is the mechanism of PACAP-stimulated
Ca2+
spike-plateau response?
The biphasic Ca2+ spike-plateau
responses to high concentrations of PACAP-38 (Fig.
1C) were analyzed with regard to the
role of Ca2+ influx. In the
absence of extracellular Ca2+,
only the Ca2+ plateau, but not the
Ca2+ spike, was blocked (Fig.
5A).
Furthermore, the Ca2+ plateau was
blocked by the addition of the
Ca2+ channel blockers (+)PN
200-110 (n = 5; Fig.
5B), nifedipine (1-10 µM;
data not shown), or NiCl2 [5
mM; (25)]. These results suggest that the
Ca2+ spike is mediated by the
release of Ca2+ from an
intracellular store, whereas the plateau phase of the response is
dependent on Ca2+ influx (25). If
PACAP-stimulated Ca2+ mobilization
were through an
Ins(1,4,5)P3-dependent mechanism, it would be expected that
Ins(1,4,5)P3 should mimic the
Ca2+ response pattern observed by
PACAP. Intracellular application of
Ins(1,4,5)P3 was achieved with the
patch-clamp technique in its whole cell configuration (23).
Ins(1,4,5)P3 at an intrapipette concentration of 20-40 µM caused a marked and rapid rise in
[Ca2+]i
starting a few seconds after break-in
(n = 8) (Fig.
5C), effectively reproducing the
pattern of the Ca2+ spike response
to PACAP-38 (100 nM) observed in the absence of extracellular
Ca2+ (Fig.
5A). Thus it is most likely that the
spike response to PACAP in T3-1 cells is mediated by
Ins(1,4,5)P3, generated by the
PACAP-stimulated activation of PLC (25, 26).
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T3-1 cells with 10 µM H-89 for 30 min
completely abolished the Ca2+
response to a low PACAP-38 concentration (100 pM) (Fig. 4; Table 1). In
contrast, at high PACAP-38 concentrations (100 nM), H-89 attenuated
(P 0.05, Student's
t-test) but did not completely block
the amplitude of the Ca2+ spike
and the plateau
[Ca2+]i
levels (Table 1).
In conclusion, it appears that low concentrations (100 pM) of PACAP
stimulate Ca2+ influx through a
cAMP/PKA-dependent mechanism. In contrast, at higher (>1 nM)
concentrations, PACAP can stimulate both
Ca2+ mobilization from an
intracellular store and Ca2+
influx. The Ca2+ mobilization is
probably mediated by an
Ins(1,4,5)P3-dependent mechanism,
whereas the Ca2+ influx (plateau)
stimulated at high (100 nM) PACAP-38 concentrations is probably
mediated by both
PLC/Ins(1,4,5)P3/DG and
AC/cAMP/PKA signaling pathways.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This is the first report, to our knowledge, to show that the cAMP/PKA
system can mediate Ca2+ changes in
gonadotroph-like cells. Such findings have important implications for
the regulation of T3-1 and gonadotroph cell activity by the
cAMP/PKA system (13, 31). We have also demonstrated that PACAP can
stimulate changes in
[Ca2+]i
in the T3-1 gonadotrophs through both
Ins(1,4,5)P3- and
cAMP/PKA-dependent mechanisms.
Regulation of
Ca2+ influx in
T3-1 cells through cAMP/PKA-dependent mechanisms.
Three independent mechanisms of increasing cytosolic cAMP levels in
T3-1 gonadotrophs, forskolin, 8-BrcAMP, and a low concentration (100 pM) of PACAP-38 stimulated
Ca2+ influx responses in such
cells. The Ca2+ responses
stimulated by forskolin and PACAP-38 were blocked by the PKA antagonist
H-89 and by the DHP-sensitive Ca2+
channel blocker PN 200-110. These results suggest that an increase in cytosolic cAMP levels in T3-1 gonadotrophs leads to an
activation of PKA and the subsequent influx of
Ca2+ through DHP-sensitive
(L-type) Ca2+ channels in the cell
membrane. However, what are the mechanisms mediating this effect? One
possibility is that cAMP/PKA activates a voltage-independent
Na+ influx, leading to membrane
depolarization and the subsequent activation of voltage-activated
Ca2+ currents. Such an effect
occurs in rat somatotrophs in response to growth hormone-releasing
hormone or the membrane-permeable cAMP analog SpcAMPS [the
stimulatory diastereoisomer of adenosine 3',5'-cyclic
monophosphorothioate (19)], and in ACTH-secreting human pituitary
adenoma cells in response to CRH or forskolin (28). Significantly, in
the latter study the CRH-stimulated inward
Na+ current was blocked by H-89
pretreatment (28). An alternative possibility is that the activated PKA
phosphorylates L-type Ca2+
channels, leading to an increase in their voltage sensitivity as has
been shown in somatotroph-like GH3 cells (3). Such an action could
theoretically lead to an increase in
Ca2+ influx at the resting
membrane potential (18) or increase the Ca2+ influx in response to
membrane depolarization. The stimulation of
Ca2+ influx by PACAP-38, 8-BrcAMP,
and forskolin in rat somatotrophs may be mediated by similar mechanisms
(21, 22).
PACAP stimulates
Ca2+ changes
through the PVR1.
We have recently demonstrated the expression of mRNA for PVR1 and PVR3
in T3-1 cells (25). However, the fact that PACAP is
significantly more potent than VIP in binding (26), stimulation of cAMP
and inositol phospholipid (PI) turnover (25, 26), and stimulation of
Ca2+ changes (this study) suggests
that the PVR1 is the dominant PACAP receptor expressed in the
T3-1 cell line. PACAP-38 and PACAP-27 were equally potent in
stimulating Ca2+ changes and PI
turnover in T3-1 cells (25, 26), whereas in rat gonadotrophs,
PACAP-38 is clearly more potent than PACAP-27 in stimulating PI
turnover-dependent Ca2+ responses
(11). One possible explanation for these differences could have been
the preferential expression in T3-1 cells of a recently
described NH2-terminal splice
variant of the PVR1 (PVR1vs for "very short"), which was shown to
couple both PACAP-38 and PACAP-27 to PLC activation with similar
potencies (20). However, reverse transcriptase-polymerase chain
reaction studies in T3-1 cells with primers common to the
PVR1vs and the standard form of the PVR1 [kind gift of L. Journot
(20)] revealed that PVR1vs mRNA was expressed at a very low level
compared with PVR1 mRNA (D. Monnier and S. R. Rawlings, unpublished
observations), and thus it is unlikely to play a major role in this
cell type. It is interesting to note that a similar dichotomy on the
relative potencies of PACAP-38 and PACAP-27 to stimulate PI turnover
has been observed in PVR1-transfected Chinese hamster ovary cells (PACAP-38 
PACAP-27) (7) or LLC-PK1 cells (PACAP-38 > PACAP-27) (14). It has been proposed that such differences in PVR1 binding and
coupling may be due to differences in G protein expression and/or receptor numbers in different cell types (7, 14).
100 pM) PACAP concentrations and
the activation of both AC and PLC at higher (1 nM) PACAP
concentrations. Similar differences in the potencies of PACAP to
stimulate cAMP production and PI turnover have also been observed in
both T3-1 cell populations (25, 26) and in cell lines
transfected with the PVR1 (7, 14). These latter results suggest that
these differences are probably due to the coupling characteristics of
the PVR1 rather than to the expression of multiple receptor subtypes.
PACAP is a potent stimulator of T3-1 cells.
The Ca2+ responses observed in the
present study are seen at PACAP concentrations (100 fM) three orders
of magnitude below those previously shown to stimulate cAMP production
in T3-1 cell populations (100 pM) (25, 26). Although this may
reflect gross differences between the standard cAMP assays on cell
populations (25, 26) [cumulative measurement over a
40- to 45-min period at 37°C in the presence of the
phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) and the
detection of cAMP-mediated effects on
Ca2+ in single cells (dynamic
responses to cAMP production over 0-5 min at room temperature in
the absence of IBMX)], a more likely explanation is that the
detection of cAMP-mediated Ca2+
influx responses may be a more sensitive assay for AC activation than
measurement of total cAMP production in populations of these cells.
Functional and ultrastructural studies have shown that ACs are often
closely associated with sites of
Ca2+ entry into a cell, and it has
been proposed that activation of such ACs would produce local changes
in cAMP concentration near to the cell membrane, where it could bind to
PKA and lead to Ca2+ channel
activation (6, 15). In such a case, a cAMP/PKA-sensitive Ca2+ channel would be a more
sensitive detector of such local increases in cAMP than the standard
cAMP assay, which measures total cellular cAMP production. Finally, it
is interesting to note that similar differences in assay sensitivity
have been observed between total cellular PI turnover measurements and
recordings of PI turnover-dependent Ca2+ responses (11, 33).
T3-1 cells, with responses being observed at PACAP-38 concentrations as low as 100 fM. In individual pancreatic -cells, 1 fM PACAP stimulated Ca2+ responses
in ~30% of cells tested, and 100 fM PACAP stimulated Ca2+ responses in 80% of cells
and insulin release from -cell populations (32). As in T3-1
cells, PACAP stimulated Ca2+
influx responses in pancreatic -cells through the activation of a
PVR1-like receptor. The low concentrations of PACAP-38 that stimulate
cAMP/PKA-dependent Ca2+ influx,
and therefore presumably cAMP production, in T3-1 gonadotrophs (100 fM to 100 pM) are within the range detected in rat hypophysial portal blood (50-100 pM) (8), thus suggesting that physiological concentrations of PACAP-38 may have effects on the cAMP/PKA system in
gonadotroph cells in vivo.
PACAP stimulates the
Ca2+ spike
plateau response through an
Ins(1,4,5)P3-dependent mechanism.
Above 100 pM, PACAP also stimulated a spike-plateau
Ca2+ response pattern, which was
seen more frequently with increasing PACAP concentrations. Similar
Ca2+ spike-plateau responses are
observed in T3-1 cells after activation of the PLC-coupled LHRH
receptor (1, 17). The PACAP-stimulated Ca2+ spike was independent of
extracellular Ca2+ and was
mimicked by microperfusion of
Ins(1,4,5)P3 into individual cells, suggesting that in T3-1 cells, as in rat gonadotrophs (23), PACAP may stimulate Ca2+
release from an intracellular
Ins(1,4,5)P3-sensitive store. In T3-1 cells, microperfusion of
Ins(1,4,5)P3 stimulated a
Ca2+ spike-like response in all
cases, and Ca2+ oscillations were
never observed. In contrast, in rat gonadotrophs, the same procedure
always produced repetitive Ca2+
oscillations (23). The reason for this difference is unclear but
possibly relates to differences in the character of the
Ins(1,4,5)P3-sensitive Ca2+ stores in these two cell
types. The Ca2+ plateau stimulated
by high PACAP concentrations is dependent on extracellular
Ca2+ and is blocked by the
Ca2+ channel antagonist PN
200-110, indicating the involvement of Ca2+ influx. This response pattern
is only partly blocked by the PKA antagonist H-89 (Table 1), suggesting
that other intracellular mechanisms may also be involved. One
possibility is the DG/PKC system. PACAP-stimulated PLC would produce
both Ins(1,4,5)P3 and DG, the
latter leading to the activation of PKC. We have observed that the
phorbol ester phorbol myristate acetate (PMA; 1 µM), which activates
PKC, also stimulates small Ca2+
step responses in ~50% of T3-1 cells tested (data not
shown), and similar effects of PMA on
[Ca2+]i
and Ca2+ channel currents have
been previously reported (1, 4). Thus the
Ca2+ plateau observed in response
to PACAP may be due to the activation of both the cAMP/PKA and DG/PKC
systems.
Function of PACAP-stimulated cAMP production and
Ca2+ influx in
T3-1 gonadotrophs.
Activation of the cAMP/PKA signaling system has been previously shown
to regulate a variety of cellular functions in both normal rat and
clonal T3-1 gonadotrophs, including the modulation of gene
expression and -subunit/LH release (31). However, the classical
physiological regulators of gonadotroph cell function, including LHRH,
do not stimulate cAMP production in such cells (12). The recent
identification of the novel hypothalamic factor PACAP and the
demonstration of its action on gonadotroph cells have led to the
proposition that PACAP may be the physiological factor regulating the
cAMP signaling system in gonadotrophs (24). In fact, PACAP has been
shown to modulate both basal and LHRH-stimulated -subunit/LH gene
expression and secretion in both T3-1 cells and normal rat
gonadotrophs (24, 31), effects that are probably mediated through its
activation of the cAMP signaling system (24, 31). However, there are
clear differences in Ca2+
signaling between normal and clonal gonadotrophs; in rat gonadotrophs the cAMP/PKA signaling system has no apparent effect on
[Ca2+]i
changes (21-23), whereas, as shown in the present study, the same
system stimulates Ca2+ influx in
clonal T3-1 gonadotrophs. Because it has been previously shown
that Ca2+ influx is an important
signal regulating immediate early gene transcription in clonal
pituitary cells (16), further studies will need to examine whether the
previously reported effects of the activation of the cAMP signaling
system in T3-1 cells are direct or whether they are mediated,
at least in part, by the stimulation of
Ca2+ influx.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. P. L. Mellon for kindly providing the T3-1
cells, Dr. Nicholas Demaurex for comments on an earlier draft of this
paper, and the group of Dr. Karl-Heinz Krause for providing the use of
their patch-clamp/microfluorimetry system.
| |
FOOTNOTES |
|---|
This work was supported by Swiss National Science Foundation Grants 32-33514.92 and 31-45830.95.
Present address of M. Hezareh: Departments of Pathology and Medicine 0679, University of California at San Diego, La Jolla, CA 92093.
Address for reprint requests: S. R. Rawlings, Fondation pour Recherches Médicales, Univ. of Geneva, 64 Ave. de la Roseraie, CH-1211 Geneva 4, Switzerland.
Received 8 April 1997; accepted in final form 21 July 1997.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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),
PACAP-27 (
), and VIP (
), producing
Ca2+ responses shown in plot as % of cells showing a Ca2+ response
to indicated concentrations of peptides. Nos. of cells tested for data
points (from lowest to highest concentration) were, for PACAP-38: 7, 7, 10, 26, 7, 8, 28; for PACAP-27: 7, 6, 6, 6, 7, 7, 10; and for VIP: 5, 5, 5, 7, 9, 12, 16.
