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The Gonadotropin-Releasing Hormone Associated Peptide Reduces Calcium

Entry in Prolactin-Secreting Cells*

Article  in  Endocrinology · February 1991

DOI: 10.1210/endo-128-1-285 · Source: PubMed

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0013-7227/91/1281-0285$02.00/0
Endocrinology
Copyright© 1991 by The Endocrine Society
The Gonadotropin-Releasing Hormone Associated
Peptide Reduces Calcium Entry in Prolactin-Secreting
Cells*
P. VACHER, P. MARIOT, L. DUFY-BARBE, K. NIKOLICS, P. H. SEEBURG,
B. KERDELHUE, AND B. DUFY
Laboratoire de Neurophysiologie, CNRS UA 1200, Uniuersite de Bordeaux II, France; Department of
Developmental Biology (K.N.), Genentech Inc., South San Francisco California, Laboratory of Molecular
Neuroendocrinology (P.H.S.), Zentrum fiir Molekulare Biologie, Universitdt Heidelberg, Heidelberg, Federal
Republic of Germany; and Laboratoire de Neurobiologie de la Reproduction INRA (B.K.), Jouy en Josas,
France
ABSTRACT. The precursor molecule to the GnRH contains
a peptide named GnRH-associated peptide (GAP) with PRL-
inhibiting properties. In this work, we have studied the electro-
physiological properties and responses to GAP of three different
types of PRL-secreting cells: 1) the rat tumor cell line GH3, 2)
normal rat pituitary cells in primary culture, and 3) human
PRL-secreting adenoma cells. Using different but complemen-
T HE decapeptide GnRH, released by hypothalamic
neurosecretory cells, controls mammalian reproduc-
tion through its direct action at the pituitary level. Mo-
lecular cloning has helped to elucidate the biosynthesis
and structure of a precursor to GnRH (1). This precursor
contains a signal sequence, the GnRH peptide with a
dibasic precursor processing site and a 56-amino acid
long peptide named GnRH-associated peptide (GAP).
GAP has been located by immunocytochemistry in rat
hypothalamic cell bodies containing GnRH and in nerve
terminal vesicles of the rat median eminence (2, 3). It is
cosecreted with GnRH into the hypophyseal portal blood
of ovariectomized sheep (4).
Several lines of evidence point to a physiological role
of GAP and/or fragments of GAP in the inhibition of
PRL secretion: GAP inhibited PRL release in vitro (5,
6). Elevated but not resting PRL levels were inhibited
by GAP in rats in vivo (7,8). Generation of gonadotropin-
releasing and PRL-inhibiting activities from the same
Received September 20, 1990.
Address correspondence and requests for reprints to: B. Dufy, La-
boratoire de Neurophysiologie, CNRS UA 1200, Universite de Bordeaux
II, Bordeaux Cedex 33076, France.
* This work was supported by grants from the Centre National de
la Recherche Scientifique (URA-1200), the Institut National de la
Sante et de la Recherche Medicale (87-4003) the Fondation pour la
Recherche Medicale, the Ligue Nationale de la Recherche contre le
Cancer (France).
285
Vol. 128, No. 1
Printed in U.S.A.
tary techniques we show that GAP reduces intracellular Ca++
levels, [Ca++]i, and inhibits Ca++ transients in these cells. This
reduction of [Ca++]i results from coordinate actions of GAP on
K+ and Ca++ conductances and may explain the inhibitory effect
of GAP on hormonal secretion by PRL-secreting cells. {Endo-
crinology 128: 285-294, 1991)
precursor protein may account for the known inverse
relationships of gonadotropin and PRL levels during
certain physiological states of reproduction and lactation
(9-11). GAP may represent a postulated peptidic PRL
release-inhibiting factor (10, 12). However, the precise
physiological role of GAP, and its mechanism of action
are still not clearly understood. There are reports that
in sheep GAP did not have any influence on PRL release
(13). The current study was undertaken to understand
whether or not GAP had an effect on normal and trans-
formed PRL-secreting cells at the level of subtle mech-
anistic changes.
A number of recent studies have demonstrated that
both normal and neoplastic pituitary cells are electrically
excitable and can fire Ca++-mediated action potentials
(14-16). The Ca++ entry associated with these action
potentials plays a critical role in regulating intracellular
Ca++ levels (17) and secretory processes (18, 19). Since
Ca++ is required for secretion, inhibition, or activation
of membrane ionic conductances resulting in modulation
of action potentials are possible mechanisms whereby
secretagogues could inhibit or stimulate hormone release
in pituitary cells (17,19). Indeed, the inhibitory effect of
dopamine is thought to result from the suppression of
action potential firing (20). We have recently shown that
another inhibitor of the PRL release, SRIF, blocks Ca++
286 GAP AND MEMBRANE ION CONDUCTANCES
action potential activity in PRL-secreting pituitary tu-
mor cells (21).
In this work, we have studied the electrophysiological
properties and responses to GAP of three different types
of PRL secreting cells: 1) the rat tumor cell line GH3, 2)
normal rat pituitary cells in primary culture, and 3)
human PRL (hPRL)-secreting adenoma cells. Using dif-
ferent but complementary techniques we show that GAP
reduces intracellular Ca++ levels and inhibits Ca++ tran-
sients in these cells. This reduction of [Ca++]i results
from coordinate actions of GAP on K+ and Ca++ con-
ductances and may explain the inhibitory effect of GAP
on hormonal secretion by PRL-secreting cells.
Materials and Methods
Pituitary cell culture
The material for this study consisted of:
GH3/B6 cells. A sub-clone of the GH3 cell line was originally
obtained from Dr. A. Tixier-Vidal (College de France, Paris,
France) and stored frozen until used or passaged. Cells were
cultured in HAM's F-10 nutrient medium supplemented with
15% heat-inactivated horse serum (IBF, Villeneuve-la-Gar-
enne, France) and 2.5% fetal bovine serum (FBS, GIBCO,
Grand Island, NY), and maintained at 37 C in a humidified
atmosphere gassed with 95% air-5% CO2. Electrophysiological
recordings were conducted 3-7 days after replating. Medium
was changed every 2-3 days.
hPRL-Secreting pituitary adenoma cells. These cells were cul-
tured as described previously (21). In brief, tumor fragments
from two patients were obtained surgically during transphen-
oidal removal of clinically identified PRL-secreting microad-
enomas. The tissue was minced and digested with 0.2% colla-
genase and 0.05% trypsin in a Ca++- and Mg++-free Hank's
balanced salt solution (HBSS). Most cells were recovered as
individual intact elements that excluded the nonvital dye try-
pan blue. The cells, cultured in HAM's F-10 containing 10%
FBS, were grown for 3-7 days. The hPRL content of the cells
was routinely verified by immunocytochemistry using as pri-
mary antibody NIDDK anti-hPRL IC4 diluted 1/3000. Briefly,
the cultures were rinsed twice with HBSS and then fixed in 4%
paraformaldehyde in 100 mM PBS, pH 7.3, for 60 min at 4 C.
The cells were rinsed in HBSS and made permeable with 0.5%
saponin. Immunocytochemical labeling was performed using
the peroxydase anti-peroxydase method. In cultures less than
2 months old, more than 70% of the cells exhibited hPRL
immunoreactivity. Cells were used for electrophysiological
studies within 1-4 weeks of culturing. Medium was changed
every 2-3 days.
Normal rat pituitary cells in primary cultures. Female rats of
the Wistar strain were housed in the laboratory under normal
lighting conditions. They had ad libitum access to a standard
diet. Vaginal smears were examined at regular intervals. Cy-
cling females in diestrus II (second day after the estrus) were
used in this study.
After decapitation, anterior pituitaries were dissected free
Endo• 1990
Voll28«Nol
from intermediate and posterior lobes, and rinsed in sterile
HBSS. Small pituitary tissue blocks were incubated at 36.5 C
in turn with 0.2% collagenase (90 min), 0.2% collagenase, and
0.01% DNAse (15 min) in Ca++-Mg++ free HBSS. The resulting
cell suspension was rinsed twice in HAM's FlO (containing 8
mM NaHCO3, pH 7.3) and final dissociation was carried out
by mechanical dispersion. The cells (2.5 x 105 cells/ml) were
resuspended in HAM's FlO containing 10% FBS and antibiotics
(penicillin 100 IU/ml, streptomycin, 100 fig/ml) and plated in
35-mm Petri dishes (NUNCLON, Poly-Labo, Strasbourg,
France). The cells were maintained at 37 C in a humidified
incubator in an air/CO2 (95/5%) atmosphere. The following
day, the medium was replaced by new medium without anti-
biotics, and half the medium was replaced every other day
thereafter. The recordings were routinely performed 3-7 days
after setting up the culture. The recorded cells were examined
for PRL immunoreactivity with the same technique as de-
scribed above using anti-rat PRL serum S9 from NIDDK at 1/
2000.
Measurement of secretory responses
For hormone release experiments, GH3/B6 and normal rat
pituitary cells were cultured in 24 well plates, 2.105 cells per
well. After culture for 4-5 days, the culture media were dis-
carded. The cells were rinsed three times with prewarmed F-
10, then exposed in quadruplicate to the different concentra-
tions of peptides dissolved in 1 ml sterile culture medium for
30, 60, and 120 min at 37 C. PRL and GH content were
determined by RIA using the reagents provided by the National
Pituitary Agency. All samples from the same experiment were
assayed in duplicate in the same assay. Hormone concentra-
tions were expressed in nanogram per ml/30 min as the mean
± SD. The data were statistically evaluated by the Student's t
test.
Electrophysiological recordings
Membrane potential and ionic currents were recorded by
using the whole cell configuration of the tight-seal recording
technique (Whole Cell Recording, WCR) described in detail
elsewhere (22). The amplifying system included a Dagan 8800
(DAGAN Corp, Minneapolis, MN) equipped with a 100 Meg-
ohm resistor in the headstage for WCR. All recordings were
made at room temperature (22 ± 1 C).
Before each experiment, the culture medium bathing the
cells was aspirated and the cells were bathed 1) in a modified
Hank's solution containing (in millimolars): NaCl, 142.6; KC1,
5.6; CaCl2, 2.5; MgCl2, 0.8; glucose, 5; and HEPES-NaOH, 10,
or 2) in HAM's FlO nutrient medium supplemented with 5%
heat-inactivated horse serum and 2% FBS. In some experi-
ments, the Ca++ concentration in the bathing solutions was
raised to 10 mM as this seemed to provide for more stable
recordings. The osmolality of these solutions was adjusted to
300-310 mOsm/kg with sucrose, and pH to 7.30 ± 0.01 with
NaOH.
Whole cell patch pipettes had tip resistances of 3-5 megohms
when filled with a salt solution (referred to as K glu) containing
(in millimolars): K gluconate, 140; MgCl2, 2; EGTA, 1.1, and
 GAP AND MEMBRANE ION CONDUCTANCES
HEPES-KOH, 5. In some experiments, Af-methyl glucamine
(NMG) gluconate, an impermeant cation, replaced K+ so as to
eliminate K+ currents which obscure the recording of Ca++
currents. NMG solutions (referred to as NMG glue) were
otherwise similar to K glue except that they were buffered to
pH 7.3 with HEPES-gluconate. Rundown of Ca++ currents (23,
24) was minimized by including in the internal solution (in
millimolars, except as noted) 1) an ATP-regenerating system
containing: MgATP, 2; creatine phosphate, 5; and creatine
phosphokinase, 20 U/ml, and 2) tris GTP, 0.04. Only cells in
which the Ca++ currents remained stable (i.e. the amplitude
declined less than 20% during the course of the experiment)
were used in this study.
All internal solutions were adjusted to pH 7.3 ± 0.01. The
osmolality was maintained at 290-295 mOsm/kg, 5-10 mOsm/
kg hypoosmolar to the external solution.
Membrane potential values were corrected for small liquid
junction potentials between the internal solution and the bath-
ing solution as already described (25).
GAP's electrical effects are dependent on serum-containing me-
dium
GAP-induced hyperpolarization and decrease in action po-
tential firing faded when the cells were recorded in serum free
external saline solution. However, change of this medium for
the culture medium (HAM's F10, supplemented with horse
serum and FBS) preserved all the electrical effects of GAP,
even several hours after the beginning of the electrophysiolog-
ical experiment. Therefore, we have used routinely the culture
medium supplemented with sera to further characterize the
effects of GAP on membrane ionic conductances.
Microfluorimetric assay of cytosolic calcium
These experiments were performed using the fluorescent
probe Indo 1 as already described (26). The cells were incubated
with 5 nM indo 1 penta-acetoxymethyl ester (Indo I/AM,
Calbiochem) and 0.02% Pluronic F-127 (Molecular Probes) in
Hank's solution for 30 min at 20 ± 1 C, then washed and
maintained at room temperature in the same saline solution
prior to the fluorescence measurements. Loading at low tem-
perature in the presence of the detergent Pluronic F-127 greatly
enhanced the percentage of hydrolysed dye and prevented Indo
I/AM trapping in endocytotic vesicles or accumulation in the
nucleus. This procedure resulted in an intracellular Indo 1
concentration of between 20 nM and 60 nM, estimated from
comparison with the loading via a patch pipette. The dual
emission microspectrofluorimeter was constructed from a Ni-
kon Diaphot inverted microscope fitted with epifluorescence
(X100 oil-immersion fluorescence objective, numerical aperture
1.3). For excitation of indo 1, a collimated light beam from a
100 W mercury arc lamp (Nikon, France) was filtered at 355
nm and reflected off a dichroic mirror (380 nm). Emitted
fluorescence signal was passed through a pinhole diaphragm
slightly larger than the selected cell and was directed to another
dichroic mirror (455 nm). Transmitted light was filtered at 480
nm, reflected light filtered at 405 nm, and the intensities
recorded by separate photometers (PI, Nikon). Single photon
287
currents were converted to voltage signals which were divided
on-line by a monolithic laser trimmed two-quadrant divider
(AD535, Analog Devices). Under these experimental condi-
tions, the ratio R = F405/F480 was recorded on-line as a voltage
signal and was expressed as [Ca++]i using the formula derived
by Grynkiewicz et al. (27). Microfluorimetric assay of cytosolic
calcium was performed at room temperature (22 ± 1 C).
Pharmacological reagents
GAP, produced as previously described (5), was diluted from
a concentrated stock solution in the bathing medium before
use. It was applied by low-pressure ejection from a micropipette
(tip diameter 3-5 /xm) positioned close to the membrane of the
recorded cell. The concentrations of test substances reported
in the text are those in the pressure pipette, but because of
dilution, the actual concentration at the cell surface may be
slightly lower. Channel blocking drugs were added directly to
bathing medium. These included tetrodotoxin (TTX, 1-5 nM)
to block the fast Na+ current (23) tetraethylammonium to block
the delayed outward current, Co++ (5 mM), Cd++ (50 /xM) and
nifedipine (50 nM) as inhibitors of Ca++ currents (28).
Statistical method
Results are expressed as means ± SD when appropriate. Each
experiment was repeated at least three times.
Results
GAP reduced basal PRL release from GH3/B6 cells and
from normal rat pituitary cells
In order to check that GAP was effective in our model
systems the effect of GAP on hormone secretion was
analyzed in GH3 cells. GAP (100 nM) was found to reduce
both PRL and GH release. At the end of the 30-min
incubation period, PRL concentration in the medium
was reduced from 14.13 ± 0.87 ng/ml-30 min to 10.12 ±
0.58 ng/ml-30 min (n = 4). GH was also slightly but
significantly reduced from 2.76 + 0.07 ng/ml • 30 min to
2.40 ± 0.07 ng/ml-30 min (n = 4). We did not observe
any significant reduction of hormone levels after 60 and
120 min; therefore, in GH3 cells, GAP had only a tran-
sient inhibitory effect.
The effect of GAP was also studied in normal rat
pituitary cells. The cells were incubated for 30 min with
different concentrations of GAP. At a dose of 1 nM GAP,
basal PRL release was not significantly affected (Table
1). Ten nanomolars GAP significantly reduced basal
PRL release by about 20% whereas 100 nM GAP reduced
PRL release by 33% (Table 1). The effect of GAP (100
nM) was also tested on the release of PRL stimulated by
TRH; we found no effect of GAP (100 nM) on TRH-
induced PRL release (Table 1).
288 GAP AND MEMBRANE ION CONDUCTANCES Endo• 1990
Voll28«Nol

TABLE 1. Effects of GAP on basal and TRH-stimulated PRL release
in normal rat pituitary cells
Gap
Controls
(ng/ml-30 min)
(lnM) (10 nM) (100 nM)
107.7 ± 12.1 90.6 ± 14.9 87.2 ± 4.2° 71.6 ± 7.3"
TRH (10 nM) + GAP
Controls TRH (10 nM)
(100 nM)
96.0 ± 14.7 419.8 ± 47.3 415.8 ± 13.0
PRL levels are expressed in ng/ml • 30 min, mean ± SD, n = 4.
" Significantly different from controls; P < 0.05.
Basal electrical properties of rat and hPRL tumor cells
and rat PRL normal cells
In order to examine the mechanism of action of GAP
on PRL-secreting cells, electrophysiological studies were
conducted. Using whole cell patch-clamp recording tech-
niques 23 GH3 cells, 36 human, and 19 rat PRL-secreting
cells were recorded in the current clamp configuration
(Table 2). Using pipettes filled with Kglu, the resting
membrane potential was measured from the zero-current
voltage after establishment of a giga seal and rupture of
the membrane to effect WCR. The resting membrane
potential of the GH3 cells, human tumor and rat normal
PRL-secreting cells bathed in the external saline solution
were -42 ± 5.2 mV (n = 23), -43.2 ± 5.8 mV (n = 36),
and -30 ± 5.6 mV (n = 19), respectively. Input resist-
ances measured during modest-sized hyperpolarizing
current pulses were 2 ± 0.3 GQ (n = 23), 1.9 ± 0.3 GQ (n
= 36), and 2.2 ± 0.4 GQ (n = 19), respectively. Immedi-
ately after rupture and throughout the extended record-
ings, the cells exhibited voltage-dependent action poten-
tials that either appeared spontaneously (83% of GH3
cells, 78% of hPRL cells, and 66% of rat PRL cells) or
could be electrically evoked (100% of tumor cells, 85%
of normal cells). These action potentials have previously
been demonstrated to be blocked by inhibitors of Ca++
currents, Co++ and dihydropyridines, and to be resistant
to TTX or replacement of external Na+ with choline.
This indicates that they were Ca++-dependent; the con-
tribution of TTX-sensitive and TTX-resistant voltage-
dependent Na+ conductances to the action potentials
being either minor or not detectable (21).
GAP reduced Ca++ action potential activity
The percentage of cells responding to an external
application of GAP differed according to the type of
PRL-secreting cells studied. Nanomolar concentrations
of GAP caused clear alterations of action potential pat-
tern in 84% of normal cells, 58% of hPRL cells and 39%
of GH3 cells. These alterations consisted of a delayed
decrease in action potential amplitude and frequency.
The latter was related to 1) an increase in the amplitude
and duration of the after hyperpolarization (AHP) after
spontaneous spikes, or 2) a modest hyperpolarization of
membrane potential, sufficient to hyperpolarize the
membrane under the threshold of spikes firing, or 3) a
direct, complete blockade of action potentials (see Table
2 for values).
These effects of GAP differed slightly from a cell type

TABLE 2. Effects of GAP on the electrical activity

Human
GH3 cells Normal rat lactotropes
prolactinoma cells
No. of cells recorded 23 36 19
% of cells responding to GAP by a de- 31 44 53
crease in AP frequency
control 0.6 ± 0.2 Hz 0.5 ± 0.1 Hz 0.4 ± 0.1 Hz
GAP 0.1 ± 0.1 Hz (n = 7) 0.1 ± 0.1 Hz (n = 16) 0.02 ± 0.06 Hz (n = 10)
% of cells responding to GAP by a de- 13 39 63
crease in AP amplitude
control 63 ± 2.5 mV 60 ± 9 mV 35 ± 8 mV
GAP 54 ± 7.5 mV (n = 3) 49 ± 14 mV (n = 14) 18 ± 5.5 mV (n = 12)
% of cells responding to GAP by a mem- 22 31 47
brane hyperpolarization amplitude of
the hyperpolarization 4 ± 1 mV (n = 5) 6 ± 3 mV (n = 11) 4 ± 3 mV (n = 9)
% of cells responding to GAP by an in- 0 36 0
crease in AHP amplitude
control 10.5 ± 2 mV
GAP 15.5 ± 4 mV (n = 13)
% of cells responding to GAP by, at least, 39 58 84
one of the effects above
These values were obtained during whole cell recording experiments. Cells were bathed in HAM's FlO supplemented with heat-inactivated
horse serum and 2% FBS. Patch pipettes were filled with K glu supplemented with an ATP regenerating system and GTP (see Materials and
Methods). AP, action potential; AHP, after hyperpolarization potential. Data are expressed as means ± SD.
 GAP AND MEMBRANE ION CONDUCTANCES
to another. In normal rat cells, the most consistent effect
of GAP (63.5% of cells) was a reduction in the peak
amplitude of spontaneous action potentials (see Table 2
for values and Fig. IB). In a few cells a complete inhibi-
tion of electrical activity was obtained (52.6% of cells).
This effect resulted from either a direct blockade of
action potential firing (5.2% of cells) or a sustained
hyperpolarization (47.3% of cells) (Fig. 1A and Table 2
for values). No modification of AHP duration and am-
plitude was observed in these cells.
GH3 cells were less responsive to GAP than normal
rat lactotrophs or hPRL cells. Like normal rat cells they
responded by a change in one of the following parame-
ters: decrease in the firing rate of spontaneous activity
(30.5% of cells), decrease in the amplitude of action
potentials (13% of cells), and hyperpolarization (21.7%
of cells) (see Table 2). The AHP duration and amplitude
of their AP were not affected.
Although the percentage of responsive cells is smaller
in hPRL cells than in normal rat cells, in the former the
effect of GAP was more pronounced. In human cells,
decrease in frequency (Fig. 2A, 44.4% of cells), AP am-
plitude (Fig. 2B, 38.9% of cells), and membrane hyper-
polarization (30.5% of cells), were often long to reverse.
In addition, we observed, in 36% of the hPRL cells, an
increase in AHP amplitude and duration (see Table 2
and Fig. 2C). Unlike the other effects, this occurred very
quickly (during peptide application) and disappeared few
seconds after the end of the application.
GAP was able to induce simultaneously several of these
effects in the same cell; for example a decrease in action
potential amplitude and an increase in AHP amplitude
and duration (Fig. 2C).
GAP enhanced a transient outward current in human
cells
To further explore the ionic mechanisms involved in
the action of GAP, voltage clamp studies were performed.
The current clamp experiments reported here above have
^ GAPdOnM)
F)10mV
10sec
FIG. 1. GAP affects Ca++ action potential pattern and frequency in
rat cells. Tight seal, whole-cell recordings (current-clamp configura-
tion) made from normal rat PRL cells (rPRL). Zero mV membrane
potential is to the left of each trace. A, GAP (10 nM) induced a transient
hyperpolarization associated with a blockade of action potential firing.
B, GAP (100 nM) decreased action potential amplitude. Hyperpolariz-
ing current pulses were periodically injected through recording elec-
trode to monitor membrane resistance.
289
10 mV
30pA
30sec
GAP Recovery
_ I 20 mV
50 msec
FIG. 2. GAP (100 nM) affects Ca++ action potential pattern and fre-
quency in human tumor PRL cells. A, GAP induced a reversible
blockade of action potential activity and a slight hyperpolarization. B,
Top: GAP decreased action potential amplitude without detectable
effect on input membrane resistance. Bottom: Display of hyperpolar-
izing current pulses injected through the recording electrode. C. Ex-
panded records on a rapid time base of spontaneous spikes in another
cell. Action potential amplitude was decreased and AHP amplitude and
duration were increased after application of GAP (right) compared to
control (left). Zero mV membrane potential is to the left of each trace.
A dashed line indicates membrane resting potential.
shown that in hPRL cells, GAP decreased the frequency
of spontaneous action potential activity coincident with
an increase in the amplitude and duration of the AHP
(Fig. 2C). Hence, we have examined the effects of GAP
on voltage-dependent K+ conductances that can be ac-
tivated during the Ca++ action potential depolarization.
Under voltage clamp, with the cell membrane held at
—40 mV, only a slowly activating delayed current re-
sponse could be elicited with depolarizing steps. In all
cell types, this current, as measured at the end of 650
msec steps was not significantly affected by GAP up to
1 ixM. The I/V plots of the delayed outward current were
not affected by the peptide (not shown).
When the cell was held more hyperpolarized (-80 mV),
in addition to the slowly activating outward current, a
transient outward current (TOC) was elicited by depo-
larizing steps. We have studied the effect of GAP on the
TOC, using cells bathed in an external medium with
supplements that abolish or minimize the other voltage -
dependent currents: inward currents by Cd++ and nife-
dipine (Ca++) and TTX (Na+), IKV by tetraethylammo-
nium chloride (see Fig. 3 legend). Thus, in the absence
of the other contaminating currents, GAP up to 1 nM did
not affect TOC amplitude in normal and tumor rat cells
(data not shown). In human tumor cells, GAP (100 nM)
increased the TOC amplitude with little if any effect on
its time constant of inactivation (Fig. 3A, 10 of 12 cells).
The I/V curves of this current were noticeably displaced
to more negative values of membrane potential (10-20
mV; Fig. 3B). Although a systematic dose response was
not carried out, subnanomolar concentrations were in-
 GAP AND MEMBRANE ION CONDUCTANCES
290
B the TOC activation curve in a hyperpolarizing direction,
OmV
]200pA
"jrecoyery
400msec
GAP3 GAP4GAP5
2000-
1000-
T min
10 20 30 50
20
\\nnj~Lnj~Lri
4y;
GAP3 GAP*. 400msec
FIG. 3. GAP( 100 nM) strongly enhances the transient outward current
in hPRL cells. In order to eliminate contaminating currents that
obscure the recording of the rapidly activating K+ current (TOC), cells
were bathed in external medium containing 0.1 i*M nifedipine, 50 /xM
Cd++, 5 MM TTX, 2 mM Ca++, and 30 mM TEA. A, A depolarization
from -80 to 0 mV elicited a TOC response that quickly reached a peak
and then progressively declined (top). GAP (100 nM) increased peak
amplitude of TOC (middle). This effect was rapidly reversible (bottom).
B, Current-voltage (I/V) relationships for the TOC before (•) and
after (O) GAP application. C and D, In another cell we have repeatedly
applied (every 1 min.) the same voltage jump (from -80 to +20 mV).
C. TOC amplitude in response to successive GAP applications. Effects
of GAP3 and GAP4 are shown in D. D, Third and fourth GAP
applications (GAP3 and GAP4) enhanced TOC amplitude to a similar
extent. The recovery to control level was complete within 3 to 4 minutes
after GAP removal.
effective, whereas 100 nM did not appear more effective
than 1-10 nM. Transient outward current returned pro-
gressively nearly to control levels after the end of GAP
application, as shown in Fig. 3, A (recovery), C, and D.
These effects of the peptide could be obtained over the
entire course of the recording (about 40 min) without
any significant decrement even when an application was
very close to the subsequent (Fig. 3, C and D).
If the TOC activated during the depolarizing phase of
an action potential, it would serve to repolarize the cell
near the K+ ion equilibrium potential (EK+). By shifting
Endo•1990
Voll28«Nol
GAP could increase the size of the AHP, and thereby
decrease Ca++ action potential frequency. This is con-
sistent with the effects of GAP in current clamp record-
ings (see Fig. 2C and Table 2) and with the observation
that AHP was increased by GAP only in the human cells.
GAP reduced voltage-dependent Ca++ currents
As in current clamp experiments, GAP was observed
to decrease the amplitude of the Ca++ action potentials,
we examined the possible effects of GAP on voltage-
dependent inward Ca++ currents. NMG gluconate was
dialyzed into the cell, instead of K+ ions, to avoid con-
tamination of inward currents by activation of outwardly
directed K+ currents. TTX (5 fiM) was added to the
external medium in order to block the fast Na+ current.
Two distinct populations of Ca++ channels that differ in
their closing time constants have been described in pi-
tuitary cells (24). They will be designated L- and T-type
channel currents after the nomenclature in use. T-chan-
nels are activated at relatively negative voltages (low
threshold) and inactivated during a 100 msec pulse. L
channels are activated in a less negative range (high
threshold) and do not inactivate within 100 msec. In
addition to their different kinetic properties, L and T
type currents can be distinguished by application of
specific inhibitors (dihydropyridines inhibit L-type cur-
-,*5mV
3-35
]200pA
recovery
B 400msec
-50 -25 25 VmV 50
\
IpA
FIG. 4. GAP (100 nM) decreases a voltage-dependent Ca++ current in
hPRL cells. In this and the subsequent figure (both of which show
whole cell voltage clamp records), outward currents were suppressed
by filling the patch-pipette with NMG gluconate. TTX was added to
the external medium to block possible Na+ current. The step potential
is shown at the top of each panel and current records are shown below.
When the cell was clamped at -35 mV, depolarizing steps of increasing
amplitudes elicited predominantly a sustained type of inwardly directed
calcium current (L current). A, Examples of current traces obtained
under control conditions (top), within 5-10 min after administration
of GAP (middle) and 20 min later, during the recovery phase (bottom).
B, Current-voltage (I/V) plots for the cell shown in A. Peak inward
currents are plotted before (filled circles) and after (open circles)
exposure to GAP (100 nM).
 GAP AND MEMBRANE ION CONDUCTANCES
rent) or on the basis of their voltage dependencies; when
the cell is held at —40 or —35 mV depolarization voltage
steps elicit only the L-type current. Figure 4A shows that
100 nM GAP depressed the amplitude of the L current
elicited by a depolarizing step from —35 to +5 mV (five
of eight cells). GAP was effective only 1-3 min after its
introduction into the bath and recovery occurred 8-12
min after washout (Fig. 4A). Voltage steps of increasing
amplitude were applied to the cell membrane to establish
the I/V curves before {closed symbols) and after {open
symbols) GAP application. GAP did not modify the cur-
rent waveform or shift the I-V curve in the hyperpolar-
izing or depolarizing directions (Fig. 4B). Thus, GAP
caused no change in the apparent reversal potential (data
not shown) whereas there was a marked decrease in Ca++
current at all potentials where it was activated.
In 5 of 8 hPRL cells, the mean maximum L current
amplitude was significantly decreased from —360 ± 105
pA (mean ± SD) under control conditions to —195 ± 85
pA during GAP application. In 6 of 11 GH3/B6 cells, the
mean maximum L current amplitude was decreased from
-420 ± 120 pA under control conditions to -292 ± 120
pA during GAP. In 6 of 9 normal rat PRL cells 100 nM
GAP reduced the mean maximum L current amplitude
from -290 ± 65 pA to -180 ± 60 pA. This effect of GAP
on L current also occurred when the cell was held at —80
mV. A depolarizing step from —80 to —20 mV elicited
both L and T type currents (Fig. 5). The steady state
current at 650 msec recorded under these conditions is
mainly derived from the L-type current. GAP depresses
,-20mV
J-80
400 msec
0 25 VmV 50
-0.25
-0.50
-0.75 InA
FIG. 5. GAP (100 nM) affects only the L current. A, Depolarizing
voltage steps from —80 to —20 mV elicited two inward current compo-
nents, a rapidly activating and inactivating component, T current, and
a sustained component, L current. The amplitude of L current (circle)
was estimated as the net inward current at the end of the command
step. The amplitude of T current (square) was obtained by substracting
L current from the peak current. GAP reduces L current but does not
have any significant effect on T type current. Plots of L current (circle)
and T current (square) are shown in B. Filled symbols are control
values, open symbols plot data obtained after exposure to GAP.
291
the amplitude of this current in the three cell types at
all potential were it was activated (3 of 5 hPRL cells:
-245 ± 85 pA, control, -150 ± 55 pA, GAP; 3 of 7 GH3/
B6 cells: -185 ± 55 pA, control -110 ± 50 pA, GAP).
On the other hand, GAP did not significantly affect the
T-type current (Fig. 5). The amplitude of this current
can be estimated by substracting the steady state current
from the peak current (Fig. 5). In the three cell types,
the mean amplitude of T-type current was unchanged at
all potentials where it was activated (Fig. 5B) (hPRL
cells: -340 ± 115 pA, control, -330 ± 110 pA, n = 5;
GH3/B6 cells: -365 ±110 pA, control, -380 ± 130 pA,
n = 7; normal rat PRL cells: —245 ± 85 pA, control,
—230 ± 90 pA, n = 7). As for the other holding potential,
the effect of GAP on L-type current occurred within 100
sec of beginning the GAP perfusion and was maintained
for as long as the peptide was continuously applied (up
to 500 sec). On termination of the GAP application, the
Ca++ current slowly returned to base line levels (within
about 200 sec). These GAP actions on Ca++ current can
be repeated several times on the same cell (data not
shown). Although a systematic dose-response curve was
not carried out, 10 nM and smaller concentrations were
less effective on Ca++ current. On the other hand, even
at 1 /iM, GAP did not affect T-type current.
Thus, GAP depresses voltage-dependent Ca++ entry in
the three cell types. This action could explain the delayed
decrease in the action potential amplitude or the com-
plete blockade of spike firing observed in current-clamp
experiments.
GAP inhibited spontaneous Ca++ oscillations and reduced
intracellular free Ca++ levels in intact cells
Intracellular free Ca++ levels were monitored in indi-
vidual PRL-secreting pituitary cells by spectrofluorime-
ICa2*]i,nM
235
130
20 sec
FIG. 6. GAP (1 nM) slows down spontaneous Ca++ oscillations in
intact, normal rat lactotrophs. Cells were loaded with the permeant,
fluorescent Ca++ probe, Indo 1AM. Fluorescence ratios (F405/F480),
related to [Ca++]i by the Grynckiewicz formula, are shown vs time. In
cells showing spontaneous Ca++ transients, GAP reduced the frequency
of Ca++ oscillations. However, the effect of GAP differed according to
the type of activity displayed by the cell; when the Ca++ transients
where unfrequent and resolvable, [Ca++]i basal level was not affected
by GAP (A). In other cells, as in B, rythmic oscillations of [Ca++]i
maintained Ca++ level well above basal level; in these cells, the effect
of GAP on Ca++ transients resulted in a decrease in [Ca++]i level.
292 GAP AND MEMBRANE ION CONDUCTANCES
try using the fluorescent Ca++ probe Indo 1. Ca++ levels
were monitored 3-7 days after cell dispersion. The cells
were loaded with the permeant probe, Indo 1 AM. The
majority of the cells analyzed exhibited an unstable
resting [Ca++]i, fluctuating from values of 60-100 nM up
to 200-300 nM (Fig. 6). These fluctuations are due to
discrete, rythmic oscillations of [Ca++]i each lasting sev-
eral seconds. These rapidly disappeared when extracel-
lular [Ca++]i was chelated with excess EGTA or voltage-
gated Ca++ channels were blocked by Co++ (5 mM) (data
not shown). These results indicate that the fluctuations
are due to Ca++ influx from the medium. Furthermore,
we have recently shown that the spontaneous oscillations
of [Ca++]i are the consequence of spontaneous Ca++-
dependent action potentials (17). In spontaneously os-
cillating cells, GAP (1 nM) decreased Ca++-oscillation
frequency (Fig. 6A). Furthermore, in cells showing fre-
quent Ca++-oscillations and a high [Ca++]i level (Fig. 6B)
GAP reduced [Ca++]i level.
Discussion
The present work shows a direct inhibition of basal
PRL release by GAP in normal rat pituitary cells in
culture. This is consistent with previous reports of a
GAP-mediated inhibition of PRL release from primary
rat (5, 7) and human pituitary cells (6). In primary rat
pituitary cells, Wormald et al. (6) obtained an inhibition
of about 38% at a dose of 100 nM GAP which is very
close to the value reported in the present study.
We also found that GAP transiently reduced PRL
release as well as GH release in the GH3 tumor cell line.
This is not surprising since GH3 cells are known to
release both PRL and GH. The decrease in PRL release
(28%) observed in this study after GAP (100 nM) admin-
istration was similar in amplitude to that reported by
Bjoro et al. (29) after SRIF (1 ^M) administration (about
20%) in another subclone (GH4/C1) of the same line of
GH3 cells. Therefore GAP appears to be more potent
than SRIF in suppressing basal PRL basal release.
Conversely we found no effect of GAP on TRH-stim-
ulated PRL release. TRH stimulation of PRL secretion
is biphasic with an initial transient phase followed by a
sustained phase of lower magnitude. The first phase,
dependent on the production of inositol 1,4,5 trisphos-
phate, is mediated through the release of intracellularly
stored Ca++. There is no evidence that GAP may affect
intracellular Ca++ pools and therefore no reason for GAP
to affect this first phase of secretion. The second phase
is thought to involve entry of Ca++ through voltage-gated
Ca++ channels. Since GAP affected membrane conduct-
ance to Ca++ ions, one might have expected a reduction
in PRL release by TRH. However, the present study was
carried out in 30-min incubation experiments where the
Endo • 1990
Voll28-Nol
two phases of secretion are pooled. The large increase in
secretion resulting from the first phase may have masked
the putative alterations of secretion ocurring during the
second phase. Perfusion experiments, where the two
phases can be clearly separated, are required to solve this
issue.
Several other studies have reported a lack of effect of
GAP in vivo. GAP inhibited elevated serum PRL con-
centrations in lactating and in ether-stressed rats, but,
the peptide did not influence resting serum PRL levels
(8). GAP was also found to be ineffective in lowering
resting serum PRL levels in sheep (13). The peptide had
no effect on prolactin release from human adenoma cells
in culture (30). Such discrepancies could be explained by
differences between in vitro and in vivo activity of the
peptide. Also, the kinetics of the effect of GAP has to be
taken into account, since our results show that GAP
reduced only transiently PRL release by GH3 cells. In
the study by Ishibashi et al. (30) adenoma cells were
incubated for 2 h and a transient effect could have been
missed. All the data reported in this study concern tran-
sient effects of the peptide.
It has long been known that secretion by pituitary
cells is a calcium-dependent process. This observation
led to the suggestion that cytoplasmic Ca++ elevations or
decreases mediate, respectively, the stimulatory or inhib-
itory effects of many agonists or antagonists. This sug-
gestion has received direct experimental support by si-
multaneous measurements of intracellular [Ca++] and
secretion (31) and by the demonstration that Ca++ can
activate secretion in permeable cells (19). Two major
sources of Ca++ have been proposed in pituitary cells.
First, Ca++ can be mobilized from intracellular pools.
There is no evidence at the present time that GAP may
affect intracellular Ca++ pools. Second, pituitary cells are
excitable. Repetitive spontaneous action potentials were
found in both normal (32) and neoplastic pituitary cells
(14-16). Recently, combining whole cell patch-clamp
recordings with Fura 2 measurements of intracellular
calcium we have demonstrated that action potentials
precede spontaneous calcium rises in the cytosol (17).
The sizes of these short lived maxima were sufficient to
evoke basal secretory activity (17,19). The entry of Ca++
during an action potential occurs primarily through volt-
age-dependent Ca++ channels. In lactotrophs two distinct
Ca++ channel species have been described: one that ac-
tivates only on depolarization from hyperpolarized po-
tentials (low threshold) and inactivates rapidly in a time-
dependent fashion (transient or T-type), and a second
that activates over a wider potential range and shows
minimal inactivation (long lasting or L-type) (24).
The Ca++ entry is also indirectly controlled by K+
conductances. Like many other secretory cells PRL-
secreting pituitary cells exhibit voltage-dependent and
 GAP AND MEMBRANE ION CONDUCTANCES
Ca++-activated K+ conductances (25, 33). These con-
ductances are thought to play critical roles both in the
repolarization phase of the action potential, thus con-
trolling action potential duration, calcium entry dura-
tion, and the probability of spontaneous action potential
generation.
Over the 10 nM-1 pM range of concentrations, GAP
eliminated spontaneous and evoked Ca++ action poten-
tial activity through effects on K+ and Ca++ conduct-
ances. The complete elimination of Ca++ action poten-
tials was due to a combination of cell hyperpolarization
away from threshold for triggering action potentials and
direct depressant effects on depolarization-activated
Ca++ conductances. The hyperpolarizing response to
GAP, which was found in a small proportion of the three
types of cells studied, was very modest (3 to 8 mV). The
hyperpolarization may result from activation of a K+
conductance at the resting potential. However, we cannot
exclude that a depression of calcium entry alone may
account for this effect.
Finally, decreases in the amplitude of the action po-
tential also occurred. The marked depression of voltage-
dependent Ca++ conductance(s) was a consistent obser-
vation in the three cell types. Two phases of depolariza-
tion-activated Ca++ entry have been characterized pre-
viously in GH3 cells as well as in primary rat and human
PRL cells. GAP depressed the amplitude of the L-type
current in the three cell types at all potentials were it
was activated. On the other hand, GAP did not signifi-
cantly affect the T-type current. In the three cell types,
the mean amplitude of T-type current was unchanged at
all potentials were it was activated. Ca++ entry through
the L-type channel should be implicated in secretion
since dihydropyridines are specific inhibitors of this type
of Ca++ channel and block basal release. This observation
is therefore consistent with the PRL-release inhibiting
effect of GAP.
In human adenoma cells, under current-clamp record-
ing conditions, GAP decreased spontaneous action po-
tential activity by intensifying the after-hyperpolarizing
phase of the action potential. Voltage clamp studies show
that, in human adenoma cells, two independent processes
are involved in the action of GAP: an increase in the
amplitude of TOC and a direct reduction of calcium
conductances.
In human adenoma cells, under voltage-clamp record-
ing conditions, GAP mainly affects a voltage-dependent
rapidly activating, rapidly inactivating transient-type K+
conductance mechanism. This conductance activates
rapidly during the depolarization induced by the Ca++-
dependent action potential that it could help to deter-
mine the amplitude and time course of the action poten-
tial and the subsequent after hyperpolarization.
GAP enhancement of TOC amplitude observed under
293
voltage clamp underlies what occurs during the voltage
excursion of the Ca++ action potential under current
clamp, and accounts for at least a part of the prolonged
after hyperhyperpolarization. By reducing the frequency
of Ca++ action potentials GAP depresses their intermit-
tent contribution to [Ca++]i, thereby maintaining a lower
level of [Ca++]i and consequently depressing PRL secre-
tion dependent on [Ca++]i.
The fact that GAP affected TOC only in human cells
and not in rat normal or tumoral cells is somewhat
remarkable. Is there a sequence in GAP which conveys
a signal that is species specific to the TOC in human
cells? SRIF was shown to decrease action potential firing
in rat and human cells, however, SRIF increased TOC
in both human and rat cells (21). Therefore GAP and
SRIF have a similar type of action in human cells. It
should be noted that human and rat SRIFs are identical
and therefore, their receptor mediated responses are
expected to be very similar if not identical. However,
human and rat GAP are only 70% identical or 79%
homologous (considering conservative substitutions)
(34). Therefore, it is conceivable that human GAP used
in the present studies may not have affected all the
responses in rat target cells.
To further analyze the mechanism of action of GAP,
intracellular free Ca++ levels were monitored in individ-
ual PRL-secreting pituitary cells by spectrofluorimetry
using the fluorescent Ca++ probe Indo 1. GAP inhibited
spontaneous Ca++ oscillations and reduced intracellular
free Ca++ levels. The reduction of intracellular Ca++
levels, as monitored by the fluorescent dye Indo 1, is
consistent with the electrophysiological observations.
In conclusion, using complementary techniques we
have demonstrated that GAP reduces intracellular Ca++
levels, inhibits Ca++ transients and reduces membrane
conductance to Ca++ ions in pituitary cells. Also, GAP
action on membrane conductances to K+ ions may con-
tribute to the reduced Ca++ entry. These effects of GAP
on membrane ion conductances may explain the inhibi-
tory effect of GAP on hormonal secretion by PRL-
secreting cells.
Acknowledgments
We thank the National Pituitary Agency, Pituitary Hormone distri-
bution program, NIDDK, for the reagents used in this study. We are
grateful to N. Vilayleck, A. M. Vacher, D. Varoqueaux, and G. Gaurier
for excellent technical assistance.
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