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Endocrinology, doi:10.1210/en.2003-0176
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Endocrinology Vol. 144, No. 12 5556-5567
Copyright © 2003 by The Endocrine Society

Neurotensin Stimulates Both Calcium Mobilization from Inositol Trisphosphate-Sensitive Intracellular Stores and Calcium Influx through Membrane Channels in Frog Pituitary Melanotrophs

Amor Belmeguenai, Laurence Desrues, Jerome Leprince, Hubert Vaudry, Marie-Christine Tonon and Estelle Louiset

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale, Unité-413, 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, Institut National de la Santé et de la Recherche Médicale, Unité-413, 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
 
Neurotensin (NT) is a potent stimulator of electrical and secretory activities in frog pituitary melanotrophs. The aim of the present study was to characterize the transduction pathways associated with activation of NT receptors in frog melanotrophs. Application of synthetic frog NT (fNT) increased the cytosolic calcium concentration ([Ca2+]c) and stimulated the formation of inositol trisphosphate (IP3). The phospholipase C inhibitor U-73122 blocked the electrophysiological and secretory effects of fNT. Intracellular application of the IP3 receptor antagonist heparin abolished fNT-induced electrical activity. Suppression of Ca2+ in the incubation medium markedly reduced the effect of NT on [Ca2+]c, firing rate, and {alpha}-melanocyte-stimulating hormone ({alpha}MSH) secretion. Similarly, the inhibitor of IP3-induced Ca2+ release and store-operated Ca2+ channels, 2-Aminoethoxydiphenylborane, and the nonselective Ca2+ channel blockers GdCl3 and NiCl2, attenuated the [Ca2+]c increase and the electrical and secretory responses evoked by fNT. Coapplication of the L- and N-type Ca2+ channel blockers nifedipine and {omega}-CgTx GVIA reduced the effects of fNT on action potential discharge, [Ca2+]c increase, and {alpha}MSH release. The protein kinase C (PKC) inhibitors, PKC-(19–31) and chelerythrine, reduced the electrophysiological and secretory responses induced by iterative applications of fNT. Collectively, these results demonstrate that, in frog melanotrophs, NT stimulates the phospholipase C/PKC pathway and increases [Ca2+]c. Both Ca2+ release from intracellular stores and Ca2+ influx through L- and N-type Ca2+ channels are involved in fNT-induced {alpha}MSH secretion. In addition, the present data indicate that PKC plays a crucial role in maintenance of the responsiveness of melanotrophs to NT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROTENSIN (NT) is a tridecapeptide originally isolated from the bovine hypothalamus and subsequently characterized in different species (1, 2, 3). NT is present in the brain (4) and in various peripheral organs, including intestinal mucosa (5), adrenal gland (6, 7), and pituitary (8, 9). NT exerts a wide range of biological effects, including vasodilation and regulation of gut motility, locomotion, temperature, nociception, and paradoxical sleep (2, 10, 11, 12). NT acts as a neurotransmitter stimulating the electrical activity of neurons (13, 14, 15). NT also regulates the release of various hormones from pancreas (16), adrenal gland (7, 17), and pituitary (3, 18, 19, 20).

Three NT receptors (NTR1, ntr2, and ntr3) with distinct pharmacological properties have been cloned to date (21). The transduction pathways associated with activation of NT receptors has been mainly investigated in cells transfected with NTR1 or ntr2 and in tumoral cell lines expressing native NTR1 receptors. These studies have shown that activation of NTR1 increases inositol trisphosphate (IP3) production and induces Ca2+ mobilization from intracellular stores (22, 23, 24, 25). It has also been reported that activation of NTR1 modulates adenylyl cyclase and/or guanylyl cyclase activities (24, 26). The signaling mechanisms associated with ntr2 receptor activation have not yet been clearly identified. Some data suggest that ntr2 is weakly coupled to the phospholipase C (PLC)/Ca2+ pathway (27, 28), but not to the adenylyl cyclase and guanylyl cyclase signaling cascades (29). In astrocytes and nerve cells, NT provokes a transient mobilization of both intracellular Ca2+ and Ca2+ influx through the plasma membrane (30, 31). There is increasing evidence for distinct roles of spatial and temporal variations in the cytosolic Ca2+ concentration ([Ca2+]c) in Ca2+-dependent cellular processes (32, 33). However, the transduction mechanisms involved in calcium signaling and the implication of each source of Ca2+ in the physiological responses of endocrine cells to NT have never been studied.

Amphibian pituitary melanotrophs provide a suitable model in which to investigate the transduction pathways activated by NT in normal endocrine cells. 1) The intermediate lobe of the frog pituitary is composed of a single population of endocrine cells, the melanotrophs, and thus these cells possess the same advantages as a cell line without the drawbacks of tumoral cells. 2) Frog melanotrophs exhibit spontaneous electrical activity underlying Ca2+ influx and actively secrete {alpha}-melanotropic hormone ({alpha}MSH) in a Ca2+-dependent manner (33, 34, 35, 36). 3) Synthetic frog NT (fNT) stimulates both the electrical and secretory activities of frog melanotrophs (3, 37). The aim of the present study was to investigate the involvement of the PLC/protein kinase C (PKC) signaling pathway and the respective contribution of IP3-sensitive and extracellular Ca2+ sources in the physiological responses of melanotrophs to neurotensin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs (Rana esculenta; body weight, 40–50 g) were obtained from a commercial source (Couétard, Saint-Hilaire de Riez, France). The animals were housed in a temperature-controlled room (8 ± 0.5 C) under running water on a 12-h dark, 12-h light regimen (lights on from 0600–1800 h). Animal manipulations were carried out according to the recommendations of the French ethical committee under the supervision of authorized investigators.

Reagents and test substances
fNT was synthesized by solid phase methodology as previously described (3). Leibovitz culture medium (L15), GTP, gramicidin D, collagenase (type IA), U-73122, U-73343, heparin, de-N-sulfated heparin, chelerythrine, nifedipine, {omega}-conotoxin GVIA ({omega}-CgTx GVIA), and synthetic {alpha}MSH were purchased from Sigma-Chemie (St. Quentin-Fallavier, France). 2-Aminoethoxydiphenylborane (2-APB) was obtained from Tocris (Bristol, UK). The PKC inhibitor peptide 19–31 [PKC-(19–31)] was from Calbiochem (La Jolla, CA). BSA (fraction V) was purchased from Roche (Meylan, France). The antibiotic-antimycotic solution and fetal bovine serum were obtained from BioWhittaker (Walkersville, MD). Indo-1/acetoxymethylester was obtained from Molecular Probes (Leiden, The Netherlands). Myo-[3H]inositol (specific activity, 100 Ci/mmol) and 125INa (specific activity, 2.3 Ci/mmol) were purchased from Amersham (Les Ulis, France).

Compounds U-73122, U-73343, 2-APB, and chelerythrine were initially dissolved in dimethylsulfoxide, and nifedipine was dissolved in ethanol. The final concentration of dimethylsulfoxide or ethanol in the incubation medium was less than 0.1% (vol/vol).

Cell culture
Neurointermediate lobes (NIL) were collected in Krebs-Ringer solution consisting of 112 mM NaCl, 2 mM KCl, 2 mM CaCl2, 15 mM HEPES, 11 mM glucose, and 0.3 mg/ml BSA (pH 7.4). The NIL were enzymatically dissociated by collagenase type IA (1 mg/ml) in a Ca2+-free Ringer’s solution (38). The cell suspension was rinsed and plated on poly-L-lysine-coated glass coverslips, in 35-mm culture dishes or in 24-well dishes. Cultured cells were maintained in L15 culture medium adjusted to Rana esculenta osmolarity (L15/water; 1:0.4, vol/vol; fL15) and supplemented with 1.1 mM glucose, 0.7 mM CaCl2, 15 mM HEPES, 1% each of the kanamycin and antibiotic-antimycotic solutions, and 10% fetal bovine serum. Cultured cells were kept at 21 C in a humidified atmosphere for 4–7 d. The culture medium was renewed every 72 h.

Cytosolic calcium ([Ca2+]c) measurement
[Ca2+]c was monitored by a dual emission microfluorimeter system as previously described (38). Briefly, melanotrophs cultured on poly-L-lysine-coated glass coverslips were incubated in the dark at room temperature for 1 h in a Krebs-Ringer solution containing 5 µM indo-1/acetoxymethylester. The fluorescence emission of indo-1, induced by excitation at 355 nm, was measured at two wavelengths (405 and 480 nm) by separate photometers (P1; Nikon, Champigny-sur-Marne, France). The three signals (405, 480, and 405/480 nm ratio) were continuously recorded using an AS1-type acquisition card with the JAD-FLUO program (Notocord Systems, Croissy-sur-Seine, France). [Ca2+]c was calculated from the formula established by Grynkiewicz et al. (39): [Ca2+]c = ß x Kd x (R - Rmin)/(Rmax - R), where Rmin represents the minimum fluorescence ratio obtained after incubation of cells with 10 mM EGTA and 10 mM ionomycin, Rmax is the maximum fluorescence ratio obtained after incubation of cells with 10 mM CaCl2 and 10 mM ionomycin, and ß is the ratio of fluorescence yields from the Ca2+min/Ca2+max indicator at 480 nm. The values for Rmin, Rmax, and ß were 0.164, 1.82, and 1.62, respectively. Kd is the dissociation constant for indo-1 (250 mM) as previously determined (40). Test substances including fNT were delivered in the vicinity of cells under study by a superfusion system. Results were expressed as the mean amplitude of [Ca2+]c increase ± SEM. Statistical significance was assessed by a two-tailed paired t test.

Quantification of IP3
Measurement of IP3 production was performed as previously described (38, 41) with minor modifications. Briefly, melanotrope cells cultured on 24-well dishes were incubated with myo-[3H]inositol (40 µCi/ml; 21 C; 18 h) in fL15 culture medium. Then the labeled medium was removed, and the cells were washed three times with Krebs-Ringer solution supplemented with 10-3 M inositol. After a 15-min preincubation with LiCl (10-2 M), fNT (10-8 M) was applied. The reaction was stopped with ice-chilled trichloroacetic acid (10% in the final volume). [3H]Inositol phosphates were analyzed by anion exchange chromatography on AG1-X8 resin. The radioactivity in the effluent was quantified using a flow scintillation detector (Radiomatic Flo-One beta A-500, Packard Instrument Co., Meriden, CT). The concentration of 3H-labeled IP3 resolved by chromatographic analysis was expressed as a percentage of the total amount of [3H]inositol phosphates eluted from each sample. Statistical significance was assessed by t test.

Electrophysiological studies
Electrical activities were recorded at room temperature by the patch-clamp technique, using either the gramicidin-perforated or the whole cell configuration. Cultured cells were continuously superfused with an extracellular solution consisting of 112 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 15 mM HEPES-NaOH (pH 7.4). For gramicidin-perforated patch-clamp recordings, the patch pipettes were filled with a solution containing 100 mM KCl, 10 mM HEPES-KOH, and 100 µg/ml gramicidin D (pH 7.4). For whole cell recordings, the intrapipette solution contained 100 mM KCl, 2 mM MgCl2, 10 mM EGTA, 0.1 mM GTP, and 10 mM HEPES-KOH (pH 7.4). In some experiments, heparin, de-N-sulfated heparin, or PKC-(19–31) was dissolved in the intrapipette solution. The electrical signals were acquired using an Axopatch 200 A (Axon Instruments, Foster City, CA) and were traced on a 2200S paper recorder (Gould, Valley View, OH). Synthetic fNT was applied in the vicinity of the cell under study by a pressure ejection system. The other test substances were applied by a superfusion system.

Perifusion experiments
The perifusion system used to determine the effects of test substances on {alpha}MSH secretion has been previously described (42). Freshly dispersed melanotrophs (equivalent to 10 NIL/perifusion column) were suspended in a Bio-Gel P2 matrix and perifused with Krebs-Ringer solution at a constant flow rate (0.3 ml/min) and temperature (24 C). After a 2-h stabilization period, the perifusion effluent from each column was collected as 7.5-min fractions during stabilization periods and as 1- or 2.5-min fractions during administration of the secretagogues. The {alpha}MSH concentration was measured in each fraction using a double-antibody RIA procedure (43). The perifusion profiles were expressed as percentages of the basal secretion rate calculated as the mean of four consecutive fractions collected just before the infusion of each compound. The figures represent the mean profiles of {alpha}MSH release (±SEM) from at least four independent experiments. To compare the net increase in {alpha}MSH production induced by fNT in control conditions and under various treatments, the areas under the curves (AUCs) were calculated using PRISM 3.00 software (GraphPad, Inc., San Diego, CA). Statistical significance was assessed by two-tailed paired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of f NT on [Ca2+]c
Under resting conditions, the mean [Ca2+]c in cultured frog melanotrophs was 24.5 ± 0.7 nM (n = 266). A 15-sec application of fNT (10-7 M) in the vicinity of melanotrophs induced an increase in [Ca2+]c, which reached 120.2 ± 4.9 nM (n = 158) within 20 sec and then decreased gradually during the next 30–60 sec (Fig. 1AGo). Repeated applications of fNT (10-7 M) to the same cells at 2-min intervals resulted in sequential increases in [Ca2+]c, with gradual attenuation of the response. Administration of graded concentrations of fNT ranging from 10-10–10-7 M provoked a dose-related increase in [Ca2+]c. The dose-response curve revealed that the half-maximum effective concentration was 3 x 10-9 M (Fig. 1BGo). Prolonged exposure of frog melanotrophs to fNT (10-7 M; 2-min) induced a transient increase in [Ca2+]c (Fig. 1CGo). Despite the continuous presence of the peptide, [Ca2+]c returned to the basal level within less than 1 min (n = 4).



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  		FIG. 1. Effect of fNT on [Ca2+]c in cultured frog melanotrophs. A, Typical profile illustrating the effect of two equimolar applications of fNT (10-7 M; 15 sec; 2-min interval) on [Ca2+]c. B, Semilogarithmic plot showing the effect of graded concentrations of fNT (10-10–10-7 M; 15 sec) on the amplitude of the [Ca2+]c transients. Each point represents the mean (±SEM) [Ca2+]c increases measured in 5–87 cells. C, Typical profile showing the effect of prolonged infusion of fNT (10-7 M; 2 min) on [Ca2+]c.

 
Involvement of the PLC pathway in responses to fNT
The effect of fNT on phosphoinositide hydrolysis was investigated by measuring IP3 production after incorporation of myo-[3H]inositol by cultured frog melanotrophs. Incubation of melanotrophs with fNT (10-8 M) for 30 sec and 1 min increased by 58.3 ± 19.1% (n = 5) and 22.1 ± 6.8% (n = 7), respectively, the formation of IP3 (Fig. 2Go).



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  		FIG. 2. Effect of fNT on IP3 formation in cultured frog melanotrophs. Cells were labeled for 18 h with myo-[3H]inositol and were preincubated for 15 min with 10 mM LiCl. Then the cells were incubated in the absence or presence of fNT (10-8 M) for 30 sec or 1 min. Each value represents the mean (±SEM) of five to 12 experiments. TRaq, Total radioactivity in the aqueous phase. **, P < 0.01; *, P < 0.05.

 
We have previously shown that fNT provokes membrane depolarization in frog melanotrophs accompanied by an increase in action potential discharge (37). Administration of the PLC inhibitor U-73122 (10-6 M; 15 min) to cultured melanotrophs suppressed the excitatory effect of fNT (10-7 M; 20 sec) on the electrical activity (n = 6; Fig. 3AGo). In contrast, U-73343 (10-6 M; 15 min), the inactive analog of U-73122, did not impair the electrophysiological response of melanotrophs to fNT (n = 6; Fig. 3BGo). In agreement with a previous report (3), fNT (10-8 M; 15 min) induced a robust stimulation of {alpha}MSH secretion by perifused frog melanotrophs (Fig. 3CGo). Application of U-73122 (10-5 M) significantly increased the spontaneous level of {alpha}MSH release (P < 0.001), but reduced by 73% the stimulatory effect of fNT (10-8 M; 15 min; Fig. 3CGo).



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  		FIG. 3. Effect of the PLC inhibitor U-73122 on fNT-induced electrical and secretory activities of frog melanotrophs. A, Typical recordings illustrating the effect of fNT (10-7 M; 20 sec) in the absence (left panel) or presence of U-73122 (10-6 M; right panel). B, Typical recordings illustrating the effect of fNT (10-7 M; 20 sec) in the absence (left panel) or presence of the inactive analog U-73343 (10-6 M; right panel). U-73122 and U-73343 were perfused 15 min before the onset of fNT application. The initial membrane potential was set at -60 mV before each recording period. Signals were recorded in the gramicidin-perforated patch configuration. C, Effect of fNT (10-8 M; 15 min) on {alpha}MSH secretion from perifused dispersed melanotrophs in the absence (left panel) or presence of U-73122 (10-5 M; right panel). U-73122 was administered 30 min before the application of f NT. The profiles represent the mean secretion pattern (±SEM) of three independent experiments. The spontaneous level of {alpha}MSH release (100% basal level) was calculated as the mean hormone secretion in the four consecutive fractions collected before the onset of fNT or U-73122 administration ({circ}). The mean basal secretion rates of {alpha}MSH in these experiments were 12.5 ± 5.3 pg/min·104 cells (left panel) and 17.1 ± 1.6 pg/min·104 cells (right panel). {alpha}MSH-LI, {alpha}MSH-like immunoreactivity.

 
Involvement of IP3-induced intracellular Ca2+ mobilization in the responses to fNT
Intracellular application of heparin (1 mg/ml; 10 min), an IP3 receptor antagonist, abrogated the electrophysiological response of cultured frog melanotrophs to fNT (10-7 M; 10 sec; n = 12; Fig. 4AGo). In contrast, dialysis of melanotrophs with de-N-sulfated heparin (1 mg/ml; 10 min), an inactive analog of heparin, did not impair the excitatory effect of fNT (n = 9; Fig. 4BGo).



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  		FIG. 4. Effect of cell dialysis with the IP3 receptor antagonist heparin on fNT-induced electrical activity of cultured frog melanotrophs. A, Typical recordings illustrating the effect of fNT (10-7 M; 10 sec) on a cell dialyzed with heparin (1 mg/ml) for 1 min (upper trace) and 10 min (lower trace). B, Typical recording illustrating the effect of fNT (10-7 M; 10 sec) on a cell dialyzed with the inactive analog de-N-sulfated heparin (D-N-SH; 1 mg/ml) for 10 min. The signals were recorded in the whole cell configuration. The elapsed time after whole cell access is indicated on the top of each trace.

 
Extracellular application of 2-APB (5 x 10-5 M; 5 min), an inhibitor of IP3-induced Ca2+ release and store-operated Ca2+ channels (44, 45), reduced fNT-induced electrical activity (n = 5; Fig. 5AGo). Similarly, exposure of cultured melanotrophs to 2-APB (5 x 10-5 M), totally abolished the effect of fNT (10-7 M; 15 sec) on [Ca2+]c (Fig. 5BGo). In contrast, the increase in [Ca2+]c evoked by KCl (30 mM; 15 sec) persisted during application of 2-APB (Fig. 5BGo). Administration of 2-APB (5 x 10-5 M) to perifused melanotrophs significantly reduced the spontaneous {alpha}MSH release (P < 0.001) and inhibited by 52% the {alpha}MSH secretion evoked by fNT (10-7 M; 15 min; Fig. 5CGo).



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  		FIG. 5. Effect of the inhibitor of IP3-induced Ca2+ release and store-operated Ca2+ channels 2-APB on fNT-induced activation of frog melanotrophs. A, Typical electrophysiological recordings illustrating the effect of fNT (10-7 M; 10 sec) in the absence (left trace) or presence (right trace) of 2-APB on a cultured cell. 2-APB (5 x 10-5 M) was perfused for 5 min before the administration of fNT. The initial membrane potential was set at -60 mV before each recording period. Signals were recorded in the gramicidin-perforated patch configuration. B, Typical profile illustrating the effect of fNT (10-7 M; 15 sec) and KCl (30 mM; 15 sec) on [Ca2+]c in the absence or presence of 2-APB (5 x 10-5 M) in cultured cells. C, Effect of fNT (10-8 M; 15 min) on {alpha}MSH secretion from perifused dispersed melanotrophs in the absence (left panel) or presence of 2-APB (right panel). 2-APB (5 x 10-5 M) was administered 15 min before the application of fNT. The profile represents the mean secretion pattern (±SEM) of four independent experiments. The mean basal secretion rates of {alpha}MSH (100% basal level) in these experiments were 71.9 ± 18.2 pg/min·104 cells (left panel) and 107.8 ± 3.6 pg/min·104 cells (right panel). See Fig. 3Go for other details.

 
Involvement of Ca2+ influx in the responses to fNT
Incubation of melanotrophs in Ca2+-free Ringer’s solution did not prevent the membrane depolarization, but abrogated the action potential discharge evoked by fNT (10-7 M; 10 sec; n = 18; Fig. 6AGo). Concurrently, suppression of calcium in the incubation medium markedly reduced the amplitude of the [Ca2+]c increase provoked by fNT (10-7 M; 15 sec; n = 15; Fig. 6BGo). Suppression of calcium in the perifusion medium inhibited by 78% (P < 0.001) the stimulatory effect of fNT (10-8 M; 15 min) on {alpha}MSH secretion (Fig. 6CGo). In the presence of gadolinium chloride (GdCl3; 5 x 10-5 M; 5 min), a nonselective Ca2+ channel blocker, the stimulatory effect of fNT (10-7 M; 10 sec) on the electrical activity of cultured melanotrophs was totally abrogated (n = 8; Fig. 7AGo). Similarly, application of nickel chloride (NiCl2; 3 mM), another nonselective Ca2+ channel blocker, reversibly suppressed the action of fNT (10-7 M; 15 sec) on [Ca2+]c in frog melanotrophs (n = 16; Fig. 7BGo). Moreover, exposure of perifused melanotrophs to NiCl2 (3 mM; 45 min) reduced by 75% (n = 4; P < 0.001) the basal {alpha}MSH release and totally blocked the response of the cells to fNT (10-8 M; 15 min; Fig. 7CGo).



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  		FIG. 6. Effect of calcium suppression in the incubation medium on fNT-induced activation of frog melanotrophs. A, Typical electrophysiological recordings illustrating the effect of fNT (10-7 M; 10 sec) in Ca2+-free Krebs-Ringer medium on a cultured cell. Ca2+-free Krebs-Ringer medium was administered 10 min before the application of fNT. The initial membrane potential was set at -60 mV before each recording period. The electrical signal was recorded in the gramicidin-perforated patch configuration. B, Typical profile illustrating the effect of fNT (10-7 M; 15 sec) on [Ca2+]c in a Krebs-Ringer solution containing 2 mM CaCl2 or in Ca2+-free Krebs-Ringer medium in cultured cells. C, Effect of fNT (10-8 M; 15 min) on {alpha}MSH secretion from perifused dispersed melanotrophs in a Krebs-Ringer solution containing 2 mM CaCl2 (left panel) or in Ca2+-free Krebs-Ringer medium (right panel). Ca2+-free Krebs-Ringer medium was infused 45 min before the application of fNT. The profile represents the mean secretion pattern (±SEM) of four independent experiments. The mean basal secretion rates of {alpha}MSH (100% basal level) in these experiments were 14.6 ± 2.9 pg/min·104 cells (left panel) and 29.3 ± 8.0 pg/min·104 cells (right panel). See Fig. 3Go for other details.

 


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  		FIG. 7. Effect of the nonselective calcium channel blockers GdCl3 and NiCl2 on fNT-induced activation of frog melanotrophs. A, Typical electrophysiological recordings illustrating the effect of fNT (10-7 M; 10 sec) in the absence (left trace) or presence (right trace) of GdCl3 on a cultured cell. GdCl3 (5 x 10-5 M) was perfused for 5 min before the administration of fNT. The initial membrane potential was set at -60 mV before each recording period. Signals were recorded in the gramicidin-perforated patch configuration. B, Typical profile illustrating the effect of fNT (10-7 M; 15 sec) on [Ca2+]c before, during, and after the application of NiCl2 (3 mM) in a cultured cell. C, Effect of NiCl2 (3 mM) on basal and fNT-stimulated {alpha}MSH secretion. All experimental values were calculated from data similar to those presented in Fig. 6Go. Each value represents the mean (±SEM) {alpha}MSH release in each condition, measured in four independent experiments. The mean basal secretion rate of {alpha}MSH (100% basal level) in these experiments was 17.7 ± 3.3 pg/min·104 cells. ***, P < 0.001.

 
Incubation of cultured melanotrophs with the L-type Ca2+ channel blocker nifedipine (10-6 M; 15 min; n = 17) or the N-type Ca2+ channel blocker {omega}-CgTx GVIA (10-6 M; 10 min; n = 11) markedly reduced both the amplitude and frequency of action potentials evoked by fNT (10-7 M; 10 sec) on melanotrophs (Fig. 8AGo). In the presence of both nifedipine and {omega}-CgTx GVIA, the action potential discharge induced by fNT was substantially inhibited, whereas the membrane depolarization was not affected (n = 6; Fig. 8AGo). Application of nifedipine (10-6 M; 30 min) and {omega}-CgTx GVIA (10-6 M; 30 min), either alone or in combination, to melanotrophs did not significantly modify basal [Ca2+]c, but reduced the amplitude of the fNT-induced [Ca2+]c increase by 30% (nifedipine alone; n = 10; P < 0.001), 51% ({omega}-CgTx GVIA alone; n = 10; P < 0.001), and 50% (nifedipine plus {omega}-CgTx GVIA; n = 9; P < 0.001; Fig. 8BGo). Exposure of perifused frog melanotrophs to nifedipine (10-6 M; 30 min), {omega}-CgTx GVIA (10-6 M; 45 min), or nifedipine plus {omega}-CgTx GVIA (10-6 M each; 45 min) reduced by 21% (n = 4; P < 0.05), 19% (n = 4; P < 0.05), or 55% (n = 4; P < 0.01) basal {alpha}MSH secretion, respectively (data not shown). Administration of nifedipine or {omega}-CgTx GVIA alone did not significantly modify the stimulatory effect of fNT (10-8 M; 15 min) on {alpha}MSH release (Fig. 9AGo). In contrast, coapplication of nifedipine plus {omega}-CgTx GVIA inhibited by 49% the stimulatory effect of fNT (10-8 M; 15 min) on {alpha}MSH secretion (n = 4; P < 0.01; Fig. 9BGo).



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  		FIG. 8. Effect of the L- and N-type calcium channel blockers nifedipine and {omega}-CgTx GVIA on fNT-induced electrical activity and [Ca2+]c in cultured frog melanotrophs. A, Typical electrophysiological recordings illustrating the effect of fNT (10-7 M; 10 sec) in the absence (left upper trace) or presence of nifedipine (right upper trace), {omega}-CgTx GVIA (left lower trace), or {omega}-CgTx GVIA plus nifedipine (right lower trace). Nifedipine (10-6 M) and {omega}-CgTx GVIA (10-6 M) were perfused for 10 min before the administration of fNT. The initial membrane potential was set at -60 mV before each recording period. Signals were recorded on two distinct cells (upper and lower panels) in the gramicidin-perforated patch configuration. B, Effects of nifedipine (10-6 M) and {omega}-CgTx GVIA (10-6 M) on basal and fNT-induced [Ca2+]c in cultured cells. The mean basal [Ca2+]c in these experiments was 28.7 ± 2.0 nM (n = 36) in control conditions, 28.3 ± 3.6 nM (n = 10) in the presence of nifedipine, 30.5 ± 5.2 nM (n = 10) in the presence of {omega}-CgTx GVIA, and 29.0 ± 4.9 nM (n = 19) in the presence of nifedipine plus {omega}-CgTx GVIA. ***, P < 0.001.

 


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  		FIG. 9. Effect of the L-type calcium channel blocker nifedipine and the N-type calcium channel blocker {omega}-CgTx GVIA on fNT-induced {alpha}MSH secretion from perifused dispersed frog melanotrophs. fNT (10-8 M) was perfused for 15 min in the absence (A and B, left panel) or presence of nifedipine (A, middle panel), {omega}-CgTx GVIA (A, right panel), or nifedipine plus {omega}-CgTx GVIA (B, right panel). Nifedipine (10-6 M) and {omega}-CgTx GVIA (10-6 M) were administered 30 and 45 min before the application of fNT, respectively. Each profile represents the mean secretion pattern (±SEM) of at least four independent experiments. The mean basal secretion rates of {alpha}MSH (100% basal level) in these experiments were 24.9 ± 3.8 pg/min·104 cells (A, left panel), 21.3 ± 7.1 pg/min·104 cells (A, middle panel), 18.5 ± 2.8 pg/min·104 cells (A, right panel), 13.1 ± 2.2 pg/min·104 cells (B, left panel), and 7.2 ± 1.5 pg/min·104 cells (B, right panel). See Fig. 3Go for other details.

 
Involvement of PKC in the responses to fNT
Three sequential ejections of fNT (10-7 M; 10 sec; 10-min intervals) in the vicinity of cultured frog melanotrophs resulted in a reproducible activation of the electrical activity (Fig. 10AGo). Intracellular application of the PKC inhibitor PKC-(19–31) (5 x 10-7 M; n = 9) gradually reduced the electrophysiological response to repeated ejections of fNT (Fig. 10BGo). In the presence of the membrane-permeant PKC inhibitor chelerythrine (5 x 10-6 M; n = 16), the electrical response to iterative applications of fNT (10-7 M; 20 sec; 10-min intervals) was also reduced (Fig. 10CGo). Concomitant application of nifedipine (10-6 M), {omega}-CgTx GVIA (10-6 M), and chelerythrine (5 x 10-6 M) totally blocked the excitatory effect of fNT (n = 9; Fig. 10DGo).



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  		FIG. 10. Effects of the PKC inhibitors PKC-(19–31) and chelerythrine on fNT-induced electrical activity of cultured frog melanotrophs. A, Typical recordings illustrating the effects of three equimolar applications of fNT (10-7 M; 10 sec; 10-min intervals) under control conditions. The signals were recorded in the gramicidin-perforated patch configuration. B, Typical recordings illustrating the effects of three equimolar applications fNT (10-7 M; 10 sec; 10-min intervals) on a cell dialyzed with PKC-(19–31) (5 x 10-7 M) for 1 min (left trace), 10 min (middle trace), and 20 min (right trace). The signals were recorded in the whole cell configuration. The elapsed time after whole cell access is indicated on the top of each trace. C, Typical recordings illustrating the effect of chelerythrine on three equimolar applications of fNT (10-7 M; 10 sec; 10-min intervals). Chelerythrine (5 x 10-6 M) was perfused for 10 min (middle trace) and 20 min (right trace) before the second and third administrations of fNT. The signals were recorded in the gramicidin-perforated patch configuration. D, Typical recordings illustrating the effect of fNT (10-7 M; 10 sec; 20-min interval) in the absence (left trace) or presence of nifedipine (10-6 M), {omega}-CgTx GVIA (10-6 M), and chelerythrine (5 x 10-6 M; right trace) on a cultured cell. The signals were recorded in the gramicidin-perforated patch configuration. The initial membrane potential was set at -60 mV before each recording period.

 
Administration of chelerythrine (5 x 10-6 M; 30 min) to cultured frog melanotrophs did not affect the calcium response to a first pulse of fNT (10-7 M; 15 sec), but completely abolished the effect of a second application of fNT (n = 9; Fig. 11AGo). Exposure of perifused melanotrophs to two sequential pulses of fNT (10-8 M; 15 min) at a 40-min interval led to similar increases in {alpha}MSH release (Fig. 11BGo, upper panel). Application of chelerythrine (5 x 10-6 M; 30 min) increased by 34% (n = 4; P < 0.01) basal {alpha}MSH secretion (data not shown). Chelerythrine did not affect the response to a first pulse of fNT, but decreased by 80% the response evoked by a second pulse of fNT (n = 4; P < 0.001; Fig. 11BGo, lower panel).



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  		FIG. 11. Effect of the PKC inhibitor chelerythrine on fNT-induced activation in frog melanotrophs. A, Typical profile illustrating the effect of two sequential applications of fNT (10-7 M; 15 sec) on [Ca2+]c in the presence of chelerythrine in cultured cells. Chelerythrine (5 x 10-6 M) was perfused for 30 min before the first administration of fNT. B, Effect of chelerythrine on fNT-induced {alpha}MSH secretion from perifused cells. Two pulses of fNT (10-8 M, 15 min) were administered at a 55-min interval in the absence (upper panel) or presence (lower panel) of chelerythrine. Chelerythrine (5 x 10-6 M) was perfused for 30 min before the first application of fNT. Each profile represents the mean secretion pattern (±SEM) of four independent experiments. The mean basal secretion rates of {alpha}MSH (100% basal level) in these experiments were 49.1 ± 13.7 pg/min·104 cells (upper panel) and 58.5 ± 17.9 pg/min·104 cells (lower panel). See Fig. 3Go for other designations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has previously been shown that NT stimulates the electrical (37) and secretory (3) activities of frog pituitary melanotrophs. The present study reveals that NT provokes an IP3-mediated increase in [Ca2+]c and a membrane depolarization that, in turn, stimulates the action potential discharge and activates Ca2+ influx through L- and N-type Ca2+ channels. NT also stimulates Ca2+ entry through Gd3+- and Ni2+-sensitive Ca2+ channels. Both Ca2+ mobilization from IP3-sensitive intracellular stores and Ca2+ influx through membrane Ca2+ channels are involved in NT-induced {alpha}MSH secretion. Our data also demonstrate that activation of PKC is required to maintain the responsiveness of frog melanotrophs to NT.

NT induces an increase in [Ca2+]c
The mean basal [Ca2+]c determined in the present experiments (24.5 ± 0.7 nM) was 4-fold lower than the [Ca2+]c measured in rat melanotrophs using either the same calibration procedure (our unpublished data) or distinct calibration methods (46). The [Ca2+]c values determined in the present study are in the same range as those previously reported in frog (35, 47). It thus appears that the low basal [Ca2+]c measured in the present study corresponds to the actual cytosolic calcium concentration in frog melanotrophs. The action of NT on frog melanotrophs was characterized by a transient rise in [Ca2+]c. The concentration of NT that provoked a half-maximal increase in [Ca2+]c was in the same range as that required to obtain half-maximal stimulation of {alpha}MSH release (3). Prolonged application of NT only induced a transient [Ca2+]c response in frog melanotrophs. A transient [Ca2+]c increase has also been observed in fibroblasts and PC12 cells transfected with rat NTR1 (22, 23). In contrast, in rat astrocytes (31) and in CHO cells expressing rat NTR1 (48), NT generates a biphasic Ca2+ response consisting of an immediate increase due to mobilization of Ca2+ from intracellular stores, followed by a sustained plateau resulting from Ca2+ influx.

NT induces IP3-mediated mobilization of [Ca2+]c
In frog melanotrophs, the electrophysiological effects of NT are mediated by activation of receptors associated with Gq/11 proteins (37), which are known to be positively coupled to PLC (49). Several lines of evidence indicate that the responses of melanotrophs to NT can be ascribed to activation of PLC and IP3-induced mobilization of cytosolic Ca2+. First, NT provoked an immediate increase in the production of IP3, and the amplitude of the response was similar to those induced by other factors that stimulate frog melanotroph activity through receptors coupled to PLC, such as TRH (41) and muscarinic M3 receptors (47). Second, the PLC inhibitor U-73122 inhibited the excitatory effect of NT on electrical and secretory activities. In agreement with previous reports showing that U-73122 stimulates exocytosis in rat anterior pituitary cells (50), we found that the PLC inhibitor provoked a marked activation of basal {alpha}MSH release in frog melanotrophs. Third, in Ca2+-free medium, NT was still able to induce membrane depolarization associated with modest [Ca2+]c and {alpha}MSH release responses. Fourth, intracellular application of the nondiffusible IP3 receptor antagonist heparin blocked the electrophysiological effect of NT. Fifth, the inhibitor of IP3-induced Ca2+ release and store-operated Ca2+ channels, 2-APB, reduced the [Ca2+]c increase, the electrical activity, and the {alpha}MSH secretion evoked by NT. Consistent with these observations, it has been shown that in rat lactotrophs, NT stimulates the PLC pathway (51) and increases 45Ca2+ efflux in the absence of extracellular Ca2+ (19).

NT induces calcium influx through membrane Ca2+ channels
The present study revealed that suppression of extracellular Ca2+ or addition of the nonselective Ca2+ channel blockers Gd3+ and Ni2+ markedly reduced the stimulatory effects of NT on action potential discharge, [Ca2+]c, and secretory activity of frog melanotrophs. These data indicate that Ca2+ influx from the extracellular medium is required for the responses of melanotrophs to NT. As NT stimulates electrical activity, we hypothesized that NT induced Ca2+ influx through L- and N-type Ca2+ channels, two types of voltage-activated Ca2+ channels expressed in frog melanotrophs (52). Indeed, the L-type channel blocker nifedipine and the N-type channel blocker {omega}-CgTx GVIA inhibited the NT-induced [Ca2+]c increase and action potential discharge without affecting depolarization. Concomitant application of nifedipine and {omega}-CgTx GVIA also decreased by approximately 50% NT-induced {alpha}MSH release. These data demonstrate that the secretory response of frog melanotrophs to NT necessitates calcium influx through L- and N-type Ca2+ channels. However, the residual secretory response to NT observed in the presence of nifedipine and {omega}-CgTx GVIA was larger than that obtained either in Ca2+-free medium or in the presence of the nonselective Ca2+ channel blocker Ni2+, indicating that NT-evoked {alpha}MSH secretion also requires Ca2+ influx through nifedipine-/{omega}-CgTx GVIA-insensitive Ni2+-sensitive channels. The fact that coadministration of nifedipine and {omega}-CgTx GVIA almost totally suppress voltage-activated calcium currents in frog melanotrophs (52) suggests that the nifedipine-/{omega}-CgTx GVIA-insensitive Ni2+-sensitive channels involved in the secretory effect of NT might be voltage-insensitive channels. Collectively, these observations support the idea that the Ca2+ influx implicated in the secretory response of melanotrophs to NT can be accounted for by activation of store-operated calcium current, which is sensitive to Ni2+, Gd3+ (53), and 2-APB (45). Consistent with this hypothesis, Gailly et al. (48) reported that in CHO cells expressing rat NTR1, NT activates store-operated calcium entry to refill intracellular Ca2+ pools. Taken together, these data demonstrate that in frog melanotrophs, the responses to NT implicate both intracellular and extracellular Ca2+ sources. The absence of a sustained plateau in the NT-induced Ca2+ increase might be ascribed to the existence of a direct flow of Ca2+ from the external medium to the intracellular stores via tunnels constituted by physical coupling between store-operated Ca2+ channels and IP3 receptors, as demonstrated in mouse pancreatic acinar cells (54) and in human platelets (55). Alternatively, active uptake of Ca2+ by endoplasmic reticulum Ca2+ pumps may attenuate the increase in [Ca2+]c in discrete microdomains near store-operated calcium channels and secretory granules, as proposed by Putney et al. (56). The different kinetics of the calcium and secretory responses observed during prolonged administration of NT would favor this latter hypothesis.

Involvement of intracellular and extracellular Ca2+ sources in NT-induced {alpha}MSH release
The fact that separate or combined applications of nifedipine and {omega}-CgTx GVIA differentially affected [Ca2+]c and {alpha}MSH release in basal conditions or in the presence of NT, whereas 2-APB reduced both spontaneous and NT-evoked hormone secretion, indicates that Ca2+ influx through voltage-gated Ca2+ channels and intracellular Ca2+ mobilization differentially contribute to exocytosis. Their respective contributions may depend on the spatial organization of Ca2+ channels and secretory granules containing {alpha}MSH. In this respect it has recently been reported that in rat melanotrophs, a large distance and a diffusion barrier separate voltage-activated Ca2+ channels and large dense cored vesicles (57, 58). In contrast, it has been proposed that in rat gonadotrophs, IP3-sensitive Ca2+ stores may be in close vicinity to exocytosis sites, as IP3-induced Ca2+ release is more efficient than Ca2+ entry through voltage-gated Ca2+ channels in triggering exocytosis (59, 60, 61). Thus, it is conceivable that in frog melanotrophs, only a few vesicles are docked near voltage-activated Ca2+ channels, whereas numerous granules are closely associated with IP3-sensitive Ca2+ channels, constituting release zones available for massive {alpha}MSH secretion.

Involvement of the PKC pathway in the maintenance of the responsiveness to NT
The PKC inhibitors PKC (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) and chelerythrine reduced the electrical, calcium, and secretory responses of frog melanotrophs to sequential applications of NT, suggesting that activation of the PKC pathway is implicated in the mechanism of action of NT. In addition, chelerythrine blocked the NT-evoked depolarization that was still observed in the presence of nifedipine and {omega}-CgTx GVIA, indicating that the membrane depolarization, leading to action potential discharge, can be ascribed to activation of PKC by Ca2+ release from intracellular stores. The lack of effect of the nonselective PKC inhibitor chelerythrine (62, 63) on the response to the first pulse of NT cannot be ascribed to a slow effect of the PKC inhibitor in frog melanotrophs. Indeed, in these cells, chelerythrine exerts a stimulatory effect on voltage-activated calcium current (52), intracellular calcium concentration (our unpublished data), and {alpha}MSH release in less than 15 min, whereas a 30-min incubation with chelerythrine did not affect the first calcium and secretory responses to NT. These data indicate that activation of PKC does not mediate the initial physiological responses to NT, but reveal the importance of PKC in the maintenance of the responsiveness of frog melanotrophs to NT. Activation of PKC generally leads to desensitization of G protein-coupled receptors (64, 65, 66). However, Turner et al. (67) reported that PKC does not mediate the NT-induced desensitization of native NTR1 in HT29 cells. In contrast, PKC exerts positive modulation on IP3 receptors (68), store-operated Ca2+ channels (69), and secretory processes (70). In addition, it has been demonstrated that NT-induced PKC activation facilitates the reloading process of Ca2+ into the endoplasmic reticulum of NTR1-transfected CHO cells (71). Whether PKC regulates IP3 receptors, store-operated Ca2+ channels, refilling of intracellular Ca2+ stores, and the secretory activity in frog melanotrophs remains to be determined. Finally, the [Ca2+]c increase and PKC activation provoked by NT might also facilitate adenylyl cyclase and mitogen-activated protein kinase activities, as reported in different cell lines (72, 73, 74).

A proposed model illustrating the mechanism of action of NT on frog melanotrophs is shown in Fig. 12Go. NT, acting through a receptor positively coupled to PLC via a Gq/11 protein, stimulates IP3 production that provokes Ca2+ release from intracellular stores and PKC activation. The activation of PKC induces membrane depolarization that, in turn, generates action potential discharge underlying Ca2+ influx through L- and N-type Ca2+ channels. Concurrently, NT also induces Ca2+ influx through nifedipine-/{omega}-CgTx GVIA-insensitive and Ni2+-/Gd3+-sensitive Ca2+ channels, possibly corresponding to store-operated Ca2+ channels that may refill intracellular Ca2+ stores, contributing to maintain the physiological responses to NT. The resulting [Ca2+]c increase triggers {alpha}MSH exocytosis. The NT-induced [Ca2+]c increase stimulates PKC activity that may regulate IP3 receptors, store-operated Ca2+ channels, and/or the secretory machinery to maintain the cell’s responsiveness to NT.



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  		FIG. 12. Proposed model depicting the mechanism of action of fNT in frog melanotrophs. Activation of NT receptor (NTR) coupled to a Gq/11-protein causes activation of PLC. Hydrolysis of phosphatidylinositol-bisphosphate (PIP2) by PLC induces the formation of diacylglycerol (DAG) and IP3, which provokes intracellular Ca2+ mobilization and triggers {alpha}MSH secretion. Ca2+ and DAG stimulate PKC activity that induces membrane depolarization ({Delta}V), leading to an increase in Ca2+ spike frequency. The action potential discharge enhances Ca2+ influx through L- and N-type Ca2+ channels and the resulting increase in [Ca2+]c is responsible for NT-induced {alpha}MSH secretion. Activation of PKC may regulate IP3 receptors, store-operated Ca2+ channels, and/or the secretory machinery to maintain the cell responsive to NT. In addition, NT provokes Ca2+ influx through Ni2+/Gd3+-sensitive channels that may contribute to refill IP3-sensitive intracellular Ca2+ stores.

 


   Acknowledgments
 
We thank Huguette Lemonnier and Colette Piard for skillful technical assistance.


   Footnotes
 
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, Unité-413, and the Conseil Régional de Haute-Normandie.

A.B. and L.D. contributed equally to this work.

Abbreviations: 2-APB, 2-Aminoethoxydiphenylborane; AUC, area under the curve; {omega}-CgTx GVIA, {omega}-conotoxin GVIA; [Ca2+]c, cytosolic calcium concentration; fL15, Leibovitz culture medium adjusted to frog osmolarity; fNT, synthetic frog neurotensin; GdCl3, gadolinium chloride; IP3, inositol trisphosphate; {alpha}MSH, {alpha}-melanocyte-stimulating hormone; NiCl2, nickel chloride; NIL, neurointermediate lobe; NT, neurotensin; NTR/ntr, neurotensin receptor; PKC, protein kinase C; PLC, phospholipase C.

Received February 6, 2003.

Accepted for publication September 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. Delarue, I. R. Jouet, M. Gras, L. Galas, A. Fournier, and H. Vaudry
Activation of EndothelinA Receptors in Frog Adrenocortical Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through L-Type Calcium Channels
Endocrinology, January 1, 2005; 146(1): 119 - 129.
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