Abstract
Gramicidin perforated patch-clamp recordings were used to study the effects of two ς 1 receptor ligands, (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO 1784) and (+)-pentazocine, on the transient outward potassium current (IA) in cultured frog melanotrope cells. (+)-Pentazocine reversibly decreased the current amplitude in a dose-dependent manner. The effects of (+)-pentazocine were mimicked by JO 1784 and were markedly reduced by the ς 1 receptor antagonist,N,N-dipropyl-2-[4-methoxy-3–2(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE 100). Inactivation rate of IA was best fitted with a double exponential function, yielding time constants of 23.7 and 112.5 ms. (+)-Pentazocine (20 μM) accelerated the current decay, decreasing the time constants to 10.7 and 59 ms, respectively. Current-voltage experiments revealed that (+)-pentazocine (20 μM) did neither modify the open-state I/V curves nor the voltage dependence of IA. However, (+)-pentazocine (20 μM) shifted the steady-state inactivation curve toward more negative potentials and increased the time constant of the time-dependent removal of inactivation. In whole-cell experiments, internal dialysis of guanosine-5′-O-(3-thiophosphate) (100 μM) irreversibly prolonged the response to (+)-pentazocine. In addition, cholera toxin pretreatment (1 μg · ml−1; 12 h) suppressed the inhibition of IAby (+)-pentazocine (20 μM). It is concluded that in frog melanotrope cells, a cholera toxin-sensitive, G protein-dependent inhibition ofIA through a ς 1 receptor activation, at least partially, underlies the excitatory effect of ς ligands.
ς Receptors were first postulated in 1976 to account for the spectrum of behaviors produced by racemic benzomorphans such as (±)-N-allylnormetazocine in dogs and humans (Martin et al., 1976). Originally, ς receptors were thought to represent a new type of opioid receptors (Martin et al., 1976). However, subsequent pharmacological studies revealed that ς binding sites were a new class of receptors, pharmacologically distinct from opioid receptors (Walker et al., 1990; Su, 1991). Whereas two types of ς receptors have been clearly described on the basis of pharmacological and binding criteria (Quirion et al., 1992), several lines of evidence suggest the existence of multiple ς receptors (Walker et al., 1990; Monnet et al., 1994). ς receptors are widely distributed in the central nervous system and more particularly in the hippocampus, hypothalamus, cerebellum, striatum, and motor nuclei of the brainstem (Su, 1991;Gonzalez-Alvear et al., 1995). The presence of ς receptors has also been demonstrated in the endocrine system, including pituitary (Jansen et al., 1991; Soriani et al., 1998), suggesting that ς receptors may participate to the regulation of hypothalamo-pituitary functions. In support of this, it has been shown that ς ligands stimulate corticosterone and prolactin secretion in rats (Gudelsky and Nash, 1992). However, because endogenous ς ligands have thus far never been definitely identified, the physiological roles of ς receptors are still unknown. In addition, the cellular transduction pathways mediating the effects of exogenous ς ligands remain largely unclear. Recently, a subtype of ς 1 receptor has been cloned in guinea pig (Hanner et al., 1996), but the mechanism of action of the corresponding protein is still mysterious. Nevertheless, in a previous work, we have demonstrated that in frog pituitary melanotrope cells, ς ligands stimulate electrical activity through a ς 1 receptor coupled to a G protein-dependent pathway by inhibiting at least two potassium conductances, i.e., a leak outward potassium current and the voltage-dependent delayed rectifier potassium conductance (Soriani et al., 1998).
The transient outward A-type potassium current (IA) has been shown to be a major current in the regulation of spiking frequency in various cell types, including neurones (Bardoni and Belluzzi, 1994) and endocrine cells (Mlinar and Enyeart, 1993; Mei et al., 1995). A necessary consequence of the IA pre-eminence in the regulation of electrical activity is that modulation of this current is expected to have important effects on cell excitability profile. In support of this, it has been demonstrated that endocrine factors such as adenosine and angiotensin II regulate electrical activity through the modulation of IA current (Mei et al., 1995; Nagatomo et al., 1995). These properties makeIA an interesting target to further investigate the mechanisms by which ς ligands modulate the electrical activity of pituitary cells. The present study focuses on the effects of two highly specific ς 1 receptor agonists onIA in cultured frog melanotrope cells.
Materials and Methods
Animals.
Adult male frogs (Rana ridibunda; body weight, 30–40 g) were obtained from a commercial supplier (Couétard, SaintHilaire de Riez, France). Frogs were housed in a temperature-controlled room (8°C) under an established photoperiod of 12 h of light/day (lights on from 6:00 AM–6:00 PM). The animals had free access to running water and were maintained in these conditions for at least 1 week 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.
Leibowitz L-15 culture medium, protease (type IX), collagenase (type IA), gramicidin D, and guanosine-5′-O-(3-thiophosphate) (GTPγS) were purchased from Sigma Chemical Co. (St. Louis, MO). HEPES was obtained from Research Organics (Cleveland, OH). Cholera toxin (CTX) was from List Biological Laboratories (Campbell, CA). Kanamycin, the antibiotic-antimycotic solution, and fetal calf serum were supplied by Boehringer Mannheim (Mannheim, Germany). Tissue culture dishes were obtained from C.M.L. (Nemours, France). (+)-Pentazocine and (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO 1784) were synthesized by the Institut de Recherche Jouveinal (Fresnes, France).N,N-Dipropyl-2-[4-methoxy-3–2(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE 100) was kindly provided by Dr. S. Okuyama (Taisho Pharmaceutical Co., Tokyo, Japan).
Cell Culture.
Eight neurointermediate lobes were dissected and washed in Leibowitz L-15 culture medium adjusted to R. ridibunda osmolality and supplemented with CaCl2 (0.1 g/liter), glucose (0.2 g/liter), and 1% (v/v) 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 (50g) for 15 min, rinsed three times, and suspended in Leibowitz 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 26°C in a humid atmosphere and used 5–10 days after plating.
Electrophysiological Procedures.
Electrophysiological recordings were performed at room temperature on cultured 5- to 10-day-old frog melanotrope cells using the perforated patch-clamp variation of the whole-cell configuration (Soriani et al., 1998). A-current was recorded by using an external solution of the following composition: 92 mMN-methyl-d-glucamine; 20 mM tetraethylammonium chloride; 3 mM KCl; 2 mM CoCl2; and 15 mM HEPES (pH adjusted to 7.4 with HCl). Soft glass patch electrodes (micro-hematocrit 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 MΩ after filling with the internal solution. Perforated patch-clamp experiments were performed with gramicidin D (Akaike, 1997). Gramicidin D was first dissolved in methanol to a concentration of 10 mg · ml−1 and then diluted in the pipette solution to a final concentration of 100 μg · ml−1 just before use. The composition of the internal pipette solution was 100 mM KCl and 10 mM HEPES. 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 achieves a stable value (4–15 mΩ) after 7–15 min following the giga-seal formation. In whole-cell experiments, GTPγS (100 μM) was 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 a pCLAMP 6.02 software (Axon Instruments). Potassium currents were recorded at a 5 kHz sampling frequency and filtered at 2 kHz. Open-state currents were acquired with a higher sampling frequency (10 kHz) and filtered at 5 kHz.
Drug Application.
(+)-Pentazocine and NE 100 were added to the external solution and sonicated (20–30 s). JO 1784 was directly solubilized in the external solution. ς Ligand solutions were administered in the vicinity of the cell under study by a pressure ejection system (76 mm Hg) 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−1 via a gravity-fed system. The excess of bathing solution was continuously aspired via a suction needle. CTX was added to the culture medium (1 μg · ml−1) 12 h before electrophysiological recordings.
Current Analysis.
Current amplitudes were determined with the pCLAMP 6.02 analysis software (Clampfit). Graphical current subtractions and exponential fits of the decaying phase of currents (calculated by Simplex method) were also performed with Clampfit. Current/voltage and current/time relationships were fitted by using Origin analysis software (Micrococal). Statistical comparisons were performed with Student’s t test, Mann and Whitney, or Wilcoxon tests, depending on the experimental and measuring conditions. Quantitative data are expressed as mean ± standard error of the mean (S.E.M.).
Results
Transient outward potassium currents were studied in the presence of extracellular tetraethylammonium (20 mM) to block the delayed rectifier outward potassium current (Mei et al., 1995). Recordings were obtained from 91 frog melanotrope cells by using the gramicidin D-perforated patch-clamp variation of the whole-cell configuration (Le Foll et al., 1998). All tested cells elicited, in response to depolarizing step pulses from potentials negative to −60 mV, a transient outward current corresponding to the A-current described previously in frog melanotrope cells (Mei et al., 1995).
Effect of ς Ligands on Amplitude ofIA.
Transient outward potassium currents were provoked by depolarizing pulses from −120 to 50 mV. In most cells, a residual sustained outward current was detected at the end of depolarizing pulses to 50 mV. Application of (+)-pentazocine (20 μM) in the vicinity of the cells produced a reversible decrease of the current amplitude (Fig.1, A and D). The percentage of inhibition of the peak current amplitude was 15.5 ± 1.0% (n= 21; Fig. 1D). Application of (+)-pentazocine at a higher concentration (200 μM) produced a more pronounced inhibition ofIA (46.6 ± 5.0%;n = 5; Fig. 1, B and D). In very much the same way as (+)-pentazocine, the ς 1 receptor agonist, JO 1784 (20 μM), reversibly reduced IA (13.2 ± 2.0%; n = 9; Fig. 1, C and D). In another set of experiments, application of the ς 1 receptor antagonist NE 100 (0.1 μm) in the extracellular solution resulted in a significant reduction (P < .01; Mann and Whitney test) of the (+)-pentazocine-induced inhibition ofIA (15.5% ± 2.2, control; 5.4% ± 2.2, NE 100; n = 10).
Effect of (+)-Pentazocine on Decay Phase ofIA.
When short depolarizing pulses (10 to 50 ms) from −120 to 50 mV were applied, the A-current decayed after a single exponential function (data not shown). However, when longer pulses were used (200 to 500 ms), a second exponential had to be introduced to fit the all-current decay (Fig.2A). Typically, the time constants of the fast (τfast) and slow (τslow) components of the current inactivation were 23.7 ± 2.3 and 112.5 ± 13.0 ms, respectively (n = 21; Fig. 2B). Application of (+)-pentazocine (20 μM) induced an acceleration of the current decay, resulting in a marked decrease of both time constants (τfast = 10.7 ± 1.4 ms; τslow = 59.0 ± 6.3 ms; Fig. 2). The time constant values measured in the presence of (+)-pentazocine (n = 17) significantly differed from those obtained in control conditions (P < .0001, Student’s t test).
In another set of experiments, voltage ramp protocols were applied to test whether the acceleration of the current decay caused by (+)-pentazocine could be ascribed to a diminution of a leak outward current. The currents evoked by ramps from −120 to 50 mV show that the inhibitory effect of (+)-pentazocine (20 μM) occurred at voltages positive to −30 mV (n = 3). By contrast, at voltages negative to −30 mV, the leak current was apparently not modified (Fig.3).
Effect of (+)-Pentazocine on Open State I/V Relationship ofIA.
To ensure that potassium was the main charge carrier of the transient outward current, the I/V relationship of open state currents was examined. Tail currents were elicited by short depolarizing pulses (3 ms) from −120 to 50 mV, followed by repolarizations to new potentials between 40 and −90 mV (n = 7). The open stateI/V relationship exhibited a marked rectification, as predicted by the constant field Goldman-Hodgkin-Katz equation (Fig.4). Zero current was observed at −85 mV, a value close to the potassium ion equilibrium potential (EK = −88 mV). Application of (+)-pentazocine (20 μM) reduced the amplitude of the tail currents (Fig. 4A) but had no effect on the open state I/Vrelationship (Fig. 4B).
Effect of (+)-Pentazocine on Voltage-Dependent Activation ofIA.
Steady-state I/V plots were obtained by using depolarizing voltage pulses from −120 mV to potentials ranging from −70 to 50 mV (Fig.5, A and B). Application of (+)-pentazocine (20 μM) induced a reversible inhibition of the A-type currents evoked by depolarizations positive to −30 mV (n = 10; Fig. 5, A and B). The voltage dependence ofIA was studied as described earlier (Mlinar and Enyeart, 1993) by calculating the ratio of the peak current amplitude (Fig. 5) versus the tail current amplitude (Fig. 4) for each tested cell. The values, normalized as the fraction of open channels, were plotted against membrane potential and fitted by a Boltzmann function (Fig. 5C). In controls, the deduced half-maximal activation and slope factor were −25.0 ± 1.5 and 15.0 ± 1.3 mV, respectively. In the presence of (+)-pentazocine (20 μM), both the half-maximal activation (24.4 ± 1.7 mV) and slope factor (17.0 ± 1.4 mV) remained unchanged (Fig. 5C).
Effects of (+)-Pentazocine on Steady-State Inactivation ofIA.
The effects of (+)-pentazocine on the voltage dependence of the steady-state inactivation of IA were studied in 14 melanotrope cells using 1-s conditioning steps between −120 and −10 mV, preceding a constant depolarizing pulse to 50 mV. The amplitude of IA decreased as the conditioning steps were more depolarized. The I/Vrelationship followed a Boltzmann function (Fig. 5). Application of (+)-pentazocine (20 μM) significantly reduced the peak current amplitudes for conditioning steps negative to −70 mV (P < .05; Student’s t test; Fig.6, A and B). In addition, (+)-pentazocine produced a marked shift of the inactivation curve toward more negative potentials (Fig. 6B). The half-maximal inactivation potentials deduced from the Boltzmann fitting equations shifted from −78.3 ± 0.6 mV (n = 14) in control to −86.0 ± 1.2 mV (n = 11) in (+)-pentazocine-challenged cells. The slope factors, before and after application of (+)-pentazocine, were 12.4 ± 0.6 mV and 14.6 ± 0.9 mV, respectively (Fig. 6B).
The effects of (+)-pentazocine (20 μM) on the time-dependent removal of inactivation were investigated in nine melanotrope cells by shortening the conditioning hyperpolarizing steps (500 to 0 ms). The amplitude of IA gradually diminished as a single exponential function of the prepulse duration, yielding a time constant of 72.4 ms (Fig. 6C). Application of (+)-pentazocine lowered the current amplitude curve with a marked augmentation of the time constant (138 ms).
Effects of GTPγS and CTX on (+)-Pentazocine-Induced Reduction ofIA.
To determine whether a G protein was involved in the reduction of IA by (+)-pentazocine (20 μM), melanotrope cells (n = 4) were challenged with GTPγS (100 μM) added in the internal solution. IA was evoked by a 500-ms depolarization to 50 mV, after a 1-s prepulse to −120 mV in the whole-cell configuration. Application of (+)-pentazocine (3 s; 20 μM) induced a nonreversible diminution of the current, even after a 5-min of washing with external solution (Fig.7). The presence of GTPγS did not modify the current amplitude in the absence of (+)-pentazocine (Fig. 7). Repetitive recordings performed before (+)-pentazocine application revealed absence of spontaneous decrease of the current (not shown). In another set of experiments, the effects of (+)-pentazocine (20 μM) were studied in six melanotrophs preincubated with CTX (1 μg · ml−1; 12 h). In each tested cell, (+)-pentazocine failed to depress the evoked current (Fig.8, A and B). In addition, the current decay was not altered by (+)-pentazocine (Fig. 8, A, C, and D).
Discussion
Previous studies have reported that frog melanotrope cells exhibit neuron-like action potentials (Louiset et al., 1988; Valentijn et al., 1991). This spontaneous electrical activity has been shown to be directly related to the α-melanocyte-stimulating hormone (α-MSH) secretion by controlling calcium influx through voltage-dependent calcium channels (Tomiko et al., 1984). In support of this, neuroendocrine factors that increase or decrease action potential frequency stimulate or inhibit α-MSH secretion in melanotrope cells (Valentijn et al., 1991, 1994; Mei et al., 1996). In a previous work, we have demonstrated that ς ligands produce an excitatory effect on the electrical activity of frog melanotrope cells through both an inhibition of the delayed-rectifier potassium current and a reduction of a leak outward potassium conductance (Soriani et al., 1998). The present study reveals for the first time that ς 1 receptors stimulate the electrical activity of pituitary cells by modulating another potassium component, i.e., the transient outward potassium current (IA), which is strongly involved in the regulation of spike frequency. It is shown that the inhibition ofIA by ς ligands is mediated through a CTX-sensitive, G protein-dependent mechanism.
Depolarizing steps from potentials negative to −50 mV provoked fast and transient outward potassium current, described earlier as the A-type current in neurones (Ficker and Heinemann, 1992; Bardoni and Belluzzi, 1993) and pituitary cells (Mlinar and Enyeart, 1993) including frog melanotrope cells (Mei et al., 1995). Both (+)-pentazocine and JO 1784 decreased the evoked current in a voltage range attributable to IA (more than −30 mV). In addition, using a graphical current subtraction protocol, it is clearly shown that the diminution of the overall current was mainly due to a reduction of the transient current independently of any effect on leak outward or on residual delayed rectifier components (Wu et al., 1991; Soriani et al., 1998).
Both tested selective ς ligands alteredIA at doses corresponding to the micromolar concentration range required for various ς ligands [including (+)-pentazocine and JO 1784] to elicit specific functional effects in cultured neurones (Wu et al., 1991; Starr and Werling, 1994;Hayashi et al., 1995; Vilner et al., 1995) and endocrine cells (Soriani et al., 1998). In addition the effects of (+)-pentazocine, which is considered as a highly specific ς 1 receptor agonist (Monnet et al., 1992; Bowen et al., 1993; Monnet et al., 1996), were antagonized by NE 100, a selective ς 1 receptor antagonist (Chaki et al., 1994; Monnet et al., 1996). Altogether, these results bring additional evidence that ς ligands likely act through the activation of a ς 1 receptor in frog melanotrophs (Soriani et al., 1998).
To further clarify the mechanisms of inhibition ofIA by ς receptors, the effects of (+)-pentazocine on the fast decaying phase of the current were analyzed. Exponential fits revealed that during a prolonged depolarization (>100 ms), the kinetics of the current decay followed a double-exponential function. This observation, which is consistent with the kinetic characteristics of A-current reported in other vertebrate neuronal and pituitary cells (Greene et al., 1990; Bardoni and Belluzzi, 1993; Mlinar and Enyeart, 1993), suggests the existence of a complex mechanism underlying the current inactivation (Mlinar and Enyeart, 1993). In the present study, (+)-pentazocine strongly decreased both time constants of the current decay, demonstrating that ς ligands also diminished IA by shortening the current duration.
The effects of ς ligands were next investigated on the voltage-dependent activation of IA. A first set of open-state current measurements revealed thatIA reversed at a voltage command corresponding to the potassium ion equilibrium potential calculated by the Nernst equation, indicating that potassium was the only charge carrier of the current. Although (+)-pentazocine reduced the amplitude of tail current, it did not modify the open state I/Vrelationship. Subsequent analysis of the steady-state voltage-dependent activation properties of the current showed that (+)-pentazocine did neither change the slope factor nor the half-maximal activation potential. These data demonstrate that the current inhibition provoked by ς ligands was not caused by a positive shift of the voltage activation threshold of IA channels.
It is well known that the rapid inactivation of the A-current can be removed by a short hyperpolarization (Greene et al., 1990; Bardoni and Belluzzi, 1993). Steady-state inactivation experiments revealed that in frog melanotrope cells, IA is half-inactivated at a membrane potential of −78 mV. This value is consistent with half-inactivation potential values reported previously in mammalian neurones (Cull-Candy et al., 1989; Bardoni and Belluzzi, 1993; Nagatomo et al., 1995) and suggests that in frog melanotrope cells, only a few number of IAchannels would be available at resting potential (varying between −55 and −45 mV (Le Foll et al., 1997a; Soriani et al., 1998)). In fact, it is likely that IA de-inactivates during the action potential after-hyperpolarization. In the present study, it was observed that (+)-pentazocine induces a pronounced shift of the inactivation curve toward more negative potentials. In addition, it was shown that removal of inactivation depends on the duration of the hyperpolarization, following a monoexponential function as described previously in neurones (Cull-Candy et al., 1989; Ficker and Heinemann, 1992; Bardoni and Belluzzi, 1993). The time constant was nearly 2-fold increased by (+)-pentazocine, suggesting that in the presence of ς ligands, the current de-inactivation requires a longer lasting hyperpolarization. Considering the neuron-like spontaneous activity of frog melanotrope cells, it can be speculated that ς ligands partially inhibit the removal ofIA inactivation occurring during the postpotential hyperpolarization in physiological conditions. The subsequent diminution of IA, associated with the accelerated current decay, likely leads to a reduction of the postpotential hyperpolarization, allowing an increase in the action potential frequency (Soriani et al., 1998).
Interestingly, we observed that the inhibition ofIA by (+)-pentazocine was irreversibly prolonged when GTPγS was dialyzed in the cellular compartment. This result clearly shows the existence of a functional coupling between ς receptors and a G protein underlying the regulation of IA channels by ς ligands and correlates previous reports suggesting that ς 1 receptors are coupled to G proteins (Connick et al., 1992; Monnet et al., 1994;Soriani et al., 1998). The recent cloning of the ς 1 receptors has indeed revealed that it appears unrelated to other known mammalian proteins. In fact, the analysis of its sequence predicts aMr 24,000 protein with a single putative transmembrane domain (Hanner et al., 1996), which is in contradiction with the hypothesis of a ς1 receptor related to the classical G protein-coupled receptor family. In the light of this, it can be hypothesized that the protein that corresponds to ς 1 receptors functionally interacts with G proteins through a mechanism that differs from that of classical metabotropic receptors.
In the course of this study, it was also demonstrated that the inhibition of IA by (+)-pentazocine was inhibited by a CTX -pretreatment, which strongly suggests that the transduction mechanism involves a Gs protein. This observation is in a good agreement with a recent study concluding that in frog pituitary, the modulation of both the delayed rectifier and a leak outward potassium conductance by ς 1 receptors was mediated through a CTX-sensitive G protein (Soriani et al., 1998). Contrasting with these results, previous works have suggested that in rodent brain, ς 1 receptors were likely coupled to Gi/o proteins (Connick et al., 1992;Monnet et al., 1994, 1995). An explanation of this apparent discrepancy might be the existence of multiple subtypes of ς 1 receptors. In this respect, several reports have demonstrated the existence of multiple ς 1 receptors subtypes associated with different coupling mechanisms (Monnet et al., 1994, 1996).
ς receptors are believed to be responsible for important regulatory functions in the endocrine system (Su et al., 1988; Su, 1991; Eaton et al., 1996a). However, because the endogenous ς ligands are still unknown, the role of ς receptors in melanotrope cells remains unclear. Together with the presence of ς receptors in rat pars intermedia, it has been shown that in vivo administration of ς receptor antagonists decreases the plasmatic α-MSH level (Eaton et al., 1996b). Our study reveals that melanotrope cells possess a mechanism of action associated with ς 1 receptors. The existence of such a mechanism, which likely modulates α-MSH secretion, suggests that endogenous ς ligand(s) would contribute, together with other endocrine factors such as dopamine, neuropeptide Y, or γ-aminobutyric acid, to the control of pituitary functions. Because steroids have been shown to interact with ς 1 receptors (Su et al., 1988; Monnet et al., 1995; Bergeron et al., 1996) and because they exhibit a significant physiological relevance in the modulation of the electrical activity of frog melanotrope cells (Le Foll et al., 1997a, 1997b), it can be hypothesized that they represent a very interesting class of endogenous ς modulators in endocrine cells.
In conclusion, the present study validates frog pituitary melanotrope cells as an appropriate model, allowing further investigation of the transduction mechanisms of ς receptors as well as the determination of the nature of endogenous ς ligand(s) that are likely to regulate pituitary endocrine functions.
Acknowledgments
We thank Catherine Buquet for excellent technical assistance.
Footnotes
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Send reprint requests to: Pr. Lionel Cazin, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale U413, Unité Associée au Centre National de la Recherche Scientificque, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail:lionel.cazin{at}univ-rouen.fr
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↵1 This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (U 413), the Institut de Recherche Jouveinal/Parke-Davis, the European Union (Human Capital and Mobility Program; ERBCHRXCT920017), and the Conseil Régional de Haute-Normandie. O.S. was a recipient of a scholarship from the Fonds de la Recherche et de la Technologie (Conventions Industrielles de Formation par la Recherche program).
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↵2 Current address: Institut de Recherche Jouveinal/Parke-Davis, 3–9 rue de la Loge, 94260 Fresnes, France.
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↵3 Current address: Institut National de la Santé et de la Recherche Médicale U 488, 80, Avenue du Général Leclerc, 94276 Le Kremlin Bicêtre, France.
- Abbreviations:
- α-MSH
- α-melanocyte-stimulating hormone
- CTX
- cholera toxin
- GTPγS
- guanosine-5′-O-(3-thiophosphate)
- IA
- transient outward potassium current
- JO 1784
- (+)-N-cyclopropylmethyl-N-methyl-1,4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride
- NE 100
- N,N-dipropyl-2[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride
- Received September 8, 1998.
- Accepted December 2, 1998.
- The American Society for Pharmacology and Experimental Therapeutics