Introduction

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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,  receptors were thought to represent a new type of opioid receptors (Martin et al., 1976). However, subsequent pharmacological studies revealed that  binding sites were a new class of receptors, pharmacologically distinct from opioid receptors (Walker et al., 1990; Su, 1991). Whereas two types of  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  receptors (Walker et al., 1990; Monnet et al., 1994).  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  receptors has also been demonstrated in the endocrine system, including pituitary (Jansen et al., 1991; Soriani et al., 1998), suggesting that  receptors may participate to the regulation of hypothalamo-pituitary functions. In support of this, it has been shown that  ligands stimulate corticosterone and prolactin secretion in rats (Gudelsky and Nash, 1992). However, because endogenous  ligands have thus far never been definitely identified, the physiological roles of  receptors are still unknown. In addition, the cellular transduction pathways mediating the effects of exogenous  ligands remain largely unclear. Recently, a subtype of  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,  ligands stimulate electrical activity through a  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 (I_A) 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 I_A 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 I_A current (Mei et al., 1995; Nagatomo et al., 1995). These properties make I_A an interesting target to further investigate the mechanisms by which  ligands modulate the electrical activity of pituitary cells. The present study focuses on the effects of two highly specific  1 receptor agonists on I_A in cultured frog melanotrope cells.

	Materials and Methods

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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) (GTPS) 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 mM N-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 M 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 · ml¹ and then diluted in the pipette solution to a final concentration of 100 µg · ml¹ 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 m) after 7-15 min following the giga-seal formation. In whole-cell experiments, GTPS (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.  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 · min¹ 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 · ml¹) 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

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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  Ligands on Amplitude of I_A. 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 of I_A (46.6 ± 5.0%; n = 5; Fig. 1, B and D). In very much the same way as (+)-pentazocine, the  1 receptor agonist, JO 1784 (20 µM), reversibly reduced I_A (13.2 ± 2.0%; n = 9; Fig. 1, C and D). In another set of experiments, application of the  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 of I_A (15.5% ± 2.2, control; 5.4% ± 2.2, NE 100; n = 10).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effects of (+)-pentazocine on IA in frog melanotrope cells. A, upper traces, superimposed currents evoked by 500-ms depolarizations to 50 mV after a 1-s conditioning prepulse from 50 to 120 mV, recorded in a cell before (Control), during (Penta), and after (Wash) application of (+)-pentazocine (3 s; 20 µM). Lower trace, subtracting the current recorded after application of (+)-pentazocine from the control current, it appears that (+)-pentazocine mainly altered the transient component. B, same protocol as in A applied to another melanotrope cell using a higher concentration of (+)-pentazocine (200 µM). C, same protocol as in A applied to another melanotrope cell with JO 1784 (20 µM). D, relative mean amplitudes (±S.E.M.; n = 21) of peak currents in the absence (Control) and presence of (+)-pentazocine (Penta; 20 or 200 µM) or JO 1784 (JO; 20 µM). The stimulation protocol was the same as in A.

Effect of (+)-Pentazocine on Decay Phase of I_A. 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 (_fast) and slow (_slow) 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 (_fast = 10.7 ± 1.4 ms; _slow = 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).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effects of (+)-pentazocine on the inactivation kinetics of IA. A, superimposed currents evoked by a 500-ms pulse to 50 mV after a 1-s conditioning prepulse at 120 mV, recorded in a melanotroph before (Control) and during (Penta) application of (+)-pentazocine (20 µM; 3 s). The current inactivation rate was best fit by a two component exponential function (continuous lines). B, histogram of the mean values (±S.E.M.) of the fast (fast, left) and slow (slow, right) inactivation time constants in the absence (n = 21; ) or presence (n = 17; ) of (+)-pentazocine (20 µM; 3 s). ***P < .0001 (Student's t test).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effects of (+)-pentazocine on instantaneous I/V ramps in a melanotrope cell. Superimposed currents evoked by a 100-ms ramp from 120 to 50 mV after a 1-s conditioning prepulse from 50 to 120 mV, recorded before (Control), during (Penta), and after (Wash, 30 s) application of (+)-pentazocine (20 µM, 3s).

Effect of (+)-Pentazocine on Open State I/V Relationship of I_A. 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 state I/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 (E_K = 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/V relationship (Fig. 4B).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of (+)-pentazocine on open state I/V relationship of IA. A, families of tail currents evoked in a melanotrope cell by the voltage protocol shown underneath, in the absence (Control) or presence of (+)-pentazocine (Penta; 20 µM). B, corresponding open-state I/V plots. The data points are mean values (±S.E.M.) of seven independent experiments. The amplitude of the tail currents was measured 1 ms after the onset of the repolarizing voltage step, while all channels were still open. The currents were expressed as the ratio of the tail current amplitudes at 0 mV. The curves were well fit by the constant field Goldman-Hodgkin-Katz equation (continuous line). Note that (+)-pentazocine did not modify the I/V relationship. The deduced reversal potential was 85 mV in the absence or presence of (+)-pentazocine.

Effect of (+)-Pentazocine on Voltage-Dependent Activation of I_A. 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 of I_A 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).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of (+)-pentazocine on the steady-state activation of IA. A, upper traces, families of currents evoked in a melanotrope cell by depolarizing pulses from 70 to 50 mV by 10-mV increments after a 1-s conditioning prepulse from 50 to 120 mV. The families of currents were recorded in the absence (Control) and in the presence of (+)-pentazocine (20 µM; Penta). A, bottom traces, corresponding subtraction currents obtained by graphically subtracting the currents recorded under (+)-pentazocine from control currents (Subtraction). B, discrete I/V plots in the absence () or presence () of (+)-pentazocine (20 µM). Data points are mean ± S.E.M (n = 10). C, steady-state activation curves obtained by dividing the peak current amplitude by the amplitude of the corresponding open-state current shown in Fig. 4, in the absence () or presence () of (+)-pentazocine (20 µM). Values are represented as the fraction of open channels (mean ± S.E.M.; n = 7). Plots were fit with a Boltzmann function (continuous line).

Effects of (+)-Pentazocine on Steady-State Inactivation of I_A. The effects of (+)-pentazocine on the voltage dependence of the steady-state inactivation of I_A 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 I_A decreased as the conditioning steps were more depolarized. The I/V relationship 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).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effects of (+)-pentazocine on the steady-state inactivation of IA. A, families of currents evoked in a melanotrope cell by pulses to 50 mV, after 1-s conditioning prepulses from 120 to 10 mV, by 10-mV increments, as indicated underneath. The currents were obtained in the absence (Control) or presence (Penta) of (+)-pentazocine (20 µM). B, corresponding steady-state inactivation curves in the absence (n = 14; ) or presence () of (+)-pentazocine (n = 11). Values were obtained by dividing the peak currents by the maximal peak (at 120 mV) measured for each cell in control conditions. Curves were best fit with a Boltzmann equation. Vertical dotted lines indicate the half-maximal inactivation potential deduced from the Boltzmann equation for each curve. Values are mean ± S.E.M. ***P < .001; **P < .01; *P < .05 (Mann-Whitney test). C, time-dependent removal of IA inactivation in the absence () or presence () of (+)-pentazocine (20 µM). Currents were evoked by depolarizing pulses from 120 to 50 mV after 500- to 0-ms conditioning prepulses (120 mV) by 50-ms increments. Data points were obtained by dividing the peak current amplitudes by the maximal peak (obtained with the 500-ms prepulse) measured for each cell in control conditions. Curves were best fit with a single exponential function. Values are mean ± S.E.M. (n = 9). ***P < .001; **P < .01; *P < .05 (Wilcoxon test).

Effects of GTPS and CTX on (+)-Pentazocine-Induced Reduction of I_A. To determine whether a G protein was involved in the reduction of I_A by (+)-pentazocine (20 µM), melanotrope cells (n = 4) were challenged with GTPS (100 µM) added in the internal solution. I_A 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 GTPS 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 · ml¹; 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).

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Effect of intracellular GTPS on the (+)-pentazocine-induced inhibition of IA. Superimposed whole-cell currents evoked in a GTPS-dialyzed (100 µM) melanotroph by applying depolarizing pulses from 120 to 50 mV after a 1-s prepulse at 120 mV. Current traces were recorded at the times indicated. Control current was evoked at t = 0. (+)-Pentazocine (3 s; 20 µM) was first applied at t = 30s, washed with external solution (t = 60, 90, and 300 s) and then applied again (t = 330 s).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Effects of CTX pretreatment on the (+)-pentazocine-induced inhibition of IA. A, superimposed currents evoked in a CTX (1 µg · ml1; 12 h)-pretreated melanotrope cell by a depolarizing pulse from 120 to 50 mV after a 1-s conditioning prepulse at 120 mV in the absence (Control) or presence (Penta; 20 µM) of (+)-pentazocine. B, percentage of (+)-pentazocine (20 µM)-induced inhibition of the peak current in untreated (n = 21; ) and in CTX-pretreated cells (n = 6; ). Currents were obtained using the same protocol as in A. Values are means ± S.E.M. (***P < .001, Mann-Whitney test). C and D, amplitude histograms of fast (fast, C) and slow (slow, D) inactivation time constants measured in the absence () or presence () of (+)-pentazocine (20 µM) in CTX-pretreated cells (n = 6). N.S., nonsignificant difference.

	Discussion

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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 -melanocyte-stimulating hormone (-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 -MSH secretion in melanotrope cells (Valentijn et al., 1991, 1994; Mei et al., 1996). In a previous work, we have demonstrated that  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  1 receptors stimulate the electrical activity of pituitary cells by modulating another potassium component, i.e., the transient outward potassium current (I_A), which is strongly involved in the regulation of spike frequency. It is shown that the inhibition of I_A by  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 I_A (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  ligands altered I_A at doses corresponding to the micromolar concentration range required for various  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  1 receptor agonist (Monnet et al., 1992; Bowen et al., 1993; Monnet et al., 1996), were antagonized by NE 100, a selective  1 receptor antagonist (Chaki et al., 1994; Monnet et al., 1996). Altogether, these results bring additional evidence that  ligands likely act through the activation of a  1 receptor in frog melanotrophs (Soriani et al., 1998).

To further clarify the mechanisms of inhibition of I_A by  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  ligands also diminished I_A by shortening the current duration.

The effects of  ligands were next investigated on the voltage-dependent activation of I_A. A first set of open-state current measurements revealed that I_A 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/V relationship. 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  ligands was not caused by a positive shift of the voltage activation threshold of I_A 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, I_A 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 I_A channels 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 I_A 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  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  ligands partially inhibit the removal of I_A inactivation occurring during the postpotential hyperpolarization in physiological conditions. The subsequent diminution of I_A, 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 of I_A by (+)-pentazocine was irreversibly prolonged when GTPS was dialyzed in the cellular compartment. This result clearly shows the existence of a functional coupling between  receptors and a G protein underlying the regulation of I_A channels by  ligands and correlates previous reports suggesting that  1 receptors are coupled to G proteins (Connick et al., 1992; Monnet et al., 1994; Soriani et al., 1998). The recent cloning of the  1 receptors has indeed revealed that it appears unrelated to other known mammalian proteins. In fact, the analysis of its sequence predicts a M_r 24,000 protein with a single putative transmembrane domain (Hanner et al., 1996), which is in contradiction with the hypothesis of a 1 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  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 I_A 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  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,  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  1 receptors. In this respect, several reports have demonstrated the existence of multiple  1 receptors subtypes associated with different coupling mechanisms (Monnet et al., 1994, 1996).

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  ligands are still unknown, the role of  receptors in melanotrope cells remains unclear. Together with the presence of  receptors in rat pars intermedia, it has been shown that in vivo administration of  receptor antagonists decreases the plasmatic -MSH level (Eaton et al., 1996b). Our study reveals that melanotrope cells possess a mechanism of action associated with  1 receptors. The existence of such a mechanism, which likely modulates -MSH secretion, suggests that endogenous  ligand(s) would contribute, together with other endocrine factors such as dopamine, neuropeptide Y, or -aminobutyric acid, to the control of pituitary functions. Because steroids have been shown to interact with  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  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  receptors as well as the determination of the nature of endogenous  ligand(s) that are likely to regulate pituitary endocrine functions.