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*NITRIC OXIDE
Endocrinology Vol. 141, No. 9 3451-3460
Copyright © 2000 by The Endocrine Society

ARTICLES

Regulation of the GABAA Receptor by Nitric Oxide in Frog Pituitary Melanotrophs1

Hélène Castel2, Sylvie Jégou, Marie-Christine Tonon and Hubert Vaudry

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale (INSERM U-413), Unité Affiliée au Centre National de la Recherche Scientifique (UA CNRS), University of Rouen, 76821 Mont-Saint-Aignan, France

Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry{at}univ-rouen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) is implicated in the regulation of various endocrine functions, but the effect of NO on GABAA receptor transmission has never been reported in endocrine cells. In the present study, we have investigated the effects of various agents acting on the NO transduction pathway on GABAA receptor function in frog pituitary melanotrophs. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against neuronal NO synthase (nNOS) revealed that nNOS is expressed in the intermediate lobe of the pituitary and in cultured melanotrophs. Whole-cell patch-clamp recordings showed that the specific substrate of NOS L-arginine (L-Arg, 10-4 M) or the NO donor sodium nitroprusside (10-5 M) provoked a long-lasting inhibition of the current evoked by GABA (5 x 10-6 M). The NOS inhibitor L-nitroarginine (10-5 M) produced a biphasic effect, i.e. a transient decrease followed by a delayed increase of the GABA-evoked current amplitude. Similarly, the specific nNOS inhibitor 7-nitroindazole and the specific inducible NOS (iNOS) inhibitor aminoguanidine (10-5 M each) provoked a transient depression of the current followed by a sustained potentiation. Formation of cGMP in neurointermediate lobes was enhanced by L-Arg (10-4 M) and by the calcium-releasing agent caffeine (10-4 M), and inhibited by the calmodulin (CaM)/Ca2+ complex blocker W7 (10-5 M). The GABA-evoked current was potentiated by the guanylyl cyclase inhibitor ODQ (10-8–10-7 M) and inhibited by the protein kinase G (PKG) activator 8pCPT-cGMP (3 x 10-7–3 x 10-5 M). The present data indicate that NO, produced by a CaM/Ca2+-dependent NOS in frog melanotrophs, exerts an autocrine inhibitory effect on the GABA-evoked current. The action of NO on the GABAA receptor function is mediated through activation of the cGMP/PKG pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NITRIC OXIDE (NO) is now recognized as an important intracellular and intercellular messenger regulating many physiological processes including vascular tone, neuronal signaling, and immunological responses (1, 2, 3, 4, 5). NO is synthesized from L-arginine (L-Arg) by three different isoforms of NO synthase (NOS), i.e. the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS), and the inducible NOS (iNOS) (6, 7, 8, 9). After diffusion from the sites of synthesis, NO exerts its effects mainly through activation of a soluble guanylyl cyclase leading to an increase in cGMP synthesis (10, 11). In turn, cGMP regulates both voltage- and ligand-gated ion channels either via direct interaction with the channel proteins (12, 13, 14) or through indirect mechanisms involving cGMP-dependent kinases (PKG) or phosphodiesterases (15).

The GABAA receptor-channel complex, which possesses consensus sites for phosphorylation by various serine/threonine and tyrosine protein kinases, is one of the targets involved in the NO/cGMP transduction pathway (16, 17, 18, 19, 20). In particular, a consensus site for PKG is present in the ß subunits of the GABAA receptor (21, 22). In addition, it has been proposed that PKG may also phosphorylate the GABAA receptor on consensus sites for phosphorylation by PKA located in the {alpha}4, {alpha}6, {gamma}1, and {gamma}3 subunits (16, 18). The effect of PKG-mediated phosphorylation depends on the cell type that expresses the receptor. For instance, cGMP or cGMP analogs provoke an increase of the GABA-evoked current in bullfrog dorsal root ganglion neurons (23) and in Xenopus oocytes expressing recombinant human GABAA receptors (24). In contrast, NO has been shown to inhibit the GABA-evoked current via a PKG in rat neurons (25, 26) and retinal cells (27).

So far, the regulation of the GABAA receptor function by NO/PKG-mediated phosphorylation has never been investigated in neuroendocrine cells. The occurrence of a nNOS in the distal and neural lobes of the pituitary suggests that NO may act as a paracrine or autocrine factor regulating the activity of pituitary cells (28, 29, 30, 31). Recent studies indicate that NO is involved in the control of melanotrope cell activity (28, 30). The intermediate lobe of the frog pituitary, which is composed of a homogenous population of endocrine cells expressing GABAA receptors (32, 33, 34, 35), constitutes a highly suitable model in which to decipher the mechanism of action of NO on the GABAA receptor. In the present study, we have used this model to investigate the effects of NO/PKG-mediated phosphorylation on the GABAA receptor function in an endocrine cell.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and test substances
Leibovitz culture medium (L-15), antibiotics, antimicotics, collagenase type IA, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7), nicotinamide adenine dinucleotide phosphate (ß-NADPH), nitroblue tetrazolium (NBT), 3-aminobenzoic acid ethyl ester (MS 222), GABA, 1,2,3,6-tetrahydro-4-pyridinecarboxylic acid hydrochloride (isoguvacine), sodium nitroprusside (SNP), L-arginine (L-Arg), H-Arg(NO2)-OH (L-NOArg), 7-nitroindazole (7-NI), aminoguanidine, caffeine and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich Corp. (Saint-Quentin Fallavier, France). 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ) and 8-(4-chlorophenylthio)guanosine-3',5'-monophosphate (8pCPT-cGMP) were obtained from Alexis Corp. (Grünberg, Germany). FBS was from Biosys (Compiègne, France). BSA (BSA, fraction V) was from Roche Molecular Biochemicals (Meylan, France).

Animals
Adult male frogs (Rana ridibunda) were purchased from a commercial supplier (Couétard, St-Hilaire de Riez, France). The animals were kept at least for 1 week in glass tanks supplied with running water, at constant temperature (8 ± 0.5 C), under an established photoperiod (lights on, 0600–1800 h).

Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

Cell culture
Frogs were killed by decapitation, and 12 neurointermediate lobes were dissected under a microscope. The lobes were incubated for 20 min at room temperature in a solution composed of 0.2% collagenase freshly dissolved in a Ca2+–free Krebs-Ringer solution (112 mM NaCl, 2 mM KCl, 15 mM HEPES, 0.5 g/liter BSA, 0.075 g/liter glucose, supplemented with 1% antimicotic and 1% kanamycine; pH 7.4). Dissociated melanotrophs were centrifuged (500 x g, 5 min) and resuspended in L-15 culture medium adjusted to R. ridibunda osmolality (L-15/water = 1:0.4), supplemented with 10% FBS and 1% antibiotics (pH 7.4), at a density of 400,000 cells/ml. Cultured cells were maintained at 22 C in a humidified atmosphere for 5–8 days.

Histochemical localization of NADPH-diaphorase activity
Frogs were anesthetized by immersion in a solution of 0.1% MS 222 and perfused transcardially with 100 ml of 0.1 M phosphate buffer (PBS; pH 7.4). The perfusion was continued with 100 ml of McLean’s fixative. The brains with the attached pituitaries were dissected and postfixed in the same fixative solution for 3 h. The tissues were rinsed overnight in 0.1 M PBS containing 15% sucrose and then transferred into a 30% sucrose solution for 24 h. The tissues were embedded in O.C.T. Tissue Tek (Reichert-Jung, Nussloch, Germany) and immediately frozen on dry ice. Parasagittal sections (12-µm thick) were cut in a cryomicrotome (Frigocut 2800C, Leica Corp., Nussloch, Germany) and mounted on glass slides coated with 0.5% gelatin-5% chromalum.

Pituitary sections or cultured cells were preincubated for 1 h at room temperature in 0.1 M Tris-HCl buffer containing 0.3% Triton X-100, (pH 8). Tissue sections or cells were then incubated, in the same solution, with 2.2 mM reduced ß-NADPH and 0.6 mM NBT, in the dark. The development of the reaction was controlled and the incubation was stopped after 1–2 h by rinsing with PBS (3 x 5 min). Finally, the preparations were dehydrated and examined on a Leitz Orthoplan microscope. Controls were performed on sections or cultured cells incubated with either NADPH or NBT alone.

Immunohistochemical localization of {alpha}-MSH and nNOS
Localization of {alpha}-MSH and nNOS in pituitary sections and cultured cells was carried out by the indirect immunofluorescence method as previously described (36). The tissues or cells were preincubated with normal goat serum (1:50) to reduce nonspecific staining, and incubated overnight at 4 C in a humid atmosphere with antisera generated in rabbits against {alpha}-MSH (37) or against nNOS purified from porcine brain (Alexis Corp.) diluted 1:100 in PBS containing 0.3% Triton X-100 and 1% BSA. The tissues or cells were rinsed for 1 h and incubated for 2 h with fluorescein isothiocyanate-conjugated goat antirabbit {gamma}-globulins (GAR/FITC; Caltag Laboratories, Inc. San Francisco, CA) at a working dilution of 1:50. Finally, the sections or cells were rinsed in PBS and mounted with PBS-glycerol (1:1). The preparations were examined under a Leitz Orthoplan microscope. Selected slices were analyzed using a confocal laser scanning microscope (CLSM; Leica Corp., Heidelberg, Germany) equipped with a Diaplan optical system and an argon/krypton ion laser.

The specificity of the immunolabeling was controlled by substituting the {alpha}-MSH or nNOS antisera with PBS or nonimmune rabbit serum. The specificity of the {alpha}-MSH immunoreaction was verified by preabsorbing the primary antiserum with 10-6 M synthetic {alpha}-MSH (38).

Measurement of NO production
Melanotrophs were cultured for 7 days in 96-well plates. The cells were washed twice and incubated during 5 h with NOS modulators. Then, the concentration of total NO derivates (nitrate plus nitrite) was determined by using the Cayman’s nitrate/nitrite assay kit (Alexis Corp.).

Determination of cGMP concentration in pituitary extracts
Intact neurointermediate lobes were preincubated for 30 min in 250 µl Krebs-Ringer solution supplemented with 10-4 M IBMX to inhibit phosphodiesterases. The solution was gassed for 15 min with moistened O2/CO2 (95:5) before use, and the pH was adjusted to 7.4. The lobes were then incubated with test substances in 250 µl of the same solution (2 lobes per test tube) at 20 C. The reaction was stopped by adding 500 µl ice-cold 20% (wt/vol) trichloroacetic acid. After homogenization, the suspension was centrifuged (13,000 x g, 10 min) and the supernatant was washed three times with water-saturated diethylether, dried and reconstituted in RIA buffer. cGMP was measured using a RIA kit (Amersham Pharmacia Biotech, Les Ulis, France). The protein content in the pellet was determined using the Lowry method.

Electrophysiological experiments
Electrophysiological studies were performed in a temperature-controlled room (21 ± 1 C) on 5- to 8-day-old cultured cells using the standard patch-clamp technique. The patch pipettes were fabricated from 1.5-mm (outer diameter) soft glass tubes on a two-step vertical pipette puller (List-Medical, L/M-3P-A, Darmstadt, Germany) and fire-polished. The pipettes were filled with filtered intracellular solution (in mM : K-glutamate, 100; CaCl2, 1; MgCl2, 2; HEPES, 10; EGTA, 10; ATP, 1; pH 7.4). The resistance of the pipette ranged from 5 to 10 M{Omega}. Before each experiment, cultured cells were bathed in 2 ml of standard extracellular solution (in mM : NaCl, 112; KCl, 2; CaCl2, 2; HEPES, 15; pH 7.4). Cell dishes were transferred to the stage of an inverted microscope (Labovert, Leica Corp.) and continuously superfused with standard solution. Current and voltage signals were recorded from an Axopatch 200 A amplifier (Axon Instruments, Foster City, CA) and filtered at 5 kHz (3 dB, four-pole, low-pass Bessel filter). For each recording, the liquid junction potential was corrected. Signals were stored on a DTR 1202 digital tape recorder (Biologic, Claix, France) at a sampling rate of 48 kHz and later replayed on a 3200 S chart recorder (Gould, Valley View, OH) for off-line analysis or digitized at 0.27 kHz using the pCLAMP version 6.0.3 suite programs through a Digidata 1200 (Axon Instruments) connected to a computer. Peak current amplitudes were determined graphically on recordings or given by the analysis software.

Drug application
GABA and isoguvacine were dissolved in the extracellular saline solution and applied in the vicinity of the cell under study by pressure ejection from a micropipette. The cells were exposed to successive 5-sec pulses of GABA at 2-min intervals to avoid tachyphylaxis. The cell permeant substances tested were driven by gravity (600 µl/min) through a plastic tubing positioned near the cell body.

Statistical analysis
Normalized currents (I/Icontrol) are expressed as the ratio of the GABA-evoked current amplitude recorded in the presence (I) or absence (Icontrol) of test substances.

All data are expressed as mean ± SEM. The statistical significance of differences was determined by using the Student’s t test or by ANOVA followed by a Student’s-Newman-Keuls multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of NOS activity and nNOS immunoreactivity in the frog pituitary
The presence and distribution of NOS were examined by using the NADPH-diaphorase reaction on parasagittal pituitary sections and cultured melanotrophs. Labeled cells were observed throughout the intermediate lobe as well as in the distal lobe, at the boundary of the intermediate lobe. Intense staining was also detected in fiber plexuses in the neural lobe (Fig. 1AGo). To determine whether the cultured melanotrophs used for electrophysiological experiments displayed NOS activity, NADPH-diaphorase reactivity was investigated in cells 7 days after plating. As shown in Fig. 1BGo, a strong reactivity was found in the cytoplasm of cultured cells.



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  		Figure 1. Microphotographs showing the distribution of NOS in frog pituitary sections and cultured melanotrophs. A, Histochemical labeling of NADPH-diaphorase activity (dark staining) in the pituitary. B, Histochemical labeling of NADPH-diaphorase activity in 7-day-old cultured melanotrophs. C, Immunofluorescence labeling of nNOS-like immunoreactivity in the pituitary. D, Immunofluorescence labeling of nNOS-like immunoreactivity in a 7-day-old cultured melanotroph analyzed with a CLSM. E and F, Double labeling of cultured melanotrophs for {alpha}-MSH immunoreactivity (E) and NADPH-diaphorase activity (F). PD, pars distalis; PI, pars intermedia; PN, pars nervosa. Scale bars: A and C, 50 µm; B, 20 µm; D, 5 µm; E and F, 10 µm.

 
Immunofluorescence labeling using a porcine nNOS antiserum, revealed intense immunoreactivity in the neural and intermediate lobes (Fig. 1CGo). In contrast, the distal lobe was virtually devoid of immunoreactivity (Fig. 1CGo). Confocal laser scanning microscope analysis showed that cultured intermediate lobe cells were intensely immunoreactive (Fig. 1DGo). Double labeling of cultured cells with the {alpha}-MSH antibody and the NADPH-diaphorase reaction revealed that all melanotrophs exhibited NADPH-diaphorase activity (Fig. 1Go, E and F).

Production of NO by frog melanotrophs
The production of NO by cultured melanotrophs was investigated by measuring the total amount of nitrate/nitrite contained in the culture medium. The NOS substrate L-arginine (L-Arg, 10-4 M) significantly increased (P < 0.05) the concentration of nitrate/nitrite in the incubation medium while the specific nNOS inhibitor 7-NI (10-5 M) significantly reduced (P < 0.05) the basal production of nitrate/nitrite (Fig. 2Go). In addition, both 7-NI and the nonspecific iNOS/nNOS inhibitor L-NOArg totally abolished (P < 0.01) the L-Arg-induced stimulation of nitrate/nitrite formation (Fig. 2Go).



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  		Figure 2. Effects of NOS modulators on nitrate/nitrite production by cultured frog melanotrophs. Seven-day-old cells were incubated with the NOS substrate L-Arg in the absence or presence of the nonspecific NOS inhibitor L-NOArg (10-5 M) or the specific nNOS inhibitor 7-NI (10-5 M). Each value represents the mean (± SEM) of triplicates from a representative experiment. Three independent experiments were performed and similar results were obtained. *, P < 0.05; **, P < 0.01 (one-way ANOVA followed by a Student’s-Newman-Keuls posttest).

 
Modulation of the GABA-evoked current by NO
The effects of NO on the electrical activity induced by GABA in cultured melanotrophs was studied by the patch-clamp technique in the whole-cell configuration. A 4-min superfusion of the cells with L-Arg (10-4 M), L-NOArg (10-5 M) or the NO-releasing compound SNP (10-5 M) did not modify the spontaneous action potential firing frequency (not shown). To investigate the effects of NO-releasing agents and NOS modulators on the whole-cell GABA-evoked current, pulses of GABA (5 x 10-6 M, 5 sec) were sequentially applied at 2-min intervals. Bath perfusion of L-Arg (10-4 M) reduced the bioelectrical response to GABA by increasing the input resistance. The maximum response was observed 8 min after the onset of the superfusion with L-Arg (Fig. 3AGo). Reciprocally, L-Arg exhibited a substantial decrease of the GABA-evoked current, which completely recovered only after 10 min washout (Fig. 3BGo). Similarly, SNP (10-5 M) gave rise to a sustained increase of the input resistance (Fig. 3CGo) and provoked a prolonged inhibition of the current amplitude (Fig. 3DGo).



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  		Figure 3. Effects of the NOS substrate L-Arg and the NO donor SNP on the GABA-evoked current in frog melanotrophs. Pulses of GABA (5 x 10-6 M, 5 sec) were applied at 2-min intervals before, during and after a 4-min superfusion with L-Arg (10-4 M, A) or SNP (10-5 M, B). A, Time-course of the effect of L-Arg (horizontal bars) on the relative input resistance measured in the presence of GABA (n = 4). The cell input resistance was monitored by administrating hyperpolarizing pulses (100 pA, 400 msec, 0.2 Hz) superimposed to the holding current (0 pA). For each data point (mean ± SEM), Rcontrol corresponds to the mean input resistance calculated from 6 successive pulses delivered in the absence of GABA, and R to 3 pulses applied in the presence of GABA. L-Arg provoked a gradual increase of the input resistance. B, Whole-cell current evoked by GABA (5 x 10-6 M) before (trace #1), during (traces #2 and 3) and after (traces #4, 5, and 6) a 4-min superfusion with L-Arg (10-4 M, n = 13, horizontal bar). The current traces shown during washout were recorded 4, 6, and 10 min after withdrawal of L-Arg. The holding potential was set at 0 mV. L-Arg provoked a long-lasting depression of the GABA-evoked current. C and D, Same protocol as in A and B, exept that L-Arg was replaced by SNP (10-5 M). SNP induced an increase of the input resistance (C, n = 3) and a decrease of the current amplitude (D, n = 15).

 
Inhibition of NOS by L-NOArg (10-5 M) resulted in a biphasic response pattern, consisting of a transient increase followed by a reversible decrease of the input resistance (Fig. 4AGo), associated with a transient decrease followed by a sustained increase in the current amplitude (Fig. 4BGo).



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  		Figure 4. Effect of the NOS inhibitor L-NOArg on the GABA-evoked current in frog melanotrophs. Pulses of GABA (5 x 10-6 M, 5 sec) were applied at 2-min intervals before, during and after a 4-min superfusion with L-NOArg (10-5 M). A, Time-course of the effect of L-NOArg (horizontal bar) on the relative input resistance measured in the presence of GABA (n = 3). The relative input resistance was monitored as indicated in Fig. 3Go. L-NOArg induced an increase of the input resistance followed by a reversible decrease. B, Current evoked by GABA (5 x 10-6 M) before (trace #1), during (traces #2 and 3) and after (traces #4, 5, and 6) a 4-min superfusion with L-NOArg (10-5 M, n = 14, horizontal bar). The current traces shown during washout were recorded 4, 6 and 10 min after withdrawal of L-NOArg. The holding potential was set at 0 mV. L-NOArg induced a transient depression followed by a reversible increase of the GABA-evoked current.

 
The selective GABAA agonist isoguvacine was also used to investigate the effect of NO on the GABAA receptor function. Repeated pulses of isoguvacine (5 x 10-6 M, 5 sec) were applied on melanotrophs during and after superfusion (2 min) with L-Arg (10-4 M) or L-NOArg (10-5 M). In all cells studied (n = 35), isoguvacine mimicked the GABA-evoked current. Application of L-Arg provoked a long-lasting inhibition of the isoguvacine-evoked current (Fig. 5AGo) while L-NOArg induced a transient inhibition followed by a reversible increase of the current amplitude (Fig. 5BGo). The effects of L-Arg and L-NOArg on the isoguvacine-evoked current vanished after 10 min washout (Fig. 5Go, A and B). Both L-Arg and L-NOArg modified the slope of the instantaneous current/voltage (I/V) curves but did not shift the reversal potential (-70 mV, a value corresponding to the chloride equilibrium potential in the present experimental conditions) (not shown).



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  		Figure 5. Effects of L-Arg and L-NOArg on the isoguvacine-evoked current in frog melanotrophs. Whole-cell current evoked by isoguvacine (5 x 10-6 M, 5 sec) before (trace #1), during (trace #2) and after (traces #3, 4, and 5) a 2-min superfusion with L-Arg (10-4 M, n = 7, A) or L-NOArg (10-5 M, n = 11, B) (horizontal bars). The holding potential was set at 0 mV. L-Arg markedly and reversibly diminished the isoguvacine-evoked current while L-NOArg provoked a transient attenuation followed by an increase of the current.

 
A 2-min superfusion with graded concentrations of L-Arg (10-6 to 5 x 10-4 M) induced a dose-dependent decrease of the current evoked by GABA (5 x 10-6 M) (Fig. 6AGo). A concentration-related inhibitory effect of L-Arg was still observed after 8 min washout. The NO donor SNP (10-6 to 5 x 10-4 M) also induced a concentration-dependent inhibition of the GABA-evoked current (not shown). A 2-min superfusion with the NOS inhibitor L-NOArg produced a transient inhibition of the GABA-evoked current followed by a potentiation of the current during the washout period (Fig. 6BGo).



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  		Figure 6. Effects of graded doses of L-Arg and L-NOArg on the GABA-evoked current in frog melanotrophs. A, A 2-min superfusion with L-Arg (10-6 M to 5 x 10-4 M) provoked a long lasting depression of the current induced by a 5-sec pulse of GABA (5 x 10-6 M). The inhibitory effect of L-Arg was still observed 8-min after the onset of the washout period. B, A 2-min superfusion with L-NOArg (10-6 M to 5 x 10-4 M) depressed the GABA-evoked current. During washout, the inhibitory effect of L-NOArg gradually vanished and potentiation of the GABA-evoked current was observed. Normalized current (I/Icontrol) is expressed as the ratio of the current amplitude recorded in the presence (I) or absence (Icontrol) of L-Arg or L-NOArg. Each point represents the mean value (± SEM) obtained from independent recordings of 6 to 13 distinct melanotrophs. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).

 
Effect of selective NOS inhibitors on the GABA-evoked current
To determine the type of NOS present in frog melanotrophs, the effect of selective NOS inhibitors on the GABA-evoked current was studied. A 10-min superfusion with the specific nNOS inhibitor 7-NI (10-5 M) induced a modest and transient decrease of the current followed by a gradual and robust potentiation (Fig. 7AGo). Similarly, the specific iNOS inhibitor aminoguanidine (10-5 M) provoked a slight inhibition followed by a gradual increase of the current (Fig. 7BGo). The nonselective NOS inhibitor L-NOArg (10-5 M) induced a biphasic response that was very similar to the sum of the individual effects of 7-NI and aminoguanidine (Fig. 7CGo).



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  		Figure 7. Effects of nNOS and iNOS inhibitors on the GABA-evoked current in frog melanotrophs. The selective nNOS inhibitor 7-NI (10-5 M, A), the selective iNOS inhibitor aminoguanidine (10-5 M, B), and the nonselective inhibitor L-NOArg (10-5 M, C) were superfused for 10 min (horizontal bars). Each NOS inhibitor induced a slight and transient decrease followed by a gradual increase of the current evoked by GABA (5 x 10-6 M). The dotted line in C corresponds to the sum of the individual effects of 7-NI and aminoguanidine shown in A and B. Normalized current (I/Icontrol) is expressed as the ratio of the current amplitude recorded in the presence (I) or absence (Icontrol) of the NOS inhibitors. Each point represents the mean value (± SEM) obtained from independent recordings of 4 to 8 distinct melanotrophs. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).

 
Effects of NOS substrate and calmodulin/Ca2+ complex modulators on cGMP production
To determine whether the action of NO on the GABA-evoked current was mediated through the NO/cGMP cascade, the effects of NOS substrate and calmodulin/Ca2+ (CaM/Ca2+) complex modulators on cGMP formation by neurointermediate lobes were investigated. A 3-min incubation with L-Arg (10-4 M) (Fig. 8AGo) or the Ca2+-releasing agent caffeine (10-4 M) (Fig. 8BGo) provoked a significant rise (P < 0.05) of the cGMP production. Conversely, exposure of neurointermediate lobes to the CaM/Ca2+ complex inhibitor W7 (10-5 M) significantly reduced the basal cGMP level (P < 0.05) and totally abolished the L-Arg-evoked increase in cGMP production (P < 0.01) (Fig. 8AGo).



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  		Figure 8. Effects of NOS substrate and CaM/Ca2+ complex modulators on cGMP production by frog neurointermediate lobes. A, Effect of the NOS substrate L-Arg (10-4 M, 3 min) in the absence or presence of the CaM/Ca2+ complex inhibitor W7 (10-5 M, 3 min) on cGMP production. B, Effect of the Ca2+-releasing agent caffeine (10-4 M, 3 min) on cGMP production. Each value represents the mean (± SEM) of triplicates from a representative experiment. 4 (A) and 2 (B) independent experiments were performed and similar results were obtained. *, P < 0.01; **, P < 0.05 (one-way ANOVA followed by a Student’s-Newman-Keuls posttest).

 
Effect of PKG modulators on the GABA-evoked current
A 2-min superfusion with the guanylyl cyclase inhibitor ODQ provoked potentiation of the current evoked by GABA (5 x 10-6 M, 5 sec). The maximum effect (P < 0.001) was observed at concentrations of 10-8 M to 10-7 M both during superfusion of ODQ and during washout (Fig. 9AGo). Reciprocally, a 2-min superfusion with the PKG activator 8pCPT-cGMP (3 x 10-7 M to 3 x 10-5 M) provoked a dose-dependent inhibition of the GABA-evoked current. A significant inhibitory effect of 8pCPT-cGMP was still observed during washout (Fig. 9BGo).



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  		Figure 9. Effects of graded concentrations of ODQ and 8pCPT-cGMP on the GABA-evoked current in frog melanotrophs. The concentration-response relationships were studied on the GABA-evoked current (5 x 10-6 M, 5 sec) after a 2-min superfusion with the guanylyl cyclase inhibitor ODQ (A) or the PKG activator 8pCPT-cGMP (B) and after a 2-min or 6-min washout period. Normalized current (I/Icontrol) is expressed as the ratio of the current amplitude recorded in the presence (I) or absence (Icontrol) of ODQ or 8pCPT-cGMP. Each point represents the mean value (± SEM) from 6 to 8 independent recordings. **, P < 0.01; ***, P < 0.001 (Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have recently shown that, in melanotrophs of the frog Rana ridibunda, phosphorylation of the ß2/ß3 subunits of the GABAA receptor causes inhibition of the GABA-evoked current (39). The present study now demonstrates that activation of the NO cascade in cultured melanotrophs stimulates the cGMP/PKG pathway leading to an inhibition of the GABAA receptor function.

Occurrence of NOS and production of NO in the neurointermediate lobe
NOSs are composed of an N-terminal oxygenase domain and a C-terminal reductase domain (40). The reductase domain, which is related to cytochrome-P450 NADPH-reductase, possesses the ability to transfer electrons from the coenzyme NADPH to various substrates such as NBT (7, 41). This NADPH-diaphorase activity has been exploited for the development of a robust histochemical technique which is widely used for the localization of NO-producing cells (42, 43).

Histochemical staining of NADPH-diaphorase activity revealed intense labeling of nerve terminals in the pars nervosa and moderate labeling of endocrine cells in the pars intermedia of the pituitary of the frog Rana ridibunda. Immunohistochemical staining using polyclonal antibodies against porcine nNOS showed the occurrence of NOS-like immunoreactivity in the neural and intermediate lobes, while the distal lobe of the pituitary appeared virtually devoid of immunolabeling. Staining of cultured cells confirmed that both NADPH-diaphorase activity and nNOS-like immunoreactivity were actually contained in melanotrophs of Rana ridibunda. In the African clawed frog Xenopus laevis, the presence of NADPH-diaphorase activity and nNOS immunoreactivity has been detected in a few cells scattered in the pars intermedia as well as in fibers innervating the intermediate lobe (28). In rat, the presence of NADPH-diaphorase staining and NOS immunolabeling has been observed in the neural lobe and in a few cells of the intermediate lobe at the boundary of the distal lobe (30). It thus appears that the distribution of NOS in the pituitary somewhat differs between vertebrate species. The occurrence of both NADPH-diaphorase activity and NOS-like immunoreactivity in most cells of the pars intermedia and in nerve plexuses of the pars nervosa in Rana ridibunda indicates that NO can be produced within the pars intermedia or may diffuse from the neural lobe to the intermediate lobe, suggesting that, in frog, NO may act as a local regulator of melanotroph activity. Besides its direct effect on pars intermedia cells, NO may also act presynaptically on nerve fibers terminating in the intermediate lobe parenchyma. In support of this hypothesis, it has been reported that, in rat, NO stimulates the release of dopamine from tubero-hypophyseal nerve terminals, which in turn causes inhibition of ß-endorphin secretion (30).

Direct measurement of nitrate and nitrite formation showed that the production of NO by the neurointermediate lobes of Rana ridibunda was stimulated by the NOS substrate L-Arg, and that the L-Arg-evoked increase in NO production was abrogated by the NOS inhibitor L-NOArg and by the selective nNOS inhibitor 7-NI. These observations indicate that the NOS-like immunoreactivity detected in the frog pars intermedia actually corresponds to an active form of the enzyme. It thus appears that NO, produced by melanotrophs of Rana ridibunda, may exert intracrine effects possibly through activation of a soluble guanylyl cyclase (10, 11). Alternatively, NO which can easily diffuse across plasma membrane may act as a paracrine factor within the frog intermediate lobe, as previously reported in the rat anterior pituitary (44, 45).

Effect of NO on GABAA receptor function
Electrophysiological studies showed that both L-Arg and the NO donor SNP provoked a marked inhibition of the GABA-evoked current associated with a long-lasting increase of the input resistance. A similar inhibition was observed when GABA was replaced by the selective GABAA receptor agonist isoguvacine, indicating that the effect of NO can be accounted for only by modulation of the GABAA receptor function. Blockage of the basal production of endogenous NO by the NOS inhibitor L-NOArg enhanced the chloride current evoked by GABA and isoguvacine, thus confirming the inhibitory effect of NO on the GABAA receptor function. In addition, L-NOArg induced a transient inhibition of the GABA/isoguvacine-evoked current mirrored by a transient increase of the input resistance, indicating that NO may exert complex effects on the GABAA receptor. In fact, studies conducted on recombinant GABAA receptors have recently shown that NO may exert either an inhibitory or a potentiating effect depending on the subunit composition and the concentration of NO (46). Taken together, our data indicate that, in frog melanotrophs, NO induces a predominant inhibition of GABAA receptor function. Endogenous NO may also exert a modest tonic stimulatory effect that can be accounted for by the diversity of the subunit composition of native GABAA receptors in melanotrophs of Rana ridibunda (36).

Type of NOS implicated in the NO-induced modulation of the GABAA receptor function
The modulatory effects of the selective nNOS inhibitor 7-NI (47) and the selective iNOS inhibitor aminoguanidine (48) on the GABA-evoked current indicated that an nNOS and, in a lesser proportion, an iNOS are present in cultured melanotrophs. It has previously been reported that both NOS isoforms possess consensus binding sites for CaM (7, 40, 49). In particular, the Ca2+-bound form of CaM has been found to activate nNOS in the rat cerebellum (50, 51). The present study shows that an increase in Ca2+ induced by caffeine activated cGMP formation. In addition, the CaM inhibitor W7 reduced both the basal cGMP level and the L-Arg-stimulated cGMP production. These data indicate that the NO-induced activation of cGMP formation is mainly attributable to activation of a neuronal NOS isoform (40). However, because the specific NOS inhibitor aminoguanidine also increased the GABA-evoked current, the inducible form of NOS may also contribute to NO formation. Expression of iNOS could be induced by various cytokines as previously reported in rodents (52). Altogether, these observations indicate that, in frog melanotrophs, NO formation can be accounted for by CaM/Ca2+ activation of nNOS and possibly transcriptional induction of iNOS.

Pathways involved in the NO-induced inhibition of GABAA receptor function
The observation that L-Arg and SNP stimulated cGMP production suggested that the effect of NO on the GABA-induced current in the melanotrophs of Rana ridibunda was mediated through activation of a soluble guanylyl cyclase, as previously shown in many tissues (10). In support of this hypothesis, the guanylyl cyclase inhibitor ODQ markedly potentiated the GABA-evoked current, whereas the PKG activator 8pCPT-cGMP induced a concentration-dependent inhibition of the current. These observations suggest that, in melanotrophs, the inhibitory effect of NO on the chloride current can be ascribed to PKG-dependent phosphorylation of GABAA receptor subunits. Indeed, it is now well established that, in mammals, various subunits of the GABAA receptor possess consensus sites for phosphorylation by PKG (18, 21, 22). In particular, the ß2/ß3, {gamma}1, and {gamma}3 subunits, which are all expressed in frog melanotrophs (36), represent potential targets for PKG-dependent phosphorylation of the GABAA receptor.

A proposed model illustrating the NO-induced cascade mediating inhibition of the GABAA receptor function in Rana ridibunda melanotrophs is shown in Fig. 10Go. Activation of an nNOS by the CaM/Ca2+ complex leads to the formation of NO, which in turn activates a soluble guanylyl cyclase. The resulting increase in cGMP production enhances PKG activity which may phosphorylate GABAA receptor ß subunits yielding to inhibition of the GABA-evoked current.



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  		Figure 10. Schematic representation summarizing the NO-induced cascade regulating the GABAA receptor function in frog pituitary melanotrophs. A neuronal NOS (nNOS), activated by a CaM/Ca2+ complex, catalyzes the formation of NO from L-arginine (L-Arg). NO activates a soluble guanylyl cyclase (sGC) leading to an increase in cGMP production. The resulting activation of a protein kinase G (PKG) induces phosphorylation of the ß2/ß3 subunits of the GABAA receptor (GABAA-R) which, in turn, causes inhibition of the chloride current.

 


   Acknowledgments
 
The authors wish to thank Mrs. Catherine Buquet for expert technical assistance.


   Footnotes
 
1 This work was supported by grants from INSERM U 413, the European Union (Human Capital and Mobility Program ERBCHRXCT 920017), and the Conseil Régional de Haute-Normandie. Back

2 Recipient of a fellowship from the Ministère de l’Education Nationale, de la Recherche et de la Technologie. Back

Received March 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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