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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . 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 MOmega . Perforated patches reached stable access resistances (20-40 MOmega ) 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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INTRODUCTION
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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 (Delta [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.

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.

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.

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.

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 GOmega (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.

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.

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.

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).

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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 282(4):C864-C872
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society




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