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Endocrinology Vol. 140, No. 7 3264-3272
Copyright © 1999 by The Endocrine Society

ARTICLES

Involvement of Protein Kinase C and Protein Tyrosine Kinase in Thyrotropin-Releasing Hormone-Induced Stimulation of {alpha}-Melanocyte-Stimulating Hormone Secretion in Frog Melanotrope Cells1

Ludovic Galas2, Marek Lamacz, Marianne Garnier2, Eric W. Roubos, 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 (L.G., M.L., M.G., M.C.T., H.V.), 76821 Mont-Saint-Aignan, France; and Department of Cellular Animal Physiology, Nijmegen Institute for Neurosciences, University of Nijmegen (E.W.R.), Toernooiveld 1, 6525 ED, Nijmegen, The Netherlands

Address all correspondence and requests for reprints to: 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
 
We have previously shown that the stimulatory effect of TRH on {alpha}-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced {alpha}-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on {alpha}-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10-6 M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced {alpha}-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7–7'-DEA did not impair the effect of TRH on {alpha}-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked {alpha}-MSH release (ED50= 1.22 x 10-5 M and ED50= 1.47 x 10-5 M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced {alpha}-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TRANSDUCTION mechanisms mediating the effects of TRH on pituitary cell activity have been extensively studied in the rat mammotrope cell line GH3 (1). These studies have revealed the importance of calcium mobilization from intracellular calcium stores and activation of protein kinase C (PKC) in the stimulatory effect of TRH on hormone secretion. In addition, TRH has been reported to stimulate adenylyl cyclase (2, 3), Ca2+/calmodulin-dependent protein kinase II (CAM KII) (4, 5), phospholipase A2 (PLA2) (6, 7) and protein tyrosine kinase (PTK) (8, 9, 10, 11). However, substantial differences have been reported between the mode of action of TRH on normal and tumor-derived pituitary cells (12). In particular, Ca2+ influx through plasma membrane channels appears to play a more crucial role in hormone release from normal anterior pituitary cells than tumoral cells (12). Studies on nontumoral endocrine cells are thus required to elucidate the intracellular effectors associated with activation of TRH receptors in normal pituitary cells.

In amphibians and fish, TRH is a potent stimulator of {alpha}-MSH secretion (13, 14, 15, 16, 17, 18, 19). The intermediate lobe of the frog pituitary, which is composed of a homogeneous population of melanotrope cells, thus represents a valuable alternative model in which to investigate the transduction pathways activated by TRH in nontumoral cells. Using this model, it has been previously shown that TRH stimulates a phospholipase C and induces a biphasic increase in cytosolic calcium concentration ([Ca2+]i) resulting from both Ca2+ influx and Ca2+ mobilization from intracellular sources (20, 21).

The aim of the present study was to investigate the possible involvement of PKC, adenylyl cyclase, CAM KII, PLA2, and PTK in the stimulatory effect of TRH on {alpha}-MSH secretion from frog melanotrope cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs (Rana ridibunda; body weight, 40–50 g) originating from Bulgary, were purchased from a commercial supplier (Couétard, St-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). The frogs were killed by decapitation and the neurointermediate lobes (NILs) were dissected under a microscope. Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators.

Reagents and test substances
Leibovitz culture (L15) medium, (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) (HEPES), collagenase (type IA), TRH, phorbol 12-myristate-13-acetate (PMA), 7,7' dimethyl-5,8-eicosadienoic acid (DEA), quinacrine and [N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide] (W-7) were purchased from Sigma Chemical Co. (St. Louis, MO). [{alpha}-cyano-(3-ethoxy-4-hydroxy-5-phenylthiomethyl)cinnamamide] (ST638), tyrphostin 23 ([AG 18; {alpha}-cyano-(3,4-dihydroxy)cinnamonitrile; RG-50810]; Tyr-A23) and N-(2-[[ (3-(4'-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4'-methoxy-benzenesulfonamide phosphate (KN-93) were obtained from Calbiochem (San Diego, CA). [9-(tetrahydro-2'-furyl)-9H-purin-6-amine] (SQ 22,536) and S-2,6-diamino-N-[[1-(1-oxotridecyl)-2-piperidinyl]methyl]-hexanamide (NPC-15437) were from RBI (Illkirch, France). Kanamycin was supplied by Life Technologies (Grand Island, NY). BSA (fraction V) was from Boehringer Mannheim (Mannheim, Germany). FBS and the antibiotic-antimycotic solution were from BioWhittaker, Inc. (Gagny, France). Indo-1 acetoxymethylester (indo-1/AM) was from Molecular Probes, Inc. (Eugene, OR). Bio-Gel P2 was from Bio-Rad Laboratories, Inc. (Hercules, CA). All other chemicals were from Sigma Chemical Co.

Cell culture
Neurointermediate lobes were collected in Ca2+-free Ringer’s solution (15 mM HEPES buffer, 112 mM NaCl, 2 mM KCl, 1 mM EGTA) supplemented with 2 mg glucose/ml, 0.3 mg BSA/ml and 1% each of the kanamycin and antibiotic-antimycotic solutions. The Ringer’s solution was gassed for 15 min with O2/CO2 (95:5; vol/vol) before use, and the pH was adjusted to 7.35. Ten NILs were enzymatically dispersed at 24 C for 20 min with a solution of collagenase (1.5 mg/ml) in a Ca2+-free Ringer’s solution. Nondissociated neural lobes were removed by sedimentation and the supernatant containing disaggregated intermediate lobes was sampled. The suspension was centrifuged (30 x g; 5 min) and rinsed three times with Ca2+-free Ringer’s medium. The digested tissues were resuspended in L15 medium adjusted to Rana ridibunda osmolality (L15-water = 1:0.4) and supplemented with 0.2 mg glucose/ml, 0.063 mg CaCl2/ml and 1% each of the kanamycin and antibiotic-antimycotic solutions (f-L15; pH 7.35). The cells were dispersed by gentle aspiration through a siliconized Pasteur pipette with a flame-polished tip. Finally, cells were plated on poly-L-lysine-coated glass coverslips (30-mm diameter) at a density of 15,000 cells per coverslip in 35-mm culture dishes. When the cells had settled, coverslips were covered with 2 ml of culture medium composed of f-L15 supplemented with 10% FBS. Cultured cells were kept at 24 C in a humidified atmosphere, and the culture medium was renewed every 48 h. Microfluorimetric measurements were performed on 3- to 5-day-old cultured cells.

Calcium measurement
Cultured cells were incubated at 24 C for 30 min in the dark with 5 µM of the fluorescent calcium probe indo-1/AM in a Ringer’s solution (15 mM HEPES buffer, 112 mM NaCl, 2 mM KCl, 2 mM CaCl2) supplemented with 2 mg glucose/ml and 0.3 mg BSA/ml. At the end of the incubation period, the cells were washed twice with 2 ml of fresh medium and placed on the stage of a Nikon Diaphot inverted microscope (Champigny-sur-Marne, France) equipped for epifluorescence with an oil-immersion objective (X100 CF Fluor series; numerical aperture, 1.3). The [Ca2+]i was monitored by a dual emission microfluorimeter system, as previously described (22). Briefly, the fluorescence emission of indo-1/AM, induced by excitation at 355 nm (Xenon lamp), was recorded at two wavelengths (405 nm, corresponding to the Ca2+-complexed form and 480 nm, corresponding to the free form) by separate photometers (P1, Nikon). The 405/480 ratio (R) was determined by using an AS1-type acquisition card (Notocord Systems, Croissy-sur-Seine, France). All three signals (405 nm, 480 nm, and the 405/480 ratio) were continuously recorded with the JAD-FLUO program (version 1.2). The [Ca2+]i was calculated according to the formula established by Grynkiewicz et al. (23): [Ca2+]i = KdXß(R-Rmin):(Rmax-R), where Rmin represents the minimum fluorescence ratio obtained after incubation of cells in Ringer’s solution containing 10 mM EGTA and 10 µM ionomycin, Rmax the maximum fluorescence ratio obtained after incubation of cells in Ringer’s solution containing 10 mM CaCl2 and 10 µM ionomycin, and ß 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. The dissociation constant for indo-1 (Kd) has been previously determined (250 nM) (24, 25). A pressure ejection system was used to deliver the test substances in the vicinity of cultured cells.

Results are expressed as the mean amplitude of [Ca2+]i increase (± SEM). Student’s t test was used for statistical comparisons.

Perifusion experiments
The perifusion technique used to determine the effect of test substances on {alpha}-MSH release has been previously described (14). For each experiment, four NILs were mixed with preswollen Bio-Gel P-2 beads and transferred into a plastic column (0.9 cm id). The tissues were perifused with Ringer’s solution at constant flow rate (0.3 ml/min) and temperature (24 C). The effluent medium was collected as 2.5-min fractions during infusion of the secretagogues and 7.5-min fractions during stabilization periods. The collected samples were immediately chilled at 4 C, and the concentration of {alpha}-MSH was measured in each fraction on the same day as the perifusion experiment by using a double-antibody RIA procedure (26). The perifusion profiles were calculated and expressed as percentages of the basal secretory level. All experiments were carried out at least three times. The basal level was calculated as the mean of five consecutive fractions (37.5 min) collected just before the infusion of each secretagogue. To compare the net increase in {alpha}-MSH production induced by TRH in control conditions and under various treatments, the areas under the curves (AUCs) were calculated by using the trapezoidal rule (27). Two-tailed paired Student’s t test was used to compare the mean secretory responses within the same set of experiments.

cAMP measurement
Whole NILs were preincubated for 30 min at room temperature with 0.1 mM 3-isobutyl-1-methyl-xanthine. The NILs were then incubated for 20 min in 0.5 ml Ringer’s solution containing 10-6 M TRH or 10-5 M forskolin. The reaction was stopped by adding 0.5 ml of ice-cold 20% trichloroacetic acid. NILs were homogenized with a glass Potter and centrifuged (10,000 x g; 10 min). Trichloroacetic acid was eliminated from the supernatant by three successive rinses with 1 ml of water-satured diethyl ether. After evaporation of the ether phase, the supernatant was lyophilized, and the cAMP content in the dried extract was measured by RIA, following the procedure recommended in the cAMP RIA kit (Amersham Pharmacia Biotech, Les Ulis, France).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of inhibition of PKC on TRH-induced {alpha}-MSH secretion and [Ca2+]i increase
Preincubation of frog NILs with 10-6 M PMA for 24 h significantly reduced (P < 0.05) the stimulatory effect of TRH on {alpha}-MSH release (Fig. 1AGo). Similarly, exposure of perifused NILs to the PKC inhibitor NPC-15437 (10-5 M; 100 min) significantly attenuated (P < 0.001) TRH-induced {alpha}-MSH release (Fig. 1BGo). As previously reported (20), application of TRH induces two types of [Ca2+]i responses: in about 2/3 of the cells, TRH caused a sustained and biphasic increase in [Ca2+]i (Fig. 2AGo), whereas in 1/3 of the cells, TRH only induced a transient response (Fig. 2BGo). Preincubation of the cells for 24 h with PMA (10-6 M) or exposure of the cells for 10 min with NPC-15437 (10-6 M) totally suppressed the sustained phase of the response i.e. in all treated cells, TRH (10-7 M; 5 sec) only elicited a transient increase in [Ca2+]i (Fig. 2Go, C and D). However, inhibition of PKC by prolonged incubation with PMA or application of NPC-15437 did not modify the amplitude of the transient Ca2+ response (Fig. 2EGo).



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  		Figure 1. Effect of PKC inhibition on the secretory response of frog NILs to TRH. A, Effect of long-term treatment with PMA on TRH-induced {alpha}-MSH release from perifused frog NILs. The NILs were incubated for 24 h in fL15 alone (top, Control), or in fL15 containing 10-6 M PMA (bottom). A single pulse of TRH (10-8 M) was administered for 10 min in the absence or presence of PMA, respectively. B, Effect of the PKC inhibitor NPC-15437 on TRH-induced {alpha}-MSH release from perifused frog NILs. Top, Control experiment showing the effect of two pulses of TRH (10-8 M; 10 min each) given at 135-min interval. Bottom, The second pulse of TRH was administered during prolonged infusion of NPC-15437 (10-5 M; 100 min). Each profile represents the mean (± SEM) secretion pattern of four independent perifusion experiments. The spontaneous level of {alpha}-MSH release (100% basal level) was calculated as the mean {alpha}-MSH concentration in the five consecutive fractions (O-O; 37.5 min) immediately preceding the administration of the first pulse of TRH. The mean basal level of {alpha}-MSH in these experiments was 163 ± 15 pg/min per NIL. The brackets indicate the limits of the peaks that were used to calculate the AUCs for statistical analysis.

 


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  		Figure 2. Effect of PKC inhibition on the Ca2+ response of melanotrope cells to TRH. A and B, Typical profiles illustrating the two types of responses that were observed after administration of a single pulse of TRH (10-7 M; 5 sec) to cultured melanotrope cells. C, The cells were preincubated with 10-6 M PMA for 24 h. D, The cells were preincubated with 10-6 M NPC-15437 for 10 min. Arrows indicate the onset of TRH administration. E, Histograms showing the mean effect of PMA and NPC-15437 on the amplitude of the [Ca2+]i transient induced by TRH. n.s., Not statistically different.

 
Effects of inhibition of the calmodulin pathway on TRH-induced {alpha}-MSH secretion
Administration of the calmodulin inhibitor W-7 (10-5 M) for 100 min to perifused frog NILs did not impair the effect of TRH (10-8 M) on {alpha}-MSH release (Fig. 3Go, A and C). Similarly, administration of the CAM KII inhibitor KN-93 (10-5 M; 100 min) did not affect the secretory response of NILs to TRH (Fig. 3Go, B and C).



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  		Figure 3. Effect of calmodulin inhibitors on the secretory response of frog NILs to TRH. A, Effect of W-7 (10-5 M; 100 min) on TRH-induced {alpha}-MSH release from perifused frog NILs. B, Effect of KN-93 (10-5 M; 100 min) on TRH-induced {alpha}-MSH release from perifused frog NILs. C, Histograms showing the mean effect of W-7 or KN-93 on the {alpha}-MSH response to TRH. The mean values were calculated as the AUCs from the perifusion profiles shown in A and B. n.s., Not statistically different. The mean basal level of {alpha}-MSH in these experiments was 124 ± 17 pg/min per NIL. See to Fig. 1Go for other details.

 
Effect of TRH on cAMP formation
Incubation of frog NILs with forskolin (10-5 M; 20 min) significantly increased (P < 0.001) the concentration of cAMP in the tissue (Fig. 4AGo). In contrast, incubation of NILs with TRH (10-6 M; 20 min) did not modify the cAMP level (Fig. 4AGo). Administration of the adenylyl cyclase inhibitor SQ 22,536 (10-4 M) for 100 min to perifused frog NILs did not affect TRH-induced {alpha}-MSH release (Fig. 4BGo).



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  		Figure 4. A, Effect of forskolin (10-5 M) and TRH (10-6 M) on cAMP formation in frog NILs. B, Effect of the adenylyl cyclase inhibitor SQ 22,536 (SQ; 10-4 M) on TRH-induced {alpha}-MSH secretion from perifused frog NILs. The mean basal level of {alpha}-MSH in these experiments was 145 ± 40 pg/min per NIL. See to Fig. 1Go for other details. ***, P < 0.001; n.s., Not statistically different.

 
Effects of inhibition of PLA2 on TRH-induced {alpha}-MSH secretion
Administration of the PLA2 inhibitor quinacrine (10-4 M; 100 min) to perifused frog NILs induced by itself a transient stimulation of {alpha}-MSH secretion (Fig. 5AGo). However, quinacrine did not affect TRH-induced {alpha}-MSH release (Fig. 5Go, A and C). Similarly, administration of 7,7'-dimethyl-5,8-eicosadienoic acid (DEA) (10-4 M; 100 min) to perifused frog NILs did not impair the stimulatory effect of TRH on {alpha}-MSH release (Fig. 5Go, B and C).



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  		Figure 5. Effect of PLA2 inhibitors on the secretory response of frog NILs to TRH. A, Effect of quinacrine (Quina; 10-4 M; 100 min) on TRH-induced {alpha}-MSH release from perifused frog NILs. B, Effect of 7,7'-DEA (DEA; 10-4 M; 100 min) on TRH-induced {alpha}-MSH release from perifused frog NILs. C, Histograms showing the mean effect of quinacrine or 7,7'-DEA on the {alpha}-MSH response to TRH. The mean values were calculated as the AUCs from the perifusion profiles shown in A and B. n.s., Not statistically different. The mean basal level of {alpha}-MSH in these experiments was 186 ± 22 pg/min per NIL. See to Fig. 1Go for other details.

 
Effects of inhibition of PTK on TRH-induced {alpha}-MSH secretion and [Ca2+]i increase
Administration of the PTK inhibitor ST638 (10-4 M) for 100 min to perifused frog NILs markedly reduced (P < 0.01) the stimulatory effect of TRH on {alpha}-MSH release (Fig. 6AGo). Similarly, infusion of the PTK inhibitor Tyr-A23 (10-4 M; 100 min) significantly inhibited (P < 0.01) the effect of TRH on {alpha}-MSH release (Fig. 6BGo). Administration of graded concentrations of ST638 or Tyr-A23 caused a dose-dependent inhibition of TRH-induced {alpha}-MSH release, with ED50 of 1.22 x 10-5 M and 1.47 x 10-5 M, respectively (Fig. 6Go, C and D).



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  		Figure 6. Effect of PTK inhibitors on the secretory response of frog NILs to TRH. A and B, Effect of ST638 (10-4 M; 100 min) or tyrphostin 23 (Tyr-A23; 10-4 M; 100 min) on TRH-induced {alpha}-MSH release from perifused frog NILs. C and D, Effect of graded doses of ST638 or Tyr-A23 on TRH-induced {alpha}-MSH release. The values were calculated as the AUCs from perifusion profiles similar to those shown in A and B. The mean basal level of {alpha}-MSH in these experiments was 132 ± 23 pg/min per NIL. See to Fig. 1Go for other details.

 
Incubation of cultured melanotrope cells with ST638 (10-5 M; 10 min) did not modify the proportion of cells exhibiting sustained and transient [Ca2+]i responses to TRH (65% and 35%, respectively) (Fig. 7Go, A–D). In addition, preincubation of cells with ST638 (10-5 M; 10 min) or Tyr-A23 (10-5 M; 10 min) did not affect the amplitude of the [Ca2+]i response induced by TRH (Fig. 7EGo).



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  		Figure 7. Effect of PTK inhibitors on the Ca2+ response of melanotrope cells to TRH. A and B, Typical profiles illustrating the two types of responses that were observed after administration of a single pulse of TRH (10-7 M; 5 sec) to cultured melanotrope cells. C and D, The cells were preincubated with ST638 (10-5 M) for 10 min. Arrows indicate the onset of TRH administration. E, Histograms showing the mean effect of ST638 and Tyr-A23 on the amplitude of the transient phase of the [Ca2+]i increase induced by TRH. n.s., Not statistically different.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that, in frog melanotrope cells, TRH activates the phospholipase C pathway (20), and we have determined the respective contribution of extracellular and intracellular Ca2+ sources in TRH-induced {alpha}-MSH secretion (21). These studies have shown that either Ca2+ entry through L-type Ca2+ channels or mobilization of Ca2+ from IP3-sensitive Ca2+ stores is sufficient to induce maximum {alpha}-MSH release. The present study has investigated the contribution of PKC, adenylate cyclase, PLA2, and PTK in TRH-induced {alpha}-MSH secretion in frog melanotrope cells.

In GH3 cells, the stimulatory effect of TRH on PRL release is associated with [Ca2+]i increase and activation of a PKC. It has been shown that, in these cells, activation of PKC is responsible for the plateau-phase of the Ca2+ response and the TRH-evoked stimulation of hormone secretion (1, 28). In contrast, in female rat pituitary cells, blockage of PKC does not affect TRH-induced PRL release (29). In the present study, we have shown that down-regulation of PKC by long term treatment of frog NILs with PMA (29) or exposure of perifused NILs to the PKC inhibitor NPC-15437 (30) reduced by 50% the effect of TRH on {alpha}-MSH release, demonstrating the involvement of PKC in the TRH-evoked stimulation of hormone secretion. In frog melanotrope cells, the sustained phase of the [Ca2+]i increase provoked by TRH is due to Ca2+ entry through N-type calcium channels (21). Treatment of cultured cells with PMA or NPC-15437 resulted in complete suppression of the sustained calcium response indicating that TRH-induced stimulation of PKC is responsible for the activation of N-type calcium channels. However, we have previously shown that, in frog melanotrope cells, the sustained phase of the [Ca2+]i increase is not involved in TRH-induced {alpha}-MSH release (21). It thus appears that the stimulatory effect of PKC on hormone release cannot be accounted for by its effect on Ca2+ mobilization. In GH4C1 cells, TRH-induced fragmentation of actin filaments is mediated through a PKC-dependent, Ca2+-independent pathway (31). Whether, in frog melanotrope cells, the effect of PKC on {alpha}-MSH release can be ascribed to rearrangement of the microfilament network remains to be determined.

It has been previously reported that the stimulatory effect of TRH on GH3 cells (4) and rat anterior pituitary cells (5) proceeds via activation of CAM KII. We have thus investigated the possible involvement of the CAM KII pathway in TRH-induced {alpha}-MSH release. The data showed that exposure of perifused frog NILs to the calmodulin inhibitor W-7 (32) or to the CAM KII inhibitor KN-93 (33) did not modify the stimulatory effect of TRH, indicating that, in frog melanotrope cells, CAM KII does not contribute to the secretory response evoked by TRH.

Most studies aimed at investigating the transduction pathways associated with activation of the TRH receptor have shown that TRH has no effect on cAMP formation (34, for review). However, a few reports suggest that TRH may stimulate adenylyl cyclase activity in normal rat anterior pituitary cells (35) and in GH tumor cells (2, 3). The present study has revealed that TRH has no effect on cAMP production in frog NILs. In addition, the adenylyl cyclase inhibitor SQ 22,536 (36) did not affect the TRH-evoked {alpha}-MSH secretion, indicating that the PKA pathway does not contribute to the secretory response of melanotrope cells to TRH. Consistent with these data, it has been shown that TRH does not affect adenylyl cyclase activity in five different cell types transfected with the mouse TRH receptor (37).

Previous reports have indicated that activation of PLA2 induces an increase in arachidonate production and PRL secretion from rat and bovine anterior pituitary cells (7, 38, 39, 40) and from GH cells (6, 41, 42). Reciprocally, PLA2 or lipoxygenase inhibitors inhibit both basal and TRH-stimulated PRL release from normal and tumoral pituitary cells (40, 42). Exposure of frog NILs to the PLA2 inhibitors quinacrine or 7–7'-DEA (43, 44) did not affect basal and TRH-induced {alpha}-MSH release. Consistent with these data, it has been previously shown that, the cyclooxygenase inhibitor indomethacin does not impair the stimulatory effect of TRH on {alpha}-MSH release (45). Taken together, these data indicate that, in contrast to mammotrope cells, arachidonic acid metabolites are not involved in basal and TRH-stimulated hormone release from frog melanotrope cells.

It is now well established that activation of various heterotrimeric G protein-coupled receptors can stimulate phosphorylation of the mitogen-activated protein (MAP) kinase pathway (46). In particular, TRH induces tyrosine phosphorylation of MAP kinase in tumoral and normal pituitary cells (8, 9, 10). TRH also activates the MAP-kinase cascade in COS-7 cells transfected with the TRH receptor (11). The present study has shown that exposure of NILs to the PTK inhibitors ST638 and Tyr-A23 (47, 48) abolished TRH-induced {alpha}-MSH release, indicating that activation of a tyrosine kinase is involved in the secretory response of melanotrope cells to TRH. Similarly, in tumoral pituitary cells, TRH-induced PRL secretion can be suppressed by ST638 or Tyr-A23 (8). The observation that ST638 and Tyr-A23 did not impair the stimulatory effect of TRH on the calcium response of melanotrope cells indicates that PTK operates downstream calcium mobilization. In support of this hypothesis, it has been shown that microtubule-associated protein 2, which contributes to association of microtubules with secretory vesicles, is one of the MAP kinase substrates (49), and it has been demonstrated that TRH increases the level of microtubule-associated phosphoproteins in rat lactotrophs (50).

In conclusion, the present study has shown that, in frog melanotrope cells, protein phosphorylation by PKC and PTK plays a pivotal role in TRH-induced {alpha}-MSH release. In contrast, calmodulin/CAM KII, adenylyl cyclase, and PLA2 do not significantly contribute to the secretory response of the cells to TRH. These data, together with two previous reports (20, 21), provide a comprehensive picture of the transduction mechanisms associated with activation of TRH receptors in frog melanotrope cells.


   Acknowledgments
 
The authors wish to thank Miss C. Buquet for technical assistance in cell culture.


   Footnotes
 
1 This work was supported by grants from INSERM (U 413), European Union Human Capital Mobility Program (Contract No. HMR-ERBCHRXCT920017), an INSERM-NWO exchange program, the Lille-Amiens-Rouen-Caen (LARC)-Neuroscience network, and the Conseil Régional de Haute-Normandie. Back

2 Recipients of fellowships from the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche. Back

Received October 27, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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