Regulation of Ca2+-Sensitive Adenylyl Cyclase in Gonadotropin-Releasing Hormone Neurons
Lazar Z. Krsmanovic,
Nadia Mores,
Carlos E. Navarro,
Melanija Tomic and
Kevin J. Catt
Endocrinology and Reproduction Research Branch National
Institute of Child Health and Human Development National Institutes
of Health Bethesda, Maryland 20892
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ABSTRACT
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In immortalized GnRH neurons, cAMP production is
elevated by increased extracellular Ca2+ and
the Ca2+ channel agonist, BK-8644, and is
diminished by low extracellular Ca2+ and
treatment with nifedipine, consistent with the expression of adenylyl
cyclase type I (AC I). Potassium-induced depolarization of GT17
neurons causes a dose-dependent monotonic increase in
[Ca2+]i and elicits a
bell-shaped cAMP response. The inhibitory phase of the cAMP response is
prevented by pertussis toxin (PTX), consistent with the activation of
Gi-related proteins during depolarization.
Agonist activation of the endogenous GnRH receptor in GT17 neurons
also elicits a bell-shaped change in cAMP production. The inhibitory
action of high GnRH concentrations is prevented by PTX, indicating
coupling of the GnRH receptors to Gi-related
proteins. The stimulation of cAMP production by activation of
endogenous LH receptors is enhanced by low (nanomolar) concentrations
of GnRH but is abolished by micromolar concentrations of GnRH, again in
a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP
production is maintained by Ca2+ entry through
voltage-sensitive calcium channels, leading to activation of
Ca2+-stimulated AC I. Furthermore, the
Ca2+ influx-dependent activation of AC I acts
in conjunction with AC-regulatory G proteins to determine basal and
agonist-stimulated levels of cAMP production.
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INTRODUCTION
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Mammalian sexual development and reproductive function are
controlled by the episodic stimulation of pituitary gonadotropin
secretion by the hypothalamic decapeptide, GnRH. GnRH is produced by a
small number (
1,500) of specialized neurosecretory cells in the
hypothalamus (1, 2, 3, 4). The secretory activity of normal and immortalized
GnRH neurons (GT17 neurons) is dependent on the
depolarization-induced entry of extracellular calcium that results from
their spontaneous firing of calcium-dependent action potentials (5, 6, 7).
GnRH secretion from hypothalamic neurons and GT-1 cells is also
stimulated by receptor-mediated increases in intracellular cAMP, and by
treatment with forskolin and cAMP analogs (8, 9, 10, 11).
Immortalized GnRH neurons (GT17 neurons) express several G
protein-coupled receptors (GPCRs), including those for GnRH and
LH/human (h) CG (9, 12, 13) as well as ß1 adrenergic (10), D1
dopaminergic (14), muscarinic (15, 16), cholinergic (4, 17, 18), and
serotonergic receptors (19). Agonist activation of specific GPCRs and
dissociation of their cognate G proteins release
- and
ß
-subunits that regulate phospholipase C-ß, adenylyl cyclase
(AC), and ion channels, which in turn control the intracellular levels
of inositol phosphates, calcium, cAMP, and other second messengers (20, 21). The regulation of AC in hypothalamic neurons by changes in
cytosolic [Ca2+]i is an
important step in the integration of the actions of the two major
second messengers, cAMP and Ca2+.
Agonist-induced activation of phospholipase C (PLC) is the major signal
transduction pathway in cells that express GnRH receptors, and the
consequent Ca2+ mobilization and activation of
protein kinase C (PKC) by GnRH are key elements in the hypothalamic
control of gonadotropin hormone secretion by the pituitary gland (22).
Activation of the GnRH receptors expressed in GT17 neurons also
increases PLC and phospholipase D activity and stimulates voltage-gated
Ca2+ entry (23). The ability of elevated
intracellular cAMP levels to increase GnRH release in GT-1 cells has
also implicated receptors that are positively coupled to AC in the
signaling cascade that regulates GnRH release (10). Recent studies in
GT-1 cells have suggested that the cAMP signaling pathway is an
essential component of the mechanism responsible for the pulsatile
release of GnRH from hypothalamic neurons (11).
In the present study, the regulation of
Ca2+-dependent AC was investigated in cultured
rat hypothalamic cells and immortalized murine GnRH neurons (GT17
neurons). The role of
[Ca2+]i in AC regulation
was analyzed during receptor-dependent and -independent changes in
Ca2+ influx and mobilization.
Receptor-independent increases in
[Ca2+]i were elicited by
high extracellular [Ca2+]; the calcium channel
agonist BK-8644; K+-induced depolarization; and
treatment with ionomycin. Conversely,
[Ca2+]i levels were
decreased by treatment with the calcium channel blocker nifedipine and
incubation in low extracellular [Ca2+]. In
addition, the consequences of agonist stimulation of the
Ca2+-mobilizing GnRH receptor, and associated
changes in cAMP production, were investigated in low and high
extracellular [Ca2+] and in the presence of
Ca2+ channel agonist and antagonist analogs. The
control of cAMP production was also analyzed during activation of
LH/hCG receptors expressed in GT17 neurons. Those studies have
demonstrated that the prevailing level of
[Ca2+]i is a critical
determinant of AC activity and cAMP production in GnRH neuronal
cells.
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RESULTS
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Dependence of Basal cAMP Production on
Ca2+ Influx
In GT17 neurons, treatment with the phosphodiesterase inhibitor,
isobutylmethylxanthine (IBMX, 1 mM), caused a 5-fold
increase in cAMP production (2.1 ± 0.3 pmol/ml vs.
13.7 ± 2.7 pg/ml). This was accompanied by increased GnRH release
(23.8 ± 3.4 pg/ml vs. 36.4 ± 4.5 pg/ml;
P < 0.01; n = 6) and had no significant effect on
[Ca2+]i (187 ± 23
nM control vs. 215 ± 37
nM IBMX; n = 8). The intracellular cAMP
concentration in the presence of IBMX reached a maximum during the
first 60 min and declined thereafter. In contrast, cAMP release into
the incubation medium showed a slower and prolonged rise between 30 and
240 min (Fig. 1
, A and B). The rate of
cAMP production by cultured GT17 neurons was highly sensitive to the
changes in extracellular Ca2+ concentration
([Ca2+]e). Increases in
[Ca2+]e caused
concentration-dependent elevations of both
[Ca2+]i and cAMP
production, with EC50 values of 1.3
mM and 2.2 mM, respectively
(Fig. 1
, C and D). Concentration-dependent increases in cAMP production
were also observed when cells were incubated with increased
[Ca2+]e in the absence of
IBMX (Fig. 1E
). AC activity, in membrane preparations from GT17
neurons, was also stimulated by Ca2+ in a
concentration-dependent manner, reaching a maximum at 10
µM Ca2+ (Fig. 1F
).

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Figure 1. Extracellular Calcium-Regulated cAMP Production in
GnRH Neurons
A and B, Time course of basal cAMP production in hypothalamic cells (A)
and GT17 neurons (B). C, Extracellular
[Ca2+]-dependence of [Ca2+]i in
GT17 neurons. D, Extracellular [Ca2+]-dependence of
cAMP production in GT17 neurons. E, Extracellular
[Ca2+]-dependence of cAMP production in the absence of
IBMX. F, [Ca2+]-dependence of AC activity assayed by cAMP
production in GT17 membrane preparations. Data are means ±
SE of three independent experiments.
Asterisks indicate significant differences
(P < 0.05) compared with basal cAMP production and
Ca2+ free-medium.
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The stimulatory action of extracellular Ca2+ on
cAMP production was highly dependent on Ca2+
entry through L-type Ca2+ channels. The
dihydropyridine Ca2+ channel agonist, BK-8644,
caused time- and dose-dependent increases in cAMP production, with an
EC50 of 9.6 nM (Fig. 2
, A and B). cAMP production was
significantly increased (12.7 ± 2.1 to 16.8 ± 1.6 pmol/ml;
P < 0.05; n = 4) after 5 min of incubation with 1
µM BK-8644 and was maximal after 15 min. The
stimulatory effect of BK-8644 on cAMP production was abolished by
incubation in low [Ca2+]e
(Fig. 2B
). Conversely, L-type channel blockade by nifedipine caused a
dose-dependent decrease in cAMP production with an
IC50 of 6.7 nM (Fig. 2D
),
and this was also maximal after 15 min (Fig. 2C
). A significant
decrease in cAMP production (12.7 ± 2.1 pmol/ml, control
vs. 8.6 ± 1.2 pmol/ml; P < 0.05;
n = 4) was observed after 5 min of incubation with 1
µM nifedipine.

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Figure 2. Effects of Dihydropyridines on cAMP Production in
GT17 Neurons
A and B, Time- and dose-dependent stimulation of cAMP production by
BK-8644. C and D, Time- and dose-dependent inhibition of cAMP
production by nifedipine. Data are means ± SE of
three independent experiments. Asterisks indicate
significant differences (P < 0.05) compared with
the basal cAMP production.
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In contrast to the stimulatory effect of increased
Ca2+ influx on cAMP production, ionomycin-induced
[Ca2+]i increases in
cells bathed in Ca2+-free medium did not lead to
increased cAMP formation. The 4-fold elevation of
[Ca2+]i induced by 400
nM ionomycin (Fig. 3A
), and
the 5-fold increase induced by 1 µM ionomycin (Fig. 3B
)
had no effect on cAMP production (Fig. 3
, C and D). The
[Ca2+]i level in cells
treated with 1 µM ionomycin was comparable to that of
[Ca2+]i in cells bathed
in 1.5 mM
[Ca2+]e, yet cAMP
production remained low despite the 5-fold increase in
[Ca2+]i during
Ca2+ mobilization from intracellular stores.

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Figure 3. Effects of Ionomycin on
[Ca2+]i and cAMP Production in GT17 Neurons
A and B, [Ca2+]i responses of GT17 neurons
treated with ionomycin (400 nM and 1 µM) in
Ca2+-free medium. C and D, cAMP production during
ionomycin-induced increases in [Ca2+]i. Data
are means ± SE of three independent experiments.
Asterisks indicate significant differences
(P < 0.01) compared with the basal
[Ca2+]i level.
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Depolarization of GT17 neurons by increased extracellular
[K+] caused a progressive,
concentration-dependent increase in
[Ca2+]i (Fig. 4A
). This was associated with a
bell-shaped cAMP response (Fig. 4B
) in which the maximal level was
elicited at 35 mM KCl, a concentration that caused only a
half-maximal increase (462 nM) in
[Ca2+]i. Higher KCl
concentrations caused further increases in
[Ca2+]i of up to 920
nM and were associated with a progressive and marked
decrease in cAMP production almost to the baseline level. As expected,
the stimulatory effect of K+ on cAMP production
was abolished by incubation in 100 nM extracellular
Ca2+ (Fig. 4B
). There was also a major decrease
in the baseline cAMP level below that observed at 1.5 mM
Ca2+.

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Figure 4. Effects of Depolarization-Induced Calcium Entry on
cAMP Production in GT17 Neurons
A, Elevation of [Ca2+]i during progressive
depolarization of GT17 neurons by increased extracellular
K+. B, The concomitant biphasic change in cAMP production,
with stimulation at K+ concentrations up to 35
mM and progressive inhibition at higher concentrations. C,
Effects of PTX on the inhibition of cAMP production by high KCl
concentrations on cAMP production. Data are means ±
SE of three independent experiments.
Asterisks indicate significant differences
(P < 0.01) compared with the basal cAMP
production.
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The inhibitory effect of 55 mM K+ on
cAMP production was prevented by pretreatment of GT17 neurons with
100 ng/ml pertussis toxin (PTX), indicating that activation of
Gi or Go was responsible
for the inhibition of cAMP production (Fig. 4C
). In this experiment,
PTX treatment significantly increased both the stimulated cAMP response
to 35 mM K+ and the inhibited
response of 50 mM K+, suggesting that
Gi-mediated impairment of AC activity increased
progressively with increasing degrees of depolarization.
Effects of GnRH Receptor Activation on cAMP Production
In addition to stimulating the inositol
phosphate/Ca2+-signaling pathway via
Gq/11, activation of the GnRH receptor has been
shown to increase cAMP production (24, 25, 26). The action of GnRH on cAMP
production in GT17 neurons was biphasic and dose-dependent. GnRH
concentrations of up to 10 nM caused progressive increases
in [Ca2+]i and cAMP
production (Fig. 5
, A and B).
Membrane-associated Gs
-subunit
immunoreactivity decreased during treatment with low nanomolar GnRH
concentrations but was unchanged after 30 min treatment with 1
µM GnRH or with a GnRH antagonist analog (Fig. 5C
).
However, higher concentrations of GnRH that continued to increase
[Ca2+]i caused a
progressive decrease in cAMP production. This inhibitory action of GnRH
on cAMP formation was prevented by treatment with PTX. The bell-shaped
dose-response curve and the reversal of the inhibitory actions of high
GnRH concentrations on cAMP production by PTX are again consistent with
the activation of Gi-related proteins (Fig. 5D
).
Both the stimulatory action of low nanomolar GnRH concentrations on
cAMP production and the inhibitory action of micromolar GnRH
concentrations were accentuated by increases in
[Ca2+]e (Fig. 6A
). Conversely, inhibition of
Ca2+ influx by nifedipine converted the biphasic
cAMP response to GnRH into a small monotonic increase (Fig. 6B
).
Furthermore, the stimulatory action of BK-8644 was enhanced by
nanomolar GnRH concentrations (Fig. 7A
)
but was abolished by micromolar concentrations of GnRH (Fig. 7B
).

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Figure 5. GnRH-Stimulated Ca2+ and cAMP Responses
in GT17 Neurons
A and B, Dose-dependent actions of GnRH on
[Ca2+]i and cAMP production. C, Effects of
GnRH on membrane-associated Gs -subunit
immunoreactivity. D, Effects of PTX on GnRH-induced changes in cAMP
production. Data are means ± SE of three independent
experiments. Asterisks indicate significant differences
(P < 0.01) compared with the basal
[Ca2+]i or cAMP production.
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Figure 6. Effects of Extracellular [Ca2+] and
Nifedipine on GnRH-Induced cAMP Production in GT17 Neurons
A, Extracellular [Ca2+] dependence of the stimulatory and
inhibitory actions of nanomolar and micromolar GnRH concentrations,
respectively, on cAMP production. B, Inhibition by nifedipine of basal
cAMP production and the biphasic GnRH-induced cAMP response. Data are
means ± SE of three independent experiments.
Asterisks indicate significant differences
(P < 0.01) compared with the basal cAMP
production.
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Figure 7. Effects of BK-8644 on GnRH-Induced cAMP Production
in GT17 Neurons
A, Enhancement of the maximal stimulatory action of BK 8644 on cAMP
production by concomitant treatment with 1 nM GnRH. B,
Inhibition of the maximum stimulatory action of BK 8644 by concomitant
treatment with 1 µM GnRH. Data are means ±
SE of three independent experiments.
Asterisks indicate significant differences
(P < 0.01) compared with the basal cAMP
production. Double asterisks indicate significant
differences (P < 0.05) compared with 1
µM BK 8644.
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Effects of LH Receptor Activation on cAMP Production
Exposure of GT17 neurons to increasing concentrations of hCG for
30 min caused dose-dependent increases in cAMP production, with
EC50 of 0.3 nM. Both basal and
hCG-stimulated cAMP production were markedly reduced in
Ca2+-free medium (Fig. 8A
) and by treatment with 1
µM nifedipine (not shown). Treatment with hCG also
decreased Gs
-subunit immunoreactivity,
consistent with coupling of the activated LH/hCG receptor to the
AC-stimulatory G protein (Fig. 8B
). The reduction of cAMP production by
inhibition of Ca2+ entry, with no change in
EC50, indicates that Ca2+
influx through voltage-sensitive calcium channels (VSCC) is necessary
to maintain hCG-induced activation of AC (Fig. 8A
). The maximal
stimulatory effect of 2 nM hCG was potentiated by
increasing [Ca2+]e, with
a similar EC50 for both control and hCG-treated
cells (Fig. 8C
). The stimulatory action of hCG was biphasically
regulated by concomitant activation of GnRH receptors, with enhancement
at low nanomolar GnRH concentrations, and inhibition at high nanomolar
and micromolar concentrations of GnRH (Fig. 8D
). In the presence of 1.5
mM [Ca2+]e,
treatment with K+ (35 mM) or hCG (2
nM) increased cAMP production from 8.8 ± 0.3 pmol/ml
to 35.9 ± 1.3 pmol/ml; n = 4; P < 0.001)
for K+, and from 8.8 ± 0.3 pmol/ml to 16.5
± 0.9 pmol/ml; n = 4; P < 0.01 for hCG. No
additive effect on cAMP production (33.2 ± 1.9 pmol/ml; n =
4) was observed during concomitant treatment with 35
mM K+ and 0.5
nM hCG. There was also no additivity between 35
mM K+ and 0.5
nM hCG in Ca2+-free medium,
since the stimulatory action of K+ was
abolished.

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Figure 8. Regulation of cAMP Production in GnRH Neurons by
hCG and GnRH
A, hCG-stimulated cAMP production of cells incubated in normal and low
extracellular [Ca2+]. B, hCG-induced reduction of
immunoreactive Gs -subunit in GT17 cell membranes. C,
Potentiation of the maximal hCG-stimulated cAMP production by increased
[Ca2+]e. D, Biphasic action of GnRH receptor
activation on hCG-induced cAMP production. Data are means ±
SE of three independent experiments.
Asterisks indicate significant differences
(P < 0.01) compared with the basal cAMP
production.
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AC Isoforms in GT17 Neurons
Western blot analysis of membrane preparations from GT17 neurons
with specific antiserum for AC I revealed that this
Ca2+-stimulated and brain-specific enzyme is
expressed in GT17 neurons (Fig. 9A
). AC
III, which was initially cloned as an AC restricted to the olfactory
neuroepithelium, was also detected in GT17 neurons by Western
blotting with specific antiserum (Fig. 9B
). AC V, which is abundant in
the brain and heart tissue, and AC VI, abundant in peripheral tissue
and low in the brain, are structurally related and are inhibited by
Ca2+. One or both of these isoforms are also
expressed in GT17 neurons as revealed by blotting with an antiserum
that cross-reacts with both enzymes (Fig. 9C
). The specificity of
immunoreactivity for each specific AC was confirmed by preadsorbtion of
primary antibody with the relevant antigenic peptide (shown as controls
in Fig. 9
, AC).

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Figure 9. Immunoblot Analysis of Ca2+-Dependent
AC Isoforms in GT17 Neurons
Solubilized GT17 membrane preparations were analyzed by SDS-PAGE and
Western blotted with specific antibodies to ACI (A), ACII (B), and
ACV/IV (C). In control samples the specificity of each antibody was
determined by preadsorbtion with the corresponding blocking peptide.
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DISCUSSION
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The episodic mode of GnRH release from perifused hypothalamic
cells and immortalized GnRH neurons is highly
[Ca2+]e dependent,
suggesting that GnRH secretion is controlled by
Ca2+ entry through plasma membrane
Ca2+ channels (27, 28). Normal GnRH neurons and
immortalized GnRH neurons express numerous plasma-membrane channels,
including tetrodotoxin-sensitive Na+ channels,
low-voltage-activated Ca2+ channels,
dihydropyridine-sensitive Ca2+ channels,
inward-rectifying K+ channels, several types of
outward K+ channels, the BK subtype of
Ca2+-sensitive K+ channels
(IK-Ca), and apamin-sensitive
IK-Ca channels (SK channels) (6, 29, 30, 31, 32, 33). An
analysis of the relationship between electrical membrane activity and
Ca2+ influx in differentiated GT17 neurons
revealed that most cells exhibit spontaneous, extracellular
Ca2+-dependent action potentials (5, 6). In
spontaneously active GT17 neurons firing regular action potentials,
each spike generates a significant increase in
[Ca2+]i. Such
[Ca2+]i increases are
consistently larger in cells exhibiting broad endocrine-like action
potentials than in those exhibiting sharp neuronal-like action
potentials (5).
The present studies have demonstrated that Ca2+
entry through VSCC and the consequent changes in
[Ca2+]i levels also
regulate AC activity and cAMP production. Under basal conditions,
Ca2+ channel blockade by nifedipine, or
incubation in Ca2+-free medium, inhibits cAMP
production in a time- and concentration-dependent manner. These
observations are consistent with the expression of
Ca2+-stimulated ACs (34, 35, 36, 37, 38, 39, 40) in GnRH cells and
other neurons. Such treatment also abolished pulsatile neuropeptide
secretion (27, 28), inhibited spontaneous electrical activity, and
reduced [Ca2+]i levels
(5). Thus, Ca2+ coordinately regulates
spontaneous electrical activity and cAMP production, and its removal or
reduction leads to abolition of pulsatile GnRH secretion.
GT17 neurons express type I AC, and increased cAMP production was
observed when [Ca2+]i was
increased as a consequence of Ca2+ channel
activation by BK-8644. The stimulatory action of
Ca2+ was evident in the
[Ca2+]i range from 100
nM to 500 nM, levels that are produced in these
excitable cells by increases in
[Ca2+]e. These
observations indicate that physiological elevations in
[Ca2+]i secondary to
Ca2+ entry can regulate cAMP production. In
contrast, mobilization of
[Ca2+]i from
intracellular stores by ionomycin in the absence of extracellular
[Ca2+] did not increase cAMP production.
The finding that only Ca2+ entry can stimulate
cAMP production indicated that AC may be functionally colocalized with
the Ca2+ entry channels in GT17 neurons, as in
other neurons (41, 42, 43). AC V and AC VI are also expressed in GnRH
neurons, but their role in the Ca2+-dependent
regulation of cAMP production appears to be minor since inhibition of
Ca2+ entry was not accompanied by increased cAMP
production.
In cerebellar granule cells, K+-induced
depolarization caused initial stimulation and subsequent inhibition of
cAMP production in the absence of extracellular
Ca2+. This biphasic response was attributed to
progressive increases in the extracellular
K+:Na+ ratio in the absence
of extracellular Ca2+. Despite the lack of direct
evidence for the modulation of cerebellar granule cell AC by membrane
potential, the results demonstrated that membrane
depolarization-induced influx of Na+, as well as
Ca2+, can elevate cAMP levels (44). In rat
brainstem synaptoneurosomes, membrane depolarization has been found to
activate PTX-sensitive G proteins (45). This action is mediated by the
voltage-gated sodium channel (VGSC), and its gating properties
determine the activation of PTX-sensitive G proteins (46). In addition,
in depolarized brainstem and cortical synaptoneurosomes, the VGSC
-subunit was most efficiently cross-linked with
Go-proteins. These findings suggest that
interactions between the VGSC
-subunit and
Go-proteins occur during membrane depolarization
(47).
In GnRH neurons, depolarization by progressive elevation of
[K+]e elicited a
bell-shaped cAMP response, with initial stimulation and subsequent
inhibition. Stimulation of cAMP production was evident for
K+ concentrations from 5 mM to 35
mM K+ and was correlated with
increased [Ca2+]i,
presumably reflecting activation of the
Ca2+-stimulated type I AC expressed in GnRH
neurons. The stimulatory effect of increased K+
was completely abolished in Ca2+-free medium,
indicating that Ca2+ entry is necessary for
activation of the endogenous Ca2+-stimulated AC
in GnRH neurons. Also, the lack of synergistic stimulation of cAMP
production by K+-induced depolarization and
activation of the Gs-coupled LH receptors in GnRH
neurons indicates the absence of voltage-sensitive AC (48). In
contrast, it appears that membrane depolarization of GnRH neurons is
linked to activation of Gi/o inhibitory proteins,
since PTX pretreatment prevented K+- induced
inhibition of cAMP production. The PTX sensitivity of the inhibitory
action of high K+ concentrations in GnRH neurons
resembles that observed in brain synaptoneurosomes during membrane
depolarization (47).
Activation of GnRH receptors expressed in GnRH neurons leads to a rapid
and concentration-dependent increase in
[Ca2+]i that consist of
an initial spike phase, driven by inositol trisphosphate-induced
Ca2+ mobilization, and a sustained
Ca2+ response that depends on
Ca2+ entry through VSCC (12). Agonist activation
of the GnRH receptors expressed in GnRH-producing neurons also
modulated cAMP production. Treatment with increasing GnRH
concentrations caused a dose-dependent and biphasic change in cAMP
production. The stimulation of cAMP production by low nanomolar GnRH
concentrations was inhibited by the Ca2+ channel
blocker, nifedipine, and was not observed in
Ca2+-free medium. In contrast, increased
[Ca2+]e, and the
Ca2+ channel agonist BK-8644, potentiated the
stimulatory actions of low nanomolar GnRH concentrations. These
findings indicate that the stimulatory actions of GnRH on cAMP
production are mediated in part by GnRH-induced
Ca2+ entry through ion channels and activation of
Ca2+-stimulated AC. The concentration of GnRH
that stimulates cAMP production was associated with a decrease in
membrane-bound G
s
immunoreactivity, consistent with Gs dissociation
and subunit redistribution during GnRH receptor activation (15, 49, 50, 51). In COS-7 cells transiently expressing the GnRH receptor,
agonist-stimulated cAMP production is dependent on specific residues in
the first intracellular loop that are not essential for activation of
the phosphoinositide signaling pathway (25). This suggests that the
GnRH receptor in GnRH neurons can regulate cAMP production by coupling
to Gs, as well as through activation of
Ca2+-stimulated AC.
The inhibition of cAMP production by high nanomolar and micromolar GnRH
concentrations and its reversal by PTX indicate that the GnRH receptors
expressed in GnRH neurons are also coupled to inhibitory
Gi/o proteins. GnRH receptors have also been
reported to couple to Gi in human reproductive
tract tumors (52, 53, 54). The inhibition of cAMP production by high GnRH
concentrations was prevented by nifedipine-induced blockade of VSCC and
was potentiated by increased
[Ca2+]e. These findings
indicate that high GnRH concentrations activate PTX-sensitive G
protein(s) that in turn inhibit types I, V, and VI ACs, causing a
decrease in cAMP production (55, 56). High concentrations of GnRH
appear to activate Gi to an extent that abrogates
the stimulatory action of Gs and
Ca2+ entry on cAMP production.
Activation of the endogenous LH receptor expressed in GnRH neurons (9, 57) causes a dose- dependent increase in cAMP production. The
stimulatory action of LH/hCG on cAMP production is highly
Ca2+-dependent and is significantly reduced in
Ca2+-free medium and by treatment with
nifedipine. In contrast, increased
[Ca2+]e and treatment
with BK-8644 significantly increased cAMP production. These results
suggest that Ca2+ entry through VSCC acts as a
conditioning factor to increase the catalytic activity of
Gs
-activated AC in GnRH neurons. Thus, VSCC-
dependent Ca2+ influx in spontaneously active
GnRH neurons provides a pathway that links Ca2+
and the cAMP signaling system triggered by activation of
Gs-coupled LH receptors. The stimulatory action
of hCG is dually regulated by concomitant activation of GnRH receptors.
The synergistic effect of hCG and low nanomolar GnRH concentrations in
GnRH neurons suggests that convergent signaling from LH and GnRH
receptors acts cooperatively on Gs to stimulate
AC activity. In contrast, higher degrees of agonist activation of the
GnRH receptor attenuate the stimulatory action of hCG by activation of
PTX-sensitive Gi/o proteins.
These observations indicate that the firing of spontaneous action
potentials in GnRH neurons causes increased
Ca2+ influx and activation of
Ca2+-stimulated AC to maintain basal cAMP
production. In addition, convergent signaling from LH/hCG and GnRH
receptors via Gs acts in conjunction with
Ca2+ influx to stimulate cAMP production. The
ability of micromolar GnRH concentrations to terminate hCG-stimulated
cAMP production in a PTX-sensitive manner indicates that the GnRH
receptor also couples to Gi/o proteins and
inhibits Gs-activated AC and cAMP production.
It is likely that this mechanism is mediated by AC I, which is
stimulated by Ca2+ entry and is also regulated by
receptor-dependent activation of Gs and
Gi. Our findings suggest that changes in GnRH
neuronal cAMP production are largely secondary to alterations in cell
excitability and Ca2+ influx. This process, in
conjunction with autocrine inhibitory actions of GnRH itself on cAMP
production, could contribute to the pulsatile neuropeptide secretion
that is characteristic of GnRH neuronal function in vivo and
in vitro.
The nature of the latter process has not yet been clarified, but may
involve more than one mechanism to provide redundancy to this essential
component of the hypothalamic control of pituitary secretion. For
example, Vitalis et al. (11) have proposed that
phosphorylation and inhibition of AC V and/or AC VI by protein kinase A
(PKA) cause oscillations in cAMP production that in turn activate
cyclic nucleotide-gated cation channels, which increase cell
excitability and promote GnRH secretion. Also, Sakakibara et
al.(58) have proposed another negative feedback limb that is
dependent on the ability of cAMP-induced activation of PKA to inhibit
phosphodiesterase activity in GT1 cells. The extent to which each of
the currently proposed mechanisms contributes to the operation of the
GnRH pulse generator, and the degree to which this depends on the
prevailing steroid milieu and other factors, has yet to be
determined.
 |
MATERIALS AND METHODS
|
---|
Tissue and Cell Culture
Hypothalamic tissue was removed from fetuses of 17-day pregnant
Sprague Dawley rats (Taconic Farms, Inc., Germantown,
NY). The borders of the excised hypothalami were delineated by
the anterior margin of the optic chiasm, the posterior margin of the
mammilary bodies, and laterally by the hypothalamic sulci. After
dissection, hypothalami were placed in ice-cold dissociation buffer
containing 137 mM NaCl, 5 mM KCl, 0.7
mM Na2HPO4, 26
mM HEPES, and 100 mg/liter gentamicin, pH 7.4. The tissues
were washed and then incubated in a sterile flask with dissociation
buffer supplemented with 0.2% collagenase, 0.4% BSA, 0.2% glucose,
and 0.05% DNase I. After 60 min incubation in a 37 C water bath with
shaking at 60 cycles/min, the tissue was gently triturated by repeated
aspiration into a smooth-tipped Pasteur pipette. Incubation was
continued for another 30 min, after which the tissue was again
dispersed. The cell suspension was passed through sterile mesh (200
µm) into a 50-ml tube, sedimented by centrifugation for 10 min at
200 x g, and washed once in dissociation buffer and
once in culture medium consisting of 500 ml DMEM containing 0.584
g/liter L-glutamate and 4.5 g/liter glucose,
mixed with 500 ml F-12 medium containing 0.146 g/liter
L- glutamine, 1.8 g/liter glucose, 100
µg/ml gentamicin, 2.5 g/liter sodium bicarbonate, and 10%
heat-inactivated FCS. Each dispersed hypothalamus yielded about
1.5 x 106 cells. Immortalized GnRH neurons
(GT17 neurons) obtained from Dr. R. I. Weiner (University of
California at San Francisco) (59) were cultured under the same
conditions as primary hypothalamic cells.
Measurement of cAMP Production
For studies on cAMP release, GnRH-producing cells were
stimulated in serum-free medium (1:1 DMEM/F-12) containing 0.1% BSA,
30 mg/liter bacitracin, and 1 mM IBMX. For measurement of
cAMP production in GT17 cell membrane preparations, cells were washed
twice with TE buffer (10 mM Tris-HCl, 1 mM EDTA
, 10 µg/ml aprotinin, 10 µg/ml leupeptin, pH 7.4), removed from the
plates by scraping, and lysed by freeze thawing. After centrifugation
at 12,000 x g for 15 min at 4 C, the pellet was
resuspended in TE buffer and stored at 70 C until assayed. Protein
contents were measured by the BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Aliquots (25 µg) of membrane
proteins were incubated with 20 mM Tris/HCl, 10
mM MgCl2, 100
µM ATP, 20 mM creatine
phosphate, 10 U/ml creatine phosphokinase, selected concentrations of
Ca2+, at 32 C for 5 min. RIA of cAMP was
performed as previously described (60), using a specific cAMP antiserum
at a titer of 1:5,000. This assay showed no cross-reaction with cGMP,
2',3'-cAMP, ADP, GDP, CTP, or IBMX. cAMP production is expressed as
picomoles/mg protein/5 min.
Measurement of Calcium Ion Concentration
For single-cell
[Ca2+]i measurements,
cultures were incubated at 37 C for 60 min with 2 µM
fura-2/AM in phenol red-free DMEM. The cells were subsequently washed
with Krebs-Ringer solution containing 1.2 mM
Ca2+ and kept for at least 30 min in this medium
before measurements. All experiments were performed in cells bathed in
Krebs-Ringer solution containing 1.2 mM
Ca2+ at room temperature. Coverslips with cells
were mounted on the stage of an Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital
Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells
were examined under a 40x oil immersion objective during exposure to
alternating 340- and 380-nm light beams, and the intensity of
light emission at 520 nm was measured. All
[Ca2+]i values were
derived from a standard curve that was constructed by addition of known
concentrations of Ca2+ to 2 µM
fura-2.
Immunoblot Analysis of Membrane-Associated G Proteins and AC
Isoforms
For immunoblot analysis, cells were washed twice with TE buffer
(10 mM Tris-HCl, 1 mM EDTA, pH 7.4), scraped
from the plates, and lysed by freeze thawing. After centrifugation at
12,000 x g for 15 min at 4 C, the pellets were
resuspended in TE buffer and stored at 70 C. Protein contents were
measured by the Pierce Chemical Co. BCA protein assay kit.
SDS-gel electrophoresis was performed on 8% acrylamide gels, followed
by blotting with polyvinyldifluoridine (PVDF) membrane of
0.45-µm pore size. The blots were incubated with first antibody
(1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
or first antibody preadsorbed with the corresponding peptide antigen
(1:1000 + 10 µg peptide), followed by peroxidase-coupled
goat-antirabbit IgG (H+L), and visualized by chemiluminescence
(Life Technologies, Inc., Gaithersburg, MD). The
immunoreactive bends were analyzed as three-dimensional digitized
images using a GS-700 Imaging Densitonometer (Bio-Rad Laboratories, Inc. Hercules, CA). The optical density (OD) of
images is expressed as volume (OD x area) adjusted for the
background, which gives arbitrary units of adjusted volume (Adj.
Volume).
Materials
Western blotting reagents and ECL were obtained from
Amersham Pharmacia Biotech, Arlington Heights, IL;
collagenase (149 U/mg) was from Worthington Biochemical Corp., Freehold, NJ; DNase I, trypsin, bacitracin, IBMX, CTP,
GDP, guanosine 3',5'-cyclic monophosphate (cGMP), adenosine
2',3'-cyclic monophosphate (2',3'-cAMP), and BSA were from
Sigma (St. Louis, MO); GnRH was from Peninsula Laboratories, Inc. (Belmont, CA);
125I-cAMP was from Covance Laboratories, Inc. (Vienna, VA); protein assay kits were from Pierce Chemical Co., and Membrane Immobilon-P was from Millipore Corp. (Bedford, MA). Peroxidase-coupled goat- antirabbit IgG
(L+H), FBS, and DMEM/F12 1:1 powder were from Life Technologies, Inc.. Antibodies to AC I, AC III, and AC V/VI, were purchased
from Santa Cruz Biotechnology, Inc., as well as the
corresponding peptide antigens. Antibodies to
G
q/11,
G
s,
G
i1,
G
i2, and
G
i3 were purchased from
Calbichem-Novabiochem Corp. (San Diego, CA), as well as
the corresponding standard peptides. Other reagents, if not specified,
were obtained from Sigma.
Data Analysis
All results are expressed as mean ± SEM, and
statistical comparisons were performed using the Kruskal-Wallis test
followed by the Mann-Whitney U test. A difference between
groups was considered to be significant if the P value
obtained from the Mann-Whitney U test was less then 0.05.
The calculations were performed on a Macintosh power PC using the
Statistica/Mac 4.1 (StatSoft, Tulsa, OK).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Kevin J. Catt, M.D., Ph.D., Endocrinology and Reproduction Research Branch, Building 49, Room 6A-36, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510. E-mail:
catt{at}helix.nih.gov
Received for publication July 19, 2000.
Revision received December 1, 2000.
Accepted for publication December 4, 2000.
 |
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