An extracellular sulfhydryl group modulates background
Na+ conductance and cytosolic Ca2+ in pituitary
cells
Rosalba I.
Fonteriz,
Carlos
Villalobos, and
Javier
García-Sancho
Instituto de Biología y Genética Molecular,
Universidad de Valladolid y Consejo Superior Investigaciones
Científicas, Departamento de Fisiología y
Bioquímica, Facultad de Medicina, 47005 Valladolid, Spain
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ABSTRACT |
Treatment of GH3 pituitary
cells with p-chloromercurybenzenesulfonate (PCMBS) increased
the cytosolic Ca2+ concentration
([Ca2+]i). This effect was reversed by
dithiothreitol and blocked by L-type Ca2+ channel
antagonists or Na+ removal. PCMBS increased membrane
conductance and depolarized the plasma membrane. Apart from minor
effects on K+ and Ca2+ channels, PCMBS
increased (6 times at
80 mV) an inward Na+ current whose
properties were similar to those of a background Na+
conductance (BNC) described previously, necessary for generation of
spontaneous electrical activity. In rat lactotropes and somatotropes in
primary culture, PCMBS also produced a Na+-dependent
[Ca2+]i increase, whereas little or no effect
was observed in thyrotropes, corticotropes, and gonadotropes. The
Na+ conductance elicited by PCMBS in somatotropes seemed to
be the same as that stimulated by the hypothalamic growth hormone
(GH)-releasing hormone, which regulates membrane excitability and GH
secretion. The BNC studied here could play a physiological role,
regulating excitability and spontaneous activity, and explains
satisfactorily the [Ca2+]i-increasing actions
of the mercurials reported previously in several excitable tissues.
GH3 cells; sodium current; mercurials; p-chloromercurybenzenesulfonate; neurotoxicity
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INTRODUCTION |
MERCURY
(Hg2+) is a common environment contaminant. Both organic
and inorganic forms are neurotoxic, and the hypothesis that the effects
of Hg2+ can be mediated by changes in the intracellular
Ca2+ concentration ([Ca2+]i) has
received considerable attention (3, 8). For example, in
PC-12 cells Hg2+ is able to produce an increase in
[Ca2+]i due to Ca2+ entry through
voltage-gated Ca2+ channels, which can lead either to cell
differentiation or to cell death, depending on the Hg2+
concentration used (31). Several effects on plasma
membrane ion channels, mainly K+ and Ca2+
channels, have been reported, but they do not explain the observed changes in [Ca2+]i (6, 13, 16, 20, 22,
25, 34). In addition, most of these studies have been carried
out with membrane-permeant mercurials, which can act, with different
time lags, at both the extracellular and the intracellular side, thus
complicating the interpretation of the results.
Here we find that the membrane-impermeant mercurial
p-chloromercurybenzenesulfonate (PCMBS) produces a large
increase of [Ca2+]i in pituitary cells. A
systematic study of the actions of PCMBS on the different plasma
membrane ionic currents reveals that, apart from minor effects on
K+ and Ca2+ channels, the action on
[Ca2+]i arises from activation of an inward
Na+ current and membrane depolarization. The activity of
this Na+ conductance controls the firing rate in
GH3 pituitary cells and in lactotropes (32,
36). Growth hormone-releasing hormone (GHRH) is known to act on
somatotropes by stimulating a similar Na+ conductance
(18, 19, 21, 29, 37). Thus, in addition to explaining the
mechanism for the stimulation of Ca2+ influx by mercurials,
our results suggest that a sulfhydryl group facing toward the
extracellular side of the membrane may be involved in regulation of the
activity of pituitary cells. It has been shown recently that regulation
of plasma membrane permeability to Na+ may be crucial for
control of membrane potential, firing rate, and secretion in other
endocrine cells (23, 24).
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METHODS |
GH3 pituitary cells and rat anterior pituitary (AP)
cells were prepared and grown as described previously (39, 40,
42). The cells were seeded over glass coverslips coated with
poly-L-lysine (0.01 mg/ml, 10 min). Enriched somatotropes
were prepared as follows: 1-3 × 106 freshly
prepared AP cells were suspended in 0.5 ml of standard medium (see
composition below) containing 40% Percoll. This cell suspension was
placed into an Eppendorf tube on top of a discontinuous Percoll
gradient formed by the following layers (from top to bottom): 40%
Percoll (0.2 ml), 50% Percoll (0.2 ml), and 80% Percoll (0.1 ml).
After a 5-min centrifugation at 3,000 g, the enriched cell population (~10% of the starting cells) was recovered at the
50-80% interphase, washed with standard medium, and plated on
coverslips as described above. Immunocytochemistry showed that 73 ± 2% of these cells stored growth hormone (mean ± SE;
n = 5).
[Ca2+]i measurements were performed by
time-resolved digital image analysis in fura 2-loaded cells as
described previously (39, 40, 42). The standard incubation
medium had the following composition (in mM): 145 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, and 10 sodium-HEPES, pH 7.35. The high-K+ (50 mM) solutions were
prepared by replacing 45 mM of NaCl by KCl. All experiments were
performed at 37°C. At the end of the [Ca2+]i measurements, AP cells present in the
microscopic field were typed by multiple sequential primary
immunocytochemistry by using antibodies against the pituitary hormones
labeled with Oregon green 488 (41, 43). This allowed
identification of up to three different cell types (e.g., somatotropes,
lactotropes, and corticotropes in Fig. 9) in the same microscope
field. Mn2+ entry was measured by quenching of fura 2 fluorescence as described previously (39, 40).
Patch-clamp experiments were performed on a Nikon Diaphot 200 inverted
microscope by using an Axopatch-1D patch-clamp amplifier (Axon
Instruments, Foster City, CA). For recording and analysis we used WCP
software (John Dempster, Dept. of Physiology & Pharmacology, Strathclyde University, Glasgow, Scotland). Coverslips with cells were
placed on a Lucite chamber (volume 200 µl) on the microscope stage
and perfused at ~2 ml/min at room temperature (22-28°C). Bath
exchanges were accomplished by switching the bath inflow line. Both
standard whole cell patch-clamp (12) and patch-perforated (15) methods were employed for electrophysiological
recording. Perforated patches were achieved by using nystatin (final
concentration 500 µg/ml). Patch electrodes had tip resistances of
4-7 M
. Access resistance was determined from the series
resistance compensation dial on the patch amplifier. In standard whole
cell recordings, access resistances ranged between 5 and 55 M
.
Perforated patches reached stable access resistances (20-40 M
)
after 5-10 min. An Ag-AgCl pellet placed in the efflux line of the
chamber was used as a ground electrode. The offset potential between
the pipette and the bath (1-4 mV) was compensated with the
patch-clamp amplifier. No series resistance compensation was used.
Corrections of the voltage drop across the access resistance for the
calculation of reversal potential were not made because the current was
very small and the voltage artifact negligible.
The standard bath solution was as described above for Ca2+
measurements. In the Na+ substitution experiments, NaCl was
replaced by equivalent amounts of
N-methyl-D-glucamine (NMDG) or
tris(hydroxymethyl)aminomethane (Tris), adjusted to neutral pH with
HCl. For measurements of Ca2+ currents, 10 mM
CaCl2 replaced an equivalent amount of NaCl. For
measurements of Na+ currents, the external solution
contained (in mM) 150 NaCl, 1 MgCl2, 10 glucose, 5 HEPES-Tris, pH 7.35, 2 NiCl2 (to block Ca2+
currents), 1 BaCl2, and 2 CsCl (to block the delayed
rectifier K+ currents). Pipette solutions for standard
whole cell recording contained (in mM) 140 KCl, 2 MgCl2,
0.7 CaCl2, 1.1 EGTA (free Ca2+ concentration
100 nM), and 10 sodium-HEPES, pH 7.2. Pipette solutions for
perforated-patch recording contained (in mM) 65 KCl, 30 K2SO4, 10 NaCl, 1 MgCl2, 50 sucrose, and 20 potassium-HEPES, pH 7.2. Pipette solution for recording
Ca2+ currents and background Na+ conductance
contained (in mM) 65 CsCl, 30 Cs2SO4, 10 NaCl,
1 MgCl2, 50 sucrose, and 20 cesium-HEPES, pH 7.2.
Antisera against pituitary hormones were the same batches cited
previously (41, 43). Furnidipine was a generous
gift from Laboratorios Alter (Madrid, Spain). Calciseptine was
purchased from Latoxan (Rosans, France). Fura 2-AM and Oregon green
488-isothiocyanate were purchased from Molecular Probes (Eugene, OR).
Other chemicals were obtained from either Sigma (Madrid, Spain) or
Merck (Darmstadt, Germany).
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RESULTS |
Treatment with PCMBS increases
[Ca2+]i in GH3
cells.
Figure 1A shows the effects of
treatment with PCMBS (50 µM) in GH3 cells loaded with
fura 2. After a lag of ~30 s, [Ca2+]i
increased from the resting value of ~100 nM to 800 nM. Washing PCMBS
did not reverse the effect. Perfusion with furnidipine (1 µM), a
dihydropyridine antagonist of L-type Ca2+ channels
(26), decreased [Ca2+]i toward
the resting values [85% decrease of the change
(
[Ca2+]i)]. When the dihydropyridine was
washed, [Ca2+]i rose again, indicating that
the [Ca2+]i-increasing ability of PCMBS was
still present. Perfusion with the sulfhydryl-reducing agent
dithiothreitol (DTT; 2 mM) at this time restored
[Ca2+]i to resting levels. When DTT was
washed, no further changes in [Ca2+]i took
place, indicating permanent reversion of the effects of the mercurial.
Single-cell analysis showed that all the GH3 cells were
similarly responsive to PCMBS. Figure 1B shows the effects of the specific L-channel blocker calciseptine (9). After
treatment with PCMBS and reversion of the
[Ca2+]i increase with DTT, the cells were
incubated with calciseptine (1 µM) for 20 min. A new treatment with
PCMBS at this time had no effect on [Ca2+]i.
After a 15-min wash of calciseptine, a new treatment with PCMBS again
produced the [Ca2+]i increase, which was
antagonized by furnidipine. Thus our results suggest that the increase
of [Ca2+]i induced by PCMBS is due to
Ca2+ entry through L-type Ca2+ channels. The
membrane-impermeant HS reagents (2-aminoethyl)- methanethiosulfonate
and (2-sulfonatoethyl)methanethiosulfonate (introducing positive and
negative net charge, respectively; Ref. 1) also induced an
increase of [Ca2+]i, which was reversed by
DTT (results not shown).

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Fig. 1.
Treatment with p-chloromercurybenzenesulfonate
(PCMBS) increases [Ca2+]i and accelerates
Mn2+ entry in GH3 pituitary cells.
Concentrations of PCMBS, furnidipine, and dithiothreitol (DTT) were 50, 1, and 2,000 µM, respectively. A: average of 44 cells
present in the same microscope field. B: average of 112 cells; cells were treated with 1 µM calciseptine for 20 min and then
washed with fresh medium for 15 min. C: MnCl2
(0.5 mM) was added 30 s before PCMBS. Furnidipine was added
30 s before Mn2+. Traces corresponding to control and
furnidipine-treated cells are superimposed. Fluorescence emission is
represented as a percentage of the value at the time PCMBS was added.
The traces shown are representative of 3-5 similar experiments.
[Ca2+]i, intracellular Ca2+
concentration; Furni, furnidipine.
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Mn2+ is able to permeate through L-type Ca2+
channels (38). In Fig. 1C, Mn2+ was
used as a tracer for Ca2+ entry. The entry of
Mn2+ was estimated from the quenching of the fura 2 fluorescence excited at 360 nm, a wavelength that is insensitive to
[Ca2+] (40). After a brief lag (~15 s),
treatment with PCMBS increased the rate of fluorescence quenching
16-fold. In the presence of furnidipine, the effect of PCMBS was nearly abolished.
Effects of PCMBS on membrane potential and excitability.
To identify the target of PCMBS, we studied its effects on the
electrical activity of GH3 cells. Figure
2 shows a typical experiment of membrane
voltage measurement in the current-clamp mode. Treatment with PCMBS
caused depolarization of the cell membrane. The lower traces in Fig. 2
show time-expanded records of spontaneous action potentials recorded
under the control condition (left), after addition of PCMBS
(middle), and after exposition to DTT (right).
PCMBS depolarized the cells, decreased the amplitude of the action
potentials, and, in most cases, increased the firing frequency. These
effects were reversed by DTT. In 22 similar experiments, the mean
depolarization (±SE), measured 3 min after treatment with PCMBS, was
15 ± 2 mV. The membrane depolarization and the increase of firing
frequency could explain the increase of
[Ca2+]i. We next investigated which membrane
current could be the one responsible for the depolarizing effect of
PCMBS.

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Fig. 2.
Effects of PCMBS on membrane potential (Vm)
and spontaneous firing of GH3 cells. Top:
perforated-patch measurements of electrical activity of
GH3 cells in current-clamp mode. Bottom:
time-expanded records of spontaneous action potentials in the control
condition (left), after addition of 50 µM PCMBS
(middle), and 3 min after washing with the reducing agent
DTT (2 mM) (right). Results are representative of 22 similar
experiments.
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Effect of PCMBS on Ca2+ and
K+ currents.
Ca2+ currents were the first candidates to explain the
observed effects. Whole cell Ca2+ currents were recorded by
using the perforated-patch technique with Cs+-loaded
micropipettes and elevated (10 mM) bath Ca2+ (see
METHODS for details). To study the effect on the
noninactivating Ca2+ currents, we held cells at
40 mV and
then depolarized to +40 mV in 10-mV voltage jumps. Figure
3A shows the average of the current-voltage (I-V) curves obtained in four similar
experiments. There was no significant difference between the mean
values obtained in control and PCMBS-treated cells (Student's
t-test, paired data). The peak Ca2+ current was
not significantly modified by PCMBS either. In another set of
experiments cells were held at
80 mV and depolarized to +10 mV. In
the average of three experiments there was no effect of PCMBS on
transient currents (Fig. 3B), although in one case there was
a small inhibition of the current (Fig. 3C). Thus the increase of [Ca2+]i cannot be explained by an
action of PCMBS on Ca2+ channels.

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Fig. 3.
A: current-voltage (I-V) curves for
Ca2+ currents elicited by 10-mV step depolarizations to 40 mV from a holding potential of 40 mV in the control condition and
after PCMBS 50 µM treatment (+PCMBS), measured by perforated patch.
Values are means ± SE of 4 experiments. B: bar graph
representing average of peak currents elicited by depolarization to +10
mV from a holding potential of 80 mV in the control condition and
after perfusion of PCMBS (n = 3). C: example
of Ca2+ current record used to calculate data in
B.
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Membrane depolarization by inhibition of K+ channels would
favor activation of voltage-gated Ca2+ channels and
Ca2+ entry. We therefore studied the effects of PCMBS on
K+ currents. Dubinsky and Oxford (10)
described two different outward K+ currents in
GH3 cells, one compatible with the delayed rectifier or
voltage-dependent K+ channel (IKv) and the
other with Ca2+-activated K+ channels
(IKCa) (see also Ref. 30). The whole outward
K+ current was measured by applying 100-ms square
depolarizing pulses in 10-mV steps from a holding potential of
50 mV.
Figure 4A shows the normalized
I-V plot constructed with the average results of seven
experiments. There was a 30% inhibition at the more depolarized potentials, but the difference was not significant (Student's t-test, paired data). In another set of experiments, 0.2 mM
CdCl2 was added to the bath to block voltage-dependent
Ca2+ channels and, consequently, Ca2+-dependent
K+ channels (Fig. 4B). Under these conditions,
no differences were observed in the outward currents recorded before
and after PCMBS treatment, suggesting that IKv is not
affected by PCMBS.

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Fig. 4.
Effects of PCMBS on K+ currents.
A: I-V representation of averaged whole cell
recordings of outward K+ currents elicited by 10-mV step
depolarizations during 100 ms, from a holding potential of 50 mV in
the control condition and after PCMBS treatment (n = 7). B: K+ currents recorded in the presence of
0.2 mM CdCl2 from a holding potential of 50 mV, elicited
by 10-mV step depolarizations to +40 mV. C: K+
currents recorded with the double pulse protocol. Cells were
depolarized to +75 mV from a holding potential of 85 mV for 500 ms,
then repolarized to 0 mV for 100 ms, and finally depolarized again to
+75 mV for 2 s. Results are from 1 of 8 similar experiments.
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To explore directly the effect of the mercurial on IKCa, we
used a double pulse protocol (35) (Fig. 4C).
Cells were first depolarized to +75 mV for 0.5 s from a holding
potential of
85 mV. During this pulse, voltage-dependent
K+ channels are activated but IKCa are not,
because very little Ca2+ enters the cell at this positive
potential. The membrane potential was then returned to 0 mV for 100 ms
to allow Ca2+ to enter the cell, and a second depolarizing
pulse to +75 mV was applied for 2 s. This results in a second
outward K+ current carried mainly through
Ca2+-dependent K+ channels (35).
The transient current elicited by the first pulse was not modified by
the mercurial. The outward current during the second pulse was
partially inhibited by PCMBS in four of eight cells studied and was
increased in the other four cells. The average of all the values was an
inhibition of 13 ± 9% (mean ± SE). This small effect of
PCMBS on K+ currents does not seem large enough to explain
the increase of [Ca2+]i. This conclusion is
further supported by the observation that treatment of the cells with
inhibitors of Ca2+-dependent K+ channels (100 nM apamine or 20 nM charybdotoxin) did not reproduce the effects of
PCMBS on [Ca2+]i (data not shown).
A human ether-á-go-go-related gene-like K+
channel has been described recently in GH3 cells
(5). To exclude a possible action of PCMBS on these
channels, we measured currents when cells were hyperpolarized to
100
mV from a holding potential of
20 mV in a high-K+ medium
in the absence and presence of 50 µM PCMBS. There was no difference
between the two conditions.
Effect of PCMBS on background Na+
conductance.
The effects of PCMBS on the cell input resistance were monitored by
injecting hyperpolarizing current pulses (Fig.
5A). The mercurial decreased
the input resistance from 4.56 ± 1.63 to 2.17 ± 0.86 G
(mean ± SE, n = 10; P < 0.01, t-test). This effect was reversed upon addition of 2 mM DTT.
These results indicate that PCMBS increases a membrane conductance.
Because Ca2+ and K+ currents were not
stimulated by the mercurial, the effects on Na+ currents
were studied.

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Fig. 5.
Effects of PCMBS on input resistance of GH3
cells. Whole cell recording was made in current-clamp mode.
Hyperpolarizing current pulses (100 pA, 100 ms) were injected every
2 s. Results are representative of 4 similar experiments.
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Only one of every four cells displayed fast Na+ currents
activated by membrane depolarization, and in these cases PCMBS had no
effect on the amplitude of the current (data not shown). Simasko (36) described in GH3 cells a background
Na+ current (BNC) necessary for spontaneous action
potential firing. Figure 6A
shows that when the membrane potential was clamped at
80 mV, there
was an small inward current that decreased when the external
Na+ was replaced by NMDG, thus evidencing a BNC. When PCMBS
was added to the Na+-containing incubation medium, the
inward current increased by
230 pA, and this current was inhibited by
removal of external Na+ (replaced by NMDG). The same
results were obtained when Na+ was replaced by Tris (data
not shown). The effect of PCMBS on Na+ conductance was
reversed by DTT (Fig. 6B).

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Fig. 6.
Effects of PCMBS on whole cell currents in
GH3 cells. Whole cell recordings were made in voltage-clamp
mode. A: the cell was held at 80 mV and perfused with
either regular, Na+-containing medium or
Na+-free medium
[N-methyl-D-glucamine (NMDG)] as indicated.
Treatment with PCMBS (50 µM) was performed in Na+ medium.
Results are representative of 5 similar experiments. B: the
cell was treated with 50 µM PCMBS in regular,
Na+-containing medium and then with the same medium
containing DTT as indicated.
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Figure 7A shows an
I-V plot of the BNC induced by a 3-s voltage ramp from
100
to +50 mV generated from a holding potential of
30 mV. The currents
were measured before (Fig. 7A, trace c) and after
(Fig. 7A, trace d) treatment with PCMBS. Leak
currents were determined in Na+-free medium with (Fig.
7A, trace b) or without (Fig. 7A,
trace a) PCMBS and subtracted from the values obtained in
the regular, Na+-containing medium (Fig. 7B).
The leak current was not modified by PCMBS. Net inward current at
80
mV was
14 ± 3 pA (mean ± SE, n = 9) in
the control condition and increased to
67 ± 12 pA after
treatment with PCMBS (P < 0.005, ANOVA). The slope
conductance measured at
80 mV was 259 ± 55 pS in the control
condition and 1,675 ± 300 pS after PCMBS. The reversal potential
of the background current was about
30 mV, suggesting incomplete
specificity for Na+. It is noteworthy that the channel
responsible for the BNC of rabbit heart sinoatrial node cells is
permeable to Cs+ ions (11). The reversal
potential we found in GH3 cells is roughly the mean of the
reversal potentials for Na+ and Cs+. The BNC
was modified by neither TTX (100 nM) nor amiloride (1 mM) (results not
shown).

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Fig. 7.
Effects of PCMBS on Na+ currents in
GH3 cells. A: I-V relationship
obtained upon application of a voltage ramp ( 100 to +50 mV in 3 s from a holding potential of 30 mV); each trace was averaged from 5 repetitive ramps. Leak currents were recorded in Tris-containing
Na+-free medium (trace a) and in
Na+-free medium plus 50 µM PCMBS (trace b).
Trace c represents the current recorded in the presence of
150 mM NaCl, and trace d represents the current recorded
after 2 min of treatment with PCMBS. B: same currents as in
A after subtraction of leak currents. Net Na+
currents before (c a) and after
(d b) treatment with PCMBS are shown.
Note that leak currents were not modified by PCMBS. Results are
representative of 9 similar experiments.
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The increase of BNC by PCMBS can explain the observed membrane
depolarization and Ca2+ entry (Figs. 1 and 2) by
recruitment of voltage-gated Ca2+ channels. To confirm this
hypothesis, we studied the effects of Na+ on the
[Ca2+]i increase induced by PCMBS in fura
2-loaded GH3 cells. Figure 8
shows that Na+ removal (replaced by NMDG) reverted
completely and reversibly the increase of
[Ca2+]i. The same trace shows that, after
reversal by DTT, a subsequent treatment with PCMBS performed in the
absence of Na+ had no effect on
[Ca2+]i and that readdition of
Na+ triggered the increase of
[Ca2+]i, which could be reverted by DTT. The
effect of depolarization with high K+ is also shown in Fig.
8 for comparison.

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Fig. 8.
The increase of [Ca2+]i induced
by PCMBS in GH3 cells is dependent on external
Na+. Na+-free medium contained NMDG as the
Na+ substitute. An average of 87 cells were present in the
same microscope field. The effect of stimulation with
high-K+ (50 mM) solution is also shown (K).
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PCMBS also increases
[Ca2+]i in rat AP cells in
primary culture.
GH3 cells are often used as a model for AP cells, but
differences in the behavior of the cell line and the primary AP cells are common. For this reason, we tested the effects of PCMBS directly in
AP cells in primary culture. Because AP contains at least five well-defined cell subpopulations, each one storing and secreting a
different hormone, the effect of PCMBS could differ among the different
AP cell types. At the end of the [Ca2+]i
measurements, the cells present in the microscope field were typed by
sequential immunocytochemistry of the hormone they store (see
METHODS) and the [Ca2+]i traces
were averaged for each cell type. Results are shown in Fig.
9. In lactotropes (Fig. 9A,
PRL+), PCMBS produced a large increase of
[Ca2+]i that was antagonized by
Na+ removal and reversed by DTT. The effect of
depolarization with high K+ is also shown for comparison.
In somatotropes (Fig. 9B, GH+) the effect of the mercurial
was also evident, although somewhat slower and weaker. Corticotropes
(Fig. 9C, ACTH+) showed little or no response. Some of the
remaining cells, which must include thyrotropes and gonadotropes,
showed a small response. In additional experiments in which thyrotropes
and gonadotropes were directly identified (by using antibodies against
thyrotropin- and follicle-stimulating hormone, respectively) we found a
very small response in thyrotropes ([Ca2+]i
increased from 124 ± 14 to 183 ± 35 nM upon PCMBS
application; mean ± SE, n = 8) and no response in
gonadotropes (n = 34).

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Fig. 9.
Effects of PCMBS on [Ca2+]i in
several rat anterior pituitary cell types. At the end of the
[Ca2+]i measurements, the cells present in
the microscope field were identified by multiple sequential primary
immunocytochemistry by using antibodies against prolactin, growth
hormone, and corticotropin (see METHODS). Each trace
corresponds to the average of all the cells of every type: lactotropes
(A; n = 49), somatotropes (B;
n = 32), and corticotropes (C;
n = 4). Other details are as described in Figs. 1 and
8.
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PCMBS stimulates the same BNC as the GHRH in rat somatotropes.
Measurements of Na+ currents similar to those performed in
GH3 cells were carried out in enriched rat somatotropes in
primary culture (see METHODS). An inward Na+
current similar to that found in GH3 cells was evidenced in
somatotropes (Fig. 10; compare with
Fig. 7). The current was measured in Na+-free medium (Fig.
10A, trace a) and in Na+-containing
medium, before (Fig. 10A, trace b) or after
treatment with either 2 nM GHRH (Fig. 10A, trace
c) or 50 µM PCMBS (Fig. 10A, trace d).
GHRH increased the inward Na+ current in three of four
cells tested (from 16 ± 7 to 33 ± 6 pA at
80 mV;
mean ± SE, n = 3). These results confirm previous observations (19, 21, 29, 37). Treatment with PCMBS
produced a much larger increase of the inward Na+ current
(to 69 ± 12 pA). The reversal potentials of the inward Na+ currents activated by either GHRH or PCMBS had the same
value, nominally about
35 mV (Fig. 10B). These results are
consistent with both agents stimulating the same current.

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Fig. 10.
Growth hormone-releasing hormone (GHRH) and PCMBS elicit
an inward Na+ current in rat somatotropes. Enriched rat
somatotropes were prepared as described in METHODS.
A: I-V relationships obtained in the same cell in
Na+-free medium (trace a) or in
Na+-containing medium before (trace b) or after
3 min of treatment with either 2 nM GHRH (trace c) or 50 µM PCMBS (trace d). GHRH was washed for 10 min before
application of PCMBS. B: same currents after subtraction of
leak currents; control current (b a) and
currents in the presence of GHRH (c a) or
PCMBS (d a) are shown. Other details are
as described in Fig. 7. Results are representative of 3 similar
experiments.
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DISCUSSION |
We describe here a BNC that can be activated by PCMBS and cause
Ca2+ entry in pituitary cells. Our results have
implications in relation to two different topics: 1) to
explain the toxic effects of mercurials and 2) to uncover a
general mechanism that can regulate the excitability and firing
frequency in excitable cells. For the second implication, the silent
BNC typical of resting cells should be susceptible to activation by
cellular signaling mechanisms. This seems to be the case for
somatotropes (see below), but it remains to be studied whether this
applies to the BNC present in other excitable tissues.
A number of studies have identified ionic conductances, mainly
K+ and Ca2+ channels, as possible targets for
the toxic action of mercurials and other sulfhydryl-oxidizing agents
(see introduction). However, several effects that cannot be explained
by actions on the studied conductances also have been reported. For
example, Jungwirth et al. (17) found a membrane
depolarization by Hg2+ in cultured renal epithelioid
(Madin-Darby canine kidney) cells that cannot arise from the increase
in K+ conductance they reported. Entry of Ca2+
through voltage-gated Ca2+ channels induced by
Hg2+ or methylmercury has been reported in PC-12 cells
(31) and in NG108-15 cells (8, 14). Leonhardt
et al. (25) found that Hg2+ induced an inward
current in dorsal root ganglion neurones held at
80 mV, and Arakawa
et al. (2) also reported slow inward currents elicited by
Hg2+ and methylmercury in the same cells. Finally, Marty
and Atchison (27) found that methylmercury induced a
Na+-dependent Ca2+ entry in cerebellar granule
neurons. This Ca2+ entry was insensitive to TTX, but it
could be inhibited by Ca2+ channel blockers. The
PCMBS-activated BNC described here could explain both the inward
current and the increase of [Ca2+]i reported
previously in several tissues. Therefore, this current may be the
target responsible for the Ca2+ overload and toxicity
induced by mercurials in excitable cells. Because the sulfhydryl group
responsible for the activation is exposed to the external milieu (see
below), the possible involvement of this conductance on the effects of
oxidizing agents present in plasma also should be considered.
Treatment with PCMBS increases BNC about sixfold (Figs. 7 and 10),
promotes membrane depolarization (Fig. 2), and activates Ca2+ entry through L-type voltage-gated Ca2+
channels (Figs. 1, 8, and 9). These effects are reversed by DTT, suggesting the involvement of a sulfhydryl group, which should face
toward the extracellular medium, since PCMBS is membrane impermeant.
This suggestion was confirmed by using membrane-impermeable methanethiosulfonate sulfhydryl reagents. Modulation of ion channel activity through interactions with sulfhydryl groups has been reported
for several channels. For example, the cardiac Na+ channel
is known to possess a cysteine facing toward the extracellular side
that, upon interaction with sulfhydryl reagents, modifies the channel
activity (4, 7). This cysteine residue confers insensitivity to TTX (33), which also was found for the
BNC described here. The cardiac Na+ channel, however, is
gated by membrane depolarization and differs in this from the BNC we
found in AP cells.
Simasko and coworkers (32, 36) have identified recently a
BNC in pituitary GH3 cells and lactotropes that is
necessary for the generation of the spontaneous depolarizations
observed in these cells. When this current is eliminated by
Na+ removal, the plasma membrane hyperpolarizes,
spontaneous firing ceases, [Ca2+]i decreases,
and the basal prolactin secretion is reduced (32, 37).
Modulation of BNC by physiological factors would be an efficient
mechanism for controlling the firing frequency and, thus, the
physiological output of the cell, but there is no information on this
topic. Sankaranarayanan and Simasko (32) speculate that an
increase of BNC might be the basis for the stimulatory action of GHRH
on somatotropes, where GHRH is known to act by stimulating a
Na+ conductance (18, 21, 29, 37), but they did
not study BNC in this cell type. Here we have found that PCMBS
treatment activates a BNC in lactotropes and somatotropes. In
somatotropes the reversal potentials of the Na+ currents
stimulated by GHRH and PCMBS were the same, suggesting that both agents
may activate the same ion channel (Fig. 10). The PCMBS-activated
current was very weak in thyrotropes and absent in corticotropes and
gonadotropes. Thus BNC seems to be most prominent in the cell types
known to show spontaneous electric activity sensitive to modulation by
secretagogues (28).
The permeability of the lipid bilayer to ionic species is too low to
explain the background conductances of the plasma membranes, suggesting
that the ion movements may take place through specific pathways,
probably involving pore-forming proteins. However, leak currents are
difficult to study because they cannot be activated during
electrophysiological protocols. It is clear, though, that changes in
these conductances may modify membrane potential and, hence, the
electrical activity of excitable cells. BNC could be the target of
extracellular or intracellular messengers, as suggested by our results
in somatotropes. In addition, a second reading of the reported effects
of mercurials (see above) is suggestive of the presence of BNC in
several excitable cells. Treatment with PCMBS offers an easy
procedure to detect BNC in a given cell type. Work is in progress to
screen the presence of BNC in different tissues and to investigate the
molecular substrate of this current.
 |
ACKNOWLEDGEMENTS |
This work was supported by Spanish Dirección General de
Enseñanza Superior Grant PB97-0474. C. Villalobos was supported by a fellowship from the Spanish Ministerio de Educación y
Cultura. Antisera to pituitary hormones were a gift from the National
Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Child Health
and Human Development, and the US Department of Agriculture.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: J. García-Sancho, IBGM, Departamento de Fisiología,
Facultad de Medicina, 47005 Valladolid, Spain (E-mail:
jgsancho{at}ibgm.uva.es).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00441.2001
Received 13 September 2001; accepted in final form 16 November 2001.
 |
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