Effect of Ca2+ and cAMP on
capacitance-measured hormone secretion in human GH-secreting
adenoma cells
Tsukasa
Takei,
Junko
Yasufuku-Takano,
Koji
Takano,
Toshiro
Fujita, and
Naohide
Yamashita
Fourth Department of Internal Medicine, Tokyo University Branch
Hospital, Tokyo 112, Japan
 |
ABSTRACT |
Membrane capacitance
(Cm) was
measured as an index of exocytosis in human growth hormone-secreting
adenoma cells using the perforated whole cell, patch-clamp technique;
the effects of membrane depolarization, growth hormone-releasing
hormone, and 8-bromoadenosine 3',5'-cyclic monophosphate
(8-BrcAMP) were examined.
Cm was increased by membrane depolarization to potentials beyond the threshold necessary
to open voltage-gated Ca2+
channels. These voltage-dependent changes in
Cm varied as a
function of both depolarization amplitude and duration and were blocked in the presence of the Ca2+
channel antagonist nitrendipine
(10
6 M). When membrane
potential was clamped at the holding potential (
78 mV),
voltage-gated Ca2+ channels were
closed, and neither application of growth hormone-releasing hormone nor
8-BrcAMP affected
Cm. However, when
these agents were applied to depolarized cells, where the voltage-gated
Ca2+ channels were open, the
increases in Cm
were augmented. From these data, it was concluded that elevation of
intracellular cAMP, per se, did not stimulate exocytosis. Rather,
Ca2+ influx through voltage-gated
channels was a prerequisite for cAMP-induced exocytosis.
growth hormone-releasing hormone; 8-bromoadenosine
3',5'-cyclic monophosphate; nitrendipine
 |
INTRODUCTION |
SECRETION OF GROWTH HORMONE (GH) from the anterior
pituitary gland is stimulated by the hypothalamic peptide GH-releasing hormone (GHRH) (28). GHRH depolarizes the plasma membranes of GH-secreting cells by activating nonselective cation channels (5, 15,
19, 25) and by enlarging voltage-gated
Ca2+ channel currents (4, 19, 26).
Both of these changes of ion channel behavior are mediated by increases
in the levels of the intracellular second messenger, cAMP (2). It is
known that the membrane depolarization and augmented
Ca2+ currents elicited by GHRH
facilitate Ca2+ influx, thereby
stimulating GH secretion. However, the precise functional roles played
by Ca2+ and cAMP during secretion
of GH have not yet been clarified because it has proven difficult to
selectively resolve their respective actions. In pancreatic
-cells
and lactotrophs, direct action of cAMP on exocytosis has been reported
(1, 8, 24). However, it remains unclear whether this cAMP-induced
exocytosis is exclusively independent of
Ca2+.
In the present study, we examined the respective effects of
Ca2+ and cAMP on GH secretion in
human GH-secreting adenoma cells. To accomplish this, membrane
capacitance
(Cm) was
measured as an index of exocytosis (17). We used the perforated whole
cell clamp technique to record
Cm (9, 11)
because it eliminates problems associated with washout of intracellular
substrates. In addition, evoked changes in
Cm differ
depending on whether they are recorded with the use of perforated patch
or conventional whole cell clamp techniques (22); for measurement of
exocytosis under the physiological conditions, perforated whole cell
clamp is preferable. Using this technique, we were able to clarify the respective roles of Ca2+ influx
and cAMP in the secretion of GH from single cells.
 |
MATERIALS AND METHODS |
Cell preparation and culture.
GH-secreting pituitary adenomas were obtained from three acromegalic
patients by transsphenoidal surgery. The Ethical Committee of Tokyo
University School of Medicine permits the use of human pituitary tissue
obtained at the surgery for experimental purposes. The adenomas were
minced into small pieces (<1 mm) and digested with 1,000 U/ml Dispase
(protease). Cells to be used in the analysis of hormone
release were seeded into 24-well dishes at a density of 1 × 105 cells/dish, and cells to be
used in electrophysiological experiments were seeded into 35-mm culture
dishes. The cells were cultured in DMEM containing 10%
heat-inactivated FCS without antibiotics and were maintained at
37°C under an atmosphere of humidified air containing 5%
CO2. Hormone release studies were
carried out after 1-2 wk of culture, and the electrophysiological
studies were performed after 1-4 wk of culture.
Electrophysiological properties and capacitance-measured exocytosis did
not change during this period.
Measurement of GH release.
Cells cultured in 24-well dishes were washed twice with serum-free DMEM
containing 0.1% BSA. They were then incubated in the serum-free medium
for 2 h with or without GHRH. After incubation, the medium was
collected and stored at
20°C until it was assayed for GH
with the use of an RIA kit (Daiichi Radioisotope Laboratories, Tokyo,
Japan). The sensitivity of the assay was 0.1 ng/ml, and the intra- and
interassay coefficients of variance were 1.1 and 2.1%, respectively.
Basal levels of GH release were 255.8 ± 46.3 ng/ml in
adenoma 1, 156.0 ± 43.9 ng/ml in
adenoma 2, and 20.9 ± 2.8 ng/ml in
adenoma 3 (mean ± SD,
n = 4). In the presence of 10
8 M GHRH, release
increased to 381.5 ± 39.4 ng/ml in adenoma
1, 791.3 ± 143.4 ng/ml in adenoma
2, and 27.6 ± 3.4 ng/ml in adenoma 3. These increases in GH release were statistically
significant when analyzed using Student's
t-test
(P < 0.05).
Perforated patch, whole cell clamp
technique.
All experiments were performed at room temperature (22-25°C)
while the cells were being continuously superfused with the use of a
peristaltic pump (~1.5 ml/min). The various agents employed in the
study were applied to the cells by changing the superfusing solution.
Cm was analyzed
using the perforated whole cell clamp technique (11); details of the
technique have been reported elsewhere (29). Briefly, patch electrodes
were prepared from 1.5-mm glass capillaries containing filaments. When
filled with solution, the resistance of the patch electrodes was
5-8 M
. A List EPC-7 amplifier was used for recording membrane
currents and potentials. Liquid junction potentials between the
extracellular and pipette solutions (
8 to
6 mV) were
measured using a 3 M KCl electrode as a reference, and all data were
corrected accordingly. When whole cell perforated patch-clamp
measurements were made, the patches were perforated using nystatin.
Shortly before beginning to record, aliquots of nystatin stock solution
(50 mg/ml DMSO), which was prepared daily, were diluted in the pipette
solution to a final concentration of 200 µg/ml. Voltage clamp
recordings were begun after the series resistance fell below 20 M
.
Because the amplitudes of the recorded currents were <150 pA, errors
caused by the series resistance were ignored. Data were filtered with a
bandwidth of 1 kHz, and the sampling frequency was 100 µs.
Measurement of Cm.
The methods used for measuring
Cm were
essentially the same as described by Maruyama (18).
Cm was measured
using a two-phase lock-in-amplifier (NF5610B, NF Instruments, Yokohama,
Japan). Initially, command pulses were applied to voltage-clamped cells from a holding potential of
78 mV, and the transient capacitive surges were offset using the cancellation circuit of the EPC-7 amplifier. Then a 200-Hz, 10-mV peak-to-peak sine wave voltage was
superimposed on the holding potential. The resultant current output was
fed into the lock-in-amplifier. The phase offset of the
lock-in-amplifier was adjusted such that when a capacitance calibration
of 1 pF was applied, there was no detectable change in 0-phase output
(membrane conductance:
G) but
maximal changes in
/2-phase output (membrane capacitance:
C). Calibrations for
C and
G were obtained by applying known
C and
G signals and rescaling them to fit
the
C and
G scales, respectively. The total
cell Cm of human
GH-secreting adenoma cells ranged from 6 to 8 pF. During application of
GHRH or 8-bromoadenosine 3',5'-cyclic monophosphate
(8-BrcAMP), the depolarized membrane potential was fixed at
18
or
28 mV; representative changes of
C and
G are shown (see Figs. 2, 4,
6-8).
Cell identification.
In early experiments, GH staining was used to identify GH-secreting
adenoma cells. Cells (n = 4) that had
membranes depolarized by application of GHRH (25) were fixed in 10%
formaldehyde and immunohistochemically stained for human GH (hGH) using
an hGH immunostaining kit (DACO, Glosprup, Denmark), which uses the
rabbit polyclonal antibodies to hGH. All of the GHRH-sensitive cells stained positive for hGH. In subsequent experiments, data were obtained
from cells that were presumed to be GH-secreting adenoma cells. Under a
phase-contrast microscope, these cells were rounded, had glittering
surfaces, and were easily discriminated from fibroblast-like cells.
Solutions and reagents.
The standard internal pipette solution contained (in mM) 95 potassium
aspartate, 47.5 KCl, 1 MgCl2, 0.1 EGTA [tetramethylammonium (TMA) salt], and 10 HEPES (TMA
salt, pH 7.2). The standard external solution was (in mM) 128 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, and 10 HEPES (Na salt, pH 7.4).
When Ca2+ channel currents were
recorded, K+ in the pipette
solution was isosmotically replaced with
Cs+, and 25 mM
Na+ in the extracellular medium was
isosmotically replaced with tetraethylammonium ion. Tetrodotoxin
(10
7 M) was also present in
the extracellular medium.
Human GHRH, 8-BrcAMP, and nystatin were purchased from Sigma (St.
Louis, MO). Dispase was purchased from Godo Shusei (Tokyo, Japan).
 |
RESULTS |
Changes in Cm caused by
Ca2+ influx
through voltage-gated channels.
Figure 1,
A and
B, depicts voltage-gated
Ca2+ channel currents recorded in
the standard extracellular medium containing 2.5 mM
Ca2+ ions. To block outward
K+ currents, cells were dialyzed
with Cs+ from the patch
electrodes, and 25 mM tetraethylammonium ion was present in the
extracellular solution. The Ca2+
currents were carried through T- and L-type
Ca2+ channels, which is consistent
with earlier recordings made using Ba2+ ions as the charge carrier
(30). The current-voltage relationships are shown in Fig.
1C. The threshold potential for T-type
Ca2+ channels was approximately
50 mV, and for L-type Ca2+
channels, the threshold was shifted by ~10 mV toward more depolarized potentials.

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Fig. 1.
Ca2+ currents in human growth
hormone (GH)-secreting adenoma cells.
A and
B:
Ca2+ currents elicited by
depolarizing membranes to 36 mV
(A) and 16 mV
(B) from a holding potential of
76 mV. Patch electrode contained 142.5 mM
Cs+, and extracellular solution
contained 25 mM tetraethylammonium ion.
C: current-voltage relationships for
T- and L-type Ca2+ currents.
Amplitudes of L-type currents were measured as amplitude of steady
current, and amplitudes of T-type currents were estimated by
subtracting L-type current amplitude from peak amplitude of total
current. Depicted currents were recorded in a cell from
adenoma 2.
|
|
When cell membranes were depolarized beyond the threshold potential for
periods of 10 s, obvious increases in
Cm were observed (Fig. 2). These changes in
Cm were dependent
on the magnitudes of the membrane depolarizations (Figs. 2 and
3); in Fig. 3, the maximal increases in
Cm, measured in
five representative cells, are plotted as a function of membrane
potential. As can be seen, the increments in
Cm appeared to
correspond to the voltage-dependent changes in the
Ca2+ currents (Figs.
1C and 3). After termination of the
membrane depolarization,
Cm declined, but
the rate did not appear to be potential dependent. When the membrane
was hyperpolarized from the holding potential of
78 mV, no
apparent changes in
Cm were seen
(data not shown).

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Fig. 2.
Effect of membrane depolarization on membrane capacitance. Holding
potential was 78 mV, and depolarizing pulse duration was 10 s. Membrane potentials (V) are shown
in bottom trace; time-dependent
changes in membrane capacitance ( C;
top) and conductance
( G;
middle) are also shown. Broken line
indicates basal conductance. Intracellular and extracellular media were
standard. Cell was from adenoma 1.
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Fig. 3.
Relationship between membrane depolarization and exocytosis. Amplitude
of maximum increase in membrane capacitance (ordinate) is plotted as a
function of membrane potential (abscissa). Means ± SD of
capacitance increases measured in 5 representative cells are shown; in
some data, bars are small and hidden by symbols. Cells were from
adenomas 1-3
(n = 2, adenoma
1; n = 2, adenoma 2; and
n = 1, adenoma
3).
|
|
It was observed that the changes in
Cm were also
dependent on the duration of the membrane depolarization (Figs.
4 and 5). When the representative cell
in Fig. 4 was depolarized to
18 mV from a holding potential of
78 mV, for periods of 1, 5 or 10 s, the amplitudes of the
changes in Cm
increased as the depolarization became more prolonged. The maximum
amplitudes of the increases in
Cm in four cells
are plotted as a function of depolarizing pulse duration in Fig. 5.
As the pulse duration became longer, the amplitudes of
Cm increased,
until they saturated at a pulse duration of 30 s.

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Fig. 4.
Effect of pulse duration of membrane depolarization on increases in
membrane capacitance. Holding potential was 78 mV, and membranes
were depolarized to 18 mV. Duration of depolarizing potentials
(V) is indicated in
bottom trace;
C
(top) and
G
(middle) are also shown. Broken line
indicates basal level of C.
Intracellular and extracellular media were standard. Cell was from
adenoma 1.
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Fig. 5.
Relationship between duration of membrane depolarization and changes in
membrane capacitance. Amplitude of maximum increase in membrane
capacitance (ordinate) is plotted as a function of time (abscissa).
Means ± SD of capacitance increases measured in 4 representative
cells are shown; in some data, bars are small and hidden by symbols.
Cells were from adenoma 2.
|
|
These data indicate that voltage-dependent
Ca2+ influxes were important
for the increases in
Cm observed in
human GH-secreting adenoma cells. To further explore this notion, we
examined the effect of a Ca2+
channel blocker, nitrendipine, on depolarization-induced changes in
Cm; it has
already been reported that nitrendipine inhibits voltage-gated
Ca2+ currents in human
GH-secreting adenoma cells (30). Figure 6 shows that in
the presence of 10
6 M
nitrendipine, membrane depolarization did not increase the Cm. Thus
Ca2+ influx through voltage-gated
channel is important for exocytosis in human GH-secreting adenoma
cells. This experiment also indicates that membrane depolarization does
not, per se, evoke exocytosis.

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Fig. 6.
Effect of nitrendipine on membrane depolarization-induced increases in
membrane capacitance. Nitrendipine
(10 6 M) was added to
extracellular medium. Holding potential was 78 mV, and
depolarized potential was 18 mV.
C and
V are shown in
top and
bottom traces, respectively. Cell was
from adenoma 2.
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Effect of GHRH and cAMP on the
Cm increase.
Figure 7A shows the
changes of the Cm
elicited by application of
10
8 M GHRH. When applied
while membrane potential was held at
78 mV, a condition under
which voltage-gated Ca2+ channels
should be closed (Fig. 1C), GHRH had
no effect on Cm. However, after the cell membrane was depolarized to
18 mV, an increase in the
Cm was observed,
similar to the case of Figs. 2 and 4. To separate the respective
effects of GHRH and depolarization, GHRH was applied after the membrane
had been depolarized to
18 mV and
Cm had plateaued
(Fig. 7B). Under these conditions,
GHRH elicited further increases in
Cm.

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Fig. 7.
Effect of GH-releasing hormone (GHRH) on membrane capacitance.
A: GHRH
(10 8 M) was applied at
holding potential of 78 mV. B:
GHRH (10 8 M) was applied
after membrane was depolarized to 18 mV from holding potential.
C and
V are shown in
top and
bottom traces, respectively. Broken
lines indicate basal capacitance before membrane depolarization or
application of GHRH. Intracellular and extracellular media were
standard. Cells were from adenoma 2.
|
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The biological actions of GHRH are mediated by cAMP (2). Therefore, we
next examined the effect of cAMP on the
Cm. Figure 8 shows the changes in
Cm elicited by
application of the membrane-permeable cAMP analog 8-BrcAMP
(10
5 M). At the holding
potential of
78 mV,
Cm was unaffected
by 8-BrcAMP (Fig. 8A). On the other
hand, after depolarization to
28 mV, 8-BrcAMP elicited a slow
rise in Cm that
was similar to that seen in the presence of GHRH (Fig.
8B). From these results, it was
concluded that GHRH-induced exocytosis was mediated by a rise in
intracellular cAMP and that GHRH (cAMP) did not stimulate exocytosis
when voltage-gated Ca2+ channels
were not activated. Therefore,
Ca2+ influx through voltage-gated
channels appeared to be a prerequisite for GHRH (cAMP)-induced
increases in
Cm.

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Fig. 8.
Effect of 8-bromoadenosine 3',5'-cyclic monophosphate
(8-BrcAMP) on membrane capacitance. A:
8-BrcAMP (10 5 M) was
applied at holding potential of 78 mV.
B: 8-BrcAMP
(10 5 M) was applied after
membrane was depolarized to 28 mV from holding potential.
C and
V are shown in
top and
bottom trances, respectively.
Intracellular and extracellular media were standard. Cells were from
adenoma 2.
|
|
 |
DISCUSSION |
The results of the present experiments revealed that
Ca2+ influx through voltage-gated
Ca2+ channels was essential for
capacitance-measured exocytosis in human GH-secreting adenoma cells.
The membrane depolarization, which activated voltage-gated
Ca2+ channels, stimulated
exocytosis, and the application of the
Ca2+ channel blocker nitrendipine
inhibited it. In human GH-secreting adenoma cells, we have reported
that nitrendipine inhibited GH secretion, as measured by RIA (30), and
that it also inhibited GHRH-induced increases in the concentration of
intracellular Ca2+
([Ca2+]i)
(26). The results of the present study are in agreement with these
earlier results.
The amplitudes of the changes in
Cm increased with
the amplitudes of the evoked Ca2+
currents (Figs. 1C and 3). This
suggests that the amount of exocytosis increased in parallel with the
increment in Ca2+ influx through
voltage-gated channels. These data are consistent with observations
made in bovine adrenal chromaffin cells, in which exocytosis was also
measured with the use of the perforated whole cell clamp technique (7).
In that study, the amount of exocytosis was strictly related to the
integral of the voltage-gated Ca2+
current. However, exocytosis tended to saturate when the duration of
the membrane depolarization was longer than 30 s (Fig. 5). It has been
known that there are two kinds of secretory vesicles, large dense-core
and small clearer vesicles; hormone-containing granules in endocrine
cells are among the large dense-core vesicles. The dependence of
exocytosis on
[Ca2+]i
is different between large dense-core vesicles and small clearer vesicles (13). Exocytosis is comparatively slow for large dense-core vesicles and requires relatively low
[Ca2+]i
compared with the small clearer vesicles. It is probable that the
increase of
[Ca2+]i
caused by longer depolarizations was sufficient to saturate the
exocytosis process in GH-secreting adenoma cells. A similar case in
which prolonged membrane depolarizations saturated exocytosis was
observed in rat posterior pituitary cells (12). In GH-secreting adenoma
cells, there are two types of voltage-gated
Ca2+ channels, T type and L type.
Because T-type channels inactivate within 200 ms, L-type channels
account for exocytosis in the case of prolonged membrane
depolarization. In fact, nitrendipine more effectively inhibits L-type
channels (30).
Application of GHRH and 8-BrcAMP did not increase
Cm when membrane
potentials were more hyperpolarized than the threshold for opening
voltage-gated Ca2+ channels.
However, these agents augmented depolarization-induced increases in
Cm when
voltage-gated Ca2+ channels were
activated. This indicates that elevation of intracellular cAMP does
not, by itself, stimulate exocytosis. Instead, cAMP only exerts its
stimulative effect in conjunction with a
Ca2+ influx through voltage-gated
channels. These results are in agreement with the report that
extracellular Ca2+ ions are
essential for GHRH-induced GH release in rat anterior pituitaries (2,
14).
A brief membrane depolarization caused an increase in
Cm that gradually
declined after repolarization (Fig. 2). The time course of the decrease
in the Cm
fluctuated and did not depend on the magnitude of the brief
depolarization. Similar fluctuations in Cm were also
observed during prolonged membrane depolarizations (Fig. 7). It has
been reported that endocytosis concomitantly occurs during exocytosis
and that endocytosis was also regulated by
[Ca2+]i
(3, 6, 10, 20, 27). Although the mechanisms of endocytosis in human
GH-secreting adenoma cells were not examined in the present experiment,
it was surmised that the observed fluctuations in
Cm were caused by
endocytosis.
Previous studies using rat or human GH-secreting cells revealed that
GHRH activates nonselective cation channels and, thereby, depolarizes
the plasma membrane (5, 15, 19, 25); this membrane depolarization
increases the firing of
Ca2+-dependent action potentials.
GHRH also enhances voltage-gated Ca2+ channel currents (4, 19, 26),
facilitating Ca2+ influx and
increasing
[Ca2+]i.
Although increases in
[Ca2+]i
are implicated in hormone secretion from anterior pituitaries (21), the
role of cAMP has not been fully elucidated in somatotrophs. In
pancreatic
-cells, cAMP enhances
Ca2+ influx through voltage-gated
Ca2+ channels and increases
[Ca2+]i
in a manner similar to that seen in human GH-secreting adenoma cells
(16). This cAMP-induced increase in
[Ca2+]i
promoted exocytosis, but in
-cells, cAMP may also stimulate exocytosis that is independent of
[Ca2+]i
(1, 8). It was suggested that cAMP may directly affect the mechanism of
insulin granule mobilization. However,
[Ca2+]i-independent
insulin release caused by cAMP disappeared when [Ca2+]i
was below 60 nM (23). Similarly, it was suggested that cAMP directly
facilitates exocytosis in bovine lactotrophs, but this effect was
inhibited when
[Ca2+]i
was chelated with 10 mM EGTA (24). In spontaneously firing human
GH-secreting adenoma cells,
[Ca2+]i
ranged from 150 to 225 nM (26). When
Ca2+ influx through voltage-gated
channels was inhibited, it decreased below this range, suggesting that
cAMP-evoked exocytosis required [Ca2+]i
>150 nM in human GH-secreting adenoma cells. Thus it appears that
physiological levels of
[Ca2+]i
are required for cAMP-induced exocytosis, but the level of [Ca2+]i
necessary for exocytosis likely differs among cell types. In the case
of human GH-secreting adenoma cells, it may be necessary for
[Ca2+]i
to be higher than in pancreatic
-cells and bovine lactotrophs.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests: N. Yamashita, Dept. of Advanced
Medical Science, Institute of Medical Science, Univ. of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Received 29 April 1998; accepted in final form 2 July 1998.
 |
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