The sigma -ligand (+)-pentazocine depresses M current and enhances calcium conductances in frog melanotrophs

O. Soriani1,2, F. le Foll1, L. Galas1, F. Roman2, H. Vaudry1, and L. Cazin1

1 European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U413, Institut Fédératif de Recherche Multidisciplinaire sur les Peptides no. 23, Unité Associée Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan; and 2 Institut de Recherche Jouveinal/Parke-Davis, 94265 Fresnes, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Gramicidin-perforated patch clamp experiments and microfluorimetric measurements were performed to study the ionic mechanisms involved in the sigma -receptor-mediated stimulation of frog (Rana ridibunda) pituitary melanotrophs. The sigma -ligand (+)-pentazocine (50 µM) depressed a sustained outward K+ current. The kinetic properties of this K+ component, investigated by analyzing tail currents, were reminiscent of those of the M current (IM), with an activation threshold close to -60 mV, a -21-mV half-maximal activation potential, and two-component exponential deactivation kinetics at -90 mV. (+)-Pentazocine (20 µM) produced a 12-mV rightward shift of the activation curve and accelerated the deactivation rate of the tail current. It is also demonstrated that (+)-pentazocine (20 µM) reversibly increased both voltage-dependent calcium conductances and internal calcium level. Altogether, these results suggest that the sigma -receptor-induced modulation of IM and calcium currents likely underlies the increase of intracellular [Ca2+].

sigma -receptors; calcium channels; patch-clamp technique; melanotrophs; (+)-pentazocine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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sigma -RECEPTORS were first described as a new type of opioid receptor mediating the psychotomimetic effects produced by N-allylnormetazocine [(+/-)-SKF 10047] and related benzomorphans (27). Subsequent studies have demonstrated that sigma -receptors represent a new class of binding sites distinct from opioid receptors. In particular, the nonselective opioid antagonist naloxone does not block the in vivo and in vitro effects of sigma -ligands (39). Furthermore, sigma -receptors are enantioselective for the (+)-isomers of opioid agonists, whereas opioid receptors are selective for the (-)-isomers (19). Although two sigma -receptors, namely sigma 1- and sigma 2-receptors, have been pharmacologically described (31), several lines of evidence suggest the existence of multiple types of sigma -receptors (3, 40). Recently, a subclass of the sigma -receptor corresponding to a 27-kDa protein has been cloned in the guinea pig and in humans (15, 18, 21). The function of this protein remains largely unclear.

sigma -Receptors are widely represented in various regions of the central nervous system, including the cerebral cortex, hippocampus, striatum, and cerebellum (12, 17). The presence of sigma -receptors has also been demonstrated in the hypothalamus and pituitary (17), suggesting that sigma -receptors regulate hypothalamopituitary functions. In support of this, it has been shown that various sigma -ligands enhance the secretion of prolactin and corticosterone in rats (14). It has also been reported that in vivo administration of sigma -antagonists causes a reduction of the plasma concentration of alpha -melanocyte-stimulating hormone in the rat (10). Although sigma -ligands have been shown to mediate a large array of biological effects in central nervous, endocrine, and immune systems (18, 20, 34, 35, 40), so far the cellular transduction mechanisms associated with sigma -receptors are still poorly understood.

The intermediate lobe of the frog pituitary is a valuable model in which to investigate the effect of regulatory substances. In contrast to the rat pars intermedia, which contains two distinct cell types, i.e., melanotrophs and corticotrophs, the frog pars intermedia is composed of a homogeneous population of cells (37). In this respect, the intermediate lobe of the frog pituitary offers the same advantages as a cell line without the drawbacks of transformed tumoral cells. In fact, amphibian melanotrophs have already been used to study the mechanism of action of biologically active compounds (for review see Ref. 37).

In a recent study, we have demonstrated that two highly specific sigma -receptor ligands, 1,2-ditolylguanidine (DTG) and (+)-pentazocine, stimulate the electrical activity of cultured frog melanotrophs through the inhibition of both a voltage-activated K+ conductance, i.e., the delayed rectifier [IK(V)], and a yet-to-be-identified outward leak potassium current (33). The aim of the present study was to further elucidate the ionic mechanisms involved in the sigma -ligand-induced stimulation of the electrical activity in pituitary cells. It is concluded that sigma -receptor activation stimulates voltage-gated calcium conductances both directly and through a depolarizing process involving the M current (IM).


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INTRODUCTION
METHODS
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Animals. Adult male frogs (Rana ridibunda; body weight 30-40 g) were obtained from a commercial supplier (Couétard, Saint-Hilaire de Riez, France). Frogs were housed in a temperature-controlled room (8°C) under an established 12:12-h light-dark photoperiod (lights on from 0600 to 1800). The animals had free access to running water and were maintained under these conditions for >= 1 wk before use. Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators.

Reagents and test substances. Leibovitz L-15 culture medium, protease (type IX), collagenase (type IA), GTP, and gramicidin D were purchased from Sigma Chemical (St. Louis, MO). HEPES was obtained from Research Organics (Cleveland, OH). Cholera toxin (CTX) was from List Biological Laboratories (Campbell, CA). Boehringer Mannheim (Mannheim, Germany) supplied kanamycin, the antibiotic-antimycotic solution, fetal calf serum, and BSA (fraction V). Indo 1 acetoxymethyl ester (indo 1-AM) was from Molecular Probes (Eugene, OR). Tissue culture dishes were obtained from CML (Nemours, France). (+)-Pentazocine and DTG were obtained from Jouveinal (Fresnes, France).

Cell culture for electrophysiological experiments. Eight neurointermediate lobes (NIL) were dissected and washed in Leibovitz L-15 culture medium adjusted to Rana ridibunda osmolality and supplemented with CaCl2 (0.1 g/l), glucose (0.2 g/l), and 1% (vol/vol) of the kanamycin and antimycotic-antibiotic solution. The tissues were enzymatically dissociated in the same medium, containing 0.15% protease and 0.15% collagenase, for 15 min at 22°C. After mechanical dispersion, the cells were centrifuged (50 g) for 15 min, rinsed three times, and suspended in Leibovitz medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics. The cells were then plated at a density of 10,000 cells per 35-mm tissue culture dish. Cultured cells were incubated at 24°C in a humid atmosphere and used 5-10 days after plating.

Cell culture for intracellular calcium measurements. NIL (10 per culture) were collected in Ca2+-free Ringer solution (in mM: 15 HEPES, 112 NaCl, 2 KCl, and 1 EGTA) supplemented with glucose (2 mg/ml), BSA (0.3 mg/ml), and 1% each of the kanamycin and antibiotic-antimycotic solutions. The Ringer solution was gassed for 15 min with O2-CO2 (95:5, vol/vol) before the experiment, and pH was adjusted to 7.4 with NaOH. NIL were enzymatically dispersed at 24°C for 20 min with a solution of collagenase (1.5 mg/ml) in a Ca2+-free Ringer solution. Nondissociated neural lobes were allowed to settle down, and the supernatant, containing mainly dispersed melanotrophs, was sampled. The cell suspension was sampled and then centrifuged three times (30 g; 5 min) with the Ca2+-free Ringer solution. Dissociated cells were resuspended in the L-15 solution already described. Cells were then dispersed and plated on poly-L-lysine-coated coverslips and recovered with L-15 culture medium supplemented with 10% fetal bovine serum. Microfluorimetric measurements were performed on 3- to 5-day-old cultured cells.

Electrophysiological procedures. Electrophysiological recordings were performed at room temperature on 5- to 10-day-old cultured frog melanotrophs by use of the perforated patch-clamp variation of the whole cell variation (33, 36). External solutions used to perform the different experiments are detailed in Table 1. Soft glass patch electrodes (microhematocrit tubes) were made on a vertical pipette puller (List Electronic, Darmstadt, Germany), and the tip of the electrode was polished with a microforge (Narishige, Tokyo, Japan) to achieve a final resistance ranging from 3 to 5 MOmega after electrodes were filled with the internal solution. Perforated patch-clamp experiments were performed with gramicidin D (2), which was first dissolved in methanol to a concentration of 10 mg/ml and then diluted in the pipette solution to a final concentration of 100 µg/ml just before use. The composition of the internal pipette solution used for recordings of potassium currents was (in mM) 100 KCl and 10 HEPES (pH adjusted to 7.4 with KOH). Barium currents were obtained with an internal solution composed (in mM) of 100 CsCl and 10 HEPES (pH adjusted to 7.4 with CsOH). A short tip-filling (2 s) of each glass electrode with an antibiotic-free internal solution was necessary just before the final back-filling with the gramicidin-containing medium. The series resistance achieved a stable value (4-15 MOmega ) after 7-15 min after the giga-seal formation. In whole cell experiments, GTP (100 µM) and EGTA (10 mM) were added in the internal pipette solution. The series resistance was compensated at a value higher than 60% for all recorded cells. Electric signals were amplified with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and acquired on an IBM-compatible personal computer with a DIGIDATA 1200 interface and pCLAMP 6.02 software (Axon Instruments). Potassium currents were recorded at a 2-kHz sampling frequency and filtered at 1 kHz. Calcium currents were sampled at 5.2 kHz and filtered at 2 kHz. All voltage-activated currents were obtained after appropriate leak correction using P/4 protocol.

                              
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Table 1.   Composition of external solutions used in electrophysiological experiments

Calcium measurements. Cultured cells were incubated at 24°C in the Ringer solution, supplemented with glucose (2 mg/ml) and BSA (0.3 mg/ml) and containing 5 µM of the fluorescent probe indo 1-AM, for 30 min in the dark. At the end of the incubation period, cells were washed with 2 ml of fresh medium and placed on the stage of a Nikon Diaphot inverted microscope (Tokyo, Japan) equipped for epifluorescence with an oil-immersion objective (*100 CF Fluor series; numerical aperture, 1.3). The variations of internal Ca2+ were monitored by a dual-emission microfluorimeter system. The fluorescence emission of indo 1-AM induced by excitation at 355 nm (Xenon lamp) was recorded at two wavelengths (405 nm, corresponding to the Ca2+-complexed form, and 480 nm, corresponding to the free form) by separate photometers (P1, Nikon). The 405-to-480-nm ratio (R, 405/480) was determined by using an analogical divider (constructed by B. Dufy, Bordeaux, France) after conversion of single photon currents to voltage signals. All three signals (405 nm, 480 nm, and 405/480) were continuously recorded with a three-channel voltage recorder (BD 100/101, Kipp & Zonen, Delft, The Netherlands). The intracellular calcium concentration was calculated according to the formula established by Grynkiewicz et al. (13): [Ca2+]i = Kd × beta (R - Rmin)/(Rmax - R), where Rmin represents the minimum fluorescence ratio obtained after incubation of cells in Ringer solution containing 10 mM EGTA and 10 µM ionomycin, Rmax is the maximum fluorescence ratio obtained after incubation of cells in Ringer solution containing 10 mM CaCl2 and 10 µM ionomycin, and beta  is the ratio of fluorescence yields from the Ca2+min/Ca2+max indicator at 480 nm. The values for Rmin, Rmax, and beta  were 0.164, 1.82, and 1.62, respectively. The dissociation constant (Kd) for indo 1 (250 nM) has been previously determined (28).

Drug application. (+)-Pentazocine was added to the external solution and sonicated for 20-30 s. DTG was first dissolved in ethanol (final concentration of ethanol <1% vol/vol) and mixed to the external solution. sigma -Ligand solutions were administered in the vicinity of the cell under study by a pressure ejection system (76 mmHg) from a glass pipette placed at a distance of 100-150 µm from the cell. The bathing medium was continuously renewed with fresh external solution at a flow rate of 3 ml/min via a gravity-fed system. The excess of bathing solution was continuously aspirated via a suction needle.

Analysis of currents. Current amplitudes were determined with the pCLAMP 6.02 analysis software (Clampfit). Exponential fits of the decaying phase of tail currents (calculated by the Simplex method) were also performed with Clampfit. Current-voltage (I/V) relationships were fitted by using Origin analysis software (Micrococal). Statistical comparisons were performed with the Student's paired or independent t-test and the Mann-Whitney or Wilcoxon test, depending on the experimental conditions. Quantitative data are expressed as means ± SE.


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INTRODUCTION
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REFERENCES

The effects of (+)-pentazocine on frog pituitary melanotrophs were studied by use of the patch-clamp and microfluorimetry techniques in a total of 79 cells.

Electrophysiological recordings were performed using the gramicidin D-perforated patch variation of the standard whole cell approach (36) to avoid dialysis of intracellular messengers, which is inherent in the whole cell configuration.

Isolation of the sustained outward K+ current. In cells bathed in a low K+ and a Ca2+- and Na+-free external solution (Table 1), long-lasting depolarizing pulses (10 s) from -90 to 50 mV elicited fast inactivating outward K+ currents prolonged by a sustained component (n = 8). The sigma 1-agonist (+)-pentazocine (50 µM) markedly inhibited both the fast inactivating and the sustained currents (Fig. 1). Whereas the sigma -ligand-induced reduction of the transient component is attributable to the inhibition of the delayed rectifier outward K+ current (33), the nature of the sustained current altered by sigma -ligands remained to be identified (33). To further characterize the properties of this latter component, open-state relaxation experiments were performed. The low K+ external solution was replaced by a high K+ solution (20 mM; Table 1). Under these conditions, reversal potential of K+ ions was shifted from -98 to -40 mV. Consequently, a tail current was observed at the end of a 10-s depolarizing step pulse because of the sudden increase in driving force of K+ ions (n = 13). The tail current occurring at -90 mV represents the deactivation of the sustained component (Fig. 2, A and B). In addition, to avoid calcium and calcium-dependent K+ currents, Mg2+ was substituted for Ca2+ (Table 1).


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Fig. 1.   Effects of (+)-pentazocine on outward potassium current in a melanotroph. Superimposed outward potassium currents evoked by long-lasting voltage steps (10 s) from -80 to 50 mV in a low K+ external solution (see Table 1). Currents were recorded before (Control), during [(+)-pentazocine], and after (Wash) application of (+)-pentazocine (50 µM). Dotted line, zero current.



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Fig. 2.   Characterization of an M-like current in frog melanotrophs. A: family of outward potassium currents recorded in response to 10-s depolarizing pulses. Voltage protocol is illustrated below. Transient inward currents (arrow) observed at offset of depolarizing pulses correspond to deactivating tail currents of sustained component. Dotted line, zero current. B: expanded traces of the deactivating tail currents observed in A. C: plot illustrating normalized current-voltage relationship (I/Imax) of peak tail currents summarized from 7 independent experiments. Continuous line, a fit to a Boltzmann equation. D: double-exponential fit (continuous line) of deactivation time course of tail current at -90 mV, recorded after a 10-s depolarizing pulse to 50 mV. For all recordings, cells were bathed in a high K+ external solution.

Properties of the sustained outward K+ current. Melanotrophs were clamped at -90 mV and depolarized to potentials between 50 and -90 mV by 20-mV steps (Fig. 2A). At depolarizing potentials ranging from -30 to 50 mV, the evoked currents presented fast activating and inactivating phases, followed by a sustained component. By contrast, at potentials ranging between -50 and -90 mV, only very weak inward currents were observed (Fig. 2A). As a consequence, the reversal potential could be estimated at a value that corresponds to the equilibrium potential of K+ ions in the present experimental conditions (EK = -40 mV). At the end of the depolarizing pulses, transient inward deactivating currents were detected (Fig. 2, A and B). The tail current increased with the depolarization in a sigmoid manner. The corresponding I/V plot obtained from seven melanotrophs fitted well with a Boltzmann function (Fig. 2C; Table 2). The deactivation rate of the tail currents recorded after depolarizations to 50 mV was fitted by a two-component exponential function, revealing the presence of a fast (tau 1) and a slow (tau 2) component in the current decay (Fig. 2D; Table 2).

                              
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Table 2.   Effects of (+)-pentazocine on kinetic characteristics of IM

Effects of (+)-pentazocine on the kinetic properties of the sustained outward K+ current. Melanotrophs were held at 90 mV, pulsed to a series of 10-s depolarizing steps from 50 to -70 mV, and then returned to -90 mV. This experimental protocol was applied to each studied cell in the absence and presence of (+)-pentazocine (20 µM). (+)-Pentazocine significantly reduced the tail currents (P < 0.05; Mann and Whitney) elicited after depolarizations positive to -50 mV (Fig. 3). Furthermore, (+)-pentazocine provoked a pronounced shift of the voltage-dependent activation curve toward more depolarized potentials (Fig. 3B; Table 2). The decrease in current amplitude was accompanied by an acceleration of the deactivating phase, yielding a significant diminution of both time constants (P < 0.01 and P < 0.05 for tau 1 and tau 2, respectively; paired t-test) deduced from the double-exponential fit of the tail current elicited after a 50-mV depolarizing step (Fig. 3A; Table 2).


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Fig. 3.   Effects of (+)-pentazocine on M current. A: families of deactivating tail currents elicited by the voltage protocol described in Fig. 2. Currents were recorded from a single melanotroph before (top, Control), during [middle, (+)-pentazocine], and after (bottom, Wash, 2 min) application of 20 µM (+)-pentazocine. B: normalized current-voltage relationship of peak tail current in absence (; n = 7) and presence of (+)-pentazocine (20 µM; open circle ; n = 5). Continuous lines represent fits to Boltzmann equations. For all recordings, the cells were bathed in a high K+ external solution. * P < 0.05; ** P < 0.01 (Mann-Whitney test).

Effects of (+)-pentazocine on the cytosolic calcium level. The effects of sigma -ligands on the intracellular calcium concentration ([Ca2+]i) were studied by use of microfluorimetric measurements. Basal [Ca2+]i was 18.8 ± 1.1 nM. Melanotrophs were challenged with (+)-pentazocine or DTG (20 µM each) after periods of 2-3 min of control recording to ensure that the tested cells did not present any spontaneous oscillatory activity. Application of (+)-pentazocine (or DTG; not shown) in the vicinity of the cells significantly elevated [Ca2+]i (68.7 ± 6.4 nM, n = 29, P < 0.001; paired t-test; Fig. 4). The response appeared within 5-25 s after the onset of the sigma -ligand application and gradually developed to reach its maximum in a period of 46.2 ± 8.5 s (n = 29).


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Fig. 4.   Effects of (+)-pentazocine on intracellular calcium concentration ([Ca2+]i). A: typical effect of (+)-pentazocine (20 µM; 15 s) on [Ca2+]i level in a melanotroph. Vertical arrows indicate onset of sigma -ligand application. B: comparison between basal [Ca2+]i and maximal level measured after application of (+)-pentazocine (20 µM). Histogram summarizes mean values obtained from 29 independent experiments. *** P < 0.001 (paired t-test). Cells were continuously perfused with fresh standard external solution.

Effects of (+)-pentazocine on voltage-activated barium currents. The effects of (+)-pentazocine (20 µM) were tested on a sample of 12 melanotrophs bathed in the BaCl2-tetraethylammonium-TTX external solution (Table 1). Depolarizing step pulses from -80 to 0 mV elicited slowly inactivating inward barium currents (IBa). Application of (+)-pentazocine gave rise to a significant (P < 0.01; paired t-test) and reversible augmentation of IBa (Fig. 5, A and B). The maximum increase in current amplitude was recorded between 20 and 120 s after the application of (+)-pentazocine (Fig. 5C). Depolarizing ramps from -80 to 60 mV evoked bell-shaped inward currents. (+)-Pentazocine clearly augmented the current amplitude for potentials ranging from -50 to 30 mV (Fig. 5D).


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Fig. 5.   Effects of (+)-pentazocine on voltage-activated barium currents. A: superimposed barium currents evoked by voltage steps from -80 to 0 mV in a single melanotroph before (Control), during [(+)-pentazocine], and after (Wash) application of (+)-pentazocine (20 µM). B: histogram of barium current amplitude (means ± SE, n = 12) measured before (solid bar) and after (open bar) application of (+)-pentazocine (20 µM). ** P < 0.01 (paired t-test). C: time course of stimulatory effect of (+)-pentazocine (20 µM; 10 s) on barium current evoked in a cell by constant pulses, as in A. Current amplitudes were measured at peak () and at end of depolarization (). D: instantaneous current-voltage (I/V) relationship elicited by 200-ms voltage ramps from -80 to 60 mV recorded from another cell in the absence (Control and Wash) or presence of (+)-pentazocine (20 µM).


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A recent study has demonstrated that sigma -ligands exert a positive control on the electrical activity of frog melanotrophs by inhibiting a voltage-dependent potassium current, namely IK(V), and a leak potassium current, both through a G protein-dependent pathway (33). The aim of the present investigations was to determine other targets that are controlled by sigma -receptors. Herein we have characterized, in frog melanotrophs, an M-like current that is inhibited by sigma -ligands. In addition, it is demonstrated for the first time that (+)-pentazocine, a highly specific and selective sigma 1-receptor ligand (4, 30), stimulates voltage-dependent calcium conductances.

We have demonstrated that, in addition to the stimulation of the electrical activity previously described in melanotrophs (33), (+)-pentazocine induces a transient increase of [Ca2+]i. Several lines of evidence have suggested that, in spontaneously active cells, calcium influx is triggered by sodium-dependent action potentials through the activation of voltage-dependent calcium channels occurring during the transient depolarizations (9, 24, 29). Moreover, it has recently been proposed that slight variations in the discharge frequency of action potentials were responsible for spontaneous cytosolic calcium spiking activity in Xenopus laevis melanotrophs (38). As a consequence, it is likely that in frog melanotrophs the sigma -ligand-induced increase of [Ca2+]i is due, at least in part, to the inhibition of IK(V), giving rise to a stimulation of an action potential discharge (33).

However, the modulation of electrical activity by sigma -ligands has also been attributed to variations of resting potential (33). As a result, the observed sigma -ligand-induced depolarizations were identified as the consequence of the clamping of a yet-to-be-identified tonic outward potassium current (33). Because the properties of such a tonic conductance were unknown in melanotrophs, we next undertook its kinetic characterization. In conditions where calcium and sodium currents were eliminated, a noninactivating potassium current was isolated. The voltage-dependent properties of this conductance revealed an activation threshold close to -60 mV, a half-maximal activation potential between -20 and -25 mV, and a nearly complete activation between 0 and 10 mV. All these characteristics fit very well those of the M current previously described in neurons (6, 7) and endocrine cells (32). In addition, it was found that this current exponentially deactivates at hyperpolarized potential with fast and slow time constants. This finding is consistent with other analyses of M currents in rat lactotrophs (32) and bullfrog sympathetic neurons (26). The values of the time constants in frog melanotrophs are in the same range as those obtained in bullfrog sympathetic neurons but are lower than in rat lactotrophs.

M current has been described in various neurons (1, 22, 25, 26) and recently in endocrine cells (32). Because IM is one of the only sustained currents at rest in each cell type, it appears as a major element in the control of membrane excitability. Again, its properties in melanotrophs strongly suggest that it contributes to the regulation of resting potential [-48 mV (23, 33)] and excitability. Another original finding provided by the present investigations is that IM is under the control of sigma -receptors in endocrine cells. It is shown that (+)-pentazocine dramatically shifted the activation curve toward more positive potentials, resulting in a diminution of the net outward potassium flux. Consequently, the sigma -ligand-mediated inhibition of IM described in this study likely participates in the previously reported inward potassium current provoked by DTG and (+)-pentazocine at potentials positive to -60 mV (33). The removal of the hyperpolarizing influence of IM at rest as well as at interspike potentials would lead to an increased excitability and to the observed sigma -ligand-induced depolarization (33). This is strongly correlated with the increase in membrane resistance occurring during the sigma -ligand-induced membrane depolarization (33). Together with the depression of IK(V) (33), the inhibition of IM by sigma -ligands would have a critical role in the augmentation of calcium influx by making cells more likely to fire action potentials (25) and by further stimulating voltage-dependent calcium channels (38).

Although our results strongly suggest that sigma -ligands indirectly activate calcium channels through the inhibition of potassium currents, the possibility of an intracellular modulation of calcium channels by sigma -receptors was investigated. It is shown here for the first time that (+)-pentazocine potentiates voltage-activated calcium conductances. This observation markedly differs from other data previously reported, demonstrating the depression of calcium influx in rat forebrain synaptosomes (5) and in mouse hippocampal pyramidal neurons (8). However, in this latter case, the high concentrations of tested compounds clearly acted as open channel blockers, independent of any sigma -receptor-mediated effect. In addition, it cannot be excluded that in rat synaptosomes, the observed decrease in calcium influx ascribed to the inhibition of calcium conductances would be the consequence of an external blockade of the channels as well (8).

In our paradigm, the sigma -ligand-induced augmentation of calcium current developed gradually in contrast to the fast stimulating effect observed on membrane potential and action potential firing (33). In very much the same way as the increase in calcium current, the maximum augmentation of cytosolic calcium level was observed after a relatively long period after the application of the sigma -ligand. It is then tempting to speculate that the early phase of calcium influx depends mainly on the voltage-dependent modulation of calcium channels through sigma -receptor-mediated closing of K+ conductances. In contrast, the late peak calcium influx is more likely supported by an intracellular pathway coupling sigma -receptors to calcium channels. The question of whether stored calcium from the internal pools also contributes to the increase of [Ca2+]i by sigma -ligands remains open. However, evidence has already been provided that inositol trisphosphate-triggered release of internal calcium provokes rapid and often sustained elevation of [Ca2+]i (11, 16), suggesting that the [Ca2+]i stores are not altered by the activation of sigma -receptors.

In conclusion, it appears that, in pituitary melanotrophs, sigma -receptors exert a complex regulation of membrane excitability through the inhibition of voltage-activated potassium conductances (33), including the newly characterized IM. In addition, it is demonstrated, for the first time to our knowledge, that sigma -ligands stimulate voltage-activated calcium conductances independently of the K+ channel pathway. Altogether, these results suggest that sigma -receptors modulate cytosolic calcium level and subsequently control hormone release. In addition, this work shows that frog melanotrophs constitute a model of choice to further determine the intracellular messengers involved in sigma -ligand-induced responses.


    ACKNOWLEDGEMENTS

We thank Catherine Buquet for excellent technical assistance.


    FOOTNOTES

This work was supported by grants from INSERM (U 413), the Institut de Recherche Jouveinal, the European Union (Human Capital and Mobility Programme; ERBCHRXCT920017), and the Conseil Régional de Haute-Normandie. 0. Soriani was the recipient of a scholarship from the Fonds de la Recherche et de la Technologie (Cifre program).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: L. Cazin, European Institute for Peptide Research (IFRMP No. 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, Univ. of Rouen, 76821 MontSaintAignan, France.

Received 25 January 1999; accepted in final form 2 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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4.   Bowen, W. D., B. R. De Costa, S. B. Hellewell, M. J. Walker, and K. C. Rice. [3H](+)pentazocine: a potent and highly selective benzomorphan-based probe for sigma-1 receptors. Mol. Neuropharmacol. 3: 117-126, 1990.

5.   Brent, P. J., L. Herd, H. Saunders, A. T. R. Sim, and P. R. Dunkley. Protein phosphorylation and calcium uptake into rat forebrain synaptosomes: modulation by the sigma ligand, 1,3-ditolylguanidine. J. Neurochem. 68: 2201-2211, 1997[Medline].

6.   Brown, D. A., and P. R. Adams. Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neuron. Nature 283: 673-676, 1980[Medline].

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