Differential Expression of Ionic Channels in Rat Anterior Pituitary Cells
Fredrick Van Goor,
Dragoslava Zivadinovic and
Stanko S. Stojilkovic
Endocrinology and Reproduction Research Branch National
Institute of Child Health and Human Development National Institutes
of Health Bethesda, Maryland 20892-4510
 |
ABSTRACT
|
---|
Secretory anterior pituitary cells are of the same
origin, but exhibit cell type-specific patterns of spontaneous
intracellular Ca2+ signaling and basal hormone
secretion. To understand the underlying ionic mechanisms mediating
these differences, we compared the ionic channels expressed in
somatotrophs, lactotrophs, and gonadotrophs from randomly cycling
female rats under identical cell culture and recording conditions. Our
results indicate that a similar group of ionic channels are expressed
in each cell type, including transient and sustained voltage-gated
Ca2+ channels, tetrodotoxin-sensitive
Na+ channels, transient and delayed rectifying
K+ channels, and multiple
Ca2+-sensitive K+
channel subtypes. However, there were marked differences in the
expression levels of some of the ionic channels. Specifically,
lactotrophs and somatotrophs exhibited low expression levels of
tetrodotoxin-sensitive Na+ channels and high
expression levels of the large-conductance,
Ca2+-activated K+
channel compared with those observed in gonadotrophs. In addition,
functional expression of the transient K+
channel was much higher in lactotrophs and gonadotrophs than in
somatotrophs. Finally, the expression of the transient voltage-gated
Ca2+ channels was higher in somatotrophs than
in lactotrophs and gonadotrophs. These results indicate that there are
cell type-specific patterns of ionic channel expression, which may be
of physiological significance for the control of
Ca2+ homeostasis and secretion in unstimulated
and receptor-stimulated anterior pituitary cells.
 |
INTRODUCTION
|
---|
The anterior pituitary is composed of the five major
hormone-secreting cell types, corticotrophs, lactotrophs, thyrotrophs,
somatotrophs, and gonadotrophs. Corticotrophs arise from a lineage that
is distinct from the other cell types, whereas the remaining cell types
share common transcription factors and frequently produce multiple
hormones, indicating that they are closely related (1). Despite their
similar origin, somatotrophs, lactotrophs, and gonadotrophs differ with
respect to their pattern of spontaneous electrical activity,
intracellular Ca2+
([Ca2+]i) signaling,
basal hormone secretion, and neuroendocrine regulation of
spontaneous Ca2+ influx and
Ca2+-dependent hormone secretion. Specifically,
somatotrophs and lactotrophs exhibit extracellular
Ca2+-dependent, high-amplitude
[Ca2+]i transients,
whereas only low-amplitude
[Ca2+]i signals have been
observed in unstimulated gonadotrophs (2, 3). In parallel to
spontaneous [Ca2+]i
signaling, somatotrophs and lactotrophs exhibit high basal secretion,
whereas basal gonadotropin secretion is low and not dependent on
extracellular Ca2+ (4). Consistent with this,
Ca2+ signaling and secretion in somatotrophs and
lactotrophs, but not in gonadotrophs, are under dual control by
positive and negative hypothalamic factors (5, 6, 7). These differences
suggest that, despite the similar origin of somatotrophs, lactotrophs,
and gonadotrophs, there may be differences in the ionic channels
expressed in the three hormone-secreting cell types.
Many of the ionic channels in native and immortalized anterior
pituitary cells have been characterized previously, including
voltage-gated Ca2+ channels (VGCCs), tetrodotoxin
(TTX)-sensitive and insensitive Na+ channels,
voltage-gated K+ channels,
Ca2+-controlled K+
channels, Cl- channels, and nonselective
cationic channels (2, 3). However, it is difficult to directly compare
the expression levels and the properties of the individual ionic
channels from the different studies, due to differences in the species,
sex, and hormonal status of the animals used, as well as the cell
cultures and recording conditions. Because of this, it is still not
known whether differences in the expression levels and/or ionic channel
properties underlie the cell type-specific patterns of voltage-gated
Ca2+ entry and hormone secretion. To address this
problem, we compared the expression levels and voltage-dependent
properties of the ionic channels in somatotrophs, lactotrophs, and
gonadotrophs from randomly cyclic female rats under identical culture
and recording conditions. A similar group of ionic channels is observed
in all three cell types. However, there was a marked difference in the
functional expression of the individual ionic channels between the
three hormone-secreting cell types. These differences likely underlie
the cell type-specific patterns of
[Ca2+]i signaling and
hormone secretion observed in unstimulated somatotrophs, lactotrophs,
and gonadotrophs.
 |
RESULTS
|
---|
Voltage-Gated Na+ Channels
The functional expression and voltage-dependent properties of the
Na+ channels in rat somatotrophs, lactotrophs,
and gonadotrophs were examined under isolated Na+
current (INa) recording conditions using the
conventional whole-cell technique. In rat somatotrophs and lactotrophs,
a rapidly activating and inactivating INa was
observed at membrane potentials more depolarized than -37 mV and
reached maximum amplitude around -7 mV (Fig. 1
, A, B, and D). A similar
INa was observed in rat gonadotrophs (Fig. 1C
),
but its peak amplitude was much greater and its current-voltage
relation was shifted approximately 10 mV in the hyperpolarizing
direction compared with that for somatotrophs and lactotrophs (Fig. 1D
). In somatotrophs, lactotrophs, and gonadotrophs, application of 1
µM TTX reduced the peak INa
amplitude by 99.4 ± 0.7% (n = 5), 98.2 ± 3.3%
(n = 5), and 98.9 ± 1.2% (n = 5), respectively.

View larger version (29K):
[in this window]
[in a new window]
|
Figure 1. Voltage-Gated Na+ Channels in
Somatotrophs, Lactotrophs, and Gonadotrophs
Representative voltage-gated INa traces in somatotrophs
(panel A, n = 5), lactotrophs (panel B, n = 5), and
gonadotrophs (panel C, n = 5) elicited by 100-msec voltage steps
from -87 mV to 83 mV form a holding potential of -117 mV. D,
Current-voltage relation of the voltage-gated INa in all
three cell types. E, Steady-state inactivation curves for the
voltage-gated INa in all three cell types were generated by
stepping the membrane potential to between -127 and -13 mV for 200
msec before stepping to a 100-msec command potential of -17 mV
(holding potential = -97 mV). The peak INa evoked
during the command potential to -17 mV in each cell type were
normalized to the maximum inward current and plotted against the
conditioning pulse potentials. In this and the following figures, the
ionic currents were normalized to the membrane capacitance of each cell
examined to compensate for the differences in the size of cells. The
dashed box in this figure and in Figs. 3 and 5
represents the range of baseline potentials commonly observed in
spontaneously active somatotrophs, lactotrophs, and gonadotrophs (54 ).
|
|
To determine the proportions of the TTX-sensitive
INa in each cell type that are available for
activation at different resting membrane potentials, the steady-state
inactivation properties of the INa in all three
cell types were examined using a two-pulse protocol. This protocol
consisted of a series of 200-msec conditioning pulses from -127 mV to
-13 mV, followed by a 100-msec test pulse to -17 mV (holding
potential = -97 mV). The peak INa evoked
during the test pulse was normalized to the maximal inward current and
plotted against the conditioning pulse potentials, and the resulting
curves were fitted with a single Boltzmann relation (Fig. 1E
). In
somatotrophs, the membrane potential at which there is 50% of the
maximal current (E1/2) available for activation
is -72 mV. In lactotrophs and gonadotrophs, the
E1/2 values were similar and were approximately
10 mV more depolarized than that observed in somatotrophs.
Voltage-Gated Ca2+ Channels
The functional expression and voltage-dependent properties of the
Ca2+ channels in rat somatotrophs, lactotrophs,
and gonadotrophs were examined under isolated
Ca2+ current (ICa)
recording conditions using the conventional whole-cell technique. The
ICa in each cell type was examined by applying a
series of 400-msec depolarizing voltage steps from -97 mV to +83 mV in
10-mV increments (holding potential = -97 mV). In all three cell
types, a transient ICa was observed in response
to the depolarizing voltage steps. In somatotrophs, the transient
ICa was activated by voltage steps more
depolarized than -77 mV and reached maximum amplitude between -27 and
-17 mV (Fig. 2
, A and B). In the other
two cell types, the activation threshold of the transient current was
shifted approximately 10 mV in the depolarizing direction (Fig. 2
, A
and B). In addition, the peak of the transient
ICa-voltage relationship was shifted more than 20
mV in the depolarizing direction compared with that in somatotrophs
(Fig. 2B
). To determine the density of the transient
ICa in each cell type, the peak
ICa evoked by voltage steps to -47 mV (holding
potential = -97 mV) was analyzed. This membrane potential was
used because it activates a transient ICa in all
three cell types with only minimal activation of a sustained
ICa (Fig. 2A
). The mean peak transient
ICa densities at -47 mV in somatotrophs,
lactotrophs, and gonadotrophs were -6.6 ± 0.8
picoamperes/picofarads (pA/pF) (n = 20), -2.2 ± 0.3
pA/pF (n = 16), and -2.7 ± 0.6 pA/pF (n = 14),
respectively.

View larger version (25K):
[in this window]
[in a new window]
|
Figure 2. Voltage-Gated Ca2+ Channels in
Somatotrophs, Lactotrophs, and Gonadotrophs
A, Representative voltage-gated ICa traces in somatotrophs
(n = 20), lactotrophs (n = 16), and gonadotrophs (n =
14) elicited by 400-msec voltage steps from a holding potential of -97
mV to -77, -47, -27, and -7 mV are shown. B, Current-voltage
relation of the peak (open circles, 025 msec) and
sustained (filled circles, 390400 msec) voltage-gated
ICa in all three cell types.
|
|
The transient ICa in somatotrophs, lactotrophs,
and gonadotrophs was followed by slow-inactivating
ICa. In all three cell types, the sustained
ICa was activated by voltage steps more
depolarized than -57 mV and reached a maximum amplitude around +3 mV
(Fig. 2
, A and B). In addition to the similar current-voltage
relationship of the sustained ICa,
dihydropyridine agonists and antagonists had a similar effect on the
sustained ICa amplitude in all three cell types
(Table 1
). To compare the density of the
slow-inactivating ICa in each cell type, the
sustained ICa (390400 msec) evoked by
voltage-steps to +3 mV was analyzed. In somatotrophs, lactotrophs, and
gonadotrophs the mean sustained ICa densities
were -6.8 ± 0.8 pA/pF (n = 20), -4.5 ± 0.9 pA/pF
(n = 16), and -9.2 ± 1.7 pA/pF (n = 14),
respectively.
View this table:
[in this window]
[in a new window]
|
Table 1. Effects of L-Type Ca2+ Channel
Agonists and Antagonists on Sustained ICa in Somatotrophs,
Lactotrophs, and Gonadotrophs
|
|
To determine the proportions of the total voltage-gated
ICa in each cell type that is available for
activation at various different resting membrane potentials, the
steady-state inactivation properties of the ICa
were examined using a two-pulse protocol. This protocol consisted of a
conditioning pulse ranging from -127 mV to -13 mV for 400 msec, after
which a 200-msec test pulse to -7 mV was applied (Fig. 3
, AC). The normalized test current in
each cell type was plotted against the conditioning pulse potentials,
and the resulting curve was fitted with a single Boltzmann relation
(Fig. 3D
). In somatotrophs, E1/2 was -56 mV
(slope factor = 6.5). The E1/2 in
lactotrophs and gonadotrophs were -34 mV (slope factor = 7) and
-40 mV (slope factor = 4), respectively, which were more than 15
mV more depolarized than that in somatotrophs. The more pronounced
steady-state inactivation of the total ICa in
somatotrophs compared with that in lactotrophs and gonadotrophs is
consistent with the greater expression of the transient
ICa in somatotrophs.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 3. Steady-State Inactivation of the total
ICa in Somatotrophs, Lactotrophs, and Gonadotrophs
AC, Steady-state inactivation curves for the voltage-gated
ICa in each cell type were generated by stepping the
membrane potential to between -127 and -3 mV for 400 msec before
stepping to a 200-msec command potential of -7 mV (holding
potential = -97 mV). Representative traces of the remaining
current elicited during the command potential after conditioning pulses
between -77 mV and -17 mV are shown. D, The peak ICa
evoked during the command potential to -7 mV in all three cell types
were normalized to the maximum inward current and plotted against the
conditioning pulse potentials. The data for each cell type were fitted
with a single Boltzmann relation.
|
|
Voltage-Gated K+ Channels
The functional expression and voltage-dependent properties of the
voltage-gated K+ channels in rat somatotrophs,
lactotrophs, and gonadotrophs were examined under isolated
K+ current (IK) recording
conditions using the perforated-patch technique. To exclude
Ca2+-sensitive IK
(IK(Ca)), extracellular
Ca2+ entry through VGCCs was blocked by addition
of 200 µM Cd2+ to the bath
solution. The total voltage-gated IK in each cell
type was examined by the application of a 500-msec holding potential to
-130 mV before giving a series of 1.5-sec depolarizing voltage steps
from -90 mV to +90 mV (Fig. 4A
;
upper panels). In rat somatotrophs, both the peak (025
msec) and sustained (1.41.5 sec) IK were
activated at membrane potentials more depolarized than -30 mV. Once
activated, the total voltage-gated IK inactivated
slowly during the 1.5-sec depolarizing voltage steps (Fig. 4
, A and D).
In lactotrophs and gonadotrophs, the peak IK
activated at membrane potentials more depolarized than -50 mV, whereas
the sustained IK activated at membrane potentials
more depolarized than -30 mV. Unlike somatotrophs, the total
voltage-gated IK in lactotrophs and gonadotrophs
was characterized by a fast- and slow-inactivating
IK (Fig. 4
, A and D).

View larger version (38K):
[in this window]
[in a new window]
|
Figure 4. Voltage-Gated K+ Channels in
Somatotrophs, Lactotrophs, and Gonadotrophs
A, Representative voltage-gated IK traces in somatotrophs
(n = 5), lactotrophs (n = 5), and gonadotrophs (n = 8)
elicited by 1.5-sec voltage steps from -90 to 90 mV in 20-mV
increments from a holding potential of -130 mV. B, Representative
voltage-gated IK traces in somatotrophs (n = 5),
lactotrophs (n = 5), and gonadotrophs (n = 8) elicited by
1.5-sec voltage steps from -90 to 90 mV in 20-mV increments from a
holding potential of -40 mV. C, A transient IK in all
three cell types was isolated by a point-by-point subtraction of the
current traces in panels A and B. D, Current-voltage relationship of
the peak (025 msec; left panel), sustained (1.491.50
sec; middle panel) and subtracted (A - B;
right panel) IK in all three cell types. To
block IK(Ca), 200 µM CdCl2 was
added to the bath solution to block voltage-gated Ca2+
entry.
|
|
To isolate the slow-inactivating IK, the
transient IK was eliminated by the application of
a 500-msec holding potential to -40 mV before the 1.5-sec depolarizing
voltage steps from -90 mV to +90 mV (Fig. 4B
). Under these conditions,
a slow-inactivating IK was observed in
somatotrophs and lactotrophs (Fig. 4B
). A similar slow-inactivating
IK was observed in gonadotrophs in response to
voltage steps between -40 and 20 mV. However, a small
fast-inactivating IK was maintained at more
depolarized membrane potentials, which may be due to incomplete
inactivation of the transient IK in gonadotrophs
by the conditioning pulse. To compare the densities of the
slow-inactivating IK in each cell type, the
sustained IK evoked by voltage steps to +90 mV
(holding potential of -90 mV) was analyzed. The sustained
IK density in lactotrophs was 147 ±
15 pA/pF (n = 5), which was significantly (P <
0.05) smaller than that in somatotrophs (254 ± 32 pA/pF; n
= 5) and gonadotrophs (282 ± 32 pA/pF; n = 8).
We next compared the transient IK in each cell
type by subtracting the IK elicited during the
various voltage steps after a holding potential to -40 mV from the
total IK (Fig. 4C
). In rat somatotrophs, a small
transient IK was evoked by membrane potential
steps more depolarized than -30 mV, reaching a peak amplitude of
67 ± 18 pA/pF at +90 mV (n = 5). In contrast, a large
transient IK was observed in lactotrophs (n
= 5) and gonadotrophs (n = 8; Fig. 4
, C and D; right
panel). The transient IK in these cell types
activated at membrane potentials more depolarized than -50 mV and
reached maximum amplitude at +90 mV of 343 ± 73 pA/pF and
389 ± 82 pA/pF at +90 mV, respectively.
To determine the proportions of the total voltage-gated
IK in each cell type that is available for
activation at various different membrane potentials, the steady-state
inactivation properties of the IK were examined
using a two-pulse protocol. This protocol consisted of a series of
1.5-sec conditioning pulses from -130 mV to -10 mV, followed by a
400-msec test pulse to +90 mV (Fig. 5A
).
The representative IK tracings evoked by the
two-pulse protocol are shown in Fig. 5
, AC. The normalized test
current in each cell type was plotted against the conditioning pulse
potentials (Fig. 5D
). A single Boltzmann relation could not be fitted
to the data, further indicating the presence of a fast- and
slow-inactivating component in each of the three cell types. However,
the magnitude of steady-state IK inactivation in
somatotrophs was much less than that observed in the other two cell
types, which is consistent with the low expression levels of the
transient IK in somatotrophs.

View larger version (20K):
[in this window]
[in a new window]
|
Figure 5. Steady-State Inactivation of the Total
IK in Somatotrophs, Lactotrophs, and Gonadotrophs
AC, Steady-state inactivation curves for the peak voltage-gated
IK in each cell type were generated by stepping the
membrane potential to between -130 and -10 mV for 1.5 sec before
stepping to a command potential of 90 mV (holding potential = -90
mV). Representative traces of the remaining current elicited during the
command potential after conditioning pulses to -130, -110, -90,
-70, -50, -30, -10, and 10 mV are shown. D, The peak IK
evoked during the command potential to +90 mV all three cell types were
normalized to the maximum inward current and plotted against the
conditioning pulse potentials.
|
|
Ca2+-Activated K+
Channels
Voltage-gated Ca2+ entry was used to examine
the expression of IK(Ca) in somatotrophs,
lactotrophs, and gonadotrophs. To activate VGCCs, a modified two-step
protocol was used. This protocol consisted of an initial 100-msec
voltage-step to -10 mV (holding potential = -90 mV) during which
VGCCs were activated (Fig. 6A
; left
panel). This Ca2+-influx step was
immediately followed by a 500-msec test pulse to +80 mV, during which
the evoked IK was monitored. As +80 mV is near
the reversal potential for Ca2+ under our
experimental conditions, there should be minimal
Ca2+ entry during this step. Consistent with
this, in the absence of the Ca2+-influx step,
there was little to no change in
[Ca2+]i, whereas in the
presence of a Ca2+-influx step alone or in
combination with the test pulse there was a marked increase in
[Ca2+]i in all three cell
types (Fig. 7
). In addition, the
IK evoked by the test pulse to +80 mV in the
absence of the Ca2+-influx step was similar in
Ca2+-containing and
Ca2+-deficient medium (Table 2
).

View larger version (26K):
[in this window]
[in a new window]
|
Figure 6. Identification of IK-Ca in
Somatotrophs, Lactotrophs, and Gonadotrophs
AC, Left panels, Representative IK traces
in somatotrophs (n = 13), lactotrophs (n = 15), and
gonadotrophs (n = 9) evoked by the two-step protocol (A,
upper panel) in Ca2+-containing
(control) and Ca2+-deficient
(-Ca2+) medium. The two-step protocol consisted of a
100-msec condition pulse to -10 mV, to activated voltage-gated
Ca2+ entry, followed by a 500-msec test pulse to +90 mV,
during which the peak IK was monitored. AC, Right
panels, The net IK-Ca activated by the two-step
protocol in each cell type was isolated by subtracting the current
evoked in the presence of Ca2+-deficient medium from that
in Ca2+-containing medium. The mean ± SEM
values of the peak IKCa evoked during the test pulse are
shown.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Figure 7. Profile of [Ca2+]i in
Cells Exposed to One- and Two-Step Protocol.
AD, Representative [Ca2+]i traces without
(solid line) and with (dotted line) the
application of a 100-msec Ca2+-influx step to -10 mV
before the application of a 500-msec step to a test potential (TP) of
+80 mV or by the Ca2+-influx step (CIS) alone (thick
line) from a holding potential = -90 mV in somatotrophs
(n = 17), lactotrophs (n = 12), and gonadotrophs (n =
12). The net change in [Ca2+]i (mean ±
SEM) evoked by the TP, CIS, and TP + CIS are shown in the
right panel. The perforated patch-clamp recording
configuration was used to control the membrane potential in cells
preloaded with 0.5 µM Indo 1-AM.
|
|
View this table:
[in this window]
[in a new window]
|
Table 2. Peak IK in Somatotrophs,
Lactotrophs, and Gonadotrophs Evoked by a 500-msec Voltage Step from
-90 mV to +80 mV in Ca2+-Containing and
Ca2+-Deficient Medium
|
|
In the presence of a 100-ms Ca2+-influx step, the
peak IK evoked during the test pulse was reduced
by extracellular Ca2+ removal in somatotrophs,
lactotrophs, and gonadotrophs (Fig. 6
, AC, and Table 3
), indicating
IK(Ca) values are expressed in all three cell
types. To compare IK(Ca) activation between the
three cell types, the IK evoked in
Ca2+-deficient medium was subtracted from that in
Ca2+-containing medium (Fig. 6
, AC, right
panels). These results clearly indicate that
IK(Ca) activation by voltage-gated
Ca2+ entry was greatest in somatotrophs and
smallest in gonadotrophs.
View this table:
[in this window]
[in a new window]
|
Table 3. Effects of Ca2+-Deficient Medium
and Ca2+-Sensitive K+ Channel Blockers and
Activators on the Peak IK in Somatotrophs, Lactotrophs, and
Gonadotrophs
|
|
The differences in IK(Ca) activation between the
three cell types may be due to differences in the ability of the
Ca2+ influx step to drive voltage-gated
Ca2+ entry and increase
[Ca2+]i. To test this, we
compared the increase in
[Ca2+]i evoked by the
two-step protocol in each cell type (Fig. 7
). In somatotrophs and
gonadotrophs, the two-step protocol increased the
[Ca2+]i by 160 ± 18
nM (n = 17) and 164 ± 19 nM (n
= 12), respectively. In lactotrophs, a smaller increase in
[Ca2+]i of 122 ± 13
nM (n = 12) was evoked by the two-step protocol. These
results are consistent with the smaller amplitude noninactivating
ICa in lactotrophs compared with the other two
cell types (Fig. 2
). Nevertheless, these results indicate that the
small amplitude IK(Ca) observed in gonadotrophs
is not due to the inability of voltage-gated Ca2+
entry to increase
[Ca2+]i. Therefore, the
differences between IK(Ca) activation in the
three cell types appears to be due to differences in channel expression
and not the capacity of the Ca2+-influx step to
drive changes in
[Ca2+]i.
The dependence of IK(Ca) activation on
voltage-gated Ca2+ influx was further confirmed
in experiments with dihydropyridine agonist and antagonists. As shown
in Fig. 8
, A and C, addition of the L
type Ca2+ channel blocker, nifedipine,
significantly decreased the amplitude of the IK
in all three cell types studied. Furthermore, Bay K 8644, an L-type
Ca2+ channel agonist, significantly increased the
amplitude of IK in all three cell types (Fig. 8
, A and C). Consistent with the differential expression of
Ca2+-activated K+ channels
in the three cell types, the effects of nifedipine and Bay K 8644 were
small in gonadotrophs, and larger in lactotrophs and somatotrophs.

View larger version (27K):
[in this window]
[in a new window]
|
Figure 8. Dihydropyridine Sensitivity of ICa and
IK-Ca in Pituitary Cells
Under isolated IK recording conditions, the effects of 1
µM nifedipine (A) and 1 µM ()-Bay K 8644
(B) on the IK amplitude evoked during the TP by 50-msec
(somatotrophs) or 100-msec (lactotrophs and gonadotrophs) CIS were
examined, and the representative current recordings are shown. C, The
peak IK amplitude during the test pulse in the presence of
1 µM nifedipine or 1 µM (-)-Bay K 8644
were normalized to the levels reached in the presence of the vehicle
alone (control) and expressed as the mean ± SEM for
somatotrophs (n = 3), lactotrophs (n = 5), and gonadotrophs
(n = 3). Asterisks indicate significant differences
(P < 0.05) compared with control values.
|
|
To determine the IK(Ca) subtype activated by
voltage-gated Ca2+ entry in somatotrophs,
lactotrophs, and gonadotrophs, we used selective blockers and
stimulators of BK and SK channels. The specific BK channel blockers,
charybdotoxin (CTX; 100 nM), iberiotoxin (IBTX; 100
nM), and paxilline (1 µM), markedly reduced
the IK in all three cell types (Fig. 9
, AC and Table 3
). In addition, the BK
channel activator, NS 1619 (30 µM), increased
IK amplitude in somatotrophs and lactotrophs, but
not in gonadotrophs (Fig. 9D
and Table 3
). Unlike the BK channel
blockers, the SK channel blocker, apamin, had no effect on the
IK evoked by the 100-msec
Ca2+-influx step in the majority of somatotrophs
(n = 27) and lactotrophs (n = 11) examined. In the remaining
somatotrophs and lactotrophs, apamin significantly reduced the
IK (Fig. 9E
and Table 3
). In all gonadotrophs
examined (n = 10), apamin had no effect on the
IK evoked by the 100-msec
Ca2+-influx step (Fig. 9E
and Table 3
). This was
not due to the absence of SK channels in these cells, as GnRH simulated
ISK in all gonadotrophs (data not shown).

View larger version (34K):
[in this window]
[in a new window]
|
Figure 9. Pharmacological Identification of BK Channels in
Pituitary Cells
AD, Representative current traces showing the effects of 100
nM IBTX (A), 100 nM CTX (B), 1 µM
paxilline (C), 30 µM NS 1619 (D), and 100 mM
apamin (E), on the IK in somatotrophs, lactotrophs, and
gonadotrophs. Averaged data for each cell type are shown in Table 3 .
|
|
In the presence of apamin and CTX, IBTX, or paxilline, no further
reduction in IK was observed when the cells were
perifused with Ca2+-deficient medium (data not
shown). These results indicate that IK(Ca)
activation by voltage-gated Ca2+ influx in
somatotrophs, lactotrophs, and gonadotrophs is predominantly mediated
by BK channels. It should be noted, however, that the inability of
apamin to significantly reduce the IK in a
majority of the cells examined is most likely due to the relatively
small current generated by its activation and not the lack of
expression in each cell type. In addition, other
Ca2+-sensitive currents that are expressed in
pituitary cells, such as Ca2+-activated
Cl- channels (8, 9), may be masked by the much
larger IK in these cells.
To determine the duration of voltage-gated Ca2+
influx required to activate IBK in each cell
type, we varied the duration of the Ca2+-influx
step and measured the peak IK amplitude during
the subsequent 500-msec voltage step to +80 mV. To isolate BK channels
from SK channels, 100 nM apamin was added to the
extracellular medium. In somatotrophs, 5-msec
Ca2+-influx steps were sufficient to activate
IBK, whereas 10-msec steps were required in
lactotrophs. In both cell types, the peak IK
increased progressively in response to incremental increases in the
duration of the Ca2+-influx step (Fig. 10
, AC). This increase was not
observed in Ca2+-deficient medium (Fig. 10
, B and
C) or in cells preloaded with BAPTA-AM (Fig. 10C
). In gonadotrophs,
Ca2+-influx steps greater than 75 msec were
required to activate IBK (Fig. 10
, AC). In
addition, no increase in IBK activation was
observed in response to further increases in the duration of
voltage-gated Ca2+ influx (Fig. 10C
). These
results suggest that brief periods of voltage-gated
Ca2+ influx are sufficient to activate BK
channels in somatotrophs and lactotrophs, whereas prolonged
Ca2+ influx is required in gonadotrophs.
Moreover, it appears that increasing the duration of voltage-gated
Ca2+ influx recruits more BK channels in
somatotrophs and lactotrophs, but not gonadotrophs.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 10. Dependence of IBK Activation on
Duration of Voltage-Gated Ca2+ Influx
The effects of increasing the duration of VGCC entry on IK
activation were examined by incremental increases in the duration of
Ca2+ influx step from 0 to 100 msec, followed by a test
pulse to +80 mV for 500 msec. Representative current tracings recorded
in Ca2+-containing (A) or Ca2+-deficient medium
(B) are shown for somatotrophs (n = 13), lactotrophs (n =
12), and gonadotrophs (n = 12). C, Peak IK amplitude
(mean ± SEM) during the test pulse after 0- to
150-msec conditioning pulses in Ca2+-containing medium
(filled circles), Ca2+-deficient medium
(open circles), and in cells preloaded with BAPTA-AM
(filled triangles). Asterisks indicate a
significant difference compared with the peak IK evoked in
the absence of a depolarizing pulse. All experiments were performed in
the presence of 100 nM apamin to block SK channel
activation.
|
|
 |
DISCUSSION
|
---|
Despite the similar origin of somatotrophs, lactotrophs,
and gonadotrophs, these cells differ with respect to their pattern of
basal hormone secretion. Specifically, basal gonadotropin is low
compared with basal GH and PRL secretion. Earlier studies have
indicated that these differences in basal hormone secretion are due to
differences in spontaneous Ca2+ signaling among
the three cell types. Unlike basal GH and PRL secretion, basal
gonadotropin secretion is not dependent on the presence of
extracellular Ca2+ or modified by increases in
the extracellular Ca2+ concentration (4). In
addition, application of voltage-gated Ca2+
channel blockers reduces basal GH and PRL secretion, but not basal LH
or FSH secretion (4, 25). Consistent with these differences in the
apparent role of voltage-gated Ca2+ channels in
controlling basal hormone secretion, the profile of spontaneous
Ca2+ signaling among the three cell types is also
different. In rat lactotrophs and somatotrophs, spontaneous and high
amplitude Ca2+ signals have been observed,
whereas only low amplitude Ca2+ signals have been
observed in spontaneously active rat gonadotrophs (2, 3, 4, 25, 35). These
differences in the extracellular Ca2+ dependency
and voltage-gated Ca2+ channel involvement in
determining the pattern of basal hormone secretion and the cell
type-specific patterns of spontaneous Ca2+
signaling suggest that there are differences in the level and/or
composition of ionic channel expression and the underlying electrical
activity between rat gonadotrophs, lactotrophs, and somatotrophs.
In this study, we compared the expression levels of different ionic
channels between pituitary somatotrophs, lactotrophs, and gonadotrophs.
As a measure of the functional expression levels of the ionic channels
in each cell type, the isolated currents were analyzed. The results
indicate a quantitative rather than qualitative difference in the
expression pattern for the major ionic currents in pituitary cells. All
three cell types express transient and sustained voltage-gated
Ca2+ channels, TTX-sensitive
Na+ channels, transient and delayed rectifying
K+ channels, and multiple
Ca2+-sensitive K+ channel
subtypes. Lactotrophs and somatotrophs exhibited low expression levels
of TTX-sensitive Na+ channels and high expression
levels of BK channel compared with those observed in gonadotrophs. The
expression of the transient voltage-gated K+
channel was much higher in lactotrophs and gonadotrophs than in
somatotrophs. Finally, the expression of the transient VGCCs was higher
in somatotrophs than in lactotrophs and gonadotrophs.
TTX-sensitive Na+ channels have been previously
identified in gonadotrophs (10, 11), lactotrophs (12), and somatotrophs
(13), as well as other pituitary cell types (14, 15, 16), indicating that
these channels are common among mammalian pituitary cells. Our results
extended these findings by demonstrating that the level of
Na+ channel expression is much greater in rat
gonadotrophs than the other two cell types. However, it is unlikely
that the difference in the expression of these channels is relevant for
the control of spontaneous electrical activity (12, 17),
Ca2+ signaling (17), and basal hormone secretion
(16, 18) in these cells. The lack of TTX-sensitive
Na+ channel involvement in controlling membrane
excitability and secretion is most likely due to the inactivation of a
large proportion of these channels at the resting membrane potential in
these cells (Fig. 1
and Ref. 10). Consistent with this, GnRH-induced
transient membrane hyperpolarization in rat gonadotrophs is required to
remove the steady-state inactivation of TTX-sensitive
Na+ channels before they can contribute to action
potential (AP) firing (10). This TTX sensitivity during agonist-induced
AP firing in gonadotrophs is important for sustained
Ca2+ signaling through VGCCs and the refilling of
endoplasmic reticulum Ca2+ stores. There are,
however, two exceptions. First, in a fraction of ovine gonadotrophs,
these channels are responsible for AP generation (19). Second, two
lactotroph subpopulations have been identified that differ with respect
to their level of Na+ channel expression, and
only in lactotrophs expressing high levels of Na+
channels did TTX application abolish basal hormone secretion (20). In
the present study, we did not observe a significant difference in the
level of Na+ channel expression in the
lactotrophs identified by our cell separation and identification
protocol.
The expression of both inactivating and noninactivating VGCCs in rat
gonadotrophs (10, 21, 22), somatotrophs (13), and lactotrophs (13), as
well as immortalized pituitary cells (14, 15, 23, 24), has been well
documented. The inactivating ICa is mediated by
the low voltage-activated or transient (T)-type
Ca2+ channel. Although we observed T-type
ICa in all three cell types examined, it was more
prominent in somatotrophs than in lactotrophs and gonadotrophs. A
similar conclusion was reached in a study that directly compared VGCC
expression between somatotrophs and lactotrophs. The prominent
expression of T-type Ca2+ channels in
somatotrophs is reflected by their contribution to the generation of
the high amplitude
[Ca2+]i transients in
spontaneously active somatotrophs (25), whereas its role in other
native anterior pituitary cells is not known. The noninactivating
ICa in pituitary cells is mediated by
dihydropyridine-sensitive (L-type) and -insensitive, high-voltage
activated Ca2+ channels (10, 21, 24, 26). Unlike
the T-type Ca2+ channel, the current
voltage-relationship of the sustained ICa in all
three cell types was similar. However, the sustained
ICa density was higher in somatotrophs and
gonadotrophs than in lactotrophs. Despite the different densities of
the sustained ICa, it is essential to the
generation of both spontaneous and agonist-induced AP-driven
Ca2+ entry in all native and immortalized
pituitary cells (4, 27, 28). In addition, the L-type VGCC has been
demonstrated to contribute to the regulation of basal and
agonist-stimulated GH and PRL secretion, as well as agonist-induced
gonadotropin secretion (28, 29).
In general, the VGCC-dependent rise in
[Ca2+]i is sufficient to
trigger activation of several Ca2+-sensitive
channels. One such channel is the BK-type K+
channel, the expression of which has been previously demonstrated in
immortalized anterior pituitary cells (30, 31, 32), and native intermediate
pituitary cells (33). Our studies indicate that BK channels are also
expressed in native anterior pituitary cells, and that they are coupled
to voltage-gated Ca2+-influx in all three cell
types examined. Moreover, by comparing BK channel activation between
somatotrophs, lactotrophs, and gonadotrophs under identical culture and
recording conditions, it was demonstrated that BK channel activation
was much greater in somatotrophs than in gonadotrophs. Due to the
similarities in the voltage-gated ICa density and
change in [Ca2+]i evoked
by the Ca2+-influx step between the three cell
types, the differences in IBK activation are most
likely due to differences in BK channel expression levels.
In other excitable cells, the colocalization of BK channels with VGCCs
facilitates spike repolarization, which limits AP-driven
Ca2+ influx. BK channel activation can also
influence the frequency of AP-driven
[Ca2+]i transients by
slowing the pacemaker depolarization (34). In native anterior pituitary
cells, the role of BK channels in shaping the frequency and duration of
AP-driven Ca2+ entry is not known. Based on our
results, we would expect that the relatively high levels of BK channel
expression in somatotrophs and lactotrophs would limit AP-driven
Ca2+ influx compared with that in gonadotrophs,
which exhibit the lowest levels of BK channel expression. However,
previous studies have demonstrated that the duration of the AP waveform
is longer in somatotrophs and lactotrophs (100500 msec) than in
gonadotrophs (10100 msec) (10, 35, 36, 37). In addition, both the
amplitude and duration of the spontaneous, extracellular
Ca2+-dependent
[Ca2+]i transients are
greater in somatotrophs and lactotrophs than in gonadotrophs (2). It is
unlikely that the prolonged duration of AP-driven
Ca2+ entry in somatotrophs and lactotrophs is due
to the inability of AP firing to activate BK channels, as short
Ca2+ influx steps (<25 msec) were sufficient to
activate IBK in both cell types (Fig. 10
). Thus,
whether BK channels have an atypical role in regulating the pattern of
AP firing and Ca2+ signaling in anterior
pituitary cells requires further studies.
All three pituitary cell types also express SK channels (31, 38, 39, 40).
However, in gonadotrophs SK channels did not appear to be coupled to
voltage-gated Ca2+ influx under the conditions
used in our study. Similarly, voltage-gated Ca2+
influx activated SK channels in only a small fraction of the
somatotrophs and lactotrophs examined. This may be due to the lack of
SK channel expression in some somatotroph and lactotroph
subpopulations. Consistent with this, activation of
Ca2+-mobilizing TRH receptors leads to activation
of SK channels and the concomitant membrane hyperpolarization in
GH3 cells (32), but only a small fraction of
lactotrophs exhibited a similar response (41). It is also possible that
the SK channels in pituitary cells, as in other cell types (reviewed in
Ref. 42), are colocalized with intracellular Ca2+
release sites and can be activated only by
Ca2+-mobilizing receptors, sustained
voltage-gated Ca2+ entry, and/or
Ca2+-induced Ca2+ release.
For example, in GnRH-secreting neurons, agonist-induced
Ca2+ mobilization and the concomitant increase in
firing frequency are needed to activate SK channels (43). Similarly, in
GH3 cells, SK channel activation requires
high-frequency firing, prolongation of APs by voltage-dependent
K+ channel inhibitors, or release of
Ca2+ from intracellular
Ca2+ stores (31). The lack of a detectable
apamin-sensitive IK in a majority of the three
cell types examined may also be due to its relatively small size
compared with the voltage-gated IK and the
IBK in these cells. Other
Ca2+-sensitive channels, such as
Cl- channels [known to be expressed in AtT-20
immortalized cells (8) and native lactotrophs (9)] may also be
masked.
Several different voltage-gated K+ channel
subtypes have been identified and characterized in somatotrophs,
lactotrophs, and gonadotrophs (44, 45), as well as immortalized
pituitary cells (14, 15). One such channel is the transient,
4-AP-sensitive (A-type) K+ channel. Direct
comparison of the three anterior pituitary cells examined in this study
indicates that the expression level of the A-type
K+ channel is much higher in lactotrophs and
gonadotrophs than in somatotrophs. In contrast, these channels have
been observed in ovine somatotrophs and may contribute to AP firing and
hormone secretion (46). The participation of the A-type
K+ channel in regulating AP firing in other
anterior pituitary cell types is also unclear. In rat lactotrophs, for
example, they do not appear to participate in AP generation (37), which
may be due to their prominent inactivation at the resting membrane
potential in these cells. The participation of the delayed rectifying
K+ channel during AP firing in anterior pituitary
cells is also not clear. In immortalized cells, inhibition of this
channel by tetraethylammonium (TEA) increased the duration of the AP
(37) and the amplitude of the spontaneous
[Ca2+]i transients (47),
whereas in native rat lactotrophs, TEA did not alter the pattern of AP
firing (37). Further studies are required to elucidate the role of both
the A-type and delayed rectifying K+ channels in
the regulation of AP-driven Ca2+ signaling in the
different anterior pituitary cell types.
Our study was focused on the ionic channels involved in the formation
of the AP waveform. Other channels have also been identified in
pituitary cells, and they contribute to pacemaking activity in these
cells. For example, a TTX-insensitive INa has
been observed in somatotrophs (48) and lactotrophs (12) and is critical
for maintaining the membrane potential near the threshold for AP
firing. In addition, inward rectifier K+
channels, including the erg-like K+ channel, have
been identified in both native (36, 49) and immortalized pituitary
cells (50). They contribute to the regulation of both spontaneous and
agonist-modulated AP-driven Ca2+ entry. The
M-type K+ channel has also been identified in
pituitary cells (41), as well as several ATP-gated P2X receptor
channels (51). Preliminary data also suggested the presence of cyclic
nucleotide-gated channels in pituitary cells, which may contribute to
pacemaking activity (25).
In summary, we examined the cell-type specific expression of the major
ionic currents in somatotrophs, lactotrophs, and gonadotrophs that
contribute to AP-driven Ca2+ entry. All
experiments were done under identical experimental conditions on cells
from randomly cycling adult female rats. This allowed us to directly
compare the expression levels and voltage-dependent properties of
several ionic channels between the three anterior pituitary cell types.
Our results demonstrate that, although these cells originate from the
same precursor cell, they exhibit cell type-specific patterns of ionic
channel expression. In particular, the marked differences in BK channel
and transient (A-type) K+ channel expression
between the three cell types are excellent candidates for future
investigations into the cell type-specific patterns of spontaneous and
receptor-controlled membrane excitability, Ca2+
signaling, and hormone secretion.
 |
MATERIALS AND METHODS
|
---|
Pituitary Cell Culture and Cell Identification
Anterior pituitary glands were excised from female Sprague
Dawley rats (Taconic Farms, Inc., Germantown, NY) and
dispersed into single cells using a trypsin/DNase (Sigma,
St. Louis, MO) cell dispersion procedure as described previously (51).
Enriched somatotroph (>90%, estimated by immunocytochemistry using
specific antibodies provided by the National Hormone and Pituitary
Program and Dr. Parlow) and lactotroph (>65%) populations were
obtained using a discontinuous Percoll density-gradient cell-separation
procedure as described previously (51). Pituitary somatotrophs and
lactotrophs were further identified using a combination of cell
separation techniques, their distinct morphology, and responses to GHRH
and TRH, respectively. Gonadotrophs were initially identified by their
distinct morphology and subsequent to experimentation by their unique
oscillatory IK and/or
[Ca2+]i response to GnRH,
the specific agonist for these cells.
Electrophysiological Recordings
Voltage-gated INa and
ICa were measured using regular whole-cell
recording techniques, whereas voltage-gated IK
and IK(Ca) were measured using perforated-patch
recording techniques (52). All voltage-clamp recordings were performed
at room temperature using an Axopatch 200 B patch-clamp amplifier (Axon
Instruments, Foster City, CA) and were low-pass filtered at 2 kHz. A
series resistance of < 20 M
was reached 10
min after the formation of a gigaohm seal (seal resistance > 5
G
). When necessary, series resistance compensation was optimized and
all current recordings were corrected for linear leakage and
capacitance using a P/-N procedure. An average membrane
capacitance (Cm) of 4.6 ± 0.2 pF (n =
48), 5.9 ± 0.2 pF (n = 47), and 7.5 ± 0.2 pF (n =
68) was recorded in somatotrophs, lactotrophs, and gonadotrophs,
respectively. Pulse generation and data acquisition were done with a PC
equipped with a Digidata 1200 A/D interface in conjunction with Clampex
8 (Axon Instruments).
Simultaneous Measurement of
[Ca2+]i and
IK
To simultaneously monitor
[Ca2+]i and
IK the cells were incubated for 15 min at 37 C in
phenol red-free medium 199 containing Hanks salts, 20 mM
sodium bicarbonate, 20 mM HEPES, and 0.5 µM
indo-1 AM (Molecular Probes, Inc., Eugene, OR). The
coverslips with cells were then washed twice with modified
Krebs-Ringers solution containing (in mM): 120 NaCl, 4.7 KCl, 2.0
CaCl2, 2 MgCl2, 0.7
MgSO4, 10 HEPES, 10 glucose (pH adjusted to 7.4
with NaOH) and mounted on the stage of an inverted epifluorescence
microscope (Nikon, Melville, NY). A photon counter system
(Nikon) was used to simultaneously measure the intensity
of light emitted at 405 nm and at 480 nm after excitation at 340 nm.
Background intensity at each emission wavelength was corrected.
Perforated-patch recording techniques (see above) were used to control
the membrane potential and monitor IK in the
voltage-clamp recording mode. The data were digitized at 4 kHz using a
PC equipped with the Clampex 8 software package in conjunction with a
Digidata 1200 A/D converter (Axon Instruments). The
[Ca2+]i was calibrated
in vivo as described by Kao (53). Briefly,
Rmin was determined by exposing the cells to 10
µM Br-A23187 in the presence of Krebs-Ringers
solution with 2 mM EGTA and 0
mM Ca2+ for 60 min; 15
mM Ca2+ was then added to
determine Rmax. The values used for
Rmin, Rmax,
Sf,480/Sb,480, and
dissociation constant (Kd) were 0.677, 2.9,
2.473, and 230 nM, respectively.
Chemicals and Solutions
For all IK recordings, the extracellular
medium contained modified Krebs-Ringer salts containing (in mM): 120
NaCl, 4.7 KCl, 2.0 CaCl2, 2
MgCl2, 0.7 MgSO4, 10 HEPES,
10 glucose and 1 µM TTX (pH adjusted to 7.4 with NaOH)
and the pipette solution contained (in mM): 50 KCl, 90 K-aspartate, 1
MgCl2, and 10 HEPES (pH adjusted to 7.2 with
KOH). To isolate the voltage-gated IK, the
extracellular medium contained 200 µM
Cd2+ and 100 µM TTX. In some
experiments, extracellular Ca2+ was replaced with
equimolar Mg2+. To isolate
INa and ICa, conventional
whole-cell recording techniques were used as previously described (52).
The extracellular medium contained Krebs-Ringers solution with 20
mM TEA and 2 mM CaCl2 (pH
adjusted to 7.4 with NaOH) and the pipette contained (in mM); 120 CsCl,
20 TEA-Cl, 4 MgCl2, 10 EGTA, 9 glucose, 20 HEPES,
0.3 Tris-GTP, 4 Mg-ATP, 14 CrPO4, and 50 U/ml
creatine phosphokinase (pH adjusted to 7.2 with Tris base). To isolate
the INa or ICa, 200
µM Cd2+ or 1 µM TTX
was added to extracellular medium, respectively. All reported membrane
potentials were corrected on line for a liquid junction potential of 10
mV between the pipette and bath solution, except for
INa and ICa, which required
a correction of 7 mV. The bath contained <500 µl of saline and was
continuously perfused at a rate of 2 ml/min using a gravity-driven
superfusion system. Stock solutions of TTX,
isobutylmethylxanthine, and CTX were prepared in
double-distilled, deionized water, whereas stock solutions of
paxilline, nifedipine and S()-Bay K 8644 were prepared in
dimethylsulfoxide. All chemicals were obtained from
Sigma-Aldrich Corp. (St. Louis, MO). In some experiments,
the cells were loaded with 1 µM BAPTA-AM
(Sigma-Aldrich Corp.) at 37 C for 45 min.
Data Analysis
Data analysis was performed using Clampfit (Axon Instruments).
In some cases, the current-voltage relations were fit with a single
Boltzmann relation: I/Imax =
Imax + exp[(E -
E1/2)/k]; where
Imax is the maximum current, E
is the command potential, E1/2 is the
membrane potential at which there is 50% of the maximal current, and
k is the slope factor. The results shown are typical tracing
or means ± SEM for at least five
recordings. Differences between groups were considered to be
significant with P < 0.05 or higher, calculated by
paired t test or ANOVA, followed by Fishers least
significant differences test.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Fredrick Van Goor, c/o Stanko S. Stojilkovic, Section on Cellular Signaling, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 6A36, 49 Convent Drive, Bethesda, Maryland 20892-4510. E-mail:
stankos{at}helix.nih.gov
Received for publication July 10, 2000.
Revision received March 27, 2001.
Accepted for publication April 2, 2001.
 |
REFERENCES
|
---|
-
Burrows HL, Douglas KR, Seasholtz AF, Camper SA 1999 Genealogy of the anterior pituitary gland: tracing a family tree.
Trends Endocrinol Metabol 10:343352[CrossRef][Medline]
-
Stojilkovic SS, Catt KJ 1992 Calcium oscillations in anterior
pituitary cells. Endocr Rev 13:256280[Medline]
-
Kwiecien R, Hammond C 1998 Differential management of
Ca2+ oscillations by anterior pituitary cells: a
comparative overview. Neuroendocrinology 68:135151[CrossRef][Medline]
-
Stojilkovic SS, Izumi S-I, Catt KJ 1988 Participation of
voltage-sensitive calcium channels in pituitary hormone secretion.
J Biol Chem 263:1305413061[Abstract/Free Full Text]
-
Bluet-Pajot M-T, Epelbaum J, Gourdji D, Hammond C, Kordon C 1998 Hypothalamic and hypophyseal regulation of growth hormone
secretion. Cell Mol Neurobiol 18:101123[CrossRef][Medline]
-
Freeman ME, Kanicska B, Lernat A, Nagy G 2000 Prolactin:
structure, function, and regulation of secretion. Physiol Rev 80:15231631[Abstract/Free Full Text]
-
Stojilkovic SS, Reinhart J, Catt KJ 1994 GnRH receptors:
structure and signal transduction pathways. Endocr Rev 15:462499[Medline]
-
Korn SJ, Bolden A, Horn R 1991 Control of action potentials
and Ca2+ influx by the
Ca2+-dependent chloride current in mouse
pituitary cells. J Physiol 439:423437[Abstract]
-
Sator P, Dufy-Barbe L, Vacher P, Dufy B 1992 Calcium-activated chloride conductance of lactotrophs; comparison of
activation in normal and tumoral cells during
thyrotropin-releasing-hormone stimulation. J Membr Biol 126:3949[Medline]
-
Tse A, Hille B 1993 Role of voltage-gated
Na+ and Ca2+ channels in
gonadotropin-releasing hormone-induced membrane potential changes in
identified rat gonadotropes. Endocrinology 132:14751481[Abstract]
-
Marchetti C, Childs GV, Brown AM 1987 Membrane currents of
identified isolated rat corticotropes and gonadotropes. Am J
Physiol 252:E340E346
-
Sankaranarayanan S, Simasko SM 1996 A role for a background
sodium current in spontaneous action potentials and secretion from rat
lactotrophs. Am J Physiol 271:C1927C1934
-
Lewis DL, Goodman MB, St.John PA, Barker JL 1988 Calcium
currents and fura-2 signals in fluorescence-activated cell sorted
lactotrophs and somatotrophs of rat anterior pituitary. Endocrinology 123:611621[Abstract]
-
Dubinsky JM, Oxford GS 1984 Ionic currents in two strains of
rat anterior pituitary tumor cells. J Gen Physiol 83:309339[Abstract]
-
Bosma MM, Hille B 1992 Electrophysiological properties of a
cell line of the gonadotrope lineage. Endocrinology 130:34113420[Abstract]
-
Mason WT, Skidar SK 1988 Characterization of voltage-gated
sodium channels in ovine gonadotrophs: relationship to hormone
secretion. J Physiol 399:493551[Abstract]
-
Stojilkovic SS, Kukuljan M, Iida T, Rojas E, Catt KJ 1992 Integration of cytoplasmic calcium and membrane potential oscillations
maintains calcium signaling in pituitary gonadotrophs. Proc Natl Acad
Sci USA 89:40814085[Abstract]
-
Mason WT, Rawlings SR 1988 Whole-cell recordings of ionic
currents in bovine somatotrophs and their involvement in growth hormone
secretion. J Physiol 405:577593[Abstract]
-
Heyward PM, Chen C, Clarke IJ 1995 Inward membrane currents
and electrophysiological responses to GnRH in ovine gonadotropes.
Neuroendocrinology 61:609621[Medline]
-
Horta J, Hiriart M, Gota G 1991 Differential expression of Na
channels in functional subpopulations of rat lactotropes. Am J
Physiol 261:C865871
-
Stutzin A, Stojilkovic SS, Catt KJ, Rojas E 1989 Characteristics of two types of calcium channels in rat pituitary
gonadotrophs. Am J Physiol 257:C865C874
-
Marchetti C, Childs GV, Brown AM 1990 Voltage-dependent
calcium currents in rat gonadotropes separated by centrifugal
elutriation. Am J Physiol 258:E589E596
-
Simasko SM, Weiland GA, Oswald RE 1988 Pharmacological
characterization of two calcium currents in GH3
cells. Am J Physiol 254:E328E336
-
Kwiecien R, Robert C, Cannon R, Vigues S, Arnoux A, Kordon C,
Hammond C 1998 Endogenous pacemaker activity of rat tumour
somatotrophs. J Physiol 508:883905[Abstract/Free Full Text]
-
Tomic M, Koshimizu T, Yuan D, Andric SA, Zivadinovic D,
Stojilkovic SS 1999 Characterization of a plasma membrane calcium
oscillator in rat pituitary somatotrophs. J Biol Chem 274:3569335702[Abstract/Free Full Text]
-
Kuryshev YA, Childs GV, Ritchie AK 1995 Three high threshold
calcium channel subtypes in rat corticotropes. Endocrinology 136:39163924[Abstract]
-
Mollard P, Theler J-M, Guerineau N, Vacher P, Chiavaroli C,
Schlegel W 1994 Cytosolic Ca2+ of excitable
pituitary cells at resting potentials is controlled by steady state
Ca2+ currents sensitive to dihydropyridines.
J Biol Chem 269:2515825164[Abstract/Free Full Text]
-
Stojilkovic SS, Iida T, Virmani MA, Izumi S-I, Rojas E, Catt
KJ 1990 Dependence of hormone secretion on activation-inactivation
kinetics of voltage-sensitive Ca2+ channels in
pituitary gonadotrophs. Proc Natl Acad Sci USA 87:88558859[Abstract]
-
Cota G, Hiriart M, Horta J, Torres-Escalante JL 1990 Calcium
channels and basal prolactin secretion in single male rat lactotropes.
Am J Physiol 259:C949959
-
Shipston MJ, Kelly JS, Antoni FA 1996 Glucocorticoids block
protein kinase A inhibition of calcium-activated potassium channels.
J Biol Chem 271:91979200[Abstract/Free Full Text]
-
Lang DG, Ritchie AK 1990 Tetraethylammonium blockade of
apamin-sensitive and insensitive Ca2+-activated
K+ channels in a pituitary cell line. J
Physiol 425:117132[Abstract]
-
Ritchie AK 1987 Thyrotropin-releasing hormone stimulates a
calcium-activated potassium current in a rat anterior pituitary cell
line. J Physiol 385:611625[Abstract]
-
Kehl SJ, Wong K 1996 Large-conductance calcium-activated
potassium channels of cultured rat melanotrophs. J Membr Biol 150:219230[CrossRef][Medline]
-
Sah P 1996 Ca2+-activated
K+ currents in neurons: types, physiological
roles and modulation. Trends Neurosci 19:150154[CrossRef][Medline]
-
Li Y-X, Rinzel J, Vergara L, Stojilkovic SS 1995 Spontaneous
electrical and calcium oscillations in pituitary gonadotrophs. Biophys
J 69:785795[Abstract]
-
Sims SM, Lussier BT, Kraicer J 1991 Somatostatin activates an
inwardly rectifying K+ conductance in freshly
dispersed rat somatotrophs. J Physiol 441:615637[Abstract]
-
Sankaranarayanan S, Simasko SM 1998 Potassium channel blockers
have minimal effect on repolarization of spontaneous action potentials
in rat pituitary lactotropes. Neuroendocrinology 68:297311[CrossRef][Medline]
-
Kukuljan M, Stojilkovic SS, Rojas E, Catt KJ 1992 Apamin-sensitive potassium channels mediate agonist-induced
oscillations of membrane potential in pituitary gonadotrophs. FEBS Lett 301:1922[CrossRef][Medline]
-
Tse A, Hille B 1992 GnRH-induced Ca2+
oscillations and rhythmic hyperpolarizations of pituitary gonadotropes.
Science 255:462464[Medline]
-
Ritchie AK 1987 Two distinct calcium-activated potassium
currents in a rat anterior pituitary cell line. J Physiol 385:591609[Abstract]
-
Sankaranarayanan S, Simasko SM 1996 Characterization of an
M-like current modulated by thyrotropin-releasing hormone in normal
rat lactotrophs. J Neurosci 16:16681678[Abstract]
-
Berridge MJ 1998 Neuronal calcium signaling. Neuron 21:1326[Medline]
-
Van Goor F, Krsmanovic LZ, Catt KJ, Stojilkovic SS 1999 Coordinate regulation of gonadotropin-releasing hormone neuronal firing
patterns by cytosolic calcium and store depletion. Proc Natl Acad Sci
USA 96:41014106[Abstract/Free Full Text]
-
Herrington J, Lingle CJ 1994 Multiple components of
voltage-dependent potassium current in normal rat anterior pituitary
cells. J Neurophysiol 72:719729[Abstract/Free Full Text]
-
Lingle CJ, Sombati S, Freeman ME 1986 Membrane currents in
identified lactotrophs of rat anterior pituitary. J Neurosci 6:29953005[Abstract]
-
Chen C, Heyward P, Zhang J, Wu D, Clarke IJ 1994 Voltage-dependent potassium currents in ovine somatotrophs and their
function in growth hormone secretion. Neuroendocrinology 59:19[Medline]
-
Charles AC, Piros ET, Evans CJ, Hales TG 1999 L-type
Ca2+ channels and K+
channels specifically modulate the frequency and amplitude of
spontaneous Ca2+ oscillations and have distinct
roles in prolactin release in GH3 cells. J
Biol Chem 274:75087515[Abstract/Free Full Text]
-
Takano K, Takei T, Teramoto A, Yamashita N 1996 GHRH activates
a nonslective cation current in human GH-secreting adenoma cells.
Am J Physiol 270:E1014E1057
-
Schafer R, Wulfsen I, Behrens S, Weinsberg F, Bauer CK,
Schwartz JR 1999 The erg-like potassium current in rat lactotrophs.
J Physiol 518:401416[Abstract/Free Full Text]
-
Bauer CK 1998 The erg inwardly rectifying
K+ current and its modulation by
thyrotrophin-releasing hormone in giant clonal rat anterior pituitary
cells. J Physiol 510:6370[Abstract/Free Full Text]
-
Koshimizu T, Tomic M, Wong AOL, Zivadinovic D, Stojilkovic
SS 2000 Characterization of purinergic receptors and receptor-channels
expressed in anterior pituitary cells. Endocrinology 141:40914099[Abstract/Free Full Text]
-
Van Goor F, Krsmanovic LZ, Catt KJ, Stojilkovic SS 1999 Control of action potential-driven calcium influx in GT1 neurons by the
activation status of sodium and calcium channels. Mol Endocrinol 13:587603[Abstract/Free Full Text]
-
Kao JPY 1994 Practical aspects of measuring
[Ca2+] with fluorescent indicators. Methods
Cell Biol 40:155181[Medline]
-
Van Goor F, Zivadinovic D, Wong AOL, Stojilkovic SS 2000 Calcium-activated, voltage-dependent K+ (BK)
channels account for differences in the spiking pattern between
spontaneously active rat somatotrophs, lactotrophs, and gonadotrophs.
30th Annual Meeting of the Society for Neuroscience, New Orleans, LA
(Abstract 689.9)